SCIENTIFIC ISSUES CONCE

EFFECTS OF C

REVIEW OF CHLORPYRIFOS BIO

RESEARCH AND INTERPRETIVE 

A

C

USEPA, OFFICE O

FIFRA SAP 
Outline

 Chlorpyrifos Biomarker Overview TCPy Biomonitoring Study Compa Biomonitoring and AChE Inhibition Risk-Based Biomonitoring Interpre

 Challenges Approaches

2
Chlorpyrifos Biomonitoring: 

Overview

3

 Chlorpyrifos biomarkers of exposure have been revie

(Barr & Angerer, 2006; Eaton et al., 2008)

 Most common chlorpyrifos biomarkers of exposure inc

metabolite TCPy and the non-specific DAP metabolite

 Although less common, chlorpyrifos has also been mon

blood and other biologic media (e.g., Columbia Coho

Biomarker/Analyte Biologic Matrix Populatio3,5,6-trichloro-2-pyridinol (TCPy)

Urine AllMeconium Pregnant 

Diethylphosphate (DEP) Urine AllDiethylthiophosphate (DETP) Urine All

Chlorpyrifos Blood All

Cord Blood Pregnant 

Common Chlorpyrifos Biomarkers of Exposure
Chlorpyrifos Biomonitoring: 

Urinary Biomarkers (DEP/DETP, 

4

Specificity DEP and DETP are non-specific biomarkers of multiple  TCPy is only metabolite of chlorpyrifos, chlorpyrifos-m Chlorpyrifos and other OP pesticides have been show

and TCPy in environmental media and food

 May overestimate chlorpyrifos exposure if assumed th

levels only attributable to chlorpyrifos

Variability & Pharmacokinetic Factors High variability observed in research suggests that sp

cannot accurately quantify pesticide exposure (Barr & 

 However, spot samples may be sufficient for assigning 

in epidemiology studies
Chlorpyrifos Biomonitoring: 

Blood Biomarkers

5

 Measurement of chlorpyrifos in blood provid

measure of internal chlorpyrifos dose

 Blood measurement is far less common in the 

urinary TCPy & DEP/DETP

 Requires more sensitive analytical method May place greater burden on study participants Blood concentrations represent more narrow per

relative to urinary biomarkers
TCPy Biomonitoring Study Comp

6

 Summarized TCPy biomarker levels in the ge

population and targeted study groups

 Intended to provide broad comparison of stu

Key Differences: Timing relative to residential phase-out Study populations (age, location, setting) Study design and research objectives

 Selected studies intended to represent major 

 NHANES  ORD observational studies Dow AgroSciences observational Studies (High-L Peer-Reviewed literature (2008 SAP; Eaton et a Children’s Health Cohorts 
TCPy Biomonitoring Study Su

7

Reference Study Population Type of Study PopulatioHill et al., 1995 General US Population 20-59 year olds NHANES General US Population 6-11 year olds, 20-59 y

olds

Egeghy et al. 2011 ORD - NHEXES, AZ 5-12 year olds 

ORD - MNCPES 3-13 year olds 

ORD - CTEPP, NC 2-5 year olds ORD - CTEPP, OH 2-5 year olds 

ORD - JAX 4-6 year olds 

ORD - CPPAES 2-4 year olds 

Lu et al., 2008 CPES-WA 3-11 year olds 

Lu et al., 2006 WA Children 3-11 year olds Alexander et al., 2006 Farm families 4-18 year olds, Adults 

Fenske et al., 2002 Farm Children 

≤ 6 years olds 

Curwin et al., 2005 Farmers Adults Curwin et al., 2007 Farm Families Children and Adults 

Berkowitz et al., 2003 Mt. Sinai Pregnant Female 

Whyatt et al., 2009 Columbia Pregnant Female Castorina et al., 2010 CHAMACOS Pregnant Female 

Eskenazi et al., 2007 CHAMACOS Pregnant Female 
8

0.1

1

10

100

1000

NHANES 1999-2000 (n=481)

NHANES 2001-2002 (n=573)

NHEXAS-AZ 1995-97, 5-12 years (n= 21)

MNCPES 1997, 3-13 years (n = 263)

CTEPP-NC 2000-2001, 2-5 years (n=129)

CTEPP-OH 2001, 2-5 years (n=123)

JAX 2001, 4-6 years (n = 9)

CPPAES 1999-2000, 2-4 (n=81)

CPES-WA, Summer 2003-2004 (n= 21) 

CPES-WA, Fall 2003-2004 (n= 21) 

CPES-WA, Winter 2003-2004 (n= 21) 

CPES-WA, Spring 2003-2004 (n= 21) 

Conventional Diet, Pre, 2003 (n=87)

CDC, 2009 (6-11 

years)

Egeghy et al, 2011 Lu et al., 2008 (3-11 years) Lu et al., 

TCPy Urinary Concentration (μg/L)

Reference 

Range

TCPy Biomonitoring Study Summary: Child Study Pop
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TCPy Biomonitoring Study Summary: Adult Study Pop

0.1

1

10

100

1000

NHANES III, 1988-1992 (n= 993)

NHANES 1999-2000 (n=832)

NHANES 2001-2002 (n=1,113)

Residential Low- Crack & Crevice/Spot (n=8)

Residential High- Turf Treatment (n=10)

Worker Low- Re-Entry (n=10)

Worker High- Mix/Load/Apply/Clean (n=15)

Nonfarmers, chlorpyrifos not sprayed (n= 45)

Farmers, chlorpyrifos not sprayed (n=45)

Farmers, chlorpyrifos self-sprayed (n=2)

Father, nonfarm (n=23)

Father, farm (n=24)

Mother, nonfarm (n=24)

Mother, farm (n=24)

Farm families—Applicators pre-appl. (n=34)

Farm families, Applicators post-appl (n=34

Farm families, Spouses pre-appl 

Farm families, Spouses post-appl (n=34)

Mother–child pair cohort 1998-2001 (n=365)

Prenatal 1 (n=95)

Hill et al, 1995; 

CDC, 2009

Dow AgroSciences Curwin et al, 2005 Curwin et al, 2007 Alexander et al., 2006 Berkowitz 

et al., 2003

TCPy Urinary Concentration (μg/L)

Reference 

Range

GM

GM

GM

GM

GM GM

GM

* NHANES data represents all survey participants aged 20-59 years old.

*
10

0.1

1

10

100

NHANES III, 1988-1992 (n= 993)

NHANES 1999-2000 (n=832)

NHANES 2001-2002 (n=1,113)

NHANES Subsample 1999-2002 (n=224)

Prenatal 1 (n=538)

Prenatal 2 (n=481)

Mother–child pair cohort 1998-2001 (n=365)

Prenatal 1 (n=95)

Prenatal 2 (n=83)

Prenatal 3 (n=54)

Hill et al, 1995; CDC, 2009 Castorina et al, 2010 Berkowitz 

et al., 2003

Whyatt et 

TCPy Urinary Concentration (μg/L)

Reference Range Maternal Co

*

TCPy Biomonitoring Study Summary: NHANES and Children’s H

* NHANES data represents all survey participants aged 20-59 years old.
TCPy Comparison Results: Findin

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 TCPy levels of mothers in the CHAMACOS, C

Mount Sinai cohorts varied in comparison witobserved in NHANES

 Median TCPy levels higher in CHAMACOS and Mo

(Eskanazi et al., 2007; Castorina et al., 2010) 

 Median TCPy levels lower in Columbia Cohort (Wh

However, in contrast to the other cohorts, data were during a time period of rapid decline in use of chlor
TCPy Biomonitoring and AChE In

12

AChE Monitoring Overview Red Blood Cell (RBC) AChE and Plasma BuCh

biomarkers of ChE activity

 RBC AChE inhibition considered an early mar

chlorpyrifos toxicity

 Plasma BuChE has been shown to be inhibited 

of exposure than RBC AChE

 As a result, Plasma BuChE may be a more sen

for monitoring potential physiological change
TCPy Biomonitoring and AChE In

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Previous Agency Findings Agency previously evaluated relationship be

RBC/Plasma AChE activity at 2008 SAP

 RBC/Plasma AChE inhibition observed in som

participants of registrant studies

 However, large variability in response to the 

dose of chlorpyrifos 

 As a result, difficult to draw definitive conclus

relationship between urinary TCPy levels and AChE inhibition.
TCPy Biomonitoring and AChE In

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Recent Study Findings (Post-2008 SAP) Two observations studies have been publishe

evaluate the relationship between TCPy on a adjusted basis and RBC/Plasma AChE inhibit

 Garabrant et al. (2009)

Evaluated group of exposed workers at a manufacturing facilities and a reference gmanufacturing workers

 Farahat et al. (2011)

Evaluated cotton field workers in Egypt fochlorpyrifos application to cotton fields
Comparison of Creatinine‐Adjusted Urinary TCPy levels (μg /g creatinine) reporte

and Farahat et al. (2011) with the General U.S. Population.

NR – Not reported

TCPy Biomonitoring and AChE In

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Population Group Time Period n

TCPy (μg 

Average Media

NHANES (CDC, 2009)General Population 1999‐2000 1,994 NR 1.5 

2001‐2002 2,508 NR 1.9 

Garabrant et al, 2009

Exposure Group

Fall 1999 53 NR 

Spring 2000 53 NR 

Turnaround 50 NR 

Fall 2000 52 NR 

Reference Group

Fall 1999 60 NR 

Spring 2000 57 NR Turnaround 57 NR 

Fall 2000 58 NR 

Farahat et al, 2011

Applicators 1‐ to 2‐ week

Exposure Period

14 6,437 

Technicians 12 184 

Engineers 12 157 
Garabrant et al. (2009) Results:

TCPy ÅÆAChE Relationship RBC AChE Plasma Bu

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Farahat et al. (2011) Results:

TCPy ÅÆAChE relationship 

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RBC AChE
TCPy Biomonitoring and AChE In

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Garabrant et al. (2009) No association observed between TCPy and RBC ACh Statistically significant association observed between 

BuChE levels above a cutpoint of 110 μg TCPy/g crea

Farahat et al. (2011)

 Identified an inflection point representing a significant 

AChE activity at TCPy levels greater than 3,148 

μg TC

 Reported that both studies observed similar inflection 

Plasma BuChE activity

 Garabrant et al. (2009) 110 μg TCPy/g crea Farahat et al. (2011) 114 μg TCPy/g crea
Risk-Based Biomonitoring Int

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NAS 2006 Biomarker Interpretation Fram

Qualitative 

Semi-Quantitative Qua
Biomonitoring Interpretation: 

ChallengesSpecificity Direct exposure to metabolites in the environment (TCP

Longitudinal Variability

 Difficult to relate single biomarker measurement Highly variable depending on sampling time/method, 

behavior, biomarker specificity, exposure routes, etc.

Toxicological Relevance

 Not directly comparable to regulatory screening valu Not always aligned with a toxicologically-relevant int

 TCPy formation reflective of detoxification and ca

chlorpyrifos-oxon 

 TCPy ÅÆAChE relationship unclear (at environme

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Biomonitoring Interpretation: 

Qualitative ApproachesEvaluate biomonitoring data without estimexposure Non-Comparative

 Urinary TCPy data for different time periods may 

temporal trends in exposure (e.g., pre- versus post-

 No link to chlorpyrifos, exposure routes, or health o

 Comparative

 Comparison to controlled human studies Primary comparison is to Nolan et al.,1984: measu

human volunteers (males) following a single oral dochlorpyrifos

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Biomonitoring Interpretation: 

Semi-Quantitative ApproachesEstimate biomarker levels likely associated exposure guidelines Biomonitoring Equivalents 

 Screening level approach for analyzing biomon Uses pharmacokinetic information and forward d

to convert an exposure guideline value (e.g., cRflevel that can be compared to biomonitoring da

 Considerations

 Biomarker Specificity: approach highly dependent on bi

 Direct exposures to TCPy take place

 Toxicological Relevance: biomarker must be aligned with a 

internal dose metric

 TCPy is reflective of detoxification of chlorpyrifos and can f

chlorpyrifos-oxon

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Biomonitoring Interpretation: 

Semi-Quantitative ApproachesEstimate chlorpyrifos exposures from biom Semi-Quantitative Reverse Dosimetry

 Utilizes creatinine-normalized urinary levels o

lifestage-changes may be incorporated

 Corrects for incomplete clearance of parent c

by biomarker 

 Considerations

 Estimates represent net absorbed doses witho

information

 Biomarker Specificity; assumes biomarker is d

chemical

 Direct exposures to TCPy take place

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Quantitatively evaluate risk using exposur

physiologically based pharmacokinetic (PB

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Biomonitoring Interpretation: Quantitative Approaches

Dow AgroSciences (2011). Source‐to‐Outcome Modeling Physiologically Based Pharmacokinetic/Pharma

Linked to a Dietary Exposure Model: Chlorpyrifos as a Case Study. FIFRA Scientific Advisory Panel, Washing
Biomonitoring Interpretation: 

Quantitative Approaches

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Chlorpyrifos PBPK Model Several groups working on chlorpyrifos PBPK  Timchalk et al. (2002) PBPK Model

 Refined since originally developed in 2002 Evaluated at 2011 FIFRA SAP Limitations

 Based on the “typical” adult human; does not incorpo

changes (e.g., pregnancy or childhood)

 Can only simulate oral exposures to chlorpyrifos with 

dietary exposures

 Several efforts to expand the model to other life-s

produce a model robust enough to inform human h
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Summary Several approaches can be used to analyze 

data associated with chlorpyrifos exposure

 Each approach has limitations that need to b

considered for any type of interpretation

 Agency soliciting recommendations from the S

regarding strengths and limitations of approalternative approaches not yet considered b
27 Questions