Evaluation of Epidemiology and Animal Data for Risk Assessment: Chlorpyrifos Developmental Neurobehavioral Outcomes

Figures & data

TABLE 1. Summary of Study Characteristics

Study Characteristic	Columbia Center for Children's Environmental Health (CCCEH)	Center for Health Assessment of Mothers and Children of Salinas (CHAMACOS)		Mt. Sinai Children's Environmental Health Cohort Study
Author (citation)	Rauh et al., 2006	Eskenazi et al., 2007	Young et al., 2005	Engel et al., 2007
Location	New York City, NY, USA	Salinas Valley, CA, USA		New York City, NY, USA
Study design	Cohort	Cohort		Cohort
Enrollment years	1998–2002	1999–2000		1998–2001
Maternal eligibility	Pregnant nonsmokers, self-identified as black or Dominican, registered at the OB-GYN clinics at NY Presbyterian Medical Center and Harlem Hospital prior to 20 weeks gestation, 18–35 years old, free from diabetes and hypertension, no diagnosis of HIV, did not use drugs, and lived in the area for ≥1 year.	Pregnant Spanish or English speaking women, receiving prenatal care at one of six community clinics that served farm workers with plans to deliver at Natividad Medical Center, <20 weeks gestation at enrollment, ≥18 years old, eligible for Medi-Cal (California's Medicaid health care program).		Pregnant, primiparous, singleton pregnancies, receiving prenatal care from Mount Sinai Hospital, no underlying health conditions that might predispose to giving birth to high risk infants
Study size ^a	254	447	381	311
Outcome measurements (assessment tool) ^b	BSID:MDI;	BSID:MDI;	BNBAS	BNBAS
	BSID:PDI;	BSID:PDI;
	CBCL	CBCL
Outcome variable format	BSID: Continuous and categorical measurement of BSID:MDI and BSID:PDI (low: ≤85; high>85)	BSID: Continuous measurement of BSID:MDI and BSID:PDI.	BNBAS: Continuous measure of BNBAS domains. Categorical measure of abnormal reflexes (>3 vs. ≤3)	BNBAS: Continuous measure of BNBAS domains.
	CBCL: Categorical, using 98^th percentile of the national normative sample in each domain as cutoff	CBCL: Categorical, using 93^rd percentile of the national normative sample in each domain as the cutoff
Timing of outcome assessment	BSID: Age 12, 24, 36 months	BSID: Age 6, 12, 24 months	BNBAS: Age ≤2 months	BNBAS: Within ≤5 days of delivery
	CBCL: Age 36 months	CBCL: Age 24 months
Testing conditions	“… each child was tested under controlled conditions in the study office by a trained bilingual research assistant, checked for reliability. Five different testers conducted a total of 1101 BSID-II assessments over the course of the 3-year study period. Every effort was made to maximize reliability in scoring by using standardized training procedures and regular quality control. Interrater reliability for the 24-month BSID-II assessments was r = 0.92, on the basis of double-scoring of a random 5% of the sample.”	“Both [BSID] scales were administered in Spanish and/or English by psychometricians blind to exposure. Psychometricians were trained using standardized protocols and 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.”	“The BNBAS was administered once to each infant and in accordance with the BNBAS protocol, by four examiners trained to reliability by a BNBAS certified trainer. Examiners were blind to the infant's exposure status. Approximately 30% of the assessments included in the final sample were completed prior to discharge from the hospital; the remainder was performed at the CHAMACOS 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 was administered before hospital discharge (n =311) by one of four examiners. Examiners were either trained and certified by the Brazelton Institute or trained by a certified examiner. Examinations took place in a quiet, semidarkened, warm room adjacent to the neonatal nursery, or in the mother's private room. The BNBAS was not administered if the infant was admitted to the Neonatal Intensive Care Unit (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).”
Exposure measurement (Chemical/Metabolite ^c & biological specimen)	CPF in umbilical cord blood ^d	TCPy in maternal urineDEPs & DAPs in maternal and child urine	DEPs & DAPs in maternal urine	DEPs & DAPs in maternal urine
Timing of exposure assessment (approx.)	At delivery	During pregnancy (maternal prenatal): 14 and 26 weeks gestation; an average of these two measures was calculated	During pregnancy (maternal prenatal): 14 and 26 weeks gestation; an average of these two measures was calculated	During pregnancy (maternal prenatal): 31 weeks gestation
		Postnatal (child): age 6, 12, and 24 months	Post-delivery (maternal): 7 days postpartum
Exposure variable format for analysis	Categorical: >6.17 pg/g vs. ≤6.17 pg/g	Continuous: (nmol/L – log10)	Continuous: (nmol/L – log10)	Continuous: (nmol/L – log10)
		Categorical: (<LOD (ref), below median, above median detectable level)
Detection limit of chemical or metabolite	LOD = 0.5–1 pg/g; 80 samples below LOD	TCPy detected in 91% of prenatal maternal samples (averaged).	DEPs and DAPs detected in 100% of prenatal samples (averaged)	DEPs detected in 89% of samples; DAPs detected 97% of samples
	Median CPF levels not provided.	Prenatal (maternal):	Prenatal (maternal):	Prenatal (maternal):
		Median TCPy = 3.5 ug/L	Median DEPs = 21 nmol/L	Median DEPs = 24.7 nm/L
		Percent with DEPs and DAPs detected not provided.	Median DAPs = 132 nmol/L	Median DAPs = 82.0 nm/L
		Geometric means: Maternal (prenatal) (average)
		DEPs = 18.1 nmol/L,
		DAPs = 114.9nmol/L
		Geometric means: Child (postnatal)
		6 mos. DEPs = 10.6 nmol/L,
		12 mos. DEPs = 15.2 nmol/L,
		24 mos. DEPs = 10.5 nmol/L,
		6 mos. DAPs = 45.5 nmol/L,
		12 mos. DAPs = 59.5 nmol/L,
		24 mos. DAPs = 70.9 nmol/L,
Information Obtained from Questionnaire / Interview	Demographic characteristics, home characteristics, lifetime residential history, history of active/passive smoking, occupational history, maternal education, income level, alcohol/drug use during pregnancy, history of residential pesticide use. Test of Nonverbal Intelligence, Home Observation for Measurement of the Environment (HOME)	Family demographics, work histories of household members, maternal behaviors (smoking, alcohol, drug use), maternal medical history (previous pregnancies) Peabody Picture Vocabulary Test (PPVT), Center for Epidemiologic Studies Depression Scale, Home Observation for Measurement of the Environment (HOME)	Family demographics, work histories of household members, maternal behaviors (smoking, alcohol, drug use), maternal medical history (previous pregnancies)	Environmental exposures, sociodemographic information, medical history, lifestyle factors.
Method of Assessing Confounding	“Covariates were included in models as possible confounders if they (1) had a significant association with level of pesticide exposure and any measure of developmental outcomes in this sample, (2) altered the estimate of chlorpyrifos effect by ≥10%, or (3) had been identified as confounders in comparable studies.”	“Covariates were selected for these analyses if they were related to conditions of testing; 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 our data.”“In addition to the variables we included, we examined the potential confounding effects of several other variables suggested by the literature but they did not markedly alter the observed associations.”“For simplicity, the same set of covariates was used for CBCL models with three exceptions: maternal depression, found to be important (p < 0.10), was added, and psychometrician and assessment location were dropped, because scores were based on maternal report. “	“Covariates were initially selected based on their predictive value of BNBAS performance reported in the literature.” “Covariates from this (initial) list were selected for final regression models if they were related to each of the seven BNBAS cluster scores, unadjusted for other variables (p < 0.15).”	“Backward elimination was used to arrive at the final adjusted models. Covariates were eliminated if their exclusion caused less than a 20 percent change in the beta coefficient of the full model.”
Study Characteristics
Name or description	Columbia Center for Children's Environmental Health (CCCEH)	Center for Health Assessment of Mothers and Children of Salinas (CHAMACOS)			Mt. Sinai Children's Environmental Health Cohort Study
Maternal Demographic Characteristics
Race/ethnicity
White					20.3%
Black	∼42% ^f				27.3%
Latina		∼100%		∼100%	51.1%
Dominican	∼58% ^f				–
Other					1.3%
Maternal age	mean years ± SD	Age group	%	mean years ± SD	Age group	%
	24.6 ± 4.9 ^f	18–24	44.5%	26 ± 5	<20	35.7
		25–29	31.1%		20–24	32.5
		30–34	16.3%		25–29	12.2
		≥ 35	8.1%		30–34	15.1
					≥35	4.5
Did not complete high school	34.6%*	80.5%		81.4%	31.5%
Married/living as married/living with infant's father	17%*	81.9%		81.1%	52.4%
^aNumber of observations in analyses may be less, depending on the infant's attained age at the time of testing, availability of exposure data, testing data, and covariate data
^bOutcome assessment tool: BNBAS: Brazelton Neonatal Behavioral Assessment Scale; BSI-MDI: Bayley Scales of Infant Development – Mental Development Index; BSI-PDI: Bayley Scales of Infant Development – Psychomotor Development Index; CBCL: Child Behavior Checklist
^cChemicals/Metabolites: DAPs = total dialkyl phosphates; DEPs = diethylphosphate; CPF: chlorpyrifos; TCPy = 3,5,6-trichloro-2-pyridionol
^dMaternal blood was used when cord blood was unavailable.
^eMaternal smoking during pregnancy or maternal residence with a smoker during pregnancy.
^fWhyatt et al. 2004
*Calculated from values presented in Table 1 (Rauh_2006) for high and low exposure categories.
Abbreviations: LOD = limit of detection

TABLE 2. Summary of results from the Bayley Scales of Infant Development II (BSID II)

	Columbia Center for Children's Environmental Health (CCCEH)	Center for Health Assessment of Mothers and Children of Salinas (CHAMACOS)
	Rauh et al., 2006	Eskenazi et al., 2007
Exposure measurement	CPF in umbilical cord blood^a	TCPy in maternal^b urine	DEPs and DAPs in maternal^b and child urine
Exposure variable format	Categorical: (>6.17 pg/g- vs. ≤6.17 pg/g )	Categorical: (<LOD (reference), below median detected level, above median detected level)	Continuous: (nmol/L – log10)
Sample size for analysis	Range: 225 – 229	Not reported. TCPy detected in 91% of 445 maternal (prenatal) urine samples	Not reported. Urine metabolite measures available for 445 women (prenatal) and 373 children at 24 mo
BSID:MDI
β (95% CI); Positive associations (β >0) indicate biomarkers associated with higher (better) MDI scores
6 mo	Not measured	<Median: 0.24 (−2.12, 2.60)	Maternal DEPs: −0.16 (−1.96, 1.65)
			Child DEPs: 0.24 (−0.78, 1.25)
		≥Median: 0.08 (−2.29, 2.44)	Maternal DAPs: −1.15 (−2.89, 0.59)
			Child DAPs: −0.17 (−1.23, 0.90)
12 mo	−0.344 (SE = 1.66)	<Median: −0.45 (−3.67, 2.76)	Maternal DEPs: −1.14 (−3.51, 1.22)
			Child DEPs: 1.89 (0.21, 3.58) *
		≥Median: −0.65 (−3.88, 2.58)	Maternal DAPs: −1.34 (−3.59, 0.92)
			Child DAPs: 1.36 (−0.05, 2.78)
24 mo	−1.480 (SE = 2.03)	<Median: −1.02 (−5.34, 3.31)	Maternal DEPs: −0.85(−3.98, 2.27)
			Child DEPs: 1.02 (−0.52, 2.57)
		≥Median: −1.94 (−6.26, 2.37)	Maternal DAPs: −3.54 (−6.59, −0.49) *
			Child DAPs: 2.37 (0.50, 4.24) *
36 mo	−3.327 (SE = 1.76)	Not measured	Not measured
Covariates included in the regression models	Race, gender, maternal education, maternal IQ, gestational age, prenatal ETS exposure	Psychometrician, location, exact age at assessment, gender, breast-feeding duration, HOME score, household income above poverty threshold, parity, maternal PPVT.	Psychometrician, location, exact age at assessment, gender, breast-feeding duration, HOME score, household income above poverty threshold, maternal PPVT.
“Mental Delay” (BSID:MDI ≤ 85)^c,
OR (95% CI); Positive associations (OR>1) indicates biomarkers associated with MDI scores ≤ 85 (worse scores)
12 mo	1.22 ( 0.48–3.06)	Not reported	Not reported
24 mo	1.75 (0.86–3.60)	Not reported	Not reported
36 mo	2.37 (1.08–5.19)*	Not measured	Not measured
Covariates included in the regression models	Race, gender, maternal education, maternal IQ, gestational age, prenatal ETS exposure, home environment
BSID: PDI
β (95% CI); Positive associations (β>0) indicates biomarkers associated with higher (better) PDI scores
6 mo	Not measured	<Median: −0.56 (−4.03, 2.91)	Maternal DEPs: 0.02 (−2.63, 2.67)
			Child DEPs: 0.60(−0.89, 2.09)
		≥Median: −0.21 (−3.69, 3.27)	Maternal DAPs: −0.71 (−3.28, 1.86)
			Child DAPs: 0.39 (−1.18, 1.97)
12 mo	−3.30 (SE = 2.11)	<Median: −0.70 (−5.26, 3.86)	Maternal DEPs: 0.30 (−3.03, 3.63)
			Child DEPs: 1.91 (−0.46, 4.27)
		≥Median: −1.62 (−6.20, 2.96)	Maternal DAPs: −0.60 (−3.77, 2.57)
			Child DAPs: 1.22 (−0.78, 3.21)
24 mo	1.17 (SE = 1.98)	<Median: −2.65 (−6.50, 1.21)	Maternal DEPs: −0.86 (−3.64, 1.92)
			Child DEPs: 0.30 (−1.07, 1.67)
		≥Median: −2.72 (−6.57, 1.12)	Maternal DAPs: −1.28 (−4.01, 1.46)
			Child DAPs: 1.06 (−0.62, 2.74)
36 mo	−6.46 (SE = 2.18) *	Not measured	Not measured
Covariates included in the regression models	Race, gender, maternal education, maternal IQ, gestational age, maternal ETS exposure	Psychometrician, location, exact age at assessment, gender, breast-feeding duration, HOME score, household income above poverty threshold, parity, maternal PPVT.	Psychometrician, location, exact age at assessment, gender, breast-feeding duration, HOME score, household income above poverty threshold, maternal PPVT.
“Psychomotor Delay” (BSID:PDI ≤ 85) ^c
OR (95% CI); Positive associations (OR>1) indicates biomarkers associated with PDI scores ≤ 85 (worse scores)
12 mo	1.88 (0.78,4.53)	Not reported	Not reported
24 mo	1.01 (0.37,2.76)	Not reported	Not reported
36 mo	4.52 (1.61,12.70) *	Not measured	Not measured
Covariates included in the regression models	Race, gender, maternal education, maternal IQ, gestational age, prenatal ETS exposure, home environment
Abbreviations: DAPs = total dialkyl phosphates; DEPs = diethylphosphates; DMPs = dimethylphosphates; ETS: environmental tobacco smoke, HOME = Home Observation for Measurement of the Environment; PPVT = Peabody Picture Vocabulary Test; OR = odds ratio, CI = confidence interval.
a Maternal blood was used when cord blood was unavailable.
b Maternal samples were collected during pregnancy.
c Threshold for mental and psychomotor “delay” corresponds to 1 standard deviation and was defined by Rauh et al. (2006).
*p<.05, significance bolded for emphasis.

TABLE 3. Summary of results from the Child Behavior Checklist (CBCL)

	Columbia Center for Children's Environmental Health (CCCEH)	Center for Health Assessment of Mothers and Children of Salinas (CHAMACOS)
	Rauh et al., 2006	Eskenazi et al., 2007
Exposure measurement	CPF in umbilical cord blood^a	TCPy in maternal^b urine	DEPs and DAPs in maternal^b and child urine
Exposure variable format	Categorical: (>6.17 pg/g vs. ≤6.17 pg/g)	Categorical: (<LOD (reference), below median detected level, above median detected level)	Continuous: (nmol/L – log10)
Sample size for analysis	228	Not reported. TCPy detected in 91% of 445 maternal urine samples	Not reported. Urine metabolite measures available for 445 women (prenatal) and 373–405 children depending on timing (age) of follow-up testing.
Age at assessment	36 mo	24 mo
CBCL^c
OR (95% CI): Positive associations (OR >1) indicate biomarkers are associated with maternal reports indicating more CBCL problems.
Attention problems	11.26 (1.79–70.99)*	No statistically significant associations, data not reported	Maternal DEPs: 0.78 (0.26, 2.31) Child DEPs: 1.02 (0.61, 1.71)
			Maternal DAPs: 0.77 (0.27–2.24)
			Child DAPs: 1.41 (0.75, 2.64)
ADHD problems	6.50 (1.09–38.69)*	No statistically significant associations, data not reported	Maternal DEPs: 0.59 (0.21, 1.68) Child DEPs: 1.18 (0.72, 1.94)
			Maternal DAPs: 1.34 (0.50, 3.59)
			Child DAPs: 1.11 (0.61, 2.03)
PDD problems	5.39 (1.21–24.11)*	No statistically significant associations, data not reported	Maternal DEPs: 0.88 (0.37, 2.07) Child DEPs: 1.72 (1.12, 2.64)*
			Maternal DAPs: 2.25 (0.99, 5.16)
			Child DAPs: 1.71 (1.02, 2.87)*
Covariates included in the regression models	Race, gender, maternal education, maternal IQ, gestational age, prenatal ETS exposure, home environment	Not reported	Gender, exact age at assessment, breast-feeding duration, HOME score, household income above poverty threshold, parity, maternal PPVT, maternal depression
Abbreviations: DAPs = total dialkyl phosphates; DEPs = diethylphosphates; DMPs = dimethylphosphates; ETS: environmental tobacco smoke, HOME = Home Observation for Measurement of the Environment; PPVT = Peabody Picture Vocabulary Test; OR = odds ratio, CI = confidence interval; ADHD = attention deficit hyperactivity disorder; PDD = pervasive developmental disorder; CPF = chlorpyrifos.
^aMaternal blood was used when cord blood was unavailable.
^bMaternal blood was collected during pregnancy.
^cMeasured at 36 mo in Rauh et al. (2006) and 24 mo in Eskenazi et al. (2007). Rauh et al. 2006 dichotomized the participants’ CBCL scores at the 98^th percentile and Eskenazi 2007 dichotomized the participants’ CBCL scores at the 93^rd percentile.
*p < .05 significance bolded for emphasis.

TABLE 4. Summary of results from the Brazelton Neonatal Behavioral Assessment Scale (BNBAS)

	Center for Health Assessment of Mothers and Children of Salinas (CHAMACOS)		Mt. Sinai Children's Environmental Health Cohort Study
	Young et al., 2005		Engel et al., 2007
Exposure measurement	DEPs in maternal urine	DAPs in maternal urine	DEPs in maternal urine	DAPs in maternal urine
Exposure variable format	Continuous: (nmol/L – log10)	Continuous: (nmol/L – log10)	Continuous: (nmol/L – log10)	Continuous: (nmol/L – log10)
Sample size for analysis	Range: 175–381 (full sample)		Range: 144–253
BNBAS (Assessment < 3 days after birth) β (95% CI)			BNBAS (within 5 days of delivery) β (95% CI)
Habituation	0.47 (–0.05, 0.99)	0.10 (–0.40, 0.60)	0.17 (–0.23, 0.57)	0.08 (–0.30, 0.46)
Orientation	−0.11 (–0.65, 0.43)	−0.02 (–0.53, 0.49)	−0.11 (–0.41, 0.20)	−0.03 (–0.34, 0.28)
Motor	0.08 (–0.17, 0.33)	0.04 (–0.20, 0.28)	0.05 (–0.08, 0.17)	0.05 (–0.08, 0.17)
Range of State	−0.21 (–0.54, 0.12)	0.11 (–0.21, 0.43)	0.04 (–0.12, 0.19)	0.02 (–0.14, 0.17)
Regulation of State	−0.08 (–0.52, 0.37)	−0.07 (–0.50, 0.36)	−0.05 (–0.30, 0.21)	−0.03 (–0.28, 0.23)
Autonomic Stability	0.31 (0.01, 0.61)*	−0.09 (–0.38, 0.20)	−0.15 (–0.38, 0.08)	−0.11 (–0.33, 0.12)
Number of Abnormal Reflexes	0.08 (–0.16, 0.32)	−0.01 (0.24, 0.22)	RR^a=1.49 (1.12, 1.98)*	RR^a=1.32 (0.99, 1.77)
BNBAS (Assessment ≥ 3 days after birth^b) β (95% CI)
Habituation	0.20 (–0.43, 0.83)	0.06 (–0.54, 0.66)	NR	NR
Orientation	−0.33 (–0.73, 0.08)	−0.13 (–0.54, 0.27)	NR	NR
Motor	0.17 (–0.05, 0.38)	−0.07 (–0.28, 0.15)	NR	NR
Range of State	0.20 (–0.21, 0.62)	−0.02 (–0.44, 0.40)	NR	NR
Regulation of State	−0.24 (–0.72, 0.24)	−0.10 (–0.58, 0.37)	NR	NR
Autonomic Stability	−0.16 (–0.47, 0.14)	−0.19 (–0.49, 0.12)	NR	NR
Number of Abnormal Reflexes	0.37 (0.09, 0.64)*	0.53 (0.23, 0.82)*	NR	NR
BNBAS (full sample^b) β (95% CI)
Habituation	0.33 (–0.06, 0.72)	0.03 (–0.34, 0.40)	–	–
Orientation	−0.32 (–0.66, 0.03)	−0.17 (–0.50, 0.17)	–	–
Motor	0.10 (–0.06, 0.27)	−0.03 (–0.19, 0.14)	–	–
Range of State	−0.02 (–0.27, 0.24)	0.09 (–0.16, 0.34)	–	–
Regulation of State	−0.15 (–0.47, 0.17)	−0.07 (–0.39, 0.24)	–	–
Autonomic Stability	0.06 (–0.15, 0.27)	−0.16 (–0.36, 0.05)	–	–
Number of Abnormal Reflexes	0.22 (0.04, 0.40)*	0.23 (0.05, 0.41)*	–	–
Covariates**
Habituation	Age at BNBAS, smoking, alcohol, method of delivery, minutes fed since BNBAS, BNBAS interviewer.		Drug use during pregnancy, examiner, PON1 enzyme tertiles, urinary creatinine.
Orientation	Age at BNBAS, BNBAS interviewer, number of prenatal care visits.		Pre-pregnancy BMI, examiner, neonatal jaundice, PON1 enzyme tertiles, urinary creatinine.
Motor	Age at BNBAS, poverty level, gestational age at initiation of prenatal care, BNBAS interviewer.		Age at BNBAS, caffeine consumption during pregnancy, drug use during pregnancy, examiner, PON1 enzyme tertiles, urinary creatinine.
Range of State	Age at BNBAS, number of prenatal care visits, gestational age at initiation of prenatal care, alcohol, BNBAS interviewer.		Age at BNBAS, examiner, PON1 enzyme tertiles, urinary creatinine.
Regulation of State	Age at BNBAS, pre-pregnancy BMI, infant sex, parity, caffeine use, BNBAS interviewer.		Maternal education, examiner, PON1 enzyme tertiles, urinary creatinine.
Autonomic Stability	Age at BNBAS, infant sex, parity, vitamin use, minutes since fed at BNBAS, BNBAS interviewer, illicit drug use during pregnancy.		Age at BNBAS, examiner, smoking during pregnancy, PON1 enzyme tertiles, urinary creatinine.
Number of Abnormal Reflexes	Age at BNBAS, maternal age at delivery, smoking, vitamin use, BNBAS interviewer, mean diastolic and systolic blood pressure.		Examiner, anesthesia during delivery, PON1 enzyme tertiles, urinary creatinine.
Abbreviations: DAPs=total dialkyl phosphates; DEPs=diethylphosphates; CI=confidence interval; RR=relative risk.
^a“Poisson regression was used to analyze the relation between biomarker levels and the number of abnormal reflexes because of the count nature of the data.”
^bIn the CHAMACOS study (Young et al. 2005), the median age at administration of the BNBAS was 3 days, with an interquartile range between 1 and 26 days post-delivery.
*p<.05, significance bolded for emphasis.
**The same covariates were included in the models for DEPs and DAPs within each paper.

TABLE 5A. Developmental landmarks/neurodevelopmental reflex in the RAT (oral and sc)

						Magnitude of Dose Tested, mg/kg-day
Author	Test	Age Exposure	Age Testing	Sample Size Per Dose Level (Yes/No litter is unit)	Measure*	0.3	1	1 to 4^a	5.0	1.5 to 6^b
Maurissen et al., 2000	Dev't landmarks	GD7-PND11	PND1	All 8 pups/ litter; 20 litters/dose	Pinna unfolding	0 ^c	0		(↑ day)^dn.s. delay
	Not blind	ORAL corn oil	PND 11 until criterion	1 M or F /litter; 20 litters	Eye opening	0	0		0
			PND 27 until criterion	Yes, litter is unit	Vaginal opening	F0	F0		F↑ daydelay
			PND 38 until criterion		Preputial Separation	M0	M0		M(↑ day)^dn.s. delay
Johnson et al., 2009	Physical and Neurodev't reflex	PND1-21	Each day beginning on PND 1	12–14 pups/litter; 20 litters/dose	Pinna detachment		0	0		0
		ORAL corn oil		Within litter design	Downy fur development		0	0		0
	Not Blind	Incremental dose		Yes, litter is unit	Hair growth		0	0		0
		regimen for mid and			Incisor eruption		0	0		0
		high dose groups			Eye opening		0	0		0
					Surface righting		0	0		0
					Negative geotaxis		0	0		0
					Cliff avoidance		0	0		0
					Free fall righting		0	0		0
					Acoustic startle		0	0		0
Dam et al., 2000	Neurodev't reflex	PND1–4	PND3–4	23–24/group; 2 pups/sex/litterLitter is not unit	Surface righting reflex (time)		M0 F↑
	Videotaped Blind	SC DMSO	PND 5–8	37–46/group;2 pups/sex/litterLitter is not unit	Negative geotaxis (# animals making 180° turn)		M0 F↓
^aIncremental dose level of 1.0 (PND1–5),2.0 (PND6–13) and 4.0 (PND14–20) mg/kg-day.
^bIncremental dose level of 1.5 (PND1–5),3.0 (PND6–13) and 6.0 (PND14–20) mg/kg-day.
^call entries without “M” and/or “F” indicates analyses is based on males and females combined.
^d( ) indicate effect in parenthesis is not statistically significant (n.s.); authors defined significance at p = 0.02 for dev't landmarks.
*If statistical analyses or measures were described in methods, then it was included and listed as “0” if not reported to be statistically significant.

TABLE 5B. Developmental landmarks/neurodevelopmental reflex in the MOUSE (oral and sc)

						Magnitude of Dose Tested, mg/kg-day
Author	Test	Age and Route of Exposure	Age Testing	Sample Size Per Dose Level (Yes/No litter is unit)	Measure*	1	3	5	6	10
Billauer-Haimovitch et al., 2009	Developmental landmarks and neurodev't reflexNot blind	GD 9–18 SC DMSO	Not stated	1 pup/sex/litter; # litters and # pups not statedLitter is not unit. M & F from same litter pooled in analysis, and litter was not a factor	Surface righting response (day)	0^a	0	0		0
					Startle response (day)	0	0	0		0
					Appearance of fur (day)	0	0	0		0
					Eye opening (day)	0	0	0		0
					Ear opening (day)	0	0	0		0
Venerosi et al., 2009	Neurodev't reflexBlind	GD 14–17 ORAL peanut oil	PND 3,6, 9, 12, 15	1 pup/sex/litter; 18 litters control and 16 litters treatedYes, litter is unitLitter was a factor in the statistical analysis	Surface righting reflex (time): shortest of 3 measures				0^a
					Surface righting reflex (time): longest of 3 measures				0
					Grasping reflex (mean angle of falling)				0
					Forelimb toothpick reflex				0
					Cliff aversion				0
					Hindlimb toothpick reflex				(↓)^b p = 0.06
					Forelimb pencil reflex				0
					Total daily score (sum of all 7 reflex scores)				(↓) p = 0.06)
^aall entries without “M” and/or “F” indicates analyses is based on males and females combined.
^b( ) indicate effect in parenthesis is not statistically significant, author defined significance to be p<0.05.
*If statistical analyses or measures were described in methods, then it was included and listed as “0” if not reported to be statistically significant.

TABLE 5C. Neonatal behaviors in the MOUSE (oral and sc)

						Magnitude of Dose Tested, mg/kg-day
Author	Test	Age and Route of Exposure	Age of Testing	Sample Size Per Dose Level (Yes/No litter is unit)	Measure	1.0	3.0	6.0
Venerosi et al., 2009	Ultrasonic vocalization 4-minAutomated confirmed by observational detection	GD15–18 ORAL Peanut Oil	PND 4, 7,10	1 male/litter; 15 litters/groupYes, litter is unit	# calls			M0 PND4&7 M↓ PND10
					Duration of calls			M0 PND4&7 M↓ PND10
					frequency			M0 PND4&7 M↑ PND10
Ricceri et al., 2003	Ultrasonic vocalization 3-minNot blind	PND1–4 SC DMSO	PND 5, 8, 11	10 males and 10 females from unstated # littersYes, litter is unitLitter was a factor in the statistical analysis	# calls	0^a	0
					Interaction with age of testing	0	0
Ricceri et al., 2003	Homing 3-min to reach goal (wood shavings from home cage) Not blind	PND 12 SC DMSO	PND 12	10 males and 10 females from unstated # littersYes, litter is unit. Litter was a factor in the statistical analysis	Time to reach nest area	0^a.	(↓)^b
					Time spent in nest area	0	0
					Locomotor activity during 5-min test	0	0
^aall entries without “M” and/or “F” indicates analyses is based on males and females combined
^bauthors state no effect, but noted that pups tended to have shorter latencies

TABLE 6A. Motor activity in the RAT (oral corn oil)

						Magnitude of Dose Tested, mg/kg-day
Author	Test	Age Exposure	Age Testing	Sample Size Per Dose Level (Yes/No litter is unit)	Measure*	0.3	1	3	5	3, 6^a	3,6,12^a
Maurissen et al., 2000	Home Cage Activity (1 hour) AutomatedTime of testing balanced across dose level and sex	GD6-PND10 Oral corn oil	PND 13	N = 20 litters; 1 pup/sex/litterYes, litter is unit, Litter was a factor in the statistical analysis	Total Activity	0^b	0		(↓)^c
			PND 17		Habituation	0	0		0
					Total Activity	0	0		(↑)
					Habituation	0	0		0
			PND 21		Total Activity	0	0		(↑)
					Habituation	0	0		0
			PND 60		Total Activity	0	0		(↑)
					Habituation	0	0		0
Carr et al., 2001	Open Field (3 min for PND 10,12; 6 min for PND 14–20) Video camera, measures from 2 technicians averaged for each rat; blind and semi-balanced	PND 1–21 (pups dosed every other day) Oral corn oil	PND 10, 12, 14, 16, 18, 20	2 males and 2 females per litter from 5 littersYes, litter is unit, Litter was a factor in the statistical analysis	Line crosses (front 2 paws)			M0 F0 for each of 6 ages		M0 F0 for each of 6 ages	M0 F0 for each of 6 ages
			PND 25					M0 F0		M↓ F↓	M↓ F↓
			PND 30					M0 F0		M↓ F↓	M↓ F↓
^aCarr 2001 dosed animals with incrementally higher doses as animals got older
^bAll entries without “M” and/or “F” indicates analyses is based on males and females combined.
^c( ) indicates changes reported by the authors were not statistically significant
* If statistical analyses or measures were described in methods, then it was included and listed as “0” if not reported to be statistically significant

TABLE 6B. Motor activity in the RAT (sc DMSO)

						Magnitude of Dose Tested, mg/kg-day
Author	Test	Age and route of Exposure	Age Testing	Sample Size per Dose Level (Yes/No Litter is Unit)	Measure*	1	5
Icenogle et al., 2004	Figure-8 maze (1 hour with 12 5-min blocks)	GD 9–12 sc DMSO	Wk 4–8	10 M +10 F; 10 litters (1 rat /sex/ reconstituted litter) Litter of origin is not unit due to randomization of pups to dams at birth.	Habituation^a (CPF x Block)	0	↑ (↓ activity time block 9, 11of 12)
	Automated				Linear trend of habituation^b,	0	↑ slope
					Quadratic trend of habituation*	0	0
Levin et al., 2002	Figure-8 maze (1 hour with 12 5-min blocks) Automated	GD 17–20 sc DMSO	Wk 4–6	10 M +10 F; 10 litters (1 rat /sex/ reconstituted litter) Litter of origin is not unit due to randomization of pups to dams at birth.	Mean activity	0	0
					Habituation (CPF x Block)	M0 F(▵)^d	M0 F(▵)^d
					Linear trend of habituation	M0 F↓ slope	M0 F↓ slope
					Quadratic trend of habituation*	0	0
Levin et al., 2001	Figure-8 maze (1 hour with 12 5-min blocks) Automated	PND 1–4 (initial pooled analysis with PND 11–14, see below) sc DMSO	Wk 4–6	10 M + 10 F; on average there was 1 rat/sex/litterLitter is not unit for initial pooled analysis combining M & F	Mean activity	0
					Habituation (CPF x Block)	0
Levin et al., 2001	Figure-8 maze (1 hour with 12 5-min blocks) Automated	PND 11–14 (initial pooled analysis with PND 1–4; see above) sc DMSO	Wk 4–6	10 M + 10 F; on average there was 1 rat/sex/litterLitter is not unit for initial pooled analysis combining M & F	Mean activity		0
					Habituation (CPF x Block)		Interaction not reported
					Linear trend of habituation		↓ slope
Dam et al., 2000	Open field (5-minute)Videotaped and blind to treatment	PND 1–4sc DMSO	PND 21	5–7 pups/sex from unstated number of litters (Methods state 2 pups/sex/litter)Litter is not unit	Number of squares crossed	M↓F0
					Rearing	M↓F0
					Self-grooming	M0 F0
			PND 30	Same as for PND 21	Number of squares crossed	M↓F0
					Rearing	M↓F0
					Self-grooming	M0 F0
Dam et al., 2000	Open field (5-minute)Videotaped and Blind to treatment	PND 11–14sc DMSO	PND 21	5–7 pups/sex from unstated number of litters (Methods state 2 pups/sex/litter)Litter is not unit	Number of squares crossed		M0 F0
					Rearing		M0 F0
					Self-grooming		M0 F0
			PND 30	Same as for PND 21	Number of squares crossed		M0 F0
					Rearing		M↑ F0
					Self-grooming		M0 F0
^aHabituation results reported for specific dose level if authors indicated statistically significant post-hoc intersession differences following significant dose x time interaction for the habituation curve. Otherwise, see footnote “d”
^bLinear trend of habituation was described by authors as “slope of decrease in activity over consecutive 5-minute block” so that ↑ slope could indicate less habituation or increased activity towards end of session.
^d▵ indicates a significant interaction between dose and 5-min intervals, but no post-hoc group differences reported.
*If statistical analyses or measures were described in methods, then it was included and listed as “0” if not reported to be statistically significant

TABLE 6C. Motor activity in the MOUSE (oral or sc)

Author	Test	Age and Route of Exposure	Age Testing	Sample Size per Dose Level (Yes/No Litter is Unit)	Measure*	Magnitude of Dose Tested, mg/kg-day
Dose to dams						0.2	1		5
Braquenier et al., 2009	Open field # line crossings (5-min daily testing for 8 days)Not blind	GD15-PND14ORAL Corn oil	Not stated, after weaningYes, litter is unit	9–10 F; 1 female pup per litter	Mean activity over 8 days	F0	F0		F0
					Habituation over 8 days	F0	F0		F0
Dose to dams									6
Venerosi et al., 2009	Open field (5-minutes)duration(d) & frequency (f) analyzedBlind	GD 14–17ORAL Peanut oil	PND 12	1 pup/sex/litter; 13 litters control and 14 litters treatedYes; litter was factor in analysis	Crossing				0
					Immobility				↑d & f^a
					Head moving				0
					Wall climbing				0
					Pivoting				↓d & f
					Self-grooming				0
Dosed dams GD 15–18 (gavage) with peanut oil, 3 or 6 mg/kg and dosed pups PND 11–14 (sc) with vehicle, 1 or 3 mg/kg:First number indicates dose during gestation/second number indicates postnatal dose						3/0	3/1	3/3	6/0	6/1	6/3
Ricceri et al., 2006	Open field (20-minute divided in 4 5-min blocks)Video duration(d) & frequency (f) analyzedNot blind	GD15–18ORAL Peanut oil & PND 11–14SC Peanut oil	PND 70	10 males from unstated number of littersYes; litter was factor in analysis	Crossing	M0	M0	M↑f first 5 min	M↑f	M0	M0
					Wall rearing	M0	M0	M0	M0	M0	M0
					Self-grooming	M0	M0	M0	M0	M0	M0
					Immobility	M0	M0	M0	M0	M0	M0
Dosed dams GD 15–18(oral) with peanut oil and dosed pups PND 11–14 (sc) with vehicle, 1 or 3 mg/kg:First number indicates dose during gestation/second number indicates postnatal dose							0/1	0/3
Ricceri et al., 2006	Open field (20 minutes divided into 4 5-min blocks) Video, Not blindduration(d) & frequency (f) analyzed	PD 11–14SC peanut oil (with oral peanut oil GD 15–18)	PD 70	10 males from unstated number of littersYes; litter was factor in analysis	Crossing		M0	M↑f first 5 min
					Wall rearing		M0	M0
					Self-grooming		M0	M0
					Immobility		M0	M0
Dose to pups							1	3	6
Ricceri et al., 2003	Automated square arena (20 minutes)	PND 1-4SC DMSO	PND 25	Control 13–17 pups1 mg/kg 8–14 pups3 mg/kg 10–16 pups#litters/group not stated.Litter not unit of analysis	Locomotion (horizontal)		0	0
					Rearing (vertical)		0	0
					Time in central area		0	0
					Time in peripheral area		0	0
Ricceri et al., 2003	Automated square arena (20 minutes)	PND 11–14SC DMSO	PND 25	Control 12–18 pups1 mg/kg 15–18 pups3 mg/kg 12–14 pups#litters/group not stated.Litter not unit of analysis	locomotion (horizontal)		(↑)^b	(↑)^b
					Rearing (vertical)		0	0
					Time in central area		0	0
					Time in peripheral area		0	0
^ad=duration; f=frequency
^bPost-hoc tests were conducted and were statistically significant, although main effect was not significant at p=0.06
*If statistical analyses or measures were described in methods, then it was included and listed as “0” if not reported to be statistically significant

TABLE 7A. Novelty/plus maze in the MOUSE (oral corn oil)

						Magnitude of Dose Tested, mg/kg-day
Author	Test	Age and Route of Exposure	Age Testing	Sample Size Per Dose Level (Yes/No litter is unit)	Measure*	0.2	1	5
Braquenier et al., 2009	Elevated plus maze (5 min)Blind review of video sequences	GD 15–PND 14 Oral Corn Oil	PND 80	9–10 F; 1 female pup per litteryes, litter is unit	Time spent in open versus closed arm	0	(↓)^a ANOVA p=0.10; Dunnett p<0.02	0
					Number of entries in open arms	0	↓	0
	Dark/Light (5 min)Not blind		PND 72	9–10 F; 1 female pup per litter	% in Dark relative to total time	0	0	0
				yes, litter is unit	% in Center of light relative to time in light	0	↓	0
					# crossings between compartments	0	(↓ )^a ANOVA p=0.08; Dunnett p<0.02	0
^a(↓) overall ANOVA not statistically significant, but post-hoc tests were statistically significant.
*If statistical analyses or measures were described in methods, it was included and listed as “0” if not reported to be statistically significant.

TABLE 7B. Novelty/plus maze in the RAT (sc DMSO)

						Magnitude of Dose Tested, mg/kg-day
Author	Test	Age and Route of Exposure	Age Testing	Sample Size Per Dose Level (Yes/No litter is unit)	Measure*	1	5
Icenogle et al., 2004	Elevated plus maze	GD 9–12	Not stated	10 M +10 F; 10 litters (1 rat /sex/ reconstituted litter)	Time spent in open versus closed arm	0	0
	Not blind	sc DMSO
				Litter of origin is not unit due to randomization of pups to dams at birth.	Center crossing	0	↑
Aldridge et al., 2005a	Elevated plus maze	PND 1–4	PND 52–53 (Wk 7)	1 pup/sex/ litter; 9 litters	Open arm time	M↑ F0 (reduced “anxiety”)
	Not blind	sc DMSO		Litter is not unit for initial pooled analysis combining M & F	Center crossing	M↑ F0
*If statistical analyses or measures were described in methods, it was included and listed as “0” if not reported to be statistically significant.

TABLE 7C. Novelty and plus maze in the MOUSE

Author	Test	Age and Route of Exposure	Age Testing	Sample Size Per Dose Level (Yes/No litter is unit)	Measure*	Magnitude of Dose Tested, mg/kg-day
Dosed GD15–18 with 3 or 6 mg/kg and Dosed PND 11–14 with vehicle: First number indicates dose during gestation to dams/second number indicates postnatal dose to pups							3/0	6/0
Ricceri et al., 2006	Elevated Plus Maze	GD 15–18 gavage CPF in peanut oil to dams	4 months	Not clear, assume 10 M and 10 F;	Total		0	0
	Not blind	PND 11–14 sc peanut oil (no CPF)		yes, litter was factor in analyses	Center crossing		0	0
					Open arm		0	0
					Closed arm		0	0
					Head dipping		M↓ F0	M0 F0
					Stretch-attend		0	0
Dosed GD 15–18 with vehicle and Dosed PND 11–14 with vehicle, 1 or 3 mg/kg: First number indicates dose during gestation to dams/second number indicates postnatal dose to pups						0/1	0/3
Ricceri et al., 2006	Elevated Plus MazeNot blind	GD 15–18 gavage peanut oil (no CPF)PND 11–14 sc CPF in peanut oil	PD 60	10 males from unstated number of littersyes, litter was factor in analyses	Total	0	0
					Center crossing	0	0
					Open arm	M0 (F↑) (reduced “anxiety”)	M0 F↑ (reduced “anxiety”)
					Closed arm	0	0
					Head dipping	0	0
					Stretch-attend	0	0
Dose to pups						1	3	6
Ricceri et al., 2003	Automated novelty test with black and white chambers (25 minute test: one 5-min block familiar; four 5-min blocks partition open)	PND 1–4sc DMSO	PND 35	Control 18 pups1 mg/kg not tested3 mg/kg 16 pups	Day 1 activity rate (25 min)		0
					Day 4 activity familiar side		0
					Day 4 activity partition open		↑(2nd 5-min block)
				# litters/ dose group not stated	Time in white		0
					Time in black		0
				litter not unit	Activity rate white		0
					Activity rate black		0
					#passages/ minute		0
					Latency to enter novel		0
Ricceri et al., 2003	Automated novelty test with black and white chambers (25 minute test: one 5-min block familiar; four 5-min blocks partition open)	PND 11–14sc DMSO	PND 35	Control 17 pups1 mg/kg not tested3 mg/kg 17 pups#litters/dose group not stated	8 measures as described above for PND 1–4 in Ricceri 2003		0 for all 8 measures described above for PND 1–4
				litter not unit	Day 4 activity partition open		↑ (4th 5-min block)
*If statistical analyses or measures were described in methods, it was included and listed as “0” if not reported to be statistically significant

TABLE 8. Spontaneous alternation in the RAT (s.c. DMSO)

						Magnitude of Dose Tested, mg/kg-day
Author	Test	Age and Route of Exposure	Age Testing	Sample Size Per Dose Level (Yes/No litter is unit)	Measure	1	5
Icenogle et al., 2004	Spontaneous Alternation T-maze Not blind	GD 9–12 s.c. DMSO	Wk 4–8	10 M +10 F; 10 litters	Spontaneous alternation	0	0
				Litter is not unit
				Litter of origin is not known due to randomization of pups to dams at birth	Latency	↓ Trial 1 of 5 ( 3 sec)	↓ Trial 1 of 5 (5 sec)
Levin et al., 2002	Spontaneous Alternation T-maze Not blind	GD 17–20 s.c. DMSO	Wk 4–6	10 M +10 F; 10 litters	Spontaneous alternation	0	0
				Litter is not unit
				Litter of origin is not known due to randomization of pups to dams at birth	Latency	↓ Trial 1 of 5	↓ Trial 1 and 2 of 5
Levin et al., 2001	Spontaneous Alternation T-maze Not blind	PND 1–4 and PND 11–14 s.c. DMSO	Wk 8–13	10 M + 10 F; “on average there was 1 rat/sex/litter”	Spontaneous alternation	0 (PND 1–4)	0 (PND 11–14)
		analysis for both periods pooled together because no dose × period interaction		Litter is not unit for initial pooled analysis combining M & F	Latency	1 and 5 mg/kg-day
					Data pooled together in different ways	↑ 3 sec / trial (average) both sexes
						↑ latency during 3^rd session in males (1 mg/kg-day PND 1–4 data pooled together with 5 mg/kg-day PND 11–14, males and females pooled together and also analyzed for each sex separately

TABLE 9A. Learning and memory (rat oral corn oil)

						Magnitude of Dose Tested mg/kg-day
Author	Test	Age and Route of Exposure	Age Testing	Sample Size per Dose Level (Yes/No litter is unit)	Measure*	0.3	1	3	5
Maurissen et al., 2000	Spatial delayed alternation	GD 6–PND10	PND 22–24	N=16 litters; 1 male OR 1 female pup per litter from 16 litters	% correct	0 ^b	0		0
			^a85% free-feeding weight		acquisition	0	0		0
					retention	0	0		0
	Blind	Oral, corn oil	PND 61–90	Yes, litter is unit	% correct	0	0		0
	No feed restrction		^a 85% free-feeding bodyweight		acquisition	0	0		0
					retention	0	0		0
Incremental dose regimen							1	1, 2 & 4^c	1.5, 3 & 6^d
Johnson et al., 2009	Radial arm maze	PND1–21	PND36–60	Within-litter design for methyl-parathion and chlorpyrifos doses	Working errors analyzed by week		M↑^e F0	M↑^e F0	M↑ F0
					Working error overall		M0F0	M0F0	M↑ F0
	Not Blind	Oral, corn oil	^agradual 95–90—80% of feed	Yes, litter is unit,
				N=10–14 rats/sex/dose group
					Reference errors analyzed by week		M0 F0	M0 F↓	M↑ F↓
	Gradual feed reduction (95%-90% to 80% over 4 weeks
					Reference errors cumulative		M0 F0	M0 F↓	M↑ F↓
					Response time (seconds per entry)		0	0	0
^aAnimals were feed restricted based on free-feeding weight or amount of feed
^bEntries without “M” and/or “F” indicate analyses was based on pooling data for males and females
^cIncremental dosing regimen from 1.0 (PND1–5) to 2.0 (PND6–13) to 4.0 (PND14–20) mg/kg-day
^dIncremental dosing regimen from 1.5 (PND1–5) to 3.0 (PND6–13) to 6.0 (PND14–20) mg/kg-day
^eIncrease errors (3.2 vs. 2.7 in controls approximated from graph) during 4th week but not first 3 weeks. No overall effect on total working errors at low and mid dose
*If statistical analyses or measures were described in methods, it was included and listed as “0” if not reported to be statistically significant

TABLE 9B. Learning and memory (rat sc DMSO or peanut oil)

						Magnitude of Dose Tested, mg/kg-day
Author	Test	Age and Route of Exposure	Age Testing	Sample Size Per Dose Level (Yes/No litter is unit)	Measure*	0.3	1	5	7
Icenogle et al., 2004	Radial Arm Maze	GD 9–12	Wk 8–13	10 M +10 F; 10 litters	Working error		0^b	↑^b (trial block1 & 3 of 6)
	Not blind	sc DMSO	85% feed^a	Litter of origin is not unit due to randomization of pups to dams at birth	Reference error		0	↑ ( trial block 1 of 6)
Levin et al., 2002	Radial Arm Maze	GD 17–20	Wk 8–13	10 M +10 F; 10 litters	Working error		M0 F↑	M0 F0
	Not blind	sc DMSO	85% feed^a	Litter of origin is not unit due to randomization of pups to dams at birth	Reference error		M0 F↑	M0 F0
Aldridge et al., 2005a	Radial Arm Maze	PND1–4	Wk 9–14	1 pup/sex/ litter; 9 litters	Working error		M0 F↓
	Blind	sc DMSO	15 g/day^a	Litter is not unit for initial pooled analysis combining M & F	Reference error		M↑ F↓
Levin et al., 2001	Radial Arm Maze	PND 1–4	Wk 8–13	10 M + 10 F; “on average there was 1 rat/sex/litter”	Working error		M↑(block1 of 6) F↓
	Not blind	sc DMSO (pooled analysis with PND 11–14)	85% feed^a	Litter is not unit for initial pooled analysis combining M & F^c	Reference error		M↑(block1 of 6) F↓
Levin et al., 2001	Radial Arm Maze	PND 11–14	Wk 8-13	10 M + 10 F; “on average there was 1 rat/sex/litter”	Working error			0
	Not blind	sc DMSO (pooled analysis with PND 1–4)	85% feed^a	Litter is not unit for initial pooled analysis combining M & F^c	Reference error			0
Jett et al., 2001	Morris Water Maze	PN 7,11,15	PND 24–28	10M and 9–10F from 2–4 litters	Escape latency	↑^b day1 only			↑
	Not blind	sc peanut oil every 4 days		litter is not unit	Probe time	0			↓ (less time in correct quad)
Jett et al., 2001	Morris Water Maze latency	PND 22,26	PND 24–28	4M + 3-4F from 2–4 litters	Escape latency	↑			↑ magnitude of change same as low dose
	Not blind	sc peanut oil		offspring dosed after weaning	Probe time	↓			↑ magnitude of change same as low dose
^aAnimals were feed restricted
^bEntries without “M” and/or “F” indicate analyses was based on males and females pooled together in a single analyses because there was no significant interaction between dose and sex.
^cPups dosed PND 1–4 1 mg/kg-day were combined with PND 11–14 5 mg/kg-day and males combined with females in the initial pooled analysis.
*If statistical analyses or measures were described in methods, it was included and listed as “0” if not reported to be statistically significant

TABLE 9C. Learning and memory (mouse sc DMSO)

Author	Test	Age Exposure	Age Testing	Sample Size Per Dose Level Yes/No litter is unit)	Measure*	1	3	5	10
Billauer-Haimovitch et al., 2009	Morris Maze Video monitored, not blind	GD9-18 sc DMSO	PD 75	1 pup/sex/litter; 7–17 litters/group	Latency	↑^a,b (deficit)	↑^a (deficit)	0^a	0^a
				Litter is not unit.
				Half pups cross-fostered at birth; males and females from same litter pooled together; litter was not a factor in the analysis
Haviland et al., 2010	Radial Arm Maze Not blind	GD17-20 sc DMSO	PD 60 Mice were fasted	8 litters/dose group	Working error	0		0
				Yes, litter is unit	Reference error	0		0
				# pups / litter was not reported
	Foraging Maze Not blind	GD17-20 sc DMSO	PD 60	8 litters/dose group	Food recognition: Food pieces found per reward baited arm	M↑^c F↓ (male improve, female deficit)		M0^c F↓ (female deficit)
				Yes, litter is unit
				# pups / litter was not reported
					Food Position : # correct looks left where bait is per total looks in baited arm	M0^c F↓ (female deficit)		M0^c F↓ (female deficit)
					Foraging activity	M0 F0		M0 F0
Ricceri et al., 2003	Passive Avoidance Not blind	PD 1–4 sc DMSO	PD 60	8 or 9 males with at least 1 male/litter; total 36 litters for 4 groups	Trials to criterion	M0	M0
					Latency to step through	M0	M0
				Pup and not litter was the unit	24 hr. retention	M0	M0
Ricceri et al., 2003	Passive Avoidance No blind	PD 11–14 sc DMSO	PD 60	7–10 males with at least 1 male/litter; total 36 litters for 4 groups	Trials to criterion	M0	M0
					Latency to step through	M0	M0
				Pup and not litter was the unit	24 hr. retention	M0	M0
^aEntries without “M” and/or “F” indicate analyses was based on males and females pooled together in a single analyses because there was no significant interaction between dose and sex.
^bInspection of data indicates significant overlap of learning curve with controls
^cIncrease (↑) means improvement in learning compared to controls. Decrease (↓) means deficit or delay in learning compared to controls
*If statistical analyses or measures were described in methods,it was included and listed as “0” if not reported to be statistically significant

TABLE 10A. Pharmacologic challenge in the MOUSE (oral peanut oil)

Author	Test	Age and Route of Exposure	Age Testing	Sample Size Per Dose Level of Challenge Drug (Yes/No litter is unit)	Challenge Drug	Measure*	Effect of Challenge Drug on Controls	Effect of CPF on the Effect of Challenge Drug Magnitude of CPF Dose6 mg/kg-day
Venerosi et al., 2010	Forced Swimming Test Videotaped and blind Not balanced	GD 15–18 Oral Peanut Oil	adulthood	12M+6F from unstated number of litters Litter included as factor in analysis	Fluvoxamine (5HT uptake inhibitor)	Swimming frequency	M↑ F0	M↓ (Attenuate)
						Swimming duration	M↑ F↑	M↓ F↓ (Attenuate)
						Struggling frequency	M↑ F0	M↓ (Attenuate)
					30 mg/kg i.p.	Struggling duration	M↑ F0	M↓ (Attenuate)
						Floating duration	M↓ F(↓) p=0.07	M and F Attenuate
Venerosi et al., 2010	Maternal Aggression Male mouse placed in home cage with the dam and her empty nest Videotaped and blind frequency(f), duration (d), and latency (l) measured for each parameter	GD 15–18 Oral Peanut Oil	Females only, 8 days after delivery of pups	5–7 F from unstated number of litters Litter included as factor in analysis	Fluvoxamine (5HT uptake inhibitor)	Fighting behavior – attack (3 different cage areas) f,d,l	F↓ f and d for 2–3 cage areas	F attenuate duration of in-nest attack Note: CPF also ↓ baseline levels
					30 mg/kg i.p.	Social behavior – social sniffing (3 subtypes) f,d,l	F↑ duration inactivity in-nest	F attenuate duration inactivity in-nest
						Non-social behavior (3 subtypes) f,d,l	F0	No effect of fluvoxamine, but CPF increased basal levels in 1 of 3 subtypes

TABLE 10B. Pharmacologic challenge in the RAT (sc DMSO)

								Effect of CPF on the Effect of Challenge Drug Magnitude of CPF dose, mg/kg-day
Author	Test	Age and Route of Exposure	Age Testing	Sample Size Per Dose Level (Yes/No litter is unit)	Challenge Drug	Measure^a	Effect of Challenge Drug on Controls	1	5
Icenogle et al., 2004	Radial Arm Maze Not blind doses for each challenge drug given in counterbalanced order	GD 9–12 sc DMSO	Wk 8–13	10 M +10 F; 10 litters (1 rat /sex/ reconstituted litter) Litter of origin is not unit due to randomization of pups to dams at birth.	Scopolamine (0, 0.04, 0.08, 0.16 mg/kg sc)	Working error	↑error	0^b	↓ (attenuate errors induced by scopolamine)
						Reference error	↑error	Not reported	Not reported
					Mecamylamine (0, 1.25, 2.5, 5 mg/kg sc)	Working error	0	0	0
						Reference error	0	0	0
Levin et al., 2002	Radial Arm Maze Not blind doses for each challenge drug given in counterbalanced order	GD 17–2 sc DMSO	Wk 4–6	10 M +10 F; 10 litters (1 rat /sex/ reconstituted litter) Litter of origin is not unit due to randomization of pups to dams at birth.	Scopolamine (0, 0.04, 0.08, 0.16 mg/kg sc)	Working error	↑error	M0 F↓ (attenuate errors induced by scopolamine based on linear trend analysis)	M0 F0
						Reference error	0	M0 F0	M0 F0
						Latency	0	↑ (enhance effect of scopolamine to increase latency – more evident in females)	0
					Mecamylamine (0, 1.25, 2.5, 5 mg/kg sc)	Working error		0	0
						Reference error		0	0
						Latency		0	0
Aldridge et al., 2005a	Radial Arm Maze Not blind doses for challenge drug given in counterbalanced order	PND1–4 sc DMSO	Wk 16–17	1 pup/sex/ litter; 9 litters Litter is not unit for initial pooled analysis combining M & F	Ketanserin (0, 0.5, 1, 2 mg/kg sc)	Working error	0	↑ errors
						Reference error	0	↑errors
Levin et al., 2001	Radial Arm Maze Not blind doses for each challenge drug given in counterbalanced order	PND 1–4 (analysis pooled with PND 11–14) sc DMSO	Wk 14–17	10 M + 10 F; “on average there was 1 rat/sex/litter” Litter is not unit for initial pooled analysis combining M & F	Scopolamine (0, 0.04, 0.08, 0.16 mg/kg sc)	Working error	↑error	0
						Reference error	↑error (graph shows M0 F↑)	0
					Mecamylamine (0, 1.25, 2.5, 5 mg/kg sc)	Working error	0	0
						Reference error	0	0
Levin et al., 2001	Radial Arm Maze Not blind doses for each challenge drug given in counterbalanced order	PND 11–14 (analysis pooled with PND 1–4) sc DMSO	Wk 14–17	10 M + 10 F; “on average there was 1 rat/sex/litter” Litter is not unit for initial pooled analysis combining M & F	Scopolamine (0, 0.04, 0.08, 0.16 mg/kg sc)	Working error	↑error		0
						Reference error	↑error		M0 F↓ (F: attenuate errors at 0.16 mg/kg scopolamine)
					Mecamylamine (0, 1.25, 2.5, 5 mg/kg sc)	Working error	0		0
						Reference error	0		0
^aRadial arm maze measures include working memory errors (entry into a formerly baited arm), reference memory errors (entry into unbaited arm), latency (total session time in seconds divided by total number of arm entries in units of seconds per entry)
^b“0” means that developmental exposure to CPF did not change the effect of the challenge drug.
*If statistical analyses or measures were described in methods, then it was included and listed as “0” if not reported to be statistically significant

TABLE 11. CPF effects on brain or RBC AChE inhibition in rats

	Selected Rat Brain or RBC AChE inhibition^a for developmental period of exposure
	GD 9–12 (dams exposed)	GD 17–20 (dams exposed)	PND 1–4 (pups exposed)	PND 10–16 or 11–14 (pups exposed)
CPF s.c. exposures (Brain AChE inhibition in rat offspring)	1 and 5 mg/kg: Not measured	1 mg/kg: 0 (24 hr) 2 mg/kg: 20% (24 hr) (Qiao et al., 2002) 5 mg/kg: 50% (24 hr) (Qiao et al., 2002)	1 mg/kg: 25% (24 hr) Song et al., 1997 1 mg/kg: 75% (2 hr after PND1 dose) Dam et al., 2000	PND 10–16 5 mg/kg 73% (4 hr) Carr and Nail, 2008PND 11–14 5 mg/kg >65% (24 hr) Song et al., 1997
CPF oral exposures (Brain AChE inhibition in rat offspring)	GD 6–20 1 mg/kg: 0 (4 hr) 5 mg/kg: 60% (4 hr) (Mattsson et al., 2000)			PND 10–16 5 mg/kg >58% (4 hr) Carr and Nail, 2008
CPF oral exposures (RBC AChE inhibition in dams)	GD 6–20 0.3 mg/kg: >25% dam RBC AChE Inhibition (4 hr) 1 mg/kg: >80% dam RBC AChE inhibition (4 hr) (Mattsson et al., 2000)
^aGreatest effect from selected publication was reported, with 0 indicating no effect

Table

5HT	serotonin
AChE	acetylcholinesterase
ADHD	attention deficit hyperactivity disorder
AUC	area under the curve
BMDL10	benchmark dose lower confidence limit for 10% effect
BNBAS	Brazelton Neonatal Behavioral Assessment Scale
BSID	Bayley Scales of Infant Development II
BSID:MDI	Bayley Scales of Infant Development II – Mental Development Index
	(sometimes abbreviated to just MDI)
BSID:PDI	Bayley Scales of Infant Development II – Psychomotor Development Index
	(sometimes abbreviated to just PDI)
BuChE	butyrylcholinesterase
CBCL	child behavior checklist
CCCEH	Columbia Center for Children's Environmental Health
CHAMACOS	Center for Health Assessment of Mothers and Children of Salina
ChAT	choline acetyl transferase
ChE	cholinesterase (used when referring to both AChE and BuChE)
CI	confidence interval
CPF	chlorpyrifos
CPO	chlorpyrifos-oxon
d	day or days
DA	Dopamine
DAPs	total group of dialkylphosphates (includes DEPs and DMPs)
DEP	diethylphosphate
DEPs	total group of diethylphosphates (DEP, DETP, DEDTP)
DETP	diethylthiophosphate
DEDTP	diethyldithiophosphate
DMPs	total group of dimethylphosphates
DMS-IV	Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition
DMSO	dimethyl sulfoxide
EPA	U.S. Environmental Protection Agency
GD	gestational day
GFAP	glial fibrillary acidic protein
HAChT	high affinity presynaptic choline transporter
min	minute or minutes
mAChR	muscarinic cholinergic receptor
MDI	mental development index (same as BSID:MDI)
mg	milligram
NBAS	Neonatal Behavioral Assessment Scale
ng	nanogram
NHANES	National Health and Nutrition Examination Survey
NIEHS	National Institute of Environmental Health Sciences
NOEL	no-observed-effect-level
NOAEL	no-observed-adverse-effect level
OP	OP insecticides
OR	odds ratio
PBPK	physiologically based pharmacokinetic (model)
PDD	pervasive development disorders
PDI	psychomotor development index (same as BSID:PDI)
PND	postnatal day
PON1	paraoxonase
RBC	red blood cell
RR	risk ratio
sc	subcutaneous(ly)
SEM	standard error of the mean
TCPy	3,5,6-trichloro-2-pyridionol
USV	ultrasonic vocalizations
VAChT	vesicular Ach transporter
μg	microgram

Rauh , V. A. , Garfinkel , R. , Perera , F. P. , Andrews , H. F. , Hoepner , L. , Barr , D. B. , Whitehead , R. , Tang , D. and Whyatt , R. W. 2006 . Impact of prenatal chlorpyrifos exposure on neurodevelopment in the first 3 years of life among inner-city children . Pediatrics , 118 : 1845 – 59 .

 PubMed Web of Science ®Google Scholar

Eskenazi , B. , Marks , A. R. , Bradman , A. , Harley , K. , Barr , D. B. , Johnson , C. , Morga , N. and Jewell , N. P. 2007 . Organophosphate pesticide exposure and neurodevelopment in young Mexican-American children . Environ. Health Perspect. , 115 : 792 – 98 .

 PubMed Web of Science ®Google Scholar

Young , J. G. , Eskenazi , B. , Gladstone , E. A. , Bradman , A. , Pedersen , L. , Johnson , C. , Barr , D. B. , Furlong , C. E. and Holland , N. T. 2005 . Association between in utero organophosphate pesticide exposure and abnormal reflexes in neonates . Neurotoxicology , 26 : 199 – 209 .

 PubMed Web of Science ®Google Scholar

Qiao , D. , Seidler , F. J. , Padilla , S. and Slotkin , T. A. 2002 . Developmental neurotoxicity of chlorpyrifos: What is the vulnerable period? . Environ. Health Perspect. , 110 : 1097 – 103 .

 PubMed Web of Science ®Google Scholar

Song , X. , Seidler , F. J. , Saleh , J. L. , Zhang , J. , Padilla , S. and Slotkin , T. A. 1997 . Cellular mechanisms for developmental toxicity of chlorpyrifos: Targeting the adenylyl cyclase signaling cascade . Toxicol. Appl. Pharmacol. , 145 : 158 – 74 .

 PubMed Web of Science ®Google Scholar

Carr , R. L. and Nail , C. A. 2008 . Effect of different administration paradigms on cholinesterase inhibition following repeated chlorpyrifos exposure in late preweanling rats . Toxicol. Sci. , 106 : 186 – 92 .

 PubMed Web of Science ®Google Scholar

Mattsson , J. L. , Maurissen , J. P. , Nolan , R. J. and Brzak , K. A. 2000 . Lack of differential sensitivity to cholinesterase inhibition in fetuses and neonates compared to dams treated perinatally with chlorpyrifos . Toxicol. Sci. , 53 : 438 – 46 .

 PubMed Web of Science ®Google Scholar