### EPA/600/R-09/057F



# Nanomaterial Case Studies:
Nanoscale Titanium Dioxide in Water Treatment and in Topical Sunscreen

November 2010

National Center for Environmen tal Assessment–RTP Division Office of Research and Development U.S. Environmental Protection Agency Research Triangle Park, NC

# Disclaimer

This document has been reviewed in accordance with U.S. Environmental Protection Agency policy and approved for publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.

ii

# Table of Contents

List of Figures _________________________________________________________ vi

List of Tables _________________________________________________________ vii
Authors, Contributors, and Reviewers ____________________________________ viii

Abbreviations ________________________________________________________ xiii

Foreword ____________________________________________________________ xix
Chapter 1. Introduction ________________________________________________ 1-1

1.1. Background _________________________________________________________________ 1-1
1.2. How to Read this Document

_____________________________________________________ 1-3
1.3. Terminology _________________________________________________________________ 1-4
1.4. Conventional TiO 2


### _____________________________________________________________ 1-7
1.5. Nano-TiO 2


### ___________________________________________________________________ 1-8
1.5.1. Drinking Water Treatment _______________________________________________ 1-12
1.5.2. Sunscr een

___________________________________________________________ 1-13
1.6. Analytical Methods ___________________________________________________________ 1-14
1.6.1. Methods for Laborat ory Research _________________________________________ 1-15
1.6.2. Methods and Instrumentation to Assess Environmental Occurrence ______________ 1-16
1.6.3. Methods and Instrumentation to Assess Workplace Exposure ___________________ 1-18
1.6.4. Summary of Analytic Methods ____________________________________________ 1-19 

# Chapter 2. Life Cycle Stages ____________________________________________ 2-1

2.1. Feedsto cks __________________________________________________________________ 2-1
2.2. Manufacturing ________________________________________________________________ 2-2
2.2.1. Drinking Water Treatment ________________________________________________ 2-3
2.2.2. Sunscr een

____________________________________________________________ 2-3
2.3. Distribution and Storage ________________________________________________________ 2-3
2.3.1. Drinking Water Treatment ________________________________________________ 2-4
2.3.2. Sunscr een

____________________________________________________________ 2-4
2.4. Use ________________________________________________________________________ 2-4
2.4.1. Drinking Water Treatment ________________________________________________ 2-4
2.4.2. Sunscr een

____________________________________________________________ 2-6
2.5. Disposal ____________________________________________________________________ 2-7
2.5.1. Drinking Water Treatment ________________________________________________ 2-7
2.5.2. Sunscr een

____________________________________________________________ 2-7 

# Chapter 3. Fate and Transport __________________________________________ 3-1

3.1. Water ______________________________________________________________________ 3-2
3.1.1. Drinking Water Treatment ________________________________________________ 3-4
3.1.2. Sunscr een

____________________________________________________________ 3-5
3.2. Soil ________________________________________________________________________ 3-5
3.2.1. Drinking Water Treatment ________________________________________________ 3-7
3.2.2. Sunscr een

____________________________________________________________ 3-7
3.3. Air _________________________________________________________________________ 3-7

iii

# Chapter 4. Exposure–Dose Characterization ______________________________ 4-1

4.1. Biota _______________________________________________________________________ 4-2
4.1.1. Aquatic Species ________________________________________________________ 4-2
4.1.2. Terrestrial Species ______________________________________________________ 4-3
4.2. Humans ____________________________________________________________________ 4-3
4.2.1. General Population _____________________________________________________ 4-3
4.2.1.1. Drinking Water Treatment ________________________________________ 4-3
4.2.1.2. Sunscr een

____________________________________________________ 4-4
4.2.2. Occupational __________________________________________________________ 4-6
4.3. Aggregate Exposure to Nano-TiO 2 

### from Multiple So urces and Pathways __________________ 4-8
4.4. Cumulative Exposure to Nano-TiO 2 

### and Other Contaminants ___________________________ 4-9
4.5. Models to Estimate Exposure

____________________________________________________ 4-9
4.6. Dose ______________________________________________________________________ 4-10
4.6.1. Uptake in Aquatic Species _______________________________________________ 4-11
4.6.1.1. Bioaccumulation _______________________________________________ 4-11
4.6.1.2. Food Web ____________________________________________________ 4-12
4.6.1.3. Cumulative Dose of Nano-TiO 2 

### and Other Pollutants __________________ 4-12
4.6.2. Respiratory (Inhalation and Instillation) _____________________________________ 4-14
4.6.3. Dermal ______________________________________________________________ 4-16
4.6.4. Ingestion ____________________________________________________________ 4-24
4.6.5. Blood Brain Barrier and Placental Transfer __________________________________ 4-24
4.6.6. Dose Me trics _________________________________________________________ 4-26 

# Chapter 5. Characterization of Effects ____________________________________ 5-1

5.1. Factors that Influence Ecological and Health Effects of Nano-TiO 2


### _______________________ 5-1
5.1.1. Nano-TiO 2 

### Physicochemical Characteristics __________________________________ 5-2
5.1.1.1. Size __________________________________________________________ 5-2
5.1.1.2. Crystallinity ____________________________________________________ 5-3
5.1.1.3. Surface Chemistry ______________________________________________ 5-3
5.1.1.4. Recommended Characterization of Nanomaterial for Ecological and Toxicologica l Studies ____________________________________________ 5-4
5.1.2. Experimental Conditions _________________________________________________ 5-6
5.1.2.1. Medium/Vehicle ________________________________________________ 5-7 5.1.2.2. Dispersion Preparation ___________________________________________ 5-8
5.1.3. Environmental Conditions ________________________________________________ 5-9
5.1.4. Summary ____________________________________________________________ 5-10
5.2. Ecological Effects ____________________________________________________________ 5-10
5.2.1. Ecological Effects of Nano-TiO 2 

### Exposure __________________________________ 5-10
5.2.1.1. Effects on Bacteria and Fungi (Terrestrial an d Aquati c) ________________ 5-16
5.2.1.2. Effects on Aquatic Organisms ____________________________________ 5-17
5.2.1.3. Effects on Terrestrial Organisms __________________________________ 5-21
5.2.1.4. Indirect and Interactive Ecological Effects ___________________________ 5-23
5.2.1.5. Summary ____________________________________________________ 5-23
5.3. Health Effects _______________________________________________________________ 5-24
5.3.1. Noncarcinogenic Effects ________________________________________________ 5-24
5.3.1.1. Studies in Humans _____________________________________________ 5-24
5.3.1.2. Animal Studies ________________________________________________ 5-25
5.3.1.3. Summary of Noncarcinogenic Effects ______________________________ 5-51
5.3.2. Carcinogenic Effects ___________________________________________________ 5-51
5.3.2.1. Studies in Humans _____________________________________________ 5-52
5.3.2.2. Animal Studies ________________________________________________ 5-52

iv

### 5.3.2.3. Modes of Action fo r Carcinog enicity

________________________________ 5-56
5.3.2.4. Summary of Carcinogenic Effects _________________________________ 5-58 

# Chapter 6. Summary __________________________________________________ 6-1

6.1. Case Study Highlights _________________________________________________________ 6-1
6.1.1. Analytical Methods _____________________________________________________ 6-2
6.1.2. Life Cycle Characterization _______________________________________________ 6-3
6.1.3. Fate and Transport _____________________________________________________ 6-5
6.1.4. Exposure and Dose Characterization _______________________________________ 6-6
6.1.4.1. Exposure Characterization ________________________________________ 6-6
6.1.4.2. Dose Characterization ___________________________________________ 6-8
6.1.5. Ecological and Health Effects ____________________________________________ 6-10
6.1.5.1. Ecological Effects ______________________________________________ 6-10
6.1.5.2. Health Effects _________________________________________________ 6-11
6.2. Role of Case Studies in Research Planning and Assessment Efforts ____________________ 6-12
6.2.1. Workshop on Research Priorities for Nano-TiO 2


### ______________________________ 6-12
6.2.2. Implications for Research Planning ________________________________________ 6-13
6.2.3. Implications for Future Assessment Efforts __________________________________ 6-13


# REFERENCES ______________________________________________________ 6-15

Annex A. Nano-TiO 2 

# in Sunscreen: Background Information _________________ A-1

REFERENCES _______________________________________________________ A-8

Annex B. Nano-TiO 2 

# in Sunscreen: Ma nufacturing Processes ________________ B-1

REFERENCES ______________________________________________________ B-10

Annex C. Nano-TiO 2 

# Exposure Control in the Wo rkplace and Labor atory ______ C-1

REFERENCES _______________________________________________________ C-4

v

# List of Figures

Figure 1-1. Basic structure of CEA as a framework for identifying and prioritizing research efforts. _________________________ 1-2

Figure 4-1. Possible pathways of nano-TiO 2 

### skin penetration. ____________________________________________________ 4-19

Figure 5-1. The pulmonary effects of TiO 2 

### or nano-TiO 2 

### exposure through inhalati on or instillation. _______________________ 5-58

Figure B-1. Generic manufacturing process for nano-TiO 2 

### for sunscreens. __________________________________________ B-1

Figure B-2. Sulfate and chloride processes for TiO 2 

### manufacture. _________________________________________________ B-2

Figure B-3. Nano-TiO 2 

### manufacturing process used by Alta ir Nanotechnologi es, Inc. __________________________________ B-3

vi

# List of Tables

Table 1-1. Examples of nano-TiO 2 

### physicochemica l properties ____________________________________________________ 1-9

Table 1-2. Characterization of three nano-TiO 2 

### particle types _____________________________________________________ 1-11

Table 1-3. Analytical met hods for characterizing nanomateria ls in aeroso l and in liquid_________________________________ 1-16

Table 1-4. Analytical methods for nanomaterials in soil, sediment, and ground wa ter for size fraction and distribution, surfac e area, and phase and structure _________________________________________________________________________ 1-18

Table 4-1. Estimated dermal exposure to nano-TiO 2 

### from sunscreen c ontaining 5% nano-TiO 2 

### for adults and 3-year-old children 4-5

Table 4-2. Tissue concentrations of various pollutants in fish after exposures to nano-TiO 2 

### in water _______________________ 4-13

Table 4-3. Nano-TiO 2 

### disposition in animals after inhalat ion or intratrac heal instilla tion _________________________________ 4-16

Table 4-4. Overview of TiO 2 

### skin absorption/ penetration studies __________________________________________________ 4-20

Table 4-5. Animal studies that measured Ti concentrations in brain after nano-TiO 2 

### exposures through injection or oral gavage_ 4-25

Table 5-1. Published recommendations for measuring nanomaterial parameters for ex posure during characte rization inhalation studies ______________________________________________________________________________________ 5-5

Table 5-2. Published recommendati ons for off-line nanomaterial characterization using noncontinuous techniques for toxicologic al studies ______________________________________________________________________________________ 5-6

Table 5-3. Summary of nano-TiO 2 

### ecological effects____________________________________________________________ 5-11

Table 5-4. Summary of health effects of nano-TiO 2 

### particles in mammalian ani mal models: dermal route __________________ 5-27

Table 5-5. Summary of health effects of nano-TiO 2 

### particles in mammalian ani mal models: oral route _____________________ 5-30

Table 5-6. Summary of health effects of nano-TiO 2 

### particles in mammalian animal models: respiratory route _______________ 5-35

Table 5-7. Summary of health effects of nano-TiO 2 

### particles in mammalian animal models: other (injec tion, ocular) route ______ 5-50

Table 5-8. Treatments and pulmonary tumor incide nces in rats exposed to fine and nano-TiO 2 

### through intratracheal instillation in Pott and Roller ( 2005) study ______________________________________________________________________ 5-54

Table 5-9. Incidence of tumor in the abdominal cavity of rats intraperitonea lly injected with photocatalytic nano-TiO 2 

### . _________ 5-56

Table 5-10. Results of nano-TiO 2 

### carcinogenicity st udies in animals ________________________________________________ 5-59

Table A-1. TiO 2 

### content in various s unscreen products. _________________________________________________________ A-7

Table B-1. Selected list of nano-TiO 2 

### particles used in sunscreen _________________________________________________ B-6

Table B-2. Formula SC-383-1 for “Weightle ss Morning Dew with Sun Protection” _____________________________________ B-8

vii

# Authors, Contributors, and Reviewers

Principal Authors

J. Michael Davis (Project Leader)—National Cent er for Environmental Assessment, Office of Research and Development, U.S. Environmental Pr otection Agency, Research Triangle Park, NC

Thomas C. Long—National Center for Environmental Assessment, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC

Jo Anne Shatkin—The Cadmus Group (currently with CLF Ventures)

Amy Wang—Oak Ridge Institute for Science and Educ ation, Postdoctoral Fellow to National Center for Environmental Assessment, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC



# Co-Authors

Judith A. Graham—Private Consultant

Maureen Gwinn—National Center for Environmental Assessment, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC

Brent Ranalli—The Cadmus Group



# Contributors

Christian Andersen—National Health and Environm ental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Pr otection Agency, Research Triangle Park, NC

Walter Cybulski—Office of Science Policy, Of fice of Research and Development, U.S. Environmental Protection Agency, Washington, DC

Genya Dana—Oak Ridge Institute for Science a nd Education, Postdoctoral Fellow to National Center for Environmental Assessment, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC

J. Allen Davis—National Center for Environm ental Assessment, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC

Steve Diamond—National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC

Kevin Dreher—National Health and Environmental Ef fects Research Laboratory, Office of Research and Development, U.S. Environmental Protec tion Agency, Research Triangle Park, NC

Patricia Gillespie—Oak Ridge Institute for Scien ce and Education, Postdoctoral Fellow to National Center for Environmental Assessment, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC

viii

### Connie Meacham—National Center for Environmental Assessment, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC

Emily Monosson—Private Consultant to the Cadmus Group

Jeffery Morris—Immediate Office of the Assist ant Administrator, Office of Research and Development, U.S. Environmental Protection Agency, Washington, DC

Christine Ogilvie-Hendren—Oak Ridge Institute fo r Science and Education, Postdoctoral Fellow to National Center for Environmental Assessment, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC

Stephanie Rosch—The Cadmus Group

Chon Shoaf—National Center for Environmental Asse ssment, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC

John Vandenberg—National Center for Environmental Assessment, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC

Debra Walsh—National Center for Environmental Assessment, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC

Sally White—Student Services Contractor to Nati onal Center for Environmental Assessment, Office of Research and Development, U.S. Environmenta l Protection Agency, Research Triangle Park, NC

William Wilson—National Center for Environmental Assessment, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC



# External Reviewers 1



### Pratim Biswas—Washington University

Bernard Goldstein—University of Pittsburgh

Fred Klaessig—Degussa

Rebecca Klaper—University of Wisconsin

Terry Medley—DuPont (with David Warheit, Gary Whiting, Scott Frerichs, and Brian Coleman)

Srikanth Nadadur—National Institute of Environmental Health Sciences

Gunter Oberdörster—University of Rochester

John A. Small—National Institute of Standar ds and Technology (with Richard Holbrook)

Jeffrey Steevens—U.S. Army Corps of Engineers

Mark Wiesner—Duke University

1 Reviewers for the November 2007 draft of case study on nano-TiO 2 in drinking water treatment.



### ix

# Peer Reviewers 1



### Steffen Foss Hansen—Technical University of Denmark

Kiril D. Hristovski—Arizona State University

Stephen J. Klaine—Clemson University

Bernd Nowack—Swiss Federal Laboratories for Materials Testing and Research (EMPA)

Annette B. Santamaria—ENVIRON International Corporation

Kathleen E. Sellers—ARCADIS 

# Workshop Participants 2



### David Andrews–Environmental Working Group

Jeff Baker–TSI Incorporated

Brenda Barry–American Chemistry Council

Catherine Barton–DuPont

Eula Bingham–University of Cincinnati

Pratim Biswas–Washington University in St. Louis

Jean-Claude Bonzongo–University of Florida

Steven Brown–Intel Corporation

Mark Bunger–Lux Research, Incorporated

Carolyn Nunley Cairns–Consumers Union

Richard Canady–McKenna, Long & Aldridge LLP

Janet Carter–U.S. Occupational Sa fety and Health Administration

Elizabeth Casman –Carnegie Mellon University

Sylvia Chan Remillard–HydroQual

Shaun Clancy–Evonik Industries AG

Ramond David–BASF Corporation

Joan Denton–California Environmental Protection Agency

Gary Ginsberg–Connecticut Department of Public Health

Pertti (Bert) Hakkinen–National Institutes of Health, National Library of Medicine

Jaydee Hanson–International Center for Technology Assessment

Patricia Holden–University of California, Santa Barbara, CA

Paul Howard–U.S. Food and Drug Administration

Sheila Kaplan–University of California, Grad uate School of Journalism, Berkeley, CA

1 Reviewers of the April 2010 draft of Nanomaterial case Studies: Nanoscale Titanium Dioxide in Water Treatment and in Topical
Sunscreen

.

2 EPA’s “Nanomaterial Case Studies Workshop: Developing a Comprehe nsive Environmental Assessment Research Strategy for Nanoscale Titanium Dioxide,” Research Triangle Park, NC, September 29-30, 2009



### x

### Fred Klaessig–Pennsylvania Bio Nano Systems, LLC

Rebecca Klaper–University of Wisconsin, Great Lakes Water Institute

Todd Kuiken–Woodrow Wilson International Center for Scholars, Project on Emerging Nanotechnologies

John LaFemina–Battelle

Thomas Lee–Minneapolis Star Tribune

Shannon Lloyd–Concurrent Technologies Corporation

Christopher Long–Gradient Corporation

Margaret MacDonell–Argonne National Laboratory

Fred J. Miller–Independent Consultant

Nancy Monteiro-Riviere–North Carolina State University

Paul Mushak–PB Associates

Srikanth Nadadur–National Institutes of Health, National Institute of Environmental Health Sciences

Michele Ostraat–Research Triangle Institute

Anil Patri–Science Applications International Corpora tion, contractor with National Cancer Institute, Nanotechnology Characterization Laboratory

Maria Victoria Peeler–Washingt on State Department of Ecology

Richard Pleus–Intertox, Incorporated

John Small–National Institute for Standards and Technology

Jeff Steevens–U.S. Army Corps of Engineers Research and Development Center

Geoffrey Sunahara–National Research Counc il, Canada, Biotechnology Research Institute

Treye Thomas–U.S. Consumer Product Safety Commission

John Veranth–University of Utah

Donald Versteeg–The Procter & Gamble Company

Nigel Walker–National Toxicology Program, Nationa l Institutes of Health, National Institute for
Environmental Health

William Warren-Hicks–EcoS tat, Incorporated

Paul Westerhoff–Arizona State University

Mark Wiesner–Duke University

xi

### xii 

# EPA Workgroup 1



### Christian Andersen # 

### Kathryn Gallagher David Olszyk
Rochelle Araujo Michael Gill Martha Otto

Fred Arnold Michael Gonzalez Scott Prothero
Ayaad Assaad Maureen Gwinn + 

### Kim Rogers

Norman Birchfield Kathy Hart Nora Savage
Deborah Burgin Tala Henry Phil Sayre

Jim Caldwell Ross Highsmith Rita Schoeny
David Cleverly Lee Hofmann Walter Schoepf

Michele Conlon Marion Hoyer Najm Shamim

Mary Ann Curran Joe Jarvis Deborah Smegal
Walter Cybulski # 

### Bernine Khan Jose Solar

J. Michael Davis* + 

### David Lai Neil Stiber

Jane Denne Wen-Hsiung Lee Timothy Taylor

Steve Diamond # 

### Laurence Libelo Susan Thorneloe
Jaimee Dong Diana Locke Dennis Utterback

Kevin Dreher # 

### Jacqueline McQueen* Amy Wang +



### Jeremiah Duncan David Meyer Eric Weber
Brian Englert Gregory Miller Randy Wentsel

Patricia Erickson J. Vincent Nabholz Doug Wolf
Cathy Fehrenbacher Nhan Nguyen

Gina Ferreira Carlos Nunez


# U.S. EPA Editorial Support

J. Sawyer Lucy—Student Services Contractor to National Center for Environmental Assessment, Office of Research and Development, U.S. Envi ronmental Protection Agency, Research Triangle Park, NC

Deborah Wales—National Center for Environm ental Assessment, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC

Barbara Wright—Senior Environmental Employee to National Center for Environmental Assessment, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC 

# Contractor Editorial Support

ICF International

1 *Co-Chair; + Co-author; # Contributor

# Abbreviations

α -HBDH Alpha-hydroxybutyrate dehydrogenase

ACGIH American Conference of Governmental Industrial Hygienists

AFM Atomic force microscopy

Al 2 

### (SO 4 

### ) 3 

### · 16H 2 

### O Alum

Al 2 

### O 3 

### Aluminum oxide, also known as alumina

ALP Alkaline phosphatase

ALT Alanine aminotransferase
As(III) Arsenite
As(V) Arsenate

AST Aspartate aminotransferase

BAL Bronchoalveolar lavage

BALF Bronchoalveolar lavage fluid

BAuA German Occupational Safety and Health (Bundesanstalt für Arbeitsschutz und Arbeitsmedizin)

BBB Blood brain barrier

BET Brunauer, Emmett, Teller meth od of calculating surface area

BrdU Bromo-deoxy-uridine

BUN Blood urea nitrogen

BW Body weight

oC Degree(s) Celsius

C 60 

### Spherical fullerene composed of 60 carbon atoms; commonly “Bucky ball”

Ca 2+ 

### Calcium cation

CCOHS Canadian Centre for Occupational Health and Safety

CE Capillary electrophoresis

CEA Comprehensive environmental assessment

CK Creatinine kinase

cm 2 

### Square centimeter(s), Centimeter(s) squared

cm 3 

### Cubic centimeter(s), Centimeter(s) cubed

CMD Count median diameter

CPC Condensation particle counter

CREM Council for Regulatory Environmental Modeling

CVD Chemical vapor deposition

DIN Deutsches Institut für Normung (German Institute for Standardization)

DLS Dynamic light scattering
DMA(V) Dimethylarsinic acid

DMEM Dulbecco’s Modified Eagle’s Medium

DPPC Dipalmitoyl phosphatidylcholine

EC3 Estimated concentration required to induce a threshold positive response, where stimulation index equals 3

EC 50 

### Half-maximal effective concentration, Effectiv e concentration 50; the concentration at which 50% of subjects show a response

EDS Energy-dispersive X-ray analysis

E-FAST V2.0 Exposure and Fate Assessment Screening Tool Version 2.0

xiii

### EHS Environmental health and safety

ELISA Enzyme-linked immunosorbent assay

ELPI Electrical low pressure impactor

EM Electron microscopy

EN European Norm

EPA U.S. Environmental Protection Agency

EU European Union

EWG Environmental Working Group

oF Degree(s) Fahrenheit

F344 Fischer 344 (Rat strain)

FDA U.S. Food and Drug Administration

FE-SEM Field emission-type scanning electron microscopy

FeTiO 3 

### Ilmenite

FFF Field flow fractionation

FHD Flame hydrolysis deposition

γ H2AX Phosphorylated form of histone H2AX (phosphorylation of H2AX at serine 139)

g Gram(s)
g/kg Gram(s) per kilogram

GFAP Glial fibrillary acidic protein

GGT γ –Glutamyltransferase

GSD Geometric standard deviation

GSH Reduced glutathione

GSH-Px Glutathione peroxidase

GST Glutathione-S-transferase

H 2 

### O 2 

### Hydrogen peroxide

H 2 

### SO 4 

### Sulfuric acid

HBSS Hank’s Basic Salt Solution

HCl Hydrochloric acid

HEPA High efficiency particulate air

HPLC High performance liquid chromatography
hprt Hypoxanthine-guanine phosphoribosyltransferase (gene)

HRTEM High resolution transmi ssion electron microscopy

Hz Hertz i.p. Intraperitoneal

i.v. Intravenous

IAEA International Atomic Energy Agency

IARC International Agency fo r Research on Cancer

IC 20 

### , IC 25 

### Concentration that results in a 20% (or 25%) change (inhibition) from the control response, Inhibitory concentration at which organisms show 20%, 25% inhibition in measured endpoints

ICP Inductively coupled plasma

ICP-AES Inductively coupled plasma atomic emission spectrometry

ICP-MS Inductively coupled plas ma-mass spectrometry

IEP Isoelectric point

IFN- γ Interferon-gamma

IL-10 Interleukin-10

xiv

### IL-1 β Interleukin-1 β

IL-4 Interleukin-4

IL-6 Interleukin-6
IL-8 (KC) IL-8 = interleukin-8, KC = chemokine (CXC motif) ligand 1 (CXCL1)

ILSI International Life Sciences Institute

IOAA (U.S. EPA) Immediate Office of the Assistant Administrator

ISO International Organization for Standardization

ITT Isopropyl titanium triisostearate

K + 

### Potassium cation
kg Kilogram(s)

L Liter(s)

LC 50 

### Concentration of a chemical that kills 50% of a sample population, Lethal concentration 50; the concentration at which 50% of subjects died

LDH Lactate dehydrogenase

LIBD Laser-induced breakdown detection

LOEC Lowest observed effect concentration

LOEL Lowest observed effect level

LPS Lipopolysaccharide

4-MBC 4-methylbenzylidene camphor

μ g Microgram(s)

μ g/g Microgram(s) per gram

μ g/kg Microgram(s) per kilogram

μ g/L Microgram(s) per liter

μ L Microliter(s)

μ m Micron(s), Micrometer(s)

μ m 2 

### /cm 3 

### Square microns per cubic centimeter, Mi crometer(s) squared per centimeter cubed

m 2 

### Square meter(s), Meter(s) squared

m 2 

### /g Square meter(s) per gram, Meter(s) squared per gram

m 3 

### Cubic meter(s), Meter(s) cubed

MARA Microbial array for risk assessment (assay)

MCL Maximum contaminant level

mg Milligram(s)
mg/cm 2 

### Milligram(s) per square centimeter, Milligram(s) per centimeter squared
mg/kg Milligram(s) per kilogram
mg/L Milligram(s) per liter
mg/m 3 

### Milligrams per cubic meter, Milligram(s) per meter cubed
mg/mL Milligram(s) per milliliter
Mg 2+ 

### Magnesium cation
MgCl 2 

### Magnesium chloride

micro-TiO 2 

### Microscale titanium dioxide
mL/kg/day Milliliter(s) per kilogram per day

mm Millimeter(s)

mM Millimolar
MMA(V) Monomethylarsonic acid

MMAD Mass median aerodynamic diameter

MMPD Multiple Path Particle Dosimetry (name of a computer model)

xv

### MPPS Maximum penetrating particle size

mSv Milliseviert

MT Metric ton(s)

MTC Microbial Toxic Concentration, in microbial array for risk assessment (MARA) assay

MTP Microsomal triglyceride

Na + 

### Sodium cation

NaCl Sodium chloride

NAG Nacetyl- β -glucosaminidase

Nano-TiO 2 

### Nanoscale titanium dioxide

Nano-TiO 2 

### F-1R Nanoscale titanium dioxide a formula containing nano-TiO 2 

### that is 3% anatase and 97% rutile

NCEA (U.S. EPA) National Center for Environmental Assessment

Nano-TiO 2 

### Nanoscale titanium dioxide
ng/mL Nanogram(s) per milliliter

NHEERL (U.S. EPA) National Health and Enviro nmental Effects Research Laboratory

NIOSH National Institute for Occupational Safety and Health

nm Nanometer(s)

NMR Nuclear magnetic resonance

NMRI Naval Medical Research Institute

NOEC No observed effect concentration

NOM Natural organic matter

NOSH Nanoparticle Occupational Safety and Health (Consortium)

O 2 - 

### Superoxide radical anion

OC Octocrylene

OECD Organization for Economic Co-operation and Development

OH Hydroxyl

· OH Hydroxyl radical(s)

OM Octyl methoxycinnamate

· OOH Hydroperoxyl radical(s)

OPC Optical particle counter

OPPT (U.S. EPA) Office of Pollu tion Prevention and Toxics

ORD (U.S. EPA) Office of Research and Development

ORISE Oak Ridge Institute for Science and Education

OSHA PEL Occupational Safety and Health Administration permissible exposure limit

OSP (U.S. EPA) Office of Science Policy

π Pi, approximately equal to 3.14159

p Pink-eyed dilution (gene)

P25 Degussa Aeroxide ® 

### P25 (uncoated nano-TiO 2 

### )

P805 Degussa Aeroxide ® 

### P805 (hydrophobic nano-TiO 2 

### )

PAM Pulse amplitude modulation

PBS Phosphate buffered saline

PEC Predicted environmental concentration
pH Measure of acidity or alkalinity of a solution
pH pzc 

### pH at the point of zero charge

PGF Placenta growth factor

PMN Polymorphonuclear neutrophil

PNEC Predicted no-effect concentration

xvi

### ppb Part(s) per billion

PPE Personal protective equipment

ppm Part(s) per million

PTFE Polytetrafluoroethylene

Pt Platinum

PTM Particle tracking model

p un 

### Pink-eyed unstable (locus)

RLE-TN Rat alveolar type II epithelial cell line

ROS Reactive oxygen species

r PTM Radius particle tracking model

RT-PCR Reverse transcription polymerase chain reaction

σ g 

### Geometric standard deviation

s.c. Subcutaneous

SAXS/WAXS Small- and wide- angle X-ray scattering

SCCNFP (European Commission) Scientific Committee on Cosmetic Products and Non-Food Products
Intended for Consumers

SCCP (European Commission) Scientific Committee on Consumer Products

SCID Severe combined immunodeficiency

SEC Size exclusion chromatography

SEM Scanning electron microscopy

SiO 2 

### Silicon dioxide

SMPS Scanning mobility particle sizer

SOD Superoxide dismutase

SPF Sunburn protection factor

SPM Scanning probe microscopy

St-C n Sunscreen standard C from the Japan Cosmetic Industry

SWCNT Single-walled carbon nanotube(s)

T805 Degussa Aeroxide ® 

### T805 (hydrophobic nano-TiO 2 

### )

TEC Threshold effect concentration

TEM Transmission electron microscopy

TEOM ® 

### Tampered element oscillating microbalance

TFF Tangential-flow ultrafiltration

TGA (Australian) Therapeutic Goods Administration

TGF- β Transforming growth factor-beta

THF Tetrahydrofuran

Ti Titanium

TiCl 4 

### Titanium tetrachloride

TiO 2 

### Titanium dioxide

TiOSO 4 

### Titanyl sulfate

TLV Threshold limit value

TNF- α Tumor necrosis factor-alpha

TRAIL Tumor necrosis factor-related apoptosis-inducing ligand

TS Technical Specification

TUNEL Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling

USP U.S. Pharmacopeia

UV Ultraviolet (light/radiation), wavelengths in the range of 10 to 400 nm

xvii

### UV-A Ultraviolet A, wavelengths in the range of 320 to 400 nm

UV-B Ultraviolet B, wavelengths in the range of 290 to 320 nm

VEDIC Video-enhanced differential interference contrast

WHMIS (Canadian) Workplace Hazardous Ma terials Information System

Wt% Weight percent

XAS X-ray absorption spectroscopy

XPS X-ray photon spectroscopy

XRD X-ray diffraction

ZnO Zinc oxide

xviii

### xix 

# Foreword

Nanoscale materials (nanomaterials) have been described as having at least one dimension on the order of approximately 1 to 100 nanometers (nm) (National Nanotechnology Initiative, 2006,
091186 ). Such materials often have unique or novel properties that arise from their small size. This document is a starting point to determine what is known and what needs to be known about selected nanomaterials as part of a process to identify and pr ioritize research to inform future assessments of the potential ecological and health implications of these materials. Two specific applications of nanoscale titanium dioxide (nano-TiO 2 

### ) are considered: (1) as an agent for removing arsenic from drinking water; and (2) as an active ingredie nt in topical sunscreen. These “case studies” do not represent completed or even preliminary assessment s, nor are they intended to serve as a basis for risk management decisions in the near term on these specific uses of nano-TiO 2 

### . Rather, the intent is to use this document in developing the scientific and technical information needed for future
assessment efforts. The case studies are organized around the comp rehensive environmental assessment (CEA) approach, which combines a product life-cycle fra mework with the risk assessment paradigm. Risk assessment relates exposure and ef fects information for a substance or stressor; CEA expands on this paradigm by including life-cycle stages and considering both indirect and direct ramifications of the substance or stressor. The organization of the document reflects the CEA approach: after Chapter 1 (Introduction), Chapter 2 highlights stages of th e product life cycle (feedstocks, manufacturing, distribution, storage, use, disposal), followed by Chap ter 3 on fate and transport processes, Chapter 4 on exposure-dose characterization, and Chapter 5 on ecological and health effects. Chapter 6 highlights the information that is currently availa ble in each of these areas, and it describes information gaps and research questions identified in the case studies. It also discusses the role of the case studies in informing research planning and fu ture assessment efforts. Appendices A through C provide supplementary information on the use of nano-TiO 2 

### in topical sunscreens, manufacturing processes for nano-TiO 2 

### and sunscreen formulations, and examples of laboratory and workplace exposure control practices, respectively. The intent of these case studies is to characterize the current state of knowledge on the environmental impacts of nano-TiO 2 

### as used in these two specific applications, as well as areas where information is missing. Note that some information gaps are specific to nano-TiO 2 

### , either as a drinking water treatment agent or as an ingredient in topical sunscreen. Other gaps may pertain more broadly to nano-TiO 2 

### irrespective of its application, and still other gaps may pertain even more widely to nanomaterials in general. In this way, the case studies may be used in developing research strategies that will support comprehensive envi ronmental assessments of nanomaterials. The case studies document has undergone a formal external peer review performed by scientists in accordance with Environmental Protection Agency (EPA) guidance on peer review (U.S. EPA, 2006, 194566 ). Six external peer reviewers reviewed the April 2010 draft of this document and provided responses to charge questions on the extent to which the case studies accurately and sufficiently characterize the state of understanding regarding the use of nano-TiO 2 

### in drinking water treatment and sunscreens. This fina l document incorporates revisions in response to the peer review comments.

# Chapter 1. I ntroduction

1.1. Background

Nanoscale materials (nanomaterials) have been described as having at least one dimension on the order of approximately 1-100 nm (National Nanotechnology Initiative, 2006, 091186 ). Engineered nanomaterials are intentionally made, as opposed to being an incidental by-product of combustion or a natural process such as erosion, and they often have unique or novel properties that arise from their small size. These materials are being used in an expanding array of consumer products (The Project on Emerging Nanotechnologies, 2009, 196052 ), and, like all technological developments, nanomaterials offer the potential for both benefits and risks. The assessment of such risks and benefits relies on information, and gi ven the nascent state of nanotechnology, much
remains to be learned about the characteristics an d impacts of nanomaterials before such assessments can be completed. This document is a starting point to identify what is known and, more importantly,
what needs to be known about selected nanomaterial applications – in this case, for nanoscale titanium dioxide (nano-TiO 2 

### ) – to assess their potential ecological and health implications. This document focuses on two specific uses of nano-TiO 2 

### : as a drinking water treatment agent, and as an active ingredient in topical sunscreen. These “case studies” do not represent completed or even preliminary assessments; rather, they present the structure for identifying and prioritizing research needed to support future assessments of nano- TiO 2 

### and an approach to study other
nanomaterials. Part of the rationale for focusing on specific app lications of selected nanomaterials is that such materials and applications can have highly varied and complex properties that make considering them in the abstract or in generalities quite diffic ult. Different materials and different applications of a given material could raise unique questions or issues, as well as some issues that are common to various applications of a given nanomaterial or ev en to different nanoma terials. After several individual case studies have been examined, refini ng a strategy for nanomaterials research to support long-term assessment efforts should be possible. The process for selecting case studies of nano-TiO 2 

### in drinking water treatment and in topical sunscreen involved a workgroup representing severa l EPA program offices, regional offices, and Office of Research and Development (ORD) la boratories and centers. The EPA workgroup
considered several candidate nanomaterials and id entified their preferences based on, among other things, apparent relevance of the nanomaterial to EPA programmatic interests. The choice of specific applications was determined by a smaller team dir ectly involved in the production of the case studies document. Among the factors guiding the selection process at each stage was the potential for exposure of ecological receptors and human populat ions to the nanomaterial as a function of a particular application. This is not to say, however, that the selection of these case studies signifies a determination that they present the greatest potential for exposure of all possible applications, or, for that matter, that any exposure actually occurs. Rather, the case studies simply provide a means to focus thinking about the types of information that would be instructive in assessing the potential ecological and health implicati ons of selected nanomaterials. The case studies follow the CEA approach, which combines a product life-cycle framework with the risk assessment paradigm (Davis, 2007, 089803 ; Davis and Thomas, 2006, 089638 ). In

Note: Hyperlinks to the reference citations throughout this doc ument will take you to the NCEA HERO database (Health and Environmental Research Online) at http://epa.gov/hero . HERO is a database of scientific litera ture used by U.S. EPA in the process of developing science assessments.



### 1-1

### essence, risk assessment relates exposure and eff ects information for a given substance or stressor, and CEA expands on this paradigm by including life-cycle stages and considering both indirect and
direct ramifications of the substance or stressor. Fi gure 1-1 illustrates the principal elements in the CEA approach. The first column of Figure 1-1 lis ts typical stages of a product life cycle: feedstock s, manufacturing, distribution, storage, use, and disposa l (including reuse or recycling, if applicable).
The second column lists environmen tal pathways or media (e.g., air, water, sediment, soil) to which nanomaterials or associated materials (e.g., manuf acturing by-products) might be released at various stages of the life cycle. Within these media, na nomaterials or associated materials can be transported and transformed, as well as interact with othe r substances in the environment, both natural and anthropogenic. Thus, a combination of primary (e.g., manufacturing by-products) and secondary (e.g., environmental transformation products) contaminants can be spatially distributed in the environment (Column 3, Figure 1-1).

Source: Adapted from Davis and Thomas (2006, 089638 ) and Davis (2007, 089803 ).



## Figure 1-1. Basic structur e of CEA as a framework for identifying and prioritizing research efforts.

The fourth column of Figure 1-1, exposure-dose, goes beyond characterizing the occurrence of contaminants in the environment, as exposure refe rs to actual contact between a contaminant and organisms (i.e., biota 1 

### as well as human populations). Under the CEA approach, exposure characterization can involve aggregate exposure across routes (e.g., inhalation, ingestion, dermal);

1 The term biota is used here to refer to all organisms other than humans.



### 1-2

### cumulative exposure to multiple contaminants (both primary and secondary); and various spatiotemporal dimensions (e.g., activity patterns, di urnal and seasonal changes). Dose is the amount of a substance that actually enters an organism by crossing a biological barrier. Conceptually, dose links exposure with the last column of Figure 1-1, which refers to ecological and human health effects that can result when an effective dose reaches a target cell or organ in a receptor organism or, in an ecological context, when a stressor is at a sufficient level to cause an adverse response in a receptor. “Effects” encompass both qualitative hazards and quantitative exposure-response relationships. The CEA framework is highly simplified in Figur e 1-1. Among the many direct and indirect impacts that could conceivably be included in a CEA are effects on other materials (e.g., damage to surfaces of structures, statuary, vehicles), hedonic or aesthetic qualities (e.g., alterations in visibility, taste, odor), and other possible large scale imp acts such as energy consumption, resource depletion, and global climate change. Although none of th ese effects are being excluded a priori from consideration here, their inclusion would depend on having a plausible premise for expecting a discernible impact. If such a premis e can be articulated for additional types of effects, the case study can be expanded to encompass their consideration within the CEA framework. CEA involves the elaboration and synthesis of information from the elements in all five columns depicted in Figure 1-1 to systematically ev aluate the direct and indirect ramifications of a nanomaterial and its by-products. Underlying the CE A elements are analytical methods that make detection, measurement, and char acterization of nanomaterials in the environment and in organisms possible. Not reflected in Figure 1-1 is an essential ingredient in making CEA effective – the inclusion of diverse technical and stakeholder pe rspectives to ensure that a holistic view is
maintained. As either an assessment tool or as a framework for developing a research strategy, CEA
is also a process that draws upon formal, structured methods to reach collective judgments by a diverse group of participants and contributors. Other efforts have been made to assess the potential risks of nanomaterials by incorporating a life-cycle perspective (e.g., Environmen tal Defense-DuPont Nano Partnership, 2007, 090565 ; Shatkin, 2008, 180065 ; Thomas and Sayre, 2005, 088085 ) or by using collective expert judgment methods (e.g., Kandlikar et al., 2007, 091626 ; Morgan, 2005, 088831 ), primarily in a risk management context. Although the present document di ffers somewhat from these other efforts in its purpose, namely to aid in developing a research strategy for the CEA of nanomaterial risks, all of these endeavors complement and reinforce one another.



# 1.2. How to Read this Document

The intent of this document is to identify system atically what is known and what needs to be
known about nano-TiO 2 

### to conduct an adequate assessment of nano-TiO 2 

### in the future. The goal is not to provide an actual CEA or to state conclusions regarding possible ecological or health risks
related to nano- TiO 2 

### , but to enable decisions on prioritizing research that would support future efforts to provide the input to policy and regulatory decision-making Although the differences between the applications of nano-TiO 2 

### as a drinking water treatment agent versus a topical sunscreen are important, the information currently available does not allow complete differentiation between the two. For example, the ecological and health effects of
nano-TiO 2 

### are described in a single chapter without regard to whether the source of nano-TiO 2 

### is drinking water treatment or sunscreen. However, wher e distinctions are possible or seem likely (e.g., in life-cycle stages such as manufacturing and use), the discussion of drinking water treatment is presented first, followed by discussion of sunscreen. In some sections, the discussions are not strictly parallel, reflecting differences in the availability of data.

1-3

### Also important to note is that these case st udies have been developed without a specific regulatory objective in mind. Although the topi cs selected for consideration, drinking water treatment and sunscreen, might be of interest in various policy and regulatory contexts, this document is not intended to serve as a basis for risk management decisions in the near term on these specific uses of nano-TiO 2 

### . Rather, the intent is to use this document in developing the scientific andtechnical information needed for future assessment efforts as input to policy and regulatory decisionmaking. Focusing on only two examples of nano-TiO 2 

### applications obviously does not represent all the possible ways in which this nanomaterial could be used or all the issues that different applications could raise. Rather, by considering the commonalit ies and differences between two applications of
nano-TiO 2 

### , research needs can be identified that appl y not only to these specific applications but generally to nano-TiO 2 

### and perhaps even more broadly to ot her nanomaterials. Also, additional case studies will be developed for other applications an d nanomaterials so that this process can continue and research strategies to support assessment efforts can be further refined. When implemented, a CEA is intended to be comparative, examining the relative risks and benefits of different technological options, for ex ample. The focus of a comparative CEA would be guided by risk management objectives. For example, the use of nano- TiO 2 

### for arsenic removal in drinking water might be compared to one or more current methods for arsenic removal; use of
nano-TiO 2 

### for topical sunscreen might be compared to sunscreen containing conventional TiO 2 

### or to sunscreen with organic ultraviolet (UV) radiation bl ocking agents. Given that a number of different options could be of interest to risk managers, c onsidering every potential option in the present case study is not feasible. Therefore, this document focuses solely on nano-TiO 2 

### in drinking water treatment and topical sunscreen, which is also consiste nt with the fact that the case studies are not intended to be assessments at this time, but rather are meant to assist in identifying and prioritizing
research needs related to nano- TiO 2 

### . Readers seeking comparative a ssessments of topical sunscreen products, with or without nano- TiO 2 

### , may wish to consult evaluations by the Scientific Committee on Consumer Products (SCCP) (2007, 196826 ) and the Environmental Working Group (EWG) (2009, 196367 ). The EWG analysis in particular takes a broad view that is consistent with the CEA approach in referring to the product life cycle and noting potential ecological as well as human
health considerations. This document is not intended to provide an e xhaustive review of the literature, and focuses instead on findings most clearly relevant to assessment objectives. The information presented in this document was obtained from a variety of publi shed and unpublished sources, including corporate Web sites and personal communications, as well as inferences based on information about other materials or applications. Such information sour ces are used because of the limited amount of published materials on nano-TiO 2 

### and its applications in the peer-r eviewed literature, coupled with the limited mechanisms for making manufacturer-specific data publicly available. This document is not an assessment but, rather, a means to identif y information gaps and research questions, thus a range of information sources was used.



# 1.3. Terminology

A number of terms used in the field of nanotechnology have specialized meanings, and definitions of certain terms could have important legal, regulatory, and policy implications. Not surprisingly, perhaps, defining such words, including the term nanomaterial itself, has often been a
matter of considerable interest and debate. For the purposes of this document, however, it is not deemed necessary to have a connotative definition that states the necessary and sufficient conditions that define a nanomaterial. Instead, a denotative approa ch is used; that is, the term “nanomaterial” in the case study denotes something that most persons would agree is (or at least appears to be) an

1-4

### example of a nanomaterial or a product that incorporates a nanomaterial, regardless of whether a consensus exists regarding what properties or characteristics qualify it as such. Although this case study focuses on “nano-TiO 2 

### ,” readers should note that this term encompasses a variety of materials that might possess a range of physicochemical properties. As a result, not all materials referred to as nano-TiO 2 

### will necessarily behave in the same manner and exert the same biological effects. Thus, caution in extrapolating from one nano-TiO 2 

### formulation to another when assessing hazards is appropriate. Conversely, until more information is available to discern more precisely how various formulations diff er in behavior and effects, pooling information from multiple sources can be useful for the purposes of this document, namely to identify potential research directions to pursue. Some other terms used throughout this document are discussed below, primarily to explain
how the terms are used here rather than to at tempt to provide a formal definition of them.



## Nano-TiO 2 

### This document focuses primarily on engineered nano-TiO 2 

### , which usually is in the form of particle s in the 1 to 100 nm size range. The term “nano-TiO 2 

### ,” as it is used in this document, refers to a variety of formulations containing titanium dioxide particles that meet this size-based definition. When reading this document, it is important to understand that the general use of this term encompasses specific formulations that can display a range of characteristics and behaviors depending on the properties of the particle, the e xperimental or environmental conditions, and other
factors. 1 

### Where information is not specific to nano-TiO 2 

### , the term “titanium dioxide” (TiO 2 

### ) is used
without the “ nano -” prefix.



## Conventional TiO 2 

### To make an explicit distinction between th e nanoscale material and other forms of TiO 2 

### not having the special characteristics of nano-TiO 2 

### , the term “conventional” is used in this document. Even so, materials described as conventional often contain a range of particle size s, including some
with nanoscale dimensions. In the scientific and technical literature, the terms “bulk” and “pigmentary” also are often used to distinguish conventional from nano-TiO 2 

### . Additionally, terms such as “ultrafine,” “PM-0.1” (whi ch means particulate matter up to 0.1 micrometer [μm] diameter), and “micronized grade ” have been used to denote nanoscale particles, but typically in a particular context or field of specialization such as aerosols and air pollution.



## Aggregate and Agglomerate

As discussed in Chapter 3, in many circumstances primary nanoscale particles can aggregate

or agglomerate into secondary particles with dimensions greater than 100 nm (a cluster that is sometimes referred to as a colloid, as describe d below). Specifically, the terms “aggregate” and “agglomerate” are used in the literature on nanomate rials and other fields to indicate the clustering of particles into a single entity of such particles. These two terms can have specific meanings. For example, the British Standards Institution (BSI, 2007, 202162 ) defines aggregate as a “particle comprising strongly bonded or fused particles wher e the resulting external surface area may be significantly smaller than the sum of calculated surface areas of the individual components” and notes that “the forces holding an aggregate together are strong forces, for example, covalent bonds, or those resulting from sintering or complex physical entanglement.” The BSI defines agglomerate as a “collection of loosely bound particles or aggregates or mixtures of the two where the resulting external surface area is similar to the sum of the surface areas of the individual components” and

1 Where sources have provided documentation on size, surface coating, extent of aggregation or agglomeration, and other salient properties or characteristics, this information is included in the case study with sources referenced appropriately.



### 1-5

### notes that “the forces holding an agglomerate t ogether are weak forces, for example van der Waals forces, as well as simple physical entangleme nt.” However, the meanings of aggregate and agglomerate have sometimes been interchanged, as noted by Nichols et al. (2002, 202114 ). This lack of consistency in terminology usage across, and som etimes within, the various fields that contribute to nanomaterials research exemplifies the challe nges posed by the multidisciplinary nature of the nanotechnology field. The nanotechnology community is an amalgam of investigators who all study nanoscale materials but whose scientific roots are in various other mature fields spanning toxicology, ecology, colloid science, materials science, an d many other disciplines. The customary terminology for aggregates and agglomerates may be well establ ished within one field, but use of these terms may elicit different interpretations within another; as a result, the definitions for these terms are not specific, nor are they consistent. Given this inconsistency in usage and, more importantly, the frequent lack of adequate information to determin e which term might be more appropriately applied in a particular study or report, the term “cluster” is used in this document to subsume both aggregates and agglomerations of na noparticles. This term has precedent within multiple disciplines and avoids confusion between potentially inconsistent c onnotations of the other terms. Note that, in addition to being used as a noun (as explained above), the word “aggregate” is used as an adjective (primarily in Chapter 5) to refer to exposure to a given material from multiple sources, pathways,
and routes.



## Colloid

The term “colloid” is used in the literature to refer to a particle or cluster of particles suspended within a given medium and that are smaller than microscale (i.e., <10 -6 

### m). Luoma (2008,
157525 ) describes a colloidal particle as containi ng multiple atoms of a substance measuring between 1 nm and 1,000 nm, and thus a colloid might or might not be a nanoparticle in that context. In this case study, although the term “colloid” is use d at times to refer to a sub-microscale particle (especially if a cited publication uses this terminology), either the more specific term “nanoscale” or a specific size range is used when the particle size is salient to the discussion. The extent to which the properties of a cluster of primary nano-TiO 2 

### particles that exceeds 100 nm are similar to the properties of conventional TiO 2 

### is unclear. For example, inhalation of
nano-TiO 2 

### (20-nm diameter) induced more pulmonary infl ammation in the rat than inhalation of fine
TiO 2 

### (approximately 250-nm diameter) at a similar mass concentration, even though particles in both groups had similarly sized agglomerates (0.71 μ m mass median aerodynamic diameter [MMAD] nano; 0.78 μ m MMAD fine) (Oberdörster, 2000, 036303 ; Oberdörster et al., 1994, 046203 ). Additional analysis revealed that effects were similar when expressed on the basis of surface area. Also unclear is the extent to which changes in conditions might initiate the formation, decomposition, or dissolution of a cluster; and there is uncertainty as well regarding what specific factors drive important changes in conditions. As discussed in Chapter 3 (Fate and Transport), disaggregation can occur under some conditions. Give n these considerations, this document does not use 100 nm as the essential and exclusive criterion for considering what might be relevant to an
evaluation of nano-TiO 2 

### . This view is consistent with a statement by the European Commission (2008, 196378 ) that extends the term “nanomaterial” to encompass “nanostructured materials,” defined by the International Organization for Standardization (ISO) (2004, 190006 ) as “[a]ggregates and agglomerates, often existing at a micro size, [t hat] may have some of the behaviour and effects of their smaller sub units, e.g., due to an increased surface area.”



## Naturally Occurring, Incidental, and Engineered Nanoparticles

In addition to distinctions based on size of particles, The Project on Emerging Nanotechnologies (2009, 196052 ) divides nanoscale materials into th ree classes based on the origin of the particles. The first class, naturally occurring nanosized particles, includes, for example, particles that originate from volcanic explosions, ocean spray, and soil and sediment weathering and

1-6

### biomineralization processes (which can result in crystals of aluminum and iron oxides with nanometer-scale dimensions). The second class is incidental nanosized particles, which are generated as by-products of processes such as combustion, cooki ng, or welding. The focus of this report is on the third class of nanoscale materials, engineered nanomaterials. This class comprises materials purposely generated for a specific function, such as the carbon nanotubes used in tennis rackets to make them lighter and stronger. In this case stud y, unless otherwise specified, references to nanoTiO 2 

### indicate engineered nanoscale materials. Nonengineered types of nanosized TiO 2 

### (from the first or second class) are referred to as “nanoscale TiO 2 

### .” Degussa Aeroxide ® 

### P25 (hereafter referred to as P25) is a commercial-grade, uncoated
nano-TiO 2 

### product (Evonik, 2008, 157578 ) that has been studied extensively and referenced in the
literature and is therefore often mentioned in late r sections of this document. As discussed below, however, P25 does not represent all nano-TiO 2 

### preparations and should not be equated with the generic term nano-TiO 2



### .



# 1.4. Conventional TiO 2

Although this document focuses on nano-TiO 2 

### , highlighting some facts about conventional
TiO 2 

### first is instructional. Also known as “titania,” TiO 2 

### has been used commercially since the early 1900s in numerous consumer and industrial applications, particularly coatings and pigments. TiO 2 

### is a naturally occurring mineral that can exist in three crystalline forms, known as rutile

, anatase , and brookite, and in amorphous form. Rutile is the most common form of TiO 2 

### found in nature.
Elemental Ti is also found in ilmenite (FeTiO 3 

### ) and other minerals and ores, and TiO 2 

### can be produced by processing of these minerals and ores. TiO 2 

### is insoluble in water, hydrochloric acid, nitric acid, and ethanol, but soluble in hot concentrated sulfuric acid, hydrogen fluoride, and alkali (NRC, 1999, 091188 ). TiO 2 

### is used to increase the whiteness or opacity of many consumer products, such as paints, coatings, plastics, paper, printing inks, roofing granules, food, medicine, toothpaste, cosmetics, and skin care products, including t opical sunscreens. In the U.S., surface-mining operations in Virginia and Florida produce concentr ated Ti-containing minerals (ilmenite and rutile)
suitable as feedstock for TiO 2 

### production (USGS, 2009, 157454 ). Other countries that produce significant amounts of Ti ores include Australia, Ca nada, China, India, Norway, and South Africa (USGS, 2009, 157454 ). With exposure to ultraviolet (UV) radiation (wavelengths less than ~400 nm), pure TiO 2 

### is photocatalytic

. 1 

### Studies suggest anatase and rutile have different photocatalytic properties, with anatase being the more reactive (Sayes et al., 2006, 090569 ; Uchino et al., 2002, 090568 ). In applications such as paints, coatings, and cosmetic s, where chemical stability is required, the photocatalytic properties of TiO 2 

### are often suppressed by coating the particles with silica and alumina layers. On the other hand, the photocatalytic properties of TiO 2 

### are increasingly exploited in a number of other experimental and commercial applications, including degradation of organic compounds, microbiological organism destruction, a nd conversion of metals to less soluble forms in wastewater, drinking water, and indoor air. For more information on conventional TiO 2 

### , see the article by Diebold (2003, 193342 ) and the Current Intelligence Bulletin published by the National Institute for Occupational Safety and Health (NIOSH) (2005, 196072 ).

1 Photocatalysis is the phenomenon by which a relatively small amount of material, called a photocatalyst, in creases the rate of chemical reaction without itself being consumed. ( adj. photocatalytic).



### 1-7

# 1.5. Nano-TiO 2

One of the main differences between nano-TiO 2 

### and conventional TiO 2 

### is the much greater surface area of a given mass or volume of nanoparticles compared to an equivalent mass or volume
of conventional TiO 2 

### particles. To illustrate, a 5-nm particle would have a volume of 65 nm 3 

### (4/3 π r 3 

### ) whereas a 500-nm particle would have a volume of 65,000,000 nm 3 

### . Therefore, one million 5-nm particles would be required to equal the vol ume of a 500-nm particle. The surface area of a 5-nm particle equals approximately 80 nm 2 

### (4 π r 2 

### ), whereas the surface area of a 500-nm particle equals approximately 800,000 nm 2 

### . Multiplying the surface area of the 5-nm particle by one million (the number of 5-nm particles needed to equal the volume of a 500-nm particle) yields a total surface area of approximately 80,000,000 nm 2 

### , which is 100-fold greater than the surface area of the 500-nm particle. This greater relative surface area of the nano-TiO 2 

### particles affords a greater potential for properties such as catalytic activity and UV absorption at certain wavelengths (Shao and Schlossman, 1999, 093301 ). Such properties have led to the development or use of nano-TiO 2 

### for a wide variety of applications, including self-cleaning surface coati ngs, light-emitting diodes, solar cells, disinfectant sprays, sporting goods, and the subjects of this document, drinking water treatment agents and topical sunscreens. Before considering specific applications of nano-TiO 2 

### , some fundamental issues
related to characterization of this material should be noted.
Surface areas of nano-TiO 2 

### primary particles, aggregates, a nd agglomerates can be expressed as total (inner and outer) surface area and external surface area. The total surface area includes the inner surface area of porous or aggregated or agglomerated nanoparticles (Scientific Committee, 2007, 157639 ), and can be measured by the Brunauer, Emmett, Teller method (BET) and other methods. The external surface area, which is inse nsitive to particle porosity, can be measured indirectly by microscopy, diffusion chargers, scan ning mobility particle sizers, and other methods (LeBlanc, 2009, 625209 ). Whether the total surface area or external surface area is more relevant for nanoparticle effects has not been determined. In one instance, humic acid caused nano-TiO 2 

### micropore blockage and consequently decreased the total surface area, but not the external surface area (Yang et al., 2009, 190513 ). Humic acid-coated nano-TiO 2 

### had lower zeta potential (i.e., increased electrostatic repulsion), which leads it to be more easily dispersed and suspended than
uncoated nano-TiO 2 

### (Yang et al., 2009, 190513 ). External surface area alone, however, does not always predict the nature and magnitude of effect s. When possible, the method of measuring surface area is provided when discussing studies in this document. Several types of nano-TiO 2 

### are available with differing physicochemical properties. Commercially available brands of nano-TiO 2 

### can vary in particle size 1 

### , surface area, purity (e.g., due to doping, coating, or quality control), surface char acteristics, crystalline form, chemical reactivity, and other properties (Table 1-1). Nano-TiO 2 

### is available in pure anatase, pure rutile, and mixtures of anatase and rutile. In general, anatase nano- TiO 2 

### is more photocatalytic than the rutile form, and nanoscale rutile is less photoreactive than either anatase and rutile mixtures or anatase alone. However, a mixture of 79% anatase and 21% rutile nano- TiO 2 

### (P25) was found to be more photocatalytic than 100% anatase nano- TiO 2 

### in some instances (Coleman et al., 2005, 089849 ; Uchino et al., 2002, 090568 ), but less effective in others (Nagaveni et al., 2004, 090578 ). Such contrasts point to the role of other factors in accounting for the behavior and effects of nano- TiO 2



### . For example, surface treatment of nano-TiO 2 

### can change nano-TiO 2 

### activity, including photoreactivity. Aeroxide ® 

### T805, which is P25 nano-TiO 2 

### that has been treated with trialkoxyoctyl silane on the surface to increase hydrophobicity, ha s lower surface reactivity than P25 as indicated

1 The sizes of nanoparticles may be reported either as crysta llite size (sometimes called primary crystallite size, such as size determined by X-ray Diffraction analysis) or as primary particle size, which is typically larger than crystallite size. (Barton, personal com munication, 2009, 625563 ).



### 1-8

### by a vitamin C oxidation assay (Degussa, 2003, 193916 ). Similarly, surface coatings of silicone and other compounds are used to decrease nano-TiO 2 

### photoreactivity so that nano-TiO 2 

### can be used to protect human skin, plastic, and other objects from UV radiation. This document presents
information on both coated and uncoated nano-TiO 2 

### , recognizing that differences in properties and environmental behavior may limit the applicability of information from one particle type to another.



## Table 1-1. Examples of nano-TiO 2 

## physicochemical properties

Agglomeration/aggregation status in
the relevant media

Bulk density/particle density

Composition/surface coatings

Crystal structure/ crystallinity (crystalline phase, crystallite size)

Dustiness

Octanol-water partition coefficient Particle size and size distribution

Photocatalytic activity

Pore density

Porosity

Purity of sample

Radical formation potential

Redox potential Shape/aspect ratio (e.g., width and length)

Surface area/specific surface area

Surface charge/zeta potential

Surface chemistry

Surface contamination

Surface reactivity

Water solubility

Source: Data from U.K. Department for Environment, Food, and Rural Affairs (2007, 195461 ); Used with permission from Oxford University Press, Powers et al. (2006, 088783 ); Used with permission from Informa Healthcare, Powers et al. (2007, 090679 ); Used with permission from Informa Healthcare, Wa rheit et al. (2007, 091305 ); and data from Organisation for Economic Co-operation and Development (OECD) (2008,
157512 ).



### External factors can also influence photoreactivity. Krishna and co-authors (2006, 193482 ), for example, found that the presence of fullerene s, which scavenge photogenerated electrons, enhances the photocatalytic efficacy of nano-TiO 2 

### . Likewise, Komaguchi and colleagues (Komaguchi et al., 2006, 193479 ) saw significant increases in photocatalytic efficiency of P25 after exposure to an oxidizing environment. Photocatalytic nano-TiO 2 

### is preferred for drinking water treatment, and photostable nano-TiO 2 

### is preferred for sunscreen use. Some sunscre ens, however, contain photoreactive nano-TiO 2



### . Although pure uncoated and undoped anatase TiO 2 

### is photocatalytic, and uncoated and undoped
rutile TiO 2 

### is generally photostable, there is no qu ick way to identify the photoreactivity of
nano-TiO 2 

### . For example, although doped rutile nano-TiO 2 

### can be extremely photostable, rutile
nano-TiO 2 

### produced by a certain specific powder-prepa ration method can be highly photocatalytic (Kim et al., 2003, 157861 ). Similarly, not all coatings decrease nano-TiO 2 

### photoreactivity. A report by Barker and Branch (2008, 180141 ) has noted that nano-TiO 2 

### in some sunscreens might not be photostable. The investigators studied the weathering of paint in contact with
sunscreen. Of five nano-TiO 2 

### sunscreens tested, four released photocatalytically generated hydroxyl radicals that accelerated the weathering of the paint. All four of those sunscreens used an
anatase/rutile mix. The one nano-TiO 2 

### sunscreen formulation that showed no appreciable effect on paint weathering used 100% rutile doped with manganese rather than surface coating with manganese (Barker and Branch, 2008, 180141 ). Due to various degrees of porosity, nano-TiO 2 

### particles with the same diameter can differ in
surface area. Because nano-TiO 2 

### reactivity and consequently behavior and effects are influenced by many nano-TiO 2 

### physicochemical properties, two nano-TiO 2 

### products with the same values for a limited set of parameters should not be assume d in fact to be equivalent. For instance, a manufacturer might use the same core nano-TiO 2 

### for surface-treated and untreated nano-TiO 2 

### , and both might have the same particle size and surface area, but differ in reactivity, as in the case of P25
and Aeroxide ® 

### T805 (T805). Another characteristic of significance is th e aggregation or agglomeration of nano-TiO 2 

### particles. According to one industrial manufactur er of nanoscale titania produced through flame

1-9

### hydrolysis (see Section 2.2 for a description of this manufacturing technique and others), “tests and calculations have shown that free primary particles with dimensions of less than 100 nm only exist in [flame] reactors for a few milliseconds” (Degussa, 2009, 193919 ). Aggregates of nano-TiO 2 

### , sometimes referred to as “ colloidal ,” are often roughly an order of magnitude greater in size than primary particle s (Dunphy Guzman et al., 2006, 090584 ; Kormann et al., 1988, 090582 ; Lecoanet et al., 2004, 089258 ). The mean aggregated particle diameter of the product P25 is claimed to be approximately 3.6 μm, with the smallest 4% of a ggregated particles reported to have an average diameter of 160 nm (Klaessig, personal communication, 2006, 196041 ). After being subjected to bath sonication for 10 minutes, the smallest 15% of P25 particles averaged an agglomerate diameter of 160 nm, while the 50th percentile diameter was 1.6 μm, roughly two orders of magnitude larger than the reported primary particle size of P25, which is 21 nm (Degussa, 2007, 193917 ; Wahi et al., 2006, 090580 ). Ridley et al. (2006, 090599 ) observed that a suspension of uncoated nano-TiO 2 

### anatase from Ishihara Techno Corporation (Osaka, Japan) with primary particles of 4-nm diameter consisted mainly of aggregates in the 1- to 30- μm diameter range, and that these size ranges persisted even under sonication and other conditions that would favor disaggregation. Reported particle size values for aggregates and agglomer ates are influenced by factors such as initial concentration, sonication power input, pH, and mea surement technique. Such variables are germane to interpreting the results of reported size distributions. Despite the presence, and sometimes the predominance, of larger aggregates and agglomerates, several researchers investigati ng laboratory-synthesized anatase and commercial
nano-TiO 2 

### products such as P25 have also found free particle s or aggregates with diameters <100 nm in varying amounts. The quantity of such particles has been found to depend on synthesis method, temperature, solution pH, and the presence of buffers (Jiang et al., 2009, 193450 ). Moreover, some preparations are specifically designed to generate dispersed particles (e.g., Seok et al., 2006, 091198 ) which would be important in using nano-TiO 2 

### as a catalyst. The pH pzc 

### of a nanoparticle (the pH at the “point of zero charge,” where the net electric charge at the particle surface is zero) has important ra mifications for aggregation, because at that pH particles will fail to electrostatically repel each other. In laboratory studies, the size range of aggregates and the presence of free nano-TiO 2 

### particles (synthesized on-site, ranging from 5 to 50 nm) were found to be pH-dependent; when the solution pH differed from the pH pzc 

### of the particles, the aggregates tended to be smaller (Dunphy Guzman et al., 2006, 090584 ; Dunphy Guzman, personal communication, 2007, 091184 ). Sampled aggregates ranged up to 150 nm in size, and contained an estimated 8-4,000 nanoparticles (Dunphy Guzman et al., 2006,
090584 ). The pH pzc 

### also depends at least in part on the crystallinity of the nano-TiO 2 

### particles; Finnegan et al. (2007, 193379 ) reported pH pzc 

### values of approximately 5.9 for rutile and approximately 6.3 for anatase. Coatings and surface treatments also affect par ticle aggregation/agglomeration behavior. A preliminary report by Wiench and colleagues (2007, 090635 ) indicated that coated nano-TiO 2 

### particles (rutile, size 50 × 10 nm, surface area of 100 square meters per gram [m 2 

### /g]; coatings included combinations of aluminum hydroxide, hydrated silica, and various polymers) had slower agglomeration and sedimentation rates and a larger fraction of primary nanoparticles remaining in the sample compared with uncoated particles (20-30 nm, anatase/rutile 80/20, surface area
48.6 m 2 

### /g). A recent study showed that a type of coated nano-TiO 2 

### used in sunscreens quickly lost its outermost coating and became easily dispersed so on after contact with water (Auffan et al., 2010,
625063 ). The tested nano-TiO 2 

### , T-LiteTM SF (manufactured by BASF company, Germany) had a mainly rutile TiO 2 

### core, coated with Al(OH) 3 

### and an outermost hydrophobic coating with polydimethylsiloxane (PDMS). The structural changes were seen within 30 minutes of aging (stirring) when the initially hydrophobic nano-TiO 2 

### started to disperse into the aqueous phase of the suspension. The PDMS coating was dissolved, and the dissolution rate was accelerated by UV exposure. Aluminum was not detected, suggesting that the Al(OH) 3 

### outer layer was more stable.

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### The complexity of nano-TiO 2 

### characterization is illustrated in Table 1-2, from Warheit et al. (2007, 091075 ). The chemical composition of three different types of ultrafine TiO 2 

### manufactured by DuPont was determined by X-ray fluorescence. The cores of all three types of nano-TiO 2 

### were TiO 2 

### , but the crystalline form and the surface coating of alumina or silica differed. Each type of particle was said to exhibit a mean diameter of approxim ately 140 nm but with (unspecified) fractions of the size distributions below 100 nm as measured by dynamic light scattering. The chloride ions on the surface of the particles were neutralized during production (Other effects these materials cause are described in Chapter 5). As shown in Table 1-2, the surface area, crystallinity, chemical reactivity, surface coating, particle size distribution, and pH varied for the materials, all three of which were nominally nano-TiO 2 

### . Even with detailed information such as provided here, additional details may be necessary to fully characteri ze the complex nature of nano-TiO 2 

### nanomaterials. In particular, the presence of a surface coating changes the nature of the interface between the nano-TiO 2 

### particle and the environment or an organism, and it is not clear whether the surface coating or the core material dominates particle-environment and particle-organism interactions.



## Table 1-2. Characterization of three nano-TiO 2 

## particle types

Median Particle Size and Size Range b pH in Deionized Water Particle Type BET Surface Area (m 2 /g) Chemical Reactivity a Chemical Composition

in Water c in PBS

Uf-A 18.2 98% TiO 2 (100% rutile), 2% alumina 10.1 136 nm ± 35% 1,990 nm ± 25% 5.64

Uf-B 35.7 88% TiO 2 (100% rutile), 5% alumina, 7% silica 1.2 149.4 nm ± 50% 2,669 nm ± 25% 7.14

Uf-C 38.5 92% TiO 2 (79% rutile; 21% anatase), 7% alumina, 1% silica 0.9 140 nm ± 44% — 4.80

BET – Brunauer, Emmett, Teller method of calculating surface area
PBS – Phosphate buffered saline

a Chemical reactivity was tested using a Vitamin C (antioxidant) yellowing assay.

b After sonication for 15 min at 60 Hertz (Hz).

c 0.1% tetrasodium pyrophosphate solution.

Source: Modified with permission from Elsevier, Warheit et al. (2007, 091075 ).



### The characteristics of a nano-TiO 2 

### product might change over time. Using a custom-made
anatase nano-TiO 2 

### formulation (uncoated) with a range of particle sizes, Kolá ř et al. (2006, 193478 ) found that average particle sizes increased over time, due to both agglomeration and re-crystallization (smaller particles dissolving in the aqueous medium and their constituent molecules then adding to the mass of the larger particles). Over the course of 8 years, average (mode) particle size increased from approximately 10 nm to approximately 14 nm. The investigators
also observed that over time relative surface area d ecreased, light energy absorbance characteristics changed, and perhaps most surprisingly, photocatalytic performance improved, even as relative
surface area decreased. As discussed in greater detail in Chapter 5 (Section 5.1.1), these and other issues have been noted in various recommendations for improving the physicochemical characterization of nanomaterials in exposure and ecological as well as health effects studies. In general, however, reports of toxicity and exposure studies of nano-TiO 2 

### , especially those conducted prior to the year 2000, have not been sufficiently attentive to th e issues described above. As discussed in Section 1.6.1, ideal characterization to support toxi cological testing would include analysis of the raw material (as received from the manufacturer or supplier), analysis of nanomaterials in the testing

1-11

### media for the duration of the experiment; and an alysis of nanomaterials (and possibly degraded products or biotransformed products) in biological sa mples. Manufacturers’ literature often has been accepted as having described their products under all conditions – an oversimplification at best. Additionally, attempts to character ize nanoscale particle sizes and size distributions in relation to toxicity and exposure evaluations have been prone to errors involving nonrepresentative sampling, agglomeration during sample preparation, contam ination and degradation during product storage, measurement methods, and conditions under which the study was conducted (Powers et al., 2007,
090679 ). Further, different particle characterization techniques can produce different estimates of particle size, suggesting that more than one techniqu e might be necessary to describe particle sizes accurately. Accurate characterization is clearly important if the behavior and effects of nano- TiO 2 

### are to be understood, predicted, and related to other materials (both nanoscale and conventional), and thetype and extent of characterization is an important consideration in interpreting the results of nanoTiO 2 

### research.



# 1.5.1. Drinking Water Treatment

This document assumes that one use of nano-TiO 2 

### would be specifically for arsenic removal in a drinking water treatment facility. In addition to arsenic removal (Li et al., 2009, 193506 ), however,
nano-TiO 2 

### could be used for disinfection of pathogens (Alrousan et al., 2009, 157461 ; Coleman et al., 2005, 089849 ; Li et al., 2008, 157538 ; Rincon and Pulgarin, 2003, 157856 ) or for remediation of ground water or wastewater contaminated with various organic and inorganic pollutants (Adams et al., 2004, 193250 ; Chen and Ray, 2001, 193310 ; Han et al., 2009, 193407 ; Kim et al., 2003, 193472 ; Lee et al., 2008, 098739 ; Lin and Valsaraj, 2003, 193511 ; Ryu and Choi, 2008, 157501 ). The latter use would pose rather different scenarios of envi ronmental releases and fate and transport, and would add considerably to the complexity of this document. Therefore, the case study of nano-TiO 2 

### for water treatment has been limited to the c onsideration to arsenic removal in drinking water
treatment facilities. Most of the relevant literature has reported laboratory tests of nano-TiO 2 

### as a photocatalytic treatment for conversion of arsenite [As(III)] to arsenate [As(V)], a species that is more easily
removed in water treatment because of its lower solubility in typical drinking water treatment conditions (e.g., Dutta et al., 2004, 157845 ; Ferguson et al., 2005, 090572 ; Pena et al., 2006,
090573 ). Although neither conventional TiO 2 

### nor nano-TiO 2 

### is known to have been used in a fullscale drinking water treatment plant, both conventional TiO 2 

### and nano-TiO 2 

### as photocatalytic agents have been pilot-tested in drinking water treatment plants (Dionysiou, personal communication, 2009,
193921 ; Pichat, 2003, 196037 ; Purifics, 2008, 196040 ; Richardson et al., 1996, 193612 ). For arsenic removal from water, both conventional and nanoscale TiO 2 

### have been developed to photocatalytically oxidize arsenic and adsorb arsenic. Studies have shown that TiO 2 

### can oxidize As(III) to As(V) and adsorb inorganic arsenic (Dutta et al., 2004, 157845 ; Fostier et al., 2008,
193381 ; Hristovski et al., 2007, 193436 ). The mechanism for TiO 2 

### photocatalytic oxidation of As(III) has been suggested to be through the gene ration of superoxide ions, and the major oxidant species might be hydroxyl radicals (·OH) (Sharma and Sohn, 2009, 193641 ). Recently, nano-TiO 2 

### was shown to mineralize methylated arsenic and to adsorb methylated arsenic (Xu et al., 2007,
193725 ; Xu et al., 2008, 193727 ). Both dimethylarsinic acid [DMA(V)] and monomethylarsonic acid [MMA(V)] were readily mineralized to As(V) by transforming the methyl group into organic compounds such as methanol, formaldehyde, and formic acid. Dimethylarsinic acid was photocatalytically oxidized into MMA(V), which was subsequently oxidized into As(V). Hydroxyl radicals could be the primary oxidant (Xu et al., 2007, 193725 ; Xu et al., 2008, 193727 ). The mechanism of arsenic adsorption onto TiO 2 

### surfaces has been demonstrated to be through the formation of bidentate inner sphere complexes for As(V), As(III), and MMA(V), and forming monodentate inner sphere complexes for DMA(V) (Jing et al., 2005, 193452 ; Jing et al., 2005,
193454 ; Pena et al., 2006, 090573 ). In ground water containing As(III), As(V), MMA(V), and

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### DMA(V), nano-TiO 2 

### adsorbed As(III) and As(V) most, followed by MMA(V), but almost no DMA(V) (Jing et al., 2009, 193453 ). The difference in competitive adsorption could be due to the increased thermodynamic stability of the bidentat e ligands formed by other arsenic species with TiO 2 

### compared with the monodentate surface structure formed between TiO 2 

### and DMA(V). However, in the presence of high concentrations of competing i ons, the other arsenic species may be forced to form monodentate rather than bidentate ligands. Photocatalytic oxidation is also the mechanism for TiO 2 

### degradation of organic pollutants in wastewater. Photocatalytic degradation is based on the formation of radicals (hydroxyl radicals [

· 

### OH], superoxide radical anions [

· 

### O 2 − 

### ], and hydroperoxyl radicals [

· 

### OOH]), which serve as oxidizing species in the photocatalytic oxidation process (Lu et al., 2009, 193528 ). Hydroxyl radicals, the most powerful oxidants TiO 2 

### produces in the photocatalysis, can act on organic contaminants present at or near the surface of TiO 2 

### (Bianco Prevot et al., 1999, 193278 ). One generally accepted mechanism of nano-TiO 2 

### antimicrobial action is the generation of reactive oxygen species (ROS), which can cause cell wall or cell membrane damage (Kuhn et al., 2003, 090597 ; Neal, 2008, 196069 ), such as lipid peroxidation (Maness et al., 1999, 193538 ). Although UV illumination increases photocatalytic nano-TiO 2 

### toxicity to bacteria and fungi, photocatalytic nano-TiO 2 

### is also toxic in the dark (Adams et al., 2006, 157782 ; Coleman et al., 2005,
089849 ). Because TiO 2 

### generates ROS (mainly highly reactive hydroxyl radicals) in the presence of UV light and oxygen (Reeves et al., 2008, 157506 ), mechanisms other than oxidative stress might
also contribute to nano-TiO 2 

### toxicity in the dark (and possibly also under UV), as suggested by a recent study indicating that anatase nano-TiO 2 

### can generate carbon-centered free radical s in the dark in the presence of dissolved oxygen (Fenoglio et al., 2009, 180383 ).



# 1.5.2. Sunscreen

Nano-TiO 2 

### formulations of sunscreen have proven popul ar because they appear transparent on the skin; formulations using conventional TiO 2 

### or other inorganics such as zinc oxide (ZnO) (Schlossman et al., 2006, 093309 ) create a milky white appearance. Nano-TiO 2 

### serves as a sunscreen in two ways, by absorption and scattering, depending on the wavelength of UV light. UV-B wavelengths are in the range of 290 to 320 nm, and are primarily absorbed by nano-TiO 2 

### ; UV-A wavelengths are in the range of 320 to 400 nm, and are primarily scattered by nano-TiO 2 

### (Shao and Schlossman, 1999, 093301 ). Optimal scattering is thought to occur when the diameter of the particles is approximately half the wavelength of the light to be scattered (Fairhurst and Mitchnick, 1997, 196248 ) also see Appendix A for more information on how nano-TiO 2 

### particle size relates to UV-A and UV-B protection). Information on chem ical and other properties of topical sunscreens containing nano-TiO 2 

### can be found in Appendix A.
Conventional TiO 2 

### absorbs and scatters UV radiation, making it an effective active ingredient in sunscreens. Like ZnO, TiO 2 

### is a “physical blocker” of UV radiation, as opposed to many chemically active ingredients that serve as “chemical filters,” such as avobenzone and benzophenone, which in some individuals can cause adverse skin reactions, including blisters, itching, and rash (U.S. FDA, 2006, 157728 ). Thus, sunscreens containing physical blockers have long been an attractive option to those with sensitive skin. Apart from this niche market, the use of
TiO 2 

### in sunscreen was historically limited because of aesthetic considerations. Because conventional
TiO 2 

### scatters visible light, it remains visible as a wh ite film when applied on skin. With the advance of technology to produce transparent nanoscale TiO 2 

### particles, which scatter very little visible light and therefore appear transparent when applied on skin, nano-TiO 2 

### has entered the mainstream as an active ingredient in sunscreens and has also been added to numerous other cosmetic products to provide UV protection. With exposure to UV radiation (wavelengths less than ~400 nm), pure
anatase nano-TiO 2 

### is photocatalytic. In sunscreen, however, photocatalysis is an undesirable property that can be addressed by applying surface treatments to the crystals, selecting a less photoreactive form (rutile), or adding antioxidant ingredients to the formula.

1-13

### The maximum concentration (by weight) of TiO 2 

### in sunscreen that the U.S. Food and Drug Administration (FDA) allows is 25% (U.S. FDA, 1999, 196374 ), but this limit does not distinguish
between conventional and nano-scale TiO 2 

### , between anatase and rutile, or between coated and uncoated particles. The concentrations actually use d, according to product labels, typically range from 2% to 15% (Table A-1, Appendix A). Europe , Australia, Canada, and South Korea also have approved the use of TiO 2 

### as a UV filter in sunscreen with a maximum concentration of 25%. Japan does not regulate TiO 2 

### as a UV filter in sunscreen (Comparative study on cosmetics legislation in the EU and other principal markets with special a ttention to so-called borde rline products, 2004,
157826 ; Oxonica, 2005, 157793 ; Steinberg, 2007, 193656 ).
Some TiO 2 

### -bearing sunscreens are explicitly labeled as containing nanoparticles. Others are labeled as containing “micronized” TiO 2 

### , a grade commonly used in cosmetics. “Micronized” implies a particle size of approximately 1 micron (or μ m, which is one order of magnitude larger than 100 nm), but how precisely manufacturers use the term is unclear. Sometimes “micronized” is taken to imply a nano-size range (e.g., Shao and Schlossman, 1999, 093301 ), as it was formerly considered distinct from nano (e.g., EWG, 2008, 196343 ) but such a distinction is no longer made by the European Working Group (EWG, 2009, 196367 ). In the latter case, TiO 2 

### with a mean particle size of several micrometers is still very likely to include a significant fraction of particles in the nano-size range. Even sunscreens using pigment-grade TiO 2 

### likely contain a proportion of nano-sized particles. When Consu mer Reports tested seven leading national sunscreens labeled as containing ZnO or TiO 2 

### or both, but with no indication on the container regarding the presence of nanoparticles (with at least one dimension less than 100 nm (La Farge, personal communication, 2007, 196047 ), they found nanoparticles in all seven products (La Farge, personal communication, 2007, 196047 ; Sunscreens: Some are short on protection, 2007, 155618 ). They also confirmed the presence of nanoparticles in an eighth brand labele d as containing nanoparticles. No information was available, however, on the quantities or sizes of th e nanoparticles detected in any of these sunscreens (La Farge, personal communication, 2007, 196047 ). FDA does not specify the use of nano or other
terms to describe TiO 2 

### (64 FR 27671), and some nano-TiO 2 

### sunscreens are therefore simply labeled as containing “titanium dioxide.”



# 1.6. Analytical Methods

Sensitive and accurate analytical methods for nano materials are critical tools for nanomaterial risk assessment, because measuremen t and characterization of nanomaterials, alone and in various media, are required for properly assessing exposure, conducting toxicological studies, estimating dose-response relationships, and understanding the be havior and effects of nanomaterials. The
standardization of characterization method and sa mple preparation protocols will also greatly facilitate the physicochemical characterization of the nanomaterials. Section 1.5 addressed some general aspects of characterization of nanomaterials, particularly
nano-TiO 2 

### . This section provides a brief overview of analytical methods that could be suitable for
nano-TiO 2 

### , with a focus on currently available methods. Different methods for measuring the same parameter may yield different results fo r the same material (Hewitt, 2009, 625212 ), and therefore stating the testing method is important. Because nano-TiO 2 

### is not radio-labeled and does not fluoresce, analytical methods base d on these two attributes are not relevant. Additionally, the importance of chemical analysis of nanomaterials is acknowledged (such as methods used for identifying their molecular components and char acterizing certain surface properties), but these methods also are not discussed in Section 1.6. Some of the chemical analysis methods suitable for nanomaterials are discussed in Powers et al. (2006, 088783 ) and U.S. EPA (2008, 157480 ). Methods for analyzing nanoparticles in the environment are summarized by Hoyt (2009, 633900 ). For detailed

1-14

### comparison of various methods, see review articles by Maynard and Aitken (2007, 090674 ), Powers et al. (2006, 088783 ; 2007, 090679 ), and Domingos et al. (2009, 193346 ).



# 1.6.1. Methods for Labo ratory Research

The physicochemical properties of nano-TiO 2 

### can change over time (Kolár et al., 2006,
193478 ) and in various milieux; therefore, the charact eristics of engineered nanomaterials at the point of production could be vastly different afte r transport, storage, and preparation for testing. Nanomaterials used in toxicological testing ideally would be characterized by analyzing the raw material (as received from the manufacturer or suppli er); nanomaterials in the testing media for the duration of the experiment; and nanomaterials (a nd possibly degraded products or biotransformed products) in the biological samples being collected and tested, such as in urine, organs, and cells. The equipment and methods for measuring nanomaterials in the laboratory are numerous and are evolving. Table 1-3 summarizes methods that can be used for characterizing nanomaterials in
aerosols and liquids (including biological fluids) (Maynard and Aitken, 2007, 090674 ; Nanosafe, 2008, 594868 ; Powers et al., 2007, 090679 ). Methods for properties relating to chemical reactivity or charge, such as surface charge and pH pzc 

### , are not included in the basic characterization methods summarized in Table 1-3. Specialized methods are also available that are specific for radio-labeled or fluorescent nanomaterials. The following methods have been used to measure various characteristics of nanomaterials in biological samples: dark field microscopy, transmission electron microscopy (TEM), energy-dispersive X-ray analysis (EDS), and inductively coupled plasma mass spectroscopy (ICP-MS) for presence and location, with additional information on size, shape, and aggregation/agglomeration state available from analysis of TEM images; dynamic light scattering (DLS) in conjunction with TEM for size (both core and shell); high resolution transmission electron microscopy (HRTEM) for crystalline structure; inductively coupled plasma atomic emissionspectroscopy (ICP-AES) for elemental composition and quantitative nanomaterial uptake; videoenhanced differential interference contrast (VEDIC) microscopy for uptake and localization (Marquis et al., 2009, 193539 ); and scanning probe microscopy (SPM) for size and threedimensional images. ICP, X-ray diffraction (XRD), and nuclear magnetic resonance (NMR) can be
used to determine chemical com position. The combination of flow FFF and ICP-AES has been used
to detect nano-TiO 2 

### in tested commercial sunscreen, with in formation on mass-size distribution and
Ti content of extracted nano-TiO 2 

### from sunscreen (Contado and Pagnoni, 2008, 157585 ). Although
combinations of these methods can be used to infer the presence of nanomaterials in tissue (e.g., by metal content), no broad-spectrum techniques are curre ntly available to measure the total amount of
nanomaterials in tissues.

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## Table 1-3. Analytical methods for character izing nanomaterials in aerosol and in liquid

Metric Method Aerosol Liquid

Number Condensation particle counter (CPC) Yes No

Scanning mobility particle sizer (SMPS) Yes No

Electrical low pressure impactor (ELPI) Yes No

Optical particle counter (OPC) Yes Maybe

Electron microscopy (EM) Yes Yes

Surface area a Scanning mobility particle sizer (SMPS) Yes No

Electrical low pressure impactor (ELPI) Yes No

SMPS and ELPI used in parallel Yes No

Diffusion charger Yes No

Specific surface area (BET, titration, diffusion charging) Yes Titration techniques only

Mass Size selective personal sampler Yes No

Size selective static sampler Yes No

Tapered element oscillating microbalance (TEOM®) Yes No

Scanning mobility particle sizer (SMPS) Yes No

Electrical low pressure impactor (ELPI) Yes No

Size Dynamic light scattering (DLS) Maybe Yes

Centrifugal sedimentation No Yes

Laser diffraction/static light scattering Yes Yes

Low pressure impactor and electrical lo w pressure impactor (ELPI) Yes No

Scanning/differential mobility analysis Yes No

Field flow fractionation (FFF) No Yes

Size exclusion chromatography (SEC) No Yes

Acoustic techniques No Yes

Electron microscopy (EM) No Possible with cryotechniques

Scanning probe microscopy (SPM) Maybe Yes

Time of flight mass spectroscopy Yes No

Atomic force microsc opy (AFM) No Maybe

a For some particle shapes, electron microscopy can be used to estimate surface area (LeBlanc, 2009, 625209 ). SMPS, ELPI, diffusion charger , and electron microscopy measure external surface area and may underestimate total surface area fo r porous particles. BET, on the other hand, will measure total surface area, which includes the inner surface area of porous or aggregated or agglomer ated particles (LeBlanc, 2009, 625209 ).

Source: Modified with permission from Info rma Healthcare, Maynard and Aitken (2007, 090674 ); Used with permission from Oxford University Press, Powers et al. (2006,
088783 ); Used with permission from Informa Healthcare, Powers et al. (2007, 090679 ); and data from Nanosafe (2008, 594868 )



# 1.6.2. Methods and Instrumentati on to Assess Environmental Occurrence

Detecting nanoparticles in the environment can be difficult because available analytical methods often are not sensitive enough for current environmentally relevant concentrations and cannot distinguish natural materials in the nanos cale size range from manufactured nanomaterials (Domingos et al., 2009, 193346 ; Englert, 2007, 193367 ; Simonet and Valcárcel, 2009, 193648 ). Also, many analytical methods require samp le treatment and extraction (Englert, 2007, 193367 ),

1-16

### which may include solvent evaporation, and conse quently could cause nanoparticle aggregation and salt precipitation (Simonet and Valcárcel, 2009, 193648 ). Detecting nanoparticles in water or soil is further complicated by the heterogeneous nature of the samples. Ideally such measurements would be done in situ to avoid changes in nanoparticles (such as agglomeration) due to differing conditions in the immediate milieu, but portable equipment suf ficiently sensitive to detect nanoparticles at environmentally relevant concentrations has not yet been developed (Simonet and Valcárcel, 2009,
193648 ). Analytical methods that are currently available for nanomaterials in soil, sediment and ground water were summarized in a recent EPA State of Science Review (U.S. EPA, 2008, 157480 ) (Table 1-4). Methods can be coupled to enable dete ction of more than one parameter at a time. For example, flow FFF can be coupled with ICP-MS for both size and chemical analysis. Methods for properties relating to chemical reactivity or charge, such as surface charge and pH pzc 

### , are not included in the basic characterization methods summarized in Table 1-4. In a study comparing six analytical methods for determining nanomaterial sizes (TEM, atomic force microscopy [AFM], DLS, fluorescence correlation spectroscopy, nanoparticle tracking analysis, and flow FFF), Domingos et al. (2009, 193346 ) concluded that the two most commonly used techniques reported in the literature (elect ron microscopy [EM] on air-dried samples and DLS) were also the two techniques that appear to be most prone to artifacts that can interfere with interpretation of results. Using multiple analytical techniques or multiple preparation techniques, or both, has been recommended (Domingos et al., 2009, 193346 ; Englert, 2007, 193367 ). The measurement and detection of engineered na noparticles across a variety of environmental media is an active and growing area of research , though an extensive review of analytical methods falls outside the scope of this case study. A gr owing body of research focuses on developing methods
to detect and characterize nanomaterials and their be havior within environmentally relevant matrices (Boxall et al., 2007, 157712 ; Hassellöv et al., 2008, 157559 ; Stone et al., 2010, 633898 ; Tiede et al., 2008, 196278 ; Tiede et al., 2009, 633895 ; Tiede et al., 2009, 193680 ).

1-17

## Table 1-4. Analytical methods for nanomateria ls in soil, sediment, and ground water for size fraction and distribution, surf ace area, and phase and structure

Metric Analytical method Sample type

Size fractionation Centrif ugation Aquatic colloids and particles extracted from soil and sediment samples. Nanoparticles must be in solution. Ultrafiltration – direct-flow ultrafiltration or tangential-flow ultrafiltration (TFF)

Field flow fractionation (FFF)

Capillary electr ophoresis (CE)

Size exclusion chromatography (SEC)

Size distribution Transmission electron microscopy (TEM)

Scanning electron microscopy (SEM)

Scanning probe microscopy (SPM)

Dynamic light scattering (DLS)

Laser-induced breakdown detection (LIBD)

Small- and wide-angle X-ra y scattering (SAXS/WAXS)

Surface area Brunauer, Emmett, Teller method (BET)

Calculation from TEM (length and widt h) and atomic force microscopy (AFM) (height) measurements, and particle nanocrystalline geometrics Only nanomaterials with a regular or pseudoregular geometry and without significant porosity

Phase and crystalline structure Electron diffraction

X-ray diffraction (XRD)

X-ray absorption spectroscopy (XAS)

Raman spectroscopy

Source: Data from U.S. EPA (2008, 157480 ).



# 1.6.3. Methods and Instrumentation to Assess Workplace Exposure

Workplace exposure monitoring thus far has focused on measuring nanoparticles in the air. Instruments that can be used for aerosol sampling are available, but most are designed for laboratory use (Nanosafe, 2008, 594868 ) and lack one or more of the foll owing desired attributes: portability, ease of use, capacity to distinguish nanoparticles from non-nanoparticles, different size bins in the 1-100 nm range, or ability to sample pe rsonal breathing zones (Ostraat, 2009, 196077 ). Several governmental and nongovernmental organi zations have begun addressing the need for instrumentation and methods for monitoring nanoma terials, particularly nanoaerosols, in the workplace. For example, NIOSH recen tly published a document titled Approach to Safe Nanotechnology–Managing the Health and Safety Concerns Associated with Engineered
Nanomaterials (NIOSH, 2009, 196073 ), in which sampling and monitoring methods and equipment are discussed. The Nanoparticle Occupational Safe ty and Health (NOSH) Consortium, an industry led consortium of participants from academia, gove rnmental, and nongovernmental organizations, is helping to define best practices for working safe ly with engineered nanoparticles (Ostraat et al., 2006, 667690 ; Ostraat et al., 2008, 193591 ). The NOSH Consortium has developed portable air monitoring methods intended for daily monitoring in nanoparticle research and development or in manufacturing settings. Maynard and Aitken (2007, 090674 ) summarized available devi ces and approaches for evaluating particle number, surface area, and ma ss concentration of na noparticles for use in monitoring aerosol exposure. In 2008, the Nano Safe2 project, a European Community-sponsored

1-18

### 1-19 

### project for safe production and use of nanomaterial s, released a report that highlighted findings in measurement methodologies for nanoparticle detec tion and measurement that use various types of on-line and off-line monitoring instruments (Nanosafe, 2008, 594868 ). The report provided examples of new nano-aerosol measurement equipm ent that is easy to transport and use. No commercially available equipment, however, is cu rrently available for long term monitoring. The report also recommended that monitoring at workplaces include not only personal sampling and measurements inside the facility, but also measur ements of nanomaterials in drains and in the exhausted air to help ensure protection of the environment. Finally, several companies are developing or have developed air monitoring devices for nanoparticle detection. The parameters that each device measures vary (Bennett, 2005, 193820 ; TRS Environmental, 2009, 196057 ; van den Brink, 2008, 196075 ).



# 1.6.4. Summary of Analytic Methods

Many techniques can be used to measure and char acterize nanomaterials in the laboratory and manufacturing workplace, and some are available fo r detecting nanomaterials in the environment. However, no single instrument can characterize all of the physicochemical properties of interest. Technical difficulties still exist in certain aspects, such as measuring and characterizing nanomaterials in organisms, and distinguishing na turally-occurring nanomaterials from engineered
nanomaterials in the environment.

# Chapter 2. Life Cycle Stages

This chapter discusses the life cycle of nano-TiO 2 

### as either a drinking water treatment agent or an ingredient in topical sunscreen. Each stage in the life cycles of the respective applications is considered from the standpoint of poten tial releases to the environment.



# 2.1. Feedstocks

Two ores, ilmenite (FeTiO 3 

### ) and rutile (TiO 2 

### ), predominate as feedstock materials for TiO 2 

### production (nano and otherwise) (Haridasan et al., 2008, 155625 ). Ilmenite and rutile are often found together, but ilmenite is found and mined in far gr eater quantities (at a ratio of more than 10:1 by weight) (Gambogi, 2008, 155622 ) and supplies approximately 90% of Ti minerals worldwide. For the rutile-based manufacturing processes, the most common manufacturing pathway for producing
TiO 2 

### of all kinds is via the chloride route using titanium tetrachloride (TiCl 4 

### ), a liquid used in approximately 60% of current manufacturing (Hext et al., 2005, 090567 ). Creating synthetic rutile
from ilmenite is often more economical than eliminating im purities from natural rutile. World ilmenite production in 2007 was approximat ely 5.6 million metric tons (MT), and world rutile production was approximately 0.5 million MT. The nations that produce the greatest quantities of ilmenite are Australia, South Africa, Canada, China, Norway, India, the U.S., and Ukraine. Significant producers of rutile include Australia, Ukraine, South Africa, India, and the U.S. (Gambogi, 2008, 155622 ). An estimated 1 billion tons of TiO 2 

### could be produced from existing world ilmenite resources, with another 230 m illion tons from rutile deposits (Mineral, 2009,
195905 ). In the U.S., ilmenite and rutile are extracted by surface mining or reprocessing of mine tailings at two sites in Florida and Virginia. Combined ilmenite and rutile production is approximately 0.3 million MT. Mine and mill employment at these sites was estimated at 229 persons in 2007, down from 344 in 2003 (Gambogi, 2008, 155622 ). Low levels of radioactive materials are present in ilmenite and natural rutile (Collier et al., 2001, 155617 ; Haridasan et al., 2008, 155625 ). A study in India found that those who work with ilmenite could be exposed to an annual dose of 1 millisievert (mSv) of gamma radiation and another 0.7 mSv of radioactivity via particle inhalation, mostly due to thorium. Thorium radioactivity in ilmenite was approximately 60% of the regulatory exemption limit established in the International Atomic Energy Agency (IAEA) Basic Safety Standar ds. Levels of radioactivity in natural rutile, ilmenite-derived synthetic rutile, and TiO 2 

### pigment (produced by the chloride route, particle size not specified) are lower than ilmenite, while levels of radioactivity (from radium as well as thorium) in solid wastes and liquid effluent are elevated co mpared with ilmenite (Haridasan et al., 2008,
155625 ).
Another common feedstock is titanium sulfate solution, which can be hydrolyzed to form
TiO 2 

### . The sulfate method begins with ground ilmenite or Ti slag.

Note: Hyperlinks to the reference citations throughout this doc ument will take you to the NCEA HERO database (Health and Environmental Research Online) at http://epa.gov/hero . HERO is a database of scientific litera ture used by U.S. EPA in the process of developing science assessments.

2-1

# 2.2. Manufacturing

Around 2005, annual global production of nano-TiO 2 

### was estimated at 2,000 MT, with an overall market value of $70 million (Dransfield, 2005, 157809 ; Osterwalder et al., 2006, 157743 ). Approximately 65% of production at that time was thought to have gone to “personal care” applications such as topical sunscreens and cosme tics, with the remainder used in industrial applications such as plastics, catalysts, a nd ceramics. Commercial production of nano-TiO 2 

### for years 2006-2010 has been estimated at 5,000 MT/year, and more than 10,000 MT/year predicted for years 2011-2014 (UNEP, 2007, 196074 ). In spite of some advantages of nano-TiO 2 

### over conventional
TiO 2 

### , nano-TiO 2 

### cannot replace all conventional uses of TiO 2 

### . For instance, as a whitening agent,
conventional TiO 2 

### , and not nano-TiO 2 

### , is needed to scatter visible light. From an economic point of view, the cheaper conventional TiO 2 

### is likely to be used in applications that can use either TiO 2 

### or nano-TiO 2 

### . Nonetheless, nano-TiO 2 

### production based on a predicted trend of graduate and a theoretical upper bound of total replacement of conventional TiO 2 

### was recently presented (Robichaud et al., 2009, 193617 ). The current and future worldwide production levels of nano-TiO 2 

### was estimated to have an upper estimate of approxi mately 2.5 million MT by 2025 (Robichaud et al., 2009, 193617 ). Thus far, nano-TiO 2 

### production has represented a small fraction of overall TiO 2 

### production, which commanded a market of 4.5 million MT and $9 billion (Dransfield, 2005, 157809 ; Osterwalder et al., 2006, 157743 ). Manufacturers and researchers report nano-TiO 2 

### synthesis by various techniques, including chemical vapor deposition (CVD) and flame hydrolysis (Wahi et al., 2006, 090580 ). Further information on manufacturing of nano-TiO 2 

### is provided in Appendix B. CVD, commonly used for production of both conventional and nanoscale TiO 2 

### , involves the conversion of a volatile compound to a nonvolatile solid that deposits on a substrate (Li et al., 2003, 090581 ; Nagaveni et al., 2004,
090578 ). A variety of techniques are used to generate the vapor and collect the particles, including plasma, high temperatures, pr essure, and injection, among others (Aitken et al., 2004, 090566 ). According to one industrial manufacturer of na noscale titania, flame hydrolysis can generate high-purity nano-TiO 2 

### using TiCl 4 

### as a feedstock (Mulenweg, 2004, 090592 ). Like CVD, flame hydrolysis can be used to deposit a thin film on a surface, a process known as flame hydrolysis deposition (FHD). In FHD , an inert gas carries TiCl 4 

### into a flame that produces hydrogen chloride (HCl) and a mixture of sizes of the metal oxide TiO 2 

### (Tok et al., 2009, 196054 ). Flame hydrolysis is used for manufacturing P25 and yields agglomer ated particles with a mean diameter of approximately 3.6 μm, with the smallest 4% of particles having an average diameter of 160 nm (Klaessig, personal communication, 2006, 196041 ). Anticipated by-products of this flame hydrolysis method of TiO 2 

### production include those resulting from chlorine contamination of the TiO 2 

### (from the TiCl 4 

### precursor). Warheit et al. (2007,
090594 ) have suggested that solutions of P25 in water are acidic (pH = 3.28) because of chloride ion artifacts on the particle surface. Manufacturer information, however, indicates that a steam washing step during the manufacturing process removes HCl adsorbed on the surface of P25 (Vormberg, 2004, 157822 ). Another production method used to manufacture pigmentary-grade TiO 2 

### is the sulfate process, although it can also be used to manufacture nano-TiO 2 

### in certain commercial settings (Medley, personal communication, 2008, 196038 ). Details on this and other processes used in producing
nano-TiO 2 

### can be found in Appendix B. When photocatalytic or other applications re quire smaller particles, additional postmanufacturing processes of sufficient energy can be utilized to break apart the aggregates/agglomerates. Surfactants or solvents can be used to help keep the smaller particles from reaggregating after separation (Hewitt, 1996, 157936 ; Porter et al., 2008, 157508 ). Also, nanoscale particles might be sonicated to increase dispersion (Bihari et al., 2008, 157593 ).

2-2

# 2.2.1. Drinking Water Treatment

No information was found on processes used in preparing or formulating nano-TiO 2 

### specifically for use in drinking water treatment. P25 is used in a commercial water treatment system (Photo-Cat from Purifics) that can be used for drinking water, ground water, and wastewater treatment (NSF International, 2009, 196092 ; Pichat, 2003, 196037 ; Purifics, 2008, 196040 ). For this treatment system, P25 is neither specially prepared nor coated (Powell, personal communication, 2009, 196056 ).



# 2.2.2. Sunscreen

Unlike drinking water treatment agents, information on the manufacture of topical sunscreens that incorporate nano-TiO 2 

### is relatively abundant. Although specific details of manufacturing protocols are typically proprietary, general informat ion on manufacturing processes and materials is readily available. The choice of a specific nano-TiO 2 

### crystalline form is a key issue in manufacturing sunscreens because various forms differ in photostability. In particular, rutile is much more photostable than anatase (Chaudhuri and Majewski, 1998, 093308 ; Maynard, 2008, 157522 ). Although less photostable, anatase appears to be in common use. Barker and Branch (2008, 180141 ) studied five sunscreens containing nano-TiO 2 

### , purchased over the counter, and found that one was pure rutile, while the other four were anatase/ruti le mixtures in which anatase predominated.
To increase nano-TiO 2 

### photostability, the particles are commonly given a surface coating such as silica, alumina, simethicone, or a variety of other compounds (see Appendix B for more information on coatings). Another technique for increasing photostability is by “doping” nano-TiO 2 

### particles by embedding minute amounts of metals within them, such as manganese, vanadium, chromium, and iron (Park et al., 2006, 193593 ). Another important consideration in the manufacture of most topical sunscreens is the use of a liquid medium, or dispersion, to ensure that nano-TiO 2 

### will be distributed evenly, thereby reducing aggregation and agglomeration. Aggregation and agglomeration can negatively impact UV scattering performance and transparency by increasing the effective particle size. Sunscreen manufacturers can purchase nano-TiO 2 

### powder and formulate their own dispersion, or they can purchase ready-made “predispersions.” Surface coatings influence the interaction of nano-TiO 2 

### with the dispersion medium, which can be water-based (aqueous), oil-based, or silicon e-based. These and many other factors figure into the manufacture of sunscreens, including pH; emulsi fiers; emollients; other physical UV blockers (e.g., ZnO, which can also be micr onized); chemical UV filters; and various inert ingredients to achieve the desired viscosity/liquidity, spray-abi lity, color/transparency, water resistance, and spreadability. More detailed information on manufactur ing processes is presented in Appendix B.



# 2.3. Distribution and Storage

Limited information is available regarding nano-TiO 2 

### distribution and storage. P25 is shipped as a powder in 10-kg “multilayer ventilated paper bags, equipped with an additional polyethylene lining when required” (Degussa, 2007, 090576 ). P25 presumably could be stored as a powder in a chemical storage facility in the original 10- kg shipping bags. Degussa recommends storing it in closed containers under dry conditions (Degussa, 2007, 090576 ). Releases could occur if bags were damaged during shipping or storage, although such releases should be minimized by proper implementation of standard good management practices. Another brand of photocatalytic nano-TiO 2 

### (KRONOS vlp 7000, 7001, and 7500) is also shipped in 10-kg paper bags (KRONOS International, 2006, 196046 ). Nano-TiO 2 

### powders from

2-3

### Sigma-Aldrich Corporation (Sigma), on the other ha nd, are shipped in amber glass bottles enclosed in foil or plastic bags, which are shrink-wrapped before being placed in cardboard boxes with shipping cushion peanuts. As a dispersion formulation, nano-TiO 2 

### is shipped in pails, drums, or totes (Klaessig, personal communication, 2008, 196042 ). Sigma ships its nano-TiO 2 

### dispersion in essentially the same way
nano-TiO 2 

### powders are shipped. Dispersion-formulated nano-TiO 2 

### presumably would require protection from freezing in cold climates. Depending on where accidental releases of such dispersions occurred, nano-TiO 2 

### could be released into water or soil during shipment or discharged into industrial or municipal wastewater treatment systems during storage.



# 2.3.1. Drinking Water Treatment

No information pertaining to the distribution and storage of nano-TiO 2 

### used specifically for water treatment agents was identified.



# 2.3.2. Sunscreen

Topical sunscreen products are generally packaged in retail-sized bottles at the production facility and shipped in large containers to whol esalers, retailers, and direct marketers. Little information is available on methods of shipping or storage. Consumers generally handle only retailsized packages. Industry data from the 1990s, although perhaps out of date, shed light on the distribution chain of sunscreens. Sales in supermarkets, drugstor es, and mass merchandise outlets accounted for approximately two-thirds of the total U.S. sun-care retail sales in 1992-1993, according to Davis (1993, 157949 ). The remaining one-third was attributed to sales in department stores and other “prestige” stores. Sun-care products are also sold by direct marketers (e.g., Avon, Amway, Mary Kay), discount stores, swimwear stores, and small variety stores (e.g., those near beaches and ski slopes) (Davis, 1993, 157949 ). At any point in the distribution-to-storage chain, accidental releases could occur. For example, a shipping accident, a dropped palette, or crushed re tail-size container(s) could lead to releases.



# 2.4. Use

2.4.1. Drinking Water Treatment

Nano-TiO 2 

### could be used in various ways to treat drinking water, as discussed in Section 1.5.1. This discussion, however, is limited to nano-TiO 2 

### that would be used to remove arsenic at drinking
water treatment facilities. Roughly 54,000 community water systems in the U. S. serve more than 95% of the population (U.S. EPA, 2006, 091194 ). Most of these systems apply some form of treatment to remove or neutralize chemical or microbial contaminants. Those that do not apply treatment serve less than 5% of the U.S. population; these systems are generally sm all or medium sized (i.e., serving no more than 10,000 people) and rely on ground water wells (U.S. EPA, 2002, 091192 ). Public water systems are required to keep arsenic concentrations in delive red water at or below a maximum contaminant level (MCL) of 0.01 mg/L (U.S. EPA, 2006, 091193 ). Approximately 5% of community water systems in the U.S. (i.e., approximately 3,000 systems serving 11 million people) have taken some action to be in compliance with the arsenic MCL (U.S. EPA, 2007, 091224 ). Likewise, approximately 5% of 20,000 nontransient noncommunity water systems that serve at least 25 of the same people for more than 6 months of the year, such as schools, churches, nursing homes, and factories (i.e.,

2-4

### approximately 1,100 systems serving 2 million people) have also taken some action to comply with the arsenic MCL (U.S. EPA, 2007, 091224 ). Altogether, approximately 13 million people use water that is treated to remove arsenic. Although it is unknown to what extent nano-TiO 2 

### might be used in any of these systems in the future, these numbers provide perspective on its potential usage for drinking water treatment. Depending on the type of drinking water treatment system, nano-TiO 2 

### might be used as powder (e.g., in a slurry) or fixed on a supporting mate rial as a component of adsorptive media. Each approach has its potential advantages and disadvantages. Powdered nano-TiO 2 

### has a large surface area and offers highly efficient photocatalytic oxidati on, but a means to filter out and/or recycle all of the photocatalyst is required (Dionysiou, personal communication, 2009, 193921 ; Pichat, 2003,
196037 ). This suggests the possibility that some amount of nano-TiO 2 

### suspended in water might pass through filters, including microfilters. Also, if nano-TiO 2 

### builds up on the filter matrix (i.e., if it is not removed by filter backwashing and hydraulic clean ing of sand), it could saturate the filtration medium, and small quantities might be released with filtered water into subsequent steps of the treatment sequence. Fixed nano-TiO 2 

### has a smaller surface area and thus is less efficient. Although the attachment to the supporting material should allow no leaching, a fixed photocatalyst might not require filters or recycling systems to remove nano-TiO 2 

### from the final product (Dionysiou, personal communication, 2009, 193921 ). Zhang et al. (2008, 193735 ) investigated the removal of nano-TiO 2 

### in a simulated conventional drinking water treatment procedure, which include d coagulation, flocculation, sedimentation, filtration, and disinfection. Two types of nano-TiO 2 

### (crystal form unspecified, primary particle sizes of 15 and 40 nm, and aggregates 200 and 500 nm, resp ectively) in 2-liter jars were subjected to the treatment procedure. Adding magnesium chloride (MgCl 2 

### ) or alum (Al 2 

### (SO 4 

### ) 3 

### ·16H 2 

### O), followed by coagulation, flocculation, and sedimentation, st ill left more than 20% of an initial 10-mg/L
concentration of nano-TiO 2 

### in the settled water. Furthermore, the removal efficiency was lower in tap water than in buffered nanopure water (pH 5.6) due to the presence of organic matter in the tap water. Membrane filtration with a pore size of 0.45 μm (450 nm) after sedimentation removed
nano-TiO 2 

### aggregates larger than 500 nm, leaving only 1-8% of the initial TiO 2 

### in the treated water. Although most, but not all, of the nano-TiO 2 

### in the initial water was removed, this level of filtration is not typical in water treatment plants (Flummer, personal communication, 2008, 157573 ; Kline, personal communication, 2008, 157545 ), nor is it available in most whole-house filtration systems (Johnson, 2005, 157799 ). At least two commercially available water treatment systems can employ nano-TiO 2 

### , although to date they are not known to be routinely used in this manner. One system uses nano-TiO 2 

### in a fixed
membrane and the other uses nano-TiO 2 

### in a slurry. A system from Matr ix Photocatalytic Inc. uses a tube covered with fiberglass mesh in which nano-TiO 2 

### is embedded; the tube contains water that circulates and UV lamps illuminate the outside (Dionysiou, personal communication, 2009, 193921 ; Pichat, 2003, 196037 ). In the Photo-Cat sys tem by Purifics, nano-TiO 2 

### (P25) circulates in a slurry inside a narrow annulus surrounded by a UV lamp (Pichat, 2003, 196037 ). A ceramic membrane
filters out nano-TiO 2 

### (Purifics, 2008, 196040 ). No empirical data are available on the life expectancy of either system or whether they can release nano-TiO 2 

### into treated water. The Purifics system was pilot-tested for two months in a community drinking water treatment facility (Purifics, 2008, 196040 ). The ceramic membrane used to filter nano-TiO 2 

### (particles as small as 12 nm) from the finished product was reported to require no servicing or cleaning during the 2-month period because the nano-TiO 2 

### particles collected in the me mbrane were removed by bursts of high-pressure air (Pichat, 2003, 196037 ; Purifics, 2008, 196040 ). Although the purpose of this pilot test was not to remove arsenic, se veral studies have bench-tested nano-TiO 2 

### in slurry systems for removal of arsenic from water (Dutta et al., 2004, 157845 ; Ferguson et al., 2005, 090572 ; Lee and Choi, 2002, 193498 ; Li et al., 2003, 090581 ; Meridian, 2006, 090595 ). Higher arsenic oxidation rates occurred using a slurry that was continuousl y stirred (compared to immobilized nano-TiO 2 

### ) (Li et al., 2003, 090581 ). In actual use, steps likely would be taken to keep nano-TiO 2 

### dispersed during

2-5

### treatment, which could affect solubility and par ticle agglomeration. Surface modification could affect dispersion and could also improve the materi al’s photocatalytic properties as described (Ryu and Choi, 2004, 193622 ). Additionally, numerous chemicals can be added for drinking water treatment (NSF International, 2009, 196092 ), any or some combination of which could affect the solubility, particle size, and behavior of the nano-TiO 2



### .



# 2.4.2. Sunscreen

The estimated use of sunscreen can vary greatly am ong surveys, but it is clear that its use is significant (Kasparian et al., 2009, 193465 ; Keeney et al., 2009, 193466 ). Four U.S. studies that collected data in the years 1995-1999, with 1,000 to more than 10,000 participants in each survey, showed that approximately one in three people said they use sunscreen regularly (Cokkinides et al., 2001, 193321 ; Geller et al., 2002, 193390 ; Santmyire et al., 2001, 193629 ; Weinstock et al., 2000,
193716 ). In three studies, 31-45% of survey respondents said they routinely or often use sunscreen (Cokkinides et al., 2001, 193321 ; Geller et al., 2002, 193390 ; Weinstock et al., 2000, 193716 ). In another study, 30% of respondents said they were very likely to use sunscreen when they were outdoors (Santmyire et al., 2001, 193629 ). More recently, data from the 2005 Health Information National Trends Survey in the U.S. showed that among a total of 496 Latino participants, 15% reported that they always use sunscreen, 9% reporte d often use of sunscreen, and 20% reported that they sometimes use sunscreen (Andreeva et al., 2009, 193252 ). In the 2007 iVillage survey, the Skin Cancer Foundation (2008, 594955 ) found that 11% of respondents use sunscreen with a sunburn protection factor (SPF) of 15 or higher “every day,” and 59% of respondents use sunscreen at least occasionally (up from 39% in a 2003 survey), where SPF is defined by the U.S. FDA (2009, 196372 ) as a “measure of how much solar energy (UV radiation) is required to produce sunburn on protected skin (i.e., in the presence of sunscreen) relative to the amount of solar energy required to produce sunburn on unprotected skin.” Of those who wear s unscreen, 74% reapply it “at least every 4-6 hours or after swimming or sweating,” and 28% reapply it every 2 hours, the Skin Cancer Foundation’s recommended rate of reapplication (Skin Cancer Foundation, 2008, 594955 ). While the use of sunscreen may be lower in young adults and adolescents than adults (Kasparian et al., 2009, 193465 ) sunscreen use is likely to be higher in young children. Robinson et al. (2000, 193618 ) surveyed 503 people in the summer of 1997, and found that 54% of parents reported that their child always or usually used a sunscreen, but only 27% of parents used sunscreen themselves during the previous weekend. This is consistent with a survey of 254 parents in June-July of 1999 by Weinstein et al. (2001, 191128 ) in Chicago, in which parents reported more frequent use
of sunscreen on their children than on themselves. The total amount of sunscreen, and more particularly the total amount of nano-TiO 2 

### in sunscreen, used in the U.S. is unknown. Furthermore, the available survey data do not differentiate between sunscreen products with or without nano-TiO 2 

### , although the percentage of sunscreen with
nano-TiO 2 

### is thought to be substantial. In 2006, the Australian Therapeutic Goods Administration (TGA) estimated that 70% of sunscreens containing Ti and 30% of sunscreens containing zinc in Australia were formulated with nanoparticles (TGA, 2006, 089202 ). As noted in Section 2.2, annual global production of nano-TiO 2 

### was estimated at 2,000 MT around 2005, with approximately 65%, or 1,300 MT, used in “personal care” products such as topical sunscreens and cosmetics (Dransfield, 2005, 157809 ; Osterwalder et al., 2006, 157743 ).
644432 A poster presentation by Johnson et al. (2009, ) at SETAC Europe’s 19th Annual Meeting suggested that possible concentrations of nano-TiO2 in water, as a result of sunscreen use, are between 2,000 and 8,000 ng/L. This range is based on modeling assumed rates of sunscreen use over the course of a day, how much is expected to wash off, and how mu ch will be removed by sewage treatment plants during various summer-time scenarios in the River Thames region of the
UK.

2-62-7 

# 2.5. Disposal

2.5.1. Drinking Water Treatment

Most community water treatment filters, with regular backwashing, have an indefinite life span. Slow sand filters are generally cleaned no t by backwashing, but by scraping and replacing the top layer of sand. Scraped sand is normally cleaned hydraulically and stockpiled for later reuse (Cleasby and Logsdon, 1999, 091181 ). This process creates wastewater, which might be recycled in the treatment train or discharged (e.g., to a municipal sewer). For processes in which nano-TiO 2 

### would be introduced prior to or during the sand filtr ation process, the eventual disposal of the filter
sand or other filter materials could result in nano-TiO 2 

### entering landfills along with the filter.
After nano-TiO 2 

### is used in drinking water treatment, a sludge material (floc) containing
nano-TiO 2 

### would likely be created. In one scenario the sludge might be taken to a landfill; this is the case with approximately 30% of sludge generate d from drinking water treatment (U.S. EPA, 2010,
635678 ). Whether TiO 2 

### could diffuse (and thus be released) from a solid matrix such as sludge is unknown. Some newly developed landfills are designed to collect and treat leachate, but leaks are still possible, and the ultimate fate of nano-TiO 2 

### in the treatment process is unknown. In addition,
some older landfills without leachate collect ion measures may still be in use. Nano-TiO 2 

### and other contaminants such as residual arsenic could become suspended in leachate and enter ground water, or they could pass through a solid waste facility liner into the subsurface. Under a different scenario, the sludge could be used for land application (U.S. EPA, 2010,
635678 ). This is the case with approximately 20% of sludge generated from drinking water treatment, which applied to land to improve soil co nditions or to fertilize the soil. The sludge is plowed directly into the soil to limit water runoff and for sanitary reasons (U.S. EPA, 2010, 635678 ).
Nano-TiO 2 

### and other contaminants such as residual arsenic would then be present in these agricultural soils.
If nano-TiO 2 

### is present in finished drinking water that reaches the tap, it would eventually enter the ambient environment or be captured by a wastewater stream, after which it could enter sewage treatment facilities.



# 2.5.2. Sunscreen

Sunscreen containers likely would be disposed of primarily as municipal solid waste and thus end up in landfills or incinerators. The potential for leaching of nano-TiO 2 

### from landfill disposal of containers would depend on many factors, including the integrity of liners and leachate collection systems, if present. Incineration of sunscreen co ntainers raises the question of whether nano-TiO 2 

### could enter the stack and be released to air, or become a trace contaminant in fly or bottom ash. Depending on the packaging, sunscreen cont ainers might be recycled, suggesting the possibility that nano-TiO 2 

### could be incorporated into recycled materials. Additional exposure pathways other than the specific handling of sunscr een containers are acknowledged as potentially important, and will be addressed as part of the fate and transport discussion in Chapter 3.

# Chapter 3. Fate and Transport

Chapter 3 explores what might happen to nano-TiO 2 

### after it is released to the environment at various stages of the product life cycles for wate r treatment agents or t opical sunscreens. Nano-TiO 2 

### could be released to air, water, or soil and th en transported or transformed through chemical or biological processes. The lack of data on the fate and transport of nano-TiO 2 

### by-products and waste produced during the manufacturing process also precludes a comprehensive discussion in this chapter. This chapter does, however, summarize what is known about the environmental pathways and transport and transforma tion processes of nano-TiO 2 

### related to the various life-cycle stages described in Chapter 2. The preceding chapter discussed life cycle stages of nano-TiO 2 

### with some considerations specific to its use in drinking water treatment for ar senic removal and in sunscreens. As this chapter focuses on the various pathways by which nano-TiO 2 

### can potentially enter and propagate through environmental compartments, information related to wastewater treatment pathways and by-products will also be pertinent. Throughout this document, it is important to note the distinction between the two types of water treatment being discusse d. The case study application of nano-TiO 2 

### used in drinking water treatment for arsenic removal is dis tinct from the potential downstream appearance of
nano-TiO 2 

### in municipal wastewater treatment plants. The former scenario deals with the use of nanoTiO 2 

### , while the latter deals with its impacts after release to the environment. Because the processes for drinking water treatment and municipal wastewat er treatment are different, they will lead to different scenarios for the fate and impacts of nano-TiO 2



### . Although most studies cited in this chapter consider nano-TiO 2 

### in aggregate or agglomerate form (as discussed in Chapter 1), it is unclear whether all constituent primary particle s remain in clusters if conditions change. Disagglomerati on, for example, can occur at certain pH pzc 

### levels. The pH pzc 

### of a nanoparticle is defined as the pH at the “ point of zero charge ,” which occurs when the net electric charge at the particle surface is zero. At the pH pzc 

### particles fail to electrostatically repel each other. In laboratory studies, the size range of aggregates and the presence of free nano-TiO 2 

### particles (ranging from 5 to 50 nm in size) were found to be pH-dependent: when the solution pH differed from the pH pzc 

### of the particles, the aggregates tended to be smaller (Dunphy Guzman et al., 2006,
090584 ; Dunphy Guzman, personal communication, 2007, 091184 ). Sampled aggregates ranged up to 150 nm in size, and contained an estimated 8-4,000 nanoparticles (Dunphy Guzman et al., 2006,
090584 ). The pH pzc 

### depends in part on the crystal form of the nano-TiO 2 

### particles. Finnegan et al. (2007, 193379 ) reported pH pzc 

### values of approximately 5.9 for rutile and 6.3 for anatase . The degree of aggregation generally increases with increases in ionic strength (Domingos et al., 2009, 193347 ; French et al., 2009, 193384 ). The interaction between natu ral organic matter (NOM) and the aggregation state of nano-TiO 2 

### is complex, and whether aggregation is enhanced or inhibited by the presence of these organic species can depend on factors such as concentration of NOM,
concentration of nano-TiO 2 

### , pH, and the presence of divalent cations such as calcium (Kim et al., 2009, 635778 ). Despite the presence, and sometimes the predominance, of large particles, several researchers investigating laboratory-synthe sized and commercial nano-TiO 2 

### products have found free particle s or aggregates with diameters less than 100 nm in varying amounts, depending on synthesis method, temperature, solution pH, and the presence of buffers (Kormann et al., 1988, 090582 ; Li et al., 2003,
090581 ; Nagaveni et al., 2004, 090578 ; Pena et al., 2006, 090573 ; Ryu and Choi, 2006, 090579 ; Sun et al., 2007, 193662 ; Wahi et al., 2006, 090580 ). Moreover, some preparations are specifically

Note: Hyperlinks to the reference citations throughout this doc ument will take you to the NCEA HERO database (Health and Environmental Research Online) at http://epa.gov/hero . HERO is a database of scientific litera ture used by U.S. EPA in the process of developing science assessments.

3-1

### designed to generate dispersed particles (e.g., Seok et al., 2006, 091198 ) to increase the efficacy of
nano-TiO 2 

### as a catalyst, increasing the potential for the presence of disagglomerated or even disaggregated nano-TiO 2 

### to occur in the environment. However, a limited number of studies of
nano-TiO 2 

### agglomeration/disagglomeration behavior under “real-world” ambient environmental conditions, irrespective of medium, have been conducted (Kiser et al., (2009, 225305 )Battin et al., 2009, 201604 ).



# 3.1. Water

Although numerous studies characterize nano -TiO 2 

### particles in aqueous solution under laboratory conditions, the fate and behavior of the particles in the environment have received less attention. One report indicated that nano-TiO 2 

### was detected in river water in Montana, but the source (natural or engineered) and the concentration of nano-TiO 2 

### were not determined (Wigginton et al., 2007, 157415 ). Several physicochemical properties of nano-TiO 2 

### can contribute directly to its environmental fate and transport in water. Long et al. (2006, 089584 ) reported that P25 rapidly aggregated in both Hank’s Basic Salt Solution (HBSS) and Dulbecc o’s Modified Eagle’s Medium (DMEM) buffer solutions, both of which are high-osmolarity fluids that contain high concentrations of the
monovalent cations Na + 

### and K + 

### [160 millimolar (mM)] and the divalent cations Ca 2+ 

### and Mg 2+ 

### (2 mM). The ionic strengths of these two solutions are approximately 155 mM and 166 mM, respectively. After 1 minute of sonication , aggregation continued for 20-45 minutes until a steadystate, stable aggregate size formed. The steady-st ate aggregate sizes ranged from 826 to 2,368 nm and the concentration of P25 ranged from 2.5 to 120 parts per million (ppm). Ridley et al. (2006, 090599 ) found that results were reproducible for classical titration procedures (with modification) to characterize th e surface charging properties of a commercially available, uncoated anatase nano-TiO 2 

### product (from Ishihara Techno Corporation, Osaka, Japan) in suspension. These findings showed that environmental pH can affect the surface charge properties. Schmidt and Vogelsberger (2006, 193634 ) studied the solubility of four types of nano-TiO 2 

### (P25 from Degussa, DT51D and G5 from Millennium Chem icals, and an original formulation – presumably all uncoated particles) in various aqueous solutions, particularly focusing on the kinetics of the dissolution process. At the beginning of the process, solubility increased rapidly over time and then reached a steady-state value. The maximum solu bility value (i.e., saturation concentration) was observed to depend on the morphology of the TiO 2 

### , the crystalline form of the nano-TiO 2 

### , and on the size of the nanoparticles exposed to dissolution. The saturation concentrations were higher in hydrolysis -generated nano-TiO 2 

### than in precipitation-generated nano-TiO 2 

### , and higher in smaller particles than larger particles. Ho wever, since the equilibrium solubilities of the four types of nanoTiO 2 

### ranged from micro-to nano moles per liter, wh ile the saturated suspensions were in the range of milligrams per liter, dissolved Ti concentrations were negligible compared with the initial TiO 2 

### input. Although many studies have demonstrated the potenti al to use the photocatalytic properties of
nano-TiO 2 

### in biocidal applications, including wa stewater treatment (Chen and Ray, 2001, 193310 ; Han et al., 2009, 193407 ; Khataee et al., 2009, 193468 ; Rincon and Pulgarin, 2003, 157856 ; Wang et al., 2008, 193705 ; Watlington, 2005, 196080 ; Xu et al., 2009, 193726 ), data on the fate of nano-TiO 2 

### in actual wastewater treatment facilities are sc arce. The Water Environment Federation released a report including the behaviors and effects of nanomaterials in wastewater treatment, although very
few studies were on nano-TiO 2 

### (Effects of nanoparticles on the wastewater treatment industry (Report No, 2008, 195800 ). Kiser et al.(2009, 225305 ), however, have reported the occurrence of
nano-TiO 2 

### at full-scale wastewater treatment plants (i n both raw and finished waters). The authors measured total Ti concentrations, which included som e nano-scale particles, on the order of 10 μg/L in tertiary effluent from wastewater treatment. Another investigator studied the effects of nano-TiO 2

3-2

### on aquatic microbial communities under environmen tal conditions, which has implications on both natural waters and on wastewater treatment environments (Battin et al., 2009, 201604 ). Several recent studies have used mass balance modeling to predict the accumulation of nanomaterials within various environmental compartments, including nano-TiO 2 

### accumulation in wastewater treatment plants (Gottschalk et al., 2009, 633897 ; Mueller and Nowack, 2008, 157519 ). Other types of nanoparticles also have been st udied in wastewater treatment plants. Limbach et al. (2008, 155628 ) studied the fate of cerium oxide nanoparticles (20- to 50-nm diameter) in a model wastewater treatment plant under a variety of conditions (e.g., with different surfactant s to stabilize dispersions, and in media with different ionic strengths and pH values). They found that surfactants stabilized dispersions under a wide range of test pH values even at high ionic strength. The model sewage treatment plant consistently reduc ed the cerium oxide nanoparticle concentration in the wastewater from 100 ppm to 2-5 ppm. M ost nanoparticles were removed via agglomeration with microorganisms in the sedimentation sludge. Comparing the physical properties and behavior of various oxides, the investigators speculated that TiO 2 

### and other insoluble oxides would behave similarly to cerium oxide, while more soluble or reactive oxides like ZnO would be even more likely to aggregate and be more amenable to removal by sedimentation. The investigators cautioned, however, that the high nanoparticle concentration (100 ppm) used in the study favors aggregation, and that at more realistic initial concentrations, a greater percentage of na noparticles are likely to break through. Kiser et al. (2010, 634458 ) investigated biosorption rates of eight nanoparticles, including
TiO 2 

### , to wastewater treatment sludge. Investigators found that different nanoparticles biosorbed at different rates when placed in solutions with varying concentrations and types of biomass, which were designed to represent wastewater treatme nt sludge. For example, 23% of nanoscale TiO 2 

### was removed via biosorption in biomass solution of 400 mg/L total suspended solids, compared to 88% of aqueous fullerenes in the same solution, 39% of functionalized Ag nanoparticles, and 13% of fullerol nanoparticles (Kiser et al., 2010, 634458 ). The authors noted that further research is needed to understand the specific mechan isms responsible for sorption.
A limited number of studies are available on nano-TiO 2 

### and its interactions with microorganisms and other NOM under “real-world” environmental conditions (Battin et al., 2009,
201604 ; Kiser et al., 2009, 225305 ; Kiser et al., 2010, 634458 ). Battin et al. (2009, 201604 ) investigated damage to microorganisms from a ggregated, agglomerated, and polydisperse nano-TiO 2 

### under natural conditions in river microcosms. Thei r toxicity results correlated poorly with lab experiment results on monodisperse nano-TiO 2 

### with monocultures, and contribute to the small but growing body of literature of nanoparticle toxici ty in natural aquatic systems. It has long been recognized that anatase TiO 2 

### can photogenerate fairly long-lived ROS such as hydrogen peroxide via photoinduced redox reactions or modification of the TiO 2 

### surface in aqueous laboratory environments (Harbour et al., 1985, 090632 ). It is not clear how relevant results from such experiments would be for anticipating nano-TiO 2 

### ’s behavior in wastewater or drinking water treatment plants.
The interaction between nano-TiO 2 

### and natural organic matter, which is ubiquitous in the environment, has been investigated in controlled conditions in the laboratory. Yang et al. (2009,
190513 ) found that humic acid, a common type of natural organic matter, is easily adsorbed onto
nano-TiO 2 

### in aqueous media. Because humic acid adsorption decreased the zeta potential (i.e., increased electrostatic repulsion) of nano-TiO 2 

### particles, humic acid-coated nano-TiO 2 

### could be more easily dispersed and suspended and thus more stable in an aqueous medium than uncoated

nano- TiO 2 

### (Yang et al., 2009, 190513 ). Sediment, the solid fragments of inorganic or orga nic material that are carried by and settle to the bottom of natural waters, is another environmental matrix that could be affected by the release of nanomaterials. One study was identified on the transport and deposition of nano-TiO 2 

### in natural streams and streambeds (Boncagni et al., 2009, 634454 ). Partitioning of nanomaterials, including

3-3

### nano-TiO 2 

### , to sediments and other environmental compartments was modeled by Gottschalk et al. (2009, 633897 ).



# 3.1.1. Drinking Water Treatment

Although the processes for using nano-TiO 2 

### for commercial water treatment are not yet well established and therefore a definitive understanding of nano-TiO 2 

### fate is not possible, nano-TiO 2 

### is not expected to be destroyed. The removal efficiencies of commercial nano-TiO 2 

### in conventional water treatment processes (coagulation, flocculati on, sedimentation, and filtration) have been reported in one study using jar testing (Zhang et al., 2008, 157462 ), although the condition was set
for nano-TiO 2 

### in source water and not as an agent in drinking water treatment. The study showed that more than 20% of initial 10 mg/L nano-TiO 2 

### remained in the water after up to 24 hours of
flocculation and 1 hour of sedimentati on (in buffered nanopure water with MgCl 2 

### ); more than 30% initial 10 mg/L nano-TiO 2 

### remained in water after alum coagul ation, flocculation, and sedimentation (in nanopure water); and filtration using a 0.45 μ m membrane as a final process was able to leave only 1% to 8 % of the total TiO 2 

### mass (in flocs smaller than 500 nm) in the water (Zhang et al., 2008, 157462 ). It is expected that the actual removal efficiencies in drinking water treatment facilities would be different from these tested conditions due to the differences in process time, source water, and other factors. For instance, under the tested conditions (Zhang et al., 2008,
157462 ), the most efficient nano-TiO 2 

### removal was seen after 8- or 24-hours flocculation and 1-hour sedimentation. Flocculation is typically less than 1 hour in drinking water treatment plants, which may result in less removal than observed in the Zhang et al. study, while sedimentation is commonly several hours, which may result in more removal (AWWA Staff, 2003, 193818 ). In addition, the removal efficiencies of nanopart icles, not limited to nano-TiO 2 

### , were lower in tap water containing natural organic matter compared to nanopure water (Zhang et al., 2008, 157462 ). Since the removal
of nano-TiO 2 

### initially received as a suspension (200-nm aggregates) was less efficient than the
removal of nano-TiO 2 

### initially received as dry powders (500-nm aggregates), the authors speculated that the removal efficiencies would be lower for small aggregates than large aggregates at the same alum (coagulation agent) concentration.
Several different waste streams that could contain nano-TiO 2 

### could be generated from drinking
water treatment facilities. For nano-TiO 2 

### that settles with floc in the sedimentation step, nano-TiO 2 

### presumably could become part of the sludge (AWWA Staff, 2003, 193818 ). The discarded sludge could be transported off-site for disposal or reuse, such as being buried in municipal solid waste landfills or directly applied to agricultural or recreational land. Theoretically, nano-TiO 2 

### might become part of the filter matrix during the filtration step of
water treatment. U.S. EPA’s Filter Back wash Recycling Rule (U.S. EPA, 2002, 644800 ) requires that, when the filter is backwashed, the water use d must be recycled back into the coagulation process. This could reintroduce nano-TiO 2 

### into the treatment process, but the implications for
concentrations of nano-TiO 2 

### in finished water are not clear. Various fate pathways could apply to nano-TiO 2 

### used as a drinking water treatment agent. For example, if nano-TiO 2 

### is not completely filtered out or ot herwise removed from the final effluent,
nano-TiO 2 

### might remain in the water as aggregates/agglomerates and enter municipal water tanks or
reservoirs. If some water were lost from the distribution system via leaks or spills, nano-TiO 2 

### could end up in surface waters or in the subsurface environment. If nano-TiO 2 

### were to enter ground water aquifers, nano-TiO 2 

### would presumably persist, given that other inorganic compounds are not readily
broken down in that environment and nano-TiO 2 

### is poorly soluble; however, particle/agglomerate
size and other characteristics could change. Conceivably, nano-TiO 2 

### could contribute to the release of (or modify the bioavailability of) other water contaminants of concern.
If nano-TiO 2 

### were present in the final drinking water product that reaches the tap, it eventually might enter the ambient environment or be capture d by a wastewater stream, after which it could reach a wastewater treatment facility. If the particular wastewater treatment method employed there

3-4

### did not completely remove nano-TiO 2 

### , some level of nano-TiO 2 

### would likely enter downstream

water sources.



# 3.1.2. Sunscreen

The environmental fate of nano-TiO 2 

### in topical sunscreens could be affected by the surface treatments and doping applied to nano-TiO 2 

### particles, by the sunscreen vehicle, or by any number of other constituents in such products (Appendix B). Nano-TiO 2 

### in emulsion, dispersion, and possibly powdered form could be present in wastewater (e .g., from equipment and site cleaning) and solid waste from sunscreen manufacturing facilities, depending on the trapping and filtration processes the facility uses. In the powdered form, nano-TiO 2 

### could escape the facility through air venting and filtration systems.
Nano-TiO 2 

### could also be released to wastewater or to natural bodies of water through showering/bathing or through laundry water draina ge following sunscreen use. Swimming after
sunscreen use could result in an accumulation of s unscreen material in the swimming pool water and potentially be a point of release into the environment as untreated wastewater. If nano-TiO 2 

### remained mobile in water, it could enter downstream wa ter sources in a manner similar to that of the
nano-TiO 2 

### used for drinking water treatment. Auffan et al. (2010, 625063 ) investigated how nano-TiO 2 

### particles formulated for use in sunscreens transform, or age, when placed in media that mimicked environmental conditions and conditions of product use. Their results showed that 90% wt of one coating constituent desorbed from the particle surfaces, that another constitue nt remained on the surface but was oxidized, and that the third constituent was chemically affected but remained sorbed at the surface. Though the remaining Al-based layer was still effective in protecting against the production of superoxide ions from the photoactive nano-TiO 2 

### particle core under their experimental conditions, these changes in coating characteristics illustrate that transformations may occur once nano-TiO 2 

### is released to the
environment. The potential for release is suggested by recent st udies that have detected topical sunscreen constituents in untreated wastewater, treated wastew ater, surface water (lakes and rivers), fish from lakes and rivers, and biosolids (Balmer et al., 2005, 157817 ; Fent et al., 2008, 157574 ; Rodil and Moeder, 2008, 157503 ). The organic compounds detected in these studies were UV filter compounds such as 4-MBC (4-methylbenzylidene camphor) and octocrylene (OC), which generally biodegrade slowly and can bioaccumulate. Some evidence also indicates that nano-TiO 2 

### can bioaccumulate (Zhang et al., 2006, 157722 ). Although nano-TiO 2 

### is unlikely to behave exactly the same way as other components of sunscreen, the observed nano-TiO 2 

### bioaccumulation in fish (Zhang et al., 2006,
157722 ) suggests the possibility of persistent presence of nano-TiO 2 

### . However, no studies to date
have documented the occurrence of nano-TiO 2 

### specifically from sunscreens in wastewater or natural
bodies of water.



# 3.2. Soil

Three studies addressed the fate and transport of nano-TiO 2 

### in soil. Dunphy Guzman et al. (2006, 090584 ) studied the effect of pH on nano-TiO 2 

### mobility in a model soil column. They found that both surface potential and aggregate size influenc e transport. In the pH range where electrostatic
forces between nano-TiO 2 

### aggregates and the experimental Pyrex surface should have been strong (pH 2.5-5.9), nano-TiO 2 

### was highly mobile. The calculated interaction energy was expected to be greatest for the largest aggregates at pH 12, but th ese were the particles that most strongly attached to microchannel surfaces. At pH 3, where c onditions were predicted to be favorable for negative/positive interaction, 84% of the particle s were transported. The authors concluded that

3-5

### current transport theory does not adequately predict transport of nanoparticles and aggregated nanoparticles. These results suggest that nano-TiO 2 

### particles and aggregates of nanoparticles in a stable dispersion might be highly mobile in the subsu rface over a wide range of conditions. This also raises the possibility that colloid transport mech anisms might be more relevant than particle transport. Lecoanet et al. (2004, 089258 ) showed that the mobility of aqueous anatase nano-TiO 2 

### particles in a porous medium was comparable to that of other types of nanoparticles when compared on the basis of particle size. Primary particles of 40-nm diameter were found to be aggregated to a diameter of 198 nm. Approximately 55% was reco vered after three pore volumes passed through the column, roughly twice the quantity of ferroxane particles with mean diameter of 303 nm and just more than half the quantity of silica particles w ith a diameter of 57 nm. After three pore volumes, approximately 95% of the 57-nm silica particles were recovered, compared with 60% of the 135-nm silica particles. Although the results were specific to the controlled experimental conditions, they suggest that particle size affects mobility of na noparticles and that anatase might be mobile in ground water (Lecoanet et al., 2004, 089258 ). A recent study using soil samples from 11 different sites found that nano-TiO 2 

### could remain suspended in soil suspensions for 10 days (Fang et al., 2009, 193371 ). Furthermore, the calculated maximum travel distance for some soil sample s was more than 30 cm, which suggested that
nano-TiO 2 

### might be transferred to deeper soil layers or even to ground water. In general, large soil particles and low ionic strength conditions favor nano-TiO 2 

### movement, while high clay content, dissolved organic carbon, and salinity conditions favor soil retention of nano-TiO 2



### . If nano-TiO 2 

### enters municipal sewage systems, liquid waste would be separated from solid
waste and nano-TiO 2 

### would likely be present in both waste streams. The solid waste, or sludge, could present a route by which nano-TiO 2 

### could enter soil media, and could be dealt with in a number of ways. In one scenario, the sludge might be sent for land disposal. The ability of TiO 2 

### to diffuse (and thus be released) from a solid matrix such as sludge is unknown. Nano-TiO 2 

### and other contaminants such as residual arsenic could become suspended in leachate and enter ground water, or they could pass through a solid waste facility liner and reach the subsurface. Under a different scenario, the sludge could be used for land application. In this case, the sludge would undergo some type of treatment, ge nerally to remove pathogenic organisms and regulated contaminants such as lead and arseni c (Ti is not regulated under U.S. EPA’s Biosolids Rule, Part 503) (see U.S. EPA, 1994, 090659 ). The treatment might include high temperature or strong alkaline pH processing, or both (U.S. EPA, 1994, 090659 ). The treated sludge could then be applied to land for agricultural use, reclamation s ites, golf courses, public parks, and other areas where nutrient-rich organic matter is useful, including forests, parks, roadsides, and in some cases, residences (U.S. EPA, 1994, 090659 ). Roughly 50% of treated sewage sludge is applied to land, and treated sewage sludge is applied to less than 1% of all U.S. agricultural land (U.S. EPA, 2006,
090658 ).
Nano-TiO 2 

### in sewage sludge could be broadly dist ributed to land used for crops or grazing, where it could enter the food chain, or to high-use areas such as parks, where people and pets could
contact nano-TiO 2 

### in soil or inhale wind-blown material. The nanomaterial could enter runoff and storm water during wet weather events, eventually returning to the aquatic medium. Ecological receptors also could also be exposed to nano-TiO 2 

### in soil by direct contact with soils or via the food web, including uptake by vegetation. Because it is an inorganic compound, nano-TiO 2 

### in soil could be expected to persist, in the same way that conventional TiO 2 

### is very thermodynamically stable, and is unlikely to undergo significant transformation in the environment. Reactivity of nano-sized TiO 2 

### might differ from conventional TiO 2 

### due to nano-TiO 2 

### particles’ greater surface area-to-volume ratio; the specifics of potential reactivity differ ences are largely unknown at this time.

3-63-7 

# 3.2.1. Drinking Water Treatment

One scenario by which nano-TiO 2 

### could enter soils would be through direct land application of sludge after specifically being used as an agent in drinking water treatment. In addition to the sewage sludge produced in wastewater treatment descri bed above, a sludge material (floc) containing
nano-TiO 2 

### would likely be created in the process of using nano-TiO 2 

### to treat drinking water. If
nano-TiO 2 

### settles with floc in the sedimentation step, it would likely become part of the sludge as well. Similarly, as described above, if nano-TiO 2 

### were present in finished drinking water, it would eventually enter sewage treatment facili ties where any remaining residual nano-TiO 2 

### could also enter the sludge. The discarded sludge would be transported off-site and could be used as daily cover in a municipal solid waste landfill or used for direct land application. Either use would result in direct application of nano-TiO 2 

### -contaminated waste to soils. Alternatively, nano-TiO 2 

### could enter soils if treated water were used to irrigate residential or agricultural vegetation. These scenarios could have implications for soil microbes and could also be noteworthy in relation to nutrient uptake by edible vegetation.



# 3.2.2. Sunscreen

As described above, nano-TiO 2 

### in topical sunscreens could end up in the sludge produced at a wastewater treatment plant. The disposal of this sludge on land seems likely to represent the primary pathway by which nano-TiO 2 

### in sunscreen could enter soil.



# 3.3. Air

Nano-TiO 2 

### manufacturing facilities could emit such particles to the ambient atmosphere. An occupational exposure study by Berges et al. (2007, 157594 ) at a European nano-TiO 2 

### manufacturing facility that supplies the nanomaterials for sunscree ns and cosmetics found that “outside the plant,”
the airborne TiO 2 

### particle concentration was approximately 13,000 particles/cm 3 

### , with nearly 94% of particles 100 nm or less in size, and approximately 52% at 40-60 nm (Berges, 2007, 157594 ; Berges, 2008, 193274 ). The authors did not specify the duration or environmental conditions of the

measurements. Some potential could exist for environmenta l or occupational atmospheric emissions and
releases of nano-TiO 2 

### if the transport or storage containers were to be compromised (e.g., due to a forklift error, train derailment, or truck accident ). Direct land application of sludge, from either drinking water or wastewater treat ment, might contribute nano-TiO 2 

### to the atmosphere if dried
material were to be re-entrained from wind turbulence. Nano-TiO 2 

### is not expected to enter air via sunscreen application or from drinking water treatment processes. The large surface area of nano-TiO 2 

### particles presents an opportunity for other co-occurring contaminants to adsorb onto their surface, potentially changing the physicochemistry of the particle
and the behavior and effects of the other cont aminant(s). Such interactions have been well documented for particulate matter and gases (U.S. EPA, 2004, 056905 ). When nano-TiO 2 

### was dispersed for 0.5 hours in the air immediately next to thermal precipitators 1.5 m above the ground in various outdoor locations in the city of El Paso, Texas, USA, the collected nano-TiO 2 

### particles were not only in agglomerate/aggregate form, but were al so associated with other airborne nanoparticles, in particular, nanosilicate particulates (Murr et al., 2004, 196310 ). Environmental conditions at the study sites were not described, other than the i nvestigators avoided collections in high-humidity
environments.

# Chapter 4. Exposure–Dose Characterization

This chapter examines the potential for biota and humans to be exposed to nano-TiO 2 

### and associated pollutants through various environmental pathways tracing back to the life cycle of two types of applications of nano-TiO 2 

### , water treatment agents and topical sunscreens. Exposure is more
than the occurrence of a substance in the enviro nment; actual contact between the substance and an organism must occur. Exposure characterization entails much more than simply identifying the
concentration of a substance in the environment. It also involves, for example, various temporal and spatial dimensions, including activity patterns and other complex variables. For nano-TiO 2 

### , even characterizing the primary material of interest, as discussed in Chapter 1, is not a simple matter. Further complications arise when considering the potential for aggregate exposure across multiple routes (e.g., inhalation, ingestion, dermal ab sorption) and for cumulative exposure to multiple contaminants that derive, either directly or indirec tly, from the life cycle of the products in question.

2 

### Dose refers to the amount of a substance that enters an organism by crossing a biological barrier such as the skin, the respiratory tract, the gast rointestinal tract, or the eyes. Dose can vary for individuals exposed to the same ambient concentrat ion of a substance. For example, an adult and a child in a room breathing the same air contai ning a contaminant would both inhale the same contaminant concentration, but the inhaled contam inant quantity and absorbed dose would differ due to differences in physiology (e.g., respiration rate s), morphology (e.g., lung volume and surface area), and other variables such as clearance. Dose can also reflect the integration of aggregate exposures across different routes of uptake. Organisms might be exposed to nano-TiO 2 

### in the environment at any stage of the manufactured product’s life cycle. In the fe edstock and manufacturing process, nano-TiO 2 

### could be present in the air exhaust, waste-water effluent, and solid waste, if appropriate control technologies
are not in use. Nano-TiO 2 

### in the air can lead to inhalation exposure to organisms in the area. The material could agglomerate or attach to other pollutants and deposit on soil and water surfaces, as well as on animals, whose grooming habits coul d then result in ingestion of nanomaterials.
Nano-TiO 2 

### in soil could become airborne when the soil is dry and windblown, or leach into bodies of
water when the soil is saturated with water. During the life cycle stages of distribution and storage, nano-TiO 2 

### could be released accidentally into the environment, and cleaning the contaminated site with water could lead to
nano-TiO 2 

### exposure to both aquatic and terrestri al organisms. The use of nano-TiO 2 

### in drinking
water treatment could result in some level of nano-TiO 2 

### in water, as described in Chapter 3, and thus potential exposure to human populations as well as biota. The use of sunscreens containing
nano-TiO 2 

### is expected to lead to nano-TiO 2 

### presence in wastewater after users bathe or shower to remove residual sunscreen on the skin and launder clothes containing traces of sunscreen. Discharges
of nano-TiO 2 

### from wastewater treatment plants are not currently regulated, and are thus not designed or operated to remove nano-TiO 2 

### , although early research suggests that some removal can occur (Kiser et al., 2009, 225305 ). Therefore, nano-TiO 2 

### might be present in the effluent and could lead to

Note: Hyperlinks to the reference citations throughout this doc ument will take you to the NCEA HERO database (Health and Environmental Research Online) at http://epa.gov/hero . HERO is a database of scientific litera ture used by U.S. EPA in the process of developing science assessments.

2 The distinction between exposure and dose in this document is consistent with risk assessment usage (U.S. EPA, 1992, 041875 ). In toxicology, however, the term dose is often used to refer to the amount of a substan ce given to test subjects, as well as the amount that enters the subjects. Applied, external, and potential dose (e.g ., on the skin, in the lung or digestive tract) in toxicology ro ughly equate to exposure in risk assessment; absorbed dose (amount entering the circulation) and target organ dose (amount taken up by a specif ic organ) in toxicology roughly equate to dose in risk assessment.



### 4-1

### aquatic species exposure. In the life cycle disposal st age, wastes from factories and research facilities containing nanomaterials are often incinerated, possibly releasing nano-TiO 2 

### into the air. Household waste containing consumer products made with nano-TiO 2 

### might be incinerated or landfilled; landfilling might lead to nano-TiO 2 

### leaching into ground water. Occupational exposure to nano-TiO 2 

### and associated contaminants (e.g., waste by-products) could occur even with appropriate safety and protective practices (see Appendix C for a more thorough discussion of occupational exposure control measures). For instance, an accident or a mechanical failure might occur in spite of good sa fety practices. Such occupational exposures could differ from exposure to the general public in vari ous ways. For example, workers could be exposed
to free nano-TiO 2 

### , whereas the public might more commonly encounter nano-TiO 2 

### embedded in a product. Exposure durations and concentration levels are likely to be higher in occupational settings. Likewise, target tissue dose levels could differ between workers and the general population or even between workers in different occupations at the same facility, depending on factors such as respiration rates in relation to sedentary or strenu ous activity in the presence of airborne nano-TiO 2



### .



# 4.1. Biota

Various scenarios and ways in which nano-TiO 2 

### from water treatment agents and topical
sunscreens could enter different environmental media were described in Chapters 2 and 3. Some of these scenarios will be further explored in th is section, specifically examining various TiO 2 

### exposure conditions and how they could affect aquatic an d terrestrial organisms. The potential for bioaccumulation and entry of nano-TiO 2 

### into the food web is discussed in Section 4.6.



# 4.1.1. Aquatic Species

Data on concentrations of nano-TiO 2 

### in sediment, whether in a laboratory or a natural environment, are limited. Nano-TiO 2 

### concentrations could be higher at the sediment surface than in the water (Handy et al., 2008, 157562 ). Settling of nano-TiO 2 

### aggregates (with nano-TiO 2 

### or with organic matter) would increase nano-TiO 2 

### exposure to benthic and benthopelagic species, such as mussels, sea cucumbers, marine worms, flatfish, a nd other species that sometimes feed at the bottom of natural bodies of water. At the same time, settling decreases nano-TiO 2 

### concentrations in the water column and would be expected to decrease exposure to suspension feeders (such as Daphnia ) and
animals that live in or drink the water. Nanoparticles can also deposit or aggregate on the surfaces of aquatic organisms. Surface aggregation can be caused by the slower flow near the interface between liquids and solids or by the viscous properties of the surface of an organism (Handy et al., 2008, 157562 ). Surface deposition or aggregation can result in a higher concentration of nano-TiO 2 

### on the organism’s surface than in the water, and might cause toxicity even if the nano-TiO 2 

### does not enter the cells (Handy et al., 2008,
157562 ). Surface-acting metal toxicity of nano-TiO 2 

### has been suggested as a cause of gill damage in rainbow trout where the Ti concentration in gill tissue was not increased (Federici et al., 2007,
091222 ).
Because water flow is also slower near the interface with air, higher concentrations of nanoparticles are also expected at the air-water interface (Handy et al., 2008, 157562 ). Consequently, organisms living at the water surface, such as zooplankton (microscopic invertebrates that float or swim in water), phytoplankton (primarily single-celled algae), and eggs of aquatic and amphibian species at the water surface, could be exposed to higher nanoparticle concentrations than organisms living throughout the water column.

4-2

# 4.1.2. Terrestrial Species

Terrestrial organisms could be exposed to nano-TiO 2 

### under various scenarios. For example, spillage during the life-cycle stages of shipping or storage, including breaching of containers by vermin, could result in contact by microbial, invert ebrate, and vertebrate species. Plants could be exposed by taking up treated or wastewater containing nano-TiO 2 

### or by growing in soil that contains
nano-TiO 2 

### , for example, as a result of application of sludge from water treatment facilities. No empirical data on the potential for such exposures to terrestrial organisms have been located.



# 4.2. Humans

As noted at the beginning of this chapter, exposure is a complex function of not only the amount of a substance in the environment but also a function of various temporal and spatial
dimensions of contact with the substance. At this ea rly stage of investigation and understanding of human exposure to nano-TiO 2 

### , however, even basic information on the potential for and amount of
human contact with this material is limited. Mo reover, exposure characterization encompasses not just the primary material but the secondary waste and transformation products related to the entire life cycle of nano-TiO 2 

### in various applications. These indirect and secondary aspects of exposure are even less well understood and therefore not discussed here. Their potential significance, however,
should not be discounted. The potential for human exposure to nano-TiO 2 

### depends first on the production and use of this material in the applications under consideration here . Generally, exposure related to life-cycle stages leading up to actual use appears more likely to occur in occupational situations, whereas exposure related to the use and disposal stages of the life cycle could occur in either occupational or nonoccupational settings. Although not absolute, this distinction provides a basis for discussing exposure with reference to either the general population or the occupational population, both of which are essential in examining the broad implications of nano-TiO 2 

### use in drinking water treatment and in topical sunscreens.



# 4.2.1. General Population

4.2.1.1. Drinking Water Treatment

Although the actual use of nano-TiO 2 

### in water treatment facilities appears to be limited at present to pilot testing (Section 2.4), the potential for general population exposure to nano-TiO 2 

### if it were to be used widely could involve sizeable numbers of people, given the number of U.S. community water suppliers that currently treat dri nking water to reduce arsenic levels. As discussed in Section 2.4.1, such water suppliers serve roughly 13 million people in the U.S. alone.
If nano-TiO 2 

### were present in potable water, exposure could involve more than just ingesting
the water. Such water could be used for bathi ng, including showering, which could imply exposure not only by dermal contact but by inhalation of water droplets and even contact through the eyes. Also, the general population includes infants and other individuals who could have relatively greater exposure to water and thus possible vulnerability if th e water were contaminated. For example, on a body weight basis, 1- to 3-month-old infants consume far more water directly and indirectly than 18- to 21-year olds. The 90th percentile consumption rate is 151 mL/kg-day for these infants versus 17 mL/kg-day for the older age group (see Table 3-9 in U.S. EPA, 2008, 196062 ). Children also have a greater water intake while swimming, so they may be more vulnerable to contaminated water in that respect as well (U.S. EPA, 2008, 196062 ).

4-3

# 4.2.1.2. Sunscreen

As discussed in Section 2.4.2, iVillage survey data from 2007 suggest that sunscreen might be used on a daily basis by 33 million people in the U.S. and on an occasional basis by another 177 million. Moreover, sunscreen use appears to be increasing. According to the Skin Cancer Foundation (2008, 594955 ), the percentage of people who use sunscreen at least occasionally rose from 39% to 59% between 2003 and 2007. Sunscreen use is presumably greatest during the warmer months of the year, in warmer climates, or dur ing outdoor recreational activities at various times during the year. No information was found regardi ng the proportion of use associated with water recreation and other specific venues or activities. Topical sunscreens are available as traditional lotions, in spray-on form, and as wipes (Jeffries, 2007, 157682 ). Nano-TiO 2 

### sunscreen powders are also available, according to the Project on Emerging Nanotechnologies at the Woodrow Wilson Center’s nanotechnology-based consumer product inventory (Woodrow Wilson International Center for Scholars, 2006, 196083 ). Another sun protection option available to consumers is “cosm eceuticals,” cosmetics that incorporate active sunscreen ingredients (Davis, 1994, 157946 ). In the mid-1990s, up to 30% of lipsticks and 20% of makeup were estimated to have SPF ratings, sunscreen claims, or both (Davis, 1994, 157946 ). Other products with active sunscreen ingredients include hair care products (e.g., hair spray, gel, mousse, and conditioner), alpha-hydroxy skin treatments, na il polish, and bath products. Sun-protective clothing is also available (Davis, 1994, 157946 ). For the general population, the principal exposure route to nano-TiO 2 

### in sunscreen is through the skin. When sunscreen is applied by spray, inhala tion presents another route, although it is not clear that the primary nanoparticles as such would be inhaled. Ingestion is also conceivable through
hand-to-mouth contact and mucocilia ry clearance of inhaled nano-TiO 2



### . 

## Dermal Exposure

Potential nano-TiO 2 

### dermal exposure from sunscreen use can be estimated by the amount of applied sunscreen. Although the recommended sunscreen application rate is 2 mg/cm 2 

### of skin (roughly 1.5 ounces or 3 tablespoons for the entire body of an average adult), most consumers use 0.5-1.5 mg/cm 2 

### skin (Srinivas et al., 2006, 157734 ). Assuming sunscreen is applied to all areas of skin exposed to sun on a day at the beach or exposed to water while swimming, an adult would use an estimated 10-46 g sunscreen/application, and a 3-year old would use an estimated 3-15 g/application (Table 4-1). Assuming that a sunscreen contains 5% nano-TiO 2 

### (the mass percent
concentrations of nano-TiO 2 

### in sunscreens range from 2% to 15%; see Table A-1 in Appendix A), the
amounts of nano-TiO 2 

### applied on the skin could range from 0.5 to 2.3 g/person/application for an adult, and 0.17 to 0.76 g/person/application for a 3-year old (Table 4-1). These exposure estimates are in line with estimates made by Hansen et al. (2008, 157560 ). Sunscreens, including the waterresistant or water-proof types, should be reapp lied every 2 hours, regardless of the SPF values. Exposure to nano-TiO 2 

### from sunscreen could range from 1.0 to 4.6 g for an adult and 0.33 to 1.5 g for a 3-year old for a half day at the beach (2 a pplications in 4 hours). As shown in Table 4-1, the ranges of applied nano-TiO 2 

### would be 12 to 55 mg/kg of body weight/application for a 3-year old and 8.0-37 mg/kg of body weight/application for an adult. This relatively higher exposure in young children could be noteworthy in relation to indications that the skin of infants and young children might have less barrier function than matured skin (Hostynek, 2003, 193435 ), although this contrasts with another report indicating that human skin is mature both structurally and functionally at 2-3 weeks of age (Makri et al., 2004, 193537 ). Although not everyone applies sunscreen at the recommended dose and frequency in real life, pa rents reported greater use of sunscreen on their children than on themselves (Weinstein et al., 2001, 191128 ).

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## Table 4-1. Estimated dermal exposure to nano-TiO 2 

## from sunscreen containing 5% nano-TiO 2 

## for adults and 3-year-old children

Applied b
nano-TiO 2 (mg/person/ application) Applied sunscreen amount (mg/person/ application) Applied nano-TiO 2 (mg/kg BW/ application) Applied sunscreen

surface density (mg/cm 2 ) Surface area a of skin (cm 2 ) Subject

3-yr-old child, total body surface (50th percentile) 6,640 0.5 3,320 166 12.0

1.5 9,960 498 35.9

2 13,280 664 47.9

3-yr-old child, total body surface (95th percentile) 7,640 0.5 3,820 191 13.8

1.5 11,460 573 41.3

2 15,280 764 55.1

Adult, body surface area subjected to water contact in swimming (50th percentile) 20,000 0.5 10,000 500 8.0

1.5 30,000 1,500 24.0

2 40,000 2,000 32.1

Adult, body surface area subjected to water contact in swimming (95th percentile) 23,000 0.5 11,500 575 9.2

1.5 34,500 1,725 27.6

2 46,000 2,300 36.9

a Body surface area values are based on Tables 6-6 and 6-16 of U.S. EPA (1997, 594981 ).

b Actual concentrations of nano-TiO 2 in commercial sunscreen on the market vary, with the high at nearly 15%. (Table A-1 in Appendix A.)
BW – Body weight. The body weights used in the calculat ion were 14 kg, the median for 36-month old females (2000, 157982 ), and 62 kg, the median for adults 18-74 years old; Table 7.5 of U.S. EPA (1997, 594981 ).



## Inhalation Exposure

Consumers could inhale water aerosol while showering or from nebulizing room humidifiers. Spray sunscreen products also present an inhala tion exposure scenario. For such products and for treated water containing nano-TiO 2 

### , the characteristics of the resulting aerosol have not been documented in the published literature. Section 4.2.2 discusses inhalation exposure from nano-TiO 2 

### for several occupational scenarios.



## Oral Exposure

Nano-TiO 2 

### from sunscreen could be ingested by accident or as a result of routine hand-tomouth contact (from residual sunscreen on hands), pa rticularly for young children. If nano-TiO 2 

### were inhaled, mucociliary clearance could lead to uptake through the gastrointestinal tract. Although no estimates of this type of nano-TiO 2 

### exposure are available, dietary intake of all sizes of TiO 2 

### from all sources (food, pharmaceuticals, etc.) has been estimated. The estimation was based on 7-day food diaries and records of pharmaceu tical, dietary supplement, and toothpaste use of 182 people in the United Kingdom. The amounts of TiO 2 

### were calculated or estimated from product labels (the listing
of food-additive TiO 2 

### is required by British law in most foods), manufacturer reports, and laboratory testing. The total median dietary intake of nano-TiO 2 

### and micro-TiO 2 

### (0.1-3 μm) has been estimated between 2.5 and 5.4 mg/individual/day (Lomer, 2000, 635672 ; Lomer et al., 2004, 157382 ). Food was the main source of dietary TiO 2 

### , followed by pharmaceuti cals, dietary supplements, and toothpaste. Individual TiO 2 

### intake varied widely (0-112 mg/individual/day), and no particle size information was provided.

4-5

# 4.2.2. Occupational

Nearly every stage of the life cycle for th e applications considered here presents some potential for occupational exposure to nano-TiO 2 

### . Moreover, no exposure route can be ruled irrelevant to these workers. Thus, assessing occupational exposure is essential to completing a CEA
of nano-TiO 2 

### in either drinking water treatment agents or topical sunscreens. As a frame of reference, NIOSH (2005, 196072 ) proposed a draft occupational exposure limit of 1.5 mg/m 3 

### for fine TiO 2 

### (primary particle <10 μ m, see below for details) and 0.1 mg/m 3 

### for
ultrafine TiO 2 

### (primary particle <0.1 μ m, see below for details), as time-weighted average concentrations for up to 10 hours/day during a 40-hour work week. The “fine” particles in this NIOSH draft were defined as all particle sizes that are collected by respirable particle sampling (i.e., 50% collection efficiency for particles of 4 μ m, with some collection of particles up to 10 μ m). “Ultrafine” particles were defined as the fraction of respirable particles with primary particle
diameter <0.1 μ m(2005, 196072 ). The NIOSH draft exposure limit was based on primary particle size, not the measured aggregate or agglomerate sizes. Agglomerates of ultrafine TiO 2 

### , which may by larger than 0.1 um, often exhibit biologi cally similar behavior to ultrafine TiO 2 

### due to the surface area of the constituent particles, and therefore the recommended exposure limits for ultrafine TiO 2 

### should apply (NIOSH, 2005, 196072 ). The draft recommended exposure limits were extrapolated from rat-based critical dose estimated to humans, using specific surface area measured by the BET method (6.68 m 2 

### /g for fine TiO 2 

### and 48 m 2 

### /g for ultrafine TiO 2 

### ) to convert particle mass to surface area dose (NIOSH, 2005, 196072 ). Because the sizes and surface areas of fine and particularly ultrafine (nano) TiO 2 

### vary, the risk estimates will vary for other particle sizes and surface areas (NIOSH, 2005, 196072 ), as well as other crystal forms of TiO 2 

### and other conditions (Section 5.1). Most information on workplace TiO 2 

### exposure relates to the production of conventional TiO 2 

### , not nano-TiO 2 

### specifically. Additionally, given that nano-TiO 2 

### tends to agglomerate or aggregate, occupational exposure conditions for nano-TiO 2 

### could involve both nanoscale and larger than
nanoscale TiO 2 

### particles. The manufacturing stage of the lif e cycle comprises multiple processes that might vary in exposure characteristics. An epidemiologic study conducted in four TiO 2 

### manufacturing facilities located in the U.S. indicated that occupational exposure to TiO 2 

### is greatest during bagging, milling, micronizing, and intern al recycling (shoveling spilled material from the floor into the processing bins) (Fryzek et al., 2003, 157864 ). However, it is possible that the levels of exposure can vary depending on the facility. The manufacturer of P25 has stated on its website that workplace inhalation exposures to TiO 2 

### are typically less than 0.5 mg/m 3 

### (Degussa, 2007, 090576 ). If such exposures are sustained for less than 2 hours/day, they would not exceed the NIOSH proposed occupational exposure limit of 0.1 mg/m 3 

### , which is expressed as a 10-hour time-weighted average. The Web site also indicated that photocatalytic P25 production occurs in a closed reactor, which presumably limits exposure. The highest exposures the manufacturer reported were less than 0.5 mg/m 3 

### and occurred during the packaging step, which is also an enclosed process. This manufacturer is said to require the use of personal protective equipment during any repair work that could lead to dust exposure (Maier, personal communication, 2007, 091185 ). Such information suggests only limited potential for inhalation exposure during P25 manufacturing, but it does not address other routes such as dermal exposure or incidental ingestion from hand-to-mouth contact.
Another manufacturer of nano-TiO 2 

### products reported that air concentrations in particle manufacturing, packaging, and distribution areas for DuPont TM 

### Light Stabilizer 210 and 220 (which protects plastic from UV damage) were less than 2 mg/m 3 

### , and in most cases were lower than the detection limit of 0.3 mg/m 3 

### (size not specified) (DuPont, 2007, 157699 ). No worker exposure data were available for materials incorporation, packing, or product fabrication for nano-TiO 2 

### -containing polymer products. Although the potential for worker exposure was stated to be low (DuPont, 2007,
157699 ), the detection limit (0.3 mg/m 3 

### ) is above the draft NIOSH recommended limit for ultrafine
or nano-TiO 2 

### of 0.1 mg/m 3 

### (NIOSH, 2005, 196072 ).

4-6

### Preliminary estimates of wo rkplace exposure in a factory that produces rutile nano-TiO 2 

### for sunscreen and cosmetics were reported by Berges (2007, 157594 ; 2008, 193274 ). Measurements were made in 2006, and then in 2007, when improvements to local exhaust systems were in operation (Berges, 2008, 193274 ). In 2007, the TiO 2 

### in the “inhalable” dust mass concentration at the bin filling station was 0.014 mg/m 3 

### , and the TiO 2 

### in the “respirable” dust mass concentration was 0.004 mg/m 3 

### . ( Inhalable refers to all particles that can enter the respiratory tract through the nose or mouth [e.g., up to approximately 100 μm]; respirable refers to particles that penetrate to the alveolar [pulmonary] region with a mass median aerosol diameter [MMAD] of approximately 4 μm 1 

### (CEN, 1993, 078032 )) In the bag filling area in 2007, the TiO 2 

### inhalable fraction was 0.028 mg/m 3 

### , and the respirable fraction was 0.022-0.042 mg/m 3 

### . Personal sampling in 2007 over a 4.87-hour period measured 0.010 mg/m 3 

### TiO 2 

### in the respirable fraction. It is not clear how applicable the results from this manufacturing facility are to occupational exp osure in facilities where, for example, sunscreen products are formulated from the raw materials. Liao et al. (2009, 157456 ) further reported and analyzed the Berges (2007, 157594 ; 2008,
193274 ) data, as well as data from several other sources to model the occupational exposure and characterize risk. In the bin filling area of the facility studied by Berges (2007, 157594 ; 2008,
193274 ), the total airborne TiO 2 

### particle number concentrations ranged from 15,000 to 156,000 particles/cm 3 

### , with a measured size range of 14 to 673 nm. More than 97% of the particles were 100 nm or less in size, and 60% were 20 -30 nm. After a leak was sealed, the high-end concentration decreased to less than 29,000 particles/cm 3 

### . Near the leak, the particle surface area concentrations reached 200 μm 2 

### /cm 3 

### for “alveolar deposited” particles and 50 μm 2 

### /cm 3 

### for “tracheobronchial deposited” particles. Under nor mal operating conditions, the particle surface area concentrations were 50 μm 2 

### /cm 3 

### for the alveolar deposited particles and 13 μm 2 

### /cm 3 

### for the tracheobronchial deposited particles. Outside the facility, the airborne TiO 2 

### particle concentration was approximately 13,000 particles/cm 3 

### . Their model indicated that the highest TiO 2 

### burdens (in terms of lung surface area) of packers were 0.174 m 2 

### (anatase) and 0.122 m 2 

### (rutile) for particles sized 10-20 nm. For particle sizes 80-300 nm, the burdens were 0.002 m 2 

### (anatase) and 0.0017 m 2 

### (rutile). Employees classified as surface treatme nt workers (involved in drying, packing, and blending operations) had a higher TiO 2 

### burden in their lung surface area. For particles 10-20 nm, the
burdens were 0.40 m 2 

### (anatase) and 0.28 m 2 

### (rutile). Using exposure data specific to particle si ze in the workplace from the Berges (2007, 157594 ; 2008, 193274 ) reports as well as conventional TiO 2 

### studies (Boffetta et al., 2004, 157849 ; Fryzek et al., 2003, 157864 ), Liao et al. (2009, 157456 ) used computer modeling to calculate that exposures to
nano-TiO 2 

### (expressed as particle surface area concentrations) were 0.1685 m 2 

### TiO 2 

### per 300 m 3 

### air (working space volume) for packers and 0.387 m 2 

### TiO 2 

### per 300 m 3 

### air for surface treatment workers.
For nano-TiO 2 

### in the 10- to 50-nm size range, the airborne concentrations (expressed as particle surface area concentrations) were higher in anatase nano-TiO 2 

### than in rutile nano-TiO 2 

### for both packers and surface treatment workers. The highest airborne concentration was anatase for surface treatment workers, followed in order by rutile for surface treatment workers, anatase for packers, and rutile for packers. Liao et al. (2009, 157456 ) also modeled the dose-response relationships from in vitro cytotoxicity studies of human dermal fibrobl asts and inflammatory responses of human lung epithelial cells. They then compared exposure le vels to the dose-response functions and concluded that packers and surface treatment workers at the studied location were “unlikely to [be at] substantial risk [of] lung inflammatory response, [but they] have significant risk [of] cytotoxicity response at relatively high airborne TiO 2 

### anatase NP [nanoparticle] concentrations at size 10-30 nm”

1 The size for respirable particles cited here is the standard used by Berges (2007, 157594 ; 2008, 193274 ), and the size used in other studies may vary by standards set by different agencies or even laboratories. Some argue that approximate 50% of 5 μm particles are deposited in the alveolar region of humans who are engaged in activities requiring moderate ventilation levels, particularly as sociated with oronasal breathing. Thus, the respirable particulate size for the alveolar region in humans is closer to 7-8 μm (F.J. Miller, p ersonal communication (2009, 625211 )).



### 4-7

### (Liao et al., 2009, 157456 ). Though these conclusions were not based on actual worker exposure, the
combination of field data on relevant TiO 2 

### size, laboratory lung cell studies and computer modeling generates data on the potential nano-TiO 2 

### burden that could be faced by people handling these
materials. In a presentation at a professiona l conference, Li et al. (2008, 196055 ) displayed photographs of a factory in Shanghai, China that mi xed, but did not manufacture, nano-TiO 2 

### . The photographs appeared to show that nano-TiO 2 

### was stored in shipping bags piled on pallets. White powder was visible on the facility floor, but its compositi on was uncertain as the factory also handled conventional “pigmentary grade” and “food grade” TiO 2 

### (Ichihara, personal communication, 2009,
196034 ). Li et al. (2008, 196055 ) reported that workers had been given masks and shirt-like protective clothing but that the masks were not always worn. The authors also noted that shirt-like protective clothing provided no protection for the forearms and legs of the workers, many of whom wore short-sleeved tops and shorts. The authors noted that this factory exhibited particularly poor conditions compared to others with similar processe s, but even so their investigation can be used to illustrate how inhalation and dermal exposure mi ght occur during the manufacturing or mixing

process. As noted in Section 2.3, nano-TiO 2 

### is routinely shipped in paper bags, which could be a source of exposure if they were to be ruptured, punctured, or otherwise compromised during distribution or storage. Nano-TiO 2 

### in dispersion form shipped in pails, drums or totes (Klaessig, personal communication, 2008, 196042 ) could be subject to accidents resulting from forklift errors, train derailments, and truck accidents, but no empirical data on such incidents specifically related to
nano-TiO 2 

### were available. The above information suggests that inhalation and dermal exposure could occur during manufacturing, packaging, shipping, and storage of nano-TiO 2 

### . To fully characterize potential risk, toxicity data at conditions comparable to be the reported exposure conditions would be useful, although extrapolation from higher concentrations can be used as well.



# 4.3. Aggregate Exposure to Nano-TiO 2 

# from Multiple Sources and Pathways

Nano-TiO 2 

### is used in various manufactured products , raising the possibility that biota and humans could be exposed to nano-TiO 2 

### from more than one source. Such sources might include drinking water treatment agents, topical sunscreens, cosmeceuticals (traditional cosmetics such as moisturizers and color cosmetics that incorporate active sunscreen ingredients containing
nano-TiO 2 

### ), sun-protective clothing, cleaning agents, air purifiers, coatings, and food packaging, among many others (The Project on Emerging Nanotechnologies, 2009, 196052 ). It should, however, be recognized that nano-TiO 2 

### particles from these different sources may have different properties such as size distribution, crystalline phase, and surface treatment. Kaegi et al. (2008, 193457 ), for example, reported nano-TiO 2 

### in water runoff from both new and naturally aged building façades painted with paint containing nano-TiO 2 

### . Hsu and Chein (2007, 193437 ) found that nano-TiO 2 

### powder-coated materials (wood, polymer, and tiles) under various conditions emitted nanoparticles to the air. Of course, merely the presence of nano-TiO 2 

### in a product does not mean that exposure will occur. For example, if nano-TiO 2 

### is firmly embedded in a product and the product remains intact, little or no exposure to nano-TiO 2 

### might actually occur. A hypothetical scenario for aggregate exposure to nano-TiO 2 

### in both treated water and sunscreen could involve a person’s ingesting the wa ter (oral route), bathing (dermal) or showering (dermal and inhalation), applying sunscreen lotion to the skin (dermal), ingestion of sunscreen through hand-to-mouth contact (oral), or uptake fro m hand-to-eye (ocular) contact. The latter two exposures pathways are particularly relevant for young children. Biota also could be subject to

4-8

### aggregate exposures. A fish, for example, could take up nano-TiO 2 

### that originated from a wastewater treatment facility and could also ingest prey whose contamination originated from ambient water, sediment, or other prey or plants that alrea dy contained sunscreen constituents. The seemingly widespread occurrence of nanoparticles of various types in aquatic media reported by Wigginton et al. (2007, 157415 ) lends plausibility to these scenarios.



# 4.4. Cumulative Exposu re to Nano-TiO 2 

# and Other Contaminants

Nano-TiO 2 

### is not the only substance relevant to the life cycle of products containing
nano-TiO 2 

### to which biota and humans could be exposed. As noted in Chapter 2, releases of other contaminants might also occur during various stages of the product life cycle, particularly waste materials during feedstock processing and during manu facturing of the primary product. Such waste materials are not necessarily nanoscale in size. As de scribed in Chapter 3, if wastes were released into the environment, they could undergo transforma tion, potentially resulting in even more types of contaminants; they might also be transported to other locations, e.g., downstream or downwind. The creation of secondary contaminants through transformation processes in various environmental media also raises the possibility of exposure to substances indirectly related to
nano-TiO 2 

### . Many nanoparticles, including nano-TiO 2 

### , tend to bind transitional metals and organic chemical pollutants (Nagaveni et al., 2004, 090578 ; Pena et al., 2006, 090573 ). With a tendency to adsorb other pollutants and an ability to absorb in to the body and cells (Sections 4.6.1, 4.6.3, and 4.6.4), the possibility cannot be ruled out that nano-TiO 2 

### could carry toxic pollutants to target sites where the pollutants would not normally go (Moore, 2006, 089839 ). Such activity could result in increased uptake of other pollutants or interactive effects that would otherwise not occur if these substances were only present individually.



# 4.5. Models to Estimate Exposure

The EPA uses various models to estimate exposures for chemical assessments, some of which
are described on the websites for the Council for Regulatory Environmental Modeling (U.S. EPA, 2009, 196065 ) and the Center for Exposure Assessment Modeling (U.S. EPA, 2009, 196064 ). For example, the Exposure and Fate Assessment Screeni ng Tool Version 2.0 (E-FAST V2.0) is a publicly available program EPA uses for screening-level as sessments of conventional industrial chemicals. The tool provides estimates of aquatic e xposure, general population exposure, and consumer exposure based on release data (U.S. EPA, 2007, 196060 ). Other fate and transport models also might be relevant, for example, the Particle Tracking Model (PTM) that the Army Corps of Engineers developed (Demirbilek et al., 2005, 193887 ); the Eulerian model that treats particles as a liquid (Kollias, 2009, 624994 ); Publicly Owned Treatment Works (POTW) models for evaluating fate in wastewater treatment plants (to sludge , effluent, and air); receiving water models for evaluating environmental transport and fate, a nd bioaccumulation models (Minerva, 2009, 625210 ); and over 20 models of aquatic fate and transport (for comparison, see a review by Paquin et al., 2003, 196867 ). However, these models were not devel oped for nanomaterials and have not been tested for their ability to estimate nanomaterial exposures, although they perhaps could be used or adapted for qualitative exposure estimation in lieu of quantitative release data. Although empirical data on nano-TiO 2 

### concentrations in the environment are currently lacking, a recent study used computer modeling to predict nano-TiO 2 

### concentrations in different environmental media. Using limited data from published literature and various assumptions,

4-9

### researchers in Switzerland developed models to estimate predicted environmental concentrations (PEC) and predicted no-effect concentr ations (PNEC) (Mueller and Nowack, 2008, 157519 ). PEC values were calculated for “realistic exposure scenarios” (based on nano-TiO 2 

### use, estimated as 25 MT per year in Switzerland) and for “high exposure scenarios” (based on 500 MT per year). The
authors estimated that more than 60% of nano-TiO 2 

### is used in cosmetics, including sunscreen, and that most of it is discharged into wastewater. To estimate PNEC, the lowest no-observed-effect concentration (based on a published study on acute toxicity to Daphnia, Hund-Rinke and Simon, 2006, 090607 ), was divided by an assessment factor of 1,000, in accordance with the Technical Guidance Document on Risk Assessment published by the European Chemicals Bureau, because, as the authors noted, the “accuracy of the data was low” (European Chemicals Bureau, 2003, 196375 ; Mueller and Nowack, 2008, 157519 ). The PEC of nano-TiO 2 

### in water was 0.7 μ g/L (“realistic scenario”) or 16 μg/L (“high scenario”), compared to a PNEC of <1 μg/L (for Daphnia ). The authors (Mueller and Nowack, 2008, 157519 ) stated that, given that the PEC was close to or greater than the PNEC, European Union authorities would consider the substance “of concern” and call for more data to validate the result (Umweltbundesamt, 2009, 196071 ). Gottschalk et al. (2009, 633897 ) followed up on this work using similar modeling procedures to predict environmental concentrations of several nanomaterials across a variety of compartmen ts and regions. The team also investigated the overall applicability of probabilistic modeling to predict environmental exposure to nanomaterials (Gottschalk et al., 2010, 635674 ). Based on available information about the applied concentration of nanoparticles in cosmetics, personal care products and paints, Boxall et al. (2007, 196111 ) used a series of algorithms to estimate the PECs of nanoparticles in soil and water. Although anticipating that 10% market penetration probably provides a conservative es timate (with the exception of sunscreens), the
researchers calculated the PEC for three scen arios assuming that 10%, 50% and 100% of the products on the market contained nanoparticles. Th e total predicted concentrations in water were found to be 24.5-245 μg/L.



# 4.6. Dose

Dose is defined as the amount of a substance th at actually enters an organism by crossing a biological barrier. Uptake of nano-TiO 2 

### by different routes has been investigated in various species. It is important to note that upon entering an or ganism, a substance may still be transported and undergo changes as it moves throughout the organism. For this reason, understanding dose includes understanding additional fate and transport conside rations specific to the media encountered by a substance once it is taken up; several investigations ha ve been identified in this area. Sager et al. (2007, 090633 ) attempted to disperse nano-TiO 2 

### , and other types of nano-sized particles in several suspension media, including phosphate-buffered saline (PBS), rat and mouse bronchoalveolar lavage fluid (BALF), and dipalmitoyl phosphatidylcholine (DPPC). Although PBS was not a satisfactory medium, BALF was an excellent medium for di spersing the particles. The dispersion was also unsatisfactory in saline containing albumin alone or DPPC alone at concentrations found in BALF. Combinations of protein and DPPC were satisfact ory, but slightly less effective, substitutes for BALF. These findings demonstrate the importance of the suspension media in determining the
behavior of nano-TiO 2 

### within a given system. Exposure to nano-TiO 2 

### in aquatic organisms has been studied mostly by measuring tissue concentrations in fish exposed to it in water. Howe ver, information related to exposure of substances
other than nano-TiO 2 

### is often also mentioned, appropriately reflecting the multiple substances to which aquatic organisms may be exposed in the na tural environment. For terrestrial organisms, including laboratory animals used for toxicological studies and used as models for human health effects, the route of exposure is important in de termining the dose that actually enters the body,

4-10

### hence information on uptake of nano-TiO 2 

### is presented here according to the route of uptake, i.e., inhalation, dermal, or ingestion. While differences in animal models, such as the differences in human and rodent nasal pathways leading to th e olfactory bulb, are known to underlie some differences in toxicological results between speci es, studies across a biological continuum are drawn upon herein to collect a spectrum of potentially informative data. Additionally, this section discusses special biological barriers (blood brain barrier [BBB] and placenta), and issues related to dose-metrics for nano-TiO 2 

### . Again, because internal transport of the
materials will influence the ultimate dose to the or ganism, it should be noted that multiple routes of exposure will be considered, even though all routes may not be equally significant. Some routes (e.g., i.v., i.p., and i.m. injections) could have relevance when internal transport is considered.



# 4.6.1. Uptake in Aquatic Species

4.6.1.1. Bioaccumulation

Zhang et al. (2006, 157722 ) found that nano-TiO 2 

### can accumulate internally in carp (Table 4-2). The authors exposed carp to photocatalytic nano-TiO 2 

### (P25) for up to 25 days. Before
dissection and TiO 2 

### analysis, carp were rinsed and wiped. The nominal concentrations of nano-TiO 2 

### in the water were 3 and 10 mg/L (based on the amount of stock nano-TiO 2 

### suspension added to the fish tank), and the authors reported that nano-TiO 2 

### concentrations were 2 and 7 mg/L after 24 hours, with most of the decreases occurring within 4 hours after the addition of stock solution. The TiO 2 

### concentration in carp tissue increased rapidly over the first 10 days and then more gradually between day 10 and day 25. TiO 2 

### concentrations were highest in viscer al organs, distantly followed by gills, and then closely followed by skin and scales (one sample), and muscle. The bioconcentration factors in the visceral organs were approximately 2,100 at 3 mg/L, and approximately 1,400 at 10 mg/L. In contrast to the finding of bioaccumulation of nano-TiO 2 

### in carp that Zhang et al. (2006,
157722 ) reported, Federici et al. (2007, 091222 ) detected no accumulation in rainbow trout exposed to up to 1 mg/L nano-TiO 2 

### for 14 days. Although the findings a ppear contradictory, each study might simply reflect the results of the specific test c onditions. For instance, the rainbow trout were exposed
to lower concentrations of nano-TiO 2 

### than were the carp. The Federici et al. (2007, 091222 ) study used photocatalytic nano-TiO 2 

### (also P25), but 80% of the water in the fish tank was changed every 12 hours. Similar to Zhang et al. (2006, 157722 ), Federici et al. (2007, 091222 ) reported that more
than 85% of the initial nano-TiO 2 

### concentrations in the tank water remained after 12 hours. Other environmental factors, such as water temperature at 14oC for rainbow trout and at 23oC for carp,
could influence the behavior or effects of nano-TiO 2 

### and contribute to the difference between these two studies. Furthermore, carp feeding behavior mainly consists of grubbing in sediments, and therefore carp could have a higher exposure to settled nano-TiO 2 

### aggregates than rainbow trout. Studies on kinetics of uptake and clearance, whic h could be valuable in understanding nano-TiO 2 

### bioaccumulation and relevant factors, were not available. Although nano-TiO 2 

### may bioaccumulate in fish, the uptake mechanism is not clear. Substances in water can enter fish through waterborne exposure (through gills and then into blood through absorption), dietary uptake, or cutane ous absorption. Handy et al. (2008, 157563 ) suggested that the absorption of nano-TiO 2 

### on the gill surface into the blood might be slow or uncertain, but that
nano-TiO 2 

### on the gut surface might be taken into ce lls by endocytosis. Although intact fish skin is unlikely to be permeable to nano-TiO 2 

### , these authors proposed that cutaneous uptake of nano-TiO 2 

### might be possible if the skin is infect ed or inflamed (Handy et al., 2008, 157563 ). Handy et al. (2008, 157563 ) did not provide experimental data to support nano-TiO 2 

### uptake through endocytosis, but a recent in vitro study indicated that an endo cytosis inhibitor, Nystatin, decreased the mutation frequencies induced by exposures to 5-nm and 40-nm nano-TiO 2 

### , but not 325-nm TiO 2 

### , in mouse embryo fibroblasts, implying that endocytosis is involved in modulating cellular response to

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### nano-TiO 2 

### exposure (Xu et al., 2009, 157452 ). The concentration of nano-TiO 2 

### or Ti in cells was not measured (Xu et al., 2009, 157452 ).



# 4.6.1.2. Food Web

Nano-TiO 2 

### could enter the food web at various levels, depending on the point and extent of its
release to the environment. If nano-TiO 2 

### were dispersed in water, for example, it could be taken up by algae, which are primary producers of chemi cal energy needed to fuel ecosystems. Many invertebrates, which are primary consumers of chemical energy in aquatic ecosystems, eat algae and in turn are consumed by larger animals such as fish. A common aquatic invertebrate is the water flea (genus Daphnia ), which is a small crustacean filter feeder (also known as suspension feeder). Daphnids use their legs to generate water flow and use the comb-like setae on their thoracic limbs to strain or catch smaller organisms (such as al gae) for consumption. Because daphnids have been reported to filter up to 120-160 mL each per day (Vanoverbeke, 2008, 157477 ), they could be exposed to quite high numbers of nanoparticles in water (Griffitt et al., 2008, 157565 ). Even if
nano-TiO 2 

### were not absorbed into tissues, nano-TiO 2 

### in the digestive tract of daphnids could still contribute to bioaccumulation in the food web. Although nano-TiO 2 

### has not been tested for trophic transfer in the food web, one study found evidence of transfer of carboxylated and biotinylated CdSe-based quantum dots to higher trophic organisms (rotifers) through eating ciliated protozoans exposed to quantum dots (Holbrook et al., 2008, 192383 ). Since quantum dot uptake in this study was inferred from quantitation of Cd 2+ 

### and assumed no dissolution of the nanoparticles, it is unclear to what extent these results are applicable to poorly soluble nano-TiO 2 

### . Biomagnification was not observed at the top predator level (rotifers), beca use biomagnifications factors (BMF) values for the quantum dots ranged from between 0.29 and 0.62 (Holbrook et al., 2008, 192383 ).



# 4.6.1.3. Cumulative Dose of Nano-TiO 2 

# and Other Pollutants

Increased uptake of other pollutants in the presence of nano-TiO 2 

### has been reported by Sun et al. (2007, 193662 ) and Zhang et al. (2006, 157722 ; 2007, 090114 ) (Table 4-2). Sun et al. (2007,
193662 ) demonstrated that arsenic as arsenate [As(V)] strongly binds to Aeroxide ® 

### P25 (P25) in water and that fish (carp) exposed to water containing 10 mg/L of this photocatalytic nano-TiO 2 

### and 200 μg/L arsenate accumulated more arsenic than fish exposed to either nano-TiO 2 

### or arsenic alone.
The bioconcentration factor of arsenic 1 

### was more than twice as high when nano-TiO 2 

### was present than when it was not (Sun et al., 2007, 193662 ). The tested arsenate concentration, 200 μg/L, is environmentally relevant, given that higher total ar senic concentrations (mainly inorganic arsenic in the forms of arsenite and arsenate) in drinking wa ter have been reported in many countries, including Bangladesh, China, Chile, and India (Basu et al., 2004, 087896 ; Feng et al., 2001, 193374 ; Moore et al., 1997, 193553 ; Tian et al., 2001, 193679 ). Although data on nano-TiO 2 

### concentrations in the
environment 2 

### are lacking, the tested nano-TiO 2 

### concentration (10 mg/L) may be higher than the likely environmental concentrations, with the exce ptions of spills or accidents. The presence of
nano-TiO 2 

### did not alter the distribution of arsenic with in fish tissues. Over various time intervals,
arsenic and TiO 2 

### accumulated significantly in the intestine, stomach, and gills, and to a lesser degree in liver, skin, and scales; the least accumulation occurred in muscle. Because the accumulation of arsenic was much greater in the presence of nano-TiO 2 

### , Sun et al. (2007, 193662 ) concluded that adsorption to nano-TiO 2 

### facilitated arsenic transport and uptake.

1 The bioconcentration factor of arsenic = 1,000 × arsenic concentration in fish (μg/g dry weight)/arsenic concentration in wate r (μg/L).

2 Limited information is available on nano-TiO 2 in the ambient air within or surrounding nano-TiO 2 production facilities, or from the runoff from structures painted with paints containing TiO 2 . In the various compartments of the environment, however, nano-TiO 2 concentrations are unknown, and current technologies have not been able to distinguish man-made nano-TiO 2 from naturally-occurring
nano-TiO 2

.



### 4-12

## Table 4-2. Tissue concentrations of various pollutants in fish after exposures to nano-TiO 2 

## in water

Protocol (no UV illumination, unless specified) Test Species Material Study Outcome Reference

Fish (carp, Cyprinus carpio ) 21-nm primary particle, 50- to 200-nm aggregates in water (P25) (photocatalytic) Up to 25-day exposure to 3 and 10 mg/L nano-TiO 2 (water changed daily, TiO 2 concentrations in water ~2 and ~7 mg/L, respectively, after the first few hr) TiO 2 accumulated in internal organs > gills > skin and scales > muscle Zhang et al. (2006, 157722 ) Bioconcentration factors were higher at 3 mg/L than at 10 mg/L

Sun et al. (2007, Fish (carp, Cyprinus carpio ) 21-nm primary particle, 40- to 500-nm aggregates in water (P25) (photocatalytic) Up to 25-day expos ure to 10 mg/L nano-TiO 2 with and without 200 μg/L arsenate Arsenate adsorbed onto nano-TiO 2 193662 Higher arsenic concent rations in tissues (skin and scales; muscle; gills; liver; stomach; intestine) with arsenate plus nano-TiO 2 exposure, compared to arsenate exposure alone )

Zhang et al. (2007, Fish (carp, Cyprinus carpio ) 21-nm primary particle, BET 50 m 2 /g (P25) (photocatalytic) Cadmium adsorbed onto nano-TiO 2 Up to 25-day expos ure to ~97 μg/L cadmium alone, cadmium with 10 mg/L nano-TiO 2 , or cadmium with 10 mg/L natural sediment particles 090114 Higher cadmium concentrations in tissues ( skin and scale; muscle; gills; viscera; whole body) with cadmium plus nano-TiO 2 exposure, compared to cadmium exposure alone, or cadmium plus natural sediment particles )

Federici et al. (2007, Fish (rainbow trout, Oncorhynchus mykiss ) 21-nm, 75% rutile: 25% anatase, sonicated (P25) (photocatalytic) 0-, 7-, or 14-day expo sure to 0, 0.1, 0.5, or 1.0 mg/L nano-TiO 2 No clear treatment or time-dependent effects on Ti levels in gill, liver, or muscle. In brain, a transient but statistically significant decrease in Ti concent rations compared to control fish on day 0, but no exposure concentration-effect. 091222 )

Respiratory distress, organ pathologies, and oxidative stress at concentrations as low as 0.1 mg/L.

BET – Brunauer, Emmett, Teller method of calculating surface area

P25 – Aeroxide® P25



### Zhang et al. (2007, 090114 ) showed that fish (carp) exposed to cadmium in water (approximately 97 μg/L) along with 10 mg/L photocatalytic nano-TiO 2 

### accumulated more cadmium than fish exposed to either nano-TiO 2 

### or cadmium alone (Table 4-2). After 20 days of exposure, the bioconcentration factor for whole-body cadmium was 64.4 in carp exposed to cadmium alone, but reached 606 in carp exposed to both cadmium and nano-TiO 2 

### . After 25 days of exposure, cadmium concentration in the whole fish was 9.07 μg/g in the cadmium-only group and 22.3 μg/g in the cadmium-plus-nano-TiO 2 

### group, indicating a 146% increase in the cadmium bioconcentration factor in the presence of nano-TiO 2 

### . When carp were analyzed after 20 days of exposure, cadmium concentrations in all groups were higher in internal organs than in gills, muscle, and skin and scale (Zhang et al., 2007, 090114 ). Unlike nano-TiO 2 

### , natural sediment particle s (19 μm) (at equivalent concentrations) did not affect cadm ium bioaccumulation. Both nano-TiO 2 

### and sediment particles adsorb cadmium and reach equilibrium within 30 minutes, but nano-TiO 2 

### adsorbed more than 5 times as much cadmium as the sediment particles. Based on the facts that nano-TiO 2 

### can adsorb
cadmium and that tissue concentrations of cadmium and nano-TiO 2 

### (measured as Ti) are positively correlated, the authors suggested that increased cadmium uptake in the presence of nano-TiO 2 

### may have been due to accumulation of cadmium adsorbed on nano-TiO 2 

### (i.e., facilitated transport). The transport routes could be from water onto the gi ll surfaces or from consumed food into internal organs. Toxicity was not measured in this study. The fact that organic disinfection by-products ca n be formed by the photocatalytic oxidation of drinking water treatment with conventional TiO 2 

### (Richardson et al., 1996, 193612 ) suggests the possibility that nano-TiO 2 

### could have the same effect . Richardson et al. (1996, 193612 ) compared

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### the organic disinfection by-products detected after using: (1) chlorine as the sole disinfectant; and (2)
TiO 2 

### /UV light treatment followed by chlorination. The authors reported detecting an additional byproduct (tentatively identified as dihydro-4,5-dichloro-2(3H)furanone) after the combined TiO 2 

### /UV and chlorine treatment compared to chlorine treatment alone. Overall, however, the numbers and concentrations of chlorinated disinfection by- products were lower after combined TiO 2 

### /UV and
chlorine treatment than after chlorination alone. Cumulative exposure to nanomaterials could also occur. Some consumer products contain more than one type of nanomaterial; e.g., nano-TiO 2 

### and nano-silver (nano-Ag) have been used together in multiple products (The Project on Emerging Nanotechnologies, 2009, 196052 ).



# 4.6.2. Respiratory (Inhalation and Instillation)

Instillation can be performed in various ways, but essentially involves the direct administration of a substance to the respiratory tract. Animal studi es have shown that inhaled or instilled nano-TiO 2 

### can translocate into the interstitium of the lung, lymph nodes (Ma-Hock et al., 2009, 193534 ; Oberdörster et al., 1992, 045110 ; Oberdörster et al., 1994, 046203 ), blood (Geiser et al., 2005,
087362 ), and the brain (Wang et al., 2005, 193703 ; Wang et al., 2007, 090290 ; Wang et al., 2008,
157473 ). Particles in the nasal cavity may enter the brain through: (1) the olfactory nerve (Elder et al., 2006, 089253 ; Oberdörster et al., 1994, 046203 ) [upper particle size limit: 200 nm (Elder et al., 2006, 089253 )]; (2) the circulating blood and then crossi ng the blood-brain barrier (Oberdörster et al., 2004, 055639 ); and (3) the olfactory mucosa and through the ethmoid bone into cerebrospinal fluid (Illum, 2000, 157897 ). One of the most visually convincing demonstrations of olfactory nerve transport, as mentioned in Oberdörster et al. (2004, 055639 ), is a study by DeLorenzo (1970,
156391 ). DeLorenzo showed sequential TEM images of intranasally instilled gold nanoparticles in the olfactory mucosa, uptake into the olfactory ro ds, retrograde translocation within the olfactory dendrites, anterograde translocation in the axons of the olfactory nerve, and appearance in the olfactory bulbs. For more discussion of nanoparticle translocation from the nasal cavity to the brain, see Oberdörster et al. (2004, 055639 ).
Intranasal instillation of three sizes of nano-TiO 2 

### particles (approximately 20, 70, and 155 nm) at approximate 0.05 g/kg BW every other day for 30 days resulted in increased Ti concentrations in the olfactory bulb of mice (Wang et al., 2005, 193703 ). Also, two forms of nano-TiO 2 

### particles (80- nm rutile and 155-nm anatase) were found to increa se Ti concentrations in the hippocampus, central cortex, and cerebrum, in addition to olfactory bulb, in mice after repeated intranasal instillation at approximate 24 mg/kg BW every other day for 30 days (Wang et al., 2008, 157473 ). The authors
noted that the fact that brain tissue Ti concen trations were higher than lung tissue concentrations suggested that the olfactory nerve was the route of transport to the brain in this study. For respiratory exposure, the deposition pattern and concentration of particles in the respiratory tract can influence the health effects of these particles. Particles of various sizes can have different mechanisms of deposition (Gebhart, 1992, 157951 ; Heyder et al., 1985, 006919 ; Oberdörster et al., 2005, 087559 ). For nanoparticles, diffusive deposition, also known as thermodynamic deposition or diffusion (due to Brownian motion), predominates, whereas for particles larger than 1 μm, aerodynamic deposition predominates. Between 0.1 and 1 μm, the combined effects of aerodynamic and diffusive deposition are important. Oberdörster et al. (2005, 087559 ) summarized the principles an d models of respiratory tract nanoparticle deposition and retention in the lung. Modeling of humans who are resting and breathing through the nose indicated that for 1-nm particles, approximately 90% will be deposited in the nasal, pharyngeal, and laryngeal region; approximately 10% in the tracheobronchial region; and almost none in the alveolar region. These results contrast with the modeling of a 5-nm particle, which is deposited roughly equally in the three regions. Appr oximately 50% of larger, 20-nm particles are deposited in the alveolar region, with approximately 15% deposition in each of the other two

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### regions. Since these simulations are based on th e International Commission on Radiological Protection (ICRP) model, which was designed for larger particles ( supplement of Ober dörster et al., 2005, 087559 ), the performance of this model regardi ng particles at the lower end of the size
distribution is unclear. In contrast to these results, a Multiple Pa th Particle Dosimetry (MPPD) model that incorporated convective flow, axial diffusion , an d convective mixing (dispersion) predicted that very few small nanoparticles would deposit in th e alveolar area (Asgharian and Price, 2007,
093119 ). Nanoparticles less than 10 nm in diameter were predicted to deposit mainly in the tracheobronchial airway, and very few nanoparticles smaller than 5 nm were predicted to reach the alveolar region (Asgharian and Price, 2007, 093119 ). Depending on particle size, consideration of axial diffusion and dispersion could result in increas ed predicted deposition in the alveolar region of up to 10%. This modified MPPD model for nanoparticles found good agreement between predicted depositions of nanoparticles with measurements reported in the literature.
Inhaled nano-TiO 2 

### persisted in the lung longer than fine TiO 2 

### in rats (Oberdörster et al., 1994,
046203 ). After 12 weeks of inhalation (6 hours/day, 5 days/week) of approximately equivalent mass
concentrations of fine TiO 2 

### (22.3 ± 4.2 mg/m 3 

### ) and nano-TiO 2 

### (23.5 ± 2.9 mg/m 3 

### ), the total retained lung burdens were 6.62 ± 1.22 mg for fine TiO 2 

### and 5.22 ± 0.75 mg for nano-TiO 2 

### . The estimated retention half-times were 174 days for fine TiO 2 

### and 501 days for nano-TiO 2 

### (Oberdörster et al., 1994, 046203 ).
In animal studies of nano-TiO 2 

### disposition (Table 4-3), 13 weeks of inhalation exposure to
nano-TiO 2 

### increased TiO 2 

### burden in lymph nodes in rats (2 and 10 mg/m 3 

### ), mice (10 mg/m 3 

### ), but not in hamsters (at up to 10 mg/m 3 

### ) (Bermudez et al., 2004, 056707 ).

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## Table 4-3. Nano-TiO 2 

## disposition in animals after inha lation or intratracheal instillation

Species/Strain Aerosol Study Pr otocol Observations Reference

Bermudez et al. (2004, Fischer 344 rats, females (6 wk) TiO 2 : 1.29-1.44 μ m MMAD Animals exposed via inhalation 6 hr/day, 5 days/wk, for 13 wk to 0.5, 2, and 10 mg/m 3

. TiO 2 pulmonary retention half-ti mes for the low-, mid-, and high-exposure groups, re spectively: 63, 132, and 365 days in rats; 48, 40, and 319 days in mice; and 33, 37, and 39 days in hamsters. Burden of TiO 2 in lymph nodes increase with ti me postexposure in midand high-dosed rats, and in high-dosed mice, but was unaffected in hamsters at any time or in any dosage group. In high-exposure groups of mice, epithelial permeability remained elevated (~2 × control groups) out to 52 wk without signs of recovery. Epithelial permeability was 3 to 4 × control in high exposure group rats through 4 wk postexposure, but approached control by 13 wk. Epithelial permeability was unaffected in all groups of hamsters. 056707 )
B3C3F1 mice, females (6 wk) ( σ g = 2.46-3.65), 21-nm primary particles Control animals exposed to filtered air. Hamsters, females (6 wk) Animals sacrificed at 0, 4, 13, 26, and 56 days (49 for hamsters) postexposure. Groups of 25 animals per species and time point.

Geiser et al. (2005, Distributions of particle s among lung compartments (airspace, epithelium/endothe lium, connective tissue, capillary lumen) were direct ly related to the volume fractions of compartments and did not differ significantly between 1- and 24 hr postexposure. On average, 79.3 ± 7.6% of part icles were on the luminal side of the airway surfaces, 4.6 ± 2.6% in epithelial or endothelial cells, 4.8 ± 4.5% in connective tissues, and 11.3 ± 3.9% within capillaries. Particles within cells were not membrane-bound. TiO 2 (22-nm CMD,

σ g = 1.7) Rats exposed 1 hr via endotracheal tube while anesthetized and ventilated at constant rate Wistar rats, 20 adult males, 250 ± 10 g 087362 ) Spark generated Lungs fixed at 1 or 24 hr postexposure 0.11 mg/m 3
7.3 × 106 particles/cm 3 (SD 0.5 × 106 particles/cm 3 )

Muhlfeld (2007, Re-evaluation of the data from Geiser et al. (2005, 087362 ) with a new statistical method in which a relative deposition index (RDI) was calculated for each compartment. When RDI=1, the particle number is the same as one would expect for the size of the compartment, if the particle distribution is random. RDI>1 suggests a preferential distribution. The new analysis suggested that at 1 hr postexposure, connective tissue was the pr eferential target for the nano-TiO 2 , while capillary lumen was the preferential target at 24 hr postexpos ure. This study suggested pulmonary clearance via microvasculature, and does not exclude clearance through exhalation or mucociliary escalator. 091106 )

WKY/NCrl (Charles River) rats, 5 young adult males, 250 ± 10 g TiO 2 (22-nm CMD,

σ g = 1.7) Rats exposed 1 hr via endotracheal tube while anesthetized and ventilated at constant rate Kapp et al. (2004, Of particles in tissues, 72% were aggregates of 2 or more particles; 93% of aggregates were round or oval; 7% were needle-like. The size distribution of particles in lung tissues (29 nm CMD, σ g = 1.7) was remarkably similar to the aerosol; the small discrepancy could have been due to differences in sizing techniques. A large 350-nm aggregate was found in a type II pneumocyte, a 37-nm particle in a capillary close to the endothelial cells, and a 106-nm particle within the surface-lining layer close to the alveolar epithelium 156624 ) Spark generated Lungs fixed immediately postexposure

CMD – Count median diameter; MMAD – Mass median aerosol diameter; σ g – Geometric standard deviation
Source: U.S. EPA (2009, 196063 )



# 4.6.3. Dermal

Because sunscreen is used on the skin, human skin penetration of nano-TiO 2 

### (as particles in a solubility vehicle or in sunscreens) has been disc ussed in several reports and reviews (NanoDerm, 2007, 157660 ; Nohynek et al., 2007, 090619 ; TGA, 2006, 089202 ). Most dermal exposure studies reviewed used human skin and pig skin (Sadrieh et al., 2010, 594511 ); several were in vivo studies in humans. Compared to other routes of exposure, de rmal exposure may be more directly relevant in assessing potential health effects associated with its use in sunscreens, at least for unflexed skin from healthy adults.

4-16

### Because of the relatively noninvasive nature of skin penetration testing, several laboratory studies have focused on skin absorption in humans, rather than animals. Human skin regulates the penetration of contaminants primarily through the stratum corneum layer, which contains keratinized
cells and has no blood vessels. The thickness of th e layer varies, ranging from approximately 60 μ m to greater thickness on the plantar and palm ar surfaces (Monteiro- Riviere et al., 1990, 625073 ). Other aspects of skin may also vary in differen t parts of the body (e.g., face versus forearm). Although published studies indicate the anatomy of stratum corneum of full-term infants and babies is comparable to that of adults (Fairley and Rasmussen, 1983, 193370 ), the physiology is not. Both the anatomy and physiology of pre-term infants’ skin are not comparable to that of adults (Kalia et al., 1998, 196039 ). Skin studies include a range of experimental conditions, including in vivo and
ex vivo /

in vitro. With few exceptions disc ussed below (Kertesz et al., 2005, 180334 ; Menzel et al., 2004, 180361 ; Sadrieh et al., 2008, 157500 ), some of which were attributed to artifacts from sample preparation, most of these human and animal studi es (Table 4-4) found clear evidence that nano-TiO 2 

### does not penetrate beyond the stratum corneum or hair follicles, and does not penetrate into living cells of healthy skin (Figure 4-1). In healthy human skin, topically applied nano-TiO 2 

### penetrates only into the upper layers of the stratum corneum (Table 4-4). The pathways of skin penetration can include in tracellular penetration, intercellular penetration, and pe netration through hair follicles (F igure 4-1) (Nohynek et al., 2007,
090619 ). Penetration through sweat glands has not been reported, according to one source (NanoDerm, 2007, 157660 ). Although increased skin penetration of other nanomaterials has been reported in flexed porcine skin (Rouse et al., 2007, 157644 ) and flexed or abraded rat skin (Zhang and Monteiro-Riviere, 2008, 193735 ) and in UV-exposed murine skin in vivo (Mortensen et al., 2008, 155612 ), studies of skin penetration in healthy flexed human skin or damaged skin have not
been identified for nano-TiO 2 

### . Similarly, studies developing transcutaneous vaccine delivery detected the presence of nanoparticles in immune cells after topical application of nanoparticles on tape stripped skin (Mahe et al., 2009, 225307 ), but nano-TiO 2 

### has not been tested in these conditions.
Nano-TiO 2 

### was observed in some hair follicles (Lekki et al., 2007, 180280 ), but did not reach the living follicle cells (with the exception of one study in hairless mice (Wu et al., 2009, 193721 ), see below). The presence of nano-TiO 2 

### in hair follicles is most likely due to mechanical force, such as the movement of the hair during sunscreen application. Nano-TiO 2 

### in hair follicles might contribute to increased Ti levels in the dermis (Sadrieh et al., 2008, 157500 ) because the shaft of the hair is exposed to the surface but the hair follic les are in the dermis. Nanoparticle loss from hair follicles is expected to be slow because the elimination occurs only by its flowing out with sebum or by its being pushed out with sebum. In a study using a hydrogel formulation containing fluorescence-labeled nanoparticles [Resomer RG 50.50 H, poly(lactide-co-glycolide)] on human skin (Lademann et al., 2007, 157678 ), approximately 15% of total nanopar ticles detected in hair follicles 30 minutes after application remained in the hair follicle for 10 days, which is at least 10 times longer than particles remain in the stratum corneum (Lademann et al., 2006, 157758 ). A recent in vitro and in vivo study using pig and hairless mice suggested that repeated in vivo dermal exposure may lead to nano-TiO 2 

### penetration into the living cells of epidermis and possibly systemic distribution (Wu et al., 2009, 193721 ). Similar to other studies, Wu and colleagues’(2009,
193721 ) 24 hour exposure in vitro of porcine skin to nano-TiO 2 

### did not show penetration beyond the stratum corneum. Thirty days of in vivo exposures to 4 nm nano-TiO 2 

### , but not larger nano-TiO 2 

### , on the ear skin of pigs, however, resulted in penetration deep into the basal cell layer of the epidermis.
No nano-TiO 2 

### was observed in the dermis. After 60 days of in vivo dermal exposure to 10-60 nm
nano-TiO 2 

### , hairless mice showed increased Ti concentr ations in multiple organs, including skin, subcutaneous muscle, heart, liver, spleen, as well as pathological changes in skin, liver, spleen and lung. The various tested sizes of nano-TiO 2 

### do not behave the same. For instance, in the heart, the
increases in Ti concentration were similar in all nano-TiO 2 

### treatments, but the pathological changes were only seen in the 10 nm nano-TiO 2 

### group. Given that hairless mouse skin has a much thinner stratum corneum than human skin (Haigh and Smith, 1994, 625322 ) and other differences, it is

4-17

### unclear to what extent the observed systemic distribution of nano-TiO 2 

### after repeated dermal exposure in the hairless mouse may occur in humans. In human skin that is diseased, nano-TiO 2 

### might penetrate more deeply. The only available study of nano-TiO 2 

### on skin with dermal lesions was completed on psoriatic skin. Psoriatic skin is a symptom of a chronic, and possibly immune-mediate d or genetic, disease called psoriasis. Unlike normal skin cells, which mature and are shed in 28-30 days, psoriatic skin cells mature in 3-4 days, accumulate on the skin surface (instead of shedding, because new skin develops faster than dead skin sheds), and develop into patches of dead skin (National Psoriasis Foundation, 2006, 157748 ; Pinheiro et al., 2007, 180160 ). Psoriatic skin has a looser corneocyte organization than healthy skin due to the loss of stratum corneum cohesion (Pinheiro et al., 2007, 180160 ). In the Pinheiro et al. (2007, 180160 ) study, nano-TiO 2 

### in a sunscreen formulation penetr ated into deeper areas of the stratum corneum in psoriatic skin than in healthy skin, but not into living cells in either psoriatic or healthy skin (Table 4-4).

4-18Source: Adapted from and used with permission from CRC Press, Monteiro-Riviere (1991, 157957 ; 2004, 157834 ); Used with permission from Informa Healthcare, Riviere and Monteiro-Riviere (1991, 625197 ); Used with permission from Informa Healthcare, Nohynek et al. (2007, 090619 )



## Figure 4-1. Possible pathways of nano-TiO 2 

## skin penetration. Top Graphic: Nanoparticles may pe netrate into skin by passing through: (1) the intercellular space between cells; (2) transcellular space; (3) opening of hair follicles; or (4) op ening of sweat glands. Nano-TiO 2 

## has been seen in the stratu m corneum and inside hair follicles, but not in sweat glands. Bottom Graphic: Skin surface (from stratum corneum to stratum basal) at a high magnification showing simplified paths of nanoparticles p assing: (1) between cells; and (2) through cells. Nanoparticles are not dr awn to scale in either graphic.

4-19

## Table 4-4. Overview of TiO 2 

## skin absorption/penetration studies

Skin Model a (Sampling Technique) Test Material Results Reference

Sunscreen Formulations Containing Nano-TiO 2

Lademann et al. (1999, Nano-TiO 2 in a sunscreen formulation Primary particle 17 nm (Kemira, 2000, Human forearm, repeated application for 4 days (tape stripping, biopsy) Most particles on and in the upper layers of stratum corneum. In the lower half of the horny layer, only in the openings of hair follicles and sebaceous glands. In deeper tissue, exclusively in the follicle channels. 157896 ), rutile, Al 2 O 3 /stearic acid coated,aggregates 150-170 nm (UVTitan M 160) in an oil-in-water emulsion, provided by L’Oréal (Clichy, France) 090591 )

No penetration into living skin.

Pinheiro et al. (2007, Sunscreen that contains nano-TiO 2 Deeper nano-TiO 2 penetration in psoriatic skin than in healthy skin. Not specified Human skin (healthy and psoriatic), in vivo, 2 hr (biopsy) 180160 ) No penetration beyond stratum corneum in both psoriatic and healthy skin.

Nano-TiO 2 in a sunscreen formulation 20-nm nano-TiO 2 , coated with silicone Human skin, in vitro, and human skin, in vivo (skin stripping) Penetration limited to upper layers of stratum corneum. Nanoparticles in skin furrows or follicular opening could be mistaken to be in the epidermal compartment. Mavon et al. (2007, 090587 )

Kertész et al. (2005, Sunscreen that contains nano-TiO 2 A commercially available sunscreen, hydrophobic emulsion containing nano-TiO 2 (Anthelios XL SPF 60, La Roche Posay, France) Human foreskin grafts transplanted onto SCID b mice; TiO 2 emulsion on the graft in occlusion for 1, 24, or 48 hr TiO 2 in the corneocyte layers of stratum

corneum. 180334 ) In two cases, penetration through the stratum corneum, to the stratum granulosum was observed.

Sunscreen that contains nano-TiO 2 A commercially available sunscreen, hydrophobic emulsion containing nano-TiO 2 (Anthelios XL SPF 60, La Roche Posay, France) Human foreskin grafts transplanted onto SCID mice; TiO 2 emulsion on the graft at 2 mg/cm 2 in occlusion for 24 hr TiO 2 in stratum corneum, not in deeper layers of the skin. Kiss et al. (2008, 157547 )

Nano-TiO 2 in

sunscreen formulation/sunscreen that contains nano-TiO 2 50-100 nm, mixture of anatase and rutile, no coating information Human abdominal skin, in vitro Penetrati on limited to upper layers of stratum

corneum. Dussert and Gooris (1997, 193359 )

Female Yucatan minipigs (in vivo), 2-mg emulsion/cm 2 skin, 5 days/wk for 4 wk (necropsy) Sadrieh et al. (2010, Various TiO 2 in

sunscreen formulations Sunscreen base formulation containing no TiO 2 or 5% of one of three types TiO 2 : Increased Ti levels in epidermis in all TiO 2 - treated groups. 594511 ) No penetration of TiO 2 particles into the dermis. Micro-sized TiO 2 No increases in Ti levels in lymph nodes or liver of any treated animals. Nano-TiO 2 , uncoated

Nano-TiO 2 , coated with aluminum hydroxide and dimethicone/methicone copolymer

Photostable nano-TiO 2 in various formulations Photostable nano-TiO 2 , needle-like shape, 45-150 nm × 17-35 nm, coated with alumina and silica (Lodén et al., 2006, Pig skin, in vitro Particles on/in the stratum corneum; minimal penetration into stratum granulosum. Menzel et al. (2004, 180361

157757 ), in the following formulations: (1) Eucerin® Micropigment Crème 15: commercial sunscreen, 5% TiO 2 concentration (Beiersdorf company); (2) a liposome dispersion: 18% TiO 2 , containing Phospholipon 90 G and Tioveil AQ-N (Tioxide Specialties Ltd., Billingham, UK); (3) formula SG110: 4.5% TiO 2 , containing Tioveil AQ-N; and(4) pure predispersion Tioveil AQ-N: 40% TiO 2 ) No penetration into living skin.



### 4-20Skin Model a (Sampling Technique) Test Material Results Reference

Photostable nano-TiO 2 in

sunscreen formulations (1) T-Lite SF-S: rutile, coated with SiO 2 and methicone; and (2) T-Lite SF: rutile, coated with methicone Pig skin, in vitro, up to 24 hr (tape stripping) No penetration beyond stratum corneum. Gamer et al. (2006, 090588

Both primary particles are needle-like: 30-60 nm × 10 nm. Aggregates and agglomerates in water phase, mostly up to 200 nm

Both are oil/water emulsions containing 10% TiO 2 Receptor solution recove ries of 0.8-1.4% of applied dose. )

Other Nano-TiO 2 Formulations

T 805 Degussa Coated with trimethyloctylsilane; ~20 nm in diameter Pig, in vivo TiO 2 found exclusively in the outermost stratum corneum layer. Traces of TiO 2 were found in the upper part of the follicle, with no evidence of uptake into the follicular epithelium. Pflucker et al. (1999, 644132 )

Human forearm, in vivo, 6 hr (biopsy) Penetration of particles into the upper layers of stratum corneum. Pflücker et al. (2001, Various nano-TiO 2 in oil-in-water emulsions Emulsions contained 4% nano-TiO 2 , only differed in nano-TiO 2 types: (1) 20-nm cubic primary particle, coated with trimethyl octylsilane, hydrophobic surface (T805, Degussa); (2) 10-15 nm primary particle, aggregated into ~100-nm needles, coated with Al 2 O 3 and SiO 2 , amphiphilic surface (Eusolex T-2000, Merck); and (3) 100- nm needles, coated with alumina and silica, hydrophilic surface (Tioveil AQ-10P, in dispersion, Solaveil) 157887 ) and Schulz et al. (2002, No penetration into living skin. 157872 )

10-100 nm, coated with SiO 2 –

, Al 2 O 3 – , Al 2 O 3 ,/SiO 2 Nano-TiO 2 Human, in vivo (biopsy) Particles on or in the outmost surface of the stratum corneum. Schulz et al. (2002, 157872 ) No penetration into living skin.

Various TiO 2 and nano-TiO 2 14 nm-200 μ m, anatase and rutile, coated and uncoated materials Pig and human skin, in vivo and in vitro (skin stripping or biopsy) No penetration beyond the stratum corneum in any study. SCCNFP (2000, 092740 )



### 4-21Skin Model a (Sampling Technique) Test Material Results Reference

(A) No penetration beyond the stratum

corneum Various nano-TiO 2 100% anatase, uncoated, nano-TiO 2 (Zhejiang Wanjin Material Technology Co., Ltd.): (A) Porcine skin, in vitro, isolated pig ear skin (without and with tape stripping) on a modified Franz equipment, nano-TiO 2 suspension (4, 10, 25, 60, or 90 nm) on the skin for up to 24 hr Wu et al. (2009, 193721 ) (B) After 30 days, nano-TiO 2 was detected in all layers of epidermis (stratum corneum, stratum granulosum, prickle cell layer, and basal cell layers), but not in the dermis of porcine skin. Only 4 nm nano-TiO 2 penetrated into the deeper layer of the epidermis (basal cell layer). (C) After 60 days, hairless mice had increased Ti in the skin, subcutaneous muscle, liver, heart, and spleen, but not in the blood or subcutaneous saccus lymphaticus in 10-, 21-, 25-, and 60-nm groups. Almost negligible changes in the brain and kidney, with the exception of increased Ti in the brain after 21 nm nano-TiO 2 exposure. Increased Ti in the lung may be significant in the 21- and 60-nm groups. 4 nm, hydrophobic surface, measured particle size 5 ± 1 nm, surface area 200 m 2 /g (B) Porcine skin, in vivo, shaved pig ear starting at age of 4 wk, approximately 24 mg of test formulation containing 5% nano-TiO 2 (4 or 60 nm) and Tween 80 was topically applied in the marked test area on the right ear skin for 30 consecutive days. Punch biopsies collected at 24 hr after the last treat ment for TEM 10 nm, hydrophobic surface, measured particle size 10 ± 1 nm, surface area 160 m 2 /g

75% anatase/25% rutile, uncoated nano-TiO 2 (P25 from Degussa, Germany): 21 nm, hydrophilic surface, surface area 50 m 2 /g (C) BALB/c hairless mice skin, in vivo, starting at age of 7-8 wk. Test formulation containing 5 % nano-TiO 2 (10 nm, 21, 25, 60, or 90 nm), carbopol 940, and triethanolamine was applied on the dorsal skin for 60 consecutive days at 8 mg emulation (or 400 μm nano-TiO 2 ) per cm 2 skin. 3 hr after application, the dressing was removed and residual nanomaterials were removed from the skin with lukewarm water and the skin was dried. 100% rutile, uncoated, nano-TiO 2 (Zhejiang Hongsheng Material Technology Co., Ltd.): 25 nm, hydrophilic surface, measured particle size 25 ± 5 nm, surface area 80 m 2 /g 60 nm, hydrophobic surface, measured particle size 60 ± 10 nm, surface area 40 m 2 /g 90 nm, hydrophobic surface, measured particle size 90 ± 10 nm, surface area 40 m 2 /g

Lansdown and Taylor (1997, TiO 2 Mixed particle sizes, mostly less than 10 μ m in aqueous solution (range from <2 μ m to >20 μ m), no coating information, 20% TiO 2 in water, castor oil, or polyethylene glycol Rabbit skin, in vivo, 4 hr for 1 day or 2 hr daily for 3 days Penetration of particles into stratum corneum and outer hair follicles. 157928 No penetration into living skin. )

Uptake of TiO 2 affected by the vehicle: in caster oil>in water>in polyethylene glycol.

Lekki et al. (2007, Nano-TiO 2 in various gels For ion microscopy study: 20- nm × 100-nm primary particles, coated (photostable UV-filter) (Eusolex® T-2000, Merck). Four formulations: hydrophobic basis gel, isopropyl myristate gel, microemulsion gel, and polyacrylate gel, each containing 5%-weight nano-TiO 2 particles After wash with water, nano-TiO 2 remains on skin, with most in stratum corneum and some in hair follicles. Porcine and human skins, for 30 min to 48 hr (biopsy) 180280 )

Nano-TiO 2 observed seen in hair follicles as deep as 400 μm, but not in living cells surrounding the follicles.

For autoradiography study: proton-irradiated 20-nm TiO 2 , rutile (R-HD2, Huntsman), coated with alumina (Huntsman, 2008, 157555 )



### 4-22Skin Model a (Sampling Technique) Test Material Results Reference

TiO 2 /Nano-TiO 2 Particles of Unknown Size

Sunscreen that contains TiO 2 Not specified Human (tape stripping) Partic les on or in the outmost layers of the stratum corneum. No penetration into living skin. Gottbath and Mueller-Goymann (2003, 193401 )

TiO 2 Not specified Mouse, pig, and human skin, in vitro TiO 2 detected in the intercellular spaced between corneocytes of the outermost layers of the stratum corneum. No penetration into living skin. Gontier et al. (2004, 193398 )

Tan et al. (1996, Sunscreen that contains TiO 2 Sunscreen containing 8% microfine TiO 2 (size, crystal form, and coating were not specified) Human skin (13 patients, 59-82 yr old), in vivo, applied TiO 2 sunscreen daily for 9-31 days until 2 days prior to surgical removal of the skin (tape stripping) Ti concentration in the dermis of patients exposed to sunscreen was higher than concentration in cadavers (controls), after exclusion of one control outlier. 157933 )

No correlation between the duration of sunscreen application and Ti concentration.

Various “microfine” TiO 2 Commercial microfine TiO 2 dispersions in octyl palmitate and in water (Tioxide Specialties Ltd, Billingham, U.K.) Human abdominal skin, in vitro, and skin equivalents (keratinocytes and fibroblasts of native human origin), in vivo Microfine TiO 2 penetrates into the human stratum corneum probably via sebum lipids of the hair follicles. Bennat and Muller-Goymann (2000, 157403 )

a Topical application unless specified.

b SCID = Severe combined immune deficiency.



### Mortensen et al. (2008, 155612 ), working with quantum dots rather than TiO 2 

### , reported greater skin penetration following UV exposure and suggested that even mildly sunburned skin might be more susceptible to penetration by nanoparticles of similar size and chemistry to the quantum dots used in their study. Though the size and chemical composition of the quantum dots differ from the
nano-TiO 2 

### used in sunscreens, this increased susceptibility to penetration is of note. The authors qualified their results by noting that under no circumstances was there evidence for massive quantum dot penetration, and that quantum dots collected pref erentially in the folds and defects in the stratum
corneum and in hair follicles. Using “microfine” TiO 2 

### , Tan et al. (1996, 157933 ) compared uptake in skin samples from 13 elderly persons (age 59-82 years) with samples from 6 control cadavers (used to determine background exposure). The authors reported some dermal uptake, although they suggested caution when interpreting their results, citing the advanced age of their participants, the fact that skin samples were taken from different locations, and the fact that TiO 2 

### concentrations were close to analytical detection limits. Kertész et al. (2005, 180334 ) reported penetration of nano-TiO 2 

### into the stratum granulosum of grafted human foreskin in two samples (of an unknown total number). Sadrieh et al. (2010, 594511 ) found elevated levels of Ti in the dermis and epidermis skin of minipigs exposed to coated and uncoated nanosize TiO 2 

### . Investigators found no evidence of Ti penetration through expected routes such as the ha ir follicles, and concluded that the very few randomized Ti particles detected in living cells of the dermis were accounted for by contamination during sample preparation, possibly by small pieces of epidermis which contained 300- to 500-fold higher Ti concentrations than the dermis (Sadrieh et al., 2010, 594511 ). Several other studies that evaluated absorption using pig skin suggest little or no absorption beyond the stratum corneum. In a study using nano-TiO 2 

### in four formulations on pig skin (Menzel et al., 2004, 180361 ), the authors
stated that nano-TiO 2 

### penetrated through the stratum corneum into the underlying stratum granulosum (but not into stratum spinosum) via inter cellular space. The presence of Ti in the dermis, however, was deemed to be an artifact of the pr eparation process. Other studies using pig skin did
not find nano-TiO 2 

### penetration beyond the stratum corneum (Gamer et al., 2006, 090588 ; Lekki et al., 2007, 180280 ; Pflücker et al., 2001, 157887 ). Some nanomaterials have been shown to penetrate deeper in damaged skin than in intact skin [quantum dots in UV-exposed murine skin (in vivo) (Mortensen et al., 2008, 155612 ) and abraded rat

4-23

### skin (in vitro) (Zhang and Monteiro-Riviere, 2008, 193735 ); nano-silver coated with polyvinylpyrrolidone in abraded human skin- skin (ex vivo) (Larese et al., 2009, 193493 )], but no experimental data on nano-TiO 2 

### dermal penetration in damaged skin were found. Preliminary data showed that two types of coated nano-TiO 2 

### topically applied on either dermabraded or intact skin of
SKH-1 hairless mice did not increase Ti concentr ations in blood, lymph nodes, liver, spleen, or kidney (Gopee et al., 2009, 193399 ; Gopee et al., 2009, 667592 ). The depth of nano-TiO 2 

### penetration in either damaged or intact skin was not reported. Hairless mice data, however, do not exclude the possibility that nano-TiO 2 

### might penetrate deeper into damaged human skin than intact human skin because relative penetration of chemicals between hairless mice and humans varies and could be chemical specific (Benavides et al., 2009, 193270 ; Simon and Maibach, 1998, 193647 ).



# 4.6.4. Ingestion

Currently only three toxicological studies of nano-TiO 2 

### through oral exposure have been reported (Section 5.3.1.2), and of these, only one (Wang et al., 2007, 090290 ) reported tissue
concentrations of nano-TiO 2 

### . In the Wang et al. (2007, 090290 ) study, male and female mice received a single oral gavage of a fixed large dose of 5,000 mg/kg TiO 2 

### as 25-nm rutile spindles, 80-nm rutile spindles, or 155-nm anatase octahedrons (10 male and 10 female mice for each type of
TiO 2 

### , and negative controls) (Table 4-5). The organs with elevated TiO 2 

### concentrations (measured only in female mice) were liver, spleen, kidney, lung, and brain. Although the liver is expected to
receive most of the TiO 2 

### absorbed from the gastrointestinal tract through the portal vein, elevated
TiO 2 

### levels in the liver were observed only in th e 80-nm group. The reason for this size-specific elevation in hepatic TiO 2 

### concentration is unknown.



# 4.6.5. Blood Brain Barrier an d Placental Transfer

The general potential for nanoparticles to cross the BBB has been investigated and developed primarily in relation to drug delivery systems (Beduneau et al., 2007, 193266 ; Emerich and Thanos, 2007, 193365 ). In addition to size (Sonavane et al., 2008, 193652 ), the surface properties of nanoparticles influence the potential for a nanomate rial to penetrate the BBB (Singh and Lillard, 2009, 193650 ). Nanoparticles developed for drug delivery often have ligands conjugated on the surface or other surface modifications to facilitate cellular uptake (Beduneau et al., 2007, 193266 ).

4-24

## Table 4-5. Animal studies that measured Ti concentrations in brain after nano-TiO 2 

## exposures through injection or oral gavage

Nano-TiO 2 Study design Findings in the brain Reference

Ti concentrations in brain were increased in all three TiO 2 treatment groups compared to negative controls. The increase was smaller in the 25-nm group than the 155-nm group, while the 80-nm group had the same increase as the 155-nm group. Nano-TiO 2 , 25 nm and 80 nm, rutile, uncoated (from Hangzhou Dayang Nanotechnology Co. Ltd. Fine TiO 2 , 155 ± 33 nm TiO 2 , anatase, uncoated, >10 wt% at <100 nm (from Zhonglina Chemical Medicine Co. (Chen, personal communication, 2008, Wang et al. (2007, Single oral gavage at 5,000 mg/kg to male and female CD-1(ICR) mice 090290 ) Ti content was measured 2 wk after gavage by ICP-MS with a detection limit of 0.074 ng/mL

Vacuoles in the neuron of hippocampus, suggesting fatty degeneration, observed in the 80-nm (but not typical) and 155-nm (frequently) groups, but not in the 25-nm
group. 157588 )

TiO 2 was not detected in the brain at any tested time points. Single i.v. injection at 5 mg/kg BW through the tail vein of male Wistar rats Nano-TiO 2 , 20-30 nm, 17% anatase, 30% rutile, uncoated, BET surface area 48.6 m 2 /g Fabian et al. (2008, 157576 )

TiO 2 concentrations in the brain were measured on days 1, 14, and 28 by ICP-AES with a Thermo Jarrell Ash “IRIS 1” spectr ometer with a detection limit of 0.5 μm/organ

Nano-TiO 2 , 15 nm, rutile, coated with silica (27.5 wt%) Single i.v. injection at approximately 60 mg/kg BW through the tail vein of male ddY mice No increase of Ti in the brain of treated mice was observed compared to negative controls at any tested time points. Sugibayashi et al. (2008, 157489 )

Ti concentrations in brain were measured at 5 min, 72 hr, and 1 mo after injection by ICP-MS with an unspecified detection limit

Ti concentrations in the brain increased with increasing nano-TiO 2 doses. All TiO 2 treatments increased Ti concentration in the brain, as compared to negative controls. At 150 mg/kg, brain Ti concentration was higher in the nano-TiO 2 group than in the conventional TiO 2 group. Multiple i.p. injection to female CD-1 (ICR) mice once per day for 14 days with nano-TiO 2 at 5, 10, 50, 100, and 150 mg/kg BW or conventional TiO 2 at 150 mg/kg BW Nano-TiO 2 , 5 nm, anatase Liu et al. (2009, 193516 Conventional TiO 2 )

Both types of TiO 2 were made from controlled hydrolysis of titanium tetrabutoxide. Ti concentration was measured 14 days after the treatment began by ICP-MS with a detection limit of 0.076 ng/mL

Nano-TiO 2 particles were seen in the brain (olfactory bulb and the cerebral cortex – frontal and temporal lobes) of the 6-wk-old mice from nano-TiO 2 -exposed dams. (Results from 4-day-old mice were not reported.) Takeda et al. (2009, Nano-TiO 2 , 25-70 nm, anatase, surface area 20-25 m 2 /g, purity 99.9% (from Sigma-Aldrich) s.c. injections of 100 μL of 1 mg/mL nano-TiO 2 (i.e., 0.1 mg nano-TiO 2 ) each time per pregnant Slc:ICP mice once per day at 3, 7, 10 and 14 days post-mating. 193667 )

Presence of nano-TiO 2 in the brain was assessed in the male offspring at age of 4 days and 6 wk by FE-SEM/ EDS Markers of apoptosis (act ivation of caspase-3 and crescent-shaped cells), occlusion of small vessels, and perivascular edema observed in the brain of 6-wk-old mice from nano-TiO 2 -exposed dams.

BET – Brunauer, Emmett, Teller method of calculating surface area ICP-MS – Inductively coupled plasma-mass spectrometry
BW – Body weight i.p. – Intraperitoneal
FE-SEM/EDS – Field emission-type scanning electron microscopy/energy dispersive Xray spectrometry i.v. – Intravenous

s.c. – Subcutaneous ICP-AES – Inductively coupled plasma atomic emission spectrometry



### Increased Ti concentrations in the brain were observed in mice 2 weeks after they were exposed to fine and nano-TiO 2 

### through a single dosage by oral gavage (Wang et al., 2007, 090290 ), and in mice following a 14-day exposure period of once-daily i.v. injections of nano-TiO 2 

### (Liu et al., 2009, 193516 ) (Table 4-5). No increase in Ti concentration in the brain was observed in rats or mice exposed to nano-TiO 2 

### via a single i.v. injection (Fabian et al., 2008, 157576 ; Sugibayashi et al., 2008, 157489 ). Due to the variations in nano-TiO 2 

### treatment regimens, and other experimental design elements, no specific characteristic of nano-TiO 2 

### or its administration has been identified as determining factors for BBB penetration. A recent study demonstrated presence of TiO 2 

### particles, and pathological changes, in the brain
of 6-week-old mice born to nano-TiO 2 

### exposed dams (Takeda et al., 2009, 193667 ) (Table 4-5), suggesting that nano-TiO 2 

### might be passed through undevelope d or developing BBB in embryos or

4-25

### young mice. Because the dams were exposed to nano-TiO 2 

### during pregnancy and the offspring were tested at 4 days and 6 weeks of age, the nano-TiO 2 

### exposure to the offspring could have been in utero (i.e., nano-TiO 2 

### could penetrate the placental barrier) or through milk, which was not tested in this study. In addition to the brain, nano-TiO 2 

### particles and pathological changes were also observed in the reproductive system of male offspring of nano-TiO 2 

### -exposed dams (female offspring were not studied) (Takeda et al., 2009, 193667 ). Although no evidence was identified from human studies for
nano-TiO 2 

### passing through the placental barrier, an ex vivo study using perfused human placentas showed that nano-gold (PEGylated gold nanoparticles at 15 and 30 nm) did not cross the placenta into the fetal circulation at the tested condition (Myllynen et al., 2008, 187028 ). Nano-gold might behave differently from nano-TiO 2 

### , given that uncoated nano-gold does not penetrate either the BBB or placental barrier in mice (Sadauskas et al., 2007, 091407 ), whereas nano-TiO 2 

### does pass through the BBB in mice (Liu et al., 2009, 193516 ; Wang et al., 2007, 090290 ).



# 4.6.6. Dose Metrics

Quantitative risk assessment relies on dose-re sponse relationships. Selecting a measurable characteristic of dosage that would be appropriat e for predicting nanoparticle toxicity has drawn
attention from both researchers and risk assessors. No single metric is recommended in this document, but supporting evidence for various selections of a dose metric is noted. The criterion for selecting a “good” dose metric is often based on generating a consistent dose-response relationship. However, an appropriate dose metric need not cons titute measurement of only one physicochemical property (such as surface area, mass, or number of particles). Although dose metrics based on one property, such as mass concentration, have been us ed successfully in toxicology, a combination of measurements of two or more physicochemical pr operties also might be appropriate for use in assessing nanomaterial toxicity. Recently, the OECD developed a guidance document on sample preparation methods and dosimetry for safety testing with nanomaterials (OECD, 2010, 644192 ). Total particle surface area, which is closely rela ted to primary particle size, has been suggested as a suitable dose metric for inhalation and instillation studies (Faux et al., 2003, 625074 ; Liao et al., 2009, 157456 ; Oberdörster et al., 2005, 087559 ). Although two distinctive dose-response curves for
fine TiO 2 

### and nano-TiO 2 

### can be drawn based on mass concentr ation, certain observed respiratory
effects of fine TiO 2 

### and nano-TiO 2 

### have been shown to fit well with a single linear dose-response curve based on primary particle surface area, even where both types of particles agglomerated to approximately 0.7 μm in diameter (Oberdörster et al., 1994, 046203 ). Hext et al. (2005, 090567 ) found that, compared to gravimetric lung burden (particle mass per lung mass), administered primary particle surface area correlated better with lung bu rdens, clearance half-lives, and certain biological responses in rats, mice, and hamsters. However, the evidence on this issue is somewhat mixed. For instance, biological responses after exposure to similarly-sized agglomerates of fine TiO 2 

### and
nano-TiO 2 

### were similar in severity according to Warheit et al. (2006, 088436 ; 2007, 091305 ); by contrast, Sager and Castranova (2009, 193625 ) found that well-dispersed nano-TiO 2 

### yielded greater effects than well-dispersed fine TiO 2



### . As mentioned previously, any one or more of various characteristics, including particle number, size (including a gglomerations or aggregati ons), shape, crystalline form, mass, surface area, and surface modifications, could play a role in nano-TiO 2 

### toxicity. Including one or more of these
factors in the dose metric could be a better choi ce than surface area alone. For instance, based on administered primary particle surface area, th e data used in the Hext et al. study (2005, 090567 ) – the increases in the numbers of pulmonary polymorphonuclear neutrophil (PMN) due to exposure to
anatase fine and nano-TiO 2 

### (Oberdörster et al., 1994, 046203 ) and rutile fine TiO 2 

### (Cullen et al., 1999, 157905 ) – would better fit two dose-response curves (one each for anatase TiO 2 

### and rutile
TiO 2 

### ), instead of one dose-response curve. Simila rly, a recent study of pulmonary effects of
intratracheal instilled rutile fine TiO 2 

### and 80% anatase/20% rutile nano-TiO 2 

### (Sager et al., 2008,
157499 ) showed that when dose was normalized to surfa ce area of the particles administered, the

4-26

### 4-27 

### dose-response curves for inflammogenic responses were not statistically different between fine and
nano-TiO 2 

### , but the anatase-rutile nano-TiO 2 

### always yielded greater (1.3- to 2-fold) responses than the
rutile fine TiO 2



### . Due to limited toxicological data from oral or dermal exposure to nano-TiO 2 

### , the choice of dose metric for these exposure routes has not be en widely discussed. For in vitro studies, nanoparticle concentration (mass or surface area) is often used to express dose. In vitro cytotoxicity, however, has been reported to be affected by both the concentration and the total mass (or total number or total surface area, since these three are closely related) of nanoparticles (Lison et al., 2008, 157530 ). In the Lison et al. study (2008, 157530 ), when cells were cultured in various volumes of a medium containing the same amount of na no-silica (same mass/number/surface area), higher toxicity occurred in a lower volume of medium, th at is, in higher nano-silica concentrations. When
the medium contained the same concentrations of nano-silica, higher toxicity occurred in cells cultured with a higher volume of medium than lower volume of medium.

# Chapter 5. Characterization of Effects

The preceding chapters have laid a foundation for the present chapter by providing an exposure context for characterizing the effects of nano-TiO 2 

### used for drinking water treatment and in topical sunscreens. This chapter provides info rmation on the factors that influence nano-TiO 2 

### ecological and health effects (Section 5.1), the ecological effects of nano-TiO 2 

### (Section 5.2), and the toxicological and human health effects of nano-TiO 2 

### (Section 5.3). Whether there are specific byproducts (e.g., waste and transformation products) or interactions with other substances that should or can be evaluated has not yet been determined. For this reason, the focus of this chapter is on
nano-TiO 2



### . Although literature exists on the effects of conventional TiO 2 

### on humans and laboratory animals (NIOSH, 2005, 196072 ), comparatively less information is available on the effects of
nano-TiO 2 

### . Consistent with studies of other na nomaterials (Ostrowski et al., 2009, 193592 ), most
nano-TiO 2 

### studies have investigated the ecological or health effects of nano-TiO 2 

### itself, and relatively few have investigated the ecological or health effects of end-use products containing
nano-TiO 2 

### or their life-cycle by-products. The physicochemical characteristics of nano-TiO 2 

### could be important to the biological effects of these materials (Section 5.1), yet those characte ristics frequently are not evaluated or reported as part of studies of such effects. This observa tion should serve as a caveat in examining and interpreting the results described throughout this chapter. The following sections are not meant to be an exhaustive review of the ecological and human
health effects literature for nano-TiO 2 

### . Instead, this chapter is intended to highlight recent work on
the effects of nano-TiO 2 

### and to identify current knowledge status and gaps in information needed for assessing potential risks of nano-TiO 2 

### in water treatment and sunscreen.


# 5.1. Factors that Influence Ecological and Health Effects of Nano-TiO 2

The large number of variables associated with nano-TiO 2 

### material itself and its ecological and health effects makes it extremely difficult to identify the primary characteristic(s) of nano-TiO 2 

### contributing to an effect or to compare the importance of different characteristics to such effects. A common statement from early studies is the announcement of size effects (or the lack of size effects)
from nano-TiO 2 

### of different crystalline forms or anatase/rutile ratios. That size alone does not
account for the effects of nano-TiO 2 

### , however, is now generally accep ted; other factors, such as shape, surface chemistry, photoreactivity, and other characteristics, could also play a role in these effects (Gonzalez et al., 2008, 157569 ; Hassellöv et al., 2008, 157559 ; Powers et al., 2006, 088783 ). With the advance of nanoparticle synthesis, the influence of different physicochemical
characteristics of nano-TiO 2 

### has been investigated using well-characterized nano-TiO 2 

### and better control of variables in recent studies (Jiang et al., 2008, 156609 ). Three categories of factors (nano-TiO 2 

### physicochemical characteristics, experimental conditions, and environmental conditions) that coul d influence the ecological and toxicological or
health effects of nano-TiO 2 

### are discussed here in Section 5.1. These are not the only factors of potential importance. As noted previously, exposure route can play a major role in the effects of
nano-TiO 2 

### , and the importance of this is reflected in th e fact that much of the information in this

Note: Hyperlinks to the reference citations throughout this doc ument will take you to the NCEA HERO database (Health and Environmental Research Online) at http://epa.gov/hero . HERO is a database of scientific litera ture used by U.S. EPA in the process of developing science assessments.



### 5-1

### chapter is organized around environmental matrices a nd routes of exposure. Host effects, particularly species differences, can also play an important role in the effects of nano-TiO 2 

### . For example, skin penetration is greatest in rabbits, followed by rats, pigs, monkeys, and humans (Nohynek et al., 2007, 090619 ). However, little information is available on these species differences or on differences in susceptibility of different cell types to nano-TiO 2 

### effects (Kiss et al., 2008, 157547 ). The phenomenon of pulmonary particle clearance “overloa d” and subsequent effects in rats and mice are
much more understood and are discussed in Section 5. 3.1.2. In the following sections, the order in which factors are presented does not imply relative importance. This section focuses on factors that have been shown to be germane to nano-TiO 2 

### , but findings related to other types of nanomaterials
are noted where relevant.



# 5.1.1. Nano-TiO 2 

# Physicochemical Characteristics

Size, crystal structure, and surface chemistry (s uch as coating) are among the factors that
influence nano-TiO 2 

### effects. Other physicochemical proper ties, such as shape (Warheit et al., 2006,
088436 ; Yamamoto et al., 2004, 157820 ), manufacturing process, dopi ng, and purity (or impurities) could also play a role in nano-TiO 2 

### toxicity, but such information is usually not reported in ecological and toxicological studies. Contributing to this lack of reported characteristics are limitations in the availability of analytic met hods for characterizing such nanomaterials. Databases describing detailed nanoparticle properties and health effects are being developed (Miller et al., 2007, 092297 ). The need for characterization of nanomaterials used in toxicity studies has been noted in reports and journal articles, with possible attributes for minimal characterization including chemical composition, size and size distribution (for primary particles and agglomerates), shape, specific surface area, and number of particles per unit mass (Department for, 2007, 195461 ; Powers et al., 2006, 088783 ; Powers et al., 2007, 090679 ; Warheit et al., 2007, 091075 ). For more information on nanomaterial physiochemical characteristics that c ould affect ecological and toxicological effects, readers are referred to reports listing recommende d information to be included in nanomaterial studies (OECD, 2008, 157512 ; Attachment 5 to Appendix D of Taylor, 2008, 157487 ; Warheit et al., 2007, 091305 ). A compilation of characterization reco mmendations from a multi-stakeholder group
can also be accessed at http://characterizationmatters.org

.



# 5.1.1.1. Size

Size is a main determining factor for the distribution of (inhaled or instilled) nano-TiO 2 

### in and outside of the respiratory tract (Oberdörster et al., 2004, 055639 ). For particles with a diameter less than 100 nm, the smaller the particles are, the more total particle deposition in the respiratory tract and deposition in nasopharyngolaryngeal regions (Oberdörster, 2000, 036303 ). Smaller sizes, however, do not always result in more deposition in other regions of the respiratory tract. For example, the highest percentages of alveolar deposition have been observed in nanoparticles of approximately 20 nm in size, and the highest percentages of tracheobronchial deposition were observed in nanoparticles 1-10 nm in size (Oberdörster, 2000, 036303 ). Furthermore, particles less than 200 nm in size can be transported from olfactor y mucosa to the olfactory bulb of the brain via the olfactory nerve (Elder et al., 2006, 089253 ). Exposures to nano-TiO 2 

### (with mean diameters of 21.05 ± 5.08 nm, 71.43 ± 23.53 nm, and 154.98 ± 32. 98 nm) through intranasal instillation increased Ti concentrations in the olfactory bulb in mice (Wang et al., 2005, 193703 ; Wang et al., 2007,
090290 ), and two types of nano-TiO 2 

### particles (80-nm rutile and 155-nm anatase) were found to increase Ti concentrations in hippocampus, central cortex, and cerebrum, in addition to olfactory bulb, in mice after repeated intranasal instillation (Wang et al., 2008, 157473 ). Jiang et al. (2008, 156609 ) investigated the size effects of nano-TiO 2 

### on the generation of ROS per unit of particle surface area. Using nine different sizes (4-195 nm) of anatase nano-TiO 2 

### , the

5-2

### investigators found that the highest levels of ROS generation per unit surface area were generated by 30 nm and larger particles. For nano-TiO 2 

### less than 30 nm, the ROS generation per surface area decreased with decreasing particle diameter down to 10 nm, below which it was constant (Jiang et al., 2008, 156609 ).



# 5.1.1.2. Crystallinity

TiO 2 

### crystalline forms also influence TiO 2 

### and nano-TiO 2 

### photoreactivity, reactive species generation, and toxicity. Nano-TiO 2 

### containing more anatase tends to generate more free radicals and induce more toxicity (e.g., cytotoxicity, inflammatory response) than nano-TiO 2 

### containing ore rutile (Hidaka et al., 2005, 157804 ; Sayes et al., 2006, 090569 ; Uchino et al., 2002, 090568 ). The influence of crystal forms of nano-TiO 2 

### on ROS generation was investigated using a fixed total surface area by Jiang et al. (2008, 156609 ), who tested 13 nano-TiO 2 

### particles of varying crystallinity, all within the size range of 42 to 102 nm. The researchers found that the ROS generation per unit surface area was highest in amorphous nano-TiO 2 

### , followed by anatase and then nano-TiO 2 

### containing both anatase and rutile, and was lowest in rutile nano-TiO 2 

### (Jiang et al., 2008, 156609 ). This finding is consistent with those of a study investigating unusually fast weathering (loss of gloss) or degradation of surface coating on steel roofing, associated with sunscreen s left by workers during installation (Barker and Branch, 2008, 180141 ). Nano-TiO 2 

### in the coating-damaging sunscreens was an anatase/rutile mixture, whereas nano-TiO 2 

### in the one sunscreen that did not accelerate loss of gloss was pure rutile (Barker and Branch, 2008, 180141 ). The cytotoxicity of anatase and anatase-mixtur es was further increased by UV illumination.
Anatase nano-TiO 2 

### can be 100 times more cytotoxic under UV than rutile of similar size (Sayes et al., 2006, 090569 ). The hydroxyl (·OH) radical production by nano-TiO 2 

### in cultured cells was found to depend on the crystalline form and size, but differences in OH radical production were not explained by the differences in UV-A absorption between anatase and rutile (Uchino et al., 2002,
090568 ). Smaller particles that contain more anatase, however, are not always more toxic either in vitro (Sayes et al., 2006, 090569 ) or in vivo (Warheit et al., 2006, 088436 ) than larger particles containing more rutile.



# 5.1.1.3. Surface Chemistry

Although coatings have been used to d ecrease the photoreactivity of nano-TiO 2 

### intended for sunscreen (Section 2.2.2), coatings affect more than photoreactivity. In particular, the presence of a surface coating changes the nature of the interface between the nano-TiO 2 

### particle and the environment or an organism, and it is not clear whether the surface coating or the core material dominates particle-environment and particle-o rganism interactions. Coatings for nano-TiO 2 

### particles can be designed to reduce agglomeration/aggregati on, which in turn affects the behavior of the particles in various matrices, including sediment ation behavior. This also affects the exposure conditions of organisms living in different parts of bodies of water or feeding on different sized particles. Particle surface modifications can also change the effects of nano-TiO 2 

### on living cells, tissues, or organisms. Using in vitro methods, Serpone et al. (2006, 157736 ) reported that a “thermally assisted” modification of the TiO 2 

### particle surface reduced photocatalytic activity, which in turn decreased (if not eliminated) toxicity to DNA plasmid, human cells, and yeast. In rats intratracheally instilled with two types of nano-TiO 2 

### having the same core material, the nano-TiO 2 

### with a hydrophobic surface (Aeroxide ® 

### T805, silanized) caused a slightly lower bioactivity than

5-31 

### hydrophilic P25, although the author s concluded that silanization did not “lead to remarkable differences in lung reaction” (Rehn et al., 2003, 090613 ).



# 5.1.1.4. Recommended Characterization of Nanomaterial for Ecological and Toxicological Studies

As noted in Chapter 1, nanomaterials, and nano-TiO 2 

### in particular, can be characterized in several ways in terms of physicochemical properties (Table 1-1). Given that the relationship between such properties and the behavior and effects of nanomaterials, including nano-TiO 2 

### , remains to be fully understood, it might seem desirable for researchers to characterize every possible property of the material they are investigating. In practice, this is not feasible. Consequently, recommendations for characterization of nanomaterials have periodically been made. For in vitro studies, Murdock et al. (2008, 193563 ) recommended characterizing nanomaterial dispersion in solution for (in no specific order) particle size and size distribution; particle morphology; particle composition; surface area; surface chemistry; particle reactivity; agglomeration; zeta potential; and impact of soni cation. For human and environmental testing, a roundtable discussion at the 2005 Society of Toxicology Annual Meeting considered the following as essential parameters in nanomaterial physicochemical properties: size distribution, agglomeration state, crystalline structure, chemical composi tion, and shape (Holsapple and Lehman-McKeeman, 2005, 088087 ). For “hazard studies with nanoparticle-types,” Warheit (2008, 193706 ) prioritized the characterization needs as (highest priority first): (1 ) particle size and size distribution (wet state) and surface area (dry state) in the relevant media; (2 ) crystal structure/crystallinity; (3) aggregation status in the relevant media; (4) composition and surfa ce coatings; (5) surface reactivity; (6) method of nanomaterial synthesis and/or preparation; and (7) purity of sample. An expert working group convened by the In ternational Life Sciences Institute (ILSI)
Research Foundation/Risk Science Institute recomme nded both off-line (i.e., not using time-resolved continuous techniques) and on-line (continuous) mea surement of mass, size distribution, surface area, and particle number for exposure characterizat ion in inhalation studies (Table 5-1), and 17 offline measurements/aspects for nanomaterial characterization for toxicological studies (Table 5-2) (Oberdörster et al., 2005, 090087 ).

1 Silanization is the covering of a surface that has hydroxyl (OH) groups with molecules that contain only silicon and hydrogen (silane),
such as SiH 4 . Silanization is one type of surface modification applicable to particles, such as metal oxides, and can render the particle surface chemically inert.

5-4

## Table 5-1. Published recommendations for m easuring nanomaterial parameters for exposure during characterizati on inhalation studies

Recommendation Metric Measurement Off-line (Discrete) a On-line (Continuous) b

Mass E (coupled with on-line) E

Size distribution E E/D

Surface area O O

Particle Number N E

a Off-line: Collected and analyzed later.

b On-line: Real-time collection and analysis during the process.

E – These measurements are considered to be essential. D – These measurements are considered to provide valuable info rmation, but are not recommended as essential due to constraints associated with complexity, cost and availability. O – These measurements are considered to provide valuable but nonessential exposure information.
N – These measurements are not considered to be of significant value to inhalation studies.

Source: Modified with permission from Bi oMed Central, Oberdörster et al. (2005, 090087 ).

5-5

## Table 5-2. Published recommendations for of f-line nanomaterial ch aracterization using noncontinuous techniques for to xicological studies

Toxicity Screening Studies

Characterization Human Exposure Supplied Material Material as Administered Material after Administration (in vivo/in vitro)

E (combine with agglomeration state) Size distribution (primary particles) E D D

Shape E E O O

Surface area D E D O

Composition E E O O

Surface chemistry D E D D/O

Surface contamination D N D N

Surface charge – suspension/solution O E E O

Surface charge – powder (use bio fluid surrogate) O E N O

Crystal structure O E O O

Particle physicochemical structure E E D D

Agglomeration state a E N E D

Porosity D D N N

Method of production E E -- -- Preparation process -- -- E -- Heterogeneity b D E E D

Prior storage c of material E E E --

Concentration E -- E D

E – These characterizations are considered to be essential.
D – These characterizations are considered to provide valuable information, but are not recommended as essential due to constra ints associated with complexity, cost and availability. O – These characterizations ar e considered to provide valuabl e but nonessential information.
N – These characterizations are not considered to be of significant value to screening studies.

a As primary particle, secondary particle (p rimary particle agglomerates and self-assemb led structures) and te rtiary structure (a ssemblies of secondary structures). When possible, material aggl omeration or de-agglomeration in different liquid media should also be characterized.

b Time and conditions, including temperature, humidity, exposure to light and atmosphere composition.

c Ratios of different components.

Source: Reprinted with permission from Bi oMed Central, Oberdörster et al. (2005, 090087 ).



### Three factors figured into these recommendations: “the context within which a material is being evaluated, the importance of measuring a sp ecific parameter within that context, and the feasibility of measuring the parameter within a specific context” (Oberdörster et al., 2005, 090087 ).



# 5.1.2. Experimental Conditions

Experimental conditions, particularly the choice of medium/vehicle in which to disperse
nano-TiO 2 

### , preparation of testing solutions or suspensions, and the formation of aggregates , can influence the behavior and effects of nano-TiO 2 

### and other nanomaterials. For example, instability of nanoparticle suspensions may cause particle settling during the experiment, leading to the effective concentration being either higher or lower than th e initial added concentration. This can result in

5-6

### improper interpretation of the dose-response relati onship. The advantages and disadvantages of various dispersion preparation methods are compar ed in a recent publication of nanomaterial ecotoxicity test methods (Crane et al., 2008, 157583 ).



# 5.1.2.1. Medium/Vehicle

Nano-TiO 2 

### in an oily dispersion penetrates deeper into skin than nano-TiO 2 

### in an aqueous dispersion, as shown in an ex vivo study using heal thy adult skin (intact samples of tissue removed from the body, and manipulated in vitr o) (Bennat and Muller-Goymann, 2000, 157403 ). Nano-TiO 2 

### did not penetrate into living cells of the skin in either aqueous dispersion or oily dispersion. When the dispersal of nano-TiO 2 

### was made in the aqueous phase of an oil-in-water emulsion, nano-TiO 2 

### did not penetrate into skin, but the emulsion was not stable. Although the stability could be improved by encapsuling the nano-TiO 2 

### into liposomes, liposome formulation increases nano-TiO 2 

### skin penetration (Bennat and Muller-Goymann, 2000, 157403 ). In another relevant phenomenon, the nanoparticles by themselves can act as a dispersan t, forming so called Pickering emulsions and essentially acting as the surfactant that helps make liposomes. An in vivo study (Lansdown and Taylor, 1997, 157928 ) in rabbits also demonstrated that uptake of TiO 2 

### particles in sizes ranging
from 2 to 20 μ m was affected by the vehicle: uptake was greatest in castor oil, followed by water, and then polyethylene glycol. Different levels of free radical production were observed in cultured cells exposed to similar
nano-TiO 2 

### but within different formulae of suspensions (Uchino et al., 2002, 090568 ). Although
nano-TiO 2 

### F-1R (a formula containing nano-TiO 2 

### that is 3% anatase and 97% rutile, with an average
size of 93 nm and a surface area of 17 m 2 

### /g) produced OH radicals after UV-A exposure, no OH radical production was detected after UV-A exposure in nano-TiO 2 

### in a different formula, St-C (sunscreen standard C from the Japan Cosm etic Industry Association containing nano-TiO 2 

### that is 2% anatase, 98% rutile, with an average size of 85 nm and a surface area of 19 m 2 

### /g). Most rutile
nano-TiO 2 

### is relatively inefficient in free radical production, and the F-1R used in this study produced more OH radicals than all four other, mainly rutile nano-TiO 2 

### forms and one of the anatase forms tested (Uchino et al., 2002, 090568 ). Although nano-TiO 2 

### has been reported to generate ROS in cell-free conditions but not in cells (a murine macrophage cell line, RAW 264.7) (Xia et al., 2006,
089620 ), whether nano-TiO 2 

### in different formulae also causes different levels of ROS production in
cells has not been verified. The purity of water affects the degree of aggreg ation, which in turn may affect exposure-dose and toxicity. The degree of aggregation generally incr eases with an increase in ionic strength, and the extent of aggregation can also be affected by th e presence of NOM and other constituents such as divalent cations (Domingos et al., 2009, 193347 ; French et al., 2009, 193384 ; Kim et al., 2009,
635778 ). Aggregation was more severe in tap water than in nanopure water (Zhang et al., 2008,
157462 ), and is likely to be more severe in fish tank water or pond water than in tap water. Because
nano-TiO 2 

### in the environment is more likely to be present in aggregated form, results from
nano-TiO 2 

### suspensions with aggregates can be informative, and as noted earlier, might even be more relevant than results from a perfectly dispersed suspension with nano-TiO 2 

### in primary particle form.
The lack of accurate measurement of nano-TiO 2 

### (e.g., size distribution, mass concentrations, numbers, and surface area) and a generally-agreed- upon choice of dose metrics, however, impede the establishment of a reliable dose-response relationship. In respiratory exposure studies, intratracheal instillation exposure typically uses saline as a
vehicle for TiO 2 

### delivery while inhalation exposure uses air. The behavior of nano-TiO 2 

### (such as agglomeration) is expected to be different in air than in solution. Furthermore, the vehicle alone can affect respiratory system responses, at least fo r a short time. Transient inflammation in the respiratory tract occurs in rats given saline alone through instillation (Driscoll et al., 1990, 087145 ; Henderson et al., 1995, 002744 ). Sager et al. (2007, 091214 ) tried to disperse several types of nanosized particles, including TiO 2 

### , in several suspension media, including: PBS; rat and mouse BAL

5-7

### fluid; and PBS containing DPPC or mouse serum albumin or both. Although the dispersion in PBS was not satisfactory, BAL fluid was an excellent ve hicle for dispersing the particles. The dispersion was also unsatisfactory in saline containing albumi n alone or DPPC alone, in concentrations found in BAL fluid. Adding protein plus DPPC in PBS, howeve r, produced satisfactory, albeit slightly less effective, substitutes for BAL fluid. The Sager et al. (2007, 091214 ) experiment demonstrates the importance of the suspension medium and suggests th at the immediate milieu (such as the BAL fluid and protein and DPPC in lung) affects not only the agglomeration of nano-TiO 2 

### , but also the consequent effects on nano-TiO 2 

### behavior and effects.



# 5.1.2.2. Dispersion Preparation

The potential importance of dispersion preparati on for nanomaterial ecotoxicity is illustrated by fullerene (C 60 

### ) studies. C 60 

### toxicity in daphnids and fishes was higher when the C 60 

### suspension was prepared with the organic solvent tetrahydrofuran (THF) than when the suspension was prepared by stirring and sonication (Henry et al., 2007, 157684 ; Zhu et al., 2006, 157721 ). Entrapped or
residual THF in the C 60 

### and THF degradation products were suspected to have contributed to toxicity (Henry et al., 2007, 157684 ). Nevertheless, no difference in toxicity to daphnids was observed
between nano-TiO 2 

### suspensions prepared with and without THF (Klaper, personal communication, 2008, 157546 ; Lovern and Klaper, 2006, 088040 ). Regardless of dispersion method, aggregation of
nano-TiO 2 

### might be unavoidable. Several studies reported that nano-TiO 2 

### formed aggregates or agglomerates in water, and that these aggregates /agglomerates could not be separated into primary particles by ultrasound or chemical dispersants (Griffitt et al., 2008, 157565 ; Jeng and Swanson, 2006, 090085 ; Zhang et al., 2008, 157462 ). Furthermore, an unfiltered nano-TiO 2 

### suspension with aggregates/agglomerates has been reported to be less toxic to daphnia than a filtered nano-TiO 2 

### suspension that mostly contained primary particles (Lovern and Klaper, 2006, 088040 ). In contrast to Lovern and Klaper’s (2006, 088040 ) reported difficulty of disagglomerating particles by sonication or chemical dispersants, Federici et al. (2007, 091222 ) reported effective dispersion of P25 by sonication in ultrapure water at final working co ncentrations up to 1 mg/L, although they did not evaluate potential agglomeration in te st tank water at these concentrations. In addition to the medium itself, the dispers ion method can affect not only the nanoparticle agglomeration or aggregation (such as the degree and size of agglomerates) but also the effects of nanoparticles (Bihari et al., 2008, 157593 ). For example, sonication with ultrasound has been used to
decrease nano-TiO 2 

### agglomeration (Bihari et al., 2008, 157593 ) and has been shown to generate particles or agglomerates in the na noparticle range (Maier et al., 2006, 090451 ). However, sonication
alone could increase the size of nano-TiO 2 

### agglomerates, as reported by Porter et al. (2008, 157508 ) who found that the mean agglomerate size of P2 5 in PBS increased from 1,930 nm before sonication to 2,849 nm immediately after sonication, while th e same sonication procedure decreased the sizes of agglomerates of P25 dispersed in BAL fluid and in a mimic BAL fluid that contained Ca 2+



### - and Mg 2+ 

### -free PBS, serum albumin, and DPPC. No explanation was provided. Furthermore, ultrasound sonication has been reported to increase nano-TiO 2 

### catalytic activity in breaking down an organic dye (acid red B) (Wang et al., 2009, 157453 ), but also to decrease changes in enzyme activity caused by ingested nano-TiO 2 

### in isopods (Jemec et al., 2008, 157552 ). Post-preparation analysis of particle size is important when comp aring laboratory studies and formulations with sunscreen preparations. Al though studies of nano-TiO 2 

### particle and agglomerate sizes are available (Delrieu et al., 2007, 157449 ), very few health effects studies have characterized nano-TiO 2 

### in sunscreen formulations and only a few studies characterized nano-TiO 2 

### in other experimental mediums. Most health effects studies have reported characteristics of only dry nano-TiO 2 

### primary particles, which are important but not representative of all exposure scenarios. Finally, without a special hydrophilic coating, nano-TiO 2 

### forms a suspension in water (rather than a solution). Standard ecotoxicological test methods are intended for soluble or poorly soluble substances, and not designed for testing suspensions (BAuA, 2007, 157694 ).

5-8

# 5.1.3. Environmental Conditions

Once nano-TiO 2 

### is released into the environment, its fate depends on abiotic and biotic conditions, which are likely to be more complex and diverse than standard ecological testing conditions. Of the many environmental factor s that might be relevant to nano-TiO 2 

### ecotoxicity, UV light exposure, purity of water (Zhang et al., 2008, 157462 ), and presence of organic matter (Domingos et al., 2009, 193347 ) have been investigated. Factors that affect nano-TiO 2 

### aggregation, such as pH value, ionic strength, and cation valence (Domingos et al., 2009, 193347 ; Dunphy Guzman et al., 2006, 090584 ; French et al., 2009, 193384 ), would influence not only
nano-TiO 2 

### fate and transport (Chapter 3), but also pote ntial exposure and possibly ecological effects. Only environmental factors that have been shown to affect toxicity in organisms used for ecological effects testing are discussed here. UV light is well known to increas e the cytotoxicity of nano-TiO 2 

### , particularly photocatalytic
nano-TiO 2 

### such as anatase or anatase/rutile mix, to cultured mammalian cells (Sayes et al., 2006,
090569 ) and fish cells (Reeves et al., 2008, 157506 ; Vevers and Jha, 2008, 157475 ) as well as microorganisms (Adams et al., 2006, 157782 ). Genotoxicity (Nakagawa et al., 1997, 157927 ) and clastogenicity (Nakagawa et al., 1997, 157927 ) of nano-TiO 2 

### to cultured mammalians cells were also increased by UV exposure. This UV-increased toxicity is at least partially due to the greater number of hydroxyl radicals (·OH) generated by anatase than by rutile under UV exposure (Sayes et al., 2006, 090569 ; Uchino et al., 2002, 090568 ). UV exposure may influence the effects of nano-TiO 2 

### in sunscreen indirectly by causing sunburn, which can ma ke skin more permeable (Mortensen et al., 2008, 155612 ). In addition to UV, visible light was shown to increase the cytotoxicity of nano-TiO 2 

### (carbon-doped TiO 2 

### and TiO 2 

### modified with platinum [IV] chlori de complexes) in bacteria and fungi (Mitoraj et al., 2007, 157665 ).
Nano-TiO 2 

### was found to form aggregates more in pond water than in pure water (MilliQ water), although no nano-TiO 2 

### toxicity to soil bacteria, green algae, or water fleas was detected in either pond water or pure water at up to 100 mg/L (Velzeboer et al., 2008, 157476 ). The adsorption
of nano-TiO 2 

### onto certified reference material sediment di d not increase the toxicity of the sediment (Blaise et al., 2008, 157592 ). Additional environmental factors that might indirectly influence the effects of TiO 2 

### nanoparticles in sunscreen include moisture; pH and water chemistry; and temperature. High humidity in the environment could increase the hydration level of the stratum corneum, and could lead to increases in skin permeability and penetr ation of both hydrophilic and lipophilic components (Benson, 2005, 193273 ; Zimmerer et al., 1986, 193744 ). For example, the level of penetration of
nano-TiO 2 

### on soaked skin, which is likely to occur af ter swimming or other water activities, has not been investigated. Similar to medium and vehicle effects on nano-TiO 2 

### , the pH and chemistry of the water in which sunscreen may be mixed, e.g., in a swimming pool versus a lake or an ocean, might
also modulate nano-TiO 2 

### effects. Finally, sunscreen is often used at much higher temperatures than typical ambient laboratory te mperatures. Although nano-TiO 2 

### itself is not expected to change in the temperature range tolerable for human beings, in creased body temperature and sweat may affect
nano-TiO 2 

### dermal penetration and thus its effects (Lu et al., 2008, 157526 ).
The influence of the immediate milieu on nano-TiO 2 

### behavior and effects is also evident when
nano-TiO 2 

### is inside an organism. For instance, in vi tro studies showed that in rat BAL, nano-TiO 2 

### formed smaller aggregates and the aggreg ates remained small longer than nano-TiO 2 

### in PBS (Porter et al., 2008, 157508 ; Sager et al., 2007, 091214 ; Sager et al., 2007, 090633 ). Because pH affects the electrostatic charge of nano-TiO 2 

### , it is plausible that nano-TiO 2 

### would behave differently in tissues and cellular organelles with different pH values, such as very acidic pH values in the stomach and in lysosomes.

5-9

# 5.1.4. Summary

Nano-TiO 2 

### physicochemical properties, experimental conditions, and the immediate environment or milieu, all can influence nano-TiO 2 

### ecological and health effects. For example,
nano-TiO 2 

### size, crystalline form, and surface characteristics all influence nano-TiO 2 

### behavior, including distribution, exposure potential, and effects. Although the influences of media, vehicles, and dispersion methods on particle aggregation and distribution have been reported, information on
these influences on health effects is very scarce (Jemec et al., 2008, 157552 ). The presence of UV and visible light often increase photocatalytic nano-TiO 2 

### activity and toxicity; other environmental factors, such as pH, ionic strength, and presence of organic matter of the aquatic environment, could
also affect nano-TiO 2 

### behavior and effects.



# 5.2. Ecological Effects

The ecological effects of nanomaterials have been gaining attention from the research and regulatory communities, and several review articles (Baun et al., 2008, 157598 ; Christian et al., 2008, 157586 ; Hassellöv et al., 2008, 157559 ; Navarro et al., 2008, 157517 ; Nowack and Bucheli, 2007, 092294 ) and conferences (such as the annual International Conference on the Environmental Effects of Nanoparticles and Nanomaterials) have addressed this topic. Although new information on nanomaterial ecotoxicity seems to emerge almost daily, available data thus far have been insufficient for a quantitative risk assessment of any particul ar nanomaterial. A thorough discussion of methods for ecotoxicity testing and characterization of nano materials (including in environmental media) is beyond the scope of these case studies, and has been reviewed elsewhere in peer-reviewed articles (Christian et al., 2008, 157586 ; Crane et al., 2008, 157583 ; Handy et al., 2008, 157562 ; Hassellöv et al., 2008, 157559 ) and in several public databases, such as those sponsored by the OECD (International Council on Nanotechnology, 2010, 644440 ; OECD, 2009, 644433 ; Project on Emerging Nanotechnologies, 2010, 644439 ). Nonetheless, a brief review of ecological effects testing and the importance of the tests are presented at the beginning of each of the following
sections for the readers’ reference.
Section 5.2.1 features a review of the ecological effects of nano-TiO 2 

### exposure. Effects on bacteria and fungi are discussed in Section 5.2.1. 1, effects on aquatic organisms are discussed in Section 5.2.1.2, effects on terrestrial organisms are discussed in Section 5.2.1.3, and indirect and interactive toxicity are discussed in Section 5.2.1.4. Section 5.2.1.5 summarizes the available ecological toxicity information.



# 5.2.1. Ecological Effects of Nano-TiO 2 

# Exposure

Most of the nano-TiO 2 

### ecological effect studies surveyed in this report (Table 5-3) used photocatalytic nano-TiO 2 

### , some of which could be suitable for water treatment purposes. Two of the studies used photostable nano-TiO 2 

### intended for topical sunscreen (Wiench et al., 2007, 090635 ) and for protecting plastic from UV degradation (Warheit et al., 2007, 091075 ). Current FDA regulation
of TiO 2 

### in topical sunscreen does not specify cryst alline form and does not require proof of photostability (or lack of photoreactivity). Pure anatase nano-TiO 2 

### is much more photoreactive than pure rutile nano-TiO 2 

### , but it is possible to have photostable anatase or an anatase/rutile mix of
nano-TiO 2 

### by using doping or surface treatments, such as coating with silica. The coating of photostable nano-TiO 2 

### is designed to endure the manufacturing process and consumer use (Lademann et al., 2000, 157895 ), but the long-term stability of coated TiO 2 

### in sunscreen remains
unclear. Once nano-TiO 2 

### is released into the environment, various environmental factors, such as high ionic strength in sea water and high acidity in landfill leachate, could compromise some

5-10

### nano-TiO 2 

### coatings. In a study presented in a professional conference, nano-TiO 2 

### from a commercially available sunscreen has an outer hydrophobic polydimethylsiloxane coating and an intermediate AlOOH coating, and the coating was gradually degraded in water, as evidenced in one study by altered dispersion and release of silicon and Al from the coated nano-TiO 2 

### product (Botta et al., 2009, 625076 ). Additionally, the by-products penetrated into aquatic organisms and were toxic at high concentrations (Botta et al., 2009, 625076 ). Therefore, the ecological effects of photocatalytic
nano-TiO 2 

### might be relevant not only for nano-TiO 2 

### used in drinking water treatment but also for
nano-TiO 2 

### in sunscreen, because photoreactive nano-TiO 2 

### can be used as the core material of photostable nano-TiO 2 

### in sunscreen. For example, the core of Aeroxide ® 

### T805 is P25, a photocatalyst, and has been used as a UV filt er in some sunscreens (Barker and Branch, 2008,
180141 ; Evonik, 2007, 157696 ). Because mass concentration is reported for all studies reviewed, this dose metric is presented in Table 5-3 and in all subsequent discussion referr ing to the literature. Whenever information on surface area of the particles (to calculate particle surface area concentration) or the measured
nano-TiO 2 

### concentration (versus calculated based on added mass) present in the final test suspension was available, it is also provided in Table 5-3. The environmental relevance of the tested
concentrations is unclear due to limited information on nano-TiO 2 

### concentrations in the environment, although even the use of elevated concentrations in laboratory studies is informative in demonstrating the potential for an effect. It should be noted that several studies reported visible turbidity in nano-TiO 2 

### stock suspension (Velzeboer et al., 2008, 157476 ; Zhang et al., 2006, 157722 ; Zhang et al., 2008, 157462 ). Because turbidity is likely caused by large aggregates of nano-TiO 2 

### , which can settle out of the liquid phase by gravity, actual concentrations of nano-TiO 2 

### in the liquid phase might be lower than concentrations calculated based on mass of nano-TiO 2 

### added.



## Table 5-3. Summary of nano-TiO 2 

## ecological effects

Protocol (No UV illumination, unless specified) Test Species (Reference) Material Study Outcome

Acute Exposure to Microorganisms

Bacteria ( Escherichia coli and Bacillus subtilis ) (Adams et al., 2006, In dark, similar growth inhibition for both bacteria 66-nm powder, ~35% rutile:65% anatase, average 330-nm in water (Sigma product 634662) (Lyon, personal communication, 2008, 6-hr exposure to: (1) 50, 100, 500, 1,000, 2,000, 5,000 ppm in medium a , in direct sunlight; or (2) 1,000 ppm in medium a , in dark In light, B. subtilis : 0%, 75%, and 99% growth inhibition at 500, 1,000, and 2,000 ppm, respectively 157782 ) 157524 )

E. coli : 0%, 15%, 44%, and 46% inhibition at 100, 500, 1,000, and 2,000 ppm, respectively

Bacterium ( Vibrio fischeri ) (Blaise et al., 2008, IC 25 >100 mg/L <100-nm powder (Sigma product 634662, Canada or France) 15-min exposure, measur e the reduction of light output from bioluminescent marine bacterium, Vibrio fischeri (Microtox® toxicity test) as an indicator of growth inhibition, tested concentrations not specified 157592 )

Mix in a 1:1 ratio with certified reference material sediment, measure light output (Microtox® toxicity test) (indirect toxicity/interaction) Nano-TiO 2 did not affect the toxicity of certified reference material sediment

Bacterium ( Vibrio fischeri ) (Heinlaan et al., 2008, 25- to 70-nm powder mixture of anatase and rutile, ratio not disclosed (Sigma product 13463- 67-7, Estonia) (Heinlaan, personal communication, 2008, 30-min exposure for up to 20,000 mg/L nano-TiO 2 and conventional TiO 2 , 8-hr exposure to 20,000 mg/L conventional TiO 2 . Measure the reduction of light output from Vibrio fischeri (Flash assay) as an indicator of growth inhibition The highest concentration tested: 20,000 mg/L nano-TiO 2 (30-min exposure) did not decrease bacterial growth 193414 ) 157558 )

Conventional TiO 2 : size and crystal form not disclosed (Sigma produc t 14027, Estonia; a former Riedel-de Haën product) (Heinlaan, personal communication, 2008, The highest concentration tested: 20,000 mg/L conventional TiO 2 (30-min and 8-hr exposure) did not decrease bacterial growth 157558 )

5-11Protocol (No UV illumination, unless specified) Test Species (Reference) Material Study Outcome

EC 50 >100 mg/L b Bacterium ( Vibrio fischeri ) (Velzeboer et al., 2008, <75-nm (primary particle) nano-TiO 2 in water suspension (Sigma product 643017, the Netherlands), mixture of rutile and anatase, ratio not reported (Velz eboer et al., 2008, 15 min, 1, 10, 100 mg/L , measure reduction of light output from bioluminescent bacteria (Microtox® method, which could be affected by turbidity of 100 mg/L TiO 2 suspension) b 157476 ) 157476 )

EC 50 >100 mg/L b Bacteria (from a soil sample, species not identified) (Velzeboer et al., 2008, <75-nm (primary particle) nano-TiO 2 in water suspension (Sigma product 643017, the Netherlands), mixture of rutile and anatase, ratio not reported (Velz eboer et al., 2008, 7 days (Biolog® test , gram positive), 100 mg/L b

157476 157476 ) )

Bacteria and yeast (proprietary information) (Blaise et al., 2008, <100-nm powder (Sigma product 634662, France), characteristics in water not reported 18-hr growth inhibition of 10 bacteria and 1 baking yeast (microbial array for risk assessment [MARA] assay), tested concentrations not specified MTC >100 mg/L

157592 ; Dando, personal communication, 2008, 18-hr exposure to the fi ltered elutriate from certified reference material sediment with and without nano-TiO 2 mixed in a 1:1 ratio (MARA assay) (indirect toxicity/interaction), tested concentrations not specified Nano-TiO 2 did not affect the toxicity of the elutriate of certified reference material sediment 157582 )

Acute Exposure to Aquatic Organisms

Alga (green alga, Desmodesmus subpicatus ) (Hund-Rinke and Simon, 2006, 72-hr growth inhibition, following the guidelines for EU standard algal assay (DIN 38412-33, 1991, EC 50 and effects of additional particle cleaning: 25-nm primary particle, 20% rutile:80% anatase (Degussa P25) (Baun et al., 2008, 157598

090607 ) ) (photocatalytic)(Product 1) 667415 ; ISA 8692, 2004, Product 1 (P25): EC 50 was not different between nano-TiO 2 washed once as manufacturer recommendation (32 mg/L) and nano-TiO 2 with an additional wash (44 mg/L), suggesting toxicity was not from contaminants 667212 ; OECD 201, 2006, 199838 ) with modifications to include pre-illumination of nano-TiO 2 dispersion with simulated sunlight (wavelength 300-800 nm) at 250 watts for 30 min; illumination alone did not affect D. subpicatus growth 100-nm primary particle, 100% anatase; (Hombikat UV100) (Baun et al., 2008, 157598 ); photocatalytic (Mehrvar et al., 2002, 193541 ) (Product 2)

Algal growth (without pre-illumination): 0, 3.1, 6.2, 12.5, 25, 50 mg/L (products 1 [P25] and 2 [UV100]) Product 2 (UV100): EC 50 >50 mg/L, both nano-TiO 2 with and without the additional wash (at up to 50 mg/L) caused less than 40% decrease in growth Shading effect: 0, 12.5, 25, 50 mg/L Algal growth (with pre-illumination): 12.5, 25, 50 mg/L (Product 1[P25]) No shading effect: when nano-TiO 2 dispersion (at up to 50 mg/L) was between algae and light source (but not in contact with algae) for 72 hr, no effects on algal growth, suggesting nano-TiO 2 effects was not due to lowered light intensity, but due to a toxicity of nano-TiO 2 Pre-illumination of nano-TiO 2 (Product 1 [P25]) did affect nano-TiO 2 effects on algal growth

EC 50 >100 mg/L b Alga (green alga, Pseudokirchneriella subcapitata ) (Velzeboer et al., 2008, <75nm (primary particle) nano-TiO 2 in water suspension (Sigma product 643017, the Netherlands), mixture of rutile and anatase, ratio not reported (Velz eboer et al., 2008, 4.5 hr, in light, 100 mg/L Photosynthesis efficiency was measured as a pulse amplitude modulation (PAM) fluorescence test, wh ich could be affected by turbidity of 100-mg/L TiO 2 suspension b 157476 157476 ) )

EC 50 21 mg/L (based on decreases in healthy cell number) Alga (green alga, Pseudokirchneriella subcapitata) (Warheit et al., 2007, 199838

091075 ) 140 nm in water, 79% rutile: 21% anatase, coated (90-wt % TiO 2 , 7% alumina, and 1% amorphous silica) (D uPont uf-C TiO 2 ) (photopassivative/ photo-stable) (Warheit, 2008, OECD 201 (2006, ) (72-hr growth), with light a
0.01, 0.1, 1, 10, and 100 mg/L (uf-C TiO 2 and fine TiO 2 ) EC 50 87 mg/L (based on inhibition of growth rate) 157470 )

Fine TiO 2 : 380 nm in water, rutile, coated (~99% TiO 2 and ~1% alumina) EC 50 16 mg/L (based on decreases in healthy cell number)

EC 50 61 mg/L (based on inhibition of growth rate)

5-12Protocol (No UV illumination, unless specified) Test Species (Reference) Material Study Outcome

Alga (green alga, Pseudokirchneriella subcapatitata) (Huang et al., 2005, Photocatalytic nano-TiO 2 : Deguassa P25 (75% anatase, 25% rutile, 30 nm) Short-term chronic toxicity – Adsorbsion onto surface of algae; concentrations not specified. Algae carried 2.3 times their own weight in TiO 2 particles on their surface Cellular weight increased by >130% 157801 )

OECD 201 (2006, Ala (green alga, ) (Aruoja et al., 2009, Nano-TiO 2 199838

193254 ) Conventional (Bulk) TiO 2 ) (algal growth inhibition test) Algal cell culture; formation of agglomerates. Nano-TiO 2 formed large agglomerates with almost all algal cells entrapped. EC50 = 5.83 mg/L NOEC = 0.98 mg/L

Conventional TiO 2 formed small agglomerates with some algal cells entrapped and some remained free. EC50 = 35.9 mg/L NOEC = 10.1 mg/L

Invertebrate (water flea, Daphnia magna ) (HundRinke and Simon, 2006, 667232

090607 ) 25-nm primary particle, 20% rutile:80% anatase (Degussa P25) (2008, ISO 6341 (1996, 157598 ) (photocatalytic); ultrasonic dispersion ), OECD 202 (2004, Pre-illumination increased toxicity compared to the same concentration 667207 ) and DIN 38412-30 (1989, 667416 ) (48-hr immobility), exposure to up to 3 mg/L, 16:8 hr light:dark cycles, compare the effects of pre-illuminated and nonilluminated nano-TiO 2 No dose-response relationship with either pre-illuminated or nonilluminated nano-TiO 2 0, 1, 1.5, 2, 2.5, 3 mg/L

100-nm primary particle, 100% anatase; (Hombikat UV100) (2008, Pre-illumination showed a trend of increasing toxicity 157598 ); photocatalytic (Mehrvar et al., 2002, 193541 ); ultrasonic dispersion No dose-response relationship with either pre-illuminated or nonilluminated nano-TiO 2

LC 50 5.5 mg/L Invertebrate (water flea, Daphnia magna ) (Lovern and Klaper, 2006, Primary particle <25 nm (smallest 5-nm), anatase, uncoated (photocatalytic) (Klaper, personal communication, 2008, EPA 48-hr tox test (U.S. EPA standard operating procedure 2024, 1991, 667211

088040 ) 157546 ); filtered through a 0.22-μm nylaflo filter, secondary particle 20-30 nm in deionized water ) (mortality) LOEC 2.0 mg/L NOEC 1.0 mg/L Filtered nano-TiO 2 : 0.2, 1, 2, 5, 6, 8, and 10 ppm

Primary particle <25 nm (smallest 5 nm), anatase, uncoated (photocatalytic) (Klaper, personal communication, 2008, Sonicated, unfiltered nano-TiO 2 : 50, 200, 250, 300, 400, and 500 ppm LC 50 >500 mg/L

157546 ) sonicated, unfiltered , secondary particle 100-500 nm in deionized water

Invertebrate (water flea, Daphnia magna ) (Wiench et al., 2007, OECD 202 (2004, 667207

090635 ) 20-30 nm, 80% anatase, 20% rutile, no surface coating, BET surface area 48.6 m 2 /g ), part 1 (48-hr immobility), tested concentrations: 0 (untreated control), 0.01, 0.1, 1.0, 10.0 and 100.0 mg/L EC 50 >100 mg/L

50 nm × 10 nm, rutile, surface coating aluminum hydroxide, dimethic one/methicone copolymer, BET 100 m 2 /g (T-LiteTM SF) (photostable UV filter) EC 50 >100 mg/L

50 nm × 10 nm, rutile, surface coating aluminum hydroxide, hy drated silica, dimethicone/methicone copolymer, BET 100

m 2 /g (T-LiteTM SF-S) (photostable UV filter) EC 50 >100 mg/L

50 nm × 10 nm, rutile, surface coating aluminum hydroxide, hy drated silica, dimethoxydiphenylsilane/ triethoxycaprylsilane crosspolymer, BET 100 m 2 /g (T-LiteTM MAX) (photostable UV filter) EC 50 >100 mg/L

~300 nm, BET surface area 6 m 2 /g (pigment grade) EC 50 >100 mg/L

Invertebrate (water flea, Daphnia magna ) (Lovern et al., 2007, 30 nm, anatase 1-hr exposure to 2.0 mg/L No changes in heart rate or behaviors

091069 )

5-13Protocol (No UV illumination, unless specified) Test Species (Reference) Material Study Outcome

EC 50 >100 mg/L Invertebrate (water flea, Daphnia magna ) (Warheit et al., 2007, 667207

091075 ) 140 nm in water, 79% rutile:21% anatase, coated (90-wt % TiO 2 , 7% alumina, and 1% amorphous silica) (D uPont uf-C TiO 2 ) (photopassivative/ photo-stable) (Warheit, 2008, OECD 202 (2004, ) (48-hr immobility) (10% immobility at 100 mg/L) 0.1, 1.0, 10, and 100 mg/L (uf-C and fine TiO 2 ) 157470 )

Fine TiO 2 : ~380-nm in water (buffered), rutile, BET surface area 5.8 m 2 /g, coated with alumina (~99% TiO 2 and ~1% alumina) EC 50 >100 mg/L (10% immobility at 10 mg/L, 0% immobility at 100 mg/L)

LC 50 >10 mg/L for both D. pulex and C. dubia Invertebrates (water flea, Daphnia pulex and Ceriodaphnia dubia ) (Griffitt et al., 2008, 48-hr mortality, 14:10 hr light:dark cycle, for D. pulex adults and C. dubia neonates (<24-hr old) 20.5-nm primary partic le, mainly 220.8 or 687.5 nm in moderately hard water, 20% rutile:80% anatase, BET surface area 45 m 2 /g; sonicated (Degussa P25) (photocatalytic) Gradient of concentra tions up to 10 mg/L (The estimated median lethal concentration (LC 50 ) from range-finder tests, and 0.6-, 0.36-, 1.67-, and 2.78-fold the estimated LC 50 . However, the estimated LC 50 was not specified.) 157565 )

EC 50 >100 mg/L b Invertebrate (water flea, Chydorus sphaericus ) (Velzeboer et al., 2008, 157476 <75 nm (primary particle) nano-TiO 2 in water suspension (Sigma product 643017, the Netherlands), mixture of rutile and anatase, ratio not reported (Velz eboer et al., 2008, 48-hr mortality, 17:7 hr light:dark cycle (Chydotox test) b

) 157476 )

NOEC >20,000 mg/L for T. platyurus ; not tested in D. magna Invertebrates (water flea, Daphnia magna ; fairy shrimp, Thamnocephalus platyurus ) (Heinlaan et al., 2008, 48-hr mortality for D. magna 25- to 70-nm powder mixture of anatase and rutile, ratio not disclosed (Sigma product 13463- 67-7, Estonia) (Heinlaan, personal communication, 2008, 24-hr immobilization for T. platyurus

157558 )

Conventional TiO 2 : size and crystal form not disclosed (Sigma produc t 14027, Estonia; a former Riedel-de Haën product) (Heinlaan, personal communication, 2008, Up to 20,000 mg/L for both nano- and conventional TiO 2 NOEC >20,000 mg/L for T. platyurus ; 60% mortality at 20,000 mg/L for D. magna 193414 )

157558 )

Invertebrate (fairy shrimp, Thamnocephalus platyurus ) (Blaise et al., 2008, LC 50 >100 mg/L <100-nm powder (Sigma product 634662, France), characteristics in water not reported 24-hr lethality (ThamnoToxkit assay), tested concentrations not specified

157592 )

Invertebrate (freshwater hydra, Hydra attenuata ) (Blaise et al., 2008, EC 50 in 10 to 100 mg/L range <100-nm powder (Sigma product 634662, France), characteristics in water not reported 96-hr morphological changes, tested concentrations not specified

157592 )

Fish cell (trout primary hepatocytes) (Blaise et al., 2008, <100-nm powder (Sigma product 634662, France), characteristics in water not reported 48-hr cytotoxicity, te sted concentrations not specified TEC in 1 to 10 mg/L range

157592 )

Neither nano-TiO 2 nor conventional TiO 2 at the tested condition caused changes in any of the three endpoints measured. Fish (zebrafish, Danio rerio ), embryo and larvae (Zhu et al., 2008, Nano-TiO 2 : uncoated anatase, purity >99.5%, primary particle in spindle shape, published size
≤ 20 nm, surface area not reported (Nanjing High Technology NANO CO., LTD, Nanjing, Jiangshu province, China); in suspension (in Milli Q water): mean measured size 230 nm, measured size range 100 to 550 nm, secondary particles formed by primary particles have irregular shapes 96-hr exposure to 0, 1, 10, 50, 100, or 500 mg/L nano-TiO 2 or conventional TiO 2 tofish eggs (started within 1.5 hr postfertilization); light cycle 14 hr light/10 hr dark; following endpoints were measured: (1) survival of embryo and larvae; (2) hatching rate at 84 hr post-fertilization; and (3) malformation (e.g., pericardial edema and tissue ulceration, body arcuation, etc.) in embryo and larvae 193742 )

Conventional TiO 2 : anatase, purity >99.0%, published size: 10,000 nm (Third Chemical Regent Factory of Tianjin, Tianjin, China); in suspension (in Milli Q water): mean measured size 1,100 nm, measured size range 330 to 2,250 nm, neither primary nor secondary particles have a uniform shape

Fish (zebrafish, Danio rerio ) (Griffitt et al., 2008, LC 50 >10 mg/L for both adults and fry 48-hr mortality on adult zebra fish and zebra fish fry (<24 hr post-hatch) at a gradient of concentrati ons up to 10 mg/L 20.5-nm primary partic le, mainly 220.8 or 687.5 nm in moderately hard water, 20% rutile:80% anatase, BET surface area 45 m 2 /g, sonicated (Degussa P25) (photocatalytic) 157565 )

5-14Protocol (No UV illumination, unless specified) Test Species (Reference) Material Study Outcome

LC 50 >100 mg/L Fish (rainbow trout, Oncorhynchus mykiss ) (Warheit et al., 2007, 667208

091075 ) 140 nm in water, 79% rutile:21% anatase, coated (90-wt % TiO 2 , 7% alumina, and 1% amorphous silica) (D uPont uf-C TiO 2 ) (photopassivative or photo-stable) (Warheit, 2008, OECD 203 (1992, ) (96 hr acute toxicity) 0.1, 1.0, 10, and 100 mg/L (uf-C and fine TiO 2 ) 157470 ).

Chronic Exposure to Aquatic Organisms

Invertebrate (water flea, Daphnia magna ) (Adams et al., 2006, 66-nm powder, ~35% rutile:65% anatase, average 330 nm in water, (Sigma product 634662) (photocatalytic) (Lyon, personal communication, 2008, 8-day exposure to sus pension at 1, 10 or 20 ppm (concentration over time was not reported) 40% mortality at 20 mg/L

157782 ) 157524 )

Invertebrate (water flea, Daphnia magna ) (Wiench et al., 2007, 667210

090635 ) 50 nm × 10 nm, rutile, surface coating aluminum hydroxide, hydrated silica, dimethicone/ methicone copolymer, BET surface area 100

m 2 /g (T-LiteTM SF-S) (photostable UV filter) OECD 211 (2008, ) (21-day reproduction), test c oncentrations: 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 mg/L NOEC 3 mg/L LOEC 10 mg/L

OECD 202 (2004, 667207 ) (Chronic toxicity, 21-day immobility) LC 0 = 30 mg/L

Fish (rainbow trout, Oncorhynchus mykiss ) (Federici et al., 2007, Respiratory distress, organ pathologies, and oxidative stress at as low as 0.1 mg/L; nano-TiO 2 could be a surface acting toxicant 21 nm, 75% rutile:25% anatase, sonicated (Degussa P25) (photocatalytic) 0-, 7-, or 14-day exposure to 0, 0.1, 0.5 or 1.0 mg/L (mean measured TiO 2 concentrations were 0.089, 0.431, and 0.853 mg/L over the 12-hr period, equating to 89, 85, and 86% of the expected concentrations, respectively) 091222 )

Acute Exposure to Terrestrial Organisms

Photosynthetic enzyme complexes isolated from spinach leaves (Blaise et al., 2008, IC 20 >100 mg/L <100-nm powder (Sigma product 634662, Canada or France), characteristics in water not reported 15 min, tested concentra tions not specified, measure the decrease in chlorophyll fluorescence emitted from the enzyme complexes as an indicator of inhibition of photosynthetic efficien cy (Luminotox assay) (Bellemare et al., 2006, 157592 ) 157779 )

Mix in a 1:1 ratio with certified reference material sediment, 15 min, tested concentrations not spec ified, measure light output (Luminotox assay) (indirect toxicity/interaction) Nano-TiO 2 did not affect the toxicity of certified reference material sediment

Nano-TiO 2 : Plant (spinach, Spinacia oleracea ) (Linglan et al., 2008, Nano-TiO 2 : 5 nm, anatase, not coated S oak the seeds in 0.25% nano-TiO 2 or conventional TiO 2 for 48 hr, and spray 0.25% nano-TiO 2 or conventional TiO 2 onto the leaves from 2-leaf stage to 8-leaf stage at 0.25% Enhanced growth (size, single plant fresh weight, single plant dry weight) 157534 )
Conventional TiO 2 Increased chlorophyll content Increased net photosynthetic rate Increased mRNA, protein concentration, and activity of Rubisco activase

Conventional TiO 2 : No significant changes

Plant (spinach, Spinacia oleracea ) (Zheng et al., 2005, Size not specified, rutile (Shanghai Chemical Co. of China product) Soak aged seeds for 48 hr at 0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 4.0, 6.0, or 8.0 mg/L Increased germination rate, intensity of photosynthesis, chlorophyll synthesis, and Rubisco activase activity in a dose response manner (at up to ~4.0 mg/L; peak effect at ~2 mg/L; higher concentrations have opposite effects) 157784 )

No statistically significant reduction in grow th, transpiration or water use efficiency rates, therefore no findings of toxicity. Adsorption of Hombikat UV100 particles more apparent than Degussa particles. Plant (willow trees) (Seeger et al., 2009, Nano-TiO 2 : Degussa P25 (20/80% rutile/anatase, average diameter 25 nm); Hombikat UV100 (100% anatase, average diameter <10 nm) 190-hr exposure to 0, 1, 10, and 100 mg/L (TiO 2 ) and 0, 1, 20, and 50 mg/L (TiO 2 ) for Degussa, and 100 mg/L (TiO 2 ) for Hombikat UV100. 644124 )

Solutions shaken. Adsorption measured by light microscopy; grow th, transpiration, and water use efficiency measured using standard willow tree acute toxicity test

5-15Protocol (No UV illumination, unless specified) Test Species (Reference) Material Study Outcome

Plant (maize, Zea mays L .) Asli and Neumann (2009, Nano-TiO 2 : Degussa P25 (mean diameter 30 nm) 5-hr root exposure to 0.3 and 1.0 g/L of either nanomaterial in hydroponic solution over 5-day period; 6-wk root exposure to 1.0g/L of either nanomaterial in clay soil Accumulation at the cell wall surfaces of primary roots and subsequent inhibition of cell wall pore size, water transport capacity, leaf growth and transpiration. No statistically significant effect observed on shoot growth rate when exposed to nanomaterials in soil. 193771 ) Bentonite clay (1-60nm)

Invertebrate (isopod, [woodlouse] Porcellio scaber ) (Jemec et al., 2008, 3-day dietary exposure to nonsonicated nano-TiO 2 at 0.1, 0.5, 1, 10, 100, 1,000, 2,000, or 3,000 μg/g food or to sonicated nano-TiO 2 at 1,000, 2,000, or 3,000 μg/g food (leaves soaked in nonsonicated or sonicated nano-TiO 2 dispersion and then dried) 15 nm in diameter, 15-75 nm in length, elongated spheroid shape, anatase, surface area 190-290 m 2 /g, 99.7% pure (Sigma product). 780- to 970-nm aggregates in nonsonicated dispersion, 350- to 500-nm aggregates in sonicated dispersion, si zes on dry leaves not reported Decreased activities of catalase and glutathione-S-transferase (GST) in digestive glands at 0.5, 2,000, and 3,000 μg/g nonsonicated nano-TiO 2 , but not in middle doses of nonsonicated nano-TiO 2 or any doses of sonicated nano-TiO 2 157552 )

No changes in feeding rate, defecation rate, food assimilation efficiency, weight, or mortality were noted up to 3,000 μg/g

Expose synchronized worms in the L1 stage to nano-TiO 2 or conventional TiO 2 in ultrapure water with pH adjusted to 7.0 with HNO 3 and NaOH Invertebrate (nematode, Caenorhabditis elegans ) (Wang et al., 2009, Nano-TiO 2 , anatase, primary particle diameter 50 nm, measured BET surface area 325 m 2 /g for primary particle, purit y >99%, hydrodynamic diameter (of aggregates in pure water) range 338 to 917 nm (medium 550 nm), zeta potential at pH 7.0 = -18.9 mV (Hongchen Material Sci & Tech, Co., China) Lethality to the vermiform nematode: 24-hr LC 50 was significantly lower for nano-TiO 2 (79.9 mg/L) than for conventional TiO 2 (135.8 mg/L) 193696 ) Exposure for 24 hr (for lethality to the vermiform nematode) or 5 days (for growth – length of the worm, and reproduction tests – number of eggs inside the worm body, and number of offspring per worm) at 24.0, 47.9, 95.9, 167.8, and 239.6 mg/L Length of the worm, number of eggs inside the worm body, and number of offspring per worm were all significantly decreased at 47.9 mg/L or higher concentrations of nano-TiO 2 and at 95.9 mg/L or higher concentrations of conventional TiO 2 Conventional TiO 2 , anatase, measured primary particle diameter 285 nm (by TEM), measured BET surface area 7.3 m 2 /g, purity >99%, hydrodynamic diameter range 158 to 687 nm (medium 494 nm), zeta potential at pH 7.0 = - 33.8 mV (ACROS, Acros Organics)

N/A – Not applicable

a Authors reported cloudy appearance or difficulty to dissolve nano-TiO 2 in preparing stock suspension. The testin g concentrations (final concentrations in medium) were calculated by the volume of 10 mg/L stock suspension added into the medium. The actual concentrations of nano-TiO 2 in medium were not reported.
b Authors reported cloudy appearance in 100 mg/L TiO 2 suspension. After centrifugation, nano-TiO 2 concentrations were no more than 10% of initial concentrations. For example, 200 μg/L nano-TiO 2 was added into pond water, and nano-TiO 2 was only 1 μg/L after centrifugation.

MTC – Microbial Toxic Conc entration, calculated by comparing the area under and above the growth curve (Gabrielson et al., 2003, BET – Surface area measured by Brunauer, Emmett, and Teller analysis DIN – Deutsches Institut für Normung (German Institute for Standardization) 157862 EC 50 – Effective concentration 50; the concentrati on at which 50% of s ubjects showed response EU – European Union

IC 20 , IC 25 – inhibitory concentration at which organi sms showed 20%, 25% inhibition in measured endpoints ISO – International Organization for Standardization

GST – Glutathione-S-transferase

LC 50 – Lethal concentration 50; the concentrati on at which 50% of subjects died

LOEC – Lowest observed effect concentration MARA – Microbial array for risk assessment (assay) ; Gabrielson et al., 2003, 157863 )

NOEC – No observed effect concentration OECD – Organization for Economic Co-operation and Development

P25 – Aeroxide® P25 PAM – Pulse amplitude modulation

TEC – Threshold effect concentration. The TEC for cytotoxicity is calculated us ing the NOEC and LOEC of cell viability reduction. TEC = (NOEC × LOEC)1/2 TEM – Transmission electron microscopy UV – Ultraviolet (light/radi ation), wavelengths in the range of 10 to 400 nm



# 5.2.1.1. Effects on Bacteria and Fungi (Terrestrial and Aquatic)

Data for the effects of photostable nano-TiO 2 

### on bacteria and fungi are lacking. On the other hand, photocatalytic nano-TiO 2 

### is known for its antibacterial and antifungal properties and has been tested for various applications, including dri nking water treatment (Coleman et al., 2005, 089849 ); surface coatings and paints (Kuhn et al., 2003, 090597 ; Tsuang et al., 2008, 157483 ); and food

5-16

### packaging (Chawengkijwanich and Hayata, 2007, 157708 ). Examples of recent studies of photocatalytic nano-TiO 2 

### in bacteria and fungi are provided in Table 5-3. Because most bacteria and fungi are nonpathogenic and are major decomposers in most terrestrial and some aquatic ecosystems, chemicals with antibacterial and antifungal properties are not necessarily beneficial when released into the environment. The health of decomposers is important for nutrient cycling in the environment, such as carbon and nitrogen cycling in soil (Neal, 2008, 196069 ). Additionally, some bacteria and fungi form a symbiotic relationship with plants. A well-known example is the nitrogen-fixing bacteria (genus Rhizobium ) that live on the roots of legumes. Legumes provide nutrients and a relatively anaerobic environment for the rhizobia, and obtain ammonia formed from atmospheric nitrogen by the rhizobia (Long, 1989, 644893 ). Thus, indiscriminant exposure to chemicals with antibacterial properties could harm plants by interfering with symbiotic bacteria. Sensitivity to photocatalytic nano-TiO 2 

### toxicity varies among species of bacteria. Adams et al. (2006, 157782 ) reported that in the presence of sunlight, gram-negative Escherichia coli were more sensitive to nano-TiO 2 

### -induced growth inhibition than gram-positive Bacillus subtilis . With 2,000 ppm of nano-TiO 2 

### in the growth medium, E. coli growth was decreased by 46% while B.
subtilis growth was inhibited by 99%. At 500 ppm, E. coli growth was decreased by only 15% and B.
subtilis growth was not inhibited (Adams et al., 2006, 157782 ). The different dose-response relationships of gram-positive and gram-negative bacteria to nano-TiO 2 

### suggest the potential for
nano-TiO 2 

### to alter microbial population balance (diversity), both in wastewater treatment plants and during various phases of use and disposal of nano-TiO 2 

### . One generally accepted explanation for
nano-TiO 2 

### -induced toxicity in bacteria and fungi is the generation of ROS, which can cause cell wall or cell membrane damage (Kuhn et al., 2003, 090597 ; Neal, 2008, 196069 ), such as lipid peroxidation (Maness et al., 1999, 193538 ). Although, as discussed ab ove, UV illumination increases photocatalytic nano-TiO 2 

### toxicity, photocatalytic nano-TiO 2 

### is also toxic in the dark (Adams et al., 2006, 157782 ; Coleman et al., 2005, 089849 ). Because TiO 2 

### generates ROS (mainly highly reactive hydroxyl radicals, ·OH) in the presence of UV light and oxygen (Reeves et al., 2008, 157506 ),
mechanisms other than oxidative st ress might also contribute to nano-TiO 2 

### toxicity in the dark and possibly also under UV light. For example, several types of nano-TiO 2 

### (anatase and a mixture of anatase/rutile) have been shown to adsorb protein and calcium (Ca 2+ 

### ) in the medium, and cause in vitro cytotoxicity in mammalian cell lines (Horie et al., 2009, 193433 ).



# 5.2.1.2. Effects on Aquatic Organisms

Data on the effects of nano-TiO 2 

### in aquatic organisms are available for freshwater algae, freshwater invertebrates (water fleas and fair y shrimp), and freshwater fish (rainbow trout) (Table 5-3). Only two aquatic organism studies in the literature involve photostable nano-TiO 2 

### (Warheit et al., 2007, 091075 ; Wiench et al., 2007, 090635 ). For other aspects of U.S. EPA Tier I aquatic toxicity testing (e.g., estuarine and marine organism acute toxicity, whole sediment acute toxicity, and bio-availability/bio-mag nification toxicity) (U.S. EPA, 2008, 157481 ), studies have not yet been reported.



## Algae

Algae are primary producers of chemical energy in ecosystems. In addition to being the food base in aquatic systems, algae provide much of the earth’s oxygen. Effects on algae are measured at the population level, for example, in terms of population growth. In algal tests, 72-hour exposures are considered acute exposure in European Un ion (EU) regulations, and 96-hour exposures are considered chronic by U.S. EPA (2008, 157481 ). A limited number of studies on the effects of either photocatalytic or photostable TiO 2 

### in algae have been completed.

5-17

### For photostable nano-TiO 2 

### , EC 50 

### values determined for 72-hour growth inhibition in green alga ( Pseudokirchneriella subcapitata ) were 21 mg/L (based on decreases in healthy cell numbers) and 87 mg/L (based on inhibition of growth rate) (Warheit et al., 2007, 091075 ). In contrast, exposure to concentrations of 0.01 to 1 mg/L of photostable nano-TiO 2 

### increased growth rate by 1-3% (green alga cell numbers increased 6-19%) (Warheit et al., 2007, 091075 ). U-shaped doseresponse relationships are not unique to nanomaterial s, and it cannot be ruled out that increased growth at the low dose was a compensatory response to low levels of toxicity (Calabrese and Baldwin, 1998, 047938 ; Davis and Svendsgaard, 1990, 048278 ). Fine (approximately 380 nm) TiO 2 

### showed almost no inhibition in growth rate (or cell number) at up to 1 mg/L, and EC 50 

### values of 16mg/L (based on decreases in healthy cell numbers) and 61 mg/L (based on inhibition of growth rate) (Warheit et al., 2007, 091075 ). Hartmann et al. (2010, 196322 ) also studied green algal growth inhibition, testing three different sizes of nano-TiO 2 

### and observing toxicity in all three cases. The authors’ primary discussion, however, was on th e difficulty of reproducing results due to the complex interactions of the systems; the de termination of a dose-response relationship was complicated by the effects of con centration-dependent aggregation of the nanoparticles, subsequent sedimentation, and possible attachment to vessel surfaces. Hartmann et al (2010, 196322 ) concluded that their research underlines the potential for in teractions with existing environmental constituents to affect the toxicity of nanoparticles. For photocatalytic nano-TiO 2 

### , the EC 50 

### values determined for 72-hour growth inhibition in green algae ranged from approximately 30 mg/L to > 50 mg/L (Blaise et al., 2008, 157592 ; HundRinke and Simon, 2006, 090607 ). Hund-Rinke and Simon (2006, 090607 ) also tested the potential
for TiO 2 

### to reduce growth by physically shading algae, an d reported that as much as 50 mg/L of photocatalytic nano-TiO 2 

### physically above the algae did not decreas e algal growth, that is, it did not cause a shading effect. When nano-TiO 2 

### and algae are in the same liquid medium, photocatalytic P25
nano-TiO 2 

### was reported to adsorb onto the surfaces of green algae ( Pseudokirchneriella subcapatitata) and to increase cellular weight by more than 130% (Huang et al., 2005, 157801 ). The concentration of P25 was not reported. If the attached nano-TiO 2 

### directly blocks sunlight that otherwise could reach the algal cell surface or if this extra weight causes algae to stay in deeper water, the consequent reduction in sunlight coul d inhibit the algal growth. Because photostable
nano-TiO 2 

### would also block UV penetration, similar effects could occur with photostable nano-TiO 2



### . Without experimental evidence, predicting the impact of nano-TiO 2 

### on photosynthesis is difficult
because nano-TiO 2 

### exposure reportedly increases photosynthesis in terrestrial plants, namely spinach, as discussed later in this section. Nano-TiO 2 

### could affect aquatic and terrestrial plants differently due to exposure routes, doses, and other factors. Although no marine organisms have been tested for nano-TiO 2 

### toxicity, the physical
attachment of nano-TiO 2 

### particles on cells could pose a risk to aquatic organisms that reproduce by external fertilization. A wide variety of marine organisms fall into this category. Attached nano-TiO 2 

### could decrease sperm cell mobility and conseque ntly reproductive success. For comparison, carbon black nanoparticles have been reported to decrease sperm frequency of seaweed (marine macroalgae) and to affect seaweed embryo development (Nielsen et al., 2008, 644828 ). As discussed earlier (Section 5.1.1), the ionic strength due to salini ty in seawater could influence the behavior and
effects of nano-TiO 2 

### , such as more aggregation as compared to pure water.
Nano-TiO 2 

### was reported to increase algal cell weight 2.3-fold by adsorbing to the algal cell surface, but the tested nano-TiO 2 

### concentrations in water were not reported (Huang et al., 2005,
157801 ). If an increase in weight forces surface algae into deeper water, photosynthesis could be
decreased 1 

### due to less sunlight available in deeper water than at the surface. The reduced light available to algae was also suggested to be the cause of more growth inhibition seen in algal cells
treated with nano-TiO 2 

### , compared to conventional TiO 2 

### (Aruoja et al., 2009, 193254 ). In Aruoja’s

1 On the other hand, nano-TiO 2 taken up by spinach increased growth and photosynthe sis by increasing the activities of enzymes important for photosynthesis (Linglan et al., 2008, 157534 ; Zheng et al., 2005, 157784 ).

5-18

### study (2009, 193254 ), nano-TiO 2 

### formed large agglomerates with almost all algal cells entrapped.
Conventional TiO 2 

### , on the other hand, formed small agglomerates with some algal cells entrapped and some algal cells free (Aruoja et al., 2009, 193254 ). Since these studies were both conducted in algal culture medium, it cannot be ruled out that some of the observed growth inhibition may be due to absorption of zinc and phosphate by nano-TiO 2 

### , decreasing the availability of these nutrients to the algae (Kuwabara et al., 1986, 625577 ).



## Aquatic Invertebrates

The endpoints used most often in ecological studies with invertebrates are mortality and immobility; other endpoints include morphological changes, heart rate changes, and reproductive effects. Fairy shrimp, Thamnocephalus platyurus , are small freshwater crustaceans and filter feeders that live in temporary water bodies that dry out or periodically experience decreased water levels (Brausch et al., 2006, 193296 ; Löhr et al., 2007, 193518 ). In the dry season, T. platyurus survives by laying resting-stage eggs (known as cysts), which ha tch into nauplii (first stage of crustacean larvae) within hours after being hydrated (Brausch and Smith, 2009, 193297 ). The lethality and
immobilization in T. platyurus larvae and adults as well as the hatch rate of T. platyurus cysts are often used as endpoints for freshwater contaminant tests. Hydras ( Hydra attenuata ) are small simple animals with a tube-shape body (usually 1-20 mm long) and tentacles on one end of the body. Intoxication of hydras can be seen in tentacle mo rphology, which can be normal, clubbed (a sign of minor intoxication), shortened (severe intoxication), or completely retracted (lethal intoxication, because this inevitably leads to death) (Environment Canada, 2007, 157697 ). Acute and chronic toxicity of nano-TiO 2 

### intended for sunscreen use was studied in Daphnia

magna and reported in a poster at a scientific meeting by Wiench et al. (2007, 090635 ). In the acute exposure study, EC 50 

### values (from 48-hour mortality tests) were above 100 mg/L for all tested forms
of TiO 2 

### , which consisted of three photostable forms (uncoated T-Lite TM 

### SF, coated T-Lite TM 

### SF-S,
and coated T-Lite TM 

### MAX), a photocatalytic nano-TiO 2 

### , and a pigment-grade TiO 2 

### (Wiench et al., 2007, 090635 ). In the chronic exposure study, photostable coated T-Lite SF-S was given to D. magna at up to 100 mg/L for 21 days, and the authors reported that the LC 50 

### was 30 mg/L. In this study, death was determined by the lack of swimming ability. For reproductive effects after 21 days, the no obs erved effect concentration (NOEC) value for T-Lite SF-S was 3 mg/L, and the lowest observed effect concentration (LOEC) value was 10 mg/L (Wiench et al., 2007, 090635 ). In a different study that used photostable nano-TiO 2 

### intended to protect plastics against UV-induced degradation, 48-hour exposure to 100 mg/L of the nano-TiO 2 

### induced 10% immobility in D. magna (Warheit et al., 2007, 091075 ). The effects of photocatalytic nano-TiO 2 

### toxicity have been studied by several research teams in four types of water fleas ( D. magna

, D. pulex

, Ceriodaphnia dubia , and Chydorus sphaericus ), one type of fairy shrimp ( T. platyurus ), and one type of freshwater hydra ( H. attenuata ). For water fleas, the 48-hour mortality or immobility EC 50 

### was generally greater than 100 mg/L (Lovern and Klaper, 2006, 088040 [unfiltered]) (Velzeboer et al., 2008, 157476 ; Wiench et al., 2007, 090635 ), with two exceptions. One study reported an LC 50 

### greater than 10 mg/L, which in this case was the highest concentration tested (Griffitt et al., 2008, 157565 ). Another study reported a 48-hour LC 50 

### of 5.5 mg/L, using filtered nano-TiO 2 

### samples, which have an average particle size of 30 nm after going through a 0.22-mm Nylaflo filter (Lovern and Klaper, 2006, 088040 ). In contrast, unfiltered
nano-TiO 2 

### samples had all sizes of nano-TiO 2 

### clumps, ranging from 100 to 500 nm in diameter, and the mortalities never exceeded 11% at up to 500 mg/L (Lovern and Klaper, 2006, 088040 ). Chronic exposure for 8 days caused 40% mortality at 20 mg/L in daphnids (Adams et al., 2006, 157782 ). For fairy shrimp, the 24-hour mortality or immobility LC 50 

### was higher than 100 mg/L (Blaise et al., 2008, 157592 ; Heinlaan et al., 2008, 193414 ). In the only study of hydra, the EC 50 

### of 96-hour morphological changes was <100 mg/L (Blaise et al., 2008, 157592 ). The relative sensitivity among

5-19

### these aquatic invertebrates to nano-TiO 2 

### cannot be determined, due to the variability of tested
nano-TiO 2 

### formulations and experimental designs.
When D. magna were exposed to photocatalytic P25 nano-TiO 2 

### in water, nano-TiO 2 

### was observed on the exoskeleton and antennae and in the digestive tract (Baun et al., 2008, 157598 ). Baun et al. (2008, 157598 ) noted that the aggregation of nanoparticles on the exoskeleton, at sufficient dose, might impede a daphnid’s mobility. Although not investigated in this study, the aggregation of nanoparticles on the antennae, a chemosensory organ important for feeding and reproductive behaviors, could adversely affect a daphnid’s growth and reproduction (Oberdörster et al., 2006, 088054 ). Because nano-TiO 2 

### primary particles are smaller than the size range of particles daphnids feed on (400 to 40,000 nm), the presence of nano-TiO 2 

### in the digestive tract suggests that daphnids feed on nano-TiO 2 

### aggregates (Baun et al., 2008, 157598 ). Whether nano-TiO 2 

### is taken up by other tissues, excreted, or transformed in daphnids is unclear (Baun et al., 2008, 157598 ). Even if
nano-TiO 2 

### is not absorbed into tissues, the presence of nano-TiO 2 

### in the digestive tract of daphnids
could still contribute to bioaccumulati on in the food web (Section 4.6.1.2.).
The behavior and heart rate of D. magna were evaluated in daphnids exposed to photocatalytic
nano-TiO 2 

### at 2.0 mg/L for 1 hour (Lovern et al., 2007, 091069 ). In this study, nano-TiO 2 

### had an average particle diameter of 30 nm, and tetrahydrofuran, the organic solvent used to prevent aggregation, was not detected in the final nano-TiO 2 

### suspension. The concentration of 2.0 mg/L was
selected because it was the lowest observed effect level (LOEL) of D. magna mortality after 48-hour exposure (Lovern and Klaper, 2006, 088040 ). Behavior (e.g., hopping frequency, appendage movement as an indicator of feeding frequency, and postabdominal claw curling) and heart rates were not affected by the 1-hour nano-TiO 2 

### exposure (Lovern et al., 2007, 091069 ).



## Fish

Fish are used in toxicity tests to represent secondary energy consumers in aquatic systems. Commonly used fish species in ecotoxicity tests include freshwater rainbow trout ( Oncorhynchus mykiss ), bluegill sunfish ( Lepomis macrochirus ), fathead minnows ( Pimephales promelas ), and estuarine species sheepshead minnows ( Cyprinodon variegatus ). Data from zebra fish ( Danio rerio ), a model organism widely used in biological and to xicological studies, can also be useful. Fish study endpoints can include concentrations of chemi cals, such as in fish bioaccumulation tests (Section 4.6.1.1, “Bioaccumulation”); mortalit y; behavioral markers (e.g., fatigue, abnormal buoyancy control, and swimming); and pathology. The toxicological studies of photostable nano-TiO 2 

### in fish are very limited. The 96-hour acute toxicity of photostable nano-TiO 2 

### (DuPont uf-C) in rainbow trout produced an LC 50 

### value of greater than 100 mg/L (Warheit et al., 2007, 091075 ). However, DuPont uf-C is designed to protect plastics from UV-induced degradation, and is not known to be used in sunscreen; no fish studies of
nano-TiO 2 

### intended for sunscreen use were found. In contrast, photocatalytic nano-TiO 2 

### , which may be used in drinking water treatment, has been tested in fish for acute effects (Griffitt et al., 2008, 157565 ; Zhu et al., 2006, 157721 ) and chronic effects (Federici et al., 2007, 091222 ). Bioaccumulation (Zhang et al., 2006, 157722 ) and interaction with other heavy metals were disc ussed previously (Table 4.2). In the acute exposure study, the LC 50 

### for a 48-hour exposure to an anatas e/rutile mixture of uncoated nano-TiO 2 

### was >10 mg/L for zebrafish (in both female adults and <24-hour post-hatch fry) (Griffitt et al., 2008,
157565 ). For zebrafish eggs (blastula stage), acute exposures for 96 hours at up to 500 mg/L of
either nano-TiO 2 

### or conventional TiO 2 

### (both uncoated anatase) did not cause developmental toxicity, as measured by survival rate of the zebrafish em bryos and larvae, hatching rate of embryos, and malformation in embryos and larvae (Zhu et al., 2008, 193742 ). In the Zhu et al. (2008, 193742 ) study, nano-Al 2 

### O 3 

### and conventional Al 2 

### O 3 

### at up to 1,000 mg/L also did not cause developmental toxicity to zebrafish eggs, but both nano-ZnO and conventional ZnO caused decreases in survival rates and hatching rate as well as increases in tissue ulceration at 1 mg/L or higher concentrations.

5-20

### Sub-lethal toxicity was observed in juvenile rainbow trout after 14 days of exposure to photocatalytic P25 nano-TiO 2 

### (Federici et al., 2007, 091222 ). Respiratory toxicity and pathological changes in the gill and intestine were seen after a 14-day exposure at concentrations as low as 0.1 mg/L. Furthermore, there were signs of oxidative stress (increased concentrations of thiobarbituric acid substances, an indicator of lipid peroxidation and oxidative stress, in multiple tissues), and activation of anti-oxidant defenses (inc reased total glutathione levels in the gill).

Na + 

### K + 

### -ATPase activity was also increased in the gill and intestine. Disturbances were observed in the metabolism of copper and zinc, but not of Na + 

### , K + 

### , Ca 2+ 

### or Mn. No major hematological disturbances were observed. Worth noting is that these effects occurred without appreciable Ti accumulation in the internal organs, suggesting no nano-TiO 2 

### accumulation, as discussed earlier in Section 4.6.1.1. The authors suggested that surface-bound TiO 2 

### (through surface adsorption) might play a role in toxicity, similar to the case of aluminum, a surface-acting toxicant that can cause systemic toxicity without significant internal accumulation. Federici et al. (2007, 091222 ) concluded that although nano-TiO 2 

### was not a major hemolytic toxicant or disruptor of ion regulation in this study, respiratory distress, organ pathologies, and oxidative stress were adverse effects.



## Summary of Effects on Aquatic Organisms

Sub-lethal effects of nano-TiO 2 

### include decreases in daphnid reproduction by photostable
nano-TiO 2 

### (Wiench et al., 2007, 090635 ), as well as respiratory distress, pathological changes in gills and intestine, and behavioral changes in fish (rainbow trout) by photocatalytic nano-TiO 2 

### (Federici et al., 2007, 091222 ). Several studies reported visible turbidity in nano-TiO 2 

### stock suspensions, and the
actual nano-TiO 2 

### concentration in the liquid phase might be different from the concentration
calculated from added nano-TiO 2 

### (Velzeboer et al., 2008, 157476 ; Zhang et al., 2006, 157722 ; Zhang et al., 2008, 157462 ). Given that natural organic matter in the environment can affect the extent of aggregation and deposition of nanoparticles or modi fy nanoparticle surface charges (Navarro et al., 2008, 157517 )(Kim et al., 2009, 635778 ), the bioavailability and behavior of nano-TiO 2 

### in the environment are likely to be different from bioavailability and behavior in pure water or simple media, although the direction of the diff erence is difficult to predict.



# 5.2.1.3. Effects on Terrestrial Organisms

Plants

Information on nano-TiO 2 

### interactions with plants is currently available for photocatalytic
uncoated nano-TiO 2 

### in spinach and willow trees (Table 5-3). Photocatalytic uncoated nano-TiO 2 

### has been shown to enhance the growth of spin ach in several studies (Lei et al., 2008, 157540 ; Linglan et al., 2008, 157534 ; Mingyu et al., 2007, 157667 ; Mingyu et al., 2007, 157666 ; Yang et al., 2006,
157723 ; Zheng et al., 2005, 157784 ). When a nano-TiO 2 

### suspension was used to soak the seeds and was sprayed on the leaves, the germination rate and growth of the plant were enhanced (Zheng et al., 2005, 157784 ). These effects were at least partially due to nano-TiO 2 

### -induced increases in the activity of several enzymes important for photosynthesis (Linglan et al., 2008), adsorption of nitrate, transformation of inorganic into organic nitrogen (Yang et al., 2006, 157723 ), and anti-oxidative stress response (Lei et al., 2008, 157540 ). Conventional TiO 2 

### suspensions showed either insignificant effects (in comparison with untreated controls) or much smaller effects than nano-TiO 2 

### did (Linglan et al., 2008, 157534 ; Zheng et al., 2005, 157784 ). Seeger et al. (2009, 644124 ) exposed willow tree roots to two types of TiO 2 

### nanoparticles (Degussa P25 and Hombikat UV100) suspended in de ionized water at various concentrations. There were no statistically significant changes in transpira tion rates, growth, or water use efficiency after 190 hours of exposure to nano-TiO 2 

### in solution. Investigators found that roots exposed to the
solution with smaller nano-TiO 2 

### particles (<10 nm average diameter) had nanoparticles compactly

5-21

### attached all over the roots’ surface, while roots in the solution with larger particles (average 25 nm diameter) showed minimally attached particles. Ho wever, the researchers did not determine whether the nanoparticles entered the trees through the xylem. The investigators concluded that these two types of nano-TiO 2 

### are not toxic to willow trees, under the experimental conditions used (Seeger et al., 2009, 644124 ).
193771 In contrast, Asli and Neumann (2009, ) found that colloidal suspensions of TiO 2 

### nanoparticles interfered with water transport capacity, leaf growth, and transpiration in maize ( Zea

mays L.) seedlings. The authors exposed maize roots to colloidal suspensions of inorganic bentonite clay (particle size 1-60 nm) and Degussa P-25 TiO 2 

### nanoparticles (mean diameter of 30 nm) in hydroponic solutions and in soil. The authors found statistically significant reductions in hydraulic conductivity (i.e. water flow through roots) when adding either material at low concentrations (1 g/L) to hydroponic solutions surrounding maize roots over a 5-hour period. Also, transpiration was rapidly inhibited (over a 3-hour period), when both materials were added to the solution at the same concentration. Colloidal nanoparticles of both materials suspended in water flowing to roots appeared to attach to root cell walls, thereby reducing cell wall pore diameters and root hydraulic conductivities. However, when the authors grew mai ze for 6 weeks in clay soil irrigated with either
bentonite or TiO 2 

### , they found no statistically significant effect on shoot growth; they hypothesized that this apparent lack of effect may be due to the fact that the total number of roots increased during this time, thereby increasing the plants' water supply capacity, which could counterbalance possible pore clogging actions by the nanoparticles.



## Terrestrial Invertebrates

The only known studies on the effects of nano-TiO 2 

### on terrestrial invertebrates include a study on an isopod, Porcellio scaber (Jemec et al., 2008, 157552 ), and a study on nematodes, Caenorhabditis elegans (Wang et al., 2009, 193696 ). Living in soil, isopods and nematodes contribute to nutrient cycling and decomposition, and have been used as indicators of soil pollutants. Jemec et al. (2008, 157552 ) investigated the effects of photocatalytic anatase nano-TiO 2 

### on the terrestrial isopod Porcellio scaber, known as woodlouse. Woodlice, approximately 16 mm long, live in the upper layer of soil and surface leaf litter. Th ey break down organic matter and contribute to soil health, and are commonly used in ecologi cal studies. In the Jemec et al. (2008, 157552 ) study, woodlice ate dry leaves that had been soaked in nano-TiO 2 

### dispersions (sonicated or nonsonicated). The sonication process decreased the mean aggl omerate size from 780-970 nm in a nonsonicated dispersion to 350-500 nm. The activities of catala se and glutathione-S-transferase (GST), two antioxidative stress enzymes in the digestive gland (h epatopancreas) were measured. The activities of both enzymes were decreased at 0.5, 2,000, and 3,000 μg/g of nonsonicated nano-TiO 2 

### , but not at middle concentrations (1, 10, 100, and 1,000 μg/g) of nonsonicated nano-TiO 2 

### or at any concentration (1,000, 2,000, and 3,000 μg/g) of sonicated nano-TiO 2 

### (Jemec et al., 2008, 157552 )

. 

### No changes in feeding rate, defecation rate, food assimilation efficiency, weight, or mortality were noted at concentrations up to 3,000 μg/g of either sonicated or nonsonicated nano-TiO 2 

### in the food. This study illustrates the importance of nano-TiO 2 

### dispersion preparation method on nano-TiO 2 

### toxicity. Wang et al. (2009, 193696 ) investigated the lethality, growth inhibition, and effects on reproduction of nano-TiO 2 

### and conventional TiO 2 

### in the nematode, C. elegans , a small free-living (i.e., not parasitic) roundworm that inhabits soil in temperate climates around the world and feeds on bacteria and fungi. In the laboratory, C. elegans is often cultured on agar plates or in liquid medium
in a Petri dish and is often fed E. coli . In the Wang et al. (2009, 193696 ) study, C. elegans strain Bristol N2 (wild-type) in L1 stage (larvae before the first molting) was exposed to anatase nano-TiO 2 

### and anatase conventional TiO 2 

### in water. In addition to lethality and growth inhibition, decreased reproduction was observed at lower mass concentrations of nano-TiO 2 

### than conventional TiO 2 

### . The tested reproduction parameters were eggs inside body and the number of offspring per worm, which

5-22

### includes offspring at all stages beyond the egg over the entire brood period. The mechanism of reproductive effects was not investigated. Due to the lack of toxicity of supernatant of nano-TiO 2 

### (obtained by centrifuging the nano-TiO 2 

### suspension), dissolution of the particle does not contribute
to observed nano-TiO 2 

### effects on C. elegans (Wang et al., 2009, 193696 ).



# 5.2.1.4. Indirect and Interactive Ecological Effects

In addition to the direct toxicity of nano-TiO 2 

### , indirect and potentially synergistic effects of
nano-TiO 2 

### could also be important. Nano-TiO 2 

### could adsorb pollutants (Nagaveni et al., 2004,
090578 ; Pena et al., 2006, 090573 ), carry the pollutants into areas in an organism that the pollutants alone would not naturally appear (Moore, 2006, 089839 ), and increase the uptake of other pollutants (a “Trojan horse” effect). Consequently, nano-TiO 2 

### could enhance pollutant toxicity, and even cause toxicities different from those caused by exposure to the pollutant alone due to differences in distribution. Also, as discussed in Se ction 4.6.1.3, co-exposure to nano-TiO 2 

### in water increased the uptake of arsenic (Sun et al., 2007, 193662 ) and cadmium (Zhang et al., 2007, 090114 ) in carp, but toxicity was not measured in these two studies.
Nano-TiO 2 

### was found to have no effect on the toxicity of sediment and its elutriate in a study using certified reference material sediment (Blaise et al., 2008, 157592 ). The effects of 11 nanomaterials on sediment toxicity (as measured in 2 direct contact assays, the Microtox solid phase

assay 1 2 

### and the Luminotox solid phase assay ) and sediment elutriate toxicity (as measured with the MARA assay 3 

### ) were studied using a mixture of each nanomaterial and the certified reference material sediment at a 1:1 ratio. Photocatalytic nano-TiO 2 

### was one of only three tested nanomaterials
that did not increase the sediment or elutriate toxi city in any of the three assays (Blaise et al., 2008,
157592 ).



# 5.2.1.5. Summary

Limited ecological toxicity information on nano-TiO 2 

### is currently available. Most ecotoxicological studies have tested photocatalytic nano-TiO 2 

### that would be suitable for water treatment, but only a few studies have used photostable nano-TiO 2 

### intended for sunscreen. Coated photostable nano-TiO 2 

### in sunscreen could lose its coating through processes such as aging, weathering, chemical alterations (e.g., change in pH), and metabolism or biotransformation in living organisms (e.g., digestion by daphnids). If so, the photocatalytic nano-TiO 2 

### core could be exposed and thus even photostable nano-TiO 2 

### could have photocatalytic properties. Effects of chronic exposure to nano-TiO 2 

### have been investigated only in water fleas and fish. Although acute exposure effects have been studied in microorganisms and various aquatic macroorganisms, these studies focused on lethality or immobility and provided little insight on modes of action. For terrestrial organisms, only acute exposure to anatase nano-TiO 2 

### was investigated and only in invertebrates ( P. scaber and C. elegans ) and spinach. Photocatalytic nano-TiO 2 

### decreased reproduction in C. elegans without affecting body length. Although increased growth in spinach following acute exposure to anatase nano-TiO 2 

### could be useful for agricultural purposes, the effects of such growth promotion in an ecological system remain unclear. Photocatalytic nano-TiO 2 

### enhanced the uptake of arsenic and cadmium in fish, indicating the possibility of interactive effects
between nano-TiO 2 

### and co-occurring toxic substances.

1 Microtox assay measures the reduction in light output from bioluminescent bacteria, Vibrio fischeri . For solid-phase assays, the concentration that causes 25% inhibition (IC 25 ) is calculated after 20 minutes of exposure.

2 Luminotox assay measures the inhibition of photosynthetic efficien cy of photosynthetic enzyme complexes isolated from spinach leaves. For the Luminotox solid-phase assay, IC 20 is calculated after 15 minutes of exposure.

3 MARA assay (microbial array for risk assessment assay) measures growth inhibition in baking yeast and ten species of bacteria. A microbial toxic concentration is calculated after 18 hours of exposure.

5-23

# 5.3. Health Effects

This section summarizes and evaluates the evidence of nano-TiO 2 

### -induced health effects from epidemiological studies, laboratory animal studies, a nd a few selected ex vivo and in vitro studies.
For a review of nano-TiO 2 

### in vitro effects, see Fond and Meyer (2006, 196337 ). Most health effects studies used pure nano-TiO 2 

### , and therefore their characteristics and effects may differ from
nano-TiO 2 

### as used in commercial products or products containing nano-TiO 2 

### . For instance,
nano-TiO 2 

### in sunscreen may include mostly agglomerat es, instead of perfectly dispersed primary particles. As discussed in Section 5.1, many other factors also influence the effects. When available, data on factors with potential influence on health effects are provided in Tables 5-4 through 5-10. The health effects evidence is organized by hu man and laboratory animal studies and route of exposure, with noncarcinogenic effects discussed in Section 5.3.1 and carcinogenic effects discussed
in Section 5.3.2.



# 5.3.1. Noncarcinogenic Effects

This section summarizes in vivo studies of nano-TiO 2 

### noncarcinogenic effects through dermal, oral, respiratory, and other routes of exposure. The presentation is organized by exposure routes, because exposure routes play a profound role in toxicokinetics, toxicodynamics, and health effects. More studies have been completed on respiratory exposure (inhalation and instillation) than on other exposure routes. Studies investigating solely skin pene tration (not health effects) are discussed in Section 4.6.3. Commercial sunscreens were tested in dermal exposure studies only. Most studies tested photocatalytic nano-TiO 2 

### , which could be suitable as an agent in drinking water treatment. Commercial sunscreens were tested in dermal exposure studies only. Known photostable nano-TiO 2 

### and rutile nano-TiO 2 

### , which is expected to be photostable, were used in some studies (Chen et al., 2006, 090139 ; Mohr et al., 2006, 097493 ; Nemmar et al., 2008, 157514 ; Oberdörster et al., 1992,
045110 ; Pott and Roller, 2005, 157790 ; Wang et al., 2007, 090290 ; Wang et al., 2007, 157616 ; Warheit et al., 2007, 091075 ; Warheit et al., 2007, 090594 ).



# 5.3.1.1. Studies in Humans

No epidemiological studies or case reports are available for nano-TiO 2 

### noncarcinogenic effects. A few case reports described noncarcinogenic effects in the respiratory system of workers exposed to TiO 2 

### particles of unspecified size. For example, exposure to conventional TiO 2 

### has been associated with pneumoconiosis (Yamadori et al., 1986, 193728 ), pulmonary fibrosis and bronchopneumonia (Moran et al., 1991, 157956 ), and pulmonary alveolar proteinosis (Keller et al., 1995, 157938 ). TiO 2 

### or Ti accumulation in the lung, sometim es years after workplace exposures, and Ti-loaded macrophages have also been reported in workers (Keller et al., 1995, 157938 ; Määttä and Arstila, 1975, 157979 ; Yamadori et al., 1986, 193728 ), as have Ti particles in the lymph nodes (Määttä and Arstila, 1975, 157979 ; Moran et al., 1991, 157956 ) and in the liver and spleen (Moran et al., 1991, 157956 ). None of these case reports, ho wever, provided quantitative TiO 2 

### exposure data or measured potentially confounding variables such as exposures to crystalline silica and tobacco
smoke. One epidemiological study (Chen and Fayerweather, 1988, 193312 ) found no consistent relationship between TiO 2 

### (size not specified) exposure and chr onic respiratory disease or fibrosis,
but no conclusions can be drawn because of seri ous limitations, including restricting subjects to workers eligible for pensions; lack of information on the duration of TiO 2 

### exposure, asbestos or other chemical exposures; and the lack of detailed information on sampling.

5-24

# 5.3.1.2. Animal Studies

For the most part (except as noted below), labora tory animal toxicity studies have investigated the effects of acute or subchronic exposure to nano-TiO 2 

### . This section presents in vivo studies of
noncancer effects nano-TiO 2 

### (Tables 5-4 to 5-7) by route of e xposure: dermal, oral, respiratory, and
others. Most animal studies of nano-TiO 2 

### focus on photocatalytic nano-TiO 2 

### , including P25. Although sunscreen nano-TiO 2 

### formulations are intended to be photostable, the coatings that impart photostability to anatase or part-anatase nano-TiO 2 

### in some sunscreen formulations are known to degrade over time (Barker and Branch, 2008, 180141 ; Dunford et al., 1997, 157929 ).



## Toxicity from Dermal Exposure

Toxicity findings from studies of dermal exposure to nano-TiO 2 

### or sunscreen that contains
TiO 2 

### are presented in Table 5-4. For healthy unflexe d skin, adverse health effects are not expected from dermal exposure to photostable nano-TiO 2 

### in sunscreen (NanoDerm, 2007, 157660 ; Scientific, 2007, 196826 ). Photocatalytic nano-TiO 2 

### , however, sometimes is used in sunscreens (Barker and Branch, 2008, 180141 ; Dunford et al., 1997, 157929 ). Photocatalytic nano-TiO 2 

### can generate ROS when exposed to UV light and can cause oxidative st ress and cytotoxicity in cells (cultured human fibroblasts) and in cell-free in vitr o experiments (Dunford et al., 1997, 157929 ; Lu et al., 2008,
157526 ). To date, the effects of long-term or repeated use of sunscreen containing nano-TiO 2 

### have not been investigated in vivo, and no case repor ts of skin damage from such use are currently available. As discussed earlier (S ection 4.6.3), most available studies indicate penetration of the outer skin layer and the stratum corneum, but not penetration of living skin cells. After a single topical application of photostable nano-TiO 2 

### , laboratory rabbits showed no skin irritation 4 hours after application or sensitization 3 days after application (Warheit et al., 2007,
091075 ). Furthermore, although some sunscreens containing TiO 2 

### (size not specified) increased mouse skin absorption of herbicides and pesticides (2,4-D, paraquat, parathion or malathion), TiO 2 

### alone actually decreased the mouse skin absorption of the tested herbicide, 2,4-D (Brand et al., 2003,
157866 ). The investigators reported that a solvent in the sunscreen caused increased skin absorption of herbicides, and this secondary effect can be avoided by substituting phenyl trimethicone as the solvent (Brand et al., 2003, 157866 ). Some researchers, such as Nohynek et al. (2007, 090619 ), have noted a discontinuity between in vitro and in vivo testing results, particularly for skin toxicity. Some in vitro cultures or preparations (other than those using intact skin samples) lack the stratum corneum layer, which according to currently available data can block penetration, such that in vitro tests might overstate toxicity of chemicals like TiO 2 

### . Of the four investigations reviewed, only three report in vivo studies of health effects after dermal exposure to TiO 2 

### (pages 16, 17, and 41-43 of NanoDerm, 2007,
157660 ; Warheit et al., 2007, 091075 ; Wu et al., 2009, 193721 ), and only one of those three used
nano-TiO 2 

### intended for sunscreen (pages 16, 17, 41, and 43 of NanoDerm, 2007, 157660 ). Warheit et al. (2007, 091075 ) used ultrafine particles, roughly 100 nm in size. Three studies used a single application, and the longest exposure was only 3 days. The NanoDerm report (2007, 157660 )
concluded that “TiO 2 

### exposure did not modify the viability, proliferation, apoptosis, and differentiation [or] adhesive properties of skin cells .” As discussed previously (Section 4.6.3), skin penetration studies have shown that some nano-TiO 2 

### can stay in hair follicles for up to 10 days. The only report with repeated dermal exposure included 30 days of daily dermal exposure on porcine skin and 60 days of daily dermal exposure on hairless mouse skin (Wu et al., 2009, 193721 ). As discussed in Section 4.6.3, the 30-day exposure of 4-nm nano-TiO 2 

### , but not larger nano-TiO 2 

### , resulted in nano-TiO 2 

### particles in the basal cells of epidermis, but not in dermis, of pigs. In addition, morphological changes at the subcellular level were seen in the basal cells in the 4 nm TiO 2 

### group. The authors also tested 60-day dermal exposur es to 10-, 21-, 25-, 60-, and 90-nm nano-TiO 2 

### with
various anatase and rutile ratios in hairless mice for nano-TiO 2 

### penetration in skin, as well as distribution, signs of oxidative stress, and pathological changes in various organs (Table 5-4). Most

5-25

### changes were seen in the 10-, 21-, and 25-nm nano-TiO 2 

### groups, and none was seen in control or
90-nm nano-TiO 2 

### group. Increased Ti concentrations were seen in skin, subcutaneous muscle, heart, liver, and spleen. Signs of oxidative stress were seen in skin and liver. Pathological changes were seen in the skin, liver, heart (only 10-nm group), spleen, and lung. While hairless mice are commonly used as a model for skin studies, hairless mice study results should be evaluated with care for human relevance. For instance, the thicknesses of stratum corneum and epidermis of hairless mice are approximately half and two thirds of that in humans, respectively; skin permeability was higher in hairless mice than in humans (Haigh and Smith, 1994, 625322 ). Furthermore, hairless mice (BALB/c nu/nu) are deficient in T cells (Ku and Lee, 2006, 625354 ), and their immune function deficiency may render them more susceptible to nano-TiO 2 

### -induced changes than other animals or
humans. With relatively few in vivo dermal exposure studies investigating nano-TiO 2 

### skin absorption and penetration (Table 4-4) and health effects (Table 5-4), several data gaps on the health effects of dermal exposure to nano-TiO 2 

### are evident. First, information on the dermal penetration and effects of
nano-TiO 2 

### in flexed skin and structurally compromised skin is lacking. Flexed healthy skin (Rouse et al., 2007, 157644 ; Zhang and Monteiro-Riviere, 2008, 193735 ) and compromised skin (Zhang and Monteiro-Riviere, 2008, 193735 ), including UV-exposed skin (Mortensen et al., 2008, 155612 ), have been shown to allow nanoparticles (other than nano-TiO 2 

### , which was not tested) to penetrate deeper than healthy nonflexed skin. Sunscreen containing nano-TiO 2 

### is expected to be used on flexed healthy skin and misused on sunburned skin or skin with micro-lesions, such as microscopic cuts due to shaving. Cytotoxicity was seen in cultured skin cells treated with nano-TiO 2 

### (Lee et al., 2009,
157457 ), and the authors postulated that, in skin with compromised epidermis structure (e.g., sunburned skin or “soaked” skin), contact could occur between nano-TiO 2 

### from sunscreen and living cells in the skin and lead to adverse effects. Second, effects from long-term, repeated dermal exposures to nano-TiO 2 

### in sunscreen, similar to real-life exposur e, have not been studied. Finally, the toxicity of the various intermediate forms of nano-TiO 2 

### in the production process (possible sources of occupational exposure, by dermal and other routes) has not been studied.

5-26

## Table 5-4. Summary of h ealth effects of nano-TiO 2 

## particles in mammalian animal models:
dermal route

Animal Testing Material Treatment Conditi ons Summary of Major Effects Reference

Brand et al. (2003, Some (not all) tested sunscreens increased transdermal penetration of herbicide/pesticide. Commercially available sunscreens, some of which contained TiO 2 (size not specified) For testing indirect dermal effect Mouse 157866 Female hairless CRL:SKH1

In vitro
exposure Single and repeated
exposures a) Commercially av ailable sunscreens, applied at 2 mg/cm 2 to skin excised from mice and placed in a diffusion chamber. 30 min after the sunscreen application, herbicide 2,4-D was applied on skin. )
Solvent, not TiO 2 or ZnO, is responsible for sunscreen-increased skin absorption of herbicide/pesticide. a) Sunscreen effect on transdermal penetration of herbicide 2,4-D: 4 out of 7 tested sunscreens that contain TiO 2 (and 1 out of 2 sunscreens that contain no TiO 2 ) increased transdermal penetration of herbicide 2,4-D. b) Combination of TiO 2 with phenyl trimethicone, ZnO, and octyl methoxycinnamate (OM) c) TiSilc untinted sunscreen, which contains TiO 2 was applied. 2,4-D was also applied. Both were applied on skin, and then again 4.5 hr after the first application. b) Formulation effects:TiO 2 alone, TiO 2 plus ZnO, and TiO 2 in trimethicone (simulation of commercial formula) decreased 2,4-D transdermal penetration. c) Repeated application of both sunscreen and herbicide: The peak penetration of 2,4-D herbicide was higher at the second application of TiSilc sunscreen and 2,4-D, compared to the first application of TiSilc and 2,4-D. However, the 2,4-D penetrations of first and second applications of TiSilc and 2,4-D were the same when skin was washed after both (but not just one) applications of TiSilc and 2,4-D. d) TiSilc untinted sunscreen and pesticides: Paraquat, Malathion, and Parathion

d) Sunscreen effect on transdermal penetration of other pesticides: Absorption of pesticides (Paraquat, Malathion, and Parathion) was also increased in skin pretreated with sunscreen TiSilc.

No effects on cell proliferation (as measured by bromo-deoxy-uridine, BrdU, labeling); apoptosis (as measured by a double-staining method of Ki67 and TUNEL, terminal deoxy nucleotidyl transferase biotin-dUTP nick end labeling); adhesive properties (as measured by the expr ession of P-cadherin, an adhesion molecule specific for basal epidermal keratinocytes); or differentiation (as measured by the expressions of keratin-1, keratin-10, and filaggrin) of epidermal keratinocytes. (NanoDerm (2007, Human foreskin grafts on SCID mice A commercially available sunscreen, hydrophobic emulsion containing nano-TiO 2 (Anthelios XL SPF 60, La Roche Posay, France) For testing dermal effects 157660

In vivo
exposure Single
exposure Sunscreen containing nano-TiO 2 applied to skin at 2 mg/cm 2 in occlusion for 1, 24, or 48 hr )

Sacrificed after exposure time; punch biopsy from the human skin graft area

Tested sunscreen containing nano-TiO 2 did not affect viability, proliferation, apoptosis, differentiation, or adhesiv e properties of skin cells.

Warheit et al. (2007, No dermal irritation effects, no clinical signs of toxicity, and no BW loss. For testing acute dermal irritation Rabbit 091075 New Zealand White

In vivo
exposure Single
exposure Nano-TiO 2 (identified as uf-C , a pre-commercial version of DuPont Light Stabilizer 210), 79% anatase/21% rutile, not coated, approximately 90 wt% TiO 2 , 7% alumina, and 1% amorphous silica, average particle size 140.0 ± 44 nm in water, average BET surface area 38.5 m 2 /g Doses – 0 or 0.5 g )

Not considered a skin irritant. Single exposure for 4 hr (nano-TiO 2 in 0.25 mL deionized water on 6 cm 2 area of skin), covered by gauze
Observation at 1, 24, 48, and 72 hr after
exposure
Increases in cell proliferation in the draining auricular lymph node of the ears treated with 50% and 100% nano-TiO 2 compared to the vehicle control group. For testing dermal sensitization (local lymph node assay) Warheit et al. (2007, Mouse 091075 Female, CBA/JHsd )
0, 5, 25, 50, or 100% nano-TiO 2 on both ears for 3 days In vivo
exposure No dermal sensitization by nano-TiO 2 : Stimulation index (mean disintegrations per minute of each experimental group/mean disintegrations per minute of the vehicle cont rol group) did not exceed 3.0 in any nano-TiO 2 treated groups. Consequently the EC3 value (the estimated concentration required to induce a thre shold positive response, i.e., where stimulation index equals 3) for nano-TiO 2 was not calculated. Positive control group: 25% hexylcinnamaldehyde in 4:1 acetone:olive oil for 3 days Repeated
exposure (Vehicle of positive control) group: 4:1 acetone:olive oil for 3 days Sacrifice on test day 5 Diluting vehicle: N,N-Dimethyl formamide

Positive control group had a dermal sensitization
response.

5-27Animal Testing Material Treatment Conditi ons Summary of Major Effects Reference

Pig: After 30 days of treatment Wu (2009, Pig Male 100% anatase, uncoated, nano-TiO 2 (Zhejiang Wanjin Material Technology Co., Ltd.): Porcine skin, in vivo, shaved pig ear starting at age of 4 wk, approximately 24 mg of test formulation containing 5% nano-TiO 2 (4 or 60 nm) and Tween 80 was topically applied in the marked test area on the right ear skin for 30 consecutive days. Punch biopsies collected at 24 hr after the last treatment for TEM. 193721

In vivo
exposure Repeated
exposure 4 nm, hydrophobic surface, measured particle size 5 ± 1 nm, surface area 200 m 2 /g 10 nm, hydrophobic surface, measured particle size 10 ± 1 nm, surface area 160 m 2 /g
75% anatase/25% rutile, uncoated nano-TiO 2 (P25 from Degussa, Germany): 21 nm, hydrophilic surface, surface area 50

m 2 /g

100% rutile, uncoated, nano-TiO 2 (Zhejiang Hongsheng Material Technology Co., Ltd.): 25 nm, hydrophilic surface, measured particle size 25 ± 5 nm, surface area 80 m 2 /g 60 nm, hydrophobic surface, measured particle size 60 ± 10 nm, surface area 40 m 2 /g 90 nm, hydrophobic surface, measured particle size 90 ± 10 nm, surface area 40 m 2 /g Nano-TiO 2 was detected in all layers of epidermis (stratum corneum, stratu m granulosum, prickle cell layer, and basal cell layers), but not in the dermis of porcine skin. Only 4 nm nano-TiO 2 penetrated into the deeper layer of the epidermis (basal cell layer). Subcellular changes (extended intercellular space, impairment of desmosome, and vacuoles around nucleus in basal cells) we re seen. No gross lesions (such as erythema or edema). )

BALB/c hairless mice skin, in vivo, dorsal region starting at age of 7-8 wk. Test formulation containing 5% nano-TiO 2 (10 nm, 21, 25, 60, or 90 nm), carbopol 940, and triethanolamine was applied on the dorsal skin for 60 consecutive days at 8 mg emulation (or 400 μg nano-TiO 2 ) per

cm 2 skin. 3 hr after application, the dressing was removed and residual nanomaterials were removed from the skin with lukewarm water and the skin was dried. Hairless mice: After 60 days of treatment Mouse Male and female BALB/c (hairless) Mice treated with 10, 21, and 25 nm nano-TiO 2 had decreased BW and increased re lative liver weight to BW. Mice in the 10- and 21-nm groups also had increased relative spleen weight to BW. In vivo
exposure Decreased SOD activities (indicator of antioxidant defense) in the skin and liver (10 and 21 nm). Increased lipid peroxidation (as measured by malondialdehyde) in the skin and liver (10, 21, 25 nm) (in skin only - 60 nm). Decreased collagen content of skin (as measured by hydroxyproline) (10, 21, 25, and 60 nm). Repeated
exposure

Increased Ti in the skin, subcutaneous muscle, liver, heart, and spleen, but not in the blood or subcutaneous saccus lymphat icus in the 10-, 21-, 25-, and 60-nm groups. Almost negligible changes in the brain and kidney, with the exception of increased Ti in the brain after 21 nm nano-TiO 2 exposure. Increased Ti in the lung may be significant in the 21- and 60-nm groups. Pathological changes in skin (excessive keratinization, thinner dermis) (particularly in the 10- and 21-nm groups, also in the 25- and 60-nm groups), liver (focal nec rosis – 21-, 25-, and 60-nm groups; liquefaction necrosis – 10-nm group), heart (small trace of white blood cells – 10-nm group), spleen (minor increase in local macrophages – 10-, 21-, 25-, and 60-nm groups), and lung (slight alveolar thickening -- in 10, 21-, 25-, and 60-nm groups). No pathological changes in the brain. 90-nm group showed no changes.

BET – Brunauer, Emmett, Teller method of calculating surface area OM – Octyl methoxycinnamate BrdU – Bromo-deoxy-uridine SOD – superoxide dismutase EC3 – Estimated concentration required to induce a threshold positive response, where stimulation index equals 3 TUNEL –Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling



## Toxicity from Oral Exposure

Currently only three toxicological studies of nano-TiO 2 

### through oral exposure are available (Table 5-5). Two of them observed the toxicity for up to 2 weeks after a single oral gavage of
nano-TiO 2 

### (Wang et al., 2007, 090290 ; Warheit et al., 2007, 091075 ), and the other investigated genomic instability after nano-TiO 2 

### exposure through drinking water for 5 or 10 days (Trouiller et al., 2008, 157484 ). The Warheit et al. study (2007, 091075 ) was intended to provide basic hazard screening information on well-characterized types of nano-TiO 2 

### through a “base set” of tests spanning mammalian toxicity, genotoxicity, and aquatic (ecol ogical) toxicity endpoints. The acute oral toxicity aspect of this project involved female rats r eceiving a single oral gavage of up to 5,000 mg/kg photostable nano-TiO 2 

### (uf-C) (3 rats per dose). The authors reported “no biologically important BW loss” and no gross lesions at necropsy 14 days after th e gavage. Given that this was a basic screening study, no information on organ weights, histologi cal examinations, or blood tests (hematological or biochemical) was obtained, and thus it was not meant to rule out systemic toxicity or functional changes. However, the study does provide evidence that up to 5,000-mg/kg nano-TiO 2 

### was not lethal
as tested.

5-28

### In the Wang et al. study (2007, 090290 ), male and female mice received a single oral gavage of 5,000 mg/kg TiO 2 

### as 25-nm rutile spindles, 80-nm rutile spindles, or 155-nm anatase octahedrons (Table 5-5 for more details). The large dose was se lected because of the expected low toxicity and was administrated according to OECD testing procedures. No obvious acute toxicity was evident over a 2-week period. However, liver and kidney t oxicity were indicated by biochemical parameters in the serum and by pathological examination. Although no abnormal pathology was observed in the heart, lung, testicle/ovary, a nd spleen tissues, myocardial damage was suggested by increases in serum lactate dehydrogenase (LDH) and alpha-hydroxybutyrate dehydrogenase ( α -HBDH), although such increases might also reflect damage to othe r organs. Morphological changes in the brain were seen in the hippocampus in both the 80-nm and 155-nm groups. The main organs with elevated TiO 2 

### concentrations (measured only in female mice) were the liver, spleen, kidneys, lungs, and brain. Although the liver is expected to receive most of the TiO 2 

### absorbed from the gastrointestinal tract through the portal vein, elevated TiO 2 

### levels in the liver were observed only in the 80-nm group. The reason for this size-specific elevation in hepatic TiO 2 

### concentration remains unknown. The preliminary results of the Trouiller et al. (2008, 157484 ) study showed increased DNA and chromosomal damage in various tissues of adult mice given 60-600 μg/mL photocatalytic
nano-TiO 2 

### (P25) in drinking water for 5 days. In a separate experiment, the offspring of mice that were given nano-TiO 2 

### in drinking water for ten days in th e second half of the pregnancy showed increases in DNA deletions in the eye-spot assay (Trouiller et al., 2008, 157484 ), which detects
reversion of the mouse pink-eyed unstable ( p un 

### ) mutation through DNA deletions of duplicated pinkeyed dilution (p) gene in the offspring of C57BL/6J p un 

### / p un 

### mice (Reliene and Schiestl, 2003,
157857 ). This study showed not only genotoxicity a nd clastogenicity, but also multi-generation effects of photocatalytic nano-TiO 2 

### through oral exposure. Although the concentrations investigated in this study are very high, the suggested modes of action and effects of exposure during pregnancy are noteworthy, particularly for photocatalytic nano-TiO 2 

### . This work is also relevant to discussions of the carcinogenicity of nano-TiO 2 

### (Section 5.3.2). The application of genotoxicity data to the question of potential carcinogenicity is based on th e premise that genetic alterations are found in all cancers. Mutagenicity/genotoxicity is the ability of chemicals to alter the genetic material in a manner that permits changes to be transmitte d during cell division. Although most tests for mutagenicity detect changes in DNA or chromosomes, some specific modifications of the epigenome including proteins associated with DNA or RNA, can also cause transmissible changes. Genetic alterations can occur via a variety of mechanisms including gene mutations, deletions, translocations, or amplification; evidence of mutagenesis provides mechanistic support for the inference of potential for carcinogenicity in humans.

5-29

## Table 5-5. Summary of h ealth effects of nano-TiO 2 

## particles in mammalian animal models: oral
route

Animal Testing Material Treatment Conditi ons Summary of Major Effects Reference

No mortality, no biologically important BW losses, and no gross lesions present in the rats at necropsy. For testing acute effects Warheit et al. (2007, Rat Female, strain/stock not specified Nano-TiO 2 (identified as uf-C, a pre-commercial version of DuPont Light Stabilizer 210), 79% anatase/21% rutile, not coated, approximately 90 wt% TiO 2 , 7% alumina, and 1% amorphous silica, average particle size 140.0 ± 44 nm in water, average BET surface area 38.5 m 2 /g 091075 Doses – 175, 550, 1,750, or 5,000 mg/kg (3 rats per dose) ) Grey colored feces were observed in rats dosed at 1,750 mg/kg (1 of 3 rats) and 5,000 mg/kg (All 3 rats).

Oral LD 50 >5,000 mg/kg for female rats. Single oral gavage Observation for 14 days postexposure

Hepatic Toxicity: Single oral gavage (acute effects) Mouse Male and female CD-1 (ICR) Nano-TiO 2 (Hangzhou Dayang Nanotechnology Co. Ltd.), rutile, uncoated, 25 nm (measured average size 21.1 ± 5.1 nm), surface area 43.0 m 2 /g, column/spindle shape, purity >99% (Chen, personal communication, 2008, Wang et al. (2007, 090290

157588 )

Nano-TiO 2 (Hangzhou Dayang Nanotechnology Co. Ltd.), rutile, uncoated, 80 nm (measured average size 71.4 ± 23.5 nm), surface area 22.7 m 2 /g, column/spindle shape, purity >99% (Chen, personal communication, 2008, 157588 )

Fine TiO 2 (Zhonglian Chemical Medicine Co.), 155 nm (measured average size 155.0 ± 33.0 nm), surface area 10.4 m 2 /g, anatase, uncoated, octahedrons, purity >99% (Chen, personal communication, 2008, 157588 ) Dose – 5,000 mg/kg

10 female and 10 male mice per TiO 2 size group Necropsy at 2 wk after the
gavage Increases in coefficients (wet organ weight/BW) of liver (females in 25-nm and 80-nm groups), serum ALT (females in 25-nm group), serum ALT/AST (females in 25-nm group and males in 155-nm groups), and serum LDH (females in 25-nm and 80-nm groups). a Decreases in AST in males in the 155-nm group (Chen, personal communication, 2008, )

157588 ). Pathological changes: hy dropic degeneration around the central vein, spotty necrosi s of hepatocytes (males and females in 80-nm and 155-nm groups). Nephrotoxicity: Increases in serum BUN (femal es in 25-nm group; no tin males) and serum LDH (females in 25-nm and 80-nm groups; male data not av ailable) (Chen, personal communication, 2008, 157588 ). a
Pathological changes: swelling in renal glomerules and proteinic liquid in renal tubule (males and females in 80-nm group). Possible Brain Toxicity: Pathological changes: increases in vacuoles in the neuron of the hippocampus (males and females in 80-nm and 155-nm groups). The vacuoles could be from reversible fatty degradation (Chen, personal communication, 2008, 157588 ). Possible Myocardial Damage:

Increase in serum LDH a (females in 25-nm and 80-nm groups; male data not available), α -HBDH (females in 25-nm and 80-nm groups; male data no t available) (Chen, personal communication, 2008, 157588 ). Based on the data in this study alone, it cannot be ruled out that LDH and α -HBDH were from kidney or liver. Pathological Results: No pathological changes in heart. No pathological changes in heart, lung, testicle/ovary or spleen in male and female mice exposed to either 80 nm or 155 nm TiO 2 . No pathological changes in any organs of mice exposed to 25 nm TiO 2

. Distribution:

TiO 2 distribution in female mice: increased Ti concentrations in liver (80-nm group), spl een (25-, 80-, and 155-nm groups), kidney (25- and 80-nm groups), lung (80-nm group) and brain (25-, 80-, and 155-nm groups). Fo r the 80-nm group, highest Ti concentration was in liver (3 ,970 ng/g), followed by spleen, kidney, and lung (~375-625 ng/g). For 25-nm group, highest Ti concentration was in spleen (~500 ng/g).

5-30Animal Testing Material Treatment Conditi ons Summary of Major Effects Reference

Increased genomic instability (adult mice): Trouiller et al. (2008, Mouse Wild-type and C57BL/6J p un / p un Nano-TiO 2 (P25), photocatalytic, 80% anatase/20% rutile, not coated For testing genotoxicity in two generations 157484 Wild-type adult mice: 60, 120, 300 and 600 μg/mL in drinking water for 5 days (Based on the assumption of 5 mL water intake per day per mouse with a BW of 30 g, the total doses would be 50, 100, 250 and 500 mg/kg BW)

C57BL/6J p un / p un mice for eye-spot assay: 10-day exposure, pregnant mice were given nano-TiO 2 in drinking water from 8.5-18.5 days post conception. Offspring were sacrificed at 20 days old. DNA damage was increased in cells in peripheral blood at 600 μg/mL. DNA damage was measured by alkaline Comet assay, which detects DNA single strand breaks, double strand breaks, alkaline liable sites, and other lesions. )

DNA double strand breaks (measured by γ H2AX immunostaining) were increased in bone marrow at all tested doses. Chromosomal damage (measured by micronucleus assay) was increased in peripheral blood at 600 μg/mL. Oxidative DNA damage (measured by HPLC) was increased in liver at 600 μg/mL. Increased genomic instability (offspring):

Increases in DNA deletions at the pink-eyed unstable (pun) locus which result from homologous recombination or double strand breaks between the DNA fragments that contain duplicated pink-eyed dilution (p) gene (Reliene and Schiestl, 2003, 157857 ) as measured by the eye-spot assay at 500 mg/kg.

Increased inflammation: Increases in (mRNA levels of) pro-inflammation markers, TNF- α , IFN- γ , and IL-8 (KC) (but not anti-inflammatory markers, TGF- β , IL-10 or IL-4) in peripheral blood at 500 mg/kg as measured by real time RT-PCR.

a LDH may be from heart, liver, kidney, skeletal muscle, brain, bl ood cells, and lungs. A test for LDH isotypes can help to narrow down the source. The primary sources for various LDH isotypes in hum ans are: LDH-1 from heart muscle and red bl ood cells; LDH-2 from white blood cells; LDH- 3 from lung; LDH-4 from kidney, placenta, and pancreas; and LDH-5 from liver and skele tal muscle (MedlinePlus, 2009, 193814 ).

IL-8 (KC) – IL-8 stands for interleuk in-8 and KC for chemokine (CXC motif) ligand 1 (CXCL1) α -HBDH – Alpha-hydroxybutyrate dehydrogenase

γ H2AX – Phosphorylated form of histon e H2AX (phosphorylation of H2AX at serine 139) IL-10 – Interleukin-10 LDH – Lactate dehydrogenase, a general marker of cell injury (Ma-Hock et al., 2009) ALT – Alanine aminotransferase AST – Aspartate aminotransferase LD50 – Lethal dose 50; the dosage that is lethal to 50% of the tested population BET – Brunauer, Emmett, Teller meth od of calculating surface area BUN – Blood urea nitrogen RT-PCR – Reverse transcripti on polymerase chain reaction HPLC – High performance liquid chromatography TGF- β – Transforming growth factor-beta IFN- γ – Interferon-gamma TNF- α – Tumor necrosis factor-alpha IL-4 – Interleukin-4



## Toxicity from Respiratory Exposure

This section discusses the health effects of nano-TiO 2 

### exposure through the respiratory tract (Table 5-6). Two methods of exposure commonly empl oyed for studies of respiratory toxicity are inhalation and instillation. Instillation can be perf ormed in various ways, but essentially involves the direct administration of a substance to the lungs ra ther than allowing the subject to inhale the material. Intratracheal instillation “can be a useful and cost-effective procedure for addressing specific questions regarding the respiratory toxicity of chemicals, as long as certain caveats are clearly understood and certain guidelines are carefully followed” (Driscoll et al., 2000, 011376 ). Among the advantages of instillation are that it pe rmits researchers to control the doses administered into the lung and allows fast administration of test material to the lower respiratory tract. Instillation studies can be useful for identifying most types of ef fects (other than upper respiratory tract effects, such as nasal effects) and for comparing the rela tive potency of compounds, and for this reason are of interest for screening different materials for toxicity. Additionally, instillation studies require smaller amounts of test material, and chances of incidental ingestion exposure (as in whole-body chamber inhalation) are lower than in i nhalation studies (Driscoll et al., 2000, 011376 ; Osier et al., 1997, 086056 ). On the other hand, instillation exposure involves invasive delivery, bypassing of the upper respiratory tract, confounding effects from the ins tilled vehicle, and the use of higher doses or dose rates than those tested in inhalation experiments. Confounding effects are also a concern from

5-31

### anesthesia (needed for instillation, but not inhalati on), which could affect the retention and clearance of the test material (Driscoll et al., 2000, 011376 ). Furthermore, studies have shown that exposure to the same particle through intratracheal instillati on and inhalation can yield different responses. For example, compared to inhalation, instillation caused more particles to be deposited in the basal regions of the lung and caused particles to be distributed less homogenously (Osier et al., 1997,
086056 ). Also, results from instillation cannot be extrapolated quantitatively for estimating inhalation results (Driscoll et al., 2000, 011376 ). Interpreting and comparing results from studies with different respiratory exposure methods (such as inhalation, instillation, and aspirati on) requires caution. Differences among exposure methods could influence uptake doses and particle distributions in the body. Also, the test material preparation required for different exposure methods (such as aerosol and suspension medium preparation) could affect nanomaterial aggregati on. Conclusions drawn from studies using different methods should disclose confounding factors to av oid misleading readers. As an illustration, consider a study that exposed mice to single-walled carbon nanotubes (SWCNT) through inhalation and pharyngeal aspiration (Shvedova et al., 2008, 157491 ). Even though the doses were designed to generate the same deposited dose in the lung, the ae rosol generation and agglomerate sizes of the test material differed. The authors carefully stated th eir conclusion at the end of discussion: “Because of exposure to smaller SWCNT structures by inhalation of a dry aerosol versus aspiration of a particle suspension containing micrometer-size agglomerates , inhalation exposure was more potent than aspiration of an equivalent mass of SWCNT.” The tendency of nano-TiO 2 

### to agglomerate raises an issue for interpreting experimental toxicology studies when the respiratory tract is the portal of entry. Upon inhalation, insoluble particles will deposit in the lung according to the aerodynamic diameter of the particulate unit (i.e., the agglomerate) and the physiological/morphometri c characteristics of the subject. Once deposited
as a result of inhalation or intratracheal instillati on, additional factors (e.g., physicochemistry of the particles, biochemistry of the fluid lining of the lung, and other pharmacokinetic factors of the subject) may impact particle size and composition and determine the ultimate dose to the target cell/molecule. The influence of the lung milieu on a gglomeration is discussed in more detail below.
It should be noted that the concentrations in ava ilable respiratory toxicity studies of nano-TiO 2 

### are presumably much higher than likely ambient or occupational exposure levels. High concentrations of fine-mode particles are known to cause the phenomenon of “particle overload.” In its simplest terms, at sufficiently high concentrati ons, the body’s ability to clear inhaled particles is severely compromised to the point that effects occur that would not occur at high-end “real-world” exposures (ISLI Risk Science Institute Workshop Participants, 2000, 002892 ). Thus, under particle overload conditions, exposure-response relationships a nd even the type of responses produced can be unreliable. However, the nanoparticle-specific expo sures evoking particle overload have not been fully described.



## Effects in Respiratory Tract

As discussed below and summarized in Table 5-6, pulmonary effects studied through
inhalation or instillation of nano-TiO 2 

### include pulmonary inflammation, recruitment of neutrophils and macrophages, nano-TiO 2 

### aggregate-loaded macrophages, disruption of alveolar spaces, alveoli enlargement, proliferation of alveolar type II pneumocytes, and increases in alveolar epithelial thickness. Selected instillation studies are highlighted here primarily for effects not investigated in inhalation studies (i.e., effects outside the respira tory tract and interactions with other factors).
Some of the factors that affect nano-TiO 2 

### respiratory tract toxicity were investigated by Oberdörster et al. (2000, 036303 ). Toxicity of nano-TiO 2 

### could be decreased by cross-tolerance to oxidative stress, because nano-TiO 2 

### given through an intratracheal instillation caused less inflammation in rats previously ex posed (and adapted) to Teflon fume s than in rats that were not adapted. Furthermore, nano-TiO 2 

### induced more severe pulmonary inflammation in compromised

5-32

### rats, which had been given an endotoxin to mimic gr am-negative bacterial infections, than in healthy

rats.

Inhalation and Instillation in the Same Study

Grassian et al. (2007, 093170 ) exposed mice to nano-TiO 2 

### through either inhalation or intranasal instillation. After instillation exposur es to similar surface area doses (based on primary particle surface areas) of 5-nm anatase nano-TiO 2 

### and 21-nm anatase/rutile nano-TiO 2 

### , mice showed a more severe inflammation response to 21-nm nano-TiO 2 

### than to 5-nm TiO 2 

### . This example shows
that surface area alone is not a sufficient dose metric in all studies (Grassian et al., 2007, 093170 ; Warheit et al., 2007, 091075 ), especially when the crystal form and other factors are not the same. In the Grassian et al. (2007, 093170 ) study, the aggregates of 21-nm and 5-nm nano-TiO 2 

### differed in both size and density, either of which could affect the surface area that would interact with the tissues. Although the same nano-TiO 2 

### was used in both inhalation and intranasal instillation, direct comparisons of exposure routes effects were not fe asible for two reasons. First, the exposure doses were not the same, whether the doses were expressed as particle concentrations in air or solution, estimated particle mass per mouse, or estimated particle surface area per mouse. Second, different vehicles (water for inhalation and saline for instillation) were used and the sizes of agglomerates were larger in inhalation aerosols than in instillation. In a study by Osier et al. (1997, 086056 ), acute intratracheal inhalation of high levels (125 mg/m 3 

### ) of fine and nano-TiO 2 

### caused less severe pulmonary response than intratracheal
instillation. Intratracheal inhalation involved deliver ing aerosols to the trachea of anesthetized rats.

Inhalation Studies

The effects in the respiratory tract after inhalation of nano-TiO 2 

### were consistent among studies. With increases in exposure duration, pulmon ary lesions in rodents evolve from reversible pulmonary inflammation (in rats, mice, and hamsters) to impaired particle clearance or overload (in rats and mice, but not hamsters) and cellular prolif eration (in rats and mice, but not hamsters). In rats, but not in mice or hamsters, chronic exposure leads to pulmonary alveolar fibrosis, metaplasia, and eventually lung tumors. In acute and subacute studies in mice and rats, the severity of pulmonary inflammation increased with increases in exposure time, and symptoms (pulmonary inflammation and increases in cell proliferation in bronchi and bronchioles) were reversible wh en exposure ended (Grassian et al., 2007, 090606 ; Ma-Hock et al., 2009, 193534 ).
In subchronic studies of nano-TiO 2 

### exposure for 12 or 13 weeks, pulmonary inflammation,pathological changes in the lung (including fibrosis), and impairment of alveolar macrophagemediated test particle clearance were reported (Baggs et al., 1997, 048642 ; Bermudez et al., 2002,
055578 ; Bermudez et al., 2004, 056707 ; Hext et al., 2002, 157878 ; Hext et al., 2005, 090567 ; Oberdörster et al., 1994, 046203 ). Similar to pulmonary lesions after acute and subacute exposure, pulmonary lesions after subchronic inhalation exposure were also decreased with recovery time, but some lesions, such as fibrotic reactions in the lung, we re not completely reversed even after 1 year of

recovery. Species differences to nano-TiO 2 

### effects were observed among rats, mice, and hamsters (Baggs et al., 1997, 048642 ; Bermudez et al., 2002, 055578 ; Bermudez et al., 2004, 056707 ; Hext et al., 2002, 157878 ; Hext et al., 2005, 090567 ; Oberdörster et al., 1994, 046203 ). Pulmonary responses after 13 weeks of exposure were generally most severe in rats, followed by mice, and least severe in hamsters. Rats and mice, but not hamsters, experienced overload at 10 mg/m 2 

### nano-TiO 2



### . Furthermore, only rats had fibroproliferative lesions and alveolar epithelial bronchiolization (a type of metaplasia).
In chronic studies of nano-TiO 2 

### inhalation in rats (Creutzenberg et al., 1990, 157963 ; Gallagher et al., 1994, 045102 ; Heinrich et al., 1995, 076637 ) and mice (Heinrich et al., 1995,

5-33

### 076637 ), lung tumors occurred in rats, but not in mi ce (for more on carcinogenicity effects in these studies, see Section 5.3.2) . In the study of Creutzenberg et al. (1990, 157963 ), decreased pulmonary clearance (overload) was clearly demonstrated by usi ng two sizes of tracer particles after nano-TiO 2 

### exposure. During the 24-month exposure to nano-TiO 2 

### (see Table 5-6 for concentrations), rats inhaled (nose-only) two types of radioactive tracer s at 3, 12, and 18 months after the beginning of the experiment. The half-times for pulmonary clea rance of the smaller tr acer particles (0.35-μm

59 

### Fe 2 

### O 3 

### ) were more than three times longer in rats exposed to nano-TiO 2 

### at all three tested time points, indicating overload. For the larger tracer particles (3.5-μm 85 

### Sr polystyrene), overload was seen at 3 and 12 months, and the clearance was ba ck to control level at 18 months, which may be due to increased lung weight, altered lung structure, and altered breathing pattern, all of which could consequently change the deposition of 85 

### Sr polystyrene particles (Creutzenberg et al., 1990, 157963 ).



## Systemic Effects and Specific Effects in Heart, Liver, Kidney, and Microvasculature

The effects of respiratory exposure to nano-TiO 2 

### are not limited to the respiratory system. In rats exposed to 5-mg nano-TiO 2 

### /kg BW of rutile nano-TiO 2 

### rods through a single intratracheal instillation, observed effects included increases in the numbers of monocytes and granulocytes in the blood (signs of systemic inflammation); decreases in the number of platelets in the blood (platelet aggregation); and cardiac edema (Nemmar et al., 2008, 157514 ). In mice exposed to rutile and
anatase nano-TiO 2 

### through intranasal instillation, pathologi cal changes were observed in the kidney, and temporary liver injury was suggested by cha nges in serum biomarkers (Wang et al., 2008,
157473 ). Endothelium-dependent arteriolar dilation wa s impaired (decreased) by both fine TiO 2 

### and
nano-TiO 2 

### inhaled by rats, more so by nano-TiO 2 

### than fine TiO 2 

### at similar lung load mass doses (Nurkiewicz et al., 2008, 156816 ). This microvascular dysfunction was attributed to fine TiO 2 

### - and
nano-TiO 2 

### -induced increases in ROS in the microvascular wall, increases in nitrotyrosine expression in spinotrapezius microcirculation, and decreases in microvascular NO production (Nurkiewicz et al., 2009, 191961 ). In both fine TiO 2 

### -and nano-TiO 2 

### -treated groups, vascular smooth muscle sensitivity to NO was not altered, but the mi crovascular NO bioavailability was compromised (Nurkiewicz et al., 2009, 191961 ).



## Effects in Brain

Since 1970, scientists have known that inha led ultrafine air pollutants and engineered nanoparticles translocate into the brain (Oberdörster et al., 2004, 055639 ). Inflammatory responses, altered neurotransmitter levels, and pathological ch anges have been observed in rodent brains after inhalation of manganese oxide (Elder et al., 2006, 089253 ); instillation of nano carbon black (Tin Tin Win et al., 2008, 157486 ); and inhalation of ultrafine elemental 13 

### C particles (Oberdörster et al., 2004, 055639 ). A few recent studies showed that anatase and rutile nano-TiO 2 

### translocate into the brain following intranasal instillations (Wang et al., 2007, 157616 ; Wang et al., 2008, 157474 ). The only available studies of nano-TiO 2 

### effects on the central nervous system are from a research group that has administered high doses of nano-TiO 2 

### to mice using intranasal instillation (Wang et al., 2007, 157616 ; Wang et al., 2008, 157474 ; Wang et al., 2008, 157473 ). These researchers have reported increased oxidative stress and inflammatory response, altered
concentrations and metabolism of neurotransmitter s, and pathological changes in the mouse brain. When mice were given 25-nm rutile, 80- nm rutile, or 155-nm anatase nano-TiO 2 

### though intranasal instillation (50 mg nano-TiO 2 

### /kg BW every 2 days for 2, 10, 20, or 30 days), changes in neurotransmitter levels in the brain were observed only in mice exposed to 80-nm and 155-nm
nano-TiO 2 

### , whereas brain TiO 2 

### concentrations were similar for all three sizes of nano-TiO 2 

### (Wang et al., 2007, 157616 ). After intranasal instillation of 80 -nm rutile or 155-nm anatase nano-TiO 2 

### (500 μg per mouse every other day for up to 30 days), the hi ghest Ti concentrations in the brain were in the hippocampus and olfactory bulb, the two regions where most pathological changes were also seen

5-34

### (Wang et al., 2008, 157474 ; Wang et al., 2008, 157473 ). The hippocampus and astrocytes seem to be the targets of nano-TiO 2 

### toxicity in the brain (Wang et al., 2008, 157474 ; Wang et al., 2008, 157473 ). At the ultra-structural level, mitochondr ia appear to be a target of nano-TiO 2 

### in nerve cells after both in vivo and in vitro exposures (Long et al., 2006, 089584 ; Wang et al., 2008, 157473 ). For the whole brain, inflammatory responses and oxidative stress, including lipid peroxidation and protein oxidation, were detected as elevated levels of oxidative markers and cytokines in mice exposed to
80-nm rutile and 155-nm anatase nano-TiO 2 

### (Wang et al., 2008, 157474 ; Wang et al., 2008, 157473 ). Levels of several neurotransmitters, incl uding norepinephrine, 5-hydroxytryptamine, homovanillic acid, 5-hydroxyindole acetic acid, dopa mine, and glutamic acid, were altered after
intranasal instillation of nano-TiO 2 

### (Wang et al., 2007, 157616 ; Wang et al., 2008, 157474 ; Wang et al., 2008, 157473 ). Nitric oxide, which serves as a neurotransmitter and an important player in inflammatory responses, was also increased in the brain of mice exposed to 80-nm and 155-nm
nano-TiO 2 

### (Wang et al., 2008, 157474 ). Additionally, the activity of cholinesterase, which inactivates the neurotransmitter acetylcholin e, increased (Wang et al., 2008, 157474 ). These changes showed
that the concentrations and metabolism of neurotrans mitters in the brain were affected by nano-TiO 2 

### given through intranasal instillations.



## Table 5-6. Summary of h ealth effects of nano-TiO 2 

## particles in mammalian animal models: respiratory route

Animal Testing Material Treatment Conditi ons Summary of Major Effects Reference

Inhalation and Instillation in the same report

Single inhalation exposure for 4 hr Increases in the numbers of total cell (high 5 nm, low and high 21 nm) and macrophage (high 5 nm and 21 nm) in BAL fluid immediately after exposure (not 20 hr after exposure). Grassian et al. (2007, Mouse Male C57BL/6 Nano-TiO 2 (Nanostructured and Amorphous Materials), anatase, 5 nm, measured BET surface area 219 ± 3 m 2 /g, surface functionalization: O, O-H, H 2 O. Aerosol size: 119 ± 1.56 nm (inhalation high dose), 122.9 ± 1.55 nm (inhalation low dose) 093170 )

Particle concentration in chamber: No changes in histology of th e lung, total protein, LDH activity, or neutrophil number in BAL fluid. 5 nm TiO 2 : Low: 0.77 mg/m 3 (necropsy immediately after exposure) Nano-TiO 2 distribution (only 4 high groups examined): agglomerates were seen in macrophages, alveolar epithelial cells, and alveolar interstitium. Little difference between 5 and 21 nm exposures or necropsy time. High: 7.22 mg/m 3 (necropsy immediately after exposure); 7.35 mg/m 3 (necropsy 20 hr after the end of exposure) Calculated/estimated particle mass per mouse (μg) and particle surface area (cm 2 ): Nano-TiO 2 (Degussa), anatase/rutile, 21 nm, BET surface area 41 ± 1.1 m 2 /g, surface functionalization: O, O-H, H 2 O. Aerosol size: 138.8 ± 1.44 m 2 /g (inhalation high dose), 152.9 ± 1.38 m 2 /g (inhalation low dose) 21 nm TiO 2 : 5 nm TiO 2 Low: 1.3 μg/mouse and 3.2 cm 2 (immediately after exposure) Low: 0.62 mg/m 3 (necropsy immediately after exposure) 5 nm TiO 2 High: 12.5 μg/mouse and 30.3 cm 2 (immediately after exposure) 12.7 μg/mouse and 30.7

cm 2 (20 hr after exposure) High: 7.16 mg/m 3 (necropsy immediately after exposure); 7.03 mg/m 3 (necropsy 20 hr after the end of exposure) 21 nm TiO 2 Low: 1.1 μg/mouse and 2.2 cm 2 (immediately after exposure)

21 nm TiO 2 High: 12.4 μg/mouse and 24.8 cm 2 (immediately after exposure) 12.2 μg/mouse and 24.4

cm 2 (20 hr after exposure)

5-35Animal Testing Material Treatment Conditi ons Summary of Major Effects Reference

21 nm TiO 2 induced more inflammation than 5 nm TiO 2 : Increases in neutrophil number (21 nm low, medium and high; 5 nm medium and high); total cell number and IL-6 (21 nm medium and high); LDH activity and IL-1 β (21 nm high) in BAL fluid. Single intra-nasal instillation

Particle concentration in instillation solutions:

5 nm TiO 2 : Low: 0.1 mg/mL No pathological changes in lung; no changes in TNF- α in BAL fluid. Medium: 0.4 mg/mL
21 nm anatase/rutile TiO 2 and 5 nm anatase TiO 2 do not share the same dose-response curve for neutrophil concentration in BAL fluid as a function to either particle mass or surface area. High: 0.6 mg/mL

21 nm TiO 2 : Low: 0.5 mg/mL Medium: 2.0 mg/mL Calculated/estimated particle mass per mouse (μg) and particle surface area (cm 2 ): High: 3.0 mg/mL 5 nm TiO 2 Low: 5 μg/mouse and 12.1 cm 2 Necropsy 24 hr after instillation 5 nm TiO 2 Medium: 20 μg/mouse and 48.4 cm 2

5 nm TiO 2 High: 30 μg/mouse and 72.6 cm 2

21 nm TiO 2 Low: 25 μg/mouse and 12.5 cm 2

21 nm TiO 2 Medium: 100 μg/mouse and 50 cm 2

21 nm TiO 2 High: 150 μg/mouse and 75 cm 2

Osier et al. (1997, Compared to fine TiO 2 , nano-TiO 2 caused more pulmonary responses and slightly higher (not significant) lung TiO 2 burden. Acute intratracheal instillation and intratracheal inhalation Rats Female F344 Fine TiO 2 (Fisher Scientific), mean primary particle size 250 nm, anatase 086056 )

Intratracheal inhalation exposure for 2 hr at 125 mg/m 3 Compared to intratracheal instillation, intratracheal inhalation to TiO 2 generally caused less severe and less persistent pulmonary respons es and slightly (not significant) higher TiO 2 lung burden. Nano-TiO 2 (Degussa), mean primary particle size 21 nm, anatase Intratracheal instillation exposure to the equivalent amount of TiO 2 as in the lung at day 0 of intratracheal inhalation (500 μg fine TiO 2 or 750 μg nano-TiO 2 in 0.2 mL saline) Increases in polymorphonuclear leukocytes in BAL cell pellet on day 1 after intratra cheal inhalation of fine TiO 2 ; on days 1, 3, and 7 after intratracheal instillation of nano-TiO 2 ; and days 0 and 1 after intratracheal inhalation of nano-TiO 2

. Necropsy 0, 1, 3 or 7 days postexposure (3 rats per group) Decreases in macrophage infla mmatory protein-2 levels in BAL supernatant on days 0, 1, and 3 after intratracheal inhalation of nano-TiO 2 ; and day 1 after intratracheal instillation of nano-TiO 2 . Increases in macrophage inflammatory protein-2 levels in BAL cell pellets on days 1, 3, and 7 after intratracheal instillation of nano-TiO 2 ; and on days 0 and 1 after intratracheal inhalation of nano-TiO 2

. Increases in TNF- α protein was detected by immunocytochemistry (but not by ELISA) on days 0 and 1 after intratracheal inhalation of water (control); days 1 and/or 3 after intratracheal instillation of fine or nano-TiO 2 and intratracheal inhalation of fine TiO 2 ; and at all time points after intratracheal inhalation of nano-TiO 2

. Inflammatory cell influx (polymorphonuclear leukocytes in BAL) was correlated with macrophage inflammatory protein-2 levels in BAL cell pellet (but not in BAL supernatant), but not correlated with TNF- α protein levels in BAL cell pellet or supernatant or in lung sections stained immunocytochemically.

Inhalation

Lung burden: SiO 2 : 0.32 mg immediately after exposure. Nano TiO 2 /fine TiO 2 : 5.33/6.62 mg, 4.15/1.2 mg, 3.14/1.66 mg immediately, 6 mo, 12 mo after exposure, respectively. Baggs et al. (1997, Subchronic inhalation Rats Male F344 Nano-TiO 2 , ~20 nm, anatase (Degussa) 048642

Fine TiO 2 , ~250 nm, anatase (Fisher Scientific) Crystalline SiO 2 , ~800 nm Nano-TiO 2 : 23.5 mg/m 3 ; fine TiO 2 : 22.3 mg/m 3 ; SiO 2 1.3 mg/m 3 )

6 mo after exposure, in the lung: SiO 2 caused moderate focal interstitial fibrosis and moderately severe focal alveolitis; nano TiO 2 caused slightly less fibrosis and fine TiO 2 caused least fibrosis. Increases in stainable collagen in all three treated groups, compared to untreated groups. 6 hr/day, 5 days/wk for 3 mo 6- or 12-mo recovery before sacrifice

12 mo after exposure, in the lung: SiO 2 – treated rats showed decreased fi brosis; nano TiO 2 and fine TiO 2 treated rats showed largely norma l amount of interstitial fibrosis but increases in alveolar macrophage number. Increases in stainable collagen only in SiO 2

.

5-36Animal Testing Material Treatment Conditi ons Summary of Major Effects Reference

Fine TiO 2 : Bermudez et al. (2002, Subchronic inhalation Rat Female CDF (F344)/CrlBR Fine TiO 2 (DuPont), rutile; aerosol 1.36 - 1.44 μm MMAD Lung burden of fine TiO 2 :
Fine TiO 2 : 0, 10, 50 or 250 mg/m 3 Immediately after exposure: lung burden of fine TiO 2 : mice > rats > hamsters at 50 and 250 mg/m 3 : rats > mice > hamsters at 10 mg/m 3 . The lung burden decreased with time after exposure. 055578 )

Nano-TiO 2 (P25), photocatalytic, average primary particle size 21 nm, 1.37 μm MMAD; aerosols: 1.29-1.44 μm MMAD Nano-TiO 2 : Bermudez et al. (2004, nano-TiO 2 : 0, 0.5, 2, or 10 mg/m 3 Mouse Female B6C3F1/CrlBR 056707

Hamster Female Syrian golden (Lak:LVG [SYR] BR) 6 hr/day, 5 days/wk for 13 wk 0 (immediately after exposure), 4, 13, 26, or 52 (up to 46 and 49 for hamsters exposed to fine TiO 2 and nano-TiO 2 , respectively) wk of recovery before sacrifice The retention in lung-associated lymph nodes: rats > mice > hamsters at all concentrations. The burden in the lymph nodes increased with time after exposure (rats of all dose groups, mice of low and mid-dose groups, and hamsters of high-dose group). ) Comparison of fine and nano-TiO 2 data reported in Bermudez et al. (2002, Pulmonary clearance kinetics of fine TiO 2 : mice and rats in high-dose groups retained 75% initial burden after 52 wk of recovery, while hamsters retained only 10% initial burden after 26 wk of recovery. Overload in rats and mice at 50 or 250 mg/m 3

. 055578 ) and Bermudez et al. (2004, 056707 ): Hext et al. (2002, Lung burden of nano-TiO 2 : 157878 ; 2005, 090567 Lung burden of nano-TiO 2 : rats ≥ mice > hamster. Immediately after exposure, at 10 mg/m 3 , rats and mice had same lung burdens for nano-TiO 2 . At 2 or 0.5 mg/m 3, rats had more lung burden. Mice and rats, but not hamsters, have pulmonary particle overload at 10 mg/m 3

. )

Pulmonary clearance kinetics of nano-TiO 2 : At 10 mg/m 3

, rats and mice had linear fashion decreases of lung burden to ~50% after 52-wk recovery, while hamsters had a biphasic fashion decr ease to 3% after 48-wk recovery. At 2 and 0.5 mg/m 3 , rats, mice and hamsters had biphasic decreases in l ung burn, and rats only had detectable nano-TiO 2 after the whole recovery period. Burden in the lymph nodes associated with lung: During the whole recovery time, burden increased with time in rats of 10 and 5 mg/m 3 groups, and in mice of 10 mg/m 3 group. No nano-TiO 2 was detected in hamster lymph nodes at any time point or treatment group.
General health of rats, mice and hamsters:

Rats and mice at all treat ed groups had decreases in weight gain after exposure, and recovery occurred 3- 4 wk postexposure. Mi ce exposed to 250 mg/m 3 fine TiO 2 had a consistent lower weight during the recovery period, but rats exposed to 250 mg/m 3 fine TiO 2 had a consistent heavier weight. Hamster exposed to fine TiO 2 had decreases in weight gain after exposure and recovery 6 wk postexposur e. Hamsters exposed to nano-TiO 2 had weight loss after exposure and a slow recovery over the remainder of the study. Hamsters had higher morbidity and mortality rates across treatmentgroups than rats and mice; this was probably due to agerelated renal diseases.

5-37Animal Testing Material Treatment Conditi ons Summary of Major Effects Reference

Pulmonary inflammation after fine TiO 2 exposure: Rats, mice and hamsters had pulmonary inflammation, and only hamsters had full recovery. Rats generally had more severe inflammation, and hamsters had the least.

Fine TiO 2 exposure: Increases in neutrophil %, lymphocyte %, and macrophage number in BAL fluid in rats and mice (in mid- and high-dose groups); increase in neutrophil % in rats at th e lowest exposure. Hamsters had increased macrophage number, neutrophil %, and lymphocyte % at the highest concentration; they had an increased neutrophil % at t he medium concentration. Within 26 wk of recovery, hamsters showed normal neutrophil % and macrophage number; within 46 wk of recovery, hamsters had normal lymphocyte %. Mice and rats showed partial recovery in neutrophil and macrophage response and no recovery in lymphocyte response after 52 wk of recovery.

Fine TiO 2 exposure: LDH levels in BAL fluid transiently increased in mice and rats Pulmonary inflammation after nano-TiO 2 exposure: Rats and mice had pulmonary inflammation.

Nano-TiO 2 exposure: Rats and mi ce, but not hamsters, in the 10 mg/m 3 groups had increased numbers of macrophage and neutrophil and concentrations of LDH and protein in BAL fluid. Pulmonary lesions were most severe in rats, and least in hamsters.

Fine TiO 2 exposure: Alveolar cell proliferation was seen in rats (0 week postexpos ure at mid- and high-dose groups, 4 and 13 wk postex posure at high-dose group) and mice (13 and 26 wk postexposure at high-dose group), but not in hamsters. Only rats had a progressive fibroproliferative lesion and alveolar epithelial metaplasia (bronchiolization).

Fine TiO 2 exposure: At 52 wk postexposure, mouse lungs had particle-laden macrophages in alveolar and relatively normal alveolar s eptal structures. Rat lungs had particle-laden macrophages inside alveolar cells, fibrosis and thickening in in terstitial tissue, and little alveolar epithelial metaplasia (bronchiolization) of lining epithelium. Hamster lungs did not show retained particle burden or macrophage accumulation.

Nano-TiO 2 exposure: Alveolar epi thelial proliferation, alveolar bronchiolization (alv eolar epithelial proliferation of metaplastic epithelial cells around macrophages loaded with particles), alv eolar septal fibrosis and interstitial particle accumulation in rats, but not mice nor hamsters, of the 10 mg/m 3 group. With increasing time postexposure, the lesions became more severe. Species and particle differences: Overload was seen in rats and mice (but not hamsters) exposed to 50 and 250 mg/m 3 fine TiO 2 or 10 mg/m 3
nano-TiO 2

. Lung TiO 2 burdens and tissue responses in mice, rat and hamsters exposed for 13 wk to 10 mg/m 3 nano-TiO 2 or to 50 mg/m 3 fine TiO 2 were similar for all three species.

5-38Animal Testing Material Treatment Conditi ons Summary of Major Effects Reference

Nano-TiO 2 (P25), photocatalytic, 80% anatase/20% rutile, primarily particle size 15-40 nm, 0.8 μ m MMAD Creutzenberg et al. (1990, Rats: Rat Female Wistar Chronic inhalation Increases in lung weight, and retention of inhaled nano-TiO 2 in lungs and lung-associated lymph nodes (mean lung retention was 39.3 mg/lung at the end of exposure). The retention sl owly decreased postexposure (from 40 mg/lung after 18 mo of nano-TiO 2 exposure to 3.3 mg/lung at 4 mo postexposure). Rats: 24 mo exposure: 7.2 mg/m 3 for the first 4 mo, followed by 14.8 mg/m 3 for 4 mo, 9.4 mg/m 3 for 16 mo, and clean air for 6 mo (concentration sometimes are reported as 7.5, 15, 10 mg/m 3 ) 157963 )

Mouse Female NMRl

Increased half-time of pulmo nary clearance of tracer particles 18 or 19 hr/day, 5 days/wk in whole body chamber For inhaled 0.35 μm labeled tracer particles Mice: 13.5-mo exposure: Same treatment as in rats for the first 8 mo, followed by 9.4 mg/m 3 for 5.5 mo, and clean air for 9.5 mo After 3-, 12-, 18-mo nano-TiO 2 exposure and 18-mo exposure plus 3-mo recovery, clearance half times were 208, 403, 357, and 368 days, respectively. The controls had 61-96 days for all time points. For inhaled 3.5 μm labeled tracer particles
After 3-, 12-, 18-mo nano-TiO 2 exposure and 18-mo exposure plus 3-mo recovery, clearance half times were 1,222, 229, 58 and 48 days, respectively. The controls had 58-70 days for all time points.

The decreases in clearanc e half time after 12- and 18-mo exposure, compared to controls, was possibly dye to increases in lung weight, altered lung structureand breathing pattern, which lead to more in the tracheobronchial region of the long and apparently higher clearance rates.

Gallagher et al. (1994, Rats did not have increases in DNA adducts in the lung: 045102 No increases in DNA adduct 2 (nuclease P1-sensitive adduct) in the lung. )

Decreases in DNA adduct 1 (age-related, putative I-compound) in peripheral l ung DNA compared to filtered air-exposed rats, probably due to adduct dilution through cell proliferation induced by particle exposure.

Heinrich et al. (1995, Rats: 076637 Increased mortality (60% vs. 42% in control) and lung wet weight, decreased mean lifetime and BW. )

Increased incidence of lung tu mors [18-mo exposure: 5 out of 20 rats exposed to TiO 2 (0 out of 18 in control) had lung tumors. 24-mo exposure: 4/9 rats in TiO 2 (0/10 in control)].

Mice: No increase lung tumors. Increased mortality (33% vs. 10% in control) and lung wet weight, decreased BW. Carcinogenic in rats, but not in mice.

5-39Animal Testing Material Treatment Conditi ons Summary of Major Effects Reference

Nano-TiO 2 (Nanostructured and Amorphous Materials), anatase, measured average primary particle size 3.5 ± 1.0 nm, BET surface area 219 ± 3 m 2 /g, surface functionalization: O, O-H, H 2 O (manufacturer reported primary particle 5 nm, surface area 210 m 2 /g) Acute inhalation Mouse Male C57BL/6 No adverse effect/Minimal pulmonary inflammation. Grassian et al. (2007, 090606

Aerosol size geometric mean 120-128 ± 1.6- 1.7 nm for acute (two concentrations) and subacute (one concentration) exposures Doses – 0, 0.77, or 7.22 mg/m 3
Single exposure of 4 hr in whole-body chamber No recovery time No treatment effects on most parameters measured to gauge inflammatory response (neutrophil number in BAL fluid, total protein, and L DH activity were not changed), and no effects on lung histopathology. )

Increased total cell count and macrophage count in BAL fluid at highest dose.

Moderate but significant pulmonary inflammatory response that lasted for at least 2 wk but resolved by wk 3 after exposure. Subacute inhalation Doses – 0 or 8.88 mg/m 3
4 hr/day for 10 days in wholebody chamber No changes in most para meters measured to gauge inflammatory response [total protein, LDH activity, and cytokine (IFN- γ , IL-6, or IL-1 β ) concentrations in BAL fluid were not changed, and no effects on lung histopathology. 0, 1, 2, or 3 wk of recovery before sacrifice

Increased macrophage count in BAL fluid in treated group at wk 0, 1, and 2 pos texposure but not at wk 3 postexposure. Macrophages in BAL fluid were loaded with TiO 2 particles and less so at wk 3 postexposure.

Absolute lung weight was increased at 50 mg/m 3 immediately after exposure, but not after 16-day
recovery. Ma-Hock et al. (2009, Short-term inhalation Rat Male Wistar Nano-TiO 2 (Baker & Collinson, Inc.), uncoated, 14% rutile/86% anatase, hydrophobic surface, average primary particle 25.1 ± 8.2 nm (range 13 to 71 nm) measured under TEM. BET surface area 51.1 ± 0.2 m 2 /g. Zeta potential was 16.5 ± 2.2 mV in 1 mM KCl. 193534

Aerosols: 0.7-1.1 μ m MMAD (geometrical standard deviations 2.3-3.4). Small and large agglomerates in the atmospheres, ranging from below 100 nm to several hundred nm. Estimated number concentrations of particles <100 nm represents only 0.1-0.4% of the total particle mass for all three atmospheres. 0, 2, 10, and 50 mg/m 3 (actual concentrations 0, 2.4, 12.1, and 50.0 mg/m 3 ), 6 hr/ for 5 days, head-nose exposures to dust aerosols )

Lung burden: 118.4, 544.9 and 1,635 μg/lung immediately after inhalation of 2, 10 and 50 mg/m 3
nano-TiO 2 , respectively. 16 days of recovery later, the lung burdens were 93.4, 400.4 and 1,340 μg/lung, respectively. Calculated clearance half-times were 47, 36 and 56 days for 2-, 10- and 50-mg/m 3 groups, respectively. No recovery (immediately after the last exposure [0 days]), 3- or 16-day recovery after the last exposure. In other words, necropsy on study days 5, 8, and 21, respectively. In the mediastinal lymph nodes, TiO 2 was only detected in the 50-mg/m 3 group, and the nano-TiO 2 concentrations were higher at 16 days after the last exposure (mean 11.01 μ g in collected lymph nodes) than immediately after exposure (mean 2.34 μg). No TiO 2 was detected in the liver, kidney, spleen or basal brain with olfactory bulb (detection limit 0.5 μg per organ).

BAL fluid: increases in to tal cell count at 50mg/m 3 and polymorphonuclear neutr ophils at 10 mg/m 3 and 50 mg/m 3 , but no changes in eosinophil, lymphocyte, or macrophage counts, total protein content, enzyme activities, and levels of 9 (out of tested 60) cell mediators. Among the 9 medi ators, effects were only observed at 10 mg/m 3 or higher immediately after exposure. After 3 days of recovery, effects were still observed, but for clusterin and haptoglobin, they were observed at 2 mg/m 3 . Cell mediator levels were the same as controls after 16 days of recovery in 2 and 10 mg/m 3 groups, but not in 50 mg/m 3 group. Clinical pathology in blood: minor effects on serum cell mediator. No increase in serum troponin I, a biomarker for myocardial damage in rodents. Increased cell replication in large/medium bronchi and terminal bronchioles at all three groups immediately after exposure and after 3 days of recovery (not after 16 days). Macrophage diffusion also decreases over time. No change in lung cell apoptosis. Changes were most prominent immediately after the last exposure or 3 days afterward, and some endpoints returned to control levels by 16 days of recovery.

5-40Animal Testing Material Treatment Conditi ons Summary of Major Effects Reference

Nano-TiO 2 caused more severe and prolonged (~1 yr) pulmonary inflammatory re sponse (i.e., increase in alveolar macrophages, polym orphonuclear neutrophils, and lavagable protein) than fine TiO 2

. Subchronic inhalation Rat Male F344 Nano-TiO 2 , 20 nm, anatase (Degussa); in aerosols: agglomerates 0.71 ± 1.9 μm MMAD Oberdörster et al. (1994, Nano-TiO 2 : 23.5 ± 2.9 mg/m 3 ; fine TiO 2 : 22.3 ± 4.2 mg/m 3 046203 )

6 hr/day, 5 days/wk, for 12 wk Fine TiO 2 , 250 nm, anatase (Fisher Scientific); in aerosols: agglomerates 0.78 ± 1.7 μm MMAD When inflammatory response was expressed as number of polymorphonuclear neutrophils and dose was expressed as surface area for retained particles (i.e., lavagable particles), nano-TiO 2 and fine TiO 2 shared the same dose response curve. Recovery for 4, 8, 12, 29 or 64 wk before sacrifice

More severe and prolonged impairment of alveolar macrophage-mediated particle clearance in rats exposed to nano-TiO 2 than rats exposed to fine TiO 2 . Seven mo after TiO 2 exposure, fine TiO 2 exposed (but not nano-TiO 2 exposed) rats showed normal clearance rates. Pathological changes in the lung: nano-TiO 2 caused greater epithelial effects (Type II cell proliferation, occlusion of pores of Kohn) and more interstitial fibrotic foci than fine TiO 2

. Dosimetry:

Nano-TiO 2 and fine TiO 2 had a similar mass deposition in the lower respiratory tract and same retention in the alveolar space up to 1 yr after exposure.

Nano-TiO 2 showed longer total pulmonary retention (retention halftime: ~500 days for nano-TiO 2 , ~170 days for fine TiO 2 ), more translocation to the pulmonary interstitium and regional ly mph nodes, a greater fraction being retained, and a larger fr action of alveolar burden in the interstitium (suggesting nano-TiO 2 depends mainly on mucocillary clearance, while fine-TiO 2 depends on clearance to the gastrointesti nal tract) than fine TiO 2

.

5-41Animal Testing Material Treatment Conditi ons Summary of Major Effects Reference

Ti and S distribution in tissues: Immediately after 5-day inhalation/after 14-day recovery Short-term inhalation: 6 hr/dayfor 5 consecutive days, headnose exposure Rat Male Wistar, Strain Crl: WI(Han) Nano-TiO 2 , 20-30 nm (measured by TEM), 70% anatase, 30% rutile, BET surface area 48.6 m 2 /g, uncoated, isoelectric point (IEP) was pH 7 in 10 mM KCl, MMAD 1.0 μm in aerosol van Ravenzwaay et al. (2009, 193689

Fine TiO 2 , median size 200 nm in ethanol (measured by DLS), rutile, BET surface area 6 m 2 /g, IEP <pH 3 in 10 mM KCl (Kronos International), MMAD 1.1 μm in aerosol
Quartz dust DQ12, median size 315 nm in ethanol, BET surface area 5.9 m 2 /g, IEP <pH 3 in 10 mM KCl (Doerentrup Quarz GmbH, Germany), MMAD 1.2 μm in aerosol Aerosol concentration (mg/m 3 ):

Nano-TiO 2 : target 100 (measured concentration 88.0 ± 6.4)

Fine TiO 2 : 250 (measured 274.0 ± 30.5)

Quartz dust DQ12: 100 (measured 96.0 ± 5.4). Count concentration of particles <100 nm (particles/cm 3 ):

Nano-TiO 2 : 205,920

Fine TiO 2 : 54.600
Quartz dust DQ12: 21.292

Calculated mass fraction measured <100 nm:

Nano-TiO 2 : 0.5%

Fine TiO 2 : 0.05%

Quartz dust DQ12: 0.03%

For distribution of the tested substance in the body, the following tissues were tested immediately after the last exposure and after 14-day recovery: lung, mediastinal lymph nodes, liver, kidney, spleen and basal brain with olfactory bulb (3 rats/group/time point) BAL at 3 or 14 days after the last exposure (5 rats/group/time point) Histological examination (6 rats/group/time point) and TEM of lung and mediastinal lymph nodes (3 rats/group/time point): immediately after the exposure and after 14-day
recovery Nano-TiO 2 : 2,025/1,547 μg TiO 2 in lung, 2.2/8.5 μg TiO 2 in mediastinal lymph nodes. )

Fine TiO 2 : 9,182/7,257 μg TiO 2 in lung, 8.2/108 μg TiO 2 in mediastinal lymph nodes. Quartz DQ 12: 2,190/1,975 μg quartz in lung, 19/56 μg quartz in mediastinal lymph nodes.

No TiO 2 or quartz were detected in any groups in liver, kidney, spleen, or basal brain with olfactory bulb (detection limits: 0.3 μg Ti = 0.5 μg TiO 2 per tissue, 5 μg Si = 11 μg SiO 2 per tissue). Deposition of inhaled fine and nano-TiO 2 in lung:

Fine and nano-TiO 2 were mainly in the lumen of the alveoli and bronchi (extracellular) and some were in the cytoplasm of alveolar macrophages.

Nano-TiO 2 was mostly agglomerates in lung, and agglomerates were roughly the same size as those in the atmosphere. No sign of disagglomeration of the inhaled agglomerates. Biological effects of fine TiO 2 , nano-TiO 2 and quartz: All treated groups:

BAL had increased total cell count (most increases in polymophonuclear neutrophils, slight increases in lymphocytes and monocytes); increased total protein; increased activity lactate dehydrogenase, alkaline phosphatase, γ -glutamyltransferase and N-acetyl- β

glucosaminidase. The changes in BAL parameters in the quartz group were not revers ible, but changes in fine and nano-TiO 2 groups were partly reversible by 14 days of recovery. Lung: diffuse histiocytosis

Nano-TiO 2 group: Reversible increases in absolute lung weight; mild neutrophilic inflammation in lung; inflammation declined by 14 days of recovery; lymphoreticulocellular hyperpl asia in the mediastinal lymph nodes.

Fine TiO 2 group: Reversible incr eases in absolute lung weight; particle-loaded macrophages in the mediastinal lymph nodes. Quartz: Increase absolute lung weight, which maintained throughout recovery; multifocal infiltration of granulocytes in lung; after recovery time, pulmonary histological changes increased severity, and mediastinal lymph nodes had increased macrophage number and granulomatous inflammation.

5-42Animal Testing Material Treatment Conditi ons Summary of Major Effects Reference

Short-term inhalation Nurkiewicz et al. (2008, Rats FemaleSpragueDawley, Hla:(SD)CVF Fine TiO 2 , primary particle <5 μm, 99% rutile (reported vendor), BET surface area 2.34 m 2 /g (reported in Sager et al., 2008, Histology of the lung: 156816

157499 ) (SigmaAldrich, product #224227); MMAD of the aerosols 402 nm with a GSD of 2.4, CMD of the aerosols 710 nm

Nano-TiO 2 (P25), primary particle 21 nm, 80% anatase, 20% rutile (reported by vendor), BET surface area 48.08 m 2 /g (reported in Sager et al., 2008, 157499 ); MMAD of the aerosols 138 nm with a GSD of 2.2, CMD of the aerosols 100 nm Whole body chamber exposure Exposures selected which do not alter BAL markers of pulmonary inflammation or lung damage Exposure to fine TiO 2 : aerosol concentration × exposure time (actual deposition in lung) 15 mg/m 3 × 480 min (90 μg) 16mg/m 3 × 300 min (67 μg) 12 mg/m 3 × 240 min (36 μg) 6 mg/m 3 × 240 min (20 μg) 3 mg/m 3 × 240 min (8 μg) Exposure of nano-TiO 2 : aerosol concentration × exposure time (calculated/actual deposition in lung) 10 mg/m 3 × 720 min that took place over 3 days (38 μg) 12 mg/m 3 × 240 min (19 μg) 6 mg/m 3 × 240 min (10 μg) 3 mg/m 3 × 480 min (10 μg) 12 mg/m 3 × 120 min (10 μg) 3 mg/m 3 × 240 min (6 μg) 1.5 mg/m 3 × 240 min (4 μg) Sham exposure (control): 0 mg/m 3 × 240 min 24 hr postexposure, sample collection, including exteriorizing spinotrapezius muscle with rats under anesthesia while leaving its nerves supply and all feed vessels intact for the test of arteriolar dilation No significant inflammation. )

Particle accumulation in alveolar macrophage. Anuclear alveolar macrophages were seen in both nano-TiO 2 and fine TiO 2 exposed rats, but not in sham-exposed rats. Anuclear alveolar macrophages are presumed to be an apoptotic change. Endothelium-dependent arteriol ar dilation as measured after intraluminal infusion of the Caionophore A23187 in exteriorized spinotrapezius muscle:

Both fine TiO 2 and nano-TiO 2 exposures impaired arteriolar dilation in a dose-dependent manner, and nano-TiO 2 exposure produced greater impairment than fine TiO 2 at similar pulmonary load doses. No-effect dose of fine TiO 2 was 8 μg (as in lung deposition), and for nano-TiO 2 was 4 μg.
On a mass base, nano-TiO 2 was approximately one order of magnitude more potent than fine TiO 2 ; on total particle surface area base calculated by BET surface area, fine TiO 2 would be more potent than nano-TiO 2 (the authors suspected overestimation of the total nano-TiO 2 surface area delivered, since no agglomeration was considered).

Additional nano-TiO 2 exposure conditions (12 mg/m 3 × 2 hr; 4 mg/m 3 × 6 hr; 8 mg/m 3 × 3 hr) yielded the same level of impairment of systemic arteriolar dilation, suggesting the response is dependent on the exposure concentration (of product) × time.

Nurkiewicz et al. (2009, Same impairment of arteriolar dilation at 67 μg fine TiO 2 and 10 μg nano-TiO 2 more than 50% decrease compared to sham treated controls after Ca 2+ ionophore A23187 injection at 20 and 40 psi ejection pressures. Same exposure conditions as above (Nurkiewicz et al., 2008, 191961 156816 ) for endogenous microvascular NO production tests, but only three groups in all other tests: aerosol concentration × exposure time (actual deposition in lung) )

No change in arteriolar dilati on in response to sodium nitroprusside (NO donor) in either 67 μg fine TiO 2 or 10 μg nano-TiO 2 exposed rats, indicating no change in vascular smooth muscle sensitivity to NO. Sham exposure (control): 0 mg/m 3 × 240 min Increased ROS amount in the microvascular wall in both 67 μg fine TiO 2 and 10 μg nano-TiO 2 groups at the same level as measured by ethidi um bromide fluorescence. Fine TiO 2 : 16mg/m 3 × 300 min (67 μg) Increased nitrotyrosine ex pression in 10 μg nano-TiO 2 treated rats (not measured in fine TiO 2 group) in lung (3 folds) and spinotrapezius microcirculation (4 folds), as compared to sham exposur e, suggesting nitrosative injury in lung and systemic microcirculation. Nano-TiO 2 : 6 mg/m 3 × 240 min (10 μg) 24 hr postexposure, sample collection, including and exteriorizing spinotrapezius muscle as described in Nurkiewicz et al. (2008, Decreased Ca 2+ ionophore A23187-stimulated endogenous microvascular NO production in fine TiO 2 and nano-TiO 2 treated groups in a dose-dependent manner: Similar to sham cont rol, the NO production was sensitive to nitric oxide synthase inhibition caused by NG-monomethyl-L-arginine. 156816 ) and excising spinotrapezius muscles from separate groups of rats for measurement of NO, microvascular oxidative stress, and nitrotyrosine staining Radical scavenging (by superoxide dismutase mimetic 2,2,6,6-tetramethylpiperid ine-N-oxyl and catalase); inhibition of NADPH oxidase (by apocynin); and inhibition of myeloper oxidase (by 4-aminobenzoic hydrazide) all restored stimulated NO production and partially restored arteriolar dilation (stimulated by Ca 2+ ionophore A23187) in 67 μg fine TiO 2 and 10 μg nano-TiO 2 groups.

5-43Animal Testing Material Treatment Conditi ons Summary of Major Effects Reference

Instillations

Chen et al. (2006, Single intratracheal instillation Gross morphology and histology of the lung: Emphysema-like lung injuries were seen at 0.1 and 0.5 mg/mouse (more severe at 0.5 mg) at 3 days, 1 wk, and 2 wk after the instillation. Mouse Male ICR Nano-TiO 2 (Degussa), rutile, highly dispersed and hydrophilic fumed nano-TiO 2 , diameter 19-21 nm (average primary particle size 21 nm), surface area of 50 ± 15 m 2 /g, purity

≥ 99.5% 090139

To avoid aggregation, the nano-TiO 2 suspension was ultrasonicated before it was used to treat animals or cells; each sample was vortexed just before an aliquot was drawn for instillation. However, authors did not report the sizes of aggregates before or after sonication. 0, 0.1, or 0.5 mg/mouse )

3 days (for hyper-acute response), 1 wk (acute) or 2 wk (chronic) of recovery before sacrifice Pulmonary changes included disruption of alveolar space, alveolar enlargement, proliferation of alveolar type II pneumocyte, increases in alveolar epithelial thickness, and accumulation of particle-laden macrophages. 1 wk after instillation, 0.1 mg/mouse increased alveolar macrophage infiltration, type II pneumocyte proliferation, and apoptosis in macrophage and type II pneumocyte. Gene expression in lung 1 wk after instillation of 0, 0.1, and 0.5 mg/mouse: cDNA microarray showed up-regulation in pathways involved in cell cycle regulation, apoptosis, chemokines, and complementary cascades. RT-PCR showed up-regulati on in plgf, chemokines (cxcl1, cxcl5, and ccl3), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and prostaglandin E receptor 4. Western blot and ELISA showed increases in placenta growth factor (PIGF) prot ein (a prechemokine that regulates the expression of several chemokines, leading to inflammatory cascade) in cells and in serum.

Increased primary benign tumo rs and malignant cancers in lung in all tested doses. Mohr et al. (2006, Rat Female Wistar (HsdCpb:WU) Nano-TiO 2 (P25), photocatalytic, hydrophilic, 80% anatase/20% rutile, primarily particle size 25 nm, BET specific surface area 52 m 2 /g Repeated weekly intratracheal instillation 097493 )

Pott and Roller (2005, Instilled doses: 5 instillations × 3 mg 5 instillations × 6 mg 10 instillations × 6 mg 157790 ) a

Nano-TiO 2 (Degussa T805 /P805), a crystal form not specified, coated with an organic silicon compound; 21 nm; 32.5 m 2 /g Repeated weekly intratracheal instillation High initial acute mortality, lowered dose to 0.5 mg. No conclusion on carcinogenicity. Instilled doses: 15 instillations × 0.5 mg 30 instillations × 0.5 mg

Fine TiO 2 , hydrophilic, anatase, primary particle 200 nm, BET specific surface area 9.9 m 2 /g Repeated weekly intratracheal instillation Increased primary benign tumo rs and malignant cancers in lung in all tested doses.

Instilled doses: 10 instillations × 6 mg 20 instillations × 6 mg

Single intratracheal instillation (acute effects) Nemmar et al. (2008, Rat Male Wistar Nano-TiO 2 , rutile, primary particle diameter 4-6 nm, rod shape (synthesized in the lab by a soft chemistry technique); BET surface for instilled nano-TiO 2 rods was 14.64 cm 2 for dose of 1 mg/kg, 82.30 cm 2 for 5 mg/kg. Aggregates appeared to be in a radial arrangement and usually less than 1 μm. Pulmonary inflammation: increases in macrophage and neutrophil numbers in BAL fluid at 5 mg/kg. most nano-TiO 2 aggregates in BAL fluid were inside macrophages. 157514 ) 1 or 5 mg/kg nano-TiO 2 or vehicle only (150 μL) Pulmonary and cardiac edema: increases in the wet weight-to-dry weight ratios of lung and of heart at 1 and 5 mg/kg. Single intratracheal instillation

nano-TiO 2 was suspended in saline containing 0.01% Tween 80 (a surfactant and emulsifier) Systemic inflammation: increases in monocyte and granulocyte (but not lympho cyte) numbers in blood at 5 mg/kg. Blood collection and necropsy at 24 hr after instillation Platelet aggregation: decreases platelet number in blood of rats exposed to 5 mg/kg nano-TiO 2 , suggesting platelet aggregation [in vitr o supporting evidence: adding 2 or 10 μg/mL (but not 0.4 μg/mL) nano-TiO 2 directly into untreated rat whole blood c aused platelet aggregation].

5-44Animal Testing Material Treatment Conditi ons Summary of Major Effects Reference

Rats Male F344 Nano-TiO 2 , ~20 nm, anatase Anatase nano-TiO 2 induced more inflammatory response and higher interstitial access in the lung than anatase fine TiO 2 of the same mass dose. Oberdörster et al. (1992, Single intratracheal instillation (acute effects) 045110 Fine TiO 2 , ~250 nm, anatase 500 μg of either anatase nano-TiO 2 or anatase fine TiO 2 )

A single intratracheal instillation, followed by 24-hr recovery

Single intratracheal instillation (acute effects) Nano-TiO 2 , ~20 nm, anatase (free anatase nano-TiO 2 ) Free anatase nano-TiO 2 and serum-exposed anatase nano-TiO 2 caused pulmonary inflammatory reaction (same level) and interstitial distribution. Free anatase nano-TiO 2 , 104 μg Phagocytized anatase nano-TiO 2 alone did not contribute significantly to inflammatory reaction, because the reaction can be explained by the alveolar macrophages and polymorphonuclear neutrophils. Alveolar macrophage collected 24 hr after donor-rat received 200 μg anatase nano-TiO 2 via intratracheal instillation (containing phagocytized anatase nano-TiO 2 ) Phagocytized anatase nano-TiO 2 104 μg + 9.5 × 106 alveolar macrophages + 3.9 × 106 polymorphonuclear neutrophils Phagocytized anatase nano-TiO 2 showed less interstitial distribution than free anatase nano-TiO 2

. Alveolar macrophages 6.8 × 106 Alveolar macrophage collected from untreated rat lung Polymorphonuclear neutrophils 2.2 × 106 PMNs from peripheral blood of untreated rats Serum-exposed anatase nano-TiO 2 100 μg Serum-exposed anatase nano-TiO 2 (incubated in rat serum for 1 hr and then washed twice) A single intratracheal instillation, followed by 24-hr recovery

When inflammatory response was expressed as number of PMN and dose was expressed as surface area for retained particles (i.e., lavagable particles), all particles shared the same dose-response curve, except anatase and rutile nano-TiO 2 at high doses. A single intratracheal instillation of 500 μg each; anatase fine TiO 2 was also tested at 1,000 μg; anatase nano-TiO 2 was also tested at 65, 107, 200, and 1,000 μg Fine TiO 2 , ~250 nm, anatase

Nano-TiO 2 , ~20 nm, anatase

Fine TiO 2 , ~220 nm, rutile (from Dr. Siegal at Argonne National Laboratory, Argonne, IL) When inflammatory response was expressed as lavage protein and dose was expressed as retained particle surface area, all particles shared the same dose
response curve. 24-hr recovery

Nano-TiO 2 , ~12 nm, rutile Higher fractions of nano-TiO 2 (anatase and rutile nano-TiO 2 ) were interstitialized (translocated into interstitium or epithelium cells) than other particles. Carbon black, ~30 nm (Cabot, 660R)

5-45Animal Testing Material Treatment Conditi ons Summary of Major Effects Reference

Single Intratracheal instillation (acute effects) Rat Nano-TiO 2 , ~20 nm, surface area is estimated to be 10 times of surface area of ~250 nm TiO 2 Pulmonary inflammation (neutrophil % in lung lavage) was seen at 24 hr postexpos ure. At the same mass dose, nano-TiO 2 induced more inflammation than fine TiO 2 . When doses are expressed as surface area, fine TiO 2 and nano-TiO 2 shared the same dose-response

curve. Oberdörster (2000, 036303 [strain/stock not specified] )

Nano-TiO 2 : 30, ~150, 500 μg

Fine TiO 2 : ~150, 500, 2,000 μg Fine TiO 2 , ~250 nm

Repeated inhalation of PTFE fume (5 × 105 particles/cm 3 = ~50 μg/cm 3 , 5 min/day for 3 days) followed by a single intratracheal instillation of 100 μ g nano-TiO 2 Nano-TiO 2 Cross tolerance: nano-TiO 2 induced less pulmonary inflammation (neutrophil % in BAL fluid) in rats that had adapted to PTFE fumes for previous 3 days than in rats that were not adapted (not exposed to PTFE fume). The author suggested this cross to lerance is from adaptation to oxidative stress. Polytetrafluoroethylene (PTFE) (Teflon) fume, count median diameter ~18 nm

Nano-TiO 2 , ~20 nm Inhalation of LPS followed by a single intratracheal instillation of nano-TiO 2 and fine TiO 2 (acute effects) LPS alone: mild pulmonary inflammation (~10% neutrophil in lung lavage at 24 hr postexposure). The treatment of LPS was to mimic an early stage of infection with gram negative bacteria (compromised host). Fine TiO 2 , ~250 nm Lipopolysaccharide (LPS), an endotoxin found in gram negative bacteria LPS: ~12 min exposure, ~70 endotoxin units (estimated alveolar dose) 50 μ g nano-TiO 2 , but not fine TiO 2 , further increased inflammatory response in compromised hosts with mild pulmonary inflammation. Nano-TiO 2 and fine TiO 2 : 50 μg Neutrophil % in rats exposed to (LPS and then nano-TiO 2 ) > (LPS and then fine TiO 2 ), LPS alone, nano-TiO 2 alone > fine TiO 2 alone, negative control. Within 30 min of inhalation of LPS or saline, intratracheal instillation of nano-or fine TiO 2 It is unclear whether fine TiO 2 at a dose that increases inflammatory response would further increase inflammatory response in compromised hosts. 24 hr of recovery

Rehn et al. (2003, Transient pulmonary inflamma tory responses to both types of nano-TiO 2 (mostly only at 1.2 mg dose, some at 0.6 mg groups) (most responses returned to normal by day 90). Single intratracheal instillation (subchronic effects) Rat Wistar Nano-TiO 2 (P25), photocatalytic, 80% anatase/20% rutile, untreated, hydrophilic surface, primarily particle size ~20 nm 090613 )
Doses: 0, 0.15, 0.3, 0.6, or 1.2 mg nano-TiO 2 (positive control: 0.6 mg quartz DQ12) in 0.2 mL saline supplemented with 0.25% lecithin P25 induced more pulmonary inflammatory responses than T805 in some tests, but T805 induced more proliferation changes in the lung (as percentage of Ki67- positive cells) than P25 on days 3 and 21. Nano-TiO 2 (Aeroxide® T805), photostable, 80% anatase/20% rutile, silanized,trimethoxyoctylsilanetreated hydrophobic surface, primarily particle size ~20 nm 3, 21, or 90 days of recovery Neither P25 nor T805 in creased oxidative DNA adduct (as 8-oxoguanine) in the lung on day 90. Quartz induced persistent inflammatory response and increased 8-oxoguanine on day 90.

Crystalline silica and quartz particles (DQ-12) as positive reference

Renwick et al. (2004, Single intratracheal instillation (acute effects) Nano-TiO 2 at 500 μ g (but not nano-TiO 2 at 125 μ g or fine TiO 2 at either 125 or 500 μ g) increased neutrophil number (inflammation), LDH ac tivity (cytotoxicity), GGT activity (epithelial da mage), total protein in bronchoalveolar lavage fluid (membrane permeability), and macrophage activity to migr ate toward chemotaxin C5a (chemotaxis). Rat Male Wistar Nano-TiO 2 (Degussa), mean diameter 29 nm, BET surface area 49.78 m 2 /g 056067 )
0, 125, and 500 μ g particles in saline Fine TiO 2 (Tioxide Ltd), mean diameter 250 nm BET surface area 6.6 m 2 /g 24 hr of recovery before sacrifice

Both nano- and fine TiO 2 (at 500 μ g, but not at 125 μ g) decreased phagocytic func tion of macrophage. Carbon black, mean diameter 260.2 nm, BET surface are 7.9 m 2 /g Carbon black caused same changes as fine TiO 2 , with the exception of increases in LDH activity at 500 μ g. Ultrafine carbon black, mean diameter 14.3 nm, BET surface 253.9 m 2 /g Ultrafine carbon black caused same changes as nano-TiO 2 , but increases in inflammation and LDH and GGT activities were significant at 125 μ g (nano-TiO 2 caused significant changes at 500 μ g only).

5-46Animal Testing Material Treatment Conditi ons Summary of Major Effects Reference

Like fine TiO 2 , nano-TiO 2 rods and nano-TiO 2 dots caused only transient pulmo nary inflammation, and not significant lung toxicity. Warheit et al. (2006, Single intratracheal instillation (subchronic effects) Rat Male Crl:CD(SD)IGS BR Fine TiO 2 (DuPont): primary particle ~300 nm, anatase, ~99 wt % TiO 2 /~1 wt % alumina, BET surface area ~6 m 2 /g (R-100) 088436 ) 0, 1 or 5 mg/kg of each testing material in PBS with polytron dispersant All 5 mg/kg TiO 2 (fine, nano rods, and nano dots), but not 1 mg/kg TiO 2 , caused transient, s hort-lived inflammation (increases in neutrophil % in BAL fluid at 24 hr postexposure only; increas es in LDH by 5 mg/kg nano-TiO 2 rods at 24 hr postexposure only). BAL fluid analysis at 24 hr, 1 wk, 1 mo, and 3 mo postexposure (5 rats per group per dose per time point) Nano-TiO 2 rods (synthesized hydrothermally): primary particle length 92-233 nm × width 20-35 nm, anatase, BET surface area 26.5 m 2 /g No changes in lung wei ght, tracheobronchial cell proliferation (measured in high dose groups only) or lung morphology (pathological changes). Morphological studies at the same time points (4 rats per group per high dose per time point; 4 rats per group per low dose for the first two time points) TiO 2 in macrophages was seen in all three types of TiO 2

. Transient lung parenchymal cell proliferation in low and high fine TiO 2 at 1 wk postexposur e (different from previous studies in similar conditions). Nano-TiO 2 dots (synthesized hydrothermally): primary particle diameter 5.8-6.1 nm, sphere, anatase, BET surface area 169.4 m 2 /g Quartz caused sustained pulmonary inflammation and early sign of pulmo nary fibrosis. Sustained pulmonary inflammation (increases in neutrophil % in BAL fluid at 1 mg/kg at 24 hr after exposure, 5 mg/kg at all time points) (increases in LDH at 5 mg/kg at all time points) (increase in neutrophils and foamy alveolar macrophages). Quartz (Min-U-Sil quartz): median primary particle ~1.5 μm (range 1 to 3 μm), crystalline silica, BET surface area 4 m 2 /g Prelude of fibrosis (thickening of lung tissue) (persistent lung parenchymal cell prolifer ation at 5 mg/kg at 1 mo and 3 mo postexposure). Absolute lung weight was increased at 5 mg/kg at 1 wk, 1 mo, and 3 mo postexposure. Increased tracheobronchial cell proliferation at 5 mg/kg (not measured in low dose) at 24 hr postexposure only.

No sustained adverse pulmonary effects for photostable nano-TiO 2 (both types of coated rutile). Single intratracheal instillation (subchronic effects) Warheit et al. (2007, Rat Crl:CD®(SD)IG S BR Nano-TiO 2 (DuPont), photostable, rutile, coated with alumina, (~98% TiO 2 , ~2% alumina), average particle size of 136 nm in water and average BET surface area of 18.2 m 2 /g (uf-1) 090594 ) 0, 1, or 5 mg/kg Pulmonary inflammation and cytotoxic effects at highest exposure of photocatalytic nano-TiO 2 increased bronchoalveolar lavage fluid LDH and BAL fluid microprotein concentrations. 90-day recovery period

Increased tracheobronchial and lung parenchymal cell proliferation rates at highes t exposure of photocatalytic nano-TiO 2

. Nano-TiO 2 (P25) (Evonik), photocatalytic, 80% anatase/20% rutile, not coated, average particle size 129.4 nm in water, average BET surface area 53.0 m 2 /g Lung inflammation/cytotoxicit y/cell proliferation and histopathological responses: quartz > nano-TiO 2 P25 (anatase and rutile) > fine TiO 2 (rutile) = nano-TiO 2 uf-1 (rutile) = nano-TiO 2 uf-2 (rutile).

Nano-TiO 2 (DuPont), photostable, rutile, coated with silica and alumina surface coating (~88 wt % TiO 2 , ~7 wt % amorphous silica and ~5 wt % alumina), average particle size of ~149.4 nm in water, average BET surface area 35.7 m 2 /g (uf-2)

Fine TiO 2 (DuPont), photostable, rutile, coated with alumina (~99% TiO 2 and ~1% alumina), an average particle size 382 nm in water, average BET surface area 5.8 m 2 /g

Quartz

5-47Animal Testing Material Treatment Conditi ons Summary of Major Effects Reference

Wang et al. (2008, Repeated intranasal instillation Mouse Female CD1(ICR) Nano-TiO 2 (Hangzhou Dayang Nanotechnology Co. Ltd.), rutile, 80 nm, measured average size 71.4 ± 23.5 nm, purity >99% TiO 2 distribution (measured after 15 instillations): first into olfactory bulb, and then to hippocampus. Ti concentrations: hippocampus, olfactory bulb > cerebellum, cerebral cortex > thalamus. 157474

Fine TiO 2 (Zhonglian Chemical Medicine Co.), anatase, 155 nm, measured average size 155.0 ± 33.0 nm, purity >99% ~500 μg TiO 2 in pure water per mouse every other day for 2, 10, 20, or 30 days (1, 5, 10 or 15 instillations, respectively) ) Wang et al. (2008, 157473

Necropsy 1 day after last instillation

For translocation of TiO 2 into brain: 6 mice per group for each time point. For effects in brain: 10 mice per
group Serum biomarkers for liver function (ALT, AST, ALP), kidney function and cholestero l levels: No consistent change. Only changes were increased ALT (80-nm group after 1 and 5 instillations, 155-nm group after 5 instillations), increased AST (80-nm group after 5 instillations) and increase ALP (155-nm group after 1 instillation). )

Pathological changes in kidney: atrophy of renal glomerulus, infiltration and dw indling of interstitially inflammatory cells in the lumen of Bowman’s capsules. No changes in organ weight. No pathological changes in heart, liver, spleen, cerebral cortex or cerebellum. No change in proinflammatory cytokine TNF- α in serum.

Brain:

Oxidative stress: GSH-Px and GST activities and GSH levels were increased in the 80-nm group after 5 instillations, but not in other groups or other time points. Malondialdehyde levels (indicator for lipid peroxidation) and soluble protein carbonyl content (indicator for protein oxidation; measured only after 15 instillations) were increased in both the 80- and 155-nm groups after 15 instillations. SOD activity was decreased in 155 nm after 15 instillations. Catalase activity (measured only after 15 instillations) was increased in the 80- and 155-nm groups. Pathological changes in olfactory bulb and C1A regions of hippocampus: Olfactor y bulbs showed increased neuron numbers, irregular a rrangement of neuron cells, and ultra-structural changes in both the 80- and 155-nm groups. CA1 region of the hippocampus showed enlarged and elongated pyramidal cell soma, dispersed arrangement and loss of neurons, fewer Nissl bodies, fewer mitochondria, and increased rough endoplasmic reticulum. Astrocytes may be damaged (only measured after 15 instillations): Hippocampus had increased glial fibrillary acidic protein (GFAP) levels, particularly in CA4 region. Activity of cholinesterase (which inactivates acetylcholine, a neurotransmitter) was increased. Both changes were in the 80- and 155-nm groups.

Neurotransmitters: Levels of glutamic acid (a neurotransmitter) and nitr ic oxide (NO, as neurotransmitter and from infl ammatory response) were increased in both the 80- and 155-nm groups (measured only after 15 instillations). Cytokines: Increased THF- α and IL-1 β , but not IL-6 (155 nm after 15 instillations).

5-48Animal Testing Material Treatment Conditi ons Summary of Major Effects Reference

Repeated intranasal instillation (subacute effects) Wang et al. (2007, Mouse CD-1(ICR) Nano-TiO 2 (Hangzhou Dayang Nanotechnology Co. Ltd.), rutile, 25 nm, purity >99% No changes in water and food consumption or BW. 157616

Nano-TiO 2 (Hangzhou Dayang Nanotechnology Co. Ltd.), rutile, 80 nm, purity >99%

Fine TiO 2 (Zhonglian Chemical Medicine Co.), anatase, 155 nm, purity >99% 10 μL of 50 mg/kg TiO 2 or water every 2 days

Blood and brain were collected from anesthetized mice after 2, 10, 20, or 30 days Brain TiO 2 content (measured in all brain samples): increased in treated mice and was highest in 25 nm treated group at 2 and 10 days; decreased slightly and was similar in all treated groups at 20 and 30 days. )

Neurotransmitters (measured in 20- and 30-day brain samples): Changed in 80 nm and 155 nm TiO 2 -treated mice compared to control, but not in 25 nm TiO 2 -treated mice. All changes were after 20 days, with the exception of decreased dopamine in 80-nm group after 30 days. After 20 days: Norepinephrine was significantly increased in 80 and 155 nm TiO 2 -treated mice; 5-hydroxytryptamine was si gnificantly increased in 155 nm TiO 2 -treated mice; homovanillic and 5-hydroxyindole acetic acid were decreased in 80 and 155 nm TiO 2 -treated mice; dopamine was decreased in 80 nm TiO 2 -treated mice.

a According to Pott and Roller (2005, 157790 ): “Titanium dioxide T805 from Degussa was ordered from Sigma-Aldrich, but the supplier only offered an amount of at least 40 kg P805. Neither Si gma-Aldrich nor Degussa answered at all cl early when questioned insistently as to the difference between T805 and P805. So, it is not proven that P805 is identical with T805 from Degussa.” The primary part icle size and surface area in th e table were from Pott and Roller (2005, 157790 ). Currently available T805 is photostable nano-TiO 2 (80% anatase, 20% rutile) that has been treated with octyls ilane to achieve a hydrophobic surface. Degussa T805 primary particle is still 21 nm, but specific surface area (BET) is 45 m 2 /g (Llames, personal communication, 2008, 157529 ).

IL-6 – Interleukin-6 ALP – Alkaline phosphatise, a marker of ty pe II epithelial cell toxicity (Ma-Hock et al., 2009, 193534 ) or liver toxicity IFN- γ – interferon-gamma ALT – Alanine transaminase LDH – Lactate dehydrogenase, a general mark er of cell injury (Ma-Hock et al., 2009, 193534 AST – Aspartate aminotransferase ) BAL – Bronchoalveolar lavage LPS – Lipopolysaccharide
BET – Brunauer, Emmett, Teller meth od of calculating surface area MMAD – Mass median aerodynamic diameter MTP – Microsomal triglyceride CMD – Count median diameter DLS – Dynamic light scattering NADPH – Nicotinamide adenine dinucleotide phosphate ELISA – Enzyme-linked immunosorbent assay P25 – Aeroxide® P25 PBS – Phosphate buffered saline F344 – Fischer 344 rat strain PIGF – Placenta growth factor GFAP – Glial fibrillary acidic protein GGT – gamma- γ –glutamyltransferase, a marker for damage to Clara and type II epithelial cells (Ma-Hock et al., 2009, PMN – Polymorphonuclear neutrophils 193534 ) PTFE – Polytetr afluoroethylene GSD – Geometric standard deviation ROS – Reactive oxygen species GSH – Reduced glutathione RT-PCR – Real-time pol ymerase chain reaction GSH-Px – Glutathione peroxidase SOD – Superoxide dismutase GST – Glutathione-S-transferase TEM – Transmission electron microscopy IEP – Isoelectric point TNF- α – Tumor necrosis factor-alpha IL-1 β – Interleukin-1 beta



## Toxicity by Other Exposure Routes

Ocular exposure, i.v., and subcutaneous (s.c.) injection have also been investigated in
nano-TiO 2 

### toxicity studies (Table 5-7). Ocular exposure to sunscreen containing nano-TiO 2 

### could occur accidentally when sunscreen spray and sunscr een lotion are applied. At least one brand of
sunscreen lotion that contains nano-TiO 2 

### is in a tear-free formula and marketed for children (Project on Emerging Nanotechnologies, 2007, 157648 ). A single ocular exposure to a photostable
nano-TiO 2 

### caused conjunctival redness for 1 or 2 days in rabbits (Warheit et al., 2007, 091075 ). One journal article and two professional meeting abstracts were identified on the effects of injected nano-TiO 2 

### in rats and mice. In the Fabian et al. (2008, 157576 ) study, an intravenous injection of 5 mg/kg nano-TiO 2 

### with unknown photoreactivity did not induce changes in blood tests diagnostic for inflammatory responses, kidney toxi city, or liver toxicity. Two meeting abstracts presented immunological effect studies in mice exposed to nano-TiO 2 

### through subcutaneous and intravenous injections (Miller et al., 2007, 157668 ; Weaver, personal communication, 2008, 157467 ). Preliminary results showed that photocatalytic nano-TiO 2 

### in suspension (Degussa W740X) appeared

5-49

### to have very limited inflammatory ability, and very high doses (560 mg/kg for intravenous injections and 5,600 mg/kg for subcutaneous injections) were needed to produce immunological effects (Weaver, personal communication, 2008, 157467 ). Prenatal exposure to nano-TiO 2 

### has been reported to affect the offspring in mice in one journal article (Takeda et al., 2009, 193667 ) and one poster at a scientific meeting (Trouiller et al., 2008,
157484 ).



## Table 5-7. Summary of h ealth effects of nano-TiO 2 

## particles in mammalian animal models: other (injection, ocular) route

Animal Testing Material Treatment Conditi ons Summary of Major Effects Reference

Injection

No inflammation, kidney toxi city, or liver toxicity detected: no changes in concentrations of cytokines, enzymes and other indicators in the blood (total of 67 parameters) for inflammatory responses, kidney function, and liver function. Fabian et al. (2008, A single intravenous injection via tail vein Rat Male Wistar (strain Crl:WI(Han) Nano-TiO 2 , primary particle 20-30 nm (measured by TEM), BET surface area 48.6 m 2 /g, 70% anatase/ 30% rutile, uncoated, IEP was pH 7 in 10 mM KCl 157576 ); van Ravenzwaay et al. (2009, Saline (control) or 5 mg/kg nano-TiO 2 193689 )

Nano-TiO 2 stock 0.5% in rat serum, then diluted in saline, injection of ~1 mL of test substance preparation/kg of rat BW TiO 2 distribution: TiO 2 concentrations 1 day after injection: liver > spleen >> lung > kidney. The time for the TiO 2 concentration to return to normal levels were in the same sequence. Liver had the same TiO 2 levels after 14 and 28 days. Spleen had slightly decreased TiO 2 levels 14 and 28 days after injection. Lung and kidney had no elevated TiO 2 14 days after injection. No TiO 2 was detected in blood cells, plasma, brain or lymph nodes (mediastinal, mesenteric, popliteal) at any three time points tested (detection limit 0.3 μg Ti = 0.5 μg TiO 2 per tissue). Fine TiO 2 (Kronos International), median size 200 nm in ethanol (measured by DLS), rutile, BET surface area 6 m 2 /g, IEP <pH 3 in 10 mM KCl Aggregates in serum are mostly <1,000 nm, with 10 wt% <100 nm Necropsy at 1, 14, and 28 days after the injection (12 rats total for 4 treatment groups)

Ti concentrations were measured in lung, liver, kidney, spleen, brain, blood cells, plasma, and popliteal lymph nodes at 1, 14, and 28 s after injection

i.v. injections Mouse Balb/c Nano-TiO 2 (Degussa W740X), dispersion of photocatalytic uncoated nano-TiO 2 (80% anatase/ 20% rutile) at 40 wt%, primary particle 4.7 nm, mean aggregate size ≤ 100 nm; (Evonik, 2007, Miller et al. (2007, Lung, liver, and spleen showed white discoloration and phagocytosis of nano-TiO 2 aggregates by macr ophages under light microscope. 157668

157577 ; Llames, personal communication, 2008, 157528 ; Weaver, personal communication, 2008, 157467 ) 5.6 mg/mouse/day for 2 days (total dose 11.2 mg/mouse) )

1 or 3 days of recovery before sacrifice

In 6-wk-old male offspring from nano-TiO 2 - exposed dams Takeda et al. (2009, Mouse Slc:ICP, pregnant female and male offspring Nano-TiO 2 , 25-70 nm, anatase, surface area 20-25 m 2 /g, purity 99.9% (from Sigma-Aldrich) s.c. injections of 100 μL of 1 mg/mL nano-TiO 2 (i.e., 0.1 mg nano-TiO 2 ) into each time-pregnant Slc:ICP mouse once per day at 3, 7, 10 and 14 days post-mating 193667 )

Nano-TiO 2 particles were seen in the testis and brain (olfactory bulb and the cerebral cortex – frontal and temporal lobes) Decreased daily sperm pr oduction, epididymal sperm motility, and the number of Sertoli cells. Abnormal testicular mo rphology (seminiferous tubules) Male offspring were weighed, and sacrificed at 4 days or 6 wk of age for evaluation. Markers of apoptosis (act ivation of caspase-3 and crescent-shaped cells), occlusion of small vessels, and perivascular edema observed in the brain

5-50Animal Testing Material Treatment Conditi ons Summary of Major Effects Reference

Subcutaneously injected mice: Weaver et al. (2007, Mouse Sex, strain/stock not specified Nano-TiO 2 (Degussa W740X), dispersion of photocatalytic uncoated nano-TiO 2 (80% anatase/ 20% rutile) at 40 wt%, primary particle 4.7 nm, mean aggregate size ≤ 100 nm; (Evonik, 2007, Subcutaneous injections: total 0 or total 5,600 mg/kg over 2 days 193713

157577 ; Llames, personal communication, 2008, 157528 ; Weaver, personal communication, 2008, 157467 ) Intravenous injections: total 0 or total 560 mg/kg over 2 days 1 or 5 days of recovery Day 1: No changes in any cell population in peripheral blood, except CD8+ T cells. )

Day 5: Increases in granulocytes in circulation and spleen; decreases in circulating lymphocyte percentages; no changes in macrophage percentages or any cell population in draining lymph nodes. Lack of Con-A stimulated T-cell proliferation in lymph nodes. Intravenously injected mice: Macrophage in the marginal zone of the spleen white pulp contained nano-TiO 2 aggregates, suggesting interaction between T-cells and nano-TiO 2

. No changes in Con-A stimulated T-cell proliferation.

Ocular exposure

Warheit et al. (2007, Acute ocular irritation Reversible conjunctival redness in the treated eye (normal by 24 or 48 hr after administration of nano-TiO 2 ). Rabbit Male New Zealand White Nano-TiO 2 (identified as uf-C , a pre-commercial version of DuPont Light Stabilizer 210), 79% anatase/21% rutile, approximately 90 wt% TiO 2 , 7% alumina, and 1% amorphous silica, average particle size 140.0 ± 44 nm in water, average BET surface area 38.5

m 2 /g 091075 Doses – 0 or 57 mg to one eye of each animal )

No corneal injury evident, no clinical signs observed, and no BW loss occurred. Single exposure (the eye remained unwashed following treatment)
Observation at 1, 24, 48, and 72 hr following administration of the nano-TiO 2

BET – Brunauer, Emmett, Teller method of calculating surface area DLS – Dynamic light scattering
BW – Body weight IEP – Isoelectric point

CD8 – Cluster of differentiation 8 P25 – Aeroxide® P25
CD8 + T cell – Cytotoxic T cell with CD8 surface protein TEM – Transmission electron microscopy



# 5.3.1.3. Summary of Noncarcinogenic Effects

Some of the noncarcinogenic effects shared by conventional and nano-TiO 2 

### were similar in the nature or type of the effects, but differed in dose-response. For example, pulmonary inflammation in laboratory animals and overload in rats were obse rved after respiratory tract exposures to either
conventional TiO 2 

### or nano-TiO 2 

### , and nano-TiO 2 

### often caused more severe or more persistent responses than conventional TiO 2 

### at the same mass concentrations/doses. Systemic effects were also observed: increased inflammatory cell numbers and decreased platelet numbers in the blood, renal pathology, potential hepatic toxicity, and change s in the brain morphology and neurotransmitters. Except for the effects in the brain, the aforementioned effects outside the lung have been reported only once and have not been confirmed by other la boratories. While topically applied photostable
nano-TiO 2 

### is not expected to cause adverse effects in h ealthy skin, data are lacking on the effects in healthy flexed human skin and damaged human skin.



# 5.3.2. Carcinogenic Effects

The carcinogenicity of TiO 2 

### to humans has been reviewed by various international health organizations and workplace regulatory agencies. Currently, TiO 2 

### (including nano-TiO 2 

### , but not considered separately) is classified as “possibly carcinogenic to humans” (Group 2B) by the International Agency for Research on Cancer (IARC) (Baan, 2007, 157717 ; IARC, 2010, 157762 ) and as “carcinogenic” (Class D2A) by the Wor kplace Hazardous Materials Information System (WHMIS), a program administered by the Canadi an Centre for Occupational Health and Safety (CCOHS) (2006, 157774 ).

5-51

### In a 2005 NIOSH draft evaluation, TiO 2 

### was not designated as a “potential occupational carcinogen,” due to insufficient evidence (NIOSH, 2005, 196072 ). For nano-TiO 2 

### , NIOSH expressed concern in the 2005 draft about the potential carcinogenicity of ultrafine TiO 2 

### (primary particle
<0.1 μ m) if exposure levels were at the current mass-based occupational limits of 1.5 mg/m 3 

### for respirable dust or 15 mg/m 3 

### for total dust, and recommended controlling exposure to as low as feasible below the recommended exposure limit (NIOSH, 2005, 196072 ). Based on an assessment of the lung tumor response in the rat and supported by consideration of the other pulmonary effects of
TiO 2 

### , NIOSH draft recommended exposure limits are 1.5 mg/m 3 

### for fine TiO 2 

### (primary particle
<10 μ m) 1 

### and 0.1 mg/m 3 

### for ultrafine (nano) TiO 2 

### . As mentioned in Section 4.2.2, these numbers were derived from converting partic le mass to surface area dose (6.68 m 2 

### /g for fine TiO 2 

### and 48 m 2 

### /g
for ultrafine TiO 2 

### ), and the risk estimates will vary for other particle sizes and surface areas (pages 60-61 of NIOSH, 2005, 196072 ), as well as crystal form and other factors not considered by NIOSH (Section 5.1) This section reviews studies in humans and in animals on carcinogenicity of nano-TiO 2 

### and briefly discusses the mode of action of conventional TiO 2 

### and nano-TiO 2 

### carcinogenicity.
Conventional TiO 2 

### has been shown to induce lung tumors through inhalation in rats at 250 mg/m 3 

### (6 hours/day, 5 days/week for 24 months) (Lee et al., 1985, 193501 ; Lee et al., 1985, 067628 ), but not at 50 mg/m 3 

### or below (Lee et al., 1985, 193501 ; Lee et al., 1985, 067628 ; Muhle et al., 1991,
063996 ). No increases in tumors were observed in mi ce receiving a single intratracheal instillation of 0.5 mg of TiO 2 

### , in mice and rats fed with TiO 2 

### in the diet at up to 5.0% daily for 103 weeks, or in hamsters given 3 mg of TiO 2 

### via intratracheal instillation weekly for 15 weeks (Baan, 2007,
157717 ). Similarly, epidemiological studies did not show increased lung cancer in people exposed to
conventional TiO 2 

### (Boffetta et al., 2001, 157891 ; Chen and Fayerweather, 1988, 193312 ; Fryzek et al., 2003, 157864 ; Ramanakumar et al., 2008, 157507 ; Siemiatycki, 1991, 157954 ). The carcinogenicity studies of conventional TiO 2 

### are not discussed in detail in this document, and readers
are referred to studies cited here and in th e IARC monographs Working Group report (Baan, 2007,
157717 ).



# 5.3.2.1. Studies in Humans

Several epidemiological studies of TiO 2 

### carcinogenicity have been reported: two populationbased case-control studies (one for lung cancer (Boffetta et al., 2001, 157891 ) and the other for 20 types of cancer (Siemiatycki, 1991, 157954 ); two retrospective cohort mortality studies (Boffetta et al., 2004, 157849 ; Fryzek et al., 2003, 157864 ); one mortality, morbidity, and case-control study (lung cancer and chronic respiratory diseases) (Chen and Fayerweather, 1988, 193312 ); and a casecontrol study (lung cancer) (Ramanakumar et al., 2008, 157507 ). Based on these studies, IARC (2010, 157762 ), the CCOHS (2006, 157774 ), and NIOSH (2005, 196072 ) concluded that the
evidence is insufficient to conclude that TiO 2 

### exposure increases the risk of lung cancer in human beings. Furthermore, none of these studies were designed for nano-TiO 2 

### exposure, and none of them provided information on TiO 2 

### particle sizes. Even if the TiO 2 

### in these studies included some particles in the nanoscale range, the risks posed by nano-TiO 2 

### (ultrafine primary particles) and the relationship between particle size and lung cancer risk in humans cannot be discerned from these studies.



# 5.3.2.2. Animal Studies

Carcinogenicity of nano-TiO 2 

### was observed in three animal studies using photocatalytic
nano-TiO 2 

### in rodents (Borm et al., 2000, 041486 ; Heinrich et al., 1995, 076637 ; Pott and Roller, 2005, 157790 ). Increased lung tumor incidences were observed in rats (Borm et al., 2000, 041486 ;

1 “Fine” particles in the NIOSH draft (2005, 196072 ) are defined as all particle sizes that are co llected by respirable particle sampling, i.e., 50% collection efficiency for particles of 4 μ m, with some collection of particles up to 10 μ m.

5-52

### Heinrich et al., 1995, 076637 ; Pott and Roller, 2005, 157790 ), but not in mice (Heinrich et al., 1995,
076637 ), exposed to P25 through inhalation or intrat racheal instillation. Photocatalytic nano-TiO 2 

### given through intraperitoneal injections did not incr ease tumors in the abdomi nal cavity in rats (Pott et al., 1987, 029823 ). Intramuscular implantation of nano-TiO 2 

### with unknown photo-reactivity also did not increase tumors at the sites of implantation in rats (Hansen et al., 2006, 090611 ). Data specifically on photostable nano-TiO 2 

### carcinogenicity are inconclusive (Pott and Roller, 2005,
157790 ). As mentioned in Chapter 4, because internal transport of the materials will influence the ultimate dose to the organism, it should be noted that multiple routes of exposure will be considered that may not be likely or possible primary exposu re routes but that could have relevance when internal transport is considered (e.g., i.v., i.p., and i.m.).



## Intratracheal Instillation

Female Wistar CRP/WU rats received fine and nano-TiO 2 

### via intratracheal instillations, and the tumor incidence and pulmonary inflammation were measured 2.5 years after administration (Borm et al., 2000, 041486 ). Fine TiO 2 

### (250 nm) was given 6 times at 10 mg each, and the photocatalytic nano-TiO 2 

### (21 nm, 80% anatase, 20% rutile, uncoated, P25) was given 5 times at 6 mg each (Borm, personal communication, 2008, 157591 ). At these total doses (60 mg for fine TiO 2 

### and 30 mg for nano-TiO 2 

### ), lung clearance might be expected to be severely compromised. The
authors found evidence of alveolar and interstitia l inflammation 2.5 years after instillation. The histologically confirmed tumor incidences were 27% for fine TiO 2 

### and 66% for nano-TiO 2 

### , while the macroscopic tumor incidences were only 20.9% for fine TiO 2 

### and 50% for nano-TiO 2 

### . In vehicletreated controls, the microscopic tumor incidences were between 5 and 6%. Although particles that induce high tumor incidences generally also cause high inflammatory cell counts, nano-TiO 2 

### caused a high tumor incidence and low inflammatory cell counts. Borm et al. (2000, 041486 ) suggested that tumor formation was directly related to high inters titialization rather than overload and subsequent tissue response, similar to the premise that lung burden is correlated to surface area of the particles (Oberdörster et al., 1994, 046203 ). Pott and Roller (2005, 157790 ) reported increases in pulmonary tumors in rats exposed to hydrophilic fine TiO 2 

### and hydrophilic nano-TiO 2 

### , but were unable to draw conclusions about the carcinogenicity of hydrophobic nano-TiO 2 

### . Female Wistar (HsdCpb:WU) rats received weekly intratracheal instillations of three types of TiO 2 

### : hydrophilic nano-TiO 2 

### (P25), hydrophobic
nano-TiO 2 

### (Aeroxide ® 

### P805/Degussa P805; see Footnote c in Table 5-8), and hydrophilic fine TiO 2 

### (232033 from Sigma). If the products used in the st udy are the same as those currently available, both the hydrophilic nano-TiO 2 

### and fine TiO 2 

### were photocatalytic and the hydrophobic nano-TiO 2 

### was photostable. The tested TiO 2 

### physicochemical properties, doses, and key results are listed in Table 5-8. The types of primary benign lung tumo r were adenoma and epithelioma, and the primary malignant tumors were adenocarcinoma and squa mous cell carcinoma. At the tested doses, 42-46 rats out of 48 rats/group survived in the hydrophilic nano-TiO 2 

### and hydrophilic fine TiO 2 

### groups, and statistically significant increases in benign or malignant lung tumors, or both, were observed in these two groups.

5-53

## Table 5-8. Treatments and pulmonary tumor in cidences in rats exposed to fine and nano-TiO 2 

## through intratracheal instillation in Pott and Roller (2005) study

Crystal form; primary particle size; specific surface area (BET) Dose (number of instillations

× mg per instillation) Lungs with primary benign tumors (%) Lungs with primary malignant tumors (%) Lungs with tumors, total (%) Lungs with metastases

of other tumors (%) Rats at start/at

risk aPhotostability Survival 50% (wk) Treatment

Nano-TiO 2 , hydrophilic (P25) Majority anatase; 25 nm b (21 nm and 30 nm were also reported); 52 m 2 /g Photocatalytic 5 × 3.0 48/42 114 21.4 31.0 52.4 14.3

5 × 6.0 48/46 114 17.4 50.0 67.4 15.2

10 × 6.0 48/46 104 23.9 45.7 69.6 15.2

Nano-TiO 2 , hydrophobic (Degussa P805) c (Sigma AL 900032) c (Data of Degussa T805) c Crystal form not specified, coated with an organic silicon compound; 21 nm; 32.5 m 2 /g (Currently available Degussa T805 is a photostable UV filter) 15 × 0.5 24/11 86 0.0 0.0 0.0 9.1

30 × 0.5 48/15 114 6.7 0.0 6.7 6.7

Fine TiO 2 , hydrophilic (Sigma AL 232033) Anatase; 200 nm; 9.9 m 2 /g (Untreated anatase isphotocatalytic) 10 × 6.0 48/44 108 15.9 13.6 29.5 11.4

20 × 6.0 48/44 113 38.6 25.0 63.6 2.3

No treatment -- -- -- 48/46 113 0.0 0.0 0.0 13.0

BET – Brunauer, Emmett, Teller method of calculating surface area

P25 – Aeroxide® P25
UV – Ultraviolet (light/radiation), wavelengths in the range of 10 to 400 nm

a Rats at risk were “sufficiently examined rats which survived at least 26 wk after first instillation” according to Pott and Rol ler (2005, 157790 ).

b Regarding particle characteristics, Pott and Roller (2005, 157790 ) noted “There are no clearly measured values or more t han one piece of information.” The value listed in the table was assumed to be close to the correct value and was used for further calculations by Pott and Roller (2005, 157790 ).

c According to Pott and Roller (2005, 157790 ): “Titanium dioxide T805 from Degussa was ordered from Sigma- Aldrich, but the supplier only offered an amount of at least 40 k g P 805. Neither Sigma-Aldrich nor Degussa answered at all clearly when questioned insistently as to the difference between T805 an d P805. So, it is not proven that P805 is identical with T805 from Degussa.” The primary particle size and surface area in the table were from the Pott and Roller (2005, 157790 ) study. Currently available T805 is photostable nano-TiO 2 (80% anatase, 20% rutile) that has been treated with octylsilane to achieve a hydrophobic surface. The primary particle size i s still 21 nm, but the specific surface area (BET) is 45 m 2 /g.



### Hydrophobic nano-TiO 2 

### (Degussa P805) showed high acute mortality in the Pott and Roller (2005, 157790 ) study. Nano-TiO 2 

### P805 was given at a much lower am ount in each instillation than
nano-TiO 2 

### P25 and fine TiO 2 

### , because instilled P805 showed acut e lethality. A single intratracheal instillation of P805 at 0.5, 1.0, and 1.5 mg caused death in 25%, 58%, and 92% female Wistar rats, respectively, within 24 hours. Pott and Roller (2005, 157790 ) originally ordered Degussa T805 for their study, and were unable to confirm that the received P805 was the same as T805. The physicochemical properties of T805, but not P805, were used for calculation and reported in the study (Pott and Roller, 2005, 157790 ). In contrast to the high acute toxicity of hydrophobic
nano-TiO 2 

### reported in the Pott and Roller (2005, 157790 ) study, very low toxicity of hydrophobic
nano-TiO 2 

### was reported in an earlier study by Rehn et al. (2003, 090613 ). Rehn et al. (2003, 090613 ) reported that a single intratracheal instillation of P805 at 0.15, 0.3, 0.6, or 1.2 mg caused no death in female Wistar rats. Furthermore, P805 induced onl y mild, reversible inflammatory responses in the lung, and was less biologically active than P25 (Rehn et al., 2003, 090613 ). The reasons for the discrepancy in the toxicity of hydrophobic nano-TiO 2 

### (P805 versus T805 manufactured by Degussa)
remain unclear.

5-54

## Inhalation

Heinrich et al. (1995, 076637 ) reported increased lung tumor rates in rats (but not in mice) that inhaled photocatalytic nano-TiO 2 

### . Animals were exposed to P25 aerosols (18 hours/day, 5 days/week) in whole-body exposure chambers. Generated by a dry dispersion technique, the
nano-TiO 2 

### aerosol had a MMAD of 0.80 μm, with a geometric standard deviation of 1.80.
For female Naval Medical Research Institute (NMRI) mice, the nano-TiO 2 

### exposure was stopped after 13.5 months and followed by clean air exposure for 9.5 months. The 13.5-month
nano-TiO 2 

### aerosol exposure was 4 months at 7.2 mg/m 3 

### , 4 months at 14.8 mg/m 3 

### , and 5.5 months at 9.4 mg/m 3 

### . Although nano-TiO 2 

### exposures decreased lifespan in mice (50% mortality at 17 months after birth, compared to 20 months in controls), the exposures did not increase lung tumor incidence at the end of the study (13.8% in nano-TiO 2 

### exposed, compared to 30% in controls). Even though the reported spontaneous lung tumor rate seemed to be hi gher than historical data (20.7% in the natural lifespan of female NMRI mice (Lohrke et al., 1984, 157978 ); 12% broncho-alveolar lung adenoma and 10% bronchiolo-alveolar lung carcinoma in female Han:NMRI mice up to 104 weeks old (Rittinghausen et al., 1997, 157924 ), 13.8% would not be considered as an increase even compared
to historical controls. For female Wistar rats, the nano-TiO 2 

### exposure was stopped after 24 months, and followed by clean air exposure for 6 months. The 24-month nano-TiO 2 

### aerosol exposure consisted of 4 months at 7.2 mg/m 3 

### , 4 months at 14.8 mg/m 3 

### , and 16 months at 9.4 mg/m 3 

### . At the end of the 30-month study,
32 of 100 nano-TiO 2 

### -exposed rats had benign or malignant lung tumors (20 benign squamous cell tumors, 13 adenocarcinoma, 4 adenoma, and 2 squamous cell carcinoma), while only 1 of 217 control rats had lung adenocarcinoma (Heinrich et al., 1995, 076637 ). The lung particle loading was 23.2 mg/lung after 6 months, and 39.2 mg/lung after 24 months (Gallagher et al., 1994, 045102 ). The exposure to nano-TiO 2 

### did not increase the levels of DNA adducts in the lung (Gallagher et al., 1994,
045102 ). This study showed that inhaled photocatalytic nano-TiO 2 

### is a lung carcinogen in female rats, but no dose-response relationship can be calculat ed due to the dosing design. In a parallel study, decreased pulmonary clearance (overload) was clearly demonstrated (Cre utzenberg et al., 1990,
157963 ). The aerosol concentrations used in the Heinrich et al. (1995, 076637 ) study, ranging from 7.2 mg/m 3 

### to 14.8 mg/m 3 

### , are occupationally relevant. For example, the OSHA PEL (Occupational Safety and Health Administration permissible exposure limit) is 15 mg/m 3 

### and the ACGIH TLV (American Conference of Governmental Industri al Hygienists threshold limit value) is 10 mg/m 3



### . 

## Intraperitoneal Injection

Pott et al. (1987, 029823 ) intraperitoneally injected Wistar and Sprague-Dawley rats with photocatalytic nano-TiO 2 

### (P25) 1 

### and examined abdominal cavities for tumors. The treatment doses ranged from a single intraperitoneal injection of 5 mg nano-TiO 2 

### to 5 injections of 20 mg nano-TiO 2 

### (for a total of 100-mg nano-TiO 2 

### ) over 5 weeks (Table 5-9). Tumor incidences were based on rats with sarcoma, mesothelioma, or carcinoma in the a bdominal cavity. Rats with uterine tumors were excluded from the rats-with-tumor count, because 5-10 % of the controls had malignant tumors of the uterus and some with metastases. Tumor incidences in the abdominal cavity in nano-TiO 2 

### -treated rats ranged from 0% to 10% in the 5 experiments using nano-TiO 2 

### (Table 5-9). Although controls were not available in all experiments, Pott et al. (1987, 029823 ) concluded there were no increases in tumor incidence (in the abdominal cavity) in nano-TiO 2 

### treated rats.

1 Data from Pott et al. (1987, 029823 ) reported the P25 as anatase and did not specify particle size in the 1987 publication. Currently available P25 is 80% anatase and 20% rutile (primary particle size approximately 21 nm), and a representative of Degussa stated that the company has never changed the formula since Degussa P25 was in troduced to the market (Clancy, personal communication, 2008,
193844 ).

5-55

## Table 5-9. Incidence of tumor in the abdominal cavity of rats intraperitoneally injected with photocatalytic nano-TiO 2



## .

Animal, age at the beginning of the experiment Rats with sarcoma, mesothelioma, or carcinoma, other than uterine tumors, in the abdominal cavity (percentage) Nano-TiO 2 treatment

Rats sacrificed when in bad health or 2.5 yr after treatment

Wistar rat, i.p. injection of 18 mg/rat, onc e per wk for 5 wk (total dose 90 mg/rat) 6 of 113 rats examined (5.3%)

9-wk old

Sprague-Dawley rats, i.p. injection of 5 mg/rat 2 of 52 rats examined (3.8%)

8-wk old

Wistar rats, i.p. injection of 5 mg/rat 0 of 47 rats examined (0%)

4-wk old

Wistar rats, i.p. injections of 2, 4, and then 4 mg/rat (tot al dose 10 mg/rat) 0 of 32 rats examined (0%)

5-wk old

Preliminary results at 28 mo after i.p. injection

Wistar rats, i.p. injection of 20 mg/rat, onc e per wk for 5 wk (total dose 100 mg/rat) 5 of 53 rats (36 rats examined and 17 rats survived) (9.4%) 8-wk old

i.p. – intraperitoneal
Source: Data from Pott et al. (1987, 029823 ).



## Intramuscular Implantation

No tumors were observed in rats receiving implantations of either conventional TiO 2 

### or nano-TiO 2 

### for up to 12 months (Hansen et al., 2006, 090611 ). Each of the 10 male Sprague-Dawley rats was surgically implanted with conventional TiO 2 

### (a 9-mm × 2-mm disk containing 100% rutile) subcutaneously on the left side, and with nano-TiO 2 

### (20-160 nm, mean size 70 nm, 90% anatase and 10% rutile) intramuscularly on the right side of pa ravertebral muscle. The implanted doses were one
disk of conventional TiO 2 

### and 0.1 mL nano-TiO 2 

### . Four rats were sacrificed after 6 months, and the remaining six were sacrificed after 12 months. Inflammation (but not granuloma) was observed at
the site of conventional TiO 2 

### implantation, and granuloma (loc alized nodular inflammation; noncancerous inflammation) was observed at the site of nano-TiO 2 

### implantation at both 6 and
12 months. No tumors were observed at either time.



# 5.3.2.3. Modes of Action for Carcinogenicity

The mode of action of lung cancer induced by poor ly soluble particles with no specific toxicity is believed to be particle deposition in respiratory epithelium, decreased lung clearance (to the degree of overload), persistent inflammation, cellular injury and persistent cell proliferation, fibrosis, and secondary genotoxicity (mutation) in the lung cells. TiO 2 

### is traditionally considered chemically inert and falls into the category of poorly soluble particles with no specific toxicity. When doseresponse is expressed as surface area (dose) to tumor proportion (response), TiO 2 

### , nano-TiO 2 

### , and other poorly soluble particles with no specific toxi city appear to share the same dose-response curve 1 

### (Dankovic et al., 2007, 157704 ). With the exception of mutation, all the events described in the previous paragraph (Baan et al., 2006, 186864 ; Muhle and Mangelsdorf, 2003, 157859 ) have been reported in rats exposed to both

1 Because the nano-TiO 2 data used in this dose-response curve were from studies using the same photocatalytic nano-TiO 2 product, this dose-response curve might not be applicable to nano-TiO 2 with a different crystalline type/ratio, purity, shape, surface treatment, or some other characteristic. Although such factors are known to affect nano-TiO 2 toxicity, their role in carcinogenicity remains unknown.

5-56

### fine TiO 2 

### and photocatalytic nano-TiO 2 

### through inhalation or instillation (Borm et al., 2000, 041486 ; Heinrich et al., 1995, 076637 ; Hext et al., 2002, 157878 ; Pott and Roller, 2005, 157790 ). Figure 5-1 illustrates that, at low or medium exposure levels , lungs with normal clearance show inflammation that diminishes over time after exposure ceases. When the exposure level is high enough to decrease clearance, rats show persistent pulmonary inflammatory responses (even after exposure ends), cell proliferation and fibrosis, and eventually tumors. In mice, when the exposure is high enough to cause decreases in clearance, pulmonary inflammatory responses gradually decrease after the exposure ceases and no persistent pathological changes or tu mors are observed in the lung. In hamsters, no overload has been observed and therefore no prediction of the outcome of overload in hamsters is presented here. Increased mutation frequency in hypoxanthine-guanine phosphoribosyl transferase (hprt) was seen in type II alveolar cells isolated from rats exposed to 100 mg/kg fine TiO 2 

### through intratracheal instillation (Driscoll et al., 1997, 053253 ). No studies that investigated mutations in lungs of rats exposed to nano-TiO 2 

### are available. In vitro studies also support the mode of action stated above. Both macrophage- and neutrophil-enriched BAL cell populations from rats exposed to high
concentrations of fine TiO 2 

### showed increased mutations in cultured cells (rat alveolar type II epithelial cell line; RLE-TN) in vitro (Driscoll et al., 1997, 053253 ). Because catalase, an enzyme that catalyzes the decomposition of hydrogen pero xide to water and oxygen, decreased BAL-cellinduced mutation in RLE-TN cells, ROS released from inflammatory cells could contribute to secondary genotoxicity and eventually to the carcinogenicity of TiO 2 

### (Driscoll et al., 1997, 053253 ). This sequence of events, however, does not appear to occur in mice. At an inhalation dose that causes overload, nano-TiO 2 

### does not appear to increase lung tumors in mice. More specifically, overload occurs in mice at an inhalation concentration of 10 mg/m 3 

### nano-TiO 2 

### (P25), based on the
increase of clearance half-life of nano-TiO 2 

### from 40 days at 2 mg/m 3 

### to 395 days at 10 mg/m 3 

### , after 13 weeks (6 hours/day, 5 days/week) of exposure (Hext et al., 2002, 157878 ). After 13.5 months of inhalation exposure to the same type of nano-TiO 2 

### (P25) at approximately 10 mg/m 3 

### (including 4 months of exposure at 14.8 mg/m 3 

### ), mice showed no increased lung tumors over the 2-year study period (Heinrich et al., 1995, 076637 ). Although the evidence available to date for nano-TiO 2 

### carcinogenesis is consistent with the mode of action of other poorly soluble particles an d suggests that particle overload is a sufficient
condition for nano-TiO 2 

### to induce lung cancer, this does not definitively establish that particle overload is a necessary condition for nano-TiO 2 

### -induced lung cancer. For example, it has been suggested that nano-TiO 2 

### -induced lung tumors are directly related to high interstitialization 1 

### rather than overload (Borm et al., 2000, 041486 ). Given the paucity of nano-TiO 2 

### cancer studies and the lack of consensus on exposure-dose metrics, the ques tion arises whether there may be other effects or modes of action unique to nano-TiO 2 

### or nanomaterials in general that are yet to be found.

1 High interstitialization can be a part of the process of part icle overload, but high interstitia lization may not lead to overl oad.

5-57

### Deposition in respiratory tract TiO 2 

### or nano-TiO 2 

### in air

Decreased clearance (overload) Normal clearance

Mice

Cellular injury, persistent cell proliferation, fibrosis Rats

Lung tumors

Mutation Inflammation diminishing over time post-exposure Persistent inflammation Deposition in respiratory tract TiO 2 

### or nano-TiO 2 

### in air

Decreased clearance (overload) Normal clearance

Mice

Cellular injury, persistent cell proliferation, fibrosis Rats

Lung tumors

Mutation Inflammation diminishing over time post-exposure Persistent inflammation



## Figure 5-1. The pulmonary effects of TiO 2 

## or nano-TiO 2 

## exposure through inhalation or instillation.

Although the carcinogenicity of TiO 2 

### and nano-TiO 2 

### in rats at high doses has been shown in inhalation and instillation studies, the relevance of this rat-specific response to human health is under debate. Rats have been suspected to be more se nsitive to poorly soluble particle-induced lung cancer because they are more prone to pulmonary inflammation (Muhle and Mangelsdorf, 2003, 157859 ). Furthermore, lung tumors induced by poorly soluble lo w-toxicity particles are limited to rats with severely compromised particle clearance in lung (overload) (Hext et al., 2005, 090567 ). In human exposures, people working in dusty environments, such as coal miners, could encounter high concentrations of particles and may have impaired lung clearance (Baan et al., 2006, 186864 ). Coal miners, however, are likely to be exposed to a mixtur e of particles (i.e., not limited to poorly soluble low-toxicity particles). Evidence of persistent or chronic inflammation in humans exposed to TiO 2 

### is suggested only by case studies of workers exposed to TiO 2 

### and other minerals (Keller et al., 1995,
157938 ; Moran et al., 1991, 157956 ; Yamadori et al., 1986, 193728 ).



# 5.3.2.4. Summary of Carcinogenic Effects

The results of nano-TiO 2 

### carcinogenicity studies in animals are summarized in Table 5-10. No
data are available for nano-TiO 2 

### carcinogenicity in humans or for photostable nano-TiO 2 

### in animals.
TiO 2 

### (not specific to nano-TiO 2 

### ) was classified as “possibly carcinogenic to humans” (Group 2B) by an IARC monographs Work Group in 2006 (Baan, 2007, 157717 ), and “carcinogenic” (Class D2A) by WHMIS (CCOHS, 2006, 157774 ). NIOSH (2005, 196072 ) proposed not designating TiO 2 

### as a “potential occupational carcinogen” because of insu fficient evidence, but expressed concern about

5-585-59 

### the potential carcinogenicity of ultrafine TiO 2 

### (nano-TiO 2 

### ) at the current exposure limits. Based on calculated lung cancer risks, the draft NIOSH recommendation was for an exposure limit of 0.1 mg/m 3 

### for ultrafine TiO 2 

### and 1.5 mg/m 3 

### for fine TiO 2 

### (less than 2.5 μm), as time-weighted average concentrations. The relevance of rat-specific nano-TiO 2 

### carcinogenicity to human health
remains to be elucidated.



## Table 5-10. Results of nano-TiO 2 

## carcinogenicity studies in animals

Exposure route Species Result Lowest effective dose (highest ineffective dose) References

Photocatalytic nano-TiO 2

Intratracheal instillation Wistar rats, female Increased lung tumors (benign and malignant) 5 instillations at 6.0 mg/instillation Borm et al. (2000, 041486 )

5 instillations at 3.0 mg/instillation Pott and Roller (2005, 157790 )

Inhalation Wistar rats, female Incr eased lung tumors Approximately 12 mg/m 3 for 24 mo a Heinrich et al. (1995, 076637 )

NMRI mice, female No increases in lung tumors (Approximately 10 mg/m 3 for 13.5 mo) b Heinrich et al. (1995, 076637 )

Intraperitoneal injection Wistar and Sprague-Dawley rats No increase in abdominal tumors (5 intr aperitoneal injections at 18 mg/rat per injection) Pott et al. (1987, 029823 )

Nano-TiO 2 with unspecified photoreactivity c

Intratracheal instillation Wistar rats, female No conclusion d (30 instillations at 0.5 mg/instillation) Pott and Roller (2005, 157790 )

Intramuscular implantation Sprague-Dawley rats, male No increases in tumor at implantation sites (not specified) H ansen et al. (2006, 090611 )

NMRI = Naval Medical Research Institute

a 7.2 mg/m 3 for 4 mo, followed by 14.8 mg/m 3 for 4 mo and then 9.4 mg/m 3 for 16 mo

b 7.2 mg/m 3 for 4 mo, followed by 14.8 mg/m 3 for 4 mo and then 9.4 mg/m 3 for 5.5 mo

c Nano-TiO 2 particles not specified or have questionable identification

d Unexpected high acute toxicity; problem with a scertaining the identity of testing material

# Chapter 6. Summary

This chapter briefly summarizes information from the preceding chapters, describing case
studies of nano-TiO 2 

### for arsenic removal in drinking water treatment and for topical sunscreens. It also highlights information gaps and research questions identified in these case studies as they might
relate to future risk assessment efforts. The case studies were developed using the CEA fram ework, which, as described in Chapter 1, combines a product life-cycle perspective with the risk assessment paradigm to provide a more holistic examination of a material’s potential envi ronmental impacts. However, the goal of this document is not to provide an actual CEA or to state conclusions regarding possible ecological or
health risks related to nano- TiO 2 

### , but to provide a foundation for a process to identify and prioritize research directions to support future efforts, and, eventually, provide input to policy and regulatory decision-making. Given that the CEA is both a framework and a process, these case studies were used as a starting point for a formal collective judgement method known as a “nominal group technique” (NGT) to identify and prioritize research questions related to nano-TiO 2 

### . The NGT process was conducted at a workshop held on September 29-30, 2009, and a summary report is available that describes the workshop and summarizes the collec tive prioritization results (U.S. EPA, 2010,
625483 ). A brief description of this process is provid ed in Section 6.2.1. Section 6.2.2 considers the information in the case studies in light of research themes identified in the U.S. EPA’s Nanomaterial Research Strategy Document (U.S.EPA, 2009, 625484 ) and discusses the potential for using this information together with collective priority ranking results to inform refined nanomaterial research strategies, not only within EPA but in the broade r scientific community. Section 6.2.3 discusses ways in which information from the case studies, collective prioritization results, and emerging research can be integrated to support future as sessment efforts for nanomaterials.



# 6.1. Case Study Highlights

This section summarizes what is known, as well as what is unknown, regarding life cycle stages (feedstocks, manufacturing, distribution, storage, use, and disposal/recycling), fate and transport in the environment, exposure and dose characterization for biota and humans, and ecological and health effects of two uses of nano-TiO 2 

### : (1) for arsenic removal from drinking water; and (2) in topical sunscreens. For each topic area, readers are referred back to the specific sections of the document where detailed discussion of the evidence and associated references are presented. In some cases, the findings and research questions are specific to nano-TiO 2 

### , while in others, they pertain to nanomaterials in general. These issues ma y be immediately useful to scientists engaged in ongoing nanomaterial research, as well as for those involved in shaping future research and
assessment efforts.

Note: Hyperlinks to the reference citations throughout this doc ument will take you to the NCEA HERO database (Health and Environmental Research Online) at http://epa.gov/hero . HERO is a database of scientific litera ture used by U.S. EPA in the process of developing science assessments.



### 6-1

# 6.1.1. Analytical Methods

Sensitive and accurate analytical methods for physicochemical characterization of nanomaterials are critical tools for CEA (Section 1.6). Measurement and characterization of nanomaterials, alone and in various media, are requi red for properly monitoring fate and transport, assessing exposure, conducting toxicological studi es, estimating dose-response relationships, and understanding the behavior and effects of nanomater ials. Minimum characterization requirements for toxicological studies have been recommended to fa cilitate interpretation and comparison of studies. Physicochemical characterization of nano-TiO 2 

### , as with other nanomaterials, is extremely important at each stage of its life cycle, because these pr operties change depending on characteristics of the surrounding matrix (e.g., pH, ionic strength, presence of natural organic matter or large biomolecules, soil or sediment composition). In conducting laboratory studies, researchers must account for changes in the properties of the raw material from the point of production during their transport, storage, and preparati on for testing. These physicochemical properties are also likely to change when nanomaterials enter the environment, making it difficult to predict their behavior based on laboratory studies. Additional challenges of characterizing nano-TiO 2 

### in the environment include low expected concentrations and the difficulty of distinguishing naturally occurring materials in the nanoscale size range from manufactu red nanomaterials. As a furt her complication, the critical properties or suite of properties that have the most influence on environmental behavior, exposure, and ecological and health effects are unknown, necessitating the use of multiple methods to
characterize nanomaterials. This need for complex characterization distinguishes nano-TiO 2 

### and other nanomaterials from chemical compounds typically studied in environmental assessments, which often may be characterized by mass concentra tion alone and maintain their chemical structure
in different matrices. Properties of nano-TiO 2 

### and other nanomaterials include particle size and size distribution, mass, surface area, particle number, crystal stru cture, chemical composition, and zeta potential. Concomitant measurements of relevant properties of liquid or solid media are also necessary for characterizing nanomaterials in those matrices, such as pH, salinity, or the presence of organic matter. Methods developed for measurements of aerosol properties are available for measuring nanomaterials in the air, including the condensation particle counter (CPC) for measuring particle number; the scanning mobility particle sizer (SMPS) and electrical low pressure impactor (ELPI) for measuring number, surface area, and mass; the opti cal particle counter (OPC) for number; direct number counts and size distribution by electron microscopy, which can be combined with EDS for elemental composition; and size-selective personal and static samplers for mass measurements of different size fractions. The BET method of gas adsorption to determine particle surface area has also been useful in characterizing nano-TiO 2 

### and other nanomaterials. Liquid-phase techniques for particle size, as presented in Table 1-3 in Section 1.6.1, include dynamic light scattering (DLS) and static light s cattering, size exclusion chromatography (SEC), acoustic techniques, SPM, and centrifugal sedimentat ion. Some of these techniques can also provide information on number and zeta potential (e.g., DLS) and mass or density (e.g., centrifugal sedimentation). Crystal structure has been mea sured by HRTEM and XRD. Field flow fractionation (FFF) to determine particle size has been combined with inductively coupled plasma atomic emission spectroscopy (ICP-AES) for detec tion and characterization of nano-TiO 2 

### in commercial sunscreen, providing information on mass, size distribution, and Ti content of extracted nano-TiO 2



### . Specific applications of other techniques to nano-TiO 2 

### in water treatment processes and sunscreens
have not been identified. Methods for characterizing nanomaterials in soil and sediment would ideally be performed in situ to avoid changes in nanoparticle properties cau sed by sample handling, but this requires portable instrumentation and techniques that can operate in complex environmental media. Therefore, liquidphase techniques are used following sample extraction. As presented in Table 1-4 in Section 1.6.2,

6-2

### these include centrifugation, ultrafiltration, FFF, and SEC for size fractionation; electron microscopy and DLS for size distribution; the BET method for surface area, and spectroscopic techniques for phase and crystalline structure. As with nanomate rials suspended in gas and liquid phases, the use of multiple techniques is recommended to pr ovide more complete characterization. Although characterization of nanomaterials is recogni zed to be a critically important aspect of evaluating their fate and effects, no consensus has been reached on a critical set of properties. Remaining questions include standardization of methodology and terminology, the potential need for improvements to existing methods or entirely new methods, the influence of coatings and other formula components on nano-TiO 2 

### properties and behavior in sunscreen, information on agglomeration of particles in drinking water treatment processes and sunscreens, and correlation among parameters measured in different phases. These questions were reflected in the collective prioritization results from the workshop (U.S. EPA, 2010, 625483 ); three of the four highest-priority topics were directly related to characterization, including the need for improved physico-chemicalcharacterization throughout life cycle stages, method development and evaluation, and productspecific characterization needs. Research to addr ess these questions may be an important part of future investigations into the ecological and health effects of nano-TiO 2 

### and other nanomaterials.



# 6.1.2. Life Cycle Characterization

Feedstocks used in TiO 2 

### production include ilmenite and rutile ores, with ilmenite accounting for approximately 90% of worldwide production of Ti minerals (Section 2.1). The presence of low
levels of radioactive materials in these ores rais es the question of the potential for risk associated with ore mining and processing. It is not clear at this time whether certain feedstocks are more suitable and widely used for production of nano-TiO 2 

### compared to conventional TiO 2 

### . Information is also lacking on the nature and magnitude of c ontaminant release associated with mining and processing of Ti-containing ores. The production quantity of conventional TiO 2 

### for use in pigments and other applications is far larger than production of nano-TiO 2 

### , with 2005 production of
conventional TiO 2 

### and nano-TiO 2 

### estimated at 4.5 million metric tons and 2,000 metric tons, respectively. The production volume of nano-TiO 2 

### is expected to grow rapidly over the next few decades, with widely varying estimates of the rate of growth. A variety of techniques are available for commercial production of nano-TiO 2 

### , many of which are adapted from manufacturing processes for conventional TiO 2 

### (Section 2.2). These include CVD, flame hydrolysis, sol-gel, calcination, aerosol pyrol ysis, and colloidal synthesis. One commercially available product (P25) is produced by flame hydrolysis using TiCl 4 

### as a feedstock, resulting in agglomerated TiO 2 

### particles with a mean diameter of approximately 3,600 nm, with the smallest 4% of particles having an average diameter of 160 nm (Section 2.2). This gas-phase chloride method generates hydrogen chloride as a by-product, w ith the potential for some residual chloride ions
adsorbed onto the TiO 2 

### particles. Specific information was not identified on processes for preparing or formulating nano-TiO 2 

### for use in arsenic removal from drinking water, although information is
available on nano-TiO 2 

### formulations used in sunscreens. Ruti le is a more photostable crystalline
form of TiO 2 

### than anatase, which should make it preferable for use in sunscreen applications, although anatase/rutile mixes are also common. Th e potential for photocatalytic action of nano-TiO 2 

### is mitigated by surface coatings applied to the par ticles, such as silica, al umina, dimethicone, or other compounds; these coatings also improve particle behavior in formulation of sunscreen dispersions. Other components of these dispersions include emulsifiers, emollients, other physical or chemical UV blockers, and ingredients to impr ove characteristics such as spreadability, water resistance, and viscosity. Among the questions rega rding the manufacturing component of the life cycles for these nano-TiO 2 

### applications are the influence of different manufacturing techniques on physicochemical properties of nano-TiO 2 

### ; the specificity of certain manufacturing techniques for

6-3

### nano-TiO 2 

### used in either drinking water treatment or sunscreen, and the potential for new techniques to emerge; the potential for release of nanoscale and larger-sized waste products from nano-TiO 2 

### manufacturing; and the potential for general population exposure to nano-TiO 2 

### in the vicinity of manufacturing facilities. Limited information was identified specifically relating to nano-TiO 2 

### distribution and storage (Section 2.3). Raw nano-TiO 2 

### in powdered form is shipped in paper bags, in some cases lined with polyethylene film, and in glass bottles enclose d in sealed bags. Dispersions of nano-TiO 2 

### are shipped in pails, drums, or totes. No specific informa tion was identified on distribution and storage of nanoTiO 2 

### formulated for drinking water treatment or sunscreen use. Some general information is available from the sunscreen industry on shipping and handling of topical sunscreens, with approximately two-thirds of sunscreen retail sales in the U.S. occurring in supermarkets, drugstores,
and mass merchandise outlets. Accide ntal releases could occur to air, water, or soil at a variety of points along the distribution chain. Inclusion of dist ribution and storage information in life cycle and comprehensive assessments will require additiona l data regarding shipping modes, distances, and quantities for nano-TiO 2 

### in various packaging, modes of storage prior to use in drinking water treatment and sunscreens, and estimates of releas es under various scenarios of distribution and storage. The drinking water treatment case study considers only the application of nano-TiO 2 

### for arsenic removal (Section 2.4.1), although it is possible that it can be used to remove other biological or chemical contaminants. Approximately 13 million pe ople in the U.S. use water that is treated to
remove arsenic. No information was identified on current use of nano-TiO 2 

### in community water systems to remove arsenic, but future use c ould affect a substantial population. At least two commercial technologies are known to be capable of using nano-TiO 2 

### in oxidative processes for water treatment, although currently they are not kn own to be used in this way. One process uses
nano-TiO 2 

### in a fixed membrane, while the other uses nano-TiO 2 

### in a slurry. In bench-scale studies,slurry applications have been shown to produce higher arsenic oxidation rates compared to fixedmatrix nano-TiO 2 

### ; however, immobilized applications presum ably would result in less release to finished water than slurries, which require filtration. A bench-scale simulation of a conventional drinking water treatment process found that more th an 20% of an initial 10 mg/L concentration of
nano-TiO 2 

### (15-40 nm particle size, 200-500 nm aggregate size) remained in water following coagulation, flocculation, and sedimentation (p rior to filtration). Filtration through a 0.45 μ m membrane reduced residual TiO 2 

### to 1-8% of the initial concentration, although this level of filtration is not typical in drinking water treatment plants or whole-house filtration systems. Another factor that could be important, but for which informati on is lacking, is the effect of drinking water treatment chemicals on the solubility, pa rticle size, and behavior of nano-TiO 2 

### . Information is also limited on the potential volume of nano-TiO 2 

### required for arsenic removal in the U.S., details on different treatment processes and the likelihood of their use to serve populations of various sizes,
release of nano-TiO 2 

### to finished water or process waste, and the effect of nano-TiO 2 

### on biofilms and corrosion in distribution systems. As discussed in Section 2.4.2, sunscreen use is substantial in the U.S., with most surveys reporting that 30-50% of respondents use sunscreen re gularly. Parents report more frequent use of
sunscreen on their children than on themselves. The amount and use of nano-TiO 2 

### in sunscreens isunknown, in part because available survey data do not distinguish between conventional and nanoTiO 2 

### , although conventional TiO 2 

### is likely to contain nanoscale TiO 2 

### . Production estimates of nanoTiO 2 

### indicate that a substantial fraction (65%) is used in personal care products such as sunscreen and cosmetics. The lack of specific information on nano-TiO 2 

### use in sunscreens represents an important gap in knowledge for life cycle and exposure assessment. Questions identified as priorities during the collective prioritization workshop (U.S. EPA, 2010, 625483 ) include the potential for
release of nano-TiO 2 

### to various media through different use patterns, the need for better
characterization of nano-TiO 2 

### as used in specific products, and the stability and behavior of surface

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### treatments during sunscreen use. Removal of surf ace coatings may increase the photocatalytic activity of the nano-TiO 2 

### particles and have implications for its effects on biota and humans. Disposal of nano-TiO 2 

### after use is likely to vary between drinking water treatment processes and sunscreen (Section 2.5). Some fraction of nano-TiO 2 

### used in drinking water treatment is likely to be associated with sludge, which may be taken to a landfill or applied to agricultural lands (U.S. EPA, 2010, 635678 ). To the extent that nano-TiO 2 

### reaches finished water, it would then be expected to enter wastewater treatment faciliti es. Disposal of used sunscreen containers, presumably with some residual product, would also result in introduction of nano-TiO 2 

### to municipal landfills or incinerators. Recycling of sunscre en containers is also possible, potentially introducing nano-TiO 2 

### into recycled materials. Remaining disposal issues for which little information is available include disposal procedures for packaging containing nano-TiO 2 

### and substandard product at manufacturing facilities, the behavior of nano-TiO 2 

### in landfills, the exact nature of waste streams from water treatment facilities, and the circumstances that might result in release of nano-TiO 2 

### from discarded sunscreen products.



# 6.1.3. Fate and Transport

Information is currently unavailable regardi ng the transport and transformation of nano-TiO 2 

### specifically from drinking water treatment processes a nd sunscreens in air, water, ground water, soil, or sediment (Chapter 3). As mentioned in Chap ter 1 and Section 6.1.1, physicochemical properties are likely to change when nanomaterials enter the environment, making it difficult to predict their behavior based on laboratory studies. One aspect of nano-TiO 2 

### that is heavily influenced by local conditions is agglomeration of particles to form la rger clusters. These agglomerates would tend to behave differently in the environment than individua l particles. Degree of agglomeration is affected by ionic strength and presence of organic matter in water, as demonstrated in laboratory studies. Agglomeration is also pH-dependent, with minimum particle attraction at the pH pzc 

### . Researchers
have also demonstrated in bench-scale studies that free particles or agglomerates with diameters less than 100 nm can be present even when the predominant form of nano-TiO 2 

### is larger clusters (Chapter 3), a finding that could have implicati ons for agglomeration in natural waters. Surface modifications to maintain dispersion may also cont ribute to the presence of nanoscale particles under conditions normally considered to promote agglomeration.
Nano-TiO 2 

### that reaches wastewater treatment plants, such as through washing off of sunscreens, has the potential to pass through the fa cility in the liquid phase and reach receiving
waters. Studies of other metal oxide nanomaterials in model wastewater treatment plants indicated that surfactants stabilized dispersions even at hi gh ionic strength, although most nanoparticles were removed by agglomeration with microorganisms and subsequent sedimentation. The low solubility
of nano-TiO 2 

### compared with other metal and metal oxide na noparticles suggests that it is likely to remain in the solid phase, although researcher s have found that crystalline form, morphology, manufacturing method, and particle size can influe nce saturation concentration. Another aspect of
nano-TiO 2 

### ’s behavior in aqueous media that should be kept in mind is its photocatalytic generation of ROS in the presence of UV light, which may be a factor in surface waters exposed to sunlight. The lack of information on environmental behavior specific to nano-TiO 2 

### used in drinking water treatment processes and sunscreens represents an open question. Substantial transformation of
nano-TiO 2 

### is not expected due to its physical and chemic al stability, so the processes likely to be relevant are transport and accumulation in various environmental compartments. Chapter 3 describes scenarios through which nano-TiO 2 

### used for arsenic removal could enter the finished water distribution system and thereby end up in surface water or the subsurface via leaks, or it could become part of sedimented sludge and enter the subsurface through landfilling. Likewise, nano-TiO 2 

### from sunscreens could be released to natural bodies of waters through wear-off, and to wastewater

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### via bathing or laundry following sunscreen use. This nano-TiO 2 

### could migrate to sediment through agglomeration with natural organic matter or micr oorganisms or could remain in the water column.
Studies have observed other sunscreen constituents in natural waters, providing plausibility for this scenario. However, no studies were identified that have documented the occurrence of nano-TiO 2 

### specifically from sunscreens in wastewater or natural waters. Laboratory studies have measured transport of nano-TiO 2 

### in synthetic porous media or model soil columns, and found nano-TiO 2 

### to be mobile in these model systems, where large soil particles and low ionic strength favored increased mobility, wh ile high clay content, dissolved organic carbon, and salinity favored retention (Section 3.2). Particle size also affected mobility, with smaller particles passing through the columns to a greater extent than larger particles. However, pH had an unanticipated effect in one model system, with high transport observed at pH values for which retention was expected. This indicates that curre nt transport theory may not accurately predict transport of nanomaterials and agglomerates. Specific information was not identified regarding pathways for nano-TiO 2 

### from arsenic removal or sunscreen use to end up in soil, although one possible scenario is accumulation in wastewater treatment sludge which is then applied to agricultural land as a soil amendment.
Information is not available on the fate of nano-TiO 2 

### that may be emitted to the atmosphere by manufacturing or distribution facili ties (Section 3.3). It is unclear whether its atmospheric fate and transport behavior would be similar to that of ultrafine particulate ma tter emitted from combustion sources, which tends to agglomerate rapidly and unde rgo phase change (condensation/volatilization) near the source, due to the differing p hysicochemical characteristics of nano-TiO 2 

### from combustion
emissions. The collective prioritization process at the worksho p identified three priority topics with fate and transport aspects (U.S. EPA, 2010, 625483 ). These include: (1) the role of physicochemical properties, including surface treatments and agglomer ation, in environmental behavior of specific
nano-TiO 2 

### products; (2) identification of pathways that pose the greatest exposure potential to nanoTiO 2 

### ; and (3) the spatial and temporal distribution of nano-TiO 2 

### in the environment. Other questions relating to fate and transport of nano-TiO 2 

### from drinking water treatment and sunscreen use include: what the effect is of environmental factors (e.g., pH, natural organic matter type and concentration)
on nano-TiO 2 

### mobility and fate; whether existing theory and models of particle transport are applicable to nano-TiO 2 

### and other nanomaterials; the extent to which knowledge regarding fate and transport of other nanomaterials may be applicable to nano-TiO 2 

### ; the potential for nano-TiO 2 

### to influence the behavior of other water and so il constituents and to bioaccumulate; whether photocatalytic activity of nano-TiO 2 

### is of concern in water treatment, wastewater treatment, or the environment; and the effect of co-o ccurring sunscreen constituents on nano-TiO 2 

### fate and transport.



# 6.1.4. Exposure and Dose Characterization

6.1.4.1. Exposure Characterization

The term exposure refers to contact between an individual and a pollutant, combining information on activity patterns and time spent in various microenvironments with concentration data in multiple environmental media. Biota a nd humans may be directly exposed to nano-TiO 2 

### used in drinking water treatment or sunscreen, or ma y receive indirect exposure through contact with
nano-TiO 2 

### in air, water, soil, or sediment. Transfer of nano-TiO 2 

### between these media is also likely to occur. As described in Chapter 4, limited evid ence is currently available regarding environmental exposures of biota and humans to nano-TiO 2 

### , although some information is available on occupational exposures associated with nano-TiO 2 

### manufacturing.

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### Aquatic organisms may be exposed to nano-TiO 2 

### present in the water column or in sediment, with exposure depending on the distribution of nano-TiO 2 

### between water and sediment as well as the tendency of the organism to feed or otherwise spend time near the bottom of water bodies. The propensity of nano-TiO 2 

### to agglomerate may also result in deposition to the surfaces of aquatic organisms, including the gills of fish. This could increase the concentration of nano-TiO 2 

### relative to the water and may result in surface toxicity even when uptake is not observed (Section 4.1.1). For terrestrial organisms, exposure scenarios may in clude contact between material spilled during shipping or storage and microbial, invertebrate, and vertebrate species, as well as potential contact
with nano-TiO 2 

### in environmental media. No specific evidence has been identified regarding actual exposures of biota to nano-TiO 2 

### in the environment. Human exposure to nano-TiO 2 

### may occur either in occupational settings or among the general population (Section 4.2). The general populati on may be exposed through use of nano-TiO 2 

### sunscreens or by drinking water with residual nano-TiO 2 

### , as well as through contact with nano-TiO 2 

### from these applications that ends up in environmental media. The use of nano-TiO 2 

### for arsenic removal in drinking water appears to be limited at present, although implementation of this technology could result in substantial exposure gi ven the sizeable population receiving finished
water that has been treated for arsenic. If nano-TiO 2 

### were present in potable water, exposures could involve pathways other than ingestion, such as de rmal contact and inhalation of droplets during bathing and showering. Potential exposures may be of greater concern for infants and children, who consume more water per body weight than adults. Sunscreen-related exposure to nano-TiO 2 

### may occur through skin contact, although dermal uptake has been found to be relatively low; other potential pathways include inhalation of spray pr oducts and ingestion via hand-to-mouth contact (particularly for children). Based on one series of assumptions, the amount of applied nano-TiO 2 

### per sunscreen application was estimated to range from 12 to 55 mg/kg body weight for a three-year-old child and 8-37 mg/kg for adults (Table 4-2). This higher exposure combined with parent reports of greater sunscreen use on their children than on th emselves could indicate an important role for exposure to nano-TiO 2 

### and related sunscreen constituents in children. Nearly every stage of the life cycle for the applications considered in these two case studies presents some potential for occupational nano-TiO 2 

### exposure. Most available information pertains to
manufacture of nano-TiO 2 

### rather than product formulation, shipping, or use by operators of drinking water treatment facilities (Section 4.2.2). Manufacturers have reported workplace inhalation exposures of less than 0.3-0.5 mg/m 3 

### , although concentrations in some areas were higher. As a frame of reference, the NIOSH has proposed a draft occupational exposure limit of 1.5 mg/m 3 

### for fine TiO 2 

### and 0.1 mg/m 3 

### for ultrafine TiO 2 

### based on relative surface area. Independent measurements in a facility producing nano-TiO 2 

### found lower mass concentrations, ranging from 0.004 to 0.042 mg/m 3 

### ; personal sampling in this facility found a concentration of 0.010 mg/m 3 

### . Number concentrations in the facility ranged from 15,000 to 29,000 particles/cm 3 

### , with 60% of the particles in the 20 to 30 nm size range; airborne TiO 2 

### concentrations outside the plant we re measured at 13,000 particles/cm 3



### . Surface area concentrations were 13-50 μ m 2 

### /cm 3 

### . A modeling study using these and other data estimated that packers were exposed to 0.39 m 2 

### TiO 2 

### per 300 m 3 

### air, while surface treatment workers had lower exposure at 0.17 m 2 

### per 300 m 3 

### . These preliminary data indicate that there is a wide range of concentrations and exposures among nano-TiO 2 

### manufacturing and handling facilities and that exposure may vary by occupation. In addition, dermal exposure may be relevant depending on the type and usage rate of personal protective equipment. As described in Chapter 4, both aggregate exposures (representing exposure to nano-TiO 2 

### through multiple routes, such as dermal, ingestion, and inhalation) and cumulative exposures (representing exposures to multiple substances associated with the use of nano-TiO 2 

### in drinking water treatment and sunscreen) are rele vant to the consideration of nano-TiO 2 

### exposure. However, limited evidence specific to nano-TiO 2 

### is currently available. Cumulative exposure may involve other sunscreen constituents, transformation products of reactions catalyzed by TiO 2 

### (in drinking water or sunscreen), or pollutants adsorbed to nano-TiO 2 

### and carried into the body as co-

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### contaminants. This latter phenomenon has been obse rved for arsenic and cadmium, as summarized in the following section on dose characterization. Although models have not been specifically developed for characterizing exposure to nano-TiO 2 

### , EPA has various models that have been used in assessments of other chemicals to provide screen ing-level estimates of aquatic exposure, general population exposure, and consumer exposure. Adaptation of these models for use with nanomaterials would likely be necessary prior to their quantitative use for nano-TiO 2 

### . Researchers in Switzerland have developed models to predict environmental concentrations of nano-TiO 2 

### and compared their estimates to no-effect concentrations in aquatic toxicity studies, although an explicit exposure component was not included. The collective priority ranking process id entified several exposure-related questions (U.S. EPA, 2010, 625483 ), including:

• which properties of nano-TiO 2 

### are most relevant for expo sure characterization, and
whether available methods are ad equate to characterize exposur e in air, water, soil, and other media;

• which pathways pose the greatest exposure for biota and human ex posure to nano-TiO 2 

### used in drinking water tr eatment and sunscreens; and

• whether certain populations of biota and humans have greater exposure potential.

Other questions include how exposure models can be developed or adapted to estimate nano-TiO 2 

### exposure, and the degree of exposure to seco ndary contaminants associated with nano-TiO 2 

### used in drinking water treatment and sunscreens. Occupational exposure can be further characterized as well, leading to questions such as:

• the size of the potentia lly exposed population;

• additional information on conc entrations and durations of exposure in various job classifications;

• which monitoring methods and properties are appropriate for measuring workplace exposure; and

• which management practices and protective equipment are appropriate for controlling exposure by various routes.



# 6.1.4.2. Dose Characterization

Dose is defined as the amount of a substance th at enters an organism by crossing a biological barrier. Various exposure routes have been investigated for uptake of nano-TiO 2 

### in studies of fish, laboratory animals, and humans (Section 4.6). Two studies investigating accumulation of nano-TiO 2 

### in fish following multi-day exposures have found mixed results, possibly due to differences in species (bottom feeder versus pela gic) and other aspects of study design. Two additional fish studies have indicated the potential for nano-TiO 2 

### to increase uptake of arsenic and cadmium, presumably by adsorption and facilitated transport. In addition to this example of cumulative dose to multiple pollutants, bioaccumulation in the food web is al so a possibility, although no studies were identified that demonstrated multi-species bioaccumulation of nano-TiO 2



### . In animal toxicological studies relevant to terrestrial biota and humans, various exposure routes have been evaluated to determine the uptake of nano-TiO 2 

### and other nanomaterials, including respiratory (inhalation and instillation), dermal, and ingestion. Animal studies have shown that inhaled or inspired nano-TiO 2 

### can translocate into the interstitium of the lung, the lymph nodes, blood, and the brain (Section 4.6.2). Deposition patterns in the respiratory tract depend on several factors, including particle size and breathi ng pattern. Model results of human lung deposition

6-8

### indicate that very few nanoparticles reach the al veolar region, having been removed by diffusive deposition in the upper airways or tracheobronchial region. Studies in rats indicate that the retention
half-life of inhaled nano-TiO 2 

### was approximately three times as long as that of fine TiO 2



### . Dermal uptake of nano-TiO 2 

### is particularly relevant for sunscreens containing nano-TiO 2 

### , and both human and animal studies are available (Section 4.6.3). These studies predominantly indicate
that nano-TiO 2 

### does not penetrate beyond the stratum corneum or hair follicles into living cells of healthy skin. In a study comparing psoriatic and healthy skin, nano-TiO 2 

### in a sunscreen formulation penetrated into deeper areas of the stratum corn eum of psoriatic skin, but still did not reach living
cells. No studies have been identified that evaluated nano-TiO 2 

### penetration in damaged skin (e.g., from sunburn), although preliminary results indicate greater penetration of quantum dots and nanosilver in damaged skin compared to healt hy skin. The extent and duration of nano-TiO 2 

### accumulation on the skin via reapplication of sunscreen and the ultimate fate of nano-TiO 2 

### from sloughed skin cells are both open questions at this time.
Evidence for accumulation of nano-TiO 2 

### following ingestion is extremely limited, with a single study reporting elevated concentrations in the liver, spleen, kidney, lung, and brain of female mice following oral gavage (Section 4.6.4). The potential for nanoparticles to cross the blood- brain barrier (BBB) has been investigated for medical applications, where in many cases the pa rticle surfaces have been modified to enhance translocation (Section 4.6.5). Mixed evidence is available for translocation of nano-TiO 2 

### across the BBB following injection or gavage, with some studies finding increased Ti concentrations in the brain and others finding no evidence of an increase. A recent study showed TiO 2 

### particles and pathological changes in the brain of mouse offspr ing following maternal exposure during gestation, although it is not clear whether nano-TiO 2 

### crossed the placenta or entered the milk to result in lactational exposure. Various metrics are possible for characterizing na noparticle dose, such as mass, surface area, or particle number, as well as crystalline fo rm, shape, and surface modifications (Section 4.6.6). Studies comparing mass and surface area to evalua te dose-response curves provide mixed results, with some evidence indicating that surface area provides a more consistent dose-response relationship for both fine and nano-TiO 2 

### . Composite metrics of two or more properties may also be useful, as suggested by a study indicating that se parate surface-area-based dose-response curves for
anatase and rutile TiO 2 

### would better fit the data than a single dose-response curve. Questions highlighted during the collective prioritization process (U.S. EPA, 2010, 625483 ) for future research on dosimetry of nanomaterials in general and nano-TiO 2 

### in particular include:

• whether certain populations (e.g ., children) may be particular ly susceptible to receiving high doses of nano-TiO 2 

### from its use in drinking water or sunscreens; and

• which dose metrics are most rele vant for characterizing nano- TiO 2 

### dosimetry.

Other questions include:

• what modifications need to be made to physiol ogically-based pharm acokinetic models so that they are appropriate for understandi ng absorption, distribution, metabolism, and
excretion of TiO 2 

### ;

• how to extrapolate received dose from animal t oxicological studies to humans; the extent
to which nano-TiO 2 

### may bioaccumulate and biom agnify in food webs; and

• whether increased uptake of copolluta nts in the presence of nano-TiO 2 

### indicates the need
for consideration of othe r substances in nano-TiO 2 

### monitoring and exposure studies.

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# 6.1.5. Ecological and Health Effects

Several factors influence the ecological and health effects of nano-TiO 2 

### , including physicochemical characteristics, experimental conditions, and environmental conditions (Section 5.1). The need for thorough characterization of nanomaterials used in toxicity studies is now well recognized. Important properties considered part of a minimum set of characteristics include: particle size, size distribution, and aggregation st atus; chemical composition and crystal structure; surface chemistry and charge; specific surface area; particle shape; and production method. Studies have found these variables to be important in de termining the chemical and biological behavior of nanomaterials. Experimental conditions also modify the effects of nano-TiO 2 

### and are therefore important to measure and report in detail. For example, skin penetration of nano-TiO 2 

### increased for an oily dispersion compared with an aqueous dispersion, although nano-TiO 2 

### did not reach living skin cells. Suspension media used in laboratory studies, such as deionized water, tap water, saline solutions, and BAL fluid, each lead to different st ates of agglomeration which can affect the uptake
and effects of nano-TiO 2 

### . Different levels of in vitro OH radi cal production have been observed in different sunscreen formulations containing similar nano-TiO 2 

### , indicating that the other components
of the mixture can affect the obs erved results. Similar issues exist for environmental conditions, such as differential effects due to changes in the aquatic chemistry of surface or ground water or the presence of natural organic matter. UV radiation is well known to increase the toxicity of nano-TiO 2 

### ; in addition, it may make the skin more permea ble by causing sunburn. Other issues that are potentially important include the influence of temperature and water saturation on skin penetration, but no studies were identified that have investigated these parameters.



# 6.1.5.1. Ecological Effects

Ecological effects on microorganisms, aquatic speci es, and terrestrial species are discussed in Section 5.2, and key studies are summarized in Tabl e 5-3. Most studies have tested photocatalytic
nano-TiO 2 

### as would be used in water treatment, with only a few studies evaluating photostable nanoTiO 2 

### intended for use in sunscreen. However, coatings used to increase the photostability of nanoTiO 2 

### could be removed in the environment through weathering or biotransformation, yielding nanoTiO 2 

### with photocatalytic properties. Studies of ac ute effects in microorganisms and higher aquatic species generally provide little evidence of toxic ity at concentrations below 10 mg/L, with several studies finding no effects at concentrations of 100 mg/L or higher. However, a longer-term (14-day) study found respiratory toxicity, injury to the gill and intestine, and evidence of oxidative stress in the gill and intestine in juvenile rainbow tr out following exposure to photocatalytic nano-TiO 2 

### at concentrations as low as 0.1 mg/L. Studies evaluating terrestrial invertebrates found no effect on behavior or mortality for P. scaber and decreased reproduction without change in body length for C. elegans . Spinach growth was enhanced by nano-TiO 2 

### in several studies, possibly due to increases in the activity of enzymes responsible for photosynthe sis, nitrogen metabolism, and oxidative stress response. Incorporation of nano-TiO 2 

### into sediment was not found to increase toxicity of sediment or elutriate, even at a 1:1 ratio. In general, the fo cus of these studies on growth and mortality provides
little information on mode of action of nano-TiO 2 

### ecotoxicity. One of the highest priority areas identified in the collective prioritization results from the workshop (U.S. EPA, 2010, 625483 ) included the question of whether standard ecotoxicity tests are appropriate for nanomaterials in general and nano-TiO 2 

### in particular. Changes in nano-TiO 2 

### properties in different matrices (raw ma terials, products containing nano-TiO 2 

### , environmental media, and biological systems) may lead to differing beha vior and make extrapolation of test results difficult. It is not currently clear whether a suit e of physicochemical properties can be used for a structure-activity relationship to predict biological effects. In addition, the interplay between physicochemical properties of nano-TiO 2 

### and changes in environmental variables (e.g., pH, oxygen

6-10

### level) is not well understood and could result in changes to both the ecotoxicity of nano-TiO 2 

### and underlying soil or aquatic chemistry. Other issues potentially relevant to changes in physicochemical properties include the effect of in vi vo biochemical processes on nano-TiO 2 

### and the potential for
interaction between nano-TiO 2 

### and associated substances resulting in increased uptake and effects of
either nano-TiO 2 

### or copollutants. The collective priority ranking results also included questions surrounding the mechanism or mode of action of nano-TiO 2 

### and whether different modes of action are important at low and high concentrations.



# 6.1.5.2. Health Effects

Health effects of nano-TiO 2 

### are discussed in Section 5.3. Both noncarcinogenic and carcinogenic effects have been examined. Noncarcinoge nic effects have been investigated in animal toxicological studies for several exposure routes, including dermal, oral, and respiratory; however, no epidemiologic studies or case reports were identified pertaining specifically to nano-TiO 2



### . Limited evidence from acute in vivo dermal exposur e studies does not indicate skin irritation or skin cell toxicity following exposure to photocatalytic nano-TiO 2 

### ; as discussed in Section 6.1.4, uptake of
nano-TiO 2 

### through healthy skin was not observed. No studies were identified that evaluated either effects in flexed or abraded skin or long-term effect s of any kind, which would be relevant to typical sunscreen usage patterns. Of the three animal studies identified that evaluated toxicity following oral
intake of nano-TiO 2 

### , two studies found no evidence of lethality or obvious acute toxicity following a single dose of 5,000 mg/kg, although brain morphologi cal changes were observed in one of these studies. The third study found DNA damage in mice in both mothers and offspring following exposure to 60-600 μ g/mL nano-TiO 2 

### in drinking water for 5 days; this is also relevant to carcinogenic effects. A larger group of studies fo cused on respiratory effects following inhalation or instillation, and found pulmonary inflammation an d impaired particle clearance, with effects generally most severe in rats, followed by mice, and least severe in hamsters. Nano-TiO 2 

### effects were often more severe or persistent than conventional TiO 2 

### at the same doses. Preliminary evidence has also been observed for systemic effects outside the lung following respiratory exposure, including changes in inflammatory cell and platel et counts in the blood, renal pathology, potential hepatic toxicity, and changes in brain morphology a nd neurotransmitter leve ls. Carcinogenic effects
of nano-TiO 2 

### have been examined in several studies due to the classification of TiO 2 

### (size unspecified) as a possible human carcinogen by IARC. The evidence indicates that inhalation or instillation of photocatalytic nano-TiO 2 

### increases lung tumor incidence in rats, but not mice. This raises the question of the human health relevance of rat-specific nano-TiO 2 

### carcinogenicity due to increased susceptibility to pulmonary inflammation and poor particle clearance in the rat strains studied. No carcinogenic effects were observed following intraperitoneal or intramuscular administration of photocatalytic nano-TiO 2



### . Another of the highest priority areas identified in the collective prioritization results from the workshop (U.S. EPA, 2010, 625483 ) included the question of whether current EPA test guidelines and assays are appropriate for determining the health effects of nano-TiO 2 

### , and, if not, which modifications, additional assays and standard re ference materials would be useful. Additional priority questions from the workshop included: what the fundamental biological responses are for
nano-TiO 2 

### interactions at the cellular level; what the important modes and mechanisms of action are
for nano-TiO 2 

### effects and whether the mode of action differs for different types of nano-TiO 2 

### (e.g.,photocatalytic versus photostable) or different organs (e.g., lung versus brain); and what the longterm effects of nano-TiO 2 

### may be. Other potentially relevant issues included: which properties are necessary and desirable for proper characterization of nano-TiO 2 

### during assays; whether nano-TiO 2 

### has the potential to penetrate compromised skin; and whether nano-TiO 2 

### has reproductive, developmental, or carcinogenic effects.

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# 6.2. Role of Case Studies in Research Planning and Assessment Efforts

These two case studies are designed to benefi t ongoing nanomaterial research efforts, the research planning process, and potential future assessment efforts on the environmental (ecological and health) effects of nano-TiO 2 

### and other nanomaterials. The currently available information presented here, along with gaps in knowledge that have been identified, should be useful in the interpretation of newly available data as well as planning for future research and assessments. As stated previously, the case studies are not intended to represent completed assessments or to serve as the basis for near-term risk management decisions regarding the use of nano-TiO 2 

### in drinking water treatment or sunscreen. In addition, othe r scenarios for potential use of nano-TiO 2 

### , such as in coatings or as a component of a solid matrix, may involve separate issues not considered in this
document. This section describes how the case studies may be used in informing ongoing and planned research on nanomaterials and in developing information useful for future assessments. It also highlights some of the information gaps identified through the case studies and the associated workshop on research priorities, described in Section 6.2.1.



# 6.2.1. Workshop on Research Priorities for Nano-TiO 2


### As part of the process of identifying areas where additional knowledge may be useful, NCEA held a workshop to identify and prioritize research directions. The workshop used a formal group decision method known as the “nominal group technique (NGT),” which is a process for a group of selected individuals to identify and rank a series of choices. Each individual presents a brief statement outlining the rationale for assigning a high priority to a particular choice. The group then discusses the priorities, with the opportunity to con solidate similar choices, and votes for the highest priority items. The result is a rank order of pr iorities based on the collective judgment of the individual participants. Research questions identified in the nano-TiO 2 

### case studies were prioritized using this technique. A summary report (U.S. EPA, 2010, 625483 ) describes the workshop and summarizes the main outcomes of the ranking process. The reader is referred to this summary report, which provides more detailed information regarding the specific quest ions used to develop research priorities and the rationale for prioritizing each of the research need s; this information is not repeated here. The NGT process identified several high-priority topic areas, with the top-ranked priorities addressing whether existing human and ecological toxicity test protocols are appropriate for use with nano-TiO 2 

### , as well as questions regarding characterization of the physicochemical properties of nano-TiO 2 

### at each stage of the product life cycle, in the environment, and in biological systems. Other priority topics included: determining what effect surface co atings and product formulations have on physicchemical properties and biological activity of nano-TiO 2 

### ; evaluating exposure pathways and populations of greatest concern, and whether ava ilable methods are appropriate for characterizing exposure to nano-TiO 2 

### ; developing a database of information on environmental concentrations of
nano-TiO 2 

### in various media, including biological systems; research into the mode of action of nanoTiO 2 

### , both at high and low doses; and determining the effects of long-term exposure to nano-TiO 2



### . Many, if not all, of these topics are relevant to other nanoparticles, although separate lines of research may be useful in characterizing diverse particle types (e.g., metal oxides and carbon-based nanoparticles).

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# 6.2.2. Implications for Research Planning

The U.S. EPA’s Nanomaterial Research Strategy document (U.S.EPA, 2009, 625484 ) outlined research themes and science questions relati ng to sources, fate, transport, and exposure to nanomaterials; human health and ecological effects, and nanomaterial risk assessment and risk management. These science questions are guiding ongoing research in EPA’s ORD and will form the basis for research planning efforts within ORD. Th e findings of these case studies are consistent with the themes emphasized in the Nanomaterial Research Strategy and should further inform interpretation of current results and planning for future research, both within EPA and among the broader scientific community. For example, the cas e studies and workshop highlighted the question of the appropriateness of health and ecological toxicity testing protocols for use with nano-TiO 2



### . This concept is included in the research strategy document as background for the key science questions on health and ecological effects of nanomaterials, and may form an overarching theme that can be used to integrate results from individual experiments using different study designs, protocols, and endpoints. Development of methods for physi cochemical characterization of nanomaterials under controlled conditions, in environmental matrices, and in biological systems is also a common priority. This highlights the potential for integrated transdisciplinary research, a focus area for ORD, to utilize contributions from materials science, engineering, and biology to fully understand nanomaterial properties and effects. One area of missing information identified in the case studies was research into the long-term
effects of nano-TiO 2 

### . Consideration of longer-term chronic effects in the context of existing research strategy themes and science questions would help address this gap in knowledge. Research into
modes of action of nano-TiO 2 

### , both at low and high doses, is another priority area identified by the case studies and workshop. The research strategy references the importance of mode of action information, and specifically discusses the potential utility of determining the physical and chemical properties responsible for biological effects of nanomate rials. Integration of critical properties with their associated modes of action could help create a more complete understanding of the health and ecological effects of nanomaterials, as well as differences between nanoscale and conventional materials and effects unique to nanomaterials. Linkages such as these between the case study findings and research planning efforts may result in more focused and effective nanomaterial research, as well as providing information useful for future risk assessments of nano-TiO 2 

### and other
nanomaterials.



# 6.2.3. Implications for Futu re Assessment Efforts

The Nanomaterial Research Strategy (U.S.EPA, 2009, 625484 ) and EPA’s Nanotechnology White Paper (U.S. EPA, 2007, 090564 ) both highlight the importance of improved information and research results to support future assessment effo rts. Their recommendations on research directions to support risk assessment for environmental fate and transport, as well as health and ecological effects, resonate with the priorities identified by the case studies and workshop. A variety of information will likely figure into future risk assessments, including characterization of nano-TiO 2 

### in multiple matrices; information on the magnitude of potential releases, environmental concentrations, and exposure pathways for nano-TiO 2 

### ; appropriateness of methods for evaluating human and ecological toxicity; information on the interacti on between physicochemical properties, dose, and mode of action; and both short-term and long-te rm health and ecological effects of nano-TiO 2



### . Information on the life cycle of nano-TiO 2 

### and products incorporating nano-TiO 2 

### , including their fate and transport in the environment, can be combined with these data to support a CEA of nano-TiO 2



### . These case studies have summarized what is known on these and other topics, as well as what remains unknown, and the accompanying workshop has pr esented a set of priorities that can be used to guide future research and assessment efforts.

6-136-14 

### An important function of environmental assess ments is the integration and synthesis of information from multiple lines of evidence. This is difficult at this stage for nano-TiO 2 

### or other
nanomaterials due to the limited and somewhat sc attered evidence in many areas along with the
near-total lack of evidence in other areas. Risk assessments for human health effects of environmental chemicals typically bring together evidence from toxicological, epidemiological, and controlled human exposure studies, which is then integrated to evaluate the likelihood of a causal relationship between the pollutant and a particular category of health effects. The CEA framework expands upon this by considering the impact of a material’s life cycle and environmental fate on health and ecological effects. CEA is particularly appropriate for engineered nanomaterials because it facilitates the transdisciplinary integration of information from materials science, engineering, and biology that is required to fully understand nanomaterial effects. CEA also can consider the multiple impacts expected to result from introduction of a new technology, compare those impacts with those from conventional technologies, and provide informa tion relevant for evaluating the sustainability of
new nanomaterials. Much work remains be fore this will be possible for nano-TiO 2 

### to the same degree as it has been accomplished for other pollutants. The information presented in these case
studies of nano-TiO 2 

### in drinking water treatment and sunscreen provides a starting point for this important work.

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193744

# Annex A. Nano-TiO 2 

# in Sunscreen: Background
Information

Nano-TiO 2 

### has been used in topical sunscreen products since approximately 1990 (EWG, 2008, 196343 ). Between 1995 and 2002, the market for inorganic sunscreen ingredients (both nanoscale and non-nanoscale) incr eased from a value of roughly $30 million to a value of approximately $38 million, and has maintained approximately a 20% share of the sunscreen ingredient market as a whole (Dransfield, 2005, 157809 ). Dransfield (2005, 157809 ) projected that the market for inorganic active ingredients in sunscreens would grow to approximately $75 million by 2010 which would account for one-third of the total market for sunscreen active ingredients. Dransfield (2005, 157809 ) suggested that the projected increase in the popularity of inorganics can be attributed to improved transparency in the produ cts, which would imply particularly rapid growth in the market for nanoscale inorganics. In 2006, the Australian Therapeutic Goods Administration (TGA) estimated that 70% of Ti sunscreens and 30% of zinc sunscreens in Australia were formulated with nanoparticles (TGA, 2006, 089202 ). The U.S. topical sunscreen market in 2000 was approximately $553 million (65%) of the $853 million “sun-care” market (a category that in cludes self-tanning produc ts, after-sun products, etc.) (Packaged Facts, 2001, 196053 ). The size of the U.S. sunscr een market had apparently not changed substantially since 1993, when retail sales were reportedly in the range of $550 to $575 million (Davis, 1994, 157946 ). The total U.S. sun-care mark et reached $1.1 billion in 2005, and is projected to reach $1.2 billion by 2010 (Jeffries, 2007, 157682 ). If sunscreens continue to account for 65% of the U.S. sun-care market, that would translate to $715 million in sunscreen sales in 2005, and a projected $780 million in sunscreen sales in 2010. Globally, sales of sun protection products that presumably include topical sunscreen s and cosmeceuticals were expected to exceed $820 million in 2006 (Newman, 2006, 157745 ). As a “mature” market in the U.S., sun protection products are expected to have a growth rate of only approximately 2% per year (Jeffries, 2007,
157682 ). Between 2005 and 2010, however, growth in the sun-care market was expected to be much faster abroad than in the U.S. (Jeffries, 2007, 157682 ). If the growth in cosmeceuticals has dampened demand for conventional sunscreen, this growth has led to even greater demand for sunscreen active ingredients, including micronized TiO 2 

### (Davis, 1994, 157946 ).


# A.1. Sunscreen Chemistry, and th e Role and Properties of Nano-TiO 2

UV radiation is classified by wavelength in to three types: UV-A (320-400 nm), UV-B (290-320 nm), and UV-C (200-290 nm). The shor ter the wavelength, the more energy the UV radiation transmits. Consequently, the shorter wave length rays can cause more damage to skin than the longer wavelength rays. Approximately 10% of th e solar radiation that reaches Earth’s surface is UV, and approximately 95% of that is UV-A. The long wavelengths of UV-A contribute to skin aging, skin wrinkling, and skin cancer. UV-B is in the middle range of UV, and contributes to burning and tanning, skin aging, and skin cancer. Although UV-C has the shortest wavelength and

Note: Hyperlinks to the reference citations throughout this doc ument will take you to the NCEA HERO database (Health and Environmental Research Online) at http://epa.gov/hero . HERO is a database of scientific litera ture used by U.S. EPA in the process of developing science assessments.



### A-1

### can be dangerous, it is blocked by ozone in th e atmosphere and does not reach Earth’s surface (Jeffries, 2007, 157682 ; Shao and Schlossman, 1999, 093301 ). The traditional SPF rating system measures protec tion against UV-B radiation only. The U.S. FDA proposed an official rating system that also takes UV-A radiation into account, awarding
sunscreens between one and four stars based on th eir UV-A protection (72 FR 49070). This system was expected to go into effect in November 2008 or later but has not been finalized as of April 2010. Various other UV-A protection ratings systems are in use or have been proposed in Australia, New Zealand, Europe, Japan, China, and Korea (Moyal, 2008, 193559 ).



# A.1.1. Size of Nano-TiO 2 

# Particles (Mean and Distribution)

The composition of nano-TiO 2 

### –based sunscreens is determined or constrained by several factors, including unique properties of nano-TiO 2 

### , general principles of sunscreen chemistry, and
aesthetic and other concerns. The size of nano-TiO 2 

### particles (both the primary particle size and the effective particle size of aggregates and agglomerates) affects protection against UV-A and UV-B radiation, the opacity of the sunscreen, and the stabi lity of the dispersions. In most cases, a range of
nano-TiO 2 

### sizes is present due to various primary particle sizes and aggregation.
The size of nano-TiO 2 

### particles affects how much UV-A and UV-B the particles transmit and scatter, and therefore, the degree of protecti on the particles provide against UV-A and UV-B radiation. Shao and Schlossman (1999, 093301 ) found that a nano-TiO 2 

### dispersion with a primary particle size of approximately 15 nm transmitted le ss UV-B and more UV-A and visible light than did dispersions with primary particle sizes of 35, 100, and 200 nm. (The particles were present in aggregates of mean sizes 125.3, 154.1, 251.1, and 263.4 nm, respectively.) The results of this study
indicated that smaller nano-TiO 2 

### particles are better for UV-B protection, and larger nano-TiO 2 

### particles are better for UV-A protection. Dransfield (2005, 157809 ) presented data indicating that
TiO 2 

### particles (not specifying whether they were prim ary or secondary particles) at 100 nm diameter provide the best UV-A protection but also significant visible light attenuation (i.e., leaving a white hue on skin if such particles are used in sunscreens), and particles in the range of 40 to 60 nm provided the best UV-B protecti on. In addition, 20-nm nano-TiO 2 

### did not provide sufficient protection against UV-A or UV-B. 200-nm TiO 2 

### particles provide poor UV -A and UV-B protection and high attenuation of visible light (Dransfield, 2005, 157809 ). According to Hewitt (2002,
093307 ), theoretical calculations suggest that the optimal mean TiO 2 

### primary particle size for good UV-B and UV-A protection is approximat ely 50 nm. Chaudhuri and Majewski (1998, 093308 ) noted
that nano-TiO 2 

### with a primary crystal size of 10-20 nm and an effective particle size of approximately 100 nm is expected to have a “very high UV scattering effect.” Particle size also determines the opacity of nano-TiO 2 

### formulations. Larger primary particles transmit less visible light (Shao and Schlossman, 1999, 093301 ). Aggregation will also make a formulation more opaque (Chaudhuri and Majewski, 1998, 093308 ). TiO 2 

### particles larger than 200 nm in sunscreen or cosmetics leave a white hue on the skin and are considered aesthetically unacceptable in many applications. Nano-TiO 2 

### particles smaller than 100 nm are generally not visible, and the sunscreen appears transparent when applied. A presentation by Schlossman et al. (2006, 093309 ) included pictures demonstrating the opacity of formulations with different particle sizes when applied to skin. Formulations with an effective agglomerated particle size of 100-120 nm (primary particle size of 10 nm) or 120-150 nm (primary particle size of 15 nm) were transparent or nearly transparent. Schlossman et al. (2006, 093309 ) noted that, in addition to particle size, two other factors affected the opacity/transparency of formulat ions: the difference between the refractive index of the particle and that of the media, and the uniformity of particle dispersion. Chaudhuri and Majewski (1998, 093308 ) noted that particle size also affects the stability of sunscreen dispersion. The reason for this was not made clear in the article, but in a discussion of pigmentary particles in paints, Himics and Pineiro (2008, 155626 ) explained that smaller pigmentary particles produce a better dispersion because the larger surface area creates a higher viscosity, which

A-2

### prevents settling and clumping. The phenome non that Chaudhuri and Majewski (1998, 093308 ) noted could have a similar explanation. A range of particle sizes provides a range of UVprotection, but too wide a range could pose a risk of opacity or of compromising the stability of the dispersion (e.g., if too many particles are too large). In the past, controlling the range of particle sizes produced by manufacturing processes was difficult, and distributions with a mean particle si ze of 50 nm included particles in the visible range. As technology has improved, creating particles of desired size and size distributions with much greater accuracy (Hewitt, 2002, 093307 ) has become possible.



# A.1.2. Active Ingredient Purity

The U.S. Pharmacopeia (USP) sets reference standards for TiO 2 

### and other active ingredients in over-the-counter and prescription drugs. The 2006 edition of the USP national formulary monographs, USP-NF 30 (U.S. Pharmacopeia, 2006, 155639 ), declares that TiO 2 

### “contains not less
than 99.0% and not more than 100.5% of TiO 2 

### .” USP specifies tests for water-soluble impurities, acid-soluble impurities, arsenic, and organic volat ile impurities, and notes that FDA also has set limits on acceptable lead, antimony, and mercury cont amination. USP also specifies that the material must be stored in well-closed containers, and that it be properly labeled as attenuation grade (with names and amounts of added coatings, stabilizers, and other treatments listed) if intended for
UV-attenuation.



# A.1.3. Photostability and Surface Coating/Doping

Nano-TiO 2 

### is a natural semiconductor with photocat alytic properties. Its electrons can easily become excited by energy absorbed from UV radiation. When the electrons return to ground state, longer wavelength radiation is emitted. Alternativel y, if the energized el ectrons escape from the particle, they can catalyze chemical reactions (oxida tion/reduction processes) in nearby molecules.
These reactions can create free radi cals, which can damage skin cells or degrade other sunscreen ingredients. The choice of nano-TiO 2 

### crystal affects photostability. In particular, rutile is much more photostable than anatase (Chaudhuri and Majewski, 1998, 093308 ; Maynard, 2008, 157522 ). Although anatase is less photostable, it appears to be in common use. Barker and Branch (2008,
180141 ) studied five TiO 2 

### sunscreens purchased over the counter and found that one was pure rutile and the other four were anatase/rutile mixes in which anatase predominated.
To increase TiO 2 

### and nano-TiO 2 

### photostability (i.e., to reduce the likelihood that excited electrons will escape), the crystals are commonl y given a surface coating. Coating TiO 2 

### with silicon dioxide and alumina (3.5% by weight) can redu ce photocatalytic activity by 99% (SCCNFP, 2000,
092740 ). Other TiO 2 

### or nano-TiO 2 

### surface coatings mentioned in the literature include inorganic oxides (Bird, 2002, 093306 ), simethicone (Chaudhuri and Majewski, 1998, 093308 ), methicone, lecithin (Schlossman et al., 2006, 093309 ), stearic acid, glycerol, silica, aluminum stearate, dimethicone (SCCNFP, 2000, 092740 ), metal soap, isopropyl titanium triisostearate (ITT), triethoxy caprylylsilane, and C9-15 fluoroalcoh ol phosphate (Schlossman et al., 2006, 093309 ). Alumina is often used in combination with other coating materials. The amount of surface coating applied varies substantially from product to product. For examples of common coating concentrations and combinations, see Appendix B, Table B-1. Another technique for increasing photostability is “doping” the TiO 2 

### or nano-TiO 2 

### particles by embedding within them minute amounts of metals such as manganese, vanadium, chromium, and iron. Doping rutile nano-TiO 2 

### with manganese is reported to in crease UV-A absorption, reduce free radical generation, and increase free radi cal scavenging behavior (Reisch, 2005, 155634 ; Wakefield et al., 2004, 193693 ). Doped TiO 2 

### is colored instead of white, which can have desirable cosmetic effects in products such as skin lighteners (Park et al., 2006, 193593 ).

A-3

### Recent research by Barker and Branch (2008, 180141 ) has found that the surface coatings on
nano-TiO 2 

### in many sunscreens might not be stable or effective. The investigators studied the weathering of paint in contact with sunscreen. Out of five nano-TiO 2 

### sunscreens tested, four released photocatalytically generated hydroxyl radicals that acc elerated the weathering of the paint. All four
of those sunscreens used an anatase/rutile mix. The one nano-TiO 2 

### sunscreen that showed no appreciable effect on paint weathering was Oxonica’s OptisolTM, which is 100% rutile, and is doped with manganese rather than surface-coated. It is not known whether nano-TiO 2 

### sunscreens generate hydroxyl radicals when applied to skin or whether such hydroxyl radicals would penetrate the skin and pose a threat to the health of the sunscreen user (Brausch and Smith, 2009, 193297 ; Maynard, 2008, 157522 ).



# A.1.4. Dispersion and pH Considerations

Nano-TiO 2 

### can exist as a dry powder, but most sunscreen applications require the particles to be suspended in a fluid medium. This liquid is calle d a “dispersion” because special care must be
taken to ensure that nano-TiO 2 

### will be distributed evenly and to minimize further aggregation and agglomeration (which could negatively impact pr operties such as UV sc attering performance and transparency by increasing the effective particle size). Sunscreen manufacturers can purchase
nano-TiO 2 

### powder and formulate their own dispers ion, or they can purchase ready-made “predispersions.” In an effective dispersion, suspended particles ar e attracted to the dispersion medium and repel each other. Surface coatings influence the interaction of nano-TiO 2 

### with the dispersion medium, which can be water-based (aqueous), oil-based, or silicone-based. Early TiO 2 

### dispersions were generally oil-based (Bird, 2002, 093306 ). Surface coatings that make TiO 2 

### dispersible in nonaqueous media can be lipophilic (e.g., metal soap, ITT, lecithin); hydrophobic (e.g., methicone, dimethicone, triethoxy caprylylsilane); or both (e.g., C9- 15 fluoroalcohol phosphate) (Shao and Schlossman, 1999, 093301 ). For methicone and C9-15 fluoroalcohol phosphate, silicone might be the preferred medium (Shao and Schlossman, 1999, 093301 ). Bird (2002, 093306 ) states that coatings have been developed to enable TiO 2 

### to be dispersed effectively in aqueous media as well, but provides no examples. Chaudhuri and Majewski (1998, 093308 ) describe one product, an “amphiphilic” powder (Eusolex® T-2000) containing approximately 80% USP-grade rutile coated with alumina and simethicone, that is easily dispersible in both water and oil. Two related concepts that are useful in discussing the dispersion of particles are the pH pzc 

### , which is the pH point at which the surface charge density of a particle is zero, and the isoelectric point (IEP), which is the pH at which the net surface el ectric charge of a particle is zero. In situations where no ions other than H+ and OH- are adsorbed at the particle surface, pH pzc 

### is identical to the

IEP. At most pH values, nano-TiO 2 

### particles suspended in a dispersion have a positive electrical charge or a negative electrical charge and repel each other. At the pH pzc 

### /IEP, however, there is no electrostatic repulsion, and particle s tend to agglomerate (Hewitt, 1995, 157939 ). To maintain electrostatic repulsion and prevent agglomeration, th e dispersed product must be maintained at a pH other than the IEP (usually at a lower pH) at every stage of production and storage. Surface coating can affect a particle’s pH pzc 

### /IEP and can potentially extend the pH range at which the dispersion can be handled. For example, uncoated nano-TiO 2 

### has an IEP of pH 6, and
nano-TiO 2 

### coated with alumina and simethicone has an IEP of pH 9 (Chaudhuri and Majewski, 1998, 093308 ). Bird (2002, 093306 ) cites lecithin as another coating that is advantageous for
electrostatic reasons. Experimental tests show additional pH considerations. Nano-TiO 2 

### performance can be adversely affected by strongly acidic formulations (effects include more agglomeration, lower SPF, and greater opacity), unless special form ulating techniques are used (Hewitt, 1995, 157939 ).

A-4

### Additional compounds can be added to the dispe rsion as “dispersants.” “[The] proper dispersant can help particles to disperse into [the] vehicle so as to shorten the dispersion time and increase the degree of dispersion. It can reduce the viscosity and yet stabilize the dispersion by either electrostatic or steric repellency” (Shao and Schlossman, 1999, 093301 ). Different dispersants are used in water- and oil- (or silicone-) based formulations. PEG-10 dimethicone is used as a dispersant
for nano-TiO 2 

### in a cyclopentasiloxane carrier in the predispersion CM3K25VM made by Kobo Products, Inc. manufactures. Polyhydroxystearic aci d is used as a dispersant in a C12-15 alkyl
benzoate carrier in Kobo’s TNP40TPPS predispersion (Shao and Schlossman, 2004, 157825 ). Mitchnick and O’Lenick (1996, 157935 ) mention lecithin and phosphate esters as potential “dispersing aids” for TiO 2 

### dispersions, but they also use language suggesting that they might actually mean surface coatings.



# A.1.5. Distribution of Active Ingredient in Emulsion

Most sunscreens are emulsions – mixtures of two fluids (called “phases”) that are immiscible (do not combine easily). For instance, water and oil, two immiscible fluids, may be mixed in an emulsion by an energetic process such as stirring or shaking. In some cases, the two fluids tend to quickly separate again. To prevent separation, an emulsifier (typically a surfactant or a polymer) can be added. In an emulsion containing two types of liquids, generally, droplets of one fluid are dispersed in a larger amount of the other fluid. The two fluids are referred to as the “dispersed phase” and the “continuous phase,” respectively. Types of emulsions used in sunscreens and other cosmetic products include oil in water (in which an oil phase is dispersed in a water phase, abbreviated “o/w”); water in oil (w/o); water in water (w/w); and occasionally water in oil in wa ter (w/o/w). In “oil-free” formulations, oil is substituted by silicones (w/S i, Si/w) (Hewitt, 2000, 157898 ). As noted above, nano-TiO 2 

### is most easily dispersed in oil, but emulsions can be formulated with nano-TiO 2 

### in a water phase, an oil phase, or a silicone phase. The nano-TiO 2 

### can be present in the dispersed phase or the continuous phase of a sunscreen emulsion (Dransfield, 2005, 157809 ). The emulsifiers used to keep the two phases from separating are typically partially hydrophilic and partially hydrophobic. By gathering on the interface between the dispersed phase and the continuous phase, emulsifiers bind the two phases (this is the principle behind soaps, shampoos, and detergents, which enable water to wash away oils and other normally hydrophobic particles), or at least prevent the two phases from repelling each other. Emulsifiers used in sunscreen emulsions include glyceryl stearate, PEG-100 stearate, and polyglyceryl-3-methyl glucose distearate (Oxonica, 2005, 157793 ).



# A.1.6. Other Ingredients – Active and Inactive

Nano-TiO 2 

### can be combined with other physical UV blockers, such as ZnO (which can also be micronized), or with chemical UV filters to impr ove the UV protection the sunscreen provides. The sunscreen formula can also include a diverse array of inactive compounds for a variety of purposes.
TiO 2 

### and ZnO can form agglomerates. This attribute presents an obstacle to using TiO 2 

### and ZnO in the same sunscreen. A solution is to put one active ingredient in the oil phase of the emulsion and the other in the water phase (Hewitt, 1995, 157939 ). Combining nano-TiO 2 

### with chemical UV filters often provides better UV-B protection than expected, based on the SPF of each ingredient. The improved protection is probably due to the scattering the physical UV blocker provides, which in creases the optical path length of the radiation and creates more opportunities for absorpti on by the chemical filter (Bird, 2002, 093306 ; Chaudhuri and Majewski, 1998, 093308 ). Emollients are often included in sunscreens to make the products feel more pleasing on the skin or to moisturize. In excessive quantiti es, emollients could break down the dispersion

A-5

### microstructure. This effect can be counteracted by using suitable surfactants or polymers (Hewitt, 1996, 157936 ). Increasingly, nano-TiO 2 

### is found in “cosmeceuticals,” products that combine a variety of active ingredients to perform multiple health and beauty functions. These products include moisturizers and color cosmetics (see below for more on cosmeceuticals). The manganese added to
some nano-TiO 2 

### formulations to prevent formation of free radicals during UV exposure can also help scavenge free radicals generated by other means, thus providing extra skin-protection benefits. Inert ingredients can be added to achieve the right viscosity or liquidity, spray-ability, color or transparency, pH, water-resistance, or spreadabil ity. Silicones and related compounds can be added to impart water-resistance, improve skin feel, se rve as emulsifiers in various formulations, and
enhance the SPF of oil-base d dispersions (Hewitt, 2000, 157898 ).


# A.2. Some Sunscreens with Nano-TiO 2 

# or Micronized TiO 2 

# as Active Ingredient

Table A-1 was compiled from information contained in the Environmental Working Group’s cosmetic database “Skin Deep” (EWG, 2008, 196343 ) and from on-line shopping sources. Products labeled as containing TiO 2 

### of unspecified particle size were excluded. The list of products provided in Table A-1 is likely not exhaustive. Also, product formulations and labels could change over time.

A-6A-7 

## Table A-1. TiO 2 

## content in various sunscreen products.

Brand/ Manufacturer Product Percentage TiO 2

Abella Solar Shade, SPF 45 N/A

Alba Botanica Chemical Free Sunscreen, SPF 18 7.0%

B. Kamins Chemist Bio-Maple Sunbar Sunscreen, SPF 30 Fragrance-Free 2.04%

BABOR High Protection Lotion, SPF 30 N/A

BABOR Moderate Protection Sun Cream, SPF 20 4.5%

BENEV Pure TiO 2 N/A

Bliss Oil-free Sunban Lotion for the Face, SPF 30 6%

California Baby SPF 30 & Fragrance Free Sunscreen; also available as Sunblock Stick, SPF 30 4.5%

California Baby Sunscreen SPF 30+ - Everyday Year Round; also av ailable as Sunblock Stick 4.5%

California Baby Water Resistant, Hy po-Allergenic Sunscreen, SPF 30 N/A

Cellex-C Sunscreen, SPF 15 2%

Cellex-C Water Resistant Sunscreen, SPF 30 2%

Cellex-C Sun Care Broad Spectrum UV-A, UV -B Sunblock & Moisturizer, SPF 15 N/A

Cellex-C Sun Care, SPF 30 2%

Colorescience SPF 30 All Clear Sparkl es Shaker Jar; SPF 30 Perfectly Clear Sparkles Shaker Jar; SPF 30 Almost Clear Sparkles Shak er Jar; these variations al so available in trial size, brushable, and rock and roller ball forms 12%

Dermalogica Oil Free Matte Block, SPF 20 4%

Dermalogica Ultra Sensitiv e FaceBlock, SPF 25 14%

EmerginC Sun 30 (and tinted version) N/A

Fallene/Total Block Total Block Clear, SPF 65 4%

Fallene/Total Block CoTZ, SPF 58 10%

Fallene/Total Block Total Block Cover-Up/Make-Up, SPF 60 10%

Fallene/Total Block Total Block Tinted, SPF 60 10%

Jan Marini Bioglycolic Facial Lotion, SPF 15 5.5%

June Jacobs Micronized Sheer, SPF 30 14.5%

Lancôme Soleil High Protection Face Cream – Gel, SPF 30 4.5%

Lancôme Soleil Soft-Touch Moisturi zing Sun Lotion, SPF 15 4.5%

Peter Thomas Roth Instant Mineral, SPF 30 15%

Pevonia Botanica Pevonia So leil Sun Block, SPF 15 N/A

ProCyte TiSilc Sheer, SPF 45 N/A

ProCyte TiSilc Sheer, SPF 45 (tinted) 3.5%

ProCyte TiSilc Sunblock, SPF 60+ 8%

ProCyte TiSilc Untinted, SPF 45 3.5%

ProCyte Z-Silc Plus S unblock, SPF 30+ 4.0%

Total Skin Care LLC pH Advantage Basics Sun Blocker, SPF 15 N/A

Wilma Schumann Wilma Schum ann Sunscreen, SPF 20 N/A

N/A – Not available.
Source: Used with permission from the Environmental Working Group, for their Skin Deep Database (EWG, 2008, 196343 ).

# REFERENCES

Barker PJ; Branch A (2008). The interactio n of modern sunscreen formulations w ith surface coatings. Progr Org Coating, 62: 313-320. http://dx.doi.org /10.1016/j.porgcoat.2008.01.008 180141

093306 Bird S (2002). Sense and stability. Soap, Perfum Cosmet, 75, 42-44.

Brausch JM; Smith PN (2009). Pesticide resistance from histor ical agricultural chemical exposure in Thamnocephalus platyurus (Crustacea: Anostraca) . Environ Pollut, 157: 481-487. 193297

Chaudhuri RK; Majewski G (1998). Amphiphilic microfine titanium dioxide: Its properties and application in sunscreen formulations. Drug Cosmet Ind, 162: 24-31. 093308

Davis DA (1994). Sunscreen odditie s. Drug Cosmet Ind, 155: 20-24. 157946

Dransfield G (2005). Manufacture of novel, transparent TiO2 based suns creens. Retrieved May 09, 2008 from http://www.wun.ac.uk/nanoman ufacturing/archive/05_06_serie s/documents/dransfield.pdf. 157809

EWG (2008). Sunscreen investigation: Skin deep-cosme tic safety reviews. Retrieved June 02, 2009 from http://www.cosmeticsdatabase.com/special/sunscreens2008/. 196343

093307 Hewitt J (2002). A moment of clarity . Soap, Perfum Cosmet, 75, 47-50.

Hewitt JP (1995). Formulating with physical sunscreens: Control of emulsion pH. Drug Cosmet Ind, 157: 28-32. 157939

157936 Hewitt JP (1996). The influence of emollients on dispersi on of physical sunscreens. Dr ug Cosmet Ind, 159: 62-65.

157898 Hewitt JP (2000). Partners in protect ion. Soap, Perfum Cosmet, 73, 85-86.

Himics R; Pineiro R (2008). The Importa nce of Particle Size in Liquid Coatings - Coating problems and solutions associated with particle size reduction. Prod Finish, 63: 46-53. http://www.pfonline.com/ articles/the-importance-ofparticle-size-in-liquid-coatings. 155626

Jeffries N (2007, February). SPF, efficacy and innovation. GCI Magazine. http://www.gcimagazine.com/marketstrends/segments/suncare/27627099.html. 157682

157522 Maynard AD (2008). Living with nanoparticles. Nano Today, 3: 64.

Mitchnick M; O'Lenick AJ Jr (1996). Patent No. 5565591 (cla ss: 556/10). United States: United States Patent Office. http://www.freepatentsonline.com/5565591.html. 157935

Moyal D (2008). How to measure UVA protection afforded by s unscreen products. Expert Review of Dermatology, 3: 307- 313. http://dx.doi.or g/10.1586/17469872.3.3.307 193559

Newman KA (2006, December). Sun protection report. GCI Magazine. http://www.gcimagazine.com/marketstre nds/segments/suncare/4829426.html?page=1. 157745

Oxonica (2005). Technical notes: Example formulation details a nd protocol tips to obtain optimal dispersion of Optisol (TM) UV Absorber. Retr ieved April 04, 2007 from http://www.oxonica.com/media/medi a_promoliterature.php?start=6. 157793

Packaged Facts (2001). The U.S. market for sun care and lipcare products. Retrieved March 31, 2001 from http://www.mindbranch.com /listing/product/R567-393.html. 196053

Park GB; Knowland JS; Flutter BR (2006). Patent No. 20060134026, class: 424/59. United States: U.S. Patent and Trademark Agency. http://www.fr eepatentsonline.com/y2006/0134026.html. 193593

Reisch MS (2005). New-wave sunscreens: active ingredient ma kers are frustrated by the long list of sunscreens and UV-A testing protocols that are still awaiting FDA decisions. Chem Eng News, 83: 18-22. http://pubs.acs.org/cen/cover story/83/8315sunscreens.html. 155634

SCCNFP (2000). Opinion of the scientific committee on cosmetic products and non-food produc ts intended for consumers concerning titanium dioxide Colipa n S75 (Report No . SCCNFP/0005/98). Brussels, Belgium: Scientific
Committee on Cosmetic Products and Non- Food Products Intended for Consumers. http://ec.europa.eu/health/ph_risk/co mmittees/sccp/documents/out135_en.pdf. 092740



### A-8A-9 Schlossman D; Shao Y; Detrieu P (2006). Perspectives on suppl ying attenuation grades of titanium dioxide and zinc oxide for sunscreen applications. Retrieved October 28, 2007 from http://www.fda.gov/nanotechnology/meet ings/kobo_files/textmostly/index.htm. 093309

Shao Y; Schlossman D (1999). Effect of particle size on pe rformance of physical sunscreen formulas. Talk presented at PCIA Conference, Shanghai, China. http://www .koboproductsinc.com/Download s/PCIA99-Sunscreen.pdf. 093301

Shao Y; Schlossman D (2004). Discovering an optimum sm all micropigment for high UV shielding and low skin whitening. Talk presented at 23d IFSCC Congress, Orlando, FL. http://www.koboproductsinc.co m/Downloads/IFSCC2004.pdf. 157825

TGA (2006). A review of the scientific literature on the sa fety of nanoparticulate titanium dioxide or zinc oxide in sunscreens. Australia: Australian Government, Depa rtment of Health and Ageing, Therapeutic Goods Administration. http://www.tga.gov.au/npmeds/sunscreen-zotd.pdf. 089202

U.S. Pharmacopeia (2006). Titanium dioxide . In U.S. pharm acopeia official monographs (p. 3364). Rockville, MD: U.S. Pharmacopeia. 155639

Wakefield G; Lipscomb S; Holland E; Knowland J (2004). The effects of manganese doping on UVA absorption and free radical generation of micronised titanium dioxide and its consequences for the photostability of UVA absorbing organic sunscreen components. Photochem Photobiol Sci, 3: 648-652. http://www.rsc.org/delivery/_ArticleLinking/Disp layArticleForFree.cfm?doi=b403697b&JournalCode=PP. 193693

# Annex B. Nano-TiO 2 

# in Sunscreen: Manufacturing Processes

B.1. Overview of Nano-TiO 2 

# Manufacturing Process

A generic manufacturing process for nano-TiO2 for sunscreen applications is outlined in Figure B-1.

Source: Dransfield (2005, 157809 )



## Figure B-1. Generic manuf acturing process for nano-TiO 2 

## for sunscreens.



# B.1.1. Titanium Dioxide Nuclei Synthesis

Commercial-scale TiO2 synthesis is mostly by sulfate or chloride processes. In this section, a sulfate process, chloride process, and patented A ltair process are described. These three processes can be used to synthesize both conventional (or pigmentary) and nanoscale TiO2. There are many new processes being developed in the laboratory, but it is outside the scope of this Appendix to cover them; see review of nano-TiO2 synthesis by Chen and Mao (2007, 193313 ). The sulfate process and

Note: Hyperlinks to the reference citations throughout this doc ument will take you to the NCEA HERO database (Health and Environmental Research Online) at http://epa.gov/hero . HERO is a database of scientific litera ture used by U.S. EPA in the process of developing science assessments.



### B-1

### the chloride process, illustrated in Figure B-2, are two common methods used to produce TiO 2 

### in a variety of grades for many different applications.

Source: Millennium Inorganic Chemicals (2007, 195899 ).



## Figure B-2. Sulfate and chloride processes for TiO 2 

## manufacture.

The sulfate process, a wet process for creating pigmentary TiO 2 

### , dates from around 1930, and it was the dominant method used to produce TiO 2 

### until the chloride process was developed in the 1950s (Hext et al., 2005, 090567 ). The chloride process now accounts for approximately 60% of
worldwide TiO 2 

### pigment production (Hext et al., 2005, 090567 ). The chloride process, a gas-phase process, is more energy efficient than the wet-ph ase sulfate process; it can produce finer particles and particles with specific morphologies (Osterwalder et al., 2006, 157743 ). The sulfate process is used primarily to create pigmentary pa rticles. Because attenuation-grade TiO 2 

### can be produced using “the same processes as larger pigmentary grades” 1 

### (Schlossman et al., 2006, 093309 ), the sulfate process and the chloride process are considered in this document as possible manufacturing techniques for nano-TiO 2 

### in sunscreen. The sulfate process and the chloride process differ in the feedstock and techniques for nuclei synthesis. In both processes, par ticles are milled and surface-treated to prepare them for the intended application. The “surface treatment” step in Figure B-2 corresponds to the “coating” step in Figure B-1. The Altair process, a patented, spray-hydrolysis- based process, is illustrated in Figure B-3. This process is used by Altair Nanotechnologies, Inc. to produce not only coated nano-TiO 2 

### for sunscreen applications, but also uncoated and larger TiO 2 

### particles and several ceramic oxides (Verhulst et al., 2003, 157854 ). The feedstock for this process is titanium oxychloride. This patented process is comparable in many respects to the sul fate process. What makes it unique, according to

1 Pigment-grade refers to a classification of particles of size 200 nm or larger. However, any grade of particles will contain a range of particle sizes, and “[a]lthough pigment-grades of TiO 2 are usually considered to consist of micron sized particles, particles below 100 nm may be present in such grades” (Scientific Committee, 2007, 157639 ).

B-2

### Verhulst et al. (2003, 157854 ), is the spray hydrolysis step, which eliminates the aqueous filtration step.

Source: Reprinted with permissi on for Verhulst et al. (2003, 157854 )



## Figure B-3. Nano-TiO 2 

## manufacturing process used by Altair Nanotechnologies,
Inc.

Details of the sulfate process, chloride proce ss, and the Altair Process (derived from spray hydrolysis) are provided in the following paragraphs . The steps unique to each process are presented first, followed by steps shared in these processes. Additionally, processes specific to manufacturing
nano-TiO 2 

### include an additional gas-phase process (TiCl 4 

### + 2H 2 

### O → TiO 2 

### + 4HCl) and three additional wet processes (TiOCl 2 

### + 2NaOH → TiO 2 

### + 2NaCl + H 2 

### O ; Na 2 

### TiO 3 

### + 2HCl → TiO 2 

### + 2NaCl + H 2 

### O ; and Ti(OR) 4 

### + 2H 2 

### O → TiO 2 

### + 4ROH) (Dransfield, 2005, 157809 ). The gas-phase process is similar to the chloride method except th at the titanium tetrachloride is hydrolyzed rather than oxidized. It is also similar in some aspects to the Altair method. These three wet processes rely on feedstocks that are not found in nature, and thus require some additional, unspecified preparatory steps. Waste products from the various processes include hydrochloric acid, salt, water, and compounds formed from impurities.



## Specific Steps in the Sulfate Process

The sulfate process begins with ilmenite ore (FeTiO 3 

### ), which is dried, ground, and treated with concentrated sulfuric acid (H 2 

### SO 4 

### ) in an exothermic digestion reac tion, producing a cake of titanyl sulfate (TiOSO 4 

### ) and other metal sulfates. This cake is then dissolved in water or a weak acid. After chemical flocculation, a clear solution and an insoluble mud are produced. The clear solution is cooled to crystallize ferrous sulfate heptahydrate (FeSO 4 

### · 7H 2 

### O, known as “copperas”). The ferrous sulfate heptahydrate is separated and sold as a by-product (Millennium Inorganic Chemicals, 2007,
195899 ).

B-3

### The insoluble mud is washed, filtered, and ev aporated to produce a concentrated TiOSO 4 

### liquor. The liquor is hydrolyzed to produce a suspensi on or “pulp” that consists mainly of colloidal hydrous titanium oxide clusters (Millenni um Inorganic Chemicals, 2007, 195899 ).
The TiO 2 

### is precipitated from the suspension, which is typically facilitated by a seeding technique to control particle size (no descripti on of the seeding technique was provided). After further washing, heat is applied to crystallize the particles in a process known as calcination, which is also used in other processes. Either anatase or rutile crystals can be pr oduced, depending on the additives applied before calcination (M illennium Inorganic Chemicals, 2007, 195899 ). The following equations represent the chemical processes involved in the sulfate process (Dransfield, 2005, 157809 ):



# FeTiO 3 

# + 2H 2 

# SO 4 

# → TiOSO 4 

# + FeSO 4 

# + 2H 2 

# O

TiOSO 4 

# + H 2 

# O → TiO 2 

# + H 2 

# SO 4


## Specific Steps in the Chloride Process

Natural or synthetic rutile is the feedstock material for the chloride process. During the chlorination step, rutile is added to chlorine and a source of carbon in a fluidized bed at 900oC. The exothermic reaction produces titanium tetrachloride (TiCl 4 

### ) plus a variety of impurities. As the gas cools, low-volatile impurities (e.g., iron, mangan ese, and chromium chlorides) condense out. A stable, very pure liquid TiCl 4 

### is achieved following condensation and fractional distillation (Millennium Inorganic Chemicals, 2007, 195899 ). The pure TiCl 4 

### is then oxidized to TiO 2 

### in a second exothermic reaction. Temperature and other reaction parameters determine the mean particle size, size distribution, and crystal type of the resulting TiO 2 

### . The TiO 2 

### is cooled, and impurities are removed. Chlorine released by the oxidation reaction is recycled for reuse (Millennium Inorganic Chemicals, 2007, 195899 ). The following equations represent the chemical processes involved in the chloride process (Dransfield, 2005, 157809 ):



# TiO 2 

# (impure) + 2Cl 2 

# + C → TiCl 4 

# + CO 2



# TiCl 4 

# + O 2 

# → TiO 2 

# + 2Cl 2


## Specific Steps in the Altair Process – Spray Hydrolysis

The patented Altair process (Verhulst et al., 2003, 157854 ) was derived from a spray hydrolysis method for TiO 2 

### synthesis. The feed is a titanium oxychloride aqueous solution. The feed solution can be produced by hydrating liquid TiCl 4 

### in a dilute hydrogen chloride (HCl) solution. In spray hydrolysis, heat (from hot air or a hot receiving surface) causes rapid and complete evaporation of the water in the feed solution as the solution is sprayed. An amorphous, homogeneous, dense, thin film remains on the re ceiving surface. The film is composed of dry, hollow, almost completely amorphous, TiO 2 

### particles containing some free or hydration water and some HCl (Verhulst et al., 2003, 157854 ).



## Calcination for Sulfate and Altair Processes

Calcination is the process of heating a solid material to a temperature high enough to change its chemical composition (though generally not high e nough to liquefy it). In wet processes like the sulfate and Altair processes, calcination generally occurs after the hydrolysis step. Verhulst et al. (2003, 157854 ) describe the calcined product as a porous crystalline structure of nanoparticles. The crystalline structure retains the shape of the original droplets from the hydrolysis step and will eventually be broken down by milling. The duration and temperature of calcination and the additives

B-4

### introduced during calcination directly influence th e structure, particle size, and particle-size distribution of the calcined product. For example, the anatase structure can be stabilized by adding phosphates during calcination (Verhulst et al., 2003, 157854 ).



## Milling and Micronizing for Sulfate, Chloride, and Altair Processes

1 

### Milling breaks apart the hollow crystalline lattice structure produced in the calcination step, but has to be mild enough not to break the individual crystallites (Verhulst et al., 2003, 157854 ). Milling also breaks down agglomerates or aggregates into smaller particles. Both a wet media mill (e.g., with zirconia be ads) and ultrasonic milling can be effective (Verhulst et al., 2003, 157854 ). After spray drying, the milled particles (“loosely agglomerated balls”) can be “further micronized to produce a dispersed powder.” While both micronizing and milling decrease the agglomerates, they are different processes. In micronizing, agglomerates collide with each ot her in a circulating stream of air or steam, and the collision breaks down the agglomerated part icles. In milling, an external grinding agent is used to decrease the size of agglomerates. For instan ce, agglomerates in a liquid medium are fed into a mill containing small ceramic beads, and the im pact from the beads on the agglomerates during mixing break the agglomerated particles.



# B.1.2. Surface Treatments and Doping

Some, but not all, nano-TiO 2 

### particles used for sunscreen undergo surface treatment to prevent the creation of free radicals, which could degrade the sunscreen or damage the skin (DuPont, 2007,
157699 ; Schlossman et al., 2006, 093309 ; Wakefield et al., 2004, 193693 ). Surface coatings for
nano-TiO 2 

### in sunscreen can include combinations of inorganic oxides, simethicone, methicone, lecithin, stearic acid, glycerol, silica, aluminum st earate, dimethicone, metal soap, isopropyl titanium triisostearate (ITT), triethoxy caprylylsilane, and C9-15 fluoroalcohol phosphate. In a patent they hold, Mitchnik and O’Lenick (1996, 157935 ) describe a sample protocol for applying a silicone surface treatment to TiO 2 

### for sunscreen. The patent does not specify the size of
the TiO 2 

### particles. A quantity of silicone compound (generally between 0.1% and 25% by weight of the total formulation) is combined with TiO 2 

### powder. The mixture is heated to 40-100oC for 2-10 hours, or long enough to remove 97% of the alcohol produced in the reaction. The patent holders claim that the resultant coated particle s provide superior performa nce because the coating “preserves the structure of the TiO 2 

### crystals, eliminates the reactivity in water, and makes them hydrophobic.”
Nano-TiO 2 

### particles can also be doped with vari ous metals such as manganese, vanadium, chromium, and iron. Park et al. (2006, 193593 ) listed examples of doping methods, including: (1) combining particles of a host TiO 2 

### lattice with a second component in solution or suspension, and then baking at no lower than 300oC. The second component is typically a salt, such as a chloride, or an oxygen-containing anion, such as a perchlorate or a nitrate; (2) mixing solutions of the dopant salt and of a titanium alkoxide, and then heating the solution to convert the alkoxide to the oxide and precipitate out the doped material; and (3) flame pyrolysis 2 

### or plasma routes (no additional detail provided).

1 Lattice is the geometrical arrangement of atoms in a crystal.

2 Flame pyrolysis is a synthesis method in which flame heat is applied to vaporize stock material (gas phase precursors) and to i nitiate chemical reaction for particle (including nanoparticles) production.

B-5

# B.2. Nano-TiO 2 

# Particles and Products Used in Sunscreens

Several commercially-available nano-TiO 2 

### particles intended for sunscreen application and some of their characteristics are summarized in Table B1 (SCCNFP, 2000, 092740 ), and an additional list is on the internet (EWG, 2009, 625314 ). Although these nano-TiO 2 

### particles were selected for their applicability to the European market , they are likely to be fairly representative of
nano-TiO 2 

### active ingredients used in the U.S.



## Table B-1. Selected list of nano-TiO 2 

## particles used in sunscreen

Average crystal size Particle name Manufacturer Crystal type Coating materials and concentrations

T805 Degussa20/80 RU/AN Degussa rutile/ anatase 21 nm silicone dioxide <2.5%

T817 Degussa79/12/2 RU/AN/Fe Degussa rut ile/ anatase 21 nm silicone dioxide <2.5 % (also doped with di-iron trioxide 2%)

UV-Titan M160 Kemira rutile 17-20 nm alumina 5.5-7.5%, stearic acid 10%

UV-Titan M212 Kemira rutile 20 nm alumina 5-6.5%, glycerol 1%

UV-Titan X161 Kemira rutile 15 nm al umina 8.5-11.5%, stearic acid 10%

UV-Titan X200 Kemira rutile 20 nm none

Eusolex T-2000 Merck unknown 14 nm alumina 8-11%, simethicone 1-3%

TTO 51A Merck rutile 35 nm alumina 11%, silica 1-7%

TTO 51C Merck rutile 35 nm alumina 11 %, silica 1-7%, stearic acid 3-7%

MT-100 AQ Mitsubishi/Tayca rutile 15 nm alumina 4-8%, silica 7-11%

MT-100 AR Mitsubishi/Tayca unknown 15 nm alumina 4-8%, silica 7-10%

MT-100 T-L-1 Mitsubishi/Tayca rutile 15 nm alumina 3.3-7.3%, stearic acid 5-11%

MT-100SA Mitsubishi/Tayca rutile 15 nm alumina 4-7.5%, silica 2-4%

MT100TV (or MT-100TV) Mitsubishi/Tayca rutile 15 nm alumina 1-15% or 3-8%; aluminum stearate 1-13% or 1-15% or stearic acid 5-11%

MT100Z (or MT-100Z) Mitsubishi/Tayca rutile 15 nm alumina 6-10%, stearic acid 10-16%

MT-500SA Mitsubishi/Tayca rutile 35 nm alumina 1-2.5%, silica 4-7%

Mirasun TiW60 Rhodia anatase 60 nm alumina 3-7%, silica 12-18%

UV-Titan M262 Rhodia and Kemira rutile 20 nm alumina 5-6.5%, dimethicone 1-4%

Tioveil dispersions Uniqema rutile 10-28 nm alumi na 10.5-12.5% or 5-15% and silica 3.5-5.5%; alumina 5-15% and aluminum stearate 5-15%

Source: Used with permission of the European Union, SSCNFP (Scientific Committee on Cosmetic Products and Non-Food Products Int ended for Consumers) (SCCNFP, 2000, 092740 ).



### Three manufacturers of USP-grade nano-TiO 2 

### for sunscreen applications provided information on their products and processes: Kobo Products In c., which specializes in powders and dispersions; Oxonica, a European nanomaterials group; and Un iqema, a manufacturing company specializing in
oleochemicals 1 

### and specialty chemicals for cosmetics and personal care products. Uniqema was acquired by Croda in 2006 (Croda, 2006, 193851 ). Kobo manufactures a line of 26 attenuation-grade TiO 2 

### dispersions containing nano-TiO 2 

### . The primary particle sizes are mostly 10-35 nm in 25 of 26 dispersions; one dispersion contains 90 nm

1 Oleochemicals, e.g., fatty acids, fatty alcohols, and fa tty esters, are derived from biological oils or fats.

B-6

### primary TiO 2 

### particles. The nano-TiO 2 

### aggregate sizes in dispersions (measured by dynamic light scattering [DLS]) are mostly 103-165 nm in 25 of 26 dispersions, including the dispersion with 90 nm primary particles; one dispersion contains 230 nm aggregates (Kobo Products Inc, 2009,
196045 ). One of the Kobo TiO 2 

### dispersions called TNP40VTTS contains nano-TiO 2 

### particles coated with alumina and an isopropyl titanium tri-isostearate/triethyl caprylysilane crosspolymer (Kobo Products Inc, 2009, 196045 ; Shao and Schlossman, 1999, 093301 ). Polyhydroxystearic acid is used to disperse the product in the solvent/carri er, C12-15 alkyl benzoate, which is an ester (Kobo Products Inc, 2009, 196045 ; Shao and Schlossman, 1999, 093301 ). The particles in another dispersion, CM3K40T4, are surface-treated with alumina and methicone and are dispersed in the cyclopentasiloxane carrier with the help of PEG-10 dimethicone (Kobo Products Inc, 2009, 196045 ; Shao and Schlossman, 1999, 093301 ). OptisolTM UV Absorber, a nano-TiO 2 

### product, is the first commercial product from Oxonica Materials (a branch of Oxonica), and the first commercial health product from Oxonica. OptisolTM is a powder composed of uncoated rutile nano-TiO 2 

### (size not specified) with approximately 0.67% manganese in the crystal lattice (Kobo Products Inc, 2009, 196045 ; Shao and Schlossman, 1999,
093301 ). Doping with manganese gives the sunscreen the advantages of increased UV-A absorption, reduced free radical generation, and increased fr ee radical scavenging behavior (Reisch, 2005,
155634 ; Umicore, 2008, 193688 ).

1 

### Uniqema/Croda manufactures several TiO 2 

### sunscreens, including a line of Solaveil TM Clarus using nano-TiO 2 

### (Chandler, 2006, 193834 ). Solaveil CT-100 and Solaveil CT-200, two of the products in the Solaveil Clarus line, are discussed here as examples. Solaveil CT-100 has more than 50% C12-C15 alkyl benzoate, 25-50% nano-TiO 2 

### , and 1-5% each of aluminum stearate, polyhydroxysteric acid, and alumina (Croda, 2007, 193875 ). Solaveil CT-200 has 15-40%
nano-TiO 2 

### , 10-30% isohexadecane, 10-30% glycerol tri( 2-ethylhexanoate), 3-7% aluminum stearate, and 1-5% each of polyhydroxysteric acid and aluminum oxide (Croda, 2008, 193878 ). The TiO 2 

### particle size distribution is very narrow, with the va st majority of particles falling in the nano-range (Croda, 2008, 193878 ). Uniqema (2004, 155637 ) recommended using CT-200 at a concentration of 2-30%. The dispersion can be included in the oil phase in an oil-in-water (o/w) emulsion, or in the water phase in water-in-oil (w/o) emulsion, or added separately to a w/o emulsion after emulsification (Uniqema, 2004, 155637 ).


# B.3. Formulations for Sunscreen Containing Nano-TiO 2

Sunscreen formulations that major manufacturer s use are proprietary. Companies that produce sunscreen ingredients, however, promote their products by publicizing suggested formulations. These suggested formulations indicate the types of ingredients and processes that might be typical in sunscreen formulation. Two such suggested formulations are discussed here. Generally, compatible ingredients are combined into a number of fluid phases. These phases are then energetically mixed in a particular sequenc e (sometimes at specified temperatures) to form
an emulsion. Formulators have to take care not to al low the pH of the mixture to reach the isoelectric point (IEP) of the nano-TiO 2 

### or any other dispersed ingredient. Table B-2 shows a sample formulation using Croda Solaveil CT-10W and Solaveil CT-200 (Croda, 2009, 193880 ). Table B-3 lists a sample formulation that uses nano-TiO 2 

### from Kobo for SPF 35 sunscreen that appears transparent when applied on skin (Kobo Products Inc, 2009, 196045 ).

1 Croda acquired Uniqema in 2006 (Croda, 2006, 193851 ). In this Appendix, information sources are cited as it was presented at the time of publication.

B-7

## Table B-2. Formula SC-383-1 for “Weight less Morning Dew with Sun Protection”

Ingredients %

Part A

Water QS

Hydroxypropyl starch phosphate a 1.00

Arlatone V-150 [steareth-100 (and) steareth-2 (and) mannan (and) xanthan gum] 0.50

Arlatone LC 2.00

PricerineTM 9088 (glycerin) 4.00

Solaveil CT-10W [water (and) TiO 2 (and) isodeceth-6 (and) oleth-10 (and) aluminum stearate (and) alumina (and) simethicone] 5.00

Part B

Solaveil CT-200 [TiO 2 (and) isohexadecane (and) 2.00 triethylhexanoin (and) aluminum stearate (and) alumina (and) polyhydroxystearic acid]

Ethyl methoxycinnamate b 4.00

BRIJTM 721 (steareth-21) 2.00

Arlamol PS15E (PPG-15 stearyl ester) 5.00

Part C

Phenoxyethanol (and) methylpar aben (and) ethylparaben (and) 1.00 propylparaben c

pH: 6.75 ± 0.5; viscosity: 223.5 ± 10% (centipoise) cps

Procedure: Disperse Arlatone V-150 in water. Then disperse the preser vative. Add Pricerine 9088 and heat to 60°C and add Arlatone LC. Continue heating to 80°C and add Solaveil CT-10W. Combine and heat Part B to 80°C. Add Part B to Part A. Homogenize for 2 minutes. Return to stirring and cool to 40°C. Add Part C. Stir to room temperature.

Note: QS means a sufficient quantity.

a Structure XL, National Starch

b Eusolex 2292, Merck KGaA

c Phenonip XB, Clariant

Source: Croda (2009, 193880 ).

B-8B-9 

## Table B-3. Formula KSL-17 for High SPF Transparent Sunscreen

Ingredients %

Part 1

Rose Talc-MS2 – Kobo Products : Talc (and) Methicone 1.00

Velvesil 125 – Momentive/Kobo Pr oducts : Cyclopentasiloxane (and) C30-45 Alkyl Cetearyl Dimethicone Crosspolymer 3.00

Net-WO – Barnet : Cyclopentasiloxane (and) PEG-10 Dimethicone (and) Disteard imonium Hectorite 0.20

CM3K40T4 – Kobo Products : C yclopentasiloxane (and) TiO 2 (and) PEG-10 Dimethicone (and) Alumina (and) Methicone 35.00

Uvinul MC80 – BASF : Ethylhexyl Methoxycinnamate 7.00

Salacos 99 – Nisshin Oil : Isononyl Isonanoate 5.00

Lexol EHP – Inolex Chemical : Ethylhexyl Palmitate 4.00

Squalane – Fitoderm : Squalane 0.20

Tocopherol – Cognis : Tocopherol 0.20

SF96-350 – Momentive/Kobo Products : Dimethicone 1.00

SF96-100 – Momentive/Kobo Products : Dimethicone 1.00

SF1202 – Momentive/Kobo Products : Cyclopentasiloxane 27.10

Propyl Paraben NF – Internati onal Sourcing : Propylparaben 0.10

Part 2

Sodium Citrate – Roche : S odium Citrate (and) Water 2.00

Net-DG – Barnet : Dipotassium Glycyrrhizinate 0.10

Sodium Hyaluronate – Cent erchem : Sodium Hyaluronate (and) Water 1.00

Keltrol CG-T – CP Kelco : Xanthan Gum (and) Water 2.00

Butylene Glycol – Ruger : Butylene Glycol 4.00

Methyl Paraben NF – International Sourcing : Methylparaben 0.10

Water 6.00

Manufacturing Procedure: *Use explosion-proof mixers and equ ipment during batching process * Mix each Part separately. Make sure Net-WO is dispersed in Part 1.

Heat both Parts to 40oC and add Part 2 to Part 1 while stirring with homogenizer at 3, 000 rotations per minute (rpm). Increase the rotation to 5,000 rpm and c ontinue to emulsify for 5 minutes. Cool down to room temperature with sweeping mixer.

Source: Kobo Products Inc. (2009, 196045 ).

# REFERENCES

Chandler M (2006). Innovative UV Protecti on. Creating your advantage in sunscreen products. Talk presented at Croda Educational eSeminar, Edison, NJ. http://www.cosmeticsandtoiletries.com/networking/eventcoverage/3729901.html. 193834

Chen X; Mao SS (2007). Titanium dioxide nanomaterials: S ynthesis, properties, modifications, and applications. Chem Rev, 107: 2891-2959. 193313

193851 Croda (2006). Croda invests in growth: Acquires Uniqema [brochure]. Cosmetics & Toiletries.

193875 Croda (2007). Product Trade Name: SOLAVEIL CT-100 [brochure].

Croda (2008). Chemical Composition of Solaveil CT-200 [brochure]. 193878

193880 Croda (2009). Weightless Morning Dew with Sun Protection (SC-383-1) [brochure].

Dransfield G (2005). Manufacture of novel, transparent TiO2 based suns creens. Retrieved May 09, 2008 from http://www.wun.ac.uk/nanoman ufacturing/archive/05_06_serie s/documents/dransfield.pdf. 157809

DuPont (2007). Nanomaterial Risk Assessment Worksheet DuPont(TM) Light Stabilizer. Retrieved June 18, 2008 from http://www.edf.org/documents/6913_TiO2_Worksheet.pdf. 157699

EWG (2009). EWG's 2009 sunscreen investigation - Section 5: Impact of nanoparticles. Retrieved April 29, 2010 from http://www.ewg.org/cosmetics/report/sunscreen 09/investigation/impact-of-nanoparticles. 625314

Hext PM; Tomenson JA; Thompson P (2005). Titanium dioxide: inhalation toxicology and epidemiology. Ann Occup Hyg,

49: 461-472. 090567

Kobo Products Inc (2009). Attenuation grade Ti O2 dispersions. Retrieved July 16, 2009 from http://www.koboproductsinc.com/Down loads/Kobo-TiO2Dispersions.pdf. 196045

Millennium Inorganic Chemicals (2007). Titanium dioxide ma nufacturing processes. Retrieved February 16, 2009 from http://www.millenniumchem.com/Products+and+Serv ices/Products+by+Type/Titanium+Dioxide+- +Paint+and+Coatings/r_TiO2+Fundamentals/Titanium+Dioxide+- +Paint+and+Coatings+TiO2+Fundamentals_EN.htm. 195899

Mitchnick M; O'Lenick AJ Jr (1996). Patent No. 5565591 (cla ss: 556/10). United States: United States Patent Office. http://www.freepatentsonline.com/5565591.html. 157935

Osterwalder N; Capello C; Hungerbühler K; Stark WJ ( 2006). Energy consumption during nanoparticle production: how economic is dry synthesis? J Nanopart Res, 8: 1-9. 157743

Park GB; Knowland JS; Flutter BR (2006). Patent No. 20060134026, class: 424/59. United States: U.S. Patent and Trademark Agency. http://www.fr eepatentsonline.com/y2006/0134026.html. 193593

Reisch MS (2005). New-wave sunscreens: active ingredient ma kers are frustrated by the long list of sunscreens and UV-A testing protocols that are still awaiting FDA decisions. Chem Eng News, 83: 18-22. http://pubs.acs.org/cen/cover story/83/8315sunscreens.html. 155634

SCCNFP (2000). Opinion of the scientific committee on cosmetic products and non-food produc ts intended for consumers concerning titanium dioxide Colipa n S75 (Report No . SCCNFP/0005/98). Brussels, Belgium: Scientific
Committee on Cosmetic Products and Non- Food Products Intended for Consumers. http://ec.europa.eu/health/ph_risk/co mmittees/sccp/documents/out135_en.pdf. 092740

Schlossman D; Shao Y; Detrieu P (2006). Perspectives on suppl ying attenuation grades of titanium dioxide and zinc oxide for sunscreen applications. Retrieved October 28, 2007 from http://www.fda.gov/nanotechnology/meet ings/kobo_files/textmostly/index.htm. 093309

Scientific Committee on Computer Products (2007). Preliminary opinion on safety of nanomaterials in cosmetic products. Brussels, Belgium: European Commission, Health and Consumer Protection Directorate-General. 157639

Shao Y; Schlossman D (1999). Effect of particle size on pe rformance of physical sunscreen formulas. Talk presented at PCIA Conference, Shanghai, China. http://www .koboproductsinc.com/Download s/PCIA99-Sunscreen.pdf. 093301



### B-10B-11Umicore (2008). Nano-sized oxide powders for UV applications. Abstract from Innovation for Sustainable Production (iSUP2008), Bruges, Belgium. 193688

Uniqema (2004). Solaveil CT-200 (Report No. PC/E/03-03/GLOB/14.5/CT200). London: Uniqema. 155637

Verhulst D; Sabacky BJ; Spitler TM; Prochazka J (2003). Pro cess for the production of nano-sized TiO2 and other ceramic oxides by spray hydrolysis. Reno, Neva da: Altair Nanomaterials Inc. 157854

Wakefield G; Lipscomb S; Holland E; Knowland J (2004). The effects of manganese doping on UVA absorption and free radical generation of micronised titanium dioxide and its consequences for the photostability of UVA absorbing organic sunscreen components. Photochem Photobiol Sci, 3: 648-652. http://www.rsc.org/delivery/_ArticleLinking/Disp layArticleForFree.cfm?doi=b403697b&JournalCode=PP. 193693

# Annex C. Nano-TiO 2 

# Exposure Control in the Workplace and Laboratory

C.1. Workplace Exposure Controls

This section provides examples of strategies that are currently in place or recommended to decrease exposures to nanomaterials in the workplace (Nanosafe, 2008, 196066 ; NIOSH, 2009,
196073 ) and to ensure the effectiveness of personal protective equipment (PPE) against nano-TiO 2 

### (Golanski et al., 2008, 196048 ; Guizard and Tenegal, 2008, 196049 ) (Nanosafe, 2008, 196066 ). Other approaches to reduce work er exposure have been developed and are undergoing further refinement; the examples provided here are intended to be illustrative rather than exhaustive. While this section focuses on workplace practice of nanoma terial manufacturers, some of the principles and use of PPE are also applicable to laboratories and other settings.
The NanoSafe Dissemination Report (Nanosafe, 2008, 196066 ) provided several tiers of approaches to decrease nanomaterial exposure in the workplace. During production, the first and preferred approach is to avoid potential exposure to fre e air flowing particles. If this avoidance is not possible, the process should be contained. If process containment is not possible, extended PPE (which includes double gloves of nitrile, a mask [FFP3 or powered respirators incorporating helmets], a protective suit, and safety shoes) and an effective local exhaust system, such as a high efficiency particulate air (HEPA) H14 filter, should be used. During loading and unloading of reactors, and while packing containers, exposure can be decreased by process containment (e.g., by using a glove box or emptying the reactor using an industrial vacuum with a HEPA filter through a liquid trap) (Nanosafe, 2008, 196066 ). Less preferred alternatives are to transfer nanoparticles within a laminar air-flow booth or extraction hood, or to conduct the transfer in an isolated area equipped with HEPA H14 filter. These alternative options would require the use of extended PPE (Nanosafe, 2008, 196066 ). During cleaning, special vacuums to avoid dust explosion can be used to trap nanoparticles. The vacuums should be cleaned in a room equipped with a HEPA H14 filter and a washer to clean the protective suites (Nanosafe, 2008, 196066 ). Alternatively, particles can be drawn into a powdercollection system using a variable-speed fan. Components should be cleaned in a hood equipped with a HEPA filter and an explosion vent panel. NIOSH has a nanotechnology program to increase safety and decrease potential exposures to nanomaterials in the workplace (NIOSH, 2009, 196073 ). In a NIOSH document for safe nanotechnology (NIOSH, 2009, 196073 ), occupational health surveillance and guidelines for working with engineered nanomaterials are discuss ed, among other topics. Some of these programs could also educate and encourage the general public to reduce environmental releases from the products into the environment. Some companies that manufacture nano-TiO 2 

### have engineering safeguards and additional programs in place to reduce or eliminate occupational and environmental exposures (e.g., BASF, 2008, 193811 ; DuPont, 2007, 157699 ). Various production methods to decrease worker exposure are also being investigated [for nano-TiO 2 

### , see Guizard and Tenegal (2008, 196049 )].

Note: Hyperlinks to the reference citations throughout this doc ument will take you to the NCEA HERO database (Health and Environmental Research Online) at http://epa.gov/hero . HERO is a database of scientific litera ture used by U.S. EPA in the process of developing science assessments.



### C-1

### With a goal toward managing nanotechnology safe ly and effectively within the industrial setting, the NOSH Consortium has investigat ed methods for monitoring workplace exposure and testing protective technologies. The NOSH Consortium has measured the effectiveness of standard respiratory filters with silicon dioxide (SiO 2 

### ) aerosol nanoparticles. With the exception of prolonged exposure (400 minutes or longer), the filter effici encies for both charged and re-neutralized SiO 2 

### aerosol nanoparticles met the specificati ons of the filter type (Ostraat, 2009, 196077 ). The longest exposure time within which the N100 filter performed at or exceeded the efficiency specified by the filter ranking (>99.97% filtration effici ency) was 210 minutes (Ostraat, 2009, 196077 ). No PPE specifically designed for nanomaterials exists or is under development (Klaessig, personal communication, 2008, 196042 ). For filter efficacy against nano-TiO 2 

### aerosol penetration tested by NanoSafe, see Section C.1.1.



# C.1.1. Personal Protective Equipment

In this section, two types of PPE are briefly discussed in terms of their protection against
nano-TiO 2 

### aerosols: (1) filters for inhalation protecti on; and (2) protective clothing and gloves for skin protection. Eye-protective gear is available as a third type of PPE commonly used for protection against nano-TiO 2 

### aerosols, but no information was found on this subject. Each type of nanomaterial is different, and the methods for testing PPE efficiency (such as using charged or neutralized particles) could greatly affect the measured barrier effectiveness. For example, fibrous filters often remove more ch arged aerosol nanoparticles than uncharged or neutralized aerosol nanoparticles (Kim et al., 2006, 193470 ; Ostraat, 2009, 196077 ). Other physicochemical properties of nanoparticles that aff ect filtration efficiency include size, chemical composition, and shape. The size of the particle that most effectively penetrates into a specific filter is called the maximum penetrating particle size (M PPS). For particles smaller than the MPPS, the particle penetrations decrease with decreasing partic le size; for particles larger than the MPPS, the particle penetrations decrease with increasing particle size. Particles smaller than the pore size of the filter may be filtered out when the Brownian move ment of the particles leads to collision of the particle and filter (McKeytta, 1984, 196036 ). Electrostatic filters are charged polypropylene fi bers, classified as FPP3 – minimum filtration efficiency 99% – based on European Norm (EN) cert ification. When an electrostatic filter was tested
with nano-TiO 2 

### aerosols, for which size ranged from 16 nm to greater than 76 nm, the MPPS was approximately 35 nm, which was very similar to graphite MPPS (Golanski et al., 2008, 196048 ). At the MPPS, however, nano-TiO 2 

### penetration was nearly five times hi gher than that for graphite. Near the MPPS, the differences between nano-TiO 2 

### and graphite particle penetration increase by an order of magnitude. HEPA filters have a minimum filtration efficiency of 99.97%, are composed of glass fibers, and are classified as H12 for particles <1 μm. Li ke electrostatic filters, HEPA filters showed one order of magnitude higher penetration of nano-TiO 2 

### (10-19 nm) than that of graphite (10-19 nm), with the highest penetration at approximately 0.2% for 19-nm TiO 2 

### (Golanski et al., 2008, 196048 ). The penetration efficacy of platinum (Pt) particles through HEPA filters was only slightly lower than
that of nano-TiO 2 

### particles. Golanski et al. (2008, 196048 ) showed that particle size alone might not
be a sufficient indicator of HEPA filter performance and suggested that nano-TiO 2 

### might penetrate fibrous filters more efficiently than other nanoma terials, namely graphite and Pt. The exposure duration of the Golanski et al. (2008, 196048 ) study was not reported, and therefore, it could be possible that the filtration efficiency of HEPA filters for nano-TiO 2 

### might decrease with prolonged exposure, as was found for the N100 filter for more than 400 minutes of exposure to SiO 2 

### aerosol nanoparticles (Ostraat, 2009, 196077 ). The efficacy of protective clothing in preventing nano-TiO 2 

### penetration by diffusion was higher for nonwoven fabric than woven cotton and polyester fabric (Golanski et al., 2008, 196048 ). Air-tight, nonwoven, polyethylene Tyvek (115 μm th ick) was more effective against nanoparticle

C-2

### penetration than woven cotton (650 μm thick) and woven polyester (160 μm thick) for 10-nm
nano-TiO 2 

### (Golanski et al., 2008, 196048 ), 10-nm nano-Pt (Golanski et al., 2008, 196048 ), and 40- and 80-nm graphite (Nanosafe, 2008, 196066 ). Nitrile, latex, and Neoprene gloves were reported to be effective against nano-TiO 2 

### aerosol penetration via diffusion for a short exposure time (minutes). No penetration through gloves was detected when the gloves were exposed to aerosols of approximately 10-nm nano-TiO 2 

### and 10-nm Pt (Golanski et al., 2008, 196048 ) or 20- to 100-nm graphite (Nanosafe, 2008, 196066 ). However, continuous flex of gloves could lead to cracks and holes in the gloves (Schwerin et al., 2002,
193636 ), so changing gloves throughout the day is recommended (Harford et al., 2007, 196051 ). 

# C.2. Manufacturer and Laboratory Practices

In 2006, the University of California-Santa Barbara completed a study of nanomaterial manufacturers and laboratories for the International Council on Nanotechnology by surveying organizations about their manufacturing and laborator y practices. Survey results indicated that only 36% of the 64 responding organizations stated that they monitored exposure to the nanomaterials in their workplace. Additionally, 38% of the organizations surveyed believed their nanomaterials posed no special risks, 40% had safety concerns, a nd 22% were unaware of whether or not the nanomaterials they worked with or manufactur ed pose safety risks (Gerritzen et al., 2006, 097620 ). Subsequently, the same research team published additional findings based on a larger sample size of 82 versus the original 64. Of the 82 respond ing firms and laboratories, 89% had a general environmental health and safety program, and 70% provided some type of special training on nanomaterial safety. Nanomaterial safety training was more prevalent in North American firms and laboratories (88%) than in European (64%) or Asian (61%) organizations. Nearly 82% of respondents made nano-specific PPE recommendations to employees. Those tended to be the same
firms and laboratories that used advanced engineering controls (i .e., beyond fume hoods) to prevent exposure. Controls included exhaust filtration, air filtration, wet scrubbers, and automated or enclosed operations. Approximately 56% of No rth American respondents practiced workplace monitoring for nanoparticles, compared to 32% of all respondents. Waste-containing nanomaterials were disposed of as hazardous waste in 78% of North American organizations, compared to 60% of all respondents (Conti et al., 2008, 155619 ). A survey of 43 New England nanotechnology firms found that larger companies (with 500 or more employees) were more likely to recognize po tential environmental h ealth and safety (EHS) risks potentially posed by nanoparticles and had EHS measures in place. Many smaller firms either did not perceive risks or did not implement EHS measures (due both to staff and resource constraints and a lack of information on how to quantify nanoparticle risks) (Lindberg and Quinn, 2007,
155629 ).

C-3C-4 

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McKeytta JJ (1984). Encyclopedia of chemical pr ocessing and design. Boca Raton, FL: CRC Press. 196036

Nanosafe (2008). Dissemination report: Are co nventional protective devices such as fibrous filter media, respirator cartridges, protective clothing and gloves also effi cient for nanoaerosols? (R eport No. DR-325/326-200801-1). France: Sixth Framework Programme. http://www.nanosafe.org/scripts/home/publigen/content/templates/show.asp?P=63&L=EN&ITEMID=13. 196066

NIOSH (2009). Approaches to safe nanotechnology: Managing the health and safety concerns associated with engineered nanomaterials. Retrieved June 02, 2009 from http://cdc.gov/niosh/docs/2009-125/. 196073

Ostraat ML (2009). Industry-led initiative for occupational h ealth and safety. In M Hull; S Friedrichs (Eds.), Risk governance of nanotechnology: Environmental, health and safety concerns (pp. 181-246). Norwich, NY: William Andrew Publishing. 196077

Schwerin MR; Walsh DL; Coleman Richardson D; Kisielewsk i RW; Kotz RM; Routson LB; David Lytle C (2002). Biaxial flex-fatigue and viral penetration of natural rubber latex gloves before and after artificial aging. J Biomed Mater Res, 63: 739-745. 193636