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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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