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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON, D.C. 20460 

OFFICE OF

CHEMICAL SAFETY AND 

POLLUTION PREVENTION

MEMORANDUM

Date: September 15, 2015

SUBJECT: Literature Review on Neurodevelopment Effects & FQPA Safety Factor 

Determination for the Organophosphate Pesticides

PC Code: See Below DP Barcode: 331251

Decision No.: Registration No.: None

Petition No.: None Regulatory Action: None

Risk Assessment Type: None Case No.: None

TXR No.: None CAS No.: See Below

MRID No.: None 40 CFR: None

 

 

FROM: Anna Lowit, Ph.D., Senior Scientist 

Khin Swe Oo, MD, DABT, Toxicologist

Elizabeth Holman, DrPH, Physical Scientist

Health Effects Division (7509P)

Office of Pesticide Programs

Virginia C Moser, PhD, DABT, Fellow ATS, Toxicologist

Toxicity Assessment Division

Office of Research and Development

THROUGH: Dana Vogel, Director

Health Effects Division (7509P)

TO: Kelly Ballard, Chemical Review Manager

Neil Anderson, Branch Chief

Risk Management and Implementation Branch 1

Pesticide Re-Evaluation Division (7508P)

This paper supports the use of the 10X FQPA Safety Factor in the individual organophosphate 

human health risk assessments. 
Page 2 of 101

Chemical PC Code CAS No. 

Dicrotophos 035201 141-66-2

Fosthiazate 129022 98886-44-3

Coumaphos 036501 56-72-4

Terbufos 105001 13071-79-9

Profenofos 111401 41198-08-7

Bensulide 009801 741-58-2

Diazinon 057801 333-41-5

Ethoprop 041101 13194-48-4

Dimethoate 035001 60-51-5

Malathion 057701 121-75-5

Phosmet 059201 732-11-6

Chlorethoxyfos 129006 54593-83-8

Acephate/

Methamidiphos

103301/ 

101201

30560-19-1/ 

10265-92-6

Pirimiphos-methyl 108102 29232-93-7

TCVP 083701 961-11-5

Tribufos 074801 78-48-8

Phorate 057201 298-02-2

Phostebupirim 129086 96182-53-5

DDVP 084001 62-73-7

Naled 034401 300-76-5

Trichlorfon 057901 52-68-6

Fenamiphos 100601 22224-92-6

AZM 058001 86-50-0

Methidathion 100301 950-37-8

Propetamphos 113601 31218-83-4

ODM 058702 301-12-2

Disulfoton 032501 298-04-4

Methyl parathion 053501 298-00-0

Temephos 059001 3383-96-8

Chlorpyrifos-methyl 059102 5598-13-0
Page 3 of 101

September 15, 2015

D331251

Office of Pesticide Programs

US Environmental Protection Agency
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Table of Contents

1.0 Introduction and Background ...................................................................................... 6

2.0 Literature Review........................................................................................................ 8

2.1 Developmental Neurotoxicity (DNT) Research on OPs other than Chlorpyrifos: 

Laboratory Animal Studies .......................................................................................... 8

2.1.1 Literature Search Strategy & Results.............................................................................. 9

2.1.2 Integration of Literature: Neurobehavioral Domains.................................................. 11

2.1.3 Integration with AChE Inhibition .................................................................................. 22

2.1.4 Summary of Findings from the Developmental Neurotoxicity (DNT) Guideline Studies

.................................................................................................................................. 23

2.1.5 Conclusions on In Vivo Laboratory Animal Studies ...................................................... 24

2.2 Epidemiology Research on OPs other than Chlorpyrifos............................................. 25

2.2.1 Overview of 2012/2014 and 2015 Literature Reviews................................................. 25

2.2.2 Literature Search Methodology.................................................................................... 25

2.2.3 Breadth and Depth of the 2015 Literature Review ...................................................... 27

Table 2.2-aStudy Designs, Exposure Measurement Methods, and Outcome Measurement 

Methods Used across the 31 2015 Review Studies..................................... 29

Table 2.2-bDetailed Neurological Outcome Measurement Methods Used across the 31 

2015 Review Studies.................................................................................... 31

2.2.4 Considerations for Study Quality Evaluation................................................................ 33

2.2.4.1 Study Designs...................................................................................................... 33

Table 2.2.4-1 Study Quality Considerations ...................................................................... 33

2.2.4.2 Exposure Measures............................................................................................. 37

Table 2.2.4-2 CDC Table of Organophosphate Pesticides and Their Dialkyl Phosphate 

Metabolites (2008) ...................................................................................... 39

2.2.4.3 Neurological and Other Outcome Measures...................................................... 40

2.2.4.4 Statistical Analysis............................................................................................... 40

2.2.4.5 Confounding........................................................................................................ 41

2.2.4.6 Risk of Bias........................................................................................................... 41

2.2.5 Review of Quality Results ............................................................................................. 42

2.2.5.1 “High” Quality Group .......................................................................................... 42

Table 2.2.5-1. High Quality Studies: Summary of Study Design Elements Impacting Study 

Quality Assignment...................................................................................... 43

2.2.5.2 “Moderate” Quality Group ................................................................................. 45

Table 2.2.5-2. Moderate Quality Studies: Summary of Study Design Elements Impacting 

Study Quality Assignment............................................................................ 46

2.2.5.3 “Low” Quality Group ........................................................................................... 49

2.2.6 Assessment of Epidemiological Studies for Relevance to Analysis .............................. 49

2.2.7 Birth Outcome Epidemiologic Studies.......................................................................... 50

2.2.8 Summary of Epidemiology Studies from 2015 Literature Review Focusing on 

Neurodevelopmental Outcomes.............................................................................. 51

2.2.9 Summary of Three US Children’s Cohort Studies (CCCEH, CHAMACOS, Mt. Sinai): 
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Focusing on Neurodevelopmental Outcomes Evaluated in 2012/2014 .................. 54

2.2.9.1 Overview ............................................................................................................. 54

2.2.9.2 Review of Study Design and Research Methods................................................. 55

Table 2.2.9Detailed Summary Tables of Children’s Environmental Health Epidemiology 

Studies (extracted from USEPA, 2014) ........................................................ 59

2.2.10 Summary & Discussion.................................................................................................. 74

3.0 Weight of Evidence Analysis: Integration Across Multiple Lines of Evidence .............. 76

3.1 Dose-Response Relationships & Temporal Concordance ............................................ 76

3.2 Strength, Consistency & Specificity............................................................................ 77

3.3 Biological Plausability & Coherence ........................................................................... 79

4.0 10X FQPA Safety Factor for Infants and Children........................................................ 80

5.0 References................................................................................................................ 81

6.0 Appendices............................................................................................................... 90

Appendix 1. Table of In Vivo Developmental Neurotoxicity Studies of OPs.................... 91

Appendix 2. Summary of Guideline DNT Studies Submitted to the Agency for OPs other 

than Chlorpyrifos. ..................................................................................................... 95

Appendix 3. Low Quality Studies: Summary of Study Design Elements Impacting Study 

Quality Assignment................................................................................................... 98
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Literature Review on Neurodevelopment Effects & FQPA Safety Factor 

Determination for the Organophosphate Pesticides 

1.0 Introduction and Background

Organophosphate pesticides (OPs), widely used in agricultural and household pesticidal 

applications, act by inhibiting acetylcholinesterase (AChE) in nerve cells. Historically the agency 

has used inhibition of AChE as the point of departure for OP human health risk assessments

(HHRAs). This science policy is based on decades of work which shows that AChE inhibition is 

the initial event in the pathway to acute cholinergic neurotoxicity. AChE inhibition is most 

often used as the regulatory endpoint for deriving points of departure (PODs) for the single 

chemical OP HHRAs. In addition, because OPs share the ability to inhibit AChE via 

phosphorylation of the active site of the enzyme leading to accumulation of acetylcholine and 

ultimately neurotoxicity, this class of pesticides is subject to assessment of cumulative risk 

(USEPA, 1999; 2006). 

Newer lines of research on OPs in the areas of potential modes of action/adverse outcome 

pathways (MOAs/AOPs),

1

in vivo animal studies, and notably epidemiological studies in 

mothers and children, have raised uncertainty about the agency’s risk assessment approach 

with regard to the potential for neurodevelopmental effects in fetuses and children. Many of 

these studies focus on chlorpyrifos and have been the subject of review by the agency over the 

last several years. 

Specific to chlorpyrifos, 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 2010a,b). 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 

 

1 Mode of action (MOA) and adverse outcome pathways (AOPs) describe a set of measureable key events that 

make up the biological processes leading to an adverse outcome and the causal linkages between such events. 
Page 7 of 101

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 2012). 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.

2

 In December, 2014, the agency 

released “Chlorpyrifos: Revised Human Health Risk Assessment for Registration Review” which 

included the use of a physiologically-based pharmacokinetic/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 2014). 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, 2014).

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, 2012) and updated for the 2014 

chlorpyrifos revised HHRA (USEPA 2014). In short, multiple biologically plausible hypotheses

and pathways are being pursued by researchers including: AChE as a morphogen; cholinergic 

system; endocannabinoid system; reactive oxygen species; serotonergic system; tubulin, 

microtubule associated proteins and axonal transport. However, no one pathway has sufficient 

data to be considered more plausible than the others. Among the available studies, there are 

effects which are either as sensitive as or more sensitive than AChE inhibition. The fact that 

there are, however, sparse data to support the in vitro to in vivo extrapolation, or the 

extrapolation from biological perturbation to adverse consequence, significantly limits their 

quantitative use in risk assessment. The SAP concurred with the agency in 2008 and 2012 

about the lack of definable key events in a MOA/AOP leading to developmental 

neurobehavioral effects. Since the 2014 literature review, there are no substantive changes in 

the ability to define and quantify steps in an MOA/AOP leading from exposure to effects on the 

developing brain. The lack of an established MOA/AOP makes quantitative use of the 

epidemiology study in risk assessment challenging, particularly with respect to dose-response, 

critical duration of exposure, and window(s) of susceptibility. The agency will continue to 

monitor the scientific literature for studies on the AOP for neurodevelopmental effects but this 

document does not include an updated literature review on this line of evidence.

This document (Section 2.0) provides the literature review of in vivo laboratory animal studies 

and epidemiology studies for OPs other than chlorpyrifos to support the single chemical HHRAs. 

It also provides an integrated weight of evidence (WOE) analysis for all the OPs to support 

retention of the 10X FQPA Safety Factor (Section 3.0).

 

2 http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPP-2008-0850-0170
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2.0 Literature Review

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 HHRAs to inform regulatory decision making3. The NRC defines 

systematic review as "a scientific investigation that focuses on a specific question and uses 

explicit, pre-specified scientific methods to identify, select, assess, and summarize the findings 

of similar but separate studies”.

4

 Consistent with NRC’s recommendations, EPA’s Office of 

Chemical Safety and Pollution Prevention (OCSPP) is currently developing systematic review 

policies and procedures. In short, OCSPP employs fit-for-purpose systematic reviews that rely 

on standard methods for collecting, evaluating and integrating the scientific data supporting 

the agency’s decisions. The literature review described here uses concepts consistent with 

systematic review such as detailed tracking of search terms and which literature have been 

included or excluded. 

2.1 Developmental Neurotoxicity (DNT) Research on OPs other than Chlorpyrifos: 

Laboratory Animal Studies

The literature on neurobehavioral effects of developmental exposure to chlorpyrifos was 

summarized and discussed at the 2012 FIFRA SAP. More recent studies were added to this 

summary for the 2014 revised HHRA. At that time, the conclusions were that the animal 

studies clearly showed neurobehavioral outcomes following developmental exposure to 

chlorpyrifos, but there were inconsistencies in the types of effects reported (neurological 

domain altered, direction of change, gender specificity). Furthermore, the studies were 

conducted with doses that most likely produced at least some amount of AChE inhibition at 

some time during the exposure based on results of guideline studies submitted for registration. 

The impact of these observations has lead the agency to evaluate whether or not these 

conclusions extend to other OP pesticides. In this review, the studies of a number of OP 

pesticides are summarized. 

The search aimed to focus on rodent studies involving prenatal/perinatal exposure to OPs in 

which the offspring were evaluated with in vivo neurobehavioral tests. The search methods 

and analytical scope for this analysis are consistent with the chlorpyrifos analysis from the 2012 

FIFRA SAP and 2014 HHRA. Preweaning measurements of behavioral development were noted 

but not compiled, since these could reflect effects of current exposure to the pesticide rather 

than long-term neuronal changes. Information on AChE inhibition in either fetuses/pups or 

dams during this exposure period was evaluated where available. Sections 2.1.1-2.1.3 describe 

the studies from the open scientific literature. Section 2.1.4 summarizes relevant results from 

the DNT guidelines studies submitted for pesticide registration (US EPA guideline 870.6300 

and/or OECD guideline 426). 

 

3 NRC 2011. “Review of the Environmental Protection Agency’s Draft IRIS Assessment of Formaldehyde” ; 

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

4 http://dels.nas.edu/Report/Review-Integrated-Risk/18764
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2.1.1 Literature Search Strategy & Results

To review and evaluate the developmentally neurotoxic effects of other OPs, a search of the 

open literature was undertaken. Due to the limited number of studies available, the agency did 

not limit the search to currently registered OPs. The agency is aware that some OPs listed 

below are no longer registered for use in the US. In addition, the data evaluation records 

(DERs) for existing guideline DNT studies were collected from OPP files and summarized. The 

literature search strategy was developed and conducted by a US EPA reference librarian. 

Databases searched were PubMed, Web of Science (WoS) and ScienceDirect using key words 

described below. Duplicates were eliminated after the total database was generated.

1. PubMed (751 results)

(((((organophos* OR Cholinesterase Inhibitors OR acetylcholinesterase inhibitor))) AND 

((prenatal OR neonatal OR perinatal OR in utero OR fetal OR foetale OR newborn OR infant* OR 

postnatal OR gestational OR pregnancy[MeSH Terms])))) AND ((neurodevelop* OR attention OR 

birth outcome* OR health outcome* OR cognitive OR cognition OR developmental disability* 

OR fetal growth OR fetal growth OR foetal development OR fetal development OR sex OR social 

OR intelligence OR memory OR neurological functioning OR psychomotor OR neurotoxicity OR 

neurobehavior OR behavior OR learning OR Nervous System[MeSH Terms]) OR Neurotoxicity 

Syndromes[MeSH Terms]) AND ((((((guinea pigs[MeSH Terms]) OR rabbits[MeSH Terms]) OR 

mice[MeSH Terms]) OR rats[MeSH Terms]) NOT fishes[MeSH Terms])

2. Web of Science (427 results)

#5 #4 AND #3 AND #2 AND #1 

DocType=All document types; Language=All languages; 

#4 TS=(guinea pig* OR rabbit* OR mice OR mouse OR rat* OR rodent*) NOT TS=(fish*)

DocType=All document types; Language=All languages; 

#3 TS=(neurodevelop* OR attention OR birth outcome* OR health outcome* OR cognitive 

OR cognition OR developmental disabilit* OR fetal growth OR fetal growth OR foetal 

development OR fetal development OR sex OR social OR intelligence OR memory OR 

neurological functioning OR psychomotor OR neurotoxicity OR neurobehavior OR behavior OR 

learning OR Nervous System OR Neurotoxicity Syndromes) 

DocType=All document types; Language=All languages; 

#2 TS=(prenatal OR neonatal OR perinatal OR in utero OR fetal OR foetale OR newborn OR 

infant* OR postnatal OR gestational OR pregnan*) 

DocType=All document types; Language=All languages; 

#1 TS=(organophos* OR Cholinesterase Inhibitors OR acetylcholinesterase inhibitor OR 

chlorpyrifos) 

DocType=All document types; Language=All languages; 

3. Science Direct (19 results)

(ALL(organophos* OR Cholinesterase Inhibitors OR acetylcholinesterase inhibitor OR 

chlorpyrifos) and ALL(prenatal OR neonatal OR perinatal OR in utero OR fetal OR foetale OR 
Page 10 of 101

newborn OR infant* OR postnatal OR gestational OR pregnan*)) AND (neurodevelop* OR 

attention OR birth outcome* OR health outcome* OR cognitive OR cognition OR developmental 

disabilit* OR fetal growth OR fetal growth OR foetal development OR fetal development OR sex 

OR social OR intelligence OR memory OR neurological functioning OR psychomotor OR 

neurotoxicity OR neurobehavior OR behavior OR learning OR Nervous System OR Neurotoxicity 

Syndromes) AND (ALL(guinea pig* OR rabbit* OR mice OR mouse OR rat* OR rodent) and not 

ALL(fish*)). 

This broad literature search identified 1012 potential papers, which were reviewed individually. 

Specific criteria were applied to select suitable studies, as was previously done with 

chlorpyrifos. Since the literature on chlorpyrifos has been previously reviewed, those papers 

were excluded here. This resulted in 19 relevant papers with the following specifications:

 Exposure occurred during gestation and/or the postnatal time frame, ending no later 

than weaning. 

 Dosing included maternal and/or pup administration. 

 Dosing was via oral or subcutaneous injection. One paper with intracisternal injection 

was excluded. 

 Behavioral testing of the offspring occurred after weaning and/or into adulthood. 

 Studies involved only single-chemical exposure, and where two or more chemicals were 

administered together, only the single-chemical data were included in the summaries. 

 Test subjects were rats or mice. Several papers in pigs and rabbits were excluded due to 

the lack of comparative database for those species. 

 The test measures of interest were neurobehavioral endpoints. At least two studies 

involved only electrophysiological measures, and those were excluded. No

neurochemical, genomic, or other molecular endpoints were included. 

The OPs examined, and the number of papers for each, are listed below. Of particular interest 

are studies from one laboratory (Duke University) that included parathion and diazinon, and 

can be directly compared to studies with chlorpyrifos using similar experimental designs. The 

majority of studies used rats (13), and exposures periods varied about evenly between 

gestational and postnatal stages.

 Parathion (5) 

 Diazinon (5)

 Methyl parathion (3) 

 Methamidophos (2) 

 Chlormephos (1)

 Dichlorvos (1)

 Fenitrothion (sumithion) (1)

 Oxydemeton-methyl (demeton-Smethyl, metasystox-R) (1)

These papers dated back to 1968, and there was a wide range in study quality. Shortcomings 

were noted in almost all papers, including cursory methodological information and 

presentation of results, inappropriate statistical analyses, contradictory statements, and 

problematic interpretation of the data. Regardless, the literature is summarized below in terms 

of the functional domains organized by each neurobehavioral evaluation. 
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Below are study descriptions and summaries organized by neurological domain. Appendix 1 is 

an overall summary for each chemical, presenting each endpoint and outcome. 

2.1.2 Integration of Literature: Neurobehavioral Domains

Cognition

Fifteen studies measured some aspect of cognition: tests included mazes (radial arm maze, 

Lashley maze, T-maze spontaneous alternation, M-maze), conditioned response (passive 

avoidance, conditioned avoidance, operant responding, T-maze), and recognition (novel 

object). Most of these showed adverse effects of OP exposure, although not always in a 

consistent or dose-responsive manner.

Rats treated with diazinon (0.5, 2 mg/kg/d, postnatal day (PND) 1-4) showed no differences 

alternating in a T-maze (Timofeeva et al., 2008a). The same rats were tested several months 

later in a radial arm maze, and showed increased working memory errors (both males and 

females), but only at the low dose (0.5 mg/kg/d) with no effect on reference memory 

performance. Diazinon (1 mg/kg/d) was given to rats on gestational day (GD) 15-18 or PND1-4, 

and there was no change in the trials to criterion in a passive avoidance test; however, there 

was clearly decreased step-down latency when tested 24 hr later (Vatanparast et al., 2013). 

This finding suggests a change in memory but not learning. After in utero exposure this effect 

was only seen in females. In contrast, both genders (greater effect in males) were affected 

following postnatal exposure. Using a novel object test, male mice (females not tested) 

exposed postnatally (PND8-11) to diazinon (0.5, 5 mg/kg/d) showed less exploration and 

discrimination of the new object (Win-Shwe et al., 2013). This was significant at both doses 

(but no dose-response) when tested at PND49, and only the high dose group showed effects at 

PND84. Mice exposed to diazinon (0.18, 9 mg/kg/d) throughout gestation were tested in a 

Lashley III maze, with no changes in the number of errors, suggesting no effect on learning 

(Spyker and Avery, 1977). Thus, these data on diazinon suggest an effect on learning and/or 

memory (radial arm maze, passive avoidance, novel object recognition) but no changes in 

learning a maze task. There was a lack of dose-response across studies.

Parathion exposure in rats (0.1, 0.2 mg/kg/d, PND1-4) produced no changes in T-maze 

spontaneous alternation (Timofeeva et al., 2008b). These rats showed decreased working 

memory errors, indicative of improvement, at the low dose only (both males and females), and 

no changes in reference memory errors when tested at about 3 months of age. However, Levin 

et al. (2008) tested littermates from the Timofeeva study beginning at 14 months, and reported 

increased working memory errors in male rats treated with the low dose only. Interestingly, 

there is a difference in direction of change and gender specificity compared to the Timofeeva 

data. Reference memory errors were also increased at both doses (but no dose-response) in 

male rats only. When the rats were tested again at 17 months, working memory errors were 

increased at both doses (but no dose-response), again only in males. There was no effect on 

reference errors at 17 months, and no change in either parameter when the rats were again 
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tested at 19 months. In a study by Stamper et al. (1988), rats (only males were tested) exposed 

postnatally to parathion (1.3, 1.9 mg/kg/d, PND5-20) showed decreased spontaneous 

alternations in a T-maze at both doses (dose-response evident), and increased working memory 

errors in a radial arm maze at both doses (but no dose-response). Reference memory errors 

were not altered. Al-Hachim and Fink (1968) administered parathion (3 mg/kg/d) during either 

the first, second or third week of gestation in mice, and reported no effect on conditioned 

avoidance (sex not mentioned) following any exposure period. Overall, the radial arm maze 

showed effects of parathion in several studies, but the direction of change, specificity of errors, 

and gender selectivity differed. The results with the T-maze were contradictory, with one study 

out of two reporting effects. 

Radial arm maze performance was affected by methyl parathion given directly to pups PND1-21 

using an incrementally increasing dose schedule (Johnson et al., 2009). The middle (0.2 to 0.6 

mg/kg/d) and high (0.3-0.9 mg/kg/d) dose groups increased both working and reference 

memory errors. The lowest dose group (0.2 mg/kg/d throughout dosing) also increased 

reference memory errors; however, on this measure all dose groups had similar averages. Only 

males were affected on all measures. Rats dosed with methyl parathion (1 mg/kg/d, GD7-15) 

were trained to go to a specific side in T-maze, and the correct side reversed five times 

(Crowder et al., 1980). Treated rats had more trials to criterion only on second and fifth switch 

(note, the text claimed there was an effect on the 4th switch, but the figure does not show it as 

significant). Effects on only certain reversals is difficult to interpret, and may be a reflection of 

the multiple t-tests used to analyze the data. Males and females were tested but data were not 

provided for each sex separately. There were no effects on passive or active avoidance in rats 

(no mention of gender) exposed to methyl parathion (1, 1.5 mg/kg/d, GD6-20) (Gupta et al., 

1985). The low-dose group only showed slower latency to bar press during operant shaping, 

and more days to asymptote; however, details of operant training and schedule were not 

provided, the sample size was extremely small (n=4/group), and high variability was mentioned. 

Thus, the most consistent effect was seen in the radial arm maze (even though there was no 

dose-response), and other tests were either negative or the data were inconclusive.

Several other OPs were tested in different cognitive tasks, but there is no more than one report 

for any specific pesticide. After single-trial passive avoidance training, male rats (females not 

tested) treated with dichlorvos (8 mg/kg/d, GD6-15) showed faster latency to cross when 

tested 7 days later, suggesting delayed retention (Lazarini et al., 2004). With fenitrothion, male 

rats (females not tested) exposed gestationally (5, 10, 15 mg/kg/d, GD7-15) were conditioned 

to climb a pole to escape shock (Lehotzky et al., 1989). The mid and high dose groups (doseresponse evident) showed more escapes, were faster, and reached criterion faster than 

controls. This same pattern seen with reacquisition after a period of extinction, during which 

time there were no group differences. There was no effect of oxydemeton methyl (0.5, 1.5, 4.5 

mg/kg/d, GD6-15) on M-maze learning or memory in rats (Clemens et al., 1990). There was 

also no effect on retention of single trial passive avoidance in mice exposed to methamidophos 

(1 mg/kg/d, PND3-9); however, the retention trial occurred at 3 hr instead of the more standard 

24 hr or greater (Lima et al., 2013).
Page 13 of 101

Table 2.1.1 Summary of Cognitive Outcomes

Early gestation

~GD1-7, ~GD8-14, 

GD6-15

GD7-15

Late gestation

~GD15-21, GD6-

20, GD15-18

Gestation

GD1-birth

Early postnatal

PND1-4,

PND3-9

Late postnatal

PND8-11, PND5-20

Postnatal 

PND1-21

Radial arm 

maze

Diazinon: cognitive 

deficit – rat, low dose 

only, M & F1

Parathion: improved 

cognition – rat, low 

dose only, M & F2

Parathion: cognitive 

deficit – rat, M not F3

Parathion: 

cognitive deficit –

rat, no doseresponse, only M 

tested4

Methyl parathion: 

cognitive deficit –

rat, dose-response, 

M only5

T-maze 

spontaneous 

alternation

Diazinon: no effect –

rat, M & F1

Parathion: no effect –

rat, M & F2

Parathion: 

cognitive deficit –

rat, dose-response, 

only M tested4

T-maze 

learning

Methyl parathion: 

cognitive deficit –

rat, sex not 

specified6

Lashley III maze Diazinon: no 

effect –

mouse, sex 

not specified7

M-maze Oxydemeton 

methyl: no effect –

rat, M & F8

Active 

avoidance

Fenitrothion: 

improved 

Methyl 

parathion: no 
Page 14 of 101

cognition – rat, 

dose-response, 

only M tested9

Parathion: no 

effect – mouse, sex 

not specified11

effect – rat, sex 

not specified10

Parathion: no 

effect – mouse, 

sex not 

specified11

Passive 

avoidance

Dichlorvos: 

cognitive deficit –

rat, only M

tested12

Diazinon: 

cognitive deficit 

– rat, F not M13

Methyl 

parathion: no 

effect – rat, sex 

not specified10

Diazinon: cognitive 

deficit – rat, M & F13

Methamidophos: no 

effect – mouse, sex 

not specified14

Novel object 

recognition

Diazinon: cognitive 

deficit – mouse, no 

dose-response, 

only M tested15

Operant 

responding

Methyl 

parathion: 

cognitive deficit 

– rat, sex not 

specified10

1 Timofeeva et al., 2008a

2 Timofeeva et al., 2008b

3

Levin et al., 2008

4

Stamper et al., 1988

5

Johnson et al., 2009

6 Crowder et al., 1980

7

Spyker and Avery, 1977

8 Clemens et al., 1990

9

Lehotzky et al., 1989

10 Gupta et al., 1985

11 al-Hachim and Fink, 1968

12 Lazarini et al., 2004

13 Vatanparast et al., 2013

14 Lima et al., 2013

15 Win-Shwe et al., 2013
Page 15 of 101

Motor Activity

Despite being a commonly tested measure in these studies, only a few of the tested OPs produced 

changes in motor activity. Rats exposed to dichlorvos (8 mg/kg/d, GD6-15) showed decreased open field 

locomotion at weaning (males not females), and decreased locomotion and increased immobility as 

adults (age not specified, only males tested) while rearing was not affected at either age (Lazarini et al., 

2004). Male rats exposed to fenitrothion (5, 10, 15 mg/kg/d, GD7-15) showed decreased horizontal 

activity in the high-dose group only on PND104, with an apparent but not significant effect at PND26, 

but no effect at PND36 (Lehotsky et al., 1989). Open field testing of rats exposed to methyl parathion (1 

mg/kg/d, GD7-15) showed what appeared to be increases only on PND23 and 54, with no differences on 

PND30, 44, 65 (also not PND18); however, the data are not compelling since statistics are not provided, 

and the text refers to the data as “a possible change” (Crowder et al., 1980). Methyl parathion-treated 

rats (1 mg/kg/d, GD6-20) showed a decrease in locomotor activity “accommodation” (apparently the 

period that is 15-30 min into the activity session) in the low dose group only (Gupta et al., 1985). 

Several OPs consistently produced no changes on motor activity, regardless of exposure or test species. 

No effects were seen with diazinon in rats exposed gestationally (1 mg/kg/d, GD15-18, Vatanparast et 

al., 2013) or postnatally (0.5, 2 mg/kg/d, PND1-4, Timofeeva et al., 2008a, or 1 mg/kg/d, PND1-4, 

Vatanparast et al., 2013), or in mice exposed gestationally (0.18, 9 mg/kg/d, GD1-birth, Spyker and 

Avery, 1977). As with diazinon, there were no motor activity changes following parathion exposure 

postnatally in rats (0.1, 0.2 mg/kg/d, PND1-4, Timofeeva et al., 2008b, or 1.3, 1.9 mg/kg/d, PND5-20, 

Stamper et al., 1988) or in mice treated during either the first, second, or third week of gestation (3 

mg/kg/d, Al-Hachim and Fink, 1968). There was no effect on measures of activity reported in rats 

treated with methamidophos (1 mg/kg/d, GD6-15), although high variability of the measures was 

discussed (deCastro et al., 2000). Methamidophos (1 mg/kg/d, PND3-9) also produced no effect in mice 

(Lima et al., 2013). Finally, there was no effect of oxydemeton methyl exposure (0.5, 1.5, 4.5 mg/kg/d, 

GD6-15) on open field activity (Clemens et al., 1990).

In this review, effects on activity are only considered in tests designed specifically for that purpose. 

During the course of other behavioral tests, e.g., radial arm or T-maze, speed or latency is often 

measured. These ancillary activity measures were sometimes altered by treatment, but are not included 

in this domain, since they are not designed to specifically target motor activity.
Page 16 of 101

Table 2.1.2. Summary of Motor Activity Outcomes

Task/Test 

Apparatus

Early gestation

~GD1-7, ~GD8-14, GD6-15

GD7-15

Late gestation

~GD15-21, GD6-20, 

GD15-18

Gestation

GD1-birth

Early postnatal

PND1-4,

PND3-9

Late postnatal

PND8-11, PND5-20

Open field Dichlorvos: decreased activity –

rat, only M tested1

Fenitrothion: decreased activity 

– rat, dose-response, only M 

tested2

Methamidophos: no effect –

rat, sex not specified3

Methyl parathion: increased 

activity – rat, sex not specified4

Oxydemeton methyl: no effect 

– rat, M & F5

Parathion: no effect – mouse, 

sex not specified6

Diazinon: no effect –

rat, M & F7

Parathion: no effect 

– mouse, sex not 

specified6

Diazinon: no effect –

mouse, sex not 

specified8

Diazinon: no effect – rat, 

M & F7

Methamidophos: no 

effect – mouse, sex not 

specified9

Parathion: no effect 

– rat, only M 

tested10

FigureEightDiazinon: no effect – rat, 

M & F11

Parathion: no effect –

rat, M & F12

Donut Methyl parathion: 

decreased activity –

rat, no doseresponse, sex not 

specified13
Page 17 of 101

1

Lazarini et al., 2004

2

Lehotzky et al., 1989

3 deCastro et al., 2000

4 Crowder et al., 1980

5 Clemens et al., 1990

6 al-Hachim and Fink, 1968

7 Vatanparast et al., 2013

8

Spyker and Avery, 1977

9

Lima et al., 2013

10 Stamper et al., 1988

11 Timofeeva et al., 2008a

12 Timofeeva et al., 2008b

13 Gupta et al., 1985
Page 18 of 101

Anxiety/Depression

In a series of tests in rats, Roegge et al. (2008) showed that early postnatal exposure to diazinon 

(0.5, 2 mg/kg/d, PND1-4) produced behaviors suggesting higher anxiety at the high dose 

(decreased open arm time in an elevated plus maze), lower fearfulness (decreased time to start 

eating in novel environment) in both dose groups, and anhedonia (decreased chocolate milk 

preference) at the low dose only, but not depression (forced swim test). These effects occurred 

only in males and did not show a clear dose-response for the novelty eating and chocolate milk 

preference tests. Using the same tests (same laboratory; Timofeeva et al., 2008b), parathion 

exposure (0.1, 0.2 mg/kg/d, PND1-4) in rats (high dose, both sexes) increased time in the open 

arm in an elevated plus maze (suggesting decreased anxiety); however, there was also an 

increase in center crossings, indicating hyperactivity, that may confound overall interpretation. 

These same rats showed no changes in novelty-suppressed feeding or in chocolate milk 

preference. Thus, in these two studies the pesticides appear to have different effects on this 

functional domain.

There are only a few reports of these behaviors with other pesticides. Rats treated with methyl 

parathion (1, 1.5 mg/kg/d, GD6-20) showed faster emergence from a cage, interpreted by the 

authors as lowered anxiety, in the low dose group only (Gupta et al., 1985). With chlormephos 

exposure (~0.06, 0.6 mg/kg/d in drinking water, one week premating to weaning), adult mice 

(both males and females) in the high-dose group showed decreased time spent in the open 

arms and increased time in the closed arms (no change in latency) of an elevated plus maze, 

suggesting increased anxiety (Ceh et al., 2012). Mice exposed to methamidophos (1 mg/kg/d, 

PND3-9) showed no differences in time spent in either arm of an elevated plus maze, but spent 

less time in center (Lima et al., 2013). This was interpreted by the authors as effect on choosing 

arms, which they say is a cognitive effect; however, this measure is often interpreted to reflect 

only activity levels. The same mice showed increased immobility in forced swim test, 

suggesting depressive-like behaviors. Overall, these results are varied and did not consistently 

show a dose-response. 
Page 19 of 101

Table 2.1.3. Summary of Anxiety/Depression Outcomes

Late gestation

~GD15-21, GD6-20,

GD15-18

Perinatal

premating-weaning

Early postnatal

PND1-4,

PND3-9

Elevated plus 

maze

Chlormephos: 

increased anxiety –

mouse, M & F1

Diazinon: increased anxiety – rat, 

dose-response, M not F2

Methamidophos: no effect –mouse, 

sex not specified3

Parathion: decreased anxiety – rat, 

dose-response, M & F4

Chocolate 

milk 

preference

Diazinon: increased anhedonia – rat, 

no dose-response, M not F2

Parathion: no effect – rat, M & F4

Novelty 

suppressed 

feeding

Diazinon: decreased fearfulness – rat, 

dose-response, M not F2

Parathion: no effect – rat, M & F4

Forced swim Diazinon: no effect – rat, M & F2

Methamidophos: increased despair –

mouse, sex not specified3

Open field 

behaviors

Methyl parathion:

decreased anxiety –

rat, no doseresponse, sex not 

specified5

1 Ceh et al., 2012

2 Roegge et al., 2008

3

Lima et al., 2013

4 Timofeeva et al., 2008b

5 Gupta et al., 1985
Page 20 of 101

Social Behavior

Only one study has used tests of social behavior using these OPs. With exposure to fenitrothion

(5, 10, 15 mg/kg/d, GD7-15), rats in mid and high dose (dose-response) spent more time 

actively interacting in conspecific pairs (Lehotsky et al., 1989). The scarcity of data on this 

measure prevents any conclusions across OPs.

Sensory Function

In a study measuring response to a tactile stimulus, with and without an acoustic prepulse, 

male (but not female) rats treated with diazinon (0.5, 2 mg/kg/d, PND1-4) showed less prepulse 

inhibition at both doses; however, no dose-response was evident (Timofeeva et al., 2008a). 

Using the same paradigm, rats treated with parathion (0.1, 0.2 mg/kg/d, PND1-4) showed a 

different pattern: lower response to the stimulus alone (high dose, both sexes), but no change 

in the inhibition produced by the prepulse (Timofeeva et al., 2008b). Mice treated with 

diazinon through gestation showed no change in response to noise (auditory startle) or smell 

(olfactory orientation), but did show change in visual cliff behavior (more steps off a “cliff”) 

which occurred only in females in the low dose group (Spyker and Avery, 1977). Additional 

studies are needed for general conclusions regarding the effects of these pesticides on sensory 

function.

Table 2.1.4. Summary of Sensory Outcomes

Gestation

GD1-birth

Early postnatal

PND1-4,

PND3-9

Auditory Diazinon: no effect – mouse, sex not 

specified1

Tactile with 

or without 

prepulse

Diazinon: decreased sensory gating 

– rat, no dose-response, M not F2

Parathion: decreased startle 

response – rat, dose-response, M & 

F

3

Visual Diazinon: decreased function – mouse, 

no dose-response, F not M1

Olfactory Diazinon: no effect – mouse, sex not 

specified1

1

Spyker and Avery, 1977

2 Timofeeva et al., 2008a

3 Timofeeva et al., 2008b
Page 21 of 101

Neuromotor Function

One study assessed neuromotor function in mice exposed to diazinon (0.18, 9 mg/kg/d, GD1-

birth), and reported some changes in motor abilities measured at about 2 months of age; 

however, these were not consistent in dose or direction of change (Spyker and Avery, 1977). 

Increased ability was suggested by a longer time to cling to a rod (both doses, no doseresponse). In contrast, there was less ability to stay on an increasingly inclined plane (both 

doses, no dose-response) or perhaps on a rotarod (group means not significant due to large 

variability, suggestive of effect at both doses). 

Rats in the high-dose group exposed to fenitrothion (5, 10, 15 mg/kg/d, GD7-15) fell off a 

rotarod faster on PND26 and PND104, but not PND36. Only males were tested (Lehotsky et al., 

1989).

There were no neuromotor changes in terms of rotarod performance in rats following exposure 

to parathion (1.3, 1.9 mg/kg/d, PND5-20) (Stamper et al., 1988) or methyl parathion (1, 1.5 

mg/kg/d, GD6-20) (Gupta et al., 1985). Overall, there is little support for conclusions of 

neuromotor outcomes following these pesticides, but more studies are needed.

Table 2.1.5. Summary of Neuromotor Outcomes

Early gestation

~GD1-7, ~GD8-14, 

GD6-15

GD7-15

Late gestation

~GD15-21, GD6-20,

GD15-18

Gestation

GD1-birth

Late postnatal

PND8-11, PND5-

20

Rotarod Fenitrothion: 

decreased 

performance – rat, 

dose-response, 

only M tested1

Methyl parathion: 

no effect – rat, sex 

not specified2

Diazinon: no effect –

mouse, sex not specified3

Parathion: no 

effect – rat, only 

M tested4

Inclined 

plane

Diazinon: decreased 

performance – mouse, 

dose-response, sex not 

specified3(Spyker)

Rod cling Diazinon: increased 

performance – mouse, 

no dose-response, sex 

not specified3

1

Lehotzky et al., 1989

2 Gupta et al., 1985

3

Spyker and Avery, 1977

4

Stamper et al., 1988
Page 22 of 101

2.1.3 Integration with AChE Inhibition

There are some data with which to compare effective doses in these DNT studies with doses 

producing AChE inhibition. A number of studies in this literature review included AChE 

inhibition (brain and/or blood) in their measurements. Information on diazinon and parathion 

can be taken from a separate (non-behavioral) study (Slotkin et al., 2006) conducted in the 

same laboratory with the same dosing paradigm used in several studies (Levin et al., 2008; 

Roegge et al., 2008, Timofeeva et al., 2008a, 2008b).

Neurobehavioral effects of diazinon were reported in rats at doses of 0.5-2 mg/kg/d (Roegge et 

al., 2008; Timofeeva et al., 2008a). Slotkin et al. (2006) reported that a dose of 0.5 mg/kg/d 

produced some (<10%, statistically significant) brain AChE inhibition when measured the day 

after the last dose. At 2 hr after a higher dose (2 mg/kg/d; lower doses were not tested at 2 hr), 

there was greater brain inhibition (25-30%) compared to 24 hr (10-20%). Thus it is probable 

that for diazinon, inhibition during and shortly after the dosing period (i.e., within hours) was

greater at lower doses. While there is no direct AChE data following diazinon exposure at 1 

mg/kg/d (Vataparast et al., 2013), it can be assumed from the Slotkin data that this dose also 

inhibited brain AChE at some time during/after dosing. No AChE activity was measured in mice 

by Spyker and Avery (1977), but the high dose of 9 mg/kg/d resulted in depressed weight gain, 

a sign of maternal toxicity. There is no mention of toxicity at the lower doses (0.5, 5 mg/kg/d) 

used in mice by Win-Shwe et al. (2013). 

The parathion studies showing effects at 0.1 and 0.2 mg/kg/d (Timofeeva et al., 2008b; Levin et 

al., 2008) are also informed by the AChE inhibition presented in Slotkin et al. (2006) in which a 

dose of 0.1 mg/kg/d inhibited brain AChE 5-15% on the day after the last dose. There are no 

AChE data available for a higher dose, 0.2 mg/kg/d, but Timofeeva noted 5% mortality in that 

dose group. Much higher doses (1.3, 1.9 mg/kg/d in rats) were reported to produced 35, 68% 

brain inhibition on the day after the last dose (PND5-20), with 26, 36% inhibition persisting a 

week later (Stamper et al., 1988).

The methyl parathion postnatal incrementing dose paradigm in rats used by Johnson et al. 

(2009) produced brain AChE inhibition in all dose groups at the end of dosing, persisting for 10 

to 20 days later; recovery was evident 30-40 days later. The low dose (0.2 mg/kg/d throughout) 

produced 13-15% inhibition. During gestational exposure to higher doses of methyl parathion 

at 1, 1.5 mg/kg/d, the dams had 20, 60% brain inhibition at the end of dosing (GD6-20) (Gupta 

et al., 1985). At the high dose, cholinergic signs and increased resorptions were noted. 

Furthermore, the offspring (fostered to control dams) showed brain inhibition as high as 50% in 

both dose groups when measured at birth and also PND7, 14, 21, and 28. The low dose showed 

recovery at PND28, but not the high dose.

In the study of rats treated with fenitrothrion, there was postnatal mortality of 16-17.5% at all 

doses (5, 10, 15 mg/kg/d, GD7-15), compared to 5% in controls (Lehotsky et al., 1989). In 

separate study of rats, 5, 25 mg/kg on GD19 produced 40, 80% brain AChE inhibition in dams, 
Page 23 of 101

and fetal brains showed about 90% inhibition (no dose response) (Sochaski et al., 2007). 

Assuming similar responses, the postnatal mortality observed in the Lehotsky study could be at 

least partly due to this high degree of AChE inhibition in both dams and fetuses. 

In rats, nonpregnant females dosed methamidophos 1 mg/kg/d for 10 days showed 16% 

plasma AChE inhibition, but brain AChE was apparently not measured. This suggests that dams 

treated at the same dose in a study by deCastro et al. (2000) most likely experienced some 

plasma inhibition. Mouse pups dosed with methamidophos 1 mg/kg/d PND3-9 showed ~36, 

46% brain inhibition 1, 4 hr after first dose, 53, 61% inhibition at 1, 4 hr after last dose, and 

~19% brain inhibition the day after last dose (Lima et al., 2013). The mice therefore 

experienced considerable brain AChE inhibition throughout dosing.

In the oxydemeton methyl study, dams showed 22-68% brain AChE inhibition on the day after 

the last dose (0.5-4.5 mg/kg/d, dose response), and 5 days later (GD20) there was 20-54% brain 

AChE inhibition. Fetal brains taken the day after dosing showed no inhibition (Clemens et al., 

1990); however, there were no fetal AChE tissues collected during or shortly after the dosing 

period when AChE inhibition would be greatest.

Overall, in the studies for which there are direct or comparable data, it is clear that the dosing 

paradigms produced AChE inhibition and in some cases maternal toxicity. Indeed, there are no 

studies reporting or even suggesting a lack of AChE inhibition in the dam and/or fetus/pup at 

any time during dosing. Thus, it is not known whether exposure paradigms that do not inhibit 

AChE would produce any neurobehavioral effects.

2.1.4 Summary of Findings from the Developmental Neurotoxicity (DNT) Guideline 

Studies

DNT studies have been submitted for 20 OPs, summarized in Appendix 2. These studies follow 

the US EPA guideline 870.6300 and/or OECD guideline 426 which require testing of motor 

activity, acoustic startle response, learning and memory, and brain morphometrics in the 

offspring around weaning and also in adulthood. In general, these studies provide exposure 

during development either via diet or oral gavage dosing, including direct dosing of the pup 

preweaning. As with the literature studies, these submitted studies have shortcomings such as

inappropriate statistical analyses, limited methodological information and presentation of 

results. Many measures tend to show high variability, which reduces their interpretability and 

utility.

In order to compare the submitted guideline and published studies under the scope identified 

under Section 2.1 and to be consistent with the chlorpyrifos 2012/2014 review, only changes 

that occurred after dosing had ended (i.e., shortly after weaning or as adults) were considered 

here. Across the seven submitted studies that reported effects, there are mostly changes in 

acoustic startle reactivity, cognitive function, and to a lesser extent, motor activity. Some OPs 
Page 24 of 101

altered multiple domains, others only one. There are both submitted guideline studies and 

literature studies for only four OPs: diazinon, methyl parathion, methamidophos, and 

dichlorvos. Diazinon produced cognitive changes but no effects on motor activity or acoustic 

startle in the DNT: these results are generally in agreement with published studies. Male

offspring in the high dose group (~33 mg/kg/d in the dam diet) showed increased errors and 

longer latency in Biel maze performance at both PND24 and PND62, and similar effects were 

seen in females but only in the middle dose group (~3.4 mg/kg/d via diet) at PND24. Following 

methamidophos exposure, female rats in the middle and high dose groups (~1.7, 5.2 mg/kg/d in 

the dam diet) showed decreased peak amplitudes of the startle response, which was 

statistically significant at PND38 and apparent but not significant at PND60. In contrast to the 

literature reports of cognitive and motor effects of methyl parathion, there were no reported 

changes in the submitted guideline study. The submitted study of dichlorvos was 

uninterpretable due to high pup mortality in all groups, including control.

In these studies, AChE activity was assessed as part of the DNT itself, or by means of a separate 

study comparing the response in pups and adults (comparative cholinesterase, or CCA, studies). 

Thus, in almost all guideline DNT studies there are adequate data describing AChE inhibition in 

the pups at some time during development. It is clear from these DNT studies that the doses 

used did produce AChE inhibition in the offspring, sometimes at all doses tested. It was noted 

that in these studies, most of the reported effects occurred before weaning, which is the period 

during which there was likely to be ongoing AChE inhibition.

Thus, as with the literature studies, there are scant data that could inform potential 

neurodevelopmental changes occurring at doses lower than those needed to inhibit AChE. 

Furthermore, there is little consistency in patterns of effects across studies or chemicals. Thus, 

there is uncertainty as to whether lower, non-inhibiting exposures are developmentally

neurotoxic; this uncertainty was described in the chlorpyrifos reviews and remains applicable 

for the available data for other OPs as well.

2.1.5 Conclusions on In Vivo Laboratory Animal Studies

For chlorpyrifos, there are >30 papers on developmental neurotoxicity; for the remaining OPs, 

the literature is sparse with very few studies for each OP (including DNT guideline studies). The 

studies span over decades, and many of the lower quality studies were the earlier ones; 

however, some very recent papers also have significant deficits. Methodological detail is 

lacking, inappropriate statistical analyses are applied, results are cursorily described and/or 

inaccurately presented, and interpretation of some behavioral changes is faulty. Overall, most 

studies have significant shortcomings and/or are of low quality.

The most commonly tested behaviors considered aspects of cognition. In the majority of 

studies, some sort of cognitive deficit was detected, especially with working memory 

performance (radial arm maze) and conditioned response retention (passive avoidance). 

However, in many cases there was no dose-response, there was some gender specificity which 
Page 25 of 101

did not replicate in multiple studies, and cognitive improvement instead of deficit was noted in 

a few papers. Changes in motor activity in offspring were generally not reported, and the 

direction of change differed in the papers reporting such effects. There is generally not enough 

information to make definitive statements about OP effects on other types of neurological 

disorders.

Few published papers included AChE measurements of the dams and/or offspring, but where 

measured, all doses used inhibited AChE to some degree. Some papers even reported overt 

maternal and fetal toxicity. This was also the case in the guideline studies, most of which 

included concurrent or supplemental data on AChE inhibition. Since there are no studies with 

low doses that definitively do not inhibit AChE, there is no information in the animal literature 

that shows whether or not there would be developmentally neurotoxic outcomes at those 

lower exposures.

2.2 Epidemiology Research on OPs other than Chlorpyrifos

2.2.1 Overview of 2012/2014 and 2015 Literature Reviews

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) Mother’s and Newborn Study of North Manhattan and South Bronx 

conducted by Columbia University, referred to in this document as “Columbia Study/Cohort;” 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.” The major findings of these cohorts are briefly summarized in Section 2.2.9 to provide 

context for integrating the findings of the 2015 review and for the WOE analysis.

In the current review, the agency has expanded its consideration of the epidemiological data to 

include studies of any OP pesticide; several different types of development and neurological, 

neurodevelopmental, and neurobehavioral health outcomes; studies performed in non-U.S. 

countries as well as US based studies; and non-cohort studies. 

2.2.2 Literature Search Methodology

To identify the epidemiological investigations of the association between OP exposure and 

adverse neurological, neurodevelopmental or neurobehavioral effects, EPA scientists queried 

PubMed/Medline and Web of Science directly. In this literature search, emphasis was placed 
Page 26 of 101

upon identification of all possible epidemiological studies available, and the ability to use the 

identical search string in both PubMed/Medline and Web of Science The following search string 

was utilized:

((Chlorpyrifos OR Organophosphates) AND (prenatal OR neonatal OR perinatal OR in 

utero OR fetal OR foetal OR newborn OR infant* OR infancy OR preschool OR child* OR 

maternal OR mother* OR pregnan*) AND (neurodevelop* OR attention OR birth 

outcome* OR health outcome* OR birth height OR birth weight OR birth length OR 

cephalometry OR head circumference OR child development OR cognitive OR cognition 

OR developmental disability* OR fetal growth OR foetal growth OR foetal development 

OR fetal development OR intelligence OR memory OR neurological functioning OR 

psychomotor) AND (human)) (Filter: English language only)

With the aid of an EPA reference librarian, EPA also searched the following databases: PsycInfo, 

Agricola, Biosis, Embase, Enviroline, Gale Health and Wellness, Global Health, Pascal and 

Pollution Abstracts. EPA used similar, but modified search terms as listed above. Upon 

identification of the final set of relevant articles (n=38), limited hand-searching of the reference 

lists and citation mapping (Science citation index) of articles deemed to be most relevant to the 

review question was performed.

EPA identified 300 articles across these several biomedical search engines. Removing 

duplicates, there were 243 articles, and 79 were determined to be epidemiological 

investigations of potential relevance. The 164 studies excluded from the analysis comprised 57 

exposure only studies; 51 review articles; 33 reports of acute OP intoxication; 20 studies in nonhuman systems; and 3 were otherwise not relevant. Among the 79 potentially relevant 

epidemiologic studies, 41 were excluded; 17 articles were previously reviewed in 2012; 16 were 

epidemiological methods papers including exposure validation studies without an original 

epidemiological risk estimate; and 8 were otherwise not relevant for various reasons. Among 

the 40 remaining studies, 2 were additionally excluded (one was a duplicate study published a 

second time; the other did not make a measure of an OP pesticide. Therefore, 38 articles are 

included in this narrative literature review (referred to herein as “2015 Literature 

Review/Studies”). The determination of relevance to the study question was made by two EPA 

epidemiologists who agreed by consensus as to article disposition in the literature search.

The following sections provide the results of this literature review. Section 2.2.3 describes the 

breadth and depth of the 2015 literature review, with Section 2.2.4 summarizing the approach 

for assigning a quality ranking and Section 2.2.5 providing the results of this quality ranking. In 

Section 2.2.6, these studies were further analyzed with focus on identification of the most 

appropriate exposure assessment and relevant outcomes for this assessment. Studies focusing 

solely on birth outcomes are discussed in Section 2.2.7. However, the emphasis in this 

assessment is on those studies focusing on neurodevelopmental outcomes, which are discussed 

in detail in Section 2.2.8 and summarized in Section 2.2.9. 
Page 27 of 101

2.2.3 Breadth and Depth of the 2015 Literature Review

Key features of each of the 38 articles in the current 2015 literature review are summarized in 

Table 2.2.5-1 and 2.2.5-2, as well as Appendix 3. These articles represent 31 distinct studies

covering a wide range of study designs, study locations and time periods, and exposure and 

outcome measurement approaches and are listed in Table 2.2-a. There were 10 different study 

designs identified; the majority of the studies utilized a cross-sectional or a prospective birth 

cohort study design.

In addition to four articles on the previously-reviewed studies (two on the Mt. Sinai Cohort and 

two on the CHAMACOS Cohort), there were a number of new birth cohorts or named studies in 

this literature, including (see Table 2.2.5-1, 2.2.5-2, and Appendix 3 for details):

 Denmark, Birth Cohort (Andersen et al. 2015)

 Saint Peter's University Hospital, New Brunswick, New Jersey, Birth Cohort (Barr et al. 

2010)

 Early Life Exposures in Mexico to Environmental Toxicants (ELEMENT), Mexico City, 

Mexico, Birth Cohort (Fortenberry et al. 2014, 2014a)

 EcoSalud Project, Cayambe-Tabacundo region, Ecuador, Infant and Young Child 

Cohort (Handal et al. 2007, 2007b, 2008)

 University Hospital of Heraklion, Crete, Greece, Birth Cohort (Koutroulakis et al. 2014)

 Children's Pesticide Survey (CPS), Yuma County, Arizona (Lizardi et al. 2008)

 Infancio y Medio Ambiente (INMA) (Environment and Childhood), Spain, Birth Cohort 

(Llop et al. 2013)

 Perturbateurs endocriniens: Étude Longitudinale sur les Anomalies de la Grossesse, 

l’Infertilité et l’Enfance (PELAGIE), Brittany, France, Birth Cohort (Petit et al. 2010)

 Health Outcomes and Measure of the Environment (HOME), Cincinnati, OH, Birth 

Cohort (Rauch et al. 2012)

 Embilipitiya Base Hospital, Southern Sri Lanka, Birth Cohort (Samarawickrema et al. 

2008)

 Ontario Farm Family Health Study, Ontario, Canada, Birth Cohort (Savitz et al. 1997)
Page 28 of 101

 Childhood Autism Risks from Genetics and the Environment (CHARGE), California, 

Child Cohort (Shelton et al. 2014)

 Secondary Exposure to Pesticides Among Infants, Children and Adolescents (ESPINA), 

Pedro Moncayo County, Pichincha, Ecuador, Child Cohort (Suarez-Lopez et al. 2012, 

2013, 2013a)

 Shenyang, China, Birth Cohort (Zhang et al. 2014)

Exposures were assessed via environmental samples, biomarkers, and/or proxy methods.

 Only one study used environmental samples as an exposure measure - methyl 

parathion in household wipe samples (Ruckart et al., 2004).

 Among the studies, 11 biomarkers were used to assess exposure. One study utilized 

a direct measure of OP pesticide – chlorpyrifos (CPF) in maternal and cord serum 

(Barr et al., 2010). Several studies looked at specific OP parent metabolites (IMPY, 

3,5,6-trichloro-2-pyridinol [TCPy], PNP). The majority of studies used non-specific OP 

biomarkers: dialkyl phosphates (DAPs), diethyl phosphates (DEPs), and dimethyl 

phosphates (DMPs). Two effect biomarkers, AChE and butyl cholinesterase (BuChE), 

were used as the exposure metric. 

 Proxy exposure methods included questionnaire and non-questionnaire 

approaches. For example, questionnaire-based exposures included maternal and or 

paternal self-report of living with an exposed worker, occupational exposure/ 

employment, and home pesticide use and child outdoor play exposure. Nonquestionnaire-based exposures included Community/area of residence, distance to 

treated area/farm, percent of area treated with pesticide, level of urbanization, 

pounds OP pesticide used/year, and pesticide spray season. 

Likewise, there were numerous outcome measures examined across the studies, falling into six 

broad categories: birth characteristics, autonomic nervous system (ANS) effects, Attention 

Deficit Hyperactivity Disorder (ADHD)/attention problems, autism, general neurodevelopment 

(cognitive, behavioral, IQ), and physiological effects. The most common outcome measures 

were birth characteristics (with birth weight, birth length, head circumference and gestational 

age being the most frequent) and neurodevelopment tests and test batteries. Table 2.2-b lists 

the many specific neurodevelopment tests employed in the studies. Most studies utilized more 

than one test, and few tests were utilized in more than one study.

Other features that varied widely among the studies include:

 Study periods ranged from 1991 to 2012 (reports were published from 1997 to 

2015).
Page 29 of 101

 Study locations included Canada (2 studies), China (3 studies), Costa Rica, Israel, 

Denmark, Ecuador (3 studies), France, Greece, Mexico (3 studies), Poland, Spain, Sri 

Lanka, and the United States [AZ, CA (2 studies), MS, NJ, NY, NC, OH (2 studies), OR, 

National (2 studies)].

 Study sizes varied from 25 to 3,159 participants.

 Children’s ages ranged from newborns to age 15 years.

Pre-natal exposures were assessed in 19 reports, post-natal exposures in 13 reports, and both 

pre- and post-natal exposures in 5 reports.

Table 2.2-a Study Designs, Exposure Measurement Methods, and Outcome Measurement 

Methods Used across the 31 2015 Review Studies

Study design

(# studies used)

Exposure measurement

(# studies used)

Outcome measurement

(# studies used)

Prospective Cohort (10)

Retrospective Cohort (3)

Case-control (2)

Cross-sectional (15)

Ecological (1)

Biomarkers

AChE - acetyl cholinesterase (maternal 1, 

child 2)

BuChE - butyl cholinesterase (child 1)

CPF -chlorpyrifos parent (maternal, cord 

serum 1)

TCPy - chlorpyrifos metabolite (maternal 1, 

child 1)

DZN – diazinon parent (1)

IMPY – diazinon metabolite (child 1)

MAL - malathion parent (0)

MDA - malathion metabolite (maternal 1)

DAP – dialkyl phosphate (maternal 5, child 

7, amniotic fluid 1)

DEP – diethyl phosphate (maternal 5, child 

7, amniotic fluid 1)

DMP – dimethyl phosphate (maternal 5, 

child 7, amniotic fluid 1)

OP - organophosphate (cord blood 1)

PNP - para-nitrophenol (child 2)

Environmental

Methyl parathion - Household wipe (1)

Proxy (questionnaire)

 Maternal occupational exposure/ 

employment (6)

 Paternal occupational exposure/ 

employment (2)

 Living with exposed worker (1)

 Home and outdoor play exposure (1)

Proxy (non-questionnaire)

Birth characteristics (10)

 Birth Weight/LBW/FGR (records 7, NP 

1, report 2)

 Birth Length (records 3)

 Head circumference (records 3, NP 1)

 Abdominal circumference (records 1)

 Gestational age (GA)/preterm (report 

2, records 3)

 Ponderal index (records 1)

 Placental maturity index (1)

 Spontaneous abortion/miscarriage 

(report 1)

 Altered sex ratio (report 1)

Autonomic Nervous System (4)

ADHD/attention problems (2)

 Diagnosis DISC-IV (1)

 Use of medication (1)

 Screening (CPRS-R, CPT, BASC-PRS) (1)

Autism (2)

 CA Department of Developmental 

Services (CDDS) reports

 US Individuals with Disabilities 

Education Act (IDEA) reports

 Autism spectrum disorders (ASD)

 Autism Diagnostic Observation 

Schedule (ADOS) combined with ADIR.

Neurodevelopmental (ND) test/battery -

US (3) – see Table 2.2-2b for details

ND test/battery - non-US (11) – see Table 

2.2-2b for details

IQ (4) - see Table 2.2-2b for details
Page 30 of 101

 Community/area of residence (3)

 Distance to treated area/farm (2)

 Percent of area treated with pesticide 

(1)

 Level of urbanization (1)

 Pounds OP used/year (1)

 Pesticide spray season (1)

Physiological (2)

 AChE activity, child

 BuChE activity, maternal

 Antioxidant status: superoxide 

dismutase (SOD) activity

 Fetal oxidative stress: 

malondialdehyde (MDA) 

concentrations)

 Fetal DNA fragmentation: 

electrophoresis
Page 31 of 101

Table 2.2-b Detailed Neurological Outcome Measurement Methods Used across the 31

2015 Review Studies

Outcome measurement (# studies used) 

Neurodevelopmental (ND) test/battery - US (4)

 Pediatric Environmental Neurobehavioral Test Battery (PENTB) : cognitive, motor, sensory, and affect domains

o Developmental Test of Visual-Motor Integration (VMI)

o Kaufman Brief Intelligence test (K-BIT)

o Purdue Pegboard

o Story Memory and Story Memory-Delay from Wide Range Assessment of Memory and Learning 

o Trail-Making test, Part A and Part B

o Verbal Cancellation test

 Children’s Memory Scale (CMS)

 Behavioral measures:

o The Child Behavior Checklist/4-18 

o The Teacher Report Form

 Developmental delay (DD):

o Mullen Scales of Early Learning (MSEL)

o Vineland Adaptive Behavioral Scale (VABS)

o Reciprocal social interaction: Social Responsiveness Scale

ND test/battery - non-US (11)

 Bayley Scales of Infant Development - mental and psychomotor development

 Behavioral Assessment and Research System (BARS): Memory and attention, response speed and coordination, visual 

memory, attention, divided attention, recall and recognition memory, dexterity, hand-eye coordination (2)

 Figure drawing task: child's perception and dexterity

 Long-term memory test

 Ages and Stages Questionnaire (ASQ) – communication, fine motor, gross motor, problem solving, and personal–social

skills

 Strengths and Difficulties Questionnaire, parent version (SDQ): behavioral problems

 NEPSY-II test (trained examiners): general assessment battery:

o attention and executive functioning 

o language

o memory and learning

o sensorimotor (visuomotor precision), 

o visuospatial processing

o Statue and Knock Tap

 Neonatal Behavioral Neurological Assessment (NBNA)

o Behavior

o Passive Tone

o Active Tone

o Primary Reflexes

o General Assessment

 Reach-and-grasp, bi-manual coordination: Prehension abilities

 UC Berkeley Preferential Looking Test Cards: Visual acuity skills

 Visual Motor Integration (VMI), Beery-Buktenica, 4th Ed.

 Finger Tapping test: Manual motor speed

 Catsys equipment: Simple reaction time

 Conners' Continuous Performance Test II (CPT II, v5): Attention

 Woodcock-Johnson III Tests of Cognitive Abilities (WJ-III) Verbal Comprehension test: Long-term memory and language 

function

 Visuospatial performance and memory functions:

o Raven's Colored Progressive Matrices 

o Stanford–Binet Copying Test, 4th ed

 Physical examination (social response, spontaneous motility, involuntary movements, Romberg's sign, walking straight line, 

standing on one leg, number hops, biceps and patellar reflexes, finger opposition, diadochokinesis, finger–nose 

coordination, hearing, vision)

 Gesell Development Schedule (GDS): motor, adaptive, language, and social

 Santa Ana Form Board: Motor coordination

 Child's developmental delay - Parent interview
Page 32 of 101

IQ (4)

 Wechsler Intelligence Scale for Children-Revised (WISC-R) Digit Span Test, Card sorting Test

 Stanford-Binet Memory for Sentences and Digit String test

 Recall and recognition test
Page 33 of 101

2.2.4 Considerations for Study Quality Evaluation

This section summarizes how specific study characteristics factored in overall quality category. 

[Note: these study quality considerations are specific to issue of relevance to this document, 

namely potential for neurodevelopmental effects of OPs. These considerations are considered 

‘fit for purpose’ under this context and could differ in another regulatory or scientific context.] 

The literature base evaluated is heterogeneous, as noted in Section 2.2.3, consisting of various 

study designs implemented in U.S. and foreign study populations. Pesticide exposure 

assessments variously relied on, for example, exposure biomarkers, maternal self-reports, and 

other proxy indicators of OP pesticide exposures. Outcome assessments were similarly varied, 

relying, for example, on biomarkers of biological effects, birth records, maternal self-reports, 

and clinical instruments designed to evaluate neurocognitive and neurobehavioral 

development. These design elements have potential impacts on study quality and relevance to 

this document. Each study was therefore judged to be of high, moderate, or low quality in each 

of the following six domains effecting study quality: Study design, exposure assessment, 

outcome assessment, confounder control, statistical analysis, susceptibility to bias (See Table 

2.2.4-1 for general considerations under each domain).

2.2.4.1 Study Designs

Four basic study designs were used in the literature reviewed for this document: cohort study, 

case-control study, cross-sectional study, and ecologic study. The first of these two constitute 

the two basic types of observational (i.e. non-interventional) studies used to evaluate relative 

incidence of health and disease outcomes by exposure status. The latter two are generally 

considered descriptive or hypothesis generating study designs, though they too can be used to 

test hypotheses regarding relative prevalence of health outcomes and, under certain 

conditions, incidence as well. 

Table 2.2.4-1 Study Quality Considerations

Parameter 

High 

(Score 8-12) 

Moderate 

(Score 4-7) 

Low 

(Score 0-3) 

Study Design Prospective, exposure 

precedes disease

Case Control Cross sectional

Ecological

Exposure assessment Exposure assessment 

includes information on 

specific OP a.i.’s (e.g., 

CPF, MAL), or urinary 

metabolite (TCPy, 

IMPy,), or high quality 

questionnaire based 

chemical specific 

exposure assessment 

during relevant exposure 

Non-specific biomarker 

of exposure (DAP), or 

effect (AChE/BuChE), or 

questionnaire based 

individual level 

information on the OP 

class, or sub-class

Low quality 

questionnaire based 

exposure assessment, 

or ecologic exposure 

assessment, with or 

without validation 
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window (pre-natal, early 

life)

Outcome Assessment Standardized tool, 

validated in study 

population; or, medical 

record review with 

trained staff (birth 

characteristics)

Standardized tool, not 

validated in population, 

or screening tool; or, 

medical record review, 

methods unstated (birth 

characteristics)

Selected sections of 

test, or maternal report, 

other; or, 

maternal/paternal selfreport (birth 

characteristics)

Confounder control Good control for 

important confounders 

relevant to OP-ND 

question, and standard 

confounders

Moderately good control 

confounders, standard 

variables, not all 

variables for OP-ND 

question

Multi-variable analysis 

not performed, no 

adjustments

Statistical Analysis Appropriate to study 

question and design, 

supported by adequate 

sample size, maximizing 

use of data, reported 

well (not selective)

Acceptable methods, 

questionable study 

power (especially subanalyses), analytic 

choices that lose 

information, not 

reported clearly 

Minimal attention to 

statistical analyses, 

comparisons not 

performed or described 

clearly 

Risk of (other) bias 

(selection, differential 

misclassification, other)

Major sources of other 

potential biases not 

likely present, present 

but analyzed, unlikely to 

influence magnitude 

and direction of the risk 

estimate

Other sources of bias 

present, acknowledged 

but not addressed in 

study, may influence 

magnitude but not 

direction of estimate

Major study biases 

present, 

unacknowledged or 

unaddressed in study, 

cannot exclude other 

explanations for study 

finding

(Adapted from Munoz-Quezada et al. 2013)

Cohort Study

A commonly used design in this literature was the cohort study (See Tables 2.2.5-1, 2.2.5-2, and 

Appendix 3 for examples). In a typical cohort study, individuals are classified according to 

exposure status (i.e., presence, absence, or magnitude of exposure), and then followed over 

time to quantify and compare the development (i.e., incidence) of the health outcome of 

interest by exposure group. Conceptually, the non-exposed comparison group in a cohort study 

provides an estimate of the incidence of the outcome among the exposed, had they, counterto-fact, not been exposed. Apart from chance variations, a valid cohort study comparing 

exposed individuals to non-exposed individuals provides an estimate of the relative risk (or 

rate) of the disease associated with exposure. Ideally, the exposed and non-exposed groups are 

exchangeable, in the sense that switching the exposed to non-exposed, and non-exposed to 

exposed would yield the same measure of association (e.g., relative risk). If this were the case 

then, apart from chance, a cohort study would yield a measure of association equivalent to that 
Page 35 of 101

produced in a corresponding (intervention) study where exposure status was randomly 

assigned. 

The chief advantage of the cohort study design is that it affords the investigator the 

opportunity to avoid and/or adjust for potential biases (i.e., selection bias, information bias, 

and confounding). Cohort studies also allow for discernment of the chronological relationship 

between exposure and outcome, and can be particularly efficient for studying uncommon 

exposures. The primary disadvantage of the cohort study design is logistical inefficiency with 

respect to the necessary time, expense, and other resources needed to conduct them. Cohort 

studies are particularly inefficient for evaluating associations with rare outcomes and diseases 

with long induction or latency periods. Though prospective studies are often logistically less 

efficient relative to other study designs (e.g., the case-control study), these logistical concerns 

can be minimized in cohort studies of short duration, such as those used to evaluate prenatal 

OP pesticide exposure effects on birth outcomes or other outcomes of neonatal development 

assessed shortly after birth. 

Two sub-categories of cohort studies – prospective and retrospective - are often applied to 

distinguish between studies in which the health outcome has occurred (retrospective study), or 

has not occurred (prospective study) at the time the investigators initiate the study. This 

distinction is important primarily as it pertains to the potential differences in the quality (e.g., 

completeness, accuracy, and precision) of information that can be ascertained by the 

investigators, and also as it relates to potential sources of bias. Although not always true, the 

prospective study design is considered the preferable of the two, as investigators can 

potentially have more choices in determining how exposure, outcome, and covariate 

information is collected. In a retrospective study conducted to evaluate the same hypothesis, 

by contrast, the investigators would have to rely on exposure information such as maternal selfreport. Such reporting is subject to (human) errors in recall. Moreover, the outcome status of 

the child (i.e., whether a child has developmental delays that are known to the mother) may 

influence the recall of prenatal OP pesticide exposure by the mother.

Case-control Study

In a typical case-control study (see, for example, Dawbrowski et al., 2003 in Appendix 3 and 

Shelton et al. 2014 in Table 2.2.5-2), individuals are classified according to their outcome status 

(i.e., cases who have developed the outcome of interest, and controls who represent the 

population from which the cases arise). The relative odds of exposure are then compared 

between cases and controls. The primary advantage of case-control studies is that they are 

logistically efficient relative to cohort studies. In fact, properly conducted case-control study 

can be conceptualized as a cohort study with efficient sampling of exposure among the cohort, 

yet they can often be conducted at a fraction of the cost, in a fraction of the time as a 

corresponding cohort study. Case-control studies can be used to examine associations between 

multiple exposures and a given health outcome. They are particularly efficient for evaluating 

rare outcomes but are inefficient for studying uncommon exposures. The primary weakness of 

the case-control study is the potential for selection bias, which arises if the exposure 

distribution among the control subjects is not representative of the exposure distribution 
Page 36 of 101

among the population that gave rise to the cases. Case-control studies that rely on selfreported exposure measures are also susceptible to information bias. 

Cross-sectional study

Cross-sectional studies (see, for example, Suarez-Lopez et al. in Table 2.2.5-2 and Grandjean et 

al. 2006 in Appendix 3) are used to evaluate associations between exposure and outcome 

prevalence in a population at a single point in (or period of) time. The primary advantage of a 

cross-sectional study is logistical efficiency; they are relatively quick and inexpensive to 

conduct, as a long period of follow-up is not required, and exposure and outcome assessments 

occur simultaneously. Cross sectional studies have three primary potential disadvantages: 1) 

potential difficulty in discerning the temporal relationships (i.e., whether the exposure 

precedes the outcome); 2) estimating outcome prevalence rather than incidence of the 

outcome; and 3) the possible overrepresentation of cases of the outcome with long duration 

relative to the average in the population, and often with a better prognosis. 

Ecological study

Ecological studies are used to evaluate associations between exposures and outcomes using 

population-level rather than individual-level data. For example, Nevison (2014, Appendix 3) 

uses annual estimates of pesticides applied to crops and population level autism prevalence to 

assess the association between OP pesticide exposure and autism. The primary advantages of 

ecological studies are related to logistical efficiency, as they often rely on pre-existing data 

sources and require no individual-level exposure, outcome, or covariate assessments. The 

primary weakness of the ecologic study is the potential for confounding and resultant 

inappropriate extrapolation of associations observed on the aggregate-level to associations on 

an individual level. The mistaken belief that associations observed at the population level exist 

at the individual level is referred to as the ecological fallacy. 

In judging an individual study’s contribution to the strength of evidence in the epidemiologic 

literature base, the following hierarchy of observational study designs was considered (from 

most to least preferred): prospective cohort study, retrospective cohort study, case-control 

study, cross-sectional study, ecological study. It is important to note, however, that this 

hierarchy of study designs reflects the potential for the collection of high quality information 

(related to exposure, outcome, confounders, and effect modifiers) and potential for efficient 

and valid estimation of the true association. Thus, in deliberating on quality, care has been 

taken to consider the circumstances and particulars of each individual study. For example, a 

well-conducted case-control study of a rare outcome can provide much higher quality evidence 

vis-à-vis the association of interest than a poorly conducted prospective cohort study of the 

same relationship. For this report, the placement of the study design in the aforementioned 

hierarchy of observational study designs was but one facet of the judgment of study design 

quality. Additional consideration was given to whether the study was well conducted, 

independent of study design type. The particulars of a study’s design, specifically the design 

elements employed to minimize and adjust for biases, were also considered. Finally, the 

relevance of each study with respect to the association of primary interest in this initiative, 
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namely the relationship between prenatal (and early life) OP pesticide exposures and fetal and 

child neurodevelopment was considered.

2.2.4.2 Exposure Measures

There were three major categories of exposure assessment employed in this literature: 

exposure biomarkers, participant-reported proxy exposure via questionnaire, and objectively 

obtained proxy indicators of exposure. Although one study included environmental wipe 

sampling results in the exposure assessment, urinary biomarker measures were also included 

and no differentiation of the two approaches was presented, so this exposure assessment 

category has not been included. The merits and the disadvantages of the three primary 

exposure assessment strategies are discussed below. 

Most of the studies reviewed herein assessed biomarkers of exposure quantified in samples of 

biological media (most often urine, but also blood, serum, and breast milk). These biomarkers 

were of three types: 1) OP pesticide residues, 2) metabolites of specific OP pesticides, and 3) 

non-specific OP metabolites. 

The most commonly measured biomarkers were urinary dialkylphosphate metabolites (DAP). 

These non-specific markers are easily quantified using gas chromatography/mass spectrometry 

and related methods. Though objective, use of urinary DAPs as biomarkers for OP pesticide 

exposures has limitations, including substantial temporal variability, often varying substantially 

over short time scales (i.e., day-to-day). Quantification of DAPs in a single urine sample may not 

represent an individual’s usual exposure to OP pesticides over the time period of interest (e.g., 

pregnancy) in their utility as biomarkers of OP pesticide exposure. Urinary DAP metabolite 

levels may also reflect exposure to ambient metabolites in addition to exposure to OP parent 

compounds. In this literature, errors in DAP as a biomarker of OP pesticide exposure are likely 

to be non-differential with respect to outcomes. Epidemiologists often distinguish between 

two mechanisms or types of misclassification – those that are non-differential (or random) and 

those that are differential (non-random). See Section 2.2.4.6 for further discussion of these 

exposure misclassifications.

Many studies used questionnaire-based exposure assessments in which study participants 

(typically mothers) self-reported their exposures, in addition to, or instead of, quantifying OP 

pesticide biomarkers in samples of biological media. These exposure assessments typically 

include querying OP pesticide exposure directly, or asked study participants to report on 

behaviors and conditions associated with pesticide use (e.g., occupation, tasks). Such reporting 

likely misclassifies actual OP pesticide exposure. If conducted as part of a prospective exposure 

assessment, these errors are likely to be non-differential with respect to the outcome(s) of 

interest. In the context of a retrospective assessment in which the mother has knowledge of 

the outcome status of the child, these errors may be differential or non-differential.

Several studies used proxy measures (including ecological indicators) of pesticide exposure. 

These included, for example questions about occupational use and exposure to pesticides, 

distance from residence to fields where pesticides were applied, the proportion of land in a 
Page 38 of 101

specified area dedicated to agricultural uses, and occurrence of pregnancy during the pesticide 

application season. Again, substantial non-differential exposure measurement 

error/misclassification of exposure is likely.

For this evaluation, studies employing exposure assessments that quantified biomarkers of 

specific OP pesticides (e.g., chlorpyrifos, malathion, diazinon), or urinary metabolites of these 

pesticides (e.g., TCPy, malathion dicarboxylic acid, 2-isopropyl-4-methyl-6-hydroxypyrimidine), 

or high-quality chemical-specific exposure quantitation during relevant exposure-time windows 

(i.e. pre-natal, early life) were given the highest weight. Studies that quantified levels of nonspecific biomarker of OP pesticide exposure (e.g., DAPs), or exposure effects (e.g., AChE, 

BuChE,) were given a moderate weight. Studies relying on high-quality survey-based individuallevel information on pesticide exposure were also assigned a moderate weight. Exposure 

assessments that only crudely or subjectively classified pesticide exposures and ecologic and 

other proxy measures of exposure were assigned a low weight.

Two studies focusing on neurodevelopmental outcomes measured OP exposure using both 

DAPs and malathion dicarboxylic acid (a metabolite of malathion) (Eskenazi et al., 2007; Engel 

et al., 2007), with TCPy exposure also being measured in one of these studies (Eskenazi et al., 

2007). Additionally, two studies focusing on birth outcomes measured OP exposure by testing 

for specific OPs, with Whyatt et al. (2004) measuring chlorpyrifos and diazinon; and Eskenazi et 

al. (2004) testing for DAPs and for seven pesticide specific metabolites (MDA - derived from 

malathion; PNP - derived from methyl parathion, parathion, and other nonpesticide chemicals; 

TCPy - from chlorpyrifos and chlorpyrifos methyl; DEAMPY - from pirimiphos methyl; IMPY -

from diazinon; CMHC - from coumaphos and coumaphos methyl; CIT - from isazophos and 

isazophos methyl). Finally, several method validation studies tested for specific OP pesticides, 

but did not evaluate the association between these exposures and specific adverse health 

outcomes (Whyatt et al., 2007; Whyatt et al., 2009; Bradman et al., 2003).

With the exception of the studies discussed above and those focusing exclusively on TCPy or 

chlorpyrifos, the majority of the epidemiological studies focus on the association of OPs 

exposure and various neurodevelopmental outcomes. The OP exposure being assessed in 

these studies used concentrations of urinary dialkyl phosphate metabolites (DAPs). Total DAPs 

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

dimethyl alkylphosphate (DMAP) molecules of DMP, DMTP, DMDTP, and three diethyl 

alkylphosphate (DEAP) molecules of DEP, DETP, and DEDTP. Each metabolite is a breakdown 

product from multiple OPs (Table 2.2.4-2). Specifically, DMP, DMTP, and DMDTP are associated 

with 18, 13, and 5 OPs, whereas DEP, DETP, and DEDTP are associated with 10, 10, and 4 OPs, 

respectively. Thus, using DAPs as an exposure measure, it is not possible to separate the 

exposure and associated effects for single, specific OPs. For studies evaluating TCPy (e.g., 

Fortenberry et al., 2014; Eskenazi et al., 2007; Whyatt et al., 2009), this molecule is a 

metabolite of chlorpyrifos, chlorpyrifos-methyl, and the herbicide triclopyr. TCPy is a primary 

environmental degradate of chlorpyrifos, chlorpyrifos-methyl, and triclopyr, and is found on 

food treated with these pesticides. Studies focusing solely on chlorpyrifos could assess 

exposure to only this OP (e.g., Lovasi et al., 2010; Whyatt et al., 2004; Rauh et al., 2011).
Page 39 of 101

Table 2.2.4-2 CDC Table of Organophosphate Pesticides and Their Dialkyl Phosphate 

Metabolites (2008)

Pesticide DMP DMTP DMDTP DEP DETP DEDTP 

Azinphos methyl | | |

Chlorethoxyphos | |

Chlorpyrifos | |

Chlorpyrifos methyl | |

Coumaphos | |

Dichlorvos (DDVP) |

Diazinon | |

Dicrotophos |

Dimethoate | | |

Disulfoton | | |

Ethion | | |

Fenitrothion | |

Fenthion | |

Isazaphos-methyl | |

Malathion | | |

Methidathion | | |

Methyl parathion | |

Naled |

Oxydemeton-methyl | |

Parathion | |

Phorate | | |

Phosmet | | |

Pirimiphos-methyl | |

Sulfotepp | |

Temephos | |

Terbufos | | |

Tetrachlorviphos |

Trichlorfon |

DMP = dimethylphosphate; DEP = diethylphosphate; DMTP = dimethylthiophosphate; DMDTP = 

dimethyldithiophosphate; DETP = diethylthiophosphate; DEDTP = diethyldithiophosphate.

The CHARGE study (Shelton et al., 2015) used a different method for exposure assessment. 

This study used geospatial analysis to focus on the residential proximity to OP exposure and the 

association of this exposure with autism spectrum disorders. OP exposure was assessed by 

Shelton et al (2015) using data from the California Department of Pesticide Regulation, with 

five OPs accounting for a total of 73% of the exposure and each accounting for 10% or more of 

the exposure (chlorpyrifos, acephate, diazinon, bensulide, and dimethoate); eight OPs 
Page 40 of 101

accounting for a total of 25% of the exposure and each accounting for 1% or more of the 

exposure (malathion, methyl parathion, azinphos-methyl, phosmet, oxydemeton-methyl, 

ethephon, naled, and methidathion); and eight OPs accounting for a total of 2% of the exposure 

and each accounting for 0.1% or more of the exposure (methamidophos, phorate, disulfoton, 

fenamiphos, coumaphos, parathion, ethoprop, and sulfotep).

2.2.4.3Neurological and Other Outcome Measures

With some exceptions, the outcomes assessed in this literature fall into three broad categories: 

1) neurobehavioral and/or neurodevelopmental status; 2) birth outcomes; and 3) 

neurodevelopmental diseases and/or disorders (see Table 2.2-b).

There is a broad body of literature available on the use and interpretation of instruments 

designed to quantify neurological status that is beyond the scope of this summary. Many 

instruments were used in this literature to assess infant neurodevelopment and neurobehavior 

(see Table 2.2-b). Importantly, performance on some of these tests can be influenced by the 

administrator of the assessment. Also of concern is whether these assessments are sensitive 

enough to distinguish potentially subtle effects of OP pesticide exposure. Some degree of error 

in the assessment of neurological outcomes is likely in all of the studies, though the errors were 

unlikely to be related to exposure status in the well conducted studies. 

The assessment of birth outcomes in this literature was primarily conducted by reviewing of 

medical records or birth certificates; these assessments are likely to have minimal errors, and 

errors that do arise are almost certainly non-differential with respect to exposure status. In 

some studies, however, birth outcomes were reported by the mother and in these instances, 

differential misclassification is possible.

The studies that evaluated specific diagnoses, autism spectrum disorders or developmental 

delay, for example, relied on existing medical records, with some effort to validate diagnoses in 

a subset of the investigations.

Studies that relied on standardized, validated instruments to assess neurodevelopment, 

medical records of birth outcomes, or validated diagnosis of disease states were weighted 

highly in the judgment of study quality. Those that used standardized instruments that had not 

been validated in the relevant population or screening assessments were given a moderate 

weight, while studies relying on selected sections of neurodevelopment assessment tools, 

maternal report of outcome status, or aggregated (ecological) outcome measures were given 

the lowest weight.

2.2.4.4 Statistical Analysis 

Statistical analyses that were appropriate to the study question and study design, supported by 

adequate sample size, maximized the use of available data, and were well characterized in the 

report were weighted most highly. Acceptable statistical methods, moderate study power, and 

analytic choices that resulted in the loss of information or that were not clearly reported were 
Page 41 of 101

given moderate weight. Reports with only minimal attention paid to the conduct and reporting 

of the statistical analyses were given the lowest weight. 

2.2.4.5 Confounding

Risk factors for early life neurodevelopment perturbations that are associated with OP pesticide 

exposure, but not caused by pesticide exposure, are potential confounders in this literature. 

Socioeconomic determinants of child development (including, for example, maternal education 

and access to prenatal and early life health care), quantity and quality of parent-child 

interactions, and exposure to other environmental toxicants (e.g., lead, PCBs, other pesticides) 

are difficult to measure and were either not accounted for, or inadequately accounted for in 

many studies. That said, some studies were relatively homogeneous with respect to these 

factors and thus limited confounding by design, while others attempted to quantify these 

factors and adjust for them. Other important potential confounders, such as the child’s sex, are 

easy to identify and adjust for analytically. 

2.2.4.6 Risk of Bias

The internal validity of the studies reviewed was judged by noting the design strategies and 

analytic methods used in each study to constrain or eliminate selection bias, information bias, 

and confounding. Selection bias occurs when the sampling of the population by the investigator 

yields a study population that is not representative of the exposure and outcome distributions 

in the population sampled. Put simply, selection bias occurs if selection of the study sample 

yields a different estimate of the measure of association than that which would have been 

obtained, had the entire target population been evaluated. Although there are numerous 

sources of selection bias, there are several mechanisms that may have induced selection bias in 

the studies reviewed: less than 100% participation rates of eligible individuals due to nonresponsiveness or refusal (self-selection bias); loss to follow-up (i.e. failure to retain all study 

participants initially enrolled in the study); and, in a case-control study, control selection bias 

arising because the exposure distribution in the control sample does not represent the 

exposure distribution of the study base (i.e., the population that gave rise to the cases or more 

formally, the person-time experience of that population).

Information bias (also referred to as observation bias) arises when study participants are 

incorrectly categorized with respect to their exposure or outcome status, or when errors arise 

in the measurement of exposure or outcome, in the case of continuously distributed measures. 

Epidemiologists often distinguish between two mechanisms or types of misclassification – those 

that are non-differential (or random) and those that are differential (non-random). Nondifferential misclassification of exposure (or non-differential exposure measurement error) 

occurs when the probability or magnitude of error in the classification or measurement of 

exposure is independent of the outcome status of the study participants. Similarly, nondifferential misclassification of outcome (or outcome measurement error) occurs when the 

probability or magnitude of error in the assignment of outcome status or level is independent 

of exposure status. In contrast, differential exposure misclassification (or measurement error) 

occurs when the error in the exposure assignment is not independent of the outcome status. 
Page 42 of 101

The mechanisms that cause non-differential misclassification in this literature include errors in 

the medical records, laboratory errors, sampling of biospecimens for biomarker assays, and 

errors in recall. The mechanisms that induce differential misclassification include recall bias, 

and interviewer/observer bias. Note that mismeasurement of confounders can result in 

residual confounding of the association of interest, even when adjustment for that confounder 

has been conducted in the analysis. 

Studies in which major sources of potential biases were not likely to be present, or in which 

potential sources of bias were present but effectively addressed and analyzed to maximize the 

study validity, and those in which sources of bias were unlikely to influence the magnitude and 

direction of the risk estimate were given a high weight. Studies where sources of bias were 

present and acknowledged by the authors but not addressed in the study and yet may influence 

the magnitude, but not direction of the association estimate received a moderate weight. A low 

weight was given to studies in which major biases were present and yet were not 

acknowledged or addressed in the study, such that they cannot be excluded as an alternative 

explanation for the study finding.

2.2.5 Review of Quality Results

Each of the studies in the 2015 review was judged to be of high, moderate, or low quality in 

each of five domains of study design and methodology effecting study quality as discussed 

above in Section 2.1. The results of the quality assessment are presented separately below for 

each group. The quality categories represent to the total evaluation. In Section 2.2.6, further 

evaluation of the study design and exposure assessment of the medium and high quality 

studies. This further evaluation led to additional studies being removed from the final analysis, 

with these excluded studies not being considered further in the remaining sections of this 

document.

2.2.5.1 “High” Quality Group

Six studies (8 articles) were assigned a high quality rating, as shown in Table 2.2.5-1. In general 

these were prospective birth cohort studies with moderate to high sample size; exposure 

assessment was based on an objective biomarker measure, the outcome measurement(s) 

utilized standardized tests and trained data collectors, appropriate statistical analyses were 

performed, considering relevant covariates, and risks of bias were minimized to the extent 

possible. For example, Fortenberry et al. (2014, Table 2.2.5-1) reported on findings in the 

ELEMENT study population of 187 mother-child pairs, assessed third trimester maternal urinary 

TCPy as a biomarker of prenatal chlorpyrifos exposure, and employed a trained and 

experienced research team to administer a battery of validated ADHD/psychometric 

assessments (Conner's’ Parental Rating Scales-Revised, Conner's’ Continuous Performance Test, 

and the Behavior Assessment System for Children-Parental Rating Scales) that had been 

translated into Spanish.
Page 43 of 101

Table 2.2.5-1. High Quality Studies: Summary of Study Design Elements Impacting Study Quality Assignment

Study Study Location

Cohort Name /

Description of Study 

Population Study Design

Exposure 

Assessment Outcome Assessment

Confounder /

Covariate Control Statistical Analysis Risk of (other) Bias

Barr et al. (2010) New Brunswick, New Jersey Mother who underwent elective 

Cesarean delivery at term and 

their newborns, Saint Peter’s 

University Hospital

Cross sectional birth 

cohort, modest sample 

numbers

N=150

Mother-infant pairs 

Objective measure, prenatal 

- parent CPF in maternal 

and cord serum at birth -

exposures do not 

necessarily precede 

outcome

Birth outcomes from medical records Generally appropriate; missing a few, 

e.g., maternal age, SES, nutrition, 

pre-natal care, race

Appropriate multivariate 

analysis

Convenience sample – deemed low 

probability of selection bias; low potential 

for exposure or outcome misclassification 

Bouchard et al. 

(2010)

U.S. National Population National Health and Nutrition 

Examination Survey (NHANES)

Cross-sectional, large 

sample size

N=1139

Age 8-15 years

Objective measure, 

postnatal – single non-OP 

specific child urinary DAPs

ADHD from DISC-IV diagnosis or 

medication in children 8-15 years old, 

standard protocol, trained 

interviewers

Appropriate: also included blood lead Appropriate, accounted for 

NHANES multistage 

probability sampling

Low probability of selection bias; some 

potential for non-differential 

misclassification of exposure

Fortenberry et al. 

(2014)

Mexico City, Mexico Early Life Exposures in Mexico 

to Environmental Toxicants 

(ELEMENT) study (3 cohorts 

1994-1997 (cohort 1), 1997-

2000 (cohort 2), and 2001-

2005 (cohort 3))

Prospective Birth Cohort 

Study – modest sample 

size

N=187 

Mother-infant pairs

Objective measure - TCPy 

concentration was 

measured in third trimester 

maternal urine samples. In 

a subset of women 

randomly selected urinary 

TCPy in samples collected 

during all three trimesters 

of pregnancy was 

measured. 

ADHD - LP - Conner's’ Parent Rating 

Scales-Revised (CPRS-R), Conner's’ 

Continuous Performance Test (CPT), 

and Behavior Assessment System for 

Children-Parental Rating Scales 

(BASC-PRS) – These are screening 

tools, not diagnostic tools. 

Standardization and quality control 

checks were conducted by reviews of 

videotaped evaluations.

Appropriate. Included continuous 

maternal IQ, education, 

socioeconomic status and blood lead 

one month after delivery, breast 

feeding (yes/no), child's sex, 

continuous age at testing, birth 

length and head circumference at 

birth.

Appropriate. Multivariable 

linear regression

History of maternal and paternal exposure 

to pesticides not included, use of a single 

urinary measure to estimate exposure, 

potential for differential exposure 

misclassification possible as mothers were 

likely aware of the neurobehavioral status 

of their children

Fortenberry et al. 

(2014a)

Mexico City, Mexico Early Life Exposures in Mexico 

to Environmental Toxicants 

(ELEMENT) study

Prospective Birth Cohort 

Study – large sample size

N=591 

Mother-infant pairs

Objective measure - blood 

samples from mother–child 

pairs were analyzed for 

PON1. OP exposure was 

not explicitly assessed. 

ADHD - LP - Conner's’ Parent Rating 

Scales-Revised (CPRS-R), Conner's’ 

Continuous Performance Test (CPT), 

and Behavior Assessment System for 

Children-Parental Rating Scales 

(BASC-PRS) - These are screening 

tools, not diagnostic tools.

Appropriate: Included continuous 

maternal IQ, education, 

socioeconomic status and blood lead 

one month after delivery, breast 

feeding (yes/no), child's sex, 

continuous age at testing, birth 

length and head circumference at 

birth.

Appropriate. Multivariable 

linear regression

Potential for differential exposure 

misclassification possible as mothers were 

likely aware of the neurobehavioral status 

of their children.

 

 
Page 44 of 101

Table 2.2.5-1. High Quality Studies: Summary of Study Design Elements Impacting Study Quality Assignment (continued)

Study

Study 

Location

Cohort Name / 

Description of 

Study Population Study Design Exposure Assessment Outcome Assessment

Confounder /

Covariate Contro Statistical Analysis Risk of (other) Bias

Quirós-Alcalá et al. (2011) Salinas Valley, 

California, USA

Center for the Health 

Assessment of Mothers and 

Children of Salinas 

(CHAMACOS)

Prospective Birth Cohort

Study Outcomes assessed 

at: 

N=149 (6 months) 

N=149 (1 year)

N=97 (3.5 years) 

N= 274 (5 years)

Objective biomarker, prenatal OP 

pesticide exposure (DAP) quantified 

in two maternal spot urine samples at 

14 and 27 weeks gestation (on 

average), and in single child urine 

sample provided at time of each 

outcome assessment

Autonomic nervous system 

dysregulation at ages 6 months, 1 

year, 3.5 years, and 5 years; 

standard protocol, trained, bilingual 

research staff, appropriate to age of 

child. May not be sensitive to resolve 

subtle effects of OP pesticide 

exposure.

Appropriate. Included maternal and 

child demographic and SES 

indicators (age, education).

Appropriate multivariate 

analysis

Selection bias possible due to 

considerable loss to follow-up; Residual 

confounding likely small in magnitude; 

some potential for non-differential 

misclassification of exposure (possible 

explanation for null findings). 

Rauch et al. (2012) Cincinnati, Ohio, 

USA

Health Outcomes and 

Measure of the 

Environment (HOME)

Prospective Birth Cohort 

Study – large sample size.

N=306

Age: newborns

Objective biomarker of prenatal OP 

pesticide exposure (DAP) quantified 

in two maternal spot urine samples 

provided at 16 and 26 weeks 

gestation 

Abstracted birth weight from medical 

records, calculated gestational age 

from mother’s self-reported date of 

last menstrual period. When 

gestational age not available, results 

of an ultrasound was used or a 

Ballard examination performed just 

after delivery.

Appropriate. Included income, 

education, maternal depressive 

symptoms, maternal IQ, insurance 

status, area of residence, prenatal 

care, PON1 genotype, and 

gestational exposure to alcohol, lead, 

and tobacco. 

Appropriate. multivariable 

regression

Unable to rule out the possibility that 

differences in DAP concentrations 

partially reflect individual variation in 

metabolism, Recall error

Wolff et al. (2007) New York City, U.S. Mount Sinai Children’s 

Environmental Health Study

Prospective Cohort –

moderate sample size

N=404

Mother-infant pairs

Objective biomarker, prenatal OP 

pesticide exposure (DAP), malathion 

(MDA) quantified in single maternal 

spot urine sample provided during 

the 3rd trimester

Birth outcomes from computerized 

perinatal database

Appropriate: different covariates for 

each outcome, e.g., weight: maternal 

age, race/ethnicity, maternal 

BMI*pregnancy weight gain, infant 

sex, gestational age, creatinine

Appropriate PROC GLM, 

with varying covariates

Selection bias unlikely; some potential 

for non-differential misclassification of 

exposure

Furlong et al. (2014) New York City, 

U.S.

Mount Sinai Children’s 

Environmental Health Study

Prospective Cohort

N=136

Age 7-9 years

Objective biomarker, prenatal OP 

pesticide exposure (DAP) quantified 

in single maternal spot urine sample 

provided during the 3rd trimester, 

simple imputation < lowest level of 

detection (LLOD)

Reciprocal social impairment at age 

7-9 years assessed using the Social 

Responsiveness Scale, completed by 

mothers. Designed to assess 

reciprocal social behaviors in 

evaluating ASDs, used here as 

general indicator of impaired social 

responsiveness.

Appropriate. Included maternal and 

child demographic and SES 

indicators (age, education) and ETS 

(not other environmental toxicants).

Appropriate multivariate 

analysis

Selection bias possible due to 

considerable loss to follow-up; Residual 

confounding likely small in magnitude; 

some potential for non-differential 

misclassification of exposure possible 

explanation for null findings). 
Page 45 of 101

2.2.5.2 “Moderate” Quality Group

Eleven studies (15 articles) were assigned a moderate quality rating, as shown in Table 2.2.5-2. 

In general, these were cross-sectional or prospective cohort studies with small to high sample 

size; exposure assessment was based on a non-specific biomarker measure or current selfreport, the outcome measurement(s) utilized standardized tests or screening tools, appropriate 

statistical analyses were performed, considering some but maybe not all relevant covariates, 

and risks of bias were minimized to some extent. For example, Guodong et al. (2012) crosssectionally evaluated the relationship between DAPs concentrations in urine sampled from 301

young children as an objective, non-specific marker of prenatal OP pesticide exposure and 

Developmental Quotients based on the Gesell Developmental Schedules adapted for a Chinese

population.
Page 46 of 101

Table 2.2.5-2. Moderate Quality Studies: Summary of Study Design Elements Impacting Study Quality Assignment

Study Study Location

Cohort Name /

Description of 

Study Population Study Design Exposure Assessment Outcome Assessment

Confounder /

Covariate Control Statistical Analysis Risk of (other) Bias

Andersen et al.(2015) Denmark School age children living 

in Denmark

Prospective Birth Cohort, 

small sample size

N=177 

(112 maternal 

occupational pesticide 

exposure during 

pregnancy; 65 without) 

Age: 6-11 years 

Prenatal occupational pesticide 

exposure ascertained by maternal 

interview at enrollment

Objective standardized clinical exam; 

neurophysiological status (heart rate 

variability); Previously validated, 

neuropsychological testing with 

demonstrated sensitivity to 

environmental pollutants; 

administered and scored by single 

neuropsychologist

Appropriate. Self-reported by mother. 

Included age, maternal demographics and 

risk factors, SES indicator (broad categories), 

maternal smoking and alcohol use. 

Possibility of recall errors (residual 

confounding). Self-reported (by mother)

Appropriate multivariate analysis. 

Evaluated numerous hypotheses 

without adjustment for multiple

comparisons. Also constructed 

structural equation model of 

interaction between child sex and 

prenatal pesticide exposure on 

general intellectual ability 

(parameterized as a latent variable)

Selection bias possible due to 

loss to follow-up; Residual 

confounding likely, small in 

magnitude; potential for nondifferential misclassification of 

exposure

Guodong et al. (2012) Shanghai, China 2-year old children 

visiting community 

hospitals

Cross-sectional, large 

sample size

N=301

Age 23-25 months

Objective biomarker, OP exposure 

(DAP) quantified in single child

spot urine sample, simple 

imputation <LLOD; OP exposure 

also assessed via questionnaire

administered to mothers after 

delivery

Developmental Quotients based on 

Gesell Developmental Schedules, 

adapted for Chinese population

Appropriate. Assessed via mother report via 

questionnaire, included child sex, maternal 

demographics and risk factors, SES 

indicators (maternal education, household 

income), maternal smoking and alcohol use; 

Possibility of recall errors

Appropriate multivariate analysis. 

Assumed linear relationship 

between log-transformed DAP level 

and DQ Scores

Selection bias unlikely; Residual 

confounding likely, small in 

magnitude; potential for nondifferential misclassification of 

exposure

Handal et al. (2007) Cayambe-Tabacundo 

region, Ecuador

EcoSalud Project: infants 

and young children 3 to 

61 months of age in 

lower-altitude 

communities A&B 

dominated largely by cutflower production and in 

more traditional rural, 

higher altitude 

community C.

Cross-sectional, modest 

sample size from census

N=283 

Age 3-61 months

- 154 high exposure

- 129 low exposure

Objective proxy measure: 

Community of residence

Ages and Stages Questionnaire (ASQ), 

adapted into local vernacular, 2 

trained testers – considered a 

screening tool

Appropriate: child health status (anemia, 

stunting) and other characteristics of the 

home environment (stimulation by 2 

methods)

Appropriate: Multiple linear 

regressions to evaluate associations 

between community of residence

and delayed development, Pairwise 

t-tests and chi-square to assess 

mean difference in ASQ score; 

Effect size Cohen's d

Selection bias unlikely; Residual 

confounding likely, small in 

magnitude; potential for nondifferential misclassification of 

exposure

Handal et al. (2008) Cayambe-Tabacundo 

region, Ecuador

EcoSalud Project: 

Children 3 to 23 months 

of age in lower-altitude 

communities A&B 

dominated largely by cutflower production and in 

more traditional rural, 

higher altitude 

community C.

Cross-sectional, modest 

sample size

N:121

Age 3-23 months

Proxy: Distance home to farm, 

parental employment, pesticide 

use on domestic crops & within 

home, child play activities

Ages and Stages Questionnaire (ASQ) 

(ages 24-61 months), a screening 

tool; Visual Motor Integration (VMI) 

Test (ages 48-61 months); two 

trained testers.

Appropriate: As above Appropriate: As above Selection bias unlikely; Residual 

confounding likely, small in 

magnitude; potential for nondifferential misclassification of 

exposure
Page 47 of 101

Table 2.2.5-2. Moderate Quality Studies: Summary of Study Design Elements Impacting Study Quality Assignment (continued)

Study Study Location

Cohort Name /

Description of 

Study Population Study Design Exposure Assessment Outcome Assessment

Confounder /

Covariate Control Statistical Analysis Risk of (other) Bias

Handal et al.(2007B) Cayambe-Tabacundo 

region, Ecuador

EcoSalud Project: Children 

24 to 61 months of age in 

lower-altitude communities

designated A & B were

dominated largely by cutflower production than in 

more traditional rural, 

higher altitude community 

C.

Cross-sectional, modest 

sample size

N:

ASQ - 142 

Age 24-61 months

VMI - 57 

Age 48-61 months

Proxy: Maternal employment during 

pregnancy, child plays outdoors, 

Pesticide use on domestic crops , 

inside home

Ages and Stages Questionnaire (ASQ) 

(3-23 months) – screening tool; Reachand-grasp, UC Berkeley Preferential 

Looking Test Cards; trained tester

Appropriate: As above Appropriate: As above;

Prehension- Logistic regression 

models, generalized estimation 

equations (GEE) to account for 

between-test dependence

Selection bias unlikely; Residual 

confounding likely, small in 

magnitude; potential for nondifferential misclassification of 

exposure

Llop et al. (2013) Spain INMA (Environment and 

Childhood) Project

Prospective Birth Cohort, 

large sample size

N=1980

Age 14 months

Maternal self-report of prenatal and 

postnatal indoor pesticide use 

(pesticide spray or use of a plug-in 

device assessed via questionnaire)

Mental and psychomotor development 

at 14 months assessed using validated 

instrument (Bayley Scales of Infant 

Development) 

Appropriate. Self-reported by mother. Included 

maternal demographics and risk factors (Age, 

BMI) SES indicators (maternal education, 

occupation), maternal smoking and alcohol use. 

Also childcare behaviors (breast feeding, 

number of siblings, day care) Possibility of recall 

errors (residual confounding)

Appropriate multivariate analysis Selection bias unlikely; Residual 

confounding likely, small in 

magnitude; potential for nondifferential misclassification of 

exposure

Oulhote and Bouchard (2013) Canadian National 

Population

Canadian Health Measures 

Survey (CHMS; cycle 1, 

2007–2009);

Cross-sectional Study, 

large national sample size

n = 779 children

Age 6-11 years

Child urinary DAP, DMP, DEP

collected within 2 weeks of survey 

questionnaire completion by the 

parents.

Behavioral problems in children based 

on the parent version of the Strengths 

and Difficulties Questionnaire (SDQ) 

(Goodman 1997) - SDQ is a validated 

screening questionnaire and accepted 

by parents. 

Appropriate: Included sex, age, race/ethnicity, 

family income, parental education, blood lead 

levels, maternal smoking during pregnancy, 

birth weight, maternal age at child's birth, child's 

BMI, and fasting status (fasting duration at 

urine collection > 10 hour/≤ 10 hour)

Appropriate: Logistic Regression Selection bias unlikely; Study 

population not mixed, single urine 

sample 

Petit et al. (2010) Brittany, France Newborn children in the 

PELAGIE Study.

Prospective Birth Cohort, 

large sample size

N= 3,159 

Age: newborns

Ecological, proxy indicator of 

exposure (proportion of municipality 

devoted to agricultural activity

Objectively measured birth outcomes 

assessed using hospital records

Appropriate. Maternal report via questionnaire. 

Included maternal demographics (age, BMI) and 

pregnancy risk factors (gestational age, 

hypertension, diabetes, season of pregnancy) 

SES indicators (district of residence, maternal 

education, occupation), maternal smoking and 

alcohol use. Also childcare behaviors (breast 

feeding, number of siblings, day care); 

Possibility of recall errors

Appropriate multivariate analysis. 

Multiple comparisons were conducted 

for this evaluation, and the authors did 

not adjust for multiple testing

Selection bias unlikely; Residual 

confounding likely, small in 

magnitude; potential for 

substantial non-differential 

misclassification of exposure; 

Misclassification of outcomes 

unlikely
Page 48 of 101

Table 2.2.5-2. Moderate Quality Studies: Summary of Study Design Elements Impacting Study Quality Assignment (continued)

Study Study Location

Cohort Name /

Description of 

Study Population Study Design Exposure Assessment Outcome Assessment

Confounder /

Covariate Control Statistical Analysis Risk of (other) Bias

Ruckart et al. (2004) Mississippi, USA (29 

counties), Ohio (one 

multi-family facility)

Children who were 6 years 

or younger in MS and OH 

when homes sprayed with 

MP

Retrospective cohort, moderate 

sample size in 2 states, 

participating/ nonparticipating 

similar in sex and age

MS: N=365 (147 exposed, 218 

unexposed)

OH: N=287 (104 exposed, 183 

unexposed)

Age: 1.9-12.5 years old at 

testing

Specific OP measures: Household 

methyl parathion (wipe) by approved 

labs, OR highest urinary PNP level in 

household, analyzed by CDC

Variety of neuro measures: PENTB, 

Parenting Stress Index (PSI), Personality 

Inventory for Children (PIC), Vineland 

Adaptive Behavior Scales (VABS)

Appropriate: Income, race, site term, ethnicity, 

mother’s use of chemicals at work, mother 

health/pregnancy conditions, report that child 

had lead or mercury poisoning (MS only); Raw 

scores were child age-adjusted

Appropriate: Linear regression 

(continuous scores), logistic 

regression (dichotomous scores)

Selection bias possible due to loss 

of follow-up; Residual confounding 

likely; substantial potential for 

differential exposure 

misclassification (time between 

spraying and testing, frequency 

and duration unknown)

Shelton et al. (2014) California, USA CHARGE Study (3 year-old 

children)

Case-control, large sample size

N = 970

Age 3 years

N=486 (ASD)

N=168 (DD)

N=316 (Controls)

Proxy indicator of prenatal OP pesticide 

exposure (residential proximity to 

agricultural pesticide applications 

defined using ecological pesticide use 

data)

Clinical outcomes – validated within the 

study

Appropriate adjustment for demographics 

(place of birth, race), SES indicators (paternal 

education) and vitamin intake during pregnancy

Appropriate multivariate analysis. Selection bias probable; Residual 

confounding likely; substantial 

potential for differential 

misclassification of exposure. 

Outcome misclassification 

unlikely.

Suarez-Lopez et al. 

(2012)

Pedro Moncayo County, 

Pichincha, Ecuador

Secondary Exposure to 

Pesticides Among Infants, 

Children and Adolescents 

(ESPINA) Study, plus new 

volunteers

Cross-sectional, moderate 

sample size, from prior census

N=277

Exposed: (n=158)

Unexposed: (n=119)

Age 4-9 years

Proxy: Cohabitation with flower worker 

>1yr, Adult questionnaire

Child AChE level using commercial kit Appropriate: Sex, age, height-for-age, 

hemoglobin concentration, income, pesticide 

use within household lot, pesticide use by 

neighbors, examination date, residence, 

distance to nearest flower plantation

Appropriate: Multiple linear regression 

(continuous) and logistic regression 

(polychotomous variables), adjusted 

models

Selection bias possible due to loss 

but deemed low; Residual 

confounding likely small in 

magnitude; potential for 

differential exposure 

misclassification

Suarez-Lopez et al. 

(2013a)

Pedro Moncayo County, 

Pichincha, Ecuador

Secondary Exposure to 

Pesticides Among Infants, 

Children and Adolescents 

(ESPINA) Study, plus new 

volunteers

Cross-sectional, moderate 

sample size, from prior census

N=307

Age 4-9 years

Non-specific measures: 

Child AChE level using commercial kit; 

Proxy: Cohabitation with flower worker 

>1yr, Adult questionnaire

Heart rate (HR), Systolic blood pressure 

(SBP), Diastolic blood pressure (DBP) by 

standard methods

Appropriate: Sex, age, height-for-age, 

hemoglobin concentration, income, pesticide 

use within household lot, pesticide use by 

neighbors, examination date, residence, 

distance to nearest flower plantation

Appropriate: Multiple linear regression 

(continuous), adjusted.

Selection bias possible due to loss 

but deemed low; Residual 

confounding likely small in 

magnitude; potential for 

differential exposure 

misclassification

Suarez-Lopez et al. 

(2013)

Pedro Moncayo County, 

Pichincha, Ecuador

Secondary Exposure to 

Pesticides Among Infants, 

Children and Adolescents 

(ESPINA) Study, plus new 

volunteers

Cross-sectional, moderate 

sample size, from prior census

N=271

Age 4-9 years

Non-specific measures: 

Child AChE level using commercial kit

NEPSY-II test (trained examiners): 

general assessment battery

Model defined a priori: hemoglobin, age, sex, 

race, height-for-age z score, household income, 

maternal education, and flower worker 

cohabitation status

Logistic models (dichotomous and 

polychotomous) and linear regression 

models, adjusted; Effect modification 

according to sex among significant 

associations; Some imputed values

Selection bias possible due to loss 

but deemed low; Residual 

confounding likely small in 

magnitude; potential for 

differential exposure 

misclassification

Wang et al. (2012) Shanghai, China Pregnant women and 

newborn children

Cross-sectional Birth Cohort 

Study

N=187

Age: newborns

Objective biomarker of prenatal OP 

pesticide exposure (DAP) in single 

maternal spot urine sample provided at 

onset of labor. Simple imputation of 

observations < LLOD. OP pesticide 

exposure also assessed via 

questionnaire administered after 

delivery

Gestational age and pre-term delivery 

appropriately defined and assessed 

using medical records

Appropriate. Included maternal 

anthropomorphic, demographic, and SES 

indicators (income, occupation), predictors of 

high-risk pregnancy (pregnancy weight gain, 

gestational age)

Appropriate multivariate analysis Selection bias unlikely; Residual 

confounding likely small in 

magnitude; potential for 

differential misclassification of 

exposure

Zhang et al. (2014) Shenyang, China Newborn children in a 

birth cohort study

Prospective Cohort, n=249 

mother-infant pairs

Age: newborns

Biomarker of prenatal OP pesticide 

exposure (DAP) quantified in single 

maternal spot urine sample provided at 

delivery. Many observations < LLOD

Neonatal neurodevelopment assessed 

using validated instrument (Neonatal 

Behavioral Neurological Assessment) by 

trained examiners

Appropriate. Included maternal demographic 

and SES indicators (age, education), predictors 

of high-risk pregnancy (BMI, gestational age) 

and environmental toxicant exposure (cord 

blood lead); Results stratified by sex

Appropriate multivariate analysis Selection bias unlikely; Residual 

confounding likely small in 

magnitude; considerable potential 

for non-differential 

misclassification of exposure 

(unlikely to account for non-null 

findings)
Page 49 of 101

2.2.5.3 “Low” Quality Group

Fourteen studies (15 articles) were assigned a low quality rating, as shown in Appendix 3. In 

general these were small or pilot studies; exposure assessment was based on a proxy 

measure(s), outcome measurement(s) utilized screening tools or self-report, limited statistical 

analyses were performed, relevant covariates were not included or discussed, or risks of bias 

were possible. For example, Acosta-Maldonado et al. (2009) conducted a cross-sectional pilot 

study among 54 women, only nine of whom were considered to have had prenatal exposure to 

pesticides (defined based on either an exposure history profile or AChE level in blood sampled 

at the time of admission into the hospital for delivery. The outcome assessed in this study was a 

standardized but partially subjective assessment of placental maturity (the Placental Maturity 

Index, PMI). Covariates adjustment of estimated associations between prenatal exposure and 

PMI in this study was minimal and limited to placental characteristics.

The remaining sections of this document do not discuss further studies identified in the ‘low’ 

category. Due to limitations in these studies, they do not provide reliable information 

evaluating associations between OP exposure and neurodevelopmental outcomes.

2.2.6 Assessment of Epidemiological Studies for Relevance to Analysis

Using the criteria summarized in Section 2.2.4, a total of 38 literature articles were identified in 

the 2015 literature review and were judged as high, moderate, or low quality. Overall, 8 

articles, 15 articles, and 15 articles were judged to be of high, moderate, or low quality, 

respectively. For the 23 high and moderate quality studies, additional evaluation was 

conducted as described in this section.

While all of the moderate quality studies had strengths including sample design and outcome 

assessment, six of these moderate quality studies did not have sufficient exposure assessment 

methods to determine whether exposure to OPs 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 are excluded from further analysis.

The focus of this epidemiological literature review is on the neurodevelopmental outcomes 

from exposures to low levels of OPs (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
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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 review and are not discussed further here.

In a retrospective cohort study of children exposed to methyl parathion (MP) before age 6 years 

in Mississippi and Ohio (Ruckart et al. 2004), as assessed by household urinary PNP or wipe 

samples, exposed children performed worse than unexposed children on a few of numerous 

neurobehavioral development tests conducted. Specifically, participants classified as having 

had MP exposure had more difficulty with short term memory and attention tasks, and parents 

reported more behavioral and motor skill problems, relative to unexposed children. However, 

upon closer inspection of the results across the MS and OH study sites, these neurobehavioral 

outcomes are not seen consistently. These inconsistencies may be due to differences in how 

the exposure occurred across the sites, including the fact that MS participants and OH 

participants were tested 2.5 and 4.5 years after MP spraying in the home. When comparing 

exposed and unexposed children using general intelligence testing, integrated visual and motor 

skills testing, and multistep processing, they did not see any differences. The exposure scenario 

associated with these observations is a critical element in assessing the utility and reliability of 

this study. Samples were collected in locations from OH and MS where illegal spraying of 

methyl parathion is known to have occurred during the 1994-1996 time period. Based on the 

“Revised Organophosphorous Pesticide Cumulative Risk Assessment” (USEPA, 2006), methyl 

parathion is known to be among the more potent OPs. It is unknown whether study 

participants were exposed to MP levels that would have induced cholinergic effects. Therefore, 

given the uncertainty around this illegal use combined with the high potency for cholinergic 

toxicity, the agency is not emphasizing this study further in the analysis.

In addition, one high quality 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, this 

study did not focus on neurodevelopmental outcomes, which is the focus of this analysis. 

Consequently, this Quirós-Alcalá et al. 2011 study is not being evaluated further.

2.2.7 Birth Outcome Epidemiologic Studies

Four identified studies, three high quality and one moderate quality, focused solely on OP 

exposure and adverse birth outcomes related to fetal growth (Barr et al., 2010; Rauch et al., 

2012; Wang et al., 2012; Wolff et al., 2007). The birth outcomes assessed included birth 

weight, birth length, head circumference at birth, and gestational age. The exposure 

assessment for these studies was conducted using objective measures or biomarkers such as 

maternal urinary DAPs (Rauch et al., 2012; Wang et al., 2012; Wolff et al., 2007), maternal 
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urinary malathion (MDA) (Wolff et al., 2007), or maternal and cord blood levels for specific OPs 

(Barr et al., 2010). The results from these studies are generally inconsistent, with some studies 

documenting statistically significant associations between OP exposure and birth outcomes 

whereas others do not. For example, maternal and cord blood serum levels of TCPy were 

inversely associated in univariate analyses conducted among participants in a prospective 

cohort study conducted in New Jersey (Barr et al. 2010), though the associations did not persist 

after adjusting for gravidity, maternal pre-pregnancy BMI, infant sex, and gestational age. 

Chlorpyrifos levels were near the lower limit of detection in this study, though detectable in 

98.6% of maternal serum and 62.8% of cord serum samples. Overall, birth length was not 

associated with third-trimester DAP in the Mount Sinai Cohort (Wolff et al. 2007). However, 

among those with slow-activity paraoxonase-1 (PON1) or PON192, urinary total DMP (but not 

total DAP or DEP) was statistically significantly associated with shorter birth length (p= 0.032). 

Birth length was also not associated with maternal urinary DAP sampled at delivery in the crosssectional investigation conducted in Shanghai, China (Wang et al. 2012). Similarly inconsistent 

results were observed across these studies for birth weight, gestational age, and head 

circumference at birth.

Overall, in this 2015 literature review, inconsistent evidence of OP exposure and association 

with adverse birth outcomes/fetal growth was observed (Barr et al., 2010; Rauch et al., 2012; 

Wang et al., 2012; Wolff et al., 2007). This lack of consistency in the literature was also 

observed for birth outcomes in the recent chlorpyrifos HHRA (USEPA, 2014) which notes that 

researchers from the three US birth cohort studies also investigated the possible role of 

prenatal OP exposure and fetal growth. These results were not consistent across these cohorts. 

Authors with Columbia Center for Children’s Environmental Health (CCCEH) Mothers and 

Newborn Study observed evidence of an inverse association, i.e., increasing cord blood 

chlorpyrifos was associated with decreased measures of birth weight and length, while authors 

with the Mt. Sinai and CHAMACOS cohorts reported either no association, or evidence of a 

positive relationship, respectively (Berkowitz et al., 2004; Eskenazi et al., 2004; Whyatt et al., 

2004). 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 evaluations for 

chlorpyrifos, EPA is focusing the remainder of this document 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.

2.2.8 Summary of Epidemiology Studies from 2015 Literature Review Focusing on 

Neurodevelopmental Outcomes

From an initial total of 23 high or moderate quality studies, with the exclusion of six studies for 

insufficient exposure assessment, four studies with measurable AChE inhibition and potential 

cholinergic toxicity which are outside the scope of this analysis, one study from an illegal use of 

a highly potent OP (MP) where cholinergic toxicity cannot be ruled out, four studies with birth 
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outcome as the only assessment, and one study assessing only PON1 genotype expression and 

neurobehavioral outcomes (Fortenberry et al, 2014a), a total of seven studies focusing on 

neurodevelopmental outcomes remain to be evaluated.

Seven total studies focused on OP exposure and either neurodevelopmental or neurobehavioral 

outcomes, with three high quality (Bouchard et al., 2010; Fortenberry et al., 2014; Furlong et 

al., 2014) and four moderate quality studies (Guodong et al., 2012; Oulhote and Bouchard,

2013; Zhang et al., 2014; Shelton et al., 2014). The results of these studies are summarized 

below. With the exception of Shelton et al. (2014), all of these studies used biomarker

measures for their exposure assessment, including child or maternal urinary DAPs (Bouchard et 

al., 2010; Furlong et al., 2014; Guodong et al., 2012; Oulhote and Bouchard, 2013; Zhang et al., 

2014), child urinary DMP and DEPs (Oulhote and Bouchard, 2013; Zhang et al., 2014; Guodong 

et al., 2012), or maternal TCPy (Fortenberry et al., 2014). Shelton et al. (2014) used pesticide 

use data from the California Department of Pesticide Regulation and geospatial methods to 

map the specific pesticide use pattern to the participant residence. The study populations 

ranged from national in scope (Bouchard et al., 2010; Oulhote and Bouchard, 2013) to mainly 

urban (Fortenberry et al., 2014; Fortenberry et al., 2014a; Furlong et al., 2014; Guodong et al., 

2012; Zhang et al., 2014; Shelton et al., 2014).

In order to summarize the results from these studies, we have separated them into three 

groups based on the outcomes assessed — two Chinese studies focusing on more generic 

neurodevelopmental outcomes (Zhang et al., 2014; Guodong et al., 2012); two studies focusing 

on social responsiveness outcomes or autism spectrum disorders (Furlong et al., 2014; Shelton 

et al., 2014); and three studies focused on ADHD, behavioral problems, and attention problems 

(Oulhote and Bouchard, 2013; Bouchard et al., 2010; Fortenberry et al., 2014).

In the first of two Chinese studies focusing on generic neurodevelopmental outcomes, Zhang et 

al. (2014) investigated prenatal exposure to OPs and neurobehavioral development of neonates 

in a birth cohort study in Shenyang, China. The authors reported that consistent statistically 

significant associations were observed between all of the quantified urinary biomarkers of 

prenatal OP pesticide exposure and neonatal neurodevelopment deficits assessed 3 days after 

birth. A 10-fold increase in total DAPs concentration was associated with an average decrease 

in Neonatal Behavioral Neurological Assessment (NBNA) summary scores of 1.78 points (95% 

CI: -2.12 to -1.45). No evidence of departure from linearity of the exposure-response 

relationship between maternal DAP concentrations and NBNA scores was observed. In the 

second Chinese study focusing on neurodevelopmental outcomes, Guodong et al. (2012) 

conducted a cross-sectional study, and did not identify any statistically significant associations 

between the children’s urinary DAP metabolite levels and any of the DQ (Developmental 

quotients) scores. The authors mentioned that their results should be interpreted with caution

since OP exposure was quantified in single spot urine sample from children, and should be 

followed up with a longitudinal study with repeated measurement of exposure levels in urine 

samples.
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Two studies focused on impaired social responsiveness, autism spectrum disorders, or 

developmental delays (Furlong et al., 2014; Shelton et al., 2014). 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 (a measure linked to many neuropsychiatric conditions that involve 

impaired social functioning (Constantino and Gruber 2005)). 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 DEP and poorer social responsiveness observed among black participants 

and boys. No association was observed among whites or Hispanics, among girls, or for DAP or 

DMP biomarker levels.

Shelton et al. (2014), investigated autism spectrum disorders (ASD) and developmental delay 

(DD) in relation to gestational residential proximity to agricultural pesticide applications utilizing 

the California population-based Childhood Autism Risks from Genetics and Environment

(CHARGE) study. The investigators reported that children with ASD were 60% more likely to 

have OPs applied near the home [1.25 km distance; adjusted OR (aOR) = 1.60; 95% CI: 1.02–

2.51] than mothers of normally developing children. They added that as the buffer distance 

grew larger, these associations became lesser, indicating an exposure-response effect. The 

authors also mentioned that each 100-lb (45.4 kg) increase in the amount applied over the 

course of pregnancy (within 1.5 km of the home) was associated with a 14% higher prevalence 

of ASD (aOR = 1.14; 95% CI: 1.0, 1.32), but no association was identified with DD.

A total of three studies focused on OP exposure and behavioral, memory, or attention/ADHD 

outcomes (Oulhote and Bouchard, 2013; Bouchard et al., 2010; Fortenberry et al., 2014). In a 

national cross-sectional study of Canadian children 2007-2009 data for age 6-11 years (Oulhote 

and Bouchard, 2013), there were no overall statistically significant associations observed 

between child urinary DAP, DMP, or DEP metabolite 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),5 observed a positive 

association between attention and behavior problems and DAPs and DMPs, but not DEPs. For 

example, even after controlling for potential confounders such as sex, age, ethnicity, and 

creatinine concentration, they found that a 10-fold increase in DMAP concentration was 

associated with a 55 to 72% increased odds of ADHD.

Fortenberry et al. (2014) evaluated the relationship between pesticide exposure and ADHD in 

school age Mexican children, recruiting 187 mother-child pairs from a prospective birth cohort, 

ELEMENT (Early Life Exposures in Mexico to Environmental Toxicants). The authors reported 

that, there were no statistically significant associations between tertiles of maternal third 

trimester urinary TCPy and measures of attention and hyperactivity in children. However, there 

was suggestive evidence for increases in the ADHD index in relation to TCPy tertiles among boys 

 

5 http://www.cdc.gov/nchs/nhanes.htm
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(the highest TCPy tertile was associated with an ADHD index score that was 5.55 points higher 

than children in the lowest tertile; p-value = 0.06). 

2.2.9 Summary of Three US Children’s Cohort Studies (CCCEH, CHAMACOS, Mt. Sinai):

Focusing on Neurodevelopmental Outcomes Evaluated in 2012/2014

Detailed summaries and evaluation, associated strengths and limitations, and accompanying 

detailed evidence table for the CCCEH, CHAMACOS, and Mt. Sinai cohorts can be found in the 

white paper for the 2012 SAP review and the 2014 chlorpyrifos revised HHRA. Limited 

summary information is provided here for comparison with the studies identified in the 2015 

literature review discussed above.

2.2.9.1Overview

In the chlorpyrifos revised HHRA, EPA included epidemiologic research results from three 

prospective birth cohort studies. These include: 1) The Mothers and Newborn Study of North 

Manhattan and South Bronx performed by the Columbia Children’s Center for Environmental 

Health (CCCEH) at Columbia University; 2) the Mt. Sinai Inner-City Toxicants, Child Growth and 

Development Study or the “Mt. Sinai Child Growth and Development Study;” and 3) the Center 

for Health Assessment of Mothers and Children of Salinas Valley (CHAMACOS) conducted by 

researchers at University of California Berkeley. In these epidemiology studies, mother-infant 

pairs were recruited for the purpose of studying the potential health effects of environmental 

exposures during pregnancy on subsequent child development. Importantly, each of these 

cohorts evaluated the association between prenatal chlorpyrifos or OP exposure with adverse 

neurodevelopmental outcomes in children through age 7 years.

These studies reflect different types of exposed groups in the total population which 

strengthens the weight of the evidence considerations regarding this stream of information. 

The CCCEH Mother’s and Newborn study 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. 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 and 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). 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 
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studies, and believes that random or systematic errors in the design, conduct or analysis of 

these studies were unlikely to fully explain observed positive associations between in utero OP 

exposure and adverse neurodevelopmental effects observed at birth and through childhood 

(age 7 years). EPA believes these are strong studies which support a conclusion that OPs likely 

played a role in these outcomes.

2.2.9.2 Review of Study Design and Research Methods

These 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. 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 potentially 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 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.

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 

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 that could be related to OP exposure and 

neurodevelopmental outcomes such as blood lead, may result in an incorrect epidemiological 

risk estimate. Across these cohort studies, investigators collected relevant information 
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concerning demographic characteristics and other environmental exposures, and were, to the 

extent possible with the existing information, able to effectively hold constant the influence of 

these other variables when estimating the association between prenatal chlorpyrifos and 

adverse neurodevelopmental outcomes. Control of these variables is important to reduce the 

chances of a false positive 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.

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. 

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 
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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).

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.

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).

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 
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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 

perform a written peer-review of this study.6 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.

In sum, across these three children’s environmental health studies, 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.

 

6 http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPP-2008-0850-0170. 
Page 59 of 101

Table 2.2.9 Detailed Summary Tables of Children’s Environmental Health Epidemiology Studies (extracted from USEPA, 2014)

Author, Year, 

Sample Size

Outcome 

Assessed

Exposure 

Measurement

Potential confounders 

considered

Primary Result Conclusions/Uncertainties Strengths and Weaknesses 

Article 1: Whyatt 

et al. (2004) 

Columbia U. 

(N=314)

Birth length, Birth 

weight, head 

circumference

4 cord plasma 

chlorpyrifos 

exposure groups 

and 4 chlorpyrifos 

and diazinon 

exposure groups. 

Chlorpyrifos only 

categories, Group 

1: levels below 

LOD (32% of 

participants); 

Group 2: lowest 

1/3 of detectable 

levels (20 %); 

Group 3: middle 

1/3 (24%), Group 

4: highest 1/3 

(25%.) 

Chlorpyrifos and 

diazinon together: 

Group 1: 26%, 

Group 2: 22 %, 

Group 3: 26%, 

Group 4: 26%.

Gestational age, maternal prepregnancy weight, maternal net 

pregnancy weight gain, gender 

of newborn, parity, 

race/ethnicity, ETS in home, 

season, cesarean section

For each log unit increase 

in cord plasma chlorpyrifos 

levels, birth weight 

decreased by 42.6 g (95% 

CI: –81.8 to –3.8) and birth 

length decreased by 0.24 

cm (95% CI: –0.47 to –

0.01). Birth weight 

averaged 186.3 g less (95% 

CI: –375.2 to –45.5) among 

newborns with the highest 

compared with lowest 26% 

of exposure levels (p = 

0.01).

Associations between birth 

weight and length and cord 

plasma chlorpyrifos were 

statistically significant (p ≤ 

0.007) among newborns 

born before the January 

2001 policy change. Among 

newborns born after 

January 2001, exposure 

levels were substantially 

lower, and no associations 

with fetal growth outcomes 

were observed (p > 0.8). 

Strengths: prospective 

nature of the study; direct 

measurement of 

chlorpyrifos in cord blood 

and personal air samples, 

rather than non-specific 

markers of 

organophosphate pesticide 

exposure; consideration of 

other pesticides and 

environmental 

contaminants as covariates 

in the multivariate models. 

Limitations: single exposure 

sampling period; the 

authors did not present nor 

discuss regression 

diagnostics to assess the 

degree to which their 

models met or violated the 

assumptions implicit in 

linear models. 

Article 2: 

Berkowitz et 

al.(2004) 

Mt. Sinai (N=404)

Birth length, birth 

weight, head 

circumference, 

gestational age

LOD: 11 ug/L 

(57% <LOD TCPy)

Race/ethnicity, infant sex, and 

gestational age. The authors also 

controlled for birth weight or 

birth length in their assessment 

of head circumference and 

pesticide exposure.

Mean levels of birth 

weight, length, head 

circumference, and 

gestational age did not 

differ between those with 

urinary pesticide 

metabolite levels below 

and above the level of 

detection.

Similarly, no statistically 

significant associations 

were observed between 

reported pesticide 

exposure and mean indices

of fetal growth and 

PON1 activity also predictor 

of smaller head 

circumference; creatinine 

corrected

Very well conducted study 

with numerous strengths 

and very few weaknesses. 

The questionnaire-based 

pesticide exposure 

questions are subject to 

imperfect recall. Errors 

would, on average, 

attenuate associations 

between these exposure 

metrics and fetal 

development. 

Recall-based exposure 

assessments were fortified 
Page 60 of 101

Author, Year, 

Sample Size

Outcome 

Assessed

Exposure 

Measurement

Potential confounders 

considered

Primary Result Conclusions/Uncertainties Strengths and Weaknesses 

gestational age. by objective measures of 

pesticides/pesticide 

metabolites.

A metabolite specific for 

chlorpyrifos (TCPy) was 

assessed. 

Statistical analysis was 

appropriate. 

Observed mean reductions 

in the outcome parameters 

appear to be small in 

magnitude and may be of 

little clinical significance. 

Assessment and control for 

confounding were 

appropriate. However, 

confounding by 

unmeasured (and 

mismeasured) risk factors 

for abnormal growth that 

are related to pesticide 

exposure would bias the 

results in this study. Such 

factors may be related to 

socioeconomic status of 

the study participants, a 

cofactor which is difficult to 

define, no less measure in 

an epidemiologic study.

Limited external validity 

(generalizability) due to the 

particular study population 

recruited and the 

numerous exclusion criteria 

applied.

Article 3: Eskenazi 

et al. (2004) 

CHAMACOS 

Birth length, birth 

weight, head 

circumference, 

Total DAPs: 

median 136 

nmol/L (range: 

Gestational age, gestational age 

squared, maternal age, 

pregnancy weight gain, week of 

Decreases in gestational 

duration associated with 

two measures of in utero 

Maternal urine collection 

averaged weeks 14, 26, not 

creatinine-corrected

Strengths in the study 

design include the 

longitudinal design, the use 
Page 61 of 101

Author, Year, 

Sample Size

Outcome 

Assessed

Exposure 

Measurement

Potential confounders 

considered

Primary Result Conclusions/Uncertainties Strengths and Weaknesses 

(N=488) Gestation Length, 

Ponderal index

10–6,854); DEP: 

median 22 nmol/L 

(range: 2–680 

nmol/L); TCPy: 

median 3.3 

nmol/L (range: 

0.2–56.1nmol/L) 

(76% >LOD)

initiating prenatal care, parity, 

infant sex, mother's country of 

birth, body mass index, family 

income, poverty level, smoking, 

alcohol, illicit drug use, 

environmental tobacco smoke, 

caffeine, history of low birth 

weight, and history of pre-term 

delivery.

pesticide exposure: levels 

of metabolites of dimethyl 

phosphate pesticide 

compounds and whole 

blood ChE.

of multiple exposure 

biomarkers, including 

quantification of nonspecific (DAPs), 

chlorpyrifos-specific (TCPy) 

metabolites, and other 

environmental coexposures. A reasonable 

set of exclusion criteria 

were applied.The selection 

of the CHAMACOS 

population, which consists 

mostly of children from 

low-income families, 

served to increase the 

relative statistical efficiency 

of the study, as this 

population is at high risk of 

neurodevelopmental 

deficits, compared to the 

general population.The 

statistical analysis used to 

assess the associations 

between the markers of 

exposure and 

neurodevelopment were 

appropriate.Errors in the 

assignment of exposure in 

this prospective study will 

likely have resulted in 

attenuation of observed 

associations.

Article 4: Harley et 

al. (2011) 

CHAMACOS 

(N=329)

Birth length, birth 

weight, head 

circumference, 

gestational age 

The geometric 

mean for the DAP 

concentrations 

during pregnancy 

(for the average 

of the two 

sampling periods) 

was 146 nmol/L 

(95% CI: 133, 

160); of this, a 

larger proportion 

Maternal intelligence (Peabody 

Picture Vocabulary Test (PPVT)), 

measures of how stimulating the 

environment is, and known or 

suspected neurotoxins were 

measured prenatally. To 

measure the quality and extent 

of stimulation available to a 

child in the home environment, 

the Infant-Toddler HOME (Home 

Observation for Measurement 

The authors observed 

evidence of an association 

between prenatal exposure 

to OP pesticides as 

measured by urinary DAP 

metabolites in women 

during pregnancy, is 

associated with decreased 

cognitive functioning in 

children at age 7.

Infants whose PON1 

genotype and enzyme 

activity levels suggested 

that they might be more 

susceptible tothe effects of 

OP pesticide exposure had 

decreased fetal growth and 

length of gestation. PON1 

may be a contributing 

factor to preterm or low 

birth weight birth.

This study has many 

strengths, the longitudinal 

design, the measurement 

of urinary DAP at multiple 

time points and following 

children to age seven when 

tests of cognitive function 

are reportedly more 

reliable. The authors were 

able to adjust for or 

consider many factors 
Page 62 of 101

Author, Year, 

Sample Size

Outcome 

Assessed

Exposure 

Measurement

Potential confounders 

considered

Primary Result Conclusions/Uncertainties Strengths and Weaknesses 

was DMP 

metabolites (GM 

= 109 nmol/L; 

95% CI =98, 120) 

than DEPs (GM= 

23 nmol/L; 95% CI 

= 21, 25). Allele 

frequencies: 

PON1 192 Q 

allele= 50%; PON1 

-108 T allele= 

46%.Mean 

arylesterase 

activity: For 

infants: 33.6 

U/mL (SD = 16) 

For mothers: 

136.6 U/mL (SD = 

44). Mean

paraoxonase 

activity: For 

infants: 256.6 U/L 

(SD = 165); For 

mothers: 989.0 

U/L (SD = 616).

of the Environment) inventory 

was completed at the 6-month, 

1, 2, 3.5, 5, and 7 year visits;

known or suspected 

neurotoxicants, polybrominated 

diphenyl ethers (PBDEs), 

polychlorinated biphenyls 

(PCBs), p,p’-

dichlorodiphenyltrichlorethane 

(DDT), p,p’-

dichlorodiphenyltrichlorethylene 

(DDE), and lead. 

related to cognitive 

function, such as prenatal 

exposure to other 

environmental agents, 

socioeconomic indicators, 

maternal intelligence and 

education, and child 

stimulation. The cohort had 

a relatively homogenous 

socioeconomic profile, 

reducing the potential for 

uncontrolled confounding. 

Article 5: Engel et 

al. (2007) Mt. 

Sinai (N=311)

Brazelton Neonatal 

Behavioral 

Assessment Scale 

(BNBAS), primitive 

reflexes 

(neurological 

integrity) measured 

before hospital 

discharge.

DEP: 24.7 nmol/L; 

Total DAP: 82 

nmol/L

Maternal age, race, marital 

status, education, cesarean 

delivery, delivery anesthesia, 

infant age at examination, infant 

gender, infant jaundice, smoking 

(yes/no), alcohol consumption, 

caffeine consumption, illicit drug 

use during pregnancy, and the 

examiner. 

No adverse associations 

were found for DAPs and 

any measured behavior. 

Relative to the first 

quartile, quartiles 2–4 of 

total DEPs, DMPs, and DAPs 

were associated with an 

increased proportion of 

abnormal reflexes, 

although the associations 

did not increase 

monotonically and varied in 

their strength and 

precision. 

Used non-specific 

biomarker DEP/DAP

This was a well conducted 

prospective study 

conducted in a young, 

predominantly minority 

population. The study 

design, analytic approach, 

and statistical analyses 

were appropriate. Pesticide 

metabolites evaluated are 

not specific for 

chlorpyrifos. The BNBAS 

was administered before 

hospital discharge only on a 

subset of children in the 

cohort (n =311/404). 

Factors related to weekend 

delivery (e.g., fewer 

inductions) would be 
Page 63 of 101

Author, Year, 

Sample Size

Outcome 

Assessed

Exposure 

Measurement

Potential confounders 

considered

Primary Result Conclusions/Uncertainties Strengths and Weaknesses 

underrepresented among 

the tested subjects, and 

may induce bias, reduce 

the degree of precision 

with which associations 

were estimated, and limit 

the generalizability of the 

study findings.The 

statistical analysis was 

largely appropriate. 

Imputing of missing data 

may affect precision of 

association estimates and 

result in attenuated effect 

estimates as a result of 

exposure measurement 

error. Assessment and 

control for confounding 

were appropriate. 

However, confounding by 

unmeasured (and mismeasured) risk factors for 

abnormal growth that are 

related to pesticide 

exposure would bias the 

results in this study. Such 

factors may be related to 

socioeconomic status of 

the study participants, a 

cofactor which is difficult to 

define, no less measure in 

an epidemiologic study.

Article 6: Young et 

al. (2005) 

CHAMACOS 

(N=381)

Neurodevelopment, 

Brazelton Neonatal 

Behavioral 

AssessmentScale 

(BNBAS), abnormal 

reflexes

DAP (average

during 

pregnancy): 

median 

222nmol/L 

(range: 7–21,867 

nmol/L); DEP 

(average during 

pregnancy): 

median 21 nmol/L 

(range: 2–680 

Maternal age, BMI, any 

smoking/alcohol/drug use 

during pregnancy, gestational 

age at which prenatal care was 

initiated, total number of 

prenatal care visits, mean 

pregnancy blood pressure, 

parity, method of delivery, 

general anesthesia used during 

delivery, breastfeeding initiated 

after delivery, poverty level, 

Among the >3 day old 

infants, increasing average 

prenatal urinary metabolite 

levels were associated with 

both an increase in number 

of abnormal reflexes (total 

DAP: adjusted beta = 0.53, 

95% CI = 0.23, 0.82; 

dimethyls: adjusted beta = 

0.41, 95% CI = 0.12, 0.69; 

diethyls: adjusted beta = 

Associations seen pre-natal 

OP, not post-natal OP 

exposure, Maternal urine 

collection averaged weeks 

14, 26

Strengths: Longitudinal 

design, measurement and 

consideration of many 

confounders, the prenatal 

exposure measures were, 

with some exceptions, the 

average of two 

measurements, and thus 

may better reflect chronic 

exposure during the 

pregnancy
Page 64 of 101

Author, Year, 

Sample Size

Outcome 

Assessed

Exposure 

Measurement

Potential confounders 

considered

Primary Result Conclusions/Uncertainties Strengths and Weaknesses 

nmol/L) infant sex, age in days at BNBAS, 

minutes since last feed at 

BNBAS, and BNBAS examiner.

0.37, 95% CI = 0.09, 0.64), 

and the proportion of 

infants with more than 

three abnormal reflexes 

(total DAP: adjusted OR = 

4.9, 95% CI = 1.5, 16.1; 

dimethyls: adjusted OR = 

3.2, 95% CI = 1.1, 9.8; 

diethyls: adjusted OR = 3.4, 

95% CI = 1.2, 9.9).

Weaknesses: Potential for 

residual confounding, 

potential for exposure 

misclassification, potential 

selection bias (not all 

children had the outcome 

measures), study 

population not 

generalizable to the whole 

US.

Article 7: Rauh et 

al. (2006) 

Columbia U. 

(N=254)

Neurodevelopment: 

The Bayley Scales of 

Infant Development 

II (BSID-II), Mental 

Development Index 

(MDI) and 

Psychomotor 

Development Index 

(PDI) at 12, 24, and 

36 months of age. 

• Behavior: Child 

Behavior Checklist 

(CBCL) at 12, 24, 

and 36 months. 

• Quality of the 

child-care 

environment: The 

Home Observation 

for Measurement of 

the Environment 

(HOME) 

Exposure levels 

were categorized 

as low (< 6.17 

pg/g) or high 

(>6.17 pg/g) 

Data were collected regarding 

lead exposure, demographics, 

education and occupational 

history, income, active and 

passive smoking, alcohol and 

drug use during pregnancy, and 

residential pesticide use. Final 

models included prenatal 

environmental tobacco smoke 

(ETS) exposure, gender, 

ethnicity, gestational age at 

birth, quality of home caretaking environment, maternal 

education, and maternal IQ.

At the 36 month milestone, 

the likelihood of highly 

exposed children 

developing mental delays 

were 2.4 times greater 

(95% CI: 1.12-5.08, p = 

0.02) and motor delays 

were 4.9 greater (95% CI: 

1.78-13.72; p = 0.002) than 

those with lower prenatal 

exposure. The GLM analysis 

for PDI scores showed a 

significant effect of 

chlorpyrifos exposure over 

time with an estimated 

deficit of approximately 7 

points by age 36 months (p 

= 0.01). 

The authors summarize 

three main findings: 1) by 

age 3, the more highly 

exposed children 

demonstrated mental and 

motor delays; 2) the 

observed developmental 

trajectories for PDI and 

MDI scores confirmed that 

the adverse impact on 

cognitive and motor 

development increased 

over time; and 3) by age 3, 

highly exposed children 

were more likely to 

demonstrate clinically 

significant attention 

problems.

• Only 53% of the children 

reached the three year 

milestone with study data 

collected. It is unclear 

what percentage of these 

children did not survive, 

were lost to follow-up, or 

too sick to participate. 

• Reliance on a single 

exposure level 

(prenatal/cord blood.) 

• No control for exposure 

over the subsequent 3 

years • Creation of a 

dichotomous exposure 

variable brings limitations 

due to the amount of 

within-group variation. 

• Limitations of the 

sensitivity and predictive 

validity of the 

developmental tests, 

especially among children 

less than 3 years of age. 

• No discussion of whether 

this 7-point deficit is 

clinically relevant. 

• Due to the pervasive, 

non-specific nature of 

neurological effects, it is 

difficult to attribute 

causality. 
Page 65 of 101

Author, Year, 

Sample Size

Outcome 

Assessed

Exposure 

Measurement

Potential confounders 

considered

Primary Result Conclusions/Uncertainties Strengths and Weaknesses 

Article 8: Lovasi et 

al. 2010 

Columbia U. 

(N=266)

Bayley scores 

(MDI/PDI) 12 

months, 24 months, 

36 months

N/A Neighborhood characteristics: 

The percentage of housing units 

without complete plumbing, the 

percentage of vacant housing 

units, the percentage of 

residents below the federal 

poverty line, the percentage of 

residents older than 25 years of 

age who completed high school, 

the percentage of households 

receiving public assistance, the 

percentage of housing units with 

one or more residents per room, 

racial composition, the 

percentage of residents born 

outside the United States, the 

percentage of Spanish-speaking 

residents, and the percentage of 

residents who were linguistically 

isolated

Neighborhood 

characteristics did not 

confound the observed 

association between 

chlorpyrifos levels and 

cognitive development.

Hierarchal regression 

analysis of potential 

confounding by SES

Direct measurement of 

chlorpyrifos.The statistical 

analyses were generally 

appropriate. Missing data 

on covariates were 

estimated using multiple 

imputation, and the 

variance estimates 

presented appropriately 

reflect the degree of 

uncertainty caused by 

missing covariate data. 

Robust standard errors 

were used.The setting of 

the investigation in a 

sample drawn from lowincome African American 

and Dominican 

communities is both a 

strength (increases the 

power, restriction of 

confounders) and a 

limitation of the study 

(reduced generalizability). 

Article 9: Engel et 

al. (2011) 

Mt. Sinai (N=276)

Bayley scores 

(MDI/PDI) at 12 

months, 24 months.

DEP: 24.7 nmol/L; 

Total DAP: 82 

nmol/L (same as 

Engel 2007)

Maternal age, race/ethnicity, 

marital status, education, 

breast-feeding, child sex, 

alcohol, smoking, or drug use 

during pregnancy, maternal IQ, a 

score based on assessment of 

the home environment (HOME), 

season of urine collection, 

language spoken in the home, 

age at testing, examiner, and 

urinary creatinine level.

An observed association 

between prenatal total 

dialkylphosphate 

metabolite level and a 

decrement in mental 

development at 12 months 

among blacks and 

Hispanics. 

Used non-specific 

biomarker DEP/DAP; some 

evidence of effect 

modification by PON1 

genotype

Limitations include use of 

non-specific markers of 

chlorpyrifos pesticide 

exposure (DAPs), use of 

only a single (thirdtrimester) urine sample, 

and the large proportion of 

loss to follow-up. Statistical 

analysis was appropriate. 

Imputing of missing data 

may affect precision of 

association estimates and 

result in attenuated effect 

estimates as a result of 

exposure measurement 

error, although these are 

offset by the further 

categorization of the 

exposure levels (at the 
Page 66 of 101

Author, Year, 

Sample Size

Outcome 

Assessed

Exposure 

Measurement

Potential confounders 

considered

Primary Result Conclusions/Uncertainties Strengths and Weaknesses 

median). However, binning 

of exposure levels reduces 

precision, relative to a 

continuously distributed 

measure of exposure. 

Assessment and control for 

confounding were 

appropriate. However, 

confounding by 

unmeasured (and 

mismeasured) risk factors 

for abnormal growth that 

are related to pesticide 

exposure would bias the 

results in this study. Such 

factors may be related to 

socioeconomic status of 

the study participants, a 

cofactor which is difficult to 

define, no less measure in 

an epidemiologic study.

Article 10: Eskenazi 

et al. (2007) 

CHAMACOS 

(N=372)

Neurodevelopment, 

Bayley Index (MDI, 

PDI), Maternal 

behavior checklist 

at 6, 12, and 24 

months

DEP: geom. Mean 

in mother 18.1 

nmol/L (95% CI = 

16.7–19.7); DEP 

geometric mean 

in child at 24 

months 10.5 

nmol/L (95% CI 

=8.8–12.6); TCPy 

median 3.54 ug/l

Psychometrician, location of 

assessment, exact age at 

assessment, sex, breast-feeding 

duration (months), HOME score, 

and household income, parity, 

maternal PPVT, maternal age, 

education, depressive 

symptoms, active/passive 

smoking exposure during 

pregnancy, regular alcohol use 

during pregnancy, marital 

status, father’s presence in 

home, housing density, maternal 

work status, ≥ 15 hours out-ofhome childcare/week, birth 

weight, gestational age, 

abnormal reflexes, PCBs, lead, 

DDT, β-hexachlorocyclohexane, 

and hexa-chlorobenzene 

DAP metabolite levels 

during pregnancy, 

particularly from dimethyl 

phosphate pesticides, may 

be negatively associated at 

24 months with mental 

development (MDI) on the 

Bayley Scales and an 

increase in risk of 

maternally reported PDD. 

No strong associations 

identified with DE or TCPy, 

Maternal urine collection 

averaged weeks 14, 26

Strengths: Longitudinal 

design, measurement and 

consideration of many 

confounders (including 

other environmental 

chemicals); the prenatal 

exposure measures were, 

with some exceptions, the 

average of two 

measurements, and thus 

may better reflect chronic 

exposure during the 

pregnancy

Weaknesses: Potential for 

residual confounding, 

potential for exposure 

misclassification, potential 

selection bias (not all 

children had the outcome 

measures), study 

population not 
Page 67 of 101

Author, Year, 

Sample Size

Outcome 

Assessed

Exposure 

Measurement

Potential confounders 

considered

Primary Result Conclusions/Uncertainties Strengths and Weaknesses 

generalizable to the whole 

US.

Article 11: Eskenazi 

et al. (2010) 

CHAMACOS 

(N=371)

Neurodevelopment, 

Bayley Index (MDI, 

PDI), Maternal 

behavior checklist 

at 6, 12, and 24 

months, PON1 gene 

and enzyme levels

The geometric 

mean for the DAP 

concentrations 

during pregnancy 

(for the average 

of the two 

sampling periods) 

was 146 nmol/L 

(95% CI: 133, 

160); of this, a 

larger proportion 

was DMP 

metabolites (GM 

= 109 nmol/L; 

95% CI =98, 120) 

than DEPs (GM= 

23 nmol/L; 95% CI 

= 21, 25). Allele 

frequencies: 

PON1 192 Q 

allele= 50%; PON1 

-108 T allele= 

46%.Mean 

arylesterase 

activity: For 

infants: 33.6 

U/mL (SD = 16) 

For mothers: 

136.6 U/mL (SD = 

44). Mean 

paraoxonase 

activity: For 

infants: 256.6 U/L 

(SD = 165); For 

mothers: 989.0 

U/L (SD = 616).

Psychometrician, location of 

assessment, exact age at 

assessment, sex, breast-feeding 

duration (months), HOME score, 

and household income, parity, 

maternal PPVT, maternal age, 

education, depressive

symptoms, active/passive 

smoking exposure during 

pregnancy, regular alcohol use 

during pregnancy, marital 

status, father’s presence in 

home, housing density, maternal 

work status, ≥ 15 hours out-ofhome childcare/week, birth 

weight, gestational age, 

abnormal reflexes, PCBs, lead, 

DDT, β-hexachlorocyclohexane, 

and hexa-chlorobenzene 

Decrease MDI (24 months) 

PON1108TT –5.7 (–9.0 to –

2.5) 9=0.01; Decrease PDI 

(24 months) PON1108TT –2.8 

(–5.7 to 0.2) p=0.07; 

increased odds PDD 2.0 

(0.8 to 5.1) p=0.14; no 

association PON1192; no 

association PON1 activity 

measured newborn, 2 

years, maternal and MDI, 

PDI, PDD. Evidence of 

decreasing MDI score by 

number of PON1108 variant 

alleles: PON1108CC –3.2 (–9.8 

to 3.5), CT –3.7 (–8.0 to 

0.6), TT –5.5 (–11.1 to 0.1), 

p-interaction 0.98.

In this study population, 

evidence PON1 may 

influence MDI score, but 

not PDI or PDD risk at twoyears. Non-significant 

evidence of decreasing MDI 

score by increasing DAP 

levels across strata of the 

number of PON1108 variant 

alleles, interaction nonsignificant. Similar trend 

with prenatal DEP levels 

and MDI, PDI by PON108 

alleles, but less 

pronounced. Overall, 

limited, non-definitive 

evidence of effect 

modification by PON1 

status in the relation 

between mental and 

psychomotor effects and 

prenatal DAPs. 

Strengths: Longitudinal 

design, measurement and 

consideration of many 

confounders (including 

other environmental 

chemicals); the prenatal 

exposure measures were, 

with some exceptions, the 

average of two 

measurements, and thus 

may better reflect chronic 

exposure during the 

pregnancy

Weaknesses: Potential for 

residual confounding, 

potential for exposure 

misclassification, potential 

selection bias (not all 

children had the outcome 

measures), study 

population not 

generalizable to the whole 

US. Study may be underpowered to evaluate effect 

modification by PON1 

status.

Article 12: Marks 

et al. (2010) 

CHAMACOS 

(N=348)

CBCL; K-CPT; ADHD 

confidence index; 

Hillside behavioral 

rating scale; 

DAP (geometric

mean) pregnancy 

109.0 nmol/L; 

DEP 17.7 nmol/L

Psychometrician, exact age at 

assessment, sex, maternal 

education, depressive 

symptoms, PPVT (continuous), ≥ 

Prenatal DAPs were nonsignificantly associated 

with maternal report of 

attention problems and 

Marked effect modification 

by gender: 11-fold increase 

ADHD composite indicator 

in boys, less than 2-fold in 

Strengths: Longitudinal 

design, measurement and 

consideration of many 

confounders (including 
Page 68 of 101

Author, Year, 

Sample Size

Outcome 

Assessed

Exposure 

Measurement

Potential confounders 

considered

Primary Result Conclusions/Uncertainties Strengths and Weaknesses 

composite ADHD 

indicator

15 hr out-of-home child 

care/week, breast feeding 

duration (months), maternal 

age, parity, marital status, 

active/passive smoking exposure 

and regular alcohol use during 

pregnancy, presence of father in 

home, maternal work status, 

and household income

ADHD at age 3.5 years, but 

were significantly related at 

age 5 years [CBCL attention 

problems: β = 0.7 points; 

95% confidence interval 

(CI), 0.2-1.2; ADHD: β = 1.3; 

95% CI, 0.4-2.1]. 

girls, however unstable 

estimates; weak evidence 

of association DAPs at 3.5, 

5 years and attention 

other environmental 

chemicals); the prenatal 

exposure measures were, 

with some exceptions, the 

average of two 

measurements, and thus 

may better reflect chronic 

exposure during the 

pregnancy

Weaknesses: Potential for 

residual confounding, 

potential for exposure 

misclassification, potential 

selection bias (not all 

children had the outcome 

measures), study 

population not 

generalizable to the whole 

US.

Article 13: Rauh et 

al. (2011) 

Columbia U. 

(N=265)

• Wechsler Scales 

of Intelligence for 

Children (WISC-IV) 

• Child Behavior 

Checklist (CBCL).

• Chlorpyrifos 

levels in umbilical 

cord blood 

samples, N=256 

newborns

• If no cord blood 

(12% of subjects), 

levels were 

imputed from 

mothers’ values. 

• Values for 

samples with nondetectable 

chlorpyrifos levels 

(N=115, 43%) 

were imputed by 

using assayspecific limit of 

detection (LOD) 

values to impute 

an approximate 

level. 

Data were collected regarding 

lead exposure, demographics, 

education and occupational 

history, income, active and 

passive smoking, alcohol and 

drug use during pregnancy, and 

residential pesticide use. Final 

models included prenatal 

environmental tobacco smoke 

(ETS) exposure, gender, 

ethnicity, gestational age at 

birth, quality of home caretaking environment, maternal 

education, and maternal IQ.

Full-Scale IQ: (B) of -0.003, 

CI = 0.006, 0.001, p= 0.064 

Working Memory Index: (B) 

of -0.006, CI = 0.009, 0.002, 

p<0.001. The investigators 

articulated these results as 

showing that a 1 pg/g 

increase in chlorpyrifos 

exposure was associated 

with a 0.006 point decrease 

in the log-transformed 

Working Memory score and 

a 0.003 point decrease in 

the log-transformed FullScale IQ score. The 

investigators concluded 

that for each standard 

deviation increase in 

exposure (4.61pg/g) there 

is a 1.4% reduction in FullScale IQ and a 2.8% 

reduction in Working 

Memory.

For each standard 

deviation increase in 

exposure (4.61pg/g) there 

is a 1.4% reduction in FullScale IQ and a 2.8% 

reduction in Working 

Memory.

Strengths

• Direct assessment of 

chlorpyrifos levels using 

maternal serum and cord 

blood.

• Analysis using a 

continuous CPF level, 

which, in contrast to 

dichotomous CPF levels, 

provides a more 

meaningful look at 

potential threshold effects 

and dose-response trends.

• The investigators 

rigorously evaluated their 

methods for imputing 

values for undetectable 

CPF levels which in the end, 

were validated.

• The authors describe an 

elegant and 

methodologically sound 

statistical analysis, 
Page 69 of 101

Author, Year, 

Sample Size

Outcome 

Assessed

Exposure 

Measurement

Potential confounders 

considered

Primary Result Conclusions/Uncertainties Strengths and Weaknesses 

addressing many of the 

potential shortcomings of 

their exposure data and 

covariates.

Weaknesses: The use of a 

single snapshot of prenatal 

chlorpyrifos exposure may 

not be an accurate 

surrogate for full prenatal 

exposure levels. 

• There is no control for 

exposure over the 

subsequent 7 years which 

may be critical, especially 

as the process of 

neurocognitive 

development is fluid and 

rapid during these early 

childhood years. • 

Possibility of that an 

increased awareness of the 

risks of pesticide exposures 

could disproportionately 

affect postnatal exposure 

behavior.• Complicating 

this analysis is the 

pervasive, non-specific 

nature of neurological 

effects and the difficulty in 

attributing causal 

pathways. • when closely 

reviewed, the 95% CI for 

Full Scale IQ for both 

techniques contain 0 

(LASSO: -0.006, 0.001, 

p=0.064; fully-adjusted: -

0.006, 0.001, p=0.048• The 

authors do not address the 

clinical relevance of the 

1.4% and 2.8% reductions 

and how this may impact a 

child or his/her 
Page 70 of 101

Author, Year, 

Sample Size

Outcome 

Assessed

Exposure 

Measurement

Potential confounders 

considered

Primary Result Conclusions/Uncertainties Strengths and Weaknesses 

psychological or 

educational plans. 

Article 9: Engel et 

al. (2011) 

Mt. Sinai (N=169)

Wechsler Preschool 

and Primary Scale 

of Intelligence, 3rd 

edition (WPPSI-III) 

at ages < 7 years; 

Wechsler-IV 

Intelligence Scale 

(verbal 

comprehension; 

perceptual 

reasoning, working 

memory, 

processing speed, 

full scale 

intelligence) at age 

7-9 years

DEP: 24.7 nmol/L; 

Total DAP: 82 

nmol/L (same as 

Engel 2007)

Maternal age, race/ethnicity, 

marital status, education, 

breast-feeding, child sex, 

alcohol, smoking, or drug use 

during pregnancy, maternal IQ, a

score based on assessment of 

the home environment (HOME), 

season of urine collection, 

language spoken in the home, 

age at testing, examiner and 

urinary creatinine level.

At age 6-9 years, nonstatistically significant 

reductions in full scale IQ, 

perceptual reasoning, 

verbal comprehension, 

working memory and 

processing speed with 

increasing DAP, more 

profound with DEP than 

DMP 

Used non-specific 

biomarker DEP/DAP; some 

evidence of effect 

modification by PON1 

genotype

Limitations include use of 

non-specific markers of 

chlorpyrifos pesticide 

exposure (DAPs), use of 

only a single (thirdtrimester) urine sample, 

and the large proportion of 

loss to follow-up.

The statistical analysis was 

largely appropriate. 

Imputing of missing data 

may affect precision of 

association estimates and 

result in attenuated effect 

estimates as a result of 

exposure measurement 

error, although these are 

offset by the further 

categorization of the 

exposure levels (at the 

median).

Assessment and control for 

confounding were 

appropriate. However, 

confounding by 

unmeasured (and mismeasured) risk factors for 

abnormal growth that are 

related to pesticide 

exposure would bias the 

results in this study. Such 

factors may be related to 

socioeconomic status of 

the study participants, a 

cofactor which is difficult to 

define, no less measure in 

an epidemiologic study.

Article 14: Wechsler-IV Total DAPs Maternal intelligence, measures The authors observed Prenatal measures taken Strengths: the longitudinal 
Page 71 of 101

Author, Year, 

Sample Size

Outcome 

Assessed

Exposure 

Measurement

Potential confounders 

considered

Primary Result Conclusions/Uncertainties Strengths and Weaknesses 

Bouchard et al. 

(2011) 

CHAMACOS 

(N=329)

Intelligence Scale 

(verbal 

comprehension; 

perceptual 

reasoning, working 

memory, 

processing speed, 

full scale 

intelligence) 

measured at age 7 

years

(quintiles): Q1 (39 

nmol/L); Q2 75 

nmol/L; Q3 126 

nmol/L; Q4 221 

nmol/L; Q5 508 

nmol/L. 

Geometric mean 

DAP 131 nmol/L

of how stimulating the 

environment is, and known or 

suspected neurotoxins were 

measured prenatally. Maternal 

intelligence was assessed via the 

Peabody Picture Vocabulary Test 

(PPVT). To measure the quality 

and extent of stimulation 

available to a child in the home 

environment, the Infant-Toddler 

HOME (Home Observation for 

Measurement of the 

Environment) inventory was 

completed at the 6-month, 1, 2, 

3.5, 5, and 7 year visits; known 

or suspected neurotoxicants, 

polybrominated diphenyl ethers 

(PBDEs), polychlorinated 

biphenyls (PCBs), p,p’-

dichlorodiphenyltrichlorethane 

(DDT), p,p’-

dichlorodiphenyltrichlorethylene 

(DDE), and lead. 

evidence of an association 

between prenatal 

exposures to OP pesticides 

as measured by urinary 

DAP metabolites in women 

during pregnancy, and 

decreased cognitive 

functioning in children at 

age 7.

later half of pregnancy 

more significantly 

associated intelligence than 

early; little evidence postnatal OP exposure 

associated with 

intelligence; 7 point 

reduction in full scale 

intelligence DAP Q5/Q1 

(SS)

design, the measurement 

of urinary DAP at multiple 

time points and following 

children to age seven when 

tests of cognitive function 

are reportedly more 

reliable. The authors were 

able to adjust for or 

consider many factors 

related to cognitive 

function, such as prenatal 

exposure to other 

environmental agents, 

socioeconomic indicators, 

maternal intelligence and 

education, and child 

stimulation. The cohort had 

a relatively homogenous 

socioeconomic profile, 

reducing the potential for 

uncontrolled confounding. 

Article 15: Whyatt 

et al. (2007) 

Columbia U. 

(N=102)

None Geometric mean, 

6.9 ± 17.0 ng/m3; 

range < 0.4–171 

ng/m3. Personal 

air monitor: 

median 2.8 

ng/m3, mean 6.2 

± 11.1 ng/m3, 

range < 0.4–83.4 

ng/m3

N/A There was little withinhome variability and no 

significant difference in air 

concentrations within 

homes over time (p ≥ 0.2); 

between-home variability 

accounted for 88% of the 

variance in the indoor air 

levels of propoxur, 92% in 

chlorpyrifos, 94% in 

diazinon, and 62% in 

piperonyl butoxide (p < 

0.001). Indoor and 

maternal personal air 

insecticide levels were 

highly correlated (r = 0.7–

0.9, p < 0.001). 

Indoor and maternal 

personal air insecticide 

levels were highly 

correlated (r = 0.7–0.9, p < 

0.001). 

Strengths: study design and 

exposure assessment 

techniques, Limitations: 

only those cohort 

participants enrolled after 

2011 were included in the 

analysis (most likely due to 

the lack of serial data from 

the earlier years.) 

Article 16: Whyatt 

et al. (2009) 

None The limit of 

detection (LOD) 

N/A Meconium TCPy 

concentrations were 

TCPy in maternal urine 

samples was not reliable, 

Comprehensive exposure 

assessment including actual 
Page 72 of 101

Author, Year, 

Sample Size

Outcome 

Assessed

Exposure 

Measurement

Potential confounders 

considered

Primary Result Conclusions/Uncertainties Strengths and Weaknesses 

Columbia U. 

(N=102)

of chlorpyrifos in 

blood samples 

was 0.5–1 pg/g 

plasma. The LOD 

of TCPy in urine 

samples was 0.26 

ng/mL urine. The 

LOD for TCPy in 

meconium was 

0.2 ng based on a 

sample weighing 

0.5 g. Exposure 

marker levels 

below the LOD 

were given a 

value of half the 

level of detection, 

and were then 

log10 

transformed.

positively correlated with 

chlorpyrifos in maternal 

and cord blood (r = 0.25–

0.33, p < 0.05) and with 

TCPy in maternal urine (r = 

0.31, p < 0.01).

but the maternal and cord 

blood chlorpyrifos as well 

as the TCPy levels in 

meconium were reliable 

measures of exposure

blood chlorpyrifos levels, 

the repeated sampling, and 

the environmental 

sampling.

Weaknesses: only included 

participants recruited in 

the post-cancellation 

period, use of 

nonparametric, rank-based 

statistics is appropriate but 

the large number of 

observations below the 

level of detection receiving 

equal rank, may be 

problematic; no dietary 

assessment 

Rauh et al.(2012), 

(n=40)

Morphological 

change in the 

pediatric brain in 

regions of the brain 

known to be 

associated with 

learning, cognition 

and social behavior

Tertile 3 ( ≥4.39 

pg/g), compared 

to Tertiles 0, 1, 2 

(<4.39 pg/g, 

including those 

not

exposed to CPF)

Age, sex Authors report differences 

in brain structure (regional

cerebral size and thickness) 

by CPF exposure groups, 

and the differences 

(high>low CPF) in regional 

brain size is likely due to 

enlargement of underlying 

white matter. Statistical 

interaction by gender 

reported.

Authors concluded that the 

evidence from the study 

illustrated changes in brain 

morphology in association 

with higher CPF exposure, 

and that changes observed 

were in areas of the brain 

that subserve those 

learning, cognition and 

social behavioral, 

supported by previous 

observational and 

experimental literature.

Study supports general 

hypothesis of CPF influence 

on brain morphology, but 

lacks specific hypotheses 

regarding particular areas 

of the cerebrum affected; 

limited and somewhat 

unbalanced depiction of 

the available rodent 

experimental data; 

statistical methods 

appropriate, correction for 

multiple statistical 

comparisons a strength; 

MRI image readers blinded 

to exposure status 

enhances study validity; 

lack of information on 

other validation practices; 

small sample size, pilot 

study, low statistical 

power; external validity 

limited; one time measure 
Page 73 of 101

Author, Year, 

Sample Size

Outcome 

Assessed

Exposure 

Measurement

Potential confounders 

considered

Primary Result Conclusions/Uncertainties Strengths and Weaknesses 

of pre-natal exposure
Page 74 of 101

2.2.10 Summary & Discussion

Across the eight studies identified in this 2015 literature review within the scope of this analysis 

and with adequate quality in design and exposure assessment, it is important to note that these 

studies used study methods that were highly variable, including different exposure 

measurement, outcome assessment, study design, and geographical location. These 

differences make it challenging to compare the results across studies. In comparison to the 

studies from the three US birth cohorts, results from the new studies (e.g., ELEMENT, CHARGE) 

provide some supportive evidence for the findings from CCCEH, Mt. Sinai and CHAMACOS but 

in general the new studies are not as robust as those from CCCEH, Mt. Sinai and CHAMACOS.

In the two Chinese studies, EPA does not know how the OP exposures from these studies relate 

to the currently registered use pattern for OPs used in the U.S./North America. In the case of 

these two Chinese studies (Guodong et al., 2012; Zhang et al., 2014), there may be differences 

in the study population and outcome measurements that may account for the observed 

differences in study results, with Zhang et al. (2014) documenting statistically significant 

associations for total DEAPs, total DMAPs, and total DAPs and Guodong et al. (2012) observing 

no association with these exposures. The Zhang et al. (2014) study was conducted in Shenyang, 

with a study population reported as 87% urban and 13% rural, whereas the Guodong et al.

(2012) study was conducted in Shanghai with a 99% urban and 1% suburban study population. 

Given the higher percentage of study participants from rural areas, the study participants from 

Zhang et al. (2014) may have had different pattern and magnitude of OP exposures compared 

to those from Guodong et al. (2012). In addition, it is noted that different outcome 

measurements were made in these studies, with Guodong et al. (2012) assessing 23-25 month 

old children using a developmental quotient score and Zhang et al. (2014) assessing 3 day old 

infants using a Neonatal Behavioral Neurological Assessment. Given the different outcome 

assessments, exposure potential, study designs (cohort vs. cross-sectional), and ages of the 

participants in these two studies, it is difficult to draw conclusions on how these study results 

compare. It is notable that the results from the Zhang et al. (2014) study focusing on neonates 

are consistent with those from other studies which reported statistically significant 

associations between delayed neurological development measured in newborns and total 

DEAP, total DMAP, and total DAP exposure (Engel et al., 2007; Young et al., 2005). However, it 

is noted that neurological development was measured within a few days of birth for Zhang et 

al. (2014) and Engel et al. (2007), whereas Young et al. (2005) conducted their measurements 

within two months of birth. 

In this 2015 literature review, two studies were identified that documented either suggestive or 

statistically significant associations between OP exposure and autism spectrum disorders 

(Furlong et al., 2014; Shelton et al., 2014). Specifically, Furlong et al. (2014) reported 

suggestive, but not statistically significant, evidence of an association between total DEAP 

exposure and reciprocal social responsiveness among black participants and boys. These 

results are consistent with previous studies conducted on the CCCEH and CHAMACOS cohorts, 

with these studies also showing statistically significant associations between OP exposure and 
Page 75 of 101

ASD (Rauh et al., 2006; Eskenazi et al., 2007).7 Specifically, Eskenazi et al. (2007) reported a 

statistically significant association between PDD and total DAP exposure, whereas Rauh et al.

(2006) showed a significant association between PDD and specifically chlorpyrifos exposure. 

Both PDD and reciprocal social responsiveness are related to the autism spectrum disorder. 

Using a different exposure assessment method (geospatial analysis and residential proximity to 

total OP exposure), Shelton et al. (2014) also documented statistically significant associations 

between total OP exposure and ASD. 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.

A total of three studies focusing on ADHD/behavioral/attention problems were identified in this 

2015 literature review (Oulhote and Bouchard, 2013; Bouchard et al., 2010; Fortenberry et al., 

2014). It is noted that the Fortenberry et al. (2014) study is a prospective cohort study on 

Mexican children, with this study adding an additional North American cohort to the previously 

identified U.S. cohort studies (CHAMACOS, Mt. Sinai, and CCEH). Of the four studies focusing 

on ADHD and OP exposure, three found statistically significant associations, with only Oulhote 

and Bouchard (2013) finding no association with total DMAP, DEAP, or total DAP exposure. In 

contrast, Bouchard et al. (2010) observed an association with total DMAP and total DAP 

exposure and ADHD. Fortenberry et al. (2014) found suggestive, but not statistically significant,

evidence of an association with TCPy and ADHD in boys. Overall, the Fortenberry and Bouchard 

study results are consistent with that of earlier studies from the CCCEH (Rauh et al., 2006) and 

CHAMACOS (Eskenazi et al., 2007; Marks et al., 2010). Specifically, statistically significant 

associations were observed by Rauh et al. (2006) with chlorpyrifos exposure and ADHD, 

Eskenazi et al. (2007) with total DMAPs and total DAPs and ADHD, and Marks et al. (2010) with 

total DEAP, DMAP, and total DAP exposure.

It is important to put into context the specific outcome measures used in the assessment of 

attention and neurobehavioral problems. For example, Bouchard et al. (2010) identified 

statistically significant associations between OP exposure and ADHD/behavioral problems, 

whereas Oulhote and Bouchard (2013) did not. It is valuable to compare these studies given 

that they are both cross-sectional studies using large population level datasets with biomarker 

information, with Oulhote and Bouchard (2013) using a Canadian dataset and Bouchard et al 

(2010) using a U.S. dataset. Bouchard et al. (2010) used criteria for ADHD from DSM-IV, 

whereas Oulhote and Bouchard (2013) used a “Strengths and Differences Questionnaire 

(SDQ),” with the SDQ being a more generic assessment of mental health status than the DSM-IV 

criteria. When Oulhote and Bouchard (2013) compared their results to Bouchard et al. (2010), 

they noted that their outcome measurements may not have been as sensitive and that this may 

account for the difference in study results.

 

7 The DSM-V defines ASD (autism spectrum disorder) which now encompasses several disorders that were 

different diagnoses in DSM-IV, including PDD (pervasive developmental disorder, a catch-all where the other 

categories didn’t fit). Depending on when the study was conducted, the authors may use the PDD or ASD criteria 

and terminology. 
Page 76 of 101

Across epidemiology studies looking at ADHD/behavioral problems, a suggestive or statistically 

significant positive association was observed in multiple studies between OP exposure and 

these neurobehavioral outcomes (Bouchard et al., 2010; Fortenberry et al., 2014; Rauh et al., 

2006, Eskenazi et al., 2007). While these studies have differences in the years that the 

exposure occurred, study design, exposure assessment, and outcome assessment, the 

commonality in their results is striking.

When all the evidence is considered together, there are uncertainties with respect to a number 

of factors such as exposure assessment, lack of ability to make strong causal linkages, and 

unknown window(s) of susceptibility. The epidemiology studies reviewed in the 2012/2014 and 

2015 literature reviews represent different investigators, locations, points in time, exposure 

assessment procedures, and outcome measurements. Despite all these uncertainties and

differences in study design, authors consistently identified associations with 

neurodevelopmental outcomes such as ADHD/behavioral problems and autism spectrum, in 

relation to OP exposure.

3.0 Weight of Evidence Analysis: Integration Across Multiple Lines of Evidence

In 2010, OPP developed a draft “Framework for Incorporating Human Epidemiologic and

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, which includes two key components: 

problem formulation and use of the MOA/AOP frameworks, was reviewed favorably by the SAP 

in 2010 (FIFRA SAP, 2010) and has recently been applied to chlorpyrifos. This document 

extends the chlorpyrifos WOE to other OPs.

One of the key components of the agency’s draft framework is the use the MOA/AOP concept 

as a tool for organizing and integrating information from different sources to inform the causal 

nature of links observed in both experimental and observational studies. Specifically, the 

modified Bradford Hill Criteria (Hill, 1965) are used to evaluate the experimental support that 

establishes key events within MOA/AOP, and explicitly considers such concepts as strength, 

consistency, dose response, temporal concordance and biological plausibility in a weight of 

evidence analysis; sections 3.1-3.3 below summarize the available evidence based on these 

principles.

3.1 Dose-Response Relationships & Temporal Concordance

Since the MOA(s)/AOP(s) is/are not established for neurodevelopmental outcomes (USEPA, 

2012, 2014), it is not possible to describe the concordance in key events or biological steps 

leading to neurodevelopmental outcomes. As such, the quantitative linkages between 

molecular initiating events (MIE)s, intermediate steps, and ultimately the adverse outcome (i.e., 

neurodevelopmental effects) cannot be determined.
Page 77 of 101

With respect to the timing of exposure, across the epidemiology database of studies the 

maternal urine, cord blood and other (meconium) measures provide evidence that exposure 

did occur to the fetus during gestation, but the actual level of such exposure during the critical 

window(s) of susceptibility is not known. While significant uncertainties remain about the 

actual exposure levels experienced by mothers and infant participants in the children’s health 

cohorts, it is unlikely that these exposures resulted in AChE inhibition. As part of the 

CHAMACOS study, Eskenazi et al. (2004) measured AChE activity and showed that no 

differences in AChE activity were observed. The biomarker data from the Columbia University 

studies are supported by the agency’s dose reconstruction analysis using the PBPK-PD model. 

Following the recommendation of the 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 (USEPA, 2014). Based on the output from the PBPK-PD model, 

for the highest exposure 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 Columbia University epidemiology studies, the relationship in time between 

prenatal chlorpyrifos exposure and adverse neurodevelopmental outcomes is concordant. The

time period under study within the Columbia University (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.

3.2 Strength, Consistency & Specificity 

Published and submitted laboratory animal studies have been reviewed for OPs. The >30 

papers on chlorpyrifos provide evidence of long-lasting neurodevelopmental disorders in rats 

and mice; however, there was no clear consistency in terms of pattern, timing, or dose 
Page 78 of 101

response for these effects. The additional toxicological literature and guideline DNT studies 

with the other OPs provide more evidence for the same conclusions, with again the same 

caveats and uncertainties. While overall cognitive function and motor activity appeared to be 

altered the most often, it is apparent that these behaviors were also the most often evaluated.

Among the epidemiology studies, two of the cohorts (CCCEH and ELEMENT) have focused on 

chlorpyrifos whereas the other studies (Mt. Sinai cohort, CHAMACOS cohort, CHARGE study, 

Bouchard et al., 2007) have focused on less specific biomarkers (i.e., DAPs) and are not specific 

to any particular OP. When considered in concert, the epidemiology studies provide consistent 

findings for some outcomes. Specifically, with regard to the three US children’s environmental 

health epidemiology studies and the ELEMENT cohort in Mexico, each of the four 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. As noted 

above, the CHAMACOS and Mt. Sinai cohorts that measured neurological effects at birth (the 

Brazelton index), observed a putative association with OPs (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), the three US cohorts each reported evidence of impaired 

mental and psychomotor development. Attentional problems and ADHD were reported by 

CCCEH, Mt. Sinai, CHAMACOS, and ELEMENT investigators with additional support from 

Bouchard et al. (2010). In addition, several studies have now documented suggestive or positive 

associations between OP exposure and autism spectrum disorders (Rauh et al., 2006; Shelton et 

al., 2014; Furlong et al., 2014; Eskenazi et al., 2007; Eskenazi et al, 2010). Finally, each of the 

three US childrens cohort study authors observed an inverse relation between the respective 

prenatal measures of chlorpyrifos and intelligence measures at age 7 years.

As stated in the EPA neurotoxicity guidelines8, direct extrapolation of developmental 

neurotoxicity results from laboratory animals to humans is limited by the lack of knowledge 

about underlying toxicological mechanisms and the relevance of these results to humans. EPA 

notes consistencies across the databases of in vivo laboratory animal studies and epidemiology 

studies, although challenges of making a direct comparison between neurodevelopmental 

domain inter-species remain. It can be assumed that developmental neurotoxicity effects in 

animal studies indicate the potential for altered neurobehavioral development in humans, 

although the specific types of developmental effects seen in experimental animal studies may 

not be the same as those that may be produced in humans. However, 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. 

Previously reviewed studies of chlorpyrifos in rats and/or mice reported impaired cognition, 

changes in locomotor activity levels, altered social interaction, and to a lesser extent, changes 

in neuromotor function (FIFRA SAP 2012; USEPA, 2014). While there are fewer studies for all 

the other OPs, behavioral effects in the same functional domains were again reported. The 

most commonly reported outcome was cognitive dysfunction, and although it was overall 

consistent there were again differences in cognitive specificity, gender differences, or dose 

 

8 http://www.epa.gov/raf/publications/pdfs/NEUROTOX.PDF
Page 79 of 101

response. Quite a few studies also report changes in motor activity and sensory function in 

offspring, but there generally fewer studies that assess social interactions for OPs other than 

chlorpyrifos. 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 these studies are sufficient to elicit ≥10% 

brain and RBC inhibition depending on the study design, age of the animal, and sampling time. 

In the epidemiology studies, based on the comparisons with biomonitoring data, reported AChE 

data from CHAMACOS and the results of the chlorpyrifos dose-reconstruction 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. 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 OP application and blood 

collections are not coupled—thus higher levels of blood OPs were likely missed.

3.3 Biological Plausability & Coherence

EPA’s cancer guidelines (2005) includes guidance which are also applicable to this current 

evaluation of OPs. In fact, 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 OPs are 

biologically active on a number of processes that affect the developing brain. Moreover, there 
Page 80 of 101

is a large body of in vivo laboratory studies which show long-term behavioral effects from early 

life exposure, albeit at doses which cause AChE inhibition. 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. When all the evidence is 

considered together, there are uncertainties with respect to a number of factors such as 

exposure assessment, lack of ability to make strong causal linkages, and unknown window(s) of 

susceptibility. The epidemiology studies reviewed in the 2012/2014 and 2015 literature 

reviews represent different investigators, locations, points in time, exposure assessment 

procedures, and outcome measurements. Despite all these uncertainties and differences in 

study design, multiple investigators have identified associations with neurodevelopmental 

outcomes such as ADHD/behavioral problems and autism spectrum, 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 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. Thus, with respect to biological plausibility and 

coherence, although uncertainties remain, these uncertainties are diminished in the context of 

the qualitative similarity between the epidemiology studies.

4.0 10X FQPA Safety Factor for Infants and Children

As section 408(b)(2)(C) of the FFDCA 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.” 

Given the totality of the evidence, there is sufficient uncertainty in the human dose-response 

relationship for neurodevelopmental effects which prevents the agency from reducing or 

removing the statutory 10X FQPA Safety Factor. For the preliminary human health risk 

assessments for the OPs, a value of 10X will be applied. Similarly, a database uncertainty factor 

of 10X will be retained for occupational risk assessments. The agency will continue to evaluate 

the epidemiology studies and pursue approaches for quantitative or semi-quantitative 

comparisons between doses which elicit AChE inhibition and those which are associated with 

neurodevelopmental outcomes prior to a revised human health risk assessment. The FQPA 10X 

Safety Factor will be retained for OPs for the population subgroups that include infants, 

children, youths, and women of childbearing age for all exposure scenarios.
Page 81 of 101

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newborns. Environ Health Perspect, 111(5), 749-756. 

Whyatt RM, Rauh V, Barr DB, Camann DE, Andrews HF, Garfinkel R, Hoepner LA, Diaz D, 

Dietrich J, Reyes A, Tang D, Kinney PL, Perera FP. Prenatal Insecticide Exposures 

and Birth Weight and Length among an Urban Minority Cohort. Environmental 
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Biomarker Validation Study of Prenatal Chlorpyrifos Exposure within an Inner-City 

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Page 90 of 101

6.0 Appendices

1. Table of In Vivo Developmental Neurotoxicity Studies of OPs.

2. Summary of Guideline DNT Studies Submitted to the Agency for OPs other than 

Chlorpyrifos.

3. Low Quality Studies: Summary of Study Design Elements Impacting Study Quality 

Assignment
Page 91 of 101

Appendix 1. Table of In Vivo Developmental Neurotoxicity Studies of OPs.

(Effects described are only those measured after weaning. Bold indicates functional domains that were reported to show treatment effects.)

OP Study Species & 

strain

Dose, route, 

vehicle

Dosing 

period

ChE inhibition Domain Age of 

testing

Outcomes NOEL, LOEL Notes & Study Problems

Chlormephos Ceh et al., 

2012

mouse 

BALB/c

3.5, 0.35 

ug/ml in 

drinking 

water of 

dams

~ 0.6, 0.06 

mg/kg/d (@ 

5ml/d, 30 g)

7 day 

premating 

to weaning

No Anxiety & 

Emotion

PND70-80 Increased time in closed arms & 

decreased time in open arms in 

elevated plus maze, ~0.6 

mg/kg/d, M&F 

NOEL=0.35 

ug/ml in 

water, ~0.06 

mg/kg/d

Litter not unit of statistical 

analysis

No pup allocation described but 

had to have used some 

littermates

M & F responses appear similar 

but not statistically compared 

Diazinon Roegge et 

al., 2008

rat 

Sprague 

Dawley

0.5, 2 

mg/kg/d sc 

DMSO 

PND1-4 Cites Slotkin et al. 

(2006): 0.5 mg/kg/d 

produced <10% brain 

inhibition on PND5, 2 

mg/kg/d produced 

25-30% brain 

inhibition 2 hr after 

dose on PND4, and 

10-20% inhibition on 

PND5

Anxiety & 

Emotion 

PND52-56 Decreased time in open arms in 

elevated plus maze, 2 mg/kg/d, 

M only

No NOEL

LOEL=0.5 

mg/kg/d

Pups & dams redistributed daily

No effect in F

Accepts p<0.1 as significant for 

interactions

No dose-response in feeding or 

milk preference studies

Abstract misstates sex differences

PND64-67, 

78-79

Decreased latency to eat in 

novelty suppressed but not 

home-cage feeding, 0.5 & 2 

mg/kg/d, M only

PND73-74 Decreased chocolate milk 

preference, 0.5 mg/kg/d only, M 

only

PND86-87 No effect on forced swim test

Diazinon Spyker and

Avery, 1977

mouse F2 

hybrid 

(NCTR 

cross 

bred)

0.18, 9 

mg/kg/d in 

peanut 

butter

GD1-birth No Sensory PND38 Increased errors on visual cliff, 

0.18 mg/kg/d only, F only

No effect acoustic startle or 

olfactory responses

No NOEL

LOEL=0.18 

mg/kg/d

No pup allocation described but 

had to have used littermates

Not clear when both sexes tested 

and/or compared

Statistics not described

Maternal weight gain lowered at 

both doses

Weight gain of high dose pups 

decreased

Preweaning testing: decreased 

contact placing, 0.18 mg/kg/d 

only

No dose-response for some 

measures

Twice as many controls as treated

Looks like decreased rotarod 

endurance PND65, not significant 

due to high variability

Neuromotor PND50, 60, 

65,75

No effect swimming ability

Increased rod cling endurance, 

0.18 & 9 mg/kg/d, sex not 

specified

Decreased inclined plane 

performance, 0.18 & 9 mg/kg/d, 

sex not specified 

Activity PND75-76 No effect open field

Cognition PND87 No effect errors in Lashley maze 

Diazinon Timofeeva 

et al., 2008

rat 

Sprague 

Dawley

0.5, 2 

mg/kg/d sc 

DMSO 

PND1-4 Cites Slotkin et al. 

(2006): 0.5 mg/kg/d 

produced <10% brain 

inhibition on PND5, 2 

mg/kg/d produced 

Activity PND28-42 No effect in figure-8 chamber, 

M&F 

No NOEL

LOEL=0.5 

mg/kg/d

Littermates of those used in 

Roegge et al. 2008

Pups & dams redistributed daily

Accepts p<0.1 as significant for 

interactions

Sensory PND77-84 Decreased prepulse inhibition, 

0.5 & 2 mg/kg/d, M only
Page 92 of 101

25-30% brain 

inhibition 2 hr after 

dose on PND4, and 

10-20% inhibition on 

PND5

Cognition PND28-35, 

91-126

No effect on T-maze 

spontaneous alternation, M&F

Increased working memory 

errors, 0.5 mg/kg/d only, M&F 

No mention of sex effects in Tmaze 

No dose-response for some 

measures

Diazinon Vatanparas

t et al., 

2013

rat Wistar 1 mg/kg/d sc 

DMSO 

GD15-18 or 

PND1-4

No Activity PND60 Gestational: No effect in open 

field, M&F 

Postnatal: No effect in open 

field, M&F

No NOEL

LOEL=1 

mg/kg/d

1 M & 1 F per litter but sex not 

nested within litter in statistics

F only affected with gestational 

exposure, both sexes affected 

with postnatal, looks like M more 

affected

Large effect sizes

Number of dams not mentioned

Discrepancy in text on pup 

sample sizes

Cognition PND60-63 Gestational: Decreased latency 

to cross and increased time 

spent in dark side on retention 

trial, no effect acquisition in 

passive avoidance, F only 

Postnatal: Decreased latency to 

cross and increased time spent 

in dark side on retention trial, 

no effect acquisition in passive 

avoidance, M&F

Diazinon Win-Shwe 

et al., 2013

mouse 

C3H/HeN

0.5, 5 

mg/kg/d sc 

DMSO

PND8-11 No Cognition PND46-49, 

81-84

PND46-49: Decreased novel 

object exploration and 

discrimination, 0.5 & 5 mg/kg/d

PND81-84: Decreased novel 

object exploration and 

discrimination, 5 mg/kg/d only 

No NOEL

LOEL=0.5 

mg/kg/d

M only tested

Separate mice at two test times

Litter allocation to dose group 

not described

Assumes that ChE inhibition 

reported by Slotkin in rats would 

be same as in mice

Larger sample size at later age

Dichlorvos Lazarini et 

al., 2004

rat Wistar 8 mg/kg po 

(dilution of 

technical 

product); 

vehicle from 

formulation

GD6-15 No Activity PND21, 

“adult”

PND21: Decreased locomotion 

open field, M only

“Adult”: Decreased locomotion 

and increased immobility, only 

M tested 

No NOEL

LOEL=8 

mg/kg/d

Data analyzed as litter but sex not 

nested within litter in statistics

No effect on physical and reflex 

preweaning development

Only M tested as after PND21

Cognition “Adult” Decreased latency to cross on Adult age not given

retention trial in passive 

avoidance

Fenitrothion

(sumithionR, 

50% ai)

Lehotzky et 

al., 1989

rat Lati 5, 10, 15 

mg/kg/d po

sunflower oil

GD7-15 No Neuromotor PND26, 36, 

104

Decreased latency to fall off 

rotarod PND26, 104, not 36, 15 

mg/kg/d

NOEL=5 

mg/kg/d

LOEL=10 

mg/kg/d

Postnatal mortality at all doses 

(16-17.5%) 

No pup allocation described but 

had to have used some 

littermates

Only M tested

Statistics not described

Measured startle, righting, 

contact placing on PND22 but no 

results given

Shorter latency in cognitive task 

hard to interpret

Nonsignificant decrease in 

activity at PND26

Activity PND26, 36, 

104

Decreased activity in open field 

PND104, 15 mg/kg/d

Cognition PND42, 104 Shorter escape latency in 

conditioned response during 

acquisition, 10 & 15 mg/kg/d

Social 

behavior

PND62 Increased time in social 

interaction, 10 & 15 mg/kg/d
Page 93 of 101

Methamidopho

s

deCastro et 

al., 2000

rat Wistar 1 mg/kg/d 

po water

GD6-15 Pilot in nonpregnant 

F dosed for 10 d 

gives 17% plasma 

inhibition at 1 

mg/kg/d

Activity PND40 No effect in open field, sex no 

mentioned

NOEL=1 

mg/kg/d

Used 2 pups/litter but no 

mention of sex, apparently used 

as independent observations

No effect on preweaning 

swimming performance

Decreased immobility time PND 

14 only 

Open field measures with really 

high variability, not reliable

Methamidopho

s

Lima et al., 

2013

mouse 

Swiss

1 mg/kg/d sc 

DMSO 

PND3-9 Pilot showed for 1 

mg/kg/d: ~19% brain 

inhibition on PND10; 

~36%, 46% brain 

inhibition 1, 4 hr 

after dosing on 

PND3; ~53, 61% 

brain inhibition 1, 4 

hr after dosing on 

PND9; no brain 

inhibition in PND60 

adults 

Activity PND61 No effect in open field, sex not 

mentioned 

No NOEL

LOEL=1 

mg/kg/d

1 M & 1 F per litter

No data for M & F separately or 

mention of statistical differences

Dosing by litter

High variability especially with 

passive avoidance 

Anxiety & 

Emotion

PND60-61 Increased immobility time in 

forced swim, sex not mentioned

No effect on elevated plus 

maze, sex not mentioned

Cognition PND63 No effect on passive avoidance, 

sex not mentioned 

Methyl 

parathion

Crowder et 

al., 1980

rat 

Sprague 

Dawley

1 mg/kg/d 

po corn oil

GD7-15 No Activity PND23, 30, 

44, 54, 65

Increased activity in open field, 

only PND23 and 54, sex not 

mentioned

to criterion 

No NOEL

LOEL=1 

mg/kg/d

Preweaning testing: possibly 

decreased wire cling time (not 

analyzed), no effect on righting, 

startle, placement response

Increased postnatal mortality 

(30%)

Littermates used

Only 3 litters used 

Small sample size for maze 

testing

Statistics not mentioned except 

for maze transfer test, just used ttest

Sex not mentioned except for 

maze transfer test, data not given 

for M & F

Methods & results cursory

Cognition >PND68 Slower transfer on 1st, 4th 

direction change in T-maze 

learning transfer, sex not 

mentioned

Methyl 

parathion

Gupta et 

al., 1985

rat 

WistarFurth F 

mated 

with F344 

M

1 mg/kg/d in 

peanut 

butter, 1.5 

mg/kg/d po 

peanut oil

GD6-20 Dams on GD19 show 

20, 60% brain 

inhibition at 1, 1.5 

mg/kg/d

Pups show brain 

inhibition up to 50% 

on PND1, 7, 14, 21, 1 

& 1.5 mg/kg/d; on 

PND28 only 1.5 

mg/kg/d 

Cognition PND60 No effect on passive avoidance

No effect on shuttle box 

avoidance

Slower latency to bar press & 

increased days to asymptote on 

operant task (no schedule 

given), 1 mg/kg/d only, sex not 

mentioned

No NOEL

LOEL=1 

mg/kg/d but 

no effects at 

1.5 mg/kg/d

High dose dams had cholinergic 

signs, increased resorptions

Pups moved to foster mothers at 

24 hr

No effect on preweaning reflexive 

behaviors

Pup allocation not described

Statistics barely described

M & F apparently tested but data 

for each not shown or mentioned 

Methods cursory

No dose-response for behavior 

but there is dose-response for 

ChE inhibition

Neuromotor PND60 No effect on rotarod

Activity PND60 Decreased activity, 1 mg/kg/d 

only, sex not mentioned

Anxiety & 

Emotion

PND60 Faster cage emergence, 1 

mg/kg/d only, sex not 

mentioned 
Page 94 of 101

Sensory PND120 No effect on acoustic startle 

response

Only 4/dose for operant testing

Methyl 

parathion

Johnson et 

al., 2009

rat 

Sprague 

Dawley

Incrementin

g doses: low 

0.2 mg/kg/d 

throughout; 

mid 0.2, 0.4, 

0.6 mg/kg/d 

every 5-6 d; 

high 0.3, 0.6, 

0.9 mg/kg/d 

every 5-6 d

PND1-21 Low dose showed 

13-15% brain 

inhibition and high 

dose showed 63, 20, 

18% brain inhibition 

on PND20, 30, 40; all 

doses recovered by 

PND50

Cognition PND29-60 Increased working memory 

errors, mid and high dose, M 

only

Increased reference memory 

errors, all doses, M only

No NOEL

LOEL=0.2 

mg/kg/d

Split-litter dose design

Included litter as random effect in 

statistics

No effect on preweaning 

measures of reflex development

No effect in F

Oxydemeton 

methyl 

(metasystoxR, 

91% ai)

Clemens et 

al., 1990

rat CD 0.5, 1.5, 4.5 

mg/kg/d 

water

GD6-15 Dams on GD16 show 

30, 54, 72% plasma 

inhibition (RBC 

similar) & 22, 52, 

68% brain inhibition 

at 0.5, 1.5, 4.5 

mg/kg/d

Dams on GD20 show 

20, 39, 54% brain 

inhibition at 0.5, 1.5, 

4.5 mg/kg/d, 40% 

RBC inhibition at 4.5 

mg/kg/d, and no 

plasma inhibition

Fetuses on GD20 

show no brain 

inhibition 

Cognition PND25, 26, 

35

No effect on M-maze NOEL=4.5 

mg/kg/d

High dose dams had tremors

1 M & 1 F per litter

No data for M & F separately or 

mention of statistical differences

Statistics barely described

No effect on preweaning reflex or 

sensory tests

Activity No effect in open field

Parathion Al-Hachim 

& Fink, 

1968

mouse 

CF1

3 mg/kg/d 

po

Corn oil

3 dosing 

times: 1st, 

2nd, or 3rd 

trimester

No Cognition PND30-37 No effect on conditioned 

avoidance learning 

NOEL=3 

mg/kg/d

Very similar experiment as other 

papers, maybe same study

Pup allocation not clear but 

littermates probably used

Inadequate statistics

No mention of sex

Parathion Al-Hachim 

& Fink, 

1968

mouse 

CF1

3 mg/kg/d 

po

Corn oil

3 dosing 

times: 1st, 

2nd, or 3rd 

trimester

No Activity PND60-66 No effect in open field NOEL=3 

mg/kg/d

Very similar experiment as other 

papers, maybe same study

Pup allocation not clear but 

littermates probably used

Inadequate stats

No mention of sex

Parathion Levin et al., 

2010

rat 

Sprague 

Dawley

0.1, 0.2 

mg/kg/d sc 

DMSO

PND1-4 Cites Slotkin et al.

2006: 0.1 mg/kg/d 

produced 5-15% 

brain inhibition on 

PND5, no data for 0.2 

mg/kg/d

Cognition PND420, 

510, 570

PND420: Increased working 

memory errors in radial arm 

maze, 0.1 mg/kg/d, M only; 

increased reference memory 

errors, 0.1 & 0.2 mg/kg/d, M 

only 

PND510: Increased working 

memory errors in radial arm 

maze, 0.1 & 0.2 mg/kg/d, M 

only

PND570: No effect in radial arm 

maze 

No NOEL

LOEL=0.1 

mg/kg/d

Littermates of those used in 

Timofeeva 2008

Pups & dams redistributed daily

5% mortality high dose

No effect in F

Accepts p<0.1 as significant for 

interactions

No dose-response for several 

measures
Page 95 of 101

Parathion Stamper et 

al., 1988

rat Long 

Evans

1.3, 1.9 

mg/kg/d sc 

corn oil

PND5-20 35, 68% brn inh 

PND21; 26, 36% brn 

in PND28

Activity PND24 No effect in open field No NOEL

LOEL=1.3 

mg/kg/d

Split-litter dose design

Pup allocation not clear but 

littermates probably used

High dose produced cholinergic 

signs, says doses are 33 and 50% 

of LD50 in PND5 rat

Decreased weight gain with both 

doses

Preweaning, increased cliff 

avoidance latency, no effect 

righting, negative geotaxis, open 

field

M only tested

No post-hoc comparison of 

groups when significant, but 

looks like effects in both doses

No dose-response in working 

memory errors

Neuromotor PND24 No effect on rotarod

Cognition PND24, 

PND 35-37

Decreased alternation rate in Tmaze, 1.3 & 1.9 mg/kg/d, only 

M tested

Increased working memory 

errors, 1.3 & 1.9 mg/kg/d, only 

M tested

Parathion Timofeeva 

et al., 2008

rat 

Sprague 

Dawley

0.1. 0.2 

mg/kg/d sc 

DMSO

PND1-4 Cites Slotkin et al.

2006: 0.1 mg/kg/d 

produced 5-15% 

brain inhibition on 

PND5, no data for 0.2 

mg/kg/d

Activity PND58-61 No effect in figure-8 chambers No NOEL

LOEL=0.1 

mg/kg/d

Pups & dams redistributed daily

5% mortality high dose

Accepts p<0.1 as significant for 

interactions

All radial arm maze effects only in 

low dose

Cognition PND35-45

PND112-

182

No effect in T-maze 

spontaneous alternation

Decreased working memory 

errors in radial arm maze, 0.1 

mg/kg/d only, M&F 

Anxiety & 

Emotion

PND50-53

PND64-72

PND81-94

Increased time in open arms in 

elevated plus maze, 0.2 

mg/kg/d, M&F

No effect on novelty suppressed 

feeding 

No effect on chocolate milk 

preference 

Sensory PND78-81 Decreased tactile startle, 0.2 

mg/kg/d, M&F

No effect on prepulse inhibition

Appendix 2. Summary of Guideline DNT Studies Submitted to the Agency for OPs other than Chlorpyrifos.

(Only changes that were observed after exposure had ended (post weaning, adult) are listed. ‘X’ indicates no significant changes on tests for each domain.)

Chemicals Cognition Motor activity Acoustic startle Neuromotor (FOB) Notes

Acephate X X X X

Azinphos-methyl X X X X

Coumaphos X X X X
Page 96 of 101

Diazinon Biel maze: increase errors & 

latency high dose (~33.1 

mg/g,/d, dam diet) M, PND24 & 

PND62; also mid dose (~3.4 

mg/kg/d, dam diet) F, PND24

X X X

Dichlorvos1-- -- -- --

Dicrotophos X X X X

Dimethoate X X X X

Disulfoton X X X X

Ethoprop M maze: increase trials to 

criterion high dose (~29.3 

mg/kg/d, dam diet) M, PND60

X X X

Fenamiphos X X X X

Malathion X increased rearing in FOB open 

field mid dose (50 mg/kg/d to 

dams & pups), F, significant at 

PND45 only (maybe also 

PND60); no change 

automated motor activity

increased peak amplitude later 

blocks (perhaps habituation effect) 

all doses (5, 50, 150 mg/kg/d to 

dams & pups), F only, PND23; 

increased peak amplitude without 

prepulse low dose only, F only, 

PND60

altered gait mid & high dose 

(50, 150 mg/kg/d to dams & 

pups), M & F, PND60 but not 

earlier

Methamidophos ~X X decreased peak amplitude early 

blocks mid & high dose (~1.65, 5.2 

mg/kg/d dam diet), F only, 

significant at PND38, looks same 

but not significant at PND60

X PA PND24: report says 

decreased latency but 

nothing significant & table 

is questionable; M maze 

PND60: report says 

increased trials to criterion 

(M) and increased (M) or 

decreased (F) errors, but 

nothing significant and 

table is questionable

Methyl parathion X X X X

Naled X X increased peak amplitude & 

decreased latency middle blocks 

low dose only (0.4 mg/kg/d to dams 

& pups), F only, PND60; report says 

decreased amplitude but not 

significant all blocks high dose (10 

mg/kg/d to dams & pups), M only, 

PND23 & PND60

X only swimming time in Y 

maze reported, varied 

significances, no mention 

of errors or other 

performance measures
Page 97 of 101

Phorate M maze: decrease number 

reaching criterion with 

relearning, low & mid dose (0.03 

& 0.1 mg/kg/d to dams & pups), 

M only significant, PND30; not 

seen in second study with 

higher dose

X decreased peak amplitude all blocks 

high dose (0.1 mg/kg/d to dams & 

pups), M only, PND60; not seen in 

second study with higher dose

X combined two studies; one 

with low & mid dose, other 

with high dose; some data 

didn't agree

Profenofos X X X X

Terbufos X X X X

Tetrachlorvinphos X X X X

Tribufos X X X X

Trichlorfon PA: decreased latency to enter 

on retention high dose (~205.1 

mg/kg/d dam diet), M only, 

PND29; M maze: increased 

average errors second trial high

dose, F only, PND60

decreased activity middle 

blocks (maybefaster 

habituation) mid dose (~76.2 

mg/kg/d dam diet) only, F 

only, PND60

decreased peak amplitude all blocks 

high dose (~205.1 mg/kg/d dam 

diet), early blocks mid dose (~76.2 

mg/kg/d dam diet), M & F, PND22; 

decreased amplitude middle blocks 

(not consistent) all doses (~23.3, 

76.2, 205.1 mg/kg/d dam diet), F 

only, PND38; for M decreased 

amplitude apparent but not 

significant high dose PND38 & 

PND60

X

1 high pup mortality in all groups, including control, negated any valid neurotoxicity assessments of dichlorvos
Page 98 of 101

Appendix 3. Low Quality Studies: Summary of Study Design Elements Impacting Study Quality Assignment

Study Study Location

Cohort Name /

Description of 

Study 

Population Study Design Exposure Assessment Outcome Assessment

Confounder /

Covariate Control Statistical Analysis Risk of (other) Bias

Acosta-Maldonado et al. 

(2009)

Chihuahua, Mexico Singleton pregnancies Cross-sectional, small 

pilot study – only 9 

women exposed, 

participant selection and 

exclusion not detailed

N=54 mothers

(9 exposed, 45 

comparison mothers)

Proxy indicator of exposure -

Residence in agricultural 

community were pesticides had 

been applied; or home located < 5 

km from a pesticide application 

zone; or cohabitating with worker 

exposed to pesticides or 

agricultural labor 

Also, AChE activity – Objective 

biomarker of exposure/altered 

function 

Standardized but partially subjective 

assessment of placental maturity

Minimal. Adjustment for placental 

characteristics. 

Appropriate multivariate analysis; 

Corrected hypothesis test results for 

multiple comparisons.

Selection bias unlikely; 

Residual confounding likely; 

substantial potential for 

differential misclassification 

of exposure.

Dawbrowski et al. (2003) Lodz, Poland Newborn children among 

Polish farmers

Case-control, large 

sample size

N=389

Age: newborns

Prenatal pesticide exposure 

assessed via questionnaire 

(retrospective self-report). Site 

visit after delivery to evaluate 

pesticide exposure

Pregnancy outcomes assessed using 

birth records

Appropriate. Included maternal 

demographics, predictors of high-risk 

pregnancy (duration, maternal weight) and 

environmental toxicant exposure (ETS). No 

adjustment for SES indicators

Appropriate multivariate analysis. Selection bias unlikely; 

Residual confounding likely 

small in magnitude; 

considerable potential for 

non-differential 

misclassification of exposure 

(unlikely to account for nonnull findings). Minimal 

misclassification of outcome. 

Grandjean et al.(2006) Tabacundo, Ecuador Healthy 2nd and 3rd 

grade children

Cross-sectional, small 

sample size

N=72 

Age <9 years

Prenatal occupational exposure 

assessed via questionnaire; Also 

recent child exposure biomarker 

assessment (DAP).

Objective anthropometric and other 

clinical outcomes; Numerous 

neurobehavioral outcomes evaluated 

using easy-to-administer screening 

instruments; Age appropriate. May be 

insensitive to subtle effects of OP 

pesticide exposure effects.

Appropriate. Homogenous population limited 

confounding by design; Included child 

demographics (age, sex, weight); SES 

indicators (maternal race, housing, running 

water, sewage), diet (meals/day), 

environmental toxicants (maternal alcohol 

and smoking) and medical history. 

Appropriate multivariate analysis; 

Numerous hypotheses evaluated 

without correction for multiple 

comparisons.

Selection bias unlikely; 

Residual confounding likely 

small in magnitude; 

considerable potential for 

non-differential 

misclassification of exposure 

(likely to account for null 

findings). Potential 

misclassification of outcome. 
Page 99 of 101

Appendix 3. Low Quality Studies: Summary of Study Design Elements Impacting Study Quality Assignment (continued)

Study Study Location

Cohort Name /

Description of 

Study Population Study Design Exposure Assessment Outcome Assessment

Confounder /

Covariate Control Statistical Analysis Risk of (other) Bias

Harari et al. (2010) Tabacundo, Ecuador 

(Andean plateau north of 

Quito)

6- to 8-year-old children 

in the two lowest grades 

(called second and third) 

of one of two schools

Cross-sectional, small sample size

n = 81

Age 6- 8 years

Child Current Exposure - DMP 

metabolites measured in a spot 

urine samples. A blood sample 

was analyzed for AChE. 

Maternal Exposure – Interview by 

skilled interviewers

Blood pressure and neurophysiologic 

measures - the instruments used for 

neurophsychologic measures were 

validated to avoid cross-cultural 

influences.

Appropriate: child’s sex, age, BMI, number of 

daily meals (only in current exposure), 

stunting, hematocrit, school grade, having 

repeated one grade, maternal education 

level, family living in a traditional house, 

drinking water supply, and paternal 

education and employment

Appropriate: Standard parametric 

tests and logistic regression

Errors in exposure 

classification status since it 

was based on the maternal 

self-report, mothers likely 

being aware of the 

neurobehavioral status of 

their children, exposure 

assessment based on a spot 

urine sample 

Kofman et al. 

(2006)

Israel (Negev region) Bedouin population 

(Children aged 6 to 12 at 

the time of the study, who 

were victims of poisoning 

before age three)

Retrospective cohort study, small 

sample size

N = 52

9-Exposed to OP; 17-Exposed to 

Kerosene/paint thinner

26-Controls 

Aged 6-12 years 

OP poisoning was confirmed by 

low serum butyrylcholinesterase 

activity based on hospital records

Neuropsychological evaluation and 

structured interview of parents. 

Errors in assessment minimized as 

psychologists were qualified 

individuals, language and cultural 

differences taken into consideration, 

each child tested on same day and in 

same place as matched control.

Age, sex, background (cultural and 

demographic)

Difference in means Errors is outcome 

classification likely since 

psychologists who 

administered the tests knew 

which children were exposed, 

small sample size 

Koutroulakis et al. 

(2014)

Crete, Greece Women with singleton 

pregnancies, permanent 

residents for at least two 

years, referral for 

amniocentesis to the 

Fetal-Maternal Unit, 

Department of Obstetrics 

and Gynecology, 

University Hospital of 

Heraklion

Prospective Cohort Study – large 

sample size, ethical issues

n = 415

Age: newborns

Objective. DAP measurement in a 

single amniotic fluid samples 

collected at either 16th or 20th 

weeks of gestation – Novel 

biomarker. Questionnaire was 

also used.

Birth weight and head circumference. 

Unclear how the outcome information 

was obtained.

Neonatal sex, maternal age, agricultural 

activities, and gestational age at 

amniocentesis – Rationale for confounder 

selection not provided

Appropriate: multiple linear 

regression

Comparing exposure 

measurements in AF against 

known biomarker (e.g., OP 

metabolite levels in urine) for 

validation of AF not 

conducted. Smoking status of 

participants before/during 

pregnancy, high risk 

pregnancies, gestational 

diabetes, PON1 enzyme 

activity in the fetuses not 

considered.
Page 100 of 101

Appendix 3. Low Quality Studies: Summary of Study Design Elements Impacting Study Quality Assignment (continued)

Study Study Location

Cohort Name /

Description of 

Study Population Study Design Exposure Assessment Outcome Assessment

Confounder /

Covariate Control Statistical Analysis Risk of (other) Bias

Lizardi et al. 

(2008)

Yuma County, Arizona, 

USA

Children's Pesticide 

Survey (CPS)

Cross-sectional, small 

sample size

N=48

Age 7 years

Objective Biomarker of prenatal 

OP pesticide exposure (DAP) 

quantified in single child spot 

urine sample provided at the 

time of the cognitive 

assessment. 

Cognitive assessment using battery of test 

instruments considered valid and reliable in 

similar populations. Spanish translation as 

necessary. 

None Correlation coefficients (type unspecified). 

Statistically significant confounders were not robust 

due to influential outliers

Selection bias unlikely; Residual 

confounding likely; considerable 

potential for non-differential 

misclassification of exposure 

Potential misclassification of 

outcome.

Lu et al. (2009) Cota Brus, Costa Rica 4-10 yr. old children 

whose parents worked in 

organic coffee farm (La 

Amistad) and 

conventional coffee 

farms (Las Mellizas)

Cross-Sectional (pilot), 

low sample numbers

N=35:

17 Organic farm

18 Conventional farms

Age 4-10 years old

Good measure (urinary PNP, 

IMPY, TCPy), but no major 

differences between exposure 

groups 

Good measure (CBARS) but different SES 

and demographic characteristics for 

exposure groups

Limited number (group, age, 

sex, handedness, grade)

Appropriate: Linear mixed effects for significant test 

outcomes from paired t-test analyses

Somewhat high - Convenience 

sample with different recruitment 

methods; exposure 

misclassification

Moreno-Banda et 

al. (2009)

Mexico (Villa Guerrero, 

Coatepec de Harinas, 

Tenancingo (Mexico); 

Cuernavaca, Cuautla, 

Jiutepec, Temixco, 

(Morelos)

Newborn children of 

floricultural workers and 

families

Cross-sectional, large 

sample size

N=328

Age: newborns

Proxy indicator of prenatal 

occupational OP pesticide 

exposure (self-reported 

floricultural occupation)

Objectively measured birth outcomes 

assessed using birth certificate (partial). 

Self-reported by mother if birth certificate 

unavailable.

Appropriate, though minimal 

– history of adverse 

reproductive outcomes, 

infant sex, maternal smoking 

and alcohol use during 

pregnancy.

Appropriate multivariate analysis Selection bias likely; Residual 

confounding likely small in 

magnitude; considerable potential 

for non-differential 

misclassification of exposure (likely 

accounts for null findings). 

Potential outcome misclassification 

(self-report in subset of 

participants).

Nevison (2014) U.S. National population Children with birth years 

1970–2005 in 1) 

California Department of 

Developmental Services 

(CDDS) reports 2) 

Individuals with 

Disabilities Education Act 

(IDEA) reports

Ecological (time trend)

N=NP (national database)

Non-specific, proxy OP exposure 

measure (lbs/yr)

Two large reporting DBs: CA Department of 

Developmental Services (CDDS), US 

Individuals with Disabilities Education Act 

(IDEA)

Limited: differences in 

autism definitions, changes 

in diagnostic criteria

Appropriate: ratio of age-resolved snapshot; tracking 

trend slopes; correlation coefficient between 

temporal trend and composite autism prevalence 

curve

Selection bias unlikely; do not 

know individual exposure
Page 101 of 101

Appendix 3. Low Quality Studies: Summary of Study Design Elements Impacting Study Quality Assignment (continued)

Study Study Location

Cohort Name /

Description of 

Study 

Population Study Design Exposure Assessment Outcome Assessment

Confounder /

Covariate Control Statistical Analysis Risk of (other) Bias

Rohlman et al. (2005) Oregon and North 

Carolina, USA

Latino children of 

immigrant parents 

living in Oregon or 

North Carolina

Cross-Sectional, small sample 

size

N= 78 

Age 48-71 months

Proxy indicator of chronic OP pesticide 

exposure - Residence in highly 

agricultural communities

Battery of neurocognitive 

development; Screening tools; 

Some instruments likely not 

appropriate for use in study 

population - Not all participants 

able to complete all evaluations. 

Poor administration. Tests 

administered twice; only 

performance on 2nd evaluation 

considered in analysis. 

Self-reported covariate information 

collected via questionnaire. Adjustment for 

age, SES indicator (maternal education). No 

environmental toxicants. 

One-sided hypothesis tests. Numerous 

hypotheses evaluated without 

correction for multiple comparisons.

Selection bias unlikely; Residual 

confounding likely; substantial 

potential for differential 

misclassification of exposure and 

outcome. Inadequate 

presentation of study results. 

Seemingly post-hoc evaluation of 

effect modification. 

Samarawickrema et al. 

(2008)

Southern Sri Lanka Pregnant women 

delivering at 

Embilipitiya Base 

Hospital

Cross-sectional Birth Cohort, 

small sample size

N=41 end of two pesticide 

spraying seasons;

N=25 at beginning of spraying 

season

Proxy indicator of prenatal OP 

pesticide exposure (delivery during 

pesticide spray season); Objective 

pesticide biomarkers assessed (OP 

pesticide residues), but detected in 

only two subject’s specimens – not 

evaluated

Objective biomarkers of early 

biological effect outcomes -

Maternal and fetal 

butyrylcholinesterase (BuChE) 

activity; antioxidant status;

fetal oxidative stress;

fetal DNA fragmentation

No adjustment for potential confounders 

(though comparison groups were 

considered to be relatively homogeneous).

Largely appropriate. Assumptions of 

some statistical tests likely violated.

Selection bias unlikely; Residual 

confounding likely; substantial 

potential for differential 

misclassification of exposure; 

Seemingly post-hoc evaluation of 

effect modification.

Savitz et al. (1997) Ontario, Canada Newborn children in 

the Ontario Farm 

Family Health Study; 

Retrospective Birth Cohort, 

large sample size

N= 1,898 couples; 3,984 

Age: newborns

Proxy indicator of pre-conception 

paternal para-occupational OP 

pesticide exposure (self-reported male 

farm activities in 3-month period prior 

to conception.

Objectively measured birth 

outcomes assessed by maternal 

self-report

Appropriate. Included family/child 

demographics (sex, weight, maternal age, 

ethnicity); SES indicators (maternal and 

paternal education and occupation, per 

capita income) race, housing, running 

water, sewage), diet (meals/day), pregnancy 

risks (maternal caffeine, alcohol and 

smoking) and medical history.

Inappropriate multivariate regression 

analysis. Likely misspecification of true 

variance; No adjustment for multiple 

comparisons.

Selection bias probable; Residual 

confounding likely; substantial 

potential for differential 

misclassification of exposure and 

outcome.

Wickerham et al. 

(2012)

Zhejiang Province,

China

Newborn children 

delivered at the Fuyang 

Maternal and 

Children's hospital

Cross-sectional, small pilot 

study

n=116

Age: Full term infants

Objective biomarker of pesticide 

exposure (pesticide residues in cord 

blood) – parameterized as number of 

pesticide residues detected. Methods 

unlikely suitable for detecting low 

levels.

Birth weight assessed using birth 

records and maternal report

Appropriate. Assessed using questionnaire 

(maternal self-report) and medical records.

Appropriate multivariate analysis. Selection bias unlikely; Residual 

confounding likely; substantial 

potential for differential 

misclassification of exposure. 

Some outcome misclassification 

unlikely .

Naksen et al (2015) Fang district, Chiang Mai

province, Thailand

Pregnant women 

delivering at Fang 

Hospital

Prospective Cohort, small pilot 

study

n=52

Age: newborns

Objective biomarkers of pesticide 

exposure (DAPs). Also AChE, BChE, 

and PON1 genotype expression 

measurement. Maternal blood and 

urinary samples taken, plus cord 

blood. Questionnaire to assess other 

exposures and covariates. 

Birth outcomes (Body weight and

length, and head circumference)

abstracted from medical records. 

Appropriate. Assessed using questionnaire 

(maternal self-report) and medical records. 

Some errors in statistical analysis were 

identified; e.g., for gestation age, log 

total DEAP at 32 weeks of pregnancy, a 

0.7 beta was reported, but the 

confidence interval is reported as (-0.1, 

-1.4)]. No adjustment for multiple 

comparisons.

Inadequate presentation of study 

results. Selection bias possible 

due to loss to follow-up; Residual 

confounding likely, small in 

magnitude; potential for nondifferential misclassification of 

exposure.