Chlorpyrifos Issue Paper:

Evaluation of Biomonitoring Data from Epidemiology Studies

EPA’s Office of Pesticide Programs

March 11, 2016
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

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Table of Contents

Table of Figures............................................................................................................................. 5

Table of Tables.............................................................................................................................. 6

1.0 Introduction............................................................................................................................ 7

2.0 Scope & Content of this 2016 Issue Paper ........................................................................... 9

3.0 Brief Summary of CCCEH Findings and Maternal & Cord Blood Levels.................... 11

4.0 Lifestages for Consideration ............................................................................................... 15

5.0 Pharmacokinetic Time Course: Considerations for Labor & Delivery......................... 17

6.0 Evaluation of CCCEH Cord Blood Data & Predicted Exposures to the Cohort .......... 21

6.1 Drinking Water Exposure to the CCCEH Cohort...........................................................................................21

6.2 Food Exposure to the CCCEH Cohort .............................................................................................................24

6.3 Residential Exposure to the CCCEH Cohort...................................................................................................27

6.3.1 Methods.........................................................................................................................................................28

6.3.2 Results...........................................................................................................................................................31

6.3.3 Discussion of Residential Exposure Characterization...................................................................................37

7.0 Deriving a Point of Departure (PoD) for Neurodevelopmental Outcomes..................... 38

7.1 Uncertainties with Using Biomarker for the PoD............................................................................................38

7.2 Options for PoD Based on the CCCEH Biomonitoring Data .........................................................................40

8.0 Assessing Extrapolation/Uncertainty................................................................................. 42

8.1 Intra-species Extrapolation................................................................................................................................44

8.2 FQPA 10X Safety Factor for Infants & Children............................................................................................46

8.2.1 Pre- and Post-Natal Toxicity Database..........................................................................................................47

8.2.2 Impact of Sample Size on CCCEH Findings.................................................................................................48

8.2.3 Conclusion on the FQPA Safety Factor ........................................................................................................49
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9.0 Proposed Approach to Deriving Internal Dose Estimates: Integration of Exposure 

Assessment & PBPK Modeling.................................................................................................. 50

9.1 Overview..............................................................................................................................................................50

9.2 Food Exposure ....................................................................................................................................................51

9.2.1 Methods.........................................................................................................................................................51

9.2.2 Food Residue Assumptions...........................................................................................................................52

9.2.3 Food Exposure Estimates..............................................................................................................................55

9.3 Drinking Water Exposure..................................................................................................................................56

9.3.1 Exposure Modeling .......................................................................................................................................57

9.3.2 Monitoring Data ............................................................................................................................................58

9.3.3 Drinking Water Treatment ............................................................................................................................60

9.3.4. Anticipated Exposure Scenarios and PBPK Model Simulations..................................................................63

9.3.4.1 Females of Childbearing Age (13-49 years old).........................................................................................63

9.3.4.2 Infants: Formula Fed (with water).............................................................................................................66

9.3.5. Drinking Water Summary ............................................................................................................................67

9.4 Worker Exposure ...............................................................................................................................................68

9.4.1 Methods.........................................................................................................................................................68

9.4.2 Results...........................................................................................................................................................71

9.4.3 Worker Exposure Summary & Preliminary Conclusions..............................................................................76

10.0 Discussion & Next Steps.................................................................................................... 76

11.0 References (for main document & appendices).............................................................. 81

Appendix 1.0: PoDs Based on 10% AChE Inhibition from the 2014 Human Health Risk 

Assessment................................................................................................................................... 97

Appendix 2.0: Summary of 2014 Dose Reconstruction Analysis.......................................... 101

Appendix 3.0: Background & Summary of Experimental and Epidemiology Studies on 

Neurodevelopmental Effects.................................................................................................... 117

A.3.1 Neurodevelopmental Effects........................................................................................................................117

A.3.1.1 Mechanistic Studies on Adverse Outcome Pathway ................................................................................118

A.3.1.2 Studies on Laboratory Animals................................................................................................................119

A.3.1.3 EPA Evaluation of Epidemiology Studies on Mothers & Children .........................................................120

A.3.1.3.1 Summary of Study Design and Scope of Evaluation.............................................................................120

A.3.1.3.2 Summary of Findings in Epidemiology Studies....................................................................................124

A.3.2 Summary of EPA’s Weight of Evidence Analysis......................................................................................130
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Appendix 4.0: Background & Summary of the Chlorpyrifos PBPK Model....................... 136

A.4.1 Introduction to the Physiologically-Based Pharmcokinetic/Dynamic Model ..........................................136

A.4.2 Summary of Metabolic Profile.....................................................................................................................139

A.4.3 Description & Structure of the Physiologically-Based Pharmcokinetic/Dynamic Model ......................140

Appendix 5.0: Verification of the lower bound of 90% CI of the estimated slope could be 

used to estimate the 95% lower limit of estimated concentration of CPF in cord blood ... 150

Appendix 6.0: Selected Figures from Poet (2015) Multi-Route, Lifestage, and Pregnancy 

PBPK/PD model for Chlorpyrifos and Chlorpyrifos-Oxon Submitted to EPA.................. 153

Appendix 7.0: Comparative analysis of NRC evaluation of methyl mercury epidemiology 

studies compared with chlorpyrifos and OP studies ............................................................. 154
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Table of Figures

Figure 1. Summary information on blood levels of chlorpyrifos provided by CCCEH to EPA (best copy available)14

Figures 2a and 2b. PBPK model-predicted time course of chlorpyrifos in blood after 8 hours/day, 5 days/week 

exposures for two weeks (2a: full profile; 2b: sub-set of Figure 2a)...................................................................18

Figure 3. New York City’s Water Supply System.......................................................................................................22

Figure 4. New York City Surface Water Intakes, Corresponding Watersheds and Agricultural Cropland Overlay ...23

Figure 5. Time course of venous blood for female of childbearing age consuming 99.9th percentile of food for 30 

consecutive days. ................................................................................................................................................25

Figure 6. Graphical representation of the PBPK model run conducted for estimated post-application exposures 

following perimeter application to carpeted floors, 8 hours exposure duration (left) and broadcast application to 

hard floors, 2 hours exposure duration (right) using Residential SOP algorithms and inputs. ...........................34

Figure 7. Graphical representation of the PBPK model run conducted for evaluation of the peak blood chlorpyrifos 

concentration on the initial day of exposure assuming a peak of 60 pg/g on the final day (30th) of indoor 

exposure; 8 hour exposure duration (left) and 2 hour exposure duration (right). ...............................................35

Figure 8. Graphical representation of the PBPK model run conducted for evaluation of peak blood chlorpyrifos 

concentrations on the final day of exposure (30th) assuming an initial blood concentration of 60 pg/g; 8 hour 

exposure duration (left) and 2 hour exposure duration (right). ...........................................................................36

Figure 9. Simulation Time Series Data for Chlorpyrifos Use on Bulb Onion (1 lb a.i./A once per year) in Georgia .58

Figure 10. Orestimba Creek Water Monitoring Data (May 1, 1996 to April 30, 1997) ..............................................59

Figure 11. Transformation of Chlorpyrifos to Chlopyrifos-oxon ................................................................................62

Figure 12. PBPK Estimated Chlorpyrifos Venous Blood Concentrations for Adult Female Based on 120-day Time 

Series Data for Chlorpyrifos Use on Bulb Onion (1 lb a.i./A once per year) in Georgia....................................64

Figure 13. PBPK Estimated Chlorpyrifos Venous Blood Concentrations for Adult Female Based on 120-day Time 

Series Data for Chlorpyrifos Monitoring Data for Orestimba Creek..................................................................65

Figure 14. PBPK Estimated Chlorpyrifos Venous Blood Concentrations for Infants Based on 120-day Time Series 

Data for Chlorpyrifos Use on Bulb Onion (1 lb. a.i./A once per year) in Georgia .............................................66

Figure 15. PBPK Estimated Chlorpyrifos Venous Concentrations for Infants Based on Time Series Data for 

Chlorpyrifos Monitoring Data for Orestimba Creek...........................................................................................67

Figure 16. PBPK modeling (full profile) estimation of chlorpyrifos venous blood concentrations (pg/g) per unit time 

(hours) resulting from the occupational exposure scenario, application via groundboom for cole crops at 1 lb 

a.i./A. ..................................................................................................................................................................72

Figure 17. PBPK modeling (sub-set of above Figure 16) of chlorpyrifos blood concentrations (pg/g) per unit time 

(hours) resulting on the final day from the example occupational exposure, application via groundboom for 

cole crops at 1 lb a.i./A .......................................................................................................................................72
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Table of Tables

Table 1. Estimated Exposure Based on Evaluation of Ambient Monitoring Data for New York City Community 

Water System Watersheds ..................................................................................................................................24

Table 2. Predicted levels of food exposure to chlorpyrifos from the 2014 revised risk assessment (Drew, 2014) and 

associated maximum and 24 hour blood concentrations in adult females ..........................................................26

Table 3. Summary of PBPK Model Runs for Analysis of Validation of Columbia Study Blood Levels....................32

Table 4. Estimated concentration of CPF in cord blood at specific percent reduction of WMI ..................................42

Table 5. Table 8-2 extracted from the 2000 NRC report on methyl mercury..............................................................44

Table 6. UFs recommended by NRC and used by EPA IRIS for methyl mercury......................................................46

Table 7. Results of Acute and Steady State Dietary (Food Only) Exposure Analysis for Chlorpyrifos......................55

Table 8. Predicted levels of food exposure to chlorpyrifos from the 2014 revised risk assessment and associated 

maximum and 24-hour blood concentrations in adult females ...........................................................................55

Table 9. Chlorpyrifos Reduction under Typical Drinking Water Treatment Conditions; Drinking Water Treatment 

Processes Utilized by Community Water System Based on Population Served.................................................61

Table 10. Summary of PBPK Model Estimated Maximum Chlorpyrifos Blood Concentrations Following Two 

Different Drinking Water Exposure Scenarios ...................................................................................................67

Table 11. Occupational dermal and inhalation exposures used for PBPK modeling of mixer/loader and applicator 

example low exposure scenarios and the maximum venous blood chlorpyrifos (pg/g) resulting from modeling.

............................................................................................................................................................................74

Table 12. Example model runs for mixing/loading EC for treatment of corn by groundboom soil incorporation at 

0.50 lb a.i./A and planting corn seed treated at 0.00058 lb a.i./lb seed...............................................................75

Table 13. Cross-reference of uncertainties identified by the FIFRA SAP (2012) on using the CCCEH biomonitoring 

data for deriving a PoD.......................................................................................................................................79
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1.0 Introduction

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

chlorinated organophosphate (OP) insecticide, acaricide, and miticide used to control a variety of 

insects. It was first registered in 1965 for control of foliage and soil-borne insect pests on a 

variety of food and feed crops. Risk mitigation actions in 1997 and 2000 led to nearly all 

residential uses being voluntarily cancelled by Dow AgroSciences. Currently, registered uses 

include food and feed crops, golf course turf, greenhouses, non-structural wood treatments (such 

as utility poles and fence posts), ant bait stations, fire ant mounds, a USDA quarantine use for 

containerized/potted ornamentals, and use as an adult mosquitocide. 

The agency has taken a stepwise, objective and transparent approach in evaluating, interpreting, 

and characterizing the strengths and uncertainties associated with all of the available lines of 

scientific information related to the human health effects of chlorpyrifos. Like other OPs, 

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

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

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

inhibiting potential of chlorpyrifos, there is a growing body of literature with laboratory animals 

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

cause persistent effects into adulthood along with high quality epidemiology studies which have 

evaluated prenatal chlorpyrifos exposure in mother-infant pairs and reported associations with 

neurodevelopment outcomes in infants and children. 

The stepwise evaluation began with the September 2008 FIFRA Scientific Advisory Panel (SAP) 

meeting involving a preliminary review of the literature for chlorpyrifos, with a particular focus 

on women and children (USEPA, 2008). In 2010, OPP developed a draft “Framework for 

Incorporating Human Epidemiologic & Incident Data in Health Risk Assessment” which 

provides the foundation for evaluating multiple lines of scientific evidence, including 

epidemiology, in the context of the understanding of the adverse outcome pathway (or mode of 

action (USEPA, 2010). The draft framework, which includes two key components: problem 

formulation and use of the modes of action/adverse outcome pathways (MOA/AOP) 

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 Programme 

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 

organization (Meek et al., 2014). 

In 2011, the agency released “Chlorpyrifos: Preliminary Human Health Risk Assessment for 

Registration Review,” focusing on the AChE inhibiting potential of chlorpyrifos (USEPA, 2011) 

and included assessment of exposures from dietary (food, water), occupational and residential 
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pathways. 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 points of 

departure (PoDs) for purposes of risk assessment. Also in 2011, the physiologically based 

pharmacokinetic-pharmacodynamic model (PBPK-PD) was reviewed by the FIFRA SAP 

(FIFRA SAP, 2011).

In 2012, the agency convened another meeting of the FIFRA SAP on chlorpyrifos which 

incorporated the newest experimental 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, the agency also performed a more in-depth 

analysis of the biomonitoring data and of epidemiological studies from three major children’s 

health epidemiology cohort studies in the U.S., as well as developed plausible hypotheses on 

MOAs/AOPs leading to neurodevelopmental outcomes (USEPA, 2012a). Following the 2012 

SAP meeting, the agency solicited additional input from federal experts in the areas of Magnetic 

Resonance Imaging (MRI) and neurobehavioral testing in children to further clarify results 

obtained by examination of the epidemiological studies.1

 In 2012, the agency also conducted an 

assessment of spray drift exposure which led to label mitigation addressing risk concerns

(USEPA, 2012b). 

In December, 2014, the agency released “Chlorpyrifos: Revised Human Health Risk Assessment 

for Registration Review” (herein called HHRA) which included the use of a PBPK-PD model 

(Appendix 2) model to derive human PoDs. This model obviated the need for the animal to 

human extrapolation factor, and refined intra-species factors for some lifestages (USEPA,

2014a). The chlorpyrifos 2014 revised HHRA also included retention of the 10X FQPA Safety 

Factor due to uncertainty regarding the degree of protection that the endpoint of AChE inhibition 

provides for potential neurodevelopmental effects (USEPA, 2014a).

In recent years, the National Academy of Sciences has encouraged the agency to move towards 

systematic review processes to enhance the transparency of scientific literature reviews that 

support chemical-specific risk assessments to inform regulatory decision making (NRC, 2011, 

2014). EPA’s Office of Chemical Safety and Pollution Prevention is currently developing 

systematic review policies and procedures. As part of the 2014 chlorpyrifos revised human 

health risk assessment and again in 2015 for all the OPs (USEPA, 2015), the agency has 

reviewed and updated the experimental toxicology literature search since the 2012 SAP using the 

concepts consistent with systematic review such as detailed tracking of search terms and which 

literature have been included or excluded. That systematic review is not repeated here but can be 

found in 2012 SAP issue paper, the 2014 revised HHRA, and updated for the 2015 literature 

review which supports risk assessments for all registered OPs. 

 1 http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPP-2008-0850-0170
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Based on the systematic review of the literature, the 2014 human health risk assessment 

identified uncertainty in the degree to which points of departure derived from AChE inhibition 

are protective for neurodevelopmental effects in humans. It is this uncertainty which provides the 

foundation for retaining the 10X FQPA Safety Factor in 2014. The agency published a 2015 

proposed tolerance revocation for chlorpyrifos based on human health risks identified in the 

2014 HHRA. If the revocation action becomes effective as proposed, it would likely lead to the 

end of chlorpyrifos use on food crops or in processed food produced in or imported into the U.S. 

In the proposed tolerance revocation, the agency noted that the evaluation of the available 

information including biomonitoring was continuing—this biomonitoring evaluation is the focus 

of this paper. While EPA would have preferred to complete that analysis prior to commencing 

rulemaking, the timing for the proposal was directed by the U.S. Court of Appeals for the 9th 

Circuit, which ordered EPA to respond to an administrative petition to revoke all chlorpyrifos 

tolerances by October 31, 2015.

The agency acknowledges that in 2012, the SAP noted that there was “general agreement that the 

data from these [epidemiology] studies alone were not sufficient to derive a point of departure 

(POD) for purposes of quantitative risk assessment (p. 50, 2012 SAP report).” However, the 

Panel further “recognized the value of these data and urged the agency to find ways to use the 

epidemiology studies, and in particular, the data from the CCCEH [Columbia Center for 

Children’s Environmental Health Mothers and Newborn Study] study, to inform the doseresponse assessment of chlorpyrifos... if one assumes that cord blood measurements reflect 

exposure levels during the critical prenatal period for induction of neurodevelopmental effects, 

then in theory, these would be the ideal data from which to derive the POD for chlorpyrifos in 

humans.” The Panel went on the state that “a [PBPK] model could be used to further 

characterize the dose estimates in the epidemiology studies, for additional dose response 

analyses. Such a PBPK model will become even more important in the event that the agency

might, at some point in the future, decide to move from using AChE inhibition to another 

outcome.” The agency has followed up on the SAP recommendations to use the PBPK model to 

further assess the epidemiological data. The status of the analysis of the biomonitoring 

evaluation is provided below. At this point in time, the agency is soliciting comment from the 

SAP on changing the critical effect for quantitative risk assessment from AChE inhibition to 

neurodevelopmental outcomes thereby changing the PoDs from doses eliciting 10% RBC AChE 

inhibition to adverse effects changes in neurodevelopment as measured by epidemiology studies 

conducted by CCCEH. 

2.0 Scope & Content of this 2016 Issue Paper

As summarized in Section 1 above, the agency has a long history of detailed technical analysis of 

various aspects of chlorpyrifos hazard and exposure assessment. In order to focus this issue 

paper on the pertinent issues--namely the transition from using AChE inhibition to adverse 

neurodevelopmental effects with the epidemiology biomarker data from CCCEH as the critical 
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effect for hazard assessment—the details of these previous evaluations are not repeated in here. 

Instead, the agency encourages the Panel and the public to also read these two documents before

reading this 2016 issue paper:

• USEPA (2014a). Chlorpyrifos: Revised Human Health Risk Assessment for Registration 

Review, December 29, 2014, D424485; 

• U.S. Environmental Protection Agency. (2015) Literature Review on Neurodevelopment 

Effects & FQPA Safety Factor Determination for the Organophosphate Pesticides. 

September 15, 2015, D331251. 

These two documents (and associated appendices) provide much of the scientific foundation for 

the current 2016 issue paper, and are available in the SAP public docket EPA-HQ-OPP-2016-

0062 at www.regulations.gov. For example, these 2014 and 2015 documents summarize the 

systematic reviews for epidemiology and experimental toxicology studies which support the 

agency’s conclusion that chlorpyrifos exposure results in long-term neurobehavioral effects and 

that chlorpyrifos contributed to the outcomes reported by the CCCEH researchers. It is noted that 

as part of these 2014 and 2015 systematic reviews, the agency has evaluated, and excluded from 

further consideration at this time, other critical effects including birth outcomes and autonomic 

nervous system impacts (see Appendix 3.0 for further details). The 2014 and 2015 documents 

also contain the agency’s reviews of the epidemiology studies, detailed summary of the PBPK 

model, and detailed analysis of contemporary exposure from food, water, and 

occupational/residential exposure. Limited summary information most pertinent to this 2016 

issue paper is contained in appendices here but for those individuals needing more details, refer 

to the citations above.

The remainder of the document is organized by the following:

• Section 3.0 Brief Summary of CCCEH Findings and Maternal & Cord Blood Levels 

provides a brief synopsis of the key findings of CCCEH regarding neurodevelopmental 

outcomes associated with chlorpyrifos exposure. This section also provides summary 

data provided by the CCCEH investigators of blood levels across time. 

• Section 4.0 Lifestages for Consideration describes the relevant lifestages for assessing the 

neurodevelopmental effects as context for the remainder of the issue paper.

• Section 5.0 Pharmacokinetic Time Course: Considerations for Labor & Delivery 

describes the PK profile of chlorpyrifos and discusses the specific lifestage context of the 

CCCEH cohort for interpreting the biomarker data.

• Section 6.0 Evaluation of CCCEH Cord Blood Data & Predicted Exposures to the 

Cohort provides a characterization of the predicted chlorpyrifos exposures to women in 

the cohort, particularly during the period of indoor residential use prior to the voluntary 

cancellation of most residential uses in 2000. This characterization includes evaluation 

of potential exposures from food, water, and indoor residential exposure and uses the 
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PBPK model to predict internal doses of chlorpyrifos. The residential exposure 

characterization provides a range of potential exposures and concludes that the 

distributions of reported blood concentrations in the CCCEH cohort are consistent with 

the exposure profile of the time period of the epidemiology studies. 

• Section 7.0 Deriving a Point of Departure (PoD) for Neurodevelopmental Outcomes of 

the document describes the conceptual approach to using the cord blood data to derive 

PoDs for women of childbearing age (13-49 years old), infants, and children. Section 7.0 

also contains the proposed internal PoD for these lifestages.

• Section 8.0 Assessing Extrapolation/Uncertainty provides an evaluation of uncertainty in 

the context of the intra-species factor and the statutory FQPA 10X Safety Factor. 

• Section 9.0 Proposed Approach to Deriving Internal Dose Estimates: Integration of 

Exposure Assessment & PBPK Modeling proposes an approach to use the PBPK model to 

estimate internal doses of chlorpyrifos from current food, water, and occupational 

exposures. Case study examples are provided for consideration.

• Section 10.0 Discussion & Next Steps describes how the analysis included in this issue 

paper addresses many of the uncertainties identified by the 2012 SAP regarding using the 

CCCEH data for deriving a PoD and describes next steps for the chlorpyrifos proposed 

tolerance revocation. 

• Section 11.0 and Appendices provide citations from the document and supplemental 

information on relevant to the science issues in this issue paper.

• As part of the package of materials for the FIFRA SAP (but in separate files from this

document), the agency has included all PBPK code needed to run the simulations 

described in the following sections and also associated output of the PBPK simulations. 

3.0 Brief Summary of CCCEH Findings and Maternal & Cord Blood Levels

EPA has conducted systematic reviews of the scientific literature on epidemiology studies on 

neurodevelopmental outcomes associated with OP exposure in 2012, 2014, and 2015. Although 

other studies exist, the most robust epidemiology studies are conducted through three major U.S.

based prospective birth cohort studies: 1) Mothers and Newborn Study of North Manhattan and 

South Bronx conducted by Columbia University, referred to in this document as “CCCEH;” 2) 

Mount Sinai Inner-City Toxicants, Child Growth and Development Study, or the “Mount Sinai 

Study/Cohort;” and 3) Center for Health Assessment of Mothers and Children of Salinas Valley 

(CHAMACOS) conducted by the University of California Berkeley, or “CHAMACOS Study/ 

Cohort.” Investigators from each study cohort utilized a similarly strong study design 

(prospective birth cohort); measured pesticide exposure using several different methods 

including environmental indicators as well as specific and non-specific biomarkers of OPs; 
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ascertained developmental outcomes using validated assessment tools well-established in both 

clinical and research settings; and, measured, analyzed, selected and statistically adjusted for 

potentially confounding variables including socio-economic status and other environmental 

exposures using reasonable and appropriate methods.

The CCCEH study measured parent chlorpyrifos in cord blood, and other indicators (e.g., air 

sampling, behavioral information), as etiologic measures of exposure, while the other two birth 

cohorts measured non-specific urinary metabolites of chlorpyrifos and other OPs (TCPy, dialkyl 

phosphate metabolites) in the mothers to estimate pesticide exposure. Therefore, EPA considers 

the CCCEH Mothers and Newborn Study research results as most relevant to the chlorpyrifos 

HHRA and is thus the focus on this analysis. The other two cohorts provide important supporting 

information.

The CCCEH Mothers and Newborn study participants were likely exposed to OPs through the 

diet and through residential use of the pesticide for indoor pest control. In the residential setting, 

study populations were most likely exposed through indoor residential use of the pesticide during 

the study time period and additionally exposed to OPs via the oral route through ingesting 

residues in the diet, from hand-to-mouth contact with in-home surfaces, as well as possible 

dermal or inhalation exposure through contact with treated areas in the home environment 

(Berkowitz et al., 2003; Whyatt et al., 2003; Whyatt et al., 2009; Whyatt et al., 2007). The time 

period under study within CCCEH study, spanned the point in time in which pesticide 

manufacturers voluntarily cancelled the use of chlorpyrifos in the home environment, and 

researchers were able to show the change in exposure before (high use period) and after (low/no 

use period) the period of removal of chlorpyrifos products from the residential marketplace. EPA 

and the FIFRA SAP (2008, 2012) have concluded that “chlorpyrifos likely played a role in the 

neurodevelopmental outcomes observed in these [epidemiology] studies.” 

Researchers across the three children’s health cohorts utilized the Bayley Scales of Infant 

Development II (BSID-II) to generate a Mental Development Index (MDI) and a Psychomotor 

Development Index (PDI) to assess neurodevelopment in early childhood. In the CCCEH 

Mothers and Newborn study, Rauh et al. (2006) investigated 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 chlorpyrifos exposure, using categories informed by results of the previous study on 

birth characteristics (Whyatt et al., 2004). Authors reported that the difference in MDI scores 

was “marginally significant” (p=0.06) between the “high” and “low” exposed groups; the high 

exposed group scoring an average of 3.3 points lower than the low exposed (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 month score was significantly related to chlorpyrifos exposure. 

Motor skill deficits observed in Rauh et al. (2006) appear to continue in Rauh et al. (2015) with hand 

tremors while writing at approximately age eleven years.
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Researchers noted that the effects were most pronounced at the 36 month testing period. Within 

the 36 month testing period, the likelihood of highly exposed children developing mental delays 

were significantly greater (MDI: 2.4 times greater (95% CI: 1.12-5.08, p=0.02) and PDI: 4.9 

times greater (95% CI: 1.78-13.72; p=0.002)) than those with lower prenatal exposure (Rauh et 

al., 2006). With respect to the findings related to the autism spectrum, from CCCEH, Rauh et al. 

(2006) reported a large odds ratio for pervasive developmental disorder (PDD) (OR=5.39; 95% 

CI: 1.21-24.11) when comparing high to low chlorpyrifos exposure groups. Regarding attention 

problems, Rauh et al. (2006) also investigated 36-month child behavior checklist (CBCL) 

(behavioral) scores. Significant differences were observed between the high and low chlorpyrifos 

exposure groups in the 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 Children, 4th edition (WISC-IV). The 

instrument measures four areas of mental functioning: the Verbal Comprehension Index, the 

Perceptual Reasoning Index, the Working Memory 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 English and Spanish-speaking children (Weschler, 

2003). In the CCCEH Mothers and Newborn Study, Rauh et al. (2011) evaluated the 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 including prenatal 

maternal interview data, prenatal chlorpyrifos marker levels from maternal and/or cord blood 

samples at delivery, postnatal covariates, and neurodevelopmental outcome data (Rauh et al., 

2011). The linear regression reported by Rauh et al. (2011) for working memory is the source of 

information used by the agency in Section 7.0 for deriving a PoD for neurodevelopmental 

effects.

Figure 1 is supplemental information summarizing the distribution of blood concentrations in 

cord blood and maternal blood across time in the CCCEH cohort. This information was 

provided to the agency upon request of Columbia in 2015 to aid in this analysis. This figure 

provides important temporal information that helps characterize the blood levels of chlorpyrifos 

in years of high indoor use (1998/1999) compared with those after the voluntary cancellation 

(2001–2004) with substantial reduction in blood levels in 2001–2004 compared to 1998–2000. 

There is a notable difference in the magnitude of chlorpyrifos reported across these years. Prior 

to the voluntary cancellation there were >80% detectable levels of chlorpyrifos in cord blood but 

in the time period after the cancellation only 16% of the measured values were greater than the 

limit of detection (LOD). In Figure 1, values listed as 0.2500 represent those below the LODs of 

0.5 pg/g or 1.0 pg/g. Whyatt et al. (2003, 2009) have shown that levels of chlorpyrifos in 

maternal blood and umbilical cord blood levels were highly correlated (r = 0.9, p<0.001, n = 64; 

r = 0.76, p<0.001, n=180 mother-child pairs). Similarly, Figure 1 shows that cord blood and 

maternal blood levels of chlorpyrifos are generally similar across years of the study and across 
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the distribution. However, the levels of chlorpyrifos in maternal blood samples are slightly 

higher for 1998/1999 compared to cord blood.

Figure 1. Summary information on blood levels of chlorpyrifos provided by CCCEH to EPA

(best copy available)
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4.0 Lifestages for Consideration

For the 2014 HHRA, the agency developed PoDs based on AChE inhibition to protect against 

cholinergic toxicity; such cholinergic toxicity could occur to any lifestage if exposure is 

sufficiently high. As such, in 2014, the agency evaluated the spectrum of lifestages from the 

fetus through adulthood (Appendix 1). At this point in time, the focus of the assessment has 

transitioned to neurodevelopmental outcomes such that the critical lifestages of concern are 

fetuses, infants (< 1 year old), and children (1–2 years old) because EPA believes these outcomes 

may occur at exposure levels below those resulting in AChE inhibition. Fetuses may be exposed 

to chlorpyrifos through the mother while infants and children may be exposed directly. As 

discussed in Appendix 3, studies in laboratory animals do not suggest any specific critical period 

or lifestage but instead suggest pre- and post-natal periods of susceptibility. The agency

acknowledges that the epidemiology literature regarding associations between post-natal 

(infancy, childhood) biomarker metrics and neurodevelopmental outcomes is limited to the 

Bouchard et al (2010) study, a cross-sectional study that observed positive association between 

attention and behavior problems and total DAPs and DMAPs, using urinary NHANES data in 

children 8–15 years old. The OP exposure being assessed in many of the epidemiology studies 

used concentrations of urinary dialkyl phosphate metabolites (DAPs) as the urinary biomarker. 

Total DAPs is a non-specific measure of OP exposure and is the sum of six separate molecules 

— three dimethyl alkylphosphate (DMAP) molecules of dimethylphosphate, 

dimethylthiophosphate, and dimethyldithiophosphate (DMP, DMTP, and DMDTP), and three 

diethyl alkylphosphate (DEAP) molecules of diethylphosphate, diethylthiophosphate, and 

diethyldithiophosphate (DEP, DETP, and DEDTP) (for further details on DAPs, see Appendix 

A.3.1.3.2). The other studies which evaluated postnatal biomarker metrics and 

neurodevelopment outcomes have found no statistically significant associations. Specifically, 

postnatal exposure to OPs (measured as DAPs) has been assessed in the CHAMACOS cohort 

(Eskenazi et al., 2007; Young et al., 2005; Bouchard et al., 2011) and two other cross-sectional 

studies (Guodong et al., 2012; Oulhote and Bouchard, 2013). Despite the limited 

epidemiological evidence from postnatal exposure, the agency is proposing to use the 

chlorpyrifos cord blood data from CCCEH as the most relevant source of information for 

deriving a PoD specific for chlorpyrifos for fetuses, infants, and children. 

The cord blood data are directly relevant to fetal exposure; however, they may be less relevant to 

older children. As described later in this document, drinking water risk was one of the areas 

identified in the 2014 HHRA as having a high exposure potential. Early infant [exposure 

through feeding via formula in bottles (made with water)] is highlighted in the case studies in 

Section 9.0. Because this group consumes formula reconstituted with water frequently and 

throughout the day and night, combined with lower body weight and lower metabolic capacity, 

this may lead to relatively high internal exposure to chlorpyrifos. The agency has not included 

any case study evaluations for children 1–2 years old, a group which often provide the highest
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food exposure. Children 1–2 years old are not included as these ages are temporally removed 

from gestational exposure; as such the relevance of such data to predict the outcomes in toddlers 

is unclear. 

The current model being used by the agency for the chlorpyrifos risk assessment does not 

include gestation or lactational exposure. With respect to lactational exposure, the agency is 

aware of breast milk monitoring data from the Pilot study for the National Children’s Study 

(NCS) Supplemental Methodological Studies (SMS)2 that can be used to evaluate infant 

exposure to chlorpyrifos through breastfeeding. These data have not been used in this issue paper 

as a case study as the breastmilk case study would be similar to the drinking water, bottle feeding 

infant scenario included in Section 9.3.4.2 and thus not provide any additional scientific 

understanding for developing the risk assessment approach for using the cord blood. Moreover, 

the agency does have information on the participants in the breast milk study to evaluate how 

different sources of exposure impact breast milk levels. The agency may consider, if 

appropriate, the breast milk monitoring data as part of the tolerance revocation. Since there are 

some (albeit limited) breast milk monitoring data from contemporary exposures, the lack of a 

lactational PBPK model does not introduce substantial uncertainty in the risk assessment.

In April 2015, DAS modified the multi-route PBPK/PD model for chlorpyrifos to include 

additional code to describe physiological changes for women during pregnancy (Poet, 2015). 

The gestational component of the model includes the following modifications: (1) placenta, 

uterine, and fetal compartments, all of which grow over the course of pregnancy; (2) pregnancyspecific changes in the fat, rapidly-perfused, and slowly-perfused compartments; (3) pregnancyspecific changes in blood composition resulting in increased blood volume and decreased 

hematocrit; and (4) pregnancy-specific changes in metabolism, both CYP450s and PON1 

enzymes based on published studies. No changes were made to the PD model since there are no 

data available to suggest cholinesterase binding changes during pregnancy. The growth of fetus, 

uterus, and placenta predicted by the model agreed with empirical data as were the changes in 

tissue and blood volume (Abduljalil, et al., 2012). While the modified model reasonably 

simulated the physiological changes during pregnancy, the model’s predictive ability to simulate 

internal dosimetry of chlorpyrifos cannot be properly evaluated since there are no chlorpyrifosspecific pharmacokinetic data available during pregnancy. As such, the agency cannot evaluate 

its predictive capacity and thus, the pregnancy model will not be used for risk assessment at this 

time. 

The agency does note, however, that the pregnancy model was built based on the best knowledge 

available and some preliminary simulations using this model suggested that when accounting for 

variability in physiology, pregnant women are not more at risk than non-pregnant women. Given 

the same oral dose, blood concentrations of chlorpyrifos in women in the third trimester were 

 2 https://www.nichd.nih.gov/research/ncs/Pages/default.aspx
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slightly lower than those in non-pregnant women (Appendix 6). While some parameter changes 

during pregnancy contributed to more chlorpyrifos in blood (e.g., smaller fat:blood partition 

during pregnancy) and other changes contributed to less chlorpyrifos in blood (e.g., increased 

tissue volumes for more storage), the combined effects from these multiple physiological 

changes during pregnancy resulted in slightly lower blood concentrations of chlorpyrifos during 

pregnancy. Similarly, RBC AChE inhibition in pregnant women at a given dose was comparable 

to non-pregnant women.

In addition, data from CCCEH suggest that maternal blood is a reasonable surrogate for cord 

blood. Figure 1 provides cord blood and maternal blood values across the CCCEH cohort and 

there is strong agreement across the years on study and across the distribution. Whyatt et al.

(2009) have shown that levels of chlorpyrifos in maternal blood and umbilical cord blood levels 

were highly correlated (r = 0.9, p<0.001, n = 64). In a second paper, Whyatt et al. (2003) also 

showed that chlorpyrifos in maternal blood and umbilical cord blood levels were highly 

correlated (r = 0.76, p<0.001, n=180 mother-child pairs). These together suggesting that 

tracking the blood concentrations of the mother is a reasonable surrogate for the fetus. Although 

the agency would prefer to have a robust gestational PBPK model parameterized with 

chlorpyrifos data, when considered together—based on the preliminary results of the pregnancy 

PBPK model and strong correlation between maternal and cord blood in the CCCEH cohort 

(Figure 1) the lack of such model robust does not add major uncertainty into the 2016 analysis. 

The agency can reliably estimate blood levels of chlorpyrifos for females of childbearing age.

5.0 Pharmacokinetic Time Course: Considerations for Labor & Delivery

An example of time course profile for chlorpyrifos in blood is shown in Figures 2a and 2b for 

current occupational exposures to pesticide handlers. The agency uses the term handlers to 

describe those individuals who are involved in the pesticide application process. Similar figures 

for food, water, and residential exposures are shown throughout the document in subsequent 

sections. As shown in Figures 2a and 2b, the time course profile shows a consistent pattern of a 

daily, rapid increase in internal dose during the exposure period followed by a rapid decline after 

the exposure period ends. The rapid decline of chlorpyrifos after exposure terminates is 

expected given the rapid metabolism of chlorpyrifos. The rapid increase periods represent rapid 

uptake during activities that lead to chlorpyrifos exposures, while the rapid decrease periods are 

primarily attributed to distribution from the central compartment (circulation) into the peripheral 

compartments (body tissues), loss to metabolism, and binding to esterase. For chlorpyrifos, the 

half-life of this initial phase is estimated to be approximately four hours. 

Figure 2b shows that after the exposure ends the internal dose curve flattens as sequestered 

chlorpyrifos slowly becomes available for clearance. During this terminal clearance phase, the 

half-life is a function of both clearance (i.e., metabolism and excretion) and distribution of 

chemical between tissues and blood; the chemical is eliminated only when it is distributed to the 
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clearing organs (e.g., liver for metabolism, kidneys for excretion) (Toutain, and BousquetMelou, 2004). Depending on the size of the peak, measuring plasma concentrations during the 

terminal elimination phase can be challenging because these levels may fall below the 

detection/quantification limits of the analytical techniques. Thus, to estimate the plasma terminal 

half-life for chlorpyrifos, the PBPK model was used to predict the time course of chlorpyrifos 

concentrations in plasma during the exposure period and 30 days after exposure. Two 

scenarios were simulated to ensure that the model estimated the same terminal half-life for 

different routes of exposure. The two scenarios were: (a) a five-day, 8 hours/day, dermal and 

inhalation exposure; and (b) a daily single oral exposure for 5 days, and the estimated terminal 

half-life from both scenarios is approximately 120 hours (i.e., 5 days). 

Figures 2a and 2b. PBPK model-predicted time course of chlorpyrifos in blood after 8 hours/day, 5 days/week 

exposures for two weeks (2a: full profile; 2b: sub-set of Figure 2a)

Figure 2a. PBPK model-predicted time course (full profile) of chlorpyrifos concentrations in

venous blood (pg/g) resulting from the occupational handler exposure scenario, mixing/loading 

liquids (EC) for cole crops at 1 lb ai/A.

0

50

100

150

200

250

300

350

0 200 400 600 800 1000 1200 1400

Blood Chlorpyrios (pg/g)

Time (hours)
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Figure 2b. PBPK model-predicted time course (sub-set of the above Figure 2a) of chlorpyrifos 

concentrations in venous blood (pg/g) after the last day of exposure resulting from the 

occupational handler exposure scenario, mixing/loading liquids (EC) for cole crops at 1 lb ai/A. 

For interpreting the CCCEH blood levels of chlorpyrifos (Figure 1), it is important to consider 

these PK time course patterns in the context of pregnant women going into labor and ultimately 

delivering a newborn in the hospital. Whyatt et al. (2003) note that a sample of umbilical cord 

blood was collected as close to delivery as possible and a sample of maternal blood was obtained 

within 2 days postpartum. Reported times for labor and delivery in a first pregnancy range from 

8-20 hours and may become faster for subsequent pregnancies 

(www.webmd.com; www.mayoclinic.org; www.parents.com; www.babycenter.com). Some of 

this time period would have been spent in labor at home while the remainder would have been at 

the hospital. The time spent at home vs. the hospital are not known and are likely to have varied 

among the women in the cohort. In a systematic review of 18 reported mean ‘active labor’ 

duration studies, Neal et al (2010) note that in contemporary medicine, women are most often 

admitted to the hospital labor unit when cervical dilation is expected to accelerate. The onset of 

the active phase generally begins at dilation between 3 cm and 5 cm and in the presence of 

regular uterine contractions. Neal et al. (2010) report than among healthy, low-risk, nulliparous 

women at term with a spontaneous labor onset, the ‘active phase’ of labor lasted an average of 

6.0 hours, and up to 13.4 hours at two standard deviations from the mean. 

Most births in the US are vaginal. The agency is aware that in the US Cesarean section rates 

have increased from approximately 20% in 1996 to 33% in 2011 (ACOG, 2014). CCCEH 

investigators have not reported the rates of vaginal vs. Caesarean deliveries in the cohort. Some 

Cesarean sections are performed after labor has started for medical reasons associated with the 

safety of the baby or the mother (and thus would be covered by the above description). 

However, in the context of this document and interpreting the chlorpyrifos biomonitoring data, it 

0

50

100

150

200

250

300

350

263 363 463 563 663 763

Blood Chlorpyrios (pg/g)

Time (hours)
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would be the planned Cesarean sections, where the mother enters the hospital and within a short 

period of time after arrival delivers her baby, which could potentially impact the current 

evaluation. Although planned Cesarean sections have similarly risen in recent years, reported 

rates are not high, ranging from 4–18% of pregnancies (Roberts et al., 2015; David et al., 2015; 

Black et al., 2015). Given the relatively low frequency of planned Cesarean sections and the 

consistency of the agency’s exposure characterization with the CCCEH cord blood and maternal 

blood biomonitoring data, the impact of short delivery times from planned Cesarean sections 

does not contribute significant uncertainty in the agency’s evaluation or proposed approach.

As shown in Figure 2b, upon ending an exposure period, there is a rapid decline in internal dose; 

this rapid decline is driven by the initial half-life of 4 hours and lasts for six to ten hours. The 

time of rapid decline (6–10 hours) is consistent with the average time of active labor in the 

hospital reported by Neal et al. (2010). In other words, at six to ten hours after the exposure 

ends, the internal dose profile is at the low point on Figure 2b. In the context of the CCCEH 

cohort, the end of chlorpyrifos exposure represents leaving the apartment and delivering a baby 

at the hospital. As such, there is a reasonable likelihood that the reported cord blood levels in the 

CCCEH publications do not represent the peak levels or levels in the rapid decline phase. 

Instead, given a labor and delivery scenario, it is more likely that the CCCEH blood samples 

were collected at or near the low points in the PK profile, especially for those individuals whose 

babies were delivered ten or more hours after chlorpyrifos exposure. Further, given that in the 

CCCEH cohort, maternal blood samples were collected “within 2 days postpartum” and not “as 

close to delivery as possible” like the cord blood, it is likely that the maternal blood samples 

were collected during the terminal clearance phase where the half-life is 120 hours and when the 

blood levels were not changing rapidly. For example, in the terminal half-life phase of the PK 

profile, it takes two days for blood concentration of chlorpyrifos to only drop from 8.8 pg/g to 

6.7 pg/g; or it takes one day to drop from 7.7 pg/g to 6.7 pg/g. 

In summary, during periods of exposure and immediately after exposure, blood concentrations 

rapidly increase and decrease which results in a daily sawtooth pattern. Upon cessation of the 

exposure, the terminal half-life (approximately 120 hours) predominates resulting in an 

asymptotic appearance for the internal dosimetry. During this terminal half-life phase, the 

internal doses are changing slowly. 

For deriving the proposed PoDs (below), the agency is assuming the CCCEH levels do not 

represent values with the rapid increase/decrease phase. Instead, the agency is assuming the 

reported values for cord blood and maternal blood are at or near the low points or likely within 

the terminal clearance period (and thus unlikely to change significantly across several days). As 

such, the agency is reporting two values in the scenarios in Sections 6.0 and 9.0 below: a) 10 

hours post-peak exposure, and b) 24 hours post-peak exposure.
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6.0 Evaluation of CCCEH Cord Blood Data & Predicted Exposures to the 

Cohort

As an initial step towards determining a PoD based on the neurodevelopmental outcomes in the 

epidemiology studies, the agency has developed an analysis:

1) to characterize the source and magnitude of chlorpyrifos exposure to the women in the 

CCCEH cohort by considering drinking water, food, and residential exposure potential; and

2) to compare PBPK model predicted blood levels across a range of exposure scenarios to 

blood levels reported by CCCEH. 

The PBPK model has been used to predict blood levels in women across exposure scenarios for 

comparison with the cord blood levels reported by the CCCEH. Drinking water exposure 

(Section 6.1), food exposure (Section 6.2), and residential exposure (Section 6.3) are described 

below. 

6.1 Drinking Water Exposure to the CCCEH Cohort

New York City has a watershed protection plan3 in order to avoid filtration requirements as part 

of the drinking water treatment process. While this watershed protection plan contains objectives 

to prevent pollution from non-agricultural and agricultural operations through a voluntary 

program (e.g., best management practices established by the USDA Natural Resources 

Conservation Service), the program does not specifically target a reduction or elimination of 

surface water contamination from pesticides.4 This protection plan may not completely prevent 

chlorpyrifos or chlorpyrifos-oxon from reaching surface water used to supply drinking water to 

New York City (specifically in the areas where participants of the CCCEH study lived: Harlem, 

Washington Heights and South Bronx). OPP has evaluated the likelihood that chlorpyrifos or its 

oxon may have been present in the drinking water of mothers in the CCCEH cohort and has 

concluded exposure to chlorpyrifos and chlorpyrifos-oxon via drinking water was unlikely.

Approximately 99 percent of New York City’s drinking water was sourced from three upstate 

watersheds (i.e., impounded in the Croton and Catskill-Delaware Systems) during the CCCEH 

study period. The watersheds are shown in Figure 3. Figure 4 shows an overlay of surface water 

intakes, corresponding watersheds and agricultural land.

 3 http://www.dos.ny.gov/watershed/nycmoa.html

4 http://www.nycwatershed.org/agriculture/planning/
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Figure 3. New York City’s Water Supply System5

 5 New York City Environmental Protection, New York City 2013 Drinking Water Supply and Quality 

Reporthttp://www.nyc.gov/html/dep/pdf/wsstate13.pdf
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Figure 4. New York City Surface Water Intakes, Corresponding Watersheds and Agricultural Cropland 

Overlay6

Many of the intakes are located in areas where large scale chlorpyrifos use (i.e., agricultural) is 

not expected, suggesting exposure would have been low. The overlap of potential nonagricultural chlorpyrifos use sites is unknown. However, the highest overlap of potential 

agricultural chlorpyrifos use sites with the intake watersheds suppling the Catskill-Delaware 

System is approximately 30 percent (Figure 3).

Examination of the USGS National Water-Quality Assessment Program (NAWQA)7, 

USEPA/USGS Pilot Reservoir Monitoring Program8, and USDA Pesticide Data Program (PDP)9

indicates a very low detection frequency of chlorpyrifos and no detections of chlorpyrifos-oxon 

for sample sites located within New York State. The low detection frequency may be an artifact 

of the program design such as not being targeted to chlorpyrifos application or not sampling 

frequently enough. Another explanation for the low detection frequency is that the watershed 

protection plan is effective at limiting chlorpyrifos in New York City drinking water sources. 

EPA’s analysis of the monitoring data for the years 1997–2004 (Table 1) suggests the highest 

potential concentration of chlorpyrifos during the CCCEH study period was 0.25 μg/L in 2003

and 2004 for the watersheds used as source drinking water for New York City. This exposure 

concentration is based on one half of the reported limit of detection. The New York City 

drinking water supply and quality reports10 report no measured concentrations of chlorpyrifos11; 

however, the detection level and the number of samples collected by the community water 

 6 Overlay developed by EPA (R. Bohaty).

7 http://water.usgs.gov/nawqa/

8 http://archive.epa.gov/pesticides/carat/web/pdf/dw5.pdf

9 https://www.ams.usda.gov/datasets/pdp/pdp-drinking-water-project 10 http://www.nyc.gov/html/dep/html/home/home.shtml 11 Chlorpyrifos sampling began in 2002

New York City Surface Water Intakes

2001 National Land Cover Dataset

New York City Surface Water Watershed
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system is not reported. Mixing of sourced water as well as drinking water treatment (described 

below) are expected to reduce the potential drinking water exposure to chlorpyrifos.

Table 1. Estimated Exposure Based on Evaluation of Ambient Monitoring Data for New York City 

Community Water System Watersheds

Year 1997 1998 1999 2000 2001 2002 2003 2004

Concentration 

(μg/L) 0.002 0.015 0.015 0.06 0.0423 0.0025 0.25 0.25

Bold font indicates measured concentrations. Other referenced concentrations are 1⁄2 the limit 

of detection. Shading indicates highest potential exposure.

The Catskill-Delaware System supplies approximately 90 percent of the surface sourced drinking 

water to New York City. Prior to 2013, the Catskill-Delaware drinking water treatment facility 

only used chlorine to disinfect water entering the drinking water distribution system. As such, 

any chlorpyrifos present is expected to have oxidized to chlorpyrifos-oxon during drinking water 

treatment and, therefore, chlorpyrifos would not have been present in drinking water and by 

extension would not have been measured in cord blood from this exposure route. In addition to 

chlorine, sodium hydroxide was added to Catskill-Delaware water to increase pH and reduce 

corrosivity.12 Examination of the New York City Drinking Water Supply and Quality Reports 

for years suggests the pH of distributed drinking water ranges from a minimum of 6.7–6.9 to a 

maximum of 8.3–9.8. The pH ranged from 6.0–8.4 during the CCCEH study period. Both 

chlorpyrifos and chlorpyrifos-oxon are more rapidly hydrolyzed under basic (pH>7) conditions. 

Mixing of sourced water from other intakes is also expected to reduce potential exposure to 

chlorpyrifos-oxon as a result of oxidation of chlorpyrifos. 

In summary, exposure to chlorpyrifos is not expected to have occurred via New York City 

drinking water. Therefore, the chlorpyrifos measured in cord blood for the CCCEH cohort would 

not have been the result of exposure to chlorpyrifos via drinking water. Exposure to chlorpyrifosoxon in drinking water was also not likely, with any exposure being very low.

6.2 Food Exposure to the CCCEH Cohort

The agency uses probabilistic exposure approaches for assessing food exposure that have had 

extensive peer review through the FIFRA SAP (2000a; 2000b) and public comment; these 

approaches and associated sources of residue data can be found in Section 9.2 below. The 

agency expects that exposure to chlorpyrifos (but not the oxon) in food may have occurred to the 

women in the cohort over the entire period of the CCCEH chlorpyrifos publications. In 2000, 

EPA required that all uses of chlorpyrifos products in the U.S. be discontinued on tomatoes. Use 

on apples was restricted to pre-bloom and dormant application. The grape tolerance was lowered 

to reflect the labeled dormant application. In 2002, EPA limited the use of chlorpyrifos on citrus 

 12 New York City Environmental Protection http://www.nyc.gov/html/dep/pdf/wsstat00b.pdf
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and tree nuts as well other crops. However, chlorpyrifos use on numerous crops remained after 

this mitigation. 

The agency performed simulations of food exposure across the distribution of food exposures 

from the 2014 HHRA. In the food exposure simulations, females of childbearing age were 

exposed once daily for 30 consecutive days. For example, Figure 5 shows the predicted blood 

profile of a women of childbearing age from once daily food exposure at the 99.9th percentile 

from the 2014 HHRA calculated using the PBPK model following 30 consecutive days of 

exposure. As shown in the graph after 30 days of exposure, the peak blood level is 7.14 pg/g, a 

value near the lower limit of the top tertile reported by CCCEH (6.17 pg/g). However, the blood 

levels drop quickly each day after the end of exposure. By eight hours post-exposure, the blood 

levels have dropped to 3.2 pg/g; and between 13–16.5 hours post-exposure, the blood levels are 

below the LOD.

Figure 5. Time course of venous blood for female of childbearing age consuming 99.9th percentile of food for 

30 consecutive days.

As shown in Table 2, from the 10–70th percentile, the peak values do not go over the LOD of 0.5 

and/or 1.0 pg/g and at the 90th percentile, the blood levels are only above the LOD for four hours.
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Table 2. Predicted levels of food exposure to chlorpyrifos from the 2014 revised risk assessment (Drew, 2014) 

and associated maximum and 24 hour blood concentrations in adult females

Percentile of 

Exposure

Exposure from 

Calendex

Max Blood Levels 

of CPFOS (pg/g) 

from Food Runs at 

Various Percentiles 

of Exposure from 

Calendex

10-hour Blood Levels 

Post Exposure Values 

of CPFOS (pg/g) 

from Food Runs at 

Various Percentiles 

of Exposure from 

Calendex

24-hour Blood Levels 

Post Exposure Values 

of CPFOS (pg/g) 

from Food Runs at 

Various Percentiles 

of Exposure from 

Calendex

10 0.003 

μg/kg/day 0.29 0.060 0.021

30 0.005 

μg/kg/day 0.48 0.099 0.034

50 0.007 

μg/kg/day 0.67 0.139 0.048

70 0.009 

μg/kg/day 0.86 0.179 0.062

90 0.014 

μg/kg/day 1.33 0.278 0.096

95 0.018 

μg/kg/day 1.71 0.358 0.124

97.5 0.023 

μg/kg/day 2.19 0.457 0.158

99 0.029 

μg/kg/day 2.76 0.576 0.200

99.5 0.037 

μg/kg/day 3.52 0.735 0.255

99.9 0.075 

μg/kg/day 7.14 1.490 0.517

The exposure period after the voluntary cancellation of indoor use would have included only 

dietary exposure. The agency’s predicted blood concentrations from food exposure in Table 2

are consistent to the pattern of reported values in the CCCEH study with respect to the number of 

samples above the LOD before and after the voluntary cancellation of the indoor uses. As noted 

above, the CCCEH study enrollment time period (and collection of cord blood) spanned the 

point in time in which pesticide manufacturers voluntarily cancelled the use of chlorpyrifos in 

the home environment but extensive agricultural use remained. Prior to the voluntary 

cancellation there were >80% detectable levels of chlorpyrifos in cord blood but in the time 

period after the cancellation only 16% of the measured values were greater than the LOD.
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Figure 1 shows that from 2000–2002, there is an overall decrease in the reported levels of 

chlorpyrifos. By 2003/2004, none of the cord blood or maternal blood samples, at least up to the 

95th percentile were above the LOD (Figure 1). Moreover, there was only one child born in the 

time period subsequent to the voluntary cancellation of chlorpyrifos in the residential 

marketplace for whom the cord blood chlorpyrifos level was in the upper-tertile of precancellation exposure levels. Given the results in Table 2 and the results reported by CCCEH 

after the voluntary cancellation (Figure 1), it is unlikely that food exposure alone leads to high 

levels of measured chlorpyrifos in blood.

It is notable that CCCEH study is underpowered to detect adverse neurodevelopmental effects at 

blood concentrations substantially lower than those reported in Rauh et al., (2006, 2011). 

Although the CCCEH investigators report before/after cancellation findings with respect to lack 

of adverse findings after the cancellation of indoor uses, it is important not to over interpret these 

findings to equate that remaining food exposure would not be associated with 

neurodevelopmental outcomes. Instead, there is uncertainty with respect to safe levels of 

remaining exposures.

6.3 Residential Exposure to the CCCEH Cohort

Based on the above evaluations for drinking water and food exposure, the agency believes the 

measured values of chlorpyrifos in the CCCEH come primarily from exposure in the home 

environment, particularly at the upper-tertile. It is important to note that the CCCEH 

investigators did not collect information on chlorpyrifos application timing; instead, the 

investigators have reported generic pesticide exposure information. Whyatt et al. (2002) 

reported that pest control measures were used by 85% of respondents, whether by housing 

superintendent, pest control operator (PCO), or by themselves. The majority reported regular use 

of a pesticide product, at least once per month. At the time of the initial recruitment period of 

pregnant mothers in the CCCEH studies (1997–1999), chlorpyrifos was being used widely for inhome pest control treatment. There were two risk mitigation actions which impact the 

interpretation of the CCCEH cord blood data (Figure 1): 

• In January 1997, the technical registrants entered into an agreement with the agency to 

reduce indoor exposure to chlorpyrifos, especially to children and other sensitive groups. 

All residential broadcast and total release aerosol/foggers uses were cancelled — indoor 

crack and crevice (perimeter) and spot treatment as a termiticide were not included. The 

following chlorpyrifos uses were also cancelled: all direct application pet products 

including sprays, shampoos, and dips (pet collars not included); and all insecticidal paint 

additives. Further, all concentrates which required mixing were eliminated limiting the 

household consumer to ready-to-use products. Although the above uses were cancelled 

in 1997, existing stocks could be phased out, or applied until depleted. 

• In June 2000, the technical registrants, entered into an agreement with the agency to 

eliminate or phase out nearly all remaining uses that resulted in residential exposure 

including home lawn, crack and crevice and other indoor uses. The only exceptions are 
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baits containerized in child resistant packaging and public health uses such as mosquito 

and fire ant control. The agreement focused first on mitigation that achieved the greatest 

reduction for children. As such, any non-residential uses where children could be 

exposed, such as schools and parks, were also cancelled. For these uses, retailers had a 

stop sale date of December 31, 2001. A phase out of existing stocks was also allowed 

following the 2001 stop sale. 

Given the lack of specific CCCEH exposure information, the agency developed a range of 

possible residential exposure scenarios resulting from residential post-application exposures to 

chlorpyrifos products available prior to the voluntary cancellation of indoor products in 2000. 

These possible residential exposure estimates were input into the PBPK model to predict a range 

of possible blood levels of chlorpyrifos in women. 

A conceptually similar analysis was undertaken by the agency following the recommendation of 

the FIFRA SAP (2012) to conduct a “dose reconstruction” analysis of indoor residential uses to 

assess potential for RBC AChE inhibition. The dose reconstruction analysis was conducted and 

presented in the 2014 HHRA. The dose reconstruction analysis, including all methods and 

inputs, is described in Appendix 2 of this document. The goal of the dose reconstruction 

exercise was to estimate upper limit, bounding level exposures to test the hypothesis whether 

RBC AChE at or above the 10% inhibition level used by the agency for typical AChE PoDs may 

have occurred in the cohort. For example, in the dose reconstruction analysis, exposure to the 

women was assumed to occur 24 hours a day without adjustments for bathing, showering, or 

leaving the residence for 14 consecutive days. In contrast, the goal of the present 2016 analysis 

is to predict typical product usage and behaviors thereby deriving more accurate and realistic 

estimates of exposure. 

The present analysis includes two parts: 1) development of six scenarios representing a range of 

possible exposure levels to women in the CCCEH cohort and 2) PBPK simulations using these 

exposure scenarios for prediction of internal dosimetry across time. These simulations are 

intended to mimic indoor application of chlorpyrifos with daily dissipation across a one month 

time period. The one-month time period was selected as the likely period between applications 

of chlorpyrifos. Since exposure information on frequency and magnitude of applications were 

not collected by CCCEH investigators, there is uncertainty in the degree to which the monthly 

application period is accurate. Similarly, in the CCCEH cohort, there is uncertainty in the timing 

of chlorpyrifos indoor application in relation to the timing for labor and delivery (i.e., when the 

cord blood was collected).

6.3.1 Methods

For the current evaluation, the agency has focused on chlorpyrifos blood levels resulting from 

post-application exposures only. Whyatt et al. (2002) reported that many women applied 

pesticide themselves and that majority who reported using a pesticide used them at least once per 

month. However, as the agency has shown in the dose reconstruction (Appendix 2) analysis, 
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

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post-application exposures are greater in magnitude than the exposure which occurs during an 

application. As such, by focusing on post-application exposure, the agency is including the 

higher exposure potential. A total of six residential post-application exposure scenarios and 

corresponding PBPK model simulations have been assessed. Two exposure scenarios mimic 

potential indoor chlorpyrifos use patterns likely to have occurred amongst the CCCEH cohort 

using EPA standard exposure assessment approaches; these two scenarios represent the high end 

exposure potential. To estimate lower exposures, four additional PBPK model simulations were 

conducted with use of reported values from the CCCEH investigators. The six post-application 

exposure scenarios allow for establishment of ranges of potential exposures and comparison of 

predicted internal dosimetry levels to those reported by the CCCEH investigators. 

All six scenarios share some exposure assumptions. For the present evaluation, the agency has 

assumed a once daily shower occurred immediately following exposure activities. All PBPK 

simulations conducted were run for 30 days post-application to be consistent with the monthly 

usage reported by CCCEH investigators. Daily exposure times for post-application adult dermal 

contact with carpets and hard surfaces were used as recommended in the 2012 Standard 

Operating Procedures for Residential Pesticide Exposure Assessment13 (herein referred to as the 

2012 Residential SOPs); specifically, how long an adult is expected to spend daily on a treated 

carpeted or hard floor. For adults, the recommended exposure times for post-application dermal 

exposure assessments are 8 and 2 hours daily for carpets and hard surfaces, respectively. These 

values are based on the EPA Exposure Factors Handbook 201114 Edition that provides 

information on the total time spent in a residence and time spent in various rooms within a 

residence. Additionally, chlorpyrifos residues were assumed to dissipate 10% daily; that is, the 

total amount of residue available for transfer from the treated floor is assumed to reduce by 10% 

for each subsequent day of exposure. 

Two residential exposure scenarios (broadcast hard surface, perimeter carpet) have been 

evaluated using the same algorithms as those described in Appendix 2 for the dose reconstruction 

analysis for estimation of daily dermal and inhalation (inhalation exposures are limited to 2 hours 

on the day of application) chlorpyrifos post-application exposures. Consistent with the dose 

reconstruction analysis, the present analysis is based on the 2012 Residential SOPs methods

which describe specific algorithms and inputs, on a scenario-specific basis; the 2012 Residential 

SOPs were subjected to peer review by FIFRA SAP in October 2009.15 Specifically, the 2012 

Residential SOPs have been used to predict the range of potential exposures which could have 

occurred to individuals in the cohort for broadcast hard surface and perimeter carpet treatments. 

Like the dose reconstruction analysis, the present analysis uses chemical-specific data inputs 

recommended in the 2012 Residential SOPS (i.e., the fraction of chlorpyrifos residues 

transferred from treated carpet and hard surfaces to the exposed individual). Similarly, both the 

 13 http://www.epa.gov/pesticides/science/residential-exposure-sop.html 14 http://cfpub.epa.gov/ncea/risk/recordisplay.cfm?deid=236252 15 http://www.regulations.gov/#!docketBrowser;rpp=50;po=0;D=EPA-HQ-OPP-2009-0516
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dose reconstruction and 2016 typical exposure analysis uses registrant submitted chemicalspecific exposures studies and exposure studies from the open literature relevant to the product 

formulations being assessed. In contrast, the present analysis uses PBPK model inputs more 

representative of typical product usage and residential behaviors than those used for the dose 

reconstruction analysis. 

Unlike the two residential exposure scenarios described above, four additional exposure 

scenarios were evaluated without use of standard agency methods (i.e., the 2012 Residential 

SOPs or chemical-specific exposure data). Instead, the highest reported blood chlorpyrifos value 

by Rauh, et al. (2011), 63 pg/g, was used as an anchor for evaluation of internal dosimetry 

following indoor chlorpyrifos exposures to women in the cohort. Two residential exposure 

scenarios were evaluated using the PBPK model to estimate, for a one month time period, what 

daily exposures would be required to result in a peak concentration of approximately 60 pg/g 

blood chlorpyrifos on the 30th (last) day modeled assuming both indoor broadcast hard surface (2 

hour daily exposure) and perimeter carpet treatment (8 hour daily exposure). Two additional 

residential exposure scenarios were evaluated using the PBPK model to estimate, for a one 

month period, what daily exposures would occur following peak concentration of approximately 

60 pg/g blood chlorpyrifos on the 1st (initial) day modeled assuming both indoor broadcast hard 

surface and perimeter carpet treatment.

The following six exposure scenarios were analyzed under the exposure and modeling conditions 

as described: 

1. Broadcast hard surface: A broadcast treatment is the application of a pesticide product 

to the entirety of the hard surface area of the use site. This exposure scenario represents 

the highest of the indoor residential exposures for the 2016 evaluation assessed since it 

results in the most conservative daily exposure estimates and corresponding PBPK model 

outputs of blood chlorpyrifos. Although these uses were cancelled in 1997, existing 

stocks could be applied until depleted. Therefore, some exposure to the broadcast 

formulation was possible for the cohort. A daily exposure time of 2 hours/day was used 

as reported in the 2012 Residential SOPs for the duration an adult spends on a treated 

hard surface. A once daily shower, 10% daily residue dissipation, and a 30 day postapplication period were assumed. 

2. Perimeter, carpet: Given the cancellation of the broadcast uses in 1997, the perimeter 

treatment was likely the predominant exposure scenario for the cohort from 1998–2001. 

A perimeter treatment is a coarse application of a liquid pesticide in a wide band or strip 

of the use site, in this case, the perimeter of the carpeted room. A daily exposure time of 

8 hours/day was used as reported in the 2012 Residential SOPs for the duration an adult 

spends on a treated carpet. Further, a once daily shower, 10% daily residue dissipation, 

and a 30 day post-application period were assumed. 

3. Approximately 60 pg/g blood level on the 30th (last) day of the simulation using 8-hour 

exposure duration (for carpet exposure). The highest reported blood chlorpyrifos value 

by Rauh, et al. (2011) is 63 pg/g. Using approximately 60 pg/g as the peak on the last 
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day of simulation, the agency back-calculated the peak at the time of application 

assuming 10% dissipation rate per day, once daily shower, and 8 hours exposure per day. 

4. Approximately 60 pg/g blood level on the 30th (last) day of the simulation using 2-hour 

exposure duration (for hard surface exposure). The highest reported blood chlorpyrifos 

value reported value by Rauh, et al. (2011) is 63 pg/g. Using approximately 60 pg/g as 

the peak on the last day of simulation, the agency back-calculated the peak at the time of 

application assuming 10% dissipation rate per day, once daily shower, and 2 hours 

exposure per day. 

5. Approximately 60 pg/g blood level on day of chlorpyrifos application (day 1 of the 

simulation) using 8-hour exposure duration (for carpet exposure). The highest 

reported blood chlorpyrifos value by Rauh, et al. (2011) is 63 pg/g. Using approximately 

60 pg/g as the peak on day of application (day 1 of the simulation). Following this peak 

value, a 10% dissipation rate per day, once daily shower, and 8 hours exposure per day 

was assumed. 

6. Approximately 60 pg/g blood level on day of chlorpyrifos application (day 1 of the 

simulation) using 2-hour exposure duration (for hard surface exposure). The highest 

reported blood chlorpyrifos value by Rauh, et al. (2011) is 63 pg/g. Using approximately 

60 pg/g as the peak on day of application (day 1 of the simulation). Following this peak 

value, a 10% dissipation rate per day, once daily shower, and 2 hours exposure per day.

6.3.2 Results

Table 3 below presents the results of the PBPK model runs for each of the six exposure 

scenarios.
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Table 3. Summary of PBPK Model Runs for Analysis of Validation of Columbia Study Blood Levels

Model Run

Daily 

Exposure 

Duration 

(hours)

Highest Peak 

Blood 

Concentration 

(pg/g)

24-Hour 

Blood 

Concentration

(pg/g)

Lowest Peak

Blood 

Concentration

(pg/g)

10 Hours 

After the Last 

Peak on

Day-30 Blood 

Concentration 

(pg/g)

24 Hours After 

the Last Peak 

on Day-30 

Blood 

Concentration

(pg/g)

Broadcast 

(Hard Floor) 2 7,179 41 396 42.6 31.7

Perimeter 

(Carpeted 

Floor)

8 1,053 13 63 12.9 10.7

60 pg/g Peak 

30th Day

2

~ 1,030

4.3

60

6.44 4.79

8 12.8 12.7 10.6

60 pg/g Peak 

on Day of 

Application

2

60

0.25

~ 3.5

0.37 0.28

8 0.74 0.73 0.61

Broadcast (hard floor) and perimeter (carpeted floor)

• The broadcast and perimeter exposure scenarios result in the highest exposure potential 

of all scenarios modeled.

• The highest peak internal dose predicted for post-application exposures to broadcast 

(hard floor, 2 hours) applications is 7,179 pg/g. For the perimeter (carpeted floors, 8 

hours) application, peak internal dose is 1,053. Peak internal dose predictions occurred 

on the day of product application. 

• Internal dose drops rapidly following the highest peak resulting in predicted 24-hour 

internal dose (blood concentrations) of 41 and 13 pg/g for broadcast and perimeter model 

runs, respectively.

• Peak internal dose drops with each subsequent day until the lowest peak is attained on the 

final day of modeled exposure resulting in predicted internal doses of 396 and 63 pg/g for 

the broadcast and perimeter runs, respectively. 

• Following the final day of modeled exposure, internal dose drops initially. Ten hours 

after the final day peak exposure, internal doses predicted from broadcast and perimeter 

exposures are 43 and 12.9 pg/g, respectively.

• Twenty-four hours following the final day of exposure predicted internal doses are 31.7 

and 10.7 pg/g for the broadcast and perimeter runs, respectively. 

• All peak levels predicted for the broadcast model run are above the 60 pg/g level. The 60 

pg/g level is predicted for the perimeter model run on the 30th (final) day. 

60 pg/g peak on the 30th day of exposure

• The highest peak internal dose predicted for post-application exposures from hard surface 

and carpet applications is 1,030 pg/g. Twenty-four hours later the internal dose rapidly 

declines to 4.3 and 12.8 pg/g for hard floor (2 hours) and carpeted floors (8 hours) 

exposures, respectively. 
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• As designed for the simulation, the (lowest) peak internal dose of 60 pg/g occurs on the 

final day of product application. 

• Following the final day of modeled exposure, internal dose drops initially. Ten hours 

after the final day peak exposure, internal doses were of 6.44 and 12.7 pg/g hard floor (2 

hours) and carpeted floors (8 hours) exposures, respectively. 

• Twenty-four hours following the final day of modeled exposure the predicted internal 

doses are 4.79 and 10.6 pg/g for the hard floor (2 hours) and carpeted floors (8 hours) 

exposures, respectively. 

60 pg/g peak on the day of application

• As designed for the simulation, the peak internal dose of 60 pg/g occurs on the day of 

product application. Twenty-four hours later the internal dose rapidly declines to 0.25 

and 0.74 pg/g for hard floor (2 hours) and carpeted floors (8 hours) exposures, 

respectively. 

• The lowest peak internal dose, ~3.5 pg/g, occurs for both the broadcast and perimeter 

runs on the final day of modeled exposure. 

• Following the final day of modeled exposure, internal dose drops initially. Ten hours 

after the final day peak exposure, the internal doses of 0.37 and 0.73 pg/g are predicted 

for hard floor (2 hours) and carpeted floors (8 hours) exposures, respectively. 

• Twenty-four hours following the final day of modeled exposure the predicted internal 

doses are 0.28 and 0.61 pg/g for the broadcast and perimeter runs, respectively. 

Figures 6 through 8 consistently show a saw-tooth pattern of daily, rapid increases in internal 

dose during the exposure period followed by a rapid decline after the exposure period. The peak 

internal dose for all modeled runs occurred during the final hour of daily exposure, just prior to 

the showering event. A rapid drop in internal dose is seen following the bathing/showering event 

and continues to decline until 24 hours following the prior day’s exposure, just before initiation 

of the exposure event for the subsequent day. Internal dose increases once again immediately 

following initiation of the exposure event. 

Further, internal dose peaks on the initial day of exposure and a step wise decline can be 

observed with each subsequent day modeled. This effect is due to the assumption of 10% daily 

dissipation of chlorpyrifos residue. Thus, the potential for exposures to chlorpyrifos are reduced 

with each subsequent day modeled. 

Figures 6 through 8 consistently show that the width of within day internal dose varies based on 

the duration of daily exposure — 2-hour exposure to hard surfaces or 8-hour exposure to a 

carpeted surface. The simulations of the 2-hour daily exposure duration results in a narrower 

width than when compared to the 8-hour exposure duration. 

A rapid decline can be observed for all figures presented below (6–8) following the 30th (final) 

day of exposure. This decline continues to a horizontal asymptote. 
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Figure 6. Graphical representation of the PBPK model run conducted for estimated post-application exposures following perimeter application to 

carpeted floors, 8 hours exposure duration (left) and broadcast application to hard floors, 2 hours exposure duration (right) using Residential SOP 

algorithms and inputs. 

-200

0

200

400

600

800

1000

1200

0 200 400 600 800 1000 1200

Blood Chlorpyrifos (pg/g)

Time (hours)

-1000

0

1000

2000

3000

4000

5000

6000

7000

8000

0 200 400 600 800 1000

Blood Chlorpyrifos (pg/g)

Time (hours)
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Figure 7. Graphical representation of the PBPK model run conducted for evaluation of the peak blood chlorpyrifos concentration on the initial day of 

exposure assuming a peak of 60 pg/g on the final day (30th) of indoor exposure; 8 hour exposure duration (left) and 2 hour exposure duration (right). 

-200

0

200

400

600

800

1000

1200

0 200 400 600 800 1000 1200

Blood Chlorpyrifos (pg/g)

Time (hours)

-200

0

200

400

600

800

1000

1200

0 200 400 600 800 1000

Blood Chorpyrifos (pg/g)

Time (hours)
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Figure 8. Graphical representation of the PBPK model run conducted for evaluation of peak blood chlorpyrifos concentrations on the final day of 

exposure (30th) assuming an initial blood concentration of 60 pg/g; 8 hour exposure duration (left) and 2 hour exposure duration (right).

-10

0

10

20

30

40

50

60

70

0 200 400 600 800 1000

Blood Chlorpyrifos (pg/g)

Time (hours)

-10

0

10

20

30

40

50

60

70

0 200 400 600 800 1000

Blood Chlopryrifos (pg/g)

Time (hours)
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6.3.3 Discussion of Residential Exposure Characterization

Table 3 and Figures 6 through 8 above provide the results of the six post-application exposure 

scenarios, which mimic indoor chlorpyrifos usage by the CCCEH cohort and were input into the 

PBPK model to predict the corresponding internal dosimetry. 

The two residential exposure scenarios, broadcast (hard surface) and perimeter (carpet), were 

evaluated with use of the algorithms and inputs described in the 2012 Residential SOPs. The 

estimated exposures and corresponding internal doses predicted with use of the PBPK model are 

well within the range of the levels reported in the CCCEH cohort and, suggest that the measured 

blood levels were likely from chlorpyrifos use in the home environment. The internal doses 

estimated for broadcast and perimeter applications are below 60 pg/g in as little as 24 hours 

following the initial (peak) day of exposure, and drop rapidly within the first 10 and 24 hours 

after the final day peak exposure. This observation supports that the women in the CCCEH 

cohort could have had internal dose levels well within the high range of those reported on any 

given day following monthly treatment. Further, for the perimeter (carpet) exposure scenario, 

the estimated peak internal dose on the 30th (final) day of exposure is approximately 60 pg/g. 

That is, even in consideration of perimeter carpet peak internal dose, levels in the range of the 60 

pg/g following exposures of this magnitude are attainable. 

Figure 1 provides a summary of the reported blood levels in CCCEH across the cohort by year; 

this information was provided to the agency in 2015. The 1998–1999 CCCEH data represent the 

time period of highest use during the epidemiology study. For the 1998–1999 CCCEH data, the 

75th and 95th percentile are reported as 9.55 pg/g and 19.35 pg/g, respectively (Figure 1). These 

values are similar to the agency’s estimates at 24 hours following the final day of peak exposure 

for the perimeter carpet (10.7 pg/g) and broadcast hard surface (31.7 pg/g) scenarios (Table 3). 

At the 24-hour time point following the final day of peak exposure, the perimeter carpet (10.7 

pg/g) simulation is approaching the lower end of the top tertile used in Rauh et al. (6.17 pg/g; 

2006, 2015). Moreover, the pattern of internal doses modeled for the perimeter (carpet) 

exposure scenario and that predicted for simulation using 60 pg/g as the peak at day 30 are very 

similar including the maximum peak, lowest peak, the 24-hour blood concentration following 

peak exposure on day of an application, and the 10- and 24-hour blood concentrations after the 

final day peak exposure for 8 hours of exposure. The results of these simulations together 

support the conclusion that the reported values in the CCCEH are driven primarily by residential 

use of the broadcast and perimeter chlorpyrifos products. These results further support the 

reasonableness of the magnitude and distribution of data reported data by the CCCEH.

For the simulations where 60 pg/g is the peak at the beginning of the simulation (i.e., day of 

application), the low values after the showering events are at or below the LOD (0.5 or 1.0 pg/g) 

at all time points. This pattern is more reflective of the CCCEH in years after the cancellation of 

the indoor uses and not reflective of the residential use. For example, in the 1998/1999 CCCEH 
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data, even at the 10th percentile, the maternal blood data are above the LOD. In contrast,

beginning in 2001, there is a dramatic increase in the number of samples below the LOD (Figure 

1) with only the 75th percentile and higher with reported values above the LOD. These results 

may suggest that this scenario is too low to represent actual residential exposure. 

In sum, the purpose of these exposure scenarios was to characterize the blood levels of 

chlorpyrifos reported by CCCEH investigators. Even using EPA’s Residential SOPs with high

end assumptions for broadcast use on hard surface (the highest possible scenario), 24 hours after 

peak exposure on day 1 of an application, 10 and 24 hours after the final day peak exposure the 

predicted values are within the range of blood values reported by CCCEH. Lower internal doses 

are achieved with lower but still high end exposure scenarios (perimeter use on carpet) and lead 

to values in the terminal half-life phase near the low end of top tertile values in the CCCEH (6.17 

pg/g) and near the 75th percentile of the maternal blood levels for the 1998/1999 time period 

when chlorpyrifos use was highest for the cohort. Taken together, this exposure characterization 

analysis suggests that the CCCEH values are plausible and reasonable based on the agency’s 

analysis and likely driven by residential exposure. Food exposure is expected to have occurred 

but not likely lead to higher levels in blood whereas drinking water exposure was unlikely. 

7.0 Deriving a Point of Departure (PoD) for Neurodevelopmental Outcomes

7.1 Uncertainties with Using Biomarker for the PoD 

In CCCEH, chlorpyrifos in blood was used as the exposure metric to evaluate the association 

between chlorpyrifos exposures and adverse health outcomes. While biomarker data are 

arguably superior to conventional exposure data in that they reflect chemicals that were absorbed 

in the body from all routes and sources, they do not provide direct measure of environmental 

exposure levels important for regulatory purposes. In general, biomarkers are expected to reflect 

aggregate exposures over a period less than five half-lives prior to sampling. For chemicals with 

short biological half-lives such as chlorpyrifos, their biomarker levels are determined by recent 

exposures. Especially for chlorpyrifos in blood, it is a snapshot of the instantaneous, internal

concentration at time of sampling. Depending on the exposure patterns, there can be large intraindividual temporal variability in spot measurements of blood concentration. When one attempts 

to use biomarker measurements as an exposure surrogate to evaluate the association between 

exposure and disease, the assumption is that higher biomarker concentrations came from higher 

exposure concentrations. But, it is not difficult to see that this assumption could be questioned in 

CCCEH by randomly selecting a time point on the PBPK model-simulated time course of 

chlorpyrifos concentrations in blood under various exposure scenarios to simulate a spot blood 

measurement in CCCEH. A specific blood level may be reached immediately after the 

termination of exposure at a lower dose, several days after the termination of exposure at a 

higher dose, or any combinations of dose and timing of exposures. Without knowing the timing 

of exposures relative to blood sample collection, a single measure of chlorpyrifos concentration 
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in blood was only evidence of exposure and absorption, and cannot be used to reconstruct 

sources, routes, frequency, duration, timing, and magnitude of exposures. For these reasons, the 

agency has not attempted to conduct reverse dosimetry to estimate doses that could have resulted 

in these observed blood levels, but has instead opted for conducting forward dosimetry through 

the development of a series of likely exposure scenarios that span a broad range of exposure 

potential to compare the predicted blood concentrations of chlorpyrifos with data observed in 

CCCEH (Section 6.3). 

With the proposed approach, the assumption is that the CCCEH cord blood data were not 

collected during the period at which blood concentrations of chlorpyrifos were at their peak or 

rapidly changing, but instead at or near the low points on PK curves and likely during the 

asymptote period where blood concentrations were fairly stable across several days. Cord blood 

was collected at delivery which suggests that volunteers in CCCEH have spent hours in labor 

and delivery in the hospital, meaning removal from their apartments and the exposure to 

chlorpyrifos had ceased. 

Besides the large uncertainty associated with linking chlorpyrifos concentrations in blood to 

chlorpyrifos doses being exposed by these volunteers, uncertainty also exists when establishing a 

quantitative relationship between chlorpyrifos concentrations in blood and adverse health 

outcomes. Ideally, such relationship can only be established when a biomarker is a reasonable 

surrogate for the toxic moiety at the target tissue. For health endpoints investigated in these 

epidemiologic studies, the adverse outcome pathways, toxic moieties, and biological targets were 

all unknown. Furthermore, it is always challenging in epidemiological studies to perform 

meaningful analysis to link biomarker measurements that reflect short-term exposures to longterm health outcomes. The key assumption is that measured biomarker levels reflect exposures 

during time windows that were critical for disease onset. It is also not clear whether cord blood 

concentrations measured at birth reflect exposure levels during the critical time window(s). 

However, there is a reasonable likelihood that chlorpyrifos was applied multiple times in the 

apartments of the women in the cohort over the course of the pregnancy (potentially once a 

month) increasing the potential for exposure during those unknown critical period(s).

In the context of the uncertainties associated with using the CCCEH blood data in quantitative 

risk assessment, there is concern that the PoDs based on AChE inhibition (Appendix 1) may not 

be adequately protective of human health. For example, given an external dose required to 

achieve 10% AChE inhibition for a female worker who was exposed dermally to chlorpyrifos 8 

hours/day, 5 day/week for 3 weeks, the blood concentration of chlorpyrifos peaked at 120,000 

pg/g, and was still above 100 pg/g at 32 days after the last exposure. Similarly, at a food 

exposure level leading to 10% AChE inhibition, chlorpyrifos concentration in blood never goes 

below 100 pg/g over the continuous 21-day exposure simulation and is around 7000 pg/g at the 

daily peaks. The agency could consider continuing to use the AChE PoDs, but additional safety 

factors beyond the FQPA Safety Factor 10X would be needed to have sufficient confidence to 
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conclude that there is a reasonable certainty of no harm under the FQPA. However, the agency

would still need to quantify such additional factors—and the analysis to quantify them would 

again require the agency to make quantitative use of the CCCEH cord blood data with the same 

uncertainties described above. Although either approach would be possible, the agency has 

elected to propose to use the cord blood directly as the PoD as the simpler, more understandable 

approach. 

7.2 Options for PoD Based on the CCCEH Biomonitoring Data 

From the CCCEH publications, there are two general options for deriving a PoD for 

extrapolating neurodevelopmental risk from chlorpyrifos exposure. 

1) Lower limit of the top tertile (>6.17 pg/g cord blood) derived from Rauh et al. (2006) and 

repeated in other CCCEH publication. Rauh et al. (2006) includes 254 children followed 

through age 3 years and used a dichotomized statistical approach of <6.17 pg/g cord blood for 

the ‘low group’ and >6.17 pg/g cord blood as the ‘high’ group. Using the same CCCEH study 

data, earlier studies (Whyatt et al., 2004; Perera et al., 2003) also documented statistically 

significant associations between birth outcomes and cord blood chlorpyrifos levels (>6.17 pg/g); 

however, these birth outcome associations are not the focus of this assessment. The Rauh et al.

(2006) study reported statistically significant deficits of 6.5 points on the Bayley Psychomotor 

Development Index (PDI) at 3 years of age when comparing high to low exposure groups. 

Notably these decrements in PDI persist even after adjustment for group and individual level 

socioeconomic variables (Lovasi et al., 2011), with this follow-up study using the same 

dichotomized statistical approach for grouping high and low exposure. These investigators also 

observed increased odds of mental delay (OR=2.4; 95% CI: 1.1–5.1) and psychomotor delay 

(OR=4.9; 95% CI: 1.8–13.7) at age 3 years when comparing high to low exposure groups (Rauh 

et al., 2006). Rauh et al. (2006) also reported extremely large odds ratios for attention disorders 

(OR=11.26; 95% CI: 1.79–70.99), ADHD (OR=6.50; 95% CI: 1.09–38.69), and PDD (OR=5.39; 

95% CI: 1.21–24.11) at age 3 years when comparing high to low chlorpyrifos exposure groups 

(Rauh et al., 2006). Furthermore, these investigators observed increased odds of mild to 

moderate tremor at age 11 when comparing high to low exposure groups (Rauh et al., 2015). As 

such, if the agency pursued these studies for deriving a PoD, the appropriate value would be 6.17 

pg/g cord blood.

2) Benchmark Dose estimates derived from linear regression reported in Rauh et al. (2011) for 

deficits in Working Memory. Rauh et al. (2011) evaluated the relationship between prenatal 

chlorpyrifos exposure and neurodevelopment among 265 of the CCCEH cohort participants who 

had reached the age of 7 years and had a complete set of data including prenatal maternal 

interview data, prenatal chlorpyrifos marker levels from maternal and/or cord blood samples at 

delivery, postnatal covariates, and neurodevelopmental outcome data. Rauh et al. (2011) 

described the log of Working Memory Index (WMI) of children as linearly associated with 

concentration of chlorpyrifos (CPF) in cord blood with a slope = -0.006
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(95% CI = -0.01, -0.002). Investigators reported that for each 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. 

For deriving potential benchmark dose (BMD) estimates from Rauh et al. (2011), the agency is 

interested in estimated concentration levels of chlorpyrifos in cord blood associated with specific 

percent(s) reduction in WMI of children. The concentration levels of chlorpyrifos in cord blood 

(and its 95% lower confidence limit, one-sided) can be estimated using the estimated slope and 

its 95% lower confidence limit (one-sided). However, neither the 95% lower confidence limit 

nor the standard error of the estimated slope (which can be used to calculate the 95% lower 

confidence limit) were available in Rauh et al. (2011). Therefore, the standard error of estimated 

slope was back-calculated as: (estimated slope – lower bound of 95% CI)/1.96 which was then 

used to calculate the 95% lower limit (one-sided) of estimated chlorpyrifos levels. The 

justification of using the 95% lower confidence limit (one-sided) of an estimated slope to 

estimate the 95% lower confidence limit of a dose level is described in Appendix 5 and 

confirmed by a simulation. The SAS code of the simulation is included in Appendix 5.

From the estimated slope = -0.006 and its 95% (two sided) CI = (-0.010, -0.002) from Rauh et al.

(2011), the 95% lower confidence limit (one-sided) of the estimated slope was calculated as 

described above to be -0.009. Using the values of estimated slope and its 95% one-sided lower 

confidence limit, the chlorpyrifos concentrations and their 95% lower limit associated with 1%, 

2%, 3%, 4% and 5% reduction in WMI were calculated. The results of calculations are 

presented in Table 4 below.

In order to make a PoD proposal based on the results presented in Table 4, the agency took 

several factors into consideration using a bounding approach. First, at the lower end, a 1% 

change in working memory is predicted which corresponds to the central and lower limit 

estimates of the internal dose (as indicated by cord blood levels) of 1.68 and 1.07 pg/g, 

respectively. These values are close to the LODs of 0.5/1.0 pg/g for measuring chlorpyrifos 

residues in cord blood (Rauh et al., 2006; 2011; 2015). Because of the proximity to the LOD, 

the lower cord blood levels have more uncertainty associated with them than higher cord blood 

levels (e.g., associated with a ≥2% loss in working memory) because they likely are based on 

partially imputed data and are thus not completely quantitatively supported. Second, at the 

higher end, there is a basis for the upper limit bound levels at which chlorpyrifos exposure has 

been associated with neurodevelopmental effects, corresponding to chlorpyrifos levels in cord 

blood that were evaluated in Rauh et al. (2006, 2015). Specifically, the estimated blood 

concentrations to achieve 3–5% reductions in working memory are near or above the 6.17 pg/g 

which represents the lower limit of the top tertile where neurodevelopmental outcomes have 

been associated with chlorpyrifos exposure (Rauh et al., 2006, 2015). As such, selecting a PoD 

based on a WMI ≥ 3% which occurs at a cord blood BMD of 5.08 pg/g (BMDL 3.26 pg/g) may 

not be protective of such outcomes. Therefore a 2% working memory reduction which 
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corresponds to an internal dose measure of 2.16 pg/g chlorpyrifos in cord blood is quantitatively 

near the value reported by Rauh (2.8% reduction in working memory) and within the range of 

levels measured in the blood samples collected by investigators. Thus, a value of 2% is proposed 

as the PoD which is supported by the existing data and it is health protective. Finally, it should 

be noted as a matter of science policy when using BMD estimates for deriving PoDs, the agency 

uses the lower limit (BMDL), not the central estimate (BMD) (USEPA, 2012c). As such, 

selection of the 2.16 pg/g value is consistent with how BMDLs are generally considered in 

agency risk assessments (i.e., since it is the BMDL and not the BMD). In summary, the agency 

is proposing to use the 2% reduction as the PoD in working memory or an internal dose of 

2.16 pg/g.

Table 4. Estimated concentration of CPF in cord blood at specific percent reduction of WMI

Estimated 

slopea

(95% CI, twosided)

Calculated by EPA\HED

95% lower 

confidence 

limit (onesided) of 

estimated 

slopeb

Percent 

Reduction 

of WMI 

(%)

Estimated 

concentration of 

CPF in cord bloodc

(pg/g):

BMD

95% lower limit 

(one-sided) of 

estimated concentration 

of CPF in cord bloodd 

(pg/g):

BMDL

-0.006 

(-0.010, -0.002) -0.009

1 1.68 1.07

2 3.37 2.16

3 5.08 3.26

4 6.80 4.36

5 8.55 5.48 a estimated slope (95% CI) was as stated in Rauh et al., 2011 study

b 95% lower confidence limit (one sided) of the estimated slope calculated by HED, using 

the Rauh’s estimated slope and its 95% CI to estimate SE c estimated CPF concentration associated with the given percent of reduction in WMI, 

calculated using the estimated slope = -0.006

d 95% lower confidence limits of CPF concentrations associated with the given percent of 

reduction in WMI, calculated using the 95% lower confidence limit = -0.009 of the 

estimated slope 

8.0 Assessing Extrapolation/Uncertainty

No risk assessment can reflect risk with absolute certainty, so it is important that uncertainties be 

accounted for in a predictable, scientifically defensible manner that is both consistent with EPA’s 

policies and responsive to the needs of decision makers (U.S. EPA, 2004). In deriving reference 

concentrations (RfCs) and reference doses (RfDs), the agency has historically used default 

uncertainty factors (UFs) to compensate for a lack of information (U.S. EPA, 2002). 
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In typical risk assessments, PoDs are derived directly from laboratory animal studies and interand intra-species extrapolation is accomplished by the use of 10X factors. In the case of 

chlorpyrifos, the proposed PoDs are derived from human information obviating the need for the 

inter-species extrapolation. The intra-species uncertainty factor (UFH) is applied to account for 

variations in susceptibility within the human population (human variability) and the possibility 

(given a lack of relevant data) that the database available is not representative of the 

dose/exposure response- relationship in the groups of the human population that are most 

sensitive to the health hazards of the chemical being assessed (U.S. EPA, 2002). As such, the 

agency still needs to consider intra-species extrapolation of the PoD from the CCCEH 

epidemiology data across the diverse human population. Moreover, the agency must consider 

the statutory requirement of the FQPA 10X Safety Factor for “potential pre- and postnatal 

toxicity and completeness of data with respect to exposure and toxicity to infants and 

children.” An evaluation of strengths and uncertainties of the existing chlorpyrifos database of 

epidemiology and laboratory studies associated with the intra-species extrapolation factor and 

the FQPA 10X Safety Factor are provided in Section 8.2.

For the evaluation of strengths and uncertainties associated with the existing epidemiology 

database, OPP first considered existing agency experience in using epidemiology studies to 

evaluate neurodevelopment in children. Specifically, OPP has used the 2000 NRC report on 

methyl mercury and the methyl mercury IRIS entry16 as a starting point for considering the key 

factors in evaluating uncertainty in the PoD and extrapolation. Table 5 was extracted from the 

2000 NRC report on methyl mercury and provides the key characteristics of importance 

considered for the intra-species factor. Appendix 7 provides a comparison of the major 

considerations from NRC for methyl mercury with the strengths and uncertainties associated 

with the CCCEH epidemiology studies. 

 16 http://cfpub.epa.gov/ncea/iris2/chemicalLanding.cfm?substance_nmbr=73
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Table 5. Table 8-2 extracted from the 2000 NRC report on methyl mercury.

8.1 Intra-species Extrapolation

The default value for UFH is 10-fold and can be apportioned into pharmacodynamic (PD) and 

PK components values, often at one half order of magnitude each.

Pharmacodynamics (PD): The AOP(s) for neurodevelopmental outcomes related to chlorpyrifos 

exposure is/are not delineated such that the molecular initiating event(s) and related key events 

are not known nor is the quantitative dose response relationships of such events. By extension, 

the window(s) of susceptibility and associated duration(s) of exposure are not known. The SAP 

also noted similar uncertainty commenting on the “Lack of specificity of a critical window of 

effect and the potential for misclassification of individual exposure measures.” Furthermore, 

given that biomarker data were taken at a single point in time (cord blood at delivery) and that 

chlorpyrifos exposure in the home was dynamic, there is uncertainty as to the representativeness 

of cord blood data for the entire pregnancy or for critical windows of susceptibility. On this 

issue, the SAP noted that with respect to “Use of a single or average sample for exposure...

Although Whyatt et al. (2009) noted moderate but significant correlations between meconium 

and cord and maternal blood and average urine TCPy, the representativeness of a single point 

exposure is still unclear. Time-varying exposures or the ability to define cumulative exposures 

would be preferable.” Similar uncertainties were noted in the methyl mercury studies with 

respect to unknown windows of susceptibility and the degree to which existing biomonitoring 
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data do or do not represent those critical periods. Moreover, given that the CCCEH investigators 

did not collect information on timing or frequency of chlorpyrifos applications, it is not known 

whether the existing cord blood encompasses the levels of chlorpyrifos near the day(s) of 

application(s). 

The SAP noted an uncertainty associated with “External generalizability of the cohorts given 

their unique racial/ethnic and socioeconomic characteristics.” The CCCEH study focused 

exclusively on women who self-identified as African-American or Dominican, were 18–35 years 

of age, and who lived in specific urban areas (northern Manhattan (Central Harlem or 

Washington Heights/Inwood) or the South Bronx) for a year or more prior to pregnancy. This 

study population was of generally low socioeconomic status, with approximately 85% of the 

population having annual incomes of $30,000 or less (Whyatt et al., 2003). However, the agency

does not have adequate data to assess the relative sensitivity of these groups compared with 

others. Given the current state of knowledge, the PD variability cannot be quantified and is 

therefore unknown. For PD variation, a data-derived factor cannot be derived and the default 3X 

is retained. 

Pharmacokinetics (PK): With respect to PK variation, although there is a robust PBPK model 

which accounts for population variability for infants, a fully vetted, validated PBPK model is not 

available for pregnant women (See Section 4.0). As such, for PK variation, a data-derived factor 

cannot be derived and the default 3X is retained. 

Intra-species Factor: As such for chlorpyrifos, the agency proposed to use a 10X intra-species 

extrapolation factor. This 10X, apportioned equally between 3X for PK variability and 3X for 

PD variability is consistent with that used for methyl mercury (Table 6). 
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Table 6. UFs recommended by NRC and used by EPA IRIS for methyl mercury

Consideration Consideration from NRC 

Report Used by EPA in IRIS Assessment

Pharmacokinetics

Interindividual toxicokinetic 

variability in dose 

reconstruction

Pharmacokinetic variability and uncertainty in 

estimating an ingested mercury dose from cordblood mercury concentration: a factor of 3 was 

applied: NRC analyses of variability in the 

pharmacokinetic factors underlying the 

conversion of a biomarker level of 

methylmercury to an ingested daily dose of 

methylmercury that corresponds to that level

Pharmacodynamics N/A

Pharmacodynamic variability and uncertainty: a 

factor of 3 was applied: the population of the 

Faroe Islands is descended from Scandinavian 

stock that settled many generations ago, and is 

extremely homogeneous. The average 

toxicodynamic response of this population 

compared with that of the United States, which is 

genetically much more diverse, is unknown. 

Similarly, the relative variability of different 

populations also is unknown. A threefold UF for 

toxicodynamic variability and uncertainty was 

applied.

Database 

Uncertainty

Data-base insufficiency (i.e., 

because of consideration of 

possible low-dose sequelae 

and latent effects, and 

immunotoxicity and 

cardiovascular effects)

N/A

Total UFs

“the committee believes that 

an overall uncertainty-factor 

adjustment of no less than 10 

is necessary and appropriate 

to provide an adequate

margin of protection.”

overall UF of 10

8.2 FQPA 10X Safety Factor for Infants & Children

The FQPA (1996) instructs EPA, in making its “reasonable certainty of no harm” finding, that in 

“the case of threshold effects, an additional tenfold margin of safety for the pesticide chemical 

residue and other sources of exposure shall be applied for infants and children to take into 

account potential pre- and postnatal toxicity and completeness of data with respect to 

exposure and toxicity to infants and children.” Section 408 (b)(2)(C) further states that “the 

Administrator may use a different margin of safety for the pesticide chemical residue only if, on 

the basis of reliable data, such margin will be safe for infants and children.” 
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8.2.1 Pre- and Post-Natal Toxicity Database

Experimental Laboratory Animals: There are numerous animal studies in the literature (see 

Appendix 3) which vary in their study design but all involve gestational and/or early postnatal 

dosing with behavioral evaluation from adolescence to adulthood. The data provide support for 

the susceptibility of the developing mammalian brain to chlorpyrifos exposure through gestation 

and early in life, with adverse outcomes in several neurological domains including cognitive, 

anxiety and emotion, social interactions, and neuromotor function. The studies have not shown 

that any specific developmental period is critical overall to the long-term outcomes, since similar 

effects are observed with different exposure periods. For example, cognitive changes in one 

laboratory using a radial arm maze were observed following gestational and early, but not late, 

postnatal exposure (Aldridge et al., 2005; Icenogle et al., 2004; Levin et al., 2002; Levin et al., 

2001), whereas other laboratories cognitive deficits in a Morris water maze were reported 

following both gestational and late-postnatal exposure (Billauer-Haimovitch et al., 2009; 

Turgeman et al., 2011). Likewise, some changes in anxiety and social behaviors were reported 

at both gestational and postnatal exposure periods. Overall, these data do not clearly show 

specific critical periods of exposure but support a conclusion that early life (pre- and post-natal) 

represent susceptible lifestages. 

Epidemiology Studies: Numerous epidemiological investigations have observed a link between 

prenatal exposure to chlorpyrifos or OPs (measured as chlorpyrifos, TCPy, or DAPs) and 

adverse effects on neurodevelopment through age seven years, with additional more limited 

evidence up through approximately age eleven. As noted previously, for these epidemiology 

studies chlorpyrifos was only assessed directly in the CCCEH study (Rauh et al., 2006, 2012,

2015), with the Mexican cohort study (Fortenberry et al., 2014) assessing the chlorpyrifos 

metabolite TCPy, and the CHAMACOS cohort study (Eskenazi et al., 2007) measuring both 

TCPy and DAPs. In contrast, all of the other epidemiology studies assessed only DAP exposure, 

and as noted previously these DAPs are metabolites of multiple OPs including chlorpyrifos.

The majority of epidemiological studies investigated only prenatal exposures. Specifically, with 

respect to biomarkers representing prenatal exposures, the three US cohorts (CCCEH, Mt. Sinai, 

CHAMACOS) each reported evidence of impaired mental and psychomotor development, albeit 

not consistent by age at time of testing (ranging from 6 month to 36 months across the three 

cohorts). Statistically significant or suggestive associations between chlorpyrifos or DAPs 

exposure and attentional problems/ADHD were reported by multiple prospective cohorts (Rauh 

et al., 2006; Eskenazi et al., 2007; Marks et al., 2010; and Fortenberry et al., 2014) with 

additional support from a case control study, Bouchard et al. (2010). In addition, the three US 

cohorts and the CHARGE study have reported suggestive or positive associations between OP 

exposure and autism spectrum disorders (Rauh et al., 2006; Shelton et al., 2014; Eskenazi et al., 

2007; Furlong et al., 2014). While these studies vary in the magnitude of the overall strength of 

association, they have consistently observed a positive association between OP exposure and 

ASD. Several studies have also documented statistically significant association between prenatal
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DAP exposure and abnormal motor development in neonates (reflexes, Brazelton score or 

similar measure; Young et al., 2005; Engel et al., 2007; Zhang et al., 2014). Finally, CCCEH, 

Mt. Sinai and CHAMACOS have reported an inverse relation between the respective prenatal 

measures of chlorpyrifos or DAPs and intelligence measures at age 7 years (Rauh et al., 2011; 

Engel et al., 2011; Bouchard et al., 2011).

A smaller number of studies have assessed postnatal exposure to DAPs. Postnatal exposures 

have not been assessed in the CCCEH and Mt. Sinai studies (Rauh et al., 2011; Engel et al., 

2011); as such, there are no studies included in this analysis which directly assessed the potential 

for postnatal chlorpyrifos exposure and associations with neurodevelopmental effects. However, 

given that the major source of exposure (residential use) was cancelled partway through the 

CCCEH study which substantially reduced and largely removed chlorpyrifos from the home 

environment, this limits the ability of this study to inform the impacts of long-term postnatal 

exposure to chlorpyrifos on neurodevelopment from the current uses of chlorpyrifos.

Postnatal exposure to DAPs has been assessed in the CHAMACOS cohort (Eskenazi et al., 2007; 

Young et al., 2005; Bouchard et al., 2011) and three cross-sectional studies (Guodong et al., 

2012; Bouchard et al., 2010; Oulhote and Bouchard, 2013). With the exception of Bouchard et 

al. (2010), no adverse neurodevelopmental associations were found between postnatal urinary 

metabolite levels and any of the developmental outcomes. Bouchard et al. (2010) looked at U.S. 

children age 8–15 years in the 2000–2004 National Health and Nutrition Examination Survey 

(NHANES), and observed a positive association between attention and behavior problems and 

total DAPs and DMAPs, but not DEAPs. 

The agency is aware that postnatal exposure to DAPs has been assessed but not found to be 

associated with neurodevelopmental outcomes in the CHAMACOS cohort. However, in sum, 

given that the extensive experimental laboratory animal database suggests that the post-natal 

period is a potential susceptible time, the lack of postnatal exposure assessment in the CCCEH 

and Mt. Sinai studies is a source of uncertainty in the epidemiology database. 

8.2.2 Impact of Sample Size on CCCEH Findings

In the 2012 SAP report, the Panel noted the CCCEH studies had:

• “Relatively modest sample sizes which limited the statistical power to classify some 

meaningful differences as statistically significant and to examine the effect of 

modification by race/ethnicity and other characteristics.”

• “Relatively moderate to large exposure differences needed to see significant effects, 

likely due to the modest sample sizes used.”

Generally, lower sample sizes limit the statistical power of a study. This in turn may lead to 

larger and less stable confidence intervals and or error bars, including not achieving statistical 

significance. In EPA’s analysis of the strengths and weaknesses of the epidemiology studies, it 

is apparent that while the chance that positive associations observed are false positives due to 
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systematic errors in the studies cannot be excluded, it is more likely that error present in these 

studies would lead to the under-estimation 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 explanations are less plausible than the 

alternative that associations have been missed or under-estimated due to non-differential 

measurement error and low sample size across exposure strata. Exposure measurement error has 

been shown to more greatly influence epidemiology study results than unknown or unmeasured 

confounding variables. Additionally, the elimination of temporal bias due to the prospective 

study design employed within each of the three children’s health cohorts assures that prenatal 

exposures preceded neurodevelopmental outcomes measured at birth, and in early and later 

childhood through age 7 years. There is additional, more limited, evidence of 

neurodevelopmental outcomes in later childhood through approximately 11 years from the Mt. 

Sinai and CCCEH studies (Furlong et al., 2014; Rauh et al., 2015). The strength of the 

associations measured was in some instances quite strong. For example, in relation to pre-natal 

chlorpyrifos exposure, measures of mental and psychomotor delays were 2–4 fold greater among 

the most highly exposed as compared to those in the lower exposure category, and the relation 

between attention-deficit symptoms (age three years) and in utero exposure was significantly 

elevated, although imprecise (Rauh et al., 2006). However, associations in many instances were 

weak to moderate, possibly due to exposure measurement error such as timing of exposure in 

relation to cord blood collection. 

Associations with neurodevelopmental outcomes were consistently identified with respect to the 

number of abnormal reflexes in the neonatal period, the presence of mental and behavioral issues 

as well as gross motor delays were pronounced especially in later toddler years of 24–36 months, 

and the observation of intelligence decrements were seen across the three cohorts using different 

measures of prenatal chlorpyrifos exposure. However, with regards to dose-response, the modest 

sample size in the CCCEH study make it difficult to say that the dose-response relationship 

between chlorpyrifos and neurodevelopmental outcomes in the overall U.S. population has been

fully characterized. The magnitude of the PoD in the general U.S. population of infants and 

children may be higher or lower than that estimated using the CCCEH study results, and the 

shape of the dose-response curve may also be different. 

8.2.3 Conclusion on the FQPA Safety Factor

The FQPA (1996) requires that the agency include “an additional tenfold margin of safety” 

unless “on the basis of reliable data, such margin will be safe for infants and children.” In other 

words, the FQPA 10X Safety Factor is present unless there are reliable data to remove it. In the 

case of chlorpyrifos, there are remaining uncertainties surrounding 1) the lack of information on 

post-natal exposure and evaluation of such exposure to neurodevelopmental outcomes in the 

CCCEH and Mt. Sinai cohorts and 2) the modest sample sizes which limited the statistical power 

and exposure differences required to see statistically significant results. As such, there is 

sufficient uncertainty which prevents the agency from reducing or removing the statutory 10X 
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FQPA Safety Factor. EPA therefore intends to continue to retain the FQPA 10X Safety Factor 

for assessing risk from chlorpyrifos for infants, children, youths, and women of 

childbearing age for all exposure scenarios.

9.0 Proposed Approach to Deriving Internal Dose Estimates: Integration of 

Exposure Assessment & PBPK Modeling

9.1 Overview

Typical risk assessments use administered dose as the PoD from an animal study, or less often,

human data and then compare PoDs to external exposure levels to estimate health risks result 

from exposures. As such, risk metrics most often used by OPP to assess human health are the 

reference dose (RfD) and margins of exposure (MOE)17. For the chlorpyrifos risk assessment, 

the agency is proposing to use internal blood concentrations of chlorpyrifos derived from the 

CCCEH cord blood data as the PoD (Section 7.0). Because of the uncertainties associated with 

reverse dosimetry due to unknown exposure timing, frequency, and magnitude, the agency

cannot confidently derive an administered/external dose from the CCCEH data. As such, the 

agency must use forward dosimetry to derive internal concentrations of chlorpyrifos from the 

most likely exposure scenarios to compare with the internal PoDs. Given the same exposure 

scenario (e.g., single daily oral dose, 2 hours/day dermal dose for 30 days), the internal 

concentrations are linearly related to external doses. 

Specifically, the agency has proposed an internal concentration PoD of 2.16 pg/g adjusted by 

100X based on the intra-species factor and 10X FQPA Safety Factor. As such the internal 

concentration RfD for assessing dietary (food and water) exposure is 0.022 pg/g. For 

exposure assessment which uses MOE as the risk metric (residential, occupational 

assessment), the internal PoD of 2.16 pg/g will be used with a target MOE of 100. 

As described earlier, the PK profile for chlorpyrifos in blood is characterized by rapid increases 

and decreases during exposure periods and immediately after exposure, followed by slower 

decrease during the terminal clearance phase. Because the CCCEH cord blood data are likely to 

represent data from the low points on the PK profile and likely in terminal clearance phase, the 

agency will, to the extent possible, compare internal concentrations of chlorpyrifos at 10 and 24 

hours after the last peak in each simulation, specifically for food exposure and 

occupational/residential exposure scenarios. 

Drinking water exposure provides a different challenge as exposure is more frequent. In 

particular, infants feed multiple times per day and night for several months. For these pathways, 

 17 RfD = Point of Departure/ Uncertainty Factors and then is compared to exposure ; MOE = Point of Departure 

/Exposure and then is compared to total uncertainty factors
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the agency is comparing the internal PoD and internal RfD to the full range of potential internal 

exposures.

The following sections provide overview information on OPP’s exposure assessment approaches 

for food, water, and occupational/residential exposure assessment followed by case study 

examples of inputing OPP exposure estimates into the PBPK model for women of childbearing 

age and infants. 

9.2 Food Exposure

9.2.1 Methods

EPA’s OPP has performed acute and 21-days and longer [i.e., steady state dietary (food)] 

exposure assessments for chlorpyrifos. This case study was performed for the purpose of 

obtaining estimated food exposure values for critical lifestages of concern (infants and females 

of childbearing age) for use with the chlorpyrifos PBPK model and were originally performed as 

part of the 2014 HHRA (USEPA, 2014a). The analyses considered combined exposure from all 

food commodities from crops or livestock for which there is a current U.S. tolerance for 

chlorpyrifos. The methods used are summarized below as are the resulting dietary exposure 

estimates for infants (<1 year old) and females (13–49 years old). The dietary assessment 

methods described below have undergone previous FIFRA SAP review (FIFRA SAP 2000a; 

FIFRA SAP 2000b) and are not subject to the current SAP review but are presented for purposes 

of assessing the new approach to integrate the exposure assessment with the PBPK model. 

Based on evidence [food monitoring data, crop field trials and metabolism studies (Trichilo, 

1988)] indicating that the metabolite chlorpyrifos oxon would be not be present in edible 

portions of the plant, it is not a residue of concern in food or feedstuff. The chlorpyrifos oxon, 

along with parent compound chlorpyrifos, is extensively tested for in a wide variety of 

commodities by the U.S. Department of Agriculture’s (USDA’s) Pesticide Data Program (PDP) 

food monitoring program. 18 While chlorpyrifos is frequently detected, the oxon is not; in fact, 

from 2007 to 2012, out of several 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 registered uses of chlorpyrifos on potato in the U.S.). The oxon 

metabolite was not found in milk or livestock tissues in cattle and dairy cow feeding studies, 

even at exaggerated feeding levels, and is not a residue of concern in livestock-based food 

commodities (Trichilo, 1988). The residue of concern included in the dietary exposure 

assessments is the parent compound chlorpyrifos. 

Chlorpyrifos acute dietary exposure analyses were conducted for the 2014 HHRA using the 

Dietary Exposure Evaluation ModelTM (DEEM) with the Food Commodity Intake Database 

 18 https://www.ams.usda.gov/datasets/pdp
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(FCID), Version 3.16, which incorporates 2003–2008 consumption data from USDA’s National 

Health and Nutrition Examination Survey, What We Eat in America, (NHANES/WWEIA). 19

The data are based on the reported consumption of more than 20,000 individuals over two nonconsecutive survey days. Foods “as consumed” (e.g., apple pie) are linked to EPA-defined food 

commodities (e.g., apples, peeled fruit — cooked; fresh or N/S; baked; or wheat flour — cooked; 

fresh or N/S, baked) using recipe translation files developed jointly by USDA/ARS and EPA. 

The diary is the entire list of foods consumed by one person on a single day. The weights of the 

food consumed in that day are multiplied by their respective pesticide residue values and added 

together to determine the quantity of pesticide consumed. That value is divided by body weight 

to arrive at the exposure estimate in terms of milligrams per kilogram body weight per day 

(mg/kg bw/day). Infants and children consume a greater quantity of food per unit body weight 

than adults. As a result, exposure estimates are generally higher for population subgroups 

comprised of infants and children. 

Individual one-day food consumption data are used on an individual-by-individual basis. The 

reported consumption amounts of each food item can be multiplied by a residue point estimate 

and summed to obtain a total daily pesticide exposure for a deterministic exposure assessment, or 

“matched” in multiple random pairings with residue values and then summed in a probabilistic 

assessment. Residue distributions are made up of individual residue values that have been 

entered into files. These files are referred to as residue distribution files, or RDFs. When a 

distribution of consumption values is used, a distribution of exposure estimates is also obtained.

Chlorpyrifos steady state dietary exposure analyses were conducted for the 2014 HHRA using 

the Calendex-FCIDTM program. Calendex provides a focus detailed profile of potential exposures 

to individuals across a calendar year. HED’s steady state assessment considers the potential risk 

from a 21-day exposure duration using a 3-week forward rolling average (sliding by day) across 

the year. Like DEEM, the Calendex software uses the NHANES/WWEIA consumption data and 

pairs one diary for each individual in the WWEIA with a randomly selected set of residue values 

for each food consumed. The same food residue values used in the acute assessment using 

DEEM were used for the 21-day (steady state) duration using Calendex. Food residues are also 

input as either a single point estimate (deterministic) or as an RDF (probabilistic).

9.2.2 Food Residue Assumptions

Food tolerances are based on crop field trial data. Crop field trials are conducted at the 

maximum label rate and the minimum pre-harvest interval stipulated on the label. The use of 

field trial residues and tolerance level residues for foods in a dietary assessment represents 

conservative assumptions.

 19 http://www.ars.usda.gov/services/docs.htm?docid=13793
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Food monitoring data are taken closer to the point of consumption than data from field trials. In 

addition, before analysis, food samples are prepared as if for consumption. As a result, they 

more adequately reflect residues present at the time foods are consumed. Residue monitoring 

data used in the chlorpyrifos dietary assessment were obtained from the USDA’s Pesticide Data 

Program (PDP), which routinely tests for chlorpyrifos. For crops/foods not tested by PDP, 

translations have been made from tested crops (USEPA, 1999). Occasionally, older PDP data 

has been used where it represented the best estimate of real residues; the overall years of PDP 

data used in the chlorpyrifos assessment are from 1998 to 2012. Field trial data or tolerances 

have been used for a few crops where translations from PDP data were not appropriate. 

Percent crop treated estimates for chlorpyrifos are provided by OPP’s Biological and Economic 

Analysis Division (BEAD) in a screening level usage analysis (SLUA) report (USEPA, 2014b). 

Percent crop treated is the ratio of base acres of a crop treated with a pesticide to the total 

number of acres planted or grown, expressed as a percentage. The SLUA gives the average 

pounds of active ingredient used per year, the average %CT, the maximum %CT, and the sources 

from which the data were obtained. These sources include the U.S. Department of Agriculture’s 

National Agricultural Statistics Service (USDA NASS), private pesticide market research data, 

and the California Department of Pesticide Regulation (DPR). 

Percent CT information, where available for a given crop, is incorporated into the chlorpyrifos 

dietary exposure assessments. Percent CT estimates are used for the mathematical purpose of 

determining the numbers of zeros and 1⁄2LODs to be entered into the RDFs, or to be averaged 

into a point estimate. When a %CT estimate is used for a commodity, the blending classification 

of the commodity determines whether a point estimate or RDF is used for the commodity. 

The maximum %CT estimates reported in the SLUA are used in the chlorpyrifos dietary 

assessments. Where no %CT data were reported for a commodity in the SLUA, a conservative 

100%CT is assumed. 

Typically, when monitoring data are used, the RDF consists of the full distribution of found 

residues, 1⁄2LODs (half the analytical method limit of detection), and zeros. A residue value of 

zero (0.0 ppm) is assigned to the percentage of the crop that is assumed to be untreated. A 

residue value of 1/2LOD is assigned to the percentage of the crop that is assumed to be treated, 

but which did not result in residues above the LOD. The remainder of the RDF consists of actual 

detected residues, if any. When 100%CT is assumed for a commodity, no zeros are included in 

the RDF. 

When monitoring data for a “not blended” (NB) or “partially blended” (PB) commodity are used 

for (translated to) a blended (B) commodity, a point estimate is used rather than an RDF. The 

average monitoring value for the NB or PB commodity is determined and that value is entered as 

a single point estimate for the blended commodity. 
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

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When using field trial data, when a commodity is blended the average field trial value is used 

along with the maximum %CT estimate; an RDF is not prepared. For NB and PB commodities, 

a RDF is prepared as for monitoring data and it is based on the maximum %CT estimate.

For some commodities, the raw agricultural commodities (RACs) are processed into other food 

forms (e.g., apple juice from apples, or refined oil from cottonseed) which may result in a 

reduction or concentration of the pesticide residues found on the RAC. Concentration or 

reduction may also occur as foods are prepared for consumption (e.g., peeled, washed, or 

cooked). In order to account for concentration or reduction of residues in an assessment, two 

types of processing factors may be used: empirical factors determined in processing studies and 

model (i.e., DEEMTM) default processing factors. For chlorpyrifos, empirical processing factors 

were available for many food forms and were applied in the dietary assessment where 

appropriate (peeling, washing, cooking, canning, and juicing). Default processing factors were 

used for foods for which the empirical processing factors are not appropriate. 

The dietary food exposure assessment for the chlorpyrifos 2014 HHRA included the following 

assumptions:

• All 47 plant-based human food commodities with tolerances in 40 CFR.342, and their 

associated food forms, were included in the dietary assessment. These include a large 

variety of the following: root vegetables, Brassica leafy vegetables, legume vegetables, 

fruiting vegetables, cucurbits, citrus fruit, pome fruit, stone fruit, nuts, grains, and other 

miscellaneous foods such as banana, grape, strawberry, cranberry, kiwifruit, asparagus, 

sunflower, fig, and mint.

• All 20 livestock-based human food commodities with tolerances in 40 CFR.342, and their 

associated food forms, were included in the dietary assessment. These include meat, fat, 

meat byproducts, milk, and eggs.

• Tolerance level residues were used for sugar beet, kiwifruit, peppermint and spearmint. 

Field trial residue data for bean (dry), cotton, sunflower, and peanut were used to 

determine anticipated residues in those food forms. PDP food monitoring data were used 

to determine residues in all other foods. 

• Processing (residue concentration or reduction) factors were used for several foods, 

where appropriate. A peeling factor was used for sweet potato, peeled apples and pears, 

and kiwifruit. A cooking and/or canning factor was applied for cooked leafy vegetables 

and to cooked pumpkin, cherry, peach, and asparagus. Most fruit juices included a 

processing factor as did dried fruit and fruit peel.

• The maximum %CT was incorporated into the anticipated residue calculations. 

Maximum %CT estimates ranged from 2.5% (when reported as <2.5% CT) to 100% 

(when there was no reported %CT value). 

• Residues were input as distribution files (RDFs) for the majority of foods, resulting in a 
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

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probabilistic assessment. Blended foods and foods for which residues were based on 

tolerance levels used a single point estimate of residue rather than an RDF, resulting in a 

deterministic assessment for those foods. 

9.2.3 Food Exposure Estimates

The results at the upper percentiles of exposure (99th, 99.5th, and 99.9th) of the chlorpyrifos acute 

and steady-state food exposure assessments for infants and females of childbearing age are 

presented in Table 7. Results of Acute and Steady State Dietary (Food Only) Exposure Analysis 

for Chlorpyrifos below. 

Table 7. Results of Acute and Steady State Dietary (Food Only) Exposure Analysis for Chlorpyrifos

Population Subgroup

Acute Exposure

(μg/kg/day)

Steady State Exposure

(μg/kg/day)

95th

Percentile

99th

Percentile

99.9th

Percentile

95th

Percentile

99th

Percentile

99.9th

Percentile)

Infants (<1 year old) 0.050 0.088 0.273 0.044 0.083 0.186

Females

(13–49 years old) 0.021 0.041 0.150 0.018 0.029 0.075

The PBPK model was used to generate time course of chlorpyrifos concentrations in blood based 

on a dietary exposure scenario in which a female adult weighing 72.9 kg following a single daily 

exposure to chlorpyrifos in food for 42 days. The PBPK simulation results at the 10th to the 

99.9th percentile of exposure for the steady state food assessment for females of childbearing age 

are presented in Table 8.

Table 8. Predicted levels of food exposure to chlorpyrifos from the 2014 revised risk assessment and 

associated maximum and 24-hour blood concentrations in adult females

(Note: Information here is the same as reported in Table 2).

Percentile of 

Exposure

Exposure from 

Calendex

Max Blood Levels of 

CPFOS (pg/g) from 

Food Runs at Various 

Percentiles of 

Exposure from 

Calendex

10-hour Blood Levels 

Post Exposure Values 

of CPFOS (pg/g) from 

Food Runs at Various 

Percentiles of Exposure 

from Calendex

24-hour Blood Levels 

Post Exposure Values 

of CPFOS (pg/g) from 

Food Runs at Various 

Percentiles of Exposure 

from Calendex

10 0.003 μg/kg/day 0.29 0.060 0.021

30 0.005 μg/kg/day 0.48 0.099 0.034

50 0.007 μg/kg/day 0.67 0.139 0.048

70 0.009 μg/kg/day 0.86 0.179 0.062

90 0.014 μg/kg/day 1.33 0.278 0.096

95 0.018 μg/kg/day 1.71 0.358 0.124

97.5 0.023 μg/kg/day 2.19 0.457 0.158
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

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Percentile of 

Exposure

Exposure from 

Calendex

Max Blood Levels of 

CPFOS (pg/g) from 

Food Runs at Various 

Percentiles of 

Exposure from 

Calendex

10-hour Blood Levels 

Post Exposure Values 

of CPFOS (pg/g) from 

Food Runs at Various 

Percentiles of Exposure 

from Calendex

24-hour Blood Levels 

Post Exposure Values 

of CPFOS (pg/g) from 

Food Runs at Various 

Percentiles of Exposure 

from Calendex

99 0.029 μg/kg/day 2.76 0.576 0.200

99.5 0.037 μg/kg/day 3.52 0.735 0.255

99.9 0.075 μg/kg/day 7.14 1.490 0.517

9.3 Drinking Water Exposure

Based on the current use profile for chlorpyrifos, the primary and dominate route of drinking 

water exposure to this pesticide in drinking water is sourced surface water.20,21 Therefore, the 

focus of this section is on chlorpyrifos exposure concentrations in surface water. Surface sourced 

drinking water is processed at local community water treatment facilities and is not nationally 

distributed. One exception is bottled water. Thus, estimated drinking water concentrations 

(EDWCs) are intended to capture exposure levels that an individual may ingest on a watershed 

basis.

The drinking water assessment methods described in this section have undergone previous 

FIFRA SAP review in the years 199822, 199923, 200024, 201025 and 201126, and are not subject 

to the current SAP review but are presented for purposes of assessing the new approach to 

integrate the exposure assessment with the PBPK model. These methods include use of exposure 

modeling as well as evaluation of available water monitoring data to fully characterize the 

potential drinking water exposure.

 20 U.S. Environmental Protection Agency (Bohaty, R.), Revised Chlorpyrifos Preliminary Registration Review 

Drinking Water Assessment, June 20, 2011, PC Code: 059101; DP Barcode: 368388, 389480

21 U.S. Environmental Protection Agency (Bohaty, R.), Chlorpyrifos: Updated Drinking Water Assessment for 

Registration Review, December 23, 2014, PC Code: 059101; DP Barcode: 424487

22 Proposed Methods for Basin-scale Estimation of Pesticide Concentrations in Flowing Water and Reservoirs for 

Tolerance Reassessment, FIFRA Scientific Advisory Panel Meeting, July 29–30, 1998 23 Office of Pesticide Programs Policy for the Use of the FQPA 10x Safety Factor; Statistical Methods for Use of 

Composite Data in Acute Dietary Exposure Assessment; Use of Watershed-derived Percent Crop Areas as 

Refinement Tool in FQPA Drinking Water Exposure Assessments for Tolerance Reassessment, FIFRA Scientific 

Advisory Panel Meeting May 25–27, 1999 24 Assessment of pesticide concentrations in drinking water and water treatment effects on pesticide removal and 

transformation, FIFRA Scientific Advisory Panel Meeting September 26–29, 2000 25 Re-evaluation of the Human Health Effects of Atrazine: Review of Experimental Animal and In-vitro Studies and 

Drinking Water Monitoring Frequency, FIFRA Scientific Advisory Panel Meeting, April 26–29, 2010 26 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, Sept 

29, 2000; SAP Report No. 2001-02. February 12, 2011.
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

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9.3.1 Exposure Modeling

Currently, EPA-OPP estimates pesticide exposure using computer models27 that simulate 

pesticide sorption to soil, in-field decay, and runoff from an agricultural field/drainage area 

following pesticide application(s) as well as resulting concentrations in an adjacent surface water 

body known as the “index reservoir.”28 Simulations account for a range of different soil, 

weather, hydrologic conditions, and management/crop use conditions. In addition, all available 

chemical specific environmental data are considered as well as different labeled application 

rates. For drinking water assessments, the estimated 1-in-10 year return frequency concentrations 

from surface water modeling, for either single-day (24-hour concentration for estimating acute 

exposures) or time-averaged periods (for estimating chronic exposures) is compared to relevant 

toxicity endpoints of concern.

To date, two drinking water assessments, which include a number of different exposure 

scenarios, have been completed for chlorpyrifos as part of the registration review process. To 

illustrate a range of upper bound EDWCs on a national basis, two maximum label rate 

application scenarios were selected to represent high and low end exposures, i.e., Michigan tart 

cherries at 5 applications totaling 14.5 pounds per acre per year, and Georgia bulb onions at a 

single application of 1 pound per acre per year, respectively. The daily (24-hour average) 

EDWCs for the entire simulation (referenced as time series data) for chlorpyrifos is provided in 

Figure 9 for the Georgia bulb onion. This simulation represents a pre-plant soil application 

occurring on December 4 every year for approximately 30 years. Multiple years of 

meteorological data are simulated to account for the variation in weather that exists over time 

(i.e., some years are wetter/drier, warmer/cooler than others). This simulation also takes into 

account required aquatic spray drift buffers for ground applications of 25 feet. Figure 9 shows a 

peak chlorpyrifos concentration of 8 μg/L (data circled in the figure). The various peak 

concentrations observed in the time series data represent the various runoff events that occur 

either as a result of a rainfall event or irrigation. The higher peak concentrations correspond to 

high rainfall events that follow shortly after application leaving little time for chlorpyrifos to 

degrade on the application site. 

 27 http://www.epa.gov/pesticide-science-and-assessing-pesticide-risks/models-pesticide-risk-assessment#aquatic. 28 See “Development and Use of the Index Reservoir in Drinking Water Exposure Assessments” at 

http://www.epa.gov/pesticide-science-and-assessing-pesticide-risks/development-and-use-index-reservoir-drinkingwater 
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

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Figure 9. Simulation Time Series Data for Chlorpyrifos Use on Bulb Onion (1 lb a.i./A once per year) in 

Georgia

As described elsewhere, the 1-in-10 year return frequency concentration is normally used in 

dietary exposure assessments. For a 30-year simulation, this value corresponds to the third 

highest peak exposure concentration (arrows indicate the second and third highest 

concentrations), and in this case, it is approximately 6 μg/L for the 24-hour concentration. 

9.3.2 Monitoring Data

The low quantity and/or quality of available monitoring data for many pesticides, including 

chlorpyrifos and chlorpyrifos-oxon, presents a serious challenge in forecasting drinking water 

exposure concentrations under various use conditions and geographic scales (national vs. 

watershed). In general, most available monitoring data are not coordinated with a particular 

pesticide application, sampling frequencies are typically insufficient to capture durations of 

exposure concern (e.g., peak), and sampling is usually temporally and spatially limited.29 It is 

important to accurately capture peak concentrations to determine upper bound exposure for acute 

exposure estimates, as well as to best estimate longer term average exposure concentrations (e.g., 

21-day average). For these reasons, monitoring data likely do not capture upper bound estimates 

of exposure for individuals on a national basis. However, a high quality monitoring data set can 

be informative as it helps elucidate what is happening under current use practices and specific 

conditions at a given location. For example, Dow AgroSciences (MRID 44711601)30 submitted 

daily monitoring data for chlorpyrifos that provides information on the variation in chlorpyrifos 

 29 Re-evaluation of the Human Health Effects of Atrazine: Review of Experimental Animal and In-vitro Studies and 

Drinking Water Monitoring Frequency, FIFRA Scientific Advisory Panel Meeting, April 26-29, 2010 30 Poletika, N.; Robb, C. (1998) A Monitoring Study to Characterize Chlorpyrifos Concentration Patterns and 

Ecological Risk in an Agriculturally Dominated Tributary of San Joaquin River: Lab Project Number: ENV96055. 

Unpublished study prepared by Dow AgroSciences and Paragon Research.

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

24

1/1/19621/1/19641/1/19661/1/19681/1/19701/1/19721/1/19741/1/19761/1/19781/1/19801/1/19821/1/19841/1/19861/1/19881/1/1990

-hour Average Chlorpyrifos 

Concentration (

μg/L)

Simulation Date
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

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concentrations in time and in space with known chlorpyrifos applications. Sampling was 

conducted for one year (May 1, 1996 to April 30, 1997) at three locations on the lower reach of 

Orestimba Creek, a tributary of the San Joaquin River in California. This study includes use 

information, percent chlorpyrifos use area, and agronomic practices such as irrigation. In 

addition, use information for chlorpyrifos is available for this watershed in the California 

Pesticide Use Reporting (PUR) database.31 Daily time-proportional composite samples were 

collected, along with weekly grab samples, and the results are presented in Figure 10.

Figure 10. Orestimba Creek Water Monitoring Data (May 1, 1996 to April 30, 1997)

In several cases, the weekly grab samples were observed to have higher concentrations of 

chlorpyrifos. This finding suggests that the composite sampling methodology used in the study 

for daily samples resulted in the dilution of peak daily concentrations. The highest measured 

daily concentration was 2.2 μg/L and was associated with a chlorpyrifos application to alfalfa 

(0.5 lb a.i./A applied aerially to a 50–70 acre field), followed by flood irrigation. The limit of 

detection reported for chlorpyrifos in this study is 0.01 μg/L in water. 

While Orestimba Creek does not have a drinking water intake, this study is one of the few 

available daily monitoring data sources with known applications of chlorpyrifos. In addition, the 

San Joaquin River, for which the Orestimba Creek is a tributary, provides source drinking 

 31 California Department of Pesticide Regulation. 2013. Pesticide Use Reporting. Retrieved from CDPR website: 

http://www.cdpr.ca.gov/docs/pur/purmain.htm

0

0.5

1

1.5

2

2.5

0 100 200 300

Measured Chlorpyrifos 

Concentration (μg/L)

Study Day

Location 1 - Weekly Grab (Discrete) Samples

Location 1 - Daily Composit Samples

Location 2 - Daily Composit Samples

Location 3 - Daily Composit Samples
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

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water32, and chlorpyrifos concentrations observed in this study are not outside the range of those 

observed in other datasets. Thus, it is reasonable to assume that the magnitude of concentrations 

observed in this study occur in other locations across the country where drinking water intakes 

are located.

9.3.3 Drinking Water Treatment

Because drinking water for a large percentage of the population is derived from community 

water systems that treat raw water33 prior to consumption, the impact of water treatment on 

pesticide removal and transformation are considered, when possible, in estimating drinking water 

exposure.34,35,36 There are a wide range of drinking water treatment processes utilized by 

community drinking water systems across the country, including coagulation/flocculation, 

sedimentation, filtration, and disinfection.37 The effect of various treatment processes have been 

investigated for a number of pesticides including chlorpyrifos. The results of one study shown in

Table 9. 38 Table 9 suggests that removal of chlorpyrifos is highly dependent on the treatment 

method employed by an individual community drinking water treatment facility. In the presence 

of free chlorine, the most common disinfection process utilized by community water systems, 

chlorpyrifos reduction is high (>90%). However, reduction of chlorpyrifos in the presence of 

monochloramines, often used as an alternative to chlorine to reduce disinfectant/disinfection byproducts, is low (<10%). These results are consistent across multiple publications.39,40,41

 32 http://www.americanrivers.org/endangered-rivers/2014-report/san-joaquin/ 33 United States Environmental Protection Agency. 1989. Technologies for Upgrading Existing or Designing New 

Drinking Water Treatment Facilities. EPA/625/4-89/023. 34 Assessment of pesticide concentrations in drinking water and water treatment effects on pesticide removal and 

transformation, FIFRA Scientific Advisory Panel Meeting September 26-29, 2000

35 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, Sept 

29, 2000; SAP Report No. 2001-02. February 12, 2011. 36 U.S. Environmental Protection Agency, Office of Pesticide Programs. The Incorporation of Water Treatment 

Effects on Pesticide Removal and Transformations in Food Quality Protection Act (FQPA) Drinking Water 

Assessment, October 25, 2001

37 U.S. EPA Office of Water 2006 Community Water System Survey, May 2009 available at EPA-815-R-09-002. 

Retrieved from USEPA website: http://nepis.epa.gov/Exe/ZyPDF.cgi?Dockey=2000JTKL.txt.

38 Chamberlain, E. Shi, H., Wang, T., Ma, Y., Fulmer, A., Adams. C. J Agric. Food Chem. 2012 60, 354-363

39 Duirk, S. W. Desetto, L. M., Davis, G. M., Lindell, C., Cornelison, C. T. Water Research 2010, 44, 761-768 40 Ormad, M. P., Miguel, N., Matesanz, J. M., Ovelleiro, J. L., Chemosphere, 2008, 71 97-106 41 Tierney, D. P.; Christensen, B. R.; Culpepper, V. C. Chlorine Degradation of Six Organophosphate Insecticides 

and Four Oxons in Drinking Water Matrix. Submitted by Syngenta Crop Protection, Inc. 2001
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

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Table 9. Chlorpyrifos Reduction under Typical Drinking Water Treatment Conditions; Drinking Water Treatment Processes Utilized by Community 

Water System Based on Population Served

Treatment Methoda

FC MCA ClO2 MnO4- UV H2O2 O3 Softening

pH 

6.6

pH 

8.6

pH 

6.6

pH 

8.6

pH 

6.6

pH 

8.6

pH 

6.6

pH 

8.6

pH 

6.6

pH 

8.6

pH 

6.6

pH 

8.6

pH 

6.6

pH 

8.6 pH 12

Percent Reductionb 90.3 85.7 8.7 9.2 34.3 27.5 15.3 5.2 14.5 1.9 7.6 3.1 60.9 30.3 100.0

System Population 

Category Percentage of Plants Performing Each Treatment Practice for Surface Waterc

100 or less 98.4 0 0 1.6 3.1 - - 0 0

101-500 79 1.2 0 9.2 1.7 - - 1.4 2.5

501-3,300 97.4 2.2 0 7.8 2.2 - - 1.5 3.4

3,301-10,000 80.8 13.7 11 24.7 1.4 - - 1.4 19.2

10,001-50,000 80.5 14.8 8.7 32.9 1.3 - - 1.2 16.9

50,001-100,000 75.1 17.1 18.5 26.8 2.6 - - 11.8 5.2

100,001-500,000 78.9 32.4 14 26.3 4.7 - - 15.8 11.8

Over-500,000 78.0 35.6 2.5 21.2 1.7 - - 14.4 21.2

a. Experimental time was representative of typical drinking water treatment condition

b. See footnote 37 (Chamberlain, 2012)

c. See footnote 36 (EPA community water system survey)

Chlorine (FC); Chlorine dioxide (ClO2); Chloramines (MCA); Lime/soda ash softener (assumed to be similar to hydrolysis at pH 12); 

Ultraviolet light (UV); Ozone (O3); Potassium permanganate (MnO4-)
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

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Oxidation (e.g., 

photooxidation, 

drinking water 

chemical 

disinfection)

Most studies in the open literature do not examine the formation of transformation products 

under the same drinking water treatment processes. Although, information on water treatment 

effects on chlorpyrifos is generally limited, there are a few available studies that investigate the 

transformation of chlorpyrifos in the presence of chlorine.38,40,42 These studies show that 

chlorpyrifos converts to chlorpyrifos-oxon. The transformation of chlorpyrifos [phosphothionate 

group (P=S)] to chlorpyrifos-oxon [phosphorus-oxygen double bond (P=O)] in the presence of 

chlorine proceeds via rapid oxidation by the oxychlorine species (see Figure 11).

Figure 11. Transformation of Chlorpyrifos to Chlopyrifos-oxon

Once formed as a disinfection by-product, chlorpyrifos-oxon is expected to be relatively stable to 

drinking water distribution conditions and times (few hours to a few days). No data on physical 

removal processes such as coagulation/ flocculation, sedimentation, and filtration are available 

for chlorpyrifos or chlorpyrifos-oxon. However, such processes, with the exception of granular 

activated carbon43, have been shown to be ineffective for select organic pesticides.35 Based on 

the physical-chemical properties of chlorpyrifos and chlorpyrifos-oxon, granular activated 

carbon likely reduces the amount of both chemicals to some extent. However, data are not 

available on the exact removal efficiency for these chemicals treated with granular activated 

carbon or powder activated carbon. It should be noted that granular activated carbon is not a 

common treatment practice for all treatment facilities. Additional, powered activated carbon, 

which is used for taste and odor control, is not used year round.

Limited monitoring data are available for chlorpyrifos and chlorpyrifos-oxon following drinking 

water treatment. Available sources include the USEPA/USGS Pilot Reservoir Monitoring 

Program and the USDA Pesticide Data Program. Chlorpyrifos and chlorpyrifos-oxon were not 

detected in finished water in either of these programs; however, when raw and finished water 

samples were collected, the correlation between samples could not be made for chlorpyrifos. 

Thus, the impact of treatment could not be conclusively determined. Nevertheless, 

 42 Wu, J.; Laird, D. A. Abiotic Transformation of Chlorpyrifos to Chlorpyrifos Oxon in Chlorinated Water. Environ. 

Toxcol. Chem., 2003, 22(2), 261-264 43 U.S. Environmental Protection Agency. 1998. Small System Compliance Technology List for the Non-Microbial 

Contaminants Regulated Before 1996. EPA 815-R-98-002.
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

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USEPA/USGS Pilot Reservoir Monitoring Program for other organophosphates (e.g., malathion) 

suggests that in the presence of chlorine treatment processes, oxon formation occurs. 

In summary, given the wide range of removal and transformation efficiencies of chlorpyrifos 

under the various drinking water treatment processes, it is assumed that it is possible, depending 

on the community water system and associated treatment processes, that source water containing 

chlorpyrifos may result in exposure to chlorpyrifos and chlorpyrifos-oxon in varying amounts. 

Therefore, in order to address the multitude of water treatment possibilities, a bounding approach 

was used. That is, to represent those facilities that use disinfectant processes other than free 

chlorine, 100 percent of the chlorpyrifos entering the facility was assumed to be unchanged in 

the finished drinking water. In addition, to represent those facilities that employ chlorine as a 

disinfectant, 100 percent of the chlorpyrifos entering the facility was assumed to convert to 

chlorpyrifos-oxon (not presented here). The treatment methods and data for the associated 

population served data are provided in Table 9. In general, chlorine is used by treatment facilities 

that serve small populations, while treatment facilities serving larger populations tend to use 

alternative disinfection processes such as monochloramine more often. Chlorpyrifos and 

chlorpyrifos-oxon are not expected to degrade during distribution, as distribution times typically 

range from a few hours to a few days. As such, these chemicals are considered residues of 

exposure concern in drinking water. 

Because the CCCEH researchers measured parent chlorpyrifos in blood as the metric of internal 

exposure and the majority of animal toxicity studies are conducted with the parent compound, 

the most robust information related to neurodevelopmental effects is associated with 

chlorpyrifos. As such, this proposed approach focuses on exposure to chlorpyrifos instead of 

chlorprifos-oxon. In short, the agency is not pursuing the use of the oxon as the moiety of 

concern for the neurodevelopmental outcomes because there are limited data to validate the 

PBPK model estimates of chlorpyrifos-oxon. As such, EPA has more confidence in the PBPK 

model estimates of chlorpyrifos blood levels compared to chlorpyrifos-oxon.

9.3.4. Anticipated Exposure Scenarios and PBPK Model Simulations

To investigate the potential chlorpyrifos exposure to people in other areas of the country that 

may be exposed to chlorpyrifos via drinking water, the cord blood levels of chlorpyrifos 

observed in the CCCEH study are compared with PBPK simulated chlorpyrifos blood 

concentrations following consumption of drinking water containing chlorpyrifos for infants and 

females of childbearing age. 

9.3.4.1 Females of Childbearing Age (13-49 years old)

Adult females may be exposed to chlorpyrifos as a result of consuming drinking water that 

contains chlorpyrifos. The PBPK model was used to predict the time course of chlorpyrifos 

concentrations in venous blood for a female adult weighing 72.9 kg (160.7 lbs.) following 
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

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exposure to chlorpyrifos in drinking water based on model EDWCs (Figure 9) and measured 

concentrations (Figure 10).

A subset (120 days) of continuous daily concentrations from time series data (Georgia Bulb 

Onion low-exposure scenario) was used as an input into the PBPK model. The 120-day time 

series used in the PBPK model simulation contained the day with the highest daily average 

concentration of chlorpyrifos (Figure 9). The entire time series was not used because running 

simulations for a longer exposure duration exceeded the runtime limit of the software, acslX 

(Aegis Technologies Lab, Inc., Huntington, AL). The simulated adult female is assumed to 

consume drinking water four times a day, with a daily consumption volume of 1.71062 L (57.8 

oz.). The results of the PBPK simulation for this low end exposure scenario are provided in 

Figure 12. The peak chlorpyrifos blood concentration is approximately 6.99 pg/g. Chlorpyrifos 

blood concentrations are greater than 1 pg/g for most of the 120-day simulation.

Figure 12. PBPK Estimated Chlorpyrifos Venous Blood Concentrations for Adult Female Based on 120-day 

Time Series Data for Chlorpyrifos Use on Bulb Onion (1 lb a.i./A once per year) in Georgia

Due to the discreet exposure scenarios that occur for the food and worker exposures, the agency

is using the 10 and 24 hours post-exposure to characterize the asymptotic portion of the PK 

profile. In the case of drinking water exposure, exposures may occur at multiple times 

throughout the day when drinking and vary widely across a year. As such, discreet exposure 

periods are more difficult to define. During time periods of higher chlorpyrifos concentrations in 

drinking water, the PK profile does not transfer from the absorption/distribution phase of PK into 

the terminal 1⁄2 life phase. As such, the agency can only compare the lowest point within a range 

of exposure days. 

Since the chlorpyrifos venous blood concentration is linearly related to the chlorpyrifos 

concentration in water within the range of these concentrations, none of the concentrations 
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

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excluded from the current simulation, due to the software limitations described above, should 

exceed the maximum blood concentration predicted using the times series containing the 

maximum daily average water concentration. While this time series contains the highest daily 

concentration, there are two other time periods that have similar peak 24-hour average 

concentrations (noted with arrows in Figure 9), thus when entered directly into the PBPK model 

would be expected to result in similar levels of chlorpyrifos in the blood. The lowest peak 24-

hour daily concentration (approximately 6 μg/L) from the three peak exposure periods 

corresponds to the 1-in-10 year return frequency concentration that is typically reported in a 

drinking water assessment. Moreover, this time series also represents the lower end of exposures 

expected for chlorpyrifos based on current maximum label rates for chlorpyrifos.

The PBPK model was also used to predict the time course of chlorpyrifos concentrations in 

venous blood for an adult female based on exposure to daily measured chlorpyrifos 

concentrations from Orestimba Creek (Figure 10). As described previously, daily concentrations 

were entered into the PBPK model. The limit of detection for chlorpyrifos in this study of 0.01 

μg/L was used when no detection was reported as the actual concentration could range between 

zero and the detection limit. The PBPK model was used to predict the time course of 

chlorpyrifos concentrations in venous blood for an adult female with body weight of 72.9 kg 

(160.7 lbs.) following exposure to chlorpyrifos in drinking water based on 120 days of measured 

chlorpyrifos concentrations from the Orestimba Creek monitoring data. The simulated adult 

female is assumed to consume drinking water four times a day, with a daily consumption volume 

of 1.71062 L (57.8 oz.). The concentration from the 120 days corresponding to the peak 

measured concentration were extracted for use (data are circled in Figure 10). Results are shown 

in Figure 13. The peak chlorpyrifos blood concentration is approximately 1.97 pg/g.

Figure 13. PBPK Estimated Chlorpyrifos Venous Blood Concentrations for Adult Female Based on 120-day 

Time Series Data for Chlorpyrifos Monitoring Data for Orestimba Creek
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9.3.4.2 Infants: Formula Fed (with water) 

A formula-fed infant may be exposed to chlorpyrifos as a result of the use of tap water to mix 

formula. The PBPK model was used to predict the time course of chlorpyrifos concentrations in 

venous blood for an infant with a fixed body weight of 4.8 kg (10.6 lbs.) following exposure to 

chlorpyrifos in drinking water based on model EDWCs (Figure 9) and measured concentrations 

(Figure 10) of chlorpyrifos. This was done by taking a subset (120 days) of continuous daily 

concentrations of chlorpyrifos from an example time series (Georgia Bulb Onion low-exposure 

scenario) as input into the PBPK model. The simulated infant is assumed to consume drinking 

water six times a day, with a daily consumption volume of 0.688557 L (23.3 oz.). The peak 

chlorpyrifos venous blood concentration is 21.6 pg/g (Figure 14). Chlorpyrifos blood 

concentrations are greater than 5 pg/g for most of the 120-day simulation.

Figure 14. PBPK Estimated Chlorpyrifos Venous Blood Concentrations for Infants Based on 120-day Time 

Series Data for Chlorpyrifos Use on Bulb Onion (1 lb. a.i./A once per year) in Georgia

As noted above, during periods of higher chlorpyrifos concentrations in drinking water, the PK 

profile does not transfer from the absorption/distribution phase of PK into the terminal 1⁄2 life 

phase. This is particularly true for infants who consume bottles at regular, frequent intervals 

throughout the day and night. 

The PBPK model was also used to predict the time course of chlorpyrifos concentrations in 

venous blood for infants based on exposure to daily measured chlorpyrifos concentrations from 

Orestimba Creek (Figure 10). As described previously, daily concentrations were entered into the 

PBPK model. In this case, the PBPK model was used to predict the time course of chlorpyrifos 

concentrations in blood for infants with a fixed body weight of 4.8 kg (10.6 lbs.) following 

exposure to chlorpyrifos in drinking water based on 120 days of measured chlorpyrifos 

concentrations from the Orestimba Creek monitoring data. The 120 days corresponding to the 
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peak observed concentration were extracted for use. The results of the PBPK simulation for this 

low end exposure scenario is provided in Figure 15. The peak chlorpyrifos blood concentration is 

approximately 6.24 pg/g.

Figure 15. PBPK Estimated Chlorpyrifos Venous Concentrations for Infants Based on Time Series Data for 

Chlorpyrifos Monitoring Data for Orestimba Creek

9.3.5. Drinking Water Summary

Given the wide range of drinking water treatment methods used across the United States, an 

individual may be exposed to both chlorpyrifos and chlorpyrifos-oxon to varying degrees via 

drinking water. This analysis focused on exposure to chlorpyrifos via drinking water and the 

associated PBPK estimated cord blood chlorpyrifos concentrations. This was done using two 

different drinking water exposure scenarios for infants and females of childbearing age. A 120-

day time series for a national low-end exposure scenario (i.e., Georgia Onion) and measured 

concentrations from a daily monitoring program conducted for Orestimba Creek in California 

were input into the PBPK model. The corresponding chlorpyrifos concentrations in blood were 

presented and are summarized in Table 10. Other use scenarios are expected to result in higher 

estimated drinking water concentrations resulting in higher blood concentrations. 

Table 10. Summary of PBPK Model Estimated Maximum Chlorpyrifos Blood Concentrations Following Two 

Different Drinking Water Exposure Scenarios

Population Exposure Scenario

Model Estimated Measured

Infants: Formula Fed 

(with water) 21.6 pg/g 6.24 pg/g

Females of 

Childbearing Age 

(13–49 years old)

6.99 pg/g 1.97 pg/g

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

1

171341511681851

10211191136115311701187120412211238125512721

PBPK Model Estimated 

Concentration in Venous Blood 

(pg/g)

Simulation (hour)

Chlorpryrifos
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9.4 Worker Exposure

The agency evaluated all potential occupational exposure scenarios as part of the 2014 HHRA. 

For this issue paper for review by the FIFRA SAP, internal dose estimates of chlorpyrifos were 

assessed for selected pesticide handler exposure scenarios which are considered among those 

with comparatively low occupational exposure potential. These ‘low’ occupational scenarios are

presented for purpose of illustrating how occupational handler exposures and PBPK modeling 

can be integrated. A brief overview of the data and methods used by the agency to evaluate 

occupational pesticide handler exposures is provided here for context. These data and methods 

have undergone extensive peer review and are not subject to the 2016 FIFRA SAP review.44 

9.4.1 Methods 

In the risk assessment process, the agency considers all potential scenarios that could lead to 

exposures associated with a pesticide’s use. As noted above, a comprehensive risk assessment 

was completed for chlorpyrifos but a select number of scenarios for pesticide handlers have been 

used to develop illustrative analyses for consideration. As such the methods described below 

focus on the types of selected exposures. The agency uses the term “handlers” to describe those 

individuals who are involved in the pesticide application process. There are distinct job 

functions or tasks related to applications and exposures can vary depending on the specifics of 

each task. Job requirements (e.g., the amount of chemical used), the kinds of equipment used, 

the target pest being treated, and the level of protection used by a handler can cause exposure 

levels to differ in a manner specific to each application event. The following represent handler 

activities which are assumed to occur by the agency:

• Mixer/Loaders: individuals perform tasks in preparation for an application. For example, 

prior to application, mixer/loaders would mix a liquid pesticide concentrate with water 

and load it into the holding tank of the airplane or groundboom equipment;

• Applicators: individuals operate application equipment during the release of a pesticide 

product onto its target pest;

• Mixer/Loader/Applicators: individuals who perform all aspects of the pesticide 

application process; and,

• Flaggers: individuals that guide aerial applicators during the release of a pesticide 

product onto its target.

The agency uses the term “unit exposure” to describe the exposure metric which serves as the 

basis for assessing handler exposures to pesticides. Unique unit exposure values have been 

developed from available data sources for each handler scenario considered and are expressed as 

mass of pesticide active ingredient exposure per unit mass of active ingredient handled (e.g., 

μg/lb a.i.). A scenario refers to a specific type of application equipment, formulation type, job 

function, and level of personal protective equipment (PPE). The agency then uses unit exposures 

 44 http://www.epa.gov/pesticide-science-and-assessing-pesticide-risks/occupational-pesticide-handler-exposure-data
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

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generically to estimate exposure for all pesticides which may have a particular exposure scenario 

associated with its use. Additionally, other key pieces of information such as the allowable uses 

on pesticide labels are also important when defining exposure scenarios and calculating exposure 

estimates (e.g., application rates on specific crops and allowable types of equipment).

As noted above, the occupational exposure methodologies and exposure data sources used have 

undergone extensive scientific peer review. Since the 1980s, the agency has required pesticide 

handler exposure data in order to assess the occupational risks of both new and existing 

pesticides. The first occupational exposure testing guidelines were established at this time. 

These guidelines were intended to standardize the methodology used to conduct the studies 

necessary to allow the agency to determine the potential exposures and consequent risks 

associated with the activities relating to the occupational use of pesticides. In the early 1990s, 

the Pesticide Handlers Exposure Database (PHED)45 was constructed in order to estimate 

exposures resulting from mixing/loading/applying pesticides. The data assembled for use in this 

database was taken from published literature as well as from industry studies submitted to the 

agency. Subsequent to PHED, the Outdoor Residential Exposure Task Force (ORETF) was 

formed in 1994 to satisfy mixer/loader/applicator exposure data required by the agency relating 

to professional lawn care operators. Additionally, the Agricultural Handler Exposure Task Force 

(AHETF) was established in 2001 to jointly address ongoing, product-specific agency exposure 

data requirements. These databases are used as the main sources for estimating occupational 

exposures to workers handling pesticides for both registration and reregistration actions. PHED 

is the basis of exposure data for most pesticide handler exposure assessments; however, given 

changes in cultural production practices over time and the limitations of the PHED data, it has 

become more appropriate to make use of the more current, AHETF data, as it becomes available 

because these data better reflect the use of technologies in present day agriculture. Currently 

recommended unit exposures for agency occupational handler exposure assessment purposes are 

available on the EPA website.46

The scenario-based approach is consistent with the agency's guidelines for exposure assessment 

which can be found on the EPA website.47 In addition, it should also be noted that generation of 

new exposure data are reviewed by the agency's Human Studies Review Board (HSRB) under 

most circumstances.48

 

45 http://www.epa.gov/pesticide-science-and-assessing-pesticide-risks/occupational-pesticide-handlerexposure-data#phed 46 http://www2.epa.gov/pesticide-science-and-assessing-pesticide-risks/exposure-surrogate-reference-table 47 http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=15263 48 http://www.epa.gov/osa/human-studies-review-board
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

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PBPK Model Usage

The use of the PBPK model is based on the exposure scenario and methods described above. 

However, specific inputs are required to use the PBPK model for estimation of chlorpyrifos 

blood levels corresponding to chlorpyrifos occupational exposures. 

• Exposure Pattern: An 8-hour workday and a 5-day work week was used which is the 

standard for this type of exposure analysis. Two days were simulated as non-exposure 

days to simulate days off from work. Worker exposure scenarios were considered for 2 

weeks of occupational exposures followed by a 30-day recovery period. 

• Use rates: Typical use rates have been used in this 2016 analysis instead of the maximum 

application rate allowable for the agricultural use site (crop) from currently registered 

product labeling. For the 2014 HHRA, maximum application rates were used. 

• Route of Exposure: For occupational workers, dermal and inhalation exposures are 

expected and were calculated as noted above. For PBPK modeling, both dermal and 

inhalation exposure inputs were input into the model and the results reflect the total of 

both types of exposures. 

• Body weight: A female, with a body weight of 69 kg was used due to concerns for 

potentially pregnant women and the potential uncertainties regarding neurodevelopmental 

effects on their children. The agency considers women of child bearing age to be 

between the ages of 13 to 49 years. 

• Dermal surface area exposed: For PBPK modeling of chlorpyrifos exposures, 100% of 

the dermal surface area was assumed to be exposed from conduct of work activities. 

• Hygiene: A daily shower was assumed following each day which means there is no 

additivity between chlorpyrifos exposure due to sequential days of work. 

• Inhalation Conversion: Because of the way exposure data are compiled, the standard 

agency occupational exposure method estimates daily inhalation exposures on a (mg/day) 

basis and not as an exposure concentration (mg/L). The coding of the PBPK model 

requires (mg/day) daily inhalation doses be converted to an air concentration for 

modeling purposes. The following equation was used to make this calculation:

Airborne concentration (mg/L) = Exposure (mg/day) / [Breathing Rate (L/min) * 60 minutes/hour * 8 hours/day]

Note: a breathing rate of 8.3 L/minute was assumed for applicator work activities and 16.7 

L/minute for mixer/loader exposure activities49. 

Occupational Handler Exposure Scenarios

For this evaluation a total of 7 ‘low’ occupational handler exposure scenarios were assessed as 

follows:

Mixer/Loader Activities

• Mixing/loading liquid (EC) for treatment of corn by groundboom soil incorporation at 

0.50 lb a.i./A

 49 NAFTA - Dept. of Pesticide Regulation (DPR), California EPA, HSM-98014, April 1998.

http://www.cdpr.ca.gov/docs/whs/memo/hsm98014.pdf
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

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• Mixing/loading liquid (EC) for treatment of cole crops50 by soil incorporation at 1.0 lb 

a.i./A

• Mixing/loading dry flowable in water soluble packets (WSP) for treatment of cole crops 

by groundboom soil incorporation at 1 lb a.i./A 

Applicator Activities

• Applying sprays via groundboom soil incorporation to corn at 0.50 lb a.i./A

• Applying sprays via groundboom soil incorporation to cole crops at 1 lb a.i./A

Seed Treatment

• Loading/applying liquid (EC) for seed treatment of beans and peas at 0.00058 lb a.i./lb 

seed

Seed Planting

• Planting corn seed treated at 0.00058 lb a.i./lb seed

9.4.2 Results 

Figures 16 and 17 present the PBPK modeling profile of a single, example ‘low’ occupational 

exposure scenario, application via groundboom equipment for cole crops at a rate of 1 lb a.i./A. 

This example exposure scenario, which is the lowest of all example scenarios evaluated, was 

selected for purpose of comparison to the internal dose profiles resulting from dietary exposures 

(i.e., food and drinking water) to chlorpyrifos. It presents the full profile of modeled internal 

dose estimated for the example occupational scenario. When modeled, all 7 occupational 

exposure scenarios result in the similar PK profile for chlorpyrifos as has been described 

previously. 

Figure 16 (full profile) below shows the sawtooth pattern as the daily, rapid increase in internal

dose during the occupational exposure period followed by a rapid decline immediately after the 

exposure period ends.

 50 Examples of cole crops include: brussels sprout, cauliflower, kale, broccoli
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Figure 16. PBPK modeling (full profile) estimation of chlorpyrifos venous blood concentrations (pg/g) per 

unit time (hours) resulting from the occupational exposure scenario, application via groundboom for cole 

crops at 1 lb a.i./A.

Figure 17 (sub-set) shows the internal dose just following the end of the modeled occupational 

exposure (i.e., a period of 2 work weeks). The internal dose curve flattens as terminal half-life is 

achieved causing an asymptotic appearance.

Figure 17. PBPK modeling (sub-set of above Figure 16) of chlorpyrifos blood concentrations (pg/g) per unit 

time (hours) resulting on the final day from the example occupational exposure, application via groundboom 

for cole crops at 1 lb a.i./A

Tables 11 and 12 present the results for all 7 example occupational exposure scenarios including 

dermal and inhalation exposures estimated with use of standard agency methods and inputs, and 

the modeled maximum, 24-hour venous blood chlorpyrifos concentrations (pg/g), and 10-hour

venous blood chlorpyrifos concentrations following the final (12th) day of peak exposure (pg/g) . 

0

50

100

150

200

250

0 200 400 600 800 1000 1200 1400

Blood Chlorpyrifos (pg/g)

Time (hours)

0

50

100

150

200

250

263 363 463 563 663 763

Blood Chlorpyrifos (pg/g)

Time (hours)
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Tables 11 and 12 present the results of the mixing/loading and application of chlorpyrifos by 

groundboom equipment, and the results of seed treatment and planting, respectively. For the 

mixing/loading and application by groundboom equipment scenarios (Table 11), the maximum 

modeled venous blood chlorpyrifos levels range from 194 to 413 pg/g, the 24-hour venous blood 

chlorpyrifos levels range from 3.0 to 8.0 pg/g, and the 10 hour venous blood chlorpyrifos level

following the final day of peak exposure range from 16.0 to 38.4 pg/g. For the seed treatment 

exposure scenario (Table 12), the maximum modeled venous blood chlorpyrifos level is 954 

pg/g, the 24-hour venous blood chlorpyrifos level is 12.4 pg/g, and the 10-hour venous blood 

chlorpyrifos level following the final day of peak exposure is 71.1 pg/g. For the seed planting 

exposure scenario (Table 12), the maximum modeled venous blood chlorpyrifos level is 214 

pg/g, the 24-hour venous blood chlorpyrifos level is 2.8 pg/g, and the 10-hour venous blood 

chlorpyrifos level following the final day of peak exposure is 16.0 pg/g. 
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Table 11. Occupational dermal and inhalation exposures used for PBPK modeling of mixer/loader and applicator example low 

exposure scenarios and the maximum venous blood chlorpyrifos (pg/g) resulting from modeling. 

Exposure Scenario App. 

Rate

Area 

Treated

Dermal 

Unit 

Exposure1

Dermal 

Exposure2

Inhalation 

Unit 

Exposure1

Inhalation 

Exposure2

Airborne 

Concentration3

Maximum 

Venous 

Blood 

Chlorpyrifos

24-Hour 

Venous 

Blood 

Chlorpyrifos

10 Hours 

After the 

Last Peak 

on Day-12

Blood 

ChlorWorker pyrifos

Activity Form.

Application 

Equipment/ 

Type

Use 

Site

Value

(lb 

a.i./A)

Value 

(Acres)

Engineering Controls

Value

(ug/lb 

a.i.)

1

Value 

(mg/day)2

Value 

(ug/lb 

a.i.)

1

Value

(pg/g)

Value 

(mg/L)

Value

(pg/g)

Value

(pg/g)

Value

(pg/g)

Mixer/

Loader

EC

Groundboom/ 

Soil 

Incorporation

Corn

(preplant)0.50 200

8.6

0.86

0.083

0.0083 1.4x10-6 413 6.6 34.4

Cole 

Crops 1.0 80

0.69 0.0066 8.3x10-7 329 5.3 27.4

Dry 

Flowable in 

WSP

0.78 0.019 2.4x10-6 404 8.0 38.4

Applicator

Spray 

(all 

starting 

forms)

Corn 

(preplant)0.50 200

5.1

0.51

0.043

0.0043 1.1x10-6 243 3.8 20.0

Cole 

Crops 1.0 80 0.41 0.0034 8.6x10-7 194 3.0 16.0

1. Based on the “Occupational Pesticide Handler Unit Exposure Surrogate Reference Table” (September 2015)

2. Dermal Exposure (mg/day) = Dermal Unit Exposure (μg/lb a.i.) × Conversion Factor (0.001 mg/μg) × Application Rate (lb a.i./acrel

) × 

Area Treated (A/day).

3. Inhalation Exposure (mg/day) = Inhalation Unit Exposure (μg/lb a.i.) × Conversion Factor (0.001 mg/μg) × Application Rate (lb a.i./acre) 

× Area Treated (A/day).

4. Airborne Concn (mg/L) = Exposure (mg/day) / (Breathing Rate (L/min) * 60 min/hr * 8 hours/day)
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

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Table 12. Example model runs for mixing/loading EC for treatment of corn by groundboom soil incorporation at 0.50 lb a.i./A and 

planting corn seed treated at 0.00058 lb a.i./lb seed. 

Exposure Scenario Applicati

on Rate

Area 

Treated

Dermal 

Unit 

Exposure1

Dermal 

Exposure2

Inhalation 

Unit 

Exposure1

Inhalation 

Exposure3

Airborne 

Concentration4

Maximum 

Venous 

Blood 

Chlorpyrifos

24-Hour 

Venous 

Blood 

Chlorpyrifos

10 Hours 

After the 

Last Peak 

on Day12 Blood

Chlorpyrifos

Worker 

Activity Form. Use Site

Value

(lb a.i./lb 

seed)

lbs seed 

treated/

day

Value

(ug/lb 

a.i.)

1

Value

(mg/day)2

Value

(ug/lb 

a.i.)

1

Value

(mg/day)2

Value

(mg/L) Value

(pg/g)

Value

(pg/g)

Value

(pg/g)

Loader/

ApplicatorEC Beans,

Peas 0.00058 200,000 18(DL/G)

2.1

(DL/G)

0.068

(PF5)

0.0079

(PF5) 1.4x10-8 954 12.4 71.1

Worker 

Activity Form. Use Site

Value

(lb 

seed/A)

Value

(Acres)

Value

(ug/lb 

a.i.)

1

Value

(mg/day)2

Value

(ug/lb 

a.i.)

1

Value

(mg/day)2

Value

(mg/L)

Value

(pg/g)

Value

(pg/g)

Value

(pg/g)

Seed 

Planting EC Corn 15 80 250(SL/G)

0.47

(DL/G)

0.068

(PF5)

0.0013

(PF5) 4.6x10-9 214 2.8 16.0

1. Unit Exposures from HED Exposure Science Advisory Council Policy 14: Standard Operating Procedures for Seed Treatment.

2. Dermal Exposure (mg/day) = Dermal Unit Exposure (μg/lb a.i.) × Conversion Factor (0.001 mg/μg) x Application Rate (lb a.i./lb seed) × 

Amount Planted (lb seed/day)

3. Inhalation Exposure (mg/day) = Inhalation Unit Exposure (μg/lb a.i.) × Conversion Factor (0.001 mg/μg) x Application Rate (lb a.i./lb seed) × 

Amount Planted (lb seed/day)

4. Airborne Concn (mg/L) = Exposure (mg/day) / (Breathing Rate (L/min) * 60 min/hr * 8 hours/day)
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

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9.4.3 Worker Exposure Summary & Preliminary Conclusions

The PK profile of chlorpyrifos in blood predicted for the ‘low’ occupational exposure scenarios 

are consistent with the other chlorpyrifos exposure scenarios modeled, dietary exposures (i.e., 

food and drinking water) and residential post-application exposures which may have occurred in 

the CCCEH cohort prior to the voluntary cancellation of indoor products. That is, the profile of 

chlorpyrifos in the blood rapidly increases during the exposure period and then rapidly decreases 

following exposure, followed by a terminal half-life phase. 

The maximum venous blood concentrations predicted for the example ‘low’ occupational 

exposure scenarios are all well in excess of those modeled for dietary exposures. For example, 

the lowest of the 7 occupational exposure scenarios, application of chlorpyrifos to cole crops via 

groundboom at an application rate of 1.0 lb a.i./A (presented in Figures 16 and 17), resulted in a 

predicted maximum blood concentration of chlorpyrifos of 194 pg/g. Even at this low level of 

occupational exposure, the blood concentration is approximately 27 times greater the maximum 

blood concentration from food at the 99.9th percentile, 7.1 pg/g (Table 8), and approximately two 

orders of magnitude greater than the maximum concentration predicted from drinking water 

based on chlorpyrifos monitoring data for Orestimba Creek (Figure 13). The agency notes that 

the majority of the occupational exposure scenarios assessed for the 2014 human health risk 

assessment result in a greater risk potential than those predicted for the 7 ‘low’ occupational 

exposure scenarios. An additional uncertainty factor is being retained to account for the same 

concerns that agency had for retaining the FQPA SF for dietary exposures to infants, children, 

youths and women of childbearing age. Similarly, this database uncertainty factor is retained for 

adult workers due to the concerns for potentially pregnant women and the uncertainties regarding 

neurodevelopmental effects. Accordingly, the same proposed internal PoD with a 100X UF/SF

will apply for both dietary and occupational exposures.

10.0 Discussion & Next Steps

In 2008 and 2012, the FIFRA SAP cautioned the agency against using the biomonitoring data 

from epidemiology studies, particularly those from CCCEH, to directly derive PoDs due, in large 

part, to uncertainties associated with a lack of knowledge about timing of indoor chlorpyrifos 

application(s) and the single measures of exposure (cord blood) that may not reflect exposure 

patterns over the course of an entire pregnancy or temporal exposure patterns could have 

coincided with unknown window(s) of susceptibility. However, the 2012 SAP also 

recommended that the agency consider use of a PBPK model to further characterize the dose 

estimates in the epidemiology studies. Since the 2012 FIFRA SAP, epidemiologists from the 

three US cohorts have continued to publish papers on the adverse effects in children up to ages 

7–11 suggesting the lack of reversibility of the outcomes; these findings have heightened the 

agency’s concern. Moreover, the PBPK model has continued to develop and mature to the point 

of use in regulatory decision making. In light of these developments since the 2012 FIFRA SAP,

the agency has followed up on the 2012 SAP recommendation and has conducted additional 
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

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characterization of the pharmacokinetic profile of simulated exposures from oral and dermal 

exposures. 

The analysis described here highlights the important context of the cohort and biomonitoring 

data-namely that when the cord blood were taken the mother would have delivered a baby and 

labor and delivery is a process that takes hours. These events would have taken sufficient time 

for the levels of chlorpyrifos in blood to drop substantially and likely plateau to the horizontal 

asymptote. The agency is explicitly assuming that the biomonitoring data reported by CCCEH 

were collected at low points on the PK profile, particularly in the terminal clearance phase where 

such blood concentrations are not changing dramatically. 

In this labor and delivery context, the exposure characterization analysis described in Section 6.0 

supports the plausibility of the CCCEH reported values across time. Specifically, the blood 

concentrations (Table 3) from the simulated perimeter, carpet scenario closely match the 

CCCEH cord blood and maternal data for 1998/1999 and 2000 (i.e., before the cancellation of 

indoor uses). Moreover, the blood concentration data in Table 2 for food exposure across the 

distribution closely match the CCCEH cord blood and maternal data for 2001–2004 (i.e., after

the cancellation of indoor uses). As such the agency’s exposure characterization analysis 

compared to the CCCEH data in Figure 1 support the conclusion that the reported biomonitoring 

data reflect the likely exposure patterns of chlorpyrifos across the time period of 1998–2004 and 

thus provide confidence in their use in risk assessment. 

Table 13 provides the notable uncertainties listed by the 2012 FIFRA SAP (p. 48 of the report) 

and provides a cross reference to the section of this document where the uncertainty is addressed 

by the agency. The CCCEH exposure characterization in Section 6.0 addresses several of the 

listed uncertainties such as the timing of pesticide applications, limited number of blood 

sampling times, lack of understanding of critical windows of susceptibility, and plausibility of 

the exposure levels. Others such as the modest sample size, moderate to large exposure 

differences need for significance, and generalizability of the cohort to other populations have 

been addressed in the agency’s proposals to retain the 10X intra-species factor and/or the FQPA 

10X Safety Factor. As described in Section 7.1, the agency is aware of these uncertainties. 

However, the agency believes these uncertainties have been sufficiently addressed and do not 

prevent the transition from using AChE inhibition as the critical effect to neurodevelopmental 

outcomes for quantitative risk assessment. Moreover, despite the listed uncertainties, there is 

different uncertainty associated with continuing to use the AChE inhibition effect for deriving 

the PoDs. Specifically, the internal blood concentrations required to achieve 10% RBC AChE 

inhibition are substantially higher than those reported by CCCEH and associated with 

neurodevelopmental outcomes suggesting that the RBC AChE inhibition PoDs are not 

sufficiently health protective.
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

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Based on human health risks identified in the 2014 human health risk assessment, the agency 

published a 2015 proposed tolerance revocation for chlorpyrifos. If the revocation action 

becomes effective as proposed, it would likely lead to the end of chlorpyrifos use on food crops 

or in processed food produced in or imported into the U.S. The agency is continuing to move 

forward with the regulatory steps necessary to revoke the chlorpyrifos tolerances. 

In summary, this issue paper has described the characterization of predicted exposures to women 

in the CCCEH prior to giving birth along with a proposed approach for using the CCCEH cord 

blood data to derive a PoD for use in quantitative risk assessment. In addition, this issue paper 

proposes an innovative approach to using the PBPK model to integrate external and internal dose 

from food, water, and occupational exposure assessment for comparison with the proposed 

internal RfD for dietary exposure and related target MOE for worker/residential exposure.

The agency is soliciting comments from the SAP and the public on the science issues raised in 

this issue paper.
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

Page 79 of 158

Table 13. Cross-reference of uncertainties identified by the FIFRA SAP (2012) on using the CCCEH biomonitoring data for deriving a PoD.

Uncertainty Identified by the FIFRA 

SAP (2012, p. 48) Section Where Considered Comment

Relatively modest sample sizes which 

limited the statistical power to classify 

some meaningful differences as 

statistically significant and to examine 

the effect of modification by 

race/ethnicity and other 

characteristics. 8.2. FQPA 10X Safety Factor for 

Infants & Children

These points increase the uncertainty that the PoD 

based on the CCCEH cord blood data are not fully 

representative and have been used as support to 

retain the FQPA 10X Safety Factor.

Relatively moderate to large exposure 

differences needed to see significant 

effects, likely due to the modest sample 

sizes used.

Exposure at one point in prenatal time 

with no additional information 

regarding postnatal exposures.

Lack of clarity regarding a linear 

dose-response instead of a potential 

threshold effect.

7.2 Options for PoD Based on the 

CCCEH Biomonitoring Data

This remains an uncertainty since CCCEH 

investigators have not released the raw data 

publicly. However, given the modest sample size 

and that the voluntary cancellation occurred in 

2000, the data may not support a different empirical 

model (i.e, non-linear model). This point adds 

support the selection of the 2% change in working 

memory as this value is within the measured data 

(albeit at the low end).

Use of a single or average sample for 

exposure. Although Whyatt et al. 

(2009) noted moderate but significant 

correlations between meconium and 

cord and maternal blood and average 

urine TCPy, the representativeness of 

a single point exposure is still unclear. 

6.3 Residential Exposure to the 

CCCEH Cohort &

7.1 Uncertainties with Using 

Biomarker for the PoD

The agency notes similar uncertainties in Section 

7.1. The agency has developed an analysis of the 

range of possible exposures to the CCCEH cohort. 

This analysis uses the best available information on 

pesticide use and chlorpyrifos applications from 

1998-2004, including Whyatt et al (2003) which 

indicates that pesticide applications occurred 
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

Page 80 of 158

Uncertainty Identified by the FIFRA 

SAP (2012, p. 48) Section Where Considered Comment

Time-varying exposures or the ability 

to define cumulative exposures would 

be preferable.

approximately monthly. The agency’s predicted

blood concentrations closely match those reported 

by CCCEH. 

Lack of specificity of a critical window 

of effect and the potential for 

misclassification of individual 

exposure measures.

External generalizability of the cohorts 

given their unique racial/ethnic and 

socioeconomic characteristics. 

However, it should be noted that their 

exposures were within the range of 

those seen in NHANES.

8.1. Intra-species Extrapolation

The agency concurs there are remaining 

uncertainties about the degree to which the CCCEH 

is more or less sensitive to other population groups. 

Thus, this issue is part of the rationale for retaining 

the 10X intra-species factor. 

Questions about biologic plausibility 

due to lack of clarity on mechanism of 

action, particularly at the low 

exposure levels seen in the cohorts and 

the limited and mixed results of animal 

studies showing neurodevelopmental 

effects.

Appendix 3.0 (3.1.1, 3.1.2, 3.2) 

Background & Summary of 

Experimental and Epidemiology 

Studies on Neurodevelopmental 

Effects &

6.3 Residential Exposure to the 

CCCEH Cohort

The agency agrees that the AOP/MOA for 

neurodevelopmental outcomes are not known. The 

agency has conducted a systematic review of the 

laboratory animal literature. There is qualitative 

similarity with respect to the three major 

neurodevelopmental domains (specifically, 

cognition, motor control, and social behavior) 

between the laboratory animal studies and the 

epidemiology findings. The CCCEH exposure 

characterization analysis provides plausibility to the 

low blood levels. 
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

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Appendix 1.0: PoDs Based on 10% AChE Inhibition from the 2014 Human 

Health Risk Assessment

Summary information is provided below. Complete analyses and more detailed narratives are 

provided in the 2014 HHRA (USEPA, 2014a).

Like other OPs, chlorpyrifos binds to and phosphorylates the enzyme acetylcholinesterase 

(AChE) in both the central (brain) and peripheral nervous systems. This can lead to 

accumulation of acetylcholine and, ultimately, at sufficiently high doses, to clinical signs of 

toxicity. In connection with EPA’s FIFRA registration review of chlorpyrifos, and to address 

issues raised in an administrative petition seeking revocation of chlorpyrifos tolerances, EPA 

presented comprehensive reviews of the literature for AChE inhibition for the 2008 and 2012 

SAP reviews along with the 2011 preliminary and 2014 revised risk assessment. AChE 

inhibition was the critical effect for the 2011 and 2014 risk assessments; this 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, 2006a) and other single chemical OP risk assessments. There are many 

chlorpyrifos studies evaluating AChE inhibition in red blood cell (RBC) or brain in multiple 

lifestages (gestational, fetal, post-natal, and non-pregnant adult), multiple species (rat, mouse, 

rabbit, dog, human), methods of oral administration (oral gavage with corn oil, dietary, gavage 

via milk) and routes of exposure (oral, dermal, inhalation via vapor and via aerosol). In addition, 

chlorpyrifos is unique in the availability of ChE data from peripheral tissues in some studies 

(e.g., heart, lung, liver). There is also scientific literature comparing the in vitro ChE response to 

a variety of tissues (Chambers, 2013) which show similar sensitivity and intrinsic activity. 

Across the database, brain AChE tends to be less sensitive than RBC AChE or peripheral ChE. 

In oral studies, RBC AChE inhibition is generally similar in response to peripheral tissues. 

In typical risk assessments, points of departures (PoDs) are derived directly from laboratory 

animal studies and inter- and intra-species extrapolation is accomplished by use of 10X factors. 

In the case of chlorpyrifos and its oxon, PBPK-PD modeling was used in 2014 HHRA to 

estimate PoDs for all age groups. The variation model was used to develop Data-Derived 

Extrapolation Factors (DDEF) for intra-species extrapolation for some groups (USEPA, 2014c) 

(See Section 4.8.4.1 in the 2014 HHRA). The agency typically uses a 10% response level for 

AChE inhibition in human health risk assessment. This response level is consistent with the 

2006 OP cumulative risk assessment (USEPA, 2006a) and other single chemical OP risk 

assessments. As such, the model was used to estimate exposure levels resulting in 10% RBC 

AChE inhibition following single day (acute; 24 hours) and repeated exposures for a variety of 

exposure scenarios. The PBPK-PD model accounts for PK and PD characteristics to derive age, 

duration, and route specific PoDs. Separate PoDs were calculated for dietary (food, drinking 

water), residential, and occupational exposures by varying inputs on types of exposures and 
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

Page 98 of 158

populations exposed. Specifically, the following characteristics have been evaluated: duration 

[acute, 21-day (steady state)]; route (dermal, oral, inhalation); body weights which vary by 

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

day (eating, drinking)]. It should also be noted that PoDs were calculated for exposure patterns 

that would be considered protective of all others within each category (e.g., children 1–2 years 

old are used to evaluate mosquito adulticide exposures for children because of their behavior 

they have high dermal exposures relative to their body weight and they also exhibit mouthing 

behaviors based on their stage of development).

 
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

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Table 1.1. Table 4.8.4 from the 2014 Revised Human Health Risk Assessment: Chlorpyrifos PBPK Modeled Doses (PoDs) Corresponding to 10% 

RBC AChE Inhibition

Table 4.8.4. Chlorpyrifos PBPK Modeled Doses (PoDs) Corresponding to 10% RBC AChE Inhibition

RA Type

Exposure 

Pathway

(all 

chlorpyrifos 

unless noted)

Infants

( <1 yr old)

Young Children 

(1–2 years old)

Children 

(Residential:6-11 

years old;

Dietary:6–12 years 

old)

Youths

(Residential:11-16 

years old;

Dietary:13–19 

years old)

Females 

(13–49 years old)

Acute

Steady 

State

(21-day)

Acute

Steady 

State 

(21-day)

Acute

Steady 

State 

(21-day)

Acute

Steady 

State 

(21-day)

Acute

Steady 

State

(21-day)

Dietary

Drinking 

Water

(oxon conc, 

ppb)

1,183 217 3,004 548 7,700 1,358 4,988 878 5,285 932

Food 

(μg/kg/day) 600 103 581 99 530 90 475 80 467 78

Residential

(Golfers)

Dermal 

(μg/kg/day) 22,750 13,950 11,890

Residential

(Mosquitocide 

Application)

Dermal 

(μg/kg/day) 134,250 23,600

Oral 

(μg/kg/day) 101

Inhalation 

(concn. in air 

mg/m3)

2.37 6.15

Occupational 

Dermal 

(μg/kg/day) 3,630

Inhalation

(μg/kg/day) 138

*PoDs and exposure and risk estimates for females 13–49 yrs covers all youths >13 yrs
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Predicted peak blood concentration at the external dose to achieve 10% RBC AChE inhibition 

for occupational dermal scenario, for example, is about 100,000 pg/g; and predicted blood 

concentration 10 hours after the peak is about 10,000 pg/g.

Table 1.2. Peak chlorpyrifos concentration in blood given the same steady state food exposure 

and worker dermal exposure scenarios used in the 2014 Revised Human Health Risk Assessment 

at the chlorpyrifos doses corresponding to 10% RBC AChE inhbition. 

RA Type Exposure pathway Infants 

(<1 year old)

Females 

(13 – 49 years old) 

Dietary Food 6375 pg/g 7572 pg/g

Worker Dermal 122,904 pg/g
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Appendix 2.0: Summary of 2014 Dose Reconstruction Analysis

On April 10–12 2012, a FIFRA SAP meeting was held in review of, Scientific Issues Concerning 

Health Effects of Chlorpyrifos. Following the SAP meeting, the Panel provided the 

memorandum, Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting 

held April 10–12, 2012 on “Chlorpyrifos Health Effects.” The following was a recommendation 

of the SAP Panel for characterization of chlorpyrifos exposures:

“Environmental monitoring and biomonitoring data in the epidemiological studies contribute to 

the overall database on estimation of exposure, including (particularly) population variability and 

(to some degree) inter-individual variability in the study populations. They also provide insight 

into more generalizable observations on temporal trends in exposure of the general population —

e.g., following the impact of withdrawal of domestic (nonagricultural) uses of chlorpyrifos. 

The biomonitoring and environmental monitoring data from the three children’s cohort studies 

should be used, then, along with exposure information from other studies and sources, to 

characterize the levels of exposure to chlorpyrifos experienced in different 56 populations 

(production workers, agricultural workers, individuals exposed via residential use, general 

population, etc.), and in similar populations over time (e.g., before and after cancellation of 

residential uses).”

Consistent with the recommendation of the SAP Panel, and in order to better understand the 

possible exposure patterns in the CCCEH study cohort and their influence on the health 

outcomes identified in the research, an analysis was conducted to supplement the exposure 

metrics reported by the CCCEH investigators. The goal was to predict potential high-end, or 

“screening level” exposures which could have occurred to pregnant women and young children 

who may have been exposed to chlorpyrifos through other pathways not addressed in the 

research then determine their potential for associated cholinesterase inhibition. Specifically, 

whether these screening level exposures could have resulted in 10% RBC AChE inhibition, the 

current regulatory endpoint for chlorpyrifos. 

Chlorpyrifos, at the time, was one of the most widely used insecticides for indoor pest control so 

there are many possible ways which exposures could 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 analysis was based on the agency’s SOPs for Residential Exposure 

Assessment51 which describe, on a scenario basis, specific algorithms and data used to predict 

the potential exposures which could have occurred to individuals in the cohort. Once complete, 

the resulting dose estimates were used to evaluate the potential level of cholinesterase inhibition 

which could be attributed to them using the PBPK-PD model. The scenarios which were 

considered include:

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• 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 residues 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 treatment which leads to exposure.

Use Profile

In order to evaluate the chlorpyrifos exposure potential for the CCCEH study cohort, it was first 

necessary to define what chlorpyrifos products would have been in use during the time of study 

conduct which spanned from 1998 to 2004. In 1997, the registrant, Dow AgroSciences, 

voluntarily agreed to cancel chlorpyrifos 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 fully contained ant baits in child resistant packaging and limited 

public health uses - were subject to voluntary phase out/cancellation. This analysis reconstructed 

the potential exposures for those in the cohort between the years 1997–2001 based on the 

chlorpyrifos uses that remained after the voluntary cancellation in 1997 and the phase out in 

2001. The exposure questionnaire-based reported use from the CCCEH publications was also 

used to inform this process. Per the publications, pest control measures were used by 85% of 

respondents, whether by housing superintendent, pest control operator (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 

month. Unfortunately, while these data help inform 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 use during this time period including, chlorpyrifos, diazinon, and 

propoxur, among others. For the purpose of the dose reconstruction exercise, it was assumed 

that only chlorpyrifos applications occurred. Considering all the available information, the 

potential for chlorpyrifos exposures from use of the following chlorpyrifos product types were 

assessed: broadcast ready to use (RTU) sprays applied by individual themselves and not 

professional applicators; surface sprays applied by individuals; crack and crevice sprays applied 

by individuals themselves or professional pest control applicators; and foggers/bombs applied by 

individuals. These chlorpyrifos residential products contained 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 Pesticide Exposure Assessment52 were used to assess the exposures that may have 

 52 http://www.epa.gov/pesticides/science/USEPA-OPP-HED_Residential%20SOPs_Oct2012.pdf
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occurred from these indoor residential chlorpyrifos uses. Based on the use patterns of the 

defined products, it is expected that exposures occurred either from the application (handling) of 

products by those in the cohort or after 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-application 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 assumption 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 CCCEH cohort 

being evaluated which was derived from the EPA Exposure Factors Handbook 2011 

Edition 53 (adult and adult female: Tables 8-3 through 8-5; body weight of pregnant 

women: Table 8-29). 

• the most protective index lifestage which serves as the basis for evaluating young 

children’s indoor exposures are children 1 to <2 years old as described in in the 2012 

Residential SOPs;

• the body weight used to evaluate exposures to children 1 to <2 years old is 11 kilograms.

For residential handler exposure assessment, the following assumptions and exposure factors 

apply:

• a broadcast application of an aerosol can is considered and the entire contents of the can 

(16 oz.) are applied at once, half the contents of the spray can are applied for crack and 

crevice applications, and a quarter of the can contents for a space spray application.

For residential post-application exposure assessment, the following assumptions and exposure 

factors apply:

• inhalation exposures are expected to occur up to 2 hours following application by which 

time the aerosolized particulate is assumed to have settled and, 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 estimate the potential level of cholinesterase 

inhibition (% RBC AChE) attributable to the daily dose estimates that result in the highest 

exposure potential. In order to model the potential health outcomes in a manner reflective of 

these exposures, a series of inputs were used that correspond to those used to quantify the daily 

dose estimates. The following exposure characteristics were used: number of days exposed; 

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body weights which vary by lifestage; daily exposure 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 was inputted into the PBPK-PD model. Dermal 

exposure was modeled assuming that chlorpyrifos residues dissipated at a rate of 10% per day. 

A 14-day period was used as the basis of the PBPK-PD modeling to ensure it adequately 

addressed the time it takes for the ACHE enzyme inhibition to reach a steady state. 

For the adult and children 1 to <2 years old age groups, the appropriate body weights were 

obtained for modeling purposes from the 2011 US EPA Exposure Factors Handbook.

54 The 

mean adult female bodyweight reflective of all trimesters of pregnancy, 75 kg, was assumed for 

pregnant women to reflect the population of interest from the CCCEH cohort being evaluated 

(Exposure Factors Handbook: adult and adult female: Tables 8-3 through 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 handler 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 assumed that 1 hour is a high end estimate of the time required to 

make an aerosol can application. 

For the evaluation of adults and children 1 to <2 years old post-application exposures (i.e., 

adults: dermal; children: 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 consistent 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 assumed 

that the exposure duration was 2 hours on the day of application (Day 0) and was not repeated

because the exposure source (i.e., aerosols from an application event) occurred only once on the 

first day of the exposure 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 drop to negligible levels by the following day.

For all dose reconstruction dermal exposures to chlorpyrifos, the fraction of skin in contact with 

chlorpyrifos was 50% which reflects uncovered skin areas for children wearing shorts and a tee 

shirt. No shower (i.e., washing off the chlorpyrifos) was assumed to occur for the 14-day 

exposure duration modeled. 

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Residential Handler Exposure Scenarios

The residential handling of chlorpyrifos residential products are expected to have occurred for 

some percentage of the CCCEH cohort population, these are anticipated to have resulted in 

dermal and inhalation exposures. Dermal and inhalation residential handler exposures are 

predicted by use of a factor known 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 mass active ingredient exposure per mass active 

ingredient handled (e.g., mg/lb a.i.). For the dose reconstruction assessment, unit exposure data 

were used as recommended in the 2012 Residential SOPs for ready-to-use (RTU) aerosol cans 

(dermal, 370 mg/ lb a.i.; inhalation, 3.0 mg/lb a.i.). Residential handler exposures from 

application of fogger formulations were not assessed which is consistent with the 2012 

Residential SOPs. Fogger product labels typically direct the user to activate the device and 

evacuate the treated area and not to re-enter for several hours. Therefore, it is assumed that 

handler exposure from fogger application is not expected. 

The exposure assessment for chlorpyrifos CCCEH cohort handler exposures is based on the 

following exposure scenarios:

• Adult dermal exposure from broadcast application of a RTU spray can;

• Adult inhalation exposure from broadcast application of a RTU spray can;

• Adult dermal exposure from crack and crevice application of a RTU spray can;

• Adult inhalation exposure from crack and crevice application of a RTU spray can;

• Adult dermal exposure from space spray application of a RTU spray can;

• Adult inhalation exposure from space spray application of a RTU spray can.

Handler Exposure Algorithm

Daily dermal and inhalation exposure (mg/day) for 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, using 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 handled (broadcast, 1 can/day; crack and crevice, 0.5 can/day; space 

spray, 0.25 can/day).
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Residential Handler Exposure/Cholinesterase Inhibition Summary 

Worst-case results of this analysis are presented below representing pregnant women who 

potentially made a broadcast application of an aerosol spray can (Table A.2.1). Peak % RBC 

AChE inhibition estimated for pregnant women from combined dermal and inhalation exposures 

occurring from product application is 0.0012%. 

Table A.2.1. Residential Handler (Pregnant Women in the CCCEH Cohort) Estimated Exposures 

and Predicted % ChE Inhibition (Route-specific and Combined)

Exposure 

Scenario Formulation Amount Handled

Dermal 

Exposure

(mg/ day)

Dermal:

Peak

% ChE 

Inhibition

Inhalation 

Exposure

(mg/day)

Inhalation:

Peak

% ChE 

Inhibition

Combined 

Peak

% ChE 

Inhibition

Broadcast 

Ready to 

Use:

1 % Aerosol

Spray Can

1 can 3.7 0.0007% 0.030 0.0008% 0.0012%

a. Dermal Exposure = Dermal Unit Exposure (370 mg/lb a.i.) × Application Rate (0.010 lb a.i./can) ×

Area Treated or Amount Handled (cans/day)

b. Inhalation Exposure = Inhalation Unit Exposure (3.0 mg/lb a.i.) × Application Rate (0.010 lb a.i./can) ×

Area Treated or Amount Handled (cans/day)

Residential Post-application Exposure Scenarios

The use of indoor residential chlorpyrifos products are anticipated to have resulted in the 

potential for post-application exposures from public housing residents in the CCCEH study 

cohort being in a previously treated environment. Based on the product use profile, residents 

may have been exposed via inhalation to the aerosols resulting from application or by means of 

contact following settling of product contents following settling on indoor surfaces or flooring. 

The exposure assessment for chlorpyrifos CCCEH cohort post-application exposures is based on 

the following exposure scenarios:

• Adult dermal contact with residues deposited on flooring (carpeted and hard) from 

broadcast application of RTU spray can;

• Adult inhalation of aerosolized particulates following broadcast 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 aerosolized 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;
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• 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 oral (hand-to-mouth) ingestion of residues deposited 

on flooring (carpeted and hard) from broadcast 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 flooring (carpeted and hard) treated by broadcast 

application of RTU spray can;

• Children 1 to <2 years old inhalation of aerosolized particulate following broadcast 

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 oral (hand-to-mouth) ingestion of residues deposited 

on flooring (carpeted and hard) from 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 flooring (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 RTU spray can or PCO applications;

• Children 1 to <2 years old incidental oral (hand-to-mouth) ingestion of residues deposited 

on flooring (carpeted 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 flooring (carpeted and hard) treated by crack and 

crevice RTU spray can or PCO applications.

Since all exposure routes (dermal, inhalation, and incidental oral) share a common toxicological 

endpoint, RBC AChE inhibition, risk estimates should been combined. The incidental oral 

scenarios (i.e., hand-to-mouth and object-to-mouth) should be considered 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 unrealistic because of the conservative nature of 

each individual assessment. Therefore, the post-application exposure scenarios that were 

combined for children 1 to <2 years old are the 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 post-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 that is included in the quantitative assessment is 

health protective for the exposures and risk estimates for any other potentially exposed lifestage.
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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 estimation of post-application indoor exposures. 

Where chemical-specific exposure data are available, these data were relied upon as a more 

accurate measure of exposures from chlorpyrifos and are described 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 Incidental Oral Exposure Assessment: No chemicalspecific deposition data were available for the indoor chlorpyrifos products assessed. Therefore, 

as recommended by the 2012 Residential SOPs, default deposited residue values were used 

based on the type of application to be made whether 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, the 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 2012 Residential SOPs. 

The transfer coefficent (TC) input used for calculation 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 typical indoor activities. Therefore, the transfer 

coefficients used for indoor scenarios are derived from information provided in three different 

studies: 1) two studies which measured exposure and surface residues while subjects performed a 

Jazzercise routine (Krieger, 2000; Selim, 2004) and 2) a study which measured biomonitoring 

doses which adults performed scripted 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 transfer input, a complete dataset was compiled for 

chlorpyrifos based on available chemical-specific data (Beamer et al., 2009). Therefore, the 

chlorpyrifos-specific residue fractions anticipated to transfer for both carpets and hard surfaces 

were used for estimation of post-application dermal and incidental oral exposures. 

Exposure Data Used for Inhalation Exposure Assessment: In order to best represent the potential 

for inhalation exposures following use of the indoor chlorpyrifos products assessed, a review of 

available literature studies and registrant-submitted exposure data was conducted. The following 

exposure data sources were identified and used: 
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Indoor broadcast applications:

• Fenske, R., et al. (1990). Potential Exposure and Health Risks of Infants following 

Indoor Residential Pesticide Applications. 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, Dislodgable 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 crevice applications:

• Byrne, S.L., Shurdut, B.A. and Saunders, D.G. (1998). Potential Chlorpyrifos Exposure 

to Residents following Standard Crack and Crevice Treatment. 106 (11): 725–731. 

• Hore, P. et al. (2005). Children’s Residential Exposures to Chlorpyrifos: Application of 

CPPAES Field Measurements of Chlorpyrifos and TCPy within MENTOR/SHEDS —

Pesticides Model. Science 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 Application in the US EPA Indoor Air Quality Research House. 

Atmospheric Environment. 37 (39–40): 5539–5549.

• EPA MRID 44458201: Byrne, S.; Saunders, 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 consistent with the 2012 Residential SOPs, the day 

of application (Day 0) air monitoring measures were significant, 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-application exposure assessment. All studies 

referenced were based on 0.5% chlorpyrifos formulated products. In order to address the 

potential for use of the 1% chlorpyrifos product formulations available at the time, all air 

monitoring measures were doubled. The broadcast exposure data are intended for use in 

assessing the potential inhalation exposures/doses anticipated to occur from both broadcast and 

space spray applications; the crack and crevice exposure data were used only for that application 

type. The resulting mean day of application air monitoring measures are presented in Table A.2.2 

below.
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Table A.2.2. Airborne Concentrations of Chlorpyrifos from Open Literature and Registrantsubmitted Studies Resulting from Indoor Broadcast and Crack and Crevice Applications

Study Chlorpyrifos Concentration (mg/m3)

Broadcast

Fenske, R., et al. (1990) 0.16

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

As described above (Use of the PBPK-PD Model), dermal post-application exposures were

modeled assuming that chlorpyrifos residues dissipated at a rate of 10% per day. This value is 

based on an evaluation of all available chlorpyrifos-specific floor residue data. Although seven 

exposure studies were identified above for sources of indoor air monitoring data, three were 

excluded from the dermal post-application exposure analysis because they were determined to be 

inadequate for the collection of daily residue dissipation from flooring. The four studies in the 

analysis included two registrant-submitted exposure studies and two exposure studies from the 

open literature. Of these four studies, only two measured floor residues beyond two subsequent 

days (i.e., the day of product application and the day immediately following). This is relevant 

since in all four exposure studies, floor residues drop markedly (43–92% dissipation) on the day 

following product application, whereas the two exposure studies where measurements of floor 

residue were taken for more than two days, residues were observed to drop markedly initially, 

and then plateau. Therefore, to include in the dissipation analysis the exposures studies with 

only two days of residue measurements would overestimate residue dissipation and have the 

result of underestimating daily post-application exposures. Further, of the two exposure studies 

which measured floor residues for more than two days, only one measured residues for 

subsequent days which is necessary for accurate quantification of daily dissipation. Ultimately, 

the assumption of 10% daily dissipation is based the exposure study, Lu and Fenske (1998). 

While the reliance on a single exposure study dataset is limiting, the selection of 10% daily 

dissipation for PBPK modeling is expected to result in a health protective estimation of daily 

post-application exposures; that is, the lower the daily dissipation, the greater the amount of floor 

residues present and, likewise, greater predicted daily exposures. Table A.2.3 below presents 

daily floor residue dissipation resulting from all exposure studies, the number of days measured, 

and for those studies excluded, the reasoning for their exclusion. 
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Table A.2.3. Percent Daily Floor Residue Dissipation from Open Literature and Registrantsubmitted Studies Resulting from Indoor Applications of Chlorpyrifos 

Study Daily Floor Residue 

Dissipation (Mean %) Days Measured

Multi-Day Floor Residue Measures

Lu, C. and Fenske, R (1998) —

Broadcast Application 10 1 – 7 (daily)

Hore, P. et al. (2005) — Crack & 

Crevice Application 33 1, 2, 3, 5, 7, 9, 11

Two-Day Floor Residue Measures

EPA MRID: 42887201 75 1, 2

Fenske, R., et al. (1990) —

Broadcast Application 80 1, 2

Excluded Exposure Studies

Byrne, S.L., Shurdut, B.A. and 

Saunders, D.G. (1998)

Floor residues collected by carpet drags were reported to 

be negligible.

EPA MRID 44458201 Carpet wipes all resulted in non-detectable residues.

Stout II, D.M. and Mason, M.A. 

(2003)

Deposition coupons were used on floors, but not via 

direct application of the coupons. 

Post-Application Algorithms for All Scenarios

Post-Application Dermal Exposure Algorithm (hard surfaces and carpets)

The algorithm to calculate exposure is as follows:

EE = TTTT ∗ TTTT ∗ EE ∗ CC1

where:

E = exposure (mg/day);

TR = indoor surface transferable residue (μg/cm2);

TC = transfer coefficient (cm2/hr); 

ET = exposure time (hr/day); and

CF1 = conversion factor (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:

TTTT = DDDDDD ∗ aaaa

where:

TR = indoor surface transferable residue (μg/cm2);

DepR = deposited residue (μg/cm2), based on (in order of preference):

(1) Chemical-specific residue deposition data (μg/cm2),

(2) Application rate (lb ai/area), or

(3) Default residue based on type of application (μg/cm2); and

Fai = fraction of a.i. available for transfer from carpet or hard surface (unitless).
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Absorbed dermal dose, normalized to body weight, are calculated as:

DD = EE ∗ AA

BBBB

where:

D = dose (mg/kg-day);

E = exposure (mg/day);

AF = absorption factor; and

BW = body weight (kg).

Table A.2.4-1. Indoor Environments (Hard Surfaces and Carpets) – Inputs for Residential 

Post-application Dermal Exposure 

Algorithm 

Notation

Exposure Factor

(units) Point Estimate(s)

TR

Transferable residue

(μg/cm2) Estimated: DepR * Fai

DepR Deposited residue

(μg/cm2)

Estimated based on default 

residue related to type of 

application

Fai

Fraction of DepR as 

TR following 

application

Carpets 0.020a

Hard surfaces 0.13a

TC Transfer Coefficient

(cm2/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 years 

old

Carpets 4

Hard 

Surfaces 2

BW Body weight (kg)

Adult 75

Children 1 < 2 years 

old 11

a. Chlorpyrifos-specific as identified (Table 7-8) in the 2012 Residential SOPs. 
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Post-application Hand-to-Mouth Exposure Algorithm

Exposure from hand-to-mouth activity is calculated as follows (based on algorithm utilized in 

SHEDS-Multimedia):

EE = �HH ∗ ( MM ∗ SS HH) ∗ (EE ∗ NN_ RR RR) ∗ �1 − (1 − SS )

FFFFFFFF_HHHH

NN_RR RRRR RR ��

where:

E = exposure (mg/day);

HR = hand residue loading (mg/cm2);

FM = fraction hand surface area mouthed / event (fraction/event);

ET = exposure time (hr/day);

SAH = surface area of one hand (cm2);

N_Replen = number of replenishment intervals per hour (intervals/hour);

SE = saliva extraction factor (i.e., mouthing removal efficiency); and

Freq_HtM = number of hand-to-mouth contacts events per hour (events/hour).

and

HH = FFFFhaa aaaa ∗ DD

SS HH ∗ 2

where:

HR = hand residue loading (mg/cm2

);

Faihands = fraction a.i. on hands compared to total surface residue from jazzercise study 

(unitless);

DE = dermal exposure (mg); and

SAH = typical surface area of one hand (cm2).

Table A.2.4-2. Indoor Environments – Inputs for Residential Post-application Hand-toMouth Exposure 

Algorithm 

Notation

Exposure Factor

(units) Point Estimate(s)

Faihands Fraction of a.i. on hands from jazzercise study

(unitless) 0.15

DE Dermal exposure calculated 

(mg) Calculated

HR Residue available on the hands

(mg/cm2) Calculated
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Table A.2.4-2. Indoor Environments – Inputs for Residential Post-application Hand-toMouth Exposure 

Algorithm 

Notation

Exposure Factor

(units) Point Estimate(s)

SAH Surface area of one 

hand (cm2) Children 1 < 2 years old 150

AR Application rate

(mass active ingredient per unit area) 1% formulation

FM 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 calculated as follows (based on algorithm utilized in 

SHEDS-Multimedia):

EE = OO ∗ CC1 ∗ SS SSOO ∗ (EE ∗ NN_ RR RR) ∗ �1 − (1 − SS )

FFFFFFFF_OO

NN_RR RRRR RR �

where:

E = exposure (mg/day);

OR = chemical residue loading on an object (μg/cm2);

CF1 = weight unit conversion factor (0.001 mg/μg);

SAMO = area of the object surface that is mouthed (cm2/event);

ET = exposure time (hr/day);

N_Replen = number of replenishment intervals per hour (intervals/hour);

SE = saliva extraction factor (i.e., mouthing removal efficiency); and

Freq_OtM = number of object-to-mouth contact events per hour (events/hour).
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and

OO = DDDDDD ∗ OO

where:

OR = chemical residue loading on the object (μg/cm2

);

DepR = deposited residue (μg/cm2); and

FO = fraction of residue transferred to an object (unitless).

a. Chlorpyrifos-specific as identified (Table 7-8) in the 2012 Residential SOPs. 

The results of this analysis are presented below for pregnant women who were potentially 

exposed from contact to residues which occur in previously treated areas such as their homes 

(Table A.2.6). Results for young children (aged 1 to < 2 years old) who are also exposed to 

residues which occur in previously treated areas such as their homes are presented in Table 

A.2.7. Peak cholinesterase inhibition for pregnant women resulting from combined dermal and 

Table A.2.5. Indoor Environments – Inputs for 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]

FO

Fraction of residue 

transferred to an 

object

Carpets 0.020a

Hard surfaces 0.13a

SAMO Surface area of object mouthed

(cm2/event) 10

N_Replen Replenishment intervals per hour

(intervals/hour) 4

SEO 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
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inhalation exposures occurring from contact with previously treated indoor areas is 0.45%. For 

children 1 to <2 years old, peak cholinesterase inhibition associated with combined dermal, 

incidental oral, and inhalation exposures from being in previously treated areas is 2.7%. 

Table A.2.6. Residential Post-application To Pregnant Women in the CCCEH Cohort Estimated 

Dermal and Inhalation Exposures and Predicted % ChE Inhibition (Route-specific and Combined)

Exposure 

Scenario Formulation

Dermal 

Exposure 

(mg/day)

Dermal:

Peak

% ChE 

Inhibition

Airborne 

Concentration 

of 

Chlorpyrifos

(mg/m3)

Inhalation:

Peak

% ChE 

Inhibition

Combined:

Peak

% ChE 

Inhibition

Broadcast

(Hard 

Surfaces)

1% PCO 

Application 

or RTU 1 

16 oz can

53 0.45% 0.092 0.0049% 0.45%

* See algorithms and inputs above used to estimate post-application dermal exposures. 

Table A.2.7. Residential Post-application (Children 1 to < 2 Years Old in the CCCEH Cohort) 

Estimated Dermal and Inhalation Exposures and Predicted % RBC AChE Inhibition (Route-specific 

and Combined)

Exposure 

Scenario Formulation

Dermal 

Exposure 

(mg/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

14 0.14% 0.10 2.2% 0.092 0.014% 2.7%

* See algorithms and inputs above used to estimate post-application dermal, (HTM) hand-tomouth, and (OTM) object-to-mouth exposures/doses. 
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Appendix 3.0: Background & Summary of Experimental and Epidemiology

Studies on Neurodevelopmental Effects

A.3.1 Neurodevelopmental Effects

The agency has taken a stepwise, objective and transparent approach in evaluating, interpreting, 

and characterizing the strengths and uncertainties associated with all of the available lines of 

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

and children. The stepwise evaluation began with the September 2008 FIFRA Scientific 

Advisory Panel (SAP) meeting involving a preliminary review of the literature for chlorpyrifos, 

with a particular focus on women and children (USEPA, 2008), followed by the draft 

“Framework for Incorporating Human Epidemiologic & Incident Data in Health Risk 

Assessment” for integration of epidemiology with other types of experimental data (USEPA, 

2010; FIFRA SAP 2010). After the draft framework (2010) was published, the agency released 

“Chlorpyrifos: Preliminary Human Health Risk Assessment for Registration Review,” focusing 

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. In 2012, the agency

convened another meeting of the FIFRA SAP focused on chlorpyrifos which incorporated the 

newest experimental 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, the agency also performed a more in-depth analysis of the 

biomonitoring data and of epidemiological studies from three major children’s health 

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

to neurodevelopmental outcomes (USEPA 2012a). Following the 2012 SAP meeting, the agency

solicited additional input from federal experts in the areas of Magnetic Resonance Imaging 

(MRI) and neurobehavioral testing in children to further clarify results obtained by examination 

of the epidemiological studies.55 In December, 2014, the agency released “Chlorpyrifos: 

Revised Human Health Risk Assessment for Registration Review” which included the use of a 

PBPK-pharmacodynamic (PBPK-PD) model to derive human PoDs, which obviated the need for 

the animal to human extrapolation factor, and refined intra-species factors for some lifestages 

(USEPA 2014a). The chlorpyrifos 2014 revised HHRA also included retention of the 10X 

FQPA Safety Factor due to uncertainty regarding the degree of protection the endpoint of AChE 

inhibition provides for potential neurodevelopmental effects (USEPA, 2014a).

 55 http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPP-2008-0850-0170
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A.3.1.1 Mechanistic Studies on Adverse Outcome Pathway

A review of the scientific literature on potential MOA/AOP leading to effects on the developing 

brain was conducted for the 2012 FIFRA SAP meeting (USEPA, 2012a) and updated for the 

2014 chlorpyrifos revised HHRA (USEPA 2014a). There is strong evidence that developmental 

neurotoxicity of chlorpyrifos may not be due to AChE inhibition per se, but to other actions on 

critical aspects of neuronal development. Following several decades of research, there is now a 

number of biologically plausible molecular events proposed for chlorpyrifos (and other OPs) 

effects on the developing nervous system, with ongoing research pursuing many of these 

potential pathways. Supporting data are derived from in vitro or ex vivo studies of chlorpyrifos, 

comparisons to other agents acting on implicated systems, and basic neurobiological research. 

Some of the more promising mechanisms (described in detail in the 2012 SAP) represent events 

at different levels of biological organization, including molecular (binding to muscarinic 

receptors, endocannabinoid enzymes, tubulin, morphogenic site of AChE), cellular (reactive 

oxygen species, neurite growth, intracellular signaling) and functional (serotonergic tone, axonal 

transport). In some cases these effects occur at chlorpyrifos oxon exposure levels at or below 

those that result in cholinesterase inhibition in the same test system, or with the parent 

chlorpyrifos that has no esterase inhibitory properties. The 2008 and 2012 SAP concurred with 

the agency about the lack of definable AOPs, and the 2014 literature review showed no 

substantive changes in this conclusion. These experimental mechanistic studies do not provide 

adequate linkages from the initiating to subsequent events leading to alterations at the molecular 

and cellular level leading to neurobehavioral effects; this significantly limits these studies for 

quantitative use in risk assessment. Thus, while there is good evidence that neurodevelopmental 

effects may not be solely a function of AChE inhibition, there are no accepted alternative AOPs 

and the use of these studies for the assessment of chlorpyrifos and/or its oxon is challenging.

Numerous studies have evaluated chlorpyrifos and its oxon for biological activity against 

relevant targets and some include TCPy. For example, using PC12 cells, Das and Barone (1999) 

examined the concentration-related effects of chlorpyrifos, chlorpyrifos oxon, and TCPy on both 

neurite outgrowth and cholinesterase inhibition. Exposure to chlorpyrifos for 24 hr inhibited 

neurite outgrowth at a concentration (3 uM) 10-fold below that which inhibited cholinesterase 

activity, while chlorpyrifos oxon inhibited both measures at equivalent concentrations (1 nM). 

TCPy, which is inactive against AChE, inhibited neurite growth at 5 uM. Similar studies of 

chlorpyrifos and its metabolites were performed in a series of experiments in the Lein laboratory 

using primary neuronal cultures derived directly from the mammalian nervous system. Using rat 

sympathetic neurons, Howard et al. (2005) showed that 24-hour exposure to chlorpyrifos and 

chlorpyrifos oxon decreased axonal outgrowth at concentrations (0.001 uM and 0.001 nM, 

respectively) well below the concentrations that inhibited AChE activity (1 uM and 1 nM). In the 

same study chlorpyrifos, chlorpyrifos oxon, and TCPy enhanced dendrite outgrowth. 
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The agency acknowledges the lack of clearly identifiable toxic moiety(ies) associated with 

neurodevelopmental effects. There are limited data on the biological activity of TCPy across 

multiple types of assays. Moreover, TCPy is not a specific biomarker to chlorpyrifos which 

makes interpretation challenging. Given the potential for the oxon to be present in chlorinated 

drinking water combined with its high potency, the agency developed an exploratory evaluation 

of using the oxon as the toxic moiety associated with neurodevelopmental outcomes. However, 

given the complete lack of measured oxon values in biomarker data and the limited oxon data in 

laboratory animals, there are limited data that validate the oxon predictions in the PBPK mode. 

As such, the agency has not pursued the oxon for purposes of the 2016 evaluation. Thus, the 

remainder of this document focuses on the parent active ingredient, chlorpyrifos, as the key 

moiety associated with neurodevelopmental outcomes. The majority of the experimental 

laboratory studies and epidemiology studies from CCCEH have evaluated chlorpyrifos and 

provide a robust database of studies associating chlorpyrifos with neurodevelopmental outcomes.

The agency notes that there are a number of known developmentally neurotoxic chemicals with 

well-established relationships between exposure and neurological disorders in humans for which 

a definitive mode of action has not been established: for example lead, methyl mercury, and 

ethanol (Alfonso-Loeches & Guerri, 2011; Castoldi et al., 2008; Farina, et al., 2011; Johansson 

et al., 2007; Verstraeten, et al., 2008). Even today, 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 possible 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 neurotoxicity has been developed for domoic 

acid in adult animals (Watanabe et al., 2011), which has a large amount of preexisting data and a 

molecular initiating event at a receptor (glutamate) with known consequences to its 

overactivation in the adult nervous system. As such, the lack of AOP for neurodevelopmental 

outcomes mediated by chlorpyrifos does not preclude the agency from using this health outcome 

for human health risk assessment. 

A.3.1.2 Studies on Laboratory Animals 

There are an ever-increasing number of experimental studies that report a range of 

neurobehavioral changes in rats and mice following developmental exposure to chlorpyrifos. 

The agency has conducted a systematic review of these studies (USEPA, 2012, 2014, 2015). 

The agency has focused on studies where exposures occurred during gestation and/or postnatally, 

and testing took place after weaning and after chlorpyrifos exposure had ended; there is a 

presumption that these effects are permanent. There are inconsistencies in effects in relation to 

functional domains, gender-specificity, and relation to dose and/or AChE inhibition. Numerous 

differences in experimental design, dosing regimens, and test methodology probably influence 

the variability in outcomes observed across studies. In some cases, less-than-optimal procedures 
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and statistical analyses serve to lower confidence in the data, but at the same time the data are 

more convincing given the sheer number of studies reporting adverse outcomes.

Overall, some generalities emerge that support conclusions of broad changes in neurological 

function. Changes in various aspects of cognitive tests indicate perturbations of learning and/or 

memory, mostly reporting deficits even though in a few cases there is improved function. 

Alterations in anxiety and depressive behaviors, and social interactions, sometimes differed in 

direction of change, but are still suggestive of impacts on normal neuronal processing. Activity 

measures provide results as varied as the different measures of assessment. Obvious species 

differences have not emerged, and effective doses are similar (1–6 mg/kg/d) in rats and mice. 

Dose-response is not always evident, since many studies only use one dose, and of those using 

two or more doses, there is not always a monotonic response. Taken together, these data provide 

evidence for global alterations in neurobehavioral function rather than a specific profile of 

effects. This broad range of neurological effects does not aid in the development of a specific 

AOP (section A.4.1.1), and while there are numerous mechanistic studies that have proposed a 

number of plausible AOPs, none have been experimentally verified. 

A.3.1.3 EPA Evaluation of Epidemiology Studies on Mothers & Children

A.3.1.3.1 Summary of Study Design and Scope of Evaluation

In April 2012, EPA presented to the FIFRA Scientific Advisory Panel (SAP) its review and 

assessment of several epidemiological investigations of the potential adverse 

neurodevelopmental outcomes of in utero and early life exposure to chlorpyrifos. In this effort, 

EPA limited its review to studies conducted within three major US based prospective birth 

cohort studies: 1) Columbia Center for Children’s Environmental Health Mothers and Newborn 

Study, referred to in this document as “CCCEH” 2) Mount Sinai Inner-City Toxicants, Child 

Growth and Development Study, or the “Mount Sinai Study/Cohort;” and 3) Center for Health 

Assessment of Mothers and Children of Salinas Valley (CHAMACOS) conducted by the 

University of California Berkeley, or “CHAMACOS Study/ Cohort.” The conclusion of EPA’s 

evaluation, supported by the FIFRA SAP (2008, 2012), was that “chlorpyrifos likely played a 

role in the neurodevelopmental outcomes observed in these studies.” 

In the 2014 chlorpyrifos revised HHRA, EPA included epidemiologic research results from three 

prospective birth cohort studies. Importantly, each of these cohorts evaluated the association 

between prenatal chlorpyrifos or OP exposure with adverse neurodevelopmental outcomes in 

children through age 7 years. In addition, more recent studies from Mt. Sinai and CCCEH have 

evaluated these associations through age 9 years and 11 years, respectively (Furlong et al, 2014; 

Rauh et al., 2015). EPA is not aware of any studies from the CHAMACOS cohort that have 

evaluated these associations in children older than 7 years old. These studies reflect different 

types of exposed groups in the total population which strengthens the weight of the evidence 

considerations regarding this stream of information. The CCCEH Mother’s and Newborn study 
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and the Mt. Sinai Child Growth and Development study participants were likely exposed to OPs 

through the diet and through residential use of the pesticide for indoor pest control which was an 

allowable use for chlorpyrifos at the time the research was conducted. Additionally, residential 

exposure to OPs may also occurred via the oral route through ingesting residues from hand-tomouth contact with in-home surfaces, as well as possible dermal or inhalation exposure through 

contact with treated areas in the home environment (Berkowitz et al., 2003; Whyatt et al., 2003, 

2007, 2009). 

In contrast, CHAMACOS cohort participants were employed as farm laborers or were residing in 

homes with farm laborers. The CHAMACOS study participants likely experienced exposure to 

OPs through the diet and from occupational exposure (primarily inhalation and dermal routes), as 

well as probable indirect take-home exposures (the “tracking in” of pesticide residues through 

shoes and clothing, augmented by poor hygiene practices) (Bradman et al., 2007). In each of the 

three US children’s health cohorts, the biological measurements in these cohorts were 

comparable to the general population NHANES. EPA has considered the strengths and 

limitations of these studies, and believes that random or systematic errors in the design, conduct 

or analysis of these studies were unlikely to fully explain the observed positive associations 

between in utero OP exposure and adverse neurodevelopmental effects observed at birth and 

through childhood (age 11 years). EPA believes these are strong studies which support a 

conclusion that OPs likely played a role in these outcomes. 

In the 2015 updated literature review, the agency conducted a systematic review expanding the 

scope of the 2012/2014 review focused on US cohort studies with particular emphasis on 

chlorpyrifos. The expanded 2015 review includes consideration of the epidemiological data on 

any OP pesticide, study designs beyond prospective cohort studies, and non-U.S. based studies. 

The updated literature review identified seven studies which were relevant (Bouchard et al., 

2010; Fortenberry et al., 2014; Furlong et al., 2014; Guodong et al., 2012; Oulhote and 

Bouchard, 2013; Zhang et al., 2014; Shelton et al., 2014). These seven studies have been 

evaluated in context with studies from the 2012/2014 chlorpyrifos review (USEPA, 2014a). This 

updated review did not change these conclusions, namely that OPs likely played a role in these 

outcomes reported in the epidemiology studies. 

The CCCEH study measured parent chlorpyrifos in cord blood, and other indicators (e.g., air 

sampling, behavioral information), as etiologic measures of exposure, while the other two birth 

cohorts measured non-specific urinary metabolites of chlorpyrifos and other OPs (TCPy, dialkyl 

phosphate metabolites) in the mothers to estimate pesticide exposure. Therefore, EPA considers 

the CCCEH study research results as most relevant to the chlorpyrifos HHRA; the other two 

cohorts provide important supporting information. 

The biomarker data from the CCCEH studies are supported by the agency’s 2014 dose 

reconstruction analysis using the PBPK-PD model. Following the recommendation of the 
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FIFRA SAP (2012), the agency conducted a dose reconstruction analysis of residential uses 

available prior to 2000 for pregnant women and young children inside the home (Appendix 2; 

USEPA, 2014a). Based on the output from the PBPK-PD model, for the highest exposure 

scenario considered (i.e., indoor broadcast use of a 1% chlorpyrifos formulation), <1% RBC 

AChE inhibition in pregnant women would be expected. While uncertainty exists as to actual 

OP exposure at (unknown) critical windows of exposure, EPA believes it is unlikely individuals 

in the epidemiology studies experienced RBC AChE inhibition.

Within the CCCEH epidemiology studies, the relationship in time between prenatal chlorpyrifos 

exposure and adverse neurodevelopmental outcomes is concordant. The time period under study 

within the CCCEH study, spanned the point in time in which pesticide manufacturers voluntarily 

cancelled the use of chlorpyrifos in the home environment, and researchers were able to show the 

change in exposure before (high use period) and after (low/no use period) the period of removal 

of chlorpyrifos products from the residential marketplace. Moreover, prior to the voluntary 

cancellation there were >80% detectable levels of chlorpyrifos in cord blood but in the time 

period after the cancellation only 16% of the measured values were greater than the level of 

detection (LOD); there was only one child born in the time period subsequent to the voluntary 

cancellation of chlorpyrifos in the residential marketplace for whom the cord blood chlorpyrifos 

level was in the upper-tertile of pre-cancellation exposure levels. The significantly reduced 

proportion of measured values greater than the LOD as well as the observation of an absence of 

an association between prenatal chlorpyrifos exposure among infants born after the voluntary 

cancellation of chlorpyrifos and neurodevelopmental effects support the hypothesis that 

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 infants born after the voluntary cancellation. It is notable that 

epidemiology studies from other research groups have not included analyses across different 

years of exposure.

Several studies have been excluded from further evaluation in this analysis. EPA considered 

studies available for the 2014 updated HHRA, the 2015 updated literature review (USEPA, 

2015), and studies published since the 2015 updated literature review. As summarized below, 

excluded studies included those related to birth outcomes, autonomic nervous system impacts, 

and those with inadequate exposure assessment methods. 

There has been inconsistent evidence of OP exposure and association with adverse birth 

outcomes/fetal growth outcomes. Authors with CCCEH 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; Whyatt et al., 2004). More recent studies evaluated in 2015 (USEPA, 

2015) also documented inconsistent evidence of OP exposure and association with adverse birth 
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outcomes/fetal growth (Barr et al., 2010; Rauch et al., 2012; Wang et al., 2012; Wolff et al., 

2007). Inconsistent results may be due to differences across study groups in exposure profiles 

as well as dissimilar methods of prenatal OP exposure assessment (Needham, 2005). Given the 

lack of consistency among cohorts for the fetal growth metrics, the proposed link between fetal 

growth and OP exposure is tenuous. Therefore, consistent with previous SAP evaluations for 

chlorpyrifos (FIFRA SAP 2008; FIFRA SAP 2012), EPA is focusing this analysis on 

neurodevelopmental outcomes. Although the agency is not evaluating these birth outcome 

studies further at this time, the agency will continue to monitor the scientific literature for 

advances in this line of research.

In the CHAMACOS study, impacts of prenatal DAP exposure and autonomic stability were also 

assessed (Young et al., 2005). When all children in the study are considered together, there were 

no statistically significant associations for prenatal DAP exposure and autonomic stability. 

Stratifying the children by age, children ≤3 days had a statistically significant association 

between total DEAP exposure and autonomic stability, but no association for total DMAP and 

total DAP exposure. Children older than 3 days had no statistically significant associations with 

prenatal DAP exposure and autonomic stability. In addition, another study on the CHAMACOS 

birth cohort (Quirós-Alcalá et al., 2011) assessed the association between DAPs and autonomic 

nervous system (ANS) outcomes at ages 6 months, 1 year, 3.5 years, and 5 years. The ANS 

outcomes assessed included heart rate and respiratory sinus arrhythmia. Overall, while there was 

some evidence of ANS dysregulation for infants at 6 months, these results were not consistently 

observed for the other assessed child (1 year, 3.5 years, and 5 years) and maternal OP exposures. 

There is not a body of literature to compare these results against, making it difficult to put them 

into context. Furthermore, these CHAMACOS studies did not focus on neurodevelopmental 

outcomes, which is the focus of this analysis. Therefore, due to the outcomes assessed and the 

lack of understanding of these findings, these studies are considered to be outside of the scope of 

this assessment and are not discussed further here. Although the agency is not evaluating this 

study data further at this time, the agency will continue to monitor the scientific literature for 

advances in this line of research.

The focus of this assessment is on the neurodevelopmental outcomes from exposures to low 

levels of chlorpyrifos (i.e. below exposures which would result in 10% or more AChE 

inhibition). Three studies conducted in Ecuador focused on child AChE inhibition and the 

potential association of AChE inhibition with other measures including parental occupation 

(Suarez-Lopez et al., 2012), as well as clinical autonomic nervous system (ANS) outcomes such 

as blood pressure and heart rate (Suarez-Lopez et al., 2013a), and neurodevelopmental outcomes 

(Suarez-Lopez et al., 2013). The range of AChE activity levels are lower in the first tertile 

(range of 1.44 to 2.93 U/mL) compared to the third tertile (range from 3.33 to 4.69 U/mL). 

Therefore, due to the outcomes assessed and the potentially toxic cholinergic effects that were 

associated with these outcomes, these studies are not considered to be relevant to this assessment 

and are not discussed further here. Although the agency is not evaluating these study data 
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further at this time, the agency will continue to monitor the scientific literature for advances in 

this line of research.

Finally, six studies identified in the 2015 updated literature review (USEPA, 2015) did not have 

sufficient exposure assessment methods to determine whether exposure to OPs (including 

chlorpyrifos) actually occurred. These studies were conducted on study populations in Spain 

(Llop et al., 2013), Ecuador (Handal et al., 2007; 2007b; 2008), Denmark (Andersen et al., 

2015), and France (Petit et al., 2010). In these studies, participants were considered exposed or 

unexposed to pesticides based on non-specific exposure measures, such as self-reported 

occupational exposure, home pesticide spraying, and proportion of municipality devoted to 

agricultural activity. For all of these proxy exposure assessments, the pesticides used may have 

included not only OPs, but also pyrethroids, fungicides, and growth regulators. Given the 

uncertainty about whether OP exposure actually occurred in these studies and whether observed 

outcomes are associated with OP exposure or with other pesticides, these studies were excluded 

from further analysis.

A.3.1.3.2 Summary of Findings in Epidemiology Studies

For the purposes of providing background and context, the study findings and study design used 

in these three cohort studies (CCCEH, CHAMACOS, and Mt. Sinai) and in the expanded 2015 

literature review are summarized below. These three cohort studies each enrolled pregnant 

women during roughly the same time period, measured both environmental exposure to the 

pesticide during pregnancy and also measured biomarkers representing internal dose during 

pregnancy and at delivery, and prospectively assessed associations in their newborns and young 

children through age 7 years. In addition, more recent studies from Mt. Sinai and CCCEH have 

evaluated these associations through approximately age 9 years and 11 years, respectively 

(Furlong et al., 2014; Rauh et al., 2015). EPA is not aware of any studies from the CHAMACOS 

cohort that have evaluated these associations in children older than 7 years old. 

Each study includes several hundred (approximately 100-400) mother-infant pairs; these sample 

sizes are sufficient to perform statistically valid analyses. Investigators from each study cohort 

utilized a similarly strong study design (prospective birth cohort); measured pesticide exposure 

using several different methods including environmental indicators as well as specific and nonspecific biomarkers of OPs; ascertained developmental outcomes using validated assessment 

tools well-established in both clinical and research settings; and, measured, analyzed, selected 

and statistically adjusted for potential confounding variables including socio-economic status and 

other environmental exposures using reasonable and appropriate methods. Limitations exist as 

well. These studies utilized a one-time measure (or the average of two measures) of chlorpyrifos 

or OP exposure to assess prenatal pesticide exposure throughout the gestational period, were 

unable to assess the influence of mixtures (co-occurring exposures in the relevant biological time 
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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 OP exposure being assessed in many of these studies used concentrations of urinary dialkyl 

phosphate metabolites (DAPs) as the urinary biomarker. Total DAPs is a non-specific measure 

of OP exposure and is the sum of six separate molecules — three dimethyl alkylphosphate 

(DMAP) molecules of dimethylphosphate, dimethylthiophosphate, and dimethyldithiophosphate 

(DMP, DMTP, and DMDTP), and three diethyl alkylphosphate (DEAP) molecules of 

diethylphosphate, diethylthiophosphate, and diethyldithiophosphate (DEP, DETP, and DEDTP). 

Each metabolite is a breakdown product from multiple OPs (CDC, 2008).56 Specifically, DMP, 

DMTP, and DMDTP are associated with 18, 13, and 5 OPs, respectively; whereas DEP, DETP, 

and DEDTP are associated with 10, 10, and 4 OPs, respectively. Thus, in using urinary DAPs 

alone as an exposure measure, it is not possible to separate the exposure and associated effects 

for single, specific OPs. 

DAPs can be found directly on food following OP applications (Zhang et al., 2008; Chen et al., 

2012). Specifically, studies have shown that DAPs may form as environmental degradates from 

abiotic hydrolysis, photolysis, and plant metabolism (Zhang et al., 2008; Chen et al., 2012; 

Racke et al., 1994). Furthermore, since these DAPs are excreted more rapidly and extensively 

than the parent OPs (Zhang et al., 2008; Forsberg et al., 2011), direct exposure to DAPs may 

lead to an overestimate of OP exposure when using urinary DAPs as a biomarker of OP 

exposure. The agency recognizes that this is a source of uncertainty when using DAPs for 

assessing OP exposure and will continue to monitor this issue in future assessments. 

CCCEH studies have largely used chlorpyrifos measured in cord blood as the specific biomarker 

(e.g., Lovasi et al., 2011; Whyatt et al., 2004; Rauh et al., 2011). However, a few studies 

including CCEH measured urinary 3,5,6-trichloro-2-pyridinol (TCPy) (e.g., Fortenberry et al., 

2014; Eskenazi et al., 2007; Whyatt et al., 2009). This metabolite must be interpreted with 

caution. TCPy is also the primary environmental degradate of chlorpyrifos, chlorpyrifos-methyl, 

and triclopyr; thus exposure can be found directly on food treated with these pesticides. 

The CHARGE study (Shelton et al., 2015) did not measure biomarkers but instead used 

geospatial analysis to focus on the residential proximity to OP exposure using data from the 

California Department of Pesticide Regulation, with five OPs accounting for a total of 73% of 

the pesticide applied near residential settings (chlorpyrifos, acephate, diazinon, bensulide, and 

dimethoate). 

As noted, two major uncertainties in environmental 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 

 56 http://www.cdc.gov/nchs/data/nhanes/nhanes_03_04/l26opd_c_met_organophosphorus_pesticides.pdf
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methodological research to address these areas to the extent possible. Across the three children’s 

health cohorts, study authors measured biomarkers of OP exposure. There is uncertainty as to the 

extent measurement of non-specific metabolites of OP or chlorpyrifos accurately reflects OP

exposure; CCCEH and Mt. Sinai studies do not estimate post-natal exposure to chlorpyrifos 

among child participants, therefore the influence of early life and childhood OP exposure is 

unaccounted for in these analyses. The CHAMACOS cohort measured urinary levels of DAPs in 

young children and did not observe negative significant associations in relation to 

neurodevelopment from post-natal exposure (Eskenazi et al., 2007). The CHAMACOS cohort 

investigators also measured AChE and butyl ChE as supplemental indicators of OP exposure.

Potential confounding bias is another major uncertainty within environmental epidemiology 

studies. Confounding variables, exposures related to OP exposure and neurodevelopmental 

outcomes, (e.g., socioeconomic status or SES), may result in an incorrect epidemiological risk 

estimate. Across these cohort studies, investigators collected relevant information concerning 

demographic characteristics and other environmental exposures, and were, to the extent possible 

with the existing information, able to effectively control the influence of these other variables 

when estimating the association between prenatal chlorpyrifos and adverse neurodevelopmental 

outcomes. Control of these variables is important to reduce the chances of a false positive (or 

negative) study result. Overall, statistical analyses were judged to be appropriate and reasonable 

(not overly large number of statistical model variables) to the research question by EPA and 

expert Panel reviews (FIFRA SAP 2008 and 2012).

Researchers with both the Mt. Sinai and CHAMACOS cohorts evaluated neonatal neurological 

functioning in association with prenatal OP exposure; CCCEH did not conduct these 

measurements. To measure indices of abnormal neonatal 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 reflexes in relation to increasing 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 associations. The authors also 

observed evidence of potential effect modification by PON1 activity level in the relation between 

DAPs and neonatal neurodevelopment in which infants of mothers who are 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 estimate of this 

statistical interaction. Similarly, a Chinese cohort study (Zhang et al., 2014) reported 

statistically significant associations between total DEAPs, total DMAPs, and total DAPs from 

prenatal OP pesticide exposure and neonatal neurodevelopment assessed 3 days after birth. 

However, another cross-sectional Chinese study, Guodong et al. (2012), observed no association 
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with postnatal child urinary DAPs and a developmental quotient score for 23–25 month old 

children.

Researchers across the three children’s health cohorts utilized the Bayley Scales of Infant 

Development II (BSID-II) to generate a Mental Development Index (MDI) and a Psychomotor 

Development Index (PDI) to assess neurodevelopment in early childhood. In the CCCEH 

Mothers and Newborn study, Rauh et al. (2006) investigated 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 chlorpyrifos exposure, using categories informed by results of the previous study on 

birth characteristics (Whyatt et al., 2004). Authors reported that the difference in MDI scores 

was “marginally significant” (p=0.06) between the “high” and “low” exposed groups; the high 

exposed group scored an average of 3.3 points lower than the low exposed (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 month score was significantly related to chlorpyrifos exposure. 

Researchers noted that the effects were most pronounced at the 36 month testing period. Within 

the 36 month testing period, the likelihood of highly exposed children developing mental delays 

were significantly greater (MDI: 2.4 times greater (95% CI: 1.12-5.08, p=0.02) and PDI: 4.9 

times greater (95% CI: 1.78-13.72; p=0.002)) than those with lower prenatal exposure (Rauh et 

al., 2006). 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 level was associated with a 

decrement in mental development at 12 months among blacks and Hispanic children; however, 

these associations either attenuated or were non-existent at the 24-month visit (Engel et al., 

2011). In the CHAMACOS cohort, Eskenazi et al. (2007) observed that prenatal DAP levels 

were adversely associated with MDI, and at 24 months of age these 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 status 

(Eskenazi et al., 2010).

Most recently, Engel et al. (2015) reports on the results of a pooled analysis from four cohorts 

(N=936) to evaluate the association between prenatal urinary DAPs and neurodevelopmental 

outcomes (MDI/PDI) at 24 months only. The four cohorts include CHAMACOS (N=377), 

HOME (N=265), Mt. Sinai (N=234), and CCCEH (N=60). The Cincinnati Children’s HOME 

Study is a newer cohort which collected study data from mothers and infants in 2003 to 2006, 

and no separate studies have been published yet on OP exposure and associations with 

neurodevelopmental outcomes. It is noted that the CCCEH participants included in this analysis 

are from women enrolled in 2000 to 2001, a time period which is during the phase out of 

chlorpyrifos in residential settings. The results of this pooled study are relatively consistent with 

those seen in the individual cohorts at 24 months. After controlling for race/ethnicity, smoking, 

and drug use during pregnancy, a statistically significant association was observed in the pooled 

population between total DAPs exposure and MDI decrements, but not with PDI decrements. 

Consistent with the results from Eskenazi et al. (2007), the strongest evidence of an association 
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was observed for the CHAMACOS cohort, with statistically significant associations for both 

total DAPs and total DMAPs exposure and MDI decrements. No significant associations were 

seen within the Mt. Sinai and CCCEH cohorts, a result which is basically consistent with the 

previous observations at 24 months in these cohorts (Engel et al., 2011; Rauh et al., 2006). The 

study authors observed significant heterogeneity from combining the cohorts, especially with 

regards to race/ethnicity, and noted that impacts on specific subpopulations may be lost when 

looking at the pooled results. 

With respect to the findings related to the autism spectrum, from CCCEH, Rauh et al. (2006) 

reported a large odds ratio for pervasive developmental disorder (PDD) (OR=5.39; 95% CI: 

1.21–24.11) when comparing high to low chlorpyrifos exposure groups. As described above, 

among 7–9 years old children in the Mount Sinai Cohort (Furlong et al. 2014), there was no 

overall statistically significant association between maternal third trimester urinary DAP 

metabolite levels and reciprocal social responsiveness. However, some evidence of modification 

of the association between prenatal OP pesticide exposure and impaired social responsiveness in 

early childhood was observed by both race/ethnicity and child sex, with an association between 

DEAP and poorer social responsiveness observed among black participants and boys. No 

association was observed among whites or Hispanics, among girls, or for DAP or DMAP 

biomarker levels. In the CHAMACOS cohort, Eskenazi et al. (2010) reported non-significant, 

but suggestive, increased odds of PDD of 2.0 (0.8 to 5.1; p=0.14), whereas Eskenazi et al. (2007) 

reported a statistically significant association between total DAP exposure and increased odds of 

PDD. Additionally, Furlong et al. (2014) documented suggestive evidence of an association 

between total DEAP exposure and reciprocal social responsiveness among blacks and boys. 

Using a different exposure assessment method (geospatial analysis and residential proximity to 

total OP exposure), Shelton et al. (2014) also showed statistically significant associations 

between total OP exposure and ASD. 

With respect to attention problems, Rauh et al. (2006) also investigated 36-month child behavior 

checklist (CBCL) (behavioral) scores. Significant differences were observed between the high 

and low chlorpyrifos exposure groups in the general category of attention-problems (p=0.010), 

and in the more specific DSM-IV scale for ADHD problems (p=0.018). The CHAMACOS 

cohort also investigated attention problems in early childhood using three different assessment 

tools: maternal report of child 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 children at age 5 years (Eskenazi et al., 2007). Additionally, in a Mexican cohort study, 

Fortenberry et al. (2014) found suggestive evidence of an association with TCPy and ADHD in 

boys. In a national cross-sectional study of Canadian children, using 2007–2009 data for 

children age 6–11 years (Oulhote and Bouchard, 2013), there were no overall statistically 

significant associations observed between child urinary DEAP, DMAP, or total DAP metabolite 
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levels and parentally reported behavioral problems. In contrast, Bouchard et al. (2010), looking 

at U.S. children age 8–15 years in the 2000–2004 National Health and Nutrition Examination 

Survey (NHANES), observed a positive association between attention and behavior problems 

and total DAPs and DMAPs, but not DEAPs. As part of their analysis, Oulhote and Bouchard 

(2013) noted that their outcome assessment for behavioral problems may not have been as 

sensitive as Bouchard et al. (2010), which may in part account for the difference in the observed 

results from these studies. 

To measure intelligence among school aged children, authors from each of the three children’s 

health cohorts used the Wechsler Intelligence Scale for Children, 4th edition (WISC-IV). The 

instrument measures four areas of mental functioning: the Verbal Comprehension Index, the 

Perceptual Reasoning Index, the Working Memory 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 English and Spanish-speaking children. In the CCCEH 

Mothers and Newborn Study, Rauh et al. (2011) evaluated the 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 including prenatal maternal interview 

data, prenatal chlorpyrifos marker levels from maternal and/or cord blood samples at delivery, 

postnatal covariates, and neurodevelopmental outcome data (Rauh et al., 2011). While models 

were developed using continuous measures of both prenatal chlorpyrifos exposure and Wechsler 

scores, for ease of interpretation, investigators reported that for each 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. In the Mt. Sinai study, prenatal maternal DEP urinary metabolite 

concentrations were associated with slight decrements in Full Scale Intelligence Quotient 

(FSIQ), Perceptual Reasoning, and Working Memory between the ages 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 evidence of an 

association between prenatal exposures to OPs as measured by urinary DAP (total DAP, DEP, 

and DMP) metabolites in women during pregnancy, and decreased cognitive functioning in 

children at age 7. In this study, children in the highest quintile of maternal DAP concentrations 

had a statistically significant 7-point difference in IQ points compared with those in the lowest 

quintile.

To ascertain whether observed differences in neurodevelopment after prenatal chlorpyrifos 

exposure may be explained by differences in brain morphology between exposed groups, 

investigators compared MRI brain images between high and low chlorpyrifos exposed child 

study participants (Rauh et al., 2012). Authors determined there were distinct morphological 

differences in brain areas associated with these neurodevelopmental outcomes. The pilot study 

included 40 child participants due to strict inclusion and exclusion criteria, and the high cost of 

performing the imaging studies on each child. EPA convened a Federal Panel of experts to 
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perform a written peer-review of this study.57 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 needed 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 epidemiologic findings, additional study is needed to make specific 

links with areas of brain development change.

Most recently, CCCEH study authors (Rauh et al., 2015) evaluated the relationship between 

prenatal chlorpyrifos exposure and motor development/movement among 263 of the cohort 

participants who had reached the age of 11 years and had a complete set of data including 

prenatal maternal interview data, prenatal chlorpyrifos marker levels from maternal and/or cord 

blood samples at delivery, postnatal covariates, and motor development/movement outcome data 

(Rauh et al., 2015). When comparing children in the upper quartile of exposure (>6.17 pg/g; 

N=43) to those in the lower quartiles, they observed statistically significant associations between 

prenatal chlorpyrifos exposure and mild to moderate tremor in the arm. These associations were 

observed even after controlling for potential confounding factors such as medication, sex, and 

ethnicity. 

In sum, across these three children’s environmental health studies (CCCEH, Mt. Sinai, and 

CHAMACOS) and in the seven studies identified in the 2015 literature review, authors 

consistently identified associations with neurodevelopmental outcomes in relation to OP 

exposure. There is evidence of delays in mental development in infants (24–36 months), 

attention problems and autism spectrum disorder in early childhood, and intelligence decrements 

in school age children who were exposed to chlorpyrifos or OPs during gestation. Investigators 

reported strong measures of statistical association across several of these evaluations (odds ratios 

2–4 fold increased in some instances), and observed evidence of exposures-response trends in 

some instances, e.g., intelligence measures.

A.3.2 Summary of EPA’s Weight of Evidence Analysis

In 2010, OPP developed a draft “Framework for Incorporating Human Epidemiologic & Incident 

Data in Health Risk Assessment” which provides the foundation for evaluating multiple lines of 

scientific evidence in the context of the understanding of the adverse outcome pathway (or mode 

of action (U.S. EPA, 2010). The draft framework 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 Programme 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 organization (Meek et al., 2014). Consistent with 

recommendations by the NRC in its 2009 report on Science and Decisions, OPP’s draft 

 57 http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPP-2008-0850-0170. 
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framework describes the importance of using problem formulation at the beginning of a complex 

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 reviews represent the problem formulation analyses for the 

weight-of-evidence (WOE) evaluation. The modified Bradford Hill Criteria (Hill, 1965) are 

used to evaluate the experimental support that establishes key events within a mode of action or 

an adverse outcome pathway, and explicitly considers such concepts as strength, consistency, 

dose response, temporal concordance and biological plausibility in a weight of evidence analysis. 

The full weight of evidence evaluation can be found in the 2014 human health risk assessment.

Dose-response relationships & temporal concordance.

Since the MOA(s)/AOP(s) is/are not established for neurodevelopmental outcomes, 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, 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 

developmental chlorpyrifos exposure may occur. Due to the dose selections in most of these in 

vivo studies evaluating effects such as behavior and cognition, it is not known whether such 

adverse effects would be shown at doses lower than those which elicit 10% RBC AChE

inhibition. 

Within the epidemiology studies, the relationship in time between prenatal chlorpyrifos exposure 

and adverse neurodevelopmental outcomes is concordant. Specifically, with regard to the 

children’s environmental health epidemiology studies, each of the three study cohorts utilized a 

prospective birth cohort study design in which mothers were recruited into study prior to the 

birth of the infants and development and identification of adverse effects; therefore, it is known 

with certainty that exposure preceded effect. In addition, because the time period under study 

within these cohorts, and specifically the CCCEH study, spanned the point in time in which 

pesticide 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 period) the period of removal of chlorpyrifos products from the residential marketplace. 

Moreover, prior to the voluntary cancellation there were >80% detectable levels of chlorpyrifos 

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 cancellation of chlorpyrifos in the residential marketplace for whom the cord blood 

chlorpyrifos level was in the upper-tertile of pre-cancellation exposure levels. The significantly 

reduced proportion of measured values greater than the limit of detection as well as the 
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observation of an absence of an association between prenatal chlorpyrifos exposure among 

infants born after the voluntary cancellation of chlorpyrifos support the hypothesis that 

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 infants born after the voluntary cancellation. 

With respect to the timing of exposure, the cord blood and other (meconium) measures from the

CCCEH study provide evidence that exposure did occur to the fetus during gestation. For these 

epidemiology studies, it is important to note that chlorpyrifos was only assessed directly in the 

CCCEH study (Rauh et al., 2006; Rauh et al., 2012), with the Mexican cohort study (Fortenberry 

et al., 2014) assessing the chlorpyrifos metabolite TCPy, and the CHAMACOS cohort study 

(Eskenazi et al., 2007) measuring both TCPy and DAPs. In contrast, all of the other 

epidemiology studies assessed only DAP exposure, and as noted previously these DAPs are 

metabolites of multiple OPs including chlorpyrifos. 

Exposures measured in the range reported in the epidemiology studies (pg/g plasma) are low 

enough that is unlikely to result in AChE inhibition. The urinary TCPy concentrations among 

mothers were comparable to the general population levels measured in NHANES. This 

biomarker data from the CCCEH studies are supported by the agency’s dose reconstruction 

analysis using the PBPK-PD model. In accordance with the recommendation of the FIFRA SAP 

(2012), the agency conducted a 2014 dose reconstruction analysis of residential uses available 

prior to 2000 for pregnant women and young children inside the home. Based on the output 

from the PBPK-PD model, for the highest exposure considered (i.e., indoor broadcast use of a 

1% chlorpyrifos formulation), <10% RBC AChE inhibition in pregnant women and young 

children would be expected from residential uses. It is noteworthy that all estimates of exposure 

based on conservative assumptions lead to predicted AChE inhibition levels <10%.

Strength, Consistency, and Specificity.

EPA notes consistencies across the databases of animal toxicology and epidemiology studies. 

Considering the toxicological and epidemiological data in the context of three major 

neurodevelopmental domains (specifically, cognition, 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 working memory; e.g., Icenogle et al., 2004, BillauerHaimovitch et al., 2009); changes in locomotor activity levels (exploration, rearing; e.g., Levin 

et al., 2002; Ricceri et al., 2003); and altered social interaction (aggression, maternal behavior; 

Venerosi et al., 2006, 2010); and effects on brain morphometrics (MRID 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 or similar measure; Young et al., 2005; Engel et al. 2007; Zhang et al., 

2014), altered social development (e.g., ADHD; Rauh et al., 2006; Bouchard et al., 2010; 

Fortenberry et al., 2014), and changes in MRI brain scans (Rauh et al., 2012). 
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It is notable that the laboratory animal studies vary in experimental designs such as species, 

strain, gender, dosing regimens (age, routes, vehicle), and test parameters (age, protocol). 

Likewise, observational epidemiology studies vary by population characteristics (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 qualitative similarity in research findings is 

striking. 

In contrast, quantitatively, there are notable differences between animals and humans. 

Specifically, in animals, the doses most often used in the behavior studies (1 and 5 mg/kg/day) 

are sufficient to elicit approximately ≥10% brain inhibition and ≥30% in RBC inhibition , 

depending on the study design, age of the animal, and sampling time. Even in one study using a 

lower dose (0.36 mg/kg/d in feed), this level was sufficient to produce a great degree of maternal 

RBC AChE inhibition (Ohishi et al., 2013). There are essentially no animal studies that have 

tested at doses shown to not inhibit AChE under the exposure conditions. In the epidemiology 

studies, based on the comparisons with biomonitoring 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; and 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 important to consider the strength of the statistical 

measures of association between prenatal chlorpyrifos exposure and adverse neurodevelopmental 

outcomes through childhood (epidemiology) and possibly into adulthood (animal studies). It is 

also important to consider the strength of the integrated qualitative and quantitative evidence, the 

consistency of the observed associations across epidemiology studies and considering both 

animal and human data that support the conclusion that chlorpyrifos plays a role in adverse 

neurodevelopmental outcomes. While it cannot be stated 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).
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The CCCEH study, which measures chlorpyrifos specifically, provides a number of notable 

associations. Regarding infant and toddler neurodevelopment, 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 high to low exposure groups (Rauh et al., 2006). 

Notably these decrements in PDI persist even after adjustment for group and individual level 

socioeconomic variables (Lovasi et al., 2011). These investigators also observed increased odds 

of mental delay (OR=2.4; 95% CI: 1.1–5.1) and psychomotor delay (OR=4.9; 95% CI: 1.8–13.7) 

at age three when comparing high to low exposure groups (Rauh et al., 2006). Rauh et al. (2006) 

also reported large odds ratios for attention disorders (OR=11.26; 95% CI: 1.79–70.99), ADHD 

(OR=6.50; 95% CI: 1.09–38.69), and PDD (OR=5.39; 95% CI: 1.21–24.11) when comparing 

high to low chlorpyrifos exposure groups (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 environmental health birth cohorts, decrements in 

intelligence measures were identified in relation to increasing levels of prenatal chlorpyrifos 

exposure. Authors from the CCCEH cohort reported statistically significant decreases of 1.4% in 

full scale IQ and 2.8% in working memory among seven-year olds for each standard deviation 

increase in chlorpyrifos exposure (Rauh et al., 2011). These results persist even when performing 

sensitivity analyses including only those with detectable chlorpyrifos levels. In addition, no 

evidence was provided of mediation by child behavior on the measure of working memory 

instrument. 

Biological plausibility and coherence. The 2012 SAP noted “Questions about biologic 

plausibility due to lack of clarity on mechanism of action, particularly at the low exposure levels 

seen in the cohorts and the mixed results of animal studies showing neurodevelopmental effects.” 

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 the associations observed in epidemiologic studies 

reflects consideration of both exposure-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 chlorpyrifos and/or its oxon are 

biologically active on a number of processes that affect the developing brain. Moreover, there is 
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

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a large body of in vivo laboratory studies which show long-term behavioral effects from early 

life exposure. EPA considers the results of the toxicological studies relevant to the human 

population, as qualitatively supported 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 effects at birth (the Brazelton index), observed an association with 

DAPs exposure (Engel et al., 2007; Young et al., 2005). Similarly, while not consistent by age at 

time of testing (ranging from 6 month to 36 months across the three cohorts), each cohort 

reported evidence of impaired mental and psychomotor 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 relation between the respective prenatal measures 

of chlorpyrifos or DAPs and intelligence measures at age 7 years. 

Overall, the results from the seven studies identified in the 2015 literature review (USEPA, 

2015), further strengthen the observations from the CCCEH study and the other two children 

cohort studies. Specifically, statistically significant associations were observed for: prenatal 

DAP exposure and neonatal development (Zhang et al., 2014); total OP exposure and autism 

spectrum disorders (Shelton et al., 2014); and a positive association between attention and 

behavior problems and total DAPs and DMAPs, but not DEAPs (Bouchard et al., 2010). 

Suggestive evidence of associations were observed for: TCPy and ADHD in boys (Fortenberry et 

al., 2014); and total DEAP exposure and reciprocal social responsiveness among blacks and boys 

(Furlong et al., 2014). No associations were observed in two cross-sectional studies for: 

postnatal child urinary DEAP, DMAP, or total DAP metabolite levels and parentally reported 

behavioral problems in Canadian children (Oulhote and Bouchard, 2013); and for postnatal child 

urinary DAPs and a developmental quotient score for 23–25 month old children from China 

(Guodong et al., 2012). It is noted that with the exception of Fortenberry et al. (2014), which 

measured TCPy exposure, these seven studies did not assess chlorpyrifos exposure directly and 

instead measured DAPs exposure. 

Although uncertainties remain as articulated above, these uncertainties are diminished in the 

context of the qualitative similarity between the databases, and the concern for long-term 

neurodevelopmental effects as a result of prenatal, perinatal and possibly early life exposure.
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Appendix 4.0: Background & Summary of the Chlorpyrifos PBPK Model

[Note: For the 2016 analysis, the agency is only using the PK portion of the PBPK model since 

the PD portion applies only to AChE inhibition.]

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 (PBPK) modelling is a scientifically sound 

and robust approach to estimating the internal dose of a chemical at a target site and as a means 

to evaluate and describe the uncertainty in risk assessments. PBPK models consist of a series of 

mathematical representations of biological tissues and physiological processes in the body that 

simulate the absorption, distribution, metabolism, and excretion (ADME) of chemicals that enter 

the body. Examples of PBPK model applications in risk assessments include interspecies 

extrapolation, intra-species extrapolation, route-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 assessment. These models can also be used to 

evaluate the relationship between an applied dose and biomonitoring data. 

Although the model includes the description of AChE inhibition, in the 2016 analysis, the 

agency is only using the PK portion of the model. The PBPK-PD model for chlorpyrifos that 

was originally developed by Timchalk and coworkers in 2002 (Timchalk et al., 2002a, b) has 

been refined 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 

reviewed 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 

and for the 2016 SAP.

A.4.1 Introduction to the Physiologically-Based Pharmcokinetic/Dynamic Model

Evaluation of PBPK-PD models intended for risk assessments includes a review of the model 

purpose, model structure, mathematical representation, parameter estimation (calibration), and 

computer implementation (USEPA, 2006b). Developers of the chlorpyrifos PBPK-PD model 

sponsored a third-party quality assurance assessment to verify model parameter 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 sources in the model parameters (USEPA, 2014d) 

and has continued to critically evaluate the code and its output. The agency also conducted a 

mass balance analysis. Minor inconsistencies were identified, and developers of the chlorpyrifos 
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PBPK-PD model have since made the corrections, or provided additional references to justify 

their parameterization choices. Recently, the agency performed additional evaluation of the code 

for dermal exposure; this evaluation did not change any aspects of the model code but supported 

agency confidence in the predictions (MRID 49830302). The mathematical equations in this 

model are adequate to reasonably predict both blood/urine dosimetry and ChE inhibition in two 

controlled, deliberate oral human dosing studies (Nolan et al., 1984; Kisicki et al., 1999) and a 

dermal human study (Nolan et al., 1984)58. The PBPK-PD model predictions for rats inhaled 

chlorpyrifos compared well with animal data (Hotchkiss et al., 2013) with respect to 

chlorpyrifos, oxon, and TCPy concentrations in plasma, and ChE in plasma, RBC and brain 

(Poet et al., 2014). 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.

Age-specific parameters are incorporated allowing for lifestage-specific evaluations from 

infancy through adulthood of chlorpyrifos, its oxon, and TCPy levels in various tissues, such as 

plasma and urine. The model can be run in two modes: deterministic and variation. In the 

deterministic mode, the output accounts for human specific metabolism and physiology thus 

obviating the need for the inter-species extrapolation 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 intraspecies extrapolation for infants, toddler, youths, and non-pregnant adults. For this 2016 issue 

paper, the focus is on deterministic evaluation; no probabilistic PBPK runs are provided in this 

issue paper. 

The current model being used by the agency for the chlorpyrifos risk assessment does not 

include gestation or lactational exposure. With respect to lactational exposure, the agency is 

aware of breast milk pilot monitoring data from the Pilot study for the National Children’s Study 

(NCS) Supplemental Methodological Studies (SMS) that can be used to evaluate infant exposure 

to chlorpyrifos through breastfeeding using. Although it would be useful to make predictions of 

breast milk exposures, since there are some (albeit limited) breast milk monitoring data from 

contemporary exposures, the lack of a lactational PBPK model does not introduce substantial 

uncertainty in the risk assessment.

In April 2015, DAS modified the multi-route PBPK/PD model for chlorpyrifos to include 

additional code to describe physiological changes for women during pregnancy. The gestational 

 

58 Two human deliberate dosing studies (Nolan et al. 1982; Kisicki et al., 1999) are 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/meetingmaterials/apr-13-14-2011/appendix1.pdf
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component of the model includes the following modifications: (1) placenta, uterine, and fetal 

compartments, all of which grow over the course of pregnancy; (2) pregnancy-specific changes 

in the fat, rapidly-perfused, and slowly-perfused compartments; (3) pregnancy-specific changes 

in blood composition resulting in increased blood volume and decreased hematocrit; and (4) 

pregnancy-specific changes in metabolism, both CYP450s and PON1 enzymes based on 

published studies. No changes were made to the PD model since there are no data available to 

suggest cholinesterase changes during pregnancy. The growth of fetus, uterus, and placenta 

predicted by the model agreed with empirical data as were the changes in tissue and blood 

volume (Abduljalil, K., et al., 2012). While the modified model reasonably simulate the 

physiological changes during pregnancy, the model’s predictive ability to simulate internal 

dosimetry of chlorpyrifos cannot be properly evaluated since there were no chlorpyrifos-specific 

pharmacokinetic data available during pregnancy. As such, the agency cannot evaluate its 

predictive capacity and thus, the pregnancy model will not be used for risk assessment at this 

time. 

The agency does note, however, that the pregnancy model was built based on the best knowledge 

available and some preliminary simulations using this model suggested that when accounting for 

variability in physiology, In addition, an initial testing of the pregnancy version of the PBPK/PD 

model showed that, given the same oral dose, blood concentrations of chlorpyrifos in women in 

the third trimester were slightly lower than those in non-pregnant women (Appendix 6). While 

some parameter changes during pregnancy contributed to more chlorpyrifos in blood (e.g., 

smaller fat:blood partition during pregnancy) and other changes contributed to less chlorpyrifos 

in blood (e.g., increased tissue volumes for more storage), the combined effects from these 

multiple physiological changes during pregnancy resulted in slightly lower blood concentrations 

of chlorpyrifos during pregnancy. Similarly, RBC AChE inhibition in pregnant women at a 

given dose was comparable to non-pregnant women. 

In addition, data from CCCEH suggest that maternal blood is a reasonable surrogate for cord 

blood. Whyatt et al. (2009) have shown that levels of chlorpyrifos in maternal blood and 

umbilical cord blood levels were highly correlated (r = 0.9, p<0.001, n = 64) with very similar 

values reported at the 90th (maternal and cord levels of 1.5 pg/g and 2.3 pg/g, respectively) and 

95th percentiles (maternal and cord levels of 2.5 pg/g) suggesting that tracking the blood 

concentrations of the mother is a reasonable surrogate for the fetus. In a second paper, Whyatt et 

al. (2003) showed that chlorpyrifos in maternal blood and umbilical cord blood levels were

highly correlated (r = 0.76, p<0.001, n=180 mother-child pairs. Although the agency would 

prefer to have a robust gestational PBPK model parameterized with chlorpyrifos data, the lack of 

such model robust does not add major uncertainty into the 2016 analysis. 
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A.4.2 Summary of Metabolic Profile

The metabolism and pharmacokinetic (PK) profiles 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. Only summary information is provided 

here. Chlorpyrifos undergoes metabolic transformations mainly by the liver microsomal 

enzymes (i.e., cytochrome P450s). The initial metabolic action of chlorpyrifos is desulfuration, 

resulting in bioactivation of the parent compound to the more toxic and potent AChE inhibitor, 

the oxon form. However, the oxon is unstable and is rapidly deactivated through hydrolytic 

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

Simultaneous with the desulfuration process, dearylation acts on both the parent chlorpyrifos as 

well as on the oxon metabolite leading to the release of TCPy. TCPy is further conjugated to 

form glycine or glucuronide conjugates and eliminated into the urine. 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 specific marker for chlorpyrifos. An important 

aspect of the chlorpyrifos PBPK model is that chlorpyrifos and its oxon are approximately 98–

99% protein bound in the blood.

There are several enzymes that have roles in the metabolism and toxicity of chlorpyrifos. In 

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

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

and carboxylesterases (highest levels in liver). These B-esterases function as a scavenger, or 

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

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

such A-esterase is paraoxonase (i.e., PON1). These 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 AChE 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 role in the secondary 

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

urine. 
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Figure A.1. Major metabolic pathways of chlorpyrifos metabolism (Reproduced from Timchalk 

et al, 2002b)

A.4.3 Description & Structure of the Physiologically-Based Pharmcokinetic/Dynamic 

Model

The chlorpyrifos PBPK-PD model includes descriptions of the ADME of chlorpyrifos, and its 

metabolites, oxon and TCPy (Figure A.2). The PBPK-PD model contains descriptions of 

metabolism to account for chlorpyrifos, its oxon, and TCPy in liver, blood, brain, small intestine, 

lungs, diaphragm, and skin. Model parameterization was achieved 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. 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, diaphragm, liver, lungs, 

plasma, and RBC. Because age-specific parameters are incorporated allowing for lifestagespecific evaluations from infant through adulthood, in the deterministic mode, the output 

accounts for human specific metabolism and physiology thus obviating the need for the interspecies extrapolation factor. The deterministic model can be used to simulate an “average 

individual” for all age groups (DAS et al., 2011; Poet et al., 2014; MRID 49074901). 
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Body weight can be calculated at any given age using a modified Gompertz growth function for 

both male and female (Smith et al., 2014). Subsequently, tissue volumes, blood flows to tissue, 

metabolism rates, ChE enzyme activities, and exposure 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 probabilistic capabilities to allow for refining the 

intra-species extrapolation factor. The FIFRA 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 performed additional analyses, including a global 

sensitivity analysis, improvements to the quantitative 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 address variation in response that occurs from 

metabolism (Dow, 2014a, b). Code is available to simulate tissue dosimetry from dermal and 

inhalation exposure routes (Poet et al., 2014). 

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 activities (Figure 4). In addition to age-related 

physiology, the model also incorporates age-specific metabolism based on in vitro data, allowing 

the model to assess changes in metabolism from infancy to adulthood. Total Vmax 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-dependent 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 agespecific Vmax was scaled to age-dependent volume of the 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 extracted from Dow 2011 and MRID 49248201. 
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Figure A.2. PBPK/PD model (Typical Adult model) structure. The shaded compartments denote 

tissues which contain B-esterases (bottom panel). Tissue volumes and enzyme activities (Vmax) 

change with age based on liver and/or blood compartmental 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 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). 
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Table A.5.3.1. 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

CPF → TCPy Km (μM) 55 Poet et al., 2003
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Parameter Value Source

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×BW1.117 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

Acetyl 220 Kousba et al. (2007)
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Parameter Value Source

Enzyme Turnover Rate (hr-1)

Butyryl 1.17x107 Maxwell et al., 1987, Timchalk 

et al., 2002b

Acetyl 3.66x106 Maxwell et al., 1987, Timchalk 

et al., 2002b

Carboxyl 1.086x105 Maxwell et al., 1987, Timchalk 

et al., 2002b

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., 2002b
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Table A.5.3.2. Compartmental Growth.

Compartment Fraction of Body 

Weight Equation1

Source for LifeStage 

Parameters

Eqn. Format

V0+V1×BW+V2×BW2+V3

×BW3

+V4×BW4+V5×BW5+

V6×BW6

(BW: Body Weight in g)

V0 V1 V2 V3 V4 V5 V6

Blood Leucke 9.15e-2 -8.59e-7 1.25e-11 -6.46e-17 - - - Young et al. 2009

Young 8.97e-2 -3.50e-7 6.54e-13

LifeStage 8.970e-2 -3.500e-7 6.540e-13 - - - -

Brain Leucke 1.19e-1 -3.51e-6 4.28 e-11 -1.82 e-16 - - Fit to Valentin et al. 

2002 (bwt≤70) & 

Young et al. 2009 data 

(body weight>70 kg) 

and extrapolated values 

based on Table 4A 

(Young et al. 2009)

Young 1.41e-1 -5.54e-6 9.30 e-11 -6.83e e-16 1.80e-21

LifeStage 1.216e-1 -3.465e-6 4.354e-11 2.463e-16 5.132e-22

Diaphragm Leucke 3.000e-4 - - - - - - Luecke et al. 2007

Young NA - - - - - -

LifeStage 3.000e-4 - - - - - -

Fat*

Female Leucke 5.91e-2 1.20e-5 -5.80e-10 1.12e-14 -6.36e-20 - - Fit to Valentin et al. 

2002 & Lafortuna et al. 

2005 data and 

extrapolated data based 

on equation in Fig 1B 

(Lafortuna et al. 2005)

Young 1.84e-2 -6.86e-6 2.46e-10 -2.11e-15 7.58e-21 -9.94e-27 -

LifeStage 9.217e-02 1.401e-05 -6.787e-10 1.540e-14 -1.558e-19 7.249e-25 1.273e-30
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Compartment Fraction of Body 

Weight Equation1

Source for LifeStage 

Parameters

Male Leucke 3.95e-2 1.59e-5 -6.99e-10 1.09e-14 -5.26e-20 - - Fit to Valentin et al. 

2002 & Lafortuna et al. 

2005 data and 

extrapolated data based 

on equation in Fig 1B 

(Lafortuna et al. 2005)

Young 1.61e-2 -3.59e-6 -8.28e-11 -3.57e-16 4.73e-22 - -

LifeStage 3.484e-2 2.803e-5 -1.422e-9 2.892e-14 -2.718e-19 1.203e-24 -2.036e-30

Liver Leucke 3.49e-2 -3.23e-7 2.13e-12 - - - - Fit to Valentin et al. 

2002 (body weight≤ 

70) & Young et al. 

2009 extrapolated 

values based on Table 

4A

Young 4.25e-2 -1.01e-6 1.99e-11 -1.66e-16 4.83e-22 - -

LifeStage 3.917e-2 -6.789e-7 1.082e-11 -7.393e-17 1.701e-22 - -

Rapid

Lung Leucke 1.67e-2 -9.96e-8 -1.09e-13 1.13e-17 - - - Young et al. 2009

Young 1.860e-2 -4.550e-8 - - - - -

LifeStage 1.860e-2 -4.550e-8 - - - - -

Kidney Leucke 7.31e-3 -8.29e-8 2.13e-12 - - - - Young et al. 2009

Young 7.26e-3 -6.69e-8 3.33e-13 - - - -

LifeStage 7.260e-3 -6.690e-8 3.330e-13 - - - -

Pancreas Leucke 1.17e-3 -1.18e-8 1.81e-13 - - - - Brown et al. 1997 and 

Young et al. 2009

Young 1.48e-3 - - - - - -

LifeStage 1.480e-3 - - - - - -

Spleen Leucke 3.05e-3 -2.09e-8 1.24e-13 - - - - Young et al. 2009

Young 3.12e-3 5.57e-9 - - - - -

LifeStage 3.120e-3 5.570e-9 - - - - -

GI Leucke 1.93e-2 -4.42e-7 9.28e-12 -4.88e-17 - - - Brown et al. 1997

Young NA - - - - - -

LifeStage 1.650e-2 - - - - - -
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Compartment Fraction of Body 

Weight Equation1

Source for LifeStage 

Parameters

Slow

Non-Fat 

adipose*

Leucke NA NA NA NA NA NA NA Fit to Valentin et al. 

2002 & Lafortuna et al. 

2005 fat data/0.8 

(Valentin 2002) data 

and extrapolated data 

based on equation in 

Fig 1A (Lafortuna et al. 

2005)

Young NA NA NA NA NA NA NA

LifeStage 2.044e-1 2.617e-5 -1.542e-9 3.268e-14 -3.116e-19 1.387e-24 -2.35e-30

Muscle Leucke 9.61e-2 -4.88e-6 3.05e-10 -3.62e-15 1.22e-20 - - Fit to Valentin et al. 

2002 & Janssen et al. 

2000 data and 

extrapolated values 

based on Fig 2A 

(Janssen et al. 2000)

Young 9.68e-1 -3.32e-6 1.83e-10 -1.24e-15 - - -

LifeStage 1.251e-1 1.458e-5 -2.927e-10 2.114e-15 -5.250e-21 - -

Skin Leucke 1.07e-1 -3.26e-6 6.11e-11 -5.43e-16 1.83e-21 - - Young et al. 2009

Young 1.03e-1 -2.56e-6 3.68e-11 -2.580e-16 8.620e-22-1.100e-27 -

LifeStage 1.030e-1 -2.560e-6 3.680e-11 -2.580e-16 8.620e-22-1.100e-27 -

Bone Marrow Leucke 5.19e-2 8.06e-7 -1.96e-10 7.63e-15 -1.08e-19 5.14e-25 NA Brown et al. 1997 (red 

only, yellow is in 

adipose)

Young NA NA NA NA NA NA NA

LifeStage 2.100e-2 - - - - - -
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As it typical of PBPK/PD modeling, parameters that have no measured values were calibrated to 

fit time course of blood/tissue concentration data. Following the calibration of the model, 

additional data not used for calibration were used to evaluate the model’s capability to predict 

other datasets and/or scenarios (e.g., different species, different body weight, different dose 

ranges, or different exposure durations). The PBPK-PD model for chlorpyrifos was calibrated 

and evaluated using rat oral data, and human oral and dermal data (Timchalk et al., 2012; Nolan 

et al., 1984; Poet et al., 2014). The rat model was calibrated using the time course of 

cholinesterase inhibition in RBC and brain, as well as chlorpyrifos concentrations in plasma. 

The human model was calibrated using the time course of plasma concentrations of chlorpyrifos 

and TCPy, urine concentrations of TCPy, and cholinesterase inhibition in plasma. The model 

predictions agree better with blood chlorpyrifos and plasma cholinesterase data for oral exposure 

than dermal exposure (Poet et al., 2014). For oral exposure, the model slightly over-predicted 

chlorpyrifos concentrations in blood, and predicted plasma cholinesterase levels agreed with 

data. For dermal exposure, the model was optimized by fitting to the TCPy data (Nolan 1984) 

following a 24-hour dermal exposure. The model over-predicted the chlorpyrifos concentrations 

in blood by five to seven fold between 6 to 24 hours, as well as the inhibition of plasma 

cholinesterase. These results suggested that some additional loss of chlorpyrifos following 

dermal exposure may be possible and not described in the current version of the model. This 

additional loss, however, was not likely to oxon because of the over-prediction of plasma 

inhibition. The model-predicted blood concentration of chlorpyrifos, however, agreed with the 

dermal data at earlier time point (at 2 hour) and after the termination of exposure (at 48 hour). 
Chlorpyrifos Issue Paper: Evaluation of Biomonitoring Data from Epidemiology Studies

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Appendix 5.0: Verification of the lower bound of 90% CI of the estimated 

slope could be used to estimate the 95% lower limit of estimated concentration 

of CPF in cord blood

Mathematical derivation

Let ΔXtrue be the true change in X given a ΔY change in Y of a general form of simple linear 

regression Y = intercept + b*X + ε. The ΔXtrue can be calculated as ΔXtrue = ΔY/btrue, where 

btrue is the true slope. 

If bl is the 95% lower confidence limit (one-sided) of a slope b estimated from a set of data, then, 

there is a 95% confidence that the true slope btrue is greater than bl. Therefore, we have 95% 

confidence that ΔXtrue = ΔY/btrue is greater than ΔY/bl if ΔY is negative.

Simulation

The 95% lower confidence limit of an estimated ΔX associated with a ΔY as described above 

and presented in Table 2. Predicted levels of food exposure to chlorpyrifos from the 2014 revised 

risk assessment (Drew, 2014) and associated maximum and 24 hour blood concentrations in 

adult females was also verified by a simulation. The following is the (defined) equation of a true 

relationship between X and Y:

Y = 4.6 – 0.006*X + ε, 

where ε~N(0, 0.1547) and X follows lognormal distribution with arithmetic mean = 3.17 and 

standard deviation = 4.61. The true value ΔXtrue associated with an arbitrary reduction ΔY = -

0.05 is calculated as -0.05/(-0.006). For this simulation, a sample of 265 data points with the 

relationship described by Rauh et al. (2011) were randomly generated and the slope and its 95% 

lower confidence limit (one-sided) was estimated. The ΔXL = -0.05/(95% lower confidence 

limit of the slope) was compared to the true value ΔXtrue. The simulation was set to have 10,000 

iterations (i.e. 10,000 samples of 265 data points per sample) with the proportion of iterations 

that ΔXL < ΔXtrue computed. If the proportion of iterations that ΔXL < ΔXtrue is about 95%, then 

the logic behind calculating the 95% lower limit of an estimated ΔX associated with a ΔY using 

95% lower confidence limit (one-sided) of the estimated slope is verified. (Note that some 

statistics from Rauh’s 2011 study were used as parameters of the simulation. Note that the 

parameters of simulation (i.e. intercept, slope, ε, range of X, ΔY, etc.) are arbitrary can be 

changed to any other values and the conclusion reached by the simulation is expected to be the 

same.

Result: the proportion of iterations that ΔXL < ΔXtrue is 95.3% which is close to the target 

proportion of 95%. Therefore, the calculation used to derive the results presented in Table 2 of a 

95% lower limit of an estimated ΔX associated with a given ΔY using the 95% lower confidence 

limit is verified.
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SAS code for simulation

%Macro DoseLevel(N =, /* number of subjects in sample*/

NSim=, /* number of iterations in simulation */

AME =, /* arithmetic mean of exposure in old study */

ASDE =, /* standard deviation of exposure (actual scale) of 

old study */

int =, /* intercept of regression line model of log-transformed Y 

*/

slope=,/* effects per unit exposure, i.e. slope of exposure */

SD0=, /* standard deviation of Y at zero exposure */

YDiff=, /* amount change in Y */

seed=);

*==> in log-transformed exposure data;

%let SDE = sqrt(log((&ASDE/&AME)**2 + 1));

%let ME = log(&AME) - 0.5*&SDE**2;

Proc datasets nolist; delete simmer results power; quit;

Data Simmer;

call streaminit(&seed);

do Sim = 1 to &NSim;

do sub = 1 to &N;

*==> create random value to derive exposure of each subject;

random=rannor(&seed);

*==> fix the random value if its value too high;

if random > 3.1 then random = 3.1 + 0.1*rand('uniform');

*==> compute the exposure for each subject;

CPF = exp(&ME + random*&SDE);

logWM = &int + &slope*CPF + rannor(&seed)*&SD0;

output;

end; *sub;

end; *Sim;

run;
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ods select none;

Proc glm data = Simmer ;

by Sim;

model logWM = CPF/solution clparm alpha=0.1;

ods output ParameterEstimates = Parm;

run;

Data Parm;

set Parm;

if Parameter = "CPF";

TrueCPF = &YDiff/&Slope;

CPF_LowerCL = &YDiff/LowerCL;

check_CPF_LB = (CPF_LowerCL < TrueCPF);

run;

ods select default;

Proc means data = Parm mean;

var check_CPF_LB ;

run;

%Mend;

%DoseLevel(N = 265, /* number of subjects in sample*/

NSim=10000, /* number of iterations in simulation */

AME = 3.17, /* arithmetic mean of exposure in old study */

ASDE = 4.61, /* standard deviation of exposure (actual scale) of old study */

int = 4.6, /* intercept of regression line model of log-transformed Y */

slope= -0.006, /* effects per unit exposure, i.e. slope of exposure */

SD0= 0.1547, /* standard deviation of Y at zero exposure */

YDiff= -0.05, /* amount change in Y */

seed=2546);
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Appendix 6.0: Selected Figures from Poet (2015) Multi-Route, Lifestage, and 

Pregnancy PBPK/PD model for Chlorpyrifos and Chlorpyrifos-Oxon 

Submitted to EPA

Figures A.3 and A.4. Predicted chlorpyrifos (A) (Figure A.3) and chlorpyrifos oxon

(B) (Figure A.4) in blood following oral doses from 0.005 to 2.5 mg/kg in pregnant

(solid lines) and non-pregnant (dashed line) women.
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Appendix 7.0: Comparative analysis of NRC evaluation of methyl mercury 

epidemiology studies compared with chlorpyrifos and OP studies

For methyl mercury, there are three epidemiological studies considered for quantitative analyses 

in the IRIS assessment. These longitudinal, developmental studies were conducted in the 

Seychelles Islands, the Faroe Islands, and New Zealand. The subjects of the Seychelles 

longitudinal prospective study were 779 mother-infant pairs from a fish-eating population 

(Myers et al., 1995a-c, 1997; Davidson et al., 1995, 1998). Infants were followed from birth to 

5.5 years of age, and assessed at various ages on a number of standardized neuropsychological 

endpoints. The Faroe Islands study was a longitudinal study of about 900 mother-infant pairs 

(Grandjean et al., 1997). The main independent variable was cord-blood mercury; maternal-hair 

mercury was also measured. The New Zealand study was a prospective study in which 38 

children of mothers with hair mercury levels during pregnancy greater than 6 ppm were matched 

with children whose mothers had lower hair mercury levels (Kjellstrom et al., 1986, 1989). The 

Seychelles study yielded little evidence of impairment related to in utero methylmercury 

exposure, whereas the other two studies found dose-related effects on a number of 

neuropsychological endpoints. 

The methyl mercury RfD is based on a benchmark dose analysis using epidemiology studies on 

different neuropsychological effects in the offspring of exposed mothers at 7 years of age 

(Grandjean et al., 1997; Budtz-Jørgensen et al., 1999a). At 7 years of age, children were tested 

on a variety of tasks designed to assess function in specific behavioral domains such as the 

Continuous Performance Test, Boston Naming Test, and California Verbal Learning Test. The 

cutoff for abnormal response was set at the lowest 5% (5th percentile) of children. Generally 

speaking, children who function at or below approximately the 5th percentile are considered 

significantly developmentally compromised for the ability that is being measured. A BMR of 

0.05 was used in the methyl mercury IRIS assessment, which would result in a doubling of the 

number of children with a response at the 5th percentile of the population. 
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Table A.7.1: Comparison of the major considerations from NRC for methyl mercury and with the chlorpyrifos studies

2000 NRC Report on Methyl mercury Chlorpyrifos & CCCEH study

Susceptible subpopulations

Interindividual toxicokinetic variability; Development of the RfD 

must consider this individual variation; in particular, any biomarkerbased measure should account for the toxicokinetic variability in the 

population. 

Similar to methyl mercury, TK variability needs to be considered.

Toxicodynamic variability

Chlorpyrifos has similar uncertainty as methyl mercury: 

AOP is not known. Thus, TD variability is unknown & cannot be 

quantified

Nutritional deficits The CCCEH cohort is made up of urban, low income population. The 

nutritional status is unknown in the CCCEH mothers.

Measures of exposure

Lack of dietary-intake data in methyl mercury studies

Dermal exposure expected to be primary exposure route from indoor, 

residential use. 

In the CCCEH studies, there is a lack of key exposure information: 

timing & frequency of applications; amount applied; and surface wipe 

samples in the CCCEH studies. 

However, EPA has developed scenarios to make predictions about dermal 

exposure and associated internal dose. The agency’s analysis reasonably 

approximates the indoor exposure to chlorpyrifos prior to the voluntary 

cancelation. 

Thus, there remains uncertainty about actual exposure to the mothers, the 

agency has reasonable estimates of such exposure.

Extrapolation from biomarker Hg content to MeHg intake

Nutritional and dietary confounders and effect modifiers (from 

exposure to fish) Not applicable
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Table A.7.1: Comparison of the major considerations from NRC for methyl mercury and with the chlorpyrifos studies

2000 NRC Report on Methyl mercury Chlorpyrifos & CCCEH study

Co-exposure to other neurotoxicants (e.g., PCBs)

The CCCEH authors were able to measure and model important 

environmental exposures including environmental tobacco smoke (ETS), 

polyaromatic hydrocarbons (PAHs), methylmercury and other ChEinhibiting pesticides such as diazinon and propoxur (Whyatt & Rauh, 

2011). Given the known relation between both lead and also methyl 

mercury and neurodevelopment, the authors performed supplemental 

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 therefore 

not considered a confounding variable in the association of interest within 

the Columbia cohort (Whyatt & Rauh, 2011; Rauh et al., 2006). This 

additional work significantly reduces uncertainty in the CCCEH research 

results.

CCCEH researchers have also evaluated the association between exposure 

to the pyrethroid permethrin and the pyrethroid synergist piperonyl 

butoxide (PBO) and neurodevelopmental outcomes (Horton et al., 2011), 

and observed no statistically significant associations with permethrin. 

The statistically significant associations with PBO were found to be 

independent of those observed with chlorpyrifos.

In addition, it is noted that the CHAMACOS study authors recently 

assessed the relationship between prenatal and postnatal polybrominated 

diphenyl ethers (PBDE) exposure and neurodevelopmental outcomes 

(Eskenazi et al., 2013). They observed associations between PBDE 

exposure and neurodevelopmental outcomes, but concluded that these 

associations were independent of those previously observed in the 

CHAMACOS cohort between OP exposure and child neurobehavioral 

development (Bouchard et al., 2011; Eskenazi et al., 2007; Marks et al., 

2010).
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Table A.7.1: Comparison of the major considerations from NRC for methyl mercury and with the chlorpyrifos studies

2000 NRC Report on Methyl mercury Chlorpyrifos & CCCEH study

Co-exposure to other forms of Hg

As shown in Whyatt et al. (2003), cord blood samples from CCCEH 

mothers included other OPs (diazinon) and other potent AChE inhibiting 

pesticides such as propoxur and carbofuran. The impact of co-exposure to 

AChE inhibiting pesticides is not clear. 

Inability to measure peak exposures: Measurement of cord blood does 

not detect temporal variability in exposure and reflects exposure 

during a period late in gestation. Therefore cord-blood concentrations 

might not correspond to the periods of greatest fetal sensitivity to Hg 

neurotoxicity

Chlorpyrifos has similar uncertainties as methyl mercury.

The temporal nature of exposure is unknown but has been predicted based 

on best available information. 

Without an AOP for neurodevelopmental effects, critical period(s) of 

exposure during development are not known.

Therefore cord-blood concentrations may or may not correspond to the 

periods of greatest sensitivity to chlorpyrifos 

Temporal matching of exposure to critical periods of susceptibility for 

the developing fetal brain: uncertainty in the linkage between the time 

and the intensity of exposure to critical periods of brain development. 

Lack of consideration of other key or most-sensitive health end points

Potential cardiovascular or immune-system effects: 

Neurodevelopmental effects are the most extensively studied sensitive 

end point for MeHg exposure, but there remains some uncertainty 

about the possibility of other health effects at low levels of exposure. 

In particular, there are indications of immune and cardiovascular 

effects, as well as neurological effects emerging later in life, that have 

not been adequately studied.

Across the CCCEH study, other endpoints also being evaluated (obesity, 

asthma, etc). Among OP studies, CHAMACOS has shown effects on 

heart & lung function but using childhood biomarkers, not in utero. A 

study in Ecuador shows autonomic nervous system impacts (i.e. heart rate 

and blood pressure) but at potentially cholinergic toxic internal dose 

levels (Suarez-Lopez et al, 2012; 2013; 2013a).

Given the number of parameters begin measured by CCCEH researchers, 

this is unlikely to be a substantial uncertainty. 

Neurological sequelae (i.e., late-emerging effects)

CCCEH investigators are making observations on the children across time 

(birth to 11 years old). Motor skill deficits observed in Rauh et al. (2006) 

appear to continue in Rauh et al. (2015) with hand tremors while writing.
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