OFFICE OF CHEMICAL SAFETY

AND POLLUTION PREVENTION

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON, D.C. 20460

RE: Meeting between Dow AgroSciences (DAS) and EPA to Discuss DAS’ Possible 

Enhancements to their Physiologically Based Pharmacokinetic Model (PBPK)

and Their Views on the Exposure and Potency of the Chlorpyrifos Oxon 

List of Documents

1. Meeting Summary Page 1

2. Meeting Sign in Sheet Page 2

3. DAS PowerPoint Presentations 

a. Using PBPK Models to Improve the Chlorpyrifos Risk Assessment Page 3

b. Chlorpyrifos-Oxon Potency: Discussion of Toxicity Adjustment Factors 

Used for Risk Assessment Page 32

c. Chlorpyrifos-Oxon Exposure in Drinking Water from Surface Water 

Sources Page 46

 

Meeting Summary 

On Wednesday, July 18, 2012, EPA’s Office of Pesticide Programs met with DAS to discuss 

their PBPK model and their views on the exposure and potency of the chlorpyrifos oxon. DAS 

provided further detail and timeline for anticipated enhancements to the PBPK model. DAS will 

discuss the progress of any enhancements with EPA as the work progresses. DAS also discussed 

their views related to the exposure and potency of the chlorpyrifos oxon in contrast to what EPA 

presented in the Chlorpyrifos Preliminary Human Health Risk Assessment released for public 

comment in July 2011. The assessment is available at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPP-2008-0850-0025. 
Name 

Vt c k~ ~-t krco 

M~~~~ 'l4t~rt~~ 

EPA and Registrant Meeting 

Chlorpyrifos and PBPK 

July 18, 2012 

Affiliation Phone Number 

3/7-337-'J?lL 

E-mail 
Using PBPK Models to 

Improve the Chlorpyrifos 

Risk Assessment 

Paul Price

Michael Bartels

July 18, 2012
• PBPK Applications – Paul Price

• PBPK Research – Michael Bartels

• Oxon – Hazard – Mike Bartels

• Oxon – Exposure – Nick Poletika

• Further Discussion - All

Speakers
Proposed Activities 

• Extend models 

– To other routes

– To oxon

– To infants and in utero exposures

– To address lactational pathway

• Develop chemical specific uncertainty factors for:

– Oral, inhalation, dermal routes

– Chlorpyrifos and oxon

• Use models to explore biomonitoring data in the Columbia study (to be 

discussed at August meeting)
Why address Inter-species and Inter-individual 

uncertainty factors?

• Refine approaches for remaining existing and emerging 

risk assessments

– MOEs for bystanders (CPF and oxon?)

– MOEs for drinking water (oxon)

– Finalize aggregate assessment
Relationship between proposed modeling and 

key issues

Development of PBPK Models

Compound Specific Uncertainty 

Factors

Current Question?

Inhalation 

Route

Dermal 

Route

Extension 

to earlier 

ages

Oxon 

model

Oral 

CPF

Oral 

Oxon

Dermal 

CPF

Inhalation 

CPF

Bystander Assessments X X X X X

Drinking water Assessment X X

Epidemiology Findings X X

X
Developing CPF-specific uncertainty factors

• Address SAP issues on developing CPF-specific uncertainty factors

• Develop a 2D oral model (separate variability and uncertainty)

• Follow the approach presented in DAS report prepared for the SAP

– Use 2D oral model to construct the human equivalent of the animal 

POD

– Use 2D oral model to determine the dose that protects a large 

fraction of the population at a specific level of confidence from an 

RBC AChEI of 10% 

– Ratios of the doses provide the basis for interindividual and 

interspecies uncertainty factors 
Proposed Timeline for PBPK model 

Enhancements

Project Q3 2012

Q4 2012Q1 2013Q2 2013Q3 2013Q4 2013Q1 2014

Oral intake of Oxon

Dermal uptake of CPF

Inhalation exposure to CPF

Late-stage Gestation / In utero- CPF

Timeline dependent on approval for use of Kisicki human volunteer study data
Details of PBPK model Enhancements

• Oral Intake of Oxon

– Model basis: CPF Life-Stage PBPK/PD

– Initial Oxon PK parameters will be estimated from CCA study TK 

data

– Sensitivity analysis will be conducted on these parameters to 

evaluate need for further Oxon kinetic studies

– Model parameters will be optimized to fit to CCA study Oxon TK 

data

– Simulations will be conducted for Oxon bioavailability and ChE

effects from Drinking Water exposure in rats & humans
Details of PBPK model Enhancements

• Dermal Uptake of CPF

– Model basis: CPF Life-Stage PBPK/PD

– Dermal uptake will be based on Surface Area x Formulation 

Concentration x Kp value, as per 2002 Timchalk model

– Dermal penetration flux values from in vitro study of Griffin (2000) 

and in vivo volunteer studies of Griffin (1999) and Nolan (1984)

– Model parameters will be optimized to fit to Griffin (1999) and Nolan 

(1984) datasets

– Model will be used to simulate occupational/residential exposure via 

dermal route
Details of PBPK model Enhancements

• Inhalation Exposure to CPF

– Model basis: CPF Life-Stage PBPK/PD

– model modifications will include incorporation of CPF and Oxon metabolism in 

lung tissue

• minimum: average metabolism rates from pooled donor tissues

• full variation evaluation: individual metabolism rates from ~20 donor tissues

• in silico only: de novo evaluation of lung metabolism via computational 

assessment of CYP isoform contributions

– evaluation of contribution and variation in CYP-based metabolism in brain tissue 

will also be conducted

same minimum/full/in silico options, as defined for lung metabolism

– validate rat version of model with 2010 Hotchkiss Inhalation toxicity study- TK data
Details of PBPK model Enhancements

• Late-stage Gestation / In Utero Exposure to CPF

– Model basis: CPF Life-Stage PBPK/PD and Lowe 2009 end-stage 

PBPK/PD model

• will include growth of fetus and maternal changes through 3rd trimester

• model will be validated against available mother/newborn datasets

– Model will be used to simulate in utero and maternal exposures 

from various routes (oral, dermal, possibly inhalation)
References

• Griffin, P., H. Mason, K. Heywood, and J. Cocker. 1999. Oral and dermal absorption of 

chlorpyrifos: a human volunteer study. Occupational and Environmental Medicine. 56:10-13. 

• Griffin, P., M. Payne, H. Mason, E. Freedlander, A.D. Curran, and J. Cocker. 2000. The in vitro 

percutaneous penetration of chlorpyrifos. Hum Exp Toxicol. 19:104-107.

• Nolan, R.J., D.L. Rick, N.L. Freshour, and J.H. Saunders. 1984. Chlorpyrifos: pharmacokinetics 

in human volunteers. Toxicology and Applied Pharmacology. 73:8-15. 

• Timchalk, C., R.J. Nolan, A.L. Mendrala, D.A. Dittenber, K.A. Brzak, and J.L. Mattsson. 2002. A 

Physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model for the 

organophosphate insecticide chlorpyrifos in rats and humans. Toxicological Sciences: An 

Official Journal of the Society of Toxicology. 66:34-53. 

Page 12
BREAK HERE MOVE TO BARTELS SLIDES

• COME BACK AFTER CAROL’s Presentation

Page 13
Understanding the Columbia Studies

• The findings raise questions:

– They suggest effects at levels 3 orders of magnitude 

lower than those from animal models and lower than 

levels shown to have effects in in vitro testing.

– The biomonitoring data are only a random snapshot of 

exposures and can not inform prior exposures
Blood levels associated developmental effects are 1000 

fold lower than animal models, 100 fold lower than ChE 

inhibition and 5-1000 fold lower than in vitro non-ChE 

effects. 

0.1

1

10

100

1000

10000

100000

1000000

10000000

100000000

Maternal levels in the 

Columbia study

Conc. in rats (1-5 mg/kg) 10% RBC AChEI Conc. 

adults and infants

Range of in vitro conc. 

Concentration in Blood pg/g

Columbia is an Outlier
Key question posed in May meeting

• Do data on chlorpyrifos suggest Columbia cohort 

mothers were exposed to levels that could have caused 

significant inhibition of RBC or brain ACHE? Considering: 

– Data published by the authors

– Data published in earlier studies of exposures from 

residential uses

– Collected by Dow as part of the registration process
What do we know?

• Biomonitoring date indicates majority of Columbia participants did not 

have body burdens that differed from the general public’s levels from 

dietary exposures

• The max blood level was 15-fold higher then the upper bound level 

predicted for the dietary exposures general population (35 vs. 2 ng/l)

• Dow workers receiving similar doses did not have RBC AChEI

• Modeling suggest that sensitive adults require blood concentrations 

that are in the range of 10,000 to 300,000 ng/l to cause RBC AChEI
Blood Conc. of Chlorpyrifos vs. RBC and Plasma AChEI

1.00E-02

1.00E-01

1.00E+00

1.00E+01

1.00E+02

1 10 100 1000 10000 100000 1000000 10000000

Maximum ChEI % of control

Maximum Blood Concentration ng/l

Blood levels associated 

with 10-100% RBC 

AChEI

Range of blood levels 

reported in Wyatt, 2004 

for years < 2002
Estimates of exposures from residential uses 

• Generally, historical estimates are combination of modeled 

risk assessment assumptions mingled with measured 

observations.

– Estimates of aggregate doses for adults in treated homes at the time of treatment 

are less than 0.5 ug/kg/d. (TAS, 1996)

– Estimates of doses from high (worst case) residential dermal and inhalation 

exposures was found to 17-37 ug/kg (DAS, 1993)

• These doses (if administered orally) would have produced 

blood levels in the 10-1000 ng/l range 

• These levels do not affect RBC AChE
Approaches to Columbia

• Option 1:

– Use the new findings of Huen et al. to test the hypothesis that 

chlorpyrifos blood levels in the range of 1-35 ng/l in blood cause 

developmental effects

– Huen et al. found higher chlorpyrifos blood levels than Whyatt

– Explore if Huen found a correlation to developmental effects
Responding to Columbia

• Option 2 Obtain data from Columbia 

– Use a PBPK model to predict blood concentrations at 

time of arrival to hospital 

• Use data in regression model (test hypothesis that 

duration of time between admittance and delivery was 

a confounder in analysis)

– Review regression model and determine if alternative 

analyses are justified and if they produce different 

results.
PBPK Capabilities Needed

• PBPK model of maternal and fetal blood levels for early 

and late stage pregnancy

• Oral, dermal, and inhalation routes of exposure

• Ability to track individuals for periods of 270 days 
Questions
EXTRA slides
Means of the highest 

and lowest quartiles of 

1999-2000 cord blood 

range from 0.5 to 13 

pg/g over all mean of 4 

pg/g

Columbia Data 

0.1

1

10

100

1000

10000

100000

1000000

10000000

100000000

Cord levels in the Columbia study

Concentration in Blood pg/g
Frank developmental and 

behavioral effects in rats are 

associated with dose of 1-5 

mg/kg/day

Blood levels in dams and 

neonates (PND 1) given an 

oral doss of 1-5 mg/kg range 

from 350,000 to 1,800,000 

pg/g (Mattsson et al., 2000) 

Rat blood levels associated with behavioral and 

developmental effects 

0.1

1

10

100

1000

10000

100000

1000000

10000000

100000000

Cord levels in the Columbia 

study

Conc. in rats (1-5 mg/kg)

Concentration in Blood pg/g
PBPK model (Timchalk 

2002; Hinderliter et al., 

2011; DAS, 2010) 

predicted blood levels 

associated with 10% RBC 

inhibition.

Age-specific metabolism 

and physiology in 5,000 

adults and 5,000 infants (6 

months)

Blood levels that cause a 10% inhibition of RBC 

AChE

0.1

1

10

100

1000

10000

100000

1000000

10000000

100000000

Cord levels in the 

Columbia study

Conc. in rats (1-5 

mg/kg)

10% RBC AChEI 

Conc. adults and 

infants

Concentration in Blood pg/g
In vitro concentrations causing initiating effects other than 

ChE inhibition.

Blood levels necessary to cause in vitro effects

0.1

1

10

100

1000

10000

100000

1000000

10000000

100000000

Cord levels in the Columbia 

study

Conc. in rats (1-5 mg/kg) 10% RBC AChEI Conc. adults 

and infants

Range of in vitro conc. 

Concentration in Blood pg/g
• Need to address: 

– Comments from the SAP on the models

– Other ages (late stage pregnancy and neonates)

– The lactation pathway

• Extend the model to oxon

– Addresses water issues

• Dermal and inhalation

– Worker/bystanders safety factors 

– Epidemiology

Development of compound-specific uncertainty 

factors 
Chlorpyrifos-Oxon Potency: Discussion 

of Toxicity Adjustment Factors used for 

Risk Assessment 

Michael J. Bartels and Daland R. Juberg 

July 18, 2012 
Chlorpyrifos-Oxon Potency 

• Overview: 

• Review underst anding of 

pharmacokinet ic differences bet ween 

chlorpyrifos (CPF) and Oxon in 

mammalian species 

• Scientific rationale for utilizing the target 

organ of brain for biological effects arising 

from exposure to Oxon in drinking water 

• Excellent data for this approach from 

recent CCA study (Marty, 2012) 

• Recommendations 
Chlorpyrifos-Oxon Potency 

GI 

Tract 

CPF

Portal vein 

 - CPF stable in blood 

 - Majority of absorbed CPF reaches 

 the liver 

Oxon -> TCP

Systemic circulation 

 - 99% TCP * 

 - 1% CPF 

 - 0.01% Oxon 

Liver 

 - high capacity for 

 CPF metabolism and 

 Oxon hydrolysis 

 - inhibition of AChE & BuChE 

 occurs from metabolic Oxon 

TCP, CPF, Oxon 

CPF is known t o undergo first pass 

met abolism via t he oral rout e 

CPF -> TCP

CPF -> Oxon 

* Timchalk, 2002 
Chlorpyrifos-Oxon Potency 

GI 

Tract 

Oxon 

Portal vein 

 - Blood flow transit time 0.35-1.7 min * 

 - oxon half-life in blood = 10 sec 

 - little or no oxon reaches the liver 

 - however, substantial AChE and BuChE 

 inhibition occurs 

Oxon -> TCP

Systemic circulation 

 - No detectable Oxon 

Liver in CCA study ** 

 - extremely high 

 capacity for Oxon 

 hydrolysis 

TCP TCP 

In cont rast , presyst emic binding of Oxon 

result s in complet e det oxificat ion of t his 

compound 

 * Munk 2003, 

** Marty, 2012 
Chlorpyrifos-Oxon Potency 

2010 CCA study: 

• Direct administration of 

Chlorpyrifos or Oxon to neonatal 

and adult rats 

• Measurements of cholinesterase 

(ChE) inhibition in plasma, RBC 

and brain tissue 

• Additional blood measurements 

of Chlorpyrifos and Oxon 
0

20

40

60

80

100

120

0 2 4 6 8 10 12

Percent of Control

Dose Level (mg/kg)

Chlorpyrifos Oxon

AChE-RBC

BuChE-Plasma

AChE Brain

0

20

40

60

80

100

120

0 5 10 15 20 25

Percent of Control

Dose Level (mg/kg)

Chlorpyrifos

AChE-RBC

BuChE-Plasma

AChE Brain

Chlorpyrifos-Oxon Potency 

 Conclusions: 

• Oxon is significantly less toxic to peripheral tissues than CPF 

• This is due to extremely rapid, and complete presystemic 

hydrolysis of Oxon by ChE and other esterases 
Chlorpyrifos-Oxon Potency 

• Summary 

• Oral exposure to Oxon results in substantial 

inhibition of RBC cholinesterase activity. 

• However, no inhibition in brain ChE occurs 

at dose levels up to 10 mg/kg bw, 

• Also, no Oxon t est mat erial was found 

in syst emic TK blood samples 

• These data show complete presystemic 

hydrolysis of this chemical in the rat. 

• This efficient hydrolysis may be due, in 

part, to slow transit time for absorbed Oxon 

through blood capillaries, coupled with the 

extremely short half-life for this compound 

in blood 
Chlorpyrifos-Oxon Potency 

• CPF- Effects via oral administration 

2-4 hours 4-6 hours 6-8 hours 2-3 Days

Key Event 1 Key Event 2 Key Event 3 Key Event 4

Dose Level 

(mg/kg/day)

Oxon metabolite 

formation *

Binding of Oxon 

to Blood 

Cholinesterase 

Enzyme(s)

Binding of Oxon 

to Brain 

Cholinesterase 

Enzyme(s)

Inhibition of Brain 

Cholinesterase 

Activity

0.05 - - - -

0.05 Repeat - - - -

0.1 slight - - -

0.1 Repeat slight slight - -

0.5 + - - -

0.5 Repeat + + + - -

2 +++ + - -

1.0 Repeat +++ + + + +

10 ++++ +++ + + + +

10Pilot ++++ +++ + + + +

3.5 Repeat ++++ +++ +++ +++

20Pilot ++++ ++++ +++ +++
Chlorpyrifos-Oxon Potency 

• Oxon- Effects via oral administration 

Immediate 2-4 hours NA NA

Key Event 1 Key Event 2 Key Event 3 Key Event 4

Dose 

(mg/kg/day)

Oxon metabolite 

formation *

Binding of Oxon 

to Blood 

Cholinesterase 

Enzyme(s)

Binding of Oxon 

to Brain 

Cholinesterase 

Enzyme(s)

Inhibition of 

Cholinesterase 

Activity

0.005 + - - -

0.01 + + - - -

0.01 Repeat + + - - -

0.05 +++ - - -

0.1 ++++ slight - -

0.5 +++++ + + - -

0.5 Repeat +++++ +++ - -

1 ++++++ +++ - -

1Pilot ++++++ ++++ - -

5Pilot +++++++ ++++ - -

10Pilot ++++++++ ++++ - -
Chlorpyrifos-Oxon Potency 

• Conclusions 

• Profile of Oxon effect s subst ant ially different 

t han t hose arising from CPF 

• Orally absorbed Oxon causes no effects in 

peripheral tissues, due to lack of systemic 

bioavailability of this chemical up to dose levels of 

10 mg/kg 

• Effects on RBC inhibition are due to presystemic 

exposure only, and are not as appropriate for Risk 

Assessment as Brain (peripheral tissue) 

• As a result, no increased Toxicity Adjustment 

factor should be applied to Oxon 

• POD > 10 mg/kg for acute- adult rat 
Chlorpyrifos-Oxon Potency 

• Proposal for Supplemental Work 

• To better understand exposure and biological 

effects from Oxon in drinking water: 

• PBPK model for CPF would be modified to 

incorporate oral exposures to Oxon 

• Additional PK studies might be conducted to 

refine Oxon kinetic parameters 

• Simulations would be conducted to evaluate 

possible systemic exposures and biological 

effects in RBC and the target organ of brain, 

from worst-case intake of Oxon in drinking water 

(0.1-13 μg/kg/day) 

• Note- these Drinking Water levels are 1000x 

lower than the relevant animal POD 
Chlorpyrifos-Oxon Potency 

• References 

• Marty, M.S., A.K. Andrus, M.P. Bell, J.K. Passage, A.W. Perala, K.A. 

Brzak, M.J. Bartels, M.J. Beck, and D.R. Juberg. 2012. Cholinesterase 

inhibition and toxicokinetics in immature and adult rats after acute or 

repeated exposures to chlorpyrifos or chlorpyrifos-oxon. Regul Toxicol 

Pharmacol. 63:209-224.

• Munk, O. L., Keiding, S. and Bass, L. (2003). Impulse-response function 

of splanchnic circulation with model-independent constraints: theory and 

experimental validation. American Journal of Physiology. Gastrointestinal 

and Liver Physiology 285, G671-680.

• Timchalk, C., R.J. Nolan, A.L. Mendrala, D.A. Dittenber, K.A. Brzak, and 

J.L. Mattsson. 2002. A Physiologically based pharmacokinetic and 

pharmacodynamic (PBPK/PD) model for the organophosphate insecticide 

chlorpyrifos in rats and humans. Toxicological Sciences: An Official 

Journal of the Society of Toxicology. 66:34-53 %U 

http://www.ncbi.nlm.nih.gov/pubmed/11861971. 
Chlorpyrifos-Oxon Potency 
Chlorpyrifos-Oxon Potency 

Summary of Chlorpyrifos (CPF) and Chlorpyrifos-Oxon Cholinesterase (ChE) Inhibition 

in Adult Female CD Rats After Single Gavage Exposure in Corn Oil (Marty, 2012) 

Dose Group 

(mg/kg)

RBC ChE (U/L; 

100 ml aliquot)

RBC ChE (U/L; 

50 ml aliquot) Mean RBC ChE Mean Plasma 

ChE (U/L)

Brain ChE

(U/L) Mean Brain ChE

0 4296 4650 4028 4014 4247 + 298 2377

48198

53568 50400 + 2812

49434

5 CPF 2614 2728 2020 1808

2293 + 448

(46% decrease) 737

53505

48840 + 6653

(3% decrease) 51794

41222

10 CPF 636 112 324 664

434 + 264

(90% decrease) 433

29675

31400 + 4216

(38% decrease) 28319

36205

20 CPF UR1 UR UR UR UR 354

14440

12669 + 1555

(75% decrease)

12038

11529

1 CPF-oxon 396 28 UR UR

212

(95% decrease) 633

53257

51422 + 3160

(0% decrease)

47773

53236

5 CPF-oxon UR UR UR UR UR 295

52353

51839 + 1221

(0% decrease) 52720

50445

10 CPF-oxon UR UR UR UR UR 362

50331

52183 + 3574

(0% decrease)

49915

56303
Chlorpyrifos-Oxon Exposure in 

Drinking Water from Surface Water 

Sources 

Nick Poletika and Cheryl Cleveland 

18 Jul 2012 

1 
Current Acute Dietary Risk 

• The 2011 CPF dietary risk for food is highly 

refined by exposure information from PDP 

with risk estimates 

– 9% aPAD for children 

• The 2011 CPF dietary risk for water is 

unrefined using modeled numbers for drinking 

water estimates 

– Infants range from 210 to 2700% of aPAD for oxon 

2 
Current Chronic Dietary Risk 

• The 2011 CPF dietary risk for food is highly 

refined by exposure information from PDP 

with risk estimates 

– 11% cPAD for children (with FHF) 

• The 2011 CPF dietary risk for water is 

unrefined using modeled numbers for drinking 

water estimates 

– Infants range from 69 to 270% of cPAD for oxon 

3 
Dilemma 

• Unrefined modeled DW numbers vs. highly refined 

monitoring Food numbers 

• Lines of evidence from water modeling and monitoring 

– Currently 

• Maximum predicted concentration range is 10.6 to 112 μg/L 

• Maximum reported concentration is 0.5 μg/L from all monitoring 

datasets reviewed 

– How can the DW contribution be refined? 

• New modeling? 

• Interpretation of existing monitoring data? 

• HED policies for actual drinking water relative to modeled surface 

water? 

• Another approach? 

4 
DAS View on Contribution 

of Water to Dietary Exposure 

• DAS continues to recommend use of the 

maximum reported chlorpyrifos concentration of 

0.5 μg/L from all monitoring datasets reviewed as 

the appropriate highest input level for acute risk 

assessment. 

• This value can be attributed to parent or to the 

oxon, depending on assumptions regarding 

oxidative conversion through disinfection 

treatment and fate during distribution. 

5 
EPA Responses to DAS Comments 

on 30 Jun 2011 DW Memo? 

• Interested in OPP views, particularly in three 

areas 

– Crops and use patterns for modeling 

– Use of oxon fate studies (hydrolysis + others in 

DCI) 

– Weight of monitoring data as a line of evidence 

• We have specific questions and more 

information in back-up slides, if helpful for 

discussion 

6 
Questions 

• What is the mechanism for EPA to respond to positions 

stated in the DAS Oct 2011 report? 

• Agreement that soybean apple, alfalfa, citrus and 

wheat are representative crops? 

• Agreement with details of current use patterns for 

model inputs? 

• Intended use of oxon fate studies? 

• Maximum concentration from Heidelberg University 

NCWQR data set corrected? 

• Utility of 2001 targeted Syngenta OP DW monitoring 

study? 

7 
Questions 

• Evaluation of survey statistics analysis of 

NAWQA/NASQAN monitoring data set? 

• View of complete PDP DW data set as an 

indicator? 

• Utility of all lines of evidence to refine DW 

exposure estimates? 

8