Document objwE5OZRknr3EvN59Ebn3vR

UNITED STA TES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON D.C. 20460 |&$82**2i OFFICE OF CHEMICAL SAFETY AMO POLLUTION PREVENTION MEMORANDUM Date; December 29, 2016 SUBJECT: Updated Literature Review on Neurodeveiopment Effects & FQPA Safety Factor Determination for the Organophosphate Pesticides PC Code: Sec Below Decision No.; 524105 Petition No.: Hone Risk Assessment Type: None TXR No.; 0057561" MRID No.: None DP Barcode: 437043 Registration No.: None Regulatory Action; None Case No.: None CAS No.: See Below 40 CFR: None FROM: Ashlee Aldridge, MPIL Epidemiologist a Health Effects Division (7509P) Office o f Pesticide Programs Anna Lowit, Ph.D., Senior Science Advisor Immediate Office Office o f Pesticide Programs (7501 P> Virginia C Moser. PhD, DABT, Fellow ATS, Toxicologist 1 Toxicity Assessment Division Office o f Research and Development THROUGH: Dana Vogel, Director Health Effects Division (7509P) TO: YU'TIng Guilaran, Director Pesticide Re-Evaluation Division (758P) /? X Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 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 Dicrotophos Fosthiazate Coumaphos Terbufos Profenofos Bensulide Diazinon Ethoprop Dimethoate Malathion Phosmet Chlorethoxyfos Acephate/ Methamidiphos Pirimiphos-methyl TCVP Tribufos Phorate Phostebupirim DDVP Naled Trichlorfon Fenamiphos AZM Methidathion Propetamphos ODM Disulfoton Methyl parathion Temephos Chlorpyrifos-methyl PC Code 035201 129022 036501 105001 111401 009801 057801 041101 035001 057701 059201 129006 103301/ 101201 108102 083701 074801 057201 129086 084001 034401 057901 100601 058001 100301 113601 058702 032501 053501 059001 059102 CAS No. 141-66-2 98886-44-3 56-72-4 13071-79-9 41198-08-7 741-58-2 333-41-5 13194-48-4 60-51-5 121-75-5 732-11-6 54593-83-8 30560-19-1/ 10265-92-6 29232-93-7 961-11-5 78-48-8 298-02-2 96182-53-5 62-73-7 300-76-5 52-68-6 22224-92-6 86-50-0 950-37-8 31218-83-4 301-12-2 298-04-4 298-00-0 3383-96-8 5598-13-0 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 2 ED 002061 00044425-00002 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00003 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-aStudy 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 2.2.4.1 Study Designs....................................................................................................... 35 2.2.4.2 Considerations for Study Quality......................................................................... 37 Table 2.2.4-1 Study Quality Considerations...................................................................... 38 2.2.4.2.1 Exposure Measures.............................................................................................. 39 Table 2.2.4-2 CDC Table of Organophosphate Pesticides and Their Dialkyl Phosphate Metabolites (2008)....................................................................................... 41 2.2.4.2.2 Neurological and Other Outcome Measures........................................................ 42 2.2.4.2.3. Statistical Analysis............................................................................................... 42 2.2.4.2.4 Confounding......................................................................................................... 43 2.2.4.2.5 Risk of B ias..........................................................................................................43 2.2.5 Review of Quality Results............................................................................................ 44 2.2.5.1 "High" Quality Group.......................................................................................... 44 Table 2.2.5.1-1.High Quality Studies from 2015 and 2016 reviews: Summary of Study Design Elements Impacting StudyQuality Assignment.............................. 45 Table 2.2.5.1-2 High Quality Studies from 2012/2014 literature search of Children's Environmental Health Epidemiology Studies.............................................. 49 2.2.5.2 "Moderate" Quality Group................................................................................... 64 Table 2.2.5.2-1.Moderate Quality Studies: Summary of Study Design Elements Impacting Study Quality Assignment........................................................................... 65 2.2.5.3 "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 EpidemiologicStudies.....................................................71 2.2.8.1 CCCEH, CHAMACOS, & Mt. Sinai Cohorts..................................................... 72 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00004 2.2.8.2 HOME Cohort...................................................................................................... 77 2.2.8.3 Meta-analysis (Engel et al, 2015)......................................................................... 78 2.2.8.4 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 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 5 ED 002061 00044425-00005 Literature Review on Neurodeveiopment Effects & FQ.PA 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),1in 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 1 Mode of action (MOA) and adverse outcome pathways (AOPs) describe a set of measureable key events that make up the biological processes leading to an adverse outcome and the causal linkages between such events. 6 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00006 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.2 In December, 2014, the agency released "Chlorpyrifos: Revised Human Health Risk Assessment for Registration Review" which included the use of a physiologically-based pharmacokinetic/pharmacodynamic (PBPK-PD) model to derive human PODs, which obviated the need for the animal to human extrapolation factor, and refined intra-species factors for some lifestages (USEPA 2014). The chlorpyrifos 2014 revised HHRA also included retention of the 10X FQPA Safety Factor due to uncertainty regarding the degree of protection the endpoint of AChE inhibition provides for potential neurodevelopmental effects (USEPA, 2014). A review of the scientific literature on potential MOA/AOP leading to effects on the developing brain was conducted for the 2012 FIFRA SAP meeting (USEPA, 2012) and updated for the 2014 chlorpyrifos revised HHRA (USEPA 2014; 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 http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPP-2008-0850-017Q 7 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00007 2.0 Literature Review In recent years, the National Academy of Sciences has encouraged the agency to move towards systematic review processes to enhance the transparency of scientific literature reviews that support chemical-specific HHRAs to inform regulatory decision making3. The NRC defines systematic review as "a scientific investigation that focuses on a specific question and uses explicit, pre-specified scientific methods to identify, select, assess, and summarize the findings of similar but separate studies".4 Consistent with NRC's recommendations, EPA's Office of Chemical Safety and Pollution Prevention (OCSPP) is currently developing systematic review policies and procedures. In short, OCSPP employs fit-for-purpose systematic reviews that rely on standard methods for collecting, evaluating and integrating the scientific data supporting the agency's decisions. The literature review described here uses concepts consistent with systematic review such as detailed tracking of search terms and which literature have been included or excluded. 2.1 Developmental Neurotoxicity (DNT) Research on OPs other than Chlorpyrifos: Laboratory Animal Studies The literature on neurobehavioral effects of developmental exposure to chlorpyrifos was summarized and discussed at the 2012 FIFRA SAP. More recent studies were added to this summary for the 2014 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). 3 NRC 2011. "Review of the Environmental Protection Agency's Draft IRIS Assessment of Formaldehyde"; NRC 2014. "Review of EPA's Integrated Risk Information System (IRIS) Process" 4 http://dels.nas.edu/Report/Review-lntegrated-Risk/18764 8 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00008 2.1.1 Literature Search Strategy & Results To review and evaluate the developmental^ 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 pregnancyfMeSH 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 SyndromesfMeSH Terms]) AND ((((((guinea pigsfMeSH 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=AII document types; Language=AII languages; #4 TS=(guinea pig* OR rabbit* OR mice OR mouse OR rat* OR rodent*) NOT TS=(fish*) DocType=AII document types; Language=AII 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=AII document types; Language=AII 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=AII document types; Language=AII languages; #1 TS=(organophos* OR Cholinesterase Inhibitors OR acetylcholinesterase inhibitor OR chlorpyrifos) DocType=AII document types; Language=AII languages;3 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 9 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00009 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. 10 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00010 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 ol., 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 ai, 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 ai, 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 ai, 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 a i (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 on1 11 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00011 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 a!., 1980). Treated rats had more trials to criterion only on second and fifth switch (note, the text claimed there was an effect on the 4th switch, but the figure does not show it as significant). Effects on only certain reversals is difficult to interpret, and may be a reflection of the multiple t-tests used to analyze the data. Males and females were tested but data were not provided for each sex separately. There were no effects on passive or active avoidance in rats (no mention of gender) exposed to methyl parathion (1,1.5 mg/kg/d, GD6-20) (Gupta et ol., 1985). The low-dose group only showed slower latency to bar press during operant shaping, and more days to asymptote; however, details of operant training and schedule were not provided, the sample size was extremely small (n=4/group), and high variability was mentioned. Thus, the most consistent effect was seen in the radial arm maze (even though there was no dose-response), and other tests were either negative or the data were inconclusive. Several other OPs were tested in different cognitive tasks, but there is no more than one report for any specific pesticide. After single-trial passive avoidance training, male rats (females not tested) treated with dichlorvos (8 mg/kg/d, GD6-15) showed faster latency to cross when tested 7 days later, suggesting delayed retention (Lazarini et al., 2004). With fenitrothion, male rats (females not tested) exposed gestationally (5,10,15 mg/kg/d, GD7-15) were conditioned to climb a pole to escape shock (Lehotzky et al., 1989). The mid and high dose groups (doseresponse evident) showed more escapes, were faster, and reached criterion faster than controls. This same pattern seen with reacquisition after a period of extinction, during which time there were no group differences. There was no effect of oxydemeton methyl (0.5,1.5, 4.5 mg/kg/d, GD6-15) on M-maze learning or memory in rats (Clemens et al., 1990). There was also no effect on retention of single trial passive avoidance in mice exposed to methamidophos (1 mg/kg/d, PND3-9); however, the retention trial occurred at 3 hr instead of the more standard 24 hr or greater (Lima et al., 2013). 12 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00012 Table 2.1.1 Summary of Cognitive Outcomes Radial arm maze Early gestation ~GDl-7, ~GD8-14, GD6-15 GD7-15 Late gestation Gestation ~GD15-21, GD6- GDl-birth 20, GD15-18 Early postnatal PND 1-4, PND3-9 Late postnatal Postnatal PND8-11, PND5-20 PND1-21 Diazinon: cognitive deficit - rat, low dose only, M & F1 Parathion: improved cognition - rat, low dose only, M & F2 Parathion: cognitive deficit rat, no doseresponse, only M tested4 Methyl parathion: cognitive deficit rat, dose-response, M only5 T-maze spontaneous alternation T-maze learning Lashley III maze Methyl parathion: cognitive deficitrat, sex not specified6 M-maze Active avoidance Oxydemeton methyl: no effect rat, M & F8 Fenitrothion: improved Methyl parathion: no Parathion: cognitive deficit - rat, M not F3 Diazinon: no effect rat, M & F1 Parathion: no effect rat, M & F2 Parathion: cognitive deficit rat, dose-response, only M tested4 Diazinon: no effect mouse, sex not specified7 13 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00013 Passive avoidance Novel object recognition cognition - rat, dose-response, only M tested9 Parathion: no effect - mouse, sex not specified11 Dichlorvos: cognitive deficitrat, only M tested12 effect - rat, sex not specified10 Parathion: no effect - mouse, sex not specified11 Diazinon: cognitive deficit - rat, F not M13 Methyl parathion: no effect - rat, sex not specified10 Operant responding 1Timofeeva eta i, 2008a 2Timofeeva etai., 2008b 3Levin et a!., 2008 4Stamper et at., 1988 5Johnson et al., 2009 6Crowder et al., 1980 7Spyker and Avery, 1977 8Clemens et al., 1990 Methyl parathion: cognitive deficit - rat, sex not specified10 Diazinon: cognitive deficit - rat, M & F13 Methamidophos: no effect - mouse, sex not specified14 Diazinon: cognitive deficit - mouse, no dose-response, only M tested15 9Lehotzky et al., 1989 10Gupta et al., 1985 11al-Hachim and Fink, 1968 12Lazarini et al., 2004 13Vatanparast et al., 2013 14Lima et al., 2013 15Win-Shwe et al., 2013 14 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00014 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, GDl-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.15 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 15 ED 002061 00044425-00015 Table 2.1.2. Summary of Motor Activity Outcomes Task/Test Apparatus Open field Early gestation ~GDl-7, ~GD8-14, GD6-15 GD7-15 Dichlorvos: decreased activity rat, only M tested1 Late gestation ~GD15-21, GD6-20, GD15-18 Diazinon: no effect rat, M & F7 Fenitrothion: decreased activity Parathion: no effect - rat, dose-response, only M - mouse, sex not tested2 specified6 Gestation GDl-birth Diazinon: no effect mouse, sex not specified8 Early postnatal PND1-4, PND3-9 Diazinon: no effect - rat, M & F7 Methamidophos: no effect - mouse, sex not specified9 Late postnatal PND8-11, PND5-20 Parathion: no effect - rat, only M tested10 Methamidophos: no effect rat, sex not specified3 Methyl parathion: increased activity - rat, sex not specified4 Oxydemeton methyl: no effect - rat, M & F5 FigureEight Parathion: no effect - mouse, sex not specified6 Diazinon: no effect - rat, M & F11 Donut Methyl parathion: decreased activityrat, no doseresponse, sex not specified13 Parathion: no effect rat, M & F12 16 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00016 1Lazarini et al., 2004 2Lehotzky et al., 1989 3deCastro et al., 2000 4Crowder et al., 1980 5Clemens et al., 1990 6al-Hachim and Fink, 1968 7Vatanparast et al., 2013 Sierra Club v. EPA 18cv3472 NDCA 8Spyker and Avery, 1977 9Lima et al., 2013 10Stamper et a!., 1988 11Timofeeva et al., 2008a 12Timofeeva et al., 2008b 13Gupta et al., 1985 Tier 3/4 17 ED 002061 00044425-00017 Anxiety/Depression In a series of tests in rats, Roegge et a!. (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 oL, 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. Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 18 ED 002061 00044425-00018 Table 2.1.3. Summary of Anxiety/Depression Outcomes Elevated plus maze Late gestation ~GD15-21, GD6-20, GD15-18 Perinatal premating-weaning Chlormephos: increased anxietymouse, M & F1 Early postnatal PND1-4, PND3-9 Diazinon: increased anxiety - rat, dose-response, M not F2 Methamidophos: no effect -mouse, sex not specified3 Chocolate milk preference Novelty suppressed feeding Forced swim Parathion: decreased anxiety - rat, dose-response, M & F4 Diazinon: increased anhedonia - rat, no dose-response, M not F2 Parathion: no effect - rat, M & F4 Diazinon: decreased fearfulness - rat, dose-response, M not F2 Parathion: no effect - rat, M & F4 Diazinon: no effect - rat, M & F2 Open field Methyl parathion: behaviors decreased anxiety - rat, no doseresponse, sex not specified5 1Ceh et al., 2012 2Roegge et al., 2008 3Lima et al., 2013 4Timofeeva tal., 2008b 5Gupta et al., 1985 Methamidophos: increased despairmouse, sex not specified3 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 19 ED 002061 00044425-00019 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 ai, 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 ai, 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 ai, 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 GDl-birth Auditory Tactile with or without prepulse Diazinon: no effect - mouse, sex not specified1 Visual Diazinon: decreased function - mouse, no dose-response, F not M1 Olfactory Diazinon: no effect - mouse, sex not specified1 1Spyker and Avery, 1977 2Timofeeva eta i, 2008a 3Timofeeva eta i, 2008b Early postnatal PND1-4, PND3-9 Diazinon: decreased sensory gating - rat, no dose-response, M not F2 Parathion: decreased startle response - rat, dose-response, M & F3 20 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00020 Neuromotor Function One study assessed neuromotor function in mice exposed to diazinon (0.18, 9 mg/kg/d, GD1birth), and reported some changes in motor abilities measured at about 2 months of age; however, these were not consistent in dose or direction of change (Spyker and Avery, 1977). Increased ability was suggested by a longer time to cling to a rod (both doses, no doseresponse). In contrast, there was less ability to stay on an increasingly inclined plane (both doses, no dose-response) or perhaps on a rota rod (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 oi, 1989). There were no neuromotor changes in terms of rota rod 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 Rotarod Inclined plane Early gestation ~GDl-7, ~GD8-14, GD6-15 GD7-15 Fenitrothion: decreased performance - rat, dose-response, only M tested1 Rod cling 1Lehotzky et al., 1989 2Gupta et al., 1985 3Spyker and Avery, 1977 4Stamper et al., 1988 Late gestation ~GD15-21, GD6-20, GD15-18 Methyl parathion: no effect - rat, sex not specified2 Gestation GDl-birth Late postnatal PND8-11, PND520 Diazinon: no effect - Parathion: no mouse, sex not specified3 effect - rat, only M tested4 Diazinon: decreased performance - mouse, dose-response, sex not specified3 (Spyker) Diazinon: increased performance - mouse, no dose-response, sex not specified3 21 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00021 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). Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00022 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 morphometries 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 23 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00023 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 24 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00024 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 InnerCity 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 25 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00025 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 2.2.8.1. Results from other studies on neurodevelopmental outcomes are summarized in 2.2.8.2. 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 26 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00026 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: Psyclnfo, 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 2.2.5.1-1, 2.2.5.1-2, and 2.2.5.2-1 as well as Appendix 3. These articles cover wide range 27 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00027 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 2.2.5.1-1 & 2.2.5.1-2) Mount Sinai Inner-City Toxicants, Child Growth and Development Study, New York, NY (See tables 2.2.5.1-1 & 2.2.5.1-2) Center for Health Assessment of Mothers and Children of Salinas Valley (CHAMACOS) conducted by the University of California Berkeley (See tables 2.2.5.1-1 & 2.2.5.1-2) 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: tude Longitudinale sur les Anomalies de la Grossesse, l'Infertilit 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) 28 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00028  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. Nonquestionnaire-based exposures included Community/area of residence, distance to treated area/farm, percent of area treated with pesticide, level of urbanization, pounds OP pesticide used/year, and pesticide spray season. Likewise, there were numerous outcome measures examined across the studies, falling into six broad categories: birth characteristics, autonomic nervous system (ANS) effects, Attention Deficit Hyperactivity Disorder (ADHD)/attention problems, autism, general neurodevelopment (cognitive, behavioral, IQ), and physiological effects. The most common outcome measures were birth characteristics (with birth weight, birth length, head circumference and gestational age being the most frequent) and neurodevelopment tests and test batteries. Table 2.2-b lists the many specific neurodevelopment tests employed in the studies. Most studies utilized more than one test, and few tests were utilized in more than one study. 29 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00029 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. Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 30 ED 002061 00044425-00030 Table 2.2-a Study Designs, Exposure Measurement Methods, and Outcome Measurement Methods Used across the 2015/2016 Study design Exposure measurement Outcome measurement (ft studies used) (ft studies used*) (ft studies used*) Biomarkers Birth characteristics (10) Prospective Cohort (16) Retrospective Cohort (3) Case-control (2) Cross-sectional (16) Ecological (1) AChE - acetyl cholinesterase (maternal 1, child 2) BuChE - butyl cholinesterase (child 1) CPF -chlorpyrlfos parent (maternal 1, cord serum 1) DAP - dialkyl phosphate (maternal 9, child 10, amnlotic fluid 1) DCCA - dlmethylvinylcyclopropane carboxylic acid (child 1) DEP - diethyl phosphate (maternal 3, child 8, amniotlc fluid 1) DETP - diethylthlophosphate (maternal 2) DEDTP - dlethyldithlophosphate (maternal 2) DMP - dimethyl phosphate (maternal 8, child 8, amniotlc fluid 1) DMTP - dlmethylthlophosphate (maternal 1) DMDTP - dlmethyldithlophosphate (maternal 2) DZN - diazlnon parent (1) IM P Y -dlazinon metabolite (child 1) MAL - malathlon parent (0) MDA - malathlon metabolite (maternal 1) OP - organophosphate (cord blood 1) PNP - para-nltrophenol (child 2) TCPy - chlorpyrifos metabolite (maternal 1, child 2) 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/mlscarriage (report 1) Altered sex ratio (report 1) Autonomic Nervous System (4) ADHD/attention problems (2) Diagnosis DISC-iV (1) Use of medication (1) Screening (CPRS-R, CPT, BASC-PRS) (1) Autism (2) CA Department of Developmental Services (CDDS) reports US Individuals with Disabilities Education Act (IDEA) reports Autism spectrum disorders (ASD) Autism Diagnostic Observation Schedule (ADOS) combined with ADIR. Neurodevelopmental (ND) test/battery (19) - see Table 2.2-2b for details Environmental IQ (57) - see Table 2.2-2b for details Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 31 ED 002061 00044425-00031 Study design {# siuiil- Uied) Exposure measurement (U studies used*) 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 Outcome measurement (# studies used*) Physiological (3) AChE activity, child BuChE activity, maternal A n tio x id a n t status: superoxide dismutase (SOD) activity Fetal oxidative stress: malondialdehyde (MDA) concentrations) Fetal DNA fragmentation: electrophoresis Respiratory symptoms, child *A single study may ine ude 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 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 32 ED 002061 00044425-00032 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 o Developmental Test of Visual-Motor Integration (VMI) o Kaufman Brief Intelligence test (K-BIT) o Purdue Pegboard o Story Memory and Story Memory-Delay from Wide Range Assessment of Memory and Learning o Trail-Making test, Part A and Part B o Verbal Cancellation test Children's Memory Scale (CMS) Bayley Scales of Infant Development, Second Edition (Bayley-ll) Mental Developmental Index (MDI) Psychomotor Developmental Index Clinical Evaluation of Language Fundamentals-Preschool, Second Edition Archimedes spirals NICU Network Neurobehavioral Scale (NNNS) Behavioral measures: o The Child Behavior Checklist/4-18 o The Teacher Report Form Developmental delay (DD): o Mullen Scales of Early Learning (MSEL) o Vineland Adaptive Behavioral Scale (VABS) o Reciprocal social interaction: Social Responsiveness Scale ND tes{/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: o attention and executive functioning o language o memory and learning o sensorimotor (visuomotor precision), o visuospatlal processing o Statue and Knock Tap Neonatal Behavioral Neurological Assessment (NBNA) o Behavior o Passive Tone o Active Tone o Primary Reflexes o General Assessment Reach-and-grasp, bl-manual coordination: Prehension abilities UC Berkeley Preferential Looking Test Cards: Visual acuity skills Visual Motor Integration (VMI), Beery-Buktenica, 4th Ed. Finger Tapping test: Manual motor speed Catsys equipment: Simple reaction time Conners' Continuous Performance Test II (CPT II, v5): Attention Woodcock-Johnson III Tests of Cognitive Abilities (WJ-III) Verbal Comprehension test: Long-term memory and language function Visuospatial performance and memory functions: o Raven's Colored Progressive Matrices 33 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00033 o Stanford-Binet Copying Test, 4th ed Physical examination (social response, spontaneous motility, involuntary movements, Romberg's sign, walking straight line, standing on one leg, number hops, biceps and patellar reflexes, finger opposition, diadochokinesis, finger-nose coordination, hearing, vision) Gesell Development Schedule (GDS): motor, adaptive, language, and social Santa Ana Form Board: Motor coordination Child's developmental delay - Parent interview 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 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 34 ED 002061 00044425-00034 2.2.4 Study Designs & Considerations for Study Quality Evaluation 2.2.4.1 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 2.2.5.1-1, 2.2.5.1-2, and Appendix 3 for examples). In a typical cohort study, individuals are classified according to exposure status (i.e., presence, absence, or magnitude of exposure), and then followed over time to quantify and compare the development (i.e., incidence) of the health outcome of interest by exposure group. Conceptually, the non-exposed comparison group in a cohort study provides an estimate of the incidence of the outcome among the exposed, had they, counterto-fact, not been exposed. Apart from chance variations, a valid cohort study comparing exposed individuals to non-exposed individuals provides an estimate of the relative risk (or rate) of the disease associated with exposure. Ideally, the exposed and non-exposed groups are exchangeable, in the sense that switching the exposed to non-exposed, and non-exposed to exposed would yield the same measure of association (e.g., relative risk). If this were the case then, apart from chance, a cohort study would yield a measure of association equivalent to that 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 35 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00035 has not occurred (prospective study) at the time the investigators initiate the study. This distinction is important primarily as it pertains to the potential differences in the quality (e.g., completeness, accuracy, and precision) of information that can be ascertained by the investigators, and also as it relates to potential sources of bias. Although not always true, the prospective study design is considered the preferable of the two, as investigators can potentially have more choices in determining how exposure, outcome, and covariate information is collected. In a retrospective study conducted to evaluate the same hypothesis, by contrast, the investigators would have to rely on exposure information such as maternal selfreport. Such reporting is subject to (human) errors in recall. Moreover, the outcome status of the child (i.e., whether a child has developmental delays that are known to the mother) may influence the recall of prenatal OP pesticide exposure by the mother. Case-control Study In a typical case-control study (see, for example, Dawbrowski et ai, 2003 in Appendix 3 and Shelton et a i 2014 in Table 2.2.5.2-1), 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 selfreported exposure measures are also susceptible to information bias. Cross-sectional study Cross-sectional studies (see, for example, Suarez-Lopez et a i in Table 2.2.5.2-land Grandjean et a i 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) 36 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00036 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 ecologie 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--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. 2.2.4.2 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 37 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00037 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 High Parameter 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) iViOUelralCP Non-specific biomarker of exposure (DAP), or effect (AChE/BuChE), or questionnaire based individual level information on the OP class, or sub-class Low Low quality questionnaire based exposure assessment, or cologie 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 selfreport (birth characteristics) Confounder control Good control for important confounders relevant to OP-ND question, and standard confounders Moderately good control confounders, standard variables, not all variables for OP-ND question Multi-variable analysis not performed, no adjustments Statistical Analysis Appropriate to study question and design, supported by adequate sample size, maximizing use of data, reported well (not selective) Acceptable methods, questionable study power (especially 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 38 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00038 (Adapted from Munoz-Quezada et al. 2013) 2.2.4.2.1 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 cologie 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 39 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00039 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 40 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00040 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 ol., 2014; Eskenazi et ol., 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 ol., 2011; Whyatt et ol., 2004; Rauh et ol., 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 | VIP = dimethylphosphate; DEP = diethylp losphate; DMTP = dimet lylthiophosphate; DMDTP = 41 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00041 dim ethyldithiophosphate; DETP = diethylthiophosphate; DEDTP = diethyldithiophosphate. 2.2.4.2.2 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. 2.2.4.2.3. 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. 42 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00042 Z.2A.2A Confounding Risk factors for early life neurodevelopment perturbations that are associated with OP pesticide exposure, but not caused by pesticide exposure, are potential confounders in this literature. Socioeconomic determinants of child development (including, for example, maternal education and access to prenatal and early life health care), quantity and quality of parent-child interactions, and exposure to other environmental toxicants (e.g., lead, PCBs, other pesticides) are difficult to measure and were either not accounted for, or inadequately accounted for in many studies. That said, some studies were relatively homogeneous with respect to these factors and thus limited confounding by design, while others attempted to quantify these factors and adjust for them. Other important potential confounders, such as the child's sex, are easy to identify and adjust for analytically. 2.2.4.2.5 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, 43 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00043 and interviewer/observer bias. Note that mismeasurement of confounders can result in residual confounding of the association of interest, even when adjustment for that confounder has been conducted in the analysis. Studies in which major sources of potential biases were not likely to be present, or in which potential sources of bias were present but effectively addressed and analyzed to maximize the study validity, and those in which sources of bias were unlikely to influence the magnitude and direction of the risk estimate were given a high weight. Studies where sources of bias were present and acknowledged by the authors but not addressed in the study and yet may influence the magnitude, but not direction of the association estimate received a moderate weight. A low weight was given to studies in which major biases were present and yet were not acknowledged or addressed in the study, such that they cannot be excluded as an alternative explanation for the study finding. 2.2.5 Review of Quality Results Each of the studies in the 2015 review was judged to be of high, moderate, or low quality in each of five domains of study design and methodology effecting study quality as discussed above in Section 2.1. The results of the quality assessment are presented separately below for each group. The quality categories represent to the total evaluation. In Section 2.2.6, further evaluation of the study design and exposure assessment of the medium and high quality studies. This further evaluation led to additional studies being removed from the final analysis, with these excluded studies not being considered further in the remaining sections of this document. 2.2.5.1 "High" Quality Group Fifteen articles assigned a high quality rating are shown in Tables 2.2.5.1-1 and 2.2.5.1-2. 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 at. (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. Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 44 ED 002061 00044425-00044 Table 2.2.5.1-1. Study Barr et ai. j2010) Bouchard et al. (2010) High Quality Studies from 2015 and 2016 reviews: Summary of Study Design Elements Impacting Study Quality Assignment Cohort Name / Description of Study Exposure Confounder/ Study Location Population Study Design Assessment Outcome Assessment Covariate Control 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 Statistical Analysis Appropriate multivariate analysis Risk of (other) Bias Convenience sample - deemed low probability of selection bias; low potential for exposure or outcome misclasslflcation 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 a!. (2016). Enge! et al. (2015) Furlong et ai. (2014) Fortenberry et al. (2014) Cincinnati, Ohio, USA Salinas Valley, California; Brown, Butler, Clermont, Hamilton, or Warren, Ohio; South Bronx, New York City; Manhattan, New York. City New York City, U.S. Mexico City, Mexico 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-i 1, MDI, and Psychomotor Development Index examinations at 1-3 years of age, and Clinical Evaluation of Language FundamentalsPreschool, 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-differentia! exposure misclassification; inability to determine exposure toxicity. 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) 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 quartile, breastfeeding at least 3 m onths after birth, m etabolite 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-differentia! exposure misclassification; inability to determine exposure toxicity; and hetergoenity of the study populations of the 4 cohorts and exposures may have effected the results Mount Sinai Children's Environmental Health Study Prospective Cohort N=136 Age 7-9 years Blood samples from the mother and child (via the umbilical chord) were also collected and genotyped for the PO N 1 genotype 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 soda! 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). Early Life Exposures in Mexico to Environmental Toxicants (ELEMENT) study (3 cohorts 1994-1997 (cohort 1), 1997- Prospective Birth Cohort Study - modest sample size Objective measure - TCPy concentration was measured in third trimester maternal urine samples. In a subset of women ADHD - LP - Conner's ' Parent Rating Scales-Revised (CPRS-R), Conner's' Continuous Performance Test (OPT), and Behavior Assessment System for Children-Parental Rating Scales Appropriate. Included continuous maternal IQ, education, socioeconomic status and blood lead one month after delivery, breast feeding (yes/no), child's sex, 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 45 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00045 Study Study Location Cohort Name / Description of Study Population 2000 (cohort 2), and 20012005 (cohort 3)) Study Design N=187 Mother-infant pairs Exposure Assessment randomly selected urinary TCPy in samples collected during all three trimesters of pregnancy was measured. Outcome Assessment (BASC-PRS) - These are screening tools, not diagnostic tools. Standardization and quality control checks were conducted by reviews of videotaped evaluations. Confounder/ Covariate Control continuous age attesting, birth length and head circumference at birth. Statistical Analysis Risk of (other) Bias likely aware of the neurobehaviora! status of their children Fortenberry et al. (2014a) Mexico City, Mexico Harley et al. (2016) Salinas Valley, California; Brown, Butler, Clermont, Hamilton, or Warren, Ohio; South Bronx, New York City; Manhattan, New York City Qulrs-Alcal et al. (2011) Salinas Valley, California, USA Raanan et al. (2015) Salinas Valley, California 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 attesting, birth length and head circumference at birth. Appropriate. Multivariable linear regression Potential for differential exposure misclassification possible as mothers were likely aware of the neurobehaviora! status of their children. 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) For each child, head circumference, birth weight, and birth length were obtained from medical records Appropriate. Included sex, race/ethnicity, cohort, country of origin, mantal 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 P O N I genotype. Lack of frequent urine spot sampling, short half-life of DAP metabolites, and the sporadic exposure to DAP may have led to non-differentia! exposure misclassification; inability to determine exposure toxicity; and hetergoenity of the study populations of the 4 cohorts and exposures may have effected the results Blood samples from the mother and child (via the umbilical chord) were also collected and genotyped for the PO N 1 genotype 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 dysregulatlon 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). 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 exerciseinduced 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 dally 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 dally fluctuations In DAP levels due to high variability In OP pesticide exposures. 46 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00046 Study Rauch et ai. (2012) Rauh et a!. (2015) Stein et al. (2016) Wolff et al. (2007) Yolton et al. (2013) Study Location Cincinnati. Ohio, USA Cohort Name / Description of Study Population Health Outcomes and Measure of the Environment (HOME) Age: newborns Study Design Prospective Birth Cohort Study - large sample size. N=306 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 Salinas Vailey, California area CHAMACOS Study (Pregnant women and 7 year-oid children) Prospective longitudinal cohort study N = 329 Mother-infant pairs Exposure Assessment Objective biomarker of prenatal OP pesticide exposure (DAP) quantified in two maternal spot urine samples provided at 16 and 26 weeks gestation Outcome Assessment 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. Confounder/ Covariate Contro 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. Statistical Analysis Appropriate, multivariable regression Risk of (other) Bias Unable to rule out the possibility that differences in DAP concentrations partially reflect individual variation in metabolism, Recall error 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 Neurodevelopmenta! assessment via Archimedes spirals, a drawing exercise, to test for tremor Models were adjusted for sex, exact age attesting, 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 Biomarker of prenatal OP pesticide exposure (DAPs) quantified in two maternal spot urine sample collected at 13 and 26 weeks of gestation. Children's IQ was analyzed using the Wechsier Intelligence Scale for Children 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 Appropriate. Ail models were adjusted for materna! IQ score at 6m assessed by Peabody Picture Vocabulary Test. Fuii-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 materna! education. Appropriate. Multivariate near regression mode! 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 New York City, U S. Cincinnati, Ohio, USA Mount Sinai Children's Prospective Cohort - Environmental Health Study moderate sample size N=404 Mother-infant pairs Health Outcomes and Measure of the Environment (HOME) Prospective Birth Cohort Study - large sample size. N=350 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: materna! age, race/ethnicity, maternal BM!*pregnancy weight gain, infant sex, gestational age, creatinine Appropriate PROC GLM, with varying covariates Selection bias unlikely; some potential for non-differentia! misclassification of exposure 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 neurobehaviora! assessment was conducted via the NICU Network Neurobehavoria! 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-!! during pregnancy and at 5 weeks post-delivery, reported marijuana and alcohol use during pregnancy, whole blood lead and Appropriate. Bivariate and multivariable regression Low concentrations of DAP detected in the urine; lack of frequent urine spot sampling during pregnancy. 47 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00047 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 folate during pregnancy, serum cotinine, and reported fruit and vegetable consumption during pregnancy 48 ED 002061 00044425-00048 Table 2.2.5.1-2 Studies High Quality Studies from 2012/2014 literature search of Children's Environmental Health Epidemiology A u th o r, Year, Sample Size O utcom e Assessed W h ya tt e t al. (2004) Colum bia U. (N = 3 1 4 ) Birth length, Birth w eight, head circum ference Berkow itz e t a l.(2004) M t. Sinai (N =404) Birth length, birth w eight, head circum ference, gestational age Exposure Measurement Potential confounders considered Primary Result Conclusions/Uncertainties Strengths and Weaknesses 4 cord plasm a chlo rpyrifo s exposure groups and 4 chlo rpyrifo s and diazinon exposure groups. C h lo rp yrifo s only categories, G roup 1: le ve ls be lo w LOD (32% of p a rtic ip a n ts ); G ro up 2: low est 1/3 o f detectable levels (20 %); G ro up 3: m iddle 1/3 (24% ), Group 4: highest 1/3 (25% .) C h lo rp yrifo s and diazinon together: G ro u p 1: 26% , G ro up 2: 22 %, G ro up 3: 26% , Group 4: 26%. G estation al age, m aternal prep regn an cy w eight, m aternal net p regn an cy w eight gain, gender o f new born, parity, ra ce /e th n icity , ETS in ho m e, season, cesarean section For each log unit increase in co rd plasm a ch lo rp y rifo s levels, birth w eight decreased by 42.6 g (95% Cl: -8 1 .8 to - 3 .8 ) and birth length decreased by 0.24 cm (95% C l:-0 .4 7 t o 0.01). Birth w eight averaged 186.3 g less (95% Cl: -3 7 5 .2 t o -4 5 .5 ) am o n g new bo rns w ith the highest com pared w ith low est 26% of expo sure levels (p = 0.01). LOD: 11 ug/L (57% <LO D TCPy) R ace/ethn icity, infant sex, and ge sta tio n al age. T h e au th o rs also co ntrolled fo r birth w e igh t or b irth le n gth in th e ir asse ssm e n t o f head circum ference and pesticide exposure. M ean levels o f birth w eight, length, head circum ference, and gestatio n al age did not d iffer betw een th o se w ith u rin ary pesticide m etab olite levels below and above the level of d e te c tio n . Sim ilarly, no statistically significant associations w ere observed between reported pesticide A sso cia tio n s betw een birth w eight and length and cord plasm a chlo rpyrifo s w ere statistically significant (p < 0.007) am ong new borns born before the January 2001 policy change. A m ong new borns born after January 2001, exposure levels w ere sub stan tially low er, and no asso ciatio ns w ith fetal grow th o utco m es w ere o bserved (p > 0.8). Stren gth s: p rospective nature of the study; direct m easurem ent of ch lo rp y rifo s in co rd blo o d and personal air sam ples, rather than no n-specific m arkers of organ oph osph ate pesticide exposure; co n sid eratio n of o ther pesticides and environm ental co ntam in ants as co variates in th e m u ltiv a ria te m o d els. Lim itations: single exposure sam p lin g period; the au tho rs did not present nor discuss regression diagnostics to assess the degree to w hich their m odels m et o r violated the a s s u m p t io n s im p lic it in linear m odels. PO N 1 activity also p red icto r of sm aller head circum ference; creatinine co rre cte d V ery w ell co nducted study w ith n u m ero us stren gth s and very few w eaknesses. The questionn aire-based pesticide exposure questions are subject to im perfect recall. Errors w ould, on average, attenuate associations betw een these exposure m etrics and fetal developm ent. Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00049 Author, Year, Sample Size Eskenazi e t al. Outcome Assessed Birth length, birth Exposure Measurement Potential confounders considered Primary Result Conclusions/Uncertainties Strengths and Weaknesses Total DAPs: exposure and m ean indices of fetal grow th and gestational age. G estation al age, gestational age D e cre a se s in g e sta tio n a l M aternal urine co llectio n R ecall-based exposure assessm ents w ere fortified by o bjective m easures of p e s tic id e s / p e s tic id e m etabolites. A m etab olite specific for chlorpyrifos (TCPy) w as assessed. Statistical an alysis w as a p p ro p ria te . O bserved m ean reductions in the o u tco m e p a ra m e te rs a p p e a r to be sm a ll in m agn itud e and m ay be of little clinical significance. Assessm ent and control for confounding w ere appropriate. How ever, confounding by unm easured (and m ism easured ) risk facto rs for abnorm al grow th that are related to pesticide exposure w ould bias the re su lts in th is stu d y . Such facto rs m ay be related to socioeconom ic status of the stu dy participants, a co fa cto r w h ich is d ifficu lt to d e fin e , no le ss m e a su re in an e p id em io lo gic study. Lim ited external validity (gen eralizability) due to the p articu lar stu dy population recru ited and the n u m ero us exclusion criteria a p p lie d . S tre n g th s in th e stu d y 50 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00050 Author, Year, Sample Size (2004) CHAM ACOS (N = 4 8 8 ) Outcome Assessed w eight, head circum ference, G estation Length, Ponderal index H arley et al. (2011) CHAM ACOS (N = 3 2 9 ) Birth length, birth w eight, head circum ference, gestational age Exposure Measurement Potential confounders considered Primary Result Conclusions/Uncertainties Strengths and Weaknesses m edian 136 nm o l/L (range: 10-6,854); DEP: m edian 22 nm ol/L (range: 2 -6 8 0 nm ol/L); TCPy: m e d ian 3.3 nm o l/L (range: 0 .2 -5 6 .1 n m o l/L) (76% >LOD) Th e geom etric m ean for the DAP c o n c e n tra tio n s during pregnancy (for the average of the two sam pling periods) w as 146 nm ol/L (95% Cl: 133, squared, m aternal age, p regn an cy w eight gain, w eek of initia tin g prenatal care, parity, infant sex, m other's co untry of birth, body m ass index, fam ily incom e, po verty level, sm oking, alco hol, illicit drug use, environm ental tobacco sm oke, caffein e , history o f low birth w eigh t, and h istory o f pre-term d e liv e ry . du ration asso ciated w ith tw o m e a su re s o f in utero p esticide exposure: levels of m etab o lites o f dim ethyl ph osph ate pesticide co m p o u n d s and w hole blood ChE. M aternal intelligence (Peabody Picture V ocabulary Test (PPVT)), m easures of how stim ulating the e n v iro n m e n t is, and kno w n o r susp ected neu ro to xin s w ere m easured pren atally. To m easure the quality and extent o f stim ulation available to a ch ild in th e h o m e e n v iro n m e n t, The authors observed evidence of an association betw een prenatal exposure to O P pesticides as m easured by u rin ary DAP m e ta b o lite s in w o m e n d u rin g p re gn an cy , is associated w ith decreased c o g n itiv e fu n c tio n in g in averaged w eeks 14, 26, not c re a tin in e -c o rr e c te d Infants w hose PON1 genotype and enzym e activity levels suggested that they m ight be m ore susceptib le tothe effects of OP pesticide exposure had decreased fetal grow th and length o f gesta tio n . PO N 1 m ay be a contributing design includ e the lon gitu dinal design, the use of m ultip le exposure bio m arkers, includ in g quantification o f non specific (DAPs), chlo rpyrifo s-specific (TCPy) m etabolites, and o ther environm ental co exposures. A reaso nab le set of exclusion criteria w ere ap plied .The selection of the CHAM ACOS population, w hich co nsists m ostly o f children from low -inco m e fam ilies, served to increase the relative statistical efficien cy of the study, as this po p u la tio n is at high risk o f n eurodevelo pm ental deficits, co m p ared to the general po pulation .The statistical analysis used to assess the associations betw een the m arkers of exposure and n eu ro d evelo p m en t w ere a p p ro p ria te .E rro rs in the a s s ig n m e n t o f e x p o s u r e in this p rospective stu d y w ill lik e ly h a ve re su lte d in attenuation o f observed a sso c ia tio n s. Th is study has m any stren gth s, the lon gitu dinal design, the m easurem en t of u rin ary DAP at m ultiple tim e p o ints and fo llow in g children to age seven w hen tests o f co gnitive function are repo rted ly m ore reliable. Th e authors w ere 51 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00051 Author, Year, Sample Size Outcome Assessed Exposure Measurement Potential confounders considered Primary Result Conclusions/Uncertainties Strengths and Weaknesses Engel e t at. (2007) Mt. Sinai (N =311) 160); o f this, a larger proportion was DMP m etab olites (GM = 109 nm ol/L; 95% Cl =98, 120) than DEPs (GM = 23 nm ol/L; 95% Cl = 21, 25). A llele fre q u e n c ie s : PON1 192 Q allele= 50% ; PO N1 -108 T allele= 46% .M ean a r y le s te ra se activity: For infants: 33.6 U /m L (SD = 16) For m others: 136.6 U /m L (SD = 44). M ean paraoxonase activity: For infants: 256.6 U/L (SD = 165); For m others: 989.0 U /L (SD = 616). Brazelton N eonatal B e h a v io ra l A ssessm ent Scale (B N B A S), prim itive re fle x e s (n e u ro lo g ic a l integrity) m easured before ho spital d isc h a rg e . DEP: 24.7 nm ol/L; Total DAP: 82 nm ol/L the Infant-To ddler HO M E (H om e O bservation for M easurem ent o f the En viron m ent) invento ry w as co m p leted at the 6-m onth, 1, 2, 3.5, 5, and 7 ye ar visits; know n or suspected n eurotoxicants, p o lybro m in ated diphenyl ethers (PBD Es), p o lychlorin ated biph en yls (PCBs), p,p'dichlo ro diphenyltrichlorethane (D D T), p,p'dichlo ro diphenyltrichlorethylene (D D E), and lead. M aternal age, race, m arital status, education, cesarean delivery, delivery anesthesia, infant age at exam ination , infant gender, infant jau n d ice , sm o kin g (yes/no ), alcohol co nsum ptio n, caffein e co n su m p tio n , illicit drug use during pregnancy, and the exam iner. children at age 7. No adverse associations w ere found for DAPs and any m easured behavior. Relative to the first qu artile, q u artiles 2 -4 of total DEPs, DM Ps, and DAPs w ere associated w ith an increased proportion of abnorm al reflexes, although the asso ciatio ns did not increase m o n o to n ic a lly and v a rie d in th e ir stren gth and p re c is io n . fa cto r to preterm or low birth w e ig h t birth. Used non-specific bio m arker D EP/D AP able to ad just fo r or co nsider m any factors related to co gnitive function, such as prenatal exposure to other environm ental agents, so cio eco n o m ic indicators, m aternal intelligence and education, and child stim ulation. Th e cohort had a re latively ho m ogeno us so cio eco n o m ic profile, redu cin g the potential for uncontrolled confounding. Th is w as a w ell co n d u cted p rospective stu dy co n d u cte d in a yo u n g, pred o m in an tly m ino rity population. Th e study design, an alytic approach, and statistical analyses w ere ap propriate. Pesticide m etab olites evaluated are not specific for chlo rp yrifo s. Th e BN BAS w as ad m in istered before ho spital d ischarge o nly on a su b se t o f ch ild re n in the co h o rt (n =31 1/4 04 ). Factors related to w eekend 52 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00052 Author, Year, Sample Size Outcome Assessed Exposure Measurement Yo u n g e t al. (2005) CHAM ACOS (N = 3 8 1 ) N eurodevelopm ent, Brazelton N eonatal B ehavioral A ssessm en tSca le (BN BAS), abnorm al re fle x e s DAP (average d u rin g pregnancy): m edian 222nm ol/L (range: 7 -2 1 ,8 6 7 nm ol/L); DEP (average during pregnancy): Potential confounders considered M aternal age, BM I, any sm oking/alco hol/d rug use du ring pregnancy, gestational age at w hich prenatal care w as initiated, total n u m b er of prenatal care visits, m ean p regn an cy blood pressure, parity, m ethod o f delivery, general anesthesia used during Primary Result Conclusions/Uncertainties Strengths and Weaknesses A m o n g th e >3 day old infants, increasing average prenatal urinary m etab olite levels w ere asso ciated w ith b o th an in cre a se in n u m b e r of ab norm al reflexes (total DAP: adjusted beta = 0.53, 95% Cl = 0.23, 0.82; dim eth yls: ad justed beta = Associations seen pre-natal OP, not post-natal OP expo sure, M aternal urine co llectio n averaged w eeks 14, 26 delivery (e.g., few er inductions) w ould be underrepresented am ong the tested subjects, and m ay induce bias, reduce the degree of precision w ith w hich asso ciatio ns w ere e stim ated , and lim it the generalizability o f the study findings.The statistical analysis w as largely appropriate. Im puting o f m issing data m ay affect precision of association estim ates and re su lt in atte n u a te d e ffe ct estim ates as a result of exposure m easurem ent error. Assessm ent and control for confounding w ere appropriate. How ever, confo un ding by un m easu red (and m ism easured) risk facto rs for abnorm al grow th that are related to pesticide exposure w ould bias the re su lts in th is stu d y . Such facto rs m ay be related to socioeconom ic status of the stu dy participants, a co facto r w hich is difficu lt to d e fin e , no le ss m e a su re in an e p id em io lo gic study. Strengths: Longitudinal design, m easurem en t and co nsideratio n o f m any confounders, the prenatal exposure m easures were, w ith som e exceptio ns, the average of two m easurem ents, and thus m ay better reflect chronic 53 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00053 Author, Year, Sample Size Outcome Assessed Exposure Measurement Potential confounders considered Primary Result Conclusions/Uncertainties Strengths and Weaknesses Rauh e ta !. (2006) C o lu m b ia U. (N = 2 5 4 ) m edian 21 nm ol/L (range: 2 -6 8 0 nm ol/L) N eurodevelopm ent: Th e Bayley Scales of Infant D evelopm ent II (B S ID -II), M e n ta l D evelop m en t Index (M D I) and Psychom otor D evelop m en t Index (PD I) at 12, 24, and 36 m onths of age. B eh avior: Child Behavior Checklist (CBCL) at 12, 24, and 36 m onths. Q u ality o f the c h ild -c a re environm ent: The H om e O bservation for M easurem ent of the Environm ent (HOM E) Exposure levels w ere categorized a s lo w (<_ 6 .1 7 pg/g) or high (> 6.17 pg/g) d elivery, b re a stfe e d in g initiated after delivery, po verty level, in fa n t se x, a ge in d a y s at BN BAS, m inutes since last feed at BN BAS, and BN BAS exam iner. Data w ere co llected regarding lead exposure, dem ograph ics, education and o ccupational history, incom e, active and passive sm oking, alcohol and drug use du ring pregnancy, and re sid e n tial p e sticid e use. Final m od els included prenatal environ m en tal tobacco sm oke (ETS) exposure, gender, eth nicity, gestatio n al age at birth, quality of hom e care takin g environm ent, m aternal e d u ca tio n , an d m a te rn a l IQ. 0.41, 95% Cl = 0.12, 0.69; dieth yls: ad justed beta = 0.37, 95% Cl = 0.09, 0.64), and the proportion of infants w ith m ore than three abnorm al reflexes (total DAP: adjusted O R = 4.9, 9 5 % Cl = 1.5, 16.1; dim eth yls: ad justed O R = 3.2, 9 5 % Cl = 1.1, 9.8; dieth yls: ad justed O R = 3.4, 9 5 % Cl = 1.2, 9.9). At the 36 m onth m ilestone, the likeliho o d o f high ly exposed children develo ping m ental delays w ere 2.4 tim es greater (9 5 % Cl: 1 .12-5.08, p = 0.02) and m otor delays w e re 4.9 g re a te r (9 5 % Cl: 1.78-13.72; p = 0.002) than those w ith low er prenatal exposure. Th e G LM analysis for PDI scores show ed a significant effect of chlorpyrifos exposure over tim e w ith an estim ated d eficit of ap p ro xim ately 7 p o in ts by age 36 m o n th s (p = 0.01). The au tho rs sum m arize th re e m ain fin d in gs: 1) by age 3, the m ore high ly exposed children dem onstrated m ental and m otor delays; 2) the observed developm ental trajecto ries for PDI and M DI scores confirm ed that the adverse im pact on co gnitive and m otor develo pm ent increased o ve r tim e; and 3) by age 3, high ly expo sed children w ere m ore likely to dem on strate clinically significant attention problem s. exposure during the pregnancy W eaknesses: Potential for residual co nfo un ding, potential for exposure m isclassificatio n, potential se le ctio n b ias (n o t all children had the o utcom e m easures), study population not gen eralizab le to the w hole US. O nly 53% of the children reached the three year m ilestone w ith stu d y data co lle cte d . It is u n cle ar w hat percentage of these children did not su rvive, w ere lost to follow -up , or too sick to participate. Reliance on a single expo sure level (p renatal/co rd blood.) No control for exposure over the subsequent 3 years Creation o f a dicho tom ou s exposure variab le brin gs lim itations due to the am ount of w ith in -gro u p variation. Lim itatio ns o f the sen sitivity and pred ictive validity o f the developm ental tests, especially am o ng children less than 3 ye ars o f age. No discussion o f w heth er th is 7 -p o in t d e ficit is clin ically relevant. Due to the pervasive, no n-specific nature of n e u ro lo g ic a l e ffe c ts, it is difficu lt to attribute 54 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00054 Author, Year, Sample Size Outcome Assessed Exposure Measurement Potential confounders considered Primary Result Conclusions/Uncertainties Strengths and Weaknesses Lovasi e t al. 2011 Bayley sco res N/A Colum bia U. (M D I/PD I) 12 (N = 2 6 6 ) m onths, 24 m onths, 36 m onths N eighbo rho o d characteristics: T h e p e rce n tage o f h o u sin g units w ith o u t co m p lete plum b in g, the percentage of vacan t housing units, the p e rce n tage of residents below the federal po verty line, the percentage of residents o ld er than 25 years of age w ho co m p leted high school, the percentage of ho useho ld s receivin g pu blic assistan ce, the percentage of ho u sin g units w ith one o r m ore residents per room , racial co m p o sitio n , the percentage o f residents born o u tsid e the U nited States, the percentage o f Sp an ish -sp eakin g residents, and the percentage of resid en ts w ho w ere lin guistically is o la te d N eighborhood ch a racteristics did not confound the observed association betw een ch lo rp yrifo s levels and cognitive developm ent H ierarchal regression analysis o f potential confounding by SES Engel e ta l. (2011) Mt. Sinai (N =276) Bayley scores (M D I/PD I) at 12 m onths, 24 m onths. DEP: 24.7 nm ol/L; Total DAP: 82 nm o l/L (sam e as Engel 2007) M aternal age, race/ethnicity, m arital status, education, breast-feed in g, child sex, alcohol, sm oking, o r drug use d u rin g p regn an cy, m aternal IQ, a score based on assessm ent of the hom e environ m en t (H O M E), season o f urine co llectio n , la n g u a g e sp o ken in th e ho m e, age at testin g, exam iner, and u rin ary cre atin in e level. An observed association betw een prenatal total d ia lk y lp h o s p h a te m etab olite level and a d e c re m e n t in m e n ta l develo pm ent at 12 m onths am ong blacks and H ispanics. Used non-specific bio m arker D EP/D AP; som e evidence of effect m odification by PON1 genotype cau sality. D irect m easurem en t of chlo rpyrifo s.The statistical analyses w ere generally appropriate. M issing data on covariates w ere estim ated using m ultip le im putatio n, and the variance estim ates presented ap pro priately reflect the degree of uncertainty caused by m issing covariate data. Robust standard errors w ere used.The settin g of th e in v e stig a tio n in a sam ple draw n from low incom e African Am erican and D o m in ican co m m u n itie s is both a strength (increases the pow er, restriction of confounders) and a lim itation of the study (reduced generalizability). Lim itatio ns include use of no n-specific m arkers of chlo rpyrifo s pesticide exposure (DAPs), use of only a single (thirdtrim ester) urin e sam ple, and the large proportion of loss to follow -up . Statistical analysis w as appropriate. Im puting o f m issing data m ay affect precision of association estim ates and re su lt in atte n u a te d e ffe ct estim ates as a result of exposure m easurem ent error, although these are offset by the further 55 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00055 Author, Year, Sample Size Eskenazi e to l. (2007) CHAM ACOS (N = 3 7 2 ) Outcome Assessed Exposure Measurement Potential confounders considered Primary Result Conclusions/Uncertainties Strengths and Weaknesses N eurodevelopm ent, Bayley Index (M D I, PDI), M aternal behavio r checklist at 6 ,1 2 , and 24 m onths DEP: geom . Mean in m o th e r 18.1 nm o l/L (95% Cl = 16.7-19.7); DEP geom etric m ean in ch ild at 24 m on th s 10.5 nm o l/L (95% Cl = 8 .8 --1 2 .6 ); T C P y m edian 3.54 ug/l Psycho m etrician, location of assessm ent, exact age at assessm ent, sex, breast-feeding duration (m onths), HO M E score, and household incom e, parity, m aternal PPVT, m aternal age, education, depressive sym pto m s, active/passive sm o kin g exposure during pregnancy, regular alcohol use du ring p regn ancy, m arital s ta tu s , f a t h e r 's p r e s e n c e in hom e, ho using density, m aternal w ork status, > 15 hours out-ofho m e ch ild care/w ee k, birth w eigh t, gestation al age, ab norm al reflexes, PCBs, lead, DDT, p-hexachlorocyclohexane, and hexa-ch lo ro benzen e DAP m etab olite levels du ring pregnancy, p a rticu larly from dim eth yl ph osph ate pesticides, m ay be negatively associated at 24 m onths w ith m ental develo p m en t (M D I) on the Bayley Scales and an in cre ase in risk o f m aternally reported PDD. No strong associations identified w ith DE o r TC Py, M aternal urine co llectio n averaged w eeks 14, 26 catego rizatio n of the expo sure levels (at the m edian). H ow ever, binning of exposure levels reduces precision , relative to a co n tin u o u sly distributed m easure of exposure. Assessm ent and control for confounding w ere appropriate. How ever, confounding by unm easured (and m ism easured ) risk facto rs for abnorm al grow th that are related to pesticide exposure w ould bias the re su lts in th is stu d y . Such facto rs m ay be related to socioeconom ic status of the stu dy participants, a co facto r w hich is difficu lt to d e fin e , no le ss m e a su re in an e p id em io lo gic study. Strengths: Longitudinal design, m easurem en t and co nsideratio n o f m any co n fo u n d ers (in clu ding o ther environm ental chem icals); the prenatal exposure m easures were, w ith som e exceptio ns, the average of two m easurem ents, and thus m ay better reflect chronic exposure during the pregnancy W eaknesses: Potential for residual co nfo un ding, potential for exposure m isclassificatio n, potential se le ctio n b ias (n o t all children had the o utcom e 56 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00056 Author, Year, Sample Size Outcome Assessed Exposure Measurement Potential confounders considered Primary Result Conclusions/Uncertainties Strengths and Weaknesses Eskenazi e t al. ( 2010) CHAM ACOS (N = 3 7 1 ) N eurodevelopm ent, Bayley Index (M D I, PDI), M aternai behavio r checklist at 6 ,1 2 , and 24 m onths, PON1 gene and enzym e levels Th e geom etric m ean for the DAP c o n c e n tra tio n s during pregnancy (for the average of the two sam pling periods) w as 146 nm ol/L (95% Cl: 133, 160); o f this, a larger proportion was DMP m etab olites (GM = 109 nm ol/L; 95% Cl =98, 120) than DEPs (GM = 23 nm ol/L; 95% Cl = 21, 25). A llele fre q u e n c ie s : PON1 192 Q allele= 50% ; PO N1 -108 T allele= 46% .M ean a r y le s te ra se activity: For infants: 33.6 U /m L (SD = 16) For m others: 136.6 U /m L (SD = 44). M ean paraoxonase activity: For infants: 256.6 U/L (SD = 165); For m others: 989.0 U /L (SD = 616). Psycho m etrician, location of assessm ent, exact age at assessm ent, sex, breast-feeding duration (m onths), HO M E score, and ho useho ld incom e, parity, m aternal PPVT, m aternal age, education, depressive sym pto m s, active/passive sm o kin g exposure during pregnancy, regular alcohol use du ring p regn ancy, m arital s ta tu s , f a t h e r 's p r e s e n c e in hom e, ho using density, m aternal w ork status, > 15 hours out-ofho m e ch ild care/w ee k, birth w eigh t, gestation al age, ab norm al reflexes, PCBs, lead, DDT, (3-hexachlorocyclohexane, and hexa-ch lo ro benzen e M arks e ta l. (2010) CHAM ACOS CBCL; K-CPT; A D H D co nfid en ce index; DAP (geom etric m ean) pregnancy Psycho m etrician, exact age at assessm ent, sex, m aternal D ecrease M DI (24 m onths) PONliosn-5.7 (-9 .0 to - 2.5) 9=0.01; D ecrease PDI (2 4 m o n th s) P O N Iiostt- 2.8 (-5 .7 to 0.2) p=0.07; increased od d s PD D 2.0 (0.8 to 5.1) p=0.14; no a s s o c ia tio n PO N I192; n o a s s o c ia tio n PON1 a c t iv it y m easured new born, 2 years, m aternal and M DI, PDI, PDD. Evidence of decreasing M DI score by n u m b e r o f PONlios v a ria n t a lle le s: PONlioscc- 3 .2 (-9 .8 to 3 .5 ), C T --3 .7 ( - 8 . 0 to 0.6), T T -5 .5 (-1 1 .1 to 0.1), p -interactio n 0.98. Prenatal DAPs w ere nonsignificantly associated In th is s tu d y p o p u la tio n , evidence PO N 1 m ay influence M DI score, but not PDI o r PDD risk at tw oyears. N on-significant evidence of d ecreasing M DI score by increasing DAP levels across strata o f the num ber o f P O N 1 TM variant alleles, in teractio n n o n significant. Sim ilar trend w ith pren atal DEP levels and M DI, PDI by PO N 108 alleles, but less pronounced. O verall, lim ited, n o n-defin itive evidence of effect m odification by PON1 sta tu s in th e re latio n betw een m ental and psychom otor effects and prenatal DAPs. M arked effect m odification by gender: 11-fold increase m easures), study population not gen eralizab le to the w hole US. Strengths: Longitudinal design, m easurem en t and co nsideratio n o f m any co n fo u n d ers (in clu ding o ther environm ental chem icals); the prenatal exposure m easures were, w ith som e exceptio ns, the average of two m easurem ents, and thus m ay better reflect chronic exposure during the pregnancy W eaknesses: Potential for residual co nfo un ding, potential for exposure m isclassificatio n, potential se le ctio n b ias (n o t all children had the o utcom e m easures), study population not gen eralizab le to the w hole US. Study m ay be under pow ered to evaluate effect m odification by PO N 1 status. Strengths: Longitudinal design, m easurem en t and 57 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00057 Author, Year, Sample Size (N = 3 4 8 ) Outcome Assessed H illside b ehavio ral rating scale; co m p osite A D H D indicato r Rauh e to l. (2011) Colum bia U. (N = 2 6 5 ) W ech sler Scales of Intelligence for Ch ild ren (W ISC-IV ) Child B eh avior Checklist (CBCL). Exposure Measurement Potential confounders considered Primary Result Conclusions/Uncertainties Strengths and Weaknesses 109.0 nm ol/L; DEP 17.7 nm o l/L education, depressive sym pto m s, PPV T (continuous), > 15 hr o u t-o f-h o m e child care/w eek, breast feeding duration (m onths), m aternal age, parity, m arital status, active/p assive sm oking exposure and regular alcohol use during p re g n a n cy , p re se n ce o f fa th e r in hom e, m aternal w ork status, and household incom e w ith m aternal report of attention problem s and A D H D at age 3.5 years, but w ere sign ifica n tly related at age 5 years [CBCL attention p r o b le m s : (3 = 0 .7 p o in ts; 95% co nfid en ce interval (C l), 0 .2-1.2; A D H D : 3 = 1.3; 95% Cl, 0.4-2.1]. A D H D co m p o site indicato r in b o ys, le ss th a n 2-fo ld in girls, ho w ever un stab le estim ates; w eak evidence o f asso ciatio n D APs at 3.5, 5 years and attention C h lo rp yrifo s le ve ls in u m b ilical cord blood sam ples, N =256 new borns If no cord blood (12% of subjects), levels w ere im puted from m others' values. V alues for sam ples w ith nond e te c ta b le ch lo rp yrifo s levels (N =115, 43% ) w ere im puted by using assaysp e cific lim it o f detection (LO D) values to im pute an approxim ate Data w ere co llected regarding lead exposure, dem ograph ics, education and o ccupational history, incom e, active and passive sm oking, alcohol and drug use du ring pregnancy, and re sid e n tial p e sticid e use. Final m od els included prenatal environm ental tobacco sm oke (ETS) exposure, gender, eth nicity, gestatio n al age at birth, quality of hom e care takin g environm ent, m aternal e d u ca tio n , an d m a te rn a l IQ. Fu ll-Scale IQ: (B ) o f -0 .00 3 , Cl = 0.006, 0.001, p= 0.064 W o rkin g M e m o ry Index: (B) of -0.006, Cl = 0.009, 0.002, pcO .001. Th e investigators articulated these results as sh o w in g that a 1 pg/g in cre ase in ch lo rp y rifo s exposure w as associated w ith a 0.006 point decrease in the lo g -tra n sfo rm e d W o rkin g M em ory score and a 0 .0 0 3 p o in t d e cre a se in the lo g-tran sfo rm e d FullScale IQ score. Th e investigators concluded that for each standard d e v ia tio n in c r e a s e in exposure (4.61pg/g)there is a 1.4% re d u ctio n in FullScale IQ a n d a 2.8% For each standard d e v ia tio n in c r e a s e in exposure (4.61pg/g) there is a 1 .4 % re d u ctio n in Fu llScale IQ and a 2.8% re d u ctio n in W o rk in g M em ory. co nsideratio n o f m any co n fo u n d ers (in clu ding o ther environm ental chem icals); the prenatal exposure m easures were, w ith som e exceptio ns, the average of two m easurem ents, and thus m ay better reflect chronic exposure during the pregnancy W eaknesses: Potential for residual co nfo un ding, potential for exposure m isclassificatio n, potential se le ctio n b ias (n o t all children had the o utcom e m easures), study population not gen eralizab le to the w hole US. Strengths D irect assessm en t of chlo rpyrifo s levels using m aternal serum and cord b lo o d . An alysis using a co n tin u o u s CPF level, w h ich , in co n tra st to dicho tom ou s CPF levels, provides a m ore m eaningful look at potential thresho ld effects and dose-response trends. The investigators rigo ro usly evaluated their m ethods for im puting values for undetectable C P F le ve ls w h ich in the end, w ere validated. Th e authors describe an elegant and 58 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00058 Author, Year, Sample Size Outcome Assessed Exposure Measurement level. Potential confounders considered Primary Result re d u ctio n in W o rk in g M em ory. Conclusions/Uncertainties Strengths and Weaknesses m ethodologically sound statistical analysis, ad dressin g m any o f the potential sh o rtcom ings of th e ir expo sure data and c o v a ria te s. W eaknesses: The use of a single snapshot o f prenatal chlorpyrifos exposure m ay not be an accurate su rro g ate fo r full prenatal e xp o su re levels. T h ere is no co ntrol for exposure over the sub seq u en t 7 ye ars w hich m ay be critical, especially as the process of n euroco gnitive d e velo p m e n t is fluid and rapid d u ring th ese early childhood years. Po ssib ility of that an increased aw areness o f the risks of p esticide expo sures could d isp ro p o rtio n ately affect postnatal exposure behavior. Co m p licatin g this a n alysis is the pervasive, non-specific nature of neurological e ffe c ts a n d t h e d if fic u lt y in attrib u tin g causal pathw ays. w hen closely review ed, the 95% Cl for Full Scale IQ fo r both tech n iq u es co ntain 0 (LASSO : -0.006, 0.001, p=0.064; fully-adjusted : 0.006, 0.001, p=0.048 The authors do not address the clinical relevance of the 1.4% and 2.8% redu ctio ns 59 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00059 Author, Year, Sample Size Outcome Assessed Exposure Measurement Potential confounders considered Primary Result Engel e t al. (2011) Mt. Sinai (N =169) W ech sler Preschool and Prim ary Scale of Intelligence, 3rd edition (W PPSI-III) at ages < 7 years; W ech sler-IV Intelligence Scale (v e rb a l co m prehension; perceptual reasoning, w orking m em ory, processin g speed, full scale intelligence) at age 7-9 years DEP: 24.7 nm ol/L; Total DAP: 82 nm o l/L (sam e as Engel 2007) M aternal age, race/ethnicity, m arital status, education, breast-feed in g, child sex, alcohol, sm oking, o r drug use d u rin g p regn an cy, m aternal IQ, a score based on assessm ent of the hom e environ m en t (H O M E), season o f urine co llectio n, la n g u a g e sp o ke n in th e ho m e, age at testin g, exam iner and u rin ary cre atin in e level. At age 6-9 years, nonsta tistica lly significant re d u c tio n s in fu ll sca le IQ, perceptual reasoning, verbal co m p rehension, w orking m em ory and processin g speed w ith increasing DAP, m ore profound w ith DEP than DMP Conclusions/Uncertalnties Strengths and Weaknesses Used no n-specific bio m arker D EP/D AP; som e evidence of effect m odification by PO N 1 g e n o ty p e and how this m ay im pact a child or his/her psychological or educational plans. Lim itatio ns include use of no n-specific m arkers of chlo rpyrifo s pesticide exposure (DAPs), use of only a single (thirdtrim ester) urin e sam ple, and the large proportion of loss to follow -up. Th e statistical analysis w as largely appropriate. Im puting o f m issing data m ay affect precision of association estim ates and re su lt in atte n u a te d e ffe ct estim ates as a result of exposure m easurem ent error, although these are offset by the further catego rizatio n of the expo sure levels (at the m edian). Assessm ent and control for confounding w ere appropriate. How ever, confounding by un m easu red (and m ism easured) risk facto rs for abnorm al grow th that are related to pesticide exposure w ould bias the re su lts in th is stu d y . Such facto rs m ay be related to socioeconom ic status of the stu dy participants, a co facto r w hich is difficu lt to d e fin e , no le ss m e a su re in an e p id em io lo gic study. 60 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00060 Author, Year, Sample Size Outcome Assessed Exposure Measurement Potential confounders considered Primary Result Conclusions/Uncertainties Strengths and Weaknesses B o u ch ard e t al. ( 2011) CHAM ACOS (N = 3 2 9 ) W ech sler-IV Intelligence Scale (v e rb a l co m prehension; perceptual reasoning, w orking m em ory, processin g speed, full scale in te llig e n c e ) m easured at age 7 years W h yatt e t al. (2007) Colum bia U. (N =102) None W h yatt e t al. None Total DAPs (q uintiles): Q1 (39 nm ol/L); Q2 75 nm ol/L; Q3 126 nm ol/L; Q 4 221 nm ol/L; Q5 508 nm ol/L. G e o m etric m ean DAP 131 nm ol/L G e o m etric m ean, 6 .9 1 7 .0 n g / m 3; range < 0 .4-171 n g / m 3. P e rs o n a l air m onitor: m e d ian 2.8 n g / m 3, m e a n 6 .2 1 1 .1 n g / m 3, range < 0 .4 -8 3 .4 ng/m 3 Th e lim it of M aternal intelligence, m easures o f how stim ulating the e n v iro n m e n t is, and kno w n o r susp ected neu ro to xin s w ere m easured pren atally. M aternal in tellige n ce w as assessed via the Peabody Picture V o ca b u lary Test (PPVT). To m easure the quality and extent of stim ulation a v a ila b le to a ch ild in th e ho m e environm ent, the Infant-Toddler HO M E (H om e O bservation for M easurem ent of the Environm ent) inventory w as co m p le te d at th e 6 -m o n th , 1, 2, 3.5, 5, and 7 year visits; know n o r suspected neurotoxicants, p o lybro m in ated diphenyl ethers (P BD Es), p o lychlorin ated b ip h e n y ls (P C B s), p,p'~ dichlo ro diphenyltrichlorethane (D D T), p,p'dichlo ro diphenyltrichlorethylene (D D E), and lead. The authors observed evidence of an association betw een prenatal expo sures to O P pesticides as m easured by urinary D A P m e ta b o lite s in w o m e n du ring pregnancy, and decreased cognitive fu n c tio n in g in c h ild re n at age 7. N /A Th ere w as little w ith in - hom e variab ility and no sig n ifica n t d iffe re n ce in air co n cen tratio n s w ith in ho m es o ver tim e (p > 0.2); betw een-ho m e variab ility accounted for 88% of the v a ria n ce in the in d o o r air le v e ls o f p r o p o x u r , 9 2 % in ch lo rp y rifo s, 9 4 % in d ia z in o n , an d 6 2 % in piperonyl bu to xide (p < 0.001). Indoor and m aternal perso nal air insecticide levels w ere high ly co rrelated (r = 0 .7 - 0.9, p < 0.001). N /A M econ iu m T C P y Prenatal m easures taken later h alf o f pregn ancy m ore significantly associated intelligence than early; little evid en ce po st natal OP exposure asso ciated w ith intelligence; 7 point re d u ctio n in full scale intelligence DAP Q 5/Q 1 (SS) Stren gth s: the lon gitu dinal design, the m easurem en t of u rin ary DAP at m ultiple tim e p o ints and fo llow in g children to age seven w hen tests o f co gnitive function are repo rted ly m ore reliable. Th e authors w ere able to ad just fo r or co nsider m any facto rs related to co gnitive function, such as prenatal exposure to other environm ental agents, so cio eco n o m ic indicators, m aternal intelligence and education, and child stim ulation. Th e cohort had a re latively ho m ogeno us so cio eco n o m ic profile, redu cin g the potential for uncontrolled confounding. Indoor and m aternal personal air insecticide levels w ere high ly co rrelated (r = 0 .7 -0 .9 , p < 0. 001) . Strengths: study design and exposure assessm ent techn iques, Lim itations: only those cohort participants enrolled after 2011 w e re in clu d ed in the analysis (m ost likely due to the lack of serial data from the earlier years.) T C P y in m a te rn a l u rin e Com prehensive exposure 61 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00061 Author, Year, Sample Size Outcome Assessed Exposure Measurement Potential confounders considered (2009) Colum bia U. (N =102) Rauh et al.(2012), (n = 4 0 ) M orphological c h a n g e in th e p e d ia tric b rain in regio ns of the brain know n to be asso ciated w ith learning, cognition and social behavior detection (LO D) o f ch lo rp y rifo s in blood sam p les w as 0 .5 -1 pg/g plasm a. Th e LOD o f T C P y in urin e sam ples w as 0.26 n g/m L urin e. Th e LO D fo r T C P y in m econium w as 0.2 ng based on a sam ple w eighing 0.5 g. Exposure m arker levels below the LOD w ere given a value of half the level o f detection, and w ere then lo g lO transform ed. Tertile 3 ( >4.39 pg/g), com pared to T e rtile s 0, 1, 2 (<4.39 pg/g, includ in g those not exposed to CPF) Age, sex Primary Result Conclusions/Uncertainties Strengths and Weaknesses co ncentrations w ere po sitively co rrelated w ith ch lo rp y rifo s in m atern al and cord blood (r = 0 .2 5 0.33, p < 0.05) and w ith T C P y in m atern al u rin e (r = 0.31, p < 0.01). sam p les w as not reliable, but the m aternal and cord blo od ch lo rp y rifo s as w ell as th e T C P y le ve ls in m econium w ere reliable m easures of exposure assessm ent includ in g actual blood ch lo rp yrifo s levels, the repeated sam pling, and the environm ental sam pling. W eaknesses: o nly included p a rtic ip a n ts re cru ite d in the post-cancellation period, use of nonparam etric, rank-based sta tistics is ap p ro p ria te but the large nu m b er of o bservatio ns below the level of detection receiving equal rank, m ay be problem atic; no dietary assessm ent A uthors report differences in brain stru ctu re (regio n a l cerebral size and thickness) by CPF exposure groups, and the differences (h ig h > lo w C P F) in re gio n al brain size is likely due to e nlargem ent of un derlyin g w hite m atter. Statistical interactio n by gen d er reported. Au tho rs concluded that the evidence from the stu dy illu stra te d ch a n g e s in brain m o rp h o lo g y in a sso cia tio n w ith high er CPF exposure, and that changes observed w e re in a re a s o f th e brain that subserve those learning, cognition and social behavioral, supported by previous o bservatio nal and experim ental literature. Study supports general hypothesis of CPF influence on brain m orpho logy, but lacks specific hypotheses regard in g p articu lar areas of the cerebrum affected; lim ited and so m e w h at unbalan ced depictio n of the available rodent experim ental data; statistical m ethods appropriate, correction for m ultip le statistical com parisons a strength; M RI im age read ers blinded to exposure status enhances study validity; lack o f inform ation on o ther validation practices; sm all sam ple size, pilot study, low statistical pow er; external validity 62 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00062 Author, Year, Sample Size Outcome Assessed Exposure Measurement Potential confounders considered Primary Result Conclusions/Uncertainties Strengths and Weaknesses lim ited; o ne tim e m easure of pre-natal exposure Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 63 ED 002061 00044425-00063 2.2.5.2 "Moderate" Quality Group Fifteen articles were assigned a moderate quality rating, as shown in Table 2.2.5.2-1. In general, these were cross-sectional or prospective cohort studies with small to high sample size; exposure assessment was based on a non-specific biomarker measure or current self-report, the outcome measurement(s) utilized standardized tests or screening tools, appropriate statistical analyses were performed, considering some but maybe not all relevant covariates, and risks of bias were minimized to some extent. For example, Guodong et al. (2012) crosssectionally evaluated the relationship between DAPs concentrations in urine sampled from 301 young children as an objective, non-specific marker of prenatal OP pesticide exposure and Developmental Quotients based on the Gesell Developmental Schedules adapted for a Chinese population. Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 64 ED 002061 00044425-00064 Table 2.2.5.2-1. Study Andersen et al.(2015) Guodongeta/. (2012) Manda! et al. (2007) Manda! et al. (2008) Moderate Quality Studies: Summary of Study Design Elements Impacting Study Quality Assignment Cohort Name / Description of Study Location Study Population Study Design Exposure Assessment Outcome Assessment Denmark Schooi age chiidren iiving Prospective Birth Cohort, in Denmark small sample size N=177 (112 maternal occupational pesticide exposure during pregnancy: 65 without) Prenatal occupational pesticide exposure ascertained by maternal interview at enrollment Objective standardized clinical exam; neurophysiological status (heart rate variability); Previously validated, neuropsychological testing with demonstrated sensitivity to environmental pollutants; administered and scored by single neuropsychologist Confounder/ Covariate Control Appropriate. Self-reported by mother. Included age, maternal demographics and risk factors, SES indicator (broad categories), maternal smoking and alcohol use. Possibility of recall errors (residua! confounding). Self-reported (by mother) Statistical Analysis Appropriate multivariate analysis. Evaluated numerous hypotheses without adjustment for multiple comparisons. Also constructed structural equation model of interaction between child sex and prenata! pesticide exposure on genera! intellectual ability (parameterized as a latent variable) Risk of (other) Bias Selection bias possible due to loss to follow-up; Residua! confounding likely, small in magnitude; potential for non differential misclassification of exposure Age: 6-11 years Shanghai, China 2-year oid chiidren visiting community hospitais Cross-sectional, large sample size N=301 Age 23-25 months Objective biomarker, OP exposure (DAP) quantified in single child spot urine sample, simple imputation <LLOD: OP exposure also assessed via questionnaire administered to mothers after delivery Developmental Quotients based on Gesel! Developmental Schedules, adapted for Chinese population Appropriate. Assessed via mother report via questionnaire, included child sex, materna! demographics and risk factors, SES indicators (maternal education, household income), maternal smoking and alcohol use; Possibility of recall errors Appropriate multivariate analysis. Assumed linear relationship between log-transformed DAP level and DQ Scores Selection bias unlikely; Residual confounding likely, small in magnitude; potential for non differential misclassification of exposure Cayambe-Tabacundo region, Ecuador EcoSaiud Project: infants and young chiidren 3 to 61 months of age in iower-aititude communities A&B dominated largely by cutflower production and in more traditional rural, higher altitude community C. Cross-sectionai, modest sample size from census N=283 Age 3-61 months -1 5 4 high exposure -1 2 9 low exposure Objective proxy measure: Community of residence Ages and Stages Questionnaire (ASQ), adapted into local vernacular, 2 trained testers - considered a screening tool Appropriate: child health status (anemia, stunting) and other characteristics of the home environment (stimulation by 2 methods) Appropriate: Multiple linear regressions to evaluate associations between community of residence and delayed development, Pairwise t-tests and chi-square to assess mean difference in ASQ score; Effect size Cohen's d Selection bias unlikely; Residual confounding likely, small in magnitude; potential for non differential misclassification of exposure Cayambe-Tabacundo region, Ecuador EcoSaiud Project: Children 3 to 23 months of age in lower-altitude communities A&B dominated largely by cutflower production and in more traditional rural, higher altitude community C. Cross-sectionai, modest sample size N:121 Age 3-23 months Proxy: Distance home to farm, parental employment, pesticide use on domestic crops & within home, child piay activities Ages and Stages Questionnaire (ASQ) (ages 24-61 months), a screening tool; Visual Motor Integration (VM!) Test (ages 48-61 months); two trained testers. Appropriate: As above Appropriate: As above Selection bias unlikely; Residual confounding likely, small in magnitude; potential for non differential misclassification of exposure Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 65 ED 002061 00044425-00065 Table 2.2.5-2. Moderate Quality Studies: Summary of Study Design Elements Impacting Study Quality Assignment (continued) Cohort Name / Description of Study Study Location Study Population Study Design Exposure Assessment Outcome Assessment M anda! et a /.(2 0 0 7 B ) Cayam b e-Tab acu n d o regio n . E cu a d o r E c o S a iu d P ro je ct: C h iid re n 2 4 to 6 1 m o n t h s o f a g e in lo w er-aititu d e c o m m u n itie s d e sig n a te d A & B w ere d o m in a te d la rge ly by cutflo w e r p r o d u c tio n t h a n in m o re tra d itio n a l rural, h igh e r altitu d e co m m u n ity C. C ro ss-se ctio n a l, m o d e st s a m p le size N: A S Q -1 4 2 Age 2 4 -6 1 m onths VMI - 57 Proxy: M atern a! e m p lo y m e n t d u rin g p re g n a n cy , ch ild p la y s o u td o o rs, P e sticid e u se on d o m e stic cro p s , in sid e hom e A g e s an d S ta g e s Q u e stio n n a ire (A SQ ) (3 -2 3 m o n th s) - s c re e n in g too!; R e a c h a n d -g ra sp , U C B e rk e le y P re fe re n tia l Lo o k in g T e s t C a rd s; tra in e d te ste r Confounder/ Covariate Control A p p ro p ria te : A s a b o ve Statistical Analysis A p p ro p ria te : A s ab o ve ; P re h e n sio n - L o g istic re g re ssio n m o d e ls, g e n e ra liz e d e stim a tio n e q u a tio n s (G E E ) to a c c o u n t for betw een-test d e p e n d en ce Age 4 8 -6 1 m onths Llo p e t a l. (2 0 1 3 ) S p a in IN M A (E n v iro n m e n t an d C h ild h o o d ) P ro je ct P ro sp e c tiv e B irth C oho rt, large sa m p le siz e N =1980 Age 1 4 m onths M ate rn al se lf-re p o rt of p re n a ta l an d p o stn a ta l in d o o r p e stic id e u se (p e s tic id e s p ra y or u se o f a p lu g-in d e v ice a s s e s s e d via q u e stio n n a ire ) M en tal an d p sych o m o to r d e v e lo p m e n t at 1 4 m o n th s a s s e s s e d u sin g v a lid a te d in stru m e n t (B a yle y S c a le s o f In fan t D e ve lo p m e n t) A p p ro p ria te . S e lf-re p o rte d by m o th e r. In clu d e d m a te rn a l d e m o g ra p h ic s an d risk fa c to rs (Age, BM I) S E S in d ic a to rs (m a te rn a l e d u ca tio n , o ccu p a tio n ), m a te rn a l s m o k in g a n d a ico h o l u se . A lso ch ild ca re b e h a vio rs (b re a stfe e d in g , n u m b e r o f s ib lin g s , d a y c a re ) P o s s ib ilit y o f re ca i! e rro rs (re sid u a l co n fo u n d in g ) A p p ro p ria te m u ltiv a ria te a n a ly s is Risk of (other) Bias S e le c tio n b ia s u n likely; R e sid u a ! c o n f o u n d in g lik e ly , s m a ll in m a g n itu d e ; p o te n tial fo r n o n d iffe re n tial m isc ia ssifica tio n of exposure S e le c tio n b ia s u n likely; R e s id u a l c o n f o u n d in g lik e ly , s m a ll in m a g n itu d e ; p o te n tial fo r n o n d iffe re n tial m isc ia ssifica tio n of exposure O u lh o te an d B o u ch a rd (2 0 1 3 ) C a n a d ia n N a tio n ai P o p u ia tio n P e tit e t a l. (2 0 1 0 ) B rittan y, F ra n ce C a n a d ia n H e a lth M e a su re s S u rv e y (C H M S: cycle 1, 2 0 0 7 -2 0 0 9 ); C ro ss-se ctio n a l Study, large n a tio n a l s a m p le size n = 7 7 9 c h iid re n Age 6 -1 1 years N e w b o rn c h ild r e n in th e P E LA G IE Study. P ro sp e c tiv e B irth C oho rt, large sa m p le siz e N= 3 ,1 5 9 Age: new borns C h ild u rin a ry D A P, D M P, D E P c o lle c te d w ith in 2 w e e k s o f s u rv e y q u e stio n n a ire co m p le tio n by th e p a re n ts. B e h a v io ra l p r o b le m s in c h ild r e n b a s e d on th e p a re n t v e rsio n of th e S tre n g th s an d D iffic u ltie s Q u e stio n n a ire (S D Q ) (G o o d m a n 1 9 9 7 ) - S D Q is a v a lid a te d s c re e n in g q u e stio n n a ire an d a c ce p te d by parents. E co lo g ica l, p roxy in d ica to r of e x p o su re (p ro p o rtio n o f m u n ic ip a lity d e vo te d to a g ricu ltu ra l activity O b je c tiv e ly m e a s u re d birth o u tc o m e s a s s e s s e d u sin g h o sp ita l re co rd s A p p ro p ria te : In c lu d e d se x, age , ra ce /e th n icity , fa m ily in co m e , p a re n ta l e d u ca tio n , b lo o d iea d le ve ls, m a te rn a l s m o k in g d u rin g p re gn a n cy, b ir t h w e ig h t , m a t e r n a ! a g e a t c h i l d 's b ir t h , c h i l d 's BM I, a n d fa s tin g s ta tu s (fa s tin g d u ra tio n at u rin e co lle ctio n > 1 0 h o u r/< 1 0 hour) A p p ro p ria te : L o g is tic R e g re s s io n S e le c tio n b ia s u n likely; S tu d y p o p u la tio n n o t m ixe d , s in g le u rin e sa m p le A p p ro p ria te . M a te rn a l re p o rt via q u e stio n n a ire . In c lu d e d m a te rn a l d e m o g ra p h ic s (a ge , BM I) an d p re g n a n cy risk fa c to rs (g e sta tio n a l age, h y p e rte n sio n , d ia b e te s, s e a s o n o f p re g n a n cy ) S E S in d ic a to rs (d istric t o f re sid e n ce , m atern a! e d u ca tio n , o ccu p a tio n ), m a te rn a l sm o k in g an d a lco h o l u se . A lso ch ild ca re b e h a vio rs (b re a st fe e d in g , n u m b e r o f sib lin g s, d a y c a re ); P o ssib ility of re ca li e rro rs A p p ro p ria te m u ltiv a ria te a n a ly s is . M u ltip le c o m p a ris o n s w e re c o n d u c te d fo r th is e v a lu a tio n , a n d th e a u th o rs did no t a d ju s t fo r m u ltip le te s tin g S e le c tio n b ia s u n likely; R e sid u a ! c o n f o u n d in g lik e ly , s m a ll in m a g n itu d e ; p o te n tial for su b s ta n tia l n o n -d iffe re n tia! m isc ia ssifica tio n of exp o su re ; M isc ia ssific a tio n of o u tco m e s u n lik e ly Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 66 ED 002061 00044425-00066 Table 2.2.5-2. Moderate Quality Studies: Summary of Study Design Elements Impacting Study Quality Assignment (continued) Study Study Location Cohort Name / Description of Study Population Study Design Exposure Assessment Outcome Assessment R u c k a r t e t al. (2 0 0 4 ) M sslsslp p , U S A (2 9 c o u n te s), O h lo (o n e m u lti-fa m ly faci ty) C h ild re n w ho w ere 6 ye a rs or y o u n g e r in M S a n d O H w h e n h o m e s s p ra y e d w ith MP R e tro sp e ctiv e co h o rt, m o d e ra te s a m p le s iz e in 2 s t a t e s , p a rtic ip a tin g / n o n p a rtic ip a tin g s im ila r in s e x a n d a g e M S: N = 3 6 5 (1 4 7 exposed, 2 1 8 unexposed) OH: N = 2 8 7 (1 0 4 exposed, 1 8 3 unexposed) A g e : 1 .9 -1 2 .5 y e a rs old at te stin g S p e c ific O P m e a su re s: H o u se h o ld m ethyi p a ra th io n (w ip e) by ap p ro ve d ia b s , O R h ig h e s t u r in a r y P N P le v e l in h o u se h o ld , a n a lyze d b y C D C V a rie ty o f neuro m e a su re s: P E N T B , P a re n tin g S tre s s in d e x (P S I), P e rso n a lity in ve n to ry fo r C h ild re n (P IC ), V in e ia n d A d a p tiv e B e h a v io r S c a le s (V A B S) S h e lt o n e t al. (2 0 1 4 ) C alifo rn ia , U SA C H A R G E S tu d y (3 ye ar-o id ch ild re n ) C ase -co n tro l, large sa m p le size N = 970 Age 3 years P ro xy in d ica to r of p re n a ta l O P p e sticid e e xp o su re (re sid e n tia l p ro xim ity to a g ricu ltu ra l p e stic id e a p p lica tio n s d e fin e d u sin g e co lo g ica l p e stic id e u se d a ta ) C lin ica ! o u tc o m e s - v a lid a te d w ith in th e stu d y Confounder/ Covariate Control A p p ro p ria te : In co m e , race , site te rm , e th n icity, m o th e r's u s e o f c h e m ic a is a t w o rk, m o th e r h e a lth /p re g n a n c y co n d itio n s, re p o rt th a t ch ild h a d le a d or m e rc u ry p o is o n in g (M S only): R a w s c o re s w ere ch ild a g e -a d ju ste d A p p ro p ria te a d ju s tm e n t fo r d e m o g ra p h ic s (p ia c e o f birth, race ), S E S in d ic a to rs (p a te rn a ! e d u c a tio n ) an d vita m in in ta k e d u rin g p re g n a n cy Statistical Analysis A p p ro p ria te : Lin e a r re g re ssio n (co n tin u o u s sc o re s), lo gistic re g re ssio n (d ich o to m o u s sc o re s) A p p ro p ria te m u ltiv a ria te a n a ly s is . N = 4 8 6 (A SD ) Risk of (other) Bias S e le c tio n b ia s p o ssib le d u e to io ss o f fo llo w -u p : R e s id u a l c o n fo u n d in g iikeiy; s u b s ta n tia l p o te n tia l fo r d iffe re n tial e xp o su re m is c ia s s ific a tio n (tim e b e tw e e n sp ra y in g an d te stin g , fre q u e n cy a n d d u ra tio n u n k n o w n ) S e le c tio n b ia s p ro b a b le ; R e s id u a l c o n fo u n d in g iikeiy; s u b s ta n tia l p o ten tial fo r d iffe re n tia l m isc ia ssifica tio n of e xp o su re . O u tco m e m is c ia s s ific a tio n u n lik e ly. N = 1 6 8 (D D ) S u a r e z -L o p e z e t al. (2 0 1 2 ) S u a r e z -L o p e z e t al. (2 0 1 3 a) S u a r e z -L o p e z e t al. (2 0 1 3 ) W a n g e t a l. (2 0 1 2 ) Z h a n g e t al. (2 0 1 4 ) Pedro M oncayo County, P ich in ch a , E cu a d o r Pedro M oncayo County, P ich in ch a , E cu a d o r S e c o n d a ry E x p o su re to P e stic id e s A m o n g in fan ts, C h ild re n an d A d o le sc e n ts (E S P iN A ) Stu d y , p lu s new vo lu n te e rs S e c o n d a ry E x p o su re to P e stic id e s A m o n g in fan ts, C h ild re n an d A d o le sc e n ts (E S P iN A ) Stu d y , p lu s new vo lu n te e rs N = 3 1 6 (C o n tro ls) C ro ss-se ctio n a l, m o d e rate s a m p le s iz e , fro m p rio r c e n s u s N=277 E x p o s e d : (n = 1 5 8 ) U nexposed: (n = 1 1 9 ) Age 4-9 years C ro ss-se ctio n a l, m o d e rate s a m p le s iz e , fro m p rio r c e n s u s N=307 Age 4-9 years Pedro M oncayo County, P ich in ch a , E cu a d o r S e c o n d a ry E x p o su re to P e stic id e s A m o n g in fan ts, C h ild re n an d A d o le sc e n ts (E S P IN A ) Stu d y , p lu s new vo lu n te e rs C ro ss-se ctio n a l, m o d e rate s a m p le s iz e , fro m p rio r c e n s u s N=271 Age 4-9 years S h a n g h a i, C h in a Pregnant w om en and n e w b o rn c h ild re n C ro s s -s e c tio n a l B irth C o h o rt Stu d y N=187 Age: new borns S h e n y a n g , C h in a N e w b o rn c h ild re n in a birth co h o rt s tu d y P ro sp e ctiv e C o h o rt, n = 2 4 9 m o th e r-in fa n t p a irs Age: new borns Proxy: C o h a b ita tio n w ith flo w e r w o rk e r > ly r , A d u it q u e stio n n a ire N o n -sp e cific m e a su re s: C h ild A C h E le v e l u s in g c o m m e r c ia l kit: Proxy: C o h a b ita tio n w ith flo w e r w o rk e r > ly r , A d u it q u e stio n n a ire N o n -sp e cific m e a su re s: C h ild A C h E le ve l u s in g c o m m e rc ia l kit C h ild A C h E le ve l u s in g c o m m e rc ia l kit A p p ro p ria te : S e x , ag e , h e ig h t-fo r-a ge , h e m o g lo b in c o n c e n tra tio n , in co m e , p e stic id e u s e w ith in h o u s e h o ld iot, p e s tic id e u s e b y n e ig h b o rs, e x a m in a tio n d a te, re sid e n ce , d ista n ce to n e a re stflo w e r p lan ta tio n H e a rt rate (H R ), S y s to lic blo o d p re ssu re (S B P ), D ia s to lic blo o d p re ssu re (D B P ) by stan d ard m ethods A p p ro p ria te : S e x , ag e , h e ig h t-fo r-a ge , h e m o g lo b in c o n c e n tra tio n , in co m e , p e stic id e u s e w ith in h o u s e h o ld iot, p e s tic id e u s e b y n e ig h b o rs, e x a m in a tio n d a te, re sid e n ce , d ista n ce to n e a re stflo w e r p lan ta tio n N E P S Y -I! te s t (tra in e d e x a m in e rs): gen eral a sse ssm e n t battery M o d e l d e fin e d a prio ri: h e m o g lo b in , a g e , se x, race , h e ig h t-fo r-a g e z sco re , h o u se h o id in co m e , m a te rn a l e d u c a tio n , an d flo w e r w o rke r co h a b ita tio n sta tu s O b je ctiv e b io m a rk e r o f p re n a ta l O P p e s t ic id e e x p o s u r e (D A P ) in s in g le m a te rn a i s p o t u rin e s a m p le p ro vid e d at o n se t of lab o r. S im p le im p u tatio n of o b se rv a tio n s < LLO D . O P p e sticid e e x p o s u re a ls o a s s e s s e d via q u e stio n n a ire a d m in iste re d afte r d e liv e ry B io m a rk e r of p re n ata l O P p e sticid e e x p o s u r e (D A P ) q u a n tifie d in s in g le m a te rn a i s p o t u rin e s a m p le p ro vid e d at d e live ry. M a n y o b s e rv a tio n s < LLO D G e sta tio n a l a g e a n d p re-term d e liv e ry a p p ro p ria te ly d e fin e d a n d a s s e s s e d u sin g m e d ica l re co rd s A p p ro p ria te . In c lu d e d m a te rn a l a n th ro p o m o rp h ic, d e m o g ra p h ic, a n d S E S in d ica to rs (in co m e , o ccu p a tio n ), p re d icto rs of h ig h -risk p re g n a n cy (p re g n a n c y w e ig h t ga in , g e sta tio n a l age) N e o n a ta l n e u ro d e v e io p m e n t a s s e s s e d u sin g v a lid a te d in stru m e n t (N e o n a ta l B e h a v io ra l N e u ro lo g ica l A s s e s s m e n t) by tra in e d e x a m in e rs A p p ro p ria te , in clu d e d m a te rn a l d e m o g ra p h ic a n d S E S in d ic a to rs (a ge , e d u c a tio n ), p re d icto rs o f h ig h -risk p re g n a n c y (B M I, g e s ta tio n a l a g e ) an d e n v iro n m e n ta l to x ic a n t e xp o su re (cord b io o d lea d ); R e s u lts s tra tifie d by s e x A p p ro p ria te : M u ltip le iin e a r re g re s sio n (c o n tin u o u s) a n d lo g istic re g re ssio n (p o ly ch o to m o u s v a ria b le s), a d ju ste d m o d e ls A p p ro p ria te : M u ltip le iin e a r re g re s sio n (co n tin u o u s), a d ju ste d . L o g istic m o d e ls (d ich o to m o u s an d p o ly c h o to m o u s) a n d iin e a r re g re ssio n m o d e ls, a d ju ste d ; E ffe ct m o d ifica tio n a c c o rd in g to s e x a m o n g sig n ifica n t a s s o c ia tio n s ; S o m e im p u te d v a lu e s A p p ro p ria te m u ltiv a ria te a n a ly s is S e le c tio n b ia s p o ssib le d u e to lo ss b u t d e e m e d low : R e s id u a ! c o n f o u n d in g iik e iy s m a ll in m a g n itu d e ; p o te n tial for d iffe re n tial e xp o su re m is c ia s s ific a tio n S e le c tio n b ia s p o ssib le d u e to lo ss b u t d e e m e d low : R e s id u a ! c o n f o u n d in g iik e iy s m a ll in m a g n itu d e ; p o te n tial for d iffe re n tial e xp o su re m is c ia s s ific a tio n S e le c tio n b ia s p o ssib le d u e to io ss b u t d e e m e d low : R e s id u a ! c o n f o u n d in g iik e iy s m a ll in m a g n itu d e ; p o te n tial for d iffe re n tial e xp o su re m is c ia s s ific a tio n S e le c tio n b ia s u n likely; R e s id u a l c o n f o u n d in g iik e iy s m a ll in m a g n itu d e ; p o te n tial for d iffe re n tial m isc ia ssifica tio n of exposure A p p ro p ria te m u ltiv a ria te a n a ly s is S e le c tio n b ia s u n likely; R e s id u a l c o n f o u n d in g iik e iy s m a ll in m a g n itu d e ; c o n sid e ra b le p o ten tial fo r n o n -d iffe re n tia! m isc ia ssifica tio n of e xp o su re (u n lik e ly to a c c o u n t fo r non-nui! fin d in g s) Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 67 ED 002061 00044425-00067 2.2.5.3 "Low" Quality Group Seventeen articles were assigned a low quality rating, as shown in Appendix 3. In general, these were small or pilot studies; exposure assessment was based on a proxy measure(s), outcome measurement(s) utilized screening tools or self-report, limited statistical analyses were performed, relevant covariates were not included or discussed, or risks of bias were possible. For example, Acosta-Maldonado et al. (2009) conducted a cross-sectional pilot study among 54 women, only nine of whom were considered to have had prenatal exposure to pesticides (defined based on either an exposure history profile or AChE level in blood sampled at the time of admission into the hospital for delivery. The outcome assessed in this study was a standardized but partially subjective assessment of placental maturity (the Placental Maturity Index, PMI). Covariates adjustment of estimated associations between prenatal exposure and PMI in this study was minimal and limited to placental characteristics. Cartier et al (2016) is from the PELAGIE prospective cohort study and used DAP biomarkers as the exposure biomarkers. However, despite these strengths, the urine samples were collected within the home instead of a laboratory or clinic and were returned via the local mail, which could have potentially led to several sampling errors due to inadequate collection and storage methods. The remaining sections of this document do not discuss further studies identified in the 'low' category. Due to limitations in these studies, they do not provide reliable information evaluating associations between OP exposure and neurodevelopmental outcomes. 2.2.6 Assessment of Epidemiological Studies for Relevance to Analysis Using the criteria summarized in Section 2.2.4, a total of 47 literature articles were identified in the 2015 literature review and were judged as high, moderate, or low quality. Overall, 15 articles, 15 articles, and 17 articles were judged to be of high, moderate, or low quality, respectively. For the 30 high and moderate quality studies, additional evaluation was conducted as described in this section. While all of the moderate quality studies had strengths including sample design and outcome assessment, six of these moderate quality studies did not have sufficient exposure assessment methods to determine whether exposure to OPs actually occurred. These studies were conducted on study populations in Spain (Llop et al., 2013), Ecuador (Handal et al., 2007; 2007b; 2008), Denmark (Andersen et al., 2015), and France (Petit et al., 2010). In these studies, participants were considered exposed or unexposed to pesticides based on non-specific exposure measures, such as self-reported occupational exposure, home pesticide spraying, and proportion of municipality devoted to agricultural activity. For all of these proxy exposure assessments, the pesticides used may have included not only OPs, but also pyrethroids, fungicides, and growth regulators. Given the uncertainty about whether OP exposure actually occurred in these studies and whether observed outcomes are associated with OP exposure or with other pesticides, these studies are excluded from further analysis. The focus of this epidemiological literature review is on the neurodevelopmental outcomes from exposures to low levels of OPs (i.e. below exposures which would result in 10% or more AChE inhibition). Three studies conducted in Ecuador focused on child AChE inhibition and the 68 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00068 potential association of AChE inhibition with other measures including parental occupation (Suarez-Lopez et al., 2012), as well as clinical autonomic nervous system (ANS) outcomes such as blood pressure and heart rate (Suarez-Lopez et al., 2013a), and neurodevelopmental outcomes (Suarez-Lopez et al., 2013). The range of AChE activity levels are lower in the first tertile (range of 1.44 to 2.93 U/mL) compared to the third tertile (range from 3.33 to 4.69 U/mL). Therefore, due to the outcomes assessed and the potentially toxic cholinergic effects that were associated with these outcomes, these studies are not considered to be relevant to this review and are not discussed further here. In a retrospective cohort study of children exposed to methyl parathion (MR) before age 6 years in Mississippi and Ohio (Ruckart et al. 2004), as assessed by household urinary PNP or wipe samples, exposed children performed worse than unexposed children on a few of numerous neurobehavioral development tests conducted. Specifically, participants classified as having had MP exposure had more difficulty with short term memory and attention tasks, and parents reported more behavioral and motor skill problems, relative to unexposed children. However, upon closer inspection of the results across the MS and OH study sites, these neurobehavioral outcomes are not seen consistently. These inconsistencies may be due to differences in how the exposure occurred across the sites, including the fact that MS participants and OH participants were tested 2.5 and 4.5 years after MP spraying in the home. When comparing exposed and unexposed children using general intelligence testing, integrated visual and motor skills testing, and multistep processing, they did not see any differences. The exposure scenario associated with these observations is a critical element in assessing the utility and reliability of this study. Samples were collected in locations from OH and MS where illegal spraying of methyl parathion is known to have occurred during the 1994-1996 time period. Based on the "Revised Organophosphorous Pesticide Cumulative Risk Assessment" (USEPA, 2006), methyl parathion is known to be among the more potent OPs. It is unknown whether study participants were exposed to MP levels that would have induced cholinergic effects. Therefore, given the uncertainty around this illegal use combined with the high potency for cholinergic toxicity, the agency is not emphasizing this study further in the analysis. In addition, two high quality studies from the CHAMACOS birth cohort (Quiros-Alcala et al. 2011; Ranaan et al., 2015) assessed the association between DAPs and autonomic nervous system (ANS) outcomes and respiratory symptoms, respectively. The ANS outcomes assessed included heart rate and respiratory sinus arrhythmia. Overall, while there was some evidence of ANS dysregulation for infants at 6 months, these results were not consistently observed for the other assessed child (1 year, 3.5 years, and 5 years) and maternal OP exposures. Ranaan et al (2015) reported that total DAPs and DEAP metabolites in urine from the second half of pregnancy were significantly associated with increased odds of respiratory symptoms in children. Ranaan et al. I (2015) also reported exercise-induced coughing at 5 and 7 years of age was significantly associated with total DAPs, DEAPs, and DMAPs in children's urine collected between the ages of 6 months and 5 years (AUC). The authors concluded that early-life exposure to OP pesticides was associated with respiratory symptoms consistent with possible asthma in childhood. There is not a body of literature to compare these results against, making it difficult to put them into context. Furthermore, these studies did not focus on 69 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00069 neurodevelopmental outcomes, which is the focus of this analysis. Consequently, Quiros-Alcala et al. (2011) and Raanan et al. (2015) are not being evaluated further. 2.2.7 Birth Outcome Epidemiologic Studies Four identified studies, three high quality and one moderate quality, focused solely on OP exposure and adverse birth outcomes related to fetal growth (Barr et al., 2010; Rauch et al., 2012; Wang et al., 2012; Wolff et al., 2007). Harley et al (2016) is a meta-analysis of data from the HOME, Mt. Sinai, CCCEH, and CHAMACOS cohorts which allowed for a better assessment of race and genetic variability given the larger sample size in the pooled analysis. The birth outcomes assessed included birth weight, birth length, head circumference at birth, and gestational age. The exposure assessment for these studies was conducted using objective measures or biomarkers such as maternal urinary DAPs (Rauch et al., 2012; Wang et al., 2012; Wolff et al., 2007), maternal urinary malathion (MDA) (Wolff et al., 2007), or maternal and cord blood levels for specific OPs (Barr et al., 2010). The results from these studies are generally inconsistent, with some studies documenting statistically significant associations between OP exposure and birth outcomes whereas others do not. For example, maternal and cord blood serum levels of TCPy were inversely associated in univariate analyses conducted among participants in a prospective cohort study conducted in New Jersey (Barr et al. 2010), though the associations did not persist after adjusting for gravidity, maternal pre-pregnancy BMI, infant sex, and gestational age. Chlorpyrifos levels were near the lower limit of detection in this study, though detectable in 98.6% of maternal serum and 62.8% of cord serum samples. Overall, birth length was not associated with third-trimester DAP in the Mount Sinai Cohort (Wolff et al. 2007). However, among those with slow-activity paraoxonase-1 (PON1) or PON 192, urinary total DMP (but not total DAP or DEP) was statistically significantly associated with shorter birth length (p= 0.032). Birth length was also not associated with maternal urinary DAP sampled at delivery in the cross-sectional investigation conducted in Shanghai, China (Wang et al. 2012). Similarly, inconsistent results were observed across these studies for birth weight, gestational age, and head circumference at birth. For the pooled analysis in Harley et al (2016), no evidence of association was observed between OPs exposure via the DAP, ]>DEP, and ]>DMP urinary metabolites and birth outcomes for birth weight, birth length, and head circumference, as all of the 95% CIs encompassed the null value of 0 and all of the p-values were > 0.05 (p-values ranged from 0.46 to 0.98), respectively. For the interaction analysis, no evidence of a statistically significant interaction between the four cohorts and exposure to urinary metabolites (JDAP, DEP, and DMP) relative to the birth outcomes (birth weight, birth length, and head circumference) were observed since all of the p for interaction values were above > 0.10, respectively. A statistically significant interaction was observed between the PON1-108 genotype (specifically the PON1-108CC gene) and the urinary metabolite JDEP relative to pooled birth weight (p = 0.08), and between the PON1-108 genotype (specifically the PON1-108CC genes) and the urinary metabolites DAP and DMP relative to pooled birth length (p for interaction = 0.07; p = 0.02) among children. With the PON1192 genotype, a statistically significant interaction was observed between the urinary 70 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00070 metabolite ]>DAP relative to pooled head circumference (p for interaction = 0.05). For mothers, a statistically significant interaction was observed between the PON1-108 genotype and the urinary metabolites DAP and DMP relative to pooled head circumference (p for interaction = 0.09 for both), respectively. No additional statistically significant interactions were observed for any other genotypes among children and mothers, and no statistically significant interactions were observed for race, sex or maternal education. Overall, in this 2015/2016 literature review, inconsistent evidence of OP exposure and association with adverse birth outcomes/fetal growth was observed (Barr et al., 2010; Rauch et al., 2012; Wang et al., 2012; Wolff et al., 2007). This lack of consistency in the literature was also observed for birth outcomes in the recent chlorpyrifos HHRA (USEPA, 2014) which notes that researchers from CCCEH, Mt. Sinai, and CHAMACOS also investigated the possible role of prenatal OP exposure and fetal growth. These results were not consistent across these cohorts. Authors with CCCEH observed evidence of an inverse association, i.e., increasing cord blood chlorpyrifos was associated with decreased measures of birth weight and length, while authors with the Mt. Sinai and CHAMACOS cohorts reported either no association, or evidence of a positive relationship, respectively (Berkowitz et al., 2004; Eskenazi et al., 2004; Whyatt et al., 2004). Inconsistent results may be due to differences across study groups in exposure profiles as well as dissimilar methods of prenatal OP exposure assessment (Needham, 2005). Given the lack of consistency among cohorts for the fetal growth metrics, the proposed link between fetal growth and OP exposure is tenuous. Therefore, consistent with previous evaluations for chlorpyrifos, EPA is focusing the remainder of this document on neurodevelopmental outcomes. Although the agency is not evaluating these birth outcome studies further at this time, the agency will continue to monitor the scientific literature for advances in this line of research. 2.2.8 Neurodevelopment Outcome Epidemiologic Studies From an initial total of 30 high or moderate quality studies, with the exclusion of six studies for insufficient exposure assessment, three studies with measurable AChE inhibition and potential cholinergic toxicity which are outside the scope of this analysis, one study from an illegal use of a highly potent OP (MP) where cholinergic toxicity cannot be ruled out, five studies with birth outcome as the only assessment, two with outcomes not related to neurodevelopmental outcomes, and one study assessing only PON1 genotype expression and neurobehavioral outcomes (Fortenberry et aI, 2014a), a total of twelve studies from the 2015/2016 literature review focusing on neurodevelopmental outcomes remain to be evaluated. Eight are considered high quality (Bouchard et al., 2010; Fortenberry et al., 2014; Furlong et al., 2014; Donauer et al. (2016), Stein et al. (2016), Rauh et aI, (2015), Engel et al (2015), and Yolton et al. (2013). Four are considered moderate quality studies (Guodong et al., 2012; Oulhote and Bouchard, 2013; Zhang et al., 2014; Shelton et al., 2014). Furlong et al (2014) is from the Mt. Sinai cohort study; Stein et al (2016) is from CHAMACOS, and Rauh et al (2015) is from CCCEH-- thus these studies share many of the same strengths and Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00071 uncertainties as other studies from these cohorts. Most of these studies used biomarker measures for their exposure assessment, including child or maternal urinary DAPs (Bouchard et al., 2010; Furlong et al., 2014; Guodong et al., 2012; Oulhote and Bouchard, 2013; Zhang et al., 2014; Yolton et al, 2013; Donauer et al, 2016; Stein et aI, 2016, Engel et al, 2015), child urinary DMP and DEPs (Oulhote and Bouchard, 2013; Zhang et al., 2014; Guodong et al., 2012), or maternal TCPy (Fortenberry et al., 2014). Shelton et al. (2014) used pesticide use data from the California Department of Pesticide Regulation and geospatial methods to map the specific pesticide use pattern to the participant residence. Engel et al. (2015) reports on the results of a pooled analysis from HOME, CCCEH, CHAMACOS, and Mt. Sinai to evaluate the association between prenatal urinary DAPs and neurodevelopmental outcomes at 24 months only. The study populations include national in scope (Bouchard et al., 2010; Oulhote and Bouchard, 2013); mainly urban (Fortenberry et al., 2014; Fortenberry et al., 2014a; Furlong et al., 2014; Guodong et al., 2012; Zhang et al., 2014; Rauh et al, 2015, Shelton et al., 2014); suburban (Yolton et al, 2013; Donauer et al, 2016); and agricultural (Stein et al, 2016). 2.2.8.1 CCCEH, CHAMACOS, & Mt. Sinai Cohorts The CCCEH, Mt. Sinai, and CHAMACOS studies reflect different types of exposed groups in the total population which strengthens the weight of the evidence considerations regarding this stream of information. The CCCEH Mother's and Newborn study and the Mt. Sinai Child Growth and Development study participants were likely exposed to OPs through the diet and through residential use of the pesticide for indoor pest control. In the residential setting, study populations were most likely exposed through indoor residential use of the pesticide during the study time period and additionally exposed to OPs via the oral route through ingesting residues in the diet and from hand-to-mouth contact with in-home surfaces, as well as possible dermal or inhalation exposure through contact with treated areas in the home environment (Berkowitz et al., 2003; Whyatt et al., 2003; Whyatt et al., 2009; Whyatt et al., 2007). In contrast, CHAMACOS cohort participants were employed as farm laborers or were residing in homes with farm laborers. The CHAMACOS study participants likely experienced exposure to OPs through the diet and from occupational exposure (primarily inhalation and dermal routes), as well as probable indirect take-home exposures (the "tracking in" of pesticide residues through shoes and clothing, augmented by poor hygiene practices) (Bradman et al., 2007). In each of these three US children's health cohorts, the biological measurements in these cohorts were comparable to the general population NHANES. These cohort studies each enrolled pregnant women during roughly the same time period, measured both environmental exposure to the pesticide during pregnancy and also measured biomarkers representing internal dose during pregnancy and at delivery, and prospectively assessed associations in their newborns and young children through age 7 years. Each study includes several hundred (approximately 100-400) mother-infant pairs; these sample sizes are 72 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00072 sufficient to perform statistically valid analyses. Investigators from each study cohort utilized a similarly strong study design (prospective birth cohort); measured pesticide exposure using several different methods including environmental indicators as well as specific and non specific biomarkers of OPs; ascertained developmental outcomes using validated assessment tools well-established in both clinical and research settings; and, measured, analyzed, selected and statistically adjusted for potentially confounding variables including socio-economic status and other environmental exposures using reasonable and appropriate methods. Limitations exist as well. These studies utilized a one-time measure (or the average of two measures) of chlorpyrifos or OP exposure to assess prenatal pesticide exposure throughout the gestational period, were unable to assess the influence of mixtures (co-occurring exposures in the relevant biological time window), and reflect a small sample size to fully evaluate the effect of more than one simultaneous exposure on neurodevelopment, i.e., evidence of effect modification. As noted, two major uncertainties in environmental epidemiology studies are the accurate and reliable measurement of exposure and potential confounding variables such as the influence of mixtures. The researchers with each of the three cohorts have provided supplemental methodological research to address these areas to the extent possible. Across the three children's health cohorts, study authors measured biomarkers of OP exposure. There is uncertainty as to the extent measurement of non-specific metabolites of OP or chlorpyrifos accurately reflects OP exposure; CCCEH and Mt. Sinai studies do not estimate post-natal exposure to chlorpyrifos among child participants, therefore the influence of early life and childhood OP exposure is unaccounted for in these analyses. The CHAMACOS cohort measured urinary levels of DAPs in young children and with the exception of pervasive developmental disorder (PDD) [per 10-fold increase in prenatal and post-natal DAP [prenatal: odds ratio (OR) = 2.3, p = 0.05; child DAPs OR = 1.7, p = 0.04], did not observe negative significant associations in relation to neurodevelopment from post-natal exposure (Eskenazi et ai, 2007; Marks et al, 2010). The CHAMACOS cohort investigators also measured AChE and butyl ChE as supplemental indicators of OP exposure. Potential confounding bias is another major uncertainty within environmental epidemiology studies. Confounding variables, exposures that could be related to OP exposure and neurodevelopmental outcomes such as blood lead, may result in an incorrect epidemiological risk estimate. Across these cohort studies, investigators collected relevant information concerning demographic characteristics and other environmental exposures, and were, to the extent possible with the existing information, able to effectively hold constant the influence of these other variables when estimating the association between prenatal chlorpyrifos and adverse neurodevelopmental outcomes. Control of these variables is important to reduce the chances of a false positive study result. Overall, statistical analyses were judged to be appropriate and reasonable (not overly large number of statistical model variables) to the research question by EPA and expert Panel reviews (FIFRA SAP 2008 and 2012). Researchers with both the Mt. Sinai and CHAMACOS cohorts evaluated neonatal neurological functioning in association with prenatal OP exposure; CCCEH did not conduct these measurements. To measure indices of abnormal neonatal behavior and/or neurological 73 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00073 integrity authors used outcome measures derived from the Brazelton Neonatal Behavioral Assessment Scale (BNBAS), a neurological assessment of 28 behavioral items and 18 primitive reflexes. This tool was administered to infants 2-5 days post-partum by trained neonatologists in the hospital setting using similar environmental conditions. The authors with both study groups observed an increased number of abnormal reflexes in relation to increasing measures of OP exposure (Engel et al., 2007; Young et al., 2005). Among the other 27 measures in the BNBAS, neither study group reported evidence of any other positive associations. The authors also observed evidence of potential effect modification by PON1 activity level in the relation between DAPs and neonatal neurodevelopment in which infants of mothers who are slower metabolizers have greater risk of abnormal reflexes (Young et al. 2005; Engel et al. 2007). However, EPA notes these studies are likely under-powered to make a statistically robust estimate of this statistical interaction. Researchers across the three children's health cohorts utilized the Bayley Scales of Infant Development II (BSID-II) to generate a Mental Development Index (MDI) and a Psychomotor Development Index (PDI) to assess neurodevelopment in early childhood. In the CCCEH Mothers and Newborn study, Rauh et al. (2006) investigated MDI and PDI at 12, 24, and 36 months of age. Children were categorized as having either high (>6.17pg/g) or low (<6.17pg/g) prenatal chlorpyrifos exposure, using categories informed by results of the previous study on birth characteristics (Whyatt et al., 2004). Authors reported that the difference in MDI scores was "marginally significant" (p = 0.06) between the "high" and "low" exposed groups; the high exposed group scoring an average of 3.3 points lower than the low exposed (Rauh et al., 2006). Regarding the PDI score (motor skills), none of the 12 or 24 month PDI scores showed significant effects, but the 36 month score was significantly related to chlorpyrifos exposure. Researchers noted that the effects were most pronounced at the 36 month testing period. CCCEH study authors (Rauh et al., 2015) evaluated the relationship between prenatal chlorpyrifos exposure and motor development/movement among 271 of the cohort participants who had reached the age of approximately 11 years. When comparing children in the upper tertile of exposure (>6.17 pg/g; N=43) to those in the lower tertiles (N=228), they observed statistically significant associations between prenatal chlorpyrifos exposure and mild to moderate tremor in the dominant arm, both arms, either arm, and a marginally statistically significant association in the non-dominant arm. The specific OR calculated associated with these elevated risks of arm tremor are as follows: dominant arm (OR=3.2; 95% CM .3-8. I; p=0.0 15); both arms (OR=3.3 ; 95% CM .1-9.4; p=0.027); either arm (OR=2.2; 95% CM .1-4.6; p=0.028); and non-dominant arm (OR=2.1; 95% 0=0.99-4.3; p=0.055). These associations were observed even after controlling for potential confounding factors such as medication, sex, and ethnicity. Within the 36 month testing period, the likelihood of highly exposed children developing mental delays were significantly greater (MDI: 2.4 times greater (95% Cl: 1.12-5.08, p = 0.02) and PDI: 4.9 times greater (95% Cl: 1.78-13.72; p = 0.002)) than those with lower prenatal exposure (Rauh et al., 2006). Within the Mt. Sinai study, authors administered the BSID-II to participating children at 12 and 24 months and observed that prenatal total DAP metabolite 74 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00074 level was associated with a decrement in mental development at 12 months among blacks and Hispanic children; however, these associations either attenuated or were non-existent at the 24-month visit (Engel et al., 2011). In the CHAMACOS cohort, Eskenazi et al. (2007) observed that prenatal DAP levels were adversely associated with MDI, and at 24 months of age these associations reached statistical significance. In this study, neither prenatal DAPs nor maternal TCPy were associated with PDI (motor skills), nor did authors observe evidence of different risk by PON1 status (Eskenazi et al., 2010). With respect to the findings related to the autism spectrum, from CCCEH, Rauh et al. (2006) reported a large odds ratio for PDD (OR=5.39; 95% Cl: 1.21-24.11) when comparing high to low chlorpyrifos exposure groups. Among 7-9 years old children in the Mount Sinai Cohort (Furlong et al. 2014), there was no overall statistically significant association between maternal third trimester urinary DAP metabolite levels and reciprocal social responsiveness. However, some evidence of modification of the association between prenatal OP pesticide exposure and impaired social responsiveness in early childhood was observed by both race/ethnicity and child sex, with an association between DEAP and poorer social responsiveness observed among black participants and boys. No association was observed among whites or Hispanics, among girls, or for DAP or DMAP biomarker levels. In the CHAMACOS cohort, Eskenazi et al. (2010) reported non-significant, but suggestive, increased odds of PDD of 2.0 (0.8 to 5.1; p=0.14), whereas Eskenazi et al (2007) reported a statistically significant association between total DAP exposure and increased odds of PDD. With respect to attention problems, Rauh et al. (2006) also investigated 36-month child behavior checklist (CBCL) (behavioral) scores. Significant differences were observed between the high and low chlorpyrifos exposure groups in the general category of attention-problems (p=0.010), and in the more specific DSM-IV scale for ADHD problems (p = 0.018). The CHAMACOS cohort also investigated attention problems in early childhood using three different assessment tools: maternal report of child behavior at 3.5 and 5 years of age; direct assessment of the child at 3.5 and 5 years; and by a psychometrician's report of the behavior of the child during testing at 5 years. In this study population, higher concentrations of OP metabolites in the urine of pregnant women were associated with increased odds of attention problems and poorer attention scores in their children at age 5 years (Marks et aI, 2010). To measure intelligence among school aged children, authors from each of the three children's health cohorts used the Wechsler Intelligence Scale for Children, 4th edition (WISC-IV). The instrument measures four areas of mental functioning: the Verbal Comprehension Index, the Perceptual Reasoning Index, the Working Memory Index, and the Processing Speed Index. A Full-Scale IQ score combines the four composite indices. WISC-IV scores are standardized against U.S. population-based norms for English and Spanish-speaking children. In the CCCEH Mothers and Newborn Study, Rauh et al. (2011) evaluated the relationship between prenatal chlorpyrifos exposure and neurodevelopment among 265 of the cohort participants who had reached the age of 7 years and had a complete set of data including prenatal maternal interview data, prenatal chlorpyrifos marker levels from maternal and/or cord blood samples at delivery, postnatal covariates, and neurodevelopmental outcome data (Rauh et al., 2011). 75 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00075 While models were developed using continuous measures of both prenatal chlorpyrifos exposure and Wechsler scores, for ease of interpretation, investigators reported that for each standard deviation increase in exposure (4.61 pg/g) there is a 1.4% reduction in Full-Scale IQ and a 2.8% reduction in Working Memory. In the Mt. Sinai study, prenatal maternal DEP urinary metabolite concentrations were associated with slight decrements in Full Scale Intelligence Quotient (FSIQ), Perceptual Reasoning, and Working Memory between the ages of 6 and 9 years, and difference in intelligence measures by putative PON1 status were also noted (Engel et al., 2011). Similarly, in the CHAMACOS cohort, Bouchard et al. (2011) observed evidence of an association between prenatal exposures to OPs as measured by urinary DAP (total DAP, DEP, and DMP) metabolites in women during pregnancy, and decreased cognitive functioning in children at age 7. In this study, children in the highest quintile of maternal DAP concentrations had a statistically significant 7-point difference in IQ points compared with those in the lowest quintile. To determine if early childhood social adversities modify the association between maternal prenatal OP exposure and cognition in children (measured using the WISC-IV results), Stein et al. (2016) showed negative associations between maternal prenatal exposure to DAP metabolites and cognition in children were observed to be stronger among children who reported more adversity, relative to children who experienced less adversity. For example, for boys who experienced a more adverse learning environment, a statistically significant decrease was observed between prenatal DAP concentrations and Full-Scale IQ ((3 = -13.3; 95% Cl: -19.9; 6.7; p < 0.01), relative to boys who lived in a less adverse learning environment ((3 = 4.2; 95% Cl: -4.2; 12.5; p < 0.01). A similar observation was reported for perceptual reasoning and processing speed ((3 = -9.8; 95% Cl: -17.4; -2.2; p < 0.01; (3= -12.7; 95% Cl: -19.5; -6.0; p = 0.01), respectively. For girls who experienced more economic adversity, a stronger effect on the association between prenatal DAP concentrations and Full-Scale IQ was observed, relative to girls who experienced less economic adversity ((3 = -8.5; 95% Cl: -16.7, -0.4 vs. (3= -4.7 95% Cl: 12.8, 3.4; p = 0.18). This was also observed for working memory for girls ((3 = -5.9; 95% Cl: 13.7, -1.8 vs. P = -2.6 95% Cl: -10.0, 4.8; p = 0.05). In addition, similar findings were observed for girls whose mother's had more maternal adversity (|3= -11.5; 95% Cl: -18.5, -4.4 vs. |3= -1.5 95% Cl: -7.9, 4.9; p = 0.07). To ascertain whether observed differences in neurodevelopment after prenatal chlorpyrifos exposure may be explained by differences in brain morphology between exposed groups, investigators compared MRI brain images between high and low chlorpyrifos exposed child study participants (Rauh et al., 2012). Authors determined there were distinct morphological differences in brain areas associated with these neurodevelopmental outcomes. The pilot study included 40 child participants due to strict inclusion and exclusion criteria, and the high cost of performing the imaging studies on each child. EPA convened a Federal Panel of experts to perform a written peer-review of this study.5The Federal Panel concurred with the authors' conclusions in general; however, the Federal Panel also noted that significantly greater and more sophisticated MRI imaging studies would be needed to link the morphological changes 5 MtPj//wwwT6&yIatL9.D.5.g^ 76 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00076 indicated in this study with specific functional outcomes noted in the CCCEH IQ study. Therefore, while generally supportive of the epidemiologic findings, additional study is needed to make specific links with areas of brain development change. In sum, across these three children's environmental health studies, authors consistently identified associations with neurodevelopmental outcomes in relation to OP exposure. There is evidence of delays in mental development in infants (24-36 months), attention problems and autism spectrum disorder in early childhood, and intelligence decrements in school age children who were exposed to chlorpyrifos or OPs during gestation. Investigators reported strong measures of statistical association across several of these evaluations (odds ratios 2-4 fold increased in some instances), and observed evidence of exposures-response trends in some instances, e.g., intelligence measures. EPA has considered the strengths and limitations of these studies, and believes that random or systematic errors in the design, conduct or analysis of these studies were unlikely to fully explain observed positive associations between in utero OP exposure and adverse neurodevelopmental effects observed at birth and through childhood (age 7 years). EPA believes these are strong studies which support a conclusion that OPs likely played a role in these outcomes. 2.2.8.2 HOME Cohort Two studies from the HOME cohort on neurodevelopmental outcomes have been reported (Yolton et al, 2013; Donauer et al, 2016). The HOME study is a prospective cohort study conducted from March 2003 to February 2006, and the study population included pregnant women living in the Cincinnati, Ohio area who successfully delivered live-born infants. The HOME cohort is a more recent US cohort compared to CHAMACOS, Mt. Sinai and CCCEH and represents exposure from post mitigation activities on many OPs which occurred during tolerance reassessment. Thus, the exposure pattern to the mothers in the HOME cohort and the study demographics (i.e. suburban, middle class study population) are likely different relative to studies from CHAMACOS, Mt. Sinai and CCCEH. Moreover, the demographic data reported in the HOME cohort indicate that mothers tended to have a higher socioeconomic status and were more likely to eat and live a healthier lifestyle relative to mothers involved in CHAMACOS, Mt. Sinai and CCCEH. As a result, these life habits were potentially protective towards the OP exposures during pregnancy. Exposure was assessed twice during pregnancy--at 16 4 weeks and 26 4 weeks of gestation--via spot urine samples that were stored and later laboratory tested. The urine samples were measured for six DAPs. In the Yolton et al (2013) study, a neurobehavioral assessment, the NICU Network Neurobehavioral Scale (NNNS), was conducted at approximately 5 weeks of age, and each infant was scored based on several categories (or subscales) of the NNNS. A total of 350 mother/ infants were included within this study, and a statistically significant association (p < 77 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00077 0.05) between maternal metabolite urinary concentrations (DE and DAP) and the following NNNS subscales for infant performance was observed: attention, lethargy, hypotonia, and autonomic stress. After adjusting the model for covariates, evidence of an association was observed between increased mean urinary DE metabolites and increased attention ((B = 0.066, p < 0.05), with no statistically significant covariates. Decreased lethargy and hypotonia were also associated with increased urinary DE metabolites at 16 weeks of gestation ((3 = -0.069, p = 0.04; P = -0.101, p = 0.03), with black race and birthweight as statistically significant covariates ((3 = 0.462, p = 0.03; (3= -0.044, p = 0.01). For DAP metabolite concentrations, evidence of an association was observed between increased DAP concentrations at 26 weeks of gestation and decreased autonomic stress ((3 coefficient = -0.010, p = 0.01), with birthweight of infants and maternal blood lead levels during gestation as statistically significant covariates ((3 = -0.003, p = 0.02; P = 0.031, p = 0.02), respectively. In Donauer et al, 2016, offspring were assessed through neurodevelopmental examinations conducted yearly, at ages 1-5 years. The Bayley-ll assessed mental and motor advancement for children ages 1-3 years, and the MDI and PDI were used to analyze cognitive, language, and motor development. For 4 year-old children, language skills were assessed by the Clinical Evaluation of Language Fundamentals-Preschool, and the WPPSI was used to assess intelligence (IQ) including verbal, performance, and full-scale for children 5 years of age. Of the 327 maternal-child cases, evidence of a significant association between maternal levels of urinary metabolites (total DAP and total DM) and verbal IQ of the child from the Wechsler Preschool and Primary Scale of Intelligence test was observed in both the unadjusted and adjusted models at the p < 0.2 level (unadjusted model: P = 0.005, p = 0.034, P = 0.003, p = 0.144; adjusted model: P = 0.003, p = 0.179, P = 0.003, p = 0.144), respectively. Although evidence of a positive association was observed for total DAP and total DM metabolites and full-scale IQ (unadjusted model: p = 0.004, p = 0.111; p = 0.004, p = 0.124, when the model was adjusted, no evidence of a positive association was observed (adjusted model: p = 0.001, p = 0.494; p = 0.002, p = 0.441). No evidence of a positive association was observed between performance IQ of the child and total DAP, DM, and DE, respectively. For Bayley-ll MDI, evidence of a positive association was observed for maternal exposure to total DM and total DE at the child's 2-year visit (unadjusted model: P = 0.003, p = 0.16; P = 0.003, p = 0.16); however, when the model was adjusted, no evidence of a positive association was observed for total DM and total DE (adjusted model: P = 0.002, p = 0.28; P = 0.002, p = 0.37). No evidence of a positive association was observed between maternal exposure (for total DAP, total DM, and total DE) and the Bayley-ll Psychomotor Developmental Index for children aged 1-3 years. 2.2.8.3 Meta-analysis (Engel et al, 2015) Engel et al. (2015) reports on the results of a pooled analysis from four cohorts (N=936) to evaluate the association between prenatal DAPs and neurodevelopmental outcomes at 24 months only. In addition, researchers in this study assessed the impact on these associations of the specific cohort, race/ethnicity, and the PON I genotype of study participants. Researchers across the four children's health cohorts utilized the BSID-11 to generate MDI and PDI to assess neurodevelopment in early childhood. The four cohorts include CHAMACOS (N=377), HOME 78 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00078 (N=265), Mt. Sinai (N=234), CCCEH (N=60), It is noted that the CCCEH participants included in this analysis are from women enrolled in 2000 to 2001, a time period which is during the phase out of chlorpyrifos in residential settings. The results of this pooled study are relatively consistent with those seen in the individual cohorts at 24 months. After controlling for race/ethnicity, smoking, and drug use during pregnancy, a statistically significant association was observed in the pooled population between total DAPs exposure and MDI decrements, but not with PDI decrements. Consistent with the results from Eskenazi et al. (2007), the strongest evidence of an association was observed for the CHAMACOS cohort, with statistically significant associations for both total DAPs and total DMAPs exposure and MDI decrements. No significant associations were seen within the Mt. Sinai and CCCEH cohorts, a result which is basically consistent with the previous observations at 24 months in these cohorts (Engel et al., 2011; Rauh et al.,2006). The study authors observed significant heterogeneity from combining the cohorts, especially with regards to race/ethnicity, and noted that impacts on specific subpopulations may be lost when looking at the pooled results. 2.2.8.4 Other Neurodevelopmental Studies In the first of two Chinese studies focusing on generic neurodevelopmental outcomes, Zhang et ol. (2014) investigated prenatal exposure to OPs and neurobehavioral development of neonates in a birth cohort study in Shenyang, China. The authors reported that consistent statistically significant associations were observed between all of the quantified urinary biomarkers of prenatal OP pesticide exposure and neonatal neurodevelopment deficits assessed 3 days after birth. A 10-fold increase in total DAPs concentration was associated with an average decrease in Neonatal Behavioral Neurological Assessment (NBNA) summary scores of 1.78 points (95% Cl: -2.12 to -1.45). No evidence of departure from linearity of the exposure-response relationship between maternal DAP concentrations and NBNA scores was observed. In the second Chinese study focusing on neurodevelopmental outcomes, Guodong et ol. (2012) conducted a cross-sectional study, and did not identify any statistically significant associations between the children's urinary DAP metabolite levels and any of the DQ (Developmental quotients) scores. The authors mentioned that their results should be interpreted with caution since OP exposure was quantified in single spot urine sample from children, and should be followed up with a longitudinal study with repeated measurement of exposure levels in urine samples. Two studies focused on impaired social responsiveness, autism spectrum disorders, or developmental delays (Furlong et al., 2014; Shelton et ol., 2014). Among 7-9 years old children in the Mount Sinai Cohort (Furlong et al. 2014), there was no overall statistically significant association between maternal third trimester urinary DAP metabolite levels and reciprocal social responsiveness (a measure linked to many neuropsychiatric conditions that involve impaired social functioning (Constantino and Gruber 2005)). However, some evidence of modification of the association between prenatal OP pesticide exposure and impaired social 79 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00079 responsiveness in early childhood was observed by both race/ethnicity and child sex, with an association between DEP and poorer social responsiveness observed among black participants and boys. No association was observed among whites or Hispanics, among girls, or for DAP or DMP biomarker levels. Shelton et al. (2014), investigated autism spectrum disorders and developmental delay (DD) in relation to gestational residential proximity to agricultural pesticide applications utilizing the California population-based Childhood Autism Risks from Genetics and Environment (CHARGE) study. The investigators reported that children with autism spectrum disorder were 60% more likely to have OPs applied near the home [1.25 km distance; adjusted OR (aOR) = 1.60; 95% Cl: 1.02-2.51] than mothers of normally developing children. They added that as the buffer distance grew larger, these associations became lesser, indicating an exposure-response effect. The authors also mentioned that each 100-lb (45.4 kg) increase in the amount applied over the course of pregnancy (within 1.5 km of the home) was associated with a 14% higher prevalence of autism spectrum disorder (aOR = 1.14; 95% Cl: 1.0,1.32), but no association was identified with DD. A total of three studies focused on OP exposure and behavioral, memory, or attention/ADHD outcomes (Oulhote and Bouchard, 2013; Bouchard et al., 2010; Fortenberry et al., 2014). In a national cross-sectional study of Canadian children 2007-2009 data for age 6-11 years (Oulhote and Bouchard, 2013), there were no overall statistically significant associations observed between child urinary DAP, DMP, or DEP metabolite levels and parentally reported behavioral problems. In contrast, Bouchard et al. (2010), looking at U.S. children age 8-15 years in the 2000-2004 National Health and Nutrition Examination Survey (NHANES),6observed a positive association between attention and behavior problems and DAPs and DMPs, but not DEPs. For example, even after controlling for potential confounders such as sex, age, ethnicity, and creatinine concentration, they found that a 10-fold increase in DMAP concentration was associated with a 55 to 72% increased odds of ADHD. Fortenberry et al. (2014) evaluated the relationship between pesticide exposure and ADHD in school age Mexican children, recruiting 187 mother-child pairs from a prospective birth cohort, ELEMENT (Early Life Exposures in Mexico to Environmental Toxicants). The authors reported that, there were no statistically significant associations between tertiles of maternal third trimester urinary TCPy and measures of attention and hyperactivity in children. However, there was suggestive evidence for increases in the ADHD index in relation to TCPy tertiles among boys (the highest TCPy tertile was associated with an ADHD index score that was 5.55 points higher than children in the lowest tertile; p-value = 0.06). 3.0 Weight of Evidence Analysis: Integration Across Multiple Lines of Evidence OPP's 2016 "Framework for Incorporating Human Epidemiologic & Incident Data in Risk 6 http://www.cdc.gov/nchs/nhanes.htm 80 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00080 Assessments for Pesticides" provides the foundation for evaluating multiple lines of scientific evidence (U.S. ERA, 2016c). OPP uses a WOE analysis for evaluating epidemiology and human incident data, such that conclusions are made on the preponderance of the information rather than relying on any one study. OPP uses the modified Bradford Hill criteria like those in the MOA/human relevance framework as a tool for organizing and integrating information from different sources (Hill, 1965; U.S. EPA, 1999, 2005; Sonich-Mullin et al., 2001; Meek et al., 2003; OECD AOP Wiki Users Handbook7). It is important to note that the Bradford Hill Criteria are not intended as a check box approach but instead are points to consider when evaluating the totality of evidence. In addition, the availability of a fully elucidated MOA/AOP is a not requirement for using epidemiology studies in human health risk assessment. The Bradford Hill Criteria explicitly considers such concepts as strength, consistency, dose response, temporal concordance and biological plausibility in a weight of evidence analysis; sections 3.1-3.3 below summarize the available evidence based on these principles. The agency's 2002 guidance on "Determination of the Appropriate FQPA Safety Factor(s) in Tolerance Assessment" contains a discussion of the relative weighing of animal and human data with respect to pre- and post-natal toxicity, dose-response, toxicokinetics, and mode of action. The guidance specifically states that "potential for pre- and postnatal toxicity can be determined from human and animal studies. Although human studies are seldom available, human data are the most relevant data for assessing potential health risks. When sufficient human data are available to judge that an adverse developmental outcome is related to exposure, the degree of concern increases (p. 33, emphasis added)." Table 1 of the 2002 FQPA Safety Factor guidance notes that when effects are found in humans related to exposure that these data receive "Increasing Weight". This table lists the following factors as receiving increasing weight in the WOE analysis with respect to pre- and post-natal toxicity: Effects found in humans related to exposure Same types of effects seen in more than one species Effects of a different type with greater potential consequences in young compared to adults Persistence or relatively longer recovery of effects in young compared to adults The agency notes that each of these factors applies to the database of studies for OPs. As described in detail above, epidemiology studies from multiple investigators representing multiple locations and different populations show associations between OP exposure and adverse neurodevelopmental outcomes. Regarding multiple laboratory species in the database of laboratory studies, a range of effects on the developing nervous system has been shown in mouse (e.g., Braquenier et al., 2010; Venerosi et al., 2015), rat (e.g., Carr et al., 2015; 7 https://aopwiki.org/wiki/index.php/Main_Page#OECD_User_Handbook 81 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00081 Vatanparas et al., 2013), and guinea pig (e.g., Mamczarz et al., 2016). Although very high levels of OP exposure can lead to coma, paralysis, or even death, the 10% AChE point of departure represents a precursor event believed to be protective of this downstream cholinergic neurotoxicity. The 10% AChE PoD is much lower than these exposure levels and thus is believed to be health protective of cholinergic neurotoxicity. In contrast, neurodevelopmental effects such as reduced IQ, autism spectrum disorder, and ADHD are adverse health outcomes of significant public health consequence. Moreover, these types of neurodevelopment effects are likely to persist through life. Further evidence of the long term consequence of such exposure is found in Rauh et al (2015) who have shown even at age 11 children exposed to chlorpyrifos in the home environment were more likely to exhibit mild or mild to moderate arm tremor. 3.1 Dose-Response Relationships & Temporal Concordance Since the MOA(s)/AOP(s) is/are not established for neurodevelopmental outcomes (USEPA, 2012, 2014), it is not possible to describe the concordance in key events or biological steps leading to neurodevelopmental outcomes. As such, the quantitative linkages between molecular initiating events (MIE)s, intermediate steps, and ultimately the adverse outcome (i.e., neurodevelopmental effects) cannot be determined. (See Appendix 6) With respect to the timing of exposure, across the epidemiology database of studies the maternal urine, cord blood and other (meconium) measures provide evidence that exposure did occur to the fetus during gestation, but the actual level of such exposure during the critical window(s) of susceptibility is not known. While significant uncertainties remain about the actual exposure levels experienced by mothers and infant participants in the children's health cohorts, it is unlikely that these exposures resulted in AChE inhibition. As part of the CHAMACOS study, Eskenazi et al. (2004) measured AChE activity and showed that no differences in AChE activity were observed. The biomarker data from the Columbia University studies are supported by the agency's dose reconstruction analysis using the PBPK-PD model. Following the recommendation of the FIFRA SAP (2012), the agency conducted a dose reconstruction analysis of residential uses available prior to 2000 for pregnant women and young children inside the home (USEPA, 2014). Based on the output from the PBPK-PD model, for the highest exposure considered (i.e., indoor broadcast use of a 1% chlorpyrifos formulation), <1% RBC AChE inhibition in pregnant women would be expected. While uncertainty exists as to actual OP exposure at (unknown) critical windows of exposure, EPA believes it is unlikely individuals in the epidemiology studies experienced RBC AChE inhibition. Preamble to the Integrated Science Assessments (ISAs) which serve as a scientific foundation for the review of EPA's National Ambient Air Quality Standards (NAAQS) notes that strong evidence for causality can be provided through "natural experiments" when a change in exposure is found to result in a change in occurrence or frequency of health (USEPA, 2015c). Within the Columbia University epidemiology studies, the relationship in time between prenatal chlorpyrifos exposure and adverse neurodevelopmental outcomes is concordant. The 82 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00082 time period under study within the Columbia University (CCCEH) study, spanned the point in time in which pesticide manufacturers voluntarily cancelled the use of chlorpyrifos in the home environment, and researchers were able to show the change in exposure before (high use period) and after (low/no use period) the period of removal of chlorpyrifos products from the residential marketplace. Moreover, prior to the voluntary cancellation there were >80% detectable levels of chlorpyrifos in cord blood but in the time period after the cancellation only 16% of the measured values were greater than the level of detection (LOD); there was only one child born in the time period subsequent to the voluntary cancellation of chlorpyrifos in the residential marketplace for whom the cord blood chlorpyrifos level was in the upper-tertile of pre-cancellation exposure levels. The significantly reduced proportion of measured values greater than the LOD as well as the observation of an absence of an association between prenatal chlorpyrifos exposure among infants born after the voluntary cancellation of chlorpyrifos and neurodevelopmental effects support the hypothesis that chlorpyrifos is related to these outcomes. However, as noted by study authors, EPA and the FIFRA SAP (2012), this could also be due to inadequate sample size to detect a small to modest effect among the group of infants born after the voluntary cancellation. It is notable that epidemiology studies from other research groups have not included analyses across different years of exposure. 3.2 Strength, Consistency & Specificity In making a weight-of-evidence analysis, it is important to consider the strength of the statistical measures of association between OP exposure and adverse neurodevelopmental outcomes through childhood (epidemiology) and possibly into adulthood (animal studies). It is also important to consider the strength of the integrated qualitative and quantitative evidence, the consistency of the observed associations across epidemiology studies and considering both animal and human data support the conclusion that chlorpyrifos plays a role in adverse neurodevelopmental outcomes. 3.2.1 Strength As noted in the 2016 epidemiology and incident framework, findings of large, precise risks increases confidence that the association is not likely due to chance, bias, or other factors (USEPA, 2016c). In the case of the CCCEH study, there are a number of notable associations. Regarding infant and toddler neurodevelopment, the CCCEH authors reported statistically significant deficits of 6.5 points on the PDI at 3 years of age when comparing high to low exposure groups (Rauh et al., 2006). Notably these decrements in PDI persist even after adjustment for group and individual level socioeconomic variables (Lovasi et al., 2011). These investigators also observed increased odds of mental delay (OR=2.4; 95% Cl: 1.1-5.1) and psychomotor delay (OR=4.9; 95% Cl: 1.8-13.7) at age three when comparing high to low exposure groups (Rauh et al., 2006). Rauh et al (2006) also reported extremely large odds ratios for attention disorders (OR=11.26; 95% Cl: 1.79-70.99), ADHD (OR=6.50; 95% Cl: 1.09-38.69), and PDD (OR=5.39; 95% Cl: 1.21-24.11) when comparing high to low chlorpyrifos exposure groups (Rauh et al., 2006). EPA notes that the magnitude of these results are so large that they 83 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00083 are unlikely to be affected by residual confounding although limited sample sizes resulted in imprecise estimates. From the CHAMACOS cohort, Eskanazi et al (2007) reported that both prenatal and postnatal DAPs were associated with risk of PDD (per 10-fold increase in prenatal DAPs: OR = 2.3, p = 0.05; child DAPs OR = 1.7, p = 0.04). Marks et al (2010) reported prenatal DAPs were associated with scores on the K-CPT ADHD Confidence Index > 70th percentile (OR = 5.1; 95% Cl: 1.7-15.7 and with a composite ADHD indicator of the various measures (OR = 3.5; 95% Cl: 1.1-10.7). Some outcomes exhibited evidence of effect modification by sex, with associations found only among boys. Children's concurrent total DAP and DMP metabolite levels at 3.5 years and 5 years were unrelated to attention outcomes, and but child DEP concentrations at 5 years were adversely associated with the composite measure of attention (OR = 2.0; 95% CI:l.l-3.6). In a recent evaluation by Stein et al (2016) from the CHAMACOS cohort, boys who experienced a more adverse learning environment, a statistically significant decrease was observed between prenatal DAP concentrations and Full-Scale IQ ((3 = -13.3; 95% Cl: -19.9; -6.7; p < 0.01), relative to boys who lived in a less adverse learning environment ((3 = 4.2; 95% Cl: -4.2; 12.5; p < 0.01). A similar observation was reported for perceptual reasoning and processing speed ([3 = -9.8; 95% Cl: -17.4; -2.2; p < 0.01; |3= -12.7; 95% Cl: -19.5; -6.0; p = 0.01), respectively. For girls who experienced more economic adversity, a stronger effect on the association between prenatal DAP concentrations and Full-Scale IQ was observed, relative to girls who experienced less economic adversity ((3 = -8.5; 95% Cl: -16.7, -0.4 vs. (3= -4.7 95% Cl: -12.8, 3.4; p = 0.18). This was also observed for working memory for girls ([3 = -5.9; 95% Cl: -13.7, -1.8 vs. (3= -2.6 95% Cl: -10.0, 4.8; p = 0.05). 3.2.2 Consistency The Preamble to the ISA's note that an inference of causality is strengthened when a pattern of elevated risks is observed across several independent studies. The reproducibility of findings constitutes one of the strongest arguments for causality. Moreover, statistical significance is not the sole criterion by which the presence or absence of an effect is determined (USEPA, 2015c). Published and submitted laboratory animal studies have been reviewed for OPs. The >30 papers on chlorpyrifos provide evidence of long-lasting neurodevelopmental disorders in rats and mice; however, there was no clear consistency in terms of pattern, timing, or dose response for these effects. The additional toxicological literature and guideline DNT studies with the other OPs provide more evidence for the same conclusions, with again the same caveats and uncertainties. While overall cognitive function and motor activity appeared to be altered the most often, it is apparent that these behaviors were also the most often evaluated. Across the various epidemiology studies within the scope of this analysis and with adequate quality in design and exposure assessment, it is important to note that these studies used study methods that were highly variable, including different exposure measurement, outcome 84 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00084 assessment, study design, and geographical location. These differences make it challenging to compare the results across studies. Among the epidemiology studies, two of the cohorts (CCCEH and ELEMENT) have focused on chlorpyrifos whereas the other studies (Mt. Sinai cohort, CHAMACOS cohort, HOME cohort, CHARGE study, Bouchard et al., 2010) have focused on less specific biomarkers (i.e., DAPs) and are not specific to any particular OP. When considered in concert, the epidemiology studies provide consistent findings for some outcomes. In comparison to the studies from CCCEH, Mt. Sinai, and CHAMACOS, results from the ELEMENT and CHARGE studies provide some supportive evidence for the findings. However, the results from the HOME cohort show different results; namely positive associations between neurodevelopmental outcomes and maternal DAPs (Yolton et al, 2013; Donauer et al, 2016). The HOME study represents a different demographic profile (higher socioeconomic status, suburban, middle class) and different time period (2003-2006) compared to CCCEH, Mt. Sinai, and CHAMACOS which are made up of women with much lower socioeconomic status and who experienced different exposure patterns. In the two Chinese studies, EPA does not know how the OP exposures from these studies relate to the currently registered use pattern for OPs used in the U.S./North America. In the case of these two Chinese studies (Guodong et at., 2012; Zhang et al., 2014), there may be differences in the study population and outcome measurements that may account for the observed differences in study results, with Zhang et al. (2014) documenting statistically significant associations for total DEAPs, total DMAPs, and total DAPs and Guodong et al. (2012) observing no association with these exposures. The Zhang et al. (2014) study was conducted in Shenyang, with a study population reported as 87% urban and 13% rural, whereas the Guodong et al. (2012) study was conducted in Shanghai with a 99% urban and 1% suburban study population. Given the higher percentage of study participants from rural areas, the study participants from Zhang et al. (2014) may have had different pattern and magnitude of OP exposures compared to those from Guodong et al. (2012). In addition, it is noted that different outcome measurements were made in these studies, with Guodong et al. (2012) assessing 23-25 month old children using a developmental quotient score and Zhang et al. (2014) assessing 3 day old infants using a Neonatal Behavioral Neurological Assessment. Given the different outcome assessments, exposure potential, study designs (cohort vs. cross-sectional), and ages of the participants in these two studies, it is difficult to draw conclusions on how these study results compare. It is notable that the results from the Zhang et al. (2014) study focusing on neonates are consistent with those from other studies which reported statistically significant associations between delayed neurological development measured in newborns and total DEAP, total DMAP, and total DAP exposure (Engel et al., 2007; Young et al., 2005). However, it is noted that neurological development was measured within a few days of birth for Zhang et al. (2014) and Engel et al. (2007), whereas Young et al. (2005) conducted their measurements within two months of birth. The CHAMACOS and Mt. Sinai cohorts that measured neurological effects at birth (the Brazelton index), observed a putative association with OPs (Engel et al., 2007; Young et al., 2005). Yolton et al (2013) used the NNNS tool at approximately 5 weeks of age in the HOME 85 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00085 cohort and reported evidence of a positive association between increased mean urinary DE metabolites and increased attention and decreased autonomic stress. Researchers across CCCEH, Mt. Sinai, CHAMACOS, and HOME studies utilized the Bayley Scales of Infant Development II (BSID-II) to generate a MDI and a PDI to assess neurodevelopment in early childhood. In the CCCEH study, Rauh et al. (2006) investigated MDI and PDI at 12, 24, and 36 months of age. Children were categorized as having either high (>6.17pg/g) or low (<6.17pg/g) prenatal chlorpyrifos exposure, using categories informed by results of the previous study on birth characteristics (Whyatt et al., 2004). Authors reported that the difference in MDI scores was "marginally significant" (p = 0.06) between the "high" and "low" exposed groups; the high exposed group scoring an average of 3.3 points lower than the low exposed (Rauh et al., 2006). Regarding the PDI score (motor skills), none of the 12 or 24 month PDI scores showed significant effects, but the 36 month score was significantly related to chlorpyrifos exposure. Within the 36 month testing period, the likelihood of highly exposed children developing mental delays were significantly greater (MDI: 2.4 times greater (95% Cl: 1.12-5.08, p = 0.02) and PDI: 4.9 times greater (95% Cl: 1.78-13.72; p = 0.002)) than those with lower prenatal exposure (Rauh et al., 2006). Within the Mt. Sinai study, authors administered the BSID-II to participating children at 12 and 24 months and observed that prenatal total DAP metabolite level was associated with a decrement in mental development at 12 months among blacks and Hispanic children; however, these associations either attenuated or were non-existent at the 24-month visit (Engel et al., 2011). In the CHAMACOS cohort, Eskenazi et al. (2007) observed that prenatal DAP levels were adversely associated with MDI, and at 24 months of age these associations reached statistical significance. In this study, neither prenatal DAPs nor maternal TCPy were associated with PDI (motor skills), nor did authors observe evidence of different risk by PON1 status (Eskenazi et al., 2010). In contrast to the findings of CCCEH, Mt. Sinai, and CHAMACOS, the HOME study (Donauer et aI, 2016) reported evidence of a positive association for MDI was observed for maternal exposure to total DM and total DE at the child's 2-year visit; however, when the model was adjusted, no evidence of a positive association was observed for total DM and total DE (adjusted model (3 = 0.002, p = 0.28; (3= 0.002, p = 0.37). No evidence of an association was observed between maternal exposure (for total DAP, total DM, and total DE) and the Bayley-ll Psychomotor Developmental Index for children aged 1-3 years. With respect to attention problems, Rauh et al. (2006) also investigated 36-month child behavior checklist (CBCL) (behavioral) scores. Significant differences were observed between the high and low chlorpyrifos exposure groups in the general category of attention-problems (p=0.010), and in the more specific DSM-IV scale for ADHD problems (p = 0.018). The CHAMACOS cohort also investigated attention problems in early childhood using three different assessment tools: maternal report of child behavior at 3.5 and 5 years of age; direct assessment of the child at 3.5 and 5 years; and by a psychometrician's report of the behavior of the child during testing at 5 years. In this study population, higher concentrations of OP metabolites in the urine of pregnant women were associated with increased odds of attention problems and poorer attention scores in their children at age 5 years (Marks et aI, 2010). 86 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00086 Of the four new studies focusing on ADHD and OP exposure, three found statistically significant associations, with only Oulhote and Bouchard (2013) finding no association with total DMAP, DEAP, or total DAP exposure. In contrast, Bouchard et al. (2010) observed an association with total DMAP and total DAP exposure and ADHD. Fortenberry et al. (2014) found suggestive, but not statistically significant, evidence of an association with TCPy and ADHD in boys. Overall, the Fortenberry and Bouchard study results are consistent with that of earlier studies from the CCCEH (Rauh et al., 2006) and CHAMACOS (Eskenazi et al., 2007; Marks et al., 2010). Specifically, statistically significant associations were observed by Rauh et al. (2006) with chlorpyrifos exposure and ADHD, Eskenazi et al. (2007) with total DMAPs and total DAPs and ADHD, and Marks et al. (2010) with total DEAP, DMAP, and total DAP exposure. It is important to put into context the specific outcome measures used in the assessment of attention and neurobehavioral problems. For example, Bouchard et al. (2010) identified statistically significant associations between OP exposure and ADHD/behavioral problems, whereas Oulhote and Bouchard (2013) did not. It is valuable to compare these studies given that they are both cross-sectional studies using large population level datasets with biomarker information, with Oulhote and Bouchard (2013) using a Canadian dataset and Bouchard et al (2010) using a U.S. dataset. Bouchard et al. (2010) used criteria for ADHD from DSM-IV, whereas Oulhote and Bouchard (2013) used a "Strengths and Differences Questionnaire (SDQ)," with the SDQ being a more generic assessment of mental health status than the DSM-IV criteria. When Oulhote and Bouchard (2013) compared their results to Bouchard et al. (2010), they noted that their outcome measurements may not have been as sensitive and that this may account for the difference in study results. Across epidemiology studies looking at ADHD/behavioral problems, a suggestive or statistically significant positive association was observed in multiple studies between OP exposure and these neurobehavioral outcomes (Bouchard et al., 2010; Fortenberry et al., 2014; Rauh et al., 2006, Eskenazi et al., 2007). While these studies have differences in the years that the exposure occurred, study design, exposure assessment, and outcome assessment, the commonality in their results is striking. Several studies have now documented suggestive or positive associations between OP exposure and autism spectrum disorders (Rauh et al., 2006; Shelton et al., 2014; Furlong et al., 2014; Eskenazi et al., 2007; Eskenazi et aI, 2010). Specifically, Furlong et al. (2014) reported suggestive, but not statistically significant, evidence of an association between total DEAP exposure and reciprocal social responsiveness among black participants and boys. These results are consistent with previous studies conducted on the CCCEH and CHAMACOS cohorts, with these studies also showing statistically significant associations between OP exposure and autism spectrum disorders (Rauh et al., 2006; Eskenazi et al., 2007).8 Specifically, Eskenazi et 8The DSM-V defines ASD (autism spectrum disorder) which now encompasses several disorders that were different diagnoses in DSM-IV, including PDD (pervasive developmental disorder, a catch-all where the other 87 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00087 al. (2007) reported a statistically significant association between PDD and total DAP exposure, whereas Rauh et al. (2006) showed a significant association between PDD and specifically chlorpyrifos exposure. Both PDD and reciprocal social responsiveness are related to the autism spectrum disorder. Using a different exposure assessment method (geospatial analysis and residential proximity to total OP exposure), Shelton et al. (2014) also documented statistically significant associations between total OP exposure and autism spectrum disorders. While these studies vary in the magnitude of the overall strength of association, they have consistently observed a positive association between OP exposure and autism spectrum disorders. Finally, CCCEH, Mt. Sinai, and CHAMACOS observed an inverse relation between the respective prenatal measures of OPs and intelligence measures at age 7 years. To measure intelligence among school aged children, authors from each of the three children's health cohorts used the WISC-IV. Rauh et al. (2011) evaluated the relationship between prenatal chlorpyrifos exposure and neurodevelopment among 265 of the cohort participants who had reached the age of 7 years investigators reported that for each standard deviation increase in exposure (4.61 pg/g) there is a 1.4% reduction in Full-Scale IQand a 2.8% reduction in Working Memory. In the Mt. Sinai study, prenatal maternal DEP urinary metabolite concentrations were associated with slight decrements in FSIQ, Perceptual Reasoning, and Working Memory between the ages of 6 and 9 years, and difference in intelligence measures by putative PON1 status were also noted (Engel et al., 2011). Similarly, in the CHAMACOS cohort, Bouchard et al. (2011) observed evidence of an association between prenatal exposures to OPs as measured by urinary DAP (total DAP, DEP, and DMP) metabolites in women during pregnancy, and decreased cognitive functioning in children at age 7. In this study, children in the highest quintile of maternal DAP concentrations had a statistically significant 7-point difference in IQ points compared with those in the lowest quintile. The CHAMACOS results with the WISC-IV were furthered in Stein et al (2016) who evaluated potential impact from adverse learning environment. In contrast, to the findings of CCCEH, Mt. Sinai, and CHAMACOS, the HOME study (Donauer et aI, 2016) reported evidence of a significant positive association between maternal levels of urinary metabolites (total DAP and total DM) and verbal IQ of the child from the WPPSI was observed in both the unadjusted and adjusted models. Although evidence of a positive association was observed for total DAP and total DM metabolites and full-scale IQ (unadjusted model: |3= 0.004, p = 0.111; (3= 0.004, p = 0.124, when the model was adjusted, no evidence of a positive association was observed (adjusted model: (3= 0.001, p = 0.494; (3= 0.002, p = 0.441). As stated in the EPA neurotoxicity guidelines9, direct extrapolation of developmental neurotoxicity results from laboratory animals to humans is limited by the lack of knowledge about underlying toxicological mechanisms and the relevance of these results to humans. EPA notes consistencies across the databases of in vivo laboratory animal studies and epidemiology studies, although challenges of making a direct comparison between neurodevelopmental domain inter-species remain. It can be assumed that developmental neurotoxicity effects in categories didn't fit). Depending on when the study was conducted, the authors may use the PDD or ASD criteria and terminology. 9 http://www.epa.gov/raf/publications/pdfs/NEUROTOX.PDF 88 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00088 animal studies indicate the potential for altered neurobehavioral development in humans, although the specific types of developmental effects seen in experimental animal studies may not be the same as those that may be produced in humans. However, considering the toxicological and epidemiological data in the context of three major neurodevelopmental domains (specifically, cognition, motor control, and social behavior), insights can be gained. Previously reviewed studies of chlorpyrifos in rats and/or mice reported impaired cognition, changes in locomotor activity levels, altered social interaction, and to a lesser extent, changes in neuromotor function (FIFRA SAP 2012; USEPA, 2014). While there are fewer studies for all the other OPs, behavioral effects in the same functional domains were again reported. The most commonly reported outcome in the laboratory animal studies was cognitive dysfunction, and although it was overall consistent there were again differences in cognitive specificity, gender differences, or dose response. Quite a few studies also report changes in motor activity and sensory function in offspring, but there generally fewer laboratory animal studies that assess social interactions for OPs other than chlorpyrifos. It is notable that the laboratory animal studies vary in experimental designs such as species, strain, gender, dosing regimens (age, routes, vehicle), and test parameters (age, protocol). Likewise, observational epidemiology studies vary by population characteristics (race/ethnicity, SES, and pesticide use/exposure profile), co-exposures (mix of chemicals, windows of exposure), and method of exposure and outcome assessment. Given the differences across laboratory animal and epidemiology studies, the qualitative similarity in research findings is striking. In contrast, quantitatively, there are notable differences between animals and humans. Specifically, in animals, the doses most often used in these studies are sufficient to elicit >10% brain and RBC inhibition depending on the study design, age of the animal, and sampling time. In the epidemiology studies, based on the comparisons with biomonitoring data, reported AChE data from CHAMACOS and the results of the chlorpyrifos dose-reconstruction analysis, it is unlikely that RBC AChE would have been inhibited by any meaningful or measurable amount, if any at all, and most likely none in the brain. This key difference in dose response between the experimental toxicology and epidemiology studies poses challenges in interpreting such data. There are a number of possible hypotheses such as: 1) limitations of experimental laboratory studies which have limited statistical power due to relatively small sample sizes; 2) humans display a broader array of behaviors and cognitive abilities than rats, thus limiting the sensitivity of the rat studies; and 3) in the epidemiology studies, the timing of OP application and blood collections are not coupled--thus higher levels of blood OPs were likely missed. 3.2.3 Specificity In considering the FQPA 10X Safety Factor, the statute indicates that both pre- and post-natal toxicity and exposure be considered. As such, it is appropriate to consider the degree specificity of with regard to lifestage. There are numerous animal studies in the literature which vary in their study design but all involve gestational and/or early postnatal dosing with behavioral evaluation from adolescence to adulthood. The data provide support for the susceptibility of the developing mammalian brain to chlorpyrifos exposure through gestation 89 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00089 and early in life, with adverse outcomes in several neurological domains including cognitive, anxiety and emotion, social interactions, and neuromotor function. The studies have not shown that any specific developmental period is critical overall to the long-term outcomes, since similar effects are observed with different exposure periods. For example, cognitive changes in one laboratory using a radial arm maze were observed following gestational and early, but not late, postnatal exposure (Aldridge et ai, 2005; Icenogle et ai, 2004; Levin et ai, 2002; Levin et ai, 2001), whereas other laboratories cognitive deficits in a Morris water maze were reported following both gestational and late-postnatal exposure (Billauer-Haimovitch et ai, 2009; Turgeman et ai, 2011). Likewise, some changes in anxiety and social behaviors were reported at both gestational and postnatal exposure periods. Overall, these data do not clearly show specific critical periods of exposure but support a conclusion that early life (pre- and post natal) represent susceptible lifestages. As discussed above, numerous epidemiological investigations have observed a link between prenatal exposure to chlorpyrifos or OPs (measured as chlorpyrifos, TCPy, or DAPs) and adverse effects on neurodevelopment through age seven years, with additional more limited evidence up through approximately age eleven. As noted previously, for these epidemiology studies chlorpyrifos was only assessed directly in the CCCEH study, with the Mexican cohort study (Fortenberry et ai, 2014) assessing the chlorpyrifos metabolite TCPy, and the CHAMACOS, HOME, and Mt. Sinai cohorts focusing on the DAPs. The majority of epidemiological studies investigated only prenatal exposures. Specifically, with respect to biomarkers representing prenatal exposures, CCCEH, Mt. Sinai, and CHAMACOS each reported evidence of impaired mental and psychomotor development, albeit not consistent by age at time of testing (ranging from 6 month to 36 months across the three cohorts). Statistically significant or suggestive associations between chlorpyrifos or DAPs exposure and attentional problems/ADHD were reported by multiple prospective cohorts (Rauh et ai, 2006; Eskenazi et ai, 2007; Marks et ai, 2010; and Fortenberry et ai, 2014) with additional support from a case control study, Bouchard et ai (2010). In addition, the three US cohorts and the CHARGE study have reported suggestive or positive associations between OP exposure and autism spectrum disorders (Rauh et a!., 2006; Shelton et ai, 2014; Eskenazi et ai, 2007; Furlong et ai, 2014). While the studies from CCCEH, Mt. Sinai, and CHAMACOS studies vary in the magnitude of the overall strength of association, they have consistently observed a positive association between OP exposure and autism spectrum disorder. Several studies have also documented statistically significant association between prenatal DAP exposure and abnormal motor development in neonates (reflexes, Brazelton score or similar measure; Young et ai, 2005; Engel et ai, 2007; Zhang et at., 2014). Finally, CCCEH, Mt. Sinai and CHAMACOS have reported an inverse relation between the respective prenatal measures of chlorpyrifos or DAPs and intelligence measures at age 7 years (Rauh et ai, 2011; Engel et ai, 2011; Bouchard et ai, 2011). With regards to effects pre-natal compared to post-natal, a small number of studies have assessed postnatal exposure to DAPs. Postnatal exposure to DAPs has been assessed in the CHAMACOS cohort (Eskenazi et ai, 2007; Young et ai, 2005; Bouchard et ai, 2011) and three 90 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00090 cross-sectional studies (Guodong et al., 2012; Bouchard et al., 2010; Oulhote and Bouchard, 2013). With the exception of Bouchard et al. (2010) and findings of PDD increase in post-natal DAP from CHAMACOS (Eskenazi et al., 2007; Marks et aI, 2010), no adverse neurodevelopmental associations were found between postnatal urinary metabolite levels and any of the developmental outcomes. Bouchard et al. (2010) looked at U.S. children age 8-15 years in the 2000-2004 National Health and Nutrition Examination Survey (NHANES), and observed a positive association between attention and behavior problems and total DAPs and DMAPs, but not DEAPs. Postnatal exposures have not been assessed in the CCCEH and Mt. Sinai studies (Rauh et al., 2011; Engel et al., 2011); as such, there are no studies included in this analysis which directly assessed the potential for postnatal chlorpyrifos exposure and associations with neurodevelopmental effects. However, given that the major source of exposure (residential use) was cancelled partway through the CCCEH study which substantially reduced and largely removed chlorpyrifos from the home environment, this limits the ability of the CCCEH study to inform the impacts of long-term postnatal exposure to chlorpyrifos on neurodevelopment from the current uses of chlorpyrifos. In sum, given that the extensive experimental laboratory animal database suggests that the post-natal period is a potential susceptible time, the lack of postnatal exposure assessment in the CCCEH and Mt. Sinai studies is a source of uncertainty in the epidemiology database 3.3 Biological Plausability & Coherence EPA's cancer guidelines (2005) includes guidance which are also applicable to this current evaluation of OPs. In fact, the Guidelines indicate: "evaluation of the biological plausibility of the associations observed in epidemiologic studies reflects consideration of both exposure-relatedfactors and toxicological evidence relevant to identification of potential modes of action (MOAs). Similarly, consideration of the coherence of health effects associations reported in the epidemiologic literature reflects broad consideration of information pertaining to the nature of the biological markers evaluated in toxicologic and epidemiologic studies, [p. 39]." The Cancer Guidelines further state that "lack of mechanistic data, however, is not a reason to reject causality [p. 41]." At this time, a MOA(s)/AOP(s) has/have not been established for neurodevelopmental outcomes. This growing body of literature does demonstrate, however, that OPs are biologically active on a number of processes that affect the developing brain. Multiple in vitro studies on endpoints relevant to the developing brain have identified outcomes in picomole concentrations, including concentrations lower than those that elicit AChE inhibition in vitro. Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 91 ED 002061 00044425-00091  1 pM chlorpyrifos oxon decreased axon length (~50%) in superior cervical ganglion cell cultures (AChE inhibition starting at lOOOpM in same cell system): Howard et al., 2005 0.03 pM oxon (30 fM) increased in CREB levels (~50%) primary cortical neuron cultures (AChE inhibition starting at 100 pM in same cell system): Schuh et al., 2002 10 pM oxon decreased axon length (~40%) in dorsal root ganglion cell cultures (AChE inhibition starting at 100 pM in same cell system): Yang et al., 2008 With regard to coherence, there is a large body of in vivo laboratory studies which show long term behavioral effects from early life exposure, albeit at doses which cause AChE inhibition. EPA considers the results of the toxicological studies relevant to the human population, as qualitatively supported by the results of epidemiology studies. The lack of established MOA/AOP pathway does not undermine or reduce the confidence in the findings of the epidemiology studies. When all the evidence is considered together, there are uncertainties with respect to a number of factors such as exposure assessment, lack of ability to make strong causal linkages, and unknown window(s) of susceptibility. The epidemiology studies reviewed in the 2012/2014 and 2015/2016 literature reviews represent different investigators, locations, points in time, exposure assessment procedures, and outcome measurements. Despite all these uncertainties and differences in study design, multiple investigators have identified associations with neurodevelopmental outcomes such as ADHD/behavioral problems and autism spectrum, in relation to OP exposure. There is evidence of delays in mental development in infants (24-36 months), attention problems and autism spectrum disorder in early childhood, and intelligence decrements in school age children who were exposed to OPs during gestation. Investigators reported strong measures of statistical association across several of these evaluations (odds ratios 2-4 fold increased in some instances), and observed evidence of exposures-response trends in some instances, e.g., intelligence measures. Thus, with respect to biological plausibility and coherence, although uncertainties remain, these uncertainties are diminished in the context of the qualitative similarity between the epidemiology studies. 4.0 10X FQPA Safety Factor for Infants and Children As section 408(b)(2)(C) of the FFDCA instructs EPA, in making its "reasonable certainty of no harm" finding, that in "the case of threshold effects, an additional tenfold margin of safety for the pesticide chemical residue and other sources of exposure shall be applied for infants and children to take into account potential pre- and postnatal toxicity and completeness of data with respect to exposure and toxicity to infants and children." Section 408 (b)(2)(C) further states that "the Administrator may use a different margin of safety for the pesticide chemical residue only if, on the basis of reliable data, such margin will be safe for infants and children." Given the totality of the evidence, there is sufficient uncertainty in the human dose-response relationship for neurodevelopmental effects which prevents the agency from reducing or removing the statutory 10X FQPA Safety Factor. For the human health risk assessments for the OPs, a value of 10X will be applied. Similarly, a database uncertainty factor of 10X will be retained for occupational risk assessments. The agency will continue to evaluate the 92 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00092 epidemiology studies and pursue approaches for quantitative or semi-quantitative comparisons between doses which elicit AChE inhibition and those which are associated with neurodevelopmental outcomes prior to a revised human health risk assessment. The FQPA 10X Safety Factor or database uncertainty factor will be retained for OPs for the population subgroups that include infants, children, youths, and women of childbearing age for all exposure scenarios. Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 93 ED 002061 00044425-00093 5.0 References Acosta-Maldonado, Brenda, Snchez-Ramrez, Blanca, Reza-Lpez, Sandra, and LevarioCarrillo, Margarita. Effects of exposure to pesticides during pregnancy on placental maturity and weight of newborns: A cross-sectional pilot study in women from the Chihuahua State, Mexico. Human and Experimental Toxicology 28(8) 451-459, 2009. Aldridge, J. E., Levin, E. D., Seidler, F. J., & Slotkin, T. A. (2005). Developmental exposure of rats to chlorpyrifos leads to behavioral alterations in adulthood, involving serotonergic mechanisms and resembling animal models of depression. Environ Health Perspect, 113(5), 527-531. al-Hachim, G.M., Fink, G.B., 1968a. Effect of DDT or parathion on open-field behavior of offspring from DDT- or parathion-treated mothers. Psychol Rep 2 2,1193-1196. al-Hachim, G.M., Fink, G.B., 1968b. Effect of DDT or parathion on condition avoidance response of offspring from DDT or parathion-treated mothers. 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Determination of The Appropriate FQPA Safety Factor(S) In Tolerance Assessment. Office of Pesticide Programs, U.S. Environmental Protection Agency, Washington, DC, February 28, 2002. U.S. Environmental Protection Agency. (2005). Guidelines for Carcinogen Risk Assessment. Risk Assessment Forum, U.S. Environmental Protection Agency. Washington, DC. EPA/630/P-03/001F. U.S. Environmental Protection Agency. (2006). Revised Organophosphorous Pesticide Cumulative Risk Assessment; July 31, 2006. Office of Pesticide Programs, U.S. Environmental Protection Agency. Washington, D.C. Available http://www.epa.gov/pesticides/cumulative/rra-op. U.S. Environmental Protection Agency. (2008). Draft Science Issue Paper: Chlorpyrifos Hazard and Dose Response Characterization. http://www.epa.gov/scipoly/sap/meetings/2008/091608_mtg.htm U.S. Environmental Protection Agency. (2010). Draft Framework for Incorporating Human Epidemiologic and Incident Data in Health Risk Assessment, January 7, 2010. U.S. Environmental Protection Agency. (2011a). Chlorpyrifos: Preliminary Human Health Risk Assessment for Registration Review. June 30, 2011. D388070. U.S. Environmental Protection Agency. (2011b). Chlorpyrifos: Occupational and Residential Exposure Assessment, EPA Barcode: D388165,. In Office of Pesticide Programs (Ed.). Washington D.C. U.S. Environmental Protection Agency. (2012). Draft Issue Paper: Scientific Issues Concerning Health Effects of Chlorpyrifos. http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPP-2012-0040-0002 U.S. Environmental Protection Agency. (2014). Chlorpyrifos: Revised Human Health Risk Assessment for Registration Review. December 29, 2014. D424485. U.S. Environmental Protection Agency. (2015) Literature Review on Neurodevelopment Effects & FQPA Safety Factor Determination for the Organophosphate Pesticides. September 15, 2015. 103 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00103 U.S. Environmental Protection Agency. 2015b. Swe, K. and Holman, E. Summary Reviews from _2015 Epidemiological Literature Review for Organophosphates. September 15, 2015. U.S. EPA (U.S. Environmental Protection Agency). 2015c. Preamble to the Integrated Science Assessments. National Center for Environmental Assessment, RTP Division, Office of Research and Development, USEPA. https://yosemite.epa.gOv/sab/sabproduct.nsf/0/33ElAD305287588F85257D2000 6BE8CC/$File/ISA_PREAM BLE_FINAL2015.PDF U.S. Environmental Protection Agency. 2016. Holman, E. and Swe, K. Summary Reviews for Additional Epidemiological Literature Studies from Prospective Birth Cohort Studies. D432184. March 25, 2016 U.S. Environmental Protection Agency. 2016b. Aldridge, A. Summary Reviews for Additional Epidemiological Literature Studies from Prospective Birth Cohort Studies. D432184. December XXX, 2016 U.S. Environmental Protection Agency. 2016c. Framework for Incorporating Human Epidemiologic and Incident Data in Health Risk Assessment, December 28, 2016. Vatanparast, J., Naseh, M., Baniasadi, M., Haghdoost-Yazdi, H., 2012. Developmental exposure to chlorpyrifos and diazinon differentially affect passive avoidance performance and nitric oxide synthase-containing neurons in the basolateral complex of the amygdala. Brain Res 1494,17-27. Venerosi A, Tait S, Stecca L, Chiarotti F, DeFelice A, Cometa MF, Volpe MT, Calamandrei G, Ricceri L. Effects of maternal chlorpyrifos diet on social investigation and brain neuroendocrine markers in the offspring - a mouse study. Environ. Health 14:3242, 2015 Wang, Pei, Tian, Ying, Wang, Xiao-Jin, Gao, Yu, Shi, Rong, Wang, Guo-Quan, Hu, GuoHua, Shen, Xiao-Ming. Organophosphate pesticide exposure and perinatal outcomes in Shanghai, China. 4 Environment International 2:100-104, 2012. Whyatt, R. M., Barr, D. B., Camann, D. E., Kinney, P. L., Barr, J. R., Andrews, H. F., .. . Perera, F. P. (2003). Contemporary-use pesticides in personal air samples during pregnancy and blood samples at delivery among urban minority mothers and newborns. Environ Health Perspect, 111(5), 749-756. Whyatt RM, Rauh V, Barr DB, Camann DE, Andrews HF, Garfinkel R, Hoepner LA, Diaz D, Dietrich J, Reyes A, Tang D, Kinney PL, Perera FP. Prenatal Insecticide Exposures and Birth Weight and Length among an Urban Minority Cohort. Environmental Health Perspectives. 2004;112:1125-1132. Whyatt RM, Garfinkel R, Hoepner LA, Holmes D, Borjas M, Williams MK, et ol. Withinand Between-Home Variability in Indoor-Air Insecticide Levels during Pregnancy among an Inner-City Cohort from New York City. Environmental Health Perspectives 2007;115:383-389. 104 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00104 Whyatt RM, Garfinkel R, Hoepner LA, Andrews H, Holmes D, Williams MK, et al. A Biomarker Validation Study of Prenatal Chlorpyrifos Exposure within an Inner-City Cohort during Pregnancy. Environmental Health Perspectives. 2009;117:559-567. Wickerham, Erin L., Lozoff, Betsy, Shao, Jie, Kaciroti, Ni ko, Xia, Yankai, Meeker, John D. Reduced birth weight in relation to pesticide mixtures detected in cord blood of full-term infants. Environment International 47:80-85, 2012. Win-Shwe, T.T., Nakajima, D., Ahmed, S., Fujimaki, H., 2012. Impairment of novel object recognition in adulthood after neonatal exposure to diazinon. Arch Toxicol 87, 753-762. Wolff, MS, Engel, S, Berkowitz, G, Teitelbaum, S, Siskind, J, Barr, DB, Wetmur, J. Prenatal Pesticide and PCB Exposures and Birth Outcomes. 2007. Pediatric Research 61(2):243-250. Yang, D., Howard, A., Bruun, D., Ajua-Alemanj, M., Pickart, C., & Lein, P. J. 2008. Chlorpyrifos and chlorpyrifos-oxon inhibit axonal growth by interfering with the morphogenic activity of acetylcholinesterase. Toxicol Appl Pharmacol, 228(1), 3241. Yolton, K., Xu, Y., Sucharew, H., Succop, P., Altaye, M., Popelar, A., ... & Khoury, J. C. (2013). Impact of low-level gestational exposure to organophosphate pesticides on neurobehavior in early infancy: a prospective study. Environmental health, 12(1), 1. Young JG, Eskenazi B, Gladstone EA, Bradman A, Pedersen L, Johnson C, Barr DB, Furlong CE, Holland NT. Association Between In Utero Organophosphate Pesticide Exposure and Abnormal Reflexes in Neonates. NeuroToxicology. 2005;26:199-209. Zhang Y., Han S., Liang D., Shi X., Wang F., et al., (2014). Prenatal exposure to organophosphate pesticides and neurobehavioral development of neonates: A birth cohort study in Shenyang, China. Plos ONE 9(2):e88491. doi:10.1371/journal. pone.0088491. Zhang X, Driver JH, Li Y, Ross JH, Krieger Rl. (2008) Dialkylphosphates (DAPs) in fruits and vegetables may confound biomonitoring in organophosphorus insecticide exposure and risk assessment. J Agric Food Chem. 2008 Nov 26;56(22):10638-45. doi: 10.1021/jf8018084. Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 105 ED 002061 00044425-00105 6.0 Appendices 1. Table of In Vivo Developmental Neurotoxicity Studies of OPs. 2. Summary of Guideline DNT Studies Submitted to the Agency for OPs other than Chlorpyrifos. 3. Low Quality Studies: Summary of Study Design Elements Impacting Study Quality Assignment 4. Table of Systematic Review Analysis: Initial Search Research & Initial Exclusion Criteria 5. Table of Systematic Review Analysis: Second Tier Exclusion Criteria 6. Plausible hypotheses on MOA/AOP for neurodevelopmental outcomes (Extracted from Section 4.4.3. RHHRA for chlorpyrifos) Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 106 ED 002061 00044425-00106 Appendix 1. Table of In Vivo Developmental Neurotoxicity Studies of OPs. (Effects described are only those measured after weaning. Bold indicates functional domains that were reported to show treatment effects.) OP C h lo rm epho s D iazinon D iazinon D iazinon Study Ceh e t al., 2012 Roegge et a/., 2008 Species & strain m ouse BALB/c rat Sprague D aw ley Dose, route, v e h ic le 3.5, 0.35 u g/m l in d rin k in g w ater of dam s ~ 0.6, 0.06 m g/kg/d (@ 5m l/d, 30 g) 0.5, 2 m g/kg/d sc DM SO D osing p e rio d 7 day prem ating to w eaning P N D 1 -4 ChE inhibition No D om ain Anxiety & Emotion C ites Slo tkin e t al. (2006): 0.5 m g/kg/d produ ced <1 0 % brain inhib ition on PN D5, 2 m g/kg/d produced 25-30% brain inhib ition 2 hr after dose on PND4, and 10-20% inhib ition on PND5 A n xiety & Emotion Age of testin g P N D 7 0 -8 0 Outcom es NOEL, LOEL Notes & Stu dy Problem s In cre a se d tim e in clo se d arm s & d e c r e a s e d tim e in o p e n a r m s in elevated plus m aze, ~0.6 m g/kg/d, M&F N O EL=0.35 ug/m l in w ater, ~0.06 m g/kg/d Litter not un it o f statistical a n a ly sis No pup allocation described but had to have used som e litterm ates M & F respo nses ap pear sim ilar but not statistically com pared P N D 5 2 -5 6 PN D64-67, 78-79 P N D 7 3 -7 4 PN D86-87 D e cre a se d tim e in o p e n a rm s in elevated plus m aze, 2 m g/kg/d, M only D e cre a se d la te n cy to eat in novelty suppressed but not h o m e-cage feeding, 0.5 & 2 m g/kg/d, M only D ecreased ch o co late m ilk p reference, 0.5 m g/kg/d only, M o n ly No effect on forced sw im test No NOEL LO EL=0.5 m g/kg/d Pups & d am s re d istrib u te d daily No e ffe ct in F A ccepts p<0.1 as sign ifican t for in te ra c tio n s No d o se -re sp o n se in fe e d in g o r m ilk preference studies A b stract m isstates sex differences S p y k e ra n d m ou se F2 0 .1 8, 9 G D l-b irth No A very, 1977 hybrid m g / k g / d in (N C T R peanut cross butter bred) Sensory Neuromotor A ctivity PND38 PND50, 60, 6 5 ,7 5 P N D 7 5 -7 6 Increased erro rs on visual cliff, 0.18 m g/kg/d o nly, F only No effect aco ustic startle or o lfacto ry respo nses No effect sw im m in g ab ility Increased rod cling endurance, 0.18 & 9 m g/kg/d, sex not sp e c ifie d D ecreased inclined plane perfo rm ance, 0.18 & 9 m g/kg/d, sex not specified No effect open field No NOEL LO EL=0.18 m g/kg/d C o g n itio n PND87 No e ffe ct e rro rs in La sh le y m aze Tim ofeeva e t a!., 2008 rat Sprague D aw ley 0.5, 2 m g/kg/d sc DM SO P N D 1 -4 C ites Slo tkin e t al. (2006): 0.5 m g/kg/d produ ced <1 0 % brain inhib ition on PN D5, 2 m g/kg/d produced A ctivity Sensory PN D28-42 P N D 7 7 -8 4 No e ffe ct in fig u re -8 ch a m b e r, M&F D ecreased prep ulse inhib ition , 0.5 & 2 m g/kg/d, M only No NOEL LO EL=0.5 m g/kg/d No pup allocation described but had to have used litterm ates Not clear w hen both sexes tested and/or com pared Statistics not described M aternal w eight gain low ered at both doses W eigh t gain of high dose pups decreased Prew eaning testin g: decreased co ntact placing, 0.18 m g/kg/d o n ly No dose-response for som e m easures Tw ice as m any co ntrols as treated Looks like decreased rota rod endurance PND65, not significant due to high variab ility L itte rm a te s o f th o se u se d in R oegge e t al. 2008 Pups & d am s re d istrib u te d daily A ccepts p<0.1 as sign ifican t for in te ra c tio n s 107 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00107 D iazinon D iazinon Vatanparas t et a l, 2013 rat W istar 1 m g/kg/d sc DM SO GD15-18 or P N D 1 -4 25-30% brain inhib ition 2 hr after dose on PND4, and 10-20% inhib ition on PND5 No Cognition A ctivity Cognition W in-Shw e m ouse 0.5, 5 P N D 8 -1 1 No e t ai., 2013 C3 H /H e N m g/kg/d sc DM SO Cognition PN D28-35, 91-126 No effect on T-m aze sp o ntaneou s alternation, M &F Increased w orking m em ory errors, 0.5 m g/kg/d only, M & F PND60 G e sta tio n a l: No e ffe ct in o p en field, M &F P o stn a tal: No e ffe ct in o p en field, M &F No NOEL LO EL=l m g/kg/d PN D60-63 PN D46-49, 81-84 G estational: Decreased latency to cro ss and in creased tim e sp e n t in d a rk sid e o n re ten tio n tria l, n o e ffe ct a cq u isitio n in passive avo id ance, F only Postnatal: D ecreased laten cy to cro ss and increased tim e spent in d a rk sid e on re te n tio n trial, no e ffe ct a cq u isitio n in p a ssiv e avo id ance, M & F PND46-49: Decreased novel object exploration and d iscrim ination , 0.5 & 5 m g/kg/d PND81-84: Decreased novel object exploration and discrim ination, 5 m g/kg/d only No NOEL LO EL=0.5 m g/kg/d D ichlorvos Lazarini et rat W istar 8 m g/kg po G D 6 -1 5 No al., 2004 (dilution of te c h n ic a l product); veh icle from form ulation F e n itro th io n Leh otzky e t rat Lati 5, 10, 15 G D 7 -1 5 No (sum ithionR, al., 1989 m g/kg/d po 50% ai) s u n flo w e r oil A ctivity Cognition Neuromotor Activity Cognition Social behavior PND21, "a d u lt" "A dult" PND26, 36, 104 PND26, 36, 104 PND42, 104 PND62 PND21: D ecreased locom otion open field, M only "A dult": D ecreased locom otion and increased im m obility, only M tested Decreased latency to cross on re te n tio n trial in p a ssiv e a v o id a n c e D ecreased laten cy to fall o ff rota rod P N D 2 6 ,104, not 3 6 ,1 5 m g/kg/d D e cre a sed a ctivity in o p e n field PND104, 15 m g/kg/d S h o r t e r e s c a p e la t e n c y in co n d itio n ed respo nse during acq uisition , 10 & 15 m g/kg/d In cre a se d tim e in so cia l interaction, 10 & 15 m g/kg/d No NOEL LOEL=8 m g/kg/d NOEL=5 m g/kg/d LOEL=10 m g/kg/d No m e n tio n o f se x e ffe cts in T m aze No dose-response for som e m easures 1 M & 1 F per litter but sex not n ested w ith in litte r in sta tistics F o nly affected w ith gestational exposure, both sexes affected w ith p o stn ata l, looks like M m ore affected Large effect sizes N um ber o f dam s not m entioned D iscre p a n cy in te xt o n pup sam ple sizes M o nly tested Sep arate m ice at tw o test tim es Litter allo ca tio n to dose group not described A ssu m es th at ChE inhibition re p o rted b y S lo tkin in ra ts w o u ld be sa m e as in m ice Larger sam ple size at later age Data analyzed as litter but se x not n ested w ith in litte r in sta tistics No effect on physical and reflex prew eanin g developm ent O nly M tested as after PND21 A dult age not given P o stnatal m o rta lity at all do ses (16-17.5% ) No pup allocation described but had to have used som e litterm ates O nly M tested Statistics not described M easured startle, righting, co ntact p lacin g on PN D22 but no results given S h o rte r la te n cy in c o g n itiv e ta sk hard to interpret N o n sig n ifica n t d e cre a se in activity at PN D26 108 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00108 M etham idopho s deCastro et al., 2000 rat W istar 1 m g/kg/d po w ater G D 6 -1 5 M etham idopho s Lim a ta l., 2013 m ouse Sw iss 1 m g/kg/d sc DM SO P N D 3 -9 M ethyl p a ra th io n Crow der et al., 1980 rat Sprague D aw ley 1 m g/kg/d po co rn oil G D 7 -1 5 M ethyl p a ra th io n Gupta et al., 1985 rat W istarFurth F m ated w ith F344 M 1 m g /k g /d in peanut b u tter, 1.5 m g/kg/d po p e a n u t oil G D 6 -2 0 Pilot in n o n p re g n a n t F dosed for 10 d gives 17% plasm a inhib ition at 1 m g/kg/d A ctivity Pilot sho w ed fo r 1 m g/kg/d: ~19% brain inhib ition on PND10; ~36% , 4 6% brain inhib ition 1, 4 hr after dosing on PND3; ~53, 61% brain inhib ition 1, 4 hr after dosing on PND9; no brain in h ib itio n in PN D 6 0 a d u lts No A ctivity Anxiety & Emotion C o g n itio n Activity Cognition Dam s on GD19 show 20, 60% brain inhib ition at 1 ,1 .5 m g/kg/d Pups sho w brain inhib ition up to 50% on PND1, 7, 14, 21, 1 & 1.5 m g/kg/d ; on PN D 28 o n ly 1.5 m g/kg/d Cognition Neurom otor Activity Anxiety & Emotion PND40 PND61 PN D60-61 PND63 PND23, 30, 44, 54, 65 >PND68 PND60 PND60 PND60 PND60 No e ffe ct in o p en fie ld , se x no m entioned No e ffe ct in o p en fie ld , se x not m entioned In cre a se d im m o b ility tim e in forced sw im , sex not m entioned No effect on elevated plus m aze, sex not m entioned No effect on p assive avoidance, sex not m entioned In cre a se d a ctivity in o p en field , o nly PN D23 and 54, sex not m entioned to criterion Slo w e r tran sfe r on 1st, 4th d ire ctio n ch a n ge in T -m a ze learning transfer, sex not m entioned No effect on p assive avoidance No effect on shuttle box a v o id a n c e Slow er latency to bar press & increased days to asym ptote on o perant task (no schedule given), 1 m g/kg/d only, sex not m entioned No effect on rotarod Decreased activity, 1 m g/kg/d only, sex not m entioned Faster cage em ergence, 1 m g/kg/d only, sex not m entioned N O EL=l m g/kg/d No NOEL LO EL=l m g/kg/d No NOEL LO EL=l m g/kg/d No NOEL LO EL=l m g/kg/d but no effects at 1.5 m g/kg/d Used 2 pups/litter but no m ention o f sex, ap p aren tly used as independ en t observatio ns No effect on prew eanin g sw im m ing perform ance D ecreased im m o b ility tim e PND 14 only O pen field m easures w ith really high va riab ility, not reliable 1 M & 1 F per litter No data for M & F separately or m ention o f statistical differences D osing by litter High va ria b ility e sp e cia lly w ith passive avo id ance Prew eaning testin g: possibly decreased w ire clin g tim e (not an alyzed), no effect on righting, startle, p lacem ent response Increased po stnatal m ortality (30% ) Litterm ates used O nly 3 litters used Sm all sam ple size fo r m aze testin g Statistics not m entioned except for m aze tran sfer test, ju st used ttest Sex not m entioned except for m aze tran sfe r test, data not given for M & F M ethods & results curso ry High dose d am s had ch o lin ergic signs, increased resorptio ns Pups m oved to foster m others at 24 hr No effect on prew eanin g reflexive b e h a v io rs Pup allocation not described Statistics barely described M & F apparently tested but data for each not show n or m entioned M ethods cursory No dose-respo nse fo r behavior but th ere is d o se-resp o n se for ChE inhibition 109 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00109 M ethyl p a ra th io n Johnson et al., 2009 rat Sprague D aw ley O xydem eton m ethyl (m etasystoxR, 91 % ai) Clem en s e t al., 1990 rat CD P a ra th io n Al-H achim & Fink, 1968 m ouse CF1 Sensory In cre m e n ts g doses: low 0.2 m g/kg/d throughout; m id 0.2, 0.4, 0.6 m g/kg/d e ve ry 5 -6 d; high 0.3, 0.6, 0.9 m g/kg/d every 5-6 d 0.5, 1.5, 4.5 m g/kg/d w ater P N D 1 -2 1 G D 6 -1 5 3 m g/kg/d po C o rn oil 3 dosing tim es: 1st, 2nd, or 3rd trim ester Low dose show ed 13-15% brain inh ib itio n and high dose show ed 63, 20, 18% brain inhibition o n P N D 2 0 , 30, 40; all doses recovered by PND50 Dam s on GD16 show 30, 54, 72% plasm a inhib ition (RBC sim ilar) & 22, 52, 68% brain inhibition at 0.5, 1.5, 4.5 m g/kg/d Dam s on GD20 show 20, 39, 54% brain in h ib itio n at 0.5, 1.5, 4.5 m g/kg/d, 40% RBC inhib ition at 4.5 m g/kg/d, and no plasm a inhibition Fetuses on GD20 sh o w no brain in h ib itio n No Cognition C o g n itio n A ctivity C o g n itio n P a ra th io n Al-H achim & Fink, 1968 m ouse CF1 3 m g/kg/d po C o rn oil 3 dosing No tim es: 1st, 2nd, or 3rd trim ester A ctivity P a ra th io n Levin e t al., 2010 rat Sprague D aw ley 0.1, 0.2 m g/kg/d sc DM SO P N D 1 -4 C ites Slo tkin e t al. 2006: 0.1 m g/kg/d produced 5-15% brain inhib ition on PN D 5, no data fo r 0.2 m g/kg/d Cognition PND120 P N D 2 9 -6 0 No effect on aco u stic startle response Increased w orking m em ory erro rs, m id and high dose, M o n ly Increased reference m em ory e rro rs, all do ses, M o n ly No NOEL LO EL=0.2 m g/kg/d PND25, 26, 35 No effect on M -m aze NO EL=4.5 m g/kg/d No e ffe ct in o p en field PN D30-37 No effect on co nditio ned avo id ance learnin g NOEL=3 m g/kg/d P N D 6 0 -6 6 No e ffe ct in o p en field NOEL=3 m g/kg/d PND420, 510, 570 PN D420: Increased w orking m e m o ry e rro rs in ra d ial arm m aze, 0.1 m g/kg/d, M only; increased reference m em ory errors, 0.1 & 0.2 m g/kg/d, M o n ly PN D510: Increased w orking m e m o ry e rro rs in ra d ial arm m aze, 0.1 & 0.2 m g/kg/d, M o n ly P N D 5 7 0 : N o e ffe ct in ra d ial arm No NOEL LO EL=0.1 m g/kg/d O nly 4/do se fo r o p eran t testin g Sp lit-litter dose design In clu d e d litte r as ra n d o m e ffe ct in s ta tis tic s No effect on prew eanin g m easures of reflex developm ent No e ffe ct in F High dose d am s had trem o rs 1 M & 1 F per litter No data for M & F separately or m ention o f statistical differences Statistics barely described No effect on prew eaning reflex or sensory tests V ery sim ilar experim ent as o ther papers, m aybe sam e study Pup allocation not clear but litterm ates probably used Inadequate statistics No m ention of sex V ery sim ilar experim ent as o ther papers, m aybe sam e study Pup allocation not clear but litterm ates probably used Inadequate stats No m ention of sex L itte rm a te s o f th o se u se d in Tim o feeva 2008 Pups & d am s re d istrib u te d daily 5% m ortality high dose No e ffe ct in F A ccepts p<0.1 as sign ifican t for in te ra c tio n s No dose-response for several m easures 110 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00110 m aze P a ra th io n P a ra th io n Stam per et al., 1988 rat Long Evans 1 3 , 1.9 m g/kg/d se co rn oil P N D 5 -2 0 35, 6 8 % brn inh PND21; 26, 36% brn in PN D 28 A ctivity PND24 Neurom otor Cognition PND24 PND24, PND 35-37 No e ffe ct in o p en field No effect on rot arod D e cre a sed a lte rn a tio n ra te in Tm aze, 1.3 & 1.9 m g/kg/d, only M tested Increased w orking m em ory errors, 1.3 & 1.9 m g/kg/d, o nly M tested No NOEL LO EL=1.3 m g/kg/d Tim ofeeva e t al., 2008 rat Sprague D aw ley 0.1. 0.2 m g/kg/d se DM SO P N D 1 -4 C ites S lo tkin e t al. 2006: 0.1 m g/kg/d produced 5-15% brain inhib ition on PN D 5, no data fo r 0.2 m g/kg/d A ctivity Cognition Anxiety & Emotion S e n so ry PN D58-61 PN D35-45 PND112182 PN D50-53 PN D64-72 P N D 8 1 -9 4 PN D78-81 No e ffe ct in fig u re -8 c h a m b e rs No e ffe ct in T -m a ze sp o ntaneou s alternation Decreased w orking m em ory e rro rs in rad ial arm m aze, 0.1 m g/kg/d only, M & F In cre a se d tim e in o p e n a rm s in elevated plus m aze, 0.2 m g/kg/d, M&F No effect on no velty suppressed fe e d in g No effect on ch o co late m ilk preference D ecreased tactile startle, 0.2 m g/kg/d, M&F No effect on prep ulse inhibition No NOEL LO EL=0.1 m g/kg/d Sp lit-litter dose design Pup allocation not clear but litterm ates probably used High dose produ ced ch o lin ergic signs, says doses are 33 and 50% o f LD 50 in PN D 5 rat D ecreased w eight gain w ith both doses Prew eaning, increased cliff avo id ance latency, no effect righting, n egative geo taxis, open field M o nly tested No post-hoc co m p ariso n of group s w hen significant, but lo o k s like e ffe cts in b o th d o ses No d o se -re sp o n se in w o rkin g m em ory errors Pups & d am s re d istrib u te d daily 5% m ortality high dose A ccepts p<0.1 as sign ifican t for in te ra c tio n s A ll r a d ia l a rm m a z e e ffe c ts o n ly in low dose Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 Ill ED 002061 00044425-00111 Appendix 2. Summary of Guideline DNT Studies Submitted to the Agency for OPs other than Chlorpyrifos. (Only changes that were observed after exposure had ended (post weaning, adult) are listed. 'X' indicates no significant changes on tests for each domain.) Chemicals Acephate Azinphos-methyl Coumaphos Diazinon Dichlorvos1 Dicrotophos Dimethoate Disulfoton Ethoprop Fenamiphos Malathion Methamidophos Methyl parathion Cognition X X X Biel maze: increase errors & latency high dose (~33.1 mg/g,/d, dam diet) M, PND24 & PND62; also mid dose (~3.4 mg/kg/d, dam diet) F, PND24 Motor activity X X X X Acoustic startle X X X X X X X M maze: increase trials to criterion high dose (~29.3 mg/kg/d, dam diet) M, PND60 X X ~X -- - X X X X X X X X X increased rearing in FOB open field mid dose (50 mg/kg/d to dams & pups), F, significant at PND45 only (maybe also PND60); no change automated motor activity X X increased peak amplitude later blocks (perhaps habituation effect) all doses (5, 50,150 mg/kg/d to dams & pups), F only, PND23; increased peak amplitude without prepulse low dose only, F only, PND60 decreased peak amplitude early blocks mid & high dose (~1.65, 5.2 mg/kg/d dam diet), F only, significant at PND38, looks same but not significant at PND60 Neuromotor (FOB) X X X X X X X X X altered gait mid & high dose (50,150 mg/kg/d to dams & pups), M & F, PND60 but not earlier X X X X X Notes PA PND24: report says decreased latency but nothing significant & table is questionable; M maze PND60: report says increased trials to criterion (M) and increased (M) or decreased (F) errors, but nothing significant and table is questionable Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 112 ED 002061 00044425-00112 Naled Phorate Profenofos Terbufos Tetrachlorvinphos Tribufos Trichlorfon X X M maze: decrease number X reaching criterion with relearning, low & mid dose (0.03 & 0.1 mg/kg/d to dams & pups), M only significant, PND30; not seen in second study with higher dose X X X X PA: decreased latency to enter on retention high dose (~205.1 mg/kg/d dam diet), M only, PND29; M maze: increased average errors second trial high dose, F only, PND60 X X X X decreased activity middle blocks (maybefaster habituation) mid dose (~76.2 mg/kg/d dam diet) only, F only, PND60 increased peak amplitude & X decreased latency middle blocks low dose only (0.4 mg/kg/d to dams & pups), F only, PND60; report says decreased amplitude but not significant all blocks high dose (10 mg/kg/d to dams & pups), M only, PND23 & PND60 decreased peak amplitude all blocks X high dose (0.1 mg/kg/d to dams & pups), M only, PND60; not seen in second study with higher dose X X X X X X X X decreased peak amplitude all blocks X high dose (~205,1 mg/kg/d dam diet), early blocks mid dose (~76.2 mg/kg/d dam diet), M & F, PND22; decreased amplitude middle blocks (not consistent) all doses (~23.3, 76.2, 205.1 mg/kg/d dam diet), F only, PND38; for M decreased amplitude apparent but not significant high dose PND38 & PND60 1high pup mortality in all groups, including control, negated any valid neurotoxicity assessments of dichlorvos only swimming time in Y maze reported, varied significances, no mention of errors or other performance measures combined two studies; one with low & mid dose, other with high dose; some data didn't agree Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 113 ED 002061 00044425-00113 Appendix 3. Low Quality Studies: Summary of Study Design Elements Impacting Study Quality Assignment Cohort Name / Description of Study Study Study Location Population Study Design Exposure Assessment Outcome Assessment A c o s ta -M a ld o n a d o e t ai. C h ih u a h u a , M exico Sin g le to n p re g n a n cie s C ro ss-se ctio n a l, sm a ll Pro xy In d icato r of e xp o su re - S ta n d a rd ize d b u t p a rtia lly su b je c tiv e (2 0 0 9 ) p ilo t s tu d y - o n ly 9 R e s id e n c e in a g r ic u lt u r a l a s s e s s m e n t of p la ce n ta l m atu rity w om en exposed, p a rticip a n t se le c tio n an d co m m u n ity w ere p e stic id e s had b e e n a p p lie d ; or h o m e lo ca te d < 5 e x clu sio n not d e ta ile d km fro m a p e stic id e a p p lica tio n N = 5 4 m others (9 e xp o se d , 4 5 zo n e ; o r c o h a b ita tin g w ith w o rk e r e x p o se d to p e stic id e s or a g ricu ltu ra l lab o r co m p ariso n m o th e rs) Confounder/ Covariate Control M in im a l. A d ju s tm e n tfo r p ia c e n ta i c h a ra cte ristic s. Statistical Analysis A p p ro p ria te m u ltiv a ria te a n a ly sis; C o rre cte d h y p o th e sis te s t re su lts for m u ltip le c o m p a riso n s. Risk of (other) Bias S e le c tio n b ia s u n lik e ly; R e s id u a l c o n fo u n d in g likely; su b s ta n tia l p o te n tial for d iffe re n tia l m isc la s s ific a tio n of exposure. D a w b ro v v sk i e t al. (2 0 0 3 ) C a rtie r e t a!. (2 0 1 6 ) F ie ld e r e t ai. (2 0 1 5 ) G ra n d je a n et a l.(2 0 0 6 ) Lo d z, P o lan d N ew b o rn ch ild re n a m o n g P o lish fa rm e rs C a se -co n tro l, large s a m p le size N=389 Age: new borns Brittan y, F ra n ce P E LA G IE stu d y (Fren ch p o p u latio n ; p re g n a n t w o m e n a n d ch ild re n ) L o n g itu n d in a ! birth cohort n = 231 M o th e r-in fan t p a irs B a n g k o k , T h a ila n d area T h a i p o p u la tio n (C h ild re n ag e d 6 to 8, w ho w ere fro m tw o fa rm in g re gio n s (a q u a c u ltu re v. rice ) Cohort, sm a ll sa m p le s iz e n = 54 Age 6- 8 years Tabacundo, Ecuador H e a ith y 2 n d an d 3 rd g ra d e ch ild re n C ro ss-se ctio n a l, sm a ll s a m p le size N=72 Age < 9 years A lso , A C h E a ctiv ity - O b je ctiv e b io m ark e r of e xp o su re /a lte re d fu n ctio n P re n a ta l p e stic id e e xp o su re a s s e s s e d via q u e stio n n a ire (re tro sp e c tiv e se lf-re p o rt). S ite v is it a fte r d e liv e ry to e v a lu a te p e stic id e e xp o su re P re g n a n cy o u tco m e s a s s e s s e d u sin g birth re co rd s A p p ro p ria te . In c lu d e d m a te rn a l d e m o g ra p h ic s, p re d icto rs o f h ig h -risk p re g n a n cy (d u ra tio n , m a te rn a l w e ig h t) an d e n v iro n m e n ta l to x ic a n t e x p o su re (E T S ). No a d ju stm e n t fo r S E S In d icato rs A p p ro p ria te m u ltiv a ria te a n a ly s is . M e ta b o lite c o n c e n tra tio n s (total DAP, DM, and D E) of prenatal OP p e s t ic id e e x p o s u r e q u a n t ifie d in tw o m a te rn a l s p o t u rin e s a m p le s p ro vid e d at 1 6 a n d 2 6 w e e k s of g e sta tio n M a te r n a l IQ a n d t h e f a m ily e n v iro n m e n t w ere a lso a s s e s s e d u s in g th e W e c h s le r A d u lt In te llig e n c e S c a le a n d th e FIO M E te st W e c h sle r In te llig e n ce S c a le a d m in iste re d to ch ild re n to d e te rm in e IQ A ll m o d e ls w e re a d ju s te d fo r FIO M E sc o re , b r e a s t f e e d in g d u r a t io n , m o t h e r s ' IQ, s c h o o l, m a te rn a l e d u c a tio n level, p sy c h o lo g ist t e s tin g th e ch ild , a n d c re a tin in e le v e ls of m o th e r an d ch ild A p p ro p ria te : Lin e a r re g re ssio n an d m u ltiv a ria te m o d e ls C h ild E x p o su re - T o tal D EA P , D AP, a n d D M A P m e ta b o lite s m e a su re d in s p o t u rin e s a m p le s , c o lle c te d a t th re e, s e p a ra te tim e p o in ts (e ve ry 6 m onths) B A R S e x a m in a tio n a d m in iste re d to ch ild re n to d e te rm in e n e u ro b e h a v o ria l p e rfo rm a n ce . A ll m o d e ls w e re a d ju s te d fo r a g e a n d th e FIO M E s c a le A p p ro p ria te : M u ltiva ria te lin e a r re g re ssio n m o d e l a n d m ix m o d e l lin e a r re g re ssio n to d e te rm in e in te ra ctio n P a re n ta l e d u ca tio n w a s a s s e s s e d via a v o c a b u la ry te st; th e h o m e e n v iro n m e n t w a s sc o re d via th e FIO M E te st P re n a ta l o ccu p a tio n a l e xp o su re a s s e s s e d via q u e stio n n a ire ; A lso re ce n t ch ild e x p o su re b io m a rk e r a s s e s s m e n t (D A P). O b je ctiv e a n th ro p o m e tric an d o th er clin ica l o u tco m e s; N u m e ro u s n e u ro b e h a vio ra l o u tco m e s e va lu a te d u sin g e a sy -to -a d m in lste r s c re e n in g in stru m e n ts; A g e a p p ro p ria te . M ay be in se n sitiv e to su b tie e ffe cts of O P p e stic id e e xp o su re e ffe cts. A p p ro p ria te . F lo m o g e n o u s p o p u la tio n lim ite d c o n fo u n d in g by d e sig n ; in clu d e d ch ild d e m o g ra p h ic s (a ge , se x, w eigh t); S E S in d ica to rs (m a te rn a l race , h o u sin g, ru n n in g w ater, se w a g e ), d iet (m e a ls/d a y ), e n v iro n m e n ta l to x ic a n ts (m a te rn a l alco h o l an d s m o k in g ) an d m e d ic a l h istory. A p p ro p ria te m u ltiv a ria te a n a ly sis; N u m e ro u s h yp o th e se s e va lu a te d w ith o u t co rre ctio n fo r m u ltip le co m p a riso n s. S e le c tio n b ia s u n lik e ly; R e s id u a l c o n fo u n d in g lik e ly s m a ll in m a g n itu d e ; c o n sid e ra b le p o te n tial for n o n -d iffe re n tial m isc la ssifica tio n of e xp o su re (u n lik e ly to a c c o u n t fo r n o n null fin d in g s). M in im al m isc la ssifica tio n of o u tco m e . U rin e co lle c tio n m e th o d s a n d sto ra g e (u rin e s a m p le s c o lle c te d a t h o m e an d m a ile d b a c k to lab o ra to ry); la c k of fre q u e n t u rin e s p o t s a m lin g a n d d u e to th e s h o rt h a lf-life o f u rin a ry m e ta b o lite s co u ld h a v e led to e x p o s u re m isc la s s ific a tio n S m a ll s a m p le s iz e (n = 5 4 ); e r ro rs in e x p o s u r e c la ssific a tio n m e th o d s, sp e c ific a lly , u rin e co lle ctio n m e th o d s a n d sto ra g e (u rin e s a m p le s c o lle cte d a t hom e); s h o rt h a lf-life of u rin a ry m e ta b o lite s co u ld h a v e led to e xp o su re m isc la s s ific a tio n S e le c tio n b ia s u n lik e ly; R e s id u a l c o n fo u n d in g lik e ly s m a ll in m a g n itu d e ; c o n sid e ra b le p o te n tial for n o n -d iffe re n tial m isc la ssifica tio n of e xp o su re (lik e ly to a c c o u n t fo r null fin d in g s). P o te n tial m isc la ssifica tio n of o u tco m e . 114 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00114 Study H a ra rj e t al. (2 0 1 0 ) Study Location Tabacundo, Ecuador (A n d e an p la te a u north of Q u ito ) Cohort Name / Description of Study Population 6 - to 8 -ye a r-o ld c h ild re n In th e tw o lo w e s t g r a d e s (ca lle d se co n d a n d th ird ) o f one of tw o sch o o ls Study Design C ro ss-se ctio n a l, sm a ll sa m p le size n = 81 Age 6- 8 years K o fm a n e t al. (2 0 0 6 ) Israe l (N e g e v re gio n ) B e d o u in p o p u la tio n (C h ild re n a g e d 6 to 1 2 at th e tim e of th e stu d y, w ho w ere v ictim s o f p o iso n in g b efo re ag e th re e) R e tro sp e ctiv e co h o rt stu d y, sm a ll s a m p le size N = 52 9 -E x p o se d to O P; 1 7 -E x p o se d to K e ro s e n e /p a in t th in n e r 2 6 -C o n tro ls Aged 6 -12 years Exposure Assessment C h ild C u rre n t E x p o su re - D M P m e t a b o lit e s m e a s u r e d in a s p o t u rin e s a m p le s . A blo o d s a m p le w a s a n alyze d fo r A C h E. M a te rn a l E x p o su re - In te rvie w by s k ille d in te rv ie w e rs Outcome Assessment B lo o d p re ssu re a n d n e u ro p h y sio lo g ic m e a su re s - th e In stru m e n ts u se d for n e u ro p h sy c h o lo g lc m e a s u re s w ere v a lid a te d to avo id cro ss-cu ltu ra l in flu e n ce s. Confounder/ Covariate Control A p p ro p ria te : c h ild 's s e x , a g e , B M I, n u m b e r o f d a ily m e a ls (o n ly In c u r r e n t e x p o s u r e ), stu n tin g , h e m ato crit, s c h o o l gra d e , h a v in g re p ea te d one gra d e , m ate rn a l e d u catio n le v e l, fa m ily liv in g in a t r a d itio n a l h o u s e , d rin k in g w ate r su p p ly, an d p a tern a l e d u ca tio n an d e m p lo y m e n t Statistical Analysis A p p ro p ria te : S ta n d a rd p a ra m e tric te sts an d lo g istic re g re ssio n O P p o iso n in g w a s co n firm e d by low s e ru m b u ty ry lc h o lin e s te ra s e a ctivity b a se d on h o sp ita l re co rd s N e u ro p sy ch o lo g ica l e v a lu a tio n an d stru c tu re d in te rv ie w o f p a re n ts. E rro rs In a s s e s s m e n t m in im iz e d a s p sy c h o lo g ists w ere q u a lifie d in d iv id u a ls, la n g u a g e an d cu ltu ra l d iffe re n c e s ta k e n Into c o n s id e ra tio n , e a c h c h ild t e s t e d o n s a m e d a y a n d in s a m e p la c e a s m a tch e d co n tro l. Age, se x, b a ck g ro u n d (cu ltu ral an d d e m o g ra p h ic) D iffe r e n c e in m e a n s Risk of (other) Bias E rro rs in e x p o s u r e c la s s if ic a t io n s t a t u s s in c e It w as b a sed on the m aternal se lf-re p o rt, m o th e rs lik e ly b e in g a w a re of th e n e u ro b e h avio ra ! s ta tu s of th e ir ch ild re n , e xp o su re a sse ssm e n t based on a sp ot u rin e s a m p le E rro rs is o u tco m e c la s s ific a tio n lik e ly sin ce p sy ch o lo g ists w ho ad m in iste re d th e te sts knew w h ich c h ild re n w ere e xp o se d , s m a ll s a m p le size K o u tro u ia k is e t al. (2 0 1 4 ) Crete, G reece W o m e n w ith s in g le to n p re gn a n cie s, p e rm a n e n t re sid e n ts fo r at le a st tw o ye a rs, referral for a m n io c e n te sis to the F e ta l-M a te rn a l U nit, D e p a rtm e n t of O b ste trics a n d G yn e co lo gy, U n ive rsity H o sp ital of H e ra kllo n P ro sp e ctiv e C o h o rt S tu d y - large s a m p le size , e th ica l Is su e s n = 415 Age: new borns O b je c tiv e . D A P m e a s u r e m e n t in a s in g le a m n lo tic flu id s a m p le s c o lle cte d at e ith e r 1 6 th or 2 0 th w e e k s of g e sta tio n - N ovel b io m a rk e r. Q u e stio n n a ire w as a lso used . B irth w e ig h t a n d h e a d c irc u m fe re n ce . U n cle a r h ow th e o u tco m e in fo rm a tio n w as o b tain ed . N e o n a ta l se x, m ate rn a l age, a g ricu ltu ra l activitie s, an d g e sta tio n a l a g e at a m n io c e n te s is - R a tio n a le fo r c o n fo u n d e r se le c tio n n o t p ro vid e d A p p ro p ria te : m u ltip ie lin e a r re gre ssio n C o m p a rin g e xp o su re m e a s u r e m e n t s in A F a g a in s t kn o w n b io m a rk e r (e.g., O P m e ta b o lite le v e ls in u rin e ) fo r v a lid a tio n of A F not co n d u cte d . S m o k in g sta tu s of p a rtic ip a n ts b e fo re /d u rin g p re g n a n cy , h igh risk p re g n a n cie s, g e sta tio n a l d iab e te s, P O N 1 en zym e a c tiv ity in th e f e t u s e s n o t co n sid e re d . Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 115 ED 002061 00044425-00115 Appendix 3. Study L iza rd j e t al. (2 0 0 8 ) Low Quality Studies: Summary of Study Design Elements Impacting Study Quality Assignment (continued) Study Location Cohort Name / Description of Study Population Study Design Exposure Assessment Outcome Assessment Y u m a C o u n ty, A rizo n a, U SA C h i l d r e n 's P e s t ic i d e Su rve y (C PS) C ro ss-se ctio n a l, sm a ll sa m p le size N=48 Age 7 years O b je ctiv e B lo m a rk e r of p re n a ta l O P p e stic id e e xp o su re (D A P) q u a n tifie d in s in g le c h ild s p o t u rin e s a m p le p ro v id e d a t th e tim e of th e co g n itive assessm en t. C o g n itiv e a s s e s s m e n t u s in g b a tte ry of te s t in s t r u m e n t s c o n s id e r e d v a lid a n d re lia b le in s im ila r p o p u la tio n s. S p a n is h tra n sla tio n a s n e ce ssa ry . Confounder/ Covariate Control None Statistical Analysis C o rre la tio n c o e fficie n ts (type u n sp e cifie d ). S ta tistic a lly sig n ific a n t co n fo u n d e rs w ere not ro b u st d u e to in flu e n tia l o u tlie rs Risk of (other) Bias S e le c tio n b ia s u n likely; R e s id u a l c o n fo u n d in g likely: c o n s id e ra b le p o ten tial fo r n o n -d iffe re n tia! m lsc la s s lfic a tio n o f e xp o su re P o te n tial m isc la ssifica tio n of outcom e. Lu e t al. (2 0 0 9 ) C o ta B ru s, C o sta R ica M o ren o -B an d a et a l. (2 0 0 9 ) M e xico (V illa G u e rre ro , C o a te p e c de H arin as, T e n a n c in g o (M e xico ); C u e rn a v a c a , C u a u tla , Jiu te p e c, Te m ixco , (M o re lo s) N e vso n (2 0 1 4 ) U S . N a tio n a l p o p u la tio n 4 -1 0 yr. old ch ild re n w h o s e p a re n t s w o r k e d in o rg a n ic co ffe e farm (La A m lsta d ) and co n ve n tio n a l co ffe e fa rm s (L a s M e lliza s) N ew b o rn ch ild re n of flo ricu ltu ra l w o rk e rs an d fa m ilie s C ro s s -S e c tio n a l (pilot), low s a m p le n u m b e rs N=35: 1 7 O rg a n ic farm 1 8 C o n v e n tio n a l fa rm s A g e 4 -1 0 y e a rs old C ro ss-se c tio n a l, large sa m p le size N=328 Age: new borns G ood m e a su re (u rin ary PN P, M PY, T C P y ), b u t no m ajo r d iffe re n ce s betw een e xp o su re g ro u p s P ro xy in d ica to r of p re n ata l o cc u p a tio n a l O P p e sticid e e xp o su re (se lf-rep o rte d flo ricu ltu ra ! o c c u p a tio n ) G ood m e a su re (C B A R S ) but d iffe re n t S E S an d d e m o g ra p h ic c h a ra cte ristic s fo r exposure groups Lim ite d n u m b e r (gro u p , age , sex, h an dedness, grade) A p p ro p ria te : L in e a r m ixe d e ffe c ts fo r s ig n ific a n t te s t o u tco m e s fro m p a ire d t-te st a n a ly s e s S o m e w h a t h igh - C o n v e n ie n ce s a m p le w ith d iffe re n t re c ru itm e n t m ethods; exposure m iscla ssifica tio n O b je c tiv e ly m e a s u re d birth o u tc o m e s a s s e s s e d u s in g birth c e rtific a te (p a rtia l). S e lf-re p o rte d b y m o th e r if b irth c e rtific a te u n a v a ila b le . A p p ro p ria te , th o u g h m in im a l - h isto ry o f a d v e rse re p ro d u ctive o u tco m e s, In fan t se x, m ate rn a l sm o k in g an d a lco h o l u se d u rin g pregnancy. A p p ro p ria te m u ltiva ria te a n a ly s is S e le c tio n b ia s likely; R e s id u a l c o n f o u n d in g lik e ly s m a ll in m a g n itu d e ; c o n s id e ra b le p o te n tial fo r n o n -d iffe re n tial m isc la s s ific a tio n o f e xp o su re (lik e ly a c c o u n ts fo r null fin d in g s). P o te n tial o u tco m e m isc la s s ific a tio n (s e lf-r e p o r t In s u b s e t o f p a rticip a n ts). C h ild re n w ith b irth y e a rs 1 9 7 0 - 2 0 0 5 in 1 ) C a lifo rn ia D e p a rtm e n t of D e ve lo p m e n ta l S e rv ic e s (C D D S ) reports 2) In d iv id u a ls w ith D isa b ilitie s E d u ca tio n A ct (ID EA ) reports E c o lo g ic a l (tim e tre n d ) N =N P (national database) N o n -sp e cific, p ro xy O P e xp o su re m e a su re (lb s/yr) Tw o large re p o rtin g D B s: C A D e p a rtm e n t of D e ve lo p m e n ta l S e rv ic e s (C D D S ), U S In d iv id u a ls w ith D is a b ilit ie s E d u c a tio n A c t (ID EA ) L im it e d : d if f e r e n c e s in a u tism d e fin itio n s, c h a n g e s in d ia g n o s t ic c rite ria A p p ro p ria te : ratio o f a g e -re s o lv e d s n a p sh o t: tr a c k in g tre n d slo p e s; co rre la tio n co e fficie n t b etw een te m p o ra l tre n d an d co m p o site a u tism p re v a le n ce cu rv e S e le c tio n b ia s u n likely; do not know in d ivid u a l e xp o su re Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 116 ED 002061 00044425-00116 Appendix 3. Low Quality Studies: Summary of Study Design Elements Impacting Study Quality Assignment (continued) Cohort Name / Description of Study Study Location Study Population Study Design Exposure Assessment Outcome Assessment R o h lm a n e t a l. (2 0 0 5 ) O rego n an d North C a ro lin a , U SA La tin o ch ild re n of im m igra n t p a re n ts liv in g in O re g o n o r N orth C a ro lin a C ro ss-S e ctio n a l, sm a ll sa m p le s iz e N= 78 Age 4 8 -7 1 m onths P ro xy In d ica to r of ch ro n ic O P p e sticid e e x p o s u r e - R e s id e n c e In h ig h ly a g ricu ltu ra l c o m m u n itie s B a tte ry of n e u ro co gn itive d e v e lo p m e n t; S c r e e n in g to o ls; S o m e In stru m e n ts lik e ly not a p p ro p r ia te fo r u s e In s tu d y p o p u la tio n - N o t all p a rtic ip a n ts ab le to co m p le te all e v a lu a tio n s. Poor ad m in istra tio n . T e sts a d m in iste re d tw ice ; o n ly p e r f o r m a n c e o n 2 nd e v a l u a t io n c o n s id e r e d In a n a ly s is . Confounder/ Covariate Control S e lf-re p o rte d c o v a ria te In fo rm atio n c o lle c te d via q u e stio n n a ire . A d ju stm e n t fo r age, S E S in d ica to r (m a te rn a l e d u ca tio n ). No e n v iro n m e n ta l to x ic a n ts. Statistical Analysis O n e -sid e d h y p o th e sis te sts. N u m e ro u s h y p o th e se s e v a lu a te d w ith o u t co rre ctio n fo r m u ltip le c o m p a riso n s. Risk of (other) Bias S e le c tio n b ia s u n likely; R e s id u a l c o n fo u n d in g likely; s u b s ta n tia ! p o ten tial fo r d iffe re n tia! m isc la sslflca tlo n of e xp o su re an d o utco m e. In ad e q u ate p re se n ta tio n o f s tu d y re su lts. S e e m in g ly p o st-h o c e va lu a tio n of e ffe ct m o d ificatio n . S a m a r a w ic k r e m a e t al. (2 0 0 8 ) Southern Srl Lanka Pregnant w om en d e liv e rin g at E m b ilip ltiy a B a s e H o s pita 1 C ro ss-se c tio n a l B irth C oho rt, s m a ll sa m p le size N = 4 1 en d of tw o p e sticid e sp ra y in g s e a so n s; N = 2 5 at b e g in n in g of sp ra y in g se a so n Pro xy In d icato r of p re n ata l O P p e stic id e e x p o su re (d e liv e ry d u rin g p e stic id e s p ra y se a s o n ); O b je ctiv e p e sticid e b io m a rk e rs a s s e s s e d (O P p e s t ic id e r e s id u e s ), b u t d e t e c t e d in o n ly tw o s u b je c t 's s p e c im e n s - n o t e va lu ate d O b je ctiv e b io m a rk e rs o f e a rly b io lo g ica l e ffe ct o u tco m e s M aternal an d feta! b u ty ry lch o lin e ste ra se (B u C h E ) activity; a n tio x id a n t sta tu s; fe ta l o xid a tiv e stre ss; fe tal D N A fra g m e n ta tio n No a d ju s tm e n tfo r p o ten tial co n fo u n d e rs (th o u gh co m p a riso n g ro u p s w ere co n sid e re d to be re la tive ly h o m o g e n e o u s). La rg e ly a p p ro p ria te . A s s u m p tio n s of so m e s ta tis tic a l te sts lik e ly vio late d . S e le c tio n b ia s u n lik e ly; R e sid u a ! c o n fo u n d in g likely; s u b s ta n tia ! p o ten tial fo r d iffe re n tia! m isc la sslflca tlo n of e xp o su re ; S e e m in g ly p o st-h o c e va lu a tio n of e ffe ct m o d ificatio n . S a v it z e t al. (1 9 9 7 ) O n tario , C a n a d a N e w b o rn c h ild r e n in th e O n tario Fa rm F a m ily H eaIth Stu d y; R e tro sp e c tiv e B irth C oho rt, large sa m p le size N = 1 ,8 9 8 co u p le s; 3 ,9 8 4 Age: new borns Pro xy In d icato r of p re -co n ce p tio n p atern al p a ra -o ccu p a tio n a l OP p e sticid e e xp o su re (se lf-re p o rte d m ale fa r m a c t iv it ie s In 3 -m o n th p e rio d p rio r to co n ce p tio n . O b je c tiv e ly m e a s u re d birth outcom es a sse sse d by m aternal se lf-re p o rt A p p ro p ria te . In c lu d e d fa m lly / c h ild d e m o g ra p h ic s (se x, w eigh t, m a te rn a l age, eth n icity); S E S in d ica to rs (m a te rn a l an d p a te rn a l e d u ca tio n an d o ccu p a tio n , per ca p ita In co m e ) race , h o u sin g , ru n n in g w ater, se w a g e ), d ie t (m e a ls/d a y ), p re g n a n cy risk s (m a te rn a l ca ffe in e , a lco h o l an d sm o k in g ) an d m e d ic a l h istory. In a p p ro p ria te m u ltiva ria te re g re ssio n a n a ly sis. Lik e ly m lssp e c ific a tio n of true v a ria n ce ; N o a d ju s tm e n tfo r m u ltip le co m p a riso n s. S e le c tio n b ia s p ro b a b le ; R e s id u a l c o n fo u n d in g likely; s u b s ta n tia l p o ten tial fo r d iffe re n tia! m isc la sslflca tlo n of e xp o su re an d outcom e. W ic k e rh a m e t al. (2 0 1 2 ) N aksen et al (2 0 1 5 ) Z h e jia n g P ro vin ce, C h in a F a n g d istrict, C h ia n g M ai p ro vin ce, T h a ila n d N e w b o rn ch ild re n d e live re d at th e F u y a n g M aternal and C h i l d r e n 's h o s p it a l C ro s s -s e c tio n a l, s m a ll p ilo t stu d y n=116 A ge: Fu ll te rm in fa n ts O b je ctiv e b lo m a rk e r o f p e stic id e e x p o s u r e ( p e s t ic id e r e s id u e s In c o rd b lo o d ) - p a ra m e te rize d a s n u m b e r of p e sticid e re sid u e s d e te cte d . M eth o d s u n lik e ly s u ita b le fo r d e te c t in g low leve ls. Pregnant w om en d e liv e rin g at F a n g Flo sp ital P ro s p e c tiv e C o h o rt, s m a ll p ilo t stu d y n= 52 Age: new borns O b je ctiv e b lo m a rk e rs o f p e stic id e e xp o su re (D A Ps). A lso A C h E . B C h E, an d P O N I ge n o typ e e xp re ssio n m e a su re m e n t. M a te rn a l b lo o d an d u rin a ry s a m p le s ta k e n , p lu s co rd blo o d . Q u e stio n n a ire to a s s e s s o th er e x p o su re s an d c o v a rla te s. B irth w e ig h t a s s e s s e d u s in g birth reco rds and m aternal report B irth o u tco m e s (B o d y w e ig h t an d len gth , an d h e a d c irc u m fe re n ce ) a b stra c te d fro m m e d ic a l re co rd s. A p p ro p ria te . A s s e s s e d u s in g q u e stio n n a ire (m a te rn a l se lf-re p o rt) an d m e d ica l re co rd s. A p p ro p ria te . A s s e s s e d u s in g q u e stio n n a ire (m a te rn a l se lf-re p o rt) an d m e d ica l re co rd s. A p p ro p ria te m u ltiv a ria te a n a ly s is . S e le c tio n b ia s u n lik e ly: R e s id u a l c o n fo u n d in g likely: s u b s ta n tia ! p o ten tial fo r d iffe re n tia! m isc la sslflca tlo n of e xp o su re . S o m e o u tco m e m is c la s s ific a tio n u n lik e ly . S o m e e rro rs in s t a t is t ic a l a n a ly s is w e re id e n tifie d ; e.g., fo r g e sta tio n a ge , log total D EA R at 3 2 w ee ks of p regnancy, a 0 .7 beta w as reported, but the c o n fid e n c e in te rv a l is re p o rte d a s (-0 .1 , -1 .4 )]. N o a d ju s tm e n t fo r m u ltip le co m p a riso n s. In a d e q u a te p re se n ta tio n o f stu d y re su lts. S e le c tio n b ia s p o ssib le d u e to lo s s to fo llo w -u p ; R e s id u a l c o n f o u n d in g lik e ly , s m a ll in m a g n itu d e ; p o te n tia l fo r n o n d iffe re n tia! m is c la s s ific a tio n of e xp o su re . Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 117 ED 002061 00044425-00117 Appendix 4. Table of Systematic Review Analysis: Initial Search Research & Initial Exclusion Criteria. Initial search yielded 299 studies. 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). Number Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-English Abdallah, M. A. E., & Covaci, A. (2014). Organophosphate flame retardants in indoor dust from 1 Egypt: implications for human exposure. [Journal article]. E n v i r o n m e n t a l S c i e n c e & a m p ; T e c h n o l o g y , 4 8 , 4782-4789. EXPO Acosta-Maldonado, B., Sanchez-Ramirez, B., Reza-Lopez, S., & Levario-Carrillo, M. (2009). 2 Effects of exposure to pesticides during pregnancy on placental maturity and weight of newborns: a cross-sectional pilot study in women from the Chihuahua State, Mexico. H u m E x p EPI 451-459. T o x i c o l , 2 8 ( 8 ) , Ali, P., Anwer, A., Bashir, B., Jabeen, R., Haroon, H., & Makki, K. (2012). Clinical pattern and 3 outcome of organophosphorus poisoning. [Article]. J o u r n a l o f t h e L i a q u a t U n i v e r s i t y o f M e d i c a l 15-18. a n d H e a l t h S c i e n c e s , 1 1 , ACR Alizadeh, A. M., Hassanian-Moghaddam, H., Shadnia, S., Zamani, N., & Mehrpour, O. (2014). Simplified acute physiology score ll/Acute physiology and chronic health evaluation ii and 4 prediction of the mortality and later development of complications in poisoned patients ACR admitted to intensive care unit. [Article]. B a s i c a n d C l i n i c a l P h a r m a c o l o g y a n d T o x i c o l o g y , 1 1 5 , 297-300. Andersen, H. R., Debes, F., Wohlfahrt-Veje, C., Murata, K., & Grandjean, P. (2015). 5 Occupational pesticide exposure in early pregnancy associated with sex-specific EPI neurobehavioral deficits in the children at school age. N e u r o t o x i c o l o g y a n d T e r a t o l o g y , 4 7 , 1-9. Attfield, K. R., Hughes, M. D., Spengler, J. D., & Lu, C. (2014). Within- and between-child 6 variation in repeated urinary pesticide metabolite measurements over a 1-year period. [Article]. 201-206. E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s , 1 2 2 , EXPO 118 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00118 Number Attfield, K. R,, Hughes, M. D., Spengler, J. D., & Lu, C. S. (2014). Within- and between-child 7 variation in repeated urinary pesticide metabolite measurements over a 1-year period. [Journal article]. E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s , 1 2 2 , 201-206. Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish DUPLICATE Babina, K., Dollard, M., Pilotto, L., & Edwards, J. W. (2012). Environmental exposure to 8 organophosphorus and pyrethroid pesticides in South Australian preschool children: A cross sectional study. [Article], E n v i r o n m e n t I n t e r n a t i o n a l , 4 8 , 109-120. DUPLICATE Babina, K., Dollard, M., Pilotto, L., & Edwards, J. W. (2012). Environmental exposure to 9 organophosphorus and pyrethroid pesticides in South Australian preschool children: A cross sectional study. [Article], E n v i r o n m e n t I n t e r n a t i o n a l , 4 8 , 109-120. EXPO Balakumar, K., Misha, K., & Milind, K. (2013). Increased fetal endocardial echogenicity 10 mimicking endocardial fibroelastosis following maternal organophosphorus poisoning and its ACR complete regression in utero. [Article], I n d i a n J o u r n a l o f R a d i o l o g y a n d I m a g i n g , 2 3 , 262-265. Baltazar, M. T., Dinis-Oliveira, R. J., de Lourdes Bastos, M., Tsatsakis, A. M., Duarte, J. A., & 11 Carvalho, F. (2014). Pesticides exposure as etiological factors of Parkinson's disease and other neurodegenerative diseases--A mechanistic approach. T o x i c o l o g y L e t t e r s , 2 3 0 , 85-103. Barr, D. B., Ananth, C. V., Yan, X., Lashley, S., Smulian, J. C., Ledoux, T. A., et al. (2010). Pesticide 12 concentrations in maternal and umbilical cord sera and their relation to birth outcomes in a population of pregnant women and newborns in New Jersey. S c i T o t a l E n v i r o n , 4 0 8 ( 4 ) , 790- 795. Bechaux, C., Zetlaoui, M. 1., Tressou, J., Leblanc, J.-C., Heraud, F., & Crepet, A. 1. (2013). 13 Identification of pesticide mixtures and connection between combined exposure and diet. [Article], F o o d a n d C h e m i c a l T o x i c o l o g y , 5 9 , 191-198. REV EPI EXPO Bedi, J. S., Gill, J. P. S., Aulakh, R. S., Kaur, P., Sharma, A., & Pooni, P. A. (2013). Pesticide 14 residues in human breast milk: Risk assessment for infants from Punjab, India. S c i e n c e o f T h e 720-726. T o t a l E n v i r o n m e n t , 4 6 3 - 4 6 4 , EXPO 119 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00119 Number Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish Bellinger, D. C. (2012a). A strategy for comparing the contributions of environmental chemicals 15 and other risk factors to neurodevelopment of children. [Article]. E n v i r o n m e n t a l H e a l t h REV 501-507. P e r s p e c t i v e s , 1 2 0 , Bellinger, D. C. (2012b). Comparing the population neurodevelopmental burdens associated 16 with children's exposures to environmental chemicals and other risk factors. [Article]. REV 641-643. N e u r o T o x i c o l o g y , 3 3 , Berkowitz, G. S., Wetmur, J. G., Birman-Deych, E., Obel, J., Lapinski, R. H., Godbold, J. H., et al. 17 (2004). In utero pesticide exposure, maternal paraoxonase activity, and head circumference. 388-391. E n v i r o n H e a l t h P e r s p e c t , 1 1 2 ( 3 ) , Berton, T., Mayhoub, F., Chardon, K., Duca, R.-C., Lestremau, F., Bach, V., et al. (2014a). 18 Development of an analytical strategy based on LC-MS/MS for the measurement of different classes of pesticides and theirs metabolites in meconium: Application and characterisation of foetal exposure in France. E n v i r o n m e n t a l R e s e a r c h , 1 3 2 , 311-320. Berton, T., Mayhoub, F., Chardon, K., Duca, R.-C., Lestremau, F., Bach, V., et al. (2014a). 19 Development of an analytical strategy based on LC-MS/MS for the measurement of different classes of pesticides and theirs metabolites in meconium: Application and characterisation of foetal exposure in France. E n v i r o n m e n t a l R e s e a r c h , 1 3 2 , 311-320. Berton, T., Mayhoub, F., Chardon, K., Duca, R.-C., Lestremau, F., Bach, V., et al. (2014b). 20 Development of an analytical strategy based on LC-MS/MS for the measurement of different classes of pesticides and theirs metabolites in meconium: Application and characterisation of foetal exposure in France. E n v i r o n m e n t a l R e s e a r c h , 1 3 2 , 311-320. Berton, T., Mayhoub, F., Chardon, K., Duca, R.-C., Lestremau, F., Bach, V., et al. (2014b). Development of an analytical strategy based on LC--MS/MS for the measurement of different 21 classes of pesticides and theirs metabolites in meconium: Application and characterisation of foetal exposure in France, [article]. E n v i r o n m e n t a l r e s e a r c h ( N e w 311- Y o r k , N . Y . : P r i n t ) , 1 3 2 , 320. EPI EXPO DUPLICATE DUPLICATE DUPLICATE Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 120 ED 002061 00044425-00120 Number 22 Bettegowda, C. (2012). Pesticides perturb prenatal brain. [Note]. S c i e n c e T r a n s l a t i o n a l M e d ic in e , 4 . Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish REV 23 Betts, K. S. (2013). Lasting impacts: Pre- and postnatal PBDE exposures linked to IQ deficits. [Note], E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s , 1 2 1 . REV 24 Bhagavath, P ., Monteiro, F. N. P ., & Gnanadev, N. C. (2012). Epidemiology of intentional self poisoning. [Article]. M e d i c o - L e g a l U p d a t e , 1 2 , 52-54. Bhaskar, R., & Mohanty, B. (2014). Pesticides in mixture disrupt metabolic regulation: In silico 25 and in vivo analysis of cumulative toxicity of mancozeb and imidacloprid on body weight of mice. 226-234. G e n e r a l a n d C o m p a r a t i v e E n d o c r i n o l o g y , 2 0 5 , Bhatnagar, S., Das, U. M., & Bhatnagar, G. (2012). Comparison of oral midazolam with oral 26 tramadol, triclofos and zolpidem in the sedation of pediatric dental patients: An in vivo study. [Article]. 109-114. J o u r n a l o f I n d i a n S o c i e t y o f P e d o d o n t i c s a n d P r e v e n t i v e D e n t i s t r y , 3 0 , 27 Biello, D. (2012). Bad for bugs and brains? A common pesticide may interfere with a child's brain development. [Article], S c i e n t i f i c A m e r i c a n , 3 0 7 , 2 2 . Bouchard, M. F., Bellinger, D. C., Wright, R. O., & Weisskopf, M. G. (2010). Attention28 deficit/hyperactivity disorder and urinary metabolites of organophosphate pesticides. [Research Support, N.I.H., Extramural]. P e d i a t r i c s , 125(6), el270-1277. Bouchard, M. F., Chevrier, J., Harley, K. G., Kogut, K., Vedar, M., Calderon, N., et al. (2011). 29 Prenatal exposure to organophosphate pesticides and IQ in 7-year-old children. E n v i r o n H e a l t h P e r s p e c t , 1 1 9 ( 8 ) , 1189-1195. ACR TOX OTH REV EPI EPI 121 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00121 Number 30 Bradman, A., Kogut, K., Eisen, E. A., Jewell, N. P., Quiros-Alcala, L., Castorina, R., et al. (2013). Variability of organophosphorous pesticide metabolite levels in spot and 24-hr urine samples collected from young children during 1 week. [Article]. E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s , 1 2 1 , 118-124. Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish EXPO 31 Brown, J. S., Jr. (2013). P s y c h i a t r i c E f f e c t s o f O r g a n i c C h e m i c a l E x p o s u r e : BLACKWELL SCIENCE PUBL, OSNEY MEAD, OXFORD OX2 0EL, UK. REV Bulgaroni, V., Lombardo, P., Rivero-Osimani, V., Vera, B., Dulgerian, L., Cerban, F., et al. (2013). 32 Environmental pesticide exposure modulates cytokines, arginase and ornithine decarboxylase expression in human placenta. [Article]. R e p r o d u c t i v e T o x i c o l o g y , 3 9 , 23-32. Burdorf, A., Brand, T., Jaddoe, V. W., Hofman, A., Mackenbach, J. P., & Steegers, E. A. (2011). 33 The effects of work-related maternal risk factors on time to pregnancy, preterm birth and birth weight: the Generation R Study. O c c u p E n v i r o n M e d , 68(3), 197-204. Burns, C. J., McIntosh, L. J., Mink, P. J., Jurek, A. M., & Li, A. A. (2013). Pesticide exposure and 34 neurodevelopmental outcomes: Review of the epidemiologic and animal studies. [Article]. 127-183. J o u r n a l o f T o x i c o l o g y a n d E n v i r o n m e n t a l H e a l t h - P a r t B : C r i t i c a l R e v i e w s , 1 6 , Butt, C. M., Congleton, J., Hoffman, K., Fang, M. L., & Stapleton, H. M. (2014). Metabolites of 35 organophosphate flame retardants and 2-ethyl hexyl tetrabromobenzoate in urine from paired mothers and toddlers. [Journal article]. E n v i r o n m e n t a l S c i e n c e & a m p ; T e c h n o l o g y , 4 8 , 10432- 10438. Butt, C. M., Congleton, J., Hoffman, K., Fang, M., & Stapleton, H. M. (2014). Metabolites of 36 organophosphate flame retardants and 2-ethyl hexyl tetrabromobenzoate in urine from paired mothers and toddlers. E n v i r o n S c i T e c h n o l , 4 8 , 10432-10438. Camann, D. E., Schultz, S. T., Yau, A. Y., Heilbrun, L. P., Zuniga, M. M., Palmer, R. F., et al. 37 (2013). Acetaminophen, pesticide, and diethylhexyl phthalate metabolites, anandamide, and fatty acids in deciduous molars: Potential biomarkers of perinatal exposure. [Article]. J o u r n a l o f 190-196. E x p o s u r e S c i e n c e a n d E n v i r o n m e n t a l E p i d e m i o l o g y , 2 3 , TOX EPI REV EXPO DUPLICATE EXPO 122 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00122 Number Cappiello, A., Famiglini, G., Palma, P., Termopoli, V., Lavezzi, A. M., & Matturri, L. (2014). 38 Determination of selected endocrine disrupting compounds in human fetal and newborn tissues by GC-MS. [Article]. A n a l y t i c a l a n d B i o a n a l y t i c a l C h e m i s t r y , 4 0 6 , 2779-2788. Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish EXPO Carmichael, S. L , Yang, W., Roberts, E. M., Kegley, S. E., Wolff, C., Guo, L., et al. (2013). 39 Hypospadias and residential proximity to pesticide applications. [Article]. P e d i a t r i c s , 1 3 2 , EPI el216-el226. Carmichael, S. L., Yang, W., Roberts, E., Kegley, S. E., Padula, A. M., English, P. B., et al. (2014). 40 Residential agricultural pesticide exposures and risk of selected congenital heart defects among offspring in the San Joaquin Valley of California. [Article]. E n v i r o n m e n t a l R e s e a r c h , 1 3 5 , EPI 133-138. 41 Carpenter, D. O. (2013). I n t e l l e c t u a l D e v e l o p m e n t a l D i s a b i l i t y S y n d r o m e s a n d O r g a n i c C h e m i c a l s : BLACKWELL SCIENCE PUBL, OSNEY MEAD, OXFORD OX2 0EL, UK. REV Castorina, R., Bradman, A., McKone, T. E., Barr, D. B., Harnly, M. E., & Eskenazi, B. (2003). 42 Cumulative organophosphate pesticide exposure and risk assessment among pregnant women living in an agricultural community: a case study from the CHAMACOS cohort. E n v i r o n H e a l t h P e r s p e c t , 1 1 1 ( 1 3 ) , 1640-1648. Cecchi, A., Rovedatti, M. G., Sabino, G., & Magnarelli, G. G. (2012a). Environmental exposure to 43 organophosphate pesticides: Assessment of endocrine disruption and hepatotoxicity in pregnant women. [Article]. E c o t o x i c o l o g y a n d E n v i r o n m e n t a l S a f e t y , 8 0 , 280-287. EXPO EPI Cecchi, A., Rovedatti, M. G., Sabino, G., & Magnarelli, G. G. (2012b). Environmental exposure 44 to organophosphate pesticides: Assessment of endocrine disruption and hepatotoxicity in pregnant women. 280-287. E c o t o x i c o l o g y a n d E n v i r o n m e n t a l S a f e t y , 8 0 , DUPLICATE Cequier, E., lonas, A. C., Covaci, A., Mare, R. M., Becher, G., & Thomsen, C. (2014). Occurrence 45 of a broad range of legacy and emerging flame retardants in indoor environments in Norway. [Journal article]. E n v i r o n m e n t a l S c i e n c e & a m p ; T e c h n o l o g y , 4 8 , 6827-6835. EXPO 123 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00123 Number Cequier, E., Mare, R. M., Becher, G., & Thomsen, C. (2014). A high-throughput method for 46 determination of metabolites of organophosphate flame retardants in urine by ultra performance liquid chromatography-high resolution mass spectrometry. [Article]. A n a l y t i c a 98-104. C h i m i c a A c t a , 8 4 5 , Chen, W.-Q., Zhang, Y.-Z., Yuan, L, Li, Y.-F., & Li, J. (2014). Neurobehavioral evaluation of 47 adolescent male rats following repeated exposure to chlorpyrifos. [Article], N e u r o s c i e n c e 76-80. L e t t e r s , 5 7 0 , Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish EXPO TOX Chen, Y., Garcia, G. E., Huang, W., & Constantini, S. (2014). The involvement of secondary 48 neuronal damage in the development of neuropsychiatric disorders following brain insults. [Article]. F r o n t i e r s i n N e u r o l o g y , 5 M A R . DUPLICATE Chen, Y., Garcia, G. E., Huang, W., & Constantini, S. (2014). The involvement of secondary 49 neuronal damage in the development of neuropsychiatric disorders following brain insults. [Article]. F r o n t i e r s i n N e u r o l o g y , 5 M A R . Cole, T. B., Li, W.-F., Co, A. L, Hay, A. M., MacDonald, J. W., Bammler, T. K., et al. (2014). 50 Repeated Gestational Exposure of Mice to Chlorpyrifos Oxon Is Associated with Paraoxonase 1 (PONI) Modulated Effects in Maternal and Fetal Tissues. [Article]. T o x i c o l o g i c a l S c i e n c e s , 1 4 1 , 409-422. Crane, A. L., Abdel Rasoul, G., Ismail, A. A., Hendy, O., Bonner, M. R., Lasa rev, M. R., et al. 51 (2013). Longitudinal assessment of chlorpyrifos exposure and effect biomarkers in adolescent Egyptian agricultural workers. [Article]. J o u r n a l o f E x p o s u r e S c i e n c e a n d E n v i r o n m e n t a l 356-362. E p i d e m i o l o g y , 2 3 , Dabrowski, S., Hanke, W., Polanska, K., Makowiec-Dabrowska, T., & Sbala, W. (2003). 52 Pesticide exposure and birthweight: an epidemiological study in Central Poland. I n t J O c c u p 31-39. M e d E n v i r o n H e a l t h , 1 6 ( 1 ) , REV TOX EXPO EPI 53 Dahlgren, J. G., Takhar, H. S., Ruffalo, C. A., & Zwass, M. (2004). Health effects of diazinon on a family. J T o x i c o l C l i n T o x i c o l , 42(5), 579-591. ACR 124 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00124 Number Das, S., Chatterjee, K., Sarkar, N., Aich, B., & Dolui, S. (2013). Cholinergic crisis, intermediate 54 syndrome and delayed polyneuropathy following malathion poisoning. [Article]. J o u r n a l o f 2,137-141. P e d i a t r i c I n t e n s i v e C a r e , Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish ACR De Cock, M., Maas, Y. G. H., & De Bor, M. V. (2012). Does perinatal exposure to endocrine 55 disruptors induce autism spectrum and attention deficit hyperactivity disorders? Review, [article]. A c t a p a e d i a t r i c a ( O s l o ) , 1 0 1 , 811-818. de Joode, B. V., Barraza, D., Ruepert, C., Mora, A. M., Cordoba, L., Oberg, M., et al. (2012). 56 Indigenous children living nearby plantations with chlorpyrifos-treated bags have elevated 3,5,6-trichloro-2-pyridinol (TCPy) urinary concentrations. [Article]. E n v i r o n m e n t a l R e s e a r c h , 1 1 7 , 17-26. Detweiler, M. B. (2014). Organophosphate intermediate syndrome with neurological 57 complications of extrapyramidal symptoms in clinical practice. [Article]. J o u r n a l o f 298-301. N e u r o s c i e n c e s i n R u r a l P r a c t i c e , 5 , Deziel, N. C., Ward, M. H., Bell, E. M., Whitehead, T. P., Gunier, R. B., Friesen, M. C., et al. 58 (2013). Temporal variability of pesticide concentrations in homes and implications for attenuation bias in epidemiologic studies. [Article], E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s , 1 2 1 , 565-571. Dhakal, A. K., Shrestha, D., Shakya, A., Shah, S. C., & Shakya, H. (2014). Clinical profile of acute 59 poisoning in children at a teaching hospital in lalitpur. [Article], J o u r n a l o f N e p a l P a e d i a t r i c S o c i e t y , 3 4 , 100-103. REV EXPO REV EXPO ACR Ding, G. D., Wang, P., Tian, Y., Zhang, J., Gao, Y., Wang, X. J., et al. (2012). Organophosphate 60 Pesticide Exposure and Neurodevelopment in Young Shanghai Children. [Article]. EPI 2911-2917. E n v i r o n m e n t a l S c i e n c e & T e c h n o l o g y , 4 6 ( 5 ) , 61 Ding, G., & Tian, Y. (2014). Organophosphate pesticide exposure and child health in China. [Article]. 759-760. E n v i r o n m e n t a l S c i e n c e a n d P o l l u t i o n R e s e a r c h , 2 1 , REV 125 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00125 Number Ding, G., Han, S., Wang, P., Gao, Y., Shi, R., Wang, G., et al. (2012). Increased levels of 862 hydroxy-2'-deoxyguanosine are attributable to organophosphate pesticide exposure among young children. [Article]. E n v i r o n m e n t a l P o l l u t i o n , 1 6 7 , 110-114. Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish EXPO Dulaurent, S., Gaulier, J. M., Blanc-Lapierre, A., Imbert, L., & Lachatre, G. (2013). Urinary 63 determination of 2-isopropyl-4-methyl-6-hydroxypyrimidine in case of non fatal poisoning with diazinon. [Article]. F o r e n s i c S c i e n c e I n t e r n a t i o n a l , 2 2 8 , e20-e24. Engel, S. M., Berkowitz, G. S., Barr, D. B., Teitelbaum, S. L., Siskind, J., Meisel, S. J., et al. (2007). 64 Prenatal organophosphate metabolite and organochlorine levels and performance on the Brazelton Neonatal Behavioral Assessment Scale in a multiethnic pregnancy cohort. A m J 1397-1404. 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P,, Barr, D. B., et al. (2004). 68 Association of in utero organophosphate pesticide exposure and fetal growth and length of EPI gestation in an agricultural population. E n v i r o n H e a l t h P e r s p e c t , 1 1 2 ( 1 0 ) , 1116-1124. Eskenazi, B., Huen, K., Marks, A., Harley, K. G., Bradman, A., Barr, D. B., et al. (2010). PON1 and 69 neurodevelopment in children from the CHAMACOS study exposed to organophosphate EPI pesticides in utero. E n v i r o n H e a l t h P e r s p e c t , 1 1 8 ( 1 2 ) , 1775-1781. 126 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00126 Number Eskenazi, B,, Kogut, K., Huen, K., Harley, K. G., Bouchard, M., Bradman, A., et al. (2014a). 70 Organophosphate pesticide exposure, PONI, and neurodevelopment in school-age children from the CHAMACOS study. E n v i r o n m e n t a l R e s e a r c h , 1 3 4 , 149-157. Eskenazi, B., Kogut, K., Huen, K., Harley, K. G., Bouchard, M., Bradman, A., et al. (2014b). 71 Organophosphate pesticide exposure, PONI, and neurodevelopment in school-age children from the CHAMACOS study. [Article]. E n v i r o n m e n t a l R e s e a r c h , 1 3 4 , 149-157. Eskenazi, B., Marks, A. R., Bradman, A., Harley, K., Barr, D. B., Johnson, C., et al. (2007). 72 Organophosphate pesticide exposure and neurodevelopment in young Mexican-American children. E n v i r o n H e a l t h P e r s p e c t , 1 1 5 ( 5 ) , 792-798. Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish EPI DUPLICATE EPI 73 Eskenazi, B., Rosas, L. G., Marks, A. R., Bradman, A., Harley, K., Holland, N., et al. (2008). Pesticide toxicity and the developing brain. B a s i c C l i n P h a r m a c o l T o x i c o l , 1 0 2 ( 2 ) , 228-236. Estevan, C., Fuster, E., Del Ro, E., Pamies, D., Vilanova, E., & Sogorb, M. A. (2014). 74 Organophosphorus pesticide chlorpyrifos and its metabolites alter the expression of biomarker genes of differentiation in D3 mouse embryonic stem cells in a comparable way to other model neurodevelopmental toxicants. [Article]. C h e m i c a l R e s e a r c h i n T o x i c o l o g y , 2 7 , 1487-1495. Fenske, R. A., Lu, C., Negrete, M., & Galvin, K. (2013). Breaking the take home pesticide 75 exposure pathway for agricultural families: Workplace predictors of residential contamination. [Article], 1063-1071. A m e r i c a n J o u r n a l o f I n d u s t r i a l M e d i c i n e , 5 6 , Ferreira, J. D., Couto, A. C. z., Pombo-de-Oliveira, M. S., & Koifman, S. (2013). In utero pesticide 76 exposure and leukemia in Brazilian children < 2 years of age. [Note]. E n v i r o n m e n t a l H e a l t h 269-275. P e r s p e c t i v e s , 1 2 1 , Feyzioglu, B. r., zdemir, M., Kutlu, N. O., Baykan, M., & Baysal, B. (2014). Pulmonary infection 77 caused by chryseobacterium indologenes in a patient with a diagnosis of organophosphate poisoning. [Article]. A n a t o l i a n J o u r n a l o f C l i n i c a l I n v e s t i g a t i o n , 8 , 137-139. REV TOX EXPO EPI ACR 127 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00127 Number Fortenberry, G. Z., Meeker, J. D., Sanchez, B. N., Barr, D. B., Panuwet, P., Bellinger, D., et al. 78 (2014). Urinary 3,5,6-trichloro-2-pyridinol (TCPY) in pregnant women from Mexico City: Distribution, temporal variability, and relationship with child attention and hyperactivity, [article]. 405-412. I n t e r n a t i o n a l j o u r n a l o f h y g i e n e a n d e n v i r o n m e n t a l h e a l t h , 2 1 7 , Fortenberry, G. Z., Meeker, J. D., Sanchez, B. N., Barr, D. B., Panuwet, P., Bellinger, D., et al. 79 (2014). Urinary 3,5,6-trichloro-2-pyridinol (TCPY) in pregnant women from Mexico City: distribution, temporal variability, and relationship with child attention and hyperactivity. I n t J 405-412. H y g E n v i r o n H e a l t h , 2 1 7 ( 2 - 3 ) , Fortenberry, G. Z., Meeker, J. D., Snchez, B. N., Barr, D. B., Panuwet, P., Bellinger, D., et al. 80 (2014). Urinary 3,5,6-trichloro-2-pyridinol (TCPY) in pregnant women from Mexico City: Distribution, temporal variability, and relationship with child attention and hyperactivity. 405-412. I n t e r n a t i o n a l J o u r n a l o f H y g i e n e a n d E n v i r o n m e n t a l H e a l t h , 2 1 7 , Fortenberry, G. Z., Meeker, J. D., Snchez, B. N., Barr, D. B., Panuwet, P., Bellinger, D., et al. 81 (2014). Urinary 3,5,6-trichloro-2-pyridinol (TCPY) in pregnant women from Mexico City: distribution, temporal variability, and relationship with child attention and hyperactivity. [Journal article]. 405-412. I n t e r n a t i o n a l J o u r n a l o f H y g i e n e a n d E n v i r o n m e n t a l H e a l t h , 2 1 7 , Fortenberry, G. Z., Meeker, J. D., Snchez, B. N., Bellinger, D., Peterson, K., Schnaas, L., et al. 82 (2014). Paraoxonase 1polymorphisms and attention/hyperactivity in school-age children from Mexico City, Mexico. [Article]. E n v i r o n m e n t a l R e s e a r c h , 1 3 2 , 342-349. Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish DUPLICATE EPI DUPLICATE DUPLICATE EPI Fortenberry, G. Z., Meeker, J. D., Sanchez, B. N., Bellinger, D., Peterson, K., Schnaas, L., et al. 83 (2014). Paraoxonase 1polymorphisms and attention/hyperactivity in school-age children from Mexico City, Mexico. E n v i r o n R e s , 1 3 2 , 342-349. DUPLICATE Fortenberry, G. Z., Meeker, J. D., Sanchez, B. N., Bellinger, D., Peterson, K., Schnaas, L., et al. 84 (2014). Paraoxonase 1polymorphisms and attention/hyperactivity in school-age children from Mexico City, Mexico. E n v i r o n m e n t a l R e s e a r c h , 1 3 2 , 342-349. DUPLICATE Fortenberry, G. Z., Meeker, J. D., Snchez, B. N., Bellinger, D., Peterson, K., Schnaas, L, et al. 85 (2014). Paraoxonase 1polymorphisms and attention/hyperactivity in school-age children from Mexico City, Mexico. E n v i r o n m e n t a l R e s e a r c h , 1 3 2 , 342-349. DUPLICATE 128 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00128 Number Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish Fortenberry, G. Z., Meeker, J. D., Snchez, B. N., Bellinger, D., Peterson, K., Schnaas, L, et al. 86 (2014). Paraoxonase 1polymorphisms and attention/hyperactivity in school-age children from Mexico City, Mexico. E n v i r o n m e n t a l R e s e a r c h , 1 3 2 , 342-349. DUPLICATE Fromme, FI., Lahrz, T., Kraft, M., Fembacher, L., Mach, C., Dietrich, S., et al. (2014). 87 Organophosphate flame retardants and plasticizers in the air and dust in German daycare centers and human biomonitoring in visiting children (LUPE 3). [Article]. E n v i r o n m e n t 158-163. I n t e r n a t i o n a l , 7 1 , EXPO Furlong, M. A., Engel, S. M., Barr, D. B., & Wolff, M. S. (2014). Prenatal exposure to 88 organophosphate pesticides and reciprocal social behavior in childhood. E n v i r o n I n t , 7 0 , 125- EPI 131. Furlong, M. A., Engel, S. M., Barr, D. B., & Wolff, M. S. (2014a). Prenatal exposure to 89 organophosphate pesticides and reciprocal social behavior in childhood. E n v i r o n m e n t 125-131. I n t e r n a t i o n a l , 7 0 , DUPLICATE Furlong, M. A., Engel, S. M., Barr, D. B., & Wolff, M. S. (2014b). Prenatal exposure to 90 organophosphate pesticides and reciprocal social behavior in childhood, [article]. E n v i r o n m e n t 125-131. i n t e r n a t i o n a l , 7 0 , DUPLICATE Gonza'lez-Alzaga, B., Lacasan, tild, a, M., Aguilar, G., o, C., et al. (2014). A systematic review of 91 neurodevelopmental effects of prenatal and postnatal organophosphate pesticide exposure. [Article], T o x i c o l o g y L e t t e r s , 2 3 0 , 104-121. Gonzalez, V., Huen, K., Venkat, S., Pratt, K., Xiang, P., Harley, K. G., et al. (2012). Cholinesterase 92 and paraoxonase (PONI) enzyme activities in MexicanAmerican mothers and children from an agricultural community. [Article]. J o u r n a l o f E x p o s u r e S c i e n c e a n d E n v i r o n m e n t a l E p i d e m i o l o g y , 2 2 , 641-648. Gonzalez-Alzaga, B., Lacasana, M., Aguilar-Garduno, C., Rodriguez-Barranco, M., Ballester, F., 93 Rebagliato, M., et al. (2013). A systematic review of neurodevelopmental effects of prenatal and postnatal organophosphate pesticide exposure. T o x i c o l L e t t , 2 3 0 , 104-121. REV EXPO DUPLICATE 129 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00129 Number Gonzlez-Alzaga, B., Lacasaa, M., Aguilar-Garduo, C., Rodrguez-Barranco, M., Ballester, F., 94 Rebagliato, M., et al. (2014). A systematic review of neurodevelopmental effects of prenatal and postnatal organophosphate pesticide exposure. T o x i c o l o g y L e t t e r s , 2 3 0 , 104-121. Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish DUPLICATE Goodman, J. E., Prueitt, R. L , & Rhomberg, L. R. (2013). INCORPORATING LOW-DOSE 95 EPIDEMIOLOGY DATA IN A CHLORPYRIFOS RISK ASSESSMENT. [Article], D o s e - R e s p o n s e , 1 1 ( 2 ) , REV 207-219. 96 Goodman, J. E,, Prueitt, R. L., & Rhomberg, L. R. (2013). Incorporating low-dose epidemiology data in a chlorpyrifos risk assessment. [Article]. D o s e - R e s p o n s e , 1 1 , 207-219. Gowda, V. K. N., Bannigidad, N. B., Kumar, P., Srikanteswara, P.-K., Shivananda, Govindraj, et 97 al. (2014). Predictors of non-traumatic coma in a pediatric cohort from a South Indian tertiary care center: Results of a multivariate analysis. [Article]. J o u r n a l o f P e d i a t r i c N e u r o l o g y , 1 2 , 35- 43. Grandjean, P., Harari, R., Barr, D. B., & Debes, F. (2006). Pesticide exposure and stunting as 98 independent predictors of neurobehavioral deficits in Ecuadorian school children. [Research Support, Non-U.S. Gov't]. P e d i a t r i c s , 1 1 7 ( 3 ) , e546-556. Guha, N., Ward, M. H., Gunier, R., Colt, J. S., Suzanne Lea, C., Buffer, P. A., et al. (2013). 99 Characterization of residential pesticide use and chemical formulations through self-report and household inventory: The northern California childhood leukemia study. [Note]. E n v i r o n m e n t a l 276-282. H e a l t h P e r s p e c t i v e s , 1 2 1 , Guiaz, N., Rena, V., Genti-Raimondi, S., Rivero, V., & Magnarelli, G. (2012). Effects of the 100 organophosphate insecticides phosmet and chlorpyrifos on trophoblast JEG-3 cell death, proliferation and inflammatory molecule production. [Article]. T o x i c o l o g y in V i t r o , 2 6 , 406-413. DUPLICATE ACR EPI EXPO TOX 101 Guodong, D., & Yixiao, B. A. O. (2014). Revisiting pesticide exposure and children's health: Focus on China, [article]. S c i e n c e o f t h e t o t a l e n v i r o n m e n t , 4 7 2 , 289-295. REV 130 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00130 Number W . Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish Guodong, D., Pei, W., Ying, T., Jun, Z., Yu, G,, Xiaojin, W., et al. (2012). Organophosphate 102 pesticide exposure and neurodevelopment in young Shanghai children. [Article]. 2911-2917. E n v i r o n m e n t a l S c i e n c e a n d T e c h n o l o g y , 4 6 , DUPLICATE 103 Guodong, D., Pei, W., Ying, T., Jun, Z., Yu, G., Xiaojin, W., et al. (2012). Organophosphate pesticide exposure and neurodevelopment in young Shanghai children. E n v i r o n S c i T e c h n o l . EPI Handal, A. J., Harlow, S. D., Breilh, J., & Lozoff, B. (2008). Occupational exposure to pesticides 104 during pregnancy and neurobehavioral development of infants and toddlers. E p i d e m i o l o g y , EPI 1 9 ( 6 ) , 851-859. Handal, A. J., Lozoff, B., Breilh, J., & Harlow, S. D. (2007a). Effect of community of residence on 105 neurobehavioral development in infants and young children in a flower-growing region of Ecuador. [Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't]. E n v i r o n EPI 128-133. H e a l t h P e r s p e c t , 1 1 5 ( 1 ) , 106 Handal, A. J., Lozoff, B., Breilh, J., & Harlow, S. D. (2007b). Neurobehavioral development in children with potential exposure to pesticides. E p i d e m i o l o g y , 1 8 ( 3 ) , 312-320. EPI Harari, R., Julvez, J., Murata, K., Barr, D., Bellinger, D. C., Debes, F., et al. (2010). 107 Neurobehavioral deficits and increased blood pressure in school-age children prenatally exposed to pesticides. E n v i r o n H e a l t h P e r s p e c t , 1 1 8 ( 6 ) , 890-896. Harley, K. G., Huen, K., Schall, R. A., Holland, N. T., Bradman, A., Barr, D. B., et al. (2011). 108 Association of organophosphate pesticide exposure and paraoxonase with birth outcome in Mexican-American women. P L o S O n e , 6(8), e23923. Hoffman, K., Daniels, J. L., & Stapleton, H. M. (2014). Urinary metabolites of organophosphate 109 flame retardants and their variability in pregnant women. [Article]. E n v i r o n m e n t I n t e r n a t i o n a l , 6 3 , 169-172. EPI EPI EXPO 131 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00131 Number Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish Hoffman, K., Daniels, J. L., & Stapleton, H. M. (2014). Urinary metabolites of organophosphate 110 flame retardants and their variability in pregnant women. [Journal article]. E n v i r o n m e n t 169-172. I n t e r n a t i o n a l , 6 3 , DUPLICATE Hore, P., Robson, M., Freeman, N., Zhang, J., Wartenberg, D., Ozkaynak, H., et al. (2005). 111 Chlorpyrifos accumulation patterns for child-accessible surfaces and objects and urinary metabolite excretion by children for 2 weeks after crack-and-crevice application. E n v i r o n H e a l t h P e r s p e c t , 113(2), 211-219. Hore, P., Zartarian, V., Xue, J., Oezkaynak, H., Wang, S.-W., Yang, Y.-C., et al. (2006). Childrens 112 residential exposure to chlorpyrifos: Application of CPPAES field measurements of chlorpyrifos and TCPy within MENTOR/SH EDS-Pesticides model. S c i e n c e o f t h e T o t a l E n v i r o n m e n t , 3 6 6 ( 2 - 3 ) , 525-537. EXPO EXPO Horton, M. K., Kahn, L. G., Perera, F., Barr, D. B., & Rauh, V. (2012). Does the home 113 environment and the sex of the child modify the adverse effects of prenatal exposure to EPI chlorpyrifos on child working memory? N e u r o t o x i c o l T e r a t o l , 3 4 ( 5 ) , 534-541. Horton, M. K., Kahn, L. G., Perera, F., Barr, D. B., & Rauh, V. (2012). Does the home 114 environment and the sex of the child modify the adverse effects of prenatal exposure to chlorpyrifos on child working memory? N e u r o t o x i c o l o g y a n d T e r a t o l o g y , 3 4 , 534-541. DUPLICATE Horton, M. K., Kahn, L. G., Perera, F., Boyd Barr, D., & Rauh, V. (2012). Does the home 115 environment and the sex of the child modify the adverse effects of prenatal exposure to chlorpyrifos on child working memory? [article]. N e u r o t o x i c o l o g y a n d t e r a t o l o g y , 3 4 , 534-541. How, V., Hashim, Z., Ismail, P., Said, S. M., Dzolkhifli, O., & Shamsul Bahri, M. T. (2014). 116 Exploring cancer development in adulthood: cholinesterase depression and genotoxic effect from chronic exposure to organophosphate pesticides among rural farm children. [Journal article]. J o u r n a l o f A g r o m e d i c i n e , 1 9 , 35-43. Huen, K., Bradman, A., Harley, K., Yousefi, P., Barr, D. B., Eskenazi, B., et al. (2012). 117 Organophosphate pesticide levels in blood and urine of women and newborns living in an agricultural community. [Article]. E n v i r o n m e n t a l R e s e a r c h , 1 1 7 , 8-16. DUPLICATE EPI EXPO 132 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00132 Number Huen, K., Bradman, A., Harley, K., Yousefi, P., Boyd Barr, D., Eskenazi, B., et al. (2012). 118 Organophosphate pesticide levels in blood and urine of women and newborns living in an agricultural community. [Article]. E n v i r o n m e n t a l R e s e a r c h , 1 1 7 , 8-16. Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish DUPLICATE 119 Huen, K., Harley, K,, Beckman, K., Eskenazi, B., & Holland, N. (2013). Associations of PON1 and Genetic Ancestry with Obesity in Early Childhood. [Article]. P L o S O N E , 8 . EPI Iftikhar, B., Siddiqui, S., & Rehman, S. (2014). Assessment of the dietary transfer of pesticides 120 to dairy milk and its effect on human health. [Journal article], A f r i c a n J o u r n a l o f B i o t e c h n o l o g y , 1 3 , 476-485. Jafarzadeh, M., Nasri Nasrabadi, Z., Sheikhazadi, A., Abbaspour, A., Vasigh, S., Yousefinejad, V., 121 et al. (2013). Is there a role for progesterone in the management of acute organophosphate poisoning during pregnancy? [Article]. M e d i c a l H y p o t h e s e s , 8 0 , 804-805. Jamkhande, P. G., Chintawar, K. D., & Chandak, P. G. (2014a). Teratogenicity: a mechanism 122 based short review on common teratogenic agents. A s i a n P a c i f i c J o u r n a l o f T r o p i c a l D i s e a s e , 4 , 421-432. Jamkhande, P. G., Chintawar, K. D., & Chandak, P. G. (2014b). Teratogenicity: A mechanism 123 based short review on common teratogenic agents. [Article]. A s i a n P a c i f i c J o u r n a l o f T r o p i c a l D i s e a s e , 4 , 421-432. Jones, K., Everard, M., & Harding, A. H. (2014). Investigation of gastrointestinal effects of 124 organophosphate and carbamate pesticide residues on young children. [Journal article]. 392-398. I n t e r n a t i o n a l J o u r n a l o f H y g i e n e a n d E n v i r o n m e n t a l H e a l t h , 2 1 7 , Kajta, M., & Wojtowicz, A. K. (2013). Impact of endocrine-disrupting chemicals on neural 125 development and the onset of neurological disorders. [Literature Review], P h a r m a c o l o g i c a l R e p o r t s , 6 5 , 1632-1639. EXPO REV REV DUPLICATE EPI REV 133 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00133 Number Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish 126 Karki, R. K,, & Risal, A. (2012). Study of poisoning cases in a tertiary care hospital. [Article], 70-73. K a t h m a n d u U n i v e r s i t y M e d i c a l J o u r n a l , 1 0 , ACR Kauffman, R., Marcus, M., Wold, J. L., & Lu, C. (2009). Pesticide exposure and 127 neurodevelopment in migrant farmworker children of South Georgia. I n t e r n a t i o n a l J o u r n a l o f C h ild a n d A d o le s c e n t H e a lth . S p e c ia l Is su e : B r e a k t h e c y c le o f a d v e r s e h e a lt h a n d 2(3), 303-312. d e v e l o p m e n t a l d i s p a r i t i e s f o r c h i l d r e n , REV Kezios, K. L., Liu, X. H., Cirillo, P. M., Cohn, B. A., Kalantzi, O. 1., Wang, Y. Z., et al. (2013). 128 Dichlorodiphenyltrichloroethane (DDT), DDT metabolites and pregnancy outcomes. [Article]. EPI 156-164. R e p r o d u c t i v e T o x i c o l o g y , 3 5 , Khan, H. A. A., & Waseem, A. (2014). The effect of temperature on the toxicity of insecticides 129 against Musca domestica L.: implications for the effective management of diarrhea. [Journal TOX article], P L o S O N E , 9 , e95636. Khan, K., Ismail, A. A., Abdel Rasoul, G., Bonner, M. R., Lasarev, M. R., Hendy, O., et al. (2014). 130 Longitudinal assessment of chlorpyrifos exposure and self-reported neurological symptoms in adolescent pesticide applicators. [Article], B M J O p e n , 4 . DUPLICATE Khan, K., Ismail, A. A., Abdel Rasoul, G., Bonner, M. R., Lasarev, M. R., Hendy, O., et al. (2014). 131 Longitudinal assessment of chlorpyrifos exposure and self-reported neurological symptoms in EPI adolescent pesticide applicators. [Article]. B M J O p e n , 4 . Kicinski, M., Vrijens, J., Vermier, G., Hond, E. D., Schoeters, G., Nelen, V., et al. (2015). 132 Neurobehavioral function and low-level metal exposure in adolescents. I n t e r n a t i o n a l J o u r n a l o f EPI 139-146. H y g i e n e a n d E n v i r o n m e n t a l H e a l t h , 2 1 8 , Kim, H.-H., Lim, Y.-W., Yang, J.-Y., Shin, D.-C., Ham, H.-S., Choi, B.-S., et al. (2013a). Health risk 133 assessment of exposure to chlorpyrifos and dichlorvos in children at childcare facilities. [Article], 441-450. S c i e n c e o f t h e T o t a l E n v i r o n m e n t , 4 4 4 , EXPO 134 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00134 Number Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish Kim, H.-H., Lim, Y.-W., Yang, J.-Y., Shin, D.-C., Ham, H.-S., Choi, B.-S., et al. (2013b). Health risk 134 assessment of exposure to chlorpyrifos and dichlorvos in children at childcare facilities. S c i e n c e 441-450. o f T h e T o t a l E n v i r o n m e n t , 4 4 4 , DUPLICATE Kofman, O., Berger, A., Massarwa, A., Friedman, A., & Jaffar, A. A. (2006). Motor inhibition and 135 learning impairments in school-aged children following exposure to organophosphate EPI pesticides in infancy. P e d i a t r R e s , 6 0 ( 1 ) , 88-92. Koirala, D. P., Rao, K. S., Malla, K. K., & Malla, T. (2013). A study of clinical features, 136 management and outcome of organophosphate and carbamate poisoning in children. [Article]. ACR 85-90. J o u r n a l o f N e p a l P a e d i a t r i c S o c i e t y , 3 3 , Koureas, M., Tsakalof, A., Tsatsakis, A., & Hadjichristodoulou, C. (2012). Systematic review of 137 biomonitoring studies to determine the association between exposure to organophosphorus and pyrethroid insecticides and human health outcomes, [article]. T o x i c o l o g y l e t t e r s , 2 1 0 , 155- REV 168. Koutroulakis, D., Sifakis, S., Tzatzarakis, M. N., Alegakis, A. K., Theodoropoulou, E., Kavvalakis, 138 M. P., et al. (2014). Dialkyl phosphates in amniotic fluid as a biomarker of fetal exposure to organophosphates in Crete, Greece; association with fetal growth. [Article]. R e p r o d u c t i v e EPI 98-105. T o x i c o l o g y , 4 6 , Kr, M. Z., Ozturk, G. 1., Gurler, M., Karaarslan, B., Erden, G. n. 1., Karapirli, M., et al. (2013). 139 Pesticide poisoning cases in Ankara and nearby cities in Turkey: An 11-year retrospective ACR analysis. [Article]. 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Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish ACR Kumar, M., Vignan Kumar, G., Babu, P., Kumar, S., Subrahmanyam, B., Veeraprasad, M., et al. 143 (2014). A retrospective analysis of acute organophosphorus poisoning cases admitted to the tertiary care teaching hospital in South India. [Article]. A n n a l s o f A f r i c a n M e d i c i n e , 1 3 , 71-75. Kumar, N., Gupta, A. K., Najotra, D. K., Digra, S. K., Gupta, S., Gupta, P. K., et al. (2013). 144 Incidence and pattern of acute poisoning among children aged 1 month to 18 years: An experience from Jammu. [Article]. I n d i a n J o u r n a l o f F o r e n s i c M e d i c i n e a n d T o x i c o l o g y , 7 , 135- 138. Lee, D., Jung, K., Choi, Y., & Cheon, Y. (2014). Body mass index as a prognostic factor in 145 organophosphate-poisoned patients. [Journal article]. A m e r i c a n J o u r n a l o f E m e r g e n c y M e d i c i n e , 3 2 , 693-696. DUPLICATE ACR ACR Lee, J., & Freeman, J. L. (2014). Zebrafish as a model for investigating developmental lead (Pb) 146 neurotoxicity as a risk factor in adult neurodegenerativo disease: A mini-review. TOX 57-64. N e u r o t o x i c o l o g y , 4 3 , Lee, W. J., Ko, Y., & Cha, E. S. (2014). Acute pesticide poisoning among children in south korea: 147 Findings from national health insurance claims data, 2006-2009. [Article]. J o u r n a l o f T r o p i c a l ACR 4-9. P e d i a t r i c s , 6 0 , 148 Li, A. A., Levine, T. E., Burns, C. J., & Kent Anger, W. (2012). Integration of epidemiology and animal neurotoxicity data for risk assessment. [Article]. N e u r o T o x i c o l o g y , 3 3 , 823-832. Li, A. A., Lowe, K. A., McIntosh, L. J., & Mink, P. J. (2012). EVALUATION OF EPIDEMIOLOGY AND 149 ANIMAL DATA FOR RISK ASSESSMENT: CHLORPYRIFOS DEVELOPMENTAL NEUROBEHAVIORAL OUTCOMES. [Literature Review]. J o u r n a l o f T o x i c o l o g y a n d E n v i r o n m e n t a l H e a l t h P a r t B C r i t i c a l R e v i e w s , 1 5 , 109-184. REV DUPLICATE 136 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00136 Number Li, A. A., Lowe, K. A., McIntosh, L. J., & Mink, P. J. (2012). Evaluation of epidemiology and 150 animal data for risk assessment: chlorpyrifos developmental neurobehavioral outcomes. J 109-184. T o x i c o l E n v i r o n H e a l t h B C r i t R e v , 1 5 ( 2 ) , 151 Li, J., Yu, N., Zhang, B., Jin, L., Li, M., Hu, M., et al. (2014). Occurrence of organophosphate flame retardants in drinking water from China. [Article]. W a t e r R e s e a r c h , 5 4 , 53-61. Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish REV DUPLICATE 152 Li, J., Yu, N., Zhang, B., Jin, L., Li, M., Hu, M., et al. (2014). Occurrence of organophosphate flame retardants in drinking water from China. [Article]. W a t e r R e s e a r c h , 5 4 , 53-61. Liu, P., Wu, C., Chang, X., Qi, X., Zheng, M., & Zhou, Z. (2014). Assessment of chlorpyrifos 153 exposure and absorbed daily doses among infants living in an agricultural area of the Province of Jiangsu, China. [Journal article]. I n t e r n a t i o n a l A r c h i v e s o f O c c u p a t i o n a l a n d E n v i r o n m e n t a l H e a l t h , 8 7 , 753-762. Lizardi, P. S., O'Rourke, M. K., & Morris, R. J. (2008). The effects of organophosphate pesticide 154 exposure on Hispanic children's cognitive and behavioral functioning. J P e d i a t r P s y c h o l , 3 3 ( 1 ) , 91-101. Llop, S., Julvez, J., Fernandez-Somoano, A., Santa Marina, L., Vizcaino, E., Iniguez, C., et al. 155 (2013). Prenatal and postnatal insecticide use and infant neuropsychological development in a multicenter birth cohort study. E n v i r o n m e n t I n t e r n a t i o n a l , 5 9 , 175-182. London, L., Beseler, C., Bouchard, M. F., Bellinger, D. C., Colosio, C., Grandjean, P., et al. (2012). 156 Neurobehavioral and neurodevelopmental effects of pesticide exposures. [Article]. 887-896. N e u r o T o x i c o l o g y , 3 3 , 157 Lopez-Carrillo, L., & Cebrian, M. E. (2013). C o g n i t i v e F u n c t i o n : BLACKWELL SCIENCE PUBL, OSNEY MEAD, OXFORD OX2 0EL, UK. EXPO EXPO EPI EPI REV REV 137 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00137 Number Lovasi, G. S., Quinn, J. W., Rauh, V. A., Perera, F. P., Andrews, H. F,, Garfinkel, R., et al. (2011). 158 Chlorpyrifos exposure and urban residential environment characteristics as determinants of early childhood neurodevelopment. A m 63-70. J P u b l i c H e a l t h , 1 0 1 ( 1 ) , Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-English EPI 159 Lozowicka, B. (2015). Health risk for children and adults consuming apples with pesticide residue. [Journal article]. S c i e n c e o f t h e T o t a l E n v i r o n m e n t , 5 0 2 , 184-198. REV Lu, C., & Andres, L. (2012). Reconstructing organophosphorus pesticide doses using the 160 reversed dosimetry approach in a simple physiologically-based pharmacokinetic model. [Article]. J o u r n a l o f T o x i c o l o g y , 2 0 1 2 . Lu, C., Essig, C., Root, C., Rohlman, D. S., McDonald, T., & Sulzbacher, S. (2009). Assessing the 161 association between pesticide exposure and cognitive development in rural Costa Rican children living in organic and conventional coffee farms. I n t J A d o l e s c M e d H e a l t h , 2 1 ( A ) , 609- 621. Marks, A. R., Harley, K., Bradman, A., Kogut, K., Barr, D. B., Johnson, C., et al. (2010). 162 Organophosphate pesticide exposure and attention in young Mexican-American children: the CHAMACOS study. E n v i r o n H e a l t h P e r s p e c t , 1 1 8 ( 1 2 ) , 1768-1774. EXPO EPI EPI 163 Marri, M. Z., Mirza, F. H., & Hasan, Q. (2012). Pattern, incidence and mortality rate of acute poisonings at Karachi- one year study. [Article]. M e d i c a l F o r u m 6-10. M o n t h l y , 2 3 , ACR Martin, 1. G., Thottempudi, S. R. K., & Frattarelli, D. A. C. (2013). Changes to Prescription Drug 164 Pediatric Labeling: Awareness by Practicing Pediatricians. [Article]. D r u g I n f o r m a t i o n J o u r n a l , OTH 4 7 , 214-218. 165 Mascarelli, A. (2013). Growing up with pesticides. [Note], S c i e n c e , 3 4 1 , 740-741. REV 138 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00138 Number McGinnis, W. R., Audhya, T., & Edelson, S. M. (2013). Proposed toxic and hypoxic impairment 166 of a brainstem locus in autism. [Article]. I n t e r n a t i o n a l J o u r n a l o f E n v i r o n m e n t a l R e s e a r c h a n d P u b l i c H e a l t h , 1 0 , 6955-7000. Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish REV Memon, A., Shaikh, J. M., Kazi, S. A. F., & Kazi, A. (2012). Changing trends in deliberate self 167 poisoning at Hyderabad. [Article]. J o u r n a l o f t h e L i a q u a t U n i v e r s i t y o f M e d i c a l a n d H e a l t h S c i e n c e s , 1 1 , 124-126. ACR Meyer-Baron, M., Kim, E. A., Nuwayhid, 1., Ichihara, G., & Kang, S.-K. (2012). Occupational 168 exposure to neurotoxic substances in Asian countries - Challenges and approaches. [Article]. REV 853-861. N e u r o T o x i c o l o g y , 3 3 , Michalakis, M., Tzatzarakis, M. N., Kovatsi, L, Alegakis, A. K., Tsakalof, A. K., Heretis, 1., et al. 169 (2014). Hypospadias in offspring is associated with chronic exposure of parents to EPI organophosphate and organochlorine pesticides. [Article]. T o x i c o l o g y L e t t e r s , 2 3 0 , 139-145. Mink, P. J., Kimmel, C. A., & Li, A. A. (2012). POTENTIAL EFFECTS OF CHLORPYRIFOS ON FETAL 170 GROWTH OUTCOMES: IMPLICATIONS FOR RISK ASSESSMENT. [Article], J o u r n a l o f T o x i c o l o g y REV 281-316. a n d E n v i r o n m e n t a l H e a l t h P a r t B C r i t i c a l R e v i e w s , 1 5 , 171 Mink, P. J., Kimmel, C. A., & Li, A. A. (2012). Potential effects of chlorpyrifos on fetal growth outcomes: implications for risk assessment. J T o x i c o l E n v i r o n H e a l t h B C r i t R e v , 15(4), 281-316. DUPLICATE Moreno-Banda, G., Blanco-Munoz, J., Lacasana, M., Rothenberg, S. J., Aguilar-Garduno, C., 172 Gamboa, R., et al. (2009). Maternal exposure to floricultura! work during pregnancy, PONI Q192R polymorphisms and the risk of low birth weight. S c i T o t a l E n v i r o n , 4 0 7 ( 2 1 ) , 5478-5485. Morgan, M. K., & Jones, P. A. (2013). Dietary predictors of young children's exposure to 173 current-use pesticides using urinary biomonitoring. [Article]. F o o d a n d C h e m i c a l T o x i c o l o g y , 6 2 , 131-141. EPI EXPO 139 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00139 Number Morgan, M. K., Wilson, N. K., & Chuang, J. C. (2014). Exposures of 129 preschool children to 174 organochlorines, organophosphates, pyrethroids, and acid herbicides at their homes and daycares in North Carolina. [Article]. I n t e r n a t i o n a l J o u r n a l o f E n v i r o n m e n t a l R e s e a r c h a n d P u b l i c H e a l t h , 1 1 , 3743-3764. Morgan, M. K., Wilson, N. K., & Chuang, J. C. (2014). Exposures of 129 preschool children to 175 organochlorines, organophosphates, pyrethroids, and acid herbicides at their homes and daycares in North Carolina. [Journal article]. I n t e r n a t i o n a l J o u r n a l o f E n v i r o n m e n t a l R e s e a r c h 3743-3764. a n d P u b l i c H e a l t h , 1 1 , Mosaddegh, M. H., Emami, F., & Asghari, G. (2014). Evaluation of residual diazinon and 176 chlorpiryfos in children herbal medicines by headspace-SPME and GC-FID. [Article]. I r a n ia n 541-549. J o u r n a l o f P h a r m a c e u t i c a l R e s e a r c h , 1 3 , Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-English EXPO DUPLICATE EXPO Munoz-Quezada, M. T., Lucero, B. A., Barr, D. B., Steenland, K., Levy, K,, Ryan, B., et al. (2013). 177 Neurodevelopmental effects in children associated with exposure to organophosphate REV pesticides: A systematic review. N e u r o t o x i c o l o g y , 3 9 , 158-168. Munoz-Quezada, M. T., Lucero, B. A., Barr, D. B., Steenland, K., Levy, K., Ryan, P. B., et al. 178 (2013). Neurodevelopmental effects in children associated with exposure to organophosphate pesticides: A systematic review, [article]. N e u r o t o x i c o l o g y ( P a r k F o r e s t S o u t h ) , 3 9 , 158-168. DUPLICATE Naseh, M., & Vatanparast, J. (2014). Enhanced expression of hypothalamic nitric oxide 179 synthase in rats developmental^ exposed to organophosphates. [Journal article]. B r a i n TOX 10-19. R e s e a r c h , 1 5 7 9 , Neal, R. E., Chen, J., Jagadapillai, R., Jang, FI., Abomoelak, B., Brock, G., et al. (2014). 180 Developmental cigarette smoke exposure: Hippocampus proteome and metabolome profiles TOX in low birth weight pups. T o x i c o l o g y , 3 1 7 , 40-49. Nevison, C. D. (2014). A comparison of temporal trends in United States autism prevalence to 181 trends in suspected environmental factors. [Article], E n v i r o n m e n t a l H e a l t h : A G l o b a l A c c e s s EPI S c ie n c e S o u rc e , 1 3 . 140 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00140 Category (EPI-epi only; EXPO- exposure only; ACR Number W. acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish Oates, L , Cohen, M., Braun, L., Schembri, A., & Taskova, R. (2014). Reduction in urinary 182 organophosphate pesticide metabolites in adults after a week-long organic diet. [Journal article]. E n v i r o n m e n t a l R e s e a r c h , 1 3 2 , 105-111. EXPO Ostrea Jr, E. M., Reyes, A., Villanueva-Uy, E., Pacifico, R., Benitez, B., Ramos, E., et al. (2012). 183 Fetal exposure to propoxur and abnormal child neurodevelopment at 2 years of age. [Article]. 669-675. N e u r o T o x i c o l o g y , 3 3 , Ostrea, E. M., Bielawski, D. M., Posecion, N. C., Corrion, M., Villanueva-Uy, E., Bernardo, R. C., 184 et al. (2009). Combined analysis of prenatal (maternal hair and blood) and neonatal (infant hair, cord blood and meconium) matrices to detect fetal exposure to environmental pesticides. 116-122. E n v i r o n R e s , 1 0 9 ( 1 ) , Ostrea, E. M., Bielawski, D. M., Posecion, N. C., Corrion, M., Villanueva-Uy, E., Jin, Y., et al. (2008). A comparison of infant hair, cord blood and meconium analysis to detect fetal 185 exposure to environmental pesticides. [Evaluation Studies Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.]. E n v i r o n R e s , 1 0 6 ( 2 ) , 277-283. Ostrea, E. M., Morales, V., Ngoumgna, E., Prescilla, R., Tan, E., Hernandez, E., et al. (2002). 186 Prevalence of fetal exposure to environmental toxins as determined by meconium analysis. 329-339. N e u r o T o x i c o l o g y , 2 3 ( 3 ) , DUPLICATE EXPO EXPO EXPO Ostrea, E. M., Reyes, A., Villanueva-Uy, E., Pacifico, R., Benitez, B., Ramos, E., et al. (2011). Fetal 187 exposure to propoxur and abnormal child neurodevelopment at 2 years of age. EPI N e u r o T o x ic o lo g y . Ostrea, E. M., Villanueva-Uy, E., Bielawski, D. M., Posecion, N. C., Corrion, M. L, Jin, Y., et al. 188 (2006). Maternal hair-an appropriate matrix for detecting maternal exposure to pesticides during pregnancy. E n v i r o n R e s , 1 0 1 ( 3 ) , 312-322. EXPO Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 141 ED 002061 00044425-00141 Number Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish Oulhote, Y., & Bouchard, M. F. (2013). Urinary metabolites of organophosphate and pyrethroid 189 pesticides and behavioral problems in Canadian children. [Article]. E n v i r o n m e n t a l H e a l t h EPI P e r s p e c t i v e s , 1 2 1 , 1378-1384. Padmanabha, T. S., Gumma, K., & Kulkarni, G. P. (2014). Study of profile of organophosphorus 190 poisoning cases in a tertiary care hospital, North Karnataka, Bidar, India. [Article]. I n t e r n a t i o n a l P332-P339. J o u r n a l o f P h a r m a a n d B i o S c i e n c e s , 5 , Papoutsis, 1., Mendonis, M., Nikolaou, P., Athanaselis, S., Pistos, C., Maravelias, C., et al. (2012). 191 Development and Validation of a Simple GC-MS Method for the Simultaneous Determination of 11 Anticholinesterase Pesticides in Blood-Clinical and Forensic Toxicology Applications. [Article]. J o u r n a l o f F o r e n s i c S c i e n c e s , 5 7 , 806-812. ACR EXPO 192 Patel, A. B., Dewan, A., & Kaji, B. C. (2012). Monocrotophos poisoning through contaminated millet flour. [Article]. A r h i v z a H i g i j e n u R a d a i T o k s i k o l o g i j u , 6 3 , 377-383. ACR Perry, L., Adams, R. D., Bennett, A. R., Lupton, D. J., Jackson, G., Good, A. M., et al. (2014). 193 National toxicovigilance for pesticide exposures resulting in health care contact-An example ACR from the UK's National Poisons Information Service. [Article]. C l i n i c a l T o x i c o l o g y , 5 2 , 549-555. Peshin, S. S., Srivastava, A., Haider, N., & Gupta, Y. K. (2014). Pesticide poisoning trend analysis 194 of 13 years: A retrospective study based on telephone calls at the National Poisons Information Centre, All India Institute of Medical Sciences, New Delhi. [Article], J o u r n a l o f F o r e n s i c a n d ACR 57-61. L e g a l M e d i c i n e , 2 2 , Petit, C., Chevrier, C., Durand, G., Monfort, C., Rouget, F., Garlantezec, R., et al. (2010). Impact 195 on fetal growth of prenatal exposure to pesticides due to agricultural activities: A prospective EPI cohort study in Brittany, France. [Research Support, Non-U.S. Gov't]. E n v i r o n H e a l t h , 9 , 71. 196 Phillips, M. L. (2006). Registering skepticism: Does the EPA'S pesticide review protect children? A592-A595. E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s , 1 1 4 ( 1 0 ) , REV 142 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00142 Number Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish Pillai, H. K., Fang, M. L., Beglov, D., Kozakov, D., Vajda, S., Stapleton, H. M., et al. (2014). Ligand 197 binding and activation of PPARy by Firemaster 550: effects on adipogenesis and osteogenesis TOX in vitro. [Journal article]. E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s , 1 2 2 , 1225-1232. Polanska, K., Jurewicz, J., & Hanke, W. (2013). REVIEW OF CURRENT EVIDENCE ON THE IMPACT 198 OF PESTICIDES, POLYCHLORINATED BIPHENYLS AND SELECTED METALS ON ATTENTION DEFICIT/HYPERACTIVITY DISORDER IN CHILDREN. [Literature Review], I n t e r n a t i o n a l J o u r n a l o f REV 16-38. O c c u p a t i o n a l M e d i c i n e a n d E n v i r o n m e n t a l H e a l t h , 2 6 , 199 Potera, C. (2012). Newly discovered mechanism for cchlorpyrifos eeffects on neurodevelopment. [Article]. E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s , 1 2 0 , A270. REV Prapamontol, T., Sutan, K., Laoyang, S., Hongsibsong, S., Lee, G., Yano, Y., et al. (2014). Cross validation of gas chromatography-flame photometric detection and gas 200 chromatography--mass spectrometry methods for measuring dialkylphosphate metabolites of organophosphate pesticides in human urine, [article]. I n t e r n a t i o n a l j o u r n a l o f h y g i e n e a n d 554-566. e n v i r o n m e n t a l h e a l t h , 2 1 7 , Prapamontol, T., Sutan, K., Laoyang, S., Hongsibsong, S., Lee, G., Yano, Y., et al. (2014a). Cross validation of gas chromatography-flame photometric detection and gas chromatography-mass 201 spectrometry methods for measuring dialkylphosphate metabolites of organophosphate pesticides in human urine. 227(4- I n t e r n a t i o n a l J o u r n a l o f H y g i e n e a n d E n v i r o n m e n t a l H e a l t h , 5), 554-566. Prapamontol, T., Sutan, K., Laoyang, S., Hongsibsong, S., Lee, G., Yano, Y., et al. (2014b). Cross validation of gas chromatography-flame photometric detection and gas chromatography-mass 202 spectrometry methods for measuring dialkylphosphate metabolites of organophosphate pesticides in human urine. I n t e r n a t i o n a l J o u r n a l o f H y g i e n e a n d E n v i r o n m e n t a l H e a l t h , 2 1 7 , 554-566. Quiros-Alcala, L., Alkon, A. D., Boyce, W. T., Lippert, S., Davis, N. V., Bradman, A., et al. (2011). 203 Maternal prenatal and child organophosphate pesticide exposures and children's autonomic function. N e u r o t o x i c o l o g y , 3 2 ( 5 ) , 646-655. EXPO DUPLICATE DUPLICATE EPI 143 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00143 Number Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish Quiros-Alcala, L., Bradman, A., Smith, K., Weerasekera, G., Odetokun, M., Barr, D. B., et al. 204 (2012). Organophosphorous pesticide breakdown products in house dust and children's urine. [Article]. 559-568. J o u r n a l o f E x p o s u r e S c i e n c e a n d E n v i r o n m e n t a l E p i d e m i o l o g y , 2 2 , EXPO Radwan, N. M., & El-Nabarawy, N. A. (2014). The role of muscle injury in organophosphate 205 poisoning and development of intermediate syndrome. [Article]. I n t e r n a t i o n a l J o u r n a l o f 61-66. P h a r m a c e u t i c a l S c i e n c e s R e v i e w a n d R e s e a r c h , 2 8 , ACR Rajanandh, M. G., Santhosh, S., & Ramasamy, C. (2013). Prospective analysis of poisoning cases 206 in a super specialty hospital in India. [Article]. J o u r n a l o f P h a r m a c o l o g y a n d T o x i c o l o g y , 8 , 60- ACR 66. 207 Rajasekharan, C., Renjith, S. W., & Jayapal, T. (2012). Opsoclonus and lingual myoclonus due to organophosphate poisoning: images in clinical medicine. [Article]. B M J c a s e r e p o r t s , 2 0 1 2 . ACR Ram, P., Kanchan, T., & Unnikrishnan, B. (2014). Pattern of acute poisonings in children below 208 15 years - A study from Mangalore, South India. [Article]. J o u r n a l o f F o r e n s i c a n d L e g a l ACR 26-29. M e d i c i n e , 2 5 , Ramanath, K. V., & Naveen Kumar, H. D. (2012). Study the assessment of poisoning cases in a 209 rural tertiary care teaching hospital by a clinical pharmacist. [Article]. A s i a n J o u r n a l o f ACR 5,138-141. P h a r m a c e u t i c a l a n d C l i n i c a l R e s e a r c h , Rauch, S. A., Braun, J. M., Barr, D. B., Calafat, A. M., Khoury, J., Montesano, M. A., et al. (2012). 210 Associations of prenatal exposure to organophosphate pesticide metabolites with gestational EPI age and birth weight. [Article]. E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s , 1 2 0 , 1055-1060. Rauh, V. A., Garfinkel, R., Perera, F. P., Andrews, H. F., Hoepner, L., Barr, D. B., et al. (2006). 211 Impact of prenatal chlorpyrifos exposure on neurodevelopment in the first 3 years of life EPI among inner-city children. P e d i a t r i c s , 1 1 8 ( 6 ) , el845-1859. 144 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00144 Number Rauh, V. A., Perera, F. P., Horton, M. K., Whyatt, R. M., Bansal, R., Hao, X. J., et al. (2012). Brain 212 anomalies in children exposed prenatally to a common organophosphate pesticide. [Article]. P r o c e e d in g s o f t h e N a t io n a l A c a d e m y o f S c ie n c e s o f th e U n it e d S ta te s o f A m e r ic a , 1 0 9 (2 0 ), 7871-7876. Rauh, V. A., Perera, F. P., Horton, M. K., Whyatt, R. M., Bansal, R., Hao, X., et al. (2012). Brain 213 anomalies in children exposed prenatally to a common organophosphate pesticide. [Article]. 7871- P r o c e e d i n g s o f t h e N a t i o n a l A c a d e m y o f S c i e n c e s o f t h e U n i t e d S t a t e s o f A m e r i c a , 1 0 9 , 7876. Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish DUPLICATE DUPLICATE Rauh, V. A., Perera, F. P., Horton, M. K., Whyatt, R. M., Bansal, R., Hao, X., et al. (2012). Brain 214 anomalies in children exposed prenatally to a common organophosphate pesticide. P r o c N a t l EPI 7871-7876. A c a d S c i U S A , 1 0 9 ( 2 0 ) , Rauh, V., Arunajadai, S., Horton, M., Perera, F., Hoepner, L., Barr, D. B., et al. (2011). Seven- 215 year neurodevelopmental scores and prenatal exposure to chlorpyrifos, a common agricultural EPI pesticide. E n v i r o n H e a l t h P e r s p e c t , 1 1 9 ( 8 ) , 1196-1201. 216 Redmond, E., Egeston, C., & Bra1ley, J. A. (2012). Low level prenatal exposure to organophosphate pesticides significantly lowers IQ in children. T o w n s e n d L e t t e r ( 342), 58-61. REV Ridano, M. E., Racca, A. C., Flores-Martin, J., Camolotto, S. A., De Potas, G. M., Genti-Raimondi, 217 S., et al. (2012). Chlorpyrifos modifies the expression of genes involved in human placental function, [article]. R e p r o d u c t i v e t o x i c o l o g y ( E l m s f o r d , N Y ) , 3 3 , 331-338. Ridano, M. E., Racca, A. C., Flores-Martin, J., Camolotto, S. A., de Potas, G. M., Genti-Raimondi, 218 S., et al. (2012). Chlorpyrifos modifies the expression of genes involved in human placental function. R e p r o d u c t i v e T o x i c o l o g y , 3 3 , 331-338. TOX DUPLICATE 219 Roberts, J. R., & Karr, C. J. (2012). Pesticide Exposure in Children. [Article]. P e d i a t r i c s , 1 3 0 , E1765-E1788. REV 145 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00145 Number Roca, M., Leon, N., Pastor, A., & Yus, V. (2014). Comprehensive analytical strategy for 220 biomonitoring of pesticides in urine by liquid chromatography-orbitrap high resolution mass spectrometry. [Article]. J o u r n a l o f C h r o m a t o g r a p h y A , 1 3 7 4 , 66-76. Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish EXPO Roca, M., Miralles-Marco, A., Ferr, J., Prez, R., & Yus, V. (2014). Biomonitoring exposure 221 assessment to contemporary pesticides in a school children population of Spain. [Article]. 77-85. E n v i r o n m e n t a l R e s e a r c h , 1 3 1 , DUPLICATE Roca, M., Miralles-Marco, A., Ferr, J., Prez, R., & Yus, V. (2014). Biomonitoring exposure 222 assessment to contemporary pesticides in a school children population of Spain. [Article]. 77-85. E n v i r o n m e n t a l R e s e a r c h , 1 3 1 , EXPO Rodriguez, T., Van Wendel De Joode, B., Lindh, C. H., Rojas, M., Lundberg, 1., & Wesseling, C. 223 (2012). Assessment of long-term and recent pesticide exposure among rural school children in Nicaragua, [article]. O c c u p a t i o n a l a n d e n v i r o n m e n t a l m e d i c i n e ( L o n d o n ) , 6 9 , 119-125. DUPLICATE Rodriguez, T., van Wendel de Joode, B., Lindh, C. H., Rojas, M., Lundberg, 1., & Wesseling, C. 224 (2012). Assessment of long-term and recent pesticide exposure among rural school children in Nicaragua. O c c u p E n v i r o n M e d , 6 9 ( 2 ) , 119-125. EXPO Rogers, T. D., McKimm, E., Dickson, P. E., Goldowitz, D., Blaha, C. D., & Mittleman, G. (2013). Is 225 autism a disease of the cerebellum?: An integration of clinical and pre-clinical research. [Article], F r o n t i e r s i n S y s t e m s N e u r o s c i e n c e . Rohitrattana, J., Siriwong, W., Tunsaringkarn, T., Panuwet, P., Ryan, P. B., Barr, D. 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Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish EPI Rohlman, D. S., Lasarev, M., Anger, W. K., Scherer, J., Stupfel, J., & McCauley, L. (2007a). 229 Neurobehavioral performance of adult and adolescent agricultural workers. N e u r o t o x i c o l o g y , EPI 2 8 ( 2 ) , 374-380. Rohlman, D. S., Lasarev, M., Anger, W. K., Scherer, J., Stupfel, J., & McCauley, L. (2007b). 230 Neurobehavioral performance of adult and adolescent agricultural workers. N e u r o t o x i c o l o g y , 2 8 ( 2 ) , 374-380. DUPLICATE Rojas, M., Agreda, O., & Infante, S. (2008). A preliminary statistical study of whether pesticide 231 use could be related to birth defects in a rural area of Venezuela. R e v S a l u d P u b l i c a ( B o g o t a ) , EPI 1 0 ( 1 ) , 85-93. Ross, M. K., Borazjani, A., Mangum, L. C., Wang, R., & Crow, J. A. (2014). Effects of 232 toxicologically relevant xenobiotics and the lipid-derived electrophile 4-hydroxynonenal on macrophage cholesterol efflux: Silencing carboxylesterase 1 has paradoxical effects on TOX cholesterol uptake and efflux. [Article], C h e m i c a l R e s e a r c h i n T o x i c o l o g y , 2 7 , 1743-1756. Ross, S. M., McManus, 1. C., Harrison, V., & Mason, O. (2013). Neurobehavioral problems 233 following low-level exposure to organophosphate pesticides: a systematic and meta-analytic REV review. [Literature Review]. C r i t i c a l R e v i e w s i n T o x i c o l o g y , 4 3 , 21-44. Roth, N., & Wilks, M. F. (2014). Neurodevelopmental and neurobehavioural effects of 234 polybrominated and perfluorinated chemicals: A systematic review of the epidemiological REV literature using a quality assessment scheme. T o x i c o l o g y L e t t e r s , 2 3 0 , 271-281. Ruckart, P. Z., Kakolewski, K., Bove, F. J., & Kaye, W. E. (2004). 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I n d i a n J o u r n a l o f F o r e n s i c M e d i c i n e a n d T o x i c o l o g y , ACR 6 , 104-106. 238 Savitz, D. A., Arbuckle, T., Kaczor, D., & Curtis, K. M. (1997). Male pesticide exposure and pregnancy outcome. A m J E p i d e m i o l , 1 4 6 ( 1 2 ) , 1025-1036. EPI Schug, T. T., Barouki, R., Gluckman, P. D., Grandjean, P., Hanson, M., & Heindel, J. J. (2013). 239 PPTOX III: Environmental stressors in the developmental origins of disease-Evidence and mechanisms. [Article]. T o x i c o l o g i c a l S c i e n c e s , 1 3 1 , 343-350. Selvam, V., Panneer Selvam, G., & Vijayanath, V. (2012). Study of death incidence by 240 insecticide poisoning in Salem. [Article]. I n t e r n a t i o n a l J o u r n a l o f M e d i c a l T o x i c o l o g y a n d 20-26. F o r e n s i c M e d i c i n e , 2 , Senarathna, L., Jayamanna, S. F., Kelly, P. J., Buckley, N. A., Dibley, M. J., & Dawson, A. H. 241 (2012). Changing epidemiologic patterns of deliberate self poisoning in a rural district of Sri Lanka. [Article]. B M C p u b l i c h e a l t h , 1 2 , 593. Sexton, K,, Salinas, J. J., McDonald, T. J., Gowen, R. M. Z., Miller, R. P., McCormick, J. B., et al. 242 (2013). Biomarkers of maternal and fetal exposure to organochlorine pesticides measured in pregnant hispanic women from brownsville, texas. [Article]. I n t e r n a t i o n a l J o u r n a l o f 237-248. E n v i r o n m e n t a l R e s e a r c h a n d P u b l i c H e a l t h , 1 0 , Shelton, J. F., Geraghty, E. M., Tancredi, D. J., Delwiche, L. D., Schmidt, R. J., Ritz, B., et al. 243 (2014). Neurodevelopmental disorders and prenatal residential proximity to agricultural pesticides: The charge study. [Article]. E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s , 1 2 2 , 1103-1109. REV ACR ACR EXPO EPI 148 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00148 Number Shelton, J. F., Geraghty, E. M., Tancredi, D. J., Delwiche, L. D., Schmidt, R. J., Ritz, B., et al. 244 (2014). Neurodevelopmental Disorders and Prenatal Residential Proximity to Agricultural Pesticides: The CHARGE Study. E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s , 122(10), 1103-1109. Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish DUPLICATE 245 Shomar, B., Al-Saad, K,, & Nriagu, J. (2014). Mishandling and exposure of farm workers in Qatar to organophosphate pesticides. E n v i r o n m e n t a l R e s e a r c h , 1 3 3 , 312-320. EXPO Simescu, M., Igna, C. P., Nicolaescu, E., Ion, 1., Ion, A. C., Caragheorgheopol, A., et al. (2014). 246 MULTIPLE PESTICIDES EXPOSURE OF GREENHOUSE WORKERS AND THYROID PARAMETERS, [article]. 15-28. 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M e d i c a l A s s o c i a t i o n , 3 0 8 , Slotkin, T. A., Card, J., & Seidler, F. J. (2013). Adverse benzoapyrene effects on 250 neurodifferentiation are altered by other neurotoxicant coexposures: Interactions with dexamethasone, chlorpyrifos, or nicotine in PC12 cells. [Article]. E n v i r o n m e n t a l H e a l t h 825-831. P e r s p e c t i v e s , 1 2 1 , Slotkin, T. A., Card, J., & Seidler, F. J. (2014). Prenatal dexamethasone, as used in preterm 251 labor, worsens the impact of postnatal chlorpyrifos exposure on serotonergic pathways. [Journal article]. B r a i n R e s e a r c h B u l l e t i n , 1 0 0 , 44-54. EPI DUPLICATE TOX REV TOX TOX 149 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00149 Number Smith, J. N., Hinderliter, P. M., Timchalk, C., Bartels, M. J., & Poet, T. S. (2014). A human life252 stage physiologically based pharmacokinetic and pharmacodynamic model for chlorpyrifos: Development and validation. [Article]. R e g u l a t o r y T o x i c o l o g y a n d P h a r m a c o l o g y , 6 9 , 580-597. Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-English TOX Smith, J. N., Hinderliter, P. M., Timchalk, C., Bartels, M. J., & Poet, T. S. (2014). A human life253 stage physiologically based pharmacokinetic and pharmacodynamic model for chlorpyrifos: Development and validation. R e g u l a t o r y T o x i c o l o g y a n d P h a r m a c o l o g y , 6 9 ( 3 ) , 580-597. DUPLICATE Snijder, C. A., Heederik, D., Pierik, F. H., Hofman, A., Jaddoe, V. W., Koch, H. M., et al. (2013). 254 Fetal growth and prenatal exposure to bisphenol A: The generation R study. [Article]. EPI 393-396. E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s , 1 2 1 , Stapleton, H. M., Misenheimer, J., Hoffman, K., & Webster, T. F. (2014). Flame retardant 255 associations between children's handwipes and house dust. [Article]. C h e m o s p h e r e , 1 1 6 , 54- 60. EXPO Suarez-Lopez, J. R., Himes, J. H., Jacobs Jr, D. R., Alexander, B. H., & Gunnar, M. R. (2013). 256 Acetylcholinesterase activity and neurodevelopment in boys and girls. [Article]. P e d i a t r i c s , 1 3 2 , EPI el649-el658. Suarez-Lopez, J. R., Jacobs Jr, D. R., Himes, J. H., & Alexander, B. H. (2013). Acetylcholinesterase 257 activity, cohabitation with floricultura! workers, and blood pressure in Ecuadorian children. EPI [Article], 619-624. E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s , 1 2 1 , Suarez-Lopez, J. R., Jacobs, D. R., Himes, J. H., Alexander, B. H., Lazovich, D., & Gunnar, M. 258 (2012). Lower acetylcholinesterase activity among children living with flower plantation EPI workers. [Article], E n v i r o n m e n t a l R e s e a r c h , 1 1 4 , 53-59. 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Exposure to persistent organic 261 pollutants in utero and related maternal characteristics on birth outcomes: A multivariate data EPI analysis approach. C h e m o s p h e r e , 74(3), 428-433. Taxvig, C., Rosenmai, A. K., & Vinggaard, A. M. (2014). Polyfluorinated alkyl phosphate ester 262 surfactants - current knowledge and knowledge gaps. [Article]. B a s i c a n d C l i n i c a l 41-44. P h a r m a c o l o g y a n d T o x i c o l o g y , 1 1 5 , Thompson, B., Griffith, W. C., Barr, D. B., Coronado, G. D., Vigoren, E. M., & Faustman, E. M. 263 (2014). Variability in the take-home pathway: farmworkers and non-farmworkers and their children. [Journal article]. J o u r n a l o f E x p o s u r e S c i e n c e a n d E n v i r o n m e n t a l E p i d e m i o l o g y , 2 4 , 522-531. Torres-Sanchez, L., Rothenberg, S. J., Schnaas, L., Cebrian, M. E., Osorio, E., Del Carmen 264 Hernandez, M., et al. (2007). 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S., Davis, M. D., Calafat, A. M., et al. 267 (2014). Urinary pyrethroid and chlorpyrifos metabolite concentrations in northern California families and their relationship to indoor residential insecticide levels, part of the Study of Use of Products and Exposure Related Behavior (SUPERB). [Article], E n v i r o n m e n t a l S c i e n c e a n d REV EXPO EPI EPI OTH DUPLICATE 151 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00151 Number T e c h n o l o g y , 4 8 , 1931-1939. Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish Trunnelle, K. J., Bennett, D. H., Tulve, N. S., Clifton, M. S., Davis, M. D., Calafat, A. M., et al. (2014). 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A revised method for determination of dialkylphosphate levels in human urine by solid-phase extraction 270 and liquid chromatography with tandem mass spectrometry: application to human urine samples from Japanese children. [Journal article]. E n v i r o n m e n t a l H e a l t h a n d P r e v e n t i v e M e d i c i n e , 1 9 , 405-413. van Balen, E. C., Wolansky, M. J., & Kosatsky, T. (2012). Increasing use of pyrethroids in 271 Canadian households: Should we be concerned? [Note]. C a n a d i a n J o u r n a l o f P u b l i c H e a l t h , 1 0 3 , 404-407. Van Dyke, M., Martyny, J. W., & Serrano, K. A. (2014). Methamphetamine Residue Dermal 272 Transfer Efficiencies from Household Surfaces. [Article]. J o u r n a l o f O c c u p a t i o n a l a n d 249-258. E n v i r o n m e n t a l H y g i e n e , 1 1 ( 4 ) , Van Thriel, C., Westerink, R. H. S., Beste, C., Bale, A. S., Lein, P. J., & Leist, M. (2012). Translating 273 neurobehavioural endpoints of developmental neurotoxicity tests into in vitro assays and readouts. [Article]. N e u r o T o x i c o i o g y , 3 3 , 911-924. DUPLICATE EXPO EXPO REV EXPO REV 152 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00152 Number Van Wendel De Joode, B., Barraza, D., Ruepert, C., Mora, A. M., Cordoba, L., 6 Berg, M., et al. 274 (2012). Indigenous children living nearby plantations with chlorpyrifos-treated bags have elevated 3,5,6-trichloro-2-pyridinol (TCPy) urinary concentrations, [article]. E n v i r o n m e n t a l : 17-26. r e s e a r c h ( N e w Y o r k , N . Y . P r i n t ) , 1 1 7 , Veale, D. J. H., Wium, C. A., & Muller, G. J. (2013). Toxicovigilance 1: A survey of acute 275 poisonings in South Africa based on Tygerberg Poison Information centre data. [Article]. S o u t h 293-297. A f r i c a n M e d i c a l J o u r n a l , 1 0 3 , Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish DUPLICATE ACR Venerosi, A., Ricceri, L., Tait, S., & Calamandrei, G. (2012). Sex dimorphic behaviors as markers 276 of neuroendocrine disruption by environmental chemicals: The case of chlorpyrifos. [Article]. TOX 1420-1426. N e u r o T o x i c o l o g y , 3 3 , Vera, B., Cruz, S. S., & Magnarelli, G. (2012). Plasma cholinesterase and carboxylesterase 277 activities and nuclear and mitochondrial lipid composition of human placenta associated with TOX maternal exposure to pesticides. [Article]. R e p r o d u c t i v e T o x i c o l o g y , 3 4 ( 3 ) , 402-407. Vera, B., Santa Cruz, S., & Magnarelli, G. (2012). Plasma cholinesterase and carboxylesterase 278 activities and nuclear and mitochondrial lipid composition of human placenta associated with maternal exposure to pesticides, [article]. R e p r o d u c t i v e t o x i c o l o g y ( E l m s f o r d , N Y ) , 3 4 , 402-407. DUPLICATE Vogt, R., Bennett, D., Cassady, D., Frost, J., Ritz, B., & Hertz-Picciotto, 1. (2012). Cancer and non 279 cancer health effects from food contaminant exposures for children and adults in California: A REV risk assessment. [Article]. E n v i r o n m e n t a l H e a l t h : A G l o b a l A c c e s s S c i e n c e S o u r c e , 1 1 . 280 Wang, P., Tian, Y., Wang, X. J., Gao, Y., Shi, R,, Wang, G. Q., et al. (2011). Organophosphate pesticide exposure and perinatal outcomes in Shanghai, China. E n v i r o n I n t . DUPLICATE Wang, P., Tian, Y., Wang, X.-J., Gao, Y., Shi, R., Wang, G.-Q., et al. (2012). Organophosphate 281 pesticide exposure and perinatal outcomes in Shanghai, China. [Article]. E n v i r o n m e n t EPI 100-104. I n t e r n a t i o n a l , 4 2 , 153 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00153 Number Wason, S. C., Julien, R., Perry, M. J., Smith, T. J., & Levy, J. I. (2013). Modeling exposures to 282 organophosphates and pyrethroids for children living in an urban low-income environment. [Article]. E n v i r o n m e n t a l R e s e a r c h , 1 2 4 , 13-22. Wason, S. C., Smith, T. J., Perry, M. J., & Levy, J. 1. (2012). Using physiologically-based 283 pharmacokinetic models to incorporate chemical and non-chemical stressors into cumulative risk assessment: A case study of pesticide exposures. [Article]. I n t e r n a t i o n a l J o u r n a l o f 1971-1983. E n v i r o n m e n t a l R e s e a r c h a n d P u b l i c H e a l t h , 9 , Whyatt, R. M., Rauh, V., Barr, D. B., Camann, D. E., Andrews, H. F., Garfinkel, R., et al. (2004). 284 Prenatal insecticide exposures and birth weight and length among an urban minority cohort. 1125-1132. E n v i r o n H e a l t h P e r s p e c t , 1 1 2 ( 1 0 ) , Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish EXPO EXPO EPI Wickerham, E. L., Lozoff, B., Jie, S., Kaciroti, N,, Yankai, X. 1. A., & Meeker, J. D. (2012). Reduced 285 birth weight in relation to pesticide mixtures detected in cord blood of full-term infants, EPI [article]. 80-85. E n v i r o n m e n t i n t e r n a t i o n a l , 4 7 , Wickerham, E. L., Lozoff, B., Shao, J., Kaciroti, N., Xia, Y. K., & Meeker, J. D. (2012). Reduced 286 birth weight in relation to pesticide mixtures detected in cord blood of full-term infants. [Article]. 80-85. E n v i r o n m e n t I n t e r n a t i o n a l , 4 7 , DUPLICATE Williams, A. L., & DeSesso, J. M. (2014). Gestational/ Perinatal chlorpyrifos exposure is not 287 associated with autistic-like behaviors in rodents. [Literature Review]. C r i t i c a l R e v i e w s in REV 523-534. T o x i c o l o g y , 4 4 , 288 Wolff, M. S., Engel, S., Berkowitz, G., Teitelbaum, S., Siskind, J., Barr, D. B., et al. (2007). Prenatal pesticide and PCB exposures and birth outcomes. P e d i a t r R e s , 6 1 ( 2 ) , 243-250. EPI 289 Xiang, H., Nuckols, J. R., & Stallones, L. (2000). A geographic information assessment of birth weight and crop production patterns around mother's residence. E n v i r o n R e s , 8 2 ( 2 ) , 160-167. EPI 154 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00154 Number Ye, X., Pierik, F. H., Hauser, R., Duty, S., Angerer, J., Park, M. M., et al. (2008). Urinary 290 metabolite concentrations of organophosphorous pesticides, bisphenol A, and phthalates among pregnant women in Rotterdam, the Netherlands: The Generation R study. [Research Support, N.I.H., Extramural]. E n v i r o n R e s , 1 0 8 ( 2 ) , 260-267. Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish EXPO Yolton, K., Cornelius, M., Ornoy, A., McGough, J., Makris, S., & Schantz, S. (2014). Exposure to 291 neurotoxicants and the development of attention deficit hyperactivity disorder and its related REV behaviors in childhood. [Journal article]. N e u r o t o x i c o l o g y a n d T e r a t o l o g y , 4 4 , 30-45. Young, J. G., Eskenazi, B., Gladstone, E. A., Bradman, A., Pedersen, L., Johnson, C., et al. (2005). 292 Association between in utero organophosphate pesticide exposure and abnormal reflexes in EPI neonates. N e u r o t o x i c o l o g y , 2 6 ( 2 ) , 199-209. Yuan, Y., Chen, C., Zheng, C., Wang, X., Yang, G., Wang, Q., et al. (2014). Residue of chlorpyrifos 293 and cypermethrin in vegetables and probabilistic exposure assessment for consumers in Zhejiang Province, China. [Article]. F o o d C o n t r o l , 3 6 , 63-68. EXPO 294 Yusa, V., Coscolla, C., & Millet, M. (2014). New screening approach for risk assessment of pesticides in ambient air. [Article]. A t m o s p h e r i c E n v i r o n m e n t , 9 6 , 322-330. REV Zhang, X., Wallace, A. D., Du, P., Lin, S., Baccarelli, A. A., Jiang, H., et al. (2012). Genome-wide 295 study of DNA methylation alterations in response to diazinon exposure in vitro. [Article]. 959-968. E n v i r o n m e n t a l T o x i c o l o g y a n d P h a r m a c o l o g y , 3 4 , Zhang, Y., Han, S., Liang, D., Shi, X., Wang, F., Liu, W., et al. (2014). Prenatal exposure to 296 organophosphate pesticides and neurobehavioral development of neonates: A birth cohort study in Shenyang, China. [Article]. P L o S O N E , 9 . Zhang, Y., Song, H., Liang, D., Shi, X., Wang, F., Liu, W., et al. (2014). Prenatal exposure to 297 organophosphate pesticides and neurobehavioral development of neonates: a birth cohort study in Shenyang, China. [Journal article]. P L o S O N E , 9 , e88491. TOX EPI DUPLICATE 155 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00155 Number Category (EPI-epi only; EXPO- exposure only; ACR acute case report or case series; TOX-animal study; REV - review or commentary, no original data; OTH - other, non-Engiish 298 Zhao, Q., Gadagbui, B., & Dourson, M. (2005). Lower birth weight as a critical effect of chlorpyrifos: A comparison of human and animal data. R e g u l T o x i c o l P h a r m a c o l , 42(1), 55-63. REV Zhou, S., Rosenthal, D. G., Sherman, S., Zelikoff, J., Gordon, T., & Weitzman, M. (2014). 299 Physical, Behavioral, and Cognitive Effects of Prenatal Tobacco and Postnatal Secondhand REV Smoke Exposure. 219-241. C u r r e n t P r o b l e m s i n P e d i a t r i c a n d A d o l e s c e n t H e a l t h C a r e , 4 4 , Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 156 ED 002061 00044425-00156 Appendix 5. Table of Systematic Review Analysis: Second Tier Exclusion Criteria. 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 literature review. Number Citations {Of 79:17 - part of 3 cohorts, previously reviewed 24excluded (NOT OP. NOT ND, NOT EPI) 38-include (9 no direct OP measure) Category (EPI-epi only; EXPO- exposure only; ACR - acute case report or case series; TOXanimal study; REV review or commentary, no original data; OTH - other, non-English include? Outcome==pediatric ND/NB/motor control/morphology/motor control; Exposure==OP (biomarker, questionnaire. env media); epi study (cohort/case control/cross sectional (not ecologic)) Include? Rationale Burdorf, A., Brand, T., Jaddoe, V. W., Hofman, A., Mackenbach, J. P., 1 & Steegers, E. A. (2011). The effects of work-related maternal risk factors on time to pregnancy, preterm birth and birth weight: the EPI NO NO OP; ALL PESTICIDE ONLY Generation R Study. O c c u p E n v i r o n M e d , 68(3), 197-204. Xiang, H., Nuckols, J. R., & Stallones, L. (2000). A geographic 2 information assessment of birth weight and crop production patterns EPI around mother's residence. E n v i r o n R e s , 8 2 ( 2 ) , 160-167. NO No pesticide measure Ferreira, J. D., Couto, A. C. z., Pombo-de-Oliveira, M. S., & Koifman, S. 3 (2013). In utero pesticide exposure and leukemia in Brazilian children < 2 years of age. [Note]. E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s , 1 2 1 , 269- EPI 275. How, V., Hashim, Z., Ismail, P., Said, S. M., Dzolkhifli, O., & Shamsul Bahri, M. T. (2014). Exploring cancer development in adulthood: 4 cholinesterase depression and genotoxic effect from chronic EPI exposure to organophosphate pesticides among rural farm children. [Journal article]. J o u r n a l o f A g r o m e d i c i n e , 1 9 , 35-43. NO NOT ND NO not ND Huen, K., Harley, K., Beckman, K., Eskenazi, B., & Holland, N. (2013). 5 Associations of PON1 and Genetic Ancestry with Obesity in Early EPI Childhood. [Article]. P L o S O N E , 8 . NO not ND 157 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00157 Number Citations {Of 79:17 part of 3 cohorts, previously reviewed 24excluded (NOT OP, NOT ND, NOT EPI) 38-include (9 no direct OP measure) Jones, K., Everard, M., & Harding, A. H. (2014). Investigation of 6 gastrointestinal effects of organophosphate and carbamate pesticide residues on young children. [Journal article]. I n t e r n a t i o n a l J o u r n a l o f 392-398. H y g i e n e a n d E n v i r o n m e n t a l H e a l t h , 2 1 7 , Khan, K., Ismail, A. A., Abdel Rasoul, G., Bonner, M. R., Lasarev, M. R., 7 Hendy, O., et al. (2014). Longitudinal assessment of chlorpyrifos exposure and self-reported neurological symptoms in adolescent pesticide applicators. [Article], B M J O p e n , 4 . Michalakis, M., Tzatzarakis, M. N., Kovatsi, L., Alegakis, A. K., Tsakalof, 8 A. K., Heretis, 1., et al. (2014). Hypospadias in offspring is associated with chronic exposure of parents to organophosphate and organochlorine pesticides. [Article], T o x i c o l o g y L e t t e r s , 2 3 0 , 139-145. Rohlman, D. S., Ismail, A. A., Abdel-Rasoul, G., Lasarev, M., Hendy, O., 9 & Olson, J. R. (2014). Characterizing exposures and neurobehavioral performance in Egyptian adolescent pesticide applicators. [Article], 845-855. M e t a b o l i c B r a i n D i s e a s e , 2 9 , Rojas, M., Agreda, O., & Infante, S. (2008). A preliminary statistical 10 study of whether pesticide use could be related to birth defects in a rural area of Venezuela. R e v S a l a d P u b l i c a ( B o g o t a ) , 10(1), 85-93. Simescu, M., Igna, C. P., Nicolaescu, E., Ion, 1., Ion, A. C., Caragheorgheopol, A., et al. (2014). MULTIPLE PESTICIDES EXPOSURE 11 OF GREENHOUSE WORKERS AND THYROID PARAMETERS, [article]. I n te r n a tio n a l jo u r n a l o f s u s ta in a b le d e v e lo p m e n t a n d p la n n in g ( P r i n t ) , 9 , 15-28. Category (EPI-epi only; EXPO- exposure only; ACR - acute case report or case series; TOXanimal study; REV review or commentary, no original data; OTH other, non-English EPI EPI EPI EPI EPI EPI Include? Outcome-- pediatric ND/NB/motor control/morphology/motor control; xposure=-QP (biomarker, questionnaire, env media); epi study (cohort/case control/cross sectional (not cologie)} NO NO NO NO NO NO include? Rationale not ND not ND not ND not ND not ND not ND Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 158 ED 002061 00044425-00158 Number Citations {Of 79:17 part of 3 cohorts, previously reviewed 24excluded (NOT OP, NOT ND, NOT EPI) 38-include (9 no direct OP measure) Cecchi, A., Rovedatti, M. G., Sabino, G., & Magnarelli, G. G. (2012a). 12 Environmental exposure to organophosphate pesticides: Assessment of endocrine disruption and hepatotoxicity in pregnant women. [Article]. 280-287. E c o t o x i c o l o g y a n d E n v i r o n m e n t a l S a f e t y , 8 0 , Rohlman, D. S., Lasarev, M., Anger, W. K., Scherer, J., Stupfel, J., & 13 McCauley, L. (2007a). Neurobehavioral performance of adult and adolescent agricultural workers. N e u r o t o x i c o l o g y , 2 8 ( 2 ) , 374-380. Category (EPI-epi only; EXPO- exposure only; ACR - acute case report or case series; TOXanimal study; REV review or commentary, no original data; OTH other, non-English EPI Include? Outcome-- pediatric ND/NB/motor control/morphology/motor control; xposure=-QP (biomarker, questionnaire, env media): epi study (cohort/case control/cross sectional (not cologie)} NO include? Rationale not ND directly; maternal ED/thyroid change->adverse fetal (unmeasured) EPI NO not ND, not OP Carmichael, S. L., Yang, W., Roberts, E. M., Kegley, S. E., Wolff, C., 14 Guo, L., et al. (2013). Hypospadias and residential proximity to EPI pesticide applications. [Article]. P e d i a t r i c s , 1 3 2 , el216-el226. Carmichael, S. L., Yang, W., Roberts, E., Kegley, S. E., Padula, A. M., English, P. B., et al. (2014). Residential agricultural pesticide 15 exposures and risk of selected congenital heart defects among EPI offspring in the San Joaquin Valley of California. [Article]. 133-138. E n v i r o n m e n t a l R e s e a r c h , 1 3 5 , Eskenazi, B., Chevrier, J., Rauch, S. A., Kogut, K., Harley, K. G., Johnson, C., et al. (2013). In Utero and Childhood Polybrominated 16 Diphenyl Ether (PBDE) Exposures and Neurodevelopment in the EPI CHAMACOS Study. [Article]. E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s , 1 2 1 ( 2 ) , 257-262. Kezios, K. L., Liu, X. H., Cirillo, P. M., Cohn, B. A., Kalantzi, O. 1., Wang, 17 Y. Z., et al. (2013). Dichlorodiphenyltrichloroethane (DDT), DDT metabolites and pregnancy outcomes. [Article]. R e p r o d u c t i v e EPI 156-164. T o x i c o l o g y , 3 5 , not ND; measured NO all pesticides, some OP not ND; measured NO all pesticides, some OP NO not OP NO not OP 159 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00159 Number Citations {Of 79:17 part of 3 cohorts, previously reviewed 24excluded (NOT OP, NOT ND, NOT EPI) 38-include (9 no direct OP measure) Kicinski, M., Vrijens, J., Vermier, G., Hond, E. D., Schoeters, G., Nelen, 18 V., et al. (2015). Neurobehavioral function and low-level metal exposure in adolescents. I n t e r n a t i o n a l J o u r n a l o f H y g i e n e a n d 139-146. E n v i r o n m e n t a l H e a l t h , 2 1 8 , Ostrea, E. M., Reyes, A., Villanueva-Uy, E., Pacifico, R., Benitez, B., 19 Ramos, E., et al. (2011). Fetal exposure to propoxur and abnormal child neurodevelopment at 2 years of age. N e u r o T o x i c o l o g y . Snijder, C. A., Heederik, D., Pierik, F. H., Hofman, A., Jaddoe, V. W., 20 Koch, H. M., et al. (2013). Fetal growth and prenatal exposure to bisphenol A: The generation R study. [Article]. E n v i r o n m e n t a l H e a l t h 393-396. P e r s p e c t i v e s , 1 2 1 , Tan, J., Loganath, A., Chong, Y. S., & Obbard, J. P. (2009). Exposure to 21 persistent organic pollutants in utero and related maternal characteristics on birth outcomes: A multivariate data analysis approach. C h e m o s p h e r e , 74(3), 428-433. Torres-Sanchez, L., Rothenberg, S. J., Schnaas, L., Cebrian, M. E., Osorio, E., Del Carmen Hernandez, M., et al. (2007). In utero p,p'-DDE 22 exposure and infant neurodevelopment: A perinatal cohort in Mexico. [Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't]. E n v i r o n H e a l t h P e r s p e c t , 1 1 5 ( 3 ) , 435-439. Torres-Sanchez, L., Schnaas, L., Cebrian, M. E., Hernandez Mdel, C., Valencia, E. O., Garcia Hernandez, R. M., et al. (2009). Prenatal 23 dichlorodiphenyldichloroethylene (DDE) exposure and neurodevelopment: a follow-up from 12 to 30 months of age. [Research Support, Non-U.S. Gov't]. N e u r o T o x i c o l o g y , 3 0 ( 6 ) , 11621165. Category (EPI-epi only; EXPO- exposure only; ACR - acute case report or case series; TOXanimal study; REV review or commentary, no original data; OTH other, non-English EPI EPI EPI EPI EPI EPI Include? Outcome-- pediatric ND/NB/motor control/morphology/motor control; xposure=-GP (biomarker, questionnaire, env media); epi study (cohort/case control/cross sectional (not cologie)} NO NO NO NO NO NO include? Rationale not OP not OP not OP not OP not OP not OP 160 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00160 Number Citations {Of 79:17 part of 3 cohorts, previously reviewed 24excluded (NOT OP, NOT ND, NOT EPI) 38-include (9 no direct OP measure) Acosta-Maldonado, B., Sanchez-Ramirez, B., Reza-Lopez, S., & Levario-Carrillo, M. (2009). Effects of exposure to pesticides during 24 pregnancy on placental maturity and weight of newborns: a cross- sectional pilot study in women from the Chihuahua State, Mexico. 451-459. H u m E x p T o x i c o l , 2 8 ( 8 ) , Andersen, H. R., Debes, F., Wohlfahrt-Veje, C., Murata, K., & 25 Grandjean, P. (2015). Occupational pesticide exposure in early pregnancy associated with sex-specific neurobehavioral deficits in the children at school age. N e u r o t o x i c o l o g y a n d T e r a t o l o g y , 4 7 , 1-9. Barr, D. B., Ananth, C. V., Yan, X., Lashley, S., Smulian, J. C., Ledoux, T. A., et al. (2010). Pesticide concentrations in maternal and umbilical 26 cord sera and their relation to birth outcomes in a population of pregnant women and newborns in New Jersey. S c i T o t a l E n v i r o n , 4 0 8 ( 4 ) , 790-795. Berkowitz, G. S., Wetmur, J. G., Birman-Deych, E., Obel, J., Lapinski, R. 27 H., Godbold, J. H., et al. (2004). In utero pesticide exposure, maternal paraoxonase activity, and head circumference. E n v i r o n H e a l t h 388-391. P e r s p e c t , 1 1 2 ( 3 ) , Bouchard, M. F., Bellinger, D. C., Wright, R. O., & Weisskopf, M. G. 28 (2010). Attention-deficit/hyperactivity disorder and urinary metabolites of organophosphate pesticides. [Research Support, N.I.H., Extramural]. P e d i a t r i c s , 1 2 5 ( 6 ) , el270-1277. Bouchard, M. F., Chevrier, J., Harley, K. G., Kogut, K., Vedar, M., 29 Calderon, N., et al. (2011). Prenatal exposure to organophosphate pesticides and IQ in 7-year-old children. E n v i r o n H e a l t h P e r s p e c t , 1 1 9 ( 8 ) , 1189-1195. Category (EPI-epi only; EXPO- exposure only; ACR - acute case report or case series; TOXanimal study; REV review or commentary, no original data; OTH other, non-English EPI EPI EPI EPI EPI EPi Include? Outcome-- pediatric ND/NB/motor control/morphology/motor control; xposure=-QP (biomarker, questionnaire, env media): epi study (cohort/case control/cross sectional (not cologie)} YES YES YES YES-3 COHO RTS YES YES-3COHORTS include? Rationale 161 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00161 Number Citations {Of 79:17 part of 3 cohorts, previously reviewed 24excluded (NOT OP, NOT ND, NOT EPI) 38-include (9 no direct OP measure) Dabrowski, S., Hanke, W., Polanska, K., Makowiec-Dabrowska, T., & 30 Sobala, W. (2003). Pesticide exposure and birthweight: an epidemiological study in Central Poland. I n t J O c c u p M e d E n v i r o n 31-39. H e a l t h , 1 6 ( 1 ) , Ding, G. D., Wang, P., Tian, Y., Zhang, J., Gao, Y., Wang, X. J., et al. 31 (2012). Organophosphate Pesticide Exposure and Neurodevelopment in Young Shanghai Children. [Article]. E n v i r o n m e n t a l S c i e n c e & T e c h n o l o g y , 4 6 ( 5 ) , 2911-2917. Engel, S. M., Berkowitz, G. S., Barr, D. B., Teitelbaum, S. L., Siskind, J., Meisel, S. J., et al. (2007). Prenatal organophosphate metabolite and 32 organochlorine levels and performance on the Brazelton Neonatal Behavioral Assessment Scale in a multiethnic pregnancy cohort. A m J 1397-1404. E p i d e m i o l , 1 6 5 ( 1 2 ) , Engel, S. M., Wetmur, J., Chen, J., Zhu, C., Barr, D. B., Canfield, R. L., 33 et al. (2011). Prenatal exposure to organophosphates, paraoxonase 1 , and cognitive development in childhood. E n v i r o n H e a l t h P e r s p e c t , 1 1 9 ( 8 ) , 1182-1188. Eskenazi, B., Harley, K., Bradman, A., Weltzien, E., Jewell, N. P., Barr, 34 D. B., et al. (2004). Association of in utero organophosphate pesticide exposure and fetal growth and length of gestation in an agricultural population. E n v i r o n H e a l t h P e r s p e c t , 1 1 2 ( 1 0 ) , 1116-1124. Eskenazi, B., Huen, K., Marks, A., Harley, K. G., Bradman, A., Barr, D. 35 B., et al. (2010). PON1 and neurodevelopment in children from the CHAMACOS study exposed to organophosphate pesticides in utero. 1775-1781. E n v i r o n H e a l t h P e r s p e c t , 1 1 8 ( 1 2 ) , Category (EPI-epi only; EXPO- exposure only; ACR - acute case report or case series; TOXanimal study; REV review or commentary, no original data; OTH other, non-English EPI EPI EPI EPI EPI EPI Include? Outcome-- pediatric ND/NB/motor control/morphology/motor control; xposure=-QP (biomarker, questionnaire, env media); epi study (cohort/case control/cross sectional (not cologie)} YES YES YES-3COHORTS YES-3COHORTS YES-3COHORTS YES-3COHORTS include? Rationale Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 162 ED 002061 00044425-00162 Number Citations {Of 79:17 part of 3 cohorts, previously reviewed 24excluded (NOT OP, NOT ND, NOT EPI) 38-include (9 no direct OP measure) Eskenazi, B., Kogut, K., Huen, K., Harley, K. G., Bouchard, M., 36 Bradman, A., et al. (2014a). Organophosphate pesticide exposure, PONI, and neurodevelopment in school-age children from the CHAMACOS study. E n v i r o n m e n t a l R e s e a r c h , 1 3 4 , 149-157. Eskenazi, B., Marks, A. R., Bradman, A., Harley, K., Barr, D. B., 37 Johnson, C., et al. (2007). Organophosphate pesticide exposure and neurodevelopment in young Mexican-American children. E n v i r o n 792-798. H e a l t h P e r s p e c t , 1 1 5 ( 5 ) , Fortenberry, G. Z., Meeker, J. D., Sanchez, B. N., Barr, D. B., Panuwet, P., Bellinger, D., et al. (2014). Urinary 3,5,6-trichloro-2-pyridinol 38 (TCPY) in pregnant women from Mexico City: distribution, temporal variability, and relationship with child attention and hyperactivity. I n t 405-412. J H y g E n v i r o n H e a l t h , 2 1 7 ( 2 - 3 ) , Fortenberry, G. Z., Meeker, J. D., Snchez, B. N., Bellinger, D., 39 Peterson, K., Schnaas, L., et al. (2014). Paraoxonase 1polymorphisms and attention/hyperactivity in school-age children from Mexico City, Mexico. [Article]. E n v i r o n m e n t a l R e s e a r c h , 1 3 2 , 342-349. Furlong, M. A., Engel, S. M., Barr, D. B., & Wolff, M. S. (2014). 40 Prenatal exposure to organophosphate pesticides and reciprocal social behavior in childhood. E n v i r o n I n t , 7 0 , 125-131. Grandjean, P., Harari, R., Barr, D. B., & Debes, F. (2006). Pesticide 41 exposure and stunting as independent predictors of neurobehavioral deficits in Ecuadorian school children. [Research Support, Non-U.S. Gov't]. P e d i a t r i c s , 1 1 7 ( 3 ) , e546-556. Category (EPI-epi only; EXPO- exposure only; ACR - acute case report or case series; TOXanimal study; REV review or commentary, no original data; OTH other, non-English Include? Outcome-- pediatric ND/NB/motor control/morphology/motor control; xposure=-QP (biomarker, questionnaire, env media): epi study (cohort/case control/cross sectional (not cologie)} EPI YES-3COHORTS EPI YES-3COHORTS EPI YES EPI YES EPI YES EPI YES include? Rationale Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 163 ED 002061 00044425-00163 Number Citations {Of 79:17 part of 3 cohorts, previously reviewed 24excluded (NOT OP, NOT ND, NOT EPI) 38-include (9 no direct OP measure) Guodong, D., Pei, W., Ying, T., Jun, Z., Yu, G., Xiaojin, W., et al. (2012). 42 Organophosphate pesticide exposure and neurodevelopment in young Shanghai children. E n v i r o n S c i T e c h n o l . Handal, A. J., Harlow, S. D., Breilh, J ., & Lozoff, B. (2008). 43 Occupational exposure to pesticides during pregnancy and neurobehavioral development of infants and toddlers. E p i d e m i o l o g y , 1 9 ( E ) , 851-859. Handal, A. J., Lozoff, B., Breilh, J., & Harlow, S. D. (2007a). Effect of community of residence on neurobehavioral development in infants 44 and young children in a flower-growing region of Ecuador. [Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't]. 128-133. E n v i r o n H e a l t h P e r s p e c t , 1 1 5 ( 1 ) , Handal, A. J., Lozoff, B., Breilh, J., & Harlow, S. D. (2007b). 45 Neurobehavioral development in children with potential exposure to pesticides. E p i d e m i o l o g y , 1 8 ( 3 ) , 312-320. Harari, R., Julvez, J., Murata, K., Barr, D., Bellinger, D. C., Debes, F., et 46 al. (2010). Neurobehavioral deficits and increased blood pressure in school-age children prenatally exposed to pesticides. E n v i r o n H e a l t h 890-896. P e r s p e c t , 1 1 8 ( 6 ) , Harley, K. G., Huen, K,, Schall, R. A., Holland, N. T., Bradman, A., Barr, 47 D. B., et al. (2011). Association of organophosphate pesticide exposure and paraoxonase with birth outcome in Mexican-American women. P L o S O n e , 6(8), e23923. Category (EPI-epi only; EXPO- exposure only; ACR - acute case report or case series; TOXanimal study; REV review or commentary, no original data; OTH other, non-English Include? Outcome-- pediatric ND/NB/motor control/morphology/motor control; xposure=-QP (biomarker, questionnaire, env media); epi study (cohort/case control/cross sectional (not cologie)} EPI YES EPI YES EPI YES EPI YES EPI YES EPI YES-3COHORTS include? Rationale Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 164 ED 002061 00044425-00164 Number Citations {Of 79:17 part of 3 cohorts, previously reviewed 24excluded (NOT OP, NOT ND, NOT EPI) 38-include (9 no direct OP measure) Horton, M. K., Kahn, L. G., Perera, F., Barr, D. B., & Rauh, V. (2012). 48 Does the home environment and the sex of the child modify the adverse effects of prenatal exposure to chlorpyrifos on child working memory? N e u r o t o x i c o l T e r a t o l , 34(5), 534-541. Kofman, O., Berger, A., Massarwa, A., Friedman, A., & Jaffar, A. A. 49 (2006). Motor inhibition and learning impairments in school-aged children following exposure to organophosphate pesticides in infancy. P e d i a t r R e s , 6 0 ( 1 ) , 88-92. Koutroulakis, D., Sifakis, S., Tzatzarakis, M. N., Alegakis, A. K., Theodoropoulou, E., Kavvalakis, M. P., et al. (2014). Dialkyl 50 phosphates in amniotic fluid as a biomarker of fetal exposure to organophosphates in Crete, Greece; association with fetal growth. [Article]. R e p r o d u c t i v e T o x i c o l o g y , 4 6 , 98-105. Kristensen, P,, Irgens, L. M., Andersen, A., Bye, A. S., & Sundheim, L. 51 (1997). Gestational age, birth weight, and perinatal death among births to Norwegian farmers, 1967-1991. A m J E p i d e m i o l , 1 4 6 ( 4 ) , 329-338. Lizardi, P. S., O'Rourke, M. K., & Morris, R. J. (2008). The effects of 52 organophosphate pesticide exposure on Hispanic children's cognitive and behavioral functioning. J P e d i a t r P s y c h o l , 3 3 ( 1 ) , 91-101. Llop, S., Julvez, J., Fernandez-Somoano, A., Santa Marina, L., Vizcaino, 53 E., Iniguez, C., et al. (2013). Prenatal and postnatal insecticide use and infant neuropsychological development in a multicenter birth cohort study. E n v i r o n m e n t I n t e r n a t i o n a l , 5 9 , 175-182. Category (EPI-epi only; EXPO- exposure only; ACR - acute case report or case series; TOXanimal study; REV review or commentary, no original data; OTH other, non-English Include? Outcome-- pediatric ND/NB/motor control/morphology/motor control; xposure=-QP (biomarker, questionnaire, env media); epi study (cohort/case control/cross sectional (not cologie)} EPI YES-3COHORTS include? Rationale NO: LONGTERM EPI YES EFFECTS OF ONE TIME POISONING EPI YES EPI YES EPI YES EPI YES Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 165 ED 002061 00044425-00165 Number Citations {Of 79:17 part of 3 cohorts, previously reviewed 24excluded (NOT OP, NOT ND, NOT EPI) 38-include (9 no direct OP measure) Lovasi, G. S., Quinn, J. W., Rauh, V. A., Perera, F. P., Andrews, H. F., 54 Garfinkel, R., et al. (2011). Chlorpyrifos exposure and urban residential environment characteristics as determinants of early childhood neurodevelopment. Am J P u b l i c H e a l t h , 1 0 1 ( 1 ) , 63-70. Lu, C., Essig, C., Root, C., Rohlman, D. S., McDonald, T., & Sulzbacher, S. (2009). Assessing the association between pesticide exposure and 55 cognitive development in rural Costa Rican children living in organic and conventional coffee farms. I n t J A d o l e s c M e d H e a l t h , 2 1 ( A ) , 609- 621. Marks, A. R., Harley, K., Bradman, A., Kogut, K., Barr, D. B., Johnson, 56 C., et al. (2010). Organophosphate pesticide exposure and attention in young Mexican-American children: the CHAMACOS study. E n v i r o n 1768-1774. H e a l t h P e r s p e c t , 1 1 8 ( 1 2 ) , Moreno-Banda, G., Blanco-Munoz, J., Lacasana, M., Rothenberg, S. J., Aguilar-Garduno, C., Gamboa, R., et al. (2009). Maternal exposure to 57 floricultural work during pregnancy, PONI Q192R polymorphisms and the risk of low birth weight. S c i T o t a l E n v i r o n , 4 0 7 ( 2 1 ) , 5478- 5485. Nevison, C. D. (2014). A comparison of temporal trends in United 58 States autism prevalence to trends in suspected environmental factors. [Article]. E n v i r o n m e n t a l H e a l t h : A G l o b a l A c c e s s S c i e n c e S o u rce , 13. Oulhote, Y., & Bouchard, M. F. (2013). Urinary metabolites of 59 organophosphate and pyrethroid pesticides and behavioral problems in Canadian children. [Article]. E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s , 1 2 1 , 1378-1384. Category (EPI-epi only; EXPO- exposure only; ACR - acute case report or case series; TOXanimal study; REV review or commentary, no original data; OTH other, non-English Include? Outcome-- pediatric ND/NB/motor control/morphology/motor control; xposure=-QP (biomarker, questionnaire, env media); epi study (cohort/case control/cross sectional (not cologie)} EPI YES-3COHORTS include? Rationale EPI YES EPI YES-3COHORTS EPI YES NO: this is a EPI YES review; ecol comparison EPI YES 166 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00166 Number Citations {Of 79:17 part of 3 cohorts, previously reviewed 24excluded (NOT OP, NOT ND, NOT EPI) 38-include (9 no direct OP measure) Petit, C., Chevrier, C., Durand, G., Monfort, C., Rouget, F,, Garlantezec, R., et al. (2010). Impact on fetal growth of prenatal 60 exposure to pesticides due to agricultural activities: A prospective cohort study in Brittany, France. [Research Support, Non-U.S. Gov't]. 71. E n v i r o n H e a l t h , 9 , Quiros-Alcala, L., Alkon, A. D., Boyce, W. T., Lippert, S., Davis, N. V., 61 Bradman, A., et al. (2011). Maternal prenatal and child organophosphate pesticide exposures and children's autonomic function. N e u r o t o x i c o l o g y , 3 2 ( 5 ) , 646-655. Rauch, S. A., Braun, J. M., Barr, D. B., Calafat, A. M., Khoury, J., Montesano, M. A., et al. (2012). Associations of prenatal exposure to 62 organophosphate pesticide metabolites with gestational age and birth weight. [Article]. E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s , 1 2 0 , 10551060. Rauh, V. A., Garfinkel, R., Perera, F. P., Andrews, H. F., Hoepner, L., 63 Barr, D. B., et al. (2006). Impact of prenatal chlorpyrifos exposure on neurodevelopment in the first 3 years of life among inner-city children. P e d i a t r i c s , 1 1 8 ( 6 ) , el845-1859. Rauh, V. A., Perera, F. P., Horton, M. K., Whyatt, R. M., Bansal, R., 64 Hao, X., et al. (2012). Brain anomalies in children exposed prenatally to a common organophosphate pesticide. P r o c N a t l A c a d S c i U S A , 1 0 9 ( 2 0 ) , 7871-7876. Rauh, V., Arunajadai, S., Horton, M., Perera, F., Hoepner, L., Barr, D. 65 B., et al. (2011). Seven-year neurodevelopmental scores and prenatal exposure to chlorpyrifos, a common agricultural pesticide. E n v i r o n 1196-1201. H e a l t h P e r s p e c t , 1 1 9 ( 8 ) , Category (EPI-epi only; EXPO- exposure only; ACR - acute case report or case series; TOXanimal study; REV review or commentary, no original data; OTH other, non-English Include? Outcome-- pediatric ND/NB/motor control/morphology/motor control; xposure=-QP (biomarker, questionnaire, env media); epi study (cohort/case control/cross sectional (not cologie)} EPI YES EPI YES EPI YES EPI YES-3 COHO RTS EPI YES-3COHORTS EPI YES-3COHORTS include? Rationale 167 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00167 Number Citations {Of 79:17 part of 3 cohorts, previously reviewed 24excluded (NOT OP, NOT ND, NOT EPI) 38-include (9 no direct OP measure) Rohlman, D. S., Arcury, T. A., Quandt, S. A., Lasarev, M., Rothlein, J., Travers, R., et al. (2005). Neurobehavioral performance in preschool 66 children from agricultural and non-agricultural communities in Oregon and North Carolina. N e u r o T o x i c o l o g y , 2 6 ( 4 Spec. Iss.), 589598. Ruckart, P. Z., Kakolewski, K., Bove, F. J., & Kaye, W. E. (2004). Long 67 term neurobehavioral health effects of methyl parathion exposure in children in Mississippi and Ohio. [Comparative Study]. E n v i r o n H e a l t h 46-51. P e r s p e c t , 1 1 2 ( 1 ) , Samarawickrema, N., Pathmeswaran, A., Wickremasinghe, R., Peiris- John, R., Karunaratna, M., Buckley, N., et al. (2008). Fetal effects of 68 environmental exposure of pregnant women to organophosphorus compounds in a rural farming community in Sri Lanka. C l i n T o x i c o l 489-495. ( P h i l a ) , 4 6 ( 6 ) , Savitz, D. A., Arbuckle, T., Kaczor, D., & Curtis, K. M. (1997). Male 69 pesticide exposure and pregnancy outcome. A m J E p i d e m i o l , 1 4 6 ( 1 2 ) , 1025-1036. Shelton, J. F., Geraghty, E. M., Tancredi, D. J., Delwiche, L. D., Schmidt, R. J., Ritz, B., et al. (2014). Neurodevelopmental disorders 70 and prenatal residential proximity to agricultural pesticides: The charge study. [Article]. E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s , 1 2 2 , 11031109. Suarez-Lopez, J. R,, Himes, J. H., Jacobs Jr, D. R., Alexander, B. H., & 71 Gunnar, M. R. (2013). Acetylcholinesterase activity and neurodevelopment in boys and girls. [Article]. P e d i a t r i c s , 1 3 2 , el649- el658. Category (EPI-epi only; EXPO- exposure only; ACR - acute case report or case series; TOXanimal study; REV review or commentary, no original data; OTH other, non-English EPI EPI EPI EPI EPi EPi Include? Outcome-- pediatric ND/NB/motor control/morphology/motor control; xposure=-QP (biomarker, questionnaire, env media); epi study (cohort/case control/cross sectional (not cologie)} YES YES YES YES YES YES include? Rationale 168 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00168 Number Citations {Of 79:17 part of 3 cohorts, previously reviewed 24excluded (NOT OP, NOT ND, NOT EPI) 38-include (9 no direct OP measure) Suarez-Lopez, J. R., Jacobs Jr, D. R., Himes, J. H., & Alexander, B. H. 72 (2013). Acetylcholinesterase activity, cohabitation with floricultural workers, and blood pressure in Ecuadorian children. [Article]. 619-624. E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s , 1 2 1 , Suarez-Lopez, J. R., Jacobs, D. R., Himes, J. H., Alexander, B. H., 73 Lazovich, D., & Gunnar, M. (2012). Lower acetylcholinesterase activity among children living with flower plantation workers. [Article]. E n v i r o n m e n t a l R e s e a r c h , 1 1 4 , 53-59. Wang, P., Tian, Y., Wang, X.-J., Gao, Y., Shi, R., Wang, G.-Q., et al. 74 (2012). Organophosphate pesticide exposure and perinatal outcomes in Shanghai, China. [Article]. E n v i r o n m e n t I n t e r n a t i o n a l , 4 2 , 100-104. Whyatt, R. M., Rauh, V., Barr, D. B., Camann, D. E., Andrews, H. F., 75 Garfinkel, R., et al. (2004). Prenatal insecticide exposures and birth weight and length among an urban minority cohort. E n v i r o n H e a l t h P e r s p e c t , 1 1 2 ( 1 0 ) , 1125-1132. Wickerham, E. L., Lozoff, B., Jie, S., Kaciroti, N., Yankai, X. 1. A., & 76 Meeker, J. D. (2012). Reduced birth weight in relation to pesticide mixtures detected in cord blood of full-term infants, [article]. 80-85. E n v i r o n m e n t i n t e r n a t i o n a l , 4 7 , Wolff, M. S., Engel, S., Berkowitz, G., Teitelbaum, S., Siskind, J., Barr, 77 D. B., et al. (2007). Prenatal pesticide and PCB exposures and birth outcomes. P e d i a t r R e s , 6 1 ( 2 ) , 243-250. Young, J. G., Eskenazi, B., Gladstone, E. A., Bradman, A., Pedersen, L., 78 Johnson, C., et al. (2005). Association between in tero organophosphate pesticide exposure and abnormal reflexes in neonates. N e u r o t o x i c o l o g y , 2 6 ( 2 ) , 199-209. Category (EPI-epi only; EXPO- exposure only; ACR - acute case report or case series; TOXanimal study; REV review or commentary, no original data; OTH other, non-English EPI EPI EPI EPI EPI EPI EPI Include? Outcome-- pediatric ND/NB/motor control/morphology/motor control; xposure=-QP (biomarker, questionnaire, env media): epi study (cohort/case control/cross sectional (not cologie)} YES YES YES YES-3COHORTS YES YES YES-3COHORTS include? Rationale 169 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00169 Number Citations {Of 79:17 part of 3 cohorts, previously reviewed 24excluded (NOT OP, NOT ND, NOT EPI) 38-include (9 no direct OP measure) Zhang, Y., Han, S., Liang, D., Shi, X., Wang, F., Liu, W., et al. (2014). 79 Prenatal exposure to organophosphate pesticides and neurobehavioral development of neonates: A birth cohort study in Shenyang, China. [Article]. P L o S O N E , 9 . Category (EPI-epi only; EXPO- exposure only; ACR - acute case report or case series; TOXanimal study; REV review or commentary, no original data; OTH other, non-English Include? Outcome-- pediatric ND/NB/motor control/morphology/motor control; xposure=-GP (biomarker, questionnaire, env media); epi study (cohort/case control/cross sectional (not cologie)} EPI YES include? Rationale Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 170 ED 002061 00044425-00170 Appendix 6. Plausible hypotheses on MOA/AOP for neurodevelopmental outcomes (Extracted from Section 4.4.3. RHHRA for chlorpyrifos) Numerous studies on the possible mechanistic aspects of neurodevelopmental effects have been published. The results have led some research groups to propose that changes in brain connectivity and/or neurochemistry may underlie the long-term in vivo neurobehavioral changes observed into adulthood. While multiple biologically plausible hypotheses are being pursued by researchers, no one pathway has sufficient data to be considered more credible than the others. The SAP concurred with the Agency in 2008 and 2012 about the lack of definable key events in a MOA/AOP leading to neurobehavioral effects. The Agency has considered the new literature since the 2012 SAP related to mechanistic hypotheses as described below (Appendix 11), and note that such a MOA/AOP still cannot be established. Acetylcholinesterase (AChE) as a morphogen: The classically understood role of AChE is the rapid hydrolysis of acetylcholine at synapses in the brain and at neuromuscular junctions, thereby regulating cholinergic neurotransmission. Consistent with this role, AChE is predominant at cholinergic synapses at neurons and in muscle, and inhibition of its catalytic activity results in the signs and symptoms of cholinergic overstimulation. Several lines of evidence suggest that AChE can also serve as a morphogen, influencing the growth of cells during neurodevelopment distinct from its role as an esterase. Alterations in the expression or structure of the AChE protein can disrupt various aspects of neuronal differentiation and growth, as has recently been shown in vitro (using NG108-15 cell line) following exposure to another OP, paraoxon (Campanha et al., 2014). While perturbation of the morphogenic activity of AChE is a plausible adverse outcome pathway for chlorpyrifos, a number of questions remain, including effective concentrations compared to those that inhibit catalytic activity of AChE. There is, however, no direct evidence showing that disruption of the morphogenic function of AChE can alter axon or dendritic growth in vivo. While limited in vivo studies using zebrafish indicate that chlorpyrifos or its metabolite chlorpyrifos oxon can disrupt axonal growth (Yang et al. 2011), it has not been demonstrated that this effect is due to alteration of the morphogenic function of AChE versus other potential mechanisms. Cholinergic system: There are several lines of evidence showing that signaling through cholinergic receptors is involved in neurodevelopment. Activation of muscarinic and/or nicotinic cholinergic receptors can regulate neural progenitor cell proliferation and differentiation (Resende & Adhikari, 2009), and in vivo studies demonstrate that cholinergic signaling is likely involved in brain morphogenesis (Hohmann & BergerSweeney, 1998). While ChE inhibitors can affect cholinergic signaling by inhibition of the catalytic activity of AChE and subsequent increase in acetylcholine, some inhibitors, including chlorpyrifos and chlorpyrifos oxon, can also directly interact with cholinergic receptors. Thus, direct interaction with cholinergic receptors by chlorpyrifos represents a potential adverse outcome pathway for disruption of neurodevelopment distinct from AChE/ChE inhibition. Some OPs have been shown to directly interact with cholinergic muscarinic receptors at relatively low concentrations. The muscarinic receptors are 171 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00171 members of the G-protein receptor family and five subtypes (ml-m5) have been identified. Ward et al. (1993) examined the relationship between ChE inhibition and direct binding to muscarinic receptors for a series of OPs and their active oxon metabolites. The results indicated a strong correlation between AChE activity of OPs, including chlorpyrifos and chlorpyrifos oxon, and the ability to compete for CD binding sites (m2 receptors) in rat brain homogenates. Binding affinities of the oxons were in the nanomolar range, at or below concentrations that inhibited AChE (Huff et a1,1994); specifically, chlorpyrifos oxon had a binding affinity of 22 nM in rat striatum and 2 nM in rat cortex (Huff, et al., 1994; Ward & Mundy, 1996). In total, these studies suggest that direct interactions with muscarinic receptors, and especially the m2 subtype, represent an alternative site of action for OPs including chlorpyrifos and chlorpyrifos oxon, with the oxon forms having high affinity. Together, the studies cited above outline a plausible adverse outcome pathway for chlorpyrifos and chlorpyrifos oxon to affect brain development via actions at the m2 subtype of muscarinic receptors. However, while there are studies showing that chlorpyrifos oxon can affect neurite outgrowth in vitro and decrease cell proliferation and differentiation both in vitro (Jameson et a l, 2006; Qiao et al,, 2001; Song, et al., 1998) and in vivo (Dam et al,, 1998; Qiao, et al., 2003), there is no experimental evidence that these effects are a result of direct actions on the m2 receptor. Endocannobinoid system: Several lines of research have suggested that disruption of the endocannabinoid (EC) system due to chlorpyrifos exposure could play a role in its acute and/or long-term toxicity, and could also be extended to potential developmental toxicity. The EC system modulates neurotransmission as well as playing a morphogenic role during development of the nervous system. Chemicals (e.g., drugs of abuse) which act on this system, produce long-term neurodevelopmental disorders in animal models and human studies. Chlorpyrifos also interacts with this system, both in vitro and in vivo. By this hypothesis, the EC system represents a possible adverse outcome pathway for developmental effects of chlorpyrifos. There is a body of studies on the interaction of OPs with relevant enzymes but only two studies have examined the effects of chlorpyrifos on the EC system in developing animals. Carr et al. (2011, 2013) has dosed preweanling rats for 5 or 7 days (1-5 mg/kg/day, p.o.), and showed that endocannabinoid-related enzymes were inhibited in rat brain tissue taken 4 to 48 hours after the last dose. Interestingly, fatty acid amid hydrolase (FAAH) showed a greater degree and more persistent inhibition compared to AChE inhibition measured in the same rats. A more recent publication (Carr et al., 2014) repeated these findings using a lower dose (0.5 mg/kg/d for 7 days), still showing significant FAAH inhibition but with no measurable AChE inhibition. This suggests a greater sensitivity of the EC system, at least in terms of the hydrolase compared to AChE activity, in the pups. However, there were no other ages tested, no downstream or correlative measure of changes in EC system function, and no subsequent neurodevelopmental effects that could be linked to the action. Additional studies along these lines are needed. 172 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00172  Reactive Oxygen Species: The production of reactive oxygen species (ROS) and resulting cellular damage has been proposed as a mechanism for a wide variety of neurotoxicants. Due to lower levels of protective enzymes and antioxidants, and relatively low numbers of glia relative to the adult, the developing brain may be particularly sensitive to neural cell damage caused by oxidative stress. In addition, recent work suggests that ROS can act as second messengers. Relatively small changes in the oxidative status of the cell (redox potential) can lead to changes in redox sensitive signaling pathways that regulate cell physiology. In the nervous system, redox signaling is involved in the regulation of neurodevelopmental processes including neural stem cell proliferation and differentiation (Le Belle et al., 2011; Vieira et al, 2011). A number of studies suggest that chlorpyrifos and chlorpyrifos oxon can induce oxidative stress in various neural cell types. Thus, generation of reactive oxygen species and/or alteration of cellular redox potential by chlorpyrifos represent a possible initiating event leading to developmental neurotoxicity. Data from both in vitro studies with neuronal cells (including neural precursors) and in vivo studies in developing brain demonstrate that chlorpyrifos can induce oxidative stress. The in vitro data suggests that this effect may not be due to AChE inhibition, since the parent compound chlorpyrifos is either equipotent or more potent than the oxon (for example, Crumpton et al., 2000). There was, however, no concurrent analysis of AChE inhibition in most of these studies. Several known developmental neurotoxicants have been shown to disrupt neural precursor cell proliferation in vitro through a common pathway that is initiated by increasing the oxidative state of the cell (Li et al, 2007), and the antioxidant vitamin E protected PC12 cells from the anti-proliferative effect of chlorpyrifos (Slotkin et al, 2007). Thus, the in vitro data suggest that chlorpyrifos can affect a critical neurodevelopmental process, at least in part, via generation of ROS. Though limited, in vivo studies show both direct evidence (lipid peroxidation) and indirect evidence (alteration in the expression of oxidative stress response genes) of oxidative stress in the developing brain after exposure to chlorpyrifos. Recent evidence suggests that oxidative stress can alter neurodevelopment in vitro and in vivo by the dysregulation of signaling pathways controlling neuroprogenitor cell function (Le Belle, et al., 2011; Vieira, et al., 2011). It has been demonstrated in vivo that antioxidant treatment can attenuate the induction of oxidative stress produced by chlorpyrifos in adult rats (Singh and Panwar, 2014), but there are as yet no such studies addressing its developmental neurotoxicity. Thus, there is the potential for initiation of an AOP via induction of oxidative stress, but supportive studies in developing animals have not been reported. Serotonergic system: Beyond its classical neurotransmitter actions, serotonin has other roles during development. In their review, Thompson and Stanwood (2009) described serotonin as a pleiotropic molecule, meaning that it can produce multiple, diverse effects, regulating different functions at different times during development. The serotonergic system is integral in many developmental processes including, but not limited to, neurogenesis, migration, and differentiation, synaptogenesis, and cardiac development before assuming its more well-known function as a neurotransmitter in the adult nervous system (reviewed in Frederick & Stanwood, 2009). Serotonin also 173 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00173 plays crucial roles in thalamocortical patterning (reviewed in (Frederick & Stanwood, 2009). As serotonin is present extremely early in development, it is thought that it modulates cellular function even before neurogenesis. Later in development, serotonin is temporarily taken up by so-called transient serotonergic neurons mainly involved in sensory processing, and is involved in activity-dependent patterning of the brain. Later in development, serotonin has also been shown to modulate differentiation in the brain. There are numerous studies of the effects of perinatal chlorpyrifos administration on the patency of the serotonergic system coming from both Duke University and Istituto Superiore di Sanita in Italy; however, there have been no additional reports since the 2012 SAP. Endpoints in various brain regions include serotonin levels, serotonin turnover, serotonin receptor levels, serotonin reuptake receptor levels, serotonin elicited second messenger activity, gene expression of serotonin receptor and metabolism related genes, serotonin related behavioral assessments, and behavior after serotonergic drug challenge. All the data indicate that there are acute, as well as permanent, effects of neonatal chlorpyrifos treatment on the maturation of the serotonergic nervous system. The effects are often gender-specific, region-specific and dose-related. There is ample evidence that chlorpyrifos exposure during development causes permanent changes in the serotonergic nervous system; there are, however, few papers that assessed concurrently the ChE inhibition (either brain or blood) in those same animals. In some cases, although ChE activity was not assessed concurrently, a dosing regimen was used that had been characterized previously with regard to ChE activity. It does appear, however, that most of the studies on the effects of chlorpyrifos on the serotonergic nervous system were conducted with doses of chlorpyrifos that likely produced inhibition of ChE activity. As many steps in this chlorpyrifos AOP are possible and plausible, and in laboratory animals the serotonergic nervous system is sufficiently sensitive to low doses of chlorpyrifos during development to alter its function, it is plausible that exposure to chlorpyrifos during development could alter brain development and the function of the serotonergic nervous system. Although chlorpyrifos effects on the serotonergic nervous system in laboratory animals likely is initiated within 24 hours (Slotkin & Seidler, 2007), the actual initiating event of this potential adverse outcome pathway is unknown. Tubulin, Microtubule Associated Proteins and Axonal Transport: Microtubules, one component of the dynamic cytoskeletal scaffolding within each cell, are composed of heterodimers of a- and (3-tubulin, as well as microtubule associated proteins. The microtubule associated proteins appear to have three main functions: (1) to stabilize the microtubules; (2) to aid in tubulin dissociation and (3) to act as motor proteins moving substances forward and backward along the microtubules (Avila et aI,, 1994; Pellegrini & Budman, 2005; Snchez et al, 2000). Not only does the microtubule cytoskeleton determine neuronal morphology (Matus, 1988,1990; Snchez, et al., 2000), but the dynamic reorganization of the microtubules and microtubule associated proteins within 174 Sierra Club v. EPA 18cv3472 NDCA Tier 3/4 ED 002061 00044425-00174 a cell may also coordinate neurite extension/retraction, as well as growth cone advancement. In addition to these integral roles in brain structure and growth, microtubules and the microtubule associated motor proteins kinesin (Hirokawa & Noda, 2008) and dynein (Vallee et aI, 2004) also provide a "railway" for transport of materials throughout the cell, i.e., axonal transport (Fukushima et al, 2009), another process which is integral to the health of the central and peripheral nervous system, playing a pivotal role in neuronal network formation and synapse maturation (Hirokawa & Takemura, 2004). The construction of an adverse outcome pathway using chlorpyrifosinduced effects on tubulin and microtubule associated proteins is still in its infancy. While it is thought that tubulin, microtubule associated proteins and axonal transport are integral to nervous system development and maintenance, there is no experimental evidence that perturbations of these endpoints by chlorpyrifos during development has neurotoxic outcomes. Overall, a definitive mode of action or adverse outcome pathway leading to effects on the developing brain cannot yet be established because of insufficient data establishing the causal linkages among different levels of biological organization to adversity. For example, while there is in vitro evidence relating binding of chlorpyrifos or the chlorpyrifos oxon to AChE and the subsequent decrease in neurite outgrowth at the cellular level, the relationship between neurite outgrowth and neurodevelopmental consequences has not been established. As described in the NRC report, "Toxicity Testing in the 21st Century" (NRC, 2007), to develop an adverse outcome pathway not only is it necessary to establish plausible relationships among the key events, but quantitative relationships also need to be established. In other words, how much of a change in one key event is needed to result in an adverse effect at the next level of biological organization? Thus, certain exposures to a chemical may impact normal physiological responses in a way that may not necessarily be adverse, and thus, the AOP concept requires an understanding of adaptive/homeostatic capacity of biological systems and their limits, relative to concentration and duration of exposure. References Cited Avila, J., Dominguez, J., & Diaz-Nido, J. (1994). Regulation of microtubule dynamics by microtubule-associated protein expression and phosphorylation during neuronal development. Int J Dev Biol, 38(1), 13-25. Campanha HM, Carvalho F, Schlosser P. 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