The geometric mean for the DAP concentrations during pregnancy (for the average of the two sampling periods) was 146 nmol/L (95% CI: 133, 160); of this, a larger proportion was DMP metabolites (GM = 109 nmol/L; 95% CI =98, 120) than DEPs (GM= 23 nmol/L; 95% CI = 21, 25). Allele frequencies: PON1 192 Q allele= 50%; PON1 -108 T allele= 46%.Mean arylesterase activity: For infants: 33.6 U/mL (SD = 16) For mothers: 136.6 U/mL (SD = 44). Mean paraoxonase activity: For infants: 256.6 U/L (SD = 165); For mothers: 989.0 U/L (SD = 616).
Maternal intelligence (Peabody Picture Vocabulary Test (PPVT)), measures of how stimulating the environment is, and known or suspected neurotoxins were measured prenatally.  To measure the quality and extent of stimulation available to a child in the home environment, the Infant-Toddler HOME (Home Observation for Measurement of the Environment) inventory was completed at the 6-month, 1, 2, 3.5, 5, and 7 year visits; known or suspected neurotoxicants, polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), p,p'-dichlorodiphenyltrichlorethane (DDT), p,p'- dichlorodiphenyltrichlorethylene (DDE), and lead.
The authors observed evidence of an association between prenatal exposure to OP pesticides as measured by urinary DAP metabolites in women during pregnancy, is associated with decreased cognitive functioning in children at age 7.
Infants whose PON1 genotype and enzyme activity levels suggested that they might be more susceptible tothe effects of OP pesticide exposure had decreased fetal growth and length of gestation. PON1 may be a contributing factor to preterm or low birth weight birth.
This study has many strengths, the longitudinal design, the measurement of urinary DAP at multiple time points and following children to age seven when tests of cognitive function are reportedly more reliable. The authors were able to adjust for or consider many factors related to cognitive function, such as prenatal exposure to other environmental agents, socioeconomic indicators, maternal intelligence and education, and child stimulation. The cohort had a relatively homogenous socioeconomic profile, reducing the potential for uncontrolled confounding.
Engel et al. (2007)  Mt. Sinai (N=311)
Brazelton Neonatal Behavioral Assessment Scale (BNBAS), primitive reflexes (neurological integrity) measured before hospital discharge.
DEP: 24.7 nmol/L; Total DAP: 82 nmol/L
Maternal age, race, marital status, education, cesarean delivery, delivery anesthesia, infant age at examination, infant gender, infant jaundice, smoking (yes/no), alcohol consumption, caffeine consumption, illicit drug use during pregnancy, and the examiner.
No adverse associations were found for DAPs and any measured behavior. Relative to the first quartile, quartiles 2 - 4 of total DEPs, DMPs, and DAPs were associated with an increased proportion of abnormal reflexes, although the associations did not increase monotonically and varied in their strength and precision.
Used non-specific biomarker DEP/DAP
This was a well conducted prospective study conducted in a young, predominantly minority population. The study design, analytic approach, and statistical analyses were appropriate. Pesticide metabolites evaluated are not specific for chlorpyrifos. The BNBAS was administered before hospital discharge only on a subset of children in the cohort (n =311/404).  Factors related to weekend delivery (e.g., fewer inductions) would be underrepresented among the tested subjects, and may induce bias, reduce the degree of precision with which associations were estimated, and limit the generalizability of the study findings.The statistical analysis was largely appropriate. Imputing of missing data may affect precision of association estimates and result in attenuated effect estimates as a result of exposure measurement error. Assessment and control for confounding were appropriate. However, confounding by unmeasured (and mis-measured) risk factors for abnormal growth that are related to pesticide exposure would bias the results in this study. Such factors may be related to socioeconomic status of the study participants, a cofactor which is difficult to define, no less measure in an epidemiologic study.
Young et al. (2005) CHAMACOS (N=381)
Neurodevelopment, Brazelton Neonatal Behavioral AssessmentScale (BNBAS), abnormal reflexes
DAP (average during pregnancy): median 222nmol/L (range: 7 - 21,867 nmol/L); DEP (average during pregnancy): median 21 nmol/L (range: 2 - 680 nmol/L)
Maternal age, BMI, any smoking/alcohol/drug use during pregnancy, gestational age at which prenatal care was initiated, total number of prenatal care visits, mean pregnancy blood pressure, parity, method of delivery, general anesthesia used during delivery, breastfeeding initiated after delivery, poverty level, infant sex, age in days at BNBAS, minutes since last feed at BNBAS, and BNBAS examiner.
Among the >3 day old infants, increasing average prenatal urinary metabolite levels were associated with both an increase in number of abnormal reflexes (total DAP: adjusted beta = 0.53, 95% CI = 0.23, 0.82; dimethyls: adjusted beta = 0.41, 95% CI = 0.12, 0.69; diethyls: adjusted beta = 0.37, 95% CI = 0.09, 0.64), and the proportion of infants with more than three abnormal reflexes (total DAP: adjusted OR = 4.9, 95% CI = 1.5, 16.1; dimethyls: adjusted OR = 3.2, 95% CI = 1.1, 9.8; diethyls: adjusted OR = 3.4, 95% CI = 1.2, 9.9).
Associations seen pre-natal OP, not post-natal OP exposure, Maternal urine collection averaged weeks 14, 26
Strengths: Longitudinal design, measurement and consideration of many confounders, the prenatal exposure measures were, with some exceptions, the average of two measurements, and thus may better reflect chronic exposure during the pregnancy
Weaknesses: Potential for residual confounding, potential for exposure misclassification, potential selection bias (not all children had the outcome measures), study population not generalizable to the whole US.
Rauh et al. (2006)    Columbia U. (N=254)
Neurodevelopment: The Bayley Scales of Infant Development II (BSID-II), Mental Development Index (MDI) and Psychomotor Development Index (PDI) at 12, 24, and 36 months of age.  :: Behavior:  Child Behavior Checklist (CBCL) at  12, 24, and 36 months.
:: Quality of the child-care environment: The Home Observation for Measurement of the Environment (HOME)
Exposure levels were categorized as low (<  6.17 pg/g) or high (>6.17 pg/g)
Data were collected regarding lead exposure, demographics, education and occupational history, income, active and passive smoking, alcohol and drug use during pregnancy, and residential pesticide use.  Final models included prenatal environmental tobacco smoke (ETS) exposure, gender, ethnicity, gestational age at birth, quality of home care-taking environment, maternal education, and maternal IQ.
At the 36 month milestone, the likelihood of highly exposed children developing mental delays were 2.4 times greater (95% CI: 1.12-5.08, p = 0.02) and motor delays were 4.9 greater (95% CI: 1.78-13.72; p = 0.002) than those with lower prenatal exposure. The GLM analysis for PDI scores showed a significant effect of chlorpyrifos exposure over time with an estimated deficit of approximately 7 points by age 36 months (p = 0.01).
The authors summarize three main findings:  1) by age 3, the more highly exposed children demonstrated mental and motor delays; 2) the observed developmental trajectories for PDI and MDI scores confirmed that the adverse impact on cognitive and motor development increased over time; and 3) by age 3, highly exposed children were more likely to demonstrate clinically significant attention problems.
:: Only 53% of the children reached the three year milestone with study data collected.  It is unclear what percentage of these children did not survive, were lost to follow-up, or too sick to participate.
:: Reliance on a single exposure level (prenatal/cord blood.)
:: No control for exposure over the subsequent 3 years :: Creation of a dichotomous exposure variable brings limitations due to the amount of within-group variation.
:: Limitations of the sensitivity and predictive validity of the developmental tests, especially among children less than 3 years of age.
:: No discussion of whether this 7-point deficit is clinically relevant.
:: Due to the pervasive, non-specific nature of neurological effects, it is difficult to attribute causality.
Lovasi et al. 2011      Columbia U. (N=266)
Bayley scores (MDI/PDI) 12 months, 24 months, 36 months
N/A
Neighborhood characteristics: The percentage of housing units without complete plumbing, the percentage of vacant housing units, the percentage of residents below the federal poverty line, the percentage of residents older than 25 years of age who completed high school, the percentage of households receiving public assistance, the percentage of housing units with one or more residents per room, racial composition, the percentage of residents born outside the United States, the percentage of Spanish-speaking residents, and the percentage of residents who were linguistically isolated
Neighborhood characteristics did not confound the observed association between chlorpyrifos levels and cognitive development.
Hierarchal regression analysis of potential confounding by SES
Direct measurement of chlorpyrifos.The statistical analyses were generally appropriate. Missing data on covariates were estimated using multiple imputation, and the variance estimates presented appropriately reflect the degree of uncertainty caused by missing covariate data. Robust standard errors were used.The setting of the investigation in a sample drawn from low-income African American and Dominican communities is both a strength (increases the power, restriction of confounders) and a limitation of the study (reduced generalizability).
Engel et al. (2011)
Mt. Sinai (N=276)
Bayley scores (MDI/PDI) at 12 months, 24 months.
DEP: 24.7 nmol/L; Total DAP: 82 nmol/L (same as Engel 2007)
Maternal age, race/ethnicity, marital status, education, breast-feeding, child sex, alcohol, smoking, or drug use during pregnancy, maternal IQ, a score based on assessment of the home environment (HOME), season of urine collection, language spoken in the home, age at testing, examiner, and urinary creatinine level.
An observed association between prenatal total dialkylphosphate metabolite level and a decrement in mental development at 12 months among blacks and Hispanics.
Used non-specific biomarker DEP/DAP; some evidence of effect modification by PON1 genotype
Limitations include use of non-specific markers of chlorpyrifos pesticide exposure (DAPs), use of only a single (third-trimester) urine sample, and the large proportion of loss to follow-up. Statistical analysis was appropriate.  Imputing of missing data may affect precision of association estimates and result in attenuated effect estimates as a result of exposure measurement error, although these are offset by the further categorization of the exposure levels (at the median). However, binning of exposure levels reduces precision, relative to a continuously distributed measure of exposure. Assessment and control for confounding were appropriate. However, confounding by unmeasured (and mismeasured) risk factors for abnormal growth that are related to pesticide exposure would bias the results in this study. Such factors may be related to socioeconomic status of the study participants, a cofactor which is difficult to define, no less measure in an epidemiologic study.
Eskenazi et al. (2007)   CHAMACOS (N=372)
Neurodevelopment, Bayley Index (MDI, PDI), Maternal behavior checklist at 6, 12, and 24 months
DEP:  geom. Mean in mother 18.1 nmol/L (95% CI = 16.7 - 19.7); DEP geometric mean in child at 24 months 10.5 nmol/L (95% CI =8.8 - 12.6); TCPy median 3.54 ug/l
Psychometrician, location of assessment, exact age at assessment, sex, breast-feeding duration (months), HOME score, and household income, parity, maternal PPVT, maternal age, education, depressive symptoms, active/passive smoking exposure during pregnancy, regular alcohol use during pregnancy, marital status, father's presence in home, housing density, maternal work status, >= 15 hours out-of-home childcare/week, birth weight, gestational age, abnormal reflexes, PCBs, lead, DDT, β-hexachlorocyclohexane, and hexa-chlorobenzene
DAP metabolite levels during pregnancy, particularly from dimethyl phosphate pesticides, may be negatively associated at 24 months with mental development (MDI) on the Bayley Scales and an increase in risk of maternally reported PDD.
No strong associations identified with DE or TCPy, Maternal urine collection averaged weeks 14, 26
Strengths: Longitudinal design, measurement and consideration of many confounders (including other environmental chemicals); the prenatal exposure measures were, with some exceptions, the average of two measurements, and thus may better reflect chronic exposure during the pregnancy

Weaknesses: Potential for residual confounding, potential for exposure misclassification, potential selection bias (not all children had the outcome measures), study population not generalizable to the whole US.
Eskenazi et al. (2010)         CHAMACOS (N=371)
Neurodevelopment, Bayley Index (MDI, PDI), Maternal behavior checklist at 6, 12, and 24 months, PON1 gene and enzyme levels
The geometric mean for the DAP concentrations during pregnancy (for the average of the two sampling periods) was 146 nmol/L (95% CI: 133, 160); of this, a larger proportion was DMP metabolites (GM = 109 nmol/L; 95% CI =98, 120) than DEPs (GM= 23 nmol/L; 95% CI = 21, 25). Allele frequencies: PON1 192 Q allele= 50%; PON1 -108 T allele= 46%.Mean arylesterase activity: For infants: 33.6 U/mL (SD = 16) For mothers: 136.6 U/mL (SD = 44). Mean paraoxonase activity: For infants: 256.6 U/L (SD = 165); For mothers: 989.0 U/L (SD = 616).
Psychometrician, location of assessment, exact age at assessment, sex, breast-feeding duration (months), HOME score, and household income, parity, maternal PPVT, maternal age, education, depressive symptoms, active/passive smoking exposure during pregnancy, regular alcohol use during pregnancy, marital status, father's presence in home, housing density, maternal work status, >= 15 hours out-of-home childcare/week, birth weight, gestational age, abnormal reflexes, PCBs, lead, DDT, β-hexachlorocyclohexane, and hexa-chlorobenzene
Decrease MDI (24 months) PON1108TT  - 5.7 ( - 9.0 to  - 2.5) 9=0.01; Decrease PDI (24 months) PON1108TT  - 2.8 ( - 5.7 to 0.2) p=0.07; increased odds PDD 2.0 (0.8 to 5.1) p=0.14; no association PON1192; no association PON1 activity measured newborn, 2 years, maternal and MDI, PDI, PDD. Evidence of decreasing MDI score by number of PON1108 variant alleles: PON1108CC  - 3.2 ( - 9.8 to 3.5), CT  - 3.7 ( - 8.0 to 0.6), TT  - 5.5 ( - 11.1 to 0.1), p-interaction 0.98.
In this study population, evidence PON1 may influence MDI score, but not PDI or PDD risk at two-years. Non-significant evidence of decreasing MDI score by increasing DAP levels across strata of the number of PON1108 variant alleles, interaction non-significant. Similar trend with prenatal DEP levels and MDI, PDI by PON108 alleles, but less pronounced. Overall, limited, non-definitive evidence of effect modification by PON1 status in the relation between mental and psychomotor effects and prenatal DAPs.
Strengths: Longitudinal design, measurement and consideration of many confounders (including other environmental chemicals); the prenatal exposure measures were, with some exceptions, the average of two measurements, and thus may better reflect chronic exposure during the pregnancy

Weaknesses: Potential for residual confounding, potential for exposure misclassification, potential selection bias (not all children had the outcome measures), study population not generalizable to the whole US. Study may be under-powered to evaluate effect modification by PON1 status.
Marks et al. (2010)          CHAMACOS (N=348)
CBCL; K-CPT; ADHD confidence index; Hillside behavioral rating scale; composite ADHD indicator
DAP (geometric mean) pregnancy 109.0 nmol/L; DEP 17.7 nmol/L
Psychometrician, exact age at assessment, sex, maternal education, depressive symptoms, PPVT (continuous), >= 15 hr out-of-home child care/week, breast feeding duration (months), maternal age, parity, marital status, active/passive smoking exposure and regular alcohol use during pregnancy, presence of father in home, maternal work status, and household income
Prenatal DAPs were non-significantly associated with maternal report of attention problems and ADHD at age 3.5 years, but were significantly related at age 5 years [CBCL attention problems: β = 0.7 points; 95% confidence interval (CI), 0.2-1.2; ADHD: β = 1.3; 95% CI, 0.4-2.1].
Marked effect modification by gender: 11-fold increase ADHD composite indicator in boys, less than 2-fold in girls, however unstable estimates; weak evidence of association DAPs at 3.5, 5 years and attention
Strengths: Longitudinal design, measurement and consideration of many confounders (including other environmental chemicals); the prenatal exposure measures were, with some exceptions, the average of two measurements, and thus may better reflect chronic exposure during the pregnancy

Weaknesses: Potential for residual confounding, potential for exposure misclassification, potential selection bias (not all children had the outcome measures), study population not generalizable to the whole US.
Rauh et al. (2011)    Columbia U. (N=265)
:: Wechsler Scales of Intelligence for Children (WISC-IV) :: Child Behavior Checklist (CBCL).
:: Chlorpyrifos levels in umbilical cord blood samples, N=256 newborns
:: If no cord blood (12% of subjects), levels were imputed from mothers' values.
:: Values for samples with non-detectable chlorpyrifos levels (N=115, 43%) were imputed by using assay-specific limit of detection (LOD) values to impute an approximate level.
Data were collected regarding lead exposure, demographics, education and occupational history, income, active and passive smoking, alcohol and drug use during pregnancy, and residential pesticide use.  Final models included prenatal environmental tobacco smoke (ETS) exposure, gender, ethnicity, gestational age at birth, quality of home care-taking environment, maternal education, and maternal IQ.
Full-Scale IQ:  (B) of -0.003, CI = 0.006, 0.001, p= 0.064 Working Memory Index: (B) of -0.006, CI = 0.009, 0.002, p<0.001.  The investigators articulated these results as showing that a 1 pg/g increase in chlorpyrifos exposure was associated with a 0.006 point decrease in the log-transformed Working Memory score and a 0.003 point decrease in the log-transformed Full-Scale IQ score.  The investigators concluded that for each standard deviation increase in exposure (4.61pg/g) there is a 1.4% reduction in Full-Scale IQ and a 2.8% reduction in Working Memory.
For each standard deviation increase in exposure (4.61pg/g) there is a 1.4% reduction in Full-Scale IQ and a 2.8% reduction in Working Memory.
Strengths
:: Direct assessment of chlorpyrifos levels using maternal serum and cord blood.
:: Analysis using a continuous CPF level, which, in contrast to dichotomous CPF levels, provides a more meaningful look at potential threshold effects and dose-response trends.
:: The investigators rigorously evaluated their methods for imputing values for undetectable CPF levels which in the end, were validated.
:: The authors describe an elegant and methodologically sound statistical analysis, addressing many of the potential shortcomings of their exposure data and covariates.

Weaknesses: The use of a single snapshot of prenatal chlorpyrifos exposure may not be an accurate surrogate for full prenatal exposure levels.
:: There is no control for exposure over the subsequent 7 years which may be critical, especially as the process of neurocognitive development is fluid and rapid during these early childhood years.   :: Possibility of that an increased awareness of the risks of pesticide exposures could disproportionately affect postnatal exposure behavior.:: Complicating this analysis is the pervasive, non-specific nature of neurological effects and the difficulty in attributing causal pathways. :: when closely reviewed, the 95% CI for Full Scale IQ for both techniques contain 0 (LASSO: -0.006, 0.001, p=0.064; fully-adjusted: -0.006, 0.001, p=0.048:: The authors do not address the clinical relevance of the 1.4% and 2.8% reductions and how this may impact a child or his/her psychological or educational plans.
Engel et al. (2011)                             Mt. Sinai (N=169)
Wechsler Preschool and Primary Scale of Intelligence, 3rd edition (WPPSI-III) at ages < 7 years; Wechsler-IV Intelligence Scale (verbal comprehension; perceptual reasoning, working memory, processing speed, full scale intelligence) at age 7-9 years
DEP: 24.7 nmol/L; Total DAP: 82 nmol/L (same as Engel 2007)
Maternal age, race/ethnicity, marital status, education, breast-feeding, child sex, alcohol, smoking, or drug use during pregnancy, maternal IQ, a score based on assessment of the home environment (HOME), season of urine collection, language spoken in the home, age at testing, examiner and urinary creatinine level.
At age 6-9 years, non-statistically significant reductions in full scale IQ, perceptual reasoning, verbal comprehension, working memory and processing speed with increasing DAP, more profound with DEP than DMP
Used non-specific biomarker DEP/DAP; some evidence of effect modification by PON1 genotype
Limitations include use of non-specific markers of chlorpyrifos pesticide exposure (DAPs), use of only a single (third-trimester) urine sample, and the large proportion of loss to follow-up.

The statistical analysis was largely appropriate. Imputing of missing data may affect precision of association estimates and result in attenuated effect estimates as a result of exposure measurement error, although these are offset by the further categorization of the exposure levels (at the median).

Assessment and control for confounding were appropriate. However, confounding by unmeasured (and mis-measured) risk factors for abnormal growth that are related to pesticide exposure would bias the results in this study. Such factors may be related to socioeconomic status of the study participants, a cofactor which is difficult to define, no less measure in an epidemiologic study.
Bouchard et al. (2011)    CHAMACOS (N=329)
Wechsler-IV Intelligence Scale (verbal comprehension; perceptual reasoning, working memory, processing speed, full scale intelligence) measured at age 7 years
Total DAPs (quintiles): Q1 (39 nmol/L); Q2 75 nmol/L; Q3 126 nmol/L; Q4 221 nmol/L; Q5 508 nmol/L. Geometric mean DAP 131 nmol/L
Maternal intelligence, measures of how stimulating the environment is, and known or suspected neurotoxins were measured prenatally. Maternal intelligence was assessed via the Peabody Picture Vocabulary Test (PPVT). To measure the quality and extent of stimulation available to a child in the home environment, the Infant-Toddler HOME (Home Observation for Measurement of the Environment) inventory was completed at the 6-month, 1, 2, 3.5, 5, and 7 year visits; known or suspected neurotoxicants, polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), p,p'-dichlorodiphenyltrichlorethane (DDT), p,p'- dichlorodiphenyltrichlorethylene (DDE), and lead.
The authors observed evidence of an association between prenatal exposures to OP pesticides as measured by urinary DAP metabolites in women during pregnancy, and decreased cognitive functioning in children at age 7.
Prenatal measures taken later half of pregnancy more significantly associated intelligence than early; little evidence post-natal OP exposure associated with intelligence; 7 point reduction in full scale intelligence DAP Q5/Q1 (SS)
Strengths: the longitudinal design, the measurement of urinary DAP at multiple time points and following children to age seven when tests of cognitive function are reportedly more reliable. The authors were able to adjust for or consider many factors related to cognitive function, such as prenatal exposure to other environmental agents, socioeconomic indicators, maternal intelligence and education, and child stimulation. The cohort had a relatively homogenous socioeconomic profile, reducing the potential for uncontrolled confounding.
Whyatt et al. (2007)      Columbia U. (N=102)
None
Geometric mean, 6.9 +- 17.0 ng/m[3]; range < 0.4 - 171 ng/m[3]. Personal air monitor: median 2.8 ng/m[3], mean 6.2 +- 11.1 ng/m[3], range < 0.4 - 83.4 ng/m[3]
N/A
There was little within-home variability and no significant difference in air concentrations within homes over time (p >= 0.2); between-home variability accounted for 88% of the variance in the indoor air levels of propoxur, 92% in chlorpyrifos, 94% in diazinon, and 62% in piperonyl butoxide (p < 0.001). Indoor and maternal personal air insecticide levels were highly correlated (r = 0.7 - 0.9, p < 0.001).
Indoor and maternal personal air insecticide levels were highly correlated (r = 0.7 - 0.9, p < 0.001).
Strengths: study design and exposure assessment techniques, Limitations: only those cohort participants enrolled after 2011 were included in the analysis (most likely due to the lack of serial data from the earlier years.)
Whyatt et al. (2009)      Columbia U. (N=102)
None
The limit of detection (LOD) of chlorpyrifos in blood samples was 0.5 - 1 pg/g plasma. The LOD of TCPy in urine samples was 0.26 ng/mL urine. The LOD for TCPy in meconium was 0.2 ng based on a sample weighing 0.5 g.  Exposure marker levels below the LOD were given a value of half the level of detection, and were then log10 transformed.
N/A
Meconium TCPy concentrations were positively correlated with chlorpyrifos in maternal and cord blood (r = 0.25 - 0.33, p < 0.05) and with TCPy in maternal urine (r = 0.31, p < 0.01).
TCPy in maternal urine samples was not reliable, but the maternal and cord blood chlorpyrifos as well as the TCPy levels in meconium were reliable measures of exposure
Comprehensive exposure assessment including actual blood chlorpyrifos levels, the repeated sampling, and the environmental sampling.

Weaknesses: only included participants recruited in the post-cancellation period, use of nonparametric, rank-based statistics is appropriate but the large number of observations below the level of detection receiving equal rank, may be problematic; no dietary assessment
Rauh et al.(2012), (n=40)
Morphological change in the pediatric brain in regions of the brain known to be associated with learning, cognition and social behavior
Tertile 3 ( >=4.39 pg/g), compared to Tertiles 0, 1, 2 (<4.39 pg/g, including those not
exposed to CPF)
Age, sex
Authors report differences in brain structure (regional cerebral size and thickness) by CPF exposure groups, and the differences (high>low CPF) in regional brain size is likely due to enlargement of underlying white matter. Statistical interaction by gender reported.
Authors concluded that the evidence from the study illustrated changes in brain morphology in association with higher CPF exposure, and that changes observed were in areas of the brain that subserve those learning, cognition and social behavioral, supported by previous observational and experimental literature.
Study supports general hypothesis of CPF influence on brain morphology, but lacks specific hypotheses regarding particular areas of the cerebrum affected; limited and somewhat unbalanced depiction of the available rodent experimental data; statistical methods appropriate, correction for multiple statistical comparisons a strength; MRI image readers blinded to exposure status enhances study validity; lack of information on other validation practices; small sample size, pilot study, low statistical power; external validity limited; one time measure of pre-natal exposure

<IP_ADDRESS>	"Moderate" Quality Group

Fifteen articles were assigned a moderate quality rating, as shown in Table <IP_ADDRESS>-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) cross-sectionally 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.

Table <IP_ADDRESS>-1.	Moderate Quality Studies: Summary of Study Design Elements Impacting Study Quality Assignment
                                     Study
                                Study Location
                 Cohort Name / Description of Study Population
                                 Study Design
                              Exposure Assessment
                              Outcome Assessment
                        Confounder /
Covariate Control
                             Statistical Analysis
                             Risk of (other) Bias
Andersen et al.(2015)
Denmark
School age children living in Denmark
Prospective Birth Cohort, small sample size
N=177
(112 maternal occupational pesticide exposure during pregnancy; 65 without)
Age: 6-11 years
Prenatal occupational pesticide exposure ascertained by maternal interview at enrollment
Objective standardized clinical exam; neurophysiological status (heart rate variability); Previously validated, neuropsychological testing with demonstrated sensitivity to environmental pollutants; administered and scored by single neuropsychologist
Appropriate. Self-reported by mother. Included age, maternal demographics and risk factors, SES indicator (broad categories), maternal smoking and alcohol use. Possibility of recall errors (residual confounding). Self-reported (by mother)
Appropriate multivariate analysis. Evaluated numerous hypotheses without adjustment for multiple comparisons. Also constructed structural equation model of interaction between child sex and prenatal pesticide exposure on general intellectual ability (parameterized as a latent variable)
Selection bias possible due to loss to follow-up; Residual confounding likely, small in magnitude; potential for non-differential misclassification of exposure
Guodong et al. (2012)
Shanghai, China
2-year old children visiting community hospitals
Cross-sectional, large sample size
N=301
Age 23-25 months
Objective biomarker, OP exposure (DAP) quantified in single child spot urine sample, simple imputation <LLOD; OP exposure also assessed via questionnaire administered to mothers after delivery
Developmental Quotients based on Gesell Developmental Schedules, adapted for Chinese population
Appropriate. Assessed via mother report via questionnaire, included child sex, maternal demographics and risk factors, SES indicators (maternal education, household income), maternal smoking and alcohol use; Possibility of recall errors
Appropriate multivariate analysis. Assumed linear relationship between log-transformed DAP level and DQ Scores
Selection bias unlikely; Residual confounding likely, small in magnitude; potential for non-differential misclassification of exposure
Handal et al. (2007)
Cayambe-Tabacundo region, Ecuador
EcoSalud Project: infants and young children 3 to 61 months of age in lower-altitude communities A&B dominated largely by cut-flower production and in more traditional rural, higher altitude community C.
Cross-sectional, modest sample size from census
N=283
Age 3-61 months
- 154 high exposure
- 129 low exposure
Objective proxy measure: Community of residence
Ages and Stages Questionnaire (ASQ), adapted into local vernacular, 2 trained testers  -  considered a screening tool
Appropriate: child health status (anemia, stunting) and other characteristics of the home environment (stimulation by 2 methods)
Appropriate: Multiple linear regressions to evaluate associations between community of residence and delayed development, Pairwise t-tests and chi-square to assess mean difference in ASQ score; Effect size Cohen's d
Selection bias unlikely; Residual confounding likely, small in magnitude; potential for non-differential misclassification of exposure
Handal et al. (2008)
Cayambe-Tabacundo region, Ecuador
EcoSalud Project: Children 3 to 23 months of age in lower-altitude communities A&B dominated largely by cut-flower production and in more traditional rural, higher altitude community C.
Cross-sectional, modest sample size
N:121
Age 3-23 months
Proxy: Distance home to farm, parental employment, pesticide use on domestic crops & within home, child play activities
Ages and Stages Questionnaire (ASQ) (ages 24-61 months), a screening tool; Visual Motor Integration (VMI) Test (ages 48-61 months); two trained testers.
Appropriate: As above
Appropriate: As above
Selection bias unlikely; Residual confounding likely, small in magnitude; potential for non-differential misclassification of exposure

Table 2.2.5-2.	Moderate Quality Studies: Summary of Study Design Elements Impacting Study Quality Assignment (continued)
                                     Study
                                Study Location
                 Cohort Name / Description of Study Population
                                 Study Design
                              Exposure Assessment
                              Outcome Assessment
                        Confounder /
Covariate Control
                             Statistical Analysis
                             Risk of (other) Bias
Handal et al.(2007B)
Cayambe-Tabacundo region, Ecuador
EcoSalud Project: Children 24 to 61 months of age in lower-altitude communities designated A & B were dominated largely by cut-flower production than in more traditional rural, higher altitude community C.
Cross-sectional, modest sample size
N:
ASQ - 142
Age 24-61 months
VMI - 57
Age 48-61 months
Proxy: Maternal employment during pregnancy, child plays outdoors, Pesticide use on domestic crops , inside home
Ages and Stages Questionnaire (ASQ) (3-23 months)  -  screening tool; Reach-and-grasp, UC Berkeley Preferential Looking Test Cards; trained tester
Appropriate: As above
Appropriate: As above;
Prehension- Logistic regression models, generalized estimation equations (GEE) to account for between-test dependence
Selection bias unlikely; Residual confounding likely, small in magnitude; potential for non-differential misclassification of exposure
Llop et al. (2013)
Spain
INMA (Environment and Childhood) Project
Prospective Birth Cohort, large sample size
N=1980
Age 14 months
Maternal self-report of prenatal and postnatal indoor pesticide use (pesticide spray or use of a plug-in device assessed via questionnaire)
Mental and psychomotor development at 14 months assessed using validated instrument (Bayley Scales of Infant Development)
Appropriate. Self-reported by mother. Included maternal demographics and risk factors (Age, BMI) SES indicators (maternal education, occupation), maternal smoking and alcohol use. Also childcare behaviors (breast feeding, number of siblings, day care) Possibility of recall errors (residual confounding)
Appropriate multivariate analysis
Selection bias unlikely; Residual confounding likely, small in magnitude; potential for non-differential misclassification of exposure
Oulhote and Bouchard  (2013)
Canadian National Population
Canadian Health Measures Survey (CHMS; cycle 1, 2007 - 2009);
Cross-sectional Study, large national sample size
n = 779 children
Age 6-11 years
Child urinary DAP, DMP, DEP collected within 2 weeks of survey questionnaire completion by the parents.
Behavioral problems in children based on the parent version of the Strengths and Difficulties Questionnaire (SDQ) (Goodman 1997) - SDQ is a validated screening questionnaire and accepted by parents.
Appropriate: Included sex, age, race/ethnicity, family income, parental education, blood lead levels, maternal smoking during pregnancy, birth weight, maternal age at child's birth, child's BMI, and fasting status (fasting duration at urine collection > 10 hour/<= 10 hour)
Appropriate: Logistic Regression
Selection bias unlikely; Study population not mixed, single urine sample
Petit et al. (2010)
Brittany, France
Newborn children in the PELAGIE Study.
Prospective Birth Cohort, large sample size
N= 3,159
Age: newborns
Ecological, proxy indicator of exposure (proportion of municipality devoted to agricultural activity
Objectively measured birth outcomes assessed using hospital records
Appropriate. Maternal report via questionnaire. Included maternal demographics (age, BMI) and pregnancy risk factors (gestational age, hypertension, diabetes, season of pregnancy) SES indicators (district of residence, maternal education, occupation), maternal smoking and alcohol use. Also childcare behaviors (breast feeding, number of siblings, day care); Possibility of recall errors
Appropriate multivariate analysis. Multiple comparisons were conducted for this evaluation, and the authors did not adjust for multiple testing
Selection bias unlikely; Residual confounding likely, small in magnitude; potential for substantial non-differential misclassification of exposure; Misclassification of outcomes unlikely

Table 2.2.5-2.	Moderate Quality Studies: Summary of Study Design Elements Impacting Study Quality Assignment (continued)
                                     Study
                                Study Location
                 Cohort Name / Description of Study Population
                                 Study Design
                              Exposure Assessment
                              Outcome Assessment
                        Confounder /
Covariate Control
                             Statistical Analysis
                             Risk of (other) Bias
Ruckart et al. (2004)
Mississippi, USA (29 counties), Ohio (one multi-family facility)
Children who were 6 years or younger in MS and OH when homes sprayed with MP
Retrospective cohort, moderate sample size in 2 states, participating/ nonparticipating similar in sex and age
MS: N=365 (147 exposed, 218 unexposed)
OH: N=287 (104 exposed, 183 unexposed)
Age: 1.9-12.5 years old at testing
Specific OP measures: Household methyl parathion (wipe) by approved labs, OR highest urinary PNP level in household, analyzed by CDC
Variety of neuro measures: PENTB, Parenting Stress Index (PSI), Personality Inventory for Children (PIC), Vineland Adaptive Behavior Scales (VABS)
Appropriate: Income, race, site term, ethnicity, mother's use of chemicals at work, mother health/pregnancy conditions, report that child had lead or mercury poisoning (MS only); Raw scores were child age-adjusted
Appropriate: Linear regression (continuous scores), logistic regression (dichotomous scores)
Selection bias possible due to loss of follow-up; Residual confounding likely; substantial potential for differential exposure misclassification (time between spraying and testing, frequency and duration unknown)
Shelton et al. (2014)
California, USA
CHARGE Study (3 year-old children)
Case-control, large sample size
N = 970
Age 3 years
N=486 (ASD)
N=168 (DD)
N=316 (Controls)
Proxy indicator of prenatal OP pesticide exposure (residential proximity to agricultural pesticide applications defined using ecological pesticide use data)
Clinical outcomes  -  validated within the study
Appropriate adjustment for demographics (place of birth, race), SES indicators (paternal education) and vitamin intake during pregnancy
Appropriate multivariate analysis.
Selection bias probable; Residual confounding likely; substantial potential for differential misclassification of exposure. Outcome misclassification unlikely.
Suarez-Lopez et al. (2012)
Pedro Moncayo County, Pichincha, Ecuador
Secondary Exposure to Pesticides Among Infants, Children and Adolescents (ESPINA) Study, plus new volunteers
Cross-sectional, moderate sample size, from prior census
N=277
Exposed: (n=158)
Unexposed: (n=119)
Age 4-9 years
Proxy: Cohabitation with flower worker >1yr, Adult questionnaire
Child AChE level using commercial kit
Appropriate: Sex, age, height-for-age, hemoglobin concentration, income, pesticide use within household lot, pesticide use by neighbors, examination date, residence, distance to nearest flower plantation
Appropriate: Multiple linear regression (continuous) and logistic regression (polychotomous variables), adjusted models
Selection bias possible due to loss but deemed low; Residual confounding likely small in magnitude; potential for differential exposure misclassification
Suarez-Lopez et al. (2013a)
Pedro Moncayo County, Pichincha, Ecuador
Secondary Exposure to Pesticides Among Infants, Children and Adolescents (ESPINA) Study, plus new volunteers
Cross-sectional, moderate sample size, from prior census
N=307
Age 4-9 years
Non-specific measures:
Child AChE level using commercial kit;
Proxy: Cohabitation with flower worker >1yr, Adult questionnaire
Heart rate (HR), Systolic blood pressure (SBP), Diastolic blood pressure (DBP) by standard methods
Appropriate: Sex, age, height-for-age, hemoglobin concentration, income, pesticide use within household lot, pesticide use by neighbors, examination date, residence, distance to nearest flower plantation
Appropriate: Multiple linear regression (continuous), adjusted.
Selection bias possible due to loss but deemed low; Residual confounding likely small in magnitude; potential for differential exposure misclassification
Suarez-Lopez et al. (2013)
Pedro Moncayo County, Pichincha, Ecuador
Secondary Exposure to Pesticides Among Infants, Children and Adolescents (ESPINA) Study, plus new volunteers
Cross-sectional, moderate sample size, from prior census
N=271
Age 4-9 years
Non-specific measures:
Child AChE level using commercial kit
NEPSY-II test (trained examiners): general assessment battery
Model defined a priori: hemoglobin, age, sex, race, height-for-age z score, household income, maternal education, and flower worker cohabitation status
Logistic models (dichotomous and polychotomous) and linear regression models, adjusted; Effect modification according to sex among significant associations; Some imputed values
Selection bias possible due to loss but deemed low; Residual confounding likely small in magnitude; potential for differential exposure misclassification
Wang et al. (2012)
Shanghai, China
Pregnant women and newborn children
Cross-sectional Birth Cohort Study
N=187
Age: newborns
Objective biomarker of prenatal OP pesticide exposure (DAP) in single maternal spot urine sample provided at onset of labor. Simple imputation of observations < LLOD. OP pesticide exposure also assessed via questionnaire administered after delivery
Gestational age and pre-term delivery appropriately defined and assessed using medical records
Appropriate. Included maternal anthropomorphic, demographic, and SES indicators (income, occupation), predictors of high-risk pregnancy (pregnancy weight gain, gestational age)
Appropriate multivariate analysis
Selection bias unlikely; Residual confounding likely small in magnitude; potential for differential misclassification of exposure
Zhang et al. (2014)
Shenyang, China
Newborn children in a birth cohort study
Prospective Cohort, n=249
mother-infant pairs
Age: newborns
Biomarker of prenatal OP pesticide exposure (DAP) quantified in single maternal spot urine sample provided at delivery. Many observations < LLOD
Neonatal neurodevelopment assessed using validated instrument (Neonatal Behavioral Neurological Assessment) by trained examiners
Appropriate. Included maternal demographic and SES indicators (age, education), predictors of high-risk pregnancy (BMI, gestational age) and environmental toxicant exposure (cord blood lead); Results stratified by sex
Appropriate multivariate analysis
Selection bias unlikely; Residual confounding likely small in magnitude; considerable potential for non-differential misclassification of exposure (unlikely to account for non-null findings)

<IP_ADDRESS>	"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 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 (MP) before age 6 years in Mississippi and Ohio (Ruckart et al. 2004), as assessed by household urinary PNP or wipe samples, exposed children performed worse than unexposed children on a few of numerous neurobehavioral development tests conducted.  Specifically, participants classified as having had MP exposure had more difficulty with short term memory and attention tasks, and parents reported more behavioral and motor skill problems, relative to unexposed children.  However, upon closer inspection of the results across the MS and OH study sites, these neurobehavioral outcomes are not seen consistently.  These inconsistencies may be due to differences in how the exposure occurred across the sites, including the fact that MS participants and OH participants were tested 2.5 and 4.5 years after MP spraying in the home.  When comparing exposed and unexposed children using general intelligence testing, integrated visual and motor skills testing, and multistep processing, they did not see any differences.  The exposure scenario associated with these observations is a critical element in assessing the utility and reliability of this study.  Samples were collected in locations from OH and MS where illegal spraying of methyl parathion is known to have occurred during the 1994-1996 time period.  Based on the "Revised Organophosphorous Pesticide Cumulative Risk Assessment" (USEPA, 2006), methyl parathion is known to be among the more potent OPs.  It is unknown whether study participants were exposed to MP levels that would have induced cholinergic effects.  Therefore, given the uncertainty around this illegal use combined with the high potency for cholinergic toxicity, the agency is not emphasizing this study further in the analysis.

In addition, two high quality studies from the CHAMACOS birth cohort (Quirós-Alcalá 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. l (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 neurodevelopmental outcomes, which is the focus of this analysis.  Consequently, Quirós-Alcalá 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 PON192, urinary total DMP (but not total DAP or DEP) was statistically significantly associated with shorter birth length (p= 0.032).  Birth length was also not associated with maternal urinary DAP sampled at delivery in the 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 (∑DAP, ∑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 ∑DEP 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 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 al, 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 al, (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 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 al, 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).

<IP_ADDRESS>	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 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 al., 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 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% CI=l .3-8. l; p=O.O 15); both arms (OR=3.3 ; 95% CI=l .1-9.4; p=0.027); either arm (OR=2.2; 95% CI=l .1-4.6; p=0.028); and non-dominant arm (OR=2. l; 95% CI=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% CI: 1.12-5.08, p = 0.02) and PDI: 4.9 times greater (95% CI: 1.78-13.72; p = 0.002)) than those with lower prenatal exposure (Rauh et al., 2006).  Within the Mt. Sinai study, authors administered the BSID-II to participating children at 12 and 24 months and observed that prenatal total DAP metabolite level was associated with a decrement in mental development at 12 months among blacks and Hispanic children; however, these associations either attenuated or were non-existent at the 24-month visit (Engel et al., 2011). In the CHAMACOS cohort, Eskenazi et al. (2007) observed that prenatal DAP levels were adversely associated with MDI, and at 24 months of age these associations reached statistical significance. In this study, neither prenatal DAPs nor maternal TCPy were associated with PDI (motor skills), nor did authors observe evidence of different risk by PON1 status (Eskenazi et al., 2010).

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% CI: 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 al, 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).  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 (β = -13.3; 95% CI: -19.9; -6.7; p < 0.01), relative to boys who lived in a less adverse learning environment (β = 4.2; 95% CI: -4.2; 12.5; p < 0.01).  A similar observation was reported for perceptual reasoning and processing speed (β = -9.8; 95% CI: -17.4; -2.2; p < 0.01; β = -12.7; 95% CI: -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 (β = -8.5; 95% CI: -16.7, -0.4 vs. β = -4.7 95% CI: -12.8, 3.4; p = 0.18).  This was also observed for working memory for girls (β = -5.9; 95% CI: -13.7, -1.8 vs. β = -2.6 95% CI: -10.0, 4.8; p = 0.05). In addition, similar findings were observed for girls whose mother's had more maternal adversity (β = -11.5; 95% CI: -18.5, -4.4 vs. β = -1.5 95% CI: -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.4F The Federal Panel concurred with the authors' conclusions in general; however, the Federal Panel also noted that significantly greater and more sophisticated MRI imaging studies would be needed to link the morphological changes indicated in this study with specific functional outcomes noted in the CCCEH IQ study. Therefore, while generally supportive of the epidemiologic findings, additional study is needed to make specific links with areas of brain development change.

In sum, across these three children's environmental health studies, authors consistently identified associations with neurodevelopmental outcomes in relation to OP exposure.  There is evidence of delays in mental development in infants (24-36 months), attention problems and autism spectrum disorder in early childhood, and intelligence decrements in school age children who were exposed to chlorpyrifos or OPs during gestation. Investigators reported strong measures of statistical association across several of these evaluations (odds ratios 2-4 fold increased in some instances), and observed evidence of exposures-response trends in some instances, e.g., intelligence measures.

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.

<IP_ADDRESS>	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 < 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 (β = 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 (β = -0.069, p = 0.04; β = -0.101, p = 0.03), with black race and birthweight as statistically significant covariates (β = -0.462, p = 0.03; β = -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 (β coefficient = -0.010, p = 0.01), with birthweight of infants and maternal blood lead levels during gestation as statistically significant covariates (β = -0.003, p = 0.02; β = 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-II 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: β = 0.005, p = 0.034, β = 0.003, p = 0.144; adjusted model: β = 0.003, p = 0.179, β = 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: β = 0.004, p = 0.111; β = 0.004, p = 0.124, when the model was adjusted, no evidence of a positive association was observed (adjusted model: β = 0.001, p = 0.494; β = 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-II 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: β = 0.003, p = 0.16; β = 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: β = 0.002, p = 0.28; β = 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-II Psychomotor Developmental Index for children aged 1-3 years.
<IP_ADDRESS>	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 (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.

<IP_ADDRESS>	Other Neurodevelopmental Studies

In the first of two Chinese studies focusing on generic neurodevelopmental outcomes, Zhang et al. (2014) investigated prenatal exposure to OPs and neurobehavioral development of neonates in a birth cohort study in Shenyang, China. The authors reported that consistent statistically significant associations were observed between all of the quantified urinary biomarkers of prenatal OP pesticide exposure and neonatal neurodevelopment deficits assessed 3 days after birth.  A 10-fold increase in total DAPs concentration was associated with an average decrease in Neonatal Behavioral Neurological Assessment (NBNA) summary scores of 1.78 points (95% CI: -2.12 to -1.45). No evidence of departure from linearity of the exposure-response relationship between maternal DAP concentrations and NBNA scores was observed.  In the second Chinese study focusing on neurodevelopmental outcomes, Guodong et al. (2012) conducted a cross-sectional study, and did not identify any statistically significant associations between the children's urinary DAP metabolite levels and any of the DQ (Developmental quotients) scores.  The authors mentioned that their results should be interpreted with caution since OP exposure was quantified in single spot urine sample from children, and should be followed up with a longitudinal study with repeated measurement of exposure levels in urine samples.
Two studies focused on impaired social responsiveness, autism spectrum disorders, or developmental delays (Furlong et al., 2014; Shelton et al., 2014).  Among 7-9 years old children in the Mount Sinai Cohort (Furlong et al. 2014), there was no overall statistically significant association between maternal third trimester urinary DAP metabolite levels and reciprocal social responsiveness (a measure linked to many neuropsychiatric conditions that involve impaired social functioning (Constantino and Gruber 2005)).  However, some evidence of modification of the association between prenatal OP pesticide exposure and impaired social responsiveness in early childhood was observed by both race/ethnicity and child sex, with an association between DEP and poorer social responsiveness observed among black participants and boys.  No association was observed among whites or Hispanics, among girls, or for DAP or DMP biomarker levels.
Shelton et al. (2014), investigated autism spectrum disorders 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% CI: 1.02 - 2.51] than mothers of normally developing children. They added that as the buffer distance grew larger, these associations became lesser, indicating an exposure-response effect. The authors also mentioned that each 100-lb (45.4 kg) increase in the amount applied over the course of pregnancy (within 1.5 km of the home) was associated with a 14% higher prevalence of autism spectrum disorder (aOR = 1.14; 95% CI: 1.0, 1.32), but no association was identified with DD.
A total of three studies focused on OP exposure and behavioral, memory, or attention/ADHD outcomes (Oulhote and Bouchard, 2013; Bouchard et al., 2010; Fortenberry et al., 2014).  In a national cross-sectional study of Canadian children 2007-2009 data for age 6-11 years (Oulhote and Bouchard, 2013), there were no overall statistically significant associations observed between child urinary DAP, DMP, or DEP metabolite levels and parentally reported behavioral problems.  In contrast, Bouchard et al. (2010), looking at U.S. children age 8-15 years in the 2000-2004 National Health and Nutrition Examination Survey (NHANES),5F observed a positive association between attention and behavior problems and DAPs and DMPs, but not DEPs.  For example, even after controlling for potential confounders such as sex, age, ethnicity, and creatinine concentration, they found that a 10-fold increase in DMAP concentration was associated with a 55 to 72% increased odds of ADHD.

Fortenberry et al. (2014) evaluated the relationship between pesticide exposure and ADHD in school age Mexican children, recruiting 187 mother-child pairs from a prospective birth cohort, ELEMENT (Early Life Exposures in Mexico to Environmental Toxicants). The authors reported that, there were no statistically significant associations between tertiles of maternal third trimester urinary TCPy and measures of attention and hyperactivity in children.  However, there was suggestive evidence for increases in the ADHD index in relation to TCPy tertiles among boys (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 Assessments for Pesticides" provides the foundation for evaluating multiple lines of scientific evidence (U.S. EPA, 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 Handbook6F).   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; 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 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% CI: 1.1-5.1) and psychomotor delay (OR=4.9; 95% CI: 1.8-13.7) at age three when comparing high to low exposure groups (Rauh et al., 2006). Rauh et al (2006) also reported extremely large odds ratios for attention disorders (OR=11.26; 95% CI: 1.79-70.99), ADHD (OR=6.50; 95% CI: 1.09-38.69), and PDD (OR=5.39; 95% CI: 1.21-24.11) when comparing high to low chlorpyrifos exposure groups (Rauh et al., 2006). EPA notes that the magnitude of these results are so large that they are unlikely to be affected by residual confounding although limited sample sizes resulted in imprecise estimates.

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% CI: 1.7 - 15.7 and with a composite ADHD indicator of the various measures (OR = 3.5; 95% CI: 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:1.1 - 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 (β = -13.3; 95% CI: -19.9; -6.7; p < 0.01), relative to boys who lived in a less adverse learning environment (β = 4.2; 95% CI: -4.2; 12.5; p < 0.01).  A similar observation was reported for perceptual reasoning and processing speed (β = -9.8; 95% CI: -17.4; -2.2; p < 0.01; β = -12.7; 95% CI: -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 (β = -8.5; 95% CI: -16.7, -0.4 vs. β = -4.7 95% CI: -12.8, 3.4; p = 0.18).  This was also observed for working memory for girls (β = -5.9; 95% CI: -13.7, -1.8 vs. β = -2.6 95% CI: -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 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 al., 2012; Zhang et al., 2014), there may be differences in the study population and outcome measurements that may account for the observed differences in study results, with Zhang et al. (2014) documenting statistically significant associations for total DEAPs, total DMAPs, and total DAPs and Guodong et al. (2012) observing no association with these exposures.  The Zhang et al. (2014) study was conducted in Shenyang, with a study population reported as 87% urban and 13% rural, whereas the Guodong et al. (2012) study was conducted in Shanghai with a 99% urban and 1% suburban study population.  Given the higher percentage of study participants from rural areas, the study participants from Zhang et al. (2014) may have had different pattern and magnitude of OP exposures compared to those from Guodong et al. (2012).  In addition, it is noted that different outcome measurements were made in these studies, with Guodong et al. (2012) assessing 23-25 month old children using a developmental quotient score and Zhang et al. (2014) assessing 3 day old infants using a Neonatal Behavioral Neurological Assessment.  Given the different outcome assessments, exposure potential, study designs (cohort vs. cross-sectional), and ages of the participants in these two studies, it is difficult to draw conclusions on how these study results compare.  It is notable that the results from the Zhang et al. (2014) study focusing on neonates are consistent with those from other studies which reported statistically significant associations between delayed neurological development measured in newborns and total DEAP, total DMAP, and total DAP exposure (Engel et al., 2007; Young et al., 2005).  However, it is noted that neurological development was measured within a few days of birth for Zhang et al. (2014) and Engel et al. (2007), whereas Young et al. (2005) conducted their measurements within two months of birth.

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 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% CI: 1.12-5.08, p = 0.02) and PDI: 4.9 times greater (95% CI: 1.78-13.72; p = 0.002)) than those with lower prenatal exposure (Rauh et al., 2006).  Within the Mt. Sinai study, authors administered the BSID-II to participating children at 12 and 24 months and observed that prenatal total DAP metabolite level was associated with a decrement in mental development at 12 months among blacks and Hispanic children; however, these associations either attenuated or were non-existent at the 24-month visit (Engel et al., 2011). In the CHAMACOS cohort, Eskenazi et al. (2007) observed that prenatal DAP levels were adversely associated with MDI, and at 24 months of age these associations reached statistical significance. In this study, neither prenatal DAPs nor maternal TCPy were associated with PDI (motor skills), nor did authors observe evidence of different risk by PON1 status (Eskenazi et al., 2010). In contrast to the findings of CCCEH, Mt. Sinai, and CHAMACOS, the HOME study (Donauer et al, 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 (β = 0.002, p = 0.28; β = 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-II 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 al, 2010).

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 al, 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).7F  Specifically, Eskenazi et al. (2007) reported a statistically significant association between PDD and total DAP exposure, whereas Rauh et al. (2006) showed a significant association between PDD and specifically chlorpyrifos exposure.  Both PDD and reciprocal social responsiveness are related to the autism spectrum disorder.  Using a different exposure assessment method (geospatial analysis and residential proximity to total OP exposure), Shelton et al. (2014) also documented statistically significant associations between total OP exposure and 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 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 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 al, 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: β = 0.004, p = 0.111; β = 0.004, p = 0.124, when the model was adjusted, no evidence of a positive association was observed (adjusted model: β = 0.001, p = 0.494; β = 0.002, p = 0.441).

As stated in the EPA neurotoxicity guidelines8F, direct extrapolation of developmental neurotoxicity results from laboratory animals to humans is limited by the lack of knowledge about underlying toxicological mechanisms and the relevance of these results to humans.  EPA notes consistencies across the databases of in vivo laboratory animal studies and epidemiology studies, although challenges of making a direct comparison between neurodevelopmental domain inter-species remain. It can be assumed that developmental neurotoxicity effects in animal studies indicate the potential for altered neurobehavioral development in humans, although the specific types of developmental effects seen in experimental animal studies may not be the same as those that may be produced in humans.  However, considering the toxicological and epidemiological data in the context of three major neurodevelopmental domains (specifically, cognition, motor control, and social behavior), insights can be gained.  Previously reviewed studies of chlorpyrifos in rats and/or mice reported impaired cognition, changes in locomotor activity levels, altered social interaction, and to a lesser extent, changes in neuromotor function (FIFRA SAP 2012; USEPA, 2014).  While there are fewer studies for all the other OPs, behavioral effects in the same functional domains were again reported.  The most commonly reported outcome 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 and early in life, with adverse outcomes in several neurological domains including cognitive, anxiety and emotion, social interactions, and neuromotor function.  The studies have not shown that any specific developmental period is critical overall to the long-term outcomes, since similar effects are observed with different exposure periods.  For example, cognitive changes in one laboratory using a radial arm maze were observed following gestational and early, but not late, postnatal exposure (Aldridge et al., 2005; Icenogle et al., 2004; Levin et al., 2002; Levin et al., 2001), whereas other laboratories cognitive deficits in a Morris water maze were reported following both gestational and late-postnatal exposure (Billauer-Haimovitch et al., 2009; Turgeman et al., 2011).  Likewise, some changes in anxiety and social behaviors were reported at both gestational and postnatal exposure periods.  Overall, these data do not clearly show specific critical periods of exposure but support a conclusion that early life (pre- and post-natal) represent susceptible lifestages.

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 al., 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 al., 2006; Eskenazi et al., 2007; Marks et al., 2010; and Fortenberry et al., 2014) with additional support from a case control study, Bouchard et al. (2010).  In addition, the three US cohorts and the CHARGE study have reported suggestive or positive associations between OP exposure and autism spectrum disorders (Rauh et al., 2006; Shelton et al., 2014; Eskenazi et al., 2007; Furlong et al., 2014).  While 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 al., 2005; Engel et al., 2007; Zhang et al., 2014).  Finally, CCCEH, Mt. Sinai and CHAMACOS have reported an inverse relation between the respective prenatal measures of chlorpyrifos or DAPs and intelligence measures at age 7 years (Rauh et al., 2011; Engel et al., 2011; Bouchard et al., 2011).

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 al., 2007; Young et al., 2005; Bouchard et al., 2011) and three cross-sectional studies (Guodong et al., 2012; Bouchard et al., 2010; Oulhote and Bouchard, 2013).  With the exception of Bouchard et al. (2010) and findings of PDD increase in post-natal DAP from CHAMACOS (Eskenazi et al., 2007; Marks et al, 2010), no adverse neurodevelopmental associations were found between postnatal urinary metabolite levels and any of the developmental outcomes.  Bouchard et al. (2010) looked at U.S. children age 8 - 15 years in the 2000 - 2004 National Health and Nutrition Examination Survey (NHANES), and observed a positive association between attention and behavior problems and total DAPs and DMAPs, but not DEAPs. 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-related factors and toxicological evidence relevant to identification of potential modes of action (MOAs). Similarly, consideration of the coherence of health effects associations reported in the epidemiologic literature reflects broad consideration of information pertaining to the nature of the biological markers evaluated in toxicologic and epidemiologic studies. [p. 39]."

The Cancer Guidelines further state that "lack of mechanistic data, however, is not a reason to reject causality [p. 41]."

At this time, a MOA(s)/AOP(s) has/have not been established for neurodevelopmental outcomes.  This growing body of literature does demonstrate, however, that OPs are biologically active on a number of processes that affect the developing brain.  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.

      ::	1 pM chlorpyrifos oxon decreased axon length (~50%) in superior cervical ganglion cell cultures (AChE inhibition starting at 1000pM 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