UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
                         WASHINGTON, D.C. 20460

                                                                     	OFFICE OF
                                                           CHEMICAL SAFETY AND
                                                           POLLUTION PREVENTION

MEMORANDUM

Date:  December 29, 2016

SUBJECT:	Updated Literature Review on Neurodevelopment Effects & FQPA Safety Factor Determination for the Organophosphate Pesticides

PC Code:  See Below
DP Barcode:  437043
Decision No.:
Registration No.: None
Petition No.: None
Regulatory Action: None
Risk Assessment Type: None
Case No.: None
TXR No.: 0057252
CAS No.: See Below
MRID No.: None
40 CFR: None

FROM:	Ashlee Aldridge
	Health Effects Division (7509P)
	Office of Pesticide Programs

              Anna Lowit, Ph.D., Senior Science Advisory
              Immediate Office
              Office of Pesticide Programs (7501P)

              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:	Nicole Zinn, 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.  This paper updates the September, 2015 paper based on comments from the public and addition of new epidemiology papers from the open literature. No additional studies have been added to the laboratory animal toxicology sections of the literature review.
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

Updated Literature Review on Neurodevelopment Effects & FQPA Safety Factor Determination for the Organophosphate Pesticides

                         Office of Pesticide Programs
                      US Environmental Protection Agency

                               December 29, 2016

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 Literature Reviews: 2012/2014, 2015, and updated in 2016	25
2.2.2	Literature Search Methodology	26
2.2.3	Breadth and Depth of the 2015/2016 Literature Review	27
Table 2.2-a	Study Designs, Exposure Measurement Methods, and Outcome Measurement Methods Used across the 2015/2016	31
Table 2.2-b	Detailed Neurological Outcome Measurement Methods Used across the  2015/2016 Review Studies	33
2.2.4	Study Designs & Considerations for Study Quality Evaluation	35
<IP_ADDRESS>	Study Designs	35
<IP_ADDRESS>	Considerations for Study Quality	37
Table 2.2.4-1	Study Quality Considerations	38
<IP_ADDRESS>.1	Exposure Measures	39
Table 2.2.4-2	CDC Table of Organophosphate Pesticides and Their Dialkyl Phosphate Metabolites (2008)	41
<IP_ADDRESS>.2	Neurological and Other Outcome Measures	42
<IP_ADDRESS>.3.	Statistical Analysis	42
<IP_ADDRESS>.4	Confounding	43
<IP_ADDRESS>.5	Risk of Bias	43
2.2.5	Review of Quality Results	44
<IP_ADDRESS>	"High" Quality Group	44
Table <IP_ADDRESS>-1.	High Quality Studies from 2015 and 2016 reviews: Summary of Study Design Elements Impacting Study Quality Assignment	45
Table <IP_ADDRESS>-2	High Quality Studies from 2012/2014 literature search of Children's Environmental Health Epidemiology Studies	49
<IP_ADDRESS>	"Moderate" Quality Group	64
Table <IP_ADDRESS>-1.	Moderate Quality Studies: Summary of Study Design Elements Impacting Study Quality Assignment	65
<IP_ADDRESS>	"Low" Quality Group	68
2.2.6	Assessment of Epidemiological Studies for Relevance to Analysis	68
2.2.7	Birth Outcome Epidemiologic Studies	70
2.2.8	Neurodevelopment Outcome Epidemiologic Studies	71
<IP_ADDRESS>	CCCEH, CHAMACOS, & Mt. Sinai Cohorts	72
<IP_ADDRESS>	HOME Cohort	77
<IP_ADDRESS>	Meta-analysis (Engel et al, 2015)	78
<IP_ADDRESS>	Other Neurodevelopmental Studies	79
3.0	Weight of Evidence Analysis:  Integration Across Multiple Lines of Evidence	80
3.1	Dose-Response Relationships & Temporal Concordance	82
3.2	Strength, Consistency & Specificity	83
3.2.1	Strength	83
3.2.2	Consistency	84
3.2.3	Specificity	89
3.3	Biological Plausability & Coherence	91
4.0	10X FQPA Safety Factor for Infants and Children	92
5.0	References	94
6.0	Appendices	106
Appendix 1.	Table of In Vivo Developmental Neurotoxicity Studies of OPs.	107
Appendix 2.	Summary of Guideline DNT Studies Submitted to the Agency for OPs other than Chlorpyrifos.	112
Appendix 3.	Low Quality Studies: Summary of Study Design Elements Impacting Study Quality Assignment	114
Appendix 4.	Table of Systematic Review Analysis:  Initial Search Research & Initial Exclusion Criteria.	118
Appendix 5.	Table of Systematic Review Analysis:  Second Tier Exclusion Criteria.	157
Appendix 6.	Plausible hypotheses on MOA/AOP for neurodevelopmental outcomes (Extracted from Section 4.4.3. RHHRA for chlorpyrifos)	171

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.  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).  Historically the agency has used inhibition of AChE as the point of departure for OP human health risk assessments (HHRAs).

Newer lines of research on OPs in the areas of potential modes of action/adverse outcome pathways (MOAs/AOPs), 0F 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 since 2008 (See Appendix 6).

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 epidemiology studies on three children's cohorts, with a particular focus on women and children and exposure to chlorpyrifos (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). In December, 2016, OPP's Framework for Incorporating Human Epidemiologic & Incident Data in
 Risk Assessments for Pesticides (USEPA, 2016c) was finalized based on input from the 2010 FIFRA SAP, public comment and experience gained over the last few years.

In 2012, the agency convened another meeting of the FIFRA SAP focused on chlorpyrifos which incorporated the newest experimental data related to AChE inhibition and both cholinergic and non-cholinergic adverse outcomes, including neurodevelopmental studies on behavior and cognition effects (FIFRA SAP 2012). Similarly, the agency also performed a more in-depth analysis of the biomonitoring data and of epidemiological studies from three major children's health epidemiology cohort studies in the U.S., as well as plausible hypotheses on MOAs/AOPs leading to neurodevelopmental outcomes (USEPA 2012; Appendix 6).  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.1F  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; Summarized in Appendix 6).  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 have been 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).  Section 4.0 states that the 10X FQPA Safety Factor is being retained for all the OPs listed in the table on page 2 above.

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 making2F.  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".3F  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 chlorpyrifos revised HHRA (USEPA, 2014).  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).

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 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-S-methyl, 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.

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 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 4[th] 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 (dose-response 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).

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 & F[1]

Parathion: improved cognition  -  rat, low dose only, M & F[2]

Parathion: cognitive deficit  -  rat, M not F[3]
Parathion: cognitive deficit  -  rat, no dose-response, only M tested[4]
Methyl parathion: cognitive deficit  -  rat, dose-response, M only[5]
T-maze spontaneous alternation

Diazinon: no effect  -  rat, M & F[1]

Parathion: no effect  -  rat, M & F[2]
Parathion: cognitive deficit  -  rat, dose-response, only M tested[4]

T-maze learning
Methyl parathion: cognitive deficit  -  rat, sex not specified[6]

Lashley III maze

Diazinon: no effect  -  mouse, sex not specified[7]

M-maze
Oxydemeton methyl: no effect  -  rat, M & F[8]

Active avoidance
Fenitrothion: improved cognition  -  rat, dose-response, only M tested[9]

Parathion: no effect  -  mouse, sex not specified[11]
Methyl parathion: no effect  -  rat, sex not specified[10]

Parathion: no effect  -  mouse, sex not specified[11]

Passive avoidance
Dichlorvos: cognitive deficit  -  rat, only M tested[12]
Diazinon: cognitive deficit  -  rat, F not M[13]

Methyl parathion: no effect  -  rat, sex not specified[10]

Diazinon: cognitive deficit  -  rat, M & F[13]

Methamidophos: no effect  -  mouse, sex not specified[14]

Novel object recognition

Diazinon: cognitive deficit  -  mouse, no dose-response, only M tested[15]

Operant responding

Methyl parathion: cognitive deficit  -  rat, sex not specified[10]

[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

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.
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 tested[1]

Fenitrothion: decreased activity  -  rat, dose-response, only M tested[2]

Methamidophos: no effect  -  rat, sex not specified[3]

Methyl parathion: increased activity  -  rat, sex not specified[4]

Oxydemeton methyl: no effect  -  rat, M & F[5]

Parathion: no effect  -  mouse, sex not specified[6]
Diazinon: no effect  -  rat, M & F[7]

Parathion: no effect  -  mouse, sex not specified[6]
Diazinon: no effect  -  mouse, sex not specified[8]
Diazinon: no effect  -  rat, M & F[7]

Methamidophos: no effect  -  mouse, sex not specified[9]
Parathion: no effect  -  rat, only M tested[10]
Figure-Eight

Diazinon: no effect  -  rat, M & F[11]

Parathion: no effect  -  rat, M & F[12]

Donut

Methyl parathion: decreased activity  -  rat, no dose-response, sex not specified[13]

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

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.

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 & F[1]
Diazinon: increased anxiety  -  rat, dose-response, M not F[2]

Methamidophos: no effect  - mouse, sex not specified[3]

Parathion: decreased anxiety  -  rat, dose-response, M & F[4]
Chocolate milk preference

Diazinon: increased anhedonia  -  rat, no dose-response, M not F[2]

Parathion: no effect  -  rat, M & F[4]
Novelty suppressed feeding

Diazinon: decreased fearfulness  -  rat, dose-response, M not F[2]

Parathion: no effect  -  rat, M & F[4]
Forced swim

Diazinon: no effect  -  rat, M & F[2]

Methamidophos: increased despair  -  mouse, sex not specified[3]
Open field behaviors
Methyl parathion: decreased anxiety  -  rat, no dose-response, sex not specified[5]

[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

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 specified[1]

Tactile with or without prepulse

Diazinon: decreased sensory gating  -  rat, no dose-response, M not F[2]

Parathion: decreased startle response  -  rat, dose-response, M & F[3]
Visual
Diazinon: decreased function  -  mouse, no dose-response, F not M[1]

Olfactory
Diazinon: no effect  -  mouse, sex not specified[1]

[1] Spyker and Avery, 1977
[2] Timofeeva et al., 2008a
[3] Timofeeva et al., 2008b

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 dose-response).  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 tested[1]
Methyl parathion: no effect  -  rat, sex not specified[2]
Diazinon: no effect  -  mouse, sex not specified[3]
Parathion: no effect  -  rat, only M tested[4]
Inclined plane

Diazinon: decreased performance  -  mouse, dose-response, sex not specified[3] (Spyker)

Rod cling

Diazinon: increased performance  -  mouse, no dose-response, sex not specified[3]

[1] Lehotzky et al., 1989
[2] Gupta et al., 1985
[3] Spyker and Avery, 1977
[4] Stamper et al., 1988

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 fenitrothion, 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, 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 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 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 Literature Reviews: 2012/2014, 2015, and updated in 2016

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 CCCEH 2) Mount Sinai Inner-City Toxicants, Child Growth and Development Study, or the "Mount Sinai Study/Cohort;" and 3) Center for Health Assessment of Mothers and Children of Salinas Valley (CHAMACOS) conducted by the University of California Berkeley, or "CHAMACOS Study/ Cohort." The conclusion of EPA's evaluation, supported by the FIFRA SAP (2008, 2012), was that "chlorpyrifos likely played a role in the neurodevelopmental outcomes observed in these studies."

In 2015, the agency 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.  This 2016 updated literature review includes edits made in response to public comments on the 2015 literature review and addition of new epidemiology papers:  Yolton et al. (2013), Cartier et al. (2015), Rauh et al (2015), Ranaan et al, (2015), Engel et al. (2015), Fiedler et al. (2015), Harley et al (2016), Stein et al (2016), Rauh et al (2015), Ranaan et al, 2015; Engel et al. (2015), and Donauer et al (2016).The agency has developed individual study reviews for each study in the 2015 and 2016 updated literature review; these study reviews contain details about study design, results, strengths, uncertainties, and interpretation.  The study reviews can be found in USEPA, 2015b, USEPA, 2016, and USEPA,2016b.  This literature review document summarizes key information, strengths and uncertainties and provides a concise, coherent weight of evidence analysis.

The 2012/2014 literature review was limited to studies from CCCEH, Mt. Sinai, and CHAMACOS; all studies from these cohorts are considered high quality.   In the CCCEH, Mt. Sinai, and CHAMACOS 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.  For those studies in 2012/2014 literature review 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 consideration with the studies identified in the 2015 and updated 2016 literature reviews.

Publication bias is a type of bias associated with the kinds of academic research likely to be published in the open literature; generally, this bias tends towards lack of publishing of null findings.  However, in the case of the neurodevelopmental epidemiology studies on OPs, as noted by commenter, there are already significant amount of null findings reported in this body of evidence.  Moreover, these studies are derived from existing cohorts of mothers and children which are well-known to the science community; these kinds of cohorts are expensive and resource intensive to develop and maintain making the number of studies from other groups unlikely.  Given the amount of null findings already reported in the literature, the agency believes that publication bias with regard to lack of publishing of null findings does not impact the interpretation of the existing studies on neurodevelopmental outcomes associated with OPs.

The findings from the birth outcome studies are summarized in 2.2.7.  The major findings of CCCEH, Mt. Sinai, and CHAMACOS on neurodevelopmental outcomes are briefly summarized in Section <IP_ADDRESS>. Results from other studies on neurodevelopmental outcomes are summarized in <IP_ADDRESS>.

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 on January 21 and January 23, 2015. In this literature search, emphasis was placed 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)) English language only((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)) English language only((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.
In 2015, EPA identified 299 articles across these several biomedical search engines. The determination of relevance to the study question was made by two EPA epidemiologists who agreed by consensus as to article disposition in the 2015 literature search.  Removing duplicates (56), 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 non-human systems; and 3 were otherwise not relevant (See Appendix 4). 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 the 2015 narrative literature review (referred to herein as "2015 Literature Review/Studies"). (See Appendix 5).
No additional formal literature search was conducted prior to the development of this 2016 update.  However, the agency maintains active inquiry of the open scientific literature on issues related to OPs and the agency believes the added studies represent the available, relevant information.  Of the 9 new epidemiology studies, two are meta-analyses.
The following sections provide the results of this literature review. Section 2.2.3 describes the breadth and depth of the 2015/2016 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.

2.2.3	Breadth and Depth of the 2015/2016 Literature Review

Key features of each of the 47 articles in the 2015/2016 literature review are summarized in Table <IP_ADDRESS>-1, <IP_ADDRESS>-2, and <IP_ADDRESS>-1 as well as Appendix 3. These articles cover wide range of study designs, study locations and time periods, and exposure and outcome measurement approaches and are listed in Table 2.2-a. The majority of the studies utilized a cross-sectional or a prospective birth cohort study design.
 Mothers and Newborn Study of North Manhattan and South Bronx conducted by Columbia University, New York, NY (See tables <IP_ADDRESS>-1 & <IP_ADDRESS>-2)
 Mount Sinai Inner-City Toxicants, Child Growth and Development Study, New York, NY (See tables <IP_ADDRESS>-1 & <IP_ADDRESS>-2)
 Center for Health Assessment of Mothers and Children of Salinas Valley (CHAMACOS) conducted by the University of California Berkeley (See tables <IP_ADDRESS>-1 & <IP_ADDRESS>-2)
 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; Cartier et al, 2015)
 Health Outcomes and Measure of the Environment (HOME), Cincinnati, OH, Birth Cohort (Rauch et al. 2012; Yolton et al, 2013; Donauer et al, 2016; Harley et al, 2016; Engel et al, 2015)
 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)
 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)
 Rice and aquaculture farming regions outside of Bangkok, Thailand (Fiedler et al, 2015)
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, MDA). 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. Non-questionnaire-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 2016).
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 23 reports, post-natal exposures in 14 reports, and both pre- and post-natal exposures in 7 reports.

Table 2.2-a	Study Designs, Exposure Measurement Methods, and Outcome Measurement Methods Used across the 2015/2016
                                 Study design
                               (# studies used)
                             Exposure measurement
                               (# studies used*)
                              Outcome measurement
                               (# studies used*)

Prospective Cohort (16)
Retrospective Cohort (3)
Case-control (2)
Cross-sectional (16)
Ecological (1)
Biomarkers
AChE - acetyl cholinesterase (maternal 1, child 2)
BuChE - butyl cholinesterase (child 1)
CPF -chlorpyrifos parent (maternal 1, cord serum 1)
DAP  -  dialkyl phosphate (maternal 9, child 10, amniotic fluid 1)
DCCA - dimethylvinylcyclopropane carboxylic acid (child 1)
DEP  -  diethyl phosphate (maternal 3, child 8, amniotic fluid 1)
DETP  -  diethylthiophosphate (maternal 2)
DEDTP  -  diethyldithiophosphate  (maternal 2)
DMP  -  dimethyl phosphate (maternal 8, child 8, amniotic fluid 1)
DMTP  -  dimethylthiophosphate (maternal 1)
DMDTP  -  dimethyldithiophosphate (maternal 2)
DZN  -  diazinon parent (1)
IMPY  -  diazinon metabolite (child 1)
MAL - malathion parent (0)
MDA - malathion metabolite (maternal 1)
OP - organophosphate (cord blood 1)
PNP - para-nitrophenol (child 2)
TCPy - chlorpyrifos metabolite (maternal 1, 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)
 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)
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 ADI-R.

Neurodevelopmental (ND) test/battery  (19)  -  see Table 2.2-2b for details

IQ (57) - see Table 2.2-2b for details

Physiological (3)
    AChE activity, child
    BuChE activity, maternal
    Antioxidant status: superoxide dismutase (SOD) activity
    Fetal oxidative stress: malondialdehyde (MDA) concentrations)
    Fetal DNA fragmentation: electrophoresis
    Respiratory symptoms, child

*A single study may include biomarkers at multiple endpoints and / or multiple outcome measurements; thus the total number of studies used may exceed the total number of studies in this manuscript

Table 2.2-b	Detailed Neurological Outcome Measurement Methods Used across the  2015/2016 Review Studies
Outcome measurement
Neurodevelopmental (ND) test/battery  (USA-based studies)
 Pediatric Environmental Neurobehavioral Test Battery (PENTB) : cognitive, motor, sensory, and affect domains
       Developmental Test of Visual-Motor Integration (VMI)
       Kaufman Brief Intelligence test (K-BIT)
       Purdue Pegboard
       Story Memory and Story Memory-Delay from Wide Range Assessment of Memory and Learning
       Trail-Making test, Part A and Part B
       Verbal Cancellation test
 Children's Memory Scale (CMS)
 Bayley Scales of Infant Development, Second Edition (Bayley-II)
    Mental Developmental Index (MDI)
    Psychomotor Developmental Index
    Clinical Evaluation of Language Fundamentals-Preschool, Second Edition
    Archimedes spirals
    NICU Network Neurobehavioral Scale (NNNS)
 Behavioral measures:
       The Child Behavior Checklist/4-18
       The Teacher Report Form
 Developmental delay (DD):
       Mullen Scales of Early Learning (MSEL)
       Vineland Adaptive Behavioral Scale (VABS)
       Reciprocal social interaction: Social Responsiveness Scale
ND test/battery  (Non-USA-based studies)
 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
 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:
       attention and executive functioning
       language
       memory and learning
       sensorimotor (visuomotor precision),
       visuospatial processing
       Statue and Knock Tap
 Neonatal Behavioral Neurological Assessment (NBNA)
       Behavior
       Passive Tone
       Active Tone
       Primary Reflexes
       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:
       Raven's Colored Progressive Matrices
       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
IQ
 Wechsler Intelligence Scale for Children-Revised (WISC-R) Digit Span Test, Card sorting Test
    Wechsler Intelligence Scale for Children, 4th edition (WISC-IV)
    Wechsler Preschool and Primary Scale of Intelligence, Third Edition
 Stanford-Binet Memory for Sentences and Digit String test
 Recall and recognition test

2.2.4	Study Designs & Considerations for Study Quality Evaluation

<IP_ADDRESS>	Study Designs
Besides the two meta-analyses, 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.

Cohort Study

A commonly used design in this literature was the cohort study (See Tables <IP_ADDRESS>-1, <IP_ADDRESS>-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, counter-to-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 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 self-report. 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 <IP_ADDRESS>-1), 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 among the population that gave rise to the cases. Case-control studies that rely on self-reported exposure measures are also susceptible to information bias.

Cross-sectional study
Cross-sectional studies (see, for example, Suarez-Lopez et al. in Table <IP_ADDRESS>-1and 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, namely the relationship between prenatal (and early life) OP pesticide exposures and fetal and child neurodevelopment was considered.

<IP_ADDRESS>	Considerations for Study Quality

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. 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 five domains effecting study quality: exposure assessment, outcome assessment, confounder control, statistical analysis, susceptibility to bias (See Table 2.2.4-1 for general considerations under each domain).

Table 2.2.4-1	Study Quality Considerations
Parameter
High

Moderate

Low

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 window (pre-natal, early life)
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
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 self-report (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 sub-analyses), 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, effect size magnification, 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)

<IP_ADDRESS>.1	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.  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 non-specific 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 individual-level 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.

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 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.

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 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).

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.

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, chlorpyrifos methyl, and triclopyr; 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).

The most commonly measured biomarkers were urinary DAPs. 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.  For example, Chen et al (2012) and Zhang et al (2008) have shown that direct exposure to DAPs can occur from consumption of DAPs in food.  Lu et al. (2005) observed that OP pesticides in fortified fruit juice samples degraded into DAPs.  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).

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 and thus is not entirely specific to chlorpyrifos.  TCPy can be found on directly food.  Studies focusing solely on chlorpyrifos could assess exposure to only this OP (i.e., measure chlorpyrifos directly) (e.g., Lovasi et al., 2011; Whyatt et al., 2004; Rauh et al., 2011).

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.

<IP_ADDRESS>.2	Neurological 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.
<IP_ADDRESS>.3.	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 given moderate weight. Reports with only minimal attention paid to the conduct and reporting of the statistical analyses were given the lowest weight.

<IP_ADDRESS>.4	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.

<IP_ADDRESS>.5	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 non-responsiveness 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). Non-differential 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, non-differential 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.

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.

<IP_ADDRESS>	"High" Quality Group
Fifteen articles assigned a high quality rating are shown in Tables <IP_ADDRESS>-1 and <IP_ADDRESS>-2. 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. All of the studies from CCCEH, Mt. Sinai, CHAMACOS, and HOME cohorts are assigned a high ranking.  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.
Table <IP_ADDRESS>-1.	High Quality Studies from 2015 and 2016 reviews: 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)
Age 8-15 years
Cross-sectional, large sample size
N=1139

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
Donauer et al. (2016).

Cincinnati, Ohio, USA
Health Outcomes and Measure of the Environment (HOME)
Prospective Birth Cohort Study  -  large sample size.
N=327
Mother-infant pairs
Metabolite concentrations (total DAP, DM, and DE) of prenatal OP pesticide exposure quantified in two maternal spot urine samples provided at 16 and 26 weeks of gestation
For children, Bayley-II, MDI, and Psychomotor Development Index examinations at 1-3 years of age, and Clinical Evaluation of Language Fundamentals-
Preschool, Second Edition, and the Wechsler Preschool and Primary Scale of Intelligence, at ages 4 and 5 were used.
Appropriate. Included household income, maternal intelligence, child's sex, maternal intelligence quotient, and maternal education; house inventory score, maternal age at birth, alcohol consumption during pregnancy, parity, and maternal lead levels
Appropriate. Bivariate and multivariable regression
Lack of frequent urine spot sampling, short half-life of DAP metabolites, and the sporadic exposure to DAP may have led to non-differential exposure misclassification; inability to determine exposure toxicity.

Engel et al. (2015)
Salinas Valley, California; Brown, Butler, Clermont, Hamilton, or Warren, Ohio; South Bronx, New York City; Manhattan, New York City
Pooled analysis of the CHAMACOS (1999-2000), Columbia (2000-2001), Mt. Sinai (1998-2002), and HOME (2003-2006) birth cohorts
Birth Cohort Study  -  large sample size
N= 936 women (377 from CHAMACOS study, 265 from HOME study, 60 from Columbia study, and 234 from Mount Sinai study)
Mother-infant pairs
Metabolite concentrations (total DAP, DMP, and DEP) of prenatal OP pesticide exposure quantified in two maternal spot urine samples provided at 13 and 26 weeks and 16 and 26 weeks of gestation (CHAMACOS and HOME studies) and in one maternal spot urine sample at 31 and 32 weeks (Mt. Sinai and Columbia studies)
Blood samples from the mother and child (via the umbilical chord) were also collected and genotyped for the PON1 genotype
For each child, Bayley Scales of Infant Development II
(BSID-11) to generate a Mental Development Index (MDI) and a Psychomotor Development Index (PDI) to assess neurodevelopment in early childhood.
Appropriate.  Included maternal education, marital status, and age at delivery, alcohol use during pregnancy, race/ethnicity, smoking and drug use during pregnancy, child sex, HOME environment score quartile, breastfeeding at least 3 months after birth, metabolite x center interaction terms, race/ethnicity, smoking and drug use during pregnancy.
Linear models with interaction analysis.
Lack of frequent urine spot sampling, short half-life of DAP metabolites, and the sporadic exposure to DAP may have led to non-differential exposure misclassification; inability to determine exposure toxicity; and hetergoenity of the study populations of the 4 cohorts and exposures may have effected the results
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).
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.
Harley et al. (2016)
Salinas Valley, California; Brown, Butler, Clermont, Hamilton, or Warren, Ohio; South Bronx, New York City; Manhattan, New York City
Pooled analysis of four CHAMACOS, Columbia, Mt. Sinai, and HOME birth cohorts
Birth Cohort Study  -  large sample size
N= 1,235 women (484 from CHAMACOS study, 328 from HOME study, 82 from Columbia study, and 341 from Mount Sinai study)
Mother-infant pairs
Metabolite concentrations (total DAP, DMP, and DEP) of prenatal OP pesticide exposure quantified in two maternal spot urine samples provided at 15.9 and 26.4 weeks of gestation (CHAMACOS and HOME studies) and in one maternal spot urine sample at 31.8 and 33.3 weeks (Mt. Sinai and Columbia studies)
Blood samples from the mother and child (via the umbilical chord) were also collected and genotyped for the PON1 genotype
For each child, head circumference, birth weight, and birth length were obtained from medical records
Appropriate. Included sex, race/ethnicity, cohort, country of origin, marital status, maternal education, smoking during pregnancy, parity, maternal age at delivery, and gestational age.
Appropriate. Multivariable linear regression and mixed-effect models to test the effect modification for sex, maternal education, race/ethnicity, and child PON1 genotype.
Lack of frequent urine spot sampling, short half-life of DAP metabolites, and the sporadic exposure to DAP may have led to non-differential exposure misclassification; inability to determine exposure toxicity; and hetergoenity of the study populations of the 4 cohorts and exposures may have effected the results
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).
Raanan et al. (2015)
Salinas
Valley, California
CHAMACOS (Mothers and their children. Children were followed from through age 7)
Prospective Birth Cohort

N = 364 children
Diethyl (DE) and dimethyl(DM) phosphate metabolites and other dialkyl phosphate (DAP) metabolites of OP pesticides measured in mother's urine during pregnancy and from children during childhood
Mother's report of child's respiratory symptoms and child's exercise-induced coughing at 5 and 7 years of age
Models were adjusted for child's sex, child's age, maternal smoking during pregnancy, secondhand tobacco smoke exposure during early childhood, season of birth, mean daily fine particulate concentration during first 3 months of life (PM2.5), breastfeeding, mold and cockroaches in home, and home distance from highway in first year.
Generalized estimating equations (GEE) with repeated measures of respiratory symptoms
Risk of misclassification of exposure, due to nonspecificity of DAP metabolite used as biomarker which may reflect exposure to parent compound (pesticide) or preformed DAPs in environment, and also due to potential for daily fluctuations in DAP levels due to high variability in OP pesticide exposures.

                                     Study
                                Study Location
                 Cohort Name / Description of Study Population
                                 Study Design
                              Exposure Assessment
                              Outcome Assessment
                         Confounder /
Covariate Contro
                             Statistical Analysis
                             Risk of (other) Bias
Rauch et al. (2012)
Cincinnati, Ohio,  USA
Health Outcomes and Measure of the Environment (HOME)
Age: newborns
Prospective Birth Cohort Study  -  large sample size.
N=306

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
Rauh et al. (2015)
New York City, New York
Columbia Center for Children's
Environmental Health (CCCEH) (Children of Black and Dominican mothers, aged 9  -  13.9 years old)
Prospective Birth Cohort Study
N = 271

Chlorpyrifos (CPF) concentrations in sample of umbilical cord blood collected at delivery
Chlorpyrifos (CPF) concentrations in sample of maternal blood collected within 2 days postpartum
Neurodevelopmental assessment via  Archimedes spirals, a drawing exercise, to test for tremor
Models were adjusted for sex, exact age at testing, ethnicity, and medications
Chi-square was to assess crude associations between high CPF exposure and presence of mild to moderate tremor.
Binary logistic regression was used to estimate associations between high exposure level and presence of tremor
Risk of misclassification of exposure, particularly due to lack of CPF exposure biomarkers to assess postnatal and childhood exposure. Misclassification may also occur in the outcome assessment due to the use of a clinical scale, based on observer's rating to assess tremor
Stein et al. (2016)
Salinas Valley, California area
CHAMACOS Study (Pregnant women and 7 year-old children)
Prospective longitudinal cohort study
N = 329
Mother-infant pairs
Biomarker of prenatal OP pesticide exposure (DAPs) quantified in two maternal spot urine sample collected at 13 and 26 weeks of gestation.

Mothers were also interviewed following birth at 6 months, 1, 2, 3.5, 5, and 7 years to collect data on early adversities and assess the home environment using the HOME test
Children's IQ was analyzed using the Wechsler Intelligence Scale for Children
Appropriate. All models were adjusted for maternal IQ score at 6m assessed by Peabody Picture Vocabulary Test.  Full-Scale and Verbal Comprehension IQ models were also adjusted for language of neurological assessment.  Models in which adversity measures did not include H.O.M.E. subscores were adjusted for long term H.O.M.E. score average; models in which adversity measures did not include parental educational attainment were adjusted for maternal education.

Appropriate. Multivariate linear regression model and three-way interaction model
Selection bias probable; adversity measures calculated via a priori hypotheses, instead of incorporating using more advanced statistical methods; lack of frequent maternal urine spot sampling and due to the short half-life of DAP metabolites may have led to exposure misclassification; low concentrations of DAP detected in the urine; lack of study details regarding the urine collection methods
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
Yolton et al. (2013)
Cincinnati, Ohio, USA
Health Outcomes and Measure of the Environment (HOME)
Prospective Birth Cohort Study  -  large sample size.
N=350
Mother-infant pairs
Metabolite concentrations (total DAP, DM, and DE) of prenatal OP pesticide exposure quantified in two maternal spot urine samples provided at 16 and 26 weeks of gestation
For children, a neurobehavioral assessment was conducted via the NICU Network Neurobehavorial Scale (NNNS) at ~ 5 weeks of age
Appropriate. Included sex, birthweight, infant weight change from birth to exam, parity, maternal age at delivery, marital status, education, employment during pregnancy, household income, body mass index (BMI) at 16 weeks gestation, weight gain per week during pregnancy, moderate to severe depression (score > 13) measured on the Beck Depression Inventory-II during pregnancy and at 5 weeks post-delivery, reported marijuana and alcohol use during pregnancy, whole blood lead and folate during pregnancy, serum cotinine, and reported fruit and vegetable consumption during pregnancy
Appropriate. Bivariate and multivariable regression
Low concentrations of DAP detected in the urine; lack of frequent urine spot sampling during pregnancy.

Table <IP_ADDRESS>-2	High Quality Studies from 2012/2014 literature search of Children's Environmental Health Epidemiology Studies

                                Author, Year,
                                  Sample Size
                                   Outcome
                                   Assessed
                             Exposure Measurement
                       Potential confounders considered
                                Primary Result
                          Conclusions/Uncertainties
                           Strengths and Weaknesses

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 pre-pregnancy 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.
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 gestational age.
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 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.
Eskenazi et al. (2004)          CHAMACOS (N=488)
Birth length, birth weight, head circumference, Gestation Length, Ponderal index
Total DAPs: median 136 nmol/L (range: 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)
Gestational age, gestational age squared, maternal age, pregnancy weight gain, week of 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.
Decreases in gestational duration associated with two measures of in utero pesticide exposure: levels of metabolites of dimethyl phosphate pesticide compounds and whole blood ChE.
Maternal urine collection averaged weeks 14, 26, not creatinine-corrected
Strengths in the study design include the longitudinal design, the use of multiple exposure biomarkers, including quantification of non-specific (DAPs), chlorpyrifos-specific (TCPy) metabolites, and other environmental co-exposures. 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.
Harley et al. (2011)              CHAMACOS (N=329)
Birth length, birth weight, head circumference, gestational age