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’ 

Completed Physiologically Based Pharmacokinetic Model (PBPK), Recently 

Conducted Chlorpyrifos Oxon Saturated Vapor Inhalation Study, and DAS’ 

Position Regarding the Toxicity Adjustment Factor for the Chlorpyrifos Oxon

List of Documents

1. Meeting Summary Page 1

2. Meeting Sign in Sheet Page 2

3. DAS PowerPoint Presentations Page 4

Meeting Summary

On Tuesday, August 27, 2013, EPA’s Office of Pesticide Programs met with DAS to discuss 

their completed PBPK model. DAS had previously presented the model enhancements related to 

dermal and oral exposure, which they have already submitted to EPA. This meeting focused on 

the complete model, which now includes an inhalation component along with the completed 

dermal and oral model components. DAS will submit the inhalation component of the PBPK

model to EPA in the coming weeks. They indicated that third party validation was currently 

underway and would be submitted at the same time as the complete model. EPA has begun

conducting its own validation of the model components already submitted. DAS also discussed 

the preliminary results of the chlorpyrifos oxon vapor inhalation study, which they expect to 

submit to EPA by mid-September. In addition, they presented their position regarding the 

appropriate toxicity adjustment factor for the chlorpyrifos oxon.

EPA and Registrant Meeting – Phone Participants

Chlorpyrifos

August 27, 2013

Name Affiliation Phone Number E-mail 

Ken Racke DAS 317-337-4654 kracke@dow.com

Jon Hotchkiss Dow 989-636-6586 JAHotchkiss@dow.com

Joseph Velovitch DAS 317-337-4179 jjvelovitch@dow.com

Brian Bret DAS 916-780-7477 blbret@dow.com

Bruce Houtman DAS 317-337-3376 bahoutman@dow.com

Carol Burns Dow 989-636-2278 CJBurns@dow.com

Torka Poet PNNL 509-371-6984 Torka.Poet@pnnl.gov

James Nguyen EPA/HED 703-347-0377 nguyen.james@epa.gov

Stephanie Padilla EPA/NHEERL 919-541-3956 padilla.stephanie@epa.gov

Ginger Moser EPA/NHEERL 919-541-5075 moser.ginger@epa.gov

Cecilia Tan EPA/NERL 919-541-2542 tan.cecilia@epa.gov

Chuck Andrews CA DPR 919-445-3984 Chuck.Andrews@cdpr.ca.gov

Jay Schreider CA DPR 919-445-4241 Jay.Schreider@cdpr.ca.gov

Randy Segawa CA DPR 916-324-4137 rsegawa@cdpr.ca.gov

Lisa Ross CA DPR 916-324-4116 Lisa.Ross@cdpr.ca.gov

Sheryl Beauvais CA DPR 919-445-4268 sbeauvais@cdpr.ca.gov

David Duncan CA DPR 916-445-3870 dduncan@cdpr.ca.gov

David Rice CA DPR 916-324-3480 drice@cdpr.ca.gov

Xiaofei Zhang CA DPR 916-445-4267 xiaofeizhang@cdpr.ca.gov
Darin Lickfeldt

August 27, 2013

Update on Chlorpyrifos Inhalation Studies 

and PBPK Model Projects
| 2

Background Information

• DAS and other CHP registrants appreciate the EPA’s 

efforts in using the best available science in the Human 

Health Risk Assessment

• CHP is ahead of many active ingredients in the reg review 

process and could potentially set precedents by which 

other active ingredients will be asked to follow

• Agricultural stakeholders have also asked for a sciencebased approach be taken in the reg review of CHP

• DAS again offers a meeting specifically with CADPR to 

provide further background and context on the topics 

presented today
| 3

Agenda

• Update on recent Chlorpyrifos Oxon Saturated Vapor 

Inhalation Study

• Chlorpyrifos Oxon: Biological Basis for Revised Toxicity 

Adjustment Factor

• Chemical Specific adjustment Factors (CSAFs) for Oral 

Exposures to Chlorpyrifos and Chlorpyrifos Oxon

• Using PBPK/PD Models to Reconstruct Aggregate 

Exposures of the Columbia University Cohort - Preliminary 

Findings
Jon Hotchkiss1

, Daland Juberg2, Michael Bartels1, 

Darin Lickfeldt2

1 The Dow Chemical Company, Midland MI

2Dow AgroSciences LLC, Indianapolis IN

August 27, 2013

CHLORPYRIFOS OXON SATURATED 

VAPOR INHALATION STUDY
| 5

Outline

• Study Overview

• Phase 1: Determination of Saturated Oxon Vapor Concentration

• Phase 2: Limited TK and Tissue ChE Inhibition Kinetics

> Blood Oxon and TCP Levels

> Lung, RBC, Brain, and Plasma Cholinesterase Activity

• Summary and Conclusions

• Questions
| 6

Purpose

• Determine oxon saturated vapor concentration

> Conflicting Vp values

• Conduct nose-only inhalation exposure to saturated oxon vapor 

atmosphere

• Determine time-dependent blood levels of oxon and 3,5,6-trichloro-2-

pyridinol (TCP) for inclusion in PBPK/PD model 

• Determine kinetics of oxon-induced inhibition of cholinesterase (ChE)

> Route of exposure = inhalation

> Exposure- and time-dependent ChE activity measured in Lung, RBC, 

Plasma and Brain 
| 7

Phase 1: Determination of Saturated Oxon Vapor Level

• New lot of oxon synthesized

> Purity = 100% 

• Identification by proton and 13C NMR, IR and MS

• Solid oxon melted (~ 45°C) and used to coat glass beads

• Oxon coated beads placed in glass J-tube to generate vapors

• Optimum flow rate of carrier gas (filtered, humidified air) determined

• J-tube output delivered to 42L flow-past nose-only exposure chamber

• Chamber oxon concentration determined by HPLC/PESI-MS/MS 

analysis of sorbent tube eluates

• Chamber monitored for aerosol formation using a real-time 

aerodynamic particle sizing (APS) spectrometer 
| 8

Determination of Saturated Vapor Concentration

• Test atmosphere generated using J-tube filled with oxon-coated beads

> Flow rate held constant (30 L/min)

> Temperature varied to adjust volatilization of solid oxon on beads

> APS used to monitor particle number, size and concentration

• Saturated vapor = inflection point where aerosol is formed

Temperature (°C)

C

o

nce

ntr

ation

O

x

o

n (

mg/m3)

P

article

C

o

n

c. (

mg/m

3

)

20 22 24 26 28 30 32 34 36 38 40 42

0

25

50

75

100

125

150

175

200

0.000

0.001

0.002

0.003

0.004

0.005

0.25

0.50

0.75

1.00

Oxon Conc.

Particle Conc.

Effect of Temperature on Oxon Concentration and

Aerosol Particle Concentration
| 9

Phase 2: Limited TK and Tissue ChE Inhibition Kinetics

• 6 h nose-only exposure to saturated oxon vapor atmosphere

> Oxon vapor concentration measured by HPLC/PESI-MS/MS

• Rats (n=8/group) sacrificed 0, 1, 2, 4, 8 or 24 h after exposure

> n = 4 blood samples analyzed for oxon and TCP at each sacrifice time

> n = 8 lung, RBC, brain and plasma samples analyzed for ChE activity at 

each sacrifice time

• Exposure start was staggered for control and oxon groups

> Groups of 6 rats were placed on chamber at 3 min intervals

> All control rats placed on chamber (~ 24 min total time) before first oxon 

group started

• Minimize potential for oxon contamination of control groups

• All rats sacrificed w/in 5 min of scheduled sacrifice time

• Blood samples for Oxon and TCP analyses were drawn and extracted 

w/in 5 min of sacrifice time
| 10

Oxon Vapor Chamber Concentration

• 6 hr Time Weighted Average (TWA) Concentration = 35.3 μg/m3

> ~ 14% of saturated CPF vapor concentration (254 μg/m3; MRID 49119501)

> ~50-fold higher than predicted (0.837 μg/m3; Vp = 4.65x10-8 mm Hg)

Sample ID Measured Amount 

(μg Oxon)

Volume Chamber 

Atmosphere Sampled

Oxon Concentration 

(μg/L)

Oxon Concentration 

(μg/m3)

Oxon Concentration 

(Mean μg/m3)

Control 1A <LLQ 85L <LLQ <LLQ

Control 1B <LLQ 85L <LLQ <LLQ

Control 2A <LLQ 77.5L <LLQ <LLQ

Control 2B <LLQ 77.5L <LLQ <LLQ

Exposure A 0.110 7.5L 0.015 14.667

Exposure B 0.027 7.5L 0.004 3.613

Exposure C 0.273 7.5L 0.036 36.400

Exposure D 0.327 7.5L 0.044 43.600

Exposure E 0.344 7.5L 0.046 45.867

Exposure F 0.308 7.5L 0.041 41.067

Exposure G 0.275 7.5L 0.037 36.667

Exposure H 0.282 7.5L 0.038 37.600

Exposure I 0.422 7.5L 0.056 56.267

Exposure J 0.320 7.5L 0.043 42.667

Exposure K 0.001 7.5L 0.000 0.175

Exposure L 0.460 7.5L 0.061 61.333

LLQ = 0.00059 μg Oxon

49.467

30.754

40.000

43.467

37.133

<LLQ

<LLQ

9.140

Chamber Oxon Concentration
| 11

Blood Levels of Oxon and 3,5,6-Trichloropyridinol (TCP)

• No oxon detected in blood at any sample time

> Oxon LLQ = 3.53 x 10-4 nmole/g blood (0.35 nM; 0.118 ng/g blood)

• Blood TCP concentration > LLQ through 8 hr post-exposure

> TCP LLQ = 1.23 x 10-2 nmole/g blood (2.44 ng/g blood)

Oxon and TCP in Blood

Time After Exposure (hours)

0 1 2 4 8 24

Blo

o

d Co

nce

ntration

(n

m

oles/g

blood)

0.0001

0.001

0.01

0.1

1

TCP LLQ

 TCP

Oxon LLQ

TCP

Oxon
| 12

Lung, RBC, Brain, and Plasma Cholinesterase Activity

RBC

Time After Exposure (hours)

RBC

C

h

E

Activity (% Control)

0

30

60

90

120

150

180

a

a = Significantly different than control (p<0.05)

0 1 2 4 8 24

Control

Oxon

Plasma

Time After Exposure (hours)

Plas

m

a

C

h

E

Activity (%

Control)

0

30

60

90

120

150

180

0 1 2 4 8 24

Control

Oxon

Brain

Time After Exposure (hours)

Brain Ch

E

Activity (% Control)

0

30

60

90

120

150

180

a

a = Significantly different than control (p<0.05)

0 1 2 4 8 24

Control

Oxon

Lung

Time After Exposure (hours)

Lung Ch

E

Activity (% Control)

0

30

60

90

120

150

180

0 1 2 4 8 24

Control

Oxon
| 13

Lung, RBC, Brain, and Plasma Cholinesterase Activity

Lung

Time After Exposure (hours)

Lung Ch

E

Activity (U/L)

0

1000

2000

3000

4000

5000

6000

0 1 2 4 8 24

Control

Oxon

RBC

Time After Exposure (hours)

RBC Ch

E

Activity (U/L)

0

1000

2000

3000

4000

5000

6000

a

a = Significantly different than control (p<0.05)

0 1 2 4 8 24

Control

Oxon

Plasma

Time After Exposure (hours)

Pla

s

m

a Ch

E

Activity (U/L)

0

1000

2000

3000

4000

5000

6000

0 1 2 4 8 24

Control

Oxon

Brain

Time After Exposure (hours)

Brain Ch

E

Activity (U/L)

0

10000

20000

30000

40000

50000

60000

a

a = Significantly different than control (p<0.05)

0 1 2 4 8 24

Control

Oxon
| 14

Summary

• The saturated vapor concentration for oxon is ~ 35.3 μg/m3

> ~ 7-fold less than saturated CPF vapor concentration (254 μg/m3; MRID # 

49119501)

• Inhaled oxon vapors are absorbed but rapidly metabolized

> No oxon detected in blood at any sample time

• Oxon LLQ = 3.53 x 10-4 nmole/g blood (0.35 nM; 0.118 ng/g blood)

> Blood TCP concentration > LLQ through 8 hr post-exposure

• TCP LLQ = 1.23 x 10-2 nmole/g blood (2.44 ng/g blood)

• 6 h nose-only exposure to saturated oxon vapor results in 

> No clinical signs of OP toxicity

> No inhibition of Lung, RBC, Brain or Plasma Cholinesterase Activity
| 15

Conclusions

• Inhaled oxon vapors are absorbed but rapidly metabolized

> Blood levels < LLQ within 5 min of a 6 hour inhalation exposure 

• TCP present in blood of oxon-exposed rats confirms oxon absorption

• 6 hr NOEC for inhaled oxon vapors ≥ 35.3 μg oxon/m3 air

> Based on no ChE inhibition in Lung, RBC, Brain or Plasma

• The results of this study demonstrate that there is no cholinesterase 

inhibition and therefore no hazard from inhalation of a saturated vapor 

concentration (35.3 μg/m3 ) of Chlorpyrifos Oxon.

• These data support and extend our previous Chlorpyrifos vapor study 

(MRID # 49119501) which reported that there was no cholinesterase 

inhibition and therefore no hazard from inhalation of the parent molecule, 

Chlorpyrifos, at a saturated vapor concentration of 254 μg/m3
16

Chlorpyrifos Saturated Vapor Study

(MRID # 49119501) 49119501)
| 17

Chamber Concentration: CPF Vapor Study

6 h TWA

17.7 ppb

0.254 mg/m3

Volume

Recovery-Adjusted 

a

Chamber Air Concentration 

b

Sample ID Measured Amount Amount Sampled Chlorpyrifos Vapor

(μg Chlorpyrifos) (μg Chlorpyrifos) (L) (ppb)

Samples taken During Exposure

 Control 1-F 

c

 <LOD 

d

<LOD 26.4 (<LOD; 0.10) e

Control 2-F <LOD <LOD 36.0 (<LOD; 0.07)

Control 3-F <LOD <LOD 36.0 (<LOD; 0.07)

Control 4-F <LOD <LOD 18.0 (<LOD; 0.15)

Exposure 1D-F 3.38 3.50 16.5 14.8

Exposure 2D-F 3.96 4.10 16.5 17.3

Exposure 3D-F 3.85 3.99 16.5 16.9

Exposure 4D-F 4.37 4.52 16.5 19.1

Exposure 5D-F 4.42 4.58 16.5 19.4

Exposure 6D-F 6.47 6.70 25.5 18.3

Chamber Homogeneity f

Sample P1-F 2.50 2.59 9.00 20.1

Sample P2-F 2.27 2.35 9.00 18.2

Sample P3-F 2.78 2.88 10.5 19.1

Sample P4-F 2.80 2.90 10.5 19.3

a

 Percent Recovery (Extraction Efficiency) based on Analysis of Spiked Sorbent Tubes was 96.6%

b

 Vapor Concentration (ppb) = [(μg CF x 24450) / (MW of CF x Liters of Air Collected)]; MW of CF = 350.6 g/mol

c

 Front and back sections of sorbent tubes were extracted and analyzed separately;

 only results from analysis of front sections are reported; extracts from back sections were all <LOD

d

Limit of Detection (LOD) = 0.0375 μg chlorpyrifos

e

Values in parentheses are LODs expressed in ppb based on mass LOD (0.0375 μg CF) and the volume of chamber air sampled

f

 Samples taken at four different exposure ports on the nose-only exposure chamber

Chlorpyrifos: Vapor Inhalation Exposure Study

Study 131040
| 18

Time After Exposure (hours)

Blo

o

d

C

o

n

c

e

n

t

ration

(n

m

oles

/

g

blo

od)

0.0001

0.001

0.01

0.1

1

10

0.254 mg CPF/m3

CPF LLQ

0.254 mg CPF/m3

TCP LLQ

0 2 4 6 12

TCP

CPF

• No oxon detected in blood

• LLQ = 0.00015 nmole/g blood

• Measurement of CPF and TCP in blood confirms absorption and 

systemic exposure to CPF vapors

Blood Levels: CPF, Oxon, TCP
| 19

Plasma

Time after exposure

0 hour 2 hour 4 hour 6 hour 12 hour

%

C

o

ntrol C

h

E

A

ctivity

(N

ormalized to

Mean C

ontrol Values)

0

20

40

60

80

100

120

140

160

0 ppb (control)

17.7 ppb CPF

Brain

Time after exposure

0 hour 2 hour 4 hour 6 hour 12 hour

%

C

o

ntrol C

h

E

A

ctivity

(N

ormalized to

Mean C

ontrol Values)

0

20

40

60

80

100

120

140

160

0 ppb (control)

17.7 ppb CPF

RBC

Time after exposure

0 hour 2 hour 4 hour 6 hour 12 hour

%

Control C

h

E

A

ctivity

(N

ormalized to

Mean C

ontrol Values)

0

20

40

60

80

100

120

140

160

0 ppb (control)

17.7 ppb CPF

Lung

Time after exposure

0 hour 2 hour 4 hour 6 hour 12 hour

%

Control C

h

E

A

ctivity

(N

ormalized to

Mean C

ontrol Values)

0

20

40

60

80

100

120

140

160

0 ppb (control)

17.7ppb CPF

Saturated CPF Vapor Study: % Control ChE 
| 20

Saturated CPF Vapor Study: ChE Activity (U/L)

Plasma

Time after exposure

0 hour 2 hour 4 hour 6 hour 12 hour

Cholin

e

steras

e

A

ctivity (U/L)

0

1000

2000

3000

4000

5000

6000

0 ppb (control)

17.7 ppb CPF

RBC

Time after exposure

0 hour 2 hour 4 hour 6 hour 12 hour

Cholin

e

steras

e

A

ctivity (U/L)

0

1000

2000

3000

4000

5000

6000

0 ppb (control)

17.7 ppb CPF

Brain

Time after exposure

0 hour 2 hour 4 hour 6 hour 12 hour

C

h

olin

e

steras

e

A

ctivity (U/L)

0

10000

20000

30000

40000

50000

60000

70000

0 ppb (control)

17.7 ppb CPF

a

a

Significantly different than 0 ppb (control) at the same post-exposure time

Lung

Time after exposure

0 hour 2 hour 4 hour 6 hour 12 hour

C

h

olinestera

s

e

A

ctivity (U/L)

0

1000

2000

3000

4000

5000

6000

0 ppb (control)

17.7 ppb CPF
| 21

Summary: Saturated Chlorpyrifos Vapor Study

• Female Sprague Dawley rats were exposed 6 h to 0 or 

17.7 ppb CPF vapor (0 or 0.254 mg/m3)

> Oxon present in exposure atmosphere @ 0.24 ppb (0.003 mg oxon/m3)

> Aerosol present (0.0036 mg/m3) with MMAD 0.72 μm (GSD = 2.1)

• No oxon detected in blood at any time point

• Maximum blood CPF levels at end of exposure

> < LLQ by 6 hours post exposure

• TCP blood levels similar at 0 and 2 hours post exposure

> > LLQ at all post exposure time points

• No inhibition of ChE activity in Lung, RBC, Plasma or 

Brain was observed – even in the presence of measured 

systemic exposure to CPF
| 22

Questions
Michael Bartels, Sue Marty, Daland Juberg

August 27, 2013

Chlorpyrifos Oxon: Biological Basis for 

Revised Toxicity Adjustment Factor
| 24

Outline - Biological Basis for Revised Toxicity 

Adjustment Factor

• Background information

• Data from Comparative Cholinesterase Assay study data, 

showing:

> preferential inhibition of RBC over brain

> complete first-pass metabolism of Chlorpyrifos Oxon

• Tissue Sensitivity Comparison- new in vitro IC50 study data

• Effects in portal-of-entry GI tissue occur only at high doses 

(≥ 5 mg/kg) and are not clinically relevant

• Proposal for brain as relevant endpoint for TAF for systemic 

exposures to Chlorpyrifos
| 25

Background Information

• EPA is interested in improving Toxicity Adjustment Factors needed to derive 

POD values from oral Oxon exposures

• Chlorpyrifos is unique in that multiple studies are available which provide data 

on ChE inhibition (ChEI) in systemic tissues

• In the 2010 DAS CCA study, Oxon was shown to elicit ChEI in RBC, but not 

brain tissue at doses ≤ 10 mg/kg

> Oxon-based ChEI in RBC tissue was more pronounced from Oxon than from 

Chlorpyrifos (76% ChEI at 1 mg/kg Oxon vs 19% ChEI at 2 mg/kg Chlorpyrifos) 

> In contrast, no Oxon-based ChEI in brain tissue vs. measureable effects from 

Chlorpyrifos (0% ChEI at 10 mg/kg Oxon vs 48% ChEI at 10 mg/kg Chlorpyrifos) 

• To inform the relevance of Oxon-based ChEI, it is important to:

> assess the impact of 1st pass metabolism 

> utilize data from non-oral route in vivo and also in vitro ChE studies that show the 

relative sensitivity of Oxon-based RBC and brain ChEI

> Summarized in 2012 Dow AgroSciences White Paper report (MRID: 48913401)
| 26

Test material comparison of ChE inhibition in RBC/plasma vs. brain tissue of adult 

female SD rats (Marty et al. 2010; 40 mg/kg Oxon brain data from Costa 1990)

CPF Oral- Gavage

Dose (mg/kg)

C

h

olin

e

steras

e

Activity (%

C

o

ntrol)

0.01 0.1 1 10 100

0

50

100 RBC

Brain

Plasma

Oxon Oral- Gavage

Dose (mg/kg)

C

h

olin

e

steras

e

Activity (%

C

o

ntrol)

0.01 0.1 1 10 100

0

50

100

RBC

Brain

Plasma

Substantially different inhibition 

profiles also seen for CPF & 

Oxon:

• CPF systemically bioavailable, 

while no Oxon seen in blood

• Oxon is significantly less toxic 

to systemic tissues than CPF

• This is due to extremely rapid, 

and complete presystemic

hydrolysis of Oxon by ChE and 

other esterases
| 27

GI 

Tract

Oxon

≤ 10 mg/kg

Sites of Presystemic Hydrolysis 

of Chlorpyrifos Oxon:

1) aqueousphaseof GI contents

2) enzymes in GI 

3) carboxylesterasesin Gut tissue

4) PON-1 esterase in Gut tissue

5) Enzymes/proteinsin portalblood

6) LiverCYPs and esterases

Oxon -> TCP

Systemic circulation

- No detectable Oxon 

(LOQ = 0.3 nM) Liver

TCP TCP

Portal vein

1

2

3

4

5

6

RBC-> RBC-inhibited RBC-inhibited

Sites of Presystemic Hydrolysis of Oxon 

ü ü

To facilitate predictions of 

PK and PD from Oxon, the 

existing PBPK model for CPF 

has been modified:

• incorporates Oxon-specific 

uptake rates

• utilizes existing rates of 

presystemic metabolism of 

Oxon

Presystemic RBC/plasma inhibition also results in Oxon hydrolysis

- Oxon blood levels below ChE inhibitory concentrations
| 28

• In Vivo Inhibition of ChE in Various Tissues of Rats following IV, IP, Dermal or Oral 

administration of CPF-Oxon (0.4, 12, 15 and 40 mg/kg, respectively; samples collected 4 hr 

post-dosing). Dose levels were selected to induce relatively similar degrees of ChE inhibition 

in plasma, RBCs, brain and diaphragm, oral dosing required the highest dose level. (data 

from Costa et al. 1990).

Dose Route

Choli

nesterase

A

ctivity (%

Control)

IV Dermal IP Oral

0

20

40

60

80

100

Plasma

RBC

Brain

E Diaphragm qual inhibition in the absence 

of 1st pass metabolism

More inhibition of RBC due to

presystemic hydrolysis of Oxon in blood

Brain effects more conserved across 

routes, due to systemic exposure
| 29

In Vivo Inhibition of ChE in Various Tissues of Rats following IP administration of 

CPF-oxon (30 mg/kg). Note- data from Carr et al. (2002).

Sampling Time (hr post-dosing)

2 hr 24 hr

0

20

40

60

80

100

Medulla-Pons

Corpus Striatum

Cerebral Cortex

Hippocampus

Skeletal Muscle

Lung

Heart

Brain representative of numerous systemic tissues 
| 30

In Vitro Inhibition of ChE in Various Tissues of Rats (Chambers 2013). 

Values in nM Chlorpyrifos Oxon concentration

Brain representative of 

numerous systemic tissues

GI tissues have 

comparable sensitivity to 

systemic tissues

RBC IC50 similar to all 

tissues

Plasma IC50 highest- due 

to presence of numerous 

esterases (including 

albumin)

ACHE Avg StDev

Brain 3.77 0.10

RBC 4.19 0.69

RBC (ghosts) 5.08 0.32

Duodenum (flushed) 3.72 0.21

Duodenum (not flushed) 4.17 0.48

Esophagus (flushed) 3.13 0.31

Esophagus (not flushed) 3.28 0.59

Stomach 4.08 0.09

Lung (perfused) 7.21 1.32

Lung (not perfused) 8.57 0.85

Heart (perfused) 3.06 0.05

Heart (not-perfused) 3.91 0.05

Diaphragm 6.64 0.28

Plasma 55.36 0.51
| 31

In vitro Inhibition of ChE in Various Tissues of Rats or Recombinant Human Enzyme 

(Data from Mortensen 1998, Chambers 2013, Moser 2011)

Brain

Duodenum

E

sophagus

Sto

m

ach

Lung

Heart

Diaphragm

RBC

H

u

man rA

ChE

Brain- Immunoppt

Pla

s

m

a- Immunoppt

Liver- Immunopppt

Pla

sma

Study Reference

IC50

(nM)

Mortensen 1998 Chambers 2013 Moser 2011

0

1

2

3

4

5

6

7

8

9

10

10

20

30

40

50

60

Brain also representative of numerous systemic tissues in vitro, 

including GI tract tissues

IC50 for human AChE comparable to purified rat ChE

NOEC 

~0.3 nM
| 32

Portal-of-Entry ChE effects following oral intake of 

Chlopyrifos Oxon

• Possible Portal-of-entry effects to GI have been reported at 5 mg/kg for Oxon 

and 20 mg/kg for Chlorpyrifos

> Supports an Oxon Toxicity Adjustment Factor for portal-of-entry effects of 4

• No GI effects at 1 mg/kg Oxon (or 0.5 mg/kg/day with repeated exposure)

> This effect level of 5 mg/kg for Oxon is 1500-fold higher than the maximum, modelpredicted oxon concentrations in drinking water of 112 ug/L

• NOAEL of 1 mg/kg Oxon is 300-fold higher than highest predicted drinking water 

concentration 

• Repeated dose NOAEL of 0.5 mg/kg is 150-fold higher 

> These effects are not expected to be clinically relevant: 

• ChEI in GI tissue does not affect nerves exiting the gut (i.e., no effects on other organ 

systems)

• GI effects by other ChEI agents (e.g., pharmaceuticals) are transient, not considered to 

have long-term clinical consequences

• OP toxicity on GI function appears minimal, and generally only seen after ingestion of 

large bolus doses of Oxon (≥ 5 mg/kg) 
| 33

Systemic ChE effects following oral intake of 

Chlopyrifos Oxon

• At extremely high oral doses of Oxon (10 mg/kg) there is no measured 

systemic bioavailability of this compound (LOQ = 0.3 nM), due to high 

first-metabolism in the GI and liver 

> no ChEI is seen in brain tissue, at doses up to 10 mg/kg

> 0.3 nM = NOEC for ChEI in Mortenson in vitro study

• Numerous in vivo and in vitro studies have shown that Oxon-induced 

ChEI in brain tissue is representative and conservative of ChE effects 

in many other tissues (including new in vitro data of Chambers et al.)

> In contrast, Chlorpyrifos causes ~ 50% brain ChEI at 10 mg/kg

> These data support an Oxon Toxicity Adjustment Factor for systemic 

effects of 0.5
| 34

Comparison of ChE inhibition in RBC and brain tissue of adult female SD rats 

administered CPF or CPF-oxon via oral gavage (Marty et al. 2010)

CPF CPF-Oxon

Key Event 1 Key Event 2 Key Event 3

Dose (mg/kg)

Cholineric 

effects in GI 

tissue

Inhibition of RBC 

Cholinesterase 

Activity

Inhibition of Brain 

Cholinesterase 

Activity

0.05 - - -

0.1 - - -

0.5 - - -

2 - + -

5-Pilot - ++ -

10 - +++ ++

10-Pilot - +++ ++

20-Pilot + * ++++ +++

* GI effects data in adult Male Long-Evan rats, from Moser et al. (1995)

Scale for ChE inhibition (% of control): ≥90% = "-", 75-90% = "+", 40-75% = "++", 

5-40% = "+++", <5% = "++++"

Key Event 1 Key Event 2 Key Event 3

Dose (mg/kg)

Cholineric 

effects in GI 

tissue

Inhibition of RBC 

Cholinesterase 

Activity

Inhibition of Brain 

Cholinesterase 

Activity

0.01 - - -

0.05 - - -

0.1 - - -

0.5 - ++ -

1 - +++ -

1-Pilot - +++ -

5-Pilot + ^ ++++ -

10-Pilot + ^ ++++ -

^ Effect seen (mucoid feces), in only 1 of 3 study animals at this dose level

Scale for ChE inhibition (% of control): ≥90% = "-", 75-90% = "+", 40-75% = "++", 

5-40% = "+++", <5% = "++++"

MOA for systemic tissue effects dependent on systemic bioavailability of CPF or Oxon

Effects seen in portal-of-entry GI tissue occur only at high doses

RBC effects not representative of either Key Event

Portal-of-Entry vs. Systemic ChE effects following oral 

intake of Chlorpyrifos or Chlopyrifos Oxon
| 35

Summary

• Chlorpyrifos and Oxon have unique pharmacokinetic profiles

> Chlorpyrifos is primarily metabolized in the liver

• but is systemically available above 0.1 mg/kg

> Oxon is more labile, being rapidly hydrolyzed by numerous tissues, 

including Intestinal lumen, portal vein RBC & plasma esterases and liver

• resulting in no systemically available Oxon at dose levels up to 1 mg/kg

• As a result, RBC is predictor of presystemic exposure to Oxon, vs. 

systemic exposure to Chlorpyrifos

• In contrast, brain sensitivity to Oxon effects has been shown to be 

comparable to numerous other systemic tissues

> Therefore, DAS proposes that the most appropriate endpoint for prediction 

of adverse systemic effects from oral exposure to Oxon, relative to 

Chlorpyrifos, is inhibition of brain ChE activity
| 36

References

• Carr, R.L., J.R. Richardson, J.A. Guarisco, A. Kachroo, J.E. Chambers, T.A. Couch, G.C. Durunna, and E.C. Meek. 

(2002). Effects of PCB exposure on the toxic impact of organophosphorus insecticides. Toxicol Sci. 67:311-321.

• Chambers, J.E. (2013). In vitro Sensitivity of Cholinesterase to Inhibition by Chlorpyrifos-oxon in Several Tissues of 

the Rat. College of Veterinary Medicine, Mississippi State University.

• Costa, L.G., B.E. McDonald, S.D. Murphy, G.S. Omenn, R.J. Richter, A.G. Motulsky, and C.E. Furlong (1990). Serum 

paraoxonase and its influence on paraoxon and chlorpyrifos-oxon toxicity in rats. Toxicology and Applied 

Pharmacology, 103:66-76. 

• Dow AgroSciences (2012). White Paper Discussion of Toxicity Adjustment Factors used for the Risk Assessment of 

Chlorpyrifos Oxon in Drinking Water. A Science Issues Paper for the US Environmental Protection Agency, 

submitted August 17, 2012 (MRID: 48913401).

• Marty, M.S., and A.K. Andrus. 2010. Comparison of Cholinesterase (AChE) Inhibition in Young Adult and Preweanling

CD Rats after Acute and Repeated Chlorpyrifos or Chlorpyrifos or Chlorpyrifos-oxon Exposures. An unpublished 

report of the Dow Chemical Company (Dow Study ID 091107) [MRID 48139301]. The Dow Chemical Company. 

• Mortensen, S.R., M.J. Hooper, and S. Padilla (1998). Rat brain acetylcholinesterase activity: developmental profile 

and maturational sensitivity to carbamate and organophosphorus inhibitors. Toxicology. 125:13-19.

• Moser, V.C., and S. Padilla (2011). Esterase metabolism of cholinesterase inhibitors using rat liver in vitro. 

Toxicology. 281:56-62.
| 37

Questions
38

BACKUP SLIDES
| 39

Outline – Gaps in Columbia Epidemiology Study 

• Corroboration/Replication 

• Exposure Measurements

• Relevant Publications

• Selected DAS and External Presentations - Epidemiology
| 40

Challenges with Evaluation of Columbia Data/Studies

• Corroboration/Replication

> No replication of Columbia cohort findings

• Inconsistent findings among 3 cohorts (Columbia, Mt. Sinai, 

Berkeley/Chamacos)

• Independent reviews/publications have confirmed this lack of 

confirmed association between exposure and effects (Eaton et al, 

Mink et al, Li et al, Prueitt, et al)

> MRI study (Columbia only) evaluated by 3 independent 

experts which reveal significant confounding/design 

challenges

> Unavailability of raw data from Columbia and other cohorts

> SAP advisement to not use Columbia for quantitative risk 

assessment
| 41

Challenges with Evaluation of Columbia Data/Studies

• Exposure Measurements

> CDC cannot confirm analytical detection method for 

Chlorpyrifos in blood which undermines the confidence 

in the reported blood measurements of most subjects

> Dose-reconstruction hindered by spot sampling and no 

information on application

> Dow exposure study demonstrates challenge with spot 

sampling for point estimates of exposure, urinary TCPY 

is appropriate biological marker and ChEI does not 

occur below doses predicted by PBPK modeling
| 42

Publications

• Eaton, D. L., Daroff, R. B., Autrup, H., Bridges, J., Buffler, P., Costa, L. G., Coyle, J., 

Mckhann, G., Mobley, W. C., Nadel, L., Neubert, D., Schulte-Hermann, R. & Spencer, 

P. S. 2008. Review of the toxicology of chlorpyrifos with an emphasis on human 

exposure and neurodevelopment. Crit Rev Toxicol, 38 Suppl 2: 1-125.

• Li, A. A., Levine, T. E., Burns, C. J. & Kent Anger, W. 2012a. Integration of 

epidemiology and animal neurotoxicity data for risk assessment. 

Neurotoxicology,33:823-832

• Li, A. A., Lowe, K. A., Mcintosh, L. J. & Mink, P. J. 2012b. Evaluation of epidemiology 

and animal data for risk assessment: chlorpyrifos developmental neurobehavioral 

outcomes. J Toxicol Environ Health B Crit Rev, 15: 109-184.

• Mink, P. J., Kimmel, C. A. & Li, A. A. 2012. Potential effects of chlorpyrifos on fetal 

growth outcomes: implications for risk assessment. J Toxicol Environ Health B Crit Rev,

15: 281-316.

• Prueitt, R. L., Goodman, J. E., Bailey, L. A. & Rhomberg, L. R. 2011. Hypothesis-based 

weight-of-evidence evaluation of the neurodevelopmental effects of chlorpyrifos. Crit

Rev Toxicol, 41: 822-903.
| 43

Selected DAS and External Presentations -

Epidemiology

• Evaluation of Bioanalytical Methods used in Epidemiological Studies of 

Chlorpyrifos Exposure – Michael J. Bartels - September 16, 2008 – EPA SAP

• Chlorpyrifos and Epidemiology – Carol J. Burns – September 16, 2008 – EPA SAP

• Chlorpyrifos Exposure and Neurobehavioral Outcomes in Infants and Pre-School 

Children – Epidemologic Interpretation and Comments Regarding Rauh et al 2006 

– Pamela Mink – September 16, 2008 – EPA SAP

• A Weight-of-Evidence Assessment of Recent Epidemiology Studies – Douglas 

Weed – September 16, 2008 – EPA SAP

• Epidemiology Regarding Children’s Health – Carol J. Burns – April 11, 2012 – EPA 

SAP

• Hypothesis-Based Weight-of-Evidence Evaluation of Chlorpyrifos and 

Neurodevelopmental Effects – Julie E. Goodman – April 11, 2012 – EPA SAP

• Human Studies, Animal Studies, MoA, and Weight of Evidence – Lorenz R. 

Rhomberg – April 11, 2012 – EPA SAP

• Epidemiology Summary – Carol J. Burns – August 21, 2012 - EPA
CHEMICAL SPECIFIC ADJUSTMENT FACTORS 

(CSAFS) FOR ORAL EXPOSURES TO 

CHLORPYRIFOS AND CHLORPYRIFOS OXON

Paul Price, Michael Bartels, and Torka Poet

August 27, 2013
| 45

History of DAS efforts on this issue

• 2007-2010 ACC LRI project (Hinderliter et al., 2011;Price et al., 2011)

• 2011 Science Advisory Panel presentation on methodology

> Recommendations on the development of variation model 

• Smith et al. (2011) publication on variation in enzyme activity

• 2008-2011 Numerous presentations at SOT, SRA, and ISES

• 2012-2013 Development of:

> More rigorous approach for construction of variation model

> 2D modeling of uncertainty 

> Revised strategy for developing CSAFs based on SAP comments

> Development of CSAFs for oxon
| 46

Chemical Specific Adjustment Factors 

• Based on model of human variation in sensitivity to CPF and oxon

• Factors are specific to the setting of acute oral standards

> The approach is relevant for other routes of exposure but separate 

analyses are required for each route

> Approach can be applied to chronic effects as well

• RBC AChE is used as the critical effect for defining the CSAFs

> While we believe that RBC is not a biologically relevant endpoint for oxon, 

we are using RBC AChEI to derive the CSAFs in this example.

> The approach can be applied to brain AChE

• Proposed approach addresses the recommendations from the 

February 2011Science Advisory Panel
| 47

Construction of the Variation Model

• Build on the LifeStage model of oral exposures (birth to adult) and address 

both CPF and oxon 

• Process and results:

1. Perform sensitivity analysis to identify critical parameters (104 parameters 

→19 parameters)

2. Identify parameters that vary across individuals (19 parameters →16 

parameters)

3. Develop distributions for the 16 parameters and build Monte Carlo model

4. Address moderate to strong correlations in inputs when they would 

increase variation in response – create linked parameters (16 parameter 

→15 parameters)

5. Determine minimum set that drives variation (15 parameters → 3 

parameters) at relevant doses (RBC AChEI > 10% for sensitive individuals)
| 48

1

10

100

0 0.2 0.4 0.6 0.8 1

P

ercent of Back

ground

A

ctivity

Fraction of the population

Population variation in RBC AChE activity after five days 

of oral doses of 0.3 and 0.1 mg/kg CPF for full (15 

parameter) and final (3 parameter) models 

Full model 0.3 mg/kg

Full Model 0.1 mg/kg

1

10

100

0 0.2 0.4 0.6 0.8 1

Fraction of the population

Population variation in RBC AChE activity after 

five days of oral doses of 0.04 and 0.015 mg/kg 

Oxon for full (15 parameter) and final (3 parameter) 

models

Full model 0.04 mg/kg

Full model 0.015 mg/kg
| 49

1

10

100

0 0.2 0.4 0.6 0.8 1

P

ercent of Back

ground

A

ctivity

Fraction of the population

Population variation in RBC AChE activity after five days 

of oral doses of 0.3 and 0.1 mg/kg CPF for full (15 

parameter) and final (3 parameter) models 

Full model 0.3 mg/kg

Final Model 0.3 mg/kg

Full Model 0.1 mg/kg

Final Model 0.1 mg/kg

1

10

100

0 0.2 0.4 0.6 0.8 1

Fraction of the population

Population variation in RBC AChE activity after 

five days of oral doses of 0.04 and 0.015 mg/kg 

Oxon for full (15 parameter) and final (3 parameter) 

models

Full model 0.04 mg/kg

Reduced model 0.04 

mg/kg

Full model 0.015 mg/kg

Reduced model 0.015 

mg/kg
| 50

Summary of Variation model 

• The variation model is an age- and gender-specific model of human 

variation

• At doses 30 ug/kg variation response to CPF can be predicted based 

on variation in three parameters and variation in response in Oxon to 

one parameter

> CPF

• PON1 in liver/plasma (linked)

• CYPs converting CPF to oxon

• CYPs converting CPF to TCPy

> Oxon

• PON1 in liver/plasma (linked)

• Global sensitivity analysis identified the same parameters for CPF
| 51

Modeling findings on age-related changes in sensitivity 

• Model predicts that age-related changes are small and dose 

dependent

> At high doses (>200 ug/kg CPF and >30 ug/kg oxon) differences in plasma 

PON1 make children slightly more sensitive (10-20%) 

> At lower doses the models suggest that adults will be slightly more 

sensitive because of differences in physiology that lead to different removal 

rates (oxon persists slightly longer in adults than infants)

• Model predicts that at doses < 200 ug/kg of CPF and >30 ug/kg of 

oxon, the liver effectively clears oxon as it is formed thus the fetus is 

protected from the oxon

> This explains the observation in rats that the fetus is not sensitive relative 

to maternal toxicity
| 52

Uncertainty 

• The variation model is extended to address uncertainty using 2-D 

Monte Carlo modeling

• The uncertainty analysis focused on the three critical model 

parameters. 

• Uncertainty in the values of these occurs as a result of limitations in 

data on variation of parameters

> Mean value and coefficient of variation are uncertain given 30 samples

> We have characterized the uncertainty in enzyme activities from Smith et 

al. using parametric bootstrap analyses 

• Other sources of uncertainty (modeling uncertainty, “surprise”, etc.) 

are addressed by:

> Long history of use of the model, extensive parameterization, and the 

evaluation of model using multiple data sets 
| 53

Description of Monte Carlo modeling 

• Population assessed:

> Six month old individuals

> Infants at birth are predicted to be less sensitive than six month olds

> Findings in in earlier work indicates that adults are similar 

• Stable estimates were obtained with model runs of 

> Each individual’s response is simulated over four days and for four doses 

> 1,000 individuals in each population

> 50 populations simulated
| 54

0

20

40

60

80

100

0.03 0.09 0.27 0.81 2.43

Percen

t

o

f

Background

A

c

tivity

Single Oral Dose (mg/kg)

Maximum RBC AChE Activity following a single 

oral dose 
| 55

0

20

40

60

80

100

0.03 0.09 0.27 0.81 2.43

Percen

t

o

f

Background

A

c

tivity

Single Oral Dose (mg/kg)

Variation in the dose that causes 10% 

inhibition of RBC AChE activity 

50th

Percentile

5

th

Percentile

1

th

Percentile

10% Inhibition
| 56

0.00

20.00

40.00

60.00

80.00

100.00

0.03 0.30

P

ercent of Back

ground

A

ctivity

Single Oral Dose (mg/kg)

Plotting an Individuals RBC AChE Dose 

Response to Determine ED10
| 57

0.00

20.00

40.00

60.00

80.00

100.00

0.03 0.30

P

ercent of Back

ground

A

ctivity

Single Oral Dose (mg/kg)

Values of ED10 for simulated individuals
| 58

0.00

20.00

40.00

60.00

80.00

100.00

0.03 0.09 0.27 0.81 2.43

P

e

rcent of Back

g

round

A

c

tivity

Single Oral Dose (mg/kg)

Variation in ED10 across a population

5

th

Percentile

1

st

Percentile 50th

Percentile
| 59

Uncertainty portion of Model creates equally probable 

descriptions of the population variability

0

20

40

60

80

100

0.03 0.09 0.27 0.81 2.43

0

20

40

60

80

100

0.03 0.09 0.27 0.81 2.43

0

20

40

60

80

100

0.03 0.09 0.27 0.81 2.43

0

20

40

60

80

100

0.03 0.09 0.27 0.81 2.43

0

20

40

60

80

100

0.03 0.09 0.27 0.81 2.43

0

20

40

60

80

100

0.03 0.09 0.27 0.81 2.43
DOW AGROSCIENCES RESTRICTED | 60

0.60 1.80 5.40

Single Oral Dose (mg/kg)

Bootstrapped Populations

Lower Confidence 

Limit of 50th percentile

Lower Confidence 

Limit of 5th percentile

Lower Confidence 

Limit of 1th percentile
| 61

Animal and human modeling results for CPF

Dose mg/kg

Best estimate of the 

dose causing 10% 

inhibition

Lower 95th percentile 

confidence limit of the 

dose causing 10% 

inhibition

Human 6 months

50th Percentile 0.20 0.17

5th Percentile 0.10 0.09

1st Percentile 0.09 0.08
| 62

Chemical Specific Adjustment Factors: Interspecies 

POD from animal study

Model prediction for typical humans Inter-species =
| 63

POD in the animal study and its equivalent in humans 

• EPA’s proposed POD for CPF is BMDL10 in male neonates by single 

oral dose milk (0.36 mg/kg) Marty et al. (2010)

• The animal BMDL10 is the estimate of a dose where there is a 95% 

confidence that the dose that causes 10% peak RBC inhibition is 

higher than the estimate

• The animal BMDL10 is treated as being equivalent to the 95% LCL of 

the estimate of dose causing 10% inhibition in the 50th percentile of the 

population of six month olds. The definition is:

> Consistent with the assumption of minimal variation across test animals

> Consistent with the use of pools of animals in the RBC AChE assay

> Consistent with the concept of a “typical” individual
| 64

Chemical Specific Adjustment Factors: Interindividual 

• Guidance on developing chemical specific adjustment factors 

suggests calculating the size of the inter-individual factor needed to 

protect 90%, 95% and 99% of the population (Meek et al. 2002, WHO, 

2005, EPA, 2011 (Draft)).

• We have focused on 95th and 99th percentiles

• Note our “typical individual” is an infant and infants are believed to be 

slightly more sensitive than adults at high doses, thus age-related 

variation is already addressed in the determination of the prediction of 

the typical human and does not need to be included in the calculation

Model prediction for sensitive human 

Model prediction for typical human Inter-individual =
| 65

Animal and human modeling results for CPF

Dose mg/kg

Best estimate of the dose 

causing 10% inhibition

Lower 95th percentile 

confidence limit of the dose 

causing 10% inhibition

Rat pup data

0.47 0.36

Human 6 months

MEDIAN 0.20 0.17

5th Percentile 0.10 0.09

1st Percentile 0.09 0.08

2.1

0.17

0.36 Inter-species = =

1.8

0.09

0.17 Inter-individual = =

2.1

0.08

0.17 Inter-individual = =
| 66

EPA’s proposed aPAD and impact of CSAFs for CPF

EPA proposal Model Based Values

Inter-species 10 2.1

Inter-individual 10 1.8 - 2.1

FQPA 1 1

Total 100 3.9 – 4.5 

Final aPAD is 360 ug/kg divided by 

3.9 to 4.5, or 

90 to 70 ug/kg
| 67

EPA’s proposed aPAD and impact of CSAFs for Oxon

EPA proposal Model Based Values

Inter-species 10 1.2

Inter-individual 10 1.65 – 1.85

FQPA 1 1

Total 100 2.0 – 2.3 

Final aPAD is 50 ug/kg divided by 2.0 

– 2.3, or 

25 to 22 ug/kg
| 68

Summary

• Dow AgroSciences believes that the science is sufficient to warrant a 

reduction in the adjustment factors for CPF and oxon

> Interspecies and interindividual factors can be much smaller

> The modeling provides evidence that infants and the developing fetus are 

not at elevated risk from CPF or oxon

• These reductions use the best science and are consistent with 

regulatory guidance on developing CSAFs and should be considered 

in setting the acute oral aPAD

• Consideration of such population modeling informing the standard 

setting process is consistent with Toxicity in the 21st Century 
| 69

Questions
USING PBPK/PD MODELS TO RECONSTRUCT 

AGGREGATE EXPOSURES OF THE COLUMBIA 

UNIVERSITY COHORT - PRELIMINARY FINDINGS

Paul Price and Torka Poet

August 27, 2013
| 71

Current model of PBPK/PBPD of CPF for multiple routes 

of exposure (aggregate exposure)

• This model is a work in process and all findings are preliminary

• This model addresses all three routes for the same individual

• Initial version is limited to:

> One day of dosing and multiple days of follow up

> Inhalation, hand to mouth, and dermal assume continuous 24 hrs exposure 

and dietary dose are assumed to occur at one meal

• Inhalation exposures are modeled as causing doses by both by the 

lung and the gut

• No human variation but gender, age, and body weight of individual 

can be specified
| 72

Use of the model to explore the impact of route on CPF 

in blood and inhibition of RBC AChE 

• Exposure parameters for each route and source were adjusted to 

result in absorbed doses of ~ 3 ug/kg (dose was selected only for the 

purpose of comparing routes)

> Inhalation 24 hours at 0.06 mg/m3

> Dermal loading of 1.5 mg of CPF over 60% of a person’s surface area for 

24 hrs

> Oral doses of 3 ug/kg from diet or hand-to-mouth exposures

• Inhalation, dermal, and hand-to-mouth exposures occurred over 24 hr 

period, but dietary doses are assumed to occur at one meal

• 85 kg adult female modeled

• Individual modeled for one day of exposure and 8 additional days of 

follow up
| 73

0.1

1

10

100

1000

10000

0 2 4 6 8

n

g

/l CPF

Days

Time course of CPF in blood after an 

absorbed dose of 3 ug/kg by various 

routes and sources

Oral (diet)

Oral (Hand to mouth)

Dermal

Inhalation
| 74

99.965

99.970

99.975

99.980

99.985

99.990

99.995

100.000

0 1 2 3 4 5 6 7 8 9

R

B

C

A

Ch

E Activity as

a

p

e

rce

n

t of baseline

Days

Time course of RBC AChE activity after a 

dose of 3 ug/kg by various routes

Oral (diet)

Oral (Hand to mouth)

Dermal

Inhalation
| 75

Conclusions 

• Route of exposure has a pronounced effect on the levels of CPF in the 

blood:

> Equivalent absorbed doses by the dermal and inhalation result in 55X and 

14X higher area under the curve (AUC), then by the oral route (diet)

• Impact of route on RBC AChE is much smaller 

> AUC of dermal is 8% larger, hand-to-mouth oral 12% lower, and inhalation 

is about 1⁄2 of AUC for oral (diet)

• Model suggests that blood levels from all routes of exposure are 

biphasic

> CPF is lipid soluble and may be slowly released from fat

> This slow release results in a second phase in the decline in blood levels 

that is much slower than the initial phase 

> As a result the model predicts that low level CFP blood concentrations (30 

to 100 fold below the peak CPF levels) will persist for a week or more
| 76

Challenge of the Columbia cohort

• Participants were potentially exposed from multiple sources (diet, 

inhalation, dermal, and hand-to-mouth)

• Participants were adults – there is limited understanding of how to 

model dermal and hand-to-mouth exposures for adults

• No data are available on use of pesticides on the days immediately 

prior to entering hospital

• Only measurements of blood levels of chlorpyrifos were performed in 

the early years of the study

• Raw data are not available – only published summaries.
| 77

Description of the Model

• Model addresses a single day of exposure and multiple days of follow 

up

• Address all routes and sources

• Determines blood levels of CPF, TCPY excretion, and plasma and 

RBC cholinesterase activity

• No variation in sensitivity but age and body-weight is specified

> (Adult female of 75 kg and 12 kg weight increase for late pregnancy) 
| 78

Data used in the analysis

• Data from Columbia University study publications

> Air levels

> Summary information on blood levels of CPF for 1999

• Vaccaro study:

> Seven individuals with a 4 hour exposures to residues on carpets – scripted 

activities to simulate high exposure infant behaviors 

• Data from Vaccaro study was used to:

> Dermal loading on hands used as a benchmark for all dermal exposure

> Estimated Kp value based on dermal loading and TCPY excretion that 

reflects pesticide formulations 

> Note pesticide formulation in the Columbia study is not specified in the 

publications

• Other studied are in the literature that could refine this assessment
| 79

Range-finding scenario

• Develop a “plausible” exposure scenario and determine how the 

predictions of blood levels match up with reported measurements

• Focus on the largest 15% of the measurements of blood as being the 

most reliable 

> Maternal blood levels 5.5 - 35 ng/l

> Cord blood 6.6 – 65 ng/l

• Exposure assumptions

> Inhalation – use the highest reported air concentration (0.345 ug/m3)

> Diet – 90th percentile of adult dietary exposure from modeling (0.017 ug/kg)

> Oral dose from hand-to-mouth is based on guidance in EPA 2012 SOPs 

• Fraction of hand and frequency are set at the low end of the ranges for infants 

and hand loading set at 1/10 of mean hand loading in Vaccaro study. 

> Dermal dose 

• Based on 1/10 of the mean hand loading in Vaccaro study applied to 15% of skin 

surface 
| 80

Results
| 81

0.1

1

10

100

1000

-1 0 1 2 3 4 5 6 7 8

C

P

F in blood ng/l

Days

Timeline for Columbia data on 

chlorpyrifos in blood
| 82

0.1

1

10

100

1000

-1 0 1 2 3 4 5 6 7 8

C

P

F in blood ng/l

Days

Timing of samples and range of top 15% 

of reported levels in cord blood

Cord Blood
| 83

0.1

1

10

100

1000

-1 0 1 2 3 4 5 6 7 8

C

P

F in blood ng/l

Days

Timing and range of top 15% of 

maternal blood levels

Maternal Blood

Cord Blood
| 84

0.1

1

10

100

1000

-1 0 1 2 3 4 5 6 7 8

C

P

F in blood ng/l

Days

Time course of chlorpyrifos in 

blood from range-finding scenario
| 85

0.1

1

10

100

1000

-1 0 1 2 3 4 5 6 7 8

C

P

F in blood ng/l

Days

Dermal drives chlorpyrifos levels in 

blood

All Routes

Diet

Hand to Mouth

Inhalation

Dermal
| 86

99.970

99.975

99.980

99.985

99.990

99.995

100.000

-1 0 1 2 3 4 5 6 7 8

RBC 

AChE 

Activity as

a

%

of baseline

Days

No significant impact on RBC AChE activity is predicted 

to occur either at home or in the hospital

All Routes

Diet

Hand to Mouth

Inhalation

Dermal

No significant 

change in 

activity
| 87

Preliminary conclusions

• The blood levels in the top 15% of Columbia study for the year 1999 are 

consistent with levels that could plausibly occur from residential uses if:

> The residence was treated some time in the week prior to the individual entering the 

hospital; and the subject had extensive dermal contact to treated surfaces 

• The blood levels of CPF could have been as much as 30-fold higher on the 

day of the application then the levels measured in the study

• These higher levels would not have caused significant RBC AChEI at any time 

(while at home or in the hospital) 

• The conclusion is that the data do not indicate an unsafe exposure to CPF 

occurred to any of the participants. Nor do the levels demonstrate that 

significant RBC AChEI occurred at any time.

• This supports Prueitt et al.’s findings that the associations reported by 

Columbia University are not due to the toxicity of chlorpyrifos and are more 

likely attributed to alternative explanations
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