Risk assessments for engineered nanomaterials: Addressing ... · Synthesis of Nanomaterials . 12/5/2012 8 . COOH COOH COOH COOH COOH HOOC COOH COOH COOH COOH COOH COOH HOOC COOH.

Risk assessments for engineered nanomaterials: Addressing the unique challenges

Harvey Clewell

Center for Human Health Assessment The Hamner Institutes for Health Sciences

Research Triangle Park, North Carolina

NIEHS Consortium Engineered Nanomaterials:

Linking Physical and Chemical Properties to Biology

NIEHS Consortium Engineered Nanomaterials:

Linking Physical and Chemical Properties to Biology

• 5 U19 Centers: – RTI / Eastern Carolina U. / Hamner – U. Washington – UCLA – Pacific NW National Laboratories – USC / Imperial College London / Rutgers

• 3 U01 Centers

– NYU – UC Davis – U Michigan

RTI / ECU / Hamner Collaborators

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Tim Fennell Susan Sumner David Ensor Anita Lewin Li Han

• Radiolabel Synthesis:

C60 and nanotubes • Nanoparticle

Characterization • ADME and

pharmacokinetics • Reproductive and

Developmental Toxicology

Jared Brown Chris Wingard Robert Lust • In vitro disposition

and effects • Cardiovascular

effects and inflammation

Harvey Clewell Miyoung Yoon • Pharmacokinetic

and pharmaco-dynamic modeling of nanoparticles

RTI / ECU / Hamner Central Hypothesis

• Pregnancy and lactation may be susceptible

conditions with respect to the effects of nanoparticles on the mothers and their offspring, and it is likely that changes in the vasculature can led to alterations in the blood flow to organs and contribute to how nanomaterials distribute to the organs of the mother, fetus, and neonates.

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RTI / ECU / Hamner Center Goal

• To develop PBPK/PD models for the disposition and effects of C60, MWCNTs, and other consortium materials to provide a firm basis for predicting exposure conditions in humans under which such materials could elicit adverse effects in the mother, fetus, or neonate.

• These models will test hypotheses related to the mechanisms of particle-induced vascular effects and describe the relationship between particle load and inflammatory cytokine production; the circulation of the cytokines to remote tissues (including fetus and pup) and the relationship between cytokine concentrations and vascular effects.

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Synthesis of Nanomaterials

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COOH

COOHCOOHCOOH

COOH

HOOC COOH

COOH

COOHCOOHCOOH

COOH

HOOC COOH

Fe3+Fe3+

Fe3+Fe3+

Fe3+

Fullerene C60 MWCNT MWCNT(COOH)n FemMWCNT(COOH)n

For simplicity, the MWCNTs are drawn as single walled tubes.

•Synthesis and purification •Characterization of materials •Characterization of dose formulations •Characterization of endotoxin contamination

Characterization by TEM and DLS

TEM Analysis

DLS: C60 in PVP

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C60

MWCNT

0

10

20

30

40

0.1 1 10 100 1000 10000

Num

ber (

%)

Size (r.nm)

Size Distribution by Number

Record 3: C60 plus PVP 1

Cellular Uptake of 14C-C60 1hr

Mouse Lung Epith

elial

Rat Lung Epith

elial

Rat Aorti

c Endotheli

al0

20

40

600.1 ug/cm20.3 ug/cm21.0 ug/cm23.0 ug/cm210 ug/cm2

14C

-C60

upt

ake

(% o

f dos

e)

3hr

Mouse Lung Epith

elial

Rat Lung Epith

elial

Rat Aorti

c Endotheli

al0

20

40

600.1 ug/cm20.3 ug/cm21.0 ug/cm23.0 ug/cm210 ug/cm2

14C

-C60

upt

ake

(% o

f dos

e)

6hr

Mouse Lung Epith

elial

Rat Lung Epith

elial

Rat Aorti

c Endotheli

al0

20

40

600.1 ug/cm20.3 ug/cm21.0 ug/cm23.0 ug/cm210 ug/cm2

14C

-C60

upt

ake

(% o

f dos

e)

24hr

Mouse Lung Epith

elial

Rat Lung Epith

elial

Rat Aorti

c Endotheli

al0

20

40

600.1 ug/cm20.3 ug/cm21.0 ug/cm23.0 ug/cm210 ug/cm2

14C

-C60

upt

ake

(% o

f dos

e)

C60 Cytotoxicity Studies

** Similar results obtained in endothelial cells

PK and distribution profiles of C60 and forms of MWCNTs in the pregnant dam (and fetuses), the lactating dam (and offspring)

Studies in pregnant and lactating rats showed significant difference in distribution to organs, and distribution to the placenta, to milk, and systemic absorption to the pup (Sumner et al. 2011).

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C60, Female Rat, 1 Day vs 30 Day

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*Less than 1% in Bladder, Ovaries, Stomach, Cecum, Large Intestine, Heart, Brain, Kidney, Marrow, Skin, Brown Fat, Mammary Tissue, Lymph Nodes, Reproductive Tract, Digestive Contents, and Pancreas

*Less than 1% in Plasma, Bladder, Ovaries, Stomach, Cecum, Large Intestine, Heart, Brain, Kidney, Marrow, Skin, Brown Fat, Mammary Tissue, Lymph Nodes, Reproductive Tract, Digestive Contents, and Pancreas

C60, Female Mouse and Rat, 30 Day

•

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*Less than 1% in Plasma, Bladder, Ovaries, Stomach, Cecum, Large Intestine, Heart, Brain, Kidney, Marrow, Skin, Brown Fat, Mammary Tissue, Lymph Nodes, Reproductive Tract, Digestive Contents, and Pancreas

*Less than 1% in Blood, Plasma, Bladder, Ovaries, Stomach, Cecum, Large Intestine, Heart, Brain, Marrow, Skin, Brown Fat, Mammary Tissue, Lymph Nodes, Reproductive Tract, Digestive Contents, and Pancreas

Nonpregnant Rats and Mice: ng/g blood and plasma following HAD

dose Female Rat

Female Mice

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Blood and Plasma Concentration in Mice Administered C60

Blood

Plasma

5 10 15 20 25 30Time (Days)

0.1

1

10

100

1000

Con

cent

ratio

n (n

g/g)

5 10 15 20 25 30Time (Days)

0.1

1

10

100

1000

Con

cent

ratio

n (n

g/g)

Currently evaluating blood, plasma, RBC, platelets, and white cells from C60 exposure: 1 and 7 days

Blood and Plasma Concentration in Rats Administered C60

Blood

Plasma

5 10 15 20 25 30Time (Days)

1

10

100

1000

10000

Con

cent

ratio

n (n

g/g)

5 10 15 20 25 30Time (Days)

1

10

100

1000

10000

Con

cent

ratio

n (n

g/g)

Cardiac I/R Injury Model

Section heart; incubate in TTC to demarcate area at risk (pink) and infarct (yellow)

Analyze Left Ventricle Area, Area at Risk, Infarct Area using digital imaging and

software

20 min ischemia, 2 hr reperfusion

Re-ligate; infuse Evan’s blue to stain all of heart except area

at risk This protocol demonstrated a significant expansion of the infarcted

areas of the left ventricle following exposure to C60

Above: C60 induces an indomethacin sensitive increase (not statistically significant) in stress production to ET-1 in coronary artery Right: C60 also induces an indomethacin sensitive depression in ET-1 relaxation response in mesenteric arteries

Altered Vascular Responses to C60

Left: C60 induces an augmented constriction of the uterine loop artery segment that is Indomethacin sensitive. Middle and Right: Mean calculated EC50 and Hill slopes for the PE dose response relationships of the Uterine artery.

Example of Altered Uterine Artery Responses from Late GD Aged Group

Hamner Multiscale Nanoparticle PBPK Model: Cellullar, Tissue, Whole Body Levels

A general modeling platform to describe in vivo disposition of nanomaterials across exposure routes and life stages

Proposed In Vitro Model Structure for C60

based on the cellular localization of C60 in HMMs cells in Porter et al., 2007

Plasma membrane

Vacuolar structure (lysosomes)

Nucleus

Epithelial or endothelial cells in vitroCulture vessel

Peri-nucleus region

Cytoplasm

Culture media

kns kinP

kreP

kinV

kdePN kinN

kreN kefC

kefV

Rate constants for the currently porposed intracellular trafficking of C60 in vitro kns : non-specific binding of C60 to the plastics of the culture vessel kinP : C60 uptake from the culture media into the plasma membrane kreP : C60 release from the plasma membrane into the cytoplasm kinV : C60 capturing into the vacuolar structure such as lysosomes kdePN : disaggregation of C60 in peri-nucleus region kinN : uptake of disaggregated C60 into the nucleus kefC : efflux of C60 from cytoplasm to the cell media kefV : efflux of C60 through lysosome mediated exocytosis

Key Features of Our PBPK modeling Approach

Cellular dosimetry/response model as a building block for whole body PBPK model

In vitro to in vivo extrapolation Tissue distribution based on kinetic behavior at the cell level determined by a number of biological processes In vivo response as an interplay among cell/tissue responses

Extending to potentially susceptible life stages of gestation and lactation

Maternal to offspring transfer at the level of cellular kinetics Potential differences for physiological/biological processes that govern nanoparticle kinetic behavior in the body between the mother and fetus/neonates

Building the Whole Body Model: Preliminary In Vivo Model Structure

at the cellular level

kin

kout

at the organ level

C60 depot in the tissue

Blood in Blood out

• At the tissue level, C60 enters into the tissue and then is distributed into the cells including tissue macrophages and/or endothelial and epithelial cells. • The cells in which C60 distributed into are represented as a compartment within the tissue, ‘C60 depot’. • Influx and efflux rates of C60 in and out of the depot (kin and kout, respectively) are loosely based on the cellular uptake and retnetion as well as efflux at the cellular level. • The composition of the depot as well as influx and efflux rate constants of C60 in each tissue differ depending on the type of tissue.

In Vivo Whole Body PBPK Model: Initial Structure for Adults

Figure 2. Preliminary structure to describe C60 in vivo disposition in adult female rats after

intravenous dosing of C60

Urine

Liver

Rest of Body

Bile

Blood IV

kinblood

koutblood

Lung

kinliver

koutliver

kinbody

koutbody

kinlung

koutlung

kbile

kexcr

Feces

Spleen kinspleen

koutspleen

Simulation results for the single IV exposure in adult female rats

Liver

Blood

A. Gestation Model B. Lactation Model

Liver

Rest of Body

Bile

BloodIV

kinblood

koutblood

Urine

Placenta

kinliver

koutliver

kinbody

koutbody

kinplacenta

koutplacenta

Pooled Fetuses

ktrans1 ktrans2

Dam

Fetus

kbile

kexcr

Feces

Liver

Rest of Body

Bile

BloodIV

kinblood

koutbloodUrine

Mammary Gland

kinliver

koutliver

kinbody

koutbody

kinplacenta

koutplacenta

Dam

Liver

Rest of Body

Bile

Blood kinblood_p

koutblood_pUrine

kinliver_p

koutliver_p

kinbody_p

koutbody_p

Pup

Milk

GI tract

ka_p

kexcr

kbile

kbile_p

kexcr_p

CLmilk

Feces

Feces

Preliminary In Vivo Whole Body PBPK Model Structure: Gestation and Lactation

Preliminary structure to describe preliminary data for C60 disposition during gestation and lactation in rats (Sumner et al., 2010).

Preliminary Modeling of C60 Perinatal IV

Pregnant Dam

Placenta Pooled Fetuses

Lactating Dam

Milk Pup Liver

Harmonization of PBPK models Collaborative effort between Hamner, RESAC, U of Washington, and PNNL to assure compatibility of PBPK models across consortium Initial focus on silver

Harmonization of QSAR models Collaborative effort between UCLA and RESAC to assure compatibility of PBPK models across consortium

Joint publication on nanomaterial risk assessment approach Organized by risk assessment paradigm Emphasis on necessary characteristics of data to inform human risk Outline completed

Proposed publication on nanomaterial risk modeling approaches

Case studies from consortium efforts

Collaborations Across Consortium

Nanosilver Modeling (20 nm vs. 110 nm)

20 nm Ag nanoparticles 110 nm Ag nanoparticles A. Single Exposure

B. Multiple Exposure

Preliminary modeling with published data in adult male Wistar rats (Lankveld et al., 2010)

PD model

Quantitative Risk Assessment for Nanomaterials

Kinetics at cellular

level in vitro to in vivo extrapolation

In vivo PK data

Physiology Adult,

Gestation Lactation

PBPK model

Cellular toxicity dose-

response in vitro to in vivo extrapolation

incorporating life stages

In vivo PD dose-response

Cross-species validation:

rat to mouse

Human cell in vitro data for PK & PD

Human PBPK/PD model for

Risk Assessment

Cross-species extrapolation

QSAR

QSAR

Human exposure data

Endpoints: pulmonary, cardiovascular, immune, CNS Susceptible populations: developmental, elderly Durations: acute, chronic

QSAR

Lessons Learned So Far – Difficulty of preparing/characterizing human exposure

relevant nanomaterials • High potential for inflammation from endotoxin

contamination – Need for studies across a wide dose-range from toxic

to environmentally relevant – Difficulty of getting nanoparticles to disperse in the

media for in vitro studies • Importance of Particokinetics

– Difficulty of characterizing cellular nanoparticle disposition with TEM

– Difficulty relating in vitro responses to in vivo effects

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