## STUDY TITLE

A POSITIVE CONTROL ACUTE NEUROTOXICITY STUDY IN RATS

STUDY NUMBER

WIL-99443

STUDY DIRECTOR
Mark T. Herberth, BS, LAT

STUDY INITIATION DATE

2 November 2007

STUDY COMPLETION DATE
5 September 2008

PERFORMING LABORATORY

WIL Research Laboratories, LLC 1407 George Road Ashland, OH 44805-8946

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## WIL-99443



# S TATEMENT 

# O F 

# N O 

# D ATA 

# C ONFIDENTIALITY 

# C LAIMS



## The purpose of the study was to validate the use of the Kinder Scientific MotorMonitor System in our laboratory and to demonstrate the ability to detect decreases in activity using this system as well as a functional observational battery (FOB). Therefore, this page has been intentionally left blank.

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## WIL-99443



# 1. A BSTRACT 

## The objective of this study was to demonstrate the ability of the Kinder Scientific MotorMonitor System (Kinder Scientific, LLC, Poway, CA, USA) to detect decreases in activity in response to chlorpyrifos within the U.S. EPA Health Effects Guideline OPPTS 870.6200 (Neurotoxicity Screening Battery) and the Organization of Economic Cooperation and Development (OECD) Guidelines for Testing of Chemicals Guideline 424 (Neurotoxicity Study in Rodents). In addition, the study evaluated the ability to detect changes in behavior using a functional observational battery (FOB). Chlorpyrifos was administered at a single dose of 20 or 100 mg/kg by oral gavage 3.5 hours prior to behavioral assessment. Male and female Sprague Dawley Crl:CD (SD) rats (6 weeks of age) were evaluated using a Neurotoxicity Screening Battery consisting of FOB assessment and locomotor behavior. The FOB detected a general decrease in the initiation of movement (time to first step in the open field), body temperature and Rotarod performance, along with impaired gait and increased catalepsy time. Motor activity (i.e., total and ambulatory activity) measured by the Kinder Scientific MotorMonitor System showed a consistent decrease in mean activity by both males and females treated with 100 mg/kg chlorpyrifos. The experimental design used in this study demonstrated that WIL Research can detect behavioral changes in an FOB, as well as locomotor effects of chlorpyrifos using the Kinder Scientific MotorMonitor System, and that the results can be considered to validate methodologies for OPPTS Guideline
870.6200 and OECD Guideline 424.

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## WIL-99443



# 2. I NTRODUCTION 

## This study was conducted in accordance with the appropriate provisions of the U.S. EPA Health Effects Guideline (OPPTS 870.6200), Neurotoxicity Screening Battery and the OECD Guidelines for Testing of Chemicals Guideline 424, Neurotoxicity Study in Rodents. The exception is that the study assessments were be completed only during pretest and at the time of peak effect on study day 0. Validation of laboratorymethodologies and demonstration of the sensitivity of the equipment to detect test articlerelated changes are requirements of these guidelines. WIL Research Laboratories, LLC chose to evaluate the use of the Kinder Scientific MotorMonitor System to measure locomotor behavior in control and test article-treated animals using chlorpyrifos, a known cholinesterase inhibitor used commonly in insecticides.
In rodents, a single dose of chlorpyrifos often results in the presence of tremors, increased lacrimation and salivation, loss of muscle tone, hypothermia, changes in gait, increased catalepsy times and inhibited pupil responsiveness (Moser 1995; Mattsson et al., 1996). When evaluated at the time of peak effect (approximately 3.5 hours post-administration) these effects, specifically regarding muscle tone and function, are consistent with the decreased motor activity observed at dose levels of 20, 50 and 100 mg/kg (Moser, 1995). This was also noted in other studies using higher dose levels (50, 100, 150 and 200 mg/kg), administered once by oral gavage, which were sufficient to cause generalized decreases in muscle tone and motor activity for both male and female adult rats (Mattsson et al., 1996). This same trend was also present in low-dose (3 to 12 mg/kg) chronic studies lasting several days to weeks using male and female rats (PND 30 and 40), where motor activity in the open field was inhibited (Carr et al., 2001). Further analysis of chlorpyrifos via alternative delivery routes identified impaired visual discrimination (following oral or subcutaneous administration at 50 mg/kg) and significantly lowered general activity in the behavioral test battery (following subcutaneous administration at 250 mg/kg) for up to 18 consecutive days (Bushnell et al., 2001).

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## WIL-99443



# 3. M ATERIALS 

# A ND 

# M ETHODS 

# 3.1. T EST 

# S PECIES 

# A ND 

# H USBANDRY 

## Sprague Dawley Crl:CD(SD) rats were obtained from Charles River Laboratories, Inc. (Raleigh, North Carolina, USA). 48 males and females (nulliparous and non-pregnant) at 3 to 5 weeks of age were ordered for the in-life testing of 12 rats/sex/group for FOB and motor activity assessment starting at approximately 6 weeks of age. Upon receipt, animals were housed 2-3/cage by sex. Following acclimation, the animals were individually housed in clean suspended wire-mesh cages until euthanasia.
Environmental controls were set to maintain temperature at 71 ± 5o F and relative humidity between 30–70%. Air handling units provided a minimum of 10 fresh air changes per hour. Fluorescent lighting, controlled by timers, provided illumination for a 12-hour light (6:00 a.m. – 6:00 p.m.)/dark photoperiod. Temperature and relative humidity were monitored continuously. Reverse osmosis-purified water and PMI Nutrition International, Inc., Certified Rodent LabDiet ® 

## 5002 were offered ad libitum during the study.



# 3.2. T EST 

# M ATERIAL AND 

# P REPARATION 

## Chlorpyrifos was purchased from Supelco, Bellefonte, PA, USA. An appropriate amount of chlorpyrifos was mixed with the vehicle (corn oil) to produce dosing concentrations of 4 and 20 mg/mL, with a corresponding dose volume of 5 mL/kg. Dosing solutions were prepared daily, stored at room temperature and protected from light prior to dispensation for dosing.



# 3.3. E XPERIMENTAL 

# D ESIGN 

## Functional observational battery assessments and locomotor activity (Kinder Scientific MotorMonitor System) were made at pretest and approximately 3.5 hours post-dose administration to 4 groups of 12 rats/sex/group on the day of chlorpyrifos administration. All rats were observed twice daily for moribundity and mortality, once in the morning and once in the afternoon until euthanasia. Body weight and clinical observations regarding general appearance were recorded on the day of dose administration. The observations included, but were not limited to evaluations for changes in appearance of skin and fur, eyes, mucous membranes, respiratory and circulatory systems, autonomic

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## WIL-99443

and central nervous systems, somatomotor activity and behavior. The following table summarizes the testing paradigm:

Group Number Test Article Dose Level (mg/kg) Number of Males Number of Females 1 Vehicle Control 0 12 12 2 Vehicle Control 0 12 12 3 Chlorpyrifos 20 12 12 4 Chlorpyrifos 100 12 12



# 3.4. B EHAVIORAL 

# T ESTING 

## Twelve rats/sex/group were evaluated in the FOB and the Kinder Scientific MotorMonitor System at pretest and on study day 0. The FOB utilized was based on previously developed protocols which included: home cage, handling, open field, sensory, neuromuscular and physiological observations (Moser et al., 1991; Irwin, 1968; Gad, 1982; Moser et al., 1988; Haggerty, 1989; O'Donoghue, 1989). All animals were observed for the following parameters as described below:



# 3.4.1. H OME 

# C AGE 

# O BSERVATIONS


## Posture Convulsions/tremors Feces consistency Biting Palpebral (eyelid) closure



# 3.4.2. H ANDLING 

# O BSERVATIONS 

## Ease of removal from cage Lacrimation/chromodacryorrhea Piloerection Palpebral closure Eye prominence Red/crusty deposits Ease of handling animal in hand Salivation Fur appearance Respiratory rate/character Mucous membranes/eye/skin color Muscle tone

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## WIL-99443



# 3.4.3. O PEN 

# F IELD 

# O BSERVATIONS 

# (E VALUATED 

# O VER 

# A 2-M INUTE 

# O BSERVATION 

# P ERIOD 

# )

Mobility Rearing Convulsions/tremors Grooming Bizarre/stereotypic behavior Time to first step (seconds) Gait Arousal Urination/defecation Gait score Backing



# 3.4.4. S ENSORY 

# O BSERVATIONS 

## Approach response Startle response Pupil response Forelimb extension Air righting reflex Touch response Tail pinch response Eyeblink response Hindlimb extension Olfactory orientation



# 3.4.5. N EUROMUSCULAR 

# O BSERVATIONS 

## Hindlimb extensor strength Hindlimb foot splay Grip strength-hind and forelimb Rotarod performance



# 3.4.6. P HYSIOLOGICAL 

# O BSERVATIONS 

## Catalepsy Body temperature Body weight

Testing was performed by the same technicians, whenever possible, who did not know the animal’s group assignment. Although data were collected and statistically analyzed for each testing parameter (when necessary) the FOB data presented in this report only include functional domains that are significantly different from the control group and/or were affected by test article administration. Locomotor activity was monitored with the automated Kinder Scientific MotorMonitor System (Kinder Scientific, Poway, CA, USA). The test session was 60 minutes in duration, consisting of 6 subintervals lasting 10 minutes each. Locomotor activity was evaluated as total and ambulatory activity counts obtained over each subinterval (0-10 minutes, 11-20 minutes, 21-30 minutes, 31- 40 minutes, 41-50 minutes and 51-60 minutes) and the entire 60-minute session.



# 3.5. S TATISTICAL 

# A NALYSIS 

## All analyses were two-tailed for minimum significance levels of 5% and 1%. All statistical tests were performed by a computer with appropriate programming as

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## WIL-99443

referenced below. Total and ambulatory counts were analyzed, by sex and session, with a repeated measures analysis of variance (RANOVA). Factors in the model included treatment group (TRT), time interval (TIME), and the interaction of time interval and treatment group (TRT*TIME). The SAS ® 

## procedure PROC MIXED was used for analysis with the random effect of animal included as the repeated measurement. The covariance structure across time was selected by comparing Akaike's Information
Criterion.
Continuous FOB data were subjected to parametric one-way analysis of variance (ANOVA) to determine intergroup differences (Snedecor, 1980). If significant differences were determined by the ANOVA, Dunnett’s test (Dunnett, 1964) was used to compare each control group with the test article-treated groups. Functional Observational Battery parameters yielding scalar or descriptive data were analyzed by Fisher’s Exact Test (Steel and Torrie, 1980).
The monotonic dose-response relationship was evaluated using sequential linear trend tests based on ordinal spacing of dose levels. The linear dose by time interaction was evaluated and, if significant at the 0.05 level, trend tests on treatment means were performed at the 0.05 level for each time interval.
Nonmonotonic dose responses were evaluated whenever no significant linear trends were detected, but TRT and/or TRT*TIME were significant at the 0.01 level. Within the framework of the RANOVA, pairwise comparisons were made for each individual test article-treated group with each control group through linear contrasts. If TRT*TIME was significant, the comparisons were conducted for each time interval. If only the TRT effect was significant, the comparisons were conducted across the pooled time intervals of the entire session. These nonmonotonic dose-response comparisons were conducted at the 0.01 significance level.
All statistical analyses for locomotor activity were conducted by BioSTAT Consultants, Inc., Portage, MI, USA, using Version 9.1 (SAS Institute, Inc., 2002-2003) software.

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## WIL-99443



# 4. R ESULTS 

# A ND 

# D ISCUSSION 

## The FOB performed prior to chlorpyrifos administration (pretest) showed no differences in handling, sensory, neuromuscular and physiological parameters. Increased urination (in the open field; p<0.05 or p<0.01, Table 4) by females assigned to the 20 mg/kg chlorpyrifos groups; as well as a longer time to first step by 20 mg/kg chlorpyrifos treated males (p<0.01), compared to control groups 1 or 2. However, approximately 3.5 hours following dose administration, chlorpyrifos-treated animals exhibited test article-related differences (compared to the control groups) in the home cage, open field, neuromuscular and physiological parameters, while handling and sensory parameters were largely
unaffected. This was not considered to be test article related since the differences were not consistent between control groups 1 and 2.



# 4.1. H OME 

# C AGE AND 

# O PEN 

# F IELD 

# A SSESSMENTS 

## The first two components of the FOB, including home cage and open field assessments identified repetitive movement of the mouth and jaw (1 female each at 20 mg/kg and 100 mg/kg and 2 males at 100 mg/kg; Table 2) in the home cage, which is an expected effect of chlorpyrifos treatment. The open field observations during the FOB test
identified a more robust test article-related effect with an increased time until the initiation of motor behavior, i.e., the first step taken, for both the 100 mg/kg group males and females (Table 4, p<0.01). This increased latency compared to both vehicle control group males and females, indicates that chlorpyrifos treatment prolonged the time to initiation of motor movement and inhibited motor function. This, along with the general decline in mean total and ambulatory activity, further suggests that chlorpyrifos impaired movement. Another aspect of motor movement, gait, was affected by chlorpyrifos administration (Table 4). The numbers of males and females with normal gait were decreased in the 100 mg/kg group (the difference was significant [p<0.01] for males compared to both control groups). Specifically, both sexes showed an increase in the incidence of hunched body posture; splayed or dragging hind limbs were also noted for 1 male, while 2 females were observed walking on tiptoes. These effects on gait are consistent with decreases in motor activity as well as the results of previous research regarding chlorpyrifos and motor behavior (Moser, 1995; Mattsson et al., 1996; Carr et al., 2001; Bushnell et al., 2001). In addition, there was a significant decrease in the

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## WIL-99443

number of rears exhibited by chlorpyrifos treated females (100 mg/kg) compared to control group 1 (Table 4, p<0.05).



# 4.2. N EUROMUSCULAR 

# A SSESSMENT 

## Neuromuscular impairment was particularly evident in males and females receiving the 100 mg/kg dose, where Rotarod performance (Table 6) was significantly decreased compared to both control groups (p<0.05 or p<0.01) and decreased compared to pretest; 1 male and 1 female in the 100 mg/kg group also exhibited slight tremors. This would indicate that there was impaired motor movement and/or balance. Since locomotor activity data also showed a decline in mean total and ambulatory activity, chlorpyrifos was indeed acting to impair motor function. In females, there was an apparent increase in forelimb grip strength (Table 6) at the 20 mg/kg dose (p<0.05); although not significant, this same group of females also had the greatest grip strength during the pretest evaluations (Table 5). The FOB data regarding impaired motor movement/balance and decreased activity are supported by published studies addressing neuromuscular impairments and chlorpyrifos (Moser 1995; Mattsson et al., 1996).



# 4.3. P HYSIOLOGICAL 

# A SSESSMENT 

## Physiological parameters, specifically catalepsy and body temperature, were also affected by chlorpyrifos treatment in males and/or females (Table 8). Catalepsy time (the length of time spent motionless) was significantly increased in 100 mg/kg group males (p<0.05). The increased catalepsy time corresponds to the previously discussed impairments in the initiation and overall motor behaviors examined. Along with the increased catalepsy time, there were significantly decreased body temperatures (p<0.01) for males and females in the 100 mg/kg group. The results are consistent with similar doses used in published research, which also show increased catalepsy time and decreased body temperature (Moser 1995; Mattsson et al., 1996; Timofeeva and Gordon, 2002).



# 4.4. M OTOR 

# A CTIVITY 

## The motor activity data over all 6 subintervals measured by the Kinder Scientific MotorMonitor System in the pretest male and female groups show that there was no difference in mean total and ambulatory activity prior to chlorpyrifos administration (Figure 1A and 2A). Examining the total and ambulatory activity during the

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## WIL-99443

6 subintervals also shows no difference prior to chlorpyrifos treatment in pretest males and females (Figure 3A and B, Figure 4A and B). In addition, there was an habituation profile present for both males and females over the 6 subintervals prior to chlorpyrifos administration. These similarities were expected, as there was no test article
administered.
There was a significant decrease (p<0.01 or p<0.001) in mean total and ambulatory activity counts compared to the vehicle control groups following delivery of 100 mg/kg chlorpyrifos to males and females and following delivery of 20 mg/kg chlorpyrifos to females (Figure 1B, Figure 2B) approximately 3.5 hours following chlorpyrifos delivery. With respect to the individual subintervals of total and ambulatory activity, there were similar decreases in activity following chlorpyrifos treatment over the first 2 subintervals (1-10 minutes and 11-20 minutes) in males at 100 mg/kg and females at 20 and 100 mg/kg (Figure 5A and B, Figure 6A and B). These decreases in activity also followed a dose-dependent profile with the highest level of chlorpyrifos (100 mg/kg) resulting in the lowest overall activity. Females appeared to be more sensitive to chlorpyrifos treatment, as both the low and high doses (20 and 100 mg/kg, respectively) resulted in decreased activity, similar to behavioral results observed in the FOB.
Because there were no differences in motor activity prior to chlorpyrifos administration, the decreased total and ambulatory activity can be attributed to chlorpyrifos. These decreases in motor activity are also consistent with the results of previous studies at similar dose levels of chlorpyrifos (Moser 1995; Mattsson et al., 1996; Carr et al., 2001; Bushnell et al., 2001).

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## WIL-99443



# 5. C ONCLUSION 

## In conclusion, the data generated using the functional observational battery and Kinder Scientific MotorMonitor System in male and female Sprague Dawley Crl:CD (SD) rats are consistent with the cited effects of chlorpyrifos on motor behavior. These procedures are therefore capable and sensitive enough to detect reductions in motor function with the positive control agent, chlorpyrifos, and can be considered to validate methodologies for
OPPTS Guideline 870.6200 and OECD Guideline 424.

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## WIL-99443



# 6. R EFERENCES 

## Bushnell, P.J.; Moser, V.C.; Samsam, T.E. Comparing cognitive and screening tests for neurotoxicity: Effects of acute chlorpyrifos on visual signal detection and a neurobehavioral test battery in rats. Neurotoxicology and Teratology 2001 , 23 , 33- 44. Carr, R.L.; Chambers, H.W.; Guarisco, J.A.; Richardson, J.R.; Tang, J.; Chambers, J.E. Effects of repeated oral postnatal exposure to chlorpyrifos on open-field behavior in juvenile rats. Toxicological Sciences 2001 , 59 , 260-267. Dunnett, C.W. New tables for multiple comparisons with a control. Biometrics 1964 , 20, 482-491. Gad, S.C. A neuromuscular screen for use in industrial toxicology. Journal of Toxicology and Environmental Health

. 1982 , 9 , 691-704. Haggerty, G.C. Development of Tier I neurobehavioral testing capabilities for incorporation into pivotal rodent safety assessment studies. Journal of American College of Toxicology 1989, 8 , 53-69. Irwin, S. Comprehensive observational assessment: Ia. A systematic quantitative procedure for assessing the behavioral physiological state of the mouse. Psychopharmacologia 1968 , 13 , 222-257.
Mattsson, J.L.; Wilmer J.W.; Shankar, M.R.; Berdasco, N.M.; Crissman, J.W.; Maurissen, J.P.; Bond D.M. (1996) Single-dose and 13-week repeated dose neurotoxicity screening studies of chlorpyrifos insecticide. Food and Chemical Toxicology 1996 , 34 , 393-405. Moser, V.C.; McCormick, J.P.; Creason, J.P.; MacPhail, R.C. Comparison of , chlordimeform and carbaryl using a functional observational battery. Fundamentals of Applied Toxicology 1988 , 11 , 189-206. Moser, V.C.; McDaniel, K.L.; Phillips, P.M. Rat strain and stock comparisons using a functional observational battery: Baseline values and effects of Amitraz Toxicology and Applied Pharmacology 1991 , 108 , 267-283. Moser, V.C. Comparisons of the acute effects of cholinesterase inhibitors using a neurobehavioral screening battery in rats. Neurotoxicology and Teratology 1995 , 17 , 617-625. O'Donoghue, J.L. Screening for neurotoxicity using a neurologically based examination and neuropathology. Journal of American College of Toxicology 1989 , 8 , 97-116.
SAS Institute, Inc. SAS ® 

## Proprietary Software Release, Version 9.1, SAS Institute, Inc.: Cary, NC, 2002-2003.

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## WIL-99443

Snedecor, G.W.; Cochran, W.G. One Way Classifications; Analysis of Variance. In Statistical Methods, 7th ed.; The Iowa State University Press: Ames, IA, 1980 , 215-237 Steel R.G.D; Torrie, J.H. Principles and Procedures of Statistics, A Biometrical Approach, Second Edition. McGraw-Hill Book Company, New York, NY, 1980 , pp. 471-472. Timofeeva, O.A.; Gordon, C.G. EEG spectra, behavioral states and motor activity in rats exposed to acetylcholinesterase inhibitor chlorpyrifos. Pharmacology Biochemistry and Behavior 2002 , 72 , 669-679.

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## WIL-99443



# 7. 

# KEY STUDY PERSONNEL AND 

# REPORT SUBMISSION Report Submitted By* 

# r!

Mark T. Herberth, BS, LAT Group Supervisor, Neurosciences Study Director
Report Prepared 

# By: 

# sad& 72 Jonathan D. Toot, PhD Staff Toxicologist, Neurosciences
Report Reviewed By:

Melissa J. Beck, PhD Assistant Director, Neurosciences Study Method Assessor Date

Project Specialist, Neurosciences



## - 15 -

## WIL-99443

FIGURES 1-6

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## WIL-99443

Pretest Mean Total Activity For Males and Females (Prior to Chlorpyrifos Treatment)

Gender Male FemaleActivity Counts (# of beam breaks)

0 500 1000 1500 2000 2500 3000 3500 Vehicle Control Group 1 Vehicle Control Group 2 Chlorpyrifos 20 mg/kg Chlorpyrifos 100 mg/kg 

# A

Study Day 0 Mean Total Activity For Males and Females (Following Chlorpyrifos Treatment)

Gender Male FemaleActivity Counts (# of beam breaks)

0 500 1000 1500 2000 2500 3000 3500 

# B

***

xxx xx

***

xx



## FIGURE 1. Mean total activity counts over all subintervals for (A) males and females at pretest and (B) males and females on Study Day 0, expressed as mean ± SEM. Significance between the treatment groups and vehicle control group 1 is indicated by *** 

## p<0.001 and between the treatment and vehicle control group 2 is indicated by xx 

## p<0.01 and xxx 

## p<0.001.

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## WIL-99443

Pretest Mean Ambulatory Activity For Males and Females (Prior to Chlorpyrifos Treatment)

Gender Male FemaleActivity Counts (# of beam breaks)

0 200 400 600 800 1000
Vehicle Control Group 1 Vehicle Control e Group 2 Chlorpyrifos 20 mg/kg Chlorpyrifos 100 mg/kg 

# A

Study Day 0 Mean Ambulatory Activity For Males and Females (Following Chlorpyrios Treatment)

Gender Male FemaleActivity Counts (# of beam breaks)

0 200 400 600 800 1000 

# B

***

xxx *

xxx

***

xxx



## FIGURE 2. Mean ambulatory counts over all subintervals for (A) males and females and (B) males and females on Study Day 0, expressed as mean ± SEM. Significance between the treatment groups and vehicle control group 1 is indicated by * 

## p<0.05 and *** 

## p<0.001 and between the treatment and vehicle control group 2 is indicated by xxx 

## p<0.001.

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## WIL-99443

Pretest Total Activity Counts For Males (Prior to Chlorpyrifos Treatment)

Subinterval 1-10 11-20 21-30 31-40 41-50 51-60Activity Counts (# beam breaks)

0 200 400 600 800 1000 1200 1400 1600 Vehicle Control Group 1 Vehicle Control Group 2 Chlorpyrifos 20 mg/kg Chlorpyrifos 100 mg/kg 

# A

Pretest Total Activity Counts For Females (Prior to Chlorpyrifos Treatment)

Subinterval 1-10 11-20 21-30 31-40 41-50 51-60Activity Counts (# beam breaks)

0 200 400 600 800 1000 1200 1400 1600 

# B

FIGURE 3. Total activity counts over each subinterval for (A) males at pretest and (B) pretest females at pretest, expressed as mean ± SEM

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## WIL-99443

Pretest Ambulatory Activity Counts For Males (Prior to Chlorpyrifos Treatment)

Subinterval 1-10 11-20 21-30 31-40 41-50 51-60Activity Counts (# beam breaks)

0 100 200 300 400 500 600

Vehicle Control Group 1 Vehicle Control Group 2 Chlorpyrifos 20 mg/kg Chlorpyrifos 100 mg/kg 

# A

Pretest Ambulatory Activity Counts For Females (Prior to Chlorpyrifos Treatment)

Subinterval 1-10 11-20 21-30 31-40 41-50 51-60Activity Counts (# of beam breaks)

0 100 200 300 400 500 600 

# B

FIGURE 4. Total ambulatory activity counts over each subinterval for (A) males at pretest and (B) females at pretest, expressed as means ± SEM

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## WIL-99443

Study Day 0 Total Activity Counts For Males (Following Chlorpyrifos Treatment)

Subinterval 1-10 11-20 21-30 31-40 41-50 51-60Activity Counts (# beam breaks)

0 200 400 600 800 1000 1200 1400 1600

Vehicle Control Group 1 Vehicle Control Group 2 Chlorpyrifos 20 mg/kg Chlorpyrifos 100 mg/kg 

# A

Study Day 0 Activity Counts For Females (Following Chlorpyrifos Treatment)

Trial Block 1-10 11-20 21-30 31-40 41-50 51-60Activity Counts (# beam breaks)

0 200 400 600 800 1000 1200 1400 1600 

# B

FIGURE 5. Total activity counts over each subinterval for (A) males on Study Day 0 and (B) females on Study Day 0, expressed as mean ± SEM

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## WIL-99443

Study Day 0 Ambulatory Activity Counts For Males (Following Chlorpyrifos Treatment)

Subinterval 1-10 11-20 21-30 31-40 41-50 51-60Activity Counts (# of beam breaks)

0 100 200 300 400 500 600
Vehicle Control Group 1 Vehicle Control Group 2 Chlorpyrifos 20 mg/kg Chlorpyrifos 100 mg/kg 

# A

Study Day 0 Ambulatory Activity Counts For Females (Following Chlorpyrifos Treatment)

Subinterval 1-10 11-20 21-30 31-40 41-50 51-60Activity Counts (# of beam breaks)

0 100 200 300 400 500 600 

# B

FIGURE 6. Total ambulatory activity counts over each subinterval for (A) males on Study Day 0 and (B) females on Study Day 0, expressed as mean ± SEM

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## WIL-99443



# FOB S UMMARY 

# T ABLES



## - 23 -TABLE 1 (HOME CAGE OBSERVATIONS - PRETEST EVALUATION) PROJECT NO.:WIL-99443 POSITIVE CONTROL NT STUDY IN RATS USING CHLORPYRIFOS PAGE 1 SPONSOR:WIL RESEARCH SUMMARY OF FUNCTIONAL OBSERVATIONAL BATTERY DATA PRETEST

HOME CAGE OBSERVATIONS MALES NUMBER OF ANIMALS SHOWING EACH OBSERVATION

----------------------------------------------------------------------------------------------------------------------------------- GROUP: 1 2 3 4

----------------------------------------------------------------------------------------------------------------------------------- NUMBER TESTED: 12 12 12 12

POSTURE SITTING OR STANDING NORMALLY 7 7 7 6 ASLEEP, LYING ON SIDE OR CURLED UP 5 5 5 6

CONVULSIONS - CLONIC ABSENT 12 12 12 12

CONVULSIONS - TONIC ABSENT 12 12 12 12

TREMORS NONE 12 12 12 12

BITING NONE 12 12 12 12

PALPEBRAL CLOSURE EYELIDS WIDE OPEN 7 8 6 6 EYELIDS SLIGHTLY DROOPING 0 0 1 0 DROOPING EYELIDS (HALF-CLOSED) 1 0 1 0 EYELIDS COMPLETELY SHUT 4 4 4 6

FECES CONSISTENCY PELLETS 11 10 8 9 PELLETS ABSENT 1 2 4 3

----------------------------------------------------------------------------------------------------------------------------------- 1- 0 MG/KG 2- 0 MG/KG 3- 20 MG/KG 4- 100 MG/KG NONE SIGNIFICANTLY DIFFERENT FROM THE CONTROLS USING FISHER’S EXACT TEST-24-TABLE 1 (HOME CAGE OBSERVATIONS - PRETEST EVALUATION) PROJECT NO.:WIL-99443 POSITIVE CONTROL NT STUDY IN RATS USING CHLORPYRIFOS PAGE 2 SPONSOR:WIL RESEARCH SUMMARY OF FUNCTIONAL OBSERVATIONAL BATTERY DATA PRETEST

HOME CAGE OBSERVATIONS FEMALES NUMBER OF ANIMALS SHOWING EACH OBSERVATION

----------------------------------------------------------------------------------------------------------------------------------- GROUP: 1 2 3 4

----------------------------------------------------------------------------------------------------------------------------------- NUMBER TESTED: 12 12 12 12

POSTURE SITTING OR STANDING NORMALLY 5 6 5 7 ASLEEP, LYING ON SIDE OR CURLED UP 6 6 7 5 ALERT, ORIENTED TOWARD OBSERVER 1 0 0 0

CONVULSIONS - CLONIC ABSENT 12 12 12 12

CONVULSIONS - TONIC ABSENT 12 12 12 12

TREMORS NONE 12 12 12 12

BITING NONE 12 12 12 12

PALPEBRAL CLOSURE EYELIDS WIDE OPEN 6 6 6 7 DROOPING EYELIDS (HALF-CLOSED) 0 0 1 1 EYELIDS COMPLETELY SHUT 6 6 5 4

FECES CONSISTENCY PELLETS 9 8 10 8 PELLETS ABSENT 3 4 2 4

----------------------------------------------------------------------------------------------------------------------------------- 1- 0 MG/KG 2- 0 MG/KG 3- 20 MG/KG 4- 100 MG/KG NONE SIGNIFICANTLY DIFFERENT FROM THE CONTROLS USING FISHER’S EXACT TEST PFOBSUv4.29 05/15/2008 R:05/15/2008-25-TABLE 2 (HOME CAGE OBSERVATIONS - DAY 0 EVALUATION) PROJECT NO.:WIL-99443 POSITIVE CONTROL NT STUDY IN RATS USING CHLORPYRIFOS PAGE 1 SPONSOR:WIL RESEARCH SUMMARY OF FUNCTIONAL OBSERVATIONAL BATTERY DATA DAY 0

HOME CAGE OBSERVATIONS MALES NUMBER OF ANIMALS SHOWING EACH OBSERVATION

----------------------------------------------------------------------------------------------------------------------------------- GROUP: 1 2 3 4

----------------------------------------------------------------------------------------------------------------------------------- NUMBER TESTED: 12 12 12 12

POSTURE SITTING OR STANDING NORMALLY 2 7 7 9a ASLEEP, LYING ON SIDE OR CURLED UP 10 5 5 1b SITTING, HEAD HELD LOW 0 0 0 2

CONVULSIONS - CLONIC ABSENT 12 12 12 10 REPETITIVE MOVEMENT OF MOUTH AND JAWS 0 0 0 2

CONVULSIONS - TONIC ABSENT 12 12 12 12

TREMORS NONE 12 12 12 12

BITING NONE 12 12 12 12

PALPEBRAL CLOSURE EYELIDS WIDE OPEN 2 8 7 10b EYELIDS SLIGHTLY DROOPING 0 0 0 1 EYELIDS COMPLETELY SHUT 10 4 5 1b

FECES CONSISTENCY PELLETS 3 7 5 9a PELLETS ABSENT 9 5 7 3a

----------------------------------------------------------------------------------------------------------------------------------- 1- 0 MG/KG 2- 0 MG/KG 3- 20 MG/KG 4- 100 MG/KG a = SIGNIFICANTLY DIFFERENT FROM CONTROL GROUP 1 AT 0.05 USING FISHER’S EXACT TEST b = SIGNIFICANTLY DIFFERENT FROM CONTROL GROUP 1 AT 0.01 USING FISHER’S EXACT TEST c = SIGNIFICANTLY DIFFERENT FROM CONTROL GROUP 2 AT 0.05 USING FISHER’S EXACT TEST-26-TABLE 2 (HOME CAGE OBSERVATIONS - DAY 0 EVALUATION) PROJECT NO.:WIL-99443 POSITIVE CONTROL NT STUDY IN RATS USING CHLORPYRIFOS PAGE 2 SPONSOR:WIL RESEARCH SUMMARY OF FUNCTIONAL OBSERVATIONAL BATTERY DATA DAY 0

HOME CAGE OBSERVATIONS FEMALES NUMBER OF ANIMALS SHOWING EACH OBSERVATION

----------------------------------------------------------------------------------------------------------------------------------- GROUP: 1 2 3 4

----------------------------------------------------------------------------------------------------------------------------------- NUMBER TESTED: 12 12 12 12

POSTURE SITTING OR STANDING NORMALLY 4 8 8 11b ASLEEP, LYING ON SIDE OR CURLED UP 8 4 3 1b ALERT, ORIENTED TOWARD OBSERVER 0 0 1 0

CONVULSIONS - CLONIC ABSENT 12 12 11 11 REPETITIVE MOVEMENT OF MOUTH AND JAWS 0 0 1 1

CONVULSIONS - TONIC ABSENT 12 12 12 12

TREMORS NONE 12 12 12 12

BITING NONE 12 12 12 11 BITING OF SELF 0 0 0 1

PALPEBRAL CLOSURE EYELIDS WIDE OPEN 4 8 9 10a EYELIDS SLIGHTLY DROOPING 1 0 0 1 DROOPING EYELIDS (HALF-CLOSED) 1 0 1 0 EYELIDS COMPLETELY SHUT 6 4 2 1

FECES CONSISTENCY PELLETS 6 5 5 7 PELLETS ABSENT 6 7 7 5

----------------------------------------------------------------------------------------------------------------------------------- 1- 0 MG/KG 2- 0 MG/KG 3- 20 MG/KG 4- 100 MG/KG a = SIGNIFICANTLY DIFFERENT FROM CONTROL GROUP 1 AT 0.05 USING FISHER’S EXACT TEST b = SIGNIFICANTLY DIFFERENT FROM CONTROL GROUP 1 AT 0.01 USING FISHER’S EXACT TEST PFOBSUv4.29 05/15/2008 R:07/25/2008-27-TABLE 3 (OPEN FIELD OBSERVATIONS - PRETEST EVALUATION) PROJECT NO.:WIL-99443 POSITIVE CONTROL NT STUDY IN RATS USING CHLORPYRIFOS PAGE 1 SPONSOR:WIL RESEARCH SUMMARY OF FUNCTIONAL OBSERVATIONAL BATTERY DATA PRETEST

OPEN FIELD OBSERVATIONS MALES NUMBER OF ANIMALS SHOWING EACH OBSERVATION

----------------------------------------------------------------------------------------------------------------------------------- GROUP: 1 2 3 4

----------------------------------------------------------------------------------------------------------------------------------- NUMBER TESTED: 12 12 12 12
TIME TO FIRST STEP (seconds) : MEAN 0.4 0.4 0.6g 0.5 S.D. 0.14 0.09 0.40 0.14

MOBILITY NORMAL 12 12 12 12

GAIT NORMAL 12 12 12 12

CONVULSIONS - CLONIC ABSENT 12 12 12 12

CONVULSIONS - TONIC ABSENT 12 12 12 12

TREMORS NONE 12 12 12 12

GAIT SCORE NORMAL 12 12 12 12

AROUSAL NORMAL: ALERT, EXPLORATORY MOVEMENTS 12 12 12 12

BIZARRE/STEREOTYPIC BEHAVIOR NONE 12 12 12 12

----------------------------------------------------------------------------------------------------------------------------------- 1- 0 MG/KG 2- 0 MG/KG 3- 20 MG/KG 4- 100 MG/KG g = SIGNIFICANTLY DIFFERENT FROM CONTROL GROUP 2 AT 0.05 USING DUNNETT’S TEST NONE SIGNIFICANTLY DIFFERENT FROM THE CONTROLS USING FISHER’S EXACT TEST-28-TABLE 3 (OPEN FIELD OBSERVATIONS - PRETEST EVALUATION) PROJECT NO.:WIL-99443 POSITIVE CONTROL NT STUDY IN RATS USING CHLORPYRIFOS PAGE 2 SPONSOR:WIL RESEARCH SUMMARY OF FUNCTIONAL OBSERVATIONAL BATTERY DATA PRETEST

OPEN FIELD OBSERVATIONS MALES NUMBER OF ANIMALS SHOWING EACH OBSERVATION

----------------------------------------------------------------------------------------------------------------------------------- GROUP: 1 2 3 4

----------------------------------------------------------------------------------------------------------------------------------- NUMBER TESTED: 12 12 12 12

REARING : MEAN 5.1 4.4 4.7 6.5 S.D. 2.64 2.50 2.50 2.20

BACKING : MEAN 0.0 0.0 0.0 0.0 S.D. 0.00 0.00 0.00 0.00

GROOMING : MEAN 0.3 0.3 0.1 0.5 S.D. 0.65 0.45 0.29 0.80

URINATION : MEAN 0.7 0.3 0.5 0.6 S.D. 0.65 0.49 0.52 0.67

DEFECATION : MEAN 0.3 0.3 0.3 0.7 S.D. 0.89 0.49 0.62 1.37

----------------------------------------------------------------------------------------------------------------------------------- 1- 0 MG/KG 2- 0 MG/KG 3- 20 MG/KG 4- 100 MG/KG NONE SIGNIFICANTLY DIFFERENT FROM THE CONTROLS USING DUNNETT’S TEST-29-TABLE 3 (OPEN FIELD OBSERVATIONS - PRETEST EVALUATION) PROJECT NO.:WIL-99443 POSITIVE CONTROL NT STUDY IN RATS USING CHLORPYRIFOS PAGE 3 SPONSOR:WIL RESEARCH SUMMARY OF FUNCTIONAL OBSERVATIONAL BATTERY DATA PRETEST

OPEN FIELD OBSERVATIONS FEMALES NUMBER OF ANIMALS SHOWING EACH OBSERVATION

----------------------------------------------------------------------------------------------------------------------------------- GROUP: 1 2 3 4

----------------------------------------------------------------------------------------------------------------------------------- NUMBER TESTED: 12 12 12 12
TIME TO FIRST STEP (seconds) : MEAN 0.5 0.5 0.4 0.4 S.D. 0.26 0.14 0.14 0.20

MOBILITY NORMAL 12 12 12 12

GAIT NORMAL 12 12 12 12

CONVULSIONS - CLONIC ABSENT 12 12 12 12

CONVULSIONS - TONIC ABSENT 12 12 12 12

TREMORS NONE 12 12 12 12

GAIT SCORE NORMAL 12 12 12 12

AROUSAL NORMAL: ALERT, EXPLORATORY MOVEMENTS 12 12 12 12

BIZARRE/STEREOTYPIC BEHAVIOR NONE 12 12 12 12

----------------------------------------------------------------------------------------------------------------------------------- 1- 0 MG/KG 2- 0 MG/KG 3- 20 MG/KG 4- 100 MG/KG NONE SIGNIFICANTLY DIFFERENT FROM THE CONTROLS USING FISHER’S EXACT TEST NONE SIGNIFICANTLY DIFFERENT FROM THE CONTROLS USING DUNNETT’S TEST-30-TABLE 3 (OPEN FIELD OBSERVATIONS - PRETEST EVALUATION) PROJECT NO.:WIL-99443 POSITIVE CONTROL NT STUDY IN RATS USING CHLORPYRIFOS PAGE 4 SPONSOR:WIL RESEARCH SUMMARY OF FUNCTIONAL OBSERVATIONAL BATTERY DATA PRETEST

OPEN FIELD OBSERVATIONS FEMALES NUMBER OF ANIMALS SHOWING EACH OBSERVATION

----------------------------------------------------------------------------------------------------------------------------------- GROUP: 1 2 3 4

----------------------------------------------------------------------------------------------------------------------------------- NUMBER TESTED: 12 12 12 12

REARING : MEAN 6.2 7.5 5.9 6.6 S.D. 3.24 3.61 2.15 3.70

BACKING : MEAN 0.0 0.0 0.0 0.0 S.D. 0.00 0.00 0.00 0.00

GROOMING : MEAN 0.2 0.1 0.3 0.3 S.D. 0.39 0.29 0.62 0.87

URINATION : MEAN 0.2 0.1 0.8eh 0.2 S.D. 0.39 0.29 0.62 0.39

DEFECATION : MEAN 0.2 0.0 0.2 0.0 S.D. 0.39 0.00 0.39 0.00

----------------------------------------------------------------------------------------------------------------------------------- 1- 0 MG/KG 2- 0 MG/KG 3- 20 MG/KG 4- 100 MG/KG e = SIGNIFICANTLY DIFFERENT FROM CONTROL GROUP 1 AT 0.05 USING DUNNETT’S TEST h = SIGNIFICANTLY DIFFERENT FROM CONTROL GROUP 2 AT 0.01 USING DUNNETT’S TEST

PFOBSUv4.29 05/15/2008 R:05/15/2008-31-TABLE 4 (OPEN FIELD OBSERVATIONS - DAY 0 EVALUATION) PROJECT NO.:WIL-99443 POSITIVE CONTROL NT STUDY IN RATS USING CHLORPYRIFOS PAGE 1 SPONSOR:WIL RESEARCH SUMMARY OF FUNCTIONAL OBSERVATIONAL BATTERY DATA DAY 0

OPEN FIELD OBSERVATIONS MALES NUMBER OF ANIMALS SHOWING EACH OBSERVATION

----------------------------------------------------------------------------------------------------------------------------------- GROUP: 1 2 3 4

----------------------------------------------------------------------------------------------------------------------------------- NUMBER TESTED: 12 12 12 12
TIME TO FIRST STEP (seconds) : MEAN 0.5 0.5 0.6 1.7fh S.D. 0.13 0.21 0.11 1.17

MOBILITY NORMAL 12 12 12 12

GAIT NORMAL 12 12 12 5bd HINDLIMBS SPLAYED OR DRAGGING, UNABLE TO SUPPORT WEIGHT 0 0 0 1 HUNCHED BODY 0 0 0 7bd

CONVULSIONS - CLONIC ABSENT 12 12 12 12

CONVULSIONS - TONIC ABSENT 12 12 12 12

TREMORS NONE 12 12 12 11 SLIGHT (1.5mm) 0 0 0 1

GAIT SCORE NORMAL 12 12 12 12

AROUSAL NORMAL: ALERT, EXPLORATORY MOVEMENTS 12 12 12 12

----------------------------------------------------------------------------------------------------------------------------------- 1- 0 MG/KG 2- 0 MG/KG 3- 20 MG/KG 4- 100 MG/KG b = SIGNIFICANTLY DIFFERENT FROM CONTROL GROUP 1 AT 0.01 USING FISHER’S EXACT TEST d = SIGNIFICANTLY DIFFERENT FROM CONTROL GROUP 2 AT 0.01 USING FISHER’S EXACT TEST f = SIGNIFICANTLY DIFFERENT FROM CONTROL GROUP 1 AT 0.01 USING DUNNETT’S TEST h = SIGNIFICANTLY DIFFERENT FROM CONTROL GROUP 2 AT 0.01 USING DUNNETT’S TEST-32-TABLE 4 (OPEN FIELD OBSERVATIONS - DAY 0 EVALUATION) PROJECT NO.:WIL-99443 POSITIVE CONTROL NT STUDY IN RATS USING CHLORPYRIFOS PAGE 2 SPONSOR:WIL RESEARCH SUMMARY OF FUNCTIONAL OBSERVATIONAL BATTERY DATA DAY 0

OPEN FIELD OBSERVATIONS MALES NUMBER OF ANIMALS SHOWING EACH OBSERVATION

----------------------------------------------------------------------------------------------------------------------------------- GROUP: 1 2 3 4

----------------------------------------------------------------------------------------------------------------------------------- NUMBER TESTED: 12 12 12 12

BIZARRE/STEREOTYPIC BEHAVIOR NONE 12 12 12 12

REARING : MEAN 1.6 1.5 1.4 1.1 S.D. 2.61 1.88 1.44 1.44

BACKING : MEAN 0.0 0.0 0.0 0.0 S.D. 0.00 0.00 0.00 0.00

GROOMING : MEAN 0.3 0.2 0.0 0.3 S.D. 0.65 0.39 0.00 0.45

URINATION : MEAN 0.4 0.0 0.5 0.4 S.D. 0.67 0.00 0.80 0.51

DEFECATION : MEAN 0.8 1.3 1.0 0.1 S.D. 0.83 1.91 1.48 0.29

----------------------------------------------------------------------------------------------------------------------------------- 1- 0 MG/KG 2- 0 MG/KG 3- 20 MG/KG 4- 100 MG/KG NONE SIGNIFICANTLY DIFFERENT FROM THE CONTROLS USING FISHER’S EXACT TEST NONE SIGNIFICANTLY DIFFERENT FROM THE CONTROLS USING DUNNETT’S TEST-33-TABLE 4 (OPEN FIELD OBSERVATIONS - DAY 0 EVALUATION) PROJECT NO.:WIL-99443 POSITIVE CONTROL NT STUDY IN RATS USING CHLORPYRIFOS PAGE 3 SPONSOR:WIL RESEARCH SUMMARY OF FUNCTIONAL OBSERVATIONAL BATTERY DATA DAY 0

OPEN FIELD OBSERVATIONS FEMALES NUMBER OF ANIMALS SHOWING EACH OBSERVATION

----------------------------------------------------------------------------------------------------------------------------------- GROUP: 1 2 3 4

----------------------------------------------------------------------------------------------------------------------------------- NUMBER TESTED: 12 12 12 12
TIME TO FIRST STEP (seconds) : MEAN 0.5 0.5 0.5 0.9fh S.D. 0.12 0.11 0.08 0.59

MOBILITY NORMAL 12 12 12 12

GAIT NORMAL 12 11 10 8 WALKS ON TIPTOES 0 1 0 2 HUNCHED BODY 0 1 2 4

CONVULSIONS - CLONIC ABSENT 12 12 12 12

CONVULSIONS - TONIC ABSENT 12 12 12 12

TREMORS NONE 12 12 12 11 SLIGHT (1.5mm) 0 0 0 1

GAIT SCORE NORMAL 12 12 12 12

AROUSAL NORMAL: ALERT, EXPLORATORY MOVEMENTS 12 12 12 12

----------------------------------------------------------------------------------------------------------------------------------- 1- 0 MG/KG 2- 0 MG/KG 3- 20 MG/KG 4- 100 MG/KG f = SIGNIFICANTLY DIFFERENT FROM CONTROL GROUP 1 AT 0.01 USING DUNNETT’S TEST h = SIGNIFICANTLY DIFFERENT FROM CONTROL GROUP 2 AT 0.01 USING DUNNETT’S TEST-34-TABLE 4 (OPEN FIELD OBSERVATIONS - DAY 0 EVALUATION) PROJECT NO.:WIL-99443 POSITIVE CONTROL NT STUDY IN RATS USING CHLORPYRIFOS PAGE 4 SPONSOR:WIL RESEARCH SUMMARY OF FUNCTIONAL OBSERVATIONAL BATTERY DATA DAY 0

OPEN FIELD OBSERVATIONS FEMALES NUMBER OF ANIMALS SHOWING EACH OBSERVATION

----------------------------------------------------------------------------------------------------------------------------------- GROUP: 1 2 3 4

----------------------------------------------------------------------------------------------------------------------------------- NUMBER TESTED: 12 12 12 12

BIZARRE/STEREOTYPIC BEHAVIOR NONE 12 12 12 12
REARING : MEAN 3.2 3.8 1.9 1.7g S.D. 2.98 3.35 1.31 1.50

BACKING : MEAN 0.0 0.0 0.0 0.0 S.D. 0.00 0.00 0.00 0.00

GROOMING : MEAN 0.1 0.0 0.0 0.4 S.D. 0.29 0.00 0.00 0.90

URINATION : MEAN 0.1 0.3 0.6 0.1 S.D. 0.29 0.62 0.90 0.29

DEFECATION : MEAN 0.3 0.2 0.3 0.3 S.D. 1.15 0.58 0.65 0.87

----------------------------------------------------------------------------------------------------------------------------------- 1- 0 MG/KG 2- 0 MG/KG 3- 20 MG/KG 4- 100 MG/KG NONE SIGNIFICANTLY DIFFERENT FROM THE CONTROLS USING FISHER’S EXACT TEST g = SIGNIFICANTLY DIFFERENT FROM CONTROL GROUP 2 AT 0.05 USING DUNNETT’S TEST PFOBSUv4.29 05/15/2008 R:05/15/2008-35-TABLE 5 (NEUROMUSCULAR OBSERVATIONS - PRETEST EVALUATION) PROJECT NO.:WIL-99443 POSITIVE CONTROL NT STUDY IN RATS USING CHLORPYRIFOS PAGE 1 SPONSOR:WIL RESEARCH SUMMARY OF FUNCTIONAL OBSERVATIONAL BATTERY DATA PRETEST

NEUROMUSCULAR OBSERVATIONS MALES NUMBER OF ANIMALS SHOWING EACH OBSERVATION

----------------------------------------------------------------------------------------------------------------------------------- GROUP: 1 2 3 4

----------------------------------------------------------------------------------------------------------------------------------- NUMBER TESTED: 12 12 12 12

HINDLIMB EXTENSOR STRENGTH HINDLIMB RESISTANCE PRESENT 12 12 12 12
GRIP STRENGTH (g) - FORE/HINDLIMB FORE: MEAN 384.7 418.1 423.9 422.3 S.D. 66.82 87.88 80.50 42.51 HIND: MEAN 182.7 190.8 207.3 182.2 S.D. 33.98 51.73 61.44 46.11
ROTAROD PERFORMANCE (seconds) : MEAN 60.9 91.4 100.5 83.0 S.D. 44.42 38.59 38.60 43.89
HINDLIMB FOOTSPLAY (mm) : MEAN 54.5 58.6 58.5 61.8 S.D. 13.79 10.00 15.99 12.04

----------------------------------------------------------------------------------------------------------------------------------- 1- 0 MG/KG 2- 0 MG/KG 3- 20 MG/KG 4- 100 MG/KG NONE SIGNIFICANTLY DIFFERENT FROM THE CONTROLS USING FISHER’S EXACT TEST NONE SIGNIFICANTLY DIFFERENT FROM THE CONTROLS USING DUNNETT’S TEST-36-TABLE 5 (NEUROMUSCULAR OBSERVATIONS - PRETEST EVALUATION) PROJECT NO.:WIL-99443 POSITIVE CONTROL NT STUDY IN RATS USING CHLORPYRIFOS PAGE 2 SPONSOR:WIL RESEARCH SUMMARY OF FUNCTIONAL OBSERVATIONAL BATTERY DATA PRETEST

NEUROMUSCULAR OBSERVATIONS FEMALES NUMBER OF ANIMALS SHOWING EACH OBSERVATION

----------------------------------------------------------------------------------------------------------------------------------- GROUP: 1 2 3 4

----------------------------------------------------------------------------------------------------------------------------------- NUMBER TESTED: 12 12 12 12

HINDLIMB EXTENSOR STRENGTH HINDLIMB RESISTANCE PRESENT 12 12 12 12
GRIP STRENGTH (g) - FORE/HINDLIMB FORE: MEAN 382.8 391.8 405.9 383.2 S.D. 55.42 63.42 71.51 88.78 HIND: MEAN 181.9 196.9 200.7 185.6 S.D. 46.04 59.21 57.78 45.55
ROTAROD PERFORMANCE (seconds) : MEAN 92.8 89.0 81.6 84.1 S.D. 38.53 45.94 42.50 39.89
HINDLIMB FOOTSPLAY (mm) : MEAN 55.5 57.6 56.3 61.1 S.D. 9.62 19.84 10.42 12.03

----------------------------------------------------------------------------------------------------------------------------------- 1- 0 MG/KG 2- 0 MG/KG 3- 20 MG/KG 4- 100 MG/KG NONE SIGNIFICANTLY DIFFERENT FROM THE CONTROLS USING FISHER’S EXACT TEST NONE SIGNIFICANTLY DIFFERENT FROM THE CONTROLS USING DUNNETT’S TEST PFOBSUv4.29 05/15/2008 R:05/15/2008-37-TABLE 6 (NEUROMUSCULAR OBSERVATIONS - DAY 0 EVALUATION) PROJECT NO.:WIL-99443 POSITIVE CONTROL NT STUDY IN RATS USING CHLORPYRIFOS PAGE 1 SPONSOR:WIL RESEARCH SUMMARY OF FUNCTIONAL OBSERVATIONAL BATTERY DATA DAY 0

NEUROMUSCULAR OBSERVATIONS MALES NUMBER OF ANIMALS SHOWING EACH OBSERVATION

----------------------------------------------------------------------------------------------------------------------------------- GROUP: 1 2 3 4

----------------------------------------------------------------------------------------------------------------------------------- NUMBER TESTED: 12 12 12 12

HINDLIMB EXTENSOR STRENGTH REDUCED HINDLIMB RESISTANCE, ANIMAL SHOWS SOME WEAKNESS 0 1 0 4 HINDLIMB RESISTANCE PRESENT 12 11 12 8
GRIP STRENGTH (g) - FORE/HINDLIMB FORE: MEAN 506.5 532.1 552.8 661.3e S.D. 123.84 175.17 170.47 88.68 HIND: MEAN 317.7 308.8 298.7 336.4 S.D. 100.42 98.90 67.93 77.86
ROTAROD PERFORMANCE (seconds) : MEAN 87.0 120.0 98.7 41.7eh S.D. 45.07 0.00 34.62 41.13
HINDLIMB FOOTSPLAY (mm) : MEAN 55.1 62.2 56.9 48.6 S.D. 10.47 10.99 14.61 15.29

----------------------------------------------------------------------------------------------------------------------------------- 1- 0 MG/KG 2- 0 MG/KG 3- 20 MG/KG 4- 100 MG/KG e = SIGNIFICANTLY DIFFERENT FROM CONTROL GROUP 1 AT 0.05 USING DUNNETT’S TEST h = SIGNIFICANTLY DIFFERENT FROM CONTROL GROUP 2 AT 0.01 USING DUNNETT’S TEST NONE SIGNIFICANTLY DIFFERENT FROM CONTROLS USING FISHER’S EXACT TEST-38-TABLE 6 (NEUROMUSCULAR OBSERVATIONS - DAY 0 EVALUATION) PROJECT NO.:WIL-99443 POSITIVE CONTROL NT STUDY IN RATS USING CHLORPYRIFOS PAGE 2 SPONSOR:WIL RESEARCH SUMMARY OF FUNCTIONAL OBSERVATIONAL BATTERY DATA DAY 0

NEUROMUSCULAR OBSERVATIONS FEMALES NUMBER OF ANIMALS SHOWING EACH OBSERVATION

----------------------------------------------------------------------------------------------------------------------------------- GROUP: 1 2 3 4

----------------------------------------------------------------------------------------------------------------------------------- NUMBER TESTED: 12 12 12 12

HINDLIMB EXTENSOR STRENGTH REDUCED HINDLIMB RESISTANCE, ANIMAL SHOWS SOME WEAKNESS 0 0 0 3 HINDLIMB RESISTANCE PRESENT 12 12 12 9
GRIP STRENGTH (g) - FORE/HINDLIMB FORE: MEAN 512.2 591.8 622.8e 537.8 S.D. 96.35 85.27 118.23 108.67 HIND: MEAN 308.6 343.7 306.3 307.3 S.D. 107.05 101.68 80.22 49.45
ROTAROD PERFORMANCE (seconds) : MEAN 103.7 113.0 102.3 71.7h S.D. 38.61 20.76 34.06 37.23
HINDLIMB FOOTSPLAY (mm) : MEAN 54.8 59.8 51.2 47.0 S.D. 9.11 19.99 11.95 16.91

----------------------------------------------------------------------------------------------------------------------------------- 1- 0 MG/KG 2- 0 MG/KG 3- 20 MG/KG 4- 100 MG/KG e = SIGNIFICANTLY DIFFERENT FROM CONTROL GROUP 1 AT 0.05 USING DUNNETT’S TEST h = SIGNIFICANTLY DIFFERENT FROM CONTROL GROUP 2 AT 0.01 USING DUNNETT’S TEST NONE SIGNIFICANTLY DIFFERENT FROM CONTROLS USING FISHER’S EXACT TEST

PFOBSUv4.29 05/15/2008 R:05/15/2008-39-TABLE 7 (PHYSIOLOGICAL OBSERVATIONS - PRETEST EVALUATION) PROJECT NO.:WIL-99443 POSITIVE CONTROL NT STUDY IN RATS USING CHLORPYRIFOS PAGE 1 SPONSOR:WIL RESEARCH SUMMARY OF FUNCTIONAL OBSERVATIONAL BATTERY DATA PRETEST

PHYSIOLOGICAL OBSERVATIONS MALES

----------------------------------------------------------------------------------------------------------------------------------- GROUP: 1 2 3 4

----------------------------------------------------------------------------------------------------------------------------------- NUMBER TESTED: 12 12 12 12
CATALEPSY (seconds) : MEAN 0.2 0.2 0.2 0.2 S.D. 0.00 0.00 0.00 0.00 N 12 12 12 12
BODY TEMPERATURE (degrees C) : MEAN 37.8 38.0 37.9 38.0 S.D. 0.26 0.30 0.27 0.33 N 12 12 12 12
BODY WEIGHT (g) : MEAN 135.3 135.8 136.0 135.5 S.D. 29.86 26.22 25.83 20.26 N 12 12 12 12

----------------------------------------------------------------------------------------------------------------------------------- 1- 0 MG/KG 2- 0 MG/KG 3- 20 MG/KG 4- 100 MG/KG NONE SIGNIFICANTLY DIFFERENT FROM THE CONTROLS USING DUNNETT’S TEST-40-TABLE 7 (PHYSIOLOGICAL OBSERVATIONS - PRETEST EVALUATION) PROJECT NO.:WIL-99443 POSITIVE CONTROL NT STUDY IN RATS USING CHLORPYRIFOS PAGE 2 SPONSOR:WIL RESEARCH SUMMARY OF FUNCTIONAL OBSERVATIONAL BATTERY DATA PRETEST

PHYSIOLOGICAL OBSERVATIONS FEMALES

----------------------------------------------------------------------------------------------------------------------------------- GROUP: 1 2 3 4

----------------------------------------------------------------------------------------------------------------------------------- NUMBER TESTED: 12 12 12 12
CATALEPSY (seconds) : MEAN 0.2 0.2 0.2 0.2 S.D. 0.03 0.00 0.00 0.00 N 12 12 12 12
BODY TEMPERATURE (degrees C) : MEAN 37.9 37.9 37.8 37.9 S.D. 0.34 0.31 0.32 0.20 N 12 12 12 12
BODY WEIGHT (g) : MEAN 118.2 114.5 113.7 115.8 S.D. 16.19 12.50 13.12 10.17 N 12 12 12 12

----------------------------------------------------------------------------------------------------------------------------------- 1- 0 MG/KG 2- 0 MG/KG 3- 20 MG/KG 4- 100 MG/KG NONE SIGNIFICANTLY DIFFERENT FROM THE CONTROLS USING DUNNETT’S TEST PFOBSUv4.29 05/15/2008 R:05/15/2008-41-TABLE 8 (PHYSIOLOGICAL OBSERVATIONS - DAY 0 EVALUATION) PROJECT NO.:WIL-99443 POSITIVE CONTROL NT STUDY IN RATS USING CHLORPYRIFOS PAGE 1 SPONSOR:WIL RESEARCH SUMMARY OF FUNCTIONAL OBSERVATIONAL BATTERY DATA DAY 0

PHYSIOLOGICAL OBSERVATIONS MALES

----------------------------------------------------------------------------------------------------------------------------------- GROUP: 1 2 3 4

----------------------------------------------------------------------------------------------------------------------------------- NUMBER TESTED: 12 12 12 12
CATALEPSY (seconds) : MEAN 0.5 0.5 0.5 1.7eg S.D. 0.21 0.16 0.17 2.01 N 12 12 12 12
BODY TEMPERATURE (degrees C) : MEAN 37.7 37.7 37.5 35.1fh S.D. 0.67 0.80 0.93 1.03 N 12 12 12 12
BODY WEIGHT (g) : MEAN 204.9 203.0 203.6 200.2 S.D. 31.80 28.79 26.93 26.38 N 12 12 12 12

----------------------------------------------------------------------------------------------------------------------------------- 1- 0 MG/KG 2- 0 MG/KG 3- 20 MG/KG 4- 100 MG/KG e = SIGNIFICANTLY DIFFERENT FROM CONTROL GROUP 1 AT 0.05 USING DUNNETT’S TEST f = SIGNIFICANTLY DIFFERENT FROM CONTROL GROUP 2 AT 0.01 USING DUNNETT’S TEST g = SIGNIFICANTLY DIFFERENT FROM CONTROL GROUP 2 AT 0.05 USING DUNNETT’S TEST h = SIGNIFICANTLY DIFFERENT FROM CONTROL GROUP 2 AT 0.01 USING DUNNETT’S TEST-42-TABLE 8 (PHYSIOLOGICAL OBSERVATIONS - DAY 0 EVALUATION) PROJECT NO.:WIL-99443 POSITIVE CONTROL NT STUDY IN RATS USING CHLORPYRIFOS PAGE 2 SPONSOR:WIL RESEARCH SUMMARY OF FUNCTIONAL OBSERVATIONAL BATTERY DATA DAY 0

PHYSIOLOGICAL OBSERVATIONS FEMALES

----------------------------------------------------------------------------------------------------------------------------------- GROUP: 1 2 3 4

----------------------------------------------------------------------------------------------------------------------------------- NUMBER TESTED: 12 12 12 12
CATALEPSY (seconds) : MEAN 0.5 0.5 0.6 0.7 S.D. 0.22 0.17 0.41 0.62 N 12 12 12 12
BODY TEMPERATURE (degrees C) : MEAN 37.6 37.8 37.1 35.8fh S.D. 0.93 0.90 1.10 1.02 N 12 12 12 12
BODY WEIGHT (g) : MEAN 157.4 152.6 149.9 150.2 S.D. 13.69 12.05 14.09 9.68 N 12 12 12 12

----------------------------------------------------------------------------------------------------------------------------------- 1- 0 MG/KG 2- 0 MG/KG 3- 20 MG/KG 4- 100 MG/KG f = SIGNIFICANTLY DIFFERENT FROM CONTROL GROUP 2 AT 0.01 USING DUNNETT’S TEST h = SIGNIFICANTLY DIFFERENT FROM CONTROL GROUP 2 AT 0.01 USING DUNNETT’S TEST PFOBSUv4.29 05/15/2008 R:05/15/2008-43-