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Chlorpyrifos: Analysis of Biomonitoring Data

Charge to the FIFRA SAP for the April 19-21, 2016 Meeting

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

organophosphate (OP) insecticide. The Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA)

Scientific Advisory Panel (SAP) has previously reviewed the human health effects of chlorpyrifos in 2008 

and 2012 and the chlorpyrifos physiologically-based pharmacokinetic/pharmacodynamic (PBPK/PD) 

model in 2011. At the 2008 and 2012 meetings of the SAP, the agency presented information on a 

variety of science issues such as: inhibition of the enzyme acetylcholinesterase (AChE) in the nervous 

system; epidemiology studies in infants and children which suggest that chlorpyrifos and other OPs 

impact neurodevelopment; and 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. Like other OPs, chlorpyrifos binds to and phosphorylates the enzyme 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. As recommended by the FIFRA SAP in 

2008 and 2012, the agency used inhibition of AChE as the critical effect to derive points of departure

(PoDs) for the 2014 human health risk assessment. However, the 2014 human health risk assessment 

identified uncertainty in the degree to which PoDs derived from AChE inhibition are protective for 

neurodevelopmental effects in humans. 

In 2008 and 2012, the FIFRA SAP cautioned the agency against using the biomonitoring data from 

epidemiology studies, particularly those from Columbia University (denoted as CCCEH herein) in this 

case, to directly derive PoDs due to uncertainties associated with a lack of knowledge about timing of 

indoor chlorpyrifos applications and a single measure of exposure (cord blood) which were collected by 

the Columbia researchers. 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 

study, to inform the dose-response 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 (2012 SAP report).” 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

(2012 SAP report).” The agency has followed up on the SAP recommendations to use the PBPK model to 

further assess the epidemiological data. 

Based on human health risks identified in the 2014 human health risk assessment, the agency published 

a 2015 proposed tolerance revocation for chlorpyrifos; in that proposal the agency noted that the 

evaluation of the available biomonitoring was continuing. 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. In any case, at this point in time, the agency’s 

analysis of biomonitoring data from cord blood collected as part of the Columbia University 

epidemiology study has progressed to a point where peer review would be useful. Specifically, the 

agency has done additional characterization of the pharmacokinetic profile of simulated exposures for 
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the CCCEH cohort from oral and post-application residential dermal exposures using the PBPK model. 

The agency is soliciting comment from the SAP on the evaluation of biomonitoring data using the PBPK 

model, proposed PoDs and extrapolation/uncertainty factors, and examples of a proposed approach to 

use the PBPK model to simulate internal doses from current exposure patterns from drinking water, 

food, and worker exposure. 

Question 1 – Lifestages for Consideration (Section 4) 

Fetuses may be exposed to chlorpyrifos through the mother, while infants and children are exposed 

directly through dietary exposure (food and drinking water). The agency has conducted case studies to 

use cord blood data as a surrogate to evaluate the health impacts on fetuses and infants from exposures 

to chlorpyrifos (Section 6). Based on the available data, there are several assumptions that are being 

made in order to assess exposure for specific lifestages. 

1.a. Fetal exposure – Without a gestational model that is parameterized with chlorpyrifos information, 

fetal exposure cannot be assessed directly. However, maternal and cord blood concentrations of 

chlorpyrifos from CCCEH are highly correlated (Figure 1), and preliminary evaluation of the Dow 

gestational PBPK model suggests little differences in blood levels between pregnant and non-pregnant 

women. Therefore, the agency has concluded that the parameterized model which is available for 

females (13-49 years old) can be used as a reasonable surrogate for estimating fetal exposure. Please 

comment on the agency’s proposal to use female blood levels as a surrogate for fetal exposure. 

1.b. Infant (<1 year old) exposure – Studies of chlorpyrifos in laboratory animals do not suggest any 

specific critical period or lifestage, but instead suggest both pre- and post-natal periods of susceptibility. 

In contrast, there are limited epidemiological evidence regarding postnatal exposure to chlorpyrifos or 

other OPs to infants and children. Because brain development continues through childhood, and due to 

the concern that AChE inhibition may not be protective of the neurodevelopment outcomes, the agency 

is proposing to use the chlorpyrifos cord blood data from CCCEH as the most relevant source of 

information for deriving a PoD for infants (see Appendix 4.0 for further details). Please comment on the 

agency’s proposal to use cord blood data as a surrogate for assessing infant exposure. 

1.c. Children (ages 1<2 years old) exposure – At this time, the agency has not included a case study for 

evaluating the health impacts on children 1<2 years old. However, the agency is aware that this age 

group often has the highest exposure from food consumption (Section 4), as is the case for some food 

commodities for chlorpyrifos exposure. Children 1<2 years old have not yet been included in the case 

studies as these ages are temporally removed from gestational exposure; as such the relevance of the 

cord blood data to predict the outcomes in toddlers is unclear. Please comment on the strength and 

uncertainties of using the CCCEH cord blood data as a surrogate for assessing (children ages 1<2 years

old) exposure to chlorpyrifos. 

 

Question 2 - Uncertainties with Using Biomarker Data from CCCEH for the PoD (Section 7.1) 

Section 7.1 describes the key uncertainties in using the cord blood biomonitoring data from CCCEH as 

the PoD. 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. Chlorpyrifos in blood represents a snapshot of the 

concentration at a particular point in time. Uncertainty also exists when establishing a quantitative 

relationship between chlorpyrifos concentrations in blood and adverse health outcomes. For 
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neurodevelopmental effects investigated in these epidemiologic studies, the adverse outcome 

pathways, toxic moieties, and biological targets were all unknown. 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 addition, 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 

days/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. Please comment on the agency’s

characterization of the uncertainty associated with using the CCCEH blood data in quantitative risk 

assessment. 

Question 3 - Pharmacokinetic (PK) Time Course: Considerations for Labor & Delivery (Section 5) 

Figure 2a-b (pg. 17-18) provides an example PK profile for chlorpyrifos for current exposures to pesticide 

applicators. Similar figures for food, water, and residential exposures are shown throughout the issue 

paper (Sections 6 and 9). As shown in Figure 2a, each PK 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 how rapidly 

chlorpyrifos is initially metabolized. The periods of rapid increase represent rapid uptake during 

activities that lead to chlorpyrifos exposures, while the periods of rapid decrease 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. Upon cessation of the exposure, the terminal halflife (approximately 120 hours) predominates resulting in an asymptotic appearance for the internal 

dosimetry. 

As summarized in Section 5, for deriving the proposed PoDs (Section 7), the agency is assuming the 

CCCEH levels do not represent values within the rapid increase/decrease phase. Instead, the agency is 

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

terminal clearance period (and thus unlikely changing significantly across several days). Although part of 

labor is spent at home (where exposure is assumed to occur), some portion was spent in the hospital—

meaning removal from the apartment caused the exposure to cease. This assumption is being made 

because labor and delivery typically requires multiple hours. Moreover, maternal blood samples for 

some mothers were taken up to two days after delivery. The agency also notes that the results from the 

agency’s exposure characterization analysis of the CCCEH (Section 6) closely match those from the 

CCCEH study, providing further support for the agency’s characterization of the PK profile. Please 

comment on the agency’s characterization of the PK profile and interpretation of the CCCEH 

biomonitoring data. Please include in your comments the agency’s proposal to use the 10 hour and 

24 hour post-peak time points on the PK profiles for assessing risk to chlorpyrifos.
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Question 4 - Evaluation of CCCEH Cord Blood Data & Predicted Exposures to the Cohort (Section 6)

The agency has used the PBPK model to predict blood levels in women across several exposure 

scenarios for comparison with the cord blood levels reported by the CCCEH (Section 6). Food exposure is 

expected to have occurred (Section 6.2), whereas drinking water exposure was unlikely (Section 6.1). 

Given the lack of specific CCCEH exposure information, the agency has developed six possible residential 

exposure scenarios representing a broad range of residential post-application exposures to chlorpyrifos 

products available prior to the voluntary cancellation of indoor products in 2000 (Section 6.3). Two 

exposure scenarios were conducted using EPA standard residential 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. These six possible residential exposure scenarios were input into the PBPK model to 

predict a range of potential exposures for comparison to the predicted internal dosimetry levels 

reported by the CCCEH investigators. Based on the results of these simulations, the agency has 

concluded that: 1) the reported higher blood levels in the CCCEH from 1998-2000 are likely driven 

primarily by residential use of the broadcast and perimeter chlorpyrifos products registered for use at 

that time; and 2) these results further support the reasonableness of the magnitude and distribution of 

data reported by CCCEH. Please comment on the agency‘s conclusions that these scenarios 

adequately capture the range of exposure. Please also comment on the agency’s simulations from 

residential and food exposures and the degree to which the estimates of internal blood levels do or do 

not match the CCCEH cohort results before and after the cancellation of indoor products in 2000. 

Question 5 - Options for Deriving a PoD for Neurodevelopmental Outcomes Based on the CCCEH 

Biomonitoring Data (Section 7.2) 

As summarized in Section 7.2, the agency has proposed a PoD for the observed neurodevelopmental 

effects and offered alternative options based on internal blood concentrations of chlorpyrifos from the 

results of the Columbia University study. 

5.a. Approach to Using the Cord Blood - The agency could consider continuing to use the AChE PoDs 

and apply additional factors over and above the Food Quality Protection Act (FQPA) 10X Safety Factor to 

reflect the level of uncertainty of protecting for neurodevelopmental outcomes when using AChE for the 

PoD. However, the agency would still need to quantify the difference between effects from AChE 

inhibition and from neurodevelopmental outcomes—and the analysis to evaluate the appropriate 

additional factor(s) would again require the agency to make quantitative use of the CCCEH cord blood 

data with the same uncertainties described above. The agency has elected to propose to use the cord 

blood directly as the PoD as the simpler, more understandable approach. Please comment on the 

agency proposal to use cord blood directly as the PoD. 

5.b. PoD Options – From the CCCEH publications, there are two general options that EPA has considered 

for deriving a PoD for extrapolating risk to chlorpyrifos: 1) Lower limit of the top tertile (>6.17 pg/g cord 

blood) derived from Rauh et al (2006) and repeated in other CCCEH publications; or 2) Benchmark Dose 

(BMD) estimates derived from linear regression reported in Rauh et al (2011) for deficits in Working 

Memory. Rauh et al (2011) 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. The agency has 

decided to use the BMD approach. Please comment on the PoD options considered by agency. 
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5.c. Agency’s Proposal for PoD – The agency proposal applies the BMD approach to the Rauh et al 

(2011) study, and the agency has selected a 2% change in working memory or an internal dose of 2.16 

pg/g as the PoD. This agency proposed value is quantitatively near the value reported by Rauh (2.8%

reduction in working memory) and thus supported by the existing data, but is still health protective and 

conservative. Please comment on the analysis/calculations used to derive these estimates as 

described in Appendix 6 and the selection of a 2% response level. 

Question 6 - Assessing Extrapolation/Uncertainty (Section 8)

In typical risk assessments, PoDs are derived directly from laboratory animal studies and inter- and intraspecies extrapolation is accomplished by the use of default 10X factors. In the case of chlorpyrifos, the 

proposed PoDs are derived from human information obviating the need for the inter-species 

extrapolation. However, the agency still needs to consider intraspecies extrapolation of the PoD from 

the CCCEH epidemiology data across the diverse human population (Section 8.1). 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” (Section 

8.2). 

6.a. Intra-species extrapolation – 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 previously by the EPA IRIS program for methyl mercury (Appendix 

8). Please comment on the agency’s scientific rationale of the proposed use of a 10X intraspecies 

extrapolation factor. 

6.b. Pre- vs. Post-natal Exposure – Numerous epidemiological investigations have observed a link 

between prenatal exposure to chlorpyrifos or OPs and adverse effects on neurodevelopment through 

age seven years, with additional more limited evidence up through approximately age eleven years. By 

contrast, epidemiological evidence is more limited for associations between postnatal exposure to 

chlorpyrifos or other OPs and neurodevelopmental effects, and the CCCEH study has specifically not 

assessed these associations. Therefore, given that the extensive experimental laboratory animal 

database suggests that the postnatal period is a potential susceptible time, the lack of postnatal 

exposure assessment in the CCCEH and other similar cohort studies is a source of uncertainty in the 

epidemiology database. Please comment on the agency’s conclusion that the lack of postnatal 

exposure assessment in the CCCEH study is a source of uncertainty in the epidemiology database. 

6.c. Impact of Sample Size on CCCEH Findings – 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 at 

ages 24-36 months, and the observation of intelligence decrements at age seven years were seen across 

the three US childrens 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 exposure to 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. Please comment on the 

agency’s conclusion that the moderate sample size of the CCCEH study is a source of uncertainty, 

given that the agency is proposing to use the CCCEH study data directly for setting a PoD.
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Question 7 - Proposed Approach to Deriving Internal Dose Estimates: Integration of Exposure 

Assessment & PBPK Modeling (Section 9)

The agency has proposed to input exposure estimates for chlorpyrifos into the PBPK model to assess 

internal blood concentrations from current exposure patterns. Several case examples were provided in 

the draft issue paper representing food exposures (Section 9.2), drinking water (Section 9.3), and worker 

exposure (Section 9.4). [Note: Exposure assumptions used in these examples have been previously 

reviewed by other SAP panels.] Please comment on the implementation of the PBPK model using such 

exposure inputs and interpretation of respective simulated blood levels.