# UNITED STATES ENVIRONMENTAL PROT ECT ION AGENCY

WASHINGTON

, D.C. 20460

OFFICE OF CHEM IC AL SAFETY AND

POLLUTION PREV ENTJON



# MEMORANDUM



# Date: December 29, 2014

SUB.fECT: Chlorpyrifos: Revised Hu ma n Health Risk Asse ssm ent for Regi str ation Review

PC Code: 05910 

# I

Decision No. : NA



# Petition No

. : NA 

# DP Barcode: 0424485



# Registration No.: NA



# Risk Assessmen t Type: Single Ch emi cal Aggregate



# TXRNo.: NA 

# Regulatory Action: Regi sh· ation Review



# Case No.: NA



# CAS No.: 292 1-88-2



# MRfD No.: NA



# FROM: 40 CF R: 40 CF R§ 

# I 80.342

Danette Drew

, Chemist, RAB Vll 

# vA**~ ~**  /1

# ~ Wade Britton

, MPH, Indu s trial H ygien i st RAB V ll 

# t/VIf<,(.( 7 

# CXJ**~**

William Irwin

, Ph

. D., DABT

, Toxi co logi st. B VII .(

Anna Lowit

, Ph.D.

, Seni or Scientist

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# ~v- 

# ..PD .( 

# UJr Carol Christensen, Ph.D .• MPH, Ep idemiologist.**TEB ~** 



# Health Effects Divisi on (7509P) A---



# [ (Rochelle Bohaty, Ph.D.

,

# Chemi**~~** '( E nvi ron m en tal Fate and Effects Divisi o n

# (75~ And

And



# THROUGH:

And



# TO: Office of.P esti cide Programs

Cecil ia Tan, Ph

. D.

# /Jr**~** #v C)

National Exposu re

# ~~**aboratory** (N E RL)

# ~ Virginia Moser

, Ph.D.

# ~ 

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# M

Natio nal Health and Environmental Effects Re searc h 

# Lab r ato 

# HEElli! )

Office of Research and Development E-2 05

- 0 I. B -l OS-04

R ese arch Triangle Par k. NC

Mi chae l Metzger, Branch Chief

Risk Ass essm ent Branch V 

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Health Effects Division (7509 P)

Dana Vogel

, Acting Division Director

Health Effects Division (7509P)

Tom Myers

, Chemical Reyiew Mana ger

Joel Wolf, Chemical Review Man ager

Risk Management and Implementation Branch II

P

est ic ide Re-evaluation Division (7508P)

# Chlorpyrifos Human Health Risk Assessment D424485

Page 2 of 531

# Chlorpyrifos Human Health Risk Assessment D424485

Page 3 of 531 Table of Contents

1.0 Executive Summary ........................................................................................................... 5

2.0 HED Recommendations .................................................................................................. 13

2.1 Data Deficiencies ............................................................................................................. . 13

2.1 Tolerance Considerations ................................................................................................ 14

2.2.1 Enforcement Analytical Method .................................................................................... 14

2.2.2 International Harmonization .......................................................................................... 14

2.2.3 Recommended/Reassessed Tolerances ........................................................................... 15

3.0 Introduction .................................................................................................................. .... 15

3.1 Chemical Identity ............................................................................................................. 16

3.2 Physical/Chemical Characteristics ................................................................................. 16

3.3 Anticipated Exposure Pathways ..................................................................................... 17

3.4 Consideration of Environmental Justice ....................................................................... 17

4.0 Hazard Characterization and Dose-Response Assessment .......................................... 18

4.1 Introduction & Background ........................................................................................... 18

4.2 Types of Studies Available for Analysis ......................................................................... 20

4.3 Absorption, Distribution, Metabol ism, & Excretion (ADME) .................................... 21

4.4 Summary of Experimental Toxicology Studies ............................................................. 24

4.4.1 AChE Inhibition in Experimental Laboratory Animal Studies .................................. 24

4.4.2 Neurodevelopmental Outcomes in Laboratory Animals ............................................. 25

4.4.3 Plausible hypotheses on MOA/AOP fo r neurodevelopmental outcomes .................... 27

4.5 Epidemiology Studies in Mothers and Children ........................................................... 32

4.5.1 Review of Children’s Environmental Health Epidemiology Studies ............................ 32

4.5.2 Review of Study Design and Research Methods ............................................................. 33

4.5.3 Neurological and Neurodevelopmental Health Effects in Children .............................. 35

4.5.3.1 The CCCEH Mothers and Newborn Study .................................................................. 35

4.5.3.2 Supporting Epidemiological Evidence: The Mt. Sinai and CHAMACOS Cohorts . 38

4.5.4 Dose Reconstruction ................................................................................................... ..... 40

4.5.4. Summary of EPA’s Conclusions Regarding Children’s Environmental Health Cohort

Studies ....................................................................................................................... ................... 42

4.6 Weight of Evidence Analysis ........................................................................................... 43

4.6.1 Summary of the draft “Framework for Incorporating Human Epidemiologic &

Incident Data in Health Risk Assessment” ............................................................................... 43

4.6.2 Integration Across Multiple Lines of Evidence ............................................................. 44

4.7 Safety Factor for Infants and Children (FQPA Safety Factor) ................................... 48

4.8 Dose-Response Assessment ............................................................................................. 49

4.8.1 Durations of Exposure, Critical Windows of Exposure, & Temporality of Effects .. 49

4.8.2. Introduction to the PBPK-PD Model ............................................................................. 50

4.8.3 Description & Structure of the PBPK-PD Model ........................................................... 51

4.8.4 Use of the PBPK-PD Model .............................................................................................. 6 1

4.8.4.1 Derivation of Human Equivalen t Doses/Concentrations ............................................ 62

4.8.4.2 Intra-species extrapolation ........................................................................................... .. 66

5.0 Dietary Exposure and Risk Assessment ......................................................................... 71

5.1 Residues of Concern Summary and Rationale .............................................................. 71

# Chlorpyrifos Human Health Risk Assessment D424485

Page 4 of 531 5.2 Food (Residue Chemistry) Profile .................................................................................. 72

5.3 Water Residue Profile...................................................................................................... 73

5.4 Dietary (Food Only) Risk Assessment............................................................................ 73

5.4.1 Description of Residue Data Used in Dietary (Food Only) Assessment ........................ 74

5.4.2 Percent Crop Treated Used in Dietary Assessment ...................................................... 74

5.4.3 Acute Dietary (Food Only) Risk Assessment ................................................................. 74

5.4.4 Steady State Dietary (Food Only) Risk Assessment ..................................................... 75

6.0 Residential (Non-Occupational) Expo sure/Risk Characterization ............................. 76

6.1 Residential Handler Exposure ........................................................................................ 77

6.2 Residential Post-Application Exposure ......................................................................... 77

6.3 Residential Bystander Post-application Inhalation Exposure ..................................... 82

6.3.1 Spray Drift ................................................................................................................... ..... 82

6.3.2 Volatilization .......................................................................................................... ............ 83

7.0 Aggregate Exposure/Risk Characterization .................................................................. 84

7.1 Acute Aggregate Risk ...................................................................................................... 85

7.2 Steady State Aggregate Risk .......................................................................................... 86

7.3 Aggregate Risk Estimates ............................................................................................... 87

8.0 Cumulative Exposure/Risk Characterization ............................................................... 96

9.0 Occupational Exposure/Risk Characterization ............................................................ 97

9.1 Steady State Occupational Handler Risk ...................................................................... 97

9.2 Steady State Occupational Post-Application Risk ...................................................... 103

9.2.1 Dermal Post-Application Risk ...................................................................................... 104

9.2.2 Occupational Post-application Dermal Expo sure/Risk Estimates: Chlorpyrifos Oxon

106

9.2.3 Inhalation Post-application Risk .................................................................................. 107

10.0 References .................................................................................................................... ... 108

11. List of Appendices ........................................................................................................ ....... 125

Appendix 1. Evaluation of Ex perimental Toxicology Data .................................................. 126

Appendix 2. Detailed Review and Synthesi s of Three Children’s Environmental Health

Cohort Studies ................................................................................................................ ........... 221

Appendix 3. Epidemiology Study Specific Evaluations ......................................................... 266

Appendix 4. Detailed Summary Tables of Ch ildren’s Environmenta l Health Epidemiology

Studies ....................................................................................................................... ................. 370

Appendix 5. Summary of OPP’s ChE Policy & Use of BMD Modeling ............................. 383

Appendix 6. Columbia Center for Child ren’s Environmental Health (CCCEH)

Epidemiology Data Acquisition “Raw Data” Request ......................................................... 384

Appendix 7. Physical/Chemical Properties............................................................................ 397

Appendix 8. Current U.S. Tolerances a nd International Residue Limits ........................... 398

Appendix 9. Master Use Summary Document ....................................................................... 402

Appendix 10. Dose Reconstruction Analysis ......................................................................... 448

Appendix 11. New Literature on Chlorpyrif os since the 2012 FIFRA SAP Meeting ........ 460

# Chlorpyrifos Human Health Risk Assessment D424485

Page 5 of 531 

# 1.0 Executive Summary

Background

This document presents the revised human h ealth risk assessment for the organophosphate

insecticide chlorpyrifos. Chlorpyr ifos is currently being evalua ted under the FIFRA section 3(g)

registration review program. The preliminar y human health risk assessment (HHRA) for

chlorpyrifos registration revi ew was completed on June 30, 2011 (USEPA, 2011). This revised

assessment addresses issues raised in the co mments received on the preliminary HHRA, as well

as incorporates new information and new approach es that have also become available since the

June 2011 risk assessment. The most si gnificant changes incl ude the following:

 Hazard

o Findings from multiple lines of eviden ce from experimental toxicology and

epidemiology with respect to acetylcho linesterase/cholinesterase (AChE/ChE)

inhibition and neurodevelopmental outcome s are used in the weight-of-evidence

determination supporting the retention of the 10X FQPA Safety Factor (SF).

o Utilization of a physiologically-bas ed, pharmacokinetic-pharmacodynamic

(PBPK-PD) model for deriving toxicologica l points of departure (PoDs) and for

determining the appropriate intra-species and inter-species uncertainty factors.

o Findings from two vapor phase, inhalation toxicity studies in dicating no adverse

effects even at the satura tion concentration for chlor pyrifos and chlorpyrifos oxon

in vapor form.

 Dietary Exposure

o Updated food monitoring data and percent crop treated information.

 Residential and Bystander Exposure

o Use of the 2012 Standard Operating Procedures for Residential Pesticide

Exposure Assessment 1 

# which prescribe updates to several elements of the

assessment.

o Use of the AgDISP model for estimation spray drift (deposition and airborne

concentrations) following aerial mo squito adulticide application.

o Updated methodology for determining the air borne concentration of chlorpyrifos

following ground-based mosquito adulticide applications.

o Completion of a dose reconstruction ev aluation to further evaluate the

epidemiological findings.

 Occupational Exposure

o Inclusion of use patterns based on a Ma ster Use Summary Document developed

for chlorpyrifos.

o Use of revised guidance for all of the currently recommended unit exposures (and

body weights) for standard EPA occupational handler exposure scenarios 2



# , Occupational Pesticide Handler Unit Exposure Surrogate Reference Table –

Revised March 2013

.

1 

# http://www.epa.gov/pesticides/science/USEPA- OPP-HED_Residential%20SOPs_Oct2012.pdf

2 

# http://www.epa.gov/opp00001/science/handler-exposure-data.html

# Chlorpyrifos Human Health Risk Assessment D424485

Page 6 of 531 o Use of the revised inputs for seed treatment use patterns.

 Aggregate Assessment

o Use of a calculated drinking water leve l of comparison (DWLOC) approach for

comparison to model estimated drinking water concentrations (EDWCs).

Use Profile

Chlorpyrifos (0,0-diethyl-0-3,5,6- trichloro -2-pyridyl phosphorot hioate) is a broad-spectrum,

chlorinated organophosphate (OP) insecticide. Regi stered use sites include a large variety of

food crops (including fruit and nut trees, many type s of fruits and vegetables, and grain crops),

and non-food use settings (e.g., golf course turf , industrial sites, greenhouse and nursery

production, sod farms, and wood products). Public health uses include aerial and ground-based

fogger adulticide treatments to control mosquitoes . There are also residen tial uses of roach bait

products and ant mound treatments. Permanent tolerances are established (40 CFR§180.342) for

the residues of chlorpyrifos in/on a variety of agricultural commodities (including meat, milk,

poultry and eggs). There are also tolerances fo r use in food handling establishments (FHE).

Chlorpyrifos is manufactured as granular, micr oencapsulated liquid, solu ble concentrate liquid,

water dispersible granular in water soluble packets (WSP), wettable powders in WSPs,

impregnated paints, cattle ear tags, insect bait stations and total re lease foggers. There is a wide

range of application rates and methods. The residues of concern for risk assessment purposes are

chlorpyrifos and chlorpyrifos oxon under some circumstances.

Hazard Characterization

The hazard characterization for ch lorpyrifos and its oxon is based on adverse health effects in

animals and humans related to two different e ndpoints - acetylcholinestera se (AChE) inhibition

and potential for neurodevelopmental effects. A weight-of-the-evidence (WOE) analysis has

been completed using the draft “Framework for Incorporating Human Epid emiologic & Incident

Data in Health Risk Assessment.” The WOE an alysis integrated quantitative and qualitative

findings from experimental t oxicology studies, epidemiology st udies and physiologically-based

pharmacokinetic-pharmacodynamic (PBPK-PD) modeli ng does not support re duction of the 10X

FQPA Safety Factor. In additi on, the Agency is explicitly usi ng the robust PBPK-PD model to

estimate human equivalent doses/concentrations wh ich are used as the toxicological points of

departure (PoDs) and also to define inter- and in tra-species factors for ch lorpyrifos and/or its

oxon from multiple exposure pathways (e.g. f ood, water, occupational, residential).

The key issues considered in the WOE are 1) wh ether chlorpyrifos causes long-term effects from

prenatal and/or early lifestage e xposure and 2) whether adverse eff ects can be attributed to doses

lower than those which elicit 10 % inhibition of RBC AChE. When taken together, the evidence

from 1) the experimental toxico logy studies evaluating outcomes su ch as behavior and cognitive

function; 2) mechanistic data on possible /m odes of action/ advers e outcome pathways

(MOA/AOP); and 3) epidemiologic and biomonitori ng studies, indicate that chlorpyrifos likely

played a role in the neurodevelopmental ou tcomes reported by the epidemiologic study

(Columbia University) inve stigators. However, uncertainties su ch as the lack of an established

MOA/AOP for neurodevelopmental effects and the potential exposure to multiple AChEinhibiting pesticides preclude definitive causal inference. However, there is sufficient

# Chlorpyrifos Human Health Risk Assessment D424485

Page 7 of 531 uncertainty in the human dose-response relations hip for neurodevelopmental effects to prevent

the Agency from reducing or removing the statut ory 10X FQPA Safety Factor. The FQPA 10X

Safety Factor will be retained for infants, chil dren, youths, and women of childbearing age for all

exposure scenarios.

EPA has applied the Data-Derived Extrapolation Factor (DDEF) guidance (USEPA, 2014), in its

use of the PBPK-PD model; the model that wa s used to calculate a DDEF intra-species

extrapolation for some populations. The PBPK-PD model estimates human RBC AChE

inhibition from exposures via oral, dermal, and i nhalation routes and thus obviates the need for

the typical inter-species factor. Administered doses leading to the response level of interest

(10% change in RBC AChE inhibition) are comp ared between a measure of average response

and response at the tail of the distribution representing sensitive individuals in order to calculate

the intra-species factor. Based on the 99 th 

# percentile of the distri butions, the DDEF for intraspecies extrapolation is 4X for chlorpyrifos and 5X for the oxon (Dow, 2014b). For this revised

risk assessment, the 99th percentile is being used to account for variation of sensitivity (i.e., the

intra-species factor used in the risk assessment is 4X for chlorpyrifos and 5X for the oxon for all

groups except women who are pregnant or ma y become pregnant for whom the 10X intraspecies factor was retained).

While the current PBPK-PD model accounts for ag e-related growth from infancy to adulthood

by using polynomial equations to describe tissue volumes and blood flows as a function of age,

the model does not include any descriptions on physiological, anatomical and biochemical

changes associated with pregnancy. Due to the uncertainty in extrapolating the current model

predictions among women of child bearing age, the Agency is applying the standard 10X intraspecies extrapolation factor for women of childbearing age. For all other relevant populations,

the Agency has reliable data to redu ce the standard 10x intra-species factor.

The PBPK-PD model has been used to estimate exposure levels resulting in 10% RBC AChE

inhibition following acute (single day, 24 hours) and steady state (21-day) exposures for a

variety of exposure scenarios for chlorpyrifos and/or chlorpyrifos oxon. For OPs, repeated

exposures generally result in more AChE inhib ition at a given administered dose compared to

acute studies. Moreover, AChE inhibition in re peated dosing guideline toxicology studies with

OPs show a consistent pattern of inhibition re aching steady state at or around 2-3 weeks of

exposure in adult laboratory animals (U.S. EPA, 2002). This pattern observed with repeated

dosing is a result of the amount of inhibition comes at equilibr ium (or steady state) with the

production of new enzyme. As such, AChE studi es of 2-3 weeks generally show the same

degree of inhibition with those of longer duration ( i.e. , up to 2 years of exposure).

Separate PoDs have been calculated for diet ary (food, drinking wate r), residential, and

occupational exposures by varying inputs on t ypes of exposures and populations exposed.

Examples of inputs include: duration; route (der mal, oral, inhalation); body weights which vary

by lifestage; exposure duration (hours per day, days per week); and exposure frequency [events

per day (eating, drinking)]. The predicted PoDs, used in this revised risk assessment can be seen

in Table 4.8.4.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 8 of 531 Exposure and Risk Assessment

The general approach for the chlorpyrifos expos ure and risk assessment can be described as

follows: The PBPK-PD model was used to calcul ate acute (24 hour) and steady state (21 day)

points of departure dose levels which corres pond to 10% RBC ChEI for the index lifestages

relevant to chlorpyrifos risk assessment (children of various ag es which differ due to exposure

pattern, and adult females of childbearing age). The PBPK-PD PoD predictions for each

exposure route and pathway were modeled sepa rately (e.g., for residential exposure dermal,

inhalation, and incidental oral calculations).

PoDs are divided by the total uncertainty fact ors to derive a populati on adjusted dose (PAD).

There are potential risks of con cern when estimated dietary risk exceeds 100% of the PAD. For

the dietary (food only) assessment, the only resi due of concern is chlorpyrifos (the oxon

metabolite is not an expected residue on foods). The chlorpyrifos total uncertainty factors are

100X for adult females (10X FQPA SF and 10X intra-species extr apolation factor) and 40X for

the other relevant populations ( 10X FQPA SF and 4X intra-species extrapolation factor). The

chlorpyrifos exposure values re sulting from dietary modeling are compared to the PAD.

For the dietary (water only) assessment, the only residue of concern assumed to occur in drinking

water is chlorpyrifos oxon. It is assumed that all chlorpyrif os in water is converted to

chlorpyrifos oxon upon treatment. The chlorpyrifos oxon total uncertainty factors are 100X for

adult females (10X FQPA SF and 10X intra-specie s extrapolation factor) and 50X for the other

relevant populations (10X FQPA SF and 5X intra-species extrapolat ion factor). A DWLOC

approach is employed to determine the risk es timates for drinking wate r. Drinking water is

addressed in the aggregate sec tions of the risk assessment.

For the residential and occupational assessment, margins of exposure (MOEs) were calculated by

comparing the PoDs to the calculated exposures for each scenario and applicable level of

personal protection (e.g., varied levels of pr otective clothing and equipment). The resulting

MOEs were then compared to the appropriate level of concern (LOC) which are 100 for adult

females and 40 for the other index lifestages evalua ted in this risk assess ment. If calculated

MOEs were less than the applicable LOC then a risk of concern was identified for that exposure

scenario.

Dietary Risk Assessment

Highly refined acute and steady state dietary (food only; pare nt chlorpyrifos only) exposure

analyses were performed using the Dietar y Exposure Evaluation Model (DEEM) and the

Calendex models, respectively. Most food residues used were based upon U.S. Department of

Agriculture’s Pesticide Data Program (PDP) mon itoring data. Percent crop treated and empirical

food processing factors were used where availabl e. Acute dietary (food only) risk estimates are

all <100 % of the acute PAD for food (aPAD food 

# ) at the 99.9 th 

# percentile of exposure. The

subgroup with the highest exposure was fe males (13-49 years old) at 3.2 % aPAD food 

# . For the

steady state dietary (food only) exposure analyses, children (1-2 years old) was the population

subgroup with the highest exposure at 9.7% of the ssPAD food 

# at the 99.9 th 

# percentile of exposure.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 9 of 531 Residential (Non-occupational) Exposures

The residential exposure assessment for chlorpyrif os has been updated to reflect the use of the

PBPK-PD model and updates to EPA’s SOPs fo r Residential Exposure Assessment. Current

chlorpyrifos residential uses include a granul ar ant mound use which is applied by commercial

applicators only and as a roach bait which is available for homeowner users in child-resistant

packaging. Additionally, chlorpyrif os is labeled for golf course turf applications and for public

health aerial and ground-based fogger ultra-low vol ume (ULV) mosquito adulticide applications.

Residential post-application expos ure is anticipated from playi ng golf on chlorpyrifos-treated

courses and from exposures which can occur following aerial and ground-based ULV mosquito

adulticide use. Chemical-specific turf transf erable residue (TTR) da ta were used in the

residential post-application assessment. The resi due of concern for resi dential post-application

exposure is the parent compound chlorpyrifos. A quantitative assessment was not performed for

the roach bait product and ant m ound treatments as residential exposures for these uses are

negligible.

The assessment of steady state golfer post-applica tion exposures (dermal only) to chlorpyrifos

treated turf for the lifestages adults (females 13- 49 years old), children (6 to < 11 years old), and

youths (11 to < 16 years old), results in no risk s of concern (i.e., for children and youths, MOEs

are ≥ 40; for adults, MOEs are ≥ 100 on the day of application) . For the assessment of postapplication exposures from public health mosqu itocide applications, no combined risks of

concern were identified, even on the day of appl ication, for adults (dermal and inhalation) and

children (dermal, incident al oral, and inhalation).

In addition to the assessment of residential exposures for golf c ourse and mosquitocide uses, the

potential for post-application exposures to reside ntial bystanders (who live on, work in, or

frequent areas adjacent to chlo rpyrifos-treated agricultural fields) from spray drift and

volatilization were also consider ed. The potential risks from sp ray drift and the impact of

potential risk reduction measures were assessed in a July 2012 3 

# memorandum. To increase

protection for children and other bystanders, chlorpyrifos technical registrant s voluntarily agreed

to lower application rates and adopt other spray drift mitigation measures such as buffer zones. 4



# The spray drift risk assessment results have been updated to incorpor ate the PBPK-PD modeling

approach. The resulting buffer di stances (feet) necessary to reach the level of concern for adults

(females 13-49 years old) and children (1 to < 2 years old) (i.e., for adults, MOEs are ≥ 100; for

children, MOEs are ≥ 40) with use of certain applicati on rates, nozzle droplet types, and

application methods range from 0 to 25 feet. The estimated buffer distances based on this

revised assessment are less than pr eviously predicted in July 2012.

3 

# J. Dawson, W. Britton, R. Bohaty, N. Mallampalli, and A. Grube. Chlorpyrifos: Eval uation of the Potential Risks

from Spray Drift and the Impact of Potential Risk Reduction Measures. 7/13/12. U.S. EPA Office of Chemical

Safety and Pollution Prevention. D399483, D399485.

4 

# R. Keigwin. Spray Drift Mitigation Decision for Chlorp yrifos (059101). 7/2012. U.S. EPA Office of Chemical

Safety and Pollution Prevention. EPA-HQ-OPP-2008-0850-0103.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 10 of 531 In January 2013, a preliminary assessment of the pot ential risks from chlo rpyrifos volatilization

was conducted. 5 

# However, this assessment was revised in June 2014 6 

# following submission of

two vapor phase inhalation toxicity studies whic h indicate no adverse effects occurred even at

the saturation concentration for ch lorpyrifos and chlorpyrifos oxon. 7 

# Because these new studies

demonstrated that no toxicity occurred even at the saturation concentration, which is the highest

physically achievable concentra tion, there are no anticipated risk s of concern from exposure to

the volatilization of either ch lorpyrifos or chlorpyrifos oxon.

Aggregate Assessment and Risk Estimates

The Agency has considered aggregate exposures and risks from combined food, drinking water,

and residential exposures to ch lorpyrifos and chlorpyrifos ox on. The acute aggregate assessment

includes only food and drinking water. The steady state aggregate assessment includes exposures

from food, drinking water and resi dential uses. Exposure to the pa rent compound chlorpyrifos is

expected for food and residential uses. Exposure to either chlorpyrifos or chlorpyrifos oxon may

be expected from drinking water sources. The drinking water assessment assumed 100%

conversion of chlorpyrifos to the more toxic ch lorpyrifos oxon (the pr edominant chlorpyrifos

transformation product formed during drinking water treatment ( e.g. , chlorination)).

For acute and steady state aggregate assessments, a DWLOC approach was used to calculate the

amount of exposure which could occur without ex ceeding the risk level of concern (i.e., the

available space in the total aggregate risk cup for exposures to chlorpyrifos oxon in drinking

water after accounting for exposures to parent chlo rpyrifos from food and re sidential uses). The

calculated DWLOCs were then compared to the estimated drinking water concentrations

(EDWCs) of oxon modeled under a variety of conditions. When the EDWC is less than the

DWLOC, there are no risk concerns for exposures to the pesticide in drinking water which also

indicates aggregate exposures are not of concer n. Conversely, when the EDWC is greater than

the DWLOC, then potential risks of concern have been identified. For chlorpyrifos, DWLOCs

were calculated for both the acute and steady stat e aggregate assessments for infants, children,

youths and adult females. However, only the steady state DWLOCs were compared to the

EDWCs as the steady state aggregate risk estimat es are protective of any acute exposures. The

lowest DWLOC calculated was 3.9 ppb for infants (<1 year old) for th e steady state exposure

duration. This water concentrati on is the concentration used fo r comparison to the EDWCs.

Drinking water concentrations of chlorpyrifos oxon above 3.9 ppb indicate a potential risk

5 

# R. Bohaty, C. Peck, A. Lowit, W. Br itton, N. Mallampalli, A. Grube. Chlo rpyrifos: Preliminary Evaluation of the

Potential Risks from Volatilization. 1/31/13. U.S. EPA Office of Chemical Safety and Pollution Prevention.

D399484, D400781.

6 

# W. Britton. W. Irwin. J. Dawson. A. Lowit. E. Mendez. Chlorpyrifos: Reevaluation of the Potential Risks from

Volatilization in Consideration of Chlorpyrifos Parent and Oxon Vapor Inhalation Toxicity Studies. 6/25/2014. U.S.

EPA Office of Chemical Safety and Pollution Prevention. D417105.

7 

# W. Irwin. Review of Nose-Only Inhalation of Chlorpyrifos Vapor: Limited Toxicokinetics and Determination of

Time-Dependent Effects on Plasma, Re d Blood Cell, Brain and Lung Cholines terase Activity in Femal CD(SD): Crl

Rats. U.S. EPA Office of Chemical Safety and Pollution Prevention. 6/25/14. D411959. TXR# 0056694. EPA

MRID# 49119501.

W. Irwin. Review of Nose-Only Inhalation of Chlorpyrifos-Oxon Vapor: Limited Toxicokinetics and Determination

of Time-Dependent Effects on Plasma, Red Blood Cell, Brain, and Lung Cholinesterase Activity in Female

CD(SD):Crl Rats. U.S. EPA Office of Chemical Safety and Pollution Prevention. 6/25/14. D415447. TXR#

0056869. EPA MRID# 49210101.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 11 of 531 concern.

EFED used modeling to perform a national screening bounding exercise to illustrate the range of

estimated EDWC. Two maximum label rate applicati on scenarios were selected to represent high

and low end exposures, i.e., tart cherries at five applications totaling 14.5 pounds per acre per

year, and bulb onions at a single application of one pound per acre per year, respectively. For

surface water, the application of chlorpyrifos to tart cherries resulted in a 21-day average

concentration that exceeded th e drinking water level of comp arison (DWLOC of 3.9 ppb). For

surface water, applications to bulb onions result ed in a 21-day average concentration below the

DWLOC. For groundwater, both ta rt cherry and onion scenarios resulted in EDWCs below the

DWLOC.

To investigate whether other chlorpyrifos applica tion scenarios may result in concentrations that

exceed the DWLOC, a screen of all available surface water modeling scenarios was completed

considering three different application dates and a single application at several different

application rates that ranged fr om one to six pounds. This analys is showed that even with only

one application, several chlorpyrifos uses may exceed the DWLOC at rates lower than maximum

labeled rates (both single as we ll as yearly), including an appl ication rate of one pound per acre

per year. The analysis also showed that the DWLOC exceedances are not expected to be

uniformly distributed across the country.

Further analysis was conducted to look at the spatial di stribution of EDWCs at a regional level,

as well as by using a drinking water intake waters hed approach. This exercise demonstrated that

chlorpyrifos applications will result in variable drinking water exposures that are highly localized

and that the highest exposures generally occur in sm all hydrologic regions where there is a high

percent cropped area on which ch lorpyrifos use could occur.

Finally, EDWCs were compared to monitoring data. This analysis showed that when modeling

scenarios are parameterized to reflect reported use and EDWCs are adjusted to reflect percent

cropped area, the EDWCs are within an order of magnitude of the measured concentrations

reported in the monitoring data. Therefore, altho ugh there are uncertainties associated with the

model input parameters for which cons ervative assumptions were made ( e.g. , one aerobic aquatic

metabolism half-life value multiplie d by the uncertainty factor of three, stable hydrolysis, 100%

of the cropped waters is treated at the same time, and use of the Index Re servoir as the receiving

waterbody), these assumptions do not appear to l ead to an overly conservative estimate of

exposure. In addition, evaluation of the monitori ng data further illustrates that exposures are

highly localized. Additional work can be done to examine EDWCs on a regional and/or

watershed scale to pinpoint community drinking water systems where exposure to chlorpyrifos

oxon as a result of chlorpyrifos appli cations may pose an exposure concern.

[For complete details of the DWA see B ohaty, R. 12/23/14, D424487, Chlorpyrifos: Updated

Drinking Water Assessment for Registration Review.]

Occupational Exposures

Based on the anticipated chlorpyrifos use patter ns and current labeling, a variety of equipment

# Chlorpyrifos Human Health Risk Assessment D424485

Page 12 of 531 and application techniques can potentially be used. These are expected to result in both

occupational handler and post- application exposures. Th e 2011 occupational exposure

assessment has been updated based on a number of changes that have occurred since that time,

including use of the PBPK-PD modeling approach and incorporation of updated metrics (e.g.,

unit exposures for some scenarios). For the occu pational risk assessment, the residue of concern

is the parent chlopyrifos. Exposure to the oxon is only an issue under very limited circumstances

in greenhouses for postapplication workers.

Current labels generally require that handlers use normal work clothing (i.e., long sleeved shirt

and pants, shoes and socks) and coveralls, chemi cal resistant gloves, and dust/mist respirators.

Also, some products are marketed in engineering controls such as water soluble packets. In

order to determine what level of personal protection is required to alleviat e risk concerns and to

ascertain if label modifications are needed, st eady state exposure and risk estimates were

calculated for occupational handlers of chlorpyrifos for a variety of scenarios at differing levels

of personal protection including en gineering controls. In this assessment, a total of 285 total

occupational handler exposure scen arios were assessed and 132 of them are not of concern (i.e.,

MOEs are ≥ 100) at current product label requirement s. Risks of concern for 27 additional

exposure scenarios could potentially be mitigated if additional personal protective equipment or

engineering controls are used. Risks of concern were estima ted for 126 exposure scenarios and

remain a concern regardless of the levels of personal protect ion and engineering controls

considered. These risks could be mitigated us ing the highest level of personal protection

available and by modification to ot her label requirements such as application rate or, in some

cases, deleting certain types of application equipment.

Additionally, occupational handl er exposure for seed treatment activities were assessed

(combined dermal and inhalation). Of the 68 e xposure scenarios assessed, 36 resulted in risk

estimates which were not of concern (i.e., MOEs are ≥ 100) at the level of personal protection

currently required by product labels. Secondary handler (seed planter) scenarios were also

evaluated and none were of con cern at current label requirem ents. The remaining 32 seed

treatment scenarios are of potential concern.

Steady state occupational post-app lication exposures and risks were assessed for any crops where

hand labor is anticipated followi ng applications of chlorpyrifos . The assessment was completed

using seven chlorpyrifos dislodgeable foliar re sidue (DFR) studies. Chlorpyrifos parent

compound is the residue of concern for occupa tional post-application exposures that occur

outdoors; however, it may be possible that the fo rmation of chlorpyrifos oxon is greater and its

degradation slower in greenhouses when compar ed to the outdoor envi ronment. Occupational

post-application assessments were performed for: 1) exposures to the parent compound

chlorpyrifos in outdoor environments (uses other than greenhouse), 2) exposures to the parent

chlorpyrifos (only) in greenhouses and 3) expos ures to both the parent and the oxon metabolite

in greenhouses.

Current labels require a Restri cted Entry Interval (REI) of 24 hours from most crops and

activities, but in some cases such as tree fruit, REIs are up to 5 days after application. All postapplication worker risks have b een updated in the current assessment. Results indicate that no

REI increase is required for the majority of outdoor environment crops and commodities with a

# Chlorpyrifos Human Health Risk Assessment D424485

Page 13 of 531 labeled REI (i.e., 43 of 55 total). However, fo r 12 crops, activities such as irrigation, hand

harvesting, scouting, and thinning re sult in risks of concern up to as many as 10 days following

application. For the assessment of post-application exposures to pa rent chlorpyrifos (only) from

ornamental production in greenhouses, 4 formulations were assessed: emulsi fiable concentrate,

microencapsulated liquid, wettable powder in WSP, and total release fogger. An REI increase of

up to 5 days may be needed to alleviate risks fr om with use of the emulsifiable concentrate,

wettable powder in WSP and tota l release fogger formulations. For the microencapsulated liquid

formulation, the estimated REIs range from 0 to > 35 days after treatment (the completion of the

monitoring period) dependent on the exposure activity considered.

Due to uncertainty regarding the formation of chlorpyrifos oxon in greenhouses, HED also

estimated risks for reentry into treated gree nhouses (all 4 formulations) for the parent

chlorpyrifos plus chlorpyrifos oxon using a total toxic residue appr oach. The total toxic residue

approach was conducted with use of chlorpyrif os DFR studies and an assumed daily fraction,

5%, of chlorpyrifos oxon. When th e total toxic residue approach is used to evaluate risks from

exposure to the oxon with use of the emulsifiable concentrate, wettable powder in WSP, and

total release fogger formulations , results indicate that an RE I increase up to 6 days may be

needed to alleviate risk concerns. For the microencapsulated liquid formulation, REIs range

from 3 to > 35 days after treatment (the comp letion of the monitoring period) dependent on the

exposure activity considered.

Tolerances and MRLs

The establishment of tolerances are being reco mmended for residues of chlorpyrifos on cotton

gin byproducts, aspirated grain fractions, milled corn byproducts, and milled wheat byproducts.

In addition, the current tolerance definition shoul d be updated to be consistent with current

policy.

U.S. tolerances and Codex maximum residue limits (MRLs) are based on the analysis of residues

of chlorpyrifos only. There are differences for some commodities between the U.S. and Codex.

Canadian MRLs are for chlorpyrifos only for some commodities and for bot h parent chlorpyrifos

and its metabolite TCP (3,5,6-trichloro-2-pyridino l; not a U.S. residue of concern) for other

commodities. The potential for tolerance harmonization will be revisited during Registration

Review.



# 2.0 HED Recommendations

2.1 Data Deficiencies

Toxicology

None.

Residue Chemistry

# Chlorpyrifos Human Health Risk Assessment D424485

Page 14 of 531 860.1500:

Separate magnitude of the residue studies for lem ons are needed after appl ication of Lorsban 4E

and 75% WDG formulations in order to reevaluate the existing tolerance for chlorpyrifos for the

citrus fruit crop group.

Magnitude of the residue studies are needed to es tablish a tolerance for residues of chlorpyrifos

on wheat hay.

860.1520:

Processing studies are needed for s oybean meal, hulls and refined oil.

Occupational/Residential

No new data requirements have been identi fied for chlorpyrifos; however, in the 2011

preliminary HHRA, additional studies to addre ss the uncertainties regard ing the formation and

degradation of chlorpyrifos oxon in greenhouses were recommended. To date, those data have

not been submitted. In the absence of the recommended data, and to account for the potential for

oxon to form in greenhouses, EPA has used a conser vative total toxic residue approach for parent

chlorpyrifos plus the chlorpyrifos oxon.



# 2.1 Tolerance Considerations

2.2.1 Enforcement Analytical Method

The methods in the PAM Volume II are adequate to analyze the residue of concern for tolerance

enforcement purposes, chlorpyrifos only. The limit of detection of these methods is adequate to

cover the lowest tolerance leve l included in the 40 CFR 180.342 fo r detection of chlorpyrifos

only, 0.01 ppm. In addition, chlorpyrifos is co mpletely recovered using FDA multiresidue

protocols D and E (nonfatty matrices) and pa rtially recovered using multiresidue method

protocol E (fatty matrices).



# 2.2.2 International Harmonization

Current U.S. tolerances for chlorpyrifos ar e listed in 40 CFR§180.342 and are summarized in

Appendix 8 of this document. The Codex Alimentarius Commission and Canada have

established Maximum Residue Limits (MRLs) for chlorpyrifos. Mexico adopts U.S. tolerances

and/or Codex MRLs for its export purposes. U. S. tolerances and Codex MRLs are based on the

analysis of residues of chlorpyrifos. Canada MRLs are for chlorpyrifos for some commodities

and for both parent chlorpyrifos and its metabo lite TCP (3,5,6-trichloro-2-pyridinol) which is not

a U.S. residue of concern, for other commodities.

With the exception of apple commodities, Canada MRLs are currently no t harmonized with the

U.S. tolerances. This is because the Canada MRL residue definition is for the combined residues

# Chlorpyrifos Human Health Risk Assessment D424485

Page 15 of 531 of chlorpyrifos and the TCP metabolite and in the U.S. the tolerance definition is just for

chlorpyrifos (TCP is not a re sidue of concern for the U.S.). Harmonization between the U.S.

tolerances and Codex MRLs is only possible for corn, field, grai n; cranberry; egg; sorghum,

grain, grain; sorghum, grain, stover; and wheat, gr ain. In addition, two commodities of the Leafy

Vegetable (CG 5) can be harmonized with the Codex, head cabbage, and Chinese cabbage (type

petsai). A summary of the U.S. and internati onal tolerances and MRLs is included in Appendix

8 of this document.



# 2.2.3 Recommended/Reassessed Tolerances

The following tolerances for chlorpyrifos on cott on gin byproducts and aspi rated grain fractions

are necessary to address resi dues found in field trials:

Cotton, gin byproducts........15 ppm

Grain, aspirated fractions.....22 ppm

The following tolerances should be reinstated to address residues of chlorpyrifos on the milled

byproducts of corn and wheat:

Corn, milled byproducts.......0.1 ppm

Wheat, milled byproducts.....1.5 ppm

Table 2.2.3 Recommended Tolerances for Chlorpyrifos

Commodity Established Tolerance

(ppm) Recommended

Tolerance (ppm) Comments

Correct Commodity Definition

Aspirated grain fractions none 22

Cotton, gin byproducts none 15

Corn, milled byproducts none 0.1

Wheat, milled byproducts none 1.5



# On 5/27/09 HED established interim guidance on writing tolerance expressions for enforcement

purposes. According to the guidance, the tole rance expression in the 40 CFR § 180.342 should

be revised to read as follows:

“(a) General. (1) Tolerances are establis hed for residues of chlorpyrifos, including

its metabolites and degradates, in or on the commodities in the table below.

Compliance with the tolerance levels sp ecified below is to be determined by

measuring only chlorpyrifos.”

The current tolerance expression reads “chlorpyrifos per se ( O,O -diethyl O -(3,5,6-trichloro-2-

pyridyl) phosphorothioate”.



# 3.0 Introduction

# Chlorpyrifos Human Health Risk Assessment D424485

Page 16 of 531 

# 3.1 Chemical Identity

Table 3.1 Chlorpyrifos Degradate/ Residues of Concern Nomenclature.

Chlorpyrifos

IUPAC name O,O -diethyl O -3,5,6-trichloro-2-pyridyl phosphorothioate

CAS name O,O -diethyl O -(3,5,6-trichloro-2-pyridinyl) phosphorothioate

CAS registry number 2921-88-2

End-use product (EP) Lorsban 75% WDG and Lorsban 50% WP

TCP Metabolite/Degradate

(Residue of Concern for

Canada)

IUPAC Name

3,5,6 Trichloro-2-pyridinol

Oxon

Metabolite/Degradate

Common Name

Chlorpyrifos Oxon

IUPAC Name

O,O-diethyl. O-3,5,6-

trichloro-2-pyridyl

phosphate



# 3.2 Physical/Chemical Characteristics

Technical chlorpyrifos is a white crystalline solid . Chlorpyrifos is stable in neutral and acidic

aqueous solutions; however, stability decreases w ith increasing pH. Chlorpyrifos is practically

insoluble in water, but is soluble in most or ganic solvents (i.e., acetone, xylene and methylene

chloride). Chlorpyrifos is moderately volatile based on its va por pressure of 1.87x10 -5 

# mmHg at

25 o 

# C. See Appendix 7.

Laboratory studies show chlorpyrifos is suscep tible to hydrolysis under alkaline conditions and

that volatilization and photo-degrad ation are not likely to play a si gnificant role in the dissipation

of chlorpyrifos in the environment. Nonetheless, chlorpyrifos has been de tected in air samples,

and so volatilization may play more of a role in dissipation than laborator y studies indicate. The

major route of dissipation a ppears to be aerobic and anaer obic metabolism, as well as

partitioning to the soil (partition co efficient of 6040). The aerobic aquatic metabolism half-life is

30.4 days (~6% remaining in 4 months). The water peak half-lives were ~1 day in a monitoring

study (MRID 44711601). Based on available data, chlo rpyrifos degrades slowly in soil under O P S

N Cl Cl

Cl OC

2 

# H

5 

# OC

2 

# H

5



# OH N Cl Cl

Cl

# Chlorpyrifos Human Health Risk Assessment D424485

Page 17 of 531 both aerobic and anaerobic conditions. Degrada tion begins with cleavage of the phosphorus

ester bond to yield 3,5,6-trichloro -2-pyridinol (TCP). Field dissipation studies show that

chlorpyrifos is moderately persistent under fi eld conditions—dissipation half-life less than 60

days. Chlorpyrifos is only slightly soluble in water (1400 ppb). However, if it reaches aquatic

environments the Log K

ow 

# (4.7) indicates that chlorpyrifos may bioaccumulate in fish and other

aquatic organisms. A fish bioaccumulation study shows that chlorpyrifos is absorbed by fish;

however, it rapidly depurate s when exposure ceases.

Oxidation of chlorpyrifos to chlorpyrifos oxon could potentially occur through photolysis,

aerobic metabolism, and chlorination as well as other oxidative processe s. Chlorpyrifos oxon is

expected to have similar fate characteristics as chlorpyrifos except chlorpyrifos oxon is more

soluble in water and undergoes hydrol ysis faster. The hydrolysis half-life of chlorpyrifos oxon is

significantly shorter than that observed for chlorpyrifos (5 days vs 81 days). Chlorpyrifos oxon

hydrolyses to form TCP. For ch lorpyrifos, water purification (chl orination) has been shown to

be a major route of chlorpyrifos oxon formation and degradation.



# 3.3 Anticipated Exposure Pathways

Chlorpyrifos applications may be made directly to growing crops (food and feedstuffs) which

may result in human exposure to chlorpyrifos in food and to chlorpyrifos oxon in drinking water

(from surface and ground water sources). Register ed uses that may result in residential (nonoccupational) exposures to chlorpyrifos in clude aerial and ground-based fogger adult

mosquitocide applications and golf course turf applications. There are al so potential exposures

for residential bystanders who live on, work in, or frequent areas adjacent to chlorpyrifos-treated

agricultural fields from spra y drift and volatiliza tion. In occupational settings, exposure may

occur while handling the pesticide prior to applic ation, as well as during application. There is

also a potential for post-app lication exposure for workers re-entering treated fields.



# 3.4 Consideration of Environmental Justice

Potential areas of environmental ju stice concerns, to the extent possi ble, were considered in this

human health risk assessment, in accordance w ith U.S. Executive Order 12898, “Federal Actions

to Address Environmental Just ice in Minority Populations an d Low-Income Populations,”

( http://epa.gov/complia nce/ej/resources/pol icy/exec_order_12898.pdf ). As a part of every

pesticide risk assessment, OPP considers a larg e variety of consumer subgroups according to

well-established procedures. In line with OPP policy, HED estimates risks to population

subgroups from pesticide exposures that are based on patterns of that subgroup’s food and water

consumption, and activities in and around the home that involve pesticide use in a residential

setting. Extensive data on food consumption pa tterns are compiled by the U.S. Department of

Agriculture (USDA) under the NHANES/WWEIA (What We Eat in America) Survey; 2003-

2008 and are used in pesticide risk assessments for all registered food uses of a pesticide. These

data are analyzed and categorized by subgroups based on age, season of the year, ethnic group,

and region of the country. Additionally, OPP is ab le to assess dietary exposure to smaller,

specialized subgroups and exposure assessments are performed when conditions or

# Chlorpyrifos Human Health Risk Assessment D424485

Page 18 of 531 circumstances warrant. Whenever appropriate , non-dietary exposures based on home use of

pesticide products and associated risks for adult applicators and for toddlers, youths, and adults

entering or playing on treated ar eas post-application are evaluate d. Potential exposures from

spray drift and via volatilization ha ve also been considered in this risk assessment. The spray

drift assessment is revised a nd previously implemented measures such as buffer zones were

refined.



# 4.0 Hazard Characterization and Dose-Response Assessment

This section provides summary information and we ight of evidence findings integrating multiple

lines of evidence from experimental toxicol ogy and epidemiology with respect to AChE/ChE

inhibition (acetylcholinesterase/c holinesterase) and neurodevelopmental outcomes. This section

also describes the use of a robust physio logically-based pharmacokinetic-pharmacodynamic

(PBPK-PD) model for deriving poin ts of departure (PoDs) and re fined intra-species factors.

Details of the science and data analysis th at support these conclu sions can be found in

Appendices 1-6, 10, and 11. Code for the PBPK-P D model can be found in the docket for this

risk assessment.

Appendix 1: Evaluation of E xperimental Toxicology Data

Appendix 2: Detailed Review and Synthesis of Three Children’s Enviro nmental Health Cohort

Studies

Appendix 3: Epidemiology Study Specific Evaluations

Appendix 4: Detailed Summary Tables of Ch ildren’s Environmental Health Epidemiology

Studies

Appendix 5. Summary of OPP’s Ch E Policy & Use of BMD Modeling

Appendix 6. Columbia Center for Children’s Environmental Health (CCCEH) Epidemiology

Data Acquisition “Raw Data” Request

Appendix 10. Dose Reconstruction Analysis

Appendix 11. New Literature on Chlorpyrifos since the 2012 FIFRA SAP Meeting



# 4.1 Introduction & Background

Mode of action (MOA) and adverse outcome path ways (AOPs) provide im portant concepts and

organizing tools for risk assessm ent (Boobis et al., 2008; Seed et al., 2005; Sonich-Mullin et al.,

2001; Meek et al, 2014; Ankley et al, 2010). MOAs/AOPs descri be a set of measureable key

events that make up the biological processes leading to an adverse outcome and the causal

linkages between such events. An AOP further de fines the initial step in the process as the

molecular initiating ev ent (MIE; Ankley, et al., 2010). Fundamentally, MOA and AOP are

different terms for basically the same con cept. Consistent with the World Health

Organization/International Progra mme on Chemical Safety, for this risk assessment, the terms

are considered equivalent and interchangeable (Meek et al, 2014). For chlorpyrifos two different

adverse outcomes provide the focus of the revi sed chlorpyrifos risk assessment: AChE/ChE

inhibition and neurodevelopmental outcomes.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 19 of 531 It is well established that AChE inhibition is the MOA/AOP for the c holinergic toxicity of

organophosphate pesticides (OPs), including chlo rpyrifos. Because AChE inhibition is the

initiating event for this MOA/AOP, using AChE i nhibition as a regulatory endpoint is protective

of downstream cholinergic effects. Moreover, gi ven the sensitivity of AChE inhibition data for

OPs, these data have historically been consider ed to be protective of other potential toxicities

and/or MOAs/AOPs for OPs.

Figure 1. Adverse outcome pathway for OPs

Newer lines of research on chlorpyrifos, notab ly epidemiological studies in mothers and

children, have raised some uncertainty about the Agency’s risk assessment approach for

chlorpyrifos with regard to the potential for neur odevelopmental effects. It is this uncertainty

which provides the foundation for retaining the 10X FQPA Safety Factor as described in Section

4.6 and 4.7.

Over the last several years, the Agency has ta ken a stepwise, objective an d transparent approach

to evaluate, interpret, and charac terize the strengths and uncertainties associated with all the lines

of scientific information related to the potential for adverse neurodevelopmental effects in infants

and children as a result of prenatal exposure to ch lorpyrifos. The stepwise evaluation began with

the September 2008 FIFRA SAP involving a prel iminary review of the literature for

chlorpyrifos, with a particular focus on wo men and children (USEPA, 2008). Subsequent

Agency activities have involved developing appro aches for performing risk assessment of semivolatile pesticides (USEPA, 2009), and developi ng the draft “Framework for Incorporating

Human Epidemiologic & Incident Data in He alth Risk Assessment ” for integration of

epidemiology with other types of experimental data (USEPA, 2010; FIFRA SAP 2010a,b). In

early 2011, the FIFRA SAP reviewed the PBPK-PD model being used in this assessment to

conduct quantitative risk assessment with estimates of human ChE inhibition following exposure

to chlorpyrifos and/or the oxon from a variety of exposure pathways (FIFRA SAP 2011). In

summer 2011, the Agency released its preliminary hu man health risk assessment that focused on

the AChE inhibiting potential of chlorpyrifos (USEPA, 2011). This focus was consistent with

the recommendation from the 2008 SAP that AChE data provide the most appropriate endpoint

and dose-response data for deriving PoDs for purposes of risk assessment. Moreover, because of

the Agency’s long experience with assessing the poten tial risk to chlorpyrifos and other OPs, and

because the dose response approaches based on AChE inhibition used in the 2011 preliminary

assessment had been vetted by numerous SAPs, there was confidence in that approach.

In 2012, the Agency convened another meeting of the FIFRA SAP focused on chlorpyrifos

which incorporated the newest e xperimental data related to AChE inhibition and both cholinergic

and non-cholinergic adverse outcomes, including neurodevelopmental studies on behavior and

cognition effects (FIFRA SAP 2012). Similarly, th e Agency also performed a more in-depth Target

Tissue dose Phosphorylation

of the active site

of AChE Neurotoxicity Accumulation of

acetylcholine

# Chlorpyrifos Human Health Risk Assessment D424485

Page 20 of 531 analysis of the biomonitoring data and of epid emiologic studies from three major children’s

health cohort studies in the U.S., as well as plausible hypotheses on MOAs/AOPs leading to

neurodevelopmental outcomes. Following th e 2012 SAP meeting, the Agency solicited

additional input from federal experts in the ar eas of MRI (Magnetic Resonance Imaging) and

neurobehavioral testing in child ren to further clarify results obtained by examination of the

epidemiological cohorts. 8 

# Also, the potential for exposure to lead and other environmental

chemicals to affect the interpretation of the results from the Columbia University studies was

investigated and EPA inquired a bout potential availability of additional information from

investigators at Columbia University (Appendi x 6). This effort has involved extensive

collaboration across EPA and also within the Federal government.

Aspects of this risk assessment represent innovat ive approaches to pesticide human health risk

assessment. For example, this risk assessmen t uses the draft “Framework for Incorporating

Human Epidemiologic & Incident Data in Health Risk Assessment” (U.S. EPA, 2010) to develop

a weight of evidence analysis integrating quantitative and qualit ative findings across many lines

of evidence, including experi mental toxicology studies, epid emiology studies and PBPK-PD

modeling, does not provide support for the reduc tion of the 10X FQPA Safety Factor. In

addition, the Agency is explicitly using th e robust PBPK-PD model to estimate human

equivalent doses/concentrations and intra-specie s factors to chlorpyrifos and/or its oxon from

multiple pathways (e.g. food, water, occupa tional, residential) of exposure.



# 4.2 Types of Studies Available for Analysis

Chlorpyrifos and its oxon are wide ly studied and thus have an ex tensive database of scientific

studies. Included in the data base are: guideline studies, sp ecial studies conducted by the

registrant, and literature studies across many scientific areas reflective of different levels of

biological organization ( e.g. metabolism, MOA/AOP, in vitro and in vivo experimental

toxicology, biomonitoring, epidemiology), vari ous species (mouse, rabbit, dog, non-rodent,

human) and across lifestages (fetal , postnatal, pregnant and non-pre gnant adult). As reflected in

the 2000 risk assessment, 2008 and 2012 draft issu e papers for the SAP, the 2011 preliminary

risk assessment, and this revised risk asse ssment, EPA has reviewed numerous studies for

chlorpyrifos and the oxon and provided those c itations in the various documents for public

evaluation.

In recent years, the National Academy of Scien ces has encouraged the Agency to move towards

systematic review processes to enhance the transp arency of scientific literature reviews that

support chemical-specific risk assessment s to inform regulatory decision making 9 

# . EPA’s Office

of Chemical Safety and Pollution Prevention is currently developing systematic review policies

and procedures. As part of th e revised human health risk assessment, the Agency has reviewed

and updated the experimental toxi cology literature sear ch since the 2012 SAP using the concepts

consistent with systematic review such as deta iled tracking of search terms and which literature

have been included or excluded. The literatur e review was conducted in PubMed for the time

8 

# http://www.regulations.gov/#!document Detail;D=EPA-HQ-OP P-2008-0 850-0170

9 

# NRC 2011. “Review of the Environmental Protection Ag ency’s Draft IRIS Assessm ent of Formaldehyde” ;

NRC 2014. “Review of EPA's Integrated Ri sk Information System (IRIS) Process”

# Chlorpyrifos Human Health Risk Assessment D424485

Page 21 of 531 period January 2012 up to August 2014 and was supplemented with ToxLine and Google

Scholar (Appendix 11).



# 4.3 Absorption, Distribution, Me tabolism, & Excretion (ADME)

The metabolism and pharmacokinetic (PK) profil es of chlorpyrifos and its oxon have been

extensively studied in in vitro systems, in vivo laboratory animals, as well as humans. This large

body of PK information is used in the PBPK-PD model which is described in more detail in

Section 4.8. Only limited summary in formation is provided here.

Chlorpyrifos undergoes metabolic transformations mainly by the liver microsomal enzymes ( i.e.

, cytochrome P450s). Although, chlorpyrifos is li pophilic, its ex tensive metabolism into water

soluble metabolites does not lead to any accumulation of the parent material or its metabolites in

the body tissues. The initial metabolic action of chlorpyrifos is desulfuration, resulting in

bioactivation of the parent co mpound to the more toxic and potent AChE inhibitor, the oxon

form (Figure 2). However, the oxon is unstabl e and is rapidly deactivated through hydrolytic

cleavage by a process called dearylation rele asing 3,5,6-trichloro-2- pyridinol (TCPy).

Simultaneous with the desulfuration process, dear ylation acts on both the pa rent chlorpyrifos as

well as on the oxon metabolite leadin g to the release of TCPy. TC Py is further conjugated to

form glycine or glucuronide conjug ates and eliminated into the urin e. TCPy is the major excreted

metabolite and used as the biomarker in PK , biomonitoring, and epidemiology studies.

Diethylphosphate (DEP) is another metabolite often used in biomonitoring studies, but since it is

produced by a number of OPs, DEP is not a spec ific marker for chlorpyrifos. An important

aspect of the chlorpyrifos PBPK model is th at chlorpyrifos and its oxon are approximately 98-

99% protein bound in the blood.

There are several enzymes that ha ve roles in the metabolism and t oxicity of chlorpyrifos. In

addition to inhibition of ChE, the oxon binds st oichometrically to B-esterases, the most

important of which are butyrylcholinesterse (BuC hE; abundant in blood, brain, and other tissues)

and carboxylesterases (highe st levels in liver). These B-esterases functi on as a scavenger, or

“sink”, of the oxon and may lessen its entry in th e brain or peripheral ta rgets to inhibit AChE.

Another group of important enzymes in the detoxifi cation of chlorpyrifos is the A-esterases; one

such A-esterase is paraoxonase (i.e., PON1). Th ese esterases are calcium- activated enzymes that

are distributed in various tissues including the liver, brain and blood. These enzymes act on the

oxon by hydrolyzing it before reaching its target AC hE enzyme. The cytochrome P450 family of

microsomal enzymes (CYPs) is also responsible for its metabolic activation and deactivation of

chlorpyrifos. Glutathione-dependent enzymes play an important ro le in the secondary

metabolism of chlorpyrifos producin g water soluble metabolites that are readily excreted into the

urine.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 22 of 531 Figure 2. Major metabolic pathways of chlorpyrif os metabolism (Reproduced from Timchalk et al,

2002)



# As summarized below and supported by the PBPK -PD model, there is lifestage sensitivity,

particularly to the very young following acute ex posures. This sensitivity is not derived from

differential inhibition of the AChE enzyme itself as supported by in vitro studies (Benke and

Murphy, 1975; Chanda et al. , 1995; Mortensen et al. , 1996; Atterberry et al. , 1997). Rat fetuses

and juveniles and human fetuses, infants, and young children have lower capacity to detoxify

chlorpyrifos than adults. Specifi cally, in rats, A-estera se activity is virtua lly nonexistent in the

fetus (Lassiter et al., 1998) and increases from birth to reach adult levels around post-natal day

21 (PND21) (Mortensen et al., 1996; Li et al., 1997). Mortenson et al (1996) showed that in the

plasma level of A-esterase in 4-day old rats was 1/11 that of adult animals. The animal data

regarding the role of carboxyleste rase in mediating OP toxicity are also quite extensive (e.g.,

Clement, 1984; Fonnum et al., 1985; Maxwell, 1992 a, b). Fetal rats and mice possess very little

carboxylesterase activity with incr easing activity as the postnatal rodent matures, reaching adult

values around puberty (Lassiter et al., 1998; Morg an et al., 1994; Moser et al., 1998; Karanth and

Pope, 2000; Zhu et al., 2009). The temporal patt ern of A-esterase and carboxylesterase activity

correlates well with studies on OP sensitivity. Several studies have shown an increased

sensitivity of newborn rats to OP compounds wh ich are detoxified via the A-esterase and/or

carboxylesterase pathways (e.g., Gagne and Br odeur, 1972; Benke and Murphy, 1975; Pope et

al., 1991; Chambers and Carr, 1993; Padilla et al., 2000; 2002; Karanth and Pope, 2000). C l C l C l

C H 3 C H 3

O O S

P

O N

O

C l C l C l

C H 3 C H 3

O O P

O N 

# AChE Inhibition

Toxicity

C H 3 C H 3

O O O

P

O H C H 3 C H 3

O O S

P

O H

C l C l C l

O H N 

# s

C l C l C l

O N 

# Conjugates- Chlorpyrifos

Chlorpyrifos-oxon A

- e

s

t e

r a

s

e

( P

O

N

- 1 ) B

- e

s

t e

r a

s

e



# Diethylphosphate Diethylthiophosphate

3,5,6-trichloro-2-pyridinol

(TCP)

Sulfate or glucuronides of TCP C l C l C l

C H 3 C H 3

O O S

P

O N

O

C l C l C l

C H 3 C H 3

O O P

O N 

# AChE Inhibition

Toxicity

C H 3 C H 3

O O O

P

O H C H 3 C H 3

O O S

P

O H

C l C l C l

O H N 

# s

C l C l C l

O N 

# Conjugates- C l C l C l

O N 

# Conjugates- Chlorpyrifos

Chlorpyrifos-oxon A

- e

s

t e

r a

s

e

( P

O

N

- 1 ) B

- e

s

t e

r a

s

e



# Diethylphosphate Diethylthiophosphate

3,5,6-trichloro-2-pyridinol

(TCP)

Sulfate or glucuronides of TCP 

# C

Y

P4 CYP450

# Chlorpyrifos Human Health Risk Assessment D424485

Page 23 of 531 While there are fewer data in human tissues wh ich could evaluate age-related maturation of

carboxylesterase and A-esterase expression, th ere are numerous studies that present age

differences in liver P-450 metabolism in humans that also play a role in OP metabolism (e.g.,

review by Hines, 2007). Studies evaluati ng maturational expression and activity of

carboxylesterases in human liver ti ssues show lowest levels in fetal tissues, with increasing

activity during childhood, although there are large individual di fferences and thus high

variability (Pope et al., 2005; Yang et al., 2009 ; Zhu et al., 2009). Adult tissues were

approximately 10-fold and 4-fold more active in hydrolyzing certain chemicals than fetal and

child (0-10 years) tissues, resp ectively, and enzyme expression differences were much greater

(Yang et al., 2009). In contrast, in Pope et al . (2005), the differences in activity were relatively

small (and not statistically si gnificant) between children ages 2–24 months and adults (20–36

years); however, that youngest ag e evaluated in the study was 2 m onths old and this individual

had the lowest level of carboxylesterase.

Serum A-esterase levels also a ppear to be very low in human infants and children compared to

adults (Augustinsson and Barr, 1962; Cole et al., 2003; Huen et al., 2009; Mueller et al., 1983;

Ecobichon and Stephens, 1973; Gonzalez et al., 2 012; Holland et al, 2006; Chen et al, 2003).

While some studies have shown that PON1 activit y reaches adult levels by about 2 years of age

(e.g., Cole et al., 2003), recent papers have report ed slightly lower levels as late as 9 years

(Gonzalez et al., 2012). Reasons for these differen ces could include type of measurement (i.e.,

genotyping vs phenotyping), assa y substrate, sample sizes , and population factors (e.g.,

ethnicity).

With respect to distribution to the fetus during gestation, there are multiple studies on

chlorpyrifos (Mattsson et al., 1998, 2000;; Qi ao et al, 2002) and other OPs (USEPA, 2006)

which show that the pregnant dam exhibits simila r or more AChE inhibition than the fetus at a

given dose to the dam. As such, for AChE inhi bition, protecting against AChE inhibition in the

pregnant female is expected to be protective for AChE inhibition in the fetus. Biomonitoring

data from rats and humans illustrate fetal exposur e levels are similar to maternal levels which

support these findings. Specifically, Whyatt et al (2003) have shown that levels of chlorpyrifos

in maternal blood are similar to the levels measur ed in human umbilical cord blood. In Hunter et

al. (1999), pregnant rats were orally dosed with chlorpyrifos for 5 days during late gestation at

doses of 3 or 7 mg/kg/day, and the amounts of chlorpyrifos, chlorpyrifos-oxon, and TCPy were

quantified in fetal and maternal brain and liver. No chlorpyrifos or its oxon were detected. The

concentration of TCPy in the fetal brain was high er than the TCPy concentration in the maternal

brain in time-course and dose-respon se studies but in liver the mate rnal levels were higher. In a

study by Mattsson et al (1998, 2000), concentrations of chlorpyrifos, the oxon, and TCPy were

measured in the blood of maternal and fetuses and TCPy levels in dam and fetal blood were

similar and chlorpyrifos levels were approximate ly 2-fold higher in maternal blood than fetal

blood. In another gavage gestational exposure study, Akhtar et al ( 2006) exposed rats to

chlorpyrifos from GD 0-20 in fetal and ma ternal tissues on GD21 at high doses (9.6-15

mg/kg/day); while there is a high degree of variability in the Akht ar et al study, dams and fetuses

showed similar levels of chlor pyrifos in liver and brain.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 24 of 531 

# 4.4 Summary of Experimental Toxicology Studies

Summarized below are key findings from experi mental toxicology studies on AChE inhibition

and neurodevelopmental outcomes. Details can be found in Appendix 1. Chlorpyrifos has also

been evaluated for other adverse outcomes such as reproductive toxicity, developmental toxicity,

cancer, genotoxicity, dermal toxi city, inhalation toxicity, and i mmunotoxicity. The summary of

these findings can be found in the 2000 risk assessment which supported the Registration

Eligibility Decision and the 2011 preliminary risk assessment. These adverse outcomes are less

sensitive than AChE inhibition and neurodevelopmental effects and are thus not discussed in

detail here.



# 4.4.1 AChE Inhibition in Experi mental Laboratory Animal Studies

For the 2008 and 2012 SAP reviews and the 2011 preliminary risk assessment, the Agency

performed comprehensive reviews of the literatur e for AChE inhibition. This risk assessment

has been updated with newer studies. A summar y of EPA’s ChE policy w ith respect to the use

of such data in risk assessment is provided in Appendix 5. AChE inhibition remains the most

robust quantitative dose response data and thus c ontinues to be the critical effect for the

quantitative risk assessment. Th is approach is consistent with the advice of the SAP from 2008

and 2012. The Agency has conducted benchmark dose (BMD) analysis of numerous studies

using empirical approaches previously accepted by the FIFRA SAP (USEPA, 2002) and

consistent with the 2006 OP cumulative risk assessment (USEPA, 2006) and other single

chemical OP risk assessments. Details on Ch E studies and related an alyses can be found in

Appendix 1 and the preliminary HHRA (USEPA, 2011).

There are many chlorpyrifos studi es evaluating AChE inhibition in red blood cell (RBC) or brain

in multiple lifestages (gestational, fetal, post-na tal, and non-pregnant adul t), multiple species (rat,

mouse, rabbit, dog, human), methods of oral admini stration (oral gavage with corn oil, dietary,

gavage via milk) and routes of exposure (oral, de rmal, inhalation via vapor and via aerosol). In

addition, chlorpyrifos is unique in the availability of ChE data fr om peripheral tissues in some

studies (e.g., heart, lung, live r). There are also literature studies comparing the in vitro ChE

response to a variety of tissues (Chambers, 2013) which show similar se nsitivity and intrinsic

activity. Across the database, brain AChE te nds to be less sensitiv e than RBC AChE or

peripheral ChE. In oral studies, RBC AChE in hibition is generally similar in response to

peripheral tissues. Thus, the in vitro data and oral studies combin ed supports the continued use

of RBC AChE inhibition as the critical effect for quantitative dose-response assessment.

As with many OPs, female rats tend to be more sensitive than males to these AChE effects. For

chlorpyrifos, there are data from multiple st udies which provide robust RBC AChE data in

pregnant, lactating, and non-pre gnant female rats from oral exposure [e.g., DNT, reproductive,

and subchronic rats], respectively. The BMD 10 

# /BMDL 10 10 

# values from these studies range from

0.05/0.04 to 0.18/0.12 mg/kg/day (Table 4 in Appendix 1). Studies ar e available in juvenile pups

which show age-dependent differences, particularly following acute exposures, in sensitivity to

10 

# BMD 10 

# is the estimated dose to yield 10% inhibition in RBC AChE inhibition compared to controls or background

levels. The BMDL 10 

# is the lower 95% conf idence limit on the BMD 10



# .

# Chlorpyrifos Human Health Risk Assessment D424485

Page 25 of 531 chlorpyrifos and its oxon (Tables 1, 2, 3, and 4 in Appendix 1). As discussed above, this

sensitivity is not derived from differences in the AChE enzyme itself but instead are derived

largely from the immature metabolic cl earance capacity in the juveniles.

Multiple route-specific studies for the dermal and inhalation routes are available. Dermal AChE

data are available from a 21-day study and 4-day probe study (MRID 40972801) in rats which

together establish a NOAEL of 5 mg/kg/day and LOAEL of 10 mg/kg/day. Two subchronic

inhalation toxicity stud ies (MRID Nos.40013901, 40166501, 40908401) in the rat are available

using vapor phase chlorpyrifos which show no ChE effects up to a concentration of 20.6 ppb

(287 μg/m 3 

# or 0.082 mg/kg/day (MRID 40013901, 40166501, 40908401). Multiple acute

inhalation studies are also available. In a special acute inhalation study, female rats were

exposed by nose only 11 

# to atmospheric concentrations of up to 53.9 mg/m 3 

# of particulate

chlorpyrifos for six hours and allowed an additional 72 hours to recover (MRID No: 48139303

Hotchkiss et al. 2010, TXR # 0055409). Consistent and significant lung ChE inhibition were

noted at the lowest concen tration tested of 3.7 mg/m 3 

# , which is a LOAEL. RBC and brain ChE

inhibition were noted at ≥ 12.9 mg/m 3 

# and 53.9 mg/m 3 

# , respectively, indicating they are less

sensitive than lung and plasma ChE inhibiti on following acute inha lation exposures.

Since the 2011 preliminary risk assessment, two acute inhalation studies on the saturated vapor

have been performed on the parent chlo rpyrifos and chlorpyrifos oxon (MRID 49119501 and

49210101, respectively). In these studies, female rats were exposed by nose only to a saturated

vapor of chlorpyrifos or its oxon for 6 hours to a time-weighted concentration of 17.7 ppb (0.254

mg/m 3 

# ) (MRID 49119501) or 2.58 ppb (35.3 μ g/m 3 ) 

# (MRID 49210101), respectively. There were

no statistically-signifi cant decreases in ChE activity in th e RBC, lung, brain or plasma tissues.

These acute studies along with the subchronic inhalation studies with vapor phase chlorpyrifos

support a conclusion that acute exposure to the sa turated vapor of chlorp yrifos or its oxon do not

result in hazard.



# 4.4.2 Neurodevelopmental Outcom es in Laboratory Animals

There is a considerable and gr owing body of literature on the e ffects of chlorpyrifos on the

developing brain of laboratory animals (rats and mice) indicating that gestational and/or

postnatal exposure may cause persis tent behavioral effects into adulthood. These data provide

support for the susceptibility of the developing mammalian brain to chlorp yrifos exposure. In

the 2008 and 2012 SAP reviews, the Agency evaluated the neurobeha vioral studies available at

that time; the literature review has been updated for the revised risk assessment (Appendix 1).

Papers considered by EPA as addressing long- term outcomes from developmental exposure

include only those where chlorpyrifos is admini stered during the pre-w eaning period (gestational

and/or postnatal) and the offspring are examined at some time after weaning. That is, papers

reporting evaluations shortly after birth or during the pre-weani ng period do not reflect long-term

consequences and may also be confounded by AChE/C hE inhibition during concurrent or recent

exposure. In addition, the Agency focused its efforts on studies using relatively low doses (e.g.,

1 mg/kg/day), that is, doses that would not be expected to produ ce a considerable degree of brain

AChE inhibition and resultant c holinergic toxicity. These cons traints aid in the unencumbered

11 

# Mass median aerodynamic diamet er/geometric standard deviati on was 1.9/1.51 , respectively

# Chlorpyrifos Human Health Risk Assessment D424485

Page 26 of 531 evaluation of longer-term effects co mpared to acute impacts of AChE inhibition. In total, the

Agency has reviewed 31 papers generated fr om 14 different laborator ies on areas such as

cognitive function, anxiety/emotion, social beha viors/interaction, and mo tor activity. Twentyfive papers were reviewed for the 2012 SAP, an d another six have been published and reviewed

by ORD since then. The review of the new papers can be found in Appendix 1.

In spite of considerable differences in study design, upon review of the published literature a

pattern of neurodevelopmental adverse outcomes em erges. Although the manifestations of these

effects differ (nature and/or severity), these diffe rences are likely due to the variability in study

designs including developmental period of exposure, dosing scenarios, testing methods, ages of

testing subjects, specific equipmen t used, choice of dependent variab le, statistical analyses--all of

which are critical features of all developmental neurotoxicity studies. Wh ile behavioral changes

were consistently reported, they were somewhat inconsistent, possibly due to experimental

design differences. Given the wide array of tes ting that has been conducte d, some variability is

not unexpected and in fact, the consistency of findi ng neurological effects is striking. At both

the 2008 and 2012 SAP meetings, the Panel agreed that exposure to doses of 1 mg/kg/d and

greater, during some developmental period, produced significant and long-term effects on animal

behavior.

These studies report a range of neurobehavi oral changes in rats and mice following

developmental exposure to chlorpyrifos. O bvious species differences have not emerged,

although effective doses are similar (1-6 mg/kg/d). Changes in various aspects of cognitive tests

indicate perturbations of lear ning and/or memory, even though in some cases these may be

manifested as improved function. Likewise, altera tions in domains such as anxiety and social

interactions may differ in dire ction of change, but are still suggestive of impacts on normal

neuronal processing. There is re plication of some e ffects across studies, a nd with some of the

newer papers, across laboratories as well. Activity measures, on the other hand, still provide

results as varied as the different measures of assessment. Taken together, these data do not

provide evidence for a specific prof ile of effects but instead sugge st more global alterations in

neurobehavioral function.

All testing was conducted at various times after weaning (adolescents to adults), and there is a

presumption that the effects are permanent; however , no study has directly addressed this issue,

and there is a range in test ag es. Dose-response is not always evident, since many studies only

use one dose, and of those using two or more dos es, there is not always a monotonic response.

While there are demonstrated differences in up take and persistence of chlorpyrifos given

subcutaneously vs. oral, and with different oils or DMSO as vehicle, the developmental literature

does not provide obvious differences in outcome based on these factors. Likewise, the

experimental literature has not shown that any specific developmental period is critical overall to

the long-term outcomes, since similar effects ar e shown with different exposure periods. For

example, cognitive changes in the radial arm maze were observed following gestational and early

postnatal (PND1-4), but not late (PND11-14), ex posure (Aldridge, et al., 2005; Icenogle, et al.,

2004; Levin, et al., 2002; Levin, et al., 2001). Ho wever, cognitive deficits were reported with

the Morris water maze following both gestationa l and late postnatal exposures (BillauerHaimovitch, et al., 2009; Jett, et al., 2001; Turgem an, et al., 2011). Similarly, some changes in

anxiety and social behaviors we re reported at both gestational and postnatal exposure periods.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 27 of 531 Overall, these data do not clearly show specifi c critical periods of e xposure, or definitive

sensitive behavioral outcomes. Unfortunate ly, no laboratory has provided systematic

comparisons across exposure period, dosing regime n, and age of testing; such studies would

improve understanding of the impact of these critical factors.

These studies have almost exclusively focu sed on doses that could produce some degree,

however minimal, of AChE inhibition. For exampl e, a number of papers from Duke University

researchers use a dose of 1 mg/kg/ d administered 1-4 days after bi rth (e.g., Aldridge et al., 2005;

Levin et al., 2001), but a recent publication (Slotkin et al., 2013) has reported that 5-10%

inhibition of brain AChE activity is measured at 2 hours after the la st dose. Given that this dose

is widely used in many studies from several la boratories, this information clearly shows that

while this dose does not induce toxic effects, it does however have a small but significant effect

on pup brain AChE. Another study of relatively low doses administer ed chlorpyrifos in feed to

pregnant rats, and even the lowest intake of 0.36 mg/kg/d produ ced about 20-25% RBC ChE

inhibition (Ohishi et al., 2013). Thus it is not possible to know wh ether effects would be present

at lower doses, since they have not been adequately studied.

Overall, across the literature on neurodevel opmental outcomes and including most recent

publications, there continue to be inconsistencies in effects in relation to functional domains,

dosing paradigms, and gender-specificity. The only studies reporting e ffects use doses that

inhibit fetal/pup brain ChE activity to some degree, even though there are also negative effects at

the same doses. The broad profile of neurological effects that have been reported do not aid in

the development of a specific AOP (AChE inhi bition or other mechanisms), and existing

experimental studies have not been designed to examine and track possible mechanisms from

early initiating events to the final neurological outcome.



# 4.4.3 Plausible hypotheses on MOA/AO P for neurodevelopmental outcomes

Numerous studies on the possible mechanistic asp ects of neurodevelopmental effects have been

published. The results have led some resear ch groups to propose that changes in brain

connectivity and/or neurochemistry may underlie the long-term in vivo neurobehavioral changes

observed into adulthood. While multiple biologically plausible hypotheses are being pursued by

researchers, no one pathway has suff icient data to be considered mo re credible than the others.

The SAP concurred with the Agency in 2008 and 2012 about the lack of definable key events in

a MOA/AOP leading to ne urobehavioral effects. The Agency has considered the new literature

since the 2012 SAP related to mechanistic hypot heses as described below (Appendix 11), and

note that such a MOA/AOP still cannot be established.

 Acetylcholinesterase (AChE) as a morphogen : The classically unde rstood role of AChE

is the rapid hydrolysis of acetylcholine at s ynapses in the brain and at neuromuscular

junctions, thereby regulating cholinergic neur otransmission. Consistent with this role,

AChE is predominant at cholinergic synapses at neurons and in musc le, and inhibition of

its catalytic activity results in the signs and symptoms of cholinergic overstimulation.

Several lines of evidence suggest that AChE can also serve as a morphogen, influencing

the growth of cells during neurodevelopment distinct from its role as an esterase.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 28 of 531 Alterations in the expression or structure of the AChE protei n can disrupt various aspects

of neuronal differentiation and growth, as has recently been shown in vitro (using

NG108-15 cell line) following exposure to another OP, paraoxon (Campanha et al.,

2014). While perturbation of the morphogenic ac tivity of AChE is a plausible adverse

outcome pathway for chlorpyrifos, a number of questions remain, including effective

concentrations compared to those that i nhibit catalytic activity of AChE. There is,

however, no direct evidence showing that disruption of the morphogenic function of

AChE can alter axon or dendritic growth in vivo . While limited in vivo studies using

zebrafish indicate that chlorpyrifos or its metabolite chlorpyrifos oxon can disrupt axonal

growth (Yang et al. 2011), it has not been demonstrated that this effect is due to alteration

of the morphogenic function of AChE versus other potential mechanisms.

 Cholinergic system: There are several lines of evid ence showing that signaling through

cholinergic receptors is involved in neurodeve lopment. Activation of muscarinic and/or

nicotinic cholinergic receptors can regulat e neural progenitor cell proliferation and

differentiation (Resende & Adhikari, 2009), and in vivo studies demonstrate that

cholinergic signaling is like ly involved in brain morphogenesis (Hohmann & BergerSweeney, 1998). While ChE inhibitors can affe ct cholinergic signali ng by inhibition of

the catalytic activity of AChE and subsequent increase in acetylcholine, some inhibitors,

including chlorpyrifos and chlo rpyrifos oxon, can also directly interact with cholinergic

receptors. Thus, direct interaction with cholin ergic receptors by chlorpyrifos represents a

potential adverse outcome pathway for disr uption of neurodevelopment distinct from

AChE/ChE inhibition. Some OPs have been sh own to directly interact with cholinergic

muscarinic receptors at relatively low c oncentrations. The muscarinic receptors are

members of the G-protein receptor family and five subtypes (m1-m5) have been

identified. Ward et al. (1993) examined the relationship between ChE inhibition and

direct binding to muscarin ic receptors for a series of OPs and their active oxon

metabolites. The results indicated a strong co rrelation between AChE activity of OPs,

including chlorpyrifos and ch lorpyrifos oxon, and the ability to compete for CD binding

sites (m2 receptors) in rat brain homogenates. Binding affinities of the oxons were in the

nanomolar range, at or below concentrations that inhibited AChE (Huff et al, 1994);

specifically, chlorpyrifos oxon had a binding affi nity of 22 nM in rat striatum and 2 nM

in rat cortex (Huff, et al., 1994; Ward & Mundy, 1996). In total, these studies suggest

that direct interactions with muscarinic receptors, and especially the m2 subtype,

represent an alternative site of action for OPs including ch lorpyrifos and chlorpyrifos

oxon, with the oxon forms having high affinity. Together, the studies cited above outline

a plausible adverse outcome pathway for chlo rpyrifos and chlorpyrifos oxon to affect

brain development via actions at the m2 subt ype of muscarinic receptors. However, while

there are studies showing that chlorpyrifos oxon can affect neurite outgrowth in vitro and

decrease cell proliferat ion and differentiation both in vitro (Jameson et al , 2006; Qiao et

al, , 2001; Song, et al., 1998) and in vivo (Dam et al, , 1998; Qia o, et al., 2003), there is

no experimental evidence that these effects are a result of direct actions on the m2

receptor.

 Endocannabinoid system: Several lines of research have suggested that disruption of the

endocannabinoid (EC) system due to chlorpyrif os exposure could play a role in its acute

# Chlorpyrifos Human Health Risk Assessment D424485

Page 29 of 531 and/or long-term toxicity, and could also be extended to poten tial developmental toxicity.

The EC system modulates neurotransmission as well as playing a morphogenic role

during development of the nervous system. Chemicals ( e.g. , drugs of abuse) which act

on this system, produce long-term neurodevelopmental disorders in animal models and

human studies. Chlorpyrifos also interacts with this system, both in vitro and in vivo . By

this hypothesis, the EC system represents a possible adverse outcome pathway for

developmental effects of chlorpyrifos. Ther e is a body of studies on the interaction of

OPs with relevant enzymes but only two studies have examined the effects of

chlorpyrifos on the EC system in developing animals. Carr et al. (2011, 2013) has dosed

preweanling rats for 5 or 7 days (1-5 mg/ kg/day, p.o.), and showed that endocannabinoidrelated enzymes were inhibited in rat brain ti ssue taken 4 to 48 hours after the last dose.

Interestingly, fatty acid amid hydrolase (F AAH) showed a greater degree and more

persistent inhibition compared to AChE inhi bition measured in the same rats. A more

recent publication (Carr et al., 2014) repeat ed these findings using a lower dose (0.5

mg/kg/d for 7 days), still showing signifi cant FAAH inhibition but with no measurable

AChE inhibition. This suggests a greater sensitivity of the EC system, at least in terms of

the hydrolase compared to AChE activity, in the pups. However, there were no other

ages tested, no downstream or correlative meas ure of changes in EC system function, and

no subsequent neurodevelopmental effects that could be linked to the action. Additional

studies along these lines are needed.

 Reactive Oxygen Species: The production of reactiv e oxygen species (ROS) and

resulting cellular damage has been proposed as a mechanism for a wide variety of

neurotoxicants. Due to lower levels of protective enzymes and antioxidants, and

relatively low numbers of glia relative to the adult, the deve loping brain may be

particularly sensitive to neural cell damage caused by oxidative stress. In addition, recent

work suggests that ROS can act as second me ssengers. Relatively small changes in the

oxidative status of the cell (redox potential) can lead to changes in redox sensitive

signaling pathways that regulate cell physio logy. In the nervous system, redox signaling

is involved in the regulation of neurodevelopm ental processes including neural stem cell

proliferation and differentiation (Le Belle et al ., 2011; Vieira et al, 2011). A number of

studies suggest that chlorpyrifos and chlo rpyrifos oxon can induce oxidative stress in

various neural cell types. Thus, generation of reactive oxygen species and/or alteration of

cellular redox potential by ch lorpyrifos represent a possible initiating event leading to

developmental neurotoxicity. Data from both in vitro studies with neuronal cells

(including neural precursors) and in vivo studies in developing brain demonstrate that

chlorpyrifos can induce oxidative stress. The in vitro data suggests that this effect may

not be due to AChE inhibition, since the parent compound chlorpyrifos is either

equipotent or more potent than the oxon (for example, Crumpton et al., 2000). There was,

however, no concurrent analysis of AChE inhi bition in most of these studies. Several

known developmental neurotoxicants have been shown to disrupt ne ural precursor cell

proliferation in vitro through a common pathway that is initiated by increasing the

oxidative state of the cell (L i et al, 2007), and the antioxi dant vitamin E protected PC12

cells from the anti-proliferativ e effect of chlorpyrifos (Slo tkin et al, 2007). Thus, the in

vitro data suggest that chlorpyrif os can affect a critical neurodevelopmental process, at

least in part, via genera tion of ROS. Though limited, in vivo studies show both direct

# Chlorpyrifos Human Health Risk Assessment D424485

Page 30 of 531 evidence (lipid peroxidation) and indirect evidence (alteration in the expression of

oxidative stress response genes) of oxidative stress in the developing brain after exposure

to chlorpyrifos. Recent evidence sugges ts that oxidative stress can alter

neurodevelopment in vitro and in vivo by the dysregulation of signaling pathways

controlling neuroprogenitor cell f unction (Le Belle, et al., 201 1; Vieira, et al., 2011). It

has been demonstrated in vivo that antioxidant treatment can attenuate the induction of

oxidative stress produced by chlorpyrifos in adult rats (Singh and Panwar, 2014), but

there are as yet no such studies addressing its developmental neurotoxicity. Thus, there is

the potential for initiation of an AOP vi a induction of oxidative stress, but supportive

studies in developing animals have not been reported.

 Serotonergic system: Beyond its classical neurotransmitter actions, serotonin has other

roles during development. In their review, Thompson a nd Stanwood (2009) described

serotonin as a pleiotropic molecule, meaning that it can produce multiple, diverse effects,

regulating different functions at different times during deve lopment. The serotonergic

system is integral in many developmen tal processes including, but not limited to,

neurogenesis, migration, and differentiati on, synaptogenesis, and cardiac development

before assuming its more well-known function as a neurotransmitter in the adult nervous

system (reviewed in Frederick & Stanwood, 2009) . Serotonin also plays crucial roles in

thalamocortical patterning (re viewed in (Frederick & Stanwood, 2009). As serotonin is

present extremely early in development, it is thought that it modul ates cellular function

even before neurogenesis. Later in developmen t, serotonin is temporarily taken up by socalled transient serotonergic neurons mainly involved in sensory processing, and is

involved in activity-dependent patterning of the br ain. Later in development, serotonin

has also been shown to modulate differentiati on in the brain. There are numerous studies

of the effects of perinatal ch lorpyrifos administration on the patency of the serotonergic

system coming from both Duke University and Istituto Superiore di Sanita in Italy;

however, there have been no additional report s since the 2012 SAP. Endpoints in various

brain regions include serotonin levels, sero tonin turnover, serotonin receptor levels,

serotonin reuptake receptor levels, serotoni n elicited second messenger activity, gene

expression of serotonin receptor and meta bolism related genes, serotonin related

behavioral assessments, and behavior after serotonergic drug challenge. All the data

indicate that there are acute, as well as permanent, effects of neonatal chlorpyrifos

treatment on the maturation of the serotonerg ic nervous system. The effects are often

gender-specific, region-sp ecific and dose-related.

There is ample evidence that chlorpyrifos exposure during development causes

permanent changes in the serotonergic ner vous system; there are, however, few papers

that assessed concurrently the ChE inhibiti on (either brain or blood) in those same

animals. In some cases, although ChE activity was not assessed concurrently, a dosing

regimen was used that had been characterized previously with regard to ChE activity. It

does appear, however, that most of the studi es on the effects of chlorpyrifos on the

serotonergic nervous system were conducted with doses of chlorpyrifos that likely

produced inhibition of ChE activity.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 31 of 531 As many steps in this chlorpyrifos AOP ar e possible and plausibl e, and in laboratory

animals the serotonergic nervous system is sufficiently sensitive to low doses of

chlorpyrifos during development to alter it s function, it is plausi ble that exposure to

chlorpyrifos during development could alter brain development and the function of the

serotonergic nervous system. Although chlorpyr ifos effects on the serotonergic nervous

system in laboratory animals likely is initiate d within 24 hours (Slotk in & Seidler, 2007),

the actual initiating event of this pot ential adverse outcome pathway is unknown.

 Tubulin, Microtubule Associated Proteins and Axonal Transport: Microtubules, one

component of the dynamic cytoskeletal scaffo lding within each cel l, are composed of

heterodimers of α - and β -tubulin, as well as microtubul e associated proteins. The

microtubule associated proteins appear to have three main fu nctions: (1) to stabilize the

microtubules; (2) to aid in tubulin dissociati on and (3) to act as motor proteins moving

substances forward and backward along the micr otubules (Avila et al , , 1994; Pellegrini

& Budman, 2005; Sánchez et al, 2000). No t only does the microtubule cytoskeleton

determine neuronal morphology (Matus, 1988, 1 990; Sánchez, et al., 2000), but the

dynamic reorganization of the microtubules and microtubule associated proteins within a

cell may also coordinate neurite extensi on/retraction, as well as growth cone

advancement. In addition to these integral roles in brain structure and growth,

microtubules and the microtubule associated motor proteins kinesin (Hirokawa & Noda,

2008) and dynein (Vallee et al, 2004) also pr ovide a “railway” for transport of materials

throughout the cell, i.e. , axonal transport (Fukushima et al, 2009), another process which

is integral to the health of the central and peripheral ne rvous system, playing a pivotal

role in neuronal network formation and synapse maturation (Hirokawa & Takemura,

2004). The construction of anadverse outcome pathway using chlorpyrifos-induced

effects on tubulin and microtubule associated pr oteins is still in its infancy. While it is

thought that tubulin, microtubule as sociated proteins and axonal transport are integral to

nervous system development and maintenance, there is no experimental evidence that

perturbations of these endpoints by chlorpyr ifos during development has neurotoxic

outcomes.

Overall, a definitive mode of action or advers e outcome pathway leading to effects on the

developing brain cannot yet be established because of insufficient data establishing the causal

linkages among different levels of biological organization to advers ity. For example, while there

is in vitro evidence relating bindi ng of chlorpyrifos or the chlo rpyrifos oxon to AChE and the

subsequent decrease in neurite outgrowth at the cellular level, the relationship between neurite

outgrowth and neurodevelopmental consequences ha s not been established. As described in the

NRC report, “Toxicity Testing in the 21 st 

# Century” (NRC, 2007), to develop an adverse outcome

pathway not only is it necessary to establish pl ausible relationships among the key events, but

quantitative relationships also need to be established. In other words, how much of a change in

one key event is needed to result in an adverse effect at the next level of biological organization?

Thus, certain exposures to a chemical may impact normal physiological responses in a way that

may not necessarily be adverse, and thus, th e AOP concept requires an understanding of

adaptive/homeostatic capacity of biological systems and their limits, relative to concentration

and duration of exposure.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 32 of 531 

# 4.5 Epidemiology Studies in Mothers and Children

4.5.1 Review of Children’s Environm ental Health Epidemiology Studies

In this chlorpyrifos human h ealth risk assessment (HHRA), EPA is including epidemiologic

research results from three prospective birth coho rt studies. These include: 1) The Mothers and

Newborn Study of North Manhattan and South Bronx performed by the Columbia Children’s

Center for Environmental Health (CCCEH) at Columbia Universi ty; 2) the Mt. Sinai Inner-City

Toxicants, Child Growth and Development St udy or the “Mt. Sinai Child Growth and

Development Study;” and 3) the Center for Health Assessment of Mothers and Children of

Salinas Valley (CHAMACOS) conducted by researcher s at University of Ca lifornia Berkeley. In

these epidemiology studies, mother-infant pairs we re recruited for the purpose of studying the

potential health effects of environmental exposures during pregnancy on subsequent child

development. Importantly, each of these cohorts evaluated the associat ion between prenatal

chlorpyrifos or OP exposure with adverse neur odevelopmental outcomes in children through age

7 years.

These studies reflect different types of exposed groups in the total population which strengthens

the weight of the evidence considerations rega rding this stream of information. The CCCEH

Mother’s and Newborn study and the Mt. Si nai Child Growth and Development study

participants were likely exposed to chlorpyrifos through the diet and through residential use of

the pesticide for indoor pest control. In the re sidential setting, study populations were most likely

exposed through indoor residential use of th e pesticide during the study time period and

additionally exposed to chlorpyrif os via the oral route through i ngesting residues in the diet and

from hand-to-mouth contact with in-home chlo rpyrifos-contaminated surfaces, as well as

possible dermal or inhalation exposure thr ough contact with treated areas in the home

environment (Berkowitz et al., 2003; Whyatt et al., 2003; Whyatt et al., 2009; Whyatt et al.,

2007). In contrast, CHAMACOS cohort participants were employed as fa rm laborers or were

residing in homes with farm laborers. The CH AMACOS study participan ts likely experienced

exposure to OPs through the diet and from occ upational exposure (pri marily inhalation and

dermal routes), as well as proba ble indirect take-home exposures (the “tracking in” of pesticide

residues through shoes and clothing, augmente d by poor hygiene practices) (Bradman et al.,

2007). Reported use of chlorpyrifos in th e CHAMACOS region was about 10% of total

pesticide use (Eskenazi et al., 2004). In each of the children’s health cohorts, the biological

measurements in these cohorts were comparable to the general populatio n (National Health and

Nutrition Examination Survey; NHANES 12 

# ).

The Columbia CCCEH study measured parent chlo rpyrifos in cord bl ood, and other indicators

(e.g., air sampling, behavioral information), as e tiologic measures of exposure, while the other

two birth cohorts measured non-specific urinar y metabolites of chlor pyrifos and other OPs

(TCPy, dialkyl phosphate metabolites) in the mother s to estimate pesticid e exposure. Therefore,

EPA considers the CCCEH Mothers and Newborn Study research results as most relevant to the

chlorpyrifos HHRA; the other two cohorts provide important supporting information.

12 

# http://www.cdc.gov/ nchs/nhanes.htm

# Chlorpyrifos Human Health Risk Assessment D424485

Page 33 of 531 EPA performed its review and critical evaluation of these epidemiology studies in an open and

transparent manner. The Agency presented an itera tive evaluation of these data at two meetings

of the FIFRA SAP, as well as discussed stre ngths and limitations of these studies in the

preliminary human health risk assessment upon which EPA sought public comment in 2011

(EPA-HQ-OPP-2008-0850). In September 2008, EPA pr esented its initial review and assessment

of the available epidemiologica l data (1999-2007) as well as s upporting experimental studies as

to the neurodevelopmental toxicity of chlorpyrifos to the Panel. 13 

# In April 2012, EPA presented

an expanded and updated review of the availabl e epidemiologic data ( 1999-2011) concerning the

effect of chlorpyrifos exposure on children’s envi ronmental health in c onjunction with a review

of recent experimental studies as well as hypothesized adverse outcome pathways (AOP). 14



# Published observational studies and supplemen tal analyses produced subsequent to the

September 2008 FIFRA SAP evaluation expanded th e knowledge base of potential long-term

sequelae of prenatal chlorpyrifos exposure. Data included the re sults of supplemental analyses

recommended by the 2008 SAP, additional data con cerning intelligence measures at age 7 years

from each study cohort, the effect of PON1 genotypic and phenotypic status on infant

neurodevelopment, and several methodological studies relating to the accur acy and reliability of

exposure and confounding variables. Both the 2008 and 2012 SAP panels agreed with EPA’s

assessment, and concluded that “chlorpyrifos likely played a role” in the observed

neurodevelopmental outcomes.

EPA has considered the strengths and limitations of these studies, and believes that random or

systematic errors in the design, c onduct or analysis of these studies were unlikely to fully explain

observed positive associations between in utero chlorpyrifos exposure and adverse

neurodevelopmental effects observed at birth and through childhood (age 7 years). EPA believes

these are strong studies which support a conclusion th at chlorpyrifos likely pl ayed a role in these

outcomes. Although it cannot be st ated the chlor pyrifos is the sole contributer to these

outcomes; co-exposure to other OPs and mixtur es of environmental exposures may also

contribute to these outcomes.



# 4.5.2 Review of Study Design and Research Methods

The study design and research methods of these prospective cohort studies are summarized in

this section. Appendices 2, 3, and 4 reflect a mo re detailed evaluation of these studies, study

specific critical evaluations, and accompanying de tailed evidence table for the included studies,

respectively. These cohort studies each enrolled pregnant women from approximately 1997

through1999, measured both environmental exposure to the pesticide during pregnancy and also

estimated internal dose during pregnancy and at delivery, and prospectively assessed associations

in their newborns and young children through ag e 7 years. Each study includes several hundred

(approximately 100-400) mother-infant pairs; th ese sample sizes are sufficient to perform

statistically valid analyses.

13 

# http://www.epa.gov/scipoly/sap/ meetings/2008/091608_mtg.htm

14 

# http://www.epa.gov/scipoly/sap/m eetings/2012/041012meeting.html

# Chlorpyrifos Human Health Risk Assessment D424485

Page 34 of 531 EPA focuses its review on research results from these three epidemiological cohort studies into

the chlorpyrifos HHRA due to th e considerable strengths in st udy design, conduct, and analyses

demonstrated in these investigations. Investig ators from each study cohort utilized a similarly

strong study design (prospective birth cohort); measured pest icide exposure using several

different methods including environmental indi cators as well as specific and non-specific

biomarkers of chlorpyrifos; ascertained deve lopmental outcomes using validated assessment

tools well-established in both cl inical and research settings; a nd, measured, analyzed, selected

and statistically adjust ed for potentially confounding variab les including socio-economic status

and other environmental exposures using reasona ble and appropriate methods. Limitations exist

as well. These studies utilized a one-time m easure (or the average of two measures) of

chlorpyrifos or OP exposure to assess prenatal pesticide exposure th roughout the gestational

period, were unable to assess the in fluence of mixtures (co-occurr ing exposures in the relevant

biological time window), and reflect a small sample size to fully evaluate the effect of more than

one simultaneous exposure on neurodevelopment, i.e. , evidence of effect modification. The lack

of understanding of the critical window of exposur e for these effects is a significant overarching

uncertainty. Balancing the strengths as well as th e limitations, EPA believes these data present an

informative body of evidence with some notable consistencies across studies.

As noted, two major uncertainties in environm ental epidemiology studies are the accurate and

reliable measurement of exposure and potential confounding variables such as the influence of

mixtures. The researchers with each of the three cohorts have provided supplemental

methodological research to address these areas to the extent possible. Across the three children’s

health cohorts, study authors measured parent chlorpyrifos, TCPy and DEP to estimate

chlorpyrifos and/or OP exposure in relation to ne urodevelopment. There is uncertainty as to the

extent measurement of non-specific metabolites of OP or chlorpyrifos accurately reflects

chlorpyrifos exposure; for this reason, the Co lumbia CCCEH study provides the most valuable

data for specifically assessing chlorpyrifos. The findings presen ted below in relation to DEP

(chlorpyrifos or diazinon) and TCPy (chlorpyrifos or chlorpyr ifos-methyl) provide supporting

information. The Columbia CCCEH study does not es timate post-natal expos ure to chlorpyrifos

among child participants, theref ore the influence of early li fe and childhood chlorpyrifos

exposure is unaccounted for in these analyses. Ho wever, the pre- and post-natal chlorpyrifos

levels would have to be strongly correlated to significantly aff ect the reported epidemiological

risk estimates; the time period of the study reflects a rapid decline in use of the pesticide due to

the voluntary cancellation of chlorpyrifos in the residential market. Further, the CHAMACOS

cohort measured early life OP exposure in rela tion to neurodevelopment and did not observe

significant associations (Esken azi et al., 2007). The CHAMACOS cohort investigators also

measured acetyl and butyl ChE as supplemental indicators of OP exposure. To overcome the

limitations of a one-time measure of chlor pyrifos exposure, CCCEH authors performed

additional analyses supporting the correlation of this measure with other measures of

chlorpyrifos during the pregnancy period (TCPy 15 

# in meconium, air concentration of

chlorpyrifos, and maternal urin ary concentration of chlorpyrif os during the third trimester).

Together, these multiple measures of exposure and internal dose improve our understanding of in

utero chlorpyrifos exposure, al though there are still uncerta inties associated with the

biomonitoring data collected at or near time of chlorpyrifos applications.

15 

# Internal measures of TCPy (3,5,6-trichloro-2-pyridinol) need to evaluated with caut ion as they may also result

from exposure to chlorpyrifos, chlorpyrifos-methyl, or TCPy in the environment.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 35 of 531 Potential confounding bias is another major un certainty within envi ronmental epidemiology

studies. Confounding variables, exposures that co uld be related to both chlorpyrifos exposure

and neurodevelopmental outcomes such as blood l ead, may result in an incorrect epidemiological

risk estimate. Across these cohort studies, in vestigators collected relevant information

concerning demographic characteri stics and other environmental exposures, and were, to the

extent possible with the existing information, able to effectively hold constant the influence of

these other variables when estimating the associat ion between prenatal ch lorpyrifos and adverse

neurodevelopmental outcomes. Contro l of these variables is importa nt to reduce the chances of a

false positive study result. Overal l, statistical analyses were judged to be appropriate and

reasonable (not overly large numbe r of statistical model variable s) to the research question by

EPA and expert Panel review s (FIFRA SAP 2008 and 2012).

EPA notes that researchers from the three birth c ohort studies also investig ated the possible role

of prenatal chlorpyrifos exposur e and fetal growth. These results were not consistent across these

cohorts. Authors with CCCEH Mothers and Ne wborn Study observed evidence of an inverse

association, i.e. , increasing cord blood chlorpyrifos was associated with decreased measures of

birth weight and length, while authors with the Mt. Sinai and CHAMACOS cohorts reported

either no association, or evidence of a positive relationship, respectively (Berkowitz et al., 2004;

Eskenazi et al., 2004; R. M. Whya tt et al., 2004). Incons istent results may be due to differences

across study groups in exposure profiles as well as dissimilar methods of prenatal chlorpyrifos

exposure assessment (Needham, 2005). The varied study results across cohorts and the limited

biological rationale between re duced fetal growth and advers e neurodevelopment reduces the

relevance of this evidence. The database of labo ratory animal studies does not suggest that fetal

growth is a key endpoint for concern with chlo rpyrifos. Given the lack of consistency among

cohorts for the fetal growth metrics and the lack of corroborating animal data, the proposed link

between fetal growth and neurodevelopment is tenuous recalling that EPA’s focus in the

chlorpyrifos HHRA is on neurodevelopmental outcomes

. These results are summarized below.



# 4.5.3 Neurological and Neurodevelop mental Health Effects in Children

4.5.3.1 The CCCEH Mothers and Newborn Study

Analysts with the CCCEH Mothers and Newborn Study have performed both etiologic studies

into the relationship between pr enatal chlorpyrifos exposure and adverse neurodevelopmental

outcomes in children from infancy through early childhood, as well as im portant methodological

research as to the validity and reliability of exposure and confounding variable measurement and

statistical modeling. The result of this body of work extends the knowledge of the long-term

sequelae of prenatal chlorpyrifos exposure and reduces uncertainty in important aspects of these

analyses. Both are briefly described in this section.

Researchers across the three children’s health co horts utilized the Bayley Scales of Infant

Development II (BSID-II) to ge nerate a Mental Development Index (MDI) and a Psychomotor

Development Index (PDI) to assess neurodevelopment in early childhood. In the CCCEH

# Chlorpyrifos Human Health Risk Assessment D424485

Page 36 of 531 Mothers and Newborn study, Rauh et al. 2006 in vestigated MDI and PDI at 12, 24, and 36

months of age. Children were categorized as having either high (>6.17pg/g) or low (<6.17pg/g)

prenatal exposure, using categories informed by results of the previous study on birth

characteristics (R. M. Whyatt et al., 2004). Author s reported that the di fference in MDI scores

was “marginally significant” (p = .06) between the “high” and “low” exposed groups; the high

exposed group scoring an average of 3.3 points lo wer than the low exposed (V. A. Rauh et al.,

2006). Regarding the PDI score (motor skills), none of the 12 or 24 month PDI scores showed

significant effects, but the 36 m onth score was significantly relate d to chlorpyrifos exposure.

Researchers noted that the eff ects were most pronounced at the 36 month testing period. Within

the 36 month testing period, the likelihood of highl y exposed children developing mental delays

were significantly greater (MDI: 2.4 times gr eater (95% CI: 1.12-5.08, p = .02) and PDI: 4.9

times greater (95% CI: 1.78-13.72; p = .002)) than those with lower prenat al exposure (V. A.

Rauh et al., 2006).

Further, in supplemental analyses suggested by the 2008 FIFRA SAP, authors illustrated that

when diazinon was added to the statistical model, the effect of chlorpyrifos on the MDI and PDI

measure was greater (increasing the magnitude ch lorpyrifos risk estimate for MDI and PDI 50-

200% in the same direction (pos itively away from the null)) suggesting diazinon is a strong

confounding variable in this asso ciation (correlation with chlor pyrifos 0.63), (R. Whyatt & Rauh,

2011). Lastly, Rauh et al. (2006) also investigated attention-problem s in infants. When analyzing

the 36-month child behavior checklist (CBCL) (beh avioral) scores, signifi cant differences were

observed between the high and low chlorpyrifos exposure groups in th e general category of

attention-problems (p=0.010), and in the more specific DSM-IV scale for ADHD problems (p

=0.018).

To measure intelligence among school aged children, authors from each of the three children’s

health cohorts used the Wechsler Intelligence Scale for Childr en, 4th edition (WISC-IV). The

instrument measures four area s of mental functioning: the Ve rbal Comprehension Index, the

Perceptual Reasoning Index, the Working Memo ry Index, and the Processing Speed Index. A

Full-Scale IQ score combines the four composite indices. WISC-IV scores are standardized

against U.S. population-based norms for Englis h and Spanish-speaking children. In the CCCEH

Mothers and Newborn Study, Rauh et al. (2011) evaluated the re lationship between prenatal

chlorpyrifos exposure and neurodevelopment among 265 of the cohort participants who had

reached the age of 7 years and had a complete se t of data including prenatal maternal interview

data, prenatal chlorpyrifos marker levels from maternal and/or cord blood samples at delivery,

postnatal covariates, and neurodevelopmental ou tcome data (Rauh et al., 2011). While models

were developed using continuous measures of bot h prenatal chlorpyrifos exposure and Wechsler

scores, for ease of interpretation, investigators reported that for e ach standard deviation increase

in exposure (4.61 pg/g) there is a 1.4% reduction in Full-Scale IQ and a 2.8% reduction in

Working Memory.

To ascertain whether observed differences in neurodevelopment by prenatal chlorpyrifos

exposure may be explained by differences in brain morphology between exposed groups,

investigators compared MRI brain images betw een high and low chlorpyrifos exposed child

study participants (Rauh et al., 2012). Authors determined ther e were distinct morphological

differences in brain areas associated with th ese neurodevelopmental outcomes. The pilot study

# Chlorpyrifos Human Health Risk Assessment D424485

Page 37 of 531 included only 40 child participants due to strict inclusion and exclusion cr iteria, and the high cost

of performing the imaging studies on each child. EPA convened a Federal Panel of experts to

perform a written peer-review of this study. 16 

# The Federal Panel concurred with the authors’

conclusions in general; however the Federal Panel also noted that significantly greater and more

sophisticated MRI imaging studies would be need ed to link the morphological changes indicated

in this study with specific functional outcomes noted in the CCCEH IQ study. Therefore, while

generally supportive of the epidem iologic findings, additional study is needed to make specific

links with areas of brai n development change.

As mentioned above, CCCEH researchers have also performed epidemiologic methods research

which in many ways reduces uncertainties rela ted to key exposure and confounding measures

within these studies. The extent to which the one (or the average of two) estimate of chlorpyrifos

or DAP exposure during pregnanc y reflects exposure(s) over cri tical windows of development

during pregnancy is an uncertainty in these da ta. To address this unc ertainty, investigators

assessed the inter- and intra- i ndividual variability in expos ure measurements over time and

determined that intra-individual variation is lo w such that the individu al exposure profiles may

be relatively stable over time (Whyatt et al., 2007). In a separate study, researchers measured the

correlation between chlorpyrifos exposure measur es taken at different time points in pregnancy

as well as between internal dose (biomarker) and external exposure. They demonstrated that the

one-time measure of cord blood chlo rpyrifos levels at birth were moderately well correlated with

measures of exposure earlier in pregnanc y (meconium, 14-weeks gestation) and with

environmental exposures (air con centration) during the last trimes ter of pregnancy (Whyatt et al.

2009). This additional informati on supports the validity of us ing the one-time measure of

chlorpyrifos in cord blood as an approximation of in utero pesticide exposure during gestation as

a method to correctly rank study particip ants by pesticide exposure category.

The appropriate control of poten tially confounding variables is vita l to an accurate assessment of

the association under study. Both important char acteristics of socio-economic status (SES) and

estimates of other environmental exposures are importa nt variables to consider in this analysis as

inaccurate measurement of modeling of either type of variable could lead to a false positive

result. With regard to both of these types of factors, the CCCEH researchers were able to

perform additional analyses to clarify the es timated association, and, therefore, to reduce

uncertainty in the interpretation of this study. SE S is difficult to measure because the effects of

variables such as race, class, education are related in comp lex ways. The CCCEH investigators

augmented the understanding of SES using regressi on methods that model individual as well as

group level variable contribution of overall model variability (L ovasi et al., 2011). This allows

researchers to better isolate the effect of chlo rpyrifos on neurodevelopmen t, holding constant the

influence of socio-demographic influences. Using this approach, investigat ors demonstrated that

the association between prenatal chlorpyrif os and neurodevelopmental outcomes remained

significantly, positively related.

In addition, the authors were ab le to measure and model impo rtant environmental exposures

including environmental tobacco smoke ( ETS), polyaromatic hydrocarbons (PAHs),

methylmercury and other ChE-inhibiting pestic ides such as diazinon and propoxur (Whyatt &

Rauh, 2011). Given the known relation between bot h lead and also methyl mercury and

16 

# http://www.regulations.gov/#!document Detail;D=EPA-HQ-OP P-2008-0 850-0170

.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 38 of 531 neurodevelopment, the authors performed supplemen tal analyses. The results of these analyses

showed that neither pre-natal nor post-natal blood lead levels or methyl mercury levels measured

in cord blood were significantly correlated with chlorpyrifos, and was ther efore not considered a

confounding variable in the associ ation of interest within th e Columbia cohort (Whyatt & Rauh,

2011; Rauh et al., 2006). This additional work si gnificantly reduces uncertainty in research

results.

Lastly, the authors demonstrated that chlorpyr ifos exposure measures in the time periods

immediately before and after th e voluntary cancellation of chlo rpyrifos for residential use

reduced substantially (Whyatt & Rauh, 2011; Whyatt et al., 2004). CCCEH researchers

illustrated that prior to the voluntary cancellati on there were >80% detectable levels of

chlorpyrifos in cord blood, in the time period after the cancella tion, only 16% of the measured

values were greater than the level of de tection (LOD). Furthermore, although reported

household use of any pesticide in general did not change over the same time period in this study,

among newborns born before 1 January 2001, 34% had combined high level exposure levels

whereas among newborns born after 1 January 2001, only one participant (1.5%) was in the

highest chlorpyrifos exposure group—a difference that was highl y significant (chi squared = 50,

p < 0.001) (Whyatt et al., 2004).

In summary, CCCEH researchers, examined se veral aspects of infa nt and early childhood

neurodevelopment in relation in chlorpyrifos exposure during th e gestational period. The authors

reported a 2-4 fold increase in reduced mental and also psyc homotor development in infants

exposed to chlorpyrifos in utero and these results are most strong at 36 months of age. At three

years of age, the authors also demonstrated statis tically significant evidence of differences in the

proportion of pervasive developmen tal disorder diagnoses between children in the high and low

exposure groups (+/- 6.17 pg/g). At seven years of age, the aut hors observed reduced measures

of intelligence by increasing meas ures of prenatal chlorpyrifos exposure (1.4-2.4% decrease in

intelligence score per 4.6 pg/g change, or 1 SD, of chlorpyrifos). Preliminary data from a small

pilot study indicate, in principle, that diffe rences in brain morphology between high and low

chlorpyrifos exposed groups may exist, howev er, it is unclear whet her these morphological

differences are directly responsible for functi onal neurodevelopmental decrements observed (a

conclusion supported by EPA’s Federal Review Panel). In addition, these investigators

performed several methodological investigations which reduce ke y uncertainties in these data,

specifically with regard to exposure measurem ent error and potential confounding bias. This

work reduces the probability that positive a ssociations reported above are inaccurate, i.e. , they

are false positive results.



# 4.5.3.2 Supporting Epidemiologica l Evidence: The Mt. Sinai and

CHAMACOS Cohorts

As previously noted, for the purpose of informing the chlorpyrifos human h ealth risk assessment,

EPA has focused its review on the Columbia Mo ther’s and Newborn Study among the three birth

cohort studies reviewed due to researcher s’ measurement of pa rent chlorpyrifos per se . However,

EPA also believes that it is valuable to comprehe nsively consider the results of all three of the

children’s environmental health cohorts togeth er, given each study cohort contributes different

# Chlorpyrifos Human Health Risk Assessment D424485

Page 39 of 531 and unique information to the chlorpyrifos HHRA assessment. The Mt. Sinai and CHAMACOS

cohorts examined other health outcomes (e.g., the Brazelton index of neonatal development and

attentional difficulties in early childhood) and performed supplemental analyses ( e.g., effect

modification by PON1 status) not reflected in the CCCEH Mothers and Newborn Study

database.

Researchers with both the Mt. Sinai and CHAMACOS cohorts ev aluated neonatal neurological

functioning in association with prenatal OP exposure; CCCEH did not conduct these

measurements. To measure indices of abnormal ne onatal behavior and/or neurological integrity

authors used outcome measures derived from the Brazelton Neonatal Behavioral Assessment

Scale (BNBAS), a neurological assessment of 28 behavioral items and 18 primitive reflexes.

This tool was administered to infants 2-5 days post-partum by trained neonatologists in the

hospital setting using similar environmental conditions. The authors with both study groups

observed an increased number of abnormal reflex es in relation to incr easing measures of OP

exposure (Engel et al., 2007; Young et al., 2005). Among the other 27 measures in the BNBAS,

neither study group reported evidence of any other positive associati ons. The authors also

observed evidence of potential effect modification by PON1 activity level in the relation between

DAPs and neonatal neurodevelopment in which infa nts of mothers who ar e slower metabolizers

have greater risk of abnormal reflexes (Young et al 2005; Engel et al 2007 ). However, EPA notes

these studies are likely under-powered to make a statistically robust estimat e of this statistical

interaction.

Each of the three children’s heal th cohorts utilized the Bayley scale (BSID-II) to generate an

MDI and PDI score to assess neurodevelopment in early childhood (6-36 months). Within the

Mt. Sinai study, authors administered the BSID-II to participating children at 12 and 24 months

and observed that prenatal total DAP metabolite le vel was associated with a decrement in mental

development at 12 months among blacks and Hisp anics children; however , these associations

either attenuated or were non-existent at the 24-month visit (Engel et al., 2011). In the

CHAMCAOS cohort, Eskenazi et al. (2007) observe d that prenatal DAP levels were adversely

associated with MDI, and at 24 months of age th ese associations reached statistical significance.

In this study, neither prenatal DAPs nor maternal TCPy were associated with PDI (motor skills),

nor did authors observe evidence of different risk by PON1 stat us (Eskenazi et al., 2010). The

CHAMACOS cohort also investigated atten tion problems in early childhood using three

different assessment tools: maternal report of ch ild behavior at 3.5 and 5 years of age; direct

assessment of the child at 3.5 and 5 years; and by a psychometrician ’s report of the behavior of

the child during testing at 5 years. In this study population, higher concentrations of OP

metabolites in the urine of pregnant women were associated with increased odds of attention

problems and poorer attention scores in their ch ildren at age 5 years (E skenazi et al., 2007).

Regarding intelligence measures, both research groups observed an inverse association between

prenatal OP exposure and intelligence measures. In the Mt. Sinai study, prenatal maternal DEP

urinary metabolite concentrations were associated with slight decrements in FSIQ, Perceptual

Reasoning, and Working Memory between the ag es of 6 and 9 years, and difference in

intelligence measures by putative PON1 status were also noted (Engel et al., 2011). Similarly, in

the CHAMACOS cohort, Bouchard et al. (2011) observed eviden ce of an association between

prenatal exposures to OPs as measured by urin ary DAP (total DAP, DEP, and DMP) metabolites

# Chlorpyrifos Human Health Risk Assessment D424485

Page 40 of 531 in women during pregnancy, and de creased cognitive functioning in children at age 7. In this

study, children in the highest quintile of mate rnal DAP concentrations had a statistically

significant 7 point difference in IQ points compared with thos e in the lowest quintile.

Results from the Mt. Sinai and CHAMACOS c ohort studies support the results of the CCCEH

study in several ways. Both of these studies also investigated infant mental and psychomotor

development. With regard to mental developm ent (as measured by the Bayley scale), both of

these epidemiology studies reported increased risk of delays in appropriate mental development

in relation to increasing DAPs, and the effects ap peared to be greater at older ages tested;

however, neither of these cohorts reported a link of psychomotor development. Similar to the

CCCEH cohort study results, the CHAMACOS i nvestigators also re ported a significant

increased odds of pervasive developmental disorder and attention difficulties in association with

prenatal OP exposure; the Mt . Sinai study did not evaluate this outcome. Each study group

reported an inverse relationship between increasing chlorpyrifos or OP exposure and intelligence

measures using the same Wechslar scale of intel ligence, when testing stud y participants at the

same age and developmental time period. Research ers with each cohort reported evidence of an

inverse exposure response rela tion, although the results were not consistently statistically

significant. Taken together, these results bolster the findings reported by CCCEH’s study

authors.



# 4.5.4 Dose Reconstruction

In 2012, the SAP recommended that the Agency c onduct a dose reconstruction analysis for the

CCCEH study to help characterize th e extent to which participants in the cohort may or may not

have experienced RBC AChE inhibition. EPA has conducted such analysis and it is summarized

here. In this analysis EPA has estimated poten tial exposures for pregna nt women who may have

used chlorpyrifos containing products and also to investigate possible po st-natal exposures for

children in the cohort. The primary goal was to estimate the degree to which adverse effects

could be attributed to doses lo wer than those below the level e liciting elicit 10% inhibition of

RBC AChE, which is the benchmark response level th at has been used to establish the points of

departure previously used for regulatory decision making.

Prior to 2000, chlorpyrifos was one of the most widely used inse cticides for indoor pest control

so there are many possible ways which exposures c ould have occurred as a result of these types

of uses (e.g., use of a total rel ease fogger, aerosol can sprays, or crack and crevice treatment by a

pest control operator). Dose estimates for thes e uses were calculated based on the Agency’s

SOPs for Residential Exposure Assessment 17



# . This SOP describes, on a scenario basis, specific

algorithms and data used to pr edict the potential exposures wh ich could have occurred in

individuals in the cohort from these now canceled uses of chlo rpyrifos. The potential for RBC

AChE inhibition was then investigated using a chlorpyrifos specific PB PK-PD model that is

described below. The scenarios which were considered represent the highest potential

exposures for each population being evaluated. They include:

17 

# See http://www.epa.gov/p esticides/science/residential-exposure-sop.html for further information. These SOPs

provide the basis for health protective risk assessments for residential scenarios used routinely by EPA for

regulatory purposes.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 41 of 531  Pregnant women who may have purchased a consumer aerosol can product and applied it

in their homes;

 Exposures to pregnant women who may have had contact with residues in their homes

after a previous treatment; and

 Exposures to young children (aged 1 to 2 years old) who may have contact with residues

in their homes after a previous treatment.

The results of this analysis are presented be low for pregnant women who potentially applied

consumer products (Table 4.5.4.1). Details of the analysis can be found in Appendix 10.

Results for pregnant women and children 1 to < 2 years old exposed from contact to residues

following broadcast applications to hard surfaces in their homes are presented in Tables 4.5.4.2

and 4.5.4.3, respectively. Peak RBC AChE inhi bition for pregnant women resulting from

combined dermal and inhalation exposure occurr ing during the application, assuming continuous

exposure for one hour, of a consumer pr oduct was 0.0012%. Similarly, peak RBC AChE

inhibition for pregnant women ( 75 kg) resulting from dermal exposure occurring from contact

with previously treated areas was 0.45% a nd from inhalation e xposure immediately after

application was 0.005%; combined peak % RBC AChE inhibition for pregnant women was

0.45%. For young children (11 kg), peak RBC AChE inhibition associated with the exposures

from being in previously treated areas result s in RBC AChE inhibition from dermal exposure of

0.14%, from mouthing behaviors of 2.2%, and from inhalation exposure of 0.014%; combined

peak inhibition for children 1 to < 2 years old was 2.7%.

This dose reconstruction analysis only includes e xposure to indoor uses of chlorpyrifos. At the

time of recruitment into the CCCEH study, women could have been exposed to additional ChEinhibiting pesticides indoors and likely were exposed to higher levels of ChE-inhibiting

pesticides in food compared to current food resi due levels of ChE-inhibiting pesticides (data

from 1998 to 2012 indicate that total annual pou nds of OPs applied to food crops has been

steadily declining). For example, Whyatt et al (2003) report that 50% of cord blood samples in

the CCCEH had detectable levels of diazinon, although all indoor us es of diazinon were removed

from product labels in December 2002 due to con cerns over human health. In addition other OPs

were detected in some women at low frequency (0.5%-2%); included among these are some of

the most potent OPs (methyl pa rathion, parathion, terbufos, phorate). The Agency’s dose

reconstruction analysis supports a qualitative conclusion that it is unlikely that > 10% RBC

AChE inhibition would have occurred in the CCCE H participants; however, it is important to

interpret the results in 4.5.4.1-4.5.4.3 with caution as it is possible that additional sources of

exposure to OPs are not accounted for in this analysis.

Overall, the dose reconstructi on results support a conclusion that indoor application of

chlorpyrifos, when used as allowed prior to cancellation from the residential marketplace in

2000, likely would not have resulted in RBC AChE inhibition greater than 10% in pregnant

women or young children. These findings are pertin ent to the weight-of-evidence discussion for

the FQPA 10X Safety Factor.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 42 of 531 Table 4.5.4.1 

# Residential Handler (Pregnant Women in the Columbia Cohort) Estimated Exposures and Predicted %

RBC AChE Inhibition (Route-specific and Combined)

Exposure

Scenario Formulation Amount

Handled Dermal Dose

(mg/kg/day) Dermal:

Peak

% RBC

AChE

Inhibition Inhalation

Dose

(mg/kg/day) Inhalation:

Peak

% RBC

AChE

Inhibition Combined Peak

% RBC AChE

Inhibition

Broadcast Ready to Use:

1 % Aerosol

Spray Can 1 can 0.049 0.0007% 0.030 0.0008% 0.0012%



# Table 4.5.4.2 

# Residential Post-application (Pregnant Women in th e Columbia Cohort) Estimated Dermal and Inhalation

Exposures and Predicted % RBC AChE 

# Inhibition (Route-specific and Combined)

Exposure

Scenario Formulation Dermal Dose

(mg/kg/day) Dermal:

Peak

% RBC AChE

Inhibition Airborne

Concentration of

Chlorpyrifos

(mg/m 3 

# ) Inhalation:

Peak

% RBC AChE

Inhibition Combined:

Peak

% RBC AChE

Inhibition

Broadcast

(Hard

Surfaces) 1% PCO 18



# Application

or RTU 1 16

oz can 0.71 0.45% 0.092 0.0049% 

# 0.45%

Table 4.5.4.3 

# Residential Post-application (Children in the Co lumbia Cohort) Estimated Dermal and Inhalation

Exposures and Predicted % RBC AChE Inhibition (Route-specific and Combined)

Exposure

Scenario Formulation Dermal

Dose

(mg/kg/day) Dermal:

Peak

% RBC

AChE

Inhibition HTM

Dose

(mg/kg/day) HTM Peak

% RBC

AChE

Inhibition

(mg/day) Airborne

Concn. of

Chlorpyrifos

(mg/m3) Inhalation:

Peak

% RBC

AChE

Inhibition Combined:

Peak

% RBC

AChE

Inhibition

Broadcast

(Hard

Surfaces) 1% PCO

Application

or RTU 1 16

oz can 1.3 0.14% 0.10 2.2% 0.092 0.014% 2.7%



# 4.5.4. Summary of EPA’s Conclusion s Regarding Children’s Environmental

Health Cohort Studies

Across these three children’s environmental heal th studies, authors consistently identified

associations with neurodevelopmental outcomes in relation to chlorpyrif os exposure. There is

evidence of delays in mental development in infants (24-36 months), attention problems and

pervasive developmental disorder in early child hood, and intelligence decrements in school age

children who were exposed to chlo rpyrifos or OP during gestati on. Investigators reported strong

measures of statistical associa tion across several of these eval uations (odds ratios 2-4 fold

increased in some instances), and observed evidence of exposures-response trends in some

instances, e.g. , intelligence measures. Researchers with CCCEH study demonstrated that

18 

# PCO: pest control oper ator; RTU: ready-to-use

# Chlorpyrifos Human Health Risk Assessment D424485

Page 43 of 531 environmental and biological measures of chlo rpyrifos exposure decrea sed significantly in the

time period subsequent to the voluntary cancellation of the pesticide.

These studies reflect several strengths and so me notable limitations ; however, upon analysis,

EPA believes that it is more like ly that research results were under-estimated, rather than overestimated. Errors (or mistakes) in the measur ement of exposure and confounding variables are

major sources of biases that may result in an inaccurate measurement of risk. Within CCCEH

study, researchers illustrate d that due to low intra-individual variability in exposure over time,

and the correlation between th e (one-time) measure of cord blood chlorpyrifos and other

measures of chlorpyrifos e xposure throughout pregnancy, a one-time measure of exposure may

accurately rank or order the study participants by chlorpyrifos exposure group in this cohort.

EPA notes that given the nature of the study design, the most likely effect of any exposure

measurement error would be to under-estimate rather th an over-estimate risk ( i.e. nondifferential exposure misclassific ation). The lack of a post-na tal estimate of chlorpyrifos

exposure in the CCCEH study is a limitation; ho wever, supporting data from the CHAMACOS

cohort indicate post-natal OP exposure is not significantly related to neurodevelopmental

outcomes. Further, both SES and other envir onmental exposures may be potential confounding

variables in these studies, i.e. , they may be related to both chlorpyrifos exposure and

neurodevelopment. However, EPA believes that au thors were able to ap propriately measure and

model the effect of these vari ables on the study outcomes.

Therefore, as a result of the Agency’s review and critical assessment of these data, including

expert elicitation to character ize known uncertainties in thes e studies, EPA concludes that

chlorpyrifos likely played a role in the neurodevelopmental outcomes observed in these

epidemiology studies. It cannot be stated with ce rtainty, however, that chlo rpyrifos is the sole

contributor to th ese effects.



# 4.6 Weight of Evidence Analysis

Sections 4.1-4.5 summarized key scientific inform ation on the PK profile of chlorpyrifos and its

oxon along with adverse health e ffects in animals and humans related to the effects of

chlorpyrifos and its oxon on two different a dverse health outcomes, AChE inhibition and

potential for neurodevelopmental effects. The Agency has conducted a WOE analysis

implementing the draft “Framework for Incorpora ting Human Epidemiologic & Incident Data in

Health Risk Assessment” focused on two key scientific questions: 1) the degree to which

scientific data suggest that ch lorpyrifos causes long- term neurodevelopmental effects from fetal

or early life exposure and 2) the degree to which adverse effects can be attributed to doses lower

than those which elicit 10% inhibition of AChE, i.e. , the dose levels previously used for

regulatory decision making.



# 4.6.1 Summary of the draft “Framework for Incorporating Human

Epidemiologic & Incident Data in Health Risk Assessment”

In 2010, OPP developed a draft “Framework for In corporating Human Epid emiologic & Incident

Data in Health Risk Assessment” which provide s the foundation for evaluating multiple lines of

# Chlorpyrifos Human Health Risk Assessment D424485

Page 44 of 531 scientific evidence in the contex t of the understanding of the a dverse outcome pathway (or mode

of action (U.S. EPA, 2010). The draft fram ework, which includes two key components:

problem formulation and use of the MOA/AOP fr ameworks, was reviewed favorably by the SAP

in 2010 (FIFRA SAP, 2010).

OPP’s draft framework is consiste nt with updates to the World H ealth Organization/International

Programme on Chemical Safety mode of acti on/human relevance framework, which highlight

the importance of problem formulation and the need to integrate information at different levels of

biological organization (Meek et al, 2014). Consistent with reco mmendations by the NRC in its

2009 report on Science and Decisions 19 

# , OPP’s draft framework describes the importance of

using problem formulation at the beginning of a complex scientific analysis. The problem

formulation stage starts with pl anning dialogue with risk managers to identify goals for the

analysis and possible risk manage ment strategies. This initial dialogue provides the regulatory

context for the scientific analysis and helps defi ne the scope of such an analysis. The problem

formulation stage also involves c onsideration of the available info rmation regarding the pesticide

use/usage, toxicological effects of concern a nd exposure pathways and duration along with key

gaps in data or scientific information. Specific to chlorpyrifos, the 2008 and 2012 SAP reviews

represent the problem formula tion analyses for the weight-o f-evidence (WOE) evaluation.

Appendix 1 specifically describes a conceptual fr amework which integrates pesticide use/usage,

toxicological effects of concern and exposure pathwa ys and duration in on effects of chlorpyrifos

to key lifestages.

MOA (Boobis et al., 2006; Boobi s et al., 2008; USEPA, 2005; Sim on et al, 2014; Meek et al,

2014) and AOP (Ankley et al., 2010) provide important concepts in this integrativ e analysis.

Both a MOA and an AOP are based on the premise th at an adverse effect caused by exposure to

a compound can be described by a series of causally linked biological key events that result in an

adverse human health or ecological outcome. On e of the key components of the Agency’s draft

framework is the use the MOA framework /AOP c oncept as a tool for or ganizing and integrating

information from different sources to inform the causal nature of li nks observed in both

experimental and observational studies. Specifica lly, the modified Bradford Hill Criteria (Hill,

1965) are used to evaluate the experimental support that establishes key even ts within a mode of

action or an adverse outcome pathway, and explic itly considers such concepts as strength,

consistency, dose response, temporal concordanc e and biological plausibility in a weight of

evidence analysis. Section 4.4.3 and Appendix 1 provide the st ate of science on MOAs/AOPs

for both AChE mediated cholinergic toxi city and neurodevelo pmental effects.



# 4.6.2 Integration Across Multiple Lines of Evidence

Dose-response relationships & temporal concordance

. Since the MOA(s)/AOP(s) is/are not establishe d for neurodevelopmental outcomes as described

in section 4.4.3, it is not possible to describe the concordance in key events or biological steps

leading to neurodevelopmental outcomes. As such, the quantitative linkages between MIEs,

19 

# NRC (National Research Council). ( 2009). Science and decisions: Advanci ng risk assessment. Washington, DC:

The National Academies Press. http://www.nap.edu/openbook.php?record_id=12209

# Chlorpyrifos Human Health Risk Assessment D424485

Page 45 of 531 intermediate steps, and ultimately the adverse outcome (i.e., neurodevelopmental effects) cannot

be determined. Experimental toxicology studies in rodents suggest that long-term effects from

chlorpyrifos exposure may occur. Due to the dose selections in most of these in vivo studies

evaluating effects such as behavi or and cognition, it is not known whether such adverse effects

would be shown at doses lower than those whic h elicit 10% RBC AChE inhi bition. It is notable,

however, that comparing the lowest NOAEL observed in the in vivo animal studies (0.2

mg/kg/day; Billauer-Haimovitch et al., 2009) for the neurodevelopmental outcomes to the

repeated dosing reliable BMDL 10 

# ranging from 0.05-0.17 mg/kg/day for RBC AChE inhibition

suggests that AChE inhibition is a sensitive endpoint.

Within the epidemiology studies, the relationship in time between prenatal chlorpyrifos exposure

and adverse neurodevelopmental outcomes is concordant. Specifi cally, with regard to the

children’s environmental health epidemiology studies, each of th e three study cohorts utilized a

prospective birth cohort study desi gn in which mothers were recr uited into study prior to the

birth of the infants and development and identifi cation of adverse effects; therefore, it is known

with certainty that exposure preceded effect. In addition, be cause the time period under study

within these cohorts, and specifically the Co lumbia University (CCCEH) study, spanned the

point in time in which pesticid e manufacturers voluntarily cancelled the use of chlorpyrifos in

the home environment, researchers were able to show the change in exposure before (high use

period) and after (low/no use pe riod) the period of re moval of chlorpyrifos products from the

residential marketplace. Moreover, prior to the voluntary cancellati on there were >80%

detectable levels of chlorpyrif os in cord blood but in the time period after the cancellation only

16% of the measured values were greater than the LOD; there was only one child born in the

time period subsequent to the voluntary cancell ation of chlorpyrifos in the residential

marketplace for whom the cord blood chlorpyr ifos level was in the upper-tertile of precancellation exposure levels. The significantly re duced proportion of measured values greater

than the limit of detection as well as the observa tion of an absence of an association between

prenatal chlorpyrifos exposure among infant s born after the voluntary cancellation of

chlorpyrifos support the hypothesis th at chlorpyrifos is related to these outcomes. However, as

noted by study authors, EPA and the FIFRA SAP ( 2012), this could also be due to inadequate

sample size to detect a small to modest effect among the group of infant s born after the voluntary

cancellation.

With respect to the timing of exposure, the co rd blood and other (meconium) measures from the

Columbia University study provide evidence that exposure did occur to th e fetus during gestation

but the actual level of such exposure during the critical window(s) of susc eptibility is not known.

While significant uncertainties remain about the actual exposure levels ex perienced by mothers

and infant participants in the three children’s he alth cohorts, particular ly during the time period

prior to the voluntary cancellation of indoor residential uses of ch lorpyrifos containing pesticide

products, exposures measured in the range repor ted in the epidemiology studies (pg/g plasma)

are likely low enough that is unlikely to re sult in AChE inhibition. The FIFRA SAP (2012)

concurred with the conclusion that measured levels of chlorpyrifos among epidemiology study

participants were unlikely to have resulted in AChE inhibition. The urinary TCPy concentrations

among mothers were comparable to the gene ral population levels measured in NHANES.

Comparing cord blood concentrations with the concentrations in which AChE inhibition was

observed in adult volunteers indica tes AChE inhibition would likely not have occurred at levels

# Chlorpyrifos Human Health Risk Assessment D424485

Page 46 of 531 observed in the epidemiology studie s (6.17 pg/g). Therefore, while uncer tainty exists as to actual

chlorpyrifos exposure at (unknown) critical windows of exposur e, EPA believes it is unlikely

mothers enrolled in the birth cohort st udies experienced RBC AChE inhibition.

This biomarker data from the Columbia Univer sity studies are supported by the Agency’s dose

reconstruction analysis using th e PBPK-PD model. In accordance with the recommendation of

the FIFRA SAP (2012), the Agency conducted a dose reconstruction analysis of residential uses

available prior to 2000 for pre gnant women and young children insi de the home. Based on the

output from the PBPK-PD model, for the highest exposure consider ed (i.e., indoor broadcast use

of a 1% chlorpyrifos formul ation), <10% RBC AChE inhibiti on in pregnant women and young

children would be expected from residential uses. It is noteworthy that a ll estimates of exposure

based on conservative assumptions lead to predicted AChE inhibition levels < 10%.

Strength, consistency, and specificity

. As stated in the EPA neurotoxicity guidelines 20 

# , direct extrapolation of developmental

neurotoxicity results from laboratory animals to humans is limited by the lack of knowledge

about underlying toxicological mechanisms and the relevance of these results to humans. EPA

notes consistencies across th ese two databases, although ch allenges of making a direct

comparison between neurodevelopmental domain inte r-species remain. It can be assumed that

developmental neurotoxicity effects in animal studies indicate the potential for altered

neurobehavioral development in humans, although the specific t ypes of developmental effects

seen in experimental animal studies may not be the same as those that may be produced in

humans. However, considering th e toxicological and epidemiologica l data in the context of three

major neurodevelopmental domains (specifically, c ognition, motor control, and social behavior),

insights can be gained. For example, chlorpyrifos studies in rats and/or mice have reported

impaired cognition (spatial learning and work ing memory; e.g., Icenogl e et al., 2004, BillauerHaimovitch et al., 2009); changes in locomotor activit y levels (exploration, r earing; e.g., Levin et

al., 2002; Ricceri et al., 2003); and altered social interaction (aggressi on, maternal behavior;

Venerosi et al., 2006, 2010); and effects on br ain morphometrics (MRI D no. 44787301; Chen et

al, 2012). Similarly, epidemiologic investigations have reported effects on cognition (Bayley

scale indices; Rauh et al. 2006; Eskenazi et al . 2007), abnormal motor development in neonates

(reflexes, Brazelton score; Young et al., 2005; Enge l et al. 2007), altered social development

( e.g., ADHD; Rauh et al. 2006; Bouchard et al. 2010), and MRI brain scans (Rauh et al., 2012).

It is notable that the laboratory animal studies vary in experimental designs such as species,

strain, gender, dosing regimens (age, routes, vehi cle), and test paramete rs (age, protocol).

Likewise, observational epidemiology studies va ry by population characteri stics (race/ethnicity,

SES, and pesticide use/exposure profile), co -exposures (mix of chemicals, windows of

exposure), and method of exposure and outcome assessment. Given the differences across

laboratory animal and epidemiology studies, the qua litative similarity in research findings is

striking.

In contrast, quantitatively, there are notable differences between animals and humans.

Specifically, in animals, the doses most often usedin the behavior studies (1 and 5 mg/kg/day)

are sufficient to elicit approximately ≥ 10% brain inhibition and ≥ 30% in RBC inhibition ,

20 

# http://www.epa.gov/raf/publi cations/pdfs/NEUROTOX.PDF

# Chlorpyrifos Human Health Risk Assessment D424485

Page 47 of 531 depending on the study design, age of the animal , and sampling time. In the epidemiology

studies, based on the comparisons with biom onitoring data and the results of the dosereconstruction analysis, it is unlikely that RBC AChE would have been inhibited by any

meaningful or measurable amount, if any at all, and most likely none in the brain (although the

Agency has not investigated the degree to which exposure to multiple AChE-inhibiting

pesticides indoors simultaneously could impact this conclusion). This key difference in dose

response between the experimental toxicology and epidemiology studies poses challenges in

interpreting such data. There are a number of possible hypotheses such as: 1) limitations of

experimental laboratory studies which have limited statistical power due to relatively small

sample sizes; 2) humans display a broader array of behaviors and cognitive abilities than rats,

thus limiting the sensitivity of the rat studies; a nd 3) in the epidemiology studies, the timing of

chlorpyrifos application and blood collections are not coupled---thus higher levels of blood

chlorpyrifos were likely missed (albeit the results of the dose reconstruction analysis reduce the

likelihood of this hypothesis).

In making a weight-of-evidence analysis, it is impor tant to consider the strength of the statistical

measures of association between prenatal chlorpyrifos exposur e and adverse neurodevelopmental

outcomes through childhood (epidemiology) and possi bly into adulthood (animal studies). It is

also important to consider the strength of the in tegrated qualitative and quantitative evidence, the

consistency of the observed associations acr oss epidemiology studies and considering both

animal and human data support the conclusion that chlorpyrif os plays a role in adverse

neurodevelopmental outcomes. While it cannot be st ated that chlorpyrifos alone is the sole

contributor to the observed outcomes (specificity ), since other environmental, demographic or

psychosocial exposures may also play a part in these outcomes, this does not obviate the

contribution of prenatal chlorpyrifos exposure in the development of adverse

neurodevelopmental outcomes as echoed by the FIFRA SAP (2012).

The CCCEH study, which measures chlorpyrifos specifically, provides a number of notable

associations. Regarding infant and toddler ne urodevelopment, the CCCEH authors also reported

statistically significant deficits of 6.5 points on the Bayley Psychomotor Development Index

(PDI) at 3 years of age when comparing hi gh to low exposure groups (Rauh et al., 2006).

Notably these decrements in PDI persist even after adjustment for gr oup and individual level

socioeconomic variables (Lovasi et al., 2011). Thes e investigators also observed increased odds

of mental delay (OR=2.4; 95% CI: 1.1-5.1) a nd psychomotor delay (OR=4.9; 95% CI: 1.8-13.7)

at age three when comparing hi gh to low exposure groups (Rauh et al., 2006). Rauh et al (2006)

also reported extremely large odds ratios fo r attention disorders (OR=11.26; 95% CI: 1.79-

70.99), ADHD (OR=6.50; 95% CI: 1.09-38.69), and PDD (OR=5.39; 95% CI: 1.21-24.11) when

comparing high to low chlorpyrifos exposure gr oups (Rauh et al., 2006). EPA notes that the

magnitude of these results are so large that they are unlikely to be affected by residual

confounding although limited sample sizes resulted in imprecise estimates.

Importantly, across the three children’s environm ental health birth cohorts, decrements in

intelligence measures were identified in relation to increasing levels of prenatal chlorpyrifos

exposure. Authors from the Columbia cohort (CCCEH) reported statis tically significant

decreases of 1.4% in full scale IQ and 2.8% in working memory among seven-year olds for each

standard deviation increase in chlorpyrifos exposure (Rauh et al. , 2011). These results persist

# Chlorpyrifos Human Health Risk Assessment D424485

Page 48 of 531 even when performing sensitivity analyses includ ing only those with detectable chlorpyrifos

levels. In addition, no evidence wa s provided of mediation by child behavior on the measure of

working memory instrument.

Biological plausibility and coherence

. EPA’s Cancer Guidelines (2005) includes guidance which

are also applicable to this current evaluation of chlorpyrifos. The Guidelines indicate:

“ evaluation of the biological plausibility of th e associations observed in epidemiologic

studies reflects consideration of both exposur e-related factors and toxicological evidence

relevant to identification of potential modes of action (MOAs). Similarly, consideration of the

coherence of health effects associations reported in the epidemiologic literature reflects

broad consideration of information pertaining to the nature of the biological markers

evaluated in toxicologic and epidemiologic studies . [p. 39].”

The Cancer Guidelines further state that “ lack of mechanistic data, however, is not a reason to reject

causality [p. 41].”



# At this time, a MOA(s)/AOP(s) has/have not been established for neurodevelopmental outcomes.

This growing body of literature does demonstrate, however, that chlorpyrif os and/or its oxon are

biologically active on a number of processes that affect the devel oping brain. Moreover, there is

a large body of in vivo laboratory studies which show long-te rm behavioral effects from early

life exposure. EPA considers th e results of the toxicological studies relevant to the human

population, as qualitatively suppor ted by the results of epidemiology studies. The lack of

established MOA/AOP pathway does not undermine or reduce the confidence in the findings of

the epidemiology studies. The CCCEH data are not considered in isolation, but rather are

strengthened when considered in concert with the results from the other two cohort studies, as

noted by the FIFRA SAP (July 2012). As noted above, the CHAMACOS and Mt. Sinai cohorts

that measured neurological eff ects at birth (the Brazelton index) , observed an association with

chlorpyrifos (Engel et al., 2007; Young et al., 20 05). Similarly, while not consistent by age at

time of testing (ranging from 6 month to 36 mont hs across the three cohorts), each cohort

reported evidence of impaired mental and psycho motor development. Attentional problems and

ADHD were reported by both Mt. Sinai and CHAMACOS investigators. Finally, each of the

three cohort study authors observed an inverse rela tion between the respectiv e prenatal measures

of chlorpyrifos and intelligen ce measures at age 7 years.

Although uncertainties remain as articulated abov e, these uncertainties are diminished in the

context of the qualitative similarity between the databases, and the concern for long-term

neurodevelopmental effects as a re sult of prenatal, perinatal an d possibly early life exposure.



# 4.7 Safety Factor for Infants an d Children (FQPA Safety Factor)

The key issues being considered by the Agency in the WOE are 1) whether chlorpyrifos causes

long-term effects from fetal or early life e xposure and 2) whether adverse effects can be

attributed to doses lower than those which elicit 10% inhibition of AChE). When taken together

the evidence from 1) the experimental toxicol ogy studies evaluating outcom es such as behavior

# Chlorpyrifos Human Health Risk Assessment D424485

Page 49 of 531 and cognitive function; 2) mechanistic data on possible adverse outcome pathways/modes of

action; and 3) epidemiologic and biomon itoring studies lead to the following:

o Qualitatively, the Agency concludes that th ese lines of evidence together support a

conclusion that exposure to chlorpyrifos results in adverse neurodevelopmental outcomes in

humans, at least under some conditions.

o Quantitatively, the dose –response relationship of AChE inhibition across different life stages

is established, but MOAs/AOPs for neurodeve lopmental outcomes are not established.

The database of in vivo animal toxicology neurodevelopmental studies on adverse outcomes

includes only a small number of studies at dos es lower than 1 mg/kg/day. Despite this

uncertainty, the Agency noted that the BMD values in adult (pregnant and nonpregnant)

female rats (0.05-0.15 mg/kg/day) are generally 10-fold or more lower than the doses where

effects on neurodevelopmental outcomes in laboratory ra ts are observed.

With respect to the mechanistic data, there are some effects wh ich are similarly sensitive or

more sensitive than AChE inhibition. The fact that there are, however, sparse data to support

the in vitro to in vivo extrapolation, or the extrapolati on from biological perturbation to

adverse consequence significantly limits thei r quantitative use in risk assessment.

As noted above, the lack of an establis hed MOA/AOP makes quantitative use of the

epidemiology study in risk assessment challe nging, particularly with respect to doseresponse, critical duration of exposure, and window(s) of susceptibil ity. Despite this

uncertainty, the cord blood and other measures (meconium) provide evidence of exposure to

the fetus during gestation. Moreover, exposur e levels in the range measured in the

epidemiology studies (pg/g) are likely low enough that is unlikely to result in AChE

inhibition, as supported by the dose reconstructi on analysis of reside ntial use prior to 2000

(although the agency has not investigated the degree to which exposure to multiple AChEinhibiting pesticides indoor s simultaneously could impact this conclusion).

Given the totality of the evidence, the agency concludes that chlor pyrifos likely played a role in

the neurodevelopmental outcomes reported by th e Columbia University investigators but

uncertainties such as the lack of an establ ished MOA/AOP for neurodevelopmental effects and

the exposure to multiple AChE-inhibiting pesticid es precludes definitive causal inference.

However, there is sufficient uncertainty in the human dose-response relationship for

neurodevelopmental effects which prevents the Agency from reduc ing or removing the statutory

10X FQPA Safety Factor. The FQPA 10X Safety Factor will be retained for infants,

children, youths, and women of childbearing age for all exposure scenarios.



# 4.8 Dose-Response Assessment

4.8.1 Durations of Exposure, Critical Windows of Exposure, & Temporality

of Effects

# Chlorpyrifos Human Health Risk Assessment D424485

Page 50 of 531 In risk assessment, exposure is evaluated in conjunction with the toxicology profile. More

specifically, a variety of toxicoki netic and toxicodynamic factors ar e considered. In the case of

chlorpyrifos, exposure can occur from a single exposure ( e.g. , eating a meal) or from repeated

days of exposure ( e.g. , worker, residential).

With respect to AChE inhibition, these effects ca n occur from a single exposure or from repeated

exposures. For OPs, repeated exposures genera lly result in more AChE inhibition at a given

administered dose compared to acute studies. Moreover, AChE inhibition in repeated dosing

guideline toxicology studies with OPs show a c onsistent pattern of inhibition reaching steady

state at or around 2-3 weeks of exposure in ad ult laboratory animals (U .S. EPA, 2002). This

pattern observed with repeated dosing is a result of the am ount of inhibition comes at

equilibrium with production of new enzyme. As such, AChE studies of 2-3 weeks generally

show the same degree of inhibiti on with those of longer duration ( i.e. , up to 2 years of exposure).

Thus, for most of the human health risk assessmen ts for the OPs, the agency is focusing on the

critical durations range from a single day up to 21 days ( i.e. , the approximate time to reach

steady state for most OPs). As described below, points of de parture for various lifestages,

routes, and scenarios have been derived at the acute and stead y state durations.

With respect to effects on the developing brai n, very little is know n about the duration of

chlorpyrifos exposure needed to precipitate adverse effects in the developing brain. There are

critical windows of vulnerabili ty (Rice & Barone, 2000; Rodier, 2004) with regard to toxicant

effects on brain development. This vulnerabl e period in humans spans early pregnancy to

adolescence (Rice & Barone, 2000). In fact, evidence shows that synapse formation peaks quite

late in human brain development at 4-8 years of age (Glantz et al, 2007). Within these

vulnerable periods there are key neurodevelopmental processes ( e.g. cell division, migration,

differentiation, synaptogenesis, and myelination) and each of these is region and stage specific.

Consequently, the time of toxicant exposure will be a major determinate in the spectrum of

neurotoxic effects. Because of the dyna mic processes in the developing brain ( i.e. , vulnerable

windows) it is difficult to determine if the effect or differences in effects is due to duration of

exposure or if different vulnerabl e windows were affected. As such, it is impossible at this time

to rule out even a single day of high exposur e to chlorpyrifos having a potential adverse

neurodevelopmental effect in humans.

For the chlorpyrifos risk assessment, PoDs fo r various lifestages, routes, and scenarios have

been derived at the acute an d steady state durations.



# 4.8.2. Introduction to the PBPK-PD Model

As described in detail in EPA’s 2006 document entitled, “ Approaches for the Application of

Physiologically Based Pharmacokinetic (PBPK) Models and Supporting Data in Risk

Assessment,” physiologically based pharmacokinetic (PBP K) modelling is a scientifically sound

and robust approach to estimating th e internal dose of a chemical at a target site and as a means

to evaluate and describe the uncertainty in risk as sessments. PBPK models consist of a series of

mathematical representations of biological tissues and physiologi cal processes in the body that

# Chlorpyrifos Human Health Risk Assessment D424485

Page 51 of 531 simulate the absorption, distribution, metabolism, and excretion (ADME) of chemicals that enter

the body. Examples of PBPK mode l applications in risk assessments include interspecies

extrapolation, intra-species extrapolation, rout e-to-route extrapolation, estimation of response

from varying exposure conditions, and high-to-low dose extrapolation. PBPK models can be

used in conjunction with an exposure assessment to improve the quantitative characterization of

the dose-response relationship and the overall risk a ssessment. These models can also be used to

evaluate the relationship between an a pplied dose and biomonitoring data.

The chlorpyrifos PBPK-PD model includes the desc ription of a molecular initiating event in the

cholinergic toxicity MOA/AOP: AChE inhibitio n. Thus, the PBPK-PD model can be used to

predict the critical dose metrics associated w ith cholinergic toxicity following chlorpyrifos

exposure: RBC and brain AChE inhibition. Also predicted by the model are chlorpyrifos, its

oxon, and TCPy levels in various tissues, such as pl asma and urine. Age-specific parameters are

incorporated allowing for lifestage-specific eval uations from infant through adulthood. The

model can be run in two modes: deterministic and variation. In the deterministic mode, the

output accounts for human specific metabolism a nd physiology thus obviating the need for the

inter-species extrapolatio n factor for all for all age groups. In variation mode, distributions for16

parameters, which are critical for determining human variations in RBC AChE inhibition, are

incorporated and thus the output accounts for intra-species extr apolation for infants, toddler,

youths, and non-pregnant adults. As described below, the agency is not using the PBPK-PD

model for pregnant females due to uncertainty associated with l ack of appropriate mathematical

equations to account for physiological changes during pregnancy that could impact population

variability.



# 4.8.3 Description & Structu re of the PBPK-PD Model

The PBPK-PD model for chlorpyrifos that was or iginally developed by Timchalk and coworkers

in 2002 (Timchalk et al., 2002a, b) has been refi ned over the years as more data has become

available (Busby-Hjerpe et al., 2010; Cole et al., 2005; Garabrant et al., 2009; Lee et al., 2009;

Lowe et al., 2009; Lu et al., 2010; Marty et al., 2007; Timchalk and Poet, 2008; Timchalk et al.,

2005; Timchalk et al., 2006). The model will not be described in detail here as it is described in

numerous publications, including a report re viewed by the FIFRA SAP in 2011; summary

information is provided here. All model code for the PBPK-PD model are provided in the public

docket for the chlorpyrifos risk assessment. Developers of the chlorpyrifos PBPK-PD model

sponsored a third-party quality a ssurance assessment to verify model parame ter values and their

respective sources. The Agency has conducted a review of this third-party assessment by

randomly checking a subset of the values and sour ces in the model paramete rs. The agency also

conducted a mass balance analysis. 

# Minor inconsistencies were id entified, and developers of the

chlorpyrifos PBPK-PD model have since made th e corrections, or provide d additional references

to justify their parameterization choices.

In recent collaborative research effort betw een Battelle Pacific Nort hwest National Laboratory

and Dow (DAS et al., 2011; Poet et al, 2014; MRIDs 49074901, 48913401), the chlorpyrifos

PBPK-PD model has been expanded from the ‘typical adult’ model to include other lifestages,

specific examples were infants (6 months), childre n (3 year-olds), and adults (30 year olds).

# Chlorpyrifos Human Health Risk Assessment D424485

Page 52 of 531 Body weight can be calculated at any given age using a modified Gompertz growth function for

both male and female (Smith et al., 2014). S ubsequently, tissue volumes, blood flows to tissue,

metabolism rates, ChE enzyme activities, and ex posure doses are calculated as a function of

body weight for infants through adulthood, which in turn is also a function of age. Furthermore,

the PBPK-PD model was refined to include probab ilistic capabilities to allow for refining the

intra-species extrapolation factor. The FIFR A SAP reviewed the initial multiple lifestage,

probabilistic model at its February 2011 meeting and recommended some additional

improvements (FIFRA SAP, 2011; Hinderliter et al, 2011; Price et al, 2011). In response to the

SAP, DAS made multiple changes and perfor med additional analyses, including a global

sensitivity analysis, improvements to the qua ntitative approach to evaluating population

variability across individuals at a given age, and an uncertainty analysis on metabolism data from

human hepatic microsomes and plasma to addr ess variation in response that occurs from

metabolism (Dow, 2014a, b). Recently, code has b een developed to simulate tissue dosimetry

from dermal and inhalation exposure routes (Poe t et al, 2014). The deterministic multi-route,

lifestage PBPK-PD model code used in the chlorpyrifos risk assessment was submitted to the

agency in 2013. The model code to evaluate pop ulation variability and to derive intra-species

data-derived extrapolation factors was mo st recently updated and submitted in 2014 (Dow,

2014a, b)

The chlorpyrifos PBPK-PD model includes descri ptions of the ADME of chlorpyrifos, and its

metabolites, oxon and TCPy (Figure 3). The metabolic profile for chlorpyrifos is provided above

in Figure 2. The PBPK-PD model contains descriptions of metabolism to account for

chlorpyrifos, its oxon, and TCPy in liver, blood, br ain, small intestine, lungs, diaphragm, and

skin. Model parameterization was achie ved most often by extrapolation from in vitro studies

(animal and human tissues), in some incidences by extrapolation from rat in vivo studies, and, for

TCPy pharmacokinetics, by fitting to human data 21 

# . The PD portion of the model is an extension

of the PBPK model and relates the prediction of the formation of oxon to activity changes in

AChE, BuChE, and carboxylesterase in brain, di aphragm, liver, lungs, plasma, and RBC.

To simulate exposures to infants and children, the model incorporates age-specific body weight

prediction (Leucke et al. (2007) and Young et al. , (2009)), which is then used to scale the tissue

volumes, blood flows to tissues, and ChE activit ies (Figure 4). In a ddition to age-related

physiology, the model also incorporates age-specific metabolism based on in vitro data, allowing

the model to assess changes in metabolis m from infancy to adulthood. Total V

max 

# of human

enzymatic metabolism of CPF to TCPy, CPF to CPF-oxon, and CPF-oxon to TCPy in the liver

over various ages were scaled to age-depe ndent volume of the liver (Table 4.8.3.1) and

concentration of microsomes in the liver, which is approximately 37 mg/g tissue (Barter et al.

, 2008). Thus, even in cases where the enzymatic measures show no in vitro age- or

physiological-dependences (on a per mg protein basis), the total enzymatic capacity of the

individual varies with age. In plasma, however, in vitro PON1 metabolism of CPF-oxon to TCPy

was age-specific on a per ml plasma volume basis, as described previously. Therefore, the age-

21 

# Two human deliberate dosing studies (Nolan et al 

# . 1982; Kisicki et al, 1999) ar e available which have been

reviewed by EPA’s Human Studies Review Board

http://www.epa.gov/osa/hsrb/files/june2009finalreport92609.pdf

http://www.epa.gov/osa/hsrb/files/meeti ng-materials/apr-13-14-2011/appendix1.pdf

These studies have been used in the development and evaluation of the PBPK-PD model.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 53 of 531 specific V

max 

# was scaled to age-dependent volume of th e blood, resulting in plasma metabolism

varying by almost 2 orders of magnitude. Values and sources of model parameters can be found

in Tables 4.8.3.1-4.8.3.2 and were ex tracted from Dow 2011 and MRID 49248201.

Figure 3. PBPK/PD model (Typical Adult model) structure. The shaded compartments denote

tissues which contain B-esterases (bottom panel). Tissue volumes and enzyme activities (V

max 

# )

change with age based on liver and/or blood comp artmental growth (Extracted from Dow, 2011).

Figure 4. Schematic of age and body weight dependences in the PBPK-PD model. All compartment volumes and

blood flows vary with age and body weight. In vivo metabolic rates are scaled based on tissue size (measured in

# Chlorpyrifos Human Health Risk Assessment D424485

Page 54 of 531 vitro 

# values scaled to describe tissue-specific (brain, blood, and liver) metabolism); in blood, PON1 metabolism of

oxon is not only blood volume but also age-dependent (Extracted from Dow, 2011).

# Chlorpyrifos Human Health Risk Assessment D424485

Page 55 of 531 Table 4.8.3.1. Pharmacokinetic model parameters.

Parameter Value Source

Tissue Ontogeny

All Scaled by age and body

weight See 

# Table 4.8.3.2

Flows (L/hr/kg tissue volume)

Cardiac Output Summed from total tissue

flow See “ Blood flows” in this section

Brain 30.6 Price et al. , 2003

Diaphragm 85.2 Luecke et al. , 2007

Fat 1.45 Luecke et al. , 2007, Cowles et al. , 1971,

Price et al. , 2003

Liver 50.4 Price et al. , 2003

Rapidly Perfused 61.8 Adrenal/Spleen average: Price et al. , 2003

Slowly Perfused 1.80 Bone: Price et al. , 2003

Partition Coefficients for CPF (tissue:blood)

Brain 16.5 Lowe et al. , 2009

Diaphragm 3.85 Lowe et al. , 2009

Fat 250 Lowe et al. , 2009

Liver 12.8 Lowe et al. , 2009

Rapidly Perfused 16.5 Lowe et al. , 2009

Slowly Perfused 3.85 Lowe et al. , 2009

Partition Coefficients for CPF-oxon (tissue:blood)

Brain 5.6 Lowe et al. , 2009

Diaphragm 1.8 Lowe et al. , 2009

Fat 75 Lowe et al. , 2009

Liver 4.5 Lowe et al. , 2009

Rapidly Perfused 5.6 Lowe et al. , 2009

Slowly Perfused 1.8 Lowe et al. , 2009

Hepatic CYP Metabolic Constants (per tissue wt)

CPF → TCPy Vmax

(nmol/hr/kg) 1580 Measured in vitro , Median: See 

# Table

4.8.3.2

CPF → TCPy Km

(μM) 53.8 Measured in vitro , Median: See 

# Table

4.8.3.2

CPF → Oxon Vmax

(μmol/hr/kg) 689 Measured in vitro , Median: See 

# Table

4.8.3.2

CPF → Oxon Km

(μM) 85.8 Measured in vitro , Median: See 

# Table

4.8.3.2

Intestinal CYP Metabolic Constants (per tissue wt)

CPF → TCPy Vmax

(μmol/hr/kg) 36.8 Poet et al. , 2003

# Chlorpyrifos Human Health Risk Assessment D424485

Page 56 of 531 Parameter Value Source

CPF → TCPy Km

(μM) 55 Poet et al. , 2003

CPF → Oxon Vmax

(μmol/hr/kg) 10.0 Poet et al. , 2003

CPF → Oxon Km (μM) 8.1 Poet et al. , 2003

Brain CYP Metabolic Constants (per tissue wt)

CPF → TCPy Vmax

(μmol/hr/kg) 3.85 Extrapolated: CPF metabolism/mechanism

of action

CPF → TCPy Km

(μM) 5.38 See Section 2: CPF metabolism/mechanism

of action

CPF → Oxon Vmax

(μmol/hr/kg) 0.91 Extrapolated: CPF metabolism/mechanism

of action

CPF → Oxon Km

(μM) 8.60 See CPF metabolism/mechanism of action

PON1 Metabolic Constants

Plasma Vmax

(μmol/hr/kg) Logistic Fit Measured in vitro , Median: See Table

4.8.3.2

Plasma Km (μM) 192 Measured in vitro , Median: See Table

4.8.3.2

Liver Vmax

(μmol/hr/kg) 3902 Measured in vitro , Median: See Table

4.8.3.2

Liver Km (μM) 498 Measured in vitro , Median: See Table

4.8.3.2

Intestine Vmax

(μmol/hr/mg) 246 Poet et al. , 2003

Intestine Km (μM) 328 Poet et al. , 2003

Oral Absorption

Stomach → Intestine 0.5 Fitted: Nolan et al. , Timchalk et al. , 2002

Intestinal Absorption 0.2 Fitted: Nolan et al. , Timchalk et al. , 2002

Plasma Protein Binding (%)

CPF 99 Lowe et al. , 2009

Oxon 99 Lowe et al. , 2009

TCPy Compartmental Model

Vd (L) 0.2×BW 1.117 

# Fitted: Nolan et al. , 1984, Timchalk et al.

,

2002

Ke (/hr) 0.013 Fitted: Nolan et al. , 1984, Timchalk et al.

,

2002

Cholinesterase Degradation Rates (hr-1)

Butyryl 0.004 Fitted (repeat dose data in rats: DOW

unpublished)

Acetyl 0.01 Standardized, Timchalk et al. , 2002b

Bimolecular Inhibition Rate (μM hr -1 

# )

Butyryl 2000 Timchalk et al. , 2002

# Chlorpyrifos Human Health Risk Assessment D424485

Page 57 of 531 Parameter Value Source

Acetyl 220 Kousba et al. (2007)

Enzyme Turnover Rate (hr -1 

# )

Butyryl 1.17x10 7 

# Maxwell et al. , 1987, Timchalk et al. , 2002b

Acetyl 3.66x10 6 

# Maxwell et al. , 1987, Timchalk et al. , 2002b

Carboxyl 1.086x10 5 

# Maxwell et al. , 1987, Timchalk et al. , 2002b

Enzyme Turnover Rate (hr-1)

Enzyme Turnover Rate (hr-1)

Butyryl 11700000 Maxwell et al. , 1987, Timchalk et al. , 2002b

Acetyl 3660000 Maxwell et al. , 1987, Timchalk et al. , 2002b

Carboxyl 108600 Albers et al. , 2010

Enzyme Activity (μmol/kg/hr)

Brain ACHE 440000 Hojring et al. , 1976

Diaphragm ACHE 77400 Maxwell et al. , 1987, Timchalk et al. , 2002b

Liver Carboxyl 1920000 Maxwell et al. , 1987, Timchalk et al.

, 2002b; Pope et al. , 2005

Plasma Carboxyl NA Li et al. , 2005

Brain Butyryl 46800 Maxwell et al. , 1987, Timchalk et al. , 2002b

Diaphragm Butyryl 26400 Maxwell et al. , 1987, Timchalk et al. , 2002b

Liver Butyryl 30000 Maxwell et al. , 1987, Timchalk et al. , 2002b

Plasma Butyryl 263000 Sidell et al. , 1975

Enzyme Reactivation Rate (hr -1 

# )

Butyryl 0.0014 Carr and Chambers, 1996, Timchalk et al.

,

2002b

Acetyl 0.014 Carr and Chambers, 1996, Timchalk et al.

,

2002b

Carboxyl 0.014 Carr and Chambers, 1996, Timchalk et al.

,

2002b

Enzyme Aging Rate (hr -1 

# )

Butyryl 0.0113 Carr and Chambers, 1996, Timchalk et al.

,

2002b

Acetyl 0.0113 Carr and Chambers, 1996, Timchalk et al.

,

2002b

Carboxyl 0.0113 Carr and Chambers, 1996, Timchalk et al.

,

2002bChlorpyrifos Human Health Risk Assessment D424485Page58of531Table 4.8.3.2. Compartmental Growth.CompartmentFraction of Body Weight Equation1Source for LifeStageParametersEqn. FormatV0+V1×BW+V2×BW2+V3×BW3+V4×BW4+V5×BW5+V6×BW6(BW: Body Weight in g)V0 V1 V2 V3 V4 V5 V6Blood Leucke 9.15e-2-8.59e-71.25e-11-6.46e-17- - - Younget al.2009Young 8.97e-2-3.50e-76.54e-13LifeStage8.970e-2-3.500e-76.540e-13- - - -Brain Leucke 1.19e-1-3.51e-64.28 e-11-1.82 e-16- - Fit to Valentinet al.2002(bwt≤70) & Younget al.2009 data (body weight>70kg) and extrapolated valuesbased on Table 4A (Youngetal.2009)Young 1.41e-1-5.54e-69.30 e-11-6.83e e-161.80e-21LifeStage1.216e-1-3.465e-64.354e-112.463e-165.132e-22Diaphragm Leucke 3.000e-4- - - - - - Lueckeet al.2007Young NA - - - - - -LifeStage3.000e-4- - - - - -Fat*Female Leucke 5.91e-21.20e-5-5.80e-101.12e-14-6.36e-20- - Fit to Valentinet al.2002 &Lafortunaet al.2005 dataand extrapolated data basedYoung 1.84e-2-6.86e-62.46e-10-2.11e-157.58e-21-9.94e-27-Chlorpyrifos Human Health Risk Assessment D424485Page59of531CompartmentFraction of Body Weight Equation1Source for LifeStageParametersEqn. FormatV0+V1×BW+V2×BW2+V3×BW3+V4×BW4+V5×BW5+V6×BW6(BW: Body Weight in g)V0 V1 V2 V3 V4 V5 V6LifeStage9.217e-021.401e-05-6.787e-101.540e-14-1.558e197.249e-251.273e-30on equation in Fig 1B(Lafortunaet al.2005)MaleLeucke 3.95e-21.59e-5-6.99e-101.09e-14-5.26e-20- - Fit to Valentinet al.2002 &Lafortunaet al.2005 dataand extrapolated data basedon equation in Fig 1B(Lafortunaet al.2005)Young 1.61e-2-3.59e-6-8.28e-11-3.57e-164.73e-22- -LifeStage3.484e-22.803e-5-1.422e-92.892e-14-2.718e191.203e-24-2.036e-30LiverLeucke 3.49e-2-3.23e-72.13e-12- - - - Fit to Valentinet al.2002(body weight≤70) & Younget al.2009 extrapolatedvalues based on Table 4AYoung 4.25e-2-1.01e-61.99e-11-1.66e-164.83e-22- -LifeStage3.917e-2-6.789e-71.082e-11-7.393e-171.701e-22- -RapidLung Leucke 1.67e-2-9.96e-8-1.09e-131.13e-17- - - Younget al.2009Young 1.860e-2-4.550e-8- - - - -LifeStage1.860e-2-4.550e-8- - - - -Kidney Leucke 7.31e-3-8.29e-82.13e-12- - - - Younget al.2009Young 7.26e-3-6.69e-83.33e-13- - - -LifeStage7.260e-3-6.690e-83.330e-13- - - -PancreaseLeucke 1.17e-3-1.18e-81.81e-13- - - - Brownet al.1997 and Younget al.2009Young 1.48e-3- - - - - -LifeStage1.480e-3- - - - - -Spleen Leucke 3.05e-3-2.09e-81.24e-13- - - - Younget al.2009Young 3.12e-35.57e-9- - - - -LifeStage3.120e-35.570e-9- - - - -Chlorpyrifos Human Health Risk Assessment D424485Page60of531CompartmentFraction of Body Weight Equation1Source for LifeStageParametersEqn. FormatV0+V1×BW+V2×BW2+V3×BW3+V4×BW4+V5×BW5+V6×BW6(BW: Body Weight in g)V0 V1 V2 V3 V4 V5 V6GI Leucke 1.93e-2-4.42e-79.28e-12-4.88e-17- - - Brownet al.1997Young NA - - - - - -LifeStage1.650e-2- - - - - -SlowNon-Fatadipose*Leucke NA NA NA NA NA NA NA Fit to Valentinet al.2002 &Lafortunaet al.2005 fatdata/0.8 (Valentin 2002) dataand extrapolated data basedon equation in Fig 1A(Lafortunaet al.2005)Young NA NA NA NA NA NA NALifeStage2.044e-12.617e-5-1.542e-93.268e-14-3.116e191.387e-24-2.35e-30Muscle Leucke 9.61e-2-4.88e-63.05e-10-3.62e-151.22e-20- - Fit to Valentinet al.2002 &Janssenet al.2000 data andextrapolated values based onFig 2A (Janssenet al.2000)Young 9.68e-1-3.32e-61.83e-10-1.24e-15- - -LifeStage1.251e-11.458e-5-2.927e-102.114e-15-5.250e21- -Skin Leucke 1.07e-1-3.26e-66.11e-11-5.43e-161.83e-21- - Younget al.2009Young 1.03e-1-2.56e-63.68e-11-2.580e-168.620e-22-1.100e-27-LifeStage1.030e-1-2.560e-63.680e-11-2.580e-168.620e-22-1.100e-27-Bone Marrow Leucke 5.19e-28.06e-7-1.96e-107.63e-15-1.08e-195.14e-25NA Brownet al.1997 (red only,yellow is in adipose)Young NA NA NA NA NA NA NALifeStage2.100e-2- - - - - -

# Chlorpyrifos Human Health Risk Assessment D424485

Page 61 of 531 

# 4.8.4 Use of the PBPK-PD Model

Evaluation of PBPK-PD models intended for risk assessments includes a review of the model

purpose, model structure, mathematical repres entation, parameter estimation (calibration), and

computer implementation (USEPA, 2006b). Th e chlorpyrifos PBPK-PD model has been

through several quality assurance reviews by various individuals or groups, including the agency,

and found that the mathematical equations in th is model were adequate to reasonably predict

both blood/urine dosimetry and ChE inhibition in two controlled, deliberate oral human dosing

studies (Nolan et al., 1984; Ki sicki et al., 1999) and a dermal human study (Nolan et al., 1984).

The PBPK-PD model predictions fo r rats inhaled chlorpyrifos compared well with animal data

(Hotchkiss et al., 2013) with resp ect to chlorpyrifos, oxon, and TCPy concentrations in plasma,

and ChE in plasma, RBC and brain (Poet et al., 2014). In addition, the agency has continued to

critically evaluate the model code and related inputs during the risk assessment process.

Significant improvements have been made to the PBPK-PD model in response to the 2008, 2011,

and 2012 SAPs, the agency, and peer reviewers from academic journals in addition to the input

of new data. The agency believes that the model is sufficiently robust for use in human health

risk assessment. Because age-specific paramete rs are incorporated allowing for lifestagespecific evaluations from infant through adu lthood, in the deterministic mode, the output

accounts for human specific metabolism and physio logy thus obviating the need for the interspecies extrapolation factor. Th e deterministic model can be us ed to simulate an “average

individual” for all age groups. As such, as de scribed below, the agency is using the PBPK-PD

model to determine the points of departure (P oDs) for all age groups (See Table 4.8.4 below).

At the 2011 SAP meeting, the Panel specifically noted the lack of maternal and fetal PK and PD

compartments in the current PBPK-PD model to inform about tissue dosimetry and AChE

inhibition during lactation (FIFRA SAP 2011). As described in detail below, the agency has

assessed exposure to bottle-feed ing infants exposed to the oxon through water used with infant

formula. With respect to chlorpyrifos or oxon exposure to infants through breast milk, any

exposure to chlorpyrifos would be far lower th an drinking water levels predicted by EFED.

Thus, the agency is already accounting for oral exposure to chlorpyrifos to infants via bottlefeeding and a lactation component in the PBPK-PD model is not necessary.

The SAP also noted the lack of maternal and fetal PK and PD compartments in the current

PBPK-PD model to inform about tissue dosimetry and AChE inhibition to pregnant women and

their fetuses (FIFRA SAP 2011). With respect to exposure to th e fetus during gestation, there

are multiple studies on chlorpyrifos (Mattss on et al., 1998, 2000) and other OPs (U.S. EPA,

2006a) which show that the pregnant dam exhibits similar or more AChE inhibition than the

fetus at a given dose to the dam. As such, for AChE inhibition, by protecting against AChE

inhibition in the pregnant female is expected to be protective for AChE inhibition in the fetus.

Biomonitoring data from rats and humans support the findings of the AChE studies.

Specifically, Whyatt et al (2003) ha ve shown that levels of chlo rpyrifos in maternal blood are

similar to the levels measured in human umbilic al cord blood (Whyatt et al, 2003). With respect

to the pregnant dam during gestation, metabolic activities and physiologi cal parameters can be

altered during pregnancy (for citations, see A ppendix 1). While the current PBPK-PD model

# Chlorpyrifos Human Health Risk Assessment D424485

Page 62 of 531 accounts for age-related growth from infancy to adulthood by using polynomial equations to

describe tissue volumes and blood flows as a f unction of age, the model does not include any

descriptions on physiological, anat omical and biochemical changes associated with pregnancy.

Due to the uncertainty in extrapolating the cu rrent model predictions among women who may be

pregnant, the agency is applying the standard 10X intra-species extrapolation factor for

women of child bearing age.



# 4.8.4.1 Derivation of Human Equi valent Doses/Concentrations

In typical risk assessments, PoDs are derived directly from labor atory animal studies and interand intra-species extrapolation is accomplishe d by use of 10X factors. In the case of

chlorpyrifos and its oxon, PBPK-P D modeling is being used as a data-derived approach to

estimate PoDs for all age groups and Data-Deriv ed Extrapolation Factors (DDEF) for intraspecies extrapolation for some groups (USEPA , 2014). The agency typically uses a 10%

response level for AChE inhibiti on in human health risk assess ment. This response level is

consistent with the 2006 OP cumulative risk assessment (USEPA, 2006a) and other single

chemical OP risk assessments. As such, the mo del has been used to estimate exposure levels

resulting in 10% RBC AChE inhibition followi ng single day (acute; 24 hours) and 21-day

exposures for a variety of exposure scenarios (F or discussion of durati ons see Section 4.8.1).

The PBPK-PD model accounts for PK and PD charac teristics to derive age, duration, and route

specific PoDs (Table 4.8.4 below). Separate PoDs have been calculated for dietary (food,

drinking water), residential, and occupational ex posures by varying inputs on types of exposures

and populations exposed. Specifically, the followi ng characteristics have been evaluated:

duration [acute, 21 day (steady state)]; route (der mal, oral, inhalation); body weights which vary

by lifestage; exposure duration (hours per day, days per week); and exposure frequency [events

per day (eating, drinking)].

For each exposure scenario, the appropriate body weight for each age group or sex was modeled

as identified from the Exposure Factor s Handbook (USEPA, 2011) for occupational and

residential exposures and from the NHANES/ What We Eat in America (WWEIA) Survey 22 

# for

dietary exposures. All body weights used are consistent with those assumed for dietary,

occupational and residential exposure assessments. For infants from birth to < 1 year old, the

agency has selected the body weight for the youngest age group, birth to < 1 month old, 4.8 kg

(Exposure Factors Handbook, Table 8-3, mean body weight for the birth to < 1 month age

group). For children between 1-2 years old, the body weight was set to 12.6 for dietary

exposures (NHANES/WWEIA) and was set to 11 kg for residential exposures (Exposure Factors

Handbook, Table 8-3, mean body weight for the 1 to < 2 year old age group). For female adults,

the body weight was set to 72.9 kg for dietary ex posures (NHANES/WWEIA) and was set to 69

kg for residential exposures (Exposure Fact ors Handbook, Table 8-5, mean body weight for

females 13 to < 49 years old).

22 

# http://www.ars.usda.gov/Services/docs.htm?docid=13793

# Chlorpyrifos Human Health Risk Assessment D424485

Page 63 of 531 The agency assesses dietary exposures for ch ildren 6-12 years old, and children between 6-11

years old for residential exposures. For purpose of aggregate assessment, these age groups are

combined. The body weight for children 6-12 y ears old was set to 37.1 for dietary exposures

(NHANES/WWEIA); the body weight for children 6-11 years old was set to 32 kg for residential

exposures (Exposure Factors Handb ook, Table 8-3, mean body weight for the 6 to < 11 year old

age group). The agency assesses dietary expos ures for youths 13-19 years old, and youths

between 11-16 years old for residential exposures . For purpose of aggregate assessment, these

age groups are combined. For youths 13-19 year s old, the body weight was set to 67.3 kg for

dietary exposures (NHANES/WWEIA); the body wei ght for youths 11-16 years old was set to

57 kg for residential exposures (Exposure F actors Handbook, Table 8-3, mean body weight for

the 11 to < 16 year old age group).

The following scenarios were evaluated: diet ary exposure to the oxon exposures via drinking

water (24-hour and 21-day exposures for infants, children, youths, and female adults); exposure

to chlorpyrifos exposures via food (24-hour and 21-day exposures for infants, children, youths,

and female adults); 21-day reside ntial exposures to chlorpyrifos via skin for children, youths, and

female adults; 21-day residential exposures to chlorpyrifos via hand- to-mouth ingestion for

children 1- 2 years old; 21-day residential exposur es to chlorpyrifos via inhalation for children 1

-2 years old and female adults.

Dietary exposure was estimated for 7 days/week . For the dietary cases in which people are

exposed to oxon via drinking water, the daily water consumption volume was set to 0.688557 L

for infants, children between 1-2 year old, a nd children 6-12 years old, and 1.71062 L for youths

13-19 years old and female adults . Infants and children were assumed to consume water six

times a day; youths and female adults were assu med to consume water four times a day. For the

dietary cases in which people are exposed to ch lorpyrifos via food, the eating event was set to

one meal per day. The daily volumes consumed a nd number of daily consumption events for all

populations are mean values by age group based on USDA What We Eat in America, NHANES

survey for dietary exposures. The mean daily water consumption for children 1- 2 years old,

0.35 L, and children 6-12 years old, 0.58 L, wa s less than that for the infants, 0.688557 L;

however, the infant daily water consumption volum e was selected to be protective for PBPK-PD

PoD derivation for these age groups. For yout hs 13-19 years old, the mean daily water

consumption, 0.93 L, was less than that for the female adults; however, the adult daily water

consumption was also selected to be protective.

For all residential dermal exposures to chlor pyrifos, the fraction of skin in contact with

chlorpyrifos was set to 50%. A daily shower (i.e., washing off the chlorpyrifos) was assumed

following chlorpyrifos exposure. All residential exposures were se t to be continuous for 21 days.

For residential exposures via golfing on treated tu rf, the daily exposure time is assumed to be 4

hours/day; for residential exposures via contact with turf follow ing public health mosquitocide

application, the daily exposure dur ation is assumed to be 1.5 hour s. For residential inhalation

exposures following public health mosquitocide application, the exposure duration was set to 1

hour per day for 21 days. The exposure times se lected are based on those recommended in the

2012 Residential SOPs.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 64 of 531 In addition to dietary and resi dential exposures, the PBPK-PD model was also used to estimate

exposure levels resulting in 10% RBC AChE inhi bition following occupational exposures (Table

4.8.4). Dermal exposures for workers assumed ev en distribution across th e entire body surface

area. A daily shower (i.e., washing off the ch lorpyrifos) was assumed following chlorpyrifos

exposure. The worker was assumed to be a female adult between the ages of 13 to 49, and had a

body weight of 69 kg. This worker is exposed to ch lorpyrifos either via i nhalation or skin for 8

hours/day, 5 days/week, for a total of 21 days.

# Ch l orpyrifos Human Health Risk Assessment D424485

Table 4.8.4. Chlorpyrifos PBPK Modeled D o ses (PoDs) Corresponding to 10% RBC AChE Inhibition

Chi l dren Youths

Infants Young C hil d r e n (Resident i a J :6- 11 years (Residential: 1 1

- 16 Females

Exposu r e 

# ( < 1 yr old) (I -2 years old) old; years o l d; ( 1 3

- 4 9 years old)

RA Type Pathway Dietary:6

- 12 years o l d) Dietary: 13

- 19 years

(all chlorpyrifos o l d)

unless note4)

S t eady State Steady Steady Steady Steady

Acute Acute State Acute State Acute State Acute State (21 day) (2 1 day) (2 1 day) (21 day) (21 day)

D i

e tar y Drinking Water 1

, 183 217 3,004 548 7,700 1 ,3 58

. 4,988 878 5,285 932 (oxon cone

, ppb)

Food (ug/kg/day) 600 103 581 99 530 90 47 5 80 467 78

Residentia l Dermal 25 ,750 13,950 1 1,89 0 (Golfers) (ug/kg/day)

Dermal 1 34,250 23,600 (ug/kg/day)

Residential Oral 1 01 (Mosq uitoc i de ( u g/kg/day)

Application) Inhalation

(concn

. in air 2.37 6

. 15

mg/m3)

Dermal 3,630 (ug/kg/day) Occupational Inhalation

( ug/kg/da y) 138

*PoDs and exposure and nsk estimates for females 13-49 yrs covers all youths> 13 yrs



# Page 65 of 531

# Chlorpyrifos Human Health Risk Assessment D424485



# 4.8.4.2 Intra -s pecies extrapolation

With respect to intra-species extrapolation, the PBPK

- PD model can be run in 'variation' mode

which allows for age-specific parameters to vary across a distribution of values. The model will

not be d escr ibed in de ta il here as it is describ ed i n multiple recent publications, includ in g a

deta il ed report reviewe d by th e FIFRA SAP in 2011; summary in formation is prov i ded here

. A ll

model code for the PBPK

- PD variation model are available to the public.

Significant improvements have been made to the PBPK-PD model in response to the 2008,2011,

and 2012 SAPs, the agency, and p eer reviewers from academic journals in addition to the input

of new data. At the 2011 SAP; the panel was critical of some aspects of how Dow proposed to

assess intra

- spec ies extrapolation. DAS made multiple changes, including the addition of a

global sensitivity analysis, improvements to the quantitative approach to evaluating population

variability across i ndividua l s at a g i ve n age, a nd an uncertainty analysis on me t abo li sm data from

human hepatic microsomes to address variation in response that occurs from metabo li sm. In

addition, Dow performed additiona l analysis l eadi n g to further refinement of th e derivation of

intra

- spec ie s extrapolation across multiple dose levels by incorporating human variat i on i n 12

additional parameters in the**pr~dictions** 

# ofRBC AChE responses to chlorpyrifos exposures.

Of the more than 120 parameters in the PBPK-PD model, 16 parameters were selec ted for

varying in the DDEF intra

- species analysis. They were selected using local and global

sensit i vity ana l yses (MRID 49248201

, Dow

, 20 14a,b )

. The distributions for these 16 are

provided in Table 4.8 .4 .2 (below). Interindividual variations for the 16 sensitive parameters

(listed above) were assumed to follow a logno rmal distribut i on. The distributions are truncated

at far extreme values only to permit the model to compute but fu nc t ionall y not truncated with

respect to assessing human variab il ity

. References cited in th e table are listed in the report

"Development of Chemical Specific Adjustment Factors for Chlorpyrifos and Chlorpyrifos

Oxon" (MRID number 49248201) and also provided in Dow, 201 4a ,b,c

.

hepatic CYP450 activation of 

# total blood

chlorpyrifos to chlorpyrifos oxon 

# volume

i 

# lnn;n•ic PONJ detoxification of

· 

# h e pat ic blood**P~!o._rp yr_i,[** **o_~ C!~.<?.!.!.H!.** **T.9...f..J!.** 

# flow

-M-'"""' '"""""""""'"'""' 

# PONJ detoxification of chlorpyrifos 

# RBCAChE

oxon to TCPy in plasma 

# inhibition

rate



# I hepatic PONJ detoxification of 

# hematocrit

chlorpyrifos oxon to TCPy

!

!



# - -·-·-·--·--· ·· -·-· · - ·· - ·

- ·· - ·· - - -· · - · 

# RBCChE

degradation rate

RBCChE

reactivation rate

........................................



# intestina l CYP

bioactivation to**c~}g~J?.Y!. i.** **f.<::> ~**  

# ...**g~g ~**  

# ......................... intestina l CYP

detoxification to

TCPy 

# I .. ................. 

# transfer rate of chi orpyrifos or

mach to th e axon from the sto

int estine

..... . ............. 

# volume of the

. liver

. ......................... .. .. .. 

# asal hepat ic carboxy l b

activity rate

.............. 

# _**~** 

# . .. .. . . 

# activation



# - 1

hepatic carboxyl re



# I ra te



# .......... ............................... J

Of these 16

, four metabolism-related parameters (hepatic CYP450 activation of chlorpyrifos to



# chlorpyrifos oxon, hepatic CYP450 

# detoxifica t ion of chlorpyrifos ox on to TCP y, hepatic PON 1

detoxification of chlorpyrifos oxon to TCPy, PONl detoxification of chlorpyrifos axon to TCPy

in plasma) were found to drive more than 80% of the total va riation in RBC AChE inhibition.

Page 66 of 531

# Chlorpyrifos Human Health Risk Assessment D424485

Page 67 of 531 The human variability for these four parameters were assessed using in vitro data from 30 human

hepatic microsome samples and 20 human plasma samples (Smith et al., 2011). Twenty of the

hepatic microsome samples came from individuals < 12 years of age; and 10 of the samples

came from adults > 17 years old. Ten of the plas ma sample came from individuals < 2 years of

age; and 10 of the samples came from adults. Because the findings from Smith et al (2011)

account for more than 80% of th e total variation in RBC AChE i nhibition, it was determined that

evaluating the uncertainty associated with the da ta (i.e., small number of samples compared to

the large U.S. population) from this study was important to having confidence in the DDEFs

derived from the variation model. Although some other in vitro studies shown in Table 4.8.4.2

also have small numbers of samples, these parame ters make relatively small contributions to the

overall variability. As such, additional quantitative uncertainty analysis on these in vitro studies

is not needed.

The uncertainty associated with these four cr itical parameters were incorporated in the

subsequent Monte Carlo analys is by generating 50 sets of unbounded parametric distributions

using the following approach. First, the parame tric bootstrap approach was used to sample 1000

values, with replacement, from the in vitro data. Then, this process was repeated for 50

iterations, and the resulting 50 sets of distribution all have equa lly probable sets of means and

coefficient of variation as the observed data, except for the coefficient of variation of the plasma

PON1 metabolism rate. Since the liver is the origin of PON1 in plasma, the variation of the

plasma PON1 metabolism rate was set to be th e same as the hepatic PON1 metabolism rate.

Even though the distributions have similar means and coefficient of variation as the observed

data, they included values outside of the range of the observed data because the distributions

were assumed to be unbounded. These 50 sets of distributions, for each of the four parameters,

were found to cover the entire range of the observed data; and the ratios of maximum value to

minimum value in the simulated distributions were at least three times the ratios of maximum

value to minimum value in the observed data.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 68 of 531 Table 4.8.4.2 Sixteen parameters in variation model. Extracted from Dow, 2014c

Parameter Mean value Standard

Deviation CV Variability Reference

Total Blood Volume (L/kg body

weight) 0.08 0.0022 0.027 P 3 M; Price et al., 2003

Plasma PON1 (μmol/hr×L) 162000 92000 0.57 Smith et al., 2011

Hepatic Blood Flow (L/hr×kg

tissue) 50 14 0.27 Materne et al., 2000

RBC ChE Inhibition Rate

(l/μmol×hr) 100 17 0.17 Dimitriadis and

Syrmos,

2011

Hepatic PON1 (μmol/hr×kg

tissue) 154000 88000 0.57 Smith et al, 2011

Hematocrit (%) 0.45 0.031 0.068 P 3 M; Price et al., 2003

RBC ChE Degradation Rate (l/hr) 0.01 0.0014 0.14 Chapman et al., 1968

Hepatic P450 Bioactivation to

Oxon (μmol/hr×kg tissue) 690 410 0.59 Smith et al., 2011

Hepatic P450 Detoxification to

TCPy (μmol/hr×kg tissue) 1500 800 0.53 Smith et al., 2011

RBC ChE Reactivation Rate

(l/hr) 0.014 0.0050 0.36 Mason et al., 2000

Intestinal CYP Bioactivation to

Oxon (μmol/hr×kg tissue) 82 43 0.52 Obach et al., 2001

Intestinal CYP Detoxification to

TCPy (μmol/hr×kg tissue) 53 28 0.52 Obach et al., 2001

Transfer Rate to Intestine (hr-1) 0.31 0.081 0.26 Singh et al., 2006

Volume of the Liver (L/kg body

weight) 0.032 0.0010 0.032 P 3 M; Price et al., 2003

Hepatic Carboxyl Basal Activity

Rate (l/hr/kg tissue) 1270000 460000 0.36 Pope et al., 2005

Hepatic Carboxyl Reactivation

Rate (l/hr) 0.014 0.0050 0.36 Mason et al., 2000

# Chlorpyrifos Human Health Risk Assessment D424485

Page 69 of 531 According to EPA’s Data-Derived Extrapolati on Factor guidance, when calculating a DDEF

intra-species extrapolation (USEPA, 2014), administ ered doses leading to the response level of

interest (10% change in RBC AC hE inhibition) are compared between a measure of average

response and response at the tail of the distribu tion representing sensitive individuals. DAS has

conducted an analysis to derive the oral dos es that cause 10% RBC AC hE inhibition in both

adults and 6-month old infants (example provided in Figure 5a, b). The ratio of the adult ED 10 

# to

the infant ED 10 

# was then used to derive intraspecies ex trapolation factors. In the subsequent

Monte Carlo simulations, the ta rget age group is six month ol d individuals. Some model

parameters are specific to this age group (e.g., PON1 metabolism in plasma), and some

parameters are scaled by body wei ght that reflect this age grou p (e.g., tissue volume). Based on

the 5 th 

# percentile of the distributions, the DDEF fo r intraspecies extrapolation is 2.8X for

chlorpyrifos and 3.1X for the oxon (Dow, 2014b). Based on the 99 th 

# percentile of the

distributions, the DDEF for intraspe cies extrapolation is 4X for chlorpyrifos and 5X for the oxon

(Dow, 2014b). For this revised HHRA, the 99 th 

# percentile is being used to account for

sensitivities (i.e., the in tra-species factor is 4X for chlo rpyrifos and 5X for the oxon for all

groups except women who are pregnant or may b ecome pregnant). As shown in Figure 5b, at

the 99 th 

# -ile, only 1% of infants will experience 10% or greater RBC AChE inhibition at the POD.

Figure 5a 

# . Simulated population of 6 month olds for intra-species extrapolation DDEF derivation. Percent RBC

AChE inhibition fr om exposure to single oral dose s of chlorpyrifos ranging from 0.05 to 5.0 mg/kg/day (X and Y

axes provided on the log scale).

0 10 20 30 40 50 60 70 80 90 100

0.05 0.50 5.00Percentinhibitioncmparedtobacground



# Single Oral Dose (mg/kg)

# Chlorpyrifos Human Health Risk Assessment D424485

Page 70 of 531 Figure 5b 

# . Simulated population of 6 month olds for intra-species extrapolation DDEF derivation. Percent RBC

AChE inhibition fr om exposure to single oral doses of chlorpyrifos ranging from 0.05 to 1.0 mg/kg/day. Green lines

represent the infant acute PoD for chlorp yrifos, the PoD adjusted for the 3X an d 4X intraspecies factors for the 95

and 99 th 

# -%ile, respectively.



# In summary, for the chlorpyrifos HHRA, the PBPK -PD model has been used to derive PODs for

RBC AChE inhibition for va rious populations, durations, and r outes (Table 4.8.4). As such, the

interspecies factor is not needed. To account for variations in sensit ivities, an intra-species factor

of 4X for chlorpyrifos and 5X for the oxon is applied for all groups except women of

childbearing age. For women of childbearing age, the typical 10X intra-species factor is being

applied due the lack of appropr iate information and algorithms to characterize physiological

changes during pregnancy. The 10X FQPA SF is being applied to women of childbearing age,

infants, youths,

# Chlorpyrifos Human Health Risk Assessment D424485

Page 71 of 531 

# 5.0 Dietary Exposure and Risk Assessment

5.1 Residues of Concern Summary and Rationale

The qualitative nature of the residue in plants and livestock is adequa tely understood based on

acceptable metabolism studies with cereal grain (cor n), root and tuber vegetable (sugar beets),

and poultry and ruminants. The residue of concer n, for tolerance expression and risk assessment,

in plants (food and feed) and livestock commodities is the parent compound chlorpyrifos.

Based on evidence (various crop field trials and metabolism studies) indicating that the

metabolite chlorpyrifos oxon would be not be presen t in edible portions of the crops (particularly

at periods longer than the currently registered PHIs), it is not a re sidue of concern in food or feed

at this time. Also, the chlor pyrifos oxon is not found on samp les in the USDA PDP monitoring

program. In fact, from 2007 to 2012, out of seve ral thousand samples of various commodities,

only one sample of potato showed presence of the oxon at trace levels, 0.003 ppm where the

LOD was 0.002 ppm, even though there are no regist ered uses of chlorpyrifos on potato in the

U.S.

The oxon metabolite was not found in milk or live stock tissues in cattle and dairy cow feeding

studies, at all feeding levels te sted, and is not a residue of co ncern in livestock commodities.

Oxidation of chlorpyrifos to chlorpyrifos oxon could potentially occur through photolysis,

aerobic metabolism, and chlorination as well as other oxidative processes. Because of the

toxicity of the oxon and data indicating that chlorpyrifos rapidly converts to the oxon during

typical drinking water treatment (chlorination), the drinking water risk assessment considers the

oxon as the residue of concern in treated dr inking water and assumes 100% conversion of

chlorpyrifos to chlorpyr ifos oxon (see DWA, D424487).

The chlorpyrifos degradate TCP is not considered a residue of concern for this assessment as it

does not inhibit cholinesterase (a separate human health risk assessment has been performed for

TCP, which has its own toxicity database. TCP (derived from triclopyr, chlorpyrifos, and

chlorpyrifos-methyl) was previously assessed on 6/6/2002 (W. Donovan, D283101)).

Table 5.1 Summary of Metabolites and Degradates to be included in the Risk Assessment and

Tolerance Expression

Matrix Residues included in Risk

Assessment Residues included in

Tolerance Expression

Plants Primary Crop Chlorpyrifos Chlorpyrifos

Rotational Crop Chlorpyrifos Chlorpyrifos

Livestock Ruminant Chlorpyrifos Chlorpyrifos

Poultry Chlorpyrifos Chlorpyrifos

Drinking Water Chlorpyrifos Oxon Not Applicable

# Chlorpyrifos Human Health Risk Assessment D424485

Page 72 of 531 

# 5.2 Food (Residue Chemistry) Profile

As mentioned in the 2011 assessment, newer crop field trial studies had been submitted for

cotton gin byproducts (MRI D 46651202), tart cherries (MRID 46651201), aspirated grain

fractions for soybean, sorghum and wheat (MRID 46640901), and grass forage and hay as part of

a data call in related to the 2002 chlorpyrifos IRED. These st udies were reviewed previously and

regulatory conclusions are included here for al l the commodities with the exception of grass

forage and hay (original petition w ithdrawn). The data are consider ed adequate to conclude that

tolerances of 15 ppm and 22 ppm would cover any residues of chlorpyrifos on cotton gin byproducts and aspirated grain frac tions, respectively, and would suppor t the current to lerance level

for tart cherries under the condition that only dormant/del ayed dormant and trunk spray

applications are allowed on the label of the 75% WDG end use product.

The dietary burden to livestock was recalculated to consider residues at tolerance level in the

feedstock commodities (aspirated grain fractions and cotton gin byproducts) and to use the most

current version of Table 1 of the OPPTS Te st Guidelines 860.1000, released on June 2008.

Based on the residues observed in the feeding study of cattle beef, dair y cow, swine and poultry

at the 1x level or higher, HED concludes th at the possible residues observed on livestock

commodities (from animals fed with feedstock th at may contain residues resulting from legal

applications) are covered by the current to lerances established in the 40 CFR §180.342.

There are some outstanding residue chemistry da ta requirements for chlorpyrifos. A magnitude

of the residue study to es tablish a tolerance for wheat hay wa s required in the previous RED and

has not been received. For soybean, processing studi es are required to determine if tolerances are

needed for chlorpyrifos in soybean meal, hulls and refined oil.

Studies submitted to support the registration of a microencapsulated formulation of chlorpyrifos

showed over-tolerance residues af ter a foliar applicati on of Lorsban 4E end use product to lemon

with a rate of 6 lb ai/A. The maximum resi due observed was 1.41 ppm while the tolerance for

citrus fruit (CG 10) is 1.0 ppm. The label of the existing Lorsban 4E end use product (44.9%

chlorpyrifos) allows a maximum application rate of 6.4 lb ai/A and addition of oil to the spray

mixture. Under these use conditions residues over tolerance may occur. Magnitude of the

residue studies are needed w ith lemon after application of Lorsban 4E and 75% WDG

formulations separately in order to reevalua te the tolerance for the citrus fruit crop group.

Since the 2011 preliminary risk assessment, co tton processing studies have been submitted

(MRID 00037455). The cotton processing study is considered scientif ically acceptable.

Following 16 broadcast foliar applica tions of the 4 lb ai/gal EC formulation of chlorpyrifos made

at a total rate of 13.0 lb ai/A using aerial equipment, quantifia ble (trail average) residues of

chlorpyrifos were 0.103 ppm in/on cottonseed and 0.074 ppm in hulls harvested at a 18-day PHI.

Residues of chlorpyrifos were nonquantifiable (<0.01 ppm) in solvent extracted meal and

refined-bleached oil. A comparison of the re sidues in cottonseed w ith those in processed

cottonseed fractions indicated that residues of chlorpyrifos do not concentrate in hulls

(processing factor of 0.7x), solven t extracted meal (<0.1x), or refined-bleached oil (<0.1x).

Separate tolerances are not needed for the cotto n processed commodities. This data requirement

is considered fulfilled.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 73 of 531 A tolerance was previously established for chlor pyrifos in wheat milling fractions at 1.5 ppm.

Also, for corn milled byproducts a tolerance of 0.1 ppm was previously recommended based on

concentration factors from 1.25x in grits to 2x in flour (D188151, S. Knizner, 8/20/1993).

Tolerances for residues of chlorpyrifos on wh eat milled byproducts (1.5 ppm) and corn milled

byproducts (0.1 ppm) should be included in the 40 CFR §180.342 as the current tolerance levels

on the associated raw agricultural commodities, wh eat grain and corn grain, are insufficient to

cover any residues that may occu r on these livestock feedstuffs.

[See Negrón-Encarnación, 5/24/11, D388164, Chlorpyrifos. Registration Review Action for

Chlorpyrifos. Summary of Anal ytical Chemistry and Residue Data for complete details

regarding label revision recommendations, ch emistry data requirements and tolerance

recommendations].



# 5.3 Water Residue Profile

EFED has provided a revised drinking water as sessment (DWA) for chlorpyrifos (Bohaty, R.,

12/23/14, D424487, Chlorpyrifos: Updated Drinking Wate r Assessment for Registration Review )

which includes the EDWCs that are used in the aggregate assessment. The DWA also serves as

the human health aggregate risk assessment since the EDWCs are compared to the DWLOC

within that document.

See the Aggregate section 7.0 below for a su mmary of the DWA including the aggregate

assessment results (comparison of DWLOCs to EDWCs). For complete details on the

assumptions, results, and characterization of the drinking water and aggreg ate assessment refer to

EFED’s DWA (2014, D424487).



# 5.4 Dietary (Food Only) Risk Assessment

The general approach for the chlorpyrifos expos ure and risk assessment can be described as

follows: The PBPK-PD model was used to predic t acute (24 hour) and steady state (21 day)

points of departure dose levels (PoDs) wh ich correspond to 10% RBC ChEI for the index

lifestages relevant to chlorpyrifos risk assessmen t (children of various ages which differ due to

exposure pattern, and adult females of childbearing age). The PoDs are then divided by the total

uncertainty factor to determine the population adjusted dose (PAD). For food, the residue of

concern is chlorpyrifos (the oxon metabolite is not an expected residue on foods). The

chlorpyrifos total uncertainty factors are 100X for adult females (10X FQPA SF and 10X intraspecies extrapolation factor) a nd 40X for the other populations (10X FQPA SF and 4X intraspecies extrapolation factor). Th e chlorpyrifos exposure values resulting from dietary modeling

are compared to the PAD. There are potential risks of concern when estimated dietary risk

exceeds 100% of the PAD.

For the dietary risk assessme nt for food only, the exposure va lues resulting from Dietary

Exposure Evaluation Model (DEEM) and the Cale ndex model are compar ed to the PBPK-PDbased acute PAD and steady state PAD, respectiv ely. When estimated dietary risk estimates

exceeds 100% of the PAD there may be a risk concern.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 74 of 531 

# 5.4.1 Description of Residue Data Us ed in Dietary (Food Only) Assessment

Acute and steady state dietary (food only) exposure analyses fo r chlorpyrifos were conducted

using the Dietary Exposure Evaluation Model (DEEM) and Calendex software with the Food

Commodity Intake Database (FCID) (D. Drew, 11/18/14, D424486, Chlorpyrifos Acute and

Steady State Dietary (Food Only) Exposure Analysis to Support Registration Review ). This

software uses 2003-2008 food cons umption data from the U.S. Department of Agriculture’s

(USDA’s) National Health and Nutrition Exam ination Survey, What We Eat in America,

(NHANES/WWEIA). The most recent previous dietary assessment was performed in 2011 to

support chlorpyrifos re gistration review (D. Soderberg, 6/30/11, D388166, Chlorpyrifos: Revised

Acute (Probabilistic) and Chronic Dietary Expos ure and Risk Assessments for Food Only (with

and without Food Handling Use included) and for Water Only for the Registration Review

Action – Typical Use Rates/Water Included) . This current analyses reflect the latest consumption

data as well as more recent food monitoring a nd percent crop treated data. These analyses was

performed for the purpose of obtaining food exposur e values for comparison to the chlorpyrifos

doses predicted by the PBPK-PD model to caus e RBC ChEI. The acute and steady state exposure

analyses do not include drinking wa ter which is assessed separately as discussed in Section 7.

All residues in food are assumed to be parent chlorpyrifos since the chlorpyrifos oxon is not

typically found in foods in monitoring data or crop field trials. Food exposures are based only

upon field and livestock use of ch lorpyrifos and do not incorporat e potential expos ure from food

handling establishment (FHE) uses since no active registered FHE uses have been identified by

BEAD. The previous (2011) diet ary risk assessment did include a chronic analysis for FHE use

based on <2% establishments treated (BEAD could not confirm that there was any actual usage

although there was a registered use at the time) and half the analytical limit of detection (1⁄2 LOD;

0.01 ppm) based on all nondetectable residues in a chlorpyrifos FHE study. The exposure from

any potential FHE uses may be considered neglig ible compared to exposures from field uses.

Both the acute and steady state dietary exposure analyses are highly refined. The large majority

of food residues used were based upon U. S. Department of Agricultu re’s Pesticide Data

Program (PDP) monitoring data except in a few instances where no appropriate PDP data were

available. In those cases, field trial data or tolerance level residues were assumed. The same data

were used for both the acute and steady state analyses. BEAD of OPP provided percent crop

treated information. Food processing factors fr om submitted studies were used as appropriate.



# 5.4.2 Percent Crop Treated Used in Dietary Assessment

The acute and steady state dietary exposure assessment used percent crop treated information

from BEAD’s Screening Level Usage Analysis ( SLUA; May 1, 2014).



# 5.4.3 Acute Dietary (Foo d Only) Risk Assessment

Chlorpyrifos acute (food only) dietary exposure assessments we re conducted using the Dietary

Exposure Evaluation Model software with th e Food Commodity Intake Database DEEMFCIDTM, Version 3.16, which incorporates consump tion data from USDA’s National Health and

# Chlorpyrifos Human Health Risk Assessment D424485

Page 75 of 531 Nutrition Examination Survey, What We Eat in America, (NHANES/WWEIA). This dietary

survey was conducted from 2003 to 2008. Acute dietar y risk estimates are presented below for

the sentinel population subgroups fo r acute risk assessment: infants (< 1 year old), children (1-2

years old), youths (6-12 years ol d) and adults (females 13-49 year s old). The assessment of these

index lifestages will be protective for the other population subgroups.

Acute dietary (food only) risk estimates are all <100 % of the acute PAD for food (aPAD food 

# ) at

the 99.9 th 

# percentile of exposure. Th e subgroup with the highest risk estimate was females (13-49

years old) at 3.2 % aPAD food



# .

Table 5.4.3 Acute Dietary (Food On ly) Exposure and Risk Estimates for

Chlorpyrifos

Population

Subgroup aPoD food 1



# (ug/kg/day) aPAD food 2



# (ug/kg/day) Food

Exposure 3



# (ug/kg/day) % of aPAD food



# Infants

(< 1 yr) 600 15 0.273 1.8

Children

(1-2 yrs) 581 14 0.423 3.0

Youths

(6-12 yrs) 530 13 0.189 1.4

Adults

(Females 13-

49 yrs) 469 4.7 0.150 3.2

1 

# acute point of departure; daily dose predicted by PBPK-P D model to cause RBC ChEI of 10% for acute dietary (food)

exposures.

2 

# aPAD= acute population adjusted dose = Po D (Dose predicted by PBPK-PD model to cause 10% RBC ChEI) ÷ total UF; Total

uncertainty factor =100X for females 13-49 yrs (10X intraspeci es factor and 10X FQPA uncertainty factor) and 40X for other

populations (4X intraspecies factor and 10X FQPA uncertainty factor).

3 

# Acute food only exposure estimates from DEEM (at 99.9 th 

# percentile). Refined with monitoring

data and %CT.



# 5.4.4 Steady State Dietary (F ood Only) Risk Assessment

A chlorpyrifos steady state diet ary (food only) exposure analysis was conducted using CalendexFCIDTM. HED’s steady state assessment consider s the potential risk from a 21-day exposure

duration using a 3-week rolling av erage (sliding by day) across the ye ar. For this assessment, the

same food residue values used in the acute assessment were used for the 21-day duration. In the

Calendex software, one diary for e ach individual in the WWEIA is selected to be paired with a

randomly selected set of residue values for each food consumed. The steady state analysis

calculated exposures for the sen tinel populations for infant, chil d, youths, and adult (infants <1

yr, children 1-2 yrs, youths 6-12 yrs, females 13-49 yrs).

Calendex reported dietary exposures for each pop ulation subgroup at several percentiles of

exposure ranging from 10 th 

# percentile to 99.9 th 

# percentile. The dietary (food only) exposures for

# Chlorpyrifos Human Health Risk Assessment D424485

Page 76 of 531 chlorpyrifos were all <100% ssPAD food 

# (all populations, at all percen tiles of exposure). Only the

99.9 th 

# percentile of exposure is presented in Table 5.4.4 below. Calendex results for other

percentiles of exposure can be found in D424486.

For the steady state dietary (food only) exposure analyses, children (1-2 years old) was the

population subgroup with the highest ri sk estimate at 9.7% of the ssPAD food 

# at the 99.9 th



# percentile of exposure.

Table 5.4.4 Steady State Dietary (Food Only) Exposure and Risk

Estimates for Chlorpyrifos

Population

Subgroup SS 

# PoD food 1



# (ug/kg/day) ssPAD food 2



# (ug/kg/day) Food

Exposure 3



# (ug/kg/day) % of

ssPAD food



# Infants

(< 1 yr) 103 2.6 0.186 7.2

Children

(1-2 yrs) 99 2.5 0.242 9.7

Youths

(6-12 yrs) 90 2.2 0.128 5.8

Adults

(Females 13-

49 yrs) 78 0.78 0.075 9.6

1 

# Steady state point of departure; daily dose predicted by PBPK -PD model to cause RBC ChEI of 10% for steady state (21 day)

dietary (food) exposures.

2 

# ssPAD= Steady state populatio n adjusted dose = PoD (Dose predicted by PBPK-P D model to cause 10% RBC ChEI) ÷ total UF;

Total uncertainty factor =100X for females 13-49 yrs (10X intraspecies factor and 10X FQPA uncertainty factor) and 40X for

other populations (4X intraspecies factor and 10X FQPA uncertainty factor).

3 

# Steady state (21 day) food only expos ure estimates from Calendex (at 99.9 th 

# percentile). Refined with monitoring data and %CT.



# 6.0 Residential (Non-Occupational) Exposure/Risk Characterization

The 2011 residential assessment has been updated 23 

# to reflect the use of the PBPK-PD modeling

approach and EPA’s SOPs for Residential Pest icide Exposure Assessment (USEPA, 2012). In

1997, the registrant, Dow AgroSciences, voluntarily agreed to cancel chlo rpyrifos registrations

for indoor broadcast use and direct pet treatme nts, except pet collars. In December 2001, the

majority of the remaining chlorpyrifos reside ntial products were subj ect to voluntary phase

out/cancellation. Current chlorpyrifos resident ial uses include a granular ant mound use

(commercial applicator only) and roach bait in child-resistant packaging (homeowner

applicator). Additiona lly, chlorpyrifos is labeled for publ ic health aerial and ground-based

fogger ULV mosquito adulticide applications and for golf course turf applications. For the

purpose of residential exposure assessment, the pa rent compound chlorpyrifos is the residue of

concern.

This assessment also includes a retrospective do se reconstruction of the exposures which could

23 

# W. Britton. Chlorpyrifos: Updated O ccupational and Residential Exposure Assessment for Regi stration Review.

12/29/2014. U.S. EPA Office of Chemical Safety and Pollution Prevention. D424484.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 77 of 531 have potentially occurred as a result of previous (now cancelled) chlorpyrifos uses in indoor

residential environments (see Section 4.5.4).



# 6.1 Residential Handler Exposure

HED uses the term “handlers” to describe those individuals who are involved in the pesticide

application process. HED believes that there are distinct tasks related to applications and that

exposures can vary depending on the specifics of each task. Residen tial (non-occupational)

handlers are addressed somewhat differently by HED as homeowners are assumed to complete

all elements of an application wit hout use of any prot ective equipment.

Based upon review of all chlorpyrifos registered uses, only the roach bait products can be applied

by a homeowner in a residential setting but the a pplication of roach bait products has not been

quantitatively assessed because these exposures ar e negligible and do not pose a risk concern.

The roach bait product is designed such that the active ingredient is c ontained within a bait

station which eliminates the potentia l for contact with the chlorpyrif os-containing bait material.



# 6.2 Residential Post-Application Exposure

There is the potential for post-application exposure s as a result of being in an environment that

has been previously treated with chlorpyrifos

. Chlorpyrifos can be used in areas frequented by

the general population including golf courses. It is also applied as an aerial and ground-based

ULV mosquito adulticide applicatio ns directly in residential areas . These uses were previously

assessed in the 2011 preliminary risk assessment. Post-application exposure from residential ant

mound treatment is not quantitatively assessed as e xposures are considered to be negligible and

do not pose a risk concern; these products can only be applied pr ofessionally and direct exposure

with treated ant mounds is not anticipated.

The residential post-application exposure scenarios and applica tion rates assessed in 2011 remain

unchanged. However, the post-application exposur e assessment has been updated to reflect the

following changes: 1) use of the PBPK-PD model for determining toxicological PoDs, 2) use of

the 2012 Standard Operating Procedures for Resi dential Pesticide Ex posure Assessment 24



# , 3)

use of the AgDISP model for estimation of airb orne concentrations a nd residue dissipation

following chlorpyrifos mosquito adulticid e applications, 4) updated methodology for

determining the airborne concen tration of active ingredient following ground-based mosquito

adulticide applications, and 5) use of updated body weights for all residential populations

assessed.

In addition, steady state durations of exposure are assumed in the updated residential assessment.

The steady state endpoint selection for chlorpyr ifos overlaps HED’s tr aditional short-term

exposure duration endpoint selecti on and is considered health protective for both short- and

intermediate- term exposures (additional explanation is av ailable in section 4.8.1 above).

The quantitative exposure/risk a ssessment for residential post-a pplication exposures is based on

24 

# http://www.epa.gov/pesticides /science/USEPA-OPP-HED_Resi dential%20SOPs_Oct2012.pdf

# Chlorpyrifos Human Health Risk Assessment D424485

Page 78 of 531 the following scenarios:

Golf Course Use (Emulsifiable concentrat e (EC) and Granular (G) formulations)

 Children 6 to < 11 years old, youths 11 to < 16 years old, and a dult post-application

dermal exposure from contact with treated turf while golfing.

Public Health Mosquito Adulticide Use (aerial and ground applications)

 Children 1 to < 2 years old and adult post-a pplication dermal expos ure from contact with

turf following the deposition of chlorpyrifos residues from public health mosquito

adulticide application.

 Children 1 to < 2 years old and adult post-a pplication inhalation exposure from airborne

chlorpyrifos following public health mosquito adulticide application.

 Children 1 to < 2 years old pos t-application incidental or al (hand-to-mouth) exposure

from contact with turf following the depos ition of chlorpyrifos residues from public

health mosquito adulticide application.

 Children 1 to < 2 years old pos t-application incidental oral (object-to-mouth) exposure

from contact with toys containing residue s from turf following the deposition of

chlorpyrifos residues from public health mosquito adulticide application.

A series of assumptions and exposure factors serv ed as the basis for completing the residential

post-application risk assessment and have been de scribed in brief. The assumptions and factors

are described in detail in the updated occupationa l and residential exposur e and risk assessment

(D424484).

Exposure Duration: Residential post-application exposures to chlorpyrifos are assumed to be

steady state (i.e., 21 da ys or longer).

The application of mosquitocide in residen tial areas may result in the potential for postapplication inhalation exposures. The aerosolized particulate re maining following application is

assumed to persist for no longer than one hour in proximity to the application source and,

accordingly, would be most appropriately defi ned as acute in duration. However, this

assessment assumes that post-applic ation inhalation exposures are st eady state, which is a highly

conservative approach given how infrequently mos quitocides are repeatedly applied to the same

locations and how rapidly aerosols dissipate after these types of applications. The parameters

used to define this exposure scenario in the PBPK-PD model conservatively reflect daily, one

hour exposures for 21 days.

Application Rates: In order to seek clarifi cation of chlorpyrifos usage, the Agency compiled a

master use summary document reflective of the use profile of all active product labels. The

document, among other information, presents al l registered uses of chlorpyrifos and

corresponding maximum single applic ation rates, equipment types, re-entry inte rvals (REIs),

etc. This assessment assumes that the detailed information on application rates and use patterns

presented in Appendix 9 (Master Use Summ ary Document) will be implemented on all

chlorpyrifos labels and is the basis of the occ upational and residential ri sk assessment. If, for

any reason, the final chlorpyrifos labels contain higher application rates, the actual risks posed by

# Chlorpyrifos Human Health Risk Assessment D424485

Page 79 of 531 those products may exceed the risks estimated in this assessment.

Body Weights: The body weights assumed for this asse ssment differ from those used in 2011

residential exposure assessment and are based on the recommendations of the 2012 Residential

SOPs. These body weights are the same as select ed for derivation of PBPK-PD PoDs for use in

assessment of residential exposures.

The standard body weights are as follows: youths 11 to < 16 years old, 57 kg; children 6 to < 11

years old, 32 kg; and children 1 to < 2 years old, 11 kg. For adults when an endpoint is not sexspecific (i.e., the endpoints are not based on deve lopmental or fetal effects) a body weight of 80

kg is typically used in risk assessment. However, in this case, a female-specific body weight of

69 kg was used. While the endpoint of concern, RBC AChE inhibition, is not sex-specific, the

female body weight was used due to concerns for neurodevelopmenta l effects related to early life

exposure to chlorpyrifos.

Post-application exposures from golfing have be en assessed using the 2012 Residential SOPs

and with use of exposure data from a chemical-s pecific turf transferab le residue (TTR) study.

The study was conducted with an emulsifiable c oncentrate, a granular, and a wettable powder

formulation. Only the emulsifiable concentrate and granular data were used because there are no

currently registered wettable powder formulations. The study was conducted in 3 states,

California, Indiana and Mississipp i, with use of the emulsifiable concentrate and wettable

powder formulations. Exposure was estimated by normalizing Day 0 TTR measures from study

application rates to the current maximum applica tion rate allowable by the label. Chlorpyrifos

oxon residues were not analyzed.

The post-application exposure potentia l from public health mosquito adulticide applications has

been considered for both ground based truck foggers and aerial applications. For assessment of

the mosquito adulticide use, the algorithms and inputs presented in the 2012 Residential SOP

Lawns/Turf section were used coupled with the available TTR data described above. The

deposition of chlorpyrifos from these applications are not based on the app lication rate alone, but

also using the AgDISP (v8.2.6) model (aerial app lications, the currently recommended model for

assessment of mosquito adulticide applications ) or empirical data (g round applications) to

determine how much pesticide is deposited on reside ntial lawns as a result of mosquito adulticide

treatments at the maximum application rates for each. The TTR data are then used to determine

the fraction of the total residue deposited following the mosqu itocide application which can

result in exposures to impacted individuals. Inhalation exposures ar e also estimated using

AgDrift for aerial application and a recently de veloped well-mixed box (WMB) model approach

for outdoor foggers.

As described above, the AgDISP (v8.2.6) model was used to estimate the deposition of

chlorpyrifos from aerial applications and the ai rborne concentration of chlorpyrifos following

public health mosquitocide application. AgDISP predicts the motion of sp ray material released

from aircraft, and determines th e amount of application volume that remained aloft and the

amount of the resulting droplets deposited on the surfaces in the treatment area, as well as

downwind from the treatment area. The mode l also allows for the estimation of air

concentrations in the breathing zones of adults and children for use in calculating the postapplication inhalation risks to indi viduals residing in areas being treated by aerial application of

# Chlorpyrifos Human Health Risk Assessment D424485

Page 80 of 531 chlorpyrifos. The aerial fraction of the mosquito adulticide applicati on rate applied (0.010 lb

ai/A) is 0.35 (i.e., 35 percent of application rate is deposited on turf); and the airborne

concentration at the breathing he ight of adults and children of chlorpyrifos 1 hour following

aerial mosquito adulticide application is 0.00060 mg/m 3



# .

Empirical data were used to derive the ground- based deposition of chlorpyrifos following public

health mosquitocide applicati on. These data, conducted by Moore et al. (1993) 25 

# and Tietze et

al. (1994 ) 26



# , measured the deposition of malath ion via ultra-low volume (ULV) ground

equipment as applied for mosquito control. Base d on these data, an off-target deposition rate of

5 percent of the application rate was used by HED to evaluate ground-based ULV applications

(i.e., 5 percent of the target application rate depo sits on turf). A value sl ightly higher than the

mean values for both studies was selected because of the variability in the data and the limited

number of data points. The adjusted applicatio n rate was then used to define TTR levels by

scaling the available TTR data as appropriate.

I 

# n order to calculate airborne c oncentrations from ULV truck f ogger applications, HED used the

2012 Residential SOPs for Outdoor Fogging/Misting Systems, with minimal modification to the

well-mixed box (WMB) model. The WMB model allo ws for the estimation of air concentrations

in the breathing zones of adults and children for use in calculating the pos t-application inhalation

exposure to individuals residing in areas being treated by ground application of chlorpyrifos.

This methodology is a modification of the previous method used in the 2011 occupational and

residential exposure assessment to evaluate post- application inhalation ex posure resulting from

truck mounted mosquito fogger. The revised methodology more accurately accounts for

dilution.

Combining Exposure and Risk Estimates

Since dermal, incidental oral, and inhalation exposure routes share a common toxicological

endpoint, RBC AChE inhibition, risk estimates ha ve been combined for those routes. The

incidental oral scenarios (i.e ., hand-to-mouth and object-to-mout h) should be considered interrelated and it is likely that they occur interspersed amongst eac h other across time. Combining

these scenarios with the dermal and inhalation exposure scenarios would be unrealistic because

of the conservative nature of each individual assessment. Therefore, the post-application

exposure scenarios that were combined for child ren 1 < 2 years old are the dermal, inhalation,

and hand-to-mouth scenarios (the highest incidental oral exposure expected). This combination

should be considered a protective estimate of children’s exposure to pesticides.

Summary of Residential Post-application Non-Cancer Exposure and Risk Estimates

The assessment of steady state golfer post-applica tion exposures (dermal only) to chlorpyrifos

treated turf for th e lifestages adults, children 6 to < 11 y ears old, and youths 11 to < 16 years old,

25 

# J.C. Moore, J.C. Dukes, J.R. Clark, J. Malone, C.F. Hallmon, and P.G. Hester. Downwind Drift and

Deposition of Malathion on Human Targets From Gr ound Ultra-Low Volume Mosquito Sprays; Journal

of the American Mosquito Control Association; Vol. 9, No. 2 (June, 1993)

26 

# N.S. Tietze, P.G. Hester, and K.R. Shaffer. Mass Recovery of Malathion in Simulated Open Field

Mosquito Adulticide Tests: Archives of Environm ental Contamination and Toxicology; 26: 473-477

(1994)

# Chlorpyrifos Human Health Risk Assessment D424485

Page 81 of 531 results in no risks of concern (i.e., children 6 to < 11 and youths 11 to < 16 years old, MOEs are

≥ 40; adults, MOEs are ≥ 100). For the assessment of post- application exposures from public

health mosquitocide applications, no combined risks of concern were identified for adults

(dermal and inhalation) and childre n 1 to < 2 years old (dermal, in cidental oral, a nd inhalation).

A summary of risk estimates is presented in Table 6.2 below

Table 6.2. Residential Post-application Non-canc er Exposure and Risk Estimates for Chlorpyrifos.

Lifestage Post-application Exposure

Scenario Application

Rate 1 

# State

(TTR

Data) Dose

(mg/kg/day) 3 

# MOEs 4 

# Combined

Routes 5 

# Combined

MOEs 6



# Use Site Route of

Exposure

Adult

(Females)

Golf Course

Turf Dermal 1.0

(Emulsifiable

Concentrate) CA 0.010 1,200

NA NA IN 0.0069 1,700

MS 0.012 1,000

Mean 0.0095 1,200

Youths 11

to < 16

years old CA 0.010 1,400

IN 0.0069 2,000

MS 0.012 1,200

Mean 0.0096 1,500

Children

6 to < 11

years old CA 0.012 1,900

IN 0.0082 2,800

MS 0.014 1,600

Mean 0.011 2,000

Adult

(Females)

1.0

(Granular) CA 0.0088 1,400

Youths 11

to < 16

years old 0.0088 1,600

Children

6 to < 11

years old 0.010 2,200

Adult

(Females)

Aerial and

Ground Based

ULV

Mosquitocide

Applications Dermal

0.010

(Aerial) MS 0.00052 46,000 X

8,400 Inhalation NA 0.00060

(mg/m 3 

# ) 10,300 X

Children

1 to < 2

years old Dermal MS 0.00088 150,000 X

2,300 Inhalation NA 2 

# 0.00060

(mg/m 3 

# ) 4,000 X

Hand-to-Mouth MS 0.000018 5,600 X

Object-to-Mouth MS 6.1x10 -6 

# 180,000 NA NA

Soil Ingestion NA 2 

# 1.3x10 -6 

# 4,900,000 NA NA

Adult

(Females) Dermal

0.010

(Ground) MS 0.000074 320,000 X

1,200 Inhalation NA 0.0051

(mg/m 3 

# ) 1,200 X

Children

1 to < 2

years old Dermal MS 0.00013 1,100,000 X

460 Inhalation NA 2 

# 0.0051

(mg/m 3 

# ) 460 X

Hand-to-Mouth MS 2.8x10 -6 

# 39,000 X

Object-to-Mouth MS 8.6x10 -7 

# 1,300,000 NA NA

Soil Ingestion NA 2 

# 1.7x10 -8 

# 34,000,000 NA NA

1 Based on the maximum applic ation rates registered for golf course tu rf and ULV mosquito adulticide uses.

2 The airborne concentrations of chlorpyrifos following ULV mos quito adulticide applications wa s determined with use of the

AgDISP (v8.2.6) model.

3 Dose (mg/kg/day) equations for golfing and mosquitocide applications are provi ded in Appendices B and C of the updated

occupational and residential expo sures assessment. For calculation of doses (i .e., dermal, hand-to-mout h, and object-to-mouth)

from exposure to ULV mosquito adulticide, TTR data was used. The MS TTR data was selected for use because it is the worst

# Chlorpyrifos Human Health Risk Assessment D424485

Page 82 of 531 case and, as a result, most protective of human health. Add itionally, the fraction of chlorpyrifos residue deposited following

mosquitocide application, 35% (0.35), was determined with use of the AgDISP (v8.2.6) model and used for dose calculation.

The fraction of chlorpyrifos deposited following ground ULV appl ication, 5% (0.050), is base d on surrogate exposure data

(malathion). For dose estimation from exposures to golfing on treated turf, on the TTR data was used. Doses have been

presented for all State sites, including the mean of all State sites.

4 MOE = PoD (mg/kg/day) ÷ Dose (mg/kg/day).

5 X indicates the exposure scenario is included in the combined MOE

6 Combined MOE = 1 ÷ (1/dermal MOE) + (1/inhalation MOE) + (1/incidental oral MOE), where applicable.

NA = Not applicable.



# 6.3 Residential Bystander Post-a pplication Inhalation Exposure

6.3.1 Spray Drift

In the 2011 occupational and residential exposure assessment the potential risks to bystanders

from spray drift were identified as possible concerns. Spray drif t is the movement of aerosols

and volatile components away from the treate d area during the appli cation process. The

chemical may then be deposited on areas such as home lawns. Post-appl ication exposures may

occur to adults (dermal) or children (dermal a nd incidental oral) during activities on treated turf.

The potential risks from spray dr ift and the impact of potential risk reduction measures were

assessed in July 2012. 27 

# This evaluation supplemented the 2011 assessment where limited

monitoring data indicate risks to bystanders. To increase protection for children and other

bystanders, chlorpyrifos technical registrants voluntarily agreed to lower application rates and to

other spray drift mitigation measures. 28 

# As of December 2012, spray drift mitigation measures

and use restrictions appear on all chlorpyrifos ag ricultural product labels. Because the use of the

PBPK-PD model impacts spray drift risks, an updated assessment was conducted.

Table 6.3.1 presents the buffer distan ces (feet) necessary to reach th e level of concern for adults

and children 1 to < 2 years old (i.e., adults, MOEs are ≥ 100; children 1 to < 2 years old, MOEs

are ≥ 40) with use of certain applic ation rates, nozzle droplet type s, and application methods. The

estimated buffer distances are less than those ag reed to by the technical registrants in July

2012. All drift risk estimates are presented in the occupati onal and residential exposure

assessment (Appendix D of D424484).

Table 6.3.1. Summary of Spray Drift Buffers for Chlorpyrifos 1



# Application

Rate

(lb ai/A) Nozzle

Droplet

Type Adult Buffer Summary Children 1 to < 2 Years Old Buffer

Summary

(Dermal + Incidental Oral)

Buffers Necessary to reach MOE of 100

(Feet) Buffers Necessary to reach MOE of 40

(Feet)

Aerial Groundboom Airblast Aerial Groundboom Airblast

6.0

Medium or

Coarse NA 2 

# NA 0 NA NA 25

4.3 NA 0 0 NA 0 10

4.0 NA 0 0 NA 0 10

3.76 NA 0 0 NA 0 10

3.0 Coarse or NA 0 0 NA 0 0

27 

# J. Dawson, W. Britton, R. Bohaty, N. Mallampalli, and A. Grube. Chlorpyr ifos: Evaluation of the Potential Risks

from Spray Drift and the Impact of Potential Risk Redu ction Measures. 7/13/12. U.S. EPA Office of Chemical

Safety and Pollution Prevention. D399483, D399485.

28 

# R. Keigwin. Spray Drift Mitigation Decision for Chlorpyrifos (059101). 7/2012. U.S. EPA Office of Chemical

Safety and Pollution Prevention. EPA-HQ-OPP-2008-0850-0103.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 83 of 531 Table 6.3.1. Summary of Spray Drift Buffers for Chlorpyrifos 1



# Application

Rate

(lb ai/A) Nozzle

Droplet

Type Adult Buffer Summary Children 1 to < 2 Years Old Buffer

Summary

(Dermal + Incidental Oral)

Buffers Necessary to reach MOE of 100

(Feet) Buffers Necessary to reach MOE of 40

(Feet)

Aerial Groundboom Airblast Aerial Groundboom Airblast

Very Coarse

3.0 Medium NA 0 0 NA 0 0

2.3 Coarse or

Very Coarse 50 0 0 0 0 0

2.3 Medium 100 0 0 10 0 0

2.0 Coarse or

Very Coarse 25 0 0 0 0 0

2.0 Medium 75 0 0 0 0 0

1.5 Coarse or

Very Coarse 25 0 0 0 0 0

1.5 Medium 50 0 0 0 0 0

1.0 Coarse or

Very Coarse 10 0 0 0 0 0

1.0 Medium 25 0 0 0 0 0



# 1 Per December 2012 spray drift mitigation memorandum, aer ial application of greater than 2 lb ai/A is only

permitted for Asian Citrus Psylla control, up to 2.3 lb ai/A.

2 NA is not allowable.



# 6.3.2 Volatilization

In January 2013, a preliminary assessment of the potential risks from volatilization was

conducted. 29 

# The assessment evaluated the potential risk s to bystanders, or those who live and/or

work in proximity to treated fields, from inha lation exposure to vapor phase chlorpyrifos and

chlorpyrifos-oxon emitted from fields following app lication of chlorpyrifos. The results of the

January 2013 assessment indicated that offsite con centrations of chlorpyrifos and chlorpyrifosoxon may exceed the target concentration based on the toxicological endpoints used at that

time. 30



# One significant area of uncertainty described in the preliminary assessment was the use of the

aerosolized chlorpyrifos inhala tion toxicity study -- as opposed to chlor pyrifos vapor -- for

evaluation of lung AChE resulti ng from field volatilization. Becau se field volatilization is the

production and release of vapor into the atmosphere after sprays have settled on treated soils and

plant canopies, the vapor, rather than the aerosol, is the relevant form for evaluation of bystander

volatilization exposures. However, EPA lacked ch lorpyrifos vapor toxici ty data at the time it

conducted the preliminary volatilization assess ment in 2013. Following the release of the

preliminary volatilization assessment, Dow AgroSciences LLC conducted, high quality nose-

29 

# R. Bohaty, C. Peck, A. Lowit, W. Br itton, N. Mallampalli, A. Gr ube. Chlorpyrifos: Prel iminary Evaluation of the

Potential Risks from Volatilization. 1/31/13. U.S. EPA Office of Chemical Safety and Pollution Prevention.

D399484, D400781.

30 

# EPA MRID# 48139303 : 

# Acute Inhalation Exposure of Adult Crl:CD( SD) Rates to Partic ulate Chlorpyrifos

Aerosols: Kinetics of Concentration-De pendent Cholinesterase (ACHE) Inhibition in Red Blood Cells, Plasma,

Brain and Lung; Authors: J. A. Hotchki ss, S. M. Krieger, K. A. Brzak, and D. L. Rick; Sponsor: Dow AgroSciences

LLC.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 84 of 531 only vapor phase inhalation toxicity studies fo r both chlorpyrifos and chlorpyrifos-oxon 31 

# to

address this uncertainty.

In June 2014, a reevaluation of the 2013 preliminary volatilizati on assessment was conducted to

present the results of the vapor st udies and their impact. In the va por studies, female rats were

administered a saturated vapor, meaning that th e test subjects received the highest possible

concentration of chlorpyrifos or chlorpyrifos-oxon which can satura te the air in a closed system.

At these saturated concentrati ons, no statistically significant inhibition of AChE activity was

measured in RBC, plasma, lung, or brain at any tim e after the six-hour exposure period in either

study. Under actual field conditions , indications are that exposures to vapor phase chlorpyrifos

and its oxon would be much lower as discusse d in the January 2013 prel iminary volatilization

assessment.

Because these new studies demonstrated that no toxicity occurred even at the saturation

concentration, which is the highest physically achievable concentra tion, then there is no

anticipated risks of concern from exposure to the volatilization of e ither chlorpyrifos or

chlorpyrifos oxon. In June 2014, the January 2013 volatilization assessment was revised to

reflect these findings. 32



# 7.0 Aggregate Exposure/Risk Characterization

In accordance with the FQPA, HED must consider and aggregate (add) pesticide exposures and

risks from three major sources: food, drinking water, and residential exposures. In an aggregate

assessment, exposures from relevant sources are added together and compared to quantitative

estimates of hazard, or the risks themselves can be aggregated. The durations of exposure

identified for chlorpyrifos uses are acute a nd steady state. The acute aggregate assessment

includes food and drinking water only. The stead y state aggregate assessment includes food,

drinking water, and residential exposures.

A drinking water level of comparison (DWLOC) approach to aggregate risk was used to

calculate the amount of exposure available in the total ‘risk cup’ fo r chlorpyrifos oxon in

drinking water after accounting for any chloropyrifos exposures from food and/or residential use.

The DWLOC approach used a reciprocal MOE calculation method for adults (females of

childbearing age) since the target MOEs are the sa me for all relevant sources of exposure, i.e.,

100X for residential dermal and for dietary food and water. This entailed calculating the MOE

31 

# W. Irwin. Review of Nose-Only Inhalation of Chlorpyrif os Vapor: Limited Toxicokinetics and Determination of

Time-Dependent Effects on Plasma, Red Blood Cell, Brain and Lung Cholineste rase Activity in Femal CD(SD): Crl

Rats. U.S. EPA Office of Chemical Safety and Pollution Prevention. 6/25/14. D411959. TXR# 0056694. EPA

MRID# 49119501.

W. Irwin. Review of Nose-Only Inhalation of Chlorpyr ifos-Oxon Vapor: Limited Toxicokinetics and Determination

of Time-Dependent Effects on Plasma, Red Blood Cell, Brain, and Lung Cholineste rase Activity in Female

CD(SD):Crl Rats. U.S. EPA Office of Chemical Safety and Pollution Prevention. 6/25/14. D415447. TXR#

0056869. EPA MRID# 49210101.

32 

# W. Britton. W. Irwin. J. Dawson. A. Lowit. E. Mendez. Chlo rpyrifos:Reevaluation of th e Potential Risks from

Volatilization in Consideration of Chlorp yrifos Parent and Oxon Vapor Inhalation Toxicity Studies. 6/25/2014. U.S.

EPA Office of Chemical Safety an d Pollution Prevention. D417105.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 85 of 531 for water (MOE water 

# ) by deducting the contributions from food (MOE food 

# ) and dermal

(MOE dermal 

# ) from the aggregate MOE (MOE agg 

# ) of 100. The aggregate MOE value is the same as

target MOE (level of concern). The DWLO C is then calculated by dividing the PoD water 

# by the

MOE water. 

# The general reciprocal MOE formula is as follows:

MOE

agg 

# = 1/ [(1/MOE

water 

# ) + (1/MOE food 

# ) + (1/MOE dermal 

# )]

MOE

water 

# = 1/ [(1/MOE

agg 

# ) – ((1/MOE food 

# ) + (1/MOE dermal 

# ))]

DWLOC= PoD

water 

# / MOE

water



# When target MOEs (levels of concern) are not th e same across the relevant sources of exposure,

the reciprocal MOE approach for calculating DW LOCs is not appropriate; instead an aggregate

risk index (ARI) method is used. This ARI method was employed for infants, children and

youths aggregate assessments for chlorpyrifos us es since the target MO Es for the relevant

sources of exposure are not the same i.e., the target MOE for dietary food and for residential

dermal exposures is 40X while the target MOE for drinking water exposure is 50X. In this

approach, the aggregate, or ‘total ’, ARI value is assigned as 1 (HED is generally concerned when

any calculated ARIs are less than 1). Similar to the reciprocal MOE approach, the ARIs for food

and dermal are deducted from the aggregate ARI to determine the ARI for water. The water ARI

is multiplied by the target MOE for water to determine the calculated water MOE (MOE water 

# ).

The DWLOC is then calculated by dividing the PoD water 

# by the MOE water. 

# The general ARI

method formula is as follows:

ARIs for food or dermal are calculated as ARI food or dermal 

# = [(MOE food or dermal 

# )/(MOE target for food or dermal 

# )].

ARI

agg 

# = 1/ [(1/ARI

water 

# ) + (1/ARI food 

# ) + (1/ARI dermal 

# )]

ARI

water 

# = 1/[(1/ARI

agg 

# ) – ((1/ARI food 

# ) + (1/ARI dermal 

# ) )]; Where ARI

agg 

# =1

MOE

water 

# = ARI

water 

# x MOE target.



# DWLOC= PoD

water 

# / MOE

water



# ARIs for food and dermal are calculated as ARI food or dermal 

# = [(MOE food or dermal 

# )/(MOE target for food or dermal 

# )].



# Aggregate risk estimate calculations and rela ted DWLOCs are based on guidance in the HED

SOP 99.5 ( Updated “Interim Guidance for Incor porating Drinking Water Exposure into

Aggregate Risk Assessments,” 8/1/99).



# 7.1 Acute Aggregate Risk

The acute aggregate assessment includes onl y food and drinking water. DWLOCs were

calculated for infants, children, youths, and adu lts. The lowest acute DWLOC calculated was for

# Chlorpyrifos Human Health Risk Assessment D424485

Page 86 of 531 infants (<1 year old) at 24 ppb.

Table 7.1 Acute Aggregate (Food and Drinking Water)

Calculation of DWLOCs 1,2



# Population Food

Exposure

(chlorpyrifos) 3 

# Drinking

Water

Exposure

(chlorpyrifos) 4 

# Acute

DWLOC 5



# (ppb chlorpyrifos

oxon) 

# MOE ARI MOE ARI

Infants 1



# (<1 yr) 2200 55 50 1.0 24

Children 1



# (1-2 yrs) 1400 35 50 1.0 60

Youths 1



# (6-12 yrs) 2800 70 50 1.0 150

Adults 2



# (Females 13-

49 yrs) 3100 NA 100 NA 53

1 

# DWLOCs for infants, children and youths are calculated using the ARI (Aggregate Risk Index) approach since

target MOEs are different for drinki ng water (chlorpyrifos oxon target MOE=50) and for food and residential

(chlorpyrifos target MOE= 40) exposure.

2 

# DWLOCs for adults (females 13-49 yrs) are calculated us ing the reciprocal MOE appro ach since the target MOEs

are the same for drinking water (chlorpyrifos oxon targ et MOE=100) and for food and residential (chlorpyrifos

target MOE= 100) exposure.

3 

# FOOD: MOE food 

# = PoD food 

# (ug/kg/day) (from Table 4.8.4)/ Food E xposure (ug/kg/day) (from Table 5.4.3).



# ARI food 

# = [(MOE food 

# )/(MOE target 

# )].

4 

# WATER (ARI approach): 

# ARI water 

# = 1/[(1/ARI

agg 

# ) – ((1/ARI food 

# ) + (1/ARI dermal 

# ) )]; Where ARI

agg 

# =1 (Note:HED is

generally concerned when calcula ted ARIs are less than 1).

MOE water 

# = ARI water 

# x MOE target. 

# WATER (Reciprocal MOE approach): MOE water 

# = 1/ [(1/MOE agg 

# ) – ((1/MOE food 

# ) + (1/MOE dermal 

# ))]; Where MOE agg = 

# Target

MOE.

5 

# DWLOC: DWLOC ppb= PoD water 

# (ppb; from Table 4.8.4) /MOE water



# 7.2 Steady State Aggregate Risk

The steady state aggregate assessment includes di etary exposures from food and drinking water

and dermal exposures from residential uses (der mal exposures represent the highest residential

exposures). DWLOCs were calculated for infa nts, children, youths, a nd adults. The lowest

steady state DWLOC calculated was for infants (<1 year old) at 3.9 ppb.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 87 of 531 Table 7.2 Steady State Aggregate (F ood, Drinking Water, Residential)

Calculation of DWLOCs 1,2



# Population Food

Exposure

(chlorpyrifos) 3 

# Dermal

Exposure

(chlorpyrifos) 4 

# Drinking

Water

Exposure

(chlorpyrifos

oxon) 5 

# Steady State

DWLOC 6



# (ppb chlorpyrifos

oxon)



# MOE ARI MOE ARI MOE ARI

Infants 1



# (<1 yr) 550 14 NA NA 55 1.1 3.9

Children 1



# (1-2 yrs) 410 10 NA NA 55 1.1 10

Youths 1



# (6-12 yrs) 700 18 1600 40 54 1.1 16

Adults 2



# (Females 13-

49 yrs) 1000 NA 1000 NA 125 NA 7.5

1 

# DWLOCs for infants, children and youths are calculated using the ARI (Aggregate Risk Index) approach since

target MOEs are different for drinki ng water (chlorpyrifos oxon target MOE=50) and for food and residential

(chlorpyrifos target MOE= 40) exposure.

2 

# DWLOCs for adults (females 13-49 yrs) are calculated us ing the reciprocal MOE appro ach since the target MOEs

are the same for drinking water (chlorpyrifos oxon targ et MOE=100) and for food and residential (chlorpyrifos

target MOE= 100) exposure.

3 

# FOOD: MOE food 

# = PoD food 

# (ug/kg/day) (from Table 4.8.4)/ Food E xposure (ug/kg/day) (from Table 5.4.4).



# ARI food 

# = [(MOE food 

# )/(MOE target 

# )].

4 

# DERMAL: MOE dermal 

# = PoD dermal 

# (ug/kg/day) (from Table 4.8.4)/ Dermal Exposure (ug/kg/day) (from Table 6.2).



# ARI dermal 

# = [(MOE dermal 

# )/(MOE target 

# )].

5 

# WATER (ARI approach): 

# ARI water 

# = 1/[(1/ARI

agg 

# ) – ((1/ARI food 

# ) + (1/ARI dermal 

# ) )]; Where ARI

agg 

# =1 (Note:HED is

generally concerned when calcula ted ARIs are less than 1).

MOE water 

# = ARI water 

# x MOE target. 

# WATER (Reciprocal MOE approach): MOE water 

# = 1/ [(1/MOE agg 

# ) – ((1/MOE food 

# ) + (1/MOE dermal 

# ))]; Where MOE agg = 

# Target

MOE.

6 

# DWLOC: DWLOC ppb= PoD water 

# (ppb; from Table 4.8.4) /MOE water



# 7.3 Aggregate Risk Estimates

The acute aggregate assessment includes f ood and drinking water only. The steady state

aggregate assessment includes food, residential and drinking water exposures. Acute and steady

state risk estimates for dietary (food only) exposur es or for residential on ly (dermal, inhaltion

and incidental oral) exposures to chlorpyrifos are not of concern. Steady state risk estimates from

food and residential exposures combined are also not of concern. For the chlorpyrifos aggregate

assessments that include drinking water along with the food and residential components, a

DWLOC approach is used to calculate the amount of exposure available in the total ‘risk cup’ for

exposures to the chlorpyrifos oxon in drinking water after accounting for any exposures from

parent compound chlorpyrifos from food and/or residential use.

Typically, for a DWLOC aggregate risk assessmen t, the calculated DWLOC is compared to the

EDWC. When the EDWC is less than the DWLOC, there are no risk concerns for exposures to

# Chlorpyrifos Human Health Risk Assessment D424485

Page 88 of 531 the pesticide in drinking water. Conversely, when the EDWC is greater than the DWLOC, there

may be a risk concern. For chlorpyrifos, DWLOCs were calculated for both the acute and steady

state aggregate assessments for infants, childre n, youths and adult females. However, the acute

DWLOCs were not compared to EDWCs. The steady state DWLOCs and risk estimates for

drinking water will be protective of any acute aggregate exposures because the oxon

concentrations expected to re sult in 10% ChEI are considerab ly higher (up to 5.7x higher) for

acute exposures than for steady state. In additi on, the steady state DWLO C calculations include

the residential exposure component. Consequent ly, the calculated acute DWLOCs are much

higher than those for the steady state. For example, for infants, the acute DWLOC is 24 ppb

while the steady state DWLOC is 3.9 ppb (Table s 7.1 and 7.2). In fact, the lowest DWLOC

calculated for any duration or population was the 3.9 ppb value (infants) and is the concentration

used for comparison to the EFED-modeled ED WCs. Drinking water concentrations of

chlorpyrifos oxon above 3.9 ppb may be of concern.

EFED has summarized the drinki ng water and aggregate assessment below (for complete details

on the assumptions, results, and char acterization of the screening an alysis refer to EFED’s DWA,

(Bohaty, R. 12/23/14, D424487, Chlorpyrifos: Updated Drin king Water Assessment for

Registration Review ).

Residue Definition

A national screening level drinki ng water assessment was complete d for the registration review

of chlorpyrifos, with focus on the agricultural uses. The primary drinking water residue of

concern is chlorpyrifos-oxon, the predominant chlorpyrifos transformation product formed

during drinking water treatment ( e.g. , chlorination). To illustra te the range of EDWCs, two

maximum label rate application scenarios were se lected to represent high and low end exposures,

i.e., tart cherries at 5 applic ations totaling 14.5 pounds per acre per year, and bulb onions at a

single application of one pound per ac re per year, respectively. The a pplication of chlorpyrifos to

tart cherries resulted in c oncentrations in surface water th at exceeded the DWLOC; whereas,

chlorpyrifos applications to bulb onions re sult in concentrations below the DWLOC.

Concentrations in groundwater are not expected to exceed the DWLOC.

To investigate whether other chlorpyrifos applica tion scenarios may result in concentrations that

exceed the DWLOC, a screen of all available surface water modeling scenarios was completed

considering three different application dates and a single application at several different

application rates that ranged fr om one to six pounds. This analys is showed that even with only

one application, several chlorpyrifos uses may exceed the DWLOC at rates lower than maximum

labeled rates (both single as we ll as yearly), including an appl ication rate of one pound per acre

per year. The analysis also showed that the DWLOC exceedances are not expected to be

uniformly distributed across the country.

Further analysis was conducted to look at the spatial di stribution of EDWCs at a regional level,

as well as by using a drinking water intake waters hed approach. This exercise demonstrated that

chlorpyrifos applications will result in variable drinking water exposures that are highly localized

and that the highest exposures generally occur in sm all hydrologic regions where there is a high

percent cropped area on which chlo rpyrifos use could occur.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 89 of 531 Finally, EDWCs were compared to monitoring data. This analysis showed that when modeling

scenarios are parameterized to reflect reported use and EDWCs are adjusted to reflect percent

cropped area, the EDWCs are within an order of magnitude of the measured concentrations

reported in the monitoring data. Therefore, altho ugh there are uncertainties associated with the

model input parameters for which cons ervative assumptions were made ( e.g. , one aerobic aquatic

metabolism half-life value multiplie d by the uncertainty factor of three, stable hydrolysis, 100%

of the cropped watershed is tr eated on the same day, and use of the Index Reservoir as the

receiving waterbody), these assump tions do not appear to lead to an overly conservative estimate

of exposure. In addition, evaluation of the monito ring data further illustrates that exposures are

highly localized. Additional work can be done to examine EDWCs on a regional and/or

watershed scale to pinpoint community drinking water systems where exposure to chlorpyrifosoxon as a result of chlorpyrifos appli cations may pose an exposure concern.

EDWCs are provided for chlorpyrifos and ch lorpyrifos-oxon. Chlorpyrifos EDWCs were

multiplied by 0.9541 (molecular weight correct ion factor) and 100% (maximum conversion

during water purification) to generate chlorpyr ifos-oxon EDWCs. Essentially, the concentration

of chlorpyrifos and chlorpyrif os-oxon are the same. A 100% conve rsion factor for the oxidation

of chlorpyrifos to chlorpyrif os-oxon was used as an approxi mation based on empirical bench

scale laboratory data that indica te chlorpyrifos rapidly oxidizes to form chlorpyrifos-oxon almost

quantitatively during typical wa ter treatment (chlorination). 33 

# There are limited data available on

the removal efficiency of chlorpyrifos prior to oxidation or the removal efficiency of

chlorpyrifos-oxon during the drinking water treatm ent process. Based on empirical data showing

that more than 75 percent of community water systems use chlo rination to disinfect drinking

water in the United States 34 

# , the assumption of exposure to ch lorpyrifos-oxon equivalent to 100%

conversion of chlorpyrifos is not considered ov erly conservative. It is possible that some

drinking water treatment procedures, such as granular activated carbon filtration and water

softening (increased rate of chlorpyrifos-oxon hyd rolysis at pH > 9) could reduce the amount of

chlorpyrifos-oxon in finished drinking water; ho wever, these treatment methods are not typical

practices across the countr y for surface water.

While there is the potential to have both chlor pyrifos and chlorpyrifos-o xon present in finished

drinking water, limited (or no) information is available to readily qua ntify how much of each

form remains in the finished water. In the abse nce of available information, all the chlorpyrifos

that enters a drinking water treat ment facility is assumed to remain after treatment, and is

converted to chlorpyrifos -oxon during treatment.

Although chlorpyrifos-oxon has a hy drolysis half-life of 5 days , the drinking water treatment

simulation half-life for chlorpyrif os-oxon is approximately 12 days. 35,36,37 

# Therefore, once

33 

# Duirk, S. E.; Collette, T. W.; Degradation of Chlorpyrifos in Aqueou s Chlorine Solutions: Pathways, Kinetics,

and Modeling. Environ. Sci. Technol. 

# , 2006, 40(2) 

# , 546-550.

34 

# Community Water System Survey 2006; U.S. Environmental Protection Agency, Washington, DC 20460 May

2009 

# (survey data)

35 

# Tunink, A. Chlorpyrifos-oxon: Determination of hydrolysis as a function of pH, 2010 (MRID 48355201;

acceptable)

36 

# Wu, J.; Laird, D. A. Abiotic Tran sformation of Chlorpyrifos to Chlorpyrifos Oxon in Chlorinated Water. Environ.

Toxcol.Chem.



# , 2003 

# , 22(2), 261-264.

37 

# Tierney, D. P.; Christensen, B. R.; Culpepper, V. C. Chlorine Degradation of Six Organophosphate Insecticides

# Chlorpyrifos Human Health Risk Assessment D424485

Page 90 of 531 chlorpyrifos-oxon forms during treatment, little tr ansformation is expected to occur before

consumption (during drinking wate r distribution). There is a wide range of treatment processes

and sequences of treatment proc esses employed at community water systems across the country

and there are limited data available on a commun ity water system specific basis to assess the

removal or transformation of chlorpyrifos during treatment. These processes are not specifically

designed to remove pesticides and pesticide tr ansformation products incl uding chlorpyrifos and

chlorpyrifos-oxon. In general, drinking water tr eatment processes, with the exception of

activated carbon, 38 

# have been shown to have little imp act on removal of pesticide residues.

Additional discussion of drinking wa ter treatment can be found in the Drinking Water

Treatment section of the DWA or in the preliminary DWA.

Aggregate

National Screen

The 2011 preliminary DWA, as well as the additional analyses completed as part of this

assessment, indicate that exposure to chlorpyr ifos-oxon in drinking wate r derived from surface

water may pose an exposure concern. Since a larg e number of chlorpyrifos uses were identified

in the preliminary DWA as tr iggering a concern, a bounding estim ate of exposure was completed

using a screening level national assessment a pproach. This was done to determine which

currently registered uses could result in expos ure to chlorpyrifos-oxon in drinking water that

exceed the DWLOC.

Use of chlorpyrifos on tart cherries is expect ed to result in the highest EDWC. EDWCs for

chlorpyrifos and chlorpyrifos-oxon 39 

# are reported below for Ti er I groundwater and Tier II

surface water model simulations. Because chlorpyrif os is used on a number of agricultural crops,

as well as turf, a drinking wate r intake percent croppe d area (PCA) adjustment factor of 1 was

used. 40,41 

# While the model input values 42 

# have been updated since the preliminary assessment,

the results presented in 7.3.1 are similar to those previously reported.

and Four Oxons in Drinking Water Matrix. Submitted by Syngenta Crop Protection, Inc. 2001



# .

38 

# Progress Report on Estimating Pesticide Concentrations in Drinking Water and Assessing Water Treatment

Effects on Pesticide Removal and Transformation: A Consultation. 

# FIFRA Scientific Advisory Panel Meeting,

September 29, 2000; SAP Report No. 2001-02 February 12, 2011.

39 

# Chlorpyrifos EDWCs were multiplied by 0.9541 (m olecular weight correction factor) and 100% (maximum

conversion during water purification) to generate chlorp yrifos-oxon EDWCs. Additiona l details on the potential

impacts on drinking water treatment on chlorpyrifos and chlorpyrifos-oxon see the Additional Analyses 

# section on

Drinking Water Treatment.

40 

# U.S. Environmental Protection Agency Brady, D. Guidance on Development and Use of Community Water

System Drinking Water Intake Percent Cropped Area Adjustment Factors for Use in Drinking Water Exposure

Assessments: 2014 Updated, September 12, 2014.

41 

# U.S. Environmental Protection Agency , Bohaty, R., Carleton, J., Crk, T., Echeverria, M., Ruhman, M., Thawley,

M., Thurman, N., Villa nueva, P., White, K., Devel opment of Community Water Syst em Drinking Water Intake

Percent Cropped Area Adjustment Factors for Use in Drinking Water Exposure Assessments: 2014 Update,

September 9, 2014.

42 

# Guidance for Selecting Input Parameters in Modeling the Environmental Fate and Transport of Pesticides



# , Version 2.1, October 22, 2009.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 91 of 531 Table 7.3.1. Estimated Drinking Water Concentrations Resulting from the Use of Chlorpyrifos

Residue Surface Water Groundwater

1-in-10 Year

Peak

Concentration

μg/L 21-day Average

Concentration

μg/L 1-in-10 Year

Annual Average

Concentration

μg/L 30 Year

Annual

Average

Concentration

μg/L SCI-GROW

Tier I

Concentration

μg/L a



# Michigan Tart Cherries



# Chlorpyrifos 129 83.8 39.2 29.7 0.16

Chlorpyrifosoxon 123 80.0 37.4 28.3 0.15

Georgia Bulb Onion



# Chlorpyrifos 6.2 3.1 1.2 0.8 0.01

Chlorpyrifosoxon 5.9 3.0 1.1 0.8 0.01

a. SCI-GROW resulted in higher EDWCs than PRZM-GW simulations.



# In order to better define the extent to which ot her chlorpyrifos use scenarios may result in an

exposure concern, the Health Effects Division de veloped a 21-day average DWLOC of 3.9 μg/L

for chlorpyrifos-oxon 43 

# that could be compared to model output values. Uncertainties in this

approach include potential tempor al aspects of relative concentr ations from day to day on AChE

inhibition, geospatial dist ribution of exposure as a result of variability in use, environmental

factors, and drinking wate r treatment processes.

Previous risk assessments 44 

# suggest that typical upper bound a pplication rates fo r chlorpyrifos

are similar to the maximum single application rate s for a wide range of crops; however, often the

number of typical applications per year is lower than the ma ximum number of applications

currently permitted on product labels ( i.e. , summarized in Master Use Summary Document ).

Considering this information, a screening analys is (see the DWA for deta ils) was completed to

determine which chlorpyrifos uses do not exceed the DWLOC, based on a single application of

chlorpyrifos per year at 1 and 4 pounds of chlo rpyrifos per acre. The results for 1 and 4 pounds

per acre are reported here as a representation of the range of potential ch lorpyrifos application

rates, bearing in mind that ch lorpyrifos can be applied at lower and higher single rates ( e.g., an

application rate of 6 pounds per acre on citrus). This analysis showed that over a 30 year period,

the current maximum application rate scenarios, as well as maximum single application rates for

a wide range of chlorpyrifos use scenarios, may result in multiple 21-day average concentrations

that exceed the DWLOC (Error! Reference source not found.).

43 

# The average 21-day concentration of chlorpyrifos-oxon necessary to cause 10% AChE inhibition was determined

by HED to be 217 ppb. This value was divided by the safe ty factors (50x; 4.3 ppb) and then the contribution from

food (0.4 ppb) was subtracted out to give to derive a DWLOC (3.9 ppb).

44 

# Dawson, J., Bohaty, R., Mallampalli, N. Evaluation of the Pote ntial Risks from Spray Drift and the Impact of

Potential Risk Reduction Measures, June 20, 2012 PC 059101 DP 399483 and 399485.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 92 of 531 Figure 7.3.1. National Screen: Exceedance Count/Percent (one application per year)



# Regional Screen

Although there are several exceedan ces of the DWLOC on a national basis, the incidence of high

exposures is expected to be highly localized. While it is cu rrently extremely challenging to

assess exposure on a local scale due to the unavailability of data and wide range of

characteristics [ i.e. , environmental such as soil, weather, etc. or otherwise ( e.g. , drinking water

treatment process)] that affect the vulnerabil ity of a given community drinking system to

chlorpyrifos-oxon contamination, a method was deve loped to examine the potential geospatial

concentration differences for two Hydrological Unit Code (HUC) 2 Regions – HUC 2 Region

17: Pacific Northwest and HUC 2 Re gion 3: South Atlantic-Gulf, in order to identify use patterns

that may result in EDWCs that exceed the DWLOC on a regional basis. 45 

# This analysis

considered all potential chlorpyrifos use site s within the HUC 2 regions based on the National

Agricultural Statistics Service cropland data layers and survey data. Due to the uncertainty

associated with the urban uses (e.g., wide area/general outdoor tr eatment) that are represented by

the impervious scenarios, modeled results from th e impervious scenarios are not included in this

analysis. Additional clarification from the registrants is needed in order to determine if these uses

pose an exposure concern.

For HUC 2 Region 17, four chlorpyrifos use pattern s were identified as a potential concern based

on maximum single application ra tes of 1 and 4 pounds per acre. However, for HUC 2 Region 3,

several chlorpyrifos use scenarios were identified that could exceed the DWLOC.

Watershed Screen

The uses that exceeded the DWLOC from the regional screening exercise for HUC 2 Region 3

were further explored for one example by utilizi ng the drinking water intake (DWI) watershed

database. This example shows an overlap of potentia l chlorpyrifos use sites that may result in an

exceedance of the DWLOC with watersheds that supply source water for community drinking

water systems. In addition, this analysis show s that exposure is not uniform within a HUC 2

Region and that some watersheds are more vulne rable than others. Wate rshed vulnerability is

45 

# http://water.usgs. gov/GIS/huc.html

# Chlorpyrifos Human Health Risk Assessment D424485

Page 93 of 531 expected to be greatest for smaller waters heds with high percent cropped areas. Smaller

community water systems are generally more vulne rable due to short distribution times and the

reliance of chlorination to treat source surface water as well as limited access to other treatment

methods such as granular activated carbon.

Monitoring Data Analysis

Water monitoring data from the USGS Nationa l Water-Quality Assessment Program (NAWQA),

USEPA/USGS Pilot Reservoir Monitoring Progra m, USDA Pesticide Data Program (PDP), and

California Department of Pesticide Regulation (C DPR) were evaluated in the preliminary DWA

with reference to an acute exposure to chlor pyrifos and its degradat ion product chlorpyrifosoxon. The monitoring data showed chlorpyrifos dete ctions at low concentrations, generally not

exceeding 0.5 μg/L. For example, USGS NAWQA, which contains an extensive monitoring

dataset for chlorpyrifos and chlorpyrifos oxon, repor ts a peak chlorpyrifos detection of 0.57 μg/L

in surface water with a detection frequency of approximately 15%. CDPR has detected

chlorpyrifos concentrations greater than 1 μg/L in surface water on several occasions, with an

observed peak chlorpyrifos concentration of 3.96 μg/L. Sampling frequencies in these

monitoring programs were sporadic and generally range from once per year to twice per month.

Since the preliminary assessment, water monitori ng data from Washington State Department of

Ecology and Agriculture (WSDE/WSDA) Coope rative Surface Water Monitoring Program 46,47



# , Dow AgroSciences (MRID 44711601), and Oregon Depa rtment of Environmental Quality were

evaluated and are presented as part of this update. The previously re ferenced data have also been

re-examined to consider short-term exposure ( i.e. , 21-day average concentrations) considering

the importance of the single day exposure and th e temporal relationship of exposure. A summary

of all surface water monitoring data examined to date for chlorpyrifos are presented in Error!

Reference source not found.. Some of the monitori ng programs analyzed for chlorpyrifos-oxon;

however, the number of detections as well as the concentrations were generally much lower.

Since the majority of the conversion of chlorpyr ifos to chlorpyrifos-oxon is assumed to occur

during drinking water treatment, and not in the environment, the monitoring data presented in

Error! Reference source not found. are limited to chlorpyrifos.

46 

# Sargeant, D, Dugger, D. Newell, E., Anderson, P, Cowles , J. Surface Water Monitoring Program for Pesticides in

Salmonid-Bearing Streams 2006-2008 Triennial Report, Febr uary 2010 (Washington State Department of Ecology

and Washington State Department of Agriculture)

https://fortress.wa.gov/ecy/publications/summarypages/1003008.html;

http://agr.wa.gov/PestFert/natres ources/docs/swm/2008_swm_report.pdf

47 

# Sargeant, D., Newell, E., Anderson, P., Cook, A. Surface Water Monitoring Program for Pesticides in SalmonidBearing Streams 2009-2011 Triennial Report, February 2013 (Washington State Department of Ecology and

Washington State Department of Agriculture) http ://agr.wa.gov/FP/Pubs/docs/377-SWM2009-11Report.pdf

# Chlorpyrifos Human Health Risk Assessment D424485

Page 94 of 531 Table 7.3.1. Surface Water Monitoring Data Summary for Chlorpyrifos

Monitoring Data Scale Years of

Sampling

(number of

samples) Detection

Frequency

(%) Maximum

Concentration

(μg/L)



# USGS NAWQA National 1991-2012

(30,542) 15 0.57

California Department of Pesticide

Regulation State 1991-2012

(13,121) 20 3.96

Washington State Department of

Ecology and Agriculture

Cooperative Surface Water

Monitoring Program State 2003-2013

(4,091) 8.4 0.4

USDA Pesticide Data Program National 2004-2009

(raw water;

1,178)

2001-2009

(finished

water; 2,918) 0 na

USGS-EPA Pilot Drinking Water

Reservoir National 1999-2000

(323) 5.3 0.034

Oregon Department of

Environmental Quality Watershed

(Clackamas) 2005-2011

(363) 13 2.4

MRID 44711601 Watershed

(Orestimba

Creek) 1996-1997

(1,089) 61 2.22



# In general, the monitoring data include sampling sites that represent a wide range of aquatic

environments including small and large wate r bodies, rivers, rese rvoirs, and urban and

agricultural locations, but are limited for some area s of the United States where chlorpyrifos use

occurs. Also, the sampling sites, as well as the nu mber of samples, vary by year. In addition, the

vulnerability of the sampling site to chlorpyrifos contamination va ries substantially due to use,

soil characteristics, weather and agronomic practices. None of the monitoring programs

examined to date were specifically designed to target chlorpyrifos use (except the Registrant

Monitoring Program MRID 44711601); therefore, peak concentrations (and likely 21-day

average concentrations) of chlo rpyrifos and chlorpyrifos-oxon likely went undetected in these

programs. See the Revised Chlorpyrifos Pre liminary Registration Re view Drinking Water

Assessment dated June 30, 2011 for further details on the monitoring programs discussed here.

http://www.regulations.gov/#!docum entDetail;D=EPA-HQ-OPP-2008-0850-0026

# Chlorpyrifos Human Health Risk Assessment D424485

Page 95 of 531 In general, sampling frequency needs to be a pproximately equal to the duration of exposure

concern. 48 

# The chlorpyrifos monitoring data evaluated thus far also show that as sample

frequency increases, so does the detection frequency. This is evident in the registrant-submitted

monitoring data, as well as examination of indivi dual sampling sites within the various datasets.

Therefore, while there are many individual sample s collected and analyzed for chlorpyrifos (or

chlorpyrifos-oxon) across the Unit ed States, it would not be appr opriate to combine these data

sources to generate exposure estimates or to use these datasets to represent exposure on a

national or even regional basis. Thus, comp aring the monitoring data results to the DWLOC

would not be a reasonable approach for the re asons given above, including limited sample

frequency, limited use information, and sampling site variability, on a nationa l or even a regional

basis. Model estimated concentrations should be considered suitable upper bound concentrations

for chlorpyrifos and chlorpyrifos-oxon.

Additionally, model simulations were completed to represent two different water monitoring

datasets - Washington State Department of Ecology and Agriculture (WSDE/WSDA)

Cooperative Surface Water Monitoring Progr am and Dow AgroSciences (MRID 44711601)

Orestimba Creek. For both of these water monitoring programs, enough information was

available, including chlor pyrifos use information as well as th e PCA, to parameterize the model.

In these simulations, the modeled EDWCs were w ithin an order of magnitude of the measured

concentrations. This suggests th at the modeling results are not overly conservative and supports

the use of the model to estimate chlorpyrif os-oxon concentrations in drinking water.

Additional modeling can be done to pinpoint regions or waters heds where EDWCs may exceed

the DWLOC. This would include completing th e regional assessment presented here for all

HUC 2 Regions and crop uses, as well as considering multiple applications per year.

Nevertheless, based on the current analysis, co ncentrations of chlorpyrifos-oxon in drinking

water are expected to be highly localized prim arily in small watersheds with high PCA.

Summary

In summary, examination of chlorpyrifos agricult ural use across the country indicates that there

are a number of uses that may result in poten tial exposure to chlorpyr ifos-oxon in finished

drinking water at levels that exceed the DWLO C. The EDWCs for tart cherries and bulb onion

reported here are expected to provide a re asonable bounding estimation of exposure based on

maximum rates included in Master Use Summary Document. This analysis showed that the

maximum use scenario for tart cherries exceeds the DWLOC, while it does not for bulb onions.

The rate used for the bulb onion simulation wa s 1 pound chlorpyrifos per acre; therefore, a

screen of all Surface Water Concentration Calcul ator modeling scenarios was done using a single

application of chlorpyrifos. This analysis show ed that exceedances are expected even for one

application of chlorpyrifos applie d at 1 pound per acre per year.

48 

# U.S. Environmental Protection Agency . Federal Insecticide, Fungicide, an d Rodenticide Act (FIFRA) Scientific

Advisory Panel: Problem Formulation for the Reassessment of Ecological Risks from the Use of Atrazine, June 12-

14, 2012, Docket Number: EPA-HQ-OPP-2012-0230.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 96 of 531 While there are uncertainties associated with the model input parameters for which conservative

assumptions were made ( e.g. , one aerobic aquatic metabolism half-life value multiplied by the

uncertainty factor of three, st able hydrolysis, 100% of the cropped waters hed is treated on the

same day, and use of the Index Reservoir as th e receiving waterbody) these assumptions do not

appear to lead to an overly conservative esti mate of exposure based on a comparison of model

estimates with measured concentrations. Compar ison of model estimated concentrations with

measured concentrations suggests that mode l estimates are consis tent with measured

concentrations when actual application rates and representative Surf ace Water Concentration

Calculator (SWCC) scenarios are considered an d a PCA adjustment factor is applied to the

model estimates. This modeling/monitoring co mparison suggests that when growers use

maximum application rates, or even rates much lower than maximum, chlorpyrifos-oxon

concentrations in drinking water could pose an ex posure concern for a wide range of chlorpyrifos

uses. However, these exposures are not expected to be uniformly distributed across the country.

Additional analyses are needed in order to pi npoint the exact community water systems where

concentrations may be of concern.



# 8.0 Cumulative Exposure/Risk Characterization

Section 408(b)(2)(D)(v) of the Federal Food, Dr ug, and Cosmetic Act (FFDCA), as amended by

the Food Quality Protection Act (199 6) stipulates that when determ ining the safety of a pesticide

chemical, EPA shall base its assessment of the risk posed by the chemical on, among other

things, available information concerning the cumula tive effects to human health that may result

from dietary, residential, or other non-occupa tional exposure to other substances that have a

common mechanism of toxicity. The reason for cons ideration of other substances is due to the

possibility that low-level exposur es to multiple chemical substa nces that cause a common toxic

effect by a common mechanism could lead to the same adverse health effect as would a higher

level of exposure to any of the other substances individually. A person exposed to a pesticide at a

level that is considered safe ma y in fact experience harm if that person is also exposed to other

substances that cause a common toxic effect by a mechanism common with that of the subject

pesticide, even if the individual exposure levels to the other substances ar e also considered safe.

Chlorpyrifos is a member of the organophosphate (OP) class of pesticid es. Other members of

this class of pesticides are numerous and incl ude azinphos methyl, diazino n, chlorpyrifos-methyl,

dichlorvos, dicrotophos, dimet hoate, disulfoton, methamidophos , methidathion, monocrotophos,

naled, oxydemeton-methyl, phorate, phosmet, pirimi phos-methyl, and trichlorfon to name a few.

EPA considers organophosphates to express toxi city through a common biochemical interaction

with cholinesterase which may lead to a myriad of cholinergic effects and, consequently the

organophosphate pesticides should be considered as a group when performing cumulative risk

assessments. HED published the final guidance that it now uses for identifying substances that

have a common mechanism of toxicity (F R 64(24) 5796-5799, February 5, 1999) “Proposed

Guidance of Cumulative Risk Assessment for Chemicals that have a Common Mechanism of

Toxicity” was made available for public comm ent in the Federal Register (65 FR 40644, June

30, 2000). The Agency presented this approach to the FIFRA Scientific Advisory Panel in late

September, 2000. The SAP reviewed revised me thods used to conduct a preliminary cumulative

risk assessment for organophosphate pest icides in 2002 (US EPA, 2002), found at

# Chlorpyrifos Human Health Risk Assessment D424485

Page 97 of 531 http://www.epa.gov/scipol y/sap/2002/index.htm.

The Agency has completed a cumulative risk assessment for OPs, (US EPA, 2001), a revised

cumulative risk assessment for OPs, (US EPA, 2002), and an updated OP cumulative risk

assessment (US EPA, August 2006) which can be found on the Agency's web site

http://www.epa.gov/pesticides/cumulative/rra-op /. The cumulative effects of exposure to

multiple OPs, including chlorpyrifos, are evaluate d in those documents. Prior to the completion

of registration review, OPP will update the OP CRA to incorporate any toxicity and exposure

information available since 2006.



# 9.0 Occupational Exposure/Risk Characterization

9.1 Steady State Occupational Handler Risk

The term handlers is used to describe those individuals who are invol ved in the pesticide

application process. There are distinct job functions or task s related to applications and

exposures can vary depending on the specifics of each task. Job requirements (amount of a

chemical used in each application), the kinds of equipment used, the target being treated, and the

level of protection used by a handl er can cause exposure levels to differ in a manner specific to

each application event. Based on the anticipated use patterns and current labeling, types of

equipment and techniques that can potentiall y be used, occupational handler exposure is

expected from chlorpyrifos use. For purpose of occupational handler assessment, the parent

chlorpyrifos is the relevant compound.

The 2011 occupational exposure assessment fo r chlorpyrifos (W. Britton, 6/27/11, D388165,

Chlorpyrifos: Occupational and Re sidential Exposure Assessment ) has been updated based on a

number of changes that have o ccurred since that time, includi ng the following: 1) use of the

PBPK-PD model for determining toxicological Po Ds, 2) inclusion of use patterns based on a

master use document developed for chlorpyrifos , 3) use of revised gui dance for all of the

currently recommended unit exposures for st andard EPA occupational handler exposure

scenarios 49



# , Occupational Pesticide Handler Unit Exposure Surrogate Reference Table –

Revised March 2013 , 4) use of the revise d seed treatment policy, Interim Guidance (SOP 15.1)

Amount of Commercial Seed Treated per Day, as was updated March 21, 2013 and 5) use of

updated adult (female) body weight.

In addition, steady state durations of expos ure are assumed in the updated occupational

assessment. HED typically classifi es exposures from 1 to 30 days as short-term and exposures 30

days to six months as intermediate-term. Th e steady state endpoint sele ction for chlorpyrifos

overlaps HED’s traditional short-term exposure dur ation endpoint selecti on and is considered

health protective for both short- and intermediate- term exposures.

Full details regarding the updated occupa tional assessment can be found in D424484 7 

# . A

summary of the assumptions and conclusions are provided below.

49 

# http://www.epa.gov/opp00001/science/handler-exposure-data.html

# Chlorpyrifos Human Health Risk Assessment D424485

Page 98 of 531 The updated quantitative exposure/risk assessment developed for occupational handlers is based

on the following scenarios:

Mixer/Loaders:

(1a) Liquids for Aerial/ Chemigation Applications

(1b) Liquids for Airblast Applications

(1c) Liquids for Groundboom Applications

(1d) Liquids for Wide Ar ea Aerial Applications

(1e) Liquids for Wide Area Ground Applications

(2a) Wettable Powder (in WSP) for Aerial/ Chemigation Applications

(2b) Wettable Powder (in WSP) for Airblast Applications

(2c) Wettable Powder (in W SP) for Groundboom Applications

(3a) Granulars for Aerial Applications

(3b) Granulars for Tractor Drawn Spreader Applications

(3c) Granulars with SmartBox® for Aerial Applications

(3d) Granulars with SmartBox® for Tr actor Drawn Spreader Applications

(4a) Water Dispersible Granulars (in WSP) for Aerial/ Chemigation Applications

(4b) Water Dispersible Granulars (i n WSP) for Airblast Applications

(4c) Water Dispersible Granulars (in WSP) for Groundboom Applications

Applicators:

(5a) Aerial Liquid Applications

(5b) Wide Area Aerial Applications

(5c) Aerial Granul ar Applications

(6a) Airblast Applications

(6b) Wide Area Ground Applications

(7a) Groundboom Applications

(7b) Tractor Drawn Gr anular Applications

(7c) Tractor Drawn Granular Applications with SmartBox®

(8) Paint Brush/Roller Applications

(9) Cattle Ear Tag Applications

(10) Bait Station Applications

(11) Total Release Fogger

Mixer/Loader/Applicators:

(12a) Liquid: Manually Pressurized Handwand

(12b) Liquid: Wettable Powder (in W SP) for Manually Pressurized Handwand

(12c) Liquid: Microencapsulated for Manually Pressurized Handwand

(12d) Liquid: Backpack Sprayer

(12e) Liquid: Wettable Powder (in WSP) for Backpack Sprayer

(12f) Liquid: Microencapsulated for Backpack Sprayer

(12g) Liquid: Mechanic ally Pressurized Handgun

(12h) Liquid: Wettable Powder (in W SP) for Mechanically Pressurized Handgun

# Chlorpyrifos Human Health Risk Assessment D424485

Page 99 of 531 (12i) Liquid: Microencapsulated for Mechanically Pressurized Handgun

(13a) Granulars: Hand Dispersal

(13b) Granulars: Rotary Spreader

(13c) Granulars: Spoon

(13d) Granulars: Belly Grinder

(13e) Granulars: Belly Grinder

(14a) Water Dispersible Granular (in WSP): Manually Pressurized Handwand

(14b) Water Dispersible Granular (in WSP): Backpack Sprayer

(14c) Water Dispersible Gr anular (in WSP): Mechanically Pressurized Handgun

Flaggers:

(15a) Flagging for Aerial Spray Applications

(15b) Flagging for Aerial Granular Applications

Occupational Handler Exposur e Data and Assumptions

A series of assumptions and exposure factors serv ed as the basis for completing the occupational

handler risk assessments.

Application Rates: In order to seek clarifi cation of chlorpyrifos usage, the Agency compiled a

master use summary document reflective of the use profile of all active product labels. The

document, among other information, presents al l registered uses of chlorpyrifos and

corresponding maximum single applic ation rates, equipment types, re-entry inte rvals (REIs),

etc. HED believes that the detailed information on application rates and use patterns presented in

Appendix 9 (Master Use Summary Document) will be implemented on all chlorpyrifos labels

and is the basis of the occupationa l and residential risk assessment . If, for any reason, the final

chlorpyrifos labels contain highe r application rates, the actual risks posed by those products may

exceed the risks estimated in this assessment.

Body weights: For adults, when an endpoint is not sex-specific a body weight of 80 kg is

typically used in occupational ri sk assessment. However, in this case, a female-specific body

weight of 69 kg was used. While the endpointof concern, RBC AChE inhibition, is not sexspecific, the female body weight was used due to concerns for neurodevelopmental effects

related to early life e xposure to chlorpyrifos.

Unit Exposures: It is the policy of HED to use the best av ailable data to assess handler exposure.

Sources of generic handler data, used as surrogate data in the absence of chemical-specific data,

include PHED 1.1, the AHETF database, the Outdoor Residential Exposure Task Force

(ORETF) database, or other registrant-submitted occupational exposure studies. Some of these

data are proprietary (e.g., AHETF data), and subject to the data protection provisions of FIFRA.

The standard values recommended for use in pred icting handler exposure that are used in this

updated assessment, known as “unit exposures”, are outlined in the “Occupational Pesticide

Handler Unit Exposure Surrogate Reference Table 50 

# ”, which, along with additional information

50 

# Available: http://www.epa.gov/opp00001/scie nce/handler-expo sure-table.pdf

# Chlorpyrifos Human Health Risk Assessment D424485

Page 100 of 531 on HED policy on use of surrogate data, including descriptions of the various sources, can be

found at the Agency website. 51



# Area Treated or Amount Handled: For the updated assessment, the daily areas treated were

defined for each handler scenario (in appropriate units) by determ ining the amount that can be

reasonably treated in a single day (e.g., acres, square feet, or gallons per da y). When possible, the

assumptions for daily areas treated are taken from the HED Science Advisory Committee on

Exposure (ExpoSAC) Policy 9.1: Standard Values fo r Daily Acres Treated in Agriculture, which

was completed on July 5, 2000. However, standa rd values are not available for numerous

scenarios. The area treated assumptions identifi ed in the 2011 occupational and residential

exposure assessment are largely the same as used for the updated except for the following

changes:

 Paint Roller/Brush: the 2011 assessment a ssumed 110 gallons for treatment of sewer

manhole covers, the current assumes 20 gallons for coverage of 50 manholes; 3 gallons

applied for all other uses as was done previously;

 Fire Ant Mounds: the previous assessment a ssumed 25 mounds were treated, the current

assumes that an area of 1,000 square feet is treated.

Exposure Duration: As described previously, the steady state approach is used and is

appropriate for chlorpyrifos gi ven the toxicological and exposu re profile. The steady state

approach accounts for the short-te rm exposure duration, as well as for workers that are exposed

over longer periods of time (i.e., intermediate-t erm exposures). Long-term exposures are not

expected based on the current chlorpyrifos uses.

Mitigation/Personal Protective Equipment: Estimates of dermal and inhalation exposure were

calculated for various levels of personal prot ective equipment (PPE). The lowest tier is

designated as the baseline exposure scenario (i.e., long-sl eeve shirt, long pants, shoes, socks, and

no respirator). If risks are of concern at base line attire, then increasi ng levels of personal

protective equipment or PPE (e.g., gloves, doubl e-layer body protection, a nd respirators) are

evaluated. If risks remain a concern with maximum PPE, then engineering controls (e.g.,

enclosed cabs or cockpits, water-soluble pack aging, and closed mixing/loading systems) are

evaluated. This approach is used to ensure that the lowest level of risk mitigation that provides

adequate protection is selected, since the addition of PPE and engineering controls involves an

additional expense to the user and – in the case of PPE – also involves an additional burden to

the user due to decreased comfort and dexterity and increased heat stress and respiratory stress.

An assessment of handler exposure from the use of chlorpyrifos total release foggers was not

performed because dermal and inhalation exposure is expected to be negligible based on the use

pattern for this method of application. A total release fogger is an ae rosol pesticide device

designed to automatically release its total cont ent in one operation for the purpose of creating a

permeating fog within a space to deliver the pes ticide throughout the space. Therefore, total

release aerosols do not need any other application equipment (PR NOTICE 98-6, 1998).

Furthermore, the total release fogger product (EPA Reg. No. 499-405) directions state that once

51 

# Available: http://www.epa.gov/pesticides/sci ence/handler-exposure-data.html

# Chlorpyrifos Human Health Risk Assessment D424485

Page 101 of 531 cans are activated to, “leave greenhouse at onc e,” and that, “all human occupants must be

removed before treatment.” In accordance with a common integrated pest management (IPM)

and preventive safety practice, if multiple fogge rs are to be activated, the chlorpyrifos total

release fogger label directs the us e to “start with the can fart hest away from the exit door.”

The assessment of handler exposure from the use of chlorpyrifos ear tag and roach bait stations

were also not conducted. Dermal exposure is exp ected to be negligible if gloves are worn, and

inhalation exposure from these uses is not anticipated.

Exposure Data for Handler Exposure Scenarios: HED uses unit exposures to assess handler

exposures to pesticides. Unit exposures are es timates of the amount of exposure to an active

ingredient a handler receives wh ile performing various handler ta sks and are expressed in terms

of micrograms or milligrams of active ingredient per pounds of active ingredient handled. HED

has developed a series of unit exposures that are unique for each scenario typically considered in

our assessments (i.e., there are different unit exposures for different types of application

equipment, job functions, and levels of protect ion). The current assessment makes use of the

document, Occupational Pesticide Handler Unit Expos ure Surrogate Reference Table – Revised

March 2013 , was publically released to provide guid ance for all of the currently recommended

unit exposures for standard EPA occupational handler exposure scenarios. 52 

# A summary of all

exposure data used to conduct the occupational handler risk assessment is presented in the

updated occupational and residential exposure assessment.

In addition to this surrogate data, one non-chem ical specific exposure study was used for the

updated assessment. The exposure study was used to assess the use of SmartBox® technology

for the mixing/loading of granular formulati ons for aerial and ground applications, and the

application by ground equipment. Separate data evaluation records (DERs) were completed for

the mixing/loading 53 

# and applicator 54 

# studies. A summary of the study is presented in the updated

occupational and residential exposure assessment.

Summary of Occupational Handler Exposure Data Gaps: Consistent with the 2011 occupational

and residential exposure assessment, no new data gaps have been identifie d for the assessment of

occupational handler exposure/risk in addition to those identified in the Registration Eligibility

Decision (RED) for chlorpyrifos (finalized 7/31/06; IRED issu ed 2/2002). Based on a 90-day

progress report submitted (August 28, 2003) by th e technical registrant, Dow AgroSciences,

many of the data requirements are anticipated to be supported by the generation of data by the

Agricultural Handlers Exposure Task Force ( AHETF). These data requirements include

exposure data for:

 seed treatment uses;

 mixing wettable powders for aerial/chemigation application;

 loading and applying granular s for aerial application;

52 

# http://www.epa.gov/opp00001/science/handler-exposure-data.html

53 

# L. LaSota. Evaluation of Loader E xposure Data Submitted as a Part of th e Conditional Registration of Fortress

5G (Chlorethoxyfos). EPA MRID: 44180401. 1/23/1997. D232274.

54 

# J. Arthur. Review of “Chlorethoxyfos Applicator Exposure Study during Application of Fortress® 5G Granular

Insecticide using the Smartbox  

# System during Corn Planting in the Midwest.” EPA MRID: 443013-01. 7/27/1998.

D238361.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 102 of 531  groundboom application; and

 human flaggers for aerial application.

AHETF data for groundboom application and for mi xing/loading liquid formulations have been

submitted and reviewed by HED and used to esti mate exposure/risk for occupational handlers

from the exposure scenario and, therefore, satisfythe data requirement. AHETF data for seed

treatment and planting treated seed has been submitted and is currently under review by HED.

The requirement for exposure data for mixing wettable powders for aerial/chemigation

applications should be waived because the formul ation is no longer support ed by the registrant.

An exposure study for loading granular formula tions is being planned under AHETF. A study

protocol was submitted and reviewed by the Hu man Studies Review Bo ard (HSRB) November

2014. It does not appear, however, that flaggers are being supported by DAS as part of their

prior response to the data requirements.

In accordance with 40CFR158, the following data are generally required for all occupational or

residential pesticide uses: dislodgeable foliar residue (DFR) data are required for all occupational

(e.g., crop, nursery, greenhouse use sites) or reside ntial (e.g., ornamental and vegetable gardens,

pick your own farms, retail tree farms) uses that could result in post-application exposure to

foliage; and turf transferable residue (TTR) data are required for all occupational (e.g., sod

farms, golf courses, parks, and recrea tional areas) or residential turf uses that could result in postapplication exposure to turf. Adequate DFR a nd TTR data are available for chlorpyrifos.

Occupational Handler Non-Cancer E xposure and Risk Estimate Equations

The algorithms used to estimat e non-cancer exposure and dose fo r occupational handlers can be

found in the updated occupa tional and residential exposure assessment (D424484).

Combining Exposures/Risk Estimates:

Dermal and inhalation risk estimates were combin ed in this assessment, since the toxicological

endpoint, RBC AChE inhibition, is the same for these exposure routes.

Summary of Occupational Ha ndler Non-Cancer Exposures and Risk Estimates

Steady state exposure and risks calculated for o ccupational handlers (other than commercial seed

treatment) for all use patterns are presented in the updated occupational and residential exposure

assessment (Appendix G of D424484).

Current labels generally require that handlers use normal work clothing (i.e., long sleeved shirt

and pants, shoes and socks) and coveralls, chemi cal resistant gloves, and dust/mist respirators.

Also, some products are marketed in engineering controls such as water soluble packets. In

order to determine what level of personal protection is required to alleviat e risk concerns and to

ascertain if label modifications are needed, st eady state exposure and risk estimates were

calculated for occupational handlers of chlorpyrifos for a variety of scenarios at differing levels

of personal protection including en gineering controls. In this assessment, a total of 285 total

occupational handler exposure scen arios were assessed and 132 of them are not of concern (i.e.,

MOEs are ≥ 100) at current product label requirement s. Risks of concern for 27 additional

# Chlorpyrifos Human Health Risk Assessment D424485

Page 103 of 531 exposure scenarios could potentially be mitigated if additional personal protective equipment or

engineering controls are used. Risks of concern were estima ted for 126 exposure scenarios and

remain a concern regardless of the levels of personal protect ion and engineering controls

considered. These risks could be mitigated us ing the highest level of personal protection

available and by modification to ot her label requirements such as application rate or, in some

cases, deleting certain types of application equipment.

Commercial Seed Treatment

Based upon the registered seed treatment uses of chlorpyrifos, occupational handler exposure is

expected for individuals involve d in commercial seed treatment. Post-application exposure for

agricultural (field) workers is not expected based on the seed treatment use of chlorpyrifos.

The 2011 occupational commercial seed treat ment assessment has been updated. All

occupational exposure scenarios a ssessed, algorithms, and unit expos ures used (taken from HED

ExpoSAC Policy 14: SOPs for Seed Treatment) are the same as we re used previously in the 2011

occupational and residential exposure assessmen t for the assessment of commercial seed

treatment. However, the assessment of occupati onal seed treatment uses has been updated to

reflect the use of the guidance document, Interim Guidance (SOP 15.1) Amount of Commercial

Seed Treated per Day, updated March 21, 2013, as well as the updated default adult (female)

body weight. Also, seed treatment risk was es timated with use of PBPK-PD modeled PoDs

(dermal and inhalation) specific for occupational assessment.

The steady state approach is appropriate for th e assessment of commercial seed treatment with

chlorpyrifos given the toxicological and exposur e profile. The steady stat e endpoint selection for

chlorpyrifos accounts for HED’s traditional short- term exposure duration endpoint selection, as

well as being appropriately health protective for workers that ar e exposed over l onger periods of

time (i.e., intermediate-term exposures).

Summary of Commercial Seed Treatment Exposures and Risk Estimates

Steady state exposure and risks calculated for occupational handlers perf orming seed treatment

activities in commercial settings and for o ccupational secondary handlers from planting

chlorpyrifos-treated seeds are presented in the updated occupa tional and residential exposure

assessment (Appendix H of D424484).

Of the 68 exposure scenarios assessed, 36 resulted in risk estimates which were not of concern

(i.e., MOEs are ≥ 100) at the level of personal pr otection currently required by product

labels. Secondary handler (seed planter) scenario s were also evaluated an d none were of concern

at current label requirements. The remaining 32 seed treatment scenarios are of potential

concern.



# 9.2 Steady State Occupational Post-Application Risk

# Chlorpyrifos Human Health Risk Assessment D424485

Page 104 of 531 

# 9.2.1 Dermal Post-Application Risk

HED uses the term post-application to describe exposures that occur when individuals are

present in an environment that has been previously treated with a pe sticide (also referred to as reentry exposure). Such exposures may occur when workers enter previously treated areas to

perform job functions, including ac tivities related to crop producti on, such as scouting for pests

or harvesting. Post-application e xposure levels vary over time and depend on such things as the

type of activity, the nature of the crop or target that was trea ted, the type of pesticide application,

and the chemical’s degradation properties. In addition, the timing of pesticide applications,

relative to harvest activities, can greatly reduce the potential for post-application exposure.

The 2011 occupational post-application exposure a ssessment has been updated to include the

following: 1) a PBPK-PD modeled dermal PoD specific for occupational assessment, 2) the

master use document, 3) the updated adult (fem ale) default body weight and 4) the changes

relating to agricultural transfer coe fficients (TC) as described in the Science Advisory Council for

Exposure (ExpoSAC) Policy 3 – Revised March 2013. 55



# Occupational Post-application Dermal Exposure Data and Assumptions

A series of assumptions and exposure factors serv ed as the basis for completing the occupational

post-application risk assessments. The majority of assumptions and exposure factor detailed in

the 2011 exposure assessment are the same, except for changes relating to agricultural transfer

coefficients (TC) as described in the Science Advisory Council for Exposure (ExpoSAC) Policy 3

– Revised March 2013 and the standard adult body weight assumptions.

Post-application Exposure Data: The 2011 occupa tional and residential exposure assessment

made use of seven chemical-specific DFR studies that encompass the use of five different

formulations and 12 different crops. Specifica lly, the DFR studies examine the use of 1)

emulsifiable concentrate formulations on sugar beet s, pecans, citrus, sweet corn, cotton, and turf;

2) wettable powder formulations on almonds, appl es, pecans, cauliflower, tomato and turf; 3)

granular formulations on sweet corn and tu rf; 4) a total release aerosol formulation on

ornamentals; and 5) a microencapsulated formulation on ornamentals. The submitted studies

were reviewed by HED. Despite limitations, HE D recommended the use of all or some of the

data in the studies to assess pos t-application risks to chlorpyrifos except for the tomato DFR

data. Summaries for all DFR studies can be referenced in the updated occupational and

residential exposure assessment (Appendix I of D424484).

The current assessment makes use of the same DF R data and crop pairings as was done for the

2011 occupational and residential exposure asse ssment. For example, DFR data for an

individual crop was applied to that specific crop, as well as to crops in the same crop grouping

(e.g., cauliflower data was used for cauliflower a nd all other cole crops). For other crops for

which no crop-specific or crop group-specific data are available, the DFR data for the crop

deemed the closest match were used as surro gates to calculate pot ential exposure (e.g.,

cauliflower data were also used for strawber ries, cranberries, and leafy vegetables).

Additionally, whenever possible, a label use was assessed using DFR data for the same

55 

# http://www.epa.gov/opp00001/science/exposac-policy-3-march2013.pdf

# Chlorpyrifos Human Health Risk Assessment D424485

Page 105 of 531 formulation type. A full descripti on of the criteria for selection of DFR data for assessment of

post-application exposures to individual crops/c rop groupings can be referenced in Section 2.4.3

of the 2011 occupational and resident ial exposure assessment (D388165).

For occupational post-application dermal expos ures, the parent compound is the residue of

concern; dermal exposure to the oxon on foliar surfaces from reentry into an outdoor

environment previously treated w ith chlorpyrifos is not anticipat ed and, therefore, has not been

assessed. However, based on study results (see Section 9.2.2), it may be possible that the

formation of the oxon is greater and its deactivati on slower in greenhouses when compared to the

outdoor environment and that an assessment may be needed for exposure to the oxon in

greenhouse settings. In the 2011 Preliminary Human Health Risk Assessme nt, additional studies

to address uncertainties regarding the formati on of chlorpyrifos oxon and its decay following

applications in greenhouses we re recommended. To date, no additional data have been

submitted. Due to uncertainty regarding the form ation of chlorpyrifos oxon in greenhouses, HED

estimated risks for reentry into (microencapsulate d and total release fogger formulation) treated

greenhouses for parent chlorpyrifos only, as well as for parent chlorpyrif os plus chlorpyrifos

oxon using a total toxic residue approach. This cons ervative approach is assumed to result in a

health protective assessment of post-applicati on exposures to chlorpyr ifos oxon in greenhouses.

Summary of Occupational Post-application Exposure Data Gaps:

No new occupational data requirements have b een identified for chlorpyrifos; however, as

described above, additional data were recommended for the evaluation of chlorpyrifos oxon and

no data have been submitted to date.

Summary of Occupational Post-applicati on Dermal Exposure and Risk Estimates

Steady state occupational post-app lication dermal risk estimates are presented in the updated

occupational and residential exposure assessm ent (Appendix J of D424484) based on the PBPKPD model predicted dermal PoD.

Current labels require a Restri cted Entry Interval (REI) of 24 hours from most crops and

activities, but in some cases such as tree fruit, REIs are up to 5 days after application. All postapplication worker risks have b een updated in the current assessment. Results indicate that no

REI increase is required for the majority of outdoor environment crops and commodities with a

labeled REI (i.e., 43 of 55 total). However, fo r 12 crops, activities such as irrigation, hand

harvesting, scouting, and thinning re sult in risks of concern up to as many as 10 days following

application.

For the assessment of post-applica tion exposures to parent chlorpyr ifos (only) from ornamental

production in greenhouses, 4 formulations were assessed: emulsifi able concentrate,

microencapsulated liquid, wettable powder in WSP, and total release fogger. An REI increase of

up to 5 days may be needed to alleviate risks fr om with use of the emulsifiable concentrate,

wettable powder in WSP and tota l release fogger formulations. For the microencapsulated liquid

formulation, the estimated REIs range from 0 to > 35 days after treatment (the completion of the

monitoring period) dependent on the exposure activity considered.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 106 of 531 

# 9.2.2 Occupational Post-application Dermal Exposure/Risk Estimates:

Chlorpyrifos Oxon

Chlorpyrifos is activated by desu lfuration, reacting in bioactivati on to the more toxic and potent

acetyl cholinesterase (AChE) inhibitor, chlo rpyrifos oxon. The oxon is highly unstable due to

rapid deactivation through hydroly tic cleavage by a process calle d dearylation which releases

TCP. Workers reentering an indoor environmen t (i.e., greenhouses) prev iously treated with

chlorpyrifos could potentially be exposed to th e oxon as chlorpyrifos degrades. Available

exposure data indicate chlorpyrifos oxon may form in indoor environments. 56 

# Toxicity

adjustment factors (TAFs) were used to estima te the potency of chlo rpyrifos oxon relative to

chlorpyrifos. HED determined the oxon to be be tween 11.9 (acute) and 18 (chronic) times more

toxic than the parent.

Dermal exposure to the oxon on foliar surfaces from reentry into an outdoor environment (e.g.,

field crops and orchards) previous ly treated with chlorpyrifos is not anticipated and, therefore,

has not been assessed. No occupational exposur e studies (handler, post- application, or DFR)

were identified that quan tified the levels of oxon present in th e environment. However, a search

of open literature for the 2011 assessment resulted in 4 plant metabolism studies which measured

surface residues. Three plant metabolism studies 57 

# measured leaf surface residues of the oxon in

outdoor environments that were either well below the parent, not detectab le, or detected at a

level just above the level of de tection (LOD). The potential fo r exposure to the oxon is further

minimized due to rapid deactivation of the oxon to TCP. Further, the dietary exposure risk

assessment 58 

# conducted in support of registration review concludes the follo wing, “all residues in

food are assumed to be parent chlorpyrifos sinc e the chlorpyrifos oxon is not typically found in

foods in monitoring data or crop field trials.”

The 4 th 

# plant metabolism study, a tomato and green bean greenhouse metabolism study, was less

definitive than the other three plant metabo lism studies regarding oxon presence. HED is

concerned that the formation of the oxon may be greater and its deactivation to TCP slower in

greenhouses when compared to the outdoor envi ronment. The study results indicate that oxon

residue is from 9 to 14X less than the parent from fruit analyzed on the day of application in flat

and asymmetric roof greenhouses. The proportion of oxon to parent is less for all days which

measurable levels were observed (all but 8 and 15 days after application). The oxon was

detected until day 5 with levels between 5 and 6X below that of the parent. It should be noted

that residues of chlorpyrifos and oxon were measured from analysis of whole fruit samples.

HED typically assesses occupational post-appli cation exposure and risk based upon the potential

for transfer from surface residues. The whole fr uit samples, which include surface residues, as

56 

# J.L. Martinez Vidal, et al. 1998. Diminution of Chlorpyrifos and Chlorpyrifos Oxon in Tomatoes and Green

Beans Grown in Greenhouses. J. of Agric. and Food Chem. 46 (4), 1440–1444

.

57 

# Iwata, Y. et al. 1983. Chlo rpyrifos Applied to Ca lifornia Citrus: Residue Levels on Foliage and On and In Fruit.

J. Agric. Food Chem. 31(3), 603-610.

H. Jin and G.R. Webster. 1997. Persistence, Penetratio n, and Surface Availability of Chlorpyrifos, Its Oxon, and

3,5,6-Trichloro-2-pyridinol in Elm Bark. 45(12), 4871-4876.

R. Putnam, et al. 2003. The Persisten ce and Degradation of Chlorthalonil and Chlorpyrifos in a Cranberry Bog. J.

Agric. Food Chem. 51(1), 170-176.

58 

# D. Drew. Chlorpyrifos: Acute and St eady State Dietary (Food Only) Exposure Analysis to Support Registration

Review. 11/18/2014. U.S. EPA Office of Chemi cal Safety and Pollution Prevention. D424486.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 107 of 531 well as residues which may have been containe d within the fruit sample, may overestimate the

amount of oxon on the fruit surface. Regardless, the 2011 occupational and residential exposure

assessment recommended additional data to meas ure the chlorpyrifos a nd oxon residues on leaf

surfaces following treatment with a liquid formul ation in greenhouses in order to address these

uncertainties and more accurately address the risk potential for exposure from occupational

reentry into greenhouses treated with chlorpyrif os. To date, no data has been submitted to

address these uncertainties. As a result, HED has conducted an assessment of occupational postapplication exposures in greenhous es that uses conservative a ssumptions of oxon formation.

In order to account for the fo rmation of and potential increas ed toxicity from exposure to

chlorpyrifos oxon, a total toxic residue approach was applied which combines chlorpyrifos and

chlorpyrifos oxon (expressed as toxicity equi valents). The total toxic residue approach 59



# estimates the chlorpyrifos oxon equivalent resi dues by 1) assuming a specific fraction of the

measured chlorpyrifos dislodgeable foliar residues are available as the oxon and 2) factoring in

the relative potency of chlorpyrif os oxon with use of a TAF. It was conservatively assumed that

5% (0.05) of the total chlorpyrifos present as DF R in greenhouses is avai lable for worker contact

during post-application activities. This assumption is based on a review of available TTR and

DFR data for other OPs where both the parent and metabolite were measured in residue

samples. Five percent was found to be the hi gh-end value for the per cent of parent that

metabolized during the course of the residue studies. The chronic TAF (which is appropriate for

steady state assessment) of 18 wa s derived from BMD analysis of inhibition of RBC AChE in

adult female rats (adult male rats not examin ed) observed in the rep eated phase of the CCA

study. Once predicted, these tota l toxic (dislodgeable foliar) residues are used to estimate

exposures from post-application activities in gree nhouse and risks are estimated with used of the

steady state PoD for occupationa l exposures, 4.9 mg/kg/day.

Summary of Occupational Post-application Dermal Exposure and Risk Estimates with Use of

Total Toxic Residue Approach

Due to uncertainty regarding the formation of chlorpyrifos oxon in greenhouses, HED also

estimated risks for reentry into treated gree nhouses (all 4 formulations) for the parent

chlorpyrifos plus chlorpyrifos oxon using a total toxic residue a pproach. When the total toxic

residue approach is used to eval uate risks from exposure to the oxon with use of the emulsifiable

concentrate, wettable powder in WSP, and total release fogger formulations, results indicate that

an REI increase up to 6 days may be need ed to alleviate risk concerns. For the

microencapsulated liquid formulation, REIs range from 3 to > 35 days after treatment (the

completion of the monitoring period) dependent on the exposure activit y considered.



# 9.2.3 Inhalation Post-application Risk

There are multiple potential sources of post-app lication inhalation exposure to individuals

performing post-applicati on activities in previously treated fields. These potential sources

include volatilization of pesticides and resuspension of dusts and/or particulat es that contain

pesticides. As described in Section 6.3.2 of this document, a quantitative risk assessment is not

59 

# Total DFR (μg/cm 2 

# ) = [ Chlorpyrifos DFR (μg/cm 2 

# ) * TAF] + [ Chlorpyrifos DFR (μg/cm 2 

# )]

# Chlorpyrifos Human Health Risk Assessment D424485

Page 108 of 531 required for chlorpyrifos or chlorpyrifos oxon due to the lack of toxicity of these chemicals when

in the vapor state, even at th e saturation concentration.

The Worker Protection Standard for Agricultural Pesticides contains requ irements for protecting

workers from inhalation exposures during and af ter greenhouse applications through the use of

ventilation requirements.[40 CFR 170.110, (3) (Restrictions a ssociated with pesticide

applications)].

A post-application inhalation exposu re assessment is not required as exposure is expected to be

negligible. Seed treatment a ssessments provide quantitative i nhalation exposure assessments for

seed treaters and secondary handlers (i.e., planters). It is expected that these exposure estimates

would be protective of any poten tial low-level post-a pplication inhalation exposure that could

result from these types of applications.



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# 11. List of Appendices

Appendix 1. Evaluation of E xperimental Toxicology Data

Appendix 2. Detailed Review and Synthesis of Three Children’s Enviro nmental Health Cohort

Studies

Appendix 3. Epidemiology Study Specific Evaluations

Appendix 4. Detailed Summary Tables of Ch ildren’s Environmental Health Epidemiology

Studies

Appendix 5. Summary of OPP’s Ch E Policy & Use of BMD Modeling

Appendix 6. Columbia Center for Children’s Environmental Health (CCCEH) Epidemiology

Data Acquisition “Raw Data” Request

Appendix 7. Physical/Chemical Properties

Appendix 8. Current U.S. Tolerances and International Residue Limits

Appendix 9. Master Use Summary Document

Appendix 10. Dose Reconstruction Analysis

Appendix 11. New Literature on Chlorpyrifos since the 2012 FIFRA SAP Meeting

# Chlorpyrifos Human Health Risk Assessment D424485

Page 126 of 531 

# Appendix 1. Evaluation of Experimental Toxicology Data

# Chlorpyrifos Human Health Risk Assessment D424485

Page 127 of 531 1.0. Introduction

Chlorpyrifos ( 0

, 0 -diethyl- 0 -3,5,6-trichloro -2-pyridyl phosphorothioate) is a broad-spectrum, chlorinated

organophosphate (OP) insecticide. In 2000, most residential uses were voluntarily cancelled by Dow

AgroSciences but agricultura l use remains. In 2011, the Agency re leased a preliminary human health risk

assessment for chlorpyrifos. The focus of the 2011 risk assessment was on the cholinesterase (ChE)

inhibiting potential of chlorpyrifos including in young an imals. Like other OPs, chlorpyrifos binds to and

phosphorylates the enzyme acetylcholinesterase (AChE) in both the central (brain) and peripheral nervous

systems. This can lead to accumulation of acetylchol ine and, ultimately, at sufficiently high doses, to

clinical signs of toxicity. Consistent with the fo cus on ChE inhibition, in the 2011 preliminary risk

assessment, EPA evaluated the extens ive database of ChE data for multiple lifestages and selected points

of departure based on consideration of all quality and reliable data.

In 2008 and 2012, the Federal Insec ticide, Fungicide and Rodenticide Act (FIFRA) Scientific Advisory

Panel (SAP) reviewed two draft science issue papers on the human health effects of chlorpyrifos which

provided a preliminary review of the scientific litera ture on experimental toxicology and epidemiology

studies available since the 2000 risk assessment. These draft issue papers considered a growing body of

literature with laboratory animals (rats and mice) i ndicating that gestational and/or early postnatal

exposure to chlorpyrifos may cause persistent effects into adulthood in addition to epidemiology studies

evaluating prenatal chlorpyrifos exposure in mother-inf ant pairs that have reported associations with birth

outcomes, childhood neurobehavioral and neurodevelopm ent outcomes in the offspring when evaluated in

neonates, infants, and young children. In 2008 and 2012, the SAP agreed with the Agency that although

the epidemiology studies were high quality with respect to design, conduct and analyses and provided

information for hazard characterization, ChE inhibitio n remained the most robust dose response data for

deriving points of departure. This appendix inco rporates components from the draft SAP issues papers,

comments from the SAP and from the public on those drafts and new studies since 2012 60



# .

In 2010, the Agency developed a draft “Framework for Incorporating Human Epidemiologic & Incident

Data in Health Risk Assessment” which provides th e foundation for evaluating multiple lines of scientific

evidence in the context of the understanding of the ad verse outcome pathway (or mode of action; (U.S.

Environmental Protection Agency, 2010). The draft framework, which includes two key components:

problem formulation and use of the mode of action/ adverse outcome pathway frameworks, was reviewed

favorably by the SAP in 2010 (FIFRA SAP, 2010). OPP’ s draft framework is consistent with updates to

the World Health Organization/International Progr amme on Chemical Safety mode of action/human

relevance framework which highlight the importance of problem formulation and the need to integrate

information at different levels of biological or ganization (Meek et al, 2014). Consistent with

recommendations by the NRC in its 2009 report, OP P’s draft framework describes the importance of

using problem formulation at the beginning of a comp lex scientific analysis. The problem formulation

stage starts with planning dialogue with risk managers to identify goals for the analysis and possible risk

management strategies. This initial dialogue provides the regulatory context for the scientific analysis

and helps define the scope of such an analysis. The problem formulation stage also involves

consideration of the available information regarding the pesticide use/usage, toxicological effects of

concern and exposure pathways and duration along with key gaps in data or scientific information.

Specific to chlorpyrifos, the 2008 and 2012 SAP review s represent the problem formulation analyses for

the weight of evidence (WOE) analysis provide d in the main body of the risk assessment.

60 

# The agency is aware of multiple review articles re lated to topics in this appendix published in the

scientific literature (Prueitt et al, 2011; Goodman et al, 2013; Li et al, 2012; Li et al, 2014; Mink et al,

2014; Reiss et al, 2012). EPA has conducted an inde pendent review of the scientific literature.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 128 of 531 Mode of action (Boobis et al., 2006; Boobis et al., 2008; U.S. EPA, 2005; Simon et al, 2014; Meek et al,

2014) and adverse outcome pathway (Ankley et al., 2010) provide important concepts in this integrative

analysis. Both a mode of action and an adverse outcome pathway are based on the premise that an

adverse effect caused by exposure to a compound can be described by a series of causally linked

biological key events that result in an adverse hum an health or ecological outcome. One of the key

components of the agency’s draft framework is the use the mode of action framework /adverse outcome

pathway concept as a tool for organizing and integrat ing information from different sources to inform the

causal nature of links observed in both experimental and observational studies. Specifically, the modified

Bradford Hill Criteria are used to evaluate the expe rimental support that establis hes key events within a

mode of action or an adverse outcome pathway, a nd explicitly considers such concepts as strength,

consistency, dose response, temporal concordance a nd biological plausibility in a weight of evidence

analysis.

The draft framework is oriented around the source to outcome pathway (Figure App 1-1) discussed in the

National Research Council’s (NRC) report on toxicity testing in the 21 st 

# Century (National Research

Council, 2007).Chlorpyrifos Human Health Risk Assessment D424485Page129of531FigureApp1‐1.Sourcetoeffectspathway(adaptedfromNRC,2007)ChemicalsMolecularTargetCellularResponseTissue/OrganIndividualPopulationPharmaco‐kineticsInvitrostudiesBiomonitoring&ExposuredataEpidemiologyStructureActivityRelationshipsToxicityPathwaysHumanIncidentsInvivostudies

# Chlorpyrifos Human Health Risk Assessment D424485

Page 130 of 531 2.0. Conceptual Framework

As an aid in organizing the available information and to identify the complex scientific issues

being considered, the agency developed a conceptual framework. This conceptual framework

(Figure App 1-2) is consistent with the sour ce-to-outcome pathway and provides the foundation

for the analysis being undertaken by the agency to delineate outcomes derived from AChE

inhibition in different lifestages and from alternative modes/mechanisms (Figure App 1-1). This

conceptual framework provides key informati on on which lifestages are thought be the most

susceptible to chlorpyrifos, the exposure pa thway by which these individuals are exposed,

possible adverse outcome pathways and related cr itical duration(s) of exposure leading from

exposure to adverse health outcome.

Figure App 1-2. Conceptual framework considering ch lorpyrifos exposure, lifestages, critical durations,

modes of action/adverse outcome pathw ays, and health outcomes of interest.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 131 of 531 2.1. Adverse Outcomes

AChE inhibition is the well-established cholinergic mode of action for OPs and is typically used

as the critical effect in hazard characterization for members of this class of pesticides. The 2008

and 2012 SAPs concurred with the agency that AChE/ChE data when taking into account

sensitive lifestages remains the most robust dose re sponse data for use in derivation of points of

departure. However, over th e last 10-15 years, experime ntal toxicology studies on

neurotoxicological as well as ep idemiology studies have been pub lished which suggest that the

developing brain of the fetus a nd young children may also be affected. There are a significant

number of literature studies evaluating neurobeha vioral outcomes in experimental animals (rats,

mice). These studies vary in their study de sign but many involve gestational and/or early

postnatal dosing with behavioral evaluation in adulthood. Epidemiological studies from three

cohorts of mothers and children, funded in part by EPA and National Institutes of Environmental

Health, have reported birth and neurodevelopm ental outcomes associated with prenatal

exposures to OPs. Two of these (Mt. Sina i and Berkeley) have focused primarily upon nonspecific urinary biomarkers of OP expos ure, the dialkyl phosphate metabolites ( i.e. , DAPs), and

thus present some uncertainty as to the ex tent these results relate to chlorpyrifos per se . The

Columbia University investigat ors are studying a cohort of moth ers and children in inner-city

New York and have published multiple papers on associations among level of prenatal

chlorpyrifos exposure and birth and neurodevelopmental outcomes at multiple time points from

birth through age seven years. With resp ect to neurodevelopmental outcomes, multiple

investigators have reported hypothesized modes/ mechanisms of action. In the conceptual

framework, both adverse health outcomes of inte rest for chlorpyrifos are being tracked—AChE

inhibition and potential for neurodevelopmental effects.

2.2. Potentially Sensitive Lifestages

The agency believes that pregnant women a nd their fetuses, newborns, and young children

represent potential sensitive lifestages for exposur e to chlorpyrifos. This conclusion is based on

multiple lines of evidence.

 Pregnant Women:

Chlorpyrifos RBC AChE data from pregnant ra ts provides the most se nsitive data for use

in deriving a point of departure from rep eated dosing studies. Although it is unclear

whether metabolic changes during pregnancy are sufficient to affect metabolism at

environmental exposure, some have suggested that there may be reduced ability to

detoxify chlorpyrifos and/ or the oxon may affect sens itivity during pregnancy.

Metabolic activities can be altered during pregnancy (Anderson, 2006; Anger & PiquetteMiller, 2008; Bologa, et al, 1991; Carpinte ro et al, 1996; Czekaj et al, 2000, 2005;

Dickmann et al., 2008a, b; Ejiri et al, 2005; Ferre et al., 2006 ; Hines, 2007; Homma et al.,

2000; Howard & Sugden, 1993; Tsutsumi et al ., 2001). For example, Chanda et al. (2002)

showed that pregnant female rats had lo wer plasma, brain, and liver carboxylesterase

activity compared to non pregnant female s. Regarding A-esterase activity, Ferre et al.

(2006) showed that the paraoxonase ( i.e. , A-esterase in serum decreased from a

nonpregnant background of 146 U/L to 111 U/L in late gestation, indicating 76% of

normal activity in late gestat ion pregnant women. Carpin tero et al. (1996), however,

# Chlorpyrifos Human Health Risk Assessment D424485

Page 132 of 531 found that phenyl acetate metabolism increased from 23.6 to 33.5  kat/g in the third

trimester, but it must be kept in mind that phenyl acetate metabolism is not necessarily a

specific measure of A-esterase activity. Data in mice support the findings of Ferre et al.

(2006) in humans suggesting a reduction in A-esterase activity during pregnancy. In

mice, Weitman et al. (1983) found that PON1 activity after exposure to parathion was 50

nmol/min/ml in non-pregnant females, but it decreased as low as 14 nmol/min/ml during

gestation (Weitman, et al., 1983). With rega rd to plasma butylch olinesterase (BuChE)

activity, Howard et al. (1978) have shown that in six healthy pregna nt women levels of

plasma BuChE activity dropped by approximate ly 30% during the first trimester, but

returned to close to pre-pregnancy levels in the third trimester. Similarly, other

investigators have also repor ted decreases in plasma BuCh E activity in pregnant women

(de Peyster et al, 1994; Venkataraman et al , 1990; Whittaker, Crawford, & Lewis, 1988).

Evans et al. (1988) showed that serum ChE levels in 39 of 44 pregnant women dropped

after conception; in 20 of those women, the decline in ChE activity continued throughout

pregnancy.

Pregnancy is a remarkably dynamic biological process in which rapid changes occurring

in both the developing system (embryo/fetus) and the mother can significantly impact the

pharmacokinetics of chemicals. In addition to the potential for decreased clearance of

chemicals due to immature metabolic systems in the developing embryo/fetus (mentioned

above), other pregnancy-related changes can also have a pronounced effect on

pharmacokinetics. For a typical human pregnanc y, total body weight gain is in the order

of 10-30% while cardiac output can in crease as much as 50% (Corley et al , 2003a, b;

Young et al. , 1997a, b). A significant fraction of the body weight gain is due to increases

in total body water and fat that can lead to a greater volume of distribution by simple

dilution. For instance, increases in total body water dilute plasma proteins which can

increase the free fraction of highl y plasma protein-bound chemicals ( e.g. , chlorpyrifos)

available for distribution. Incr eases in fat, on the other hand, can provide a la rger storage

volume for lipophilic chemicals (Corley et al. , 2003). Additionally, significant changes

take place in the maternal circulatory sy stem to accommodate the development of

placental blood flow which undergoes consid erable increases throughout pregnancy

along with embryo/fetus which increases in volume over a billion-fold from conception

to birth (Young et al. , 1997).

 Fetuses, Infants, Toddlers & Young Children:

With respect to distribution to the fetus during gestation, there are multiple studies on

chlorpyrifos (Mattsson et al. , 1998, 2000; Qiao et al ., 2002) and other OPs (USEPA,

2006) which show that the pre gnant dam exhibits similar or more AChE inhibition than

the fetus at a given dose to the dam. As such, for AChE inhibition, protecting against

AChE inhibition in the pregnant female is e xpected to be protective for AChE inhibition

in the fetus. Biomonitoring data from rats and humans illustrate fetal exposure levels are

similar to maternal levels which sup port these findings. Specifically, Whyatt et al.

, (2003) have shown that levels of chlorpyrifos in maternal blood are similar to the levels

measured in human umbilical cord blood. In Hunter et al. , (1999), pregnant rats were

orally dosed with chlo rpyrifos for 5 days during late gestation at doses of 3 or 7

mg/kg/day, and the amounts of chlorpyrif os, chlorpyrifos-oxon, and TCPy were

quantified in fetal and matern al brain and liver. No chlo rpyrifos or its oxon were

# Chlorpyrifos Human Health Risk Assessment D424485

Page 133 of 531 detected. The concentration of TCPy in the fetal brain was higher than the TCPy

concentration in the maternal brain in time- course and dose-response studies but in liver

the maternal levels were higher. In a study by Mattsson et al. , (1998, 2000),

concentrations of chlorpyr ifos, the oxon, and TCPy were measured in the blood of

maternal and fetuses and TCPy levels in dam and fetal blood were similar and

chlorpyrifos levels were approximately 2-fold higher in maternal blood than fetal blood.

In another gavage gestati onal exposure study, Akhtar et al. , (2006) exposed rats to

chlorpyrifos from GD 0-20 in fetal and ma ternal tissues on GD21 at high doses (9.6-15

mg/kg/day); while there is a high degree of vari ability in the Akhtar et al study, dams and

fetuses showed similar levels of ch lorpyrifos in liver and brain.

With respect to neurodevelopmental outcomes, numerous epidemiological investigations

have observed a link between ear ly life exposure to chlorpyr ifos and adverse effects on

neurodevelopment through age seven years. Epidemiology studies focused on exposure

during gestation but the impact of post-natal exposure can no t be ruled out. Overall,

these studies are strong, well-c onducted studies in which likel y sources of error or bias

would more likely tend to underestimate, ra ther than overestimate an effect size ( e.g.

, relative risk measure). In addition, there a growing body of literature with laboratory

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

chlorpyrifos may cause persiste nt effects into adulthood.

Potential sensitivity, particularly observed in acute, single dose exposures, is largely

derived from immature metabolic systems that have less capacity to detoxify the parent

OP and its toxicologically active metabolite chlorpyrifos oxon. This sensitivity is not

derived from differential inhibition of the AChE en zyme itself as supported by in vitro

studies (Benke and Murphy, 1975; Chanda et al. , 1995; Mortensen et al. , 1996;

Atterberry et al. , 1997). Rat fetuses and juveniles a nd human fetuses, infants, and young

children have lower capacity to detoxify chlorpyr ifos than adults. Specifically, in rats, Aesterase activity is virtually nonexistent in the fetus (Lassiter et al. , 1998) and increases

from birth to reach adult levels around PND21 (Mortensen et al ., 1996; Li et al. , 1997).

Mortenson et al. , (1996) showed that in the plasma le vel of A-esterase in 4-day old rats

was 1/11 that of adult animals. The animal data regarding the role of carboxylesterase in

mediating OP toxicity are also quite extensive (e.g., Clement, 1984; Fonnum et al. , 1985;

Maxwell, 1992 a, b). Fetal rats and mice posse ss very little carboxyles terase activity with

increasing activity as the postnatal rodent matures, reaching adult values around puberty

(Lassiter et al. , 1998; Morgan et al. , 1994; Moser et al. , 1998; Karanth and Pope, 2000;

Zhu et al. , 2009). The temporal pattern of A- esterase and carboxylesterase activity

correlates well with studies on OP sensitivity. Several studies have shown an increased

sensitivity of newborn rats to OP compounds which are detoxified via the A-esterase

and/or carboxylesterase pa thways (e.g., Gagne and Brode ur, 1972; Benke and Murphy,

1975; Pope et al. , 1991; Chambers and Carr, 1993; Padilla et al. , 2000; 2002; Karanth

and Pope, 2000).

While there are fewer data in human tissues which could evaluate age-related maturation

of carboxylesterase and A-esterase expressi on, there are numerous st udies that present

age differences in liver P-450 metabolism in humans that also play a role in OP

metabolism ( e.g. , review by Hines, 2007). Studies ev aluating maturational expression

and activity of liver carboxyleste rases in human liver tissues s how lowest levels in fetal

# Chlorpyrifos Human Health Risk Assessment D424485

Page 134 of 531 tissues, with increasing activ ity during childhood, although th ere are large individual

differences and thus hi gh variability (Pope et al. , 2005; Yang et al. , 2009; Zhu et al.

, 2009). Adult tissues were approximately 10-f old and 4-fold more active in hydrolyzing

certain chemicals than fetal and child (0- 10 years) tissues, respectively, and enzyme

expression differences were much greater (Yang et al. , 2009). In contrast, in Pope et al.

, (2005), the differences in activity were rela tively small (and not statistically significant)

between children ages 2–24 months and adu lts (20–36 years); howev er, that youngest age

evaluated in the study was 2 months old and this individual had th e lowest level of

carboxylesterase.

Serum A-esterase levels also appear to be very low in human infants and children

compared to adults (Augus tinsson and Barr, 1963; Cole et al. , 2003; Huen et al. , 2009;

Mueller et al

., 1983; Ecobichon and Stephens, 1973; Gonzalez et al. , 2012; Holland et

al. , 2006; Chen et al. , 2003). While some studies have shown that PON1 activity reaches

adult levels by about 2 years of age (e.g., Cole et al. , 2003), recent papers have reported

slightly lower levels as late as 9 years (Gonzalez et al., 2012). Reasons for these

differences could include type of measur ement (i.e., genotyping vs phenotyping), assay

substrate, sample sizes, and popula tion factors (e.g., ethnicity).

There are critical windows of vulnerabil ity (Rice & Barone, 2000; Rodier, 2004) with

regard to toxicant effects on brain developmen t. This vulnerable period in humans spans

early pregnancy to adolescence (Rice & Bar one, 2000). In fact, evidence shows that

synapse formation peaks quite late in huma n brain development at 4-8 years of age

(Glantz et al. , 2007). Within these vulnerable periods there are key neurodevelopmental

processes ( e.g. cell division, migration, differentiati on, synaptogenesis, and myleination)

and each of these is region and stage speci fic. Consequently, the time of toxicant

exposure will be a major determinate in the spectrum of neurotoxic effects

# Chlorpyrifos Human Health Risk Assessment D424485

Page 135 of 531 3.0. Adverse Outcome Pathways: AChE Inhibi tion & Plausible Pathways Leading to

Neurodevelopmental Outcomes

Mode of action and adverse outcome pathways provide important concepts in the draft

“Framework for Incorporating Human Epidemio logic & Incident Data in Health Risk

Assessment” (U.S. EPA, 2010). As mentioned above, both a mode of action and an adverse

outcome pathway (AOP) include a set of measurea ble key events that describe the biological

processes leading to an apical effect. Figure App1-3 is a graphical presentation of a generic

adverse outcome pathway (Ankley et al. , 2010). This figure is an extension of the source-tooutcome pathway and provides additional detail on the types of scientific information from

various levels of biological orga nization used in establishing an adverse outcome pathway. As

shown in the conceptual framework, two differe nt adverse outcomes in laboratory animals are

being evaluated: AChE/ChE inhibition and neur odevelopmental outcomes. Each of these is

discussed below.

Figure App1-3. Generic Advers e Outcome Pathway (Adapted from Ankley et al., 2010)



# 3.1. Adverse Outcome Pathway: AChE Inhibition

3.1.1. Initiating Event & Health Outcomes

AChE inhibition is the well-established mode of action for OPs and is typically used as the

critical effect in hazard characte rization for members of this class of pesticides. Specifically, the

initiating event for OP mediated neurotoxicity is in teraction at the serine re sidue at the active site

of the AChE. This interaction leads to accumula tion of acetylcholine and ultimately to clinical

signs of neurotoxicity at high doses . In experimental toxicity st udies in laboratory animals, high

acute doses produce signs of autonomic stimulation, ataxia, fasciculations, tremors, respiratory

# Chlorpyrifos Human Health Risk Assessment D424485

Page 136 of 531 difficulties, and convulsions. The Agency’s recent review of human incident reporting databases

(Recore & Oo, 2011) indicates that humans exposed to high levels of chlorpyrifos may complain

of gastrointestinal and neurologi cal effects consistent with acut e AChE poisoning. Specifically,

individuals have reported: naus ea, vomiting, diarrhea, tremors, headaches, dizziness, numbness

and tingling sensations, muscle spasm, shortness of breath, and seizures (Recore & Oo, 2011).

Although inhibition of AChE as the initiating ev ent in this adverse outcome pathway has long

been established, the quantitative dose respons e linkage between AChE inhibition and toxicity

has been shown to be more variable. In other words, the amount of AChE inhibition required to

elicit such responses from OP e xposure is not consistent but inst ead varies among different OPs,

different study designs, and different neurotoxic outcomes. Specific to chlorpyrifos, there are

limited human data which inform the quantit ative dose-response linkage between AChE

inhibition and apical neurotoxic effects. Th ere are two human deliber ate dosing studies in

human subjects (Nolan et al. , 1982) 61 

# which provides plasma ChE and RBC AChE data from a

single oral dose (0.5 mg/kg). AChE/ChE activ ity (plasma and RBC) was monitored at 2, 6, 12,

24 hours, and 2, 3, 4, 8, 14, 22, 27, and 30 days post dose. Despite plasma ChE inhibition of 83-

89% at the peak time of effect, no clinical signs of toxicity or inhibition of RBC ChE activity

were observed. Kisicki et al. (1999) provides data from si x human subjects exposed to

chlorpyrifos in a gelatin capsule at dose levels of 0 (lactose-filled gelatin capsule, 2 control

groups), 0.5, 1.0, or 2.0 mg/kg; RBC AChE activity was monitored at 2, 4, 8, 12, 24, 36, 48, 72,

96, 120, 144, and 168 hours post dose. In the Kisicki et al. (1999) study RBC AChE inhibition

changes were only seen in one person.

3.1.2. Dose Response Analysis for AChE Inhibition

In the 2011 preliminary risk asse ssment, the agency used typical reference dose and margin of

exposure approaches where points of departure are derived from toxicology studies in laboratory

animals and standard 10X factors for inter- and in tra-species extrapolation. In the revised risk

assessment, the agency is using a more sophistic ated approach with the PBPK-PD model. As

part of the 2008 SAP, the agency performed a comprehensive review of the literature on

available AChE data across multiple lifestages, durations, and routes (FIFRA SAP, 2008b). This

literature review was considered by the SAP at that time and pr ovided part of the scientific

rationale for the Panel’s recommendation that the ag ency continue to use AChE data in the most

sensitive lifestages for dose-re sponse analysis. The literature review was updated prior to the

2011 preliminary risk assessment, the 2012 SAP review, and again for the 2014 revised risk

assessment. Section 3.1.2.1 provides a summar y of the benchmark dose (BMD) modeling on

selected oral AChE studies conducted for the preliminary HHRA (2011). Section 3.1.2.2

provides additional dose-reponse data extracted fr om the 2008 draft issue paper reviewed by the

FIFRA SAP.

3.1.2.1. Summary of BMD Modeling Results

61 

# Nolan (1982) and Kisicki et al (1999) has been review ed by the Human Studies Revi ew Board (HSRB) and found

to be ethically and scientifically conducted ( http://www.epa.gov/osa/hsrb/files/june2009finalreport92609.pdf;

http://www.epa.gov/osa/hsrb/files/meeti ng-materials/apr-13-14-2011/appendix1.pdf ).

# Chlorpyrifos Human Health Risk Assessment D424485

Page 137 of 531 For the 2011 preliminary risk as sessment, the Agency conducted BMD modeling on selected oral

AChE studies 62 

# . These studies were selected based on th e availability of at least two treatment

groups in addition to a control group and those th at showed good dose response. The agency has

also focused on those studies where AChE was m easured at or near the time of peak effect,

typically within one to several hours after dosing. AChE data measured 24 hours or longer after

dosing will underestimate the am ount of AChE inhibition and are thus not appropriate for

deriving points of departure. In addition, the st udies used in the dose response assessment were

selected as they represented a variety of ages, lifestages, and durations. The agency focused on

those studies representing singl e and repeating dosing in post-natal exposure to rat ages (PND 10

and older); PND10 and older is considered to be approxima tely concordant with human

postnatal exposure (Benjamins & McKhann, 1981; Dobbing & Smart, 1974). AChE data from

adult laboratory animals (both pregnant and non- pregnant) from single dose and repeating dosing

studies has also been collected. The focus ha s been on AChE data from rats as these are

generally the most robust and sens itive studies. Data in fetuses for chlorpyrifos have not been

evaluating using BMD techniques as these studies tend to be less sensitive than those in postnatal juveniles and adults. The agency has c onducted BMD analysis on available data for blood

(RBC), brain (whole or sections as appropriate), a nd peripheral ( i.e. , heart) tissues.

Specifically, the agency used the decreasing e xponential dose-response m odel similar to that

used for the OP and N -methyl carbamate cumulative risk assessments and multiple risk

assessments for individual AChE inhibiting pest icides (U.S. EPA, 2006, 2007). The use of this

empirical dose-response procedure has been pr eviously reviewed and supported by the FIFRA

SAP on several occasions (2001, 2002, 2005a, 2005b, 2008bThe agency typically uses a 10%

decrease in brain and periphera l ChE inhibition as the response level in deriving points of

departure. This 10% response level is called th e benchmark response level (BMR). This level

has been shown to be a level which is protectiv e for cholinergic outcomes for OPs and can also

be reliably measured in most guideline experi mental toxicology studies (U.S. EPA, 2002, 2006).

The agency has calculated BMD 10 

# s and BMDL 10 

# s where the BMD 10 

# is the estimated dose where

AChE is inhibited by 10% compared to background and the BMDL 10 

# is the lower confidence

bound on the BMD 10 

# . As a matter of science policy, the agency uses the BMDL, not the BMD,

for use as the point of departure since the BMDL accounts for variability of the data (U.S. EPA,

2012).

For the assessment of infants and children, th e most robust studies are 1) the comparative

cholinesterase 63 

# (CCA) study (MRID 48139301, also referred to as Marty et al. , 2012) which

provides brain and RBC data in PND11 pups and 2) (Moser et al ., 2006) which provides data

from brain and whole blood ChE data in PND17 pups . In both of these studies, the rat pups were

dosed directly and ChE was measured at the tim e of peak effect. In addition, two studies

(Timchalk et al ., 2006; Zheng et al ., 2000) provide supporting data. These two studies show

AChE data in post-natal rats which are consiste nt with the comparative cholinesterase study and

Moser et al. (2006). However, Timchalk et al. (2006) and Zheng et al. (2000) are not robust

datasets for dose-response m odeling (U.S. EPA, 2011). For the assessment of adults, single

dosing AChE data from the comparative ch olinesterase study (MRID 48139301) provide the

62 

# Note: This appendix fo cuses on chlorpyrifos studies. See the 2011 preliminary HHRA for oxon results.

63 

# Comparative cholinesterase (CCA) studies are specially de signed studies in juvenile and adult rats which provide

direct comparative ChE/AChE data for evaluating lifestage sensitivit y. For OPs, data are typically collected from

acute dosing (typically PND11, young adult) and from 11-days repeated dosing (PND11-21 and young adults).

# Chlorpyrifos Human Health Risk Assessment D424485

Page 138 of 531 most robust data for BMD analysis 64 

# . Tables App 1-1 and 1-2 pr ovide a summary of the BMD

results for the single dosing studies in post-natal rat pups and adu lt rats, respectively. Generally

for the both the pups and the adults, the BMD and BMDL estimates for the blood measures are

lower than those for the brain. In addition, the results from th e pups are approximately 2-fold

lower than those for adults ( i.e. , when comparing the same compartment, administration vehicle).

Table App1-2. Results of BMD Modeling of Rat Pup Brain and Blood ChE Inhibition following a

Single Oral Dose of Chlorpyrifos

Table App1-3. Results of BMD Modeling of Adul t Rat Brain and RBC AChE Inhibition following

a Single Oral, Dose of Chlorpyrifos

Reference Sex &

Administration

Method Endpoint BMD 10 

# (mg/kg) BMDL 10 

# (mg/kg)

CCA

(MRID 48139301) Female

Gavage Brain AChE 4.11 2.26

CCA

(MRID 48139301) Female

Gavage RBC AChE 1.9 1.2

64 

# Only adult female rats were tested in MRID 48139301. 

# Reference Sex & Age at

Dosing Endpoint BMD 10 

# (mg/kg) BMDL 10 

# (mg/kg)

Moser et al.,

2006 Male PND 17

Gavage Brain AChE 1.89 1.54

Moser et al.,

2006 Male PND 17

Gavage Whole Blood

AChE 0.62 0.43

CCA Study

(MRID 48139301) Male PND 11

Gavage Brain AChE 2.13 1.53

CCA Study

(MRID 48139301) Male PND 11

Gavage RBC AChE 0.82 0.65

CCA Study

(MRID 48139301) Female PND 11

Gavage Brain AChE 2.18 1.56

CCA Study

(MRID 48139301) Female PND 11

Gavage RBC AChE 0.96 0.75

CCA Study

(MRID 48139301) Male PND 11

Milk Brain AChE 4.4 2.4

CCA Study

(MRID 48139301) Male PND 11

Milk RBC AChE 0.47 0.36

CCA Study

(MRID 48139301) Female PND 11

Milk Brain AChE 1.42 0.91

CCA Study

(MRID 48139301) Female PND 11

Milk RBC AChE 0.5 0.36

# Chlorpyrifos Human Health Risk Assessment D424485

Page 139 of 531 CCA

(MRID 48139301) Female

12 hr diet Brain AChE 4.47 3.30

CCA

(MRID 48139301) Female

12 hr diet RBC AChE 1.03 0.6



# For BMD analysis of repeated dosing studies, in the preliminary risk assessment, the Agency

selected the repeated dosing portions of the comparative ch olinesterase study (MRID 48139301)

in juvenile and non-pregnant adul t rats;, the data from pregnant dams in the developmental

neurotoxicity rat study (Maurissen et al ., 2000) (MRID 44556901) and the data from pregnant

dams in the special cholinesterase study in wh ich dams where administered chlorpyrifos from

gestation day 6 through lactation day 10 (GD6 – LD10) (Mattsson et al ., 2000; MRID

44648101). Although the Zheng et al . (2000) data are not robust data for BMD modeling, the

repeated dosing portion of this study provides additional supportive data for the findings from

the comparative cholinesterase study in both pups and adults.

Table App1-4. Results of BMD Modeling of Pup Rat Brain and RBC AChE Inhibition following

11-days Repeated Oral, Gavage Doses of Chlorpyrifos

Reference Sex &

Age/Duration of

Dosing Endpoint BMD 10 

# (mg/kg/day) BMDL 10 

# (mg/kg/day)

CCA

(MRID 48139301) Female PND 11

‐ 21

Gavage Brain AChE 0.80 0.69

CCA

(MRID 48139301) Female PND 11

‐ 21

Gavage RBC AChE 0.17 0.15

CCA

(MRID 48139301) Male PND 11

‐ 21

Gavage Brain AChE 0.63 0.52

CCA

(MRID 48139301) Male PND 11

‐ 21

Gavage RBC AChE 0.11 0.09

Table App1-5. Results of BMD Modeling of Adult, Female Rat Brain, RBC and Heart

Cholinesterase Inhibition following Repeat Oral Doses of Chlorpyrifos

Reference Sex, Age/Duration &

Administration method Endpoint BMD 10 

# (mg/kg/day) BMDL 10 

# (mg/kg/day)

Dow (MRID 44556901;

Maurissen et al., 2000) Dams, GD6

‐ 20,

Gavage Brain AChE 0.65 0.54

Dow (MRID 44556901;

Maurissen et al., 2000) Dams, GD6

‐ 20,

Gavage RBC AChE 0.06 65 

# 0.03

Dow (MRID 44648101;

Mattsson et al., 2000) Dams, GD6

‐ 20, Gavage Brain AChE 1.1 0.8

Dow (MRID 44648101;

Mattsson et al., 2000) Dams, GD6

‐ 20, Gavage RBC AChE 0.14 0.08

65 

# The BMD results from this dataset are not considered re liable due to high variability in the data & BMD values

below the tested doses.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 140 of 531 Dow (MRID 44648101;

Mattsson et al., 2000) Dams, GD6

‐ 20, Gavage Heart ChE 0.85 0.22

Dow (MRID 44648101;

Mattsson et al., 2000) Dams, GD6

‐ LD10

Gavage Brain AChE 1.13 0.89

Dow (MRID 44648101;

Mattsson et al., 2000) Dams, GD6

‐ LD10

Gavage RBC AChE 0.050 0.044

Dow (MRID 44648101;

Mattsson et al., 2000) Dams, GD6

‐ LD10

Gavage Heart ChE 0.21 0.18

CCA (MRID 48139301) Adult, non

‐ pregnant

11 daysGavage Brain AChE 1.03 0.95

CCA (MRID 48139301) Adult, non

‐ pregnant

11 days Gavage RBC AChE 0.45 0.35



# Subchronic Oral

(MRID 40952801) Dietary, non

‐ pregnant

female, Day 44 RBC AChE 0.180 0.117



# Subchronic Oral

(MRID 40952801) Dietary, non-pregnant

female, Day 91 RBC AChE 

# 0.15 0.090



# As shown in Tables App 1-3 and 1-4, ther e is no meaningful difference between BMD 10 

# s and

BMDL 10 

# s from pups and adults in repeated dosing st udies. This is not unexpected given that

over the duration of the repeated dosing study, the metabolic system in pups is maturing closer to

adult levels. Thus, it is not unexpected that as the metabolic system matures that the degree of

sensitivity decreases. Typically, studies submitt ed for pesticide registration and most studies

from the public literature only measure brain an d/or blood ChEs. It is rare for data from

peripheral tissues to be available for considerat ion. Chlorpyrifos is unique in that multiple

studies are available which provide such peripheral data. It is notable that the BMDs and

BMDLs for heart ChE are more sensitive than brai n and closer in magnitude than those for RBC

AChE. This finding supports the use of the blood measures for extrapolating risk.

There is good concordance in the estimates across the BMD 10 

# and BMDL 10 

# estimates across the

adult studies evaluated in th is analysis. Notably, brain AChE estimates range from

approximately 0.5 to 1 mg/kg/day and the RBC AChE estimates range from 0.03 to 0.35

mg/kg/day. In addition, for the OP cumulative risk assessment (U.S. EPA, 2006), the agency

performed BMD modeling using a sophisticated me ta-analysis approach on brain AChE data

from adult, non-pregnant female and male rats from one subchronic oral toxicity study and two

chronic oral toxicity studies (MRI D nos. 40952801, 40952802, and 42172802). The BMD results

reported in the OP cumulative risk assessment for chlorpyrifos are BMD 10 

# s of 1.48 and 1.50

mg/kg/day and BMDL 10 

# s of 1.26 and 1.27, mg/kg/day respectively in females and males. The

values for brain AChE inhibition reported in the cumulative assessment from subchronic and

chronic dosing are quite similar to those reported in Table 1-4 above for both pregnant and nonpregnant females and males in the selected st udies and add further s upport to the analysis

conducted for the preliminary risk assessment for chlorpyrifos.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 141 of 531 3.1.2.2. Summary of Additional AChE/ChE Data (Extracted from USEPA, 2008)

Table App1-5. Summary of repeated studies evaluating gestational exposure to maternal rats and

fetuses (Extracted from USEPA, 2008).



# Study Route

(vehicle) Time of

exposure Time of

measurement

post-dosing Dose Fetal

inhibition Maternal

inhibition Compartment

Mattsson

et al.,

1998,

2000 oral

gavage

(corn

oil) GD6-20 4 hrs 1

mg/kg/day 8% (NS) 10%/7%

(p<0.02) forebrain

0% 12%/7%

(NS) hindbrain

5% (NS) 87%/85%

(p<0.02) RBC

4% (NS) 77%/60%

(p<0.02) plasma

0% 49%/50%

(p<0.02) heart

Hoberman

et al.,

1998a,b;

Maurissen

et al.,

2000 oral

gavage

(corn

oil) GD6-20 4-5 hrs 1

mg/kg/day N/A 68.9% plasma

N/A 84.4% RBC

N/A 17.9% brain

Mattsson

et al.,

1998,

2000 oral

gavage

(corn

oil) GD6-

PND1 2 hrs 1

mg/kg/day 5% (NS) 6% (NS) forebrain

0% 6% (NS) hindbrain

0% 90%

(p<0.02) RBC

5% (NS) 80%

(p<0.02) plasma

2% (NS) 40%

(p<0.02) heart

Qiao et

al., 2002 s.c.

injection

(DMSO) GD 17-

20 GD 21 1

mg/kg/day 3% (NS) brainstem N/A

6% (NS) forebrain N/A

# Chlorpyrifos Human Health Risk Assessment D424485

Page 142 of 531 Table App1-6. Summary of acute studies evalua ting post-natal exposure to juvenile rats

(Extracted from USEPA, 2008).



# Study Route

(vehicle) Age Time of

measurement

post-dosing a 

# Dose Inhibition Compartment

Dam et al.

(2000) s.c. injection

(DMSO) PND 1 2 hrs 1 mg/kg 70% (M); 25%

(F) brainstem

80% (M); 10%

(F) cerebellum

60% (M); 35%

(F) forebrain



# Betancourt

and Carr

(2004) oral gavage

(corn oil) PND 1 12 hrs 1.5 mg/kg 58% forebrain



# Timchalk et

al. (2006) oral gavage PND 5 3 hrs 1 mg/kg 22.1% brain

45.7% RBC

62.1% plasma



# Zheng et al,

2000 oral gavage

(peanut oil) PND 7 4 hrs 1.5 mg/kg 17% frontal cortex

32% RBC

51% plasma



# Timchalk et

al. (2006) or al gavage PND 12 6 hrs 1 mg/kg 5.2% brain

27.0% RBC

33.4% plasma



# Timchalk et

al. (2006) oral gavage PND 17 24 hrs 1 mg/kg 2.1% brain

15% RBC

21.9% plasma



# Moser et al.

2006 oral gavage

(corn oil) PND17 4.5 hrs 0.5 mg/kg 0%

brain

2 mg/kg 10%

0.5 mg/kg 10%

whole blood

2 mg/kg 40%

a. Reported time of peak effect

# Chlorpyrifos Human Health Risk Assessment D424485

Page 143 of 531 Table App1-7. Summary of repeated studies evaluati ng post-natal exposure to juvenile rats

(Extracted from USEPA, 2008).

Study Route

(vehicle) Time of

exposure Time of

measurement

post-dosing Dose Inhibition Compartment

Guo-Ross et

al. (2007) 

# oral gavage

(corn oil) PND 1-4 4 hrs 1

mg/kg/day 25%

brain

1.5

mg/kg/day 45%

Betancourt

and Carr

(2004) 

# oral gavage PND 1-3 24 hrs 1.5

mg/kg/day 27% forebrain

Song et al.

(1997) 

# s.c. injection

(DMSO) PND 1-4 24 hrs 1

mg/kg/day 24% brainstem

Richardson

and Chambers

(2005) 

# oral gavage

(corn oil) PND 1-6 6 hrs 1.5

mg/kg/day 49% brain (excluding

cerebellum and

medulla-pons)

Betancourt

and Carr

(2004) 

# oral gavage PND 1-6 24 hrs 1.5

mg/kg/day 28% forebrain

Betancourt

and Carr

(2004) 

# oral gavage PND1-11 24 hrs 1.5

mg/kg/day None forebrain

Richardson

and Chambers

(2005) 

# oral gavage

(corn oil) PND 1- 12 12 hrs 1.5

mg/kg/day 43% brain (excluding

cerebellum and

medulla-pons)

Richardson

and Chambers

(2005) 

# oral gavage

(corn oil) PND 1-21 24 hrs 1.5

mg/kg/day 36% brain (excluding

cerebellum and

medulla-pons)

Zheng et al

(2000) 

# oral gavage

(peanut oil) PND7-20 4 hrs 1.5

mg/kg/day 42% frontal cortex

57% RBC

59% plasma

# Chlorpyrifos Human Health Risk Assessment D424485

Page 144 of 531 3.2. Adverse Outcome Pathway: Neurodevelopmental Outcomes

With respect to modes of action/adverse outcome pathways leading to neurodevelopmental

effects, at the present time, there is no establishe d series of causal key events at a biological level

of organization relevant to the risk assessment ( i.e. , adverse neurodevelopmental effects from

gestational and/or postnatal e xposure). For the 2014 revised HHRA, the agency conducted an

updated literature review on the experimental toxicology studies for chlorpyrifos (Appendix 11)

for studies published since the 2012 SAP meeting. Some of the new st udies since 2012 have

been integrated in this section. Despite the newest st udies, the agency does not believe that any

of the current lines of research support a coherent set of key even ts and that much work remains

to elucidate the modes of acti on and adverse outcome pathways of chlorpyrifos toxicity.

Even though a rigorous demonstration of an adve rse outcome pathway for chlorpyrifos has not

been constructed at this time, there are multiple studies from numerous laboratories that show

that exposure to a variety of OPs (including ch lorpyrifos, other pesticid es, nerve gases) during

the developmental period affects events in nervous system development (see below). These

studies conducted to date genera lly report a correlation between OP exposure and a tested effect

without consideration of temporal concordance ( i.e. , the sequence of biologi cal events leading to

effects) and quantitative linkages ( i.e. , degree of change that re sults in neurodevelopmental

consequences). Some of this experimental work specific to chlorpyrifos is summarized below,

and may be shown in the future actually to be steps in the same adverse outcome pathways and

not discreet pathways. However, at this time, the experimental studies below do not provide all

the information needed to demonstrate linkages among initiating event(s), subsequent events at

the molecular and cellular level, and the advers e outcome of interest and are thus described

separately.

3.2.1. Biologically Plausible Toxicity Pathways Leading to Neurodevelopmental Outcomes

3.2.1.1. Acetylcholinesterase (AChE) as a morphogen

The classically understood role of AChE is the ra pid hydrolysis of acetylcholine at synapses in

the brain and at neuromuscular junctions, th ereby regulating cholin ergic neurotransmission.

Consistent with this role, AChE is predominant at cholinergic synapses at neurons and in muscle,

and inhibition of its catalytic activity result s in the signs and symptoms of cholinergic

overstimulation. Several lines of evidence, however, suggest that AChE can also serve as a

morphogen, influencing the growth of cells dur ing neurodevelopment. Alterations in the

expression or structure of th e AChE protein can disrupt various aspects of neuronal

differentiation and growth. Because chlorpyrifos can interact with AChE, perturbation of this

morphogenic activity represents a plausible adverse outcome pathway leading to developmental

neurotoxicity. Additional for the plausibility of this adverse outcome pathways is found in the

chlorpyrifos developmental neurotoxicity study (MRID 44556901) where PND66 pups exposed

during gestation and lactation at the 1 and 5 mg/kg/day groups exhibited significant dose- and

treatment-related decreases in measurements of the parietal cortex in female offspring.

One of the first indications that AChE had ro les other than the termination of cholinergic

# Chlorpyrifos Human Health Risk Assessment D424485

Page 145 of 531 neurotransmission was the timing and distribution of its expre ssion in the developing nervous

system. In certain brain regions containing non-cholinergic neurons and few cholinergic

synapses, AChE levels are still high. In additi on, AChE is highly expres sed throughout the brain

during periods of active axonal outgrowth in the ab sence of other cholinergic markers and before

synaptic connections are made (Bigbee et al ., 1999; Brimijoin & Koenigsberger, 1999). For

example, AChE levels are high during early cereb ellar development while there is very little

evidence for the presence of cholinergic cells or cholinergic neurotransmission (Appleyard &

Jahnsen, 1992; Parvari, 1983). A second line of ev idence for a morphogenic role arose after the

cloning and sequencing of rode nt and human AChE. Surprisi ng sequence homologies were

found with a family of proteins l acking a catalytically ac tive esterase site but pocessing a similar

extacellular domain. Several of the homologs of AChE are extracellular matrix components

essential to neuronal adhesion, axon guidan ce, and synapse formation (Brimijoin &

Koenigsberger, 1999; Grisaru et al ., 1999). Thus, the sequence of AChE is similar to other

proteins which have a morphogeni c role during the development of the nervous system. Finally,

experimental evidence indicates that manipulatio n of AChE levels or activity can influence

neuronal growth in vitro . Studies in cultured neuroblas toma cells (Koenigsberger et al ., 1997) or

PC12 cells (Grifman et al ., 1998) have shown that antisense suppression of AChE decreased

neurite outgrowth, and that neurite outgrowth coul d be rescued or increased by transfection with

sense AChE. In cultures of rat dorsal root gang lion cultures, treatment w ith an antibody against

AChE (which did not inhibit the catalytic ac tivity) decreased neurite outgrowth, and neurite

outgrowth was restored upon removal of the antibody (Bigbee et al ., 1999). Expression in frog

embyros of a recombinant human AChE lacking th e ability to hydrolyze acetylcholine resulted in

increased neurite outgrowth from cultured spinal neurons (Sternfeld et al ., 1998). Thus, the

catalytic ability of AChE does not seem to be required for its morphogenic properties.

Studies using pharmacologic inhibi tion of AChE activity to alter neurite outgrowth also suggest

a dissociation of the catalytic and morphogenic activities. Several laborat ories using different in

vitro systems have demonstrated that some pot ent AChE inhibitors including BW284c51 supress

neurite outgrowth while other, equally potent AC hE inhibitors like ecoth iophate do not (Bigbee

et al ., 1999; Koenigsberger, et al ., 1997; Layer et al ., 1993). These differential effects may be

related to the degree of chemical binding to separate sites on the AChE enzyme. There are at

least two sites that can bind inhibitors: the catalytic (active) site and the peripheral anionic site

(Grisaru et al ., 1999). It has been suggested that inhibito rs that bind directly to the peripheral

anionic site, or that bind to the catalytic site in such a way that change s the conformation of the

peripheral anionic site, w ill alter the morphogenic f unction of AChE (Bigbee et al ., 1999; Yang

et al ., 2008). Thus inhibitors that bi nd only to the cat alytic site ( e.g. edrophonium, tacrine) did

not affect neurite outgrowth, while ligands that bind to the peripheral an ionic site (propidium,

gallamine) or both sites (BW284c51) inhibi ted neurite outgrowth (Koenigsberger et al ., 1997;

Munoz et al ., 1999). At least one study indicates that chlorpyrifos oxon can bind at both sites

(Kousba et al ., 2004).

Data showing a potential action of chlorpyr ifos on the morphogenic function of AChE are

derived primarily from in vitro studies. Chlorpyrifos (as well as the oxon and TCPy) has been

examined for effects on neurite outgrowth using a number of cell lines and in primary neuronal

cultures. In some cases concurrent assessments of cholinesterase inhibiti on or cytotoxicity were

performed. Li and Cassida (1998) examined the e ffect of chlorpyrifos oxon on neurite outgrowth

# Chlorpyrifos Human Health Risk Assessment D424485

Page 146 of 531 in the PC12 cell line. In this study cells were e xposed prior to, but not dur ing the active phase of

neurite growth, which was assessed after 5 days . Chlorpyrifos oxon inhib ited neurite outgrowth

by 50%, but only at a relativel y high concentration (200 uM) that was both cytotoxic and

decreased cholinesterase activity. Flaskos et al . (2011) showed that neurite outgrowth in

differentiating N2a cells was observed at μM con centrations of the oxon which also resulted in

inhibition of AChE in the same cell line. Sachana et al . (2005, 2008) examined the effect of 3

uM chlorpyrifos on the initiation of neurite growth in mouse N2a neuroblastoma cells. The

number of cells exhibiting neurit es after 4 or 8 hours of exposur e (considered as a measure of

cell differentiation) was decreased by approxima tely 50% in the abse nse of cytotoxicity.

Cholinesterase activity was not measured. Axelrad et al. (2002) exposed mouse NB2a cells to

chlorpyrifos for 24 hr and directly measured neurite length. Chlorpyrif os inhibited neurite

outgrowth by 50% at 25 uM. Again, cholinesterase activity was not measured. While the studies

cited above suggested that chlor pyrifos and chlorpyrifos oxon coul d affect measures related to

neuronal differentiation and neurit e growth, they were not specifi cally designed to examine the

morphogenic role of AChE.

Using PC12 cells, Das and Barone (1999) examin ed the concentration-related effects of

chlorpyrifos, chlorpyrifos oxon, and TCPy on both neurite outgrowth and cholinesterase

inhibition. Exposure to chlorpyrifos for 24 hr inhi bited neurite outgrowth at a concentration (3

uM) 10-fold below that which inhibited cholineste rase activity, while chlo rpyrifos oxon inhibited

both measures at equivalent concentrations (1 nM). TCPy, which is inactive against AChE,

inhibited neurite growth at 5 uM. Similar studi es of chlorpyrifos and its metabolites were

performed in a series of experiments in the Lein laboratory using pr imary neuronal cultures

derived directly from the mammalian nervous sy stem. Using rat sympathetic neurons, Howard et

al . (2005) showed that 24 hr exposure to chlor pyrifos and chlorpyrifos oxon decreased axonal

outgrowth at concentrations (0.001 uM and 0.001 nM, respectively) well below the

concentrations that inhibited AC hE activity (1 uM and 1 nM). In the same study chlorpyrifos,

chlorpyrifos oxon and TCPy enhanced dendrit e outgrowth. A follow-up study from the same

laboratory used sensory neurons from the dorsal root ganglion, which extend only axons (Yang,

et al., 2008). Similar results we re observed, with both chlorp yrifos and chlorpyrifos oxon

decreasing axonal outgrowth at concentrations be low those that inhibited AChE activity. To

establish whether the target of chlorpyrifos and chlorpyrifos oxon was AChE or some other

molecule, Yang et al. (2008) repeated the experiments in cultures from AChE-null animals.

Chlorpyrifos and chlorpyrifos oxon had no effect in the AChE-null cultu res, suggesting that

inhibition of neurite outgrowth required AChE. These later three studies provide the most

convincing evidence for selective inhibition of the morphogenic ac tivity of AChE. It is not yet

clear whether the effects are a direct action of chlorpyrifos on AChE, or the result of in vitro

conversion to the active oxon form. It should be mentioned that studies from the Slotkin

laboratory suggest an effect of chlorpyrifos on neurite outgrowth in vitro (Song et al ., 1998). In

those experiments, however, neur ite outgrowth is inferred from biochemical measurements and

cholinesterase activity is not assessed concurrently.

Testing the hypothesis that exposure to chlor pyrifos (or any chemical) can inhibit the

morphogenic function of AChE in vivo and alter brain development is difficult. Neuronal

differentiation and the subsequent development of axonal and dendritic networks occurs in a

temporal- and region-specific manner. In the ab sence of prior informa tion extensive studies

# Chlorpyrifos Human Health Risk Assessment D424485

Page 147 of 531 would be required to survey brain morphology du ring both the prenatal a nd postnatal period. In

addition, the methods needed to assess the morphology of axonal and dendritic growth in vivo

and detect potentially subtle chemical-indu ced changes are tedious and time consuming.

Nevertheless, some attempts have been made to detect chlorpyrifos-indu ced changes in neurite

growth in vivo . In particular, several laboratories have examined the effect of chlorpyrifos and

chlorpyrifos oxon on neuronal morphogenesi s in developing zebrafish. Jacobson et al . (2010)

exposed zebrafish embryos at 3 hr post fe rtilization to 300 nM ch lorpyrifos oxon. When

examined 24 hr later (1 day post fertilization), AChE was inhibited by 50%. Gross morphology

was only slightly affected and muscular deve lopment (including the neuromuscular junction)

was normal; however, there was a decrease in the number of Rohon-B eard sensory neurons

accompanied by abnormal extension of their axons. As a follow up to this study and to their in

vitro work described above, Yang et al . (2011) exposed zebrafish embryos from 24 to 72 hr post

fertilization to chlorpyrifos a nd chlorpyrifos oxon. Chlorpyrifos had no effect, but chlorpyrifos

oxon significantly decreased AChE activity (30 nM), decrease d touch-induced swimming (100

nM) and inhibited axon growth from both sens ory neurons and motor neurons (100 – 1000 nM).

These two studies show that exposure to chlorpyr ifos oxon in an intact, developing organism can

alter neurite outgrowth. However, because catalytic activity was also inhibited, it is not clear that

this effect was solely due to disrup tion of the morphogeni c role of AChE.

As noted above, neurite outgrowth has been assessed indirectly in vitro . This method has also

been applied in vivo in mammals. Qiao et al . (2003) exposed rats to 1 or 5 mg/kg chlorpyrifos on

gestational days 17-20. In a previous study from the same laboratory this exposure paradigm

resulted in a non-significant 5% decrease in AChE activity afte r 1 mg/kg and a significant 50%

decrease in AChE activity after 5 mg/kg wh en assessed 24 hr after the last dose (Qiao et al

., 2002). The ratio of membrane protein to total prot ein in several brain regions was assessed as a

surrogate index of neurite outgrowth. The results indicated that there was no effect of treatment

during early postnatal development (PND 4-21), but that both doses decreased this ratio on PND

30 and 60. While the authors interpret this bioche mical measurement as a decrease in neurite

growth, there has not been data presented to co rrelate the changes in th e membrane protein to

total protein ratio with actual quantification of ne urite length. In addi tion, no effects were

observed early in development when axons and dendrite growth is high.

In summary, while perturbation of the morphogenic activity of AChE is a plausible adverse

outcome pathway for chlorpyrifos, a number of questions remain. There is substantial evidence

for a morphogenic role of AChE in nervous system development distinct from its role as an

esterase to hydrolyze acetylcholine. In vitro evidence suggests that AC hE can regulate neurite

outgrowth, and that cholinestera se inhibitors (incl uding chlorpyrifos and its metabolites) can

interfere with this process at concentrations that do not inhibit the esterase activity. There is,

however, no direct evidence showing that disrup tion of the morphogenic function of AChE can

alter axon or dendritic growth in vivo . While limited in vivo studies using zebrafi sh indicate that

chlorpyrifos or its metabolite chlorpyrifos oxon can disrupt axonal gr owth, it has not been

demonstrated that this effect is due to alte ration of the morphogenic function of AChE versus

other potential mechanisms.

3.2.1.2. Cholinergic system

There are several lines of ev idence showing that signaling th rough cholinergic receptors is

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Page 148 of 531 involved in neurodevelopment. Activation of mus carinic and/or nicotinic cholinergic receptors

can regulate neural progenitor cell proliferation and differentia tion (Resende & Adhikari, 2009),

and in vivo studies demonstrate that cholinergic signaling is likely involved in brain

morphogenesis (Hohmann & Berger-Sweeney, 1998). Wh ile cholinesterase inhibitors can affect

cholinergic signaling by inhibition of the catalytic activity of AC hE and subsequent increase in

acetylcholine, some, including chlorpyrifos and chlo rpyrifos oxon, can also di rectly interact with

cholinergic receptors. Thus, direct interaction with cholinergic receptors by chlorpyrifos

represents a potential adverse outcome pathwa y for disruption of neur odevelopment distinct

from AChE/ChE inhibition.

Some OPs have been shown to directly inter act with cholinergic muscarinic receptors at

relatively low concentrations. The muscarinic re ceptors are members of the G-protein receptor

family and five subtypes (m1-m5) have been identified. Volpe et al . (1985) reported that

nanomolar concentrations of several OPs decr eased binding of quinuclidinyl benzilate (QNB),

which binds equally to all five subtypes. This effect was noncompetitive and occurred at only a

small fraction of the total QNB binding sites. A further study from the same laboratory proposed

these receptor subtypes to be m2 and/or m3 ( Katz & Marquis, 1989). Bakry et al . (1988) found

that a number of OP nerve agents and an OP therapeutic agent inhib ited QNB binding to rat

brain membranes with low potency, but competitiv ely inhibited binding of cis-methyldioxolane

(CD) with high potency. In rat car diac tissue, OPs also competitively inhibited CD binding with

very high potency (Silveira et al ., 1990). CD is a muscarinic a gonist that binds to the high

affinity state of the m2 receptor in mammalian brain and heart (Closse et al ., 1987; Vickroy et

al. , 1984; Watson, 1986). Subsequent research us ing Chinese hamster ovary (CHO) cells

transfected with cDNA for the five distinct genes for muscarinic receptors (ml-m5) has identified

the CD binding site as the high affinity state (GDP bound G-protein) of the m2 receptor (Huff &

Abou-Donia, 1994). In light of these findings, Ward et al. (1993) examined the relationship

between cholinesterase inhibition and direct binding to muscarinic receptors for a series of OPs

and their active “oxon” metabolites. The resu lts indicated a strong correlation between

anticholinesterase activity of OP s, including chlorpyrifos and ch lorpyrifos oxon, and the ability

to compete for CD binding sites (m2 receptors) in rat brain homogenates. Binding affinities of

the oxons were in the nanomolar range, at or be low concentrations that inhibited AChE (Huff et

al. , 1994); specifically, chlorpyrifos oxon had a binding affinity of 22 nM in rat striatum and 2

nM in rat cortex (Huff et al ., 1994; Ward & Mundy, 1996). In tota l, these studies suggest that

direct interactions with muscarinic receptors , and especially the m2 subtype, represent an

alternative site of act ion for OPs including chlorpyrifos and chlorpyrifos oxon, with the oxon

forms having high affinity.

Further studies determined whether binding of OPs to the m2 receptor had functional

consequences on downstream second messenger signa ling. The m2 and m4 receptors are coupled

to an inhibitory G-protein (G i 

# ) and activation inhibits adenyl ate cyclase and decreases cAMP

formation. In contrast, m1, m3, and m5 receptors increase PI hydrolysis via the stimulatory Gprotein G p 

# (Lameh et al ., 1990). Jett et al. (1991) reported that paraoxon could inhibit both CD

binding and cAMP formation in rat striatum. The effects of paraoxon were similar to the

classical muscarinic agonist carbachol, and were blocked by the muscarinic antagonist atropine.

Similar results were observed in rat frontal cortex for paraoxon and malaoxon (Ward & Mundy,

1996). In addition, Ward and Mundy (1996) obser ved that the OPs had no effect on PI

# Chlorpyrifos Human Health Risk Assessment D424485

Page 149 of 531 hydrolysis. These data suggest that OPs can act as agonists at the m2 receptor (but not the m1,

m3, or m5 receptor) and decr ease cAMP formation. Huff et al . (1994) extended these findings to

chlorpyrifos oxon, which had an IC 50 

# for inhibition of cAMP of 155 nM. Unlike the other OPs,

however, the effects of chlor pyrifos oxon were not complete ly blocked by atropine; an

observation confirmed by Ward and Mundy (1996) . Thus, like other OPs chlorpyrifos oxon can

act directly as an agonist at the m2 receptor, but may also act at another site downstream of the

receptor to inhibit adenylate cyclase.

Cholinergic receptor signaling has been shown to be involved in apoptosis, cell proliferation and

neuronal differentiation (Resende & Adhikari, 20 09). While the m2 receptor subtype is widely

expressed in proliferatin g neuroepithelial cells in the ventricular zone of embryonic brain (Ma et

al ., 2004), evidence for a specific role of in th e modulation of neurodevelopment is derived

primarily from in vitro studies. Neural precursor cells de rived from embryonic rat cortex

expressed the m2 receptor, and exposure to mus carinic agonists increased cell proliferation and

enhanced differentiation into neurons (Ma et al ., 2000). Using a P19 embryonic cell line as a

model for neurogenesis, Resende et al. (2008) used pharmacologi c agonists and antagonists to

demonstrate the ability of m2 receptors to i nduce cell differentiation. The m2 receptor is highly

expressed in developing cells of the dorsal root ganglia, and is thought to regulate both neuronal

and non neuronal cell differentiation (Biagioni et al ., 2000; Tata et al ., 2003). Another line of

evidence for involvement of m2 receptor signaling in neurodevelopment is the role of cAMP in

neurite outgrowth. Studies in bot h cell lines and primary neuronal cultures show that activation

of adenylate cyclase and subsequent formation of cAMP stimulate neurite outgrowth (Kamei &

Tsang, 2003; Mattson et al ., 1988), while inhibition of adenyl ate cyclase decreases neurite

outgrowth (Tam et al ., 2006; Wong et al ., 1991). Thus, in vitro evidence supports a role of m2

receptor signaling in neurodevelopment at the ce llular level. However, evidence showing that

manipulation of m2 receptors can alter brain development in vivo is lacking.

Because they can also influence neurodevelopmen t, it should be noted that there are several

studies demonstrating that OPs in cluding chlorpyrifos and chlorpyr ifos oxon can bind directly to

and desensitize nicotinic receptors (Katz et al ., 1997; Smulders et al ., 2004). This binding,

however, occurs at relatively high concentrations (5 – 30 uM) and has not been demonstrated in

neuronal tissue.

In summary, in vitro studies have shown that chlorpyrif os oxon can bind to and activate m2

receptors at levels similar to those that inhibit AChE activity. Other work has shown that m2

receptor signaling can regulate various aspects of neurodevelopment. Together, the studies cited

above outline a plausible adverse outcome path way for chlorpyrifos and chlorpyrifos oxon to

affect brain development via actio ns at the m2 subtype of muscar inic receptors. However, while

there are studies showing that chlorpyrifos oxon can affect neurite outgrowth in vitro and

decrease cell proliferat ion and differentiation both in vitro (Jameson et al ., 2006; Qiao et al

., 2001; Song et al ., 1998) and in vivo (Dam et al ., 1998; Qiao, et al., 2003), there is no

experimental evidence that these effects are a result of direct actions on the m2 receptor.

3.2.1.3. Endocannabinoid system

Several lines of research have suggested that disruption of the endocannabinoid (EC) system due

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Page 150 of 531 to chlorpyrifos exposure could play a role in its acute and/or l ong-term toxicity, and could also

be extended to potential developm ental toxicity. The EC system modulates neurotransmission as

well as playing a morphogenic role during deve lopment of the nervous system. Chemicals e.g.

, drugs of abuse, which act on this system, pr oduce long-term neurodevelopmental disorders in

animal models and human studies. Chlorpyrif os also interacts with this system, both in vitro and

in vivo . By this reasoning, the EC system represents a possible adverse outcome pathway for

developmental effects of chlorpyrifos.

The EC system includes inhibitory G-protei n-coupled receptors (CB1 and CB2), endogenous

ligands (2-arachidonoylglycerol, or 2-AG, and anadamide, or AEA), and hydrolases

(monoacylglycerol, or MAG, and fatty acid amide hydrolase, or FAAH) th at end the receptor

actions of these ligands. CB1 receptors are pr edominant in certain brain regions, and the EC

system modulates neuronal transmission and is involved in several physiological processes

including appetite, pain sensation, mood, a nd cognition (Wilson & Nicoll, 2002). Emerging

lines of research indicate that during development, this system plays a major role in controlling

the morphological and functiona l specification of neurons ( e.g. , Fride, 2008; Harkany et al

., 2007; Harkany et al ., 2008a, b; reviewed in Campolongo et al ., 2009). Its involvement in

neurogenesis, proliferation, and axon guidance sugge sts a role in overall ne uronal connectivity.

There are epidemiological data on neurodevelopm ental outcomes following prenatal exposure to

cannabinoids such as cannabis and THC through maternal drug a buse, but interpretations are

often somewhat difficult given co-e xposures to other neurotoxicants ( e.g. , ethanol). Despite

limitations, an overall picture has emerged of long-lasting impaired cognitive processes,

including attention and problem-sol ving deficits, as well as anxiet y and depressive symptoms in

humans ( e.g. , Campolongo et al ., 2009; Fried & Smith, 2001; Jutras-Aswad et al ., 2009; Trezza

et al ., 2008). In addition, animal models have indicated various changes in neurotransmission of

catecholaminergic and indolaminergic system s, ontogeny of motor function, and cognitive

function in adults developmentally exposed to cannabis as well as other CB1 agonists ( e.g.

, WIN55,212-2) (reviewed in Campolongo et al ., 2011; Marco et al ., 2009). This supports the

possibility that other chemicals, unrelated to cannabis, that also act on the EC system early in

development could lead to long-te rm neurological dysfunction. De spite the number of studies in

this area, however, the specific responsible cellular responses have not been delineated, and most

studies have not evaluated basic dose-response su ch as correlations of the degree of receptor

binding with any structural or functional outcomes.

In addition to drugs of abuse ( e.g. , cannabis), there are several cl asses of chemicals that act on

the EC system, including organosul fonyl fluorides and some OPs ( e.g. , Casida & Quistad, 2004;

Segall et al ., 2003). OPs phosphorylate and thereby bl ock the action of th e hydrolases FAAH

and MAG. This inhibition of the hydrolysis of endogenous cannabinoids could prolong their

actions on the receptor, which inhibits several neurotransmitter systems, including ACh. OPs

also bind the CB1 receptor and block the bindi ng of specific receptor ligands. The OPs do not

bind at the agonist site, however, and may have no inherent agonist or antagonists consequences;

however, this has not been adequately studied. Thus, prolongation of the ligand residence time,

plus blockade of ligand binding at the recepto r, could lead to downstream modulation of

cholinergic activity and signaling, separate from AChE inhibition. It is unclear which actions

could predominate in vivo

.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 151 of 531 Casida and colleagues have produced several pape rs relating FAAH inhib ition to that of AChE

and neurotoxic esterase (NTE), and consider ed its involvement in acute cholinergic,

intermediate, and delaye d toxicity syndromes ( e.g. , Casida & Quistad, 2004; Quistad et al , 2006;

Quistad et al, 2002a, b; Quistad et al 2001; Segall, et al., 2003). Several studies have included

chlorpyrifos. They have reported that in vitro chlorpyrifos oxon inhi bits FAAH and MAG with

IC50s of 40 nM and 34 nM, respectively, compar ed to an AChE IC50 of 19 nM. Other OPs

(paraoxon, dichlorvos, diazoxon) have hi gher hydrolase inhibitory actions in vitro (IC50s 540-

14,000 nM). In vitro receptor binding assays of CB1 a gonists reveal an IC50 of 14 nM for

chlorpyrifos oxon, and higher IC50s (>64 nM) for other OPs (chlor pyrifos methyl oxon,

diazoxon, dichlorvos).

Casida’s group has also dosed adult mice w ith OPs and evaluated motor behavior and

cholinergic signs, followed by ex vivo biochemical assays. These studies report inhibition of

FAAH activity and CB1 binding only at highly toxi c doses of chlorpyrifos and/or chlorpyrifos

oxon. Chlorpyrifos dose-response differs somewh at across studies, but in general, FAAH is

inhibited more than MAG and AChE, and CB1 bindi ng is moderately inhibi ted, but only at doses

producing cholinergic signs. Chlorpyrifos inhi bition of FAAH does not correlate with the

hypomotility (akinesia, rigidity) produced by the EC agonist amandamide. Furthermore,

chlorpyrifos does not potentiate the effects of amandamide, sugges ting that it does not appear to

have functional impacts in the EC system. In contrast, treatment with other chemicals that do

produce marked (>76%) FAAH inhi bition does indeed potentiate anandamide effects in mice;

however, chlorpyrifos doses that would be requir ed to produce this level of FAAH inhibition are

likely to be lethal. This line of research has led Casida and colleag ues to conclude that

chlorpyrifos actions on the EC system in vivo are less important to its acute toxicity than AChE

inhibition.

A series of studies from the laboratories of P ope and colleagues has evaluated the influence of

CB1 agonists administered in vivo on the signs of toxicity produced by several OPs (Baireddy et

al, 2011; Nallapaneni et al, 2006, 2008; Pope et al, 2010; Wright et al, 2010). They reported that

CB1 agonists and other cannabinomimetics block the acute signs of e xposure to paraoxon or

DFP (chlorpyrifos was not tested). Studies with chlorpyrifos show increas ed extracellular levels

of 2-AG but not amandamide in hippocampus. Somewhat different c onclusions were drawn,

however, in a paper showing that the toxicity and AChE inhib ition produced by an acute dose of

chlorpyrifos is not altered in mice without the CB1 receptor (knockout) compared to their wild

type littermates. Pope et al. also measured chlorpyrifos oxon-induced ACh release from these

tissues and saw only a small difference. These somewhat contradictory results suggest that

potential EC involvement in the acute toxi city of certain OPs is still uncertain.

A limited number of studies have examined chlor pyrifos effects on the EC system in developing

animals. Carr et al. (2011) dosed preweanling rats for 5 days (1-5 mg/kg/day, p.o.) and took

brain tissue 4 hr after the last dose. The lowest dose pr oduced 40% inhibition of FAAH, 14%

inhibition of MAG, and only 18% inhibition of AChE; in addition, the highest dose eliminated

FAAH activity and produced about 52-55% inhibition of AChE. This suggests a greater

sensitivity of the EC system, at least in terms of the hydrolase compared to AChE activity, in the

pups. Carr et al (2013) confirmed the findings in 2011 paper and showed that FAAH recovers

# Chlorpyrifos Human Health Risk Assessment D424485

Page 152 of 531 more faster than brain AChE and MAGL. Anothe r recent publicati on (Carr et al., 2014) repeated

these findings using a lower dose (0.5 mg/kg/d for 7 days), still showing significant FAAH

inhibition but with no measurable AChE inhibi tion. Additional studies along these lines are

needed.

In summary, most of the research in this area has evaluated acute res ponses in the EC system,

even though such changes during a critical pe riod of development could have long-term

consequences. While some steps along a ch lorpyrifos adverse outcome pathway for

developmental neurotoxicity are pl ausible, there are remaining que stions as to whether the EC

system is sufficiently sensitive to low doses of chlorpyrifos to alter its function during

development. Despite the incr easing understanding of the EC sy stem, there are no data during

development on 1) inhibition of the CB1 recep tor at any time during development (animal

studies used only agonists); 2) inhibition of FAAH during de velopment; 3) time-course and

dose-response of OPs in vivo compared to AChE inhibition; a nd 4) whether these actions on the

receptors and/or hydrolases actually change EC signaling to a point that would impact

downstream nervous system development.

3.2.1.4. Reactive Oxygen Species

The production of reactive oxygen species (ROS) and resulting cellular damage has been

proposed as a mechanism for a wide variety of neur otoxicants. Due to lower levels of protective

enzymes and antioxidants, and relatively low number s of glia relative to th e adult, the developing

brain may be particularly sensitive to neural cell damage caused by oxidative stress. In addition,

recent work suggests that ROS can act as se cond messengers. Relatively small changes the

oxidative status of the cell (redox potential) ca n lead to changes in redox sensitive signaling

pathways that regulate cell physio logy. In the nervous system, redox signaling is involved in the

regulation of neurodevelopmental processes in cluding neural stem ce ll proliferation and

differentiation (Le Belle et al ., 2011; Vieira et al, 2011). A num ber of studies suggest that

chlorpyrifos and chlorpyrifos oxon can induce oxidat ive stress in various neural cell types. Thus,

generation of reactive oxygen speci es and/or alteration of cellula r redox potential by chlorpyrifos

represent a possible initiating event le ading to developmental neurotoxicity.

Both chlorpyrifos and chlorpyrifos oxon have b een shown to induce oxidative stress in cells in

vitro (Ojha and Srivastava, 2014; Ki et al, 2013; Chiapella et al, 2013). Lee et al (2012)

exposed PC12 cells to chlorpyrifos and suggest th at neuronal cell death is partly due to MAPK

activation via ROS generation. In addition, a seri es of studies using PC12 cells were performed

by the Slotkin laboratory. Crumpton et al. (2000) reported that acute exposure of undifferentiated

PC12 cells to 1.5 – 150 uM chlorpyrifos for 10 min resulted in a rapid increase in ROS

production. Exposure to 30 uM chlorpyrifos oxon fo r 10 min had no effect. Consistent with the

generation of ROS, further studies demonstrat ed an increase in oxidative damage (lipid

peroxidation) in both undifferentiated and differentiated PC12 cells after exposure to

chlorpyrifos (3 – 100 uM) for 24 hr (Qiao, et al , 2005). Finally, Slotkin a nd Seidler (2009) used

microarrays to examine the expression of genes involved in the response to oxidative stress after

exposure of PC12 cells to 30 uM chlorpyrifos for 24 or 72 hr. They observed a significant effect

on transcription of genes including glutathione peroxidase, glutathi one-S-transferase, and

superoxide dismutase, which wa s greater in differentiated cells compared to undifferentiated

# Chlorpyrifos Human Health Risk Assessment D424485

Page 153 of 531 cells. PC12 cells were also used by a separate gr oup of researchers who reported that very high

concentrations of chlorpyrif os (1.4 – 14 mM) increased ROS production during a 5 hr exposure

period (Geter et al., 2008). ROS production has also been reported in ot her neuronal cell types.

Giodano et al. (2007) examined the effects of ch lorpyrifos and chlorpyr ifos oxon on ROS levels

and lipid peroxidation in primary cultures of cerebellar granule cells from mice. Exposure to 1

uM chlorpyrifos for 60 min increased both ROS and lipid peroxidation, and a similar response

was observed with 1 uM chlorpyrifos oxon. Saulsbur y et al. (2009) examined oxidative stress in

oligodendrocyte progenit or cells, which are the precursors to glial cell types in the brain.

Exposure to 15 – 120 uM chlorpyrifos for 3 hr resulted in a significant increase in ROS

production. These in vitro studies suggest that ex posure of cells to the mi cromolar concentrations

of the parent compound chlorpyr ifos (which is not a potent AChE inhibitor) can result in

oxidative stress. The active metabolite chlorpyr ifos oxon was found to be active in cerebellar

granule cells but not in PC12 ce lls. Inhibition of AChE was not determined in these studies.

A limited number of studies have examined the oxidative stress response in rat brain after in vivo

exposure to chlorpyrifos. Bagchi et al . (1995) dosed adult female rats p.o. twice with 41 mg/kg

chlorpyrifos, once at 0 hr and once at 21 hr. Animals were sacrificed at 24 hr. Both ROS

production and lipid peroxidation were increased in homogenates from wh ole brain. In a followup study, the same dosing paradigm resulted in an up-regulation of heat shock protein, which in

some cases can be a response to oxidative stress (Bagchi et al ., 1996). There are two studies of

oxidative stress in response to chlorpyrifos expo sure in developing animals. Slotkin et al., (2005)

used three dosing paradigms: s.c. exposure of pregnant rats on GD 17-20 followed by collection

of the fetal brain on GD 21, s.c. exposure of ra t pups on PND 1-4 followed by brain collection on

PND 5, or s.c. exposure of rat pups on P ND 11-14 followed by brain collection on PND 15.

Lipid peroxidation was measured in brain regi ons including the brainstem, forebrain and

cerebellum. There was a 20% increase in lipid per oxidation in the brain only after exposure to 5

mg/kg chlorpyrifos during the later postnatal period (PND 11-14) and only in males. AChE

inhibition was not assessed. Ray et al. (2010) dosed 7 day old rat pups p.o. with 0.1, 0.5, 1, or 2

mg/kg chlorpyrifos and examined gene expression in the forebrain 24 hr later using microarrays.

Only the highest dose inhibited AChE (25%). Ge ne expression changes were observed at all

doses, and ‘oxidative stress’ was one of the six canonical pathways that was significantly

affected. In a recent study, Ambali and Ayo (2012) showed that in vivo treatment to adult rats

with vitamin C and chlorpyrifos together mediated the muscle ChE inhibition and

neurobehavioral effects of chlorpyrifos followi ng 17 weeks of gavage dosing at 10.6 mg/kg/day.

Kalender et al . (2012) also showed that chlorpyrifos e ffects at a dose of 5 mg/kg/day could be

modulated by co-treatment with catechin and que rcetin. In recent pape rs (Basha and Poojary,

2012a, b; Poojary and Basha, 2012), it is hypothesized that cold temperatures induces oxidative

stress which increases the toxicity from chlorpyrif os. The oral doses used by these investigators

are relatively high; replication of these findings is needed by another laboratory, particularly at

lower doses, to provide additiona l credence to the hypothesis.

Data from both in vitro studies with neuronal cells (inc luding neural precursors) and in vivo

studies in developing brain demonstrate chlorpyrifos can induce oxidative stress. The in vitro

data suggests that this effect may not be due to AChE inhibition, si nce the parent compound

chlorpyrifos is either equi potent or more potent than th e oxon. There was, however, no

concurrent analysis of AChE inhibition in most of these studies. Seve ral known developmental

# Chlorpyrifos Human Health Risk Assessment D424485

Page 154 of 531 neurotoxicants have been shown to disr upt neural precurso r cell proliferation in vitro through a

common pathway that is initiated by increasing the oxidative state of the cell (Li et al, 2007), and

the antioxidant vitamin E protected PC12 cells from the anti-proliferative effect of chlorpyrifos

(Slotkin et al, 2007). Thus, the in vitro data suggest that chlorpyrifos can affect a critical

neurodevelopmental process, at least in part, via generation of ROS. Though limited, in vivo

studies show both direct evidence (lipid peroxi dation) and indirect evidence (alteration in the

expression of oxidative stress res ponse genes) of oxidative stre ss in the developing brain after

exposure to chlorpyrifos. Recent evidence suggests that oxidative stress can alter

neurodevelopment in vitro and in vivo by the dysregulation of signaling pathways controlling

neuroprogenitor cell function (Le Belle, et al., 2011; Vieira, et al., 2011). Thus, while there is the

potential for initiation of an adverse outcome pa thway via induction of oxid ative stress, it has yet

to be demonstrated in vivo that treatments preventing the induction of oxidative stress can

ameliorate the developmental ne urotoxicity of chlorpyrifos.

3.2.1.5. Serotonergic system

The serotonergic system (reviewed in Rho & Storey, 2001; Serotonin Receptors in

Neurobiology , 2007) includes 7 distinct families of serotonin receptors regulating diverse

functions such as neuronal exc itability, appetite control, memo ry, learning, circadian rhythm,

sexual behavior, anxiety, aggres sion, and respiration. While the majority of the receptors are

either negatively or positively coup led to adenylate cyclase, the 5-HT 3 

# serotonergic receptor is a

ligand-gated ion channel. Serotonin receptors are distributed thr oughout the brain and body.

Synthesized from L-tryptophan, the rate limiting st ep in serotonin biosynthesis is catalyzed by

tryptophan hydroxylase. Afte r release, the acti on of serotonin is primarily terminated by reuptake

into the presynaptic terminal via monoamine tran sporters. Various drugs inhibit the reuptake of

serotonin, effectively increasing the available synaptic concentrations and actions: Ecstasy,

amphetamine, cocaine, tricyclic antidepressants and the newer antidepressant s: SSRIs (selective

serotonin reuptake inhibitors).

Beyond its classical neurotransmitter actions (descr ibed above), serotonin has other roles during

development. In their revi ew, Thompson and Stanwood (2009) described serotonin as a

pleiotropic molecule, meaning that it can produce multiple, diverse effects, regulating different

functions at different times duri ng development. The serotonergic system is integral in many

developmental processes including, but not limited to, neurogenesis, migration, and

differentiation, synaptogenesis, and cardiac development before assuming its more well-known

function as a neurotransmitter in the adult nerv ous system (reviewed in Frederick & Stanwood,

2009). Serotonin also plays crucial roles in thal amocortical patterning (r eviewed in Frederick &

Stanwood, 2009). As serotonin is present extremel y early in development, it is thought that it

modulates cellular function even before neurog enesis. Later in development, serotonin is

temporarily taken up by so-called transient sero tonergic neurons mainly involved in sensory

processing, and is involved in ac tivity-dependent patterning of th e brain. Later in development,

serotonin has also been shown to mo dulate differentiation in the brain.

Perturbation of the development of the seroto nergic system in laboratory animals causes

permanent changes in the neurochemical, behavioral and structural aspects of the adult nervous

system (reviewed in Borue et al, 2007; Da ubert & Condron, 2010; Fred erick & Stanwood, 2009;

# Chlorpyrifos Human Health Risk Assessment D424485

Page 155 of 531 Oberlander et al, 2009). Moreover, it is generally recognized th at the development stage of

serotonergic perturbation determines the developm ental outcome (reviewed in Oberlander, et al.,

2009; Olivier et al, 2011), meaning that the time at which the deve loping animal experienced the

insult to the serotonergic nervous system would determine th e degree and the result. The review

by Daubert and Condron (2010) in cludes a table summarizing ma ny laboratory studies where

different aspects of the serotonergic syst em were manipulated (either through knock-out,

conditional knock-out, or chemical treatme nt) during development accompanied by the

behavioral and morphological re sults of that manipulation. Fo r example, SERT (serotonin

reuptake receptor) knock-out mice had alterations in sensory patterning, sleep pattern

abnormalities, and long-term behavioral deficits (Li, 2006), and animal s treated with SSRIs

during development show many si milarities to the SERT kno ck-out mice. Developmental

exposure to SSRIs in mice produced permanent (a dulthood) effects on behavior (Lisboa et al,

2007; Noorlander et al., 2008) and brain chemistr y (Noorlander, et al., 2008) and changes in

morphology in the hippocampus (Zheng et al., 2 011) and somatosensory cortex (Lee, 2009).

Because of the extensive usage of SSRI antidepre ssants in the human population, there is a rich

literature SSRI effects during pregnancy and la ctation (reviewed in Alwan & Friedman, 2009;

Ellfolk & Malm, 2010; Gentile, 2005; Oberlander, et al., 2009; Olivier, et al., 2011; Tuccori et

al., 2009). The main effects of SSRI usage are on cardiac development, and while there is some

support for concluding that actions on the serotonergic system early in development can lead to

long-term neurological dysfunction, there are only few studies that have studied this relationship

in humans in detail. A recent study has noted that children born to mothers who used SSRIs have

increased likelihood for social-behavioral abnormalities (Klinge r et al., 2011).

There are numerous studies of the effects of pe rinatal chlorpyrifos admi nistration on the patency

of the serotonergic system. Endpoints in various brain regions include serotonin levels (Aldridge

et al, 2005a; Slotkin & Seidler, 2007b, 2007c), serotonin turnover (Aldridgeet al., 2005a; Slotkin

& Seidler, 2007b, 2007c), serotonin receptor levels (Aldridge et al, 2005b; Aldridge et al, 2003;

Aldridge et al, 2004; Slotkin & Seidler, 2005), serotonin reuptake receptor levels (Aldridge, et

al., 2005b; Aldridge, et al., 2003; Aldridge, et al., 2004; Sl otkin & Seidler, 2005), serotonin

elicited second messenger activity (A ldridge et al., 2005b; Al dridge et al., 2003; Aldridge et al.,

2004), gene expression of serotonin receptor and metabolism related genes (Slotkin & Seidler,

2007a; Xu et al, 2012), serotonin related behavioral assessments (Aldridge et al, 2005; Chen et

al., 2011; Venerosi et al, 2010), a nd behavior after serotonergic drug challenge (Aldridgeet al.,

2005; Venerosi, et al., 2010). Chlo rpyrifos was administered du ring gestation (Aldridgeet al.,

2005a; Aldridge et al., 2003; Aldr idge et al., 2004; Slotkin & Se idler, 2007c; Venerosi et al.,

2010), postnatally (Aldridge, et al., 2005; Aldridge et al., 20 05a, 2005b; Aldridge et al., 2003;

Aldridge et al., 2004; Slotkin & Seidler, 2005, 2007a, 2007b) or during ad olescent (Chen, et al.,

2011) and the animals were assessed either shor tly after dosing ceased (Aldridge et al., 2003;

Slotkin & Seidler, 2007a), or af ter a matter of weeks (Aldridgeet al., 2005b; Aldridge et al.,

2003; Chen, et al., 2011; Slotkin & Seidler, 2007 b, 2007c) or in adulthood (Aldridge et al.,

2005a; Aldridge et al., 2004; Slotki n & Seidler, 2005; Venerosi et al., 2010). All the data indicate

that there are acute, as well as permanent, e ffects of neonatal chlor pyrifos treatment on the

maturation of the serotonergic nervous system . The effects are often gender-specific, regionspecific and dose-related. For example, a group of papers found permanent effects of late

gestational chlorpyrifos administ ration in rats (Aldridge, et al ., 2005a; Aldridge et al., 2004) or

# Chlorpyrifos Human Health Risk Assessment D424485

Page 156 of 531 mice (Venerosi et al., 2010) on the development of the serotonergic nervous system. In rats

(Aldridge et al., 2005a; Aldridge et al., 2004), chlorpyrifos was ad ministered on GD17-20 (1 or 5

mg/kg, sc in DMSO), and the adult brain showed changes in brain regional serotonin content (5

mg/kg group), increased serotonin turnover (1 and 5 mg/kg groups), increases in 5-HT 1A 

# and 5-

HT 2 

# receptor populations (1 mg and 5 mg/kg groups), increases in serotonin reuptake receptor (1

and 5 mg/kg groups), and changes in the adenylat e cyclase response to se rotonin in the cerebral

cortex and mid brain (1 mg/kg group)—all indicative of permanen t changes in the serotonergic

tone of the adult brain. In a related study in mi ce (Venerosi et al., 2010), chlorpyrifos was also

administered during late gestation, but at a hi gher dose and by a different route (GD14-17; 6

mg/kg, peanut oil, gavage); the offspr ing were assessed upon reaching adulthood (  90 days).

The chlorpyrifos-treated rats show ed minor differences in the behavi oral tests, but when the tests

were combined with a fluvoxamine (serotonin reuptake inhibitor) pharm acological challenge,

there were marked differences in responses be tween the animals that had been exposed to

chlorpyrifos during gestation and those that had not—another indication that developmental

chlorpyrifos exposure had perman ently altered the serotonergic tone of the adult brain.

There is ample evidence that chlorpyrifos exposure during development causes permanent

changes in the serotonergic nervous system; th ere are, however, few papers that assessed

concurrently the cholinesterase in hibition (either brain or blood) in those same animals. In some

cases, although cholinesterase activity was not as sessed concurrently, a dosing regimen was used

that had been characterized previously with re gard to cholinesterase activity. It does appear,

however, that most of the studi es on the effects of chlorpyrif os on the serotonergic nervous

system were conducted with doses of chlorpyrif os that likely produced marked inhibition of

cholinesterase activity. There is, however, one of the dosin g regimens [GD17-20 (daily

subcutaneous dosing with chlorpyrifos in DMSO ] that has been shown to produce no fetal brain

cholinesterase inhibition measur ed 24 hours after the last dose at the 1 mg/kg , and inhibition

(~20%) at 2 mg/kg (Qiao et al., 2002). As noted above, this level of gestational exposure to

chlorpyrifos permanently altered the development of the serotonergic ne rvous system (Aldridge

et al., 2005a; Aldridge et al., 2004).

In summary, with regard to development of th e chlorpyrifos adverse outcome pathway for the

serotonergic nervous system: the serotonergic system is intregal to mammalian brain

development and function; perturbation of that system during development leads to lasting

effects in mammals; and exposure to chlorpyrif os during the perinatal period can permanently

change the function of serotonergic system. Theref ore, as many steps in th is chlorpyrifos adverse

outcome pathway are possible and pl ausible, and in laboratory an imals the serotonergic nervous

system is sufficiently sensitive to low doses of chlorpyrifos during development to alter its

function, it is plausible that exposure to chlo rpyrifos during development could alter brain

development and the function of the serotonergic nervous system. Although chlorpyrifos effects

on the serotonergic nervous system in laborat ory animals likely is in itiated within 24 hours

(Slotkin & Seidler, 2007a) , the actual initiating ev ent of this potential adverse outcome pathway

is unknown.

3.2.1.6. Tubulin, Microtubule Associated Proteins and Axonal Transport

# Chlorpyrifos Human Health Risk Assessment D424485

Page 157 of 531 Microtubules, one component of the dynamic cyto skeletal scaffolding within each cell, are

composed of heterodimers of α - and β -tubulin, as well as microtubule associated proteins. The

microtubule associated proteins appear to have three main fu nctions: (1) to stabilize the

microtubules; (2) to aid in tubulin dissociati on and (3) to act as motor proteins moving

substances forward and backward along the micr otubules (Avila et al, 1994; Pellegrini &

Budman, 2005; Sánchez et al, 2000). Not onl y does the microtubule cytoskeleton determine

neuronal morphology (Matus, 1988, 1990; Sánchez, et al., 2000), but the dynamic reorganization

of the microtubules and microtubule associated proteins within a cel l may also coordinate neurite

extension/retraction, as well as grow th cone advancement. In addi tion to these integral roles in

brain structure and growth, micr otubules and the microtubule associ ated motor proteins kinesin

(Hirokawa & Noda, 2008) and dynein (Vallee et al, 2004) also provide a “railway” for transport

of materials throughout the cell, i.e. , axonal transport (Fukushima et al, 2009), another process

which is integral to the health of the central and peripheral nervous system, playing a pivotal role

in neuronal network formation and synapse maturation (Hirokawa & Takemura, 2004).

During brain development, the pattern of e xpression of microtubules (Bond & Farmer, 1983;

Denouletet al, 1986; Meininger & Binet, 1989; Moskowitz & Oblinger, 1995), the posttranslational modification of the tubulin (Fukushima, et al., 2009; Lee et al, 1990), and the

spectrum of microtubule associated proteins (Fischer & Romano-Clarke, 1990; Nunez, 1986;

Sánchez, et al., 2000) are all developmentally regulated. Because the phosphorylation of both

microtubules and microtubule associ ated proteins are integral to their functional roles in the

nervous system (Avila, et al., 1994; Fischer & Romano-Clarke, 1990; Fukushima, et al., 2009;

Sánchez, et al., 2000), there is concern that exposure to OP s may affect the phosphorylation

status and consequently the function of these molecules.

One of the shortcomings of constructing this adverse outcome pathway is that there are no

studies on tubulin dynamics, microtubule associated proteins or axonal tr ansport in developing

animals, but because tubulin dynamics, microtubule associated proteins and axonal transport are

so integral to the development and maintenance of the nervous system, the available adult data

are first given in detail and then summarized below.

Adult rats treated for 14 days with chlorpyrifos (sc in pean ut oil; 2.5, 10, 18, or 25 mg/kg; all

doses produced plasma cholinesterase inhibiti on) show depressed ante rograde and retrograde

axonal transport in the sciatic ne rve 6 days after cessation of trea tment (Terry et al., 2003). This

same laboratory followed up (Terry et al., 2007) w ith another, slightly different, study where

axonal transport was assessed in adult rats give n chlorpyrifos repeatedly (sc, 3% DMSO, 97%

peanut oil vehicle, every other day for 30 da ys; 18 mg/kg). Depressed bidirectional axonal

transport was noted after 1 dose, after the 30 days of dosing or 2 weeks after cessation of

treatment. This dose produced about 30-50% brai n cholinesterase inhibition 14 days after last

dose. In that same year, the Terry laboratory published an in vitro paper (Gearhart et al, 2007)

studying the effect of chlorpyrifos or chlo rpyrifos oxon on kinesin-dependent microtubule

motility (the mechanism of ante rograde axonal transport). Using tubulin and kinesin prepared

from bovine brain, they assessed kinesin-mediated movement of microtubules. This was done by

either preincubating the tubulin or the kinesin fraction with chlor pyrifos or chlorpyrifos oxon and

then combining the fractions and assessing move ment of the microtubules. They obtained the

same interesting pattern using chlorypyrifos or ch lorpyrifos oxon: there was no effect if

# Chlorpyrifos Human Health Risk Assessment D424485

Page 158 of 531 preincubated with microtubules, bu t marked effect (increased det achment of the microtubules) if

preincubated with kinesin. This assay contains no cholineste rase activity, thereby precluding

inhibition assessment. A recently published in vitro paper (Middlemore-Risher et al, 2011)

assessed the effect of chlorpyrif os or chlorpyrifos oxon transport of mitochondria in rat cortical

neurons. They found dose-related e ffects of chlorpyrifos or ch lorpyrifos oxon on total distance

moved and number of moving mito chondria. All doses produced chol inesterase inhibition except

lowest dose of chlorpyrifos (1 uM) or chlorpyr ifos oxon (5 nM), and those doses still affected

movement. Interestingly the toxic effect was not altered by co-treat ment of either a nicotinic or

muscarinic receptor blocker, an indication that the depressed transport was not mediated through

cholinergic receptors. Subsequent to demonstrating that in vitro chlorpyrifos oxon appeared to

alter tubulin dynamics (Grigorya n & Lockridge, 2009) and also covalently bound to tubulin

(Grigoryan et al., 2008), the Lo ckridge laboratory assessed tubu lin and microtubule associated

proteins in the brains of mice that had been dosed in vivo with chlorpyrifos or chlorpyrifos oxon

(Jiang et al., 2010). Repeated (14 days) dosing with chlorpyrifos (sc, 3% DMSO/97% peanut oil,

3 mg/kg) produced a transient decrease in blood AChE activity, no change in blood

carboxylesterase activity a nd a steady decline in blood BuChE activity. Brain β -tubulin from the

animals treated with chlorpyrif os had been covalently modified (diethosyphosphorylated) on

tyrosine 281 (the residue also found in the in vitro study (Grigoryan et al., 2009). This same

paper also described treating adult mice with a si ngle dose of chlorpyrifos oxon (3 mg/kg; ip);

this dose produced toxic signs and marked AC hE and BuChE inhibition in the plasma. Brain

tubulin was then purified, polymerized, the prot eins separated by gel electrophoresis and the

proteins identified by mass spectrophometry. The polymerized tubulin was also subjected to

atomic force microscopy nanoimaging to assess the structure (width and height) of the

microtubules. Microtubules from chlorpyrifos ox on- treated brains we re missing 6 microtubule

associated proteins. This observation was supported by the nanoimaging showing that

microtubules from chlorpyrifos oxon-treated anim als were narrower and shorter than control

brain microtubules and also had fewer attached (decorated) proteins than do the microtubules

from control brains. It inte resting to note that an in vitro assessment (Prendergast et al., 2007) of

the effects of chlorpyrifos oxon tubulin/microtubule associated protein dynamics in a 8 day old

rat hippocampal slice preparati on showed all concen trations of chlorpyrifos oxon decreased

mircrotubule associated protein- staining and decreased microtubul e associated pr otein mediated

tubulin polymerization by about 45%; all chlorpyrifos-oxon doses inhibited AChE.

In summary, in the late 2000s, a num ber of papers were published on the in vitro modification of

various proteins by chlorpyrifos or chlorpyrifos oxon (Grigoryan, Li , et al., 2009; Li et al., 2009),

including tubulin (Grigoryan, Li, et al., 2009; Grigoryan & Lockridge, 2009; Grigoryan,

Schopfer, et al., 2009; Grigorya n, et al., 2008). Although interes ting and provocative, these

studies were usually conducte d with exceedingly high concentr ations (high micromolar to

millimolar) of the OP compound, making the conn ection to a “real world” human exposure

tenuous. In 2010, however, Jiang and coworkers published an in vivo study in which they found

covalently labeled tubulin in th e brains of mice treated with ch lorpyrifos or chlorpyrifos oxon,

showing the real-world possibility of chlorpyrifos-induced covalent modification of brain tubulin

in mammals at doses that produced minimal blood cholinesterase inhibiti on (Jiang, et al., 2010).

Other studies have shown perturbations in tubu lin dynamics, perturbations in the amount of

tubulin protein or microtubule a ssociated proteins, and changes in axonal transport elicited by

treatment with chlorpyrifos or its oxon. Submicromolar concen trations of chlorpyrifos oxon

# Chlorpyrifos Human Health Risk Assessment D424485

Page 159 of 531 markedly inhibited tubulin polymerization in vitro (Prendergast et al ., 2007), while in vitro

treatment with chlorpyrifos oxon (but not chlorpyrifos) reduced that amount tubulin in rat C6

glioma cells (Sachana et al ., 2008). Decreases in the amount of microtubule associated proteins

after chlorpyrifos or chlorpyrif os oxon treatment has been reporte d by three separa te studies: two

in vitro studies both showed decreases after ch lorpyrifos oxon trea tment (Prendergast et al

., 2007; Sachana et al ., 2008), and one in vivo study showed extensive reduction in specific

microtubule associated proteins in the brains of mice treated with chlorpyrifos oxon (Jiang et al

., 2010). Depressed axonal transport has b een noted by many laboratories both in vivo (Terry et al

., 2007; Terry et al ., 2003) and in vitro (Gearhart et al ., 2007; Middlemore-Ri sher, et al., 2011)

using both chlorpyrifos ( in vivo and in vitro studies) and chlorpyrifos oxon ( in vitro studies). In

two instances (Gearhart et al ., 2007; Middlemore-Risher et al ., 2011), the effects of the

chlorpyrifos and oxon were noted at concentrations of ch lorpyrifos or chlorpyrifos oxon that did

not inhibition cholinesterase activ ity (Middlemore-Risher, et al., 2011) or in systems that were so

basic (purified microtubule/ki nesin) that no cholinestera se was present (Gearhart et al ., 2007).

The construction of an adverse outcome pathwa y using chlorpyrifos-induc ed effects on tubulin

and microtuble associated proteins is still in its infancy mainly because although it is thought that

tubulin, microtubule associated proteins and axon al transport are integral to nervous system

development and maintainence, there is no experi mental evidence that perturbations of these

endpoints by chlorpyrifos during deve lopment has neurotoxic outcomes.

3.2.1.7 Summary/Conclusions

There are a number of known developmentally neurotoxic chemicals with well-established

relationships between exposure and neurological disorders in humans for which a definitive

mode of action has not been established: fo r example lead, methyl mercury, and ethanol

(Alfonso-Loeches & Guerri, 2011; Castoldi et al ., 2008; Farina et al ., 2011; Johansson et al

., 2007; Verstraeten et al ., 2008). Even today, despite with thousands of published papers on these

three, accepted, developmental neurotoxicants, no coherent adverse outcome pathway or

pathways can be constructed, because there are a multitude of initiating toxic events and cellular

responses put forth, and they are not positively connected with one another. The only adverse

outcome pathway available for any form of neur otoxicity has been deve loped for domoic acid in

adult animals (Watanabe et al ., 2011).

As can be seen from the section above, ther e are several lines of evidence for actions of

chlorpyrifos distinct from the classical mode of action of cholines terase inhibition. This

information has been generated from model syst ems representing different levels of biological

organization, and provide support for molecular initiating events (binding to the morphogenic

site of AChE, muscarinic receptors, or tubulin ), cellular responses (a lterations in neuronal

proliferation, differentiation, neur ite growth, or intracellular signa ling) and responses at the level

of the intact nervous system (s erotonergic tone, axonal transport) . In some cases these effects

occur at chlorpyrifos exposure levels below or equi valent to those which result in cholinesterase

inhibition in the same test system. When taken in aggregate, data from individual studies can be

used to develop plausible hypotheses for a mode of action leading to developmental

neurotoxicity ( e.g. inhibition of the morphogenic action of AChE leading to reduced neurite

outgrowth and subsequent miswiring or dysmorphol ogy of the brain); however, as is the case for

# Chlorpyrifos Human Health Risk Assessment D424485

Page 160 of 531 many other developmental neurotoxicants, most of these studies have not b een designed with the

specific goal of construction or testing an adverse outcome pathway. Thus, there are not

sufficient data available to test rigorously the causal relationship between effects of chlorpyrifos

at the different levels of biologi cal organization in the nervous sy stem. Moreover, there is still

lack of data on the toxic moiety (ies) im pacting neurodevelopment (i.e., chlorpyrifos,

chlorpyrifos oxon and/or TCPy) (Flaskos, 2012). Th is is not surprising in light of the complex,

intricate, and interrelated dyna mic processes ongoing in the developing brain and multiple

critical windows for exposure.

3.2.2. Adverse Outcomes in Laboratory Animal s: Effects on the Developing Brain

There is a considerable and gr owing body of literature on the e ffects of chlorpyrifos on the

developing brain of laboratory animals (rats and mice) indicating that gestational and/or

postnatal exposure may cause persis tent behavioral effects into adulthood. These data provide

support for the susceptibility of the developing mammalian brain to chlorp yrifos exposure. The

literature was summarized and a preliminary re view was provided by the Agency for the 2008

SAP with a more extensive review in 2012 and upda ted in 2014. The Panel agreed that exposure

to doses of 1 mg/kg/d and great er, during some developmental pe riod, produced significant and

long-term effects on behavior. While behavioral changes were consistently reported, they were

somewhat inconsistent, most likel y due to experimental design diffe rences. Such factors include

route of exposure, developmental period of exposur e, test methods, choice of dependent variable,

statistical analyses, and so on, which are critical features of all developmental neurotoxicity

studies.

In this section the agency reviews newer literature, i.e. , papers published since 2008, to build on

the earlier SAP review. The new information pr ovided is evaluated in terms of concordance

with, or divergence from, the previous, pre-2008 fi ndings. Overall, these new studies serve to

strengthen the findings on which the 2008 SAP ma de their conclusions and further documents

the scope of long-term behavioral effects re sulting from chlorpyr ifos exposure during

development.

Text and tables in 3.2.2.2 were extracted from the 2012 draft SAP issue paper. Section 3.2.2.3

provides a summary of the newest st udies published between 2012 and 2014.

Papers considered herein by the Agency as a ddressing long-term outcomes from developmental

exposure include only those wher e chlorpyrifos is administer ed during the preweaning period

(either gestational and/or postnat al) and the offspring are examined at some time after weaning.

That is, papers reporting evalua tions shortly after birth or duri ng the pre-weaning period do not

reflect long-term consequences and may also be confounded by AChE/ChE inhibition during

concurrent or recent exposure; thus, those data are not discusse d further here. In addition, the

Agency focused its efforts on studi es using relatively low doses, and that did not (or would not

be expected to) produce a consider able degree of brain AChE inhi bition and resultant cholinergic

toxicity. These constraints ai d in the unencumbered evaluation of longer-term effects compared

to acute impacts of AChE inhibition, but it does limit the use of some papers.

At the time of the 2012 SAP meeting, the followi ng papers were identified and evaluated by the

# Chlorpyrifos Human Health Risk Assessment D424485

Page 161 of 531 Agency; (Abou-Donia et al., 2006; Aldridge, Levi n, et al., 2005; Billau er-Haimovitch et al.,

2009; Braquenier, Quertemont, Tirelli, & Plumier, 2010; Carr et al., 2001; Chakraborti, Farrar, &

Pope, 1993; Chanda & Pope, 1996; Dam et al, 2000; Haviland, Butz, & Port er, 2010; Icenogle et

al., 2004; D. A. Jett, Navoa, Beckles, & McLemore, 2001; Johnson, Chambers, Nail,

Givaruangsawat, & Carr, 2009; Laviola, Adrian i, Gaudino, Marino, & Kell er, 2006; Levin et al.,

2002; Levin et al., 2001; Mauris sen, et al., 2000; Muto, Lobell e, Bidanset, & Wurpel, 1992;

Ricceri et al., 2003; Ricceri et al., 2006; Venerosi , Calamandrei, & Ricceri, 2006; Venerosi et al.,

2008; Venerosi, et al., 2010; Ve nerosi, Ricceri, Scattoni, & Ca lamandrei, 2009; Zhang et al.,

2011). It is important to appr eciate the various experimental designs, encompassing species,

gender, exposure scenarios, ages of assessment, a nd test apparatuses. Of the papers included in

the 2012 analysis, four papers and one study from a fifth paper did not meet the criteria stated

above, and were excluded from further consideration herein.

Specifically, these excluded studies are:

1) Muto et al. (1992) used a chlorpyrifos pr oduct that was formulat ed in xylene, a known

developmental and reproductive toxicant, yet the vehicle c ontrol group received saline,

not xylene. Thus, the findings of that study cannot be attributed to chlorpyrifos alone.

Furthermore, all testing took place be fore weaning, no later than PND16;

2) A gestational-exposure study (GD12-19) used a dose (25 mg/kg/d sc) that produced 90%

brain inhibition in the dams at the end of dosing, and ~20% brain i nhibition in offspring

on PND3 (Chanda & Pope, 1996). The reporte d motor effects are confounded with the

concurrent cholinesterase inhibition on PND1 and 3, and no testing was conducted at

later ages;

3) A postnatal study (Chakraborti, et al., 1993) used a very hi gh dose (40 mg/kg/d sc) given

directly to the pups postnat ally (PND7, 11, 15, 19), with up to 60% brain inhibition

evident four days after dosing;

4) A gestational study (Venerosi, et al., 2009) only tested pups up to PND12, reporting

altered motor ontogeny and changes in ultrasonic vocalizations; and

5) The “postnatal” study described by ( Jett, et al., 2001) involved dosing after weaning,

while the rats were being tested in the Mo rris water maze. Note, part of this paper

remains in consideration, since the “prewean ing” study from that paper met the criteria

for inclusion.

3.2.2.1 Developmental Impacts on Neurological Domains



# Because many of these papers report a number of positive as well as negative findings, the

agency had previously taken the approach of co mparing responses that were observed following

various exposures to a common dose, 1 mg/kg/d (FIFRA SAP, 2008a; U.S. EPA, 2011). A more

robust approach is taken here, to include importa nt factors such as dose-response and differences

in exposure scenarios. In order to evaluate e ffects on domains of neurological functions, broken

# Chlorpyrifos Human Health Risk Assessment D424485

Page 162 of 531 down by exposure period, the papers have been su mmarized as such in Section 3.2.2.1.5. It is

well-known that exposures during di fferent critical periods of development can result in very

different outcomes (Adams et al., 2000; Rice & Barone, 2000). Unfortunately, development of

the nervous system does not occur in distinct non-overlapping temporal periods with which to

attribute the apical behavioral ch anges observed. In the text and tables below, the papers from

2008 to 2012 are described and evaluated in terms of their contribution to and concordance with,

previous papers using similar measures. The ag ency’s evaluation follows several Bradford Hill

criteria including consistency of findings acr oss domains and dose response. The latter was

considered as either graded ma gnitude of effects across doses or significant findings at a higher

but not lower dose; unfortunately, many of the chlorpyrifos studies ha ve evaluated only one

dose.

These studies include measures that evaluate the following neur obehavioral domains: cognitive

function, anxiety and emotion, soci al behavior and maternal interactions, and motor activity.

These are discussed in greater detail below; ove rall, the newer data were in general agreement

with previous studies.

3.2.2.1.1 Cognitive Function

A few of the new studies evaluated effects on cognitive function, using some of the same

procedures used in the earlier studies (radial ar m maze, Morris water maze). The earlier findings

are summarized below, along with co mparisons to the newer studies.

Radial Arm Maze



# The radial arm maze is a spatial task requiring the subject to enter different arms for food

reinforcer, which is located in only certain arms . A widely used test of learning and memory,

both working errors (re-entering baited arms after taking the food) and refe rence errors (entering

arms that are never baited) ar e recorded over days of training. From 2001-2005, a series of

behavioral studies from a single laboratory (D uke University) described effects on radial arm

maze performance in adult rats that were treated with chlorpyrifos during gestational or postnatal

development. Both working and reference memory are altered in rats trea ted during gestation or

early postnatally, and gender differences were described in all but one study. Across these

studies, significant differences were detected in one or the other t ype of error, or both, and error

selectivity was not fully consistent across studi es. Following early gestational exposure (GD9-

12), high-dose (5 mg/kg/d) rats of both sexes showed increased working and reference memory

errors early in training (Icenogle, et al., 2004). With later gestational exposure (GD17-20), only

females showed more errors; how ever, this was observed only at the low dose (1 mg/kg/d, not 5

mg/kg/d) (Levin, et al., 2002). W ith early postnatal exposure (P ND1-4, 1 mg/kg/d), two separate

studies showed similar results in that error rate was increased in males (worse performance) but

decreased in females (better pe rformance) (Aldridge, Levin, et al., 2005; Levin, et al., 2001).

Only the late postnatal exposure (PND11-14, 5 mg /kg/d) was without effect on this measure

(Levin, et al., 2001).

Several new studies have confirmed chlorpyrif os impacts on learning and memory. In a new

# Chlorpyrifos Human Health Risk Assessment D424485

Page 163 of 531 study using the radial arm maze and longer pos tnatal exposures (PND1-21), male, but not

female, rats showed more working memory e rrors at all doses (lowest dose, 1 mg/kg/d)

(Johnson, et al., 2009). This was evid ent later in training, indicating that the task was not learned

as well as in controls. Reference memory errors showed the same gender-s pecific pattern as seen

in two Duke studies (Aldridge, Levin, et al., 2005; Levin, et al ., 2001), in that chlorpyrifostreated females showed fewer errors during tr aining, and males showed more. The altered

reference memory pattern was only seen in the mid and high dose groups, which had

incrementally increasing doses (up to 4 or 6 mg /kg/d) during the exposur e period. In this study,

the low-dose group had experienced about 14% brain AChE inhibition on PND20, with no

inhibition evident when radial arm maze traini ng began (PND36). On the other hand, the mid

and high-dose rats showed some residual AChE inhibition at that time , making it challenging to

separate the memory deficits from co ncurrent AChE holinesterase inhibition.

A study using mice exposed gesta tionally (GD17-20, 1 or 5 mg/kg/ d) also used a radial arm

maze, although comparisons are difficult due to differences in length of training and data

presentation (Haviland, et al., 2010 ). Reference memory errors showed some differences across

trial, dose, and sex; these are not interpreted by the authors as a meaningful effect, and may

indeed be spurious. The author s used these data to compare with their novel foraging task,

which used a modification of a radial arm maze a nd examined the rate of learning that a reward

(food) is present (recognition) as well as its lo cation (positional learni ng). Both of these

parameters were altered by chlorpyrifos exposur e. The low dose (1 mg/kg/d) females were

delayed in learning to recognize th e reward, but there were no stat istically significant differences

at that dose in positional learning (despite the aut hors’ claim of an effect). On the other hand,

low-dose males showed accelerated food recognition, and increased positional learning that was

evident during only two sessions. The high-dose groups (both sexes) showed the same pattern of

changes, with somewhat greater magnitude of differences, indicating dose-response. Thus,

effects were observed on a spatial learning task, albeit a different a pparatus and procedure. Note

that the gender effect was reversed from that reported above for rats in the radial arm maze –

these differences could be due to species or testing apparatus.

Morris Water Maze



# The Morris water maze is a different type of spatia l learning task, which can be varied to assess

different types of learning and me mory. In this test, the animals are trained over days to swim to

the location of a submerged platform to escape fr om the water, and learning is evident by faster

latencies and other measures of memory fo r the platform position. A recent paper using

gestational exposure (GD9-18) in mice resulted in slower learning in the offspring, although a

clear dose-response was not evid ent (Billauer-Haimov itch, et al., 2009). Th e authors report an

overall effect of chlorpyrifos tr eatment, but state that only the lower doses (1, 3 mg/kg/d, but not

5, 10 mg/kg/d) were individually si gnificant. While the magnitude of effect does not appear to

be pronounced in the first study (visual inspection of Figure 3 in the paper), the finding was

repeated, and was more obvious, in two additiona l studies using a single dose (3 mg/kg/d).

Another recent paper from the same laboratory again reported slower learning in mice exposed

during gestation (3 mg/kg/d; GD9-18) (Turgema n et al., 2011). Thus, there are internal

replications of this finding across several studies, albeit in th e same laboratory.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 164 of 531 These recent findings, plus anot her study available only in Chinese 66 

# , extend an earlier report of

rats that showed deficits in the Morris wa ter maze (beginning on PND23) following exposure on

PND7, 11, and 15 (0.3, 7 mg/kg/d ) (Jett, et al., 2 001). The 2008 SAP, however, was critical of

this paper, for reasons described below.

Summary



# Taken together, these studies in rats and mice show altered cognitive function using wellaccepted tests of spatial learning and memory (radial arm maze, Morris water maze). The

direction of change may be sex-specific and de pendent on the timing of exposure. Often these

changes suggest impaired learning and/or memory. While enhanced function is also apparent in

some studies, such changes are evidence of alterations in me mory processes nonetheless.

Several of these findings have re cently been replicated across st udies and laborator ies. Effects

were also reported in a spatial foraging task (Haviland, et al., 2010), but direct comparison

between that and the radial arm maze is difficult. Earlier papers have reported that other

cognitive tasks (spontaneous or delayed alternatio n, passive avoidance) are not altered, but there

are no new studies using these ot her tasks. These outcomes are summarized in App 1-8.

66 

# Another new study also evaluated Mo rris water maze learning, using rats exposed postnatally (PND11-14, 5

mg/kg/d, route uncertain) (Zhang et al., 2011). The abst ract states that learning and memory impairments were

observed, but the rest of the paper cannot be critically evalua ted at this time (article is in Chinese) and so cannot be

combined with other findings.Chlorpyrifos Human Health Risk Assessment D424485Page165of531Table App. 1-8. Summary of Outcomes onCognitive Tests in Male (M) and Female (F) Rodents (Extractedfrom USEPA, 2012)Early gestationGD9-12; 9-18Late gestationGD 17-20PerinatalGD6-LD10Early postnatalPND1-4Late postnatalPND11-14;7,11,15PostnatalPND1-21Radial Arm MazeCognitive deficit -rat, M&F, doseresponse6Cognitive deficit -rat, F not M, nodose-response4Cognitive deficitin M, improvedfunction in F - rat3Cognitive deficitin M, improvedfunction in F - rat7No effect – rat,M&F3Cognitive deficitin M, improvedfunction in F - rat,dose-response9Morris WaterMazeCognitive deficit -mouse, M&F, nodose-response8Cognitive deficit –mouse, M&F11Cognitive deficit -rat, M&F, doseresponse2Foraging MazeCognitive deficitin F, improvedfunction in M –mouse, doseresponse in F notM10T-mazeSpontaneousAlternationNo effect – rat,M&F6No effect – rat,M&F4No effect – rat,M&F3No effect – rat,M&F3Delayed SpatialAlternationNo effect – rat,M&F1Passive AvoidanceNo effect – mouse,only M tested5No effect – mouse,only M tested51Maurissen et al., 20002Jett et al., 2001Chlorpyrifos Human Health Risk Assessment D424485Page166of5313Levin et al., 20014Levin et al., 20025Ricceri et al., 20036Icenogle et al., 20047Aldridge et al., 20058Billauer-Haimovitch et al., 20099Johnson et al., 200910Haviland et al., 201011Turgeman et al., 2011

# Chlorpyrifos Human Health Risk Assessment D424485

Page 167 of 531 3.2.2.1.2 Anxiety/Emotion

Anxiety and despair/affect have been modeled in animals usin g a number of different tests,

including those that measure avoidance of aversi ve areas, investigati on of novel areas, response

to forced activity, and response to preferred substances. Several new studies assessed anxiety,

despair, and affect. While di fferent procedures were used in most cases, there was some

concordance in outcomes. These are described below.

Tests of Anxiety



# Several studies have suggested that chlorpyrifos alters measures of anxiety. The early studies

came from the laboratories of Duke University a nd ISS (Italy). Three newe r studies, all in mice,

add to the findings in this area: two were fr om the ISS laboratory and one came from a third

laboratory (Belgium). Earlier studies employed an elevated plus maze, which has open and

enclosed areas: being fearful of bright open areas, rodents tend to stay in the enclosed arms.

Decreased anxiety is inferred from changes such as increased time in the open arms, decreased

head dipping, and other measures. Using this paradigm, lower anxiety was reported in rats

(males, not females) exposed postnatally to 1 mg/kg/d (PND1-4)(Aldridge, Levin, et al., 2005;

Ricceri, et al., 2003) and in mice (females, no t males) exposure postnatally to 3 mg/kg/d

(PND11-14) (Ricceri, et al., 2006). On the other hand, increased a nxiety was measured in mice

exposed gestationally (GD15-18) , but only in the low-dose (3 mg/kg/d, not 6 mg/kg/d) males

(not females) (Ricceri, et al., 2006). There were no changes in a nxiety behaviors in rats exposed

gestationally (GD9-12, 1 or 5 mg /kg/d) (Icenogle, et al., 2004).

Three new studies have used a di fferent apparatus, the light-dar k box, to measure anxiety, but the

principle is the same in that rodents typically pr efer the dark chamber. In one study (Braquenier,

et al., 2010), both the elevated plus maze and the light-dark box were used, providing direct

comparisons between these tests. Mice were exposed both gestationally and lactationally

(GD15-LD14, 0, 0.2, 1, or 5 mg/kg/d) , and only female offspring were tested. When tested in

the light-dark box, the middle dose group only (1 mg/kg/d) showed decreased time in the center

of the light side, which was cons idered to reflect increased anxiety. The same dose group moved

back and forth between the sides less often, whic h could reflect general activity levels but was

also interpreted by the authors as greater anxiety. The time in th e dark and light sides, however,

did not differ. Littermates were tested in th e elevated plus maze, and again the middle dose

group showed less time spent in the open arms and fewer open arm entries, supporting a

conclusion of increased anxiety, bu t only at the middle dose. The lack of dose-response was not

addressed by the authors.

A study in which mice were treated gestationally (GD14-17, 6 mg/ kg/d) (Venerosi, et al., 2010)

showed no difference in time spent on either side of the light-dark box, but females (not males)

spent more time in the tunnel connecting the sides. This finding, along with a few other

measures that did not reach sta tistical significance, was interpreted by the authors as increased

anxiety. Another study by this group (Venerosi, et al., 2008) exposed mice postnatally (PND11-

14, 3 mg/kg/d), then bred the female offspring as adults and allowed them to deliver normally.

On postpartum day 2, the dams were removed fr om the pups and tested in the light-dark box.

They reported a decreased latency and higher proportion of mice entering the light side,

indicating decreased anxiety. Time in th e dark or light sides was not reported.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 168 of 531 Despair/Affect



# Only a few studies have also in cluded measures of despair and affect, and the results are not

completely consistent. Most recently, behavi oral despair was measured using a forced

swimming procedure (Venerosi, et al., 2010). In that study, gestational ex posure to chlorpyrifos

did not alter any baseline respons es, and there are no other studie s using similar measures with

which to compare. There have been no new studies that could add to, and aid in the

interpretation of, the findings re ported by others: decreased prefer ence for chocolate milk in rats,

or no effect on novelty exploration in mice (Aldri dge, Levin, et al., 2005; Ri cceri, et al., 2003).

Summary



# Taken together, these assessments suggest that, in both rats and mice, changes in anxiety are

dependent on exposure period. Specifically, postnatal exposure decr eases anxiety, whereas

increased anxiety is observed following late gest ational exposure or a l onger gestational plus

postnatal exposure. Some inconsistencies are ev ident, such as lack of dose-response in a few

studies and effects on one sex or the other (same laboratory, si milar dosing regimen). Thus,

while the data are not fully consistent, overall th ere is evidence for long-term changes in anxiety

behavior following chlorpyrifos e xposure, as shown in Table App 1-9.Chlorpyrifos Human Health Risk Assessment D424485Page169of531Table App 1-9 Summary of Anxiety/Emotion Outcomes in Male(M) and Female (F) Rodents (Extracted from USEPA, 2012)Early gestationGD9-12Late gestationGD14-17; GD15-18Early postnatalPND1-4Late postnatalPND11-14PerinatalGD15-LD14Elevated Plus Maze(anxiety)No effect – rat, M&F1Decreased anxiety -mouse, M not F, nodose-response5Decreased anxiety -rat, M not F2Decreased anxiety -mouse, F not M, doseresponse5Increased anxiety -mouse, only F tested,no dose-response3Light-Dark box(anxiety)Increased anxiety -mouse, F not M7Decreased anxiety -mouse, dams6Increased anxiety -mouse, only F tested,no dose-response3DespairNo effect – mouse,M&F7Novelty/preferenceDecreased anxiety -rat, M&F2No effect – mouse,M&F4No effect – mouse,M&F41Icenogle et al., 20042Aldridge et al., 20053Braquenier et al., 20104Ricceri et al., 20035Ricceri et al., 20066Venerosi et al., 20087Venerosi et al., 2010

# Chlorpyrifos Human Health Risk Assessment D424485

Page 170 of 531 3.2.2.1.3 Social Behaviors/Interactions

Conspecific behaviors are not ty pically studied in the context of developmental neurotoxicity

studies, and methods for such a ssessments are not well-standardi zed (Cory-Slechta, et al., 2001).

Aggressive, social, and parental behaviors have been studied following developmental exposure

to chlorpyrifos, all of which were conducted in the ISS laboratory using mice. Even within this

one laboratory, however, various te sting methods have been use d. These include: 1) social

investigation of nulliparous female:female pairs; 2) social investigation and agonistic behaviors

in male:male pairs; 3) maternal behaviors towards pups, induced by placing nulliparous females

with foster litters; 4) maternal behaviors in lactating dams towards their own pups, and 5)

agonistic behaviors in lactating dam:male pairs. In most st udies, measures include social

(interactive) as well as nonsocial behaviors ( e.g. , grooming, exploration). Within each grouping,

there are often many measures take n, and treatment effects have so metimes been reported on just

a few, suggesting that the changes are subtle . There are few systematic comparisons of

chlorpyrifos effects across these varied be haviors using similar dosing regimens.

Maternal Behavior



# Two recent papers focused on maternal behavior, measuring actions of the lactating dam towards

her pups, nesting activity, and agonistic behavior s towards an intruder male. In one study

(Venerosi, et al., 2008), mice were exposed postn atally (PND11-14, 3 mg/kg/d), mated as adults,

and the dams were tested. They reported that the treated dams showed delayed start of nesting,

decreased latency to lick pups, as well as fewer de fensive postures and more social investigation

of an intruder male. This latter effect was repli cated in that decreased aggressive behaviors were

also reported for lactating dams that had b een exposed in their own fetal period (GD14-17, 6

mg/kg/d) (Venerosi, et al., 2010). These findings were interpreted by th e authors as impaired

maternalistic behaviors.

Social/Agonistic Behavior



# Evaluations of male or female same-sex social behaviors generally show no effect of postnatal

(PND1-4 or PND11-14) chlorpyrifos exposures (Ricceri, et al., 2003; Ve nerosi, et al., 2006), a

finding that was recently confirmed (Venerosi, et al., 2008; Venerosi, et al., 2010). On the other

hand, increased investigation of the stranger mouse was reported following gestation exposure

(GD15-18)(Venerosi, et al., 2006) , and increased solicitation beha viors were observed following

late postnatal exposure (PND11-14) (Ricceri, et al., 2003). Thus, there are contradictory reports

of changes in female social behavior, being ei ther increased or not al tered. The only study of

male social behavior showed no effect.

Earlier studies reported that male agonistic be haviors were consistently increased in mice

exposed either postnatally (PND1-4, PND11-14) or gestationally (GD15-18); the lowest

effective dose was 1 mg/kg/d administered during either postnatal peri od (Ricceri, et al., 2003;

Ricceri, et al., 2006). None of the newer studies addressed male behaviors. These findings are

summarized in Table 1-10.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 171 of 531 Summary



# Overall, decreased aggressive be haviors of dams that had been exposed during their development

are a common finding across several exposure periods, as are increased male agonistic behaviors.

Social and/or maternal behaviors in females are less consistently or convincingly altered.Chlorpyrifos Human Health Risk Assessment D424485Page172of531Table App 1-10. Summary of Social/Interactive Behavior Outcomes in Male (M) and Female (F) Mice (Extracted from USEPA, 2012)Exposure/effectLate gestationGD14-17, 15-18Early postnatalPND1-4Late postnatalPND11-14Female:female socialIncreased investigation, doseresponse3No effect1Increased solicitation, doseresponse1No effect3No effect4Male:male socialIncreased aggressive postures,dose-response2Increased agonistic behavior,differs across time-course, dose-response1Increased solicitation, doseresponse1Increased agonistic behavior, overtime, no dose-response1Increased attack behaviors, doseresponse2No effect social4,aInduced maternal No effect2Increased maternalism, doseresponse2Natural maternal Decreased maternalism4Natural maternal:male Decreased aggression5Decreased defensiveness4DR dose-response, if not mentioned, only one dose testedaapparently only social, no agonistic, behaviors measured1Ricceri et al., 20032Ricceri et al., 20063Venerosi et al., 20064Venerosi et al., 20085Venerosi et al., 2010

# Chlorpyrifos Human Health Risk Assessment D424485

Page 173 of 531 3.2.2.1.4 Motor activity

Most of the developmental studies of chlorpyrif os have evaluated motor activity of some sort;

these were presented and summarized in detail in the 2008 SAP report. In general, activity levels

have been recorded in terms of: 1) locomotion; 2) response latencies or other activity measures

in the course of testing in various apparatuses (t-maze, radial arm maze, etc); and 3) habituation

of activity over the session in activity chambers. In general, the earlier studies reported that

activity is increased, decreased, or not altered, in either both or just one sex. Recent studies add

to this literature, but none provide information to better explain these varied and contradictory

outcomes.

Activity Devices



# Evaluations of locomotor activity in open fields or other activity devices have been made in

numerous studies across quite a few laboratories. Looking acr oss developmental period and

gender, there have been similar reports of increased, d ecreased, or no change in various measures

of activity ( e.g. , exploration, rearing, etc). Generalizations of effect could not be made in the

2008 preliminary review.

Two recent studies have used an open field to meas ure activity. Braquenier et al. (2010) reported

no change of activity levels in female mice that had been exposed from late gestation through

late lactation (GD15-LD14). Wh ile Zhang et al. (Zhang, et al., 2011) reported decreased activity

in rats, those data cannot be evaluated at this time due to the language barrier.

Ancillary Activity Measures



# As with activity measured specifically in activ ity devices, various measures of activity during

other tests have shown increases , decreases, or no effect, in both or only one sex, with no

obvious association to exposure period. It is impo rtant to consider, for example, that the time it

takes to visit the arms of a radial arm maze is a somewhat different behavior than exploratory

movement in a novel chamber. Thus, these activity measures may not be fully comparable, and

the evaluations described here shoul d be interpreted with caution.

Three recent papers reported assessments of activ ity during cognitive and social behavior testing.

There was no change noted in postnatally expo sed rats during radial arm maze testing (PND1-

21) (Johnson, et al., 2009), or in gestationally exposed mice dur ing foraging testing (GD17-20)

(Haviland, et al., 2010). Mice e xposed postnatally (PND11-14) s howed decreased exploratory

behaviors during the acclimatiza tion phase of social testing ( i.e. , before being exposed to another

mouse); however, this was only significant in the first block of the test session (Venerosi, et al.,

2008).

# Chlorpyrifos Human Health Risk Assessment D424485

Page 174 of 531 Habituation



# Habituation, or decrease in act ivity level during the course of a test session, has only been

specifically evaluated in the a fe w laboratories. As with other activity outcomes, the data are

specific to exposure period and gender. Within th e same laboratory, habituation is either faster

(early gestation, both sexes) (Ice nogle, et al., 2004), sl ower (late gestation, females only; late

postnatal, both sexes) (Levin, et al., 2002; Levin, et al., 2001), or not altered (early postnatal)

(Levin, et al., 2001). A perinatal study (GD6-LD 10) also reported no change in habituation;

however, the data were not statis tically analyzed in the same way, so results are not comparable

(Maurissen et al., 2000). This measure has not been addressed in more recent studies.

Summary



# There remains to be inconsistencies in the variou s measures of motor activity, but it is important

to note the numerous differences in procedures and apparatuses in which activity was measured.

Given this, generalizable conclusions and summaries cannot be made at this time.Chlorpyrifos Human Health Risk Assessment D424485Page175of531Table App 1-11. Summary of Motor Activity Outcomes in Male (M) and Female (F) Rodents (Extracted from USEPA, 2012)Early gestationGD9-12Late gestationGD14-16; 14-17;15-18; 17-20PerinatalGD6-LD10; GD15-LD14Early postnatalPND 1-4Late postnatalPND11-14Postnatal PND1-21ActivitychambersNo effect – rat,M&F6Increased activity -mouse, only Mtested, doseresponse13No effect - mouse8No effect – rat,M&F10No effect – rat,M&F11No effect – mouse,only F tested2No effect - mouse,M&F12Decreased activity -rat, M not F4No effect – rat,M&F9Increased activity -mouse, only Mtested, doseresponse13Increased activity -mouse, M&F, doseresponse12Increased activity -rat, M&F4No effect – rat,M&F9Decreased activity -rat, M&F, doseresponse3ActivitymeasuresIncreased activity -rat, M&F, doseresponse6No effect - mouse,only F tested14No effect - mouse,M&F5Increased activity -rat, M&F, DR10No effect - mouse,M&F13No effect – mouse,only F tested2Increased activity -rat, M not F1Increased activity -mouse, M&F12Decreased activity -rat, M not F9Decreased activity -mouse, M&F14Decreased activity -rat, M not F9Increased activity -mouse, M&F12No effect – mouse,M&F13No effect – rat,M&F7Habituation rateIncreasedhabituation - rat,M&F, doseresponse6Decreasedhabituation - rat, Fnot M, doseresponse10No effect – mouse8No effect – rat,M&F11No effect – rat,M&F9Decreasedhabituation - rat,M&F91Aldridge et al., 20052Braquenier et al., 20103Carr et al., 20014Dam et al., 2000Chlorpyrifos Human Health Risk Assessment D424485Page176of5315Haviland et al., 20106Icenogle et al., 20047Johnson et al., 20098Laviola et al., 20069Levin et al., 200110Levin et al., 200211Maurissen et al., 200012Ricceri et al., 200313Ricceri et al., 200614Venerosi et al., 2008Chlorpyrifos Human Health Risk Assessment D424485Page177of5313.2.2.1.5 Detailed information on behavioralstudies (Extracted from USEPA, 2012).Table App 1-12.In vivostudies of chlorpyrifosadministered during gestation; effects described are only those measured afterweaning. Bold indicates functional domain that showed effectsStudy &laboratorySpecies &strainDose, route,vehicleDosing periodAChEinhibitionDomainAge of testingOutcome,effective dose &genderNotesaDoseresponseb?Abou-Donia etal., 2006DukeRat SD 1 mg/kg/ddermal in 70%ethanolGD4-20 0-30% increasebrain (regions)activity atPND90, F notMNeuromotorfunctionPND90 Decreased griptime, M&F;Decreasedperformance oninclined plane, Fnot M;Used2/sex/litter, (5litters/treatment), didnot control forlitter;CerebellarpathologyNAIcenogle et al.,2004DukeRat SD 1, 5 mg/kg/d scin DMSOGD9-12 Not measuredMotor activityPND28-42 Faster habituationin figure-8, 5mg/kg/d, M&F10/sex/dose,no more than 1of each sexfrom eachlitter;ACh systeminvolvementYesLOEL=1mg/kg/dPND28-56Lower latencies(faster) in t-maze,early in testing,1&5 mg/kg/d,M&FPND>91 Increased centercrosses inelevated plusmaze (moreactive), 5mg/kg/d, M&FCognitionPND28-56 No effect on tmazespontaneousalternation, M&FPND56-91 Increased radialarm mazeworking &referencememory errors,early in training,5 mg/kg/d, M&FNeuromotorfunctionPND>91 No effect onacoustic startleChlorpyrifos Human Health Risk Assessment D424485Page178of531Study &laboratorySpecies &strainDose, route,vehicleDosing periodAChEinhibitionDomainAge of testingOutcome,effective dose &genderNotesaDoseresponseb?reflex or prepulseinhibition, M&FAnxiety &EmotionPND>91 No effect on timein sides ofelevated plusmaze, M&FBillauerHaimovitch etal., 2009IsraelMouse HS/lbg 1, 3, 5, 10, 20mg/kg/d sc inDMSOGD9-18 Not measuredCognitionPND75 Longer latencies(slower learning)in Morris watermaze, 1&3mg/kg/d, M&F1/sex/litter;N varied,14-34/dose group;All damsdosed at 20mg/kg/d diedbeforedelivery;Nicotine &stem cell astreatmentsNoLOEL=1mg/kg/dMaximal effectat 3 mg/kg/d;Higher doseshad no effectTurgeman etal., 2011ItalyMouse HS/lbg 3 mg/kg/d GD9-18 Not measuredCognitionPND80 Longer latencies(slower learning)in Morris watermaze, M&F1/sex/litter;N=13-25/dosegroup;Stem cells astreatmentsNALaviola et al.,2006ItalyMouse, wildtype onC57/Bl6background;Study includedreeler homoandheterozygousmiceOxon 5mg/kg/d sc inosmostic pumpwith DMSOGD14-16 Not measured Motor activity PND>70 No difference inactivity levelsduringhabituation,before drugchallengesOnly data fromwild typeshown here;Nevermentionsgenders;Oxon notparent;Individual, asphenotyped,used; Total of6litters/treatment;ACh & DAsysteminvolvement;Also someNAChlorpyrifos Human Health Risk Assessment D424485Page179of531Study &laboratorySpecies &strainDose, route,vehicleDosing periodAChEinhibitionDomainAge of testingOutcome,effective dose &genderNotesaDoseresponseb?preweaningtestingVenerosi et al.,2010ItalyMouse CD-1 6 mg/kg/d po inpeanut oilGD14-17 Not measuredAnxiety &emotionPND90 Increased time intunnel betweensides in light-darkbox, F not M;Decreasedlatency to enterdark side(p=0.05)(increasedanxiety), F not M;Litter as unit;N=6-13/dose;5HT systeminvolvementNANo effect onforced swimmingtask, M&FSocialbehavior &maternalinteractionAdult F, aftermating onpostpartumday 8Decreased attackbehavior,increased socialsniffing behaviorto intruder maleHaviland et al.,2010UWMMouse SwissWebster1, 5 mg/kg/d scin DMSOGD17-20 Not measuredCognitionPND60-81 Slower learningof foodrecognition &position, 1&5mg/kg/d, F notM;Faster foodrecognitionlearning, 1 not 5mg/kg/d, M notLitter as unit;N=8/sex/dose;ThyroidinvolvementYes (F only)LOEL=1mg/kg/dIn males, 5mg/kg/d had noeffectChlorpyrifos Human Health Risk Assessment D424485Page180of531Study &laboratorySpecies &strainDose, route,vehicleDosing periodAChEinhibitionDomainAge of testingOutcome,effective dose &genderNotesaDoseresponseb?F;Spurious changesin radial armmaze (says noeffect, only 9trials)Motor activity PND60-81 No effect onforaging rate,M&FLevin et al.,2002DukeRat SD 1, 5 mg/kg/d scin DMSOGD17-20 Not measuredMotor activityPND28-42 Lower latencies(faster) in t-maze,early in testing,1&5 mg/kg/d,M&F1/sex/dosefrom 10 littersYes LOEL=1mg/kg/dSlowerhabituation infigure-8, 1&5mg/kg/d, F onlyPND56-91 Lower latencies(faster) in radialarm maze duringcertain blocksonly, 1&5mg/kg/d, M&FCognitionPND28-42 No effect on tmazespontaneousalternation, M&FPND56-91 Increased radialarm mazeworking &referencememory errors, 1mg/kg/d only, Fnot MNoRadial armmaze effect at 1mg/kg/d onlyChlorpyrifos Human Health Risk Assessment D424485Page181of531Study &laboratorySpecies &strainDose, route,vehicleDosing periodAChEinhibitionDomainAge of testingOutcome,effective dose &genderNotesaDoseresponseb?Ricceri et al.,2006ItalyMouse CD-1 3, 6 mg/kg/d poin peanut oilPlus vehicle onPND11-14GD15-18 Dam on GD19(pilot study):40% brain ChEinhibition at 6mg/kg/d, seruminhibition atboth dosesPup on GD19:no braininhibition,seruminhibition atboth dosesPup on PND15:no brain orseruminhibitionMotor activityPND70 Increasedcrossing in openfield, 6 mg/kg/d,only M testedLitter as unit;10 litters/gestationaldoseYesActivityNOEL= 3mg/kg/dPND120 No effect onelevated plusmaze activity,M&FAnxiety&emotionPND120 Decreased headdips in elevatedplus maze(decreasedanxiety), 3mg/kg/d only, Mnot FNoElevated plusmaze effectonly at 3mg/kg/dSocialbehavior &maternalinteractionPND75-80 Increasedoffensive posturebehaviors, 6mg/kg/d, only Mtested;No effect on nonsocial behaviorsYes AggressionNOEL=3mg/kg/dPND90 No effect oninduced maternalbehaviorsVenerosi et al.,2006ItalyMouse CD-1 3, 6 mg/kg/d scin peanut oilPlus vehicle onPND11-14GD15-18 Cites Ricceri2006 findingsSocialbehavior &maternalinteractionPND120 Increasedultrasonicvocalizations, 6mg/kg/d; only FtestedIncreased socialinvestigation, 6mg/kg/dLitter as unit;10 litters/gestationaldoseYesNOEL=3mg/kg/daSample sizes and analyses refer only to behavioral measuresbGraded response across doses or elseeffect at higher but not lower dose(s)M, F, Male, FemaleChlorpyrifos Human Health Risk Assessment D424485Page182of531Table App 1-13.In vivostudies of chlorpyrifos administered postnatally. Effects described are only those measured afterweaning. Bold indicates functional domain showed effects.Study &laboratorySpecies& strainDose, route,vehicleDosingperiodAChEinhibitionDomainAge oftestingOutcome,effective dose &genderNotesaDoseresponseb?Dam etal., 2000DukeRat SD 1 mg/kg/d scin DMSOPND1-4 ~60% brain(regions)inhibition inM, ~20% in F;more at 2 hrthan 4 hr aftersingle dose onPND1MotoractivityPND21,30Decreased openfield activity &rearing, M not F5-7/sex/dose;Maybe split-litterdosing but notexplicit;Also somepreweaningtestingNALevin etal., 2001DukeRat SD 1 mg/kg/d scin DMSOPND1-4 Not measuredMotoractivityPND28-42Longer latencies(slower) in t-maze,M not F10/sex/dose;Split-litter dosing;No ACh systeminvolvementNANo effect onhabituation infigure-8, M&FCognitionPND28-42No effect on tmaze spontaneousalternation, M&FPND56-91Increased radialarm maze working& reference errors,early in training,M not F;Decreased radialarm maze working& reference errors,F not MAdridgeet al.,2005DukeRat SD 1 mg/kg/d scin DMSOPND1-4 Not measuredCognitionPND64+ Increased radialarm maze working& referencememory errors, Mnot F;Decreasedworking memory9/sex/dose;Probably splitlitter dosing butnot explicitNAChlorpyrifos Human Health Risk Assessment D424485Page183of531errors in radialarm maze, F notMAnxiety &emotionPND52-53Increased openarm time inelevated plusmaze (decreasedanxiety), M not FPND54 Decreasedchocolate milkpreference(anhedonia), M&FMotoractivityPND52-53Increased centercrossings inelevated plusmaze, M not FPND64+ No differences inradial arm mazelatencies, M&FRicceri etal., 2003ItalyMouseCD11, 3 mg/kg/dsc in DMSOPND1-4 20, 23% braininhibition atlow & highdose 1 hr afterdose onPND4; noinhibition at 4,24 hrMotoractivityPND25No effect on openfield activitySplit-litter dosing;n=8-18/sex/dose;says all mice gotall tests but thereare unexplaineddifferences insample sizeacross tests;Bonferronicorrected posthocs followedANOVA maineffects withp>0.05;Also somepreweaningtestingNo, ChE &agonisticbehaviorssimilar atboth doses,LOEL=1mg/kg/dPND35-38Increased activityat door opening in2-chamber box,only 3 mg/kg/dtested, M&FAnxiety &emotionPND38 No effect onnovelty preferencein 2-chamber box,only 3 mg/kg/dtested, M&FSocialbehavior &maternalinteractionPND45 No effectinvestigative,affiliative, orsolicitingbehaviors, M&F;Decreased selfgrooming, 1&3Chlorpyrifos Human Health Risk Assessment D424485Page184of531mg/kg/d, M&FIncreasedfrequency &duration agonisticbehavior sum,differs acrosstime, 1&3mg/kg/d, M not FCognition PND60 No effect onpassive avoidance,only M testedDam etal., 2000DukeRat SD 5 mg/kg/d scin DMSOPND11-14 ~20-30% brain(regions)inhibition,M&F, more at4 hr than 2 hrafter singledose onPND11MotoractivityPND21,30Increased openfield rearing &grooming, M&F5-7/sex/dose;Maybe split-litterdosing but notexplicit;Figure 3 saysmale only butgenderdifferences notsignificantNALevin etal., 2001DukeRat SD 5 mg/kg/d scin DMSOPND11-14 Not measuredMotoractivityPND28-42Longer latencies(slower) in t-maze,M not F10/sex/dose;Split-litter dosing;ACh systeminvolvementNADecreasedhabituation(slower) in figure8, M&FCognition PND28-42No effect on tmaze spontaneousalternation, M&FPND56-91No effect on radialarm maze workingor referencememory, M&FRicceri etal., 2003ItalyMouseCD11, 3 mg/kg/dsc in DMSOPND11-14 No braininhibition at 1,4, 24 hr afterdoseMotoractivityPND25 Increased distancetraveled in openfield, 1&3mg/kg/d, M&FSplit-litter dosing;n=8-18/sex/dose;says all mice gotall tests but thereYes & NoDoseresponse onactivity, 1Chlorpyrifos Human Health Risk Assessment D424485Page185of531PND35-38Increased activityfollowing dooropening in 2-chamber box, only3 mg/kg/d tested,M&Fare unexplaineddifferences insample sizeacross tests;Bonferronicorrected posthocs followedANOVA maineffects withp>0.05has moreeffect onagonisticbehaviorsLOEL=1mg/kg/dAnxiety &emotionPND38 No effect onnovelty preferencein 2-chamber box,only 3 mg/kg/dtested, M&FSocialbehavior &maternalinteractionPND45 No effect oninvestigative oraffiliativebehaviors, M&F;Increasedsolicitingbehavior, 3mg/kg/d, M&FIncreasedfrequency &duration agonisticbehaviorsthroughout, 1&3mg/kg/d, M not FCognition PND60 No effect onpassive avoidance,only M testedRicceri etal., 2006ItalyMouseCD-11, 3 mg/kg/dsc in peanutoilPlus vehicleto damGD15-18PND11-14 24 hr after lastdose (PND15):no braininhibition,seruminhibition bothdosesMotoractivityPND70 Increased crossingin open field, only1st5 min, 3mg/kg/d, only MtestedLitter as unit;10litters/gestationaldose, postnatalsplit-litter dosing;Says anxietyeffect in bothsexes but notsignificant inmalesYesNOEL=1mg/kg/d(mostendpoints)PND120 No effect onelevated plusmaze activity,M&FSocialbehavior &maternalPND75-80Increased attackbehaviors, 3mg/kg/d, only MYesmaternalLOEL=1Chlorpyrifos Human Health Risk Assessment D424485Page186of531interactiontested;No effect on Mnon-socialbehaviorsmg/kg/dPND90 Increased inducedmaternalbehaviors, 1&3mg/kg/dAnxietyPND120 Increased time inopen arms inelevated plusmaze (decreasedanxiety), 3mg/kg/d, F onlyYesNOEL=1mg/kg/dVenerosiet al.,2006ItalyMouseCD-11, 3 mg/kg/dsc in peanutoilPlus vehicleto damGD15-18PND11-14 Not measured,cites Ricceri2006 findingsSocialbehavior &maternalinteractionPND120 No effect onultrasonicvocalizations orsocialinvestigation, onlyF testedLitter as unit10litters/gestationaldose, postnatalsplit-litter dosingYesNOEL=3mg/kg/dVenerosiet al.,2008ItalyMouseCD-13 mg/kg/d scin peanut oilPND11-14 Not measuredSocialbehavior &maternalinteractionPND40-45No effect on socialinteraction withsame-sexconspecific, M&FLitter as unit;Split-litter dosing,2/sex/dose/litter;N=15 litters;N=9-14/dose forsome maternaltests;Female offspringmated on PND60,maternal testsafter giving birthNALD1-7In lactating dams:Longer latency tobuild nest; Shorterlatency to lickpups;Increased timedigging (nonmaternal behavior)LD7 In lactating dams:Decreaseddefensive,increased socialinvestigative, &increased diggingbehaviors whenChlorpyrifos Human Health Risk Assessment D424485Page187of531exposed tointruder maleAnxiety &emotionLD2 In lactating dams:Faster entry &more miceentering light side(decreasedanxiety)MotoractivityPND40-45Decreaseddistance movedduring acclimationbefore social test,1sttime block,M&FJohnsonet al.,2009MSURat SD Incrementingdoses: low, 1mg/kg/d;mid, 1 up to4 mg/kg/d;hi, 1.5 up to6 mg/kg/dPo in corn oilPND1-21 PND20 (timeafter dose notgiven) – 14-53% brain(hippocampal)inhibition, alldosessignificant;PND30-40,mid & highdoses stillshowinhibition (17-23%);No inhibitionPND50;M&F sameCognitionPND36-60Increased radialarm maze workingmemory errors, hidose throughouttraining, all dosesduring last week,M only;Decreased radialarm mazereference memoryerrors, middle toend of training,mid & hi doses, Fonly;Increased radialarm mazereference errors,middle week ontraining, mid & hidoses, M onlySplit-litter dosing;n=10-14/sex/dose;Table 1 saysPND20, not 21YesLOEL=lowdose (1mg/kg/dPND1-21)MotoractivityPND36-60No effect onresponse time inradial arm mazeCarr etal., 2001MSURat SD Incrementingdoses: low, 3mg/kg/d;PND1-21,every otherdayPND25, 30 –14-26% brain(regions)MotoractivityPND25,30Decreased openfield activity, mid& hi, M&FBetween-litterdosing;N=5 litters/dose,YesActivityNOEL=lowChlorpyrifos Human Health Risk Assessment D424485Page188of531mid, 3 up to6 mg/kg/d;hi, 3 up to 12mg/kg/dPo in corn oilinhibition atlow dose; alldosessignificant,M&F same2/sex from eachlitter;Used within-litterfactor in analysis;Also somepreweaningtestingdose (3mg/kg/dPND1-21)Jett et al.,2001JHURat LE 0.3, 7mg/kg/d scin peanut oilPND7, 11,15“preweaningstudy”No braininhibition onPND7 (3 hrafter dose), 8,16, or 28CognitionPND24-28Longer latenciesto find platform inMorris watermaze, 7 mg/kg/dthroughout, 0.3mg/kg/d on day 1(p=0.05), M&F;Less time spent intraining quadrant,7 mg/kg/d, M&FBetween-litterdosing afterrandom culling;N=8-10/sex/dose,“2 or more littersused”;“Postweaning”study notpresentedYesNOEL orLOEL=0.3mg/kg/d(p=0.05)Authors sayboth waysaSample sizes and analyses refer only to behavioral measuresbGraded response across doses or elseeffect at higher but not lower dose(s)M, F, Male, FemaleChlorpyrifos Human Health Risk Assessment D424485Page189of531Table App 1-14.In vivostudies of chlorpyrifos administered during gestation and postnatally, continuous or discontinuousexposures. Effects described are only those measured after weaning. Bold indicates functional domain showed effects.Study &laboratorySpecies &strainDose,route,vehicleDosingperiodAChEinhibitionDomainAge oftestingOutcome, effectivedose & genderNotesaDoseresponseb?Maurissenet al., 2000DowRat SD 0.3, 1, 5mg/kg/dpo in cornoilGD6-LD10Dam onGD20, 4-5 hrafter dose,45% bloodbut no braininhibition at0.3; greaterblood andbraininhibition athigher dosesNeuromotorfunctionPND22 Increased auditorystartle latency did notmeet lower p-value, 5mg/kg/d, M&F;“effects may havebeen real” – p-value=0.03Litter as unit;1/sex/litter;N=8/sex/dose fordelayedalternation (samerats at both ages);N=20/sex/dosefor startle andmotor activity(same rats at bothages);alpha set at 0.02;Also somepreweaning tests;Maternal toxicityat 5 mg/kg/dNo dosesshowedeffectsusinghigherstatisticalthresholdPND61 No effect on auditorystartle measures,M&FMotoractivityPND21andPND60No effect on totalcounts or habituationat either age, M&F;High dose “may havehad ..slight increase inactivity on PNDs 17,21, and 60”Cognition PND22-24 andPND61-91No effect on spatialdelayed alternationlearning at either age,M&FBraquenieret al., 2010BelgiumMouseCD-10.2, 1, 5mg/kg/dpo in cornoilGD15-LD14Pup onPND1, 3 hrafter dose todam, 14%braininhibition,only 5mg/kg/dgroup testedMotoractivity“Adult” No effect onlocomotor activity1/litter for eachtest; only Ftested;N=8-10/dose;Dunnetts posthoc after maineffect p<0.10NoEffect onlyat 1mg/kg/dNOEL=0.2mg/kg/dPND80 No effect on elevatedplus maze total armentriesAnxiety &emotion“Adult”Decreased time inchamber center inlight-dark box anddecreased boxswitches (increasedanxiety), 1mg/kg/donly;Chlorpyrifos Human Health Risk Assessment D424485Page190of531Study &laboratorySpecies &strainDose,route,vehicleDosingperiodAChEinhibitionDomainAge oftestingOutcome, effectivedose & genderNotesaDoseresponseb?PND80 Decreased time &entries in elevatedplus maze open arm(increased anxiety), 1mg/kg/d onlyRicceri etal., 2006ItalyMouseCD-13, 6mg/kg/dpo inpeanut oilPlus 1, 3mg/kg/d scin peanutoil onPND11-14GD15-18“pre”plusPND11-14 “post”Dam onGD19 (pilotstudy): 40%brain ChEinhibition at6 mg/kg/d,seruminhibition atboth dosesPup onGD19: nobraininhibition,seruminhibition atboth dosesPup onPND15: nobrain orseruminhibitionMotoractivityPND70 Increased crossing inopen field, 6 mg/kg/dprenatal plus 1, not 3mg/kg/d, postnatalNAPND120 No pre-postinteraction onelevated plus mazeactivity, M&FAnxiety &emotionPND120 No pre-postinteraction onelevated plus maze,M&FSocialbehavior &maternalinteractionPND75-80No pre-postinteraction on socialbehaviors, M&FPND90 No pre-postinteraction on inducedmaternal behaviorsVenerosi etal., 2006ItalyMouseCD-13, 6mg/kg/d scin peanutoilPlus 1, 3mg/kg/d scin peanutoil onPND11-14GD15-18“pre”plusPND11-14 “post”Cites Ricceri2006findingsSocialbehavior &maternalinteractionPND120 Increased ultrasonicvocalizations & socialinvestigation at 6mg/kg/d prenatallyreversed by 1&3mg/kg/d postnatallyNAChlorpyrifos Human Health Risk Assessment D424485Page191of531aSample sizes and analyses refer only to behavioral measuresbGraded response across doses or elseeffect at higher but not lower dose(s)M, F, Male, Female

# Chlorpyrifos Human Health Risk Assessment D424485

Page 192 of 531 3.2.2.2. Update on Developmental Neurotoxicity St udies of Chlorpyrifos as of August 2014

There are six new studies since early 2012, and five of these are from labor atories that have not

published behavioral studies of this type before. Note, however, th at most of these laboratories

have published neurochemical, molecular, or in vitro studies with chlorpyrifos or other OPs.

Most of these studies use the same dosing paradigms as earlier studies.

Of particular interest is a recent publication from the Duke laboratory (Slotkin et al., Neurotox.

Teratol. 37:1-12, 2013) that reported 5-10% inhibition of fetal brain ChE activity at 2 hr after the

last dose of 1 mg/kg/d sc in DMSO administered PND1-4. This dose is widely used in many of

these studies, and this information clearly show s that while this dose does not induce toxic or

developmental effects, it does have a sma ll but significant effect on fetal brain ChE.

A critical review of the six papers follow:

1) Chen X-P, Chen W-Z, Wang F-S, Liu J-X. ( 2012) Selective cognitive im pairments are related

to selective hippocampus and pref rontal cortex deficits after prenatal ch lorpyrifos exposure.

Brain Res. 1474:19-28.

This study represents a new laboratory to the fi eld. ICR mice were used, no information on how

many pregnant females they started with. Chlor pyrifos (1, 5 mg/kg/d) was given GD13-17 sc in

DMSO. The paper does not specify if litters were culled or sex distribution within litters.

Littermates were randomly re-distributed across litters every 7 days, there is no information

given as to whether littermates were allocated to di fferent dams or if the litter stayed as a unit.

On PND20, 8 litters were chosen for testing. It says that littermates were divided into groups,

but it is not clear if a ll pups in a litter tested together – figure legend does say n=8 mice per

group. It is also not clear how pups were sampled, whether there were equal numbers of males

and females, or whether the sta tistics accounted for littermates.

Behavioral testing was conducted in a T-maze using an alternating pr ocedure, i.e., for each trial

the baited arm alternated. They conducted 10 trials/session, 2 sessions/ day (does not say how

far apart), for 3 days for each inter-trial interval (ITI). Training used the shortest ITI (no time

between trials), then used 10, 20, 40, and 60 sec ITIs . It does not give th e order of presentation

of the different ITIs. Win-shift failure, or erro rs, are described as going to the wrong arm, which

triggered training that is not cl early described. Lose-shift failu re, or errors, was defined as

continuing to go to the wrong arm. Training bega n on PND45 (after 4-5 days acclimation to the

apparatus) and continued 15 days.

Adult offspring showed no difference in percent co rrect on first choice. It reports a sex effect

and then analyzes them separately, but not clear if there was a sex*trial interaction. Lose-shift

errors were significant in males, showed a trend in females – they reported male effects at longer

delays (40, 60 sec) and effect in females only at 10 sec. Only the high dose was significant.

Procedural details are not well-ex plained, especially the training that was initiated after each

choice error. Figure legend states 8 mice pe r group, which could imply one mouse from each

litter, but unclear if they were st ill littermates. Additional uncerta inties in the statistics include

how littermates and sex were handled and whether there were significant dose*trial, sex*dose, or

dose*sex*trial interact ions. Even in controls, there is not an evident delay-rete ntion gradient, in

that 60 sec performance looks similar to 0 sec, suggesting that even 60 sec may have been too

# Chlorpyrifos Human Health Risk Assessment D424485

Page 193 of 531 short a delay. T-maze data are usually somewhat variable, yet the data show remarkable, and too

much, consistency. This raises questions as to the actual procedure used, and whether the

apparent treatment effects are real.

2) Cole TB, Fisher JC, Burbacher TM, Costa LG, Furlong CE. (2012) Neurobehavioral

assessment of mice following repe ated postnatal exposure to ch lorpyrifos-oxon. Neurotoxicol.

Teratol. 34:311-322.

This study represents another new laboratory to conduct DNT studies, although they are an

experienced laboratory for studies of PON1 a nd OPs. This study used only the oxon in PON1-

knockout mice, thus it is not exactly comparable to other chlorpyrifos studies. However, given

that the knockout mice were assumed to be more sensitive, and the curr ent interest in the oxon,

the study is relevant.

This was part of a larger study with PON -/-



# , +/+ 

# , and humanized PONs, but only the PON -/- 

# mice

were tested behaviorally. The study was done in two cohorts and onl y findings that were seen in

both were reported. On PND4 mice were culled to three males and three females. Pups were

directly dosed (sc in DMSO; 0.15, 0.18, 0.25 mg/kg/d) on PND4-21: all mice in a litter received

the same dose. One male and one female per litter were allocated to a set of tests, another male

and female per litter allocated to another set of tests, designa ted sets “A” and “B”. Before

weaning, all mice were tested for ontogeny of ri ghting reflex, cliff avoidance, and negative

geotaxis. Tests for set “A” were: rotarod PND 23, pre-pulse acoustic startle reflex PND25 and

50, fear conditioning PND62-64, and water radial arm maze PND77-116. Set “B” tests were:

open field PND25, Morris water maze PND70-98, a nd served as no-shock controls in fear

conditioning. All procedures were standard and appropriate for these tests. It is not clear

whether sex was nested or analy zed as a within-litter variable.

Littermates of these mice were used to measure brain AChE activity on PND22 (day after last

dose), and showed 10%, 16%, and 27% inhibition at the three increasing doses. Body weight

was decreased at 0.25 mg/kg/d, which appeared during dosing and persisted through adulthood.

Hyperkinetic responses (jumping, darting) were noted in all dos e groups within 5 min of dosing

beginning PND15. Of all the neur obehavioral and cognitive tests, only decreased startle latency

was detected in the 0.18 and 0.25 groups on PND50. Also on that day only the 0.18 group

showed an effect on pre-pulse amplitude, but no e ffect of the pre-pulse inhibition. The high dose

of 0.25 mg/kg/d showed no behavioral effects.

The study was conducted appropriate ly, and generally analyzed correctly except that it appears

that sex was not nested. The gr aphs do not show males and female s separately (and it is unclear

which it is, or if it’s combined ) but supplemental data show sex -related differences in some of

the tests where it’s expected (e.g., fear conditioning). The aut hors discuss the shorter startle

latency as representing stronger synaptic connecti ons, but do not adequately address the lack of

dose response. Generally the lack of oxon effects stands in contra st to many of the studies that

report effects of chlorpyrifos us ing the same behavioral tests.

3) Vatanparast J, Naseh M, Baniasadi M, Haghdoost-Yazdi H. (2013) Developmental exposure

to chlorpyrifos and diazinon differentially affect passive avoidance performance and nitric oxide

synthase-containing neurons in the basolateral complex of the amygdala. Brain Res. 1494:17-27

# Chlorpyrifos Human Health Risk Assessment D424485

Page 194 of 531 This paper is from another laborato ry that is new to this field. Wistar rats received 1 mg/kg/d sc

in DMSO either on GD15-18 or directly to pups on PND1-4. Adult offspring (PND60) were

tested in an open field followed by passive avoidance. After two habituation trials, rats were

subjected to shock when they crossed over. Th is was repeated every 2 min until they did not

cross in 120 sec. At 24 hr they were retested. No te that rats that did no t go into the dark side

during the habituation trials were eliminated, this results in a selection bias that could have been

related to dose but no information is given. Depe ndent variables were tria ls to criterion on the

day of training, and step-through late ncy and time in dark side (dir ectly correlated with latency)

at 24 hr. Litters were culled and 1 male and 1 female per litter were tested with at least 12

litters/treatment. Analysis uses sex as a factor but not nest ed within litter.

There were no changes in activity, nor were there ch anges in trials to crit erion. In the retention

test, prenatally dosed rats showed decreased late ncy, similarly in males and females. There were

no effects in postnatally treated rats.

Group means for trials to criterion are very close to 1, meaning that most rats met it in 1-2 trials;

therefore, there was not much dynamic range with which to detect effects. However, the 24-hr

retention data show an obvious treatment eff ect. Passive avoidance has not been tested

previously in rats, but there were no effects in mice treated postnata lly (Ricceri et al., 2013).

4) Mullen BR, Khialeeva E, Hoffman DB, Ghiani CA, Carpenter EM. (2013) Decreased reelin

expression and organophosphate pesticide expos ure alters mouse behaviour and brain

morphology. ASN NEURO 5:27-42.

This study is from a new laboratory, and focuse s on chlorpyrifos intera ctions in mice having

decreased or normal expression of the reelin ge ne which has been associated with autism.

Results of chlorpyrifos treatment in the reelin +/+ 

# mice (normal expression ) are evaluated here.

Osmotic minipumps were implanted on GD13.5, releasing 6 mg/ml oxon in PBS, apparently

over 3 days. There was no extrapola tion to administered dose. Reelin +/+ 

# and +/- 

# were bred,

resulting in litters with mixed genotypes. Th ere were 9 oxon and 13 vehi cle litters, and there

were at least 6 mice per treatment/genotype grou p. It is unclear how many males and females

tested came from the same litter. AChE was measured in fetal heads on GD16.5. On PND7,

litters were separated from the dam and USVs were measured. On PND30 there was a 30-min

open field session, also a social interaction test. Marble bu rying was measured on PND60.

Measured were collected using an overhead vide o system. Outliers were rejected (no criterion

given) and data were analyzed with t-te sts and “aggregate” ANOVAs (no information where

each approach was used).

AChE activity was decreased by about 15% (not stat ed whether this was significant). There was

a decrease in the number and duration of USVs, similarly in males and females. Males only

showed decreased sniffing duration in the open field. Females only showed an increased in

sniffing at stranger 1 in the social interac tion test, but no differen ce when stranger 2 was

introduced. There was no change in marble burying. There were seve ral other non-significant

differences which they inte rpret as being effects.

The only statistically significant changes were seen with sniffing, and this was not consistent in

# Chlorpyrifos Human Health Risk Assessment D424485

Page 195 of 531 males vs females. It is not described what algor ithm was used to define sniffing in the software

using an overhead camera, and this information is not available at the ma nufacturer’s website.

Sniffing is a questionable measure of activity, and in the social interaction test, a better measure

is time spent with the other mice, yet this wa s not altered. The data are not particularly

convincing.

5) Ohishi T, Wang L, Akane H, Itahashi M, Naka mura D, Yafune A, Mitsumori K, Shibutani M.

(2013). Reversible effect of mate rnal exposure to chlorpyrifos on the intermediate granul cell

progenitors in the hippocampal dentate gyrus of rat offspring. Reprod. Toxicol. 35:125-136.

This is a new laboratory to do these studies. SD rats were given chlorpyrifos in the diet from

GD10-PND21, at 2.8, 14, 70 ppm. AChE was meas ured in dams at weaning and in male

offspring on PND21 and PND77. Preweaning phys ical and righting reflex milestones were

measured. Detailed clinical observations we re noted at PND29, 48, 71. Also on PND71, rats

were subject to FOB-type measur es: sensory responses (click, visu al approach, touch, tail pinch,

pupil), righting reflex, landing f oot splay, and forelimb and hindlimb grip strength. Motor

activity was also measured that day in 1-hr sess ion but it does not specif y type of chamber or

cage. Biel maze testing on PND45-58 looked at la tencies and choice errors. Behavioral tests

used 1 male and 1 female per litter, and litter was used as the unit of measure for stats. However,

it does not appear that sex was c onsidered as a factor in the analyses, and repeated measures

were not used where it should have been. Th is study design is generally consistent with

developmental neurotoxicity test guidelines, and is comparable to the Maurissen et al. (2000)

paper.

Maternal intakes over exposur e were calculated to be 0.36, 1.86, and 9.18 mg/kg/d. In dams

(PND21), AChE was decreased in RBC (all doses, up to ~80% inhibition), plasma (14 and 70

ppm, similar magnitude), and br ain (70 ppm only, similar magnitude ). On PND21 in offspring,

AChE was inhibited in RBC and plasma (14 and 70 ppm, ~70% inhibition) and brain (70 ppm

only, ~50%). There were no treatment effects on milestones or FOB-like measures. There was

increased body weight in both sexes during pos tnatal period but only in 14 ppm group, along

with faster eye opening (2.8 and 14 ppm) and fast er incisor eruption (14 ppm). Motor activity

was different in 2 intervals in 14 ppm males, otherwise there were no effects. There were

decreased errors in one trial in 2.8 ppm males in the Biel maze, and increased latency in the

straight channel on the first tr ial only in the 2.8 and 14 ppm groups. The high dose of 70 ppm

had no effects.

This study had no doses where AChE was not at a ll inhibited, but brain ChE was inhibited only

at the high dose in both dams and pups. Magn itude of inhibition was quite high, but there was

no mention of toxic signs, and dams gained we ight normally (Note: this finding is somewhat

unexpected). The pre-weaning weight and deve lopment differences at 14 ppm are in the

opposite of the expected direction, and suggest differences in actu al gestational age. The few

significant intervals and trials we re not dose-related and most lik ely were spurious due to the

multiple tests run, instead of doing a repeated-m easures ANOVA. If the appropriate statistics

were used, it is doubtful there woul d be any significant differences.

6) Levin ED, Cauley M, Johnson JE, Cooper EM, Stapleton HM, Ferguson PL, Seidler FJ,

Slotkin TA. (2014). Prenatal dexamethasone augments the neurobehavioral teratology of

# Chlorpyrifos Human Health Risk Assessment D424485

Page 196 of 531 chlorpyrifos: Significance for ma ternal stress and preterm labor. Neurotoxicol. Teratol. 41:35-

42.

This laboratory published extensively in this area in the early 2000s. The focus of this study is

interactive effects of prenatal dexamethasone and postnatal chlorpyrif os; however, for this

review only the effects in the ch lorpyrifos alone group compare to control are considered. Dams

received saline sc GD17-19, then chlorpyrifos 1 mg/kg/d sc was give n to pups on PND1-4.

Litters were culled after birth and redistributed within treatment group every few days. Figure

legend (only) states 11-12 per treatment, but ne ver states that one male and one female came

from each litter; however, that is the usual procedure used in this laboratory. The same offspring

went through all behavioral tests: T-maze e xploration in week 4, motor activity (figure 8

chamber) week 5, novel environment feeding in week 7, novel object recognition in week 9.

There were no effects of chlorpyrifos on T-maze or the novel environment feeding, but note that

while there were no significant differences, the abstract and discussion say that there were

effects. Chlorpyrifos increased motor activity in males only, but the chan ge in habituation did

not reach significance (although the authors clai m there is an effect). On novel object

recognition, there was an increased overall exploration in males only, but no effect on

differential time between novel and familiar objects.

The study was generally conducted properly, usin g many of the same methods from earlier

publications. Effects are somewhat exaggerated in that non-significant di fferences are presented

as effects. The increased moto r activity likely accounts for increas ed activity in the novel object

test, and there is no evidence of a cognitive effect.

The effects described in this study disagree with more than half of the findings from earlier

studies in this laboratory usi ng the same dosing paradigm. Sp ecifically, the effects and the

associated functional domains here are: 1) Ac tivity – Increased figure-8 activity and increased

exploration in novel object test, males only, and no effect on T-maze exploration, which agree

with increased elevated plus maze activity, ma les only (Aldridge et al., 2005) but not with

decreased T-maze activity in males (Levin et al., 2001) or decreased open field activity in males

(Dam et al., 2000); 2) Cognition – No effect on T-maze alternation or novel object recognition,

which agree with no effect on T-maze alternation (Lev in et al., 2001) but disagree with deficit in

radial arm maze performance in males, and impr oved function in females; 3) Anxiety/Emotion –

No effect on novel environment feeding which disa grees with evidence of decreased anxiety in

elevated plus maze and anhedonia suggested by d ecreased chocolate milk preference (Aldridge

et al., 2005). The authors do not discuss these discrepancies.

Overall summary:

For half of the studies, the lowest dose was 1 mg/kg/d, and two studies used the oxon,

making it difficult to compare dose levels. Only one study used a lower dose, 0.36 mg/kg/d, in

feed, and even this level was sufficient to pr oduce a great degree of RBC ChE inhibition. The

functional effects are summarized below (See Table App 1-15).

1) Cognition – Of these studies, cognition was ge nerally unaffected except for an effect on

passive avoidance; interestingly, previous studies reported no passi ve avoidance effects. There

were no effects on other cognitive tests that did show changes in earlier studies, as well as no

effects in new tests being used for the first time. Thus, what appeared to be fairly consistent

reports of cognitive changes from earlier studies is not so conclusive.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 197 of 531 2) Anxiety & emotion – The only study which eval uated this domain reported no effect, in

contrast to previous reports, incl uding one from the same laboratory.

3) Social & interactive behavior – Only one study measured this and reported increased

interaction with a stranger mous e (albeit with a questionable measure), whereas earlier studies

showed mixed effects on other te sts of social investigation.

4) Activity – Most of these studies report no chan ge in activity, with only one report of increased

activity and another of d ecreased sniffing (a questionable measure of activity). Combined with

the previous literature, this cont inues to show inconsistent cha nges in motor activity levels and

habituation.

5) Neuromotor development and function were generally unaltere d except for one measure that

did not show a dose-response. Few other studies have addressed this.

Conclusions: There continue to be inconsistencie s in effects in relation to functional domains,

dosing paradigms, and gender-specificity. The only studies reporting eff ects used doses that

inhibited fetal/pup brain ChE ac tivity to some degree, even though there were many negative

effects at these same doses.Chlorpyrifos Human Health Risk Assessment D424485Page198of531Table App 1-15.Summary of newest studies. Bolded domains indicatesignificance in some measure within that domain.Study &laboratorySpecies &strainDose,route,vehicleDosingperiodAChEinhibitionDomainAge of testingOutcome, effective dose,genderNotesDose-response?Chen et al.,2012HangzhouChinaMouseICR1, 5mg/kg/dsc inDMSOGD13-17 NotmeasuredCognition PND45-60 Increasedlose-shift errors inT-maze delayed alternation at5 mg/kg/d M, trend FLitter allocationand statisticaltreatment not clear.Control data don’tshow expectedretention decay atlonger ITIsYesNOEL=1mg/kg/dLevin etal., 2014Rat SD 1 mg/kg/dsc inDMSOPND1-4(salinevehicleGD17-19)NotmeasuredCognition PND28;PND63No effect on T-mazealternation;No effect on differentialexploration in novel objectrecognition12-14/dose, nomore than 1 ofeach sex from eachlitter; littersredistributed everyfew daysDid not next sex inlitterStudy addressesCPF anddexamethasoneinteractions;Presents nonsignificant data aseffectsNAActivityPND28;PND35;PND63No effect on T-mazealternation;Increased total activity infigure-8, M only;Increased overall activity innovel object recognition, MonlyAnxiety &emotionPND49 No effect on novelenvironment feedingVatanparast et al.,2013Rat Wistar 1 mg/kg/dsc inDMSOGD15-18 NotmeasuredActivity PND60 No effect in open field, M&F 1 male and 1female each litter,but sex not nestedin litter foranalysesNACognitionPND60-63 Decreased step-throughlatency at 24 hr in passiveavoidance, M&FPND1-4 Activity PND60 No effect in open field, M&FCognition PND60-63 No effect in passiveavoidance, M&FMullen etal., 2013Reelin+/+mice6 mg/mloxon inPBS, withosmoticminipmp~GD13.5-16.5~15%inhibitionin fetalheadsGD16.5ActivityPND30 Decreased sniffing in openfield, F not MSampling of M andF from litters notclear, sex notnested in statsNASocialPND30 Increased sniffing of firststranger in social interactiontest, F not MAnxiety &emotionPND60 No change in marble burying,M&FChlorpyrifos Human Health Risk Assessment D424485Page199of531Ohishi etal., 2013Rat SD 2.8, 14, 70ppm indiet,giving0.36, 1.86,1.98mg/kg/dGD10-PND21Dams onPND21:inhibition(max~80%) inRBC (alldoses),plasma(14 and70), brain(70)Pups onPND21:inhibition(max~70%) inRBC andplasma(14 and70), brain(70)Noinhibitionon PND77Activity PND71 Changes in activity in twointervals only, 14 ppm, M notF, likely spuriousDid not nest sex inlitter; Appears thatsex was not factorin analysis andrepeated measureswas not usedLOEL=2.8 ppm(0.36 mg/kg/d)(RBC ChE)Cognition PND56-58 Decreased errors in one trialonly in Biel maze, 2.8 ppm,M not F, likely spuriousNeuromotorPND56-58Preweaning& PND29,48, 71Increased latency in Bielmaze in straight channel, 2.8and 14 only, M not F, likelyspuriousNo effect on many measuresof development,sensorimotor, neuromuscularfunctionCole et al.,2012PON-/-mice0.15, 0.18,0.25mg/kg/doxon sc inDMSOPND4-21 10%,16%, 27%inhibitioninlittermateson PND22Cognition PND62-64;PND77-116;PND70-98No effect on fearconditioning, M&F;No effect on water radial armmaze, M&F;No effect on Morris watermaze, M&FNoSimilar effects at0.18, 0.25mg/kg/dNOEL=0.15 mg/kg/dMotoractivityPND25 No effect in open fieldNeuromotor functionPreweaning;PND23;PND25, 50;No effect on reflex ontogeny,M&F;No effect on rotarod, M&F;Decreased acoustic startlelatency on PND50, 0.18 &0.25 mg/kg/d, M&F; Alteredpre-pulse startle, 0.18mg/kg/d only, M&F

# Chlorpyrifos Human Health Risk Assessment D424485

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# Appendix 2. Detailed Review and Synthesis of Three Children’s

Environmental Health Cohort Studies

NOTE: Appendix 2 contains the Detailed Revi ew and Synthesis of the Three Children’s

Environmental Health Cohort Studies. This information was presented to the FIFRA

Scientific Advisory Panel (SAP) in April of 2012; however, it is supplemented by both an

external Federal panel review performed June –September 2012, and also the results of a

on-site meeting with staff of the Columbia Children’s Center for Environmental Health

(CCCEH) in April 2013. The CCCEH is one of the three children’s environmental health

cohorts included in the EPA review. EPA has considered the FIFRA SAP input regarding

the analysis and interpretation of these epidemiologic studies in the completion of the

revised chlorpyrifos HHRA (April 2012 FIFRA SA P review of this information can be

found at: http://www.epa.gov/scipoly/sap/m eetings/2012/april/041012minutes.pdf .)

Information concerning the Federal letter re view of a subsequent study published by

CCCEH researchers in addition to other questio ns can be found in the Federal docket at:

http://www.regulations.gov/#!docum entDetail;D=EPA-HQ-OPP-2008-0850-0170 . The

Federal panel review considered the resu lts of a Magnetic Resonance Imaging (MRI)

study which detailed alterations in brain morphology in children by in utero chlorpyrifos

exposure level. Within the Federal letter review, EPA also specifically requested

feedback upon areas of child neurodevelopment testing for which the April 2012 FIFRA

SAP expressed a lack of expertise to fully consider. Lastly, in April 2013, EPA scientists

met with CCCEH researchers to address a seri es of questions genera ted by the April 2012

FIFRA SAP, the June 2013 Federal letter re view, and EPA deliber ations including the

need for obtaining certain “raw data” or origin al analytic data files to address important

uncertainties in the use of these data in the HHRA. Specifically, questions regarding

whether additional information may be av ailable through the CCCEH data collection

efforts to further characterize chlorpyrifos exposure of CCC EH study participants and to

better characterize other environmental exposures within the study population. The

results of this meeting are encapsulated in the EPA Memorandum “Columbia Center for

Children’s Environmental Health (CCCEH) Epidemiology Data Acquisition (“Raw Data”

Request) and is included as Appendix 6 to the revised chlorpyrif os HHRA. The input

provided from the two reviews and the re sults of further inquiry of the CCCEH

researchers by EPA is encapsulated herein. The combined force of this material informed

EPA’s deliberations concerni ng the content of the revi sed chlorpyrifos HHRA.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 222 of 531 1.0 Scope and Purpose

In September 2008, EPA presented to the FIFRA SAP its preliminary review of available

epidemiologic investigations of pr enatal exposure to chlo rpyrifos in association with measures of

fetal growth and adverse neurodeve lopmental effects in three major prospective children’s health

cohorts in the U.S. 67 

# These are: 1) The Mother’s and Newborn Study of North Manhattan and

South Bronx performed by Columbia University researchers referred in this document as

“Columbia Mother’s and Newborn Study;” 2) the Mt. Sinai Inner-City Toxicants, Child Growth

and Development Study or the “Mt. Sinai child growth and development study;” and, 3) Center

for Health Assessment of Mother’s and Child ren of Salinas Valley (CHAMACOS) conducted by

researchers at University of California Berkeley, the “CHAMACOS study.” In this document,

EPA updates and expands its targeted evaluation of this important line of evidence regarding

chlorpyrifos developmental neurotoxicity.

At the previous meeting, the Panel agreed with EPA’s conclusions that “chlorpyrifos likely

played a role in the birth and developmental out comes noted in the thr ee cohort studies” (pp. 37

Meeting Minutes). In support of th is statement, the Panel offered that investigations performed

within these three epidemiologi cal cohorts utilized a similarl y strong study design (prospective

cohort); measured exposure using several differe nt methods including specific and non-specific

biomarkers of chlorpyrifos; ascertained deve lopmental outcomes using validated assessment

tools common to both clinical and research settin gs; and, analyzed, selected and statistically

adjusted for potentially confounding variable s using reasonable and appropriate methods.

Overall, the Panel noted that the epidemiological database at that time presented an informative

body of evidence with some notable consistencie s across studies. Areas of inconsistency were

also observed, and judged, in part, to be due to differing methods of measurement and

evaluation, as well as dissim ilar exposure profiles ( i.e. , residential versus direct and indirect

occupational (farm laborer) exposure). Important ly, the Panel at the 2008 FIFRA SAP meeting

also stated that it could not conc lude that chlorpyrifos was the so le contributor to these outcomes,

as co-exposure to other organophos phate pesticides and mixtures of environmental exposures

may also have played a part in these outcomes.

For the purpose of informing the chlorpyrifos risk assessment, the Panel concurred with the

Agency with respect to the primacy of the Columbia Moth er’s and Newborn Study among the

three birth cohort studies, alt hough the Panel also encouraged the Agency to comprehensively

consider the results of the thre e children’s health cohorts. Wh ile the Columbia Mother’s and

Newborn Study researchers measured the pare nt compound chlorpyrifos, as opposed to nonspecific organophosphate metabolites, the other cohorts examined health outcomes (the

Brazelton index of neonatal development) and performed supplemental analyses ( e.g., effect

modification by PON1 status) not reflected in the Columbia Mother’s and Newborn Study

database. Therefore, the Panel expected EPA c ould strengthen its unders tanding of the potential

developmental neurotoxicity of chlorpyrifos by considering the three children’s health cohort

studies together. Additionally, the Panel in 2008 suggested supplem ental statistical analyses to

enhance understanding of epidemiological study results in the risk assessment context (See

Attachment 1). The Panel also generally noted both strengths and limitatio ns of these studies,

and offered that random or systematic errors in the design, conduct or anal ysis of these studies

67 

# See Meeting Minutes at: http://www.epa.gov/sci poly/sap/meetings/2008/september/sap0908report.pdf.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 223 of 531 were unlikely to fully explain observed associati ons. However, the Panel also noted that absent

the available toxicological and epidemiologi cal databases was a well understood and defined

mode of action as to the role of chlorpyrifos exposure in the e tiology of adverse infant and child

neurodevelopmental outcomes.

In the revised chlorpyrifos HHRA, EPA expa nds and updates its review of the available

epidemiologic data concerning the effect of ch lorpyrifos exposure on children’s environmental

health in conjunction with a review of recent experimental studies and hypothesized adverse

outcome pathways (AOP). Observational studi es published subsequent to the September 2008

FIFRA SAP evaluation extend the knowledge base of potential long-term sequalae of prenatal

chlorpyrifos exposure. Specifically, in April 2011, researchers with each of the three prospective

children’s health cohort studies c oncurrently published re sults of their respec tive investigations

of prenatal chlorpyrifos exposure and measur es of intelligence am ong school aged children

approximately 7-years (Bouchard et al., 2011; Engel et al., 2011; V. Rauh et al., 2011).

Additionally, researchers with the Mt. Sinai Ch ild Growth and Development study contributed

their evaluation of organophosphate exposure and mental and psychomotor development, and

authors with the CHAMACOS study published an evaluation of chlorpyrifos exposure and both

fetal growth and neurodevelopment measures in young children as modified by paraoxonase-1

( PON1 ) genotype and phenotype (Eskenazi et al., 2010; Harley et al., 2011).

Researchers have also performed epidemiologic methods research which in many ways reduces

uncertainties related to key measures within these studies. Within the Columbia Mothers and

Newborn study, investigators publish ed results of analyses evalua ting the validity of prenatal

chlorpyrifos exposure measures in the time periods immediately after the voluntary cancellation

of chlorpyrifos for residential use (R. M. Whyatt et al., 2009; R. M. Whyatt et al., 2007), as well

as employed innovative statistical techniques to further assess th e potential confounding bias of

socio-economic status (SES) in the relation between prenatal ch lorpyrifos exposure and adverse

neurodevelopmental health outcomes (Lovasi et al., 2011). Overall, these additions to the

epidemiologic database concerning children’s health effects in combinati on with other lines of

evidence, add to the body of know ledge available to inform the ways in which chlorpyrifos

exposure may be related to adverse ne urodevelopment outcomes in children.

2.0 Summary of Epidemiology Findings

In this section, EPA summarizes and critically reviews epidemiologic studies of prenatal

chlorpyrifos exposure and subsequent fetal grow th and child development evaluated within the

three prospective children’s heal th cohorts described above. Speci fically, in this section the

design, conduct and methods of analysis of each cohort study are briefly presented; individual

study results are summarized by type of hea lth outcome investigated; and strengths and

limitations of these investigations are discusse d. Appendix 3 of the chlo rpyrifos human health

risk assessment (HHRA) includes detailed st udy reviews and critical analysis of each

investigation and Appendix 4 includes extensiv e evidence tables summarizing details of each

investigation. The following section 3.0 reflects EP A’s synthesis and evaluation of the current

chlorpyrifos epidemiology database from the three children’s health cohorts. The results of the

April 2012 FIFRA SAP review, the June 2012 Federal letter review , and the April 2013 on-site

meeting with CCCEH study staff are integrated throughout the document where appropriate. In

# Chlorpyrifos Human Health Risk Assessment D424485

Page 224 of 531 accordance with the draft “Framework for Inco rporating Epidemiology in Risk Assessment,” 68



# this analysis considers the stre ngths and limitations reflected in each cohort and research study as

well as modified Bradford Hill considerations for the synthesis of these data.

2.1 Overview of Design and Methods of Children’s Health Studies

These cohorts were recruited for the purpose of studying the potential health effects of

environmental exposures during pregnancy on su bsequent child development: The Columbia

University’s Mother’s and New born Cohort (Lovasi et al., 2011; V. Rauh et al., 2011; V. A.

Rauh et al., 2006; R. M. Whyatt et al., 2009; R. M. Whyatt et al., 2007; R. M. Whyatt et al.,

2004); The Mount Sinai Hospital Children’s Envir onmental Health Cohort (Berkowitz et al.,

2004; Engel et al., 2007; Engel et al., 2011); and UC Berkeley’s the Center for the Health

Assessment of Mothers and Children of Salin as (CHAMACOS) Cohort (Bouchard et al., 2011;

Eskenazi et al., 2004b; Eskenazi et al., 2010; Eskenazi et al., 2007; Harley et al., 2011; Marks et

al., 2010; Young et al., 2005). These studies en rolled pregnant women at baseline and

prospectively assessed associations in their newborns and young children. Multiple reports have

been published based on the findings in each cohort.

The three study populations reflec t different exposure profiles. The Columbia Mother’s and

Newborn study and the Mt. Sinai Child Growth a nd Development study participants were likely

exposed to chlorpyrifos through residential use of the pesticide for indoor pest control. In the

residential setting, chlorpyrifos was among the most widely used household pesticides in the US

during the time period of these epidemiologic investigations. However, pesticide companies

voluntary cancelled indoor residential uses of chlorpyrifos-containi ng pesticide products on

December 31, 2000, during the time period of thes e cohort investigations. In the agricultural

setting, chlorpyrifos was registered for use on commonly consumed crops such as corn, almonds,

apples and oranges. Therefore, these study popula tions were most likely additionally exposed to

chlorpyrifos via the oral rout e through ingesting residues in the diet and from hand-to-mouth

contact with in-home chlorpyrifos-contaminate d surfaces, as well as possible dermal exposure

through contact with treated areas in the home environment (R. M. Whya tt et al., 2003; R. M.

Whyatt et al., 2009; R. M. Whyatt et al., 2007). In contrast, CHAMACOS cohort participants

were employed as farm laborers or were residing in homes with fa rm laborers. These participants

likely experienced either occupational exposure th rough the inhalation and dermal routes, as well

as probable indirect exposure through drinking water and take-home exposures (Bradman et al.,

2007). In each of the children’s health cohorts, the prevalence of pesticide exposure was high;

however, reported use of chlorpyrifos in the CHAMACOS region was modest (10% of total

pesticide use) (Eskenazi et al., 2004a).

2.1.1. The Mother’s and Newborns Study of No rth Manhattan and South Bronx (Columbia

University)

Researchers with the Columbia Mother’s and Newborn Study evaluated the association between

prenatal exposure to pesticides including chlo rpyrifos and developmental outcomes in children

through age 7 years. In this birt h cohort study, participants were recruited during early pregnancy

( ≤ 20th week) among African-American and Dominican women age 18-35 years, and registered

68 

# http://www.epa.gov/scipoly/ sap/meetings/2010/020210meeting.html#materials

# Chlorpyrifos Human Health Risk Assessment D424485

Page 225 of 531 for prenatal care and delivery at either New York Pr esbyterian Medical Center or Harlem City

hospitals. Women who smoked, had a history of drug abuse, diabetes, hypertension, or HIV

infection were excluded from participation in the study, as were women who resided in New

York City for less than 1 year. The study sample s represented in the reports reviewed were

recruited between 1998 and 2004, a period whic h overlaps the voluntary cancellation of

chlorpyrifos use in the re sidential environment.

In this cohort, authors measured chlorpyrifos exposure in se veral different biological and

environmental matrices. These include chlorp yrifos parent compound in cord blood; the

chlorpyrifos metabolite 3,5,6-trichl oro-2-pyridinol (TCPy) in ma ternal and infant urine and

meconium; and chlorpyrifos in personal and sta tionary air monitoring samples. In epidemiologic

analyses, investigators c onsistently utilized cord blood measures of chlorpyrifos as the measure

of prenatal exposure. Chlorpyrifos levels in um bilical cord blood samples were sampled as close

to the time of delivery as po ssible, and within 2 days pos t-partum. Cord blood plasma

chlorpyrifos levels were imputed from matern al blood levels for newborns for whom no cord

blood sample was obtained because correlation was high (>80%). Quantification of chlorpyrifos

levels in plasma were conducted by the Center s for Disease Control and Prevention (CDC).

Information regarding basic demographics, so cio-economic status, and pregnancy related

measures, among other factors was collected th rough self-report questionna ire at the time of

enrollment.

2.1.2. Inner-City Toxicants, Child Growth an d Development Study (Mt. Sinai Hospital)

Researchers with the Mt. Sinai Children’ s Health and Development study conducted a

prospective birth cohort study in which they enrolled primiparous women presenting for prenatal

care with singleton pregnancies at the Mount Sinai prenatal clinic and two private practices and

who delivered their infants at Mount Sinai Ho spital in New York City between May 1998 and

July 2001. Mothers were excluded if they had a ny of the following characteristics: an initial

prenatal visit after 26 weeks of gestation, serious chronic diseases, a serious pregnancy

complication that could affect fetal growth a nd development. Additional, participants were

excluded for risky health beha viors including alcohol consumed greater than two alcoholic

beverages per day or illicit drug use. Mothers and infants were also excluded if the child was

born with a congenital malforma tion or severe prematurity.

To measure prenatal pesticide exposure, resear chers implemented a self -report questionnaire to

solicit information regarding pesticide usage, in-home pest pressure and other exposure

characteristics. In addition, in the early third trim ester, participants were asked to provide a urine

sample at the time of a routin e clinical blood draw. Using th is biological sample, authors

measured urinary concentration of pesticide me tabolites including both TC Py (Berkowitz et al.,

2004), and also non-specific measures of orga nophosphate exposure, dialkyl phosphates (DAPs)

(Engel et al., 2007; Engel et al., 2011). Authors adapted analytical met hods to conduct laboratory

measurement of TPCy; DAPs were measured by CDC using published methods (Barr et al.,

2002). Using maternal and infant (cord blood) blood sample, researchers measured PON1

enzymatic activity levels, and conducted genoty ping analysis to determine prevalence of PON1

variant alleles.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 226 of 531 Potentially confounding variables we re measured through self-report questionnaire and included

in final statistical models if variables were known to be associated with either pesticide exposure

or fetal growth. Authors also measured concentra tion of other pesticides in biological matrices

including metabolites of pyrethr oid exposure (PBA), pentachlor ophenol (Berkowitz et al., 2004),

as well as other organophosphates (malathi on) and organochlorines compounds including

polychlorinated biphenyls (PCBs) (Engel et al ., 2007). However, authors did not present

analyses concerning potentia l confounding effect of co-exposure to other environmental

chemicals.

2.1.3 Center for Health Assessment of Mother’s and Children of Salinas Valley,

CHAMCOS (University of California/Berkley)

The Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS) cohort

is comprised by participants who live and work in the Salinas Valley, CA. The Salinas Valley is

a major center of agricultural production in the United States with approximately 500,000

pounds of organophosphate pesticides applied annually (California EPA. Pesticide Use

Reporting 2001 Summary Data, 2002. ( www.cdpr.ca.gov/docs/pur/pur01rep/01_pur.htm) ),

chlorpyrifos, however, was not frequently used in agriculture in this area during the time period

of this study. This cohort is comprised of low-income, predominantly Mexican-American (or

Mexican immigrant) women employed as farm la borers or living with someone employed as a

farm laborer. Enrollment of the Center for the Health Assessment of Mothers and Children of

Salinas (CHAMACOS) cohort took place at regiona l community clinics. Women considered

eligible for the study were less than 20 weeks gestation, aged 18 years or older, Medi-Cal

eligible, fluent in English and/or Spanish, and planning to deliver at Na tividad Medical Center.

Excluded from analyses were women with gestati onal or preexisting diabetes, hypertension, twin

births, or stillbirths.

Data collection was performed using several diffe rent tools. Authors ad ministered self-report

questionnaires to study participants to obtain info rmation regarding demographic characteristics,

work history and health behaviors. To assess exposure, participants were asked to provide

biological samples (urine, blood). Specifically, ma ternal and fetal exposure to organophosphate

pesticides was assessed by measurement of organophosphate dialkyl phosphate metabolites

(DAPs) and seven different pesticide-specific metabolites, including TCPy, in maternal urine

during two periods in the pregna ncy. Maternal urine samples collected between 5 and 27 weeks

gestation and again between 18 and 39 weeks. Th e post-delivery urines were collected within 1

week of delivery for 73% of the sample, with the remainder obtained up to 176 days afterwards.

Total DAP, including dimethyl phosphates (DMP), and diethyl phosphates (DEP) levels were

determined for each participant for each of the two pregnancy urine samples, and because the

measures did not differ substantially, these values were averaged in epidemiologic analyses to

estimate prenatal pesticide exposure. Quan tification of organophosphate metabolites was

conducted by the Centers for Disease Control an d Prevention (CDC) labs (Barr et al., 2002).

In this cohort, authors also measured cho linesterase (ChE) in whole blood and butyl

cholinesterase (BChE) in plasma as a surroga te for organophosphate pesticide exposure. PON1

was also measured in blood. Researchers also m easured concentration of other environmental

chemicals including PCBs, lea d, DDT/DDE, HCB, and PBDEs; however, these compounds were

# Chlorpyrifos Human Health Risk Assessment D424485

Page 227 of 531 not included in final models as potential confoundi ng variables.

2.2 Summary of Research Results

Across these three children’s he alth cohorts, authors have assessed the relation between

measures of prenatal chlorpyrif os exposure and various measures of fetal growth and infant and

child neurodevelopment. In this section, rese arch results are briefl y summarized by health

outcome category. A more detailed description of the study design, methods and analysis, as well

as research results and a criti que of individual study strengths and limitations are presented in

Appendix 3. An evidence table delineating key st udy features is presented in Appendix 4. Table

1 briefly summarizes these results.

2.2.1 Measures of Fetal Growth

Authors with each of the three respective ch ildren’s health cohorts measured prenatal

chlorpyrifos exposure in associa tion with fetal growth including birth weight, birth length, head

circumference, gestational age and Ponderal inde x. To ascertain birth characteristics, authors

linked with medical records at respective partic ipating hospitals. Across these children’s health

cohorts, authors observed inconsis tent evidence of an association; however, differing exposure

profiles as well as dissimilar methods of prenatal chlorpyrifos exposure assessment likely played

a role in this obser vation (Needham, 2005).

# Chlorpyrifos Human Health Risk Assessment D424485

Page 228 of 531 Table 1 Summary of Findings in the Columbia Mothers and Newborn Cohort, the Mount Sinai Hospital

Children’s Environmental Health Cohort, and the UC Berkeley Center for the Health Assessment of Mothers

and Children of Salinas Studies of Prenatal Organophosphate Pesticide Exposure and Child Development



# Columbia

Cohort Mount Sinai Cohort UC Berkeley Cohort

Markers of

Exposure: Chlorpyrifo

s TCPy DAPS TCPy DAPS

Birth Length Inverse

(Null post

2000) Null

‐‐ Positive

(NS) Positive

Birth Weight Inverse

(Null post

2000) Null

‐‐ Positive

(NS) Null

Head Circumference

at Birth Null Null

(Inverse*) ‐‐ Null Positive

(Inverse*)

Gestational Age

‐‐ Null

‐‐ Null Null

(Inverse*)

BNBAS Newborn

(Abnormal reflexes) ‐‐  ‐‐ Positive*

‐‐ Positive

BNBAS Newborn

(Neurodevelopment

) ‐‐  ‐‐ Null

‐‐ Null

Bayley Scores

6 months (MDI/PDI) ‐‐  ‐‐  ‐‐ Null/

Inverse (NS) Inverse

(NS)/

Inverse

(NS)

Bayley Scores

12 months

(MDI/PDI) Null / Null

‐‐ Inverse**/

Inverse (NS) Inverse

(NS)/

Inverse (NS) Inverse

(NS)/

Inverse

(NS)

Bayley Scores

24 months

(MDI/PDI) Null / Null

‐‐ Inverse

(NS)/Null Inverse(NS)

/

Inverse (NS) Inverse/

Inverse

(NS)

Bayley Scores

36 months

(MDI/PDI) Inverse/

Inverse ‐‐  ‐‐  ‐‐  ‐‐

Pervasive

Development

Disorder (PDD) Positive

(36 mo.) ‐‐  ‐‐  ‐‐ Positive

(24 mo.)

Mental

Development ‐‐  ‐‐ Null

‐‐  ‐‐

# Chlorpyrifos Human Health Risk Assessment D424485

Page 229 of 531 Columbia

Cohort Mount Sinai Cohort UC Berkeley Cohort

Markers of

Exposure: Chlorpyrifo

s TCPy DAPS TCPy DAPS

(WPPSI

‐ III, age 6

years)

Mental

Development

(WISC

‐ IV, age 7

‐ 9

years) Inverse

(Full

‐ scale

IQ and

Working

Memory) ‐‐ Inverse (NS)

(Full scale IQ,

perceptual

reasoning,

working

memory and

processing

speed) ‐‐ Inverse

(FSIQ,

working

memory,

processin

g speed,

verbal

perceptua

l

reasoning

)

Odds of ADHD/

attention and

behavior problems Positive (36

mo.) ‐‐  ‐‐  ‐‐ Positive

(NS 3

years)

Positive (5

years)

*Interaction observed between pesticide markers and PON1 activity or genotype

**Interaction observed between pesticide markers and race

NS=Not statistically significant

Inverse= Higher levels of exposure associated with adverse health outcomes (measurement value or score decreased)

Positive = Higher levels of exposure associated with adverse health outcome (measurement value or score increased)

Null

= No association observed

MDI= Mental Development Index

PDI = Psychomotor Development Index

# Chlorpyrifos Human Health Risk Assessment D424485

Page 230 of 531 Researchers with the Columbia Mothers and Ne wborn study observed an association between

decreased fetal growth and prenatal chlor pyrifos measures among 314 mother-infant pairs

selected for this study (R. M. Whyatt et al., 2004). Controlling for pot ential confounders, for

each log unit increase in cord plasma chlorpyrif os levels, birth weight decreased by 42.6 g (95%

CI: –81.8 to –3.8) and birth length decreased by 0.24 cm (95% CI: –0.47 to –0.01). Whyatt et al.

(2004) did not report an association with head circumference. Combined exposure to both

chlorpyrifos and diazinon (adjusted for rela tive potency using US EPA cumulative risk

assessment methods) were also significantly invers ely associated with birt h weight and length (p

< 0.05). When births were stratified by time period prior to or after the vo luntary cancellation of

chlorpyrifos for residential use, researchers no longer observed evidence of an association (p >

0.8) among births which took place in the cancella tion period (n=77). In supplementary analyses,

authors replicated this analysis with additiona l post-cancellation era births (total n=193) and

similarly did not observe evidence of an associati on with chlorpyrifos in the later time period (R.

Whyatt & Rauh, 2011) (See Attachment 1). Overall, authors suggest that pr enatal chlorpyrifos

exposures may have impaired fetal growth among this inner-city, low income cohort. While

suggestive of a conclusion that cessation of ch lorpyrifos exposure explains the different

associations with fetal growth before and after the period of the voluntary cancellation of

chlorpyrifos, EPA notes that this finding may also be suggestive of a possible threshold effect, or

it could also be due to a lack of statistical power to asse ss associations by time period.

Within the Mt. Sinai Child Growth and Develo pment study, Berkowitz et al. (2004) assessed the

association between prenatal ch lorpyrifos exposure measured as urinary TCPy and subsequent

risk of impaired fetal growth (B erkowitz et al., 2004). Authors also evaluated potential effect

modification by PON1 genotype and phenotype in the relation of interest. Among 404 births

which took place between 1998 and 2002, authors found no statistically signi ficant associations

between fetal growth including birth weight, or bi rth length or head circumference and

chlorpyrifos (estimated as TCPy) exposure. Ho wever, Berkowitz et al . (2004) did observe

evidence of heterogeneity of effect by PON1 activity level. Specifically, researchers observed a

small, but statistically significant reduction in head circumference among children of mothers

with levels of chlorpyrifos above the limit of detection and also in the lowest tertile of PON1

activity (least able to metabolize exogenous exposu res such as OP pesticides). In the subgroup of

infants whose mothers had TCPy levels greater than the level of det ection, those with low

maternal PON1 had an average (SD) head circumference of 33.3cm (1.5cm) which was

significantly smaller than t hose with medium (34.0cm ( 1.5cm)) and high (34.1cm (1.6cm))

maternal PON1 activity after adjusting for race/ ethnicity, infant sex, and gestational age (p =

0.014), although the statistic al interaction was not significant. Authors did not observe evidence

of heterogeneity of effect by PON1 in the re lation between other meas ures of birth outcomes

including birth weight and birth length and pesticide exposure. Neither maternal PON1 genetic

polymorphisms nor infant paraoxonase levels we re associated with reduced head size.

Within the CHAMACOS cohort, among 488 part icipants enrolled be tween 1999-2000 Eskenazi

et al. (2004) did not observe a significant adverse relationship between fetal growth and any

measure of in utero organophosphate pesticide exposure (Eskenazi et al., 2004b). On the

contrary, investigators repor ted positive associations be tween birth length and head

circumference associated with non-specific organophosphate exposure measures (DAPs).

Researchers observed decreases in gestati onal age associated with measures of in utero pesticide

# Chlorpyrifos Human Health Risk Assessment D424485

Page 231 of 531 exposure: urinary DMP metabolites ( β = –0.41 weeks per log10 un it increase; 95% CI: –0.75––

0.02; p = 0.02), which reflects exposure to di methyl organophosphate compounds such as

malathion, but not chlorpyrifos. Authors did not ob serve an association us ing TCPy as a measure

of chlorpyrifos exposure; however they did repo rt an increased risk of preterm delivery with

decreasing cholinesterase concentrations, a th ird biomarker of organophosphate exposure. To

identify “critical windows” of fetal developmen t when exposure may have a greater impact, the

authors analyzed the associations of outcomes and metabolite levels m easured during moving 6-

week windows of pregnancy ( e.g. , 5–10 weeks, 6–11 weeks, 7–12 weeks) using a series of

multiple regression analyses. No period of greater impact was observed in this largely null study.

In a follow-up analysis within th is cohort, Harley et al. (2011) performed the same analysis

however evaluating the potential effect modifying role of PON1 genotype and phenotype in the

relation between prenatal pesticide and chlorpyrif os exposure (DAP, TCPy, ChE ) and fetal

growth (Harley et al., 20 11). In this study, infants’ (but not mother's) PON1 genotype and PON1

activity modified the association between gestati onal age and head circumference. Infants with

the susceptible PON1 -108 

# TT genotype had shorter gestationa l age (beta =-0.5 weeks, 95%CI: -

0.9, 0.0) and smaller head circumference (beta= -0.4 cm, 95% CI: -0.7, 0.0 ) than those without

the susceptible genotype ( PON1 -108 

# CC genotype). Infants’ aryleste rase and paraoxonase activity

were positively associated with gestational age. Maternal DAP concentrations were associated

with shorter gestational age am ong infants of the susceptible PON1 -108 

# TT genotype, although

only the interaction between PON1 -108 

# genotype and DEP metabolite concentrations was

statistically significant (p-value for interaction = 0.09). However, maternal DAP concentrations

were associated with larger bi rth weight (p-value for interac tion = 0.06) and head circumference

(p-value for interaction =0.01) in infants with non-s usceptible genotypes. The authors conclude

that infants with certain PON1 genotypes ( e.g.

, PON1 -108 

# TT) may be more susceptible to effects

of in utero organophosphate pesticide exposure.

2.2.2 Brazelton Neonatal Neurological Functioning

Researchers with both the Mt. Sinai Child Growth and Develo pment study and the CHAMACOS

cohort evaluated neonatal neurological functioning in association with pr enatal chlorpyrifos

exposure. To measure indices of abnormal ne onatal behavior and/or neurological integrity

authors used outcome measures derived from the Brazelton Neonatal Behavioral Assessment

Scale (BNBAS). The BNBAS includes 28 behavi oral items and 18 primitive reflexes which

assesses the infant across several different developmental areas. This tool was administered to

infants within days after birth and before th ey left the hospital (2-5 days post-partum).

Examinations were conducted by trained neonato logists in the hospital setting using similar

environmental conditions. The Mt. Sinai Chil d Development study and the CHAMACOS cohort

evaluated this outcome measure; researchers with the Columbia Mother’s and Newborn study

did not measure the relation.

Among the 438 infants eligible to participate in the Mt. Sinai evalua tion, Engel et al. 2007

observed an association between generic (DAPS) biomarkers of prenatal organophosphate

pesticides and an increased number of abnorma l primitive reflexes which are considered a

critical marker of neurologic integrity. Controlling for confounding factors, subjects with

prenatal total diethylphosphates levels above the median (24.7 nmol/L) delivered infants who

# Chlorpyrifos Human Health Risk Assessment D424485

Page 232 of 531 were 2.3 times more likely to have at l east two abnormal reflexes (95% CI: 1.1, 5.0).

Associations with other DAPs we re also reported. Notably, no st atistically significant adverse

associations were found for any of the dialkyl phosphate metabolites and other domains of the

Brazelton assessment tool in the author’s primary analyses, e.g. , habituation, range of state,

orientation. Authors also observed eviden ce of potential effect modification by PON1 activity

level in the relation between DAPs and neonatal neurodevelopment. Specifically, in the first

tertile of paraoxonase 1 expression (slowest me tabolizers), the relative risks (RR) of having

abnormal reflexes were significantly related to DAP and DMP levels; the risk estimate for DEP

and the number of abnormal reflexes within the lowest PON1 level was not significant. In the

CHAMACOS cohort, Young et al. (2005) observed a statistically significant association between

organophosphate pesticide exposure and the reflex cluster of the BNBAS (Young et al., 2005).

The proportion of infants with more than thre e abnormal reflexes was 3-fold increased among

those with prenatal DEP exposure (DEP: OR = 3.4, 95% CI = 1.2, 9.9). Similar to Engel et al.

(2007), no other associations between prenatal DAPs and other aspects of the Brazelton

assessment were noted (other neurodevelopmen tal domains) (Engel et al., 2007). No adverse

associations were found between postnatal urinary metabolite levels and any of the

developmental outcomes.

2.2.3. Bayley Scale of Mental and Psychomotor Development

Researchers across the three children’s health co horts utilized the Bayley Scales of Infant

Development II (BSID-II) to ge nerate a Mental Development Index (MDI) and a Psychomotor

Development Index (PDI) to assess neurodevelopm ent in early childhood. As a complement to

this measure of mental development and behavior , authors also used the 99-item Child Behavior

Checklist (CBCL). In addition, beca use the quality of the home e nvironment is a key determinant

of child development, authors also used the Home Observation for Measurement of the

Environment (HOME) instrument. This instrume nt collects data regarding the physical and

interactive qualities of the child’s home as a measure of mental stimul ation and interactions.

Results from each of these measurement tools were used to model the relation between prenatal

chlorpyrifos exposure and adverse neurodevelopm ental outcomes in infancy and toddlerhood (6-

36 months of age).

Rauh et al . (2006) investigated Mental Develo pment Index (MDI) and a Psychomotor

Development Index (PDI) at 12, 24, and 36 months of age within the Columbia Mother’s and

Newborn Study. Children were categorized as having either hi gh (>6.17pg/g) or low

(<6.17pg/g) prenatal exposure, using categories in formed by results of the previous study on

birth characteristics (R. M. W hyatt et al., 2004). Authors report ed that the difference in MDI

scores was “marginally significant” (p = .06) wi th the exposed group scoring an average of 3.3

points lower. When the same multivariate regr ession models were calculated regarding the PDI

scores, none of the 12 or 24 month PDI scores sh owed significant effects, but the 36 month score

was significantly related to chlo rpyrifos exposure. Investigator s also calculated estimates of

adjusted risk for developmental delays (MDI and PDI) related to chlorpyrifos exposure and

illustrated that before 36 months of age, delays were no more likely in the highly exposed group,

but, at the 36 month milestone , the likelihood of highly expose d children developing mental

delays were 2.4 times greater (95% CI: 1.12-5.08, p = .02) and motor delays were 4.9 times

greater (95% CI: 1.78-13.72; p = .002) than those w ith lower prenatal expos ure. Using general

# Chlorpyrifos Human Health Risk Assessment D424485

Page 233 of 531 linear modeling (GLM), authors an alyzed developmental trajectorie s of the effects and results

were consistent with the 12, 24, and 36 month mile stone analyses indicating that the age effects

most significantly occurred at the later phase of the 3 year period. In supplemental analyses

suggested by the 2008 FIFRA SAP, authors il lustrated that diazinon was a strong confounding

variable in this association (correlation with chlorpyrifos 0.63), in creasing the magnitude

chlorpyrifos risk estimate for MDI and PDI 50-200% in the same direction (away from the null)

(R. Whyatt & Rauh, 2011) (Attachment 1).

Within the Mt. Sinai Children’s Environmental Health study, authors administered the BSID-II

to participating children at 12 and 24 months. Among 404 women originally enrolled between

May, 1998 and July, 2001 (n = 404), children of these mothers returned for neurodevelopment

assessments at ages 12 months (n = 200), 24 mont hs (n = 276) of age. Using generalized linear

models, authors found that prenatal total dialkylphosphate metabolite level was associated with a

decrement in mental development at 12 m onths among blacks and Hi spanics children. The

associations appeared to be strongest among children of mothers who carried the PON1 Q192R

QR/RR genotype. The authors concluded that th eir findings are suggestive of an association

between prenatal exposure to organophosphates and decrements in cognitive development,

particularly perceptual reas oning, with evidence of effects beginning at 12 months and

continuing through early childhood, with PON1 being a potentially impor tant susceptibility

factor for these deleterious effects. The author s did not observe any eff ect modification by PON1

enzyme activity level. In general, associati ons observed during the 12-month follow-up were

either attenuated or non-exi stent at the 24-month visit.

In the CHAMACOS cohort, Eskenazi et al 2007 , the authors report on the relationship between

prenatal and child urinary organophosphate meta bolite levels and child neurodevelopment at

ages 6, 12, and 24 months of age. Controlling for indicators such as the psychometrician

conducting the assessment and the location of asse ssment, age at assessment, sex, duration of

breast-feeding, HOME score, and household in come, parity and indi cator of maternal

intelligence (PPVT score), authors observed that pr enatal DAP levels were adversely associated

with MDI, while early life DAP levels were pos itively associated with MDI. At 24 months of

age, these associations reached statistical significance (per 10-f old increase in prenatal DAPs: β

= –3.5 points; 95% CI: –6.6 to –0.5; child DAPs: β = 2.4 points; 95% CI: 0.5 to 4.2). In a

subsequent study, investigators did not obs erve evidence of effect modification by PON1 status

in the relation between prenatal DAPs and chil d neurodevelopment as measured by the Bayley

Scale (Eskenazi et al., 2010). Neither prenatal nor child DAPs were associated with PDI or

CBCL attention problems. Both prenatal and pos tnatal DAPs were associated with risk of

pervasive developmental disorder (per 10-fold increase in prenatal DAPs: OR = 2.3, p = 0.05;

child DAPs OR = 1.7, p = 0.04). TCPy as a biom arker of chlorpyrifos exposure was not

significantly associated with any neur odevelopment outcome in this study.

2.2.4 Attention Problems

Also within the CHAMACOS cohort, Marks et al . (2010) conducted a study to investigate the

association between urinary dial kyl phosphate (DAP) metabolites in pregnant women and their

children, as a marker of organophosphate expos ure, and attention-related outcomes among 348

children who had available data at 3.5 and/or 5 years and met in clusion criteria (Marks et al.,

# Chlorpyrifos Human Health Risk Assessment D424485

Page 234 of 531 2010). Attention-related health outcomes were measured thro ugh maternal report of child

behavior at 3.5 and 5 years of ag e; direct assessment of the ch ild at 3.5 and 5 years; and by a

psychometrician’s report of the behavior of the child during te sting at 5 years. To identify

children whose behaviors were most sugges tive of possible ADHD w ithin the cohort, a

composite ADHD variable was defined that combined the results of the maternal report (CBCL),

child testing (K-CPT), and the psychometrician report (Hillside). In this study population, higher

concentrations of organophosphate metabolites in the urine of pre gnant women were associated

with increased odds of attention problems and poorer attention scores in th eir children at age 5

years. Prenatal DAPs were non-significantly asso ciated with maternal report of attention

problems and ADHD at age 3.5 years but were significantly related at age 5 years (CBCL

attention problems: β = 0.7 points; 95% CI: 0.2–1.2; ADHD: β = 1.3; 95% CI: 0.4–2.1). Prenatal

DAPs were associated with scores on the K-CPT ADHD Confidence Index > 70th percentile

(OR = 5.1; 95% CI: 1.7–15.7 and with a composite ADHD indicator of the various measures

(OR = 3.5; 95% CI: 1.1–10.7). Some outcomes exhibi ted evidence of effect modification by sex,

with associations found only among boys. Childre n’s concurrent total DAP and DMP metabolite

levels at 3.5 years and 5 years were unrelate d to attention outcomes, and but child DEP

concentrations at 5 years were adversely associated with the co mposite measure of attention (OR

= 2.0; 95% CI:1.1–3.6). The result s of this investigation by Marks et al. (2010) in the

CHAMACOS cohort are sugges tive of a detrimental asso ciation between prenatal

organophosphate exposure, as measured by maternal urinary metabolite le vels, and attentional

difficulties at age 5 years using three different measures of this neurodevelopmental outcome.

2.2.5. Intelligence Measures

To measure intelligence among school aged children, authors from each of the three children’s

health cohorts used the Wechsler Intelligence Scale for Childr en, 4th edition (WISC-IV). The

instrument measures four area s of mental functioning: the Ve rbal Comprehension Index, the

Perceptual Reasoning Index, the Working Memo ry Index, and the Processing Speed Index.

These indices are associated with, but distinct from, overall intelligence quotient (IQ) and are

sensitive to cognitive deficits related to learning and workin g memory. A Full-Scale IQ score

combines the four composite indices. A Gene ral Ability Index score is a summary score of

general intelligence, similar to Full-Scale IQ , but excludes contributi ons from both Working

Memory Index and Processing Speed Index. WISC -IV scores are standardized against U.S.

population-based norms for English and Spanish-speaking children.

Rauh et al. (2011) evaluated th e relationship between prenatal chlorpyrifos exposure and

neurodevelopment among 265 of the cohort participants who had reached the age of 7 years and

had a complete set of data incl uding prenatal maternal intervie w data, prenatal chlorpyrifos

marker levels from maternal and/or cord blood samples at delivery, postnatal covariates, and

neurodevelopmental outcome data (V. Rauh et al., 2011). While models were developed using

continuous measures of both pren atal chlorpyrifos exposure and Wechsler scores, for ease of

interpretation, investigators repo rted that for each standard deviation increase in exposure

(4.61pg/g) there is a 1.4% reduction in Full-Scale IQ and a 2.8% re duction in Working Memory.

In the Mt. Sinai Child Growth and Developmen t study, Engel et al. 2011 used generalized linear

models to analyze the rela tionship between total diet hylphosphates (DEPs), total

# Chlorpyrifos Human Health Risk Assessment D424485

Page 235 of 531 dimethylphosphates (DMPs), total dialkylphosphate metabolites (DAPs) measured in maternal

urine during the third trimester, and subsequent cognitive/intelligence development evaluated at

age 7 years. In this study, prenatal maternal DEP urinary metabolite concentrations were

associated with slight decrements in FSIQ, Perceptual Reasoning, and Working Memory

between the ages of 6 and 9 years. Among children of mothers with the susceptible PON1

genotype, DAP and DMP urinary metabolite concentra tions were associated with poorer scores

on Perceptual Reasoning and FSIQ.

In the CHAMACOS cohort, Bouchard et al. (20 11) observed evidence of an association between

prenatal exposures to organophosphate pesticides as measured by urinary DAP metabolites in

women during pregnancy, and decreased cognitive f unctioning in children at age 7 (Bouchard et

al., 2011). Authors observed this finding usi ng total DAP, DMP, and DEP metabolites to

estimate pesticide exposure. Children in the highest quintile of maternal DAP concentrations had

an average deficit of 7.0 IQ point s compared with those in the lowest quintile. Authors reported

the associations were linear with no thres hold. Urinary DAP concentrations in childhood

(postnatal) were not associated with cognitive sc ores in this cohort of children. Of note, the

following known or suspected neurotoxicants were measured: polybrominated diphenyl ethers

(PBDEs), polychlorinated biphenyls (PCBs), p,p ’-dichlorodiphenyltrichl orethane (DDT), p,p’-

dichlorodiphenyltrichlorethylene (DDE), and lead. Lead was meas ured in maternal blood at 26

weeks gestation, in cord blood for a subset of participants, and ch ildren’s blood a 2 years of age.

These other environmental expos ures including blood lead con centration did not change the

magnitude and direction of the associations observed between DAP/D EP and IQ measures

among school-aged children.

2.2.6 MRI Imaging Study

In April 2012, CCCEH researchers published a study utilizing magnetic resonance imaging

(MRI) technology to identify and contrast anatomical characteristics of the cortical region of the

brain including cortical surface ar ea and thickness in relation to prenatal pesticide exposure (V.

A. Rauh et al., 2012). Between June and Se ptember 2012, EPA sought additional expertise

within the Federal government as to the quality of the MRI data collection procedures used in

this study as well as the appropriateness of the authors’ interpretation of the MRI studies in

association with chlorpyrifos e xposure. EPA’s critical review and analysis of this MRI study can

be found in Appendices 3 and 4; the results of the Federal Panel review concerning EPA’s

questions about the study can be found at:

http://www.regulations.gov/#!docum entDetail;D=EPA-HQ-OPP-2008-0850-0170

.

In this study, authors CCCEH birth cohort st udy evaluated whether there were areas of

morphological change in the pediat ric brain in regions of the brai n known to be associated with

learning, cognition and social behavi or in association with prenat al chlorpyrifos (CPF) exposure

(V. A. Rauh et al., 2012). These are functiona l neurodevelopmental outcomes that have been

adversely associated with chlorpyrifos exposure in previous observationa l research (V. A. Rauh

et al., 2006; V. Rauh et al., 2011), as well as some experimental st udies, as noted by the authors.

Specifically, in this study, aut hors evaluated 1) whether mor phological changes in the brain

# Chlorpyrifos Human Health Risk Assessment D424485

Page 236 of 531 including cortical thickness and differences in the cerebral brain surface area and volume were

related to prenatal chlorpyrifos exposure (the main effect), an d, 2) whether the relation between

the exposure and morphological changes in th e brain were modified by either full scale

intelligence quotient (FSIQ) score (evidence of effect modificati on by IQ) and, 3) in preliminary

analyses, whether changes in the normal sexua l differentiation of the brain exist by exposurelevel (V. A. Rauh et al., 2012).

Authors concluded that while overa ll brain size did not differ by prenatal chlorpyrifos exposure,

certain areas of the brain were enlarged among those more highly exposed to chlorpyrifos

including superior temporal, pos terior middle temporal, and in ferior post-central gyri, and

superior frontal gyrus, gyrus re ctus, cuneus, and pre-cuneus in the mesial views of the right

hemisphere of the cerebrum. Further, they not ed a difference exposure- response relation between

cerebral surface distances and CPF exposure, by FSIQ wherein the relation between brain area

enlargements and FSIQ were positively correlated in the low CPF group (greater regional brain

size correlated with greater IQ), but an inverse relati on among the high CPF group. In

supplemental analyses, authors illustrated that areas of regional brain enlargement among those

more highly exposed to chlorpyrifos were due to underlying white matter enlargements.

Researchers also noted sporadic differences in cortical thickness wherein higher CPF exposure

was related to reduced cortical thickness. Prel iminary analyses also displayed a disruption of

normal male-larger-than-female areas as well as fe male-larger-than-male in certain areas of the

brain known to differ by child sex in normal br ain. Overall, research ers concluded that

differences in brain structure (r egional cerebral size and thickn ess) exist between CPF exposure

groups, and the differences (high>low CPF) in re gional brain size may be due to enlargement of

underlying white matter. However, authors did no t speculate as to th e underlying biological

explanation of the effect of C PF exposure on intelligence or w ith morphological changes in key

areas of the brain linked with lear ning, cognition and social behavior.

2.3. Strength and Limitations of Child ren’s Health Cohort Studies

There are several strengths reflected in the design, conduct and analys is decisions made by

investigators with each of the three children’s he alth studies, as well as some limitations to

consider. Strengths of these studies in clude the prospective nature of these investigations,

extensive data collection includi ng several different measures of prenatal chlorpyrifos exposure

which have been validated, and use of neurodevelopmental outcome ascertainment tools which

have been validated in different populations a nd commonly used in both clinical and research

settings, among other strengths noted in this se ction. Key limitations reflect the difficulty in

measuring multi-dimensional characteristics such as socio-economic status accurately, the

challenge of conducting environmental epidemiol ogy studies in populations exposed to several

different compounds and mixtures, and the ability to assess e ffect modification ( e.g. , genetic

variability) with precision and accuracy. Considering both the strengths and limitations of the

respective investigations, EPA puts forward epidem iologic inference at th e conclusion of this

section.

Study authors from each of the respective long-term studies hypothesized a role for

environmental exposures, including but not limite d to pesticides generally and chlorpyrifos

specifically, in the etio logy of adverse fetal growth and neurodevelopmental outcomes. Study

# Chlorpyrifos Human Health Risk Assessment D424485

Page 237 of 531 authors stated their motivation was the knowledge that these populations we re highly exposed to

pesticides and other environmental contamin ants, and that prior research had linked in utero

environmental exposure to adverse neurodevelo pmental outcomes. Cohorts were each funded

through the EPA and National Institute of Envi ronmental Health Science (NIEHS) Children’s

Environmental Health and Disease Prevention Research Centers 69 

# . Cohort studies were

comprised of several hundred mother-infant pairs (range: n=102 to n= 488) and were likely

sufficiently statistically powered to detect hypothesized main effects, but studies may have

lacked statistical power to perform stratified analyses, e.g., effect modification by genetic

variant. This is especially true for the MRI imaging study performed within the CCCEH (Rauh et

al. 2012). The pilot nature of th e study, inclusion of only 40 child participants from the overall

cohort, severely limited the evalua tion of statistical in teractions. Similarly, statistical methods

utilized across these investigati ons were appropriate to the data collected and research question

hypothesized, however statistical analyses were discussed in varying degrees of detail by

separate study groups. EPA notes that in some in stances, additional information is provided in

supporting methods paper, e.g., approaches to handling non-detects in large datasets (Arunajadai

& Rauh, 2012). Statistical model selection wa s generally parsimonious in nature, and

investigators performed multiple sensitivity analys es (not always shown) within each study to

further explore and eliminate alternativ e explanations for observed results, e.g. , confounding by

blood lead, variable transformati ons. Concerning the main effects hypothesized, both design and

analysis decisions made by researchers enhanced the probability of identifying an association, if

an association exists. For the association under study, m easurement errors likely occurred due to

the challenge of estimating exposure duri ng the critical window of development.

Selection criteria were clearly defined and appr opriate across the three cohorts. Investigators

limited participants to individuals within a simila r racial and ethnic group characteristics, income

and education level, and geographic area, and who reported low prevalence of risky health

behaviors (alcohol use, smoking during pregnancy), and who did not report major medical comorbidities which may have adversely a ffected fetal growth and development ( e.g. , gestational

diabetes). Therefore, authors s ought to control through design (re striction) variability in the

sample population to isolate the effect of chlor pyrifos exposure on fetal outcomes. However, the

many selection criteria applied may have aff ected the generalizability of study results. The

selected study population is narrowly defined and dissimilar to the general U.S. population

across many demographic and exposure related char acteristics. However, in comparison with

biomonitoring levels in the U.S. general popu lation, exposure among these cohorts was greater,

but within a similar range. In addition, the external validity of the study findings may be limited

if the organophosphate pesticide exposure-fetal development association is modified by factors

that are more, or less, prevalent in the study popu lations relative to the population(s) to which

inference is being made, or to populations with a substantially differe nt exposure range.

All three studies are prospectiv e cohort studies in which expos ure measurements were obtained

prenatally and/or at delivery, and fetal growth and neurodevelopmental outcomes were assessed

subsequently. This study design eliminates or reduces several potential sources of bias. This

design eliminates temporal bias, i.e. , uncertainty as to whether exposure precedes the adverse

outcome, and, and also reduces the effect of differ ential exposure measurement error (a type of

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# Chlorpyrifos Human Health Risk Assessment D424485

Page 238 of 531 information bias) as it is unlikely exposure measurement would be differentially assessed by

(unmeasured, future) health outcome.

Researchers utilized similar tools to measure a dverse health outcomes across the studies. The use

of similar outcome measures across studies aids comparability of studies and assessment of the

consistency of results. Neurodevelopmental outcome assessment tools utilized were common to

both clinical and research setti ngs and most have been validated in both English and Spanish

speaking populations. While measures of fetal growth are somewhat objective to measure,

neurodevelopmental outcomes measur ed within these studies are more difficult to assess. While

administered consistently within these investig ations, tests of mental development, cognition,

psychomotor development, and intelligence tools are somewhat subjective in nature, and may be

affected by child’s anxiety (Bayley Scales) a nd influenced by cultural factors (Wechsler).

However, the homogeneity of the study populatio n may reduce the potenti al influence of a

cultural bias or differences in assessment tool response based on external factors, and these are

among the best (gold standard) of tools to assess th ese types of developmental issues. Authors

employed appropriate quality assurance and c ontrol measures such as training of test

administrators, periodic evaluation of adjudicators, consistent envi ronments in which tests were

administered, and in many instan ces included an indica tor variable for test administrator in

statistical analysis to adjust for slight differences in examiner effect. . Fe deral letter reviewers in

September 2012 concurred that the neurodevelopmen tal testing utilized in these studies were

reasonable and appropriate 70 

# . With the exception of the child be havior checklist (CBCL), errors

in the measurement of health outcomes were li kely non-differential in nature, leading to an

attenuation of the risk estimates. Overall, acro ss these studies researchers utilized the best

available outcome measurement tools for neurodev elopment health effects, and implemented the

evaluations in a consistent, stan dardized manner with trained h ealth professionals and/or study

staff. The results of the Federal Panel review of June-September 2012 supported this conclusion.

Federal panelists agreed that these are acceptable tools and appropriate to the age range of the

child participants in these cohort studies. The to ols allowed researchers to address the question as

to whether neurodevelopment scores differed by chlorpyrifos exposure. 71



# Across the three children’s health cohorts, study authors measured parent chlorpyrifos, TCPy and

diethyl phosphate (DEP) to estimate chlorpyrifos and/or organophosphate exposure. Chlorpyrifos

metabolites (TCPy and DAPs) are likely mo re accurate and objective indicators of

organophosphate pesticide exposure than other exposure ascertainment methods such as selfreport, but uncertainty remains as to the extent measurement of non-specific metabolites reflects

chlorpyrifos exposure. TCPy is a metabolite specif ic to chlorpyrifos, but can also be produced as

a result of chlorpyrifos-methyl exposure or en vironmental exposure to TCPy itself (Morgan et

al., 2005; Wilson, Chuang, Lyu, Menton, & Morg an, 2003). Urinary dialkyl phosphate (DAP)

metabolites of organophosphorus insecticides are markers of exposure to organophosphate

pesticides generally; chlorpyrifos and diazinon are metabolized to diethyl phosphates (DEP),

while other organophosphates are metabolized to dimethyl phosphate (DMP). For risk

assessment purposes, it is difficult to infer chlor pyrifos effects specifically from urinary DAPs

and to a lesser extent the TCPy metabolite. Diffe rent exposure measurements may in part explain

inconsistencies across study findings, as noted by others (Needham, 2005). The three birth

70 

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# Chlorpyrifos Human Health Risk Assessment D424485

Page 239 of 531 cohorts recruited participants who likely have chlorpyrifos exposures th at are higher than the

general population, and the exposures in these cohorts may also be more consistent over time.

Inner-city populations report fre quent use of pesticides, most applied at least once per week,

during the time period of these studies (R. M. Wh yatt et al., 2003; R. M. Whyatt et al., 2002). In

New York City, location of two of the three children’s health cohort studies, housing authority

reported common use of chlorpyrifos for indoor pest control during the time period of these

studies, prior to the time period of the voluntary cancellation of these uses. In the CHAMACOS

cohort, chlorpyrifos metabolite (TCPy) was detect ed in 77% of urine samples (Bradman et al.,

2005; Eskenazi et al., 2004a). Despite being c onsidered to have gr eater opportunities for

exposure relative to the general pop ulation, the average levels of ch lorpyrifos biomarkers in the

three cohorts were generally comparable to the U.S. general population, which may indicate a

difference between actual and measured exposure. Ne vertheless, it is not cl ear to what extent the

use of one or two biomarker measurements conducte d in the reported studie s reflects exposure(s)

over critical windows of devel opment during pregnancy. This remains an uncertainty within

these studies. As previously mentioned, chlorpyr ifos exposure was assessed by quantification of

biomarkers primarily in one- or two-samples of maternal urine taken during the third trimester,

and also, in one cohort, in a si ngle maternal blood or cord blood sample obtained at the time of

delivery. If the exposure is chronic, a biomarke r measured at a single time point may provide a

representative dosimeter, even if the toxicant has a short half-life, as is the case for chlorpyrifos

and organophosphate pesticides generally. Howeve r, if pesticide exposures are sporadic or

otherwise vary over short time scales, the biomar ker measurement may not be representative of

“usual” exposure or of the exposure during critic al periods of fetal deve lopment. In the UC

Berkeley CHAMACOS cohort (Eskenazi et al., 2004a ), prenatal urine was collected between 5

and 27 weeks gestation and again between 18 and 39 weeks. The within-person standard

deviation in the DAP metabolites was approximately three times larger th an the between-person

standard deviation, and concluded that a single biomarker assessment, as was conducted in the

other two cohorts, “may not accurately reflec t exposures over the entire pregnancy.”

This potential limitation was also assessed in two validation studies conducted by Whyatt et al

within the Columbia Mother’s and Newborn Study. The first of these stud ies (R. M. Whyatt et

al., 2007) assessed within- and between-home variabil ity in indoor-air insect icides over the final

2 months of pregnancy among a subset of part icipants. Authors obser ved little w ithin-home

variability and no significant di fference in air concentrations within homes over time (p ≥ 0.2);

between-home variability accounted for 92% of the variance in the i ndoor air levels of

chlorpyrifos. Indoor and maternal personal air ins ecticide levels were hi ghly correlated (r = 0.7–

0.9, p < 0.001). While this study provides some evidence that assessment of chlorpyrifos

exposure at a single time point may be reasonab le, the study took place during a period of rapid

decreasing indoor use of chlorpyrifos, and may not reflect variability in indoor air concentration

prior to the period of cancellati on. In addition to inhalational expos ure, participants were likely

also exposed by ingesting residues in the diet and from hand-to-mouth contact with surfaces

contaminated with the pesticide. If true, then high correlation of chlorpyrifos air exposure over

time may not indicate a true cons istency in exposure levels.

The variability in exposure measur es and the validity of different biomarkers of exposure was

evaluated in a second study (R. M. Whyatt et al. , 2009) which evaluated trends over time in

multiple biomarkers of prenatal chlorpyrifos exposure. Authors measured TCPy levels in repeat

# Chlorpyrifos Human Health Risk Assessment D424485

Page 240 of 531 prenatal urine samples and determined they were positively, but only moderately correlated (r =

0.23–0.56), and within-subject variability exceeded between-subject vari ability (intraclass

correlation coefficient = 0.43). This indicates that variability in individu al exposure over time

may be considerable. Indoor air levels explaine d only 19% of the varian ce in prenatal urine

TCPy (p = 0.001) in this study. However, these re searchers also demonstrated the presence of

TCPy in meconium; meconium is fecal matter acc umulated in intestines of developing fetus

from week 13 of gestation and re leased immediately prior to or w ithin a few days after delivery.

This matrix is considered to be an integrated measure of exogenous exposures to the fetus during

the gestational period. Importantl y, investigators reported a mode rate and significant correlation

between maternal blood and urine co llected in the later part of th e pregnancy period, infant cord

blood collected at delivery, and meconium (R. M. Whyatt et al., 2009). As noted in earlier

sections, the critical windows of effect for th ese specific outcomes is unknown at this time, and

may span the period of early pregnancy thr ough early childhood (Rice & Barone, 2000; Rodier,

2004). The correlation among several biomarkers of chlorpyrifos exposure reflecting different

windows of exposure from week 13 through delivery ( e.g. , correlation between meconium and

infant cord blood r=0.33, p=0.01, n=56), sugge sts a one-time measure of exposure may

accurately rank participants with re spect to prenatal exposures, at l east within a 2- to 3-fold level

of variation as suggested by authors (R. M. Whyatt et al., 2009). Additionally, the correlation

between the analytic measure of exposure and a measure reflective of e xposure during a large

proportion of the gestational pe riod suggests the exposure measur es may also reflect exposure

during the several likely critical windows of development in the mid- to latter period of

gestation. However, to the extent participants experienced “peak” exposur es at key periods of

development, this information would not likely be reflected in the one-time measure, and likely

lead to non-differential measurement error.

Bias due to confounding occurs primarily when risk factors for the outcome are unequally

distributed among exposure groups, but are not themselves caused by the exposure, and these

factors are not controlled in eith er the design or the analysis of the study. The greatest concern is

for unmeasured confounders, those variables not measured within the study that may be related

to both the exposure and diseas e of interest but are unknown to the investigator. Residual

confounding can also arise due to errors in th e measurement or categorization of confounders,

even after apparent adjustment for these factors in the study, i.e. , these are factors not measured

well enough. Among the major poten tial unmeasured or poorly me asured confounding variables

in the chlorpyrifos and neurodevelopmental associat ion are: 1) difficult to measure aspects of the

social environment ( e.g. , chronic stress, socioeconomic cond itions), and 2) environmental coexposures that may be determinants of neurodevelopment ( e.g. , lead exposure, mercury

exposure, air pollution, tobacco smoke exposure, ma ternal alcohol intake, and exposure to other

pesticides). The selection of cohorts that are homogenous with respect to many demographic and

social factors significantly reduces the potenti al for residual or unmeasured confounding factors

related to socio-economic status to have influenced these results as they have been controlled

through restriction (another method of adjustment ). For example, socioeconomic status cannot

confound the main association in a cohort of i ndividuals living under th e same socioeconomic

condition, i.e. , there is little variation in the study population on this factor. However, close

examination of descriptive tables in these publ ications indicates some degree of variability

remains. Therefore, authors appropriately eval uated and included in final models several

individual-level markers for socio-economic status such as education, income, and race.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 241 of 531 Additionally, within th e Columbia Mother’s and New born study, authors performed new

analyses using hierarchal regre ssion techniques to model variabil ity due to SES in the relation

between prenatal chlorpyrifos exposure and ment al and motor development (the Bayley Scale)

(Lovasi et al., 2011). These techni ques utilize individual- as we ll as group-level variables to

model SES. In this research, au thors did not observe a significan t difference in risk estimates,

indicating that the role of SES as a major confounder in the Co lumbia cohort is appropriately

adjusted. However, others argue that the multi-dime nsional nature of a characteristic such as SES

can never be fully captured through commonly used va riables, and suggest w ithin a term such as

SES there could be components that act as both positive and negative confounding variables

(Bellinger, 2011). However, at this time, there is no compelling evidence that lack of control of

the confounding influence of SES significantl y biased the reported risk estimates.

Additionally, authors have meas ured several other environmen tal chemicals and in some

instances considered these other exposures as potential confounding variables (not always

shown). Researchers collected information from participants regarding biomarkers of other

organophosphates, environmental tobacco smoke ( ETS), blood lead, PAH, methylmercury (R.

Whyatt & Rauh, 2011) (See Attachment 1), pyrethroi ds/pyrethrins, organochlorines such as

DDT/DDE and hexachlorobenzene, PBDE, and polychlorinated biphenyls. While authors

evaluated these environmental exposures in re lation to both chlorpyrifos and outcomes, none

were included as confounders in final models due to lack of statistical evidence of confounding.

Within the Columbia Mothers and Newborn study, authors performed additional analyses

suggested by the 2008 FIFRA SAP to evaluate th e role of other organophosphate exposures,

specifically diazinon and propoxur, in the rela tion between chlorpyrifos and both birth

characteristics and mental and motor delays (Bayley scores) (R. Whyatt & Rauh, 2011) (See

Attachment 1). Chlorpyrifos and diazinon biomar kers were highly correlated in this cohort

(r=63%). While the effect sizes reported for th e reported relation between both birth length and

also birth weight remained unchanged from pub lished reports when diazinon was added to the

model, significant differences we re observed in the relation with neurodevelopmental functions

(mental (MDI) an PDI (motor) scores). Specifi cally, authors reported a 50-200% increase in

effect sizes with MDI and PDI, respectively, i.e. , observed adverse effects became more

pronounced with diazinon added to the model (See Attachment 1). Limited evidence of

confounding by propoxur was also observed, but th e correlation between chlorpyrifos and

propoxur was moderate (23%), the confounding eff ect was not significant. In addition, neither

pre- nor post-natal blood lead le vels or methyl mercury levels measured in cord blood were

significantly correlated with chlorpyrifos, and was ther efore not considered a confounding

variable in the association of interest within the Columbia c ohort (V. A. Rauh et al., 2006; R.

Whyatt & Rauh, 2011).

The appropriate measurement and controlling of potentially confounding variables was a major

line of inquiry in the EPA initiat ed Federal Panel review in 2012. On this point, reviewers agreed

with EPA that factors relati ng to socio-economic status we re adequately measured and

characterized by authors. In addition, reviewer s did not note other major potential confounders

inadequately measured or contro lled in analysis. With respect to blood lead, reviewers of the

MRI study indicated the sample size may have been inadequate to fully cont rol for the effect of

low-levels of lead exposure. As a result of th is comment, EPA contacted researchers with the

CCCEH to gain additional information as to the association between chlo rpyrifos and blood lead

# Chlorpyrifos Human Health Risk Assessment D424485

Page 242 of 531 in the cohort (additiona l data collection beyond the published st udy data). In further written and

oral communication with the CCCEH staff, EPA learned that when the correlation between the

two environmental exposures was assessed among a la rger sub-set of the c ohort than previously

published (n=300), the correlation be tween chlorpyrifos and cord blood lead remained extremely

low (See Appendix 6 to the revised chlorpyrifos HHRA). This information reinforces EPA’s

understanding that lead expos ure is not likely a confounding variable in this study.

However, uncertainties remain as to the role of other (unmeasured) environmental compounds as

potential confounding variables in this association. None of the authors evaluated the possible

effect modifying role of other environmental ch emicals in the relation between chlorpyrifos and

neurodevelopment, nor were studi es suited to examining the eff ect of mixtures (simultaneous

combined exposures at potentially releva nt critical windows of development). New

investigations are needed to address these hypotheses , if relevant. While the possibility of

unknown, unmeasured positive confounding bias can never be completely excluded, given the

evidence available it is unlikely this potential bias may entirely explai n observed associations.

Selection bias occurs when study pa rticipants are either selected or lost to observation as a result

of a third, unmeasured factor that is associated with both the expos ure and outcome of interest. In

these prospective cohort studies, se lection bias is most likely i nduced not by selection into the

study, but by selection out of the study, i.e. due to attrition of study pa rticipants and missing data.

Because data are often not obtained on those that are lost to follow-up or for whom data is

otherwise missing, it is difficult to determine whethe r or not, and to what extent, the observed

associations are biased due these factors. The remedy is simple, in theory – follow the entire

cohort and obtain all relevant data – but often difficult in practice, particularly for long-term

cohort studies such as those reviewed here. Ac ross these studies, the amount of missing data

varied but was great in some studies. To addre ss missingness in data, authors imputed values in

several instances, and performed se nsitivity analyses with and without imputed data to illustrate

comparability in reported results. In addition, authors were able to illustrate differences between

those included and excluded were comparable on several major characteristics, but some were

not evaluated such as blood lead levels. Therefore, while it is difficult to ascertain the degree to

which selection bias due to missing data may have influenced these study results, authors

employed appropriate analytic tools to addres s missingness in data to the extent possible.

Regarding external validity, the inclusion a nd exclusion criteria employed may limit the

generalizability of study results. This is particularly true of the MRI imaging study, which was a

pilot effort.

Information bias arises due to misclassificati on or error in the measurement of either the

exposure or outcome of interest, or the accura te measurement of confounding variables.

Qualities of the outcome ascertainment me thod, the exposure assessment method and the

measurement of confounding variables were revi ewed above. In summary, regarding exposure

measurement error the true relevant exposure in these studies is prenatal chlorpyrifos to the

developing fetus. Sources of exposure measurem ent errors include 1) the single measurement

chlorpyrifos or its metabolites during the thir d trimester, 2) error arising from differences

between measured biomarker levels and actual ch lorpyrifos or chlorpyrifos oxon exposure, even

at the one time point, 3) unmeasured time- a nd space- dependent patterns of chlorpyrifos

exposure, 4) uncertainty regardi ng the critical period for chlorpyr ifos effects on development, 5)

# Chlorpyrifos Human Health Risk Assessment D424485

Page 243 of 531 missing exposure data, 6) laboratory errors, a nd 7) imputation of missing exposure levels.

Measurement errors in the ascertainment of chlorpyr ifos exposure are likely to have occurred in

the studies reviewed. However, because of the prospective st udy designs employed, the errors

are unlikely to result in falsely positive findings , because the probability and magnitude of these

errors are likely to be independent of the outcome status of part icipants. Measurement errors in

the ascertainment of the health outcomes were al so likely to have occurred, perhaps more for the

neurocognitive outcomes, than for the indicators of fetal growth and development, and also are

unlikely to have spuriously positive findings. Thes e measurement errors ar e more likely to have

resulted in false negative findings, if a causal association truly exists.

While the MRI study by Rauh et al. 2012 follows upon both previous epidemiological research

and experimental toxicol ogy studies that suggest an association between ch lorpyrifos exposure in

the pre-natal and early life periods with a dverse neurodevelopmental outcomes observed in

children through school age, EPA believes that the use of thes e data to inform specific

underlying biological mechanisms or pathways of the potential toxic action of CPF is limited;

additional and more sophisticated imaging would ha ve to be performed to determine links to the

very specific types of neurodevelopmental effect s posited by authors in the Rauh et al. (2012)

study.

2.3.1. Epidemiologic Inference of Comb ined Children’s Health Cohorts

As stated earlier, the three children’s health cohorts considered herein have several strengths as

well as limitations to consider in the interpretatio n of these studies. Within these studies, there

are several factors that would tend to under-estimate th e actual association (possibly leading to

false negative association), as well as some characteristics that may lead to over-estimation

(possibly leading to a false positive associa tion). However, it must also be noted that

methodological research and supplem ental analyses (primarily with in the Columbia Mothers and

Newborn study) performed subsequent to the 2008 FIFRA SAP delibera tions concerning these

children’s health cohort studies have reduced and further charact erized important sources of

uncertainty. As noted in the Intr oduction to this paper, currently scientists cannot determine with

accuracy the critical window of exposure fo r these outcomes (early gestation through early

childhood). Across the children’s he alth cohorts, researchers used one or two measure(s) of

exposure to estimate gestational exposure durin g the critical window of development, and

investigators assessed the main exposure usi ng non-specific biomarke rs of chlorpyrifos.

Undoubtedly, exposure measuremen t error occurred. However, exposure validation studies

illustrate some degree of correlation across exposure measures made at different periods of

gestation ( e.g., meconium, week 13 through delivery; mate rnal urine during last 8-weeks of

pregnancy; and cord blood at delivery). Add itionally, given the prospective nature of these

studies, it is unlikely the measuremen t error was differential by outcome, i.e., non-differential

exposure misclassification leading to biased estimates toward the null is anticipated. Finally, as

noted above the degree of missingness in some key variables across these studies may have

resulted in a form of selection bias which may ha ve also lead to an unde r-estimation of effects.

However, given the data are missi ng, it is difficult to assess the magnitude and direction of this

error on study results. Low sample size may have limited the ability of researcher to identify

sources of effect modification or perf orm stratified analyses with accuracy.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 244 of 531 Conversely, there may have been factors at play which led to an inflated estimate of the true

association. Factor that may lead to false positive associations include unmeasured or poorly

measured confounding factors which are positively associated with both chlorpyrifos use and

adverse neurodevelopmental outcomes (positiv e confounding variables). The measurement of

socio-economic factors and other environmental exposures experien ced either pre- or post-natal

environment are most likely among these potential factors, although others may exist. However,

when additional adjustment was made for thes e factors using both individual- and group-level

variables, the magnitude and di rection of the associations re mained stable (diazinon, propoxur,

blood lead, methyl mercury, SES, post-natal exposure) (Lovasi et al., 2011; V. A. Rauh et al.,

2006; R. M. Whyatt et al., 2004; R. Whyatt & Ra uh, 2011) (See Attachment 1). In the instances

in which authors assessed the role of early childhood exposure to chlorpyrifos or other

environmental contaminants as po tentially confounding variables in the relation between prenatal

exposure and neurodevelopmental outcomes, c onfounding bias was not observed. However, not

all investigations eval uated the role of post-natal pestic ide exposure, and authors note the

limitation. Selection bias due to missing or drop ou ts from the study could also have influenced

the observation of a false positive association, and this is again di fficult to assess in the absence

of data. However, researchers uniformly disc ussed and described the comparison of those

included and excluded from individual analyses based in past of missing data using several

important factors, and in many in stances reported a le vel of comparability which is reassuring.

The observation of positive associations as a result of multiple comparisons may also be a factor

to consider; however the a priori identification of research qu estions of interest and the

consistency of findings across se veral neurodevelopmental domain s argues against a large role

for multiple statistical testing to explain positive findings.

Overall, these are well performed studies which ar e shielded from several major sources of bias

in the interpretati on of results due to the strong design, c onduct and analyses utilized in these

investigations. While factors are present across th ese studies which may have led to either false

positive or negative associations, it is notable that positive associations were observed as EPA

believes the possibility of under-estimation of eff ect size is more likely th an factors that would

lead to over-estimation of effect size. Author s have taken significant steps to address major

sources of uncertainty that may l ead to over-estimation of effects, i.e. , positive confounding bias

as a result of poorly measured f actors related to socio-economic status and other environmental

chemical exposures.

3.0. Chlorpyrifos Epidemiology Synthesis and Evaluation

In this chapter, EPA reviewed the results of epidemiological investigations of the association

between prenatal chlorpyrifos exposure and adve rse effects upon fetal growth and neonatal and

early childhood neurodevelopmental outcomes across three major prospectiv e children’s health

cohort studies in the U.S. In accordance with the OPP’s draft “Framework for the Incorporation

of Epidemiology into Regulatory Risk Assessment” 72 

# , including the use of the modified

Bradford Hill considerations in judging the poten tial causal nature of observed associations, in

this section EPA considers the totality of th e epidemiological evidence from these cohorts. To

perform this analysis, EPA considered the strength of the associations observed and the presence

of exposure-response trends, the temporality of the observed associations, and the degree to

72 

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# Chlorpyrifos Human Health Risk Assessment D424485

Page 245 of 531 which alternative explanations have been consid ered and eliminated as explanatory factors,

among other considerations. Issues of biological plausibility and specific mechanisms of action

which may explain a causal role for chlorpyrifos in adverse neurodevelopmental outcomes, while

broadly considered within the context of these investigations, are disc ussed in depth in the

revised chlorpyrifos HHRA and supporting documents. EPA notes that while the pilot MRI study

by the CCCEH researchers supports other finding s from these cohorts, additional and more

sophisticated imaging would have to be performe d to determine links to the very specific types

of neurodevelopmental effects posited by au thors in the Rauh et al. (2012) study.

As noted previously, each investigation is a pr ospective cohort study in which prenatal exposures

occurred prior to the developmental of either fetal growth anomalies or neonatal or early

childhood neurological delays. Theref ore, in each study exposure pre ceded the health effect, risk

may be directly calculated, and measurement error is more likely non-differential in nature, i.e.

, likely under-estimation rather than over-estimat ion of risk effect. Table 1 summarizes the

associations observed for chlorpyrifos marker s and neurodevelopmental outcomes in the three

cohorts. Within this database, moderately strong associations have been observed, and in many

instances measured with precision. The relation between both neonatal neurological development

as measured using the Brazelton index (number of abnormal reflexes) and also mental and

psychomotor development among toddlers (Bayle y Scale for 24-36 months) with prenatal

chlorpyrifos exposure is elevated approximately 2- to 4- fold among those more highly exposed.

This observation was consistent across cohorts w ith respect to the neonatal period, but not across

measures using the Bayley Scale. Considering adverse effect in the 24-36 months period of

development, the effects became more pronounced over time in one cohort (the Columbia

Mother’s and Newborn study), and became less pronounced over time in the Mt. Sinai Child

Development study using non-specific biomarker of chlorpyrifos exposure (DAPs). However, it

is notable the significant eff ects were seen when chlorpyrif os parent compound was measured

directly; and, the effect size became significan tly greater when diazinon was considered in the

final statistical model. Notable decrements in in telligence measures were consistently observed

across all three cohorts. While eff ect sizes measured as beta-coeffi cients are reflective of average

change in intelligence score per unit change in chlorpyr ifos exposure, and may appear modest in

effect size, EPA notes that according to these m odels, the adverse influence at the extremes of

the chlorpyrifos exposure distri bution, if truly presen t in nature, would be more deleterious.

Strong, consistent evidence of a positive association between prenatal chlorpyrifos and

attentional problems, pervasive developmen tal disorder, and ADHD-like symptoms was

observed in these studies, although not always measured with precision.

An association is consistent when a similar magn itude and direction of re sults are replicated in

studies in different settings usi ng different methods. If a relationshi p is causal, one may expect to

observe it consistently in diffe rent studies and among different populations. Several consistencies

were noted across these studies. Higher levels of the chlorpyrifos-specific metabolite TCPy were

associated with prevalence of abnormal refl exes among newborns, as assessed using the

Brazelton Neonatal Behavioral Assessment Scale (BNBAS), in both the Mount Sinai Hospital

and UC Berkeley (CHAMACOS) Cohorts. The BNBAS assessment was not conducted in the

Columbia Mothers and Newborns cohort. TCPy le vels were consistently not associated with

BNBAS indicators of newborn neurodevelopment su ch as behavioral domains other than those

observed for number of abnormal reflexes. In ad dition, associations betw een pesticide markers

# Chlorpyrifos Human Health Risk Assessment D424485

Page 246 of 531 and the Mental Development Index (MDI) portion of the Bayley Scores at 12, 24, and 36 months

varied between small, largely non-statistically signifi cant decrements, and null. However, in

supplemental (post-publication) analyses, auth ors illustrated that adjustment for other

organophosphate pesticides in the relation between prenatal chlo rpyrifos and MDI strengthened

the magnitude of the effect significantly at the 36 months time point (R. Whyatt & Rauh, 2011)

(See Attachment 1). Associati ons between early life orga nophosphate pesticide exposure

biomarkers and decrements in mental developm ent in childhood (age 7- 9 years) were observed

in all three studies, although the relationships were not consiste ntly statistically significant.

Some notable inconsistencies were also observe d. These may be explai ned by differing exposure

biomarkers, timing of biomarker collection, or other limitations of study design and

measurement, e.g., the magnitude of non-differential e xposure misclassification. However,

inconsistency in results may also argue against a true causal a ssociation. Associ ations between

markers of prenatal pesticide exposure and fetal growth were not consistent across the three

studies, as noted in the FIFR A SAP 2008 meeting. In the Mount Sinai Child Growth and

Development cohort, no statistica lly significant associations were observed in primary analyses,

although a modest decrease in average head ci rcumference with increasing TCPy was noted

among those with low PON1 activity. In the Columbia Mothers and Newborn Study (R. M.

Whyatt et al., 2004), chlorpyrifos levels in ma ternal blood were modestly associated with

decreased fetal growth. In contrast, among the UC Berkeley CHAMACOS study participants,

modest increased birth weight and head circumfe rence were associated with in utero DAP

concentrations (Eskenazi et al., 2004; Harl ey et al., 2011). In fact, stratification by PON1 status

seemed to enhance these positive associations , such that in the non-susceptible groups ( i.e.

, PON1 -108 

# CC, PON1 192 

# RR, and high arylesterase activity), increasing DAP concentrations were

significantly associated with increased birth we ight and head circumference. The pesticide

biomarkers differed between the two studies; cord blood chlorpyrifos levels were assessed in the

Columbia Mothers and Newborn study, while DAPs were assessed in the UC Berkeley

CHAMCOS cohort. It is possible that high urinary DAP concentra tions may be an indication of

rapid detoxification and excreti on of organophosphate pesticides, ra ther than a marker of high

exposure, particularly among the participants that are less potentially less susceptible to pesticide

effects due to their PON1 genotype or PON activity level, as suggested by study authors.

Although consistency of associations across studi es set in different populations, and employing

different methods has been used as a criterion for causality, it is not the only compelling factor.

In fact, heterogeneity across studies is expected to occur between studies in the presence of effect

modification, if the prevalence of the effect modifier varies between the populations being

assessed. Apparent inconsistencies could also be due to chance differences, and due to the

presence of biases operating in the studies under consideration.

There was evidence for an exposure-response rela tionship in several of the studies reviewed,

whereby increasing levels of exposure were asso ciated with increasingl y large decrements in

measures of neurodevelopment (V . Rauh et al., 2011; V. A. Rauh et al., 2006). For the analyses

in which continuous distributions of exposure mark ers were used, the leve ls were often, but not

always, log transformed prior to entry in statistical models. Th e estimated association between

the biomarker level and the outcome in these cases is not linear; rather, the association between

the log-transformed exposure and the outcome is lin ear. In such cases, results are interpretable

# Chlorpyrifos Human Health Risk Assessment D424485

Page 247 of 531 as changes in mean level/risk of the outcome of interest for a given percentage increase in the

exposure. Examination of departures from lin earity of exposure-response relationships was

reported in only a small subset of the reviewed articles. For example, Ra uh et al. (2011) reported

that, in the Columbia cohort, “the dose-effect relationships between C PF [chlorpyrifos] exposure

and log-transformed Working Memory Index and Fu ll-Scale IQ scores are linear across the range

of exposures in the study popul ation, with no evidence for a th reshold” (V. Rauh et al., 2011).

Departures from linearity were not statistically significant. Two possible explanations for this

finding are that 1) the exposure resp onse is linear on the scale asse ssed, or that 2) the studies did

not have sufficient power to detect departures from linearity in the shape exposure-outcome

relationship. The smoothed expos ure-response curve superimposedupon the scatter plot of logtransformed working memory appears to be mono tonically decreasing; however, a similar curve

superimposed upon the scatter plot of log-transfor med IQ score and chlorp yrifos levels appears

flat if not slightly positive, before turning ne gative beyond an inflection point at about 5 pg/gram

of chlorpyrifos. As such, it may be that bot h explanations for the null finding regarding

departures from linearity are operati ng in this study. As another ex ample, Bouchard et al used

cubic splines to evaluate the shape of dose-res ponse curves, test the linearity assumption, and

investigate potential threshol ds (Bouchard et al., 2011). Agai n, no statistically significant

departures from linearity were observed.

Due to the voluntary cancellation by registrants of indoor reside ntial uses of chlorpyrifoscontaining pesticide products, ch ildren born after the year 2000 we re likely exposed to far lower

levels of chlorpyrifos, on average, than childre n born prior to 2001. In their report, Whyatt et al.

(2004) took advantage of this “n atural experiment” by stratifyi ng their analyses on birth date

prior to, or after, January 1, 2001. Although reported household use of pesticides in general did

not change over the same time period in this study , maternal chlorpyrifos in maternal air samples

were significantly lower after th e voluntary cancellation (4.9 ng/m 3 

# ), relative to those taken

before 2001 (8 ng/m 3 

# ; p<0001). Associations between birth outcomes and organophosphate

insecticide levels in maternal personal air samp les were not statistically significant in both unstratified analyses and among subgroups stratified by birth pr ior to or after January, 2001.

However, the associations between birth weight and le ngth and cord plasma chlorpyrifos were

highly significant (p ≤ 0.007) among newborns born before the 2001 cancellation of registrants.

Among newborns born after January 2001, no associat ion with fetal growth was observed (p >

0.8) (R. M. Whyatt et al., 2004). Supplemental anal ysis suggested this observation persisted

among additional number of infants born after th e period of the voluntary cancellation (n=193)

(R. Whyatt & Rauh, 2011) (Attachment 1). This fi nding by Whyatt et al provides evidence that

the association “can be altered (prevented or ameliorated) by an appropriate experimental

regimen,” as advocated by AB Hill in the assessment of causality in observational studies.

Changes in exposure were re fl ected in decreasing chlorpyrifos levels, and appear to have been

associated with changes in developmental ou tcomes. However, as noted by the 2008 FIFRA SAP

Panel in their report, this study was not designed to test this hypothesis, specifically.

It must also be considered that given the many selection criteria applied within each of the three

children’s health cohorts, the ge neralizability of study results may have been affected. These

studies reflect a somewhat na rrowly defined sub-population w ho likely experienced a greater

range of exposures and higher peak exposures th an the general U.S. population; a comparison of

maternal urinary concentration of TCPy in the three children’s health cohorts with the general

# Chlorpyrifos Human Health Risk Assessment D424485

Page 248 of 531 population (NHANES) illustrates higher exposures occurred among the cohort participants. The

exception is with the urinary c oncentration data collected by W hyatt et al. (2009) which were

lower than the U.S. general population; howev er, these data were collected 2001-2004, a period

after the voluntary cancellation of indoor residential uses of chlorpyrifos. In addition, the

external validity of the study findings may be limited if the organophosphate pesticide exposurefetal development association is modified by factor s that are more, or less, prevalent in the study

populations relative to the population( s) to which inference is bei ng made, or to populations with

a substantially different exposur e range. Generalizability may also be affected by low sample

size such as in the brain imagi ng (MRI) study. In the studies revi ewed, the exploration of geneby-environment, and phenotype-by-environment in teractions is a strength of many of the

analyses, although as noted above the studies ar e uniformly underpowered to assess such

interactions. There was limited assessment of interaction between ch lorpyrifos and other

environmental co-exposures conducted in these investigations includ ing blood lead levels.

4.0 Conclusions

The FIFRA SAP panel convened in September 2008 c oncluded that given the strengths of these

studies, the effect of biases likely present, a nd rigorous statistical an alyses performed, the

observation of associations acr oss these studies can be inte rpreted as a conclusion that

chlorpyrifos likely played a role in the ne urodevelopmental outcomes observed among children

more highly exposed to chlorpyrifos in the in utero environment. Since that time, researchers

with each of the three children’s health cohor ts have published several new etiologic and

methodological studies and performed supplementa l analyses as suggest ed by the 2008 Panel

which extend the knowledge base, and reduce severa l major sources of uncertainty present in the

2008 database ( i.e. , confounding by SES, exposure validati on in the Columbia Mothers and

Newborn study, further evaluation of PON1 genotype and PON activity level, pilot MRI imaging

study). In the current document, EPA utilized the draft “Framework for Incorporating

Epidemiology into Risk Assessment” 73 

# , including use of modified Bradford Hill considerations,

to assess the strengths and limitations of the studies in this database on the association of interest,

and to clarify the epidemiologic evidence in suppor t of a causal role for chlorpyrifos in infant

and child adverse neurodevelopmental outcomes.

In EPA’s analysis of the strengths and weaknesses of these studies, the chance that positive

associations observed are false positives due to systematic errors in the studies cannot be

excluded; however, it is more lik ely that error presen t in these studies would lead to the underestimation of the true association. Therefore, while alternative explanations for positive

association can be hypothesized ( e.g. , additional unmeasured or poorly measured positive

confounding variables), these explan ations are judged to be less pl ausible than the alternative

that associations have been missed or under-es timated due to non-differential measurement error

and low sample size across exposur e strata. In occupational setti ngs, exposure measurement error

has been shown to more greatly influen ce epidemiology study results than unknown or

unmeasured confounding variables (B lair et al., 2011). Additionally, the elimination of temporal

bias due to the prospective st udy design employed within each of the three children’s health

cohorts assures that prenatal exposures preceded neurodevelo pmental outcomes measured at

birth, and in early and later ch ildhood through age 7 years. The st rength of the associations

73 

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Page 249 of 531 measured in some studies was notably strong. Ho wever, associations in many instances were

weak to moderate, possibly due to measurement error. Associations w ith neurodevelopmental

outcomes were consistently identif ied with respect to the number of abnormal reflexes in the

neonatal period, the presence of mental and behavior al issues as well as gross motor delays were

pronounced especially in later t oddler years of 24-36 months, and the observation of intelligence

decrements were seen across the three cohorts usi ng different measures of prenatal chlorpyrifos

exposure, although not consistently statisti cally significant. EPA notes that other

organophosphates may be involved as well as the CHAMACOS and Mt. Sinai Child

Development study utilized dial kyl phosphates which measure e xposure to several different

organophosphates, and that use of chlorpyrifos in the CHAMACOS study region was low at the

time of the study. However, links with diethyl phospha te which result from either chlorpyrifos or

diazinon exposure were observed as well.

In summary, while the strengths and limitations of th e studies would be more likely to lead to an

under-estimation of the true effect, the possibility of false positive associati ons cannot be entirely

ruled out. The temporal associati on, strength of the sta tistical associations observed and presence

of exposure response across some (but not all) of the investigations, as well as some notable

consistencies in findings within this epidemiologic database tend to support a role for

chlorpyrifos in this relation. It is true that the Columbia Mo thers and Newborn study is the only

cohort to have measured chlorpyrifos parent co mpound directly; replication in other cohorts that

use this exposure metric would aid causal infe rence. The presence of factors which may have

over-estimated effects, the lack of consistency in many domains ( e.g. , fetal growth), and the lack

of a clear mechanism of action may argue agains t a true association. Overall, the current

database supports the conclusion from 2008 Panel th at “chlorpyrifos likely played a role in the

birth and developmental outcomes noted in th e three cohort studies.” Additionally, the

subsequent studies made available since the time of the 2008 evaluation helps to clarify the

potential role for chlorpyrifos in adve rse neurodevelopmental health effects.

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Health Perspect, 110( 5), 507-514.

Whyatt, R. M., Garfinkel, R., Hoepner, L. A., Andrews, H., Holmes, D., Williams, M. K., . . . Barr, D. B.

(2009). A biomarker validation study of prenatal chlorpyrifos exposure within an inner-city

cohort during pregnancy. E nviron Health Perspect, 117( 4), 559-567. doi: 10.1289/ehp.0800041

Whyatt, R. M., Garfinkel, R., Hoepner, L. A., Holmes , D., Borjas, M., Williams, M. K., . . . Camann, D.

E. (2007). Within- and between-home variability in indoor-air insecticide levels during pregnancy

among an inner-city cohort from New York City. E nviron Health Perspect, 115( 3), 383-389. doi:

10.1289/ehp.9546

Whyatt, R. M., Rauh, V., Barr, D. B., Camann, D. E., Andrews, H. F., Garfinkel, R., . . . Perera, F. P.

(2004). Prenatal insecticide exposures and birt h weight and length among an urban minority

cohort. E nviron Health Perspect, 112( 10), 1125-1132.

Wilson, N. K., Chuang, J. C., Lyu, C., Menton, R., & Morgan, M. K. (2003). Aggregate exposures of nine

preschool children to persistent organic pollutants at day care and at home. JExpo Anal Environ

Epidemiol, 13( 3), 187-202. doi: 7500270 [pii] 10.1038/sj.jea.7500270

# Chlorpyrifos Human Health Risk Assessment D424485

Page 252 of 531 Young, J. G., Eskenazi, B., Gladstone, E. A., Bradman, A., Pedersen, L., Johnson, C., . . . Holland, N. T.

(2005). Association between in utero organophos phate pesticide exposure and abnormal reflexes

in neonates. Ne urotoxicology, 26(2 ), 199-209. doi: 10.1016/j.neuro.2004.10.004

# Chlorpyrifos Human Health Risk Assessment D424485

Page 253 of 531 Appendix 2. Attachment 1

# Chlorpyrifos Human Health Risk Assessment D424485

Page 254 of 531 UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON, D.C. 20460

OFFICE OF CHEMICAL SAFETY AND

POLLUTION PREVENTION



# MEMORANDUM

Date: June 30, 2011

SUBJECT: Chlorpyrifos Correspondence with Co lumbia Researchers: (1) Responses to

Scientific Advisory Panel (SAP) comments (Whyatt and Rauh 2010), and

(2) Responses to Dow AgroSciences inquiries (Whyatt 2010).

PC Code: 059101 DP Barcode: D391257

Decision No.: NA Registration No.: NA

Petition No.: NA Regulatory Action: NA

Risk Assessment Type: Single Chemical Aggregate Case No.: NA

T XR No.: 0056066 CAS No.: NA

MRID No.: NA 40 CFR: NA

FROM: Deborah Smegal, MPH,

Immediate Office (IO)

Health Effects Division (7509P)

TO: Danette Drew, Risk Assessor

RABV

Health Effects Division (7509P)

Backrgound

On September 16-19, 2008, the Agency held a FIFR A Scientific Advisory Panel (SAP) meeting

(SAP 2008) to discuss the Agency's Evaluation of the Toxicity Profile of Chlorpyrifos, which

includes more recent and extensive research on various aspects of chlorpyrifos including its

neurological effects in animals and humans fo llowing gestational and post-natal exposures, its

pharmacokinetics, and mechanism of action. Deta ils can be found at the Scientific Advisory

Panel website ( http://www.epa.gov/scipoly/ sap/meetings/2008/091608_mtg.htm ), and in the

Chlorpyrifos Final SAP report

( http://www.epa.gov/scipoly/sap/mee tings/2008/september/sap0908report.pdf .)

Attached is a response to the SAP comments on the “Epidemiologic Appendix: Columbia Study”

that Deborah Smegal summarized and sent to R obin Whyatt and Virginia Rauh, dated 1/28/2010.

In addition, the Columbia researchers prepar ed responses to questions raised by Dow

AgroSciences.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 255 of 531 

# Columbia Response to the SAP Queries

2/19/2010

The following is a response to the Scie ntific Advisory Panel comments on the

“Epidemiologic Appendix: Columbia Study” that Deborah Smegal summarized and sent

to Robin Whyatt and Virginia Rauh, dated 1/28/2010.

1. SAP comment: EPA should continue its collaboration with Columbia researchers in

analyzing the epidemiologic data ...[and] sh ould seek clarification of the reported

exposure levels . . . EPA sh ould use the epidemiological cohort data for bounding

exposure levels and in conjunction with PB PK models, address current or potential

human exposures and to determine the fi nal RfD/RfC . . . PBPK models enable

estimation of exposure dose metric for mu ltiple sources (e.g., air, food, water etc).

EPA should pursue simpler PBPK model in short-term.

Response: We are happy to continue the collaboration. In addition, as described in our letter, Dr.

Dale Hattis at Clark University is conducting PB PK modeling of the Colu mbia data under a U.S.

EPA Star Grant. The grant is entitled “Use of Biomarkers and Physiologically Based

Pharmacokinetic [PBPK] Modeling in Risk Analysis for Developmental Effects of Chlorpyrifos”.

We believe that the PBPK model being develope d by Dr. Hattis will prove extremely helpful to

EPA in addressing the SAP comments and in cl arifying the exposure concentrations that

correspond to the chlorpyrifos levels in umbilical cord blood at which we be gin to see statistically

significant adverse effects on fetal growth and child neurocognitive development. Thus this data

should be helpful in bounding th e exposure data as recommended by the SAP. We recommend

that EPA contact Dr. Hattis.

2. SAP comment: Determine if the chlorpyrifos effect on birth weight would result in

more infants in “low birth weight” category.

Response: We are not able to addre ss this question as our cohort was predominantly full term by

design. This is because we did not conduc t our environmental monitoring until the 3 rd 

# trimester of

pregnancy and completion of the environmental m onitoring was a prerequisite for enrollment. For

example, of the subjects in our 2004 anal yses (Whyatt et al., EH P, 112:1125-1132, 2004), only

12/314 (3.8%) of the women deliver ed prior to 37 weeks gestati on and only 12/314 (3.8%) of the

infants were <2500 grams at birth. We , therefore, do not have adequate statistical power to evaluate

the relationship between prenatal chlor pyrifos exposure and low birth weight.

3. SAP comment: Ascertain whether there are st atistically significant differences in

neurodevelopmental outcomes at 3 yrs betw een chlorpyrifos levels in cord blood

below LOD and those with chlorpyrifos >6.1 7 pg/g . . . Verify/c larify there is no

suggestion of an effect as the most high ly exposed group and the group with values

below the LOD had lower MDI and PDI sc ores than did the middle levels.

Response: Table 1 below provide the same type of informa tion as Table 2 in the 2006 Pediatrics

paper (Rauh et al., Pediatrics, 118:e1845-e1859, 2006) with respect to the % of subjects whose

tests scores indicated developmental delay with re spect to the PDI and the MDI, as a function of

# Chlorpyrifos Human Health Risk Assessment D424485

Page 256 of 531 chlorpyrifos exposure. However, here we prov ide the % delayed for all four exposure groups,

rather than just the ‘high’ and ‘low’ exposure groups shown in the paper. For both the PDI and

MDI the rate of delay is clearl y highest in the high chlorpyrif os group (Group 4).For the 36 month

PDI, the rate of developmental delay is relative ly low in all 3 low-exposure groups, and is about

three times higher in the highe st exposure group. And for th e 36 month MDI, while the <LOD

group experienced a higher rate of delay than group 2 and 3, the very highest rate of delay is among

the most exposed children. In univariate analyses , the difference in % delay between the highest

exposure (>6.17pg/g) group versus the lowest expos ure (<LOD) group, was statistically significant

for PDI (chi-square p=.018, 1 sided; p=.031 two-si ded), but not significant for MDI. However,

importantly, there was no significant difference in the rate of delay between groups 1, 2 and 3 for

either the PDI (chi-squared= 0.97, p=0.61) or MD I (chi-squared= 4.4, p=0.11). And the rate of

developmental delay in terms of both PDI and MDI in group four was signifi cantly greater than in

groups 1-3 combined, as re ported in our paper.

Table 1. The proportion of children at age 36 month wh o were at risk of delay (score < 85) on the

Psychomotor Development Index (PDI) or Mental Development Index (MDI) of the Bayley Scales of Infant

Development by chlorpyrifos exposure group

Group 1

(<LOD) Group 2 (1 st 

# tertile >

LOD) Group 3 (2 nd 

# tertile >

LOD) Group 4 (3 rd 

# tertile >

LOD)

PDI 

# 7/80 (8.8%) 3/65 (4.6%) 3/38 (7.95) 11/45 (24.4%)

MDI 

# 30/80 (37.5%) 14/65 (21.5%) 11/39 (28.2%) 20/44 (45.5%)



# 4. SAP comment: Conduct an analysis of exposu re levels as dependent variable and year

(an ordinal variable) as the primary inde pendent variable, while adjusting for any

factors associated with both exposu re levels and year of study.

Response: We have already published on changes in indoor and personal air levels, as well as in

multiple biomarker levels by year of monitoring a nd have enclosed the most relevant articles (

Whyatt et al., 2007; Whyatt et al., 2009). In add ition, Dr. Hattis has also recently completed

analyses looking at changes in chlorpyrifos levels by year in the context of other factors such as

season of monitoring. As stated above, we recommend that EPA cont act Dr. Hattis to review his

results.

5. SAP comment: EPA should request additional details on the loss to follow-up in each

of the 4 exposure groups and provide additi onal details on how the exposure groups

were combined.

Response: Subjects were eligible for inclusion in the Pediatric paper if they had been born by

7//12/02 and thus had reached age three years by the time of the analys es and also had: (1) measures

of chlorpyrifos in blood at bi rth; (2) neurologic assessments at least once between ages 12-36

months and (3) data on model c ovairates. A total of 381 subjects in the cohort had been born by

7/12/02 and had birth chlorpyrifos blood measure; of these 254 ( 67%) met the above criteria for

inclusion, including 22 8 who had developmental outcomes at age 36 months. The remaining

subjects had either been lost to follow-up (accounting for approximately 14%) or were missing

outcome and/or covariate data. Ta ble 2 below shows the 254 subjects included in the analyses over

all 381 subjects by the chlorpyrifos groups. (The numbers per group just fo r the 228 subjects with

outcome data at 36 months are given in the Pedi atric manuscript.) Group 1 includes all subjects

# Chlorpyrifos Human Health Risk Assessment D424485

Page 257 of 531 with chlorpyrifos blood levels below LOD; the re maining subjects were ranke d into tertiles based

on ascending levels (Groups 2-4). However, as de scribe in the Pediatric manuscript, this grouping

was done on subjects born prior to 1/1/01 to be cons istent with our prior publication (see Whyatt

et al. 2004). This was essential in order to compare results acro ss analyses moving forward, given

that chlorpyrifos concentrations were changi ng rapidly following the ba n and thus we needed

consistent cutpoints. Groups 1-3 were combined and compared with group 4 for the analyses

presented in the Pediatrics manuscript.

Table 2. Subjects included in the Pediatric paper over all cohort subjects born at or before 7/12/02 who had

chlorpyrifos measured in cord blood at birth

Group 1 (<LOD) Group 2 (1 st 

# tertile > LOD) Group 3 (2 nd 

# tertile > LOD) Group 4 (3 rd 

# tertile > LOD)



# 88/121 (72%) 72/127 (56.7%) 44/63 (69.8%) 50/70 (71.4%)



# 6. SAP comment: Post ban declines in effects ar e based on 1 subject being classified as

highly exposed, which lacks statistical pow er and may be sensitive to the cutoff value

chosen to classify subjects as highly exposed. Can an anal ysis be conducted to look at

relationships between more equally sized ex posure groups that separate between the

lower exposure levels?

0 2 4 6 8 10

chlopyrifos 

# 1999 68/71 (96%)

2001 26/86 (30%) 2000 101/120 (84%)

2002 18/59 (31%) 

# Figure 1. Chlorpyrifos levels in maternal and umbilical cord

blood samples (pg/gram) at delivery by year of birth

2003 0/58 (0%)

2004 0/30 (0%)



# Cord blood (N=395) Year N > LOD (%)

0 2 4 6 8 10

chlopyrifos 

# Maternal blood (N=424) Year N > LOD (%)

1999 93/110 (85%)

2001 18/71 (25%) 2000 88/110 (80%)

2002 14/36 (39%)

2003 0/43 (0%)

2004 0/25 (0%)



# Response: We believe the SAP has misunderstood that our post ban declines were based not on

the change in the number of subjects that fe ll into the highly exposure group but on the decrease

we had seen in chlorpyr ifos concentrations in both mate rnal and cord bl ood among all cohort

subjects by year of birth (see Figur e 1). As can be seen in the a bove figure, chlorpyrifos levels

had dropped dramatically by 2001. Our 2004 study cate gorized the full cohort available at that

time (most born prior to1/1/01) into four groups ba sed on ascending levels of chlorpyrifos in

umbilical cord blood. The only significant differen ce we saw was between infants in the highest

exposure group (group 4) compared to those with le vels below the limit of detection (group 1). By

contrast, there was no significant difference in birth outcomes betw een subjects in the intermediate

# Chlorpyrifos Human Health Risk Assessment D424485

Page 258 of 531 exposure groups (2 or 3) compared to group 1 (see Whyatt et al. EHP, 112: 1125-1132. 2004). At

the time we conducted the analyses for our 2004 pa per, only n=77 infants we re born after 1/1/01,

which limited our power to determine whether or not there was any continuing effect of prenatal

chlorpyrifos exposure on birth outcomes afte r2001. In April 23, 2008, we presented undated

analyses to EPA that included more infants (n=1 93) born after 1/1/01. Results are shown in Table

3 below and are consistent w ith our 2004 publication. Specifica lly, we saw a highly significant

decrease in birth weight for each l og unit increase in umbilical cord

Table 3

. Change in birth weight for each log unit increase

inchlorpyrifos concentrations in umbilical cord plasma among

babies born before and after 1/1/01

B (95% CI) P-value

Born before 1/1/01 (n=233) -71/4 (-121/1, -21.8 P=0.005

Born after 1/1/01 (n=193) 32.7 (-59.3, 124.7) P=0.48

chlorpyrifos concentrations among babies bor n before 1/1/01, but no association between

chlorpyrifos and birth outcomes among infants bor n after 1/1/01. Of those infants born after 1/1/01,

only 16% had chlorpyrifos levels in cord blood above the LOD and only one of these was in the

previously defined ‘high exposure’ group. It is obvi ous that this marked reduction in chlorpyrifos

exposure, and the virtual eliminati on of high exposure leve ls after 1/1/01, is due to the chlorpyrifos

ban , . To address the SAP comments further, we have conducted analyses that categorize infants

born after 1/1/01 into those withchlorpyrifos above and below the LOD. Keeping in mind that

there was only one child with hi gh (group 4) chlorpyrif os exposure in this post-ban sample, it is

not surprising that there was no significant diffe rence in birth weight between the <lod group and

the small group with some exposure during this post-ban period. These findings thus support the

hypothesis that chlorpyrifos exposures following the ban had dropped below concentrations that

have adverse effects on fetal growth. However, it is also possible that our sample size is simply

too small to detect relatively small but real negati ve effects on birth outcomes associated with the

much reduced exposure levels after 1/1/01.

7. SAP comment: The panel felt it might be help ful to have a longitudinal component to the

study to determine if the adv erse effect persisted in child ren who exhibited poor birth

outcomes or neurobehavioral deficits.

Response: As described in the attached letter, we ha ve just completed statistical analyses on the

relationship between prenatal ch lorpyrifos exposure a nd outcomes on the Wechsler Intelligence

Scale for Children (Fourth Edition) and the Ch ild Behavior Check List, which have been

administered to cohort children at age 7 years. We anticipate that a ma nuscript on these findings

will be completed and ready for submission within several months.

8. SAP comment: It would be useful to examine the results of a statis tical analysis that

includes all three AChE-inhibiting insecticides in the analysis model as dichotomous

variables (above or below the LOD) in combin ation with continuous measurements for these

variables. This is not likely to change the results but could be helpful in illustrating threshold

or dose response effects . . . Are all 3 AChE i nhibiting insecticides correlated (if lack of

correlation—less likely would confound cyp and neuro outcomes) . . . Conduct additional

# Chlorpyrifos Human Health Risk Assessment D424485

Page 259 of 531 analysis controlling for multiple exposures examin ing additive effects to elucidate the role of

other Ops. This is important for AChEI to be considered as mode of action.

Response : It should be noted that our 2004 paper (Whyatt et al., EHP, 112: 1125-1132) looked at

the additive effects of chlorpyr ifos and diazinon on birth outcomes using the U. S. EPA guidelines

for conducting cumulative risk assessments for the organophosphates. However, in response to the

SAP comments, we have now conducted a number of additional analyses both to evaluate the

correlations among the three AChE inhibiting in secticides (chlorpyrif os, diazinon and propoxur)

detected in our cohort and to l ook at the effects of chlorpyrifos on both birth outcomes and child

neurocognitive development after controlling for diazinon and propoxur. Specifically we have

devloped 4 additional models for birth weight an d birth length and 4 models for the psychomotor

development index (PDI) and mental development index (MDI) on the Bayley Scales of Infant

Development at age 36 months. All models controlled for the same covariates as in our 2004 and

2006 publications. Model 1 includes only chlorpyrifos as the independent variable (i.e. is identical

to the data in our initial publi cations); model 2 includes chlo rpyrifos and diazinon; model 3

includes chlorpyrifos and 2-isopropoxyphenol (the chemical-specific metabolite of propoxur); and

model 4 includes all three acetylc holinesterase inhibiting insecticidess Results are presented

below:

Correlations among pesticides : For infants born prior to the chlor pyrifos phase out (the relevant

time period), there was a modest correlation betw een 2-isopropoxyphenol (the chemical specific

metabolite of propoxur) and both chlorpyrifos (r=0.25, p<0.001, n=222) and diazinon (r=0.21,

p=0.002, p=227) in umbilical cord blood samples. However, there was a higher correlation

between chlorpyrifos and diazinon (r=0.63, p<0.0001, n=222), incdicating that approximately 33%

shared variabilitybetween these two pesticides. Gi ven this relationship be tween chlorpyrifos and

diazinon, the question raised by the SAP—whether the chlorpyrifos e ffects we report could be due

to confounding by diazinon—is therefore very appropria te. This question is addressed in the new

analyses reported below.

Results on birth outcomes. Results are presented in Tables 4 and 5 below. As can be seen,

chlorpyrifos remained inversely associated with birth weight and length in all models after

controlling for diazinon and 2-is opropoxyphenol, and the magnitude of the significant negative

effect of chlorpyrifos on infant size remained re latively constant across th e models, indicating that

neither of these two other acetylcholinestera se inhibitors was c onfounding the relationship

between chlorpyrifos and birt h outcomes. Further neither di azinon nor 2-isopropoxyphenol, either

individually or in combination, was associated with birth weight , although the inverse association

reported previously between 2-isopropoxyphenol and birth length (see Whyatt et al., 2004)

remains of borderline significant (p=0.05). Consis tent with our prior findings, birth weight

decrease significantly by 198 grams among infants in the highest compared to the lowest

chlorpyrifos groups (Group 4 versus Group 1) after controlling for both diazinon and 2-

isopropoxyphenol, as well as the other model covari ates (p<0.05). These analyses thus indicate

that it is primarily prenatal chlorpyrifos and not the other two acetylcholinesterase-inhibiting

insecticides that is having an adverse effect on fetal growth. Further, the findings suggest that the

effect of chlorpyrifos on fe tal growth may well be mediat ed by mechanisms other than

acetylcholinesterase inhibition, given the lack of an association between the other two

acetylchlolinesterase inhibitors and birth weight.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 260 of 531 Table 4. Decrease in birth weight fo r each log-unit increase in insecticide levels in umbilical cord blood at

birth among infants born prior to 1/01/01. Values shown are regression coeffi cient (B), 95% confidence

interval, and p-value

Model 1 Model 2 Model 3 Model 4

(ln)Chlorpyrifos 

# -67.3 (-17.8, -

116.8)

p=0.008 -65.9 (-5.7, -

126)

p=0.03 -60.9 (-10.0, -

111.8)

p=0.02 -60.2 (0.9, -121.3)

p=0.054

(ln)Diazinon 

# -4.7 (107.7, -

117.1)

p=0.94 -2.5 (110, -114)

p=0.96

(ln)2-isopropoxyphenol 

# -50.9 (46.1, -

147.8)

p=0.30 -50.8 (46.5, -114.9)

p=0.30

All models controlled for gestational age, parity, maternal pre-pregnancy weight and net weight gain during

pregnancy, maternal self-reported environmental tobacco smoke in the home, gender of the newborn and

season of delivery.

Table 5. Decrease in birth length for each log-unit increase in insecticide levels in umbilical cord blood at

birth among infants born prior to 1/01/01. Values shown are regression coeffi cient (B), 95% confidence

interval, and p-value

Model 1 Model 2 Model 3 Model 4

(ln)Chlorpyrifos 

# -0.43 (-0.14,-

0.73)

p=0.004 -0.46 (-0.1, -

0.82)

p=0.01 -0.36 (-0.06,, -

0.66)

p=0.02 -0.40 (-0.75, -

0.35)

p=0.03

(ln)Diazinon 

# 0.09 (0.76, -

0.58)

p=0.80 0.12 (-0.78, 055)

p=0.73

(ln)2-isopropoxyphenol 

# -0.57 (0.002, -

1.14)

p=0.051 -0.58 (-.001, -

1.15)

p=0.05

All models controlled for gestational age, parity, maternal pre-pregnancy weight and net weight gain during

pregnancy, maternal self-reported environmental tobacco smoke in the home, gender of the newborn and

season of delivery



# .

Results on PDI and MDI are presented in Table 6 and 7 below. The findings were quite

surprising. There was no significant associati on between 2-isopropoxyphenol and delay on either

the PDI or MDI. However, when diazinon was a dded to the model, the negative effect of

chlorpyrifos on developmental delay for bot h the PDI and MDI became much stronger.

Specifically, the odds ratio almost doubled for PD I and increased by 40%-60% for the MDI. These

results again indicate that it is chlorpyrifos a nd not the other two acetylcholinesterase insecticides

that is having an adverse effect on child mental and motor development and further that the effect

size appears considerably greater than we had estimated before. The findings again support the

suggestion, as discussed by the SAP, and as is s uggested by the experimental evidence, that the

effects of chlorpyrofos on child development may well be by a mode of action other than

acetylcholinesterase inhibition.

Table 6. Effects of chorpyrifos on the odds of ps;ychomotor delay on the Bayley Scales of Infant Development

at age 36 month. Values shown are regression coefficient (B), 95% confidence interval, and p-value s

Model 1 Model 2 Model 3 Model 4

Chlorpyrifos 2 

# 4.5 (1.6, 12.7) 8.4 (2.4, 30.2) 4. 0 (1.4, 11.8) 7. 9 (2.1, 29.1 )

# Chlorpyrifos Human Health Risk Assessment D424485

Page 261 of 531 p=0.004 p=0.001 p=0.01 p=0.002

(ln)Diazinon 

# 0.45 (0.18, 1.13)

p = 0.09 0.4 (0.2, 1.1)

p=0.08

(ln)2-isopropoxyphenol 

# 1.3 (0.65, 2.8)

p=0.4 1.4 (0.67, 3.0)

p=0.37

All models controlled for race/ethni cally, gender, ETS, gestational age, maternal IQ, maternal education,

HOME score 1 

# OR = Odds ratio

2 

# >6.17 pg/g versus ≤ 6.17 pg/g in umbilical cord blood

Table 7. Effects of chorpyrifos on the odds of mental delay on the Bayley Scales of Infant Development at

age 36 months. Values shown are regression coe fficient (B), 95% confidence interval, and p-value

Model 1 Model 2 Model 3 Model 4

Chlorpyrifos 2 

# 2.3 (1.1, 5.2)

p=0.03 3.8 (1.5, 9.4)

p=0.004 2.0 (0.9, 4.5)

p=0.11 3.2 (1.3, 8.2 )

p=0.02

(ln)Diazinon 

# 0.57 (0.35, 0.95)

p = 0.03 0.6 (0.3, 0.9)

p=0.03

(ln)2-isopropoxyphenol 

# 1.5 (0.9, 2.5)

p=0.12 1.5 (0.9, 2.6)

p=0.11

All models controlled for race/ethni cally, gender, ETS, gestational age, maternal IQ, maternal education,

HOME score 1 

# OR = Odds ratio

2 

# >6.17 pg/g versus ≤ 6.17 pg/g in umbilical cord blood



# 9. SAP comment: Is it possible to determine quantitativ e contribution of chlorpyrifos in

exposure to mixture by accounting for differen ces in chemicals, potency, pharmacokinetics

and possible chemical interactions

.

Response: Dr. Hattis can address this question after his PK model is implemented and calibrated.

10. SAP comment: Look at interm ediate markers of biologically effective dose and early

effect that further link exposure and outcome and or a clearer idea of mode of action from

exposure studies.

Response: As described in our letter, we have been conducting Magnetic Resonance Imaging

(MRI) scans on cohort children with either high prenatal chlorpyr ifos exposure (n=20) or no

exposure to any of the contaminants that have been associated with adverse child cognitive

development among the CCCEH cohort (n=20). The MRI results will provide a marker of biologic

effect. These analyses are being undertaken under the direction of Dr. Bradley Peterson, Director

of Child and Adolescent Psychiatry at the New York State Psychiatric Institute. We anticipate that

a manuscript on the results will be submitted to a peer-reviewed journal by Aug 1 2010.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 262 of 531 

# COLUMBIA CENTER

F OR C HI LD REN ' S

EN VI RON M E NT A L

HE AL TH

MAILMAN SCHOOL OF PUBLIC HEALTH

Co lum b i a U niver s i t y



# Memo to: Deborah Smegal

From: Robin Whyatt

Date: March 8, 2010

We are pleased that our answers to the questions raised by the Science Advisory Panel (SAP)

will be of value to EPA as you move forward to update the chlorpyrifos risk assessment. Below

are our responses to the questions raised by Dow Agrosciences which you also asked us to

address in your email of March 5,2010. Pl ease let us know if you need any additional

information or if we can be of more help.

Question one: The Columbia birth cohort includes cord blood chlorpyrifos levels for most

children and urine from many mothers (Whyatt et aL. 2005,2009). However, the authors state

that "we found no association between chlorpyrifos levels in maternal and cord blood and TCPy

levels in maternal urine samples during pregnancy or after delivery." (Whyatt et aL. 2009, pg

563). Since the health effect papers are based solely upon chlorpyrifos levels in blood, and

because blood chlorpyrifos measurements have not been reported in any other human exposure

study at these trace-level concentrations, this is a critical point to evaluate with full transparency.

Specific questions include:

a. Why is there no association between blood chlorpyrifos and urinary TCPy?

b. How many mother infant pairs in 1999 have both urine TCPy and blood chlorpyrifos?

c. Are the 1999 urine and blood levels correlated?

d. Are the 2000 urine and blood levels correlated?

e. Are the 2001 urine and blood levels correlated?

f. With personal monitoring, air monitoring, blood chlorpyrifos and urinaryTCPy, what is the

estimated internal dose for the infant-mother pairs in each year of measurement?

Answer: Please note that the primary biomarker we have measured in our cohort is chlorpyrifos

concentrations in maternal and umbilical cord blood. We did not measure TCPy in maternal

urine samples except for a small subset of subj ects. Specifically, as described in our 2009

manuscript (Whyatt et aI., EHP, 117;559-567, 2009), we measured TCPy in repeat urine

samples collected from only n=97 women during pregnancy. These urine samples were collected

among women who delivered between 2001-2004. Thus the answer to la through 1d above is

that no mother/infant pairs have both chlorpyrifos in blood and TCPy in urine in either 1999 or

# Chlorpyrifos Human Health Risk Assessment D424485

Page 263 of 531 2000 and we thus do not have the data to look at the correlation between TCPy in urine and

chlorpyrifos in blood among subjects who delivered prior to 2001. For subjects who delivered in

2001, we can look at this relationship. It should be noted, however, that these data are not

particularly helpful in evaluating the correlati ons between urinary TCPY and blood chlorpyrifos

concentrations, due to small sample size. But more importantly, chlorpyrifos in blood samples

had already dropped substantially by 2001 following the residential ban. Specifically, by 2001

among subjects that had both TCPy measured in maternal urine and chlorpyrifos measured in

blood, chlorpyrifos was only detected in 24% of maternal blood samples and 19% of umbilical

cord blood samples (See Figure 2, 

# Whyatt et at., 2009). By contrast, as shown in Figure 1 

# from

in the Columbia Response to the SAP Queries (2/19/2010), chlorpyrifos was detected in 98% and

85% of maternal and umbilical cord blood samples, respectively, during 1999 and 84% and 80%

during 2000. Given these caveats, the results for 2001 are as follows: For these subjects, the

average chlorpyrifos concentrations in two-week integrated indoor air samples collected over



# the final 6-8 weeks of pregnancy were positiv ely correlated with average TCPy levels in

maternal prenatal spot urine samples (r=0.5, p=0. 005). However, there was no correlation

(p>0.2) between TCPy levels and chlorpyrifos in either maternal blood samples (n=29) or cord

blood samples (n=21) at delivery. Maternal and umbilical cord blood levels were highly

correlated (r=0.85, p<0.001, n=21).

Regarding question "If" above, please note, that as discussed in our answers to the SAP

querries, Dr. Dale Hattis at Clark University is conducting PBPK modeling of the Columbia

data under a Us. EPA Star Grant. The grant is en titled “Use of Biomarkers and Physiologically

Based Pharmacokinetic [PBPK] Modeling in Risk Analysis for Develop mental Effects of

Chlorpyrifos ". We believe that the PBPK model being developed by Dr. Hattis will prove helpful

in clarifying the exposure concentrations that correspond to the chlorpyrifos levels in umbilical

cord blood at which we begin to see statistically significant adverse effects on fetal growth and

child neurocognitive development and also in estima ting the internal dose for the infant/mother

pairs in each year of measurement. Dr. Hattis es timates that his analyses should be completed

relatively shortly. We recommend that you contract Dr. Hattis.

Question Two: In the Rauh et al. (2006) paper the authors note a nonlinear dose trend with using

4 exposure groups. The final analyses are limited to two exposure groups. An analysis of 4

exposure groups is more information from a risk perspective and is critical for full transparency

of the data. Analysis with 4 exposure groups for the results presented in Tables 2-8 should be

replicated and made publically available.

Answer: We have already addressed this comment in our answer 

# # 3 from the Columbia

Response to the SAP Queries (2/19/2010). To reiterate, we prepared a new table (Table 1 below)

which provided the same type of information as Table 2 in the 2006 Pediatrics paper (Rauh et

at., Pediatrics, 11 8:el 845..;el 859, 2006) with respect to the 

# % of subjects whose tests scores

indicated developmental delay with respect to th e PDI and the MDI, as a function of chlorpyrifos

exposure. However, we provided the % 

# delayed for all four exposure groups, rather than just

the 'high' and 'low' exposure groups shown in the paper. For both the PDI and MDI the rate of

delay is clearly highest in the high chlorpyrifos group (Group 4). For the 36 month PDI, the rate

of developmental delay is relatively low in all 3 low-exposure groups, and is about three times



# higher in the highest exposure group. And for the 36 month MDI, while the <LOD group

experienced a higher rate of delay 

# than 2 and 3

, the very highest rate of delay is among

the most exposed children. In univariate analyses, the difference in 

# % delay between highest

# Chlorpyrifos Human Health Risk Assessment D424485

Page 264 of 531 exposure (>6.17pg/g) group versus the lowest exposur e group (<LOD) was statistically significant

for PDI (chi-square p=0.018, 1 sided; p-0.031 two-sided), but not significant for MDI

However, importantly, was no significant difference in the rate of delay between groups 1



# , 2 and 

# 3 for either the PDI (chi-squared=0.97, p=0. 

# 61) or MDI (chi-squared= 

# 4.4, p=0.11).

And the rate of developmental delay in terms of both PDI and MDI in group four was significantly

greater than in groups 1 -3 combined, as reported in our paper.

Table 1. The proportion of children at age 36 month wh o were at risk of delay (score < 85) on the

Psychomotor Development Index (PDI) or Mental Development Index (MDI) of the Bayley Scales of Infant

Development by chlorpyrifos exposure group

Group 1

(<LOD) Group 2 (1 st 

# tertile >

LOD) Group 3 (2 nd 

# tertile >

LOD) Group 4 (3 rd 

# tertile >

LOD)

PDI 

# 7/80 (8.8%) 3/65 (4.6%) 3/38 (7.95) 11/45 (24.4%)

MDI 

# 30/80 (37.5%) 14/65 (21.5%) 11/39 (28.2%) 20/44 (45.5%)



# Question Three: In 

# the Rauh et al. (2006) 

# paper, the authors compar e results of the Bayley

Mental Development Index (MDI) and Bayley Psychomotor Development (PDI) in different

years. Using significance testing only, they claim there is a “preban” diffe rence and no "postban"

difference. This natural experiment is impor tant to control for the many confounding factors

associated with the home environment of these children. A more comprehensive and transparent

analysis of these data over would provide info rmation about role of ch lorpyrifos and MDI and

PDI this unique population.

Answer: Please note that most cohort children in the 2006 paper by Rauh et at. were born prior

to the residential ban on chlorpyrifos. Specifically , as described in our answers to SAP queries,

subjects were eligible for in clusion in the Pediatric paper if they had been born by 7/12/02 and

thus had reached age three by the time of the analyses and also had: (1) measures of

chlorpyrifos in blood at birth; (2) neurologic assessments at least once between ages 12-36

months and (3) data on model covariates. Therefore, we were not able to undertake statistical

analyses relating the changes in chlorpyrifos concentrations to changes in MDI and PDI

following the residential ban. However, authors conc urred that this is an important research

question and state that “large r epidemiologic studies will to assess the full impact of

the ban (see page e1854 of the Pediatric paper)." As discussed in our February 17 th 

# letter to

Dr. Jack Fowle, we are currently preparing a manuscript on the relationship between prenatal

chlorpyrifos exposure and outcomes on the Weschler Intelligence Scale for Children (Fourth

Edition) and the Child Behavior Check List, which have been administered to cohort children at

age 

# 7 years. However, we also plan to undertake additional analyses of the 36 th 

# month MDI and

PDI in relationship to prenatal chlorpyrifos exposures following the ban, since many more

children in our cohort have now completed the 3 year developmental assessments and we

have the sample size to undertake the analysis outlined above, while controlling for the

many potential confounders.

Question Four: The exposure estimates in the Columb ia and CHAMACOS studies are reported

differently, thus preventing a meta-analysis from being undertaken. Both studies reported Bayley

MDI and Bayley PDI results at 24 months and neither was statistically significant (Eskenazi et al.

2007). This can be interpreted as a tendency but underpowered, or as a chance finding. Obtaining the

# Chlorpyrifos Human Health Risk Assessment D424485

Page 265 of 531 raw data and pooling the results should be undertaken and has several benefits.

a. The exposure estimates can be unified (either by calculating an estimated dose or

estimating a TCP level from the blood data). The dose cut points can be set to at least 3

doses to determine the presence of a dose response relationship.

b. The power will doubled by combining the two cohorts, thereby increasing the statistical

power.



# Answer: As discussed in our Februar y 17th letter to Jack Fowle, a multi-site study has been

funded the National Institute of Environmen tal Health Sciences ($568,000 total costs)

that will pool the children's centers at the University of California-Berkeley,

Columbia University (our children’s center) and the Mount School of Medicine to address

questions about relationships across the three cohorts between prenatal organophosphate

exposure, fetal growth and child neurocognitive development. Dr. Brenda Eskenazi at Berkeley

is the principal inve stigator on project.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 266 of 531 

# Appendix 3. Epidemiology Study Specific Evaluations

APPENDIX 3

Study Specific Evaluations of Child ren’s Health Epidemiology Studies



# Article 1. Whyatt et al. (2004)

Prenatal Insecticide Exposures and Birth We ight and Length among an Urban Minority

Cohort. Robin M. Whyatt, Virginia Rauh, Da na B. Barr, David E. Camann, Howard F.

Andrews, Robin Garfinkel, Lori A. Hoepner, Diurka Diaz, Jessica Dietrich, Andria Reyes,

Deliang Tang, Patrick L. Kinney, and Fred erica P. Perera. Environmental Health

Perspectives. 112:1125–1132 (2004).

Study Summary

In this report, Whyatt et al. report on the relationship between pe sticide exposures and

developmental outcomes in newborns in the Co lumbia Mother’s and Newborn Study to include

additional insecticides (the organophosphate diazinon and the carbamate propoxur), a larger

sample size (n = 314 mother–newborn pairs), and in secticide measurements in maternal personal

air sampled in the home, as well as in um bilical cord plasma sampled at delivery.

Study participants were recru ited during early pregnancy ( ≤ 20th week) among African-American

and Dominican women age 18-35 y ears, and registered at Ne w York Presbyterian Medical

Center and Harlem City. Wo men who smoked, had a history of drug abuse, diabetes,

hypertension, or HIV infection were excluded fro m participation in the study, as were women

who resided in New York City for less than 1 year . The study sample presented in this report was

recruited between 1998 and 2002, a period whic h straddles the December, 2000 voluntary

cancellation by registrants of indoor residential chlor pyrifos use.

Insecticides, including chlorpyrifos, were meas ured in 48-hr personal air samples collected

during the third trimester of pre gnancy. Analyses of air samples fo r pesticide levels were carried

out at the Southwest Research Institute.

Chlorpyrifos levels in umbilical cord blood sa mples were available for 256 newborns, sampled

as close to the time of delivery as possible, a nd within 2 days post-partum. Cord blood plasma

chlorpyrifos levels were imputed from maternal blood levels for 31 newborns for whom no cord

blood sample was obtained. Quantification of chlo rpyrifos levels in plasma were conducted by

the Centers for Disease Control and Prevention (CDC).

Insecticide levels in both maternal personal air and blood samples were av ailable for 259 of the

314 mother–newborn pairs (82%). Measures in either personal air or blood were available for the

remaining 55 pairs (18%). Pesticide levels in personal air and blood samples below the level of

detection (LOD) were assigned a value of half the LOD; levels were then log 2 

# -transformed for

statistical analysis. For pesticid es in blood, 31% of chlorpyrifos levels were below the LOD.

Infants with below LOD cord blood chlorpyrifos levels were used as a comparison group and

compared to groups with increasing levels, ca tegorized by tertile of above LOD levels.

Infants’ weight, length, and head circumference were assessed at birth and obtained for this study

# Chlorpyrifos Human Health Risk Assessment D424485

Page 267 of 531 through medical record abstraction.

Multiple linear regression analyses were performe d to evaluate associations between prenatal

insecticide exposure and the indicat ors of infant development at bi rth. Covariates were selected

from variables known to be associated with insecticide exposure or fetal growth. Effect

modification by race/ethnicity wa s evaluated, but not observed.

Controlling for potential conf ounders, the researchers found no a ssociation between maternal

personal air insecticide levels and birth we ight, length. Head circumference was not

significantly associated with mate rnal personal air or cord blood chlorpyrifos levels. However,

for each log unit increase in co rd plasma chlorpyrifos levels , birth weight decreased by 42.6 g

(95% CI: –81.8 to –3.8) and birth length decreased by 0.24 cm (95% CI: –0.47 to –0.01).

Combined measures of (ln)cord plasma chlorpyrifos and diazinon (adjusted for relative potency)

were also inversely associated with birth weight a nd length (p < 0.05). Birth weight averaged

186.3 g less (95% CI: –375.2 to –45.5) among newborns with the highest compared with lowest

26% of exposure levels (combined p-value = 0.01). When stratified by date of birth prior to or

after 2001 (before and after the voluntary can cellation by registrants of indoor residential

chlorpyrifos use was implemented), associatio ns between birth weight and length and cord

plasma chlorpyrifos were statistically significant (p ≤ 0.007) among newborns born before the

cancellation. Among newborns born after January 2001, exposure levels were substantially

lower, and no associations with fetal growth outcomes were observed (p > 0.8). Results suggest

that prenatal chlorpyrifos exposures may have impaired fetal growth among this inner-city, low

income cohort.

Study Review

This was a very well conducted study with numer ous strengths, and some noted limitations. The

prospective nature of the study limits the infl uence of systematic e rrors in the exposure

assessment on the association observed, such th at mismeasurement of chlorpyrifos exposure

would likely be non-differential with respec t to the indicators of fetal growth.

A notable strength of this study, relative to inve stigations in other c ohorts, is the direct

measurement of chlorpyrifos in cord blood and personal air samples, rather than non-specific

markers of organophosphate pesticid e exposure. Errors in estimati on of cord blood chlorpyrifos

levels were likely to have been non-differen tial with respect to th e indicators of fetal

development. However, authors did not formally assess the variability in the exposure estimate

resulting from the imputation of missi ng cord blood chlorpyrifos levels.

A limitation of the specific biomarker exposure in dicators in this study is the single sampling

period (at delivery). It is not cl ear to what extent this single measurement reflects exposures over

critical windows of fetal growth and development during pregnancy. If the exposure is chronic, a

biomarker measured at a single time point can pr ovide a representative dosimeter, even if the

toxicant has a short half-l ife, as is the case for chlorpyrifos. However, if pesticide exposures are

sporadic or otherwise vary over short time scales, the biomarker measurement will not be

representative of “usual” expos ure, or of the exposure durin g critical periods of fetal

development. An observation effect could also influence the validity of the 48-hour personal air

# Chlorpyrifos Human Health Risk Assessment D424485

Page 268 of 531 sampling if, for example, the ac t of wearing the dosimeter inhibited participants from using

pesticides or otherwise altered th eir behavior patterns in a manner that affected exposure levels.

These limitations are assessed in subsequent va lidation studies conducte d within this cohort

(Whyatt, 2007 and Whyatt, 2009).

The health outcomes in this study, birth weig ht, birth length, and head circumference were

obtained through direct linkage w ith the mothers’ and infants’ computerized medical records,

and were likely to have been accurately measur ed. Any errors in outcome measurement would

have occurred in the clinic, and are likely to be small and likely to be non-differential with

respect to pesticide exposure.

The investigators collect ed data on, and adjusted for, impor tant potential conf ounders. Covariate

selection proceeded appropriately by assessi ng relationships between potential confounders and

the indicators of fetal growth, change-in-estimate of the main effect of chlorpyrifos exposure,

and assessments of model fit. Covariates included in final models were race/ethnicity,

gestational age, parity, maternal pre-pregnanc y weight, and net weight gain during pregnancy,

maternal self-reported environmental tobacco sm oke in the home, sex of the newborn, and

season of delivery. Models for head circumference also included an indicator for delivery type

(vaginal versus cesarean section).

Maternal exposure to polycyclic aromatic hydrocarbons (PAHs) during pregnancy was not

included in the final models because it did not im prove model fit. However, to ensure that PAH

exposure did not confound the pesticide associations with fetal development, models including

PAHs were evaluated. The authors’ considerat ion of other pesticid es and environmental

contaminants as covariates in their multivariate models is a notable strength of this study.

Confounding by maternal smoking behavior s was controlled by restriction ( i.e., excluding

smokers from the study sample). Confounding by un measured factors may have been similarly

controlled by design, this being a relativel y homogeneous population with respect to

determinants of fetal growth, compared to the general population. For example, the authors

found it unnecessary to adjust for maternal alcoho l consumption (which was measured), partly

because few women in the study drank alcohol during pregnancy.

Bias due to confounding by unmeasured determinan ts of fetal growth is possible in this

observational study, for example, confounding by f actors related to socioeconomic condition.

Annual household income, maternal education, matern al marital status, material hardship during

pregnancy, and degree of housing disrepair were evaluated as possible markers for

socioeconomic status but were not included in fi nal models because they were not associated

with birth outcomes, did not improve the model fit, and did not substa ntially alter parameter

estimates, confidence intervals (CIs), or significa nce levels of variable s included in the final

models. This observation that i ndicators of socioeconomic cond ition were not associated with

the health outcomes is somewhat unexpecte d, although perhaps explainable by the relative

homogeneity of these factors in this study population (contro l through restriction of study

population).

In categorical analyses, infants with the lowest tertile of obser vable cord blood levels had, as a

# Chlorpyrifos Human Health Risk Assessment D424485

Page 269 of 531 group, larger heights and weights at birth, compared to infants with chlorpyrifos levels below the

level of detection, although the increases were not statistic ally significant. Among those with

higher cord blood levels, increasing tertiles of chlorpyrifos were significantly a ssociated with

decreasing birth weight and height. This is sugg estive of a threshold effect (a hockey stick doseresponse curve). Departures from linearity in the exposure-response relationship were not

assessed in models including (ln)chlorpyrifos entered as continuous variables.

The authors observed an association between ch lorpyrifos and fetal growth only when the

biomarkers were used as indicators of prenatal exposure and not when environmental levels (in

maternal 48-hr personal air sampling conducte d during the third trimester) were used.

Researchers note several possible explanations for this discrepancy, includ ing the notion that the

biomarkers are integrating dosimeters of exposur e. This may be particularly important for

insecticide exposure by routes othe r than inhalation in the home ( e.g. , dermal, ingestion, and

occupational exposures). In addition, the author s speculate that the biomarkers may provide

better dosimeters of the dose to the developing fetus, relative to the airborne exposures.

The voluntary cancellation by registrants of indo or residential chlor pyrifos use introduces

exogenous variation in the exposure of interest. No associations were obs erved between the fetal

growth indicators and chlorpyrifos levels am ong infants born after the implementation of the

voluntary cancellation of chlorpyrifos containing in home pesticide products. This finding is

similarly suggestive of a possible threshold eff ect (decreasing dose-levels before and after

voluntary cancellation), although it c ould also be due to a lack of statistical power to assess

associations at lower levels of exposure in the relatively small subsample (n=77).

The statistical analysis appeared to be conventional and appropr iate. However, the authors did

not present nor discuss regression diagnostics to assess the degree to which their models met or

violated the assumptions implicit in linear models ( i.e. homoscedasticity, normality of error term

distribution, independence of erro r terms, and linearity of the exposure-response relationship),

other than to state that inve stigators log transformed chlor pyrifos levels. As discussed

previously, there is some evidence in the da ta to suggest a non-lin ear exposure response

relationship ( i.e. , threshold) between the na tural log of cord blood chlo rpyrifos levels and birth

weight and length, but no formal st atistical testing of this possibil ity was presented or discussed.

Bias due to differential errors in the selection of study par ticipants is unlikely to have occurred in

this prospective study with low loss to follow-up. Appropriate exclusion criteria were applied,

although these design decisions may reduce extern al validity (generalizability) when making

inference to other populations us ing the associations observed in this narrowly defined study

population.

The results presented in this expanded analysis c onfirm the authors’ earlier findings of an inverse

association between chlorpyrifos levels in umbilic al cord plasma and birth weight and length in

this study population of non-smoking African -American and Dominican women, age 18-35

years, living in New York City (Whyatt et al., 2003). Systematic errors in the selection of study

participants or in the measurem ent of key analytic variables (exposure and outcome) are unlikely

to explain this observed association. The po ssibility of unmeasured or poorly measured

potentially confounding variables cannot be completely excluded, although study authors

# Chlorpyrifos Human Health Risk Assessment D424485

Page 270 of 531 thoroughly evaluated this possibility in the data av ailable. Data suggest a threshold effect may be

possible, although authors did not formally ev aluate this characte ristic of the data.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 271 of 531 Works Cited

Whyatt, R. M., Barr, D. B., Camann, D. E., Kinney, P. L., Barr, J. R., Andrews, H. F., . . . Perera,

F. P. (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 RM, Garfinkel R, Hoepner LA, Holmes D, Borjas M, Williams MK, Reyes A, Rauh VA,

Perera FP, Camann DE. Within- and Between-Home Variability in Indoor-Air Insecticide Levels

during Pregnancy among an Inner-City Cohort from New York City. En vironmental Health

Perspectives. 115:383–389 (2007).

Whyatt RM, Garfinkel R, Hoepner LA, Andrews H, Holmes D, Williams MK, Reyes A, Diaz D,

Perera FP, Camann DE, Barr DB. A Biomarker Validation Study of Prenatal Chlorpyrifos

Exposure within an Inner-City Cohort during Pregnancy. Environal H ealth Perspectives.

117:559–567 (2009).

# Chlorpyrifos Human Health Risk Assessment D424485

Page 272 of 531 

# Article 2. Berkowitz et al. (2004)

In Utero Pesticide Exposure, Maternal Pa raoxonase Activity, and Head Circumference.

Gertrud S. Berkowitz, James G. Wetmur, Elen a Birman-Deych, Josephine Obel, Robert H.

Lapinski, James H. Godbold, Ian R. Holzma n, Mary S. Wolff. Environmental Health

Perspectives. 112:388–391 (2004)

Study Summary

To assess the relationship of exposure to pestic ides during pregnancy an d subsequent risk of

impaired growth and neurodevelopment, Be rkowitz and colleagues (Mount Sinai Child

Development Study) investigated the relationship between prenatal pesticide exposure and infant

growth and neurodevelopment in a prospective, multiethnic cohort study of mothers and infants

delivered at Mount Sinai Hosp ital in New York City.

The study population was drawn primarily from East Harlem and consisted largely of young

Hispanic women (predominantly Puerto Rican) , but also included African-American and

Caucasian women. Mothers were enrolled during early pregnancy from a prenatal clinic and two

private practices at Mount Sinai Hospital fr om March 1998 to March 2002. The study included

only primiparous with singleton births. Mothers were excluded if they had any of the following:

an initial prenatal visit after 26 weeks of gesta tion; serious chronic dis eases; a serious pregnancy

complication that could affect fetal growth and development; alcohol cons umed greater than two

alcoholic beverages per day; or, illicit drug use. Mothers and infant s were also excluded if the

child was born with a congenita l malformation or severe premat urity. A total of 479 prenatal

patients were recruited; 75 were excluded because of medical complications, premature birth, an

infant with birth defects, inability to collect bi ologic specimens before birth, change of hospital

or residence outside New York City, or refusal to continue to participate.

Pesticide exposure was assessed by a prenatal questionnaire and measurement of urinary

concentration of 3,5,6-trichloro-2- pyridinol (TCPy), a metabolite of chlorpyrifos exposure. The

survey was administered, and maternal urine and blood specimens were collected, during the

third trimester. Cord blood samples were also obtained at birth. Measurement of paraoxanase-1

( PON1 ) genotype and phenotype was made using mate rnal and infant cord blood samples. PON1

is an enzyme that can detoxify chlorpyrifos be fore it can inhibit acetylcholinesterase in the

peripheral and central nervous systems. The i nvestigators assessed birt h weight, length, head

circumference, and gestational age of the new borns for 404 participating mother-child pairs.

Almost half of the participants in the study, (46%) reported that the y, or a household member,

had used indoor pesticides dur ing the pregnancy; 72% were cl assified as exposed using a

composite index defined as indoor pesticide use in the household or exte rminator application,

fumigation, or pesticide use in common areas. De tectable levels of urinary 3,5,6-trichloro-2-

pyridinol (TCPy; a metabolite of chlorpyrifos) we re observed in 44% of the study participants.

The authors constructed generali zed linear models to evaluate the associations among pesticide

exposure and PON1 activity with average birth weight, birth length, head circumference, and

gestational age, adjusting for selected covariates (race/ethnicity, infant sex, and gestational age).

The authors found no statistically significant a ssociations between pesticide exposure based

# Chlorpyrifos Human Health Risk Assessment D424485

Page 273 of 531 either on questionnaire responses or biomarkers of pesticide expos ure (TCPy) on fetal growth or

gestational age.

Researchers also evaluated the pot ential effect modifying role of PON1 genotype and phenotype

in the relation of interest. Stratifying participants by presence of PON1 polymorphisms and

tertiles of maternal PON1 ac tivity, researchers reported a small but statistically significant

reduction in head circumference am ong children of mothers with leve ls of chlorpyrifos above the

limit of detection and also in the lowest tertile of PON1 activity, i.e. , slowest metabolic activity.

However, the interaction between TCPy level and PON1 activity was not statistically significant.

In the subgroup of infants whose mothers had TCPy levels greater than the level of detection,

those with low maternal PON1 had an averag e (SD) head circumference of 33.3cm (1.5cm)

which was significantly smaller than those w ith medium (34.0cm (1.5cm)) and high (34.1cm

(1.6cm)) maternal PON1 activity af ter adjusting for race/ethnicity, infant sex, and gestational age

(p = 0.014). A similar trend was observed with head circumference when maternal PON1 activity

was considered alone; the adjusted means were 33.5 cm for the low PON1, 33.9 cm for medium

PON1, and 34.1 cm for high PON1 activity (p = 0.004). Maternal PON1 genetic polymorphisms

were not associated with reduced head size. No tr ends were seen for birt h weight or birth length

for TCPy or the other metabolite levels when maternal paraoxonase level was taken into account.

Infant paraoxonase activity ha d no association with any of the fetal growth measures.

Because small head size has been found to be pr edictive of subsequent cognitive ability, the

authors conclude that chlorpyrifos may have a detrimental effect on fetal neurodevelopment

among mothers who exhibit low PON1 activity.

Study Review

The largely null study had notable strengths a nd some limitations; notably authors did not

discuss any limitations of their study in the publis hed manuscript. The prospective nature of the

study limits the influence of errors in the self-reported exposure classification on the association

observed; as such errors would likely be non-differential with re spect to determinants of the

health outcome under investigation.

The authors assessed pesticide exposure using bo th subjective questionnaire-based methods and

biomarkers of exposure which are objective, sensitive and specific surrogates of pesticide

exposure. Biomarkers of specific pesticides ( i.e., TCPy) were assessed in a single maternal urine

sample provided during the third trimester. The authors do not discuss the impact of policies

aimed at reducing use of chlorpyrifos on the pr evalence and magnitude of pesticide exposures

during the time period of this study; it is likely that use of ch lorpyrifos exposure was decreasing

over the time period of the study, as noted in othe r similar studies of the same time period. The

authors do not deliberate on the degree to wh ich the biomarkers assessed represent usual

exposure to pesticides versus recent, acute e xposure(s), nor do they discuss the relative

importance of assessment of exposure during the third trimester vis-à-vis fetal growth and

development. The critical window of exposure for fetal growth and neurodevelopment health

effects is not fully characterized. If sources and patterns of exposure remain static, a single

measurement would be a good indicator of pest icide exposure during pregnancy, even if the

pesticides have short half-lives in vivo . If, however, sources and e xposure patterns change over

# Chlorpyrifos Human Health Risk Assessment D424485

Page 274 of 531 short time-spans, perhaps on the order of days to weeks, the biomarker assessment would not

necessarily provide a good indicator of the relevant exposure. The authors classified biomarker

levels according to the assays’ levels of de tection (above versus below). Falsely negative

exposure characterization would occur if mothers were exposed only early in pregnancy, and the

pesticides were fully metabolized prior to the third trimester. Incorrect classification of

participants as exposed, when in fact they were not exposed, was unlikely to have occurred using

the biomarkers. Like other sources of measuremen t error in this study, er rors in the biomarker

indicators of exposure in this study are like ly to have resulted in a non-differential

misclassification, with respect to fetal growth and development.

The health outcomes in this study, birth length, bi rth weight, head circumfe rence, and gestational

age were likely to have been accurately measur ed in this study, as they were obtained through

direct linkage with the computer ized medical record. Any errors in outcome measurement would

have occurred in the clinic, and are likely to be small and certainly non-di fferential with respect

to pesticide exposure.

To some extent, the recruitment process resu lted in a population that was likely homogeneous

with respect to confounders of the in utero pesticide exposure-fetal growth relationship

(compared to the general population), and the potential for confounding was thus limited by

design. The authors were also able to collect data on, and adjust for, important covariates,

including race/ethnicity, infant se x, and gestational age. The authors also controlled for birth

weight or birth length in their assessment of head circumference and pesticide exposure.

Covariates used in the analyses were selected, accordi ng to the authors, from variables known to

be associated with either pesticide exposure or fetal growth. Howeve r, factors related to

socioeconomic condition of those assessed and co-contaminants to which the study participants

may have been exposed were not addr essed in detail in the article. To the extent that such factors

may be determinants of fetal growth and deve lopment and correlated with (but not caused by)

pesticide exposure, they may conf ound the associations of interest in the study. If these factors

are positively associated with bot h pesticide exposure and dimini shed fetal development, they

would bias the true associati on upward (towards larger mean differences associated with

exposure). However, this is unlikely to have occurred in this largely null study.

Only factors that were statistic ally significant (p < 0.05) were in cluded in the final models. The

use of a statistical test as a means to select cova riates from a larger set of potential confounders is

discouraged in some epidemiologic circles (Miettinen 1976, Breslow and Day 1980, Greenland

1989). The assessment of confounding is generally not considered to be a st atistical endeavor, in

part because the association be tween the confounder and the outcome is just one factor that

determines the degree of confounding, and also b ecause the statistical significance of an

association is a function not only of the magnitude of the associat ion but also of other factors

( e.g. , sample size, and sample variability) whic h are irrelevant to bias due to confounding.

Moreover, the use of significan ce testing in the selection of confounders treats false negative

errors ( i.e. the deleting a confounder) as secondary to false positive error ( i.e. , inclusion of a nonconfounder). This implicit ranking of error is back ward, as deleting a confounder introduces bias

and is justified only if the bias induced by its exclusion is tolerabl y small or deemed to be worth

the precision gain, whereas including a non-c onfounder only (potentially) reduces precision by

increasing the size of the model. This is a sm all consideration in this study, and different

# Chlorpyrifos Human Health Risk Assessment D424485

Page 275 of 531 approaches are not likely to qualitatively cha nge the study findings or conclusions. Notably,

smoking was not included as a potentially confounding va riable in this analys is as the prevalence

of smoking was low in this cohort. According to the authors, the inclusion of smoking in final

models did not alter risk es timates and added to variance as stated by the authors.

Bias due to differential errors in the selection of study par ticipants is unlikely to have occurred in

this prospective study with no loss to follow-up at the time of this study. Appropriate exclusion

criteria were applied, including mothers with no prenatal medical visit prior to 26 weeks of

gestation, mothers with se rious chronic diseases ( e.g. , diabetes, hypertensi on, thyroid disease),

mothers who developed a serious pregnancy comp lication that could affe ct fetal growth and

development, and mothers who consumed more than two alcoholic beverages per day or who

used illicit drugs, mothers with multiple births, and infants born with a congenital malformation

or severe prematurity. The subjective, survey-b ased pesticide exposure assessment was fortified

by objective indicators of exposure ( i.e. urinary biomarkers).

The generalizability of the study may be limited as consequence of the particulars of the study

population assessed and the numerous exclusion cr iteria applied. Making valid inference using

these results requires an assumption that the pes ticide exposure-fetal development association is

the same in this population as the population to which the results are being generalized. This

study population consisted of primiparous women, r ecruited early on in their pregnancies from

the prenatal clinic and two priv ate practices at Mount Sinai Hospital in New York from March

1998 to March 2002, consisted predominantly of young Hispanic women with low education.

Sixty-eight percent were less than 25 years old. Just under half were Hispanic (49.8%), although

substantial numbers of White (21.0%) and Afri can-American (27.7%) women also participated.

Just under half (46.8%) classified themselves as being either single, divorced, widowed, or

separated, and just over half (50.1%) had no more than a high school education. The exclusion

criteria applied to potential st udy participants further focused th e population assessed, relative to

the general population of pregnant women. In the likely situation that the prevalence of

important modifiers of the exposure-fetal deve lopment association in this focused study

population is different from that in other populations of concern, ge neralizability will be limited.

Important modifiers to consider may include time spent in the home, patte rns of food preparation

and consumption, health consciousne ss, the baseline health status of the mother (and father), and

a host of socioeconomic determinants. The invest igators finding of effect modification by PON1

activity is illustrative of limits on the genera lizability of the study; populations who have high

levels of paraoxonase activity may vary by genotypic and phenotypic determinants. These

factors were not discussed in the report, but it has been noted prev iously that there is 10- to 40-

fold inter-individual variability in rates of paraoxon hydrolysis (Playfer et al 1976). The

potential limitation vis-à-vis gene ralizability is offset by a st rength; the study has increased

statistical efficiency, relative to one set in a gene ral population.

Because small head size has been found to be pr edictive of subsequent cognitive ability, the

authors conclude that chlorpyrifos may have a detrimental effect on fetal neurodevelopment

among mothers who exhibit low PON1 activity. Imp licit in this conclusion is the notion that

small head size is a good surrogate for fetal ne urodevelopment. To support their conclusion, the

authors note that head circumference has been shown to correlate with brain weight, and that

brain size and head circumferences are both pr edictive of IQ and cognitive ability. They

# Chlorpyrifos Human Health Risk Assessment D424485

Page 276 of 531 additionally cite research associating brain volu me with attention-deficit/hyperactivity disorder.

Although head circumference was significantly asso ciated with cognitive ability, the differences

in head size across tertiles of exposure were notably small ( i.e. less than one centimeter); the

clinical relevance of such sma ll changes in the context of even a small amount of measurement

error in assessment of he ad size is unclear.

The authors were not surprised that infant paraoxonase levels in their study were not associated

with birth outcomes because PON1 is not well expr essed until after birth, and infant PON1 levels

are much lower during the first year of life than maternal levels. The expression of PON1 in

infants is approximately one-third that of their mothers (Chen et al. 2003). They speculate that

lower PON1 activity in infants may make them more susceptible to PON1-related toxicity,

including exposure to organophosphate pesticides, rela tive to adults.

The net effects of confounding, selection bias, and information bias, are unlikely to have

qualitatively changed the results of this we ll conducted, prospective cohort study. Non-specific

biomarkers of exposure, as were assessed in th is study, are likely more objective, sensitive, and

likely also more accurate and reliable indicator s of pesticide exposure, relative to exposure

assessment methodologies that rely on self-report . However, to the extent that prenatal

chlorpyrifos is the true expos ure of interest, the assessmen t of non-specific organophosphate

pesticide metabolites in a single sample of pren atal urine is a primary potential source of

exposure measurement error. Almost certainly, su ch biases would not have resulted in the

observation of a falsely positive association betw een pesticide exposure and fetal growth. More

likely to have occurred is an attenuation of the true (unobserved) associat ions, toward (observed)

associations that are relatively sm aller, or null. Indeed, the findi ngs of this study are either null,

or associations of small magnitude. To contextu alize these findings, the authors cite literature

describing the finding that even small increases in prenatal or early childhood exposures to other

environmental toxicants (lead a nd polychlorinated biphe nyls) are associated with significant

decrements in IQ and academic achievement up to 11 years of age. The validity of this analogy is

speculative. The external validity of the study findings may be limited if the organophosphate

pesticide exposure-fetal development association is modified by factors that are more, or less,

prevalent in this young, urban, minority populati on, relative to the population(s) to which

inference is being made. That sa id, the decision to focus on this population is reasonable, as it is

a population likely to be highly exposed, and pe rhaps also at high ri sk of cognitive and

behavioral deficits enhancing th e likelihood of observing an associa tion if an association exists.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 277 of 531 Works Cited

Breslow NE, Day NE. Statistical Methods in Ca ncer Research: Volume II: The Design and

Analysis of Cohort Studies. Lyon: IARC, 1987.

Chen J, Kumar M, Chan W, Berkowitz G, Wetmur J. Increased influence of genetic variation on

PON1 activity in neonates. Environt al Health Persp ectives. 2003;111:1403–1409.

Engel SM, Berkowitz GS, Barr DB, Teitelbaum SL , Siskind J, Meisel SJ, Wetmur JG, Wolff

MS. Prenatal Organophosphate Metabolite and Or ganochlorine Levels and Performance on the

Brazelton Neonatal Behavioral Assessment Scale in a Multiethnic Pregnancy Cohort. American

Journal of Epidemiology. 2007;165:1397-1404.

Engel SM, Wetmur J, Chen J, Zhu C, Barr DB, Canfield RL, Wolff MS. Prenatal Exposure to

Organophosphates, Paraoxonase 1, and Cognitive Development in Childhood. Environmental

Health Perspectiv es 2011;119:1182-1188

Greenland, S. Modeling and Variable Selection in Epidemiologic Analysis. American Journal of

Public Health. 1989; 79:340-349.

Miettinen OS. Stratification by a multivariate confounder score. American Journal of

Epidemiology 1976;104:609- 620.

Playfer J, Eze LC, Bullen MF, Evans DAP. Genetic polymorphism and inter- ethnic variability of

plasma paraoxonase activity. J Med Genet. 1976;13:337–342.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 278 of 531 

# Article 3. Eskenazi et al. (2004)

Association of in Utero Organophosphate Pesticide Exposure and Fetal Growth and

Length of Gestation in an Agricultural Po pulation. Brenda Eskenazi, Kim Harley, Asa

Bradman, Erin Weltzien, Nicholas P. Jewell, Dana B. Barr, Clement E. Furlong, Nina T.

Holland. Environmental Health Perspectives. 115:792–798 (2007).

Study Summary

In this article by Eskenazi et al, the authors report on the relati onship between prenatal urinary

organophosphate metabolite levels and fetal growth and gestational duration in the CHAMACOS

cohort of Mexican-American mothers and their children living Salinas Valley, an agricultural

region of California.

Enrollment of the Center for the Health A ssessment of Mothers and Children of Salinas

(CHAMACOS) cohort took place at community clin ics. A total of 601 out of 1,130 eligible

women agreed to participate and were enrolled between October, 1999 and October, 2000, with

538 being followed through to delivery of a live born infant, whose birt h weight was measured.

The study populations consisted la rgely of low-income, agricultura l workers. Women considered

eligible for the study were less than 20 weeks gestation, aged 18 years or older, Medi-Cal

eligible, fluent in English and/or Spanish, and planning to deliver at Na tividad Medical Center.

Excluded from analyses were women with gest ational or preexisti ng diabetes (n = 26),

hypertension (n = 15), twin births (n = 5), or stillbirths (n = 3). One additional woman for whom

birth weight information was out of range (< 500 g) was also excluded. The final analytic sample

consisted of 488 mother-infant pairs.

Maternal/fetal exposure to organophosphate pesticides was assessed by measurement of

organophosphate dialkyl phosphate metabolites (DAP s) and seven different pesticide-specific

metabolites in maternal urine during pregnancy, including 3,5,6-trichloro-2-pyridinol (TCPy), a

metabolite specific to chlorpyrifos. Cholin esterase (ChE) in whole blood and butyryl

cholinesterase (BChE) in plasma collected from mothers during pregnancy, at delivery, and from

umbilical cord blood was also assessed. Matern al urine samples were collected between 5 and

27 weeks gestation and again between 18 and 39 weeks. The dialkyl phosphates measured were

dimethylphosphate, dimethyldithi ophosphate, dimethylthiophospha te (DMPs), diethylphosphate,

diethyldithiophosphate, and diethy lthiophosphate (DEPs). Total DAPs and DMPs were available

for 485 women; DEP metabolite levels were available for 486 women. Quantification of

organophosphate metabolites using gas chromat ography-tandem mass spectrometry and isotope

dilution calibration was conducted by the Center s for Disease Control and Prevention (CDC)

labs.

Total DAP metabolite level was defined as the sum of the molar concentrations of the six DAP

metabolites, dimethylphosphate (DMP) metabolite le vel as the sum of the molar concentrations

of the three dimethylphosphate metabolites only, and diethylphosphate (DEP ) metabolite level as

the sum of the molar concentrations of the th ree diethylphosphate meta bolites. Total DAP, DMP,

and DEP levels were determined for each par ticipant for each of the two pregnancy urine

samples. Individual metabolite levels below the limit of detection (LOD) were assigned a value

# Chlorpyrifos Human Health Risk Assessment D424485

Page 279 of 531 of the LOD divided by the square root of two, and this value was included in each sum. Total

DAP, DMP, and DEP metabol ite levels were log 10 

# transformed. DAP, DMP, and DEP values

were created by averaging the two log-tran sformed pregnancy measures. For specific

metabolites, if a woman was below the LOD for bot h measurements, she was classified as below

LOD for the average. If she was above the LOD at either or both measurements, her

measurements were averaged and classified in relation to the median. For 19 women with only

one DAP measurement in pregnancy, the single measure was used.

Similar to the analytic treatment of dialkyl phosphate metabolite, authors assigned a value of the

LOD divided by the square root of two for TC Py measurements below LOD. Because a large

proportion of women had non-detectable levels of TCPy, the chlorpyrif os-specific metabolite

was categorized into three groups: TCPy less th an LOD for both pregnancy measurements, and,

for those with at least one detectable level, subdivided below and above the median of the

average pregnancy level.

Infant birth weight, crown–heel length, and head circumference were obtained from hospital

delivery logs and medical records. Infant pondera l index, a measure of pr oportionality of growth,

was calculated as (birth weight in grams × 100) /(length in centimeter s). Gestational age was

obtained from medical records and was based on ultrasound procedures for 25% of women. Low

birth weight was defined as < 2,500 g. Preterm delivery was defi ned as birth at less than 37

completed weeks of gestation. A “small for gest ational age” (SGA) birth was defined as birth

weight less than the 10 th 

# percentile for gestational age accordi ng to ethnicity, parity, and infant

sex.

Multiple linear regression models were developed to assess associations between the markers of

pesticide exposure and length of gestation, birt h weight, length, head circumference, and

Ponderal index. Multivariate logi stic regression was used to ev aluate associations between

exposure measurements and low birth weight, pret erm delivery, and SGA birt hs. Covariates in

the models included including maternal age, pregnancy weight gain, week of initial prenatal care,

parity, infant sex, mother’s country of birth, body mass index, and family income. All models of

birth weight, length, head circumference, and Ponderal index were adjusted for linear and

quadratic terms for gestational age.

The women in this study averaged 25 years of age (SD = 5); two-thirds were multiparous, 80%

were married, 79% had not graduated from hi gh school, 58% were overweight or obese, 88%

preferred to speak Spanish, and 84% were born in Mexico, with more than half residing in the

United States for < 5 years. Almost all of th e women were living within 200% of the poverty

level. Very few women reported smoking (6%), drug use (2%), or alcohol consumption (1%)

during pregnancy. Approximately 28% of the wo men had worked in the fields during the

pregnancy, and another 14% had worked at other j obs in agriculture. Eighty-five percent of the

women in the study had agricultu ral workers living in their ho mes during their pregnancy. The

median total DAP, DMP, DEP and total DAP me tabolite levels for the study population were

136 nmol/L, 101 nmol/L, and 22 nmol/L, respectiv ely. Only one woman had no detectable DAP

levels and TCPy was detected in 77% of maternal urine samples. The median level for TCPy was

3.3 μ g/L.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 280 of 531 The mean (± SD) duration of gestation was 38.9 ± 1.7 weeks; mean birth weight was 3,449 ± 516

g; mean body (crown–heel) length was 50.2 ± 2. 7 cm; mean head circumference was 34.1 ± 1.5

cm; and mean Ponderal index was 2.7 ± 0.3 g/m3. A to tal of 3.7% (n = 18) of children were born

of low birth weight; 4.8% (n = 23) were SGA births, and 6.6% (n = 32) were preterm.

The authors did not observe an adverse relations hip between fetal growth and any measure of in

utero organophosphate pesticide exposure. On th e contrary, they found positive associations

between body length and head circumference associ ated with some exposure measures, but not

chlorpyrifos. They did observe decreases in ge stational duration associated with two measures

of in utero pesticide exposure: urinary DMP metabolites ( β = –0.41 weeks per log 10 

# unit

increase; 95% CI: –0.75––0.02; p = 0.02), which refl ects exposure to dimethyl organophosphate

compounds such as malathion, not chlorpyrifos.

Study Review

The study has numerous strengths; most notab ly the prospective design the longitudinal

assessment of both exposure and outcomes. Strengths in the study design al so include the use of

multiple exposure biomarkers including quantification of non-specific (DAPs), chlorpyrifosspecific (TCPy) metabolites, cholinesterase ( acetyl and butyl), and other environmental coexposures.

The ascertainment and analysis of urinary or ganophosphate pesticide markers was appropriate.

Quantification was conducted at the CDC, using published methods. DAP metabolites are likely

more accurate and objective indicators of organophos phate pesticide exposu re, relative to other

ascertainment methods such as self-report. Altho ugh they are sensitive indi cators of exposure, it

is difficult to infer chlorpyrifos effects specifically from urinary DAPs. Urinary metabolite levels

may reflect exposure not only to a host of organophosphate parent compounds, but also to

potentially less toxic environmental metabolites. Also, because exposure to pesticides varies

considerably from day to day, DAPs from s pot urine samples may not represent average

exposure over time. However, the prenatal exposu re measures were, with some exceptions, the

average of two measurements, and thus may bette r reflect chronic exposur e during the pregnancy

than a one-time exposure measure.

Errors in the assignment of exposure in this prospective study will likely have resulted in

attenuation of observed associatio ns. The likelihood of these errors is high, and the magnitude of

the bias induced difficult to assess. Under an assumption of no other biases, the negative

findings, those for the association between childre n DAP levels and fetal development outcomes,

for example, could have resulted from exposure measurement error.

To identify “critical windows” of fetal development when expos ure may have a greater impact,

the authors analyzed the associ ations of outcomes and metabolit e levels measured during moving

6-week windows of pregnancy ( e.g. , 5–10 weeks, 6–11 weeks, 7–12 weeks) using a series of

multiple regression analyses. No period of greater impact was observed in this largely null study.

The indicators of fetal growth and developm ent (infant birth weight, length, and head

circumference, Ponderal index, and gestational age) were obtained from hospital delivery logs

# Chlorpyrifos Human Health Risk Assessment D424485

Page 281 of 531 and medical records, or calculated using data from these records. They are likely to have been

accurately and objectively measure d. Although partially self-reported, any errors in gestational

age are unlikely to be associated with pe sticide exposures in this prospective study.

The statistical analysis used to assess the asso ciations between the markers of exposure and fetal

growth and development were appropriate. Howe ver, the authors did not present nor discuss

regression diagnostics to assess th e degree to which their models me t or violated the assumptions

implicit in linear models ( i.e. homoscedasticity, normality of error term distribution,

independence of error terms, and linearity of th e exposure-response relationship), other than to

state that they log transformed exposure marker levels. The authors also conduct secondary and

sensitivity analyses to assess influence of modeling decisions on their findings, and generally

found their results to be robust.

A reasonable set of exclusion criteria were applied which likely resulted in recruitment of a study

population that was relatively homogeneous with respect to confounders of the in utero pesticide

exposure-fetal growth relationship (compared to the general population) , and the potential for

confounding was thus limited by design. For exampl e, smoking, alcohol use, and illicit drug use

were not included in the models because very few women reported use and controlling for these

variables did not al ter the results.

Bias due to confounding was also co ntrolled in the analysis, by a ssessing and adjusting for risk

factors for impaired fetal growth and development that are potentially associated with pesticide

exposure, including maternal age, pregnancy weight gain, week of initial prenatal care, parity,

infant sex, mother’s country of birth, body ma ss index, and family income. Variables were

selected as potential confounders for the multivaria te models based on associations reported in

the literature, and included in final models if they changed the coefficient associated with the

exposure by 10% or more. Inclusion of indicat ors for environmental tobacco smoke exposure,

consumption of caffeinated beverages, history of low birth weight a nd history of preterm

delivery, did not alter the relati onship of pesticide me tabolites and birth outcome and were not

included in final models. In addition to restrict ion of confounding by design, the selection of the

CHAMACOS population, which consists mostly of children from low-income families, also

served to increase the relative statistical efficiency of the study, as this population is at high risk

of developmental deficits, compared to the general population.

The results of this study by Eskenazi et al (2004) in the CHAMAC OS cohort failed to

demonstrate an adverse relations hip between fetal growth and in utero organophosphate

pesticide exposure as assessed by multiple measures of prenatal pesticide exposure. The

prospective study design and reasonable methods used protect from certain false-positive

inducing biases, although it is possible that ( non-differential) exposure measurement error

attenuated the associations. Misc lassification of the fetal growth outcomes was unlikely to have

biased the results. Residual c onfounding by mismeasured or unmeas ured factors is possible in

this observational study, although it is unlikely that such confounding would have resulted in the

null finding.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 282 of 531 

# Article 4. Harley et al. (2011)

Association of Organophosphate Pestic ide Exposure and Paraoxonase with Birth

Outcome in Mexican-American Women. Kim G. Harley, Karen Huen, Raul Aguilar

Schall1, Nina T. Holland, Asa Bradman, Dana Boyd Barr, Brenda Eskenazi. PLoS ONE

6(8)1-10 (2011).

Study Summary

In this article by Harley et al, the authors present a follow- up assessment of the relationship

between prenatal urinary organophosphate metabolite levels and fetal growth and gestational age

in the CHAMACOS cohort of Mexican-American mothers and their children living Salinas

Valley, an agricultural region of California.(Esk enazi et al, 2004) They evaluate whether

paraoxonase (PON1), a key enzyme involved in detoxification of organophosphate pesticides,

could be an effect modifier of the associati on between indicators of maternal organophosphorus

pesticide exposure (DAP metabolite concentrations) and poorer fetal growth previously reported

in this cohort. They additionally examine the main effects of maternal and child PON1 genotypes

and PON1 activity on birth outcomes.

Enrollment of the Center for the Health A ssessment of Mothers and Children of Salinas

(CHAMACOS) cohort took place at community clin ics. A total of 601 out of 1,130 eligible

women agreed to participate and were enrolled between October, 1999 and October, 2000, with

538 being followed through to delivery of a live born infant, whose birt h weight was measured.

The study populations consisted la rgely of low-income, agricultura l workers. Women considered

eligible for the study were less than 20 weeks gestation, aged 18 years or older, Medi-Cal

eligible, fluent in English and/or Spanish, and planning to deliver at Na tividad Medical Center.

Excluded from analyses were women with gest ational or preexisti ng diabetes (n = 26),

hypertension (n = 15), twin births (n = 5), or stillbirths (n = 3), and one infant with a birth < 500

grams. In addition, mother-infant pairs with missing PON1 genotype and activity levels were

excluded (n =21), leaving a fina l sample size of 467. Of these, PON1 genotype information was

available for 451 mothers and 436 infants, and PON1 activity data were available for 371

mothers and 324 infants.

Maternal/fetal exposure to organophosphate pesticides was assessed by measurement of

organophosphate dialkyl phosphate metabolites (DAPs) . Maternal urine samples were collected

between 5 and 27 weeks gestati on and again between 18 and 39 weeks. The dialkyl phosphates

measured were dimethylphosphate, dimethyld ithiophosphate, dimethylthiophosphate (DMPs),

diethylphosphate, diethyld ithiophosphate, and diethylth iophosphate (DEPs).

Quantification of organophosphate metabolite s using gas chromatography-tandem mass

spectrometry and isotope dilution calibration wa s conducted by the Centers for Disease Control

and Prevention (CDC) labs. Total DAP metabolit e level was defined as the sum of the molar

concentrations of the six DAP metabolites, dimethylphosphate (DMP) metabolite level as the

sum of the molar concentrations of the three dimethylphosphate metabolites only, and

diethylphosphate (DEP) metabolite level as the sum of the molar concentrations of the three

diethylphosphate metabolites. For eight women, missing metabolite values were imputed using

# Chlorpyrifos Human Health Risk Assessment D424485

Page 283 of 531 linear regression to predict the missing meta bolite level as a function of the other known

metabolites for a given participant at a given time point.

The variables for total DAP, DMP, and DEP concen trations were log-transformed to reduce the

impact of outlier points. Me tabolite measurements from the two sampling time points were

averaged to create a summary measure of in utero organophosphate pesticide exposure.

The primary analyses were conducted using DAP concentrations unadjusted for creatinine,

although sensitivity analyses incl uded creatinine as a covariate in the models. Total DAPs and

DMPs were available for 485 women; DEP meta bolite levels were available for 486 women.

Maternal and umbilical cord blood samples were collected at the time of delivery. Genotyping

of the PON1 -108 

# and PON1 192 

# SNPs was performed using ge nomic DNA extracted from blood

clots and Taqman PCR methods.

Two PON1 enzyme activity assays (arylester ase and paraoxonase) were performed using

spectrophotometric methods. Aryl esterase activity measures the rate of hydrolysis of the

substrate phenyl acetate, and reflects the qua ntity of PON1 enzyme . Paroaxonase activity

measures the rate of hydrolysis of paraoxon, and thus reflects a combination of the catalytic

efficiency and quantity of the PON1 enzyme.

Birth outcome data, including infa nt birth weight, length, head circumference, and gestational

age, were abstracted from medical records by a registered nurse.

Linear regression models were constructe d to examine the main associations of PON1 genotype

and activity with length of gest ation, infant birth weight, birt h length, and head circumference,

independent of pesticide e xposure. Maternal and child PON1 genotypes were entered into

models as categorical variables; for PON1 -108 

# , the CC genotype was the reference, and for

PON1 192 

# , the RR genotype was the reference. Aryleste rase and paraoxonase activity in maternal

and umbilical cord blood were examined as co ntinuous variables, normalized by dividing by the

standard deviation.

The associations of PON1 activity with birth outcomes were assessed within each PON1 192 

# genotype by stratifying by genotype and by testing for interaction between activity and genotype.

The presence of effect modification of associat ions between maternal DAP metabolite and birth

outcomes by PON1 genotype and activity was assessed by stratification, and us e of interaction

terms. PON1 genotype (as a categorical variable) or act ivity (as a continuous variable) and crossproduct terms were included in models previously used to assess pesticide associations in the

2004 report by Eskenazi et al. (Eskenazi et al, 2004) For PON1 genotype, two cross-product

terms were included in the model ( e.g. QR*DAP concentrations a nd RR*DAP concentrations).

Similar cross-product terms were generated us ing tertiles of PON1 activity. Statistical

significance for interaction was set at p=0.1. In addition, effect modi fication by PON1 was

assessed by conducting linear regr essions on data stratified by PON1 genotype and by tertiles of

enzyme activity.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 284 of 531 Models of PON1 genotype included maternal pre-pre gnancy BMI and maternal weight gain

during pregnancy as covariates. Mo dels of PON1 enzyme activity also included maternal age and

country of birth, assa y temperature, and PON1 192 

# genotype. In models of birth weight, length and

head circumference, the authors also controlle d for linear and quadratic gestational age to

examine associations independe nt of gestational duration.

As previously summarized, women in this coho rt were predominantly Spanish-speaking and

born in Mexico; 41% of the women worked in ag riculture during pregnancy. More than 60% of

families were living below the federal povert y threshold, and 89% of women had never

completed high school. The mean maternal ag e was 25.5 years (SD = 5.0). All women had

detectable levels of organophosphate pesticide metabolites in their urin e during pregnancy. The

geometric mean for the DAP concentrations du ring pregnancy (for th e average of the two

sampling periods) was 146 nmol/L (95% CI: 133 , 160); of this, a larger proportion was DMP

metabolites (GM = 109 nmol/L; 95% CI =98, 120) than DEPs (GM= 23 nmol/L; 95% CI = 21,

25). The allele frequencies of the PON1 192 

# Q allele and the PON1 -108 

# T allele were 50% and

46%, respectively, and did not differ between mother s and newborns. The mean arylesterase and

paraoxonase activity was 33.6 U/mL (SD = 16) and 256.6 U/L (SD = 165), respectively, for

infants and 136.6 U/mL (SD = 44) and 989.0 U/L (SD = 616) for mothers.

In this study, infants’ (but not mother's) PON1 genotype and PON1 activity were associated with

gestational age and head circumfere nce: Infants with the susceptible PON1 -108 

# TT genotype had

shorter gestational age ( =-0.5 weeks, 95%CI: -0.9, 0.0) and smaller head circumference ( =-

0.4 cm, 95% CI: -0.7, 0.0) than those with the PON1 -108 

# CC genotype. Infants’ arylesterase and

paraoxonase activity were positively associated with gestational age. Maternal DAP

concentrations were associated with shorter gestational age among infants of the susceptible

PON1 -108 

# TT genotype, although only the interaction between PON1 -108 

# genotype and DEP

metabolite concentrations was st atistically significant (p-value for interaction = 0.09). However,

maternal DAP concentrations were associated with larger birth weight (p -value for interaction =

0.06) and head circumference (p-value for inte raction =0.01) in infant s with non-susceptible

genotypes. The authors conclude that infants with certain PON1 genotypes ( e.g.

, PON1 -108 

# TT)

may be more susceptible to effects of in utero organophosphate pesticide exposure.

Study Review

This study improves on the work by Eskenazi et al (2004) by incl uding an assessment of effect

modification of the association be tween prenatal pesticide marker levels and birth outcomes by

PON1 genotype and activity. Additional strengths of this study include the prospective design,

the assessment of pesticide mark ers at two time-points during pr egnancy, and the assessment of

PON1 activity by two different methods.

The authors speculate that a possible reason that they did not observe an association between

DEPs and gestational age in the earlier study by Eskenazi is that it was “masked” by the effects

of PON1 . They note that the observati on of an interaction only for DEP concentrations is

plausible, since the known biol ogically relevant action of PON1 is with two DEP pesticides

(chlorpyrifos and diazinon).

# Chlorpyrifos Human Health Risk Assessment D424485

Page 285 of 531 The finding of an independent effect of PON1 genotype on birth outcomes is consistent with

finding of reductions in average ge stational age and head circumfere nce at birth associated with

the PON1 -108 

# TT genotype in the Mount Sinai cohort, as reported by Berkowitz et al. (Berkowitz

et al, 2004). The association betw een infant PON1 (arylesterase) activity and gestational age was

null in the Mount Sinai cohort. This is in contra st to the positive associ ation reported by Harley

et al. Maternal PON1 (arylesterase) activity was not associated with birth outcomes in the Haley

study, but positively associated with head circumfe rence in the Berkowitz et al study. Finally, the

interactions observed in the two studies differed. Berkowitz et al reported a decrease in head

circumference associated with chlorpyrifos mark er levels among newborns of mothers with low

arylesterase activity in th e Mount Sinai cohort. Harley et al, in contrast, observed a statistically

significant interaction between DEP levels and ge stational age among infant s in the UC Berkeley

CHAMACOS cohort with the TT PON1 genotype, but they did not observe significant

interaction between PON1 activity and birth outcomes. In ad dition to measuring different

indicators of pesticide exposure (TCPy), the PON1 genotypes and activity assessed for the

Mount Sinai cohort differed some what from those assessed in the UC Berkeley cohort;

Berkowitz et al did not assess PON1 192 

# (only PON1 -108 

# ) and paraoxonase activity (only

arylesterase activity)

In the Columbia Mothers and Newborn Study (Whyatt et al, 2004), organophosphate pesticide

levels in maternal blood were associated with decreased fetal growth, while in the UC Berkeley

CHAMACOS study, modest increases in birth weight and head circumference were associated

with in utero DAP concentrations. In fact, stratificati on by PON1 status seemed to enhance these

positive associations, such that in the non-susceptible groups ( e.g. PON1 -108 

# CC, PON1 192 

# RR,

and high arylesterase activity), increasing DAP con centrations were significantly associated with

increased birth weight and head circumference.

The finding of a positive association between DAP s and DMPs and both head circumference and

birth weight among those with the CT or CC PON1 genotypes, and a positive association

between head circumference and DAPs among infants with RR PON1 genotypes is perplexing.

The authors speculated that it is possible that , among the less susceptible groups, high urinary

DAP concentrations may be an indication of rapid detoxification and excretion of

organophosphate pesticides, rather th an a marker of high exposure.

The ascertainment and analysis of urinary or ganophosphate pesticide markers was appropriate.

Quantification was conducted at the CDC, usi ng published methods. However, the use of DAP

metabolites to measure exposure has some limitati ons, and these were discussed by the authors.

Although they are sensitive and relatively specifi c indicators of organophosphate exposure, it is

difficult to infer chlorpyrifos e ffects specifically from urinary DAPs. Urinary metabolite levels

may reflect exposure not only to a host of or ganophosphate parent comp ounds, but also exposure

to the less toxic metabolites themselves. Because e xposure to pesticides varies considerably from

day to day, DAPs from spot ur ine samples may not represent average exposure over time.

However, the prenatal exposure measures were , with some exceptions, the average of two

measurements taken early and late pregnancy, and thus may better reflect chronic exposure

during the pregnancy. Additionally, DAP metabolites in urine may overestimate

organophosphate pesticide exposure if they reflect exposure to both pre-formed DAPs in the

environment and the parent organophosphate compounds.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 286 of 531 Errors in the assignment of exposure in this prospective study will likely have resulted in

attenuation of observed associatio ns. The likelihood of these errors is high, and the magnitude of

the bias induced difficult to assess. Under an assumption of no other biases, the negative

findings, those for the association between child ren’s DAP levels and a ttention outcomes, for

example, could have resulted from exposure measurement error.

The indicators of fetal growth and developm ent (infant birth weight, length, and head

circumference, and gestational age) were obtained from hospital medical records, and

ascertained by a registered nurse. They are lik ely to have been accurately and objectively

measured, although the potential for errors in their measurement was not assessed. Although

partially self-reported, any errors in gestational age are unlikely to be associated with pesticide

exposures, PON1 genotype, or PON1 activity in this prospective study.

The statistical analysis used to assess the asso ciations between the markers of exposure and fetal

growth and development were appropriate. Howe ver, despite a reasonable sample size for

assessing main effects, the Harley et al study lik ely lacked sufficient statistical power to assess

effect modification by PON1 genotype and PON1 activity, as the CHACAMOS Cohort study

was not originally designed to a ssess interactions between prenat al pesticide exposures and these

co-factors. As a result, the analysis is at risk of failing to detect effect modification if effect

modification is truly present.

The authors did not present nor di scuss regression diagnostics to as sess the degree to which their

models met or violated the assump tions implicit in linear models ( i.e. homoscedasticity,

normality of error term distribut ion, independence of error terms, and linearity of the exposureresponse relationship), other than to state that they log-transf ormed DAP levels to reduce the

impact of outlier observations. The authors di d conduct secondary and sensitivity analyses to

assess influence of modeling decisions on their findings, including adjustment for creatinine.

A reasonable set of exclusion criteria were applied which likely resulted in recruitment of a study

population that was relatively homogeneous with respect to confounders of the relationship

between in utero pesticide exposure and fetal growth (compared to the general population), and

the potential for confounding was thus limited by design. For example, smoking, alcohol use,

and illicit drug use were not included in the m odels because very few women reported use and

controlling for these variables did not alter the results.

The authors reported that factors known, a priori , to be associated with birth outcomes in this

population were considered pot ential confounders, although they did not enumerate these

variables. Covariates were retained in final models if they were also associated with PON1

genotype or activity (in the study population, presumably) or if their exclusion from the model

changed the main effect coefficient by more th an 10%. The authors’ final models for birth

weight and head circumference adjusted for ti ming of urine collection, timing of entry into

prenatal care, maternal age, parity, country of birth, household income, pre-pregnancy body mass

index, maternal weight gain, infant sex, and gestational age using bot h linear and quadratic

terms. Gestational age models adjusted for tim ing of urine collection, timing of entry into

prenatal care, maternal age, parity, country of birth, a nd household income.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 287 of 531 The authors controlled for gestational age in models of birth weight, length, and head

circumference to examine associations independen t of gestational duratio n. It is possible that

gestational age is an intermediate on the causal path way. If this is the case, the coefficients from

these models can be interpreted as estimates of the ‘direct-effect’ of prenatal urinary DAPs and

fetal growth outcomes due to causal pathways ot her than those mediated by gestational age.

These pathway-specific analyses have a poten tial limitation that wa s not discussed by the

authors. Namely, there is the potential for induci ng bias if gestational ag e and fetal growth have

unmeasured common causes ( i.e. an unknown co-exposure) (C ole and Hernan, 2002).

In addition to restriction of confounding by design, the selec tion of the CHAMACOS population,

which consists mostly of children from low-income families, also served to increase the relative

statistical efficiency of the study, as this popul ation is at high risk of neurodevelopmental

deficits, compared to the general population.

The results of this study by Harl ey et al in the CHAMACOS c ohort demonstrate modest adverse

associations between some meas ures of PON1 and fetal growt h, as well as providing some

evidence of effect modificati on of the relationship between organophosphate pesticide exposure

markers and fetal growth. The prospective st udy design and reasonable methods used protect

from certain false-positive inducing biases, alth ough it is possible that exposure measurement

error attenuated the associations. Misclassification of the fetal growth outcomes was unlikely to

have biased the results. Resi dual confounding by mismeasured or unmeasured factors, including

bias induced by adjusting for an intermediate variable (gestationa l age), is possible in this study,

and may account for the observed associations.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 288 of 531 Work Cited

Berkowitz GS, Wetmur JG, Birman-Deych E, Ob el J, Lapinski RH, Godbold JH, Holzman IR,

Wolff MS. In Utero Pesticide Exposure, Maternal Paraoxonase Activity, and Head

Circumference. Environmental Health Perspectives. 2004;112:388–391.

Cole SR and Hernan MA. Fallibility in Estimating Di rect Effects. Int. J. Epidemiol. 2002; 31 (1):

163-165.

Eskenazi B, Harley K, Bradman A, Weltzie n E, Jewell NP, Barr DB, Furlong CE, Holland NT.

Association of in Utero Organophosphate Pestic ide Exposure and Fetal Growth and Length of

Gestation in an Agricultural Population. Environmental Health Perspectives. 2004;115:792–798.

Whyatt RM, Rauh V, Barr DB, Camann DE, Andrew s HF, Garfinkel R, Hoepner LA, Diaz D,

Dietrich J, Reyes A, Tang D, Kinney PL, Perera FP. Prenatal Insectic ide Exposures and Birth

Weight and Length among an Ur ban Minority Cohort. Environmental Health Perspectives.

2004;112:1125–1132.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 289 of 531 

# Article 5. Engel et al. (2007)

Prenatal Organophosphate Metabolite and Organ ochlorine Levels and Performance on the

Brazelton Neonatal Behavioral Assessment Scale in a Multiethnic Pregnancy Cohort.

Stephanie M. Engel, Gertrud S. Berkowitz, Dana B. Barr, Susan L. Teitelbaum, Jodi

Siskind, Stefanie J. Meisel, James G. Wetmu r and Mary S. Wolff. American Journal of

Epidemiology. 165:1397-1404, 2007.

Study Summary

Engel et al conducted a study leveraging the Child ren’s Environmental Health Study cohort data

to investigate the impact of pesticide exposure on pregnancy outcomes and child

neurodevelopment in an inner-ci ty multiethnic cohort of women recruited during pregnancy. The

study enrolled primiparous women presenting for pren atal care with single ton pregnancies at the

Mount Sinai prenatal clinic and two private practices and who de livered their infants at Mount

Sinai Hospital between May 1998 and July 2001. Part icipants in this study were predominantly

young, unmarried, Hispanic and African American women with low educational attainment.

This is the second of three published analyses of prenatal pesticide e xposure and child growth

and development in the Mount Sinai Hospital Ch ildren’s Environmental Health Study. The first

(Berkowitz et al 2004) reported on assessments of birth weight, le ngth and head circumference.

The third (Engel et al 2011) reports on assessm ents of infant development at age 12 and 24

months and child development at ages 6-9 years.

The study population was drawn primarily from East Harlem and consisted largely of young

Hispanic women (predominantly Puerto Rican) , but also included African-American and

Caucasian women. Mothers were enrolled during ear ly pregnancy from the Prenatal Clinic and

two private practices at Mount Sinai Hospital from May 1998 to July 2001. The study included

only primiparous with singleton births. Mothers were excluded if they had any of the following:

an initial prenatal visit after 26 weeks of gesta tion, serious chronic dis eases, a serious pregnancy

complication that could affect fetal growth and development; alcohol cons umed greater than two

alcoholic beverages per day, illicit drug use. Mother s and infants were also excluded if the child

was born with a congenital malformation or severe prematurity. A total of 479 prenatal patients

were recruited; 75 were excluded because of medical complications , premature birth, an infant

with birth defects, inability to collect biologic specimens before birth, change of hospital or

residence outside New York City, or re fusal to continue to participate.

The authors assessed the relationship betw een total diethylphos phates (DEPs), total

dimethylphosphates (DMPs), total dialkylphosphate metabolites (DAPs), as well as malathion

dicarboxylic acid (MDA) and indices of abnorma l neonatal behavior and/or neurological

integrity using outcome measures derived from the Brazelton Neonatal Behavioral Assessment

Scale (BNBAS), which was administered to the infa nts of participating mothers within days after

birth and before they left the hospital. The aut hors also assessed modificat ion of the associations

between pesticide metabolite leve ls and metrics of neurodevelopment by maternal paraoxonase 1

activity.

Environmental exposures, soci odemographic characteristics, medical history, and lifestyle

# Chlorpyrifos Human Health Risk Assessment D424485

Page 290 of 531 factors were assessed by a prenatal questionnaire administered to the mothers during the third

trimester. Participating mothers provided urine samples during the 3rd tr imester prenatal visit

which were provided to the CDC in order to assay six dialkylphos phate metabolites and

malathion dicarboxylic acid (MDA). The investigat ors imputed values for samples with one or

more missing values using methods described by Eskenazi et al. (2004), extending the method to

also include imputation of metabolite values fo r women who were missing two metabolites in a

class. Diethyl- and dimethylphosphate metabolites were summed on a molar basis (as nm/liter) to

obtain total diethylphosphates (D EPs) and total dimethylphospha tes (DMPs), respectively, and

together to obtain total dialkylphosphates ( DAP) levels. All biomarkers except MDA were

included in analyses as log 10 

# linear terms in the initial model selection stage. Results were

reported for DEPs dichotomized at the median level. MDA was dichotomized at the limit of

detection because only 21.6% of the population had detectable values. Maternal plasma

paraoxonase 1 activity was cl assified in tertiles.

A high prevalence of pesticide us e was reported in this cohort of pregnant women residing in

New York City. The study cohort was enrolled between 1998 and 2002, which, the authors point

out, overlaps with a period when chlorpyrifos an d diazinon were being phase d out of residential

use. However, the authors note that in 1997, chlor pyrifos was the most heavily used insecticide

by pest control operators at the New York City Housing Authority. The aut hors noted that all of

the women who participated in personal air mon itoring of their home had detectable levels of

diazinon, chlorpyrifos, carbamate propoxur, and o-phenylphenol. 46% of women in the cohort

reported that pesticides had been applied in their home during their pregnancy. Total

diethylphosphates were calculate d as the sum of di ethyldithiophosphate, diethylphosphate, and

diethylthiophosphate. In this coho rt, the DEPs were detectable in 88.8% of the mothers, with a

median (IQR) of 24.7 nm/liter (8.9–52.8 nm/liter). Tota l dimethylphosphates were calculated as

the sum of dimethyldithiophosphate, dimethyl phosphate, dimethylthiophosphate. DMPs were

detectable in 90.2% of the mothers, with a median (IQR) of 47.8 nm/liter (15.6–149.2 nm/liter).

Total dialkylphosphates were dete ctable in 96.5% of the mothers, with a median (IQR) of 82.0

nm/liter (35.2–194.7 nm/liter).

To assess infants’ neurodevelopment, the inves tigators conducted a beha vioral assessment using

the Brazelton Neonatal Behavioral Assessment Scale (BNBAS). All BNBAS examinations were

completed within 5 days of delivery, and 86.1% were conducted within tw o days of delivery.

The BNBAS includes 28 behavioral items and 18 primitive reflexes. The exam does not yield a

single score but instead assesses the infant acr oss different developmental areas; infants'

behaviors were scored acco rding to seven domains:

1) Habituation - the ability to respond to and inhibit di screte stimuli while asleep;

2) Orientation -attention to vi sual and auditory stimuli and quality of overall alertness;

3) Motor - motor performance and quality of movement and tone;

4) Range of state - a measure of in fant arousal and state lability;

5) Regulation of state - the ability to regulate state in th e face of increasing levels of

stimulation;

6) Autonomic stability - signs of stress rela ted to homeostatic adjustments of the central

nervous system;

7) The number and type of abnormal primitive reflexes

# Chlorpyrifos Human Health Risk Assessment D424485

Page 291 of 531 Of the 308 infants evaluated, all 18 primitive refl exes assessed in the BNBAS, 64% had all reflexes

within the normal range, 18% had one abnormal refl ex, 8% had two abnormal reflexes, and 10% had

three or more abnormal reflexes. The majority of abnormal reflexes were due to hypotonicity (which

includes a reflex not elicited after stimulation). Authors do not discuss the prevalence of other common

causes of hypotonicity such as hypothyroidism, hypoglycemia, hypoxic encephalopathy.



# The authors observed an association between both generic and pesticide-specific biomarkers of

prenatal organophosphate pesticides and an in creased number of abnormal primitive reflexes

which are considered a critical marker of neurologic integrity. No tably, no statistically

significant adverse associations were found for the dialkylpho sphate metabolites and any

measured behavior in the au thor’s primary analyses.

They did observe, however, an interaction be tween paraoxonase 1expres sion levels and total

dimethylphosphates on risk of abnormal reflexes, su ch that infants born to women in the first

(interaction p = 0.002) and second (interaction p = 0.01) tertiles (slowe r metabolizers) had a

greater risk of abnormal reflexes than infants of those in the highe st tertile (fast metabolizers). In

the first tertile of paraoxonase 1 expression, the relative risks ( RR) of having abnormal reflexes

were 1.78 (1.01, 3.14) associated with total diethylphosphates, 1.96 (1.27, 3.03) for total

dialkylphosphates, and total dialkylphosphates 2.38 (1.37, 4.15). In the second tertile of

paraoxonase 1 expression, the corresponding rela tive risks (RR) were 1.42 (0.85, 2.35) for total

diethylphosphates, 1.66 (1.03, 2.65), for total dialkylphosphates, a nd total dialkylphosphates 1.75

(0.96, 3.17). No increased risk of abnormal re flexes with increasing exposure was found for

women in the highest tertile of paraoxonase 1 expression, nor was a simi lar interaction observed

between total diethylphos phates and paraoxonase 1.

Higher levels of total diethylphospha tes and total dialkylphosphates, too, were associated with an

increase in the risk of having abnormal reflexes . Subjects with prenatal total diethylphosphates

levels above the median delivered infants who we re 2.3 times more likely to have at least two

abnormal reflexes (95% CI: 1.1, 5.0). Among women with lower tertiles of paraoxonase 1

expression ( i.e. , slow organophosphate pesticide metabo lizers) the authors also observed a

significantly increased risk of abnorma l reflexes with increasing exposure.

Relative to the first quartile, qua rtiles 2–4 of total diethylphospha tes, total dimethylphosphates,

and total dialkylphosphates were as sociated with an increased pr oportion of abnormal reflexes,

although the associations did not increase monotonically and vari ed in their strength and

precision. The authors speculated that th is may due to a threshold effect.

With the exception of MDA, there appeared to be stronger associations between metabolite

levels and abnormal reflexes for examinations pe rformed after the first day of life, although only

the total dimethylphosphat es interaction p-value interaction 

# = 0.10. In contrast, an interaction

(p interaction 

# = 0.10) with MDA showed that, for infa nts examined before day 2, the relation

between MDA and abnormal reflexes was st ronger (RR= 2.51, 95% CI: 1.61,3.90) compared

with that for infants examined later (RR= 1.34, 95%CI: 0.72, 2.49).

The authors noted that their findi ngs were remarkably consistent with those of Young et al.

(Neurotoxicology, 2005) who reported that for each log 10 

# unit increase in total

# Chlorpyrifos Human Health Risk Assessment D424485

Page 292 of 531 dialkylphosphates, there was a 26% increased ri sk of abnormal reflexes, (compared to 32%

increased risk in this study). The magnitudes of association were also similar for total

diethylphosphates (RR Young 

# = 1.25, RR current study 

# = 1.49) and total dimethylphosphates (RR Young 

# =

1.20, RR current study 

# ) = 1.13). Both studies reported stronge r associations between dialkylphosphate

metabolites and abnormal reflexes for older children, although, in the present study, the

interaction with day of administrati on was not statistically significant.

The association between MDA, a malathion-specif ic metabolite, and abnormal reflexes appeared

to be age dependent in this study, with th e most pronounced effect observed among infants

evaluated within the first day of life. The aut hors noted that this fi nding conflicts with the

suggestion of later effects for dialkylphospha te metabolites, including dimethylphosphates,

which encompasses nonspecific metabolites of ma lathion. The authors concluded that it is

unclear what pesticides may be driving this disparity.

Study Review

This was a well conducted prospective study conducted in a young, predominantly minority

population. The study design, analytic approach, and statistical anal yses were appropriate. The

authors used a sensitive albeit non-specific marker of chlorpyrif os pesticide exposure

(DEP/DAP). While the analytic methods appear sound, authors assessed these markers using

only a single maternal urine specimen taken dur ing the third trimester. However, in their

discussion, the authors note that organophosphate pe sticide metabolites have short half-lives. If

sources and patterns of exposure remain static, a single measurement would be a good indicator

of pesticide exposure during preg nancy. If, however, sources and exposure patterns change over

short time-spans, perhaps on the order of days to weeks, such an exposure assessment would not

necessarily provide a good indica tor of the relevant exposure. Like the many other sources of

error possible in this study, this, too is likely to result in a non-differential misclassification of

the exposure, with respect to the disease outcome. Serial measurements of urinary biomarkers of

pesticide exposure in the third tr imester would shed light on thes e patterns, alt hough the benefits

of such information are offset by the added cost , logistical complexity expense, and burden on

the study participants.

The Neonatal Behavioral Assessment Scale (NBAS), used to ascertain the health endpoints in

this study, is designed to examine indivi dual differences in newborn behavior

( http://www.brazelton-institute.com/intro.html ). Examinations took place in a quiet, semidarkened, warm room adjacent to the neonatal nursery, or in the mother’s private room.

Examiners in this study were either trained and certified by the Brazelton Institute or trained by a

certified examiner. It was not specified how ma ny examinations were conducted by the certified

examiners, nor how recently the certified examiners had received their certifica tion. Procedures

for tracking the quality of the a ssessment process were also not discussed in the report. The

health outcome assessment methodology used in th is study is less than ob jective; assessments

rely on substantial interaction between the infant and the examiner. Procedures for ensuring that

the examiners were blinded to th e participants’ exposures were not discussed in the article.

However, authors did include examin er as an indicator variable in statistical analyses to control

for slight differences in examiner methodology.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 293 of 531 Moreover, the behavioral assessment was admini stered before hospital discharge only on a

subset of children in the cohor t (n =311/404). The BNBAS was not administered if the infant

was admitted to the NICU (n = 21); if the infant was delivered and discharged over a weekend (n

= 43); if the parent refused (n = 5); if the infant was not testable (n = 2); or if study personnel

were unavailable (n = 22). Fact ors related to weekend delivery ( e.g. , fewer inductions) would be

underrepresented among the tested subjects. This missing data has the pote ntial to result in a

selection bias, reduce the degree of precision with which associations were estimated, and limit

the generalizability of the study findings. However, the authors’ explanation for the incomplete

ascertainment is presented and seems appropr iate and largely logistical in nature.

The authors employed a reasonable set of exclusion cr iteria that likely resulted in recruitment of

a study population that was rela tively homogeneous with re spect to confounders of the in utero

pesticide exposure-neurodevelopment relationshi p (compared to the general population). The

potential for confounding was thus limited by de sign (restriction). Bias due to confounding was

also controlled in the analysis, by assessing and adjusting for risk factors for neuromuscular

functioning that are potentially asso ciated with pesticide exposure, including maternal age, race,

marital status, education, cesarean delivery, delivery anesthesia, infant age at examination, infant

gender, infant jaundice, and sm oking, alcohol consumption, caffein e consumption, or illicit drug

use during pregnancy, and the exam iner conducting the BNBAS.

The use of an automated statistical procedure (backward elimination a nd change of effect

measure) as a means to eliminate potential cova riates is discouraged in some epidemiologic

circles on the basis that assessmen t of confounding is generally not considered to be a statistical

endeavor, although they continue to be used by others, larg ely on the grounds that it is

‘objective’. The authors used a change-in-estima te approach, rather than a reliance on the

statistical significance (p-value) asso ciated with a covariate; their variable selection rule has been

shown to produce more reliable models than va riable selection methods based on statistical

significance (Greenland, 1989). Becaus e they used a backward elimination procedure, they

considered confounding by a given risk factor in th e context of adjustment for other covariates.

The approach to covariate selection is a mi nor consideration in th is study, and different

approaches are not likely to qualitatively cha nge the study findings or conclusions. A robust

discussion of covariate selection concepts can be found in Chapter 15 of Modern Epidemiology

(Kenneth J. Rothman, Sander Greenland, Timothy L. Lash).

The authors noted that post-entry exclusions/loss-to follow-up in the study may have biased their

findings if they were associated with both organophosphate pesticide levels and neonatal

behavior. Missing data has the potential to induc e a selection bias if the reasons for missingness

are determined by exposure (or a predictor of exposure) and are also not independent of

neurodevelopment. Most of the excluded wome n were those who moved out of the study area,

were lost-to-follow up, or lacked prenatal biologic specimens, which largely reflect

socioeconomic condition. Participants included and excluded differed slighted with regard to

maternal age and race; however, these factors were not associated w ith number of abnormal

reflexes in this study. These considerations may, however, limit the generalizability of the study

findings.

The statistical analysis used in the study wa s appropriate, although seve ral subtleties in the

# Chlorpyrifos Human Health Risk Assessment D424485

Page 294 of 531 analytic procedures are worthy of mention. The main finding of the study was a statistical

interaction between biomarker levels of orga nophosphate pesticides and paraoxon activity. It was

not clear whether this effect modification was consideration a priori . Also, the authors imputed

missing data, and this may 1) affect the precisi on of association estimat es and 2) result in

attenuated effect estimates as a result of expos ure measurement error. The description of the

imputation procedure used was insufficient for dr awing strong conclusions regarding its validity

with respect to the magnitude of measurement erro r/bias, and its effect on estimates of precision.

The generalizability of the study may be limited as consequence of the particulars of the study

population assessed and the numerous exclusion criteria applied. This study population consisted

of primiparous women, recruited early on in their pregnancies from the pr enatal clinic and two

private practices at Mount Sinai Hospital in New York from March 1998 to March 2002,

consisted predominantly of young Hispanic women with low education. Sixt y-eight percent were

less than 25 years old. Just over half were Hispanic (51.1%), although substantial numbers of

White (20.3%) and African-American (27.3%) women also particip ated. Just under half (47.6%)

classified themselves as being single. The exclusion criteria a pplied to potential study

participants further focused the population assessed, relative to the general population of

pregnant women. In the likely situation that the prevalence of important modifiers of the

exposure-fetal development association in this focused study population is different from that in

other populations of concern, gene ralizability will be limited. Im portant modifiers to consider

may include time spent in the home, patterns of food preparation a nd consumption, health

consciousness, the baseline health status of the mother (and father), and a host of socioeconomic

determinants including access to health care. Th e investigators finding of effect modification of

the biomarker-fetal growth relationship by PON1 activity is illustrative of limits on the

generalizability of the study; popul ations who have high levels of paraoxonase activity may vary

by genetotypic and phenotypic determinants. These factors were not discu ssed in the report, but

it has been noted previously that there is 10- to 40-fold inter-individual variability in rates of

paraoxon hydrolysis (Playfer et al 1976).

The primary finding was an interaction betw een high paraoxonase 1expression levels and

organophosphate pesticide exposure, whereby fast metabolizers had lower risk of abnormalities

associated with prenatal pesticide metabolite leve ls in maternal urine samples. As with their

previous report, bias due to c onfounding, and selection of study part icipants is unlikely to have

large and qualitative impact on the association obse rved. To the extent that prenatal chlorpyrifos

is the true exposure of inte rest, the assessment of non-specific organophosphate pesticide

metabolites in a single sample of prenatal ur ine is a primary potential source of exposure

measurement error. Because of th e prospective design, information bi as is expected to result in

attenuation of the true (unobser ved) associations, toward (obs erved) associations that are

relatively smaller, or null.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 295 of 531 Works Cited

Berkowitz GS, Wetmur JG, Birman-Deych E, Ob el J, Lapinski RH, Godbold JH, Holzman IR,

Wolff MS. In Utero Pesticide Exposure, Maternal Paraoxonase Activity, and Head

Circumference. Environmental Health Perspectives. 2004;112:388–391.

Breslow NE, Day NE. Statistical Methods in Ca ncer Research: Volume II: The Design and

Analysis of Cohort Studies. Lyon: IARC, 1987.

Engel SM, Wetmur J, Chen J, Zhu C, Barr DB, Canfield RL, Wolff MS. Prenatal Exposure to

Organophosphates, Paraoxonase 1, and Cognitive Development in Childhood. Environmental

Health Perspectiv es 2011;119:1182-1188

Greenland, S. Modeling and Variable Selection in Epidemiologic Analysis. American Journal of

Public Health. 1989; 79:340-349.

Miettinen OS. Stratification by a multivariate confounder score. American Journal of

Epidemiology 1976;104:609- 620.

Playfer J, Eze LC, Bullen MF, Evans DAP. Genetic polymorphism and inter- ethnic variability of

plasma paraoxonase activity. J Med Genet. 1976;13:337–342.

Rothman KJ, Greenland S, Lash TL. Modern Ep idemiology. Third Edition. Lippincott, Williams,

& Wilkins, 2008.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 296 of 531 

# Article 6. Young et al. (2005)

Association Between In Utero Organophosp hate Pesticide Exposure and Abnormal

Reflexes in Neonates. Jessica G. Young, B renda Eskenazi, Eleanor A. Gladstone, Asa

Bradman, Lesley Pedersen, Caroline Johnson, Dana B. Barr, Clement E. Furlong, Nina T.

Holland. NeuroToxicology. 26:199–209 (2005).

Study Summary

Young et al. assessed th e relationship between in utero and early postnatal organophosphate

pesticide exposure and neonatal neurobehavior in the Center for the Health Assessment of

Mothers and Children of Salinas (CHAMACOS) c ohort. Urinary levels of dialkylphosphate

(DAP) metabolites, including dimethyl and diethylphosphate metabolites, measured at

approximately fourteen and twenty-six weeks gestation, and once post-delivery, measured at

approximately seven days postpartum were asse ssed as markers of exposure to organophosphate

pesticides. Cognitive development was assessed using the Brazelton Neonatal Behavioral

Assessment Scale (BNBAS).

The CHAMACOS study is a long itudinal birth cohort study of e nvironmental exposures and the

health of pregnant women and their children livin g in the Salinas Valley, California. The Salinas

Valley is a major center of agricultural produc tion in the United States with approximately

500,000 pounds of organophosphate pesticides app lied annually, according to the CDC.

(California EPA. Pesticide Use Reporting 2001 Summary Data, 2002.

(www.cdpr.ca.gov/docs/pur/pur01rep/ 01_pur.htm) Pregnant women initiating prenatal care at

the Natividad Medical Center, a county hospital locat ed in the city of Salinas, were recruited to

participate in the Center for the Health A ssessment of Mothers and Children of Salinas

(CHAMACOS) project. Both the source and study populations consisted la rgely of low-income,

Latina agricultural workers. Women considered e ligible for the study were less than 20 weeks

gestation, aged 18 years or olde r, Medi-Cal e ligible, fluent in Eng lish and/or Spanish, and

planning to deliver at Natividad Medical Center. A total of 6 01 women were enrolled between

October, 1999 and October, 2000, with 528 followed through delivery of a live born infant. The

BNBAS was performed on 421 infants. Eight twins and 27 preterm infants were excluded from

analysis.

Exposure was assessed by measurement of organophosphate dialkylphosphate (DAP)

metabolites in maternal urine samples collected between 5 and 27 weeks gestation and again

between 18 and 39 weeks. Maternal urine specimens were generally collected at the time of the

prenatal and post-delivery interviews. The post-deliv ery urines were collected within 1 week of

delivery for 73% of the sample, with the re mainder obtained up to 176 days afterwards.

Quantification of metabolites was conducted by Centers for Di sease Control and Prevention

(CDC) labs. Total DAP metabolite level was defined as the sum of the molar concentrations of

the six DAP metabolites, dimethylphosphate (DMP ) metabolite level as the sum of the molar

concentrations of the three dimethylphosphate metabolites only, and diethylphosphate (DEP)

metabolite level as the sum of the molar co ncentrations of the three diethylphosphate

metabolites. Total DAP, DMPT, and DEP levels were determined for each participant for each of

the two pregnancy urine samples and for the pos t-delivery urine sample. Individual metabolite

levels below the limit of detection (LOD) were assigned a value of the LOD divided by the

# Chlorpyrifos Human Health Risk Assessment D424485

Page 297 of 531 square root of two, and this value was incl uded in each sum. A small number of missing

metabolite values were imputed using regressi on to predict the missing metabolite level as a

function of the other known metabolites for give n participant at a give n time point. The total

DAP, DMP, and DEP metabolite levels were then log 10 

# transformed.

For 30% of participating infants, developmental outcomes were assessed prior to discharge from

the hospital; the remainder was assessed at the CHAMACOS research office or in the

participant’s home. Assessments were performed aw ay from the mother in a private room with

low-level light and noise control. The BNBAS, consists of 28 be havioral measurements scored

on a nine-point scale and 18 reflex items scored on a four-point scale. These scores were

classified into seven clusters : habituation, orientation, motor performance, range of state,

regulation of state, autonomic stability, and reflex. Higher scor es on the behavioral clusters

indicate more optimal functioning while higher scor es on the reflex cluster indicate less optimal

functioning. Missing individual BNBAS items for the six behavioral cl usters were imputed as the

median value of known scores within the cluster a ssociated with the missing item. If all items in

a particular cluster were missing, then the cluster score was considered missing. Missing

individual reflex items comprising th e reflex cluster were not imputed.

Separate linear regression models were fit for si x of the seven clusters of the BNBAS. Poisson

regression was used to model the association be tween urinary metabolite levels and number of

abnormal reflexes. Independent vari ables included the average of prenatal metabolite levels, the

postnatal level, and relevant covariates. Models relating metabolite levels to each cluster score

were initially fit including the postnatal metabol ite level and the averag e of the two prenatal

metabolite levels. For eighteen mothers missing one of the two pregnancy urine samples, the one

remaining was used to represent the average. Al l models adjusted for covariates, and were rerun

with creatinine-adjusted metabolite levels. Analys es were performed for the entire study sample

and stratified by the median age at BNBAS assessment (3 days).

Of the 378 infants included in this analysis, 47.5% were male. The mean maternal age was 26±5

years. Most mothers lived at or below the povert y line (64%), were born in Mexico (85%), had

previous live births (68%), we re married (81%), and had not completed high school (81%). Few

women smoked (5%) or drank alcohol (16%) dur ing pregnancy, although most reported caffeine

use (75%). Only 1% of the sample reporte d any illicit drug use during pregnancy.

The authors observed a statistically significant association between or ganophosphate pesticide

exposure and the reflex cluster of the BNBAS, both for the entire sample and for infants assessed

at greater than 3 days of age. Among the >3 da y old infants, increasing average prenatal urinary

metabolite levels were associated with both an increase in number of abnormal reflexes (total

DAP: β = 0.53, 95% CI = 0.23, 0.82; DMP: β = 0.41, 95% CI = 0.12, 0.69; DEP: β = 0.37, 95%

CI = 0.09, 0.64), and the proportion of infants with more than three abnormal reflexes (total

DAP: OR = 4.9, 95% CI = 1.5, 16.1; DMP: OR = 3.2, 95% CI = 1.1, 9.8; DEP: OR = 3.4, 95%

CI = 1.2, 9.9). No other associations between eith er prenatal or postnat al DAPs with any other

developmental outcomes were observed.

Study Review

# Chlorpyrifos Human Health Risk Assessment D424485

Page 298 of 531 Overall, authors performed a we ll-conducted study of pre- and pos t-natal pesticid e exposure and

adverse neurodevelopmental outcomes. The prosp ective nature of the study, use of multiple

biomarkers of exposure, and extensive data co llection allowing for adju stment of potentially

confounding variables are key strengths of this wo rk. For the purpose of informing the nature of

the relation between chlorpyrifos per se and these outcomes, use of non-specific OP metabolites

is a disadvantage. Other limitations of this work are also noted.

The ascertainment and analysis of urinary or ganophosphate pesticide markers was appropriate.

Quantification was conducted at the CDC, us ing published methods. DAP metabolites are

sensitive albeit non-specific markers of orga nophosphate pesticide exposure. They are likely

more accurate and objective indicators of organophos phate pesticide exposu re, relative to other

ascertainment methods such as self-report. In th eir discussion, the authors note that, “presently,

there are no better methods of exposure a ssessment to capture exposure to multiple

organophosphate compounds.” It is difficult to infer chlorpyrifos effects specifically using

metabolite levels, as they also indicate exposure to other organophosphates. The study

population was likely sustained many other potentia lly relevant exposures to multiple chemicals

and other pesticides which may affect development. It is not possible to disentangle the various

effects from these potential co-exposures and chlo rpyrifos in this study. Moreover, the degree to

which metabolites in prenatal maternal urine samples reflect ‘usual’ levels, and thus usual

exposure, is uncertain. The author s’ observation of large within-m other variability in pesticide

marker levels between the two prenatal sampli ng periods is evidence that a single sample may

not be representative of longer-te rm levels in this population, pa rticularly when exposures are

transient and highly variable. Av eraging of the two prenatal exposure measures may ameliorate

this issue somewhat.

Metabolite levels in postnatal urines likely reflect early postnatal, rather than in utero exposure,

given the short half -life of organophosphate pesticides. This is particularly likely for the 27% of

postnatal samples collected greater than one week after delivery. The effect of errors in the

assessment of organophosphate pesticide exposur e, whether due to mismeasurement of the

metabolites, imperfect correlation between meta bolite levels and pesticide exposure, or

imputation of missing data, is likely to be an at tenuation of the associa tion (a bias toward no

association) in this prospective study.

The BNBAS was appropriately administered by one of four examiners trained by a BNBAS

certified trainer. BNBAS is considered to pr ovide an appropriate ev aluation of newborn

development when administered up to the second month of life, and is considered a valid tool,

when properly conducted. The cluster approach to classification of BNBAS scores has

precedence in the literature. Missing BNBAS data points were imputed, if possible. Indeed, there

was a substantial amount of missing outcome data, due largely to the par ticular requirements of

the assessment ( e.g. that the infant be asleep immediatel y prior to the assessment of habituation

and that the infant by crying prior to assessment of consolability and self-quieting). For example,

fifty-four percent of the sample was missing all habituation items and an additional 22% required

at least one imputation. Forty per cent of the sample required at l east one imputation of the four

items comprising the regulation of state cluste r, with 13, 25, and 0.5% of the sample requiring

one, two, and three items imputed, respectively. Indi vidual items with the most missing values

for the regulation of state cluster were consolab ility and self-quieting. Two infants were missing

# Chlorpyrifos Human Health Risk Assessment D424485

Page 299 of 531 all items comprising the orient ation cluster score. The numbe r of abnormal reflexes was

calculated based on non-missing values only. Reflex es with the most mi ssing values included

incurvation (9%), nystagmus (7%), and tonic neck reflex (7%). Missing values did not exceed

4% for the remaining reflex items. Again, the effect of errors in the ascertainment of

developmental outcomes is likely to result in attenu ation of observed associ ations as it is unlikely

that missingness in outcome measur e is related to pesticide exposur e, as examiners were blind to

the infant’s exposure status and the imputati on procedures were unlikely to induce bias.

The analyses presented were appropriate to the data collected and the research question posed.

The authors averaged participants’ pesticide mark er levels from the two prenatal urine samples

(if available), and, as a separate variable, the pos tnatal metabolite level. Sixteen mothers were

missing the post-delivery urine. In order not to exclude those with missing post-delivery urines

from the statistical models, results were re-exami ned with the variable representing post-delivery

metabolite level excluded from all models. The eff ect of the missing exposure data is a decrease

in the precision, and also likely at tenuation of observed associati ons due to measurement error.

Potential confounders considered in this investigation included primarily predictors of BNBAS

performance, including: maternal age, pr e-pregnancy body mass index, self-reported smoking,

alcohol, caffeine, and illicit drug use duri ng pregnancy, vitamin use during pregnancy,

gestational age at which prenatal care was initiated, total number of prenatal care visits, average

blood pressure during pregnancy, parity, method of delivery, general anesthesia use during

delivery, breastfeeding initiated after delivery, and poverty level. Othe r covariates included

infant sex, age in days at BNBAS assessment, minutes since last fed at BNBAS, and BNBAS

examiner. Missing covariate values were imputed by random selection of an individual among

all participants with known values for the covariate and assigning the missing value the selected

participant’s value.

Covariates were selected for in clusion in final models if they were statistically significantly

associated with each of the seven cluster sc ores in univariate mode ls, (alpha = 0.15). The

authors used a less conservative alpha level as a cut-off for selection of covariates, a somewhat

more conservative approach to c ovariate selection, rela tive to use of the c onventional alpha level

of 0.05. However, the use of a statis tical test as a means to select covariates from a larger set of

potential confounders is discour aged in some epidemiologic circles (Miettinen 1976, Breslow

and Day 1987, Greenland 1989). The assessment of confounding is generall y not considered to

be a statistical endeavor, in part because the association between the confounder and the outcome

is just one factor that determines the degr ee of confounding, and also because the statistical

significance of an association is a function not only of the magnit ude of the association but also

of other factors ( e.g. , sample size, and sample variability) which are irrelevant to bias due to

confounding. Moreover, the use of significance tes ting in the selection of confounders treats

false negative errors ( i.e. the deleting a confounder) as secondary to false positive error

(inclusion of a non-confounder). This implicit ra nking of error is backward, as deleting a

confounder introduces bias and is ju stified only if the bias induced by its exclusion is tolerably

small or deemed to be worth the precis ion gain, whereas incl uding a non-confounder only

(potentially) reduces precision by in creasing the size of the model. This is a small consideration

in this study, and different approa ches are not likely to qualitative ly change the study findings or

conclusions. Birth weight and gest ational age were considered potential causal intermediates in

# Chlorpyrifos Human Health Risk Assessment D424485

Page 300 of 531 this relation, and not confounders.

Regardless of the confounder evaluation conduct ed by the investigator s, residual confounding

due to poorly measured or unmeasured confounders is likely to have influenced the observed

associations. Missing covariate da ta were imputed. The resulting an alysis is relatively more

precise, owing to the larger sample size analyzed, but this statistical effici ency comes at the trade

off of bias due to residual confounding by impe rfectly measured covariates. Socioeconomic

conditions and other difficult to measure determ inants of early life de velopment may further

confound the reported associations in the direction of mo re deleterious effect s associated with

the organophosphate metabolites and with unknown ma gnitude. The potential for this bias may

be mitigated somewhat due to restriction in th e variability of these c onfounding factors in this

relatively homogeneous study population.

The generalizability of the findi ngs to other populations may be limited, due to the particular

nature of this study population with respect to fa ctors which may modify the association between

organophosphate exposures and infant development.

The results of this investigati on are suggestive of a detrimenta l association between prenatal

organophosphate exposure, as measured by matern al urinary metabolite levels, and abnormal

reflexes, as measured by the BNBAS, particularly in infants assessed after th e first 3 days of life.

This study may be limited in its direct contribu tion to the literature base regarding the specific

association between chlorpyrif os and developmental outcomes; although approximately 500,000

pounds of organophosphate pesticides are applied annually in Salinas Valley, chlorpyrifos

constitutes only a small percentage of the pesticides applied. Alt hough the prospective study

design and reasonable methods protect from certain false-positive inducing biases, there remains

the potential of residual confounding in the direct ion of an adverse effect on development by

unmeasured factors.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 301 of 531 

# Article 7. Rauh et al. (2006)

Impact of Prenatal Chlorpyrifos Exposure on Neurodevelopment in the First 3 Years of

Life Among Inner-City Children. Virginia A. Rauh, Robin Garfinkel, Frederica P. Perera,

Howard F. Andrews, Lori Hoepner, Dana B. Barr, Ralph Whitehead, Deliang Tang, Robin

W. Whyatt. Pediatri cs. 18(6): 1835-1859.

Study Summary

To evaluate the affect/impact of prenatal e xposure to ambient and indoor pollutants including

pesticides, the investigators conducted a pr ospective cohort study among inner-city (NYC)

mothers and their babies born between Februa ry 1998 and May 2002. A major aspect of the

protocol was to assess pesticide exposure, specif ically chlorpyrifos, and its impact on pregnancy

outcome, neurodevelopment, and behavior. Prev ious publications presented outcomes of this

cohort such as birth weight and length findings (Whyatt, et al, 2004 and Perera et al, 2003.) This

publication presents findings focused on the neur odevelopmental assessments made at the 12, 24,

and 36 month time points.

The women enrolled into the study were identified during thei r pregnancy, through the New

York Presbyterian Medical Center and Harlem Hosp ital. To be eligible for the study they were

required to be between the ages of 18 and 35 year s, non-smokers (later verified by blood cotinine

levels,) and without a history of diabetes, hypertension, HIV infecti on, or drug abuse. They must

have sought prenatal care by the 20 th 

# week of pregnancy and self-i dentified as African American

or Dominican. At the time of this report, the authors expl ain that out of the original 648 pregnant

women consented, 536 (83%) of them and 254 children were still actively enrolled at the 3 year

milestone. Of these children, 228 completed evalua tions at 12 months of age, 227 at 24 months,

and 228 at 36 months, with 189 children ha ving full data at all 3 time points.

The primary source of chlorpyrifos measuremen ts were samples of cord blood collected at

delivery as well as samples of maternal blood collect ed shortly after (within 2 days of) delivery.

These serum levels were supplemented by maternal self-report of resident ial pesticide use during

the third trimester of pregnancy. Using findings fr om previous analyses of chlorpyrifos effects

on birth weight (Whyatt, et al, 2004 and Perera et al, 2003,) the investigators chose a cord blood

chlorpyrifos limit of 6.17pg/g to define the e xposure groups. Children were categorized as

having either high (>6.17pg/g) or lo w (<6.17pg/g) prenatal exposure.

Two instruments were used to evaluate neur odevelopment and child behavior. The Bayley

Scales of Infant Development II (BSID-II) was us ed to generate a Mental Development Index

(MDI) and a Psychomotor Development Index (P DI) at 12, 24, and 36 months of age. Second,

the behavior of the children was described by the mother using the 99-item Child Behavior

Checklist (CBCL) and the resulti ng syndrome scale scores. Fina lly, the Home Observation for

Measurement of the Environment (HOME) instru ment was used to assess the quality of the

child-care environment. This instrument collects data regard ing the physical and interactive

qualities of the child’s home as a measure of mental stimulation and interactions.

The maternal and cord blood samples also provided cotinine levels to conf irm maternal report of

environmental tobacco smoke (ETS) exposures a nd lead levels to understand the role lead

# Chlorpyrifos Human Health Risk Assessment D424485

Page 302 of 531 exposure may play interacting with the outcomes of measure. During their third trimester of

pregnancy, the women completed a 45-minute questionnaire to co llect demographics, education

and occupational history, income, active a nd passive smoking, alcohol and drug use during

pregnancy, and residential pesticide use. From this self-report questionnaire and additional

analyte measurement in biological sample, aut hors obtained information regarding potentially

confounding variables.

Multivariate linear regression and logistic regression were used to estimate associations between

prenatal chlorpyrifos exposure and developmen tal outcomes. General linear modeling of the

repeated measures (12, 24, 36 mont h evaluations) was also used to estimate the effect over time

that chlorpyrifos had on neurodeve lopment. All models included prenatal ETS exposure, gender,

ethnicity, gestational age at bi rth, quality of home care-taking envi ronment, maternal education,

and maternal IQ. Other measured co-variables in cluding blood lead levels were evaluated as to

the potential confounding influence on the main eff ect, and were not included in final models

due to lack of statis tical or biological ev idence of confounding bias.

The descriptive statistics pr esent the cohort as relativel y homogenous with respect to

socioeconomic variables but showing a wide range of chlorpyrifos exposure. Chlorpyrifos levels

ranged from undetectable to 63 pg/g with 20.6% of the cord blood results falling into the high

exposure category. Significant differences in chlorpyrifos exposure were seen across ethnic

categories with 24.2 % of African American part icipants and 14.9% of Do minican participants

classified as highly exposed (p =.02). Birth weight and prenat al exposure to ETS were also

significantly associated with th e exposure categories with in fants in the high exposure group

weighing approximately 211g less than babies in the low exposure group (p <.05) and 56% of

those in the high exposure group reporting prenatal ETS versus 32% in the low exposure group

(p = .002).

When the BSID-II results were analyzed in te rms of exposure group, no significant differences

were seen at the 12 and 24 month time points. At the 36-month milestone, although MDI scores

did not show a significant diffe rence across the exposure groups, PDI scores were significantly

lower among children in the highly exposed group (95.69 vs . 101.63, p = .006). Statistically

significant differences were also seen at the 36 month milestone, in mental (cognitive) and

psychomotor delays (p = .048 and .002 respectively.)

When analyzing the 36-month child behavior checklist (CBCL) (beh avioral) scores, results from

the 12 and 24 month milestones were not signifi cantly associated with exposure status. At 36

months, significant differences were observed in the general category of attention-problems

(p=.010) and in the more specific DSM-IV scale for ADHD problems (p = .018).

The multivariate regression models included gender, ethnicity, gestational age at birth, home

environment, prenatal ETS exposure, maternal e ducation, and maternal intelligence. The authors

reported that the difference in MDI scores wa s “marginally significant” (p = .06) with the

exposed group scoring an average of 3.3 points lo wer. Among the significant covariants were

race/ethnicity (at 24 and 36 months), gender of in fant (at 12, 24, and 36 months), gestational age

(at 24 and 36 months), low maternal educati on (at 36 months), and HOME score (at 36 months)

with Dominicans, male children, low gestational age, low maternal education (< high school,)

# Chlorpyrifos Human Health Risk Assessment D424485

Page 303 of 531 and low HOME score all linked to lower MDI scores . It was also report ed that environmental

exposures (chlorpyrifos and ETS) and sociodemogr aphic covariables accounted for 25% of the

variance seen in the 36-month MDI scores. When the same multivariate regression models were

calculated regarding the PDI scor es, none of the 12 or 24 month PDI scores showed significant

effects. The 36 month scores did show significa nt effects for chlorpyrifos exposure, race, and

gestational age.

Next, investigators calculated estimates of adju sted risk for developmental delays (MDI and

PDI) related to chlorpyrifos exposure. Adjust ments were made for gestational age, gender,

ethnicity, maternal education, maternal intel ligence, and home environment. The findings

showed that before 36 months of age, delays were no more likely in the highly exposed group.

But, at the 36 month milestone, the likelihood of highly exposed childr en developing mental

delays were 2.4 times greater (95% CI: 1.12-5.08, p = .02) and motor delays were 4.9 times

greater (95% CI: 1.78-13.72; p = .002) than th ose with lower prenatal exposure.

General linear models (GLM) were fit to descri be longitudinal developm ental trajectories for

high and low chlorpyrifos exposure groups, adjusti ng for race/ethnicity, gender, gestational age,

maternal educational level, HOME score, and ET S exposure. The GLM results were consistent

with the 12, 24, and 36 month linear regression anal yses indicating that associations between

developmental scores and chlorpyrifos were mo st pronounced at 3 years of age. The authors

describe an initial decrease in scores in both groups between the ages of 12 and 24 months, but

remark that such decreases are commonly observed among low-income groups followed by a

rebound in scores after 24 months of age. The rate of increase in the MDI scores after 24 months

was greater among the low exposure group th an the high exposure group but was not

significantly greater (p = .23). The GLM analysis for PDI scores showed a significant effect of

chlorpyrifos exposure over time with an esti mated deficit of approximately 7 points by age 36

months (p = .01).

The authors summarize three main findings from the study: 1) by age 3, the more highly

exposed children demonstrated mental and mo tor delays; 2) the observed developmental

trajectories for PDI and MDI scores confirmed th at the adverse impact on cognitive and motor

development increased over time; and 3) by age 3, highly exposed children were more likely to

demonstrate clinically signi ficant attention problems.

Study Review

The investigators conducted a well-designed prospective cohort st udy and retained a high

percentage (83%) of thei r participating mothers over the three year follow-up period. With very

narrow inclusion criteria, the i nvestigators selected a homogenous cohort of minority, inner-city

women, most of whom fall into low income and low education categories. This homogeneity

serves to equalize the study comparison groups with regard to potential measured and

unmeasured confounders, but may also limit the generalizability of the findings.

While the high retention rate of the mothers provides as stable study population, the authors do

not address that only 53% of the children from these mothers reached the three year milestone

with study data collected. It is unclear what, if any, pe rcentage of these children did not survive,

# Chlorpyrifos Human Health Risk Assessment D424485

Page 304 of 531 were lost to follow-up, or were too sick/affected by neurodevelopmen tal delays to participate.

Authors did not provide inform ation concerning the degree of missingness by chlorpyrifos

exposure category in the published analyses, however in supplemental analyses suggested by the

2008 FIFRA SAP on chlorpyrifos authors did prov ide this information (See Appendix 4-1). In

these additional analyses, authors concluded the degree of missingness was relatively equal by

exposure category and therefore not likely to have imposed a selection bias.

As discussed by the authors, one weakness of the study is the reliance on a single exposure level

(prenatal/cord blood.) It is acknowledged that this snapshot of exposure may not accurately

reflect both pre- and post-natal exposure levels, although in genera l, for chronic exposures with a

high prevalence and consistent frequency of use as indicated in this study population, a single

measure may be sufficient to accurately rank part icipant exposure levels. The authors further

explain that previous analyses of this cohort showed that exposure s to chlorpyrifos did not show

significant variation throughout preg nancy (Whyatt et al., 2007) and therefore concluded that this

single measure can accurately represent prenatal exposure. While this may accurately represent

prenatal exposure, there is no control for expos ure over the subsequent 3 years which may be

critical, especially as the author s note that their GLM analyses show a critical developmental rebound period between 24 and 36 months. From this study, it is unclear if an increase or decrease

in exposure during the period of early childhood could also infl uence the neurodevelopment of

these children. Finally, th e creation of a dichotomous exposure variable also brings limitations

to the interpretation of the study results due to the amountof within-group variation likely

present using this method of exposure classifica tion. By setting the cut-off at 6.17pg/g, there are

members of their “low exposur e” group who may approximate high exposure levels and vice

versa, resulting in non-differential measurement error.

The authors also address limitations to the se nsitivity and predictive validity of the

developmental tests, especially among children less than 3 years of age. Although, these tests

are considered to be most valid among school-aged children they are also used as indicators for

early intervention to prevent cont inued developmental delay. The au thors describe a statistically

significant reduction in scores as sociated with high exposure le vels but they do not discuss

whether this 7-point deficit is clin ically relevant in terms of dia gnosing or treating a child at this

stage of development.

Also complicating this analysis is the pervasiv e, non-specific nature of neurological effects and

the difficulty in attributing causal pathways re lated to just one environmental exposure.

Although the authors site previous research linking chlorpyrifos expos ure to inhibition of neural

growth (Dam, 1998) it is still difficult to attrib ute developmental and behavioral effects to a

single exposure variable measured at one point in time. In future studies it would be

advantageous to include other pote ntial covariates such as family history of developmental delay

or attention disorders (especially among parents and siblings) as well as other co-morbidities not

adjusted for by design ( i.e , restriction using eligibility criteri a to exclude co-morbidities). Not

only is the etiology of attention and related behavi oral disorders not well understood but also the

diagnosis of these disorders is often influenced by complex outside forces.

This well-designed study provides suggestive fi ndings linking prenatal chlorpyrifos exposure

with neurodevelopmental deficits at the three year s of age. The lack of outcome information for

# Chlorpyrifos Human Health Risk Assessment D424485

Page 305 of 531 a high percentage of cohort children is a concern which is not directly addressed by the authors.

The use of a single exposure estimate (which likely led to non-differential exposure

misclassification bias in this study) and an imprecise outc ome characterization (although a

clinical tool, neurodevelopmenta l milestones are ‘sof t’ outcomes to measure) may have an

accumulative effect to underestimate the true effect (bias results toward to null value), if a true

effect exists. As mentioned previously, at th e time of this publication, these analyses covered

only a snapshot of the findings of the ongoing cohor t study. The authors all ude to the fact that

they will use the subsequent evaluations, especial ly the 7-year scores to further describe the

effects of chlorpyrifos exposure on neurodevelopment and behavior.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 306 of 531 Works Cited

Whyatt RM, Rauh V, Barr DB, et al. Prenatal ins ecticide exposure and birth weight and length

among an urban minority cohort. Environ Health Perspect. 2004;112:1125–1132

Perera F, Rauh VA, Tsai WY, et al. Effects of transplacental exposure to environmental

pollutants on birth outcomes in a multi-ethnic population. Environ Health Perspect. 2003;111:

201–205

Whyatt, R. M., Garfinkel, R., Hoepner, L. A., Holmes, D., Borjas, M., Williams, M. K., . . .

Camann, D. E. (2007). Within- and between-home va riability in indoor-air insecticide levels

during pregnancy among an inner-ci ty cohort from New York City. Environ Health Perspect,

115 (3), 383-389. doi: 10.1289/ehp.9546

Dam K, Seidler FJ, Slotkin TA. Developmental ne urotoxicity of chlorpyr ifos: delayed targeting

of DNA synthesis after repeated administration. Brain Res. 1998;108:39–45

# Chlorpyrifos Human Health Risk Assessment D424485

Page 307 of 531 

# Article 8. Lovasi et al. (2010)

Chlorpyrifos Exposure and Urban Reside ntial Environment Characteristics as

Determinants of Early Childhood Neurodevel opment. Gina S. Lovasi, James W. Quinn,

Virginia A. Rauh, Frederica P. Perera, Ho ward F. Andrews, Robin Garfinkel, Lori

Hoepner, Robin Whyatt, Andrew Rundle. American Journal of Public Health. 101(1):63-70

(2005).

Study Summary

Lovasi et al evaluated asso ciations between neighborhood char acteristics and early-life

neurodevelopment in the Columbia Mother’s an d Newborn Study Cohort, and assessed whether

such characteristics confound th e association between exposure to chlorpyrifos and

neurodevelopment previously re ported. US Census data (2000) were used to estimate

neighborhood-level measures of physical infras tructure, socioeconomic status, crowding,

demographic composition, and linguistic isolati on, augmenting previously reported prenatal

chlorpyrifos exposure, psychomotor development, and mental development data on a New York

City birth cohort.

Study participants were recru ited during early pregnancy ( ≤ 20 th 

# week) among African-American

and Dominican women age 18-35 y ears, and registered at Ne w York Presbyterian Medical

Center and Harlem Hospital, in New York City. Women who smoked, had a history of drug

abuse, diabetes, hypertension, or HIV infection we re excluded from participation in the study, as

were women who resided in New Yo rk City for less than 1 year.

Cord blood plasma chlorpyrifos levels were asse ssed, and dichotomized as high (greater than the

third tertile level of 6.17 pg/g) or low (<6.17pg/g). Maternal blood levels of chlorpyrifos were

used to estimate cord blood levels for the 12% of infants for whom cord blood was not obtained.

The Bayley Scales of Infant Intelligence–Revi sed (BSID-II) were used to assess cognitive and

psychomotor development at age 36 months. De velopment was reported using a Psychomotor

Development Index (PDI) and a Mental Develo pment Index (MDI), which served as the

continuous outcome measurements in the study.

Participants’ neighborhoods were defined using a 1-kilometer network buffer. Using 2000 US

Census data, network buffer characteristics were determined, including: the percentage of

housing units without complete plumbing, the per centage of vacant housing units, the percentage

of residents below the federal pover ty line, the percentage of resi dents older than 25 years of age

who completed high school, the percentage of households receiving public assistance, the

percentage of housing units with one or more residents per room, racial composition, the

percentage of residents born outside the Unite d States, the percentage of Spanish-speaking

residents, and the percentage of residents w ho were linguistically is olated (all household

members aged 14 years and older have at least some difficulty with English).

Indicators of building disrepair were self-repo rted by mothers during pr enatal interviews, and

summed to create an index of disrepair that included one point for each of the following

problems: holes in ceilings or wa lls, peeling or flaking paint, wa ter damage, leaking pipes, and

# Chlorpyrifos Human Health Risk Assessment D424485

Page 308 of 531 lack of gas or electricity in the prior 6 months.

Generalized estimating equation models were created for the continuous outcomes (PDI and

MDI) and Bayley Scores of Intelligence at 36 m onths. Robust standard errors were used to

correct for possible autocorrelati on within community district areas. All models included the

following potential confounders: gender, gestati onal age at birth, Dominican versus African

American ethnicity, maternal education, mate rnal intelligence quotient, the presence of

secondhand smoke in the home during pregnancy, and an index of the quality of the home

environment with respect to caretaking. Using hi erarchal regression tec hniques which allow for

control of individual level as well as group-level variables, author s were able to further clarify

the nature of the association between mental and psychomotor development (Bayley scales of

infant development) and prenatal chlorpyrifos exposure. Other variab les including blood lead,

other environmental exposures among other fact ors were not considered confounders in the

prenatal chlorpyrifos and Bayley score association.

Of 266 children included as participants, 47% we re male, 59% were Dominican, and 41% were

African American. After adjust ing for potentially confounding va riables, high chlorpyrifos

exposure (greater than 6.17 pg/g) was associated with a 6.5-point decrease in PDI and a 3.3-point

decrease in MDI at age 36 months, as previo usly reported. These associations remained

statistically significant and similar in magnitude in models additionally adjusting for dilapidated

housing and neighborhood characteristics.

Study Review

This study helps clarify the nature of the rela tion between prenatal ch lorpyrifos exposure and

childhood neurodevelopment by controlling for the pot ential influence of socioeconomic status

(SES) using advanced statistical techniques. Unique to this re search, authors were able to

measure variability due to SES using several diffe rent variables measured at both the individuallevel and the group-level. This work enhances the precision and accuracy of the epidemiologic

measures previously reported.

The authors note that participants lost to fo llow-up were not signifi cantly different from

continuing participants with respect to maternal age, ethnicity, marital st atus, education, income,

or gestational age and birth weight of the newbor n; authors cited a retent ion rate at the 3-year

follow-up of 83%. How they arrived at that num ber is unclear. Originally, 648 pregnant women

consented to participate and 327 children completed a developmental assessment at

approximately 36 months of age. In this re port, analyses were conducted among 266 children

with available chlorpyrifos expos ure data, yielding an effective retained proportion of 41%. This

leaves open the possibility of selection bias resulting from loss to follow-up; one may only

speculate as to the magnitude and direction of such bias.

The direct measurement of cord blood chlorpyrif os in this study is a substantial strength,

compared to the assessment of non-specific ma rkers as assessed in similar studies of

organophosphate pesticide and child neurodevelopm ent. Umbilical cord blood was obtained at

delivery for 88% of the children in the study. For the remaining 12% for whom no cord blood

was obtained, cord blood plasma chlorpyrifos was estimated from maternal plasma levels using

# Chlorpyrifos Human Health Risk Assessment D424485

Page 309 of 531 blood sampled two days prior to delivery. Any ch lorpyrifos measurement error resulting from

this estimation is almost assuredly non-differential with respect to cognitive development

outcomes. Children excluded for missing chlorpyrifos exposure data were similar to the analytic

sample except that excluded children had more indicators of building dilapidation, so the

likelihood of selection bias of large magnitude is unlikely. The decision to dichotomize cord

blood chlorpyrifos le vels using the highest tertile as a cu t-point reduces the ability to assess

exposure-response relationships, and may somewhat decrease statistical efficiency relative to

using a continuous measure. If a large number of participants had levels of chlorpyrifos below

the level of detection, however, using a categorical approach dispenses with the need to estimate

or otherwise define exposure at low levels. The prenatal exposures assessed in this study are

likely correlated with other pren atal exposures and factors in the postnatal environment which

themselves predict cognitive development. This presents a challenge for a causal interpretation

of prenatal chlorpyrifos exposure per se , as the agent and time period responsible for observed

cognitive deficits.

The Bayley Scales of Infant Development–Revise d (BSID-II) is a widely used norm-referenced

developmental test for young child ren, it can be used to diagnose developmental delay, and it is

known to be sensitive to the eff ects of toxic exposures such as low-level intrauterine lead. The

assessment was appropriately administered, with each child being evaluated under controlled

conditions by a trained bilingual research assist ant. Inter-observer reliability was assessed but

data were not presented in this report. The authors note that, when administered at age 3 years,

the BSID-II demonstrates only moderate predicti ve power for subsequent intelligence and school

performance, but cite previous literature as eviden ce that the test is nevertheless clinically useful

for children performing in the subnormal range.

The statistical analyses were ge nerally appropriate. Missing data on covariates were estimated

using multiple imputations, and the variance estimates presented appropriately reflect the degree

of uncertainty caused by missing covariate data. Robust standard errors were estimated to

account for potential autocorrelation. The author s presented multiple model results, each with a

different indicator of neighborhood character. Only neighborhood covariates representing

socioeconomic conditions ( i.e. percentage of indivi duals living in poverty, and percentage with

high school education) were sta tistically significant predictors of PDI and MDI. Interestingly,

and without explanation, the auth ors did not present the results of a model which simultaneously

included all of the neighborhood char acteristics as covariates. Presumably, this was done for the

sake of parsimony, as one could argue against the inclusion of covariates considered not to be

predictors of cognitive development. Alternatively, these factors were, a priori

, considered

potential confounders, and the use of statistic al significance tests in the assessment of

confounding is discouraged in many epidemiologic circles.

The observation that neighborhood characteristics did not conf ound the observed association

between chlorpyrifos levels and cognitive de velopment in this study has several possible

explanations. This was a rela tively homogenous study population liv ing in the New York City

Area, and it is possible that confounding is limited by the small degree of variability in

neighborhood characteristics be tween those with and without high exposure levels of

chlorpyrifos. It is also possibl e that the neighborhood characterist ics are measured with error,

such that they fail to capture aspects of socioeconomic condition, neighborhood composition,

# Chlorpyrifos Human Health Risk Assessment D424485

Page 310 of 531 and neighborhood condition that effect cognitive de velopment, even within this context of

measurement of several individual-level as well as group-level SES measures. This residual

bias, if it exists, could (at least partially) explain the decrement in cognitive development

associated with chlorpyrifos exposure, assu ming that poorer neighborhood conditions are both

predictors of reduced cognitive function and co mparatively more prevalent among those with

high chlorpyrifos exposure. The first of these tw o explanations has no eff ect on internal validity;

under the assumption of no other bias, the observed association is unbiased, but external validity

may be limited due to the restricted study popul ation. The investigators’ methods to assess

neighborhood characteristics were well conceived and well impl emented, and reflective of

commonly utilized variables to measure important aspects of the multi-dimensional characteristic

of socio-economic status. However, such expos ures are notoriously di fficult to measure well

even using these advanced statis tical methods and approaches.

The authors did not assess effect modification of the association between prenatal chlorpyrifos

exposure and cognitive development by neighbor hood conditions, which is an equally, if not

more, interesting area of inquiry than assessm ent of confounding. The present study simply does

not have the sample size/statistic al power to address these effect s, but, in the discussion, the

authors speculate that large st udies that collect both neighbo rhood context and exposure data

could reveal patterns of eff ect modification. The relative hom ogeneity of the study population

may have precluded statistically robust examinati on of effect modification by factors related to

SES in this study.

The setting of the investigati on in a sample drawn from low-income African American and

Dominican communities is both a strength and a limitation of the study. The population is likely

at high risk of developmental defi cits, which increases the power of the study relative to a similar

study set in a population at low risk. The assessment of confounding by demographic and

neighborhood characteristics is limited by the re striction of the study sa mple to low-income

communities (a weakness), but so too is the bias induced by these potential confounders (a

strength). The generalizability of the study results may also be limited because participants were

recruited from a restricted range of neighborhood s in New York City (a weakness), but the study

population is likely among the most highly expose d and also at risk for cognitive impairment

(Landrigan, 1990a, 1990b; Landr igan et al., 1999).

This study represents an extension of previously published work with additional adjustment for

multiple neighborhood characteristics. The work was well conceived and conducted,

representing perhaps the best assessment of the association between chlorpyrifos and cognitive

development in the epidemiologic literature. Chie f among its strengths is th e direct assessment of

cord blood plasma chlorpyrifos, the prospect ive design, and the assessment and control of

potential confounders. Using the methods of data collection, statistical analyses, and enhanced

methods to evaluate the role of SES as a pot entially confounding variables, authors report no

substantial difference in the reported associati on between prenatal chlo rpyrifos exposure and

child neurodevelopment as previously reported (Rauh et al. 2006).

# Chlorpyrifos Human Health Risk Assessment D424485

Page 311 of 531 Works Cited:

Landrigan, P. J. (1990a). Health effects of environmental toxins in deficient housing. Bull N Y

Acad Med, 66 (5), 491-499.

Landrigan, P. J. (1990b). Housing and health: conclusions and challenges for the future. Bull N Y

Acad Med, 66 (5), 587-591.

Landrigan, P. J., Claudio, L., Markowitz, S. B., Be rkowitz, G. S., Brenner, B. L., Romero, H., . .

. Wolff, M. S. (1999). Pesticides and inner-ci ty children: exposures, risks, and prevention.

Environ Health Perspect, 107 Suppl 3 , 431-437.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 312 of 531 

# Article 9. Engel et al. (2011)

Prenatal Exposure to Organophosphates, Pa raoxonase 1, and Cognitive Development in

Childhood. Stephanie M. Engel, James Wetmur, Jia Chen, Chenbo Zhu, Dana Boyd Barr,

Richard L. Canfield, and Mary S. Wolff. Environmental Health Perspectives 2011;

119:1182-1188

Study Summary

This is the third of three published analyses of prenatal pesticide exposure and child growth and

development in the Mount Sinai Hospital Child ren’s Environmental Health Study. The first

(Berkowitz et al 2004) reported on assessments of birth weight, le ngth and head circumference.

The second (Engel et al 2007) reported on assessme nts of infant neurocognitive development and

reflexes shortly after birth. In this study, Engel and colleague s extended their 2007 to include

analysis of PON1 activity, PON1 polymorphisms, and cognitive development at ages 12 and 24

months and 6–9 years.

Of the 404 women originally enrolled between May, 1998 and July, 2001 (n = 404), a subset (n =

360) had organophosphate metabolite levels quantif ied in urine samples collected during the

third trimester. Prenatal maternal blood and co rd blood samples were analyzed for PON1 activity

and genotype. Children of these mothers returned for neurodevelopment assessments at ages 12

months (n = 200), 24 months (n = 276), and 6–9 (n = 169) years of age. Participants in this study

were predominantly young, unmarried, Hispanic wo men with low educational attainment. Most

women delivered term infants of normal birth wei ght, as prematurity and very low birth weight

were exclusion criteria for study enrollment.

The study population was drawn primarily from East Harlem and consisted largely of young

Hispanic women (predominantly Puerto Rican) , but also included African-American and

Caucasian women. Mothers were enrolled during ear ly pregnancy from the Prenatal Clinic and

two private practices at Mount Sinai Hospital from May 1998 to July 2001. The study included

only primiparous with singleton births. Mothers were excluded if they had any of the following:

an initial prenatal visit after 26 weeks of gesta tion, serious chronic dis eases, a serious pregnancy

complication that could affect fetal growth and development; alcohol cons umed greater than two

alcoholic beverages per day, illicit drug use. Mother s and infants were also excluded if the child

was born with a congenital malformation or severe prematurity. A total of 479 prenatal patients

were recruited; 75 were excluded because of medical complications , premature birth, an infant

with birth defects, inability to collect biologic specimens before birth, change of hospital or

residence outside New York City, or re fusal to continue to participate.

A questionnaire was administered to participants during their th ird trimester of pregnancy to

obtain information on environmental exposures, so ciodemographic characteristics, obstetrical

and medical history, a nd lifestyle factors.

Generalized linear models were used to analyze the relationship between total diethylphosphates

(DEPs), total dimethylphosphates (DMPs), tota l dialkylphosphate metabolites (DAPs), and

subsequent cognitive development evaluated at age 12 and 24 months using the Bayley Scales of

Infant Development, 2nd edition (BSID-II) and us ing the Wechsler Preschool and Primary Scale

# Chlorpyrifos Human Health Risk Assessment D424485

Page 313 of 531 of Intelligence, 3rd edition (WPPS I-III) at approximately age 7 year s. The authors also assessed

modification of pesticide metabol ite - cognitive development asso ciations by maternal and child

PON 1 activity and PON 1 polymorphisms.

The authors presented their results stra tified by level of race/ethnicity and PON1 genotype,

having observed apparent effect modification by th ese variables. The aut hors found that prenatal

total dialkylphosphate metabolite level was associat ed with a decrement in mental development

at 12 months among blacks and Hispanics. The a ssociations appeared to be strongest among

children of mothers who carried the PON1 Q192R QR/RR genotype. In later childhood,

increasing prenatal total DAP and DMP metabol ites were associated with decrements in

perceptual reasoning in the maternal PON1 Q192R QQ genotype (imp arting slow catalytic

activity for chlorpyrifos oxon), w ith a monotonic trend consistent with greater decrements with

increasing prenatal exposure. The authors conclu ded that their findings are suggestive of an

association between prenatal exposure to organophosphates and decrements in cognitive

development, particularly perceptual reasoning, with evidence of effects beginning at 12 months

and continuing through early childhood, with PON1 being a potentially important potential

susceptibility factor for these deleterious eff ects. The authors did not observe any effect

modification by PON1 enzyme activity.

The authors noted that the asso ciations they observed betwee n the organophosphate pesticide

metabolites and cognitive development are similar to those reported in a similar prospective

cohort study set in an agricultur al area of California (the Center for the Health Assessment of

Mothers and Children of Salinas St udy, Eskenazi et al. 2010).

Urine samples obtained during the 3rd trimester pr enatal visit were provided to the CDC to

quantify levels of six dialkylphosphate metabolites. In cases where individual dialkylphosphate

metabolite levels were missing because of analytic interference, metabolit e levels were imputed

using regression analysis to predict the missing metabolite on the basis of the other non-missing

metabolites measured for that woman within the group of correlated metabolites (Eskenazi et al.

Environ Health Perspect, 2004). Samples below the limit of detection (LOD) were defined as

LOD/ √ –2. Diethyl- and dimethylphosphate metabolites were then summed on a molar basis (as

nm/liter) to obtain total diethylphosphates (D EPs) and total dimethylphosphates (DMPs),

respectively, and together to obtain total dialky lphosphates (DAP) levels. Tw enty-six very dilute

urine samples with < 20 μ g/dL creatinine were exclude d from organophosphate metabolite

analyses. Approximately 97%, 89%, and 90% of the cohort had detectable le vels of DAP, DEP,

and DMP metabolites, respectively. Prenatal orga nophosphate marker levels were dichotomized

using median exposure as the cut-off.

Plasma was separated from samples of prenatal maternal peripheral blood and cord blood

and used to measure PON1 activity. PON1 polymorphisms in both maternal and child DNA

were also assessed using the blood samples provi ded by participants. Prenatal and cord blood

PON1 activity levels were classified in tertil es for analysis of eff ect modification of the

relationship between pesticide marker levels and subsequent cognitive development.

The Bayley Scales of Infant Development, 2nd edition (BSID-II), was administered to

participating children at 12 and 24 months of age, approximately, to evaluate mental and

# Chlorpyrifos Human Health Risk Assessment D424485

Page 314 of 531 psychomotor development. The Mental Develo pment Index (MDI) portion of the assessment

rates a child’s cognitive ability ( i.e. memory, habituation, problem solving, early number

concepts, generalization, clas sification, vocalizations, langua ge, and social skills). The

Psychomotor Development Index (PDI) rates fine and gross motor coordination. Between the

ages of 6 and 9 years of age, a subset of participating children was again assessed using

psychometric intelligence tests. Before age 7, children were admini stered Block Design,

Information, Matrix Reasoning, Vocabulary, Pi cture Concepts, Symbol Search, Word

Reasoning, and Coding subtests of the Wechsler Preschool and Primary Scale of Intelligence,

3rd edition (WPPSI-III). Composite Verbal, Perfor mance, Processing speed, and Full-Scale IQ

(FSIQ) scores were derived using age-standa rdized WPPSI-III norms. Children between the ages

of 7 and 9 years were administered the Block De sign, Similarities, Digit Span, Picture Concepts,

Coding, Vocabulary, Letter-Number Sequence, Ma trix Reasoning, Comprehension, and Symbol

Search subtests of the Wechsler Intelligence Scale for Children, 4th edition (WISC-IV). Working

Memory, Processing Speed, and FSIQ scores were derived using age-standardized WISC-IV

norms.

The BSID-II was administered at Mount Sinai Hospital. Interviews and examinations were

conducted in English or Spanish. The WPPSI-III wa s administered in English or Spanish by one

of four examiners, as was the WISC-IV. by one of four examiners Both the WPPSI-III and the

WISC-IV were administered in a private room, without the parent being present.

Of the 200 children who were administered th e BSID-II at approximately 12 months of age

(mean ± SD, 13.1 ± 1.6 months), one was excluded from analyses because they refused to

compete a substantial portion of the examination; two children were excluded from the 12-month

analysis because their parent reported a diagnos is of pervasive developmental disorder and 20

children were excluded because their correspondi ng maternal urine samples were deemed to

dilute to analyze. Of the 177 eligible ch ildren who completed 12-month exams, 174 had

organophosphate metabolites measured in prenatal urine. At the 24-month BSID-II (mean ± SD,

27.4 ± 4.5 months), 276 children co mpleted the exam. Two were excluded from the 24-month

analysis because the parent reported a diagnosis of pervasive developmental disorder. Children

of mothers who submitted very dilute samples of urine (less than 20 μ g/dL creatinine) were also

excluded from analysis (n = 23). Of the remaining 251 eligible observations, 247 had

organophosphate metabolites measured in prenatal urine. MDI scores for 10 children were not

usable due to extensive refusals; they were incl uded in the PDI analyses because their scores

were valid. The authors reporte d an association between pr enatal total dialkylphosphate

metabolite level and a decrement in mental development at 12 months among blacks and

Hispanics. These associations appeared to be enhanced among children of mothers who carried

the PON1 Q192R QR/RR genotype. In later childhood, increasing pren atal total dialkyl- and

dimethylphosphate metabolites were associated w ith decrements in per ceptual reasoning among

those with maternal PON1 Q192R QQ genotype, which impart s slow catalytic activity for

chlorpyrifos oxon, with a monotonic trend consiste nt with greater decrements with increasing

prenatal exposure.

Study Review

This was a well conducted prospective study conducted in a young, predominantly minority

# Chlorpyrifos Human Health Risk Assessment D424485

Page 315 of 531 population. The study design, analytic approach, and statistical an alyses were appropriate.

Differential measurement error of exposure and outcomes is likely to be minimal, although other

sources of bias may have influenced the study findings.

The dramatic loss-to-follow-up over time is a possi ble cause of selection bias in this study. The

authors noted that mothers returning for follow- up assessments tended to be older, with a

disproportionately low fraction of women in the youngest age category; they also had

disproportionately higher level of education, on average, relativ e to the original cohort.

However, the subset of participants who returned for follow-up visits did not differ substantially

from the originally enrolled cohort with resp ect to racial/ethnic composition, breast-feeding

behaviors, and self-reported alcohol use during pregnancy.

The authors employed a reasonable set of exclusion cr iteria that likely resulted in recruitment of

a study population that was rela tively homogeneous with re spect to confounders of the in utero

pesticide exposure - cognitive development relationship (compared to the general population),

and the potential for confounding was thus lim ited by design (restriction). Bias due to

confounding was also controlled in the analys is, by assessing and ad justing for known risk

factors for cognitive development that are poten tially associated with pesticide exposure,

including maternal age, race/e thnicity, marital status, educ ation, breast-feeding, child sex,

alcohol, smoking, or drug use dur ing pregnancy, maternal IQ, a sc ore based on assessment of the

home environment (HOME), season of urine co llection, language spoken in the home, age at

testing, examiner and urinary creatinine level. The authors considered gestat ional age at delivery

and birth weight, not as confounders , but as potential causal interm ediates. They used a changein-estimate approach to covariate selection, begi nning with a full model, which allowed them to

consider confounding by a given risk factor in the context of ad justment for other covariates.

Given the limited sample size in this study, a full model with many covariates may be unstable,

although it is unclear from the article wh ether such instability was observed.

Residual confounding by unmeasured or poorly measured confounders is likely in this study.

Socioeconomic determinants, which are notoriously difficult to measure well, are likely

predictive of pesticide exposure, and risk fact ors for poor cognitive development. The authors

adjusted for some factors which may be surrogates for socioeconomic status ( e.g. , race/ethnicity,

marital status, maternal education), but adjustment for these factors is unlikely to have fully

accounted for confounding by socioeconomic status. If such residual confounding exists, it is

likely to result in effect estimates which overe stimate the effect of organophosphate pesticide

exposure. However, the extent of this conf ounding was likely limited by the recruitment of a

relatively homogenous study population, vis-à-vis socioeconomic conditions.

Presence of environmental contaminants in the ho me, either during pregna ncy or after birth, may

confound the relationship under study. The authors do not discuss co-contaminants present in the

home during the pregnancy; such factors would li kely result in the observa tion of a greater than

effect of pesticide exposure on cognitive developm ent if they are positiv ely associated with

pesticide exposure and predictive of deficits in cognitive development. The authors stated that

they considered a “Home Observation for Measur ement of the Environment” (HOME) score as a

potential confounder, and, in fact, they included this score in their multivariate models. The

degree to which this “HOME” score is a good proxy for relevant co-exposures is unclear, and

# Chlorpyrifos Human Health Risk Assessment D424485

Page 316 of 531 leaves open the possibility of re sidual confounding by environmental contaminants in the home.

The authors observed apparent effect modification by maternal race/ethnicity. For example,

among non-whites, increasing DAP and DMP tertile s of exposure were associated with a

decrease in the MDI. However, among white s, the opposite was observed; higher metabolite

levels were associated with an increase in MDI scores. Such an observation may be indicative of

a true interaction betwee n race/ethnicity and prenatal pesticide exposure, but it may also be

explained by residual and differential confounding by co-contaminants that are correlated with

pesticide exposure and race. Indeed, in this study population, race/ethnicity was strongly

associated with housing type ( i.e. public versus private housing), and the authors did, indeed,

observe similar trends in the effect estimate s when stratifying by hous ing type rather than

race/ethnicity. The authors speculated that this observed heterogeneity ma y indicate that whites,

or those living in private or ow ner-occupied housing, may have expe rienced a different source of

exposure to pesticides or their metabolites that may have contributed substantially to their

urinary concentrations, and identify pestic ide residues from fresh fruit and vegetable

consumption as one such source. The authors adjusted for seasonal variation in exposure,

although they note that other time-related vari ability may influence exposure assessment.

Although they observed heterogeneity by PON1 genotype, the authors did not observe any effect

modification by PON1 enzyme activity in blood samp led during the third trimester, as they had

in their earlier assessments of pesticide meta bolites and cognitive development assessed shortly

after birth. They speculated that this was because genotype is a more stable, long-term predictor

of metabolism potential, relative to prenatal phenotype as measured by PON1 activity.

The metabolites assessed in this study are sensi tive, but non-specific, markers for chlorpyrifos.

The analytic methods appear sound. However, th ey assessed these markers using only a single

maternal urine specimen taken during the th ird trimester – a critical period for

neurodevelopment. If sources and patterns of exposure remain static, a single measurement

would be a good indicator of pesticide exposur e during pregnancy. If, however, sources and

exposure patterns change over short time-spans, pe rhaps on the order of days to weeks, such an

exposure assessment would not necessarily provid e a good indicator of the relevant exposure.

Like the many other sources of error possible in th is study, this, too is likely to result in a nondifferential misclassification of the exposure, with respect to the c ognitive outcome. Serial

measurements of urinary biomarkers of pesticide exposure in the third trimester would shed light

on these patterns, although the benefits of such information are offset by the added cost,

logistical complexity expense, and burden on the study participants.

The instruments used to assess ne urodevelopment in this study ( i.e., the Bayley Scales of Infant

Development, 2nd edition (BSID-II), the Wechsler Preschool and Primary Scale of Intelligence,

3rd edition (WPPSI-III), and the Wechsler Intell igence Scale for Children, 4th edition (WISCIV)) are commonly used in both a c linical and research setting a nd are widely accepted tools.

In this study, prenatal maternal DEP urinary me tabolite concentrations were associated with

slight decrements in FSIQ, Perceptual Reasoni ng, and Working Memory between the ages of 6

and 9 years. Among children of QQ mothers, DAP and DMP urinary metabolite concentrations

were associated with poorer scores on Per ceptual Reasoning and FSIQ. The authors reported

# Chlorpyrifos Human Health Risk Assessment D424485

Page 317 of 531 stronger Q192R interactions for DAP and DMP ur inary metabolites compared to that for the

DEP metabolites, although the expl anation is unclear and may have been influenced by small

sample size. The authors note that PON1 Q192R exhibits substrate sp ecificity, but the strongest

interactions were observed for DMP, not DE P (into which chlorpyrifos and diazinon both

metabolize). Interestingly, researchers state th at malathion spraying for mosquitoes carrying

West Nile virus was a well-publicized source of exposure during the period of enrollment which

may have increased the levels of DMP in this population.

The statistical analyses used in the study appear to have been appropria te, although they are not

explained in detail. The aut hors stratified their findings by both race/ethnicity and PON 1

genotype, although it is unlikely that the study wa s sufficiently powered to provide optimal

statistical power to assess pest icide effects in the presence of such effect modification. To

maximize the number of particip ants included in the analysis , the investigators defined a

combined population of children who came for at least one of the Wechsler psychometric

intelligence exams. The WISC-IV composite scores were incl uded preferentially, and WPPSI-III

composite scores substituted if the child did not return for the later exam . Regarding the use of a

composite population for the outcome evaluations at 6-9 years, the aut hors report ‘convergent

validity’ between the WISC-IV and WPPSI-III. In a previous report, the correlation between

WPPSI-III and WISC-IV FSIQ scor es was 0.89; WPPSI-III Verbal IQ and WISC-IV Verbal

Comprehension, 0.83; and WPPSI-III Performanc e IQ and WISC-IV Perceptual Reasoning,

0.79. In the present study, correlations between the composite scores on the WPPSI-III and

WISC-IV were likewise strong: FSIQ = 0.83, Ve rbal IQ/Verbal Comprehension = 0.84, and

Performance IQ/Perceptual Reasoni ng = 0.78. In their statistical m odeling, the authors included a

variable to indicate whether th e WISC-IV and WPPSI-III was used.

Regarding the measurement of developmental out comes using the Bayley Score, in general,

associations observed during the 12-month follow-up were either attenuated or non-existent at

the 24-month visit, including the observation of race-based heterogeneity. The authors

speculated that, although exposures likely decr eased over time, the 0- to 12-month exposure

window may have included less hand-to-mout h childhood exposure (from crawling exposure to

house dust, and/or fresh fruit a nd vegetable consumption) than in the 12- to 24-month window;

thus, more childhood exposure in th e 12- to 24-month window may modi fy the effect of prenatal

exposure.

The prospective nature of the study is a prim ary strength. Pesticide exposure indicators and

outcomes metrics are likely to have been measured with some error, but these errors are likely to

be non-differential. On average, such non-differen tial measurement error results in attenuation of

the measures of association, and are thus unlikely to result in false-positiv e findings. A further

strength attributable in part to the prospective design was the inve stigators’ ability to consider

potential confounders and effect-modifiers a priori , measure them by design, and adjust

for/assess their influence in the analysis.

Importantly, this study was conducted at a time during which important regulatory changes in

residential use of chlorpyrifos were bei ng implemented (occurring approximately midway

through the study). As a result, th e participating children’s exposur es to chlorpyrifos may have

decreased over time. The investigators report ed that they did not observe any time-period

# Chlorpyrifos Human Health Risk Assessment D424485

Page 318 of 531 interactions, but they did not present data on th e presence or absence of temporal changes in

participants’ pesticide metabolite levels ( i.e. comparing those that were enrolled earlier versus

later in time). In the discussion, they did men tion that it was likely that childhood exposure to

chlorpyrifos and diazinon was lower post-2000 (following the voluntary cancellation of indoor

residential chlorpyrifos use). To the extent that post-natal exposures we re largely reduced, the

associations observed can be more definitivel y linked to prenatal exposures. If however,

pesticide exposures persisted into childhood, it would be less clear whether deficits in cognitive

functioning observed in this study were caused specifically by pre- natal exposures, rather than

subsequent exposure early in life , assuming that pesticide exposure is causally associated with

such decrements.

In this third study of organophosphate pesticide markers in maternal urine samples collected

during the third trimester and subs equent cognitive development in children partic ipating in the

“Mt. Sinai Child Growth and Development St udy”, the authors report on findings out to age

nine. The specter of selection bias induced by lo ss-to-follow-up is a marked weakness in an

otherwise well conducted study. Unfortunately, it is difficult to assess the magnitude and

direction of such bias. Confounding by unmeasured or poorly measured factors is yet another

limitation, specifically with refere nce to the measurement of socio-economic factors and other

environmental co-exposures. Non-specific biomarke rs of exposure, as were assessed in this

study, are likely more objective, sensitive, and lik ely also more accurate and reliable indicators

of pesticide exposure, relative to exposure as sessment methodologies that rely on self-report.

However, to the extent that prenatal chlorpyrifos is the true exposure of interest, the assessment

of non-specific organophosphate pesticide metabolites in a single sample of prenatal urine is a

primary potential source of e xposure measurement error. Because of the prospective design,

information bias is expected to result in attenuation of the true (unobserved) associations, toward

(observed) associations that ar e relatively smaller, or null. The finding of apparent effect

modification by maternal race/ethnicity in this study could result from differential loss to followup, measurement error in the assessment of actual pesticide exposure, and/ or differential residual

confounding.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 319 of 531 Works Cited

Berkowitz GS, Wetmur JG, Birman-Deych E, Ob el J, Lapinski RH, Godbold JH, Holzman IR,

Wolff MS. In Utero Pesticide Exposure, Maternal Paraoxonase Activity, and Head

Circumference. Environmental Health Perspectives. 2004;112:388–391.

Engel SM, Berkowitz GS, Barr DB, Teitelbaum SL , Siskind J, Meisel SJ, Wetmur JG, Wolff

MS. Prenatal Organophosphate Metabolite and Or ganochlorine Levels and Performance on the

Brazelton Neonatal Behavioral Assessment Scale in a Multiethnic Pregnancy Cohort. American

Journal of Epidemiology. 2007;165:1397-1404.

Eskenazi B, Huen K, Marks A, Harley KG, Bradman A, Barr DB, Holland N. PON1 and

neurodevelopment in children from the CHAMACOS study exposed to organophosphate

pesticides in utero . Environmental Health Perspectives 2010;118:1775–1781.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 320 of 531 

# Article 10. Eskenazi et al. (2007)

Organophosphate Pesticide Exposure and Neur odevelopment in Young Mexican-American

Children. Brenda Eskenazi, Amy R. Marks, Asa Bradman, Kim Harley, Dana B. Barr,

Caroline Johnson, Norma Morga, Nicholas P. Je well. Environmental He alth Perspectives.

115:792–798 (2007).

Study Summary

In this article by Eskenazi et al, the authors report on the relati onship between prenatal and child

urinary organophosphate metabolite levels and child neurodevelopment up to two years of age in

the CHAMACOS cohort.

Enrollment for the CHAMACOS took place from 1999–2000 at community clinics, and included

women who were ≥ 18 years old, pregnant at < 20 w eeks gestation, Spanish- or Englishspeaking, eligible for low-income health insuran ce, and planning to deli ver at the local public

hospital. Researcher followed 531 women to delivery of a live-born, surviving, neonate.

Excluded from the analysis were children that did not have a neurodevelopmental assessment,

(n=71), who did not have prenatal and relevant concurrent DAP metabolite measurements (n =

3), were not singletons (n=8), had a medical condition that c ould affect performance on the

neurobehavioral assessment (n = 3; deafness, Down syndrome, hydrocephalus). Also excluded

from analysis were Bayley results with raw scor es which were too low for standardization (n=3,

1, and 1, for the 6-month, 12-month, and 24-month assessments, respectively).

Maternal/fetal exposure was assessed by measurement of si x non-specific organophosphate

dialkylphosphate (DAP) metabolit es in maternal urine sample s collected between 5 and 27

weeks gestation and again between 18 and 39 week s. Maternal urine was also analyzed for 3,5,6-

trichloro-2-pyridinol (TCPy), a metabolite specif ic to chlorpyrifos. Post-natal early childhood

urine samples were also collected, and DAP metabol ites quantified, for the participating children

at the time of the neurodevelopmental asse ssments. Quantification of organophosphate

metabolites using gas chromatography-tandem mass spectrometry and isotope dilution

calibration was conducted by the Centers for Di sease Control and Prevention (CDC) labs.

To asses exposure, investigators measured si x nonspecific organophosphate DAP metabolites in

maternal and child urine: three dimethyl (DMP) phosphate metabolites (dimethylphosphate,

dimethylthiophosphate, dimethyldithiophospha te); and three diethyl (DEP) phosphate

metabolites (diethylphosphate, diethylthiophosphate , and diethyldithiophosphate) (Bradman et

al. 2005). These six metabolites represent the by-pr oducts of approximately 80% of OPs used in

the Salinas Valley. Values below the limit of detection (LOD) were assigned a value of

LOD/( √ 2). The urine analysis was completed by th e CDC. Maternal prenatal urine was also

analyzed for specific metabolites including TCP y, a metabolite specific ch lorpyrifos. Because a

large proportion of women had non- detectable levels of TCP y, the chlorpyrifos-specific

metabolite was categorized into three groups : TCPy less than LOD for both pregnancy

measurements, and, for those with at least one de tectable level, subdivided below and above the

median of the average pregnancy level.

The outcome, child neurodevelopment, was asse ssed using the Bayley Scales of Infant

# Chlorpyrifos Human Health Risk Assessment D424485

Page 321 of 531 Development (Mental Development (MDI) and Psychomotor Development (PDI) Indices)

(Bayley, 1993) and the Child Behavior Checkli st (CBCL). The Bayley Scales of Infant

Development were completed at the 6 months (n = 396), 12 months (n = 395), and 24 months (n

= 372) of age. Both scales were administered in Spanish and/or E nglish by psychometricians

blinded to exposure. Psychometricians were traine d using these standardized protocols and were

supervised for quality assurance by a clinical neuropsychologist. Assessments were performed in

a private room at the CHAMACOS research office or in a recreation vehicle (RV) modified to be

a mobile testing facility. Scores > 1 SD below the mean ( i.e. , < 85) indicate possible

developmental delay.

Mother’s completed the Child Behavior Checklist (CBCL) at the 24 month visit (n = 356). From

the CBCL report, the authors asse ssed three scales as indicators of child neurodevelopment: The

Attention Problems syndrome scale, which includes such items as “can’t concentrate” and “can’t

sit still”; the DSM-oriented Attention-Defici t/Hyperactivity Disorder (ADHD) scale, which

additionally includes such items as “gets into everything”; and the DSM-oriented Pervasive

Developmental Disorder (PDD) scal e, which includes such items as “avoids eye contact,” “rocks

head, body,” and “unresponsive to affection”. E xperts consider a score greater than the 98th

percentile of a national normative sample of clini cal significance; they consider a score greater

than the 93rd percentile of borderline clinical significance.

Many confounders were ascertained including, intell igence of the mother, maternal depression, a

measure of how stimulating the child’s environmen t is, and prenatal exposure to some known or

suspected neurotoxins. Maternal intelligence wa s assessed via the Peabody Picture Vocabulary

Test (PPVT). Maternal depression was assessed using the Center for Epidemiologic Studies

Depression Scale (CES-D). To measure the quality and extent of stimulation available to a child

in the home environment, the Infant-Toddler HOME (Home Observation for Measurement of the

Environment) inventory was completed at the 6-month, 12-month, and part ially at the 24-month

visit. The following known or suspected neur otoxicants were measured: polybrominated

diphenyl ethers (PBDEs), polychl orinated biphenyls (PCBs), p,p ’-dichlorodiphenyltrichlorethane

(DDT), p,p’- dichlorodiphenyltrichlorethylene ( DDE), and lead. Lead was measured in cord

blood. PBDEs, PCBs, DDT, and DDE were measured in maternal serum samples collected at 26

weeks gestation.

Statistical analyses of the rela tionship between metabolite levels and Bayley performance were

conducted using multiple linear regression models and generalized estimating equations ( i.e.

, longitudinal models) for MDI and PDI were deve loped for each of the three assessment time

points ( i.e. , 6, 12, and 24 months). Neurodevelopmenta l scores were modeled as continuous

variables. Covariates in the models included indicators for the psychom etrician conducting the

assessment and the location of assessment, age at assessment, sex, duration of breast-feeding,

HOME score, and household income, parity and indi cator of maternal intelligence (PPVT score).

Other covariates that were considered but not included in the analysis were: maternal age,

education, depressive symptoms, active/passi ve smoking exposure durin g pregnancy, regular

alcohol use during pregnancy, marital status, father’s presence in home, housing density,

maternal work status, ≥ 15 hours out-of-home childcare/week, birth weight, gestational age,

abnormal reflexes, PCBs, lead, DDT, β -hexachlorocyclohexane, and hexachlorobenzene.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 322 of 531 Furthermore, the authors examined interactions between child DAPs and child sex and, because

they previously observed an association w ith maternal DDT and 24-month MDI, between

maternal DAPs and DDT. Finally, authors also reran the models us ing log-transformed

creatinine-adjusted metabolit es for comparative purposes.

Child behavior checklist score at the 24-m onth assessment was modeled using separate

multivariate logistic regression models for th e five child behavior checklist indices,

dichotomized at the clinical significance level an d at the borderline clinical significance level.

The same covariates that were included in th e final Bayley models were used for the CBCL

models with three exceptions: maternal depression was added, and psychometrician and

assessment location were dropped, because sc ores were based on maternal report.

Of the 348 infants included in this analysis, 49.4% were male. Most mothers were less than 30

years old at baseline (75.6), born in Mexico and Spanish-spea king (89%), had household income

below the poverty line (62%). Ma ny of the participating mother s worked in agriculture during

pregnancy (43%) and had symptoms of depressi on (51%). Most of the women were married,

non-smokers, and did not drink alcohol during pregnancy.

In this study population, prenatal DAP levels were adversely asso ciated with MDI, while early

life DAP levels were positively associated with MDI. At 24 months of age, these associations

reached statistical significance (per 10 -fold increase in prenatal DAPs: β = –3.5 points; 95% CI:

–6.6 to –0.5; child DAPs: β = 2.4 points; 95% CI: 0.5 to 4.2). Neither prenatal nor child DAPs

were associated with PDI or child behavior ch ecklist attention problem s. Both prenatal and

postnatal DAPs were associated with risk of pervasive developmental disorder (per 10-fold

increase in prenatal DAPs: OR = 2.3, p = 0.05; child DAPs OR = 1.7, p = 0.04). TCPy was not

associated with any neurodevelopment outcome in this study.

Study Review

This study tested whether in utero or postnatal exposures to or ganophosphate pesticides have a

detrimental impact on neurodevelopment, as assessed by the Bayley Scales of Infant

Development and the child beha vior checklist (CBCL). The st udy population was appropriate to

the hypothesis, given that there were a range of exposure levels to OPs and there was some

homogeneity in the population, e.g. , mostly low SES, Hispanic, agricultural workers. The

homogeneity of the population restricted conf ounding by design. For example, another paper

reported that smoking, alcohol use, and illicit dr ug use were rare the CHAMACOS cohort. The

homogeneous population also increa sed the relative statistical e fficiency of the study because it

is likely at higher risk of defi cits in neurodevelopment than th e general population (Landrigan,

1990; Landrigan et al., 1999). However, th e exclusion of chil dren who lacked a

neurodevelopmental assessment (n = 71, approxim ately 13% of the study population), has the

potential to induce a selection bi as if the reasons for the missing data are determined by exposure

(or a predictor of exposure) and are also not independent of neurodevelopment. For example,

participants with high pesticide exposures co uld have missed neurodevelopmental assessments

for reasons related to uncontrolled determinan ts of exposure which are also related to

neurodevelopment. Comparisons of maternal DAP metabolite levels between those included in

this analysis and those not included in the analysis were not presented.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 323 of 531 The exposure assessment methods in this study were good. Quantification of the metabolite

levels was conducted at the CDC, using published methods. The study measured the

organophosphate biomarker, DAP metabolites, in mate rnal and child spot urine samples, and the

chlorpyrifos specific biomarker, TCPy, in mate rnal urine only. Measurement of biomarkers in

urine is a more objective and likely more accu rate indicator of organophosphate pesticide

exposure than other ascertainment methods such as self-report. However, these biomarkers do

present a few limitations, because study participants could be exposed to less toxic preformed

DAP or TCPy metabolites (Lu et al. 2005; Wils on et al. 2003; Morgan et al. 2004), and exposure

to pesticides varies over time and the half-life of chlorpyrifos in the body is short. However, the

prenatal exposure measures were, with some exceptions, the average of two measurements, and

thus may better reflect chronic exposure during pregnancy than in studies where only a single

spot urine sample was collected. Under an assu mption of no other biases, the negative findings

could have resulted from e xposure measurement error.

The outcome measurements, the Bayley Scales of Infant Development and the CBCL, were also

appropriate. The Bayley Scales of Infant Development is consider ed a gold standard assessment.

The assessments were reportedly appropriately ad ministered. Both the MDI and PDI scales were

administered in Spanish and/or English by psycho metricians masked to exposure status of the

children. Psychometricians were trained using sta ndardized protocols and were supervised for

quality assurance by a clinical neuropsychologi st. Assessments were performed in a private

room. The influence of inter-observer reliability was controlled by inclusion for an indicator for

the assessed but data were not presented in this report.

The Child Behavior Checklist (CBCL) has been widely used in cross-cultural research and

collects data on a range of beha vior problems, yielding scores for several syndrome scales and

five scales designed to be consis tent with Diagnostic and Statisti cal Manual of Mental Disorders

(DSM) diagnoses. The three scales used in this assessment were select ed by the investigators a

priori because the effects they measured were simila r to effects seen in relation to DAP exposure

in animal models. The child behavior checklist is limited in that it is reliant on maternal report

and the authors note the findings from the child behavior checklist ar e not directly equivalent to a

DSM diagnosis.

The statistical analysis used to assess the a ssociations between the markers of exposure and

neurodevelopment were appropriate. However, the authors did not present nor discuss regression

diagnostics to assess the degree to which their models met or violated the assumptions implicit in

linear models ( i.e. homoscedasticity, normality of error te rm distribution, independence of error

terms, and linearity of the exposure-response rela tionship), other than to state that they log

transformed DAP levels. The authors also conduc ted a host of sensitivity analyses to assess

influence of modeling decisions on their findings, and generally f ound their results to be robust.

For example, they controlled for birth weight, ge stational age, and abnormal reflexes which were

considered causal intermediates, re-ran models excluding low birt h weight and preterm infants,

and ran models with and without adjustment of biomarkers for cr eatinine levels. The authors also

assessed effect modification by gender and DDT exposure because population of primarily

immigrant women who may have been exposed to DDT and other U.S. banned substances in

country of origin; associations between pesticide markers and the indicators of

# Chlorpyrifos Human Health Risk Assessment D424485

Page 324 of 531 neurodevelopment did not differ by either of these co-variables.

To increase the statistical e fficiency of the study by preservi ng the size of the analytic

population, missing covariate values were im puted by randomly selecting a value from

participants with non-missing valu es. Maternal depression had the largest percenta ge of values

requiring imputation (5%). Of remaining covariat es, between 0% and 1.8% of values were

imputed. Although a reasonable appro ach, poorly measured confounders ( i.e. residual

confounding) may also be influenced by imputed covariate data, alt hough perhaps to a lesser

degree because imputed data may still be preferable to no data at all.

Bias due to confounding was also co ntrolled in the analysis, by a ssessing and adjusting for risk

factors for neurodevelopment that are potentially associated with pestic ide exposure, including

age at assessment, sex, duration of breast-feed ing, HOME score, and household income, parity

and indicator of maternal intellig ence. In addition to the variable s included in the final models,

the authors examined potential confounding by severa l other variables sugges ted by the literature

( i.e. , maternal age, education, depressive sy mptoms, active/passive smoking exposure during

pregnancy, regular alcohol use dur ing pregnancy, marital status, father’s presence in home,

housing density, maternal work status, childcare), but they did not marked ly alter the observed

associations. Covariates were selected for inclusi on in the final analyses if they were related to

conditions of testing, related to neurodevelopment in the literature and asso ciated (p < 0.10) with

most outcomes or consistently related to neurodev elopment in the literature, even if not in the

data. This is a reasonable appro ach to assessment of confounding.

The authors’ quantification of envi ronmental co-exposures (DDT, DDE, β

hexachlorocyclohexane, hexachlorobenzene, PCBs, and lead) is a significant strength of this

study. Children and mothers, especially those from economically impoverished environments

and émigrés from home countries in which thes e compounds were frequently use in the recent

past, may be exposed to other environmental agen ts that may affect neurodevelopment. Potential

confounding by these exposures, as well as by in dicators of fetal development (shortened

gestation and poorer reflexes) was assessed, and observed associations in these models were

consistent with the primary findings.

This study has apparent inconsistencies in th e findings which are worth noting. First, while

DAPs were associated with an increased risk of PDD in this population, post-natal DAPs were

positively associated with MDI. No explanati on was presented by the authors. One possible

explanation is that children w ith higher cognitive functioning may be more interactive with their

environment, leading to higher exposure to pesticide residues, an example of “reverse

causation”, whereby the temporal order of the exposure and outcome are reversed. The

measurements of prenatal pesticide markers do not suffer from the same issues of unknown

temporal order; they clearly preceded the meas ures of post-natal child development. Another

possible explanation suggested by the authors is confounding by diet. Children who eat more

fruits and vegetables may have higher DAP le vels, but because of better diets have higher

functioning. Second, although the investigat ors observed associations between

neurodevelopment and the non-specific metabo lite markers of organophosphate pesticides

(DAPs), no such associations were observed for metabolites specific to chlorpyrifos (TCPy), nor

for a malathion-specific metabolite (MDA). The organophosphate metabolites (DAPs) cannot be

# Chlorpyrifos Human Health Risk Assessment D424485

Page 325 of 531 traced back to individual pestic ides, and the observed associations with DAPs may be attributed

to other pesticides, oxydemeton-methyl for ex ample, for which no specific metabolite is

measureable, or may be due to th e short-half life and lower storage stability of the pesticide

specific metabolites as compared to the non-specific metabolites.

The study has numerous strengths, most notab ly the prospective design the longitudinal

assessment of both exposure and outcomes. Stre ngths in the study desi gn also include the

quantification of non-specific ( DAPs), chlorpyrifos-specific (T CPy) metabolites, and other

environmental co-exposures. The results of this investigation by Eskenazi et al in the

CHAMACOS cohort are sugges tive of a detrimental asso ciation between prenatal

organophosphate exposure, as measured by ma ternal urinary metabolite levels and

neurodevelopment up to 24 months of age. Although the prospec tive study design and reasonable

methods protect from certain false-positive i nducing biases, there remains the potential of

residual confounding in the direction of an adve rse effect on development by unmeasured factors

leading to the observation of a false-positive as sociation. The authors reasonably suggest that

their results should be interp reted with caution because, a lthough they observed adverse

associations between prenatal DAPs and ment al development and pervasive developmental

problems at 24 months of age, they also observed a positive relationship between

neurodevelopment and postnatal DAPs.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 326 of 531 

# Article 11. Eskenazi et al. (2010)

PON1 and Neurodevelopment in Children from the CHAMACOS Study Exposed to

Organophosphate Pesticides in utero. Brenda Eskenazi, Karen Huen, Amy Marks, Kim G.

Harley, Asa Bradman, Dana Boyd Barr, and Nina Holland

. 

# Environmental Health

Perspectives. 118:1775-1781 (2010).

In this article by Eskenazi et al. (2010), the authors repo rt on the relationship between PON1

genotype and PON1 activity level and child ne urodevelopment, and the potential effect

modifying role of PON1 status in the re lation between prenatal and child urinary

organophosphate metabolite levels and child neur odevelopment up to two years of age in the

CHAMACOS cohort.

Methods and analyses employed in this investigation were similar to Eskenazi et al. 2007, with

the exception of the measurement of PON1 stat us in association with this outcome, and

assessment of interaction between PON1 and DAPs with child neurodevelopment. Enrollment

for the CHAMACOS took place from 1999–2000 at community clinics, and included women

who were ≥ 18 years old, pregnant at < 20 weeks gestation, Spanish- or English-speaking,

eligible for low-income health insurance, and planning to deliver at th e local public hospital.

Researcher followed 528 women to delivery of a live-born, surviving, neonate. Excluded from

the analysis were children that did not have a neurodevelopmental assessment, (n=71), who did

not have prenatal and relevant concurrent DAP metabolite measurements (n = 3), were not

singletons (n=8), had a medical condition that could affect perf ormance on the neurobehavioral

assessment (n = 3; deafness, Down syndrome, hydr ocephalus). Also excluded from analysis were

Bayley results with raw scores which were too low for standardization (n=3, 1, and 1, for the 6-

month, 12-month, and 24-month assessments, respect ively), and participants for whom PON1

information was not available. The study included 371 mother-infant pairs.

Maternal/fetal exposure was assessed by measurement of si x non-specific organophosphate

dialkylphosphate (DAP) metabolit es in maternal urine sample s collected between 5 and 27

weeks gestation and again between 18 and 39 w eeks. Specifically, investigators measured six

nonspecific organophosphate DAP metabolites in ma ternal and child urine: three dimethyl

(DMP) phosphate metabolites (dimethylphosphate, dimethylthiophosphate,

dimethyldithiophosphate); and three diethyl (DEP) phosphate metabolites (diethylphosphate,

diethylthiophosphate, and diethy ldithiophosphate) (Bradman et al . 2005). These six metabolites

represent the by-products of approximately 80% of OPs used in the Salinas Valley. Chlorpyrifos

and diazinon were the most commonly used or ganophosphates in the region that metabolize to

the diethyl phosphate metabolite. Values below the limit of detection (LOD) were assigned a

value of LOD/( √ 2). Quantification of organophosphate metabolites using gas chromatographytandem mass spectrometry and isotope diluti on calibration was conducted by the Centers for

Disease Control and Prevention (CDC) labs us ing published, validated, and accepted methods.

Maternal and umbilical cord blood samples were collected at the time of delivery. Genotyping

of the PON1 -108 

# and PON1 192 

# SNPs was performed using ge nomic DNA extracted from blood

clots and Taqman PCR methods. Two PON1 en zyme activity assays (arylesterase and

paraoxonase) were performed using spectrophotometr ic methods. Arylesterase activity measures

the rate of hydrolysis of the substrate phenyl ace tate, and reflects the qua ntity of PON1 enzyme.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 327 of 531 Paroaxonase activity measures the rate of hydrolysis of pa raoxon, and thus reflects a

combination of the catalytic efficiency and qua ntity of the PON1 enzyme. Using triplicate

samples, quality assurances methods illustrated the coefficient of variation was within the

acceptable range (<10%).

The outcome, child neurodevelopment, was asse ssed using the Bayley Scales of Infant

Development (Mental Development (MDI) and Psychomotor Development (PDI) Indices)

(Bayley, 1993) and the Child Behavior Checkli st (CBCL). The Bayley Scales of Infant

Development were completed at the 6 months (n = 396), 12 months (n = 395), and 24 months (n

= 372) of age. Both scales were administered in Spanish and/or E nglish by psychometricians

blinded to exposure. Psychometricians were traine d using these standardized protocols and were

supervised for quality assurance by a clinical neuropsychologist. Assessments were performed in

a private room at the CHAMACOS research office or in a recreation vehicle (RV) modified to be

a mobile testing facility. Scores > 1 SD below the mean ( i.e. , < 85) indicate possible

developmental delay.

Mother’s completed the Child Behavior Checklist (CBCL) at the 24 month visit (n = 356). From

the CBCL report, the authors asse ssed three scales as indicators of child neurodevelopment: The

Attention Problems syndrome scale, which includes such items as “can’t concentrate” and “can’t

sit still”; the DSM-oriented Attention-Defici t/Hyperactivity Disorder (ADHD) scale, which

additionally includes such items as “gets into everything”; and the DSM-oriented Pervasive

Developmental Disorder (PDD) scal e, which includes such items as “avoids eye contact,” “rocks

head, body,” and “unresponsive to affection”. E xperts consider a score greater than the 98th

percentile of a national normative sample of clini cal significance; they consider a score greater

than the 93rd percentile of borderline clinical significance.

Many confounders were ascertained including, intell igence of the mother, maternal depression, a

measure of how stimulating the child’s environmen t is, and prenatal exposure to some known or

suspected neurotoxins. Maternal intelligence wa s assessed via the Peabody Picture Vocabulary

Test (PPVT). Maternal depression was assessed using the Center for Epidemiologic Studies

Depression Scale (CES-D). To measure the quality and extent of stimulation available to a child

in the home environment, the Infant-Toddler HOME (Home Observation for Measurement of the

Environment) inventory was completed at the 6-month, 12-month, and part ially at the 24-month

visit. The following known or suspected neur otoxicants were measured: polybrominated

diphenyl ethers (PBDEs), polychl orinated biphenyls (PCBs), p,p ’-dichlorodiphenyltrichlorethane

(DDT), p,p’- dichlorodiphenyltrichlorethylene ( DDE), and lead. Lead was measured in cord

blood. PBDEs, PCBs, DDT, and DDE were measured in maternal serum samples collected at 26

weeks gestation. However, these variables were not included in final models. The following

variables were adjusted in the interaction analys is: age at assessment, sex, parity, breast-feeding

duration, HOME score, maternal PPVT, and hous ehold poverty status. For the MDI and PDI

models an indicator variable for the psychom etrician was included, and maternal depression

included in the model for PDD.

Statistical analyses of the re lationship between PON1 status and Bayley performance were

conducted using multiple logistic regression mode ls; interaction terms a nd stratified analyses

were presented to evaluate the potential effect modifying role of PON1 status in the DAPs and

# Chlorpyrifos Human Health Risk Assessment D424485

Page 328 of 531 child neurodevelopment association. Neurodevelo pmental scores were modeled as continuous

variables. PON1 genotype was modeled as a three-level va riable, using the wild type as the

referent. PON1 enzyme levels were categorized as tertiles. Authors log 10 

# transformed DAP

levels, and averaged the two measures of urin ary DAPs taken during pregnancy in the analyses,

when available (n=22 for which only one sample available). Covariates in the models included

indicators for the psychometric ian conducting the assessment and th e location of assessment, age

at assessment, sex, duration of breast-feeding, HOME score, and household income, parity and

indicator of maternal intelligence (PPVT score) . Authors imputed missing values to preserve

sample size, particularly in the stratified analys es. Statistical significance of the main effect was

considered a p-value of 0.05, and significance of an interaction te rm was considered a p-value of

0.15.

Among the selected participants, mothers were generally young age (average 26.4 years, sd 5.2),

born in Mexico, were predominantly married or living as married, and were poor, 60% living at

or below the poverty line. Forty- four percent of the women re ported working in agriculture

during pregnancy, 82% resided wi th a household member who wo rks in agriculture, and 23%

live near an agricultura l operation. Most mothers reported breast feeding, and the duration of

breast feeding was on average 6 months. Authors detected dimethyl phosphates more frequently

than diethyl phospahtes. The av erage score for the MDI was 85 and PDI 98; 14% of children

evaluated exceeded the concern level of the CBCL.

Overall, infants with the variant PON1 108TT 

# allele who produced lower amounts of PON1 enzyme

had reduced scores on the MDI and PDI, and increased risk of PDD using the CBCL. Authors

observed no association with PON1 192QQ 

# genotypes in the relation w ith MDI, PDI or PDD.

Similarly, authors observed no association betwee n enzyme levels and any measure of child

neurodevelopment in this study. The in teraction between maternal DAP and PON1 genotype

with the Bayley assessed outcomes was non-signif icant (p=0.98), however researchers observed

a trend of lower scores by the number of PON1 108 

# variants alleles. For each 10-fold increase in

total DAPs, the MDI score reduced by 3.2 PON1 108CC 

# , 3.7 PON1 108CT 

# , and 5.5 PON1 108TT 

# (pinteraction 0.98 ). A similar trend was observed for the di ethyl phosphates, but neither strataspecific estimates nor trend tests were signifi cant. No similar trend was apparent across the

number of variant alleles at the PON1 192 

# genomic locus. No associa tions were observed between

DAPs and PDI by level of the PON1 108 

# genotype. However, there was limited evidence of

heterogeneity by PON1 192 

# genotype in the relation between DAPs and PDI, but results were not

significant.

Study Review

Overall, researchers offer some evidence of a link between infant PON1 status and child

neurodevelopment state, and author s’ state they cannot conclude the study provides evidence of

effect modification by PON1 genotype or phenotype status in the relation between DAPs and

MDI, PDI or PDD in this study. Similarly to the accompanying investigation (Eskenazi et al.

2007), the study population was appropr iate to the hypothesis, given that there were a range of

exposure levels to OPs and there was some homogeneity in the population, e.g. , mostly low SES,

Hispanic, agricultural workers. The homogene ity of the population restricted confounding by

design. For example, another paper reported that smoking, alcohol use, and illicit drug use were

# Chlorpyrifos Human Health Risk Assessment D424485

Page 329 of 531 rare the CHAMACOS cohort. The homogeneous population also increa sed the relative statistical

efficiency of the study because it is likely at higher risk of defi cits in neurodevelopment than the

general population (Landrigan, 1990; Landrigan et al ., 1999). Authors did not share whether

DAP levels were similar in mothers of infants for whom the MDI and PDI scores were not

available; the potential for sele ction bias therefore cannot be assessed. The measurement of both

urinary DAPs and PON1 status were well condu cted, however limitations previously noted

regarding the non-specific nature of a DAP measurement to the question of chlorpyrifos toxicity

and the use of one- or two measur es during the latter part of pregnancy as an estimate for

gestational exposure apply equa lly to this investigation.

The outcome measurements, the Bayley Scales of Infant Development and the CBCL, were also

appropriate. The Bayley Scales of Infant Development is consider ed a gold standard assessment.

The assessments were reportedly appropriately ad ministered. Both the MDI and PDI scales were

administered in Spanish and/or English by psycho metricians masked to exposure status of the

children. Psychometricians were trained using sta ndardized protocols and were supervised for

quality assurance by a clinical neuropsychologi st. Assessments were performed in a private

room. The influence of inter-observer variability was controlled by inclusion of an indicator

variable for the psychomatrician.

The Child Behavior Checklist (CBCL) has been widely used in cross-cultural research and

collects data on a range of beha vior problems, yielding scores for several syndrome scales and

five scales designed to be consis tent with Diagnostic and Statisti cal Manual of Mental Disorders

(DSM) diagnoses. The three scales used in this assessment were select ed by the investigators a

priori because the effects they measured were simila r to effects seen in relation to DAP exposure

in animal models. The child behavior checklist is limited in that it is reliant on maternal report,

and the authors note the findings from the child behavior checklist ar e not directly equivalent to a

DSM diagnosis.

Again, the statistical analysis us ed to assess the associations between the markers of exposure

and neurodevelopment were appropriate. Howeve r, the authors did not present nor discuss

regression diagnostics to assess th e degree to which their models me t or violated the assumptions

implicit in linear models ( i.e. homoscedasticity, normality of error term distribution,

independence of error terms, and linearity of th e exposure-response relationship), other than to

state that they log transformed DAP levels. Th e authors also conducted a host of sensitivity

analyses to assess influence of modeling deci sions on their findings, and generally found their

results to be robust. For example, investigators controlled for maternal depression in the relation

between pesticide exposure and PDD, and ran models with and without adjustment of

biomarkers for creatinine levels.

To increase the statistical e fficiency of the study by preservi ng the size of the analytic

population, missing covariate values were im puted by randomly selecting a value from

participants with non-missing valu es. Maternal depression had the largest percenta ge of values

requiring imputation (5%). Of remaining covariat es, between 0% and 1.8% of values were

imputed. Although a reasonable appro ach, poorly measured confounders ( i.e. residual

confounding) may also be influenced by imputed covariate data, alt hough perhaps to a lesser

degree because imputed data may still be preferable to no data at all.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 330 of 531 Bias due to confounding was also co ntrolled in the analysis, by a ssessing and adjusting for risk

factors for neurodevelopment that are potentially associated with pestic ide exposure, including

age at assessment, sex, duration of breast-feed ing, HOME score, and household income, parity

and indicator of maternal intellig ence. In addition to the variable s included in the final models,

the authors examined potential confounding by severa l other variables sugges ted by the literature

( i.e. , maternal age, education, depressive sy mptoms, active/passive smoking exposure during

pregnancy, regular alcohol use dur ing pregnancy, marital status, father’s presence in home,

housing density, maternal work status, childcare), but they did not marked ly alter the observed

associations. Covariates were selected for inclusi on in the final analyses if they were related to

conditions of testing, related to neurodevelopment in the literature and asso ciated (p < 0.10) with

most outcomes or consistently related to neurodev elopment in the literature, even if not in the

data. This is a reasonable appro ach to assessment of confounding.

The authors’ quantification of envi ronmental co-exposures (DDT, DDE, β

hexachlorocyclohexane, hexachlorobenzene, PCBs, and lead) is a significant strength of this

study. Children and mothers, especially those from economically impoverished environments

and émigrés from home countries in which thes e compounds were frequently use in the recent

past, may be exposed to other environmental agen ts that may affect neurodevelopment. Potential

confounding by these exposures, as well as by in dicators of fetal development (shortened

gestation and poorer reflexes) was assessed, and observed associations in these models were

consistent with the primary findings.

Similarly to the previous inve stigation by Eskenazi et al. (2007), the study has numerous

strengths, most notably the prospective design the longitudinal assessmen t of both exposure and

outcomes. Strengths in the study design also in clude the quantification of non-specific (DAPs),

PON1 status, and other environmental co-exposur es. The results of this investigation by

Eskenazi et al. (2007) in the C HAMACOS cohort were suggestiv e of a detrimental association

between prenatal organophosphate exposure, as measured by maternal urinary metabolite levels

and neurodevelopment up to 24 months of age, and this investigation presents some additional,

albeit limited evidence of potential effect modi fication by PON1 status. While results were not

significant, sample size within strata of PON1 genotype and question as to the relevance of

measurement of just PON1 among the suit e of enzymes involved in organophosphate

metabolism remain. Although the prospective stu dy design and reasonable methods protect from

certain false-positive inducing biases, there rema ins the potential of residual confounding in the

direction of an adverse effect on development by unmeasured factors leading to the observation

of a false-positive association, however less so with respect to PON1 status as these factors

would not likely be correlate d. The authors reasonably suggest that their results should be

interpreted with caution as the evidence for true effect modification in this relation is weak.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 331 of 531 

# Article 12. Marks et al. (2010)

Organophosphate Pesticide Exposure and Attention in Young Mexican-American

Children: The CHAMACOS Study. Amy R. Marks, Kim Harley, Asa Bradman, Katherine

Kogut, Dana Boyd Barr, Caroline Johnson , Norma Calderon, and Brenda Eskenazi.

Environmental Health Perspectives. 118:1768–1774 (2010).

Study Summary

Using data collected as part of the CHAMAC OS Study, Marks et al conducted a study to

investigate the association between urinary di alkyl phosphate (DAP) metabolites in pregnant

women and their children, as a marker of or ganophosphate exposure, and attention-related

outcomes among Mexican-American children livi ng Salinas Valley, an agricultural region of

California.

Enrollment for the CHAMACOS took place from 1999–2000 at community clinics, and included

women who were ≥ 18 years old, pregnant at < 20 w eeks gestation, Spanish- or Englishspeaking, eligible for low-income health insuran ce, and planning to deli ver at the local public

hospital. The investigators followed 526 women to delivery of a live born, surviving singleton.

Excluded from analyses were children who did not have a prenatal DAP metabolite measurement

(n = 2), had a medical condition that could affect performance on the neurobehavioral

assessment (n = 3; deafness, Down syndrome, hydr ocephalus), were lost to follow-up, or did not

participate at the 3.5- or 5-year study visit (n = 173) were excluded from the study. The study

population consisted of the remaining 348 children w ho had available data at 3.5 and/or 5 years.

Exposures were assessed by measurement of organophosphate dialkylphosphate (DAP)

metabolites in maternal and child urine samples. Maternal urine was collected between 5 and 27

weeks gestation and again between 18 and 39 w eeks. The post-delivery ur ines were collected

within 1 week of delivery for 73% of the sample , with the remainder obtained up to 176 days

afterwards. Urine samples were also collected from participati ng children at the 3.5 year and 5

year visits. Quantification of organophosphate metabolites was conducted by Centers for Disease

Control and Prevention (CDC) labs. Total DAP metabolite level was defined as the sum of the

molar concentrations of the six DAP metabolite s, dimethylphosphate (DMP) metabolite level as

the sum of the molar concentrations of the three dimethylphosphate metabolites only, and

diethylphosphate (DEP) metabolite level as the sum of the molar concentrations of the three

diethylphosphate metabolites. Total DAP, DMP, and DEP levels were determined for each

participant for each of the two pregnancy urine samples and for the children’s urine samples.

Individual metabolite levels below the limit of detection (LOD) were assigned a value of the

LOD divided by the square root of two, and this value was included in each sum. Total DAP,

DMP, and DEP metabolite levels were log 10 

# transformed. “Pregnancy” DAP, DMP, and DEP

values were created by averag ing the two log-transformed pr egnancy measures. For 19 women

with only one DAP measurement in pregnancy, the single measure was used. Several children

were missing DAP measures at 3.5 year s (n = 58) and 5 years (n = 14).

Attention-related outcomes were assessed in three ways: 1) maternal report of child behavior at

3.5 and 5 years of age; 2) direct assessment of the child at 3.5 and 5 years; 3) and by a

psychometrician’s report of the behavior of the child dur ing testing at 5 years.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 332 of 531 Mothers completed the Child Behavior Checklist (CBCL) for 1.5–5 years of age as part of the

maternal interview at the 3.5- and 5-year visits to assess emotional/behavioral problems and

competencies of the children. Attention was eval uated as continuous raw scores on these scales,

and by the proportion above a cutoff score greater than the 93rd percentile (a borderline clinical

range).

Children completed a battery of neurodevelopmen tal assessments at each visit: The visual

attention subtest of the NEPSY-II at the 3.5-y ear visit, and the C onners’ Kiddie Continuous

Performance Test (K-CPT), during the 5-year neurodevelopmental assessment. The NEPSY-II

was administered to children by trained psychometri cians. Children were asked to circle specific

images on a page of pictures that included dist racters. For data analysis, the authors used

continuous scores scaled to a normative sample of U.S. children; the age-standardized mean ±

SD for this subtest is 10 ± 3. The K-CPT is a 7-mi n computerized test that assesses reaction time,

accuracy, and impulse control. Briefly, children were instructed to press the space bar when they

saw any image on the computer screen except a ba ll. The computer program yields T-scores agestandardized to a general U.S. population (mean ± SD = 50 ± 10) for errors of commission ( i.e.

, the child responds when he or she should not), errors of omission ( i.e. , the child fails to respond

when he or she should), and hit reaction time. Th e authors examined T-scores continuously and

categorically using the cutoff of T-score > 65, whic h is reportedly considered markedly atypical.

The program used also combined measures to generate a clinical ADHD Confidence Index score

(range, 0–100). For statistical analyses, the authors used continuous Confidence Index

percentiles and selected a cu t-point of > 70th percentile (meaning that 70% of children

performing similarly on the test could be co rrectly classified as having clinical ADHD).

Following the 5-year neurodevelopmental assess ment, psychometricians blinded to exposure

status answered several subjectiv e questions evaluating the behavior of the child during the 2-hr

visit, including four questions derived from the seven-item Hillside Behavior Rating Scale. The

authors summed responses to two questions assessi ng motor activity and distractibility to create

an adapted ADHD symptoms scale. The Hillside S cale is reportedly associated with parent and

teacher ratings and has been found to add significa ntly to the clinical prediction of ADHD. For

analysis the authors created a dichotomized Hillside outcome variable with scores ≥ 7 of 12

possible points (representing < 10 % of children) to flag childr en displaying a higher degree of

attention problems based on ps ychometricians’ observation.

Statistical analyses were conducted using multivaria te linear and logistic regression models to

evaluate the associations be tween the markers of organophosph ate pesticide e xposure and the

indicators of attention deficits summarized a bove. Separate models were run with maternal

DAPs only, child DAPs only, and the two together in the same model. Odds ratios greater than

one and β -estimates greater than ze ro indicate positive associa tions between exposure and

increased risk of adverse outcome, except for the NEPSY visual attention subtest, where a β

estimate greater than zero indicates better perf ormance. All models adjusted for the following

covariates: an indicator of the evaluating psycho metrician (not included in CBCL models), age at

assessment, sex, maternal education, depressive symptoms, maternal inte lligence, out-of-home

child care, and breast feeding duration. The covari ates maternal age, parity, marital status,

active/passive smoking exposure and regular alcohol use during pregna ncy, presence of father in

# Chlorpyrifos Human Health Risk Assessment D424485

Page 333 of 531 home, maternal work status, and household income were also cons idered but not included in the

final models because they did not markedly alte r the observed associations and were therefore

not used in final models. To preserve the si ze of the analytic popul ation, remaining missing

covariate values (six instances for maternal depression and three for maternal PPVT) were

imputed by simple random selection of a value from participants with non-missing values.

The authors also conducted a number of sensitiv ity analyses. They reran all models using

creatinine-adjusted DAP metabolite concentrations. In addition, they added to final models some

factors potentially on the causal pathway ( i.e. , birth weight, gestationa l duration). The authors

also considered whether controlling for lead (log 10 

# -transformed), a known neurotoxicant, altered

results for DAP concentrations in the subsamples with cord (n = 229) or 2-year (n = 296) lead

values. Finally, because rates of ADHD vary considerably by sex in the general population

(Pastor and Reuben 2008) and effects of other toxicants have been found to vary by sex

(Delaney-Black et al. 2004), the authors tested for interactions between DAP concentrations and

child sex, using p < 0.15 for the interaction term to determine whether associations of DAP

concentrations with measures of at tention differed for boys and girls.

Of the 378 infants included in this analysis , 47.5% were male. The mean maternal age was

26.5±5.2 years. Most mothers were born in Mexi co (86%), spoke only Spanish in the home

(90%), had low income (97% within 200% of the federal threshold for poverty; 63% within

100%), had not completed high school (79%), were nonsmokers (96%), had previous live births

(66%), and were married (82%). Many of the pa rticipating mothers had symptoms of depression

(44%).

In this study population, higher co ncentrations of organophosphate metabolites in the urine of

pregnant women were associated with increased odds of attenti on problems and poorer attention

scores in their children at ag e 5 years. Prenatal DAPs were non-significantly associated with

maternal report of attention problems and ADHD at age 3.5 years but were significantly related

at age 5 years (CBCL attention problems: β = 0.7 points; 95% CI: 0.2–1.2; ADHD: β = 1.3; 95%

CI: 0.4–2.1). Prenatal DAPs were a ssociated with scores on th e K-CPT ADHD Confidence Index

> 70th percentile (OR = 5.1; 95% CI: 1.7–15.7 and with a composite ADHD indicator of the

various measures (OR = 3.5; 95% CI: 1.1–10.7). So me outcomes exhibited evidence of effect

modification by sex, with associations found onl y among boys. Children’s concurrent total DAP

and DMP metabolite levels at 3.5 years and 5 year s were unrelated to attention outcomes, and

but child DEP concentrations at 5 years were adve rsely associated with th e composite measure of

attention (OR = 2.0; 95% CI:1.1–3.6).

Study Review

This study tested whether in utero or postnatal exposures to or ganophosphate pesticides have a

detrimental impact on attention. The study populati on was appropriate to the hypothesis, given

that there were a range of exposure levels to OPs and there was some homogeneity in the

population, e.g. , mostly low SES, Hispanic, agricultural workers. The homogeneity of the

population restricted confounding by design. For exam ple, another paper reported that maternal

smoking, alcohol use, and illicit drug use were rare this cohort. The homogeneous population

also increased the relative statistical efficiency of the study becau se it is likely at higher risk of

# Chlorpyrifos Human Health Risk Assessment D424485

Page 334 of 531 deficits in neurodevelopment than the general population. The exclusion of children who did not

complete the 3.5- or 5-year assessment (n = 173), has the potential to indu ce a selection bias if

the reasons for the missing data are determined by exposure (or a predicto r of exposure) and are

also not independent of the development of atten tion disorders. However, the authors stated that

the levels of DAPs were similar in the mothers of the children which were not included relative

to those included in the analysis . The authors also report that di stributions of other covariates

measured before 3.5 years were also similar, exce pt that more boys than girls dropped from the

study. This provides some indication th at selection bias may not be a la rge factor in this analysis.

The ascertainment and analysis of urinary or ganophosphate pesticide markers was appropriate.

Quantification was conducted at the CDC, using published methods. DAP metabolites are likely

more accurate and objective indicators of organophos phate pesticide exposu re, relative to other

ascertainment methods such as self-report. Altho ugh they are sensitive indi cators of exposure, it

is difficult to infer chlorpyrifos effects specifically from the metabolite levels, as they also

indicate exposure to other organophosphates. The study population likel y sustained many other

potentially relevant exposures to multiple chem icals and other pesticides which may affect

development. The six organophosphate metabolites cannot be traced back to individual

pesticides but, according to the authors, they represent the breakdown products of approximately

80% of the total organophosphate pesticides used in the Salinas Valley. It is not possible to

disentangle the various effects fr om these potential co-exposures a nd chlorpyrifos in this study.

Because organophosphate pesticides have short ha lf-lives, the degree to which metabolites in

urine samples reflect ‘usual’ levels, and thus us ual exposure, is uncertai n. The prenatal exposure

measures were, with some exceptions, the averag e of two measurements, and thus may better

reflect chronic exposure during th e pregnancy. The absence of findings associated with child

levels may be due, in part, to th e fact that the child’s levels are poor indicators of exposure

during critical windows of exposure with respect to development of attentional difficulties.

Errors in the assignment of exposure in this prospective study will likely have resulted in

attenuation of observed associatio ns. The likelihood of these errors is high, and the magnitude of

the bias induced difficult to assess. Under an assumption of no other biases, the negative

findings, those for the association between child ren’s concurrent DAP levels and attention

outcomes, for example, could have result ed from exposure measurement error.

The outcome in this study is very difficult to measure. Instead of opting for a single best

instrument/indicator of attention deficits, the inve stigators chose to use multiple instruments, and

also to combine the results of these various instruments to form a composite indicator of

attentional difficulties. There is no evidence presen ted in the report that would suggest that the

outcome assessments were improperly conducted. Nevertheless, outcome measurement error is

likely. As with the exposure assessm ent, errors in the assignment of attention deficits in this

prospective study will likely ha ve resulted in attenuation of ob served associations. Again, the

likelihood of these errors is high, and the magnitude of the bias induced difficult to assess. The

authors did not report on an assessment of cha nges in attentional difficu lties between the 3.5 and

5 year visits. If such changes did occur, it would have been interesting to the reader to present

results of a longitudinal analysis between this change in the outcome, a nd the child DAP levels

over time.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 335 of 531 The statistical analysis used to assess the a ssociations between the markers of exposure and

attention deficits were appropriate. Model diagnostics were performed and, according to the

authors, found acceptable. The results of the model diagnostics were not presented. The authors

also conducted a host of sensitivity analyses to assess influence of m odeling decisions on their

findings, and generally found thei r results to be robust.

In addition to those included in final models, the following covariates we re considered, but not

included in multivariate models: maternal age, parity, marital status, active/passive smoking

exposure, regular alcohol use duri ng pregnancy, presence of father in the home, maternal work

status, and household income at the time of assessment. Confounding by blood lead levels was

also assessed in a subset of children with measur ements of cord blood (n = 229) and 2-year (n =

296) lead levels, and found not to influe nce the DAP and attentional outcomes.

Covariates were selected for these analyses if they were related to conditions of testing or related

to attention deficits in the literature and/or a ssociated with more than one outcome (p < 0.10 or

changes to the main effect coefficient by ≥ 10%). The authors used a less conservative alpha

level as a cut-off for selection of covariates , a somewhat more conservative approach to

covariate selection, relative to use of the convent ional alpha level of 0.05. However, the use of a

statistical test as a means to select covariates from a larger set of potential confounders is

discouraged in some epidemiologic circle s (Miettinen 1976, Breslow and Day 1980, Greenland

1989). The assessment of confounding is generally not considered to be a st atistical endeavor, in

part because the association be tween the confounder and the outcome is just one factor that

determines the degree of confounding, and also b ecause the statistical significance of an

association is a function not only of the magnitude of the associat ion but also of other factors

( e.g. , sample size, and sample variability) whic h are irrelevant to bias due to confounding.

Moreover, the use of significan ce testing in the selection of confounders treats false negative

errors ( i.e. the deleting a confounder) as secondary to false positive error (inclusion of a nonconfounder). This implicit ranking of error is back ward, as deleting a confounder introduces bias

and is justified only if the bias induced by its exclusion is tolerabl y small or deemed to be worth

the precision gain, whereas including a non-c onfounder only (potentially) reduces precision by

increasing the size of the model. This is a sm all consideration in this study, and different

approaches are not likely to qualitatively change the study findings or conclusions. Despite the

confounder evaluation conducted by the investigators, residual confounding due to mismeasured

or unmeasured confounders is likely to have somewhat biased the observed associations.

To preserve the sample size the authors appropriately imputed or replace data for missing

covariates. Data on maternal depression were missing for 5% of obser vations and maternal

intelligence scores for less than 1%. Values of maternal depression measured when the child was

1 year old were available and substituted for 13 instances where maternal depression when the

child was 3.5 years was missing. Other missing cova riate values (six instances for maternal

depression and three for maternal PPVT) were imputed by simple random selection of a value

from participants with non-missing values. The re sulting analysis is relatively more precise,

owing to the larger sample size analyzed, but this statistical efficiency comes at the tradeoff of

bias due to residual confounding by imperfectly measured covari ates. Socioeconomic conditions

and other difficult to measure determinants of early life development may further confound the

# Chlorpyrifos Human Health Risk Assessment D424485

Page 336 of 531 reported associations in the direction of mo re deleterious effects associated with the

organophosphate metabolites and with unknown magnit ude. The potential for this bias may be

mitigated somewhat due to restriction in the variability of these confounding factors in this

relatively homogeneous study population.

The generalizability of the findi ngs to other populations may be limited, due to the particular

nature of this study population with respect to fa ctors which may modify the association between

organophosphate exposures and infant developm ent. The organophosphate pesticide exposure

markers and the prevalence of attention deficits in this low-income Mexican-American

population sampled from an agricultural area are almost certainly not generalizable to the U.S.

population, although exposure levels are within a comparable range to the U.S. general

population (see Section 5.0).

The study has numerous strengths, most notably the prospective design and the longitudinal

assessment of both exposure and outcomes. The positive association between organophosphate

metabolites in the urine of pregnant women and increased odds of attention problems and poorer

attention scores in their offspring was relativ ely consistent using di fferent assessments of

attentional difficulties ( i.e. by maternal report, psyc hometrician observation, and

neuropsychological testing). Asso ciations were stronger at ag e 5 years compared to age 3.5

years, and there was ev idence of a stronger association am ong boys, compared to girls. The

results of this investigation by Marks et al in the CHAMACOS cohor t are suggestive of a

detrimental association between prenatal orga nophosphate exposure, as measured by maternal

urinary metabolite levels, and attentional difficul ties at age 5 years. This study may be limited in

its direct contribution to the literature base regarding the specific association between

chlorpyrifos and developmental outcomes because the assessment of non-specific

organophosphate pesticide metabolites (DAPs) is a primary potential source of exposure

measurement error. Because of th e prospective design, information bi as is expected to result in

attenuation of the true (unobser ved) associations, toward (obs erved) associations that are

relatively smaller, or null.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 337 of 531 

# Article 13. Rauh et al. (2011)

Seven-Year Neurodevelopmental Scores a nd Prenatal Exposure to Chlorpyrifos, a

Common Agricultural Pesticide. Virginia Rauh, Srikesh Arunajadai, Megan Horton,

Frederica Perera, Lori Hoepner, Dana B. Ba rr, and Robin Whyatt. Environmental Health

Perspectives. 119:1196–1201 (2011).

Study Summary

Having previously reported an association betw een prenatal exposure to chlorpyrifos and

neurodevelopmental problems at three years of age in the Columbia Center for Children’s

Environmental Health, Rauh et al now evaluate the relationship between prenatal chlorpyrifos

exposure and neurodevelopment among cohort children at age seven.

Study participants were recru ited during early pregnancy ( ≤ 20th week) among African-American

and Dominican women age 18-35 y ears, and registered Hospital, in New York at New York

Presbyterian Medical Center and Harlem City. Women who smoked, had a history of drug abuse,

diabetes, hypertension, or HIV inf ection were excluded from partic ipation in the study, as were

women who resided in New York City for less th an 1 year. The study sample presented in this

report was recruited between 1998 and 2002, a period which straddles the January, 2001

implementation of a voluntary cancellation by regist rants of indoor residen tial chlorpyrifos use.

Of originally 725 consenting women, 535 were ac tive participants in the ongoing cohort study at

the time of this report. This report uses data from 265 of their children who had reached the age

of 7 years and had a complete set of data includ ing prenatal maternal interview data, prenatal

chlorpyrifos marker levels fr om maternal and/or cord blood samples at delivery, postnatal

covariates, and neurodevelopmental outcome data.

Chlorpyrifos levels in umbilical cord blood sa mples were available for 256 newborns, sampled

as close to the time of delivery as possible, a nd within 2 days post-partum. In cases where the

umbilical cord blood sample was not collected (12% of subjects), cord blood chlorpyrifos levels

were imputed from mothers’ values. Quantifica tion of chlorpyrifos levels in plasma were

conducted by the Centers for Diseas e Control and Prevention (CDC).

Neurodevelopment at age 7 years was assessed using the Wechsler Intelligence Scale for

Children, 4th edition (WISC-IV). Th e instrument measures four ar eas of mental functioning: the

Verbal Comprehension Index, the Perceptual Reasoning Index, the Working Memory Index, and

the Processing Speed Index. These indices are as sociated with, but distinct from, overall

intelligence quotient (IQ) and are sensitive to cogn itive deficits related to learning and working

memory, which have been linked to chlorpyrifos exposure in rodent st udies. A Full-Scale IQ

score combines the four composite indices. A Gene ral Ability Index score is a summary score of

general intelligence, similar to Full-Scale IQ , but excludes contributi ons from both Working

Memory Index and Processing Speed Index.

Multivariate linear regression models were used to estimate associ ations. Chlorpyrifos exposure

level (picograms per gram) was entered as a continuous variable. The WISC-IV Composite

Index scores were natural log (l n) transformed to stabilize thei r variances and to improve the

linear model fit, based on regression diagnostics . Linear and non-linear functional relationships

# Chlorpyrifos Human Health Risk Assessment D424485

Page 338 of 531 between chlorpyrifos exposure and each of the log-transformed WISC-IV indices were assessed

and compared.

Participating mothers were predominantly from low income households, with 31% failing to

complete high school by child’s age 7 years, a nd 66% were never married . The children in the

study were largely full term and included ve ry few low-birth-wei ght infants through study

design. Chlorpyrifos exposure levels as measur ed in cord blood ranged from non-detectable to

63 pg/g.

Unadjusted correlations showed significant invers e associations between ch lorpyrifos levels and

Working Memory (r=-0.21, p=<0.0001); and Full-sca le IQ (r=-0.13, p=0.02). Because cord lead

levels were not significantly co rrelated with chlorpyrifos levels , and in an effort to avoid

excluding those with missing lead levels, lead wa s not included in the regression models. ETS

was significantly correlated w ith chlorpyrifos levels (Spearman coefficient 0.113, p=0.01) but

not with any of the WISC-IV indices. In addi tion, birth weight was not significantly associated

with any of the WISC scales and not included in the final models.

The investigators used both fully-adjusted line ar regression modeling an d the Least Absolute

Shrinkage and Selection Operator (LASSO) t echnique, described as a more parsimonious

selection of covariates that a ddresses unstable estimates while avoiding overfitting (Houwelingen

2001; Tibshirani, 1996.) Both me thods yielded very similar estimates, finding associations

between chlorpyrifos exposure and Full-Scale IQ as well as the Working Memory Index. For

Full-Scale IQ both techniques generated a coeffi cient (B) of -0.003 with matching CIs of -0.006,

0.001 but the P-values were calculated as 0.064 for the LASSO and 0.048 for the fully-adjusted

model. The fact that both CI’s contain zero wh ile the fully-adjusted model’s P-value is lower,

suggests that the LASSO technique does in fact shrink unstable es timates towards zero. For the

Working Memory Index, both techniques generate d a coefficient (B) of -0.006 with the LASSO

calculating a CI of -0.009 to -0.002, p<0.001 and the fully-adjusted model calculating a CI of -

0.010 to -0.002, p=0.003. The investigators articula ted these results as showing that a 1 pg/g

increase in chlorpyrifos exposure was associ ated with a 0.006 point decrease in the logtransformed Working Memory score and a 0.003 poi nt decrease in the log-transformed FullScale IQ score. In order to understand the magnitude of these eff ects, the investigators

calculated the neurodevelopmenta l deficit associated with the increase in exposure and

concluded that for each standard deviation in crease in exposure (4.61pg/g) there is a 1.4%

reduction in Full-Scale IQ and a 2.8% reduction in Working Memory. Authors explain that their

full model results did not show any significant in teractions between ETS or polycyclic aromatic

hydrocarbons (PAH) with measures of prenatal chlorpyrifos expos ure during the prenatal period

and intelligence measures in this study.

The investigators conducted a sensitivity analys is to determine if th e association between

chlorpyrifos levels and Working Memory Inde x was partially due to the effect on general

intelligence. To do this, they looked at the Ge neral Ability Index of the WISC-IV, a general

intelligence scale calculated without the inclusion of the items included in the Working Memory

Index or Processing Speed Index. When the Ge neral Ability Index was added to the linear

regression model, the estimated chlorpyrifos eff ect did not change and they did not observe an

interaction between chlorpyrifos and General Abi lity Index. From this they concluded that the

# Chlorpyrifos Human Health Risk Assessment D424485

Page 339 of 531 Working Memory Index is a targeted measur e, independent of ge neral intelligence.

The investigators further expl ored their findings by conducting a supplemental analysis to

evaluate whether the observed associations between chlorpyrifos and the Working Memory

Index were related to underlyi ng behavioral problems. They used the results of the Child

Behavior Checklist and it s clinically-oriented behavioral scal es and concluded that there was no

evidence of mediation caused by behavioral issues.

Lastly the investigators addressed the possible influence of th eir method and frequency of use of

LOD imputations. To do this th ey recalculated all of the associ ation estimates using only those

records that had detectable chlo rpyrifos levels and explained th at this method would provide an

unbiased estimate of the associa tion (Little, 1992.) They did not find any consistent differences

in estimates between the full sample (including im puted exposure levels) versus those calculated

without those imputed levels.

Study Review

This was a very well conducted study with numerous strengths including se lection of a relevant

and appropriate cohort, the prospective study design, direct measurement of chlorpyrifos,

comprehensive consideration of confounding, an d analytic design choices which maximize

statistical efficiency. However, some limitations are also noted.

The direct measurement of chlorpyrifos in cord blood and personal air samples, rather than selfreported indirect exposure assessment or use of biomarkers of organophosphate pesticide

exposure that are not specific to chlorpyrifos exposure is a not able strength of the Columbia

Mother’s and Newborn Study cohort. Unlike previ ous analyses among this cohort that used a

dichotomous variable (low vs. high exposure) to classify expos ure status, these findings employ

a continuous chlorpyrifos level, which provides a more meaningful look at the data including the

ability to assess potential threshold effects and linear and non-linear dos e-response trends. The

investigators rigorously evaluated their met hods for imputing values for undetectable

chlorpyrifos levels; analyses il lustrated imputation methods were valid. However, regarding the

LOD estimations, the authors did no t describe the methodology used ( e.g. bootstrap estimation),

if any, to incorporate the added uncertainty ( i.e. added imprecision to estim ates) inherent in the

use of imputed data into the es timation of the corresponding variance estimates. However, errors

in estimation of cord blood chlorpyrifos levels were likely to have b een non-differential with

respect to the indicators of childhood neurodevelopment and intelligence.

A limitation of the specific biomarker exposure in dicators in this study is the single sampling

period (at delivery). It is not cl ear to what extent this single measurement reflects exposures over

critical windows during pregnancy. If the exposure is chronic, a biomarker measured at a single

time point can provide a representative dosimeter, ev en if the toxicant has a short half-life, as is

the case for chlorpyrifos. However, if pesticide exposures are s poradic or otherwise vary over

short time scales, the biomarker measurement will not be representative of “usual” exposure, or

of the exposure during critical pe riods of fetal neurodevelopment. In previous publications from

this same cohort, the investig ators explain that analyses we re performed showing that, among

these women, exposures to chlorpyrifos did not show significant variat ion throughout pregnancy

# Chlorpyrifos Human Health Risk Assessment D424485

Page 340 of 531 (Whyatt et al, 2007) and therefore concluded that this single meas ure can accurately represent

prenatal exposure. This assumption is furt her assessed in subsequent validation studies

conducted by Whyatt et al (2007 and 2009) within this cohort.

Whether or not these cord blood measurements accu rately represent prenat al exposure, there is

no control for exposure over the subsequent 7 year s which may be critical, especially as the

process of neurocognitive development is fluid and rapid during these early childhood years.

From this study, it is unclear if an increase or decrease in ex posure during this influential

postnatal period, caused by age-related or season al dietary changes or changes in pesticide

application, could also impact the neurodevelopmen t of these children. Because this imprecise

measure of exposure is unlikely to vary different ially, this error would bias towards the null.

Another influence unaccounted in the analysis is the effect of incr eased awareness of the risks of

pesticide exposures over the time period of cohort follow-up in this study (1998-2004) and the

potential for differential exposure or outcome (CBCL) measures. It is feasible to assume that

those parents of children with suspected or diagnosed neurodevelopmental issues in early

childhood would be more aware of issues concer ning organophosphate exposure, and thus more

likely to subsequently reduce their child’s expos ures, especially through a reduction in pesticide

or dietary sources in childhood. If this is true, and post-natal e xposures play a role in childhood

neurodevelopment measured in this study, those children with neurodevelopmental deficits may

be less likely to experience post-natal exposures than those not diagnosed, thus making the prenatal effects appear weaker. Similarly, mothers of infants in the high exposure category may

differentially report behavioral issues on the mate rnal-reported child behavior checklist . Either

type of differential measurement error may lead to information bias, with unknown

consequences on the estimate of risk in these studies. It would be meaningful to assess the

mother’s awareness of the effects of organophospha te exposures as well as details regarding

dietary exposures and how they may ha ve changed over the 7-year period.

The prospective nature of the study limits the infl uence of errors in the exposure assessment on

the association observed, such that mismeasurem ent of chlorpyrifos exposure would likely be

non-differential with respect to the indicators of neurodevelopment and intelligence at age 7

years. The voluntary cancellation of the resident ial use of chlorpyrifos introduces exogenous

variation in the exposure of inte rest. After the cancellation, leve ls of chlorpyrifos in personal

and indoor air samples in this cohort decreased by more than 65%, and plasma blood levels

dropped by more than 80%.

The Wechsler Intelligence Scale for Children (W ISC-IV) instrument meas ures four areas of

mental functioning: the Verbal Co mprehension Index is a measure of verbal concept formation, a

good predictor of school readine ss; the Perceptual Reasoning Index measures nonverbal and

fluid reasoning; the Working Memory Index assesses children’s ability to memorize new

information, hold it in short-term memory, conc entrate, and manipulate information; and the

Processing Speed Index assesses ability to focus attention and quickly scan, discriminate, and

sequentially order visual information. The author s explain that the sensitivity of the WISC-IV

among 6 to 7.5 year old children has been validate d in previous neurodevelopmental studies of

environmental lead exposure, even at low-dose levels (Chiodo et al. 2004). However, WISC-IV

scores are also known to be influenced by so cioeconomic background and/or child behavior

# Chlorpyrifos Human Health Risk Assessment D424485

Page 341 of 531 problems, particularly those rela ted to anxiety (Wechsler, 2003). The narrowly restricted cohort

and evaluation of behavioral issu es, which may in part reflect a nxiety issues, within this study

somewhat limits this potential bias of th e outcome assessment tool in this study.

One potential weakness is that the investigators do not give the details of the administration of

the WISC-IV; specifically the credentials of the interviewers, whether or not they were blinded

to the child’s exposure status, any trainings or ce rtifications, and whether or not intra- or interinterviewer reliability or quality assurance was conducted. These types of administration issues

can result in less-sensitive measures and misclassi fication, but there is no reason to believe that

there would be a differential eff ect based on exposure level.

Also complicating this analysis is the pervasiv e, non-specific nature of neurological effects and

the difficulty in attributing causal pathways to one agent. In future studies it would be

meaningful to include other potential co-varia nts such as family history (especially among

parents and siblings) of developmental delay or attention disorders as well as other comorbidities. Not only is the etiology of attention and relate d behavioral disorders not well

understood but also the diagnosis of these disorders is often infl uenced by complicated outside

forces. Because the study population is relative ly homogenous with respect to socio-economic

variables, it is unlikely that diffe rences in co-morbidities or fam ily history would be associated

with exposure status, thus biasing the association towards the null.

The authors describe an elegan t and methodologically sound statis tical analysis, addressing many

of the potential shortcomings of their exposure da ta and co-variables. Out of the five WISC-IV

scores, two were described as being significantly influenced by chlorpyrifos exposure. After

they conducted both full-adjusted linear regressi on and LASSO techniques, they concluded that

both Full Scale IQ and Working Memory index were affected by prenatal ch lorpyrifos exposure.

For each standard deviation increase in exposur e (4.61pg/g), there was a 1.4% reduction in FullScale IQ and a 2.8% reduction in Working Memo ry. The Working Memory Index conclusions

seem reasonable, but the associations for Full Scale IQ were not statistically significant (LASSO:

-0.006, 0.001, p=0.064; fully-adjusted: -0.006, 0.001, p= 0.048.) In addition, the authors do not

address the clinical relevance of the 1.4% and 2.8% reductions and how this may or may not

influence a child or his/her psycholog ical or educational success.

In their confirmatory analyses they used the General Ability Index (the intelligence score

calculated after removing Working Memory and Pr ocessing Speed items) to verify that the

association seen between chlorpyrifos exposure and Working Memory was not an artifact of the

association with Full Scale IQ. With the Genera l Ability Index included in the linear regression

model, the estimated chlorpyrifos effect did not change and they did not observe an interaction

between chlorpyrifos and General Ability Index. Subsequently th ey concluded that the deficits

in Working Memory Index were associated with chlorpyrifos exposure, independent of general

intelligence but analyses could have also taken an additional step. These results could also be

used to conclude that since the General Ab ility Index was not found to be affected by

chlorpyrifos exposure, the Full Scale IQ findings were caused by the influence of the Working

Memory module of the instrument. The influe nce of chlorpyrifos exposure on the working

memory aspect of the Full Scale IQ score was strong enough to overcome the non-significant

results from the other modules of the instrument.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 342 of 531 Confounding by maternal smoking behavior s was controlled by restriction ( i.e. , excluding

smokers from the study sample). Confounding by un measured factors may have been similarly

controlled by design, this being a relativel y homogeneous population with respect to

determinants of neurodevelopment, compared to the general population. Authors evaluated

several potentially confounding va riables including exposure to ot her environmental chemicals

including blood lead, and only included thos e variables which di splayed evidence of

confounding.

While controlling confounders by restriction, the choice to study low income, urban dwellers

likely resulted in a relative incr ease in statistical efficiency ( i.e. increased power) because this

population likely experiences a greater variability of pesticide exposure and is at higher risk of

neurodevelopmental deficits, re lative to the general population.

Although blood lead levels were not included as covariates in the multivariate models, the

authors reported that umbilical cord lead was not significantly co rrelated with chlorpyrifos level

or WISC-IV scores among the subgroup of children in this population for whom blood lead was

assessed (n=89). Polyaromatic hydrocarbons were weakly correlate d with chlorpyrifos in this

cohort but were not significantly associated with WISC-IV sc ores. Birth weight was not

significantly associated with a ny of the WISC-IV indices and was not included in the final

models.

Bias due to confounding by unmeasured determinan ts of neurodevelopment is possible in this

observational study, for example, confounding by f actors related to socioeconomic condition.

The urban dwelling, minority children assessed in this study may experience other unmeasured

exposures or underlying health problems th at could potentially confound (or modify)

associations with pesticide exposure. Howeve r, both adjustment thr ough restriction of study

population, as well as control for several measures of SES in multi-variable models, as well as

additional methodological analyses of the role of SES in this associa tion (Lovasi et al. 2011),

does not suggest a significant additional role of unmeasured confounding due to variation in SES

within this study population. Effect modification by all of the covariates was assessed, including

for environmental tobacco smoke and polycyclic aromatic hydrocarbons, but no interactions

were observed.

The authors state that the retention rate for the full cohort was 82% at th e 7-year follow-up, and

reported that there were no signi ficant sociodemographic differenc es between subjects retained

in the study and those lost to follow-up, although these data were not provided. However, the

265 children included in this an alysis were born to a 36.6% s ubsample of the 725 women who

originally consented to partic ipate in the study. The resulting large proportion of missing data

has the potential to i nduce a selection bias if the reasons for missingness of outcome data are

determined by exposure (or a predictor of e xposure) and are also not independent of

neurodevelopment. For example, participants wi th high pesticide exposures could have missed

neurodevelopmental assessments for reasons rela ted to uncontrolled determinants of exposure

which are also related to neurodeve lopment. This was not discussed.

A comprehensive set of sensitivity analyses and regression diagnostics were conducted,

# Chlorpyrifos Human Health Risk Assessment D424485

Page 343 of 531 including examination of residuals for normality and homoscedasticity, influential observations,

and the linearity of the exposure- response relationship, and assessmen t of the influence of design

choices ( e.g. imputation) on observed results. Models we re determined to be consistent with

modeling assumptions.

Non-linearity of the exposure response curves were assessed by superimposing smoothed cubic

spline regression curves over sca tterplots of the data. The lo g-transformed Working Memory

Index and Full-Scale IQ were approximately linea r, whereas the other functions demonstrated

some curvature across exposure levels, with sp arse observations at the highest exposures.

However, formal goodness of fit testing did not demonstrate that non-linear models provided a

better fit to the data, compared to models th at assume a linear exposure-response relationship

( i.e. no observed threshold effect).

Results of this study showed that higher prenatal chlorpyrifos exposure, as measured in umbilical

cord blood plasma, was associated with decr eases in cognitive functioning on two different

WISC-IV indices, in this cohort of urban minority children at 7 years of age. For each SD

increase in exposure (4.61 pg/g) , Full-Scale IQ declined, on av erage, by 1.4% (0.94–1.8 points)

and Working Memory Index scores declined by 2.8% (1.6–3.7 points). These results are

consistent with earlier findings of an inverse association between chlorpyrifos levels in umbilical

cord plasma and neurodevelopment in the same cohort. The study represents perhaps the most

rigorous study design and analysis of the studies reviewed.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 344 of 531 Works Cited

Bradley RH and Corwyn RF. Socioeconomic Status and Child Development. Annual Review of

Psychology. 53:371-399 (2002).

Chiodo LM, Jacobson SW, Jacobson JL. 2004. Neurodevelopmental effects of postnatal lead

exposure at very low levels. Neur otoxicol and Teratol, 26:359-371.

Houwelingen JCv. 2001. Shrinkage and penalized lik elihood as methods to improve predictive

accuracy. Stat Neerlandica. 55(1):17-34.

Lovasi, G. S., Quinn, J. W., Rauh, V. A., Perera, F. P., Andrews, H. F., Garfinkel, R., . . .

Rundle, A. (2011). Chlorpyrifos exposure and ur ban residential environment characteristics as

determinants of early childhood neurodevelopment. Am J Public Health, 101 (1), 63-70. doi:

10.2105/ajph.2009.168419

Little RJA. 1992. Regression with missing X’s: a review. J Amer Stat Assoc. 87:1227–1237.

Wechsler D. Wechsler Intel ligence Scale for Children. 4rd Edition. San Antonio, Texas:

Psychological Corporation. 2003.

Whyatt RM, Rauh V, Barr DB, et al. Prenatal ins ecticide exposure and birth weight and length

among an urban minority cohort. Environ Health Perspect. 2004;112:1125–1132

Whyatt RM, Garfinkel RS, Hoepner LA, Borjas M, Camann DE. Within and between home

variability in indoor-air insecticide levels during pregnancy [abstract W2A–3]. Presented at the

15th Annual Conference of the International Society of E xposure Analysis; October 30–

November 3, 2005; Tucson, AZ

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Page 345 of 531 

# Article 14. Bouchard et al. (2011)

Prenatal Exposure to Organophosphate Pesticides and IQ in 7-Year-Old

Children. Maryse F. Bouchard, Jonathan Chev rier, Kim G. Harle y, Katherine Kogut,

Michelle Vedar, Norma Calderon, Celina Trujillo, Caroline Johnson, Asa Bradman, Dana

Boyd Barr, Brenda Eskenazi. Environmen tal Health Perspect ives. 119:1189–1195 (2011)

Study Summary

Researchers with the Center for the Health Asse ssment of Mothers and Children of Salinas study

(CHAMACOS study) published an analysis which i nvestigated the relationship between prenatal

and early life exposures to organophosphate pest icides (OPs) and cognitive development. The

CHAMACOS study is a prospective birth cohor t study of environmental exposures and the

health of pregnant women and their children livin g in the Salinas Valley, an agricultural area of

California.

A 2007 report from this cohort suggested that pr enatal, but not postnatal, exposure to OPs was

associated with increased odds of pervasive deve lopmental disorder and lower scores of mental

development at two years (Eskenazi B 2007). A dditionally, a 2010 paper reported an association

between organophosphate pesticide exposure and poor er attention skills as well as hyperactive

behaviors at 5 years (Marks AR 2010). This 2011 study by Bouchard et al was conducted to

determine whether cognitive deficits associat ed with prenatal exposure to organophosphate

pesticides are persistent, since previous cohort studies have not followed children to school-age,

when cognitive deficits may have greater imp lications for school performance. This paper

reports the association between prenatal and postnatal exposure to organophosphate pesticides,

indicated by urinary dialkyl phosphate (DAP) meta bolite concentrations, a nd cognitive abilities

of 7-year olds.

Enrollment took place from 1999–2000 at community clinics, and included women who were ≥

18 years old, pregnant at < 20 weeks gestation, Spanish- or English-speaking, eligible for lowincome health insurance, and planning to deli ver at the local public hospital. The initial 601

women who were enrolled into the study delivered 526 infants. From this analysis, 2 participants

with missing DAP concentration measurements were excluded, 4 children with a medical

condition that would affect the a ssessment, children who were lost to follow-up or who did not

participant in the 7-year vis it were excluded (72 moved, 59 refu sed, 24 unable to trace, 21 unable

to schedule, 2 deceased), and children missing the cognitive assessment at the 7-year visit (13)

were also excluded.

Prenatal urine was collected between 5 and 27 weeks gestation and again between 18 and 39

weeks. Urine was collected from the children at 6 months and 1, 2, 3 1⁄2 and 5 years of age. Six

nonspecific organophosphate DAP metabolites (3 DEP metabolites and 3 DMP metabolites)

were measured in maternal and child urine which represent the brea kdown products of about

80% of the total organophosphate pesticides us ed in the Salinas Valle y (CDC 2009). The most

commonly used organophosphate pesticides in the Salinas Valley include chlorpyrifos and

diazinon (which are metabolized to DEP) as we ll as malathion and oxydemeton-methyl (which

devolve to DMP). Concentrations below the li mit of detection (LOD) were randomly imputed

based on a log-normal probability distribution whos e parameters were estimated using maximum

# Chlorpyrifos Human Health Risk Assessment D424485

Page 346 of 531 likelihood estimation. The DAP metabolite concentr ations were expressed on a molar basis and

summed to yield total DEP, DMP, and DAP concentrations.

Cognitive abilities were measured at the 7-year study visit using th e Wechsler Intelligence Scale

for Children - Fourth Edition (WISC-IV) (Wech sler 2003). Scores for four domains were

calculated based on the following subtests: Verbal Comprehension (comprised of the Vocabulary

and Similarities subtests), Perceptual Reasoning (B lock Design and Matrix Reasoning subtests),

Working Memory (Digit Span and Letter-Number Sequencing subtests), and Processing Speed

(Coding and Symbol Search subtests). All subtes ts were administered in the dominant language

of the child, which was determined through admini stration of the Oral Vocabulary subtest of the

Woodcock-Johnson/Woodcock-Muñoz Tests of Cogn itive Ability in both English and Spanish

(Woodcock and Johnson 1990). Ultimately, 67% of ch ildren were tested in Spanish, and 33% in

English. WISC-IV scores are standardized agai nst U.S. population-based norms for English and

Spanish-speaking children.

Many confounders were measured, including intel ligence of the mother, measures of how

stimulating the environment is, and known or su spected neurotoxins were measured prenatally.

Maternal intelligence was assessed via the Peabody Picture Vocabulary Test (PPVT). To

measure the quality and extent of stimulation available to a child in the home environment, the

Infant-Toddler HOME (Home Observation for Meas urement of the Environment) inventory was

completed at the 6-month, 1, 2, 3.5, 5, and 7 ye ar visits. The following known or suspected

neurotoxicants were measured: polybrominated diphenyl ethers (PBD Es), polychlorinated

biphenyls (PCBs), p,p’-dichlorodiphe nyltrichlorethane (DDT), p,p’-

dichlorodiphenyltrichlorethylene (DDE), and lead. Lead was meas ured in maternal blood at 26

weeks gestation, in cord blood for a subset of participants, and ch ildren’s blood a 2 years of age.

PBDEs, PCBs, DDT, and DDE were measured in maternal serum samples collected at 26 weeks

gestation.

Nonspecific total DAP, DEP, and DMP metabol ites (nmol/L) were log10-transformed. The

authors examined the association between non-cr eatinine adjusted urinar y DAP concentrations

and cognitive scores using multiple linear regr ession, with point estimates representing the

change in cognitive scores for each 10-fold increase in DAP concentrations. For prenatal

exposure, they examined associations with the DA P concentrations measured separately for urine

collected during the 1st a nd 2nd half of pregnancy ( ≤ 20 vs. >20 weeks gestation). However,

since they found similar relations between cogniti ve scores and DAPs meas ured earlier and later

than 20 weeks gestation, they averaged the two DAP measures for further analyses. For 20

children (6%) only one prenatal measure was available for the analyses. Because DMP and DEP

metabolites might have different relationships to the outcomes, the authors examined them

separately. For postnatal exposure, they examin ed the cross-sectional association of cognitive

scores with DAP concentrations measured in ch ildren’s urine collected at different ages in

separate models.

They also calculated the cumulative DAP leve l between 6 and 60 months using the area under

the curve (AUC), calculated using the trapez oidal method. For 46 child ren with 1 missing DAP

measurement at 1, 2, or 31⁄2 years, they imputed th e mean of the 2 measures closest in time for

the AUC calculation. Forty-nine children who were missing DAP measures at either the 6-month

# Chlorpyrifos Human Health Risk Assessment D424485

Page 347 of 531 or 5-year visit, or missing more than 1 DAP measure from the 3 other time points were excluded

from the AUC analysis. For comparison with prenat al exposure, they calcu lated the mean urinary

DAP concentrations measured during childhood for ch ildren with at least 3 out of 5 measures

(taken at 6 months, 1, 2, 3 1⁄2, and 5 years); this excluded 20 children.

The authors compared effect estimates for urin ary DAPs measured in ea rly vs. late pregnancy

and in the prenatal vs. postnatal periods using seemingly unrelated estimation and they used the

mean postnatal DAP concentrations (as opposed to AUC) for these analyses in order to compare

metrics with similar units. They used generali zed additive models with 3-degree-of-freedom

cubic splines to evaluate the shape of dose-res ponse curves, test the linearity assumption, and

investigate potential thresholds while controlling for covariates. They did not observe evidence

of departure from linearity, or th reshold for effect, thus, they retained the simpler models based

on linear regression. For illustration purposes, they grouped DAP concentrations into quintiles,

entered this categorical variable in the multip le regression model with the same covariates

described above, and obtained the mean IQ score for each quintile.

The authors explored potential confounding eff ects for the following variables: maternal

intellectual abilities, maternal education, HOME score at 6 months, 1, 2, 3 1⁄2, and 5 years,

breastfeeding duration, mate rnal age, birth order, poverty cate gory, marital status, children’s age

at WISC testing, and maternal levels of PBDEs, PCBs, DDE, DDT, and lead during pregnancy.

They added each of these variables added indi vidually to the final model. However only

maternal intellectual abi lities (PPVT score, continuous), matern al education (3 categories), and

continuous HOME score at 6 months were retained in the final models. Other variables were

dropped from the final models because they did not change the magnitude of the coefficient for

urinary DAP concentrations by >10%.

In separate analyses, the author s also investigated potential confounding and effect modification

by variables possibly on the causal pathway ( i.e. birth weight and gestational age, assessed

continuously). Because the majority of children (67%) were tested in Spanish, the authors reran

the analyses restricted to this subset. The aut hors also examined the in teraction between sex and

DAP concentrations, based on previous fi ndings in this cohort (Marks AR, 2010).

To explore possible synergistic effects between pre- and postnatal DAP concentrations, the

authors included an interaction term for mean prenatal DAP concentrations×AUC. However,

since this term was not statistic ally significant (p > .15) they did not include it in the final

models.

The authors observed evidence of an association between prenatal exposur es to organophosphate

pesticides as measured by urinary DAP metabol ites in women during pregnancy, and decreased

cognitive functioning in children at age 7. This finding was seen for total DAP, DMP, and DEP

metabolites. Children in the highest quintile had of maternal DAP concentrations had an average

deficit of 7.0 IQ points compared with those in the lowest quintile. The associations were linear

with no threshold. Urinary DAP concentrations in childhood were not associated with cognitive

scores in this cohort of children.

Study Review

# Chlorpyrifos Human Health Risk Assessment D424485

Page 348 of 531 This study tested whether in utero or postnatal exposures to or ganophosphate pesticides have a

detrimental impact on cognitive abilities at age seven years. The study population was

appropriate to the hypothesi s, given that there were a range of exposure levels to OPs and there

was some homogeneity in the population, e.g. , mostly low SES, Hispanic, agricultural workers.

The homogeneity of the population restricted co nfounding by design. For example, another paper

reported that maternal smoking, al cohol use, and illicit drug us e were rare this cohort. The

recruitment of an inner-city cohort may also have increased the relative statistical efficiency of

the study if the study population is at higher risk of neurodevelopmen tal deficits compared to the

general population. Selection bias may have been a factor in this analysis as only 329 families

out of 601 who were originally en rolled into the study were incl uded. The authors state that the

families included in this analysis did not di ffer on most attributes, including urinary DAP

concentrations during pregnancy, maternal measur es of cognitive abilit y, maternal education,

marital status, poverty category, a nd child’s birth weight. However, mothers of children included

in the present study were slightly older and breas tfed longer than the initial cohort. Differences

between maternal blood lead and other environm ental exposures which are potential confounders

were not discussed.

The ascertainment and analysis of urinary or ganophosphate pesticide markers was appropriate.

Quantification was conducted at the CDC, using published methods. DAP metabolites are likely

more accurate and objective indicators of organophos phate pesticide exposu re, relative to other

ascertainment methods such as self-report. Altho ugh they are sensitive indi cators of exposure, it

is difficult to infer chlorpyrifos effects specifically from the metabolite levels, as they also

indicate exposure to other organophosphates. The study population likel y sustained many other

potentially relevant exposures to multiple chem icals and other pesticides which may affect

development. The six organophosphate metabolites cannot be traced back to individual

pesticides but, according to the authors, they represent the breakdown products of approximately

80% of the total organophosphate pesticides used in the Salinas Valley. It is not possible to

disentangle the various effects fr om these potential co-exposures a nd chlorpyrifos in this study.

The IQ test used the WISC-IV is very widely util ized in both clinical and research setting, and

has a long history of use, and was appropriately administered by psychom etrician. Additionally,

the test was administered at age 7 years, whic h is above the floor for administration (age 6)

(Wechsler 2003). However, IQ tests are notorious ly susceptible to possible biases due to

differences in cultural and/or ethnic compositi on of the population, althou gh the homogeneity of

the population may ameliorate the effect of this potential bias. The authors did perform the

analysis both by looking at only th e tests that were administered in Spanish and with the Spanish

and English included. However, language may not ha ve controlled for all potential biases which

are inherent in IQ testing, e.g. , degree of acculturation.

The statistical models used are appropriate a nd well powered. For exposure measures that were

less than the laboratory analytic limit of detec tion, the measures were imputed. Imputation is a

better method than substitution of the LOD/ √ 2 or LOD/2, which is the method used in many

studies. It appears that the investigators sel ected parsimonious models by only including terms

which changed the outcome measure and were sta tistically significant. Additionally, the authors

tested the linearity assumption which is inhere nt in linear regression using cubic splines.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 349 of 531 Residual confounding by unmeasured or poorly meas ured confounders is possible in this study.

The authors did measure a large number of poten tial confounders, but no t all were evaluated

notably, childhood exposure to neurotoxins, and maternal drug used during pregnancy. A

number of environmental chemicals were cons idered, although only measurements of exposure

during pregnancy were included in the analysis. Additionally, exposure to carbamates, which are

also potentially neurotoxic, was not included in the analysis. The authors also did not consider

maternal alcohol, tobacco, or drug use in this an alysis. However, another study with this cohort

reported that these confounders are ve ry rare in this study population.

The authors did not discuss paternal intelligence or intelligence of other caregivers. This is

probably because only the mothers and children were enrolled into the study, and it is difficult to

enroll fathers and other caregive rs into birth cohort studies. Although it is understandable that

these measures were not addressed, it seems highl y possible that intelligence of the fathers and

other caregivers could be associated with th e exposure, organophosphate pesticides, and the

disease, cognitive impairment of the child. T hus these factors could have confounded the study

results, and it is a limitation that they were not included in the meas urement or analysis.

A potential limitation with respect to generalizability is the high level of e xposures observed in

the study cohort, compared to the US population. Th e mean level of urinary DAP metabolites in

this cohort is higher than the mean which has been seen among women of child bearing years in

NHANES. However, the investigators report th at 25% of the US population have urinary DAP

levels exceeding the median in this study. So, the c oncentrations in this cohort are not out of the

range of normal for the US as a whole. Genera lizing beyond the range of exposure observed in

this study requires an assumption that the linear exposure-outcome relationship observed in the

study is applicable to populations wi th different levels of exposure.

This study has many strengths, the longitudinal de sign, the measurement of urinary DAP at many

time points and following children to age seven when tests of cognitive function are reportedly

more reliable. The authors were able to adjust for or consider many factors related to cognitive

function, such as prenatal expos ure to other environmental agen ts, socioeconomic indicators,

maternal intelligence and education, and ch ild stimulation. The c ohort had a relatively

homogenous socioeconomic profile, reducing the potential for uncontrolled confounding.

Additionally, the authors noted their findings were consistent with findings from previous reports

from this cohort (Eskenazi, 2007) and other investigations of adverse associations between

prenatal exposure to organophosphate pesticides and cognition (Rauh, 2006). They also noted

that although they did not see an associati on between childhood exposures to organophosphate

pesticides, the other studies that have differed in the measurement of exposure and/or the specific

outcomes found to be associated with exposur e to organophosphate pesticides (Lizardi, 2008,

Ruckart, 2004). The many advantages of this st udy, the plausibility of the findings, and that

similar association have been s een in other studies support the hypothesis that prenatal exposure

to OPs reduces cognitive function of children. However, to the extent that prenatal chlorpyrifos

is the true exposure of inte rest, the assessment of non-specific organophosphate pesticide

metabolites is a primary potential source of exposure measurement error. Because of the

prospective design, information bias is expected to result in attenuation of the true (unobserved)

associations, toward (observed) associations that are relatively smaller, or null.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 350 of 531

# Chlorpyrifos Human Health Risk Assessment D424485

Page 351 of 531 Works Cited

Bradman A, Barr DB, Claus Henn BG, Drumheller T, Curry C, Eskenazi B. "Measurement of

pesticides and other toxicants in amniotic fluid as a potential bi omarker of prenatal exposure: a

validation study." Environ Hea lth Perspect, 2003: 1779-1782.

Bradway DE, Shafik TM, Lores EM. "Comparison of cholinesterase activ ity, residue levels, and

urinary metabolite excretion of rats exposed to organophosphorus pesticides." J Agric Food

Chem, 1977: 1353-1358.

CDC. "Fourth National Report on Human Exposur e to Environmental Chemicals. Chemical

Information. Organophosphorus Insecticides : Dialkyl Phosphate Metabolites." 2009.

http://www.cdc.gov/exposurereport/data_tables /OP-DPM_ChemicalInformation.html (accessed

March 29, 2011).

Eskenazi B, Marks AR, Bradman A, Harley K, Barr DB, Johnson C, et al. "Organophosphate

pesticide exposure and neurodevelopment in young Mexican-American." Environ Health

Perspect, 2007: 792-798.

Lizardi PS, O'Rourke MK, Morris RJ. "The eff ects of organophosphate pesticide exposure on

Hispanic children's cognitive and behavioral functioning." J Pediatr Psychol, 2008: 91-101.

Lu C, Kedan G, Fisker-Andersen J, Kissel JC, Fenske RA. "Multipathway organophosphorus

pesticide exposures of preschool children living in agricultural and nonagricultural

communities." Environ Res, 2004: 283-289.

Marks AR, Harley K, Bradman A, Kogut K, Barr DB, Johnson C, et al. "Organophosphate

pesticide exposure and attention in young Me xican-American children." Environ Health

Perspect, 2010: 1768-1774.

Muto MA, Lobell F Jr, Bidanset JH, Wurpel JN . "Embyrotoxicity and ne urotoxicity in rats

associated with prenatal exposure to DURS-BAN." Vet Hum Toxicology, 1992: 498-501.

Rauh VA, Garfinkel R, Perera FP, Andrews H, Barr D, Whitehead D, Tang D, Whyatt RM.

"Impact of prenatal chlorpyrifos exposure on neurodevelopment in the first three years of life

among inner-city children." Pediatrics, 2006: 1845-1859.

Ruckart PZ, Kakolewski K, Bove FJ, Kaye WE. "Long-term neurobehavioral health effects of

methyl parathion exposure in ch ildren in Mississippi and Ohio." Environ Health Perspect, 2004:

46-51.

Wechsler, D. Wechsler Intelligence Scale for Children-IV: Administration and Scoring Manual.

San Antonio, TX: Harcourt Assessment Inc, 2003.

Whyatt RM, Garfinkel R, Hoepner LA, Andrew s H, Holmes D, Williams MK, et al. "A

biomarker validation study of pren atal chlorpyrifos e xposure within an inne r-city cohort during

pregnancy." Environ Hea lth Perspect, 2009: 559-567.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 352 of 531

# Chlorpyrifos Human Health Risk Assessment D424485

Page 353 of 531 

# Article 15. Whyatt et al. (2007)

Within- and Between-Home Variability in Indoor -Air Insecticide Levels during Pregnancy

among an Inner-City Cohort from New York City. Robin M. Whyatt, Robin Garfinkel,

Lori A. Hoepner, Darrell Holmes, Mejico Bo rjas, Megan K. Williams, Andria Reyes,

Virginia Rauh, Frederica P. Perera, and David E. Camann. Environmental Health

Perspectives. 115:383–389 (2007).

Study Summary

Whyatt et al conducted a study to assess within- and between-home variab ility in indoor-air

insecticides over the final 2 mont hs of pregnancy among a subset of participants in the Columbia

Mother’s and Newborn Study, a cohort of women fr om New York City. Participants (n = 102)

assessed in this investigati on were enrolled between February, 2001 and May, 2004, after the

January, 2001 implementation of a voluntary can cellation by registrants of indoor residential

chlorpyrifos use. Expectant mothers were recruited during early pregnancy ( ≤ 20 th 

# week) among

African-American and Dominican women age 18- 35 years, and registered at New York

Presbyterian Medical Center and Harlem Hospita l, in New York City. Women who smoked, had

a history of drug abuse, diabetes, hyperten sion, or HIV infection were excluded from

participation in the study, as were women who resi ded in New York City for less than 1 year or,

for this validation study, who were employed outside the home at th e time of enrollment. Most

of the mothers (96%) in the pres ent study lived in residential ap artment buildings; the remaining

lived in multifamily housing (n = 3) or in combin ed residential/commercial buildings (n = 1).

Nine insecticides (the organophosphates chlorpyrif os, diazinon, malathion, a nd methyl parathion;

the carbamates propoxur, bendiocarb, and carbofuran; and the pyrethorids cis and transpermethrin) and piperonyl butoxi de, an adjuvant that is fr equently added to pyrethroid

formulations, were measured in 48-hr personal air samples and 2-week integrated indoor air

samples collected sequentially for 7.0 ± 2.3 week s (n = 337 air samples). Indoor air monitoring

occurred around the 32nd week of pregnancy, an d air samples were collected every 2 weeks

throughout the monitoring period. Personal air samp les were obtained for mothers over a 48 hour

period during the 32nd week of pregnancy.

Arithmetic means ± SDs were calculated for pesticid es detected in greater than 45% of samples.

Before statistical analyses, pest icide values were log-transforme d to normalize their distributions.

Values below the level of detec tion (LOD) were assigned a value of half of the LOD. Pearson

correlation coefficients or Spearman’s correlation co efficients, as appropriate , were calculated to

assess the relationship between pest icide levels in indoor air and pe rsonal air samples. Analysis

of variance (ANOVA) was used to test whether pesticides levels varied significantly by season

and year of monitoring and among the following groups: women not using any pest control

methods; women using “nonspray” methods only; a nd women using higher-toxicity pesticides.

If levels differed significantly among the groups, the least significan t difference test was used to

determine which groups varied significantly. T-te sts were employed to compare differences in

mean pesticide levels by ethnicity. Chi-square an alyses were used to te st whether the proportion

of women using pest control meas ures over the final 2 months of pregnancy was related to pest

sightings in the home. Finally, a mixed-effect s model was used to assess within-home and

between-home variability in repeat indoor air sa mples, after controlling for season and year of

# Chlorpyrifos Human Health Risk Assessment D424485

Page 354 of 531 delivery. Except for the Spearman’s rank and chi-s quare analyses, all st atistical testing was

conducted on log-transformed data.

Sixty-one percent of the participating wome n reported using pest control during the air

samplings. Chlorpyrifos, diazinon, and propoxur we re detected in 99–100% of personal and

indoor samples (range, 0.4–641 ng/m 3 

# ). Piperonyl butoxide (a pyret hroid adjuvant) was detected

in 45.5–68.5% (0.2–608 ng/m 3 

# ). There was little within-hom e variability and no significant

difference in air concentrati ons within homes over time (p ≥ 0.2); between- home variability

accounted for 88% of the variance in the indoor air levels of pr opoxur, 92% in chlorpyrifos, 94%

in diazinon, and 62% in piper onyl butoxide (p < 0.001). Indoor and maternal personal air

insecticide levels were hi ghly correlated (r = 0.7– 0.9, p < 0.001). Diazinon and chlorpyrifos

levels declined 5-fold between 2001 and 2004 but were detected in all homes at 1.5 and 2.5

years, respectively, after implementation of a voluntary cancellation by registrants of indoor

residential chlorpyrifos use. No previously unr eported associations between chlorpyriphos and

health outcomes were presented in this report. The aut hors concluded that th e insecticides were

persistent in the home with little variability in air concentrations over the 2 months and likely

contributed to chronic maternal in halation exposures during pregnancy.

Study Review

Although the Columbia Mother’s an d Newborn Study enrolled particip ants in years prior to the

implementation of the 2001 voluntary cancellati on by registrants of indoor residential

chlorpyrifos use, only data for participants recruited in the post- cancellation period were

considered in this validation study analysis. Ex planation for this desi gn choice was not provided,

but is likely that appropriate repeated sampling was not conducted during earlier study periods.

The authors noted that the mothers in the pres ent study were comparable with women in the

larger cohort in terms of matern al age, ethnicity, marital stat us, annual household income, and

reported use of pest control methods during pr egnancy. However, a greater proportion of women

in the present study compared with those in th e larger cohort reported having less than a high

school education, which could be due to the fact that, for these analys es, the investigators

excluded women who worked outside the home.

The exposure assessment in this study was we ll conducted using appropr iate and previously

published methodology. The semivolatile insecticides were readily detected in indoor air after

residential use. Indoor air levels of these chlo rpyrifos and the other organophosphate pesticides

in the 2-week integrated indoor air samples rema ined remarkably stable within most homes over

6–8 weeks of sequential sampling. The two- week integrated sampling methodology was

targeted towards evaluation of chronic exposur e during the latter preg nancy period, and thus

precludes assessment of changes in concentrations in indoor air at times scales shorter than twoweeks ( e.g. day-to-day changes).

In this study, pesticide levels in indoor air were remarkably stable. Correspondingly, almost all

(90%) of the total variance in the indoor air levels was explained by between-home variability.

The degree to which this finding in the post-canc ellation period informs variability in airborne

pesticide levels prior to the cancellation was beyond the sc ope of the investigation.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 355 of 531 The authors did not observe an association between indoor air levels of chlorpyrifos and

maternal self-reported pesticide use, a finding wh ich indicates that most of the chlorpyrifos

concentrations detected in the indoor air samples li kely resulted from prior, rather than current,

pesticide use. However, that chlorpyrifos was de tected in indoor air samples from all the homes

assessed, including those evaluated 2.5 years afte r implementation of the voluntary cancellation

of indoor residential chlorpyrifos use, demonstrates the persis tence of the compound in certain

indoor environments.

No associations between pesticide exposures and health outcomes were assessed in this

validation study.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 356 of 531 

# Article 16. Whyatt et al. (2009)

A Biomarker Validation Study of Prenatal Ch lorpyrifos Exposure within an Inner-City

Cohort during Pregnancy. Robin M. Whyatt, Robin Garfinkel, Lori A. Hoepner, Howard

Andrews, Darrell Holmes, Megan K. Williams, Andria Reyes, Diurka Diaz, Frederica P.

Perera,1 David E. Camann, and Dana B. Barr. Environal Health Perspectives. 117:559–567

(2009).

Study Summary

Whyatt et al conducted a biomarker validation study within the Colu mbia Mother’s and

Newborn Study to evaluate trends over time in multiple biomarkers of prenatal chlorpyrifos

exposure. The group previously documented signifi cant decreases in chlor pyrifos concentrations

in maternal personal and indoor air samples am ong pregnant African-A merican and Dominican

women from New York City after the 2001 vol untary cancellation by registrants of indoor

residential chlorpyrifos use. Participants (n = 102) were enrolled be tween February, 2001 and

May, 2004. 3,5,6-trichloro-2-pyridinol (TCPy) was measured in newborn meconium (n = 83),

repeat prenatal maternal urine samples (n = 253) , and postnatal urine from mothers (n = 73) and

newborns (n = 59). Chlorpyrifos was directly measured in postnatal maternal (n = 92) and

umbilical cord (n = 65) blood plasma and in personal and indoor air samples. No previously

unreported associations between chlorpyriphos and health outcomes were presented in this

report.

Study participants were recru ited during early pregnancy ( ≤ 20 th 

# week) among African-American

and Dominican women age 18-35 y ears, and registered at Ne w York Presbyterian Medical

Center and Harlem Hospital, in New York City. Women who smoked, had a history of drug

abuse, diabetes, hypertension, or HIV infection we re excluded from participation in the study, as

were women who resided in New York City for less than 1 year or who were employed outside

the home at the time of enrollment (for the purpose s of this validation study). The study sample

presented in this report was recruited between 2001 and 2004, after the January, 2001

implementation of a voluntary cancellation by regist rants of indoor residen tial chlorpyrifos use.

Most of the mothers (96%) in the present study lived in residential apartment buildings; the

remaining lived in multifamily housing (n = 3) or in combined residential/commercial buildings

(n = 1).

Indoor air monitoring occurred ar ound the 32nd week of pregnanc y using 2-week integrated

indoor air samples collected every 2 weeks th roughout the monitoring period. Personal air

samples were obtained for mothers over a 48 hour period during the 32nd week of pregnancy.

Repeat prenatal maternal urine samples were co llected from mothers at the end of each 2-week

indoor air sampling period, beginning at the 34 th week of pregnancy and continuing until

delivery. Women who went into la bor before the end of the first 2 weeks of indoor air sampling

did not provide urine samples. Urine samples we re collected from 97 of 102 women in the study;

more than half of the women provided three or more urine samples, 29 of 97 (30%) two samples,

and 15 of 97 (15%) provided one sample. An a dditional urine sample was obtained on the day

after delivery for 73 women and 59 newborns.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 357 of 531 An umbilical cord blood sample was collected fr om 65 newborns after delivery. A sample of

maternal blood was obtained from 92 mothers wi thin 2 days postpartum. Paired maternal and

cord blood samples were available for 64 of the mothers and newborns.

Meconium samples were collected from 83 newbor ns during the postpartum hospital stay. In 53

(64%) of the births, the meconium samples were collected within 1 day of delivery.

Biologic samples were analyzed for organophos phate pesticide markers by the CDC using

previously established methods using both positiv e and negative controls. The limit of detection

(LOD) of chlorpyrifos in blood samples was 0.5–1 pg/g plasma. The LOD of TCPy in urine

samples was 0.26 ng/mL urine. The LOD for TCPy in meconium was 0.2 ng based on a sample

weighing 0.5 g.

Exposure marker levels below th e LOD were given a value of ha lf the level of detection, and

were then log 10 

# transformed. Nonparametric statistics were used when distributions remained

non-Gaussian. Spearman rank correlation coefficients were used to assess relationships between

levels of chlorpyrifos or TCPy in biological sa mples and to examine correlations between levels

of chlorpyrifos or TCPy in the biological samp les and chlorpyrifos levels in air samples.

Multiple linear regression was employed to evaluate the amount of variance in prenatal maternal

urine TCPy levels explained by indoor air chlo rpyrifos levels among s ubjects enrolled in 2001–

2002.

Analysis of variance (ANOVA) or Kruskal–Wallis tests with post-hoc testing as appropriate,

were used to assess whether pesticide levels va ried by year of birth (between 2001 and 2004), or

among the following groups: women not using any pest control methods; women using

“nonspray” pest abatement methods only; and wome n using can sprays, pest bombs, or sprays by

an exterminator, with or w ithout nonspray methods. Authors performed t-tests corresponding

nonparametric Mann–Whitney U-test were used to assess whether pesticide levels differed by

ethnicity. Chi-square analyses were used to test whether the freque ncies of detection of

chlorpyrifos and TCPy in biological samples vari ed by year of assessment as well as to compare

detection frequencies of chlorpyr ifos in maternal and cord blood samples. A mixed effects model

was used to evaluate the within- and between-subj ect variability in TCPy levels in the repeat

maternal urine samples collected during pregna ncy, and to calculate an intraclass correlation

coefficient (ICC) for women enrolled in 2001–2002 who had three repeat urine samples (TCPy

was detected in only one-third of samples amon g subjects enrolled after 2002). The ICC is a

measure of reliability of repeated measures ove r time, defined as the ratio of between-subject

variance to total variance.

The authors did not detect TCPy in infant ur ine, but all other biom arkers showed a highly

significant decrease in detection frequencies ( χ 2 = 7.8–34.0, p ≤ 0.005) and mean ranks (p ≤

0.006) among subjects enrolled in 2003–2004 co mpared with those enrolled in 2001–2002.

Chlorpyrifos in maternal persona l and indoor air declined 2- to 3-fold over the same period (p <

0.05). In 2001–2002 samples, TCPy levels in repeat prenatal urine samples were positively

correlated (r = 0.23–0.56), although within-subject variability exceeded between-subject

variability (intraclass correla tion coefficient = 0.43); indoor ai r levels explained 19% of the

variance in prenatal urine TCPy (p = 0.001). Me conium TCPy concentrations were positively

# Chlorpyrifos Human Health Risk Assessment D424485

Page 358 of 531 correlated with chlorpyrifos in maternal and cord blood (r = 0.25–0.33, p < 0.05) and with TCPy

in maternal urine (r = 0.31, p < 0.01).

Study Review

Although the Columbia Mother’s an d Newborn Study enrolled particip ants in years prior to the

implementation of the 2001 voluntary cancellati on by registrants of indoor residential

chlorpyrifos use, only data for participants recruited in the post- cancellation period were

considered in this analysis. Explanation for this design choice was not prov ided, but is likely that

appropriate repeated sampling was not conducted during earlier study periods. The authors

noted that the mothers in the present study were comparable with women in the larger cohort in

terms of maternal age, ethnicity, marital stat us, annual household income, and reported use of

pest control methods during pregnancy. However, a greater proportion of women in the present

study compared with those in th e larger cohort reported having less than a high school education,

which could be due to the fact that, for thes e analyses, the investigators excluded women who

worked outside the home.

The investigators’ use of nonparametric, rank-base d statistics is appropri ate for analysis of nonnormally distributed data. However, the large number of ties among marker levels due to

observations below the level of detection recei ving equal rank, may be problematic. When

ranked values have the same numeric number ( e.g. , half the level of detection), the rank is 0.5

times the number of ties. Whether, and at what point a large propor tion of ties begins to limit the

inferential power of rank based statistics is unclear, although at some point, a non-continuous

classification of the data ( e.g. , classification of observations into bins at a cut-point equal to the

level of detection) seems to make more sense if the number of below the level of detection

observations is large.

The exposure assessment conducted in this invest igation is the most comprehensive of any

epidemiologic study of chlorpyrifos to date. St rengths of the exposure assessment include the

quantification of actual blood chlo rpyrifos levels in addition to sensitive, but non-specific

markers of organophosphate pestic ide exposure in urine, the re peated sampling of urinary

markers, measures in meconium and the e nvironmental sampling. The exposure assessments

were appropriately timed with respect to criti cal window of gestation for fetal growth and

neurodevelopment outcomes which li kely occurs, in part, during th e third trimester of pregnancy,

and appear to have been well conducted. Althou gh inhalation exposures ar e likely a major route

of exposure, no direct dietary assessment was included in this investigation.

The study was set during a period of rapidly declining use of chlo rpyrifos, and the findings are

demonstrative of decreasing exposure over time. The investigators obser ved a highly significant

decrease in the detection freque ncy and mean ranks of all biom arkers among subjects enrolled

during years 2001–2004, with the exception of TCPy in newborn urine, which was not detected.

Maternal and umbilical cord blood plasma chlo rpyrifos levels were below the LOD in all

samples collected after 2002. The results suggest that chlorpyrifos from residential use was the

main contributor to the chlorpyrifos levels detected in both the maternal and umbilical cord

blood among study subjects in the larger coho rt (1998-2004, both before and after voluntary

# Chlorpyrifos Human Health Risk Assessment D424485

Page 359 of 531 cancellation of chlorpyrifos containing compounds).

While no association was observed between matern al and umbilical blood chlorpyrifos levels

measured at delivery and TCPy in maternal ur ine samples collected through the latter third

trimester, TCPy levels in meconium (an integrative measure week 13-delivery) were

significantly correlated w ith chlorpyrifos levels in both maternal and umbilical cord blood

measured at delivery, and with TCPy levels in maternal urine samples collected in third

trimester. These results suggest that TCPy in meconium may provide a valid biomarker of

prenatal exposure, possibly both prior to and subsequent to th e cancellation period.

The finding that within-subject variability exceeded between-subject variability in maternal urine

TCPy (intraclass correlation coefficient, ICC= 0.43), again emphasizes the point that a single

prenatal urine sample is likely insufficient to capture ‘usual’ exposure. The authors noted that

despite the significant within-sub ject variability seen in their study, the ICC observed was higher

than that observed in another population (Meeker et al., 2005).

The authors concluded that their re sults suggest that pesticide biomar kers utilized in these studies

are reliable dosimeters to differentiate between groups with prenatal chlorpyrifos exposures

varying by a factor of 2 or more, and that cord blood measures are reasonable well correlated

with exposure measures taken throughout pre gnancy (meconium correla ted with cord blood

(delivery) and maternal urine (3 rd 

# trimester). Authors also support that these data illustrate the

efficacy of the voluntary cancellation of indoor reside ntial chlorpyrifos use to reduce the internal

dose during pregnancy.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 360 of 531 Works Cited

Meeker, J. D., Barr, D. B., Ryan, L., Herrick, R. F., Bennett, D. H., Bravo, R., & Hauser, R.

(2005). Temporal variability of urinary levels of nonpersistent insectic ides in adult men. J Expo

Anal Environ Epidemiol, 15 (3), 271-281. doi: 7500402 [pii]

10.1038/sj.jea.7500402

# Chlorpyrifos Human Health Risk Assessment D424485

Page 361 of 531 

# Article 17. Rauh et al. (2012)

Rauh, V. A., Perera, F. P., Horton, M. K., Whya tt, R. M., Bansal, R., Hao, X., . . . Peterson,

B. S. (2012).

Brain anomalies in children exposed prenatal ly to a common organophosphate pesticide.

Proc

Natl Acad Sci U S A, 109(20), 7871-7876.

Study Summary

In this study, authors with the Columbia Cent er for Children’s Environmental Health (CCCEH)

birth cohort study evaluated whethe r there were areas of morphologi cal change in the pediatric

brain in regions of the brain known to be associ ated with learning, cognition and social behavior

in association with prenatal chlorpyrifos (C PF) exposure (V. A. Rauh et al., 2012). These are

functional neurodevelopmental outcomes that have been adversely associat ed with chlorpyrifos

exposure in previous observationa l research (V. A. Rauh et al., 2006; V. Rauh et al., 2011), as

well as some experimental studies, as noted by th e authors. Specifically, in this study, authors

evaluated 1) whether morphological changes in the brain including co rtical thickness and

differences in the cerebral brain surface area and volume were related to prenatal chlorpyrifos

exposure (the main effect), and, 2) whether th e relation between the exposure and morphological

changes in the brain were modified by either full scale intelligence quotient (FSIQ) score

(evidence of effect modification by IQ) and, 3) in preliminary analyses, whether changes in the

normal sexual differentiation of the brain exis t by exposure-level (V. A. Rauh et al., 2012).

Authors concluded that while overa ll brain size did not differ by prenatal chlorpyrifos exposure,

certain areas of the brain were enlarged among those more highly exposed to chlorpyrifos

including superior temporal, pos terior middle temporal, and in ferior post-central gyri, and

superior frontal gyrus, gyrus re ctus, cuneus, and pre-cuneus in the mesial views of the right

hemisphere of the cerebrum. Further, they not ed a difference exposure- response relation between

cerebral surface distances and CPF exposure, by FSIQ wherein the relation between brain area

enlargements and FSIQ were positively correlated in the low CPF group (greater regional brain

size correlated with greater IQ), but an inverse relati on among the high CPF group. In

supplemental analyses, authors illustrated that areas of regional brain enlargement among those

more highly exposed to chlorpyrifos were due to underlying white matter enlargements.

Researchers also noted sporadic differences in cortical thickness wherein higher CPF exposure

was related to reduced cortical thickness. Prel iminary analyses also displayed a disruption of

normal male-larger-than-female areas as well as fe male-larger-than-male in certain areas of the

brain known to differ by child sex in normal br ain. Overall, research ers concluded that

differences in brain structure (r egional cerebral size and thickn ess) exist between CPF exposure

groups, and the differences (high>low CPF) in re gional brain size may be due to enlargement of

underlying white matter. However, authors did no t speculate as to th e underlying biological

explanation of the observed qualitative inte raction between CPF exposure and FSIQ with

morphological changes in key areas of the br ain linked with learning, cognition and social

behavior.

Design and Methods

Children selected for this MRI study (n=40) were participants in the larger Columbia Center for

Children’s Environmental Health (CCCEH) birt h cohort study (n=369). The study was initiated

in the late 1990’s to evaluate the relation between air pollution and pesticide exposure and

# Chlorpyrifos Human Health Risk Assessment D424485

Page 362 of 531 neurodevelopmental effects in ch ildren. The cohort is comprised of a low income, inner-city

population primarily of Dominican and African American ethnicity. Mothers were recruited

among those who sought prenatal care by 20 weeks of pregnancy at one of the participating

study hospitals. Participants were non-smoking women, ages 18-35, with no other co-morbidities

including diabetes, hypertensi on, known HIV infection, and no doc umented drug use. Therefore,

the cohort represents a population of women at low-risk for adverse neurodevelopmental

outcomes among their children. Participants were resi dents of NYC for at least one year prior to

enrollment in the research study. EPA’s review of all the epidemiological studies from the

CCCEH (the Columbia studies) is contained in a recent presentati on to the Federal Insecticide,

Fungicide, and Rodenticide Act (FIFRA) Scie nce Advisory Panel (FIFRA SAP), April 2012

(FIFRA Scientific Advisory Panel, 2012).

Authors measured chlorpyrifos and other organo phosphate pesticides, cotinine, lead and mercury

in umbilical cord blood samples taken at delive ry using CDC analytic methods with published

quality assurance and control procedures. Pren atal environmental t obacco smoke (ETS) was

measured as maternal self-report, an d verified through cotinine levels ( ≤ 15 ng/mL). Polycyclic

aromatic hydrocarbon (PAH), another environmen tal chemical possibly related to adverse

neurodevelopmental outcomes, wa s measured through personal air monitoring in the third

trimester of pregnancy. In the current (MRI) st udy, high chlorpyrifos e xposure was defined as

the upper tertile of the exposed group, ≥ 4.39 pg/g, and low chlorpyrifos exposure was defined as

values below the upper tertil e, <4.39 pg/g, including those not exposed to CPF.

To measure intelligence among school aged children, authors used the Wechsler Intelligence

Scale for Children, 4th edition (WISC-IV). The in strument measures four areas of mental

functioning: the Verbal Compre hension Index, the Perceptual Reasoning Index, the Working

Memory Index, and the Processing Speed Index. Th ese indices are associat ed with, but distinct

from, overall intelligence quotient (IQ) and are se nsitive to cognitive deficits related to learning

and working memory. A Full-Scale IQ score combines the four composite indices. A General

Ability Index score is a summary score of gene ral intelligence, similar to Full-Scale IQ, but

excludes contributions from both Working Memo ry Index and Processing Speed Index. WISCIV scores are standardized against U.S. population-based norms for English and Spanishspeaking children.

MRI

This section provides some broad detail as to the method of MRI data collection employed in this

research paper; this is not a cr itical review of met hods. Health outcomes of interest in this study

were morphological changes in the brain in regi ons of the pediatric brain that are related to

learning, cognition and social behavior, as no ted in the traditional epidemiology studies.

Researchers performed brain imaging studies using Magnetic Resonance Imaging (MRI)

technology among a sub-sample of the cohort, and measured and compared morphological

characteristics by chlorpyrifos exposure status. Details concerning the MRI imaging procedures,

processing and pre-processing image modifications , methods to measure cortical thickness and

methods used to identify the “template” brain ag ainst which individual ch ild participant brain

images were compared are presente d in the Supplemental Information.

Briefly, researchers used a high-resolution MR I, whole body scanner, with 8-channel head

scanner for this study. Authors processed MRI images using biomedical imaging software

(ANALYZE 8.0) while technicians were blinded to participant characteristi cs, and whether left

or right hemisphere of the brain was imaged. Before morphological anal yses were performed,

# Chlorpyrifos Human Health Risk Assessment D424485

Page 363 of 531 images in the dataset were re-sliced to correct for residual head rotation, tilt, and

flexion/extension by par ticipants during imaging

. MRI images were pre-processed to correct for

large-scale variation in color intensity. Extra- cerebral tissues captured in the MRI brain image

were manually removed from the cerebral image before analyzing differences in brain

morphology among participants by chlorpyrifos exposure; authors hypothe sized difference in

regional brain areas of the cereb rum only, other parts of the brai n were therefore excised from

the image before processing. Other similar modi fications to the brain image were made to

enhance measurement of key/pre-identified endpoints (cerebral surface morphology and cortex

thickness) (See Supplemental Information). Variat ion in cortical grey matter was assigned by

authors as four categories of co lor changes across images; author s selected color categories that

adequately grouped the participants as to degree of cortical grey matter ( i.e. , creation of fourlevel categorical variable to measure cortical thickness) and to allow for a robust statistical

analysis, particularly consideri ng the small study sample (n=40).

To measure morphological characteristics within cerebral areas associated with learning,

cognition and social behavior, au thors identified a “template” brain image against which all

participant images were compared and measured . Using a two-step process, authors initially

identified the individual in the study sample that most closely approximated average

demographic features (child age, height and weight ). Using this brain image, all other participant

brain image studies were co-registered, i.e. , similar points on the cere bral surface were matched

according to the International Consortium on Brain Imaging. Researchers then measured

distances of correlated points on the cerebral su rface between the “template” brain image and all

participant images. Using these measured distance s, authors then identified a final “template”

brain based upon the brain image series which most closely matched the average distances

among cortical surface measures. Authors advocated that use of one “template” brain as opposed

to an amalgamated “average” brain is superior because of the enhanced clarity and more well

defined tissue interface with whic h to make the measurements for the study. All sets of brain

image studies were then re-calibrated to the fina l “template” brain image fo r statistical analyses.

Statistical Analyses

Regarding the statistical analyses employed in this paper, the an alyses involved comparison of

the Euclidean distances measured between correlated points on th e cerebral surface or cortical

thickness by chlorpyrifos (high/ low) exposure level, adjustin g for age and sex. Supplemental

information reported that thousands of statistical tests were performed. Au thors used generalized

linear equations and linear regression modeling to estimate average differences in morphological

characteristics by change in chlorpyrifos expos ure level (high/low). Because authors performed

multiple statistical tests, they utilized the False Discovery Rate (FDR) methods to adjust the pvalue against Type I error. Aut hors tested the statistical signi ficance of intera ction terms of

cerebral surface measures and FSIQ, and by child sex at p<0.05, adjusted for multiple testing.

Study Results & Authors’ Interpretation

For this study, authors selected child participants who had comp lete measures for CPF, PAH,

ETS, and who completed the WISC IV testing. Ther e were 70 participants who met these criteria

and who also had cord blood levels in the uppe r tertile of 4.39 pg/g (High CPF exposure), and

among these 28 met the additional criteria of having low or no exposure to PAH and ETS.

Twenty of these participants completed the MRI seri es (20 of 28 eligible participants enrolled in

# Chlorpyrifos Human Health Risk Assessment D424485

Page 364 of 531 study). Similarly, there were 99 participants wh o met the enrollment criteria (completed the

environmental exposure and IQ testing battery ) and were in the lower exposure group (<4.39

pg/g) and had no or low ETS and PAH. Among th is group of low CPF exposed participants, 38

were randomly selected to partic ipate in the MRI study, and 20 part icipants completed the series

(20 of 38 eligible participan ts enrolled in study).

Authors reported no significant differences be tween those in the high and low chlorpyrifos

exposure group, or among the larger cohort acco rding the common socioeconomic indicators

including maternal income and e ducation, maternal IQ, race, gesta tional age and birth weight of

child, and blood lead measure (See Table 1, main text). The correlation between chlorpyrifos and

blood lead was low ( ρ <0.20) and non-significant (p>0.20).

Authors concluded that while overa ll brain size did not differ by prenatal chlorpyrifos exposure,

certain areas of the brain were enlarged among those more highly exposed to chlorpyrifos

including superior temporal, pos terior middle temporal, and in ferior post-central gyri, and

superior frontal gyrus, gyrus re ctus, cuneus, and pre-cuneus in the mesial views of the right

hemisphere of the cerebrum. Further, they not ed interaction, or a di fferent exposure response

relation between cerebral surf ace distances and FSIQ, by CPF exposure wherein the relation

between brain area enlargements and FSIQ we re positively correlated in the low CPF group

(greater regional brain size equated to greater IQ), but an inverse relation among the high CPF

group (greater regional brain si ze related to lower FSIQ scores ). Authors noted statistical

interactions in the superior temporal, inferior frontal, inferior pre-ce ntral, and inferior post

central gyri bilaterally, and the pre- cuneus of the left hemisphere.

In supplemental analyses, authors illustrated that areas of regional brain enlargement among

those more highly exposed to chlorpyrifos we re due to underlying white matter enlargements.

This conclusion was supported by the observation of sporadic diffe rences in cortical thickness

wherein higher CPF exposure was related to reduced cortical thickness, or thinning of the cortex

in certain areas. The qua litative interaction between CPF expos ure and FSIQ with cerebral brain

region enlargements may be explained by size of underlying white matter of the cerebrum, but

authors did not speculate as to a biological explanation of the observed qualitat ive interaction.

Preliminary analyses also displa yed a disruption of normal male-lar ger-than-female areas as well

as female-larger-than-male in certain areas of the brain known to differ by child sex in normal

brain.

Overall, researchers conclude that differences in brain structure (regi onal cerebral size and

thickness) exist between CPF e xposure groups, and the differences (high>low CPF) in regional

brain size is likely due to enla rgement of underlying white matter. Authors also observed an

inverse relation between regiona l brain enlargements and IQ among those highly exposed to

CPF, and positive association between regiona l brain size and IQ among those in the low/no

exposed group. While authors do no t speculate as to a possibly bi ological explanation of this

observed statistical interaction, EPA notes that given that tota l brain size did not differ by

exposure group, the enlargement of the white matte r in certain areas could have been at the

decrement of the size, quality or other morphologi cal changes in the grey matter in other areas;

neurons in grey matter of cerebrum affect learni ng and cognition. Observed inward deformations

(reduced brain area) in the dorsal and mesial surfaces of the left superior frontal gyrus as also

# Chlorpyrifos Human Health Risk Assessment D424485

Page 365 of 531 reported the authors suggest may also be part of the explanation of th e qualitative interaction.

However, the authors indicate that additional stu dy is needed to elucidate these possibilities.

EPA Review

This study by Rauh et al. 2012 follows upon both previous epidemiological research and

experimental toxicology studies th at suggest an association between chlorpyrifos exposure in the

pre-natal and early life periods with adverse neurodevelopmental outcomes observed in children

through school age, including intelligence deficits . Within this CCCEH cohort, authors reported

links between prenatal chlorpyrifos exposure (c oncentration of CPF in cord blood at delivery

(pg/g)) with attentional difficulties, behavioral problems and measures of intelligence

particularly working memory (V. A. Rauh et al ., 2006; V. Rauh et al., 2011). EPA’s review of

these previous epidemiology studies from the CC CEH cohort were presented to the FIFRA SAP

in April 2012 (FIFRA Scientif ic Advisory Panel, 2012). Regarding this study, EPA notes a

limited and somewhat unbalanced depiction of th e available rodent experimental data was

presented in the paper. A brief review of re ferences seems to provide some support for the

authors’ assertion that the epidemiological findings are consistent with those in the experimental

literature; however, a fuller evaluation of the da ta concerning animal to human concordance is

needed. For example, the authors’ stated suppor t for their finding regarding enlargements in

underlying white matter is not strongly supported by existing experimental data concerning CPF

induced glial scarring.

In this study, authors evaluate d morphological characteristics of cortical brain regions which

have been linked to disrupted learning, cognition and social behavior in previous epidemiologic

and experimental studies. The cer ebrum has been described as the “seat of intelligence” and

provides the ability to read, write, and speak; to make calculations ,and to compose music; and to

remember the past, to plan for the future, and to imagine things that have never existed before

(Tortora & Grabowski, 2003). However, while the cerebrum’s influence upon human

intelligence is generally supported, the study lack ed specific hypotheses regarding particular

areas of the cerebrum affected (the largest of th e four components of th e brain). Therefore, as

noted in supplemental information, many thousands of statistical tests we re performed evaluating

areas of the cerebrum that ha ve been mapped. Authors approp riately employed methods to

control for Type I error, however in many ways this research re quires replication, as noted by the

authors. Therefore, use of these data to info rm specific underlying biological mechanisms or

pathways of the potential toxic action of CPF is limited.

EPA is seeking external peer review of this specific aspect of th e current study to verify that the

MRI methods are sound and appropriate. Briefl y, however, the main text and supplemental

information states that technicians (MRI image r eaders) were blinded as to exposure status, as

well as right or left hemisphere. This design feat ure adds to the validity and reliability of the

study. However, EPA notes authors do not stat e whether multiple MRI image readers were

employed in identifying and measuring cortical su rface thickness and cerebr al surface area, etc.

of interest in this study, and whet her quality control sta tistics such as the in ter-rater reliability

coefficient were calculated and considered. S upplemental information notes that validation

studies were performed, however does not describe the aspects of methods that were validated.

EPA particularly seeks input from experts in this area as to wh ether these methods and validation

studies, if known, are sound.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 366 of 531 Authors appropriately described the inclusion and exclusion criteria used in this MRI study.

Authors excluded participants for who measureab le levels of both PAH and ETS were reported

in the total cohort study. In th is way, by design, authors elimin ated the chance of confounding by

some other environmental chemicals known to eff ect neurodevelopment in the relation between

chlorpyrifos and brain morphology. Authors also included only those participants who provided

full information regarding prenatal chlorpyrif os exposure, other environmental chemical

exposure and who completed the WISC intelligen ce testing. Given the relatively small sample

size (n=40) for this relatively expensive imagi ng study these decisions eliminated the issue of

missing data, and the need to add degrees of freed om to statistical models for control of other

measured environmental chemicals. These decision s added to the validity a nd statistical power of

the study. Although inclusion and exclusion criter ia were somewhat rigorous in this study,

authors still achieved a 60% part icipation rate among those eligib le to participant (HIGH: 20/28,

LOW: 20/38). Further, Table 1 indicates adequa te comparability between the exposure groups

and the total cohort, although those in the high exposed group were slightly al beit not statistically

significantly more likely to be in families in whic h maternal education and income were slightly

greater than the low exposure group. Therefore, in general, EPA believes that authors were able

to identify an unbiased sample of cohort, repr esentative of population sample of low income,

inner city kids. However, given the rigorous enroll ment criteria, the extern al validity of the study

findings may be limited if the organophosphate pe sticide exposure-brain morphology association

is modified by factors that are more, or less, prevalent in the study populat ions relative to the

population(s) to which inference is being made, or to populations with a substantially different

exposure range.

In the larger cohort, authors meas ured concentration of chlorpyrif os parent compound in the cord

blood of infants at delivery. Ge nerally, biomarkers are consider ed superior measures than

questionnaire based self report or environmenta l monitoring. The assays used in the study were

highly sensitive allowing detecti on of very low levels of chlor pyrifos, in the pg/g range (CDC

published methods). However, these measures are comparable to those in other samples of the

general population, as noted by auth ors. Uncertainties remain however, as to the extent the use of

one biomarker measurement reflects exposure(s) over critical windows of development during

pregnancy, the temporality or periodicity of expo sure, over the relevant ti me period of gestation.

These cord blood measures have been used in se veral studies to estimate pre-natal exposure to

chlorpyrifos, i.e. , during the entire period of gestation. Validations studies performed within the

CCCEH cohort support that use of a one-time measur e is an accurate and reliable measure with

which to rank participants (high/low). In this study, authors categorized chlorpyrifos exposure as

high (greater than or equal to the upper tertile of 4.39 pg/g) a nd low (all values below 4.39 pg/g

including those values below the LOD) which exposure methodology can accurately and reliably

support. PAH in the ambient air was measured using stationary air monitors and ETS was

measured through maternal self-report, validated by cotinine level. A noted limitation in the

measurement of pesticide exposure in these studi es is the lack of measurement during the postnatal period through the time period of the epidemiologic evaluations, i.e. , school age. Chemical

exposure in the post-natal period may confound or alter the relation between prenatal

chlorpyrifos and neurodevelopmental outcomes, as noted in EPA’s evaluation (FIFRA Scientific

Advisory Panel, 2012). In this study, child age an d sex were the only co-variables considered;

these variables are simple to accurately and re liably measure using linkage with birth/medical

# Chlorpyrifos Human Health Risk Assessment D424485

Page 367 of 531 records.

The selection of few co-variables for the statistical models used to evaluate the relation between

prenatal chlorpyrifos exposure and brain mo rphology changes is appropriate. For another

variable to be a confounder in th is relation, the other variable w ould have to be related to both

chlorpyrifos exposure and (causa lly) related to brain morphology ( e.g. , surface area, cortical

thickness, gyri dimensionality). Child age and sex are appropriate conf ounders as these factors

would influence brain size, growth and differe ntiation. While some research has noted a link

between social characteristics and brain development and possibl y morphological ch aracteristics,

the authors’ decision to rest rict CCCEH participation to t hose with very similar SES

characteristics reduces that possi bility of confounding the associa tion of interest in this MRI

study by SES. Further, the small sample size supports the use of few additional co-variables in

the statistical model to maxi mize statistical power. Similar, within the MRI study, authors

restricted participation to those without ot her co-exposures such as PAH and ETS thus

eliminating the need to consid er co-adjustment for these variables. Authors additionally

measured blood lead in this sample and the ove rall, total cohort and observe the correlation

between chlorpyrifos and blood lead (necessary factor for positive confounding bias to occur)

was very low ( ρ <0.20) and not statistically significant (p>0.20).

Other biases that may be present in epidemio logy studies include selection and information

biases. Selection bias in prosp ective cohort studies may occur if participants drop out of a study

and the decision to drop out was related to ei ther the CPF exposure or neurodevelopmental

outcome. In the CCCEH study the retention ra te is high for a birth cohort study. The

participation rate in the MRI study was good (60%). Authors illust rated that those included and

excluded were comparable, and the sample and total cohort were also comparable. This

ameliorates the concern. Temporal bias, a questi on as to whether exposure preceded disease, is

not a large concern in a prosp ective study. The prospective natu re of the cohort study also

reduces that chance of differential informati on bias or the misclassification of exposure,

confounder, or outcome information by key study factors. Non-differential exposure

measurement error may have occurred: 1) the si ngle measurement chlorpyrifos during the third

trimester, 2) error arising from differences between measured biomarker levels and actual

chlorpyrifos at the one-time point, 3) unmeas ured time- and space- dependent patterns of

chlorpyrifos exposure, 4) uncerta inty regarding the cr itical period for chlorpyrifos effects on

development, 5) missing exposure data, 6) la boratory errors, and 7) imputation of missing

exposure levels. Measurement errors in the ascer tainment of chlorpyrifos exposure are likely to

have occurred. However, because of the pros pective study design employed, the errors are

unlikely to result in falsely positive findings, b ecause the probability a nd magnitude of these

errors are likely to be independent of the outcome status of part icipants (MRI characteristics).

The degree to which measurement error in the imaging studies performed using MRI technology

are of keen interest to EPA.

Authors employed generalized linear models to estimate the association between CPF exposure

(high/low) and “voxels” or thre e-dimensional data reflecting mo rphological characteristics of the

cerebral cortex, specifically cortical thickness, distances on the cerebrum, size and dimension of

key gyri important to learning, c ognition, and social behavioral, ad justing for child age and sex.

Use of a two-level exposure variab le is preferable as it conser ves study power, and is supported

# Chlorpyrifos Human Health Risk Assessment D424485

Page 368 of 531 by the one-time measure of CPF at delivery (c ord blood CPF concentra tion pg/g). Researchers

utilized a method to correct for multiple stat istical tests (FDR) which was reasonable and

appropriate in this instance. Au thors acknowledged low statistical power to evaluate statistical

interaction was likely including ge ne by environment interactions. Linear modeling is a preferred

method when sample size is small as the met hod conserves statistical power. Authors did not

present a table of model results, only graphical depictions of areas of morphological differences

of the cerebrum by CPF exposure, color coded to reflect the st atistical significance of the

differences in specific, region brain size or volume or area. Additional information reflecting

model fit statistics, as well as display of evidence of effect mo dification in both exposure strata

(high and low CPF exposure) woul d enhance clarity and presenta tion of the research results.

While authors appropriately characterized the ev aluation of the interaction with child sex and

CPF and brain morphology as preliminary, given th e number of statistical tests performed and

lack of hypotheses regarding partic ular areas of the cerebrum and cortical surface, results require

replication before firm conclusi ons regarding underlyi ng biological mechanisms at play can be

posited.

Authors concluded that the evidence from the study illustrated changes in brain morphology in

association with higher CPF exposur e, and that changes observed were in areas of the brain that

sub-serve those learning, cognition and social behavioral, supported by previous observational

and experimental literature.

# Chlorpyrifos Human Health Risk Assessment D424485



# References

FIFRA Scientific Advisory Panel. (2012). Draft Issue Paper Scientific I s sues Concerning the

Health E ffec ts of Chlorp y rifo s. http: // www.regulations.go v / # !documentD e tail;D

= EPA

H 

# Q

- 0 PP- 2012-0040-0002

.

Rauh

, V. 

# A.

, Garfmkel

, R.

, Per e ra, F. 

# P.

, Andr e ws, H. F

., Hoepner

, L.

, Barr

, D. B

. , ... Whyatt,



# R. W. (2006). Impact of prenatal chlorpyrifos exposure on neur odeve lop ment in the first

3 year s of life among inner

- city children. 

# Pediatrics

, 11 8 ( 6), e 1845-1859

. doi:

1

0.1542 / ped s .2006-0 33 8

Rauh

, V. A., Perera

, F. P.

, Horton, M. K.

, Whyatt

, R. M.

, Ban s al

, R

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# X.

, . . . Peterson

, B.S.

(20 1 2 ). Brain anomali e s in c hil dren exposed prenatally to a common organopho s phate

pesticide. 

# Pro c Nat/ A c ad Sci U S A

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10.1 073 / pnas.I2033961 09

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# Wh y att, R . ( 2011).

Seven-year neurodevelopmental score s and prenatal exposure to chlorpyrifos, a common

agricultural pesticide

. 

# E nviron Health P e rspect

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Ph ys i o logy (pp. pag e 467). U nited State s of America: John Wiley & 

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# of 531

# Ch l orpyrifos Human Health Risk Assessment D424485



# Appendix 4. Detailed Summary Tables of Children's Env ironmental Health Epidemiology Stud ies

Aut h o r

, \' ear

, O u tco m e Exposure Potent i a l co nfounder s Primary Result Conclusions/Uncertainties St r engths and Weaknesses

Sa m(!le S ize Assessed Measurement cons ider e d

Article I : Whyatt Birth length, Birth Four cord plasma Gestational age

, maternal pre- For each log unit increase in Associations between birth Strengths: prospective

et al. (2004) weight

, head ch l orpyri fos pregnancy weight, maternal net co rd plasma ch l orpyrifos weight and length and cord nat ure of the st u dy; direct

Co l umbia U. circumference exposure groups pregnancy weight gain, gender levels, birth weight plasma chlorpyrifos were measure m e nt of

(N ; 314) and four of newborn, parity

, decreased by 42.6 g (95% statistically sign ifi cant (p 

# :5 chlorpyrifos in cord b l ood

chlorpyrifos and race/ethnicity

, ETS in home

, Cl : -81.8 to

- 3

. 8) and birth 0

. 007) among newborns and personal air samp l es

, diazinon expos ur e season, cesarean section length decreased by 0.24 born before the Jan u ary rather than non-specific

groups

. em (95% CI: -0.47 to

- 200 I policy change. Among markers o f

Chlorpyrifos only 0

. 0 1 ). Birth weight newborns born after organophosphate pesticide

ca

t egor ie s, Group averaged 186.3 g less (95% January 200 I, exposure exposure ; consideration of

I : levels below CI: -3 75.2 to -45.5) among levels were substantially other pesticides and

LOD (32%of newborns wi th the highest lower, and no associations environmental co n ta minant s participants) ; compared w ith lowest 26% with fe tal growth outcomes as covar iat es in the

Group 2 : lowest of exposure levels (p ; were observed (p > 

# 0

. 8)

. mult i va riat e models

. J / 3 of d etectable 0

. 01). Limitations : single

levels (20 %): exposu r e sampling period ; Group 3: middle the authors did not present

1/3 (24%)

, Group nor discuss regression

4: highest 1/3 dia gnostics to assess the

(25%.) degree to which the i r Chlorpyrifos and models met or vio lat ed the

diazinon together: assumptions implicit in

Group 1 : 2 6%, linear models

. Group 2: 22 %,

Group 3: 26%

, Group 4: 26%.

Article 2: Birth length

, birth LOD: I I ug/L Race/ethnicity, infant sex, and Mean levels of birth weight

, PON I activity also Very well conducted study

Berkowitz et weight

, head (57%<LOD gestational age

. The autho r s also length, head circumference

, predictor of smaller head with numerous strengths

aL(2004) Mt. circumfe r ence, TCPy) controlled for birth weight or and gestat i onal age did not circumfe r ence; c r eatinine and very few weakhesses.

Sinai**(N~04)** 

# gestational age birth length in their assessment differ between those with co rre cted

of head ci rcumfe rence and urinary pesticide meta bolite The question nair e-based

pesticide exposure. leve ls b e l ow and above the pesticide exposure

level of detection. questions are subject t o imperfect re call. Errors

S imilarl y

, no s t atis t ically would, on average

, significant associations attenuate associations

were observed between betwe en th ese expos ur e

reporte d pesticide exposure metrics and fetal

and mean indices of fetal development.



# Pag e 370 

# of 531Chlorpyrifos Human Health Risk Assessment D424485Page371of531Author, Year,Sample SizeOutcomeAssessedExposureMeasurementPotential confoundersconsideredPrimary Result Conclusions/Uncertainties Strengths and Weaknessesgrowth and gestational age. Recall-based exposureassessments were fortifiedby objective measures ofpesticides/pesticidemetabolites.A metabolite specific forchlorpyrifos (TCPy) wasassessed.Statistical analysis wasappropriate.Observed mean reductionsin the outcome parametersappear to be small inmagnitude and may be oflittle clinical significance.Assessment and control forconfounding wereappropriate. However,confounding byunmeasured (andmismeasured) risk factorsfor abnormal growth thatare related to pesticideexposure would bias theresults in this study. Suchfactors may be related tosocioeconomic status of thestudy participants, acofactor which is difficultto define, no less measurein an epidemiologic study.Limited external validity(generalizability) due to theparticular study populationrecruited and the numerousexclusion criteria applied.Article 3: Eskenaziet al. (2004)CHAMACOS(N=488)Birth length, birthweight, headcircumference,Gestation Length,Ponderal indexTotal DAPs:median 136nmol/L (range: 10–6,854); DEP:median 22 nmol/LGestational age, gestational agesquared, maternal age,pregnancy weight gain, week ofinitiating prenatal care, parity,infant sex, mother's country ofDecreases in gestationalduration associated withtwo measures of in uteropesticide exposure: levels ofmetabolites of dimethylMaternal urine collectionaveraged wks.14, 26, notcreatinine-correctedStrengths in the studydesign include thelongitudinal design, the useof multiple exposurebiomarkers, includingChlorpyrifos Human Health Risk Assessment D424485Page372of531Author, Year,Sample SizeOutcomeAssessedExposureMeasurementPotential confoundersconsideredPrimary Result Conclusions/Uncertainties Strengths and Weaknesses(range: 2–680nmol/L); TCPy:median 3.3 nmol/L(range: 0.2–56.1nmol/L) (76%>LOD)birth, body mass index, familyincome, poverty level, smoking,alcohol, illicit drug use,environmental tobacco smoke,caffeine, history of low birthweight, and history of pre-termdelivery.phosphate pesticidecompounds and wholeblood ChE.quantification of nonspecific (DAPs),chlorpyrifos-specific(TCPy) metabolites, andother environmental coexposures. A reasonable setof exclusion criteria wereapplied.The selection of theCHAMACOS population,which consists mostly ofchildren from low-incomefamilies, served to increasethe relative statisticalefficiency of the study, asthis population is at highrisk of neurodevelopmentaldeficits, compared to thegeneral population.Thestatistical analysis used toassess the associationsbetween the markers ofexposure andneurodevelopment wereappropriate.Errors in theassignment of exposure inthis prospective study willlikely have resulted inattenuation of observedassociations.Article 4: Harley etal. (2011)CHAMACOS(N=329)Birth length, birthweight, headcircumference,gestational ageThe geometricmean for the DAPconcentrationsduring pregnancy(for the average ofthe two samplingperiods) was 146nmol/L (95% CI:133, 160); of this,a larger proportionwas DMPmetabolites (GM =109 nmol/L; 95%CI =98, 120) thanDEPs (GM= 23nmol/L; 95% CI =21, 25). AlleleMaternal intelligence (PeabodyPicture Vocabulary Test(PPVT)), measures of howstimulating the environment is,and known or suspectedneurotoxins were measuredprenatally. To measure thequality and extent of stimulationavailable to a child in the homeenvironment, the Infant-ToddlerHOME (Home Observation forMeasurement of theEnvironment) inventory wascompleted at the 6-month, 1, 2,3.5, 5, and 7 year visits; knownor suspected neurotoxicants,polybrominated diphenyl ethersThe authors observedevidence of an associationbetween prenatal exposureto OP pesticides asmeasured by urinary DAPmetabolites in womenduring pregnancy, isassociated with decreasedcognitive functioning inchildren at age 7.Infants whose PON1genotype and enzymeactivity levels suggestedthat they might be moresusceptible tothe effects ofOP pesticide exposure haddecreased fetal growth andlength of gestation. PON1may be a contributing factorto preterm or low birthweight birth.This study has manystrengths, the longitudinaldesign, the measurement ofurinary DAP at multipletime points and followingchildren to age seven whentests of cognitive functionare reportedly morereliable. The authors wereable to adjust for orconsider many factorsrelated to cognitivefunction, such as prenatalexposure to otherenvironmental agents,socioeconomic indicators,maternal intelligence andChlorpyrifos Human Health Risk Assessment D424485Page373of531Author, Year,Sample SizeOutcomeAssessedExposureMeasurementPotential confoundersconsideredPrimary Result Conclusions/Uncertainties Strengths and Weaknessesfrequencies: PON1192 Q allele= 50%;PON1 -108 Tallele= 46%.Meanarylesteraseactivity: Forinfants: 33.6 U/mL(SD = 16) Formothers: 136.6U/mL (SD = 44).Mean paraoxonaseactivity: Forinfants: 256.6 U/L(SD = 165); Formothers: 989.0U/L (SD = 616).(PBDEs), polychlorinatedbiphenyls (PCBs), p,p’-dichlorodiphenyltrichlorethane(DDT), p,p’-dichlorodiphenyltrichlorethylene(DDE), and lead.education, and childstimulation. The cohort hada relatively homogenoussocioeconomic profile,reducing the potential foruncontrolled confounding.Article 5: Engel etal. (2007) Mt.Sinai (N=311)Brazelton NeonatalBehavioralAssessment Scale(BNBAS) ,primitive reflexes(neurologicalintegrity) measuredbefore hospitaldischarge.Diethylphosphates(DEP): 24.7nmol/L; TotalDAP: 82 nmol/LMaternal age, race, maritalstatus, education, cesareandelivery, delivery anesthesia,infant age at examination, infantgender, infant jaundice, smoking(yes/no), alcohol consumption,caffeine consumption, illicitdrug use during pregnancy, andthe examiner.No adverse associationswere found for DAPs andany measured behavior.Relative to the first quartile,quartiles 2–4 of total DEPs,DMPs, and DAPs wereassociated with an increasedproportion of abnormalreflexes, although theassociations did notincrease monotonically andvaried in their strength andprecision.Used non-specificbiomarker DEP/DAPThis was a well conductedprospective studyconducted in a young,predominantly minoritypopulation. The studydesign, analytic approach,and statistical analyses wereappropriate. Pesticidemetabolites evaluated arenot specific forchlorpyrifos. The BNBASwas administered beforehospital discharge only on asubset of children in thecohort (n =311/404).Factors related to weekenddelivery (e.g., fewerinductions) would beunderrepresented among thetested subjects, and mayinduce bias, reduce thedegree of precision withwhich associations wereestimated, and limit thegeneralizability of the studyfindings.The statisticalanalysis was largelyappropriate. Imputing ofmissing data may affectprecision of associationChlorpyrifos Human Health Risk Assessment D424485Page374of531Author, Year,Sample SizeOutcomeAssessedExposureMeasurementPotential confoundersconsideredPrimary Result Conclusions/Uncertainties Strengths and Weaknessesestimates and result inattenuated effect estimatesas a result of exposuremeasurementerror.Assessment andcontrol for confoundingwere appropriate. However,confounding byunmeasured (and miasured) risk factors forabnormal growth that arerelated to pesticideexposure would bias theresults in this study. Suchfactors may be related tosocioeconomic status of thestudy participants, acofactor which is difficultto define, no less measurein an epidemiologic study.Article 6: Young etal. (2005)CHAMACOS(N=381)Neurodevelopment,Brazelton NeonatalBehavioralAssessmentScale(BNBAS),abnormal reflexesDAP (ave. duringpregnancy):median 222nmol/L(range: 7–21,867nmol/L); DEP(ave. pregnancy):median 21nmol/L(range: 2–680nmol/L)Maternal age, BMI, anysmoking/alcohol/drug useduring pregnancy, gestationalage at which prenatal care wasinitiated, total number ofprenatal care visits, meanpregnancy blood pressure,parity, method of delivery,general anesthesia used duringdelivery, breastfeeding initiatedafter delivery, poverty level,infant sex, age in days atBNBAS, minutes since last feedat BNBAS, and BNBASexaminer.Among the >3 day oldinfants, increasing averageprenatal urinary metabolitelevels were associated withboth an increase in numberof abnormal reflexes (totalDAP: adjusted beta = 0.53,95% CI = 0.23, 0.82;dimethyls: adjusted beta =0.41, 95% CI = 0.12, 0.69;diethyls: adjusted beta =0.37, 95% CI = 0.09, 0.64),and the proportion ofinfants with more than threeabnormal reflexes (totalDAP: adjusted OR = 4.9,95% CI = 1.5, 16.1;dimethyls: adjusted OR =3.2, 95% CI = 1.1, 9.8;diethyls: adjusted OR = 3.4,95% CI = 1.2, 9.9).Associations seen pre-natalOP, not post-natal OPexposure, Maternal urinecollection averaged wks.14,26Strengths: Longitudinaldesign, measurement andconsideration of manyconfounders, the prenatalexposure measures were,with some exceptions, theaverage of twomeasurements, and thusmay better reflect chronicexposure during thepregnancyWeaknesses:Potential for residualconfounding, potential forexposure misclassification,potential selection bias (notall children had theoutcome measures), studypopulation notgeneralizable to the wholeUS.Article 7: Rauh etal. (2006)Columbia U.(N=254)Neurodevelopment:The Bayley Scalesof InfantDevelopment IIExposure levelswere categorizedas low (< 6.17pg/g) or highData were collected regardinglead exposure, demographics,education and occupationalhistory, income, active andAt the 36 month milestone,the likelihood of highlyexposed childrendeveloping mental delaysThe authors summarizethree main findings: 1) byage 3, the more highlyexposed children• Only 53% of the childrenreached the three yearmilestone with study datacollected. It is unclear whatChlorpyrifos Human Health Risk Assessment D424485Page375of531Author, Year,Sample SizeOutcomeAssessedExposureMeasurementPotential confoundersconsideredPrimary Result Conclusions/Uncertainties Strengths and Weaknesses(BSID-II), MentalDevelopment Index(MDI) andPsychomotorDevelopment Index(PDI) at 12, 24, and36 months of age.• Behavior: ChildBehavior Checklist(CBCL) at 12, 24,and 36 months . •Quality of thechild-careenvironment: TheHome Observationfor Measurement ofthe Environment(HOME)(>6.17 pg/g) passivesmoking, alcohol anddrug use during pregnancy, andresidential pesticide use. Finalmodels included prenatalenvironmental tobacco smoke(ETS) exposure, gender,ethnicity, gestational age atbirth, quality of home caretaking environment, maternaleducation, and maternal IQ.were 2.4 times greater (95%CI: 1.12-5.08, p = .02) andmotor delays were 4.9greater (95% CI: 1.78-13.72; p = .002) than thosewith lower prenatalexposure. The GLManalysis for PDI scoresshowed a significant effectof chlorpyrifos exposureover time with an estimateddeficit of approximately 7points by age 36 months (p= .01).demonstrated mental andmotor delays; 2) theobserved developmentaltrajectories for PDI andMDI scores confirmed thatthe adverse impact oncognitive and motordevelopment increased overtime; and 3) by age 3,highly exposed childrenwere more likely todemonstrate clinicallysignificant attentionproblems.percentage of these childrendid not survive, were lost tofollow-up, or too sick toparticipate. • Reliance on asingle exposure level(prenatal/cord blood.) • Nocontrol for exposure overthe subsequent 3 years •Creation of a dichotomousexposure variable bringslimitations due to theamount of within-groupvariation. • Limitations ofthe sensitivity andpredictive validity of thedevelopmental tests,especially among childrenless than 3 years of age. •No discussion of whetherthis 7-point deficit isclinically relevant. • Due tothe pervasive, non-specificnature of neurologicaleffects, it is difficult toattribute causality.Article 8: Lovasi etal. 2010Columbai U.(N=266)Bayley scores(MDI/PDI) 12 mo.,24 mo., 36 mo.N/A Neighborhood characteristics:The percentage of housing unitswithout complete plumbing, thepercentage of vacant housingunits, the percentage of residentsbelow the federal poverty line,the percentage of residents olderthan 25 years of age whocompleted high school, thepercentage of householdsreceiving public assistance, thepercentage of housing units withone or more residents per room,racial composition, thepercentage of residents bornoutside the United States, thepercentage of Spanish-speakingresidents, and the percentage ofresidents who werelinguistically isolatedNeighborhoodcharacteristics did notconfound the observedassociation betweenchlorpyrifos levels andcognitive development.Hierarchal regressionanalysis of potentialconfounding by SESDirect measurement ofchlorpyrifos.The statisticalanalyses were generallyappropriate. Missing dataon covariates wereestimated using multipleimputation, and thevariance estimatespresented appropriatelyreflect the degree ofuncertainty caused bymissing covariate data.Robust standard errors wereused.The setting of theinvestigation in a sampledrawn from low-incomeAfrican American andDominican communities isboth a strength (increasesthe power, restriction ofconfounders) and aChlorpyrifos Human Health Risk Assessment D424485Page376of531Author, Year,Sample SizeOutcomeAssessedExposureMeasurementPotential confoundersconsideredPrimary Result Conclusions/Uncertainties Strengths and Weaknesseslimitation of the study(reduced generalizability).Article 9: Engel etal. (2011) Mt.Sinai (N=276)Bayley scores(MDI/PDI) at 12mo., 24 mo.DEP: 24.7 nmol/L;Total DAP: 82nmol/L (same asEngel 2007)Maternal age, race/ethnicity,marital status, education, breastfeeding, child sex, alcohol,smoking, or drug use duringpregnancy, maternal IQ, a scorebased on assessment of thehome environment (HOME),season of urine collection,language spoken in the home,age at testing, examiner, andurinary creatinine level.An observed associationbetween prenatal totaldialkylphosphate metabolitelevel and a decrement inmental development at 12months among blacks andHispanics.Used non-specificbiomarker DEP/DAP; someevidence of effectmodification by PON1genotypeLimitations include use ofnon-specific markers ofchlorpyrifos pesticideexposure (DAPs), use ofonly a single (thirdtrimester) urine sample, andthe large proportion of lossto follow-up. Statisticalanalysis was appropriate.Imputing of missing datamay affect precision ofassociation estimates andresult in attenuated effectestimates as a result ofexposure measurementerror, although these areoffset by the furthercategorization of theexposure levels (at themedian). However, binningof exposure levels reducesprecision, relative to acontinuously distributedmeasure of exposure.Assessment and control forconfounding wereappropriate. However,confounding byunmeasured (andmismeasured) risk factorsfor abnormal growth thatare related to pesticideexposure would bias theresults in this study. Suchfactors may be related tosocioeconomic status of thestudy participants, acofactor which is difficultto define, no less measurein an epidemiologic study.Article 10:Eskenazi et al.(2007)Neurodevelopment,Bayley Index(MDI, PDI),DEP: geom. Meanin mother 18.1nmol/L (95% CIPsychometrician, location ofassessment, exact age atassessment, sex, breast-feedingDAP metabolite levelsduring pregnancy,particularly from dimethylNo strong associationsidentified with DE or TCPy,Maternal urine collectionStrengths: Longitudinaldesign, measurement andconsideration of manyChlorpyrifos Human Health Risk Assessment D424485Page377of531Author, Year,Sample SizeOutcomeAssessedExposureMeasurementPotential confoundersconsideredPrimary Result Conclusions/Uncertainties Strengths and WeaknessesCHAMACOS(N=372)Maternal behaviorchecklist at 6, 12,and 24 months16.7–19.7); DEPgeom mean inchild at 24 months10.5 nmol/L (95%CI 8.8–12.6);TCPy median 3.54ug/lduration (months), HOMEscore, and household income,parity, maternal PPVT, maternalage, education, depressivesymptoms, active/passivesmoking exposure duringpregnancy, regular alcohol useduring pregnancy, marital status,father’s presence in home,housing density, maternal workstatus,≥15 hours out-of-homechildcare/week, birth weight,gestational age, abnormalreflexes, PCBs, lead, DDT,βhexachlorocyclohexane, andhexa-chlorobenzenephosphate pesticides, maybe negatively associated at24 months with mentaldevelopment (MDI) on theBayley Scales and anincrease in risk ofmaternally reported PDD.averaged wks.14, 26 confounders (includingother environmentalchemicals); the prenatalexposure measures were,with some exceptions, theaverage of twomeasurements, and thusmay better reflect chronicexposure during thepregnancyWeaknesses: Potential forresidual confounding,potential for exposuremisclassification, potentialselection bias (not allchildren had the outcomemeasures), study populationnot generalizable to thewhole US.Article 11:Eskenazi et al.(2010)CHAMACOS(N=371)Neurodevelopment,Bayley Index(MDI, PDI),Maternal behaviorchecklist at 6, 12,and 24 months,PON1 gene andenzyme levelsThe geometricmean for the DAPconcentrationsduring pregnancy(for the average ofthe two samplingperiods) was 146nmol/L (95% CI:133, 160); of this,a larger proportionwas DMPmetabolites (GM =109 nmol/L; 95%CI =98, 120) thanDEPs (GM= 23nmol/L; 95% CI =21, 25). Allelefrequencies: PON1192 Q allele= 50%;PON1 -108 Tallele= 46%.Meanarylesteraseactivity: Forinfants: 33.6 U/mL(SD = 16) Formothers: 136.6Psychometrician, location ofassessment, exact age atassessment, sex, breast-feedingduration (months), HOMEscore, and household income,parity, maternal PPVT, maternalage, education, depressivesymptoms, active/passivesmoking exposure duringpregnancy, regular alcohol useduring pregnancy, marital status,father’s presence in home,housing density, maternal workstatus,≥15 hours out-of-homechildcare/week, birth weight,gestational age, abnormalreflexes, PCBs, lead, DDT,βhexachlorocyclohexane, andhexa-chlorobenzeneDecrease MDI (24 mo.)PON1108TT–5.7 (–9.0 to –2.5) 9=0.01; Decrease PDI(24 mo.) PON1108TT–2.8 (–5.7 to 0.2) p=0.07;increased odds PDD 2.0(0.8 to 5.1) p=0.14; noassociation PON1192; noassociation PON1 activitymeasured newborn, 2 years,maternal and MDI, PDI,PDD. Evidence ofdecreasing MDI score bynumber of PON1108variantalleles: PON1108CC–3.2 (–9.8 to 3.5), CT –3.7 (–8.0 to0.6), TT –5.5 (–11.1 to 0.1),p-interaction 0.98.In this study population,evidence PON1 mayinfluence MDI score, butnot PDI or PDD risk at twoyears. Non-significantevidence of decreasing MDIscore by increasing DAPlevels across strata of thenumber of PON1108variantalleles, interaction nonsignificant. Similar trendwith prenatal DEP levelsand MDI, PDI by PON108alleles, but less pronounced.Overall, limited, nondefinitive evidence of effectmodification by PON1status in the relationbetween mental andpsychomotor effects andprenatal DAPs.Strengths: Longitudinaldesign, measurement andconsideration of manyconfounders (includingother environmentalchemicals); the prenatalexposure measures were,with some exceptions, theaverage of twomeasurements, and thusmay better reflect chronicexposure during thepregnancyWeaknesses:Potential for residualconfounding, potential forexposure misclassification,potential selection bias (notall children had theoutcome measures), studypopulation notgeneralizable to the wholeUS. Study may be underpowered to evaluate effectmodification byPON1status.Chlorpyrifos Human Health Risk Assessment D424485Page378of531Author, Year,Sample SizeOutcomeAssessedExposureMeasurementPotential confoundersconsideredPrimary Result Conclusions/Uncertainties Strengths and WeaknessesU/mL (SD = 44).Mean paraoxonaseactivity: Forinfants: 256.6 U/L(SD = 165); Formothers: 989.0U/L (SD = 616).Article 12: Markset al. (2010)CHAMACOS(N=348)CBCL; K-CPT;ADHD confidenceindex; Hillsidebehavioral ratingscale; compositeADHD indicatorDAP (geom mean)pregnancy 109.0nmol/L; DEP 17.7nmol/LPsychometrician, exact age atassessment, sex, maternaleducation, depressivesymptoms, PPVT (continuous),≥15 hr out-of-home childcare/week, breast feedingduration (months), maternal age,parity, marital status,active/passive smoking exposureand regular alcohol use duringpregnancy, presence of father inhome, maternal work status, andhousehold incomePrenatal DAPs were nonsignificantly associatedwith maternal report ofattention problems andADHD at age 3.5 years, butwere significantly related atage 5 years [CBCLattention problems:β= 0.7points; 95% confidenceinterval (CI), 0.2-1.2;ADHD:β= 1.3; 95% CI,0.4-2.1].Marked effect modificationby gender: 11-fold increaseADHD composite indicatorin boys, less than 2-fold ingirls, however unstableestimates; weak evidence ofassociation DAPs at 3.5, 5yrs. and attentionStrengths: Longitudinaldesign, measurement andconsideration of manyconfounders (includingother environmentalchemicals); the prenatalexposure measures were,with some exceptions, theaverage of twomeasurements, and thusmay better reflect chronicexposure during thepregnancyWeaknesses: Potential forresidual confounding,potential for exposuremisclassification, potentialselection bias (not allchildren had the outcomemeasures), study populationnot generalizable to thewhole US.Article 13: Rauh etal. (2011)Columbia U.(N=265)• Wechsler Scalesof Intelligence forChildren (WISCIV) • ChildBehavior Checklist(CBCL).• Chlorpyrifoslevels in umbilicalcord bloodsamples, N=256newborns• If nocord blood (12% ofsubjects), levelswere imputed frommothers’ values. •Values for sampleswith nondetectablechlorpyrifos levels(N=115, 43%)were imputed byData were collected regardinglead exposure, demographics,education and occupationalhistory, income, active andpassive smoking, alcohol anddrug use during pregnancy, andresidential pesticide use. Finalmodels included prenatalenvironmental tobacco smoke(ETS) exposure, gender,ethnicity, gestational age atbirth, quality of home caretaking environment, maternaleducation, and maternal IQ.Full-Scale IQ: (B) of -0.003, CI -0.006, 0.001, p=0.064 Working MemoryIndex: (B) of -0.006, CI -0.009, -0.002, p<0.001.The investigatorsarticulated these results asshowing that a 1 pg/gincrease in chlorpyrifosexposure was associatedwith a 0.006 point decreasein the log-transformedWorking Memory score anda 0.003 point decrease inthe log-transformed FullFor each standard deviationincrease in exposure(4.61pg/g) there is a 1.4%reduction in Full-Scale IQand a 2.8% reduction inWorking Memory.Strengths• Directassessment of chlorpyrifoslevels using maternal serumand cord blood • Analysisusing a continuous CPFlevel, which, in contrast todichotomous CPF levels,provides a more meaningfullook at potential thresholdeffects and dose-responsetrends.• The investigatorsrigorously evaluated theirmethods for imputingvalues for undetectable CPFlevels which in the end,Chlorpyrifos Human Health Risk Assessment D424485Page379of531Author, Year,Sample SizeOutcomeAssessedExposureMeasurementPotential confoundersconsideredPrimary Result Conclusions/Uncertainties Strengths and Weaknessesusing assayspecific limit ofdetection (LOD)values to impute anapproximate level.Scale IQ score. Theinvestigators concluded thatfor each standard deviationincrease in exposure(4.61pg/g) there is a 1.4%reduction in Full-Scale IQand a 2.8% reduction inWorking Memory.were validated.• Theauthors describe an elegantand methodologically soundstatistical analysis,addressing many of thepotential shortcomings oftheir exposure data andcovariates.Weaknesses• Theuse of a single snapshot ofprenatal chlorpyrifosexposure may not be anaccurate surrogate for fullprenatal exposure levels. •There is no control forexposure over thesubsequent 7 years whichmay be critical, especiallyas the process ofneurocognitivedevelopment is fluid andrapid during these earlychildhood years. •Possibility of that anincreased awareness of therisks of pesticide exposurescould disproportionatelyaffect postnatal exposurebehavior.• Complicatingthis analysis is thepervasive, non-specificnature of neurologicaleffects and the difficulty inattributing causal pathways.• when closely reviewed,the 95% CI for Full ScaleIQ for both techniquescontain 0 (LASSO: -0.006,0.001, p=0.064; fullyadjusted: -0.006, 0.001,p=0.048• The authors donot address the clinicalrelevance of the 1.4% and2.8% reductions and howthis may impact a child orhis/her psychological oreducational plans.Chlorpyrifos Human Health Risk Assessment D424485Page380of531Author, Year,Sample SizeOutcomeAssessedExposureMeasurementPotential confoundersconsideredPrimary Result Conclusions/Uncertainties Strengths and WeaknessesArticle 9: Engel etal. (2011)Mt. Sinai (N=169)WechslerPreschool andPrimary Scale ofIntelligence, 3rdedition (WPPSIIII) at ages < 7years; Wechsler-IVIntelligence Scale(verbalcomprehension;perceptualreasoning, workingmemory,processing speed,full scaleintelligence) at age7-9 yearsDEP: 24.7 nmol/L;Total DAP: 82nmol/L (same asEngel 2007)Maternal age, race/ethnicity,marital status, education, breastfeeding, child sex, alcohol,smoking, or drug use duringpregnancy, maternal IQ, a scorebased on assessment of thehome environment (HOME),season of urine collection,language spoken in the home,age at testing, examiner andurinary creatinine level.At age 6-9 years, nonstatistically significantreductions in full scale IQ,perceptual reasoning, verbalcomprehension, workingmemory and processingspeed with increasing DAP,more profound with DEPthan DMP;Used non-specificbiomarker DEP/DAP; someevidence of effectmodification by PON1genotypeLimitations include use ofnon-specific markers ofchlorpyrifos pesticideexposure (DAPs), use ofonly a single (thirdtrimester) urine sample, andthe large proportion of lossto follow-up.The statistical analysis waslargely appropriate.Imputing of missing datamay affect precision ofassociation estimates andresult in attenuated effectestimates as a result ofexposure measurementerror, although these areoffset by the furthercategorization of theexposure levels (at themedian).Assessment and control forconfounding wereappropriate. However,confounding byunmeasured (and mismeasured) risk factors forabnormal growth that arerelated to pesticideexposure would bias theresults in this study. Suchfactors may be related tosocioeconomic status of thestudy participants, acofactor which is difficultto define, no less measurein an epidemiologic study.Article 14:Bouchard et al.(2011)CHAMACOS(N=329)Wechsler-IVIntelligence Scale(verbalcomprehension;perceptualreasoning, workingmemory,Total DAPs(quintiles): Q1 (39nmol/L); Q2 75nmol/L; Q3 126nmol/L; Q4 221nmol/L; Q5 508nmol/L. GeometricMaternal intelligence, measuresof how stimulating theenvironment is, and known orsuspected neurotoxins weremeasured prenatally. Maternalintelligence was assessed via thePeabody Picture VocabularyThe authors observedevidence of an associationbetween prenatal exposuresto OP pesticides asmeasured by urinary DAPmetabolites in womenduring pregnancy, andPrenatal measures takenlater half of pregnancymore significantlyassociated intelligence thanearly; little evidence postnatal OP exposureassociated with intelligence;This study has manystrengths, the longitudinaldesign, the measurement ofurinary DAP at multipletime points and followingchildren to age seven whentests of cognitive functionChlorpyrifos Human Health Risk Assessment D424485Page381of531Author, Year,Sample SizeOutcomeAssessedExposureMeasurementPotential confoundersconsideredPrimary Result Conclusions/Uncertainties Strengths and Weaknessesprocessing speed,full scaleintelligence)measured at age 7yearsmean DAP 131nmol/LTest (PPVT). To measure thequality and extent of stimulationavailable to a child in the homeenvironment, the Infant-ToddlerHOME (Home Observation forMeasurement of theEnvironment) inventory wascompleted at the 6-month, 1, 2,3.5, 5, and 7 year visits; knownor suspected neurotoxicants,polybrominated diphenyl ethers(PBDEs), polychlorinatedbiphenyls (PCBs), p,p’-dichlorodiphenyltrichlorethane(DDT), p,p’-dichlorodiphenyltrichlorethylene(DDE), and lead.decreased cognitivefunctioning in children atage 7.7 point reduction in fullscale intelligence DAPQ5/Q1 (SS)are reportedly morereliable. The authors wereable to adjust for orconsider many factorsrelated to cognitivefunction, such as prenatalexposure to otherenvironmental agents,socioeconomic indicators,maternal intelligence andeducation, and childstimulation. The cohort hada relatively homogenoussocioeconomic profile,reducing the potential foruncontrolled confounding.Article 15: Whyattet al. (2007)Columbia U.(N=102)None Geometric mean,6.9 ± 17.0 ng/m3;range < 0.4–171ng/m3. Personal airmonitor: median2.8 ng/m3, mean6.2 ± 11.1 ng/m3,range < 0.4–83.4ng/m3N/A There was little withinhome variability and nosignificant difference in airconcentrations withinhomes over time (p≥0.2);between-home variabilityaccounted for 88% of thevariance in the indoor airlevels of propoxur, 92% inchlorpyrifos, 94% indiazinon, and 62% inpiperonyl butoxide (p <0.001). Indoor and maternalpersonal air insecticidelevels were highlycorrelated (r = 0.7–0.9, p <0.001).Indoor and maternalpersonal air insecticidelevels were highlycorrelated (r = 0.7–0.9, p <0.001).Strengths: study design andexposure assessmenttechniques, Limitations:only those cohortparticipants enrolled after2011 were included in theanalysis (most likely due tothe lack of serial data fromthe earlier years.)Article 16: Whyattet al. (2009)Columbia U.(N=102)None The limit ofdetection (LOD) ofchlorpyrifos inblood samples was0.5–1 pg/g plasma.The LOD of TCPyin urine sampleswas 0.26 ng/mLurine. The LODfor TCPy inmeconium was 0.2N/A Meconium TCPyconcentrations werepositively correlated withchlorpyrifos in maternal andcord blood (r = 0.25–0.33, p< 0.05) and with TCPy inmaternal urine (r = 0.31, p <0.01).TCPy in maternal urinesamples was not reliable,but the maternal and cordblood chlorpyrifos as wellas the TCPy levels inmeconium were reliablemeasures of exposureComprehensive exposureassessment including actualblood chlorpyrifos levels,the repeated sampling, andthe environmentalsampling.Weaknesses: only includedparticipants recruited in thepost-cancellation period,use of nonparametric, rank-Chlorpyrifos Human Health Risk Assessment D424485Page382of531Author, Year,Sample SizeOutcomeAssessedExposureMeasurementPotential confoundersconsideredPrimary Result Conclusions/Uncertainties Strengths and Weaknessesng based on asample weighing0.5 g. Exposuremarker levelsbelow the LODwere given a valueof half the level ofdetection, and werethen log10transformed.based statistics isappropriate but the largenumber of observationsbelow the level of detectionreceiving equal rank, maybe problematic; no dietaryassessmentRauh (2012),(n=40)Morphologicalchangeinthepediatricbraininregionsofthebrainknowntobeassociatedwithlearning,cognitionandsocialbehaviorTertile3(≥4.39pg/g),comparedtoTertiles0,1,2(<4.39pg/g,includingthosenotexposedtoCPF)Age, sexAuthorsreportdifferencesinbrainstructure(regionalcerebralsizeandthickness)byCPFexposuregroups,andthedifferences(high>lowCPF)inregionalbrainsizeislikelyduetoenlargementofunderlyingwhitematter.Statisticalinteractionbygenderreported.AuthorsconcludedthattheevidencefromthestudyillustratedchangesinbrainmorphologyinassociationwithhigherCPFexposure,andthatchangesobservedwereinareasofthebrainthatsubservethoselearning,cognitionandsocialbehavioral,supportedbypreviousobservationalandexperimentalliterature.StudysupportsgeneralhypothesisofCPFinfluenceonbrainmorphology,butlacksspecifichypothesesregardingparticularareasofthecerebrumaffected;limitedandsomewhatunbalanceddepictionoftheavailablerodentexperimentaldata;statisticalmethodsappropriate,correctionformultiplestatisticalcomparisonsastrength;MRIimagereadersblindedtoexposurestatusenhancesstudyvalidity;lackofinformationonothervalidationpractices;smallsamplesize,pilotstudy,lowstatisticalpower;externalvaliditylimited;onetimemeasureofpre‐natalexposure

# Chlorpyrifos Human Health Risk Assessment D424485

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# Appendix 5. Summary of OPP’s ChE Policy & Use of BMD Modeling

OPP’s ChE policy (USEPA, 2000 74 

# ) describes the manner in which ChE data are used in human

health risk assessment. The following text provi des a brief summary of that document to provide

context to points of departure selected.

AChE inhibition can be inhibited in the central or peripheral nervous tissue. Measurements of

AChE or cholinesterase (ChE) i nhibition in peripheral tissues (e .g., liver, diaphragm, heart, lung

etc) are rare. As such, experi mental laboratory studies genera lly measure brain (central) and

blood (plasma and red blood cell, RBC) ChE. Blood measures do not represent the target tissue,

per se, but are instead used as surrogate meas ures for peripheral toxicity in studies with

laboratory animals or for periph eral and/or central toxicity in humans. In addition, RBC

measures represent AChE, whereas plasma me asures are predominately BuChE. Thus, RBC

AChE data may provide a better representation of th e inhibition in target tissues. As part of the

dose response assessment, evaluations of neurobe havior and clinical si gns are performed to

consider the dose response linkage between AChE inhibition and apical outcomes.

Refinements to OPP’s use of ChE data have come in the implementation of BMD approaches in

dose response assessment. Beginning with the OP CRA, OPP has incr eased its use of BMD

modeling to derive PoDs for AChE inhibi ting compounds. Most often the decreasing

exponential empirical model has been used.

OPP does not have a defined benchmark response (BMR) for OPs. Howeve r, the 10% level has

been used in the majority of dose response analyses conducte d to date. This 10% level

represents a 10% reduction in AC hE activity (i.e., inhibition) compared to background (i.e.,

controls). Specifically, the BMD 10 

# is the estimated dose where ChE is inhibited by 10%

compared to background. The BMDL 10 

# is the lower confidence bound on the BMD 10



# .

The use of the 10% BMR is derived from a combination of statis tical and biological

considerations. A power analysis was conducte d by the Office of Research and Development

(ORD) on over 100 brain AChE datasets across mo re than 25 OPs as part of the OP CRA

(USEPA, 2002). This analysis demonstrated that 10% is a level that can be reliably measured in

the majority of rat toxicity studi es. In addition, the 10% level is generally at or near the limit of

sensitivity for discerning a statistically significant decrease in ChE activity in the brain

compartment and is a response le vel close to the background brain ChE level. With respect to

biological considerations, a change in 10% brai n AChE inhibition is pr otective for downstream

cholinergic clinical signs a nd apical neurotoxic outcomes . With respect to RBC AChE

inhibition, these data tend to be more variable than brain AChE data . OPP begins its BMD

analyses using the 10% BMR for RBC AChE inhibition but BMRs up to 20% could be

considered on a case by case basis as long as su ch PoDs are protective fo r brain AChE inhibition,

potential peripheral inhib ition, and clinical signs of cholinergic toxicity.

74 

# USEPA (2000) Office of Pesticide Programs, US Environmental Protection Agency, Washington DC 20460.

August 18, 2000 Office of Pesticide Programs Science Policy of Th e Use of Data on Cholin esterase Inhibition for

Risk Assessments of Organophosphorous and Carbamate Pesticides.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 384 of 531 

# Appendix 6. Columbia Center fo r Children’s Environmental Health

(CCCEH) Epidemiology Data Acquisition “Raw Data” Request

I. ACTION REQUESTED

To fulfill identified information needs for the purposes of incorporating the Columbia Center for

Children’s Environmental Health (CCCEH) epid emiology data into the Human Health Risk

Assessment (HHRA) for chlorpyrifos, the agency sought to obtain certain “raw data” from

CCCEH researchers. Specifically, EPA requested the original analytic data file used to support

analyses presented in the peer-reviewe d, published epidemiology studies concerning in utero

chlorpyrifos exposure (V. Rauh et al., 2011; V. A. Rauh et al., 2006; Whyatt et al., 2004).

CCCEH researchers did not agree to provide these data, however, the researchers met with EPA

and discussed the agency’s questi ons about the data to help dete rmine whether further review of

the raw data might assist EPA in resolving uncerta inties. As a result of new information gathered

through an on-site meeting and other sources, EP A is no longer pursuing the request for the

original analytic data file from CCCEH re searchers. This memorandum details the new

information gained that resolves or renders unobtainable the previously identified information

needs.

II. BACKGROUND

EPA considers many different type s of scientific information wh en performing a human health

risk assessment (HHRA) of pesticide exposure in the human population. Tr aditionally, EPA uses

toxicology, product and residue ch emistry, and industrial hygiene studies as well as measured

and modeled human and environmental exposu re information to support assessment of

environmental risks. In its prep aration of the HHRA for chlorpyr ifos, the agency has evaluated

environmental epidemiology studies of the poten tial risk of long-term neurodevelopmental

effects such as delayed motor sk ill acquisition or reduced intell igence quotient (IQ) measures

among children who experienced pe sticide exposure during gestati onal development. There are

three prospective birth cohort studies in the U. S. that examine pestic ide exposure (as well as

other environmental toxicants) to the pregnant mother and fetus, and then measure neurological

and neurodevelopmental performance in children as they grow older. EPA has provided some of

the funding support for each of thes e studies. Authors hypothesize that in utero and early life

# Chlorpyrifos Human Health Risk Assessment D424485

Page 385 of 531 exposure may influence brain development and effect neurological f unctioning in children.

These studies include the CHAMACOS study in th e Salinas Valley, CA, the Mt. Sinai children’s

environmental health study (Mt. Sinai study) , and the Columbia Center for Children’s

Environmental Health (CCCEH).

The CCCEH study is the only one of the three studies that measures maternal and fetal exposure

to chlorpyrifos specifically; the other two cohorts meas ure exposure to organophosphate

pesticides generally. Authors with the CCCEH study reported redu ced birth weight and birth

length among neonates more highly exposed to ch lorpyrifos during gestation (as measured by

cord blood concentration of chlorpyrifos) (W hyatt et al., 2004). Similarly, authors observed

slower motor skill acquisition and reduced mental capacity among infants w ho were more highly

exposed to the chemical in utero (V. A. Rauh et al., 2006). In 2011, authors from all three birth

cohort studies concurrently reported evidence of reduced measures of intelligence (Wechslar

intelligence scale scores) by increasing in utero chlorpyrifos and/or organophosphate exposure

(M. F. Bouchard et al., 2011; Enge l et al., 2011; V. Rauh et al., 2011).

Given the value of this information to the ag ency’s HHRA for chlorpyrifos, EPA requested the

FIFRA SAP to provide external pe er review of the strengths a nd limitations of the epidemiology

data for use in the chlorpyrifos HHRA (F IFRA SAP September 2008 and April 2012). The

agency identified two major areas in which additional information was needed to fully

incorporate these data into the HHRA: additi onal measures of environmental exposure to

chlorpyrifos in the CCCEH cohort to discern whet her acetyl cholinesterase inhibition was likely

to have occurred in connection with reported adverse outcomes, and also the role of other

environmental chemicals (lead, polycyclic ar omatic hydrocarbon (PAH), other organophosphate

pesticides) in the observed adverse neurol ogical effects reported in relation to in utero

chlorpyrifos exposure.

To fulfill these information needs for the purposes of incorporating the epidemiology data into

the chlorpyrifos HHRA, the ag ency sought to obtain certain “raw data” from the Columbia

Center for Children’s Environmental Health (CCCEH) study. Specifically, EPA requested the

original analytic data file us ed to support analyses presented in the peer-reviewed, published

# Chlorpyrifos Human Health Risk Assessment D424485

Page 386 of 531 epidemiology studies concerning in utero chlorpyrifos exposure (V. Rauh et al., 2011; V. A.

Rauh et al., 2006; Whyatt et al., 2004). CCCEH di d not agree to provide the data based upon

these initial inquiries and they asserted that because EPA di d not fund the pesticide exposure

component of their cohort study EPA was not le gally entitled to review their underlying data.

CCCEH did agree, however, to meet and discu ss EPA’s questions about the data to help

determine whether further review of the raw data might assist EPA in resolving uncertainties. As

a result on April 15 th 

# , 2013, EPA scientists and CCCEH resear chers held an all-day meeting at

the CCCEH data center (Mailman School of Pub lic Health, New York City, NY) to discuss

EPA’s information needs and whether acquisition of the full analytic data would be necessary or

valuable to EPA’s assessment. Addendum 1 de lineates the questions EPA posed to CCCEH

study staff at this all-day meeting.

III. RESOLUTION OF INFORMATION NEEDS

A. EPIDEMIOLOGY STUDY EXPO SURE CHARACTERIZATION

The primary rationale supporting EPA’s request for “raw dat a” from the CCCEH researchers

relates to the agency’s need to determine whet her the levels of chlor pyrifos exposure in the

environment (apartments, apartment building or ot her outdoor environment, or dietary exposure)

of CCCEH study participants were above or below levels that may elicit a greater than 10%

inhibition of acetylcholinesterase enzyme leve ls, the current regulatory endpoint. During the

April 2013 meeting, EPA learned that this type of information is ne ither available nor obtainable.

CCCEH researchers estimated relative pesticid e exposure using several different exposure

methods including 48-hour air sampling with persona l monitor, 2-week integrated stationary air

monitoring, maternal urinary con centration of TCPy (urinary me tabolite of chlopryrifos) during

the last trimester of pregnanc y, maternal urinary concentratio n of TCPy at delivery, and

umbilical cord blood and meconium at delivery. To determine whether a significant change in

acetyl cholinesterase levels may have occurred as a result of actual environmental exposure,

temporal concordance between pesticide use an d the chlorpyrifos measurement is needed, i.e.

,

exposure estimation at the time of pesticide appl ication is optimal. The CCCEH study design did

not incorporate pre- and post- pesticide use/exposure measurement in the study protocol.

Therefore, this information was not collected and is not retrospectively obtainable.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 387 of 531 In addition, EPA requested any additional inform ation obtained by resear chers as to specific

pesticide products used to better understa nd the pattern and frequency of organophosphate

pesticide use among cohort participants. This info rmation was solicited from participants in a

written questionnaire administered during a fo llow-up period (unpublished copy of questionnaire

obtained by EPA Oct. 2012). In response to th e EPA inquiry, researchers recalled that the

Whyatt (2002) publication described the challenges of collect ing pesticide product information

in etiologic epidemiology studies , and in the on-site meeting in April 2013 confirmed that the

information quality in the CCCEH written questionna ire responses is very low. This information

was deemed of such poor quality by CCCEH data analysts that the data were not coded or

entered into the analytic data file. Therefore, EPA learned that this specific request for “raw

data” concerning pesticide produc t use is not available.

As a surrogate for this information, CCCEH res earchers suggested EPA contact the New York

City Department of Health to obtain a linked dataset of CCCE H study participant residential

address and public housing pesticide usage. The linked dataset provides aggregated pesticide

usage data at the cohort participant building- level only. EPA has obtained and reviewed these

data (June 2013) and determined that pursuing a data reconstruction exercise is the most

appropriate way to estimate en vironmental pesticide exposure that would have to occur among

CCCEH study participants. EPA has conducted such analysis and included it in the revised

human health risk assessment.

B. CO-EXPOSURE TO OTHER E NVIRONMENTAL CONTAMINENTS

A second major concern raised by EPA, FIFRA SA P peer reviewers, and public commenters is

the ability of the CCCEH study authors to accu rately measure and statistically model the

relationship between other environmental chem icals (lead and PAH, specifically) or other

pesticides (diazinon, propoxur) that may influence fetal br ain development and childhood

neurodevelopmental performance, and also be re lated to chlorpyrifos exposure (these are

“potentially confounding” exposures ). EPA’s concern stems from the understanding that if these

other exposures are not sufficiently considered in the epidemiologi cal analysis, then an incorrect

# Chlorpyrifos Human Health Risk Assessment D424485

Page 388 of 531 inference and conclusion may result ( i.e. , a potential false positive association). For example,

prenatal and early life exposure to lead in the environment has been causally linked to adverse

neurodevelopmental outcomes similar to those measured in the CCCEH cohort study including

intelligence measures. EPA was concerned about th e potential error in the CCCEH study if lead

levels were not appr opriately considered, i.e. , the apparent chlorpyrifos effect on

neurodevelopment observed in the study may have been due to the lead exposure.

However, EPA has confirmed with study authors that lead levels and chlorpyr ifos levels in cord

blood are not statistic ally associated in this population. Pl otting blood lead levels against cord

blood chlorpyrifos levels illustrates that the tw o exposures are extremely weakly (linearly)

correlated in this cohort ( ρ <1%) (V. A. Rauh et al., 2006). Further, EPA learned from

unpublished, supplemental analyses performed by CCCEH researchers upon EPA request that

postnatal blood lead levels and prenatal chlorpyr ifos levels are also no t strongly statistically

associated (Andrews, January 21, 2013). This is plausible because of intensive lead abatement

programs on-going in New York City during the ti me period of this study. According to the New

York City Department of Health, the number of children with elevat ed blood lead levels declined

92% between 1995 and 2008. 75 

# Therefore, because the two expos ures are not related, it is not

likely that pre- or postnatal blood lead exposur e could explain the observed association with

chlorpyrifos.

Furthermore, during the April 2013 meeting CC CEH researchers pointed out that based upon

available information it appears that lead and chlorpyrifos may affe ct the brain differently. It is

well understood that lead affects the neurode velopmental sub-domain leading to outward

motivation and aggression; while research with in the CCCEH cohort indicates chlorpyrifos may

affect inward motivation, information processing and organization (V. Rauh et al., 2011; V. A.

Rauh et al., 2006; Wright et al., 2008). Additiona lly, MRI imaging studies of lead affected

persons and preliminary brain imaging studies of chlorpyrifos affected persons show different

MRI patterns, grey matter as opposed to white matter compositional pa tterns, respectively

(Brubaker, Dietrich, Lanphear, & Cecil, 2010; Brubaker et al., 2009; Cecil et al., 2008; Cecil et

al., 2011; V. A. Rauh et al., 2012). Therefore, given that neither pre- nor postnatal lead levels

75 

# http://www.nyc.gov/html/doh/htm l/data/stats-childlead.shtml

# Chlorpyrifos Human Health Risk Assessment D424485

Page 389 of 531 and chlorpyrifos levels are not statistically a ssociated with one another in the CCCEH study, and

the different ways through which lead and chlorp yrifos appear to influence neurodevelopmental

domains EPA concludes that lead exposure did not likely confound (bias or render incorrect) the

observed association between chlorpyrifos e xposure and neurodevelopment in this study

population.

Peer review panelists participating on the Ap ril 2012 FIFRA SAP panel identified the concern

that authors had not fully considered the long- term effects of polycyclic aromatic hydrocarbon

(PAH) exposure, a ubiquitous air pollutant in inner-city areas such as NYC, in the observed

association between chlorpyrif os and neurodevelopmental outco mes. Specifically, panelist

argued that ‘a shift in environm ental exposures over time’ such that postnatal PAH exposure may

have combined with the measured in utero pesticide exposure to result in the observed ND

outcomes. During the April 2013 meeting, authors cl arified that the study design did not include

a repeat measure of exposures over time, so an analysis of postnatal PAH exposures is not

possible. In the published studies, authors were able to control fo r the effect of prenatal PAH

through statistical adjustment. In addition, aut hors examined the possible modifying role of

prenatal PAH in this epidemiological associatio n and did not observe any evidence of a different

risk estimate between chlorpyrifos and ND among those more highly exposed to PAH.

Concerning the role of postnatal environmental exposures, CCCEH researchers also stated their

belief that their overall study resu lts illustrate that it is gesta tional exposure, and not early life

exposure, that influences neurodevelopment in the study population. They state that the

longitudinal analyses of infant and child neurodevelopment in relation to in utero chlorpyrifos

exposure illustrates a persistent effect of the prenatal environm ent (M. Bouchard et al., 2003; M.

F. Bouchard et al., 2011; Engel et al., 2007; Enge l et al., 2011; Eskenazi et al., 2004; Eskenazi et

al., 2007; V. Rauh et al., 2011; V. A. Rauh et al ., 2006; Whyatt et al., 2004). EPA concluded that

CCCEH researchers utilized best practices in st atistical analysis of epidemiological data

concerning the role of prenatal PAH in neur odevelopmental outcomes, and that a study of

repeated, postnatal PAH exposure was beyond th e scope of the current CCCEH study, and would

require a follow-up stud y not yet undertaken.

EPA was also interested to learn more about the co-exposure to other organophosphate pesticides

# Chlorpyrifos Human Health Risk Assessment D424485

Page 390 of 531 among CCCEH study participants. Sp ecifically, EPA as well as exte rnal peer review panelists

noted the uncertainty as to the degree to which exposure to multiple acetyl cholinesterase

inhibiting pesticides exposures over time and/or concurrent in time may have influenced study

results. CCCEH researchers agreed that a more clear understanding of the role of mixtures –

exposure to multiple OP pesticides overall or co ncurrent in time – on these neurodevelopmental

outcomes is desirable; however they also recognized that the current sample size is too small to

perform this type of analysis. To better understa nd the role of exposure to a mixture of OP

pesticides a new cohort study with a larger sample size and di fferent design is required.

Therefore, EPA concluded that co-exposure to multiple organophosphate mixtures is not

currently obtainable.

For risk characterization purposes, EPA was al so interested in understanding the relative

contributions of various environm ental exposures on ND outcomes, ( e.g. , PAH, environmental

tobacco smoke, chlorpyrifos). Researchers noted that a preliminary indication of the relative

contribution of various risk factors for intelligen ce measures in these cohorts can be seen through

examination of supplemental tables published by CCCEH researchers, i.e. , the beta-coefficients

provided in published supplemental tables provide an indication of the relative contribution of

each risk factor (V. Rauh et al., 2011). Howeve r, CCCEH researchers indicated that to gain a

true reflection the causal model in the population a series of studies in ot her study populations is

needed. EPA and CCCEH researchers agreed that these studies will likely accumulate over time,

however they are not currently available.

IV. CONCLUSIONS

In the past, EPA sought to obtain the original analytic data file used to support certain

epidemiological analysis of in utero exposure to chlorpyrifos and subsequent adverse

neurodevelopmental health outcomes in childr en generated by the Columbia Center for

Children’s Environmental Health (CCCEH) to support the Human Health Risk Assessment

(HHRA) of chlorpyrifos. EPA believed these datawere important to both clarify the exposureresponse relationship observed in the epidemio logy study relative to th e current regulatory

endpoint (acetylcholinesterase i nhibition), and also to resolv e uncertainties regarding study

# Chlorpyrifos Human Health Risk Assessment D424485

Page 391 of 531 participants co-exposure to other environm ental contaminants, among other areas of

uncertainties. CCCEH researchers did not agree to provide these data, howe ver, the researchers

met with EPA and discussed the agency’s questi ons about the data to help determine whether

further review of the raw data might assist EPA in resolving uncertainties . As a result of this

meeting and additional discussi ons with CCCEH staff, EPA conc luded that access to the raw

data would either not provide answers to EPA’ s questions or that the information EPA sought

could be obtained without analyzing the raw data. Indeed, based on discussions in that meeting

as well as further work conducted by agency st aff, EPA has gained ad ditional information to

better clarify and characterize the major issue areas identified as uncertainties. For these reasons,

EPA decided that it would not further pursue its request for the analytic data file from the

CCCEH researchers.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 392 of 531 Works Cited

Andrews, H. F. (January 21, 2013). [Clarifica tion of Relation between Blood Lead and Cord Blood

Levels of Chlorpyrifos in the Columbia Cent er for Children's Environmental Health (CCCEH)

Studies (Electronic mail communication)].

Bouchard, M., Gosselin, N. H., Brunet, R. C., Samu el, O., Dumoulin, M. J., & Carrier, G. (2003). A

toxicokinetic model of malathi on and its metabolites as a tool to assess human exposure and risk

through measurements of urinary biomarkers. Toxicol Sci, 73 (1), 182-194. doi:

10.1093/toxsci/kfg061

Bouchard, M. F., Chevrier, J., Harley, K. G., Kogut, K. , Vedar, M., Calderon, N., . . . Eskenazi, B. (2011).

Prenatal exposure to organophosphate pes ticides and IQ in 7-year-old children. Environ Health

Perspect, 119 (8), 1189-1195. doi: 10.1289/ehp.1003185

Brubaker, C. J., Dietrich, K. N., Lanphear, B. P., & Cecil, K. M. (2010). The influence of age of lead

exposure on adult gray matter volume. Neurotoxicology, 31 (3), 259-266. doi:

10.1016/j.neuro.2010.03.004

Brubaker, C. J., Schmithorst, V. J., Haynes, E. N., Diet rich, K. N., Egelhoff, J. C., Lindquist, D. M., . . .

Cecil, K. M. (2009). Altered myelination and a xonal integrity in adults with childhood lead

exposure: a diffusion tensor imaging study. Neurotoxicology, 30 (6), 867-875. doi:

10.1016/j.neuro.2009.07.007

Cecil, K. M., Brubaker, C. J., Adler, C. M., Dietrich, K. N., Altaye, M., Egelhoff, J. C., . . . Lanphear, B.

P. (2008). Decreased brain volume in adults with childhood lead exposure. PLoS Med, 5 (5), e112.

doi: 10.1371/journal.pmed.0050112

Cecil, K. M., Dietrich, K. N., Altaye, M., Egelhoff, J. C., Lindquist, D. M., Brubaker, C. J., & Lanphear,

B. P. (2011). Proton magnetic resonance spectrosc opy in adults with childhood lead exposure.

Environ Health Perspect, 119 (3), 403-408. doi: 10.1289/ehp.1002176

Engel, S. M., Berkowitz, G. S., Barr, D. B., Teitelbaum, S. L., Siskind, J., Meisel, S. J., . . . Wolff, M. S.

(2007). Prenatal organophosphate metabolite and organochlorine levels and performance on the

Brazelton Neonatal Behavioral Assessment Scale in a multiethnic pregnancy cohort. Am J

Epidemiol, 165 (12), 1397-1404. doi: 10.1093/aje/kwm029

Engel, S. M., Wetmur, J., Chen, J., Zhu, C., Barr, D. B., Canfield, R. L., & Wolff, M. S. (2011). Prenatal

exposure to organophosphates, paraoxonase 1, and cognitive development in childhood. Environ

Health Perspect, 119 (8), 1182-1188. doi: 10.1289/ehp.1003183

Eskenazi, B., Harley, K., Bradman, A., Weltzien, E., Jewe ll, N. A., Barr, D. B., . . . Holland, N. T. (2004).

Association of in utero organophosphate pestic ide exposure and fetal growth and length of

gestation in an agricultural population. Environmental Health Perspectives, 112 (10), 1116-1124.

doi: 10.1289/ehp.6789

Eskenazi, B., Marks, A. R., Bradman, A., Harley, K., Barr, D. B., Johnson, C., . . . Jewell, N. P. (2007).

Organophosphate pesticide exposure and neur odevelopment in young Mexican-American

children. Environ Health Perspect, 115 (5), 792-798. doi: 10.1289/ehp.9828

Rauh, V., Arunajadai, S., Horton, M., Perera, F., Hoe pner, L., Barr, D. B., & Whyatt, R. (2011). Sevenyear neurodevelopmental scores and prenatal exposure to chlorpyrifos, a common agricultural

pesticide. Environ Health Perspect, 119 (8), 1196-1201. doi: 10.1289/ehp.1003160

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(2006). Impact of prenatal chlorpyrifos exposure on neurodevelopment in the first 3 years of life

among inner-city children. Pediatrics, 118 (6), e1845-1859. doi: 10.1542/peds.2006-0338

Rauh, V. A., Perera, F. P., Horton, M. K., Whyatt, R. M., Bansal, R., Hao, X. J., . . . Peterson, B. S.

(2012). Brain anomalies in children exposed prenat ally to a common organophosphate pesticide.

Proceedings of the National Academy of Sc iences of the United States of America, 109 (20), 7871-

7876. doi: 10.1073/pnas.1203396109

Whyatt, R. M., Camann, D. E., Kinney, P. L., Reyes, A., Ramirez, J., Dietrich, J., . . . Perera, F. P. (2002).

Residential pesticide use during pregnanc y among a cohort of urban minority women. Environ

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Whyatt, R. M., Rauh, V., Barr, D. B., Camann, D. E., Andrews, H. F., Garfinkel, R., . . . Perera, F. P.

(2004). Prenatal insecticide exposures and birt h weight and length among an urban minority

cohort. Environ Health Perspect, 112 (10), 1125-1132.

Wright, J. P., Dietrich, K. N., Ris, M. D., Hornung, R. W., Wessel, S. D., Lanphear, B. P., . . . Rae, M. N.

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# Chlorpyrifos Human Health Risk Assessment D424485

Page 394 of 531 Appendix 6. Addendum 1:

Columbia University Epidemiology Studies

The agency is obligated to review and a ddress peer review comments in support of

regulatory decisions. The following is a lis t of key issues abou t the epidemiological

studies carried out by researchers at Colu mbia University that were raised in peer

review comments. These issues require EP A to have access to the raw data for

additional analyses by the agency.

1) Further analysis of other chemical exposures ( e.g., lead, PAHs, other pesticides)

to address, if possible, their impact or contribution as modulating factors on the

measured outcomes

 2012 SAP -- “it should be noted th at it cannot be stated th at chlorpyrifos is the

sole contributor to the observed outcomes.”

 2012 SAP -- “In an earlier examination of the same cohort, Perera et al. (2009)

reported an association between a decrease in full-scale IQ and verbal IQ in

5year-olds with prenatal polycyclic aromatic hydrocarbons (PAH) exposure

rather than chlorpyrifos, thus, raising an issue of the shift in chemical exposure

association with increase in age. In each of these analyses, statistical modeling

showed that the exposures were inde pendently associated with IQ, and no

significant interaction was observed with the other chemical. While this is a

statistically sound approach to determin e independent responses, panel members

noted that it is very difficult to identi fy the independent physiological effects of

a single chemical in this type of multi-chemical exposure scenario.”

 2012 Federal Peer Review

-- “even low levels of lead can impact

neurodevelopment, and even that the observed neurobehavioral deficits are

more pronounced at lower blood lead leve ls when compared with higher blood

lead levels”.

 2008 SAP -- “In order to eliminate the possi ble causes of neurodevelopmental

effects by other pesticides in the Co lumbia study, it is suggested that EPA

should repeat the pre-post residential can cellation analysis don e for chlorpyrifos

using other pesticide measurements, such as malathion diacid (MDA), a specific

metabolite of malathion. The outcomes fr om those additional analyses will

either confirm or reject EP A’s preliminary conclusion that chlorpyrifos is likely

to play a role in the neur odevelopmental outcomes.”

 2008 SAP -- ““It would be useful to examine th e results of a statistical analysis

that includes all three AChE-inhibiting in secticides in the analysis model as

dichotomous variables (above or below LOD) in combination with continuous

measurements for these variables. This t ype of analysis would likely not change

# Chlorpyrifos Human Health Risk Assessment D424485

Page 395 of 531 the results, but it could be helpful in illustrating threshold or dose response

effects.”

2) Further analysis and information to addre ss and, if possible, better characterize

uncertainty around outcome measu res on learning/memory/IQ

 2012 SAP -- Alternative considerat ions for non-quantified sa mples: “little use

was made of techniques to integrate n on-quantified samples into the statistical

test.... Various methods were reviewed by the July 2010 SAP that can be applied

to either normally or lognormally distribu ted data that include a significant (even

a majority) of non-detectable sample . . . . Specifically, the use of ’probability

plots’ was described that can yield an estimate of the geometric mean of the

distribution [GM], the geometric standa rd deviation [GSD], and corresponding

percentiles.”

 Federal Peer Review -- “There is a scatterplot show ing the raw scores for overall

IQ and for each of the subtests, but it is not possible to obtain the necessary

information to compare the distributions of these scores with the norms for the

test or with any other study sample. Id eally, the means and standard deviations

for these scores should be presented fo r either a non-exposed or a non-exposed

combined with low exposed group and these should be compared to a moderate or

high-exposed group as was done for the BS ID-II in the Rauh et al., 2006 paper.

Here the uncertainties stem from the a ssumptions that are made when regression

analyses are performed. The main issue he re is that outliers can greatly influence

the slope of the function.”

 Federal Peer Review-- A between group analysis using inferential statistics, as

was done for the Bayley Scales of Infa nt Development II in the Rauh et al.,

2006 paper, should be performed on each vari able in both studies (i.e., the Child

Behavior Checklist in Rauh et al., 2006, and the full scale IQ and subscales for

the WISC-IV in the Rauh et al., 2011 study) . This would be the most direct and

least problematic method for determini ng whether exposure to chlorpyrifos

resulted in significant decreases in IQ or significant increas es in behavioral

problems “..... no information was provided regarding the qualifications of the

individuals who administered and scored the tests. “

3) Further analysis to assess, if possible , whether individual cohort members had

the potential for exposure to chlorpyrif os and/or other acetylcholinesterase

(AChE) inhibiting pesticides (e.g., di azinon, propoxur), prenatally and /or

postnatally, at levels leading to grea ter than 10% AChE inhibition (the level

used to derive the regulatory point of departure).

# Chlorpyrifos Human Health Risk Assessment D424485

Page 396 of 531  2012 SAP-- recommended conducting a dose reconstruction analysis—“data on

the concentration of chlor pyrifos in various media ( i.e . house dust, air and

water) while market basket data exists on the concentration of chlorpyrifos on

food. These data provide the main tool s for developing an effective exposure

assessment and a subsequent reconstructi on of potential dose.” The agency has

begun such analysis but the current draft analysis is limited without data on the

exposure information relevant to indi vidual women such that environmental

chlorpyrifos exposure can then be linked to measures of blood chlorpyrifos.

 2012 SAP-- recommended the agency consider issues related to multiple

chemical exposure (i.e., mixtures) to chlorpyrifos and other key AChE

inhibiting pesticides identif ied by the Columbia Univer sity studies (diazinon,

propoxur). Assumptions of co-exposure wi ll likely be grossly overestimated

without access to the raw data ; such raw data may enable the agency to evaluate

actual co-exposure information for indi viduals from air monitoring samples and

blood samples.

# Chlorpyrifos Human Health Risk Assessment D424485

Page 397 of 531 

# Appendix 7. Physica l/Chemical Properties

Physical/Chemical Properties of Chlorpyrifos.

Parameter Value 

# Reference

Melting point/range 41.5-42.5 oC 

# Chlorpyrifos

IRED pH NR

Density (21oC) 1.51 g/mL

Water solubility (25°C) 1.05 mg/L

Solvent solubility (20°C) Acetone >400 g/L

Dichloromethane >400 g/L

Methanol 250 g/L

Ethyl acetate >400 g/L

Toluene >400 g/L

n-hexane >400 g/L

Vapor pressure, (25°C)

1.87x10 -5 

# torr 1



# Dissociation constant, pK

a 

# NR

Octanol/water partition

coefficient, Log(K O W 

# ) 4.7

UV/visible absorption

spectrum NR

NR – not reported.

1 

# R. Bohaty, June 2011, D368388 and D389480, Chlorpyrifos Drinking Water A ssessment for Registration Review (CRF

assessment, Oct. 16, 2009 product chemistry BC 2062713)

# Chlorpyrifos Human Health Risk Assessment D424485

Page 398 of 531 

# Appendix 8. Current U.S. Tolerances a nd International Residue Limits

Chlorpyrifos (059101)

Summary of US and International Tol erances and Maximum Residue Limits

Residue Definition:

US Canada Mexico 2 

# Codex 3



# 40CFR180.342

chlorpyrifos per se ( O,O

- diethyl O -(3,5,6-trichloro2-pyridyl) phosphorothioate O,O -diethyl- O -(3,5,6-trichloro-2-

pyridyl) phosphorothioate

(apples, grapes, tomatoes)

O,O -diethyl- O -(3,5,6- trichloro2-pyridyl) phosphorothioate,

including the metabolite 3,5,6-

trichloro-2-pyridinol

(citrus fruits; fat, kidney, and

liver of cattle; kiwifruit; peppers;

rutabagas; meat and meat

byproducts of cattle (calculated

on the fat content)) Chlorpyrifos. The

residue is fat

soluble.

Commodity 1, 

# Tolerance (ppm) /Maximum Residue Limit (mg/kg)

US Canada Mexico 2 

# Codex 3



# Alfalfa, forage 3.0

Alfalfa, hay 13 5 alfalfa fodder

Almond 0.2 0.05

Almond, hulls 12

Apple 0.01 0.01 1 pome fruits

Apple, wet pomace 0.02

Banana 0.1 2

Beet, sugar, dried pulp 5.0

Beet, sugar, molasses 15

Beet, sugar, roots 1.0 0.05

Beet, sugar, tops 8.0

Cattle, fat 0.3 1.0

Cattle, meat 0.05 1.0 1 (fat)

Cattle, meat byproducts 0.05 1.0 0.01 cattle,

kidney and liver

Cherry, sweet 1.0

Cherry, tart 1.0

Citrus, dried pulp 5.0

Citrus, oil 20

Corn, field, forage 8.0

Corn, field, grain 0.05 0.05 0.05 maize

Corn, field, refined oil 0.25 0.2 maize oil,

edible

Corn, field, stover 8.0 10 maize fodder

(dry)

Corn, sweet, forage 8.0

Corn, sweet, kernel plus

cob with husk removed 0.05 0.05 0.01 sweet corn

(corn-on-the-cob)

Corn, sweet, stover 8.0

# Chlorpyrifos Human Health Risk Assessment D424485

Page 399 of 531 Summary of US and International Tol erances and Maximum Residue Limits

Residue Definition:

US Canada Mexico 2 

# Codex 3



# Cotton, undelinted seed 0.2 0.3 cotton seed

Cranberry 1.0 1

Cucumber 0.05 0.05

Egg 0.01 0.01 (*)

Fig 0.01

Fruit, citrus, group 10 1.0 1.0 1

Goat, fat 0.2

Goat, meat 0.05

Goat, meat byproducts 0.05

Hazelnut 0.2

Hog, fat 0.2

Hog, meat 0.05 0.02 (fat)

Hog, meat byproducts 0.05 0.01 (*) pig,

edible offal

Horse, fat 0.25

Horse, meat 0.25

Horse, meat byproducts 0.25

Kiwifruit 2.0 2.0

Milk, fat (Reflecting 0.01

ppm in whole milk) 0.25 0.02 milk

Nectarine 0.05 0.05

Onion, bulb 0.5 0.2

Peach 0.05 0.05 0.5

Peanut 0.2

Peanut, refined oil 0.2

Pear 0.05 1 pome fruits

Pecan 0.2 0.05 (*)

Pepper 1.0

1.0 2 peppers sweet

including pimento

or pimiento); 20

peppers chili,

dried

Peppermint, tops 0.8

Peppermint, oil 8.0

Plum, prune, fresh 0.05 0.5 plums

(including prunes)

Poultry, fat 0.1

Poultry, meat 0.1 0.01 (fat)

Poultry, meat byproducts 0.1 0.01 (*) poultry,

edible offal

Pumpkin 0.05

Radish 2.0

Rutabaga 0.5 0.5

Sheep, fat 0.2

Sheep, meat 0.05 1 (fat)

# Chlorpyrifos Human Health Risk Assessment D424485

Page 400 of 531 Summary of US and International Tol erances and Maximum Residue Limits

Residue Definition:

US Canada Mexico 2 

# Codex 3



# Sheep, meat byproducts 0.05 0.01 sheep, edible

offal

Spearmint, tops 0.8

Spearmint, oil 8.0

Sorghum, grain, forage 0.5

Sorghum, grain, grain 0.5 0.5

Sorghum, grain, stover 2.0 2 sorghum straw

and fodder, dry

Soybean, seed 0.3 0.1 soya bean

(dry)

Strawberry 0.2 0.3

Sunflower, seed 0.1 0.1

Sweet potato, roots 0.05

Turnip, roots 1.0

Turnip, tops 0.3

Vegetable, brassica, leafy,

group 5 1.0 2 Broccoli

1 Cabbages, head

0.05 Cauliflower

1 Chinese

cabbage (type petsai)

Vegetable, legume, group 6

except soybean 0.05 0.05 lentils 0.01 common

bean (pods and/or

immature seeds);

peas (pods and

succulent=immat

ure seeds)

Walnut 0.2 0.05 (*)

Wheat, forage 3.0

Wheat, grain 0.5 0.5

Wheat, straw 6.0 5 wheat straw and

fodder, dry

MRLs with No US Equivalents

Grapes 0.01 0.5

Tomatoes 0.01

Carrot 0.1

Coffee beans 0.05

Cotton seed oil, crude 0.05 (*)

Cotton seed oil, edible 0.05 (*)

Dried grapes (=currants,

raisins and sultanas) 0.1

Potato 2

Rice 0.5

Soya bean oil, refined 0.03

Tea, green, black (black,

fermented and dried) 2

# Chlorpyrifos Human Health Risk Assessment D424485

Page 401 of 531 Summary of US and International Tol erances and Maximum Residue Limits

Residue Definition:

US Canada Mexico 2 

# Codex 3



# Wheat flour 0.1

1 

# Includes commodities listed in the CFR as of 4/12/11. The 40C FR 180.342 (a) (3) also stipulates that “a tolerance of 0.1 part

per million is established for residues of chlorpyrifos, per se, in or on food commodities (other than those already covered by a

higher tolerance as a result of use on grow ing crops) in food service establishments where food an d food products are prepared

and served, as a result of the application of chlorpyrifos in microencapsulated form.”

2 

# Mexico adopts US tolerances and/or Codex MRLs for its export purposes.

3 

# * = absent at the limit of quant itation; Po = postharvest treatment, such as treatment of stored grains. PoP = processed

postharvest treated commodity, such as pro cessing of treated stored wheat. (fat) = to be measured on the fat portion of the

sample. MRLs indicated as pr oposed have not been finali zed by the CCPR and the CAC.



# (c) Tolerances with regional registrations. Tolerances with regional re gistration, as defined in

180.1(m), are established for residues of the pesticide chlorpyrifos per se ( O,O -diethyl- O

- (3,5,6-trichloro-2-pyridyl) phosphorothioate) in or on the follow ing food commodities:

Commodity Parts per million Canada Codex

Asparagus 5.0

Grape 0.01 0.01 0.5



# In addition, the following tolerances for ch lorpyrifos are recommended under registration

review:

Table 8A. Recommended Tolerances for Chlorpyrifos

Commodity Established Tolerance

(ppm) Recommended

Tolerance (ppm) Comments

Correct Commodity Definition

Aspirated grain fractions NA 22

Cotton, gin byproducts NA 15

Corn, milled byproducts NA 0.1

Wheat, milled byproducts NA 1.5

# Chlorpyrifos Human Health Risk Assessment D424485

Page 402 of 531 

# Appendix 9. Master Use Summary DocumentHealth Risk Assessment D424485nt Chlorpyrifos UsageTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2Indoorgeneralsurface spraybackpacksprayer; highand low sprayer(pressure orvolume)0.075 lb a.i./1000 ft sq1.2EC, ME[14.4]NSNA 12 NA NA NS NSOnly permittedfor use inpoultry housesAt plant groundboom1.0G1.0 1.0[1]NS1 21 24[10]NSMissourionlyLower PHIpermitted forEC rates 0.33 lba.i./A (7 d) and0.67 lb a.i./A(14 d) e.g. Reg.No. 62719-591Stand is inproduction 3-5years. Planted1⁄4” to 1⁄2” deep.aerial or1 0[4 0][4]Lower PHIChlorpyrifos Human Health Risk Assessment D424485Page404of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2broadcast,chemigationEC rates 0.33 lba.i./A (7 d) and0.67 lb a.i./A(14 d)e.g., Reg.No. 62719-591Multipleharvests (orcuttings) peryear when usedfor feed/fodderand 1 harvestper year whengrown for seed.Cuttings occurabout every 30days.Only 1 cropcycle per yearbut up to 9cuttings, variesby geography.Total1.05.05.0[5]NS52124[10]NSRepresentsMissouriscenariootherwise 4.0 lba.i./A per ismax.ALMONDdormant/delayedaircraft, airblast2.0WDG, WP2.0 NA 1 NA NA 24 10Restricteduse inChlorpyrifos Human Health Risk Assessment D424485Page405of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2dormant;broadcastCalifornia.foliar;broadcastaircraft, airblast2.0WDG,WP6.0 NA 3 NA 14 10pre-plant,foliar;trunkspray/drench or preplant diphandheld,backpack,drench/dip,handgun, andlow pressurehand wand2.5(3.0/100 gal)WDG2.5 NA 1 NA 14 NSDormant/delayeddormant;foliar;orchardfloorsbroadcastground boom,handgun,chemigation4.0EC*4.0 NA 2 NA 14 10Restricteduse inCalifornia.Only onedormantapplicationcan be made.Total--4.014.5NA7NA14NSExcludesnurseryapplications(See general“Fruits” listing)APPLEdormant/delayeddormant;broadcastaircraft, airblast2.0EC2.0WDG1.5WP2 2.0 1 1 NA24/4 d10dReflects spraydrift mitigationmeasures.pre-plant,foliar;handheld,backpack,1.5(1.5 lb ai/1001.5 NA 1 1284d NSUsepermitted inChlorpyrifos Human Health Risk Assessment D424485Page406of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2trunkspray/drench or preplant dip;grounddrench/dip,handgun, andlow pressurehand wandgal)WDGstates east ofthe RockiesexceptMississippi.Total2.03.52ASPARAGUSFoliar, preharvest;broadcastaircraft, groundboom1.0EC, WDG1.0 1.0 1 1 1 24 10Postharvest,broadcastaircraft, groundboom1.0EC, WDG2.0 2.0 2 1 1 24 10granular soilband treatmentground boom1.5G3.0 3.0 2 2 180 24[10]NSPermitted inCalifornia,the Midwest,and thePacificNorthwest19713-505,19713-521,5481-525,62719-34,83222-34Do not applymore than 3.0 lba.i./A betweenharvests.Total1.5G3.0 G2.03.0 G2.03312410BEANSPreplant;SeedSeed Treatment0.016-0.3480.000798 lbNS[0.348]NSNS[1]NSNS NS NSME is SLNonly for IDItalics highlightthe range ofChlorpyrifos Human Health Risk Assessment D424485Page407of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2treatment ai/lb seedME0.013-0.2720.000625 lbai/lb seedWP0.012-0.2530.00058 lbai/lb seedECapplicationrates dependingon the numberof seeds per lband the numberof seeds plantedper acre.Seeding rateinformationprovide byBEAD.4BEEF/RANGE/FEEDERCATTLE(MEAT)/DAIRYCATTLE (NONLACTATING)Summer, latefall, spring;impregnatedcollar/tagAnimaltreatment (eartag)0.0066lb/animal[0.0099]NSNA 3 NA NS NS NSReg. No.39039-6Cattle ear tagsare assumed tolast 4-6 monthsTwo tags peranimal at0.0033 lba.i./tag in thesummer andone tag peranimal at0.0033 lb a.i./A.BEETS(UNSPECIFIED;TABLE ORSUGAR)“grown for seed”At plant, soilbandtreatmentGround boom1.0ECNS 1 NS 1 24Allowed inOregonCourtorderedbuffer of 60ft for groundMinimumIncorporation: 2inchesChlorpyrifos Human Health Risk Assessment D424485Page408of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2chlorpyrifosapplicationis requiredfor “affectedwaterways”.Preplant, soilincorporatedtreatmentBroadcast/ground boom1.9ECNS(2.8 ID)NS 1 NSAllowed inOregon andIdahoOR-09007;62719-591ID-090002;62719-591Total1.9NSNSNSNS24One or the othertype ofapplication.SUGAR BEETSPreplant, soilincorporatedtreatmentBroadcast/ground boom1.0EC2.0G3.0 2.0 11NA 24 10MinimumIncorporation: 1inchAt plant, soilbandtreatmentBroadcast/ground boom1.0EC, WDG2.0G3.0 2.0 1 1 30 24 10Postplant,soil bandBroadcast/ground boom2.0G3.0 2.0 1 1 30 24 10Postemergencebandtreatment;broadcastBroadcast/ground boom1.0EC, WDG3.0 1.0 3 1 30 24 10broadcastAircraft, groundboom,1.0EC, WDG3.0 1.0 3 1 30 24 10EC is not foruse in MSChlorpyrifos Human Health Risk Assessment D424485Page409of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2chemigationTotal1.0EC2.0G4.0[4.0]NS3[3]NS302410One granularapplication at2.0 a.i./A andtwo liquidapplications at1.0 a.i./A peryear. Alsoassumed percrop cycle.CARROTGrown for Seed(INCLUDINGTOPS)Foliar prebloombroadcastaircraft, groundboom0.94EC0.94 1 1 1 7 24 NAOregon andWashingtonCourtorderedbuffer of 60ft for groundand 300 ftfor aerialapplicationis requiredfor “affectedwaterways”.OR090011SLN Expires:12/31/2018WA090011SNL Expires:12/31/2016Carrots taketwo years toproduce seed.All commercialproduction ofthe carrot(vegetable)takes place inthe first yearwhen the plantis nowhere nearblooming.Chlorpyrifos Human Health Risk Assessment D424485Page410of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2CHERRIESdormant/delayeddormant;broadcastaircraft, airblast2.0WDG, EC1.5WP2.0 NA 1 NA NS 24 10foliar;broadcastairblast4.0EC10.0 NA 5 NA 14 24 10Tart cherry onlyaircraft 2.0Reflects spraydrift mitigationFoliar, postharvest;trunkspray/drenchhandheld,backpack,drench/dip,handgun, andlow pressurehand wand2.5(3.0/100 gal)WDG, EC2.5 NA 1 NA 2 24[10]NSOnly somelabels specify a10 d MRI.Total--4.04.5(sweet)14.5(tartonly)6Excludesnurseryapplications(See general“Fruits” listing)The foliarapplicationsonly apply totart cherries,thus, sweetcherry scenarios(e.g., PacificNW) annualapplication ratewould be 4.5 lbChlorpyrifos Human Health Risk Assessment D424485Page411of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2total a.i./year.CHRISTMASTREEPLANTATIONSfoliar;broadcasthelicopter,orchard blast1.0EC, WDG, WP3.0 NA 3 NA[0]NS247Aerialapplicationsviahelicopterare onlypermitted inWashingtonand Oregon.post harvest;StumpTreatmenthandheld,backpack,drench/dip,handgun, andlow pressurehand wand2.5(3.0/100 gal)EC, WDG2.5 NA 1 NA NA 7Total2.55.54CITRUSfoliar;broadcastairblast, groundboom6.0WP, WSP, EC7.5NA 2 NA35(21forlowrates)5d30(10forlowrates)6.0 lb a.i. /Ais onlypermitted inCaliforniaand Arizona.The maxsingle rate inother statesis restrictedto 4 lb a.i./A.aircraft2.3WP, WSP, EC21 5 10Florida,California,andpotentiallyAerialapplication usedto controlpsyllid, theChlorpyrifos Human Health Risk Assessment D424485Page412of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2Texas vector for citrusgreening.Reflects spraydrift mitigationfoliar;orchardfloorsbroadcastground boom,chemigation,handheld,backpack,drench/dip,handgun, andlow pressurehand wand1.0G*, WSP, EC3.0 NA 3 NA 2824/5 d10Total--6.010.55Registeredlabels permitboth foliar andsoil applicationsin the sameorchard.Total excludesnurseryapplications(See general“Fruits” listing)CLOVER(GROWN FORSEED)Preplant Ground boom1.9EC1.9 1.9 1 1 NS 24 NAUse onlypermitted inOregon.OR-0900100;master label:62719-591Post-PlantFoliaraircraft andground boomEither apreplant or postplantChlorpyrifos Human Health Risk Assessment D424485Page413of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2application isallowed.COLE CROPS(EXCLUDESCAULIFLOWER ANDPreplant, soilincorporatedtreatmentGround boom2.0EC, WDG, G4.0 2.0 21302410Min.incorporation:2 inchesBRUSSELSSPROUTS)At plant, soilbandtreatmentGround boom1One granularapplicationpermitted peryear.Post plant Ground boom 1FoliarEstablishedPlantings,soil sidedresstreatmentGround boom 1Foliar,broadcastAircraft, groundboom,chemigation1.0EC, WDG, WP4.0 3.0 4 3 21 10Multiple cropsper year arepossible insome locations.Total8.0564Some labelsrestrict theyearlyapplication rateto 3 lb a.i./A.The maximumnumber of cropsper year is 2.BRUSSELSAt plant, soil Ground boom 2.0 2.0 [2.0] 2 1 21Chlorpyrifos Human Health Risk Assessment D424485Page414of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2SPROUTSbandtreatmentEC; G NS2410Preplant, soilincorporatedtreatmentGround boomMinimumincorporation is2 inchesPostplant,soilapplicationGround boom 2.25 EC, G 2.25[2.25]NSFoliarbroadcastAircraft,Ground boom1.0EC[5.3]NS3.0 NS 3 1083222-20,84930-7,86363-3 specifya 7 day MRI.All other labelsspecify a 10 dayMRI.The PHI stated84930-7 isconflicting [p. 4(21 days and p.19 (30 days)]Total2.35.3NS21247Assume oneapplication ofeither at plant,preplant, orpostplantfollowed withadditional foliarapplications.CAULIFLOWERAt plant, soilbandGround boom2.0EC2.0ECNS[1]NS1 21 3d 10Only onegranularChlorpyrifos Human Health Risk Assessment D424485Page415of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2treatment2.3G2.25Gapplication.Preplant, soilincorporatedtreatmentGround boom2.3G2.0EC2.3 NS[1]NS130,EC,21GMinimumincorporation is2 inchesPostplant,soilapplicationGround boomFoliarbroadcastaircraft, groundboom1.0EC[5.3]NS3.0 NS 3 21 10Total2.35.3[5.3]NSNS[4]NS212410Assume oneapplication ateither plant,preplant, orpostplantfollowed withadditional foliarapplications.COMMERCIAL/INSTITUTIONAL/INDUSTRIALPREMISES/EQUIP.(INDOOR)Non-food areas ofmanufacturing,industrial, andfood processingplants;BroadcastProductContainer0.4373 lba.i./100 sq ft190.5GNS NA NS NA NA NS NSFor treatment offire antsCrack andCrevice/VoidSprayer/Injection0.0625 lba.i./1000 sq ft2.7MENS NA NS NA NA NS NS 499-419Crack andCrevice/SpotSprayer/Injection0.0424 lb/galMENS NA NS NA NA NS 7Chlorpyrifos Human Health Risk Assessment D424485Page416of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2warehouses; shipholds; railroadboxcars.COMMERCIAL/INSTITUTIONAL/INDUSTRIALPREMISES/EQUIP.(OUTDOOR)Outdoorcommercial usearound non-foodareas of manufacturing, industrial,and foodprocessing plants;warehouses; shipholds; railroadboxcarsSoilbroadcastLow and HighPressure,Backpack,HandgunSprayers0.0247 lba.i./1000 sq ft1.1MENS NA NS NA NA NS NSDirectedspray0.1132 lba.i./1000 sq ft4.9MENS NA NS NA NA NS NSSpecific to:Inside andoutsidedumpsters andother trashholdingcontainers, trashcorrals andother trashstorage areas.Crack andCrevice/void/generaloutdoor0.0424 lb/galMENS NA NS NA NA NS 7CONIFERSANDDECIDUOUSTREES;?foliar;broadcastGround boom1.0EC3 NA 6 NA 7 24 7PLANTATION,NURSERY?foliar;stumptreatmentbackpack,drencher, lowpressure handwand0.3EC0.3 NA 1 NA 7 24 7Total1.03NA6NA7247The totalChlorpyrifos Human Health Risk Assessment D424485Page417of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2number ofapplicationsassumed iseither 3 foliarapplications or2 foliarapplicationswith one stumptreatment.CORN (ALL)Preplantground/soilincorporatedconservationtillage, infurrow,broadcast,chemigation,soil band3.0EC2.0G3.0 3.0 NS 3 NA24/5EC1019713-520,19713-599,33658-26,34704-857,72693-11,83222-20The minimumincorporationdepth is 2inches.soilincorporatedaerialconservationtillage2.0EC, Gground/conservationtillage, infurrow,broadcast,1.0EC2.0G3.0 3.0 NS 3 21 10 19713-520Chlorpyrifos Human Health Risk Assessment D424485Page418of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2chemigation,soil bandStorage orpreplant seedtreatmentSeed treatment0.001-0.0210.000625 lba.i./ lb seedWP0.1-1.90.058 lb a.i./ lbseedFC[?]NS[1.9]NS[?]NS1 NS NS NSItalics highlightthe range ofapplicationrates dependingon the numberof seeds per lband the numberof seeds plantedper acre.Seeding rateinformationprovide byBEAD.4At plantsoilincorporated,conservationtillage2.0G[?]NS3.0[?]NS3 21 24 10PostemergenceAerial orground,broadcast,chemigation1.5EC1.0WDGNS 3.0 NS 3 2124/5d(EC10A brush on maxsingle rate ispermitted at 1.0lb ai/a (72693-11)FoliarAerial orground/broadcast,granule, seedandchemigation1.5EC3.0 3.0 NS 3 21 10Chlorpyrifos Human Health Risk Assessment D424485Page419of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2Total3.08.18.1NS42110Two granularapplications areallowed with amaximumsingle rate of1.0 lb a.i./A orone granularapplication at 2lb a.i./A.Total with seedtreatmentPHI: 21 dexceptDelaware andFlorida (7 d)COTTONStorage orpreplant seedtreatmentSeed treatment0.8-2.20.00116 lb/lbseedEC[2.2]NS[2.2]NS[1]NS1 NS NS NS264-932Rates in italicshighlight thepotential rangeof applicationrates dependingon the numberof seeds per lband the numberof seeds plantedper acre.Seeding rateinformationprovide byBEAD.2Chlorpyrifos Human Health Risk Assessment D424485Page420of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2Foliaraerial,chemigation,ground boom1.0EC, WDGP3 3.0 3 3 14 24 10Except MSTotal1.03.23.2331424101.6 lb a.i./A ismax single rate(seed treatment)Total with seedtreatment1 crop cycle peryear assumedCRANBERRYFoliaraircraft, groundboom/broadcast andchemigation1.5EC, WDG3.0 NA 2 NA 60 24 10Not for useinMississippi.Do not apply tobogs whenflooded.CUCUMBERStorage orpreplant seedtreatmentCommercialseed treatment0.40.00058 lb/lbseedECNS 0.1 2 1 NS NS NSSeeding rateinformationprovide byBEAD.2264-932,62719-221,CA040004Per registrant 2CCs per yearFIGSdormant/delayeddormant;soilapplicationground boom2.0WDG, EC2.0 NA 1 NA 217 4 d NSUse isrestricted toCaliforniaonly.Incorporation to3 inches issuggested butnot requiredfollowingapplication.FILBERTS/dormant/aircraft, airblast 2.0 2.0 NA 1 NA 14 24 10Chlorpyrifos Human Health Risk Assessment D424485Page421of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2HAZELNUT delayeddormant;broadcastWPfoliar;broadcastaircraft, airblast2.0WDG, WP, EC6.0 NA 3 NA 14 10Some labelsspecify aretreatmentinterval of 10days.Total2.06.0NS3.0NA142410Excludesnurseryapplications(See general“Fruits” listing)FOODPROCESSINGPLANTPREMISES(NONFOODCONTACT)Whenneeded, crackand crevicetreatment,spottreatment0.0424 lb/ galMENS NA NS NA NA NS 753883-264,84575-3Spot Treatment:Do not exceedtwo square feetper individualspot.FORESTPLANTINGS(REFORESTATIONPROGRAMS)(TREE FARMS,TREEFoliar,broadcastground boom1.0EC6.0 NA 6 NA247PLANTATION,ETC.)Foliar, stumptreatmentdirect spray,drencher0.34EC6.0 NA[18]NSNA 7FORESTFoliar, ground boom, 0.61 3.6 NA NS NA 24 7Chlorpyrifos Human Health Risk Assessment D424485Page422of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2TREES(SOFTWOODS,CONIFERS)broadcast drencher ECFoliar, stumptreatmentdirect spray[3.6]2.4 lb a.i./100galEC3.6 NA NS NA 7Application rateis provided as adilution factor.FRUITS &NUTSNon-bearing (notto bear fruitwithin 1 year)fruit trees innurseries(includes:almonds, citrus,filbert, apple,cherry, nectarine,peach, pear, plum,prune).Foliar-NonbearingnurserybroadcastHigh/lowvolume spay/hand heldsprayer/powersprayer4.0EC4.0 NA NS NA 14 NS 7For nectarinesand peaches,the use isrestricted to oneapplication ofno more than 3lb a.i./A per cc.For apples, themax rate is 2 lba.i./A per cropcycle and theuse is restrictedto 1 application(either canopyor trunk drench)per year.Example label,62719-254Foliar-Nonbearingnursery trunkdrenchdrencher, highand lowpressure sprayer2.0WDG2.0 NA NS114 7Total4.06.0MaximumChlorpyrifos Human Health Risk Assessment D424485Page423of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2Single Rates:3.0 (nectarinesand peaches)2.0 (apples)MaximumYearly Rates:3.0 (nectarinesand peaches)2.0 (apples)GINSENG(MEDCINAL)Preplant,postemergenceGround, soilbroadcast2.0G2.0 NA 1 NA 365 24 NAPermitted inMichiganandWisconsinMI110006,WI110003)Minimumincorporation: 4inchesApplicationshould befollowed byrainfall oroverheadwatering.Valid until June29, 2016.GOLF COURSETURFWhenneeded, soilbroadcast/spottreatmentGround, lowpressure1.0EC2.0 NA 2 NA24NSFoliar,broadcast,Ground boom,handgun, low1.0EC, G, B2.0 NA 2 NA NSChemigationnot allowed forChlorpyrifos Human Health Risk Assessment D424485Page424of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2pressure andbackpackthe ECformulation.Tractor drawnspreader, pushtype spreader,belly grinder1.0G[24]NS7MoundtreatmentGranuleapplicator1.0G2.0 NS 2 NS NS 7Total2.02.0NA2NANSNSGRAPESDormant/DelayedDormant(pre-bloom)Ground boom,broadcast,drenchhigh/low sprayvolume1.0WDG, EC1.0 1 1 NA 3524NSEast of thecontinentaldivide only.Do not use inconjunctionwith soilsurfaceapplications forgrape borercontrol.2.0EC2.0 1 1 NA 35Permitted inColorado,Idaho, andWashingtonCO080008,ID090004,WA090002Master label:62719-591FoliarGround/broadcast, basalspray anddrench (soiltreatment)2.25EC2.25 1 1 NA 35 NSPermittedeast of thecontinentaldivide.1.0EC3.0 3 3 NA 35 NS California CA080010Chlorpyrifos Human Health Risk Assessment D424485Page425of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2Postharvest,dormant/delayeddormantGround boom,broadcast2.0EC2.0 1 1 NA NS NS California CA080009Total2.252.2513524NSPermittedeast of thecontinentaldivide.2.05.04NSNSCaliforniaGRASSFORAGE/FODDER/HAYFoliar,broadcastAircraft, groundboom,chemigation1.0EC3.0 NA 3 NA NS 24Permitted inNevada,Oregon,Washington,and IdahoNV080004,NV940002,OR090009,WA090010,ID090003GREENHOUSEearlyevening,aerosol, fogor fumigationTotal releasefogger0.0290.0066 lba.i./1000 sq. ftPLNS NA NS NA NS NS 2HOUSEHOLD/DOMESTICDWELLINGSINDOORPREMISESWhen needed Bait station0.0003 lb/baitstationNS NA NS NA NA NS NS 9688-67HYBRIDCOTTONWOOD/ POPLARPLANTATIONSFoliar,dormant,delayeddormant;broadcastHigh volume(dilute)Low volume(concentrate)1.9EC[2.0]NS6.0[1]NS3 24 7 WashingtonWA090004Energy woodplantations maybe harvested asoften as every2-3 years;Chlorpyrifos Human Health Risk Assessment D424485Page426of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2pulpwood 5-10years; and sawtimber 15-20years.(Arkansasproductionguide). InWashington thecrop takes 2-8yearsLEGUMEVEGETABLESPreplant, soiltreatmentGround boom1.0EC, WDG1.0 NA 1 NA NS24NANo MRIbecauseapplication onlyonce a yearAt planting,soil treatmentGround boom1.0EC, WDG1.0 NA 1 NA NS NATotal1.01.0NA1NANS24NSAssumed eithera preplant or anat planttreatment.MINT/PEPPERMINT/SPEARMINTPreplant soilincorporatedAerial orground/broadcast2.0EC, WDG[2.0]NS2.0[1]NS1 90 24 NANo use inMississippi.19713-599,33658-26,34704-857,67760-28,84229-25,84930-7,OR940027MRI NA due toonce per cropcycleapplicationChlorpyrifos Human Health Risk Assessment D424485Page427of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2Postemergence,Postharvest,FoliarChemigation,ground/ airblast2.0EC2.0 2.0[1]NS2 90 NSNo use inMississippi.Postharvestapplicationretreatment notspecified onsome labels.Total2.04.04.02.039024NSLabels allowone growingseasonapplicationincluding preplant and onepost-harvestapplication perseason.MOSQUITOCONTROL;HOUSEHOLD/DOMESTICDWELLINGSOUTDOORPREMISES;RECREATIONAL AREASWhenneeded;broadcastUltra lowvolume air andground0.01EC0.26 NA 26 NS NA NS 24 hIn Florida:Do not applyby aircraftunlessapproved bythe FloridaDept of Ag.Aerialapplicationsmay be made ataltitudesranging from75-300 ft (seelabels forspecifics).For use byfederal, state,tribal or localgovernmentofficials or bypersonscertified in theappropriateChlorpyrifos Human Health Risk Assessment D424485Page428of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2category orauthorized bythe state ortribal leadregulatoryagency.NECTARINEdormant/delayeddormantbroadcastairblast,handgun3.0WDG, EC3.0 NA 1 NA NS24/4d1083222-20 othersat 2 lb a.i./aAircraft2.0WDG, ECUpdated toreflect spraydrift mitigation.pre-plant,foliar;trunkspray/drench or preplant dipHandgun, lowpressurebackpack, dip2.5(3.0/100 gal)WDG, EC2.5 NA 1 NA 14 5There is noapplicationretreatmentintervalspecified onsome of thelabel. Theapplication rateis provided as adilution factor.Total3.05.5NA2NASome labelslimit theamount a.i./Aper year.Multiple typesof applicationscan occur suchas preplant,trunk drenchChlorpyrifos Human Health Risk Assessment D424485Page429of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2and dormant,delayeddormantapplications.Excludesnurseryapplications(See general“Fruits” listing)NONAGRICULTURALOUTDOORBUILDINGS/STRUCTURESto and aroundoutside surfacesof nonresidentialbuildings andstructures.Permitted areas ofuse includefences, preconstructionfoundations,refuse dumps,outside of walls,and other areaswhere pestscongregate orhave been seenOutdoorgeneralsurface/ Band(may bebetter ifcalledperimeter)Ground sprayer/band sprayer1.0ECNS NA NS NA NA NS NSChlorpyrifos Human Health Risk Assessment D424485Page430of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2NURSERYSTOCK: :Ornamentalnursery stockannuals,perennials andwoody plantsbeing grown inthe field, in balland burlap or incontainersoutdoor and ingreenhousesDormant/DelayedDormanthigh spray3.0EC3.0 NA 1 NA 24 NSPreplantGround boom,soilincorporated4.0EC, WPNS NA NS NAfoliar, soildirectedTractor drawnspreader, pushtype spreader,belly grinder,gravity fedbackpack,spoon1.1GTotal4.0CBD3ONIONSPost plant(seeding)BroadcastGround boom1.0EC1.0NS2NS 60 24 NSAt plant, soildrench orbasal sprayGround boom1.0EC, WDG, G1.0 12 inchincorporationChlorpyrifos Human Health Risk Assessment D424485Page431of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2Total2.02.026024NSORNAMENTALAND/ORSHADE TREES,HERBACEOUSPLANTSFoliarbroadcastGround boom,air blast,handgun, lowand highpressure handwands2.0EC, WP1.0G, B2.0 NA[2]NSNA NS24NSSome labelsinclude a MRIof 7 days.Dormant/DelayedDormantHandgun, lowpressure andbackpack3.0EC3.0 NA 1 NA NS 7Low volumespray permittedfor concentratedsolutions andlower rates.ORNAMENTALLAWNS ANDTURF, SODFARMS (TURF)Whenneeded,broadcast,soil or spottreatmentground boom(WP only), highpressure handwand3.76EC, WP7.52 NA 2 NA NS 24 NSNSTractor drawnspreader, pushtype spreader,belly grinder1.0B2.0 NA 2 NA NS 24 NSBait is used forfire ant control.ORNAMENTALNONFLOWERINGPLANTSFoliar,broadcast,soil drenchChemigation,ground boom,low and highpressurehandwand,handgun,backpacksprayer,sprinkling can0.007/galMENS NA 12 NA NA 24 NSApplication rateprovided as adilution factor.Restricteduse—occupationalonlyChlorpyrifos Human Health Risk Assessment D424485Page432of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2ORNAMENTALWOODYSHRUBS ANDVINESFoliarbroadcastGround boom,air blast,handgun, lowand highpressuresprayer,backpack2.0EC, WDG0.01 lb/galEC2.00.01lb/galNA[1]NSNA NS 24 NSSeveral labelsdo not restrictthe applicationrate in lb a.i./A.Examplesinclude 16.5lb/100 gal (228-625) and 1.0lb/100 gal (829-280).Dormant/delayeddormant1.0EC0.005 lb/galEC1.0 NA[1]NSNAPreharvestTractor drawnspreader, pushtype spreader,belly grinder6.0G6.0 NA[1]NSNAPreplant,potted,bailed-andburlapped,containerizedgroundboom,handgun, lowand highpressuresprayer,backpack,drench1.0ECNS 1 NS 1Pretransplant groundboom4.0WP[48.0]NS4 12 4Total6.0G4.0CBDCBDChlorpyrifos Human Health Risk Assessment D424485Page433of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2WPPEACHdormant/delayeddormantbroadcastairblast3.0EC2.0WDG3.0 NA 1 NA 1024/4dNS83222-20 (allother labelsrestrict to 2 lbai/a)aircraft,2.0EC2.0WDGNSUpdated toreflect spraydrift mitigation.Post-harvestbroadcastairblast2.5(3.0/100 gal)EC2.5NA 1 NA NA NSPermitted inGeorgia andSouthCarolinaGA0400001,SC040001SLN Expires:aircraft2.0(3.0/100 gal)EC2.0GA0400001,SC040001SLN Expires:Updated toreflect spraydrift mitigationpre-plant,foliar;trunkspray/drench or preplant dip;groundhandheld,backpack,drench/dip,handgun, andlow pressurehand wand2.5(3.0/100 gal)WDG2.5 NA 1 NA 14 5 NSSome labels donot specifyminimumretreatmentinterval.Total3.05.5NA3NANA24NSIt is possiblethat multipletypes ofapplications canoccur such as3.08.0NA3NANA24NSPermitted inGeorgia andSouthCarolinaChlorpyrifos Human Health Risk Assessment D424485Page434of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2soil, foliarand/or postharvest anddormant/delayeddormantapplications.Excludesnurseryapplications(See general“Fruits” listing)PEANUTPreplantAerial orground/broadcast2.0EC, WDG[4.0]NS4.0[2]NS2 NA 24 10Do not applyaerial inMississippiAssumes onecrop cycle peryear.At plant,postplant4.0G[4.0]NS4.0 2 2 21 24 10At pegging2.0GEC, WDG[4.0]NS4.0 2[2]NS21 24 10Total4.0G2.0EC, WDG4.04.022102410PEARdormant/delayeddormantbroadcastaircraft, airblast2.0WDG, EC2.0 NA 1 NA NA 24 NARestricteduse inCalifornia.83222-20allows 3.0 lba.i./ A;however, thisdoes not matchthe 2001 RED.Chlorpyrifos Human Health Risk Assessment D424485Page435of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2Post-harvestbroadcastaircraft, airblast2.0WDG, EC2.0 NA 1 NA NA 24 NSPermitted inCalifornia,Oregon andWashington.Total2.0WDG, EC4.0NA2NANA24NSMultiple typesof applicationsmay occur inwithin a year inCalifornia,Oregon andWashingtonsuch as a postharvestapplication anda dormant,delayeddormant.Excludesnurseryapplications(See general“Fruits” listing)PEASPreplant SeedtreatmentSeed Treatment0.300.000625 lb/lbseedWP0.280.00058 lb/lbseedNS NS NS NS NS NS NSThere is a rangeof potentialapplicationrates dependingon the numberof seeds per lband the numberof seeds plantedChlorpyrifos Human Health Risk Assessment D424485Page436of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2ECper acre.Seedinginformationprovide byBEAD.2PECANSdormant/delayeddormantbroadcastaircraft, airblast2.0EC, WDG2.0 NA 1 NA 14241066222-19 and66222-233foliar;broadcastairblast4.3EC, WDG6.3 NA 3 NA 14 10Some labelsrequire a 28 dPHIaircraft2.0EC, WDGUpdated toreflect spraydrift mitigation.foliar;orchardfloorsbroadcastGround boom,chemigation4.3EC, WDG4.3 NA 2 NA 14 10Total4.312.6NA6NA142410Considersmultiple type ofapplications(e.g., dormant,foliar broadcast,and orchardfloor) butexcludingnurseryFor nurseryapplicationsChlorpyrifos Human Health Risk Assessment D424485Page437of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2(See general“Fruits” listing)PEPPERFoliarGroundbroadcast1.0WDG[8]NS8.0[8]NS8 7 24 10Permitted inFloridaFL040005; 1crop cycle peryear.PINEAPPLEPostplantGround boom,broadcast2.0EC6.0 6.0 3 NA 365 24 30Permitted inHawaiiHI090001SNL Expires:March 29,2014.Do not makeapplicationsbeyond threemonths afterplanting.PLUM/PRUNEdormant/delayeddormant;broadcastAircraft,airblast2.0EC, WDG2.0 NA 1 NA NA24/4d10foliar;trunkspray/drenchhandheld,backpack,drench/dip,handgun, andlow pressurehand wand2.53.0/100 galWDG2.5 NA 1 NA NA 10Total2.54.5NA2NAExcludesnurseryapplications(See general“Fruits” listing)POULTRYWhen Sprayer 0.07126 NS NA NS NA NA NS 53883-264,Chlorpyrifos Human Health Risk Assessment D424485Page438of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2LITTERneeded,animalbedding/littertreatment.a.i./1000 sq ft3.1ME84575-3PUMPKINPreplant SeedtreatmentSeed treatment0.30.00058 lb /lbseedWP[0.3]NS[1]NS[1]NS1 NS NS NSCaliforniamaximumsingle rate0.000625 lba.i./lb.There is a rangeof potentialapplicationrates dependingon the numberof seeds per lband the numberof seeds plantedper acre.Seedinginformationprovide byBEAD.4RADISHFoliarBroadcastground1.0ECNS 1 NS 1 NS 24 NSpermitted inOregonOR090012 onradish grownfor seed.Label validuntil December31, 2012. (perregistrant SLNstill valid)PreplantSoilincorporationground3.0EC12.0 3 4 1 NS NS 10At plant/postplantIn furrowdrench/3.0EC[15.0]NS3[5]NS130,EC,24 10Only onegranularChlorpyrifos Human Health Risk Assessment D424485Page439of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2treatment 2.8G7,Gapplicationpermitted.Total3.0[22.0]NS2[9]NSOnly onepreplant or atplantapplication isassumed.RIGHTS OFWAY, ROADMEDIANSWhenneeded, soilbroadcastGranular or lowpressure wand1.0EC, G, Bait[2.0]NSNA 2 NA NA NS 7Apply whenneededRUTABAGAPreplantChemigation,Groundboom2.4EC, WDG[4.8]NS2.4[2]NS1 30 24 10Aerial2.0EC, WDG2.0Updated toreflect spraydrift mitigation.At plant/postplantIn furrowdrench/treatment2.4EC, G WDG4.8 2.4[2]NS1 7 24 10Disallowedin Californiaand Arizona.Two cropcycles per yearTotal2.4[9.6]NS4.8[4]NS22410SEWERMANHOLECOVERS ANDWALLSWhen needed Low pressure0.31lb/manholeRTUNS NA NS NA NA NA NS3 pints product/manholeSEEDORCHARDTREESfoliar;broadcastGround boom1.0EC3.0 3.0 NS NA 30 24 762719-575,62719-615High volume 2.5 2.5 NS [1] NA 30 24 7 Cone wormChlorpyrifos Human Health Risk Assessment D424485Page440of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2sprayer 0.01a.i./tree0.02 ECNS treatment(62719-575 and62719-615)Treatment of1000 trees peracre wouldresults in ansingleapplication rateof 10 lb a.i./a.DAS: 1000 is abit high,typically fororchards 312trees per acrefoliar;stumptreatmentbackpack,drencher, lowpressure handwand,0.3EC0.3 1.0 NS NA 30 24 762719-575,62719-615Total1.05.83NSNA30247The totalnumber ofapplicationsassumed iseither threefoliarapplications ortwo foliarapplicationswith one stumptreatment.Chlorpyrifos Human Health Risk Assessment D424485Page441of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2SORGHUMGRAINSeedTreatmentSeed treatment[0.0009]0.01-0.0024 lb ai/100 lbs seedEC0.01 0.01[1]NS1 NA NS NS264-932Preplant SoilDirectedGroundSpreader/TBand1.5G1.5 1.5[1]NS1 60 24 10Foliar/PostemergentGround, Aerial,Chemigation1.0EC, WDG1.5[1.5]NS[1]NS3 30 24 10PHI variesacross labelsTotal3.3G1.0EC, WDG3.013.01[3]CBD3302410One crop cycleper year.SOYBEANfoliar , postemergencesoil broadcastbroadcastground, aerial,chemigation1.0EC, WDG3.0 3.0 3 3 28 24 14One crop cycleper year.At plant/postplanttreatment;soil bandground boom2.2G1.0 EC3.0 3.01 (G),3 (EC)1 (G),3(EC)28 24 10Total1.0EC, WDG2.2G3.03.033One crop cycleper year.STRAWBERRIESPre-plantAerial orground/broadcast2.0EC2.0NS1 NS NA 24 10No use inMississippi33658-26Chlorpyrifos Human Health Risk Assessment D424485Page442of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2FoliarAerial orground/broadcast, foliarspray1.0EC, WDG2.0NS2NS212410Twoapplications (2lb ai) for allproducts per cc.Post harvestGround directedspray1.0EC, WDG2.0NS2NS21 14Total2.04.03One preplantapplication andtwo foliarand/orpostharvestapplicationpermitted peryear.SUNFLOWERAt plantAerial/ground2.0G3.0 3.0[1]NS1 422410Per registrant 1cc per yearPreplant2.0EC, WDG3.0 3.0[1]NS1 42 102 inches minincorporationPostemergent orfoliar1.5EC, WDG3.0 3.0[2]NS2 42 10Total2.05.05.033Assumed eitheran at plant orpreplantapplicationfollowed withtwo foliarapplications.Chlorpyrifos Human Health Risk Assessment D424485Page443of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2One crop cycleper yearSWEETPOTATOPreplant, soilbroadcastAircraft, groundboom2.1G, EC, WDG2.1 NS 1 1 125 24LA090002,MS080007,NC090001permits 60PHIAircraft2.0G, EC, WDGUpdated toreflect spraydrift mitigation.TOBACCOPreplantAircraft, groundboom2.0EC, G, WDG2.0 NS 1 1 7 24 NATRITICALEStorageCommercialSlurry SeedTreatmentSeed treatment0.0030.0024 lb ai/100 lbs seedEC[0.003]NS[1]NS[1]NS[1]NSNA[10]NS[10]NS264-932Seedinginformationprovide byBEAD.4One crop cycleper year.TURNIPPreplantsoilincorporation/ground boom,handgun2.3G, WDG[4.6]NS2.3[2]NS1 30 24 10Minimumincorporation:2 inches.PostplantSoilincorporation/ground boom,handgun2.3G, WDGP[4.6]NS2.3[2]NS1 30 24 10Minimumincorporation:2 inches.Total2.34.62.321302410Assumed eithera preplant orpostplantapplication.Two cropcycles per yearChlorpyrifos Human Health Risk Assessment D424485Page444of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2UTILITIESFor use in andaroundtelecommunications, power, utilitiesand railroadsystemsequipment:Buried cables,cable televisionpedestals, cables,pad-mountedelectric powertransformers,telephone cables,undergroundvaults,telecommunications equipment,power and utilitiesequipmentWhenneeded,broadcastProductcontainer190.5G0.44 lba.i./100sq ft(seecomments)NS NS NS NS NS NS NSApplicationspermitted asneeded. Reg.Nos. 13283-14,13283-17Broadcastproduct onto thegroundcovering thearea of the padlocation, plus atwo footperimeteraround theoutside of thepad location.WALNUTSdormant/delayeddormant;broadcastAircraft,airblast2.0EC, WDG2.0 NA 1 NA 14241062719-301 (12lb a.i./A)foliar;broadcastaircraft,airblast,chemigation2.0EC, WDG4.0 NA 2 NA 14 10Some labels donot specifyretreatmentinterval.foliar; Ground boom, 4.0 4.0 NA 1 NA 14 10Chlorpyrifos Human Health Risk Assessment D424485Page445of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2orchardfloorsbroadcastchemigation EC, WDGTotal4.010.04Excludingnurseryapplications;includesdormant, foliarbroadcast, andorchard floor.For nurseryapplications(See general“Fruits” listing)WIDE AREA/GENERALOUTDOORTREATMENTFor ants and othermisc. pests.when needed,BroadcastGround sprayer0.5084 lbai/100 galEC[1.02]NSNA 2 NA NANSNS 66222-19when needed,DrenchDrench1 NS NA NS NA NA NS 228-624[1]8.2 lb a.i/100gal ECNS NA NS NA NA NS 228-625Total[1]NSNANSNANAWHEATSlurry SeedTreatmentSeed treatment0.0030.0024 lb ai/100 lbs seedEC[0.006]NS1[2]NS1 NA NA NAOnly for usein AZ, CA,CO, ID, KS,MN, MO,NE, NM,NV, ND,OK, OR,Seedinginformationprovide byBEAD.4Foliar, soiltreatmentGround,broadcast0.5EC[8.0]NS4.0[2]NS114/2824 14PHI: 14 forageor hay, 28 grainor strawChlorpyrifos Human Health Risk Assessment D424485Page446of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC2PostemergencefoliarGround, Aerial,Chemigation1.0EC[4.0]NS2.0[4]NS214/28NSSD, TX, UT,WA and WYLabel states 1.0lb ai/A forcereal leafbeetles and thenstate max rate0.5 lb ai/A inrestriction).Some labelsrestrict no morethan 2applications percrop/seasonPHI 14 forageor hay, 28 grainor strawTotal[1]4.0EC[12.006][6.003]5.0[8]NS[4]2MO otherwise2.0 plus seedtreatmentWOODPROTECTIONTREATMENTTOBUILDINGS/PRODUCTSOUTDOORWhenneeded,WoodsurfacetreatmentLow pressurehandwand,backbacksprayer,paintbrush16.65lb/10,000 sq ft0.17 lb a.i./galECNS NA NS NA NS NS NS0.08 lb ai/galEC, RTU EC,MENS NA NS NA NS NS NSApply 1 gal per100 sq ft ofwoodChlorpyrifos Human Health Risk Assessment D424485Page447of531Table 9.1. Summary of Current Chlorpyrifos UsageCrop/Site Residential Agricultural ForestryTiming;ApplicationTypeMethod/EquipmentMaximumSingleApplicationRate byFormulation1(lb a.i./A)MaximumApplication RateMaximumApplicationNumber PHI (days)

3 REI (hours)

3 MRI (days)

3GeographicRestrictionsCommentsPerYearlba.i./APerCC2lb a.i./APerYearPerCC21.EC - emulsifiable concentrate; WDG – waterdispersible granular in water soluble packet; WP – wettable power in water soluble packet; B – bait (granular), G – granular; ME –microencapsulated; RTU – ready to use.2.Reported as per crop cycle or per season3.PHI – Preharvest interval; REI – reentry interval; MRI – Minimum retreatment interval4.Becker, J.; Ratnayake, S. Acres Planted per Day and Seeding Rates of Crops Grown in the United States, U.S. EPA OPP/BEAD, 2011;example calculations provided below:Beans: 0.00058 lb a.i./lb seed / 960 seeds/lb seedx 418,176 seeds/A [pgs. 19, 81 (beans, succulent)]Corn: 0.000625 lb a.i./lb seed / 1,800 seeds/lb seed x 59,739 seeds/A [pgs. 24, 81 (corn, sweet)]Cotton: 0.00116 lb a.i./lb seed / 4,500 seeds/lb seed x 85,00 seeds/A [pgs. 13, 81]Cucumber: 0.00058 lb a.i./lb seed / 12,000 seeds/lb seed x 80,418seeds/A [pgs. 25, 81]Peas: 0.000625 lb a.i./lb seed / 1,361 seeds/lb seed x 653,400 seeds/A [pgs. 34, 82]Pumpkin: 0.00058 lb a.i./lb seed / 1,600 seeds/lb seed x 7,260 seeds/A [pgs. 37, 82]Sorghum: 0.001 lb a.i./lb seed / 11,000 seeds/lb seed x 100,000 seeds/A [pgs. 16, 39]Triticale: 0.003 lb a.i./100 lb seed / 109 lb seed/A [pg.16]Wheat: 0.003 lb a.i./100 lb seed /116 lb seed/A [pg. 16][ ] indicate assumptions that are made when the information is not specified but can be inferred

# Chlorpyrifos Human Health Risk Assessment D424485

Page 448 of 531 

# Appendix 10. Dose Reconstruction Analysis

Introduction

In order to better understand the possible exposure patterns in the Columbia University study

cohort and their influence on the health outcomes identified in the research, an analysis was

conducted to supplement the exposure metrics repor ted by the Columbia investigators. The goal

was to predict potential exposures which coul d have occurred to pregnant women and young

children who may have been exposed to chlorpyr ifos through other path ways not addressed in

the research then determine their potential for associated cholinesterase inhibition.

Chlorpyrifos, at the time, was one of the most wide ly used insecticides fo r indoor pest control so

there are many possible ways which exposures coul d have occurred as a result of these types of

uses (e.g., use of a total release fogger, aerosol can sprays, or crack and crevice treatment by a

pest control operator). This anal ysis was based on the Agency’s SOPs for Residential Exposure

Assessment 76 

# which describe, on a scenario basis, specifi c algorithms and data used to predict the

potential exposures which could have occurred to individuals in the coho rt. Once complete, the

resulting dose estimates were used to evaluate the potential level of c holinesterase inhibition

which could be attributed to them using th e PBPK-PD model. The scenarios which were

considered include:

 Pregnant mothers who may have purchased a consumer aerosol can product and applied

it in their homes;

 Exposures to pregnant mothers who may have had contact with re sidues in their homes

after a previous treatment which leads to exposure; and

 Exposures to young children (aged 1 to 2 years old) who may have contact with residues

in their homes after a previous tr eatment which leads to exposure.

This appendix describes the details of those calculations.

Use Profile

In order to evaluate the chlorpyrifos exposur e potential for the Colu mbia study cohort, it was

first necessary to define what chlorpyrifos pr oducts would have been inuse during the time of

study conduct which spanned from 1998 to 2004. In 1997, the registrant, Dow AgroSciences,

voluntarily agreed to cancel chlo rpyrifos registrations for indoor broadcast use and direct pet

treatments, except pet collars. In December 2001, the majority of the remaining chlorpyrifos

residential products -- except for fu lly contained ant baits in child resistant packaging and limited

public health uses -- were subject to volunt ary phase out/cancellation. This analysis

reconstructed the potential expos ures for those in the cohort between the years 1997-2001 based

on the chlorpyrifos uses that remained after the voluntary can cellation in 1997 and the phase out

in 2001. The exposure questionnaire-based repor ted use from the Columbia publications was

also used to inform this process. Per the publ ications, pest control m easures were used by 92%

of respondents, whether by housing superinte ndent, pest control ope rator (PCO), or by

themselves. Of these, respondents reported that 39% were by PCO, 26% by spray can, and 5%

foggers/bombs. The majority reported regular use of a pesticide product, at least once per

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Page 449 of 531 month. Unfortunately, while these data help info rm the types of products used, they do not allow

for the determination of what pesticide active ingredient(s) were used. Multiple active

ingredients were in regular us e during this time period incl uding, chlorpyrifos, diazinon, and

propoxur, among others. For the purpose of the dose reconstruction exercise, it was assumed

that only chlorpyrifos applic ations occurred. Considering al l the available information, the

potential for chlorpyrifos exposures from use of the following chlorpyrifos product types were

assessed: broadcast ready to use (RTU) spra ys applied by individua l themselves and not

professional applicators; surface sp rays applied by individuals; cr ack and crevice sprays applied

by individuals themselves or professional pest co ntrol applicators; and foggers/bombs applied by

individuals. These chlorpyrif os residential products containe d either 0.5% or 1% active

ingredient. The latter was assumed to ensure an assessment reflective of the highest potential

exposures, or those most likely to result in the greatest % RBC AChE inhibition.

Exposure Assessment Methods

With the products and application rates defined, the 2012 Standard Operating Procedures for

Residential Pesticid e Exposure Assessment 77 

# were used to assess the exposures that may have

occurred from these indoor re sidential chlorpyrifos uses. Based on the use patterns of the

defined products, it is expected that exposures occurred either from the applic ation (handling) of

products by those in the cohort or af ter applications from being in contact with previously treated

indoor environments.

A series of assumptions and exposure factors serve as the basis for completing residential

handler and post-applica tion exposure assessment:

 maximum application rates allowed by labels in its risk assessments are assumed;

 the use of personal protective equipment is not considered in residential risk

assessments;

 residential assessments are based on the assu mption that individuals are wearing shorts,

short-sleeved shirts, socks, and shoes;

 adults are assumed to perform all pesticide applications;

 the mean female bodyweight reflective of all trimesters of pregnancy, 75 kg, was

assumed for pregnant women to reflect the population of interest from the Columbia

cohort being evaluated which was derive d from the EPA Expo sure Factors Handbook

2011 Edition 78 

# (adult and adult female: Tables 8-3 through 8-5; body weight of pregnant

women: Table 8-29).

 the most protective index lifestage whic h serves as the basis for evaluating young

children’s indoor exposures are children 1 to < 2 years old as descri bed in in the 2012

Residential SOPs;

 the body weight used to evaluate exposures to children 1 to < 2 year s old is 11 kilograms.

For residential handler exposure assessment, the following assumptions and exposure factors

apply:

 a broadcast application of an aerosol can is c onsidered and the entire contents of the can

(16 oz.) are applied at once, half the contents of the spra y can are applied for crack and

crevice applications, and a quarter of the can contents for a space spray application.

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# Chlorpyrifos Human Health Risk Assessment D424485

Page 450 of 531 For residential post-application exposure assessm ent, the following assumptions and exposure

factors apply:

 inhalation exposures are exp ected to occur up to 2 hours following application by which

time the aerosolized particulate is assumed to have settled an d, as a result, after that time

frame exposures are assumed to be negligible;

Use of the PBPK-PD Model

Once complete, the PBPK-PD model was used to es timate the potential level of cholinesterase

inhibition (% RBC AChE) at tributable to the daily dose estim ates that result in the highest

exposure potential. In order to model the pote ntial health outcomes in a manner reflective of

these exposures, a series of input s were used that correspond to those used to quantify the daily

dose estimates. The following exposure character istics were used: number of days exposed;

body weights which vary by lifestage; daily expos ure duration (hours per day, days per week);

and the fraction of the dermal surface area exposed.

The dose reconstruction exercise was modeled for a subsequent 14 day post-application duration.

The daily dose estimated with the 2012 Standard Operating Procedures for Residential Pesticide

Exposure Assessment for the day of application wa s inputted into the PBPK-PD model. Dermal

exposure was modeled assuming that chlorpyrifos re sidues dissipated at a rate of 10% per day.

A 14 day period was used as the basis of th e PBPK-PD modeling to ensure it adequately

addressed the time it takes for the ACHE enzy me inhibition to reach a steady state.

For the adult and children 1 to < 2 years old age groups, the appropria te body weights were

obtained for modeling purposes from the Exposure Factors Handbook 79 

# (USEPA, 2011). The

mean adult female bodyweight reflective of all trimesters of pregnancy, 75 kg, was assumed for

pregnant women to reflect the population of inte rest from the Columbia cohort being evaluated

(Exposure Factors Handbook: adult and adult fema le: Tables 8-3 throug h 8-5; body weight of

pregnant women: Table 8-29). For children 1 to < 2 years old, the body weight was set to 11 kg

(Exposure Factors Handbook, Table 8-3, mean body weight for the 1 to < 2 year old age group).

For the evaluation of adult hand ler exposures (dermal and inhalation) it was assumed that the

application event continued for 1 hour daily and that it occurred each day of the 14 day exposure

period. The 2012 Residential SOPs makes no recommendation regarding the daily exposure

duration for handlers. It was a ssumed that 1 hour is a high end estimate of the time required to

make an aerosol can application. For the evaluation ofadults and children 1 < 2 years old postapplication exposures (i.e., adults: dermal; child ren: dermal and incidental oral), the daily

exposure duration was assumed to be 2 hours daily and that it occurs each day in the 14 day

exposure period. This exposure duration is c onsistent with that recommended in the 2012

Residential SOPs for exposures on hard surfaces. For adults and children 1 to < 2 years old

inhalation exposures, it was a ssumed that the exposure duration was 2 hours on the day of

application (Day 0) and was not repeated becaus e the exposure source (i.e., aerosols from an

application event) occurred only once on the first day of the exposur e period. This is consistent

with the available monitoring data and consistent with the 2012 Residential SOPs, the Day 0 air

monitoring measures were followed by a marked dr op to negligible levels by the following day.

For all dose reconstruction dermal exposures to chlorpyrifos, the fr action of skin in contact with

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Page 451 of 531 chlorpyrifos was 50% which reflected uncovered skin areas for children wearing shorts and a tee

shirt. No shower (i.e., washing off the chlo rpyrifos) was assumed to occur for the 14 day

exposure duration modeled.

Residential Handler Exposure Scenarios

The residential handling of chlorpyrifos resident ial products are expected to have occurred for

some percentage of the Columbia cohort populati on, these are anticipated to have resulted in

dermal and inhalation exposures. Dermal and inhalation residential handler exposures are

predicted by use of a factor know n as the unit exposure. Unit exposure is the ratio, for a given

formulation and application equipment, of an individual’s exposure the amount of active

ingredient handled (AaiH), expressed as ma ss active ingredient e xposure per mass active

ingredient handled (e.g., mg/lb ai). For the dos e reconstruction assessment, unit exposure data

were used as recommended in the 2012 Residen tial SOPs for ready-to-use (RTU) aerosol cans

(dermal, 370 mg/ lb ai; inhalation, 3.0 mg/lb ai). Residential handl er exposures from application

of fogger formulations were not assessed which is consistent with the 2012 Residential SOPs.

Fogger product labels typically di rect the user to activate the de vice and evacuate the treated area

and not to re-enter for several hours. Therefore, it is assume d that handler exposure from fogger

application is not expected.

The exposure assessment for chlorpyrifos Columb ia cohort handler exposures is based on the

following exposure scenarios:

 Adult dermal exposure from broadcas t application of a RTU spray can;

 Adult inhalation exposure from broadcas t application of a RTU spray can;

 Adult dermal exposure from crack and cr evice application of a RTU spray can;

 Adult inhalation exposure from crack and crevice applica tion of a RTU spray can;

 Adult dermal exposure from space spray application of a RTU spray can;

 Adult inhalation exposure from space sp ray application of a RTU spray can.

Handler Exposure Algorithm

Daily dermal and inhalation exposure (mg/day) fo r residential pesticide handlers, for a given

formulation-application method combination, is estimated by multiplying the formulationapplication method-specific unit exposure by an estimate of the amount of active ingredient

handled in a day, usi ng the equation below:

E = UE * AR * A

where:

E = exposure (mg/day);

UE = unit exposure (dermal, 370 mg/ lb ai; inhalation, 3.0 mb/lb ai);

AR = application rate (e.g., lb ai/can); and

A = area treated or amount ha ndled (broadcast, 1 can/day; cr ack and crevice, 0.5 can/day;

space spray, 0.25 can/day).

Residential Handler Exposure/Cholin esterase Inhibition Summary

# Chlorpyrifos Human Health Risk Assessment D424485

Page 452 of 531 Worst-case results of this analysis are pres ented below representing pregnant women who

potentially made a broadcast ap plication of an aerosol spray can (Table 10.1). Peak % RBC

AChE inhibition estimated for pregnant women from combined dermal and inhalation exposures

occurring from product app lication is 0.0012%.

Table 10.1. Residential Handler (Pregnant Women in the Columbia Cohort) Estimated Exposures and

Predicted % ChE Inhibition (Route-specific and Combined)

Exposure

Scenario Formulation Amount

Handled Dermal Dose

(mg/kg/day) Dermal:

Peak

% ChE

Inhibition Inhalation

Dose

(mg/kg/day) Inhalation:

Peak

% ChE

Inhibition Combined

Peak

% ChE

Inhibition

Broadcast Ready to Use:

1 % Aerosol

Spray Can 1 can 0.049 0.0007% 0.030 0.0008% 0.0012%

a. Dermal Exposure = Dermal Unit Exposur e (370 mg/lb ai) × Application Rate (0 .010 lb ai/can) × Area Treated or Amount

Handled (cans/day)

b. Inhalation Exposure = Inhalation Unit Exposur e (3.0 mg/lb ai) × Applicat ion Rate (0.010 lb ai/can ) × Area Treated or Amount

Handled (cans/day)



# Residential Post-application Exposure Scenarios

The use of indoor residential ch lorpyrifos products are anticipa ted to have resulted in the

potential for post-application exposures from public housing residents in the Columbia study

cohort being in a previously treated environmen t. Based on the product use profile, residents

may have been exposed via inhalation to the aero sols resulting from app lication or by means of

contact following settling of produc t contents following settling on i ndoor surfaces or flooring.

The exposure assessment for chlorpyrifos Columb ia cohort post-application exposures is based

on the following exposure scenarios:

 Adult dermal contact with residues deposit ed on flooring (carpeted and hard) from

broadcast application of RTU spray can;

 Adult inhalation of aerosolized particulates following broadc ast application of RTU spray

can;

 Adult dermal contact with residues deposited on flooring (carpeted and hard) from space

spray application of RTU spray can;

 Adult inhalation of aerosoli zed particulates following space spray application of RTU

spray can;

 Adult dermal contact with residues deposited on flooring (carpeted and hard) from crack

and crevice RTU spray can or PCO application;

 Adult inhalation of aerosolized particulates following crack and crevice application of

RTU spray can;

 Children 1 to < 2 years old dermal contact with residues deposited on flooring (carpeted

and hard) from broadcast application of RTU spray can;

 Children 1 to < 2 years old incidental or al (hand-to-mouth) ingestion of residues

deposited on flooring (carpete d and hard) from broadcast a pplication of RTU spray can

that have transferred to children’s hands;

 Children 1 to < 2 years old incidental oral (object-to-mouth) ingestion of residues

deposited on toys in contact with floo ring (carpeted and hard ) treated by broadcast

application of RTU spray can;

 Children 1 to < 2 years old inhalation of aerosolized particulate following broadcast

# Chlorpyrifos Human Health Risk Assessment D424485

Page 453 of 531 application of RTU spray can;

 Children 1 to < 2 years old dermal contact with residues deposited on flooring (carpeted

and hard) from space spray application of RTU spray can;

 Children 1 to < 2 years old incidental or al (hand-to-mouth) ingestion of residues

deposited on flooring (carpeted and hard) fr om space spray application of RTU spray can

that have transferred to children’s hands;

 Children 1 to < 2 years old incidental oral (object-to-mouth) ingestion of residues

deposited on toys in contact with floo ring (carpeted and hard) treated by space spray

application of RTU spray can;

 Children 1 to < 2 years old inhalation of aerosolized particulate following space spray

application of RTU spray can;

 Children 1 to < 2 years old dermal contact with residues deposited on flooring (carpeted

and hard) from crack and crevice RT U spray can or PCO applications;

 Children 1 to < 2 years old incidental or al (hand-to-mouth) ingestion of residues

deposited on flooring (carpete d and hard) from crack and crevice RTU spray can or PCO

applications that have transferred to children’s hands;

 Children 1 to < 2 years old incidental oral (object-to-mouth) ingestion of residues

deposited on toys in contact with floo ring (carpeted and hard ) treated by crack and

crevice RTU spray can or PCO applications;

Since all exposure routes (dermal, inhalation, an d incidental oral) shar e a common toxicological

endpoint, RBC AChE inhibition, risk estimates sh ould been combined. The incidental oral

scenarios (i.e., hand-to-mouth and object-to-mouth) should be consid ered inter-related and it is

likely that they occur interspersed amongst each other across time. Combining these scenarios

with the dermal exposure scenario would be unrea listic because of the conservative nature of

each individual assessment. Therefore, the post-application exposure scenarios that were

combined for children 1 < 2 years old are th e dermal and hand-to-mouth scenarios. This

combination should be considered a protective estimate of children’s exposure to pesticides used

indoors.

The lifestages selected for each pos t-application scenario are based on an analysis provided as an

Appendix in the 2012 Residential SOPs. While not the only lifestage potentially exposed for

these post-application scenarios, the lifestage th at is included in the quantitative assessment is

health protective for the exposures and risk esti mates for any other poten tially exposed lifestage.

Post-application Exposure Data

The 2012 Residential SOPs relied on exposure data from Agency submitted studies in order to

establish many of the inputs necessary for es timation of post-application indoor exposures.

Where chemical-specific exposure data are availa ble, these data were relied upon as a more

accurate measure of exposures from chlorpyrifos and are describe d below. In the absence of

these data, exposure inputs are based on the use of available exposure data as recommended in

the 2012 Residential SOPs.

Exposure Data Used for Dermal and Incident al Oral Exposure Assessment: No chemicalspecific deposition data were available for the in door chlorpyrifos products assessed. Therefore,

as recommended by the 2012 Residential SOPs, de fault deposited residue values were used

# Chlorpyrifos Human Health Risk Assessment D424485

Page 454 of 531 based on the type of application to be made whethe r it be a broadcast, crack and crevice, or space

spay application. For broadcast applications only, residue values were influenced by the percent

spray applied (i.e., the higher the percent spray, th e higher the residue values). The higher, 1%

chlorpyrifos spray formulation was assumed for post-application assessment of broadcast

exposures. The default values used are based on an analysis of available residue deposition data

from Agency submitted studies and literature studies. A summary of the recommended values

are presented in the 201 2 Residential SOPs.

The transfer coefficent (TC) input used for calcu lation of post-application exposures is a measure

of surface-to-skin residue transfer derived from concurrent measures of exposure and surface

residue. Per the 2012 Residential SOPs, no studies were available that measure both exposure

and surface residue with subjects performed typi cal indoor activities. Therefore, the transfer

coefficients used for indoor scenarios are deri ved from information provi ded in three different

studies: 1) two studies which m easured exposure and surface resi dues while subjects performed a

Jazzercise routine (Krieger , 2000 and Selim, 2004) and 2) a study which measured

biomonitoring doses which adults performed scri pted activities for 4 hours on carpets (Vaccaro,

1991). Of these studies, the Krieger study was conducted using chlorpyrifos.

As for the fraction of residue available for tran sfer input, a complete dataset was compiled for

chlorpyrifos based on available chemical-specifi c data (Beamer et al., 2009). Therefore, the

chlorpyrifos-specific residue fract ions anticipated to transfer fo r both carpets and hard surfaces

were used for estimation of post-application de rmal and incidental oral exposures.

Exposure Data Used for Inhalation Exposure Assessmen t: In order to best represent the potential

for inhalation exposures following use of the indo or chlorpyrifos products assessed, a review of

available literature studies and registrant-s ubmitted exposure data was conducted. The following

exposure data sources were identified and used:

Indoor broadcast applications –

 Fenske, R., et al (1990). Potential Exposure and Health Risks of Infants

following Indoor Residential Pesticide Applic ations. American Journal of Public

Health. 80 (6): 689-693.

 Lu, C. and Fenske, R (1998). Air and Surface Chlorpyrifos Residues following

Residential Broadcast and Aerosol Pesticide Applications. Environmental Science

and Technology. 32 (10): 1386 -1390.

 EPA MRID: 42887201. Contardi, J. (1993). An Evaluation of the Appropriate

Drying Time via Air Monitoring, Dislodgabl e Residue Determination, and Carpet

Weight Loss, After Applying Dursban LO Insecticide to a Carpeted Surface:

Unpublished study prepared by Dow Chemical Co., Health and Environmental

Sciences. 29 pp.

Indoor crack and crev ice applications –

 Byrne, S.L., Shurdut, B.A. and Saunders, D.G. (1998). Potential Chlorpyrifos

Exposure to Residents following Standa rd Crack and Crevice Treatment. 106

(11): 725 -731.

 Hore, P. et al. (2005). Children’s Resi dential Exposures to Chlorpyrifos:

Application of CPPAES Fiel d Measurements of Chlor pyrifos and TCPy within

# Chlorpyrifos Human Health Risk Assessment D424485

Page 455 of 531 MENTOR/SHEDS – Pesticides Model. Scie nce of the Total Environment. 336 (2

-3): 525-537.

 Stout II, D.M. and Mason, M.A. (2003) . The Distribution of Chlorpyrifos

following a Crack and Crevice Type A pplication in the US EPA Indoor Air

Quality Research House. Atmospheric Environment. 37 (39 -40): 5539 -5549.

 EPA MRID 44458201: Byrne, S.; Saunde rs, D.; Cook, W. et al. (1998)

Residential Exposure to Chlorpyrifos from Reentry to Structures Treated with

Crack and Crevice and Spot Applications of Dursban Pro. Unpublished study

prepared by Dow AgroSciences. 133 pp.

Based on a review of the available data and c onsistent with the 2012 Re sidential SOPs, the day

of application (Day 0) air mon itoring measures were significan t, followed by a marked drop to

negligible levels by the following day. Therefore, HED made use only of day of application air

monitoring measures for purpose of post-app lication exposure assessment. All studies

referenced were based on 0.5% ch lorpyrifos formulated products. In order to address the

potential for use of the 1% chlorpyrifos produc t formulations available at the time, all air

monitoring measures were doubled. The broadc ast exposure data are intended for use in

assessing the potential inhalati on exposures/doses anticipated to occur from both broadcast and

space spray applications; the crack and crevice expos ure data were used only for that application

type. The resulting mean day of application air monitoring measures are presented in Table 10.2

below.

Table 10.2. Airborne Concentrations of Chlorpyrifos from Open Literature and Registrant-submitted

Studies Resulting from Indoor Broadcast and Crack and Crevice Applications

Study Chlorpryrifos Concentration (mg/m 3 

# )

Broadcast



# Fenske, R., et al (1990) 0.18

Lu, C. and Fenske, R (1998) 0.088

EPA MRID: 42887201 0.031

Average (All Studies) 0.092

Crack and Crevice



# Byrne, S.L., Shurdut, B.A. and Saunders, D.G. (1998) 0.0010

Hore, P. et al. (2005). 0.00057

Stout II, D.M. and Mason, M.A. (2003) 0.00086

EPA MRID 44458201 0.0011

Average (All Studies) 0.00089



# Post-Application Algorithms for All Scenarios

Post-Application Dermal Exposure Algor ithm (hard surfaces and carpets)

The algorithm to calculate exposure is as follows:

1ܨܥ ∗ ܶܧ ∗ ܥܶ ∗ ܴܶ ൌ ܧ

where:

E = exposure (mg/day);

# Chlorpyrifos Human Health Risk Assessment D424485

Page 456 of 531 TR = indoor surface transferable residue (μg/cm 2 

# );

TC = transfer coefficient (cm 2 

# /hr);

ET = exposure time (hr/day); and

CF1 = conversion fact or (0.001 mg/μg).

If chemical-specific TR data are available, this is preferred and should be used to calculate

exposure. However, if chemical-specific TR data are not available, then TR can be calculated

using the following formula:

ܨ ∗ ܴ݌݁ܦ ൌ ܴܶ ௜௔



# where:

TR = indoor surface transferable residue (μg/cm 2 

# );

DepR = deposited residue (  g/cm 2 

# ), based on (in order of preference):

(1) Chemical-specific residue deposition data (  g/cm 2 

# ),

(2) Application ra te (lb ai/area), or

(3) Default residue base d on type of application (  g/cm 2 

# ); and

F ai 

# = fraction of ai available for transfer from carpet or hard surface (unitless).

Absorbed dermal dose, normalized to body weight, ar e calculated as:

ൌ ܦ ∗ ܧ ܣ ܨ

ܹܤ

where:

D = dose (mg/kg-day);

E = exposure (mg/day);

AF = absorption factor; and

BW = body weight (kg).

Table 10.3. Indoor Envi ronments (Hard Surfaces and Carpets) – Inputs for Residentia l Post-application

Dermal Exposure

Algorithm

Notation Exposure Factor

(units) Point Estimate(s)



# TR Transferable residue

(  

# g/cm 2 

# ) Estimated: DepR * F ai



# DepR Deposited residue

(  

# g/cm 2 

# ) Estimated based on default residue

related to type of application

F ai 

# Fraction of DepR as TR

following application Carpets 0.020 a



# Hard surfaces 0.13 a



# TC Transfer Coefficient

(cm 2 

# /hr) Adult 6,800

Children 1 < 2 years old 1,800

ET Exposure Time (hrs/day) Adults Carpets 8

Hard

Surfaces 2

Children 1 < 2 Carpets 4

# Chlorpyrifos Human Health Risk Assessment D424485

Page 457 of 531 Table 10.3. Indoor Envi ronments (Hard Surfaces and Carpets) – Inputs for Residentia l Post-application

Dermal Exposure

Algorithm

Notation Exposure Factor

(units) Point Estimate(s)



# years old Hard

Surfaces 2

BW Body weight (kg) Adult 80

Children 1 < 2 years old 11

a. Chlopyrifos-specific as identified (Table 7-8) in the 2012 Residential SOPs.



# Post-application Hand-to-Mouth Exposure Algorithm

Exposure from hand-to-mouth activity is calculat ed as follows (based on algorithm utilized in

SHEDS-Multimedia):

ܧ ൌ ቈ

ܪܴ 	∗ ሺ ܨ ெ 

# ∗ 	 ܵܣ ு 

# ሻ ∗ ሺ ܧܶ ∗ ܰ

_ ܴ݁݌݈݁݊ ሻ ∗ ቆ1 െ ሺ 1 െ ܵܧ ሻ ி௥௘௤

_ ு௧ெ

ே

_ ோ௘௣௟௘௡ 

# ቇ ቉

where:

E = exposure (mg/day);

HR = hand residue loading (mg/cm 2 

# );

F M 

# = fraction hand surface area mout hed / event (fraction/event);

ET = exposure time (hr/day);

SA H 

# = surface area of one hand (cm 2 

# );

N_Replen = number of replenishmen t intervals per hour (intervals/hour);

SE = saliva extraction factor (i.e ., mouthing removal efficiency); and

Freq_HtM = number of hand-to-mouth cont acts events per hour (events/hour).

and

ൌ ܴܪ ܽ݅ܨ ௗ௦௛௔௡ 

# ܧܦ ∗

ܣܵ ு 

# 2 ∗

where:

HR = hand residue loading (mg/cm 2 

# );

Fai hands 

# = fraction ai on hands compared to total surface residue from jazzercise

study (unitless);

DE = dermal exposure (mg); and

SA H 

# = typical surface area of one hand (cm 2 

# ).

# Chlorpyrifos Human Health Risk Assessment D424485

Page 458 of 531 Table 10.4. Indoor Environments – Inputs fo r Residential Post-application Hand-to-Mouth

Exposure

Algorithm

Notation Exposure Factor

(units) Point Estimate(s)



# Fai hands 

# Fraction of ai on hands from jazzercise study

(unitless) 0.15

DE Dermal exposure calculated in Section Error! Reference source

not found.



# (mg) Calculated

HR Residue available on the hands

(mg/cm 2 

# ) Calculated

SA H 

# Surface area of one hand

(cm 2 

# ) Children 1 < 2 years old 150

AR Application rate

(mass active ingredient per unit area) 1% formulation

F M 

# Fraction of hand mouthed per event

(fraction/event) 0.13

N_Replen Replenishment intervals per hour

(intervals/hr) 4

ET Exposure time

(hours per day) Children 1 < 2

years old Carpets 4

Hard Surfaces 2

SE Saliva extraction factor

(fraction) 0.48

Freq_HtM Hand-to-mouth events

per hour

(events/hr) Children 1 < 2 years old 20

BW Body Weight

(kg) Children 1 < 2 years old 11



# Post-application Object-to-Mouth Exposure Algorithm

Exposure from object-to-mouth activity is calculate d as follows (based on algorithm utilized in

SHEDS-Multimedia):

ܧ ൌ ܱܴ ∗ ܨܥ1 	∗ ܵܯܣ ை 

# ∗ ሺ ܧܶ ∗ ܰ

_ ܴ݁݌݈݁݊ ሻ ∗ ቆ1 െ ሺ 1 െ ܵܧ ሻ ி௥௘௤_ை௧ெ

ே

_ ோ௘௣௟௘௡ 

# ቇ

where:

E = exposure (mg/day);

OR = chemical residue loading on an object (  g/cm 2 

# );

CF1 = weight unit conversion factor (0.001 mg/  g);

SAM O 

# = area of the object surface that is mouthed (cm 2 

# /event);

ET = exposure time (hr/day);

N_Replen = number of replenishmen t intervals per hour (intervals/hour);

SE = saliva extraction factor (i .e., mouthing removal efficiency); and

# Chlorpyrifos Human Health Risk Assessment D424485

Page 459 of 531 Freq_OtM = number of object-to-mouth contac t events per hour (events/hour).

and

ܨ ∗ ܴ݌݁ܦ ൌ ܱܴ ை



# where:

OR = chemical residue loading on the object ( μ g/cm 2 

# );

DepR = deposited residue ( μ g/cm 2 

# ); and

F O 

# = fraction of residue transfe rred to an object (unitless).

Table 10.5. Indoor Environments – Inputs fo r Residential Post-application Object-to-Mouth

Exposure

Algorithm

Notation Exposure Factor

(units) Point Estimate(s)



# AR Application rate

(mass active ingredient per unit area) [input]



# F O 

# Fraction of residue

transferred to an

object Carpets 0.020 a



# Hard surfaces 0.13 a



# SAM O 

# Surface area of object mouthed

(cm 2 

# /event) 10

N_Replen Replenishment intervals per hour

(intervals/hour) 4

SE O 

# Saliva extraction factor 0.48

ET Exposure Time

(hours per day) Children 1 <

2 years old Carpets 4

Hard Surfaces 2

Freq_OtM Object-to-mouth events

per hour

(events/hour) Children 1 < 2 years old 14

BW Body Weight

(kg) Children 1 < 2 years old 11

a. Chlopyrifos-specific as identified (Table 7-8) in the 2012 Residential SOPs.



# Residential Post-Application Exposure/Ch olinesterase Inhibition Summary

The results of this analysis are presented below for pregnant who were pot entially exposed from

contact to residues which occur in previously treated areas such as their homes (Table 10.6).

Results for young children (aged 1 to < 2 years old) who are also expos ed to residues which

occur in previously treated areas such as their homes are presented in Table 10.7. Peak

cholinesterase inhibition for pregnant women resulting from combined dermal and inhalation

exposures occurring from contact with previously treated indoor areas is 0.45%. For children 1

# Chlorpyrifos Human Health Risk Assessment D424485

Page 460 of 531 to < 2 years old, peak cholinestera se inhibition associated with co mbined dermal, incidental oral,

and inhalation exposures from being in previously treated areas is 2.7%.

Table 10.6. Residential Post-application To Pregnant Women in the Columbia Cohort Estimated Dermal and

Inhalation Exposures and Predicted % ChE Inhibition (Route-specific and Combined)

Exposure

Scenario Formulation Dermal Dose

(mg/kg/day) Dermal:

Peak

% ChE

Inhibition Airborne

Concentration of

Chlorpyrifos

(mg/m 3 

# ) Inhalation:

Peak

% ChE

Inhibition Combined:

Peak

% ChE

Inhibition

Broadcast

(Hard

Surfaces) 1% PCO

Application

or RTU 1 16

oz can 0.71 0.45% 0.092 0.0049% 0.45%

* See algorithms and inputs above used to es timate post-app lication dermal exposures.



# Table 10.7. Residential Post-application (Children 1 to < 2 Years Old in the Columbia Cohort) Estimated Dermal

and Inhalation Exposures and Predicted % RBC AC hE Inhibition (Route-specific and Combined)

Exposure

Scenario Formulation Dermal

Dose

(mg/kg/day) Dermal:

Peak

% ChE

Inhibition HTM

Dose

(mg/kg/day) HTM Peak

% ChE

Inhibition

(mg/day) Airborne

Concn. of

Chlorpyrifos

(mg/m3) Inhalation:

Peak

% ChE

Inhibition Combined:

Peak

% ChE

Inhibition

Broadcast

(Hard

Surfaces) 1% PCO

Application

or RTU 1 16

oz can 1.3 0.14% 0.10 2.2% 0.092 0.014% 2.7%

* See algorithms and inputs above used to estimate post-applic ation dermal, (HTM) hand-to-mouth, and (OTM) object-to-mouth

exposures/doses.



# Appendix 11. New Literature on Chlorpyrifos since the 2012 FIFRA SAP

Meeting

# Ch l orp y rifo s Human Health Risk Ass essm e nt 

# 0424485

Experimental toxicology

Literature search for Revi s ed

Appendix 11

. chlorpyrifos HHRA

, 2014

PubDate PubDate Full Citation

Comments Authors (Year) (Month)

Acker Cl, Souza AC, DosSantos MP, Mazzanti CM, Nogueira

CW.Dipheny l d i selenide attenuates hepatic a n d hemato l ogic

Acker Cl

, Souza AC

, Dos Santos toxicity induced by chlorpyrifos acute exposure in rats.

Acute effects MP, Mazzanti CM, Nogueira CW 2012 Sep Environ Sci Pollut Res Int. 2012 Sep; 19(8):3481-90.

Cardona D

, 

# LA 3 

# pez

- Granero C, CaA±adas F

, Llorens J, Flores P

,

Pancetti F, SAi nchez

- Santed F

. Dose

- dependent regional

Cardona D, lt\lpez

- Granero C, brain acetylcholinesterase and acyl peptide hydro l ase

CaA±adas F, Llorens J

, Flores P, inhibition without cell death after chlorpyrifos

Acute effects Pancetti F, SAinchez-Santed F 2013 administration. J Toxicol Sci. 2013; 38(2):193-203.

Cardona D, lA 3 

# pez

- Granero C, CaA ± adas F, Llorens J, Flores P,

Pancetti F, SAi nchez-Santed F.Dose

- dependent regional

Cardona D

, lA 3 

# pez

- Granero C, brain acetylcholinesterase and acylpeptide hydrolase

CaA±adas F, Llorens J, Flores P, inhibition without cell death after chlorpyrifos

Acute effects Pancetti F, SAi n c h ez-Sa n ted F 2013 admin i stratio n

. J Tox i co l Sci. 2013; 38(2):193-203.

Jayawardane P, Senanayake N

, Buckley NA

, Dawson

AH.E i ectrophysiological correlates of respiratory failure in

acute organophosphate poisoning: evidence for differential

Jayawardane P, Senanayake N, roles of muscarini c and nicotinic stimulation

. Clin Toxicol

Acute effects Buckley NA

, Dawson AH 2012 Apr (Ph il a). 2012 Apr; 50(4):250

- 3.

li B, Eyer P

, Eddleston M

, Jiang W

, Schopfer lM

, lockridge

O.Protein tyrosine adduct in humans se l f

- poisoned by

li B

, Eyer P

, Eddleston M, Jiang chlorpyrifos. Toxicol Appl Pharma c al. 2013 Jun 15;

Acute effects W

, Schopfer lM, lockridge 

# 0 2013 Jun 269(3):215

- 25

.

liu J, Parsons l, Pope C.Comparative effects of parathion and

chlorpyrifos on extracellular endocannabinoid levels in rat

hippocampus: influence on cho li nergic toxicity. T oxico l Appl

Acute effects liu J

, Parsons l, Pope C 2013 Nov Pharmacal. 2013 Nov 1; 272(3):608

- 15.



# Page 461 of 531

# C hlorpyrifos Human H e alth Ri s k Assessment 

# 0424485

Lopez-Granero C

, Canadas F

, Cardona D, Yu Y

, Gimenez E

,

Lozano R, Avila DS, Aschner M, SAinchez

- Santed

Lopez-Granero C, Canadas F, F .Chlorpy rifos

- , diisopropylphosphorofluoridate-, and

Cardona D, Yu Y, Gimenez E, parathion

- induced behavioral and oxidative stress effects:

Lozano R, Avila DS, Aschner M, are they mediated by analogous mechanisms of act ion ?

Acute effects Sanchez-Santed, F. 2013 Jan Toxicol Sci. 2013 Jan; 131(1):206-16

.

Lukaszewicz-Hussain A

. [Serum glucose concentration in

subacute intoxication wi t h chlorpyrifos- organophosphate

Acute effects Lukaszewicz- H ussain A 2013 insecticide]

. Med Pr. 2013; 64(4):527-31.

Ma P

, Wu Y, Zeng 

# Q, Gan Y, Chen J, Ye X, Yang X.Oxidative

damage induced by chlorpyrifos in the hepatic and renal

Ma P, Wu Y

, Zeng Q, 

# Gan Y, Chen tissue of Kunming mice and the antioxidant role of vitamin E.

Acute effects J, Ye X, Yang X 2013 Aug Food Chern Toxicol. 2013 Aug; 58:177

- 83.

MontesdeOca L

, Moreno M, Cardona D, Campa L, Sunol C,

Montes de Oca L

, Moreno M, Galofre© M, Flores P, Sanchez-Santed F

. Long term

Cardona D, Campa L, SuA±ol C

, compulsivity on the 5-choice serial reaction time task after

GalofrA© M

, Flores P

, SA i nchez- acute Chlorpyrifos exposu re

. Toxicol Lett

. 2013 Feb 4 ; 216(2

-

Acute effects Santed F 2013 Feb 3):73-85

.

Park JH, Lee JE, Shin IC

, Koh HC.Autophagy regulates

chlorpyrifos-induced apoptosis in SH-SY5Y cells. Toxico l Appl

Acute e ffects Park JH, Lee JE, Shin IC, Koh HC 2013 Apr Pharmacal. 2013 Apr 1; 268(1):55-67.

Rajasekharan C, Renjith SW, Jayapal T.Opsoclonus and

Rajasekharan C, Renjith SW

, lingual myoclonus due to organophosphate poisoning :

Acute effects Jayapal T 2012 Sep images in clinical medicine. BMJ Case Rep

. 2012 Sep 21; 2012

Starks SE

, Gerr F, Kamel F, Lynch CF

, Jones MP

, Alavanja MC

,

Sandler DP

, Hoppin JA

. Neurobehavioral fun c tion and

Starks SE

, Gerr F, Kamel F, Lynch organophosphate insecticide use among pesti c ide

CF, Jones MP, Alavanja MC, applicators in the Agricu ltura l Health Study. Neurotoxicol

Acute effects Sandler DP, Hoppin JA 2012 J an

- Feb Te ratol. 2012 Jan

- Feb; 34(1):168-76.



# P age 

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D424485Page471of531Epidemiology/ExposureQuirÃ3s‐AlcalÃ¡L,BradmanA,SmithK,WeerasekeraG,OdetokunM,BarrDB,NishiokaM,CastorinaR,HubbardAE,NicasM,HammondSK,McKoneTE,EskenaziB2012NovQuirÃ3s‐AlcalÃ¡L,BradmanA,SmithK,WeerasekeraG,OdetokunM,BarrDB,NishiokaM,CastorinaR,HubbardAE,NicasM,HammondSK,McKoneTE,EskenaziB.Organophosphorouspesticidebreakdownproductsinhousedustandchildren'surine.JExpoSciEnvironEpidemiol.2012Nov;22(6):559‐68.Epidemiology/ExposureRauhVA,PereraFP,HortonMK,WhyattRM,BansalR,HaoX,LiuJ,BarrDB,SlotkinTA,PetersonBS2012MayRauhVA,PereraFP,HortonMK,WhyattRM,BansalR,HaoX,LiuJ,BarrDB,SlotkinTA,PetersonBS.Brainanomaliesinchildrenexposedprenatallytoacommonorganophosphatepesticide.ProcNatlAcadSciUSA.2012May15;109(20):7871‐6.Epidemiology/ExposureWangN,YiL,ShiL,KongD,CaiD,WangD,ShanZ2012WangN,YiL,ShiL,KongD,CaiD,WangD,ShanZ.PollutionlevelandhumanhealthriskassessmentofsomepesticidesandpolychlorinatedbiphenylsinNantongofSoutheastChina.JEnvironSci(China).2012;24(10):1854‐60.Epidemiology/ExposureWuN,HaoF,YuX2012JulWuN,HaoF,YuX.PeripheralnerveandskindamageassociatedwithworkinginaSTCPfactory:reportoffourcases.ClinToxicol(Phila).2012Jul;50(6):514‐7.Epidemiology/ExposureCostaLG,GiordanoG,ColeTB,MarsillachJ,FurlongCE2013MayCostaLG,GiordanoG,ColeTB,MarsillachJ,FurlongCE.Paraoxonase1(PON1)asageneticdeterminantofsusceptibilitytoorganophosphatetoxicity.Toxicology.2013May10;307:115‐22.ExposureAbdulra'ufLB,TanGH2013DecAbdulra'ufLB,TanGH.Multivariatestudyofparametersinthedeterminationofpesticideresiduesinapplebyheadspacesolidphasemicroextractioncoupledtogaschromatography‐massspectrometryusingexperimentalfactorialdesign.FoodChem.2013Dec15;141(4):4344‐8.ExposureAffamAC,ChaudhuriM2013NovAffamAC,ChaudhuriM.Degradationofpesticideschlorpyrifos,cypermethrinandchlorothalonilinaqueoussolutionbyTiO2photocatalysis.JEnvironManage.2013Nov30;130:160‐5.Chlorpyrifos 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D424485Page472of531ExposureAgboSO,KeinÃ¤nenM,Keski‐SaariS,LemmetyinenJ,AkkanenJ,LeppÃ¤nenMT,MayerP,KukkonenJV2013SepAgboSO,KeinÃ¤nenM,Keski‐SaariS,LemmetyinenJ,AkkanenJ,LeppÃ¤nenMT,MayerP,KukkonenJV.ChangesinLumbriculusvariegatusmetabolitesunderhypoxicexposuretobenzo(a)pyrene,chlorpyrifosandpentachlorophenol:consequencesonbiotransformation.Chemosphere.2013Sep;93(2):302‐10.ExposureAgostiniMG,KacolirisF,DemetrioP,NataleGS,BonettoC,RoncoAE2013MayAgostiniMG,KacolirisF,DemetrioP,NataleGS,BonettoC,RoncoAE.AbnormalitiesinamphibianpopulationsinhabitingagroecosystemsinNortheasternBuenosAiresProvince,Argentina.DisAquatOrgan.2013May27;104(2):163‐71.ExposureAgudeloCRM,JaramilloML,PeÃ±uelaG2012AugAgudeloCRM,JaramilloML,PeÃ±uelaG.Comparisonoftheremovalofchlorpyrifosanddissolvedorganiccarboninhorizontalsub‐surfaceandsurfaceflowwetlands.SciTotalEnviron.2012Aug1;431:271‐7.ExposureAguilera‐LuizMM,Romero‐GonzÃ¡lezR,Plaza‐BolaÃ±osP,MartÃnezVidalJL,GarridoFrenichA2013AugAguilera‐LuizMM,Romero‐GonzÃ¡lezR,Plaza‐BolaÃ±osP,MartÃnezVidalJL,GarridoFrenichA.Wide‐scopeanalysisofveterinarydrugandpesticideresiduesinanimalfeedbyliquidchromatographycoupledtoquadrupole‐time‐of‐flightmassspectrometry.AnalBioanalChem.2013Aug;405(20):6543‐53.ExposureAkkadR,SchwackW2012Sep‐OctAkkadR,SchwackW.Determinationoforganophosphorusandcarbamateinsecticidesinfreshfruitsandvegetablesbyhigh‐performancethin‐layerchromatography‐multienzymeinhibitionassay.JAOACInt.2012Sep‐Oct;95(5):1371‐7.ExposureAlcÃ¡ntara‐ConcepciÃ3nV,CramS,GibsonR,PoncedeLeÃ3nC,Mazari‐HiriartM2013Jul‐AugAlcÃ¡ntara‐ConcepciÃ3nV,CramS,GibsonR,PoncedeLeÃ3nC,Mazari‐HiriartM.Methoddevelopmentandvalidationforthesimultaneousdeterminationoforganochlorineandorganophosphoruspesticidesinacomplexsedimentmatrix.JAOACInt.2013Jul‐Aug;96(4):854‐63.Chlorpyrifos Human Health Risk Assessment D424485Page473of531ExposureAlvesAC,GonÃ§alvesMM,BernardoMM,MendesBS2012OctAlvesAC,GonÃ§alvesMM,BernardoMM,MendesBS.Dispersiveliquid‐liquidmicroextractionoforganophosphorouspesticidesusingnonhalogenatedsolvents.JSepSci.2012Oct;35(19):2653‐8.ExposureAndersonB,PhillipsB,HuntJ,SieglerK,VoorheesJ,SmallingK,KuivilaK,HamiltonM,RanasingheJA,TjeerdemaR2014MarAndersonB,PhillipsB,HuntJ,SieglerK,VoorheesJ,SmallingK,KuivilaK,HamiltonM,RanasingheJA,TjeerdemaR.ImpactsofpesticidesinaCentralCaliforniaestuary.EnvironMonitAssess.2014Mar;186(3):1801‐14.ExposureArmstrongJL,FenskeRA,YostMG,Tchong‐FrenchM,YuJ2013JulArmstrongJL,FenskeRA,YostMG,Tchong‐FrenchM,YuJ.ComparisonofpolyurethanefoamandXAD‐2samplingmatricestomeasureairborneorganophosphoruspesticidesandtheiroxygenanalogsinanagriculturalcommunity.Chemosphere.2013Jul;92(4):451‐7.ExposureAroraS,MukherjeeI,KumarA,GargDK2014JanAroraS,MukherjeeI,KumarA,GargDK.Comparativeassessmentofpesticideresiduesingrain,soil,andwaterfromIPMandnon‐IPMtrialsofbasmatirice.EnvironMonitAssess.2014Jan;186(1):361‐6.ExposureBabinaK,DollardM,PilottoL,EdwardsJW2012NovBabinaK,DollardM,PilottoL,EdwardsJW.EnvironmentalexposuretoorganophosphorusandpyrethroidpesticidesinSouthAustralianpreschoolchildren:acrosssectionalstudy.EnvironInt.2012Nov1;48:109‐20.ExposureBagheriH,Es'haghiA,Es‐haghiA,MesbahiN2012AugBagheriH,Es'haghiA,Es‐haghiA,MesbahiN.Ahigh‐throughputapproachforthedeterminationofpesticideresiduesincucumbersamplesusingsolid‐phasemicroextractionon96‐wellplate.AnalChimActa.2012Aug31;740:36‐42.ExposureBaldimIM,SouzaMC,SouzaJC,FigueiredoEC,MartinsI2012OctBaldimIM,SouzaMC,SouzaJC,FigueiredoEC,MartinsI.Applicationofthemolecularlyimprintedsolid‐phaseextractiontotheorganophosphateresiduesdeterminationinstrawberries.AnalBioanalChem.2012Oct;404(6‐7):1959‐66.Chlorpyrifos Human Health Risk Assessment D424485Page474of531ExposureBeamerPI,CanalesRA,FergusonAC,LeckieJO,BradmanA2012JanBeamerPI,CanalesRA,FergusonAC,LeckieJO,BradmanA.Relativepesticideandexposureroutecontributiontoaggregateandcumulativedoseinyoungfarmworkerchildren.IntJEnvironResPublicHealth.2012Jan;9(1):73‐96.ExposureBediJS,GillJP,AulakhRS,KaurP,SharmaA,PooniPA2013OctBediJS,GillJP,AulakhRS,KaurP,SharmaA,PooniPA.Pesticideresiduesinhumanbreastmilk:riskassessmentforinfantsfromPunjab,India.SciTotalEnviron.2013Oct1;463‐464:720‐6.ExposureBhattacharjeeS,FakhruddinAN,ChowdhuryMA,RahmanMA,AlamMK2012AugBhattacharjeeS,FakhruddinAN,ChowdhuryMA,RahmanMA,AlamMK.Monitoringofselectedpesticidesresiduelevelsinwatersamplesofpaddyfieldsandremovalofcypermethrinandchlorpyrifosresiduesfromwaterusingricebran.BullEnvironContamToxicol.2012Aug;89(2):348‐53.ExposureBootharajuMS,PradeepT2012FebBootharajuMS,PradeepT.Understandingthedegradationpathwayofthepesticide,chlorpyrifosbynoblemetalnanoparticles.Langmuir.2012Feb7;28(5):2671‐9.ExposureBrunEM,PuchadesR,MaquieiraÃ2013AprBrunEM,PuchadesR,MaquieiraÃ.Gold,carbon,andaluminumlow‐reflectivitycompactdiscsasmicroassayingplatforms.AnalChem.2013Apr16;85(8):4178‐86.ExposureCaiD,WangL,ZhangG,ZhangX,WuZ2013SepCaiD,WangL,ZhangG,ZhangX,WuZ.Controllingpesticidelossbynaturalporousmicro/nanocomposites:strawash‐basedbiocharandbiosilica.ACSApplMaterInterfaces.2013Sep25;5(18):9212‐6.ExposureCarlsonJC,ChallisJK,HansonML,WongCS2013FebCarlsonJC,ChallisJK,HansonML,WongCS.Stabilityofpharmaceuticalsandotherpolarorganiccompoundsstoredonpolarorganicchemicalintegrativesamplersandsolid‐phaseextractioncartridges.EnvironToxicolChem.2013Feb;32(2):337‐44.Chlorpyrifos Human Health Risk Assessment D424485Page475of531ExposureCatalinaRodriguezD,CarvajalS,PenuelaG2013JulCatalinaRodriguezD,CarvajalS,PenuelaG.Effectofchlorpyrifosontheinhibitionoftheenzymeacetylcholinesterasebycross‐linkinginwater‐supplysamplesandmilkfromdairycattle.Talanta.2013Jul15;111:1‐7.ExposureCavalcanteRM,LimaDM,FernandesGM,DuavÃWC2012MayCavalcanteRM,LimaDM,FernandesGM,DuavÃWC.Relationfactor:anewstrategyforqualitycontrolinthedeterminationofpesticidesinenvironmentalaqueousmatrices.Talanta.2012May15;93:212‐8.ExposureCavariY,LandauD,SoferS,LeibsonT,LazarI2013MayCavariY,LandauD,SoferS,LeibsonT,LazarI.Organophosphatepoisoning‐inducedacuterenalfailure.PediatrEmergCare.2013May;29(5):646‐7.ExposureChaiLK,WongMH,BruunHansenHC2013AugChaiLK,WongMH,BruunHansenHC.Degradationofchlorpyrifosinhumidtropicalsoils.JEnvironManage.2013Aug15;125:28‐32.ExposureChenC,QianY,LiuX,TaoC,LiangY,LiY2012FebChenC,QianY,LiuX,TaoC,LiangY,LiY.RiskassessmentofchlorpyrifosonriceandcabbageinChina.RegulToxicolPharmacol.2012Feb;62(1):125‐30.ExposureChishtiZ,HussainS,ArshadKR,KhalidA,ArshadM2013JanChishtiZ,HussainS,ArshadKR,KhalidA,ArshadM.Microbialdegradationofchlorpyrifosinliquidmediaandsoil.JEnvironManage.2013Jan15;114:372‐80.ExposureChowdhuryMA,BanikS,UddinB,MoniruzzamanM,KarimN,GanSH2012SepChowdhuryMA,BanikS,UddinB,MoniruzzamanM,KarimN,GanSH.OrganophosphorusandcarbamatepesticideresiduesdetectedinwatersamplescollectedfrompaddyandvegetablefieldsoftheSavarandDhamraiUpazilasinBangladesh.IntJEnvironResPublicHealth.2012Sep11;9(9):3318‐29.ExposureCliftonMS,WargoJP,WeathersWS,ColÃ3nM,BennettDH,TulveNS2013JanCliftonMS,WargoJP,WeathersWS,ColÃ3nM,BennettDH,TulveNS.Quantitativeanalysisoforganophosphateandpyrethroidinsecticides,pyrethroidtransformationproducts,polybrominateddiphenylethersandbisphenolAinresidentialsurfacewipesamples.JChromatogrA.2013Jan18;1273:1‐11.Chlorpyrifos Human Health Risk Assessment D424485Page476of531ExposureDabrowskiJM,BalderacchiM2013NovDabrowskiJM,BalderacchiM.DevelopmentandfieldvalidationofanindicatortoassesstherelativemobilityandriskofpesticidesintheLourensRivercatchment,SouthAfrica.Chemosphere.2013Nov;93(10):2433‐43.ExposureDemeP,AzmeeraT,PrabhavathiDeviBL,JonnalagaddaPR,PrasadRB,VijayaSarathiUV2014JanDemeP,AzmeeraT,PrabhavathiDeviBL,JonnalagaddaPR,PrasadRB,VijayaSarathiUV.Animproveddispersivesolid‐phaseextractionclean‐upmethodforthegaschromatography‐negativechemicalionisationtandemmassspectrometricdeterminationofmulticlasspesticideresiduesinedibleoils.FoodChem.2014Jan1;142:144‐51.ExposureDuD,WangJ,WangL,LuD,LinY2012FebDuD,WangJ,WangL,LuD,LinY.Integratedlateralflowteststripwithelectrochemicalsensorforquantificationofphosphorylatedcholinesterase:biomarkerofexposuretoorganophosphorusagents.AnalChem.2012Feb7;84(3):1380‐5.ExposureDubeyKK,FulekarMH2012AprDubeyKK,FulekarMH.ChlorpyrifosbioremediationinPennisetumrhizospherebyanovelpotentialdegraderStenotrophomonasmaltophiliaMHFENV20.WorldJMicrobiolBiotechnol.2012Apr;28(4):1715‐25.ExposureDuffnerA,IngwersenJ,HugenschmidtC,StreckT2012Jul‐AugDuffnerA,IngwersenJ,HugenschmidtC,StreckT.PesticidetransportpathwaysfromaslopedLitchiorchardtoanadjacenttropicalstreamasidentifiedbyhydrographseparation.JEnvironQual.2012Jul‐Aug;41(4):1315‐23.ExposureEstÃ©vezE,CabreraMdelC,Molina‐DÃazA,Robles‐MolinaJ,Palacios‐DÃazMdelP2012SepEstÃ©vezE,CabreraMdelC,Molina‐DÃazA,Robles‐MolinaJ,Palacios‐DÃazMdelP.Screeningofemergingcontaminantsandprioritysubstances(2008/105/EC)inreclaimedwaterforirrigationandgroundwaterinavolcanicaquifer(GranCanaria,CanaryIslands,Spain).SciTotalEnviron.2012Sep1;433:538‐46.Chlorpyrifos Human Health Risk Assessment D424485Page477of531ExposureFanS,DengK,YuC,ZhaoP,BaiA,LiY,PanC,LiX2013SepFanS,DengK,YuC,ZhaoP,BaiA,LiY,PanC,LiX.Influenceofdifferentplantingseasonsofsixleafvegetablesonresiduesoffivepesticides.JAgricFoodChem.2013Sep25;61(38):9036‐44.ExposureFanS,ZhangF,DengK,YuC,LiuS,ZhaoP,PanC2013MarFanS,ZhangF,DengK,YuC,LiuS,ZhaoP,PanC.Spinachoramaranthcontainshighestresidueofmetalaxyl,fluazifop‐P‐butyl,chlorpyrifos,andlambda‐cyhalothrinonsixleafvegetablesuponopenfieldapplication.JAgricFoodChem.2013Mar6;61(9):2039‐44.ExposureFemiaJ,MarianiM,ZalazarC,TiscorniaI2013FemiaJ,MarianiM,ZalazarC,TiscorniaI.PhotodegradationofchlorpyrifosinwaterbyUV/H2O2treatment:toxicityevaluation.WaterSciTechnol.2013;68(10):2279‐86.ExposureFuentesMS,BriceÃ±oGE,SaezJM,BenimeliCS,DiezMC,AmorosoMJ2013FuentesMS,BriceÃ±oGE,SaezJM,BenimeliCS,DiezMC,AmorosoMJ.EnhancedremovalofapesticidesmixturebysingleculturesandconsortiaoffreeandimmobilizedStreptomycesstrains.BiomedResInt.2013;2013:392573.ExposureGarrisonVH,MajewskiMS,ForemanWT,GenualdiSA,MohammedA,MasseySimonichSL2014JanGarrisonVH,MajewskiMS,ForemanWT,GenualdiSA,MohammedA,MasseySimonichSL.PersistentorganiccontaminantsinSaharandustairmassesinWestAfrica,CapeVerdeandtheeasternCaribbean.SciTotalEnviron.2014Jan15;468‐469:530‐43.ExposureGebremariamSY,BeutelMW,FluryM,HarshJB,YongeDR2012JanGebremariamSY,BeutelMW,FluryM,HarshJB,YongeDR.Nonsingularadsorption/desorptionofchlorpyrifosinsoilsandsediments:experimentalresultsandmodeling.EnvironSciTechnol.2012Jan17;46(2):869‐75.ExposureGebremariamSY,BeutelMW,YongeDR,FluryM,HarshJB2012GebremariamSY,BeutelMW,YongeDR,FluryM,HarshJB.Adsorptionanddesorptionofchlorpyrifostosoilsandsediments.RevEnvironContamToxicol.2012;215:123‐75.ExposureGhoshdastidarAJ,SaundersJE,BrownKH,TongAZ2012GhoshdastidarAJ,SaundersJE,BrownKH,TongAZ.Membranebioreactortreatmentofcommonlyusedorganophosphatepesticides.JEnvironSciHealthB.2012;47(7):742‐50.Chlorpyrifos Human Health Risk Assessment D424485Page478of531ExposureGonzÃ¡lez‐CurbeloMÃ,HernÃ¡ndez‐BorgesJ,Borges‐MiquelTM,RodrÃguez‐DelgadoMÃ2012GonzÃ¡lez‐CurbeloMÃ,HernÃ¡ndez‐BorgesJ,Borges‐MiquelTM,RodrÃguez‐DelgadoMÃ.DeterminaƟonofpesticidesandtheirmetabolitesinprocessedcerealsamples.FoodAdditContamPartAChemAnalControlExpoRiskAssess.2012;29(1):104‐16.ExposureGonzÃ¡lez‐CurbeloMÃ,Herrera‐HerreraAV,HernÃ¡ndez‐BorgesJ,RodrÃ‐guez‐DelgadoMÃ2013FebGonzÃ¡lez‐CurbeloMÃ,Herrera‐HerreraAV,HernÃ¡ndez‐BorgesJ,RodrÃguez‐DelgadoMÃ.AnalysisofpesƟcidesresiduesinenvironmentalwatersamplesusingmultiwalledcarbonnanotubesdispersivesolid‐phaseextraction.JSepSci.2013Feb;36(3):556‐63.ExposureGuS,LuY,DingY,LiL,ZhangF,WuQ2013SepGuS,LuY,DingY,LiL,ZhangF,WuQ.Droplet‐basedmicrofluidicsfordose‐responseassayofenzymeinhibitorsbyelectrochemicalmethod.AnalChimActa.2013Sep24;796:68‐74.ExposureHallLJr,AndersonRD2012HallLJr,AndersonRD.Historicaltrendsanalysisof2004to2009toxicityandpesticidedataforCalifornia'scentralvalley.JEnvironSciHealthAToxHazardSubstEnvironEng.2012;47(6):801‐11.ExposureHarnpicharnchaiK,ChaiearN,CharerntanyarakL2013NovHarnpicharnchaiK,ChaiearN,CharerntanyarakL.Residuesoforganophosphatepesticidesusedinvegetablecultivationinambientair,surfacewaterandsoilinBuengNiamSubdistrict,KhonKaen,Thailand.SoutheastAsianJTropMedPublicHealth.2013Nov;44(6):1088‐97.ExposureHarwoodAD,LandrumPF,WestonDP,LydyMJ2013FebHarwoodAD,LandrumPF,WestonDP,LydyMJ.UsingSPMEfibersandTenaxtopredictthebioavailabilityofpyrethroidsandchlorpyrifosinfieldsediments.EnvironPollut.2013Feb;173:47‐51.Chlorpyrifos Human Health Risk Assessment D424485Page479of531ExposureHaywardDG,WongJW,ShiF,ZhangK,LeeNS,DiBenedettoAL,HengelMJ2013MayHaywardDG,WongJW,ShiF,ZhangK,LeeNS,DiBenedettoAL,HengelMJ.Multiresiduepesticideanalysisofbotanicaldietarysupplementsusingsalt‐outacetonitrileextraction,solid‐phaseextractioncleanupcolumn,andgaschromatography‐triplequadrupolemassspectrometry.AnalChem.2013May7;85(9):4686‐93.ExposureIsmailM,KhanHM,SayedM,CooperWJ2013OctIsmailM,KhanHM,SayedM,CooperWJ.Advancedoxidationforthetreatmentofchlorpyrifosinaqueoussolution.Chemosphere.2013Oct;93(4):645‐51.ExposureJyotG,MandalK,BattuRS,SinghB2013JulJyotG,MandalK,BattuRS,SinghB.Estimationofchlorpyriphosandcypermethrinresiduesinchilli(CapsicumannuumL.)bygas‐liquidchromatography.EnvironMonitAssess.2013Jul;185(7):5703‐14.ExposureKÃ¶ck‐SchulmeyerM,OlmosM,LÃ3pezdeAldaM,BarcelÃ3D2013AugKÃ¶ck‐SchulmeyerM,OlmosM,LÃ3pezdeAldaM,BarcelÃ3D.Developmentofamultiresiduemethodforanalysisofpesticidesinsedimentsbasedonisotopedilutionandliquidchromatography‐electrospray‐tandemmassspectrometry.JChromatogrA.2013Aug30;1305:176‐87.ExposureKaranasiosEC,TsiropoulosNG,KarpouzasDG2013SepKaranasiosEC,TsiropoulosNG,KarpouzasDG.Quantitativeandqualitativedifferencesinthemetabolismofpesticidesinbiobedsubstratesandsoil.Chemosphere.2013Sep;93(1):20‐8.ExposureKarpuzcuME,SedlakDL,StringfellowWT2013JanKarpuzcuME,SedlakDL,StringfellowWT.Biotransformationofchlorpyrifosinriparianwetlandsinagriculturalwatersheds:implicationsforwetlandmanagement.JHazardMater.2013Jan15;244‐245:111‐20.ExposureKongZ,ShanW,DongF,LiuX,XuJ,LiM,ZhengY2012AugKongZ,ShanW,DongF,LiuX,XuJ,LiM,ZhengY.Effectofhomeprocessingonthedistributionandreductionofpesticideresiduesinapples.FoodAdditContamPartAChemAnalControlExpoRiskAssess.2012Aug;29(8):1280‐7.Chlorpyrifos Human Health Risk Assessment D424485Page480of531ExposureKorbaK,PelitL,PelitFO,OzdokurKV,ErtaÅŸH,EroÄŸluAE,ErtaÅŸFN2013JunKorbaK,PelitL,PelitFO,OzdokurKV,ErtaÅŸH,EroÄŸluAE,ErtaÅŸFN.Preparationandcharacterizationofsodiumdodecylsulfatedopedpolypyrrolesolidphasemicroextractionfiberanditsapplicationtoendocrinedisruptorpesticideanalysis.JChromatogrBAnalytTechnolBiomedLifeSci.2013Jun15;929:90‐6.ExposureKouzayhaA,AlAshiA,AlAkoumR,AlIskandaraniM,BudzinskiH,JaberF2013NovKouzayhaA,AlAshiA,AlAkoumR,AlIskandaraniM,BudzinskiH,JaberF.OccurrenceofpesticideresiduesinLebanon'swaterresources.BullEnvironContamToxicol.2013Nov;91(5):503‐9.ExposureKuangY,QiuF,KongW,LuoJ,ChengH,YangM2013NovKuangY,QiuF,KongW,LuoJ,ChengH,YangM.Simultaneousquantificationofmycotoxinsandpesticideresiduesinginsengwithone‐stepextractionusingultra‐highperformanceliquidchromatography‐electrosprayionizationtandemmassspectrometry.JChromatogrBAnalytTechnolBiomedLifeSci.2013Nov15;939:98‐107.ExposureKumarA,NayakAK,ShuklaAK,PandaBB,RajaR,ShahidM,TripathiR,MohantyS,RathPC2012AprKumarA,NayakAK,ShuklaAK,PandaBB,RajaR,ShahidM,TripathiR,MohantyS,RathPC.Microbialbiomassandcarbonmineralizationinagriculturalsoilsasaffectedbypesticideaddition.BullEnvironContamToxicol.2012Apr;88(4):538‐42.ExposureKusvuranE,YildirimD,MavrukF,CeyhanM2012NovKusvuranE,YildirimD,MavrukF,CeyhanM.Removalofchloropyrifosethyl,tetradifonandchlorothalonilpesticideresiduesfromcitrusbyusingozone.JHazardMater.2012Nov30;241‐242:287‐300.ExposureLariSZ,KhanNA,GandhiKN,MeshramTS,ThackerNP2014JanLariSZ,KhanNA,GandhiKN,MeshramTS,ThackerNP.Comparisonofpesticideresiduesinsurfacewaterandgroundwaterofagricultureintensiveareas.JEnvironHealthSciEng.2014Jan7;12(1):11.Chlorpyrifos Human Health Risk Assessment D424485Page481of531ExposureLavinKS,HagemanKJ,MarxSK,DillinghamPW,KamberBS2012JanLavinKS,HagemanKJ,MarxSK,DillinghamPW,KamberBS.Usingtraceelementsinparticulatemattertoidentifythesourcesofsemivolatileorganiccontaminantsinairatanalpinesite.EnvironSciTechnol.2012Jan3;46(1):268‐76.ExposureLeeKG,LeeSK2012DecLeeKG,LeeSK.Monitoringandriskassessmentofpesticideresiduesinyuzafruits(CitrusjunosSieb.exTanaka)andyuzateasamplesproducedinKorea.FoodChem.2012Dec15;135(4):2930‐3.ExposureLiH,WeiY,LydyMJ,YouJ2014JulLiH,WeiY,LydyMJ,YouJ.Inter‐compartmentaltransportoforganophosphateandpyrethroidpesticidesinSouthChina:implicationsforaregionalriskassessment.EnvironPollut.2014Jul;190:19‐26.ExposureLinharesAG,AssisCR,SiqueiraMT,BezerraRS,CarvalhoLBJr2013AugLinharesAG,AssisCR,SiqueiraMT,BezerraRS,CarvalhoLBJr.Developmentofamethodforextractionandassayofhumanerythrocyteacetylcholinesteraseandpesticideinhibition.HumExpToxicol.2013Aug;32(8):837‐45.ExposureLuD,QiuX,FengC,JinY,LinY,XiongL,WenY,WangD,WangG2012MayLuD,QiuX,FengC,JinY,LinY,XiongL,WenY,WangD,WangG.Simultaneousdeterminationof45pesticidesinfruitandvegetableusinganimprovedQuEChERSmethodandon‐linegelpermeationchromatography‐gaschromatography/massspectrometer.JChromatogrBAnalytTechnolBiomedLifeSci.2012May1;895‐896:17‐24.ExposureLuHY,ShenY,SunX,ZhuH,LiuXJ2013SepLuHY,ShenY,SunX,ZhuH,LiuXJ.Washingeffectsoflimoneneonpesticideresiduesingreenpeppers.JSciFoodAgric.2013Sep;93(12):2917‐21.ExposureMaliyekkalSM,SreeprasadTS,KrishnanD,KouserS,MishraAK,WaghmareUV,PradeepT2013JanMaliyekkalSM,SreeprasadTS,KrishnanD,KouserS,MishraAK,WaghmareUV,PradeepT.Graphene:areusablesubstrateforunprecedentedadsorptionofpesticides.Small.2013Jan28;9(2):273‐83.ExposureMastMA,AlvarezDA,ZauggSD2012MarMastMA,AlvarezDA,ZauggSD.DepositionandaccumulationofairborneorganiccontaminantsinYosemiteNationalPark,California.EnvironToxicolChem.2012Mar;31(3):524‐33.Chlorpyrifos Human Health Risk Assessment D424485Page482of531ExposureMishraRK,IstamboulieG,BhandS,MartyJL2012OctMishraRK,IstamboulieG,BhandS,MartyJL.Detoxificationoforganophosphateresiduesusingphosphotriesteraseandtheirevaluationusingflowbasedbiosensor.AnalChimActa.2012Oct1;745:64‐9.ExposureMoreno‐GonzÃ¡lezR,CampilloJA,GarcÃaV,LeÃ3nVM2013JulMoreno‐GonzÃ¡lezR,CampilloJA,GarcÃaV,LeÃ3nVM.SeasonalinputofregulatedandemergingorganicpollutantsthroughsurfacewatercoursestoaMediterraneancoastallagoon.Chemosphere.2013Jul;92(3):247‐57.ExposureMoreno‐GonzÃ¡lezR,CampilloJA,LeÃ3nVM2013DecMoreno‐GonzÃ¡lezR,CampilloJA,LeÃ3nVM.InfluenceofanintensiveagriculturaldrainagebasinontheseasonaldistributionoforganicpollutantsinseawaterfromaMediterraneancoastallagoon(MarMenor,SESpain).MarPollutBull.2013Dec15;77(1‐2):400‐11.ExposureMoscosoF,TeijizI,DeiveFJ,SanromÃ¡nMA2013SepMoscosoF,TeijizI,DeiveFJ,SanromÃ¡nMA.Approachingchlorpyrifosbioeliminationatbenchscalebioreactor.BioprocessBiosystEng.2013Sep;36(9):1303‐9.ExposureMoussaouiY,TuduriL,KerchichY,MeklatiBY,EppeG2012JulMoussaouiY,TuduriL,KerchichY,MeklatiBY,EppeG.AtmosphericconcentrationsofPCDD/Fs,dl‐PCBsandsomepesticidesinnorthernAlgeriausingpassiveairsampling.Chemosphere.2012Jul;88(3):270‐7.ExposureMugniH,DemetrioP,ParacampoA,PardiM,BulusG,BonettoC2012JulMugniH,DemetrioP,ParacampoA,PardiM,BulusG,BonettoC.Toxicitypersistenceinrunoffwaterandsoilinexperimentalsoybeanplotsfollowingchlorpyrifosapplication.BullEnvironContamToxicol.2012Jul;89(1):208‐12.ExposureMuhammadF,AwaisMM,AkhtarM,AnwarMI2013JanMuhammadF,AwaisMM,AkhtarM,AnwarMI.Quantitativestructureactivityrelationshipandriskanalysisofsomepesticidesinthegoatmilk.IranianJEnvironHealthSciEng.2013Jan4;10(1):4.Chlorpyrifos Human Health Risk Assessment D424485Page483of531ExposureMunoz‐QuezadaMT,IglesiasV,LuceroB,SteenlandK,BarrDB,LevyK,RyanPB,AlvaradoS,ConchaC2012OctMunoz‐QuezadaMT,IglesiasV,LuceroB,SteenlandK,BarrDB,LevyK,RyanPB,AlvaradoS,ConchaC.PredictorsofexposuretoorganophosphatepesticidesinschoolchildrenintheProvinceofTalca,Chile.EnvironInt.2012Oct15;47:28‐36.ExposureNagarajanG,KhanZS,UttureSC,DasguptaS,BanerjeeK2013NovNagarajanG,KhanZS,UttureSC,DasguptaS,BanerjeeK.Ensuringselectivityandsensitivitybytimed‐andultra‐selectivereactionmonitoringduringgaschromatography‐tandemmassspectrometricdeterminationofpesticides.JChromatogrA.2013Nov29;1318:226‐33.ExposureNavarroP,MoraisS,GabaldÃ3nJA,PÃ©rezAJ,PuchadesR,MaquieiraA2013JunNavarroP,MoraisS,GabaldÃ3nJA,PÃ©rezAJ,PuchadesR,MaquieiraA.Arraysondiscforscreeningandquantificationofpollutants.AnalChimActa.2013Jun19;784:59‐64.ExposureNavarroP,PÃ©rezAJ,GabaldÃ3nJA,NÃoÃ±ez‐DelicadoE,PuchadesR,MaquieiraA,MoraisS2013NovNavarroP,PÃ©rezAJ,GabaldÃ3nJA,NÃoÃ±ez‐DelicadoE,PuchadesR,MaquieiraA,MoraisS.Detectionofchemicalresiduesintangerinejuicesbyadupleximmunoassay.Talanta.2013Nov15;116:33‐8.ExposureNougadÃ ̈reA,SirotV,KadarA,FastierA,TruchotE,VergnetC,HommetF,BaylÃ©J,GrosP,LeblancJC2012SepNougadÃ ̈reA,SirotV,KadarA,FastierA,TruchotE,VergnetC,HommetF,BaylÃ©J,GrosP,LeblancJC.TotaldietstudyonpesticideresiduesinFrance:levelsinfoodasconsumedandchronicdietaryrisktoconsumers.EnvironInt.2012Sep15;45:135‐50.ExposureOdabasiM,CetinB2012DecOdabasiM,CetinB.Determinationofoctanol‐airpartitioncoefficientsoforganochlorinepesticides(OCPs)asafunctionoftemperature:applicationtoair‐soilexchange.JEnvironManage.2012Dec30;113:432‐9.ExposureOelligC,SchwackW2012OctOelligC,SchwackW.Planarsolidphaseextractionclean‐upforpesticideresidueanalysisinteabyliquidchromatography‐massspectrometry.JChromatogrA.2012Oct19;1260:42‐53.Chlorpyrifos Human Health Risk Assessment D424485Page484of531ExposureOliverDP,PanYF,AndersonJS,LinTF,KookanaRS,DouglasGB,WendlingLA2013JanOliverDP,PanYF,AndersonJS,LinTF,KookanaRS,DouglasGB,WendlingLA.Sorptionofpesticidesbyamineralsandminingby‐product,neutralisedusedacid(NUA).SciTotalEnviron.2013Jan1;442:255‐62.ExposureOmerogluPY,AmbrusA,BoyaciogluD2013OmerogluPY,AmbrusA,BoyaciogluD.Estimationoftheuncertaintiesofextractionandclean‐upstepsinpesticideresidueanalysisofplantcommodities.FoodAdditContamPartAChemAnalControlExpoRiskAssess.2013;30(2):308‐20.ExposureOsmanR,SaimN,JuahirH,AbdullahMP2012JanOsmanR,SaimN,JuahirH,AbdullahMP.ChemometricapplicationinidentifyingsourcesoforganiccontaminantsinLangatriverbasin.EnvironMonitAssess.2012Jan;184(2):1001‐14.ExposureOtienoPO,OwuorPO,LalahJO,PfisterG,SchrammKW2013MarOtienoPO,OwuorPO,LalahJO,PfisterG,SchrammKW.Impactsofclimate‐inducedchangesonthedistributionofpesticidesresiduesinwaterandsedimentofLakeNaivasha,Kenya.EnvironMonitAssess.2013Mar;185(3):2723‐33.ExposureOtienoPO,OwuorPO,LalahJO,PfisterG,SchrammKW2013DecOtienoPO,OwuorPO,LalahJO,PfisterG,SchrammKW.ComparativeevaluationofELISAkitandHPLCDADforthedeterminationofchlorpyrifosethylresiduesinwaterandsediments.Talanta.2013Dec15;117:250‐7.ExposureOtienoPO,SchrammKW,PfisterG,LalahJO,OjwachSO,ViraniM2012AprOtienoPO,SchrammKW,PfisterG,LalahJO,OjwachSO,ViraniM.Spatialdistributionandtemporaltrendinconcentrationofcarbofuran,diazinonandchlorpyrifosethylresiduesinsedimentandwaterinLakeNaivasha,Kenya.BullEnvironContamToxicol.2012Apr;88(4):526‐32.ExposurePelitFO,ErtaÅŸH,SeyraniI,ErtaÅŸFN2013MayPelitFO,ErtaÅŸH,SeyraniI,ErtaÅŸFN.AssessmentofDFG‐S19methodforthedeterminationofcommonendocrinedisruptorpesticidesinwinesampleswithanestimationoftheuncertaintyoftheanalyticalresults.FoodChem.2013May1;138(1):54‐61.Chlorpyrifos Human Health Risk Assessment D424485Page485of531ExposurePelitFO,PelitL,ErtaÅŸH,NilErtaÅŸF2012SepPelitFO,PelitL,ErtaÅŸH,NilErtaÅŸF.DevelopmentofagaschromatographicmethodforthedeterminationofChlorpyrifosanditsmetaboliteChlorpyrifos‐oxoninwinematrix.JChromatogrBAnalytTechnolBiomedLifeSci.2012Sep1;904:35‐41.ExposurePelitL,DizdaÅŸTN2013OctPelitL,DizdaÅŸTN.Preparationandapplicationofapolythiophenesolid‐phasemicroextractionfiberforthedeterminationofendocrine‐disruptorpesticidesinwellwaters.JSepSci.2013Oct;36(19):3234‐41.ExposurePhungDT,ConnellD,MillerG,ChuC2012JulPhungDT,ConnellD,MillerG,ChuC.ProbabilisticassessmentofchlorpyrifosexposuretoricefarmersinVietNam.JExpoSciEnvironEpidemiol.2012Jul;22(4):417‐23.ExposurePhungDT,ConnellD,MillerG,HodgeM,PatelR,ChengR,AbeyewardeneM,ChuC2012AprPhungDT,ConnellD,MillerG,HodgeM,PatelR,ChengR,AbeyewardeneM,ChuC.BiologicalmonitoringofchlorpyrifosexposuretoricefarmersinVietnam.Chemosphere.2012Apr;87(4):294‐300.ExposureQuerejetaGA,RamosLM,FloresAP,HughesEA,ZaltsA,MontserratJM2012AprQuerejetaGA,RamosLM,FloresAP,HughesEA,ZaltsA,MontserratJM.Environmentalpesticidedistributioninhorticulturalandfloriculturalperiurbanproductionunits.Chemosphere.2012Apr;87(5):566‐72.ExposureRaniM,SainiS,KumariB2013SepRaniM,SainiS,KumariB.Persistenceandeffectofprocessingonchlorpyriphosresiduesintomato(LycopersiconesculantumMill.).EcotoxicolEnvironSaf.2013Sep;95:247‐52.ExposureRaniM,SainiS,KumariB2014JanRaniM,SainiS,KumariB.Leachingbehaviourofchlorpyriphosandcypermethrininsandyloamsoil.EnvironMonitAssess.2014Jan;186(1):175‐82.Chlorpyrifos Human Health Risk Assessment D424485Page486of531ExposureRobles‐MolinaJ,MartÃndeVidalesMJ,GarcÃa‐ReyesJF,CaÃ±izaresP,SÃ¡ezC,RodrigoMA,Molina‐DÃazA2012NovRobles‐MolinaJ,MartÃndeVidalesMJ,GarcÃa‐ReyesJF,CaÃ±izaresP,SÃ¡ezC,RodrigoMA,Molina‐DÃazA.Conductive‐diamondelectrochemicaloxidationofchlorpyrifosinwastewaterandidentificationofitsmaindegradationproductsbyLC‐TOFMS.Chemosphere.2012Nov;89(10):1169‐76.ExposureRocaM,Miralles‐MarcoA,FerrÃ©J,PÃ©rezR,YusÃV2014MayRocaM,Miralles‐MarcoA,FerrÃ©J,PÃ©rezR,YusÃV.BiomonitoringexposureassessmenttocontemporarypesticidesinaschoolchildrenpopulationofSpain.EnvironRes.2014May;131:77‐85.ExposureRodrÃguezT,vanWendeldeJoodeB,LindhCH,RojasM,LundbergI,WesselingC2012FebRodriguezT,vanWendeldeJoodeB,LindhCH,RojasM,LundbergI,WesselingC.Assessmentoflong‐termandrecentpesticideexposureamongruralschoolchildreninNicaragua.OccupEnvironMed.2012Feb;69(2):119‐25.ExposureRojasR,MorilloJ,UseroJ,Delgado‐MorenoL,GanJ2013AugRojasR,MorilloJ,UseroJ,Delgado‐MorenoL,GanJ.Enhancingsoilsorptioncapacityofanagriculturalsoilbyadditionofthreedifferentorganicwastes.SciTotalEnviron.2013Aug1;458‐460:614‐23.ExposureRotariuL,ZamfirLG,BalaC2012OctRotariuL,ZamfirLG,BalaC.Arationaldesignofthemultiwalledcarbonnanotube‐7,7,8,8‐tetracyanoquinodimethansensorforsensitivedetectionofacetylcholinesteraseinhibitors.AnalChimActa.2012Oct20;748:81‐8.ExposureSamadiS,SereshtiH,AssadiY2012JanSamadiS,SereshtiH,AssadiY.Ultra‐preconcentrationanddeterminationofthirteenorganophosphoruspesticidesinwatersamplesusingsolid‐phaseextractionfollowedbydispersiveliquid‐liquidmicroextractionandgaschromatographywithflamephotometricdetection.JChromatogrA.2012Jan6;1219:61‐5.Chlorpyrifos Human Health Risk Assessment D424485Page487of531ExposureSangchanW,BannwarthM,IngwersenJ,HugenschmidtC,SchwadorfK,ThavornyutikarnP,PansombatK,StreckT2014FebSangchanW,BannwarthM,IngwersenJ,HugenschmidtC,SchwadorfK,ThavornyutikarnP,PansombatK,StreckT.MonitoringandriskassessmentofpesticidesinatropicalriverofanagriculturalwatershedinnorthernThailand.EnvironMonitAssess.2014Feb;186(2):1083‐99.ExposureSarkouhiM,ShamsipurM,HassanJ2012DecSarkouhiM,ShamsipurM,HassanJ.Â3Â1P‐NMRevaluationoforganophosphoruspesticidesdegradationthroughmetalionpromotedhydrolysis.EnvironMonitAssess.2012Dec;184(12):7383‐93.ExposureSasikalaC,JiwalS,RoutP,RamyaM2012MarSasikalaC,JiwalS,RoutP,RamyaM.Biodegradationofchlorpyrifosbybacterialconsortiumisolatedfromagriculturesoil.WorldJMicrobiolBiotechnol.2012Mar;28(3):1301‐8.ExposureSeebunruengK,SantaladchaiyakitY,SrijaranaiS2012SepSeebunruengK,SantaladchaiyakitY,SrijaranaiS.Studyontheeffectofchain‐lengthcompatibilityofmixedanionic‐cationicsurfactantsonthecloud‐pointextractionofselectedorganophosphoruspesticides.AnalBioanalChem.2012Sep;404(5):1539‐48.ExposureShahpouryP,HagemanKJ,MatthaeiCD,MagbanuaFS2013OctShahpouryP,HagemanKJ,MatthaeiCD,MagbanuaFS.Chlorinatedpesticidesinstreamsedimentsfromorganic,integratedandconventionalfarms.EnvironPollut.2013Oct;181:219‐25.ExposureSharmaI,BhardwajR,PatiPK2012NovSharmaI,BhardwajR,PatiPK.Mitigationofadverseeffectsofchlorpyrifosby24‐epibrassinolideandanalysisofstressmarkersinaricevarietyPusaBasmati‐1.EcotoxicolEnvironSaf.2012Nov;85:72‐81.ExposureShiH,ZhaoG,LiuM,FanL,CaoT2013SepShiH,ZhaoG,LiuM,FanL,CaoT.Aptamer‐basedcolorimetricsensingofacetamipridinsoilsamples:sensitivity,selectivityandmechanism.JHazardMater.2013Sep15;260:754‐61.Chlorpyrifos Human Health Risk Assessment D424485Page488of531ExposureShinHM,McKoneTE,TulveNS,CliftonMS,BennettDH2013JanShinHM,McKoneTE,TulveNS,CliftonMS,BennettDH.Indoorresidencetimesofsemivolatileorganiccompounds:modelestimationandfieldevaluation.EnvironSciTechnol.2013Jan15;47(2):859‐67.ExposureShingWL,HengLY,SurifS2013MayShingWL,HengLY,SurifS.Performanceofacyanobacteriawholecell‐basedfluorescencebiosensorforheavymetalandpesticidedetection.Sensors(Basel).2013May14;13(5):6394‐404.ExposureSilvaE,PereiraAC,EstalagemSP,Moreira‐SantosM,RibeiroR,CerejeiraMJ2012Sep‐OctSilvaE,PereiraAC,EstalagemSP,Moreira‐SantosM,RibeiroR,CerejeiraMJ.AssessingthequalityoffreshwatersinaprotectedareawithintheTagusRiverbasindistrict(centralPortugal).JEnvironQual.2012Sep‐Oct;41(5):1413‐26.ExposureSmallingKL,KuivilaKM,OrlandoJL,PhillipsBM,AndersonBS,SieglerK,HuntJW,HamiltonM2013AugSmallingKL,KuivilaKM,OrlandoJL,PhillipsBM,AndersonBS,SieglerK,HuntJW,HamiltonM.Environmentalfateoffungicidesandothercurrent‐usepesticidesinacentralCaliforniaestuary.MarPollutBull.2013Aug15;73(1):144‐53.ExposureSreeprasadTS,GuptaSS,MaliyekkalSM,PradeepT2013FebSreeprasadTS,GuptaSS,MaliyekkalSM,PradeepT.Immobilizedgraphene‐basedcompositefromasphalt:facilesynthesisandapplicationinwaterpurification.JHazardMater.2013Feb15;246‐247:213‐20.ExposureSrinivasPS,BanerjeeK,JadhavMR,GhasteMS,LawandeKE2012SrinivasPS,BanerjeeK,JadhavMR,GhasteMS,LawandeKE.Bioefficacy,dissipationkineticsandsafetyevaluationofselectedinsecticidesinAlliumcepaL.JEnvironSciHealthB.2012;47(7):700‐9.ExposureStarnerK,GohKS2013SepStarnerK,GohKS.Chlorpyrifos‐treatedcropsinthevicinityofsurfacewatercontaminationintheSanJoaquinValley,California,USA.BullEnvironContamToxicol.2013Sep;91(3):287‐91.ExposureSuciuNA,FerrariF,VasileiadisS,MerliA,CapriE,TrevisanM2013SuciuNA,FerrariF,VasileiadisS,MerliA,CapriE,TrevisanM.Pesticideswaterdecontaminationinoxygen‐limitedconditions.JEnvironSciHealthB.2013;48(9):793‐9.Chlorpyrifos Human Health Risk Assessment D424485Page489of531ExposureSunX,CaoY,GongZ,WangX,ZhangY,GaoJ2012DecSunX,CaoY,GongZ,WangX,ZhangY,GaoJ.Anamperometricimmunosensorbasedonmulti‐walledcarbonnanotubes‐thionine‐chitosannanocompositefilmforchlorpyrifosdetection.Sensors(Basel).2012Dec13;12(12):17247‐61.ExposureSunX,ZhaiC,WangX2013MarSunX,ZhaiC,WangX.Anovelandhighlysensitiveacetyl‐cholinesterasebiosensormodifiedwithhollowgoldnanospheres.BioprocessBiosystEng.2013Mar;36(3):273‐83.ExposureSwarnamTP,VelmuruganA2013JulSwarnamTP,VelmuruganA.PesticideresiduesinvegetablesamplesfromtheAndamanIslands,India.EnvironMonitAssess.2013Jul;185(7):6119‐27.ExposureTiwariMK,GuhaS2013OctTiwariMK,GuhaS.Simultaneousanalysisofendosulfan,chlorpyrifos,andtheirmetabolitesinnaturalsoilandwatersamplesusinggaschromatography‐tandemmassspectrometry.EnvironMonitAssess.2013Oct;185(10):8451‐63.ExposureTortellaGR,RubilarO,CastilloMd,CeaM,Mella‐HerreraR,DiezMC2012JunTortellaGR,RubilarO,CastilloMd,CeaM,Mella‐HerreraR,DiezMC.Chlorpyrifosdegradationinabiomixtureofbiobedatdifferentmaturitystages.Chemosphere.2012Jun;88(2):224‐8.ExposureUrrutiaC,RubilarO,TortellaGR,DiezMC2013AugUrrutiaC,RubilarO,TortellaGR,DiezMC.Degradationofpesticidemixtureonmodifiedmatrixofabiopurificationsystemwithalternativeslignocellulosicwastes.Chemosphere.2013Aug;92(10):1361‐6.ExposurevanWendeldeJoodeB,BarrazaD,RuepertC,MoraAM,CÃ3rdobaL,ObergM,WesselingC,MerglerD,LindhCH2012AugvanWendeldeJoodeB,BarrazaD,RuepertC,MoraAM,CÃ3rdobaL,ObergM,WesselingC,MerglerD,LindhCH.Indigenouschildrenlivingnearbyplantationswithchlorpyrifos‐treatedbagshaveelevated3,5,6‐trichloro‐2‐pyridinol(TCPy)urinaryconcentrations.EnvironRes.2012Aug;117:17‐26.Chlorpyrifos Human Health Risk Assessment D424485Page490of531ExposureVogtR,BennettD,CassadyD,FrostJ,RitzB,Hertz‐PicciottoI2012NovVogtR,BennettD,CassadyD,FrostJ,RitzB,Hertz‐PicciottoI.Cancerandnon‐cancerhealtheffectsfromfoodcontaminantexposuresforchildrenandadultsinCalifornia:ariskassessment.EnvironHealth.2012Nov9;11:83.ExposureWalorczykS,DroÅ1⁄4dÅ1⁄4yÅ„skiD2012AugWalorczykS,DroÅ1⁄4dÅ1⁄4yÅ„skiD.Improvementandextensiontonewanalytesofamulti‐residuemethodforthedeterminationofpesticidesincerealsanddryanimalfeedusinggaschromatography‐tandemquadrupolemassspectrometryrevisited.JChromatogrA.2012Aug17;1251:219‐31.ExposureWangC,ZhouQ,ZhangL,ZhangY,XiaoE,WuZ2013WangC,ZhouQ,ZhangL,ZhangY,XiaoE,WuZ.Variationcharacteristicsofchlorpyrifosinnonsterilewetlandplanthydroponicsystem.IntJPhytoremediation.2013;15(6):550‐60.ExposureWangP,DaiW,GeL,YanM,GeS,YuJ2013FebWangP,DaiW,GeL,YanM,GeS,YuJ.VisiblelightphotoelectrochemicalsensorbasedonAunanoparticlesandmolecularlyimprintedpoly(o‐phenylenediamine)‐modifiedTiO2nanotubesforspecificandsensitivedetectionchlorpyrifos.Analyst.2013Feb21;138(3):939‐45.ExposureWangQ,YangJ,LiC,XiaoB,QueX2013WangQ,YangJ,LiC,XiaoB,QueX.InfluenceofinitialpesticideconcentrationsinwateronchlorpyrifostoxicityandremovalbyIrispseudacorus.WaterSciTechnol.2013;67(9):1908‐15.ExposureWangS,WangZ,ZhangY,WangJ,GuoR2013JunWangS,WangZ,ZhangY,WangJ,GuoR.PesticideresiduesinmarketfoodsinShaanxiProvinceofChinain2010.FoodChem.2013Jun1;138(2‐3):2016‐25.ExposureWangZ,HuangJ,ChenJ,LiF2013AugWangZ,HuangJ,ChenJ,LiF.Effectivenessofdishwashingliquidsinremovingchlorothalonilandchlorpyrifosresiduesfromcherrytomatoes.Chemosphere.2013Aug;92(8):1022‐8.Chlorpyrifos Human Health Risk Assessment D424485Page491of531ExposureWangZW,HuangJ,ChenJY,LiFL2013JulWangZW,HuangJ,ChenJY,LiFL.Time‐dependentmovementanddistributionofchlorothalonilandchlorpyrifosintomatoes.EcotoxicolEnvironSaf.2013Jul;93:107‐11.ExposureWasonSC,JulienR,PerryMJ,SmithTJ,LevyJI2013JulWasonSC,JulienR,PerryMJ,SmithTJ,LevyJI.Modelingexposurestoorganophosphatesandpyrethroidsforchildrenlivinginanurbanlow‐incomeenvironment.EnvironRes.2013Jul;124:13‐22.ExposureWeiW,ZongX,WangX,YinL,PuY,LiuS2012DecWeiW,ZongX,WangX,YinL,PuY,LiuS.Adisposableamperometricimmunosensorforchlorpyrifos‐methylbasedonimmunogen/platinumdopedsilicasol‐gelfilmmodifiedscreen‐printedcarbonelectrode.FoodChem.2012Dec1;135(3):888‐92.ExposureWeschlerCJ,NazaroffWW2012OctWeschlerCJ,NazaroffWW.SVOCexposureindoors:freshlookatdermalpathways.IndoorAir.2012Oct;22(5):356‐77.ExposureWestonDP,DingY,ZhangM,LydyMJ2013JanWestonDP,DingY,ZhangM,LydyMJ.Identifyingthecauseofsedimenttoxicityinagriculturalsediments:theroleofpyrethroidsandnineseldom‐measuredhydrophobicpesticides.Chemosphere.2013Jan;90(3):958‐64.ExposureWoffordP,SegawaR,SchreiderJ,FederighiV,NealR,BrattesaniM2014MarWoffordP,SegawaR,SchreiderJ,FederighiV,NealR,BrattesaniM.Communityairmonitoringforpesticides.Part3:usinghealth‐basedscreeninglevelstoevaluateresultscollectedforayear.EnvironMonitAssess.2014Mar;186(3):1355‐70.ExposureYangL,WangG,LiuY,WangM2013SepYangL,WangG,LiuY,WangM.DevelopmentofabiosensorbasedonimmobilizationofacetylcholinesteraseonNiOnanoparticles‐carboxylicgraphene‐nafionmodifiedelectrodefordetectionofpesticides.Talanta.2013Sep15;113:135‐41.ExposureYangL,WangGC,LiuYJ,AnJJ,WangM2013MarYangL,WangGC,LiuYJ,AnJJ,WangM.DevelopmentofastablebiosensorbasedonaSiO2nanosheet‐Nafion‐modifiedglassycarbonelectrodeforsensitivedetectionofpesticides.AnalBioanalChem.2013Mar;405(8):2545‐52.Chlorpyrifos Human Health Risk Assessment D424485Page492of531ExposureYaoGH,LiangRP,HuangCF,WangY,QiuJD2013DecYaoGH,LiangRP,HuangCF,WangY,QiuJD.Surfaceplasmonresonancesensorbasedonmagneticmolecularlyimprintedpolymersamplificationforpesticiderecognition.AnalChem.2013Dec17;85(24):11944‐51.ExposureZhaiC,SunX,ZhaoW,GongZ,WangX2013AprZhaiC,SunX,ZhaoW,GongZ,WangX.Acetylcholinesterasebiosensorbasedonchitosan/prussianblue/multiwallcarbonnanotubes/hollowgoldnanospheresnanocompositefilmbyone‐stepelectrodeposition.BiosensBioelectron.2013Apr15;42:124‐30.ExposureZhanY,ZhangM2012AugZhanY,ZhangM.PURE:aweb‐baseddecisionsupportsystemtoevaluatepesticideenvironmentalriskforsustainablepestmanagementpracticesinCalifornia.EcotoxicolEnvironSaf.2012Aug;82:104‐13.ExposureZhangQ,WangB,CaoZ,YuY2012JunZhangQ,WangB,CaoZ,YuY.Plasmid‐mediatedbioaugmentationforthedegradationofchlorpyrifosinsoil.JHazardMater.2012Jun30;221‐222:178‐84.ExposureZhangW,TangY,DuD,SmithJ,TimchalkC,LiuD,LinY2013SepZhangW,TangY,DuD,SmithJ,TimchalkC,LiuD,LinY.DirectanalysisoftrichloropyridinolinhumansalivausinganAunanoparticles‐basedimmunochromatographicteststripforbiomonitoringofexposuretochlorpyrifos.Talanta.2013Sep30;114:261‐7.ExposureZhangX,ShenY,YuXY,LiuXJ2012AprZhangX,ShenY,YuXY,LiuXJ.Dissipationofchlorpyrifosandresidueanalysisinrice,soilandwaterunderpaddyfieldconditions.EcotoxicolEnvironSaf.2012Apr;78:276‐80.ExposureZhangX,StarnerK,SpurlockF2012NovZhangX,StarnerK,SpurlockF.AnalysisofchlorpyrifosagriculturaluseinregionsoffrequentsurfacewaterdetectionsinCalifornia,USA.BullEnvironContamToxicol.2012Nov;89(5):978‐84.Chlorpyrifos Human Health Risk Assessment D424485Page493of531ExposureZhangY,AnJ,YeW,YangG,QianZG,ChenHF,CuiL,FengY2012SepZhangY,AnJ,YeW,YangG,QianZG,ChenHF,CuiL,FengY.Enhancingthepromiscuousphosphotriesteraseactivityofathermostablelactonase(GkaP)fortheefficientdegradationoforganophosphatepesticides.ApplEnvironMicrobiol.2012Sep;78(18):6647‐55.ExposureZhaoX,WuC,WangY,CangT,ChenL,YuR,WangQ2012FebZhaoX,WuC,WangY,CangT,ChenL,YuR,WangQ.AssessmentoftoxicityriskofinsecticidesusedinriceecosystemonTrichogrammajaponicum,aneggparasitoidofricelepidopterans.JEconEntomol.2012Feb;105(1):92‐101.ExposureZhaoY,WangC,WendlingLA,PeiY2013AugZhaoY,WangC,WendlingLA,PeiY.Feasibilityofusingdrinkingwatertreatmentresidualsasanovelchlorpyrifosadsorbent.JAgricFoodChem.2013Aug7;61(31):7446‐52.ExposureZhongG,XieZ,CaiM,MÃ¶llerA,SturmR,TangJ,ZhangG,HeJ,EbinghausR2012JanZhongG,XieZ,CaiM,MÃ¶llerA,SturmR,TangJ,ZhangG,HeJ,EbinghausR.Distributionandair‐seaexchangeofcurrent‐usepesticides(CUPs)fromEastAsiatothehighArcticOcean.EnvironSciTechnol.2012Jan3;46(1):259‐67.ExposureZhouJ,LuQ,TongY,WeiW,LiuS2012SepZhouJ,LuQ,TongY,WeiW,LiuS.DetectionofDNAdamagebyusinghairpinmolecularbeaconprobesandgrapheneoxide.Talanta.2012Sep15;99:625‐30.ExposureGonzÃ¡lez‐CurbeloMA,Asensio‐RamosM,Herrera‐HerreraAV,HernÃ¡ndez‐BorgesJ2012JulGonzÃ¡lez‐CurbeloMA,Asensio‐RamosM,Herrera‐HerreraAV,HernÃ¡ndez‐BorgesJ.Pesticideresidueanalysisincereal‐basedbabyfoodsusingmulti‐walledcarbonnanotubesdispersivesolid‐phaseextraction.AnalBioanalChem.2012Jul;404(1):183‐96.ExposureLinYZ,ZengGM,ZhangY,ChenM,JiangM,ZhangJC,LuLH,LiuLF2012MarLinYZ,ZengGM,ZhangY,ChenM,JiangM,ZhangJC,LuLH,LiuLF.[BiodegradationmechanismofDDTandchlorpyrifosusingmolecularsimulation].HuanJingKeXue.2012Mar;33(3):1015‐9.Exposure/BiomarkersAdolfsson‐EriciM,Ã...KermanG,McLachlanMS2012JunAdolfsson‐EriciM,Ã...KermanG,McLachlanMS.In‐vivopassivesamplingtomeasureeliminationkineticsinbioaccumulationtests.Chemosphere.2012Jun;88(1):62‐8.Chlorpyrifos 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D424485Page494of531Exposure/BiomarkersDingY,LandrumPF,YouJ,HarwoodAD,LydyMJ2012SepDingY,LandrumPF,YouJ,HarwoodAD,LydyMJ.Useofsolidphasemicroextractiontoestimatetoxicity:relatingfiberconcentrationstotoxicity‐‐partI.EnvironToxicolChem.2012Sep;31(9):2159‐67.Exposure/BiomarkersSunH,SiC,BianQ,ChenX,ChenL,WangX2012AugSunH,SiC,BianQ,ChenX,ChenL,WangX.Developinginvitroreportergeneassaystoassessthehormonereceptoractivitiesofchemicalsfrequentlydetectedindrinkingwater.JApplToxicol.2012Aug;32(8):635‐41.HighDoseNutterTJ,CooperBY2014JunNutterTJ,CooperBY.PersistentmodificationofNav1.9followingchronicexposuretoinsecticidesandpyridostigminebromide.ToxicolApplPharmacol.2014Jun15;277(3):298‐309.HighDoseYildirim,E.,BaydanE,KanburM,KulO,CÄ±narM,EkiciH,AtmacaN2013Yildirim,E.,BaydanE,KanburM,KulO,CinarM,EkiciH,AtmacaN.Theeffectofchlorpyrifosonisolatedthoracicaortainrats.BiomedResInt.2013;2013:376051.HighDose/MixtureNutterTJ,JiangN,CooperBY2013DecNutterTJ,JiangN,CooperBY.PersistentNa+andK+channeldysfunctionsafterchronicexposuretoinsecticidesandpyridostigminebromide.Neurotoxicology.2013Dec;39:72‐83.HighDose/MixtureOjhaA,SrivastavaN2012JanOjhaA,SrivastavaN.Redoximbalanceinrattissuesexposedwithorganophosphatepesticidesandtherapeuticpotentialofantioxidantvitamins.EcotoxicolEnvironSaf.2012Jan;75(1):230‐41.HighDoses/Acute/AdultsSandhuMA,SaeedAA,KhiljiMS,AhmedA,LatifMS,KhalidN2013SandhuMA,SaeedAA,KhiljiMS,AhmedA,LatifMS,KhalidN.Genotoxicityevaluationofchlorpyrifos:agenderrelatedapproachinregulartoxicitytesting.JToxicolSci.2013;38(2):237‐44.Chlorpyrifos Human Health Risk Assessment D424485Page495of531MethodsPaper(BiosensorandBiomonitorApproach)CamannDE,SchultzST,YauAY,HeilbrunLP,ZunigaMM,PalmerRF,MillerCS2013MarCamannDE,SchultzST,YauAY,HeilbrunLP,ZunigaMM,PalmerRF,MillerCS.Acetaminophen,pesticide,anddiethylhexylphthalatemetabolites,anandamide,andfattyacidsindeciduousmolars:potentialbiomarkersofperinatalexposure.JExpoSciEnvironEpidemiol.2013Mar;23(2):190‐6.MethodsPaper(BiosensorandBiomonitorApproach)HendersonJD,GlucksmanG,LeongB,TigyiA,AnkirskaiaA,SiddiqueI,LamH,DePetersE,WilsonBW2012JanHendersonJD,GlucksmanG,LeongB,TigyiA,AnkirskaiaA,SiddiqueI,LamH,DePetersE,WilsonBW.Pyridostigminebromideprotectionagainstacetylcholinesteraseinhibitionbypesticides.JBiochemMolToxicol.2012Jan;26(1):31‐4.MethodsPaper(BiosensorandBiomonitorApproach)JiangW,DuysenEG,LockridgeO2012MayJiangW,DuysenEG,LockridgeO.Micetreatedwithanontoxicdoseofchlorpyrifosoxonhavediethoxyphosphotyrosinelabeledproteinsinbloodupto4dayspostexposure,detectedbymassspectrometry.Toxicology.2012May16;295(1‐3):15‐22.MethodsPaper(BiosensorandBiomonitorApproach)JolyC,Gay‐QuÃ©heillardJ,LÃ©kÃ©A,ChardonK,DelanaudS,BachV,Khorsi‐CauetH2013MayJolyC,Gay‐QuÃ©heillardJ,LÃ©kÃ©A,ChardonK,DelanaudS,BachV,Khorsi‐CauetH.ImpactofchronicexposuretolowdosesofchlorpyrifosontheintestinalmicrobiotaintheSimulatoroftheHumanIntestinalMicrobialEcosystem(SHIME)andintherat.EnvironSciPollutResInt.2013May;20(5):2726‐34.MethodsPaper(BiosensorandBiomonitorApproach)JonesOA,SwainSC,SvendsenC,GriffinJL,SturzenbaumSR,SpurgeonDJ2012FebJonesOA,SwainSC,SvendsenC,GriffinJL,SturzenbaumSR,SpurgeonDJ.PotentialnewmethodofmixtureeffectstestingusingmetabolomicsandCaenorhabditiselegans.JProteomeRes.2012Feb3;11(2):1446‐53.MethodsPaper(BiosensorandBiomonitorApproach)MaravgakisG,TzatzarakisMN,AlegakisAK,StivaktakisPD,TsatsakisAM2012MayMaravgakisG,TzatzarakisMN,AlegakisAK,StivaktakisPD,TsatsakisAM.Diethylphosphatesaccumulationinrabbits'hairasanindicatoroflongtermexposuretodiazinonandchlorpyrifos.ForensicSciInt.2012May10;218(1‐3):106‐10.Chlorpyrifos 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D424485Page496of531MethodsPaper(BiosensorandBiomonitorApproach)MultariRA,CremersDA,ScottT,KendrickP2013MarMultariRA,CremersDA,ScottT,KendrickP.Detectionofpesticidesanddioxinsintissuefatsandrenderingoilsusinglaser‐inducedbreakdownspectroscopy(LIBS).JAgricFoodChem.2013Mar13;61(10):2348‐57.MethodsPaper(BiosensorandBiomonitorApproach)PapoutsisI,MendonisM,NikolaouP,AthanaselisS,PistosC,MaraveliasC,SpiliopoulouC2012MayPapoutsisI,MendonisM,NikolaouP,AthanaselisS,PistosC,MaraveliasC,SpiliopoulouC.DevelopmentandvalidationofasimpleGC‐MSmethodforthesimultaneousdeterminationof11anticholinesterasepesticidesinblood‐‐clinicalandforensictoxicologyapplications.JForensicSci.2012May;57(3):806‐12.MethodsPaper(BiosensorandBiomonitorApproach)SchopferLM,LockridgeO2012Mar‐AprSchopferLM,LockridgeO.Analyticalapproachesformonitoringexposuretoorganophosphorusandcarbamateagentsthroughanalysisofproteinadducts.DrugTestAnal.2012Mar‐Apr;4(3‐4):246‐61.MethodsPaperforDermalAbsorptionMircioiuC,VoicuVA,IonescuM,MironDS,RadulescuFS,NicolescuAC2013MayMircioiuC,VoicuVA,IonescuM,MironDS,RadulescuFS,NicolescuAC.Evaluationofinvitroabsorption,decontaminationanddesorptionoforganophosphorouscompoundsfromskinandsyntheticmembranes.ToxicolLett.2013May23;219(2):99‐106.MethodsPaper/AlternativesforneurodevelopmentVisanA,HayessK,SittnerD,PohlEE,RiebelingC,SlawikB,GulichK,OelgeschlÃ¤gerM,LuchA,SeilerAE2012OctVisanA,HayessK,SittnerD,PohlEE,RiebelingC,SlawikB,GulichK,OelgeschlÃ¤gerM,LuchA,SeilerAE.Neuraldifferentiationofmouseembryonicstemcellsasatooltoassessdevelopmentalneurotoxicityinvitro.Neurotoxicology.2012Oct;33(5):1135‐46.MethodsPaper/Alternativesforneurodevelopment/NotreadytoestablishAOPAmaroliA,AluigiMG,FalugiC,ChessaMG2013FebAmaroliA,AluigiMG,FalugiC,ChessaMG.Effectsoftheneurotoxicthionophosphatepesticidechlorpyrifosondifferentiatingalternativemodels.Chemosphere.2013Feb;90(7):2115‐22.Chlorpyrifos 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D424485Page497of531MethodsPaper/Alternativesforneurodevelopment/NotreadytoestablishAOPCulbrethME,HarrillJA,FreudenrichTM,MundyWR,ShaferTJ2012DecCulbrethME,HarrillJA,FreudenrichTM,MundyWR,ShaferTJ.Comparisonofchemical‐inducedchangesinproliferationandapoptosisinhumanandmouseneuroprogenitorcells.Neurotoxicology.2012Dec;33(6):1499‐510.MethodsPaper/Alternativesforneurodevelopment/NotreadytoestablishAOPEstevanC,VilanovaE,SogorbMA2013FebEstevanC,VilanovaE,SogorbMA.Chlorpyrifosanditsmetabolitesaltergeneexpressionatnon‐cytotoxicconcentrationsinD3mouseembryonicstemcellsunderinvitrodifferentiation:considerationsforembryotoxicriskassessment.ToxicolLett.2013Feb13;217(1):14‐22.MethodsPaper/Alternativesforneurodevelopment/NotreadytoestablishAOPGuiÃ±azÃoN,RenaV,Genti‐RaimondiS,RiveroV,MagnarelliG2012AprGuinaza,N,RenaV,Genti‐RaimondiS,RiveroV,MagnarelliG.EffectsoftheorganophosphateinsecticidesphosmetandchlorpyrifosontrophoblastJEG‐3celldeath,proliferationandinflammatorymoleculeproduction.ToxicolInVitro.2012Apr;26(3):406‐13.MicrobialAbrahamJ,ShankerA,SilambarasanS2013DecAbrahamJ,ShankerA,SilambarasanS.RoleofGordoniaspJAAS1inbiodegradationofchlorpyrifosanditshydrolysingmetabolite3,5,6‐trichloro‐2‐pyridinol.LettApplMicrobiol.2013Dec;57(6):510‐6.MixturesCole,T.B.,Jansen,Karen,Park,Sarah,Li,Wan‐Fen,Furlong,ClementE.,Costa,LucioG.Cole,T.B.,Jansen,Karen,Park,Sarah,Li,Wan‐Fen,Furlong,ClementE.,Costa,LucioG.TheToxicityofMixturesofSpecificOrganophosphateCompoundsisModulatedbyParaoxonase1Status.S.T.Reddy(ed.),ParaoxonaseinInflammation,Infection,andToxicology,AdvancesinExperimentalMedicineandBiology660,DOI10.1007/978‐1‐60761‐350‐3_6,HumanaPress,LLC2010Chlorpyrifos 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D424485Page498of531MixturesSlotkinTA,CardJ,SeidlerFJ2013JulSlotkinTA,CardJ,SeidlerFJ.Adversebenzo[a]pyreneeffectsonneurodifferentiationarealteredbyotherneurotoxicantcoexposures:interactionswithdexamethasone,chlorpyrifos,ornicotineinPC12cells.EnvironHealthPerspect.2013Jul;121(7):825‐31.Mixtures/AcuteeffectsWilsonBW,RusliFJ,YanTamMK,DePetersE,HendersonJD2012DecWilsonBW,RusliFJ,YanTamMK,DePetersE,HendersonJD.CarbamateprotectionofAChEagainstinhibitionbyagriculturalchemicals.JBiochemMolToxicol.2012Dec;26(12):506‐9.Nobodyofevidence;SmallnumberofstudieswithoutmechanistichypothesistooutcomeLiW,EhrichM2013OctLiW,EhrichM.Transientalterationsoftheblood‐brainbarriertightjunctionandreceptorpotentialchannelgeneexpressionbychlorpyrifos.JApplToxicol.2013Oct;33(10):1187‐91.Nobodyofevidence;SmallnumberofstudieswithoutmechanistichypothesistooutcomePoteraC2012JulPoteraC.Newlydiscoveredmechanismforchlorpyrifoseffectsonneurodevelopment.EnvironHealthPerspect.2012Jul;120(7):a270‐1.Nobodyofevidence;SmallnumberofstudieswithoutmechanistichypothesistooutcomeRidanoME,RaccaAC,Flores‐MartÃnJ,CamolottoSA,dePotasGM,Genti‐RaimondiS,Panzetta‐DutariGM2012JunRidanoME,RaccaAC,Flores‐MartÃnJ,CamolottoSA,dePotasGM,Genti‐RaimondiS,Panzetta‐DutariGM.Chlorpyrifosmodifiestheexpressionofgenesinvolvedinhumanplacentalfunction.ReprodToxicol.2012Jun;33(3):331‐8.Chlorpyrifos 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D424485Page499of531Nobodyofevidence;SmallnumberofstudieswithoutmechanistichypothesistooutcomeVera,Berta,SantaCruz,Silvia,Magnarelli,Gladis2012AprilVera,Berta,SantaCruz,Silvia,Magnarelli,GladisPlasmacholinesteraseandcarboxylesteraseactivitiesandnuclearandmitochondriallipidcompositionofhumanplacentaassociatedwithmaternalexposuretopesticides.ReproductiveToxicology34(2012)402‐407.NotinEnglishÅukaszewicz‐HussainA2012Åukaszewicz‐HussainA.[Paraoxonaseactivityandlipidperoxidesconcentrationinserumofratssubchronicallyintoxicatedwithchlorpyrifos‐‐organophosphateinsecticide].MedPr.2012;63(5):559‐64.NotinEnglishTanQT,BaiHS,LiuW2012DecTanQT,BaiHS,LiuW.[DetectionofchlorpyrifosinairofworkplacewithHPLC].ZhonghuaLaoDongWeiShengZhiYeBingZaZhi.2012Dec;30(12):953‐5.NotrelatedtoneurodevelopmentalAmbaliSF,AyoJO2012MayAmbaliSF,AyoJO.VitaminCAttenuatesChronicChlorpyrifos‐inducedAlterationofNeurobehavioralParametersinWistarRats.ToxicolInt.2012May;19(2):144‐52.NotrelatedtoneurodevelopmentalDuttaAL,SahuCR2013DuttaAL,SahuCR.EmblicaofficinalisGartenfruitsextractamelioratesreproductiveinjuryandoxidativetesticulartoxicityinducedbychlorpyrifosinmalerats.Springerplus.2013;2:541.NotrelatedtoneurodevelopmentalJubergDR,GehenSC,CoadyKK,LeBaronMJ,KramerVJ,LuH,MartyMS2013AugJubergDR,GehenSC,CoadyKK,LeBaronMJ,KramerVJ,LuH,MartyMS.Chlorpyrifos:weightofevidenceevaluationofpotentialinteractionwiththeestrogen,androgen,orthyroidpathways.RegulToxicolPharmacol.2013Aug;66(3):249‐63.NotrelatedtoneurodevelopmentalMandalTK,DasNS2012AprMandalTK,DasNS.Testiculargametogenicandsteroidogenicactivitiesinchlorpyrifosinsecticide‐treatedrats:acorrelationstudywithtesticularoxidativestressandroleofantioxidantenzymedefencesystemsinSprague‐Dawleyrats.Andrologia.2012Apr;44(2):102‐15.Chlorpyrifos 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D424485Page500of531NotrelatedtoneurodevelopmentalNoroY,TomizawaM,ItoY,SuzukiH,AbeK,KamijimaM2013OctNoroY,TomizawaM,ItoY,SuzukiH,AbeK,KamijimaM.Anticholinesteraseinsecticideactionatthemurinemalereproductivesystem.BioorgMedChemLett.2013Oct1;23(19):5434‐6.NotrelatedtoneurodevelopmentalRichJD,GabrielSM,Schultz‐NortonJR2012RichJD,GabrielSM,Schultz‐NortonJR.Invitroeffectsofherbicidesandinsecticidesonhumanbreastcells.ISRNToxicol.2012;2012:232461.NotrelatedtoneurodevelopmentalSlotkinTA,CooperEM,StapletonHM,SeidlerFJ2013SepSlotkinTA,CooperEM,StapletonHM,SeidlerFJ.Doesthyroiddisruptioncontributetothedevelopmentalneurotoxicityofchlorpyrifos?EnvironToxicolPharmacol.2013Sep;36(2):284‐7.NotrelatedtoneurodevelopmentalSvenssonRU,BannickNL,MarinMJ,RobertsonLW,LynchCF,HenryMD2013SvenssonRU,BannickNL,MarinMJ,RobertsonLW,LynchCF,HenryMD.ChronicchlorpyrifosexposuredoesnotpromoteprostatecancerinprostatespecificPTENmutantmice.JEnvironPatholToxicolOncol.2013;32(1):29‐39.NotrelatedtoneurodevelopmentalTripathiS,SuzukiN,SrivastavAK2013JulTripathiS,SuzukiN,SrivastavAK.Responseofserumminerals(calcium,phosphate,andmagnesium)andendocrineglands(calcitonincellsandparathyroidgland)ofWistarratafterchlorpyrifosadministration.MicroscResTech.2013Jul;76(7):673‐8.NotrelatedtoneurodevelopmentalYaduvanshiSK,SrivastavaN,MarottaF,JainS,YadavH2012SepYaduvanshiSK,SrivastavaN,MarottaF,JainS,YadavH.Evaluationofmicronucleiinductioncapacityandmutagenicityoforganochlorineandorganophosphatepesticides.DrugMetabLett.2012Sep1;6(3):187‐97.NotrelatedtoneurodevelopmentaleffectsÅozowickaB2013NovÅozowickaB.Thedevelopment,validaƟonandapplicaƟonofaGC‐dualdetector(NPD‐ECD)multi‐pesticideresiduemethodformonitoringbeepoisoningincidents.EcotoxicolEnvironSaf.2013Nov;97:210‐22.NotrelatedtoneurodevelopmentaleffectsAckerCI,NogueiraCW2012OctAckerCI,NogueiraCW.Chlorpyrifosacuteexposureinduceshyperglycemiaandhyperlipidemiainrats.Chemosphere.2012Oct;89(5):602‐8.Chlorpyrifos 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D424485Page501of531NotrelatedtoneurodevelopmentaleffectsAhmadF,IqbalS,AnwarS,AfzalM,IslamE,MustafaT,KhanQM2012OctAhmadF,IqbalS,AnwarS,AfzalM,IslamE,MustafaT,KhanQM.Enhancedremediationofchlorpyrifosfromsoilusingryegrass(Lolliummultiflorum)andchlorpyrifos‐degradingbacteriumBacilluspumilusC2A1.JHazardMater.2012Oct30;237‐238:110‐5.NotrelatedtoneurodevelopmentaleffectsAhmadM,AkhtarS2013AprAhmadM,AkhtarS.DevelopmentofinsecticideresistanceinfieldpopulationsofBrevicorynebrassicae(Hemiptera:Aphididae)inPakistan.JEconEntomol.2013Apr;106(2):954‐8.NotrelatedtoneurodevelopmentaleffectsAlasbahiRH,MelzigMF2012JanAlasbahiRH,MelzigMF.ForskolinandderivativesastoolsforstudyingtheroleofcAMP.Pharmazie.2012Jan;67(1):5‐13.NotrelatedtoneurodevelopmentaleffectsAlviAH,SayyedAH,NaeemM,AliM2012AlviAH,SayyedAH,NaeemM,AliM.FieldevolvedresistanceinHelicoverpaarmigera(Lepidoptera:Noctuidae)toBacillusthuringiensistoxinCry1AcinPakistan.PLoSOne.2012;7(10):e47309.NotrelatedtoneurodevelopmentaleffectsAnsariMA,ButtTM2012OctAnsariMA,ButtTM.Evaluationofentomopathogenicfungiandanematodeagainstthesoil‐dwellingstagesofthecraneflyTipulapaludosa.PestManagSci.2012Oct;68(10):1337‐44.NotrelatedtoneurodevelopmentaleffectsArainMS,HuXX,LiGQ2014FebArainMS,HuXX,LiGQ.Assessmentoftoxicityandpotentialriskofbutene‐fipronilusingDrosophilamelanogaster,incomparisontonineconventionalinsecticides.BullEnvironContamToxicol.2014Feb;92(2):190‐5.NotrelatedtoneurodevelopmentaleffectsAsselmanJ,JanssenCR,SmaggheG,DeSchamphelaereKA2014MayAsselmanJ,JanssenCR,SmaggheG,DeSchamphelaereKA.EcotoxicityofbinarymixturesofMicrocystisaeruginosaandinsecticidestoDaphniapulex.EnvironPollut.2014May;188:56‐63.Chlorpyrifos 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