Appendix 4
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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY AND

 POLLUTION PREVENTION

MEMORANDUM

Date: June 30, 2011

SUBJECT: Chlorpyrifos Correspondence with Columbia Researchers: (1) Responses to 

Scientific Advisory Panel (SAP) comments (Whyatt and Rauh 2010), and 

(2) Responses to Dow AgroSciences inquiries (Whyatt 2010). 

PC Code: 059101 DP Barcode: D391257

Decision No.: NA Registration No.: NA

Petition No.: NA Regulatory Action: NA

Risk Assessment Type: Single Chemical Aggregate Case No.: NA

TXR No.: 0056066 CAS No.: NA

MRID No.: NA 40 CFR: NA

FROM: Deborah Smegal, MPH, 

Immediate Office (IO)

Health Effects Division (7509P)

TO: Danette Drew, Risk Assessor

RABV

Health Effects Division (7509P)

Backrgound

On September 16-19, 2008, the Agency held a FIFRA Scientific Advisory Panel (SAP) meeting 

(SAP 2008) to discuss the Agency's Evaluation of the Toxicity Profile of Chlorpyrifos, which 

includes more recent and extensive research on various aspects of chlorpyrifos including its 

neurological effects in animals and humans following gestational and post-natal exposures, its 

pharmacokinetics, and mechanism of action. Details can be found at the Scientific Advisory 

Panel website (http://www.epa.gov/scipoly/sap/meetings/2008/091608_mtg.htm), and in the 

Chlorpyrifos Final SAP report 

(http://www.epa.gov/scipoly/sap/meetings/2008/september/sap0908report.pdf.) 

Attached is a response to the SAP comments on the “Epidemiologic Appendix: Columbia Study” 

that Deborah Smegal summarized and sent to Robin Whyatt and Virginia Rauh, dated 1/28/2010. 
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In addition, the Columbia researchers prepared responses to questions raised by Dow 

AgroSciences. 
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Columbia Response to the SAP Queries

2/19/2010

The following is a response to the Scientific Advisory Panel comments on the 

“Epidemiologic Appendix: Columbia Study” that Deborah Smegal summarized and sent 

to Robin Whyatt and Virginia Rauh, dated 1/28/2010.

1. SAP comment: EPA should continue its collaboration with Columbia researchers in 

analyzing the epidemiologic data ...[and] should seek clarification of the reported 

exposure levels . . . EPA should use the epidemiological cohort data for bounding 

exposure levels and in conjunction with PBPK models, address current or potential 

human exposures and to determine the final RfD/RfC . . . PBPK models enable 

estimation of exposure dose metric for multiple sources (e.g., air, food, water etc). 

EPA should pursue simpler PBPK model in short-term. 

Response: We are happy to continue the collaboration. In addition, as described in our letter, Dr. 

Dale Hattis at Clark University is conducting PBPK modeling of the Columbia data under a U.S. 

EPA Star Grant. The grant is entitled “Use of Biomarkers and Physiologically Based 

Pharmacokinetic [PBPK] Modeling in Risk Analysis for Developmental Effects of 

Chlorpyrifos”. We believe that the PBPK model being developed by Dr. Hattis will prove 

extremely helpful to EPA in addressing the SAP comments and in clarifying the exposure 

concentrations that correspond to the chlorpyrifos levels in umbilical cord blood at which we 

begin to see statistically significant adverse effects on fetal growth and child neurocognitive 

development. Thus this data should be helpful in bounding the exposure data as recommended 

by the SAP. We recommend that EPA contact Dr. Hattis.

2. SAP comment: Determine if the chlorpyrifos effect on birth weight would result in 

more infants in “low birth weight” category. 

Response: We are not able to address this question as our cohort was predominantly full term by 

design. This is because we did not conduct our environmental monitoring until the 3rd trimester 

of pregnancy and completion of the environmental monitoring was a prerequisite for enrollment. 

For example, of the subjects in our 2004 analyses (Whyatt et al., EHP, 112:1125-1132, 2004), 

only 12/314 (3.8%) of the women delivered prior to 37 weeks gestation and only 12/314 (3.8%) 

of the infants were <2500 grams at birth. We, therefore, do not have adequate statistical power to 

evaluate the relationship between prenatal chlorpyrifos exposure and low birth weight.

3. SAP comment: Ascertain whether there are statistically significant differences in 

neurodevelopmental outcomes at 3 yrs between chlorpyrifos levels in cord blood 

below LOD and those with chlorpyrifos >6.17 pg/g . . . Verify/clarify there is no 

suggestion of an effect as the most highly exposed group and the group with values 

below the LOD had lower MDI and PDI scores than did the middle levels. 

Response: Table 1 below provide the same type of information as Table 2 in the 2006 Pediatrics 

paper (Rauh et al., Pediatrics, 118:e1845-e1859, 2006) with respect to the % of subjects whose 

tests scores indicated developmental delay with respect to the PDI and the MDI, as a function of 
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chlorpyrifos exposure. However, here we provide the % delayed for all four exposure groups, 

rather than just the ‘high’ and ‘low’ exposure groups shown in the paper. For both the PDI and 

MDI the rate of delay is clearly highest in the high chlorpyrifos group (Group 4).For the 36 

month PDI, the rate of developmental delay is relatively low in all 3 low-exposure groups, and is 

about three times higher in the highest exposure group. And for the 36 month MDI, while the 

<LOD group experienced a higher rate of delay than group 2 and 3, the very highest rate of delay 

is among the most exposed children. In univariate analyses, the difference in % delay between 

the highest exposure (>6.17pg/g) group versus the lowest exposure (<LOD) group, was 

statistically significant for PDI (chi-square p=.018, 1 sided; p=.031 two-sided), but not 

significant for MDI. However, importantly, there was no significant difference in the rate of 

delay between groups 1, 2 and 3 for either the PDI (chi-squared= 0.97, p=0.61) or MDI (chisquared= 4.4, p=0.11). And the rate of developmental delay in terms of both PDI and MDI in 

group four was significantly greater than in groups 1-3 combined, as reported in our paper. 

Table 1. The proportion of children at age 36 month who were at risk of delay (score < 85) on the 

Psychomotor Development Index (PDI) or Mental Development Index (MDI) of the Bayley Scales of Infant 

Development by chlorpyrifos exposure group

Group 1 

(<LOD)

Group 2 (1st tertile > 

LOD)

Group 3 (2nd tertile > 

LOD)

Group 4 (3rd tertile > 

LOD)

PDI 7/80 (8.8%) 3/65 (4.6%) 3/38 (7.95) 11/45 (24.4%)

MDI 30/80 (37.5%) 14/65 (21.5%) 11/39 (28.2%) 20/44 (45.5%)

4. SAP comment: Conduct an analysis of exposure levels as dependent variable and 

year (an ordinal variable) as the primary independent variable, while adjusting for 

any factors associated with both exposure levels and year of study. 

Response: We have already published on changes in indoor and personal air levels, as well as in 

multiple biomarker levels by year of monitoring and have enclosed the most relevant articles ( 

Whyatt et al., 2007; Whyatt et al., 2009). In addition, Dr. Hattis has also recently completed 

analyses looking at changes in chlorpyrifos levels by year in the context of other factors such as 

season of monitoring. As stated above, we recommend that EPA contact Dr. Hattis to review his 

results.

5. SAP comment: EPA should request additional details on the loss to follow-up in 

each of the 4 exposure groups and provide additional details on how the exposure 

groups were combined.

Response: Subjects were eligible for inclusion in the Pediatric paper if they had been born by 

7//12/02 and thus had reached age three years by the time of the analyses and also had: (1) 

measures of chlorpyrifos in blood at birth; (2) neurologic assessments at least once between ages 

12-36 months and (3) data on model covairates. A total of 381 subjects in the cohort had been 

born by 7/12/02 and had birth chlorpyrifos blood measure; of these 254 (67%) met the above 

criteria for inclusion, including 228 who had developmental outcomes at age 36 months. The 

remaining subjects had either been lost to follow-up (accounting for approximately 14%) or were 

missing outcome and/or covariate data. Table 2 below shows the 254 subjects included in the 

analyses over all 381 subjects by the chlorpyrifos groups. (The numbers per group just for the 

228 subjects with outcome data at 36 months are given in the Pediatric manuscript.) Group 1 
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includes all subjects with chlorpyrifos blood levels below LOD; the remaining subjects were 

ranked into tertiles based on ascending levels (Groups 2-4). However, as describe in the 

Pediatric manuscript, this grouping was done on subjects born prior to 1/1/01 to be consistent 

with our prior publication (see Whyatt et al. 2004). This was essential in order to compare results 

across analyses moving forward, given that chlorpyrifos concentrations were changing rapidly 

following the ban and thus we needed consistent cutpoints. Groups 1-3 were combined and 

compared with group 4 for the analyses presented in the Pediatrics manuscript. 

Table 2. Subjects included in the Pediatric paper over all cohort subjects born at or before 7/12/02 who had 

chlorpyrifos measured in cord blood at birth 

Group 1 (<LOD) Group 2 (1st tertile > LOD) Group 3 (2nd tertile > LOD) Group 4 (3rd tertile > LOD)

88/121 (72%) 72/127 (56.7%) 44/63 (69.8%) 50/70 (71.4%)

6. SAP comment: Post ban declines in effects are based on 1 subject being classified as 

highly exposed, which lacks statistical power and may be sensitive to the cutoff value

chosen to classify subjects as highly exposed. Can an analysis be conducted to look 

at relationships between more equally sized exposure groups that separate between 

the lower exposure levels? 

0

2

4

6

8

10

chlopyrifos

1999 68/71 (96%)

2001 26/86 (30%)

2000 101/120 (84%)

2002 18/59 (31%)

Figure 1. Chlorpyrifos levels in maternal and umbilical cord 

blood samples (pg/gram) at delivery by year of birth

2003 0/58 (0%)

2004 0/30 (0%)

Cord blood (N=395) Year N > LOD (%)

0

2

4

6

8

10

chlopyrifos

Maternal blood (N=424) Year N > LOD (%)

1999 93/110 (85%)

2001 18/71 (25%)

2000 88/110 (80%) 

2002 14/36 (39%)

2003 0/43 (0%)

2004 0/25 (0%)

Response: We believe the SAP has misunderstood that our post ban declines were based not on 

the change in the number of subjects that fell into the highly exposure group but on the decrease 

we had seen in chlorpyrifos concentrations in both maternal and cord blood among all cohort 

subjects by year of birth (see Figure 1). As can be seen in the above figure, chlorpyrifos levels 

had dropped dramatically by 2001. Our 2004 study categorized the full cohort available at that 

time (most born prior to1/1/01) into four groups based on ascending levels of chlorpyrifos in 

umbilical cord blood. The only significant difference we saw was between infants in the highest 

exposure group (group 4) compared to those with levels below the limit of detection (group 1). 

By contrast, there was no significant difference in birth outcomes between subjects in the 
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intermediate exposure groups (2 or 3) compared to group 1 (see Whyatt et al. EHP, 112: 1125-

1132. 2004). At the time we conducted the analyses for our 2004 paper, only n=77 infants were 

born after 1/1/01, which limited our power to determine whether or not there was any continuing 

effect of prenatal chlorpyrifos exposure on birth outcomes after2001. In April 23, 2008, we 

presented undated analyses to EPA that included more infants (n=193) born after 1/1/01. Results 

are shown in Table 3 below and are consistent with our 2004 publication. Specifically, we saw a 

highly significant decrease in birth weight for each log unit increase in umbilical cord 

Table 3. Change in birth weight for each log unit increase 

inchlorpyrifos concentrations in umbilical cord plasma among 

babies born before and after 1/1/01

B (95% CI) P-value

Born before 1/1/01 (n=233) -71/4 (-121/1, -21.8 P=0.005

Born after 1/1/01 (n=193) 32.7 (-59.3, 124.7) P=0.48

chlorpyrifos concentrations among babies born before 1/1/01, but no association between 

chlorpyrifos and birth outcomes among infants born after 1/1/01. Of those infants born after 

1/1/01, only 16% had chlorpyrifos levels in cord blood above the LOD and only one of these was 

in the previously defined ‘high exposure’ group. It is obvious that this marked reduction in 

chlorpyrifos exposure, and the virtual elimination of high exposure levels after 1/1/01, is due to 

the chlorpyrifos ban , . To address the SAP comments further, we have conducted analyses that 

categorize infants born after 1/1/01 into those with chlorpyrifos above and below the LOD. 

Keeping in mind that there was only one child with high (group 4) chlorpyrifos exposure in this 

post-ban sample, it is not surprising that there was no significant difference in birth weight 

between the <lod group and the small group with some exposure during this post-ban period. 

These findings thus support the hypothesis that chlorpyrifos exposures following the ban had 

dropped below concentrations that have adverse effects on fetal growth. However, it is also 

possible that our sample size is simply too small to detect relatively small but real negative 

effects on birth outcomes associated with the much reduced exposure levels after 1/1/01. 

7. SAP comment: The panel felt it might be helpful to have a longitudinal component to the 

study to determine if the adverse effect persisted in children who exhibited poor birth 

outcomes or neurobehavioral deficits.

Response: As described in the attached letter, we have just completed statistical analyses on the 

relationship between prenatal chlorpyrifos exposure and outcomes on the Wechsler Intelligence 

Scale for Children (Fourth Edition) and the Child Behavior Check List, which have been 

administered to cohort children at age 7 years. We anticipate that a manuscript on these findings 

will be completed and ready for submission within several months.

8. SAP comment: It would be useful to examine the results of a statistical analysis that 

includes all three AChE-inhibiting insecticides in the analysis model as dichotomous 

variables (above or below the LOD) in combination with continuous measurements for 

these variables. This is not likely to change the results but could be helpful in illustrating 

threshold or dose response effects . . . Are all 3 AChE inhibiting insecticides correlated (if 

lack of correlation—less likely would confound cyp and neuro outcomes) . . . Conduct 
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additional analysis controlling for multiple exposures examining additive effects to 

elucidate the role of other Ops. This is important for AChEI to be considered as mode of 

action. 

Response: It should be noted that our 2004 paper (Whyatt et al., EHP, 112: 1125-1132) looked at 

the additive effects of chlorpyrifos and diazinon on birth outcomes using the U. S. EPA 

guidelines for conducting cumulative risk assessments for the organophosphates. However, in 

response to the SAP comments, we have now conducted a number of additional analyses both to 

evaluate the correlations among the three AChE inhibiting insecticides (chlorpyrifos, diazinon 

and propoxur) detected in our cohort and to look at the effects of chlorpyrifos on both birth 

outcomes and child neurocognitive development after controlling for diazinon and propoxur. 

Specifically we have devloped 4 additional models for birth weight and birth length and 4 

models for the psychomotor development index (PDI) and mental development index (MDI) on 

the Bayley Scales of Infant Development at age 36 months. All models controlled for the same 

covariates as in our 2004 and 2006 publications. Model 1 includes only chlorpyrifos as the 

independent variable (i.e. is identical to the data in our initial publications); model 2 includes 

chlorpyrifos and diazinon; model 3 includes chlorpyrifos and 2-isopropoxyphenol (the chemicalspecific metabolite of propoxur); and model 4 includes all three acetylcholinesterase inhibiting 

insecticidess Results are presented below:

Correlations among pesticides: For infants born prior to the chlorpyrifos phase out (the relevant 

time period), there was a modest correlation between 2-isopropoxyphenol (the chemical specific 

metabolite of propoxur) and both chlorpyrifos (r=0.25, p<0.001, n=222) and diazinon (r=0.21, 

p=0.002, p=227) in umbilical cord blood samples. However, there was a higher correlation 

between chlorpyrifos and diazinon (r=0.63, p<0.0001, n=222), incdicating that approximately 

33% shared variabilitybetween these two pesticides. Given this relationship between chlorpyrifos 

and diazinon, the question raised by the SAP—whether the chlorpyrifos effects we report could 

be due to confounding by diazinon—is therefore very appropriate. This question is addressed in 

the new analyses reported below.

Results on birth outcomes. Results are presented in Tables 4 and 5 below. As can be seen, 

chlorpyrifos remained inversely associated with birth weight and length in all models after 

controlling for diazinon and 2-isopropoxyphenol, and the magnitude of the significant negative 

effect of chlorpyrifos on infant size remained relatively constant across the models, indicating 

that neither of these two other acetylcholinesterase inhibitors was confounding the relationship 

between chlorpyrifos and birth outcomes. Further neither diazinon nor 2-isopropoxyphenol, 

either individually or in combination, was associated with birth weight, although the inverse 

association reported previously between 2-isopropoxyphenol and birth length (see Whyatt et al., 

2004) remains of borderline significant (p=0.05). Consistent with our prior findings, birth weight 

decrease significantly by 198 grams among infants in the highest compared to the lowest 

chlorpyrifos groups (Group 4 versus Group 1) after controlling for both diazinon and 2-

isopropoxyphenol, as well as the other model covariates (p<0.05). These analyses thus indicate 

that it is primarily prenatal chlorpyrifos and not the other two acetylcholinesterase-inhibiting

insecticides that is having an adverse effect on fetal growth. Further, the findings suggest that the 

effect of chlorpyrifos on fetal growth may well be mediated by mechanisms other than 
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acetylcholinesterase inhibition, given the lack of an association between the other two 

acetylchlolinesterase inhibitors and birth weight. 

Table 4. Decrease in birth weight for each log-unit increase in insecticide levels in umbilical cord blood at 

birth among infants born prior to 1/01/01. Values shown are regression coefficient (B), 95% confidence 

interval, and p-value

Model 1 Model 2 Model 3 Model 4

(ln)Chlorpyrifos -67.3 (-17.8, -

116.8)

p=0.008

-65.9 (-5.7, -126)

p=0.03

-60.9 (-10.0, -

111.8)

p=0.02

-60.2 (0.9, -121.3)

p=0.054

(ln)Diazinon -4.7 (107.7, -

117.1)

p=0.94

-2.5 (110, -114) 

p=0.96

(ln)2-isopropoxyphenol -50.9 (46.1, -

147.8)

p=0.30

-50.8 (46.5, -114.9)

p=0.30

All models controlled for gestational age, parity, maternal pre-pregnancy weight and net weight gain during 

pregnancy, maternal self-reported environmental tobacco smoke in the home, gender of the newborn and 

season of delivery.

Table 5. Decrease in birth length for each log-unit increase in insecticide levels in umbilical cord blood at 

birth among infants born prior to 1/01/01. Values shown are regression coefficient (B), 95% confidence 

interval, and p-value

Model 1 Model 2 Model 3 Model 4

(ln)Chlorpyrifos -0.43 (-0.14,-0.73)

p=0.004

-0.46 (-0.1, -

0.82)

p=0.01

-0.36 (-0.06,, -

0.66)

p=0.02

-0.40 (-0.75, -

0.35)

p=0.03

(ln)Diazinon 0.09 (0.76, -0.58)

p=0.80

0.12 (-0.78, 055)

p=0.73

(ln)2-isopropoxyphenol -0.57 (0.002, -1.14)

p=0.051

-0.58 (-.001, -

1.15)

p=0.05

All models controlled for gestational age, parity, maternal pre-pregnancy weight and net weight gain during 

pregnancy, maternal self-reported environmental tobacco smoke in the home, gender of the newborn and 

season of delivery. 

Results on PDI and MDI are presented in Table 6 and 7 below. The findings were quite 

surprising. There was no significant association between 2-isopropoxyphenol and delay on either 

the PDI or MDI. However, when diazinon was added to the model, the negative effect of 

chlorpyrifos on developmental delay for both the PDI and MDI became much stronger. 

Specifically, the odds ratio almost doubled for PDI and increased by 40%-60% for the MDI. 

These results again indicate that it is chlorpyrifos and not the other two acetylcholinesterase 

insecticides that is having an adverse effect on child mental and motor development and further 

that the effect size appears considerably greater than we had estimated before. The findings again 

support the suggestion, as discussed by the SAP, and as is suggested by the experimental 

evidence, that the effects of chlorpyrofos on child development may well be by a mode of action 

other than acetylcholinesterase inhibition. 
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Table 6. Effects of chorpyrifos on the odds of ps;ychomotor delay on the Bayley Scales of Infant Development at 

age 36 month. Values shown are regression coefficient (B), 95% confidence interval, and p-value s 

Model 1 Model 2 Model 3 Model 4

Chlorpyrifos2 4.5 (1.6, 12.7)

p=0.004

8.4 (2.4, 30.2)

p=0.001

4.0 (1.4, 11.8)

p=0.01

7.9 (2.1, 29.1 )

p=0.002

(ln)Diazinon 0.45 (0.18, 1.13)

p = 0.09

0.4 (0.2, 1.1)

p=0.08

(ln)2-isopropoxyphenol 1.3 (0.65, 2.8)

p=0.4

1.4 (0.67, 3.0)

p=0.37

All models controlled for race/ethnically, gender, ETS, gestational age, maternal IQ, maternal education, 

HOME score1

OR = Odds ratio 2

>6.17 pg/g versus ≤ 6.17 pg/g in umbilical cord blood

Table 7. Effects of chorpyrifos on the odds of mental delay on the Bayley Scales of Infant Development at age 

36 months. Values shown are regression coefficient (B), 95% confidence interval, and p-value

Model 1 Model 2 Model 3 Model 4

Chlorpyrifos2 2.3 (1.1, 5.2)

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 1.5 (0.9, 2.5)

p=0.12

1.5 (0.9, 2.6)

p=0.11

All models controlled for race/ethnically, gender, ETS, gestational age, maternal IQ, maternal education, 

HOME score1

OR = Odds ratio 2

>6.17 pg/g versus ≤ 6.17 pg/g in umbilical cord blood

9. SAP comment: Is it possible to determine quantitative contribution of chlorpyrifos in 

exposure to mixture by accounting for differences in chemicals, potency, pharmacokinetics 

and possible chemical interactions. 

Response:

10. SAP comment: Look at intermediate markers of biologically effective dose and early 

effect that further link exposure and outcome and or a clearer idea of mode of action from 

exposure studies. 

 Dr. Hattis can address this question after his PK model is implemented and 

calibrated. 

Response: As described in our letter, we have been conducting Magnetic Resonance Imaging 

(MRI) scans on cohort children with either high prenatal chlorpyrifos exposure (n=20) or no 

exposure to any of the contaminants that have been associated with adverse child cognitive 

development among the CCCEH cohort (n=20). The MRI results will provide a marker of 

biologic effect. These analyses are being undertaken under the direction of Dr. Bradley Peterson, 

Director of Child and Adolescent Psychiatry at the New York State Psychiatric Institute. We 

anticipate that a manuscript on the results will be submitted to a peer-reviewed journal by Aug 1 

2010. 
Page 11 of 14

COLUMBIA CENTER

FOR CHILDREN'S 

ENVIRONMENTAL 

HEALTH 

MAILMAN SCHOOL OF PUBLIC HEALTH 

Columbia University 

Memo to: Deborah Smegal

From: Robin Whyatt

Date: March 8, 2010

We are pleased that our answers to the questions raised by the Science Advisory Panel (SAP)

will be of value to EPA as you move forward to update the chlorpyrifos risk assessment. Below

are our responses to the questions raised by Dow Agrosciences which you also asked us to

address in your email of March 5,2010. Please let us know if you need any additional

information or if we can be of more help.

Question one: The Columbia birth cohort includes cord blood chlorpyrifos levels for most

children and urine from many mothers (Whyatt et aL. 2005,2009). However, the authors state

that "we found no association between chlorpyrifos levels in maternal and cord blood and TCPy

levels in maternal urine samples during pregnancy or after delivery." (Whyatt et aL. 2009, pg

563). Since the health effect papers are based solely upon chlorpyrifos levels in blood, and

because blood chlorpyrifos measurements have not been reported in any other human exposure

study at these trace-level concentrations, this is a critical point to evaluate with full transparency.

Specific questions include:

a. Why is there no association between blood chlorpyrifos and urinary TCPy?

b. How many mother infant pairs in 1999 have both urine TCPy and blood chlorpyrifos?

c. Are the 1999 urine and blood levels correlated?

d. Are the 2000 urine and blood levels correlated?

e. Are the 2001 urine and blood levels correlated?

f. With personal monitoring, air monitoring, blood chlorpyrifos and urinaryTCPy, what is the 

estimated internal dose for the infant-mother pairs in each year of measurement?

Answer: Please note that the primary biomarker we have measured in our cohort is chlorpyrifos

concentrations in maternal and umbilical cord blood. We did not measure TCPy in maternal

urine samples except for a small subset of subjects. Specifically, as described in our 2009

manuscript (Whyatt et aI., EHP, 117;559-567, 2009), we measured TCPy in repeat urine

samples collected from only n=97 women during pregnancy. These urine samples were collected

among women who delivered between 2001-2004. Thus the answer to la through 1d above is
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that no mother/infant pairs have both chlorpyrifos in blood and TCPy in urine in either 1999 or

2000 and we thus do not have the data to look at the correlation between TCPy in urine and

chlorpyrifos in blood among subjects who delivered prior to 2001. For subjects who delivered in

2001, we can look at this relationship. It should be noted, however, that these data are not

particularly helpful in evaluating the correlations between urinary TCPY and blood chlorpyrifos

concentrations, due to small sample size. But more importantly, chlorpyrifos in blood samples

had already dropped substantially by 2001 following the residential ban. Specifically, by 2001

among subjects that had both TCPy measured in maternal urine and chlorpyrifos measured in

blood, chlorpyrifos was only detected in 24% of maternal blood samples and 19% of umbilical

cord blood samples (See Figure 2, Whyatt et at., 2009). By contrast, as shown in Figure 1 from

in the Columbia Response to the SAP Queries (2/19/2010), chlorpyrifos was detected in 98% and

85% of maternal and umbilical cord blood samples, respectively, during 1999 and 84% and 80%

during 2000. Given these caveats, the results for 2001 are as follows: For these subjects, the

average chlorpyrifos concentrations in two-week integrated indoor air samples collected over

the final 6-8 weeks of pregnancy were positively correlated with average TCPy levels in

maternal prenatal spot urine samples (r=0.5, p=0. 005). However, there was no correlation

(p>0.2) between TCPy levels and chlorpyrifos in either maternal blood samples (n=29) or cord

blood samples (n=21) at delivery. Maternal and umbilical cord blood levels were highly

correlated (r=0.85, p<0.001, n=21).

Regarding question "If" above, please note, that as discussed in our answers to the SAP

querries, Dr. Dale Hattis at Clark University is conducting PBPK modeling of the Columbia

data under a Us. EPA Star Grant. The grant is entitled “Use of Biomarkers and Physiologically

Based Pharmacokinetic [PBPK] Modeling in Risk Analysis for Developmental Effects of

Chlorpyrifos ". We believe that the PBPK model being developed by Dr. Hattis will prove helpful

in clarifying the exposure concentrations that correspond to the chlorpyrifos levels in umbilical

cord blood at which we begin to see statistically significant adverse effects on fetal growth and

child neurocognitive development and also in estimating the internal dose for the infant/mother

pairs in each year of measurement. Dr. Hattis estimates that his analyses should be completed

relatively shortly. We recommend that you contract Dr. Hattis.

Question Two: In the Rauh et al. (2006) paper the authors note a nonlinear dose trend with using

4 exposure groups. The final analyses are limited to two exposure groups. An analysis of 4

exposure groups is more information from a risk perspective and is critical for full transparency

of the data. Analysis with 4 exposure groups for the results presented in Tables 2-8 should be

replicated and made publically available.

Answer: We have already addressed this comment in our answer # 3 from the Columbia

Response to the SAP Queries (2/19/2010). To reiterate, we prepared a new table (Table 1 below)

which provided the same type of information as Table 2 in the 2006 Pediatrics paper (Rauh et

at., Pediatrics, 118:el 845..;el 859, 2006) with respect to the % of subjects whose tests scores

indicated developmental delay with respect to the PDI and the MDI, as a function of chlorpyrifos

exposure. However, we provided the % delayed for all four exposure groups, rather than just

the 'high' and 'low' exposure groups shown in the paper. For both the PDI and MDI the rate of

delay is clearly highest in the high chlorpyrifos group (Group 4). For the 36 month PDI, the rate

of developmental delay is relatively low in all 3 low-exposure groups, and is about three times

higher in the highest exposure group. And for the 36 month MDI, while the <LOD group

experienced a higher rate of delay than 2 and 3, the very highest rate of delay is among
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the most exposed children. In univariate analyses, the difference in % delay between highest

exposure (>6.17pg/g) group versus the lowest exposure group (<LOD) was statistically significant 

for PDI (chi-square p=0.018, 1 sided; p-0.031 two-sided), but not significant for MDI

However, importantly, was no significant difference in the rate of delay between groups 1,

2 and 3 for either the PDI (chi-squared=0.97, p=0. 61) or MDI (chi-squared=4.4, p=0.11).

And the rate of developmental delay in terms of both PDI and MDI in group four was significantly 

greater than in groups 1-3 combined, as reported in our paper.

Table 1. The proportion of children at age 36 month who were at risk of delay (score < 85) on the 

Psychomotor Development Index (PDI) or Mental Development Index (MDI) of the Bayley Scales of Infant 

Development by chlorpyrifos exposure group

Group 1 

(<LOD)

Group 2 (1st tertile > 

LOD)

Group 3 (2nd tertile > 

LOD)

Group 4 (3rd tertile > 

LOD)

PDI 7/80 (8.8%) 3/65 (4.6%) 3/38 (7.95) 11/45 (24.4%)

MDI 30/80 (37.5%) 14/65 (21.5%) 11/39 (28.2%) 20/44 (45.5%)

Question Three: In the Rauh et al. (2006) paper, the authors compare results of the Bayley

Mental Development Index (MDI) and Bayley Psychomotor Development (PDI) in different years. Using 

significance testing only, they claim there is a “preban” difference and no "postban" difference. This 

natural experiment is important to control for the many confounding factors associated with the home 

environment of these children. A more comprehensive and transparent analysis of these data over would 

provide information about role of chlorpyrifos and MDI and PDI this unique population.

Answer: Please note that most cohort children in the 2006 paper by Rauh et at. were born prior

to the residential ban on chlorpyrifos. Specifically, as described in our answers to SAP queries, 

subjects were eligible for inclusion in the Pediatric paper if they had been born by 7/12/02 and

thus had reached age three by the time of the analyses and also had: (1) measures of

chlorpyrifos in blood at birth; (2) neurologic assessments at least once between ages 12-36

months and (3) data on model covariates. Therefore, we were not able to undertake statistical

analyses relating the changes in chlorpyrifos concentrations to changes in MDI and PDI 

following the residential ban. However, authors concurred that this is an important research

question and state that “larger epidemiologic studies will to assess the full impact of

the ban (see page e1854 of the Pediatric paper)." As discussed in our February 17th letter to

Dr. Jack Fowle, we are currently preparing a manuscript on the relationship between prenatal

chlorpyrifos exposure and outcomes on the Weschler Intelligence Scale for Children (Fourth

Edition) and the Child Behavior Check List, which have been administered to cohort children at

age 7 years. However, we also plan to undertake additional analyses of the 36th month MDI and

PDI in relationship to prenatal chlorpyrifos exposures following the ban, since many more

children in our cohort have now completed the 3 year developmental assessments and we

have the sample size to undertake the analysis outlined above, while controlling for the

many potential confounders.

Question Four: The exposure estimates in the Columbia and CHAMACOS studies are reported

differently, thus preventing a meta-analysis from being undertaken. Both studies reported Bayley 

MDI and Bayley PDI results at 24 months and neither was statistically significant (Eskenazi et al. 

2007). This can be interpreted as a tendency but underpowered, or as a chance finding. Obtaining the 

raw data and pooling the results should be undertaken and has several benefits.
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a. The exposure estimates can be unified (either by calculating an estimated dose or

estimating a TCP level from the blood data). The dose cut points can be set to at least 3

doses to determine the presence of a dose response relationship.

b. The power will doubled by combining the two cohorts, thereby increasing the statistical 

power.

Answer: As discussed in our February 17th letter to Jack Fowle, a multi-site study has been 

funded the National Institute of Environmental Health Sciences ($568,000 total costs)

that will pool the children's centers at the University of California-Berkeley,

Columbia University (our children’s center) and the Mount School of Medicine to address

questions about relationships across the three cohorts between prenatal organophosphate

exposure, fetal growth and child neurocognitive development. Dr. Brenda Eskenazi at Berkeley

is the principal investigator on project.