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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 AF TER 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 48674THE DOW CHEMICAL COMPANY PAGE 2 OF 7



### SUMMARY
In the acute comparative cholinesterase study, initial studies were conducted to determine the time-of-peak inhibition for red blood ce ll (RBC), brain and plasma cholinesterase (ChE) in pups and adults. Groups of 4-6 P ND 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 comp are the impact of vehi cle on ChE inhibition. Similarly, adult female CD rats (4/group) we re administered a si ngle gavage dose (corn oil vehicle) of 0 or 10 mg/kg chlorpyrif os 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 inhibi tion 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 andadults, 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 C PF in corn oil and CPF in milk only) and ChE inhibition was determined using a m odified Ellman method. There were no
treatment-related clinical obs ervations in either pups or adults using these dosing scenarios. Using experimental data, pub lished literature and physiologically-based pharmacokinetic/pharmacodynamic (PBPK/PD) m odel, 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 chlo rpyrifos in corn oil, 4 h post-dosing for chlorpyrifos-oxon in corn oil, and 8 h post-e xposure for chlorpyrifos in diet (i.e., after conclusion of the 12-h dietary exposur e to chlorpyrifos-containing diet).
The definitive acute compara tive 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 Ch E inhibition at doses > 2 mg/kg (Summary Table 1). TheTHE DOW CHEMICAL COMPANY PAGE 3 OF 7



### 0.5 mg/kg chlorpyrifos dose was considered a no-observed-effect-lev el (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 a nd its metabolites; however, pups are equally
sensitive or less sensitive than adults at lowe r chlorpyrifos levels (e.g., 0.5 mg/kg). Thus, there was no evidence of age-related differe ntial 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 pu ps were analyzed separately, but are combined in this table; treatment-related effects shaded)



### For the definitive acute comparative cholin esterase study with chlorpyrifos-oxon, ChE activity was measured in PND 11 male and fe male CD rat pups (8/sex/dose) and adult female CD rats (8/dose) exposed to a si ngle 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 activ ity in male or female PND 11 pups. There was no significant inhibition of brain ChE in e ither pups or adults at any dose level of chlorpyrifos-oxon (Summary Table 2). RBC Ch E inhibition was seen at the same dose level (0.5 mg/kg) in PND 11 pups and adu lts, but PND 11 pups exhibited significant plasma ChE inhibition at a slig htly lower dose than adults ( 0.1 mg/kg), a finding that mayTHE DOW CHEMICAL COMPANY PAGE 4 OF 7



### 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; ch lorpyrifos-oxon was be low the lower limit of quantitation in pups and a dults). 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 female s with acute exposure.

Summary Table 2. ChE Inhibition in P ND 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 = trichloropyridino l; <LLQ = Less than the lowest level quantified * Significantly different from controls at alpha = 0.05 using Dunnett’s test when raw ChE data wereanalyzed (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 chlorpyrif os treatment. Compared to corn oil, the maximum ChE inhibition at the time-of-peak effect for chlorpyr ifos was slightly later in milk (8 h) than corn oil (6 h). At the time-of-peak inhibi tion, 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 ch lorpyrifos at doses of 0, 0.05, 0.1, 0.5, 2 or 10THE DOW CHEMICAL COMPANY PAGE 5 OF 7



### mg/kg and acute ChE inhibition was compared to levels seen in adult females exposed to the same chlorpyrifos doses by gavage in co rn oil. With a 12 h dietary exposure to chlorpyrifos, adult females exhibited signi ficant brain ChE inhi bition at the same concentration as chlorpyrifos given via gava ge in corn oil (10 mg/kg); however, the magnitude of brain ChE inhibition was le ss 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 us ing the dietary exposure scenario. In both dosing scenarios, ChE inhibition was not signif icantly altered in a ny 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) fema le 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 duri ng the exposure period. Neurotoxicity was assessed at the time-of-peak ChE inhibition after the 10 th 

### exposure by performing a functional observational battery and examin ing 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 neurobe havioral toxicity measures in eith er 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 ad ults at any dose of chlorpyrifos or chlorpyrifos-oxon during the treatment peri ods. There were no discernable gender differences in ChE activity in male or fe male PND 11 pups in response to repeated chlorpyrifos treatment. At the high dos e (3.5 mg/kg/day), pups had similar ChE
inhibition to adults across all tissues, despite having hi gher 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. pu ps (6 h) relative to the proposed C max 

### for gavage chlorpyrifos in corn oil (2-3 h) and a some what higher metabolic rate in adults. At 1 mg/kg/day, ChE was significantly de creased in both pups and adu lts across all tissues. At 0.5 mg/kg/day, there was no signifi cant 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 dosesTHE DOW CHEMICAL COMPANY PAGE 6 OF 7



### decreased below 1 mg/kg/day, there was no evid ence 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 pu ps 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 chlor pyrifos-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 Ta ble 4). ChE was not i nhibited in either of these tissues at 0.01 mg/kg/day in either adul ts 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.THE DOW CHEMICAL COMPANY PAGE 7 OF 7



### 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 = trichloropy ridinol; <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 pu ps were analyzed separately, but are combined in this table; treatment-related effects shaded)



### Overall, both the acute and repeated-dose data collected in th is 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 sens itive to chlorpyrifos-induced ChE inhibition at environmentally relevant doses.