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: NIA 

### 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 Assessment Branch VNII 

# f1**~**

# M{ /
.**~** Health Effects Division (7509P) · 

### 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

### 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 ofpeak 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 ofFOB parameters was observed in the 2000 mg/kg bw group. Home cage observations revealed clonic convulsions (repetitive movement of mouth andjaws) (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 ofhead 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 control s (35.8 v s. 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 of3Similar 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 ofthe 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 ofdepressed 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 ofcumulative ambulatory activity counts was considered evidence of an adverse response at the m id 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 oflow 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 3of3Fluridone / 112900 Acute Neurotoxicity Study (ra ts) (201 2) I Page I of2 7 OCSPP 870
. 6200/ DACO 4.5
. 12/ OEC D 424

DATA EVALUATION RECORD FLURIDONE STUDY TYPE: ACUTE NEUROTOXICITY

- RATS OCSPP 870.6200a 1§81

-8); OECD 424 MRID 48939603 Prepared for Health Effects Division Office ofPesticide 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

# ~~ture**~** Claudia Troxel
. Ph
.D
. Secondary Reviewers : 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 

# ,,,
Signatun: :

## ~ 

# "1·**~** Date:7/,i Quality Assurance: Angela M. Edmonds. B.S.

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-11-014Fluridone / 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



### DATA EV ALUATION 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 of0 (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 ofpeak 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
.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 offluridone
. These changes were transient, occurring only on Day 0 , and often disappeared by Day 7 or 14
. Anomalies in a multitude ofFOB parameters was observed in the 2000 mg/kg bw group. Home cage observations revealed clonic convulsions (repetitive movement ofmouth andjaws) (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 ofhead 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 ofthe 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 ofan adverse response at the mid and high doses. In the absence ofa corroborative effect on motor activity, the FOB effects at the low doseFluridone 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 oflow 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
.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, 2012Fluridone 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 ofthe 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) ofthe test substance in the vehicle at concentrations of 30 and 400 mg/mL were previously established. However, additional homogeneity and stability as sessments were performed as part ofthe 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 ofthe 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.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 ofthe 25 mg/mL test formulation revealed a mean concentration of24.3 mg/mL (97.2% oftarget) and a% RSD of0
. 89%. Stability analysis: Stability analysis of duplicate samples taken from the top and bottom ofthe 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 ofthese 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 % ofnominal, 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. Ifthe 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 ofmultiple 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 oftime 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)
.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. Ifthe 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 ofthe RANOVA, pairwise comparisons were made for each individual test substance-treated group with the control group through linear contrasts. IfTRT*TIME was significant, the comparisons were conducted for each time interval. Ifonly 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 ofthe 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 ofpeak 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 ofthe 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 ofthe 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

# I Fluridone I 112900 Acute Neurotoxicity Study (rats) (20 12) 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 ofthe FOB on all animals prior to the initiation of dose administration (Day -6), at the time ofpeak 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 oftreatment 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.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 offine motor skills (i
.e., grooming, interruption of 1 photobeam) and ambula tory motor activity (interruption of2 or more consecutive photobeams). 6. Sacr ifice and p athology : 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 centra l 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 co loration or lesions ofthe 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 tiss ues 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

-th igh 

### x Center of cerebrum 

### x At Sciatic Notch 

### x Midbrain 

### 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 s welling (T13-u) 

### x Lumbar dorsa l root fibers (T13-£A) Thoracic swell ing 

### x Lumbar ve ntral root fibers (T1 3 -L4) OTHER 

### x Cervical dorsal root gangla (C3-C 1) Gasserian Ganglion 

### x Cervi cal dorsal root fib ers (C3-C1) 

### x Trigeminal nerves plus ganglia 

### x Cervical ve ntral root fibers (C3

- C 1 ) 

### x Optic nerves 

### x Olfactory bulbs 

### x Eyes 

### x Hippocampus/ dentate gym s 

### x Gastrocnemius muscle 

### x Basa l ganglia 

### x Cervical spinal nerve 

### x Thalamus 

### x Lumbar sp inal nerve 

### x Hypothalamus
7
. Positive controls: Positive control data were provided in Appendix F ofMRID 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 durati on, with 6 subintervals lasting 10 minutes each. Locomotor activity was evaluated as total and ambulatory activity counts obtained over each subinterval and theFluridone I 112900 Acute Neurotoxicity Study (rats) (2012) I Page 11 of 27 OCSPP 870
. 6200/ DACO 4 .5.12/ OECD 424 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 of0, 1 or 3 mg/kg bw (males and females) on PND 61 and at doses of0, 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 ofmovement (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 ofFOB 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 ofthe same technicians. There were three phases oftesting : Phase I consisted ofFOB assessments on naive stock animals in order to ensure consistent and accurate use of all equipment and gainFluridone I 112900 Acute Neurotoxicity Study (rats ) (2012) I Page 12 of27 OC SPP 870
.6200/ DACO 4.5.12/ OECD 424 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 ofthree animals/group administered a positive control or vehicle and observed at a time designated by the Study Director; each technician individually recorded observations ofthe 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 ofthe technicians , the Study Director concluded that all but two ofthe technicians fulfilled the criteria for reliably observing FOB parameters to assess behavioral changes in rats. One ofthe two technicians was to receive additional training and demonstrated proficiency in the evaluation ofthe 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 of4/24/01 to 7

1 51

06, while the locomotor activity studies ranged over a period from 11 / 12 /07 to 10/22/ 10
. A description oftesting methods was not included. 

### II. RESULTS: 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 ab solute 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 femal e rats at any dose
. Male and female body weight gain was depre ss ed (5-2 5% 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.Fluridone I 112900 Acute Neurotoxicity Study (rats) (2012) I Page 13 of27 OCSPP 870
.6200/ DACO 4.5.12/ OECD 424 

# 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 ofmouth and jaws) and cage biting. In females, 5 animals at 2000 mg/kg bw developed clonic convulsions, with 3 ofthe 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 ofthe 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).Fluridone I I I 2900 Acute Neurotoxicity Study (rats) (2012) I Page 14 of27 OCSPP 870.6200/ DACO 4.5.12/ OECD 424

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

Convulsio ns - C lonic -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 ofcage 0 -Day 7 and Day 14 : None 10 Convu lsions

- 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 -Da y O Ab sent 10 Biting ofcage 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 oftreated 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 ofhead from side to side).Fluridone I 112900 Acute Neurotoxicity Study (rats) (2012) I Page 15 of27 OCSPP 870.6200 / DACO 4.5.12/ OECD 424



## I Open-field observations offemales 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 ofmale 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 ofresponse to stimuli in the groups oftreated rats are generally considered an effect oftreatment 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), w ith 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
.

## I Fluridone I 112900 Acute Neurotoxic ity Study (rats) (2012) I Page 16 o f 27 OCSPP 870.6200/ DACO 4.5.12/ OECD 424
TA BLE S
. F unc tional obser vat io na l batterl'. r es ults: Sen s orl'. ob servatio ns in males •,b,c 

## I D ose level m!!lki!: bw) O b servation Control Low dose M id d ose High dose (0 ) (125) (650) (2000) Approach response -Pretest: S low 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 S low approach, sniffing or turning awa y 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 s lowly 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 1 0 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 S light reaction, ear flick/evidence s nap heard 10 10 10 10 -Day 14 No reaction 0 0 0 I S light reaction , ear flick/evidence snap heard 10 10 10 9 Tail pinch response

- Pretest: Animal may s lowly 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 vocali zation 3 3 0 0

- Day 7: Animal may s lowly turn, walk away 8 9 9 9 More energetic response, with/without vocalization 2 I I I -Day 14 : Animal may slow ly turn , walk away 8 8 10 8 More energetic response, with/without vocalization 2 2 0 2

## I Fluridone I J J2900

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 Acute Neurotoxicity Study (rats) (20 12) I Page 17 of27 OCSPP 870.6200 / DACO 4.5. J2/ OECD 424

JO JO JO 9 JO 7 0 0 2 I 0 I JO JO 10

TABLE6
. Fu nctional observational batter:!'. res u lts : Sen sor:!'. o bservatio ns in females a,b,c 

## I Dose level me:/ke: bw) Observation Co ntro l Low dose Mid dose High d ose (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 -Da y 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 JOFluridone I 112900 Tail pinch respo nse -Pretest: Animal ma y slo wly tum, walk away More e nergeti c , with/without vocalization -Day 0: No reaction Animal ma y s lowly tum, walk away More energetic response, with/without vocali zation -Day 7: Animal may slowly turn , walk away More energetic response, with/without vocalization -Day 14 : Animal ma y s lowly 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 Acute Neurotoxicity Study (rats) (2012) I Page 18 of27 OCSPP 870.6200/ DACO 4
. 5 .12/ OECD 424

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 oftreated male and female rats compared to controls (Table 7). It was unclear ifthe reductions in body temperature were an adverse effect oftreatment 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 ofthe treated males and females were not indicative of an adverse effect with the exception ofthe high-do se 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 ofthe 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.Fluridone I 1 12900 Acute Neurotoxicity Study (rats) (2012) I Page 19 of27 OCSPP 870.6200/ DACO 4.5 .1 2/ OECD 424 TA B LE 7. Functio nal o bservat ional batterv res ults: Phvsioloeical observations in males a nd fe males •,b Home c age observatio n
Body temperature (0 C):

-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 Contro l (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 H ig h 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. 1 1 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±0.27Fluridone I 112900 Acute Neurotoxicity Study (rats) (2012) I Page 20 of 27 OCSPP 870.6200/ DACO 4.5.12/ OECD 424 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 oftreatment-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
. S ummary 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

Fe males 

### x x x x Other Ob servations

vocalization and energetic respon se to tail pinchFluridone I 112900 Acute Neurotoxi c ity Study (rats) (2012) I Page 21 of 27 OCSPP 870.6200/ DACO 4.5
. 12 / OECD 424

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= I0 males and I0 fe males Biting Hunched Cage Body



### x

x No Reaction/Response to: Air Righting Reflex Approach Touch Startle Tail Uncoo rdinated Lands Pinch on s ide 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)Fluridone I 112900 Acute Neurotoxicity Study (rats) (2012) I Page 22 of27 OCSPP 870.6200/ DACO 4.5.12/ OECD 424
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

Fema les 

### x x x x 

### x x x x 

### x 

### x x x x

x x Other Observa tion s

head search

behavior

j catalepsy ( 1.3 secs) j catalepsy (19
. 3 secs) energetic tai l pinch response j catalepsy (1.5 secs)

no hindlimb

extensionFluridone / 112900 Acute Neurotoxicity Study (rats) (2012) I Page 23 of27 OCSPP 870
.6200 / DACO 4.5.12/ OECD 424 

### 2. Motor activitv: Statistically significant differences were observed in total and ambulatory motor activity ofmale and female rats on the day of dosing (Day 0). A summary ofthe 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 ofhabituation 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 ofthe 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.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 (cumu lative 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 3 1-40 120 ± 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 ± 847.9 266 1 ±975.8 Interval (min) 0-10 1496± 170.9 1376 ± 339.8 11-20 76 1 ±3 42.7 504* ±330.8 21-30 284 ± 350.3 248 ± 298
.0 3 1-40 119 ± 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 ± 9 16.6 Day 14 3467 ± I I30.3 3059 ± 1083.0 •Da ta were extracted from MRID 48939603 , pp. pp . 169

-200 b Values re present mean ± SD c n= lO *Statistically different from controls ; p<0.05 Mid (650)
2690 ± I 15 3.9

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 2 120 ± 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 ±25 6 .9 33 1 ± 295 .2 3507 ± 1089.7 3466 ± 897.3Fluridone 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 Contro l 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 2 1-3 0 65 :!: 97
. 1 2 1 ±36. 2 31-40 10 ±2 1.9 16 ± 35
.9 41-50 I ± 2.8 27 :!: 43 .6 5 1-60 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 2 18 ± 117
. 8 109 * ±90
.4 2 1-3 0 51 ± 113
.4 42 ± 84.8 3 1-40 16±46.0 10 ± 18
. 1 41-50 0 ± 0.4 16 ± 43
. 1 5 1-60 4 1 ± 130.0 45 ± 124
.0 Day7 1103 ±3 48
.9 1002 ± 369.4 Day 14 922 ± 320.4 846 ± 389.3 •Data were extracted from MRID 48939603 , pp . pp. 1 69-200 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 ± 1 1.9 28 ± 32.3 22 ± 36.8 568 :!: 13 9.0 5 1

4 :!: 148.9
535 ± 11 4.8 432* :!: 183.4 253* :I: 82
. 6 44 * :I: 46 .2 19 ± 27
. 1 50 ± 10 4.2 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: 2 12
.4 341 *:I: 121.9

44* :!: 47 .3 21 ±28.9 3 1 ± 34.0 46 ± 63
. 8 38 ±29.2 9 18 ± 303 .0 913 ± 270.3Fluridone I 112900 

### F. SACRIFICE AND PATHOLOGY: Acute Neurotox ici ty Study (rats) (2012) I Page 26 of27 OCSPP 870
. 620 0 / DACO 4
. 5.12 / OECD 424

1. 

### Gross pathology: Gross necropsy findings were limited to one control male with a dark red discoloration ofthe 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 ofthe cerebellum and olfactory bulbs. In females , changes were limited to one control animal with minimal degeneration ofthe gastrocnemius muscle, and one high-dose female with mild congestion of the olfactory bulbs, another female with mild angiectasis ofthe cerebellum, cerebral cortex, and cervical and lumbar spinal cord.


### III. DISCUSSION AND CONCLUSIONS: A. INVESTIGATORS' CONCLUSIONS: Based on the results ofthe FOB evaluations at the time ofpeak 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 andjaws , 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 oftesting and when the overall session (ambulatory only) was evaluated at the time ofpeak 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 ofpeak 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 oftreatment-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 cumulativeFluridone / 112900 Acute Neurotoxicity Study (rats) (20 12) I Page 27 o f 27 OCS PP 870 .620 0 / DACO 4
. 5
. 12 / OEC D 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
.