UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON D.C., 20460 

OFFICE OF 

PREVENTION, PESTICIDES AND TOXIC 

SUBSTANCES 

December 17, 2008 

MEMORANDUM 

SUBJECT: 

TO: 

FROM: 

THRU: 

Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting 

Held September 16-18, 2008 on the Agency's Evaluation of the Toxicity Profile 

of Chlorpyrifos 

Debbie Edwards, Ph.D. 

Director 

Office of Pesticide Programs 

Sharlene R. Matten, Ph.D. 

Designated Federal Official 

FIFRA Scientific Advisory Panel 

Office of Science Coordination and Policy ,1 

Steven Knott, Executive Secretary i~>'-'.fd/. Tf}. yf,;z--;:ft· 

FIFRA Scientific Advisory Panel -

Office of Science Coordination and Policy 

....-:-,(-~IS F~ank Sander~ r ~~<" --f!cr . Duector '; '·, ... ____ _ 

Office of Science Coordination and Policy 

Attached, please find the meeting minutes of the FIFRA Scientific Advisory Panel open 

meeting held in Aflington, VA on September 16-18, 2008. This report addresses a set of 

scientific issues regarding the Agency's Evaluation of the Toxicity Profile ofChlorpyrifos. 

Attachments 
cc: 

James B. Gulliford 

James J. Jones 

Anne Lindsay 

William Jordan 

Margie Fehrenbach 

Donald Brady 

Janet Andersen 

Steven Bradbury 

William Diamond 

Joan Harrigan-Farrelly 

Tina Levine 

Lois Rossi 

Richard Keigwin 

Enesta Jones 

Vanessa Vu (SAB) 

Anna Lowit 

Deborah Smegal 

Ginger Moser 

Jack Housenger 

OPP Docket 

FIFRA SAP Members 

John R. Bucher, Ph.D., D.A.B.T. 

Janice E. Chambers, Ph.D., D.A.B.T., 

A.T.S. 

Kirby C. Donnelly, Ph.D 

Steven G. Heeringa, Ph.D. 

CareyN. Pope, Ph.D 

Kenneth M. Portier, Ph.D. (FIFRA SAP 

Session Chair) 

Daniel Schlenk, Ph.D. 

FQPA Science Review Board Members 

Sophie J. Balk, M.D. 

Laura Beane Freeman, Ph.D. 

John F. Bowyer, Ph.D .. 

Russell L. Carr, Ph.D. 

Michael DiBartolomeis, Ph.D., DABT 

John Doull, M.D., Ph.D., ATS 

Paul B. English, Ph.D., M.P.H. 

Gaylia Jean Harry, Ph.D 

Wendy J. Heiger-Bemays, Ph.D. 

Antonio Hernandez Jerez, M.D., Ph.D. 

Bette Meek, M.S. 

John D. Meeker, Sc.D., CIH 

Andrew C. Povey, Ph.D. 

Nu-may Ruby Reed, Ph.D., DABT 

Sonya K. Sobrian, Ph.D. 

Heather A. Young Durick, Ph.D., MPH 
SAP Minutes No. 2008-04 

A Set of Scientific Issues Being Considered by the 

Environmental Protection Agency Regarding: 

The Agency's Evaluation of the Toxicity Profile of 

Chlorpyrifos 

September 16-18, 2008 

FIFRA Scientific Advisory Panel Meeting 

held at the 

Holiday Inn - Rosslyn 

Arlington, Virginia 
NOTICE 

These meeting minutes have been written as part of the activities ofthe 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 ofthese 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 (FQP A) 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. FQP A 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 Sharlene R. Matten, Ph.D., SAP Designated Federal Official, via e-mail at 

matten.sharlene@epa. gov. 

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

presented by EPA, as well as information presented in public comment. This document 

addresses the information provided and presented by EPA within the structure of the charge. 

2 
TABLE OF CONTENTS 

NOTICE ............................................... ~ .............. ~·········································································· 1 

TABLE OF CONTENTS ................................. ' ............................................................................ 3 

PUBLIC COMMENTERS ..................................................................................... ~ ..................... 9 

SUMMARY OF PANEL DISCUSSION AND RECOMMENDATIONS ............................. 10 

DETAILED RESPONSES TO CHARGE QUESTIONS ........................................................ 17 

1. METABOLISM & TOXICOKINETICS .......................................................................................... 17 

2. CHOLINESTERASE INHIBITION ................................................................................................ 21 

3. LABORATORY STUDIES ON THE DEVELOPING BRAIN .............................................................. 24 

4. EPIDEMIOLOGY STUDIES IN CHILDREN AND MOTHERS ........................................................... 31 

5. HUMAN lNFORMA TIONA V AILABLE FOR RISK ASSESSMENT ................................................... 44 

6. POINTS OF DEPARTURE (PoD) FOR RISK 

ASSESSMENT ................................................................................................................... 50 

7. EXTRAPOLATION/UNCERTAINTY FACTORS ............................................................ 55 

REFERENCES ............................................................................................................................ 64 

3 
SAP Minutes No. 2008-04 

A Set of Scientific Issues Being Considered by the 

Environmental Protection Agency Regarding: 

The Agency's Evaluation of the Toxicity Profile of 

Chlorpyrifos 

September 16-18, 2008 

FIFRA Scientific Advisory Panel Meeting 

held at the 

Holiday Inn - Rosslyn 

Arlington, Virginia 

Kenneth M. Portier, Ph.D. 

FIFRA SAP Session Chair 

FIFRA Scientific Advisory Panel 

Date: t "l-/ Frjo q 

S arlene R. Matteo, Ph.D. 

Designated Federal Official 

FIFRA Scientific Advisory 

Panel Staff 

Date: t 2- j1r(D f? 
Federal Insecticide, Fungicide, and Rodenticide Act 

Scientific Advisory Panel Meeting 

September 16-18,2008 

The Agency's Evaluation of the Toxicity Profile of Chlorpyrifos 

PARTICIPANTS 

FIFRA SAP Chair 

Steven G. Heeringa, Ph.D., Research Scientist & Director for Statistical Design, University of 

Michigan, Institute for Social Research, Ann Arbor, MI 

FIFRA SAP Session Chair 

Kenneth M. Portier, Ph.D., Program Director, Statistics, American Cancer Society, Statistics 

and Evaluation Center, Atlanta, GA 

Designated Federal Official 

Sharlene R. Marten, Ph.D., FIFRA Scientific Advisory Panel, Office of Science Coordination 

and Policy, EPA 

FIFRA Scientific Advisory Panel Members 

John R. Bucher, Ph.D., DABT, Associate Director, National Toxicology Program, National 

Institute of Environmental Health Sciences, Research Triangle Park, NC 

Janice E. Chambers, Ph.D., D.A.B.T., A.T.S., Director, Center for Environmental Health 

Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 

Kirby C. Donnelly, Ph.D., Professor and Head, Department of Environmental and Occupational 

Health, School of Rural Public Health, Texas A&M University System Health Science Center, 

College Station, TX . 

··Carey N. Pope, Ph.D., Professor, Head & Sitlington Chair of Toxicology 

Department of Physiological Sciences, Oklahoma State University College ofVeterinary 

Medicine, Stillwater, OK 

Daniel Schlenk, Ph.D., Professor, Aquatic Toxicology, Department of Environmental Sciences, 

University of California, Riverside, CA 

5 
FQPA Science Review Board Members 

Sophie J. Balk, M.D., Attending Pediatrician, Professor of Clinical Pediatrics, Children's 

Hospital at Montefiore, Albert Einstein College ofMedicine,Bronx, NY 

Laura Beane Freeman, Ph.D., Occupational and Environmental Epidemiology Fellow, 

National Cancer Institute, Bethesda, MD 

John F. Bowyer, Ph.D., Research Pharmacologist, National Center for Toxicological 

Research/FDAJHHS, Jefferson, AR 

Russell L. Carr, Ph.D., Associate Professor, College ofVeterinary Medicine, Center for 

Environmental Health Sciences, Mississippi State University, Mississippi State, MS 

Michael DiBartolomeis,_Ph.D., DABT, Research Scientist Supervisor II, Occupational Health 

Branch, California Department of Public Health, Richmond, CA 

John Doull, M.D., Ph.D., ATS, Professor Emeritus of Pharmacology & Toxicology, 

Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical 

School, Kansas City, KS 

Paul B. English, Ph.D., M.P.H., Scientific Advisor, Environmental Health Investigations 

Branch, California Department of Public Health, Richmond, CA 

Gaylia Jean Harry, Ph.D., Biologist, Head ofNeurotoxicology Group, Division of Intramural 

Research, National Institute of Environmental Health Sciences, Research Triangle Park, NC 

Wendy J. Heiger-Bernays, Ph.D., Associate Professor, Department of Environmental Health 

Boston Uni\rersity School of Public Health, Boston, MA 

Antonio Hernandez Jerez, M.D., Ph.D., Senior Lecturer in Toxicology, Department of Legal 

Medicine and Toxicology, University of Granada, Granada, SPAIN 

Bette Meek, M.S., Associate Director of Chemical Risk Assessment, McLaughlin Institute for 

Population Health Risk Assessment, University of Ottawa, Ottawa, Ontario, CANADA 

John D. Meeker, Sc.D., CIH, Assistant Professor, Department of Environmental Health 

Sciences, University of Michigan School ofPublic Health, Ann Arbor, MI 

Andrew C.Povey, Ph.D., Reader in Molecular Epidemiology, Occupational & Environmental 

Health Research Group, Faculty of Medical and Human Sciences University of Manchester, 

Manchester, England, UNITED KINGDOM 

Nu-may Ruby Reed, Ph.D., D.A.B.T., StaffToxicologist, Department of Pesticide Regulation, 

California EPA, Sacramento, CA 

6 
Sonya K. So brian, Ph.D., Associate Professor, Department of Pharmacology, Howard 

University College of Medicine, Washington, DC 

Heather A. Young Durick, Ph.D., MPH, Associate Professor, Department of Epidemiology 

and Biostatistics, The George Washington University, Washington, DC 

7 
INTRODUCTION 

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

(SAP) has completed its review ofThe Agency's Evaluation of the Toxicity Profile of 

Chlorpyrifos. Advance notice of the SAP meeting was published in the Federal Register on 

July 9, 2008 and August 28, 2008. The review was conducted in an open panel meeting 

September 16-18, 2008 held at the Holiday Inn-Rosslyn, Arlington, Virginia. Dr. Kenneth M. 

Portier chaired the meeting. Dr. Sharlene R. Matten served as the Designated Federal Official. 

Dr. Tina Levine, Director, Health Effects Division, Office of Pesticide Programs (OPP), 

provided opening remarks at the meeting. Presentations of technical background materials were 

provided by Dr. Anna Lowit and Ms. Deborah Smegal, MPH, Health Effects Division, OPP and 

by Dr. Ginger Moser, EP A-ORD-N ational Health and Environmental Effects Research 

Laboratory (NHEERL). Additional technical assistance was provided by Dr. John Liccione and 

Dr. John Doherty of the Health Effects Division, OPP. 

Chlorpyrifos (0,0-diethyl-0-3,5,6-trichloro -2-pyridinyl phosphorothioate) is a broad-spectrum, 

chlorinated organophosphorus (OP) insecticide. Chlorpyrifos is one of the most widely used OPs 

in the U.S. In 2000, nearly all residential uses were voluntarily cancelled by Dow AgroSciences, 

LLC. However, chlorpyrifos continues to be used extensively in commercial agriculture. Since 

2000, there has been extensive research on various aspects of chlorpyrifos toxicity, particularly 

on effects in animals and humans from gestational and postnatal exposure. Many new studies in 

rats investigating different endpoints including acetylcholinesterase (AChE) inhibition and 

adverse effects on the developing brain are now available. In addition, manuscripts have recently 

been published from three cohorts of pregnant women and children exposed in utero to 

organophosphates (OPs). At this time, the Agency is re-evaluating the extent to which toxicity 

endpoints and extrapolation/uncertainty factors for chlorpyrifos require updating based on this 

new information. The Agency's issue paper and associated appendices contain the proposed 

updates and the scientific foundation for the proposed revisions. The contents and conclusions 

drawn in the issue paper and appendices are preliminary. The ultimate goal of the Agency's ongoing work is to improve the scientific support for the Agency's risk assessment. This will be 

accomplished by 1) evaluating new data on potentially susceptible subpopulations and 2) 

incorporating improved approaches, e.g., benchmark dose modeling instead of relying on noobserved-adverse-effect levels (NOAELs) for points of departure; and using extrapolation factors 

based on data instead of relying on default factors to account for differences in animals and 

humans and among humans. The Agency has progressed to a point in the review that feedback 

from the FIFRA SAP would be helpful. 

8 
PUBLIC COMMENTERS 

Oral statements were presented by: 

1) Daland Juberg, Ph.D., Dow AgroSciences, LLC 

2) Charles Timchalk, Ph.D., DABT, Battelle Center for BiologicalMonitoring and 

Modeling 

3) Carol Bums, Ph.D., Dow Chemical Company 

4) Pamela Mink, Ph.D., MPH, Department of Epidemiology, Emory University 

5) Michael Bartels, Ph.D., Dow Chemical Company 

6) Douglas Weed, M.D., MPH, Ph.D., DLW Consulting Services 

7) Robert Sielken, Ph.D., Sielken & Associates Consulting, Inc. 

8) Michael Dourson, Ph.D., DABT, ATS, Toxicology Excellence for Risk Assessment 

(TERA) 

9) Mr. Ray McAllister, Crop Life America 

1 0) Elliot Gordon, Ph.D., Elliot Gordon Consulting, LLC 

11) Jennifer Sass, Ph.D., Natural Resources Defense Council (NRDC) and on behalf of 

Pesticide Action Network North America (PANNA) 

12) Michael Fry, Ph.D., American Bird Conservancy 

13) Robin M. Whyatt, Ph.D., and Virginia A. Rauh, Ph.D., Columbia Center fo(Children's 

Environmental Health, Mailman School of Public Health, Columbia University 

Written statements were provided by: 

1) Torka S. Poet, Ph.D. and Charles Timchalk, Ph.D., DABT, Battelle Center for Biological 

Monitoring and Modeling, Battelle 

2) Charles Timchalk, Ph.D., Battelle Center for Biological Monitoring and Modeling 

3) David, Eaton, Ph.D., DABT, FATS, Center for Ecogenetics and Environmental Health 

and Associate Vice Provost for Research, University of Washington on behalf of the 

authors of Eaton et al. 2008 

4) Theodore Slotkin, Ph.D., Professor of Pharmacology and Cancer Biology and Psychiatry 

and Behavioral Sciences and Neurobiology and Director of Graduate Studies, Integrated 

Toxicology and Environmental Health Program, Duke University Medical Center 

5) Scott Phillips, M.D., Department of Medicine, University of Colorado Health Sciences 

Center 

6) Pamela Mink, Ph.D., MPH, Department of Epidemiology, Emory University 

7) Douglas Weed, M.D., MPH, Ph.D., Founder and Managing Member, DLW Consulting 

Services, LLC 

8) Kenneth D. Racke, Ph.D., Dow AgroSciences, LLC 

9) Gary J. Mihlan, Ph.D., CIH and Walter Schmitt, Ph.D., Bayer CropScience 

1 0) Michael Dourson, Ph.D., DABT, A TS, Bernard Gadagbui, Ph.D., DABT, and Lynne 

Haber, Ph.D., DABT, Toxicology Excellence for Risk Assessment (TERA) 

9 
SUMMARY OF PANEL DISCUSSION AND RECOMMENDATIONS 

The Panel acknowledged the extent of the chlorpyrifos database and coinmended the Agency for 

preparing a comprehensive document considering the scientific evidence as a whole. Some of 

the more significant new data available to the Agency since the preparation of the 2000 

chlorpyrifos risk assessment comes from three large prospective cohort studies of pregnant 

women and their children. Throughout the two-day discussion, ~anel members referred to the 

results of these studies in an attempt to integrate their findings with the toxicology data from 

laboratory animal experiments. Despite the large volume of data available to address the risks of 

chlorpyrifos exposure from agricultural uses, several Panel members were concerned that a high 

degree of uncertainty is evident in the available data, particularly under low dose, chronic 

conditions. Uncertainty was expressed in attributing observed adverse effects to chlorpyrifos in 

the epidemiological studies where exposure was to metabolites of chlorpyrifos or to a mixture of 

chlorpyrifos and two additional anticholinesterase insecticides. The Panel agreed that the 

epidemiological studies have utility for risk assessment, but not as the principal basis for 

characterization of the point of departure (PoD). There was extensive discussion on 

experimental and epidemiological studies, e.g., oflow-dose exposure in animals, of exposures in 

agricultural and pesticide handlers, and of the mode of action including the need to develop 

chlorpyrifos-specific PBPK models, that could provide the information needed to address critical 

data gaps and reduce uncertainty. 

1. Metabolism & Toxicokinetics 

The Panel concluded that a weight of evidence evaluation of available information supports the 

Agency's conclusions that 1) sensitivity to the adverse effects of chlorpyrifos is influenced by 

age, with young animals having lesser total ability to detoxify chlorpyrifos and many other 

organophosphorus compounds, and 2) that the age-dependent sensitivity observed in 

experimental animals is due mostly to toxicokinetic (TK) differences between juveniles and 

adults instead of only being the result oftoxicodynamic (TD) differences. 

The Panel agreed that current scientific data on chlorpyrifos uptake, transport, sequestration and 

excretion suggest that individual differences in metabolism and transformation will explain much 

of the variability seen in these factors, but that other potential TK differences between juveniles 

and adults should not be dismissed. For instance, the high respiratory rate of children may 

enhance the absorption of chlorpyrifos present in the air .. Children have a greater cardiac output 

compared to adults, together with their less developed blood-brain barrier; facilitate some of the 

chlorpyrifos-oxon reaching the brainS of exposed infants. 

Some panel members questioned whether detoxication of the oxon plays a significant role in 

explaining the differential susceptibility between adults and juveniles at lower levels of 

chlorpyrifos exposure. Toxicokinetic differences are less likely to be relevant under low dose 

conditions compared to high dose conditions where enzyme systems may become saturated. 

Human tissue data specific to enzyme-mediated detoxication are minimal, however. While blood 

levels of detoxifying enzymes (e.g., PONt, carboxylesterases) have been studied, data on 

chlorpyrifos detoxication in specific organs (e.g., the liver) from humans are limited, making it 

10 
difficult to draw firm conclusions about the role of activation and detoxication in explaining 

differences in susceptibility to chlorpyrifos toxicity between human infants, children, and adults. 

The Panel also agreed with the Agency's conclusion that "pregnant animals and humans may be 

somewhat more sensitive to chlorpyrifos than non-pregnant adults" based on the expected role of 

key detoxication enzymes (e.g., paraoxonase, P450 isozymes) in modulating levels of 

chlorpyrifos in animal studies. However, the Panel noted that there are relatively small 

differences in levels of these key enzymes between pregnant and non-pregnant animals. At dose 

levels expected from environmental exposures the importance of such small differences in 

enzyme levels is unknown, but the data suggest a reduced capacity to detoxify chlorpyrifos 

during pregnancy. 

2. Cholinesterase Inhibition 

Panel members agreed with the Agency's conclusion that postnatal rats are more sensitive than 

adult rats to increased cholinesterase inhibition and lethality following acute exposures. 

Increased susceptibility appears to be the result of the juvenile animals' lower capacity to 

detoxify either chlorpyrifos or the chlorpyrifos-oxon. The Panel was uncertain about how to 

interpret the data describing differences in cholinesterase inhibition between post-natal animals 

and adults following repeated chlorpyrifos dosing. The evidence suggests that younger animals 

are less sensitive than adults to cholinesterase inhibition from low dose repeated exposures, 

likely due to a lack of enzymes capable of converting chlorpyrifos to the oxori form in younger 

animals. In essence, the total acetylcholinesterase activity in the younger animal's tissues 

increases more effectively in between exposures, leading to lesser accumulation of inhibition 

over time than in adults. However, the role of the activation and detoxication pathways in 

producing differential cholinesterase inhibition and cholinergic effects at low dose repeated 

exposures is not clearly defined by the available data. 

The Panel agreed with the Agency's preliminary conclusion that data showing less cholinesterase 

inhibition after repeated dosing during gestation may not reflect the true potential toxicity to the 

fetus. The data indicate, however, that the level of cholinesterase inhibition depends on the time 

of sampling following exposure. Developing rats would be expected to show less cholinesterase 

inhibition than would adults, with increasing length of time of measurements vs. dosing. Two 

mechanisms may explain this phenomenon: 1) a higher rate of recovery of the inhibited 

cholinesterase molecules and/or 2) an increase in cholinesterase enzyme synthesis, in the 

developing rat. There is little experimental evidence to indicate differences in enzyme 

reactivation following inhibition in cholinesterases from either young or adult tissues. The 

difference in rate of synthesis of new enzyme molecules is likely the dominant factor. A more 

accurate comparison of cholinesterase inhibition between the developing animal and the adult 

could be determined if sample collection coincided with the time of the peak cholinergic effect. 

3. Laboratory Studies on the Developing Brain 

In general, the Panel agreed with the Agency's conclusion that gestational or early postnatal 

exposures can lead to neurochemical and behavioral alterations that persist into adulthood. A 

number of rodent studies suggest that prenatal or postnatal exposures may lead to long-term 

11 
neurobehavioral changes in motor and cognitive behaviors. The Panel question~d the meaning of 

the phrases "at sufficiently high exposures" and "persisting into adulthood after any initial AChE 

inhibition has reversed." Some panelists stated that a "sufficiently high exposure" should be 

defined on the basis of a dose that induces brain cholinesterase inhibition. They noted that the 

majority of experimental animal studies reviewed by the Agency used doses of about 1 mg/kg. 

This dose (exposure level) should be sufficient to inhibit brain cholinesterase activity and at the 

same time causes neurobehavioral effects in most age groups. 

Some members questioned the experimental methods used in some of the animal studies as well 

as the interpretation and application of the results of neurobehavioral testing in animals for risk 

assessment. Several Panel members felt that while behavioral findings are consistently reported 

in the literature, the types of behavioral activities reported as significant were not always the 

same. It was acknowledged that the study outcomes could be affected by 1) the route of 

administration of chlorpyrifos, 2) the developmental period of exposure, 3) the methods used to 

measure changes in behavioral domains, and 4) the choice of dependent variables. Panel 

members agreed with the Agency's expressed caution on the use of dimethyl sulfoxide (DMSO) 

as a vehicle because of its intrinsic toxicity and potential influence on absorption. In addition, 

uncertainly was expressed about potential interactions between DMSO and low doses of 

· chlorpyrifos and the effect of this interaction on the developing animal. 

The Panel agreed with the Agency's determination that insufficient data exist to clearly identify a 

specific mode of action for effects in the developing nervous system for use in risk assessment. 

Some panel members believed that studies cited by Eaton et al. (2008) were indicative of 

possible non-cholinergic modes of action. Other panel members disagreed with this conclusion. 

They stated that the effects on blood cholinesterase are considered protective for the observed 

effects (i.e., occurring at lower doses); however, nothing is implied about mode of action, except 

in the context of species and interindividual variability in toxicokinetics. Most of the Panel stated 

that the available information does not allow behavior to be considered as a point of departure. 

These panelists stated that cholinesterase inhibition should continue to be used for PoD until, at 

such time; an alternative mode of action is identified and validated. Collaboration between EPA 

and developmental neurotoxicologists would help identify enzyme inhibition occurring in 

discrete brain sites at critical periods of development. 

4. Epidemiology Studies in Children and Mothers 

The Panel agreed with the Agency's conclusions regarding the potential utility of the 

epidemiological data from the three cohort studies (Columbia University in New York, Mt. Sinai 

Hospital in New York, and the University of California at Berkeley) in broadly characterizing 

risk of chlorpyrifos exposure to pregnant women, fetuses, infants, and children. They also 

concurred that these studies should not be considered as the principal basis for characterization 

of the PoD. The Columbia study was considered the most epidemiologically-sound and to have 

adequately addressed selection and information biases to the best extent possible. It was also 

considered the most robust and appropriate in assessing chlorpyrifos developmental toxicity 

because specific measurements of exposure to chlorpyrifos in neonates and children (i.e., the 

study populations) were obtained. Confounding factors in the Mt. Sinai and Berkeley studies, 

particularly the fact that exposures were based on OP and carbamat~ metabolites and that 

12 
chlorpyrifos was not specifically measured, reduce their utility in a quantitative context for risk 

assessment. Several panel members pointed out that although the Agency classified the study 

population's exposure as "high" for a residential setting, it would be considered a "low'~ exposure 

for a population of agricultural workers or pesticide handlers. 

The weaknesses inherent to observational studies were discussed. Given the paucity of human 

epidemiological studies of OP insecticides in children, the Mt. Sinai and Berkeley studies do 

provide useful information for risk assessment. For example, these studies provide data 

demonstrating abnormal reflexes in neonates that is not available from the Columbia cohort 

study. In addition, the Mt. Sinai cohort study considered paraoxonase activity as a factor in the 

analyses that may have relevance for human toxicity. There is uncertainty in the exposure 

estimates in the Columbia study because measurements were derived from a single time point 

· (maternal and/or cord blood). Notwithstanding this uncertainty, the Panel felt that the results 

from the Columbia study raise concerns for adverse neurodevelopmental effects in children 

exposed in utero to environmental levels of chlorpyrifos although there was confounding by the 

presence of other anticholinesterase insecticides. The majority of issues raised by the Panel were 

points of clarification or issues that ifaddressed could strengthen the analysis and provide the 

Agency with better and more useful data for risk assessment. 

Overall, the Agency's conclusion that chlorpyrifos could have contributed to the birth and 

neurodevelopmental outcomes noted in the three cohort studies was supported. The Panel agreed 

with the Agency that: a) exposures to multiple cholinesterase-inhibiting insecticides in 

combination cannot be ruled out as contributing to the birth and neurodevelopmental outcomes; 

b) a potential synergistic and/or additive effect of these compounds does not rule out a role for 

chlorpyrifos in contributing to the adverse outcomes; and c) it cannot be determined if 

chlorpyrifos is the sole contributor to the observed outcomes. These conclusions are supported 

by the Columbia study finding of a crude dose-response relationship to chlorpyrifos levels which 

persisted even after controlling for exposure to other pesticides. 

When the results ofthe three cohort studies (with an emphasis on the Columbia study) are 

considered along with the findings from experimental studies in animals, the Panel concluded 

that maternal chlorpyrifos exposure would likely be associated with adverse neurodevelopmental 

outcomes in humans. Some panelists indicated that if the associations between chlorpyrifos 

exposure and the reported outcomes are causally related, then it is possible to conclude that the 

mechanism of action might be independent of cholinesterase inhibition. Exposures to multiple 

cholinesterase-inhibiting pesticides or other neurotoxicants may result in additive or interactive 

effects. This information might be useful when considering cumulative exposures to these 

pesticides. 

5. Human Information Available for Risk Assessment 

The Panel generally agreed with the Agency's conclusion that due to their limitations, the 

epidemiological data currently available are useful primarily for hazard identification. The Panel 

. disagreed on whether the current epidemiological data provide sufficient evidence to suggest that 

the uncertainty factor for the cholinesterase inhibition endpoint be changed to accommodate the 

possibility of neurodevelopmental effects from low-level in utero exposures of chlorpyrifos. The 

13 
majority of the Panel agreed that the current epidemiological data do not provide sufficient 

evidence to increase the uncertainty factor for the cholinesterase inhibition to accommodate the 

possibility of neurodevelopmental effects. However, some panel members felt strongly that the 

current epidemiological data do provide evidence to indicate that the margin of safety should be 

increased. The Panel recommended that the Agency conduct a full formal weight of evidence 

evaluation for causality of the reported associations between exposure to chlorpyrifos and 

neurodevelopmental outcomes in the existing epidemiological database. 

The Panel recommended that the Agency continue its collaboration with Columbia University 

researchers in analyzing the epidemiological data. The Agency is encouraged to continue open 

discussions with the study researchers (and the Centers for Disease Control and Prevention, as 

appropriate) to seek clarification on the level of confidence in the reported exposure levels. The 

Agency should then attempt to use the cohort data quantitatively to inform the risk assessment 

process, such as in a boundary setting exercise. 

The use of physiologically-based pharmacokinetic (PBPK) modeling would enable estimation of 

exposure dose metric for multiple sources of exposure, e.g., air, food, water. The Panel also 

agreed with the concept of using a PBPK model to examine individual intra-species variations as 

well as inter-species differences. While the potential contribution of the chlorpyrifos PBPK 

model being developed was recognized, some panelists believed that the Agency should also 

pursue a simpler PBPK model specifically applicable to the chlorpyrifos data that would be 

available in a relatively short timeframe. The Panel concluded that the epidemiological data may 

be used for bounding exposure levels, and in conjunction with PBPK models, address current or 

potential human exposures and to determine the final reference dose or reference concentrations. 

The Panel agreed with the Agency's approach to deriving a dermal absorption factor using data 

from the deliberate dosing in human studies. This approach is especially valuable if no other 

sources of data are available. However; the current 3% absorption factor should be further 

refined by considering different exposure scenarios and adjusting for expected underestimation 

of exposure. The Panel also agreed with the Agency's scientific analysis that these delibe:J;"ate 

dosing studies cannot be used to directly establish aPoD or UFs, but indicated that these data 

might be used as bounding levels similar to what was suggested by the Panel concerning the data 

from the epidemiological studies. 

6. Points of Departure (PoD) for Risk Assessment 

The Panel was presented with three options for deriving points of departure for acute and chronic 

chlorpyrifos exposure from animal studies for extrapolating human risk. Panel members 

expressed difficulty in separating the discussion of points of departure from that ofuncertainty 

factors with the result that the discussion of these two issues overlapped in many areas. 

The Panel agreed that Option 3 was the most favorable option for deriving points of departure for 

acute and chronic exposure. There was also general agreement among Panel members with the 

Agency's proposal to use the dermal and inhalation studiesfrom their 2000 assessment as a basis 

to develop points of departure for these exposure routes, taking into account sensitive life stages 

and adjusting for dosing regimen. Option 3 was chosen principally on the basis that the 

proposed point of departure for acute exposure takes into account all life stages, is based on 

14 
benchmark doses (an advantage over the NOAEL or LOAEL), and represents the results of 

several studies. 

Based on available data, most of the Panel members (a few members disagreed) stated that the 

PoD presented in Option 3 is also believed to be protective for effects on the developing brain, 

although it is based on cholinergic effects. :tJowever, this conclusion has associated 

uncertainties; the lack of information on the mode of action in inducing the observed behavioral 

effects and evidence from in vivo and in vitro studies that show non-cholinergic modes of action 

are likely to be involved in the adverse developmental neurotoxicity and behavior endpoints. 

The Panel encourages EPA to explicitly address these uncertainties when deriving the reference 

dose/concentrations. 

The effects on neurobehavioral development observed in the three epidemiological studies, the 

uncertainties surrounding the mode(s) of action following longer term exposure, and the need to 

account for potential cumulative effects support the lower point of departure for repeated 

exposures used by the Agency in its 2000 risk assessment. The Panel also recommended that the 

Agency investigate the possibility of conducting a benchmark dose assessment for the chronic 

point of departure determination. 

The Panel recommended that the Agency consider development of an appropriate study using 

doses spanning and below those expected to produce cholinergic effects with the most sensitive 

administration method and the most sensitive life stage. Such a study would examine 

developmental neurotoxicity and behavior outcomes along with cholinesterase inhibition. This 

would likely help elucidate non-cholinergic modes of action for developmental neurotoxicity and 

behavior effects. I 

7. Extrapolation/Uncertainty Factors 

After review of available data, the Panel concurred with the Agency that paraoxonase-1 (PONI) 

status cannot be ruled out as a determinant of chlorpyrifos toxicity, particularly for the fetus and 

the young child. Actual human exposures vary between bystanders and applicators (and 

everyone in between) and it would be difficult to define what constitutes a low level of exposure 

in humans. The Panel disagreed on whether the PONI data alone could be used to address 

uncertainty. The majority of panel indicated that these data should not be used out of context 

until rate limiting step(s) is/are identified. The use of the PONI data without such information 

was considered by some to be a misinterpretation or misuse of the IPCS guidance for 

determining chemical-specific adjustment factors (CSAFs). · 

Some of the Panel indicated that chlorpyrifos may,produce effects in the fetus that would be 

manifest later at the juvenile stage or later oh in life (e.g. adulthood). They stated that adverse 

neurodevelopmental effects in the fetus, neonates, and young children should be considered the 

most important endpoints for assessing chlorpyrifos toxicity. The Panel noted that there does 

seem to be a different susceptibility between fetuses and neonates compared to adults. Because 

specific modes of action have yet to be identified for these effects, available data are inadequate 

to inform inter-species and intra-species differencesin TD and TK. Therefore, the application of 

default uncertainty factors for TD and TK considerations was recommended by the Panel.. 

15 
The Panel noted the uncertainties surrounding the role ofPON1 and its genetic polymorphisms 

· in humans exposed to chlorpyrifos and other cholinesterase inhibitors. Some panel members 

emphasized the .lack of data in animals and humans on the involvement ofPON1 in detoxication 

pathways, especially in situations oflow dose exposures. Most ofthe Panel encouraged the 

Agency to use PBPK modeling to assess the overall impact ofPON1 on toxicity over a range of 

chlorpyrifos exposures. While other panel members recommended that the Agency gather data 

(both in vivo and in vitro data) on animal and human enzyme kinetics and analyze carefully the 

effects as related to the PON-192 Q/R polymorphism. The information on the overall 

contribution ofPON1 Q192R polymorphism on the deactivating pathways and potentially ratelimitanting components will be very useful when examined in light of future PBPK models. 

The Agency proposed two options for deriving an intra-species TK (human variation) 

uncertainty factor: 1) a 12-fold data-derived factor based on the PON1 data or 2) the default 

three-fold factor. The majority1

ofthe Panel supported the use of the default three-fold factor. 

These panel members disagreed with the Agency's approach to use PONl genetic 

polymorphisms to derive intra-species uncertainty factor. They stressed that PONl is only one 

downstream enzyme in a complex metabolic pathway and that the PON1 genotype alone is 

insufficient to predict human variability. One panel member added that if chlorpyrifos acts 

directly on certain brain targets to elicit developmental neurotoxicity without the need of 

activation to the chlorpyrifos oxon (the substrate for PON1), then- the role ofPON1 for 

neurodevelopmental toxicity would be irrelevant. On the other hand, several panelists stated that 

the available PON1 data support an uncertainty factor of 12 for the intra-species factor to 

account for potential developmental neurotoxicity of chlorpyrifos as shown in the 

epidemiological studies. These members preferred the UF HK of 12-fold, rather than the default of 

3-fold, because these were the only two <;hoices proposed. However, none of the panel members 

endorsed the CSAF approach used by the Agency to identify the factor of"12-fold" calculated 

based on chlorpyrifos-oxonase and encouraged the Agency to pursue other approaches based on 

the mode of action (i.e., 2005 IPCS CSAF guidelines). 

The Panel did not reach consensus on one specific uncertainty factor for inter-species TK 

differences. Most ofthe panel concurred with the Agency's proposal to stay with the default 

three-fold factor for inter-species TK differences and add no additional uncertainty factor for 

developmental and behavior neurotoxicity. These panel members noted that the most sensitive 

effect appears to be AChE inhibition with a BMDL10 (i.e., lower confidence limit on the 

benchmark dose calculated to a 10 percent effect level) for the red blood cell (RBC) AChE 

inhibition after repeated exposure at 0.03 mg/kg/day; whereas, the lowest dose tested in the 

developmental neurotoxicity studies in dams (0.3 mg/kg/day) was 10-fold greater and failed to 

produce observable behavioral effects in the offspring. On the other hand, a few panel members 

· disagreed with this assessment and recommended that the Agency should apply a default 

uncertainty factor of 100 to the points of'departure based on the cholinesterase inhibition 

endpoint and further consider the use of an additional uncertainty factor to address the concerns 

for developmental and behavioral neurotoxicity as observed in both the animal and 

epidemiological studies. 

16 
The Panel generally favored the use of a PBPK model to integrate key TK and TD factors and 

evaluate their contributions to the endpoints of interest (e.g~, cholinesterase inhibition) for 

various chlorpyrifos exposure scenarios and for various life stages. In addition, the dose 

response relationships used in these models need to reflect the understanding gained from animal 

studies, what is known about inter-species and intra-species differences, and be validated against 

data from deliberate dosing studies in humans and epidemiological studies. The Panel 

encouraged the Agency to continue examining the importance of all enzymes in the metabolic 

pathway for chlorpyrifos, and acknowledged the data gaps for carboxylesterases and P450 

enzymes. 

The Panel discussed alternative approaches for calculating the uncertainty factors in the 

chlorpyrifos risk assessment. · 

DETAILED RESPONSES TO CHARGE QUESTIONS 

1. Metabolism & Toxicokinetics (Issue Paper Section 3.1, Appendix A): 

The Agency has performed a literature review of in vivo and in vitro studies on the metabolic 

profile and toxicokinetic (TK) properties of chlorpyrifos with particular focus on age-dependent 

and lifestage sensitivity. 

a. The Agency has concluded that age-dependant sensitivity, at least in part, is derived based 

on toxicokinetic (TK) differences between juveniles and adults. These TK differences lead 

to reduced ability to detoxify chlorpyrifos or the ox on in juvenile animals. Please comment 

on the Agency 's conclusion and the scientific support for or against this conclusion. 

Panel Response 

The Panel supported the Agency's conclusion. A weight of evidence evaluation of the available 

information shows that sensitivity to the adverse effects of chlorpyrifos is influenced by age, 

with young animals having lesser total ability to detoxify chlorpyrifos as well as many other 

OPs. The Panel agreed with the Agency's conclusion that the age-dependent sensitivity is mostly 

based on TK differences between juveniles and adults rather than so ley on TD differences. I 

Chlorpyrifos is activated by oxidative desulfuration to the oxon which is further detoxified by 

cytochrome P450-mediated dearylation. A second detoxication mechanism involves A-esterases 

(PONl activity towards chlorpyrifos-oxon) and B-esterases (carboxylesterases, cholinesterases) 

operating only on oxon that is available to them following the bioactivation reaction, and hence 

these are the second enzymes in the pathway. These reactions take place mainly in the liver, 

although they .can also occur to a minor extent in other tissues. 

The Panel agreed that, based on the current scientific data, differences in metabolism and 

biotransformation seem to play a more prominent role than age-dependent differences in the 

uptake, transport, sequestration, and/or excretion of chlorpyrifos. They also indicated that other 

potential TK differences between juveniles and adults should not be dismissed. For instance, it 

17 
was mentioned that the high respiratory rate of children enhances their absorption of any 

chlorpyrifos present in the air. Children have a higher skin surface-to-weight ratio that favors 

dermal absorption and a higher permeability of the small intestine epithelium that facilitates 

absorption by the oral route. The relatively greater cardiac output of juveniles, together with 

their less developed blood-brain barrier may facilitate some of the circulating chlorpyrifos-oxon 

eventually reaching the brain. Children have a greater cardiac output compared to adults, 

facilitating some of the chlorpyrifos-oxon reaching the brain. The blood-brain barrier in humans 

is not complete until 6 months of age (Rodier, 2004 ), facilitating some of the circulating 

chlorpyrifos-oxon in reaching the brains of exposed infants 

Polymorphisms in several of the enzymes involved in chlorpyrifos metabolism can lead to 

variability in an individual's ability to detoxify the chlorpyrifos-oxon. Some enzymes are also 

inducible which may also contribute to variability in response to chlorpyrifos (and its 

.metabolites). Most of the studies evaluating biotransformation capacity with acute sensitivity are 

correlative in nature, however, and are not mechanistic. While isoform expression of cytochrome 

P450 (CYP450) can be markedly different between juvenile and adult animals, the relative roles 

these isoforms play in sensitivity to chlorpyrifos are not clear. Juveniles appear to be less 

efficient than adults at activating chlorpyrifos to its oxon metabolite using CYP450. 

Nevertheless, the dearylation-to-desulfuration ratio in adults and juveniles is unknown, but may 

differ as a function of age. 

Choi et al. (2006) stated that the adult (human) liver plays an important role in detoxication of 

chlorpyrifos and that in the liver the oxon does not accumulate to be released into the 

bloodstream. Hunter et al. (1999), however, suggested that the fetal liver does not play a 

significant role in detoxication. Some· studies suggest that chlorpyrifos ox on formed in the liver 

does not escape to enter the bloodstream (Sultatos et al., 1984). Poet et al. (2003) stated that at 

low oral doses of chlorpyrifos, CYP/PON1 in the intestine and liver may effectively remove the 

oxon from circulation prior to systemic exposures. By contrast, individuals with lower levels 

and/or activity of the detoxication enzymes may have these pathways become saturated at high 

exposure levels. At high acute dosages, as is frequently the case in laboratory animal 

experiments, the detoxication systems are operating at maximal levels and the differences 

between adults and juveniles are more readily apparent. However these saturating conditions are 

less likely to occur at environmental exposure levels. If the detoxication mechanisms are not 

saturated, then their effectiveness in adults and juveniles may not be as different as experimental 

animal data with high exposures suggest. The lack of available data on maturational differences 

in tissue (non-blood) detoxication capacity by A-esterases and other pathways limits 

extrapolation of relative sensitivity in infants based on differential detoxication. 

Timchalk et al. (2006) reported that the rate of detoxication (measured as the formation of the 

chlorpyrifos specific metabolite, 3,4,6-trichloro-2-pyridinol, referred to as TCP) exceeded the 

rate of activation of chlorpyrifos (measured as chlorpyrifos oxon formation) in rats across age 

groups from 5 days to adulthood, with exposures ranging from 1-1 0 mglkg. This would suggest 

that at similar exposures, inactivation processes may outweigh the net activation. At higher 

exposures, inactivation might become compromised. At low chlorpyrifos concentrations, the 

formation of non-toxic metabolites is highly favored in the fetus (Buratti et al., 2006). 

18 
In evaluating the role oftoxicokinetics and more specifically the lower detoxication potential in 

juveniles compared to adults, the Panel proposed that the relatively inefficient bioactivation 

reaction, especially in juveniles, would not likely lead to high concentrations of the oxon at 

environmental exposure levels. The detoxication enzymes would not be at saturating substrate 

concentrations for efficient inactivation. Age differences in toxicokinetics are likely to be 

important at high dosage levels, but less likely to be relevant at lower dosages where enzymes 

are not saturated. 

The two detoxication pathways studied the most, in relation to age-related differences in 

sensitivity; are carboxylesterases and A-esterases (primarily paraoxonase-1 referred to as PON1 ). 

Specific activities ofliver carboxylesterases (CarbE) increased with age in male rats (Atterberry 

et al., 1997) which indicated the presence of more protective esterases in the adult as compared 

to the juvenile animal. The lower carboxylesterases activity in blood and tissues in juveniles 

appears to play a role in the differential sensitivity of juvenile animals to chlorpyrifos compared 

to adults (Morgan et al., 1994; Moser et al., 1998; Karanth and Pope, 2000). However, humans 

do not have carboxylesterases in the plasma (Li et al., 2005) and some data suggest relatively 

minimal differences in liver carboxylesterases between infants and adults (Pope et al., 2005). On 

the other hand, juveniles contain low serum albumin levels as compared to adults, and albumin 

has been reported to hydrolyze chlorpyrifos-oxon (Sogorb et al., 2008), and is capable of 

protecting AChE (acetyl cholinesterase) "in vitro" from inhibition at low concentrations of this 

toxic metabolite ( ~ 0.1 11M). 

Although BChE -/- (butyrylcholinesterase knockout) mice showed relatively similar toxic 

responses as BChE +/+mice following chlorpyrifos oxon exposure (Duysen et al. 2007), the 

contribution of BChE to the metabolism of chlorpyrifos-oxon in humans remains to be 

determined. This finding can be explained by the high carboxylesterase activity in mouse 

plasma. Given that carboxyl esterase is lacking in human plasma, the protective role of BChE 

cannot be disregarded. 

The Panel was not aware of data specifically demonstrating that chlorpyrifos can be bioactivated 

in the brain, the main target organ of this compound. However, there are data from "in situ" 

experiments in rats that show bioactivation of parathion (another insecticide of the 

organophosphorothioate class) in the brain in the intact organism (Chambers et al., 1989 and 

1991). Buratti et al. (2005) hypothesized that independent ofthe chemical structures, 

organophosphorothioates are bioactivated by the same CYP450s. Since chlorpyrifos is 

bioactivated less efficiently than parathion by rat liver microsomes (Ma and Chambers, 1994, 

1995), it is certainly possible that bioactivation of chlorpyrifos occurs in the brain since very low 

desulfuration activity ofvariousphosphorothioate insecticides, including chlorpyrifos, has been 

reported in both microsomal and crude mitochondrial fractions from brain (Chambers and 

Chambers, 1989). The brain desulfuration activities of these phosphorothioates generally 

correlate well with the toxicity and may be important in determining their overall acute toxicity 

levels (Chambers and Chambers, 1989). Thus, extrahepatic sites of activation likely play an 

important role in mediating the acute toxicity of chlorpyrifos (Sultatos et al., 1984). 

PON1 is differentially expressed throughout maturation with lower levels in younger animals 

being associated with higher acute sensitivity (Mortensen et al., 1996; Li et al., 1997; Karanth 

19 
and Pope, 2000). The Panel noted that there is less information suggesting that human infants are 

more sensitive than adults to chlorpyrifos toxicity because of age differences in PON1-mediated 

detoxication. In humans, blood A-esterase is very low at birth and increases for the first two 

years oflife'(Augustinsson and Barr, 1962; Ecobichon and Stephens, 1973; Burlina et al., 1977) 

or up to 4 years according to more recent data from large cohorts (Holland et al., 2006). 

Errors in the metabolism section of the Agency's background materials provided to the FIFRA 

SAP for this review were pointed out by one Panel member. 

b. There are limited data on the meta~olic capacity of pregnant animals and pregnant 

humans. Theselimited data on metabolism are supported by some toxicity data in rats. The 

Agency believes these studies suggest that pregnant animals and humans may be somewhat 

more sensitive to chlorpyrifos than non-pregnant adults to chlorpyrifos. Please comment on 

the Agency's preliminary conclusion and the scientific support for or against this 

conclusion. 

Panel Response 

The Panel agreed with the Agency's preliminary conclusion that "pregnant animals and humans 

may be somewhat more sensitive to chlorpyrifos than non-pregnant adults" based on metabolic 

capacity and their effect on levels ofkey enzymes in modulating chlorpyrifos toxicity. However, 

panel members noted that only relatively small differences in levels of the key detoxication 

enzymes involved in chlorpyrifos metabolism have been reported. Some panel members 

recommended that a direct comparison of the metabolic capacity of pregnant versus nonpregnant females be performed. Other panel members strongly disagreed with this 

recommendation. 

The Panel noted that it is not clear why pregnant females would be more susceptible to the 

adverse effects of chlorpyrifos than non-pregnant females. The information on toxicity of 

chlorpyrifos in pregnant animals is limiteq and the data on biotransformation of chlorpyrifos in 

pregnant females are inadequate. Reports from the American Association of Poison Control 

Centers (AAPCC) do not suggest increased susceptibility of pregnant women to chlorpyrifos and 

other OPs. The physiological response to pregnancy includes an increased vascular volume 

resulting in relevant hemodilution, along with higher cardiac output and increase in liver weight. 

These physiological changes may impact liver and serum enzymes involved in chlGrpyrifos 

metabolism that contribute to some extent in the variability of activity observed during 

pregnancy. Most of the gestational studies published so far report that there is greater concern for 

the fetus rather t)1an the dam. These studies are inadequate to determine whether the fetus is 

more sensitive than postnatal pups. The sensitivity to chlorpyrifos clearly decreases when dosing 

occurs with increasing age in postnatal pups. However, the increased sensitivity does not seem 

to occur when pups are repeatedly exposed to low doses. In fact, when cholinesterase activity, 

muscarinic receptor binding, and motor activity changes in response to the antimuscarinic drug 

scopolamine were compared in juvenile and adult rats treated repeatedly with chlorpyrifos; 

adults showed more extensive changes (Chakraborti et al., 1993). As noted before, this maybe 

due to more rapid synthesis of acetylcholinesterase molecules in tissues from younger animals 

20 
between exposures, thus leading to lesser accumulation of inhibition across exposures in the 

younger animals. 

The Panel discussed the biological significance of the possible changes in metabolic activity 

during pregnancy relative to expected toxicology outcomes. In humans, the main three CYP450 

isozymes that metabolize chlorpyrifos are CYP2B6, CYP2C19, and CYP3A4. The net effect of 

these isozymes on the balance of desulfuration/dearylation is unknown since CY,P2B6, CYP3A4 

and CYP 1 A2 promote the activation of chlorpyrifos to chlorpyrifos-oxon whereas CYP2C 19 

detoxifies the oxon by means of a dearylation reaction. Whereas, CYP3A4 increases during 

pregnancy, CYP2C19 decreases (Anderson, 2006) and no data on CYP2B6 in pregnant women 

are available. Pregnancy-induced decreases in levels of the CYP2B1/2 protein, which is the 

orthologous form of human CYP2B6, has been reported in rat liver but this is not accompanied 

by a statistically significant decrease in pentoxyresorufin 0-dealkylase (PROD) activity (Czekaj 

et al., 2000). 

Review of the literature indicates that there appears to be a reduction in the activities of several 

enzymes potentially important in the detoxication of chlorpyrifos associated with pregnancy; 

including PON1, carboxylesterases and blood cholinesterases. The Panel agreed with the 

Agency that the "importance of the decreases (in detoxifying enzymes) is unknown at 

environmental exposures." Although the importance of these decreases under low environmental 

exposures is not known, decreases during pregnancy may indicate a corresponding reduced 

capacity to detoxify chlorpyrifos. While the enzymes mentioned above are expected to have 

lesser (or perhaps no significant) role in modulating toxicity with low level exposures, the 

limited evidence does suggest a potential for an overall reduced capacity to detoxify chlorpyrifos 

during pregnancy. 

2. Cholinesterase Inhibition (Issue Paper Section 3.2, Appendix B): 

The Agency has reviewed numerous studies submitted for pesticide registration and from the 

literature in animals and human on the AChE-inhibiting effects of chlorpyrifos in blood and in 

the peripheral and central nervous system. 

a. Regarding inhibition of AChE, the Agency has preliminarily concluded that post-natal 

studies in rat support the conclusion that juveniles are more sensitive than adults. The Agency 

has further concluded that sensitivity is greatest in younger pups and decreases as pups mature 

towards adulthood: Please comment on these Agency's preliminary conclusions and the scientific 

support for or against these conclusions. 

Panel Response 

The Panel agreed with the Agency's conclusion that post-natal rats are more sensitive than adult 

rats with respect to increased AChE inhibition and lethality following acute exposures. A · 

number of studies have consistently shown that juvenile rats are more sensitive than adults to 

cholinesterase inhibition and cholinergic signs of toxicity following acute doses of chlorpyrifos 

exposure. In addition, several studies show a gradient of sensitivity to acute chlorpyrifos in rats 

during postnatal maturation. This greater sensitivity appears due to a lower detoxication capacity 

and as these capacities develop, the rats become more aligned with the lower adult sensitivity. 

21 
However, this. greater sensitivity may be true mainly for high dosages because of the saturation 

of the detoxication mechanisms. This difference in sensitivity (measured as acetylcholinesterase 

inhibition) between juveniles and adults is less likely to be apparent at low environmentally 

relevant dose levels because the detoxication mechanisms will not be saturated. Some on the 

Panel felt that physiologically-based pharmacokinetic/pharmacodynamic (PBPK/PD) models are 

likely to be of greatest value in predicting such age-related differences over a span of dose levels. 

The Panel, however, did not support the suggestion that post-natal rats are more sensitive than 

adult rats under repeated exposures. The timing of the measurement of cholinesterase inhibition, 

as well as, the timing between exposures can be pivotal in the degree of inhibition observed. 

Results in juveniles can be confounded by their higher capacity to synthesize of new AChE 

molecules. For example, Chakraborti et al. (1993) reported that juvenile rats are less sensitive 

than adults under subacute chlorpyrifos dosing (i.e., 40 mg/kg/day, every four days for 4 doses). 

Four days after the last exposure, adult rats showed 90-92% inhibition of brain cholinesterase 

while juvenile rats show only 54-59% inhibition. Greater inhibition of brain cholinesterase in 

adults was correlated with more extensive reductions in muscarinic receptor binding. These 

receptor changes were correlated with enhanced locomotor responses to the antimuscarinic drug 

scopolamine in adults only, out to eight weeks after the end of dosing. Thus, under these 

conditions, adults appear markedly more sensitive to cholinesterase inhibition and cholinergic 

effects of chlorpyrifos. Liu et al., (1999) report that repeated, daily chlorpyrifos exposures (5 

mg/kg/day, for 14 days) were associated with relatively similar degrees ofbrain AChE inhibition 

between juveniles and adults 1 day after either the 7th or 14th dose, but more extensive 

inhibition was noted in adults 8 days after termination of dosing. Relatively similar changes in 

muscarinic receptor binding were also noted between age groups following daily exposures. 

Together, these studies illustrate the ability of brain cholinesterase to more effectively recover 

between repeated doses in pups and the possibility of relatively more extensive cholinergic 

responses in adults following repeated chlorpyrifos dosing under the same conditions (repeated, 

"low" dose exposures). 

Overall, the Panel decided that a conclusion of greater sensitivity to cholinesterase inhibition in 

juvenile rats-compared to adult rats only holds for acute high exposure situations. When acute 

exposures occur at environmental concentrations and/or under repeated (low-level) exposures; 

however, many on the Panel believed that higher sensitivity in younger animals under 

"environmental concentrations" would be unlikely. Other members stated that this conclusion 

cannot be made without having a better idea of the meaning of"high exposure level situations" 

or "environmental concentrations." 

b. There are multiple gestational studies available which provide AChE data in dams 

and/or fetuses. These gestational studies have consistently shown that AChE inhibition observed 

in the dam is greater than in the fetus. These studies suggest that the dam serves to protect the 

fetus. However, TK gestational studies have shown that fetal tissues have similar or higher levels 

of chlorpyrifos and/or its metabolites than the dam. In addition:, multiple studies have shown that 

recovery from AChE inhibition is more rapid in juveniles compared to adults. This rapid 

recovery combined with production of the AChE enzyme as the rats mature leads to less AChE 

inhibition observed in the juveniles. The Agency has preliminarily concluded that AChE 

inhibition data from repeated dosing gestational studies showing less inhibition in fetuses may 

22 
not reflect the true potential toxicity to the fetus. Please comment on the Agency's preliminary 

conclusions and the scientific support for or against this conclusion. 

Panel Response 

The Panel agreed with the Agency's preliminary conclusion regarding observations oflower 

brain cholinesterase inhibition in pups (rats) compared to dams following gestational OP 

exposures. However, as noted in the previous question on the findings from the juvenile versus 

adult studies, the cholinesterase activity measured depends on the time of sampling relative to 

time of exposure. Whether one considers gestational or postnatal exposures, the longer the time 

from dosing to measurement of cholinesterase inhibition, the lesser the inhibition expected in 

younger animals. If the time between dosing and measurement is held constant and the same for 

dams and pups, one would expect lesser inhibition in pups compared to the dam. This is thought 

to be due to the more rapid synthesis of new proteins including cholinesterase in tissues of 

younger animals. Thus, the addition of newly synthesized (uninhibited) cholinesterase molecules 

in younger individuals contributes to more effective and rapid recovery ~f activity. 

The Panel also agreed with the Agency's preliminary conclusion that "AChE inhibition data 

from repeated dosing gestational studies showing less inhibition in fetuses may not reflect the 

true potential toxicity to the fetus." The Panel based its decision after review of those studies 

indicating the sensitivity to cholinesterase inhibition at dosages of chlorpyrifos sufficient to elicit 

AChE inhibition. One Panel member cautioned about equating AChE inhibition to toxicity, as is 

implied in the Agency's question. Inhibition is a sensitive biomarker of exposure and is a 

precursor to at least some, if not all, of the adverse acute effects of AChE organophosphates. In 

addition, there can be no cholinergic toxicity without previous AChE inhibition. Lassiter et al. 

(1998) confirmed less inhibition ofbrain cholinesterase of pups treated daily with chlorpyrifos (7 

mglkg/day) from GD14-18, when assayed from 2 to 48 hours after the last dose compared to 

peak inhibition measured at 5 hours after a single dose of chlorpyrifos (1 0 mglkg) on GD18. 

Relatively similar inhibition was noted in both dam and fetus (approximately 40% in the fetus 

and 50% in the dam) for the acute dose treatment. Hunter et al. (1999) also found that peak 

AChE inhibition was 5 hours for the dam and fetus and this paralleled the time of peak TCP 

levels in the brain. 

Lassiter et al. (1998) considered several mechanisms for differential ChE inhibition, but 

concluded that more effective recovery of enzyme activity following each daily exposure in the 

fetal tissues prevented accumulation of ChE inhibition, while slower recovery of activity in the 

maternal brain allowed increased ChE inhibition throughout the dosing period. This is essentially 

the same mechanism described earlier in postnatal pups to explain the lesser' cholinesterase 

inhibition and effects on other. cholinergic markers in pups as compared to adults that were 

treated at subactute doses with chlorpyrifos (Chakraborti et al., 1993). The recovery of inhibited 

AChE, i.e., the apparently lower level of AChE inhibition in juveniles compared to adults, is 

very likely due in large measure to greater synthesis of new AChE molecules during growth. In 

animal studies, it would be unlikely that significant "synthesis of new AChE" would occur in a 5 

hour window to a level such that it would alter AChE activity and give the impression ofless 

inhibition. In fact, AChE activity in control animals changed very little between ·2, 5, and 10 

hours following exposure as reported in Lassiter et al. (1998). Thus, AChE measurements prior 

23 
to 5 hours may be an accurate reflection of the amount of AChE inhibition, but AChE 

measurements beyond that point are subject to question .. 

The Panel agreed that the timing of the AChE assay following exposure may be less important 

when evaluating the degree of cumulative inhibition in dams following repeated dosing, but 

critical when evaluating the more rapid inhibition and recovery in the fetus or pup. A more 

accurate assessment of AChE inhibition in the growing juvenile could be determined ifthe 

timing of the sample collection were closer to the time of peak effect and if more interim 

sacrifices were used in these experiments. Some on the panel recommended more studies to 

examine AChE inhibition resulting from lower doses of chlorpyriphos in dams and developing 

neonates/pups, at more time points, as well as mdre carefully quantifying AChE protein levels as 

well as AChE inhibition. 

Regardless of whether there was recovery of AChE inhibition or increased AChE synthesis, this 

event is expected to be of little consequence at levels of exposure that are below those inhibiting 

the target enzyme. It was pointed out that the human fetus is slower developing than the rat, so 

rapid synthesis would not be present. Some Panel members felt that in the human fetus there 

would be no inhibition at all at environmentally relevant levels of exposure. But another 

member indicated that inhibition might be a concern for exposures experienced by agricultural 

workers. Many agricultural workers are women and some of those may be pregnant when 

exposed to higher levels. One Panel member stated that predicting the toxicological effects in 

humans based on the available data is too risky based solely on animal studies. 

3. Laboratory Studies on the Developing Brain (Issue Paper Section 3.3, Appendix 

C): 

The Agency has performed a literature review of in vivo and in vitro studies on the effects of 

chlorpyrifos on the developing brain. 

a. From a review oflaboratory animal studies, the Agency has preliminarily concluded 

that gestational and early postnatal exposure at sufficiently high exposures to chlorpyrifos can 

lead to neurochemical and behavioral alterations persisting into adulthood after any initial AChE 

inhibition has reversed. The Agency has put particular emphasis on the behavioral data because 

studies are available from multiple laboratories. Please comment on the Agency's preliminary 

conclusions and the scientific support for or against this conclusion. 

Panel Response 

In general, the Panel was in agreement with the preliminary conclusions made by the Agency. 

The preliminary scientific findings and conclusions are supported by the review of the in vivo 

neurobehavioral data from multiple laboratories on two mammalian species (i.e., rat and mice) of 

gestational exposure and/or early postnatal exposure to chlorpyrifos. The Panel concluded that 

independent of the exact time frame of the gestational or postnatal exposure, only doses of 

lmg/kg or greater demonstrate significant effects on behavior. However, only two studies to 

date (Maurissen et al., 2000; Jett et al., 2001)-have examined developmental effects at the lower 

dose level of0.3 mg/kg, and neither study is considered sufficient (see below). The Panel noted 

24 
that the inclusion of some more recent studies would add to the number of laboratories involved, 

as well as increase the number of neurobehavioral endpoints examined. One panel member 

provided a table of studies that supplements the information provided in Table 3 ofthe Agency's 

issue paper (see Appendix 1). 

Two areas of concern were identified and discussed. The first involved methodological issues in 

the studies reviewed. The second was conceptual, and involved the use of the phrase 

"sufficiently high exposures" in the Agency's charge question. 

The methodological issues centered on the consistency of behavioral endpoints reported as 

significant. The Panel noted that although behavioral findings were consistently found, (refer to 

Table 3, Agency Issue Paper), a somewhat inconsistent set ofbehavioral changes were reported. 

In some cases, motor activity is increased and in others, it is decreased. In learning and memory 

tests, error rate can be increasing, decreasing or not affected under similar treatment conditions. 

Increased and decreased habituation was reported with different gestational exposure paradigms. 

Gender differences are also reported in some studies. In studies comparing exposure level, lower 

dosages sometimes caused greater effects. These inconsistencies likely reflect differences in 

route of administration, developmental period during exposure to chlorpyrifos, method used to 

measure changes in the same behavioral domain, and/or choice of dependent variable. For 

example, motor activity can be measured by distance traveled in an open field and electronically 

by beam breaks. Moreover, different studies use different types of testing chambers, or number 

of crossings in an elevated plus maze. The use of several detection methods for motor activity 

may present difficulty in comparing across studies, especially with respect to younger animals. 

Concerns were also raised about the high level of variability of measurements and its effect on 

such endpoints as the mean startle response (see Maurissen et al., 2000). 

While the Agency put particular emphasis on the behavioral data, primarily because it is 

available from multiple laboratories, all related data were considered in identifying long-term 

changes in the nervous system. With respect to the Maurissen et al. (2000) study, the Panel 

raised concerns on the use of static morphometries to evaluate a dynamically changing brain 

structure. Measurement of the hippocampal dentate granule cell layer at postnatal day 12 would 

potentially capture a structural assessment of a region with poorly defined landmarks at a time 

when the neurons are undergoing an active period of migration. In the adult brain, morphometric 

measurements of the cortical regions displayed about 10% variability, a level expected to be 

within the normal variability for such crude measurements. Unbiased stereology is 

recommended by the Panel to address questions of regional volume or cell number. 

Several Panel members specifically noted methodological shortcomings in the Jett et al. (2001) 

study. Although it is only one of two studies to investigate behavioral changes after a dose of 0.3 

mglkg of chlorpyrifos, there were significant problems with respect to the training paradigm i.e., 

the Morris water inaze [MWM]. Animals were not tested to asymptote and only 60% learning 

was evident in the control group. Neurochemical data indicated that AChE inhibition was not 

observed at a dose of 7 mglkg, a finding at odds with other published studies. In addition, 

behavioral changes were noted for the 0.3 mglkg/dose only when dosing continued during 

testing. This represents a confounding factor in determining the acute versus development 

effects of chlorpyrifos. 

25 
The Panel agreed with theAgency's expressed caution about the use ofDMSO as a vehicle 

because of its intrinsic toxicity and influence on absorption. In addition, the "observed signs of 

discomfort" in pups injected with DMSO reported by Marty et al. (2000), and more recently by 

Carr and Nail (2008), raised concerns about the use of this vehicle in developmental studies. An 

additional concern was raised by the Panel concerning the influence ofDMSO on the toxicity of 

the compound under study. Ballough et al (2008) reported that DMSO, administered at levels 

below those used in many developmental chlorpyrifos studies, enhanced the toxicity of soman by 

exacerbating the neuropathology caused by soman-induced seizures. The interaction of DMSO 

and soman, an OP, raised concerns about a possible DMSO-chlorpyrifos (al~o an 

organophosphate) interaction in developmental studies. Additional concerns were raised 

regarding both DMSO and ethanol as vehicles used not only in the in vivo studies, but also in the 

in vitro studies. 

There was also discussion about the varied routes of administration (ROA) of chlorpyrifos used 

for developmental exposure. The Panel noted that each ROA used presented drawbacks and 

most if not all were stressful. It was generally agreed that the subcutaneous ROA was valid for 

gestational exposure. Moreover, despite a variety of routes used in the literature, there are , 

consistent reports of long-term behavioral changes in offspring after recovery of cholinesterase 

inhibition. , 

The Panel noted that many of the papers cited by the Agency do not link exposure periods to 

chlorpyrifos with structural changes occurring in the developing brain. While the EPA 

developmental neurotoxicity guideline studies defined a broader period of developmental 

exposure, more recent studies from the peer-reviewed literature have attempted to demonstrate 

that there are distinct developmental windows of vulnerability. While effects of chlorpyrifos can 

be detected in the older animal a direct linkage between the developmental window with regard 

to the stage ofbrain development and endpoints examined was ill-defined. It was suggested that 

a more specific examination of the critical events occurring at the time of exposure and the 

specific targeted endpoints of interest would be a valuable contribution to the literature. Such 

data would be particularly important to distinguish cholinergic versus non-cholinergic modes of 

action for specific adverse effects. Clancy et al. (2007) and the website, 

http://www.translatingtime.net, were suggested as potential resources for the design of such 

studies with respect to timing of brain development. 

The Panel indicated that the phrase "at sufficiently high exposures" is open to interpretation. 

Overall, it was interpreted by the Panel in this meeting to refer to studies in which the exposure 

level was within a dose range expected to inhibit brain AChE. The majority of studies reviewed 

by the Agency focused on an exposure near 1 mg/kg/day, a level generally sufficient to inhibit 

brain acetylcholinesterase in most treatment/age groups studied, and which produce behavioral' 

alterations. However, measurements were often obtained at a single time point post-dosing and 

at a time when the period of peak inhibition may be missed. The availability of dose-response 

data was limited and in some studies the calculated levels of inhibition were not consistent with 

the predicted levels of inhibition for high dose exposure. Studies examining critical 

developmental periods for exposure were primarily focused on dose levels expected to produce 

AChE inhibition. Given the nature of the gestational exposure studies, the use of fetuses for 

26 
neurochemical analysis is limited. Given the critical role ofthe cholinergic system for brain 

development, the lack of sufficient data to support the absence or presence of enzyme inhibition 

is a significant problem in the interpretation of the results from many of these developmental 

studies. The inherent problems in assessing brain AChE inhibition during development and the 

type of measurements needed for the assessment were noted in the Agency's document. The 

Panel noted that it is possible that the morphogenic role of AChE may alter aspects ofbrain 

development and neurogenesis, even in the absence of detectable levels of inhibition. 

The critical point stressed was that dosages considered "sufficiently high" to elicit . 

neurobehavioral disruption into adulthood would also be anticipated to affect the target enzyme 

for the common mechanism of toxicity for chlorpyrifos and other organophosphate insecticides. 

Whether any suchchanges were initiated by early inhibition of acetylcholinesterase remain 

unclear. 

Errors in the Agency's Issue Paper described below were noted . 

. 1) Section 3.3 .1, page 44, paragraph 3. The behavioral effects in the Maurissen et al. (2000) 

study are only limited to an increased latency to peak response in the auditory startle 

response that was independent of the dose of chlorpyrifos. The Agency interpreted the 

data to mean that there was both a decreased amplitude and increased latency at PND 22. 

2) Section 4, page 16, paragraph 4.There is no dose-response for effects ofketanserin on 

reference memory since increased errors were observed in the low and high dosages but 

not the medium dosage. 

b. Consideration of the mode of toxic action is an important component of risk 

assessment. The International Programme of Chemical Safety (IPCS) and International Life 

Sciences Institute Risk Science Institute (ILSI RSI) have developed a Mode of Action 

(MOA)/Human Relevance Framework which provides structure and transparency to MOA 

analyses (Meek et al., 2003; Seed et al., 2005 and Boobis et al., 2006). IPCS have combined and 

extended these components to produce a unified Human Cancer Relevance Framework (IPCS 

HRF). In this approach, involvement of a series of key events in the MOA is established on 

weight-of-evidence, using criteria based on those described by Bradford Hill, taking account of 

factors such as dose-response and temporal concordance, biological plausibility, coherence and 

consistency. Other MOAs that logically present themselves also should be considered. Once an 

MOA is established, qualitative and quantitative comparison of each key event between the 

experimental animal and humans enables a conclusion as to likely relevance of the MOA for 

human risk. In the case of chlorpyrifos, the Agency has considered the available mechanistic data 

but has not evaluated these data in the context ofMOA/human relevance framework. It has been 

initially determined that there are insufficient data to develop a series of supportable key events 

(as in a mode of action analysis 1

) for neurodevelopmental toxicities other than AChE inhibition. 

The Agency notes a particular lack of data on dose response and temporal concordance that are 

critical in a MOA framework analysis. There may be other mechanisms which lead to effects on 

the developing brain but a supportable mode of action(s) can not be elucidated at this time. 

Please comment on the Agency's preliminary conclusions and whether there is sufficient 

1 For information on the Mode of Action Framework, see U.S. EPA ,1999, 2005; Sonich-Mullin eta!., 2001; Meek 

eta!., 2003 ; Seed et a12005 and Boobis, eta!, 2006 

27 
scientific information to merit a full mode of action framework analysis. If a mode of action 

framework analysis is pursued, what would be the biologically plausible hypotheses to evaluate? 

Panel Response 

There was a consensus of the Panel that available data were inadequate to support a weight of 

evidence evaluation for non-cholinergic mode(s) of action for the behavioral alterations 

following gestational and eady postnatal exposure to chlorpyrifos that persisted into adulthood. 

The Panel agreed that the available information does not allow for behavioral endpoints to be 

considered as a point of departure and recommended, based upon currently available data, that 

cholinesterase inhibition be used as the PoD. 

Pointed out at the beginning of the discussion of this charge question was the fact that a mode of 

action/human framework analysis requires consideration of testable hypotheses with identified 

key events in a causal pathway. It then requires comparison of systematic analyses of traditional 

criteria for weight of evidence for each of these hypotheses including consistency, dose-response 

. and temporality among early key and end events; biological plausibility and coherence; all of 

which requires expert multidisciplinary input. The framework, therefore, focuses attention early 

on key events that can be compared between species and how they are quantified, ultimately 

critical points in driving the dose-response relationship. While the structured analysis contributes 

to increased transparency in weight of evidence in risk assessment, it is also helpful in framing 

research relevant to risk assessment and permits iterative dialogue between the research and risk 

assessment communities, as a basis to generate more appropriate data. 

The data presented in Appendix C of the Agency's issue paper summarizes the studies on 

persistent behavioral changes in adults following gestational and early postnatal exposure as well 

as selected {n vivo and in vitro studies on the interaction of chlorpyrifos with several 

neurochemical parameters. The Agency noted in its issue paper that during development of the 

brain, acetylcholine plays a role in morphogenesis, and AChE may alter aspects of neurogenesis 

in the absence of a detectable level of inhibition using current methods. The Panel indicated that 

there is a large amount of variance presented in many of the studies, a lack of dose response for 

many of the proposed targets, and a lack of reports of altered targets from more than one lab. 

The Panel concluded that these studies were not inclusive, coordinated, nor conducted in the 

context of hypothesis testing for mode of action. Rather they were conducted principally for 

generating testable hypotheses. Without clearly defined and testable hypotheses we are left with 

uncertainty about whether the long term behavioral changes occur downstream from AChE 

inhibition or independently of it. 

Specific points were raised regarding the question of mode of action. In Figure 5 of the issue 

paper, the Agency cites Slotkin et al. (2006) to suggest possible mechaiJisms for how 

chlorpyrifos may elicit neurodevelopmental effects. The Panel did not find enough information 

from the Slotkin et al. (2006) paper to determine if chlorpyrifos directly interacts with the 

molecular components ofthe proposed pathways that could then lead, ina causal manner, to 

subsequent neurobehavioral changes. Panel members determined that it was generally unclear in 

what way the animal studies exhibit "qualitative similarities" to findings reported in children. 

The aninial studies do report neurobehavioral changes in rats and mice following prenatal or 

28 
postnatal chlorpyrifos exposures at levels that would be expected to inhibit cholinesterase. These 

"sufficiently high" exposures may lead to persistent neurobehavioral effects under some 

conditions, but "qualitative" similarities to neurodevelopmental findings reported in children are 

difficult to equate. 

There are several facets of data evaluation that may need to be examined with respect to effects 

on serotonin, G-proteins, macromolecules, neurotrophic factors etc. by developmental exposure 

to chlorpyrifos. For the most part, the effects seen on these systems following chlorpyrifos 

exposure are modest, and many represent less than a 25% change from control values/levels. 

These changes were often sex specific and dependent upon the time of exposure and 

measurement. Although there is always the possibility that such modest changes in 

neurochemistry and biochemistry will have a significant impact on behavior, replication and 

validation across laboratories are required. Some Panel members agreed with the Agency's 

conclusion that, given the effects on learning and memory, characterization of glutamate Nmethyl-D-aspartic acid (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic 

acid (AMP A) receptors would be important endpoints in assessing the developmental 

neurotoxicity of chlorpyrifos. 

The use ofmorphometric measurements ofbrain regions was questioned with regard to age, i.e., 

regional measurements were conducted during an active period of brain development resulting in 

the lackof discrete structures due to neuronal migration. The diffuse structural features at early 

postnatal ages, e.g., PND12, would confound the ability to obtain an accurate measurement. In 

addition, in the mature animal a change within the range of 1 0% for such a crude measurement 

would be expected to be within the normal variability of control tissue. One panel member 

indi.cated a preference for the use of unbiased stereology in obtaining structural measurements of 

discrete brain regions. However, this approach would also be hindered during early postnatal 

development. Unbiased stereo logy is a well established method for determining cell number and 

tissue volume and has been used in developmental, aging, and pharmacological studies. An 

additional concern was raised in that changes often occur in the network organization of the 

brain rather than gross cell death. A number of different neural and vascular cell types can be 

influenced by acetylcholine during development and such effects may not involve cell death but 

rather a structural or functional alteration. While standard histological assessement of 

hematoxylin and eosin stained sections accurately detect cell death and general organizational 

features of the brain, neither it nor morphometric measurements would detect less than gross cell 

loss occurring prior to such assessment. Nor would such a st~dard approach detect changes in 

the non-neuronal cells within the brain, such as the various glial populations. Additionally, 

developmental neurotoxicity can be associated with changes in the synaptic organization of the 

various brain regions and the normal temporal pattern of apoptosis and synaptic pruning, 

differentiation of radial glia, neuronal migration, synapse stripping; maturation of the resfdent 

brain monocytes, the microglia, and the brain vascular system. Thus, to determine low-dose 

effects for any chlorpyriphos will require more sophisticated methods of analysis. 

One Panel member took issue with the material in Section 9: Problems with studies from the 

literature, and in particular with Paragraph 5, Page 28 (see Agency's issue paper) which states 

"An aspect of some ofthe in vivo studies discussed above is that no AChE inhibition was 

detected during the windows of exposure at the dose level used. This is especially true for · 

29 
gestational ~xposures. This suggests that some other target was affected by chlorpyrifos or its 

oxon." As stated repeatedly in Appendix B, AChE activity increases rapidly in gestational and 

neonatal animals and, in many studies, especially the gestational studies, the time of assay does 

not coincide with the time of peak inhibition. Thus, unless a time course of AChE inhibition is 

performed and clearly demonstrates that there is no AChE inhibition, the claim ofno inhibition 

or low levels of inhibition may not be accurate. 

Some panel members suggested that the recent review of chlorpyriphos toxicity by Eaton et al. 

(2008) provided a basis for considering possible non-cholinergic modes of action. The serine 

hydrolase enzyme KIAA1363 is important in an ether lipid signaling network involving platelet 

activating factor, and is highly expressed in cancer cells. In vitro studies demonstrated that this 

enzyme was inhibited by chlorpyrifos-oxon (IC502 

= 8 nM) within the range of AChE inhibition. 

in vivo, a 98% inhibition of AChE and lethality due to chlorpyrifos-oxon administration resulted 

in a39% inhibition of brain KIAA1363 (Nomura et al., 2006). The relative changes have not 

been reported for lower dose levels thus, a direct causative role in the toxicity of chlorpyrifos has 

not been identified. So while KIAA1363 may serve as an important detoxication mechanism, as 

is supported by the increased toxicity in knockout mice (Nomura et al., 2006), it does not 

necessarily indicate that this enzyme is a target for "mode of action." 

At dose levels significantly lower than any previously reported effects, chlorpyrifos induced the 

phosphorylation of cAMP response element binding (CREB). This transcription factor plays a 

role in synaptic plasticity and in cell survival and differentiation. In primary cortical or 

hippocampal neurons, pCREB was elevated following dosing with chlorpyrifos, 0.06 nM and 1-

1 OnM, respectively (Schuh et al., 2002). No effect was observed in primary cortical astrocytes. 

Schuh et al. (2002) speculated that, rather than indicative of neurotoxicity, the elevation in 

pCREB represents a neuroprotective response to metabolic stress in neurons. Some Panel 

members felt that the studies cited in Eaton et al. (2008) (specifically Table 14 and related text) 

could be useful to the Agency in evaluating potential alternative modes of action. 

Collaboration between EPA and developmental neurotoxicologists may help define research 

needs for identifying alternative neurotoxic processes involved in the developmental 

neurotoxicity of chlorpyrifos. At a minimum, such studies would require testing multiple doses 

of chlorpyrifos between 0.05 and 1.0 mg/kg to set the high dose for expected AChE inhibition. 

Such data can then be used to determine level of cholinesterase inhibition and related changes in 

brain development. This may require additional methods development for detection of targeted 

brain regions or cellular localization. The data set can then be used to identify cholinesterase 

inhibition or an alternative mode of action for developmental neurotoxicity of chlorpyrifos. Low 

dose level effects reported for in vivo developmental effects require replication as well as the 

extremely low-dose in vitro activation of CREB. Inclusion of data on multiple carboxyl esterase 

activities and in localized brain regions are major requirements for future studies. 

2 IC50 (Isolated Cortical) refers to the concentration of chlorpyrifos in cell culture that produced the observed effect 

(in this case an increase in CREB) in 50%·ofthe neurons. 

30 
4. Epidemiology Studies in Children and Mothers (Issue Paper Section 3.4, 

Appendix D): 

The Agency has evaluated epidemiology studies from three major cohorts: the study sites are: (1) 

Columbia University, NYC, (2) Mt Sinai, School of Medicine, NYC, both with multi-ethnic 

urban low income women and infants, and (3) University of California at Berkeley (Center for 

Health Assessment ofMothers and Children of Salinas, CHAMACOS) with women and their 

children from farm worker populations. 

a. The Agency believes that all three studies provide valuable information on the effects 

in children of high exposures to pesticides, particularly OPs. For purposes of e~aluating human 

health effects of chlorpyrifos, the Columbia University studies provide more robust information 

for evaluating the human healtli effects of chlorpyrifos because it measured chlorpyrifos rather 

than a metabolite in both environmental (air) and biologic media (maternal and cord blood) and 

showed that chlorpyrifos was significantly associated With birth outcomes (low birth weight and 

length) and neurodevelopmental outcomes that were no longer present when the residential uses 

were cancelled (i.e., conducted a pre- and post-residential cancellation analysis). Although the 

results reported by the Mount Sinai group are informative with regard to evaluating the relevance 

ofPONl status in health outcomes, this study is limited because the neurodevelopmental 

outcomes were linked to non-specific OP maternal urinary metabolites (DAP, DEP and DMP), 

rather than the chlorpyrifos-specific metabolite TCP. The exposure of the CHAMA COS to many 

OPs reduces its usefulness in the chlorpyrifos risk assessment because the outcomes can not be 

specifically linked to chlorpyrifos exposure. Please comment on the Agency's preliminary 

conclusions on each of the three cohorts regarding the degree to which the data informs the 

chlorpyrifos human health risk assessment. Please also comment on the scientific support for or 

against these preliminary conclusions. 

Panel Response 

Overall, the Panel agreed with the Agency's conclusion that although each of the three studies 

(Columbia, Mt. Sinai, and Berkeley) provide valuable information, the Columbia study is the 

most robust and appropriate for informing risk assessment with respect to the exposure 

(chlorpyrifos), outcomes (birth and neurodevelopmental), and population (neonates and children) 

being addressed by the Agency's evaluation. To be precise, the Columbia study was not 

necessarily a more robust epidemiological study, but it directly measured chlorpyrifos exposure 

as opposed to the Mt. Sinai and Berkeley studies where metabolites not specific to chlorpyrifos 

exposure were measured and reported, including those of other OPs and carbamates. The use of 

the term "high exposure" in the phrasing of the question was questioned because although the 

exposures in these studies may be high for residential settings, they are not high when compared 

to agricultural worker and pesticide handler populations. 

The Panel noted that the conclusions of the Agency are supported by the following information: 

• All three studies are prospective cohorts with relatively low loss to follow-up and are 

conducted by respected researchers using widely accepted protocols with results 

published in peer-reviewed journals. 

31 
• The Columbia study has the strength of having used blood measures (both maternal and 

cord) of chlorpyrifos specifically as opposed to the non-specific urinary metabolites (e.g., 

diethylphosphate (DEP) and dimethylphosphate (DMP)) utilized in the other two cohorts. 

There is also high correlation (r=0.76; p<O.OOOl) between maternal and cord blood 

chlorpyrifos levels noted in the Whyatt et al. (2003) study. In addition, the Columbia 

study has exposure measurements from air monitoring which correlate with both 

maternal and cord blood (Whyatt et al., 2003). 

• A particularly convincing strength ofthe Columbia cohort is that it spans the residential 

cancellation date allowing the researchers to evaluate changes in birth and 

neurodevelopmental outcomes pre and post chlorpyrifos residential cancellation. It was 

noted,that although the pre and post analysis validates exposure measurements, the 

overall results are more interesting epidemiologically because it looks at the entire 

spectrum of exposure. 

• The initial reference group for the birth outcomes (those whose chlorpyrifos levels in 

cord blood were below the level of detection (LOD) with similar demographic and 

socioeconomic characteristics) in the Columbia study was also judged to be appropriate. 

• The methodology for exposure measurement was considered good by the Panel and the 

quality assurance and quality control procedures used by the Centers for Disease Control 

(CDC) are extensive. The Columbia study included both environmental and biological 

measurements of chlorpyrifos and biological measurements were made in both maternal 

and cord blood. It was noted that there are concerns about values near or below the 

detection limits; however, the precision of the data are acceptable while accuracy is 

reduced. In other words, the Panel felt confident that the chemical was present when 

detected in a sample although the reported value may be a poor measure of the actual 

concentration. The difference between the true and measured amount could be 

appreciable. 

• Raub and Whyatt presentations to the Agency in 2007 and 2008 (presentations found in 

the docket, EPA-HQ-OPP-2008-0274) showed intelligence quotient (IQ) results at five 

years of age and indicated intent to extend the follow-up for this cohort until age 7. This 

work provides the longest follow-up to date for a population of children who were 

exposed to chlorpyrifos in utero. · 

The Panel concluded that the Columbia study is epidemiologically sound and that there is 

minimal selection and information bias. Additionally, any information bias would likely be nondifferential sinQe any misclassification of outcome is unlikely to depend on exposure status and 

misclassification of exposure is unlikely to depend on outcome status. However, even the bestdesigned epidemiology studies are susceptible to weaknesses inherent to observational studies, 

and a few of these possible weaknesses are addressed below: 

• The possibility of residual confounding in the analysis of the Columbia study data on 

birth outcomes (Whyatt et al., 2004) was discussed. In particular, variables such as 

32 
presence of prenatal care, sexually transmitted diseases, history of substance abuse and of 

cigarette smoking, low pre-pregnancy body mass index (BMI) and inadequate weight 

gain are potential confounders. Review of the model co variates found that a complete list 

of standard variables which are commonly available were controlled for in the analysis 

including passive smoking, ethnicity, parity, maternal pre-pregnancy weight, weight gain, 

newborn gender, and season of delivery. In addition, the Columbia cohort was restricted 

to exclude women who smoked during pregnancy, used illicit drugs, had human 

immunodeficiency virus (HIV), or had their first prenatal visit after the 20th week of 

pregnancy (Whyatt et al., 2004). The Panel agreed that these factors limit the possibility 

of residual confounding. In addition, the authors of the Columbia study further addressed 

the potential for residual confounding by including a variety of other variables in the 

analysis. The results as presented in the Rauh et al. 2008 paper are stable and not 

sensitive enough to eliminate the various confounders. Although there may be other 

variables not included in the analyses that are associated with either exposure or outcome, 

in: order for a variable to be a confounder it must be associated with both exposure and 

outcome. 

• The Panel also noted that the infants in the Whyatt study were of normal weight. The 

Panel questioned the significance of lower birth weights and lengths in infants more 

highly exposed to chlorpyrifos. Low birth weight is defined as a weight of2500 grams (5 

pounds 8 ounces) or less. Low birth weight infants are known to be at increased risk for 

serious health problems as newborns, lasting disabilities and death (March of Dimes; 

http://www.marchofdimes.com/professionals/14332 1153.asp. Accessed September 21, 

2008). Although infants most affected by chlorpyrifos exposure in the Whyatt study fell 

within normal parameters for birth weight, there could still be cause for concern. Infants 

who are born smaller than expected by their genetic potential would have increased risk 

of perinatal mortality, neurological morbidity and morbidity in general as compared to 

infants born at their predicted birth weight (Figueras et al., 2007). Reductions in birth 

weight are also associated with marked increase in chronic diseases in adulthood 

including diabetes, cardiovascular disease, stroke, and hypertension (Barker, 2004). One 

issue of concern is whether chlorpyrifos' effect on reducing birth weight would result in 

more infants falling into the category of low birth weight. 

• Concerns raised by public commentators before the Panel about neurobehavioral testing, 

including the specific tests used, the method of administration, and the training of the 

individuals administering the test were discussed. The Panel concluded that the 

Columbia study authors used neurobehavioral tests that are vyidely accepted in the 

scientific community and used experienced and well-trained examiners to administer the 

tests. The Panel also concluded that the results obtained were probably as good as could 

be obtained given all of the limitations associated with neurobehavioral testing. 

• Concerns with the three year period between the sampling of cord blood and the 

neurobehavioral findings in Rauh et al. (2006) were discussed. The exposure 

classification is based on a one-time measurement that provides a snapshot of exposure 

and should not be taken as an absolute representation of total chlorpyrifos exposure 

throughout pregnancy. Many researchers have argued that chlorpyrifos in blood and 

33 
adipose tissue are in steady state (see Appendix I, p. 25, Eaton et al., 2008) so that cord 

blood provides a reasonable dosimeter for the amount transferred to the fetus. This 

assertion does not take into account the changes produced in lipid metabolism during 

pregnancy. In spite of maternal fat stores accumulated in early and mid pregnancy, in 

late pregnancy human chorionic somatomammotropin (HCS) promotes lipolysis and fat 

mobilization (Butte, 2000) to meet the.increased fetal demand at that time; therefore, fat 

reservoirs are gradually emptying of the accumulated chemicals. Accordingly, the single 

measurement of cord blood chlorpyrifos may not be representative of the total exposure 

during pregnancy, but only reflects exposure happened in the few days before delivery. 

The Panel noted that exposure events, whether chemical or environmental, that could 

have occurred in these households over the three year time period are not known. These 

unmeasured exposure events could influence the neurobehavioral test results, especially 

given the fact that neurodevelopmental deficits may be multifactorial in origin. It would 

be important to ascertain whether there are statistically significant differences in 

neurodevelopmental outcomes at 3 years of age between children with chlorpyrifos levels 

in cord blood below the LOD and those with a chlorpyrifos level of 6.17 pg/g. Rauh et 

al. (2006) stated that the Bayley Scales of Infant Development scores (Mental 

Development Index (MDI) and Psychomoter Development Index (PDI)) were similar in 

both groups of children. If so, chlorpyrifos levels in cord blood at the time of delivery 

would not be expected to be associated with neurodevelopmental outcomes. 

• It is difficult to compare exposure levels in the Columbia study with the US population 

(as measured in the National Health and Nutrition Examination Survey (NHANES) or 

with other biomonitoring studies since urinary metabolite levels were not measured. 

• Although the data on post-ban declines in exposure are compelling, limitations must be 

kept in mind when using these results in the weight of evidence. The study' was not 

designed to asses~ the effect of the ban, so data are essentially cross-sectional (i.e. 

exposures among the same women were not measured over time). Additionally, the data 

presented for exposure levels by year have been crude in nature. It is not likely to make a 

large difference, but an analysis should be conducted looking at exposure level as the 

dependent variable and year (an ordinal variable) as the primary independent variable, 

while also adjusting for any factors that may be associated with both exposure levels and 

year of study. 

• There was discussion about the cut-off values and exposure groupings used in the 

Columbia studies (Rauh et al., 2006; Whyatt et al., 2004). There was also discussion 

about the numbers in the various exposure groups in 2004 compared to 2006. Concern 

was expressed that loss to follow-up may have occurred in more severely affected 

children or less severely affected children as opposed to a non-differential loss. Some 

felt that more attention should be given to .the sensitivity of the results to the selected 

"highly exposed" group. For example, the authors said these groups were first based on 

tertiles, but according to Rauh et al. (2006) the groups were unequal in size. The authors 

did clarify via personal communication on September 18, 2008 (found in the docket, 

EPA-HQ-OPP-2008-0274) that these groups were unequal in the 2006 study because 

only children who reached three years of age were analyzed, suggesting that the origi~al 

exposure groupings were not balanced. Some panel members noted that the Agency 

34 
should explore this· further as the loss of children in the high exposure group could not be 

due to length of follow up as all the children in the high exposure group (bar one) had to 

have been born prior to 2001 (and hence should have been at least three years old) as 

after the ban there was only one child in the high exposure group. The Panel felt that it 

would be useful if the authors could provide additional details on the loss to follow-up in 

each of the four exposure groups and provide additional details on how the exposure 

groups were combined. However, it was also noted that the inclusion of both the children 

below LOD and with low exposures values into the reference group would be expected to 

attenuate any association, if indeed there is an effect at such low exposures. However, in 

Rauh et al. (2006), there is no suggestion of such an effect as the most highly exposed 

group and the group with values below LOD had lower mean MDI and PDI scores than 

did the two middle levels. There was disagreement on the Panel about the mode of 

action; therefore, there is no compelling reason to believe that the epidemiologic data 

should reflect either a linear association or a threshold effect. 

• The post-ban declines in effect are based on only one subject being classified as highly 

exposed in post-ban years. This analysis lacks statistical power and may be sensitive to 

the cutoff value chosen to classify subjects as highly exposed. It would be interesting to 

see what the relationships looked like among more equally sized exposure groups that 

separate between the lower exposure levels. 

• The Panel felt that it might be helpful to have a longitudinal component to the Columbia 

study to determine if the adverse effects persisted in those children who exhibited poor 

birth outcomes or neurobehavioral deficits. 

• It would be useful to examine the results of a statistical analysis that includes all three 

AChE-inhibiting insecticides in the analysis model as dichotomous variables (above or 

below LOD) in combination with continuous measurements for these variables. This 

type of analysis would likely not change the results, but it could be helpful in illustrating 

threshold or dose response effects. 

Despite the questions and discussion noted above, it was affirmed that the Columbia study was 

indeed quite strong and provided extremely valuable information. Most of the points addressed 

above are points of clarification or points that if addressed could strengthen the analysis and 

provide the Agency with better data to use in the risk assessment process. 

Given the paucity of human epidemiological studies of organophosphates in children, the other 

two cohorts (Mt. Sinai and Berkeley) should not be completely discounted. Both studies provide 

information on abnormal reflexes in neonates, and this data is not available in the Columbia 

cohort. Additionally, the Mt. Sinai cohort considers PONl as a factor in the analyses, something 

that may·have some relevance to risk assessment (Engel et al., 2007). More information may be 

gleaned from the Berkeley cohort (Eskenazi et al., 2004). A close comparison of OP metabolite 

concentrations in this study with those reported in NHANES may be useful. It is interesting to 

note that median 3,5,6-trichloro-2-pyridinol (TCP, a specific metabolite of chlorpyrifos) levels in 

this.cohort were similar to those reported in NHANES; whereas, DEP (diethylphosphate, one of 

the non-specific urinary metabolites) levels were much higher. This suggests chlorpyrifos 

35 
exposures in the study are similar to "background level" and that exposures to other OPs (besides 

chlorpyrifos) that are metabolized to DEP are proportionally much higher in this cohort (relative 

to chlorpyrifos) than the general population. Other OPs may very well be driving the 

associations between DEP/DMP (non-specific organophosphate urinary metabolites) levels and 

adverse outcomes. However, because chlorpyrifos is likely to contribute very little to the overall 

DAP levels in the Berkeley cohort TCP and DAP levels may be poorly correlated. Thus, in the 

TCP analysis, it may (with some caution exercised) be possible to additionally adjust for DAPs 

in an attempt to assess more chlorpyrifos (and/or chlorpyrifos-methyl) specific associations. The 

presence of these other OP pesticides may overshadow any TCP associations, but may still be 

worthwhile to investigate. 

The Panel acknowledged that there are potential confounders and issues that reduce the utility of 

both the Mt. Sinai and Berkeley cohorts for risk assessment. For example, both studies measure 

organophosphate metabolites in urine but chlorpyrifos is not specifically measured. The 

Berkeley study, in particular, has the least relevance to chlorpyrifos risk assessment because only 

a small percentage (10%) of the pesticides applied in the Salinas Valley are chlorpyrifos; 

therefore, the Panel assumed that chlorpyrifos would make only a small contribution to the nonspecific metabolites measured in the study, and as a result might be expected to have small 

impact on the study outcomes, although this assumption has not been verified. As such, it is 

difficult to ascribe the effects seen to chlorpyrifos, in particular, rather than OPs in general. 

A recently published study by Samarawickrema et al. (2008) entitled Fetal effects of 

environmental exposure of pregnant women to organophosphorus compounds in a rural farming 

community in Sri Lanka was brought to the attention of the Panel and suggested for use in 

clarifying the issue of whether maternal and cord blood are differentially distributed to the fetus. 

It was suggested that the results from this study could be compared to the results observed in 

Raub et al. (2004) and Mattsson et al. (2000). Not having seen the Samarawickrema et al. study, 

the Panel could only recommend that the Agency consider this study when this issue is revisited. 

The Panel also encouraged the Agency to identify and review any other published studies on this 

topic, even if of lesser size and scope, since they may also contribute to the total weight of 

evidence analysis of the potential neurobehavioral effects of chlorpyrifos. 

b. Data from Whyatt et al. {2003) show that 100% of air samples detected three AChE 

inhibiting pesticides ( chlorpyrifos, diazinon and propoxur). Similarly, all three pesticides were 

found in 48-49% of umbilical cord samples at lower levels than chlorpyrifos. The investigators 

reported that chlorpyrifos was significantly associated with decreased birth weight and length, 

even after statistically controlling for these two OPs; a similar analysis has not been conducted 

for the neurodevelopmental outcomes. The Agency can not rule out that exposures to all three 

AChE-inhibiting pesticides in combination resulted in the neurodevelopmental health outcomes 

reported in the studies. However, this possibility does not rule out the potential role of 

chlorpyrifos in contributing to the reported health outcomes, particularly given the reported 

findings pre- and post-voluntary cancellation. In balance, given, that 1) measured levels of 

chlorpyrifos have been statistically associated with multiple birth and neurodevelopmental 

outcomes; 2) these associations are correlated in time prior to the cancellation of indoor uses of 

chlorpyrifos when exposures were much greater (and thereby show some degree of doseresponse); and 3) there are animal data which support neurobehavioral effects resulting from 

36 
gestational exposure, the Agency has preliminarily concluded that chlorpyrifos likely played a 

role in these outcomes. Please comment on the Agency's preliminary conclusion and the 

scientific support for or against this conclusion. 

Panel Response 

Overall, the Panel agreed with the Agency's conclusion that chlorpyrifos likely played a role in 

the birth and neurodevelopmental outcomes noted in the three cohort studies. The Panel agreed 

with the Agency on the following points: a) exposures to all three AChE-inhibiting pesticides in 

combination cannot be ruled out as contributing to the birth and neurodevelopmental outcomes, 

b) the potential of combination and/or additive effects of these three compounds does not rule 

out the role of chlorpyrifos in contributing to the outcomes, and c) it cannot be stated that 

chlorpyrifos is the sole contributor to the observed outcomes. These conclusions are supported 

by the effects observed at various age groups across the three cohorts as summarized in Table 1. 

ll Strength and significance of association as reflected by the following results from the 

Columbia cohort: 

a. Extremely large odds ratios (OR) for attention disorders (OR~11.26; 95% CI: 1.79-

70.99), attention deficit hyperactivity disorder (ADHD OR=6.50; 95% CI: 1.09-38.69), 

and pervasive developmental disorder (PDD OR=5.39; 95% CI: 1.21-24.11) were seen 

when comparing high to low chlorpyrifos exposure groups (Raub et al., 2006). Although 

limited sample sizes resulted in fairly large confidence intervals, the magnitude ofthese 

results is so large that they are unlikely to be affected by residual confounding. 

b. _There were statistically significant deficits in birth weight of 150 grams when comparing 

high exposure to exposure that was below the level of detection (LOD); and, decreases of 

43 grams in birth weight per log unit increase in chlorpyrifos in cord blood. Statistically 

significant deficits in birth length were also noted (Whyatt et al., 2004). 

c. There were statistically significant deficits of6.5 points on PDI at 3 years of age when 

comparing high to low exposure groups (Raub et al., 2006). 

d. There were 2.4 times increased odds (95% CI: 1.1-5.2) of mental delay and 4.5 times 

increased odds (95% Cl: 1.6-12.7) of psychomotor delay when comparing high to low 

exposure groups at age 3 (Raub et al, 2006). 

e. There were deficits of 5.6 points on verbal IQ and 5.1 points on full IQ at age 5 (Whyatt, 

EPA presentation, 2007) . 

.ll Consistency of association between the three cohorts is that they all found some 

developmental effect in a population exposed to elevated levels of OPs. The Berkeley and 

Mt. Sinai cohort studies measured non-specific urinary DAP metabolites (e.g., 

diethylphosphates (DEPs), dimethylphosphates (DMPs) produced by many OP pesticides 

including chlorpyrifos. However, urinary levels of 3,5,6-trichloro-2-pyridinol (TCP) 

were not measured in these two studies, but only in the Columbia cohort study. 

Therefore, the conclusions below are based on the non-specific DAP metabolites rather 

37 
than the more specific indicator, TCP. The Panel noted, however, that urinary TCP does 

not absolutely correlate with exposure to chlorpyrifos, but could be an indicator of 

exposure to other pesticides including methyl chlorpyrifos and the herbicide trichlopyr 

(3,5,6-trichloro-2-pyridinyloxyacetic acid, Remedy®) or even to TCP itself (as an 

environmental degradate of chlorpyrifos, methyl chlorpyrifos or trichlorpyr). However, 

developmental effects were consistently observed in children whose mothers exhibited 

biomarkers of exposure to OPs in each of these studies. 

a. There were increased abnormal reflexes in neonates associated with maternal urinary 

DAP measurements in both the Berkeley and Mt. Sinai cohorts (Young et al., 2005; 

Engel et al., 2007). 

b. There were increases in MDI associated with increases in DEP metabolites at 1 year 

(Eskenazi et al. 2007). Although Eskenazi and colleagues found decreases in MDI 

associated with prenatal DEP metabolites at 2 years, they were not statistically significant 

(see Table 4, Eskenazi et al., 2007). · 

c. Results from cohort studies conducted by Eskenazi. et al. (2007) indicated total urinary 

prenatal DAP metabolite measurements were associated with increased odds ratios of 

pervasive developmental disorder (PDD) at 2 and 3.5 years (Eskenazi et al., 2007) and 

child urinary DAP metabolites were associated with increased odd ratios of PDD at 2 and 

3.5 years (Eskenazi et al., 2007; Eskenazi et al., in preparation as cited in the Whyatt 

presentation made to EPA in 2007). 

d. Results indicated prenatal urinary DAP measurements were associated with decreased 

verbal IQ and full-scale IQ at 3.5 years and decreased verbal IQ at 5 years (Eskenazi et 

al., in preparation, Whyatt presentation to EPA in 2007). However, since DMP is 

definitely not a metabolite of chlorpyrifos, the associations stated here and in all of the 

previous points must be viewed with caution so as to not overinterpret the significance of 

the finding 

~ A crude dose-response relationship as evidenced by the following results from the pre. 

and post cancellation analyses in the Columbia cohort: 

a. There were statistically significant deficits in birth weight (211 grams when comparing 

high to LOD groups) in children born before the residential cancellation, but there were 

no significant deficits in these outcomes in children born after the residential cancellation 

(Whyatt, EPA presentation, 2008). 

b. Statistically significant decreases in birth weight and birth length per log unit increase of 

cord blood chlorpyrifos were evident in children born before residential cancellation but 

the association was not significant for children born post-cancellation (Whyatt et al., 

2004). 

c. There were statistically significant reductions in maternal personal air samples and cord 

blood chlorpyrifos levels pre- and post-cancellation ahd a statistically significant 

38 
reduction in the numbers of newborns who were categorized in the highly exposed group 

(Whya!t et al., 2004). 

d. There were statistically significant increases in maternal personal air samples and cord 

blood chlorpyrifos levels in 3 year MDI and PDI scores when comparing pre-cancellation 

to post-cancellation periods (Rauh et al., 2006). 

~ Persistence of strong statistically significant associations after controlling for the 

effects of other compounds as evidenced by the following: 

a. Additional analyses on birth weight and neurodevelopmental outcomes (MDI and PDI) 

controlling for diazinon did not reduce the effect of chlorpyrifos (Whyatt, August 12, 

2008, personal communication). 

b. Association between birth weight and length remained statistically significant after 

controlling for diazinon and isopropoxyphenol (Whyatt et al., 2004). The authors noted 

that blood concentrations for chlorpyrifos were higher than those for diazinon and 

propoxur. The Panel had some questions about these calculations. 

39 
Table 1. Neurodevelopmental Effects with Chlorpyrifos and Its Metabolites and the Metabolites ofOP Insecticides in Three Epidemiological Human Cob.ort Studies 

Source: Appendix D, USEP A; Eaton et al., (2008); referenced studies 

Age CHAMACOS (Non-specific OP metabolites+ TCP Mt. Sinai (Non-specific OP metabolites measured) Columbia (chlorpyrifos, TCP, propoxur, diazinon) 

measured) 

Neonate Abnormal reflexes (maternal urinary DAPs): During Abnormal reflexes (maternal urinary DAP metabolites): 

pregnancy, total DAPs (summed DEP metabolites and Increased total DAPs, (summed DEP and DMP metabolites) 

DMP metabolites) with increased number of abnormal with increased proportion and number of abnormal reflexes 

reflexes and presence of 3 or more abnormal reflexes. (BNBAS) (Engel et al., 2007) 

(BNBAS: Brazelton Neonatal Behavioral Assessment 

Scales) (Young et al., 2005) 

6 months No association with prenatal TCP and MDI (Motor 

Development Index)/PDI (Psychomoter Development 

Index) ( BSID-II: Bayley Scale of Infant Development 

-II) 

(Eskenazi et al., 2007) 

I year • Child DEPs associated with increases in MDI 

(BSID-II) (Eskenazi et al., 2007) 

• No association with prenatal TCP and MDI/PDI 

(BSID-II) (Eskenazi et al., 2007) 

2 years • Total urinary prentatal DAP metabolites Prenatal DAPs associated with decrease in MDI (BSID-II) 

associated with increases in POD (Psychomoter (Engel et al., in prep) 

Development Delay) { CBCL: Child Behavior 

Checklist)) (Eskenazi et al., 2007) 

• Child DAPs (measured DMPs, and 

DEPs)associated with increase in POD (CBCL) 

(Eskenazi et al., 2007) 

• Prenatal DAPs (measured DMP, one DAP) 

associated with decrease in MDI (BSID-II) 

(Eskenazi et al., 2007) 

• Child DAPs (measured DMPs) associated with 

increases in MDI (BSID-II) (Eskenazi et al., 2007) ' 

• No significant association ofTCP with MDI or 

POI. (BSID-II) (Table 6, Eskenazi et al., 2007) 

3 years IQ deficits associated with prenatal DAPs at 3.5 years Increased chlorpyrifos with decreased mean PDI, 

(Ezkenazi et al., in prep) increased percent with cognitive (MDI) and motor 

problems (PDI), increased risk of mental and motor 

delay, increased problems with attention and ADHD 

(Attention Deficit Hyperactivity Disorder) and POD. 

Decreased levels of chlorpyrifos after January 2001 

associated with improved scores on MDI and POI. 

(BSID-II and CBCL) (Raub et al., 2006) 

5,7 years IQ deficits associated with prenatal DAPs (Ezkenazi et IQ deficits associated with chlorpyrifos (Raub 2008, 

al., in prep) presentation at SAP meeting, see Docket: EPA-HQOPP-2008-0274) 

40 

I 

I 

I 
Some caveats and areas for further analysis and clarification were noted by various panel 

members: 

1) In the Berkeley cohort, there was a finding of increased head circumference with DEP 

levels (3 DEPs were measured) in maternal urine and increased length and head 

circumference with DMP levels (3 DMPs were measured) (Ezkenazi et al., 2004). This 

could be a chance finding or it may be due to the exposure profile in this cohort. 

However, the Mt Sinai study found an association between decreased head circumference 

and maternal urinary TCP levels above the limit of detection (Engel et al., 2007). Due to 

the nature of the exposure in the Berkeley cohort, it was noted that perhaps these urinary 

levels do not reflect significant exposures to chlorpyrifos given that DAP metabolites 

have two potential origins: breakdown of products arising from OPs in general and direct 

· absorption from environmental sources and diet. As measurements of DAP metabolites 

are non-specific in nature, the particular chemical involved may be one or more of diethyl 

phosphate insecticides in use. With regard to the Mt. Sinai study, the results indicated 

that maternal urinary TCP levels reflected not only direct chlorpyrifos exposure, but also 

exposure to TCP residues directly from the environment or diet. In addition, the Panel 

noted that PON1 polymorphism may potentially affect those urinary metabolite levels 

because women with the higher activity phenotype will eliminate higher levels ofTCP in 

urine as compared to those with the lower activity phenotype. However, this statement is 

not true if exposure levels are low and below enzyme saturation in the case of carriers of 

a low activity phenotype. 

2) In interpreting the Columbia studies, the Agency should bear in mind that diazinon and 

propoxur as well as other unmeasured potential developmental neurotoxicants were 

present along with chlorpyrifos, and the exact roles of all of these components in the 

mixture cannot be determined. In Table 3 ofWhyatt et al. (2004) it is seen that the beta 

coefficients for the association with birth weight are -42.6 for chlorpyrifos and -44.2 for 

diazinon. The beta coefficient for chlorpyrifos is statistically significant while the 

coefficient for diazinon shows large variability and is not statistically significant. One 

panel member questioned whether selecting chlorpyrifos over diazinon is biologically 

justified although it may be statistically justified. When the effects of chlorpyrifos and 

diazinon are summed using U.S. EPA methodology (U.S. EPA, 2001), there is a slightly 

greater reduction in birth weight (b= -49.1 ). This may indicate that the effect of the 

combined chemicals is slightly greater than the individual chemicals alone and that there 

could be potential interaction between the two chemicals with respect to the association. 

Given these results, some on the Panel questioned whether one should single out just one 

of the two chemicals when both are present and there is a potential interaction. Rauh et 

al. (2006) did not consider diazinon or mixtures in the neurobehavioral study but justified 

the selection of chlorpyrifos based on the birth weight data, ignoring the similarities of 

diazinon with chlorpyrifos and the significance of the mixtures. There was disagreement 

among the Panel with this interpretation. Confidence limits as well as the beta 

coefficients must be considered when interpreting statistical analysis results. The Panel 

was in agreement that there was potential for interaction between chemicals, but this did 

not preclude chlorpyrifos as a contributor. The Panel also noted that Rauh did perform an 

41 
analysis of the neurodevelopmental effects while controlling for diazinon and the mixture 

of diazinon and chlorpyrifos (Raub personal communication to the Agency dated August 

12, 2008, found in docket, EPA-HQ-OPP-2008-0274). In this analysis, the authors stated 

that the combination of chlorpyrifos and diazinon produced slightly more significant 

effects for MDI than were seen with chlorpyrifos alone. Diazinon alone did not produce 

significant changes in MDI. This interaction is critically important in interpreting the 

results. 

3) More analysis controlling for multiple exposures and examining additive effects would 

help to elucidate the role of other organophosphates, but these analyses were not 

necessary for the panel to affirm that chlorpyrifos likely played a role in the outcomes 

that were observed. These results are important for AChE inhibition to be considered as 

a mode of action. 

4) Whyatt and Raub (Columbia University) in their comments to the Panel (September16, 

2008) described how they separated the neurodevelopmental effects of chlorpyrifos 

exposure from those of diazinon exposure. Some panelists questioned whether the 

biological effects of these anticholinesterase insecticides could be separated. 

5) The Agency's use of the word "likely" at the end of the paragraph needs to be considered 

carefully. The Agency should ensure that the use of the word is not based heavily on the 

Columbia study in the absence of intermediate markers of biologically effective dose and 

early effect that further link exposure and outcome and or a clearer idea ofmode of action 

from experimental studies. 

6) It is unclear whether the blood levels of the three AChE-inhibiting insecticides are highly 

correlated. If there is a lack of correlation, it is less likely that they would confound or 

be responsible for the observed relationships between chlorpyrifos and neurological 

outcomes. However, it would take a much larger and more detailed study to elucidate the 

more complex issues involving chemical mixtures (the possibility of synergism, 

antagonism, potentiation, activation, etc.) Some panelists suggested that chlorpyrifos (or 

chlorpyrifos oxon) may inhibit certain xenobiotic metabolizing CYP isoforms which may 

make some individuals more susceptible to exposure to other chemicals (e.g. other OPs, 

carbamates, pyrethroid insecticides, or other environmental contaminants). This is may 

be due to the resulting declined efficiency in CYP metabolism in response to these _ 

exposures when preceded by exposure to chlorpyrifos (Note: work by Ernest Hodgson's 

group at North Carolina State University was mentioned). Organophosphorothiate 

insecticides, including chlorpyrifos, act as "suicide substrates" leading to inhibition of 

CYP450 (DeMatteis1974; Halpert et al. 1980). Thus, exposure to these other compounds 

may also affect the metabolism of chlorpyrifos. 

7) Other issues with the Columbia cohort study (Whyatt et al., 2004) were identified. The 

level of diazinon in the homes was 8 times higher than that of chlorpyrifos suggesting 

that the exposure level to diazinon was 8 times higher. Diazinon is less toxic than 

chlorpyrifos, and chlorpyrifos is more lipophilic than diazinon. With the difference in 

lipophilicity, it was not surprising to see more chlorpyrifos than diazinon detected in the 

42 
cord blood. There was an additional OP insecticide (methyl parathion) as well as 5 

carbamates (including propoxur) present in the homes. These anticholinesterases could 

be interacting with each other. 

8) The following are some clarifications provided by one Panelist on the material presented 

in Appendix D: 

a. The Agency stated that exposure is underestimated by use of blood levels. The 

Panelist agrees that this is true for peak exposures and a single blood sample 

would likely miss the peaks. However, because exposure is multi-media and 

multi-route, for most women in the Columbia cohort (as well as in the general 

population) single high insults are likely limited, and exposure may more likely 

· be a series of contact events that are lower in magnitude over time via various 

routes and media. Thus, spot samples may be a useful way to estimate average 

exposure for women in the study since there is much lower within-home 

variability of pesticide levels as compared to between-home variability (Whyatt et 

al., 2007). . 

b. Description of blood levels and underestimation of exposure for diazinon and 

propoxur on page 50 of the Agency's issue paper should be edited for clarity in 

the document. As it appears now, the document reads as though these pesticide 

levels would be more heavily underestimated as compared to chlorpyrifos levels 

in the same blood samples. 

In conclusion, notwithstanding the fact that the three studies are not totally comparable, the 

Panel found that there were more similarities than discrepancies across them. When considered 

along with animal data, chlorpyrifos is likely associated with adverse neurodevelopmental 

outcomes. Although the Mt. Sinai and Berkeley cohorts are less specific than the Columbia 

study, they support the overall findings ofthe latter. The Panel supported the statement that 

exposures to all three AChE-inhibiting insecticides may act in combination to produce the 

observed effects. The Panel agreed that there may, in fact, be additive effects or effects 

generated by a mixture of the agents. Although the authors of the Columbia studies have 

attempted to isolate the effects that would be associated with chlorpyrifos, the Panel noted it is 

difficult to quantifythe contribution of other neurotoxic compounds in such simultaneous 

exposures. It was also noted by one panelist that other OPs, as well as other chemicals, may 

play a role in neurotoxic effects in light of a recent study by Eskenazi et al. (2008). The Panel 

concluded that although a mixture of chemicals may produce the observed effects, this did not 

rule out chlorpyrifos as plaYing a role. However, the fact that mixtures were involved in the 

measurements of all three studies does limit the conclusions relative to the specific role of 

chlorpyrifos in observed outcomes. 

The majority of the Panel agreed that although the exposures in the Columbia study occurred at 

·levels below the U.S. EPA reference dose and likely underestimate true exposures, the results of 

this study are of concern regarding adverse effects on normal neurodevelopment of children 

exposed 'in utero to chlorpyrifos. This is particularly true in light of evidence demonstrating that 

low levels of exposure to toxicants once thought to have adverse neurodevelopmental effects 

only at high levels (i.e. lead, mercury, and PCBs) are now known to produce significant effects at 

43 
lower levels. Organophosphates are known neurologic poisons designed to kill insects. People 

exposed to high levels of OPs may suffer seizures, coma and death (Etzel, 2003). There was 

disagreement among the Panel as to what conclusions can be drawn from observed 

neurodevelopmental outcomes regarding the cholinesterase inhibition mechanism of action. 

Many other associations have been found between DAP, DMP, DEP and neurodevelopmental 

and neonatal outcomes that indicate a role for other organophosphates. This can be supported by 

a cholinesterase inhibition mechanism of action or perhaps some different mechanism. Some 

panel members believed more strongly that there was likely a different mechanism of action 

other than acetylcholinesterase inhibition mediating these neurodevelopmental effects. 

5. Human Information Available for Risk Assessment: 

Ultimately, the Agency will assess potential risk to humans from current exposures to 

chlorpyrifos. Thus, data in humans provide a valuable tool for considering human outcomes, 

metabolism, and dose response. Under this context, the Agency has considered the extent to 

which data in the epidemiology studies and the deliberate dosing studies can be used 

quantitatively in the chlorpyrifos risk assessment (See Issue Paper, Section 2.3). 

a. The epidemiology studies provide important information about potential human 

outcomes related to the potential effects of OPs on the developing brain. Moreover, they provide 

data which supports the human relevance of outcomes observed in animal studies. However, at 

this time, they have not been proposed for use in directly deriving the PoDs or UFs. 

Each of the cohorts has been exposed to chlorpyrifos to some extent. However, in addition to 

chlorpyrifos, each cohort has been exposed to multiple pesticides, including other OPs. 

Determining the quantitative contribution of chlorpyrifos to the reported outcomes separate from 

the other OPs is challenging. This determination would be highly uncertain given the current 

state of the science on the dose response relationships for mechanisms (other than AChE 

inhibition) leading to effects on the developing brain. As the science evolves in this area, the 

understanding of TK and TD factors which impact toxicity to the developing brain will improve 

as will the dose response information in animals. With this improved understanding, the Agency 

may, in the future, be able to better characterize the linkage between blood or urinary levels of 

chlorpyrifos and/or its metabolites with health outcomes. At this time, the Agency has used the 

reported levels of chlorpyrifos and its metabolites simply as markers of exposure without an 

attempt to estimate actual exposure or dose to the tissues. The Agency is aware of an effort by 

Drs. Dale Hattis and Robin Whyatt to develop a physiologically-based pharmacokinetic (PBPK) 

model which includes a placental compartment for assessing tissue dosimetry to the fetus and 

which accounts for intra-species TK variability. Tbe investigators then plan to use that model to 

estimate a human PoD from the blood biomarker reported in Whyatt et al (2003). This work has 

only just begun and will likely take several years. Please comment on the Agency's conclusion to 

use the epidemiological studies primarily for purposes of hazard characterization and not for 

dose response assessment. Please also comment on the scientific support for or against this 

preliminary conclusion. 

44 
Panel Response 

The Panel concurred with the Agency that there are key limitations to the three epidemiological 

studies and that they should be used primarily for hazard identification. The Panel disagreed on 

whether the current epidemiological data provide sufficient evidence to suggest that the 

uncertainty factor for the cholinesterase inhibition endpoint be changed to accommodate the 

possibility of neurodevelopmental effects from low-'level in utero exposures of chlorpyrifos. The 

majority position was that current epidemiological data do not provide sufficient evidence to 

increase the uncertainty factor for the cholinesterase inhibition to accommodate the possibility of 

neurodevelopmental effects. However, some panel members felt strongly that the current 

epidemiological data do provide evidence to indicate that the margin of safety should be 

increased. The Panel recommends that the Agency conduct a full formal weight of evidence 

evaluation for causality of the reported associations between exposure to chlorpyrifos and 

neurodevelopmental outcomes in the existing epidemiological database. 

One of the key limitations of the epidemiological studies is that the exposure data were collected 

at single time point and lack information on the long-term exposure level and duration. A 

second limitation is that the subjects in two of the cohort studies (conducted by the Mt. Sinai and 

University of California-Berkeley researchers) had multiple chemical exposures including 

multiple AChE-inhibiting insecticides (see discussion for previous question). The Columbia 

cohort study (Whyatt et al., 2002) also had multiple chemical exposures,for example, to the · 

insecticides, DDT (detected at 68% ofthe samples) and chlordane (detected in 78% ofthe 

samples). One panel member added that it may be more difficult to use neurobehavioral 

endpoints in dose-response assessment as they are qualitative in nature. Another limitation is that 

neurodevelopmental changes are caused by multiple factors, precluding an accurate doseresponse relationship. The Panel disagreed on whether the epidemiological data, and in particular 

the neurodevelopmental findings; provide sufficient data to support changes to the default 

uncertainty factor 

The Panel stated that data from human studies, in general, are of great value for assessing risks to 

human health. Besides epidemiological studies and deliberate dosing studies potential sources 

for data on the effects of chlorpyrifos in humans may come from poison control information on 

accidental and intentional exposure (decreased since 2001 when indoor uses of chlorpyrifos were 

voluntarily cancelled) as, well as from occupational exposure data from workers in manufacture, 

formulation, transportation, and application of chlorpyrifos (e.g., pest control operators, pilots, 

flagmen, pickers). 

Confounders are unavoidable in epidemiological studies (see Panel response to Charge Question 

4). Confounders regarding the exposure agent may include impurity, mixture 

(synergism/antagonism with other pesticides or agents), vehicle etc. Confounders regarding the 

exposure are most commonly related to dose, route, and duration of exposure. Confounders 

associated with the subjects enrolled in the study may include age, gender, health/disease status, 

personal environment, and genetic factors such as isoforms and transporters in key toxicological 

events. Within the chlorpyrifos epidemiological datasets, determining the quantitative 

contribution of chlorpyrifos in exposures to mixtures may be possible by accounting for 

differences in chemical potency, pharmacokinetics and possible chemical interactions. Further 

45 
resolution of dose-response relationships may also come from the on-going data analysis by the 

Columbia University study group and any other long-term studies with improved exposure 

information and more quantitative measure of chlorpyrifos toxicity. 

The Panel recommended that the Agency conduct a full weight of evidence evaluation for the 

neurodevelopmental outcomes. This generally requires a much wider scope of review that 

encompasses all available data; not only data for the given chemical, but also data for chemicals 

of similar structure and activities, e.g., OPs. To increase transparency and defensibility of the 

conclusions concerning the potential role of chlorpyrifos in contributing to neurodevelopmental 

outcomes, the weight of evidence analysis should specifically address criteria for causality 

including consistency, and take into account the results not only of the studies considered here 

but others and the supporting biological data. Such an exercise requires explicit consideration of 

criteria such as strength, consistency, specificity related to chlorpyrifos or to its anticholinesterase effects common to OPs as a whole, dose-response, temporal concordance and 

biological plausibility in a framework analysis similar to that which is conducted currently for 

hypothesized modes of action. This allows comparative analysis across assessments of 

consistency of weight of evidence determinations. The weight of evidence analysis might 

increase confidence in this case and potentially identify additional relevant analyses to address 

unc~rtainties such as the role of other pesticides in the observed associations. To the extent 

possible, once a hazard is established, an array of dose-response data in both animals and 

humans in the context of their relative associated uncertainties may also be helpful, recognizing 

that the magnitude of these uncertainties for the dose-response may generally be greater for 

animal species, owing to interspecies differences. Outliers need to be excluded, and the relative 

magnitude of uncertainties explicitly weighted in considering the relative contribution of 

epidemiological versus other types of data. 

The Panel agreed with the Agency that there were limitations in the three epidemiological studies 

that precluded them from being used to directly derive the PoD or the uncertainty factor. The 

Columbia University cohort study could be used to determine bounding values for the levels of 

chlorpyrifos that might cause a measurable effect. In a similar way, data from epidemiological 

studies can also be used in risk assessment. .The use of a PBPK model would enable estimation 

of an exposure dose metric for multiple sources of exposure, e.g., air, food, water. 

For example, one Panel member estimated the blood concentrations of chlorpyrifos from the 

PBPK model provided by Dr. Timchalk ( Battelle Center for Biological Monitoring and 

Modeling -Public comments dated Aug 28, 2008, submitted to docket EPA-HQ-OPP-2008-

0274)) at the 50th, 75th, 95th percentile at the maximum of the California air concentrations of 

chlorpyrifos as stated in oral public comments provlded by Dr. Sass of the Natural Resources 

Defense Council (NRDC and representing the Pesticide Action Network- North America 

(PANNA), September 16, 2008). Based on this analysis, the simulated chlopryrifos blood 

concentration was approximately 1x10-4 f.tmol/L, or 35 ng/L, when calculated using a peak air 

concentration of 1.34 ug/m3

. Some panel members raised concerns about this computation.3 In 

3 One panel member provided some additional notes to clarify the vague concerns expressed at the SAP meeting. 

This information will be useful to the Agency. Review of public comments in the docket (EPA-HQ-OPP-2008-

0274-0056,) indicated that the 24-hour average concentrations were below the adult RELs (recommended exposure 

levels). This finding raises a concern about the calculations made by one panel member. A lag time is needed to 

46 
this illustration, potential exposures to a blood concentration of 35 ng/L chlorpyrifos would be 

nearly 6 times greater in comparison with the measured chlorpyrifos blood concentration, of 6.17 

ng/L reported in the Columbia University study. In the Columbia University study, levels of 

6.17ng/L chlorpyrifos in blood were associated with neurobehaviornal effects. The Panel urged 

the Agency to more carefully analyze these current data to determine whether individuals 

exposed to potentially higher levels of chlorpyrifos in air would show signs of 

neurodevelopmental effects. In a similar fashion, data from the epidemiological studies could be 

used in risk assessment as bounding values to evaluate exposure standards, i.e., reference 

concentrations or doses. Based on the level of analysis that can be performed, Panel members 

suggested that the Agency might be able to use the cohort data in a more quantitative fashion as 

part of the risk assessment, such as in the aforementioned boundary setting exercise. 

While the potential contribution of the chlorpyrifos PBPK model being developed was 

recognized, some panelists believed that the Agency should also pursue a simpler PBPK model 

specifically applicable to the chlorpyrifos data that would be available in a relatively short 

timeframe. Some panel members urged the Agency to not delay in studying whether the level of 

chlorpyrifos in air, as noted in the NRDC and PANNA comments, poses a concern for human 

health. 

The Panel recommended the consideration of a fetal or placental compartment in a PBPK model 

should there be any indication from the paired maternal and cord blood data that chlorpyrifos and 

its active metabolite(s) are differentially distributed to the fetuses. In addition, the PBPK model 

can also simulate inter-individual (intraspecies) TK variations as well as inter-species 

differences. Furthermore, the Panel noted that additional work as a basis to consider more 

robustly interspecies differences (using central estimates) may be more fruitful over the shorter 

term. 

b. Three deliberate dosing studies in adult (non-pregnant) humans are available which 

measure AChE activity and urinary levels of chlorpyrifos and/or its metabolites (See Appendix 

G). The Agency has determined that the deliberate dosing studies in adults are not appropriate 

for use in PoD or UF derivation in the current proposal. This determination is based on several 

factors. 

• There are experimental laboratory animal data that indicate that the susceptibility of the 

developing nervous system to chlorpyrifos may be related to cholinergic and 

noncholinergic mechanisms. Findings in epidemiology studies in children support the 

animal studies. The human studies do not include the potentially susceptible populations 

being evaluated in the current effort, namely pregnant woman and children and thus do 

not consider toxicity endpoints other than AChE inhibition (and related clinical signs). 

reach a steady-state between air concentration of chlorpyrifos and blood levels, so that peak concentrations lasting 

one hour should not be used for calculating blood concentrations. A time-weighted average must be used instead, 

which would lead to lower blood levels. 

47 
• Nolan et al. (1982) and Griffin et al. (1999) only include a single dose group for a 

particular route (a study design issue previously criticized by the Human Studies Review 

Board (HSRB) with respect to other human studies). 

' 

• Griffin et al. (1999) report no changes in AChE inhibition and in Kisicki et al. (1999) 

changes were only seen in one person leading to the characterization of these studies as 

NOAEL only studies (a type of study not supported by the HSRB for use in risk 

assessment since absence of an effect (LOAEL) raises questions about whether the 

investigators were able to detect an effect or that it was possible given the study design). 

However, the Agency has determined that the human studies do provide valuable information on 

correlating oral or demial exposure with levels of chlorpyrifos and/or TCP in blood and urine. In 

addition, these studies also provide information on time course of absorption, metabolism, and 

excretion. Kisicki et al. (1999) also includes PON1 genotype information. Due to the availability 

of quality TK information, these studies have been used in the past by the Agency to aid in 

interpreting biomonitoring data. Specifically, results from Nolan et al. (1982) have been used 

previously by the Agency in estimating (i.e., back-calculating) chlorpyrifos exposure based on 

urinary levels ofTCP. Nolan et al. (1982) has also been used to derive a dermal absorption 

factor in humans. If the Agency wishes to continue this use of the human studies to assist in 

characterizing and interpreting epidemiology and biomonitoring data, the studies will be brought 

to the Agency's HSRB for review of their scientific and ethical conduct. To assist the Agency in 

preparing for this review by the HSRB, please comment on the clarity and completeness of the 

Agency's scientific analysis of the human studies. In particular, please focus on whether the 

Agency has identified the key scientific issues and whether other information or studies are 

available that should be considered in formulating the Agency's preliminary conclusion to use 

these studies for purposes of characterizing and interpreting the epidemiology and 

biomonitoring data and not for deriving PoDs or UFs. 

Panel Response 

The Agency's issue paper clearly laid out EPA's scientific analysis of the human studies and 

identified desirable components that are missing in these human studies. One panel member 

noted that to enhance clarity for readers not intimately familiar with EPA procedures, 

information about human subjects and the conduct ofthe study should accompany the synopses 

of the studies provided to the Panel. 

Overall, the Panel agreed that the human deliberate dosing studies contain scientifically useful 

information for risk assessment, but not for directly establishing PoD or uncertainty factors. The 

following major limitations of these data were noted by the Panel. Collectively, these limitations 

preclude their direct use for establishing PoDs. 

• These studies do not include the potentially susceptible populations (e.g., women of 

childbearing age, pre- and post natal stages, teenagers and young adults before brain 

development is complete in the mid-20's). 

• The single dose regimen does not provide information to evaluate dose-response 

relationship and span the range of environmental exposure levels. 

48 
• These studies do not provide information on any other toxicological endpoints besides 

cholinesterase inhibition. 

• These studies do not include the inhalation route, a major route of exposure for workers 

and bystanders alike. 

One panelist provided the following information to the Panel from his unpublished study 

conducted on a small cohort of children (n = 50) examining cholinesterase activity and PON1 

genotype as a result of chlorpyrifos exposure (hand wash measurements for exposure). Two 

children with comparable exposures showed a 100-fold cholinesterase difference at the highest 

hand rinse concentrations, but different (slow versus fast) polymorphisms in the PON-1 

genotype. On the other hand, two other chil~ren showed a 10-fold variation in cholinesterase 

activity even though they had the same PON-1 genotype. These studies used a single dose that 

may not reflect environmental concentrations and examined only dermal exposure. A significant 

portion of the observed differences in cholinesterase activity may be due to differences in 

inhalation or dietary exposures rather than dermal exposure (the route of exposure measured in 

this study). Given the small population size, use oftwo isolated comparisons, and the uncertainty 

·associated with exposures, this study does not provide enough information to influence a choice 

of uncertainty factor. However, these unpublished results do confirm that genetic differences 

between individuals in a population may alter the exposure to toxic chlorpyrifos metabolites. 

In principle, the Panel agreed with the Agency's scientific approach to derive a dermal 

absorption factor from the human studies. These human studies contain valuable scientific 

information especially if no other source of data is available. However, the current 3% 

absorption factor should be further refined by considering the different exposure scenarios. 

Taken together, these studies provide more robust database than what each single study can 

offer. They span a wide range of exposures, between 0.06 mg/kg (5 mg for 67-81 kg subjects in 

the Meuling et al. (2005) study) and 5 mg/kg (subjects B-F in the Nolan et al. (1982) study), 

different wash-off durations (as short as 4 hours in the Meuling et al. (2005) study), and using 

different vehicles. 

The Panel recommended that if the Agency wishes to use these studies to derive dermal 

absorption factors for use in risk assessment, then they should adjust for any underestimation 

using the apparent inverse relationship between dermal absorption and the concentration of 

exposure (see Nolan et al., 1982 and Meuling et al., 2005.) The Panel also noted that when 

deriving the dermal absorption from each study, the estimated oral absorption in the same study 

should be used as a reference point instead of the 100% oral absorption that was assumed in 

extrapolating data from oral and dermal routes. When necessary, material balance and 

elimination kinetics from each study should also be accounted for. Other limitations of these 

studies should be considered as uncertainties, e.g., exposure only on forearms and shorter 

duration of skin contact than the anticipated human exposures. The Panel was familiar with how 

the 3% dermal absorption factor used by the Agency (see EPA's 2000 chlorpyrifos risk 

assessment) was derived based on the Nolan et al. (1982) study. The Agency compared this 

factor to the 0.03 ratio between the oral LOAEL of0.3 mg/kg/day from the rat DNT study to the 

21-day dermal LOAEL of 10 mg/kg/day in rats. Because it is not likely that the dermal 

absorption factor derived in humans would closely coincide with the values for rats, and given 

49 
that the comparison of toxicity at the LOAELs is crude, the support from this comparison would 

not be crucial. 

The Panel agreed with the Agency scientific analysis that these deliberate dosing studies cannot 

be used to directly establish a PoD or UFs. The Panel indicated that these data might be used as 

bounding levels similar to what was suggested by the Panel concerning the data from the 

epidemiological studies (see Panel response to Question 5(a)). · 

The Panel appreciated the Agency's scientific analysis to compare the blood levels in the 

deliberate dosing and epidemiological studies,. and considered it critically important to 

maximally use the information from these studies. These studies· could be used for purposes of 

interpreting (at least crudely) the nature of dose-response in the epidemiological data and as a 

basis to "bound" the reference doses/concentrations. The Panel encouraged the Agency to 

consider the use of a PBPK model to widen the application of these bounding data for current or 

potential human exposures and for the final reference dose or reference concentrations. In 

addition, these human studydata may contribute to an array of the dose-response data in both 

animals and humans mentioned under question 5a, but in the context of their relative associated 

uncertainties. 

6. Points of Departure (PoD) for Risk Assessment (Issue Paper, Section 2.5): 

a. Based on the results of the extensive literature review, the Agency has proposed 

updated PoDs derived from the laboratory animal studies for extrapolating human risk. The 

Agency has posed three options for the PoDs for chlorpyrifos in its issue paper. When the 

Agency derives PoDs for assessing the risk from exposures to pesticides, it needs to consider all 

relevant routes (oral, dermal, inhalation), durations (ranging from acute to chronic), and all 

exposed populations (including adult, pregnant women of child bearing age and children). 

The first option proposes to use the PoDs which were based on rat RBC and plasma 

cholinesterase inhibition in the 2000 risk assessment for acute oral exposures and blood AChE 

for chronic oral exposures. The 2000~risk assessment included a weight of the evidence 

discussion primarily on adult rat and dog AChE guideline studies and adult data from Zheng et 

al. (2000). This option would involve application of the no-observed-adverse-effect-levels 

(NOAELS) for blood AChE inhibition from route specific studies (oral, dermal, inhalation) in 

rats or dogs to all populations. The acute oral PoD would be 0.5 mglkg/day and the repeated oral 

PoD would be 0.03 mg/kg/day. The dermal and inhalation NOAELs would be 5 and 0.1 

mglkg/day, respectively. 

The second option proposes to use a value of 0.1 mglkg/day derived from multiple studies and 

lifestages. This proposed PoD would be applied to all populations and all durations. The 

proposed value of 0.1 mglkg/day was derived using benchmark dose estimates from brain and 

RBC AChE in young pups (PND1 and 12) following acute dosing and from peripheral (heart) 

AChE following repeated gestational studies with dams. As such, multiple lifestages are 

considered in the proposed PoD: pregnant dams, PND1 pups, and PND12 pups. Furthermore, the 

proposed value is 3 to 10 fold lower than causing effects on the developing brain reported in 

other laboratory animal studies and thus is expected to be protective for those effects. 

50 
The third option is a blend of options 1 and 2. This option proposes to use a value of 0.1 

mg/kglday derived from the acute post-natal rat brain and RBC AChE data for all populations 

but only for the acute duration for oral exposures. Exposure scenarios invo~ving repeated 

exposures would use the PoD of 0.03 mg/kglday from the 2000 risk assessment for oral 

exposures. The value of 0.03 mg/kglday was derived for the 2000 risk assessment based on 

blood AChE from multiple adult nit and dog studies. The lower PoD for repeated exposures in 

option 3 is proposed to account for potential accumulation of toxicity which can occur following 

repeating doses of chlorpyrifos in adult studies. Please comment on the strengths and weaknesses 

of each proposed approach. Is there another option or a variation of one of the three options that 

the Agency should consider? 

Panel Response 

General comments: 

The principal focus of the questions from the Agency on PoD relates to whether PoD used in the 

2000 chlorpyrifos risk assessment will be altered given more recent data on neurodevelopmental 

effects associated with early exposure to chlorpyrifos. Recommendations here are predicated on 

some general principles in selecting PoDs for risk assessments, including making maximal use of 

route specific data as a basis to minimize uncertainty, to rely to the extent possible on benchmark 

doses (BMDs) versus no- or lowest-observed-(adverse)-effect levels (NOAEL or LOAEL) and to 

take into consideration the sensitivity of all life stages. 

Members of the panel also noted that it was difficult to separate fully the discussion of points of 

departure from that of uncertainty factors so that the content here has relevance to the responses 

to Question 7 and vice versa (i.e., the adequacy of proposed uncertainty factors is necessarily 

related to the nature of the point of departure). As a result, comment is also included herein on 

residual uncertainty associated with proposed or preferred points of departure including that 

related particularly to potential lack of adequate consideration of all life stages. 

The Panel agreed with the Agency's decision not to .use the human deliberate dosing studies to 

establish ·poDs due to their limitations, in particular their lack of account of potentially sensitive 

life stages. 

Specific Comments: 

There was consensus that of the three options proposed by the Agency, Option 3 is preferred, 

based on the available data. This is predicated principally on the basis that the proposed PoD for 

acute exposure takes into account all life stages, is based on benchmark doses which offer an 

advantage over the NOAEL or LOAEL, and represents the results of several studies, for which 

results converge around the same value. Based on available data, most of Panel members (a few 

disagreed) stated that the PoD presented in Option 3 is also believed to be protective for effects 

on the developing brain, although it is based on cholinergic effects. However, this conclusion is 

highly uncertain, given the lack of information on the mode of induction of the observed 

behavioral effects and in light of evidence from in vivo and in vitro studies that non-cholinergic 

mode of action(s) are likely involved in the adverse developmental neurotoxicity (DNT) and 

51 
behavior endpoints. The Agency is encouraged, therefore, to explicitly address this uncertainty 

in their additional deliberations in derivation of the reference dose/concentrations. 

The Panel suggested that if the Agency wanted to test whether the cholinergic pathways are an 

appropriate endpoint for the PoD then an appropriate temporal dose-response study using the 

most sensitive administration method and the most sensitive life-stage comparing DNT/behavior 

with cholinesterase inhibition would be needed. This would test the hypothesis that cholinergic 

pathways are an appropriate endpoint for the PoD, even though such a study would likely not 

elucidate specific non-cholinergic mode of action(s) for DNT/behavior effects. Relevant design 

would need to take into account differing sensitivity in various species to detect effects on the 

developing brain. 

The neurobehavioral developmental effects seen in the three epidemiological studies supported 

the retention in Option 3 of the lower PoD for repeated exposures used in EPA's 2000 

chlorpyrifos risk assessment to account for potential cumulative effects. For repeated exposures, 

PoD of 0.03 mg/kg/day is supported by a BMDLw of 0.03 mg/kg/day for RBC AChE inhibition 

in pregnant dams (see Agency issue paper, p. 18, Table 2). The Panel also recommended that the 

Agency additionally investigate developing appropriate benchmark doses (BMDs) for the 

chronic PoD determination. 

More detailed comments: 

Option 1: Option 1 uses the rat RBC and plasma ChE inhibition in the Agency's 2000 risk 

assessment (Table 1 of the Agency's issue paper), weight of evidence (WQE) approach based 

primarily on adult rat and dog AChE guideline studies, NOAELs for various routes and durations 

of exposure with an acute oral dose of 0.5 mg/kg/day and a repeated oral dose of 0.03 

mg/kg/day. 

Option 1 is the status quo used in the Agency's 2000 chlorpyrifos risk assessment based on 

studies conducted solely in adults (blood AChE for acute and chronic oral exposures). The 

additional data on neurodevelopmental effects would have no impact at this time. As per other 

options, it has the advantage that it avoids uncertainties associated with interroute extrapolation. 

However, it is based on no-or lowest-observed-advese-effect-levels rather than benchmark doses; 

moreover, the dermal and inhalation NOAELs were determined in mature animals and did not 

include protection for pre- and post-natal sensitivity. 

Option 2: Option 2 uses studies that provide data amenable to benchmark dose (BMD) 

modeling; RBC AChE studies in repeated gestational studies in dams (heart) and acute post-natal 

pups (brain and RBC AChE) provide BMDs (BMD and BMDL10) in the same range of0.06-

0.12 mg/kg/day. The Agency proposes to use the weight of evidence approach, a PoD of 0.1 

mg/kg/day for oral exposure, all age groups and all durations. 

For Option 2, a value of 0.1 mg/kg/day is being proposed for all populations and durations. It is 

based on benchmark doses/concentrations which offer some advantage over NOAELs/LOAELs 

though it needs to be recognized that these advantages can be small in relation to relative 

uncertainties associated with interspecies and intraspecies differences. It does draw from 

52 
multiple studies covering several life stages (brain and RBC) AChE in young pups following 

acute dosing- PNDs 1, 7 and 12 and peripheral AChE from repeated gestational studies with 

dams; also pups). The Panel was reassured that the values for several studies fall within the same 

range and the approach offers considerable advantage in drawing maximally on the available 

data in animals at several life stages. Some panel members stated that 0.3 mg/kg/day was 

protective because it is 3- to 1 0-times lower than levels causing effects on the developing brain. 

The lowest dose tested for learning/memory was 0.3 mg/kg/day so proposed PoD was 3-fold 

lower and 10-fold lower than lowest dose used in many gestational and post-natal studies which 

evaluated toxicities other than AChE inhibition. Nevertheless, the degree of protectiveness is 

uncertain because the dose-response relationship cannot be obtained from these studies reporting 

substantial effects at 0.3 to 1.0 mg/kg/day. Another consideration is that the 0.1 mg/kg/day dose 

does not take into account potential for cumulative effects following repeated exposure. 

Option 3: Option 3 separates the PoD for acute and repeated exposures. A PoD of0.1 

mg/kg/day was used for acute and short term exposures. For chronic dietary exposure, a value of 

0.03 mg/kg/day was used. The value for repeated exposure is based on NOAELs and LOAELs 

for plasma and RBC ChE inhibition in 5 studies. This option is also supported by the BMD in 

. pregnant dams. 

These PoDs (0.1 and 0.03 mg/kg/day) are 3 and 10 'times, respectively, lower than the lowest 

dose tested across all neurodevelopmental studies (0.3 mg/kg/day). The latter dose was 

administered to dams through gestation and even several postnatal days without observing 

behavioral effects at any time in the offspring exposed to this dose level (see Agency issue paper, 

p. 19). The PoDs for acute and repeated exposures might be considered 3-fold and 10-fold 

higher than the uncertainty factor (TD), respectively, for neurodevelopmental effects. Option 3, 

is essentially Option 2, but introduces a lower value for repeated exposures from the 2000 

assessment to account for potential accumulation of toxicity which can occur following repeated 

doses. This option was preferred bythe Panel, on the basis of potential for different or perhaps 

multiple actions of chlorpyrifos at repeated low level exposures and results of the recent 

epidemiological studies. Most of panel members (a few disagreed) stated that this option was 

protective of effects on the developing brain, although it is based solely on cholinergic effects. 

However, this conclusion has associated uncertainties; the lack of information on the mode of 

action in inducing the observed behavioral effects and evidence from in vivo and ili vitro studies 

that show non-cholinergic mQdes of action are likely to be involved in the adverse developmental 

neurotoxicity and behavior endpoints. The Panel encourages EPA to explicitly address these 

uncertainties when deriving the reference dose/concentrations. 

b. Route specific data are preferred because such data accounts for potential differences 

in absorption, distribution, or metabolism. In the case of chlorpyrifos, dermal and inhalation 

studies are available which identify NOAELs for these routes in adult rats. Withrespect to 

inhalation exposure, there are two nose only studies with vapor chlorpyrifos which provides a 

NOAEL of 287 ug/m3 or 20 ppb (0.1 mg/kg/day). Similarly, there are two dermal studies which 

together provide a dermal NOAEL in adult rats of 5 mg/kg/day. These studies do not include 

pregnant dams, fetuses or post-natal pups and therefore do not consider potentially susceptible 

populations. In the absence of data in these groups, the Agency will continue to use route 

specific studies, as appropriate. An alternative for dermal exposure is to use an oral Pod derived 

53 
from susceptible populations (as discussed above) with a dermal absorption factor. Specifically, 

the Agency could use a dermal absorption of 3% from human subjects (Nolan et al., 1982). 

Please comment on the strengths and weaknesses associated with use of the adult dermal and 

inhalation studies in the chlorpyrifos human health risk assessment. The Agency also requests 

the SAP toprovide suggestions on potential toxicity and/or toxic kinetic studies (if any) in 

pregnant dams, fetuses, and/or post-natal pups which could be conducted to better inform the 

dermal and inhalation risk assessments. 

Panel Response 

The Panel was in general agreement with the Agency's proposed option to use the dermal and 

inhalation studies as a basis for development of the points of departure for these routes for 

repeated exposures, as per the 2000 chlorpyrifos risk assessment given the uncertainty associated 

with route to route extrapolations based on available data, though residual uncertainty 

concerning sensitivelife stages would need to be taken into account. The Panel also noted that 

the proposed oral, inhalation and dermal PODs are roughly equivalent (0.1 mg/kglday), taking 

into account estimated absorption from the Agency's 2000 chlorpyrifos risk assessment based on 

the Nolan et al. (1982) study. However, proper adjustment ofthedermal absorption factor for 

use in oral to dermal extrapolation is needed when the in-study oral absorption factor is less than 

1 00%. Other considerations for deriving and using the dermal absorption factor in response to 

question 5(b ). Inhalation Pod of 0.1 mg/kglday should also be adjusted for repeated exposures 

based on the 5 days per week dosing regimen. 

Also, rather than relying on the potentially proposed value of 3% for dermal exposure derived in 

the deliberate dosing study in humans by Nolan et aL (1982), the Agency is encouraged to 

review the entire database from all doses and all studies to obtain quantitative understanding of 

age sensitivity specific to the endpoint of choice, as a basis to apply it across all routes of 

exposure consistently. Physiologically based pharmacokinetic modeling may be helpful in this 

context. While the Agency is pursuing development of a PBPK model that includes the fetal 

compartment, the Panel also encourages consideration of simpler models which might 

additionally inform comparison of age- and route sensitivity in the near term future. In relation 

to dermal exposure, the Panel suggested a more relevant species, e.g., minifies, may be helpful. 

Detailed Comments: 

Given the magnitude of the uncertainties to extrapolate between routes, use of route specific data 

for inhalation and dermal exposure as a basis for points of departure was supported. However, 

the residual uncertainty for the lack of relevant data in pregnant dams, fetuses or post-n.atal pups 

exposed by these routes needs to be additionally addressed. 

Inherent in calculating the absorbed dose from the inhalation NOAEL is the recognition that the 

dose level should be somewhat comparable between routes for the same systemic endpoint, 

although rout-specific pharmacokinetic differences can be a significant factor. Some 

comparisons between dermal or inhalation NOAELs and the oral PoD are presented below. I 

54 
Dermal NOAEL of 5 mg/kg 

Using the Agency's suggested 3% dermal absorption factor derived from the deliberate dosing 

study in humans of Nolan et al (1982), the absorbed dose for the dermal NOAEL is 0.15 

mglkg/day, not significantly different from the 0.1 mglkg/day value in Option 2. However, 

because the extent of oral human absorption estimated from the same study (i.e., Nolan et al., 

1982) is 73%, the oral NOAEL of 0.1 mglkg/day would need to be adjusted by 73% of its 

original value for this comparison. Thus, the acute dermal NOAEL of 5 mglkg is approximately 

2-fold higher than the oral BMD. 

The Panel commented that it is important to recognize that when used in human risk assessment, 

the magnitude of the absorption factor is dependent on the dermal exposure conditions. For 

example, in the Nolan et al (1982) study, the absorption factor was 3% at 0.5 mglkg, but only 1% 

at 5 mglkg. The Panel noted that it is important to consider other factors that could affect 

dermal absorption, e.g., variation of rate of penetration on different skin surfaces, moisture on 

the skin under the cover of clothing, contact duration before wash off. 

Inhalation NOAEL of 0.1 mg/kg/day 

According to the Agency's Issue Paper, the NOAEL of20 ppb from the two inhalation studies in 

rats result.ed in an absorbed dose of 0.1 mglkg/day, the same value as in Option 2. The value, as 

calculated, is therefore applicable to acute exposures. For intermediate and chronic inhalation 

exposure, the absorbed dose of 0.1 mglkg/day should be adjusted down by·a factor of 5/7 to 

account for the 5 days/week dosing regimen. This is slightly lower than the 0.1 mglkg/day value 

as proposed in Option 2. 

Overall, the Panel encourages the Agency to review the entire database from all doses and all 

studies (not just the Nolan et al. (1982) study) to obtain quantitative understanding of age 

sensitivity specific to the endpoint of choice and apply it across all routes of exposure 

consistently. Physiologically-based pharmacokinetic modeling may be helpful in this context. 

While the Agency is pursuing development of a PBPK model that includes the fetal 

compartment, the Panel also encourages consideration of simpler models which might 

additionally inform comparison of age- and route sensitivity in the near term future. 

7. Extrapolation/Uncertainty Factors (Issue Paper, Section 2.6, Appendix E): 

In risk assessment, once PoDs are selected, extrapolation from animals to humans (inter-species) 

and within human variability is performed. Historically, the Agency has used default 1 0-fold 

factors to account 'for inter- and intra-species extrapolation. More recently, emphasis on the 

derivation of extrapolation factors from TK and toxicodynamic (TD) data instead of default 

factors has increased. With the intent of improving the scientific basis for the chlorpyrifos risk 

assessment, in this issue paper the Agency has considered the availability of current PBPK . 

models, TK, and TD data for chlorpyrifos to use in animal to huinan and within human 

extrapolations. Overall, the available PBPK models, although well-developed and supported for 

n<?n-pregnant adults, do not include calculations for dose during pregnancy (e.g., no placental 

compartment) and for young children less than 5 years old and thus cannot be used in a 

quantitative manner for this effort. As such, the Agency has used the 2005 IPCS guidance on 

Chemical-Specific Adjustment Factors to evaluate available TK and TD data in animals and 

55 
humans and to determine the extent to which such data support data-derived or chemical-specific 

extrapolation factors. 

a. Inter-species and Intra-species Toxicodynamic Extrapolation: The Agency has 

preliminarily concluded that with regard to TD characteristics, due to the likelihood of several 

possible modes of action of neurodevelopmental toxicity of chlorpyrifos and lack of identifiable 

and quantifiable key events for MOAs not related to AChE inhibition, the Agency cannot 

confidently refine the TD component of the animal to human and within human variability 

factors (i.e., UF AD and UFHo). Please comment on the scientific support for or against the use of 

default factors of inter- and intra-species TD extrapolation. 

Panel Response 

In response to the question of whether the Agency can refine the toxicodynamic (TD) component 

with respect to uncertainty of the animal to human and within-human variability, the Panel was 

in agreement with the Agency that there is not sufficient information to confidently refine the TD 

component ofthe animal to human and within human variability factors (i.e., UFAo and UFHo). 

The Panel agreed that neurodevelopmental effects in the fetus and neonate are important 

endpoints for chlorpyrifos toxicity, but that there are several possible modes of action of 

neurodevelopmental toxicity of chlorpyrifos and there is a clear lack of identifiable key events 

for mode of actions not related to AChE inhibition. One panel member commented that the 

Agency should also consider AChE inhibition rather than behavioral outcomes, 0.03 mg/kg/day 

versus 0.3 mg/kg/day, respectively, because of its higher level of sensitivity. Overall, the lack of 

identified mode of action, lack of correlation of acetylcholinesterase inhibition with possible 

non-cholinergic mechanisms and developmental outcomes, taken together, do not support a dataderived inter-species and intra-species TD extrapolation. All panel members concurred that the 

application of uncertainty factors is linked to the effect and dose at the PoD and the issues related 

to PoD described in Question 6 are relevant here. 

Based on consensus around sensitivity discussed in Questions 1 and 2, panel members agreed 

that animal and human sensitivities are greater for fetuses and neonates, although the current data 

do not allow for determination of the magnitude of the sensitivity. Until this is known, the Panel 

recommended application of default factors for the TD uncertainty extrapolation; A few 

members of the panel suggested that the Agency search the open literature for specific 

information and perhaps seek additional data regarding xenobiotic chemical effects on the 

developing nervous system. This information might show variations in response among 

developing brains due to genetics, time of exposure, pre-existing health status and other factors 

and support the adequacy of a 3-fold uncertainty factor (UF) for intra-species variation for these 

endpoints. 

In conclusion, until a mode of action is identified and supported (i.e., primary target other than 

AChE activity) for neurodevelopmental toxicity is identifiable and consistently documented in 

vivo, the default uncertainty factors for the toxicodynamic component should be used. That is, 3-

fold and 1 0-fold, for acute and chronic exposures, respectively, for neurodevelopmental effects. 

56 
b. Inter-species and Intra-species Toxicokinetic Extrapolation: As discussed in detail in 

Appendix E, the Agency evaluated the extent to which data on carboxylesterases, P450s, and 

paraoxonase (PON1, or A-esterase) support development ofDDEFs of inter- and intra- TK 

extrapolation (i.e., UF AK and UFHK). Based on differences in rat and humans with regard to 

maturation of metabolic processes, there are uncertainties Surrounding appropriate metabolic 

parameters for animal to human extrapolation of juveniles. This uncertainty in combination with 

limited data precludes the development of a DDEF for inter-species TK extrapolation (i.e., 

UF AK). Thus, the Agericy proposes to apply the default 3X for UF AK· Data on carboxylesterases 

are not sufficiently robust fot intra-species TK extrapolation (i.e., UFHK). Data on P450s are 

complicated by multiple enzymes each with its own maturation profile. Others have evaluated 

the P450 literature for use in derivation of child specific UFs with poor success (Ginsberg et al., 

2004a). Please comment on the scientific support for or against the use of default factors of 

inter-species TK extrapolation. Please further comment on the Agency's preliminary conclusions 

on the utility of carboxy/esterase and P45 0 data to refine the intra-species extrapolation factor. 

Panel Response 

The Panel concurred with the Agency that there is scientific support to use a default factor for 

inter-species TK extrapolation. The Panel agreed with EPA's decision to apply the default UF AK· 

The Panel unanimously encouraged the Agency to continue to consider the importance of all the 

enzymes in chlorpyrifos' metabolic pathway; the P450s, the carboxylesterases and PON1 as 

chlorpyrifos' toxicity appears to be dependent on the active metabolite, chlorpyrifos-oxon. The 

PON1 activity towards the chlorpyrifos-oxon, i.e., chlorpyrifos-oxonase is involved in the 

catalytic activity of the enzyme, but displays genetic polymorphism. There are important 

differences in the rates of hydrolysis (enzyme activity) across genotypes (192RR > 192QR > 

192QQ), with PON1 R192 allele hydrolyzing chlorpyrifos with a higher catalytic efficiency than 

PON1 Q192 allele (Furlong, 2007). However, PON1 activity and polymorphism at position 192 

(referred to as PON1 status) is not the only determinant of chlorpyrif9s toxicity, since other 

metabolic pathways may modulate potential deficits in detoxication capacity. Thus, at least 

theoretically, the level of chlorpyrifos-oxon present in serum will protect against the circulating 

active metabolite. However, at low dose exposures, most active metabolites generated in the liver 

are also detoxified immediately in the liver, either catalytically (PON1 and CYP450) or 

stoichiometrically (carboxyl esterase and butyryl cholinesterase, BChE) ). One panel member 

commented that research by Sogorb et al. (2008) indicated that even if a small fraction of 

chlorpyrifos-oxon leaks from the liver without being detoxified, it is rapidly bound and 

inactivated by serum BChE and also by albumin, demonstrating the hydrolytic activities of 

BChE against chlorpyrifos oxons at concentrations lower than 0.5 ).1M (Sogorb et al, 2008). 

Mattsson et al. (2000)detected chlorpyrifos-oxon in blood of fetuses at a concentration of 1 ng/g 

(that equals to 0.003 ).1M) only after the administration of high doses of chlorpyrifos 

(5mg/kg/day) by gavage to dams at gestational day 20. ·Such a low concentration of 

chlorpyrifos-oxon can be efficiently detoxified by serum albumin regardless ofPONl. On the 

other hand, no chlorpyrifos-oxon was detected in blood samples from human volunteers exposed 

to single oral doses of chlorpyrifos ranging from 0 to 2 mg/kg (Kisicki et al., 1999). Even in the 

case that some chlorpyrifos-oxon escapes from binding to blood proteins or enzymes, and from 

being hydrolyzed by PON1 in the blood; the possibility that it reaches the· brain is scarce since 

chlorpyrifos-oxon is a highly reactive metabolite and undergoes spontaneous hydrolysis (its half57 
life is less than one minute in blood, see Brzak et al., 1998), so there is not enough time to 

complete that journey intact. Thus, PON1 probably fails to play a relevant role at low 

environmental concentrations (nM to low J.tM). The maximum daily intake of chlorpyrifos is 

0.11 Jlg/k:g/day for infants and 0.24 Jlg/k:g/day for toddlers (see review by Eaton et al., 2008; 

Table 21 ). At middle and higher concentrations, chlorpyrifos-oxon may be formed in the brain 

because of the low desulfuration activity of brain microsomes and mitochondria (Chambers and 

Chambers, 1989). However, the brain has no PON1 activity and, therefore, the oxon produced 

"in situ" interacts with its target molecules unless it undergoes stoichiometric binding to BChE 

or other proteins acting as "scavengers". 

Lassiter at al. (1998) demonstrated that maternal blood chlorpyrifos-oxonase activity is variable 

before birth, and the placenta has about 20% the activity of the liver, which is consistent during 

late pregnancy. Fetal liver exhibited minimal activity during gestation, but increased after 

delivery. Mortensen et al. (1996) showed that maternal and fetal brains had no detectable 

chlorpyrifos-oxonase activity (Mortensen et al, 1996). Although, the Km of chlorpyrifos-oxonase 

activity is high (Km=210-380 J.tM), chlorpyrifos-oxonase in plasma and liver from adult animals 

has be shown to be capable of hydrolyzing physiologically relevant concentrations of 

chlorpyrifos-oxon (nM to low J.tM) whereas the young animal has, in tum, less capacity to 

detoxify such concentrations of chlorpyrifos-oxon via chlorpyrifos-oxonase (Mortensen et al., 

1996). 

One panel member pointed out that the lack of tissue specific data for both species under similar 

dose and timing considerations e.g., liver, is a major gap in the ability to extrapolate from animal 

data to humans. Others pointed out that there are human data on human liver carboxylesterases 

during maturation, but these data were not considered "sufficiently robust" to be useful in TK 

extrapolations. 

c. Intra-species Toxicokinetic Extrapolation (Within Human Variability): There are 

extensive data on PON1 from many populations worldwide and for different age groups. Using 

these data, the Agency has performed a preliminary analysis for within TK human variability for 

PON1 activity. These calculations were done in a manner consistent with the IPSC CSAF 

guidance. The calculations show that the largest variability in PON1 activity is between 

newborns and their mothers and is thus likely related to age-dependant maturation. 

There is some debate as to the extent to which PON1 status plays a role in toxicity at low 

environmental concentrations. Some have suggested that significant amount of OP (active oxons, 

not the parent components) must be present in the blood or brain for PON1 activity to affect 

toxicity based on generally low affinity (Km, 0.1-10 mM). Others believe that PON1 status is a 

key determinant in chlorpyrifos toxicity. The Agency has evaluated the available in vivo and in 

vitro data from animals and humans relevant to this issue. The Agency has preliminarily 

concluded that the available data suggest that PON1 status can not be ruled out as a determinant 

in chlorpyrifos toxicity, particularly for the fetus or young child. However, uncertainties remain, 

particularly regarding the degree to which other metabolic pathways modulate potential deficits 

in detoxication capacity. Please comment on the science which does and does not support PONJ 

status as a determinant in toxicity at low environmental concentrations. 

58 
Panel Response 

The Panel unanimously concurred with the Agency, based on the data, that PON1 status cannot 

be ruled out as a determinant in chlorpyrifos toxicity, particularly for t4e fetus or young child. 

Several panel members stated that actual human exposures vary between bystander and 

applicators (and everyone in-between) and it is difficult to define what exactly a low level of 

exposure is for humans. Some in the Panel suggested that the PON1 dataset could be used to 

address data uncertainty. However, many in the Panel cautioned that these data should not be 

used out of context until the rate limiting step is identified based on a PBPK model. Such a 

model does not yet exist, but is under development. These panelists believed that the use of the 

PON1 data set without such information would be a misuse of the IPCS guidance for 

determining a CSAF (chemical-specific adjustment factor). 

Several members of the Panel encouraged the Agency to obtain an independent peer review for 

the Timchalk PBPK model in order to assess the overall impact ofPON1 relative to the toxicity 

of a range of CPF, including what may be the choice of dose metric for such comparison, e.g., 

BChE inhibition or blood or tissue level chlorpyrifos, chlorpyrifos-oxon, or TCP. 

One panel member reminded the panel that there have been no direct experiments looking at 

what we might consider to be low dose exposures in animals in reference to the involvement of 

PONl. This panel member discussedtwo published model systems that might be of use to 

answer this question: the PON1 knockout (ko) and the PON1-Q192 humanized mice. In the 

PONI ko model if there any effects ofPON1 at low dose exposures then the panelist suggested 

that these effects would be manifested in this model. The Shih et al. (1998) paper shows that the 

presence ofPON1 prevents the inhibition of brain acetylcholinesterase at a dose of chlorpyrifos 

of300 mglkg (see discussion in Appendix A (Metabolism), p. 26, Figure 8, Panel E ofthe figure 

reprinted from Shih et al., 1998). However, the magnitude of the difference between the wild 

type and the PON1 ko mice is on further reflection not that great. By this panel member's 

calculations the loss of acetylcholinesterase activity is ~60% in the ko mice and 40% in wt mice. 

This would then suggest that at lower chlorpyrifos exposures and in people with varying levels 

of PONI activity (and not a complete lack of activity) there might be very little effect. 

In the second model system, the PON1 humanized mouse model, if there are higher chlorpyrifosoxon doses then there would be a significant difference in the inhibition of brain 

acetylcholinesterase in mice with either the R192 or Q192 polymorphism (see Appendix E, 

figure 4, p21). At low doses, it may not be so clear, particularly as there are no data for the R192 

mice at the lowest dose used. The question then remains whether this results in a functional 

effect that can be identified. The original paper by Cole et al. (2005) also presents morbidity data 

in the same mice following these chlorpyrifos-oxon doses (Panel C of the same figure). Again 

there would appear to be missing data (namely there does not appear to be any data for the R192 

mice below 1.5 mg/kg. However at 1.5 mg/kg there is no morbidity in the R-192 mice and with 

the Q192 there is no morbidity at doses below 1.0 mg/kg. Again this would suggest at "low" 

doses there might not be significant differences between the R/Q forms in preventing 

chlorpyrifos-oxon induced toxicity. This panel member concluded that further data are needed 

59 
but that current evidence is suggestive that PON1 is not a determinant of toxicity at low 

environmental concentrations. 

d. The Agency's PON1 calculations have focused on the PON-192Q/R polymorphism 

based largely on the extensive data available. No calculations have so far been performed on 

other genotypes. Please comment on scientific support for or against focusing on 'the PON192QIR polymorphism. 

Panel Response 

The Panel concluded that the use of the PON192Q/R polymorphism in the Agency's calculations 

for PON1 intra-human variability is appropriate. This conclusion was based on the available data 

(Furlong et al., 2005; Brophy et al., 2001). However, the analysis of the individual contribution 

of each polymorphism in the promoter region on serum paraoxonase activity/levels is 

complicated because of the pronounced linkage disequilibrium between the promoter and the 

coding region polymorphisms (Draganov and La Du, 2004). Thus, it cannot be ruled out that the 

variation observed for one of the promoter polymorphisms (PON1 C-1 08T) may be in part due to 

linkage disequilibrium with the PON1 Q192R polymorphism (Brophy et al.; 2001; Sirivarasaia et 

al., 2007). Other studies have actually excluded a significant effect ofthis polymorphism on 

serum PON1 (Phuntuwate et al., 2005). 

The 2005 IPCS CSAF guidelines state that the sensitive subgroup should be evaluated as a 

distinct population (bi-modal distribution). The data from Holland et al. (2006) on mothers and 

infants are the two important sensitive subgroups (infants and those with the Q/R 

polymorphism). Intra-individual variability could be underestimated because this population is of 

similar ethnic descent (i.e., Latina mothers and infants, primarily of Mexican descent). 

In the case ofPON1, the sensitive group is the neonates with the QQ genotype. They should be 

(and are) compared back to the mothers of the QR genotype. If data from other studies exist 

(could be done now for adults with the data from Brzak et al. (1998), it would be appropriate to 

combine geometric means and geometric standard deviations by a weighted average approach. 

This would provide a more diverse group to be considered as the reference group (i.e. QR 

genotype, Latina mothers, white women and white men). 

Several panel members made a strong recommendation that the Agency gather and analyze the 

data (in vivo & in vitro) from animal and human enzyme kinetics to look more closely at the 

PON -192Q/R polymorphism. Once a PBPK model is developed then the impact of activating 

and deactivating pathways and potentially rate-limiting components will be identified and 

information regarding PON1 Q/R polymorphism could be put into context. Such information, if 

attainable, might be used to modify the Agency's proposed approach. 

e. The Agency's calculations conducted on PON1 activity follow the 2005 IPCS CSAF 

guidance for developing intra-species extrapolation factors for TK. The preliminary analysis 

suggests that within human variability is larger than the default 3X when newborns and adults 

are considered together. Specifically a value of 12X has been calculated for chlorpyrifos-oxonase 

activity. The Agency has proposed two options for these calculations: 1) use the value calculated 

60 
for chlorpyrifos-oxonase derived from newborn and mother values in Holland et al. (2006) and 

2) use the default factor of 3X. Please comment on the strengths and weaknesses of each 

proposed approach. Please include comments on the statistical approach used in the analysis as 

a component of your response. 

Panel Response 

The panel reviewed the two approaches and the majority of the panel concluded that the 

currently available data indicate that the UFHK should be the default value of 3-fold when 

newborns and adults are considered together. On the other hand, a few panel members 

concluded that the currently available data indicate that the overall UF (UFHK x UFHo) should be 

greater than 10 based on PON1 data (not other enzymes involved in the metabolism of 

chlorpyrifos) and the potential for developmental neurotoxicity of chlorpyrifos as noted in the 

epidemiological studies (Rauh et al). These members preferred the UFHK of 12-fold, rather than 

the default of3-fold, because these were the only two choices proposed. However, none.ofthe 

panel members endorsed the CSAF approach used by the Agency to identify the factor of"12-

fold" calculated based on chlorpyrifos-oxonase and encouraged the Agency to pursue other 

approaches based on the mode of action (see 2005 IPCS CSAF guidelines). Many panel 

members did not endorse the 12-fold uncertainty factor because it focused strictly on PON1 as 

the focal point of toxicity, i.e., focused on only one enzyme out of a very complex system. 

Similarly, one panel member stated that use of the 12-fold uncertainty factor would be a 

complete misuse of the 2005 IPCS CSAF guidelines in which there is a clear need to define 

uncertainty factors based on the endpoint linked to the mode of action, neither of which is 

defined in this case. 

Members of the panel expressed concern about lack of data for non-plasma enzymes, no 

biologica1 reason or data given support the notion that maturational differences in one tissue 

relate to another. The data on the relative contributions of P450, carboxylesterases and 

butyrylcholinesterase to the ultimate concentration of oxon in the target tissue(s) is insufficient to 

derive a data-derived uncertainty factor, as EPA has concluded. Members of the panel stated 

that the information on PON1 polymorphisms should not be used as the sole factor in a dataderived uncertainty factor for two main reasons: 1) it is only one enzyme in a complex pathway, 

and is subsequent to the bioactivation reaction; therefore, it can only function on the amount of 

bioactivation product (i.e., chlorpyrifos-oxon) that is delivered to it by CYP450): and 2) the 

genotype of PON 1 alone is insufficient to predict vulnerability because the overall level of 

enzyme activity is ultimately what determines detoxication potential from that pathway; thus, it 

is better to use PON1 status because it provides information regarding both PON1 genotype and 

activity. Some of the data from laboratory animal studies in PON knockout animals are using an 

unrealistic animal model and frequently very high dose levels, and do not reflect what might 

happen in humans. 

When all of the kinetic parameters including those for PON1 genotype were placed into the 

Timchalk and Poet PBPK model, PON1 genotype only influenced chlorpyrifos-oxon 

concentration at the higher doses, but did not appreciably influence chlorpyrifos-oxon levels at 

the lower more environmentally relevant levels. Therefore several panel members concluded that 

61 
it is not reasonable to base an uncertainty factor on PON1 genotype alone and the suggested 

factor of 12 is unreasonable. 

One panelist stated the EPA-derived CSAF for UF HK underestimated the UF HK and that a 

statistical approach is recommended to correct for this problem. This panelist suggested an 

alternative approach to calculate UF HK would be to use the 99th (or 1st) percentile rather than 

the 95th (or 5th) to protect a greater portion of the population, and correct for the bias in 

estimating the GSD from the arithmetic summary statistics. The justification and approach to 

correcting the bias introduced when using arithmetic summary statistics is available in a 

manuscript currently in preparation by this panelist (Lynch et al., (2008)). An example of how 

this method can be applied is provided in Table 2 below. The Panel recognized that the 

justification for using the 99th rather than the 95th percentile is a policy decision. 

Table 2: Comparison ofUFHK Factors (Lynch et al. 2008) 

Default 501

n %tile QR 501

n %tile QR 

Mothers/ Mothers/ 

51

h%tile QQ 1st%tile QQ 

neonates neonates 

Current Method 3 11.6 16.5 

Corrected 3 12.2 17.6 

Method 

Additional Recommendations Regarding UFs 

Fallowing discussion of Question #7 (a-e), several panel members recommended the following: 

1. Establish the UFs for chlorpyrifos based on the PoD as given in any of the three options 

presented in question 6, and the concern for developmental neurotoxicity as indicated in the 

epidemiological studies. 

2. Because the Agency's choices of PoD are all based on cholinesterase inhibition (AChE 

inhibition), the Panel stated that it would be most appropriate that the UFs be established for 

this endpoint and not for the developmental neurotoxicity endpoints. Separate from 

establishing a set ofUF for AChE inhibition endpoints, the Agency is encouraged to 

establish an UF specific for addressing the concerns for developmental neurotoxicity based 

on the evidence in the epidemiological studies. 

3. Given the complexity ofinter-relationship among all the TK and TD components leading to 

AChE inhibition, it is difficult to derive individual UFs (i.e., UFAn, UFHn, UFAT, UFHT) in a 

distinct and separate manner. Therate-limiting step in the formation/removal of the oxon is 

necessary to adequately predict the concentration of active metabolite in target tissues at 

environmental do~es. Because ofthe complexity of the pathway, focusing on one enzyme 

that is likely not the rate-limiting step won't allow for the best, nor entire picture, of intra62 
human variability estimates. Instead, the Agency is encouraged to pursue the use of a 

"simple" PBPK model that can integrate all key toxicokinetic and toxicodynamic factors and 

evaluate their contributions to the overall o~tcome of endpoint (e.g., AChE inhibition) or 

related dose metric of interest (e.g., profiles of chlorpyrifos, chlorpyrifos-oxon, and the 

chlorpyrifos-specific urinary metabolite, TCP) at various chlorpyrifos exposure scenarios and 

for various life stages. The Panel noted that the Agency has made substantial progress in 

exploring the use of PBPK model with a few remaining pieces yet to be completed for a 

preliminary review (Timchalk, 2008; comments to the docket, EPA-HQ-OPP-2008-0274). 

The Agency is encouraged to bring this model to a stage ready for independent peer review 

in the very near future, without waiting for the newly funded model. Such a model is 

expected to be revised as needed, and as new information becomes available in the future. 

Data-derived extrapolation factors (i.e., uncertainty factors, UFs) are predicated on at least 

some understanding ofthe mode of action for the critical effect. DDEFs (also called, 

CSAFs) need to be based on quantitative data. At a minimum, we need to understand the 

rate-limiting step in delivery to the target tissue and the active metabolite. One panel member 

suggested this approach should be done particularly in two tissues, liver (before any amount 

of chlorpyrifos-oxon reaches the blood stream) and brain. There is uncertainty about whether 

the effect is mediated by the AChE inhibition pathway and even if so, a PBPK model is 

needed to characterize the relevant activation and detoxication at relevant concentrations or 

doses, as a basis to replace defaults. 

One panel member stated that the replacement of default uncertainty factors is more often 

achievable for interspecies differences because of the attempt to capture ratios of central 

estimates for the relevant animal species and humans vs. population variability in the human 

population, which requires considerably more data. This panel member emphasized how 

misleading it is use the PON 1 activity data as a basis for replacement of default, in this case, 

given that it is an inappropriate surrogate for human variability. 

Sever~l panel members recommended "bounding" the reference doses developed on the basis 

of the animal studies, taking into account the dose-response information from the human 

deliberate dosing and epidemiological studies, but not in relation to the points of departure. 

This is because the dose-response information needs to be interpreted in the context of what 

we know about interspecies and intraspecies differences, such that the bounding exercise 

using animal studies can be tested using human data. 

One panel member expressed overall concern about using PBPK models due to lack of 

transparency. This panelist believed that there is a blurring of inputs/outputs based on what 

is known (i.e., data are missing) versus what is predicted. Only when the predictions of the 

models can be verified with empirical data should they be used. 

In the interim, the majority of the Panel recommended that the Agency apply a default UF of 

100 to the PoD based on AChE inhibition; i.e., 10-fold for interspecies, 10-fold for interindividual variation of sensitivity (i.e., intra-species differences). Some panel members 

recommended that the Agency should consider the use of additional UFs to address the 

concerns for developmental neurotoxicity as indicated in epidemiological studies. The 

63 
default UF may be modified in the future when information on the mode of action for the 

developmental neurotoxicity becomes available. 

64 
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73 
APPENDIX 1: Summary of NeurobE!havioral Effects of Developmental Exposure to Chlorpyrlfos In Rodents: 2000-2008 

Article 

1 Maurlssen et al, 2000 

Slotkln's Group 

2 Levin et al, 2002 

Species Exposure Period 

Rat (S-0) GD6- LD 10 

Rat(S·D) GO 17-20 

Pkperiod 

neurogenesis 

Dose (mglkg/day) RoA 

0.0 

0.3 

1.0 

5.0 

Gavage 

{com oil) 

DMS0(1 ~1/kg) sc (DMSO) 

1.0 

5.0 

Maternal Toxicity Pup Toxicity AChEI 

5 mglkg: l Wgth 5 mglkg: Delay in vaginal GO 20 Dams: 

Gain (End of Gest) opening 1 & 5 mglkg 

Dams: High dose: 

muscle twitches, 

hypemea, Decreased BW: Males 

hyperactivity PND 22-65 0.3 mg/kg 

Controls&low 

dose:sporalic 

hyperactivity 

No change in 

maternal care taking N.D. 

Task Age atTest 

Delayed Spatial 

l Brain, Plasma, Alternation: Repeated 

RBC Testing 

!Plasma 

Apparatus:Wire 

7 x 12 PND 23-24 

4x12PND24 

6 x 10 PND 72-83 

6 x6 PND 85-90 

bottom cages Motor Activity 

(Habituation) 

PND 13 

PND 17 

PND21 

PND60 

(Beam Breaks) 1 Hr 

NoGenderDiff 

Auditory Startle 

50b1s: 120dB 

Spontaneous Alternation 

A. rT-Maze) 

Percent Alternation 

Latency to Choice 

5x5 

B. Fig 8 Locomol Act 

3 x1 hr 

PND22 

PND61 

4-Swks 

1 &5mglkg 

4 -6wks 

Motor Activity No Rx Effect 

Habituation 1 & 5 mg/kg 

C. RAM (16 arms) 

18 sessions 

Working Memory Errors 

Reference Memory Errs. 

Response Latercies 

8-13wks 

1 mglkg 

1mglkg 

1 &5mglkg 

Drug Challenge(RAM) 14-17wks 

Scopolamine T in WME & RME 

Mecamylamine No RxEffect 

Results 

Acquisition: No Rx Effect 

'Retention: No Rx Effect 

Acquisition: No Rx Effect · 

Retention: No Rx Effect 

NoRx 

No Rx 

NoRx 

NORK 

T lat to peak response (p=.03) [overall effect] 

NoRxeffects 

No RxEffect 

T during Trails 1 and 2 (Hyperactivity) 

!(Females) 

T (Females) 

T(Females) 

I 

(Difference in WME bet1 & 5. mglkg gps) 
Article Species Exposure Period Dose (mg/kg/day) RcA Maternal Toxicity Pup Toxicity AChE! Task Age atTest Results 

No effects of Spontaneous Alternation 

3 Icenogle et al, 2004 Rat(S-0) GD9-12 1.0 sc(DMSO) None growth/viability N.D. A. IT-Maze) 4-8Wks 

t-omuL:1osNeura1 

Tube 5.0 Percent Alternation Not Reported 

Latency to Choice CPF1&5 1(Trial1) 

B. Fig 8 Locomol Act 4-8Wks 

PND 1-4, & PND 

SMA 11-14 

Habituation CPF 5 

C. RAM (16 arms) 8-13wks 

18sessions 

Working Memory Errors CPF 5 t(EarlyTrials) 

Reference Memory Errs. CPF 5 t(EarlyTrials) 

Drug Challenge(RA.Ml 14-17wks 

Scopolamine CPF 5 Attenuated response 

Mecamylamine No Effects 

D Startle/PPI No Rx Effects 

65 Db Bckg: 11 OdB SS 

PP 68,71,77dB 

E Elevated Plus Maze 

Center crosses CPFS 

Article Species Exposure Period Dose (mg/kglday) RoA Maternal Toxicity AChE! Task Age atTest Results 

Spontaneous AJternatlon 

4 Levin et al, 2001 Rat(S·D) PND1-4 1.0 sc (DMSO) None N.D. A. (T-Maze) 4-6Wks 

PND 1-4, PND 11· 

PND 11·14 5.0 sc(DMSO) Percent Alternation 14 No RxEffect 

PNO 1-4, &PNO 

Latency to Choice 11-14 1 (Males) 

B. Fig 8 Locomot Act 4-6Wks 

PND 1-4, &PND 

SMA 11-14 No Rx Effect 

HabituatiOn PND11-14 1 (For first session) 

C. RAM (16 arms) 8-13wks 

18 sessions 

Working Memory Errors PNO 1-4(CPF1) l(Fems) tMales 

Reference Memory Errs. PND1-4 !(Ferns) tMales Initial phase 

PN011-14 No RxEffect 

Drug Challenge(RAM) 14-17wl<s' 

Scopolamine No scop impair (CPF1 Ferns) WME 

Mecamylamine NoRXeffect 
MatemaUPup 

Article Species Exposure Period Dose (mgfkgfday) RoA Toxicity AChE! Task Age atTest Results 

5 Aldridge et al, 2005 Rat(S·D) PNO 1-il 1.0 sc(DMSO) None A. EleVated Plus Maze PND 52-53 

Time in Open Arms t(Males) 

Center crosses t(Males) 

B Choc Milk Consumpt PN054 

2 choice/2 hr5 Preference 1( M &F) 

CRAM PNO 64-97 

Working Memory Error5 !(Ferns) tMales 

Reference Memory Errs. !(Ferns) T Males 

Drug Challenge(RAM) 16-Hwks 

Ketaserin WME t(M &F) 

5-HT2 antagonist - RME T (M &F) 

Acute Study 

6 Dam et al, 2000 Rat(S·D) PND 1-4 1.0 ~(DMSO) 

2 hr aft lir5t Rx PNO 

None 1 Reflexes 

!60% (CBM, BS, 

PNO 11-14 5.0 sc (OMSO) 1 mg/kgMales FBr}. Rec. 4 hr Righting Reflex PND 3-4 

!20% (CBM, BS, 

1 mg/kg Females FBr) Time to Right CPF 1 T Females 

2 hr aftCPF Rx 

PND11 Negative Geotaxis PND 5-8 

!20% (CBM, BS, 

5rng/kg FBr)M&F. Pups meeting ctiteria CPF 1 l Females 

Open FJeld PND21 

SMA !Males 

Rearing !Males 

PND30 

SMA !Males 

Rearing !Males 

Calamandrei's Group 

MatemaUPup 

Article Species Exposure Period Dose (mg/kg/day) RoA Toxicity AChE! Task Age atTest Results 

20-40% I serum 

AchE (at 24 but 

Gavage not4Bhr5after 

7 Venerosl et al, 2006 CD-1 Mice GO 15·16 and 0,3.0, or 6.0 & (peanut oil) None G019:CPY3&6 Rx) Social Recognition 4months 

PND 11-14 0,1.0, 3.0 Test GO CPF6 t Rate ofUSV 

Retest Same Partner GO CPF6 1 Rate ofUSV 

Retest Different Partner. GO CPF6 T Rate ofUSV 

Social Investigation 

Test GO CPF6 t Social investigation 
Article 

8 Venerosl et al, 2008 

9 Rlccerl et al, 2006 

10 Rlccerl et al, 2003 

Individual Studies 

Article 

11 Moser, 2000 

Species Exposure Period 

CD-1 Mice PND 11-14 

CD-1 Mice GO 15-18 and 

PNO 11-14 

CD-1 Mice PNO 1-4, or 

PNO 11·14 

PNO 32-35 

Species Exposure Period 

Rat (Long· 

Evans) PNO 17 

PND27 

PND70 

Dose (mg/kgfday) ROA 

$C 

3.0 (peanut oil) 

Gavage 

0,3.0, or 6.0 & (peanut oil) 

0,1.0, 3.0 

1.0, 3.0 

1.0, 3.0 

1.0, 3.0 

$C 

(peanut oil) 

sc (OMSO) 

Dose (mglkgfday) RoA 

Oral gavage 

4, 10,20 mglkg (com oil) 

10, 25, 50 mgfkg 

10, 50, 100 mgfkg 

MaternaUPup 

.Toxicity 

Maternal Behavior. 

PND 1: Decreased latencies to first lick 

AChEI 

PND 2:Light-Dark Test Decreased lal to enter light comp 

PND 7: Dec aggression: Inc investigation of intruder 

PND 1-7: Nest Building: Increases latencies 

PND7: Maternal Agression 

None Pups 

GO 1g: Dose 

dependent 

decrease(Serum) 

No Rx effect Brain 

PND 15: Same as GO 19 

Non dose-depend. 

AChe! (total & 

soluble AChE) 1 hr 

PND4:CPF1 &' butnotat4&24 

Task 

Social Approach 

Social Novelty 

Open Field (SMA) 

Isolation-induced 

aggression 

Maternal Behavior 

Elevated Plus Maze 

Age atTest 

PND45 

Results 

l Socialcontact(Session 1) 

No RxEffect 

PNO 70(Ma!e5) l CPF 6 

PNO 75-80 

(Males) T Our Attack (GO CPF3) 

T Off posture (CPF6) 

Def post No Rx effect 

PNO 90 (Ferns) flicking dura & crouching (PN CPF 1 &3) 

PND 120 (M&F) 

Time in Open 

. !licking freq (PN CPF 1&3) 

arms T PN CPF3 Ferns 

Head Dipping 1 PNCPF 3 Males 

No overt signs of 

chlinergicintoxication 

at either age CPF 3 hrs Neonates 

Maternal Toxicity Pup Toxicity AChEI 

Weight losss in all 

NIA groups N.D. 

USV (Social) PNO 5,8, 11 

Homing (Olfactory) PNO 10 

locomotor Activity PND 25 

No Rx Effect CPF 1-4 or CPF 11-14 

No RxEffect CPF 1-4 orCPF 11-14 

No RxEffect CPF 1-4 orCPF 11-14 

Adolescents 

Adult 

Task 

Novelty Seeking PND 35 

Sociallnter'!lction PND 45 

Agonist Behavior PND 45 Males 

CPF1-4 

CPF 11-14 

CPF1-4 

CPF 11-14 

CPF 1-4 

CPF 11-14 

f Activity No Rx effect novelty seeking 

f Activity No Rx effect novelty seeking 

l Self Grooming 

T Soliciting resp. 

fAgonisticresp. 

fAgonisticresp. 

Passive Aviodance PND 60 Males CPF 1-4 No Rx effect 

CPF 11-14 No Rx effect 

Age atTest Results 

FOB (Functional Forelimb 

Observational Battery) 

Neuromuscular PND 70 

Grip Foot Splay 

I (F) (CPF 50) 

·Activity/reactivity 

Sensorimotor 

PND 17 

PND27 

T (F)(CPF 100) 

Total Activity Rearing Arousal 

I (M & F) 

10) 

liM &F) I(M&F) (CPF I(M&F) 

(CPF 25) 25) 50125) 

Hand!. React 

(CPF I(F) 

(CPF 20) 

(CPF I(F) 

(CPF 50) 

I(M & F) I (M & f) (CPF I(M & F) (CPF I(M) 

PN070 (CPf; 10/50) 50) 50/100) (CPF 100) 

Tail-pinch 

Response Click Response Touch Response 

I (M & F) I (M) (CPF 

PND 17 (CPF 10/4) 20) 

I (M & F) I (M) (CPF 

PNO 27 (CPF 50125) 25) l(F) (CPF 25) 

l (M & F) l (M & F) (CPF l (M) (CPF 

PNO 70 (CPF 10/4) 50) 50) 

Weight Temperature 

Physiological PND 17 

PND27 

PN070 

I( F) 

(CPF 25) 

1(M&F) 

(CPF 50) 

I(M&F) 

(CPF25) 

l(M&F) (CPF 

50) 
Article Species Exposure Period 

12 Carr et at, 2001 Rat(S-0) PND 1-21 

PND 1-5, 7-21 

Dose (mg/kg/day) RoA 

LOW:3/mg/kg 

(Every 2nd Day) 

MED: 

mgfkg/day, 

mglkg/day 

HIGH: 

mgfkg/day, 

mgfkg/day, 

3 

6 

3 

6 

12 

Gavage 

(com oil) 

PND 1-5,7-13, 15-21 mgfkg/day 

Article Species Exposure Period 

Rat (Long13 Jett et at, 2001 Evans) PND7,11,15 

PN~ 22,26 

14 Abou-Donla et at, 2006 Rat (S-0) GO 4-20 

Article Species Exposure Period 

Wild-type 

15 Lavlola et al, 2006 Reeler mice GO 14, 15, 16 

Onlydatafromwilcltypemice 

N.B.: are presented. 

Dose (mg/kg/day) RoA 

0.3 or 7.0 

0.3or 7.0 

1.0 

S.C. 

(peanut oil) 

Dermal in 

70% Ethanol 

Dose (mgfkgfday) RoA 

Osmotic mini 

pump 

5 mglkg CPF-oxon (DMSO): 

Inserted 

under neck 

skin of dam 

Maternal Toxicity Pup Toxicity AChE I Task Age atTest Results Heart: L;nt:l 

PND 6, 10, 16 (Lew Dose: PND 

20,25, 30: -50% 6,10,16,20). 

lnhihbition in (Med Dose: PND 

forebrain and 10, 16, 20, 25). 

l Body Weight, PND hindbrain in all 3 Rx (High Dose: PND 

N/A 13-21 (HIGH Dose) groups 16, 20, 25). Open Field Activity PND 10-20 No RxEffect 

r 

Maternal Toxicity Pup Toxicity 

N/A 

No overt signs of 

cholinergic toxicity 

No Overt signs of tocicity in dams or pups 

No difference in litter No differences in body 

Diaphragm: (Low 

Dose:PND 

Skeletal Muscle: 6,1 0,16). 

ChEI on PND 6 (Med Dose: PND 

·(Low Dose); PND 10, 16, 20). 

10, 16( Low & Med (High Dose: PND 

Doses) 16, 20, 25). · 

SerumAChEI: 

(Low Dose: PND 

6,10). (Med 

Lung: ChEI on PND Dose: PND 10, 16, 

6 (Low Dose); PND 20). (High 

10, 16,20 (Low Dose: PND 16, 

& Med Doses) 20). 

AChE! 

Cortex, 

hippocampus, 

cerebellum: PND 

7,8 16, 28 No ChEI due to 

Task 

(Pre/Post) CPF Rx Morris water maze 

Mucarinic Receptor 

Binding No Rx Effects 

PND 90: 

PND 25 & 30 

Age atTest 

PND 24-28 

l High& 

MedDoses 

M&F(Doserelated) 

Results 

Acquisition Preweaning CPF 7.0: tescape latency Day 5 

1 percentage of rats findinQ platform by Day 5 

Probe Preweaning CPF 7.0:1 time in platform training quadrant 

PND 24-28 

Acquisition Postweaning CPF 0.3 & 7.0: tescape latency Day 5 

!percentage of rats finding platform by Day 5 

' !timeinplatform 

Probe Postweaning CPF 0.3 & 7.0: training quadrant 

PND90 

Beam Walking Walk time: No Rx 

size weight Midbrain No Rx Effect (Coordination) Effect 

Maternal Toxicity Pup Toxicity 

None (WT only) None (WT only) 

Cerebellum 

Brainstem 

AChE! 

PND 10-12 

WTtypevaluse 

used as the 100% 

level 

t in AChE! (F) . 

t in AChEI (F) Inclined Plane 1 in fall angle (F) 

{Sensorimotor Reflexes) 

Forepaw Grip Time 

(Motor Strength) 

1 in grip time (M ~F) 

Task Age atTest Results 

Ultrasonic Vocalizations 

(Social Assessment) PND3, 7,11 NoRxeffect 

Grasping Reflex 

(Sensory/Motor) PND 3, 7,11 Tin fall angle 

Righting Renex (M~tor) PND 3, 7,11 NoRxeffect 

locomotor Activity: Open 

Field PND >70 No Rxetfect 

Scopc~amine Challenge PND >70 1 in Seep-induced locomotor stimulation 

Amphetamine Challgene PND >70 1 in Amph. induced locomotor stimulation