UNITED STATES ENVIRONMENTAL PROTECTIONAGENCY WASHINGTON D.C., 20460

OFFICE OF PREVENTION, PESTICIDES AND TOXIC SUBSTANCES

December 17, 2008
MEMORANDUM
SUBJECT:

TO:

FROM:

THRU: Transmittal ofMeeting Minutes ofthe FIFRA Scientific Advisory Panel Meeting Held September 16-18, 2008 on the Agency's Evaluation ofthe ToxicityProfile ofChlorpyrifos

Debbie Edwards, Ph.D. Director Office ofPesticide Programs
Sharlene R. Matten, Ph.D. Designated Federal Official FIFRA Scientific Advisory Panel Office ofScience Coordination and Policy
,1 Steven Knott, Executive Secretary

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Attached, please find the meeting minutes ofthe 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 ofthe ToxicityProfile ofChlorpyrifos.

Attachments

### cc: James B. Gulliford James J. Jones Anne Lindsay William Jordan Margie Fehrenbach Donald Brady JanetAndersen Steven Bradbury William Diamond Joan Harrigan-Farrelly Tina Levine Lois Rossi Richard Keigwin EnestaJones VanessaVu (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 BoardMembers 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-mayRuby Reed, Ph.D., DABT Sonya K. Sobrian, Ph.D. Heather A. Young Durick, Ph.D., MPH

# SAP Minutes No. 2008-04

A Set ofScientific Issues Being Considered by the Environmental ProtectionAgency Regarding:

The Agency's Evaluation ofthe Toxicity Profile of Chlorpyrifos

September 16-18, 2008 FIFRA Scientific Advisory PanelMeeting held at the Holiday Inn

- Rosslyn Arlington, VirginiaNOTICE
These meeting minutes havebeen written as part ofthe activities oftheFederal Insecticide, Fungicide, and Rodenticide Act (FIFRA), Scientific Advisory Panel (SAP). The meeting minutes represent the views and recommendations ofthe FIFRA SAP, not the United States Environmental Protection Agency (Agency). The content ofthe meeting minutes does not represent information approved or disseminated bythe Agency. Themeetingminutes have not been reviewed for approval bythe Agency and, hence, the contents ofthesemeeting minutes do not necessarilyrepresent the views and policies ofthe Agency, nor ofother agencies inthe Executive Branch ofthe Federal government, nor does mention oftrade names or commercial products constitute arecommendation for use. ·
The FIFRA SAP is a Federal advisory committee operating in accordance with the Federal Advisory Committee Act and established under theprovisions ofFIFRA as amended bythe Food Quality Protection Act (FQPA) of 1996. The FIFRA SAP provides advice, information, and recommendations to the Agency Administrator on pesticides and pesticide-related issues regarding the impact ofregulatory actions onhealth and the environment. The Panel serves as the primary scientificpeerreview mechanism ofthe Environmental Protection Agency, Office of Pesticide Programs (OPP), and is structured to providebalanced expert assessment ofpesticide and pesticide-related matters facing the Agency. FQPA Science Review Board members serve the FIFRA SAP on an ad hoc basis to assist inreviews conducted bythe FIFRA SAP. Further information about FIFRA SAP reports and activities canbe 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 byEPA, as well as informationpresented in public comment. This document addresses the informationprovided and presented byEPA within the structure ofthe charge.

2TABLE 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 VAILABLE FOR RISKASSESSMENT

................................................... 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 ofScientific Issues Being Considered by the EnvironmentalProtection Agency Regarding:

The Agency's Evaluation ofthe Toxicity Profile of Chlorpyrifos

September 16-18, 2008 FIFRA Scientific Advisory PanelMeeting held atthe 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(Df?

### FederalInsecticide, Fungicide, andRodenticide Act Scientific Advisory PanelMeeting September 16-18,2008

TheAgency's Evaluation ofthe Toxicity Profile ofChlorpyrifos

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 PanelMembers
John R. 

### Bucher, Ph.D., DABT, Associate Director, National Toxicology Program, National Institute ofEnvironmental 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 ofVeterinary Medicine, Mississippi State University, Mississippi State, MS Kirby C. Donnelly, Ph.D., Professor and Head, Department ofEnvironmental and Occupational Health, School ofRural Public Health, Texas A&M University System Health Science Center, College Station, TX

. ··Carey N. Pope, Ph.D., Professor, Head & Sitlington Chair ofToxicology Department ofPhysiological Sciences, Oklahoma State University College ofVeterinary Medicine, Stillwater, OK


### Daniel Schlenk, Ph.D., Professor, Aquatic Toxicology, Department ofEnvironmental Sciences, University ofCalifornia, Riverside, CA

5FQPA Science Review Board Members Sophie J. Balk, M.D., Attending Pediatrician, Professor ofClinical Pediatrics, Children's Hospital at Montefiore, Albert Einstein College ofMedicine,Bronx, NY

Laura Beane Freeman, Ph.D., Occupational and Environmental EpidemiologyFellow, 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 ofVeterinaryMedicine, 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 ofPublic Health, Richmond, CA

John Doull, M.D., Ph.D., ATS, Professor Emeritus ofPharmacology & Toxicology, Department ofPharmacology, Toxicology & Therapeutics, University ofKansas Medical School, Kansas City, KS

Paul B. English, Ph.D., M.P.H., Scientific Advisor, Environmental Health Investigations Branch, California Department ofPublic Health, Richmond, CA Gaylia Jean Harry, Ph.D., Biologist, Head ofNeurotoxicology Group, Division ofIntramural Research, National Institute ofEnvironmental Health Sciences, Research Triangle Park, NC Wendy J. Heiger-Bernays, Ph.D., Associate Professor, Department ofEnvironmental Health Boston Uni\rersity School ofPublic Health, Boston, MA

Antonio Hernandez Jerez, M.D., Ph.D., Senior Lecturer in Toxicology, Department ofLegal Medicine and Toxicology, University ofGranada, Granada, SPAIN

Bette Meek, M.S., Associate Director ofChemical Risk Assessment, McLaughlin Institute for Population Health Risk Assessment, University ofOttawa, Ottawa, Ontario, CANADA

John D. Meeker, Sc.D., CIH, Assistant Professor, Department ofEnvironmental Health Sciences, University ofMichigan School ofPublic Health, Ann Arbor, MI

Andrew C.Povey, Ph.D., Reader in Molecular Epidemiology, Occupational & Environmental Health Research Group, FacultyofMedical and Human Sciences University ofManchester, Manchester, England, UNITED KINGDOM Nu-may Ruby Reed, Ph.D., D.A.B.T., StaffToxicologist, Department ofPesticide Regulation, California EPA, Sacramento, CA

6Sonya K. Sobrian, Ph.D., Associate Professor, Department ofPharmacology, Howard University College ofMedicine, Washington, DC

Heather A. Young Durick, Ph.D., MPH, Associate Professor, Department ofEpidemiology and Biostatistics, The George Washington University, Washington, DC

7INTRODUCTION
The Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) Scientific Advisory Panel (SAP) has completed its review ofThe Agency's Evaluation ofthe Toxicity Profile of Chlorpyrifos. Advance notice ofthe SAP meeting was published in theFederalRegister on July9, 2008 and August 28, 2008. Thereview was conducted in an openpanel 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 ofPesticide Programs (OPP), provided opening remarks at the meeting. Presentations oftechnical background materials were provided byDr. Anna Lowit and Ms. Deborah Smegal, MPH, Health Effects Division, OPP and byDr. Ginger Moser, EPA-ORD-National Health and Environmental Effects Research Laboratory (NHEERL). Additional technical assistance was provided byDr. John Liccione and Dr. John Doherty ofthe Health Effects Division, OPP. Chlorpyrifos (0,0-diethyl-0-3,5,6-trichloro -2-pyridinyl phosphorothioate) is abroad-spectrum, chlorinated organophosphorus (OP) insecticide. Chlorpyrifos is one ofthe most widely used OPs in the U.S. In 2000, nearly all residential uses were voluntarily cancelledbyDow AgroSciences, LLC. However, chlorpyrifos continues to beused extensively in commercial agriculture. Since 2000, there has been extensive research on various aspects ofchlorpyrifos 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 beenpublished from three cohorts ofpregnant 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. Theultimate goal ofthe 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 ofrelying onnoobserved-adverse-effect levels (NOAELs) for points ofdeparture; and using extrapolation factors based on datainstead ofrelying on default factors to account for differences in animals and humans and among humans. The Agencyhas progressed to a point in the review that feedback from the FIFRA SAP wouldbehelpful.

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 ofEpidemiology, 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. RayMcAllister, Crop Life America 10) Elliot Gordon, Ph.D., Elliot Gordon Consulting, LLC 11)Jennifer Sass, Ph.D., Natural Resources Defense Council (NRDC) and on behalfof 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 ofPublic Health, ColumbiaUniversity


### 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 ofWashington on behalfofthe authors ofEaton et al. 2008 4) Theodore Slotkin, Ph.D., Professor ofPharmacology and Cancer Biology and Psychiatry and Behavioral Sciences and Neurobiology and Director ofGraduate Studies, Integrated Toxicology and Environmental Health Program, Duke UniversityMedicalCenter 5) Scott Phillips, M.D., Department ofMedicine, University ofColorado Health Sciences Center 6) PamelaMink, Ph.D., MPH, Department ofEpidemiology, EmoryUniversity 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, ATS, Bernard Gadagbui, Ph.D., DABT, and Lynne Haber, Ph.D., DABT, Toxicology Excellence for Risk Assessment (TERA)

9SUMMARY OF PANEL DISCUSSION AND RECOMMENDATIONS

The Panel acknowledged the extent ofthe chlorpyrifos database and coinmended the Agency for preparing a comprehensive document considering the scientific evidence as a whole. Some of the more significantnew data available to the Agency since the preparation ofthe 2000 chlorpyrifos risk assessment comes from three largeprospective cohort studies ofpregnant women and their children. Throughout the two-day discussion,**~anel** members referred to the results ofthese studies in an attempt to integrate their findings with the toxicology data from laboratory animal experiments. Despite the large volume ofdata available to address the risks of chlorpyrifos exposure from agriculturaluses, several Panel members were concerned that a high degree ofuncertaintyis evident in the available data, particularlyunder low dose, chronic conditions. Uncertainty was expressed in attributing observed adverse effects to chlorpyrifos in the epidemiological studies where exposure was to metabolites ofchlorpyrifos orto 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 ofthe point ofdeparture (PoD). There was extensive discussion on experimental and epidemiological studies, e.g., oflow-dose exposure in animals, ofexposures in agricultural and pesticide handlers, and ofthe mode ofaction including the need to develop chlorpyrifos-specific PBPKmodels, that could provide the information needed to address critical data gaps and reduce uncertainty.

1. Metabolism & Toxicokinetics

The Panel concluded that a weight ofevidence evaluation ofavailable information supports the Agency's conclusions that 1) sensitivityto the adverse effects ofchlorpyrifos is influencedby 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 mostlyto toxicokinetic (TK) differences betweenjuveniles and adults instead ofonlybeing 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 ofthe variability seen in these factors, butthat otherpotential TK differences betweenjuveniles and adults should notbe dismissed. For instance, the highrespiratoryrate ofchildren may enhance the absorption ofchlorpyrifos present in the air

..Children have a greater cardiac output compared to adults, together with their less developed blood-brainbarrier; facilitate some ofthe chlorpyrifos-oxon reaching the brainS ofexposed infants.

Some panel members questioned whether detoxication ofthe oxon plays a significant role in explaining the differential susceptibilitybetween adults andjuveniles at lower levels of chlorpyrifos exposure. Toxicokinetic differences are less likelyto berelevant under low dose conditions compared to high dose conditions where enzyme systems maybecome saturated. Human tissue data specific to enzyme-mediated detoxication are minimal, however. While blood levels ofdetoxifying enzymes (e.g., PONt, carboxylesterases) havebeen studied, data on chlorpyrifos detoxication in specific organs (e.g., the liver) from humans are limited, making it

10difficult to draw firm conclusions about the role ofactivation and detoxication in explaining differences in susceptibilityto chlorpyrifos toxicitybetweenhuman infants, children, and adults.

The Panel also agreed with the Agency's conclusion that "pregnant animals and humans maybe somewhat more sensitive to chlorpyrifos thannon-pregnant adults" based on the expected role of key detoxication enzymes (e.g., paraoxonase, P450 isozymes) inmodulating levels of chlorpyrifos in animal studies. However, the Panel noted that there are relatively small differences inlevels ofthese key enzymes betweenpregnant and non-pregnant animals. At dose levels expected from environmental exposures the importance ofsuch small differences in enzyme levels is unknown, but the data suggest a reduced capacityto 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 ofthejuvenile animals' lower capacityto detoxify either chlorpyrifos orthe chlorpyrifos-oxon. The Panel was uncertain about how to interpret the data describing differences in cholinesterase inhibitionbetween 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 ofenzymes capable ofconverting 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 ofinhibition
over time than in adults. However, the role ofthe activation and detoxication pathways in producing differential cholinesterase inhibition and cholinergic effects at low dose repeated exposures is not clearly defined bythe available data.

The Panel agreed with the Agency's preliminary conclusion that data showing less cholinesterase inhibition after repeateddosing during gestation maynot reflect the true potential toxicityto the fetus. The data indicate, however, that the level ofcholinesterase inhibition depends onthe time ofsampling following exposure. Developing rats would be expected to show less cholinesterase inhibition than would adults, with increasing length oftime ofmeasurements vs. dosing. Two mechanisms may explainthis phenomenon: 1) a higher rate ofrecovery ofthe 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 inrate ofsynthesis ofnew enzyme molecules is likelythe dominant factor. A more accurate comparison ofcholinesterase inhibition between the developing animal and the adult couldbe determined ifsample collection coincided with the time ofthe peak cholinergic effect.
3. Laboratory Studies on the Developing Brain

In general, the Panel agreed with the Agency's conclusion that gestational or earlypostnatal exposures can lead to neurochemical and behavioral alterations that persist into adulthood. A number ofrodent studies suggest that prenatal orpostnatal exposures maylead to long-term

11neurobehavioral changes in motor and cognitivebehaviors. The Panel**question~d** the meaning of the phrases "at sufficientlyhigh exposures" and "persisting into adulthood after any initial AChE inhibition has reversed." Somepanelists stated that a "sufficientlyhigh exposure" shouldbe defined on the basis ofa dose that induces brain cholinesterase inhibition. Theynoted that the majority ofexperimental animal studies reviewed bythe Agencyused doses ofabout 1mg/kg. This dose (exposure level) should be sufficient to inhibitbrain cholinesterase activity and at the
same time causes neurobehavioral effects in most age groups.
Some members questioned the experimental methods used in some ofthe animal studies as well
as the interpretation and application ofthe results ofneurobehavioral testing in animals for risk assessment. Several Panel members felt that whilebehavioral findings are consistentlyreported in the literature, the types ofbehavioral activities reported as significant were not always the
same. Itwas acknowledged that the study outcomes could be affected by 1) the route of administration ofchlorpyrifos, 2) the developmental period ofexposure, 3) the methods used to
measure changes inbehavioral domains, and 4) the choice ofdependent variables. Panel members agreed with the Agency's expressed caution onthe use ofdimethyl sulfoxide (DMSO)
as a vehiclebecause ofits 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 ofthis interaction onthe developing animal.

The Panel agreed with the Agency's determination that insufficient data exist to clearlyidentify a specific mode ofaction for effects in the developing nervous system for use inrisk assessment. Somepanel members believed that studies cited by Eaton et al. (2008) were indicative of possible non-cholinergic modes ofaction. Otherpanel members disagreed with this conclusion. They stated that the effects onblood cholinesterase are considered protective for the observed effects (i.e., occurring at lower doses); however, nothing is implied about mode ofaction, except in the context ofspecies and interindividual variabilityin toxicokinetics. Most ofthe Panel stated that the available information does not allow behavior to be considered as a point ofdeparture. These panelists stated that cholinesteraseinhibition should continue to beused for PoD until, at such time; an alternative mode ofaction is identified and validated. Collaboration between EPA and developmental neurotoxicologists would help identify enzyme inhibition occurring in discrete brain sites at critical periods ofdevelopment.

4. Epidemiology Studies in Children and Mothers

The Panel agreed with the Agency's conclusions regarding the potential utility ofthe epidemiological data from the three cohort studies (ColumbiaUniversity in New York, Mt. Sinai Hospital inNew York, and the University ofCalifornia at Berkeley) inbroadly characterizing risk ofchlorpyrifos exposure to pregnant women, fetuses, infants, and children. They also concurred that these studies should notbe considered as the principal basis for characterization ofthe PoD. The Columbia study was considered the most epidemiologically-sound and to have adequately addressed selection and information biases to the best extent possible. Itwas also considered the mostrobust and appropriate in assessing chlorpyrifos developmental toxicity because specific measurements ofexposure to chlorpyrifos in neonates and children (i.e., the studypopulations) were obtained. Confounding factors in the Mt. Sinai and Berkeley studies, particularlythe fact that exposures werebased on OP and**carbamat~** metabolites and that



### 12chlorpyrifos was not specificallymeasured, reduce their utilityin 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 wouldbe considered a**"low'~** exposure for a population ofagricultural workers orpesticidehandlers.

The weaknesses inherent to observational studies were discussed. Given thepaucity ofhuman epidemiological studies ofOP insecticides in children, the Mt. Sinai and Berkeley studies do provideuseful 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 mayhave relevance for human toxicity. There is uncertainty in the exposure estimates in the Columbia studybecausemeasurements were derived from a single time point

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

from the Columbia studyraise concerns for adverse neurodevelopmental effects in children exposed in utero to environmental levels ofchlorpyrifos although there was confoundingbythe presence ofother anticholinesterase insecticides. The majority ofissues raised bythe Panel were points ofclarification or issues that ifaddressed could strengthen the analysis and provide the Agency withbetter and more useful data for risk assessment. Overall, the Agency's conclusion that chlorpyrifos could have contributed to the birth and neurodevelopmental outcomes noted inthe three cohort studies was supported. The Panel agreed with the Agency that: a) exposures to multiplecholinesterase-inhibitinginsecticides in combination cannotberuled out as contributing to thebirth and neurodevelopmental outcomes; b) a potential synergistic and/or additive effect ofthese compounds does not rule out a role for chlorpyrifos in contributing to the adverse outcomes; and c) it cannotbe determined if chlorpyrifos is the sole contributorto the observed outcomes. These conclusions are supported bythe Columbia study finding ofa crude dose-response relationship to chlorpyrifos levels which persisted even after controlling for exposure to otherpesticides.

When the results ofthe three cohort studies (with an emphasis onthe Columbia study) are considered along with the findings from experimental studies in animals, the Panel concluded that maternal chlorpyrifos exposure would likelybe associated with adverse neurodevelopmental outcomes in humans. Some panelists indicated that ifthe associations between chlorpyrifos exposure and the reported outcomes are causallyrelated, then it is possible to conclude that the mechanism ofaction mightbe independent ofcholinesterase inhibition. Exposures to multiple cholinesterase-inhibiting pesticides or other neurotoxicants mayresult in additive orinteractive effects. This information mightbeuseful when consideringcumulative exposures to these pesticides.

5. Human Information Available for RiskAssessment

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 dataprovide sufficient evidence to suggest that the uncertainty factor for the cholinesterase inhibition endpointbe changed to accommodate the possibility ofneurodevelopmental effects from low-level in utero exposures ofchlorpyrifos. The

13majority ofthe 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 ofneurodevelopmental effects. However, somepanel members felt stronglythat the current epidemiological data do provide evidence to indicate that the margin ofsafety should be increased. The Panel recommended that the Agency conduct a full formal weight ofevidence evaluation for causality ofthe reported associations between exposure to chlorpyrifos and neurodevelopmental outcomes in the existing epidemiological database.

The Panel recommended that the Agency continue its collaboration with ColumbiaUniversity researchers in analyzing the epidemiological data. The Agencyis encouraged to continue open discussions with the studyresearchers (and the Centers for Disease Control and Prevention, as appropriate) to seek clarification on the level ofconfidence in the reported exposure levels. The Agency should then attempt to use the cohort data quantitatively to inform therisk assessment process, such as in aboundary setting exercise.

The use ofphysiologically-based pharmacokinetic (PBPK) modeling would enable estimation of exposure dose metric for multiple sources ofexposure, e.g., air, food, water. The Panel also agreed with the concept ofusing a PBPKmodel to examine individual intra-species variations as well as inter-species differences. While the potential contribution ofthe chlorpyrifos PBPK model being developed was recognized, some panelists believed that the Agency should also pursue a simplerPBPK model specifically applicable to the chlorpyrifos data that wouldbe available in a relatively short timeframe. The Panel concluded that the epidemiological datamay beused for bounding exposure levels, and in conjunction with PBPKmodels, address current or potential human exposures and to determine the final reference dose orreference concentrations.
The Panel agreed with the Agency's approach to deriving a dermal absorption factor using data from the deliberate dosing inhuman studies. This approach is especiallyvaluable ifno other
sources ofdata are available. However; the current3% absorption factor shouldbe further refined by considering different exposure scenarios and adjusting for expected underestimation ofexposure. The Panel also agreed with the Agency's scientific analysis that these delibe:J;"ate dosing studies cannotbeused to directly establish aPoD orUFs, butindicated that these data mightbe used as bounding levels similar to what was suggestedbythe Panel concerning the data from the epidemiological studies.



### 6. Points ofDeparture (PoD) for Risk Assessment

The Panel was presented with three options for deriving points ofdeparture for acute and chronic chlorpyrifos exposure from animal studies for extrapolating human risk. Panel members expressed difficulty in separating the discussion ofpoints ofdeparture from that ofuncertainty factors with the result that the discussion ofthese two issues overlapped in many areas.
The Panel agreed that Option 3 was the most favorable option for deriving points ofdeparture 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 ofdeparture for these exposure routes, taking into account sensitive life stages and adjusting for dosing regimen. Option 3 was chosen principally on the basisthatthe proposed point ofdeparture for acute exposure takes into account all life stages, is based on



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

Based on available data, most ofthe Panel members (a few members disagreed) stated that the PoD presented in Option 3is also believed to beprotective for effects onthe developingbrain, although itis based on cholinergic effects. :tJowever, this conclusionhas associated uncertainties; the lack ofinformation on the mode ofaction in inducing the observed behavioral effects and evidence from in vivo and in vitro studies that show non-cholinergic modes ofaction
are likelyto 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) ofaction following longer term exposure, and the need to account for potential cumulative effects support the lowerpoint ofdeparture for repeated exposures usedbythe Agencyin its 2000 risk assessment. The Panel also recommended thatthe Agencyinvestigate the possibilityofconducting a benchmark dose assessment for the chronic point ofdeparture determination.

The Panel recommended that the Agency consider development ofan appropriate studyusing 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 likelyhelp elucidate non-cholinergic modes ofaction for developmental neurotoxicity and behavior effects.

I 

### 7. Extrapolation/Uncertainty Factors

Afterreview ofavailable data, the Panel concurred with the Agency that paraoxonase-1 (PONI) status cannotberuled out as a determinant ofchlorpyrifos toxicity, particularly for the fetus and the young child. Actual human exposures varybetweenbystanders and applicators (and everyone in between) and it would be difficult to define what constitutes a low level ofexposure in humans. The Panel disagreed on whether the PONI data alone couldbeused to address uncertainty. The majority ofpanel indicated that these data shouldnotbeused out ofcontext until rate limiting step(s) is/are identified. The use ofthe PONI data without such information
was consideredby some to be a misinterpretation ormisuse ofthe IPCS guidance for determining chemical-specific adjustment factors (CSAFs). ·
Some ofthe Panel indicated that chlorpyrifos may,produce effects in the fetus that would be manifest later at thejuvenile stage orlater oh in life(e.g. adulthood). They stated that adverse neurodevelopmental effects inthe fetus, neonates, and young children shouldbe considered the most important endpoints for assessing chlorpyrifos toxicity. The Panel noted that there does
seem to be a different susceptibilitybetween fetuses and neonates compared to adults. Because specific modes ofaction 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 bythe Panel..

15The Panel noted the uncertainties surrounding the role ofPON1 andits genetic polymorphisms

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

emphasized the .lack ofdata in animals and humans on the involvement ofPON1 in detoxication pathways, especiallyin situations oflow dose exposures. Most ofthe Panel encouraged the Agencyto use PBPKmodeling to assess the overall impact ofPON1 on toxicity over a range of chlorpyrifos exposures. While otherpanel 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. Theinformation on the overall contribution ofPON1 Q192Rpolymorphism on the deactivatingpathways and potentiallyratelimitanting components will beveryuseful when examined in light offuture PBPKmodels.

The Agencyproposed two options for deriving an intra-species TK (human variation) uncertainty factor: 1) a 12-fold data-derived factor based onthe PON1 data or 2) the default three-fold factor. The majority 1ofthe Panel supported the use ofthe 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 

### acomplex metabolic pathway and that the PON1 genotype alone is insufficient to predict human variability. Onepanel member added that ifchlorpyrifos acts directly on certainbrain 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 wouldbe irrelevant. On the otherhand, 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 ofchlorpyrifos as shown in the epidemiological studies. These members preferred the UFHK of 12-fold, rather than the default of 3-fold, because these were the onlytwo <;hoices proposed. However, none ofthepanel members endorsed the CSAF approach used bythe Agencyto identifythe factor of"12-fold" calculated based on chlorpyrifos-oxonase and encouraged the Agency to pursue other approaches based on the mode ofaction (i.e., 2005 IPCS CSAF guidelines).

The Panel did notreach consensus on one specific uncertainty factor for inter-species TK differences. Most ofthepanel concurred with the Agency's proposal to stay with the default three-fold factor for inter-species TKdifferences and add no additional uncertainty factor for developmental and behaviorneurotoxicity. These panel members noted that the most sensitive effect appears to beAChE 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 observablebehavioral effects in the offspring. On the otherhand, 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 onthe cholinesterase inhibition endpoint and further considerthe use ofan additional uncertainty factor to address the concerns for developmental and behavioral neurotoxicity as observed in both the animal and epidemiological studies.

16The Panel generally favored the use ofa PBPKmodel to integrate keyTK and TD factors and evaluate their contributions to the endpoints ofinterest**(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 bevalidated against data from deliberate dosing studies inhumans and epidemiological studies. The Panel encouraged the Agencyto continue examiningthe importanceofall 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 TOCHARGE QUESTIONS
1. Metabolism & 

### Toxicokinetics (Issue Paper Section 3.1, Appendix A):

The Agencyhas performed a literature review ofin vivo and in vitro studies on the metabolic profile and toxicokinetic (TK) properties ofchlorpyrifos with particular focus on age-dependent and lifestage sensitivity.

a. The Agencyhas concluded that age-dependant sensitivity, at least in part, is derived based
ontoxicokinetic (TK) differences betweenjuveniles and adults. These TKdifferences lead to reduced ability to detoxify chlorpyrifos orthe ox oninjuvenile animals. Please comment
on theAgency 's conclusion andthe scientific support for or against this conclusion.


### Panel Response
The Panel supported the Agency's conclusion. A weight ofevidence evaluation ofthe available information shows that sensitivityto the adverse effects ofchlorpyrifos is influencedby 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 TKdifferences between juveniles and adults rather than soley on TD differences.
I Chlorpyrifos is activated byoxidative desulfuration tothe 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 occurto 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 ofchlorpyrifos. They also indicated that other potential TK differences betweenjuveniles and adults should notbe dismissed. For instance, it

17was mentioned that the high respiratoryrate ofchildren enhances their absorption ofany chlorpyrifos present in the air. Childrenhave a higher skin surface-to-weightratio that favors dermal absorption and a higherpermeability ofthe small intestine epithelium that facilitates absorption bythe oral route. Therelatively greater cardiac output of juveniles, together with their less developed blood-brain barrier may facilitate some ofthe circulating chlorpyrifos-oxon eventuallyreaching the brain. Childrenhave a greater cardiac output compared to adults, facilitating some ofthe chlorpyrifos-oxon reaching the brain. Theblood-brainbarrier in humans is not completeuntil 6 months ofage (Rodier, 2004), facilitating some ofthe circulating chlorpyrifos-oxon in reaching the brains ofexposed infants Polymorphisms in several ofthe enzymes involved in chlorpyrifos metabolism can lead to variabilityin an individual's ability to detoxify the chlorpyrifos-oxon. Some enzymes are also inducible which may also contribute to variabilityinresponse to chlorpyrifos (and its .metabolites). Most ofthe studies evaluatingbiotransformation capacity with acute sensitivity are correlative in nature, however, and are notmechanistic. While isoform expression ofcytochrome P450 (CYP450) canbe markedly different betweenjuvenile and adult animals, the relative roles these isoforms playin sensitivityto 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 andjuveniles is unknown, but may differ as a function ofage.
Choi et al. (2006) stated thatthe adult (human) liverplays an important role in detoxication of chlorpyrifos and that in the liver the oxon does not accumulate to be released into the bloodstream. Hunter etal. (1999), however, suggested that the fetal liver does not play a significant role in detoxication. Some·studies suggest that chlorpyrifos oxon 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 ofchlorpyrifos, CYP/PON1 in the intestine and liver may effectivelyremove the
oxon from circulation prior to systemic exposures. Bycontrast, individuals with lower levels and/or activity ofthe detoxication enzymes mayhave 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 andjuveniles are more readily apparent. Howeverthese saturating conditions are less likelyto occur at environmental exposure levels. Ifthe detoxication mechanisms are not saturated, then their effectiveness in adults andjuveniles maynotbeas different as experimental animal data withhigh exposures suggest. The lack ofavailable data on maturational differences in tissue (non-blood) detoxication capacitybyA-esterases and otherpathways limits extrapolation ofrelative sensitivityininfants based on differential detoxication.

Timchalk et al. (2006) reported that the rate ofdetoxication (measured as the formation ofthe chlorpyrifos specific metabolite, 3,4,6-trichloro-2-pyridinol, referred to as TCP) exceeded the rate ofactivation ofchlorpyrifos (measured as chlorpyrifos oxon formation) inrats across age groups from 5 days to adulthood, with exposures ranging from 1-10 mglkg. This would suggest that at similar exposures, inactivationprocesses may outweigh the net activation. Athigher exposures, inactivation mightbecome compromised. At low chlorpyrifos concentrations, the formation ofnon-toxic metabolites is highly favored in the fetus (Buratti et al., 2006).

18In evaluatingthe role oftoxicokinetics and more specificallythe lower detoxication potential in juveniles compared to adults, the Panel proposed that the relativelyinefficientbioactivation reaction, especiallyinjuveniles, would not likely lead to high concentrations ofthe oxon at environmental exposure levels. The detoxication enzymes would notbe at saturating substrate concentrations for efficient inactivation. Age differences in toxicokinetics are likely to be important at high dosage levels, but less likely to berelevant at lower dosages where enzymes are not saturated.

The two detoxication pathways studied the most, inrelation to age-related differences in sensitivity; are carboxylesterases and A-esterases (primarilyparaoxonase-1 referred to as PON1). Specific activities ofliver carboxylesterases (CarbE) increased with age in male rats (Atterberry et al., 1997) which indicated the presence ofmore protective esterases in the adult as compared to thejuvenile animal. The lower carboxylesterases activity in blood and tissues injuveniles 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 nothave carboxylesterases in the plasma (Li et al., 2005) and some data suggestrelatively 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 (Sogorbet al., 2008), and is capable of protecting AChE (acetyl cholinesterase) "invitro" from inhibition at low concentrations ofthis 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 ofBChE to the metabolism ofchlorpyrifos-oxon in humans remains to be determined. This finding canbe explained bythe high carboxylesterase activity in mouse plasma. Given that carboxylesterase is lacking in human plasma, the protective role ofBChE cannotbe disregarded.

The Panel was not aware ofdata specificallydemonstrating that chlorpyrifos canbebioactivated in the brain, the main target organ ofthis compound. However, there are data from "in situ" experiments in rats that showbioactivation ofparathion (another insecticide ofthe 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 bioactivatedbythe same CYP450s. Since chlorpyrifos is bioactivated less efficientlythan parathionbyrat liver microsomes (Ma and Chambers, 1994, 1995), it is certainlypossible that bioactivation ofchlorpyrifos 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). Thebrain desulfuration activities ofthese phosphorothioates generally correlate well with the toxicity and maybeimportant in determining their overall acute toxicity levels (Chambers and Chambers, 1989). Thus, extrahepatic sites ofactivation likelyplay an importantrole inmediating the acute toxicity ofchlorpyrifos (Sultatos et al., 1984).

PON1 is differentially expressed throughout maturation with lower levels in younger animals being associated withhigher acute sensitivity (Mortensen et al., 1996; Li et al., 1997; Karanth

19and Pope, 2000). The Panel noted that there is less information suggesting that human infants are more sensitive than adults to chlorpyrifos toxicitybecause ofage differences in PON1-mediated detoxication. In humans, blood A-esterase is very low at birthand increases for the first two years oflife'(Augustinsson and Barr, 1962; Ecobichon and Stephens, 1973; Burlina et al., 1977)
orup to 4 years according to more recent data from large cohorts (Holland et al., 2006).

Errors in the metabolism section ofthe Agency'sbackground materials provided to the FIFRA SAP for this review were pointed outbyone Panel member.

b. There are limited data onthe**meta~olic** capacity ofpregnant animals and pregnant humans. Theselimited data on metabolism are supported by some toxicity data in rats. The Agencybelieves these studies suggest thatpregnant animals and humansmaybe somewhat
more sensitive to chlorpyrifos than non-pregnant adults to chlorpyrifos. Please comment on theAgency'spreliminary conclusion and the scientific support for oragainst this conclusion.

Panel Response
The Panel agreed with the Agency's preliminary conclusion that "pregnant animals andhumans maybe somewhat more sensitive to chlorpyrifos than non-pregnant adults" based onmetabolic capacity and their effect on levels ofkey enzymes in modulating chlorpyrifos toxicity. However, panel members noted that onlyrelatively small differences in levels ofthekeydetoxication enzymes involved in chlorpyrifos metabolism havebeen reported. Some panel members recommended that a direct comparison ofthe metabolic capacity ofpregnant versus nonpregnant females beperformed. Otherpanel members strongly disagreed with this recommendation.

The Panel noted that it is not clear whypregnant females wouldbemore susceptible to the adverse effects ofchlorpyrifos than non-pregnant females. The information on toxicity of chlorpyrifos in pregnant animals is limiteq and the data onbiotransformation ofchlorpyrifos in pregnant females are inadequate. Reports from the American Association ofPoison Control Centers (AAPCC) do not suggest increased susceptibility ofpregnant women to chlorpyrifos and other OPs. Thephysiological 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 mayimpact liver and serum enzymes involved in chlGrpyrifos metabolism that contribute to some extent in the variability ofactivity observed during pregnancy. Most ofthe gestational studies published so far report that there is greater concern for the fetus rather t)1an the dam. These studies areinadequate to determine whetherthe 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 sensitivitydoes not seem to occur when pups are repeatedly exposed to low doses. In fact, when cholinesterase activity, muscarinic receptorbinding, and motor activity changes in response to the antimuscarinic drug scopolamine were compared injuvenile 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 ofacetylcholinesterase molecules in tissues from younger animals



### 20between exposures, thus leading to lesser accumulation ofinhibition across exposures inthe younger animals.

The Panel discussed the biological significance ofthe possible changes in metabolic activity during pregnancyrelative to expected toxicology outcomes. Inhumans, the main three CYP450 isozymes thatmetabolize chlorpyrifos are CYP2B6, CYP2C19, and CYP3A4. The net effect of these isozymes on thebalance ofdesulfuration/dearylation is unknown since CY,P2B6, CYP3A4 and CYP1 A2 promotethe activation ofchlorpyrifos to chlorpyrifos-oxon whereas CYP2C19 detoxifies the oxonbymeans ofa dearylation reaction. Whereas, CYP3A4 increases during pregnancy, CYP2C19 decreases (Anderson, 2006) and no data on CYP2B6 inpregnant women are available. Pregnancy-induced decreases in levels ofthe CYP2B1/2 protein,which is the orthologous form ofhuman CYP2B6, has beenreported in rat liverbut this is not accompanied by a statistically significant decrease in pentoxyresorufin 0-dealkylase (PROD) activity (Czekaj et al., 2000).

Review ofthe literature indicates that there appears to be a reduction inthe activities ofseveral enzymes potentially important inthe detoxication ofchlorpyrifos associated with pregnancy; including PON1, carboxylesterases andblood cholinesterases. The Panel agreed with the Agency that the "importance ofthe decreases (in detoxifying enzymes) is unknown at environmental exposures." Although the importance ofthese decreases under low environmental exposures is not known, decreases during pregnancymayindicate a corresponding reduced capacityto detoxify chlorpyrifos. While the enzymes mentioned above are expected to have lesser (orperhaps no significant) role in modulating toxicitywith low level exposures, the limited evidence does suggest a potential for an overall reduced capacity to detoxify chlorpyrifos duringpregnancy.
2. Cholinesterase Inhibition (Issue Paper Section 3.2, Appendix B):

The Agencyhas reviewed numerous studies submitted for pesticide registration and from the literature in animals and human on the AChE-inhibiting effects ofchlorpyrifos in blood and in theperipheral and central nervous system.

a. Regarding inhibition ofAChE, the Agencyhas preliminarily concluded that post-natal studies inrat support the conclusion thatjuveniles are more sensitive than adults. The Agency has further concluded that sensitivityis greatest inyoungerpups and decreases as pups mature towards adulthood: Please comment on theseAgency'spreliminary conclusions and the scientific support for oragainst these conclusions.

Panel Response

The Panel agreed with the Agency's conclusion thatpost-natal rats are more sensitive than adult rats with respect to increased AChE inhibition and lethality following acute exposures. A · number ofstudies have consistently shown thatjuvenilerats are more sensitive than adults to cholinesterase inhibition and cholinergic signs oftoxicity following acute doses ofchlorpyrifos exposure. In addition, several studies show a gradient ofsensitivityto acute chlorpyrifos inrats during postnatal maturation. This greater sensitivity appears due to a lower detoxication capacity and as these capacities develop, the rats becomemore aligned with the lower adult sensitivity.

21However, this. greater sensitivitymaybetrue mainly for high dosages because ofthe saturation ofthe detoxication mechanisms. This difference in sensitivity (measured as acetylcholinesterase inhibition) betweenjuveniles 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 thatphysiologically-basedpharmacokinetic/pharmacodynamic (PBPK/PD) models are likelyto be ofgreatest value inpredicting such age-related differences over a span ofdose levels.

The Panel, however, did not support the suggestion that post-natal rats are more sensitive than adult rats underrepeated exposures. The timing ofthe measurement ofcholinesterase inhibition,
as well as, the timing between exposures canbepivotal in the degree ofinhibition observed. Results injuveniles canbe confounded bytheir higher capacityto synthesize ofnew AChE molecules. For example, Chakraborti et al. (1993) reported thatjuvenile rats are less sensitive than adults under subacute chlorpyrifos dosing (i.e., 40 mg/kg/day, every four days for 4 doses). Fourdays after the last exposure, adult rats showed 90-92% inhibition ofbrain cholinesterase whilejuvenile rats show only 54-59% inhibition. Greater inhibition ofbrain cholinesterase in adults was correlated with more extensive reductions in muscarinic receptorbinding. These receptor changes were correlated with enhanced locomotorresponses to the antimuscarinic drug scopolamine in adults only, out to eight weeks after the end ofdosing. Thus, under these conditions, adults appear markedlymore sensitive to cholinesterase inhibition and cholinergic effects ofchlorpyrifos. Liu et al., (1999) report that repeated, daily chlorpyrifos exposures (5 mg/kg/day, for 14 days) were associated with relatively similar degrees ofbrainAChE inhibition between juveniles and adults 1 day after either the7th or 14th dose, butmore extensive inhibition was noted in adults 8 days after termination ofdosing. Relatively similar changes in muscarinicreceptorbinding were also notedbetween age groups following daily exposures. Together, these studies illustrate the ability ofbrain cholinesterase to more effectivelyrecover between repeated doses in pups and the possibility ofrelativelymore extensive cholinergic responses in adults following repeated chlorpyrifos dosing under the same conditions (repeated, "low" dose exposures).
Overall, the Panel decided that a conclusion ofgreater sensitivityto cholinesterase inhibition in juvenilerats-compared to adult rats onlyholds 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 sensitivityin younger animals under "environmental concentrations" would beunlikely. Othermembers stated that this conclusion cannotbe made withouthaving a better idea ofthe meaning of"high exposure level situations"
or "environmental concentrations."

b. There are multiple gestational studies available which provideAChE 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 orhigher levels ofchlorpyrifos and/or its metabolites than the dam. In addition:, multiple studies have shown that recovery from AChE inhibition is more rapid injuveniles compared to adults. This rapid recovery combined with production ofthe AChE enzyme as the rats mature leads to less AChE inhibition observed in thejuveniles. The Agencyhas preliminarily concluded that AChE inhibition data from repeated dosing gestational studies showing less inhibition in fetuses may



### 22notreflect the true potential toxicity to the fetus. Please comment on theAgency'spreliminary conclusions andthe scientific support for oragainst this conclusion.

Panel Response
The Panel agreed with the Agency'spreliminary conclusion regarding observations oflower brain cholinesterase inhibition inpups (rats) compared to dams following gestational OP exposures. However, as noted in the previous question on the findings from thejuvenile versus adult studies, the cholinesterase activity measured depends onthe time ofsamplingrelative to time ofexposure. Whether one considers gestational orpostnatal exposures, the longerthe time from dosing to measurement ofcholinesterase inhibition, the lesserthe inhibition expected in younger animals. Ifthe timebetween 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 bedue to the more rapid synthesis ofnew proteins including cholinesterase in tissues of younger animals. Thus, the addition ofnewly 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 maynot reflect the true potential toxicity to the fetus." The Panel based its decision after review ofthose studies indicating the sensitivityto cholinesterase inhibition at dosages ofchlorpyrifos sufficient to elicit AChEinhibition. One Panel member cautioned about equating AChE inhibition to toxicity, as is implied in the Agency's question. Inhibition is a sensitive biomarker ofexposure and is a precursorto at least some, ifnot all, ofthe adverse acute effects ofAChE organophosphates. In addition, there canbe no cholinergic toxicity without previous AChE inhibition. Lassiter et al. (1998) confirmed less inhibition ofbrain cholinesterase ofpups 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 5hours after a single dose ofchlorpyrifos (10 mglkg) on GD18. Relatively similar inhibition was noted inboth dam and fetus (approximately40% 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 ofpeak TCP levels in the brain.

Lassiter et al. (1998) considered several mechanisms for differential ChE inhibition, but concluded that more effective recovery ofenzyme activity following each daily exposure in the fetal tissues prevented accumulation ofChE inhibition, while slowerrecovery ofactivity in the maternal brain allowed increased ChE inhibition throughout the dosing period. This is essentially the same mechanism described earlier inpostnatal pups to explain the lesser'cholinesterase inhibition and effects on other. cholinergic markers inpups as compared to adults that were treated at subactute doses with chlorpyrifos (Chakraborti et al., 1993). Therecovery ofinhibited AChE, i.e., the apparently lower level ofAChE inhibition injuveniles compared to adults, is very likely due in large measure to greater synthesis ofnew AChE molecules during growth. In animal studies, it would be unlikely that significant "synthesis ofnew AChE" would occur in a 5 hour window to a level such that it would alter AChE activity and give the impression ofless inhibition. Infact, AChE activity in control animals changed verylittle between·2, 5, and 10 hours following exposure as reported in Lassiter et al. (1998). Thus, AChE measurements prior



### 23to 5 hours maybe an accuratereflection ofthe amount ofAChE inhibition, but AChE measurements beyond that point are subject to question

.. The Panel agreed that the timing ofthe AChE assay following exposure maybe less important when evaluatingthe degree ofcumulative 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 ofAChE inhibition in the growingjuvenile couldbe determined ifthe timing ofthe sample collection were closerto the time ofpeak effect and ifmore interim sacrifices were used in these experiments. Some on the panel recommended more studies to examine AChE inhibition resulting from lower doses ofchlorpyriphos in dams and developing neonates/pups, at more timepoints, as well as mdre carefully quantifying AChE protein levels as well as AChE inhibition. Regardless ofwhether there was recovery ofAChE inhibition or increased AChE synthesis, this event is expected to be oflittle consequence at levels ofexposure that are below those inhibiting the target enzyme. Itwas pointed out that the human fetus is slower developing than the rat, so rapid synthesis would notbepresent. Some Panel members felt that in the human fetus there wouldbeno inhibition at all at environmentallyrelevant levels ofexposure. But another member indicated that inhibition mightbe a concern for exposures experienced by agricultural workers. Many agricultural workers are women and some ofthose maybe pregnant when exposed to higher levels. One Panel member stated that predicting the toxicological effects in humans based on the available data is too riskybased solely on animal studies.

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

### C):

The Agencyhas performed a literature review ofin vivo and in vitro studies onthe effects of chlorpyrifos onthe developingbrain.

a. From a review oflaboratory animal studies, the Agencyhas preliminarily concluded that gestational and earlypostnatal exposure at sufficientlyhigh exposures to chlorpyrifos can lead to neurochemical andbehavioral alterations persisting into adulthood after any initial AChE inhibitionhas reversed. TheAgencyhas put particular emphasis on thebehavioral databecause studies are available from multiple laboratories. Please comment on theAgency'spreliminary conclusions andthe scientificsupport for or against this conclusion.

Panel Response

In general, the Panel was in agreement with the preliminary conclusions madebythe Agency. The preliminary scientific findings and conclusions are supported bythereview ofthe in vivo neurobehavioral data from multiple laboratories on two mammalian species (i.e., rat and mice) of gestational exposure and/or earlypostnatal exposure to chlorpyrifos. The Panel concluded that independent ofthe exact time frame ofthe gestational orpostnatal exposure, onlydoses of lmg/kgor greater demonstrate significant effects onbehavior. However, onlytwo 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 studyis considered sufficient (see below). The Panel noted

24that the inclusion ofsomemorerecent studies would add to the number oflaboratories involved,
as well as increase the number ofneurobehavioral endpoints examined. One panel member provided a table ofstudies that supplements the information provided in Table 3 oftheAgency's issue paper (see Appendix 1).

Two areas ofconcern were identified and discussed. The first involved methodological issues in the studies reviewed. The second was conceptual, and involved the use ofthe phrase "sufficientlyhigh exposures" in the Agency's charge question.

The methodological issues centered on the consistency ofbehavioral endpoints reported as significant. ThePanel 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, errorrate canbeincreasing, 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 likelyreflect differences in route ofadministration, developmental period during exposure to chlorpyrifos, method used to measure changes in the samebehavioral domain, and/or choice ofdependentvariable. For example, motor activity canbemeasuredbydistance traveled in an open field and electronically bybeambreaks. Moreover, different studies use different types oftesting chambers, ornumber ofcrossings in an elevated plus maze. The use ofseveral detection methods for motor activity maypresent difficulty incomparing across studies, especially with respect to younger animals. Concerns were also raised about the high level ofvariability ofmeasurements and its effect on such endpoints as the mean startle response(see Maurissen et al., 2000).

While the Agencyputparticular emphasis onthe behavioral data, primarilybecause it is available from multiple laboratories, all related data were considered in identifying long-term changes in the nervous system. Withrespect to the Maurissen et al. (2000) study, the Panel raised concerns on the use ofstatic morphometries to evaluate a dynamically changingbrain structure. Measurement ofthe hippocampal dentate granule cell layer at postnatal day 12 would potentially capture a structural assessment ofa region withpoorly defined landmarks at a time when the neurons are undergoing an active period ofmigration. Inthe adultbrain, morphometric measurements ofthe cortical regions displayed about 10% variability, a level expected to be within the normal variability for such crude measurements. Unbiased stereology is recommendedbythe Panel to address questions ofregional volume or cell number.

Several Panel members specificallynoted methodological shortcomings inthe Jett et al. (2001) study. Although it is only one oftwo studies to investigatebehavioral changes after adose of0.3 mglkg ofchlorpyrifos, there were significantproblems with respect to the training paradigm i.e., the Morris waterinaze [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 of7 mglkg, a finding at odds with otherpublished studies. In addition, behavioral changes were noted for the 0.3 mglkg/dose onlywhen dosing continued during testing. This represents a confounding factor in determining the acute versus development effects ofchlorpyrifos.



### 25The Panel agreed with theAgency's expressed caution about theuse ofDMSO as a vehicle because ofits intrinsic toxicity and influence on absorption. In addition, the "observed signs of discomfort" in pups injected with DMSO reported byMarty et al. (2000), and more recentlyby Carr and Nail (2008), raised concerns about the use ofthis vehicle in developmental studies. An additional concern was raised bythe Panel concerning the influence ofDMSO on the toxicityof the compound under study. Ballough et al (2008) reported that DMSO, administered at levels below those used inmany developmental chlorpyrifos studies, enhanced the toxicity ofsomanby exacerbatingthe neuropathology caused by soman-induced seizures. The interaction ofDMSO 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 ofadministration (ROA) ofchlorpyrifos used for developmental exposure. The Panel noted that each ROA used presented drawbacks and most ifnot all were stressful. 

### It was generally agreed that the subcutaneous ROA was valid for gestational exposure. Moreover, despite a variety ofroutes used in the literature, there are , consistent reports oflong-termbehavioral changes in offspring after recovery ofcholinesterase inhibition. , The Panel noted that many ofthe papers cited bythe Agencydo not link exposure periods to chlorpyrifos with structural changes occurring in the developingbrain. While the EPA developmental neurotoxicity guideline studies defined a broaderperiod ofdevelopmental exposure, more recent studies from the peer-reviewed literature have attempted to demonstrate that there are distinct developmental windows ofvulnerability. While effects ofchlorpyrifos can be detected in the older animal a direct linkagebetween the developmental window with regard to the stage ofbraindevelopment and endpoints examined was ill-defined. 

### It was suggested that
a more specific examination ofthe critical events occurring at the time ofexposure and the specific targeted endpoints ofinterest would be a valuable contribution to the literature. Such data wouldbeparticularly 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 ofsuch studies with respect to timing ofbrain development.

The Panel indicated that thephrase "at sufficientlyhigh exposures" is open to interpretation. Overall, it was interpreted bythe Panel in this meetingto refer to studies in which the exposure level was within a dose range expected to inhibitbrain AChE. The majority ofstudies reviewed bythe Agency focused on an exposure near 1mg/kg/day, a level generally sufficient to inhibit brain acetylcholinesterase inmost treatment/age groups studied, and which produce behavioral' alterations. However, measurements were often obtained at a singletimepoint post-dosing and at a time when the period ofpeak inhibition maybemissed. The availability ofdose-response data was limited and in some studies the calculated levels ofinhibition were not consistent with the predicted levels ofinhibition for high dose exposure. Studies examining critical developmental periods for exposure wereprimarily focused on dose levels expected to produce AChE inhibition. Given the nature ofthe gestational exposure studies, theuse offetuses for



### 26neurochemical analysis is limited. Given the critical role ofthe cholinergic system for brain development, the lack ofsufficient data to support the absence orpresence ofenzyme inhibition is a significantproblem in the interpretation ofthe results from many ofthese developmental studies. Theinherent problems in assessing brain AChE inhibition during development and the type ofmeasurements needed for the assessment were noted in the Agency's document. The Panel noted that it is possible that the morphogenic role ofAChE may alter aspects ofbrain development and neurogenesis, even in the absence ofdetectable levels ofinhibition.

The critical point stressed was that dosages considered "sufficientlyhigh" to elicit

. neurobehavioral disruption into adulthood would also be anticipated to affect the target enzyme for the common mechanism oftoxicity for chlorpyrifos and other organophosphate insecticides. Whether any suchchanges were initiated by early inhibition ofacetylcholinesterase remain unclear.

Errors in the Agency's Issue Paperdescribed below were noted

.

. 1) Section 3.3.1, page 44, paragraph 3. Thebehavioral effects in the Maurissen et al. (2000) study are only limited to an increased latencyto peak response in the auditory startle response that was independent ofthe dose ofchlorpyrifos. 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 ofthe mode oftoxic action is an important component ofrisk assessment. The International Programme ofChemical Safety (IPCS) and International Life Sciences Institute Risk Science Institute (ILSI RSI) have developed a Mode ofAction (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, involvementofa series ofkey events in the MOA is established on weight-of-evidence, using criteriabased on those described byBradford Hill, taking account of factors such as dose-response and temporal concordance, biological plausibility, coherence and consistency. Other MOAs that logicallypresentthemselves also should be considered. Once an MOA is established, qualitative and quantitative comparison ofeach key eventbetween the experimental animal and humans enables a conclusion as to likelyrelevance ofthe MOA for human risk. In the case ofchlorpyrifos, the Agencyhas considered the available mechanistic data but has not evaluated these data inthe context ofMOA/humanrelevance framework. Ithas been initiallydetermined that there are insufficient data to develop a series ofsupportable key events (as in a mode ofaction analysis 1) for neurodevelopmental toxicities otherthan AChE inhibition. The Agencynotes a particular lack ofdata on dose response and temporal concordance that are critical in a MOA framework analysis. There maybe othermechanisms which lead to effects on the developingbrainbut a supportable mode ofaction(s) can notbe elucidated at this time. Please comment on theAgency'spreliminary conclusions andwhether there is sufficient

1For information on the Mode ofAction Framework, see U.S. EPA ,1999, 2005; Sonich-Mullin eta!., 2001; Meek eta!., 2003 ; Seed et a12005 and Boobis, eta!, 2006

27scientific information to meritafull mode of action frameworkanalysis. 

## If a mode of action framework analysis ispursued, whatwouldbe the biologicallyplausible hypotheses to evaluate?

Panel Response

There was a consensus ofthe Panel that available data were inadequate to support a weight of evidence evaluation for non-cholinergic mode(s) ofaction for the behavioral alterations following gestational and eadypostnatal exposure to chlorpyrifos thatpersisted into adulthood. The Panel agreed that the available information does notallow for behavioral endpoints to be considered as a point ofdeparture and recommended, based upon currently available data, that cholinesterase inhibitionbeused as the PoD.

Pointed out at thebeginning ofthe discussion ofthis charge question was the fact that a mode of action/human framework analysis requires consideration oftestable hypotheses with identified key events in a causal pathway. Itthen requires comparison ofsystematic analyses oftraditional criteria for weight ofevidence for each ofthese hypotheses including consistency, dose-response

.and temporality among earlykey 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 comparedbetween 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 ofevidence inrisk 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 ofthe Agency's issue paper summarizes the studies on persistent behavioral changes in adults following gestational and earlypostnatal exposure as well
as selected {n vivo and in vitro studies on the interaction ofchlorpyrifos with several neurochemical parameters. The Agencynoted in its issuepaper that during development ofthe brain, acetylcholine plays a role in morphogenesis, and AChE may alter aspects ofneurogenesis in the absence ofa detectable level ofinhibition using current methods. ThePanel indicated that there is a large amount ofvariance presented in many ofthe studies, a lack ofdose response for many ofthe proposed targets, and a lack ofreports ofaltered targets from more than one lab. The Panel concluded that these studies were not inclusive, coordinated, nor conducted in the contextofhypothesis testing for mode ofaction. 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 orindependently ofit. Specific points were raised regarding the question ofmode ofaction. 

### In Figure 5 ofthe issue paper, the Agency cites Slotkin et al. (2006) to suggest possiblemechaiJisms for how chlorpyrifos mayelicit neurodevelopmental effects. The Panel did not find enough information from the Slotkin et al. (2006) paper to determine ifchlorpyrifos directlyinteracts with the molecular components oftheproposed pathways that could then lead, ina causal manner, to subsequent neurobehavioral changes. Panel members determined that it was generallyunclear in what waythe animal studies exhibit "qualitative similarities" to findings reported in children. The aninial studies do report neurobehavioral changes in rats and mice following prenatal or



### 28postnatal chlorpyrifos exposures at levels that wouldbe expected to inhibit cholinesterase. These "sufficientlyhigh" exposures maylead to persistent neurobehavioral effects under some conditions, but "qualitative" similarities to neurodevelopmental findings reported in children are difficult to equate.
There are several facets ofdata evaluation that mayneed to be examined with respect to effects
on serotonin, G-proteins, macromolecules, neurotrophic factors etc. bydevelopmental exposure to chlorpyrifos. Forthe mostpart, the effects seen onthese systems following chlorpyrifos exposure are modest, and manyrepresent less than a 25% change from control values/levels. These changes were often sex specific and dependent uponthetime ofexposure and measurement. Although there is always the possibilitythat such modest changes in neurochemistry and biochemistrywill 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 ofglutamate Nmethyl-D-aspartic acid (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors would be important endpoints in assessing the developmental neurotoxicity ofchlorpyrifos.

Theuse ofmorphometricmeasurements ofbrainregions was questioned withregard to age, i.e., regional measurements were conducted during an active period ofbrain development resulting in the lackofdiscrete 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 bewithin the normal variability ofcontrol tissue. One panel member indi.cated a preference for the use ofunbiased stereologyin obtaining structural measurements of discrete brain regions. However, this approach would also behindered during earlypostnatal development. Unbiased stereologyis a well established methodfor determining cell number and tissue volume and has beenused in developmental, aging, and pharmacological studies. An additional concern was raised in that changes often occur in the network organization ofthe brain rather than gross cell death. A number ofdifferent neural and vascular cell types can be influencedby acetylcholine during development and such effects maynot 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 ofthe brain, neither it nor morphometric measurements would detect less than gross cell loss occurringpriorto 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 ofthe various brain regions and the normal temporal pattern ofapoptosis and synapticpruning, differentiation ofradial glia, neuronal migration, synapse stripping; maturation ofthe resfdent brain monocytes, the microglia, and the brain vascular system. Thus, to determine low-dose effects for any chlorpyriphos will require more sophisticatedmethods ofanalysis.

One Panel member took issue with the material in Section 9: Problems with studies from the literature, and inparticular with Paragraph 5, Page 28 (see Agency's issue paper) which states "An aspect ofsome ofthe in vivo studies discussed above is that no AChE inhibition was detected during the windows ofexposure at the dose level used. This is especiallytrue for ·



### 29gestational**~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 ofassay does not coincide with the time ofpeak inhibition. Thus, unless a time course ofAChE inhibition is performed and clearly demonstrates that there is no AChE inhibition, the claim ofno inhibition
or low levels ofinhibition maynotbe accurate.
Some panel members suggested that the recentreview ofchlorpyriphos toxicitybyEaton et al. (2008) provided a basis for consideringpossible non-cholinergic modes ofaction. The serine hydrolase enzyme KIAA1363 is important in an ether lipid signaling network involvingplatelet 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 ofAChE inhibition. in vivo, a 98% inhibition ofAChE and lethality due to chlorpyrifos-oxon administrationresulted in a39% inhibition ofbrain KIAA1363 (Nomura et al., 2006). Therelative changeshave not beenreported for lower dose levels thus, a direct causative role in the toxicity ofchlorpyrifos has notbeen identified. So while KIAA1363 may serve as an important detoxication mechanism, as is supportedbythe increased toxicity in knockout mice (Nomura et al., 2006), it does not necessarily indicate that this enzyme is a target for "mode ofaction."

At dose levels significantly lowerthan any previouslyreported effects, chlorpyrifos induced the phosphorylation ofcAMPresponse elementbinding (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 ofneurotoxicity, 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) (specificallyTable 14 and related text) could beuseful to the Agencyin evaluatingpotential alternative modes ofaction.

Collaboration between EPA and developmental neurotoxicologists mayhelp define research needs for identifying alternative neurotoxicprocesses involved in the developmental neurotoxicity ofchlorpyrifos. At a minimum, such studies would require testing multiple doses ofchlorpyrifos between 0.05 and 1.0 mg/kg to set the high dose for expected AChE inhibition. Such data can then beused to determine level ofcholinesterase inhibition and related changes in brain development. This mayrequire additional methods development for detection oftargeted brain regions or cellular localization. The data set can thenbeused to identify cholinesterase inhibition or an alternative mode ofaction for developmental neurotoxicity ofchlorpyrifos. Low dose level effects reported for in vivo developmental effects require replication as well as the extremely low-dose in vitro activation ofCREB. Inclusion ofdata on multiple carboxylesterase activities and in localized brain regions are majorrequirements for future studies.

2 IC50 (Isolated Cortical) refers to the concentration ofchlorpyrifos incell culture that produced the observed effect (in this case an increase in CREB) in 50%·ofthe neurons.



### 304. Epidemiology Studies in Children and Mothers (Issue Paper Section 3.4, Appendix D):

The Agencyhas evaluated epidemiology studies from three major cohorts: the study sites are: (1) ColumbiaUniversity, NYC, (2) Mt Sinai, School ofMedicine, NYC, both withmulti-ethnic urban low income women and infants, and (3) University ofCalifornia at Berkeley (Center for Health Assessment ofMothers and Children ofSalinas, CHAMACOS) with women and their children from farm workerpopulations.

a. The Agencybelieves that all three studies provide valuable information on the effects in children ofhigh exposures to pesticides, particularly OPs. Forpurposes of**e~aluating** human health effects ofchlorpyrifos, the ColumbiaUniversity studies provide more robust information for evaluating thehuman healtli effects ofchlorpyrifos because it measured chlorpyrifos rather than a metabolite in both environmental (air) and biologic media (maternal and cordblood) 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 bythe Mount Sinai group are informative with regard to evaluating the relevance ofPONl status inhealth outcomes, this studyis limited because the neurodevelopmental outcomes were linked to non-specific OP maternal urinarymetabolites (DAP, DEP and DMP), rather than the chlorpyrifos-specificmetabolite TCP. The exposure ofthe CHAMACOS 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 theAgency'spreliminary conclusions on each of the three cohortsregarding the degree to which the data informs the chlorpyrifos human health riskassessment. Please also comment on the scientificsupport for or against thesepreliminary conclusions.

Panel Response
Overall, the Panel agreed with the Agency's conclusion that although each ofthe three studies (Columbia, Mt. Sinai, and Berkeley) provide valuable information, the Columbia studyis the most robust and appropriate for informingrisk assessment withrespect to the exposure (chlorpyrifos), outcomes (birth and neurodevelopmental), and population (neonates and children) being addressed bythe Agency's evaluation. To be precise, the Columbia study was not necessarily a more robust epidemiological study, butit 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 ofother OPs and carbamates. The use of the term "high exposure" in the phrasing ofthe question was questionedbecause although the exposures in these studies maybe high for residential settings, they are not high when compared to agricultural worker and pesticide handlerpopulations.

The Panel noted that the conclusions ofthe Agency are supported bythe following information:

• All three studies are prospective cohorts with relatively low loss to follow-up and are conducted byrespected researchers using widely accepted protocols with results published in peer-reviewedjournals.

31• The Columbia studyhas the strength ofhaving usedblood measures (both maternal and cord) ofchlorpyrifos specifically as opposed to the non-specific urinarymetabolites (e.g., diethylphosphate (DEP) and dimethylphosphate (DMP)) utilized in the othertwo cohorts. There is also high correlation (r=0.76; p<O.OOOl) between maternal and cord blood chlorpyrifos levels noted intheWhyatt et al. (2003) study. In addition, the Columbia studyhas exposure measurements from air monitoring which correlate withboth maternal and cordblood (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. Itwas noted,that although the pre and post analysis validates exposure measurements, the overall results are more interesting epidemiologicallybecause itlooks at the entire spectrum ofexposure.

• The initial reference group for thebirth outcomes (those whose chlorpyrifos levels in cord blood were below the level ofdetection (LOD) with similar demographic and socioeconomic characteristics) in the Columbia study was alsojudged to be appropriate.

• The methodology for exposure measurement was considered good bythe Panel and the quality assurance and quality control procedures used bythe Centers for Disease Control (CDC) are extensive. The Columbia study includedboth environmental and biological measurements ofchlorpyrifos and biological measurements were made inboth maternal and cordblood. Itwas noted that there are concerns about values near orbelow the detection limits; however, the precision ofthe data are acceptable while accuracy is reduced. In other words, the Panel felt confidentthat the chemical was presentwhen detected in a sample although the reported value maybe a poormeasure ofthe actual concentration. The difference between the true and measured amount couldbe appreciable.

• Raub and Whyatt presentations to the Agencyin 2007 and 2008 (presentations found in the docket, EPA-HQ-OPP-2008-0274) showed intelligence quotient (IQ) results at five years ofage 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 ofchildren who were exposed to chlorpyrifos 

### in utero. ·
The Panel concluded that the Columbia studyis epidemiologically sound and that there is minimal selection and informationbias. Additionally, any information bias would likelybe nondifferential sinQe any misclassification ofoutcome is unlikely to depend on exposure status and misclassification ofexposure is unlikely to depend on outcome status. However, even the bestdesigned epidemiology studies are susceptible to weaknesses inherent to observational studies, and a few ofthese possible weaknesses are addressed below:

• The possibility ofresidual confounding in the analysis ofthe Columbia study data on birth outcomes (Whyatt et al., 2004) was discussed. Inparticular, variables such as



### 32presence ofprenatal care, sexuallytransmitted diseases, history ofsubstance abuse and of cigarette smoking, low pre-pregnancybodymass index (BMI) and inadequate weight gain are potential confounders. Review ofthe model covariates found that a complete list ofstandard variables which are commonly available were controlled for in the analysis includingpassive smoking, ethnicity, parity, maternal pre-pregnancyweight, weight gain, newborn gender, and season ofdelivery. In addition, the Columbia cohort was restricted to exclude women who smoked during pregnancy, used illicit drugs, had human immunodeficiency virus (HIV), orhad their first prenatal visit after the 20th week of pregnancy (Whyatt et al., 2004). The Panel agreed that these factors limit the possibility ofresidual confounding. 

### In addition, the authors ofthe Columbia study further addressed the potential for residual confoundingbyincluding a variety ofother variables in the analysis. Theresults as presented inthe Rauh et al. 2008 paper are stable and not sensitive enough to eliminate the various confounders. Although there maybe other variables not included in the analyses that are associated with either exposure or outcome, in: order for a variable to be a confounder itmustbe associated withboth exposure and outcome.

• The Panel also noted that the infants in the Whyatt study were ofnormal weight. The Panel questioned the significance oflowerbirth 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 ofDimes; 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 areborn smaller than expected bytheir genetic potential would have increased risk ofperinatal mortality, neurological morbidity and morbidity in general as compared to infants born at their predicted birth weight (Figueras et al., 2007). Reductions inbirth weight are also associated with marked increase in chronic diseases in adulthood including diabetes, cardiovascular disease, stroke, and hypertension (Barker, 2004). One issue ofconcern is whether chlorpyrifos' effect on reducing birth weight would result in
more infants falling into the category oflowbirth weight.

• Concerns raised bypublic commentators before the Panel about neurobehavioral testing, including the specific tests used, the method ofadministration, and the training ofthe 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 obtainedwereprobably as good as could be obtained given all ofthe limitations associated with neurobehavioral testing.

• Concerns with the three year period between the sampling ofcord 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 ofexposure and should notbetaken as an absolute representation oftotal chlorpyrifos exposure throughout pregnancy. Many researchers have argued that chlorpyrifos inblood and

33adipose 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 ofmaternal fat stores accumulated in early and mid pregnancy, in late pregnancyhuman 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 ofthe accumulated chemicals. Accordingly, the single measurement ofcordblood chlorpyrifos maynot berepresentative ofthe total exposure during pregnancy, but onlyreflects 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 maybemultifactorial in origin. It would be important to ascertain whether there are statistically significant differences in neurodevelopmental outcomes at 3 years ofage between children with chlorpyrifos levels in cordblood below the LOD and those with a chlorpyrifos level of6.17 pg/g. Rauh et al. (2006) stated that the Bayley Scales ofInfant Development scores (Mental Development Index (MDI) and Psychomoter Development Index (PDI)) were similar in both groups ofchildren. Ifso, chlorpyrifos levels in cordblood at the time ofdelivery would notbe expected to be associated with neurodevelopmental outcomes.

• It is difficult to compare exposure levels in the Columbia studywith the US population (as measured in the National Health and Nutrition Examination Survey (NHANES) or with otherbiomonitoring studies sinceurinarymetabolite levels were not measured.

• Although the data on post-ban declines in exposure are compelling, limitations mustbe kept inmind when using these results in the weight ofevidence. The study'was not designed to**asses~** the effect ofthe ban, so data are essentially cross-sectional (i.e. exposures among the same women were not measured overtime). Additionally, the data presented for exposure levels by year have been crude in nature. It is not likelyto 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 primaryindependent variable, whilealso adjusting for any factors that maybe associated with both exposure levels and year ofstudy.

• There was discussion about the cut-offvalues 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 mayhave occurred in more severely affected children or less severely affectedchildren as opposed to a non-differential loss. Some felt that more attention shouldbe given to .the sensitivity ofthe 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 viapersonal communication on September 18, 2008 (found in the docket, EPA-HQ-OPP-2008-0274) that these groups were unequal in the 2006 studybecause only children who reached three years ofage were analyzed, suggestingthat the**origi~al** exposure groupings were notbalanced. Some panel members noted that the Agency

34should explore this· further as the loss ofchildren in the high exposure group could notbe due to length offollow up as allthe children in the high exposure group (bar one) had to have been bornpriorto 2001 (and hence should havebeen at least three years old) as after thebanthere was onlyone child in the high exposure group. The Panel felt that it wouldbeuseful ifthe authors couldprovide additional details onthe loss to follow-up in each ofthe four exposure groupsandprovide additional details onhow the exposure groups were combined. However, it was also noted that the inclusion ofboth the children below LOD and with low exposures values into the reference group would be expected to attenuate any association, ifindeed there is an effect at such low exposures. However, in Rauh et al. (2006), there is no suggestion ofsuch an effect as the mosthighly exposed group and the group with values below LOD had lowermean 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 compellingreason 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 subjectbeing classified as highly exposed in post-ban years. This analysis lacks statistical power and maybe sensitive to the cutoffvalue 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 separatebetween the lower exposure levels.

• The Panel felt that itmight be helpful to have a longitudinal component to the Columbia studyto determine ifthe adverse effects persisted in those children who exhibited poor birth outcomes orneurobehavioral deficits.



### • It wouldbeuseful to examine theresults ofa 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 ofanalysis would likely not change the results, but it couldbehelpful in illustrating threshold or dose response effects. Despite the questions and discussion noted above, itwas affirmed that the Columbia study was indeed quite strong and provided extremely valuable information. Most ofthepoints addressed above are points ofclarification orpoints that ifaddressed could strengthen the analysis and provide the Agency with better data to use in the risk assessment process.
Given the paucity ofhuman epidemiological studies oforganophosphates in children, the other two cohorts (Mt. Sinai and Berkeley) should notbe completely discounted. Both studies provide information on abnormal reflexes in neonates, and thisdata is not available in the Columbia cohort. Additionally, the Mt. Sinai cohort considers PONl as a factor inthe analyses, something that may·have some relevance to risk assessment (Engel et al., 2007). More information maybe gleaned from the Berkeley cohort (Eskenazi et al., 2004). A close comparison ofOP metabolite concentrations in this study with those reported in NHANES maybeuseful. 

### It is interesting to note that median 3,5,6-trichloro-2-pyridinol (TCP, a specific metabolite ofchlorpyrifos) levels in this.cohort were similar tothosereported in NHANES; whereas, DEP (diethylphosphate, one of the non-specific urinary metabolites) levels were muchhigher. This suggests chlorpyrifos

35exposures in the study are similarto "background level" and that exposures to other OPs (besides chlorpyrifos) that are metabolized to DEP are proportionallymuch higher in this cohort (relative to chlorpyrifos) than the general population. Other OPs mayverywell be driving the associations between DEP/DMP (non-specific organophosphate urinarymetabolites) levels and adverse outcomes. However, because chlorpyrifos is likelyto contribute very little to the overall DAP levels inthe Berkeley cohort TCP and DAP levels maybepoorly correlated. Thus, inthe TCP analysis, it may (with some caution exercised) bepossible to additionally adjust for DAPs in an attempt to assessmore chlorpyrifos (and/or chlorpyrifos-methyl) specific associations. The presence ofthese other OP pesticides may overshadow anyTCP associations, butmay still be worthwhile to investigate.

The Panel acknowledged that there are potential confounders and issues thatreduce theutility of boththe Mt. Sinai and Berkeleycohorts for risk assessment. For example, both studies measure organophosphate metabolites inurine but chlorpyrifos is not specificallymeasured. The Berkeley study, in particular, has the least relevance to chlorpyrifos risk assessmentbecause only
a small percentage (10%) ofthepesticides applied in the Salinas Valley are chlorpyrifos; therefore, the Panel assumed that chlorpyrifos would make only a small contribution to the nonspecific metabolites measured inthe study, and as a result mightbe expected to have small impact onthe study outcomes, although this assumption has notbeen verified. As such, it is difficult to ascribe the effects seen to chlorpyrifos, inparticular, rather than OPs in general.

A recentlypublished studyby Samarawickrema et al. (2008) entitledFetal effects of environmental exposure of pregnant women to organophosphorus compounds in a rural farming community in SriLanka was brought to the attention ofthe Panel and suggested for use in clarifying the issue ofwhethermaternal and cord blood are differentially distributed to the fetus. Itwas 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 onlyrecommend that the Agency consider this studywhen this issue is revisited. The Panel also encouraged the Agencyto identify and review any otherpublished studies on this topic, even ifoflesser size and scope, since theymay also contribute to the total weight of evidence analysis ofthe potential neurobehavioral effects ofchlorpyrifos.

b. Data from Whyatt et al. {2003) show that 100% ofair samples detected three AChE inhibitingpesticides (chlorpyrifos, diazinon and propoxur). Similarly, all three pesticides were found in 48-49% ofumbilical 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 notbeen conducted for the neurodevelopmental outcomes. The Agencycan notrule out that exposures to all three AChE-inhibiting pesticides in combination resulted inthe neurodevelopmental health outcomes reported in the studies. However, this possibilitydoes not rule outthepotential 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 havebeen statistically associated with multiple birth and neurodevelopmental outcomes; 2) these associations are correlated in time priorto the cancellation ofindooruses of chlorpyrifos when exposures were much greater (and thereby show some degree ofdoseresponse); and 3) there are animal data which support neurobehavioral effects resulting from

36gestational exposure, the Agencyhas preliminarily concluded that chlorpyrifos likelyplayed a role in these outcomes. Pleasecomment on theAgency'spreliminary conclusion and the scientific support for or against this conclusion.

Panel Response
Overall, the Panel agreed with the Agency's conclusion that chlorpyrifos likelyplayed a role in the birth and neurodevelopmentaloutcomes noted in the three cohort studies. The Panel agreed with the Agency on the following points: a) exposures to all three AChE-inhibitingpesticides in combination cannotberuled out as contributing to the birth and neurodevelopmental outcomes, b) the potential ofcombination and/or additive effects ofthese three compounds does not rule out the role ofchlorpyrifos in contributing to the outcomes, and c) it cannotbe stated that chlorpyrifos is the sole contributor to the observed outcomes. These conclusions are supported bythe effects observed at various age groups across the three cohorts as summarized in Table 1. 

## ll Strength and significance ofassociation as reflected bythe 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 byresidual confounding.

b. _There were statistically significant deficits in birth weight of 150 grams when comparing high exposure to exposure that was below the level ofdetection (LOD); and, decreases of 43 grams in birth weight per log unit increase in chlorpyrifos in cord blood. Statistically significant deficits inbirth length were also noted (Whyatt et al., 2004).

c. There were statistically significant deficits of6.5 points on PDI at 3 years ofage 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) ofmental delay and 4.5 times increased odds (95% Cl: 1.6-12.7) ofpsychomotor delay when comparing high to low exposure groups at age 3 (Raub et al, 2006).

e. There were deficits of5.6 points on verbalIQ and 5.1 points on full IQ at age 5 (Whyatt, EPA presentation, 2007)

. 

## .ll Consistency ofassociation between the three cohorts is that they all found some developmental effect in a population exposed to elevated levels ofOPs. The Berkeley and Mt. Sinai cohort studies measured non-specific urinary DAP metabolites (e.g., diethylphosphates (DEPs), dimethylphosphates (DMPs) producedbymany OP pesticides including chlorpyrifos. However, urinary levels of3,5,6-trichloro-2-pyridinol (TCP)
were not measured in these two studies, but onlyin the Columbia cohort study. Therefore, the conclusions below are based onthe non-specific DAP metabolites rather

37than 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 otherpesticides including methyl chlorpyrifos and the herbicide trichlopyr (3,5,6-trichloro-2-pyridinyloxyacetic acid, Remedy®) or even to TCP itself(as an environmental degradate ofchlorpyrifos, methyl chlorpyrifos ortrichlorpyr). However, developmental effects were consistently observed in children whosemothers exhibited biomarkers ofexposure to OPs in each ofthese 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 1year (Eskenazi et al. 2007). Although Eskenazi and colleagues found decreases in MDI associated withprenatal 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 ofPDD 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., inpreparation, Whyattpresentation to EPA in 2007). However, since DMP is definitelynot a metabolite ofchlorpyrifos, the associations stated here and in all ofthe previous points mustbe viewed with caution so as to not overinterpret the significance of the finding**~** A crude dose-response relationship as evidenced bythe following results from the pre. and post cancellation analyses in theColumbia cohort:

a. There were statistically significant deficits in birth weight (211 grams when comparing high to LOD groups) in childrenborn before the residential cancellation, butthere were no significant deficits in these outcomes in childrenborn after the residential cancellation (Whyatt, EPA presentation, 2008).

b. Statistically significant decreases inbirth weight and birth lengthperlogunit increase of cord blood chlorpyrifos were evident in children born before residential cancellationbut the association was not significant for childrenbornpost-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

38reduction in the numbers ofnewborns who were categorized inthe 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-cancellationperiods (Rauh et al., 2006).**~** Persistence ofstrong statistically significant associations after controlling for the effects ofother compounds as evidenced bythe following:

a. Additional analyses on birth weight and neurodevelopmental outcomes (MDI and PDI) controlling for diazinon did notreduce the effect ofchlorpyrifos (Whyatt, August 12, 2008, personal communication).

b. Associationbetweenbirth 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.



### 39Table 1. Neurodevelopmental Effects with Chlorpyrifos and Its Metabolites and the Metabolites ofOP Insecticides in Three Epidemiological Human Cob.ort Studies Source: Appendix D, USEPA; 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 ofabnormal with increased proportion and number ofabnormal reflexes reflexes and presence of3 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 ofInfant 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 ofmental and motor delay, increased problems with attention and ADHD (Attention Deficit Hyperactivity Disorder) and POD. Decreased levels ofchlorpyrifos 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

ISome caveats and areas for further analysis and clarification were noted byvarious panel members:
1) In the Berkeley cohort, there was a finding ofincreased head circumference with DEP levels (3 DEPs weremeasured)inmaternal urine and increased length and head circumference with DMP levels (3 DMPs weremeasured) (Ezkenazi et al., 2004). This couldbe a chance finding oritmaybe due to the exposure profile inthis cohort. However, the Mt Sinai study found an associationbetween decreased head circumference and maternal urinaryTCP levels above the limit ofdetection (Engel et al., 2007). Due to the nature ofthe exposure inthe Berkeley cohort, it was noted thatperhaps these urinary levels do not reflect significant exposures to chlorpyrifos given that DAP metabolites have two potential origins: breakdown ofproducts arising from OPs in general and direct

· absorption from environmental sources and diet. As measurements ofDAP metabolites

are non-specific in nature, the particular chemical involved maybe one ormore ofdiethyl 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 maypotentially affect those urinarymetabolite levels because women with the higher activityphenotype will eliminate higher levels ofTCP in urine as compared to those with the lower activityphenotype. However, this statement is not true ifexposure levels are low and below enzyme saturation inthe case ofcarriers of
a low activityphenotype.
2) In interpreting the Columbiastudies, the Agency shouldbearinmind that diazinon and propoxur as well as otherunmeasured potential developmental neurotoxicants were present along with chlorpyrifos, and the exactroles ofall ofthese components inthe mixture cannotbe determined. In Table 3 ofWhyatt et al. (2004) it is seen that thebeta coefficients for the association withbirth weight are -42.6 for chlorpyrifos and -44.2 for diazinon. Thebeta 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 maybe statisticallyjustified. Whenthe effects ofchlorpyrifos and diazinon are summed using U.S. EPAmethodology (U.S. EPA, 2001), there is a slightly greaterreduction in birth weight (b= -49.1). This mayindicate that the effect ofthe combined chemicals is slightly greater than the individual chemicals alone and that there could bepotential interactionbetween the two chemicals with respect to the association. Given these results, some on the Panel questioned whether one should single outjust one ofthe two chemicals whenboth are present and there is a potential interaction. Rauh et al. (2006) did not consider diazinon ormixtures in the neurobehavioral studybutjustified the selection ofchlorpyrifos based on the birth weight data, ignoring the similarities of diazinon with chlorpyrifos and the significance ofthe mixtures. There was disagreement among the Panel with this interpretation. Confidence limits as well as the beta coefficients mustbe considered when interpreting statistical analysis results. ThePanel
was in agreement that there was potential for interactionbetween chemicals, butthis did notpreclude chlorpyrifos as a contributor. ThePanel also noted that Rauh did perform an

41analysis ofthe neurodevelopmental effects while controlling for diazinon and the mixture ofdiazinon 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 ofchlorpyrifos and diazinon produced slightlymore significant effects for MDI than were seen with chlorpyrifos alone. Diazinon alone did notproduce significant changes in MDI. This interaction is criticallyimportant in interpreting the results. 3) More analysis controlling for multiple exposures and examining additive effects would help to elucidate the role ofother organophosphates, butthese analyses were not necessary for the panel to affirm that chlorpyrifos likelyplayed a role inthe outcomes that were observed. Theseresults are important for AChE inhibition to be considered as a mode ofaction. 4) Whyatt and Raub (ColumbiaUniversity) in their comments to the Panel (September16, 2008) described how they separatedtheneurodevelopmental effects ofchlorpyrifos exposure from those ofdiazinon exposure. Somepanelists questioned whether the biological effects ofthese anticholinesterase insecticides could be separated. 5) The Agency's use ofthe word "likely" at the end oftheparagraph needs to be considered carefully. TheAgency should ensure that the use ofthe word is notbased heavily on the Columbia studyin the absence ofintermediatemarkers ofbiologically effective dose and early effect that further link exposure and outcome and or a clearer idea ofmode ofaction from experimental studies. 6) Itis unclear whether the blood levels ofthe three AChE-inhibiting insecticides are highly correlated. Ifthere is a lack ofcorrelation, it is less likelythat they would confound or beresponsible for the observed relationships between chlorpyrifos and neurological outcomes. However, it would take a much larger and more detailed studyto elucidate the
more complex issues involving chemical mixtures (the possibilityofsynergism, antagonism, potentiation, activation, etc.) Somepanelists suggested that chlorpyrifos (or chlorpyrifos oxon) mayinhibit 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 whenprecededbyexposure to chlorpyrifos (Note: workbyErnest 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 ofchlorpyrifos. 7) Other issues with the Columbia cohort study (Whyatt et al., 2004) were identified. The level ofdiazinon in the homes was 8 times higher than that ofchlorpyrifos 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

42cord blood. There was an additional OP insecticide (methyl parathion) as well as5 carbamates (includingpropoxur) present in the homes. These anticholinesterases could beinteracting with each other. 8) The following are someclarifications provided byone Panelist on the material presented in Appendix D:

a. The Agency stated that exposure is underestimated byuse ofblood 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 maymore likely

· be a series ofcontact events that are lower in magnitude over time via various

routes and media. Thus, spot samples maybe a useful wayto estimate average exposure for women in the study since there is much lower within-home variability ofpesticide levels as compared to between-home variability (Whyatt et al., 2007).

. b. Description ofblood levels and underestimation ofexposure for diazinon and propoxur on page 50 ofthe 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 bemoreheavilyunderestimated as compared to chlorpyrifos levels in the sameblood 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 oreffects generatedby a mixture ofthe agents. Although the authors ofthe Columbia studies have attempted to isolate the effects that would be associated with chlorpyrifos, the Panel noted it is difficult to quantifythe contribution ofother neurotoxic compounds in such simultaneous exposures. Itwas also noted byone panelist that other OPs, as well as other chemicals, may play a role in neurotoxic effects in light ofa recent studybyEskenazi et al. (2008). The Panel concluded that although a mixture ofchemicals mayproduce the observed effects, this did not rule out chlorpyrifos as plaYing a role. However, the fact that mixtures were involved in the measurements ofall three studies does limit the conclusions relative to the specific role of chlorpyrifos in observed outcomes.
The majority ofthe Panel agreed that although the exposures in the Columbia study occurred at ·levels below the U.S. EPA reference dose and likelyunderestimate true exposures, the results of this study are ofconcern regarding adverse effects on normal neurodevelopment ofchildren exposed 'in utero to chlorpyrifos. This is particularlytrue in light ofevidence demonstrating that low levels ofexposure 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

43lower levels. Organophosphates are known neurologic poisons designed to kill insects. People exposed to high levels ofOPs 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 ofaction. Many other associations havebeen found between DAP, DMP, DEP and neurodevelopmental and neonatal outcomes that indicate a role for other organophosphates. This canbe supportedby
a cholinesterase inhibition mechanism ofaction orperhaps some different mechanism. Some panel members believed more stronglythat there was likely a different mechanism ofaction other than acetylcholinesterase inhibitionmediating 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 Agencyhas considered the extent to which data in the epidemiology studies and the deliberate dosing studies canbeused 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 ofOPs on the developing brain. Moreover, theyprovide data which supports the human relevance ofoutcomes observed in animal studies. However, at this time, theyhave notbeen proposed for use in directly deriving the PoDs or UFs.

Each ofthe cohorts has been exposed to chlorpyrifos to some extent. However, in addition to chlorpyrifos, each cohorthas been exposed to multiple pesticides, including other OPs. Determining the quantitative contribution ofchlorpyrifos tothe reported outcomes separate from the other OPs is challenging. This determination would behighlyuncertain giventhe current state ofthe science onthe dose response relationships for mechanisms (otherthan AChE inhibition) leading to effects onthe developing brain. As the science evolves in this area, the understanding ofTK and TD factors which impact toxicityto the developingbrain 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 linkagebetweenblood orurinary levels of chlorpyrifos and/or its metabolites withhealth outcomes. At this time, the Agencyhas used the reported levels ofchlorpyrifos and its metabolites simply as markers ofexposure without an attempt to estimate actual exposure or dose to the tissues. The Agency is aware ofan effortby 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 inWhyatt et al (2003). This work has onlyjustbegun and will likelytake several years. Please comment on theAgency's conclusion to
use the epidemiologicalstudiesprimarily forpurposes of hazardcharacterization andnot for dose response assessment. Please also comment on the scientific support for or against this preliminary conclusion.

44Panel Response

The Panel concurred with the Agencythat there are key limitations to the three epidemiological studies and that they shouldbeused 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 ofneurodevelopmental effects from low-'level in utero exposures ofchlorpyrifos. The majorityposition was that current epidemiological datado notprovide sufficient evidence to increase the uncertainty factor for the cholinesterase inhibition to accommodate the possibility of neurodevelopmental effects. However, somepanel members felt strongly that the current epidemiological data do provide evidence to indicate that the margin ofsafety shouldbe increased. The Panel recommends that the Agency conduct a full formal weight ofevidence evaluation for causality ofthe reported associations between exposure to chlorpyrifos and neurodevelopmental outcomes in the existing epidemiological database.

One ofthe key limitations ofthe epidemiological studies is that the exposure data were collected at single timepoint and lack information onthe long-term exposure level and duration. A second limitation is that the subjects in two ofthe cohort studies (conducted bythe Mt. Sinai and University ofCalifornia-Berkeleyresearchers) had multiple chemical exposures including multiple AChE-inhibitinginsecticides (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 maybemore difficult to use neurobehavioral endpoints in dose-response assessment as they are qualitative in nature. Another limitation is that neurodevelopmental changes are causedbymultiple 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 ofgreat value for assessing risks to human health. Besides epidemiological studies and deliberate dosing studies potential sources for data on the effects ofchlorpyrifos in humans may come from poison control information on accidental and intentional exposure (decreased since 2001 when indooruses ofchlorpyrifos were voluntarily cancelled) as, well as from occupational exposure data from workers in manufacture, formulation, transportation, and application ofchlorpyrifos (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 otherpesticides or agents), vehicle etc. Confounders regarding the exposure are most commonlyrelated to dose, route, and duration ofexposure. Confounders associated with the subjects enrolled in the study mayinclude age, gender, health/disease status, personal environment, and genetic factors such as isoforms and transporters inkeytoxicological events. Within the chlorpyrifos epidemiological datasets, determining the quantitative contribution ofchlorpyrifos in exposures to mixtures maybepossibleby accounting for differences in chemical potency, pharmacokinetics and possible chemical interactions. Further

45resolution ofdose-response relationships may also come from the on-going data analysis bythe ColumbiaUniversity study group and any otherlong-term studies with improved exposure information and more quantitative measure ofchlorpyrifos toxicity.

The Panel recommended that the Agency conduct a full weight ofevidence evaluation for the neurodevelopmental outcomes. This generally requires a much wider scope ofreview that encompasses all available data; not onlydata for the given chemical, but also data for chemicals ofsimilar structure and activities, e.g., OPs. To increase transparency and defensibility ofthe conclusions concerning thepotential role ofchlorpyrifos in contributing to neurodevelopmental outcomes, the weight ofevidence analysis should specifically address criteria for causality including consistency, and take into account the results not only ofthe studies considered here but others and the supportingbiological data. Such an exerciserequires explicit consideration of criteria such as strength, consistency, specificityrelated to chlorpyrifos orto its anticholinesterase effects common to OPs as a whole, dose-response, temporal concordance and biological plausibility in a framework analysis similarto that which is conducted currently for hypothesized modes ofaction. This allows comparative analysis across assessments of consistency ofweight ofevidence determinations. The weight ofevidence analysis might increase confidence in this case and potentially identify additional relevant analyses to address**unc~rtainties** such as the role ofotherpesticides in the observed associations. To the extent possible, once a hazard is established, an array ofdose-response data inboth animals and humans inthe context oftheir relative associated uncertainties may also behelpful, recognizing that the magnitude ofthese uncertainties for the dose-response may generallybe greater for animal species, owing to interspecies differences. Outliers need to be excluded, and the relative magnitude ofuncertainties explicitly weighted in considering the relative contribution of epidemiological versus other types ofdata.

The Panel agreed with the Agencythat there were limitations in the three epidemiological studies thatprecluded them from beingused to directly derive the PoD or the uncertainty factor.The ColumbiaUniversity cohort study couldbeused to determine bounding values for the levels of chlorpyrifos that might cause a measurable effect. In a similarway, data from epidemiological studies can also beused in risk assessment. .The use ofa PBPK model would enable estimation ofan exposure dose metric for multiple sources ofexposure, e.g., air, food, water.

For example, one Panel member estimated the blood concentrations ofchlorpyrifos from the PBPKmodel provided byDr. 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 ofthe California air concentrations of chlorpyrifos as stated in oral public comments provlded byDr. Sass ofthe Natural Resources Defense Council (NRDC and representing the Pesticide Action Network- North America (PANNA), September 16, 2008). Based onthis analysis, the simulated chlopryrifos blood concentration was approximately 1x10-4 f.tmol/L, or 35 ng/L, when calculatedusing a peak air concentration of 1.34 ug/m3

. Somepanel members raised concerns about this computation.3 In

3 One panel memberprovided some additional notes to clarify the vague concerns expressed at the SAP meeting. This information will be useful to the Agency. Review ofpublic comments in the docket (EPA-HQ-OPP-2008- 0274-0056,) indicated that the 24-hour average concentrations were below theadult RELs (recommended exposure levels). This finding raises a concern about the calculations made by one panel member. A lag time is needed to

46this illustration, potential exposures to a blood concentration of35 ng/L chlorpyrifos would be nearly 6 times greater in comparison with the measured chlorpyrifos blood concentration, of6.17 ng/L reported in the Columbia University study. In the ColumbiaUniversity study, levels of 6.17ng/L chlorpyrifos inblood were associated with neurobehaviornal effects. The Panel urged the Agencyto more carefully analyze these current data to determine whetherindividuals exposed to potentiallyhigher levels ofchlorpyrifos in air would show signs of neurodevelopmental effects. 

### In a similar fashion, data from the epidemiological studies couldbe used inrisk assessment as bounding values to evaluate exposure standards, i.e., reference concentrations or doses. Based on the level ofanalysis that can beperformed, Panel members suggested that the Agencymightbe able to use the cohort data in a more quantitative fashion as part ofthe risk assessment, such as in the aforementioned boundary setting exercise.

While the potential contribution ofthe chlorpyrifos PBPKmodel being developed was recognized, some panelists believed that the Agency should also pursue a simpler PBPKmodel specifically applicable to the chlorpyrifos data that wouldbe available in a relatively short timeframe. Some panel members urged the Agency to not delay in studying whetherthe level of chlorpyrifos in air, as noted in the NRDC and PANNA comments, poses a concern for human health.

The Panel recommended the consideration ofa fetal orplacental compartment in a PBPKmodel 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) TKvariations as well as inter-species differences. Furthermore, the Panel noted that additionalwork as a basis to consider more robustly interspecies differences (using central estimates) maybemore fruitful over the shorter term.
b. Three deliberate dosing studies in adult (non-pregnant) humans are available which
measure AChE activity and urinarylevels ofchlorpyrifos and/or its metabolites (See Appendix G). The Agencyhas 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 ofthe developing nervous system to chlorpyrifos mayberelated to cholinergic and noncholinergic mechanisms. Findings in epidemiology studies in children support the animal studies. The human studies do not include the potentially susceptiblepopulations being evaluated in the current effort, namelypregnant 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 ofchlorpyrifos and blood levels, so that peak concentrations lasting
one hour should notbe used for calculating blood concentrations. A time-weighted average mustbeused instead, which would lead to lowerblood levels.

47• Nolan et al. (1982) and Griffin et al. (1999) onlyinclude a single dose group for a particularroute (a study design issue previously criticizedbythe Human Studies Review Board (HSRB) with respect to otherhuman 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 ofthese studies as NOAEL only studies (a type ofstudynot supported bythe HSRB for use in risk assessment since absence ofan effect (LOAEL) raises questions about whether the investigators were able to detect an effect orthat it was possible given the study design).
However, the Agencyhas determined that the human studies do provide valuable information on correlating oral or demial exposure with levels ofchlorpyrifos and/or TCP inblood and urine. In addition, these studies also provide information on time course ofabsorption, metabolism, and excretion. Kisicki et al. (1999) also includes PON1 genotype information. Due to the availability ofquality TKinformation, these studies havebeenused inthe pastbythe Agencyto aid in interpretingbiomonitoring data. Specifically, results from Nolan et al. (1982) havebeen used previouslybythe Agency in estimating (i.e., back-calculating) chlorpyrifos exposurebased on urinary levels ofTCP. Nolan et al. (1982) has also been used to derive a dermal absorption factor in humans. Ifthe Agency wishes to continue this use ofthehuman studies to assist in characterizing and interpreting epidemiology and biomonitoring data, the studies will bebrought to the Agency's HSRB for review oftheir scientific and ethical conduct. To assist theAgency in preparing for this review by the HSRB, please comment on the clarity and completeness of the Agency'sscientific analysis of the human studies. Inparticular,please focus on whether the Agency has identified the key scientific issues andwhether other information orstudies are available that shouldbe considered informulating theAgency'spreliminary conclusion to use these studiesforpurposes of characterizing and interpreting the epidemiology and biomonitoringdata andnot for deriving PoDs or UFs.

Panel Response

The Agency's issuepaper clearlylaid out EPA's scientific analysis ofthehuman studies and identified desirable components that are missinginthese human studies. Onepanel 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 ofthe studies provided to the Panel.
Overall, the Panel agreed that the human deliberate dosing studies contain scientificallyuseful information for risk assessment, but not for directly establishing PoD or uncertainty factors. The following major limitations ofthese data were noted bythe 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 beforebrain 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 ofenvironmental exposure levels.

48• These studies do notprovide information on any other toxicological endpoints besides cholinesterase inhibition.
• These studies do not include the inhalation route, a major route ofexposure for workers and bystanders alike.

One panelistprovided the following information to the Panel from his unpublished study conducted on a small cohortofchildren (n = 50) examining cholinesterase activity and PON1 genotype as a result ofchlorpyrifos 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 otherhand,two other**chil~ren** showed a 10-fold variation in cholinesterase activity even though theyhad 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 ofthe observed differences in cholinesterase activity maybedue to differences in inhalation or dietary exposures rather than dermal exposure (theroute ofexposure 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 ofuncertainty factor. However, these unpublished results do confirm that genetic differences between individuals in a populationmay alter the exposure to toxic chlorpyrifosmetabolites.

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 ifno other source ofdatais available. However, the current 3% absorption factor should be further refined byconsidering the different exposure scenarios. Taken together, these studies provide more robust database than what each single study can offer. They span a wide range ofexposures, 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-offdurations (as short as 4 hours in the Meuling et al. (2005) study), and using different vehicles.

The Panel recommended that ifthe 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 shouldbeused as a reference point instead ofthe 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 ofthese studies should be considered as uncertainties, e.g., exposure onlyon forearms and shorter duration ofskin contact than the anticipated human exposures. The Panel was familiar with how the 3% dermal absorption factor used bythe Agency (see EPA's 2000 chlorpyrifos risk assessment) was derived based onthe 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 studyto the 21-day dermal LOAEL of 10 mg/kg/day in rats. Because it is not likelythat the dermal absorption factor derived in humans would closely coincide with the values for rats, and given

49that the comparison oftoxicity at the LOAELs is crude, the support from this comparison would notbe crucial.

The Panel agreed with the Agency scientific analysis that these deliberate dosing studies cannot beused to directly establish a PoD orUFs. ThePanel indicated that these datamightbeused as bounding levels similar to what was suggested bythe 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 theblood levels in the deliberate dosing and epidemiological studies,.and considered it criticallyimportant to maximallyuse the information from these studies. These studies·couldbeused forpurposes of interpreting (atleast crudely) the nature ofdose-response in the epidemiological data and as a basis to "bound" the reference doses/concentrations. The Panel encouraged the Agencyto consider the use ofa PBPKmodel to widen the application ofthese bounding data for current or potential human exposures and for the final reference dose or reference concentrations. In addition, thesehuman studydatamay contribute to an array ofthe dose-response data inboth animals and humans mentionedunder question 5a, but in the context oftheirrelative associated uncertainties.



### 6. Points ofDeparture (PoD) for RiskAssessment (Issue Paper, Section 2.5):

a. Based on the results ofthe extensive literature review, the Agency has proposed updated PoDs derived from the laboratory animal studies for extrapolatinghuman risk. The Agencyhas posed three optionsfor 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 ofchildbearing age and children).


### The first option proposes to use the PoDs which were based on rat RBC andplasma 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 ofthe evidence discussion primarily on adultrat and dog AChE guideline studies and adult data from Zheng et al. (2000). This option would involve application ofthe 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 of0.1 mglkg/day derived from multiple studies and lifestages. This proposed PoD would be applied to all populations and all durations. The proposed value of0.1 mglkg/day was derived usingbenchmark 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 onthe developing brain reported in other laboratory animal studies and thus is expected to beprotective for those effects.



### 50The third option is a blend ofoptions 1 and 2. This option proposes to use a value of0.1 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 of0.03 mg/kglday from the 2000 risk assessment for oral exposures. The value of0.03 mg/kglday was derived for the 2000 risk assessmentbased 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 oftoxicity which can occur following repeating doses ofchlorpyrifos in adult studies. Please comment on the strengths andweaknesses of eachproposedapproach. Is there another option or a variation of one of the three options that theAgencyshouldconsider?

Panel Response
General comments: Theprincipal focus ofthe questionsfrom 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 minimizeuncertainty, to rely to the extentpossible onbenchmark doses (BMDs) versus no- orlowest-observed-(adverse)-effect levels (NOAEL or LOAEL) and to take into consideration the sensitivity ofall life stages.
Members ofthe panel also noted that it was difficult to separate fully the discussion ofpoints of departure from that ofuncertainty factors so that the contentherehas relevance to the responses to Question 7 and vice versa (i.e., the adequacy ofproposed uncertainty factors is necessarily related to the nature ofthe point ofdeparture). As a result, comment is also included herein on residual uncertainty associated with proposed or preferred points ofdeparture including that related particularlyto potential lack ofadequate consideration ofall 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 ofaccount ofpotentially sensitive life stages. Specific Comments: There was consensus that ofthe three options proposed bythe 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 ofseveral studies, for which results converge around the same value. Based on available data, most ofPanel members (a few disagreed) stated that the PoD presented in Option 3 is also believed to beprotective for effects
on the developingbrain, although it is based on cholinergic effects. However, this conclusion is highlyuncertain, given the lack ofinformation onthe mode ofinduction ofthe observed behavioral effects and in light ofevidence from in vivo and in vitro studies that non-cholinergic mode ofaction(s) are likelyinvolved in the adverse developmental neurotoxicity (DNT) and

51behavior endpoints. The Agencyis encouraged, therefore, to explicitly address this uncertainty in their additional deliberations in derivation ofthe reference dose/concentrations.

The Panel suggested that ifthe Agency wanted to test whetherthe cholinergic pathways are an appropriate endpoint for the PoD then an appropriate temporal dose-response studyusing the most sensitive administration method and the most sensitive life-stage comparing DNT/behavior with cholinesterase inhibition wouldbeneeded. This would test thehypothesis that cholinergic pathways are an appropriate endpoint for the PoD, even though such a studywould likelynot elucidate specific non-cholinergic mode ofaction(s) for DNT/behavior effects. Relevant design would need to take into account differing sensitivity in various species to detect effects on the developingbrain.

The neurobehavioral developmental effects seen in the three epidemiological studies supported the retention in Option 3 ofthe lower PoD for repeated exposures used in EPA's 2000 chlorpyrifos risk assessment to account for potential cumulative effects. Forrepeated exposures, PoD of0.03 mg/kg/day is supportedby a BMDLw of0.03 mg/kg/day for RBC AChE inhibition inpregnant dams (see Agency issuepaper, p. 18, Table 2). ThePanel also recommended that the Agency additionally investigate developing appropriatebenchmark doses (BMDs) for the chronic PoD determination.



### More detailed comments: Option 1: Option 1uses therat RBC and plasma ChE inhibition in the Agency's 2000 risk assessment (Table 1 ofthe Agency's issue paper), weight ofevidence (WQE) approachbased primarily on adult rat and dog AChE guideline studies, NOAELs for various routes and durations ofexposure with an acute oral dose of0.5 mg/kg/day and a repeated oral dose of0.03 mg/kg/day. Option 1 is the status quo used inthe Agency's 2000 chlorpyrifos risk assessmentbased 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 itavoids uncertainties associated with interroute extrapolation. However, it is based onno-or lowest-observed-advese-effect-levels rather thanbenchmark doses; moreover, the dermal and inhalationNOAELs were determined in mature animals and did not includeprotection for pre- and post-natal sensitivity. Option 2: Option 2 uses studies that provide data amenable to benchmark dose (BMD) modeling; RBC AChE studies inrepeated gestational studies in dams (heart) and acute post-natal pups (brain and RBC AChE) provide BMDs (BMD and BMDL10) in the samerange of0.06- 0.12 mg/kg/day. TheAgencyproposes to use the weight ofevidence approach, a PoD of0.1 mg/kg/day for oral exposure, all age groups and all durations.

For Option 2, a value of0.1 mg/kg/dayis beingproposed for all populations and durations. It is based onbenchmark doses/concentrations which offer some advantage overNOAELs/LOAELs though it needs to berecognized that these advantages canbe small inrelation to relative uncertainties associated with interspecies and intraspecies differences. It does draw from



### 52multiple 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 withinthe same range and the approach offers considerable advantage in drawing maximally onthe available data in animals at several life stages. Some panel members stated that 0.3 mg/kg/day was protective because itis 3- to 10-times lower than levels causing effects on the developingbrain. Thelowest dose tested for learning/memory was 0.3 mg/kg/day so proposed PoD was 3-fold lower and 10-fold lowerthan lowest dose used in many gestational and post-natal studies which evaluated toxicities otherthan AChE inhibition. Nevertheless, the degree ofprotectiveness is uncertainbecause the dose-response relationship cannotbe 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 onNOAELs and LOAELs for plasma and RBC ChE inhibition in 5 studies. This option is also supportedbythe 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 mightbe 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 oftoxicity which can occur following repeated doses. This option was preferred bythe Panel, on thebasis ofpotential for different orperhaps multiple actions ofchlorpyrifos at repeated low level exposures and results ofthe recent epidemiological studies. Most ofpanelmembers (a few disagreed) stated that this option was protective ofeffects on the developingbrain, although it is based solely on cholinergic effects. However, this conclusion has associated uncertainties; the lack ofinformation on the mode of action in inducing the observedbehavioral effects and evidence from in vivo and ili vitro studies that show non-cholinergic mQdes ofaction are likelyto beinvolved 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, ormetabolism. Inthe case ofchlorpyrifos, dermal and inhalation studies are available which identifyNOAELs for these routes in adult rats. Withrespectto inhalation exposure, there are two nose only studies with vapor chlorpyrifos whichprovides a NOAEL of287 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 of5 mg/kg/day. These studies do not include pregnant dams, fetuses orpost-natal pups and therefore do not considerpotentially susceptible populations. In the absence ofdatain these groups, the Agency will continue to useroute specific studies, as appropriate. An alternative for dermal exposure is to use an oral Pod derived

53from susceptiblepopulations (as discussed above) with a dermal absorption factor. Specifically, the Agency could use a dermal absorption of3% from human subjects (Nolan et al., 1982). Please comment on the strengths andweaknesses associatedwith use of the adult dermal and inhalation studies in the chlorpyrifos human health risk assessment. The Agency also requests the SAP toprovide suggestions onpotential toxicity and/or toxic kinetic studies (if any) in pregnantdams,fetuses, and/orpost-natalpups which couldbe conducted to better inform the dermal and inhalation riskassessments.
Panel Response

The Panel was in general agreement withthe Agency's proposed option to use the dermal and inhalation studies as a basis for development ofthe points ofdeparture for these routes for repeated exposures, as perthe 2000 chlorpyrifos risk assessment given the uncertainty associated with route to route extrapolations based on available data, thoughresidual uncertainty concerning sensitivelife stages would need to be taken into account. The Panel also noted that theproposed oral, inhalation and dermal PODs are roughly equivalent (0.1 mg/kglday), taking into account estimated absorption from the Agency's 2000 chlorpyrifos risk assessmentbased 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 of0.1 mg/kglday should also be adjusted for repeated exposures based on the 5 days per week dosing regimen.
Also, rather than relying on thepotentiallyproposed value of3% for dermal exposure derived in the deliberate dosing studyin humans byNolan 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 ofchoice, as a basis to applyit across all routes of exposure consistently. Physiologicallybased pharmacokinetic modeling maybehelpful in this context. While the Agency is pursuing development ofa PBPK model that includes the fetal compartment, the Panel also encourages consideration ofsimplermodels which might additionally inform comparison ofage- and route sensitivityin the nearterm future. In relation to dermal exposure, the Panel suggested a more relevant species, e.g., minifies, maybehelpful.
Detailed Comments:
Giventhe magnitude oftheuncertainties to extrapolatebetween routes, use ofroute specific data for inhalation and dermal exposure as a basis for points ofdeparture was supported. However, the residual uncertainty for the lack ofrelevant data in pregnant dams, fetuses orpost-n.atal pups exposed bythese routes needs to be additionally addressed.

Inherent in calculating the absorbeddose from the inhalation NOAEL is the recognition that the dose level should be somewhat comparablebetween routes for the same systemic endpoint, although rout-specific pharmacokinetic differences canbe a significant factor. Some comparisons between dermal orinhalationNOAELs and the oral PoD are presented below.

I

54

### DermalNOAEL of5 mg/kg Using the Agency's suggested 3% dermal absorption factor derived from the deliberate dosing studyinhumans ofNolan 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 oforal human absorption estimated from the same study (i.e., Nolan et al., 1982) is 73%, the oral NOAEL of0.1 mglkg/day would need to be adjusted by 73% ofits original value for this comparison. Thus, the acute dermal NOAEL of5 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 ofthe 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 ofrate ofpenetration on different skin surfaces, moisture on the skin under the cover ofclothing, contact duration before wash off.


### Inhalation NOAEL of0.1 mg/kg/day Accordingto the Agency's Issue Paper, the NOAEL of20 ppb from the two inhalation studies in rats result.ed in an absorbed dose of0.1 mglkg/day, the same value as in Option 2. The value, as calculated, is therefore applicable to acute exposures. Forintermediate and chronic inhalation exposure, the absorbed dose of0.1 mglkg/day shouldbe adjusted down by·a factor of5/7 to account for the 5 days/week dosingregimen. This is slightlylower than the 0.1 mglkg/day value
as proposed in Option 2.
Overall, the Panel encourages the Agencyto review the entire database from all doses and all studies (notjustthe Nolan et al. (1982) study) to obtain quantitative understanding ofage sensitivity specific to the endpoint ofchoice and apply it across all routes ofexposure consistently. Physiologically-based pharmacokinetic modeling maybehelpful in this context. While the Agency is pursuing development ofa PBPK model that includes the fetal compartment, the Panel also encourages consideration ofsimplermodels which might additionally inform comparison ofage- and route sensitivity inthe 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 variabilityis performed. Historically, the Agencyhasused default 10-fold factors to account 'for inter- and intra-species extrapolation. More recently, emphasis on the derivation ofextrapolation factors from TK and toxicodynamic (TD) data instead ofdefault factors has increased. With the intent ofimproving the scientificbasis for the chlorpyrifos risk assessment, in this issue paperthe Agencyhas considered the availability ofcurrent PBPK

. models, TK, and TD data for chlorpyrifos to use in animal to huinan and withinhuman extrapolations. Overall, the available PBPK models, although well-developed and supported for n<?n-pregnantadults, do not include calculations for dose during pregnancy (e.g., no placental compartment) and for young children less than 5 years old and thus cannotbeused in a quantitative manner for this effort. As such, the Agencyhas used the 2005 IPCS guidance on Chemical-Specific Adjustment Factors to evaluate available TK and TD datain animals and



### 55humans 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: TheAgencyhas preliminarily concluded that with regard to TD characteristics, due to the likelihood ofseveral possible modes ofaction ofneurodevelopmental toxicity ofchlorpyrifos and lack ofidentifiable and quantifiable key events for MOAs not related to AChE inhibition, the Agency cannot confidently refine the TD component ofthe animal to human and within human variability factors (i.e., UFAD and UFHo). Please comment on the scientific support for oragainst the use of default factors of inter- and intra-species TD extrapolation.

Panel Response

In response to the question ofwhether the Agency can refine the toxicodynamic (TD) component with respect to uncertainty ofthe animal to human and within-human variability, the Panel was in agreementwiththe Agencythat there is not sufficient information to confidentlyrefine 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 ofaction of neurodevelopmental toxicity ofchlorpyrifos and there is a clear lack ofidentifiablekey events for mode ofactions not related to AChE inhibition. One panel member commented that the Agency should also considerAChE inhibitionrather thanbehavioral outcomes, 0.03 mg/kg/day
versus 0.3 mg/kg/day, respectively, because ofits higherlevel ofsensitivity. Overall, the lack of identified mode ofaction, lack ofcorrelation ofacetylcholinesterase inhibition with possible non-cholinergic mechanisms and developmental outcomes, takentogether, do not support a dataderived inter-species and intra-species TD extrapolation. All panel members concurred that the application ofuncertainty 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 sensitivitydiscussed in Questions 1 and 2, panel members agreed that animal and human sensitivitiesare greater for fetuses and neonates, although the current data do not allow for determination ofthe magnitude ofthe sensitivity. Until this is known, the Panel recommended application ofdefault factors for the TD uncertainty extrapolation; A few members ofthe 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 informationmight show variations inresponse among developingbrains due to genetics, time ofexposure, pre-existinghealth status and other factors and support the adequacy ofa 3-fold uncertainty factor (UF) for intra-species variation for these endpoints.

In conclusion, until a mode ofaction is identified and supported (i.e., primary target otherthan AChE activity) for neurodevelopmental toxicityis identifiable and consistentlydocumented in vivo, the default uncertainty factors for thetoxicodynamic component should beused. That is, 3- fold and 10-fold, for acute and chronic exposures, respectively, for neurodevelopmental effects.

56b. 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 ofinter- and intra- TK extrapolation (i.e., UFAKand UFHK). Based on differences in rat and humans with regard to maturation ofmetabolic processes, there are uncertainties Surrounding appropriate metabolic parameters for animal to human extrapolation of juveniles. This uncertainty in combination with limited dataprecludes the development ofa DDEF for inter-species TK extrapolation (i.e., UFAK). Thus, the Agericy proposes to apply thedefault 3X for UFAK· Data on carboxylesterases
are not sufficiently robust fot intra-species TK extrapolation (i.e., UFHK). Data on P450s are complicated bymultiple enzymes each with its own maturationprofile. Others have evaluated the P450 literature for use in derivation ofchild specificUFs with poor success(Ginsberg et al., 2004a). Please comment on the scientific support for oragainst the use of default factors of inter-species TKextrapolation. Please further comment on theAgency'spreliminary conclusions
on the utility of carboxy/esterase andP450 data to refine the intra-species extrapolationfactor.

Panel Response

The Panel concurred with the Agencythat 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 UFAK· The Panel unanimously encouraged the Agency to continue to consider the importance ofall 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 ofthe enzyme, but displays genetic polymorphism. There are important differences in the rates ofhydrolysis (enzyme activity) across genotypes (192RR > 192QR > 192QQ), with PON1 R192 allele hydrolyzing chlorpyrifos with a higher catalytic efficiencythan PON1 Q192 allele (Furlong, 2007). However, PON1 activity and polymorphism at position 192 (referred to as PON1 status) is notthe only determinant ofchlorpyrif9s toxicity, since other metabolic pathways maymodulate potential deficits in detoxication capacity. Thus, at least theoretically, the level ofchlorpyrifos-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 (carboxylesterase and butyrylcholinesterase, BChE)). Onepanelmember commented that researchby Sogorb et al. (2008) indicated that even ifa small fraction of chlorpyrifos-oxon leaks from the liver withoutbeing detoxified, it is rapidly bound and inactivatedby serum BChEand 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 inblood offetuses at a concentration of 1ng/g (that equals to 0.003 ).1M) only after the administration ofhigh doses ofchlorpyrifos (5mg/kg/day) by gavage to dams at gestational day 20. ·Such a low concentration of chlorpyrifos-oxon canbe 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 ofchlorpyrifos 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 byPON1 in the blood; the possibilitythat it reaches the· brain is scarce since chlorpyrifos-oxon is a highlyreactive metabolite and undergoes spontaneous hydrolysis (its half57life is less than one minute in blood, see Brzak et al., 1998), so there is not enough time to complete thatjourneyintact. Thus, PON1 probably fails to play a relevantrole at low environmental concentrations (nM to low J.tM). The maximum daily intake ofchlorpyrifos is 0.11 Jlg/k:g/day for infants and 0.24 Jlg/k:g/day for toddlers (see reviewbyEaton et al., 2008; Table 21). At middle and higher concentrations, chlorpyrifos-oxon maybe formed in the brain because ofthelow desulfuration activity ofbrain microsomes and mitochondria (Chambers and Chambers, 1989). However, the brain has no PON1 activity and, therefore, the oxonproduced "in situ" interacts with its target molecules unless itundergoes stoichiometricbinding to BChE
or otherproteins acting as "scavengers".

Lassiter at al. (1998) demonstrated that maternal blood chlorpyrifos-oxonase activity is variable beforebirth, and the placentahas about 20% the activity ofthe 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 ofchlorpyrifos-oxonase activity is high (Km=210-380 J.tM), chlorpyrifos-oxonase in plasma and liverfrom adult animals has be shown to be capable ofhydrolyzingphysiologicallyrelevant concentrations of chlorpyrifos-oxon (nM to low J.tM) whereas the young animal has, in tum, less capacityto detoxify such concentrations ofchlorpyrifos-oxon via chlorpyrifos-oxonase (Mortensen et al., 1996).

One panel memberpointed out that the lack oftissue specific data for both species under similar dose and timing considerations e.g., liver, is a major gap inthe ability to extrapolate from animal data to humans. Others pointed out that there are human data onhuman liver carboxylesterases during maturation, but these data were not considered "sufficientlyrobust" to beuseful in TK extrapolations.

c. Intra-species Toxicokinetic Extrapolation (Within Human Variability): There are extensive data onPON1 from manypopulations worldwide and for differentage groups. Using these data, the Agencyhas performed a preliminary analysis for within TKhuman variability for PON1 activity. These calculations were done in a manner consistent with the IPSC CSAF guidance. The calculations show that the largest variabilityin PON1 activity is between newborns and their mothers and is thus likelyrelated 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 ofOP (active oxons, not the parent components) mustbepresent inthe blood orbrain for PON1 activity to affect toxicitybased on generally low affinity (Km, 0.1-10 mM). Others believe that PON1 status is a key determinant in chlorpyrifos toxicity. The Agencyhas evaluated the available in vivo and in vitro data from animals and humans relevant to this issue. The Agencyhas preliminarily concluded that the available data suggest that PON1 status can notberuled out as a determinant in chlorpyrifos toxicity, particularly for the fetus oryoung child. However, uncertainties remain, particularlyregardingthe degree to which other metabolicpathways modulate potential deficits in detoxication capacity. Please comment on the science which does anddoes notsupportPONJ status as a determinant in toxicity at low environmental concentrations.

58Panel 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 oryoung child. Several panel members stated that actual human exposures varybetweenbystander and applicators (and everyone in-between) and itis difficult to define what exactly a low level of exposure is for humans. Some in the Panel suggested that the PON1 dataset couldbe used to address datauncertainty. However, manyin the Panel cautioned that these data should notbe used out ofcontext until the rate limiting step is identified based on a PBPKmodel. Such a model does not yet exist, but is under development. These panelists believed that the use ofthe PON1 data set without such information would be a misuse ofthe IPCS guidance for determining a CSAF (chemical-specific adjustment factor).

Several members ofthe Panel encouraged the Agencyto obtain an independentpeerreview for the Timchalk PBPKmodel in orderto assess the overall impact ofPON1 relative to the toxicity ofa range ofCPF, including what maybethe choice ofdose metric for such comparison, e.g., BChE inhibition orblood or tissue level chlorpyrifos, chlorpyrifos-oxon, or TCP.

Onepanel memberreminded the panel that there have been no direct experiments looking at what we might considerto below dose exposures in animals in reference to the involvement of PONl. This panel member discussedtwo published model systems that might be ofuse to answer this question: the PON1 knockout (ko) and the PON1-Q192 humanized mice. Inthe PONI ko model ifthere any effects ofPON1 at low dose exposures then thepanelist suggested that these effects wouldbemanifested in this model. The Shih et al. (1998) paper shows that the presence ofPON1 prevents the inhibition ofbrain acetylcholinesterase at a dose ofchlorpyrifos of300mglkg (see discussion in Appendix A (Metabolism), p. 26, Figure 8, Panel E ofthe figure reprinted from Shih et al., 1998). However, themagnitude ofthe differencebetween the wild type and the PON1 ko mice is on further reflection not that great. Bythis panel member's calculations the loss ofacetylcholinesterase 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 ofPONI activity (and not a complete lack ofactivity) there mightbe very little effect.

In the second model system, the PON1 humanized mouse model, ifthere are higher chlorpyrifosoxon doses then there would be a significant difference inthe inhibition ofbrain acetylcholinesterase in mice with either the R192 or Q192 polymorphism (see Appendix E, figure 4, p21). At low doses, it maynot be so clear, particularly as there are no data for the R192 mice atthelowest dose used. The question then remains whether this results in a functional effect that canbe identified. The original paperby Cole et al. (2005) also presents morbidity data in the samemice following these chlorpyrifos-oxon doses (Panel C ofthe same figure). Again there would appear to bemissing data (namelythere does not appear to be any data for the R192 micebelow 1.5 mg/kg. However at 1.5 mg/kg there is no morbidityin 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 notbe significant differences between the R/Q forms inpreventing chlorpyrifos-oxon induced toxicity. This panel member concluded that further data are needed

59butthat current evidence is suggestive that PON1 is not a determinant oftoxicity at low environmental concentrations.

d. The Agency's PON1 calculations have focused on the PON-192Q/Rpolymorphism based largely onthe extensive data available. No calculations have so far beenperformed on other genotypes. Please comment on scientific support for or against focusing on 'the PON192QIRpolymorphism.

Panel Response

The Panel concludedthat the use ofthe PON192Q/R polymorphism in the Agency's calculations for PON1 intra-human variability is appropriate. This conclusion was based onthe available data (Furlong et al., 2005; Brophy et al., 2001). However, the analysis ofthe individual contribution ofeach polymorphism in the promoterregion on serum paraoxonase activity/levels is complicated because ofthe pronounced linkage disequilibriumbetween the promoter and the codingregion polymorphisms (Draganov and La Du, 2004). Thus, it cannot beruled out that the variation observed for one ofthe promoterpolymorphisms (PON1 C-108T) maybe 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) onmothers and infants are the two important sensitive subgroups (infants and those with the Q/R polymorphism). Intra-individual variability could beunderestimated because this population is of similar ethnic descent (i.e., Latina mothers and infants, primarily ofMexican descent).

In the case ofPON1, the sensitive group is the neonates with the QQ genotype. They shouldbe (and are) comparedback to the mothers ofthe QR genotype. Ifdata from other studies exist (could be done now for adults with the data from Brzak et al. (1998), it wouldbe appropriate to combine geometric means and geometric standard deviations by a weighted average approach. This wouldprovide 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 strongrecommendation 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 PBPKmodel is developed then the impact ofactivating and deactivatingpathways and potentiallyrate-limiting components will be identified and information regarding PON1 Q/R polymorphism could beput into context. Such information, if attainable, mightbeused 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.Thepreliminary analysis suggests that within human variabilityis larger than the default 3X when newborns and adults
are considered together. Specifically a value of 12Xhas been calculated for chlorpyrifos-oxonase activity. The Agencyhas proposed two options for these calculations: 1) use the value calculated



### 60for chlorpyrifos-oxonase derived from newborn and mothervalues in Holland et al. (2006) and 2) use the default factor of3X. Please comment on the strengths andweaknesses of each proposedapproach. Please include comments on the statistical approach used in the analysis as a component of your response.
Panel Response

Thepanel reviewed the two approaches and the majorityofthepanel concluded that the currently available data indicate that the UFHK should be the default value of3-fold when newborns and adults are considered together. On the otherhand, 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 ofchlorpyrifos as noted in the epidemiologicalstudies (Rauh et al). Thesemembers 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 bythe Agencyto identify the factor of"12- fold" calculatedbased on chlorpyrifos-oxonase and encouraged the Agency to pursue other approaches based onthe mode ofaction (see 2005 IPCS CSAF guidelines). Manypanel members did not endorse the 12-folduncertainty factor because it focused strictly onPON1 as the focal point oftoxicity, i.e., focused on onlyone enzyme out ofa very complex system. Similarly, onepanel member stated thatuse ofthe 12-folduncertainty factor would be a complete misuse ofthe 2005 IPCS CSAF guidelines in whichthere is a clearneed to define uncertainty factors based onthe endpoint linked to the mode ofaction, neither ofwhich is defined in this case.
Members ofthe panel expressed concern about lack ofdata for non-plasma enzymes, no biologica1 reason or data given supportthe notion that maturational differences in one tissue relate to another. The data onthe relative contributions ofP450, carboxylesterases and butyrylcholinesterase to the ultimate concentration ofoxon inthe target tissue(s) is insufficient to derive a data-derived uncertainty factor, as EPA has concluded. Members ofthe panel stated that the information on PON1 polymorphisms should notbeused as the sole factor in a dataderived uncertainty factor for two main reasons: 1) it is onlyone 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 itbyCYP450): and 2) the genotype ofPON1 alone is insufficient to predict vulnerabilitybecause the overall level of enzyme activityis ultimately what determines detoxicationpotential from that pathway; thus, it is better to use PON1 status because itprovides informationregardingboth PON1 genotype and activity. Some ofthe data from laboratory animal studies in PON knockout animals are using an unrealistic animal model and frequently veryhigh dose levels, and do not reflect what might happen inhumans.

When all ofthe kinetic parameters including those for PON1 genotype were placed into the Timchalk and Poet PBPKmodel, PON1 genotype only influenced chlorpyrifos-oxon concentration at the higher doses, but did not appreciably influence chlorpyrifos-oxon levels at the lower more environmentallyrelevant levels. Therefore several panel members concluded that

61it is notreasonable to base an uncertainty factor on PON1 genotype alone and the suggested factor of 12 is unreasonable.

Onepanelist 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 beto use the 99th (or 1st) percentilerather than the 95th (or 5th) to protect a greaterportion ofthe population, and correct for thebias in estimatingthe GSD from the arithmetic summary statistics. Thejustification and approach to correcting thebias introduced whenusing arithmetic summary statistics is available in a manuscript currentlyinpreparation bythis panelist (Lynch et al., (2008)). An example ofhow this method canbe 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 501n %tile QR 501n %tile QR Mothers/ Mothers/ 51h%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 ofQuestion#7 (a-e), several panel membersrecommended the following:

1. Establish the UFs for chlorpyrifos based on the PoD as given in any ofthe three options presented in question 6, and the concern for developmental neurotoxicity as indicated in the epidemiological studies.

2. Because the Agency's choices ofPoD are all based on cholinesterase inhibition (AChE inhibition), the Panel stated that it would bemost 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 neurotoxicitybased
onthe 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 inthe formation/removal ofthe oxon is necessaryto adequatelypredictthe concentration ofactive metabolite in target tissues at environmental**do~es.** Because ofthe complexity ofthe pathway, focusing on one enzyme that is likelynot the rate-limiting step won't allow for thebest, nor entirepicture, ofintra62humanvariability estimates. Instead, the Agency is encouraged to pursue theuse ofa "simple" PBPKmodel that can integrate all keytoxicokinetic and toxicodynamic factors and evaluatetheir contributions to the overall**o~tcome** ofendpoint (e.g., AChE inhibition) or related dose metric ofinterest (e.g., profiles ofchlorpyrifos, chlorpyrifos-oxon, and the chlorpyrifos-specific urinarymetabolite, TCP) at various chlorpyrifos exposure scenarios and for various life stages. The Panel noted that the Agencyhas made substantial progress in exploring the use ofPBPKmodel with a few remaining pieces yet to be completed for a preliminaryreview (Timchalk, 2008; comments to the docket, EPA-HQ-OPP-2008-0274). The Agency is encouraged to bring this model to a stage ready for independentpeer review inthe very near future, without waitingfor the newly funded model. Such a model is expected to berevised as needed, and as new informationbecomes available in the future.

Data-derived extrapolation factors (i.e., uncertainty factors, UFs) are predicated on at least
someunderstanding ofthemode ofaction for the critical effect. DDEFs (also called, CSAFs) need to bebased 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 ofchlorpyrifos-oxon reaches the blood stream) and brain. There is uncertainty about whether the effect is mediatedbythe AChE inhibition pathway and even ifso, a PBPKmodel is needed to characterize therelevant activation and detoxication at relevant concentrations or doses, as a basis to replace defaults.

Onepanel member stated that the replacement ofdefault uncertainty factors is more often achievable for interspecies differences because ofthe attempt to capture ratios ofcentral estimates for the relevant animal species and humans vs. population variabilityin the human population, which requires considerablymore data. This panel member emphasizedhow misleading it is use the PON 1 activity data as a basis for replacement ofdefault, in this case, given that it is an inappropriate surrogate for human variability.**Sever~l** panel members recommended "bounding" the reference doses developed onthe basis ofthe animal studies, taking into account the dose-response information from the human deliberate dosing and epidemiological studies, butnot inrelation to the points ofdeparture. This is because the dose-response information needs to be interpreted in the context ofwhat
weknow about interspecies and intraspecies differences, such that thebounding exercise using animal studies can be tested using human data.

One panel member expressed overall concern about using PBPKmodels due to lack of transparency. This panelistbelieved thatthere is a blurring ofinputs/outputs based onwhat is known (i.e., data are missing) versus what is predicted. Onlywhen the predictions ofthe models can be verified with empirical data should theybeused.

In the interim, the majority ofthe 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 ofsensitivity (i.e., intra-species differences). Some panel members recommended that the Agency should considerthe use ofadditional UFs to address the
concerns for developmental neurotoxicity as indicated in epidemiological studies. The

63default UF maybemodified in the future when information onthe mode ofaction for the developmental neurotoxicitybecomes available.

64

### REFERENCES
Abou-Donia, M.B., W.A. Khan, A.M. Dechkovskaia, L.B. Goldstein, S.L. Bullman, S.L., A. Abdel-Rahman. 2006. In utero exposure to nicotine and chlorpyrifos alone, and in combinationproduces persistent sensorimotor deficits and Purkinjeneuron loss in the cerebellum ofadult offspringrats. Arch. Toxicol. 80:620-631. Aldridge, J.E., E.D. Kevub, F.J. Seidler, T.A. Slotkin. 2005. Developmental Exposure of Rats to Chlorpyrifos Leads to Behavioral Alterations in Adulthood, Involving Serotonergic Mechanisms and Resembling Animal Models ofDepression. Environment. Health Perspect. 113: 527-531.
Anderson, G.D. 2006. Usingpharmacokinetics to predict the effects ofpregnancy and maternalinfant transfer ofdrugs during lactation. Expert Opin. Drug Metab.Toxicol. 2:947-960. Atterberry, T.T., W.T. Burnett, J.E. Chambers. 1997. Age-related differences in parathion and chlorpyrifos toxicityinmale rats: Target and nontarget esterase sensitivity and cytochrome P450-mediated metabolism. To.xicol. Appl. Pharmacol. 147:411-418. Augustinsson, K.B. and M. Barr. 1963. Age variation in plasma arylesterase activity in children. Clin. Chim. Acta 8:568-573. Ballough, G. P.H., R.K. Kan, J.D. Nicholson, D.M. Fath, C.P. Tompkins, G.M. Moffa, M.G. Filbert. 2008. Brain damage from soman-induced seizures is greatly exacerbated bydimethyl sulfoxide (DMSO): Modest neuroprotectionby2-aminoethyl diphenylborinate (2-APB), a transient receptor potential channel inhibitor and inositol 1,4,5-triphosphate receptor antagonist. J. Med. CBRDef. 6: 1-20. http://www.jmedcbr.org/issue 0601/Ballough/Ballough 03 08.html. Accessed December 5, 2008.
Barker, D.J.P. 2004. The Developmental Origins ofAdultDisease. J. Amer. Col. Nutrition23: 588S-595S.
Boobis, A.R., S.M. Cohen, V. Dellarco, D. 'McGregor, M.E. Meek, C. Vickers, D. Willcocks, and W. Farland. 2006. IPCS framework for analyzing the relevance ofa cancermode of action for humans. Crit. Rev. Toxicol. 36: 781-792. Brophy, V.H., R.L Jampsa, J.B. Clendenning, L.A. McKinstry, G.P. Jarvik, C.E. Furlong. 2001. Effects of5' regulatoryregion polymorphisms onparaoxonase-gene (PONt) expression. Am.J. Hum.Genet. 68:1428-1436.

- Brzak, K.A., D.W. Harms, M.J. Bartels, R.J. Nolan. 1998. Determination ofchlorpyrifos, chlorpyrifos oxon, and 3,5,6-trichloro-2-pyridinol inrat and humanblood. J. Anal. Toxicol. 22: 203-210.

65Buratt,i F.M, A. D'Aniello, M.T. Volpe, A. Meneguz, E. Testai. 2005. Malathionbioactivation in the human liver: the contribution ofdifferent cytochromep450 isoforms. DrugMetab. Dispos. 33:295-302.
Buratti, F.M., C. Leoni, E.Testai. 2006. Foetal and adult human CYP3A isoforms in the bioactivation oforganophosphorothionate insecticides.Toxicol. Lett. 167:245-255.
Burlina, A., E. Michielin, L. Galzigna, 1977. Characteristics and behavior ofarylesterase in human serum and**liver~** Eur. J. Clin. Invest. 7(1): 17-'20.
Butte, N.F. 2000. Carbohydrate and lipid metabolism inpregnancy: normal compared with gestational diabetes mellitus. Am. J. Clin. Nutr. 71(5S):1256S-61S.
Carr, R.L. and C.A. Nail. 2008. Effect ofdifferent administrationparadigms on cholinesterase inhibition following repeated chlorpyrifos exposure inlate preweanlingrats. Toxicological Sciences. In press. PMID: 18703558
Carr, R.L., H.W. Chambers, J.A. Guarisco, J.R. Richardson, J. Tang, J.E. Chambers. 2001. Effects ofrepeated oral postnatal exposure to chlorpyrifos on open-field behavior injuvenile rats. Toxicol. Sci. 59: 260-267.
Chambers, J.E., and H.W. Chambers. 1989. Oxidative desulfuration ofchlorpyrifos, chlorpyrifos-methyl, and leptophos byratbrain and liver. J. Biochem. Toxicol. 4:201-203.
Chambers, J.E., H.W. Chambers, J.E. Snawder. 1991. Target site bioactivation ofthe neurotoxic organophosphorus insecticide parathion in partiallyhepatectomized rats. Life Sci. 48:1023-1029.**Chambers~** J.E., J.R. Munson, H.W. Chambers. 1989. Activation ofthephosphorothionate insecticide parathionbyratbrain in situ. Biochem. Biophys. Res. Comm. 165:327-333.
Chakraborti, T.K., J.D. Farrar, C.N. Pope, C.N. 1993. Comparative neurochemical and neurobehavioral effects ofrepeated chlorpyrifos exposures in young and adult rats. Pharmacol. Biochem. Behav. 46:219-224.
Choi, K., H. Joo, R.L. Rose, E. Hodgson. 2006. Metabolism ofchlorpyrifos and chlorpyrifos
oxon byhumanhepatocytes. J. Biochem. Mol. Toxicol. 20:279-291. Clancy, B., Finlay B.L., R.B. Darlington, K.J.S. Anand. 2007. Extrapolatingbrain development from experimental species to human. Neurotoxicology 28: 931-937.
Cole, T.B., B.J. Walter, D.M. Shih, A.D. Tward, A.J. Lusis, C. Timchalk, R.J. Richter, L.G. Costa, and C.E. Furlong. 2005. Toxicityofchlorpyrifos and chlorpyrifos oxon in a transgenic
mouse model ofthehuman paraoxonase (PON1) Q192R polymorphism. Pharmacogenet Genomics 15: 589-598.



### 66Czekaj, P., A. Wiaderkiewicz , E. Florek, R. Wiaderkiewicz. 2000. Expression ofcytochrome CYP2B112 in nonpregnant, pregnant and fetal rats exposed to tobacco smoke. Acta Biochim. Pol. 47:1115-1127.
Dam, K., F.J. Seidler, T.A. Slotkin. 2000. Chlorpyrifos exposure during a critical neonatal period elicits gender-selective deficits in the development ofcoordination skills and locomotor activity. Dev. Brain Res. 121: 79-189.
DeMatteis, F. 197 4. Covalentbinding ofsulphur to microsomes and loss ofcytochrome P450 during oxidative desulphuration ofseveral chemicals. Mol. Pharmacol. 10: 849-854.
Draganov, D.I. and.B.N. La Du. 2004. Pharmacogenetics ofparaoxonases: a briefreview. Arch. Pharmacol. 369: 78-88.
Duysen, E.G., B. Li, S. Darvesh, 0. Lockridge. 2007. Sensitivityofbutyrylcholinesterase knockoutmice to (-)-huperzine A and donepezil suggests humans with butyrylcholinesterase deficiencymaynot tolerate these Alzheimer's disease drugs and indicates butyrylcholinesterase function inneurotransmission. Toxicology233:60-69.
Eaton, D.L, R.B. Daroff, H. Autrup, J. Bridges, P. Buffler, L.G. Costa; J. Coyle, G. McKhann, W. C. Mobley, L. Nadel, D. Neubert, R. Schulte-Hermann, P.S. Spencer. 2008. Review of the toxicology ofchlorpyrifos with an emphasis onhuman exposure and neurodevelopment. Crit. Rev. Toxicol. 38 (S2):1-125.
Ecobichon, D.J., and D.S. Stephens. 1973. Perinatal development ofhumanblood esterases. Clin. Pharmacol. Ther. 14:41-47. Engel, S.M., G.S. Berkowitz, D.B. Barr, S.L. Teitelbaum, J. Siskind, S.J. Meisel, J.G. Wetmur, M.S. Wolff. 2007. Prenatal organophosphate metabolite and organochlorine levels and performance onthe BrazeltonNeonatal Behavioral Assessment Scale in a multiethnic pregnancy cohort. Am. J. Epidemiol. 165 (12):1397-404.
Eskenazi, B., K. Harley, A. Bradman, E. Weltzien, N.P. Jewell, D. B. Barr, C. E. Furlong, N.T. Holland. 2004. Association ofin utero organophosphate pesticide exposure and fetal growth and length ofgestation in an agricultural population. Env. Health Perspect. 112 (10): 1116- 1124.
Eskenazi, B., A.R. Marks, A. Bradman, K. Harley, D.B. Barr, C. Johnson, N. Morga, N.P. Jewell. 2007. Organophosphatepesticide exposure and neurodevelopment in young MexicanAmerican children. Environ Health Perspect. 115 (5): 792-798.
Eskenazi, B., L.G. Rosas, A.R. Marks, A. Bradman, K. Harley, N. Holland, C. Johnson, L. Fenster, D.B. Barr. 2008. Pesticide toxicity and the developingbrain. Basic Clin Pharmacol Toxicol. 102: 228-236.



### 67Etzel, RA, ed. 2003. Pediatric Environmental Health. 2nd EditionAmerican Academy of Pediatrics. Elk Grove Village IL. Figueras, F., J. Figueras, E. Meier, E. Eixarch, 

### 0. Coll, E. Gratacos, J. Gardosi, X. Carbonell. 2007. Customised birthweight standards accurately predict perinatal morbidity.Arch. Dis. Child. Fetal Neonatal Ed. 92(4): F277-F280. Furlong, C.E. 2007. Genetic variability in the cytochrome P450-paraoxonase (PON1) pathways for the detoxication oforganophosphorus compounds. J. Biochem. Mol. Toxicol. 21:197- 205. Furlong, C.E., T.B. Cole, G.P. Jarvik, C. Pettan-Brewer, G.K. Geiss, R. J. Richter, D. M. Shih, A. D. Tward, A. J. Lusis, L.G. Costa. 2005. Role ofParaoxonase (PON1) Status in Pesticide Sensitivity: Genetic and Temporal Determinants. NeuroToxicology 26(4): 651-659. Ginsberg, G., D. Hattis, B. Sonawane. 2004. Incorporatingpharmacokinetic differences between children and adultsin assessing children's risk to environmental toxicants. Toxicol. Appl. Pharmacal. 198: 164-183.
Griffin, P., H. Mason, K. Heywood, J. Cocker, J. 1999. Oral and dermal absorption of chlorpyrifos: A human volunteer study. J. Occup. Environ. Med. 56:10-13. Halpert, J., D. Hammond, R.A. Neal. 1980. Inactivation ofpurified rat liver cytochrome P450 during the metabolism ofparathion (diethyl p-nitrophenyl phophorothionate). J. Biol. Chern. 255: 1080-1089.
Holland, N., C. Furlong, M. Bastaki, R. Richter, A. Bradman, K. Huen, K. Beckman, and B. Eskenazi, B. 2006. Paraoxonase polymorphisms, haplotypes, and enzyme activity in Latino · mothers and newborns. Environ. Health Perspect. 114:985-991.
Hunter, D.L., T.L. Lassiter, S. Padilla. 1999. Gestational exposure to chlorpyrifos: comparative distribution oftrichloropyridinol in the fetus and dam. Toxicol. Appl. Pharmacal. 158: 16- 23. Icenogle, L.M., C. Christopher, W.P. Blackwelder, D.P. Caldwell, D. Qiao, F.J. Seidler, T.A. Slatkin, E.D. Levin. 2004. Behavioral alternations in adolescent and adult rats caused by a briefsubtoxic exposure to chlorpyrifos during neurulations. Neurotoxicol. Teratol. 26: 95- 101.
Jett, D.A., R.V. Navoa, R.A. Beckles, G.L. McLemore. 2001. Cognitive function and cholinergic neurochemistryin weanling rats exposed to chlorpyrifos. Toxicol. Appl. Pharmacal. 174:89- 98.
Karanth, S. and C. Pope, C. 2003. Age-related effects ofchlorpyrifos and parathion on acetylcholine synthesis inrat striatum. Neurotoxicol. Teratol. 25:599-606.

68Karanth, S. and C. Pope. 2000. Carboxylesterase and A-esterase activities during maturation and aging: Relationship to the toxicity ofchlorpyrifos and parathion in rats. Toxicol. Sci. 58:282- 289.
Kisicki, J.C., C.W. Seip, M.L. Combs. 1999. A rising dose toxicology study to determine the noobservable effect levels (NOEL) for erythrocyte acetylcholinesterase (AChE) inhibition and cholinergic signs and symptoms ofchlorpyrifos at three dose levels. DowAgroSciences LLC.
Lassiter, T.L., S. Padilla, S.R. Mortensen, S.M. Chanda, V.C. Moser, S. Barone, S., Jr. 1998. Gestational exposure to chlorpyrifos: Apparent protection ofthe fetus? Toxicol. Appl. Pharmacal. 152:56-65.
Laviola, G. W. Adriani, C. Gaudino, R. Marino, F. Keller. 2006. Paradoxical effects ofprenatal acetylcholinesterase blockade on neuron-behavioral development and drug-induced stereotypes inreeler mutant mice. Psychopharmacol. 187: 331-344.
Levin, E.D., N. Addy, A. Baruah, A. Elias, N.C. Christopher, F. J. Seidler, T.A. Slotkin. 2002. Prenatal chlorpyrifos exposure in rats causes persistentbehavioral alternations. Neurotoxicol. Teratolog. 24: 733-741.
Levin, E.D., N. Addy, A. Nakajima, N.C. Christopher, F.J. Seidler, T.A. Slotkin. 2001. Persistent behavioral consequences ofneonatal chlorpyrifos exposure in rats. Dev. Brain Res. 130: 83-89.
Li,W.F., C. Matthews, C.M. Disteche, L.G. Costa, C.E. Furlong. 1997. Paraoxonase (PON1) gene in mice: Sequencing, chromosomal localization and developmental expression. Pharmacogenetics 7:137-144.
Li, B., M. Sedlacek, I. Manoharan, R. Boopathy, E.G. Duysen, P. Masson, P., 0. Lockridge. 2005. Butyrylcholinesterase, paraoxonase, and albumin esterase, butnot catboxylesterase, are present in human plasma. Biochem. Pharmacal. 70:1673-1684.
Liu, J., K. Olivier, C.N. Pope. 1999. Comparative neurochemical effects ofrepeated methyl parathion or chlorpyrifos exposures in neonatal and adult rats. Toxicol. Appl. Pharmacal. 158:186-196. Lynch M.K., W. Heiger-Bemays, A. Ozonoff, 2008. Quantification and Correction ofthe Bias in the Estimated Geometric Standard Deviation Implications for the Derivation ofthe Reference Dose. Manuscript in Preparation
Ma, T. and J.E. Chambers. 1994. Kinetic parameters ofdesulfuration and dearylation of parathion and chlorpyrifos byrat liver microsomes. Food Chern. Toxicol. 32:763-767.
Ma, T. and J.E. Chambers. 1995. A kinetic analysisofhepaticmicrosomal activation of parathion and chlorpyrifos in control and phenobarbital-treated rats. J. Biochem. Toxicol. 10:63-68.



### 69March ofDimes; http://www.marchofdimes.com/professionals/14332 1153.asp. Accessed September 21, 2008. Marty, M.S., J.Y. Domoradzki, S.C. Hansen, C. Timchalk, M.J. Bartels, J.L. Mattsson. 2007. The effect ofrol,!te, vehicle, and divided doses on the pharmacokinetics ofchlorpyrifos and its metabolite trichloropyridinol in neonatal Sprague-Dawleyrats. Toxicol. Sci. 100:360-373.
Mattsson, J.L., J.P. Maurissen, R.J. Nolan, K.A. Brzak, K.A. 2000. Lack ofdifferential sensitivityto cholinesterase inhibition in fetuses and neonates compared to dams treated perinatally with chlorpyrifos. Toxicol. Sci. 53:438--446.
Maurissen, J.P., A.M. Hoberman, R.H. Garman, R.H., T.R. Hanley Jr. 2000. Lack ofselective developmental neurotoxicityin rat pups from dams treated by gavage with chlorpyrifos. Toxicol. Sci. 57:250-263.
Meek, M.E., J.R. Bucher, S.M. Cohen, V. Dellarco, R.N. Hill, L.D. Lehman-McKeeman, D.G. Longfellow, 

### 1. Pastoor, J. Seed, D.E. Patton. 2003. A framework for humanrelevance analysis ofinformation on carcinogenicmodes ofaction. Crit. Rev. Toxicol. 33: 591-653. Meuling, W.J.A., L.C. Ravensberg, L.Roza, J.J. van Hemmen. 2005. Dermal absorption of chlorpyrifos inhuman volunteers. Int. Arch. Occup. Environ. Health 78:44-50. Morgan, E.W., B. Yan, D. Greenway, A. Parkinson. 1994. Regulation oftwo rat liver microsomal carboxylesterase isozymes: species differences, tissue distribution, arid the effects ofage, sex, and xenobiotic treatment ofrats. Archives Biochem. Biophysics 315(2): 513-526.**Morte~sen,** S.R., S.M. Chanda, M.J. Hooper, S. Padilla. 1996. Maturational differences in chlorpyrifos-oxonase activitymay contribute to age-related sensitivity to chlorpyrifos. J. Biochem. Toxicol. 11:279-287.
Moser, V.C. 2000. Dose-response and time-course ofneurobehavioral changes following oral chlorpyrifos inrats ofdifferent ages. Neurotoxicol. Teratol. 22:713-723.
Moser, V.C., S.M. Chanda, S.R. Mortensen, S. Padilla. 1998. Age- and gender-related ,differences in sensitivityto chlorpyrifos in theratreflect developmental profiles ofesterase activities. Toxicol. Sci. 46:211-222.
Nolan, R.J., D.L. Rick, N.L. Freshour, H. Saunders. 1984. Chlorpyrifos: Pharmacokinetics in human volunteers.Toxicol. Appl. Pharmacol. 73:8-15.
Nomura, D. K., K.A. Durkin, K.P. Chiang, G.B. Quistad, B.F. Cravatt and J.E. Casida. 2006. Serinehydrolase KIAA1363: Toxicological and structural features with emphasis on organophosphate interactions. Chem.Res. Toxicol. 19 , pp; 1142-1150.

70Phuntuwate, W., C. Suthisisang, B. Koanantakul, M. Mackness, B. Mackness. 2005. Paraoxonase 1 status in thre Thai population. J Human Genetics 50(6): 293-300.
Poet, T.S., H. Wu, A.A. Kousba and C. Timchalk. 2003. In vitro hepatic and intestinal metabolism ofthe organophosphate pesticides chlorpyrifos and diazinon. Toxicol. Sci. 72: 193-200.
Rauh, V.A, R. Garfinkel, F.P. Perera, H.F. Andrews, L. Hepner, D.B. Barr, R.Whitehead, D. Tang, R.M. Whyatt. 2006. Impact ofprenatal chlorpyrifos exposure on neurodevelopment in the first 3 years oflife among inner-city children. Pediatrics 118: e1845-e1859. Rauh, V.A. 2008. Discussion ofanalyses ofprenatal chlorpyrifos exposure and neurodevelopmental outcomes. Presentation to US EPA (April 23, 2008). Ricceri, L., A. Venerosi, F. Capone, M.P. Cometa, P. Lorenzini, S. Fortuna, G. Calamandrei. 2006. Developmental neurotoxicity oforganophosphorous pesticides: Fetal and neonatal exposure to chlorpyrifos alters sex-specificbehaviors at adulthood in mice. Toxicol. SCi. 93:105-113.
Ricceri, L., N. Markina, A. Valanzano, S. Fortuna, M.P. Cometa, A. Meneguz, G. Calamandrei, 2003. Developmental exposure to chlorpyrifos alters reactivity to environmental and social
cues in adolescent mice. Toxicol. Appl. Pharmacal. 191:189-201.
Rodier, P.M. 2004. Environmental Causes ofCentral Nervous System. Pediatrics 113: 1076- 1083 Samarawickrema, N., A. Pathmeswaran, R. Wickremasinghe, R. Peiris-John, M. Karunaratna, N. Buckley, A. Dawson, J. de Silva. 2008. Fetal effects·ofenvironmental exposure ofpregnant women to organophosphorus compounds in a rural farming communityin Sri Lanka. Clin Toxicol (Phila). 46(6): 489-95. Schuh, R.A., P.J. Lein, A. Randell, A. Beckles D. A. Jett. 2002. Noncholinesterase Mechanisms ofChlorpyrifos Neurotoxicity: Altered Phosphorylation ofCa 2+/cAMP Response Element Binding Protein in Cultured Neurons. Toxicology and Applied Pharmacology 182(2): 176- 185. Seed, J., E. Carney, R. Corley, K. Crofton, J. DeSesso, P. Foster, R. Kavlock, G. Kimmel, J. Klaunig, E. Meek, J. Preston, W. Slikker, S. Tabacova, G. Williams. 2005. Overview: Using mode ofaction and life stage information to evaluate the human relevance ofanimal toxicity data. Crit. Rev. Toxicol. 35: 663..,672. Shih, D.M., L. Gu, Y.R. Xia, M. Navab, W.F. Li, S. Hama, L.W. Castellani , C.E. Furlong, L.G. Costa, A.M. Fogelman, A.J. Lusis. 1998. Mice lacking serum paraoxonase are susceptible to organophosphate toxicity and atherosclerosis. Nature 394: 284-287.

71Sirivarasaia, J., S. Kaojarerna, K. Yoovathawornb, T. Surac. 2007. Paraoxonase (PON1) polymorphism and activity as the determinants ofsensitivityto organophosphates in human subjects. Chemica-Biological Interactions 168 (3): 184-192. Slotkin, T.A., E.D. Levin, F.J. Seidler. 2006. Comparative developmental neurotoxicity of organophosphate insecticides: Effects on brain development are separable from systemic toxicity. Environ. Health Perspect.**114:746~751.** Sogorb, M.A., S. Garcia-Argiielles, V. Carrera, E. Quesada, E. Vilanova. 2008. Humanserum albumin hydrolyzes the organophosphorus insecticides paraoxon, diazoxon, chlorpyrifosoxon and 0-hexyl 0-2,5-dichlorophenyl phosphoramidate. Tox. Letters. 180(Supplement 1 ): S29. Sultatos, L.G., M. Shao, S.D. Murphy. 1984. Therole ofhepaticbiotransformation inmediating the acute toxicity ofthe phosphorothionate insecticide chlorpyrifos. Toxicol. Appl. Pharmacal. 73:60-68. Timchalk, C., T.S. Poet, A.A. Kousba. 2006. Age-dependentpharmacokinetic and pharmacodynamic response inpreweanling rats following oral exposure to the organophosphorus insecticide chlorpyrifos. Toxicology. 220(1): 13-25. Timchalk, C., A. Busby, J.A. Campbell, L.L. Needham, D.B. Barr. 2007. Comparative pharmacokinetics ofthe organophosphorus insecticide chlorpyrifos and its major metabolites diethylphosphate, diethylthiophosphate and 3,5,6-trichloro-2-pyridinol in the rat. Toxicology 237:145-157.
U.S. EPA. 2001. Organophosphate Pesticides: Preliminary OP Cumulative Risk Assessment. Washington, DC: U.S. Environmental Protection Agency, Office ofPesticide Programs. Available: http://www.epa.gov/pesticides/cumulative/pra-op [accessed 30 July 2003]. Venerosi, A. et al. 2008. Neonatal exposure to chlorpyrifos affects maternal responses and maternal aggression offemale mice in adulthood. Neurotoxicol. Teratol. 11 July 2008. [Epub ahead ofprint] Venerosi, A., G. Calamandrei, L. Ricceri. 2006. A social recognition test for female mice reveals behavioral effects ofdevelopmental chlorpyrifos exposure. Neurotoxicol. Teratol. 28: 466- 471. Whyatt, R.M., D.B. Barr, D.E. Camann, P.L. Kinney, J.R. Barr, H.F. Andrews, L.A. Hoepner, R. Garfinkel, Y. Hazi, A. Reyes, J. Ramirez, Y. Cosme, F.P. Perera. 2003. Contemporary-use pesticides in personal air samples during pregnancy and blood samples at delivery among urban minority mothers and newborns. Environ Health Perspect. 111 (5): 749-756. Whyatt, R.M., D.E. Camann, P.L. Kinney, A. Reyes, J. Ramirez, J. Dietrich, D. Diaz, D. Holmes, F.P. Perera. 2002. Residdential pesticide use during pregnancy among a chohort of urban minority women. Environ. Health Perspect. 110: 507-514.

72Whyatt, R.M, R. Garfinkel, L.A. Hoepner, D. Holmes, M. Borjas, M.K. Williams, A. Reyes, V. Rauh, F.P. Perera, D.E. Camann. 2007. Within- and between-home variabilityin indoor-air insecticide levels during pregnancy among an inner-city cohort from New York City. Environ Health Perspect. 115(3): 383-389. Whyatt, R.M, V. Rauh, D.B. Barr, D.E. Camann, H.F. Andrews, R. Garfinkel, L.A. Hoepner, D. Diaz, J. Dietrich, A. Reyes, D. Tang, P.L. Kinney, F.P. Perera. 2004. Prenatal insecticide exposures and birth weight and length among an urban minority cohort. Environ Health Perspect. 112:1125-1132. Whyatt, R.M: Discussion ofanalyses ofprenatal chlorpyrifos exposure andbirth outcomes. Presentation to US EPA(December 13, 2007). Whyatt, R.M. Discussion ofanalyses ofprenatal chlorpyrifos exposure and birth outcomes. Presentation to US EPA (April 23, 2008). Whyatt, R.M. Personal communication to D. Smegal (EPA) via email, August 12,2008. Whyatt, R.M. Personal communication to D. Smegal (EPA) and H.A.Young (GWU, member of the SAP) viatelephone, September 18, 2008. Young, J.G., B. Eskenazi, E.A. Gladstone, A. Bradman, L. Pedersen, C. Johnson, D.B. Barr, C.E. Furlong, N.T. Holland. 2005. Associationbetween in utero organophosphate exposure and abnormal reflexes in neonates. Neurotoxicology 26 (2): 199-209. Zheng, Q., K. Olivier, Y.K. Won, C.N. Pope. 2000. Comparative cholinergic neurotoxicity of oral chlorpyrifos exposures in preweanling and adult rats. Toxicol. Sci. 55:124--132.

73APPENDIX 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
MotorActivity 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 Tin 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
Tlat to peak response (p=.03) [overall effect] NoRxeffects

No RxEffect Tduring 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 SpontaneousAlternation 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: 11OdB 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 SpontaneousAJternatlon 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 NoRXeffectMatemaUPup 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) TMales

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 TFemales

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 TRate ofUSV
Social Investigation Test GO CPF6 t Social investigationArticle

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) 1hr 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) TOurAttack (GO CPF3) TOff posture (CPF6) Def post No Rx effect
PNO 90 (Ferns) flicking dura & crouching (PN CPF 1&3)

PND 120 (M&F) Time in Open . !lickingfreq (PN CPF 1&3)

arms T PN CPF3 Ferns Head Dipping 1 PNCPF 3 Males

No overtsigns 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 TSoliciting 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 etat, 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 Jettetat, 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 AChEI 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 oftocicity 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& MedDosesM&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 ofrats 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 1in fall angle (F) {Sensorimotor Reflexes) Forepaw Grip Time (Motor Strength) 1in 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
locomotorActivity: Open Field PND >70 No Rxetfect**Scopc~amine** Challenge PND >70 1in Seep-induced locomotor stimulation Amphetamine Challgene PND >70 1in Amph. induced locomotor stimulation