EPA Scientific Advisory Panel, Washington, DC, February 15-18, 2011

Sue Marty, Ph.D., D.A.B.T.

The Dow Chemical Company

Utility of New Animal Study 

Data for Model Development
EPA Scientific Advisory Panel, Washington, DC, February 15-18, 2011 2

Information in this presentation addresses:

 Chapter in the report

» 10. Using the source-to-outcome model to estimate 

compound and age-specific uncertainty factors 

 Charge question

» 6.1 Comment on the strengths and limitations of DAS’s 

proposed approach to estimate an animal-to-human 

extrapolation factor using a single animal study as 

opposed to a weight of the evidence approach when 

multiple studies are available.
EPA Scientific Advisory Panel, Washington, DC, February 15-18, 2011 3

Outline

 Comparative Cholinesterase Assay (CCA): 

Rationale for presenting study data

 Study Design

– Probe study: Time of peak ChE inhibition

– Definitive study: D-R for ChE inhibition

» Route of exposure

» RBC as a biomarker

 Benchmark Dose Modeling
EPA Scientific Advisory Panel, Washington, DC, February 15-18, 2011 4

CCA: Rationale for Presenting Study Data 

 CCA is the cornerstone data set for modeling

– Study design drove some model prediction scenarios

» Model similar sample sizes

– Acute exposure relevant

– Samples taken at peak ChE inhibition after acute CPF

 CCA study as part of EPA data call-in

– Fulfilled data gaps not addressed by other studies 

– Study included a larger dose range
EPA Scientific Advisory Panel, Washington, DC, February 15-18, 2011 5

Chlorpyrifos Comparative Cholinesterase (ChE) 

Study

 CCA – Standard design to evaluate age-related sensitivity to ChEI

– Young (PND 11) vs. Adults (PND 70)

– Gender differences in young animals

– Acute vs. Repeated dose

– Different vehicles

– TK analyses

Age Gender n vehicle Dose Time Sampled Tissues for ChE

4-6/dose Corn oil 0, 3 mg/kg Females 6/dose Milk 0, 5 mg/kg 2, 4, 6, 8, 12, 24, 48 h

Corn oil 6 h 8/dose Milk 5 doses (0.05-5 mg/kg) 8 h

PND 11 Pups

Males & Females

8/dose Corn oil 5 doses (0.05-3.5 mg/kg/day) PND 11-21

Brain, RBC and 

Plasma

Corn oil 4/dose Diet (12 h) 0, 10 mg/kg 2, 4, 6, 8, 12, 24 h

Corn oil 8/dose Diet (12 h) 5 doses (0.05-10 mg/kg) 8 h PND 70 Adults Females

8/dose Corn oil 5 doses (0.05-3.5 mg/kg/day) 70-80 days of age

Brain, RBC and 

Plasma

Chlorpyrifos-oxon dose groups not shown

ChE measured by a modified Ellman method.
EPA Scientific Advisory Panel, Washington, DC, February 15-18, 2011 6

Probe Study: Time of Peak Inhibition in PND 11 Pups

Females with 8 female pups/litter

♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀

Dosed 0.75 mg/kg CPF 

in milk 4 times at 6 h 

intervals

Group 1: single gavage 3 mg/kg 

CPF in corn oil

Group 2: single gavage 3 mg/kg CPF 

in milk

Group 4: single gavage 0.5 mg/kg CPF-oxon in 

corn oil

Group 5: single 0 mg/kg gavage corn oil 

vehicle (control)

Group 6: single 0 mg/kg gavage milk vehicle 

(control)

Extra pup not used in acute study

Group 3: divided dose gavage 0.75 mg/kg 

CPF in milk

Group 7: divided dose 0 mg/kg gavage milk 

vehicle (con)

All pups euthanized and samples collected at 2, 4, 6, 8, 12, or 24 h after single exposure

or after last administered dose (Groups 3 and 7)
EPA Scientific Advisory Panel, Washington, DC, February 15-18, 2011 7

Time of Peak Inhibition in PND 11 Pups (Corn Oil)
8

Estimated Time of Peak AChEI in PND 11 Pups Using PBPK 

Model

RBC

Brain
EPA Scientific Advisory Panel, Washington, DC, February 15-18, 2011 9

Probe Study: Time of Peak Inhibition in 

Adult Female Rats

~ 70-day-old female rats Group 8: single gavage 

10 mg/kg CPF in corn oil

Group 9: single gavage 0.3 mg/kg CPF-oxon in 

corn oil

Group 10: single gavage 

0 mg/kg CPF in corn oil

Adult females euthanized and samples collected at 2, 4, 6, 8, 12, or 24 h 

after single exposure on postnatal day 70
EPA Scientific Advisory Panel, Washington, DC, February 15-18, 2011 10

Definitive CPF CCA Study in PND 11 Pups

Females with 3 male & 5 female pups/litter

♂ ♂ ♂ ♀ ♀ ♀ ♀ ♀

Group 1: single gavage CPF in 

corn oil1

Group 3: Pup used for analysis of CPF, CPF-oxon, and 

TCP in blood

Group 2: single gavage CPF-oxon in 

corn oil

Group 2: single gavage CPF-oxon in 

corn oil

Group 3: Pup used for analysis of CPF, CPF-oxon, and TCP 

in blood

Group 1: single gavage CPF 

in corn oil1

Group 4 (Pilot): single gavage 5 mg/kg CPF (milk) or 3 mg/kg 

CPF (corn oil) (48 h only)

Group 4 (Pilot): single 0 mg/kg gavage milk or corn oil 

vehicle (48 h only)

Group 1, 2 and 3 pups euthanized and samples collected at time of peak ChEI based on probe study data

Group 4 pups euthanized and samples collected at 2, 4, 6, 8, 12, 24 or 48 h after single exposure

1Dose levels: 0, 0.05, 0.1, 0.5, 2 or 5 mg/kg chlorpyrifos
EPA Scientific Advisory Panel, Washington, DC, February 15-18, 2011 11

Dose-response for AChEI in PND 11 Male and Female 

Pups (Corn Oil)

PND 11 Male Pups PND 11 Female Pups 

0

20

40

60

80

100

120

140

0.05 0.1 0.5 2 5

CPF (mg/kg)

RBC ChE Activity (% Control)

*

*

0

20

40

60

80

100

120

140

0.05 0.1 0.5 2 5

CPF (mg/kg)

RBC ChE Activity (% Control)

*

*
EPA Scientific Advisory Panel, Washington, DC, February 15-18, 2011 12

~ 70-day-old female rats Group 8: single gavage 

CPF in corn oil1

Group 9: single gavage 0.3 mg/kg CPF-oxon in 

corn oil

Group 10: 12-h Dietary CPF

Definitive CPF CCA Study in Adult Rats

Samples collected at time of peak ChE inhibition based on pilot study data

1Dose levels: 0, 0.05, 0.1, 0.5, 2 or 10 mg/kg chlorpyrifos
EPA Scientific Advisory Panel, Washington, DC, February 15-18, 2011 13

Dose-response for AChEI in Adult Female Rats vs. PND 11 Male 

Pups (Corn Oil)

Adult Females PND 11 Male Pups 

0

20

40

60

80

100

120

140

0.05 0.1 0.5 2 5

CPF (mg/kg)

RBC ChE Activity (% Control)

*

*

0

20

40

60

80

100

120

140

0.05 0.1 0.5 2 10

CPF (mg/kg)

RBC ChE Activity (% Control)

*

*
EPA Scientific Advisory Panel, Washington, DC, February 15-18, 2011 14

CPF & CPF-Oxon vs. AChE Inhibition in PND 11 Pups

PND-11 Acute CPF (corn oil)

0.001 0.01 0.1 1 10

0.1

1

10

100

0

25

50

75

100

Chlorpyrifos (ng/g)

RBC ChE (% Control)

Brain ChE (% Control)

Chlorpyrifos-oxon (ng/g)

Dose Level (mg/kg bw)

Concentration (ng/g)

Percent of Control ChE Activity

Peripheral Tissue Inhibition when CPF-Oxon is Measurable.
EPA Scientific Advisory Panel, Washington, DC, February 15-18, 2011 15

Dietary Results in Adult Females

 Dose rate has some effects on response

 Diet had:

» Slightly greater RBC AChE inhibition than Corn Oil

» Slightly less brain AChE inhibition than Corn Oil

 Corn Oil modeling is more conservative

 Blood levels of CPF were decreased in diet
16

Benchmark Dose Analysis (Reiss et al.)

0 1 2 3 4 5

400 600 800 1000 1200 1400 1600

Dose (mg/kg)

Cholinesterase Activity
EPA Scientific Advisory Panel, Washington, DC, February 15-18, 2011 17

BMD Results

Subpopulati

on Dosing Vehicle

RBC AChe Brain AChe

BMD10 BMD20 BMD10

PND 11 

males Gavage Corn oil 0.45 (0.36) 0.94 (0.76) 2.1 (0.71)

PND11 

females Gavage Corn oil 0.53 (0.41) 1.1 (0.87) 2.0 (0.70)

Adult 

females Gavage Corn oil 0.65 (0.52) 1.4 (1.1) 2.9 (0.92)

Adult 

females Diet n/a 0.30 (0.26) 0.63 (0.55) 4.6 (2.7)

With Acute CPF, no significant differences between:

 Male and Female PND 11 Pups

 Pups and Adults

Significant Difference with Dietary Exposure
EPA Scientific Advisory Panel, Washington, DC, February 15-18, 2011 18

Using the CCA Data Set for Modeling 

• CCA data set is optimal for modeling purposes

– Data quality analysis conducted across studies (Reiss)

– Advantages of this data set:

– Data collected at the time of peak ChE inhibition

– Broad dose-response range examined

– Study supplies data in the range of the BMD

– Allows gavage and dietary comparisons

– Raw data were available

– Limitations of other data sets:

– Reliability of data sets not well known (variability? TOPI?)

– Not enough dose groups

– Different ages of animals were evaluated

– Lower BMD limit (AChEI10): 10-fold lower than lowest dose

– Provide info needed to match source-to-outcome models
EPA Scientific Advisory Panel, Washington, DC, February 15-18, 2011 19

CCA and Other Published CPF Data (PND 11-17)

Chlorpyrifos Brain AChE Inhibition (PND 11-17)

0.01 0.1 1 10

0

25

50

75

100

125

Marty & Andrus - CCA (PND 11)

CPF (mg/kg)

Brain ChE Activity (% Control)

Chlorpyrifos RBC AChE Inhibition (PND 11-17)

0.01 0.1 1 10

0

25

50

75

100

125

Marty & Andrus - CCA (PND 11)

CPF (mg/kg)

RBC ChE Activity (% Control)
EPA Scientific Advisory Panel, Washington, DC, February 15-18, 2011 20

CCA and Other Published CPF Data (PND 11-17)

Chlorpyrifos Brain AChE Inhibition (PND 11-17)

0.01 0.1 1 10

0

25

50

75

100

125

Marty & Andrus - CCA (PND 11)

Timchalk et al., 2006 (PND 17)

Moser et al., 1998 (PND 17)

CPF (mg/kg)

Brain ChE Activity (% Control)

Timchalk et al., 2006 (PND 12)

Chlorpyrifos RBC AChE Inhibition (PND 11-17)

0.01 0.1 1 10

0

25

50

75

100

125

Marty & Andrus - CCA (PND 11)

Timchalk et. al, 2006 (PND 17)

Moser et. al, 1998 (PND 17)

Timchalk et. al, 2006 (PND 12)

CPF (mg/kg)

RBC ChE Activity (% Control)
EPA Scientific Advisory Panel, Washington, DC, February 15-18, 2011 21

Applying CCA Data to CARES Model

 Study design and results drove some model variables:

– Model uses 8 individuals/dose (CCA study)

– Modeling acute exposures is relevant

– RBC AChEI is a conservative endpoint

– Corn oil gavage is useful for modeling