APPENDIX 6 Detailed Reviews of Children’s Health Epidemiology StudiesPage 2 of 105 Table of Contents

Article 1. Whyatt et al. (2004)

................................ ................................ ....................... 3

Article 2. Berkowitz et al. (2004)

................................ ................................ ................... 9

Article 3. Eskenazi et al. (2004)

................................ ................................ ................... 16

Article 4. Harley et al. (2011)

................................ ................................ ....................... 21

Article 5. Engel et al. (2007)

................................ ................................ ........................ 29

Article 6. Young et al. (2005)

................................ ................................ ....................... 37

Article 7. Rauh et al. (2006)

................................ ................................ ......................... 42

Article 8. Lovasi et al. (2010)

................................ ................................ ....................... 48

Article 9. Engel et al. (2011)

................................ ................................ ........................ 54

Article 10. Eskenazi et al. (2007)

................................ ................................ ................... 63

Article 11. Eskenazi et al. (2010)

................................ ................................ ................... 69

Article 12. Marks et al. (2010)

................................ ................................ ....................... 75

Article 13. Rauh et al. (2011)

................................ ................................ ......................... 82

Article 14. Bouchard et al. (2011)

................................ ................................ ................. 90

Article 15. Whyatt et al. (2007)

................................ ................................ ..................... 98

Article 16. Whyatt et al. (2009)

................................ ................................ ................... 101Page 3 of 105 Article 1. Whyatt et al. (2004)

Prenatal Insecticide Exposures and Birth Weight and Length among an Urban Minority Cohort. Robin M. Whyatt, Virginia Rauh, Dana B. Barr, David E. Camann, Howard F. Andrews, Robin Garfinkel, Lori A. Hoepner, Diurka Diaz, Jessica Dietrich, Andria Reyes, Deliang Tang, Patrick L. Kinney, and Frederica P. Perera. Environmental Health Perspectives. 112:1125

– 1132 (2004).

Study Summary

In this report, Whyatt et al

. report on the relationship between pesticide exposures and developmental outcomes in newborns in the Columbia Mother’s and Newborn Study to include additional insecticides (the organophosphate diazinon and the carbamate propoxur), a larger sample size (n = 314 mo ther

– newborn pairs), and insecticide measurements in maternal personal air sampled in the home, as well as in umbilical cord plasma sampled at delivery.

Study participants were recruited during early pregnancy ( ≤20th week) among African

- American and
Domi nican women age 18

- 35 years, and registered at New York Presbyterian Medical Center and Harlem City. Women who smoked, had a history of drug abuse, diabetes, hypertension, or HIV infection were excluded from participation in the study, as were women who re sided in New York City for less than 1 year. The study sample presented in this report was recruited between 1998 and 2002, a period which straddles the December, 2000 voluntary cancellation by registrants of indoor residential chlorpyrifos use.

Insectic ides, including chlorpyrifos, were measured in 48

- hr personal air samples collected during the third trimester of pregnancy. Analyses of air samples for pesticide levels were carried out at the Southwest Research Institute.

Chlorpyrifos levels in umbilic al cord blood samples were available for 256 newborns, sampled as close to the time of delivery as possible, and within 2 days post

- partum. Cord blood plasma chlorpyrifos levels were imputed from maternal blood levels for 31 newborns for whom no cord blood sample was obtained. Quantification of chlorpyrifos levels in plasma were conducted by the Centers for Disease Control and Prevention (CDC).

Insecticide levels in both maternal personal air and blood samples were available for 259 of the 314 mother

– newb orn pairs (82%). Measures in either personal air or blood were available for the remaining 55 pairs (18%). Pesticide levels in personal air and blood samples below the level of detection (LOD) were assigned a value of half the LOD; levels were then log 2 - t ransformed for statistical analysis. For pesticides in blood, 31% of chlorpyrifos levels were below the LOD. Infants with below LOD cord blood chlorpyrifos levels were used as a comparison group and compared to groups with increasing levels, categorized by tertile of above LOD levels.Page 4 of 105 Infants’ weight, length, and head circumference were assessed at birth and obtained for this study through medical record abstraction.

Multiple linear regression analyses were performed to evaluate associations between pren atal insecticide exposure and the indicators of infant development at birth. Covariates were selected from variables known to be associated with insecticide exposure or fetal growth. Effect modification by race/ethnicity was evaluated, but not observed.

Controlling for potential confounders, the researchers found no association between maternal personal air insecticide levels and birth weight, length. Head circumference was not significantly associated with maternal personal air or cord blood chlorpyrif os levels. However, for each log unit increase in cord plasma chlorpyrifos levels, birth weight decreased by 42.6 g (95% CI:

– 81.8 to

– 3.8) and birth length decreased by 0.24 cm (95% CI:

– 0.47 to

– 0.01). Combined measures of (ln)cord plasma chlorpyrifos an d diazinon (adjusted for relative potency) were also inversely associated with birth weight and length (p < 0.05). Birth weight averaged 186.3 g less (95% CI:

– 375.2 to

– 45.5) among newborns with the highest compared with lowest 26% of exposure levels (com bined p

- value = 0.01). When stratified by date of birth prior to or after 2001 (before and after the voluntary cancellation by registrants of indoor residential chlorpyrifos use was implemented), associations between birth weight and length and cord plasm a chlorpyrifos were statistically significant (p ≤ 0.007) among newborns born before the cancellation. Among newborns born after January 2001, exposure levels were substantially lower, and no associations with fetal growth outcomes were observed (p > 0.8). Results suggest that prenatal chlorpyrifos exposures may have impaired fetal growth among this inner

- city, low income cohort.

Study Review

This was a very well conducted study with numerous strengths, and some noted limitations. The prospective nature o f the study limits the influence of systematic errors in the exposure assessment on the asso ciation observed, such that mis measurement of chlorpyrifos exposure would likely be non

differential with respect to the indicators of fetal growth.

A notable str ength of this study, relative to investigations in other cohorts, is the direct measurement of chlorpyrifos in cord blood and personal air samples, rather than non

- specific markers of organophosphate pesticide exposure. Errors in estimation of cord blood c hlorpyrifos levels were likely to have been non

- differential with respect to the indicators of fetal development. However, authors did not formally assess the variability in the exposure estimate resulting from the imputation of missing cord blood chlorpyr ifos levels.

A limitation of the specific biomarker exposure indicators in this study is the single sampling period (at delivery). It is not clear to what extent this single measurement reflects exposures over critical windows of fetal growth and develop ment during pregnancy. If the exposure is chronic, a biomarker measured at a single time point can provide a representative dosimeter, even if the toxicant has a short half

- life, as isPage 5 of 105 the case for chlorpyrifos. However, if pesticide exposures are sporadic or otherwise vary over short time scales, the biomarker measurement will not be representative of “usual” exposure, or of the exposure during critical periods of fetal development. An observation effect could also influence the validity of the 48

- hour pe rsonal air sampling if, for example, the act of wearing the dosimeter inhibited participants from using pesticides or otherwise altered their behavior patterns in a manner that affected exposure levels. These limitations are assessed in subsequent validat ion studies conducted within this cohort (Whyatt, 2007 and Whyatt, 2009).

The health outcomes in this study, birth weight, birth length, and head circumference were obtained through direct linkage with the mothers’ and infants’ computerized medical recor ds, and were likely to have been accurately measured. Any errors in outcome measurement would have occurred in the clinic, and are likely to be small and likely to be non

- differential with respect to pesticide exposure.

The investigators collected data o n, and adjusted for, important potential confounders. Covariate selection proceeded appropriately by assessing relationships between potential confounders and the indicators of fetal growth, change

- in

- estimate of the main effect of chlorpyrifos exposure, a nd assessments of model fit. Covariates included in final models were race/ethnicity, gestational age, parity, maternal pre

- pregnancy weight, and net weight gain during pregnancy, maternal self

- reported environmental tobacco smoke in the home, sex of the newborn, and season of delivery. Models for head circumference also included an indicator for delivery type (vaginal versus cesarean section).

Maternal exposure to polycyclic aromatic hydrocarbons (PAHs) during pregnancy was not included in the final models because it did not improve model fit. However, to ensure that PAH exposure did not confound the pesticide associations with fetal development , models including PAHs were evaluated. The authors’ consideration of other pesticides and environmental contaminants as covariates in their multivariate models is a notable strength of this study.

Confounding by maternal smoking behaviors was controlled by restriction ( i.e.
, excluding smokers from the study sample). Confounding by unmeasured factors may have been similarly controlled by design, this being a relatively homogeneous population with respect to determinants of fetal growth, compared to the ge neral population. For example, the authors found it unnecessary to adjust for maternal alcohol consumption (which was measured), partly because few women in the study drank alcohol during

pregnancy.

Bias due to confounding by unmeasured determinants of fe tal growth is possible in this observational study, for example, confounding by factors related to socioeconomic condition. Annual household income, maternal education, maternal marital status, material hardship during pregnancy, and degree of housing disr epair were evaluated as possible markers for socioeconomic status but were not included in final models because they were not associated with birth outcomes, did not improve the model fit, and did not substantially alter parameter estimates, confidence int ervals (CIs), or significance levels of variables included in the final models. This observation that indicators of socioeconomic condition werePage 6 of 105 not associated with the health outcomes is somewhat unexpected, although perhaps explainable by the relative h omogeneity of these factors in this study population (control through restriction of study population).

In categorical analyses, infants with the lowest tertile of observable cord blood levels had, as a group, larger heights and weights at birth, compare d to infants with chlorpyrifos levels below the level of detection, although the increases were not statistically significant. Among those with higher cord blood levels, increasing tertiles of chlorpyrifos were significantly associated with decreasing birt h weight and height. This is suggestive of a threshold effect (a hockey stick dose

- response curve). Departures from linearity in the exposure

- response relationship were not assessed in models including (ln)chlorpyrifos entered as continuous variables.

Th e authors observed an association between chlorpyrifos and fetal growth only when the biomarkers were used as indicators of prenatal exposure and not when environmental levels (in maternal 48

- hr personal air sampling conducted during the third trimester) w ere used. Researchers note several possible explanations for this discrepancy, including the notion that the biomarkers are integrating dosimeters of exposure. This may be particularly important for insecticide exposure by routes other than inhalation in t he home ( e.g. , dermal, ingestion, and occupational exposures). In addition, the authors speculate that the biomarkers may provide better dosimeters of the dose to the developing fetus, relative to the airborne exposures.

The voluntary cancellation by regi strants of indoor residential chlorpyrifos use introduces exogenous variation in the exposure of interest. No associations were observed between the fetal growth indicators and chlorpyrifos levels among infants born after the implementation of the volunta ry cancellation of chlorpyrifos containing in home pesticide products. This finding is similarly suggestive of a possible threshold effect (decreasing dose

- levels before and after voluntary cancellation), although it could also be due to a lack of statist ical power to assess associations at lower levels of exposure in the relatively small subsample (n=77).

The statistical analysis appeared to be conventional and appropriate. However, the authors did not present nor discuss regression diagnostics to asses s the degree to which their models met or violated the assumptions implicit in linear models ( i.e. homoscedasticity, normality of error term distribution, independence of error terms, and linearity of the exposure

- response relationship), other than to stat e that investigators log transformed chlorpyrifos levels. As discussed previously, there is some evidence in the data to suggest a non

- linear exposure response relationship ( i.e. , threshold) between the natural log of cord blood chlorpyrifos levels and bi rth weight and length, but no formal statistical testing of this possibility was presented or discussed.

Bias due to differential errors in the selection of study participants is unlikely to have occurred in this prospective study with low loss to follo w

- up. Appropriate exclusion criteria were applied, althoughPage 7 of 105 these design decisions may reduce external validity (generalizability) when making inference to other populations using the associations observed in this narrowly defined study population.

The results presented in this expanded analysis confirm the authors’ earlier findings of an inverse association between chlorpyrifos levels in umbilical cord plasma and birth weight and length in this study population of non

- smoking African

- American and Domini can women, age 18

- 35 years, living in New York City (Whyatt et al., 2003) . Systematic errors in the selection of study participants or in the measurement of key analytic variables (exposure and outcome) are unlikely to explain this observed association. Th e possibility of unmeasured or poorly measured potentially confounding variables cannot be completely excluded, although study authors thoroughly evaluated this possibility in the data available. Data suggest a threshold effect may be possible, although au thors did not formally evaluate this characteristic of the data.Page 8 of 105 Works Cited

Whyatt, R. M., Barr, D. B., Camann, D. E., Kinney, P. L., Barr, J. R., Andrews, H. F., . . . Perera, F. P. (2003). Contemporary

- use pesticides in personal air samples during pregnancy and blood samples at delivery among urban minority mothers and newborns. Environ Health Perspect, 111 (5), 749

- 756.

Whyatt RM, Garfinkel R, Hoepner LA, Holmes D, Borjas M, Williams MK, Reyes A, Rauh VA, Perera FP, Camann DE. Within

- and Between

- Home Variability in Indoor

- Air Insecticide Levels during Pregnancy among an Inner

- City Cohort from New York City. Environmental Health Perspectives. 115:383

– 389 (2007).

Whyatt RM, Garfinkel R, Hoepner LA , Andrews H, Holmes D, Williams MK, Reyes A, Diaz D, Perera FP, Camann DE, Barr DB. A Biomarker Validation Study of Prenatal Chlorpyrif os Exposure within an Inner

City Cohort during Pregnancy. Environal Health Perspectives. 117:559

– 567 (2009).Page 9 of 105 Article 2. Berkowitz et al. (2004) In Utero Pesticide Exposure, Maternal Paraoxonase Activity, and Head Circumference. Gertrud S. Berkowitz, James G. Wetmur, Elena Birman

- Deych, Josephine Obel, Robert H. Lapinski, James H. Godbold, Ian R. Holzman, Mary S. Wolff. Environmental Health Perspectives. 112:388

– 391 (2004)

Study Summary

To assess the relationship of exposure to pesticides during pre gnancy and subsequent risk of impaired growth and neurodevelopment, Berkowitz and colleagues (Mount Sinai Child Development Study) investigated the relationship between prenatal pesticide exposure and infant growth and neurodevelopment in a prospective, mu ltiethnic cohort study of mothers and infants delivered at Mount Sinai Hospital in New York City.

The study population was drawn primarily from East Harlem and consisted largely of young Hispanic women (predominantly Puerto Rican), but also included Afr ican

- American and Caucasian women. Mothers were enrolled during early pregnancy from a prenatal clinic and two private practices at Mount Sinai Hospital from March 1998 to March 2002. The study included only primiparous with singleton births. Mothers were excluded if they had any of the following: an initial prenatal visit after 26 weeks of gestation; serious chronic diseases; a serious pregnancy complication that could affect fetal growth and development; alcohol consumed greater than two alcoholic beverag es per day; or, illicit drug use. Mothers and infants were also excluded if the child was born with a congenital malformation or severe prematurity. A total of 479 prenatal patients were recruited; 75 were excluded because of medical complications, prematu re birth, an infant with birth defects, inability to collect biologic specimens before birth, change of hospital or residence outside New York City, or refusal to continue to participate.

Pesticide exposure was assessed by a prenatal questionnaire and me asurement of urinary concentration of 3,5,6

- trichloro

- 2

- pyridinol (TCPy), a metabolite of chlorpyrifos exposure. The survey was administered, and maternal urine and blood specimens were collected, during the third trimester. Cord blood samples were also ob tained at birth. Measurement of paraoxanase

- 1 ( PON1 ) genotype and phenotype was made using maternal and infant cord blood samples. PON1 is an enzyme that can detoxify chlorpyrifos before it can inhibit acetylcholinesterase in the peripheral and central ner vous systems. The investigators assessed birth weight, length, head circumference, and gestational age of the newborns for 404 participating mother

- child pairs.

Almost half of the participants in the study, (46%) reported that they, or a household member , had used indoor pesticides during the pregnancy; 72% were classified as exposed using a composite index defined as indoor pesticide use in the household or exterminator application, fumigation, or pesticide use in common areas. Detectable levels of urina ry 3,5,6

- trichloro

- 2

- pyridinol (TCPy; a metabolite of chlorpyrifos) were observed in 44% of the study participants.Page 10 of 105 The authors constructed generalized linear models to evaluate the associations among pesticide exposure and PON1 activity with average bir th weight, birth length, head circumference, and gestational age, adjusting for selected covariates (race/ethnicity, infant sex, and gestational age). The authors found no statistically significant associations between pesticide exposure based either on q uestionnaire responses or biomarkers of pesticide exposure (TCPy) on fetal growth or gestational age.

Researchers also evaluated the potential effect modifying role of PON1 genotype and phenotype in the relation of interest. Stratifying participants by p resence of PON1 polymorphisms and tertiles of maternal PON1 activity, researchers reported a small but statistically significant reduction in head circumference among children of mothers with levels of chlorpyrifos above the limit of detection and also in the lowest tertile of PON1 activity, i.e. , slowest metabolic activity. However, the interaction between TCPy level and PON1 activity was not statistically significant. In the subgroup of infants whose mothers had TCPy levels greater than the level of detec tion, those with low maternal PON1 had an average (SD) head circumference of 33.3cm (1.5cm) which was significantly smaller than those with medium (34.0cm (1.5cm)) and high (34.1cm (1.6cm)) maternal PON1 activity after adjusting for race/ethnicity, infant sex, and gestational age (p = 0.014). A similar trend was observed with head circumference when maternal PON1 activity was considered alone; the adjusted means were 33.5 cm for the low PON1, 33.9 cm for medium PON1, and 34.1 cm for high PON1 activity (p = 0.004). Maternal PON1 genetic polymorphisms were not associated with reduced head size. No trends were seen for birth weight or birth length for TCPy or the other metabolite levels when maternal paraoxonase level was taken into account. Infant
paraoxonase activity had no association with any of the fetal growth measures.

Because small head size has been found to be predictive of subsequent cognitive ability, the authors conclude that chlorpyrifos may have a detrimental effect on fetal neurodevelopment amo ng mothers who exhibit low PON1 activity.

Study Review

The largely null study had notable strengths and some limitations; notably authors did not discuss any limitations of their study in the published manuscript. The prospective nature of the study limi ts the influence of errors in the self

- reported exposure classification on the association observed; as such errors would likely be non

- differential with respect to determinants of the health outcome under investigation.

The authors assessed pesticide exp osure using both subjective questionnaire

- based methods and biomarkers of exposure which are objective, sensitive and specific surrogates of pesticide exposure. Biomarkers of specific pesticides ( i.e.
, TCPy) were assessed in a single maternal urine sample provided during the third trimester. The authors do not discuss the impact of policies aimed at reducing use of chlorpyrifos on the prevalence and magnitude of pesticide exposures during the time period of this study; it is likely that use of chlorpyrifos exposure was decreasing over the time period of the study, as noted in other similar studies of the same time period. The authors do not deliberate on the degree toPage 11 of 105 which the biomarkers assessed represent usual exposure to pesticides versus recent, acute e xposure(s), nor do they discuss the relative importance of assessment of exposure during the third trimester vis

- à

vis fetal growth and development. The critical window of exposure for fetal growth and neurodevelopment health effects is not fully characte rized. If sources and patterns of exposure remain static, a single measurement would be a good indicator of pesticide exposure during pregnancy, even if the pesticides have short half

- lives in vivo . If, however, sources and exposure patterns change over sh ort time

- spans, perhaps on the order of days to weeks, the biomarker assessment would not necessarily provide a good indicator of the relevant exposure. The authors classified biomarker levels according to the assays’ levels of detection (above versus belo w). Falsely negative exposure characterization would occur if mothers were exposed only early in pregnancy, and the pesticides were fully metabolized prior to the third trimester. Incorrect classification of participants as exposed, when in fact they were not exposed, was unlikely to have occurred using the biomarkers. Like other sources of measurement error in this study, errors in the biomarker indicators of exposure in this study are likely to have resulted in a

non

- differential misclassification, with respect to fetal growth and development.

The health outcomes in this study, birth length, birth weight, head circumference, and gestational age were likely to have been accurately measured in this study, as they were obtained through direct linkage with the computerized medical record. Any errors in outcome measurement would have occurred in the clinic, and are likely to be small and certainly non

- differential with respect to pesticide exposure.

To some extent, the recruitment process resulted in a popu lation that was likely homogeneous with respect to confounders of the in utero pesticide exposure

- fetal growth relationship (compared to the general population), and the potential for confounding was thus limited by design. The authors were also able to co llect data on, and adjust for, important covariates, including race/ethnicity, infant sex, and gestational age. The authors also controlled for birth weight or birth length in their assessment of head circumference and pesticide exposure. Covariates used i n the analyses were selected, according to the authors, from variables known to be associated with either pesticide exposure or fetal growth. However, factors related to socioeconomic condition of those assessed and co

- contaminants to which the study part icipants may have been exposed were not addressed in detail in the article. To the extent that such factors may be determinants of fetal growth and development and correlated with (but not caused by) pesticide exposure, they may confound the associations o f interest in the study. If these factors are positively associated with both pesticide exposure and diminished fetal development, they would bias the true association upward (towards larger mean differences associated with exposure). However, this is unl ikely to have occurred in this largely null study.

Only factors that were statistically significant (p < 0.05) were included in the final models. The use of a statistical test as a means to select covariates from a larger set of potential confounders is discouraged in some epidemiologic circles (Miettinen 1976, Breslow and Day 1980, Greenland 1989). The assessment of confounding is generally not considered to be a statistical endeavor, in part because the association between the confounder and the outcom e is just one factor that determines the degree of confounding, and also because the statistical significance of an association is a function not only of thePage 12 of 105 magnitude of the association but also of other factors ( e.g. , sample size, and sample variability) which are irrelevant to bias due to confounding. Moreover, the use of significance testing in the selection of confounders treats false negative errors ( i.e. the deleting a confounder) as secondary to false positive error ( i.e. , inclusion of a non

- confounder). This implicit ranking of error is backward, as deleting a confounder introduces bias and is justified only if the bias induced by its exclusion is tolerably small or deemed to be worth the precision gain, whereas including a non

- confounder only (potentially) reduces precision by increasing the size of the model. This is a small consideration in this study, and different approaches are not likely to qualitatively change the study findings or conclusions. Notably, smoking was not included as a potentially confounding variable in this analysis as t he prevalence of smoking was low in this cohort. According to the authors, the inclusion of smoking in final models did not alter risk estimates and added to variance as stated by the authors.

Bias due to differential errors in the selection of study participants is unlikely to have occurred in this prospective study with no loss to follow

- up at the time of this study. Appropriate exclusion criteria were applied, including mothers wit h no prenatal medical visit prior to 26 weeks of gestation, mothers with serious chronic diseases ( e.g. , diabetes, hypertension, thyroid disease), mothers who developed a serious pregnancy complication that could affect fetal growth and development, and mo thers who consumed more than two alcoholic beverages per day or who used illicit drugs, mothers with multiple births, and infants born with a congenital malformation or severe prematurity. The subjective, survey

based pesticide exposure assessment was fort ified by objective indicators of exposure ( i.e. urinary biomarkers).

The generalizability of the study may be limited as consequence of the particulars of the study population assessed and the numerous exclusion criteria applied. Making valid inference u sing these results requires an assumption that the pesticide exposure

- fetal development association is the same in this population as the population to which the results are being generalized. This study population consisted of primiparous women, recruited early on in their pregnancies from the prenatal clinic and two private practices at Mount Sinai Hospital in New York from March 1998 to March 2002, consisted predominantly of young Hispanic women with low education. Sixty

- eight percent were less than 25

y ears old. Just under half were Hispanic (49.8%), although substantial numbers of White (21.0%) and African

- American (27.7%) women also participated. Just under half (46.8%) classified themselves as being either single, divorced, widowed, or separated, and just over half (50.1%) had no more than a high school education. The exclusion criteria applied to potential study participants further focused the population assessed, relative to the general population of pregnant women. In the likely situation that the prevalence of important modifiers of the exposure

- fetal development association in this focused study population is different from that in other populations of concern, generalizability will be limited. Important modifiers to consider may include time spen t in the home, patterns of food preparation and consumption, health consciousness, the baseline health status of the mother (and father), and a host of socioeconomic determinants. The investigators finding of effect modification by PON1 activity is illustr ative of limits on the generalizability of the study; populations who have high levels of paraoxonase activity may vary by genotypic and phenotypic determinants. These factors were notPage 13 of 105 discussed in the report, but it has been noted previously that there is 10

- to 40

- fold inter

- individual variability in rates of paraoxon hydrolysis (Playfer et al 1976). The potential limitation vis

- à

- vis generalizability is offset by a strength; the study has increased statistical efficiency, relative to one set in
a genera l population.

Because small head size has been found to be predictive of subsequent cognitive ability, the authors conclude that chlorpyrifos may have a detrimental effect on fetal neurodevelopment among mothers who exhibit low PON1 activity. Implicit in this conclusion is the notion that small head size is a good surrogate for fetal neurodevelopment. To support their conclusion, the authors note that head circumference has been shown to correlate with brain weight, and that brain size and head circumfere nces are both predictive of IQ and cognitive ability. They additionally cite research associating brain volume with attention

- deficit/hyperactivity disorder. Although head circumference was significantly associated with cognitive ability, the differences i n head size across tertiles of exposure were notably small ( i.e. less than one centimeter); the clinical relevance of such small changes in the context of even a small amount of measurement error in assessment of head size is unclear.

The authors were no t surprised that infant paraoxonase levels in their study were not associated with birth outcomes because PON1 is not well expressed until after birth, and infant PON1 levels are much lower during the first year of life than maternal levels. The expression of PON1 in infants is approximately one

- third that of their mothers (Chen et al. 2003). They speculate that lower PON1 activity in infants may make them more susceptible to PON1

- related toxicity, including exposure to organophosphate pesticides, relative to adults.

The net effects of confounding, selection bias, and information bias, are unlikely to have qualitatively changed the results of this well conducted, prospective cohort study. Non

- specific biomarkers of exposure, as were assessed in this study, are likely more objective, sensitive, and likely also more accurate and reliable indicators of pesticide exposure, relative to exposure assessment methodologies that rely on self

- report. However, to the extent that prenatal chlorpyrifos is the true exposur e of interest, the assessment of non

- specific organophosphate pesticide metabolites in a single sample of prenatal urine is a primary potential source of exposure measurement error. Almost certainly, such biases would not have resulted in the observation o f a falsely positive association between pesticide exposure and fetal growth. More likely to have occurred is an attenuation of the true (unobserved) associations, toward (observed) associations that are relatively smaller, or null. Indeed, the findings o f this study are either null, or associations of small magnitude. To contextualize these findings, the authors cite literature describing the finding that even small increases in prenatal or early childhood exposures to other environmental toxicants (lead and polychlorinated biphenyls) are associated with significant decrements in IQ and academic achievement up to 11 years of age. The validity of this analogy is speculative. The external validity of the study findings may be limited if the organophosphate
p esticide exposure

- fetal development association is modified by factors that are more, or less, prevalent in this young, urban, minority population, relative to the population(s) to which inference is being made. That said, the decision to focus on this pop ulation is reasonable, as it is a population likely to be highlyPage 14 of 105 exposed, and perhaps also at high risk of cognitive and behavioral deficits enhancing the likelihood of observing an association if an association exists.Page 15 of 105 Works Cited

Breslow NE, Day NE. Statistical Methods in Cancer Research: Volume II: The Design and Analysis of Cohort Studies. Lyon: IARC, 1987.

Chen J, Kumar M, Chan W, Berkowitz G, Wetmur J. Increased influence of genetic variation on PON1 activity in neonates. Environtal Health Persp ectives. 2003;111:1403

– 1409.

Engel SM, Berkowitz GS, Barr DB, Teitelbaum SL, Siskind J, Meisel SJ, Wetmur JG, Wolff MS. Prenatal Organophosphate Metabolite and Organochlorine Levels and Performance on the Brazelton Neonatal Behavioral Assessment Scale in a Multiethnic Pregnancy Cohort. American Journal of Epidemiology. 2007;165:1397

- 1404.

Engel SM, Wetmur J, Chen J, Zhu C, Barr DB, Canfield RL, Wolff MS. Prenatal Exposure to Organophosphates, Paraoxonase 1, and Cognitive Development in Childhood. Environ mental Health Perspectives 2011;119:1182

- 1188

Greenland, S. Modeling and Variable Selection in Epidemiologic Analysis. American Journal of Public Health. 1989; 79:340

- 349.

Miettinen OS. Stratification by a multivariate confounder score. American Journal of Epidemiology 1976;104:609

- 620.

Playfer J, Eze LC, Bullen MF, Evans DAP. Genetic polymorphism and inter

- ethnic variability of plasma paraoxonase activity. J Med Genet. 1976;13:337

– 342.Page 16 of 105 Article 3. Eskenazi et al. (2004) Association of in Utero Organophosphate Pesticide Exposure and Fetal Growth and Length of Gestation in an Agricultural Population. Brenda Eskenazi, Kim Harley, Asa Bradman, Erin Weltzien, Nicholas P. Jewell, Dana B. Barr, Clement E. Furlong, Nina T. Holland. Environmental Health Perspectives. 115:792

– 798 (2007).

Study Summary

In this article by Eskenazi et al, the authors report on the relationship between prenatal urinary organophosphate metabolite levels and fetal growth and gestational duration in the CHAMACOS cohort of Mexic an

- American mothers and their children living Salinas Valley, an agricultural region of California.

Enrollment of the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS) cohort took place at community clinics. A total of 601 out of 1,130 eligible women agreed to participate and were enrolled between October, 1999 and October, 2000, with 538 being followed through to delivery of a live born infant, whose birth weight was measured. The study populations consisted largely of low

- inc ome, agricultural workers. Women considered eligible for the study were less than 20 weeks gestation, aged 18 years or older, Medi

- Cal eligible, fluent in English and/or Spanish, and planning to deliver at Natividad Medical Center.

Excluded from analyses were women with gestational or preexisting diabetes (n = 26), hypertension (n = 15), twin births (n = 5), or stillbirths (n = 3). One additional woman for whom birth weight information was out of range (< 500 g) was also excluded. The final analytic sample consisted of 488 mother

- infant pairs.

Maternal/fetal exposure to organophosphate pesticides was assessed by measurement of organophosphate dialkyl phosphate metabolites (DAPs) and seven different pesticide

- specific metabolites in maternal urine during pr egnancy, including 3,5,6

- trichloro

- 2

- pyridinol (TCPy), a metabolite specific to chlorpyrifos. Cholinesterase (ChE) in whole blood and butyryl cholinesterase (BChE) in plasma collected from mothers during pregnancy, at delivery, and from umbilical cord blo od was also assessed. Maternal urine samples were collected between 5 and 27 weeks gestation and again between 18 and 39 weeks. The dialkyl phosphates measured were dimethylphosphate, dimethyldithiophosphate, dimethylthiophosphate (DMPs), diethylphosphate , diethyldithiophosphate, and diethylthiophosphate (DEPs). Total DAPs and DMPs were available for 485 women; DEP metabolite levels were available for 486 women. Quantification of organophosphate metabolites using gas chromatography

- tandem mass spectrometry and isotope dilution calibration was conducted by the Centers for Disease Control and Prevention (CDC) labs.

Total DAP metabolite level was defined as the sum of the molar concentrations of the six DAP metabolites, dimethylphosphate (DMP) metabolite lev el as the sum of the molar concentrations of thePage 17 of 105 three dimethylphosphate metabolites only, and diethylphosphate (DEP) metabolite level as the sum of the molar concentrations of the three diethylphosphate metabolites. Total DAP, DMP, and DEP levels were det ermined for each participant for each of the two pregnancy urine samples. Individual metabolite levels below the limit of detection (LOD) were assigned a value of the LOD divided by the square root of two, and this value was included in each sum. Total DAP , DMP, and DEP metabolite levels

were log 10 transformed. DAP, DMP, and DEP values were created by averaging the two log

- transformed pregnancy measures. For specific metabolites, if a woman was below the LOD for both measurements, she was classified as belo w LOD for the average. If she was above the LOD at either or both measurements, her measurements were averaged and classified in relation to the median. For 19 women with only one DAP measurement in pregnancy, the single measure was used.

Similar to the analytic treatment of dialkyl phosphate metabolite, authors assigned a value of the LOD divided by the square root of two for TCPy measurements below LOD

. Because a large proportion of women had non

- detectable levels of TCPy, the chlorpyrifos

- specific metabolite was categorized into three groups: TCPy less than LOD for both pregnancy measurements, and, for those with at least one detectable level, subdivided below and above the median of the average pregnancy level.

Infant birth weight, crown

– heel leng th, and head circumference were obtained from hospital delivery lo gs and medical records. Infant p onderal index, a measure of proportionality of growth, was calculated as (birth weight in grams × 100)/(length in centimeters). Gestational age was obtained f rom medical records and was based on ultrasound procedures for 25% of women. Low birth weight was defined as < 2,500 g. Preterm delivery was defined as birth at less than 37 completed weeks of gestation. A “small for gestational age” (SGA) birth was define d as birth weight less than the 10 th percentile for gestational age according to ethnicity, parity, and infant sex.

Multiple linear regression models were developed to assess associations between the markers of pesticide exposure and length of gestation, birth weight, l ength, head circumference, and Ponderal index. Multivariate logistic regression was used to evaluate associations between exposure measurements and low birth weight, preterm delivery, and SGA births. Covariates in the models included includ ing maternal age, pregnancy weight gain, week of initial prenatal care, parity, infant sex, mother’s country of birth, body mass index, and family income. All models of birth weight, l ength, head circumference, and Ponderal index were adjusted for linear and quadratic terms for gestational age.

The women in this study averaged 25 years of age (SD = 5); two

- thirds were multiparous, 80% were married, 79% had not graduated from high school, 58% were overweight or obese, 88% preferred to speak Spanish, and 84 % were born in Mexico, with more than half residing in the United States for < 5 years. Almost all of the women were living within 200% of the poverty level. Very few women reported smoking (6%), drug use (2%), or alcohol consumption (1%) during pregnancy. Approximately 28% of the women had worked in the fields during the pregnancy, and another 14% had worked at other jobs in agriculture. Eighty

- five percent of the women in the study had agricultural workers living in their homes during their pregnancy. The median total DAP, DMP, DEP and total DAP metabolite levels for the studyPage 18 of 105 population were 136 nmol/L, 101 nmol/L, and 22 nmol/L, respectively. Only one woman had no detectable DAP levels and TCPy was detected in 77% of maternal urine samples. The median le vel for TCPy was 3.3 μg/L.

The mean (± SD) duration of gestation was 38.9 ± 1.7 weeks; mean birth weight was 3,449 ± 516 g; mean body (crown

– heel) length was 50.2 ± 2.7 cm; mean head circumference was 34.1 ± 1.5 cm; and me an P onderal index was 2.7 ± 0.3 g /m3. A total of 3.7% (n = 18) of children were born of low birth weight; 4.8% (n = 23) were SGA births, and 6.6% (n = 32) were preterm.

The authors did not observe an adverse relationship between fetal growth and any measure of in utero organophosphate pe sticide exposure. On the contrary, they found positive associations between body length and head circumference associated with some exposure measures, but not chlorpyrifos. They did observe decreases in gestational duration associated with two measures of in utero pesticide exposure: urinary DMP metabolites (β =

– 0.41 weeks per log 10 unit increase; 95% CI:

– 0.75

–– 0.02; p = 0.02), which reflects exposure to dimethyl organophosphate compounds such as malathion, not chlorpyrifos.

Study Review

The study has numerous strengths; most notably the prospective design the longitudinal assessment of both exposure and outcomes. Strengths in the study design also include the use of multiple exposure biomarkers including quantification of non

- specific (DA Ps), chlorpyrifos

- specific (TCPy) metabolites, cholinesterase (acetyl and butyl), and other environmental co

- exposures.

The ascertainment and analysis of urinary organophosphate pesticide markers was appropriate. Quantification was conducted at the CDC, using published methods. DAP metabolites are likely more accurate and objective indicators of organophosphate pesticide exposure, relative to other ascertainment methods such as self

- report. Although they are sensitive indicators of exposure, it is difficu lt to infer chlorpyrifos effects specifically from urinary DAPs. Urinary metabolite levels may reflect exposure not only to a host of organophosphate parent compounds, but also to potentially less toxic environmental metabolites. Also, because exposure to pesticides varies considerably from day to day, DAPs from spot urine samples may not represent average exposure over time. However, t he prenatal exposure measures were, with some exceptions, the average of two measurements, and thus may better reflect chro nic exposure during the pregnancy than a one

- time exposure measure.

Errors in the assignment of exposure in this prospective study will likely have resulted in attenuation of observed associations. The likelihood of these errors is high, and the magnitude of the bias induced difficult to assess. Under an assumption of no other biases, the negative findings, those for the association between children DAP levels and fetal development outcomes, for example, could have resulted from exposure measurement error.Page 19 of 105 To identify “critical windows” of fetal development when exposure may have a greater impact, the authors analyzed the associations of outcomes and metabolite levels measured during moving 6

- week windows of pregnancy ( e.g. , 5

– 10 weeks, 6

– 11 weeks, 7

– 12 w eeks) using a series of multiple regression analyses. No period of greater impact was observed in this largely null study.

The indicators of fetal growth and development (infant birth weight, length, and head circumference, Ponderal index, and gestational age) were obtained from hospital delivery logs and medical records, or calculated using data from these records. They are likely to have been accurately and objectively measured. Although partially self

- reported, any errors in gestational age are unlikely to be associated with pesticide exposures in this prospective study.

The statistical analysis used to assess the associations between the markers of exposure and fetal growth and development were appropriate. However, the authors did not present nor dis cuss regression diagnostics to assess the degree to which their models met or violated the assumptions implicit in linear models ( i.e. homoscedasticity, normality of error term distribution, independence of error terms, and linearity of the exposure

- respon se relationship), other than to state that they log transformed exposure marker levels. The authors also conduct secondary and sensitivity analyses to assess influence of modeling decisions on their findings, and generally found their results to be robust

.

A reasonable set of exclusion criteria were applied which likely resulted in recruitment of a study population that was relatively homogeneous with respect to confounders of the in utero pesticide
exposure

- fetal growth relationship (compared to the gen eral population), and the potential for confounding was thus limited by design. For example, smoking, alcohol use, and illicit drug use were not included in the models because very few women reported use and controlling for these variables did not alter th e results.

Bias due to confounding was also controlled in the analysis, by assessing and adjusting for risk factors for impaired fetal growth and development that are potentially associated with pesticide exposure, including maternal age, pregnancy weight gain, week of initial prenatal care, parity, infant sex, mother’s country of birth, body mass index, and family income. Variables were selected as potential confounders for the multivariate models based on associations reported in the literature, and incl uded in final models if they changed the coefficient associated with the exposure by 10% or more. Inclusion of indicators for environmental tobacco smoke exposure, consumption of caffeinated beverages, history of low birth weight and history of preterm de livery, did not alter the relationship of pesticide metabolites and birth outcome and were not included in final models. In addition to restriction of confounding by design, the selection of the CHAMACOS population, which consists mostly of children from l ow

- income families, also served to increase the relative statistical efficiency of the study, as this population is at high risk of developmental deficits, compared to the general population.

The results of this study by Eskenazi et al (2004) in the CHAMA COS cohort failed to demonstrate an adverse relationship between fetal growth and in utero organophosphate pesticide exposure asPage 20 of 105 assessed by multiple measures of prenatal pesticide exposure. The prospective study design and reasonable methods used protect from certain false

- positive inducing biases, although it is possible that (non

- differential) exposure measurement error attenuated the associations. Misclassification of the fetal growth outcomes was unlikely to have biased the results. Residual confoundin g by mismeasured or unmeasured factors is possible in this observational study, although it is unlikely that such confounding would have resulted in the null finding.Page 21 of 105 Article 4. Harley et al. (2011) Association of Organophosphate Pesticide Exposure and Paraoxonase with Birth Outcome in Mexican

- American Women. Kim G. Harley, Karen Huen, Raul Aguilar Schall1, Nina T. Holland, Asa Bradman, Dana Boyd Barr, Brenda Eskenazi. PLoS ONE 6(8)1

- 10 (2011).

Study Summary

In this article by Harley et al, the authors present a follow

- up assessment of the relationship between prenatal urinary organophosphate metabolite levels and fetal growth and gestational age in the CHAMACOS cohort of Mexican

- American mothers and their chi ldren living Salinas Valley, an agricultural region of California.(Eskenazi et al, 2004) They evaluate whether paraoxonase (PON1), a key enzyme involved in detoxification of organophosphate pesticides, could be an effect modifier of the association between indicators of maternal organophosphorus pesticide exposure (DAP metabolite concentrations) and poorer fetal growth previously reported in this cohort. They additionally examine the main effects of maternal and child PON1 genotypes and PON1 activity on bir th outcomes.

Enrollment of the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS) cohort took place at community clinics. A total of 601 out of 1,130 eligible women agreed to participate and were enrolled between October, 1999 and October, 2000, with 538 being followed through to delivery of a live born infant, whose birth weight was measured. The study populations consisted largely of low

- income, agricultural workers. Women considered eligible for the study were less than 20 w eeks gestation, aged 18 years or older, Medi

- Cal eligible, fluent in English and/or Spanish, and planning to deliver at Natividad Medical Center.

Excluded from analyses were women with gestational or preexisting diabetes (n = 26), hypertension (n = 15), t win births (n = 5), or stillbirths (n = 3), and one infant with a birth < 500 grams. In addition, mother

infant pairs with missing PON1 genotype and activity levels were excluded (n =21), leaving a final sample size of 467. Of these, PON1 genotype informat ion was available for 451 mothers and 436 infants, and PON1 activity data were available for 371 mothers and 324 infants.

Maternal/fetal exposure to organophosphate pesticides was assessed by measurement of organophosphate dialkyl phosphate metabolites ( DAPs). Maternal urine samples were collected between 5 and 27 weeks gestation and again between 18 and 39 weeks. The dialkyl phosphates measured were dimethylphosphate, dimethyldithiophosphate, dimethylthiophosphate (DMPs), diethylphosphate, diethyldithiop hosphate, and diethylthiophosphate (DEPs).

Quantification of organophosphate metabolites using gas chromatography

- tandem mass spectrometry and isotope dilution calibration was conducted by the Centers for Disease Control and Prevention (CDC) labs. Total DAP metabolite level was defined as the sum of the molar concentrations of the six DAP metabolites, dimethylphosphate (DMP) metabolite level as the sum of the molar concentrations of thePage 22 of 105 three dimethylphosphate metabolites only, and diethylphosphate (DEP) metabolite level as the sum of the molar concentrations of the three diethylphosphate metabolites. For eight women, missing metabolite values were imputed using linear regression to predict the missing metabolite level as a function of the other known me tabolites for a given participant at a given time point.

The variables for total DAP, DMP, and DEP concentrations were log

- transformed to reduce the impact of outlier points. Metabolite measurements from the two sampling time points were averaged to crea te a summary measure of in utero organophosphate pesticide exposure.

The primary analyses were conducted using DAP concentrations unadjusted for creatinine, although sensitivity analyses included creatinine as a covariate in the models. Total DAPs and DMP s were available for 485 women; DEP metabolite levels were available for 486 women.

Maternal and umbilical cord blood samples were collected at the time of delivery. Genotyping of the
PON1

- 108 and PON1 192 SNPs was performed using genomic DNA extracted fr om blood clots and Taqman PCR methods.

Two PON1 enzyme activity assays (arylesterase and paraoxonase) were performed using spectrophotometric methods. Arylesterase activity measures the rate of hydrolysis of the substrate phenyl acetate, and reflects the quantity of PON1 enzyme. Paroaxonase activity measures the rate of hydrolysis of paraoxon, and thus reflects a combination of the catalytic efficiency and quantity of the PON1 enzyme.

Birth outcome data, including infant birth weight, length, head circu mference, and gestational age, were abstracted from medical records by a registered nurse.

Linear regression models were constructed to examine the main associations of PON1 genotype and activity with length of gestation, infant birth weight, birth length , and head circumference, independent of pesticide exposure. Maternal and child PON1 genotypes were entered into models as categorical variables; for PON1

- 108 , the CC genotype was the reference, and for PON1 192 , the RR genotype was the reference. Arylester ase and paraoxonase activity in maternal and umbilical cord blood were examined as continuous variables, normalized by dividing by the standard deviation.

The associations of PON1 activity with birth outcomes were assessed within each PON1 192 genotype by stratifying by genotype and by testing for interaction between activity and genotype.

The presence of effect modification of associations between maternal DAP metabolite and birth outcomes by PON1 genotype and activity was assessed by stratification, and use of interaction terms.
PON1 genotype (as a categorical variable) or activity (as a continuous variable) and cross

- product terms were included in models previously used to assess pesticide associations in the 2004 report by Eskenazi et al. (Eskenazi et al, 2004) For PON1 genotype, two cross

- product terms were included in the modelPage 23 of 105 ( e.g. QR*DAP concentrations and RR*DAP concentrations). Similar cross

- product terms were generated using tertiles of PON1 activity. Statistical significance for interaction was set at p=0.1. In addition, effect modification by PON1 was assessed by conducting linear regressions on data stratified by PON1 genotype and by tertiles of enzyme activity.

Models of PON1 genotype included maternal pre

- pregnancy BMI and maternal weight g ain during pregnancy as covariates. Models of PON1 enzyme activity also included maternal age and country of birth, assay temperature, and PON1 192 genotype. In models of birth weight, length and head circumference, the authors also controlled for linear an d quadratic gestational age to examine associations independent of gestational duration.

As previously summarized, women in this cohort were predominantly Spanish

- speaking and born in Mexico; 41% of the women worked in agriculture during pregnancy. More than 60% of families were living below the federal poverty threshold, and 89% of women had never completed high school. The mean maternal age was 25.5 years (SD = 5.0). All women had detectable levels of organophosphate pesticide metabolites in their urine during pregnancy. 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). The allele frequencies of the PON1 192 Q allele and the PON1

- 108 T allele were 50% and 46%, respectively, and did not differ between mothers and newborns. The mean arylesterase and paraoxonase activity was 33.6 U/mL (SD = 16) and 256.6 U/L (SD = 165), respectively, for infants and 136.6 U/mL (SD = 44) and 989.0 U/L (S D = 616) for mothers.

In this study, infants’ (but not mother's) PON1 genotype and PON1 activity were associated with gestational age and head circumference: Infants with the susceptible PON1

- 108 TT genotype had shorter gestational age (  =

- 0.5 weeks, 95% CI:

- 0.9, 0.0) and smaller head circumference (  =

- 0.4 cm, 95% CI:

- 0.7, 0.0) than those with the PON1

- 108 CC genotype. Infants’ arylesterase and paraoxonase activity were positively associated with gestational age. Maternal DAP concentrations were associ ated with shorter gestational age among infants of the susceptible PON1

- 108 TT genotype, although only the interaction between PON1

- 108 genotype and DEP metabolite concentrations was statistically significant (p

- value for interaction = 0.09). However, mate rnal DAP concentrations were associated with larger birth weight (p

value for interaction = 0.06) and head circumference (p

- value for interaction =0.01) in infants with non

susceptible genotypes. The authors conclude that infants with certain PON1 genotype s ( e.g. , PON1

- 108 TT) may be more susceptible to effects of in utero organophosphate pesticide exposure.

Study Review

This study improves on the work by Eskenazi et al (2004) by including an assessment of effect modification of the association between p renatal pesticide marker levels and birth outcomes by PON1 genotype and activity. Additional strengths of this study include the prospective design, the assessmentPage 24 of 105 of pesticide markers at two time

- points during pregnancy, and the assessment of PON1 activi ty by two different methods.

The authors speculate that a possible reason that they did not observe an association between DEPs and gestational age in the earlier study by Eskenazi is that it was “masked” by the effects of PON1 . They note that the observ ation of an interaction only for DEP concentrations is plausible, since the known biologically relevant action of PON1 is with two DEP pesticides (chlorpyrifos and diazinon).

The finding of an independent effect of PON1 genotype on birth outcomes is consi stent with finding of reductions in average gestational age and head circumference at birth associated with the PON1

- 108 TT genotype in the Mount Sinai cohort, as reported by Berkowitz et al. (Berkowitz et al, 2004). The association between infant PON1 (ar ylesterase) activity and gestational age was null in the Mount Sinai cohort. This is in contrast to the positive association reported by Harley et al. Maternal PON1 (arylesterase) activity was not associated with birth outcomes in the Haley study, but posi tively associated with head circumference in the Berkowitz et al study. Finally, the interactions observed in the two studies differed. Berkowitz et al reported a decrease in head circumference associated with chlorpyrifos marker levels among newborns of m others with low arylesterase activity in the Mount Sinai cohort. Harley et al, in contrast, observed a statistically significant interaction between DEP levels and gestational age among infants in the UC Berkeley CHAMACOS cohort with the TT PON1 genotype, but they did not observe significant interaction between PON1 activity and birth outcomes. In addition to measuring different indicators of pesticide exposure (TCPy), the PON1 genotypes and activity assessed for the Mount Sinai cohort differed somewhat fr om those assessed in the UC Berkeley cohort; Berkowitz et al did not assess PON1 192 (only PON1

- 108 ) and paraoxonase activity (only arylesterase activity)

In the Columbia Mothers and Newborn Study (Whyatt et al, 2004), organophosphate pesticide levels in maternal blood were associated with decreased fetal growth, while in the UC Berkeley CHAMACOS study, modest increases in birth weight and head circumference were associated with in utero DAP concentrations. In fact, stratification by PON1 status seemed to enhance these positive associations, such that in the non

- susceptible groups ( e.g. PON1

- 108 CC, PON1 192 RR, and high arylesterase activity), increasing DAP concentrations were significan tly associated with increased birth weight and head circumference.

The finding of a positive association between DAPs and DMPs and both head circumference and birth weight among those with the CT or CC PON1 genotypes, and a positive association between h ead circumference and DAPs among infants with RR PON1 genotypes is perplexing. The authors speculated that it is possible that, among the less susceptible groups, high urinary DAP concentrations may be an indication of rapid detoxification and excretion of organophosphate pesticides, rather than a marker of high exposure.Page 25 of 105 The ascertainment and analysis of urinary organophosphate pesticide markers was appropriate. Quantification was conducted at the CDC, using published methods. However, the use of DAP

meta bolites to measure exposure has some limitations, and these were discussed by the authors. Although they are sensitive and relatively specific indicators of organophosphate exposure, it is difficult to infer chlorpyrifos effects specifically from urinary D APs. Urinary metabolite levels may reflect exposure not only to a host of organophosphate parent compounds, but also exposure to the less toxic metabolites themselves. Because exposure to pesticides varies considerably from day to day, DAPs from spot urine samples may not represent average exposure over time. However, the prenatal exposure measures were, with some exceptions, the average of two measurements taken early and late pregnancy, and thus may better reflect chronic exposure during the pregnancy. Ad ditionally, DAP metabolites in urine may overestimate organophosphate pesticide exposure if they reflect exposure to both pre

- formed DAPs in the environment and the parent organophosphate compounds.

Errors in the assignment of exposure in this prospectiv e study will likely have resulted in attenuation of observed associations. The likelihood of these errors is high, and the magnitude of the bias induced difficult to assess. Under an assumption of no other biases, the negative findings, those for the

assoc iation between children’s DAP levels and attention outcomes, for example, could have resulted from exposure measurement error.

The indicators of fetal growth and development (infant birth weight, length, and head circumference, and gestational age) were obtained from hospital medical records, and ascertained by a registered nurse. They are likely to have been accurately and objectively measured, although the potential for errors in their measurement was not assessed. Although partially self

- reported, any errors in gestational age are unlikely to be associated with pesticide exposures, PON1 genotype, or PON1 activity in this prospective study.

The statistical analysis used to assess the associations between the markers of exposure and fetal growth and dev elopment were appropriate. However, despite a reasonable sample size for assessing main effects, the Harley et al study likely lacked sufficient statistical power to assess effect modification by PON1 genotype and PON1 activity, as the CHACAMOS Cohort stud y was not originally designed to assess interactions between prenatal pesticide exposures and these co

- factors. As a result, the analysis is at risk of failing to detect effect modification if effect modification is truly present.

The authors did not pre sent nor discuss regression diagnostics to assess the degree to which their models met or violated the assumptions implicit in linear models ( i.e. homoscedasticity, normality of error term distribution, independence of error terms, and linearity of the exp osure

- response relationship), other than to state that they log

- transformed DAP levels to reduce the impact of outlier observations. The authors did conduct secondary and sensitivity analyses to assess influence of modeling decisions on their findings, in cluding adjustment for creatinine.Page 26 of 105 A reasonable set of exclusion criteria were applied which likely resulted in recruitment of a study population that was relatively homogeneous with respect to confounders of the relationship between in
utero pesticide e xposure and fetal growth (compared to the general population), and the potential for confounding was thus limited by design. For example, smoking, alcohol use, and illicit drug use were not included in the models because very few women reported use and co ntrolling for these variables did not alter the results.

The authors reported that factors known, a priori , to be associated with birth outcomes in this population were considered potential confounders, although they did not enumerate these variables. Covariates were retained in final models if they were also associated with PON1 genotype or activity (in the study population, presumably) or if their exclusion from the model changed the main effect coefficient by more than 10%. The authors’ final models for birth weight and head circumference adjusted for timing of urine collection, timing of entry into prenatal care, maternal age, parity, country of birth, household income, pre

- pregnancy body mass index, maternal weight gain, infant sex, and gestational age using both linear and quadratic terms. Gestational age models adjusted for timing of urine collection, timing of entry into prenatal care, maternal age, parity, country of birth, and household
income.

The authors controlled for gestational age in mod els of birth weight, length, and head circumference to examine associations independent of gestational duration. It is possible that gestational age is an intermediate on the causal pathway. If this is the case, the coefficients from these models can be
in terpreted as estimates of the ‘direct

- effect’ of prenatal urinary DAPs and fetal growth outcomes due to causal pathways other than those mediated by gestational age. These pathway

- specific analyses have a potential limitation that was not discussed by the authors. Namely, there is the potential for inducing bias if gestational age and fetal growth have unmeasured common causes ( i.e. an unknown co

exposure) (Cole and Hernan, 2002).

In addition to restriction of confounding by design, the selection of the C HAMACOS population, which consists mostly of children from low

- income families, also served to increase the relative statistical efficiency of the study, as this population is at high risk of neurodevelopmental deficits, compared to the general population.

The results of this study by Harley et al in the CHAMACOS cohort demonstrate modest adverse associations between some measures of PON1 and fetal growth, as well as providing some evidence of effect modification of the relationship between organophosphate pesticide exposure markers and fetal growth. The prospective study design and reasonable methods used protect from certain false

- positive inducing biases, although it is possible that exposure measurement error attenuated the associations. Misclassificati on of the fetal growth outcomes was unlikely to have biased the results. Residual confounding by mismeasured or unmeasured factors, including bias induced by adjusting for an intermediate variable (gestational age), is possible in this study, and may accou nt for the observed
associations.Page 27 of 105Page 28 of 105 Work Cited

Berkowitz GS, Wetmur JG, Birman

- Deych E, Obel J, Lapinski RH, Godbold JH, Holzman IR, Wolff MS. In Utero Pesticide Exposure, Maternal Paraoxonase Activity, and Head Circumference. Environmental Health Perspectives. 2004;112:388

– 391.

Cole SR and Hernan MA. Fallibility in Estimating Direct Effects. Int. J. Epidemiol. 2002; 31 (1): 163

- 165.

Eskenazi B, Harley K, Bradman A, Weltzien E, Jewell NP, Barr DB, Furlong CE, Holland NT. Association of
in Utero Or ganophosphate Pesticide Exposure and Fetal Growth and Length of Gestation in an Agricultural Population. Environmental Health Perspectives. 2004;115:792

– 798.

Whyatt RM, Rauh V, Barr DB, Camann DE, Andrews HF, Garfinkel R, Hoepner LA, Diaz D, Dietrich J, Reyes A, Tang D, Kinney PL, Perera FP. Prenatal Insecticide Exposures and Birth Weight and Length among an Urban Minority Cohort. Environmental Health Perspectives. 2004; 112:1125

– 1132.Page 29 of 105 Article 5. Engel et al. (2007) Prenatal Organophosphate Metabolite an d Organochlorine Levels and Performance on the Brazelton Neonatal Behavioral Assessment Scale in a Multiethnic Pregnancy Cohort. Stephanie M. Engel, Gertrud S. Berkowitz, Dana B. Barr, Susan L. Teitelbaum, Jodi Siskind, Stefanie J. Meisel, James G.
Wetmur and Mary S. Wolff. American Journal of Epidemiology. 165:1397

- 1404, 2007.

Study Summary

Engel et al conducted a study leveraging the Children’s Environmental Health Study cohort data to investigate the impact of pesticide exposure on pregnancy outcomes and child neurodevelopment in an
inner

- city multiethnic cohort of women recruited during pregnancy. The study enrolled primiparous women presenting for prenatal care with singleton pregnancies at the Mount Sinai prenatal clinic and two private practices an d who delivered their infants at Mount Sinai Hospital between May 1998 and July 2001. Participants in this study were predominantly young, unmarried, Hispanic and African American women with low educational attainment.

This is the second of three published analyses of prenatal pesticide exposure and child growth and development in the Mount Sinai Hospital Children’s Environmental Health Study . The first (Berkowitz et al 2004) reported on assessments of birth weight, leng th and head circumference. The third (Engel et al 2011) reports on assessments of infant development at age 12 and 24 months and child development at ages 6

- 9 years.

The study population was drawn primarily from East Harlem and consisted largely of youn g Hispanic women (predominantly Puerto Rican), but also included African

- American and Caucasian women. Mothers were enrolled during early pregnancy from the Prenatal Clinic and two private practices at Mount Sinai Hospital from May 1998 to July 2001. The s tudy included only primipar ous with singleton births. Mothers were excluded if they had any of the following: an initial prenatal visit after 26 weeks of gestation, serious chronic diseases, a serious pregnancy complication that could affect fetal growth a nd development; alcohol consumed greater than two alcoholic beverages per day, illicit drug use. Mothers and infants were also excluded if the child was born with a congenital malformation or severe prematurity. A total of 479 prenatal patients were recrui ted; 75 were excluded because of medical complications, premature birth, an infant with birth defects, inability to collect biologic specimens before birth, change of hospital or residence outside New York City, or refusal to continue to participate.

The authors assessed the relationship between total diethylphosphates (DEPs), total dimethylphosphates (DMPs), total dialkylphosphate metabolites (DAPs), as well as malathion dicarboxylic acid (MDA) and indices of abnormal neonatal behavior and/or neurologica l integrity using outcome measures derived from the Brazelton Neonatal Behavioral Assessment Scale (BNBAS), which was administered to the infants of participating mothers within days after birth and before they left thePage 30 of 105 hospital. The authors also assessed modification of the associations between pesticide metabolite levels and metrics of neurodevelopment by maternal paraoxonase 1 activity.

Environmental exposures, sociodemographic characteristics, medical history, and lifestyle factors were assessed by a p renatal questionnaire administered to the mothers during the third trimester. Participating mothers provided urine samples during the 3rd trimester prenatal visit which were provided to the CDC in order to assay six dialkylphosphate metabolites and malathi on dicarboxylic acid (MDA). The investigators imputed values for samples with one or more missing values using methods described by Eskenazi et al. (2004), extending the method to also include imputation of metabolite values for women who were missing two metabolites in a class. Diethyl

- and dimethylphosphate metabolites were summed on a molar basis (as nm/liter) to obtain total diethylphosphates (DEPs) and total dimethylphosphates (DMPs), respectively, and together to obtain total dialkylphosphates (DAP) l evels. All biomarkers except MDA were included in analyses as log 10 linear terms in the initial model selection stage. Results were reported for DEPs dichotomized at the median level. MDA was dichotomized at the limit of detection because only 21.6% of the population had detectable values. Maternal plasma paraoxonase 1 activity was classified in tertiles.

A high prevalence of pesticide use was reported in this cohort of pregnant women residing in New York City. The study cohort was enrolled between 1998 a nd 2002, which, the authors point out, overlaps with a period when chlorpyrifos and diazinon were being phased out of residential use. However, the authors note that in 1997, chlorpyrifos was the most heavily used insecticide by pest control operators

at t he New York City Housing Authority. The authors noted that all of the women who participated in personal air monitoring of their home had detectable levels of diazinon, chlorpyrifos, carbamate propoxur, and o

- phenylphenol. 46% of women in the cohort report ed that pesticides had been applied in their home during their pregnancy. Total diethylphosphates were calculated as the sum of diethyldithiophosphate, diethylphosphate, and diethylthiophosphate. In this cohort, the DEPs were detectable in 88.8% of the mot hers, with a median (IQR) of 24.7 nm/liter (8.9

– 52.8 nm/liter). Total dimethylphosphates were calculated as the sum of dimethyldithiophosphate, dimethylphosphate, dimethylthiophosphate. DMPs were detectable in 90.2% of the mothers, with a median (IQR) of 4 7.8 nm/liter (15.6

– 149.2 nm/liter). Total dialkylphosphates were detectable in 96.5% of the mothers, with a median (IQR) of 82.0 nm/liter (35.2

– 194.7 nm/liter).

To assess infants’ neurodevelopment, the investigators conducted a behavioral assessment using the Brazelton Neonatal Behavioral Assessment Scale (BNBAS). All BNBAS examinations were completed within 5 days of delivery, and 86.1% were conducted within two days of delivery. The BNBAS includes 28 behavioral items and 18 primitive reflexes. The exam does not yield a single score but instead assesses the infant across different developmental areas; infants' behaviors were scored according to seven domains:

1) Habituation

- the ability to respond to and inhibit discrete stimuli while asleep; 2) Or ientation

- attention to visual and auditory stimuli and quality of overall alertness;Page 31 of 105 3) Motor

- motor performance and quality of movement and tone; 4) Range of state

- a measure of infant arousal and state lability; 5) Regulation of state

- the abi lity to regulate state in the face of increasing levels of stimulation; 6) Autonomic stability

- signs of stress related to homeostatic adjustments of the central nervous system; 7) The number and type of abnormal primitive reflexes

Of the 308 infants evaluated, all 18 primitive reflexes assessed in the BNBAS, 64% had all reflexes within the normal range, 18% had one abnormal reflex, 8% had two abnormal reflexes, and 10% had three or more abnormal reflexes. The majority of abnormal reflexes were due to hypotonicity (which includes a reflex not elicited after stimulation). Authors do not discuss the prevalence of other common causes of hypotonicity such as hypothyroidism, hypoglycemia, hypoxic encephalopathy.

The authors observed an association betwee n both generic and pesticide

- specific biomarkers of prenatal organophosphate pesticides and an increased number of abnormal primitive reflexes which are considered a critical marker of neurologic integrity. Notably, no statistically significant adverse

ass ociations were found for the dialkylphosphate metabolites and any measured behavior in the author’s primary analyses.

They did observe, however, an interaction between paraoxonase 1expression levels and total dimethylphosphates on risk of abnormal refle xes, such that infants born to women in the first (interaction p = 0.002) and second (interaction p = 0.01) tertiles (slower metabolizers) had a greater risk of abnormal reflexes than infants of those in the highest tertile (fast metabolizers). In the firs t tertile of paraoxonase 1 expression, the relative risks (RR) of having abnormal reflexes were 1.78 (1.01, 3.14) associated with total diethylphosphates, 1.96 (1.27, 3.03) for total dialkylphosphates, and total dialkylphosphates 2.38 (1.37, 4.15). In the second tertile of paraoxonase 1 expression, the corresponding relative risks (RR) were 1.42 (0.85, 2.35) for total diethylphosphates, 1.66 (1.03, 2.65), for total dialkylphosphates, and total dialkylphosphates 1.75 (0.96, 3.17). No increased risk of abnorm al reflexes with increasing exposure was found for women in the highest tertile of paraoxonase 1 expression, nor was a similar interaction observed between total diethylphosphates and paraoxonase 1.

Higher levels of total diethylphosphates and total dialk ylphosphates, too, were associated with an increase in the risk of having abnormal reflexes. Subjects with prenatal total diethylphosphates levels above the median delivered infants who were 2.3 times more likely to have at least two abnormal reflexes (95% CI: 1.1, 5.0). Among women with lower tertiles of paraoxonase 1 expression ( i.e. , slow organophosphate pesticide metabolizers) the authors also observed a significantly increased risk of abnormal reflexes with increasing exposure.

Relative to the first quartile, quartiles 2

– 4 of total diethylphosphates, total dimethylphosphates, and total dialkylphosphates were associated with an increased proportion of abnormal reflexes, althoughPage 32 of 105 the associations did not increase monotonically and varied in their streng th and precision. The authors speculated that this may due to a threshold effect.

With the exception of MDA, there appeared to be stronger associations between metabolite levels and abnormal reflexes for examinations performed after the first day of life, although only the total dimethylphosphates interaction p

- value interaction = 0.10. In contrast, an interaction (p interaction = 0.10) with MDA showed that, for infants examined before day 2, the relation between MDA and abnormal reflexes was stronger (RR= 2. 51, 95% CI: 1.61,3.90) compared with that for infants examined later (RR= 1.34, 95%CI: 0.72, 2.49). The authors noted that their findings were remarkably consistent with those of Young et al. (Neurotoxicology, 2005) who reported that for each log 10 unit i ncrease in total dialkylphosphates, there was a 26% increased risk of abnormal reflexes, (compared to 32% increased risk in this study). The magnitudes of association were also similar for total diethylphosphates (RR Young = 1.25, RR current study = 1.49) an d total dimethylphosphates (RR Young = 1.20, RR current study ) = 1.13). Both studies reported stronger associations between dialkylphosphate metabolites and abnormal reflexes for older children, although, in the present study, the interaction with day of adm inistration was not statistically significant.

The association between MDA, a malathion

- specific metabolite, and abnormal reflexes appeared to be age dependent in this study, with the most pronounced effect observed among infants evaluated within the firs t day of life. The authors noted that this finding conflicts with the suggestion of later effects for dialkylphosphate metabolites, including dimethylphosphates, which encompasses nonspecific metabolites of malathion. The authors concluded that it is uncle ar what pesticides may be driving this disparity.

Study Review

This was a well conducted prospective study conducted in a young, predominantly minority population. The study design, analytic approach, and statistical analyses were appropriate. The author s used a sensitive albeit non

- specific marker of chlorpyrifos pesticide exposure (DEP/DAP). While the analytic methods appear sound, authors assessed these markers using only a single maternal urine specimen taken during the third trimester. However, in th eir discussion, the authors note that organophosphate pesticide metabolites have short half

- lives. If sources and patterns of exposure remain static, a single measurement would be a good indicator of pesticide exposure during pregnancy. If, however, source s and exposure patterns change over short time

- spans, perhaps on the order of days to weeks, such an exposure assessment would not necessarily provide a good indicator of the relevant exposure. Like the many other sources of error possible in this study, t his, too is likely to result in a non

- differential misclassification of the exposure, with respect to the disease outcome. Serial measurements of urinary biomarkers of pesticide exposure in the third trimester would shed light on these patterns, although t he benefits of such information are offset by the added cost, logistical complexity expense, and burden on the study participants.Page 33 of 105 The Neonatal Behavioral Assessment Scale (NBAS), used to ascertain the health endpoints in this study, is designed to exami ne individual differences in newborn behavior ( http://www.brazelton

institute.com/intro.html ). Examinations took place in a quiet, semi

- darkened, warm room adjacent to the neonatal nursery, or in the mother’s private room. Examiners in this study were either trained and certified by the Brazelton Institute or trained by a certified examiner. It was not specified how many examinations were conducted by the certified examiners, nor how recently the c ertified examiners had received their certification. Procedures for tracking the quality of the assessment process were also not discussed in the report. The health outcome assessment methodology used in this study is less than objective; assessments rely on substantial interaction between the infant and the examiner. Procedures for ensuring that the examiners were blinded to the participants’ exposures were not discussed in the article. However, authors did include examiner as an indicator variable in st atistical analyses to control for slight differences in examiner methodology.

Moreover, the behavioral assessment was administered before hospital discharge only on a subset of children in the cohort (n =311/404). The BNBAS was not administered if the in fant was admitted to the NICU (n = 21); if the infant was delivered and discharged over a weekend (n = 43); if the parent refused (n = 5); if the infant was not testable (n = 2); or if study personnel were unavailable (n = 22). Factors related to weekend d elivery ( e.g. , fewer inductions) would be underrepresented among the tested subjects. This missing data has the potential to result in a selection bias, reduce the degree of precision with which associations were estimated, and limit the generalizability o f the study findings. However, the authors’ explanation for the incomplete ascertainment is presented and seems appropriate and largely logistical in nature.

The authors employed a reasonable set of exclusion criteria that likely resulted in recruitment o f a study population that was relatively homogeneous with respect to confounders of the in utero pesticide
exposure

- neurodevelopment relationship (compared to the general population). The potential for confounding was thus limited by design (restriction). Bias due to confounding was also controlled in the analysis, by assessing and adjusting for risk factors for neuromuscular functioning that are potentially associated with pesticide exposure, including maternal age, race, marital status, education, cesarea n delivery, delivery anesthesia, infant age at examination, infant gender, infant jaundice, and smoking, alcohol consumption, caffeine consumption, or illicit drug use during pregnancy, and the examiner conducting the BNBAS.

The use of an automated stat istical procedure (backward elimination and change of effect measure) as a means to eliminate potential covariates is discouraged in some epidemiologic circles on the basis that assessment of confounding is generally not considered to be a statistical ende avor, although they continue to be used by others, largely on the grounds that it is ‘objective’. The authors used a change

- in

estimate approach, rather than a reliance on the statistical significance (p

- value) associated with a covariate; their variable s election rule has been shown to produce more reliable models than variable selection methods based on statistical significance (Greenland, 1989). Because they used a backward elimination procedure, they considered confounding by a given risk factor in the context of adjustmentPage 34 of 105 for other covariates. The approach to covariate selection is a minor consideration in this study, and different approaches are not likely to qualitatively change the study findings or conclusions. A robust discussion of covariate sele ction concepts can be found in Chapter 15 of Modern Epidemiology (Kenneth J. Rothman, Sander Greenland, Timothy L. Lash).

The authors noted that post

- entry exclusions/loss

- to follow

- up in the study may have biased their findings if they were associated wi th both organophosphate pesticide levels and neonatal behavior. Missing data has the potential to induce a selection bias if the reasons for missingness are determined by exposure (or a predictor of exposure) and are also not independent of neurodevelopmen t. Most of the excluded women were those who moved out of the study area, were lost

- to

- follow up, or lacked prenatal biologic specimens, which largely reflect socioeconomic condition. Participants included and excluded differed slighted with regard to mate rnal age and race; however, these factors were not associated with number of abnormal reflexes in this study. These considerations may, however, limit the generalizability of the study findings.

The statistical analysis used in the study was appropriate , although several subtleties in the analytic procedures are worthy of mention. The main finding of the study was a statistical interaction between biomarker levels of organophosphate pesticides and paraoxon activity. It was not clear whether this effect m odification was consideration a priori . Also, the authors imputed missing data, and this may 1) affect the precision of association estimates and 2) result in attenuated effect estimates as a result of exposure measurement error. The description of the imp utation procedure used was insufficient for drawing strong conclusions regarding its validity with respect to the magnitude of measurement error/bias, and its effect on estimates of precision.

The generalizability of the study may be limited as consequen ce of the particulars of the study population assessed and the numerous exclusion criteria applied. This study population consisted of primiparous women, recruited early on in their pregnancies from the prenatal clinic and two private practices at Mount Si nai Hospital in New York from March 1998 to March 2002, consisted predominantly of young Hispanic women with low education. Sixty

- eight percent were less than 25 years old. Just over half were Hispanic (51.1%), although substantial numbers of White (20.3%) and African

- American (27.3%) women also participated. Just under half (47.6%) classified themselves as being single. The exclusion criteria applied to potential study participants further focused the population assessed, relative to the general population of pregnant women. In the likely situation that the prevalence of important modifiers of the exposure

- fetal development association in this focused study population is different from that in other populations of concern, generalizability will be limited. Important modifiers to consider may include time spent in the home, patterns of food preparation and consumption, health consciousness, the baseline health status of the mother (and father), and a host of socioeconomic determinants including access to heal th care. The investigators finding of effect modification of the biomarker

- fetal growth relationship by PON1 activity is illustrative of limits on the generalizability of the study; populations who have high levels of paraoxonase activity may vary by genet otypic and phenotypic determinants. These factors were not discussed in the report, but it hasPage 35 of 105 been noted previously that there is 10

- to 40

- fold inter

- individual variability in rates of paraoxon hydrolysis (Playfer et al 1976).

The primary finding was a n interaction between high paraoxonase 1expression levels and organophosphate pesticide exposure, whereby fast metabolizers had lower risk of abnormalities associated with prenatal pesticide metabolite levels in maternal urine samples. As with their previo us report, bias due to confounding, and selection of study participants is unlikely to have large and qualitative impact on the association observed. To the extent that prenatal chlorpyrifos is the true exposure of interest, the assessment of non

- specific organophosphate pesticide metabolites in a single sample of prenatal urine is a primary potential source of exposure measurement error. Because of the prospective design, information bias is expected to result in attenuation of the true (unobserved)
associ ations, toward (observed) associations that are relatively smaller, or null.Page 36 of 105 Works Cited

Berkowitz GS, Wetmur JG, Birman

- Deych E, Obel J, Lapinski RH, Godbold JH, Holzman IR, Wolff MS. In Utero Pesticide Exposure, Maternal Paraoxonase Activity, and Head Circumference. Environmental Health Perspectives. 2004;112:388

– 391.

Breslow NE, Day NE. Statistical Methods in Cancer Research: Volume II: The Design and Analysis of Cohort Studies. Lyon: IARC, 1987.

Engel SM, Wetmur J, Chen J, Zhu C, Barr DB, Canf ield RL, Wolff MS. Prenatal Exposure to Organophosphates, Paraoxonase 1, and Cognitive Development in Childhood. Environmental Health Perspectives 2011;119:1182

- 1188

Greenland, S. Modeling and Variable Selection in Epidemiologic Analysis. American Journal of Public Health. 1989; 79:340

- 349.

Miettinen OS. Stratification by a multivariate confounder score. American Journal of Epidemiology 1976;104:609

- 620.

Playfer J, Eze LC, Bullen MF, Evans DAP. Genetic polymorphism and inter

- ethnic variability o f plasma paraoxonase activity. J Med Genet. 1976;13:337

– 342.

Rothman KJ, Greenland S, Lash TL. Modern Epidemiology. Third Edition. Lippincott, Williams, & Wilkins,
2008.Page 37 of 105 Article 6. Young et al. (2005) Association Between In Utero Organophosphate Pestic ide Exposure and Abnormal Reflexes in Neonates. Jessica G. Young, Brenda Eskenazi, Eleanor A. Gladstone, Asa Bradman, Lesley Pedersen, Caroline Johnson, Dana B. Barr, Clement E. Furlong, Nina T. Holland. NeuroToxicology. 26:199

– 209 (2005).

Study Summary

Young et al. assessed the relationship between in utero and early postnatal organophosphate pesticide exposure and neonatal neurobehavior in the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS) cohort. Urinary levels of dialky lphosphate (DAP) metabolites, including dimethyl and diethylphosphate metabolites, measured at approximately fourteen and twenty

- six weeks gestation, and once post

- delivery, measured at approximately seven days postpartum were assessed as markers of exposu re to organophosphate pesticides. Cognitive development was assessed using the Brazelton Neonatal Behavioral Assessment Scale (BNBAS).

The CHAMACOS study is a longitudinal birth cohort study of environmental exposures and the health of pregnant women and their children living in the Salinas Valley, California. The Salinas Valley is a major center of agricultural production in the United States with approximately 500,000 pounds of organophosphate pesticides applied annually, according to the CDC. (Califor nia EPA. Pesticide Use Reporting 2001 Summary Data, 2002. (www.cdpr.ca.gov/docs/pur/pur01rep/01_pur.htm) Pregnant women initiating prenatal care at the Natividad Medical Center, a county hospital located in the city of Salinas, were recruited to participa te in the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS) project. Both the source and study populations consisted largely of low

- income, Latina agricultural workers. Women considered eligible for the study were less than 20 weeks gestation, aged 18 years or older, Medi

- Cal eligible, fluent in English and/or Spanish, and planning to deliver at Natividad Medical Center. A total of 601 women were enrolled between October, 1999 and October, 2000, with 528 followed through deliver y of a live born infant. The BNBAS was performed on 421 infants. Eight twins and 27 preterm infants were excluded from analysis.

Exposure was assessed by measurement of organophosphate dialkylphosphate (DAP) metabolites in maternal urine samples collected between 5 and 27 weeks gestation and again between 18 and 39 weeks. Maternal urine specimens were generally collected at the time of the prenatal and post

- delivery interviews. The post

- delivery urines were collected within 1 week of delivery for 73% of th e sample, with the remainder obtained up to 176 days afterwards. Quantification of metabolites was conducted by Centers for Disease Control and Prevention (CDC) labs. Total DAP metabolite level was defined as the sum of the molar concentrations of the six DAP metabolites, dimethylphosphate (DMP) metabolite level as the sum of the molar concentrations of the three dimethylphosphate metabolites only, and diethylphosphate (DEP) metabolite level as the sum of the molar concentrations of the three diethylphospha te metabolites. Total DAP, DMPT, and DEP levels were determined for each participantPage 38 of 105 for each of the two pregnancy urine samples and for the post

- delivery urine sample. Individual metabolite levels below the limit of detection (LOD) were assigned a value o f the LOD divided by the square root of two, and this value was included in each sum. A small number of missing metabolite values were imputed using regression to predict the missing metabolite level as a function of the other known metabolites for given p articipant at a given time point. The total DAP, DMP, and DEP metabolite levels were then log 10 transformed.

For 30% of participating infants, developmental outcomes were assessed prior to discharge from the hospital; the remainder was assessed at the CHA MACOS research office or in the participant’s home. Assessments were performed away from the mother in a private room with low

- level light and noise control. The BNBAS, consists of 28 behavioral measurements scored on a nine

- point scale and 18 reflex
items scored on a four

- point scale. These scores were classified into seven clusters: habituation, orientation, motor performance, range of state, regulation of state, autonomic stability, and reflex. Higher scores on the behavioral clusters indicate more opti mal functioning while higher scores on the reflex cluster indicate less optimal functioning. Missing individual BNBAS items for the six behavioral clusters were imputed as the median value of known scores within the cluster associated with the missing item . If all items in a particular cluster were missing, then the cluster score was considered missing. Missing individual reflex items comprising the reflex cluster were not imputed.

Separate linear regression models were fit for six of the seven clusters of the BNBAS. Poisson regression was used to model the association between urinary metabolite levels and number of abnormal reflexes. Independent variables included the average of prenatal metabolite levels, the postnatal level, and relevant covariates. Mod els relating metabolite levels to each cluster score were initially fit including the postnatal metabolite level and the average of the two prenatal metabolite levels. For eighteen mothers missing one of the two pregnancy urine samples, the one remaining w as used to represent the average. All models adjusted for covariates, and were rerun with creatinine

- adjusted metabolite levels. Analyses were performed for the entire study sample and stratified by the median age at BNBAS assessment (3 days).

Of the 378 infants included in this analysis, 47.5% were male. The mean maternal age was 26±5 years. Most mothers lived at or below the poverty line (64%), were born in Mexico (85%), had previous live births (68%), were married (81%), and had not completed high scho ol (81%). Few women smoked (5%) or drank alcohol (16%) during pregnancy, although most reported caffeine use (75%). Only 1% of the sample reported any illicit drug use during pregnancy.

The authors observed a statistically significant association betwee n organophosphate pesticide exposure and the reflex cluster of the BNBAS, both for the entire sample and for infants assessed at greater than 3 days of age. 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: β = 0.53, 95% CI = 0.23, 0.82; DMP: β = 0.41, 95% CI = 0.12, 0.69; DEP: β = 0.37, 95% CI = 0.09, 0.64), and the proportion of infants with more than three abnormal reflexes (total DAP: OR = 4.9, 95% CI = 1.5, 16.1; DMP: OR =Page 39 of 105 3.2, 95% CI = 1.1, 9.8; DEP: OR = 3.4, 95% CI = 1.2, 9.9). No other associations between either prenatal or postnatal DAPs with any other developmental outcomes were observed. Study Review

Overall, authors performed a w ell

- conducted study of pre

- and post

- natal pesticide exposure and adverse neurodevelopmental outcomes. The prospective nature of the study, use of multiple biomarkers of exposure, and extensive data collection allowing for adjustment of potentially confoun ding variables are key strengths of this work. For the purpose of informing the nature of the relation between chlorpyrifos per se and these outcomes, use of non

- specific OP metabolites is a disadvantage. Other limitations of this work are also noted.

Th e ascertainment and analysis of urinary organophosphate pesticide markers was appropriate. Quantification was conducted at the CDC, using published methods. DAP metabolites are sensitive albeit

non

- specific markers of organophosphate pesticide exposure. Th ey are likely more accurate and objective indicators of organophosphate pesticide exposure, relative to other ascertainment methods such as self

- report. In their discussion, the authors note that, “presently, there are no better methods of
exposure assessm ent to capture exposure to multiple organophosphate compounds.” It is difficult to infer chlorpyrifos effects specifically using metabolite levels, as they also indicate exposure to other organophosphates. The study population was likely sustained many oth er potentially relevant exposures to multiple chemicals and other pesticides which may affect development. It is not possible to disentangle the various effects from these potential co

- exposures and chlorpyrifos in this study. Moreover, the degree to which metabolites in prenatal maternal urine samples reflect ‘usual’ levels, and thus usual exposure, is uncertain. The authors’ observation of large within

- mother variability in pesticide marker levels between the two prenatal sampling periods is evidence that a single sample may not be representative of longer

- term levels in this population, particularly when exposures are transient and highly variable. Averaging of the two prenatal exposure measures may ameliorate this issue somewhat.

Metabolite levels in po stnatal urines likely reflect early postnatal, rather than in utero exposure, given the short half

- life of organophosphate pesticides. This is particularly likely for the 27% of postnatal samples collected greater than one week after delivery. The effect o f errors in the assessment of organophosphate pesticide exposure, whether due to mismeasurement of the metabolites, imperfect correlation between metabolite levels and pesticide exposure, or imputation of missing data, is likely to be an attenuation of the association (a bias toward no association) in this prospective study.

The BNBAS was appropriately administered by one of four examiners trained by a BNBAS certified trainer. BNBAS is considered to provide an appropriate evaluation of newborn development when administered up to the second month of life, and is considered a valid tool, when properly conducted. The cluster approach to classification of BNBAS scores has precedence in the literature. Missing BNBAS data points were imputed, if possible. Indeed, there was a substantial amount of missing outcome data, due largely to the particular requirements of the assessment ( e.g. that the infant be asleep immediately prior to the assessment of habituation and that the infant by crying prior to assessment of co nsolabilityPage 40 of 105 and self

- quieting). For example, fifty

- four percent of the sample was missing all habituation items and an additional 22% required at least one imputation. Forty percent of the sample required at least one imputation of the four items comprisin g the regulation of state cluster, with 13, 25, and 0.5% of the sample requiring one, two, and three items imputed, respectively. Individual items with the most missing values for the regulation of state cluster were consolability and self

- quieting. Two in fants were missing all items comprising the orientation cluster score. The number of abnormal reflexes was calculated based on non

- missing values only. Reflexes with the most missing values included incurvation (9%), nystagmus (7%), and tonic neck reflex ( 7%). Missing values did not exceed 4% for the remaining reflex items. Again, the effect of errors in the ascertainment of developmental outcomes is likely to result in attenuation of observed associations as it is unlikely that missingness in outcome measu re is related to pesticide exposure, as examiners were blind to the infant’s exposure status and the imputation procedures were unlikely to induce bias.

The analyses presented were appropriate to the data collected and the research question posed. The

au thors averaged participants’ pesticide marker levels from the two prenatal urine samples (if available), and, as a separate variable, the postnatal metabolite level. Sixteen mothers were missing the post

delivery urine. In order not to exclude those with missing post

- delivery urines from the statistical models, results were re

- examined with the variable representing post

- delivery metabolite level excluded from all models. The effect of the missing exposure data is a decrease in the precision, and also like ly attenuation of observed associations due to measurement error.

Potential confounders considered in this investigation included primarily predictors of BNBAS performance, including: maternal age, pre

- pregnancy body mass index, self

- reported smoking, al cohol, caffeine, and illicit drug use during pregnancy, vitamin use during pregnancy, gestational age at which prenatal care was initiated, total number of prenatal care visits, average blood pressure during pregnancy, parity, method of delivery, general a nesthesia use during delivery, breastfeeding initiated after delivery, and poverty level. Other covariates included infant sex, age in days at BNBAS assessment, minutes since last fed at BNBAS, and BNBAS examiner. Missing covariate values were imputed by random selection of an individual among all participants with known values for the covariate and assigning the missing value the selected participant’s value.

Covariates were selected for inclusion in final models if they were statistically significantly associated with each of the seven cluster scores in univariate models, (alpha = 0.15). The authors used a less conservative alpha level as a cut

- off for selection of covariates, a somewhat more conservative approach to covariate selection, relative to us e of the conventional alpha level of 0.05. However, the use of a statistical test as a means to select covariates from a larger set of potential confounders is discouraged in some epidemiologic circles (Miettinen 1976, Breslow and Day 1987, Greenland 1989) . The assessment of confounding is generally not considered to be a statistical endeavor, in part because the association between the confounder and the outcome is just one factor that determines the degree of confounding, and also because the statistical significance of an association is a function not only of the magnitude of the association but also of other factors ( e.g. , sample size, and sample variability) whichPage 41 of 105 are irrelevant to bias due to confounding. Moreover, the use of significance testing in t he selection of confounders treats false negative errors ( i.e. the deleting a confounder) as secondary to false positive error (inclusion of a non

- confounder). This implicit ranking of error is backward, as deleting a confounder introduces bias and is just ified only if the bias induced by its exclusion is tolerably small or deemed to be worth the precision gain, whereas including a non

- confounder only (potentially) reduces precision by increasing the size of the model. This is a small consideration in this study, and different approaches are not likely to qualitatively change the study findings or conclusions. Birth weight and gestational age were considered potential causal intermediates in this relation, and not confounders.

Regardless of the confounder e valuation conducted by the investigators, residual confounding due to poorly measured or unmeasured confounders is likely to have influenced the observed associations. Missing covariate data were imputed. The resulting analysis is relatively more precise, owing to the larger sample size analyzed, but this statistical efficiency comes at the trade off of bias due to residual confounding by imperfectly measured covariates. Socioeconomic conditions and other difficult to measure determinants of early life deve lopment may further confound the reported associations in the direction of more deleterious effects associated with the organophosphate metabolites and with unknown magnitude. The potential for this bias may be mitigated somewhat due to restriction in the variability of these confounding factors in this relatively homogeneous study population.

The generalizability of the findings to other populations may be limited, due to the particular nature of this study population with respect to factors which may m odify the association between organophosphate exposures and infant development.

The results of this investigation are suggestive of a detrimental association between prenatal organophosphate exposure, as measured by maternal urinary metabolite levels, a nd abnormal reflexes, as measured by the BNBAS, particularly in infants assessed after the first 3 days of life. This study may be limited in its direct contribution to the literature base regarding the specific association between chlorpyrifos and develop mental outcomes; although approximately 500,000 pounds of organophosphate pesticides are applied annually in Salinas Valley, chlorpyrifos constitutes only a small percentage of the pesticides applied. Although the prospective study design and reasonable me thods protect from certain false

- positive inducing biases, there remains the potential of residual confounding in the direction of an adverse effect on development by unmeasured factors.Page 42 of 105 Article 7. Rauh et al. (2006) Impact of Prenatal Chlorpyrifos Exposure on Neurodevelopment in the First 3 Years of Life Among
Inner

- City Children. Virginia A. Rauh, Robin Garfinkel, Frederica P. Perera, Howard F. Andrews, Lori Hoepner, Dana B. Barr, Ralph Whitehead, Deliang Tang, Robin W. Whyatt. Pediatrics. 18(6): 1835

- 1859.

Study Summary

To evaluate the affect/impact of prenatal exposure to ambient and indoor pollutants including pesticides, the investigators conducted a prospective cohort study among inner

- city (NYC) mothers and

t heir babies born between February 1998 and May 2002. A major aspect of the protocol was to assess pesticide exposure, specifically chlorpyrifos, and its impact on pregnancy outcome, neurodevelopment, and behavior. Previous publications presented outcomes of this cohort such as birth weight and length findings (Whyatt, et al, 2004 and Perera et al, 2003.) This publication presents findings focused on the neurodevelopmental assessments made at the 12, 24, and 36 month time points.

The women enrolled into the study were identified during their pregnancy, through the New York Presbyterian Medical Center and Harlem Hospital. To be eligible for the study they were required to be between the ages of 18 and 35 years, non

- smokers (later verified by blood cotini ne levels,) and without a history of diabetes, hypertension, HIV infection, or drug abuse. They must have sought prenatal care by the 20 th week of pregnancy and self

- identified as African American or Dominican. At the time of this report, the authors expl ain that out of the original 648 pregnant women consented, 536 (83%) of them and 254 children were still actively enrolled at the 3 year milestone. Of these children, 228 completed evaluations at 12 months of age, 227 at 24 months, and 228 at 36 months, w ith 189 children having full data at all 3 time points.

The primary source of chlorpyrifos measurements were samples of cord blood collected at delivery as well as samples of maternal blood collected shortly after (within 2 days of) delivery. These serum levels were supplemented by maternal self

- report of residential pesticide use during the third trimester of pregnancy. Using findings from previous analyses of chlorpyrifos effects on birth weight (Whyatt, et al, 2004 and Perera et al, 2003,) the in vestigators chose a cord blood chlorpyrifos limit of 6.17pg/g to define the exposure groups. Children were categorized as having either high (>6.17pg/g) or low ( < 6.17pg/g) prenatal exposure.

Two instruments were used to evaluate neurodevelopment and ch ild behavior. The Bayley Scales of Infant Development II (BSID

- II) was used to generate a Mental Development Index (MDI) and a Psych omotor Development Index (PDI) at 12, 24, and 36 months of age. Second, the behavior of the children was described by the mother using the 99

- item Child Behavior Checklist (CBCL) and the resulting syndrome scale scores. Finally, the Home Observation for Measurement of the Environment (HOME) instrument was used to assess the quality of the child

- care environment. This instru mentPage 43 of 105 collects data regarding the physical and interactive qualities of the child’s home as a measure of mental stimulation and interactions.

The maternal and cord blood samples also provided cotinine levels to confirm maternal report of environmental toba cco smoke (ETS) exposures and lead levels to understand the role lead exposure may play interacting with the outcomes of measure. During their third trimester of pregnancy, the women completed a 45

- minute questionnaire to collect demographics, education a nd occupational history, income, active and passive smoking, alcohol and drug use during pregnancy, and residential pesticide use. From this self

- report questionnaire and additional analyte measurement in biological sample, authors obtained information reg arding potentially confounding variables.

Multivariate linear regression and logistic regression were used to estimate associations between prenatal chlorpyrifos exposure and developmental outcomes. General linear modeling of the repeated measures (12, 24, 36 month evaluations) was also used to estimate the effect over time that chlorpyrifos had on neurodevelopment. All models included prenatal ETS exposure, gender, ethnicity, gestational age at birth, quality of home care

- taking environment, maternal education, and maternal IQ. Other measured co

- variables including blood lead levels were evaluated as to the potential confounding influence on the main effect, and were not included in final models due to lack of statistical or biological evidence of conf ounding bias.

The descriptive statistics present the cohort as relatively homogenous with respect to socioeconomic variables but showing a wide range of chlorpyrifos exposure. Chlorpyrifos levels ranged from undetectable to 63 pg/g with 20.6% of the cord blood results falling into the high exposure category. Significant differences in chlorpyrifos exposure were seen across ethnic categories with 24.2 % of African American participants and 14.9% of Dominican participants classified as highly exposed (p=.0 2). Birth weight and prenatal exposure to ETS were also significantly associated with the exposure categories with infants in the high exposure group weighing approximately 211g less than babies in the low exposure group (p <.05) and 56% of those in the high exposure group reporting prenatal ETS versus 32% in the low exposure group (p = .002).

When the BSID

- II results were analyzed in terms of exposure group, no significant differences were seen at the 12 and 24 month time points. At the 36

- month milest one, although MDI scores did not show a significant difference across the exposure groups, PDI scores were significantly lower among children in the highly exposed group (95.69 vs. 101.63, p = .006). Statistically significant differences were also seen

at the 36 month milestone, in mental (cognitive) and psychomotor delays (p = .048 and .002 respectively.)

When analyzing the 36

- month child behavior checklist (CBCL) (behavioral) scores, results from the 12 and 24 month milestones were not significantly associated with exposure status. At 36 months, significant differences were observed in the general category of attention

- problems (p=.010) and in the more specific DSM

- IV scale for ADHD problems (p = .018).Page 44 of 105 The multivariate regression models included gender, ethnicity, gestational age at birth, home environment, prenatal ETS exposure, maternal education, and maternal intelligence. The authors reported that the difference in MDI scores was “marginally significant” (p = .06) with the exposed group
scori ng an average of 3.3 points lower. Among the significant covariants were race/ethnicity (at 24 and 36 months), gender of infant (at 12, 24, and 36 months), gestational age (at 24 and 36 months), low maternal education (at 36 months), and HOME score (at 36 months) with Dominicans, male children, low gestational age, low maternal education (< high school,) and low HOME score all linked to lower MDI scores. It was also reported that environmental exposures (chlorpyrifos and ETS) and sociodemographic covariab les accounted for 25% of the variance seen in the 36

- month MDI scores. When the same multivariate regression models were calculated regarding the PDI scores, none of the 12 or 24 month PDI scores showed significant effects. The 36 month scores did show s ignificant effects for chlorpyrifos exposure, race, and gestational age.

Next, investigators calculated estimates of adjusted risk for developmental delays (MDI and PDI) related to chlorpyrifos exposure. Adjustments were made for gestational age, gende r, ethnicity, maternal education, maternal intelligence, and home environment. The findings showed that before 36 months of age, delays were no more likely in the highly exposed group. But, at the 36 month milestone, the likelihood of highly exposed chil dren developing mental delays were 2.4 times greater (95% CI: 1.12

- 5.08, p = .02) and motor delays were 4.9 times greater (95% CI: 1.78

- 13.72; p = .002) than those with lower prenatal exposure.

General linear models (GLM) were fit to describe longitudin al developmental trajectories for high and low chlorpyrifos exposure groups, adjusting for race/ethnicity, gender, gestational age, maternal educational level, HOME score, and ETS exposure. The GLM results were consistent with the 12, 24, and 36 month lin ear regression analyses indicating that associations between developmental scores and chlorpyrifos were most pronounced at 3 years of age. The authors describe an initial decrease in scores in both groups between the ages of 12 and 24 months, but remark th at such decreases are commonly observed among low

- income groups followed by a rebound in scores after 24 months of age. The rate of increase in the MDI scores after 24 months was greater among the low exposure group than the high exposure group but was no t significantly greater (p = .23). 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 = .01).

The authors summarize three main findings from the study: 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 o ver time; and 3) by age 3, highly exposed children were more likely to demonstrate clinically significant attention problems.

Study ReviewPage 45 of 105 The investigators conducted a well

- designed prospective cohort study and retained a high percentage (83%) of their participating mothers over the three year follow

- up period. With very narrow inclusion criteria, the investigators selected a homogenous cohort of minority, inner

- city women, most of whom fall into low income and low education categories. This homogeneit y serves to equalize the study comparison groups with regard to potential measured and unmeasured confounders, but may also limit the generalizability of the findings.

While the high retention rate of the mothers provides as stable study population, the authors do not address that only 53% of the children from these mothers reached the three year milestone with study data collected. It is unclear what, if any, percentage of these children did not survive, were lost to follow

- up, or were too sick/affecte d by neurodevelopmental delays to participate. Authors did not provide information concerning the degree of missingness by chlorpyrifos exposure category in the published analyses, however in supplemental analyses suggested by the 2008 FIFRA SAP on chlorp yrifos authors did provide this information (See Appendix 4

- 1). In these additional analyses, authors concluded the degree of missingness was relatively equal by exposure category and therefore not likely to have imposed a selection bias.

As discussed by the authors, one weakness of the study is the reliance on a single exposure level (prenatal/cord blood.) It is acknowledged that this snapshot of exposure may not accurately reflect both pre

- and post

- natal exposure levels, although in general, for chron ic exposures with a high prevalence and consistent frequency of use as indicated in this study population, a single measure may be sufficient to accurately rank participant exposure levels. The authors further explain that previous analyses of this cohort showed that exposures to chlorpyrifos did not show significant variation throughout pregnancy (Whyatt et al., 2007) and therefore concluded that this single measure can accurately represent prenatal exposure. While this may accurately represent prenatal exposure, there is no control for exposure over the subsequent 3 years which may be critical, especially as the authors note that their GLM analyses show a critical developmental re

- bound period between 24 and 36 months. From this study, it is unclear if an increase or decrease in exposure during the period of early childhood could also influence the neurodevelopment of these children. Finally, the creation of a dichotomous exposure variable also brings limitations to the interpretation of the study resul ts due to the amount of within

- group variation likely present using this method of exposure classification. By setting the cut

- off at 6.17pg/g, there are members of their “low exposure” group who may approximate high exposure levels and vice versa, result ing in non

- differential measurement error.

The authors also address limitations to the sensitivity and predictive validity of the developmental tests, especially among children less than 3 years of age. Although, these tests are considered to be most v alid among school

- aged children they are also used as indicators for early intervention to prevent continued developmental delay. The authors describe a statistically significant reduction in scores associated with high exposure levels but they do not dis cuss whether this 7

- point deficit is clinically relevant in terms of diagnosing or treating a child at this stage of development.Page 46 of 105 Also complicating this analysis is the pervasive, non

- specific nature of neurological effects and the difficulty in attribu ting causal pathways related to just one environmental exposure. Although the authors site previous research linking chlorpyrifos exposure to inhibition of neural growth (Dam, 1998) it is still difficult to attribute developmental and behavioral effects t o a single exposure variable measured at one point in time. In future studies it would be advantageous to include other potential covariates such as family history of developmental delay or attention disorders (especially among parents and siblings) as we ll as other co

- morbidities not adjusted for by design ( i.e , restriction using eligibility criteria to exclude co

- morbidities). Not only is the etiology of attention and related behavioral disorders not well understood but also the diagnosis of these disor ders is often influenced by complex outside forces.

This well

- designed study provides suggestive findings linking prenatal chlorpyrifos exposure with neurodevelopmental deficits at the three years of age. The lack of outcome information for a high
percen tage of cohort children is a concern which is not directly addressed by the authors. The use of a single exposure estimate (which likely led to non

- differential exposure misclassification bias in this study) and an imprecise outcome characterization (altho ugh a clinical tool, neurodevelopmental milestones are ‘soft’ outcomes to measure) may have an accumulative effect to underestimate the true effect (bias results toward to null value), if a true effect exists. As mentioned previously, at the time of this publication, these analyses covered only a snapshot of the findings of the ongoing cohort study. The authors allude to the fact that they will use the subsequent evaluations, especially the 7

- year scores to further describe the effects of chlorpyrifos exp osure on neurodevelopment and behavior.Page 47 of 105 Works Cited

Whyatt RM, Rauh V, Barr DB, et al. Prenatal insecticide exposure and birth weight and length among an urban minority cohort. Environ Health Perspect. 2004;112:1125

– 1132

Perera F, Rauh VA, Tsai WY, et al. Effects of transplacental exposure to environmental pollutants on birth outcomes in a multi

- ethnic population. Environ Health Perspect. 2003;111: 201

– 205

Whyatt, R. M., Garfinkel, R., Hoepner, L. A., Holmes, D., Borjas, M., Williams, M. K., . . . Camann, D. E. (2007). Within

- and between

- home variability in indoor

- air insecticide levels during pregnancy among an
inner

- city cohort from New York City. Environ Health Perspect, 115 (3), 383

- 389. doi: 10.1289/ehp.9546

Dam K, Seidler FJ, Slotkin TA. Developmental neurotoxicity of chlorpyrifos: delayed targeting of DNA synthesis after repeated administration. Brain Res. 1998;108:39

– 45Page 48 of 105 Article 8. Lovasi et al. (2010) Chlorpyrifos Exposure and Urban Residential Environment Characteristics as Determinants of Early Childhood Neurodevelopment. Gina S. Lovasi, James W. Quinn, Virginia A. Rauh, Frederica P. Perera, Howard F. Andrews, Robin Garfinkel, Lori Hoepner, Ro bin Whyatt, Andrew Rundle. American Journal of Public Health. 101(1):63

- 70 (2005).

Study Summary

Lovasi et al evaluated associations between neighborhood characteristics and early

- life neurodevelopment in the Columbia Mother’s and Newborn Study Cohort, and assessed whether such characteristics confound the association between exposure to chlorpyrifos and neurodevelopment previously reported. US Census data (2000) were used to estimate neighborhood

- level measures of physical infrastructure, socioeconomic status, crowding, demographic composition, and linguistic isolation, augmenting previously reported prenatal chlorpyrifos exposure, psychomotor development, and mental development data on a New York City birth cohort.

Study participants were recruited du ring early pregnancy ( ≤20 th week) among African

- American and Dominican women age 18

- 35 years, and registered at New York Presbyterian Medical Center and Harlem Hospital, in New York City. Women who smoked, had a history of drug abuse, diabetes, hypertensio n, or HIV infection were excluded from participation in the study, as were women who resided in New York City for less than 1 year.

Cord blood plasma chlorpyrifos levels were assessed, and dichotomized as high (greater than the third tertile level of 6.17 pg/g) or low (<6.17pg/g). Maternal blood levels of chlorpyrifos were used to estimate cord blood levels for the 12% of infants for whom cord blood was not obtained.

The Bayley Scales of Infant Intelligence

– Revised (BSID

- II) were used to assess cogniti ve and psychomotor development at age 36 months. Development was reported using a Psychomotor Development Index (PDI) and a Mental Development Index (MDI), which served as the continuous outcome measurements in the study.

Participants’ neighborhoods were defined using a 1

- kilometer network buffer. Using 2000 US Census data, network buffer characteristics were determined, including: the percentage of housing units without complete plumbing, the percentage of vacant housing units, the percentage of residen ts 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 compo sition, the percentage of residents born outside the United States, the percentage of Spanish

- speaking residents, and the percentage of residents who were linguistically isolated (all household members aged 14 years and older have at least some difficulty with English).Page 49 of 105 Indicators of building disrepair were self

- reported by mothers during prenatal interviews, and summed to create an index of disrepair that included one point for each of the following problems: holes in ceilings or walls, peeling or flaking paint, water damage, leaking pipes, and lack of gas or electricity in the prior 6 months.

Generalized estimating equation models were created for the continuous outcomes (PDI and MDI) and Bayley Scores of Intelligence at 36 months. Robust standard error s were used to correct for possible autocorrelation within community district areas. All models included the following potential confounders: gender, gestational age at birth, Dominican versus African American ethnicity, maternal education, maternal intell igence quotient, the presence of secondhand smoke in the home during pregnancy, and an index of the quality of the home environment with respect to caretaking. Using hierarchal regression techniques which allow for control of individual level as well as gr oup

- level variables, authors were able to further clarify the nature of the association between mental and psychomotor development (Bayley scales of infant development) and prenatal chlorpyrifos exposure. Other variables including blood lead, other environ mental exposures among other factors were not considered confounders in the prenatal chlorpyrifos and Bayley score association.

Of 266 children included as participants, 47% were male, 59% were Dominican, and 41% were African American. After adjusting for potentially confounding variables, high chlorpyrifos exposure (greater than 6.17 pg/g) was associated with a 6.5

- point decrease in PDI and a 3.3

- point decrease in MDI at age 36 months, as previously reported. These associations remained statistically sign ificant and similar in magnitude in models additionally adjusting for dilapidated housing and neighborhood characteristics.

Study Review

This study helps clarify the nature of the relation between prenatal chlorpyrifos exposure and childhood neurodevelop ment by controlling for the potential influence of socioeconomic status (SES) using advanced statistical techniques. Unique to this research, authors were able to measure variability due to SES using several different variables measured at both the individ ual

- level and the group

- level. This work enhances the precision and accuracy of the epidemiologic measures previously reported.

The authors note that participants lost to follow

- up were not significantly different from continuing participants with respec t to maternal age, ethnicity, marital status, education, income, or gestational age and birth weight of the newborn; authors cited a retention rate at the 3

- year follow

- up of 83%. How they arrived at that number is unclear. Originally, 648 pregnant women c onsented to participate and 327 children completed a developmental assessment at approximately 36 months of age. In this report, analyses were conducted among 266 children with available chlorpyrifos exposure data, yielding an effective retained proportion of 41%. This leaves open the possibility of selection bias resulting from loss to follow

- up; one may only speculate as to the magnitude and direction of such bias.Page 50 of 105 The direct measurement of cord blood chlorpyrifos in this study is a substantial strength, compared to the assessment of non

- specific markers as assessed in similar studies of organophosphate pesticide and child neurodevelopment. Umbilical cord blood was obtained at delivery for 88% of the children in the study. For the remaining 12% for whom no cord blood was obtained, cord blood plasma chlorpyrifos was estimated from maternal plasma levels using blood sampled two days prior to delivery. Any chlorpyrifos measurement error resulting from this estimation is almost assuredly non

- differential with respect to cognitive development outcomes. Children excluded for missing chlorpyrifos exposure data were similar to the analytic sample except that excluded childre n had more indicators of building dilapidation, so the likelihood of selection bias of large magnitude is unlikely. The decision to dichotomize cord blood chlorpyrifos levels using the highest tertile as a cut

- point reduces the ability to assess exposure

- r esponse relationships, and may somewhat decrease statistical efficiency relative to using a continuous measure. If a large number of participants had levels of chlorpyrifos below the level of detection, however, using a categorical approach dispenses with the need to estimate or otherwise define exposure at low levels. The prenatal exposures assessed in this study are likely correlated with other prenatal exposures and factors in the postnatal environment which themselves predict cognitive development. This presents a challenge for a causal interpretation of prenatal chlorpyrifos exposure per se , as the agent and time period responsible for observed cognitive deficits.

The Bayley Scales of Infant Development

– Revised (BSID

- II) is a widely used norm

- reference d developmental test for young children, it can be used to diagnose developmental delay, and it is known to be sensitive to the effects of toxic exposures such as low

- level intrauterine lead. The assessment was appropriately administered, with each child b eing evaluated under controlled conditions by a trained bilingual research assistant. Inter

- observer reliability was assessed but data were not presented in this report. The authors note that, when administered at age 3 years, the BSID

- II demonstrates only moderate predictive power for subsequent intelligence and school performance, but cite previous literature as evidence that the test is nevertheless clinically useful for children performing in the subnormal range.

The statistical analyses were generally appropriate. Missing data on covariates were estimated using multiple imputations, and the variance estimates presented appropriately reflect the degree of uncertainty caused by missing covariate data. Robust standard errors were estimated to account for potential autocorrelation. The authors presented multiple model results, each with a different indicator of neighborhood character. Only neighborhood covariates representing socioeconomic conditions ( i.e. percentage of individuals living in poverty, and percentage with high school education) were statistically significant predictors of PDI and MDI. Interestingly, and without explanation, the authors did not present the results of a model which simultaneous ly included all of the neighborhood characteristics as covariates. Presumably, this was done for the sake of parsimony, as one could argue against the inclusion of covariates considered not to be predictors of cognitive development. Alternatively, these fa ctors were, a priori
, considered potential confounders, and the use of statistical significance tests in the assessment of confounding is discouraged in many epidemiologic circles.Page 51 of 105 The observation that neighborhood characteristics did not confound the o bserved association between chlorpyrifos levels and cognitive development in this study has several possible explanations. This was a relatively homogenous study population living in the New York City Area, and it is possible that confounding is limited by the small degree of variability in neighborhood characteristics between those with and without high exposure levels of chlorpyrifos. It is also possible that the neighborhood characteristics are measured with error, such that they fail to capture aspects of socioeconomic condition, neighborhood composition, and neighborhood condition that effect cognitive development, even within this context of measurement of several individual

- level as well as group

- level SES measures. This residual bias, if it exists, could (at least partially) explain the decrement in cognitive development associated with chlorpyrifos exposure, assuming that poorer neighborhood conditions are both predictors of reduced cognitive function and comparatively more prevalent among those wit h high chlorpyrifos exposure. The first of these two explanations has no effect on internal validity; under the assumption of no other bias, the observed association is unbiased, but external validity may be limited due to the restricted study population. The investigators’ methods to assess neighborhood characteristics were well conceived and well implemented, and reflective of commonly utilized variables to measure important aspects of the multi

- dimensional characteristic of socio

- economic status.
Howev er, such exposures are notoriously difficult to measure well even using these advanced statistical methods and approaches.

The authors did not assess effect modification of the association between prenatal chlorpyrifos exposure and cognitive development by neighborhood conditions, which is an equally, if not more, interesting area of inquiry than assessment of confounding. The present study simply does not have the sample size/statistical power to address these effects, but, in the discussion, the authors speculate that large studies that collect both neighborhood context and exposure data could reveal patterns of effect modification. T he relative homogeneity of the study population may have precluded statistically robust examination of effect modification by factors related to SES in this study

.

The setting of the investigation in a sample drawn from low

- income African American and Dominican communities is both a strength and a limitation of the study. The population is likely at high risk of developmenta l deficits, which increases the power of the study relative to a similar study set in a population at low risk. The assessment of confounding by demographic and neighborhood characteristics is limited by the restriction of the study sample to low

- income co mmunities (a weakness), but so too is the bias induced by these potential confounders (a strength). The generalizability of the study results may also be limited because participants were recruited from a restricted range of neighborhoods in New York City (a weakness), but the study population is likely among the most highly exposed and also at risk for cognitive impairment (Landrigan, 1990a, 1990b; Landrigan et al., 1999)

.

This study represents an extension of previously published work with additional ad justment for multiple neighborhood characteristics. The work was well conceived and conducted, representing perhaps the best assessment of the association between chlorpyrifos and cognitive development in thePage 52 of 105 epidemiologic literature. Chief among its stren gths is the direct assessment of cord blood plasma chlorpyrifos, the prospective design, and the assessment and control of potential confounders. Using the methods of data collection, statistical analyses, and enhanced methods to evaluate the role of SES a s a potentially confounding variables, authors report no substantial difference in the reported association between prenatal chlorpyrifos exposure and child neurodevelopment as previously reported (Rauh et al. 2006).Page 53 of 105 Works Cited:

Landrigan, P. J. (1990 a). Health effects of environmental toxins in deficient housing. Bull N Y Acad Med,
66 (5), 491

- 499. Landrigan, P. J. (1990b). Housing and health: conclusions and challenges for the future. Bull N Y Acad Med, 66 (5), 587

- 591. Landrigan, P. J., Claudio, L., Markowitz, S. B., Berkowitz, G. S., Brenner, B. L., Romero, H., . . . Wolff, M. S. (1999). Pesticides and inner

- city children: exposures, risks, and prevention. Environ Health Perspect, 107 Suppl 3 , 431

- 437.Page 54 of 105 Article 9. Engel et al. (2011) Prenatal Exp osure to Organophosphates, Paraoxonase 1, and Cognitive Development in Childhood. Stephanie M. Engel, James Wetmur, Jia Chen, Chenbo Zhu, Dana Boyd Barr, Richard L. Canfield, and Mary S. Wolff. Environmental Health Perspectives 2011; 119:1182

- 1188

Study Summary

This is the third of three published analyses of prenatal pesticide exposure and child growth and development in the Mount Sinai Hospital Children’s Environmental Health Study . The first (Berkowitz et al 2004) reported on assessments of birth weight, length and head circumference. The second (Engel et al 2007) reported on assessments of infant neurocognitive development and reflexes shortly after birth. In this study, Engel and colleagues extended their 2007 to include analysis of PON1 activity , PON1 polymorphisms, and cognitive development at ages 12 and 24 months and 6

– 9 years.

Of the 404 women originally enrolled between May, 1998 and July, 2001 (n = 404), a subset (n = 360) had organophosphate metabolite levels quantified in urine samples collected during the third trimester. Prenatal maternal blood and cord blood samples were analyzed for PON1 activity and genotype. Children of these mothers returned for neurodevelopment assessments at ages 12 months (n = 200), 24 months (n = 276), and 6

– 9 (n = 169) years of age. Participants in this study were predominantly young, unmarried, Hispanic women with low educational attainment. Most women delivered term infants of normal birth weight, as prematurity and very low birth weight were exclusion crit eria for study enrollment.

The study population was drawn primarily from East Harlem and consisted largely of young Hispanic women (predominantly Puerto Rican), but also included African

- American and Caucasian women. Mothers were enrolled during early p regnancy from the Prenatal Clinic and two private practices at Mount Sinai Hospital from May 1998 to July 2001. The study included only primipar ous with singleton births. Mothers were excluded if they had any of the following: an initial prenatal visit aft er 26 weeks of gestation, serious chronic diseases, a serious pregnancy complication that could affect fetal growth and development; alcohol consumed greater than two alcoholic beverages per day, illicit drug use. Mothers and infants were also excluded if the child was born with a congenital malformation or severe prematurity. A total of 479 prenatal patients were recruited; 75 were excluded because of medical complications, premature birth, an infant with birth defects, inability to collect biologic specim ens before birth, change of hospital or residence outside New York City, or refusal to continue to participate.

A questionnaire was administered to participants during their third trimester of pregnancy to obtain information on environmental exposures, s ociodemographic characteristics, obstetrical and medical history, and lifestyle factors.Page 55 of 105 Generalized linear models were used to analyze the relationship between total diethylphosphates (DEPs), total dimethylphosphates (DMPs), total dialkylphosphate metabo lites (DAPs), and subsequent cognitive development evaluated at age 12 and 24 months using the Bayley Scales of Infant Development, 2nd edition (BSID

- II) and using the Wechsler Preschool and Primary Scale of Intelligence, 3rd edition (WPPSI

- III) at approxi mately age 7 years. The authors also assessed modification of pesticide metabolite

- cognitive development associations by maternal and child PON 1 activity and PON 1 polymorphisms.

The authors presented their results stratified by level of race/ethnicity and PON1 genotype, having observed apparent effect modification by these variables. The authors found that prenatal total dialkylphosphate metabolite level was associated with a decrement in mental development at 12 months among blacks and Hispanics. The associations appeared to be strongest among children of mothers who carried the PON1 Q192R QR/RR genotype. In later childhood, increasing prenatal total DAP and DMP metabolites were associated with decrements in perceptual reasoning in the maternal PON1 Q1 92R QQ genotype (imparting slow catalytic activity for chlorpyrifos oxon), with a monotonic trend consistent with greater decrements with increasing prenatal exposure. The authors concluded that their findings are suggestive of an association between prena tal exposure to organophosphates and decrements in cognitive development, particularly perceptual reasoning, with evidence of effects beginning at 12 months and continuing through early childhood, with PON1 being a potentially important potential susceptib ility factor for these deleterious effects. The authors did not observe any effect modification by PON1 enzyme activity.

The authors noted that the associations they observed between the organophosphate pesticide metabolites and cognitive development are similar to those reported in a similar prospective cohort study set in an agricultural area of California (the Center for the Health Assessment of Mothers and Children of Salinas Study, Eskenazi et al. 2010).

Urine samples obtained during the 3rd trimes ter prenatal visit were provided to the CDC to quantify levels of six dialkylphosphate metabolites. In cases where individual dialkylphosphate metabolite levels were missing because of analytic interference, metabolite levels were imputed using regression analysis to predict the missing metabolite on the basis of the other non

- missing metabolites measure d for that woman within the group of correlated metabolites (Eskenazi et al. Environ Health Perspect, 2004). Samples below the limit of detection (LOD) were defined as LOD/ √

– 2. Diethyl

- and dimethylphosphate metabolites were then summed on a molar basis ( as nm/liter) to obtain total diethylphosphates (DEPs) and total dimethylphosphates (DMPs), respectively, and together to obtain total dialkylphosphates (DAP) levels. Twenty

- six very dilute urine samples with < 20 μg/dL creatinine were excluded from

organop hosphate metabolite analyses. Approximately 97%, 89%, and 90% of the cohort had detectable levels of DAP, DEP, and DMP metabolites, respectively. Prenatal organophosphate marker levels were dichotomized using median exposure as the cut

- off.

Plasma was se parated from samples of prenatal maternal peripheral blood and cord bloodPage 56 of 105 and used to measure PON1 activity. PON1 polymorphisms in both maternal and child DNA were also assessed using the blood samples provided by participants. Prenatal and cord blood PO N1 activity levels were classified in tertiles for analysis of effect modification of the relationship between pesticide marker levels and subsequent cognitive development.

The Bayley Scales of Infant Development, 2nd edition (BSID

- II), was administered to participating children at 12 and 24 months of age, approximately, to evaluate mental and psychomotor development. The Mental Development Index (MDI) portion of the assessment rates a child’s cognitive ability ( i.e. memory, habituation, problem solving, early number concepts, generalization, classification, vocalizations, language, and social skills). The Psychomotor Development Index (PDI) rates fine and gross motor coordination. Between the ages of 6 and 9 years of age, a subset of participating childr en was again assessed using psychometric intelligence tests. Before age 7, children were administered Block Design, Information, Matrix Reasoning, Vocabulary, Picture Concepts, Symbol Search, Word Reasoning, and Coding subtests of the Wechsler Preschool an d Primary Scale of Intelligence, 3rd edition (WPPSI

- III). Composite Verbal, Performance, Processing speed, and Full

- Scale IQ (FSIQ) scores were derived using
age

- standardized WPPSI

- III norms. Children between the ages of 7 and 9 years were administered the Block Design, Similarities, Digit Span, Picture Concepts, Coding, Vocabulary, Letter

- Number Sequence, Matrix Reasoning, Comprehension, and Symbol Search subtests of the Wechsler Intelligence Scale for Children, 4th edition (WISC

- IV). Working Memory, Proce ssing Speed, and FSIQ scores were derived using
age

- standardized WISC

- IV norms.

The BSID

- II was administered at Mount Sinai Hospital. Interviews and examinations were conducted in English or Spanish. The WPPSI

- III was administered in English or Spanish b y one of four examiners, as was the WISC

- IV. by one of four examiners Both the WPPSI

- III and the WISC

- IV were administered in a private room, without the parent being present.

Of the 200 children who were administered the BSID

- II at approximately 12 mont hs of age (mean ± SD, 13.1 ± 1.6 months), one was excluded from analyses because they refused to compete a substantial portion of the examination; two children were excluded from the 12

- month analysis because their parent reported a diagnosis of pervasive developmental disorder and 20 children were excluded because their corresponding maternal urine samples were deemed to dilute to analyze. Of the 177 eligible children who completed 12

- month exams, 174 had organophosphate metabolites measured in prenatal ur ine. At the 24

- month BSID

- II (mean ± SD, 27.4 ± 4.5 months), 276 children completed the exam. Two were excluded from the 24

- month analysis because the parent reported a diagnosis of pervasive developmental disorder. Children of mothers who submitted very d ilute samples of urine (less than 20 μg/dL creatinine) were also excluded from analysis (n = 23). Of the remaining 251 eligible observations, 247 had organophosphate metabolites measured in prenatal urine. MDI scores for 10 children were not usable due to extensive refusals; they were included in the PDI analyses because their scores were valid. The authors reported an association between prenatal total dialkylphosphate metabolite level and a decrement in mental development at 12 months among blacks and His panics. These associations appeared to be enhanced among children of mothers who carried the PON1 Q192RPage 57 of 105 QR/RR genotype. In later childhood, increasing prenatal total dialkyl

- and dimethylphosphate metabolites were associated with decrements in perceptual r easoning among those with maternal
PON1 Q192R QQ genotype, which imparts slow catalytic activity for chlorpyrifos oxon, with a monotonic trend consistent with greater decrements with increasing prenatal exposure.

Study Review

This was a well conducted prospective study conducted in a young, predominantly minority population. The study design, analytic approach, and statistical analyses were appropriate. Differential measurement error of exposure and outcomes is likely to be min imal, although other sources of bias may have influenced the study findings.

The dramatic loss

- to

- follow

- up over time is a possible cause of selection bias in this study. The authors noted that mothers returning for follow

- up assessments tended to be old er, with a disproportionately low fraction of women in the youngest age category; they also had disproportionately higher level of education, on average, relative to the original cohort. However, the subset of participants who returned for follow

- up visit s did not differ substantially from the originally enrolled cohort with respect to racial/ethnic composition, breast

- feeding behaviors, and self

- reported alcohol use during pregnancy.

The authors employed a reasonable set of exclusion criteria that likely resulted in recruitment of a study population that was relatively homogeneous with respect to confounders of the in utero pesticide
exposure

- cognitive development relationship (compared to the general population), and the potential for confounding was t hus limited by design (restriction). Bias due to confounding was also controlled in the analysis, by assessing and adjusting for known risk factors for cognitive development that are potentially associated with pesticide exposure, including maternal age, r ace/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 te sting, examiner and urinary creatinine level. The authors considered gestational age at delivery and birth weight, not as confounders, but as potential causal intermediates. They used a change

- in

- estimate approach to covariate selection, beginning with a f ull model, which allowed them to consider confounding by a given risk factor in the context of adjustment for other covariates. Given the limited sample size in this study, a full model with many covariates may be unstable, although it is unclear from the article whether such instability was observed.

Residual confounding by unmeasured or poorly measured confounders is likely in this study. Socioeconomic determinants, which are notoriously difficult to measure well, are likely predictive of pesticide expo sure, and risk factors for poor cognitive development. The authors adjusted for some factors which may be surrogates for socioeconomic status ( e.g. , race/ethnicity, marital status, maternal education), but adjustment for these factors is unlikely to have f ully accounted for confounding by socioeconomic status. If such residual confounding exists, it is likely to result in effect estimates which overestimate the effect of organophosphate pesticide exposure. However, the extent of thisPage 58 of 105 confounding was likely limited by the recruitment of a relatively homogenous study population, vis

- à

- vis socioeconomic conditions.

Presence of environmental contaminants in the home, either during pregnancy or after birth, may confound the relationship under study. The authors do not discuss co

- contaminants present in the home during the pregnancy; such factors would likely result in the observation of a greater than effect of pesticide exposure on cognitive development if they are positively associated with pesticide exposure and predictive of deficits in cognitive development. The authors stated that they considered a “Home Observation for Measurement of the Environment” (HOME) score as a potential confounder, and, in fact, they included this score in their multivariate models . The degree to which this “HOME” score is a good proxy for relevant co

- exposures is unclear, and leaves open the possibility of residual confounding by environmental contaminants in the home.

The authors observed apparent effect modification by materna l race/ethnicity. For example, among

non

- whites, increasing DAP and DMP tertiles of exposure were associated with a decrease in the MDI. However, among whites, the opposite was observed; higher metabolite levels were associated with an
increase in MDI scor es. Such an observation may be indicative of a true interaction between race/ethnicity and prenatal pesticide exposure, but it may also be explained by residual and differential confounding by co

- contaminants that are correlated with pesticide exposure and race. Indeed, in this study population, race/ethnicity was strongly associated with housing type ( i.e. public versus private housing), and the authors did, indeed, observe similar trends in the effect estimates when stratifying by housing type rather than race/ethnicity. The authors speculated that this observed heterogeneity may indicate that whites, or those living in private or owner

- occupied housing, may have experienced a different source of exposure to pesticides or their metabolites that may have c ontributed substantially to their urinary concentrations, and identify pesticide residues from fresh fruit and vegetable consumption as one such source. The authors adjusted for seasonal variation in exposure, although they note that other time

- related var iability may influence exposure assessment.

Although they observed heterogeneity by PON1 genotype, the authors did not observe any effect modification by PON1 enzyme activity in blood sampled during the third trimester, as they had in their earlier assess ments of pesticide metabolites and cognitive development assessed shortly after birth. They speculated that this was because genotype is a more stable, long

- term predictor of metabolism potential, relative to prenatal phenotype as measured by PON1 activity

.

The metabolites assessed in this study are sensitive, but non

- specific, markers for chlorpyrifos. The analytic methods appear sound. However, they assessed these markers using only a single maternal urine specimen taken during the third trimester

– a cr itical period for neurodevelopment. If sources and patterns of exposure remain static, a single measurement would be a good indicator of pesticide exposure during pregnancy. If, however, sources and exposure patterns change over short time

- spans, perhaps o n the order of days to weeks, such an exposure assessment would not necessarily provide a good indicator of the relevant exposure. Like the many other sources of error possible in this study, this,Page 59 of 105 too is likely to result in a non

- differential misclassific ation of the exposure, with respect to the cognitive outcome. Serial measurements of urinary biomarkers of pesticide exposure in the third trimester would shed light on these patterns, although the benefits of such information are offset by the added cost, logistical complexity expense, and burden on the study participants.

The instruments used to assess neurodevelopment in this study ( i.e.
, the Bayley Scales of Infant Development, 2nd edition (BSID

- II), the Wechsler Preschool and Primary Scale of Intellig ence, 3rd edition (WPPSI

- III), and the Wechsler Intelligence Scale for Children, 4th edition (WISC

- IV)) are commonly used in both a clinical and research setting and are widely accepted tools.

In this study, prenatal maternal DEP urinary metabolite concen trations were associated with slight decrements in FSIQ, Perceptual Reasoning, and Working Memory between the ages of 6 and 9 years. Among children of QQ mothers, DAP and DMP urinary metabolite concentrations were associated with poorer scores on Perceptua l Reasoning and FSIQ. The authors reported stronger Q192R interactions for DAP and DMP urinary metabolites compared to that for the DEP metabolites, although the explanation is unclear and may have been influenced by small sample size. The authors note tha t PON1 Q192R exhibits substrate specificity, but the strongest interactions were observed for DMP, not DEP (into which chlorpyrifos and diazinon both metabolize). Interestingly, researchers state that malathion spraying for mosquitoes carrying West Nile vi rus was a well

- publicized source of exposure during the period of enrollment which may have increased the levels of DMP in this population.

The statistical analyses used in the study appear to have been appropriate, although they are not explained in deta il. The authors stratified their findings by both race/ethnicity and PON 1 genotype, although it is unlikely that the study was sufficiently powered to provide optimal statistical power to assess pesticide effects in the presence of such effect modificatio n. To maximize the number of participants included in the analysis, the investigators defined a combined population of children who came for at least one of the Wechsler psychometric intelligence exams. The WISC

- IV composite scores were included preferenti ally, and WPPSI

- III composite scores substituted if the child did not return for the later exam. Regarding the use of a composite population for the outcome evaluations at 6

- 9 years, the authors report ‘convergent validity’ between the WISC

- IV and WPPSI

- II I. In a previous report, the correlation between WPPSI

- III and WISC

- IV FSIQ scores was 0.89; WPPSI

- III Verbal IQ and WISC

- IV Verbal Comprehension, 0.83; and WPPSI

- III Performance IQ and WISC

- IV Perceptual Reasoning, 0.79. In the present study, correlation s between the composite scores on the WPPSI

- III and WISC

- IV were likewise strong: FSIQ = 0.83, Verbal IQ/Verbal Comprehension = 0.84, and Performance IQ/Perceptual Reasoning = 0.78. In their statistical modeling, the authors included a variable to indicate whether the WISC

- IV and
WPPSI

- III was used.

Regarding the measurement of developmental outcomes using the Bayley Score, in general, associations observed during the 12

- month follow

- up were either attenuated or non

- existent at the 24

- month visit, includi ng the observation of race

- based heterogeneity. The authors speculated that, although exposures likely decreased over time, the 0

- to 12

- month exposure window may have included less hand

- to

- mouthPage 60 of 105 childhood exposure (from crawling exposure to house dust, an d/or fresh fruit and vegetable consumption) than in the 12

- to 24

- month window; thus, more childhood exposure in the 12

- to 24

- month window may modify the effect of prenatal exposure.

The prospective nature of the study is a primary strength. Pesticide ex posure indicators and outcomes metrics are likely to have been measured with some error, but these errors are likely to be non

differential. On average, such non

- differential measurement error results in attenuation of the measures of association, and are thus unlikely to result in false

- positive findings. A further strength attributable in part to the prospective design was the investigators’ ability to consider potential confounders and effect

- modifiers a priori , measure them by design, and adjust for/ass ess their influence in the analysis.

Importantly, this study was conducted at a time during which important regulatory changes in residential use of chlorpyrifos were being implemented (occurring approximately midway through the study). As a result, the participating children’s exposures to chlorpyrifos may have decreased over time. The investigators reported that they did not observe any time

- period interactions, but they did not present data on the presence or absence of temporal changes in participants ’ pesticide metabolite levels ( i.e. comparing those that were enrolled earlier versus later in time). In the discussion, they did mention that it was likely that childhood exposure to chlorpyrifos and diazinon was lower post

- 2000 (following the voluntary c ancellation of indoor residential chlorpyrifos use). To the extent that post

natal exposures were largely reduced, the associations observed can be more definitively linked to prenatal exposures. If however, pesticide exposures persisted into childhood, it would be less clear whether deficits in cognitive functioning observed in this study were caused specifically by pre

- natal exposures, rather than subsequent exposure early in life, assuming that pesticide exposure is causally associated with such decremen ts.

In this third study of organophosphate pesticide markers in maternal urine samples collected during the third trimester and subsequent cognitive development in children participating in the “Mt. Sinai Child Growth and Development Study”, the authors report on findings out to age nine. The specter of selection bias induced by loss

- to

- follow

- up is a marked weakness in an otherwise well conducted study. Unfortunately, it is difficult to assess the magnitude and direction of such bias. Confounding by unmeasured or poorly measured factors is yet another limitation, specifically with reference to the measurement of socio

- economic factors and other environmental co

- exposures. Non

- specific biomarkers of exposure, as were assessed in this study, are likely more objec tive, sensitive, and likely also more accurate and reliable indicators of pesticide exposure, relative to exposure assessment methodologies that rely on self

- report. However, to the extent that prenatal chlorpyrifos is the true exposure of interest, the as sessment of non

- specific organophosphate pesticide metabolites in a single sample of prenatal urine is a primary potential source of exposure measurement error. Because of the prospective design, information bias is expected to result in attenuation of the true (unobserved) associations, toward (observed) associations that are relatively smaller, or null. The finding of apparent effect modification by maternal race/ethnicity in this study could result from differential loss to follow

-Page 61 of 105 up, measurement error in the assessment of actual pesticide exposure, and/or differential residual confounding.Page 62 of 105 Works Cited

Berkowitz GS, Wetmur JG, Birman

- Deych E, Obel J, Lapinski RH, Godbold JH, Holzman IR, Wolff MS. In Utero Pesticide Exposure, Maternal Paraoxonase Ac tivity, and Head Circumference. Environmental Health Perspectives. 2004;112:388

– 391.

Engel SM, Berkowitz GS, Barr DB, Teitelbaum SL, Siskind J, Meisel SJ, Wetmur JG, Wolff MS. Prenatal Organophosphate Metabolite and Organochlorine Levels and Performance o n the Brazelton Neonatal Behavioral Assessment Scale in a Multiethnic Pregnancy Cohort. American Journal of Epidemiology. 2007;165:1397

- 1404.

Eskenazi B, Huen K, Marks A, Harley KG, Bradman A, Barr DB, Holland N. PON1 and neurodevelopment in children fro m the CHAMACOS study exposed to organophosphate pesticides in utero . Environmental Health Perspectives 2010;118:1775

– 1781.Page 63 of 105 Article 10. Eskenazi et al. (2007) Organophosphate Pesticide Exposure and Neurodevelopment in Young Mexican

- American Children.
Bre nda Eskenazi, Amy R. Marks, Asa Bradman, Kim Harley, Dana B. Barr, Caroline Johnson, Norma Morga, Nicholas P. Jewell. Environmental Health Perspectives. 115:792

– 798 (2007).

Study Summary

In this article by Eskenazi et al, the authors report on the relationship between prenatal and child urinary organophosphate metabolite levels and child neurodevelopment up to two years of age in the CHAMACOS cohort.

Enrollment for the CHAMACOS took place from 1999

– 2000 at community clinics, and included women who were ≥ 18 years old, pregnant at < 20 weeks gestation, Spanish

- or English

- speaking, eligible for low

- income health insurance, and planning to deliver at the local public hospital. Researcher followed 531 women to delivery of a live

- born, surviving, neonat e. Excluded from the analysis were children that did not have a neurodevelopmental assessment, (n=71), who did not have prenatal and relevant concurrent DAP metabolite measurements (n = 3), were not singletons (n=8), had a medical condition that could affe ct performance on the neurobehavioral assessment (n = 3; deafness, Down syndrome, hydrocephalus). Also excluded from analysis were Bayley results with raw scores which were too low for standardization (n=3, 1, and 1, for the 6

- month, 12

- month, and 24

- month assessments, respectively).

Maternal/fetal exposure was assessed by measurement of six non

- specific organophosphate dialkylphosphate (DAP) metabolites in maternal urine samples collected between 5 and 27 weeks gestation and again between 18 and 39 weeks . Maternal urine was also analyzed for 3,5,6

- trichloro

- 2

- pyridinol (TCPy), a metabolite specific to chlorpyrifos. Post

- natal early childhood urine samples were also collected, and DAP metabolites quantified, for the participating children at the time of th e neurodevelopmental assessments. Quantification of organophosphate metabolites using gas chromatography

- tandem mass spectrometry and isotope dilution calibration was conducted by the Centers for Disease Control and Prevention (CDC) labs.

To asses exposu re, investigators measured six nonspecific organophosphate DAP metabolites in maternal and child urine: three dimethyl (DMP) phosphate metabolites (dimethylphosphate, dimethylthiophosphate, dimethyldithiophosphate); and three diethyl (DEP) phosphate metabo lites (diethylphosphate, diethylthiophosphate, and diethyldithiophosphate) (Bradman et al. 2005). These six metabolites represent the by

- products of approximately 80% of OPs used in the Salinas Valley. Values below the limit of detection (LOD) were assigne d a value of LOD/( √2). The urine analysis was completed by the CDC. Maternal prenatal urine was also analyzed for specific metabolites including TCPy, a metabolite specific chlorpyrifos. Because a large proportion of women had non

- detectable levels of TCPy , the chlorpyrifos

- specific metabolite was categorized into three groups: TCPy less than LOD forPage 64 of 105 both pregnancy measurements, and, for those with at least one detectable level, subdivided below and above the median of the average pregnancy level.

The outc ome, child neurodevelopment, was assessed using the Bayley Scales of Infant Development (Mental Development (MDI) and Psychomotor Development (PDI) Indices) (Bayley, 1993) and the Child Behavior Checklist (CBCL). The Bayley Scales of Infant Development wer e completed at the 6 months (n = 396), 12 months (n = 395), and 24 months (n = 372) of age. Both scales were administered in Spanish and/or English by psychometricians blinded to exposure. Psychometricians were trained using these standardized protocols an d were supervised for quality assurance by a clinical neuropsychologist. Assessments were performed in a private room at the CHAMACOS research office or in a recreation vehicle (RV) modified to be a mobile testing facility. Scores > 1 SD below the mean ( i. e. , < 85) indicate possible developmental delay.

Mother’s completed the Child Behavior Checklist (CBCL) at the 24 month visit (n = 356). From the CBCL report, the authors assessed three scales as indicators of child neurodevelopment: The Attention Problem s syndrome scale, which includes such items as “can’t concentrate” and “can’t sit still”; the
DSM

- oriented Attention

- Deficit/Hyperactivity Disorder (ADHD) scale, which additionally includes such items as “gets into everything”; and the DSM

- oriented Pervasi ve Developmental Disorder (PDD) scale, which includes such items as “avoids eye contact,” “rocks head, body,” and “unresponsive to affection”. Experts consider a score greater than the 98th percentile of a national normative sample of clinical significance ; they consider a score greater than the 93rd percentile of borderline clinical significance.

Many confounders were ascertained including, intelligence of the mother, maternal depression, a measure of how stimulating the child’s environment is, and prenatal exposure to some known or suspected neurotoxins. Maternal intelligence was assessed via the Peabody Picture Vocabulary Test (PPVT). Maternal depression was assessed using the Center for Epidemiologic Studies Depression Scale (CES

- D). 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, 12

- month, and partially at the 24

- month visit. The following known or suspected neurotoxicants were measured: polybrominated diphenyl ethers (PBDEs), polychl orinated biphenyls (PCBs), p,p’

- dichlorodiphenyltrichlorethane (DDT), p,p’

- dichlorodiphenyltrichlorethylene (DDE), and lead. Lead was measured in cord blood. PBDEs, PCBs, DDT, and DDE were measured in maternal serum samples collected at 26 weeks gestation

.

Statistical analyses of the relationship between metabolite levels and Bayley performance were conducted using multiple linear regression models and generalized estimating equations ( i.e.
, longitudinal models) for MDI and PDI were developed for each of the three assessment time points ( i.e.
, 6, 12, and 24 months). Neurodevelopmental scores were modeled as continuous variables. Covariates in the models included indicators for the psychometrician conducting the assessment and the location of assessment, a ge at assessment, sex, duration of breast

- feeding, HOME score, and household income, parity and indicator of maternal intelligence (PPVT score).Page 65 of 105 Other covariates that were considered but not included in the analysis were: maternal age, education, depress ive 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, ab normal reflexes, PCBs, lead, DDT, β

hexachlorocyclohexane, and hexachlorobenzene. Furthermore, the authors examined interactions between child DAPs and child sex and, because they previously observed an association with maternal DDT and 24

- month MDI, betwe en maternal DAPs and DDT. Finally, authors also reran the models using log

- transformed creatinine

- adjusted metabolites for comparative purposes.

Child behavior checklist score at the 24

- month assessment was modeled using separate multivariate logistic reg ression models for the five child behavior checklist indices , dichotomized at the clinical significance level and at the borderline clinical significance level. The same covariates that were included in the final Bayley models were used for the CBCL models with three exceptions: maternal depression was added, and psychometrician and assessment location were dropped, because scores were based on maternal report.

Of the 348 infants included in this analysis, 49.4% were male. Most mothers were less than 30 ye ars old at baseline (75.6), born in Mexico and Spanish

- speaking (89%), had household income below the poverty line (62%). Many of the participating mothers worked in agriculture during pregnancy (43%) and had symptoms of depression (51%). Most of the women were married, non

- smokers, and did not drink alcohol during pregnancy.

In this study population, prenatal DAP levels were adversely associated with MDI, while early life DAP levels were positively associated with MDI. At 24 months of age, these associat ions reached statistical significance (per 10

- fold increase in prenatal DAPs: β =

– 3.5 points; 95% CI:

– 6.6 to

– 0.5; child DAPs: β = 2.4 points; 95% CI: 0.5 to 4.2). Neither prenatal nor child DAPs were associated with PDI or child behavior checklist atten tion problems. Both prenatal and postnatal DAPs were associated with risk of pervasive developmental disorder (per 10

- fold increase in prenatal DAPs: OR = 2.3, p = 0.05; child DAPs OR = 1.7, p = 0.04). TCPy was not associated with any neurodevelopment outc ome in this study.

Study Review

This study tested whether in utero or postnatal exposures to organophosphate pesticides have a detrimental impact on neurodevelopment, as assessed by the Bayley Scales of Infant Development and the child behavior checklis t (CBCL). The study population was appropriate to the hypothesis, given that there were a range of exposure levels to OPs and there was some homogeneity in the population, e.g. , mostly low SES, Hispanic, agricultural workers. The homogeneity of the populat ion restricted confounding by design. For example, another paper reported that smoking, alcohol use, and illicit drug use were rare the CHAMACOS cohort. The homogeneous population also increased the relative statistical efficiency of the study because it i s likely at higher risk of deficits in neurodevelopment thanPage 66 of 105 the general population (Landrigan, 1990; Landrigan et al., 1999) . However, the exclusion of children who lacked a neurodevelopmental assessment (n = 71, approximately 13% of the study population) , has the potential to induce a selection bias if the reasons for the missing data are determined by exposure (or a predictor of exposure) and are also not independent of neurodevelopment. For example, participants with high pesticide exposures could have missed neurodevelopmental assessments for reasons related to uncontrolled determinants of exposure which are also related to neurodevelopment. Comparisons of maternal DAP metabolite levels between those included in this analysis and those not included in t he analysis were not presented.

The exposure assessment methods in this study were good. Quantification of the metabolite levels was conducted at the CDC, using published methods. The study measured the organophosphate biomarker, DAP metabolites, in mater nal and child spot urine samples, and the chlorpyrifos specific biomarker, TCPy, in maternal urine only. Measurement of biomarkers in urine is a more objective and likely more accurate indicator of organophosphate pesticide exposure than other ascertainmen t methods such as self

- report. However, these biomarkers do present a few limitations, because study participants could be exposed to less toxic preformed DAP or TCPy metabolites (Lu et al. 2005; Wilson et al. 2003; Morgan et al. 2004), and exposure to pes ticides varies over time and the half

- life of chlorpyrifos in the body is short. However, t he prenatal exposure measures were, with some exceptions, the average of two measurements, and thus may better reflect chronic exposure during pregnancy than in stud ies where only a single spot urine sample was collected. Under an assumption of no other biases, the negative findings could have resulted from exposure measurement error.

The outcome measurements, the Bayley Scales of Infant Development and the CBCL, we re also appropriate. The Bayley Scales of Infant Development is considered a gold standard assessment. The assessments were reportedly appropriately administered. Both the MDI and PDI scales were administered in Spanish and/or English by psychometricians m asked to exposure status of the children. Psychometricians were trained using standardized protocols and were supervised for quality assurance by a clinical neuropsychologist. Assessments were performed in a private room. The influence of inter

observer re liability was controlled by inclusion for an indicator for the assessed but data were not presented in this report.

The Child Behavior Checklist (CBCL) has been widely used in cross

- cultural research and collects data on a range of behavior problems, yie lding scores for several syndrome scales and five scales designed to be consistent with Diagnostic and Statistical Manual of Mental Disorders (DSM) diagnoses. The three scales used in this assessment were selected by the investigators a priori because the effects they measured were similar to effects seen in relation to DAP exposure in animal models. The child behavior checklist is limited in that it is reliant on maternal report and the authors note the findings from the child behavior checkli st are not directly equivalent to a DSM diagnosis.

The statistical analysis used to assess the associations between the markers of exposure and neurodevelopment were appropriate. However, the authors did not present nor discuss regressionPage 67 of 105 diagnostics to a ssess the degree to which their models met or violated the assumptions implicit in linear models ( i.e. homoscedasticity, normality of error term distribution, independence of error terms, and linearity of the exposure

- response relationship), other than to state that they log transformed DAP levels. The authors also conducted a host of sensitivity analyses to assess influence of modeling decisions on their findings, and generally found their results to be robust. For example, they controlled for birth weight , gestational age, and abnormal reflexes which were considered causal intermediates, re

ran models excluding low birth weight and preterm infants, and ran models with and without adjustment of biomarkers for creatinine levels. The authors also assessed eff ect modification by gender and DDT exposure because population of primarily immigrant women who may have been exposed to DDT and other U.S. banned substances in country of origin; associations between pesticide markers and the indicators of neurodevelopmen t did not differ by either of these co

- variables.

To increase the statistical efficiency of the study by preserving the size of the analytic population, missing covariate values were imputed by randomly selecting a value from participants with non

- missing values. Maternal depression had the largest percentage of values requiring imputation (5%). Of remaining covariates, between 0% and 1.8% of values were imputed. Although a reasonable approach, poorly measured confounders ( i.e. residual confounding) may al so be influenced by imputed covariate data, although perhaps to a lesser degree because imputed data may still be preferable to no data at all.

Bias due to confounding was also controlled in the analysis, by assessing and adjusting for risk factors for

n eurodevelopment that are potentially associated with pesticide exposure, including age at assessment, sex, duration of breast

- feeding, HOME score, and household income, parity and indicator of maternal intelligence. In addition to the variables included in the final models, the authors examined potential confounding by several other variables suggested by the literature ( i.e. , 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, childcare), but they did not markedly alter the observed associations. Covariates were selected for inclusion in the final analyses if they were related to conditions of te sting, related to neurodevelopment in the literature and associated (p < 0.10) with most outcomes or consistently related to neurodevelopment in the literature, even if not in the data. This is a reasonable approach to assessment of confounding.

The authors’ quantification of environmental co

- exposures (DDT, DDE, β

- hexachlorocyclohexane, hexachlorobenzene, PCBs, and lead) is a significant strength of this study. Children and mothers, especially those from economically impoverished environments and émi grés from home countries in which these compounds were frequently use in the recent past, may be exposed to other environmental agents that may affect neurodevelopment. Potential confounding by these exposures, as well as by indicators of fetal development (shortened gestation and poorer reflexes) was assessed, and observed associations in these models were consistent with the primary findings.

This study has apparent inconsistencies in the findings which are worth noting. First, while DAPs were associated with an increased risk of PDD in this population, post

- natal DAPs were positively associatedPage 68 of 105 with MDI. No explanation was presented by the authors. One possible explanation is that children with higher cognitive functioning may be more interactive with th eir environment, leading to higher exposure to pesticide residues, an example of “reverse causation”, whereby the temporal order of the exposure and outcome are reversed. The measurements of prenatal pesticide markers do not suffer from the same issues of unknown temporal order; they clearly preceded the measures of post

- natal child development. Another possible explanation suggested by the authors is confounding by diet. Children who eat more fruits and vegetables may have higher DAP levels, but because of better diets have higher functioning. Second, although the investigators observed associations between neurodevelopment and the non

- specific metabolite markers of organophosphate pesticides (DAPs), no such associations were observed for metabolites specif ic to chlorpyrifos (TCPy), nor for a malathion

- specific metabolite (MDA). The organophosphate metabolites (DAPs) cannot be traced back to individual pesticides, and the observed associations with DAPs may be attributed to other pesticides, oxydemeton

- methy l for example, for which no specific metabolite is measureable, or may be due to the short

- half life and lower storage stability of the pesticide specific metabolites as compared to the non

- specific metabolites.

The study has numerous strengths, most nota bly the prospective design the longitudinal assessment of both exposure and outcomes. Strengths in the study design also include the quantification of non

specific (DAPs), chlorpyrifos

- specific (TCPy) metabolites, and other environmental co

- exposures. The results of this investigation by Eskenazi et al in the CHAMACOS cohort are suggestive of a detrimental association between prenatal organophosphate exposure, as measured by maternal urinary metabolite levels and neurodevelopment up to 24 months of age. Alt hough the prospective study design and reasonable methods protect from certain false

- positive inducing biases, there remains the potential of residual confounding in the direction of an adverse effect on development by unmeasured factors leading to the obs ervation of a false

- positive association. The authors reasonably suggest that their results should be interpreted with caution because, although they observed adverse associations between prenatal DAPs and mental development and pervasive developmental pro blems at 24 months of age, they also observed a positive relationship between neurodevelopment and postnatal DAPs.Page 69 of 105 Article 11. Eskenazi et al. (2010) PON1 and Neurodevelopment in Children from the CHAMACOS Study Exposed to Organophosphate Pesticides in u tero

. Brenda Eskenazi, Karen Huen, Amy Marks, Kim G. Harley, Asa Bradman, Dana Boyd Barr, and Nina Holland

. Environmental Health Perspectives. 118:1775

- 1781 (2010).

In this article by Eskenazi et al. (2010), the authors report on the relationship between PON1 genotype and PON1 activity level and child neurodevelopment, and the potential effect modifying role of PON1 status in the relation between prenatal and child ur inary organophosphate metabolite levels and child neurodevelopment up to two years of age in the CHAMACOS cohort.

Methods and analyses employed in this investigation were similar to Eskenazi et al. 2007, with the exception of the measurement of PON1 statu s in association with this outcome, and assessment of interaction between PON1 and DAPs with child neurodevelopment. Enrollment for the CHAMACOS took place from 1999

– 2000 at community clinics, and included women who were ≥ 18 years old, pregnant at
< 20 we eks gestation, Spanish

- or English

- speaking, eligible for low

- income health insurance, and planning to deliver at the local public hospital. Researcher followed 528 women to delivery of a live

born, surviving, neonate. Excluded from the analysis were child ren that did not have a neurodevelopmental assessment, (n=71), who did not have prenatal and relevant concurrent DAP metabolite measurements (n = 3), were not singletons (n=8), had a medical condition that could affect performance on the neurobehavioral as sessment (n = 3; deafness, Down syndrome, hydrocephalus). Also excluded from analysis were Bayley results with raw scores which were too low for standardization (n=3, 1, and 1, for the 6

- month, 12

- month, and 24

- month assessments, respectively), and partici pants for whom PON1 information was not available. The study included 371 mother

- infant pairs.

Maternal/fetal exposure was assessed by measurement of six non

- specific organophosphate dialkylphosphate (DAP) metabolites in maternal urine samples collected b etween 5 and 27 weeks gestation and again between 18 and 39 weeks. Specifically, investigators measured six nonspecific organophosphate DAP metabolites in maternal and child urine: three dimethyl (DMP) phosphate metabolites (dimethylphosphate, dimethylthio phosphate, dimethyldithiophosphate); and three diethyl (DEP) phosphate metabolites (diethylphosphate, diethylthiophosphate, and diethyldithiophosphate) (Bradman et al. 2005). These six metabolites represent the by

- products of approximately 80% of OPs used in the Salinas Valley. Chlorpyrifos and diazinon were the most commonly used organophosphates in the region that metabolize to the diethyl phosphate metabolite. Values below the limit of detection (LOD) were assigned a value of LOD/( √2). Quantification of organophosphate metabolites using gas chromatography

- tandem mass spectrometry and isotope dilution calibration was conducted by the Centers for Disease Control and Prevention (CDC) labs using published, validated, and accepted methods.

Maternal and umbil ical cord blood samples were collected at the time of delivery. Genotyping of the
PON1

- 108 and PON1 192 SNPs was performed using genomic DNA extracted from blood clots and TaqmanPage 70 of 105 PCR methods. Two PON1 enzyme activity assays (arylesterase and paraoxonase) w ere performed using spectrophotometric methods. Arylesterase activity measures the rate of hydrolysis of the substrate phenyl acetate, and reflects the quantity of PON1 enzyme. Paroaxonase activity measures the rate of hydrolysis of paraoxon, and thus refl ects a combination of the catalytic efficiency and quantity of the PON1 enzyme. Using triplicate samples, quality assurances methods illustrated the coefficient of variation was within the acceptable range (<10%).

The outcome, child neurodevelopment, was assessed using the Bayley Scales of Infant Development (Mental Development (MDI) and Psychomotor Development (PDI) Indices) (Bayley, 1993) and the Child Behavior Checklist (CBCL). The Bayley Scales of Infant Development were completed at the 6 months (n

= 396), 12 months (n = 395), and 24 months (n = 372) of age. Both scales were administered in Spanish and/or English by psychometricians blinded to exposure. Psychometricians were trained using these standardized protocols and were supervised for quality as surance by a clinical neuropsychologist. Assessments were performed in a private room at the CHAMACOS research office or in a recreation vehicle (RV) modified to be a mobile testing facility. Scores > 1 SD below the mean ( i.e. , < 85) indicate possible deve lopmental delay.

Mother’s completed the Child Behavior Checklist (CBCL) at the 24 month visit (n = 356). From the CBCL report, the authors assessed three scales as indicators of child neurodevelopment: The Attention Problems syndrome scale, which includes such items as “can’t concentrate” and “can’t sit still”; the
DSM

- oriented Attention

- Deficit/Hyperactivity Disorder (ADHD) scale, which additionally includes such items as “gets into everything”; and the DSM

- oriented Pervasive Developmental Disorder (PDD) scale, which includes such items as “avoids eye contact,” “rocks head, body,” and “unresponsive to affection”. Experts consider a score greater than the 98th percentile of a national normative sample of clinical significance; they consider a score greater than the 93rd percentile of borderline clinical significance.

Many confounders were ascertained including, intelligence of the mother, maternal depression, a measure of how stimulating the child’s environment is, and prenatal exposure to some known or suspected neurotoxins. Maternal intelligence was assessed via the Peabody Picture Vocabulary Test (PPVT). Maternal depression was assessed using the Center for Epidemiologic Studies Depression Scale (CES

- D). 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, 12

- month, and partially at the 24

- month visit. The following known or suspected neurotoxicants were measured: polybrominated diphenyl ethers (PBDEs), polychl orinated biphenyls (PCBs), p,p’

- dichlorodiphenyltrichlorethane (DDT), p,p’

- dichlorodiphenyltrichlorethylene (DDE), and lead. Lead was measured in cord blood. PBDEs, PCBs, DDT, and DDE were measured in maternal serum samples collected at 26 weeks gestation . However, these variables were not included in final models. The following variables were adjusted in the interaction analysis: age at assessment, sex, parity, breast

- feeding duration, HOME score, maternal PPVT, and household pov erty status. For the MDI and PDI models an indicator variable for the psychometrician was included, and maternal depression included in the model for PDD.Page 71 of 105 Statistical analyses of the relationship between PON1 status and Bayley performance were conducted using multiple logistic r egression models; interaction terms and stratified analyses were presented to evaluate the potential effect modifying role of PON1 status in the DAPs and child neurodevelopment association. Neurodevelopmental scores were modeled as continuous variables. PO N1 genotype was modeled as a three

- level variable, using the wild type as the referent. PON1 enzyme levels were categorized as tertiles. Authors log 10 transformed DAP levels, and averaged the two measures of urinary DAPs taken during pregnancy in the analy ses, when available (n=22 for which only one sample available). Covariates in the models included indicators for the psychometrician conducting the assessment and the location of assessment, age at assessment, sex, duration of breast

- feeding, HOME

score, a nd household income, parity and indicator of maternal intelligence (PPVT score). Authors imputed missing values to preserve sample size, particularly in the stratified analyses. Statistical significance of the main effect was considered a p

- value of 0.05, and significance of an interaction term was considered a p

- value of 0.15.

Among the selected participants, mothers were generally young age (average 26.4 years, sd 5.2), born in Mexico, were predominantly married or living as married, and were poor, 60% living at or below the poverty line. Forty

- four percent of the women reported working in agriculture during pregnancy, 82% resided with a household member who works in agriculture, and 23% live near an agricultural operation. Most mothers reported breast f eeding, and the duration of breast feeding was on average 6 months. Authors detected dimethyl phosphates more frequently than diethyl phospahtes. The average score for the MDI was 85 and PDI 98; 14% of children evaluated exceeded the concern level of the CBCL.

Overall, infants with the variant PON1 108TT allele who produced lower amounts of PON1 enzyme had reduced scores on the MDI and PDI, and increased risk of PDD using the CBCL. Authors observed no association with PON1 192QQ genotypes in the relation with MDI, PDI or PDD. Similarly, authors observed no association between enzyme levels and any measure of child neurodevelopment in this study. The interaction between maternal DAP and PON1 genotype with the Bayley assessed outcom es was non

significant (p=0.98), however researchers observed a trend of lower scores by the number of PON1 108 variants alleles. For each 10

- fold increase in total DAPs, the MDI score reduced by 3.2 PON1 108CC , 3.7
PON1 108CT , and 5.5 PON1 108TT (p

- interactio n 0.98 ). A similar trend was observed for the diet h yl phosphates, but neither strata

- specific estimates nor trend tests were significant. No similar trend was apparent across the number of variant alleles at the PON1 192 genomic locus. No associations were observed between DAPs and PDI by level of the PON1 108 genotype. However, there was limited evidence of heterogeneity by PON1 192 genotype in the relation between DAPs and PDI, but results were not significant.

Study Review

Overall, researchers offer som e evidence of a link between infant PON1 status and child neurodevelopment state, and authors’ state they cannot conclude the study provides evidence of effectPage 72 of 105 modification by PON1 genotype or phenotype status in the relation between DAPs and MDI, PDI or PDD in this study. Similarly to the accompanying investigation (Eskenazi et al. 2007), the study population was appropriate to the hypothesis, given that there were a range of expos ure levels to OPs and there was some homogeneity in the population, e.g. , mostly low SES, Hispanic, agricultural workers. The homogeneity of the population restricted confounding by design. For example, another paper reported that smoking, alcohol use, and illicit drug use were rare the CHAMACOS cohort. The homogeneous population also increased the relative statistical efficiency of the study because it is likely at higher risk of deficits in neurodevelopment than the general population (Landrigan, 1990; La ndrigan et al., 1999)

. Authors did not share whether DAP levels were similar in mothers of infants for whom the MDI and PDI scores were not available; the potential for selection bias therefore cannot be assessed. The measurement of both urinary DAPs and P ON1 status were well conducted, however limitations previously noted regarding the non

- specific nature of a DAP measurement to the question of chlorpyrifos toxicity and the use of one

- or two measures during the latter part of pregnancy as an estimate for gestational exposure apply equally to this investigation.

The outcome measurements, the Bayley Scales of Infant Development and the CBCL, were also appropriate. The Bayley Scales of Infant Development is considered a gold standard assessment. The

assessm ents were reportedly appropriately administered. Both the MDI and PDI scales were administered in Spanish and/or English by psychometricians masked to exposure status of the children. Psychometricians were trained using standardized protocols and were supe rvised for quality assurance by a clinical neuropsychologist. Assessments were performed in a private room. The influence of inter

observer variability was controlled by inclusion of an indicator variable for the psychomatrician.

The Child Behavior Check list (CBCL) has been widely used in cross

- cultural research and collects data on a range of behavior problems, yielding scores for several syndrome scales and five scales designed to be consistent with Diagnostic and Statistical Manual of Mental Disorders (DSM) diagnoses. The three scales used in this assessment were selected by the investigators a priori because the effects they measured were similar to effects seen in relation to DAP exposure in animal models. The child behavior checklist is limited in th at it is reliant on maternal report, and the authors note the findings from the child behavior checklist are not directly equivalent to a DSM diagnosis.

Again, the statistical analysis used to assess the associations between the markers of exposure and

ne urodevelopment were appropriate. However, the authors did not present nor discuss regression diagnostics to assess the degree to which their models met or violated the assumptions implicit in linear models ( i.e. homoscedasticity, normality of error term di stribution, independence of error terms, and linearity of the exposure

- response relationship), other than to state that they log transformed DAP levels. The authors also conducted a host of sensitivity analyses to assess influence of modeling decisions on their findings, and generally found their results to be robust. For example, investigators controlled for maternal depression in the relation between pesticide exposure and PDD, and ran models with and without adjustment of biomarkers for creatinine levels

.Page 73 of 105 To increase the statistical efficiency of the study by preserving the size of the analytic population, missing covariate values were imputed by randomly selecting a value from participants with non

- missing values. Maternal depression had the largest pe rcentage of values requiring imputation (5%). Of remaining covariates, between 0% and 1.8% of values were imputed. Although a reasonable approach, poorly measured confounders ( i.e. residual confounding) may also be influenced by imputed covariate data, alt hough perhaps to a lesser degree because imputed data may still be preferable to no data at all.

Bias due to confounding was also controlled in the analysis, by assessing and adjusting for risk factors for neurodevelopment that are potentially associated with pesticide exposure, including age at assessment, sex, duration of breast

- feeding, HOME score, and household income, parity and indicator of maternal intelligence. In addition to the variables included in the final models, the authors examined potenti al confounding by several other variables suggested by the literature ( i.e. , maternal age, education, depressive symptoms, active/passive smoking exposure during pregnancy, regular alcohol use during pregnancy, marital status, father’s presence in home, ho using density, maternal work status, childcare), but they did not markedly alter the observed associations. Covariates were selected for inclusion in the final analyses if they were related to conditions of testing, related to neurodevelopment in the liter ature and associated (p < 0.10) with most outcomes or consistently related to neurodevelopment in the literature, even if not in the data. This is a reasonable approach to assessment of confounding.

The authors’ quantification of environmental co

- exposur es (DDT, DDE, β

- hexachlorocyclohexane, hexachlorobenzene, PCBs, and lead) is a significant strength of this study. Children and mothers, especially those from economically impoverished environments and émigrés from home countries in which these compounds w ere frequently use in the recent past, may be exposed to other environmental agents that may affect neurodevelopment. Potential confounding by these exposures, as well as by indicators of fetal development (shortened gestation and poorer reflexes) was asse ssed, and observed associations in these models were consistent with the primary findings.

Similarly to the previous investigation by Eskenazi et al. (2007), t he study has numerous strengths, most notably the prospective design the longitudinal assessment of both exposure and outcomes. Strengths in the study design also include the quantification of non

- specific (DAPs), PON1 status, and other environmental co

- exposures. The results of this investigation by Eskenazi et al

. (2007) in the CHAMACOS cohort were suggestive of a detrimental association between prenatal organophosphate exposure, as measured by maternal urinary metabolite levels and neurodevelopment up to 24 months of age, and this investigation presents some additional, albeit limited evidence of potential effect modification by PON1 status. While results were not significant, sample size within strata of PON1 genotype and question as to the relevance of measurement of just PON1 among the suite of enzymes involved in organophosphate metabolism rema in. Although the prospective study design and reasonable methods protect from certain false

- positive inducing biases, there remains the potential of residual confounding in the direction of an adverse effect on development by unmeasured factors leading to the observation of a false

- positive association, however less so with respect to PON1 status as these factors would notPage 74 of 105 likely be correlated. The authors reasonably suggest that their results should be interpreted with caution as the evidence for true effe ct modification in this relation is weak.Page 75 of 105 Article 12. Marks et al. (2010) Organophosphate Pesticide Exposure and Attention in Young Mexican

- American Children: The CHAMACOS Study. Amy R. Marks, Kim Harley, Asa Bradman, Katherine Kogut, Dana Boyd Barr,
Ca roline Johnson, Norma Calderon, and Brenda Eskenazi. Environmental Health Perspectives.
118:1768

– 1774 (2010).

Study Summary

Using data collected as part of the CHAMACOS Study, Marks et al conducted a study to investigate the association between urinary d ialkyl phosphate (DAP) metabolites in pregnant women and their children, as a marker of organophosphate exposure, and attention

- related outcomes among Mexican

- American children living Salinas Valley, an agricultural region of California.

Enrollment for the CHAMACOS took place from 1999

– 2000 at community clinics, and included women who were ≥ 18 years old, pregnant at < 20 weeks gestation, Spanish

- or English

- speaking, eligible for low

- income health insurance, and planning to deliver at the local public hospital. The investigators followed 526 women to delivery of a live born, surviving singleton. Excluded from analyses were children who did not have a prenatal DAP metabolite measurement (n = 2), had a medical condition that could affect per formance on the neurobehavioral assessment (n = 3; deafness, Down syndrome, hydrocephalus), were lost to follow

- up, or did not participate at the 3.5

- or 5

- year study visit (n = 173) were excluded from the study. The study population consisted of the remai ning 348 children who had available data at 3.5 and/or 5 years.

Exposures were assessed by measurement of organophosphate dialkylphosphate (DAP) metabolites in maternal and child urine samples. Maternal urine was collected between 5 and 27 weeks gestatio n and again between 18 and 39 weeks. The post

- delivery urines were collected within 1 week of delivery for 73% of the sample, with the remainder obtained up to 176 days afterwards. Urine samples were also collected from participating children at the 3.5 ye ar and 5 year visits. Quantification of organophosphate metabolites was conducted by Centers for Disease Control and Prevention (CDC) labs. Total DAP metabolite level was defined as the sum of the molar concentrations of the six DAP metabolites, dimethylph osphate (DMP) metabolite level as the sum of the molar concentrations of the three dimethylphosphate metabolites only, and diethylphosphate (DEP) metabolite level as the sum of the molar concentrations of the three diethylphosphate metabolites. Total DAP, DMP, and DEP levels were determined for each participant for each of the two pregnancy urine samples and for the children’s urine samples. Individual metabolite levels below the limit of detection (LOD) were assigned a value of the LOD divided by the squar e root of two, and this value was included in each sum. Total DAP, DMP, and DEP metabolite levels were log 10 transformed. “Pregnancy” DAP, DMP, and DEP values were created by averaging the two log

- transformed pregnancy measures. For 19 women with only one DAP measurement in pregnancy, the single measure was used. Several children were missing DAP measures at 3.5 years (n = 58) and 5 years (n = 14).Page 76 of 105 Attention

- related outcomes were assessed in three ways: 1) maternal report of child behavior at 3.5 and 5 yea rs of age; 2) direct assessment of the child at 3.5 and 5 years; 3) and by a psychometrician’s report of the behavior of the child during testing at 5 years.

Mothers completed the Child Behavior Checklist (CBCL) for 1.5

– 5 years of age as part of the mate rnal interview at the 3.5

- and 5

- year visits to assess emotional/behavioral problems and competencies of the children. Attention was evaluated as continuous raw scores on these scales, and by the proportion above a cutoff score greater than the 93rd percen tile (a borderline clinical range).

Children completed a battery of neurodevelopmental assessments at each visit: The visual attention subtest of the NEPSY

- II at the 3.5

- year visit, and the Conners’ Kiddie Continuous Performance Test (K

CPT), during the 5

- year neurodevelopmental assessment. The NEPSY

- II was administered to children by trained psychometricians. Children were asked to circle specific images on a page of pictures that included distracters. For data analysis, the authors used continuous score s scaled to a normative sample of U.S. children; the age

- standardized mean ± SD for this subtest is 10 ± 3. The K

- CPT is a 7

- min computerized test that assesses reaction time, accuracy, and impulse control. Briefly, children were instructed to press the sp ace bar when they saw any image on the computer screen except a ball. The computer program yields T

- scores age

- standardized to a general U.S. population (mean ± SD = 50 ± 10) for errors of commission ( i.e. , the child responds when he or she should not), er rors of omission ( i.e. , the child fails to respond when he or she should), and hit reaction time. The authors examined T

- scores continuously and categorically using the cutoff of T

- score > 65, which is reportedly considered markedly atypical. The program u sed also combined measures to generate a clinical ADHD Confidence Index score (range, 0

– 100). For statistical analyses, the authors used continuous Confidence Index percentiles and selected a cut

- point of > 70th percentile (meaning that 70% of children per forming similarly on the test could be correctly classified as having clinical ADHD).

Following the 5

- year neurodevelopmental assessment, psychometricians blinded to exposure status answered several subjective questions evaluating the behavior of the chil d during the 2

- hr visit, including four questions derived from the seven

- item Hillside Behavior Rating Scale. The authors summed responses to two questions assessing motor activity and distractibility to create an adapted ADHD symptoms scale. The Hillside Scale is reportedly associated with parent and teacher ratings and has been found to add significantly to the clinical prediction of ADHD. For analysis the authors created a dichotomized Hillside outcome variable with scores ≥ 7 of 12 possible points (repr esenting < 10% of children) to flag children displaying a higher degree of attention problems based on psychometricians’ observation.

Statistical analyses were conducted using multivariate linear and logistic regression models to evaluate the associations between the markers of organophosphate pesticide exposure and the indicators of attention deficits summarized above. Separate models were run with maternal DAPs only, child DAPs only, and the two together in the same model. Odds ratios greater than one an d β

- estimates greaterPage 77 of 105 than zero indicate positive associations between exposure and increased risk of adverse outcome, except for the NEPSY visual attention subtest, where a β

- estimate greater than zero indicates better performance. All models adjusted for the following covariates: an indicator of the evaluating psychometrician (not included in CBCL models), age at assessment, sex, maternal education, depressive symptoms, maternal intelligence, out

- of

- home child care, and breast feeding duration. The covari ates 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 were also considered but not included in the final models because th ey did not markedly alter the observed associations and were therefore not used in final models. To preserve the size of the analytic population, remaining missing covariate values (six instances for maternal depression and three for maternal PPVT) were im puted by simple random selection of a value from participants with non

- missing values.

The authors also conducted a number of sensitivity analyses. They reran all models using creatinine

adjusted DAP metabolite concentrations. In addition, they added to f inal models some factors potentially on the causal pathway ( i.e. , birth weight, gestational duration). The authors also considered whether controlling for lead (log 10 - transformed), a known neurotoxicant, altered results for DAP concentrations in the subsam ples with cord (n = 229) or 2

- year (n = 296) lead values. Finally, because rates of ADHD vary considerably by sex in the general population (Pastor and Reuben 2008) and effects of other toxicants have been found to vary by sex (Delaney

- Black et al. 2004), the authors tested for interactions between DAP concentrations and child sex, using p < 0.15 for the interaction term to determine whether associations of DAP concentrations with measures of attention differed for boys and girls.

Of the 378 infants includ ed in this analysis, 47.5% were male. The mean maternal age was 26.5±5.2 years. Most mothers were born in Mexico (86%), spoke only Spanish in the home (90%), had low income (97% within 200% of the federal threshold for poverty; 63% within 100%), had not co mpleted high school (79%), were nonsmokers (96%), had previous live births (66%), and were married (82%). Many of the participating mothers had symptoms of depression (44%).

In this study population, higher concentrations of organophosphate metabolites i n the urine of pregnant women were associated with increased odds of attention problems and poorer attention scores in their children at age 5 years. 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% CI: 0.2

– 1.2; ADHD: β = 1.3; 95% CI: 0.4

– 2.1). Prenatal DAPs were associated with scores on the K

- CPT ADHD Confidence Index > 70th percentile (OR = 5.1; 9 5% 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 metab olite 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).Page 78 of 105 Study Review

This study tested whether i n utero or postnatal exposures to organophosphate pesticides have a detrimental impact on attention. The study population was appropriate to the hypothesis, given that there were a range of exposure levels to OPs and there was some homogeneity in the popul ation, e.g. , mostly low SES, Hispanic, agricultural workers. The homogeneity of the population restricted confounding by design. For example, another paper reported that maternal smoking, alcohol use, and illicit drug use were rare this cohort. The homogen eous population also increased the relative statistical efficiency of the study because it is likely at higher risk of deficits in neurodevelopment than the general population. The exclusion of children who did not complete the 3.5

- or 5

- year assessment (n = 173), has the potential to induce a selection bias if the reasons for the missing data are determined by exposure (or a predictor of exposure) and are also not independent of the development of attention disorders. However, the authors stated that the l evels of DAPs were similar in the mothers of the children which were not included relative to those included in the analysis. The authors also report that distributions of other covariates measured before 3.5 years were also similar, except that more boys than girls dropped from the study. This provides some indication that selection bias may not be a large factor in this analysis.

The ascertainment and analysis of urinary organophosphate pesticide markers was appropriate. Quantification was conducted at the CDC, using published methods. DAP metabolites are likely more accurate and objective indicators of organophosphate pesticide exposure, relative to other ascertainment methods such as self

- report. Although they are sensitive indicators of exposure, it i s difficult to infer chlorpyrifos effects specifically from the metabolite levels, as they also indicate exposure to other organophosphates. The study population likely sustained many other potentially relevant exposures to multiple chemicals and other pes ticides which may affect development. The six organophosphate metabolites cannot be traced back to individual pesticides but, according to the authors, they represent the breakdown products of approximately 80% of the total organophosphate pesticides used in the Salinas Valley. It is not possible to disentangle the various effects from these potential co

- exposures and chlorpyrifos in this study.

Because organophosphate pesticides have short half

- lives, the degree to which metabolites in urine samples refl ect ‘usual’ levels, and thus usual exposure, is uncertain. The prenatal exposure measures were, with some exceptions, the average of two measurements, and thus may better reflect chronic exposure during the pregnancy. The absence of findings associated wit h child levels may be due, in part, to the fact that the child’s levels are poor indicators of exposure during critical windows of exposure with respect to development of attentional difficulties.

Errors in the assignment of exposure in this prospective study will likely have resulted in attenuation of observed associations. The likelihood of these errors is high, and the magnitude of the bias induced difficult to assess. Under an assumption of no o ther biases, the negative findings, those for thePage 79 of 105 association between children’s concurrent DAP levels and attention outcomes, for example, could have resulted from exposure measurement error.

The outcome in this study is very difficult to measure. Inste ad of opting for a single best instrument/indicator of attention deficits, the investigators chose to use multiple instruments, and also to combine the results of these various instruments to form a composite indicator of attentional difficulties. There is no evidence presented in the report that would suggest that the outcome assessments were improperly conducted. Nevertheless, outcome measurement error is likely. As with the exposure assessment, errors in the assignment of attention deficits in this prosp ective study will likely have resulted in attenuation of observed associations. Again, the likelihood of these errors is high, and the magnitude of the bias induced difficult to assess. The authors did not report on an assessment of changes in attentional difficulties between the 3.5 and 5 year visits. If such changes did occur, it would have been interesting to the reader to present results of a longitudinal analysis between this change in the outcome, and the child DAP levels over time.

The statistical analysis used to assess the associations between the markers of exposure and attention deficits were appropriate. Model diagnostics were performed and, according to the authors, found acceptable. The results of the model diagnostics were no t presented. The authors also conducted a host of sensitivity analyses to assess influence of modeling decisions on their findings, and generally found their results to be robust.

In addition to those included in final models, the following covariates we re considered, but not included in multivariate models: maternal age, parity, marital status, active/passive smoking exposure, regular alcohol use during pregnancy, presence of father in the home, maternal work status, and household income at the time of a ssessment. Confounding by blood lead levels was also assessed in a subset of children with measurements of cord blood (n = 229) and 2

- year (n = 296) lead levels, and found not to influence the DAP and attentional outcomes.

Covariates were selected for th ese analyses if they were related to conditions of testing or related to attention deficits in the literature and/or associated with more than one outcome (p < 0.10 or changes to the main effect coefficient by ≥ 10%). The authors used a less conservative a lpha level as a cut

- off for selection of covariates, a somewhat more conservative approach to covariate selection, relative to use of the conventional alpha level of 0.05. However, the use of a statistical test as a means to select covariates from a larger set of potential confounders is discouraged in some epidemiologic circles (Miettinen 1976, Breslow and Day 1980, Greenland 1989). The assessment of confounding is generally not considered to be a statistical endeavor, in part because the association betwe en the confounder and the outcome is just one factor that determines the degree of confounding, and also because the statistical significance of an association is a function not only of the magnitude of the association but also of other factors ( e.g. , samp le size, and sample variability) which are irrelevant to bias due to confounding. Moreover, the use of significance testing in the selection of confounders treats false negative errors ( i.e. the deleting a confounder) as secondary to false positive error ( inclusion of a non

-Page 80 of 105 confounder). This implicit ranking of error is backward, as deleting a confounder introduces bias and is justified only if the bias induced by its exclusion is tolerably small or deemed to be worth the precision gain, whereas including a non

- confounder only (potentially) reduces precision by increasing the size of the model. This is a small consideration in this study, and different approaches are not likely to qualitatively change the study findings or conclusions. Despite the confounder evaluation conducted by the investigators, residual confounding due to mismeasured or unmeasured confounders is likely to have somewhat biased the observed associations.

To preserve the sample size the authors appropriately imputed or replace data for mi ssing covariates. Data on maternal depression were missing for 5% of observations and maternal intelligence scores for less than 1%. Values of maternal depression measured when the child was 1 year old were available and substituted for 13 instances where maternal depression when the child was 3.5 years was missing. Other missing covariate values (six instances for maternal depression and three for maternal PPVT) were imputed by simple random selection of a value from participants with non

- missing values. T he resulting analysis is relatively more precise, owing to the larger sample size analyzed, but this statistical efficiency comes at the tradeoff of bias due to residual confounding by imperfectly measured covariates. Socioeconomic conditions and other dif ficult to measure determinants of early life development may further confound the reported associations in the direction of more deleterious effects associated with the organophosphate metabolites and with unknown magnitude. The potential for this bias may be mitigated somewhat due to restriction in the variability of these confounding factors in this relatively homogeneous study population.

The generalizability of the findings to other populations may be limited, due to the particular nature of this study population with respect to factors which may modify the association between organophosphate exposures and infant development. The organophosphate pesticide exposure markers and the prevalence of attention deficits in this low

- income Mexican

- American popul ation sampled from an agricultural area are almost certainly not generalizable to the U.S. population, although exposure levels are within a comparable range to the U.S. general population (see Section 5.0).

The study has numerous strengths, most notably the prospective design and the longitudinal assessment of both exposure and outcomes. The positive association between organophosphate metabolites in the urine of pregnant women and increased odds of attention problems and poorer attention scores in their offspring was relatively consistent using different assessments of attentional difficulties ( i.e. by maternal report, psychometrician observation, and neuropsychological testing). Associations were stronger at age 5 years compared to age 3.5 years, and th ere was evidence of a stronger association among boys, compared to girls. The results of this investigation by Marks et al in the CHAMACOS cohort are suggestive of a detrimental association between prenatal organophosphate exposure, as measured by maternal urinary metabolite levels, and attentional difficulties at age 5 years. This study may be limited in its direct contribution to the literature base regarding the specific association between chlorpyrifos and developmental outcomes because the assessment o f non

- specific organophosphate pesticide metabolites (DAPs) is a primary potential source of exposure measurementPage 81 of 105 error. Because of the prospective design, information bias is expected to result in attenuation of the true (unobserved) associations, toward (observed) associations that are relatively smaller, or null.Page 82 of 105 Article 13. Rauh et al. (2011) Seven

- Year Neurodevelopmental Scores and Prenatal Exposure to Chlorpyrifos, a Common Agricultural Pesticide. Virginia Rauh, Srikesh Arunajadai, Megan Horton, Fr ederica Perera, Lori Hoepner, Dana B. Barr, and Robin Whyatt. Environmental Health Perspectives. 119:1196

– 1201 (2011).

Study Summary

Having previously reported an association between prenatal exposure to chlorpyrifos and neurodevelopmental problems at th ree years of age in the Columbia Center for Children’s Environmental Health, Rauh et al now evaluate the relationship between prenatal chlorpyrifos exposure and neurodevelopment among cohort children at age seven.

Study participants were recruited during early pregnancy ( ≤20th week) among African

- American and Dominican women age 18

- 35 years, and registered Hospital, in New York at New York Presbyterian Medical Center and Harlem City. Women who smoked, had a history of drug abuse, diabetes, hypertension, o r HIV infection were excluded from participation in the study, as were women who resided in New York City for less than 1 year. The study sample presented in this report was recruited between 1998 and 2002, a period which straddles the January, 2001 implem entation of a voluntary cancellation by registrants of indoor residential chlorpyrifos use. Of originally 725 consenting women, 535 were active participants in the ongoing cohort study at the time of this report. This report uses data from 265 of their chi ldren 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 outcom e data.

Chlorpyrifos levels in umbilical cord blood samples were available for 256 newborns, sampled as close to the time of delivery as possible, and within 2 days post

- partum. In cases where the umbilical cord blood sample was not collected (12% of subj ects), cord blood chlorpyrifos levels were imputed from mothers’ values. Quantification of chlorpyrifos levels in plasma were conducted by the Centers for Disease Control and Prevention (CDC).

Neurodevelopment at age 7 years was assessed using 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. These indic es are associated with, but distinct from, overall intelligence quotient (IQ) and are sensitive to cognitive deficits related to learning and working memory, which have been linked to chlorpyrifos exposure in rodent studies. A Full

- Scale IQ score combines the four composite indices. A General Ability Index score is a summary score of general intelligence, similar to Full

- Scale IQ, but excludes contributions from both Working Memory Index and Processing Speed Index.Page 83 of 105 Multivariate linear regression models wer e used to estimate associations. Chlorpyrifos exposure level (picograms per gram) was entered as a continuous variable. The WISC

- IV Composite Index scores were natural log (ln) transformed to stabilize their variances and to improve the linear model fit, based on regression diagnostics. Linear and non

- linear functional relationships between chlorpyrifos exposure and each of the log

- transformed WISC

- IV indices were assessed and compared.

Participating mothers were predominantly from low income households , with 31% failing to complete high school by child’s age 7 years, and 66% were never married. The children in the study were largely full term and included very few low

- birth

- weight infants through study design. Chlorpyrifos exposure levels as measured i n cord blood ranged from non

- detectable to 63 pg/g.

Unadjusted correlations showed significant inverse associations between chlorpyrifos levels and Working Memory (r=

- 0.21, p=<0.0001); and Full

- scale IQ (r=

- 0.13, p=0.02). Because cord lead levels

were n ot significantly correlated with chlorpyrifos levels, and in an effort to avoid excluding those with missing lead levels, lead was not included in the regression models. ETS was significantly correlated with chlorpyrifos levels (Spearman coefficient 0.113 , p=0.01) but not with any of the WISC

- IV indices. In addition, birth weight was not significantly associated with any of the WISC scales and not included in the final models.

The investigators used both fully

- adjusted linear regression modeling and the Least Absolute Shrinkage and Selection Operator (LASSO) technique, described as a more parsimonious selection of covariates that addresses unstable estimates while avoiding overfitting (Houwelingen 2001; Tibshirani, 1996.) Both methods yielded very simila r estimates, finding associations between chlorpyrifos exposure and Full

Scale IQ as well as the Working Memory Index. For Full

- Scale IQ both techniques generated a coefficient (B) of

- 0.003 with matching CIs of

- 0.006, 0.001 but the P

- values were calcul ated as 0.064 for the LASSO and 0.048 for the fully

- adjusted model. The fact that both CI’s contain zero while the fully

adjusted model’s P

- value is lower, suggests that the LASSO technique does in fact shrink unstable estimates towards zero. For the Wor king Memory Index, both techniques generated a coefficient (B) of

- 0.006 with the LASSO calculating a CI of

- 0.009 to

- 0.002, p<0.001 and the fully

- adjusted model calculating a CI of

- 0.010 to

- 0.002, p=0.003. The investigators articulated these results a s 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. In order to understand the magnitude of these effects, the investigators calculated the neurodevelopmental deficit associated with the increase in exposure and 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% reduct ion in Working Memory. Authors explain that their full model results did not show any significant interactions between ETS or polycyclic aromatic hydrocarbons (PAH) with measures of prenatal chlorpyrifos exposure during the prenatal period and intelligenc e measures in this study.

The investigators conducted a sensitivity analysis to determine if the association between chlorpyrifos levels and Working Memory Index was partially due to the effect on general intelligence. To do this,Page 84 of 105 they looked at the Ge neral Ability Index of the WISC

- IV, a general intelligence scale calculated without the inclusion of the items included in the Working Memory Index or Processing Speed Index. When the General Ability Index was added to the linear regression model, the est imated chlorpyrifos effect did not change and they did not observe an interaction between chlorpyrifos and General Ability Index. From this they concluded that the Working Memory Index is a targeted measure, independent of general intelligence.

The inve stigators further explored their findings by conducting a supplemental analysis to evaluate whether the observed associations between chlorpyrifos and the Working Memory Index were related to underlying behavioral problems. They used the results of the Ch ild Behavior Checklist and its clinically

- oriented behavioral scales and concluded that there was no evidence of mediation caused by behavioral issues.

Lastly the investigators addressed the possible influence of their method and frequency of use of LOD imputations. To do this they recalculated all of the association estimates using only those records that had detectable chlorpyrifos levels and explained that this method would provide an unbiased estimate of the association (Little, 1992.) They did not find any consistent differences in estimates between the full sample (including imputed exposure levels) versus those calculated without those imputed levels.

Study Review

This was a very well conducted study with numerous strengths including selection of a relevant and appropriate cohort, the prospective study design, direct measurement of chlorpyrifos, comprehensive consideration of confounding, and analytic design choices which maximize statistical efficiency. However, some limitations are also noted.

The direct measurement of chlorpyrifos in cord blood and personal air samples, rather than self

reported indirect exposure assessment or use of biomarkers of organophosphate pesticide exposure that are not specific to chlorpyrifos exposure is a notable s trength of the Columbia Mother’s and Newborn Study cohort. Unlike previous analyses among this cohort that used a dichotomous variable (low vs. high exposure) to classify exposure status, these findings employ a continuous chlorpyrifos level, which provide s a more meaningful look at the data including the ability to assess potential threshold effects and linear and non

- linear dose

- response trends. The investigators rigorously evaluated their methods for imputing values for undetectable chlorpyrifos levels; analyses illustrated imputation methods were valid. However, regarding the LOD estimations, the authors did not describe the methodology used ( e.g. bootstrap estimation), if any, to incorporate the added uncertainty ( i.e. added imprecision to estimates) inherent in the use of imputed data into the estimation of the corresponding variance estimates. However, errors in estimation of cord blood chlorpyrifos levels were likely to have been non

- differential with respect to the indicators of childhood neurodev elopment and intelligence.Page 85 of 105 A limitation of the specific biomarker exposure indicators in this study is the single sampling period (at delivery). It is not clear to what extent this single measurement reflects exposures over critical windows during pregn ancy. If the exposure is chronic, a biomarker measured at a single time point can provide a representative dosimeter, even if the toxicant has a short half

- life, as is the case for chlorpyrifos. However, if pesticide exposures are sporadic or otherwise var y over short time scales, the biomarker measurement will not be representative of “usual” exposure, or of the exposure during critical periods of fetal neurodevelopment. In previous publications from this same cohort, the investigators explain that analys es were performed showing that, among these women, exposures to chlorpyrifos did not show significant variation throughout pregnancy (Whyatt et al, 2007) and therefore concluded that this single measure can accurately represent prenatal exposure. This ass umption is further assessed in subsequent validation studies conducted by Whyatt et al (2007 and 2009) within this cohort.

Whether or not these cord blood measurements accurately represent prenatal exposure, there is no control for exposure over the subs equent 7 years which may be critical, especially as the process of neurocognitive development is fluid and rapid during these early childhood years. From this study, it is unclear if an increase or decrease in exposure during this influential postnatal pe riod, caused by age

related or seasonal dietary changes or changes in pesticide application, could also impact the neurodevelopment of these children. Because this imprecise measure of exposure is unlikely to vary differentially, this error would bias tow ards the null.

Another influence unaccounted in the analysis is the effect of increased awareness of the risks of pesticide exposures over the time period of cohort follow

- up in this study (1998

- 2004) and the potential for differential exposure or outcome (CBCL) measures. It is feasible to assume that those parents of children with suspected or diagnosed neurodevelopmental issues in early childhood would be more aware of issues concerning organophosphate exposure, and thus more likely to subsequently redu ce their child’s exposures, especially through a reduction in pesticide or dietary sources in childhood. If this is true, and post

- natal exposures play a role in childhood neurodevelopment measured in this study, those children with neurodevelopmental def icits may be less likely to experience post

- natal exposures than those not diagnosed, thus making the pre

- natal effects appear weaker. Similarly, mothers of infants in the high exposure category may differentially report behavioral issues on the maternal

reported child behavior checklist . Either type of differential measurement error may lead to information bias, with unknown consequences on the estimate of risk in these studies. It would be meaningful to assess the mother’s awareness of the effects of or ganophosphate exposures as well as details regarding dietary exposures and how they may have changed over the 7

- year period.

The prospective nature of the study limits the influence of errors in the exposure assessment on the association observed, such that mismeasurement of chlorpyrifos exposure would likely be non

differential with respect to the indicators of neurodevelopment and intelligence at age 7 years. The voluntary cancellation of the residential use of chlorpyrifos introduces exogenous variati on in the exposure of interest. After the cancellation, levels of chlorpyrifos in personal and indoor air samples in this cohort decreased by more than 65%, and plasma blood levels dropped by more than 80%.Page 86 of 105 The Wechsler Intelligence Scale for Children (W ISC

- IV) instrument measures four areas of mental functioning: the Verbal Comprehension Index is a measure of verbal concept formation, a good predictor of school readiness; the Perceptual Reasoning Index measures nonverbal and fluid reasoning; the Working Memory Index assesses children’s ability to memorize new information, hold it in short

term memory, concentrate, and manipulate information; and the Processing Speed Index assesses ability to focus attention and quickly scan, discriminate, and sequentially order visual information. The authors explain that the sensitivity of the WISC

- IV among 6 to 7.5 year old children has been validated in previous neurodevelopmental studies of environmental lead exposure, even at low

- dose levels (Chiodo et al. 2004). How ever, WISC

- IV scores are also known to be influenced by socioeconomic background and/or child behavior problems, particularly those related to anxiety (Wechsler, 2003). The narrowly restricted cohort and evaluation of behavioral issues, which may in part r eflect anxiety issues, within this study somewhat limits this potential bias of the outcome assessment tool in this study.

One potential weakness is that the investigators do not give the details of the administration of the
WISC

- IV; specifically the cred entials of the interviewers, whether or not they were blinded to the child’s exposure status, any trainings or certifications, and whether or not intra

- or inter

- interviewer reliability or quality assurance was conducted. These types of administration iss ues can result in less

- sensitive measures and misclassification, but there is no reason to believe that there would be a differential effect based on exposure level.

Also complicating this analysis is the pervasive, non

- specific nature of neurological e ffects and the difficulty in attributing causal pathways to one agent. In future studies it would be meaningful to include other potential co

- variants such as family history (especially among parents and siblings) of developmental delay or attention disor ders as well as other co

- morbidities. Not only is the etiology of attention and related behavioral disorders not well understood but also the diagnosis of these disorders is often influenced by complicated outside forces. Because the study population is relatively homogenous with respect to socio

- economic variables, it is unlikely that differences in co

- morbidities or family history would be associated with exposure status, thus biasing the association towards the null.

The authors describe an elegant an d methodologically sound statistical analysis, addressing many of the potential shortcomings of their exposure data and co

- varia bles . Out of the five WISC

- IV scores, two were described as being significantly influenced by chlorpyrifos exposure. After the y conducted both full

- adjusted linear regression and LASSO techniques, they concluded that both Full Scale IQ and Working Memory index were affected by prenatal chlorpyrifos exposure. F or each standard deviation increase in exposure (4.61pg/g), there was a 1.4% reduction in Full

- Scale IQ and a 2.8% reduction in Working Memory. The Working Memory Index conclusions seem reasonable, but the associations for Full Scale IQ were not statistically significant (LASSO:

- 0.006, 0.001, p=0.064; fully

- adjusted:

- 0.00 6, 0.001, p=0.048.) In addition, the authors do not address the clinical relevance of the 1.4% and 2.8% reductions and how this may or may not influence a child or his/her psychological or educational

success.Page 87 of 105 In their confirmatory analyses they used t he General Ability Index (the intelligence score calculated after removing Working Memory and Processing Speed items) to verify that the association seen between chlorpyrifos exposure and Working Memory was not an artifact of the association with Full Scal e IQ. With the General Ability Index included in the linear regression model, the estimated chlorpyrifos effect did not change and they did not observe an interaction between chlorpyrifos and General Ability Index. Subsequently they concluded that the de ficits in Working Memory Index were associated with chlorpyrifos exposure, independent of general intelligence but analyses could have also taken an additional step. These results could also be used to conclude that since the General Ability Index was

not found to be affected by chlorpyrifos exposure, the Full Scale IQ findings were caused by the influence of the Working Memory module of the instrument. The influence of chlorpyrifos exposure on the working memory aspect of the Full Scale IQ score was stro ng enough to overcome the non

significant results from the other modules of the instrument.

Confounding by maternal smoking behaviors was controlled by restriction ( i.e. , excluding smokers from the study sample). Confounding by unmeasured factors may have been similarly controlled by design, this being a relatively homogeneous population with respect to determinants of neurodevelopment, compared to the general population. Authors evaluated several potentially confounding variables including exposure to oth er environmental chemicals including blood lead, and only included those variables which displayed evidence of confounding.

While controlling confounders by restriction, the choice to study low income, urban dwellers likely resulted in a relative increase in statistical efficiency ( i.e. increased power) because this population likely experiences a greater variability of pesticide exposure and is at higher risk of neurodevelopmental deficits, relative to the general population.

Although blood lead levels w ere not included as covariates in the multivariate models, the authors reported that umbilical cord lead was not significantly correlated with chlorpyrifos level or WISC

- IV scores among the subgroup of children in this population for whom blood lead was as sessed (n=89). Polyaromatic hydrocarbons were weakly correlated with chlorpyrifos in this cohort but were not significantly associated with WISC

- IV scores. Birth weight was not significantly associated with any of the WISC

- IV indices and was not included in the final models.

Bias due to confounding by unmeasured determinants of neurodevelopment is possible in this observational study, for example, confounding by factors related to socioeconomic condition. The urban dwelling, minority children assessed i n this study may experience other unmeasured exposures or underlying health problems that could potentially confound (or modify) associations with pesticide exposure. However, both adjustment through restriction of study population, as well as control for several measures of SES in multi

- variable models, as well as additional methodological analyses of the role of SES in this association (Lovasi et al. 2011), does not suggest a significant additional role of unmeasured confounding due to variation in SES wi thin this study population. Effect modification by allPage 88 of 105 of the covariates was assessed, including for environmental tobacco smoke and polycyclic aromatic hydrocarbons, but no interactions were observed.

The authors state that the retention rate for the ful l cohort was 82% at the 7

- year follow

- up, and reported that there were no significant sociodemographic differences between subjects retained in the study and those lost to follow

- up, although these data were not provided. However, the 265 children include d in this analysis were born to a 36.6% subsample of the 725 women who originally consented to participate in the study. The resulting large proportion of missing data has the potential to induce a selection bias if the reasons for missingness of outcome d ata are determined by exposure (or a predictor of exposure) and are also not independent of neurodevelopment. For example, participants with high pesticide exposures could have missed neurodevelopmental assessments for reasons related to uncontrolled deter minants of exposure which are also related to neurodevelopment. This was not discussed.

A comprehensive set of sensitivity analyses and regression diagnostics were conducted, including examination of residuals for normality and homoscedasticity, influent ial observations, and the linearity of the exposure

- response relationship, and assessment of the influence of design choices ( e.g. imputation) on observed results. Models were determined to be consistent with modeling assumptions.

Non

- linearity of the exposure response curves were assessed by superimposing smoothed cubic spline regression curves over scatterplots of the data. The log

- transformed Working Memory Index and Full

Scale IQ were approximately linear, whereas the other functions demonstrated so me curvature across exposure levels, with sparse observations at the highest exposures. However, formal goodness of fit testing did not demonstrate that non

- linear models provided a better fit to the data, compared to models that assume a linear exposure

- r esponse relationship ( i.e. no observed threshold effect).

Results of this study showed that higher prenatal chlorpyrifos exposure, as measured in umbilical cord blood plasma, was associated with decreases in cognitive functioning on two different WISC

- IV indices, in this cohort of urban minority children at 7 years of age. For each SD increase in exposure (4.61 pg/g), Full

- Scale IQ declined, on average, by 1.4% (0.94

– 1.8 points) and Working Memory Index scores declined by 2.8% (1.6

– 3.7 points). These resu lts are consistent with earlier findings of an inverse association between chlorpyrifos levels in umbilical cord plasma and neurodevelopment in the same cohort. The study represents perhaps the most rigorous study design and analysis of the studies reviewe d.Page 89 of 105 Works Cited

Bradley RH and Corwyn RF. Socioeconomic Status and Child Development. Annual Review of Psychology.
53:371

- 399 (2002).

Chiodo LM, Jacobson SW, Jacobson JL. 2004. Neurodevelopmental effects of postnatal lead exposure at very low levels. Neurotoxicol and Teratol, 26:359

- 371.

Houwelingen JCv. 2001. Shrinkage and penalized likelihood as methods to improve predictive accuracy. Stat Neerlandica. 55(1):17

- 34.

Lovasi, G. S., Quinn, J. W., Rauh, V. A., Perera, F. P., Andrews, H. F., Garfinkel, R., . . . Rundle, A. (2011). Chlorpyrifos exposure and urban residential environment characteristics as determinants of early childhood neurodevelopment. Am J Public Health, 101 (1), 63

- 70. doi: 10.2105/ajph.2009.168419 Little RJA. 1992. Regression with mis sing X’s: a review. J Amer Stat Assoc. 87:1227

– 1237.

Wechsler D. Wechsler Intelligence Scale for Children. 4rd Edition. San Antonio, Texas: Psychological Corporation. 2003.

Whyatt RM, Rauh V, Barr DB, et al. Prenatal insecticide exposure and birth weight and length among an urban minority cohort. Environ Health Perspect. 2004;112:1125

– 1132

Whyatt RM, Garfinkel RS, Hoepner LA, Borjas M, Camann DE. Within and between home variability in indoor

- air insecticide levels during pregnancy [abstract W2A

– 3]. Pres ented at the 15th Annual Conference of the International Society of Exposure Analysis; October 30

– November 3, 2005; Tucson, AZPage 90 of 105 Article 14. Bouchard et al. (2011) Prenatal Exposure to Organophosphate Pesticides and IQ in 7

- Year

- Old Children. Maryse F. Bo uchard, Jonathan Chevrier, Kim G. Harley, Katherine Kogut, Michelle Vedar, Norma Calderon, Celina Trujillo, Caroline Johnson, Asa Bradman, Dana Boyd Barr, Brenda Eskenazi. Environmental Health Perspectives. 119:1189

– 1195 (2011)

Study Summary

Researchers with the Center for the Health Assessment of Mothers and Children of Salinas study (CHAMACOS study) published an analysis which investigated the relationship between prenatal and early life exposures to organophosphate pesticides (OPs) and cognitive develo pment. The CHAMACOS study is a prospective birth cohort study of environmental exposures and the health of pregnant women and their children living in the Salinas Valley, an agricultural area of California.

A 2007 report from this cohort suggested that p renatal, but not postnatal, exposure to OPs was associated with increased odds of pervasive developmental disorder and lower scores of mental development at two years (Eskenazi B 2007). Additionally, a 2010 paper reported an association between organophos phate pesticide exposure and poorer attention skills as well as hyperactive behaviors at 5 years (Marks AR 2010). This 2011 study by Bouchard et al was conducted to determine whether cognitive deficits associated with prenatal exposure to organophosphate p esticides are persistent, since previous cohort studies have not followed children to school

- age, when cognitive deficits may have greater implications for school performance. This paper reports the association between prenatal and postnatal exposure to or ganophosphate pesticides, indicated by urinary dialkyl phosphate (DAP) metabolite concentrations, and cognitive abilities of 7

- year olds.

Enrollment took place from 1999

– 2000 at community clinics, and included women who were ≥ 18 years old, pregnant at < 20 weeks gestation, Spanish

- or English

- speaking, eligible for low

- income health insurance, and planning to deliver at the local public hospital. The initial 601 women who were enrolled into the study delivered 526 infants. From this analysis, 2 participan ts with missing DAP concentration measurements were excluded, 4 children with a medical condition that would affect the assessment, children who were lost to follow

- up or who did not participant in the 7

- year visit were excluded (72 moved, 59 refused, 24 u nable to trace, 21 unable to schedule, 2 deceased), and children missing the cognitive assessment at the 7

- year visit (13) were also excluded.

Prenatal urine was collected between 5 and 27 weeks gestation and again between 18 and 39 weeks. Urine was colle cted from the children at 6 months and 1, 2, 3 1⁄2 and 5 years of age. Six nonspecific organophosphate DAP metabolites (3 DEP metabolites and 3 DMP metabolites) were measured in maternal and child urine which represent the breakdown products of about 80% of the total organophosphate pesticides used in the Salinas Valley (CDC 2009). The most commonly used organophosphate pesticides in the Salinas Valley include chlorpyrifos and diazinon (which arePage 91 of 105 metabolized to DEP) as well as malathion and oxydemeton

- methyl (which devolve to DMP). Concentrations below the limit of detection (LOD) were randomly imputed based on a log

- normal probability distribution whose parameters were estimated using maximum likelihood estimation. The DAP metabolite concentrations were expre ssed on a molar basis and summed to yield total DEP, DMP, and DAP concentrations.

Cognitive abilities were measured at the 7

- year study visit using the Wechsler Intelligence Scale for Children

- Fourth Edition (WISC

- IV) (Wechsler 2003). Scores for four do mains were calculated based on the following subtests: Verbal Comprehension (comprised of the Vocabulary and Similarities subtests), Perceptual Reasoning (Block Design and Matrix Reasoning subtests), Working Memory (Digit Span and
Letter

- Number Sequencing subtests), and Processing Speed (Coding and Symbol Search subtests). All subtests were administered in the dominant language of the child, which was determined through administration of the Oral Vocabulary subtest of the Woodcock

- Johnson/Woodcock

- Muñoz Tes ts of Cognitive Ability in both English and Spanish (Woodcock and Johnson 1990). Ultimately, 67% of children were tested in Spanish, and 33% in English. WISC

- IV scores are standardized against U.S. population

based norms for English and Spanish

- speaking ch ildren.

Many confounders were measured, including intelligence of the mother, measures of how stimulating the environment is, and known or suspected neurotoxins were measured prenatally. Maternal intelligence was assessed via the Peabody Picture Vocabular y 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 vis its. The following known or suspected neurotoxicants were measured: polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), p,p’

- dichlorodiphenyltrichlorethane (DDT), p,p’

- dichlorodiphenyltrichlorethylene (DDE), and lead. Lead was measur ed in maternal blood at 26 weeks gestation, in cord blood for a subset of participants, and children’s blood a 2 years of age. PBDEs, PCBs, DDT, and DDE were measured in maternal serum samples collected at 26 weeks gestation.

Nonspecific total DAP, DEP, and DMP metabolites (nmol/L) were log10

- transformed. The authors examined the association between non

- creatinine adjusted urinary DAP concentrations and cognitive scores using multiple linear regression, with point estimates representing the change in cogn itive scores for each 10

- fold increase in DAP concentrations. For prenatal exposure, they examined associations with the DAP concentrations measured separately for urine collected during the 1st and 2nd half of pregnancy ( ≤20 vs. >20 weeks gestation). Howe ver, since they found similar relations between cognitive scores and DAPs measured earlier and later than 20 weeks gestation, they averaged the two DAP measures for further analyses. For 20 children (6%) only one prenatal measure was available for the anal yses. Because DMP and DEP metabolites might have different relationships to the outcomes, the authors examined them separately. For postnatal exposure, they examined the cross

- sectional association of cognitive scores with DAP concentrations measured in ch ildren’s urine collected at different ages in separate models.Page 92 of 105 They also calculated the cumulative DAP level between 6 and 60 months using the area under the curve (AUC), calculated using the trapezoidal method. For 46 children with 1 missing DAP measurem ent at 1, 2, or 31⁄2 years, they imputed the mean of the 2 measures closest in time for the AUC calculation. Forty

nine children who were missing DAP measures at either the 6

- month or 5

- year visit, or missing more than 1 DAP measure from the 3 other time poi nts were excluded from the AUC analysis. For comparison with prenatal exposure, they calculated the mean urinary DAP concentrations measured during childhood for children with at least 3 out of 5 measures (taken at 6 months, 1, 2, 3 1⁄2, and 5 years); this

e xcluded 20 children.

The authors compared effect estimates for urinary DAPs measured in early vs. late pregnancy and in the prenatal vs. postnatal periods using seemingly unrelated estimation and they used the mean postnatal DAP concentrations (as opposed to AUC) for these analyses in order to compare metrics with similar units. They used generalized additive models with 3

- degree

- of

- freedom cubic splines to evaluate the shape of dose

- response curves, test the linearity assumption, and investigate potential thresholds while controlling for covariates. They did not observe evidence of departure from linearity, or threshold for effect, thus, they retained the simpler models based on linear regression. For illustration purposes, they grouped DAP concentrations into quintiles, entered this categorical variable in the multiple regression model with the same covariates described above, and obtained the mean IQ score for each quintile.

The authors explored potential confounding effects for the following variables: maternal intellectual abilities, maternal education, HOME score at 6 months, 1, 2, 3 1⁄2, and 5 years, breastfeeding duration, maternal age, birth order, poverty category, marital status, children’s age at WISC testing, and maternal levels of PBDEs, PCBs, DD E, DDT, and lead during pregnancy. They added each of these variables added individually to the final model. However only maternal intellectual abilities (PPVT score, continuous), maternal education (3 categories), and continuous HOME score at 6 months wer e retained in the final models. Other variables were dropped from the final models because they did not change the magnitude of the coefficient for urinary DAP concentrations by >10%.

In separate analyses, the authors also investigated potential confound ing and effect modification by variables possibly on the causal pathway ( i.e. birth weight and gestational age, assessed continuously). Because the majority of children (67%) were tested in Spanish, the authors reran the analyses restricted to this subset. The authors also examined the interaction between sex and DAP concentrations, based on previous findings in this cohort (Marks AR, 2010).

To explore possible synergistic effects between pre

- and postnatal DAP concentrations, the authors included an inter action term for mean prenatal DAP concentrations×AUC. However, since this term was not statistically significant (p > .15) they did not include it in the final models.

The authors observed evidence of an association between prenatal exposures to organopho sphate pesticides as measured by urinary DAP metabolites in women during pregnancy, and decreased cognitive functioning in children at age 7. This finding was seen for total DAP, DMP, and DEPPage 93 of 105 metabolites. Children in the highest quintile had of maternal DA P concentrations had an average deficit of 7.0 IQ points compared with those in the lowest quintile. The associations were linear with no threshold. Urinary DAP concentrations in childhood were not associated with cognitive scores in this cohort of childre n.

Study Review

This study tested whether in utero or postnatal exposures to organophosphate pesticides have a detrimental impact on cognitive abilities at age seven years. The study population was appropriate to the hypothesis, given that there were a r ange of exposure levels to OPs and there was some homogeneity in the population, e.g. , mostly low SES, Hispanic, agricultural workers. The homogeneity of the population restricted confounding by design. For example, another paper reported that maternal

smo king, alcohol use, and illicit drug use were rare this cohort. The recruitment of an inner

- city cohort may also have increased the relative statistical efficiency of the study if the study population is at higher risk of neurodevelopmental deficits compare d to the general population. Selection bias may have been a factor in this analysis as only 329 families out of 601 who were originally enrolled into the study were included. The authors state that the families included in this analysis did not differ on m ost attributes, including urinary DAP concentrations during pregnancy, maternal measures of cognitive ability, maternal education, marital status, poverty category, and child’s birth weight. However, mothers of children included in the present study were s lightly older and breastfed longer than the initial cohort. Differences between maternal blood lead and other environmental exposures which are potential confounders were not discussed.

The ascertainment and analysis of urinary organophosphate pesticide m arkers was appropriate. Quantification was conducted at the CDC, using published methods. DAP metabolites are likely more accurate and objective indicators of organophosphate pesticide exposure, relative to other ascertainment methods such as self

- report. Although they are sensitive indicators of exposure, it is difficult to infer chlorpyrifos effects specifically from the metabolite levels, as they also indicate exposure to other organophosphates. The study population likely sustained many other potentiall y relevant exposures to multiple chemicals and other pesticides which may affect development. The six organophosphate metabolites cannot be traced back to individual pesticides but, according to the authors, they represent the breakdown products of approxi mately 80% of the total organophosphate pesticides used in the Salinas Valley. It is not possible to disentangle the various effects from these potential co

- exposures and chlorpyrifos in this study.

The IQ test used the WISC

- IV is very widely utilized in both clinical and research setting, and has a long history of use, and was appropriately administered by psychometrician. Additionally, the test was administered at age 7 years, which is above the flo or for administration (age 6) (Wechsler 2003). However, IQ tests are notoriously susceptible to possible biases due to differences in cultural and/or ethnic composition of the population, although the homogeneity of the population may ameliorate the effect of this potential bias. The authors did perform the analysis both by looking at only the tests thatPage 94 of 105 were administered in Spanish and with the Spanish and English included. However, language may not have controlled for all potential biases which are inhere nt in IQ testing, e.g. , degree of acculturation.

The statistical models used are appropriate and well powered. For exposure measures that were less than the laboratory analytic limit of detection, the measures were imputed. Imputation is a better method than substitution of the LOD/ √2 or LOD/2, which is the method used in many studies. It appears that the investigators selected parsimonious models by only including terms which changed the outcome measure and were statistically significant. Additionally, t he authors tested the linearity assumption which is inherent in linear regression using cubic splines.

Residual confounding by unmeasured or poorly measured confounders is possible in this study. The authors did measure a large number of potential confou nders, but not all were evaluated notably, childhood exposure to neurotoxins, and maternal drug used during pregnancy. A number of environmental chemicals were considered, although only measurements of exposure during pregnancy were included in the analysi s. Additionally, exposure to carbamates, which are also potentially neurotoxic, was not included in the analysis. The authors also did not consider maternal alcohol, tobacco, or drug use in this analysis. However, another study with this cohort reported th at these confounders are very rare in this study population.

The authors did not discuss paternal intelligence or intelligence of other caregivers. This is probably because only the mothers and children were enrolled into the study, and it is difficult to enroll fathers and other caregivers into birth cohort studies. Although it is understandable that these measures were not addressed, it seems highly possible that intelligence of the fathers and other caregivers could be associated with the exposure, orga nophosphate pesticides, and the disease, cognitive impairment of the child. Thus these factors could have confounded the study results, and it is a limitation that they were not included in the measurement or analysis.

A potential limitation with respect to generalizability is the high level of exposures observed in the study cohort, compared to the US population. The mean level of urinary DAP metabolites in this cohort is higher than the mean which has been seen among women of child bearing years in NHAN ES. However, the investigators report that 25% of the US population have urinary DAP levels exceeding the median in this study. So, the concentrations in this cohort are not out of the range of normal for the US as a whole. Generalizing beyond the range of exposure observed in this study requires an assumption that the linear
exposure

- outcome relationship observed in the study is applicable to populations with different levels of
exposure.

This study has many strengths, the longitudinal design, the measure ment of urinary DAP at many 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,Page 95 of 105 reducing the potential for uncontrolled confounding. Additionally, the autho rs noted their findings were consistent with findings from previous rep orts from this cohort (Eskenazi, 2007) and other investigations of adverse associations between prenatal exposure to organophosphate pesticides and cognition (Rauh
, 2006). They also not ed that although they did not see an association between childhood exposures to organophosphate pesticides, the other studies that have differed in the measurement of exposure and/or the specific outcomes found to be associated with exposure to organo phosp hate pesticides (Lizardi, 2008, Ruckart, 2004). The many advantages of this study, the plausibility of the findings, and that similar association have been seen in other studies support the hypothesis that prenatal exposure to OPs reduces cognitive functio n of children. However, t o the extent that prenatal chlorpyrifos is the true exposure of interest, the assessment of non

- specific organophosphate pesticide metabolites is a primary potential source of exposure measurement error. Because of the prospective design, information bias is expected to result in attenuation of the true (unobserved) associations, toward (observed) associations that are relatively smaller, or null.Page 96 of 105 Works Cited

Bradman A, Barr DB, Claus Henn BG, Drumheller T, Curry C, Eskenazi B. "Measurement of pesticides and other toxicants in amniotic fluid as a potential biomarker of prenatal exposure: a validation study." Environ Health Perspect, 2003: 1779

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- IV: Administration and Scoring Manual. San Antonio, TX: Harcourt Assessment Inc, 2003.Page 97 of 105 Whyatt RM, Garfinkel R, Hoepner LA, Andrews H, Holmes D, Williams MK, et al. "A biomarker validation study of pren atal chlorpyrifos exposure within an inner

- city cohort during pregnancy." Environ Health Perspect, 2009: 559

- 567.Page 98 of 105 Article 15. Whyatt et al. (2007) Within

- and Between

- Home Variability in Indoor

- Air Insecticide Levels during Pregnancy among an
Inner

- City Cohort from New York City. Robin M. Whyatt, Robin Garfinkel, Lori A. Hoepner, Darrell Holmes, Mejico Borjas, Megan K. Williams, Andria Reyes, Virginia Rauh, Frederica P. Perera, and David E. Camann. Environmental Health Perspectives. 115:383

– 389 (2007).

Study Summary

Whyatt et al conducted a study to assess within

- and between

- home variability in indoor

- air insecticides over the final 2 months of pregnancy among a subset of participants in the Columbia Mother’s and Newborn Study, a cohort of women from N ew York City. Participants (n = 102) assessed in this investigation were enrolled between February, 2001 and May, 2004, after the January, 2001 implementation of a voluntary cancellation by registrants of indoor residential chlorpyrifos use. Expectant mo thers were recruited during early pregnancy ( ≤20 th week) among African

- American and Dominican women age 18

- 35 years, and registered at New York Presbyterian Medical Center and Harlem Hospital, in New York City. Women who smoked, had a history of drug abuse , diabetes, hypertension, or HIV infection were excluded from participation in the study, as were women who resided in New York City for less than 1 year or, for this validation study, who were employed outside the home at the time of enrollment. Most of the mothers (96%) in the present study lived in residential apartment buildings; the remaining lived in multifamily housing (n = 3) or in combined residential/commercial buildings (n = 1).

Nine insecticides (the organophosphates chlorpyrifos, diazinon, ma lathion, and methyl parathion; the carbamates propoxur, bendiocarb, and carbofuran; and the pyrethorids cis and trans

- permethrin) and piperonyl butoxide, an adjuvant that is frequently added to pyrethroid formulations, were measured in
48

- hr personal air s amples and 2

- week integrated indoor air samples collected sequentially for 7.0 ± 2.3 weeks (n = 337 air samples). Indoor air monitoring occurred around the 32nd week of pregnancy, and air samples were collected every 2 weeks throughout the monitoring perio d. Personal air samples were obtained for mothers over a 48 hour period during the 32nd week of pregnancy.

Arithmetic means ± SDs were calculated for pesticides detected in greater than 45% of samples. Before statistical analyses, pesticide values were lo g

- transformed to normalize their distributions. Values below the level of detection (LOD) were assigned a value of half of the LOD. Pearson correlation coefficients or Spearman’s correlation coefficients, as appropriate, were calculated to assess the relat ionship between pesticide levels in indoor air and personal air samples. Analysis of variance (ANOVA) was used to test whether pesticides levels varied significantly by season and year of monitoring and among the following groups: women not using any pest control methods; women using “nonspray” methods only; and women using higher

- toxicity pesticides. If levels differed significantly among the groups, the least significant difference test was used to determine which groups varied significantly. T

- tests we re employed to compare differences in mean pesticide levels by ethnicity. Chi

- square analyses were usedPage 99 of 105 to test whether the proportion of women using pest control measures over the final 2 months of pregnancy was related to pest sightings in the home. Fin ally, a mixed

- effects model was used to assess within

- home and between

- home variability in repeat indoor air samples, after controlling for season and year of delivery. Except for the Spearman’s rank and chi

- square analyses, all statistical testing was

con ducted on log

- transformed data.

Sixty

- one percent of the participating women reported using pest control during the air samplings. Chlorpyrifos, diazinon, and propoxur were detected in 99

– 100% of personal and indoor samples (range,
0.4

– 641 ng/m 3 ). Pipero nyl butoxide (a pyrethroid adjuvant) was detected in 45.5

– 68.5% (0.2

– 608 ng/m 3 ). 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 th e 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). Diazinon and chlorpyrifos levels declined 5

- fold between 2001 and 2004 but were detected in all homes at 1.5 and 2.5 years, respectively, after implementation of a voluntary cancellation by registrants of indoor residential chlorpyrifos use. No previously unreported associations b etween chlorpyriphos and health outcomes were presented in this report. The authors concluded that the insecticides were persistent in the home with little variability in air concentrations over the 2 months and likely contributed to chronic maternal inhal ation exposures during pregnancy.

Study Review

Although the Columbia Mother’s and Newborn Study enrolled participants in years prior to the implementation of the 2001 voluntary cancellation by registrants of indoor residential chlorpyrifos use, only data for participants recruited in the post

- cancellation period were considered in this validation study analysis. Explanation for this design choice was not provided, but is likely that appropriate repeated sampling was not conducted during earlier study peri ods. The authors noted that the mothers in the present study were comparable with women in the larger cohort in terms of maternal age, ethnicity, marital status, annual household income, and reported use of pest control methods during pregnancy. However, a greater proportion of women in the present study compared with those in the larger cohort reported having less than a high school education, which could be due to the fact that, for these analyses, the investigators excluded women who worked outside the home.

The exposure assessment in this study was well conducted using appropriate and previously published methodology. The semivolatile insecticides were readily detected in indoor air after residential use. Indoor air levels of these chlorpyrifos and the other organophosphate pesticides in the 2

- week integrated indoor air samples remained remarkably stable within most homes over 6

– 8 weeks of sequential sampling. The two

- week integrated sampling methodology was targeted towards evaluation of chronic ex posure during the latter pregnancy period, and thus precludes assessment of changes in concentrations in indoor air at times scales shorter than two

- weeks ( e.g. day

- to

- day changes).Page 100 of 105 In this study, pesticide levels in indoor air were remarkably stable. Correspondingly, almost all (90%) of the total variance in the indoor air levels was explained by between

- home variability. The degree to which this finding in the post

- cancellation peri od informs variability in airborne pesticide levels prior to the cancellation was beyond the scope of the investigation.

The authors did not observe an association between indoor air levels of chlorpyrifos and maternal self

reported pesticide use, a find ing which indicates that most of the chlorpyrifos concentrations detected in the indoor air samples likely resulted from prior, rather than current, pesticide use. However, that chlorpyrifos was detected in indoor air samples from all the homes assessed, including those evaluated 2.5 years after implementation of the voluntary cancellation of indoor residential chlorpyrifos use, demonstrates the persistence of the compound in certain indoor environments.

No associations between pesticide exposures and he alth outcomes were assessed in this validation study.Page 101 of 105 Article 16. Whyatt et al. (2009) A Biomarker Validation Study of Prenatal Chlorpyrifos Exposure within an Inner

- City Cohort during Pregnancy. Robin M. Whyatt, Robin Garfinkel, Lori A. Hoepner, Howar d Andrews, Darrell Holmes, Megan K. Williams, Andria Reyes, Diurka Diaz, Frederica P. Perera,1 David E. Camann, and Dana B. Barr. Environal Health Perspectives. 117:559

– 567 (2009).

Study Summary

Whyatt et al conducted a biomarker validation study within the Columbia Mother’s and Newborn Study to evaluate trends over time in multiple biomarkers of prenatal chlorpyrifos exposure. The group previously documented significant decreases in chlorpyrifos concentrations in maternal personal and indoor air samples among pregnant African

- American and Dominican women from New York City after the 2001 voluntary cancellation by registrants of indoor residential chlorpyrifos use. Participants (n = 102) were enrolled between February, 2001 and May, 2004. 3,5,6

- trichloro

- 2

- pyridinol (TCPy) was measured in newborn meconium (n = 83), repeat prenatal maternal urine samples (n = 253), and postnatal urine from mothers (n = 73) and newborns (n = 59). Chlorpyrifos was directly measured in postnatal maternal (n = 92) and umbilical cord (n = 65) blood plasma and in personal and indoor air samples. No previously unreported associations between chlorpyriphos and health outcomes were presented in this report.

Study participants were recruited during early pregnancy ( ≤20 th week) among A frican

- American and Dominican women age 18

- 35 years, and registered at New York Presbyterian Medical Center and Harlem Hospital, in New York City. Women who smoked, had a history of drug abuse, diabetes, hypertension, or HIV infection were excluded from pa rticipation in the study, as were women who resided in New York City for less than 1 year or who were employed outside the home at the time of enrollment (for the purposes of this validation study). The study sample presented in this report was recruited between 2001 and 2004, after the January, 2001 implementation of a voluntary cancellation by registrants of indoor residential chlorpyrifos use. Most of the mothers (96%) in the present study lived in residential apartment buildings; the remaining lived in multifamily housing (n = 3) or in combined residential/commercial buildings (n = 1).

Indoor air monitoring occurred around the 32nd week of pregnancy using 2

- week integrated indoor air samples collected every 2 weeks throughout the monitoring period. Per sonal air samples were obtained for mothers over a 48 hour period during the 32nd week of pregnancy.

Repeat prenatal maternal urine samples were collected from mothers at the end of each 2

- week indoor air sampling period, beginning at the 34th week of pre gnancy and continuing until delivery. Women who went into labor before the end of the first 2 weeks of indoor air sampling did not provide urine samples. Urine samples were collected from 97 of 102 women in the study; more than half of the women provided t hree or more urine samples, 29 of 97 (30%) two samples, and 15 of 97 (15%) provided onePage 102 of 105 sample. An additional urine sample was obtained on the day after delivery for 73 women and 59 newborns.

An umbilical cord blood sample was collected from 65 newborns after delivery. A sample of maternal blood was obtained from 92 mothers within 2 days postpartum. Paired maternal and cord blood samples were available for 64 of the mothers and newborns.

Meconium samples were collected from 83 newborns during the postpar tum hospital stay. In 53 (64%) of the births, the meconium samples were collected within 1 day of delivery.

Biologic samples were analyzed for organophosphate pesticide markers by the CDC using previously established methods using both positive and negati ve controls. 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 log 10 transformed. Nonparametric statistics were used when distributions remained non

- Gaussian. Spearman rank correlation coefficients were used to assess relationships between level s of chlorpyrifos or TCPy in biological samples and to examine correlations between levels of chlorpyrifos or TCPy in the biological samples and chlorpyrifos levels in air samples. Multiple linear regression was employed to evaluate the amount of variance in prenatal maternal urine TCPy levels explained by indoor air chlorpyrifos levels among subjects enrolled in 2001

– 2002.

Analysis of variance (ANOVA) or Kruskal

– Wallis tests with post

- hoc testing as appropriate, were used to assess whether pesticide lev els varied by year of birth (between 2001 and 2004), or among the following groups: women not using any pest control methods; women using “nonspray” pest abatement methods only; and women using can sprays, pest bombs, or sprays by an exterminator, with or without nonspray methods. Authors performed t

- tests corresponding nonparametric Mann

– Whitney U

- test were used to assess whether pesticide levels differed by ethnicity. Chi

- square analyses were used to test whether the frequencies of detection of chlorpyrif os and TCPy in biological samples varied by year of assessment as well as to compare detection frequencies of chlorpyrifos in maternal and cord blood samples. A mixed effects model was used to evaluate the within

- and between

- subject variability in TCPy le vels in the repeat maternal urine samples collected during pregnancy, and to calculate an intraclass correlation coefficient (ICC) for women enrolled in 2001

– 2002 who had three repeat urine samples (TCPy was detected in only one

- third of samples among subj ects enrolled after 2002). The ICC is a measure of reliability of repeated measures over time, defined as the ratio of between

- subject variance to total
variance.

The authors did not detect TCPy in infant urine, but all other biomarkers showed a highly s ignificant decrease in detection frequencies (χ2 = 7.8

– 34.0, p ≤ 0.005) and mean ranks (p ≤ 0.006) among subjects enrolled in 2003

– 2004 compared with those enrolled in 2001

– 2002. Chlorpyrifos in maternal personalPage 103 of 105 and indoor air declined 2

- to 3

- fold over t he same period (p < 0.05). In 2001

– 2002 samples, TCPy levels in repeat prenatal urine samples were positively correlated (r = 0.23

– 0.56), although within

- subject variability exceeded between

- subject variability (intraclass correlation coefficient = 0.43); indoor air levels explained 19% of the variance in prenatal urine TCPy (p = 0.001). 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).

Study Review

Although the Columbia Mother’s and Newborn Study enrolled participants in years prior to the implementation of the 2001 voluntary cancellation by registrants of indoor residential chlorpyrifos use, only data for participants recruit ed in the post

- cancellation period were considered in this analysis. Explanation for this design choice was not provided, but is likely that appropriate repeated sampling was not conducted during earlier study periods. The authors noted that the mothers i n the present study were comparable with women in the larger cohort in terms of maternal age, ethnicity, marital status, annual household income, and reported use of pest control methods during pregnancy. However, a greater proportion of women in the prese nt study compared with those in the larger cohort reported having less than a high school education, which could be due to the fact that, for these analyses, the investigators excluded women who worked outside the home.

The investigators’ use of nonparam etric, rank

- based statistics is appropriate for analysis of non

- normally distributed data. However, the large number of ties among marker levels due to observations below the level of detection receiving equal rank, may be problematic. When ranked values h ave the same numeric number ( e.g. , half the level of detection), the rank is 0.5 times the number of ties. Whether, and at what point a large proportion of ties begins to limit the inferential power of rank based statistics is unclear, although at some poi nt, a non

- continuous classification of the data ( e.g. , classification of observations into bins at a cut

- point equal to the level of detection) seems to make more sense if the number of below the level of detection observations is large.

The exposure ass essment conducted in this investigation is the most comprehensive of any epidemiologic study of chlorpyrifos to date. Strengths of the exposure assessment include the quantification of actual blood chlorpyrifos levels in addition to sensitive, but non

- spec ific markers of organophosphate pesticide exposure in urine, the repeated sampling of urinary markers, measures in meconium and the environmental sampling. The exposure assessments were appropriately timed with respect to critical window of gestation for f etal growth and neurodevelopment outcomes which likely occurs, in part, during the third trimester of pregnancy, and appear to have been well conducted. Although inhalation exposures are likely a major route of exposure, no direct dietary assessment was
i ncluded in this investigation.

The study was set during a period of rapidly declining use of chlorpyrifos, and the findings are demonstrative of decreasing exposure over time. The investigators observed a highly significantPage 104 of 105 decrease in the detection freq uency and mean ranks of all biomarkers among subjects enrolled during years 2001

– 2004, with the exception of TCPy in newborn urine, which was not detected.

Maternal and umbilical cord blood plasma chlorpyrifos levels were below the LOD in all samples col lected after 2002. The results suggest that chlorpyrifos from residential use was the main contributor to the chlorpyrifos levels detected in both the maternal and umbilical cord blood among study subjects in the larger cohort (1998

- 2004, both before and after voluntary cancellation of chlorpyrifos containing compounds).

While no association was observed between maternal and umbilical blood chlorpyrifos levels measured at delivery and TCPy in maternal urine samples collected through the latter third trime ster, TCPy levels in meconium (an integrative measure week 13

- delivery) were significantly correlated with chlorpyrifos levels in both maternal and umbilical cord blood measured at delivery, and with TCPy levels in maternal urine samples collected in third trimester. These results suggest that TCPy in meconium may provide a valid biomarker of prenatal exposure, possibly both prior to and subsequent to the cancellation period.

The finding that within

- subject variability exceeded between

- subject variability in maternal urine TCPy (intraclass correlation coefficient, ICC= 0.43), again emphasizes the point that a single prenatal urine sample is likely insufficient to capture ‘usual’ exposure. The authors noted that despite the significant within

- subject variabi lity seen in their study, the ICC observed was higher than that observed in another population (Meeker et al., 2005).

The authors concluded that their results suggest that pesticide biomarkers utilized in these studies are reliable dosimeters to different iate between groups with prenatal chlorpyrifos exposures varying by a factor of 2 or more, and that cord blood measures are reasonable well correlated with exposure measures taken throughout pregnancy (meconium correlated with cord blood (delivery) and mat ernal urine (3 rd trimester). Authors also support that these data illustrate the efficacy of the voluntary cancellation of indoor residential chlorpyrifos use to reduce the internal dose during pregnancy.Page 105 of 105 Works Cited Meeker, J. D., Barr, D. B., Ryan, L ., Herrick, R. F., Bennett, D. H., Bravo, R., & Hauser, R. (2005). Temporal variability of urinary levels of nonpersistent insecticides in adult men. J Expo Anal Environ Epidemiol,
15 (3), 271

- 281. doi: 7500402 [pii] 10.1038/sj.jea.7500402