REVIEW OF CHILDREN’S HEALTH 

EPIDEMIOLOGY COHORT STUDIES

CAROL H. CHRISTENSEN, PH. D., MPH

EPIDEMIOLOGIST

USEPA, OFFICE OF PESTICIDE PROGRAMS

FIFRA SAP April 10, 2012
Background and History 

 September 2008 EPA presented targeted review of

three children’s health cohort studies

 FIFRA SAP Report:

 “Chlorpyrifos likely played a role”

Cannot conclude “chlorpyrifos is the sole contributor” due to 

2

 Cannot conclude “chlorpyrifos is the sole contributor” due to 

other co-exposures

 However, epidemiology studies "should not be 

considered ...basis for characterization of PoD” 

 Lack biologically plausible MOA, exposure uncertainties

 Supplemental analyses recommended
Background and History 

 April 2012 EPA updates and expands its targeted review of

these data: 

 New etiologic papers

 Intelligence Quotient (IQ)

 New methodological papers

 Socioeconomic status (SES)

3

 Exposure characterization and validation

 Supplemental analyses (unpublished) (Appendix 4)

 Draft Framework Analysis approach 

 Working toward WOE Evaluation

 Panel guidance regarding qualitative and/or quantitative 

use these studies in risk assessment
Overview of Cohorts

4

 Columbia cohort (1998-2002)

 NYC, Dominican and African -American Mothers-Infant pairs

 Cord Blood Chlorpyrifos

 Validation studies (air, maternal blood, urine, meconium)

 Mt. Sinai cohort (1998-2001)

 NYC, multi-ethnic (African (African American, American, Hispanic, Hispanic, White)

 Measured TPCy (fetal growth only) and DAPs

 PON1 genotype and phenotype

 U of CA Berkeley Cohort (CHAMACOS) (1999-2002)

 Mexican-American, agricultural worker population

 Measured TPCy and DAPs

 PON1 genotype and phenotype
Overview of Cohorts

5

 Three prospective birth cohorts to examine environmental 

exposures and adverse health outcomes 

 Chlorpyrifos/OP studies focus of this review

 Birth outcomes

 Neurodevelopment

 Enrolled women during pregnancy, follow children through 

time, multiple measures

 Children’s Environmental Health and Disease Prevention 

Research Centers (funded by EPA and NIEHS)
Population Characteristics

 Sought prenatal care (<20-

26 weeks)

Residency requirement

 Multi-ethnic populations: 

African-American, 

Dominican, Hispanic, 

6

General Eligibility Cohort Characteristics

 Residency requirement

 Deliver participating 

hospitals 

 No diabetes, hypertension, 

HIV, history birth defects

 No smoking, illegal drug 

use

Dominican, Hispanic, 

Mexican-American, White

 Younger mothers, ~25 

years 

 Low prevalence risky health 

behaviors, co-morbidities 

(criteria)
Exposure Measurement, Confounding Adjustment, 

Statistical Analyses

7 Methods and Analysis
Exposure Assessment

8

 Cohorts differing exposure profiles, pathways, routes

 Inner-city, agricultural and both residential, occupational

 Multiple biomarkers reflect different windows of exposure:

 Chlorpyrifos in cord blood (Columbia)

 TCPy in urine (eac in urine (each cohor h cohort)

 DAPs in urine (CDC methods) (Mt. Sinai, CHAMACOS)

 TCPy meconium (validation study) (Columbia)

 AChE and BuChE in cord blood (CHAMACOS)

 PON1 genotype and phenotype

 Chlorpyrifos in air (validation study) (Columbia)

 Self-report questionnaire (each cohort)
Exposure Assessment

9

 Ideal Measures

 Estimates exposure during critical window of susceptibility

 Repeat samples

 periodicity, variability of exposure

 Reflectiv flective of true rank of ue rank of actual e actual exposure xposure

 Timing of sample collection with application

 Actual Measures

 One-time, or average of two samples

 Third trimester (unknown window of effect)

 Specific and non-specific biomarkers of chlorpyrifos

Adapted D. Savitz 2002
Exposure Measurement Error

10

 External exposure versus measured internal dose

 Reflects total exposure (all routes)

 Single measure, third trimester

 Temporal-spatial patterns of exposure unclear

 Exposure Exposure during critical windo during critical window of development elopment

 Likely third trimester important

 Missing exposure data

 Imputation methods, data

 Laboratory errors

 Extensive QA/QC measures, CDC laboratories
Exposure Validation Study

11

 Questionnaire Data:

 85% report use pesticide, most report at least 

monthly use

 Evidence consistent use prior to 2000 

 External Data

 NYC Housing Authority reported use 

 Total usage in NYC housing greater than NY state Ag uses 

prior to voluntary cancellation
Exposure Validation Study

 Air monitoring

 Last 8-wks, stationary 

air 

 Personal (48-hr) and

 Biomarker Collection:

 Maternal Urine

 Last trimester

 At delivery

 Maternal blood 

12

 Personal (48-hr) and

 Intra<inter variability

 Maternal blood 

 Meconium 

 Neonate urine

 Compare Cord blood

Pe  Etiologic measure riod of Rapid 

Decline of Use

Whyatt et al. 2007, 2009
Exposure Validation Study

13

Meconium week 14 thru delivery 

Conception Week 14 Week 20 Week 24 Cord Blood

First trimester Second Trimester Third Trimester 

Week 32 thru delivery: biweekly air monitor and 

maternal urine sample 

Week 32 

(pilot): 48 hr. 

air

Conception Week 14 Week 20 Week 24

Cord blood, 

meconium, 

maternal urine

Cord Blood
Exposure Validation Study

14

Cord Blood Measures Correlate with Meconium (exposure period)

Whyatt et al. 2009
Exposure Assessment

15

 Uncertainties Remain

 Limited window of exposure measured 

 One-time measure (Columbia, Mt. Sinai)

 CHAMACOS averages two exposures

 Validation studies suggest some degree of correlation

 Unknown periodicity of exposure

 Peaks, constant

 Prevalence of pesticide exposure high

 Median concentration TCPy within range of general 

population (NHANES) 

 May indicate difference actual and measured
Methods to Control Confounding

 Restriction (design)

 Through recruitment techniques, similar population 

characteristics 

 Statistical Adjustment (individual-level)

16

 Statistical Adjustment (individual-level)

 Race, education, income, housing quality

 Tested for statistical interaction

 Multilevel Model (SES, Columbia only)

 Account group-level and individual-level variability
Methods to Select Potentially 

Confounding Variables

 Methods

 Searching literature, other studies in area

 Tested univariable and multivariable models

 Included if observed +/- 10% change in estimate

 Evidence of statistical confounding

17

 Evidence of statistical confounding

 Forward/Backward step-wise selection procedures

 Include if known risk factor

 Evidence of biological confounding 

 Many known or suspected confounding variables 

measured and modeled

 Emphasis on Parsimony
Confounding Variables

 Demographic

 Health-related

 Other environmental 

chemicals: Lead, Mercury, 

Diazinon, Propoxur

18

General Factors Major Potential (residual) 

Confounding Variables

 Environmental 

exposures PAH, ETS, 

DDT/DDE, PCB, 

OC/HCB

 Postnatal environment

Diazinon, Propoxur

 Socio-economic status

 In general, challenging to 

measure
When Confounding Bias?

Confounder CPF Exposure

19

Lead, Mercury* 

correlation <0.10

*Personal Communication R. Whyatt

ρ<0.20 – likely no effect

0.20<ρ<0.40 – little chance

0.40<ρ<0.60- some effect

0.60<ρ<0.80 – strong effect

ρ>0.80 – collinearity

ND Outcomes
Confounding: Other OPs

20

Effects of Chlorpyrifos on the odds of Mental Development Delay on the Bayley

Scales of Infant Development at age 36 months. (β coefficient, 95% CI, p-value)

Model 1 Model 2 Model 3 Model 4

Chlorpyrifos2 Chlorpyrifos 2.3 (1.1, 5.2) 2.3 (1.1, 5.2) 3.8 (1.5, 9.4) 2.0 (0.9, 4.5) 3.2 (1.3, 8.2 )

p=0.03

3.8 (1.5, 9.4)

p=0.004

2.0 (0.9, 4.5)

p=0.11

3.2 (1.3, 8.2 )

p=0.02

(ln)Diazinon 0.57 (0.35, 

0.95)

p = 0.03

0.6 (0.3, 0.9)

p=0.03

(ln)2-

isopropoxyphenol

(propoxur)

1.5 (0.9, 2.5)

p=0.12

1.5 (0.9, 2.6)

p=0.11

Appendix 4: Whyatt and Rauh, 2011 (unpublished)
SES: Multilevel Analysis

 The study of the effects of collective or group 

characteristics (as well as individual ones) on 

individual-level outcomes

 Not all associations explained by individual level 

21

Not all associations explained by individual level 

characteristics

 Group-level variables may affect individuals directly 

and also constrain the choices that individuals make

 Improve accuracy and precision of statistical models 

include Group and Individual level 
SES: Multilevel Analysis (cont.)

Group level 

CPF estimates

22

Group level 

variables

Individual level variables Lovasi et al. 2010
Statistical Analysis

23

 Statistical modeling decisions appropriate to the 

research question, characteristics of data etc.

 Missing data, <LOD

 (imputed, sensitivity analyses)

 Sample size adequate main effects

 Likely under-powered for effect modification

 Sensitivity analyses to evaluate alternative 

modeling options
Fetal Growth, Neonatal Neurological 

Development, Infant Motor and Mental 

24 Results

Development, Attention Disorders, Intelligence 

Measures
Fetal Growth and Characteristics

25

 Authors hypothesized link between pesticide and 

effects on fetal growth

 Hypothesized link to neurodevelopment

 Medical record linkage

 Birth weight, length, length, ponderal index

 Gestational age, preterm births

 Head circumference

 Effect modification evaluated

 PON1 genotype and phenotype 
Fetal Growth and Characteristics

26

Columbia 

Cohort

Mount Sinai 

Cohort U of CA Berkeley Cohort

Markers of 

Exposure: 

Chlorpyrifos

TCPy (fetal 

growth) TCPy DAPS

Birth Length Inverse Birth Length Null Positive (n.s.) Positive

Inverse

(Null post 2000) Null Positive (n.s.) Positive

Birth Weight

Inverse

(Null post 2000) Null Positive (n.s.) Null

Head 

Circumference 

at Birth

--

Null

(Inverse (NS))* Null Positive(Inverse*)

Gestational Age -- Null Null

Null

(Inverse*)

* PON1 genotype or phenotype
Neonatal Neurodevelopment

27

 Brazelton Neonatal Behavioral Assessment Scale 

(BNBAS)

 Goal: provide information parents/caregivers

 28 behavior behavioral items-habituation, habituation, range of state, 

orientation

 18 primitive reflexes

 Assessed 2-5 days post-partum

 Trained neonatologists administered

 Hospital setting
Neonatal Neurodevelopment

28

Consistent, 

reflexes

Columbia 

Cohort

Mount Sinai 

Cohort

U of CA 

Berkeley 

Cohort

Exposure 

Measure 

CPF DAPS DAPS reflexes

Outcome 

measure good 

quality

DAPs only, 

limitation

Measure 

BNBAS 

(Abnormal 

reflexes)

-- Positive* Positive

BNBAS (Other 

Behavioral) -- Null Null

* PON1 genotype or phenotype
Infant Neurodevelopment

29

 Bayley Scales of Infant Development II

 Mental development index (MDI)

 Psychomotor development index (PDI)

 Measured 6-36 months

 High reliability and validity as screening tool for early intervention

 Child Behavior Checklist

 Designed to assess "social competence" and "behavior problems" in children 

(Maternal report)

 Race/ethnicity small proportion of score variance

 99-item questionnaire to ascertain behavior and/or attentional

 Home Observation Measurement of the Environment (HOME)

 Stimulating environment for child development

 Effect modification evaluated

 PON1 genotype and phenotype
Infant Neurodevelopment

30

Columbia Cohort

Mount Sinai 

Cohort

U of CA Berkeley Cohort

Markers of 

Exposure: 

Chlorpyrifos DAPS TCPy DAPS

Bayley Scores 

6 months 

(MDI/PDI)

-- -- Null/Null Inverse (NS)/Null

(MDI/PDI)

Bayley Scores 

12 months 

(MDI/PDI)

Null / Null Inverse(*/**) / null 

(NS) Null/Null Null / Null

Bayley Scores 

24 months 

(MDI/PDI)

Null / Null Inverse(n.s.)/Null Null/ Null Inverse / Null

Bayley Scores 

36 months 

(MDI/PDI)

Inverse/ Inverse -- --

*PON1 genotype or phenotype; **Among blacks and Hispanics 
Attention Problems

31

 Measurement Tools

 Combination Maternal report and direct observation

 Child Behavior Checklist (maternal report)

 Direct Assessment of child (K-CPT)

 Hillside Psychometrician report

 DSVM-IV ADHD symptoms

 Assessed at 3, 3.5 and 5 years in two cohorts
Attention Problems

32

Sensitivity of

test at 3 yrs 

limited

Consistent 

results , two 

Columbia 

Cohort

Mount 

Sinai 

Cohort

U of CA 

Berkeley 

Cohort

Exposure: CPF DAPS DAPS results , two 

different 

exposures

Prenatal lead, 

lack postnatal

(PDD) Positive 

(36 mo.) --

Positive 

(24 mo.)

Odds of ADHD/ 

attention 

problems

Positive

(36 mo.) --

Positive

(NS 3 years)

Positive (5 

years)
Intelligence Measures 

33

 Wechslar Intelligence Scale for Children IV

 Verbal comprehension

 Perceptual Reasoning

 Working Memory (WM)

 Processing Speed

 Full Scale IQ (FSIQ)

 Standardized US population, norms English and 

Spanish speaking populations

 Cultural Bias has been noted

 Restricted sample population
Intelligence Measures

34

Consistent results 

across studies 

(WM)

Columbia 

Cohort

Mount Sinai 

Cohort

U of CA 

Berkeley 

Cohort

Exposure CPF DAPS DAPS

(WM)

Large number 

missing tests, 

include/exclude 

comparable Mental 

Development

(WISC-IV)

Inverse 

(FSIQ, WM)

Inverse (NS) 

(FSIQ, 

perceptual 

reasoning, 

WM)

Inverse (FSIQ, 

WM, 

processing 

speed, verbal, 

perceptual 

reasoning) 
Draft Framework Analysis

35 Evaluation and Synthesis
Strengths and Limitations

 Measurement outcome and 

exposure extensive, validated, 

reliable

 Important confounding variables 

 Unknown factor may correlate 

with CPF 

 However, among known or 

hypothesized factors with 

36

Strengths Limitations

 Important confounding variables 

measured and modeled

 Design and analysis decisions aid 

comparability between groups

 Reduce many sources of bias

 Peer-reviewed publications 

hypothesized factors with 

potential positive confounding 

bias, lack evidence explain 

observed associations

 In general, difficult to tease out 

SES in any epidemiology study

 Restriction, individual and grouplevel multi-variable analysis (R2

moderately high)

 Role of postnatal environment

 Limited instances in which 

measured, not confounder 
Study Design Issues

 A priori hypotheses 

 Statistical modeling 

appropriate

 Discussed varying degrees 

 Selection Criteria 

extensive

 Enhance internal validity

 Reduce external validity 

(generalizability)

37

 Discussed varying degrees 

across studies

 Missing data imputed when 

possible

 High prevalence exposure, 

outcome enhanced study 

power

 Limited evaluation of

interaction with other 

chemicals, OPs
Statistical Analysis and Grouping

 Several Methods Used across studies:

 Motivated by sample size, study hypothesis

 Grouping straight-forward, fully explained

 Analysis issues not always discussed in full

38

 Analysis issues not always discussed in full

 Several sensitivity analyses performed

 QA/QC measures

 Alternative hypotheses explored

 Model fitting exercises, shape of data
Exposure Measurement

39

 Chlorpyrifos (parent), non-specific (TCPy, DAP)

 Lack specificity, direct comparability

 May explain heterogeneity 

 Unknown temporal, spatial pattern of exposure

 Constant, intermittent, and/or sporadic

 One-time measure correlated with longer-term marker

 Meconium=maternal urine (ave.)=cord blood

 Cohorts’ exposures greater than, but comparable to 

population median

 Generalizable to US population
Outcome Ascertainment

40

 Outcome Measures

 Similar, aids comparison

 Clinical, research settings

 Spanish/English

 Quality Assurance and Control adequate

 Stated observers blinded in some, not all studies

 Limitations

 Neurodevelopmental assessments subjective 

 Cultural bias –Wechslar tool

 Non-differential error likely

 Multiple exposure ameliorates potential differential bias
Confounding Bias

41

 Multiple methods of control

 Restriction, individual- and group-level (SES)

 Cannot exclude possibility positive confounding bias 

of unknown factor

 Lead, mercury mercury, other OP, OC, PCB , OC, PCB, PAH

 SES

 Multi-dimensional, both +/- confounding biases

 Hierarchal regression analysis utilized to assess

 Lack examination of heterogeneity by other 

environmental exposures
Threats to Validity

 Retention rate 

moderately high for 

birth cohorts (60-80% 

 Errors in outcome and 

exposure measures 

occurred

42

Selection Bias Information Bias

birth cohorts (60-80% 

at 7 years)

 Included and excluded 

comparable

 Measured variables 

 Missing Data imputed

occurred

 Design reduces 

differential bias

 Many exposures

 Non-differential may 

have reduced effect 

sizes
Synthesis of Evidence

43

 Strength of observed associations high in some cases, 

but not all

 Non-differential exposure measurement error could reduce 

magnitude

 Some evidence evidence of exposure xposure-response response in studies in studies

 Consistency in Brazelton (abnormal reflexes), ADHD-like 

behavior problems, Bayley (particularly MDI), and IQ 

(WM)

 Alternative explanations investigated

 Modeling, transformations, confounding variables
Synthesis of Evidence

44

 Prospective studies

 Temporal relation

 Differential misclassification less likely 

 Biological plausibility

 See other sections, generally plausible, lack specific 

details MOA

 Influence of cessation of residential exposure on 

association

 Limited evidence (low power), not designed to assess
Conclusions

45

 Current database strengthens conclusion 2008 

“chlorpyrifos likely plays a role” in observed 

adverse effects on child neurodevelopment

 Given stud en study conduct, design, methods EP conduct, design, methods EPA believ A believes 

more likely under- than over-estimate associations

 Integration across streams of information will 

continue
Section 4.0

Appendices 4-6

46 Questions
Appendix 4: Supplementary AnalysesColumbia U. Cohort

 Bounding

 See Hattis PBPK

 How many “LBW?”

 Not designed to 

address

 Loss to follow-up

 Non-differential by

exposure group (4)

 Pre-cancellation 

clarification

47

address

 Bayley Scores

 Expanded two to four 

categories, highest 

category greatest risk

clarification

 Exposure level decline

 Confounding analysis

 Diazinon, propoxur