### 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 oil 1 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 oil 1 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 1 

### Dose 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 oil 1 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 1 

### Dose 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 54006008001000120014001600

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 BMD 10 BMD 20 BMD 10 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 (AChEI 10 

# ): 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