Considerations for Characterizing the Potential Health Effects from

Exposure to Nanomaterials

David B. Warheit, PhD.

DuPont Haskell Laboratory

Newark, Delaware, USA

NNI-NIST Workshop

Gaithersburg, MD

September 13, 2007

Outline• Particle characterization as it relates to

• particle deposition, macrophage interactions, particle translocation

• Particle characterization for 5 studies

• Fine/Ultrafine TiO2 particle types;

• Fine/Nanoscale Quartz particle-types;

• Summary - Recommendations

Rat Lung Microdissection

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

Iron Particle Deposition at Bronchoalveolar Junction

Iron Particle () Deposition in the Lungs of Exposed Rats

Iron Particle Deposition at Bronchoalveolar Junction

(Backscatter Image)

Alveolar Macrophage Clearance of Inhaled Iron Particles

Alveolar Macrophage Clearance of Inhaled Iron Particles

(Backscatter Image)

Alveolar Macrophage Migration to Iron Particle Deposition and Phagocytosis

Alveolar Macrophage Migration to Iron Particle Deposition and Phagocytosis

(Backscatter Image)

Macrophage phagocytosis of TiO2 particles

Two Alveolar Macrophages (M) Sharing a Chrysotile Asbestos Fiber () with an Alveolar Epithelial Cell (E)

MM E

TEM demonstrating pathways for possible translocation of particles

Translocation of chrysotile asbestos fibers from airspace to epithelium

1) Pulmonary Instillation Studies with Nanoscale TiO2 Rods and Dots in Rats: Toxicity is not dependent upon

Particle Size and Surface Area. Toxicol Sci., 2006

• Material characterization employed in this study:• synthesis method• crystal structure • particle size • surface area • composition/surface coating • aggregation status • cryo TEM • crystallinity • purity (TGA)

2) Pulmonary bioassay studies with nanoscale and fine quartz particles in rats: Toxicity is not dependent upon particle size but on surface characteristics. Toxicol Sci.

2007

• Material characterization employed in this study:

• synthesis method • crystal structure/crystallinity (XRD)• median particle size - particle size (range) • purity (% Fe content)– ICP-AES • surface area • TEM • aggregation status• purity• surface reactivity (erythrocyte hemolysis)• reactive oxygen species (ESR) •

3) Pulmonary Toxicity Study in Rats with Three Forms of ultrafine-TiO2 Particles: Differential Responses

related to Surface Properties Toxicology, 2007

• Material characterization employed in this study:• crystal phase• median particle size and size distribution in water and

PBS• pH in water and PBS• surface area (BET)• TEM • aggregation status, • chemical (surface) reactivity – (Vitamin C assay) • surface coatings/composition, purity

4) Assessing toxicity of fine and nanoparticles: Comparing in vitro measurements to in vivo pulmonary toxicity

profiles. Toxicol Sci. 2007.• Particle-types utilized in this study:• Fine-sized carbonyl iron• Fine-sized crystalline silica• Fine-sized amorphous silica• Nano ZnO• Fine ZnO

• Particle characterizations conducted both in the “dry state” and “wet state”

• Material characterization employed in this study:• Particle characterization in the dry state• particle size - surface area – density - crystallinity• calculated size in dry state (based on surface area

determinations) • purity

4) Assessing toxicity of fine and nanoparticles: Comparing in vitro measurements to in vivo pulmonary toxicity

profiles. Toxicol Sci. 2007. (cont)

• Particle characterization in the wet state• particle size in solutions – PBS, culture media, water

• average aggregated size in solutions,

• % distribution

• surface charge

• aggregation status

•

• Conversion and comparisons of in vitro and in vivo doses for dosimetric comparisons

5) Comparative Pulmonary Toxicity Assessments of C60 Water Suspensions in Rats: Few Differences in Fullerene

Toxicity In Vivo in Contrast to In Vitro Profiles. Nano Lett. 2007.

• Material characterization employed in this study:• particle size and size distribution• surface charge • crystallinity • TEM • composition • oxidative radical activity (ESR measurements) • surface reactivity (erythrocyte hemolytic potential)

Recommendations for Minimal Essential Material Characterization for Hazard

Studies with Nanomaterials

• Particle size and size distribution (wet state) and surface area (dry state) in the relevant media being utilized – depending upon the route of exposure;

• Crystal structure/crystallinity;• Aggregation status in the relevant media;• Composition/surface coatings;• Surface reactivity;• Method of nanomaterial synthesis and/or

preparation including post-synthetic modifications (e.g., neutralization of ultrafine TiO2 particle-types);

• Purity of sample;

Studies to Assess Pulmonary Hazards to Nanoparticulates

Ultrafine TiO2 Studies

Pulmonary Toxicity Study in Rats with Three Forms of ultrafine-TiO2

Particles: Differential Responses related to Surface Properties

Toxicology 230: 90-104, 2007

Characterization of Ultrafine TiO2 Particle-types - 1

uf-3

C

300 nm

uf-2

B

300 nm

uf-1

A

300 nm

Characterization of Ultrafine TiO2 Particle-types - 2

SampleCrystalline

phase

Median size and width distribution

(nm) Surface area

(m2/g)

pHChemical reactivity

in water* in PBSdeionized water

in PBS

delta b*

F-1 rutile382.0± 36%

2667.2 ± 35% 5.8 7.49 6.75 0.4

uf-1 rutile136.0± 35%

2144.3± 45% 18.2 5.64 6.78 10.1

uf-2 rutile149.4± 50%

2890.7± 31% 35.7 7.14 6.78 1.2

uf-380/20

anatase/ rutile

129.4± 44%

2691.7± 31% 53.0 3.28 6.70 23.8

Protocol for ultrafine TiO2 Pulmonary Bioassay Study

Exposure Groups• PBS (vehicle control)• Particle-types (1 and 5 mg/kg)

o rutile-types uf-1 TiO2

o rutile-type uf-2 TiO2

o anatase/rutile-type uf-3 TiO2

o rutile-type F-1 fine TiO2 (negative control)o α-Quartz particles (positive control)

Instillation Exposure

24 hr 1 wk 1 mo 3 mo

Postexposure Evaluation via BAL and Lung Tissue

RESULTSBiomarkers

Pulmonary InflammationPulmonary CytotoxicityLung cell Proliferation

Pulmonary Inflammation

BAL Fluid LDH Values (cytotoxicity)

Pulmonary Cell Proliferation Rates

Lung Sections of Rats exposed to uf-1 (A); uf-2 (B); or F-1 (C)- 3 months pe

Lung Section of Rat exposed to uf-3 3 months postexposure

Lung Section of Rat exposed to Quartz particles - 3 months postexposure

Nanoscale Quartz

Pulmonary Bioassay Studies with Nanoscale and Fine Quartz Particles

in Rats: Toxicity is not Dependent upon Particle Size but on Surface

Characteristics

Toxicol Sci. 95:270-280, 2007

Nanoscale Quartz Particles

Characterization of Nanoscale Quartz Particles

Sample

Average size (nm)

Size range

(nm)

Surface area (m2/g) Crystallinity

ICP-AES (% Fe content)

Nanoquartz I 50 30-65 31.4 α-Quartz 0.080%

Nanoquartz II 12 10-20 90.5α-Quartz

0.034%

Fine quartz 300 100-500 4.2α-Quartz

0.011%

Min-U-Sil 534 300-700 5.1α-Quartz

0.042%

Pulmonary Inflammation – Nanoscale Quartz study

Percent Neutrophils in BAL Fluids of Rats exposed to Fine and Nano-sized Quartz Particles (Study #2)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

5 mg/kg 1 mg/kg 5 mg/kg 1 mg/kg 5 mg/kg 1 mg/kg 5 mg/kg

PBS CarbonylIron

particles

Min-U-Sil quartz particles Nano quartz II particles Fine quartz particles

Exposure Groups

% P

MN

s

24 Hour 1 Week 1 Month 3 Month

**

*

***

*

*

***

*

*

BAL Fluid LDH Values – Nanoscale Quartz study

BAL Fluid LDH Values in Rats exposed toFine and Nano-sized Quartz Particles (Study #2)

0

100

200

300

400

500

600

700

800

0.5 mls 5 mg/Kg 1 mg/Kg 5 mg/Kg 1 mg/Kg 5 mg/Kg 1 mg/Kg 5 mg/Kg

PBS CarbonylIron

particles

Min-U-Sil quartz particles Nano quartz II particles Fine quartz particles

Exposure Groups

BA

L f

luid

LD

H v

alu

es

(u/L

)

24 Hour 1 Week 1 Month 3 Month

*

*

*

*

**

*

*

Lung Parenchymal Cell Proliferation– Nanoscale Quartz study

Lung Parenchymal Cell Proliferation rates of rats exposed to Nano-Quartz and other particulates

0.00%

0.20%

0.40%

0.60%

0.80%

1.00%

1.20%

5 mg/kg 1 mg/kg 5 mg/kg 1 mg/kg 5 mg/kg 1 mg/kg 5 mg/kg

PBS CarbonylIron

Min-U-Sil quartz particles Nano quartz II particles Fine quartz particles

Exposure Groups

Pe

rce

nt

Pro

life

rati

ng

Ce

lls

24 Hour 1 Week 1 Month 3 Month

*

*

**

Lung Tissue Sections – Control (A); Min-U-Sil (B); NanoQ II (C); Fine Quartz (D).

A B

C D

The hemolytic potential of the four -quartz samples used in the study. The samples, including:

These samples show a similar trend as the inflammation, cytotoxicity, and cell proliferation data.

Hemolytic Potential of -Quartz Samples

nano-quartz II = Min-U-Sil > fine-quartz > nano-quartz I

Hemolytic potential is a measure of surface reactivity.

• Min-U-Sil• fine-quartz• nano-quartz I• nano-quartz II

Nano-quartz II (NQ-2) 12 nm

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Blank PBS TritonX100

15.000 7.500 3.750 1.875 0.938 0.469 0.234 0.117 0.059

Concentration (mg/mL)

AB

S @

540

nm

Nano-quartz I (NQ-1) 50 nm

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Blank PBS TritonX100

15.000 7.500 3.750 1.875 0.938 0.469 0.234 0.117 0.059

AB

S @

540 n

m

Fine-quartz (FQ-1) Silica 300 nm

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Blank PBS TritonX100

15.000 7.500 3.750 1.875 0.938 0.469 0.234 0.117 0.059

AB

S @

54

0 n

m

Crystalline Silica (Min-U-Sil) 534 nm

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Blank PBS TritonX100

15.000 7.500 3.750 1.875 0.938 0.469 0.234 0.117 0.059

AB

S @

54

0 n

m

Crystalline Silica (Min-U-Sil 5) 534 nm

Fine Quartz 300 nm

Nano Quartz I 50 nm

Nano Quartz II 12 nm

Concentration (mg/mL)

AB

S @

540

nm

Summary of α-Quartz Results

Endpoint Min-U-Sil Nanoquartz I Nanoquartz II Fine quartzParticle size ++++ ++ + +++

Surface area + +++ ++++ ++

Fe content ++ +++ ++ +

Crystallinity ++++ ++++ ++++ ++++

Radical content ++++ ++ +++ -

Hemolytic content +++ + +++ ++

Lung inflammation +++ ++ +++ ++

Cytotoxicity +++ ++ +++ +

Airway BrdU ++ N/A ++ +

Lung parenchymal BrdU

++ N/A ++ +

Histopathology +++ N/A ++++ ++

Fullerene Water Suspensions Characterization

Nano-C60 C60(OH)24

FWSSize and Size Distribution

Surface Charge Crystallinity

nano-C60 160 ± 50 nm - 36 mV simple hexagonal

C60(OH)24 <2 nm 0 not crystalline

OHOH

OH

OH

HO

HO

OH

OHHO

HO

HOOH

OHHO

OHOH

200 nm

Nano-C60 characterization

50 100 150 200 2500

50

100

150

200

Pop

ulat

ion

Size (nm)

Fullerene Water Suspensions Characterization

Recommendations for Minimal Essential Material Characterization for Hazard

Studies with Nanomaterials

• Particle size and size distribution (wet state) and surface area (dry state) in the relevant media being utilized – depending upon the route of exposure;

• Crystal structure/crystallinity;• Aggregation status in the relevant media;• Composition/surface coatings;• Surface reactivity;• Method of nanomaterial synthesis and/or

preparation including post-synthetic modifications (e.g., neutralization of ultrafine TiO2 particle-types);

• Purity of sample;

Acknowledgments• This study was supported by DuPont

Central Research and Development. • Tom Webb and Ken Reed provided the

pulmonary toxicology technical expertise for the study. Dr. Christie Sayes – postdoctoral fellow. Denise Hoban, Elizabeth Wilkinson and Rachel Cushwa conducted the BAL fluid biomarker assessments. Carolyn Lloyd, Lisa Lewis, John Barr prepared lung tissue sections and conducted the BrdU cell proliferation staining methods. Don Hildabrandt provided animal resource care.