Potential Effects of Chlorpyrifos on Fetal Growth Outcomes: Implications for Risk Assessment

Figures & data

FIGURE 1. Major metabolic pathways of chlorpyrifos metabolism. CPF is metabolized by cytochrome P-450 (CYP). CPO is the primary toxic metabolite and is detoxified by esterases including carboxylases and paraoxonase (PON1) (color figure available online).

TABLE 1. Biomarkers of Organophosphate Exposures and Their Relevance to Chlorpyrifos

Chemical name	Common acronym	Chemical structure	Not a CPF metabolite ^a	DEPs ^b	DMPs ^c	DAPs ^d
Chlorpyrifos	CPF
Chlorpyrifos-oxon	CPO
3,5,6-Trichloropyridinol	TCPy
Diethylthiophosphate	DETP			√		√
Diethylphosphate	DEP			√		√
Diethyldithiophosphate	DEDTP		√	√		√
Dimethylthiophosphate	DMTP		√		√	√
Dimethylphosphate	DMP		√		√	√
Dimethyldithiophosphate	DMDTP		√		√	√
^aUrinary biomarkers that cannot be formed from CPF.
^b“DEPs” refers to broad class of urinary OP metabolites containing ethyl groups.
^c“DMPs” refers to broad class of urinary OP metabolites containing methyl groups that are not biomarkers of CPF.
^d“DAPs” refers to DEPs and DMPs collectively and include OP metabolites that are not biomarkers of CPF.

TABLE 2. Characteristics of the Epidemiologic Studies Reporting Associations Between Chlorpyrifos or Relevant Metabolites and Fetal Growth Outcomes

Study Cohort and Location	Author (sample size; enrollment or birth dates)	Consideration of tobacco, ethanol, drug exposure	Chlorpyrifos measure or metabolite ^a	Detection limit/mean or median exposure levels	Source and timing of sample	Head circumference	Birth weight	Birth length	Ponderal index	Abdominal circumference
Columbia Center for Children's Environmental Health (New York, USA)	Perera et al. 2003 (n = 263; n = 113 for plasma) Enrolled 9/98–11/99 ^b	Illicit drug users and active smokers were excluded based on self-reported history and plasma cotinine concentrations >15 ng/ml. Covariates included maternal self-reported environmental tobacco smoke in the home. Cotinine and alcohol consumption were not significant predictors of outcomes and were not included.	CPF (parent compound)	Detected in 94% of samples, arithmetic mean = 7.6 pg/g	Umbilical Cord Plasma ^c	X	X	X
	Whyatt et al. 2004 (n = 314; n = 286 ^f cord blood) Births 3/98–7/02		CPF (parent compound)	31% of CPF samples were below LOD (LOD not reported) Mean = 4 pg/g (umbilical cord plasma)	Umbilical Cord Plasma ^c Personal air samples were collected in the 3rd trimester for two days	X	X	X
	Whyatt et al. 2005 (n = 571; n = 341 cord blood) Enrolled 1/98–1/04		CPF ^d (parent compound)	Detected in 64% of cord blood samples, mean = 3.7 pg/g; detected in 99.7% of air samples; mean = 14.3 ng/m^3	Umbilical Cord Plasma ^c Personal air samples were collected in the 3rd trimester for two days	X	X	X
	Rauh et al. 2006 (n = 254) Births 2/98–5/02		CPF (parent compound)	LOD = 0.5–1 pg/g; 80 samples below LOD Median levels not provided	Umbilical Cord Plasma ^c	X	X	X
Mt. Sinai Center for Children's Environmental Health and Disease Prevention Research (New York, USA)	Berkowitz et al. 2004 (n = 404) Enrolled 3/98–3/02	Women consuming more than 2 alcoholic beverages per day or using illegal drugs were excluded. “Active and passive cigarette smoking were not included in the final models because they did not affect the results and only increased the variance. (Berkowitz et al. 2004).	TCPy	LOD level = 11.0 μg/L; 57% of samples were below LOD	Maternal urine collected in 3rd trimester ^e	X	X	X
	Wolff et al. 2007 (n = 404) Enrolled 3/98–3/02		DEP, DAP	DEP detected in 88.1%, DAP detected in 97.2%; Median = 18.1 nm/L DEP, 75.9 nm/L DAP	Maternal urine collected during the 3rd trimester	X	X	X	X
The Center for Health Assessment of Mothers and Children of Salinas (California, USA)	Eskenazi et al. 2004 (n = 488) 10/99–10/00	“Smoking, alcohol, and illicit drug use were not included in the models because very few women reported use and controlling for these variables did not alter the results.” Environmental tobacco smoke and caffeinated beverages also did not alter the results and were not included.	TCPy, DEP, DAP	TCPy detected in 77% of samples; median 3.3 μg/L DEP and DAP detected in 99.8% of samples; median = 22 nmol/L for DEP, 136 nmol/L for DAP	Maternal urine collected at mean = 13 wks (range 4–29 wks) and mean = 26 wks (range 18–39 wk)	X	X	X	X
New Jersey Cohort (New Jersey, USA)	Barr et al. 2010 (n = 150) 7/03–5/04	“The vast majority of the population was non-smoking (96%).” Women were excluded if they were taking medications that could interfere with metabolism of environmental chemicals. No information on controlling for smoking, alcohol or illicit drug use.	CPF (parent compound)	Maternal Blood: Mean = 0.09ng/g (SD = 0.87), Median = 0.0007 ng/g; detected in 98.6% of samples Cord Blood: Mean = 0.55ng/g (SD = 0.73), Median = 0.0007; detected in 62.8% of samples	Maternal blood (collected immediately prior to birth) and umbilical serum	X	X	X		X
* LOD = limit of detection; SD = standard deviation; See footnotes in Table 2 for additional covariates included in the final statistical models.
^aCPF = chlorpyrifos; DAP = total dialkyl phosphates; DEP = diethylphosphate; TCPy = 3,5,6-trichloro-2-pyridionol.
^bBased on Whyatt et al. 2002 as cited by Perera et al. 2003.
^cMaternal blood was used when cord blood was unavailable.
^dChlorpyrifos was also measured in maternal air samples during the third trimester of pregnancy.
^eMaternal blood and cord blood was also used to assess PON1 activity at the PON1 polymorphisms.
^fBased on 256 cord blood samples plus 31 imputed values based on maternal cord levels.

TABLE 3. Summary of growth indices by metabolite

CPF in Blood or Air Samples	TCPy in Urine Samples	DEP and DAP in Urine Samples
HEAD CIRCUMFERENCE (cm)
COLUMBIA COHORT (CCCEH) Perera et al. 2003 ^a (cord plasma) Ln- CFP (All Participants): Ln-HC β = −0.005, n = 113 Ln- CFP (African American Participants): Ln-HC β = −0.003, n = 57 Ln- CFP (Dominican Participants): Ln-HC β = −0.005, n = 56 Whyatt et al. 2004 ^c (cord plasma) Ln-CPF in cord blood samples: β = −0.01 (95% CI = −0.13, 0.11), n = 287 Ln-CPF in maternal personal air samples: β = −0.04 (95% CI = −0.18, 0.10), n = 271 Whyatt et al. 2005 ^c (cord plasma) No association observed between Ln- CFP in blood or air samples and head circumference (data not shown) Rauh et al. 2006 ^f (cord plasma) CPF exposure initially categorized into undetectable (n = 80) and 3 tertiles in detectable range (n = 65, 39 and 44). Lower 3 groups were combined and compared with highest group: Ln-CPF≤6.17pg/g: mean = 34.35 (SD = 1.84), n = 204 Ln-CPF >6.17pg/g: mean = 34.03(SD = 1.69), n = 50	CHAMACOS Eskenazi et al. 2004 ^b No detectable levels (referent), n = 41 TCPy <3.3ug/L: β = 0.06 (95% CI = −0.37, 0.49), n = 220 TCPy ≥3.3ug/L: β = 0.04 (95% CI = −0.39, 0.47), n = 221 MT. SINAI COHORT Berkowitz et al. 2004 ^d TCPy<11.0 μg/L: mean = 33.8 (SD = 1.7), n = 216 TCPy >11.0 μg/L: mean = 33.8 (SD = 1.7), n = 171	CHAMACOS Eskenazi et al. 2004 ^b Log10-DEP: β = 0.28 (95% CI: −0.02, 0.59), n = 486 Log 10 -DAP: β = 0.32 (95% CI: 0.03, 0.62), n = 485 MT. SINAI COHORT Wolff et al. 2007 ^e Log10-DEP (no creatinine adjustment): β = −0.067 (SE = 0.12), n = 318 Log10-DEP (creatinine adjustment): β = −0.052 (SE = 0.12), n = 318 Log10-DAP (no creatinine adjustment): β = −0.26 (SE = 0.13), n = 318 Log10-DAP (creatinine adjustment): β = −0.25 (SE = 0.13), n = 318
NEW JERSEY COHORT Barr et al. 2010 ^g Maternal Serum: CPF ≤0.0007ng/g: 35.0 in (SD = 1.3), n = 34 CPF >0.0007ng/g: 33.4 in (SD = 0.6), n = 104 (75^th percentile of 138) Cord Serum: CPF ≤1.32ng/g: 35.0 in (SD = 1.2), n = 37 CPF >1.32ng/g: 34.9 in (SD = 1.4), n = 111 (75^th percentile of 148)
BIRTH WEIGHT (g)
COLUMBIA COHORT Perera et al. 2003 ^a (cord plasma) Ln- CFP (All Participants): Ln-BW β = −0.04, n = 113 Ln- CFP (African American Participants): Ln-BW β = −0.05, n = 57 Ln- CFP (Dominican Participants): Ln-BW β = −0.02, n = 56 Whyatt et al. 2004 ^c (cord plasma) Ln-CPF in cord blood samples: β = −42.6 (95% CI = −81.8, −3.8),n = 287 Newborns were categorized into 4 exposure groups: Group 1 <LOD and Groups 2,3,4 >LOD divided into low, mid and high tertile exposure groups. Sample size reported to be 32, 20, 24, 25% for Groups 1,2,3,4, respectively. Group 1 vs. 2: β = 39.2 (95% CI = −107.3, 185.7), n = 57 Group 1 vs. 3: β = −50.9 (95% CI = −188.2, 86.3), n = 69 Group 1 vs. 4: β = −150.1 (95% CI = −287.7, −12.5), n = 72 Ln-CPF in maternal personal air samples: β = −17.7 (95% CI = −64.2, 28.9), n = 271 Whyatt et al. 2005 ^c (cord plasma) Ln-CPF in participants born before 1/1/01: β = −67.3 (95% CI = −116.6, −17.8), n = 237 Ln-CPF in participants born after 1/1/01: β = 30.7 (95% CI = −108.6, 169.9), n = 77 No association between air samples and birth weight (data not shown)	CHAMACOS Eskenazi et al. 2004 ^b No detectable levels (referent), n = 41 TCPy <3.3ug/L: β = −6.0 (95% CI = −138, 126), n = 220 TCPy ≥3.3ug/L: β = 27.0 (95% CI = −106, 159), n = 221 MT. SINAI COHORT Berkowitz et al. 2004 ^d TCPy <11.0 μg/L: mean = 3284 (SD = 441), n = 216 TCPy >11.0 μg/L: mean = 3296 (SD = 434), n = 171	CHAMACOS Eskenazi et al. 2004 ^b Log10-DEP: β = 52.0 (95% CI: −40.0, 144.0), n = 486 Log10-DAP: β = 42.0 (95% CI: -46.0, 131.0), n = 485 MT. SINAI COHORT Wolff et al. 2007 ^e Log10-DEP (no creatinine adjustment): β = −52.0 (SE = 32.0), n = 318 Log10-DEP (creatinine adjustment): β = −56.0 (SE = 32.0), n = 318 Log10-DAP (no creatinine adjustment): β = −25.0 (SE = 34.0), n = 318 Log10-DAP (creatinine adjustment): β = −27.0 (SE = 34.0), n = 318
BIRTH WEIGHT (g) Continued
COLUMBIA COHORT (cont.) Rauh et al. 2006 ^f (cord plasma) CPF exposure initially categorized into undetectable (n = 80) and 3 tertiles in detectable range (n = 65, 39 and 44). Lower 3 groups were combined and compared with highest group: Ln-CPF≤6.17pg/g: mean = 3450.93 (SD = 448.30), n = 204 Ln-CPF >6.17pg/g: mean = 3239.58(SD = 558.09), n = 50 NEW JERSEY COHORT Barr et al. 2010 ^g Maternal Serum: CPF ≤0.0007ng/g: 3548 (SD = 448), n = 34 CPF >0.0007ng/g: 3053 (SD = 111), n = 104 (75^th percentile of 138) Cord Serum: CPF ≤1.32ng/g: 3544 (SD = 433), n = 37 CPF >1.32ng/g: 3581 (SD = 422), n = 111 (75^th percentile of 148)
BIRTH LENGTH (cm)
COLUMBIA COHORT Perera et al. 2003 ^a (cord plasma) Ln-CPF (All Participants): Ln-BL β = −0.02, n = 113 Ln-CPF (African American Participants): Ln-BL β = −0.01, n = 57 Ln-CPF (Dominican Participants): Ln-BL β = −0.02, n = 56 Whyatt et al. 2004 ^c (cord plasma) Ln-CPF in cord blood samples: β = −0.24 (95% CI = −0.47, −0.01), n = 287 Newborns were categorized into 4 exposure groups: Group 1 <LOD and Groups 2,3,4 >LOD divided into low, mid and high tertile exposure groups. Sample size reported to be 32, 20, 24, 25% for Groups 1,2,3,4, respectively. Group 1 vs. 2: β = 0.17 (95% CI = −0.70, 1), n = 57 Group 1 vs. 3: β = −0.21 (95% CI = −1, 0.61), n = 69 Group 1 vs. 4: β = −0.75 (95% CI = −1.6 to 0.06), n = 72 Ln-CPF in maternal personal air samples: β = −0.02 (95% CI = −0.28, 0.25), n = 271	CHAMACOS Eskenazi et al. 2004 ^b No detectable levels (referent), n = 41 TCPy <3.3ug/L: β = 0.09 (95% CI = −0.70, 0.87), n = 220 TCPy >3.3ug/L: β = 0.44 (95% CI = −0.35, 1.22), n = 221 MT. SINAI COHORT Berkowitz et al. 2004 ^d TCPy <11.0 μg/L: mean = 50.4 (SD = 2.4), n = 216 TCPy >11.0 μg/L: mean = 50.8 (SD = 2.4), n = 171	CHAMACOS Eskenazi et al. 2004 ^b Log10-DEP: β = 0.40 (95% CI: −0.15, 0.94), n = 486 Log10-DAP: β = 0.52 (95% CI: -0.01, 1.05), n = 485 MT. SINAI COHORT Wolff et al. 2007 ^e Log10-DEP (no creatinine adjustment): β = −0.02 (SE = 0.18), n = 318 Log10-DEP (creatinine adjustment): β = 0.017 (SE = 0.18), n = 318 Log10-DAP (no creatinine adjustment): β = −0.13 (SE = 0.19), n = 318 Log10-DAP (creatinine adjustment): β = −0.13 (SE = 0.19), n = 318
Whyatt et al. 2005 ^c (cord plasma) Ln-CPF in participants born before 1/1/01: β = −0.43 (95% CI = −0.73, −0.14), n = 237 Ln-CPF in participants born after 1/1/01: β = 0.07 (95% CI = −0.65, 0.79), n = 77 No association between air samples and birth length (data not shown) Rauh et al. 2006 ^f (cord plasma) CPF exposure initially categorized into undetectable (n = 80) and 3 tertiles in detectable range (n = 65, 39 and 44). Lower 3 groups were combined and compared with highest group: Ln-CPF≤6.17pg/g: mean = 51.05 (SD = 3.60); n = 204 Ln-CPF >6.17pg/g: mean = 50.02 (SD = 2.41); n = 50 NEW JERSEY COHORT Barr et al. 2010 ^g Maternal Serum: CPF ≤0.0007ng/g: mean = 51.3 (SD = 3.0), n = 34 CPF >0.0007ng/g: mean = 49.8 (SD = 0.2), n = 104 (75^th percentile of 138) Cord Serum: CPF ≤1.32ng/g: mean = 51.4 (SD = 3.1), n = 37 CPF >1.32ng/g: mean = 50.9 (SD = 1.7), n = 111 (75^th percentile of 148)
PONDERAL INDEX (g/cm^3)
None reported	CHAMACOS Eskenazi et al. 2004 ^b No detectable levels (referent), n = 41 TCPy <3.3ug/L: β = −0.01 (95% CI = −0.12, 0.11), n = 220 TCPy ≥3.3ug/L: β = −0.04 (95% CI = −0.16, 0.08), n = 221	CHAMACOS Eskenazi et al. 2004 ^b Log10-DEP: β = −0.01 (95% CI = −0.09, 0.07), n = 486 Log10-DAP: β = −0.04 (95% CI = −0.12, 0.04), n = 485 MT. SINAI COHORT Wolff et al. 2007 ^e Log10-DEP (no creatinine adjustment): β = −0.04 (SE = 0.02), n = 318 Log10-DEP (creatinine adjustment): β = −0.04 (SE = 0.02), n = 318 Log10-DAP (no creatinine adjustment): β = −0.002 (SE = 0.023), n = 318 Log10-DAP (creatinine adjustment): β = −0.003 (SE = 0.023), n = 318
ABDOMINAL CIRCUMFERENCE (in.)
NEW JERSEY COHORT Barr et al. 2010 ^g Maternal Serum: CPF ≤0.0007ng/g: mean = 32.0 (SD = 2.7), n = 34 CPF >0.0007ng/g: mean = 29.2 (SD = 0.8), n = 104 (75^th percentile of 138) Cord Serum: CPF ≤1.32ng/g: mean = 32.0 (SD = 2.7), n = 37 CPF >1.32ng/g: mean = 32.5 (SD = 2.3), n = 111 (75^th percentile of 148)	None reported	None reported
Note: bold text indicates p<0.05.
BMI: body mass index; BL: birth length; BW: birth weight; CPF = chlorpyrifos; CI = confidence interval; DAPs = total dialkyl phosphates; DEPs = diethylphosphates; HC = head circumference; Ln = log transformed; SD = standard deviation; SE = standard error; TCPy = 3,5,6-trichloro-2-pyridionol.
^aAdjusted for BMI, parity, cotinine, sex of baby, and gestational age.
^bAdjusted for timing of urine collection, timing of entry into prenatal care, maternal age, parity, infant sex, country of birth, weight gain, BMI, poverty level, gestational age, and (gestational age)^2.
^cAdjusted for gestational age of newborn, maternal prepregnancy weight and net weight during pregnancy, newborn sex, parity, race/ethnicity, environmental tobacco smoke in the home, season of delivery, and for head circumference whether or not the delivery was by cesarean section.
^dAdjusted for race/ethnicity, infant sex, and gestational age.
^eAdjusted for maternal age, race/ethnicity, maternal BMI x pregnancy weight gain (interaction), infant sex, and gestational age.
^fNo adjustments reported.
^gAdjusted for maternal age, primigravida, race, prepregnancy BMI, infant sex, and gestational age.

TABLE 4. Summary of PON1 results presented by the Mt. Sinai Cohort that are related to potential CPF biomarker exposure

	Head circumference (HC; cm)	Birth weight (BW; g)	Birth length (cm)	Ponderal index (g/cm^3)
Berkowitz, et al. 2004 ^a	PON1 Enzyme Activity ^b	PON1 Enzyme Activity	PON1 Enzyme Activity	Not reported
	TCPy < LOD:	TCPy < LOD:	TCPy < LOD:
	Tertile1 PON1 activity ^c : mean = 33.6 (SD = 1.8), n = 76	Tertile1 PON1 activity ^c : mean = 3237 (SD = 456), n = 76	Tertile1 PON1 activity ^d : mean = 50.3 (SD = 2.3), n = 75
	Tertile2 PON1 activity: mean = 33.7 (SD = 1.7), n = 62	Tertile2 PON1 activity: mean = 3255 (SD = 436), n = 62	Tertile2 PON1 activity: mean = 50.1 (SD = 2.2), n = 62
	Tertile3 PON1 activity: mean = 34.1 (SD = 1.7), n = 70	Tertile3 PON1 activity: mean = 3337 (SD = 444), n = 71	Tertile3 PON1 activity: mean = 50.3 (SD = 2.3), n = 71
	TCPy > LOD:	TCPy>LOD:	TCPy>LOD:
	Tertile1 PON1 activity: mean = 33.3 (SD = 1.5), n = 47	Tertile1 PON1 activity: mean = 3278 (SD = 395), n = 47	Tertile1 PON1 activity: mean = 50.9 (SD = 2.3), n = 46
	Tertile2 PON1 activity: mean = 34.0 (SD = 1.5), n = 57	Tertile2 PON1 activity: mean = 3327 (SD = 406), n = 57	Tertile2 PON1 activity: mean = 51.0 (SD = 2.3), n = 57
	Tertile3 PON1 activity: mean = 34.1 (SD = 1.6), n = 55	Tertile3 PON1 activity: mean = 3270 (SD = 409), n = 55	Tertile3 PON1 activity: mean = 50.8 (SD = 2.4), n = 55
	(p-value for trend in HC across PON1 tertile levels among participants with TCPy>LOD = 0.014)	(No statistically significant trends) PON 1 genotypes Q192R, L55M, −909, −162, −108 No statistically significant results with TCPy, data not shown	(No statistically significant trends) PON 1 genotypes Q192R, L55M, −909, −162, −108 No statistically significant results with TCPy, data not shown
	(p-value for interaction between PON1 activity and TCPy >0.05)
	PON 1 genotypes Q192R, L55M, −909, −162, −108
	No statistically significant results with TCPy, data not shown
Wolff, et al. 2007 ^d	No interactions between PON1 enzyme or PON192 genotype and head circumference by DAPs (data analyzed but not shown; DEPs not tested)	PON1 Enzyme Activity	Interactions between PON192 genotype and birth length by DAPs or DEPs were tested but results were not reported	No statistically significant association between PON1 enzyme or PON192 and Ponderal Index by DEP levels (data not shown)
		Maternal Log10-DEPs < Median Level:
		Tertile1 PON1 activity^c: mean = 3305 (SE = 53), n = 60
		Tertile2 PON1 activity: mean = 3348 (SE = 57), n = 53
		Tertile3 PON1 activity: mean = 3396 (SE = 64)^†, n = 45
		Maternal Log10-DEPs > Median Level:
		Tertile1 PON1 activity: mean = 3233 (SE = 56)^†, n = 53
		Tertile2 PON1 activity: mean = 3282 (SE = 57), n = 51
		Tertile3 PON1 activity: mean = 3279 (SE = 54), n = 56
		(p-value for interaction between PON1 and DEPs = 0.88)
		(† p-value for BW among participants in 3rd tertile (higher PON1 enzyme activity) and DEPs< median level versus participants in 1st tertile (lower PON1 enzyme activity) and DEPs>median level = 0.042, 164g difference)
Wolff, et al. 2007 ^d (continued)		PONQ192R Genotype
		Maternal Log10-DEPs < Median Level:
		PON192RR (slow): mean = 3346 (SE = 69), n = 39
		PON192RQ (medium): mean = 3278 (SE = 46), n = 84
		PON192QQ (fast): mean = 3453 (SE = 60)‡ ǁ, n = 33
		Maternal Log10-DEPs > Median Level:
		PON192RR (slow): mean = 3254 (SE = 63)‡, n = 55
		PON192RQ (medium): mean = 3285 (SE = 50), n = 66
		PON192QQ (fast): mean = 3232 (SE = 52) ǁ, n = 42
		(p-value for interaction between PON192 and DEPs = 0.076)
		(‡p-value for BW among participants with PON192QQ and DEPs< median level versus participants with PON192RR and DEPs>median levels = 0.020, 199g difference)
		(ǁ p-value for BW among participants with PON192QQ and DEPs < median level versus participants with PON192QQ and DEPs > median levels = 0.005)
Note: Bold text indicates statistical significance for trends across PON1 tertiles subdivided by CPF related biomarker exposure, or statistically significant interactions between PON1 or PON genotype and relevant biomarkers (DEP, DAP, TCPy).
^a Adjusted for race/ethnicity, infant sex, and gestational age.
^bPhenylacetate was used as substrate for all PON1 enzyme assays in this table.
^cMaternal PON1 activity was categorized based on tertile distribution. Wolff et al. 2007 describes tertiles as tertile 1: <96 mg/m-mL (slow activity); tertile 2: 97–116.6 mg/m-mL; tertile 3: 116.7–200 mg/m-mL (fast activity). Berkowitz et al. 2004 did not provide details on tertile distribution, but we assume the tertiles are defined similarly because of nearly identical cohort (both have sample size of 404 with only minor discrepancies in the reported characteristics of population).
^dAdjusted for race, sex, gestational age, and creatinine level. Urinary metabolites included samples with creatinine >20 mg/dL. PON activity was cut at >96 and <116.7 μg/mL/min for the second tertile.

TABLE 5. Birth outcomes and other related measures in animal CPF studies

Reference Type of Study	Exposure route, duration, and dose levels (mg/kg-d)	Species and number of litters (litters/dose group)	NOEL fetal or pup birth weight	NOEL pup mortality or dead fetuses	NOEL maternal toxicity	NOEL maternal RBC or brain ChEI	NOEL pup RBC or brain ChEI
Breslin et al. 1996 Developmental	Gavage GD6-15 0, 0.1, 3, 15	Fischer 344 rats N=24–29	0.1 mg/kg-d based on ↑fetal weight (however, authors consider NOAEL >15 based on historical control data)	>15 mg/kg-d	3 mg/kg-d based on clinical signs of ChEI	0.1 based on RBC ChEI (sacrificed GD15, 4 h after dosing)	Not measured
Breslin et al. 1996 Reproduction	Diet 10 wk prior to mating for 2 – generations 0, 0.1, 1, 5	Sprague-Dawley rats F1 N=24–30 F2 N=22–26	F1: 1 mg/kg-d based on ↓ pup weight at 5 mg/kg-d in male and female pups on Day 1 (not statisitically significant).F2: 0.1 mg/kg-d based on ↓ pup weight in F2 male on Day 1 (not stat sig; 1 mg/kg-d not considered by authors to be treatment related)	F1: 1 mg/kg-d F2: 1 mg/kg-d (however, authors consider loss of 5 litters at 5 mg/kg-d compared to 3 in controls not treatment-related)	1 mg/kg-day based on, histopathology in adrenal cortex, evidence of maternal neglect, slight decreases in feed consumption during last 2 wk of lactation>5 mg/kg-d based on clinical findings during gestation	F1 and F2: 0.1 mg/kg-d RBC ChEI; 1 mg/kg-day brain ChEI (sacrificed 25–27 wk of age following 19–21 wk of exposure)	Not measured
Maurissen et al. 2000 and Mattsson et al. 2000 Developmental Neurotoxicity	Gavage GD6-LD10 0, 0.3, 1, 5	Sprague-Dawley rats N=20	1 mg/kg-d based on ↓ birth weight in males and females	1 mg/kg-d pup body weight on day of birth, and pup death prior to PND 4 culling	1 mg/kg-d based on clinical signs of ChEI, body weight gain decreases GD17-20	< 0.3 mg/kg-d RBC ChEI GD20,; 0.3 brain ChEI (sacrificed 2-4 h after dosing GD20)	1 mg/kg-d at GD20, (sacrificed 2-4 h after dosing)
Farag et al. 2003 Developmental	Gavage GD6-15 0, 5, 15, 25	Fischer 344 rats N=24–28	15 mg/kg-d based on ↓ fetal weight at 25 mg/kg-d	15 mg/kg-d	5 mg/kg-d based on clinical signs of ChEI (tremors) and reduced body weights	5 mg/kg-d based on brain ChEI (sacrificed GD 21, 6 d after last dose)	>25 mg/kg-d fetal brain
Akhtar et al. 2006 Developmental	Gavage GD0-20 0, 9.6, 12, 15	Wistar rats N=20	12 mg/kg-d based on ↓ fetal weight at 15 mg/kg-d	12 mg/kg-d	9.6 mg/kg-d based on reduced body weight gain at 12 mg/kg-day and clinical signs of ChEI at 15 mg/kg-d	Not measured	Not measured
Lassiter et al. 2008 ^a Developmental	Gavage GD7-PND21 0, 2.5	Long Evans rats N=9–10	>2.5 mg/kg-d, based on no effects on birth weight and body weights prior to weaning (after PND 51 ↑body weight in males only)	>2.5 mg/kg-d	>2.5 mg/kg-d	Not measured	Not measured
Deacon et al. 1980 Developmental (initial study)	Gavage GD6-15 0,1, 10, 25	CF-1 Mouse 29–36	10 mg/kg-d based on ↓ fetal weight at 25 mg/kg-day Fetal crown-rump length NOEL=10 mg/kg-d	>25 mg/kg-d	1 mg/kg-d based on clinical signs of ChEI	<1 mg/kg-d RBC ChEI (sacrificed 5 h after last dose)	1 mg/kg-d fetal homogenate (sacrificed 5 h after last dose)
Deacon et al. 1980 Developmental (repeat study at lower doses)	Gavage GD 6-15 0, 0.1, 1, 10	CF-1Mouse 23–30	>10 mg/kg-d based on no effects on fetal weight Fetal crown-rump length NOEL=10 mg/kg-d	>10 mg/kg-d	>10 mg/kg-d	0.1 mg/kg-d RBC ChEI (sacrificed 5 h after last dose)	1 mg/kg-d fetal homogenate (based on non-statistically significant ChEI; sacrificed 5 h after last dose)
Note: NOAEL, no-observed-adverse-effect level; NOEL, no-observed-effect level; GD, gestation day; PND, postnatal day; ChEI cholinesterase inhibition.
^aThis study did not meet our a priori inclusion criteria of n = 20. It was included because the primary focus of this study was body weight.

FIGURE 2. Comparison of NOAELs and LOAELs for fetal weight, birth weight, and maternal toxicity from animal studies with human exposure estimates. Human exposure estimates were calculated from McCone et al. (2007) based on CHAMACOS study. McKone et al. (2007) estimated total CPF exposures (dietary, inhalation, dermal, nondietary ingestion) among pregnant women in the CHAMACOS cohort to be 1.43 to 6.73 nmol/d, which is equivalent to 0.007 to 0.031μg/kg-d, assuming a 75.8-kg pregnant female (U.S. EPA 2011a, from NHANES). Lowe et al. (2009) estimated exposures based on umbilical cord blood levels from the CCCEH cohort (Whyatt et al. 2005). The NOAEL of 1 mg/kg-d for pup birth weight is based on non-statistically significant treatment-related decreases at 5 mg/kg-d Breslin et al. (2006) and Maurissen et al. (2000) (see Table 5). The LOAEL of 5 mg/kg-d for maternal toxicity (e.g., tremors) and pup death is based on Maurissen et al. (2000). The NOAEL of 12 mg/kg-d for fetal birth weight is the highest NOAEL for effects in multiple studies as summarized in Table 5.

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