UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

MEMORANDUM 

Date: December 29, 2015 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

SUBJECT: Fluridone: Review of acute neurotoxicity study 

PC Code: 112900 

Decision No.: 494948 

Petition No.: NI A 

Risk Assessment Type: NI A 

TXR No.: 0057317 

MRID No.: 48939603 

DP Barcode: D429841 

Registration No.: NI A 

Regulatory Action: NI A 

Case No.: NIA 

CAS No.: 59756-60-4 

40 CFR: NIA 

FROM: Austin Wray, Ph.D., Toxicologist A-~ !///<-? ___ Risk Assessment Branch IV, ~ 

Health Effects Division (7509P) 

THROUGH: Michael S. Metzger, Branch Chietf j /) /J 0 J ---­ Risk 

Health 

Assessment 

Effects Div

Branch 

ision (7509P) 

V NII f1 ~ M{ · / . ~ 

TO: Sarah Meadows, Risk Manager Reviewer 

Kathryn Montague, Risk Manager 

Herbicide Branch 

Registration Division (7505P) 

I. CONCLUSIONS: 

The acute neurotoxicity study in rats (MRID 48939603) is classified as Acceptable/Guideline. 

The LOAEL was 650 mg/kg/day based on decreased ambulatory counts and the prevalence of 

functional observational battery anomalies in males and females. The NOAEL was 125 

mg/kg/day. 

Page I of3 

Page 1 of 30
II. ACTION REQUESTED: 

Review acute neurotoxicity study to support the fluridone registration review and registration 

for new use on cotton. 

III.BACKGROUND: 

The acute neurotoxicity study was submitted to fulfill a data deficiency requirement for 

fluridone registration review and registration for new use on cotton. 

IV. RESULTS/DISCUSSION: 

In an acute neurotoxicity study (MRID 48939603), groups of 6-week-old Crl:CD(SD) rats 

(10/sex/dose) were given a single oral gavage dose offluridone (98.02% a.i.; Lot no. 

MARl lBEOl 1) in 5% Acacia in deionized water at doses of 0 (vehicle control), 125, 650, or 

2000 mg/kg bw and observed for 14 days. Functional observation battery (FOB) and locomotor 

activity data were recorded for all animals prior to the initiation of dose administration, on Day 0 

at the time of peak effect (approximately 4 hours following dose administration), and on Days 7 

and 14. Animals were sacrificed on Day 15 and perfused in situ. Brain weights and brain 

dimensions were recorded. A neuropathological evaluation of selected tissues from the central 

(CNS) and peripheral nervous systems was performed on 5 animals/sex in the control and 2000 

mg/kg bw groups. 

Treatment with fluridone did not affect clinical signs, mortality, gross necropsy findings, 

neuropathology, brain weight, or brain dimensions. 

Dose-related changes in FOB parameters were observed in males and females at 125, 650 and 

2000 mg/kg bw offluridone. These changes were transient, occurring only on Day 0, and often 

disappeared by Day 7 or 14. 

Anomalies in a multitude of FOB parameters was observed in the 2000 mg/kg bw group. Home 

cage observations revealed clonic convulsions (repetitive movement of mouth and jaws) (2 

males; 5 females) and cage biting (2 males; 3 females). Open-field observations included gait 

abnormality (hunched body: 2 males; 5 females) and one male also exhibiting bizarre/stereotypic 

behavior (repetitive turning of head from side to side). Sensory observations revealed a number 

of animals had no reaction or response to approach (3 rats each), touch (3 rats each), startle ( 4 

males; 2 females), and/or tail pinch (4 males; 3 females). Air righting reflex was also impaired 

(slightly uncoordinated: 2 males; 2 females; landing on their side: 1 male; 2 females). One highdose female exhibited no hindlimb extension. Physiological observations found a longer 

catalepsy time in males only (2.4 vs. 0.4 seconds for controls), mainly due to 2 rats with long 

times. Females had reduced body temperature compared to controls (35.8 vs. 37.1°C for 

controls). Lastly, motor activity was reduced. Although mean cumulative total activity counts 

were not affected, mean cumulative ambulatory activity counts were decreased (p<0.05) in males 

(325 vs. 500 counts for controls) and females (521 vs. 828 counts for controls). 

Page 2 of3 

Page 2 of 30
Similar effects were observed in male and female rats at 650 mg/kg bw on Day 0 but generally 

occurred at a much lower incidence and were mostly limited to minor FOB anomalies. These 

effects included: clonic convulsions (1 female), cage biting (1 female), gait abnormality 

(hunched body; 1 male), no response/reaction to approach (4 males; 1 female), touch (4 males; 1 

female), startle (3 males), and/or tail pinch (3 males), and abnormal righting reflex (landing on 

its side; 1 female). Males from the 650 mg/kg bw dose group also exhibited longer catalepsy 

time outside the historical control range, which was considered a more serious neurotoxic 

response relative to the other FOB anomalies. Both males and females experienced a reduction in 

mean ambulatory counts concurrent with the behavioral anomalies. 

Changes in FOB parameters on Day 0 were also noted in males and females at 125 mg/kg bw 

although many of the changes, similar to the 650 mg/kg bw dose group, occurred at a low 

incidence and were relatively minor anomalies. These effects included gait abnormality (hunched 

body) and uncoordinated air righting reflex (both in same female), and no reaction/response to 

approach (4 males; 1 female), touch (3 males; 2 females), startle (2 males), and/or tail pinch (1 

male). At the 125 mg/kg bw dose, cumulative total and ambulatory motor activity counts were 

not affected. 

Male and female rats from all dose groups exhibited depressed body weight gain (5-25% less 

than controls) during the two weeks after treatment. The impact of depressed body weight gain 

on absolute body weight was negligible (all dose groups were comparable to controls on Day 0, 

7 and 14); therefore, the body weight gain depression was not considered adverse. Food 

consumption was not recorded. 

The changes in FOB parameters in combination with significant depression of cumulative 

ambulatory activity counts was considered evidence of an adverse response at the mid and high 

doses. In the absence of a corroborative effect on motor activity, the FOB effects at the low dose 

were not considered adverse because the effects were limited to minor behavior aberrations not 

anticipated to impact quality of life and were of low incidence. 

The LOAEL is 650 mg/kg bw based on decreased ambulatory counts and the prevalence of 

FOB anomalies in males and females. The NOAEL is 125 mg/kg bw. 

This neurotoxicity study is classified as Acceptable/Guideline and satisfies the guideline 

requirement for an acute neurotoxicity study in rats (OCSPP 870.6200; OECD 424). 

Page 3of3 

Page 3 of 30
Fluridone / 112900 

Acute Neurotoxicity Study (rats) (201 2) I Page I of27 

OCSPP 870.6200/ DACO 4.5.12/ OECD 424 

DATA EVALUATION RECORD 

FLURIDONE 

STUDY TYPE: ACUTE NEUROTOXICITY - RA TS 

OCSPP 870.6200a 1§81-8); OECD 424 

MRID 48939603 

Prepared for 

Health Effects Division 

Office of Pesticide Programs 

U.S. Environmental Protection Agency 

One Potomac Yard 

2777 South Crystal Drive 

Arlington. VA 22202 

Prepared by 

Swnmitec Corporation 

9724 Kingston Pike, Suite 602 

Knoxville, TN 37922 

Task Order No.: 6-142 

Primary Reviewer: ' (J/3 

Secondary 

Claudia Troxel

Reviewers

. Ph.D. 

: 

~~ture ~ 

T. Borges. Ph.D .• MTCASCP>. D.A.B.T. 0994-2014) 

Robert H. Ross. M.S. Program Manager 

Signature: r;~llS Date: D1~0l5 

Signature: R ~ i.l. R Ct>'> 

Date: 7/"0/S 

,,, 

Quality 

Angela M. 

Assurance: 

Edmonds. B.S. Signatun:

Date:7/,i 

: ~ "1· ~ 

Disclaimer 

This review may have been altered subsequent to the contractor's signatures above. 

Summitec Corp. for the U.S. Environmental Protection Agency under Contract No. EP-W-11-014 

Page 4 of 30
Fluridone / 112900 

Acute Neurotoxicity Study (rats) (2012) I Page 2 of27 

OCSPP 870.6200/ DACO 4.5.12/ OECD 424 

EPA Reviewer: Austin Wrav. Ph.D. Signature: A-~ v~ Risk Assessment Branch IV, Health Effects Division (7509P) Date: --=-'~z.. ...... /_1 __ s ...... /_1s __ _ EPA Secondary Reviewer: Monique M. Perron, Sc.D. Signature: ~ ~ 

Risk Assessment Branch I, Health Effects Division (7509P) Date: __ J_2.....:.~....:.1=..r:-=-/,1s--=---- Temptate version 09/11 

TXR#: 0057317 

DAT A EV ALU A TI ON RECORD 

STUDY TYPE: Acute Neurotoxicity- Rats OCSPP 870.6200 [§81-8]; OECD 424. 

PC CODE: 112900 DP BARCODE: D42984 l 

TEST MATERIAL (PURITY): Fluridone, 98.02% a.i. 

SYNONYMS: None provided 

CITATION: Herberth, M.T. (2012). An oral (gavage) acute neurotoxicity study offluridone in 

rats. WIL Research, 1407 George Rd., Ashland OH and WIL Research, 310 

Millstone Dr., Hillsborough, NC. Laboratory Project ID: WIL-867005, 

September 6, 2012. MRID 48939603. Unpublished. 

SPONSOR: SePRO Corporation, 11550 North Meridian Ave., Suite 300, Carmel, IN 

EXECUTIVE SUMMARY: 

In an acute neurotoxicity study (MRID 48939603), groups of 6-week-old Crl:CD(SD) rats 

(10/sex/dose) were given a single oral gavage dose of fluridone (98.02% a.i.; Lot no. 

MARl lBEOl 1) in 5% Acacia in deionized water at doses of 0 (vehicle control), 125, 650, or 

2000 mg/kg bw and observed for 14 days. Functional observation battery (FOB) and locomotor 

activity data were recorded for all animals prior to the initiation of dose administration, on Day 0 

at the time of peak effect (approximately 4 hours following dose administration), and on Days 7 

and 14. Animals were sacrificed on Day 15 and perfused in situ. Brain weights and brain 

dimensions were recorded. A neuropathological evaluation of selected tissues from the central 

(CNS) and peripheral nervous systems was performed on 5 animals/sex in the control and 2000 

mg/kg bw groups. 

Treatment with fluridone did not affect clinical signs, mortality, gross necropsy findings, 

neuropathology, brain weight, or brain dimensions. 

Page 5 of 30
Fluridone / 112900 

Acute Neurotoxicity Study (rats) (2012) I Page 3 of27 

OCSPP 870.6200/ DACO 4.5.12/ OECD 424 

Dose-related changes in FOB parameters were observed in males and females at 125, 650 and 

2000 mg/kg bw of fluridone. These changes were transient, occurring only on Day 0, and often 

disappeared by Day 7 or 14. 

Anomalies in a multitude of FOB parameters was observed in the 2000 mg/kg bw group. Home 

cage observations revealed clonic convulsions (repetitive movement of mouth and jaws) (2 

males; 5 females) and cage biting (2 males; 3 females). Open-field observations included gait 

abnormality (hunched body: 2 males; 5 females) and one male also exhibiting bizarre/stereotypic 

behavior (repetitive turning of head from side to side). Sensory observations revealed a number 

of animals had no reaction or response to approach (3 rats each), touch (3 rats each), startle ( 4 

males; 2 females), and/or tail pinch (4 males; 3 females). Air righting reflex was also impaired 

(slightly uncoordinated: 2 males; 2 females; landing on their side: 1 male; 2 females). One highdose female exhibited no hindlimb extension. Physiological observations found a longer 

catalepsy time in males only (2.4 vs. 0.4 seconds for controls), mainly due to 2 rats with long 

times. Females had reduced body temperature compared to controls (35.8 vs. 37.l °C for 

controls). Lastly, motor activity was reduced. Although mean cumulative total activity counts 

were not affected, mean cumulative ambulatory activity counts were decreased (p<0.05) in males 

(325 vs. 500 counts for controls) and females (521 vs. 828 counts for controls). 

Similar effects were observed in male and female rats at 650 mg/kg bw on Day 0 but generally 

occurred at a much lower incidence and were mostly limited to minor FOB anomalies. These 

effects included: clonic convulsions (1 female), cage biting (1 female), gait abnormality 

(hunched body; 1 male), no response/reaction to approach ( 4 males; 1 female), touch ( 4 males; 1 

female), startle (3 males), and/or tail pinch (3 males), and abnormal righting reflex (landing on 

its side; 1 female). Males from the 650 mg/kg bw dose group also exhibited longer catalepsy 

time outside the historical control range, which was considered a more serious neurotoxic 

response relative to the other FOB anomalies. Both males and females experienced a reduction in 

mean ambulatory counts concurrent with the behavioral anomalies. 

Changes in FOB parameters on Day 0 were also noted in males and females at 125 mg/kg bw 

although many of the changes, similar to the 650 mg/kg bw dose group, occurred at a low 

incidence and were relatively minor anomalies. These effects included gait abnormality (hunched 

body) and uncoordinated air righting reflex (both in same female), and no reaction/response to 

approach (4 males; 1 female), touch (3 males; 2 females), startle (2 males), and/or tail pinch (1 

male). At the 125 mg/kg bw dose, cumulative total and ambulatory motor activity counts were 

not affected. 

Male and female rats from all dose groups exhibited depressed body weight gain (5-25% less 

than controls) during the two weeks after treatment. The impact of depressed body weight gain 

on absolute body weight was negligible (all dose groups were comparable to controls on Day 0, 

7 and 14); therefore, the body weight gain depression was not considered adverse. Food 

consumption was not recorded. 

The changes in FOB parameters in combination with significant depression of cumulative 

ambulatory activity counts was considered evidence of an adverse response at the mid and high 

doses. In the absence of a corroborative effect on motor activity, the FOB effects at the low dose 

Page 6 of 30
Fluridone I 112900 

Acute Neurotoxicity Study (rats) (2012) I Page 4 of27 

OCSPP 870.6200/ DACO 4.5.12/ OECD 424 

were not considered adverse because the effects were limited to minor behavior aberrations not 

anticipated to impact quality of life and were of low incidence. 

The LOAEL is 650 mg/kg bw based on decreased ambulatory counts and the prevalence of 

FOB anomalies in males and females. The NOAEL is 125 mg/kg bw. 

This neurotoxicity study is classified as Acceptable/Guideline and satisfies the guideline 

requirement for an acute neurotoxicity study in rats (OCSPP 870.6200; OECD 424). 

COMPLIANCE: Signed and dated GLP, Quality Assurance, and Data Confidentiality 

statements were provided. 

Page 7 of 30
Fluridone I 112900 

Acute Neurotoxicity Study (rats) (2012) I Page 5 of27 

OCSPP 870.6200/ DACO 4.5.12/ OECD 424 

I. MATERIALS AND METHODS: 

A. MATERIALS: 

1. Test material: Fluridone 

Description: Off-white powder 

Lot/batch #: MARllBEOll 

Purity: 98.02% 

CAS # ofTGAI: 59756-60-4 

Structure: 

F-~-F 

I F 

2. Vehicle and/or oositive control: 5% Acacia (Lot no. 2AC0916, exp. date: 15 December 

2012, manufactured by Spectrum Chemical Manufacturing Corporation, New Brunswick, 

NJ; stored at room temperature) in deionized water 

3. Test animals: 

Species: 

Strain: 

Age/weight at dosing: 

Source: 

Housing: 

Diet: 

Water: 

Environmental 

conditions: 

Acclimation period: 

B. STUDY DESIGN: 

Rat 

Crl:CD(SD) 

- 6 weeks old; males: 199-254 g; females: 138-197 g 

Charles River Laboratories, Inc., Raleigh, NC. 

All animals were housed 2-3 per cage by sex for approximately 6 days; 

then housed individually in clean, stainless steel, wire-mesh cages 

suspended above cage-board 

PM! Nutrition International, LLC Certified Rodent LabDiet® 5002, ad 

libitum 

Reverse osmosis-treated drinking water ad libitum 

Temperature: 71°F±5°F (22°C ± 3°C) 

Humidity: 50 ± 20%, 

Air changes: 10 fresh air changes per hour 

Photoperiod: 12 hrs dark/ 12 hrs light 

Minimum of 13 days 

1. In life dates: Start: January 31, 2012; End: March 2, 2012 

Page 8 of 30
Fluridone I 112900 

Acute Neurotoxicity Study (rats) (2012) I Page 6 of27 

OCSPP 870.6200/ DACO 4.5.12/ OECD 424 

2. Animal assignment and treatment: Animals were randomly assigned by computer to the 

test groups noted in Table 1 based on body weight stratification in a block design. The 

animals were randomized into 4 study replicates to facilitate neurobehavioral observations. 

Each dose group and sex was approximately equally represented within each study replicate. 

The vehicle and test substance formulations were administered as a single oral gavage dose 

in a dose volume of 5 mL/kg. Animals were not fasted prior to dose administration. 

Dose levels were selected based on the results of a previous dose range-finding study in 

which the test substance was administered as a single oral dose to 3 groups of 3 Crl:CD(SD) 

rats/sex/group at dose levels of 550, 1750, and 2000 mg/kg. There were no significant 

clinical observations noted at any dose level. As a result, a high dose of 2000 mg/kg bw 

(limit dose for acute neurotoxicity studies) was chosen for the current study. 

TABLE I. Study desi2n 

Dose 2roup (m2/k2 bw) 

Experimental parameter Control Low dose Mid dose High dose 

(0) (125) (650) (2000) 

Total number of animals/sex/2roup 10/sex IO/sex 10/sex 10/sex 

Behavioral testin2 (FOB, Motor Activity) IO/sex 10/sex 10/sex 10/sex 

Necropsy 10/sex 10/sex IO/sex IO/sex 

Neuropathology 5/sex - - 5/sex 

3. Test Substance preparation and analysis: 

The vehicle suspension was prepared once for administration to the control group (Group 1) 

and for preparation of the test substance formulations; aliquots were prepared for 

dispensation to the control group and stored refrigerated. The vehicle was mixed throughout 

the preparation, sampling, and dose administration procedures. The test substance 

formulations used for dose administration were prepared once as single formulations for each 

dose level, divided into aliquots for dispensation, and stored refrigerated (2°C to 8°C). The 

test substance formulations were stirred continuously throughout the preparation, sampling, 

and dose administration procedures. 

Homogeneity, resuspension homogeneity, and stability (following 4 days ofrefrigerated 

storage) of the test substance in the vehicle at concentrations of 30 and 400 mg/mL were 

previously established. However, additional homogeneity and stability assessments were 

performed as part of the current study at 25 mg/rnL. Prior to the initiation of dose 

administration, duplicate samples for homogeneity determination were collected from the 

top, middle, and bottom of a 25 mg/mL non-dosing formulation. In addition, a sufficient 

volume of the same 25 mg/mL non-dosing formulation, similar in size to the amount needed 

for 1 day of dosing, was stored refrigerated (2°C to 8°C) for 4 days. Following the 4-day 

storage period, the formulation was mixed using a magnetic stirrer for a minimum of 30 

minutes and samples were collected from the top and bottom strata and assessed for stability. 

Samples for concentration analyses were collected from the middle stratum of each dosing 

formulation (including the control group). All analyses were conducted using a validated 

high performance liquid chromatography method using ultraviolet absorbance detection. 

Page 9 of 30
Fluridone I 112900 

Results: 

Acute Neurotoxicity Study (rats) (2012) I Page 7 of27 

OCSPP 870.6200/ DACO 4.5.12/ OECD 424 

Homogeneity analysis: Homogeneity analyses of duplicate samples taken from the top, middle, 

and bottom of the 25 mg/mL test formulation revealed a mean concentration of24.3 mg/mL 

(97.2% of target) and a% RSD of 0.89%. 

Stability analysis: Stability analysis of duplicate samples taken from the top and bottom of the 

25 mg/mL test substance formulation and stored refrigerated for 4 days revealed mean 

concentrations of25.7, 26.1, 25.5, and 25.8 mg/mL. The mean of these values was 106% of 

the initial concentration of25 mg/mL. 

Concentration analysis: Concentration analyses of duplicate samples taken from the 25, 130, 

and 400 mg/mL test formulations revealed mean concentrations of24.0, 113, and 403 

mg/mL, respectively, resulting in concentrations that were 95.9, 87.1, and 101 % of nominal, 

respectively. The test substance was not detected in the vehicle formulation that was 

administered to the control group. 

The analytical data indicated that the mixing procedure was adequate and that the variance 

between nominal and actual dosage to the animals was acceptable. 

4. Statistics: Each mean was presented with the standard deviation (S.D.) and the number of 

animals (N) used to calculate the mean. In addition, standard error (S.E.) was presented for 

body weight, brain measurements, and locomotor activity data. 

Statistics Conducted by WIL Research: All statistical tests were performed using 

WTDMSTM unless otherwise noted. Analyses were done using two-tailed tests (except as 

noted otherwise) for minimum significance levels of 1 % and 5%, comparing each test 

substance-treated group to the control group by sex. Body weight, body weight change, 

continuous FOB, and brain weight and measurement data were subjected to a parametric oneway ANOVA to determine intergroup differences. If the ANOVA revealed significant 

(p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated 

groups to the control group. FOB parameters that yielded scalar or descriptive data, necropsy 

findings, and non-graded histopathologic findings were analyzed using Fisher's Exact Test. 

Statistical analysis of graded histopathologic findings was not conducted on this study due to 

the absence of multiple severities (i.e., those findings with more than 2 distinct severities 

including none/not remarkable). 

Statistics Conducted by BioSTAT Consultants, Inc.: All statistical analyses were done 

using SAS® version 9.2 or higher. All repeated measures analysis of variance (RANOVA) 

statistical analyses for total and ambulatory locomotor activity counts recorded during pretest 

and after dosing were conducted as follows. Each analysis endpoint was analyzed, by sex 

and session, with a RANOVA. Factors in the model included treatment group (TRT), time 

interval (TIME), and the interaction of time interval and treatment group (TR T*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 (AIC). 

Page 10 of 30
Fluridone I 112900 

Acute Neurotoxicity Study (rats) (2012) I Page 8 of27 

OCSPP 870.6200/ DACO 4.5.12/ OECD 424 

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 (LinTrt*Time) 

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. If the linear dose by time interaction was 

not significant, the trend test was conducted across the pooled time intervals for the entire 

session only. 

Nonmonotonic dose responses were evaluated whenever no significant linear trends were 

detected but TRT and/or TRTTIME interaction was significant at the 0.01 level. Within the 

framework of the RANOVA, pairwise comparisons were made for each individual test 

substance-treated group with the control group through linear contrasts. If TRT*TIME was 

significant, the comparisons were conducted for each time interval. If only the TR T effect 

was significant, the comparisons were conducted across the pooled time intervals for the 

entire session. These nonmonotonic dose-response comparisons were conducted at the 0.01 

significance level. 

The reviewer considered the statistical analyses to be adequate. 

C. METHODS: 

1. Mortality and clinical observations: All animals were observed twice daily for mortality 

and moribundity. 

Clinical examinations were performed once daily on all animals. On the days of the FOB, no 

additional clinical findings were recorded. 

2. Body weight: Individual body weights were recorded weekly, beginning 1 week prior to test 

substance administration (Day -7). 

3. Food consumption: Food consumption was not measured. 

4. Cholinesterase determination: Cholinesterase activity was not determined. 

5. Neurobehavioral assessment: 

a. Functional Observational Battery (FOB): FOB findings were recorded for all animals 

prior to the initiation of dose administration (Day -6), at the time of peak effect 

(approximately 4 hours post-dosing) on Day 0, and on Days 7 and 14. Testing was 

performed by the same biologists, to the extent possible, without knowledge of the 

animal's group assignment. The FOB was performed in a sound-attenuated room 

equipped with a white-noise generator set to operate at 70 ± 10 dB. 

Scoring criteria were included for the measured parameters and are provided in Appendix 

G of the study report. For the open-field observations, the animal was placed in a 

standard arena (24" x 24" x 6"; constructed from black Plexiglas®) and observed for a 

Page 11 of 30
I 

Fluridone I 112900 

Acute Neurotoxicity Study (rats) (2012) I Page 9 of27 

OCSPP 870.6200/ DACO 4.5.12/ OECD 424 

two-minute recording period. Forelimb and hindlimb grip strength were measured using 

a T-shaped grip bar and a Mark-IO series EG digital force gauge. For the rotarod test, 

animals were tested for up to 2 minutes on a 7.0-cm-diameter rod rotating at 12 rpm. 

The CHECKED (X) parameters were examined. 

x HOME CAGE OBSERVATIONS x HANDLING OBSERVATIONS x OPEN FIELD OBSERVATIONS 

x Posture* x Reactivity* x Mobility 

x Biting x Lacrimation*/chromodacryorrhea x Rearing+ 

x Convulsions* x Salivation* x Arousal/ general activity level* 

x Tremors* x Piloerection* x Convulsions* 

x Abnormal Movements* x Fur appearance x Tremors* 

x Palpebral closure* x Palpebral closure* x Abnormal movements* 

x Feces consistency x Respiratory rate+ x Urination I defecation* 

x Red/crusty deposits• x Grooming 

I SENSORY OBSERVATIONS I x Mucous membranes/eye /skin x Gait abnormalities/posture* 

colour 

x Approach response+ x Eye prominence* x Gait score* 

x Touch response+ x Muscle tone* x Bizarre/stereotypic behavior• 

x Startle response• x Backing 

x Pain response*(tail pinch) PHYSIOLOGICAL x Time to first step OBSERVATIONS 

I NEUROMUSCULAR x Pupil response* x Body weight* OBSERVATIONS 

x Eyeblink response x Body temperature+ x Hindlimb extensor strength 

x Forelimb extension x Forelimb grip strength* 

x Hindlimb extension OTHER OBSERVATIONS x Hindlimb grip strength* 

x Air righting reflex+ x Catalepsy x Landing foot splay* 

x Olfactory orientation x Rotarod performance 

*Required parameters; +Recommended parameters 

Note: Open field observations were evaluated over a 2-minute observation period. 

b. Locomotor activity: Locomotor activity was assessed after completion of the FOB on 

all animals prior to the initiation of dose administration (Day -6), at the time of peak 

affect (approximately 4 hours post-dosing) on Day 0, and on Days 7 and 14. Locomotor 

activity was measured automatically using a computer-controlled system that utilized a 

series of infrared photobeams surrounding an amber plastic, rectangular cage to quantify 

the motor activity of each animal. Four-sided black plastic enclosures were used to 

surround the transparent plastic boxes and decrease the potential for distraction. The 

locomotor activity assessment was performed in a sound-attenuated room equipped with 

a white-noise generator set to operate at 70 ± 10 dB. The testing of treatment groups was 

conducted according to replicate sequence. Each animal was tested separately. Data 

were collected in 5-minute epochs, and the test session duration was 60 minutes. These 

data were compiled as six 10-minute subintervals for tabulation. 

Page 12 of 30
Fluridone I 11 2900 

Acute Neurotoxicity Study (rats) (2012) I Page 10 of27 

OCSPP 870.6200/ DACO 4.5. 12/ OECD 424 

Total motor activity was defined as a combination of fine motor skills (i.e., grooming, 

interruption of 1 photobeam) and ambulatory motor activity (interruption of2 or more 

consecutive photobeams). 

6. Sacrifice and pathology: On Day 15, all animals were anesthetized by an intraperitoneal 

injection of sodium pentobarbital and then perfused in situ with a 4.0% paraformaldehyde in 

a 0.1 M phosphate buffered solution. The central and peripheral nervous system tissues were 

dissected and preserved. Fixed brain weight and brain dimensions (length [excluding 

olfactory bulbs] and width) were recorded. Any observable gross changes and abnormal 

coloration or lesions of the brain and spinal cord were recorded. 

I 

Additionally, the CHECKED (X) tissues from five males and five females from the control 

and 2000 mg/kg bw groups were processed and evaluated histologically. The tissues were 

prepared for a histopathological examination by embedding in paraffin (central nervous 

system tissues) or plastic (peripheral nervous system tissues), sectioning, and staining with 

hematoxylin and eosin. 

x I CENTRAL NERVOUS SYSTEM II x I PERIPHERAL NERVOUS SYSTEM 

BRAIN SCIATIC NERVE 

x Forebrain x Mid-thigh 

x Center of cerebrum x At Sciatic Notch 

x Mid brain 

x Cerebellum OTHER 

x Pons x Surat Nerves 

x Medulla oblongata x Tibial Nerves 

SPINAL CORD x Peroneal Nerves 

x Cervical swelling (C3-C1) x Lumbar dorsal root gangla (T13-L4) 

x Lumbar swelling (T13-u) x Lumbar dorsal root fibers (T13-£A) 

Thoracic swelling x Lumbar ventral root fibers (T13-L4) 

OTHER x Cervical dorsal root gangla (C3-C1) 

Gasserian Ganglion x Cervical dorsal root fibers (C3-C1) 

x Trigeminal nerves plus ganglia x Cervical ventral root fibers (C3-C1) 

x Optic nerves x Olfactory bulbs 

x Eyes x Hippocampus/dentate gyms 

x Gastrocnemius muscle x Basal ganglia 

x Cervical spinal nerve x Thalamus 

x Lumbar spinal nerve x Hypothalamus 

7. Positive controls: Positive control data were provided in Appendix F of MRID 48939603. 

I 

Studies WIL-99435 (MRID 48939603, pp. 352-375) and WIL-99441(MRID48939603, pp. 

376-404) were positive control validation studies of locomotor activity in Crl:CD (SD) rats. 

Locomotor activity was recorded using a Kinder Scientific MotorMonitor (Kinder Scientific, 

LLC, Poway, CA USA). The test sessions were conducted in a sound-attenuated room and 

were 60 minutes in duration, with 6 subintervals lasting 10 minutes each. Locomotor activity 

was evaluated as total and ambulatory activity counts obtained over each subinterval and the 

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entire 60-minutes sessions. In study WIL-99435, groups of20 rats/sex were administered 

Haloperidol at dose levels of 0, 0.05, 0.1 , and 0.5 mg/kg bw by a single IP injection 30 

minutes prior to behavioral testing on postnatal day (PND) 13, 17, 21, or 61. Overall, 

haloperidol caused a dose-dependent decrease in locomotor activity (total activity counts and 

ambulatory counts) for both male and female rats at PND 13, 17, 21 and 61. Habituation was 

noted on PND 17, 21, and 61, but not on PND 13, males and females. In study WIL-99441 , 

groups of 20 rats/sex were administered nicotine at dose levels of 0, 0.1 or 0.5 mg/kg bw by a 

single IP injection 3 minutes prior to behavioral testing on PND 13, 17, and 21. Also, groups 

of 20 young adult rats were administered amphetamine by a single IP injection 15 minutes 

prior to behavioral testing at dose levels of 0, 1 or 3 mg/kg bw (males and females) on PND 

61 and at doses of 0, 0.2 and 1 mg/kg (females only) on PND 61. Nicotine caused an 

increase in locomotor activity (mean total and ambulatory activity) in male and female 

neonatal rats at PND 13, 1 7, and 21 . In a similar manner, amphetamine stimulated locomotor 

behavior in male and female rats at PND 61 , causing both total and ambulatory activity to 

increase. The subsequent female-only groups (0.2 and 1 mg/kg bw amphetamine) exhibited 

a dose-response with increased total and ambulatory activity compared to controls. In the 

PND 61 males and females (given 1 or 3 mg/kg bw amphetamine), the habituation profile 

was only present in the control and 3 mg/kg bw amphetamine treatment groups, whereas the 

habituation profile was present in the female-only PND 61 control and 0.2 mg/kg bw 

amphetamine groups. The experimental design used in these studies (WIL-99435 and WIL99441) demonstrated that WIL Research can detect locomotor effects ofhaloperidol 

(decrease in motor activity) and nicotine and amphetamine (increases in motor activity) using 

the Kinder Scientific MotorMonitor System. It was noted that these validation studies used 

20 rats/sex/group for evaluation, while the acute neurotoxicity study used only 10 

rats/sex/group. Also, the acute neurotoxicity study did not specify that the Kinder Scientific 

MotorMonitor System was used. 

In WIL-99443 (MRID 48939603, pp. 405-447), groups of 12 Crl:CD (SD) rats/sex/group 

were administered chlorpyrifos at a single dose of 0, 20 or 100 mg/kg bw by oral gavage 3.5 

hours before FOB assessment and locomotor activity measurement. The methodology for 

the FOB assessment and locomotor activity determination appeared to be the same as that 

used in the main acute neurotoxicity study (MRID 48939603). The FOB detected a general 

decrease in the initiation of movement (time to first step in the open field), number of rears, 

body temperature and rotarod performance, along with impaired gait (hindlimbs splayed or 

dragging, walks on tip toes, hunched body), and increased catalepsy time. Motor activity 

(both 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 bw 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. 

The other two studies submitted for validation of FOB methodology, WIL-99263 (MRID 

48939603 , pp. 448-461) and WIL-99310 (MRID 48939603, pp. 462- 497), were conducted 

to train personnel on the conduct the FOB and to assess inter-observer reliability of the same 

technicians. There were three phases of testing: Phase I consisted of FOB assessments on 

naive stock animals in order to ensure consistent and accurate use of all equipment and gain 

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knowledge of stereotypic behavior of naive animals; Phase II consisted of an open discussion 

of each animal's observations conducted in a group setting (2 rats/group dosed with positive 

control or vehicle); and Phase III consisted of three animals/group administered a positive 

control or vehicle and observed at a time designated by the Study Director; each technician 

individually recorded observations of the animals and the resulting inter-observer reliability 

was evaluated. Substances evaluated in study WIL-99263 included Parathion, 3,3 ' -

Iminodipropionitrile (IDPN) or deionized water, and study WIL-99310 included IDPN, 

Parathion, d-Amphetamine, and com oil. After reviewing the responses of the technicians, 

the Study Director concluded that all but two of the technicians fulfilled the criteria for 

reliably observing FOB parameters to assess behavioral changes in rats. One of the two 

technicians was to receive additional training and demonstrated proficiency in the evaluation 

of the pupillary response, while the other observer was not considered to have met acceptable 

criteria for performing FOB testing. The technicians in the other study (WIL-99263) were 

considered to have met the acceptable criteria for evaluation. 

No positive control studies demonstrating the ability to detect central and peripheral nervous 

system pathology were included. 

Acute FOB historical control data and acute motor activity historical control data for 

Crl:CD(SD) rats were provided for a variety of vehicles (MRID 48939603; Appendix J and 

Appendix K). The FOB studies that were cited ranged over a period oftime of 4/24/01 to 

715106, while the locomotor activity studies ranged over a period from 11/12/07 to 10/22/10. 

A description of testing methods was not included. 

II. RES UL TS: 

A. OBSERVATIONS: 

1. Clinical signs: No clinical signs related to treatment were observed. Findings were limited 

to low incidences of alopecia in males and females that were not related to dose. 

2. Mortality: All rats survived to study termination. 

B. BODY WEIGHT AND BODY WEIGHT GAIN: 

Mean absolute body weight and body weight gain data are summarized in Table 2. No 

statistically significant, treatment-related changes in mean absolute body weight or body 

weight gain were noted in male or female rats at any dose. Male and female body weight gain 

was depressed (5-25% less than controls) in the two weeks after treatment, but did not have a 

biologically significant impact on absolute body weight indicating the effect was not adverse. 

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I TABLE 2. Body weight and body weight gain (g ± SD) •, b 

Observation Dose level (mg/kg bw) 

Control Low 

(0) (125) 

Body weight-males 

Day-7 170± 15.4 169± 14.8 

DayO 232 ± 17.8 232±15.I 

Day7 286±22.2 280± 15.5 

Dav 14 329± 29.3 322 ± 24.5 

Body wei2ht-females 

Day-7 141 ±9.7 139 ± 11.7 

DayO 171±12.9 172 ± 11.1 

Day7 195 ± 17.0 192± 13.5 

Day 14 214 ± 17.0 213 ± 16.7 

Body wei ~ht gain-males 

Day 0-7 54 ± 6.9 48±7.5U11%) 

Day 0-14 97 ± 14.8 90 ± 19.3 (! 7%) 

Body weieht 2ain-females 

Day 0-7 24 ± 7.8 21±7.2 UJ2%) 

Day0-14 43 ± 10.7 41 ± 9.5 (!5%) 

• Data extracted from MRID 48939603, pp. 84-87. 

b n = 10 

Number in parenthesis is the percent difference from controls. 

Mid 

(650) 

169 ± I I.I 

227 ± 14.1 

274 ± 20.5 

315±29.3 

140±7.4 

172±9.8 

191 ± 11.7 

211±13.9 

47 ± 11.0 UJ3%) 

88 ± 19.7 (!9%) 

19 ± 5.7 (!21%) 

38 ± 6.4 (112%) 

C. FOOD CONSUMPTION: Food consumption was not measured. 

D. NEUROBEHA VIORAL RESULTS: 

1. FOB findings: 

Home cage observations: 

High 

(2000) 

170± 14.5 

230± 16.8 

275± 17.4 

319± 23.9 

138 ± 12.4 

169± 14.0 

187 ± 21.3 

206± 26.0 

45 ± 8.2 (l 17%) 

89 ± 19.0 (18%) 

18 ± 9.1 (125%) 

37 ± 13.7 (114%) 

Home cage observations revealed a treatment-related effect in males and females in the highdose group on Day 0 (Table 3). Two males at 2000 mg/kg bw exhibited clonic convulsions 

(repetitive movement of mouth and jaws) and cage biting. In females, 5 animals at 2000 

mg/kg bw developed clonic convulsions, with 3 of the 5 additionally biting the cage. 

I 

One male at 125 mg/kg bw exhibited clonic convulsions and one female at 650 mg/kg bw 

exhibited clonic convulsions and cage biting; however both were considered spontaneous and 

not treatment related based on similar behavior reported in several animals from the 

historical controls and the lack of dose response between the low and mid doses. 

Single incidences ofrepetitive mouth and jaw movement at 125 and 650 mg/kg bw were not 

considered an adverse response to treatment based on the nature of the effect and low 

incidence. This finding in animals from the 2000 mg/kg bw dose group was attributed to 

treatment due to higher frequency, particularly in the females (5/10 animals). 

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TABLE 3. Functional observational battery results: Home cage observations a,b,c 

Home cage observation Control 

Convulsions - Clonic 

-Pretest: Absent 10 

-DayO 

Absent 10 

Repetitive movement of mouth/jaws 0 

-Day 7 and Day 14: Absent 10 

Biting 

-Pretest: None 10 

-DayO 

None IO 

Biting of cage 0 

-Day 7 and Day 14: None 10 

Convulsions - Clonic 

-Pretest: Absent 10 

-Day 0 

Absent 10 

Repetitive movement of mouth/jaws 0 

-Day 7 and Day 14: Absent 10 

Biting 

-Pretest: None 10 

-Day O 

Absent 10 

Biting of cage 0 

-Day7 

Absent 10 

Biting of cage 0 

-Day 14: None 10 

• Data were extracted from 48939603, pp. 88-96. 

b Values represent number of animals affected 

c n= IO 

*=p<.05 compared with controls 

Handling observations: 

(0) 

Dose level (m2/k2 bw) 

Low dose Mid dose High dose 

(125) (650) (2000) 

Males 

10 10 10 

9 10 8 

1 0 2 

IO 10 10 

10 JO 10 

10 10 8 

0 0 2 

10 10 10 

Females 

IO IO 10 

10 9 5* 

0 1 5* 

10 10 IO 

10 10 10 

10 9 7 

0 I 3 

9 10 10 

I 0 0 

10 10 10 

There were no dose-related differences in the handling observations of treated male or female 

rats compared to controls. 

Open field observations: 

A summary of selected open field observations is presented in Table 4. In males, findings on 

Day 0 included 1, 1, and 2 animals at the low-, mid-, and high-dose with a hunched posture, 

and 1 male at the high-dose with bizarre/stereotypic behavior (repetitive turning of head from 

side to side). 

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I 

Open-field observations of females on Day 0 revealed a treatment-related effect on gait with 

5 females exhibiting a hunched body at the high dose. Additionally, one female at the low 

dose had a hunched body. No other significant observations were noted. 

TABLE 4. Functional observational batterl'. results: O~en field observations a,b,c 

Dose level (ml?fkf? bw) 

Home cage observation Control Low dose Mid dose High dose 

(0) (125) (650) (2000) 

Males 

Gait: 

-Pretest: Normal 10 10 10 10 

-DayO 

Normal 10 9 9 8 

Hunched body 0 I I 2 

-Day 7 and Dav 14: Normal 10 10 10 10 

Bizarre/StereO!YQic behavior 

-Pretest: None 10 10 10 10 

-DayO 

None 10 10 10 9 

Head search/repetitive turning side to side 0 0 0 I 

-Dav 7 and Dav 14: Absent 10 10 10 10 

Females 

Gait: 

-Pretest: Normal 10 10 10 10 

-DayO 

Normal 10 9 10 5* 

Hunched body 0 I 0 5* 

-Day 7 and Day 14: Normal 10 10 10 10 

I 

• Data were extracted from 48939603, pp. 121-136. 

b Values represent number of animals affected 

c n= IO 

*=p<.05 compared with controls 

Sensory observations: 

Sensory observations of male and female rats on Day 0 revealed a general decrease in 

response to outside stimuli (Tables 5 and 6). Although not always following a clear doseresponse, the lack of response to stimuli in the groups of treated rats are generally considered 

an effect of treatment because animals from the control groups always exhibited a reaction. 

Male rats in the low-, mid-, and high-dose groups exhibited no reaction to approach response 

(4, 4, and 3 rats, respectively), touch response (4, 4, and 3 rats, respectively), startle response 

(2, 3, and 4 rats, respectively), and/or tail pinch response (1, 3, and 4 rats, respectively). Air 

righting reflex was also impaired in high-dose males, with 2 rats being slightly uncoordinated 

and 1 rat landing on its side. 

Females in the low-, mid-, and high-dose groups exhibited no reaction to approach response 

(1, 1, and 3 rats, respectively), touch response (2, 1, and 3 rats, respectively), with high-dose 

females additionally showing no startle response (2 rats), and/or tail pinch response (3 rats). 

Air righting reflex was impaired in mid- and high-dose females, with 2 high-dose rats being 

slightly uncoordinated and 1 mid-dose and 2 high-dose female rats landing on their side. 

Another high-dose female had no hind limb extension. 

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TABLE S. Functional obser vat iona l batterl'. results: Sensorl'. observations in males •,b,c I 

Dose level m!!lki!: bw) 

Observation Control Low dose Mid dose High dose 

(0) (125) (650) (2000) 

Approach response 

-Pretest: Slow approach, sniffing or turning away 10 10 10 9 

More energetic response, with/without vocalization 0 0 0 I 

-DayO 

No reaction 0 4 4 3 

Slow approach, sniffing or turning away 10 4* 6 7 

More energetic response, with/without vocalization 0 2 0 0 

-Day7 

Slow approach, sniffing or turning away 10 9 10 10 

More energetic response, with/without vocalization 0 I 0 0 

-Day 14 

Slow approach, sniffing or turning away 10 10 10 10 

Touch response 

-Pretest: Animal may slowly tum/walk away 10 10 9 10 

More energetic response 0 0 I 0 

-DayO 

No reaction 0 4 4 3 

Animal may slowly tum/walk away 10 4* 6 7 

More energetic response 0 2 0 0 

-Day 7 and Day 14 

Animal may slowly tum/walk away 10 10 10 10 

Startle response 

-Pretest: Slight reaction, ear flick/evidence snap heard 10 10 10 10 

-Day 0 

No reaction 0 2 3 4 

Slight reaction, ear flick/evidence snap heard 10 7 7 6 

More energetic response, with/without vocalization 0 I 0 0 

-Day 7 

Slight reaction, ear flick/evidence snap heard 10 10 10 10 

-Day 14 

No reaction 0 0 0 I 

Slight reaction, ear flick/evidence snap heard 10 10 10 9 

Tail pinch response 

-Pretest: Animal may slowly tum, walk away 10 9 10 10 

More energetic, with/without vocalization 0 I 0 0 

-Day 0: 

No reaction 0 I 3 4 

Animal may slowly turn, walk away 7 6 7 6 

More energetic response, with/without vocalization 3 3 0 0 

-Day 7: 

Animal may slowly turn, walk away 8 9 9 9 

More energetic response, with/without vocalization 2 I I I 

-Day 14: 

Animal may slowly turn, walk away 8 8 10 8 

More energetic response, with/without vocalization 2 2 0 2 

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Air righting reflex 

-Pretest: Normal 

-DayO 

Normal 

Slightly uncoordinated 

Lands on side 

-Day 7 and 14: 

Normal 

• Data were extracted from 48939603, pp. 137- I 52. 

b Values represent number of animals affected 

< n=lO 

*=p<.05 compared with controls 

JO 

10 

0 

0 

10 

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JO JO JO 

9 JO 7 

0 0 2 

I 0 I 

JO JO 10 

TABLE6. Functional observational batter:!'. results: Sensor:!'. observations in females a,b,c I 

Dose level me:/ke: bw) 

Observation Control Low dose Mid dose High dose 

(0) (125) (650) (2000) 

Approach response 

-Pretest: Slow approach, sniffing or turning away JO 9 JO JO 

More energetic response, with/without vocalization 0 I 0 0 

-Day O 

No reaction 0 J J 3 

Slow approach, sniffing or turning away 8 8 9 7 

More energetic response, with/without vocalization 2 J 0 0 

-Day 7 

Slow approach, sniffing or turning away 9 JO 10 9 

More energetic response, with/without vocalization I 0 0 I 

-Day J4 

Slow approach, sniffing or turning away 9 8 9 8 

More energetic response, with/without vocalization J 2 J 2 

Touch response 

-Pretest: Animal may slowly tum/walk away JO JO 10 10 

-Day 0 

No reaction 0 2 J 3 

Animal may slowly turn/walk away JO 8 9 7 

-Day 7 and Day 14 

Animal may slowly tum/walk away JO 10 10 10 

Startle res12onse 

-Pretest: Slight reaction, ear flick/evidence snap heard 10 10 10 10 

-Day 0 

No reaction 0 0 0 2 

Slight reaction, ear flick/evidence snap heard 10 JO JO 8 

-Day 7 

Slight reaction, ear flick/evidence snap heard 10 10 10 10 

-Day 14 

No reaction 0 0 I 0 

Slight reaction, ear flick/evidence snap heard 10 10 9 JO 

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Tail pinch response 

-Pretest: Animal may slowly tum, walk away 

More energetic, with/without vocalization 

-Day 0: 

No reaction 

Animal may slowly tum, walk away 

More energetic response, with/without vocalization 

-Day 7: 

Animal may slowly turn, walk away 

More energetic response, with/without vocalization 

-Day 14: 

Animal may slowly tum, walk away 

More energetic response, with/without vocalization 

Air righting reflex 

-Pretest: Normal 

-Day 0 

Normal 

Slightly uncoordinated 

Lands on side 

-Day 7 and 14: 

Normal 

• Data were extracted from 48939603, pp. 137-152. 

b Values represent number of animals affected 

c n= lO 

Neuromuscular observations: 

9 

I 

0 

8 

2 

9 

1 

9 

1 

10 

10 

0 

0 

10 

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8 8 10 

2 2 0 

0 0 3 

8 8 6 

2 2 I 

6 5 6 

4 5 4 

6 7 6 

4 3 4 

10 10 10 

9 9 6 

1 0 2 

0 1 2 

10 10 10 

The neuromuscular parameters were unaffected by treatment. 

Physiological observations: 

A transient, statistically significant decrease in body temperature was observed in all groups 

of treated male and female rats compared to controls (Table 7). It was unclear if the 

reductions in body temperature were an adverse effect of treatment at all dose levels. The 

historical control data of 329 males reported a mean body temperature of 38.1 ± 0.85°C 

[minimum: 36.0°C; maximum: 39.2°C], while the historical control data of 249 females 

reported a mean of 38.3 ± 0.70°C [minimum: 36.5°C; maximum: 39.4°C] (MRID 48939603, 

p. 562 and 577, respectively). Compared to the historical control data and the pre-test values, 

the mean body temperatures of the treated males and females were not indicative of an 

adverse effect with the exception of the high-dose females. 

Body temperature was not affected during the pretest or on Day 7 or 14. 

Catalepsy in the mid- and high-dose males was increased on Day 0 compared to controls. An 

examination of the individual data revealed the increases were due to a few individual 

animals per group. In the mid-dose group, one male each had a time of 9.3 and 1.6 seconds, 

while in the high-dose group, one male each had a time of 19.3 seconds and 1.3 seconds and 

one female had a time of 1.5 seconds. Other catalepsy times were comparable to controls. 

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TABLE 7. Functional observational batterv results: Phvsioloeical observations in males and females •,b 

Home cage observation 

Body temperature (

0C): 

-Pretest: 

-Day 0: 

-Day 7: 

-Day 14: 

Catalepsy (sec): 

-Pretest: 

-DayO: 

-Day 7: 

-Day 14: 

Body temperature (

0

C): 

-Pretest: 

-Day 0: 

-Day 7: 

-Day 14: 

•Data were extracted from 48939603, pp. 161-168. 

b n=IO 

*=p<0.01 compared with controls 

Control 

(0) 

Males 

36.9 ± 0.24 

37.4 ± 0.34 

37.0 ± 0.40 

37.5 ± 0.69 

0.5 ± 0.1 0 

0.4 ± 0.14 

0.4±0.09 

0.5±0.10 

Females 

36.6 ± 0. 12 

37.1±0.35 

37.9 ± 0.29 

38.0 ± 0.43 

Dose level (m2/k2 bw) 

Low dose Mid dose High dose 

(125) (650) (2000) 

36.7 ± 0.37 36.8 ± 0.31 36.8± 0.27 

36.7** ± 0.42 36.5** ± 0.34 36.4** ± 0.42 

37.0 ± 0.49 37.0 ± 0.33 37.1±0.20 

37.5 ± 0.73 37.3 ± 0.50 37.4± 0.79 

0.4* ± 0.07 0.4 ± 0.11 0.5 ± 0.08 

0.4 ± 0.05 1.4 ± 2.82 2.4 ± 5.95 

0.5 ± 0.15 0.6 ±0.38 0.5 ± 0.13 

0.4 ± 0.11 0.5 ±0.11 0.5 ±0.20 

36.6±0.46 36.7 ±0.26 36.7 ±0.39 

36.6** ± 0.49 36.3** ± 0.26 35.8** ± 0.40 

37.7 ± 0.34 37.6 ± 0.33 37.6 ± 0.41 

38.3 ± 0.42 37.8 ± 0.46 38.0±0.27 

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All relevant FOB observations for individual animals have also been summarized in Tables 8-10 to display frequency of effects in 

each animal. These tables show that the incidence of treatment-related effects on sensory and open field parameters were similar in 

the low and mid dose groups. At the high dose, similar FOB signs were noted at a higher frequency along with additional effects 

that were not observed at lower doses. 

Table 8. Summary of Day 0 FOB Observations for Individual Animals in 125 mg/kg bw dose group 8 

Animal Rearing Repetitive mouth and Biting 

Number jaw movement 

40581 

40590 

40600 

40613 

40614 x 

40595 x 

40641 

40648 

•Data were extracted from 48939603, pp. 647-993. 

n= I 0 males and I 0 females 

Cage 

Hunched 

Body 

x 

x 

No Reaction/Response to: Air Righting Reflex 

Lands 

Approach Touch Startle Tail Uncoordinated Pinch on 

side 

Males 

x x x 

x x x 

x x 

x x x 

Females 

x x 

x x 

Other 

Observations 

vocalization and 

energetic 

response to tail 

pinch 

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Table 9. Summary of Day 0 FOB Observations for Individual Animals in 650 mg/kg bw Dose Group a 

Animal Repetitive mouth and jaw 

Number Rearing movement 

40575 

40580 

40584 

40588 

40597 

40610 

40611 

40618 

40631 x 

•Data were extracted from 48939603, pp. 647-993 . 

n= I 0 males and I 0 females 

Biting Hunched 

Cage Body 

x 

x 

No Reaction/Response to: Air Righting Reflex 

Approach Touch Startle Tail Uncoordinated Lands 

Pinch on side 

Males 

x x 

x x 

x x 

x x x 

x x 

x x 

x 

Females 

x x 

x 

Other 

Observations 

j catalepsy ( 1.6 

secs) 

j catalepsy (9.3 

secs) 

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Table 10. Summary of Day 0 FOB Observations for Individual Animals in 2000 mg/kg bw Dose Group a 

Animal Repetitive mouth and 

Number Rearing jaw movement 

40585 

40586 

40589 

40602 x 

40604 

40605 

40582 x 

40591 x 

40608 

40620 

40638 x 

40643 x 

40649 

40655 x 

40633 x 

40636 

40653 

40646 • x 

• Data were extracted from 48939603, pp. 647-993 

n= I 0 males and I 0 females 

Biting Hunched 

Cage Body 

x 

x 

x 

x 

x 

x 

x 

x 

x x 

x 

x 

No Reaction/Response to: Air Righting Reflex 

Tail Lands on Approach Touch Startle Pinch Uncoordinated side 

Males 

x 

x x x 

x x 

x x x 

x x x 

x x x x 

x 

x 

Females 

x x 

x x 

x x x 

x x x 

x x x 

x 

x 

Other 

Observations 

head search 

behavior 

j catalepsy 

( 1.3 secs) 

j catalepsy 

(19.3 secs) 

energetic tail 

pinch 

response 

j catalepsy 

(1.5 secs) 

no hindlimb 

extension 

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2. Motor activitv: Statistically significant differences were observed in total and ambulatory 

motor activity of male and female rats on the day of dosing (Day 0). A summary of the 

motor activity data is presented in Tables 11 and 12. 

In male rats, the linear dose by time interaction (LinTrt*Time) was significant for both total 

and ambulatory counts indicating at least one time interval in both motor activity measures 

exhibited a monotonic dose response relationship. A within-session linear trend analysis 

revealed significantly decreased total activity counts at the mid-and high-dose over intervals 

0-10 and 11-20 minutes, and statistically increased at 41-50 minutes and 51-60 minutes 

(high-dose only) compared to controls. A similar pattern was observed for within-session 

cumulative ambulatory activity counts; activity counts were statistically significantly 

decreased at the mid-and high-dose over intervals 0-10 and 11-20 minutes, and statistically 

increased in high-dose males at 41-50 and 51-60 minutes. Low dose males also exhibited 

significantly decreased ambulatory activity counts over the first interval of 0-10 minutes 

whereas total activity counts in the low dose males were comparable to controls at all time 

intervals. Despite significant differences from control in the within session analysis, 

treatment did not have a significant impact on cumulative total activity counts. Meanwhile, 

cumulative ambulatory activity counts were statistically significantly decreased at the midand high-dose (281 and 325 counts, respectively, vs. 500 counts for controls). The low dose 

cumulative ambulatory counts were comparable to controls indicating depressed activity in 

the 0-10 minute interval was not significant to the cumulative counts. 

As in male rats, the linear dose by time interaction (LinTrt*Time) was significant for both 

total and ambulatory counts. When conducting within-session analyses, mean cumulative 

total activity counts and ambulatory activity counts were statistically significantly decreased 

in females at the mid-and high-dose over intervals 0-10 and 11-20 minutes and in low-dose 

females at 11-20 minutes. Similar to the males, mean cumulative total activity counts in 

female rats were not affected by treatment, while the mean cumulative ambulatory activity 

counts were statistically significant decreased at the mid- and high-dose ( 432 and 521 counts, 

respectively, vs. 828 counts for controls) but not in the low dose. 

The cumulative mean values for total or ambulatory motor activity in males or female rats 

were not affected at the pre-test evaluation or on Days 7 or 14. No significant shifts in the 

pattern of habituation occurred in male or female dose groups on Days 7 and 14. 

Overall, within-session total and ambulatory counts were significantly different from controls 

for at least one time interval in all dose groups; however, the number of sessions affected and 

magnitude of the effect was greater at mid and high doses compared to the low dose. 

Furthermore, cumulative ambulatory counts for the entire observation period were 

significantly depressed at the mid dose and above. 

Page 26 of 30
Fluridone I 11 2900 

Acute Neurotoxicity Study (rats) (2012) I Page 24 of27 

OCSPP 870.6200/ DACO 4.5 .12/ OECD 424 

TABLE 11. Motor activity (cumulative total activity counts for session) •,b,c 

Dose level (mg/kg bw) 

Test day Control Low 

(0) (125) 

Males 

Pre-test 2105 ± 597.3 2472±909.5 

Day 0-mean 2216 ± 811.1 1994 ± 708.5 

Interval (min) 

0-10 1069 ± 202.3 959 ± 291.2 

11-20 580 ± 332.5 436 ± 274.3 

21-30 328 ± 369.3 152 ± 225.9 

31-40 120 ± 105.3 120 ± 132.8 

41-50 51 ± 50.4 207 ± 258.1 

51-60 69 ± 87.5 121 ± 128.2 

Day7 2575 ± 588.7 3084 ± 998.5 

Day 14 2496 ± 711.1 2905 ± 464.7 

Females 

Pre-test 2240 ± 478.2 2904 ± 695.7 

Day 0- mean 2856 ± 847.9 2661±975.8 

Interval (min) 

0-10 1496± 170.9 1376 ± 339.8 

11-20 761±342.7 504* ±330.8 

21-30 284 ± 350.3 248 ± 298.0 

31-40 119 ± 221.4 151 ± 221.7 

41 -50 45 ± 29.6 143 ± 227.4 

5 1-60 152 ± 360.3 239 ± 329.2 

Day7 3774± 979.2 3518 ± 916.6 

Day 14 3467 ± I I 30.3 3059 ± 1083.0 

•Data were extracted from MRID 48939603, pp. pp. 169-200 

b Values represent mean ± SD 

c n= lO 

*Statistically different from controls; p<0.05 

Mid 

(650) 

2690 ± I 153.9 

1961 ± 738.6 

823* ±239.7 

270* ± 249.1 

207±266.7 

164 ± 166.7 

275* ± 233.2 

222± 262.I 

2589 ± 523.0 

2369 ± 729.0 

2028 ±427.7 

2120 ± 724.3 

1005* ± 293.6 

293* ±260.9 

168 ± 152.5 

220 ± 373.3 

215 ± 282.5 

220 ± 245.9 

3053 ± 1071.2 

2596 ± 485.3 

High 

(2000) 

2407 ± 549.7 

2071±555.8 

775* ± 135.8 

218* ± 175.8 

222 ± 194.3 

300* ± 218.7 

264* ± 227.4 

291 * ± 291.0 

2791±1 154.0 

2536 ± 286.5 

2764 ± 955.2 

2675 ± 837.7 

1201*±293.l 

309* ± 230.2 

240± 272.4 

3 14 ± 292.2 

281 ±256.9 

33 1± 295.2 

3507 ± 1089.7 

3466 ± 897.3 

Page 27 of 30
Fluridone I 112900 

Acute Neurotoxicity Study (rats) (2012) I Page 25 of27 

OCSPP 870.6200/ DACO 4.5.12/ OECD 424 

TABLE 12. Motor activity (cumulative ambulatory activity counts for session) •,b,c 

Dose level (mg/kg bw) 

Test day Control Low 

(0) (125) 

Males 

Pre-test 489 ± 175.9 584 ± 329.5 

Day 0- mean 500 ± 268.8 365 ± 178.3 

Interval (min) 

0-10 312±96.6 235* :!: 98.7 

11-20 112 :!: 111.3 57 ± 71.0 

21-30 65 :!: 97.1 21±36.2 

31-40 10 ±21.9 16 ± 35.9 

41-50 I ± 2.8 27 :!: 43.6 

51-60 0 ± 0.3 10 ±20.7 

Day 7 566 ± 173.3 657 ± 245.2 

Day 14 544 ± 206.4 630 ± 155.3 

Fema les 

Pre-test 596± 125.9 853 :!: 303.2 

Day 0- mean 828 ± 265.I 638 ± 332.8 

Interval (min) 

0-10 501 ±71.7 41 7± 159.4 

11 -20 218 ± 117.8 109* ±90.4 

21-30 51 ± 113.4 42 ± 84.8 

31-40 16±46.0 10 ± 18.1 

41-50 0 ± 0.4 16 ± 43.1 

51-60 41 ± 130.0 45 ± 124.0 

Day7 1103 ±348.9 1002 ± 369.4 

Day 14 922 ± 320.4 846 ± 389.3 

•Data were extracted from MRID 48939603, pp. pp. 169-200 

b Values represent mean ± SD 

< n= 10 

*Statistically different from controls; p<0.05 

Mid 

(650) 

631±344.8 

281* :!: 117.0 

184* :!: 65.6 

20* ±24.3 

20 ± 37.0 

8 ± 11.9 

28 ± 32.3 

22 ± 36.8 

568 :!: 139.0 

514 :!: 148.9 

535 ± 11 4.8 

432* :!: 183.4 

253* :I: 82.6 

44* :I: 46.2 

19 ± 27.1 

50 ± 104.2 

42 ± 60.7 

25 ± 50.8 

845 ± 325.4 

702 ± 163.5 

High 

(2000) 

537 ± 213.4 

325* :!: 127.8 

162* ± 55.5 

29* :!: 31.0 

19 ± 20.2 

35 :!: 35.2 

38* :!: 36.5 

42* ± 57.6 

63 1± 391.0 

574 ± 324.6 

753 ± 327.1 

521* :I: 212.4 

341 *:I: 121.9 

44* :!: 47.3 

21 ±28.9 

31 ± 34.0 

46 ± 63.8 

38 ±29.2 

918 ± 303.0 

913 ± 270.3 

Page 28 of 30
Fluridone I 112900 

F. SACRIFICE AND PATHOLOGY: 

Acute Neurotoxicity Study (rats) (2012) I Page 26 of27 

OCSPP 870.6200/ DACO 4.5.12/ OECD 424 

1. Gross pathology: Gross necropsy findings were limited to one control male with a dark red 

discoloration of the brain. 

2. Brain weight, length, and width: No statistically significant, treatment-related changes 

were observed in brain weight, length, or width in male or female rats treated with fluridone. 

3. Neuropathology: No treatment-related changes were observed during microscopic 

examination. Findings in males were limited to a control male with minimal congestion of 

the cerebral cortex and one high-dose male with minimal congestion of the cerebellum and 

olfactory bulbs. In females, changes were limited to one control animal with minimal 

degeneration of the gastrocnemius muscle, and one high-dose female with mild congestion of 

the olfactory bulbs, another female with mild angiectasis of the cerebellum, cerebral cortex, 

and cervical and lumbar spinal cord. 

III. DISCUSSION AND CONCLUSIONS: 

A. INVESTIGATORS' CONCLUSIONS: 

Based on the results of the FOB evaluations at the time of peak effect (approximately 4 hours 

following dose administration) on study day 0, CNS activity, neuromuscular, sensorimotor, 

and/or physiological functional domains were affected at dose levels of 650 and 2000 mg/kg 

bw. FOB findings included repetitive movement of mouth and jaws, altered gait, biting of 

the cage, abnormal air righting reflex, bizarre/stereotypic behavior, no hindlimb extension, 

lack of response to various stimuli, longer catalepsy times, and/or lower mean body 

temperatures for males and females in the 650 and 2000 mg/kg bw groups. In addition, 

decreased total and ambulatory locomotor activity were noted for males and females in the 

650 and 2000 mg/kg bw groups during the first 20-30 minutes of testing and when the overall 

session (ambulatory only) was evaluated at the time of peak effect on study day 0. No test 

substance-related effects were noted for males and females at a dose level of 125 mg/kg bw. 

Therefore, the NOAEL for male and female neurotoxicity was considered to be 125 mg/kg 

bw. 

B. REVIEWER CONCLUSION'S: 

Treatment-related changes in FOB parameters were observed in males and females at all 

doses. These changes were transient, occurring only at the time of peak effect ( 4 hours after 

dosing on Day 0) and resembling pre-test observations on subsequent days. Animals from the 

low and mid dose groups exhibited similar incidence of treatment-related effects on minor 

FOB sensory and open field parameters. At the high dose, similar FOB signs were noted at a 

higher frequency and additional effects not observed at lower doses were seen. Disruption of 

physiological (increased catalepsy time, decreased body temperature) and locomotor 

(reduced cumulative ambulatory counts) function only occurred at the mid and high doses. 

The changes in FOB parameters in combination with a significant depression of cumulative 

Page 29 of 30
Fluridone / 112900 

Acute Neurotoxicity Study (rats) (20 12) I Page 27 of 27 

OCSPP 870.6200/ DACO 4.5.12/ OECD 424 

ambulatory activity counts was considered evidence of an adverse response at the mid and 

high doses. In the absence of a corroborative effect on motor activity, the FOB effects at the 

low dose were not considered adverse because the effects were limited to minor behavior 

aberrations not anticipated to impact quality of life and were of low incidence. 

The LOAEL is 650 mg/kg bw based on decreased ambulatory counts in combination 

with the prevalence of FOB anomalies in males and females. The NOAEL is 125 mg/kg 

bw. 

C. STUDY DEFICIENCIES: 

No relevant study deficiencies were noted. 

Page 30 of 30