the hamner institutes for health sciences | june 7, 2011 integration of dosimetry, human exposure...

1The Hamner Institutes for Health Sciences | June 7, 2011

Integration of Dosimetry, Human Exposure and High-Throughput Screening Data in the Toxicity Assessment of Environmental Chemicals

Barbara A. WetmoreThe Hamner Institutes for Health Sciences

June 7, 2011DRSG Teleseminar Series


Broad-Based Movement in Toxicology Towards In Vitro Testing and Hazard Prediction

The Hamner Institutes for Health Sciences | June 7, 2011 3

ToxCast: Forecasting the Toxicity of Environmental Chemicals

Collins et al., Science 319:906, 2008

Convergence of diverse fields and technologies to predict toxicity of chemicals and to aidin prioritization efforts

-- high-throughput screening (HTS)-- bioinformatics-- computational toxicology


Judson et al. Env Health Perspect 118:485-92, 2010.

ToxCast Assays~500 HTS Assays: Multiple Cell Types, Pathways Assessed


Difficulty Translating Nominal Testing Concentrations into In Vivo Doses

Knudsen et al. Toxicology 282:1-15, 2011


Is there a way to incorporate human dosimetry and exposure information with AC50 or LEC data to better understand the impact of a chemical on human health?

Question


Defining Dosimetry and Exposure in High Throughput Toxicity Screens

Provided by ToxCast

Data Generated In Vitro

~500 In Vitro ToxCast Assays

ToxCast AC50 or LEC Values

Estimated Target Tissue Bioactivity

Concentration

Hepatic ClearancePlasma Protein

Binding

Toxicokinetic Parameters

Human ExposureEstimates

Predicted Assay Oral Equivalent

Doses

Data Obtained from Registration Documents

In Vitro-to-In Vivo Extrapolation

Chemicals with Potential to Perturb Cellular Pathways at

Relevant Human Exposure LevelsComputational Modeling


High-Throughput PharmacokineticsPerformed on ToxCast Phase I Chemicals

Human Hepatocytes

(10 donor pool)

HumanPlasma

(6 donor pool)

-5

-4

-3

-2

-1

0

1

2

3

0 50 100 150

Ln C

onc

(uM

)

Time (min)

Nifedipine

1 uM initial

10 uM initial

Hepatic Clearance

Plasma Protein Binding

Population-Based In Vitro-to-In Vivo

Extrapolation

Plasma Concentration at Steady State for 100

Healthy Individuals of Both Sexes from 20 to 50

Yrs Old

Rotroff et al., Toxicol Sci 117:348, 2010Wetmore et al., In preparation.


Distribution of In Vitro Pharmacokinetic Data for ToxCast Phase I Chemicals

Hepatic Clearance (µl/min/106 cells)Percent Unbound

Distribution Summary Statistics

Median 2.44Upper Quartile 18.41Lower Quartile 0.50


Median 6.21Upper Quartile 17.96 Lower Quartile 0.00


Estimating Steady State Plasma Concentrations Using the In Vitro Assay Results

-5

-4

-3

-2

-1

0

1

2

3

0 50 100 150

Ln C

onc

(uM

)

Time (min)

Nifedipine

1 uM initial

10 uM initial

Hepatic Clearance

Plasma Protein Binding

Estimated Renal

Clearance

Population-BasedIn Vitro to In Vivo

Extrapolation Software

Plasma Concentration at Steady State for 100

Healthy Individuals of Both Sexes from 20 to 50

Yrs Old


Estimating Steady State Plasma Concentrations Using the In Vitro Assay Results

[Conc]SS =Dose Rate * Body Weight

CLWholeBody

CLR CLH+

CSS = DR * BW / (CLR + CLH) where DR = 0.042 mg/kg/hr and BW = 70 kg

CLR = fU * GFR where GFR ≈ 6.7 L/hr

CLH = where QL ≈ 90 L/hrfU * QL * ClInt

QL + fU * ClInt

CLInt = HPGL * VL * Clinvitro

where HPGL ≈ 137 million cells/g, VL ≈ 1820 g

100% oral bioavailability assumed for both CLR and CLH

Kinetics are assumed to be linear


Chemical

PK- or PBPK-Derived

Css (µM)

IVIVE

Cssa,b (µM)

IVIVE

Caco-2c

Cssa,b µM)

IVIVE

Fub=0.99

Css (M)

IVIVE

Fub=0.99,

Caco-2c

Css (M)2,4-dichlorophenoxyacetic acid 9.05-90.05 39.25 40.34 39.25 40.34

Bisphenol-A < 0.13d 0.35 0.40 0.06 0.07

Cacodylic acid 1.80 3.06 --e 3.06 --e

Carbaryl 0.03 0.07 0.07 0.03 0.03Fenitrothion 0.03 17.91 --e 0.10 --e

Lindane 0.46 13.21 --e 0.07 --e

Oxytetracycline dehydrate 0.36 2.00 0.44 2.00 0.44Parathion 0.17 24.63 --e 0.14 --e

Perfluorooctane sulfonic acid 19,990f 160.78f 179.96f 160.78 f 179.96 f

Perfluorooctanoic acid 20,120 f 55.34f 58.19 f 0.40 f 0.40f

Picloram 0.27 57.63 32.01 0.37 0.19Thiabendazole 0.45 13.76 15.20 13.76 15.20Triclosan 2-10 1.56 1.59 0.01 0.01

Assessment of IVIVE-derived values using Published PK and PBPK Models

a Css, concentration at steady state for 1 mg/kg/day dose; predicted using the 1 M metabolic clearance rate.c IVIVE performed incorporating Caco-2 data into the simulation. d Css value in (Volkel et al., 2002) represented total bisphenol A, of which 99% is glucuronidated.

The published value was divided by 100 to estimate the free concentration for this table.e Caco-2 assay to be verified for these chemicals.fPFOS and PFOA undergo active renal resorption (Andersen et al., 2006) and may explain the discrepancy in the listed values.

Restrictive CL Non-restrictive CL

Bioavailabilityan issue in 2 of13 compounds

Restrictive CL:6/13 agree; Css for

5 others are overpredicted

Non-restrictive CL:8/13 agree; Css for 4 others

are underpredicted

Css overprediction afforded by restrictive clearance model generates lower and more conservative estimates for oral equivalent values, which would be more protective

of human health.


Reverse Dosimetry Modeling for Interpreting In Vitro Assay Results

Upper 95th Percentile Css Among 100 Healthy

Individuals of Both Sexes from 20 to 50 Yrs Old

Reverse Dosimetry

Plasma Concentration

ToxCast AC50 Value

Assay X (e.g., ACE inhibition)

Oral Exposure

Oral Dose Required to Achieve Steady

State Plasma Concentrations

Equivalent to AC50

Oral Equivalent (mg/kg/day)

ToxCast AC50 (uM)

1 mg/kg/day

Upper 95th Percentile Css (uM)

=

1 mg/kg/dayOral Exposure

Metabolic and binding

parameters


Reverse Dosimetry Modeling for Interpreting In Vitro Assay Results

Upper 95th Percentile Css Among 100 Healthy

Individuals of Both Sexes from 20 to 50 Yrs Old

Reverse Dosimetry

Oral Exposure

Plasma Concentration

ToxCast AC50 Value

Oral Dose Required to Achieve Steady

State Plasma Concentrations

Equivalent to In Vitro Bioactivity

500 In Vitro ToxCast Assays

Represented as a Box

Plot

Ora

l E

qu

ival

ent

Do

se (

mg

/kg

/day

)

What are humans exposed to?

?

?

?

Chemical

In VitroBioactivity


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Assessment of ToxCast Phase I Chemicals

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Approximately 12% of ToxCast Phase I chemicals have in vitro bioactivity at oral equivalent doses that overlap with estimated human exposures.


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Compound

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100

1000

10000

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Ora

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Do

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lT

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Dis

ulf

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hlo

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eny

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ate

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roto

ph

os

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qu

at m

eth

yl s

ulf

ate

Ab

amec

tin

Oxy

tetr

acyc

lin

e d

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dra

teM

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ate

Dim

eth

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siso

pro

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zin

eT

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Fe

no

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Se

tho

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olu

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e

0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

10000

100000

Compound

Ora

l Eq

uiv

alen

t D

ose

or

Est

imat

ed E

xpo

sure

(mg

/kg

/day

)Triclosan

2-phenylphenol



Ch

loro

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Pac

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Mes

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Fen

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0.001

0.01

0.1

1

10

100

1000

10000

100000

Compound

Ora

l Eq

uiv

alen

t D

ose

or

Est

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xpo

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(mg

/kg

/day

)Dicamba Fenhexamid

Fluroxypyr-meptyl



Incorporation of Dosimetry Provides Greater Context to AC50 Data

Fe

nti

n h

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am

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PF

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pe

tam

ph

os0.001

0.01

0.1

1

10

100

1000

Compound

AC

50

or

LE

C (

M)

Cyprodinil

Prometon

FenbuconazoleDifenoconazole

Dichloran

FludioxonilQuinclorac

TriflumizolePyriproxyfen

AcifluorfenSpiroxamine

Isoxaben

PFOS

Pyraclostrobin

Buprofezin


Chemical Assay Endpoint AC50 (M)

Oral Equivalent

Dose (mg/kg/day)a

Human Exposure

(mg/kg/day)b

2-Phenylphenol BSK_LPS_PGE2_down Prostaglandin E receptor 2 (subtype EP2) downregulation 4.4444 0.056900 0.2500

2-Phenylphenol NVS_GPCR_hPY2 Competitive binding of GPCR P2RY1 4.9400 0.063300 0.2500

Acifluorfen CLZD_SLCO1B1_24 Change in SLCO1B1 expressn, 24hr 0.00636 0.000056 0.00013

Chlorpropham BSK_BE3C_uPA_down Plasminogen activator, urokinase (PLAU) downregulation 1.4815 0.002900 0.005

Cyprodinil ACEA_LOC2 Change in cell growth kinetics 33.111 0.0136296 0.0257 Cyprodinil ACEA_LOCdec Change in cell growth kinetics 33.113 0.0136296 0.0257 Cyprodinil CLM_MitoMass_24hr Mitochondrial Function 26.94 0.0110887 0.0257 Cyprodinil CLM_MitoticArrest_72hr HCS Mitotic Arrest 50.48 0.0207779 0.0257 Cyprodinil CLM_p53Act_72hr Activation of p53 42.07 0.0173163 0.0257 Cyprodinil CLM_StressKinase_24hr Stress kinase activation 56.94 0.0234369 0.0257 Cyprodinil CLZD_CYP1A1_24 Change in CYP1A1 expressn. 13.922 0.0057304 0.0257 Cyprodinil CLZD_CYP1A2_24 Change in CYP1A2 expressn. 14.631 0.0060222 0.0257 Dicamba CLZD_SLCO1B1_24 Change in SLCO1B1 expresn, 24hr 0.0168 0.001200 0.0297 Dichloran NVS_ADME_rCYP2A2 CYP2A2 inhibition (rat) 0.0365 6.923E-05 0.000375

Difenoconazole NVS_ADME_hCYP2C19 CYP2C19 inhibition 0.0173 0.0011996 0.0044 Difenoconazole NVS_ADME_hCYP3A5 CYP3A5 inhibition 0.0338 0.0023437 0.0044

Isoxaben CLZD_CYP3A4_24 Change in CYP3A4 expressn. 0.22457 0.006046 0.0083 PFOS NVS_ADME_hCYP2C9 CYP2C9 inhibition 0.078 0.00034 0.00043 PFOS NVS_ADME_rCYP2C11 CYP2C11 inhibition 0.059 0.00026 0.00043

Piperonyl butoxide NVS_ADME_hCYP2J2 CYP2J2 inhibition 0.0387 0.0079741 0.0185 Triflumizole NVS_ADME_rCYP2A2 CYP2A2 inhibition (rat) 0.0245 0.000869 0.00513

ToxCast Assays with Oral Equivalent Values Overlapping Human Exposures

Are these related to adverse effects in vivo?Is the AC50 value the right basis for calculating the oral equivalent dose?


Chemical Use Pattern In vivo Effects Assay Endpoint Hits MOE

Value/Range

2-Phenylphenol Microbicide, bactericide fungicide household items; citrus, pears

Bladder carcinogen (rat) Liver carcinogen

(mouse)

Prostaglandin E receptor 2 (subtype EP2) downregulation

Competitive binding of GPCR P2RY1 0.23-0.25

Acifluorfen Diphenyl ether herbicide

Soybeans

Liver carcinogen (mouse)

Kidney lesions Change in SLCO1B1 expression, 24 hr 0.4310

Buprofezin Insect growth regulator

Citrus, cucumbers, tomatoes Thyroid effects,

Mouse liver carcinogen Change in cell growth kinetics 0.1174

Chlorpropham Plant growth regulator (herbicide)

potatoes Thyroid toxicity

Plasminogen activator urokinase (PLAU) downregulation

0.5800

Cyprodinil Fungicide

Almonds, grapes, pome and stone fruits Liver and kidney effects Several (see Table 2) 0.14-0.99

Dicamba Benzoic acid post-emergent herbicide Broadleaf weeds; asparagus, corn, oats Reproductive toxicity Change in SLCO1B1 expression, 24 hr 0.0404

Dichloran Pre- and post-harvest fungicide; lettuce,

peaches, sweet potatoes

Neuropathology; liver, kidney, spleen, and hematologic effects

CYP2A2 inhibition (rat) 0.1846

Difenoconazole Triazole fungicide Wheat, citrus, grape, onion

Liver carcinogen CYP2C19 inhibition CYP3A5 inhibition 0.27-0.53

Fenbuconazole Triazole fungicide Wheat, barley, apple, pear

Thyroid carcinogen Liver carcinogen

Change in cell growth kinetics CYP2A2 inhibition (rat) CYP2B1 inhibition (rat)

0.23-0.77

Fenhexamid Fungicide

Grapes, strawberries, ornamentals Hematologic effects

Adrenal changes Change in CYP 3A4 expression, 48 hr 0.3660

Fludioxonil Pyrrole fungicide; citrus, apple, root vegetables

Decr. in body weight; liver, kidney effects

CYP1A2 inhibition Change in cell growth kinetics

0.10-0.72

Fluroxypyr-meptyl

Pyridine herbicide Barley, wheat, apple, pear

Kidney effects Decr. in body weight

Change in CYP2B6 expression, 6 hr Several bioseek assays; 1 Novascreen

0.35-0.89

Isoxaben Benzamizole herbicide Non-bearing fruit/nut trees, Christmas trees

Liver effects, enzyme induction

Change in CYP3A4 expression 0.7284

PFOS Formerly a stain repellent; fire-fighting foam, metal plating, photography uses

Liver effects, toxicity CYP2C9 and CYP2C11 Inhibition 0.60-0.79

Piperonyl butoxide

Synergist; CYP450 and non-specific esterase inhibitor

Hematologic effects, hepatotoxicity

CYP2J2 inhibition 0.4310

Summary of use patterns, in vivo effects and assay hits for flagged Phase I Chemicals


Log10 Transformed Margin-of-ExposureMost Highly Exposed Subpopulation

Log10 Transformed Margin-of-ExposureGeneral US Population


Median 2.07 (117.49)Upper Quartile 2.90 (794.33)Lower Quartile 0.97 (9.33)


Median 1.59 (38.90)Upper Quartile 2.67 (467.74)Lower Quartile 0.70 (5.01)

Distribution of Ad hoc Margin of Exposure Values for the ToxCast Phase I Chemicals

Of the 24 chemicals that overlapped using the upper bound of exposure estimates 16 also overlapped with oral equivalent values when general U.S. population exposure estimates

were employed.


Reverse Dosimetry Project has Generated Two Manuscripts Thus Far


In progress…Analysis of 350 Phase II Compounds Underway

http://www.epa.gov/pesticides/ppdc/testing/feb09/toxcast-presentation.pdf


• Integration of in vitro pharmacokinetic assays with computational modeling allows estimation of oral equivalent doses required to produce steady state in vivo concentrations equivalent to AC50 or LEC values in HTS assays.

• Of the 239 chemicals tested thus far, only 23 had overlapping human exposure estimates and oral equivalent doses.

• For 16 of these 23 chemicals, exposure estimates for the general U.S. population – and not just the most highly exposed subpopulation – displayed overlap with the oral equivalent values.

• Incorporation of dosimetry and exposure information with AC50/LEC values provides a necessary context for interpretation of in vitro toxicity screening data.

• The pharmacokinetic approaches presented in this study have the potential to move beyond a hazard identification paradigm towards the use of in vitro data in a risk assessment context.

Conclusions


Acknowledgements

Harvey ClewellMel AndersenMark SochaskiBrittany AllenKatherine CantwellEd LeCluyse

David Dix (EPA)John Wambaugh (EPA)Daniel Rotroff (EPA)Richard Judson (EPA)Keith Houck (EPA)Bob Kavlock (EPA)Matt Martin (EPA)David Reif (EPA)Stephen Ferguson (CellzDirect)Kimberly Freeman (CellzDirect)Cornelia Smith (CellzDirect)

Institute Collaborators

External Collaborators

American Chemistry Council

Funding

Rusty ThomasFrank BoellmannEric HealyReetu SinghLing-Chieh Tsai

Thomas Lab