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

STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

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. ABSTRACT

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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2. INTRODUCTION

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 laboratory

methodologies 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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3. MATERIALS AND METHODS

3.1. TEST SPECIES AND HUSBANDRY

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. TEST MATERIAL AND PREPARATION

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. EXPERIMENTAL DESIGN

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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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. BEHAVIORAL TESTING

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. HOME CAGE OBSERVATIONS

Posture

Convulsions/tremors

Feces consistency

Biting

Palpebral (eyelid) closure

3.4.2. HANDLING OBSERVATIONS

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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3.4.3. OPEN FIELD OBSERVATIONS (EVALUATED OVER A 2-MINUTE

OBSERVATION PERIOD)

Mobility

Rearing

Convulsions/tremors

Grooming

Bizarre/stereotypic behavior

Time to first step (seconds)

Gait

Arousal

Urination/defecation

Gait score

Backing

3.4.4. SENSORY OBSERVATIONS

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. NEUROMUSCULAR OBSERVATIONS

Hindlimb extensor strength

Hindlimb foot splay

Grip strength-hind and forelimb

Rotarod performance

3.4.6. PHYSIOLOGICAL OBSERVATIONS

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. STATISTICAL ANALYSIS

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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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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4. RESULTS AND DISCUSSION

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. HOME CAGE AND OPEN FIELD ASSESSMENTS

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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number of rears exhibited by chlorpyrifos treated females (100 mg/kg) compared to

control group 1 (Table 4, p<0.05).

4.2. NEUROMUSCULAR ASSESSMENT

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. PHYSIOLOGICAL ASSESSMENT

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. MOTOR ACTIVITY

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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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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5. CONCLUSION

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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6. REFERENCES

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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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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7. KEY STUDY PERSONNEL AND REPORT SUBMISSION 

Report Submitted r! By* 

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 

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FIGURES 1-6

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WIL-99443

Pretest Mean Total Activity For Males and Females 

(Prior to Chlorpyrifos Treatment)

Gender

Male Female

Activity 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 Female

Activity 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 xxp<0.01 and xxxp<0.001.

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Pretest Mean Ambulatory Activity For Males and Females 

(Prior to Chlorpyrifos Treatment)

Gender

Male Female

Activity Counts (# of beam breaks)

0

200

400

600

800

1000

Vehicle Control Group 1

Vehicle Controle 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 Female

Activity 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 xxxp<0.001.

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Pretest Total Activity Counts For Males 

(Prior to Chlorpyrifos Treatment)

Subinterval

1-10 11-20 21-30 31-40 41-50 51-60

Activity 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 51-60

Activity 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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Pretest Ambulatory Activity Counts For Males 

(Prior to Chlorpyrifos Treatment)

Subinterval

1-10 11-20 21-30 31-40 41-50 51-60

Activity 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 51-60

Activity 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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Study Day 0 Total Activity Counts For Males 

(Following Chlorpyrifos Treatment)

Subinterval

1-10 11-20 21-30 31-40 41-50 51-60

Activity 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 51-60

Activity 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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Study Day 0 Ambulatory Activity Counts For Males

(Following Chlorpyrifos Treatment)

Subinterval

1-10 11-20 21-30 31-40 41-50 51-60

Activity 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 51-60

Activity 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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FOB SUMMARY TABLES

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 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 

 

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 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 

 

 

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 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 

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