THE DOW CHEMICAL COMPANY 

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Summary 

(In accordance with 40 CFR Part 152, this summary is available for 

public release after registration) 

Study Title 

COMPARISON OF CHOLINESTERASE (ChE) INHIBITION IN YOUNG ADULT 

AND PREWEANLING CD RATS AFTER ACUTE AND REPEATED 

CHLORPYRIFOS OR CHLORPYRIFOS-OXON EXPOSURES 

Data Requirement 

None 

Author(s) 

M. S. Marty, Ph.D. 

A. K. Andrus, M.S. 

Report Date 

29 June 2010 

Sponsor 

Dow AgroSciences LLC 

9330 Zionsville Road 

Indianapolis, Indiana 46268 

Performing Laboratory 

Toxicology & Environmental Research and Consulting 

The Dow Chemical Company 

Midland, Michigan 48674 
THE DOW CHEMICAL COMPANY 

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SUMMARY 

In the acute comparative cholinesterase study, initial studies were conducted to determine 

the time-of-peak inhibition for red blood cell (RBC), brain and plasma cholinesterase 

(ChE) in pups and adults. Groups of 4-6 PND 11 female CD rat pups were administered 

a single gavage dose (corn oil vehicle) of 0 or 3 mg/kg chlorpyrifos or 0 or 0.5 mg/kg 

chlorpyrifos-oxon. A separate group of PND 11 female pups (6/dose) were exposed to 0 

or 5 mg/kg chlorpyrifos in rat milk to compare the impact of vehicle on ChE inhibition. 

Similarly, adult female CD rats (4/group) were administered a single gavage dose (corn 

oil vehicle) of 0 or 10 mg/kg chlorpyrifos or 0 or 0.3 mg/kg chlorpyrifos-oxon. A 

separate group of adult females were exposed for 12-h to dietary chlorpyrifos (dose levels 

of 0 or 10 mg/kg) to compare acute ChE inhibition by a more relevant exposure scenario. 

Clinical observations were recorded pre- and post-dosing; body weights and feed 

consumption were monitored for adults exposed to dietary chlorpyrifos. In both pups and 

adults, samples (RBC, brain, plasma) were collected at 2, 4, 6, 8, 12 , 24 or 48 h postdosing (48 h included for PND 11 pups given CPF in corn oil and CPF in milk only) and 

ChE inhibition was determined using a modified Ellman method. There were no 

treatment-related clinical observations in either pups or adults using these dosing 

scenarios. Using experimental data, published literature and physiologically-based 

pharmacokinetic/pharmacodynamic (PBPK/PD) model, time–to-peak ChE inhibition was 

determined for each dosing scenario. For PND 11 pups, the times-to-peak inhibition 

were 6 h post-dosing for chlorpyrifos in corn oil, 4 h post-dosing for chlorpyrifos-oxon in 

corn oil and 8 h post-dosing for chlorpyrifos in milk. For adult female rats, the times-topeak inhibition were 8 h post-dosing for chlorpyrifos in corn oil, 4 h post-dosing for 

chlorpyrifos-oxon in corn oil, and 8 h post-exposure for chlorpyrifos in diet (i.e., after 

conclusion of the 12-h dietary exposure to chlorpyrifos-containing diet). 

The definitive acute comparative cholinesterase study examined age-related differential 

sensitivity across multiple dose levels of chlorpyrifos. ChE activity was measured in 

PND 11 male and female CD rat pups (8/sex/dose) and adult female CD rats (8/dose) 

exposed to a single gavage dose (corn oil vehicle) of 0, 0.05, 0.1, 0.5, 2, 5 (pups only) or 

10 (adults only) mg/kg chlorpyrifos. There were no discernable gender differences in 

ChE activity in male or female PND 11 pups in response to chlorpyrifos treatment. Both 

pups and adults had significant and comparable decreases in brain ChE activity at the 

highest dose of chlorpyrifos (5 mg/kg in PND 11 pups and 10 mg/kg in adults) and 

significant RBC and plasma ChE inhibition at doses >2 mg/kg (Summary Table 1). The 
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0.5 mg/kg chlorpyrifos dose was considered a no-observed-effect-level (NOEL) for ChE 

inhibition across all tissues in both age groups with acute exposure. Thus, pups may be 

more sensitive to ChE inhibition at higher dose levels of chlorpyrifos (e.g., 5 mg/kg) due 

to higher plasma levels of chlorpyrifos and its metabolites; however, pups are equally 

sensitive or less sensitive than adults at lower chlorpyrifos levels (e.g., 0.5 mg/kg). Thus, 

there was no evidence of age-related differential sensitivity to acute chlorpyrifos 

exposure over the lower portion of the dose-response curve. 

Summary Table 1. ChE Inhibition in PND 11 Pups and Adult Females 

Following an Acute Dose of Chlorpyrifos in Corn Oil 

ADULTS – 

CPF in Corn 

Oil 

Dose 

RBC ChE 

(% of 

Control 

Activity) 

Brain ChE 

(% of 

Control 

Activity) 

Plasma ChE 

(% of 

Control 

Activity) 

[CPF] 

(ng/g) 

[TCP] 

(ng/g) 

0.05 mg/kg 95.1 99.5 108.2 <LLQ 10.53 

0.1 mg/kg 101.6 93.4 90.7 <LLQ 26.83 

0.5 mg/kg 110.4 95.5 87.5 <0.15 121.50 

2.0 mg/kg 80.6* 95.4 45.8* 1.10 494.25 

10 mg/kg 15.7* 42.5* 13.2* 32.46 2803.50 

PND 11 PUPS – 

CPF in Corn 

Oil

Dose 

RBC ChE 

(% of 

Control 

Activity) 

Brain ChE 

(% of 

Control 

Activity) 

Plasma ChE 

(% of 

Control 

Activity) 

[CPF] 

(ng/g) 

[TCP] 

(ng/g) 

0.05 mg/kg 103.0 107.9 100.7 0.19 57.28 

0.1 mg/kg 102.9 104.0 101.5 0.37 72.30 

0.5 mg/kg 96.8 106.9 91.4 1.79 304.00 

2.0 mg/kg 66.7* 95.7 51.0* 10.83 1100.25 

5.0 mg/kg 12.7* 46.8* 22.4* 88.48 1879.25 

CPF = chlorpyrifos; TCP = trichloropyridinol; <LLQ = Less than the lowest level quantified 

* Significantly different from controls at alpha = 0.05 using Dunnett’s test when raw ChE data 

were analyzed (male and female pups were analyzed separately, but are combined in this table; 

treatment-related effects shaded) 

For the definitive acute comparative cholinesterase study with chlorpyrifos-oxon, ChE 

activity was measured in PND 11 male and female CD rat pups (8/sex/dose) and adult 

female CD rats (8/dose) exposed to a single gavage dose (corn oil vehicle) of 0, 0.005 

(pups only), 0.01, 0.05, 0.1, 0.5, or 1 (adults only) mg/kg chlorpyrifos-oxon. There were 

no discernable gender differences in ChE activity in male or female PND 11 pups. There 

was no significant inhibition of brain ChE in either pups or adults at any dose level of 

chlorpyrifos-oxon (Summary Table 2). RBC ChE inhibition was seen at the same dose 

level (0.5 mg/kg) in PND 11 pups and adults, but PND 11 pups exhibited significant 

plasma ChE inhibition at a slightly lower dose than adults (0.1 mg/kg), a finding that may 
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be related to the 1.6X higher plasma TCP levels in pups at this dose level (both adult and 

pup samples collected at 4 h post-exposure; chlorpyrifos-oxon was below the lower limit 

of quantitation in pups and adults). Thus, the 0.1 mg/kg chlorpyrifos-oxon dose was 

considered a NOEL for ChE inhibition in RBC and brain in PND 11 pups and a NOEL in 

all tissues for adult females with acute exposure. 

Summary Table 2. ChE Inhibition in PND 11 Pups and Adult Females Following 

an Acute Dose of Chlorpyrifos-Oxon in Corn Oil 

ADULTS – 

CPF-O in Corn 

Oil 

Dose 

RBC ChE 

(% of 

Control 

Activity) 

Brain ChE 

(% of 

Control 

Activity) 

Plasma ChE 

(% of 

Control 

Activity) 

[CPF-O] 

(ng/g) 

[TCP] 

(ng/g) 

0.01 mg/kg 97.9 99.4 91.3 <LLQ 3.43 

0.05 mg/kg 103.5 100.7 94.2 <LLQ 22.33 

0.1 mg/kg 100.4 98.0 99.2 <LLQ 50.13 

0.5 mg/kg 63.4* 96.9 44.2* <LLQ 230.00 

1 mg/kg 23.8* 96.6 23.8* <LLQ 724.75 

PND 11 PUPS – 

CPF-O in Corn 

Oil

Dose 

RBC ChE 

(% of 

Control 

Activity) 

Brain ChE 

(% of 

Control 

Activity) 

Plasma ChE 

(% of 

Control 

Activity) 

[CPF-O] 

(ng/g) 

[TCP] 

(ng/g) 

0.005 mg/kg 95.9 96.9 95.1 <LLQ 16.93 

0.01 mg/kg 91.9 95.0 98.9 <LLQ 28.28 

0.05 mg/kg 90.2 101.2 93.2 <LLQ 45.60 

0.1 mg/kg 86.9 96.9 80.6* <LLQ 82.58 

0.5 mg/kg 53.4* 94.9 49.9* <LLQ 387.50 

CPF-O = chlorpyrifos-oxon; TCP = trichloropyridinol; <LLQ = Less than the lowest level quantified 

* Significantly different from controls at alpha = 0.05 using Dunnett’s test when raw ChE data were 

analyzed (male and female pups were analyzed separately, but are combined in this table; treatmentrelated effects shaded) 

To examine a more relevant dosing scenario for pups, ChE activity was measured in 

PND 11 male and female CD rat pups (8/sex/dose) exposed to a single gavage dose (corn 

oil vehicle) of 0, 0.05, 0.1, 0.5, 2, or 5 mg/kg chlorpyrifos. When using milk as a vehicle, 

there were no discernable gender differences in ChE activity in male or female PND 11 

pups in response to chlorpyrifos treatment. Compared to corn oil, the maximum ChE 

inhibition at the time-of-peak effect for chlorpyrifos was slightly later in milk (8 h) than 

corn oil (6 h). At the time-of-peak inhibition, the plasma chlorpyrifos and chlorpyrifosoxon levels, as well as the magnitude of ChE inhibition across tissues, were similar with 

both milk and corn oil vehicles. 

To examine a more relevant dosing scenario for adults, females (~ 70 days of age, 

8/dose) were exposed for 12-h to dietary chlorpyrifos at doses of 0, 0.05, 0.1, 0.5, 2 or 10 
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mg/kg and acute ChE inhibition was compared to levels seen in adult females exposed to 

the same chlorpyrifos doses by gavage in corn oil. With a 12 h dietary exposure to 

chlorpyrifos, adult females exhibited significant brain ChE inhibition at the same 

concentration as chlorpyrifos given via gavage in corn oil (10 mg/kg); however, the 

magnitude of brain ChE inhibition was less by the dietary route, where plasma 

chlorpyrifos and TCP levels were 7.4X and 2.4X lower, respectively. This result 

suggests that dose rate impacts plasma levels of chlorpyrifos and its metabolites and 

subsequently, the magnitude of brain ChE inhibition. In contrast RBC ChE inhibition 

appeared to be more pronounced at 2 mg/kg using the dietary exposure scenario. In both 

dosing scenarios, ChE inhibition was not significantly altered in any tissue at the 0.5 

mg/kg dose. 

In the repeated dose studies, ChE activity was measured in PND 21 male and female CD 

rat pups (8/sex/dose) and adult (~PND 80) female CD rats (8/dose) following exposure to 

eleven daily gavage doses (corn oil vehicle) of 0, 0.05, 0.1, 0.5, 1, 3.5 mg/kg/day 

chlorpyrifos or 0.01 or 0.5 mg/kg/day chlorpyrifos-oxon. Animals were given daily 

clinical observations and body weights during the exposure period. Neurotoxicity was 

assessed at the time-of-peak ChE inhibition after the 10th exposure by performing a 

functional observational battery and examining motor activity in PND 20 pups and ~PND 

79 adults. 

Eleven-day exposures with chlorpyrifos or chlorpyrifos-oxon did not significantly affect 

clinical signs or neurobehavioral toxicity measures in either pups or adults, including no 

treatment-related effects on FOBs and motor activity assessments. There were no 

significant effects on body weights in pups or adults at any dose of chlorpyrifos or 

chlorpyrifos-oxon during the treatment periods. There were no discernable gender 

differences in ChE activity in male or female PND 11 pups in response to repeated 

chlorpyrifos treatment. At the high dose (3.5 mg/kg/day), pups had similar ChE 

inhibition to adults across all tissues, despite having higher plasma levels of CPF 

(Summary Table 3). The higher plasma levels in pups were likely related to a later 

sampling time in adults (8 h) vs. pups (6 h) relative to the proposed Cmax for gavage 

chlorpyrifos in corn oil (2-3 h) and a somewhat higher metabolic rate in adults. At 1 

mg/kg/day, ChE was significantly decreased in both pups and adults across all tissues. At 

0.5 mg/kg/day, there was no significant brain ChE inhibition in either adults or pups. 

Both age groups had significant decreases in RBC and plasma ChE activity with no 

apparent difference in sensitivity. Thus, for a given plasma chlorpyrifos level, pups were 

equally sensitive or less sensitive than adults for ChE inhibition. As chlorpyrifos doses 
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decreased below 1 mg/kg/day, there was no evidence of increased sensitivity in pups to 

chlorpyrifos-induced ChE inhibition. The 0.1 mg/kg/day chlorpyrifos dose was 

considered a no-observed-effect-level (NOEL) for ChE inhibition across all tissues in 

both age groups with repeated exposure. 

Summary Table 3. ChE Inhibition in PND 11-21 Pups and Adult (~PND 70-80) 

Females Following Repeated Doses of Chlorpyrifos in Corn Oil 

ADULTS – 

CPF in Corn 

Oil 

Dose 

RBC ChE 

(% of 

Control 

Activity) 

Brain ChE 

(% of 

Control 

Activity) 

Plasma ChE 

(% of 

Control 

Activity) 

[CPF] 

(ng/g) 

[TCP] 

(ng/g) 

0.05 mg/kg/day 94.9 99.9 128.3 <LLQ 23.20 

0.1 mg/kg/day 84.0* 100.0 116.1 <LLQ 26.45 

0.5 mg/kg/day 80.5* 99.5 54.1* <0.13 196.08 

1.0 mg/kg/day 27.2* 91.1* 30.7* 0.54 351.75 

3.5 mg/kg/day 2.7* 31.0* 11.6* 2.21 1813.00 

PND 11 PUPS – 

CPF in Corn 

Oil

Dose 

RBC ChE 

(% of 

Control 

Activity) 

Brain ChE 

(% of 

Control 

Activity) 

Plasma ChE 

(% of 

Control 

Activity) 

[CPF] 

(ng/g) 

[TCP] 

(ng/g) 

0.05 mg/kg/day 96.7 103.3 102.1 0.12 11.66 

0.1 mg/kg/day 91.9 100.8 96.4 0.20 30.65 

0.5 mg/kg/day 72.5*& 97.8 74.1* 0.60 125.80 

1.0 mg/kg/day 47.4* 76.4* 56.2* 1.69 251.75 

3.5 mg/kg/day 10.3* 36.6* 24.8* 8.83 950.75 

CPF = chlorpyrifos; TCP = trichloropyridinol; <LLQ = Less than the lowest level quantified 

* Significantly different from controls at alpha = 0.05 using Dunnett’s test when raw ChE data 

were analyzed (male and female pups were analyzed separately, but are combined in this table; 

treatment-related effects shaded) 

& Only males were statistically identified 

The response of pups and adults to chlorpyrifos-oxon was similar with significant 

decreases in RBC and plasma ChE at 0.5 mg/kg/day in both age groups, although adults 

had 2X the levels of TCP as pups (Summary Table 4). ChE was not inhibited in either of 

these tissues at 0.01 mg/kg/day in either adults or pups. Brain ChE was not altered at 

either dose of chlorpyrifos-oxon; thus, the 0.01 mg/kg/day chlorpyrifos-oxon dose was 

considered a no-observed-effect-level (NOEL) for ChE inhibition across all tissues in 

both age groups with repeated exposure. 
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Summary Table 4. ChE Inhibition in PND 11-21 Pups and Adult (~PND 70-80) 

Females Following Repeated Doses of Chlorpyrifos-Oxon in Corn Oil 

ADULTS – 

CPF-O in Corn 

Oil 

Dose 

RBC ChE 

(% of 

Control 

Activity) 

Brain ChE 

(% of 

Control 

Activity) 

Plasma ChE 

(% of 

Control 

Activity) 

[CPF-O] 

(ng/g) 

[TCP] 

(ng/g) 

0.01 mg/kg/day 106.6 99.0 124.8 <LLQ 4.84 

0.5 mg/kg/day 12.6* 98.4 24.0* <LLQ 300.75 

PND 11 PUPS – 

CPF-O in Corn 

Oil

RBC ChE 

(% of 

Control 

Activity) 

Brain ChE 

(% of 

Control 

Activity) 

Plasma ChE 

(% of 

Control 

Activity) 

[CPF-O] 

(ng/g) 

[TCP] 

(ng/g) 

0.01 mg/kg/day 89.0 102.6 100.6 <0.102 7.32 

0.5 mg/kg/day 14.8* 95.6 38.7* <LLQ 144.00 

CPF-O = chlorpyrifos-oxon; TCP = trichloropyridinol; <LLQ = Less than the lowest level 

quantified 

* Significantly different from controls at alpha = 0.05 using Dunnett’s test when raw ChE data 

were analyzed (male and female pups were analyzed separately, but are combined in this table; 

treatment-related effects shaded) 

Overall, both the acute and repeated-dose data collected in this study support the 

conclusion that young pups (PND 11, and PND 11-21) are not more sensitive to ChE 

inhibition over the lower portion of the dose-response curves. These data suggest that 

infants and children would not be more sensitive to chlorpyrifos-induced ChE inhibition 

at environmentally relevant doses.