Hypothesis-based weight-of-evidence evaluation of the neurodevelopmental effects of chlorpyrifos

Figures & data

Table 1.  Comparison of frameworks for integrating human data into risk assessment.

Key aspects of a weight-of-evidence evaluation	US EPA framework	ECETOC framework	HBWoE framework
1. Systematically review individual studies potentially relevant to causal question at hand (e.g., epidemiology, mode of action, pharmacokinetic, toxicology), both negative and positive, and of varying quality.	Yes (focus on epidemiology)	Yes (focus on epidemiology and animal toxicity studies)	Yes
2. Within a realm of investigation (e.g., epidemiology, animal toxicology, or mode of action studies), systematically examine the data for particular endpoints across studies, evaluating consistency, specificity, and reproducibility of outcomes.	Yes (focus on epidemiology)	Yes (focus on epidemiology and animal toxicity studies)	Yes
3. Identify and articulate lines of argument (or “overarching hypotheses”), newly proposed or those already put forth (if available), that bear on the available data.	No explicit guidance	No explicit guidance	Yes
4. Evaluate the logic of the proposed hypotheses with respect to each line of evidence or realm of investigation (e.g., separate evaluation of epidemiology, animal toxicology, and mode of action data).	No explicit guidance	No explicit guidance	Yes
5. Evaluate the logic of the proposed hypotheses with respect to all lines of evidence holistically so that all of the data are considered and integrated and allowed to inform interpretation of one another.	No explicit guidance (although importance of integrating is discussed)	No explicit guidance (although importance of integrating is discussed)	Yes
6. Describe and compare (if more than one hypothesis has been put forth) the various alternate accounts of the observations at hand. That is, describe how well each overarching hypothesis is supported by all of the available data, discussing the uncertainties and inconsistencies in the data set and ad hoc assumptions required to support each hypothesis.	No explicit guidance	No explicit guidance	Yes
7. Propose next steps (e.g., sharpening of proposed hypothesis already put forth, propose additional testing to clarify data gaps).	Yes	Yes	Yes

Table 2.  Overview of cohort studies of chlorpyrifos exposure and neurodevelopmental outcomes.

Study	Subjects	Exposure Metric							Outcome assessed
		Chlorpyrifos in cord blood	Chlorpyrifos in maternal blood	Urinary TCPy	Urinary DEPs	Cholinesterases in cord blood	Cholinesterases in maternal blood	Personal air monitoring	Age at outcome assessment	Head circumference	Infant neurobehavior (BNBAS)	Cognitive and motor development (BSID-II)	Cognitive development (WISC-IV)	Behavioral outcomes (ADHD, PDD, and attention problems) (CBCL)
Columbia Cohort (Infants born between 1998–2002)
Perera et al., 2003	113 African-American and Dominican mother-newborn pairs living in inner-city (NYC)	Plasma levels, collected at delivery	Plasma levels, collected within 1 day postpartum						Birth	X
Whyatt et al., 2004	Expanded number of mother-newborn pairs (n = 287) from Perera et al. (2003)	Plasma levels, collected at delivery	Plasma levels, collected within 2 days postpartum					Collected for 48 hours during third trimester	Birth	X
Rauh et al., 2006	254 children from Whyatt et al. (2004)	Plasma levels, collected at delivery	Plasma levels, collected within 1 day postpartum						12 months 24 months 36 months			X X X		X
Rauh et al., 2011	265 children from Whyatt et al. (2004)	Plasma levels, collected at delivery	Plasma levels, collected within 2 days postpartum						7 years				X
CHAMACOS Cohort (Infants born between 2000–2001)
Eskenazi et al., 2004	488 mother-newborn pairs living in agricultural community (Salinas Valley, CA)			Maternal levels, collected at 13 and 26 weeks gestation	Maternal levels, collected at 13 and 26 weeks gestation	ChE activity in whole blood; BuChE activity in plasma; collected at delivery	ChE activity in whole blood; BuChE activity in plasma; collected at 26 weeks gestation and delivery		Birth	X
Young et al., 2005	381 mother-newborn pairs from Eskenazi et al. (2004)				Maternal levels, collected at 14 and 26 weeks gestation and within one week of delivery				0 to 62 days		X
Eskenazi et al., 2007	396 children from Eskenazi et al. (2004)			Maternal levels, collected at 14 and 26 weeks gestation	Maternal levels, collected at 14 and 26 weeks gestation; child levels, collected at 6, 12, and 24 months of age				6 months			X
									12 months			X
									24 months			X		X
Mount Sinai Cohort (Infants born between 1998–2002)
Berkowitz et al., 2004	404 mother-newborn pairs living in NYC			Maternal levels, collected during third trimester					Birth	X
Wolff et al., 2007	404 mother-newborn pairs from Berkowitz et al. (2004)				Maternal levels, collected during third trimester		BuChE activity in plasma; collected during third trimester		Birth	X
Engel et al., 2007	311 mother-newborn pairs from Berkowitz et al. (2004)				Maternal levels, collected during third trimester				0 to 5 days		X
Engel et al., 2011	360 children from Berkowitz et al. (2004)				Maternal levels, collected during third trimester				12 months 24 months 7–9 years			X X	X
UMDNJ Cohort (Infants born between 2003–2004)
Barr et al., 2010	150 mother-newborn pairs scheduled for elective cesarean delivery at Saint Peter’s University Hospital, New Brunswick, New Jersey	Serum levels, collected at delivery	Serum levels, collected just prior to delivery						Birth	X

DEPs = diethyl phosphates; TCPy = 3,5,6-trichloro-2-pyridinol; ChE = cholinesterase; BuChE = butyrylcholinesterase.

Table 3.  Results of cohort studies assessing head circumference.

Cohort	Study	Exposure metric/group	Exposure	N	Estimate	95% CI	p-value	Statistical method
Columbia	Perera et al., 2003	Chlorpyrifos in cord plasma	Mean: 7.5 ± 7.5 pg/g	113	β =  –0.005	NR	0.28	Multiple regression with log-transformed chlorpyrifos as a continuous variable
	Whyatt et al., 2004	Chlorpyrifos in cord plasma	Mean: 4.0 ± 6.1 pg/g	287	β =  –0.01	(−0.13, 0.11)	0.86	Multiple regression with log-transformed chlorpyrifos as a continuous variable
		Chlorpyrifos in maternal air	Mean: 15.3 ± 31.8 ng/m3	271	β =  –0.04	(−0.18, 0.10)	0.59
CHAMACOS	Eskenazi et al., 2004	Maternal urinary TCPy	Median: 3.3 µg/L					Linear regression with TCPy analyzed as categorical variables
		Detectable levels < median		220	β = 0.06	(−0.37, 0.49)	0.78
		Detectable levels > median		221	β =  0.04	(−0.39, 0.47)	0.85
		Maternal urinary DEPs	Median: 22 nmol/L	486	β =  0.28	(−0.02, 0.59)	0.07	Linear regression with log-transformed DEP levels as a continuous variable
		ChE (µmol/min/mL)						Linear regression with log-transformed cholinesterase levels as a continuous variable
		Maternal blood, pregnancy	Median: 5.7 µmol/min/mL	340	β =  0.06	(−0.09, 0.21)	0.45
		Maternal blood, delivery	Median: 5.1 µmol/min/mL	357	β = –0.07	(−0.19, 0.05)	0.27
		Cord blood	Median: 3.8 µmol/min/mL	292	β =  –0.04	(−0.23, 0.14)	0.65
		BuChE (µmol/min/mL)
		Maternal blood, pregnancy	Median: 1.4 µmol/min/mL	340	β = 0.12	(−0.31, 0.56)	0.58
		Maternal blood, delivery	Median: 1.4 µmol/min/mL	357	β =  –0.07	(−0.45, 0.31)	0.73
		Cord blood	Median: 1.2 µmol/min/mL	292	β =  –0.03	(−0.50, 0.45)	0.91
Mount Sinai	Berkowitz et al., 2004	Maternal urinary TCPy	Median: 7.6 µg/L					Generalized linear models with TCPy analyzed as categorical variables
		TCPy < LOD		216	NR	NR	NS
		TCPy > LOD		171
		TCPy > LOD, Low PON1		47	NR	NR	0.014
		TCPy > LOD, Medium PON1		57	NR	NR	NS
		TCPy > LOD, High PON1		55	NR	NR	NS
	Wolff et al., 2007	Maternal urinary DEPs	Median: 18.8 nmol/L	318	β ± SE = –0.052 ± 0.12		0.67	Generalized linear models with log-transformed DEP levels or BuChE activity as continuous variables
		BuChE enzymatic activity	Range: <1.9–13.8 mg/ml * min	NR	β ± SE = 0.44 ± 1.27		0.728
UMDNJ	Barr et al., 2010	Chlorpyrifos in maternal serum	Mean: 0.09 ng/g					Multivariable linear regression with exposure indicators > versus ≤ 75th percentile
		> 75th percentile versus ≤ 75th percentile		138	NR	NR	0.229
		Chlorpyrifos in cord serum	Mean: 0.55 ng/g
		> 75th percentile versus ≤ 75th percentile		148	NR	NR	0.989

Table 4.  Results of cohort studies assessing infant neurobehavior using the BNBAS.

Cohort	Study	Exposure metric/group	Exposure concentration	N	Estimate	95% CI	p-value	Statistical method
CHAMACOS	Young et al., 2005	Maternal urinary DEPs	Median: 21 nmol/L	175	Habituation; β = 0.33	(−0.06, 0.72)		Regression models with log-transformed DEP levels as a continuous variable for all clusters except abnormal reflexes; Poisson regression for abnormal reflexes
				379	Orientation; β = –0.32	(−0.66, 0.03)
				381	Motor Performance; β = 0.10	(−0.06, 0.27)
				381	Range of State; β = –0.02	(−0.27, 0.24)
				381	Regulation of State; β = –0.15	(−0.47, 0.17)
				381	Autonomic Stability; β = 0.06	(−0.15, 0.27)
				381	Reflexes; β = 0.22	(0.04, 0.40)	< 0.05
		Maternal urinary DEPs, ≤ 3 days at assessment		109	Habituation; β = 0.47	(−0.05, 0.99)		Regression estimates stratified by age at BNBAS assessment (≤ 3 days)
				197	Orientation; β = –0.11	(−0.65, 0.43)
				197	Motor Performance; β = 0.08	(−0.17, 0.33)
				197	Range of State; β = –0.21	(−0.54, 0.12)
				197	Regulation of State; β = –0.08	(−0.52, 0.37)
				197	Autonomic Stability; β = 0.31	(0.01, 0.61)	< 0.05
				197	Reflexes; β = 0.08	(−0.16, 0.32)
		Maternal urinary DEPs, > 3 days at assessment		66	Habituation; β = 0.20	(−0.43, 0.83)		Regression estimates stratified by age at BNBAS assessment (> 3 days)
				182	Orientation; β = –0.33	(−0.73, 0.08)
				184	Motor Performance; β = 0.17	(−0.05, 0.38)
				184	Range of State; β = 0.20	(−0.21, 0.62)
				184	Regulation of State; β = –0.24	(−0.72, 0.24)
				184	Autonomic Stability; β = –0.16	(−0.47, 0.14)
				184	Reflexes; β = 0.37	(0.09, 0.64)	< 0.05
		Maternal urinary DEPs, > 3 days at assessment, Quintile 1	0.51 to 0.90 nmol/L	37	χ ^2 trend = 4.0		0.05	Regression estimates for proportion of > 3 abnormal reflexes, stratified by age at BNBAS assessment (> 3 days) with log-transformed DEP levels categorized by quintiles
		Quintile 2	0.90 to 1.10 nmol/L	37
		Quintile 3	1.11 to 1.27 nmol/L	37
		Quintile 4	1.28 to 1.58 nmol/L	37
		Quintile 5	1.58 to 2.35 nmol/L	36
		Maternal urinary DEPs		N = 381	Total DEPs, OR = 3.4	(1.2, 9.9)		Logistic regression for > 3 abnormal reflexes versus ≤ 3 abnormal reflexes; OR per 10-fold increase in log-transformed DEP levels
Mount Sinai	Engel et al., 2007	Maternal urinary DEPs	Median: 24.7 nmol/L	144	Habituation; β = 0.168	(−0.23, 0.57)		Generalized linear models with log-transformed DEP levels as a continuous variable for all clusters except abnormal reflexes; Poisson regression for abnormal reflexes
				233	Orientation; β = –0.106	(−0.41, 0.20)
				249	Motor Performance; β = 0.049	(−0.08, 0.17)
				253	Range of State; β = 0.035	(−0.12, 0.19)
				253	Regulation of State; β = –0.047	(−0.30, 0.21)
				253	Autonomic Stability; β = –0.154	(−0.38, 0.08)
				239	Reflexes; RR = 1.49	(1.12, 1.98)
		Maternal urinary DEPs > median versus ≤ median		239	RR = 2.3	(1.1, 5.0)		Multivariable logistic regression with number of abnormal reflexes dichotomized at two or more

Table 5.  Results of cohort studies assessing cognitive and motor development using the BSID-II or WISC-IV.

Outcome assessed	Cohort	Study	Exposure metric/group	Exposure concentration	Age at assessment	N	Estimate	95% CI	p-value	Statistical method
MDI scores	Columbia	Rauh et al., 2006	Chlorpyrifos in cord blood	Range: ND to 63 pg/g						Multivariate linear regression with log-transformed, dichotomized exposure variable
			High exposure (>6.17 pg/g) compared to low exposure (≤ 6.17 pg/g)		12 months	229	β = –0.344 ± 1.66		0.836
					24 months	225	β = –1.480 ± 2.03		0.466
					36 months	228	β = –3.327 ± 1.76		0.6
			High exposure (>6.17 pg/g) compared to low exposure (≤ 6.17 pg/g)		12 months	228	OR = 1.22	(0.48, 3.06)		Logistic regression with log-transformed, dichotomized exposure variable; odds of mental delay
					24 months	228	OR = 1.75	(0.86, 3.60)
					36 months	228	OR = 2.37	(1.08, 5.19)
	CHAMACOS	Eskenazi et al., 2007	Maternal urinary TCPy	Median: 3.54 µg/L						Multiple linear regression with log-transformed metabolite levels as continuous variables
			< Median detected		6 months	395	β = 0.24	(−2.12, 2.60)
			≥ Median detected				β = 0.08	(−2.29, 2.44)
			< Median detected		12 months	393	β = –0.45	(−3.67, 2.76)
			≥ Median detected				β = –0.65	(−3.88, 2.58)
			< Median detected		24 months	369	β = –1.02	(−5.34, 3.31)
			≥ Median detected				β =  –1.94	(−6.26, 2.37)
			Maternal urinary DEPs	Geometric mean: 18.1 nmol/L
					6 months	395	β =  –0.16	(−1.96, 1.65)
					12 months	393	β = –1.14	(−3.51, 1.22)
					24 months	369	β = –0.85	(−3.98, 2.27)
			Concurrent child urinary DEPs	Geometric mean: 10.6 nmol/L	6 months	395	β =  0.24	(−0.78, 1.25)
				Geometric mean: 15.2 nmol/L	12 months	393	β = 1.89	(0.21, 3.58)	≤ 0.05
				Geometric mean: 10.5 nmol/L	24 months	369	β = 1.02	(−0.52, 2.57)
	Mount Sinai	Engel et al., 2011	Maternal urinary DEPs	Median: 20.2 nmol/L	12 months	149	β = 0.03	(−2.23, 2.29)		Generalized linear models with log-transformed DEP levels as a continuous variable
					24 months	208	β = –1.47	(−3.99, 1.04)
PDI scores	Columbia	Rauh et al., 2006	Chlorpyrifos in cord blood	Range: ND to 63 pg/g						Multivariate linear regression with log-transformed, dichotomized exposure variable
			High exposure (>6.17 pg/g) compared to low exposure (≤ 6.17 pg/g)		12 months	228	β =  –3.30 ± 2.11		0.12
					24 months	227	β =  1.17 ± 1.98		0.56
					36 months	228	β = –6.46 ± 2.18		0.003
			High exposure (>6.17 pg/g) compared to low exposure (≤ 6.17 pg/g)		12 months	228	OR = 1.88	(0.78, 4.53)		Logistic regression with log-transformed, dichotomized exposure variable; odds of psychomotor delay
					24 months	228	OR = 1.01	(0.37, 2.76)
					36 months	228	OR = 4.52	(1.61, 12.70)
	CHAMACOS	Eskenazi et al., 2007	Maternal urinary TCPy	Median: 3.54 µg/L						Multiple linear regression with log-transformed metabolite levels as continuous variables
			< Median detected		6 months	395	β =  –0.56	(−4.03, 2.91)
			≥ Median detected				β = –0.21	(−3.69, 3.27)
			< Median detected		12 months	393	β =  –0.70	(−5.26, 3.86)
			≥ Median detected				β =  –1.62	(−6.20, 2.96)
			< Median detected		24 months	369	β = –2.65	(−6.50, 1.21)
			≥ Median detected				β =  –2.72	(−6.57, 1.12)
			Maternal urinary DEPs	Geometric mean: 18.1 nmol/L
					6 months	395	β = 0.02	(−2.63, 2.67)
					12 months	393	β =  0.30	(−3.03, 3.63)
					24 months	369	β = –0.86	(−3.64, 1.92)
			Concurrent child urinary DEPs	Geometric mean: 10.6 nmol/L	6 months	395	β =  0.60	(−0.89, 2.09)
				Geometric mean: 15.2 nmol/L	12 months	393	β =  1.91	(−0.46, 4.27)
				Geometric mean: 10.5 nmol/L	24 months	369	β = 0.30	(−1.07, 1.67)
	Mount Sinai	Engel et al., 2011	Maternal urinary DEPs	Median: 20.2 nmol/L	12 months	149	β = –0.20	(−3.28, 2.87)		Generalized linear models with log-transformed DEP levels as a continuous variable
					24 months	210	β = 0.67	(−1.72, 3.06)
WISC-IV scores	Columbia	Rauh et al., 2011	Chlorpyrifos in cord blood	Range: ND to 63 pg/g	7 years	265	Full–Scale IQ; β = –0.003	(−0.006, 0.001)	0.048	Multivariate linear regression with exposure as a continuous variable and ln-transformed WISC-IV scores
							Working Memory; β = –0.006	(−0.010, –0.002)	0.003
							Verbal Comprehension; β = –0.002	(−0.005, 0.001)	0.208
							Perceptual Reasoning; β = –0.002	(−0.006, 0.002)	0.29
							Processing Speed; β = 0.001	(−0.004, 0.005)	0.728
	Mount Sinai	Engel et al., 2011	Maternal urinary DEPs	Median: 20.2 nmol/L	7–9 years	114	Full–Scale IQ; β = –3.15	(−7.19, 0.89)		Generalized linear models with log-transformed DEP levels as a continuous variable
							Working Memory; β = –3.48	(−7.29, 0.34)
							Verbal Comprehension; β = –0.08	(−3.91, 3.76)
							Perceptual Reasoning; β = –4.37	(−9.10, 0.36)
							Processing Speed; β = –2.11	(−6.81, 2.59)

Table 6.  Results of cohort studies assessing child behavioral outcomes using the CBCL.

Outcome assessed	Cohort	Study	Exposure metric/group	Exposure concentration	Age at assessment	N	Estimate	95% CI	p-value	Statistical method
ADHD	Columbia	Rauh et al., 2006	Chlorpyrifos in cord blood	Range: ND to 63 pg/g						Logistic regression with log-transformed, dichotomized exposure variable
			High exposure (> 6.17 pg/g) compared to low exposure (≤ 6.17 pg/g)		36 months	228	OR = 6.50	(1.09, 38.69)
	CHAMACOS	Eskenazi et al., 2007	Maternal urinary DEPs	Geometric mean: 18.1 nmol/L	24 months	356	OR = 0.59	(0.21, 1.68)		Multiple regression; OR per 10-fold increase in log-transformed DEP levels
			Concurrent child urinary DEPs	Geometric mean: 10.5 nmol/L	24 months	356	OR = 1.18	(0.72, 1.94)
Attention problems	Columbia	Rauh et al., 2006	Chlorpyrifos in cord blood	Range: ND to 63 pg/g						Logistic regression with log-transformed, dichotomized exposure variable
			High exposure (> 6.17 pg/g) compared to low exposure (≤ 6.17 pg/g)		36 months	228	OR = 11.26	(1.79, 70.99)
	CHAMACOS	Eskenazi et al., 2007	Maternal urinary DEPs	Geometric mean: 18.1 nmol/L	24 months	356	OR = 0.78	(0.26, 2.31)		Multiple regression; OR per 10-fold increase in log-transformed DEP levels
			Concurrent child urinary DEPs	Geometric mean: 10.5 nmol/L	24 months	356	OR = 1.02	(0.61, 1.71)
PDD	Columbia	Rauh et al., 2006	Chlorpyrifos in cord blood	Range: ND to 63 pg/g						Logistic regression with log-transformed, dichotomized exposure variable
			High exposure (> 6.17 pg/g) compared to low exposure (≤ 6.17 pg/g)		36 months	228	OR = 5.39	(1.21, 24.11)
	CHAMACOS	Eskenazi et al., 2007	Maternal urinary DEPs	Geometric mean: 18.1 nmol/L	24 months	355	OR = 0.88	(0.37, 2.07)		Multiple regression; OR per 10-fold increase in log-transformed DEP levels
			Concurrent child urinary DEPs	Geometric mean: 10.5 nmol/L	24 months	355	OR = 1.72	(1.12, 2.64)	≤ 0.05

Table 7.  Neurodevelopmental tests used in animal studies of chlorpyrifos.

Test	Description	Psychobiological significance	References
Social behavior
Sociability	An unfamiliar animal of the same strain, sex, and age is placed in a side chamber to serve as a social stimulus; this allows nose contact through the bars, but prevents fighting. The other side chamber contains an empty wire cage identical to that enclosing the social stimulus, serving as a control for exploring a novel object/environment.	Sociability and social preference	Venerosi et al. (2008)
Socioagonistic behavior	The animal undergoes an agonistic encounter with an unfamiliar partner of the same sex and age and the behavior is video-recorded. Nonsocial and social behaviors are scored; these usually include general activity, such as exploring, rearing, and digging, as well as social responses, including attack, often accompanied by biting attempts; aggressive grooming; tail rattling; offensive upright posture; defensive upright posture; and submissive upright posture.	Social aggression	Ricceri et al. (2006)
Social novelty preference	The test animal is restricted in a chamber and an unfamiliar partner is introduced. The test mouse is allowed to choose between a familiar partner and the novel, unfamiliar partner. The mouse is observed for choice of partner and time spent with partner.	Sociability and social preference	Venerosi et al. (2008)
Maternal behavior
Maternal aggressive behavior	After delivery, pups are removed and dam is exposed to an intruder male in its home cage. Maternal behaviors are recorded.	Maternal aggression against an intruder	Venerosi et al. (2008)
Pup-directed behavior	Maternal behavior with pups, including pup retrieval, sniffing, crouching over the pups, and nest-building is observed and recorded.	Maternal behavior	Ricceri et al. (2006); Venerosi et al. (2008)
Emotion/Anxiety
Light/dark box test	The light/dark box test consists of a box with two compartments: a small, opaque, dark compartment closed by a red glass and a large, transluscent and illuminated white compartment. The floor of the light compartment is divided into identical squares to allow determination of exploratory behavior. The compartments are connected by an opening to allow mice to freely move between them. A spotlight illuminates the apparatus to provide an aversive stimulus. Animals are individually placed in the center of the light compartment and allowed to explore the compartments. The time spent in each compartment, the time spent in the central square of the light compartment, and the number of crossings between compartments is recorded. This apparatus provides an unfamiliar environment for rodents where a natural conflict occurs between the tendency to explore a novel environment (spontaneous exploratory behavior) and the innate aversion to an open field and brightly illuminated area. As the light, but not the dark, compartment provides stress-like conditions for mice, the percentage of time spent by the mice in the dark compartment is used as a measure of anxiety in mice. In addition, exploratory behavior in the light compartment (time spent in the central square relative to the entire time spent in the light compartment) is also indicative of anxiety. The number of crossings between compartments provides an estimation of the anxiety level if measured without changes of spontaneous locomotor activity.	Anxiety	Venerosi et al. (2008, 2010); Braquenier et al. (2010)
Ultrasonic vocalization	During the postnatal period, pups emit ultrasonic calls to solicit parental care from the dam.These calls peak at PND 7–9. The most frequently used paradigm to elicit ultrasonic calls is to isolate a pup from the dam for a few minutes. Parameters that are analyzed include number, duration, and frequency of calls.	Homing/Anxiety	Venerosi et al. (2009); Laviola et al. (2006)
Elevated plus-maze	The elevated plus-maze is a conflict-avoidance test. Rodents prefer a dark, enclosed, small space over a brightly lit, open, large space, but they are also highly exploratory. This maze represents the natural conflict between the tendency of mice to explore a novel environment and the tendency to avoid a brightly lit open field. The behavior is also influenced by the fear of heights. The elevated plus-maze is in the shape of a “+”. The four arms extend from a central platform. Two alternate arms are dark and enclosed, while two alternate arms are open, lit, and available with or without ledges. Additionally, the surface of all arms is raised 1 m above the floor. The intensity of the fear drive (anxiety) is measured by the ratio of entries in open and enclosed arms. The open and enclosed arms of the maze generate exploratory behavior and the avoidance of elevated open arms is an indication of the intensity of anxiety. Furthermore, head dipping over the edges of the open arms, risk-assessment, self grooming, and defecation can be recorded.	Anxiety	Braquenier et al. (2010); Ricceri et al. (2006); Icenogle et al. (2004); Aldridge et al. (2004)
Forced swimming test	The animal is observed during a swimming session and behavioral parameters such as floating (the animal floats in the water making only those movements necessary to keep its head above water), struggling (vigorous movements of all four limbs with the forelimbs breaking the surface of the water) and swimming (all four limbs move under the surface of the water with the head kept above water) are observed. The frequency and duration of each pattern are scored.	Mood or adaptation/learning	Venerosi et al. (2010)
Reflexes and motor activity
Open field locomotor test	Assessment takes place in either a circular platform arena or square arena. The animal is placed in the center of the open field arena and allowed to freely move about while being measured by an automated tracking system. Measurements taken include, but are not limited to, general path length, rearing (standing on hind limbs) behaviors, and exploratory behaviors.	Locomotor activity (general activity levels, gross locomotor activity, and exploration habits. Assessment over several days allows habituation to the environment to be evaluated.)	Carr et al. (2001); Ricceri et al. (2003, 2006)
Geotaxic response	The animal is placed head-downward and given a period of time to turn itself 180 degrees. Animals are scored for completing the 180 degree turn.	Sensorimotor reflexes	Dam et al. (2000)
Cliff avoidance	This test assesses pup withdrawal from the edge of a flat surface when its snout and forepaws are placed over a real or virtual cliff; time of aversion can be recorded.	Sensorimotor reflexes	Venerosi et al. (2009)
Acoustic startle test	The animal is subjected to a loud sound or click. A subsequent freezing reaction and blink indicate functional hearing abilities.	Auditory reflexes (hearing ability)	Icenogle et al. (2004); Johnson et al. (2009)
Free-fall and surface righting	Free-fall: The ability of a pup to correct its posture such that it lands on all four feet when released from a supine position above a soft surface.	Posture reflex	Dam et al. (2000); Johnson et al. (2009)
	Surface: The ability of a pup to right itself to an upright position from a supine position.
Inclined plane	The animal is placed on a flat plane in the horizontal position with its head facing the side of the board to be raised. The angle at which the animal begins to slip downward is recorded.	Neuromotor performance	Abou-Donia et al. (2006)
Grip strength and forepaw grip time	Forelimb strength is measured as tension force using a computerized grip strength meter (GSM). The animal is lifted over the baseplate of the GSM by the tail so that its forepaws are allowed to grasp onto the steel grip. The mouse is then gently pulled backward by the tail until the grip is released. The GSM measures the maximal force before the mouse releases the bar. Forepaw strength is assessed by having animals grip a wooden dowel that is held horizontally and raised so that the animal supports its weight. The time to release grip is recorded in seconds.	Neuromuscular function and muscular strength	Abou-Donia et al. (2006)
Beam-walking	This test assesses the animal’s performance in crossing a narrow wooden beam.	Motor coordination and balance	Abou-Donia et al. (2006)
Rotorod test	This test assesses the number of times pups fall off a rotating rod during a 2-minute period.	Neuromotor performance	Muto et al. (1992)
Memory and Learning
T-maze	The animal is placed in the start arm of the T-maze and is allowed to explore one of the two goal arms. The mouse is then returned to the start for the next trial and will typically choose to explore the alternate arm. Because this behavior is not reinforced it is considered “spontaneous.”	Exploratory behavior and/or memory (measures exploratory behavior when the intervals between the trials are short, but also serves a spatial memory test when the interval between trials is lengthened)	Maurissen et al. (2000); Icenogle et al. (2004); Levin et al. (2001, 2002)
Multi-arm radial maze	Animals are trained to visit a pattern of arms in a multi-arm arm maze to receive a reward (e.g., food). To perform well, the animal must keep the egocentric or allocentric/spatial information regarding which arms of the maze it has already visited during the course of the task. Assessment of choice accuracy for information presented within the day’s test session and learning that has accumulated across days of testing are often referred to as working memory and reference memory tasks, respectively.	Working and reference memory errors	Johnson et al. (2009); Icenogle et al. (2004); Levin et al. (2001, 2002); Haviland et al. (2010)
Morris swim test	Animals are trained to locate a hidden platform within a pool of opaque water using spatial cues around the pool as a guide. The tank is surrounded by curtains that contain contrasting shapes to serve as spatial cues. Free swims (trials in which the platform is removed) are given throughout training to assess learning. Cued or visible platform trials are typically given at the end of training and serve as a sensorimotor index for each animal.	Spatial learning and memory	Jett et al. (2001)
Figure-8 apparatus	The apparatus consists of a continuous, enclosed alley in the shape of a Figure-8, and two blind alleys extending from either side. A single photobeam is located in each blind alley and three photobeams are located in the loops of the Figure-8 for a total of eight photobeams. Animals are allowed to roam in the maze for a set time and photobeam breaks are recorded to measure locomotor activity. To assess the speed of habituation, the blocks can be analyzed for linear and quadratic trends.	Motor activity and habituation	Icenogle et al. (2004)
Foraging maze	The foraging maze consists of a central platform elevated from the floor on a small movable table, containing eight arms projecting radially. At the end of each radial arm, two choice arms project in a ‘Tee’ format. At the end of each Tee, a low barrier is placed on both sides that serves as a food cup, and conceals a large bait under a screen to mask olfactory clues. The maze has opaque sides and clear overhead plastic and is housed in a lighted room with multiple extra-maze visual cues overhead. A fasted animal is placed in a cylinder in the central starting area and then allowed to roam freely about the maze for a set time or until all baits have been discovered. Radial arm choice and pre-reward and post-reward arm entries are recorded.	Learning, memory, and foraging behavior	Haviland et al. (2010)
Passive avoidance	This task exploits the rodent’s natural preference for dark environments. In this task the animal has to choose between responding to obtain a positive reinforcement (entering into a dark chamber) and not responding to avoid an electric footshock (not entering the dark chamber). The latency to refrain from entering into the dark compartment serves as an index of conditioned suppression and the ability to avoid, and allows memory to be assessed.	Conditioned suppression of instrumental behavioral responses (memory)	Ricceri et al. (2003)
Acclimatization	The test animal is first placed in a middle chamber and the doorways into side chambers are opened to allow exploration of the whole cage. Animals are observed for time of exploration of the chamber.	Exploration and habituation	Venerosi et al. (2008)

Table 8.  Rodent studies of social and maternal behavior

Study	Species	Animals per dose	Exposure			Outcome						Effects on AChE activity in brain
			Route	Concentration (mg CPF/kg b.w./day)	Developmental period	Sex	Test period	Endpoint examined	Statistically significant results	Null endpoints	Implication of effects
Ricceri et al. (2006)	Mouse, CD-1	10	Oral gavage	0, 3, 6	GD 15-18	Male	PND 75-80	Socioagonistic behavior	Increased offensive upright posture	Defensive or submissive upright posture; frequency and duration of attack response; aggressive grooming of partner, tail rattling	Increase in one marker of socioagonistic behavior with 6 mg/kg-day	Null
			s.c.	0, 1, 3	PND 11-14	Male	PND 75-80	Socioagonistic behavior	Increased frequency and duration of attack response	Offensive upright posture; defensive upright or submissive posture; aggressive grooming of partner, tail rattling	Increase in two markers of socioagonistic behavior with 3 mg/kg-day
			Oral gavage	0, 3, 6	GD 15-18	Male	PND 75-80	Social interactions	None	Rearing; digging; moving around cage; self-grooming	Null
			s.c.	0, 1, 3	PND 11-14
			Oral gavage	0, 3, 6	GD 15-18	Female	PND 90	Maternal behavior	None	Nest building latency, frequency, and duration; pup retrieval to nest; crouch response frequency and duration; pup licking frequency and duration; pup sniffing	Null
			s.c.	0, 1, 3	PND 11-14	Female	PND 90	Maternal behavior	Increased frequency and duration of crouch response; decreased licking frequency; increased licking duration; decreased sniffing	Nest building latency, frequency, and duration; pup retrieval to nest	Alterations of 5 markers of maternal behavior with 1 and 3 mg/kg-day
Ricceri et al. (2003)	Mouse, CD-1	7 to 10	s.c.	0, 1, 3	PND 1-4	Male, Female	PND 45	Social interactions	None	Social interactions with an unfamiliar untreated mouse for 20 minutes	Null	∼20% inhibition with 1 and 3 mg/kg-day
					PND 11-14							Null
					PND 1-4	Male, Female	PND 45	Social investigative behaviors	None	Anogenital sniffing; nose sniffing; body sniffing; following partner around cage; mutual sniffing of anogenital area)	Null	∼20% inhibition with 1 and 3 mg/kg-
					PND 11-14							Null
					PND 1-4	Male, Female	PND 45	Affiliative behaviors	None	Social rest, usually next to partner; grooming partner’s body)	Null	∼20% inhibition with 1 and 3 mg/kg-day
					PND 11-14							Null
					PND 1-4	Male, Female	PND 45	Soliciting behaviors	None	Push-under response (pushing snout or whole anterior body under partners’); crawl behavior(crawling over partner)	Null	∼20% inhibition with 1 and 3 mg/kg-day
					PND 11-14				Increased frequency of the push-under response	Crawl behavior	Alteration of one marker of soliciting behavior with 3 mg/kg-day	Null
					PND 1-4	Male	PND 45	Socioagonistic behavior	Increased frequency of aggressive grooming of partner; increased frequency of pooled aggressive responses in two of four observation blocks	Individual aggressive behaviors: attack response; tail rattling; offensive upright posture; defensive upright or submissive posture	Increase in one marker of socioagonistic behavior and transient increase in overall socioagonistic behavior with 1 and 3 mg/kg-day	∼20% inhibition with 1 and 3 mg/kg-day
					PND 11-14				Increased frequency of pooled aggressive responses in all observation blocks	Individual aggressive behaviors: attack response; aggressive grooming of partner; tail rattling; offensive upright posture; defensive upright or submissive posture	Increased socioagonistic behavior with 1 and 3 mg/kg-day	Null
Venerosi et al. (2008)	Mouse, CD-1	12 to 15	s.c.	0, 3	PND 11-14	Male, Female	PND 40-45	Acclimatization	Decreased distance moved when allowed to freely explore cage in first of three sessions	None	Altered acclimatization with 3 mg/kg-day	Not examined
							PND 40-45	Social novelty preference and sociability	None	Time spent with partner 1 vs. partner 2; time spent with a partner vs. empty chamber	Null
						Female	Birth to PPD 7	Nest-building activity	Longer latency to nest building	Amount of materials used for nest; nest qualitative features	Alteration of one marker of maternal behavior with 3 mg/kg-day
							PPD 1	Maternal behavior	Shorter latency to licking	Licking duration; licking frequency; time spent with pups; nursing; pup retrieval; pup sniffing	Alteration of one marker of maternal behavior with 3 mg/kg-day
							PPD 7	Maternal aggression against male intruder	Decreased defensive posture duration; increased digging duration; increase in pooled investigative Behaviors	Attack frequency; individual investigative behaviors: circling, social sniffing, and following)	Decrease in 3 markers of maternal aggression with 3 mg/kg-day

Notes:

CPF = chlorpyrifos; GD = ge stational da y; PND = postn atal day; PPD = pos tpartum day; s.c. = subcutaneous injection

Table 9.  Rodent studies of effects on emotion and anxiety.

Study	Species	Animals per dose	Exposure			Outcome						Effects on AChE activity in brain
			Route	Concentration (mg CPF/kg b.w./day)	Developmental period	Sex	Test period	Endpoint examined	Statistically significant results	Null endpoints	Implication of effects
Venerosi et al. (2009)	Mouse, CD-1	15 per sex	Oral gavage	0, 6	GD 15-18	Male, Female	PND 4, 7, 10	Anxiety via ultrasonic vocalization	Fewer calls, shorter call duration, longer latency to calls, and higher peak frequency of calls (PND 10 only)	Peak amplitude of calls	Increased anxiety on PND 10 with 6 mg/kg-day	NR
Venerosi et al. (2010)	Mouse, CD-1	10 to 12	Oral gavage	0, 6	GD 15-18	Male, Female	PND 90	Anxiety via the light/dark box test	Increased percentage of time spent in the tunnel connecting the two compartments of the apparatus (females only)	Latency to enter dark compartment; time spent in dark or light compartments; risk assessment and exploratory behavior in light compartment	Increase in one marker of anxiety for females only, with 6 mg/kg-day	NR
		5 to 7					PND 90	Mood via the forced swim test	None	Swimming, struggling, and floating in water	Null
Braquenier et al. (2010)	Mouse, CD-1	8 to 10	Oral gavage	0, 0.2, 1, 5	GD 15 - PND 14	Female	PND 80	Anxiety via the elevated plus-maze	Decreased percentage of time spent in open arms; decreased proportion of entries in open arms	None	Increased anxiety with 1 mg/kg-day	14% inhibition with 5 mg/kg-day
							PND 72	Anxiety via the light/dark box test	Decreased percentage of time spent in center of light compartment; decreased number of compartment switches	Percentage of time in dark compartment	Increased anxiety with 1 mg/kg-day
Ricceri et al. 2006	Mouse, CD-1	10	Oral gavage	0, 3, 6	GD 15-18	Male, Female	4 months	Anxiety via the elevated plus-maze	Decreased head dipping frequency (males only)	Frequency of arm entries; duration of time spent in open, central, and closed arms; frequency and duration of risk assessment posture	Decreased anxiety in males with 3 mg/kg-day	Null
			s.c.	0, 1, 3	PND 11-14	Male, Female	4 months	Anxiety via the elevated plus-maze	Increased percentage of time spent in open arms (females only)	Frequency of arm entries; duration of time spent in central and closed arms; head dipping frequency; frequency and duration of risk assessment posture	Decreased anxiety in females with 3 mg /kg-day
Icenogle et al. (2004)	Rat, Sprague-Dawley	10	s.c.	0, 1, 5	GD 9-12	Male, Female	4-8 weeks	Anxiety and hyperactivity via the elevated plus-maze	Increased number of center crosses	Duration of time spent in open and closed arms	Increased hyperactivity with 5 mg/kg-day	NR
Laviola et al. (2006)	Mouse, C57/B16	4 to 6	s.c.	0, 5 (chlorpyrifos-oxon)	GD 14-16	Male, Female	PND 3, 7, 11	Anxiety via ultrasonic vocalization	None	Number of calls	Null	NR
Aldridge et al. (2005)	Rat, Sprague-Dawley	9 per sex	s.c.	0, 1	PND 1-4	Male, Female	PND 52-53	Anxiety via the elevated plus-maze	Increased percentage of time spent in open arms and increased number of center crosses (males only)	Duration of time spent in closed arms	Decreased anxiety and increased hyperactivity in males with 1 mg/kg-day	NR
Ricceri et al. (2003)	Mouse, CD-1	10	s.c.	0, 1, 3	PND 1-4	Male, Female	PND 5, 8, 11	Anxiety via ultrasonic vocalization	None	Number of calls	Null	~20% inhibition with 1
							PND 10	Homing	None	Latency to reach nest area; time spent in nest area; locomotor activity during test	Null	and 3 mg/kg-day
Venerosi et al. (2008)	Mouse, CD-1	9 to 11	s.c.	0, 3	PND 11-14	Female	PPD 2 (after mating on PND 60)	Anxiety via the light/dark box test	Increased proportion of dams entered light compartment; decreased latency to enter light compartment	Percentage of time in dark or light compartment; number of transitions across tunnel connecting the two compartments	Decreased anxiety in dams with 3 mg/kg-day	NR

Notes: CPF = chlorpyrifos; GD = gestational day; NR = not reported; PND = postnatal day; PPD = postpartum day; s.c. = subcutaneous injection.

Table 10.  Rodent studies of effects on sensorimotor reflexes and motor activity.

Study	Species	Animals per dose	Exposure			Outcome
			Route	Concentration (mg CPF/kg b.w./day)	Developmental period	Sex	Test period	Endpoint examined	Statistically significant results	Null endpoints	Implication of effects	Effects on AChE activity in brain
Venerosi et al. (2009)	Mouse, CD-1	13 to 18 per sex	Oral gavage	0, 6	GD 15-18	Male, Female	PND 12	Spontaneous motor behavior	Decreased frequency and duration of pivoting; increased duration of immobility	Frequency and duration of crossing (locomotor activity), head movement, wall climbing, and grooming	Decrease in three markers of spontaneous motor behavior with 6 mg/kg-day	NR
							PND 3, 6, 9, 12, 15	Sensorimotor maturation	None	Righting reflex; grasping reflex; cliff aversion; forelimb and hind limb reflexes; pole grasping	Null
Ricceri et al. (2006)	Mouse, CD-1	10	Oral gavage	0, 3, 6	GD 15-18	Male	PND 70	Locomotor activity via open field test	Increased crossing frequency	Crossing duration; rearing; wall rearing; self-grooming	Increase in one marker of locomotor activity with prenatal exposure to 6 mg/kg-day followed by postnatal exposure to 0 or 1 mg/kg-day, and with prenatal exposure to 0 or 3 mg/kg-day followed by postnatal exposure to 3 mg/kg-day	Null
			s.c.	0, 1, 3	PND 11-14
Maurissen et al. (2000)	Rat, Sprague-Dawley	20 per sex	Oral gavage	0, 0.3, 1, 5	GD 6 - PND 10	Male, Female	PND 13, 17, 21, 60	Locomotor activity	None	Locomotor activity	Null	NR
							PND 22, 61	Auditory reflexes via acoustic startle test	None	Auditory reflexes	Null
Braquenier et al. (2010)	Mouse, CD-1	8 to 10	Oral gavage	0, 0.2, 1, 5	GD 15 - PND 14	Female	> PND 21 for 8 days	Locomotor activity	None	Locomotor activity	Null	14% inhibition with 5 mg/kg-day
Moser et al. (1998)	Rat, Long Evans hooded	5 per sex	Oral gavage	0, 5, 20	PND 17	Male, Female	3.5 or 6.5 hours post-exposure	Locomotor activity via Figure-8 apparatus	Decreased locomotor activity	None	Decreased locomotor activity with 20 mg/kg-day	50–90% inhibition with 5 and 20 mg/kg-day
								Sensorimotor reflexes	Righting deficits; gait changes	None	Deficits in sensorimotor reflexes with 20 mg/kg-day
Carr et al. (2001)	Rat, Sprague-Dawley	10	Oral gavage	Low: 3 (PND 1–21)	PND 1–21 (every other day)	Male, Female	PND 10, 12, 14, 16, 18, 20, 25, 30	Locomotor activity via open field test	Decreased locomotor activity (PND 25 and 30 only; medium and high dose groups only)	None	Decreased locomotor activity with 6 and 12 mg/kg-day	17–70% inhibition with 3, 6, and 12 mg/kg-day
				Medium: 3 (PND 1–5), 6 (PND 7–21)
				High: 3 (PND 1–5), 6 (PND 7–13), 12 (PND 15–21)
Johnson et al. (2009)	Rat, Sprague-Dawley	9 to 14 per sex	Oral gavage	Low: 1 (PND 1–5), 1 (PND 6–13), 1 (PND 14–20)	PND 1–20	Male, Female	NR	Sensorimotor reflexes	None	Surface righting; free-fall righting; negative geotaxis; cliff avoidance; auditory reflexes	Null	14–53% inhibition with 1 to 6 mg/kg-day
				Medium: 1 (PND 1–5), 2 (PND 6–13), 4 (PND 14–20)
				High: 1.5 (PND 1–5), 3 (PND 6–13), 6 (PND 14–20)
Chanda and Pope (1996)	Rat, Sprague- Dawley	7 to 8	s.c.	25	GD 12–19	Male, Female	PND 1, 3	Sensorimotor reflexes	Decreased cliff avoidance; surface righting deficits	None	Deficits in sensorimotor reflexes with 25 mg/kg-day	60% inhibition with 25 mg/kg-day
Icenogle et al. (2004)	Rat, Sprague- Dawley	10 per sex	s.c.	0, 1, 5	GD 9–12	Male, Female	4–6 weeks	Locomotor activity via Figure-8 apparatus	Decreased locomotor activity (2 of 12 test sessions only)	None	Transient decrease in locomotor activity with 5 mg/kg-day	NR
							NR	Auditory reflexes via acoustic startle test	None	Auditory reflexes	Null
Levin et al. (2002)	Rat, Sprague- Dawley	10 per sex	s.c.	0, 1,	GD 17–20	Male, Female	4–6 weeks	Locomotor activity via Figure-8 apparatus	None	Locomotor activity	Null	NR
Laviola et al. (2006)	Mouse, C57/B16	4 to 6	s.c.	0, 5 (chlorpyrifos-oxon)	GD 14–16	Male, Female	PND 3,7,11	Neuromuscular function via grasping reflex test	Increased fall angle (PND 3 only)	None	Increased maturation of grasping reflex at 5 mg/kg-day chlorpyrifos-oxon	NR
								Sensorimotor reflexes	None	Righting reflex	Null
Ricceri et al. (2003)	Mouse, CD-1	10	s.c.	0, 1, 3	PND 1-4	Male, Female	PND 25	Locomotor activity via open field test	None	Locomotor activity	Null	~20% inhibition with 1 and 3 mg/kg-day
					PND 11-14				Increased locomotor activity	None	Increased locomotor activity with 1 and 3 mg/kg-day	Null
					PND 1-4	Male, Female	PND 45	Motor behavior	Decreased self-grooming	Exploring; rearing; digging	Decrease in one marker of motor behavior at 1 and 3 mg/kg-day	~20% inhibition with 1 and 3 mg/kg-day
					PND 11-14				None	Exploring; rearing; digging; self-grooming	Null	Null
Dam et al. (2000)	Rat, Sprague-Dawley	23 to 24 per sex	s.c.	0, 1	PND 1-4	Male, Female	PND 3, 4	Sensorimotor reflexes	Deficits in reflex righting (females only)	None	Deficit in one marker of sensorimotor reflexes in females with 1 mg/kg-day	20-60% inhibition with 1 mg/kg-day
		23 to 24 per sex	s.c.	0, 1	PND 1-4	Male, Female	PND 5, 8	Sensorimotor reflexes	Deficits in geotaxic response (females only)	None	Deficit in one marker of sensorimotor reflexes in females with 1 mg/kg-day	20-60% inhibition with 1 mg/kg-day
		5 to 7 per sex	s.c.	0, 1	PND 1-4	Male, Female	PND 21, 30	Locomotor activity via open field test	Decreased locomotor activity and rearing (males only)	Grooming	Decrease in two markers of locomotor activity in males with 1 mg/kg-day	20-60% inhibition with 1 mg/kg-day
				0, 5	PND 11-14				Increased rearing (males on PND 30 only)	Locomotor activity; grooming	Increase in one marker of locomotor activity in males with 5 mg/kg-day	20-60% inhibition with 5 mg/kg-day
Levin et al. (2001)	Rat, Sprague-Dawley	10	s.c.	0, 1	PND 1-4	Male, Female	4-6 weeks	Locomotor activity via Figure-8 apparatus	None	Locomotor activity	Null	NR
				0, 5	PND 11-14
Chakraborti et al. (1993)	Rat, Sprague-Dawley	10	s.c.	0, 40	PND 7-10	Male, Female	2, 4, 6, 8 weeks post-exposure	Locomotor activity via open field test	None	Locomotor activity	Null	55-60% inhibition with 40 mg/kg-day
Muto et al. (1992)	Rat, species unspecified	12 to 20	i.p.	0, 0.03, 0.1, 0.3 (as Dursban)	GD 0-7	Not specified	PND 16	Neuromotor function via the rotorod test	Increased number of falls	None	Decreased neuromotor function with 0.03 to 0.3 mg/kg-day
					GD 7-21			Neuromotor function via the rotorod test	Increased number of falls	None	Decreased neuromotor function with 0.3 mg/kg-day
					PND 3, 10, or 12			Neuromotor function via the rotorod test	Increased number of falls	None	Decreased neuromotor function with 0.1 and 0.3 mg/kg-day	NR
								General motor behavior	None	General motor behavior	Null
								Neuromuscular function via inclined plane test	None	Neuromuscular function	Null
Abou-Donia et al. (2006)	Rat, Sprague-Dawley	10	Dermal	0, 1	GD 4-20	Male, Female	PND 90	Motor coordination/balance via beam-walking test	None	Beam-walking score	Null
								Neuromuscular function via inclined plane test	Decreased incline angle (females only)	None	Deficits in one marker of neuromuscular function in females with 1 mg/kg-day	25% increase in females with 1 mg/kg-day
								Neuromuscular function	Decreased forepaw grip time	None	Deficits in one marker of neuromuscular function with 1 mg/kg-day

Notes: CPF = chlorpyrifos; GD = gestational day; NR = not reported; PND = postnatal day; s.c. = subcutaneous injection.

Table 11.  Rodent studies of effects on cognitive function.

Study	Species	Animals per dose	Exposure			Outcome
			Route	Concentration (mg CPF/kg b.w./day)	Developmental period	Sex	Test period	Endpoint examined	Statistically significant results	Null endpoints	Implication of effects	Effects on AChE activity in brain
Maurissen et al. (2000)	Rat, Sprague-Dawley	8 per sex	Oral gavage	0, 0.3, 1, 5	GD 6 - PND 10	Male, Female	PND 22–24, PND 61–91	Learning and memory via the T-maze test	None	Spatial delayed alternation (learning acquisition; short- term retention; non- mnemonic factors)	Null	NR
Johnson et al. (2009)	Rat, Sprague-Dawley	9 to 14 per sex	Oral gavage	Low: 1 (PND 1–5), 1 (PND 6–13), PND 14–20L	PND 1–20	Male, Female	PND 36–60	Memory via the 12-arm radial maze	Increased number of working memory errors (males only; one of four testing weeks)	Reference memory errors	Transient deficit in one marker of memory in males with 1 mg/kg-day	14–53% inhibition with 1 to 6 mg/kg- day
				Medium: 1 (PND 1–5), 2 (PND 6–13), 4 (PND 14–20)					Fewer working and reference memory errors (females only); increased number of working and reference memory errors (males only; one of four testing weeks)	None	Transient memory deficits in males and memory gains in females and with 1 to 4 mg/kg- day Memory deficits in males and memory gains in females and with 1.5 to 6 mg/kg-day
				High: 1.5 (PND 1–5), 3 (PND 6–13), 6 (PND 14–20)					Increased number of working memory errors (males only); fewer reference memory errors (females only); increased number of reference memory errors (males only; one of four testing weeks)
Icenogle et al. (2004)	Rat, Sprague- Dawley	10 per sex	s.c.	0, 1, 5	GD 9–12	Male, Female	4–8 weeks	Exploratory behavior via the T-maze test	Shorter spontaneous alternation latency (first of five sessions only)	None	Increased hyperactivity with 1 and 5 mg/kg-day	NR
							8–13 weeks	Memory via the 16-arm radial maze	Increased working memory errors (two of six sessions); increased reference memory errors (one of six sessions)	None	Transient memory deficits with 5 mg/kg-day
							4–8 weeks	Habituation via Figure-8 apparatus	Faster linear trend of habituation	None	Faster habituation with 5 mg/kg day
Levin et al. (2002)	Rat, Sprague- Dawley	10 per sex	s.c.	0, 1, 5	GD 17–20	Male, Female	4–6 weeks	Exploratory behavior via the T-maze test	Decrease in alternation latency (one or two of five test trials)	Alternation between maze arms	Transiently increased hyperactivity with 1 and 5 mg/kg-day	NR
							8–13 weeks	Memory via the 16-arm radial maze	Increased mean number of working and reference memory errors across 18 sessions (females only)	Memory error frequency	Memory deficits in females with 1 mg/kg-day
							4–6 weeks	Habituation via Figure-8 apparatus	Slower linear trend of habituation (females only)	Habituation time	Slower habituation in females with 1 and 5 mg/kg-day
Haviland et al. (2010)	Mouse, Swiss Webster	8	s.c.	0, 1, 5	GD 17–20	Male, Female	PND 60–81	Learning via the foraging maze	Decreased food recognition learning (two or three of nine sessions; females only); increased food recognition learning (four of nine sessions; males only); decreased food position learning (two of nine sessions; females only); increased food position learning (two of nine sessions; males only)	Foraging rate	Transiently decreased foraging ability in females with 1 and 5 mg/kg-day; transiently increased foraging ability in males with 1 and 5 mg/kg-day	NR
								Memory via the 8-arm radial maze	Increased reference memory errors (two of nine sessions); decreased reference memory errors (two of nine sessions; females only)	None	Transient memory deficits in males with 1 or 5 mg/kg-day; transient memory deficits in females with 1 mg/kg-day; transient memory gains in females with 1 or 5 mg/kg-day
Aldridge et al. (2005)	Rat, Sprague-Dawley	9 per sex	s.c.	0, 1	PND 1-4	Male, Female	PND 64-99	16-arm radial maze	Increased mean number of reference memory errors across 18 sessions (males only)	Working memory errors	Memory deficits in males with 1 mg/kg-day	NR
Levin et al. (2001)	Rat, Sprague-Dawley	10	s.c.	0, 1	PND 1-4	Male, Female	4-6 weeks	Exploratory behavior via the T-maze test	None	Alternation frequency; alternation latency	Null	NR
				0, 5	PND 11-14				Increase in alternation latency (one of 12 sessions; males only)	Alternation frequency	Transiently decreased hyperactivity in males with 5mg/kg-day
				0, 1	PND 1-4		8-13 weeks	Memory via the 16-arm radial maze	Increased number of working and reference memory errors (over first three of 18 sessions; males only); increased mean number of working and reference memory errors (females)	None	Memory deficits with 1 mg/kg-day
				0, 5	PND 11-14				None	Working and reference memory errors	Null
				0, 1	PND 1-4		4-6 weeks	Habituation via Figure-8 apparatus	None	Habituation	Null
				0, 5	PND 11-14				Slower linear trend of habituation	None	Slower habituation with 5 mg/kg-day
Ricceri et al. (2003)	Mouse, CD-1	10	s.c.	0, 3	PND 1-4	Male, Female	PND 35-38	Novelty-seeking behavior	Increased activity rate in novel compartment (one of five sessions)	Novelty preference; latency to enter novel compartment; activity rate in familiar compartment	Transient alteration in one marker of novelty-seeking behavior with 3 mg/kg-day	~20% inhibition with 1 and 3 mg/kg-day
					PND 11-14				Increased activity rate in novel compartment (two of five sessions)	Novelty preference; latency to enter novel compartment; activity rate in familiar compartment		Null
				0, 1, 3	PND 1-4	Male, Female	PND 60-81	Passive avoidance learning	None	Memory	Null	~20% inhibition with 1 and 3 mg/kg-day
					PND 11-14							Null
Venerosi et al. (2008)	Mouse, CD-1	9 to 11	s.c.	0, 3	PND 11-14	Male, Female	PND 40-45	Acclimatization	Decreased exploration (one of three sessions)	None	Transient decrease in exploration with 3 mg/kg-day	NR
Jett et al. (2001)	Rat, Long Evans	8 to 10 per sex	s.c.	0, 0.3, 7	PND 7, 11, 15 (pre weaning)	Male, Female	PND 24-28	Cognitive function via the Morris swim test	Increased escape latency (two of four testing days); learning deficiency in probe test	Swimming speed	Transient learning deficits with 7 mg/kg-day	Null
		3 to 4 per sex			PND 22, 26 (post-weaning)				Increased escape latency (two of four testing days); learning deficiency in probe test	Swimming speed	Transient learning deficits with 0.3 and 7 mg/kg-day	Null

Notes: CPF = chlorpyrifos; GD = gestational day; NR = not reported; PND = postnatal day; s.c. = subcutaneous injection.

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