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SAP Minutes No. 2012-04

A Set of Scientific Issues Being Considered by the 

Environmental Protection Agency Regarding Chlorpyrifos Health 

Effects 

April 10 – 12, 2012 

FIFRA Scientific Advisory Panel Meeting

Held at the 

Environmental Protection Agency Conference Center

Arlington, VA
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NOTICE

These meeting minutes have been written as part of the activities of the Federal 

Insecticide, Fungicide, and Rodenticide Act (FIFRA), Scientific Advisory Panel (SAP). 

The meeting minutes represent the views and recommendations of the FIFRA SAP, not 

the United States Environmental Protection Agency (Agency). The content of the 

meeting minutes does not represent information approved or disseminated by the Agency. 

The meeting minutes have not been reviewed for approval by the Agency and, hence, the 

contents of these meeting minutes do not necessarily represent the views and policies of 

the Agency, nor of other agencies in the Executive Branch of the Federal Government, 

nor does mention of trade names or commercial products constitute a recommendation 

for use.

The FIFRA SAP is a Federal advisory committee operating in accordance with the 

Federal Advisory Committee Act and established under the provisions of FIFRA as 

amended by the Food Quality Protection Act (FQPA) of 1996. The FIFRA SAP provides 

advice, information, and recommendations to the Agency Administrator on pesticides and 

pesticide-related issues regarding the impact of regulatory actions on health and the 

environment. The Panel serves as the primary scientific peer review mechanism of the 

Environmental Protection Agency, Office of Pesticide Programs (OPP), and is structured 

to provide balanced expert assessment of pesticide and pesticide-related matters facing 

the Agency. FQPA Science Review Board members serve the FIFRA SAP on an ad hoc 

basis to assist in reviews conducted by the FIFRA SAP. Further information about 

FIFRA SAP reports and activities can be obtained from its website at 

http://www.epa.gov/scipoly/sap/ or the OPP Docket at (703) 305-5805. Interested 

persons are invited to contact Fred Jenkins, Jr., Ph.D., SAP Designated Federal Official, 

via e-mail at jenkins.fred@epa.gov.

In preparing these meeting minutes, the Panel carefully considered all information 

provided and presented by EPA, as well as information presented by public commenters.
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TABLE OF CONTENTS

PARTICIPANTS ...................................................................................................................6

INTRODUCTION .................................................................................................................10

PUBLIC COMMENTS..........................................................................................................11

SUMMARY OF PANEL DISCUSSION AND RECOMMENDATIONS...........................12

DETAILED PANEL DELIBERATIONS AND RESPONSE TO CHARGE.......................27

REFERENCES ......................................................................................................................77

APPENDIXES.......................................................................................88

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Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) Scientific Advisory 

Panel (SAP)

Chlorpyrifos Health Effects

April 10 – 12, 2012 

PARTICIPANTS

FIFRA SAP Chair

Kenneth Portier, Ph.D.

Program Director, Statistics

American Cancer Society

National Home Office

Atlanta, GA 

Designated Federal Official

Fred Jenkins, Jr., Ph.D.

US Environmental Protection Agency

Office of Science Coordination & Policy

FIFRA Scientific Advisory Panel

EPA East Building, MC 7201M

1200 Pennsylvania Avenue, NW 

Washington, DC 20460

Phone (202) 564-3327

Fax (202) 564-8382

jenkins.fred@epa.gov

FIFRA Scientific Advisory Panel Members

Stephen Klaine, Ph.D.

Professor

Environmental Toxicology & Biological Sciences

Clemson Institute of Environmental Toxicology

Pendleton, SC 

Carey Pope, Ph.D.

Professor

Head & Sitlington Chair of Toxicology

Department of Physiological Sciences

Center for Veterinary Health Sciences

Oklahoma State University College of Veterinary Medicine

Stillwater, OK
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Martha Sandy, Ph.D.

Senior Toxicologist and Chief

Cancer Toxicology and Epidemiology Section

Reproductive and Cancer Hazard Assessment Branch

California Environmental Protection Agency

Oakland, CA

FQPA Science Review Board Members

Michael Aschner, Ph.D.

Director

Division of Pediatric Toxicology

Medical Center

Vanderbilt University Medical Center

Nashville, TN

Brian Buckley, Ph.D.

Executive Director of Laboratories

Environmental & Occupational Health Sciences Institute

Rutgers University

Piscataway, NJ 

Russell Carr, Ph.D.

Associate Professor

College of Veterinary Medicine 

Center for Environmental Health Sciences

Mississippi State University

Mississippi State, MS 

Abby Collier, Ph.D.

Assistant Professor of Pharmacology

John A. Burns School of Medicine

University of Hawaii at Manoa, Kaka'ako Campus

Honolulu, HI 

Ellen Gold, Ph.D. 

Professor and Chair

Department of Public Health Sciences

University of California-Davis

Davis, CA 
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Gaylia Jean Harry, Ph.D.

Biologist, Head of Neurotoxicology Group 

Division of Intramural Research

National Institute of Environmental Health Sciences

Research Triangle Park, NC 

Stella Koutros, Ph.D.

Research Fellow

Division of Cancer Epidemiology and Genetics 

Epidemiology Branch

National Institutes of Health 

Rockville, MD 

Chensheng (Alex) Lu, Ph.D.

Associate Professor 

Department of Environmental Health

Exposure, Epidemiology and Risk Program

Harvard School of Public Health 

Boston, MA 

Bette Meek, Ph.D. 

Associate Director Chemical Risk Assessment

McLaughlin Institute for Population Health Risk Assessment

University of Ottawa

Ottawa, Canada

William Popendorf, Ph.D.

Professor, Emeritus

Department of Biology

Utah State University

Logan, UT 

Barry Ryan, Ph.D.

Professor

Exposure Assessment & Environmental Chemistry

Department of Environmental and Occupational Health

Rollins School of Public Health

Emory University

Atlanta, GA 

Alvin Terry, Ph.D.

Professor

Pharmacology and Toxicology

Georgia Health Sciences University

Augusta, GA 
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John Vena, Ph.D.

UGA Foundation Professor & Department Head 

B.S. Miller Hall Room 105

Health Sciences Campus

University of Georgia

Athens, GA

Charles Vorhees, Ph.D.

Professor

Department of Pediatrics, Div. of Neurology

College of Medicine 

University of Cincinnati and

Cincinnati Children’s Hospital

Cincinnati, OH 

Heather Young, Ph.D.

Associate Professor

Associate Chair

Department of Epidemiology & Biostatistics

School of Public Health & Health Services

George Washington University

Washington, DC 
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INTRODUCTION

Chlorpyrifos (0,0-diethyl--3,5,6-trichloro -2-pyridyl phosphorothioate) is a broadspectrum, chlorinated organophosphate (OP) insecticide. Like other OPs, chlorpyrifos 

binds to and phosphorylates the enzyme acetylcholinesterase (AChE) in both the central 

(brain) and peripheral nervous systems. This can lead to accumulation of acetylcholine 

and, ultimately, at sufficiently high doses, to clinical signs of toxicity. In 2011, the 

Agency released a preliminary human health risk assessment for chlorpyrifos. The focus 

of this assessment was on the cholinesterase (ChE) inhibiting potential of chlorpyrifos. 

Consistent with this focus, EPA evaluated the extensive database of ChE data for 

multiple lifestages and selected points of departure (PoDs) based on consideration of all 

quality and reliable data. There is, however, a growing body of literature with laboratory 

animals (rats and mice) indicating that gestational and/or early postnatal exposure to 

chlorpyrifos may cause persistent effects into adulthood. The results of both in vivo and 

in vitro studies on chlorpyrifos have led some research groups to propose that changes in 

brain connectivity and/or neurochemistry may underlie these changes into adulthood. In 

addition, there are epidemiology studies evaluating pre- and post-natal chlorpyrifos or 

other OP exposure in mother-infant pairs that have reported associations with birth 

outcomes, childhood neurobehavioral and neurodevelopment outcomes in the offspring 

when evaluated in neonates, infants, and young children. 

In 2008, the FIFRA Scientific Advisory Panel (SAP) reviewed a draft science issue paper 

on the human health effects of chlorpyrifos which provided a preliminary review of the 

scientific literature on experimental toxicology and epidemiology studies available at that 

time. In 2010, the Agency developed a draft “Framework for Incorporating Human 

Epidemiologic & Incident Data in Health Risk Assessment” which provides the 

conceptual foundation for evaluating multiple lines of scientific evidence in the context 

of the understanding of the adverse outcome pathway (or mode of action). This draft 

framework uses modified Bradford Hill Criteria to evaluate the sufficiency of evidence to 

establish key events within a mode of action(s) and explicitly considers such concepts as 

strength, consistency, dose response, temporal concordance and biological plausibility. 

Since the 2008 SAP on chlorpyrifos, the Agency has performed further analyses on the 

existing and new epidemiology results in mothers and children, available biomonitoring 

data, and experimental toxicology studies evaluating proposed adverse outcome pathways 

in the context of human health risk assessment. Specifically, the Agency is evaluating 

available literature on the potential for chlorpyrifos to cause long term adverse effects 

from early life exposure, in vivo and in vitro studies evaluating mechanistic aspects of 

chlorpyrifos, and the potential for adverse effects below doses established from ChE 

inhibition that are used for regulatory purposes. At this time, the Agency is working 

towards a weight of evidence evaluation integrating the epidemiology studies with the 

experimental toxicology studies for the neurodevelopmental outcomes. This analysis is 

complex and multifaceted as it involves different lines of scientific evidence (i.e., in vivo

& in vitro experimental toxicology studies, explicit consideration of adverse outcome 

pathways, exposure, epidemiology, and biomonitoring data). As such, the Agency 

believes that peer review on the status of the current analysis is important. 
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Opening remarks at the meeting were provided by:

Steven Bradbury, Ph.D., Director, Office of Pesticide Programs (OPP), EPA; Karen 

Whitby, Ph.D., Acting Director, Health Effects Division, OPP, EPA; Anna Lowit, Ph.D., 

OPP, EPA; William R. Mundy, Ph.D., Office of Research and Development (ORD), 

EPA; Ginger Moser, Ph.D., DABT, Fellow ATS, ORD, EPA; Carol H. Christensen, 

Ph.D., MPH, OPP, EPA; Lieutenant Aaron Niman, US Public Health Service, OPP EPA

Public Comments

Public comments were provided by: 

Dow AgroSciences 

Julie E. Goodman Ph.D. and Lorenz Rhomberg, Ph.D. of Gradient on behalf of Dow 

Agrosciences

Abby Li, Ph.D. of Exponent on behalf of Dow Agrosciences

Jennifer Sass, Ph.D. of Natural Resources Defense Council (NRDC)

Dale Hattis, Ph.D. of Clark University on behalf of himself
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Summary of Panel Discussion and Recommendations

Charge 1: Mode of action/adverse outcome pathway: Acetylcholinesterase (AChE) 

inhibition

Question 1.0 

It is well established that AChE inhibition is the primary mode of action/adverse outcome 

pathway for OPs, like chlorpyrifos. Because AChE inhibition is the initiating event for 

this mode of action/adverse outcome pathway, using AChE inhibition as a regulatory 

endpoint is protective of downstream cholinergic effects. Moreover, historically, given 

the sensitivity of AChE inhibition data for OPs, these data have been considered to be 

protective of other potential toxicities and/or modes of action for OPs. In 2008, the 

Agency performed a comprehensive review of the available AChE data from multiple 

lifestages. This review has been supplemented with the newest studies. Consistent with 

the recommendations from the 2008 SAP, the Agency believes that AChE data remain 

the most robust dose-response data for deriving points of departure in in vivo

experimental toxicology studies with laboratory animals. Please comment on the 

Agency’s preliminary conclusion that AChE data remain the most robust source of data 

for deriving points of departure for chlorpyrifos. Please include a discussion of the 

strengths and uncertainties of this preliminary conclusion.

The Panel concurs with the Agency’s position that AChE data continue to be the 

strongest resource of data for deriving points of departure for chlorpyrifos. The Panel’s 

conclusion is based on the premise that all studies reporting neurobehavioral changes 

following in vivo prenatal or postnatal exposures to chlorpyrifos have been accompanied 

by AChE inhibition when measured at an appropriate time following administration of 

chlorpyrifos. 

The Panel additionally notes that studies evaluating neurodevelopmental effects entailed 

experimental designs that do not permit an efficient means of determining a point of 

departure for chlorpyrifos. Thus, just as the in the 2008 SAP, this Panel advises that the 

Agency continue to use AChE data at the most sensitive lifestages for dose-response 

analysis and deriving points of departure. Also in keeping with the 2008 SAP, this Panel 

expresses concern about the use of Dimethyl Sulfoxide (DMSO) as a vehicle because of 

its intrinsic toxicity, its potential influence on absorption and interaction with 

chlorpyrifos, and the impact of this interaction on the developing organism. 
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Charge 2.0: Mode(s) of action/adverse outcome pathway(s): Plausible pathways 

leading to potential neurodevelopmental outcomes

Question 2.1 

As discussed in Section 3.2.1, although there are numerous mechanistic studies in the 

scientific literature, the research on different hypotheses does not provide sufficient data 

to establish causal linkages among different levels of biological organization to show 

how effects lead to adversity. As such, a mode of action or adverse outcome pathway 

leading to effects on the developing brain cannot be established at this time. Moreover, 

although multiple biologically plausible hypotheses are being pursued by researchers, 

based on the current state of the science, no one pathway has sufficient data to be 

considered more credible than the others. Please comment on the Agency’s preliminary 

conclusion that although there are multiple biologically plausible hypotheses being 

evaluated by research scientists, the mechanistic experimental toxicology data do not yet 

support a coherent set of key events in a mode of action/adverse outcome pathway.

The Panel agrees with the Agency’s conclusion that based on the current state of the 

science, no one pathway has sufficient data to be considered more credible than the 

others with respect to a causal link between chlorpyrifos exposure and

neurodevelopmental outcome.

In regard to the Agency’s case study demonstrating domoic acid’s adverse outcome 

pathway, the Panel contends that the linear connections of the pathway demonstrated in 

this case study appear likely to be rare and unique. They also note that it is more likely 

that other such neurotoxicological pathways are non-linear. Expectations of a linear

pathway specifically in the case of chlorpyrifos may be artificially elevated and 

potentially unrealistic for risk assessment.

Question 2.2 

Although a mode of action/adverse outcome pathway has not been established, 

qualitatively, the growing body of mechanistic studies does demonstrate that chlorpyrifos

and/or its oxon are biologically active on a number of processes that affect the developing 

brain. Some mechanistic studies provide evidence of possible effects which are similarly 

sensitive or more sensitive than AChE inhibition (e.g., neurite outgrowth, binding to 

muscarinic receptors, axonal transport; serotonergic nervous system development). Some 

of these comparisons must be considered with caution since the amount of change in the 

in vitro systems required to elicit an adverse effect in vivo is unknown. Moreover, 

extrapolation from in vitro perturbations to in vivo effects has not been established, which 

introduces additional uncertainties. Given the doses/concentrations evaluated in the in 

vitro and in vivo mechanism studies, please comment on the degree to which these studies 

suggest that endpoints relevant to evaluating potential neurodevelopmental outcomes 

may or may not be more sensitive than AChE inhibition. Please include in your 

comments a discussion of the strengths and uncertainties. Please also include in your 

comments a discussion of the scientific understanding of dose-response relationships for 

biological perturbations and the magnitude, frequency and/or duration of such 

perturbations that can lead to adverse effects at higher levels of biological organization 
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to support characterization of the likelihood of adverse outcomes in a human health risk 

assessment (as articulated in NRC 2007).

The Panel concurs with the Agency that caution should be applied in interpreting the invivo significance of the changes observed across the various in vitro studies. Several 

uncertainties and limitations are associated with the translation of in vitro study results to 

in vivo effects. The inherent complexity of the nervous system presents significant 

challenges to accomplishing this translation. An additional example of uncertainty is that 

cells that are isolated in culture within an in vitro experiment may be affected differently 

than they would if they were within their in vivo environment.

The Panel recommends continued literature review and analysis of published data with 

the goal of developing additional hypotheses linking in vitro findings to in vivo relevance. 

As an example, the analytical studies of the Lockridge group indicating that chlorpyrifos

oxon can covalently modify key cytoskeletal proteins such as tubulin and motor proteins 

like kinesin, provide information that can contribute to the interpretation of findings of 

alterations in neurite outgrowth and axonal transport, respectively.

The Panel also recommends that the Agency consider other areas that might be added to 

the review such as the effect of chlorpyrifos on neurotrophins (growth factors). Several 

researchers have found early evidence of the potential for these effects (Pope et al., 1995;

Slotkin et al., 2007; Betancourt and Carr, 2004). 

The Panel cautions the Agency concerning their examination of the dose-response

relationships. They particularly note that when evaluating these relationships, 

pharmocodynamic (PD) analyses should not be uncoupled from pharmacokinetic (PK)

models given that PK differences can affect active site concentrations and hence, PD 

effects. Thus, PK models can significantly affect the magnitude and duration of an 

effect.

Lastly, the Panel raises concerns about the equivalency of developmental stages between

ages of rodents to human. These are not well defined with regard to cell type

compositions, brain region, cellular architecture, and physiological or biochemical 

processes. This lack of equivalence further limits the translation to the in vivo situation 

and the ability to provide a quantitative dose-response relationship that can be compared 

to that for AChE inhibition.

.
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Charge 3.0 Neurodevelopmental data from laboratory animals

Question 3.1 

As discussed in Section 3.2.2, the experimental toxicology data in laboratory rodents 

show neurobehavioral effects following developmental exposure with changes in a 

number of neurological domains. In 2008, the SAP agreed to this preliminary 

conclusion, and the nine additional studies available since 2008 add further support. 

Please comment on the degree to which these studies show changes in a number of 

neurological domains and support the qualitative conclusion that chlorpyrifos exposure 

during gestation and/or early post-natal period may result in long-term adverse effects 

on the developing nervous system. What evidence does and does not support this 

conclusion? Please also include in your comments a discussion of the strengths and 

uncertainties. Please also include in your comments a discussion of the scientific 

understanding of dose-response relationships for biological perturbations and the 

magnitude, frequency and/or duration of such perturbations that are can lead to adverse 

effects at higher levels of biological organization to support characterization of the 

likelihood of adverse outcomes in a human health risk assessment (as articulated in NRC 

2007). 

The Panel agrees with the 2008 SAP conclusions that developmental neurobehavioral 

studies demonstrate adverse effects from chlorpyrifos exposure. However, the number of 

available neurobehavioral studies is limited leading to caution concerning this finding. 

Also many of these studies are statistically under-powered and prone to Type I errors and 

should be discounted in formulating the weight of evidence for or against 

neurobehavioral effects from developmental exposure to chlorpyrifos. The Panel also 

expressed caution with the significance of some of the experimental neurotoxicological 

outcomes that have not been validated. These included the tests of anxiety, depression, 

and social interactions. The Panel recommends these experimental outcomes be regarded 

as exploratory, and hypothesis-generating, as opposed to being evidence of toxicity. The 

lack of specificity in the direction of the neurobehavioral dose response findings is a

problematic issue. 

Despite the issues raised by the Panel about these studies, the overall evidence across 

these studies is persuasive in indicating that there are enduring effects on the Central 

Nervous System (CNS) from chlorpyrifos exposure at or above 1.0 mg/kg. The Panel 

recommends that future neurodevelopmental studies be focused on testing chlorpyrifos

levels below 1.0 mg/kg/day and that these studies be geared towards identifying the 

correct testing paradigm and neural substrates for detecting possible effects. The Panel 

advises that cross-laboratory or collaborative studies may provide systematic comparison 

of the effects of chlorpyrifos on neurodevelopmental domains using unified exposure 

periods, dosing, age of testing, and methods, combined with urinary analysis of 

chlorpyrifos’ metabolites, and accurate assessments of AChE inhibition.
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Question 3.2 

The dose-response data in the in vivo experimental neurodevelopmental toxicity studies 

are not amenable to empirical dose-response modeling as many studies use only one or 

two doses, and in some cases the lower dose, but not higher dose level, produced 

significant effects. Many studies report effects at a dose of 1 mg/kg/d-- a dose that 

produces some amount of brain ChE inhibition when given directly to the pups postnatally, but may or may not alter fetal brain ChE activity when given to the dams 

gestationally. One study (Braquenier et al., 2010) using lower doses, administered to the 

dam on GD15-LD14, reported a NOEL of 0.2 mg/kg/d. Comparing the NOEL of 0.2 

mg/kg/d to a repeated dosing AChE inhibition BMDL10 of 0.03 mg/kg/d suggests that 

AChE inhibition is a sensitive and protective endpoint. 

a. Please comment on the scientific quality and robustness of the animal 

neurodevelopmental toxicity studies.

The Panel notes that the quality of these studies vary. The “high quality” studies use

multiple doses, adequate sample sizes, controls for litter effects, sound behavioral 

methods, and appropriate statistical methods to analyze the data. Since all these studies 

demonstrate long-term neurobehavioral effects, the data generated by them (especially 

the findings that occurred at doses > 1 mg/kg of chlorpyrifos) can be considered robust. 

The Panel has some concerns even with the high quality studies. For example, the rat 

strains used in some of the studies are considered by the Panel to be less than preferable. 

The Panel advises that studies that are considered to support regulatory decisions should 

be those that use a mainstream rat strain such as Sprague-Dawley from Charles River or 

Harlan because much more is known about their behavioral characteristics and they do 

not perform at the extremes of the distribution.

 

Despite the concerns expressed about the studies, the Panel concurs with the conclusions 

of the 2008 SAP findings and the EPA White Paper background document, and 

concludes that the collective weight of evidence from these studies demonstrate that it is

probable that there are significant long-term adverse effects from chlorpyrifos exposure.

 

b. Please comment on the degree to which studies that measured AChE inhibition 

and those that measured neurodevelopmental outcomes can be integrated to 

evaluate whether points of departure based on 10% AChE inhibition provide 

more sensitive endpoints than endpoints measured in the experimental 

neurodevelopmental studies (as reviewed in Section 3.2.2). Please include in 

your comments a consideration of the strengths and uncertainties associated with 

this assessment. 

The Panel concludes that since AChE inhibition recovers quickly the data are 

insufficiently refined to allow for a linkage between the mode of action and the 

neurodevelopmental effects (acute vs. chronic, respectively). They note that since the 

mode of action of these effects is not established and cannot be presumed to be related to 

AChE inhibition, these studies do not exclude the possibility that other mechanisms may 
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be involved, especially concerning long-term effects that may be unmasked at later lifestages. Additionally, since the neurodevelopmental effects may be independent of AChE 

inhibition, the Agency should consider whether AChE inhibition represents the critical 

marker for derivation of points of departure for chronic studies.

Charge 4.0 Epidemiology Regarding Children’s Health

Question 4.1 

Section 4.0 and Appendices 5 and 6 provide the Agency’s review of the available 

epidemiology studies from the Columbia Mothers and Newborn study, the Mt. Sinai 

Child Development study, and the Center for Health Assessment of Mothers and 

Children of Salinas Valley (CHAMACOS) study. Consistent with the 2008 SAP 

recommendations, the Agency has considered information offered from each of the three 

cohort investigations; however EPA acknowledges the primacy of the Columbia cohort 

data for the purposes of informing risk assessment because researchers measured 

chlorpyrifos parent compound directly in this study. Please comment on the sufficiency, 

clarity, and quality of the Agency’s epidemiology review as contained in Section 4.0 and 

Appendices 5 and 6 of the draft issue paper with respect to identifying the major 

strengths and limitations of each study.

The Panel considers the Agency’s epidemiology review to be very clearly written,

accurate, and to generally provide a very thorough review of the epidemiology literature. 

In addition, the Panel commends the Agency for putting their epidemiology review in the 

context of the modified Bradford Hill criteria, as recommended by the 2010 SAP. The 

Panel believes that the epidemiology review appropriately concludes that the studies 

show some consistent associations relating exposure measures to abnormal reflexes in the 

newborn, pervasive development disorder at 24 or 36 months, mental development at 7-9 

years, and attention and behavior problems at 3 and 5 years of age, in addition to less 

consistent results for reduced mental and psychomotor development at 12 and 24 months. 

Inconsistent results are found for associations between exposure and measures of fetal 

growth. 

The Panel views the Agency’s epidemiology review as an excellent description of the

strengths and limitations of the studies conducted to examine the relation of chlorpyrifos 

to children’s growth and neurodevelopment. It is noted that studies of this nature are 

logistically difficult to implement because of the potentially large burden imposed on the 

study participants in terms of time and effort, often with little or no specific benefit to the 

participants.

Although in agreement with the Agency that chlorpyrifos could have played a role in the 

neurodevelopmental outcomes observed in the Columbia cohort, some panel members 

expressed concern about associating the observed deficits in neurodevelopmental 

outcomes in children with a single chemical. This is because the studies entail a multichemical exposure spanning a multi-year period that encompasses an important period of 

sequential developmental processes necessary for brain maturation. Thus, panel 

members caution that it is very difficult to attribute the independent physiological effects 

to a single chemical in this type of multi-chemical exposure scenario. An additional 
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concern raised by the Panel is the modest sample sizes of the studies. They deem 

inadequate sample size as one of the most important limitations of these studies. 

Question 4.2 

Similar to the initial conclusions from 2008, the Agency has preliminarily concluded that, 

qualitatively, chlorpyrifos likely played a role in the neurodevelopmental outcomes 

reported in the epidemiologic studies, and that information available since 2008, 

including both new etiologic investigations as well as epidemiologic methods papers, 

strengthens this conclusion. Please comment on the Agency’s preliminary, qualitative 

conclusion that chlorpyrifos likely played a role in the neurodevelopmental outcomes 

observed in the epidemiologic studies. Please include in your comments a discussion of 

the strengths and uncertainties associated with this preliminary conclusion.

Overall, the Panel concurs with the 2008 SAP and the Agency in concluding that 

chlorpyrifos likely plays a role in impacting the neurodevelopmental outcomes examined 

in the three cohort studies. Although exposures to other AChE-inhibiting compounds 

cannot be excluded as contributing to neurodevelopmental (adverse) outcomes, the 

potential combination and/or additive effects of these compounds does not rule out the 

role of chlorpyrifos. As a result, it cannot be concluded that chlorpyrifos is the only 

contributor to the observed outcomes.

The strengths of the three studies support the Panel’s conclusion. There are nine 

strengths identified by the Panel which are discussed in the detailed response section of 

this report. Some of the strengths noted include, but are not limited to: 1) the longitudinal 

designs which permit clear indications of the temporal relation of chlorpyrifos exposure 

to adverse neurodevelopmental outcomes, 2) the inclusion of biomarkers of exposure as 

well as self reported exposure, and 3) the relative consistency of findings in different 

populations while using similar standardized exposure and outcome measures. 

Question 4.3 

As discussed in Question 2.0, a mode of action/adverse outcome pathway has not yet 

been fully elucidated for the potential neurodevelopmental outcomes as a result of 

prenatal chlorpyrifos exposure. Although this does not undermine the qualitative 

interpretation of these studies, and the preliminarily conclusion stated above (Question 

4.2), the identification of the dose-response for neurodevelopmental effects based on 

mode of action is not possible. Further, given the urine and cord blood sampling 

frequency in the study there is a large degree of uncertainty in estimating absolute 

exposure-response relationships, as opposed to establishing relative exposure groups for 

evaluating associations. With respect to dose-response, critical durations of exposure, 

and windows of susceptibility are unknown. In 2008, the SAP cautioned against using 

the Columbia cohort data for deriving a point of departure due, in part, to only measuring 

biomarkers (3rd trimester maternal, cord blood, meconium) at one point in time, and 

because they cannot exclude possibility that the effects seen were due to chlorpyrifos in 

combination with other pesticides. In 2008, the SAP advised against using data from the 

epidemiology studies (including the Columbia Mothers and Newborn study which 

measured chlorpyrifos directly) for deriving a point of departure due to limitations of the 
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exposure assessment in these epidemiology studies for the purpose of risk assessment, 

e.g., lack of repeated exposure estimates to ascertain more specifically the variability and 

periodicity of exposure over time (i.e., predominant use of one-time exposure estimate). 

Question 4.3

a. Due to the limitations of exposure assessment performed in the epidemiologic 

investigations for the purposes of quantitative risk assessment, the Agency has 

concluded that the epidemiologic data are not sufficient for deriving points of 

departure for quantitative risk assessment. The Agency proposes that AChE 

inhibition data from laboratory animals remain the most appropriate data to use for 

dose-response modeling and the derivation of points of departure. Please comment 

on the scientific evidence that does and does not support this conclusion, as well as 

the strengths and limitations of the evidence.

The Panel recognizes the limitations of estimating chlorpyrifos exposures based on the 

exposure measures collected in the three longitudinal children’s cohort studies (i.e., the 

Columbia study, the Mt. Sinai study, and the CHAMACOS study). Consequently, the

Panel largely concurs with EPA that the data generated from these studies alone are not 

adequate enough to obtain a point of departure (POD) for the purposes of quantitative 

risk assessment. 

However, despite the limitations of the exposure assessment of these three cohort studies, 

the Panel recognizes the significance of these data, and advises the Agency to explore 

additional ways of using these studies, especially the data from the Columbia study, to 

inform the dose response assessment of chlorpyrifos. This recommendation is 

underscored by the Panel’s concerns regarding the proposed use of dose-response data on 

AChE inhibition in laboratory animals to derive points of departure for the chlorpyrifos

risk assessment. The Panel notes that multiple lines of evidence suggest chlorpyrifos can 

affect neurodevelopment at levels lower than those associated with AChE inhibition. 

These multiple lines of evidence include: 1) the collective findings of the three cohort 

studies, 2) in vivo animal neurodevelopmental studies summarized in the Draft Issue 

Paper that report differential expression of oxidative stress genes and altered serotonergic 

tone in rat brain associated with early life chlorpyrifos exposures at doses below which 

acetylcholinesterase inhibition was detected, and 3) several in vitro mechanistic studies 

reported in the Draft Issue Paper demonstrating interference with neurite and axon 

outgrowth, reduced axonal transport, and increased oxidative stress in a variety of cell 

types exposed to chlorpyrifos concentrations that do not or are not expected to inhibit 

AChE. As noted in the response to Charge Question 2.2, additional evidence comes from 

studies not included in the Draft Issue Paper, reporting effects of chlorpyrifos on nerve 

growth factors and mitochondrial morphology at concentrations below which 

acetylcholinesterase inhibition is expected.

The Panel recommends that the Agency consider developing a functional physiologically 

based pharmacokinetic (PBPK) model for chlorpyrifos for pregnancy and the prenatal 

lifestage. The PBPK model could be utilized for additional dose-response analyses to 

further characterization of the dose estimates in the epidemiology studies. This model 
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could also become important in the future if the Agency decides to transition from using 

AChE inhibition to another outcome.

The Panel suggests additional research that may answer the key question of whether 

chlorpyrifos induces neurodevelopmental effects in humans at doses that do not cause 

AChE inhibition. More specifically the Panel suggests conducting studies that test 

whether red blood cell or brain AChE inhibition occurs as a result of chlorpyrifos

concentrations in cord blood being associated with neurodevelopmental effects. 

Additional Panel concerns about the use of AChE inhibition dose-response data to protect 

against neurodevelopmental effects is based on the potential for AChE inhibition and 

adverse neurodevelopmental effects to be two separate events. AChE inhibition is the 

result of an acute exposure scenario and neurodevelopmental effects likely being caused 

by chronic low level exposure to chlorpyrifos in utero. 

Lastly, the Panel cautions the Agency dose-response data for AChE inhibition by 

chlorpyrifos in the pregnant rat may not be predictive of AChE inhibition in the human 

fetus given known interspecies differences in CYP450 isoforms, substrate affinities, fetal 

expression levels, and degree of polymorphism. 

Question 4.3

b. The Agency does, however, believe that the epidemiologic data are useful to 

informing other key aspects of the chlorpyrifos risk assessment including hazard 

characterization, exposure characterization, and quantitative uncertainty 

characterization and analysis. Please suggest approaches/analyses for potentially 

using the epidemiology data in different parts of the chlorpyrifos risk assessment 

including those noted above. (Note: Some of these may also be covered in 

Question 5.4 below.) 

The Panel agrees that the epidemiologic data are useful to inform key aspects of the 

chlorpyrifos risk assessment including exposure characterization, hazard characterization, 

and quantitative uncertainty characterization and analysis.

In regard to the exposure characterization, the Panel notes that environmental monitoring 

and biomonitoring data in these epidemiology studies can contribute to the overall 

database on estimation of exposure, including (particularly) population variability. These

data can also enable the Agency to characterize exposure levels over time among diverse 

populations including production workers, agricultural workers, individuals exposed via 

residential use, general population, etc.

With respect to toxicological hazard characterization, the Panel suggests that these data 

can serve as the key source of support for the identification of prenatal exposures to 

chlorpyrifos as a cause of neurodevelopmental effects in humans. These data have many 

strengths. First there are consistencies in the findings of neurodevelopmental effects 

across the three cohort studies. Second, the levels of chlorpyrifos exposure experienced 

in these cohorts are comparable and well-characterized, being based on biomonitoring 
21

(blood and urine measurements of chlorpyrifos, metabolites, etc.) and environmental 

monitoring measures (e.g., personal air monitoring in the Columbia study) and having 

similar levels observed in data collected from other studies of the general U.S. 

population (e.g., National Health and Nutrition Examination Survey (NHANES), for 

similar time periods (i.e., pre- and post-cancellation of residential uses).

In reference to the epidemiology data being used to support the quantitative uncertainty 

characterization and analysis, the Panel agrees with the 2008 SAP suggestion that at a

minimum the Agency should use available data from these studies to at least “bound” 

reference doses developed on the basis of animal data. Given the potential significance 

of the epidemiological findings, the Panel advises the Agency to consider the potential 

impact of factors of study design and interpretation to bound the dose-response 

relationship from the human studies. For example, it would be useful to consider 

systematically (and at least semi-quantitatively) the potential impact of exposure 

measurement error, outcome ascertainment, confounding variables and statistical analysis

methodology on the reported dose-response analysis.

To increase the confidence in the selected point of departure, the Panel recommends a 

simple experimental protocol to determine whether chlorpyrifos levels measured in the 

cord blood in the Columbia study inhibit either red blood cells or brain AChE. The 

results of such an exercise could potentially contribute to the essential question of 

whether or not a causal association between chlorpyrifos exposure and 

neurodevelopmental effects in the absence of AChE inhibition is plausible for humans. 

Charge 5.0 Exposure Profile & Biomonitoring Research

Question 5.1 

a. Section 5 of the draft issue paper presents an overview of the principal chlorpyrifos

biomarkers and a comparison of biomonitoring studies that measured urinary TCPy 

levels in a range of study populations involving both the general population and 

potentially vulnerable populations, including children, workers, and farm families.

Please comment on the degree to which the Agency identified the primary 

chlorpyrifos biomarkers of exposure, appropriately discussed the strengths and 

limitations of such biomarkers, and how the strengths and limitations affect the 

interpretation of the chlorpyrifos biomonitoring data. 

The Panel notes that the Agency was thorough in its coverage of the literature on 

biomarkers of chlorpyrifos exposure. The Panel recommends that chlorpyrifos in blood 

be the first choice for a biomarker, particularly because of its specificity, the availability 

of standard methods for measuring it, the relevance of its concentration levels, and the 

number of laboratories that are capable of conducting the measurements. However, the 

Panel acknowledges that this is a most challenging assay, and has been used in only a 

small percentage of published research. The second biomarker of choice is 3,5,6-

trichloro-2-pyridinol (TCPy) followed by diethylthiophosphate/diethylphosphate

(DETP/DEP), both measured in urine. These have roughly the same equivalence and 
22

neither is equivalent to measuring chlorpyrifos directly in blood because of the frequent 

presence of these environmental degradates of the active ingredient. Total DAPs (as 

DMP and DEP) are not selective enough to be a useful biomarker for chlorpyrifos

although DAPs may be more appropriate in a global risk calculation model when all 

AChE inhibiting chemicals are considered together when evaluating risk.

The Panel suggests that more importance should be afforded to the direct intake of TCPy

which is mainly present in foods. A growing body of research developing since the 

1990s, has established the significance of this factor.

The Panel also acknowledges the capability of measuring AChE and BuChE as 

biomarkers of exposure. However, inhibitions of these enzymes are even less specific 

than DAPs, although they are more indicative of potential health risk. Unfortunately, the 

ability to measure small changes in these enzymes differs widely among laboratories and 

among study designs. 

 

The Panel also recommends including in future considerations the phase II conjugation 

products of chlorpyrifos (namely, glucuronidase and sulfonates). Quantifying the 

conjugative metabolism will ensure that levels of biomarkers are correctly interpreted 

with respect to biomonitoring data and for performing reverse dosimetry. 

b. Section 5 of the draft issue paper compares biomonitoring findings from the three 

children’s health cohorts with other major observational exposure studies in the 

United States. Based on comparison with NHANES 2001-2002, median TCPy 

levels in the CHAMACOS and Mount Sinai cohorts were slightly higher than in 

the general population. It should be noted that the exposures experienced by the 

CHAMACOS and Mount Sinai cohorts overlapped the start of the residential 

chlorpyrifos phase-out. By contrast, median TCPy levels in the Columbia cohort, 

for which sampling occurred when chlorpyrifos use should have rapidly declined 

due to the voluntary cancelation, were slightly lower than the levels measured by 

NHANES in the general population. Please comment on the adequacy of the 

Agency’s comparison for the purposes of evaluating chlorpyrifos exposure levels 

in the three children’s health cohorts. Are there any additional biomonitoring 

studies that should be included in the Agency’s comparison?

The Panel concurs with EPA that the human studies discussed in this section are currently

the best available, primarily because they are carefully designed and well executed. The

Panel recommends the following additional biomonitoring studies listed in the ordered 

that they should be considered: 1) NHANES 1999-2004, 2) Barr et al. 2010, 3) Bradman 

et al. 2005 (because the families studied are likely to continue to see significant exposure

which should be validated by the next round of NHANES data), 4) the Children's 

Pesticide Exposure Study (CPES) by Lu et. al. 2008 and Children's Post-Pesticide 

Application Exposure Study (CPPAES) studies, and 5) studies that are either currently in 

process or completed and will be published soon. This last group includes: 1) The 

Children’s Pesticide Exposure Study that focuses on dietary intake of children and related 

pesticide exposures being conducted at the Harvard School of Public Health. 2) The 
23

Children’s Exposure to Environmental Pesticides study which is evaluating the utility of 

biomarkers of pesticide exposures, e.g., DAPs and pesticide-specific markers of OP and 

pyrethroid exposures, and environmental levels measured in soil, house dust, and food 

being conducted by Emory University, and 3) the SAWASDEE cohort study, that is 

examining pesticide biomarker concentrations in pregnant mothers and similar markers in 

their newborn children, run by Emory University and Chiang Mai University in Northern 

Thailand. The Panel advises that in comparing the results among these different studies, 

it is important to verify that analytical results from the studies are directly analogous, 

especially in the methods used to control for the effect of small day-to-day variations in a 

laboratory’s AChE results when trying to quantify small changes in an exposed 

population.

Question 5.2 

In Section 5.0 of the draft issue paper, the Agency summarized the 2008 preliminary 

findings on the association between urinary TCPy levels and AChE/BuChE inhibition 

and discussed two recent studies involving manufacturing workers in the US and Egypt. 

Please comment on the scientific quality of these studies and their findings. Please 

include a discussion of their strengths and limitations. Please comment on the strengths 

and limitations of the evidence from this research to show an association between TCPy 

and AChE/BuChE inhibition at exposure levels experienced by occupational populations.

The Panel notes that both studies were in general well designed and implemented. They 

both have adequate power to demonstrate an association between TCPy and AChE or/and 

BuChE inhition at exposure levels experienced by occupational populations. In addition 

the cholinesterase data from the studies verify the ability of the PBPK model to predict 

that once chlorpyrifos is absorbed, it interacts first with BuChE and only starts to inhibit 

RBC AChE and AChE in the central nervous system after BuChE is more than 50% 

inhibited. 

The Panel points out several primary weaknesses in both studies as it relates to using 

them within the weight of evidence paradigm. These include high levels of TCPy in preexposure samples indicative of prior exposure to chyorpyrifos or environmental 

degradates either from food or accumulated residues in the workplace. Neither study 

reported analyses that adjusted for levels in control groups. 

The Panel recommends that the Agency separate the scenarios for occupational 

exposures, as reported in these two studies, from exposures to residential sources. The 

extrapolation of these data to the population as a whole is subject to criticism. The 

subjects in these two studies are adults and issues such as the “healthy worker effect” and 

the notion that low-level exposure and high-level exposures are likely to be detoxified by 

differing mechanisms make extrapolation difficult. Additionally, the Panel suggested

that studies of agricultural workers and their families could provide a better avenue of 

investigation that compares “occupational-levels” exposure with other members of their 

families likely to see slightly “elevated” but lower levels of exposure, and to study the 

potential impact on the offspring in such cohorts either in utero or otherwise. The Panel 
24

recommends that in the future, the Agency consider the quality of ChE measurements 

before pursuing further uses of these data in exposure and risk assessments.

Question 5.3 

Several approaches ranging from qualitative to the most sophisticated PBPK/PD 

modeling approach were introduced as potential options for analyzing the chlorpyrifos

biomonitoring data. Please comment on the strengths and limitations of these 

approaches. In addition, please suggest, if appropriate, alternative approaches or 

analyses not identified by the Agency. 

There are a rising number of data-informed options for interpreting biomonitoring data. 

Choosing the adequate option relies on the extent of the data available, on the 

toxicokinetics of the relevant population subset, on the mode of action, and on the 

integration of these data. Integrating these data through a verified PBPK model has the 

potential to be the most informative approach while also being the most data intensive.

The Panel advises EPA that at the very least the chlorpyrifos biomonitoring data should 

be utilized as a means of “ground truthing” total external exposures under a variety of use 

conditions. Considering the availability of chlorpyrifos biomonitoring data on the 

general population, and as a basis to support its maximal consideration of public health, 

the Panel also recommends that the Agency seriously consider developing a 

“biomonitoring equivalent” at the same time the reference dose for chlorpyrifos is 

derived. The biomonitoring equivalent is defined as a calculated level of a biomarker 

associated with exposures consistent with health protective guidance values for the 

general population. The Panel also recommends that the Agency utilize a verified PBPK 

model which will provide a robust opportunity to integrate the considerable available data 

on external and internal exposures (i.e., biomonitoring) to chlorpyrifos at different life 

stages under different conditions of exposure. With respect to a specific PB/PK model 

for the Agency to consider, the Panel recommends a sophisticated model such as the 

SimCYP pediatric model (SimCYP Company, Sheffield, UK) for children that is 

currently available.

Question 5.4 

Characterization of chlorpyrifos exposure experienced by women in the Columbia cohort, 

particularly during the pre-cancellation period, remains an important uncertainty in using 

these data in quantitative risk assessment. Exposure levels in the range measured in the 

cord blood data from the epidemiology studies (pg/g plasma) are probably low enough 

that is unlikely that the cohort mothers were experiencing AChE inhibition at the time of 

delivery; however, the biomonitoring data were taken after birth and not necessarily 

associated in time with an application of chlorpyrifos. As such, the actual level of such 

exposure particularly during any critical window(s) of susceptibility is not known, and a 

better understanding of the range of possible exposures and the degree to which they may 

or may not have elicited inhibition of AChE, remains a key scientific question. In light of 

Panel discussions of Questions 4.3 and 5.3, please suggest approaches and/or analyses 

which would inform the understanding of the degree to which exposure levels 

experienced by the Columbia cohort participants may or may not have been below doses 
25

which result in 10% inhibition of AChE in the most sensitive lifestage. Please discuss the 

strengths and uncertainties associated with such analyses. Please include in your 

discussions approaches involving chlorpyrifos and its metabolites and also chlorpyrifos

plus other AChE-inhibiting pesticides (propoxur, diazinon) which the cohort participants 

were exposed too.

The Panel notes that it is important to realize that the short half-life of chlorpyrifos and its 

metabolites in the body calls into question any ”spot data” that might be used. Large 

cross-sectional studies may capture some exposure but they do not put these exposures

into context. Longitudinal investigations with frequent samplings are more likely to 

provide data that are more useful. Thus, the Panel recommends that a longitudinal study 

with measurement throughout the pregnancy (rather than a few samples in the last 

trimester) would fill many of the data gaps that currently exist for this group. Such a 

study is needed given the potential for neurodevelopmental effects on the fetus as well as 

the metabolic differences in pregnant women versus the workers from the 1984 study.

As discussed in the response to the previous question, the Panel again recommends that a 

more sophisticated PBPK model may provide better data particularly if the model is 

pertinent to the population being studied, i.e. pregnant women and small children. 

Studies discussed in Question 5.1 provide data on the concentration of chlorpyrifos in 

various media (i.e. house dust, air and water) while market basket data exists on the 

concentration of chlorpyrifos on food. These data provide the main tools for developing 

an effective exposure assessment and a subsequent reconstruction of potential dose. 

Dose reconstruction can be used to evaluate the efficacy of the PBPK model since its 

prediction of excretion rates can potentially be validated with an accurate estimate of 

dose. This assessment of the PBPK model through reconstructed dose may bridge some 

of the data gaps in assessing risk by validating the PBPK model.

The Panel discusses the issue of mixtures of chlorpyrifos + Diazinon /chlorpyrifos + 

Propoxur or chlorpyrifos/Propoxur/Diazinon. The Panel recommends that the Agency 

address the following questions: “Do mixture components affect each other’s half lives,

distributions and clearance through metabolic competition?” “Are the net AChE effects

of mixtures additive or multiplicative?, and “Do they share mechanistic pathways?”

Lastly, the Panel expresses concern over the Agency’s focus on a 10% AChE activity 

reduction. They point out that to their knowledge there is no proposed mechanism 

whereby a 10% AChE activity reduction in pregnant women would be responsible for a 

cognitive defect or developmental delay in their offspring.
26

Charge 6: Characterizing the range of potential risks.

The 2009 NRC report, Science and Decisions, focused on improving the technical 

analysis through the development and use of scientific knowledge and information to 

promote more accurate characterizations of risk, and thus improving the utility of risk 

assessment for risk-management decisions. The NRC report also pointed out that 

regulatory risk assessment does not routinely approach public health and environmental 

problems by arraying a wide range of options for dealing with them. In the case of 

chlorpyrifos, in light of the discussions of Questions 1-5, please provide guidance for 

assessing and presenting the range of plausible responses at given doses, and the effect 

of the overall uncertainty and variability around that range. 

With regard to characterizing the probable response at given doses, the Panel 

recommends that the Agency use the dose-response data to establish multiple points of 

departure. For instance, it would be informative for risk management purposes to fully 

characterize the nature of the risk above the reference dose. The Panel also recommends 

that the Agency maximizes its use of available data on dose response from the 

epidemiology studies as a basis to at least “bound” reference doses developed on the 

basis of points of departure from animal data. As advised by the Panel, options for doseresponse analysis for acute effects should be considered independent from those based on 

long term exposures, i.e., measures representing acute adverse neurological outcomes 

(ChE inhibition) commonly associated with occupational exposure versus those 

potentially related to lower level long term exposure in the general population, such as 

neurobehavioral disorders. 

The Panel suggests that the Agency focus on the data of chlorpyrifos in the cord blood as 

a means for creating the point of departure for chronic exposures to chlorpyrifos based on 

the PBPK/PD model.

Another consideration that the Panel deems important is the degree to which 

epidemiological data on neurotoxicity is consistently used within EPA to establish points 

of departure for chemicals with the potential for neurotoxic outcomes (e.g., mercury and 

lead). The Panel asks: “How is the evidence from epidemiological studies weighted in 

the assessment for these compounds, and how does this compare with what is proposed

for chlorpyrifos?” 
27

DETAILED PANEL DELIBERATIONS AND RESPONSE TO CHARGE

It is well established that acetylcholinesterase (AChE) inhibition is the primary mode of 

action/adverse outcome pathway for organophosphorus chemicals (OPs) such as 

chlorpyrifos. In June 2011, consistent with the recommendations from the Scientific 

Advisory Panel (SAP) in 2008, the Agency performed a risk assessment utilizing AChE 

inhibition data in laboratory animals for deriving points of departure and for doseresponse analysis as the Agency believes these data remain the most robust and most 

sensitive information available for regulatory risk assessment. However, newer lines of 

research on chlorpyrifos such as epidemiological studies in mothers and children, have 

posed the issue of whether AChE inhibition is the most sensitive health outcome, leading 

to questions about the chlorpyrifos risk assessment. 

In order to determine the degree to which these recent studies are appropriate for 

incorporation into risk assessment (qualitatively and/or quantitatively), the Agency is 

taking a stepwise, objective and transparent approach to evaluate and interpret all the 

lines of scientific information related to the potential for adverse neurodevelopmental 

effects in infants and children as a result of their prenatal exposure to chlorpyrifos, as 

well as to characterize thoroughly the strengths and uncertainties associated with these 

studies. The issue paper entitled “Scientific Issues Concerning Health Effects of 

Chlorpyrifos” extends the Agency’s September 2008 review of the available 

experimental toxicology and observational epidemiology data. This 2012 review 

incorporates experimental data available since the time of the last review relating to 

AChE inhibition and both cholinergic and non-cholinergic adverse outcomes, including 

neurodevelopmental studies on behavior and cognition effects. Similarly, the Agency 

also performed a more in-depth analysis of the epidemiologic studies from three major 

children’s health cohort studies in the U.S., plausible hypotheses on modes of 

action/adverse outcome pathways (MOA/AOP) leading to neurodevelopmental outcomes, 

along with biomonitoring and physiologically-based pharmacokinetic/pharmacodynamic 

(PBPK/PD) modeling than was conducted in 2008. Overall, the Agency has updated and 

extended its evaluation of multiple lines of evidence informing the chlorpyrifos risk 

assessment.

As discussed the 2012 issue paper, two of the key scientific questions are: 1) the degree 

to which scientific data suggest that chlorpyrifos causes long-term neurodevelopmental 

effects from fetal or early life exposure and 2) the degree to which adverse effects can be 

attributed to doses lower than those which elicit 10% inhibition of AChE, i.e., the dose 

levels previously used for regulatory decision making. The evaluation of these scientific 

questions requires integration numerous types of data, and consideration of the nature and 

degree of the uncertainties surrounding the data, including the extent to which alternative 

interpretations may be supported. This step is vital to robust risk characterization and 

uncertainty analysis. The 2011 preliminary risk assessment noted that a full weight of the 

evidence analysis that explicitly considers uncertainty and implications of experimental 

and epidemiologic lines of evidence using factors such as biological plausibility, strength, 

consistency, and dose-response and temporal concordance, will be conducted in the 
28

future. A full weight of the evidence and full uncertainty characterization has not yet 

been conducted; the 2012 SAP is an important step toward this effort. 

Question 1.0 Mode of action/adverse outcome pathway: Acetylcholinesterase (AChE) 

inhibition

Question 1.0 

It is well established that AChE inhibition is the primary mode of action/adverse outcome 

pathway for OPs, like chlorpyrifos. Because AChE inhibition is the initiating event for 

this mode of action/adverse outcome pathway, using AChE inhibition as a regulatory 

endpoint is protective of downstream cholinergic effects. Moreover, historically, given 

the sensitivity of AChE inhibition data for OPs, these data have been considered to be 

protective of other potential toxicities and/or modes of action for OPs. In 2008, the 

Agency performed a comprehensive review of the available AChE data from multiple 

lifestages. This review has been supplemented with the newest studies. Consistent with 

the recommendations from the 2008 SAP, the Agency believes that AChE data remain 

the most robust dose-response data for deriving points of departure in in vivo

experimental toxicology studies with laboratory animals. Please comment on the 

Agency’s preliminary conclusion that AChE data remain the most robust source of data 

for deriving points of departure for chlorpyrifos. Please include a discussion of the 

strengths and uncertainties of this preliminary conclusion. 

Response

The Panel agreed with the Agency’s conclusion that the AChE data remain the most 

robust source of data for deriving points of departure for chlorpyrifos. This is based on 

the observation that all studies reporting neurobehavioral changes following in vivo 

prenatal or postnatal exposures to chlorpyrifos have been accompanied by AChE 

inhibition when measured at an appropriate time following administration of 

chlorpyrifos. Moreover many studies reporting persistent neurobehavioral changes used 

a potentially confounding vehicle (e.g., DMSO). Most importantly, the experimental 

design for essentially all experimental studies evaluating neurodevelopmental effects of 

chlorpyrifos do not allow for the effective determination of a point of departure. There 

are candidates that may replace AChE as a more sensitive indicator but, at this juncture, 

these have not been fully validated and their alteration has not been determined to result 

in a well-defined, measurable neurotoxic outcome.

As in 2008, the Panel recommended that the Agency continue to use AChE data at the 

most sensitive lifestages for dose-response analysis and deriving points of departure.

When looking at obstetric outcomes and pediatric exposures, life-stage levels of red 

blood cells (RBC) AChE activity in humans, which has been reported as significantly 

lower in fetal cord blood than in adults (de Peyster et al. ,1994), needs to be taken into 

account to eliminate potential uncertainties.

The Panel concurred with the 2008 Panel in expressed caution on the use of DMSO as a 

vehicle because of its intrinsic toxicity and potential influence on absorption. Again, 

uncertainty was expressed about potential interactions between DMSO and low doses of 
29

chlorpyrifos and the effect of this interaction on the developing organism. In addition to 

the three papers cited by the 2008 SAP (FIFRA Scientific Advisory Panel, 2008b), more 

recent evidence is available to support the potential toxicity of DMSO. Hanslick et al.

(2009) reported that following acute intraperitoneal injection of DMSO into 7 day-old 

mice, there was a significant increase in the number of apoptotic neurons at dosages as 

low as 0.3 ml/kg. An increased number of apoptotic neurons was also observed at 1 

ml/kg which is the most frequent volume of DMSO administered in the cited studies 

using DMSO as a vehicle. Recent reports from the zebrafish literature suggest that 

DMSO has the capacity to directly induce neurobehavioral effects. Exposure to 0.05% 

DMSO induces anxiolytic behavior in adult zebrafish (Sackerman et al., 2010) and 

exposure to 0.01% DMSO alters locomotor activity in larval zebrafish exposed 

embryonically (Chen et al., 2011). Also, based on earlier studies observing that DMSO 

induces a stress protein response in zebrafish embryos (Hallare et al., 2004; 2006), 

Turner et al., (2012) reported that levels of DMSO as low as 25 μl/L (0.0025%) were 

sufficient to induce gene expression changes in embryonic zebrafish. While altered gene 

expression does not indicate a toxic response, it suggests disruption of homeostasis by 

low levels of this solvent. While the experimental studies reviewed in the White Paper 

all had controls with DMSO only, there is no way to rule out the potential for an 

interaction between DMSO and the OP. For example, Fossum et al. (2008) reported that 

2% DMSO had no effect when microinjected into the periaquaductal gray region of rat 

brain, but it enhanced the potency of morphine when co-administered. In this case, if 

morphine was dissolved in 2% DMSO and the controls received DMSO only, the 

interpretation of the findings are confounded. It should be noted that the concentration of 

100% DMSO is approximately 14 M. Because of the potential biological/cellular 

changes noted above, the lack of evaluation of potential interactions between DMSO and 

chlorpyrifos, and the well-known effects of DMSO on membrane permeability 

(Gurtovenko and Anwar, 2007), caution should be exercised in the use of data for 

quantitative risk assessment from in vivo (or in vitro) studies using DMSO as a solvent. 

Question 2.0 Mode(s) of action/adverse outcome pathway(s): Plausible pathways 

leading to potential neurodevelopmental outcomes

Question 2.1 

As discussed in Section 3.2.1, although there are numerous mechanistic studies in the 

scientific literature, the research on different hypotheses does not provide sufficient data 

to establish causal linkages among different levels of biological organization to show 

how effects lead to adversity. As such, a mode of action or adverse outcome pathway 

leading to effects on the developing brain cannot be established at this time. Moreover, 

although multiple biologically plausible hypotheses are being pursued by researchers, 

based on the current state of the science, no one pathway has sufficient data to be 

considered more credible than the others. Please comment on the Agency’s preliminary 

conclusion that although there are multiple biologically plausible hypotheses being 

evaluated by research scientists, the mechanistic experimental toxicology data do not yet 

support a coherent set of key events in a mode of action/adverse outcome pathway. 
30

Response 

The Panel acknowledged the efforts EPA has taken to review all of the relevant data 

addressing the various cellular and mechanistic based studies on chlorpyrifos and 

relevant associated neurobiologically-based studies. Research scientists are examining 

multiple biologically plausible hypotheses regarding cellular mechanisms of chlorpyrifos

neurotoxicity. Over the past approximately 15 years a number of studies have evaluated

changes in neurite outgrowth, axonal transport, dendritic growth, and other cellular 

processes following chlorpyrifos or chlorpyrifos oxon exposure that could potentially 

disrupt the development of the nervous system. In no case, however, is there a defined, 

coherent set of events from alteration of any of these cellular functions to disrupted 

development of the nervous system sufficient to explain a variety of neurobehavioral 

changes. There is also limited evidence that these current research efforts are directed in 

such a manner to link the in vitro findings to a structural or functional change in the 

animal. The Panel agreed with the Agency that, based on the current state of the science, 

no single pathway has sufficient data to be considered more credible than the others with 

respect to a causal link between chlorpyrifos exposure and toxicological outcome.

As defined, the progression of events from molecular initiation to adverse outcome 

requires a logical sequence of changes in the mode of action/adverse outcome pathway. 

The Panel raised the issue that the example of domoic acid as a linear connection is likely 

to be a unique case that can provide components at each level of the pathway but may 

also generate a non-linear pattern. A linear pathway from mode of action to adverse 

outcome appears rare. Thus, the Panel agreed that while laudable, expectations of the 

existence of such a pathway may be artificially elevated and potentially unrealistic for 

risk assessment.

Question 2.2 

Although a mode of action/adverse outcome pathway has not been established, 

qualitatively, the growing body of mechanistic studies does demonstrate that chlorpyrifos

and/or its oxon are biologically active on a number of processes that affect the developing 

brain. Some mechanistic studies provide evidence of possible effects which are similarly 

sensitive or more sensitive than AChE inhibition (e.g., neurite outgrowth, binding to 

muscarinic receptors, axonal transport; serotonergic nervous system development). Some 

of these comparisons must be considered with caution since the amount of change in the 

in vitro systems required to elicit an adverse effect in vivo is unknown. Moreover, 

extrapolation from in vitro perturbations to in vivo effects has not been established, which 

introduces additional uncertainties. Given the doses/concentrations evaluated in the in 

vitro and in vivo mechanism studies, please comment on the degree to which these studies 

suggest that endpoints relevant to evaluating potential neurodevelopmental outcomes 

may or may not be more sensitive than AChE inhibition. Please include in your 

comments a discussion of the strengths and uncertainties. Please also include in your 

comments a discussion of the scientific understanding of dose-response relationships for 

biological perturbations and the magnitude, frequency and/or duration of such 

perturbations that can lead to adverse effects at higher levels of biological organization 

to support characterization of the likelihood of adverse outcomes in a human health risk 

assessment (as articulated in NRC 2007). 
31

Response

The Panel agreed with EPA that caution must be exercised in interpreting the in-vivo

relevance of the changes observed across the various in vitro studies. There are a number 

of cellular processes such as, neurotransmitter receptor activation, and others that are 

currently under study; however, the studies lack the necessary data on the relevance of 

these changes observed in vitro and effects occurring in vivo. Much of this work is 

speculative because the underlying process being studied is assumed a process critical to 

brain development. In vitro work is best suited for testing hypotheses that can be further 

explored in vivo, but given their reductionist approach, they lack the experimental power 

and demonstrated predictive validity of an in vivo effect at this stage to be of scientific 

value for risk assessment considerations. The in vitro models that have been utilized to 

address the effects of chlorpyrifos and oxon such as neurite outgrowth, M2 acetylcholine 

receptor binding, mitochondrial morphology and axonal transport, as well as oxidative 

stress, may potentially provide information on non-cholinesterase related mechanisms. 

The Panel discussed the limitations of the cellular in vitro systems to translate to in vivo

systems as well as to identify a human health risk. They noted that the inherent 

complexity of the nervous system cannot be replicated in a cellular in vitro system. The 

majority of these studies were conducted to explore the potential for chemical exposure 

to induce a change in cell physiology and to further examine underlying mechanisms. 

Thus, while the reductionist approach provides information on possible mechanisms of 

action for chlorpyrifos, it does not translate to in vivo effects. The extrapolation of data 

from in vitro to in vivo is filled with uncertainty factors. Adding to this uncertainty is the 

possibility that although chlorpyrifos has a well-established molecular target (AChE) for 

cholinergic toxicity, it may not be the only toxicologically relevant target. In addition, 

due to the inherent complexity of the nervous system, which contains multiple regions 

and systems that are connected and interact with one another, effects on the molecular 

target could induce downstream effects that can result in a neurotoxic response that may 

not be directly attributable to the molecular target. This complexity and these 

connections cannot be replicated in vitro.

Issues of concern raised by the Panel were not specific to chlorpyrifos, but rather these 

concerns were directed toward the use of in vitro systems in general. Such concerns

entailed the isolated nature of the cells in culture and the inability to address critical 

regulatory components of the in vivo environmental niche. For instance, the Panel noted 

that the in vitro model system can influence the effects observed in isolated cells, but the 

responses may be changed or absent when these same cells are co-exposed with other 

cells normally within their in vivo environment. The Panel also raised questions about 

how the isolated nature of the various model systems could deal with altered homeostasis 

or dose response differences that may occur as a function of differential recruitment of 

processes in vivo. In addition, the actual amount of compound or AChE inhibition at the 

target site is one critical factor toward determining if a toxicological event observed in 

vitro can occur in vivo. The Panel mentioned that that until such in vivo translation can 

be established, it is difficult to determine what level, magnitude, or duration of change is 

required within each model system to be indicative of a change that may occur in vivo. 
32

The Panel also discussed the likelihood that using such models may not provide a linear 

dose-response relationship for chlorpyrifos or for AChE inhibition. Thus, providing a 

direct assessment of the relative sensitivity to AChE inhibition is difficult. Changes in 

neurite outgrowth, dendritic spine development, axonal transport and other cellular 

responses that have been seen with chlorpyrifos or chlorpyrifos oxon exposure in vitro

have been reported to occur at levels of exposure below those necessary to inhibit AChE. 

These comparisons must be made with caution. For example, adding chlorpyrifos oxon 

directly to hippocampal neurons in vitro in tissue culture medium is unrealistic with 

regards to how the oxon would reach a neuron in vivo. Many detoxifying/binding 

proteins are not included in in vitro conditions, potentially modifying the interaction of 

the chemical with the cell. Similarly, if acetylcholinesterase in a disrupted tissue (e.g., a 

homogenate) such as liver and is inhibited in vitro by chlorpyrifos oxon, its sensitivity is 

much higher than if that same enzyme is immunoprecipitated and then inhibited by 

chlorpyrifos oxon in vitro under similar conditions. Such effects of tissue components, in 

the relative potency of chlorpyrifos or chlorpyrifos oxon, make extrapolation of in vitro

effects to in vivo settings difficult. In general, changes in neurodevelopmental endpoints 

require relatively higher exposures in in vivo models. Reviews of mechanistic/cellular 

studies with neurodevelopmental outcomes following chlorpyrifos exposure suggest that 

such responses may occur at dose levels at, near, or above those necessary to induce

AChE inhibition. The development of a physiologically-based pharmacokinetic (PBPK)

model that can estimate the in vivo dosage required to reach toxicant concentration at the 

target site that is similar to the in vitro concentration at which the effects were observed

would be especially valuable. Such a model would assist in determining the plausibility 

that the effect observed in vitro also occurs during an in vivo exposure.

The Panel was in agreement that further mining of the published literature may provide 

significant information on how one might utilize the available data obtained for 

chlorpyrifos to generate plausible hypotheses for future evaluation. As an example, the 

analytical studies of the Lockridge group indicating that chlorpyrifos (chlorpyrifos) oxon 

can covalently modify key cytoskeletal proteins such as tubulin and motor proteins like 

kinesin provide information that can contribute to the interpretation of findings of 

alterations in neurite outgrowth and axonal transport, respectively. Such an integrated 

effort may allow for the design of specific targeted studies to test the hypothesis in vivo

as an effort to obtain predictive validity (Jiang et al., 2010; Grigoryan et al., 2009). 

The Panel considered items that might be added to the review. One topic is the effect of 

chlorpyrifos on neurotrophins (growth factors). Pope et al. (1995) provided the first 

evidence that OPs might alter the activity of growth factor-like (neurotrophic) molecules. 

Data suggesting that growth factors could be altered within the brain tissue, in vivo, has 

recently been provided in neonatal rats across low dose levels of chlorpyrifos. 

Alterations were observed in mRNA levels for specific members of the fibroblast growth 

factor (FGF) superfamily of neurotrophic factors (Slotkin et al., 2007). Further, early 

postnatal exposure to chlorpyrifos has also been associated with decreases in nerve 

growth factor (NGF) in the rat forebrain (Betancourt and Carr, 2004). These effects are 

not limited to the immature rodent in that adult exposure to chlorpyrifos can result in 

protracted alterations in NGF-related signaling proteins (e.g., the high affinity nerve 
33

growth factor receptor TrkA and its activated form, phospho-TrkA in the prefrontal 

cortex) (Terry et al., 2007). Also, as a note, in the Middlemore-Risher et al. (2011)

study, alterations in mitochondrial morphology and decreases in axonal transport were 

observed in primary cortical neurons exposed to chlorpyrifos and chlorpyrifos oxon at 

concentrations including ones that did not inhibit acetylcholinesterase.

The Panel raised the issue that oxon and protein adducts likely serve as a potentially 

important pathway for cellular/protein damage. The oxon has an incredibly rapid half life

and despite a relatively high affinity for ChE, one would expect that they would 

occasionally bind non-ChE cellular components 3,5,6-trichloro-2-pyridinol (TCPy) and 

diethylthiophosphate (DETP) are conjugated by -O-sulfotransferases or 

gluruconosyltransferases; and as presented in public comments from Dow Chemicals, 

both sulfonylates and glucuronides are “equally prevalent” but relative affinities (Km) 

appear to be unknown. In translating from animal to human, it was considered of 

importance by the Panel that Uridine 5'-diphospho-glucuronosyltransferase enzymes

(UGTs) develops late in children. In this case the sulfotransferase (SULTS) are usually 

present during gestation, despite the fact that the AChE adducting oxon would not be 

conjugated. One must consider that if glucuronidation is the rate-limiting pathway in 

children, then other metabolites may accumulate to toxic levels due to ontogenetic 

inadequacy of UGTs. This could result in a potential for error in biomarker analysis and 

generate errors in dosimetry estimations.

The Panel discussed the types of dose-response relationships that may be observed and 

allowed for non-linear patterns or no clear dose-response association to be observed. The 

Panel cautioned that when examining the dose-response relationship, one should not 

uncouple PD analyses from PK models too far given that PK differences can affect active 

site concentrations and hence, PD effects. To this end PK can significantly affect the 

magnitude and duration of an effect. It was noted that the p450s and PON1 were well 

integrated into the PB/PK/PD models and this was considered a major strength. There 

were, however a number of weaknesses discussed including the fact that there is a 

substantial lack of knowledge about the high capacity of phase II conjugation for 

chlorpyrifos in humans. For example, there are species differences in Phase II where 

UGTs and SULTS may be non-orthologous between humans and rodents; hence any 

extrapolation of animal data to humans must take this into consideration. The Panel 

noted that this was highlighted by the public presentation from Dr. Hattis who noted that 

the human data he subsequently used and modeled demonstrated lower clearance than 

seen in rat data. An issue was raised by the Panel that pregnancy can be considered as a 

specific state and that information is needed relative to how PK differs in a pregnancy 

scenario relative to exposure and toxicity. In addition to maternal influences such as 

metabolism by the liver, the placenta was also raised as a unique component requiring 

attention in such PB/PK/PD models for gestational exposure. 

The Panel raised a concern that equivalency developmental stages between ages of 

rodents to human are not well defined with regards to cell type compositions, brain 

region, cellular architecture, and physiological or biochemical process. This is not a 

problem that the Agency needs to address but rather the Panel emphasized that specific 
34

developmental periods in which perturbation may occur appear to be ill defined and may 

not translate between rodent and human species. The numerous in vitro mechanistic 

studies suggest that chlorpyrifos can alter numerous biological processes in normal brain 

development. However, these data do not permit translation to the in vivo situation nor 

do they provide a quantitative dose-response relationship that can be compared to AChE 

inhibition.

Question 3.0 Neurodevelopmental data from laboratory animals

Question 3.1 

As discussed in Section 3.2.2, the experimental toxicology data in laboratory rodents 

show neurobehavioral effects following developmental exposure with changes in a 

number of neurological domains. In 2008, the SAP agreed to this preliminary conclusion, 

and the nine additional studies available since 2008 add further support. Please comment 

on the degree to which these studies show changes in a number of neurological domains 

and support the qualitative conclusion that chlorpyrifos exposure during gestation and/or 

early post-natal period may result in long-term adverse effects on the developing nervous 

system. What evidence does and does not support this conclusion? Please also include 

in your comments a discussion of the strengths and uncertainties. Please also include in 

your comments a discussion of the scientific understanding of dose-response

relationships for biological perturbations and the magnitude, frequency and/or duration 

of such perturbations that are can lead to adverse effects at higher levels of biological 

organization to support characterization of the likelihood of adverse outcomes in a 

human health risk assessment (as articulated in NRC 2007). 

Response

In order to address the first part of this charge question, the Panel critically reviewed 

these toxicology data in laboratory rodents. This review included a total of 21 

developmental neurobehavioral effects studies which also entailed the nine studies 

published since the 2008 SAP review (These studies are identified in Appendix 3 of the 

Agency’s Draft Issue Paper: Scientific Issues Concerning Health Effects of 

Chlorpyrifos.). Based upon their review, the Panel agreed with the 2008 SAP 

conclusions that developmental neurobehavioral experiments show adverse effects of 

chlorpyrifos exposure. However, the Panel cautioned that the existing neurobehavioral 

studies are limited and a number are under-powered and prone to Type I error (meaning 

the null hypotheses may have been falsely rejected) and therefore should be discounted in 

determining the weight of evidence for or against neurobehavioral effects from 

developmental exposure to chlorpyrifos. An additional concern was raised by the Panel 

in the inclusion of tests that have not been validated as to neurotoxicological significance. 

Such assessments included anxiety tests, depression tests, or social interactions. The 

Panel concluded that, in the current state, such outcomes should be regarded as 

exploratory, and hypothesis-generating, rather than evidence of toxicity. The Panel 

considered that the lack of observable effects below dosages equal to or exceeding 1.0 

mg/kg/day in some studies could be due to the possibility that the effects at lower 

dosages might not be the same as those observed at higher dosages. In addition, low dose 

exposure may lack a sufficient level of response to elicit a physiological compensation 
35

response that would occur with higher dose levels. In this scenario, the toxic effects of 

the higher and lower dosages may manifest themselves in different ways. If the response 

is through the same physiological target, the expected dose-response curve may be 

altered (such as a U-shaped or inverted U-shaped curve). In contrast, the response of the 

lower dosages may be quite different from that of the higher dosages and require a 

different behavioral paradigm.

Many of the 21 studies reviewed included 2 or 3 dose levels of chlorpyrifos (ranging 

from 0.2 in one case up to as high as 10 mg/kg in another but more commonly from 1-7 

mg/kg), but dose-effect outcomes were, more often than not, not observed. Some of the 

most consistent effects are from the Slotkin-Levin experiments (see below) where radial 

arm maze deficits and several other effects have been replicated but the studies are 

seldom designed with three doses levels and even when two dose levels were included 

the findings were not dose-dependent in most cases. The Panel agreed with the Agency 

that the lack of specificity of direction of the neurobehavioral findings is problematic. A 

statistically significant change in isolated markers of certain behaviors may not be 

supported by other studies under similar dosing paradigms thereby raising concern 

regarding the biological significance of the observed change. The Panel questioned the 

Agency’s interpretation that a change in either direction or a specific behavior is 

necessarily indicative of an adverse effect. Rather, such discrepancies may suggest 

methodological error, problems in study execution, or a predilection toward searching 

data for positive rather than negative findings. Dose-response and attention to 

methodological issues should play a role in evaluating the weight of evidence within and 

across the different studies. The Panel agreed that the overall evidence across these 

studies is persuasive, indicating that there are enduring effects from chlorpyrifos

exposure at 1.0 mg/kg or above on the CNS. Future neurodevelopmental studies need to 

focus on levels below 1.0 mg/kg/day and to expand the studies to identify the correct 

testing paradigm to detect these effects and possibly identify a neural substrate. The 

Panel also considered the possibility that additional negative data exists at lower dose 

levels but is not available in the published literature. The Panel suggested that crosslaboratory or collaborative studies could provide systematic comparison of the effects of 

chlorpyrifos on neurodevelopmental domains using unified exposure periods, dosing, age 

of testing, and methods, combined with urinary analysis of chlorpyrifos’ metabolites, and 

accurate assessments of AChE inhibition.

In evaluating the inconsistencies among these studies, the Panel suggested that the 

Agency should consider factors such an the distinct ontogeny of the various brain 

regions, cellular components, and neurotransmitter systems in the fetal/gestational 

exposures and that the structural and functional maturation of each system is unique and 

thus may be at different stages during the age of exposure. In addition, the redundancy 

and compensatory capability of each system should be considered as the level of insult 

may be required to reach a substantial level before it manifests as a neurobehavioral 

change. 
36

The Panel’s detailed review of each these developmental neurobehavioral effects studies 

is provide in Appendix A of this report. However, the following provides a collective 

summary of the Panel’s observations and conclusions regarding the studies. 

Among the 21 reviewed articles (which include more than 21 experiments), many effects 

are reported at chlorpyrifos doses ranging from at 1.0 to 7.0 mg/kg (and in one study 10 

mg/kg). However, these are dose levels known to significantly inhibit cholinesterase in 

RBC, therefore, based on these 21 studies, cholinesterase inhibition is an adequate 

threshold as no credible evidence of neurobehavioral effects below 1.0 mg/kg were 

found.

Three studies tested doses <1.0 mg/kg chlorpyrifos. Two studies used 0.3 mg/kg and one 

used 0.2 mg/kg (Jett et al., 2001;Braquenier et al., 2010;Maurissen et al., 2000). Of 

these, two found no effects at these lower doses (Maurissen et al., 2000;Braquenier et al., 

2010). Only one study found effects at 0.3 mg/kg (Jett et al., 2001), however, this study 

contains serious methodological flaws which are of sufficient magnitude to cast serious 

doubt on the credibility of the findings. While the data from Jett et al. (2001) raise the 

possibility of neurobehavioral effects at 0.3 mg/kg/d, these data require replication in a 

study that is properly designed, adequately powered, and appropriately analyzed. Until 

such time as new data at such lower doses become available, it is concluded that no dose 

<1.0 mg/kg in any neurodevelopmental behavioral studies shows evidence of adverse 

effects (or of any effects, even including those outcome measures of

indeterminate/unknown toxicological significance).

In addition, effects of chlorpyrifos at 1.0 mg/kg are difficult to interpret because of 

methodological limitations, inconsistencies, and variation in study design, sometimes 

lack of control for litter effects, oversampling issues, behavioral methods used, and lack 

of dose-response findings.

At doses exceeding 1.0 mg/kg, the data show somewhat more consistency, but even here, 

dose-response experiments are the exception. A 5.0 mg/kg of chlorpyrifos, reduced body 

weight is sometimes seen, and at doses above 5.0 mg/kg increased mortality may occur 

along with other evidence of toxicity. Given this, it is a significant gap in the literature 

that more dose-response studies are not available in the range downward toward 0.2 

mg/kg and extending up to and including doses previously tested of 1.0-2.0 mg/kg in 

order to determine what, if any, dose-effect curve occurs in this range for 

neurobehavioral effects.

It appears that prenatal and prenatal-neonatal exposures are more sensitive than neonatal 

exposure alone on neurobehavioral outcomes. This implies that prenatal exposure may 

be the exposure period contributing to this observation, but unfortunately, most of the 

pre- and neonatal studies are not entirely informative because the neonatal exposure was 

to the dam rather than directly to the progeny. This makes it unclear what the exposure to 

the offspring actually was or whether it was at similar levels to those reaching the embryo 

and fetus. More studies, especially dose-response studies, in the lower dose ranges with 

exposure from implantation to the end of major neurogenesis (approximately P20) are 
37

needed, again with doses below 1.0 mg/kg and with concomitant measurement of 

maternal, fetal, and neonatal cholinesterase activity.

Many of the existing studies expose for only a narrow interval during gestation or the 

neonatal period. Prenatal exposures should be from E6-20 to 21 for rats, and E6-18 or 19 

in mice in order to span most of early brain development (equivalent to human first and 

part of second trimester). And for neonatal treatment, exposures should be from shortly 

after birth to approximately P20 (equivalent to the latter half of second and all of third 

trimester equivalent brain development comparable to that for humans). If the critical 

period or most sensitive period is within this range, then such comprehensive exposure 

should cover the entire span of CNS development that represents the species being 

modeled, i.e., human beings.

In the prenatal studies, the use of timed-pregnant females shipped from breeders is 

problematic for behavioral studies because maternal stress, even if regarded as equivalent 

across dams assigned to the treated and control groups, introduces a variable that has the 

potential to interact with the independent variable. If maternal stress were to interact 

with chlorpyrifos, it would confound the outcome and make a result difficult to interpret 

(which is exactly what is found in many of the reviewed studies). Since no one has tested 

for this, it is currently impossible to rule it out.

Many studies use diurnal and some nocturnal testing. If additional dose-response studies 

are undertaken, this factor should be held constant so that results can be better compared.

Question 3.2 

The dose-response data in the in vivo experimental neurodevelopmental toxicity studies 

are not amenable to empirical dose-response modeling as many studies use only one or

two doses, and in some cases the lower dose, but not higher dose level, produced

significant effects. Many studies report effects at a dose of 1 mg/kg/d-- a dose that 

produces some amount of brain ChE inhibition when given directly to the pups postnatally, but may or may not alter fetal brain ChE activity when given to the dams 

gestationally. One study (Braquenier et al., 2010) using lower doses, administered to the 

dam on GD15-LD14, reported a NOEL of 0.2 mg/kg/d. Comparing the NOEL of 0.2 

mg/kg/d to a repeated dosing AChE inhibition BMDL10 of 0.03 mg/kg/d suggests that 

AChE inhibition is a sensitive and protective endpoint. 

a. Please comment on the scientific quality and robustness of the animal 

neurodevelopmental toxicity studies.

Response

The quality of the studies in this category varies, but there are some of high quality. 

Overall, the studies by Slotkin (Dam et al., 2000; Icenogle et al., 2004) and Levin (Levin 

et al. 2001 and 2002), those by Carr et al. (Carr et al. 2001 and Johnson et al., 2009), the 

study by Maurissen (Maurissen et all, 2000), and several of those from the Ricerri and 

Venerosi group (Ricerri et al., 2003 and 2006 and Venerosi et al., 2006, 2008, and 2010), 

are among the better ones because they generally used multiple doses, had adequate 

sample sizes, controlled for litter effects, used sound behavioral methods, and used 
38

appropriate statistical methods to analyze the data. Because each of these studies found 

long-term neurobehavioral effects, these data may be regarded as robust within the limits 

of what was tested. These more persuasive findings only occurred at doses of 1.0 mg/kg 

of chlorpyrifos and above. This data set is, therefore, moot concerning effects at <1.0 

mg/kg of chlorpyrifos. Among these studies, however, several concerns remain. In the 

Slotkin and Levin experiments, the use of commercially supplied timed-pregnant rats is a 

concern as is the use of Zivic-Miller (ZM) Sprague-Dawley rats. The ZM rat is known to 

be different from other Sprague-Dawley rats on some behavioral tests where it often 

performs as an outlier. For studies with regulatory implications, it is preferable to use a 

mainstream rat strain such as Sprague-Dawley from Charles River or Harlan where much 

more is known about their behavioral characteristics and they do not perform at the 

extremes of the distribution. Also, the concern about the RAM method as noted in Panel 

review in Appendix A should be taken into account with regard to robustness. 

Notwithstanding these caveats, the weight of evidence from the neurobehavioral studies 

is that there are too many long-term effects for them all to be attributable to Type I errors, 

hence, it is more likely than not that there are significant long-term adverse effects and in 

this the Panel concurs with the conclusions of the 2008 SAP findings and the EPA White 

Paper background document.

c. Please comment on the degree to which studies that measured AChE inhibition 

and those that measured neurodevelopmental outcomes can be integrated to 

evaluate whether points of departure based on 10% AChE inhibition provide 

more sensitive endpoints than endpoints measured in the experimental 

neurodevelopmental studies (as reviewed in Section 3.2.2). Please include in 

your comments a consideration of the strengths and uncertainties associated with 

this assessment. 

Response

Data in the available studies, including the nine additional studies reported since 2008, 

provide qualitative (emphasis on qualitative) support for the effect of chlorpyrifos

exposure during gestation and/or early post-natal period and long-term adverse effect on 

the developing nervous system. Several of these studies examined AChE activity in the 

brain after oral and/or subcutaneous chlorpyrifos exposures during postnatal periods, and 

inhibition of AChE within one day of exposure was observed in these studies at doses as 

low as 1 mg/kg/day. Since AChE inhibition recovers quickly the data are insufficiently 

refined to allow for a linkage between the mode of action and the neurodevelopmental 

effects (acute vs. chronic, respectively).

Since the mode of action of these effects is not established and cannot be presumed a 

priori to be related to AChE inhibition, these studies do not exclude the possibility that 

other mechanisms may be involved, especially long-term effects where functional 

characteristics may be unmasked at later life-stages due to neuroplasticity. A few studies 

have reported AChE inhibition when a dose of 1 mg/kg/d was administered directly to the 

pup postnatally (Dam, et al., 2000; Johnson, et al., 2009; Ricceri, et al., 2003). However, 

none of the neurobehavioral studies described in the Panel’s review tested for fetal AChE 

inhibition when 1 mg/kg/d was given during gestation. A companion study to Maurissen 
39

et al. (2000) reported no cholinesterase inhibition in samples taken from fetuses 4 h after 

dosing the dam when 1 mg/kg/d had been administered daily since E6 (Mattsson, et al., 

2000). Qiao et al. (2002) also reported no brain AChE inhibition in fetuses 24 h after the 

last dose of 1 mg/kg/d to the dam on E17-20. No other time points or days were assessed 

in either study. These results suggest, but do not confirm, that the fetus would not 

experience AChE inhibition at 1 mg/kg/d to the dam, further suggesting that the 

behavioral effects reported in those studies were not due to AChE inhibition.

The studies published since 2008 demonstrate alterations in a number of 

neurodevelopmental and biochemical outcomes. The amount of AChE inhibition 

required to elicit the various endpoints was however inconsistent and varied, because of

differences in study designs, analysis of different endpoints, and how long animals were 

followed-up. Many of the studies measured AChE inhibition 24 hours or longer after 

dosing, which can underestimate the amount of AChE inhibition. Furthermore, since the 

neurodevelopmental effects may be independent of AChE inhibition, it needs to be 

considered whether AChE inhibition represents a critical marker for derivation of points 

of departure when considering chronic studies.

Finally, the Panel notes that there has been little consideration of the relationship to 

genetic variability on experimental outcomes, the exception being paraoxonase 1

(PON1). Recovery of AChE activity is linked to changes in AChE gene expression. It 

has been previously reported that molecular and behavioral effects may be attributable to 

alternative splicing of the AChE gene. Within the brain there are 2 variants: AChE-S 

(synaptic) and AChE-R (read-through splice variant) mRNA. Under normal conditions 

variant AChE-S dominates; however, under stress conditions, such as OP exposure 

(chlorpyrifos has yet to be studied), the transcription of AChE-R increases. Following 

stressful events, AChE-R increases to a level that is no longer adaptive and result in 

varied physiological changes. Of the neurobehavioral effects reported in the reviewed 

experiments that assessed AChE inhibition, no studies were identified that showed effects 

on behavior at low levels of AChE inhibition, including at 1.0 mg/kg of chlorpyrifos. 

Doses below 1.0 mg/kg/day chlorpyrifos did not show convincing evidence of 

neurobehavioral effect; hence, no extrapolation to lower doses in terms of AChE 

inhibition is possible from the data reviewed herein.

Question 4.0 Epidemiology Regarding Children’s Health

Question 4.1 

Section 4.0 and Appendices 5 and 6 provide the Agency’s review of the available 

epidemiology studies from the Columbia Mothers and Newborn study, the Mt. Sinai 

Child Development study, and the Center for Health Assessment of Mothers and 

Children of Salinas Valley (CHAMACOS) study. Consistent with the 2008 SAP 

recommendations, the Agency has considered information offered from each of the three 

cohort investigations; however EPA acknowledges the primacy of the Columbia cohort

data for the purposes of informing risk assessment because researchers measured 

chlorpyrifos parent compound directly in this study. Please comment on the sufficiency, 

clarity, and quality of the Agency’s epidemiology review as contained in Section 4.0 and 
40

Appendices 5 and 6 of the draft issue paper with respect to identifying the major 

strengths and limitations of each study.

Response

The Panel believes that the epidemiology section of the draft issue paper is very wellwritten, clear, accurate and fairly complete. The Panel commends the Agency staff on 

the thorough review of the epidemiology literature, for putting their epidemiology review 

in the context of the modified Bradford Hill criteria, as recommended by the 2010 SAP, 

and for reviewing the potential for selection and information biases in each of the studies. 

In particular, the Panel commends the Agency staff for the tremendous amount of work 

and thoughtfulness that went into Appendices 5 and 6. The Panel believes that the 

epidemiology review appropriately concludes that the studies show some consistent 

associations relating exposure measures to abnormal reflexes in the newborn (using the 

Brazelton Neonatal Behavioral Assessment Scale), pervasive development disorder at 24 

or 36 months, mental development at 7-9 years, and attention and behavior problems at 3 

and 5 years of age , in addition to less consistent results for reduced mental and 

psychomotor development (measured by Bayley scores) at 12 and 24 months. 

Inconsistent results were found for associations between exposure and measures of fetal 

growth. 

The Agency’s epidemiology review provided an excellent description of the strengths 

and limitations of the studies conducted to examine the relation of chlorpyrifos to 

children’s growth and neurodevelopment. Epidemiologic studies such as these require 

that large numbers of mothers and infants/children be followed longitudinally for an 

extended period with extensive data collection at regular intervals to ascertain exposure 

measures, potential confounders, and health outcomes, all of which can change over time. 

These studies are logistically difficult to implement and require great commitment by the 

researchers and a potentially large burden in terms of time and effort on the part of the 

study participants over a lengthy period of time, often with little or no specific benefit or 

return to themselves. 

The Agency’s review adequately summarizes the challenges and scientific contributions 

of each of three studies: one conducted in an inner city sample of African American or 

Dominican initially pregnant women and their infants/children by Columbia University 

investigators; one conducted by Mt. Sinai investigators in a predominately Hispanic and 

African American sample in New York City, and one conducted in the Salinas Valley in 

California led by University of California Berkeley investigators. As noted in the review, 

all three studies had the significant strength of being longitudinal, prospective designs, 

the most effective design for establishing the temporal sequence in relating exposure to 

health outcomes, specifically in these studies relating exposure measures obtained 

prenatally and/or at delivery to outcomes measured at six months up to nine years of age. 

Regarding exposure assessment, only the study by the Columbia investigators measured 

chlorpyrifos parent compound in cord blood, and conducted an exposure validation study 

and looked at correlations of cord blood measures with mothers blood and meconium, 

and used the cord blood as the measure of exposure in relation to scores on standard 
41

neurodevelopmental test batteries for children at age 7 years (Rauh et al., 2011). The 

Panel agrees with the Agency that because this study included the most specific exposure 

measure, particular attention should be focused on its results. The Panel disagrees with 

some of the public comments claiming that because the observed associations with 

adverse neurodevelopmental outcomes occurred at levels below those required for AChE

inhibition, the results should be discounted. Instead, the Panel believes that these 

findings are derived from a well-designed and conducted study and thus suggest that the 

mechanism may be other than that of AChE inhibition. Further, the very large effect 

observed in this study for the relationship of exposure to attention deficits, reduced birth 

weight prior to the voluntary withdrawal of chlorpyrifos, reduced mental and 

psychomotor development, and reduced Intelligence Quotient (IQ) are unlikely to be due 

to important misclassification of or bias in assessing exposure or to uncontrolled 

confounding. [The Panel notes that in Appendix 5, some of the directional signs of 

effect, i.e., betas, and their 95% confidence intervals (CIs), are missing or incorrect so 

that all of them should be double-checked and corrected.] Finally, the review might 

additionally note that the timing of exposure may be important, although the critical time 

window of exposure is not known with certainty.

Although, the other two studies only used metabolites as markers of chlorpyrifos

exposure, all three studies used TCPy, a metabolite specific to chlorpyrifos, in relation to 

at least some of the outcomes assessed. (Even though TCPy is a better measure than 

dialkylphosphates (DAPs), the other indirect biomarker, the TCPy measure has 

limitations as noted by the review. The potential misclassification of exposure should be 

more explicitly stated. The value of using TCPy as a biomarker also hinges on the mode 

of action, which is not established with certainty.) As the epidemiology review 

appropriately notes, all three studies were strengthened in design by using biomarkers of 

exposure instead of relying on self-reports of exposure, which would be likely to result in 

much greater misclassification of exposure. All three studies also used similar standard, 

validated measures of non-verbal and general intelligence, behavior and home 

environment, which enhance the quality of the outcome assessments and the ability to 

assess the consistency of the findings regarding these outcomes across studies. In 

addition, as appropriately noted in the Agency’s review, all three studies collected 

extensive data on potential confounding variables, used appropriate multivariate 

statistical techniques to control for confounding effects of socioeconomic factors, 

lifestyle and behavioral factors as well as additional environmental exposures, and 

conducted sensitivity analyses to determine if assumptions made about missing data, for 

example, were appropriate.

While agreeing that chlorpyrifos could have played a role in the neurodevelopmental 

outcomes observed in the Columbia cohort, some panel members raised concern about 

associating the observed deficits in neurodevelopmental outcomes in children with a 

single chemical, given that this was a multi-chemical exposure spanning a multi-year 

period that encompassed an important period of sequential developmental processes 

necessary for brain maturation. Rauh et al. (2011) reported that decreased working 

memory and full-scale IQ in 7 year-olds were statistically significantly associated with 

prenatal chlorpyrifos exposure. In an earlier examination of the same cohort, Perera et al.
42

(2009) reported an association between a decrease in full-scale IQ and verbal IQ in 5 

year-olds with prenatal polycyclic aromatic hydrocarbons (PAH) exposure rather than 

chlorpyrifos, thus, raising an issue of the shift in chemical exposure association with 

increase in age. In each of these analyses, statistical modeling showed that the exposures 

were independently associated with IQ, and no significant interaction was observed with 

the other chemical. While this is a statistically sound approach to determine independent 

responses, panel members noted that it is very difficult to identify the independent 

physiological effects of a single chemical in this type of multi-chemical exposure 

scenario. This identification is further complicated by limitations in exposure assessment 

with respect to on-going and post-natal exposures and the potential for chemical 

interactions during the exposure period. In addition, developmental progression of the 

children and the level of skills examined by the tests employed may have been 

confounding factors. Maturation of the brain is a critically timed sequence of events with 

each subsequent event dependent upon the successful completion of the previous one. 

Thus, appropriate brain function at age 7 is dependent on completion of maturation 

processes that occur at earlier ages. Panel members noted that, while this statistical 

approach could be used in studies examining the exact same endpoint at a single age, this 

brain maturation process would need to be taken into consideration prior to determining 

that at 5 years of age the cognitive deficit was due to one exposure and at 7 years of age it 

was due to a different chemical. The ever-changing aspect of any developmental study is 

further demonstrated in the assessments of this cohort of children at earlier ages. At 36 

months of age, the deficits in the Bayley Mental Development Index scores were 

associated with exposures to prenatal chlorpyrifos (Lovasi et al., 2011), prenatal 

phthalates (Whyatt et al., 2012), prenatal PAHs (Perera et al., 2006), and prenatal 

piperonyl butoxide (Horton et al., 2011). Thus, panel members cautioned about 

identifying any one specific chemical as the main one associated with the cognitive 

deficits observed at 7 years of age in the Columbia cohort.

One additional concern is that in general, the sample sizes of the three studies were only 

moderately large, ranging from just over 100 to slightly under 500, depending on which 

subset of data from mothers and children were analyzed. The more recent papers had 

fewer participants, ranging from just under 200 to just over 300. The epidemiology 

review correctly notes that the modest samples sizes were a limitation in having sufficient 

statistical power to detect as statistically significant possible modest relations of exposure 

to outcomes or interactions with other variables. Thus, modest sample sizes were one of 

the most important limitations of these studies, which is reflected in the wide confidence 

intervals for some of the effect estimates and the use of moderate (e.g. 1 standard 

deviation) or large (e.g., 10-fold) increases in exposure measures (which did not seem to 

be mentioned in the Agency’s epidemiology review) to see statistically significant 

effects, e.g., in IQ (Rauh et al. 2011; Bouchard et al. 2011). However, some evidence of 

interaction with paraoxonase 1 (PON 1) genotype and/or phenotype was provided in 

some of the studies (Berkowitz et al. 2004, Engel et al 2007; Engel et al. 2011; Harley et 

al. 2011), and some examinations of interactions with other exposures were presented in 

the studies and summarized in the review. Future examination of potential epigenetic 

effects might also be informative. The Panel also recommended that investigators of the 
43

three studies consider possible pooling of samples and data to enhance the ability to 

investigate effect modification and possible roles for other agents.

Two other items that might be added to the review are: 1) replacing “null” and “positive 

(ns)” with point estimates and 95% CIs for effect estimates (to the extent possible, 

realizing that quantiles or betas might have to be used) to Table 10 on page 59; and 2) 

noting in the text that other interactions (e.g., with sex of the child, gestational age at 

measurement of exposure, length of breastfeeding, use of alcohol, etc.) were not 

consistently described in the three studies, and in most cases sample sizes were 

inadequate to have sufficient statistical power to detect meaningful effect modification as 

statistically significant. Providing the point estimates and 95% CIs for effects in Table 

10 will permit assessment of the magnitude, variability, and direction of the effects, 

which are more important in assessing consistency than statistical significance. The 

second point concerning interaction is important because it means that the potential for 

stronger associations (larger effects) in subgroups with potentially enhanced 

susceptibility could not be or were not adequately examined or reported. In addition, the 

Agency’s epidemiology review mentions that the restriction of some of the study samples 

by race/ethnicity and/or to low risk pregnancies (e.g., nonsmokers, women without 

comorbidities) reduced the potential for confounding (which was a plus) but also reduced 

the generalizability of the results. However, the review perhaps did not sufficiently 

emphasize that this limitation also meant that modification of effect by race/ethnicity or 

other risk factors could not be examined with these study sample restrictions, and the 

sample sizes in general were inadequate to examine interactions with such factors. Thus, 

differential effects for subgroups with other risk factors or characteristics could not be 

determined.

The Agency’s epidemiology review also examined the potential for misclassification and 

bias in each of the studies and mentions the likelihood that any such misclassification 

and/or biases that operated were non-differential and thus likely to result in an underestimation of effect. For example, chlorpyrifos exposures, particularly when the parent 

compound was not measured, could have been misclassified, especially because some 

analyses indicated greater within-person than between-person variability in exposure 

measures. However, this was unlikely to be differential with respect to the 

neurodevelopmental outcomes measured and thus would likely have resulted in bias to 

the null or under-estimation of effect measures. Similarly, although not explicitly stated 

in the publications, it was unlikely that those who were assessing outcomes using 

standardized measurement instruments knew the exposure levels of the participants, 

which could have biased their assessments. Thus, again, misclassification of outcomes 

could have occurred but were likely non-differential with respect to exposure levels and 

thus were likely to have resulted in bias to the null or under-estimation of effects. 

The Panel also felt they should respond to the issue of multiple comparisons that was 

raised in the public comments. The Panel feels it is important to note that all the 

comparisons made in the three studies were hypothesis-driven and dealt with related 

outcomes, rather than reflecting “fishing expeditions” that would have been likely to 

result in significant findings by chance due to multiple comparisons. The Panel thus 
44

believes that the multiple comparisons issue is not an important concern regarding the 

findings of the three studies over the years.

The Agency’s epidemiology review reflects the authors’ views from the three studies that 

among the statistically significant effects seen, most appeared to have a linear relation 

with exposure with no evidence of a threshold. However, upon examination of some of 

the graphs and other results presented in some of the papers, it would appear that this 

point requires some further data and examination. For example, the graphs in the 

Columbia study seem to suggest no threshold for the effect on working memory but do 

suggest a threshold for the full-scale IQ (Rauh et al. 2011). In the California sample, the 

graphs presented in the most recent paper (Bouchard et al. 2011) suggest a drop in IQ 

beginning generally with the second quintile of exposure level (depending on which 

outcome is examined) and seem not to worsen greatly in higher quintiles of exposure 

levels, which is also suggestive of a threshold effect. The graphs presented in the recent 

publication from the Mt. Sinai study (Engel et al. 2011) seem to indicate no threshold 

when using tertiles, but the confidence intervals were quite wide. Thus, the Agency’s 

epidemiology review appropriately notes that, due to the modest sample sizes, statistical 

power may have been inadequate to detect departures from linearity with log transformed 

exposures or outcomes. It might also be mentioned that modest sample sizes limited 

statistical power to assess dose-response adequately, which is one of the key postulates 

promulgated by Bradford Hill, so that such attempts sometimes resulted in wide 

confidence intervals around effect measures in each quantile, making adequate 

assessment of dose-response difficult, and that different quantiles were used across the 

studies, making direct comparisons difficult. Additional analyses of dose-response in 

both animal and human data and particularly at lower levels of exposure would be very 

helpful in informing inferences from the epidemiologic studies.

In conjunction with the modified Bradford Hill criteria, the epidemiology review states 

that a biologically plausible role for chlorpyrifos in relation to adverse 

neurodevelopmental outcomes is believed to involve inhibition of AChE. While this is a 

reasonable assumption, the recent papers from the epidemiologic studies noted that 

noncholinergic mechanisms may play roles in the associations of exposure with the 

neurodevelopmental outcome measures (Bouchard et al. 2011; Rauh et al. 2011). In 

addition, the observed effect modification also suggests other mechanisms, including 

oxidative stress and lipid peroxidation. The mode of action is discussed in more detail 

elsewhere in the draft issue paper, but these additional potential mechanisms might be 

added in the epidemiology review. As noted above, just because the significant effects 

are observed at exposure levels below which (acetyl cholinesterase) AChE inhibition 

occurs does not mean that the observed associations are not real, but rather that the 

mechanism(s) in humans may be other than by AChE inhibition. Further mechanistic 

work needs to be done to clarify this issue.

In summary, the epidemiology review contained in the draft issue paper is very clearly 

written, accurate and generally provides a very thorough review in the context of the 

modified Bradford Hill criteria. As noted above, a few additions would enhance the 

completeness of the review.
45

Question 4.2 

Similar to the initial conclusions from 2008, the Agency has preliminarily concluded that,

qualitatively, chlorpyrifos likely played a role in the neurodevelopmental outcomes

reported in the epidemiologic studies, and that information available since 2008,

including both new etiologic investigations as well as epidemiologic methods papers,

strengthens this conclusion. Please comment on the Agency’s preliminary, qualitative

conclusion that chlorpyrifos likely played a role in the neurodevelopmental outcomes 

observed in the epidemiologic studies. Please include in your comments a discussion of 

the strengths and uncertainties associated with this preliminary conclusion. 

Response

Overall, the Panel reiterates the 2008 SAP’s conclusion and the Agency’s concurrence 

with the statement that chlorpyrifos likely plays a role in neurodevelopmental outcomes 

in the three cohort studies. The qualitative conclusion of the epidemiology review seems 

well-justified. The Panel agrees with the Agency that although exposures to other AChEinhibiting compounds cannot be ruled out as contributing to neurodevelopmental 

outcomes, the potential combination and/or additive effects of these compounds do not 

rule out the role of chlorpyrifos. However, it should be noted that it cannot be stated that 

chlorpyrifos is the sole contributor to the observed outcomes.

The conclusion is enhanced by the strengths of the three studies reviewed, specifically: 

the longitudinal designs which permitted clear indications of the temporal 

relation of chlorpyrifos exposure to adverse neurodevelopmental outcomes; 

the inclusion of biomarkers of exposure as well as self reported exposure;

the relative consistency of findings in different populations but using similar 

standardized exposure and outcome measures; 

the strength of the associations found; 

the use of objective measures of exposure and standardized, validated 

measures of outcomes; 

the control of multiple confounding variables including other environmental 

exposures and other pesticides;

the suggestion of a dose-response effect;

minimization in bias in assessing outcomes and exposures and the likelihood 

that biases and misclassification of exposures and outcomes resulted in a bias 

to the null, i.e., under-estimation of effect; and 

attempts to investigate genetic and phenotypic effect modification and doseresponse effects. 

The conclusion is further supported by the following details of strength and consistency 

of association and a crude exposure response relationship. Some of these details were 

previously presented in the 2008 SAP report, but they are reiterated here to include all 

epidemiologic evidence in one place. It should be noted that studies published since 

2008 have continued to show associations between neurodevelopmental outcomes and 

potential exposure to chlorpyrifos and have strengthened the available epidemiologic 
46

evidence. Recent analyses have looked at neurodevelopmental outcomes in older 

children and addressed some of the issues of confounding by socioeconomic status, other 

pesticides, and issues of exposure measurement validation. 

Strength of association: This criterion focuses on the Columbia cohort because this 

cohort specifically measured chlorpyrifos directly from cord blood and therefore has the 

most robust exposure measurement. Although the results from the other cohorts are 

useful even if these studies were negated due to non-specific exposure measurement, the 

Columbia cohort provides a number of strong associations. The effects as described 

below are seen as early as fetal growth and continue through early childhood with recent 

evidence of neurodevelopmental effects until age seven. 

a) Fetal growth: Statistically significant deficits of birth weight of 186 grams 

when comparing high exposure to lowest quartile of exposure and decreases 

of 43 grams in birth weight per log increase in chlorpyrifos in cord blood 

(Whyatt et al., 2004).

b) Infant neurodevelopment: Statistically significant deficits of 6.5 points on 

Bayley Psychomotor Development Index (PDI) at 3 years of age when 

comparing high to low exposure groups (Rauh et al., 2006). Notably these 

decrements in PDI persist even after adjustment for group and individual level 

socioeconomic variables (Lovasi et al., 2010).

c) Increased odds of mental delay (OR=2.4; 95% CI: 1.1-5.1) and psychomotor 

delay (OR=4.9; 95% CI: 1.8-13.7) at age three when comparing high to low 

exposure groups (Rauh et al., 2006). When controlling for diazinon and 

propoxur exposures, chlorpyrifos still showed significant increased odds of 

mental (OR=3.2; 95% CI: 1.3-8.2) and psychomotor delay (OR=7.9; 95% 

CI: 2.1-29.1) (Appendix 4, Whyatt & Rauh, 2011 unpublished)

d) Attention problems: Extremely large odds ratios for attention disorders 

(OR=11.26; 95% CI: 1.79-70.99), ADHD (OR=6.50; 95% CI: 1.09-38.69), 

and PDD (OR=5.39; 95% CI: 1.21-24.11) were seen when comparing high to 

low chlorpyrifos exposure groups (Rauh et al., 2006). The magnitude of these 

results as so large that they are unlikely to be affected by residual confounding 

although limited sample sizes resulted in imprecise estimates.

e) Intelligence measures: Statistically significant decreases of 1.4% in full scale 

IQ and 2.8% in working memory among seven-year olds for each standard 

deviation increase in chlorpyrifos exposure (Rauh et al., 2011). These results 

persist even when performing sensitivity analyses including only those with 

detectable chlorpyrifos levels. In addition, no evidence was provided of 

mediation by child behavior on the measure of working memory instrument.

Consistency of association: This criterion outlines the results from the Berkeley 

and Mt. Sinai cohorts which were consistent with or supportive of the conclusions 
47

of the Columbia cohort. It should be noted that the Berkeley and Mt. Sinai 

cohorts did not replicate the effects on fetal growth that were seen in the 

Columbia cohort. Although the cohorts had similar composition and study 

design, it should be noted that the Berkeley and Mt. Sinai cohorts used nonspecific measures of general organophosphate exposure (TCPy and DAPs). 

However, the internal validity across cohorts gives confidence in the consistency 

of the results for the neurodevelopmental outcomes. It should also be noted that 

neurodevelopmental effects are seen in both of these cohorts beginning at 

neonatal development and extending to early childhood.

a) Neonatal neurodevelopment: Increased abnormal reflexes in neonates were 

significantly associated with maternal and urinary DAPs in both the Berkeley 

and Mt. Sinai cohorts (Young et al., 2005; Engel et al., 2007).

b) Infant neurodevelopment: In the Mt. Sinai cohort, prenatal DAP was 

significantly associated with deficits in Bayley mental development index 

(MDI) at 12 months among blacks and Hispanics. This association was 

enhanced among children with maternal carriers of PON1 QR/RR, i.e. fast 

metabolizers (Engel et al., 2011). In the Berkeley cohort significant decreases 

in MDI at 24 months were associated with increased prenatal and infant 

urinary DAP measures (Eskenazi et al. 2007). Examination by PON1 status 

also showed evidence of poorer MDI scores at 2 years among those children 

with the PON1-108T allele (Eskenazi et al., 2010).

c) Attention problems: In the Berkeley cohort, total urinary prenatal and 

postnatal DAP measures were associated with significantly increased odds of 

PDD at 2 (Eskenazi et al., 2007). In addition, prenatal DAP was associated 

with ADHD and Child Behavior Checklist attention problems at 5 years. 

Child concentrations of diethylphosphate (DEP) were also adversely 

associated with a composite measure of attention (Marks et al., 2010).

d) Intelligence measures: In the Berkeley cohort, a significant deficit of 7 points 

in full scale IQ was seen among seven year olds when comparing the highest 

quintile of maternal DAP to the lowest level (Bouchard et al., 2011). In the 

Mt. Sinai cohort, there were slight but not significant decrement in full scale 

IQ, perceptual reasoning and working memory associated with prenatal 

maternal urinary DEP in 6 to 9 year olds. Increased prenatal maternal urinary 

DAP was also associated with decreases in perceptual reasoning in maternal 

QQ carriers. This association showed a monotonic trend (Engel et al., 2011).

e) Crude exposure response relationship: This was demonstrated in the pre-post 

residential cancellation analyses in the Columbia cohort in the outcomes of 

birth weight, birth length, and three year MDI and PDI scores (Whyatt et al., 

2004; Rauh et al, 2006). In addition a significant reduction in cord blood 

chlorpyrifos and maternal personal air samples was seen when comparing pre 

and post cancellation levels (Whyatt et al., 2004). The effectiveness of a 
48

prevention measure can often be shown when reductions in effect can be 

measured subsequent to a reduction in exposure. This was the case in the 

natural ‘experiment’ that occurred during the course of the Columbia cohort. 

Although the study was not designed to test an exposure-response 

relationship, decreases in both outcomes and exposure following the 

residential ban argue for a crude dose-response relationship.

The following uncertainties should be noted: 

Relatively modest sample sizes which limited the statistical power to classify

some meaningful differences as statistically significant and to examine the effect 

of modification by race/ethnicity and other characteristics. 

Relatively moderate to large exposure differences needed to see significant 

effects, likely due to the modest sample sizes used.

Exposure at one point in prenatal time with no additional information regarding 

postnatal exposures.

Lack of clarity regarding a linear dose-response instead of a potential threshold 

effect.

Use of a single or average sample for exposure. Although Whyatt et al. (2009) 

noted moderate but significant correlations between meconium and cord and 

maternal blood and average urine TCPy, the representativeness of a single point 

exposure is still unclear. Time-varying exposures or the ability to define 

cumulative exposures would be preferable.

Lack of specificity of a critical window of effect and the potential for 

misclassification of individual exposure measures.

External generalizability of the cohorts given their unique racial/ethnic and 

socioeconomic characteristics. However, it should be noted that their exposures 

were within the range of those seen in NHANES.

Questions about biologic plausibility due to lack of clarity on mechanism of 

action, particularly at the low exposure levels seen in the cohorts and the limited 

and mixed results of animal studies showing neurodevelopmental effects.

One panel member suggested that before the Agency could conclude that chlorpyrifos is 

likely to play a role in the neurodevelopmental outcomes observed in epidemiologic 

studies, particularly in the Columbia study, additional analyses need to be conducted. 

In order to eliminate the possible causes of neurodevelopmental effects by other 

pesticides in the Columbia study, it is suggested that EPA should repeat the pre-post 

residential cancellation analysis done for chlorpyrifos using other pesticide 

measurements, such as malathion diacid (MDA), a specific metabolite of malathion. The 

outcomes from those additional analyses will either confirm or reject EPA’s preliminary 

conclusion that chlorpyrifos is likely to play a role in the neurodevelopmental outcomes.

While one panelist agreed with the overall statement, the Panelist also endorsed changes 

in the phrasing from "chlorpyrifos likely played a role ..." to "chlorpyrifos may [or could] 

have played a role ." That Panelist noted that TCPy has some serious limitations as a 
49

quantitative indicator of exposure to chlorpyrifos due primarily to its common occurrence 

in foods. In addition, triethylphosphate (TEP) has some similar limitations particularly 

within the Berkeley cohort because the usage rate of diazinon in Monterrey County is at 

least10 times more than the use rate of chlorpyrifos; and diazinon also produces TEP. 

This ratio presents a dilemma between the characterization of this cohort as farm laborers 

and the attribution of their higher levels of urinary TCPy to exposure to chlorpyrifos. 

In conclusion although the three studies were not comparable in all regards, more 

similarities than discrepancies were found across them. The Panel concludes that the 

additional literature since the 2008 SAP continues to support and strengthens the 

evidence for the conclusion that chlorpyrifos plays a likely role in the adverse effects in 

child neurodevelopment. 

Question 4.3 

As discussed in Question 2.0, a mode of action/adverse outcome pathway has not yet 

been fully elucidated for the potential neurodevelopmental outcomes as a result of 

prenatal chlorpyrifos exposure. Although this does not undermine the qualitative 

interpretation of these studies, and the preliminarily conclusion stated above (Question 

4.2), the identification of the dose-response for neurodevelopmental effects based on 

mode of action is not possible. Further, given the urine and cord blood sampling 

frequency in the study there is a large degree of uncertainty in estimating absolute 

exposure-response relationships, as opposed to establishing relative exposure groups for 

evaluating associations. With respect to dose-response, critical durations of exposure, 

and windows of susceptibility are unknown. In 2008, the SAP cautioned against using 

the Columbia cohort data for deriving a point of departure due, in part, to only measuring 

biomarkers (3rd trimester maternal, cord blood, meconium) at one point in time, and 

because they cannot exclude possibility that the effects seen were due to chlorpyrifos in 

combination with other pesticides. In 2008, the SAP advised against using data from the 

epidemiology studies (including the Columbia Mothers and Newborn study which 

measured chlorpyrifos directly) for deriving a point of departure due to limitations of the 

exposure assessment in these epidemiology studies for the purpose of risk assessment, 

e.g., lack of repeated exposure estimates to ascertain more specifically the variability and 

periodicity of exposure over time (i.e., predominant use of one-time exposure estimate). 

a. Due to the limitations of exposure assessment performed in the epidemiologic 

investigations for the purposes of quantitative risk assessment, the Agency has 

concluded that the epidemiologic data are not sufficient for deriving points of 

departure for quantitative risk assessment. The Agency proposes that AChE 

inhibition data from laboratory animals remain the most appropriate data to use for 

dose-response modeling and the derivation of points of departure. Please comment 

on the scientific evidence that does and does not support this conclusion, as well as 

the strengths and limitations of the evidence. 

Response

The Panel acknowledged the limitations in the three longitudinal children’s cohort studies 

of estimating chlorpyrifos exposures (i.e., the Columbia study, the Mt. Sinai study, and 
50

the CHAMACOS study), based on the exposure measures collected, and was in general 

agreement that the data from these studies alone were not sufficient to derive a point of 

departure (POD) for purposes of quantitative risk assessment. As a panel member noted, 

these three epidemiologic studies were primarily focused on assessing health outcomes 

associated with a variety of environmental factors, and were not designed to conduct a 

quantitative exposure assessment for chlorpyrifos. In addition, the use by the three 

studies of different exposure matrices (urine, maternal blood, cord blood, and meconium) 

and different targeted analytes (TCPy, DAPs, and chlorpyrifos) makes the effort of 

deriving a definitive POD based on those data alone impossible. 

Despite the exposure assessment limitations noted for these three epidemiology studies, 

the Panel recognized the value of these data and urged the Agency to find ways to use the 

epidemiology studies, and in particular, the data from the Columbia study, to inform the 

dose-response assessment of chlorpyrifos. Only the Columbia study provided data on 

measurements of chlorpyrifos in cord blood coupled with neurodevelopmental 

measurements. As noted by the Panel, if one assumes that cord blood measurements 

reflect exposure levels during the critical prenatal period for induction of 

neurodevelopmental effects, then in theory, these would be the ideal data from which to 

derive the POD for chlorpyrifos in humans. Specific Panel suggestions included using 

the Columbia data “as an exercise” to derive a POD for neurodevelopmental effects in 

infants, and analyzing the data from each of the cohorts to put some bounds on the range 

of chlorpyrifos doses associated with the observed neurodevelopmental effects. 

The Panel also recognized the value in developing a functional PBPK model for 

chlorpyrifos for pregnancy and the prenatal lifestage. Such a model could be used to 

further characterize the dose estimates in the epidemiology studies, for additional doseresponse analyses. Such a PBPK model will become even more important in the event 

that the Agency might, at some point in the future, decide to move from using AChE 

inhibition to another outcome. In particular, such a tool could not only relate a dose of 

chlorpyrifos to a non-AChE outcome but it could also link a dose to the chlorpyrifos

or/and chlorpyrifos oxon concentration in vitro to a non-AChE target-site in vivo. 

The Panel expressed concerns regarding the Agency’s proposal to use the dose-response 

data on AChE inhibition in laboratory animals to derive points of departure for the 

chlorpyrifos risk assessment, and referred to multiple lines of evidence suggesting that 

adverse neurodevelopmental effects may be attributed to chlorpyrifos doses lower than 

those that elicit a 10% inhibition of AChE. 

This evidence comes from the epidemiological data derived from the three longitudinal 

children’s cohort studies i.e., the Columbia study, the Mt. Sinai study, and the 

CHAMACOS study. A number of findings of neurodevelopmental outcomes associated 

with chlorpyrifos are consistent across these three cohorts. For example, there is a 

consistent association between chlorpyrifos exposure and deficits in mental development 

at age 7 as ascertained by decrements in full-scale IQ and Working memory using the 

Wechsler Intelligence Scale for Children (WISC-IV) (Engel et al., 2011; Bouchard et al., 
51

2011; Rauh et al., 2011). (See responses to Charge Questions 4.1 and 4.2 for more 

detailed discussion and assessment of the findings from these studies.)

There are limitations to the exposure assessment in these three cohorts. The Columbia 

study has the most direct measure of exposure to chlorpyrifos, measuring the compound 

in cord and maternal blood at time of delivery (Rauh et al., 2011). This study also has 

48-hr personal air measurements of chlorpyrifos for pregnant women, air chlorpyrifos

measurements (stationary samples) collected during the last 8 weeks of pregnancy, 

urinary metabolite data (TCPy) during the last trimester (up to 4 measurements for some 

participants) and at delivery for mom and baby, and TCPy in meconium (Whyatt et al., 

2007; 2009). In an exposure validation study conducted by the Columbia researchers, the 

levels of TCPy in meconium and maternal urine correlated with cord blood chlorpyrifos

levels (Whyatt et al., 2009). This suggests that cord blood levels can be used as a 

representative measure of exposure. Overall, the estimates of chlorpyrifos exposure in 

the Columbia cohort (based on measured levels of maternal urinary TCPy) were slightly 

lower, but generally comparable with the levels of urinary TCPy measured in adults in 

the general U.S. population at that time, based on the NHANES data for 1999-2000 and 

2001-2002 (CDC, 2009). The estimates of chlorpyrifos exposure (based on measured 

levels of maternal urinary TCPy) in the Mt. Sinai (Berkowitz et al., 2003) and 

CHAMACOS (Eskenazi et al., 2007; Castorina et al., 2010) cohorts were slightly higher, 

but generally also comparable to the 1999-2000 and 2001-2002 NHANES data for the 

U.S. adult population. 

The Panel suggested that while there are no data on AChE inhibition in either the 

Columbia study participants (e.g., Rauh et al., 2006; Whyatt et al. 2007; 2009; Rauh et 

al., 2011)or the NHANES participants (CDC, 2009), the measured levels of chlorpyrifos

exposure are not anticipated to produce AChE inhibition. Specifically, as noted in the 

Draft issue paper, neurodevelopmental effects seen in the Columbia cohort were 

associated with cord blood chlorpyrifos levels > 6.17 pg/g (Rauh et al., 2006). Based on 

AChE inhibition studies in adult men dosed with chlorpyrifos (Nolan, 1984), in which 

AChE inhibition was associated with peak blood levels of 0.01-0.03 μg/ml (more than 

104 more), blood levels of 6.17 pg/g are unlikely to elicit AChE inhibition.

Additional evidence suggesting that adverse neurodevelopmental effects may be 

attributed to chlorpyrifos doses lower than those that elicit a 10% inhibition of AChE 

comes from the in vivo animal neurodevelopmental studies. 

As discussed in response to Charge Question 3, the Panel concluded there are only 3 

animal neurobehavioral studies that evaluated doses below 1 mg/kg and also assessed 

AChE inhibition—2 found no effects at doses below 1 mg/kg (Maurissen et al., 2000; 

Braquenier et al., 2010), and one reported effects at 0.3 mg/kg, but had serious 

methodological flaws (Jett et al., 2001). In addition, as discussed in the Agency’s Draft 

Issue Paper and the SAP public meeting presentation entitled “Adverse Outcome 

pathway: Data for Chlorpyrifos at Varying Levels of Biological Organization”, there are

another three in vivo neurodevelopmental studies conducted in rats that report effects at 

doses below those at which acetylcholinesterase inhibition was detected (Ray et al., 2010; 
52

Aldridge et al., 2004; Aldridge et al., 2005). These additional three studies are briefly 

summarized below.

The study, of Ray et al 2010, reported differential expression of oxidative stress genes in 

rat pup forebrain 24 hours after administration of chlorpyrifos, in the absence of AChE 

inhibition. Briefly, chlorpyrifos was administered via gavage to 7 day old rat pups at 

doses of 0, 0.1, 0.5, 1, or 2 mg/kg, and forebrain gene expression and AChE activity 

assessed after 24 hours. Gene expression changes, including differential expression of 

genes associated with oxidative stress, were observed at all doses, while inhibition of 

brain AChE was observed only at the highest dose tested (2 mg/kg). As noted on p. 30 of 

the Agency’s Draft Issue Paper, it has been suggested that oxidative stress can result in 

dysregulation of signaling pathways controlling neuroprogenitor cell function. 

The studies of Aldridge and colleagues (Aldridge et al., 2004; 2005) reported a number 

of molecular, biochemical, and functional changes associated with altered serotonergic 

tone in the brains of adult rats exposed prenatally to chlorpyrifos at doses shown in 

separate studies employing the same (Qiao et al., 2002) or similar (Mattsson et al., 2000) 

experimental designs to not result in fetal AChE inhibition. Briefly, in the Aldridge 

studies rats were administered chlorpyrifos (0, 1, or 5 mg/kg in DMSO) by subcutaneous 

injection on gestation days 17-20, and assessed in adulthood for a number of brain 

parameters on postnatal day 60. Developmental chlorpyrifos exposure at the 1 mg/kg 

dose level was associated with increases in serotonin receptors (5-HT1A and 5-HT2)

(Aldridge et al., 2004; 2005), increases in serotonin reuptake receptors (Aldridge et al., 

2004; 2005), increased serotonin turnover (Aldridge et al., 2005), and changes in the 

adenylate cyclase response to serotonin in the cerebral cortex and mid-brain (Aldridge et 

al., 2004; 2005). While neither of the Aldridge studies measured AChE, another study 

from this group reported that subcutaneous injection of 1 mg/kg chlorpyrifos on gestation 

days 17-20 had no significant effect on fetal rat AChE levels, which were measured 24 

hours after the last administered dose (Qiao et al., 2002). The Panel noted that the 

measurement of AChE activity 24 hours after the last chlorpyrifos dose, and the use of 

DMSO as the vehicle, raises some concerns about the validity of the Qiao et al. 2002 

findings regarding fetal AChE activity. These concerns are tempered somewhat by the 

study of Mattsson et al. (2000), in which pregnant rats were dosed with 0, 0.3, 1, or 5 

mg/kg chlorpyrifos (in corn oil) from gestation day 6 through gestation day 20, after 

which fetal rat AChE activity was assessed 4 hours post-gavage. No inhibition of fetal 

AChE activity was observed at either the 0.3 or 1 mg/kg dose levels (Mattsson et al., 

2000). 

Evidence that adverse neurodevelopmental effects may be attributed to chlorpyrifos doses 

lower than those which elicit a 10% inhibition of AChE also comes from the several in 

vitro mechanistic studies that have been summarized in the Draft Issue Paper,

demonstrating a variety of effects at the molecular and cellular level, including 

interference with neurite and axon outgrowth (Das and Barone, 1999; Howard et al.et al., 

2005; Yang et al.et al., 2008), reduced axonal transport (Middlemore-Risher et al., 2011), 

and increased oxidative stress (Crumpton et al., 2000; Qiao et al., 2005; Giodano et al ., 

2007; Saulsbury et al., 2009). Briefly, the study of Das and Barone (1999) in PC12 cells 
53

shows that chlorpyrifos interferes with neurite outgrowth at concentrations that do not 

inhibit AChE, and the studies of Howard et al. (2005) in rat sympathetic neurons and 

Yang et al. (2008) in dorsal root ganglion sensory neurons show that chlorpyrifos

decreases axonal outgrowth at concentrations that do not inhibit AChE. The studies of 

Middlemore-Risher et al. (2011) show that incubation of rat cortical neurons with 

chlorpyrifos or chlorpyrifos oxon reduces axonal transport of mitochondria at

concentrations that do not inhibit AChE. The studies of Crumpton et al. (2000), Qiao et 

al. (2005), Giodano et al. (2007) and Saulsbury et al. (2009) demonstrate that exposures 

of a variety of cell types (i.e., primary cerebellar granule cells, oligodendrocyte 

progenitor cells, PC12 cells) to chlorpyrifos at concentrations thought to be so low as not 

to inhibit AChE result in increased levels of reactive oxygen species and oxidative 

damage (measured as lipid peroxidation). 

As mentioned in the response to Charge Question 2.2, there are additional effects that 

should be included in the EPA review, namely, the effects of chlorpyrifos on nerve 

growth factors (Pope et al., 1995; Slotkin et al., 2007; Betancourt and Carr, 2004; Terry 

et al., 2007) and mitochondrial morphology (Middlemore-Risher et al., 2011). Many of 

these effects have been observed in the absence of AChE inhibition, or at concentrations 

below which acetylcholinesterase inhibition would be predicted.

In summary, these lines of evidence suggest that chlorpyrifos can affect 

neurodevelopment at levels lower than those associated with AChE inhibition, and that 

the use of AChE inhibition data may not be the most appropriate for dose-response 

modeling and derivation of a point of departure for assessment of the 

neurodevelopmental risks of chlorpyrifos.

The Panel suggested additional research that could answer the critical question of 

whether chlorpyrifos induces neurodevelopmental effects in humans at doses that do not 

cause AChE inhibition. This suggestion was to test whether the chlorpyrifos levels 

measured in cord blood that were associated with neurodevelopmental effects in the 

Columbia study would result in either red blood cell or brain AChE inhibition. This 

study could be easily performed by EPA researchers, or by others.

Additional concern about the use of AChE inhibition dose-response data to protect 

against neurodevelopmental effects was based on the potential for the outcomes of AChE 

inhibition and adverse neurodevelopmental effects to be two separate observations, in 

which the former is the result of an acute exposure scenario and the latter is likely to be 

caused by chronic low level exposure to chlorpyrifos in utero. All 3 cohort studies report 

neurodevelopmental outcomes associated with maternal or in utero chlorpyrifos exposure 

measures, which are considered to be representative of chronic exposures during the 

prenatal period. None of these studies assessed AChE inhibition or other acute responses 

to recent chlorpyrifos exposures. 

Additional questions and concerns about the use of the rodent AChE inhibition doseresponse data were raised. The AChE inhibition study that serves as the basis for 

selecting 0.03 mg/kg/day as the POD (BMDL10) for a benchmark response of 10% 
54

AChE inhibition is Maurissen et al (2000). In this repeat dosing study pregnant dams 

received daily doses of chlorpyrifos by oral gavage from gestation day (GD) 6 to 20, and 

red blood cell AChE inhibition was measured 4-5 hours after the last dose of chlorpyrifos

was administered. One question raised by the Panel is whether the time of AChE 

assessment was optimal to detect the peak inhibition effect. The Draft Issue paper 

provides no information on how the time of AChE assessment in this study was justified 

by the study investigators, although a general statement on p. 17 indicates that the peak 

inhibitory effect on AChE activity is typically within one to several hours after dosing. 

A second question broached by the Panel regarding Maurissen et al (2000) was whether 

inhibition of AChE had reached steady state in this study. The Panel noted that a similar 

BMDL10 for RBC AChE inhibition of 0.044 mg/kg/day was obtained from a companion 

study (Mattsson et al , 2000) that dosed dams for a longer period of time, i.e., from GD6 

to lactation day 10. This comparison suggests that steady state inhibition of RBC AChE 

likely had been reached in the Maurissen et al (2000) study.

A more important question is whether the dose-response for AChE inhibition in the 

pregnant rat is predictive of AChE inhibition in the human fetus. The Panel cautions the 

Agency on using pregnant rodent and rodent neonatal/juvenile data as the basis for 

deriving a point of departure for quantitative calculation of dose-response and risk 

assessment in human pregnancy and human children for the following reasons:

The AChE inhibition is caused by an oxon of chlorpyrifos that is produced metabolically 

by CYP450 (P450) metabolism. The isoforms involved include P450 1A2, a 2B isoform, 

3A4, 2C9 and 2C19 (there may be others). This presents the following problems with 

extrapolation from rodents to humans:

Several of these P450s are highly polymorphic in humans, which will cause 

considerable variation in human responses.

The polymorphisms existing in humans may be different from those in rodents.

Since rodents have different homologues and orthologues, metabolic activation rates 

and extents may differ between rodents and humans based on differing enzyme 

affinities for chlorpyrifos.

Several of these P450s are not active (or only active at very low levels) in the human 

fetal liver and arise in months-to-years after birth, yet their corresponding rodent 

P450s are commonly present in the fetal rodent liver. 

A positive suggestion in this respect is that much of the ontogeny work in humans and 

rodents has already been performed and ontogenetic differences are known. For human 

pediatric CYP ontogenies, the Panel recommended that the Agency explore the work of

Professors Ron Hines and J. Steven Leeder to determine qualitative and quantitative 

differences ((e.g., de Wildt et al, 1999; Pearce et al, 2001; Koukouritaki et al , 2004; 

Nong et al , 2006; Blake et al, 2007, Hines, 2007; Hines, 2008; Stevens et al, 2008)

Question b. The Agency does, however, believe that the epidemiologic data are useful to 

informing other key aspects of the chlorpyrifos risk assessment including hazard 

characterization, exposure characterization, and quantitative uncertainty characterization 

and analysis. Please suggest approaches/analyses for potentially using the epidemiology 
55

data in different parts of the chlorpyrifos risk assessment including those noted above.

(Note: Some of these may also be covered in Question 5.4 below.) 

Response

The framework for integrative analysis to evaluate multiple lines of evidence in the 

context of understanding the AOP/MOA proposed by the Agency is extremely helpful as 

a basis for framing thoughts on the weight of evidence and the integration of increasingly 

varied types of information, including epidemiological data. 

In relation to the specific use of the epidemiological data to inform key aspects of the 

chlorpyrifos risk assessment, this is likely best expanded beyond the scope included in 

the question – – i.e., “hazard characterization, exposure characterization, and quantitative 

uncertainty characterization and analysis,” since the epidemiological data are also 

informative in the context of dose-response analysis.

Although the panel was not explicitly charged with making a FQPA safety factor 

determination, one panel member suggested that the epidemiologic data, which represent 

a significant portion of the evidence base demonstrating increased sensitivity of early 

lifestages to the neurodevelopmental effects of chlorpyrifos, be used in selecting the Food 

Quality Protection Act (FQPA) factor to be applied in the risk assessment. The Panel 

recognizes that it is constituted as a technical advisory body, not a panel intended to 

provide policy advice. However, the choice to apply particular FQPA safety factors in 

the EPA’s risk assessment involves both policy and science. The FQPA safety factor 

recommendation is based on the scientific evidence provided to the panel. As discussed 

in detail in the responses to Charge Questions 4.1, 4.2, and 4.3.a, the strengths of the 

three longitudinal children’s cohort studies, the consistency of associations of 

chlorpyrifos with neurodevelopmental outcomes across these studies, and the large effect 

measures observed for serious long-term neurological effects (e.g., attention problems), 

coupled with data indicating that chlorpyrifos exposures in these cohorts were generally 

comparable with those of the general U.S population and unlikely to be associated with 

AChE inhibition, all suggest that in the event that the Agency continues to use doseresponse data for AChE inhibition to derive a point of departure, a FQPA factor of 

10=fold is recommended to protect sensitive early lifestages, 

Exposure Characterization: 

Environmental monitoring and biomonitoring data in the epidemiological studies 

contribute to the overall database on estimation of exposure, including (particularly) 

population variability and (to some degree) inter-individual variability in the study 

populations. They also provide insight into more generalizable observations on temporal 

trends in exposure of the general population – e.g., following the impact of withdrawal of 

domestic (nonagricultural) uses of chlorpyrifos.

The biomonitoring and environmental monitoring data from the three children’s cohort 

studies should be used, then, along with exposure information from other studies and 

sources, to characterize the levels of exposure to chlorpyrifos experienced in different 
56

populations (production workers, agricultural workers, individuals exposed via residential 

use, general population, etc.), and in similar populations over time (e.g., before and after 

cancellation of residential uses). 

Data available from the epidemiological studies also provide unique opportunity to 

investigate the relationship between environmental levels and results of biomonitoring 

(e.g., dose reconstruction as described by public commenter, Dr. Dale Hattis) since for 

some of the studies, both types of data (including air monitoring in the Columbia study) 

are available.

To some degree, the epidemiological studies can also provide sources of data to consider 

the suitability of the various biomarkers as measures of short and/or long term exposure 

to chlorpyrifos.

Toxicological Hazard Characterization:

The epidemiological data contribute to an evolving database on potential toxicological 

hazards to humans. They have contributed and continue to contribute to hypothesis 

generation for targeted investigations of developmental neurotoxicity in animal studies. 

To (limited) degree, they also confirm expectations concerning potentially susceptible 

subgroups based on mode of action – i.e., the PON 1 genotype. They also provide some 

information on the extent of impact of other factors, which in combination with 

chlorpyrifos, may have an impact on the observed effects. 

These studies represent the key datasets that support the identification of chlorpyrifos

prenatal exposures as causing neurodevelopmental effects in humans. Important 

elements to discuss in their evaluation include i) consistency in the findings of 

neurodevelopmental effects across these three studies, and ii) comparison of the levels of 

chlorpyrifos exposure experienced in these cohorts based on biomonitoring (blood and 

urine measurements of chlorpyrifos, metabolites, etc.) and environmental monitoring 

measures (e.g., personal air monitoring in the Columbia study) with data collected in 

other studies of the general U.S. population (e.g., NHANES), for similar time periods 

(i.e., pre- and post-cancellation of residential uses). 

Quantitative uncertainty characterization and analysis: 

It seems important to address this aspect in the context of dose-response analysis, given 

particularly, that EPA has concluded that the current epidemiological database 

strengthens the 2008 SAP conclusion that “chlorpyrifos likely plays a role” in observed 

adverse effects on child neurodevelopment (specifically those reported by Columbia 

University). There is also a need to address the consistent epidemiologic findings of 

significant, long-term neurodevelopmental effects across the three cohorts at levels 

within the same range as those in the general population since this would seem to suggest 

that these effects occurred at exposures below those associated with AChE inhibition. 
57

As a minimum, then, it seems important to maximally utilize available data on doseresponse from these studies to at least “bound” reference doses developed on the basis of 

animal data (Given that this was also recommended by the 2008 SAP, prioritization of 

this work seems critical.). However, the scientific weight given to the different measures 

of dose and of response necessarily needs to take into consideration that most of the 

effort in the epidemiological studies has been directed to the assessment of outcome 

rather than exposure. In addition, the use of different exposure matrices (urine, maternal 

blood, cord blood, and meconium) and the difference in the targeted analytes (TCPy, 

DAPs, and chlorpyrifos) complicates derivation of the POD based on epidemiological 

data, uncertainties which need to be assessed in dose-response evaluation and risk 

characterization. 

In addition, given the potential significance of the observations in the epidemiological 

studies, it is also clearly desirable to consider at least semi-quantitatively the potential 

impact of factors of study design and interpretation that bound the dose-response 

relationship from the human studies. It would be helpful, for example, to consider 

systematically (and at least semi-quantitatively) the potential impact on the reported 

dose-response analysis of exposure measurement error, outcome ascertainment, 

confounding variables and statistical analysis.

For example, in relation to limitations of data on exposure in the epidemiological studies, 

a Panelist noted that despite a fairly high portion of the samples whose results were 

below the limit of detection or quantification for whatever was being analyzed, little use 

was made of techniques to integrate non-quantified samples into the statistical test. [One 

of the studies utilized a method described by Richardson and Ciampi (2003).] Various 

methods were reviewed by the July 2010 SAP that can be applied to either normally or 

lognormally distributed data that include a significant (even a majority) of non-detectable 

sample Specifically, the use of “probability plots” was described that can yield an 

estimate of the geometric mean of the distribution [GM], the geometric standard 

deviation [GSD], and corresponding percentiles. Various aspects of the technique are 

described in publications such as Cunane (1978), Haas and Scheff (1990), Travis and 

Land (1990), Helsel (1990), Hattis and Burmaster (1994), and Hattis et al. (1999). 

Another method called the “maximum likelihood estimate” is not recommended for data 

sets with a large number of measurable values (Cohen, 1961; Perkins et al., 1990).]

As a basis to increase the confidence in the selected point of departure, a relatively simple 

experimental protocol to determine whether chlorpyrifos levels measured in the cord 

blood in the Columbia study inhibit either red blood cells or brain AChE inhibition would 

be helpful. This seems to be an important priority, given that human data (e.g., coupling 

of chlorpyrifos measurements in cord blood with neurodevelopmental measurements 

from the Columbia study) would typically be preferred in estimating dose-response 

relationships (and particularly for potentially susceptible age groups, such as infants). 

The outcomes from the above exercise should contribute to consideration of the critical 

question of whether or not “a causal association between chlorpyrifos and 

neurodevelopmental effects in the absence of AChE inhibition is plausible for humans.” 
58

Given that AChE inhibition results from acute exposure and adverse neurodevelopmental 

effects are likely to be caused by chronic low levels of chlorpyrifos, it is important to 

verify whether or not maintaining long-term exposure to levels below those likely to 

cause AChE inhibition is likely to be sufficiently protective to prevent 

neurodevelopmental effects. 

With regard to quantitative uncertainty characterization, the results and the uncertainties 

associated with the dose-response analysis of the neurodevelopmental epidemiology 

findings should be taken into consideration, along with uncertainties in the dose-response 

assessment for acetylcholinesterase inhibition, data gaps and database uncertainties 

regarding whether neurodevelopmental effects or acetylcholinesterase inhibition is the 

most sensitive endpoint in humans, and uncertainties associated with pharmacokinetic 

differences due to lifestage and genetic polymorphisms in metabolic enzymes.

Question 5.0 Exposure Profile & Biomonitoring Research

Question 5.1 

c. Section 5 of the draft issue paper presents an overview of the principal chlorpyrifos

biomarkers and a comparison of biomonitoring studies that measured urinary TCPy 

levels in a range of study populations involving both the general population and 

potentially vulnerable populations, including children, workers, and farm families.

Please comment on the degree to which the Agency identified the primary 

chlorpyrifos biomarkers of exposure, appropriately discussed the strengths and 

limitations of such biomarkers, and how the strengths and limitations affect the 

interpretation of the chlorpyrifos biomonitoring data. 

Response

The draft paper was thorough in its coverage of the literature on chlorpyrifos and its 

biomarkers of exposure. Considering the availability of standard methods, the specificity 

of the biomarker, the number of laboratories capable of making the measurement, and the 

relevant concentration levels, the first choice for a biomarker would be chlorpyrifos in 

blood. The Panel recognizes that this is the most difficult assay and represents only a 

small percentage of the literature, but it s deemed to be the highest priority because of its 

specificity.

The next biomarker of choice is TCPy, then DETP/DEP in urine. These have roughly the 

same equivalence and neither is close to the validity of measuring chlorpyrifos directly in 

blood because they are both present in the environment as degradates of the active 

ingredient. Total DAPs (as DMP and DEP) are not selective enough to be a useful

biomarker for chlorpyrifos although it may be more appropriate in a global risk 

calculation model because all AChE inhibiting chemicals should be considered together 

when evaluating risk. The Panel recognizes the inability of urinary TCPy to distinguish 

between exposure of chlorpyrifos, chlorpyrifos-methyl, trichlopyr as well as direct 

exposure to TCPy (a chlorpyrifos degradatein the open environment). However, TCPy is 

more selective than any of the DAPs and currently is the most selective of the urinary 

metabolites but questions remain about its efficacy because the Panel believes there could 
59

be significant contribution from environmental and dietary TCPy as a chlorpyrifos

degradate.

More emphasis should be placed on the direct intake of the environmental degradate 

TCPy, mainly present in foods. As early as the late 1990s, the Ryan group (See 

MacIntosh, et al., 1999) had identified an anomaly in that the amount of TCPy found in 

urine was substantially greater than the measured likely intake of chlorpyrifos. This work 

has continued with the papers by Morgan, et al., Wilson, et al., and Lu, et al. 2005, 2008, 

indicating the presence of degradates in foods. Radford et. al., 2012 have continued this 

work on the kinetics of this process. While this work has not yet been published, and the 

other works are mentioned in the Issues Paper, insufficient emphasis has been placed on 

the presence of TCPy in food or other exposure media (dust, air particulate, etc.) putting 

into question the utility of urinary TCPy as a useful measure of exposure to the parent 

compound. During the discussion it was pointed out that Lu et. al., 2005 found that 

roughly 30% of the TCPy measured in urine could be coming from TCPy directly,

present in foods.

The Panel also recognizes the ability to measure AChE and BuChE as biomarkers of 

exposure, but they are even less specific than DAPs. They are however more indicative 

of potential health risk and are more than just a biomarker of exposure. Unfortunately the 

ability to measure these enzymes is likely to vary widely from lab to lab and method to 

method as they are difficult to calibrate. Changes in cholinesterase activity after an 

exposure should probably be evaluated more within a laboratory (especially via the use of 

an unexposed control group) than across laboratories or from study to study. 

From its earliest years, measuring AChE has been subject to unresolved inter-day 

variability (Gage, 1967). For instance, Grob and Harvey (1958) could measure AChE in 

replicate samples on one day with a standard deviation of ±3% but only to within ±5% on 

separate days following storage of hemolyzed RBC. The literature dating as far back as 

Gage (1967) has recommended that in order to measure small changes within an exposed 

group's cholinesterase activity, researchers should collect blood from an unexposed group 

of controls, measure the cholinesterase activity in their blood at the same time as the 

exposed group, and apply a correction factor based on the daily change in the mean of the 

measured activity in the unexposed group of controls. Yager et al. (1976) collected 10 

blood samples from 10 unexposed people over five weeks and found that the measured 

intra-individual coefficient of variation for RBC enzyme activity of ±10% could be 

reduced to ±6% by controlling the day-to-day component of the variance (i.e., accounting 

for a shift in the average laboratory results from one day to the next). They also found 

that plasma activity is more variable between individuals but less variable day-to-day. 

Similar findings for plasma ChE were reported by Trundle and Marcial (1988) and Brock 

and Brock (1990). 

In an occupational (or other repeated exposure) dose-response study, it is generally costeffective to adjust the blood ChE results of each member of the exposed or "test" group 

for the change in the laboratory's reported mean blood ChE of an unexposed or "control" 

group analyzed at the same time (typically the same day as the post-exposure group or in 
60

the same batch if they were stored). This adjustment has traditionally been made in 

proportion to the change in the mean AChE of the unexposed controls; however, this 

form of adjustment could also be applied to plasma ChE except using ChE values. The 

more accurate fraction of inhibited enzyme [ΔAChE] would be calculated for each 

subject using this adjusted activity. The final variance of the group would decrease in 

proportion to the square-root of the number of subjects within the study.

In the future the phase II conjugation products of chlorpyrifos (namely, glucuronides and 

sulfonates) should be considered. Quantifying conjugative metabolism will ensure that 

levels of biomarkers are correctly interpreted with respect to biomonitoring data and for 

performing reverse dosimetry. Even though the AChE adducting oxon is not conjugated, 

the TCPy and DETP metabolites are extensively biotransformed by the glucuronosyl 

transferases and sulfotransferases, although the precise isoform pathways are not yet 

known. Therefore, particularly in the fetus and child, if glucuronidation or sulfonation 

are saturated and/or ontogenetically deficient, then TCPy and DETP may accumulate. 

This would almost certainly cause error in biomarker analysis through overestimates of 

exposure. Moreover, accumulation of these metabolites may present the opportunity for 

direct metabolite toxicity. Panelists noted there has not been significant effort to look at 

either the glucuronide or sulfate metabolites possibly because these metabolites have only 

recently been evaluated both from a physiological and analytical perspective. 

The oxon is believed to be the most toxic of the metabolites of chlorpyrifos and is not an 

environmental degredate. While the oxon does not exist long in the blood, a method to 

directly measure the oxon in blood is likely to be available in the near future. As the 

most toxic form and an exclusive measure of exposure to chlorpyrifos, the chlorpyrifosoxon may be the most predictive biomarker of risk, once a method is published. 

When evaluating any of the biomarkers in blood, the EPA will need to consider that some 

of these biomarkers will differ in concentrations between cord blood and maternal blood 

as they will have different lipophilicity. 

Measuring multiple metabolites simultaneously and then taking ratios of metabolites such 

as TCPy/DETP represents an untested route to provide greater discrimination between 

exposure to chlorpyrifos and its degradation product TCPy. However, the Panel could 

find no direct studies on the stability of these two degradates in the environment. By 

using this ratio and assuming that the ratio of degradates-to-active in the environment is 

different from the 1:1 ratio that results from metabolism, it may even be possible to do a 

source apportionment and separate exposure to the degredate from exposure to the active

ingredient. 

Other considerations:

The Agency suggested in their public presentation that meconium could be used as a 

biomarker of fetal chlorpyrifos exposure throughout pregnancy. chlorpyrifos in 

meconium represent the unmetabolized pesticide. The metabolized form oxon or TCpy 

may have arrived at the fetus in that form rather than having been metabolized by the 
61

fetus itself. Although there should be chlorpyrifos or metabolites in amniotic fluid if it is 

found in meconium, no studies exist as to the residence time, flow or amount. It is 

conceivable (if not totally likely) that chlorpyrifos and metabolites are sent directly into 

the fetal blood (across the placenta) and that meconium picks it up from sloughed cells. 

More importantly, the utility of meconium as a cumulative biomarker is uncertain. More 

specifically, the Panel suggests that this is not (currently) a good idea for the following 

reasons:

1) No studies of chlorpyrifos in amniotic fluid have been performed. The ratio of 

aminotic fluid chlorpyrifos to metabolite may add evidence that the fetus is actually 

metabolizing the chlorpyrifos (if they are developed enough to metabolize the 

chlorpyrifos). 

2) The diffusion and/or transport of chlorpyrifos across the placenta (in either 

direction) is unknown, but since it is rather fat soluble, equilibration with maternal serum 

might be postulated. This does not seem to be the case with at least one umbilical:

maternal serum study presented at this meeting (Yan, 2010).

3) Metabolism across the placenta is unknown. Are the metabolites passed or only 

the chlorpyrifos, possibly the oxon?

4) The contribution of umbilical tissue, including any adducting of cord tissue for 

example by the oxon is unknown. However, since umbilical tissue is so well perfused, it 

may be expected to be a target for oxon binding. Umbilical cord tissue consists of a 

polymatrix of Wharton's jelly, which is made up of mucopolysaccharides. (Kliman, 

1998). Based on these characteristics, the very fat-soluble nature of chlorpyrifos and its 

relatively fat soluble metabolites, the umbilical cord would not be expected to function as 

a good reservoir of the parent compound or metabolites, but may be a target for oxon 

binding and deregulation of pregnancy homeostasis.

5) Meconium, being composed mostly of intestinal epithelia, lanugo, mucus, 

amniotic fluid, bile, and water, is reasonably hydrophilic and thus should also be 

considered a poor matrix (reservoir) for chlorpyrifos and other fat-soluble xenobiotics to 

accumulate. It may be marginally better for the metabolites TCPy and DETP, but these 

molecules would also, in addition to diffusion, have some net flow in the paracellular 

pathway. Thus, they may over represent the exposure to chlorpyrifos (as described 

above).

6) Human placental studies of chlorpyrifos metabolism and transport have not, to the 

best current knowledge been published, and this is a limitation of data available to the 

Agency. Several important points are already known from the illicit drug literature and 

should be considered when attempting the same type of monitoring for chlorpyrifos (or 

other xenobiotics). For example, antipyrine (an amphetamine derivative) is used in 

placental perfusion experiments as a marker of pure diffusive transport with effectively 

no barrier (Schneider et. al. 1972). In contrast, cocaine and cotinine show differing and, 

slightly less fat-soluble profiles. For example, cocaine is transferred across the placenta 
62

at only 80% the rate of antipyrine (Schenker et. al.,1993) and some studies have 

suggested that the placenta acts as a depot for cocaine accumulation preventing transfer 

to the fetus (Simone et.al. 1994). Additionally, previous studies have indicated that while 

nicotine (again highly fat soluble) is transferred into the fetal compartment up to 5 times 

the concentration in the maternal blood, cotinine concentrations in the fetal compartment 

were considerably lower than corresponding maternal serum levels (Luck et. al. 1985). 

Again, it has been suggested that cotinine adducts the placenta, preventing equilibration 

of concentrations between maternal and fetal systems. These studies support the need for 

greater consideration of the trans-placental characteristics of chlorpyrifos, and since 

placental characteristics change drastically by term (the placental barrier becomes 

increasingly “leaky” after ~36 weeks), placental studies need to consider each trimester. 

In the first trimester, the placenta is perfused only after ~8 weeks; prior to 8 weeks only 

active transport or diffusion across the placenta can occur because villi are being blocked. 

Analgous studies for chlorpyrifos are recommended before extrapolating fetal exposure 

and may be included as part of a longitudinal study in pregnancy. Such a longitudinal 

study may present additional problems. 

The real question for the Agency is almost certainly not related to the fetal load of 

chlorpyrifos or its metabolites at birth or even at discrete pregnancy time points. The 

exposure information (fetal load) needs to be correlated to a time in fetal development 

when the fetus is susceptible to effects of chlorpyrifos, perhaps during critical points of 

neurodevelopment. Unless the time of these exposures can be definitively correlated with 

specific adverse health effects, then consensus on how to relate fetal effects to a 

biomarker concentration is unlikely. Rather, the Agency seems to be seeking to quantify 

the amount of maternal chlorpyrifos ingestionor/exposure is subsequently experienced by 

the fetus. In general, the half-lives of chlorpyrifos and its metabolites are rather short. 

This means that even in the case of TCPy, which has the longest systemic residence time, 

the terminal half-life (i.e.,. complete clearance of TCPy from the fetal compartment) 

would occur within several days. Therefore, unless the pregnant woman is exposed to 

chlorpyrifos either chronically or acutely exposed but within a few days of testing, 

quantifying the chlorpyrifos exposure of the fetus would be difficult. It would require

collection of samples from pre-term as well as [full or near full term fetal tissues or 

sampling directly from placentas (such as chorionic villus sampling), amniotic fluid 

(amniocentesis), or umbilical blood. A longitudinal study would almost certainly be 

needed to determine exposure over pregnancy, which may not be cumulative but 

pulsatile. 

These points highlight the uncertainty of using meconium as a measure of exposure over 

the course of pregnancy at this time. Essentially, production of meconium is from fetal 

swallowing of amniotic fluid as well as some sloughing of intestinal epithelia, and 

meconium should not be thought of as a matrix into which chlorpyrifos or its metabolites 

may accumulate by simple diffusion through fetal tissues. 

d. Section 5 of the draft issue paper compares biomonitoring findings from the three 

children’s health cohorts with other major observational exposure studies in the United 

States. Based on comparison with NHANES 2001-2002, median TCPy levels in the 
63

CHAMACOS and Mount Sinai cohorts were slightly higher than in the general 

population. It should be noted that the exposures experienced by the CHAMACOS and 

Mount Sinai cohorts overlapped the start of the residential chlorpyrifos phase-out. By 

contrast, median TCPy levels in the Columbia cohort, for which sampling occurred when 

chlorpyrifos use should have rapidly declined due to the voluntary cancelation, were 

slightly lower than the levels measured by NHANES in the general population. Please 

comment on the adequacy of the Agency’s comparison for the purposes of evaluating 

chlorpyrifos exposure levels in the three children’s health cohorts. Are there any 

additional biomonitoring studies that should included in the Agency’s comparison?

Response

The human studies discussed in this section are the best available. They were carefully 

designed and well implemented. They do, however, look at specific types of exposure: 

agriculturally based exposure and exposures in city dwelling units likely treated for 

insects on a regular basis. Further, they span a range of times from when chlorpyrifos use 

was ubiquitous through the phase-out of indoor uses of the insecticide. Because of this, 

there are “inconsistencies” in the data that are indicative of changes in use patterns. 

Current use in indoor settings is dominated by pyrethroids rather than chlorpyrifos. 

Agricultural settings are still likely to see large exposures to chlorpyrifos (although 

apparently not in the county surrounding Salinas, CA). There appears to be inconsistent 

recognition of this change, especially in light of comparisons with “group norms” via, for 

example, the NHANES studies. It would be to no one’s surprise if the 1990-2000 

NHANES data indicate higher exposures to chlorpyrifos in residential settings than the 

later data. Among these three studies the Panel believes the Columbia study has a 

particular importance because it has data collected before and after the indoor use “ban,” 

and the results reflect the pathway from exposure to biomarker concentration and health 

outcome.

The Panel recommended the following order in which the studies should be considered. 

They believe that the next NHANES data set may be the most important as it is likely to 

reflect the decrease in exposure caused by the voluntary removal of chlorpyrifos from the 

home market. If the levels progress in a manner similar to those predicted by the 

decrease demonstrated in the Columbia study, the risk from chlorpyrifos might also 

decline as rapidly. Even if this is true, chlorpyrifos as a model compound for a risk 

paradigm that includes epidemiological, dose reconstruction, PBPK modeling, and 

exposure dosimetry, requires a much broader consideration of studies. The Agency 

seemed to concentrate on studies that include a reported health outcome, and the Panel

wonders why these studies were the principal focus as many studies provide data on 

exposure and dose. For example to be protective the agency should consider the National 

Human Exposure Assessment Survey, (NHEXAS-Az, summarized in Egeghy et al. (2011)

study many of the participants from Arizona were exposed through agricultural 

application of chlorpyrifos and this represents the highest non manufacturing level of 

exposure and may continue to represent direct or indirect agricultural exposure.

Many of the studies listed in the draft paper but not directly discussed should be 

considered when estimating dose and subsequently risk. The NJ studies where cord 
64

blood measurements were used as the principal sample type are important because that is 

likely to be the desirable biomarker and used more frequently in future studies. Farmworkers studies are important because their families are likely to be one of the remaining 

populations that continue to see significant exposure, again an expectation to be validated 

by the next round of NHANES data. They should however probably be considered 

primarily in relation to farm workers’ families. The Children's Pesticide Exposure Study

(CPES) by Lu, 2009 and Children's Post-Pesticide Application Exposure Study 

(CPPAES) studies are important because they provide data on multiple exposure 

vehicles/media and will be especially useful in dose reconstruction. Dose reconstruction 

will be paramount in validating PBPK models using media (dust, food, air particulate) 

measured concentrations and estimated exposure levels, to be subsequently discussed 

sections 5.3. Among the current studies those that look at both the urinary concentrations 

and the media where the exposure is likely to occur, will provide the best models for 

closing the knowledge gap between exposure and dose; and studies where urine was 

collected within one half-life after a fresh exposure may provide the most useful 

information.

Although not ready for this report, studies now underway that are longitudinal in design 

will afford a better understanding of actual exposure profiles when compared to crosssectional approaches. Due to the short biological half-lives of the metabolites of 

chlorpyrifos in the body, a spot check of a relatively small number of people may not be 

enough to represent the exposure of a vulnerable population at key time periods. Only a 

longitudinal investigation can get at these important data.

Several new studies of interest to this group have been completed and will be published 

in the near future that are. The Children’s Pesticide Exposure Study, led by Dr. Alex Lu 

of Harvard School of Public Health, focuses on dietary intake of children and related 

pesticide exposures. The Children’s Exposure to Environmental Pesticides, led by Dr. P. 

Barry Ryan of Emory University, evaluated the utility of biomarkers of pesticide 

exposures, e.g., DAPs and pesticide-specific markers of OP and pyrethroid exposures, 

and environmental levels measured in soil, house dust, and food. The target population is 

children ages 3-6. The SAWASDEE study, led by Drs. Dana Boyd Barr and Ryan, and 

Dr. Tippawan Prapamontol of Chiang Mai University in Northern Thailand, examined 

pesticide biomarker concentrations in pregnant mothers, and similar markers in their 

newborn children. Multiple measurements in both urine and serum have been made 

throughout pregnancy giving a better longitudinal picture of exposure. Several smaller 

investigations are underway designed to evaluate the direct intake of pesticide degradates 

and to evaluate the kinetics of the degradation process in environmental media, including 

food.

In comparing the results from study to study, it is important confirm that analytical 

results are directly comparable. Data quality of some studies has been called into 

question due to apparent changes in limits of detection associated with two analytical 

methods developed by Center for Disease Control (CDC) used to evaluate serum 

chlorpyrifos concentrations. The questions arose due to a misunderstanding of the 

methods. There is an apparent 20-fold difference in the limits of detection (LOD)
65

between the two methods. This can be accounted for in three ways. First, the “newer” 

method uses a sample size one-half as large as the “older” method, and injects one-half as 

large an aliquot thereby accounting for a factor of four difference in LOD. Second, 

although both methods are multi-contaminant, the newer method spans a much larger 

range of analyte polarities. In order to obtain adequate recoveries for some of the less 

polar compounds, there is some sacrifice in sensitivity toward more polar compounds, 

such as chlorpyrifos. 

Third, the newer method was developed with the expectation that higher concentrations 

would be evident in the samples analyzed, hence precluding the need for a lower limit of 

detection; a listed limit of detection of 10 ppb was adequate for the purposes of the study. 

Attribution to the new, higher limit of detection to samples analyzed by the older, more 

sensitive method, is therefore not warranted. The value for the LOD determined for the 

earlier method should be viewed as appropriate for the samples analyzed by that methods 

and deemed useful for presentation in any other work.

In addition to analytical differences these studies (Columbia, CHAMACOS and Mount 

Sinai) are all cross-sectional in design with some repeated measurements during the 

pregnancy period. Because of the cross-sectional design coupled with the short 

biological half-life of chlorpyrifos, the spot urine measurement would be highly affected 

by daily chlorpyrifos exposure, as well as the timing of sample collection. It would be 

great if all three epidemiologic studies were using the identical sampling protocol so the 

outcome measurements could be compared across the board they weren’t. Generally 

speaking, it should not be surprising either to see the similarities in the CHAMACOS and 

Mount Sinai cohorts during the period they overlapped the Columbia study, before the 

residential chlorpyrifos phase-out. It is likely that dietary exposure to chlorpyrifos in 

these two cohorts may represent a portion of the overall exposure. However, the 

Columbia cohorts may differ from the CHAMACOS and Mount Sinai cohorts because 

the Columbia study reported the reduction of chlorpyrifos in the indoor air after the 

phase-out.

Question 5.2 

In Section 5.0 of the draft issue paper, the Agency summarized the 2008 preliminary 

findings on the association between urinary TCPy levels and AChE/BuChE inhibition 

and discussed two recent studies involving manufacturing workers in the US and Egypt. 

Please comment on the scientific quality of these studies and their findings. Please 

include a discussion of their strengths and limitations. Please comment on the strengths 

and limitations of the evidence from this research to show an association between TCPy 

and AChE/BuChE inhibition at exposure levels experienced by occupational populations.

Response

Both of the occupational exposure studies were observational in nature. Garabrant et al. 

(2009) involved 53 workers manufacturing chlorpyrifos in Michigan, while Farahat et al. 

(2001) involved 38 field workers applying chlorpyrifos onto cotton plants in Egypt. Both 

of these studies contain data that have multiple sources of imprecision (as will be detailed 

below), but they both included enough participants that their overall results match PBPK 
66

model predictions quite well. In many ways both studies were well designed and 

implemented. Both studies had sufficient power to show an association between TCPy 

and AChE or/and BuChE inhibition at exposure levels experienced by occupational 

populations. In fact, the PBPK model and cholinesterase data confirm that chlorpyrifos

once absorbed interacts first with BuChE and only starts to inhibit RBC AChE and AChE 

in the central nerve system after BuChE is more than 50% inhibited. 

Perhaps the most unique feature of the Farahat study was the extremely high levels of 

TCPy found in urine from these field workers after applying chlorpyrifos to the target 

cotton fields. For example, the mean post-exposure values of urinary TCPy were about 

25× more than the TCPy from the manufacturers reported by Garabrant et al. and over 

1000× more than those in the women and children cohorts discussed in Section 4. On the 

one hand, the Panel pointed out that this contrast made this study less relevant to our 

discussion. On the other hand, a major strength of the study is that not only were the 

qualitative patterns of both BuChE and AChE activities when paired to urinary TPCy 

from the same individuals qualitatively similar to the patterns predicted using the PBPK 

model described by Timchalk et al. (2002) and used by Garabrant et al. (2009), but the 

“inflection points” within the paired data closely match those predicted by the PBPK 

model. This correspondence between the measured and predicted TCPy excretions and 

cholinesterase inhibitions is strong evidence for the robustness of the PBPK model over a 

wide range of exposures. 

The Panel pointed out five weaknesses within the Farahat study for use within the weight 

of evidence. First, virtually all of these field workers had high levels of TCPy in their 

pre-exposure urine samples. These background concentrations (with sub-group means 

ranging from 10 to 2000 μg TCPy/g creatinine) were up to three orders of magnitude 

higher than the levels in the women and children epi cohorts. The source of this 

background is unknown but seems likely to have been due in large part to these workers’ 

prior use of chlorpyrifos outside of the jobs being studied and possible contributions from 

TCPy on chlorpyrifos treated food and from TCPy or/and chlorpyrifos within homes 

treated with chlorpyrifos. Second, the urine samples were collected from morning voids 

that a study by Lu et al. (2006) found to be less reliable than evening voids. Nonetheless, 

these high background TCPy levels jumped about 30× after the applications began. 

Thirdly, the cholinesterase values were measured by the battery-powered kit based on the 

Ellman method. Prior publications (including one by the same researchers who 

participated in the Farahat study) concluded that cholinesterase activities measured by 

those field test kits are not as reproducible either from kit-to-kit or as a function of 

temperature as those using more robust clinical methods (Oliveira et al, 2002; Hofmann 

et al., 2008). Fourth, the study was not designed to analyze blood samples from an 

unexposed control group concurrent with blood from their field workers; the importance 

of such control was discussed in the Panel’s response to Charge Question # 5.1. This 

deficiency further weakens the precision of their cholinesterase results which was offset 

somewhat by having 38 participants. Lastly, for reasons not stated, the authors chose to 

report (and plot) individual cholinesterase activities rather than inhibitions in comparison 

to individual baseline values. Thus, the reader is led to believe that the ratio of an 

activity of 2 U/g Hgb for the individual with the lowest AChE and the highest TCPy to 
67

about 25 U/g Hgb for the cluster of individuals with the highest AChE and lowest TCPy 

measurements (in their Figure 3) represents an inhibition of almost 90%. While some 

found this degree of inhibition incredulous, Grob et al. (1947) and Grob and Harvey 

(1958) showed that a sequence of small oral doses of an OP (DFP) delivered over three to 

five days can cause someone's AChE to be reduced down to about 1% of their normal 

level or to be 99% inhibited) but still not cause symptoms if delivered slowly enough. 

But of course, a fractional ΔAChE inhibition of 30-50% in one day can cause acute 

symptoms (e.g., Gage, 1967; Reigart and Roberts, 1999). Thus, the idea that any 

particular level of ΔAChE either is or is not clinically important depends on more than 

just its numeric value. Another troubling observation in Farahat et al. (2011) is the 

persistent elevated TCPy measurements in some of the workers and the persistent 

depressed RBC AChE 14 days post-application; perhaps these lingering effects are linked 

to the high preexposure levels or the inhibition may have “aged.” With these caveats, not 

only does the pattern of paired levels of AChE activity and concentrations of TCPy in 

urine qualitatively match the pattern predicted by the Timchalk PBPK model, but also the 

value of the mean of four measured AChE inflection points at 3161 μg TCPy/g creatinine 

quantitatively matches the inflection point predicted for AChE by that model.

The study reported by Garabrant et al. (2009) has some broadly similar and some 

different weaknesses for use within the weight of evidence. One different weakness is 

the greater potential for a proportion of the chlorpyrifos employees’ urinary TCPy to 

have come from doses of residues of TCPy that might have accumulated within the 

manufacturing workplace (Burns et al., 2006). This study added urine collection to an 

on-going occupational health monitoring program that involved monthly blood samples 

that were analyzed for cholinesterase via a proprietary system (Vitros by Johnson & 

Johnson) with which the Panel was not familiar. The time at which the pre-exposure 

cholinesterase was measured was not stated but could have been some years earlier. 

Despite the study having a “referent group,” there is no indication that the cholinesterase 

results for the chlorpyrifos workers were adjusted for variations in the results of blood 

samples from an unexposed control group (again see CQ#5.1). The three urine samples 

per person collected in this study were also collected in the morning (first voids in this 

case); however, an additional source of uncertainty was introduced into the results of 

Garabrant et al. because the blood and urine samples were collected between 5 and 14 

days apart. The authors concluded that conducting paired analyses using only the 48% of 

the urine results that were collected within 7 days of a blood sample was optimum; 

however, this interval spans several half-lives for TCPy within the human body. The 

range of ΔAChEs reported in this study slightly exceeded ±20% but, as predicted by the 

PBPK model, showed no correlation with TCPy. Only the BuChE inhibition could be 

attributed to chlorpyrifos exposures. Indeed, the inflection point for ΔBuChE found by 

this study (110 μg TCPy/g creatine)) not only matched that found by Farahat et al. (114 

μg TCPy/g creatinine) but also matched that predicted by the PBPK model.

In the responses to Charge Questions 4.2 and 4.3, the Panel suggested that the Agency 

should separate scenarios for occupational exposures, as reported in these two studies, 

from exposures from environmental sources. Indeed, one panel member suggested that 

data from Farahat et al. (2011) should not be considered for any further uses. It should 
68

be noted that the subjects in these two studies were adults. Although participants in 

Farahat’s study were as young as 15 and roughly 25% of participants in Garabrant’s 

study were females, none are directly comparable to newborn infants. Even the 

extrapolation of any working population to the population as a whole is subject to 

criticism. Such criticisms include the “healthy worker effect” and the idea that low-level 

exposure and high-level exposures are likely to be detoxified by differing mechanisms. 

Studies of agricultural workers and their families could offer a better avenue of 

investigation that compares “occupational-levels” exposure with other members of their 

families likely see slightly “elevated” but lower levels of exposure, and to study the 

potential impact on the offspring in such cohorts exposed either in utero or otherwise. In 

the future, the Agency should take into account the quality of ChE measurements prior to 

further uses in the exposure and risk assessments.

Question 5.3 

Several approaches ranging from qualitative to the most sophisticated PBPK/PD

modeling approach were introduced as potential options for analyzing the chlorpyrifos

biomonitoring data. Please comment on the strengths and limitations of these 

approaches. In addition, please suggest, if appropriate, alternative approaches or 

analyses not identified by the Agency. 

Response

The increasingly data-informed options for interpreting biomonitoring data presented by 

the Agency range from qualitative (non-comparative, looking at trends or comparative, 

taking into consideration controlled human studies data where ACN inhibition has been 

measured) to semi quantitative approaches (estimating biomarker levels associated with 

regulatory exposure guidelines or estimating exposures from biomarker levels using 

reverse dosimetry or a PBPK model). 

Presentation of a number of options in an increasingly data-informed construct of this 

nature has potential to maximize the use of biomonitoring data for different applications 

(accounting internally for more factors contributing to variability in exposure than do 

external estimate), taking into account (relative) uncertainty depending on: availability 

and specific nature of biomonitoring data, and the intent of use (i.e., what degree of 

uncertainty is acceptable for the intended purpose; what population; and what 

application?).

The selection of appropriate options is necessarily dependent on the extent of the data 

available on toxicokinetics relevant to the population subset and mode of action, and their 

integration, with a verified PBPK model having the potential to be the most informative, 

but being the most data intensive. In relation to intended application, for example, if the 

objective is media specific assessment or management, dose reconstruction (reverse 

dosimetry) from biomonitoring data is required. 

As a minimum, currently, the biomonitoring data on chlorpyrifos should be helpful in 

“ground truthing” total external exposure estimates under various use conditions, which 
69

are necessarily based on many more assumptions such as activity patterns and intakes and 

concentrations in various media.

Given the availability of biomonitoring data on chlorpyrifos in the general population, 

and as a basis to encourage its maximal consideration in a public health risk context, the 

Agency is also encouraged to seriously consider the development of a value akin to a 

“biomonitoring equivalent” concurrently with the derivation of a reference dose for 

chlorpyrifos. (A biomonitoring equivalent (BE), is a calculated level of a biomarker 

associated with exposures consistent with health protective guidance values for the 

general population). This BE would provide a valuable addition for interpretation of 

population biomonitoring data with limited additional effort, drawing efficiently on the 

existing process for review and consultation for the regulatory assessment (i.e., BEs are 

based on similar considerations as the reference dose but incorporating toxicokinetic 

translation to internal doses).

Clearly, a verified PBPK model provides the most robust opportunity to integrate the 

considerable available data on external and internal exposure (i.e., biomonitoring) to 

chlorpyrifos at different life stages under different conditions of exposure. 

As indicated in the response to Q. 5.4a), prediction of excretion by the PBPK model can 

potentially be validated or verified with an accurate estimate of dose, through dose 

reconstruction based on data from the epidemiological studies on the concentration of 

chlorpyrifos in media such as house dust, air and water combined with market basket data 

on the concentration of chlorpyrifos on food. This would permit the effective prediction 

of exposure at the critical windows of maximum effect (i.e. AChE suppression) with 

measured urine concentrations. However, it’s somewhat unclear currently based on input 

at the meeting from Dr. Bartels of Dow Chemical and Agency staff whether or not the 

developed PBPK model is life-stage specific. In the interest of addressing this need, the 

following recommendations are offered: If an adult PK or PBPK model is used, simple 

allometric scaling (3/4 power) or scaling based on Wang’s modification of the Dubois 

and Dubois equation (Wang et al., 1992) can be useful, relatively accurate and robust for 

extrapolating to children (Anderson et al., 2009; Anderson, 2010). This is a simple way 

to improve prediction for pediatric populations. Moreover, plasma proteins differ 

drastically in infants (and in pregnant women); since chlorpyrifos is so highly protein 

bound, this should be taken into account, but may be less important for TCPy or DETP.

A sophisticated PB/PK model for children is also available that allows for flexible inputs 

(i.e., SimCYP pediatric (SimCYP Company, Sheffield, UK). Although building a 

pregnancy PK or PBPK model is challenging and ambitious, it was extremely gratifying 

to see Dr. Hattis’ progress on development of a multi-compartment model where the fetal 

compartment (including the fetus, amniotic sac/fluid and placenta) is separately 

considered. While it is acknowledged that this will affect outcomes from Dr. Hattis’ 

current oral exposure model (but less so the inhalational) by altering first pass, it’s 

important to recognize that at term, the placenta is perfused to ~600 mL/minute of 

maternal blood (i.e., the equivalent of the entire mother’s blood supply passes through 

the placenta in about 8 minutes) and has an average surface area of 11 m2. Moreover it 
70

expresses significant CYPs, UGTs and SULTs that have been implicated in chlorpyrifos

metabolism (Benirschke et al., 2006). These considerations are relevant to the 

importance of the feto-placental unit as a separate compartment which is both well 

perfused and metabolic.

Additionally, while passage from maternal blood, to placenta and fetal blood may be 

bidirectional, distribution into amniotic fluid is uncertain; it would be helpful, then, to 

confirm whether or not placental effects might be negligible, retaining the placenta as 

part of the “liver metabolism.” Based on similar scenarios for bisphenol A (BPA), this is 

not at all certain.

In response to a request from the Agency, it was clarified that the uncertainties in any 

PBPK model cannot be estimated at this time, since working model parameters [for the 

Agency’s assessment] are not yet defined. In response to a further request from the 

Agency, Panelists suggest that a more expeditious path to attaining reasonable estimates 

of fetal exposure would be to generate an equation or algorithm that describes the 

relationship between maternal serum levels and cord blood levels of chlorpyrifos or its 

metabolites. Although less certain than the output of a verified PBPK model, this would 

enable basic dose reconstruction that can then be validated or verified by comparison to 

parameters in urine and blood reported in epidemiological studies. This may also be a 

starting point for assessing fetal exposure by defining “flow” and for continuing to build 

a more sophisticated model. The main limitation in using an equation describing the 

maternal: fetal ratio (and hence the flow) of chlorpyrifos and/or its metabolites is their 

short systemic residence time (i.e. the blood may only reflect exposures up to a few days 

prior to blood sampling). As a result, this method will not necessarily reflect cumulative 

exposure or acute exposures in earlier prenatal periods.

It was also noted that reported relationships between chlorpyrifos in maternal and cord 

blood warrant reconsideration. In particular, the ratio of 1.05 between the mean values 

3.9 pf chlorpyrifos / g maternal blood to 3.7 pf chlorpyrifos / g cord blood in Table 2 of 

Whyatt et al. 2005 differs widely from the ratio of 1.49 between the mean value of 5.96 

pg chlorpyrifos/g maternal blood derived from the regression equation Cord = 1.03 

Maternal 0.76 given in Whyatt et al. 2004 and 4.0 pg chlorpyrifos/g cord blood in Table 

1 of that publication. Only the former ratio was referred to in discussions of current 

PBPK models. Independent of whether or not these ratios represent the same 

populations, the broad range of this relationship needs to be defined. 

Question 5.4 

Characterization of chlorpyrifos exposure experienced by women in the Columbia cohort,

particularly during the pre-cancellation period, remains an important uncertainty in using 

these data in quantitative risk assessment. Exposure levels in the range measured in the 

cord blood data from the epidemiology studies (pg/g plasma) are probably low enough 

that is unlikely that the cohort mothers were experiencing AChE inhibition at the time of 

delivery; however, the biomonitoring data were taken after birth and not necessarily 

associated in time with an application of chlorpyrifos. As such, the actual level of such 

exposure particularly during any critical window(s) of susceptibility is not known, and a
71

better understanding of the range of possible exposures and the degree to which they may 

or may not have elicited inhibition of AChE, remains a key scientific question. In light of 

Panel discussions of Questions 4.3 and 5.3, please suggest approaches and/or analyses 

which would inform the understanding of the degree to which exposure levels 

experienced by the Columbia cohort participants may or may not have been below doses 

which result in 10% inhibition of AChE in the most sensitive lifestage. Please discuss the 

strengths and uncertainties associated with such analyses. Please include in your 

discussions approaches involving chlorpyrifos and its metabolites and also chlorpyrifos

plus other AChE-inhibiting pesticides (propoxur, diazinon) which the cohort participants 

were exposed too.

Response

It is important to realize that the short half-life of chlorpyrifos and its metabolites in the 

body calls into question any” spot data” that might be used. Large cross-sectional 

investigations may “catch” some exposure, but do not put them in context. Only 

longitudinal investigations, with frequent sampling are likely to give results that are of 

real use. 

What is called for in estimating the peak dose is prediction of the dose-response curve 

that would correspond to the vulnerable populations that were exposed. Understanding 

the limitations of the data available, a PBPK model having the potential to estimate dose 

given a fixed time since exposure, may provide some information. Additional 

information that would still be required for a reasonable estimation of maximum dose 

includes, whether the exposure/dose was steady state or bolus and approximately how 

long after the bolus exposure was the sample collected. With a very simple one 

compartment model and a time after exposure a reasonable estimate of the maximum 

dose can be calculated as well as whether the AChE inhibition threshold was reached. A 

more sophisticated PBPK model may provide even better data assuming that the PBPK 

model is applicable to the population being studied, specifically to pregnant women and 

small children.

Previous Panels have noted the decided lack of a realistic PBPK model for chlorpyrifos

for all populations. An effective PBPK model that is applicable to target groups such as 

pregnant women and infants/small children should be used for these vulnerable 

populations. An effective commercial version has already been identified for 

infants/small children (SimCYP pediatric from the SimCYP Company, Sheffield, UK) 

and should be used for a more comprehensive risk assessment model. Children are 

potential targets for any developmental issues related to exposure and while there are 

effective PBPK models for children, they have yet to be discussed here. Utilizing PBPK 

models designed for the individual and unique demographic (e.g. children and pregnant 

women) means more than the adjustment of body mass within the model designed for 

adult males.

In assessing both exposure and dose, a significant data gap exists for the population as a 

whole but especially for pregnant women which should be addressed with a longitudinal 

study. A single dose PK study like Clement (1984) provides the foundation for like 
72

populations (adult males) but does not address steady state (or the approximation that is 

our real world exposures) or populations with different metabolic conditions such as 

pregnant women or children. As discussed in Section 5.2, progress on this front has 

already been made as a compartment model with the fetus as a compartment currently 

exists. 

A longitudinal study throughout the pregnancy rather than a few samples in the last 

trimester would fill many of the data gaps that currently exist for this group. The 

potential for neurodevelopmental affects on the fetus as well as the metabolic differences 

in pregnant women versus the workers from the 1984 study, necessitate such a study. 

Placental tissue might provide more information on the metabolism and the delivered 

dose to the fetus as the concentration of chlorpyrifos going into the fetus cannot be 

measured directly from the cord blood or from the difference between cord blood and 

maternal blood. The tissue concentration may provide information on the chlorpyrifos

stores. This information will be vital in creating an effective PBPK model for pregnant 

women. For any PBPK model used in a comprehensive risk assessment, validation 

would add confidence to the predictions derived from its use.

Many of the studies discussed in Q 5.1 provide data on the concentration of chlorpyrifos

in the media such as house dust, air and water while market basket data exists on the 

concentration of chlorpyrifos on food. These are the primary tools for generating an 

effective exposure assessment and a subsequent reconstruction of potential dose. Dose 

reconstruction can be used to evaluate the efficacy of the PBPK model since its 

prediction of excretion rates can potentially be validated with an accurate estimate of 

dose. This assessment of the PBPK model through reconstructed dose may bridge some 

of the data gaps in assessing risk by validating the PBPK model. A validated model 

allows for effective prediction of exposure at the critical windows of maximum effect 

(AChE suppression) with measured urine concentrations. More data exists on 

chlorpyrifos than other pesticides in the environment, and this may be the best 

opportunity for utilizing exposure data to evaluate a PBPK model. It is noted however 

that is there is a significant difference between the predicted urine or blood 

concentrations that both the PBPK modelers and those that produce the exposure 

estimate, will point to the other for using “bad” assumptions. In this case both models 

should be reevaluated for the assumptions used.

The effects mixtures of chlorpyrifos + Diazinon /chlorpyrifos + Propoxur or 

chlorpyrifos/Propoxur/Diazinon have not previously been considered. Like from all 

mixtures both constructive and destructive interference can occur. Questions will have to 

be addressed; do they affect each other’s half lives and distributions and clearance

through metabolic competition (Coughli et. al. 2012)? Are their net AChE effects 

additive or multiplicative? Do they share mechanistic pathways? To address these 

questions, the Panel recommends further studies described in 5.3 to improve estimates of 

effects when mixtures of xenobiotics are used compared to single agents. In particular 

PK parameters such as distribution, half-life and clearance/elimination can be altered if 

admixtures of chemicals interfere with the absorption or metabolism of another 

component of the admixture. Using currently available data, other than improving in 
73

silico PK or PBPK approaches (again described above in 5.3), the Panel is not sure there 

is more that the Agency can do to reanalyze or transform the available data into more 

meaningful studies. However, any estimation of effect should have an additive dose 

effect as a minimum and perhaps greater protective factors until mechanistic studies can 

be done. 

Other considerations

A further criticism is in the focus on 10% AChE activity reduction. While certainly a 

benchmark, the fact that no mechanism has been proposed that would tie such a reduction 

to any specific outcome begs the question; what is the role of the 10% reduction of 

AChE in predicting negative health outcomes. The Panel noted that to their knowledge 

there is no proposed mechanism whereby a 10% reduction in AChE activity in a pregnant 

woman, even at a specific point in pregnancy, is responsible for cognitive deficit or 

neurodevelopmental delay of the fetus? The current proposed mechanisms focus on 

correlation; the deficit in AChE in the mother is assumed to be associated with some 

other activity, e.g., transport of parent chlorpyrifos (or TCPy for that matter) across the 

placenta and the nascent blood-brain barrier in the developing fetus? Since no one knows

whether this occurs, the utility of the measurement of maternal AChE reduction is 

unknown. AS is often the case, “more research is needed.”

Some on the Panel feel that the 10% figure is merely a marker of some level of exposure. 

This level may differ in its impact depending on the association of the AChE inhibition 

with the parent pesticide concentration in the serum. If the Panel assumes that each OP 

produces exactly the same level of AChE inhibition on a, say, molar basis, does that 

imply that there is an identical effect of each? Focusing again on chlorpyrifos, is the 

parent, the oxon, or some other metabolite that is responsible for some of the effects seen 

in the Columbia study? Only with a better understanding of exposure to chlorpyrifos at 

various gestational ages will the Agency be able to determine what exposures are causing 

the effects. The mouse studies do not seem to help all that much.

Question 6: Characterizing the range of potential risks.

The 2009 NRC report, Science and Decisions, focused on improving the technical 

analysis through the development and use of scientific knowledge and information to 

promote more accurate characterizations of risk, and thus improving the utility of risk 

assessment for risk-management decisions. The NRC report also pointed out that 

regulatory risk assessment does not routinely approach public health and environmental 

problems by arraying a wide range of options for dealing with them. In the case of 

chlorpyrifos, in light of the discussions of Questions 1-5, please provide guidance for 

assessing and presenting the range of plausible responses at given doses, and the effect 

of the overall uncertainty and variability around that range. 
74

Response

Part of the value of the framework for integrative analysis to evaluate multiple lines of 

evidence in the context of the AOP/MOA is to enable us to draw inference on the weight 

of evidence from the totality of the data.

In characterizing the range of plausible responses at given doses, it seems important to 

draw maximally on the dose-response data, beyond a single or several points of 

departure. For example, for risk management purposes, characterization to the extent 

possible, of the nature of potential risks above the reference dose would be informative. 

Presentation, then, of an array of points of departure for various endpoints for different 

types of effects bounded by their relative uncertainty, would more meaningfully 

characterize that value selected for the Reference Dose in the context of the range of 

effects reported in the broader database. It should also promote reliance on more certain 

rather than the most conservative data. As a minimum, it would be helpful in 

communicating the relative degree of protection provided by the selected point of 

departure. 

As indicated, in response to previous questions, the maximal use of the available doseresponse data from the epidemiological studies is recommended as a basis to at least, 

“bound” reference doses developed on the basis of points of departure from animal data.

To the extent possible, this step should take into account at least the semi-quantitative 

bounding of the dose-response relationship from human studies based on the impact of 

identified uncertainties. This would perhaps clarify the basis for (the seeming) 

conclusion that the uncertainties associated with the exposure–response relationship in 

the epidemiological studies are greater than those associated with the POD derived on the 

basis of the animal data (i.e., the basis for relying on the latter for dose-response 

analysis). 

Similarly, options for dose-response analysis for acute effects should be considered 

separately from those based on long term exposures – i.e., measures representing acute 

adverse neurological outcomes (ChE inhibition) commonly associated with occupational 

exposure versus those potentially related to long term exposure in the general population, 

such as neurobehavioral disorders. This separation would underscore the significant 

variation in the range of exposures in the population associated with these different types 

of effects, as reflected in reported TCPy levels measured in the three birth cohort studies 

and the recent occupational studies. Reconciliation of variability and uncertainty for 

these different options will likely require additional, focused study (see response to 

Question 4.3b). 

It is further suggested that the Agency focus on the data of chlorpyrifos levels in the cord 

blood samples as the base to develop the POD for chronic exposures to chlorpyrifos

based on a PBPK/PD model. This preliminary work would not only identify priorities for 

the acquisition of additional data, but would also reduce overall uncertainty and 

variability.
75

It seems important, also, to consider comparability within the Agency across compounds 

for which epidemiological data on neurotoxicity have served as the basis of points of 

departure – e.g., mercury and lead. How does the weight of evidence from 

epidemiological studies for these compounds compare with that for chlorpyrifos? For 

example, in the background paper, it is stated that: ”There are a number of known 

developmentally neurotoxic chemicals with well established relationships between 

exposure and neurological disorders in humans for which a definitive mode of action has 

not been established: for example, lead, methyl mercury and ethanol.” While 

documentation of an MOA is, then, not a prerequisite for basing points of departure on 

human epidemiological data, the nature of the weight of evidence that distinguishes 

chlorpyrifos from these cases, as a basis for reliance on animal rather than 

epidemiological data to characterize the point of departure, is unclear. 

 

In relation to the databases of studies which underlie considerations related to weight of 

evidence including consistency, specificity and biological plausibility, it would also be 

extremely helpful to have a priori criteria (to be presented initially) as the basis for 

evaluation of the individual studies on, for example, neurodevelopmental effects in 

animals and humans. While it is recognized that these criteria cannot be prescriptive, an

upfront discussion of the factors taken into account in judging the adequacy of individual 

studies and hence, the weighting of their contribution within the weight of evidence, 

would be valuable. 

The most susceptible lifestage(s), populations that would be expected to be more 

vulnerable to the effects of the chemical, and the effects of background exposures on 

these risks need also to be addressed in risk characterization. 

Background exposures would include:

exposures to other sources of chlorpyrifos

exposures to other chemicals that affect 

o key steps thought to be involved in chlorpyrifos’ neurodevelopmental 

adverse outcome pathway, e.g., exposures to other chemicals competing 

with chlorpyrifos for metabolism by the same enzyme system (see 

response to Charge Question 5.4).

o the same apical neurodevelopmental endpoints, e.g., decrements in 

working memory or full scale IQ. 

In assessing the range of plausible responses at a given dose of chlorpyrifos, variability in 

response within the population of concern should be taken into account, and uncertainties 

in estimation of the response should be addressed. 

Sources of variability in response to chlorpyrifos include:

differences in biological susceptibility, such as differences in lifestage, health and 

disease status, and genetics (i.e., polymorphisms in Phase I and Phase II 

metabolism)

differences in background exposures
76

Significant uncertainty in the draft chlorpyrifos risk assessment, arises from multiple 

sources. These include uncertainties:

estimation of chlorpyrifos exposures in the children’s cohort studies,

whether protecting against AChE inhibition is protective against 

neurodevelopmental effects,

whether the dose-response data for AChE inhibition in the most susceptible 

animal model, pregnant rats, can be used to derive a dose at which AChE

inhibition would not occur in humans exposed prenatally,

fully characterizing the neurodevelopmental effects of chlorpyrifos,

translation of in vitro concentration–response relationships for neurotoxicity to in 

vivo dose-response relationships,

dose-response for neurodevelopmental effects,

identifying the neurodevelopmental adverse outcome pathway(s) of chlorpyrifos,
77

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Appendixes

Appendix A to Question 3.1- Evaluation of Individual Studies

The strengths and weaknesses of the studies are outlined as are the principal findings and 

study designs that are also summarized in a different way in the EPA Draft Issue Paper in 

Appendix 3. In general, the studies fall into two major groups: (a) those that have 

adequate group sizes controlled for litter effects by sampling either only 1 

offspring/sex/litter or over-sampled only slightly by testing 2 offspring/sex/litter and 

analyzed the data using appropriate statistical models, e.g., ANOVA with factors of 

group and sex, optimally with litter as a block factor in a randomized block design in 

which treatment and sex are fixed effect factors within blocks and with within subject 

factor(s) for trial, day, or interval where the same subjects is assessed on the same 

parameter repeatedly; and (b) those that failed to control for litter effects, tested too few 

litters and/or offspring, were under-powered and prone to Type I error, and therefore 

should be given less weight. 

Some studies included appropriate down-stream statistical analyses and some did not. In 

general, most studies used factorial ANOVA or MANOVA models, but the follow-up 

methods used varied significantly. Most ANOVA models should be further analyzed by 

some method that deconstructs interactions and all experiments should include aposteriori group comparison methods than control for multiple comparisons while 

holding alpha-constant. There was much use of the Fisher Least Significant Difference 

(LSD) test for post hoc group comparisons among the above reviewed studies, which is 

only appropriate when there are not more than three groups. In a number of studies there 

were only three groups therefore this method is acceptable in those cases. However, 

there are a number of studies where there were more than three groups and the LSD or 

Protected Least Significant Difference (PLSD) tests were used despite their drawbacks. 

The use of Tukey tests in the absence of significant F-tests was also reported in several 

studies. This approach requires clearer justification and reporting of which results used 

Tukey T-Tests irrespective of a significant F-test and which were used after a significant 

F-test as a follow-up in experiments where both approaches are used. 

The Radial-arm maze (RAM) has been used extensively in the experiments reviewed. A 

significant concern is that none of those reviewed above controlled for the response 

pattern known as chaining, i.e., where the animal learns a strategy such as entering each 

adjacent arm successively or approximately successively by, for example, learning to 

always turn right or always turn left. When this occurs, working memory is not assessed. 

What is assessed is more likely habit formation, which is learning, but more rudimentary 

than working memory which is closely tied to higher cognitive functions such as 

attention and executive functions and hence assesses higher order processes. It is 

difficult, therefore, in all the RAM data in this group of reviewed experiments to 

determine what the chlorpyrifos-related effects were measuring. There is no doubt that 

chlorpyrifos has effects on RAM performance, but they may not be working memory 

effects unless it can be shown that more rudimentary forms of learning were not utilized. 

As for the reference memory effects, these are more likely to be as they appear but even 
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here one cannot be sure without better test procedures that have an inter-trial interval 

delay before each new trial to ensure that animals are not entering non-baited arms 

because the cost of doing so does not outweigh the overall retrieval of the rewards 

regardless of the small cost of running down an empty arm. One experiment used spatial 

delayed alternation in a T-maze (Maurissen et al., 2000). This experiment appropriately 

imposed a delay between the sample and test trials, which is the appropriate way to test 

working memory. This paper also showed an appropriate short-term memory decay 

function that was dependent on the length of the delay interval. This is a valuable 

internal control to prove that working memory was assessed. Some delay between arm 

choices in the RAM is similarly needed, even if a full decay function is not demonstrated. 

This would ensure that trial-dependent memory is being assessed rather than some other 

strategy. Not one experiment that used the RAM in this group of experiments imposed 

this basic requirement rendering interpretations difficult at best. 

There were many other test method issues among these studies that raise further 

concerns, including that methods were used that have no known neurotoxicological 

significance. The functional significance of increases or decreases in time in open arms 

of the elevated-plus maze is unclear as a toxicological end point. Which change 

represents an adverse outcome: an increase or a decrease? Or is any change from control 

regarded as an adverse effect? What is the meaning of greater or lesser social 

investigation of a stranger mouse in neurotoxicological terms? What does it mean that a 

female mouse has a more upright posture when presented with a male intruder? Is it 

more adverse that she stands more or stands less in such a defensive posture? With no 

validation as to the neurotoxicological significance of anxiety tests, depression tests, or 

social interactions, such outcomes should be regarded as exploratory, and hypothesisgenerating, rather than evidence of toxicity.

The bidirectional neurodevelopmental changes that were found in many of the studies 

presents challenges to interpretation. These effects included a slower habituation trend in 

female animals in one study, but not another study with the same doses also administered 

during a prenatal period, and a few transient effects in some of the cognitive tests that 

were also observed to occur in opposite directions. The lack of specificity of the

direction of the neurobehavioral findings is problematic. In some cases isolated markers 

of certain behaviors were determined to be statistically significant, but these findings 

were sometimes not supported by other studies reporting no effects (or effects in the other 

direction) in similar dose ranges using similar routes of exposure. It is difficult to 

reconcile effects that are bidirectional in given domains of neurodevelopment, or 

inconsistent across doses and sex, and in the absence of specific hypotheses. In addition, 

the lack of dose-response among test outcomes (especially those that took doses below 1 

mg/kg/d such as Braquenier, et al., 2010, that only found positive responses in the middle 

dose group) is not reassuring.

Prenatal Studies

This study (Abou-Donia et al., 2006) was a prenatal study of chlorpyrifos. They used 

timed-pregnant Sprague-Dawley rats from Charles River (evidence of conception = E1). 
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The authors’ indicate that dams were treated transdermally on E4-20 with chlorpyrifos in 

a 70% ethanol vehicle or were given ethanol vehicle alone. Dams were assigned to 

groups as follows: 5 mg/kg of chlorpyrifos, 5 mg/kg of nicotine, 5 mg/kg of chlorpyrifos

+ 5 mg/kg of nicotine, or saline. It is stated that 2 M/2 F per litter were sampled for 

testing. No statement of whether litter was included in the statistical model was 

provided; therefore, presumably it was not; hence there was no control for litter effects. 

Pairwise comparisons were performed using Fisher’s LSD test, but they had 4 groups and 

this test is not appropriate for >3 groups. No litter culling to standardize litter size was 

done; hence, postnatal rates of growth between litters was not equalized. On P90, rats 

were tested for Beam walking, inclined plane, and forelimb hang time. Results: They 

report no CFP effects on beam walking; a female-only effect on the inclined plane 

(females slipped at lower angles than controls as the plane was tilted). Hang time: Both 

sexes in the chlorpyrifos group had shorter hang times than controls. Strengths: Used a 

transdermal route of exposure; they exposed animals from shortly prior to implantation to 

near-term. Weaknesses: Only one dose of chlorpyrifos was used (estimated to be 

equivalent to ~1 mg/kg/day). Used 2-way ANOVA but in the results they give no Fvalues; they provide no indication if reported effects were main effects or interactions; 

and they moved from ANOVA to LSD tests with no sorting of interactions (although 

which reported effects were from interactions and which main effects is unclear). They 

slightly oversampled per litter, but the most significant weaknesses are the small group 

sizes that results in an under-powered design and they did not analyze the data by litter. 

Additionally, they used timed-pregnant dams from the supplier for use in a prenatal study 

for which treatment started very early on E4. To do this, they would have had to have 

purchased rats that were plug-positive (without confirmation) and received them within 

1-2 days of mating, leaving no more than 1-2 days to acclimate to their vivarium before 

treatment began.

This study (Icenogle et al., 2004) used timed-pregnant SD Charles River dams for a 

prenatal study, again raising concerns about shipping stress. They dated pregnancy as 

counting evidence of conception = E1. They treated on E9-12 (the rationale for these 

days was not given, but seems to be a very narrow period of exposure). They randomized 

among dams on P1 and then again “every several days” thereafter, introducing unknown 

stressors in the experiment for both the dams and the offspring. They culled to 10 pups 

per litter then selected no more than 1 M and 1 F per (artificial) litter and assigned 10 

litters to each treatment group. Groups: 0, 1, 5 mg/kg/day chlorpyrifos administered s.c 

in DMSO on E9-12. They tested offspring for T-maze spontaneous alternation. The Tmaze was elevated with 1.5 cm curbs to prevent falling off the edge, and animals were 

given 5 trials/day with 30 seconds post-choice confinement. The test was given for 5 

successive days. The apparatus is non-standard in the field and is designed more akin to 

the elevated plus maze (EPM), which is a test designed to induce anxiety so that it can be 

measured. The T-maze for spontaneous alternation is not intended to induce anxiety and 

for that reason is normally an enclosed maze. The elevated T-maze used in this study 

probably tests alternation AND anxiety, but one cannot determine how the resulting 

measurements can be attributed to memory versus anxiety. They also tested rats in a 

figure-eight locomotor activity monitor for 1 h with data recorded in 5 min intervals and 

repeated the test three times each spaced one week apart and in a 16-arm radial-arm maze 
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(RAM) with 12 arms baited daily, 4 arms never baited; the test sessions were 10 min or 

until 12 baited arms had been entered; they tested twice per week for 18 sessions; then 

gave a scopolamine (muscarinic antagonist) challenge at doses of 0.04, 0.08, 0.16 mg/kg 

or to separate animals a drug challenge of mecamylamine (nicotinic antagonist) at doses 

of 1.25, 2.5, 5.0 mg/kg. They also tested acoustic startle response with prepulse 

inhibition (ASR/PPI). They first conducted ASR-only trials and later intermixed ASR 

and PPI trials. Lastly, they tested animals in the elevated plus maze (EPM). For this they 

used a standard method (standard size apparatus for the typical 5 min. test). Note: this 

test was given AFTER all preceding tests whereas in most labs it is given before other 

tests based on the fact that this test is sensitive to prior experience. The statistical 

approach was MANOVA; interactions were further analyzed and pairwise comparisons 

were made by Fisher’s PLSD. Findings: Spontaneous alternation: chlorpyrifos decreased 

shortened choice latency on early trials; no effects on alternation but failed to show 

whether they got alternation rates typical of this test to establish validity in their 

laboratory. Figure-eight test: They obtained a significant treatment x interval interaction. 

The chlorpyrifos 5 mg/kg showed faster habituation on two out of four of the last 5-min 

intervals, with one interval with higher activity in the chlorpyrifos 1.25 mg/kg group; 

they also note that the linear trend in this analysis was significant for treatment group, but 

it is noteworthy that the effects observed on this test were very small even if significant. 

RAM findings: The data were blocked into three sessions per block for analysis; hence, 

there were six blocks for the repeated measure factor. They found increased reference 

memory errors in block 1 and increased working memory errors in blocks 1 and 3 in the 

CFP 5 mg/kg group only with males and females combined. They found no effects on 

RAM performance after mecamylamine challenge. They found an effect of scopolamine 

challenge that was complex: Scopolamine increased errors with increasing scopolamine 

dose in controls but in the chlorpyrifos 1 mg/kg group it increased baited arm errors at 

lower doses more than in controls but less than in controls at the highest scopolamine 

dose. For the chlorpyrifos 5 mg/kg group scopolamine increased errors more than in 

controls after saline but less than in controls after scopolamine. They report no ASR/PPI 

effects and no EPM effects for time in the open (the principal index of anxiety in this 

test); the CFP 5 mg/kg group crossed center more than controls, however, suggesting a 

slight increase in activity. Strengths: Groups sizes were adequate and the data were 

analyzed by (artificial) litter. The factorial MANOVA models were appropriate. They 

tested two doses of chlorpyrifos and used many standard methods. Weaknesses: Used 

timed-prenatal females for a prenatal study thereby introducing prenatal shipping stress. 

RAM: they ran trials continuously with no intertrial interval (ITI) delay to ensure that 

working memory was being assessed. While there is no doubt they obtained RAM 

effects, it cannot be distinguished as to whether these were working memory or habit 

learning effects. The small reference memory effect they obtained is more likely to be a 

real reference memory change, but this cannot be certain without observation of the 

animals’ performance or a method to ensure an ITI delay. In this test, a control for 

chaining (moving sequentially around the maze from one arm to the next or every other 

arm) or for random selection is important. Without a confinement period in the center 

between each arm choice, there is no way to rule out that rats obtained the food without 

relying upon working memory instead of another strategy. For reference memory 

findings, what one needs to determine is whether rats found the cost of entering even 
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empty arms insufficiently aversive so that is was more effective to check them all versus 

remembering which ones were unbaited. There is a basis for this concern in the data. 

Reference memory errors among controls improved from about 6.5 to about 5 across the 

18 sessions. Given that there are 4 unbaited arms, the data suggest that even controls 

never actually acquired memory for the unbaited arms. If they had, one would expect 

well under 4 reference memory errors per trial block, assuming they blocked the data by 

averages rather than by sums; but the paper is moot on how the blocked data were 

formed. For these reasons, center confinement between trials is an important control 

especially for the working memory assessments. The authors would have greatly 

strengthened their experiment by either using center confinement between trials or having 

an observer map the problem solving strategy during testing to see that the animals were 

using alternate strategies. The chlorpyrifos vehicle used was DMSO, which was raised as 

a significant concern in response to another charge question.

In this experiment (Billauer-Haimovitch et al., 2009), heterozygous HB/Igb mice were 

used and bred in-house (conception = E1). Mice were treated on E9-18 with chlorpyrifos

at doses of 1, 3, 5, 10, 20 mg/kg given s.c in DMSO. Half the litters were fostered, half 

were not; they report no differences in outcome in preliminary analyses and state that the 

data were therefore pooled for subsequent analyses. They sampled 1 M/1 F for testing 

per litter. At P75, mice were tested in the Morris water maze (MWM). The pool was 87 

cm diameter, the platform 8x10 which is a search ratio of 74:1 (Note: this is less than 

optimal for a test of spatial navigation in mice). They ran 2 blocks of 4 trials per day for 

4 days. After completing hidden platform trials, they ran cued trials with the platform 

made visible above the water line. Data were analyzed by MANOVA, log transformed, 

with Tukey a-posteriori tests. They report no differential effects between males and 

females, so they combined sexes for presentation (but in the statistical analyses ). They 

report that in the chlorpyrifos20 group all animals died. They found a chlorpyrifos main 

effect on MWM latency, which they report was significant for the chlorpyrifos 1 and 

chlorpyrifos 3 mg/kg groups but not for the chlorpyrifos 5 or 10 mg/kg groups. In a 

second experiment they used only the chlorpyrifos 3 mg/kg group and found somewhat 

larger MWM latency effects and these were reversed by nicotine treatment prior to each 

daily test session. In a third experiment with chlorpyrifos 3 mg/kg, they again found 

MWM latency effects and these were reversed by cell implantation from neonatal cells 

grown in neurospheres. Found no speed differences in MWM on trials where latency 

was significantly increased. They found no chlorpyrifos effect on developmental reflexes 

(surface righting, startle emergence, age of fur appearance, day of pinna unfolding, or day 

of eye opening). Strengths: They used the MWM which is one of the most well-validated 

spatial learning/reference memory tests in neuroscience; they used 14-34 litters per 

group. A major strength was that they replicated the MWM effect in three separate 

experiments. Weakness: It is unclear why effects were seen in the MWM at chlorpyrifos

1 and 3 mg/kg but not at 5 and 10 mg/kg. The vehicle used was DMSO. 

In this experiment (Turgeman et al., 2011), heterozygous HB/Igb mice were used and 

bred in-house; Conception = E1 and mice were treated on E9-18. The exposure was CFP 

3 mg/kg given s.c in DMSO. Half the litters were fostered, half not; they reported no 

differences in outcome between fostered and non-fostered litters; therefore, they pooled 
93

data across this factor for later analyses. They tested 1 M/1 F per litter. At P75, mice 

were tested in a MWM (with the same 87 cm diameter, platform 8x10, and 74:1 search 

area as in the study by Billauer-Haimovitch et al. (2009) and has raised the same concern 

as above). Testing was in two blocks of four trials/day for four days. After hidden 

platform trials they ran cued trials. Data were analyzed by factorial MANOVA, log 

transformed, with Tukey a-posteriori tests. They report no M/F differential effects, so 

they combined sexes for presentation (but not statistically, they note). They report that 

chlorpyrifos 3 mg/kg increased MWM latency which was reversed by stem cell 

transplantation, just as in the previous experiment by Billauer-Haimovitch et al. (2009) 

(the stem cell methods were also identical). They found no effect on swim speed, but this 

experiment included no cued trials to ensure the absence of proximal cue learning 

problems. They also ran no probe trial to assess reference memory. Strengths: Found 

clear MWM effects, used 13-25 litters per group, they tested only 1 sex per litter. 

Weaknesses: Tested only one dose of chlorpyrifos, used a small maze, and did not 

conduct a test of reference memory or include cued trials as a control or include a 

reversal component to verify that the effects were hippocampally-dependent. The vehicle 

used was DMSO. 

This study (Laviola et al., 2006) used heterozygous Reeler KO mice on a C57BL/6 

background. The mice were bred in-house by het x het crosses and conception = E0. 

chlorpyrifos-oxon (chlorpyrifos-O) was tested, not chlorpyrifos. chlorpyrifos-O was 

given on E14-16 (the rationale for this embryonic period was not given but seems very 

narrow for a compound whose exposure would be expected to be chronic). The 

compound was delivered by implanted osmotic minipump at a rate equaling 5 mg/kg/d. 

Offspring were tested on P3, 7, 11 for ultrasonic vocalizations, wire mesh hang time 

when the mesh was gradually rotated 180 degrees until animal was hanging upside down, 

and surface righting. As adults (>P70), they tested locomotor activity (30x30 cm 

apparatus) for 45 min., then administered 2 mg/kg scopolamine and retested for another 

45 min.; 1 week later they re-tested the same animals in another apparatus (40x30 cm) for 

10 min then removed them and gave a high dose (10 mg/kg) of amphetamine and retested 

for 50 min.; movements were video recorded and scored later using the Noldus Observer 

system to rate specific behaviors. Within litters, they had 3 genotypes (KO, het, WT). 

Treatment and genotype were regarded as between subject factors and other factors as 

within-subject factors. They used factorial ANOVA with pairwise comparisons by 

Tukey tests. On P7, the lowest ultrasonic calls were found in KO mice; they were 

intermediate in hets, and highest in WT but no such pattern was found in chlorpyrifos-Otreated mice; in chlorpyrifos-treated mice they observed high call frequencies in all 

genotypes. For angle to fall on the wire screen grasping task, they found no genotype 

effect in controls but fall angle was higher in Reeler chlorpyrifos-O exposed groups and it 

decreased from WT to het to KO, but even KOs were better than Control KO mice. They 

reported a chlorpyrifos-O effect on righting only in KO mice on P 7 and 11 (longer 

latency) but this effect was seen only on the worst of the three daily test trials; no effect 

was seen on the best or intermediate test trial given each day. In the 30x30 cm activity 

chambers, they found control KO mice were hyperactive, an effect which according to 

the text chlorpyrifos-O exposure ‘normalized’ but they did not show these data. After 

they gave scopolamine, all groups showed the expected hyperactivity response, but for all 
94

genotypes a main effect was seen of greater hyperactivity in the chlorpyrifos-O groups 

but only when the 45 min. test session was subdivided such that the difference occurred 

only in the first 25 min. of the test session and after subtraction of pre-drug activity data 

(in an effort to adjust for the innate hyperactivity of the KO mice). In the 30x40 cm test 

chamber, no differences during the 10 min habituation prior to amphetamine challenge 

were seen. Post-amphetamine, KO chlorpyrifos-O treated mice showed increased 

activity that was greater than in KO controls, but for stereotypy, the opposite occurred, 

i.e, chlorpyrifos-O exposed mice were less stereotypic than KO controls; similar but 

weaker trends were seen in WT and hets. Strengths: This is the only study among the 

neurobehavioral papers to test the oxon. Weaknesses: N’s were given for progeny but 

not by litter; the number of litters used was vague: They state that 12 breeding pairs were 

used, so presumably 6 litters were treated with chlorpyrifos-O and 6 with the vehicle, but 

the Panel was given no information on how the genotypes were distributed among the 

litters within treatment groups. There does not appear to be any control for litter effects. 

Sex was not described as a factor in ANOVAs. Only one dose was tested. The study was 

under-powered given only six litters per group with each litter subdivided by genotype 

and sex. Given the expected Mendelian ratios, it would be expected that half of the 

offspring would be hets, leaving 25% as KO and 25% as WT. Given that C57BL mice 

typically have 6-8 pups per litter, the sample sizes per genotype per sex per litter would 

be quite small and not likely to be evenly distributed.

In this study (Venerosi et al., 2010), they tested the effects of chlorpyrifos 6 mg/kg given 

by gavage on E14-17 to CD-1 mice (bred in-house), but the date of inferred conception 

was not provided. They treated 18 litters with oil and 16 litters with chlorpyrifos. 

Shortly after birth, they culled to 4M/4F. Testing began on ~ P90. They evaluated the 

offspring in a Light/Dark (L/D) test for 5 min and also did observer scoring. They tested 

the mice in the Forced swim test (FST) either with an injection of saline or of the 

selective serotonin reuptake inhibitor (SSRI) fluvoxamine (30 mg/kg) 30 minutesebefore 

the test. They scored immobility, struggling, and swimming and testing was during the 

dark cycle. Females were grown to adulthood and bred, and on P8, a stranger male 

intruder was put in their cage (pups were removed) and scored for aggression. Data were 

analyzed by ANOVA or Mann-Whitney using litter as a factor; ANOVAs were followed 

by Tukey tests but in some cases in the absence of a significant F-test. They do not 

define the reason for this but cite a reference that it may be used in the absence of a 

significant F-test. They also state that they used non-parametric tests in some cases 

including analyzing interactions by Mann-Whitney U tests. It is unclear how it is used to

detect interactions. They report finding a significant U-test for females for time in the 

tunnel connecting the two sides in the L/D test, with chlorpyrifos-exposed females 

spending more time in the tunnel than controls. For the FST, they report an interaction 

between chlorpyrifos x fluvoxamine in which chlorpyrifos eliminated the increased 

swimming induced in controls given fluvoxamine; this pattern was repeated for 

immobility time, i.e, fluvoxamine reduces immobility in controls, but this effect was 

dampened in chlorpyrifos-treated animals enough that the change in this group was not 

significant. In the intruder test, they also reported a chlorpyrifos x fluvoxamine 

interaction. They found that fluvoxamine reduced the duration of attack and increased 

inactivity in controls, but this pattern was largely eliminated in the chlorpyrifos exposed 
95

group. Strengths: chlorpyrifos was given in oil, not DMSO. They used litter in the 

analysis and tested 6-13 offspring per group. Weaknesses: They had only one dose of 

chlorpyrifos; they do not say which outcomes they assessed by direct Tukey tests and 

which by ANOVA followed by Tukey comparisons. The meaning of the intruder test 

outside of basic research or as an index of neurotoxicity is not known.

In this study (Haviland et al., 2010), they used 0, 1, 5 mg/kg chlorpyrifos given s.c. in 

DMSO to Swiss-Webster on E17-20 where conception was termed E0.5 (but after stating 

this, they never used half days again, so it is unclear of E0.5 was rounded to E0 or E1). 

Mice were bred in-house and litters were culled to 8 and balanced for sex. Testing began 

on P60 using a modified 8-arm RAM with a T at the end of each arm with food always in 

the left arm of the T (they name it the Foraging maze) and they compared animals tested 

in this maze to a group tested in a standard 8-arm RAM. They tested animals for 3 

sessions per week for 3 weeks for a total of 9 sessions. For some reason that they do not 

explain, in the RAM they baited 6 of the 8 arms on each trial, but for the Foraging maze 

they baited 4 of the 8 arms on each trial. Strengths: Testing two groups in two mazes that 

presumably assess the same functions is a strength (potentially). They analyzed the data 

by litter, but the results are not well described. They state that RAM data in standard 

terms of errors by type (entries into baited versus unbaited arms), but they did not use an 

ITI delay to ensure that working memory was being assessed. Data were analyzed by 

ANOVA, but no mention was given of how pairwise comparisons were done, but it was 

probably by LSD since they used the LSD for pairwise comparisons for their thyroid 

assay data. They report finding only reference memory errors in the RAM, but all groups 

showed poor learning; they do not show working memory data at all and the reference 

memory differences are scattered across the test sessions and between doses nonsystematically. In the foraging maze, they report their findings differently. It is 

noteworthy that they state that the entrance of each of the baited arms were marked with a 

0.5 cm radius peg (or 1 cm in diameter) (Note: this is an extraordinary procedure; the 

provision of an obvious cue to which arms are baited and which arms is not, defeats the 

purpose of the test which is to remember which arms are baited and which are not; cuing 

the rat at the entrance to each arm provides evidence only that the rat can recognize the 

cue, requiring little memory). They report the data as the proportion of correct choices 

out of total choices, such that perfect performance results in a score of 1.0. Clear 

improvement toward 1.0 was seen across test sessions and chlorpyrifos caused a slower 

increase toward 1.0 at both doses in females but not in males. In males, chlorpyrifos

exposure cased a more rapid increase toward 1.0 at 1 mg/kg but not at 5 mg/kg. 

Strengths: Use of two learning tests and the effort to develop an improved version of the 

RAM are noteworthy. Weaknesses: The vehicle used was DMSO. Poor learning in the 

RAM, performance on the Foraging maze not shown in terms of working and reference 

memory errors, and the use of an in-maze cue to which arms were baited seems to defeat 

the purpose of the test; findings were not dose-dependent or consistent across sessions. 

No ITI delay was used. It is not clear what the foraging maze adds to a more standard 

RAM. Why would a mouse learn better if it goes down a cued arm and turns left rather 

than going down an unmarked arm to find food when the arm is straight?
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This study (Levin et al., 2002) used timed pregnant SD Zivic-Miller (ZM) rats; date of 

inferred conception was not stated. They tested 0, 1, 5 mg/kg chlorpyrifos given s.c. in 

DMSO administered on E17-20. Litters were culled to 10 and randomized to dams. The 

rationale for randomizing the pups is not stated, nor is there any published data this 

reviewer is aware of that establishes that this cancels out litter effects although it clearly 

randomizes within litter genetic factors, but whether that improves or confounds 

outcomes is unknown. Moreover, the degree of stress this induces in the pups or the 

dams is unknown. Testing was done during the dark cycle. Litters were culled to 10 

M/10 F per group per artificial litter. Spontaneous alternation used an elevated T-maze 

with no walls (see above) and was tested on P28-42, 5 trials per session (with 

confinement) for 5 daily sessions. They tested rats in a figure-eight locomotor system on 

P28-42 for 1 h/session, and did this three times each spaced one week apart; also used a 

16-arm RAM with testing on P57-91. The testing was for 3 days/week for a total of 18 

sessions. Their procedure was to use 4 unbaited arms and 12 baited arms. Each session 

lasted up to 10 min or until all baits were taken. They continued RAM testing after this 

on P98-119 with either a scopolamine challenge (muscarinic antagonist) at doses of 0.04, 

0.08, 0.16 or mecamylamine (nicotinic antagonist) challenge at doses of 1.25, 2.5, 5.0 

mg/kg given prior to each test session. Data were analyzed by ANOVA with factors of 

treatment and sex as between factors and test interval or day as within-subject factors. 

Main effects were taken as significant if they occurred at P<0.05 but interactions were 

followed-up if P<0.10. Pairwise comparisons were by Fisher’s PLSD. They found no 

effects on spontaneous alternation frequency but report finding that chlorpyrifos

decreased latencies to choose one of the arms (Note: latency to choose is not a typical 

outcome measure in this test). For the figure-eight test, they found no effects on the 

omnibus ANOVA, but then analyzed the first session and report finding a treatment x sex 

interaction in females in which they habituated slower than both chlorpyrifos groups. 

They used a trend analysis in which the linear trend was different in both chlorpyrifos

groups compared with controls, but the effect was small and linearity did not appear to be 

a good fit to the data. In the RAM, they report a significant main effect of treatment and 

a treatment x error type and a treatment x error type x sex interaction. They followed 

these up and found female working memory effects at chlorpyrifos 1 mg/kg but not at 

chlorpyrifos 5 mg/kg. They report the same pattern for reference memory errors but less 

pronounced. After scopolamine, it was again found that female chlorpyrifos1 animals 

showed differential effects on RAM on working memory errors (not on reference 

memory errors or latency). For females, they found the slope of improvement across 

trials was lower in the chlorpyrifos1 group than for controls or the chlorpyrifos5 group 

for total errors using a P<0.07 trend to justify the trend analysis. When only working 

memory errors were analyzed, the female chlorpyrifos 1 effect was not seen. No effects 

were seen in males. For reference memory errors, there was again a significant linear 

trend for females, but the effect was that the CFP 1 mg/kg females made fewer errors 

than controls but this was because scopolamine caused controls to make more errors 

whereas it did not cause this in the chlorpyrifos 1 mg/kg females, which might make 

sense except that a higher dose of scopolamine did not cause controls to make more 

errors, making these data largely uninterpretable. Strengths: Sample size was adequate 

and they used litter by selecting only 1 male and 1 female per (artificial) litter. They used 

two doses of chlorpyrifos. Weaknesses: They did not use an ITI during RAM testing to 
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ensure that working memory was being assessed. They use an unorthodox elevated Tmaze to assess spontaneous alternation. They used ZM rats. They used timed-pregnant 

rats for a prenatal study introducing a potential prenatal stressor. They provide no 

rationale for the short E17-20 exposure. Given that environmental exposure to 

chlorpyrifos might be chronic this short exposure window does not appear to be a very 

appropriate choice.

In this experiment (Ricceri et al., 2006), CD-1 mice were gavaged with chlorpyrifos at 

doses of 0, 3, 6 mg/kg/d in peanut oil on E15-18 (in-house breeding with date of inferred 

conception = E0). At birth, litters were culled to 4M/4F. Within each litter, one 

male/female pair was treated postnatally on P11-14 with 0, 1, or 3 mg/kg chlorpyrifos, 

creating permutations totaling 9 pre/post-natal treatment group combinations. On P70, 

they tested males for 20 min. in an open-field under red light, video recorded the animals, 

and later used the Noldus system to categorize behavior. On P75-80, males were tested 

for 20 min. in a novel cage with a stranger male. On P90, females were given 3 pups for 

20 min. to observed induced maternal behavior. On P120, mice were tested for 5 min. in 

the EPM. Data were analyzed by litter using ANOVA models, but the statistical section 

is difficult to follow because the run-on sentence says “prenatal treatment as block with 

respect to postnatal treatment, sex, and repeated measure as within-litter treatment 

factors, postnatal treatment, and sex as fixed-effect factors within litter, and repeated 

measure as fixed factor within subjects.” From this, it is somewhat difficult to determine 

whether sex was treated as a between or within factor, but the analysis does indicate that 

litter was handled as a blocking factor within the ANOVA and it appears that prenatal 

treatment was a between factor and postnatal treatment a within factor. Variables where 

the same subject was tested repeatedly were handled as repeated measure factors. 

Significant F-tests were followed up using Tukey with Bonferroni correction. Results: In 

the open-field, they found increased activity in the chlorpyrifos6 males (females were not 

tested). In the test with a stranger mouse, the principal finding was in the postnatal 

chlorpyrifos3 group that showed increased attack behaviors against the stranger; they also 

found that the prenatal chlorpyrifos6 group showed a significant increase “upright 

postures” during this test. In the test of induced maternal behavior, the postnatal CFP 1 

& 3 groups showed decreased licking frequency but increased licking duration, along 

with increased crouching frequency over the pups and for longer intervals but decreased 

pup sniffing. In the EPM, the significant finding was in the postnatal chlorpyrifos3 group 

that showed increased time in open arms. Strengths: They controlled for litter effects, 

and then did factorial ANOVAs; they controlled for multiple comparisons; they used 

adequate numbers of litters; and they used sound behavioral methods. The complex 

prenatal x postnatal treatment design could be strength to the extent that it identifies 

critical periods of exposure but could be a weakness as it makes the experiment 

logistically complex and difficult to manage; the study included multiple doses, 2 of 

which were prenatal and 2 of which were postnatal. Weaknesses: The choice of the 

narrow exposure windows is not well justified and appears arbitrary. Changing the dose 

levels used for the prenatal and postnatal exposures adds another complication. The fact 

that significant outcomes appear only in some dose groups treated prenatally and in some 

treated only postnatally, but not in those treated both pre- and postnatally which 

cumulatively had greater chlorpyrifos exposure, is difficult to reconcile. The effects were 
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not generally dose-dependent even within the prenatal, postnatal, or prenatal-postnatal 

combination groups. More fundamentally, the interpretation of the anxiety and social 

interaction tests as indices of neurotoxicity is uncertain. While these tests are interesting 

and identify areas for further investigation, they are difficult to use in risk assessment 

until they have an established neurotoxicological basis. For example, is it more adverse 

to show a modest increase in anxiety or a decrease? It is known that in humans 

anxiety/stress is an inverted U-shaped function. Low stress and anxiety leads to poor 

performance whereas high stress and anxiety interferes with performance; moderate 

stress and anxiety produce optimal performance. This is true in all mammalian species. 

Chronic stress and anxiety follow the pattern; many studies have shown that moderate 

developmental stress in rodents leads to increased cortical thickness and greater 

arborization and improves learning. Anxiety tests, such as the EPM, have a wellvalidated basis in the context of antidepressants. They are valid when used as intended to 

assess the effects of acute or subchronic exposure to SSRI, tricyclic and atypical 

antidepressants. They are increasingly used in gene targeting studies where candidate 

genes suspected of involvement in fear, anxiety, and stress are being assessed, but they 

have never been validated in neurotoxicology. This is also the case with social 

interaction tests. These tests are still being developed and their meaning in basic 

neuroscience research, as for example, in genetic models of autism spectrum disorders 

(ASD), are not yet established. Trying to interpret such methods in neurotoxicology is 

premature. Again, one must pose the question: Is it worse or better that the females given 

pups in this experiment, lick them more or less if they were exposed pre- and/or postnatally to chlorpyrifos? Unfortunately, no one can answer this question based on 

currently available data.

This experiment (Venerosi et al., 2006) is identical in design to that of (Ricceri et al., 

2006). The Ns are the same as are all the major experimental design features. In terms of 

test outcomes, they conducted a social recognition test in which females were placed in 

single cages for three days and then introduced to stranger females for three min.; 45 min. 

later, they were re-exposed to stranger mouse-1 for another 3 min. and 45 min. later 

exposure to stranger mouse-2 and ultrasonic calls were recorded. Vocalizations in 

controls went down on retest-same and up on retest-different, whereas vocalization in the 

prenatal CFP3 group changed slightly, and in the CFP6 group it changed dramatically, 

causing retest-same to go up and retest-different to go up more than in controls. Postnatal 

CFP largely reversed the pattern. Social investigation: prenatal CFP increased social 

investigation, had no effect on retest-same or retest-different (latter not shown in figures). 

Here again, neither social interaction induced vocalizations nor social investigation of 

other animals has a known neurotoxicology interpretation. Strengths: This experiment 

has the same strengths as Ricceri et al. (2006). Weaknesses: It has the same weaknesses 

as Ricceri et al. (2006).

Postnatal Studies

This study (Dam et al., 2000) comes from the Slotkin lab and has most of the features of 

this lab’s previous work. As before, Sprague-Dawley Zivic-Miller rats were used. As 

before, litters were culled after birth to 10 pups and randomized across dams on P1 and 
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every 3 days thereafter until weaning. chlorpyrifos was administered s.c. in DMSO on 

P1-4 at doses of 0 or 1, and other animals were treated with chlorpyrifos on P11-14 with 

0 or 5 mg/kg/day. Prior to weaning, offspring were tested for surface righting on P3-4 

and on the inclined plane (20 degree angle) on P5-8 for those treated on P1-4. Open-field 

activity was manually tested in a large 100x100 cm field on P21 and P30 for 5 min. each 

time. Results: They found delayed surface righting and inclined plane rotation times in 

the chlorpyrifos1 females treated on P1-4, but not in males. In the open-field, they report 

that males in the CFP1 group had decreased square crossings and rearing frequency with 

no change in grooming frequency. In the P11-14 groups, they report no change in P21 or 

P30 in open-field line crossings, but chlorpyrifos males showed increased rearing at P30 

and no other changes. Strengths: They used adequate sample sizes and analyzed the data 

taking litter, albeit artificial litter, into account. Weaknesses: They sampled 2 offspring 

per sex per litter, so there was slight over-sampling. The artificial litter technique 

remains unverified as a technique, and it could introduce stress on the pups and the dams 

being shuffled every three days. The 5-minute open-field test is generally regarded as 

inadequate by current standards, including those from 2000 when this study was 

published. The Zivic-Miller rat is less than ideal for behavioral studies. The vehicle used 

was DMSO. The findings were not dose-dependent.

This study (Levin et al., 2001) was in collaboration with the Slotkin lab and has many of 

the common experimental design features noted above from this group. They used 

Sprague-Dawley rats from Zivic-Miller. Litters were culled to 10 pups, randomized and 

re-randomized every several days. Offspring were treated with CFP on P1-4 with 1 

mg/kg, or on P11-14 with 5 mg/kg s.c. as in the previous study by Dam et al. (2000) 

dissolved in DMSO. However, this experiment shows the influence of the Levin lab: 

Adult offspring were tested for spontaneous alternation in the elevated T-maze referred to 

previously, including significant concerns about using a non-standard way of conducting 

this test. Locomotor activity was tested in the figure-eight system and RAM testing was 

as reviewed above with pharmacological challenges given after initial learning. All 

testing was done during the dark cycle. Spontaneous Alternation was conducted on P28-

42, 5 trials with confinement after an arm choice for five sessions; figure-eight testing 

was done on P28-42, 1 h per session, 3 sessions spaced one week apart. The RAM was 

the 16-arm system tested on P57-91 with testing conducted three 3 days per week for a 

total of 18 sessions (4 arms unbaited; 12 arms baited; 10 min per session). RAM with 

drug challenge was conducted on P98-119 with scopolamine at doses of 0.04, 0.08, 0.16 

or mecamylamine at doses of 1.25, 2.5, 5.0 mg/kg. ANOVA models were treatment and 

sex as between factors, and interval or day as within factors at P<0.05 except interactions 

which were taken as significant at P<0.10; the method of doing pairwise comparisons 

was not indicated in this paper. Results: They reported no effect on spontaneous 

alternation frequency and a small effect on latency in chlorpyrifos-exposed males on this 

test. In the figure-eight test, no effects were seen in the chlorpyrifos P1-4 exposed group, 

but reduced habituation slope was noted in the P11-14 CFP group. In the RAM test, the 

P1-4 CFP group showed effects on working and reference memory errors in males in the 

first block of trials but not thereafter and in females across blocks; no effect of 

chlorpyrifos exposure was seen after P11-14 exposure on learning the task. Treatment 

with 0.16 mg/kg of scopolamine increased reference memory errors in P11-14 
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chlorpyrifos-exposed males, with a larger effect in females that occurred at the lower 

scopolamine doses but not at the highest dose (0.16 mg/kg) of. Strengths: They used 

adequate sample sizes, litter was accounted for in the analyses, there was no litter oversampling, and the behavioral methods were mostly sound. Weaknesses: Not a doseresponse study; the vehicle for chlorpyrifos was DMSO; the use of pup randomization; 

issues concerning interpretation of the RAM data given the absence of an ITI delay 

interval; and the use of the elevated T-maze for spontaneous alternation.

This study (Aldridge et al., 2005) also comes from the Slotkin lab. This time they used 

Sprague-Dawley CD rats from Charles River. They obtained timed pregnant rats and 

culled litters shortly after birth to 10, randomized pups among dams and re-randomized 

them every several days. Pups were treated with 1 mg/kg chlorpyrifos on P1-4 by s.c. 

injection in DMSO. No more than 1M/1F per artificial litter were sampled for a total 

9M/9F per treatment (hence 36 rats were used altogether). Rats were tested during the 

dark cycle starting. Tested consisted of the EPM on P52-53, a two-bottle sweetness 

preference test on P54, and starting on P64 RAM learning with ketanserin challenges on 

weeks 16-17 with the drug given 20 min prior to testing at doses of 0, 0.5, 1.0 or 1.5 

mg/kg (5HT2 antagonist). RAM was tested for 18 sessions (as above). Results: In the 

EPM, they report that CFP-exposed males had increased time in open arms (indicative on 

reduced anxiety) with no effect in females. In the sweetness preference test, CFP 

exposure reduced preference for the sweet choice in both sexes from about 4:1 to about 

3:1. In the RAM, chlorpyrifos caused treatment x sex interactions on both working and 

reference memory. For working memory, chlorpyrifos exposure increased working 

memory errors in males and decreased these errors in females; similarly reference 

memory errors were increased in chlorpyrifos-exposed males and were decreased in 

females. While several of these changes from the interaction were individually short of 

being statistically significant, when errors types were combined, the male and female 

changes in errors were significant. Ketanserin had no effect on errors of either type in 

controls, but increased both error types in chlorpyrifos exposed rats at all doses for 

working memory and at the high and low doses for reference memory. Strengths: 

Adequate sample sizes, analyses that took litter into account, well-conducted behavioral 

methods. The use of ketanserin to show an effect of a 5-HT2 antagonist tested at 

multiple dose levels of the challenge drug was a major strength of this study and is a 

finding worthy of future investigation. Weaknesses: They used only one dose of 

chlorpyrifos, they used DMSO as the vehicle, and they did not use a delayed ITI during 

RAM testing making it difficult to determine if working memory or habit learning was 

actually what was affected.

In this study (Ricceri et al., 2003) CD-1 mice (bred in-house, conception = E0), were 

culled to 5/5 M/F per litter and 30 litters were used. chlorpyrifos was administered s.c. in 

DMSO at doses of 0, 1 or 3 mg/kg on P1-4 or P11-14. On P1, 5 and 11 ultrasonic 

vocalizations were recorded; on P10 homing behavior to home cage scent was tested; on 

P25 locomotor activity was tested; on P35 mice were tested in a box divided into white 

and black compartments; social interaction with stranger mice was tested at P45; and at 

P60, passive avoidance (males only) were tested for up to 10 trials to remain in light side 

for 2 min. with a 24 h retention test. They sampled only 1 mouse per sex per litter and 
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analyses took litter into account. Results: No effect of chlorpyrifos on ultrasonic 

vocalizations or pup homing to home cage scent were obtained, or on locomotor activity 

but the authors noted a p<.06 trend in the P11-14 chlorpyrifos3 mg/kg group to be more 

active. In the white/black box, effects of P1-4 chlorpyrifos exposure were significant on 

1 out of 5 test intervals (interval-2), whereas in the P11-4 chlorpyrifos exposed animals 

activity changes were significant on 1 out of 5 test intervals (internval-4). Several 

borderline effects and several significant interactions on different social interaction 

measures were found in the CFP groups but a clear pattern was not evident. There were 

no significant CFP-related effects on passive avoidance acquisition or retention. 

Strengths: Sample sizes were adequate and litter was taken into account. Behavioral 

methods were appropriately conducted. Weaknesses: Dissolved chlorpyrifos in DMSO, 

effects were not dose-dependent nor exposure period-dependent. Most of the effects 

were small.

In this study (Venerosi et al., 2008) CD-1 mice (bred in-house, conception = E0) were 

treated S.C. with CFP at 0 or 3 mg/kg dissolved in peanut oil on P11-14. Litters were 

culled 4M/4F. Mice were evaluated in a social interaction test at P40-45. Female 

offspring bred and after delivery tested for nest building on P1-7 and other maternal 

behavior on P1 and later tested in the light/dark test of anxiety. They also did put 

retrieval test and a test for maternal aggression. These authors did a power calculation 

and sample size determination; they controlled for litter effects and used mixed model 

ANOVAs for most data analyses, but for some data they used non-parametric methods. 

They had 15 litters using a split-litter design. Results: they found no effects on ultrasonic 

vocalizations or social investigation. They found females exposed to chlorpyrifos did not 

build nests as well or defend their territory as much against a stranger male mouse, and 

took less time to emerge from the dark side of the light/dark box, but no other measure on 

this test was affected. Strengths: This was one of the most rigorous experiments in terms 

of sample size, control for litter effects and statistical methods for analyzing the data. 

Weakness: The relevance of the tests as indices of neurotoxicity are entirely speculative 

as none have been validated in this context, or in any context as strong evidence of 

developmental abnormality no matter what the independent variable.

The study by (Johnson et al., 2009) used Sprague-Dawley CD IGS rats (Charles River) 

bred in-house. Twenty litters were used in a split litter design. Litter size was adjusted to 

12-14 balanced for sex, with 7 groups per litter to the extent possible. Exposure was on 

day P1-5, 6-13, 14-20 as follows: Controls received oil from P1-20, and the low dose 

group received chlorpyrifos at 1.0 mg/kg from P1-2, but the mid and high doses groups 

received escalating doses: the mid dose received chlorpyrifos 1.0, 2.0, and 4.0 mg/kg 

during each of the aforementioned exposure ages, and the high dose received doses of 

1.5, 3.0, and 6.0 mg/kg, respectively. The remaining three groups were exposed to 

methyl parathion at doses of 0.2 mg/kg throughout, or escalating doses of 0.2, 0.4, and 

0.6 mg/kg/day (mid dose) or 0.3, 0.6, or 0.9 mg/kg/day (high dose) in oil by gavage. 

Results: The authors’ report no effects of physical landmarks of development (pinna 

unfolding, fur appearance, day of eye opening, or day that incisors erupted) and no 

effects on early reflexes (surface righting, air righting, startle emergence, cliff avoidance, 

or inclined plane). Ad adults, they tested rats in a 12-arm RAM (8 baited, 4 unbaited 
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arms). They found no significant working memory effects in females but a significant 

increase in working errors in the high dose chlorpyrifos males across sessions and at 

lower doses in final week only. For reference memory, the female mid and high dose 

chlorpyrifos groups made fewer errors, whereas for males in the mid and high dose 

groups made significantly more errors. Strengths: Used a split-litter design and 

controlled for litter effects, had adequate sample sizes, included multiple doses of 

chlorpyrifos and tested two OPs (methyl parathion), had a strong statistical approach. 

Weaknesses: Did not include an ITI delay in the RAM test. Overall, this was one of the 

stronger studies.

In this study (Carr et al., 2001) Sprague-Dawley CD rats (Charles River) were used and 

bred in-house. Rats were assigned to four groups with whole litters assigned to each 

group with a total of 5 litters per group with two offspring tested per sex per litter, i.e., 

final numbers were 10 per sex per treatment group. Rats were gavaged on P1-21 every 

other day with corn oil, or corn oil containing a lower dose CFP 3 mg/kg, a mid-dose P1-

5 of 3 mg/kg, P7-21 of 6 mg/kg, or a higher dose of P1-5 of 3 mg/kg, P7-13 of 6 mg/kg, 

and P15-21 of 12 mg/kg. The offspring were tested in an open-field on P10 and P12 for 

3 min. each time and on P14, 16, 18, 20, 25, and 30 for 6 min each time). Statistically, 

they used a general linear model ANOVA and they set a significance level at p<0.01. 

The pairwise method used was not mentioned. They found reduced locomotion at P25 

and P30 at the mid and high dose levels in both males and females. Strengths: They 

accounted for litter effects in the design and used appropriate statistical methods. They 

included three doses levels of chlorpyrifos plus control. Weaknesses: The sample size of 

five litters per group made the study under-powered and they slightly over-sampled per 

litter by using two per sex per litter.

This study (Jett et al., 2001) used Long-Evans (Charles River) rats and obtained timedpregnant animals. They culled litters to 10 and randomized pups among dams (no 

mention of sex balancing). Offspring were treated with chlorpyrifos on P7, 11, 15 at 

doses of 0.3 or 7 mg/kg given S.C. in oil to entire litters. A major concern is this 

sentence: “2 or more litters were used for random selection of pups used in behavioral 

studies”. This suggests that severe over-sampling from a few as two litters were used. 

Another group was treated with chlorpyrifos (same doses) on P22 & 26). They tested 

offspring in the MWM on P24-28 hence in postweaning treatment group one dose was 

given in the middle of the testing regimen. The MWM was 90 cm in diameter and the 

goal platform was 25 cm2; hence search ratio was 245:1 (which is within the range 

typically used for mice). They tested for 5 days, 2 trials on day-1, then 1 trial per day on 

days 2-5 with a probe trial 30 min after last training trial. They gave cued trials on day-5 

the method used was not described. Statistically, they used ANOVAs but the details are 

not provided. They report a main effect on MWM latency for both CFP exposed groups 

but the method of pairwise comparisons is not given, and they report a high dose effect 

on the probe trial for time spent in the target quadrant. There is no mention of path 

length; no differences were reported on cued performance or on swim speed. Their final 

Ns were (M/F): Control 10/10, low dose 10/9, high dose 9/8. For the postweaning 

chlorpyrifos treatment, they report a treatment main effect which they report sorting by 

day by an unspecified statistical method. Looking at the figure, most of the effect 
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appears to be on days 3-5; also both CFP groups spent less time in target quadrant on the 

probe trial. They report no speed differences and state that no cued differences were 

found but the data are not shown. For the postnatal experiment the Ns are (M/F): Control 

4/3; low dose 4/3; high dose 4/4. Strengths: They did most of the procedures in the 

MWM that should be included, such as assessing swim speed and cued performance. 

Weaknesses: There appears to be no control for litter effects and the number of litter used 

was as low as two, indicating a severely under-powered and potentially fatally flawed 

design. This is unfortunate because this is one of the few studies to test a lower dose of 

chlorpyrifos (0.3 mg/kg). Also, giving the probe trial for the MWM shortly after the last 

training trial provides somewhat ambiguous information. Changes may be attributed to 

either working or reference memory since the interval was too short to rule out working 

memory as a principal contribution.

Pre- and Postnatal Studies

This study (Maurissen et al., 2000) used Sprague-Dawley CD rats (Charles River) bred 

in-house. They used 20 litters per treatment group (conception = E0). Treatment was by 

gavage on E6-P10. Doses given were 0, 0.3, 1.0, 5.0 mg/kg/day, but note that the 

postnatal exposure was to the dams, not the pups. On P4 litters were culled to 5/5 M/F. 

They used different subsets of pups per litter for different tests as follows: Set-1: Brain 

morphometry on P11; Set-2: Delayed spatial alternation on P22-24 and again on P61-90 

(but using only 8/sex/group from 16 litters rather than 10/sex/group from all 20 litters); 

Set-3: Locomotor activity on P13, 17, 21, 60, and ASR on P22 and 61 (used 1 M/1 F 

from all 20 litters/group for these tests); Set-4: Developmental landmarks; body weight, 

and on P65-70 brained dissected and fixed for neuropathology. For the delayed spatial 

alternation test they used 3 delay intervals at each test age. For locomotion they used a 

40 x 25 photocell system. For ASR they gave 50 trials with acoustic signals of 120 dB 

with ITI = 10 s. Data were analyzed by ANOVA with litter taken into account. P-values 

were considered significant at P<0.02. Where treatment main effect occurred, follow up 

was by a stepwise approach by first removing of high dose and re-analyzing the data, if 

still significant, then removal of the mid dose and reanalyze, etc. Interactions were 

followed up using simple-effect ANOVAs. Results: They report delayed vaginal patency 

at the high dose; delayed pinna detachment and prenuptial separation at p<0.03 and P<.05 

neither of which reached their P<.02 cut-off. However these would be more commonly 

regarded as significant. They found no significant effects on the delayed spatial 

alternation test. They found no significant effects on locomotor activity. They found a 

trend on ASR latency at P<.03 but no effect on startle amplitude (the principal measure 

on this test). Strengths: This study has the most robust sample size in this entire group of 

21 articles. They controlled for litter effects and used appropriate statistical methods. 

They delayed spatial alternation test was a particular strength and in their Fig. 7 they 

show the working memory decay as a function of the length of the delay interval, proving 

that they are measuring working memory. They included control plus 3 dose levels of 

chlorpyrifos and this is one of the only studies to test a low dose (0.3 mg/kg 

chlorpyrifos). They also avoided the use of DMSO as the vehicle. Weaknesses: The 

delayed spatial alternation test used much reduced sample sizes compared to the study as 
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a whole (8 per sex per group rather than 20). While this is of some concern, there were 

no trends in the results suggesting that a latent effect might have been missed. For the 

ASR, they performed a simple startle habituation test rather than a PPI procedure which 

is more informative. Note: It may be significant given the discussion at the meeting that 

they used an oral rather than subcutaneous route of exposure. Given the first pass 

metabolism of chlorpyrifos, this may have implications for the total amount of exposure 

the rats received.

This study (Braquenier et al., 2010) used CD-1 mice (Charles River) but the number of 

dams was not given nor was how conception was dated (a relevant factor in studies with 

prenatal exposure). Doses of chlorpyrifos were 0, 0.2, 1.0, 5 mg/kg/d given on E14-P14. 

For the postnatal CFP exposure the compound was given to dams (not pups). Litters 

were culled to 4M/4F with one female per litter used for testing. They tested offspring 

for locomotor activity (5 min/session) for 8 days (scored manually). They also did a 5 

min light/dark test (mice started on the light side). EPM was assessed (standard 5-min 

procedure). Data were analyzed by ANOVA with follow-up by Dunnett’s test. Results: 

They found no significant effects on locomotor activity. For the light/dark test, they 

found no effect on the percent of time spent in the dark compartment but a significant 

decrease in the percent of time in the light compartment in the chlorpyrifos1 group, but 

not in the chlorpyrifos0.2 or 5 mg/kg groups. Side transitions showed trend (P<.08) 

which they followed-up anyway and report a significant reduction in the CFP1 group by 

Dunnett. In the EPM they report a trend at p<.10, did Dunnett follow up tests anyway 

and found reduced time in open arms in the CFP1 group but not the other dose groups. 

They also found that the percentage of arm entries into open arms was significantly 

decreased in the CFP1 group. Total transitions were not significantly affected. 

Strengths: Tested three dose levels of chlorpyrifos, including a low dose (0.3 mg/kg), and 

gave the compound in oil rather than DMSO, and used standard methods. Weaknesses: 

No indication that litter effects were accounted for in the design or statistical analyses. 

Effects were found only at the 1 mg/kg dose and were not dose-dependent, they did 

follow-up tests on many trends that were not statistically significant and declared these 

follow-up effects to be significant findings.

Summary

Among these 21 reviewed articles (which include more than 21 experiments), many 

effects are reported at chlorpyrifos doses ranging from at 1.0 to 7.0 mg/kg (and in one 

study 10 mg/kg). However, these are dose levels known to significantly inhibit 

cholinesterase in RBC; therefore, based on these 21 studies, cholinesterase inhibition is 

an adequate threshold as no credible evidence of neurobehavioral effects below 1.0 

mg/kg were found.

Three studies tested doses < 1.0 mg/kg chlorpyrifos. Two studies used 0.3 mg/kg and 

one used 0.2 mg/kg (Jett et al., 2001; Braquenier et al., 2010; Maurissen et al., 2000, 

respectively). Of these, two found no effects at these lower doses (Maurissen et al., 

2000;Braquenier et al., 2010). Only one study found effects at 0.3 mg/kg (Jett et al., 

2001); however, this study contains serious methodological flaws which are of sufficient 
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magnitude to cast serious doubt on the credibility of the findings. While the data from 

Jett et al. (2001) raise the possibility of neurobehavioral effects at 0.3 mg/kg/d, their data 

require replication in a study that is properly designed, adequately powered, and 

appropriately analyzed. Until such time as new data at such lower doses become 

available, it is concluded that no dose <1.0 mg/kg in any neurodevelopmental behavioral 

study shows evidence of adverse effects (or of any effects, even including those outcome 

measures whose effect is indeterminate or unknown).

In addition, effects of chlorpyrifos at 1.0 mg/kg are difficult to interpret because of 

methodological limitations, inconsistencies, and variation in study design, sometimes 

lack of control for litter effects, oversampling issues, behavioral methods used, and lack 

of dose-response findings.

Above 1.0 mg/kg, the data show somewhat more consistency, but even here, doseresponse experiments are the exception. At 5.0 mg/kg of chlorpyrifos, reduced body 

weight is sometimes seen, and at doses above 5.0 mg/kg, increased mortality may occur 

along with other evidence of toxicity. Given this, a significant gap in the literature of 

dose-response studies exists in the range downward toward 0.2 mg/kg and extending up 

to and including doses previously tested of 1.0-2.0 mg/kg that is needed in order to 

determine what, if any, dose-effect curve for neurobehavioral effects occurs in this range.

It appears that neurobehavioral outcomes are more sensitive to prenatal and prenatalneonatal exposures than to neonatal exposure alone . This implies that prenatal exposure 

may be the exposure period contributing to this observation, but unfortunately, most of 

the pre- and neonatal studies are not entirely informative because the neonatal exposure 

was to the dam rather than directly to the progeny. This makes it unclear what the dose 

to the offspring actually was . More studies, especially dose-response studies, in the 

lower dose ranges with exposure from implantation to the end of major neurogenesis 

(approximately P20) are needed, again with doses below 1.0 mg/kg and with concomitant 

measurement of maternal, fetal, and neonatal cholinesterase activity.

Exposures in many of the existing studies are for only a narrow interval during gestation 

or the neonatal period. Prenatal exposures should be from E6-20 to 21 for rats, and E6-

18 or 19 in mice in order to span most of early brain development (equivalent to human 

first and part of the second trimester). And for neonatal treatment, exposures should be 

from shortly after birth to approximately P20 (equivalent to latter half of the second and 

all of the third trimester equivalent brain development for humans). If the critical period 

or most sensitive period is within this range, then such comprehensive exposure should 

cover the entire span of CNS development that represents the species being modeled, i.e, 

human beings.

Exposures in many of the existing studies are for only a narrow interval during gestation 

or the neonatal period. Prenatal exposures should be from E6-20 to 21 for rats, and E6-

18 or 19 in mice in order to span most of early brain development (equivalent to human 

first and part of the second trimester). And for neonatal treatment, exposures should be 

from shortly after birth to approximately P20 (equivalent to latter half of the second and 
106

all of the third trimester equivalent brain development for humans). If the critical period 

or most sensitive period is within this range, then such comprehensive exposure should 

cover the entire span of CNS development that represents the species being modeled, i.e, 

human beings.

In the prenatal studies, the use of timed-pregnant females shipped from breeders is 

problematic for behavioral studies because maternal stress, even if regarded as equivalent 

across dams assigned to the treated and control groups, introduces a variable that has the 

potential to interact with the independent variable. Were maternal stress to interact with 

chlorpyrifos, it would confound the outcome and make a result difficult to interpret 

(which is exactly what is found in the 21 reviewed studies). Since no one has tested for 

this, it is currently impossible to rule it out.

Many studies use diurnal and some nocturnal testing. If additional dose-response studies 

are undertaken, this factor should be held constant so that results can be better compared.