### A3

- 1 

## Appendix 3. Weiner Study DER

DATA EVALUATION RECORD

12 PYRETHROIDS

OPPTS

STUDY TYPE: Acute Neurotoxicity (non

- guideline)

MRID 47050505 and 47050504 (range

- finding)

Prepared for

Health Effects Division

Office of Pesticide Programs

U.S. Environmental Protection Agency

One Potomac Yard

2777 S. Crystal Drive

Arlington, VA 22202

Prepared by

Toxicology and Hazard Assessment Group

Life Sciences Division

Oak Ridge National Laboratory

Oak Ridge, TN 37831

Task Order No. 206

- 2009

Primary Reviewer:

Carol S. Wood , Ph.D. , D.A.B.T.

Date: Signature:

Secondary Reviewers:

Sylvia S. Talmage , Ph.D., D.A.B.T.

Date: Signature:

Robert H. Ross, M.S., Group Leader

Date: Signature:

Quality Assurance:

Kimberly Slusher, M.S

. Date: Signature:

Disclaimer

This review may have been altered subsequent to the contractor

= s signatures above.

Oak Ridge National Laboratory managed and operated by UT

- Battelle, LLC., for the U.S. Department of

Energy under Contract No. DE

- AC05

- 00OR22725.Acute Neurotoxicity Study (rats) ( 2006 ) / Page 2 of 27

12 PYRETHROIDS OPPTS 870.6200a/( Non

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### A3

- 2 EPA Reviewer: Marquea D. King, Ph.D.

Registration Action Branch IV , Health Effects Division (7509P) Date: Signature :

EPA Work Assignment Manager : Lori Brunsman

Science Information Management Branch , Health Effects Division (7509P) Date: Signature :

Template version 0 2 /0 6



### TXR : 0055106

DATA EVALUATION RECORD

STUDY TYPE : A cute Neurotoxicity

- Rats (non

- guideline)

PC CODE : See Table Below DP BARCODE : 363024

Pyrethroid PC Code CAS No.

Beta

- Cyfluthrin 118831 68359

- 37

- 5

S

- Bioallethrin 004004 28434

- 00

- 6

Fenpropathrin 127901 39515

- 41

- 8

Lambda

- Cyhalothrin 128897 91465

- 08

- 6

Cypermethrin 109702 52315

- 07

- 8

Permethrin 109701 52645

- 53

- 1

Resmethrin 097801 10453

- 86

- 8

Deltamethrin 097805 52918

- 63

- 5

Pyrethrins 069001 8003

- 34

- 7

Tefluthrin 128912 79538

- 32

- 2

Esfenvalerate 109303 66230

- 04

- 4

Bifenthrin 128825 82657

- 04

- 3



### TEST MATERIAL (PURITY) : β

- cyfluthrin; S

- bioallethrin; f enpropathrin; λ

- cyhalothrin;

cypermethrin; permethrin; resmethrin; deltamethrin;

pyrethrins; tefluthrin; esfenvalerate; bifenthrin (purity of each

responsibility of sponsor).

SYNONYMS : none given

CITATION : Nemec, M.D. ( 200 6 ) An Acute Functional Observational Battery Comparison

Study in Rats. WIL Research Laboratories, LLC, 1407 George Road, Ashland,

OH 44805

- 9281

. Laboratory report number s: 02

- PWG

- 001 and WIL

- 409002

, April 28, 2006

. MRID 47050505

. Unpublished.

Nemec, N.D. (2005) A Range

- Finding Acute Functional Observational Battery

Study in Rats. WIL Research Laboratories, LLC, 1407 George Road, Ashland,

OH. November 3, 2005. MRID 47050504. Unpublished.

SPONSOR : Pyrethroid Working Group, Consortium No. 64977, Bev eridge and Diamond,

1350 I Street, NW, Washington, DC 20005Acute Neurotoxicity Study (rats) ( 2006 ) / Page 3 of 27

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### A3

- 3 EXECUTIVE SUMMARY :

In an acute neurotoxicity study ( MRID 47050505 )

, Groups of 10 male Crl:CD ® 

### (SD)IGS BR rats

received one of 12 pyrethroid compounds by oral gavage followed by testing in a modified

Functional Observational Battery (FOB) at the time of peak effect for each compound. Dose

levels and times of peak effect were established in a range

- finding study (MRID 4750504).

Testing of the chemicals was staggered with several chemicals tes ted at a time along with a

concurrent vehicle control group. The vehicle control was Mazola ® 

### corn oil. The dose volume

was 5 mL/kg. A complete necropsy was performed on all animals found dead or euthanized in

extremis (via carbon dioxide inhalation); su rviving animals were euthanized and discarded

without examination following the FOB evaluation. Time to peak effect and dosing was as

follows:

2 hours, β

- cyfluthrin (12.5, 25, 45 mg/kg), S

- bioallethrin (150, 200, 300 mg/kg), fenpropathrin

(15, 30 mg/kg); 4 hours, λ

- cyhalothrin (10, 20 mg/kg), cypermethrin (65, 100, 150 mg/kg),

permethrin (200, 250 mg/kg), resmethrin (350, 500, 700 mg/kg), deltamethrin (12.5, 25, 35

mg/kg), pyrethrins (400, 800 mg/kg);

8 hours, tefluthrin (10, 15 mg/kg), esfenvalerate (15, 25, 50 mg/kg), bifenthrin (40, 55 mg/kg).

β

- Cyfluthrin : One high

- dose animal was found dead; all remaining rats dosed with this chemical

survived to scheduled termination. Dose

- related weight loss was found in the mid

- and high

dose groups (

- 9 and

- 11 g , respectively versus +2 g for controls). During home cage

observations, an increased number of animals with abnormal posture and drooping eyelids were

seen at the mid

- and high

- dose; four animals of the low

- dose group were observed with clonic

convulsion s but only one each in the mid

- and high

- dose groups. In

- hand observations revealed

an increased number of animals with salivation and wet or stained fur at the mid

- and high

- dose;

two high

- dose animals also exhibited lacrimation. Dur ing open field obser vations, an increased

number of animals were seen with abnormal gait at all three dose levels and clonic convulsions,

low arousal and stereotypic behavior at the mid

- and high

- dose groups ; the number of rears was

slightly decreased in all treated groups

. All three treated groups had an increase in the number of

animals with a more energetic response to tail pinch and the mid

- and high

- dose groups had a

decrease in the air righting reflex (slightly uncoordinated). Hind limb weakness was found in

two animals in each of the mid

- and high

- dose groups and corresponded with reduced hind limb

grip strength for these groups.

S

- Bioallethrin : Two high

- dose animals were found dead; all remaining rats dosed with this

chemical survived to scheduled termination. No effects on body weight were seen. During

home cage observations, an increase in the incidences of abnormal posture, clonic convulsions,

and tremors in all three dose groups was found. An increase in the incidences of resist ance to

removal and resistance to handling in all three groups was noted during in

- hand observations.

During open field observations, an increase in the incidences of abnormal gait, clonic

convulsions, and tremors in all three dose groups were found. All three treated groups showed a

more energetic startle response, and the high

- dose group had a decreased air righting reflex.

Reduced hind limb grip strength was found in the high

- dose group.

Fenpropathrin : All animals survived to scheduled termination. No effects on body weight were

seen. During home cage observations, slightly increased incidences of abnormal posture andAcute Neurotoxicity Study (rats) ( 2006 ) / Page 4 of 27

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### A3

- 4 tremors were seen in both treated groups. While handling

, slightly increased incidences of

resistance to removal and salivation were seen in both treated groups; the low

- dose group also

had three animals with resistance to handling but none in the high

- dose group. During open field

observations, i ncreased incidences of abnormal gait, clonic convulsions, tremors, and stereotypic

behavi ors were seen in both treated groups, but were more pronounced at the high dose ; the

number of rears was slightly decreased in the high

- dose group. Both groups showed an increase

in the number of animals with a more energetic response to tail pinch. Redu ced hind limb grip

strength was found in the high

- dose group.

λ

- Cyhalothrin : All animals survived to scheduled termination. Dose

- related weight loss was

found in both groups (

- 7 and

- 9 g, respectively versus +4 g for controls). During home cage

observat ions, a dose

- related increased number of animals were found with abnormal posture and

drooping eyelids in both treated groups and clonic convulsions at the high

- dose. In

- hand

observations found a dose

- related increased number of animals with resistance to removal and

handling, salivation, and wet or stained fur in both treated groups. During open field

observations, a dose

- related increased number of animals with abnormal gait and stereotypic

behavior in both treated groups and low arousal at the high

- dos e were seen ; the number of rears

was slightly decreased in the treated groups. At the high dose, an increased number of animals

showed no reaction to startle response, touch response, approach response, olfactory orientation,

decreased hind limb extension , and greatly reduced air righting reflex. In contrast, the high

- dose

group had an increased number of animals with a more energetic reaction to tail pinch. Four

animals in the high

- dose group showed hind limb weakness and mean fore

- and hind limb grip

strengths, landing footsplay , and rotarod performance time were decreased in the high

- dose

group. Mean body temperature was decreased in the high

- dose group.

Cypermethrin : All animals survived to scheduled termination. Dose

- related weight loss was

found in the mid

- and high

- dose groups (

- 2 and

- 7 g, respectively versus +4 g for controls).

During home cage observations, the mid

- and high

- dose groups had a slight increase in the

incidences of abnormal posture, clonic convulsions, and drooping eyelid s. The mid

- and high

dose cypermethrin groups had increased incidences of resistance to removal and handling (high

dose) , salivation, wet or stained fur, and lacrimation during handling. During open field

observations, all three groups had increased inci dences of abnormal gait and low arousal and the

mid

- and high

- dose groups had a slight increase in the incidences of stereotypic behaviors ; the

number of rears was slightly decreased in all treated groups. The high

- dose group had a more

energetic reaction to startle response and tail pinch but a decrease in olfactory orientation; both

mid

- and high

- dose groups had a decrease in number of animals with hind limb extension and

normal air righting reflex. Fore

- and hind limb grip strengths were decreased in t he high

- dose

group. Rotarod performance time was reduced in the mid

- and high

- dose groups. Mean body

temperature was decreased in the high

- dose group.

Permethrin : All animals survived to scheduled termination. No change in body weight,

compared to a ga in in the controls, was observed in the high

- dose group. During home cage

observations, both treated groups showed an increase in clonic convulsions and tremors. Both

treated groups showed an increase in resistance to removal from the cage. During open field

observations, both groups showed an increase in abnormal gait, clonic convulsions, and tremors;

two high

- dose animals also displayed stereotypic behavior ; the number of rears was slightlyAcute Neurotoxicity Study (rats) ( 2006 ) / Page 5 of 27

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### A3

- 5 decreased in all treated groups. In the high

- dose group, an i ncreased number of animals showed

more energetic approach and startle responses.

Resmethrin : One high

- dose animal was found dead; all remaining rats dosed with this chemical

survived to scheduled termination. No change in body weight, compared to a gain in the

controls, was observed in the mid

- and high

- dose groups. During home cage observations, an

increased incidence was found of abnormal posture at the mid

- and high

- dose, and clonic

convulsions and tremors at all three doses. An increased incidence o f red deposits around the

nose in the mid

- and high

- dose groups was seen during handling. During open field

observations, an increased incidence of abnormal gait, clonic convulsions, and tremors at all

three doses was found ; the number of rears was slight ly decreased in all treated groups. An

increase in the number of animals with a more energetic startle response was observed at all

three doses. Rotarod performance time was reduced in the mid

- and high

- dose groups.

Deltamethrin : All animals survived to scheduled termination. Dose

- related weight loss was

found in all three treated groups (

- 3,

- 16, and

- 18 g, respectively versus +4 g for controls).

During home cage observations, increased incidences of abnormal posture and clonic

convulsions at the mid

- and high

- doses, and biting and drooping eyelids at the high dose were

observed. In

- hand observations showed that incidences of resistance to removal and handling

were increased in all three dose groups, and salivation, wet or stained fur, and red deposit s

around the nose in the mid

- and high

- dose groups were increased. During open field

observations, incidences of abnormal gait were increased in all three groups and clonic

convulsions, low arousal, and stereotypic behavior were increased at the mid

- and high

- doses ;

the number of rears was slightly decreased in all treated gr oups. The high

- dose group showed a

marked increase in the mean time to first step. The high

- dose group had an increased number of

animals with more energetic touch and startle respon ses as well as an absence of hind limb

extension and all three groups had an increase tail pinch response ; greatly diminished air righting

reflex was found in the mid

- and high

- dose groups . Two mid

- dose and seven high

- dose animals

showed hind limb weakness and mean fore

- and hind limb grip strengths were decreased in both

groups and landing footsplay was reduced in the high

- dose group. Rotarod performance time

was reduced in all groups. Mean body temperature was decreased in the mid

- and high

- dos e

groups. An increase in the mean catalepsy time was measured in the high

- dose group.

Pyrethrins : All animals survived to scheduled termination. No effects on body weight were seen.

During home cage observations, all high

- dose animals showed clonic con vulsions. Both treated

groups had increased incidences of resistance to removal and handling. During open field

observations, high

- dose animals showed tremors, and both treated groups had a low incidence of

animals with abnormal gait and clonic convulsio ns. Both groups had a slight increase in the

number of animals with a more energetic startle response.

Tefluthrin : All animals survived to scheduled termination. No change in body weight, compared

to a gain in the controls, was observed in both groups. During home cage observations, slight

increases in the incidence of abnormal posture, clonic convulsions, and tremors were found in

the high

- dose group. The high

- dose group had an increased incidence of resistance to removal

from the cage. During open f ield observations, both treated groups had increases in the

incidences of abnormal gait, clonic convulsions, and tremors and the high

- dose group had twoAcute Neurotoxicity Study (rats) ( 2006 ) / Page 6 of 27

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### A3

- 6 animals with stereotypic behavior ; the number of rears was slightly decreased in all treated

groups.

Esfenvalerate : All animals survived to scheduled termination. No change in body weight,

compared to a gain in the controls, was observed in the mid

- and high

- dose groups. During

home cage observations, slight increases in the incidence of abnormal posture, clonic

convulsions, and tremors were found in high

- dose group. During in

- hand observations, increases

in the incidence of resistance to handling and removal, and saliv ation were noted in the mid

- and

high

- dose groups and wet or stained fur was seen in the high

- dose group. During open field

observations, treatment resulted in abnormal gait at all three doses, and clonic convulsions,

tremors, and stereotypic behaviors in the mid

- and high

- dose groups; additionally three high

dose animals had low arousal ; the number of rears was slightly decreased in the mid

- and high

dose groups. Forelimb grip strength was reduced in the high

- dose group.

Bifenthrin : One high

- dose animal was found dead; all remaining rats dosed with this chemical

survived to scheduled termination. Dose

- related weight loss was found in both dose groups (

- 7

and

- 13 g, respectively versus + 5 g for controls). During home cage observations, both treated

grou ps showed marked dose

- related increased incidences of abnormal posture, clonic

convulsions, and tremors. Both treated groups showed dose

- related increased incidences of

resistance to removal and handling, salivation, and wet or stained fur. During open f ield

observations, both treated groups showed marked dose

- related increased incidences of abnormal

gait, clonic convulsions, tremors, and stereotypic behavior; two high

- dose animals had low

arousal ; the number of rears was slightly decreased in the high

- dose group. Both groups had an

increase in the number of animals with a more energetic startle response and an uncoordinated

air righting reflex. Forelimb grip strength was reduced in both groups.

A NOAEL/LOAEL could not be established for most compounds as effects were observed at all

doses tested. However

, it was not the intent of the study to establish a NOAEL or to clearly

define the dose

- response for each compound. The results of the study d emonstrated that the

FOB can be used to evaluate the acute neurotoxic effects of pyrethroid compounds.

This neurotoxicity study is classified as Acceptable/N on

- guideline and does not satisfy the

guideline requirement for an acute neurotoxicity study in ra ts (870.6200; OECD 424). The study

fulfilled the purpose for which it was intended, evaluation of the acute neurotoxic potential of the

test articles in a FOB comparison using the same methodology and dosing regimen

.

COMPLIANCE : Signed and dated GLP, Qu ality Assurance, and Data Confidentiality

statements were provided

.Acute Neurotoxicity Study (rats) ( 2006 ) / Page 7 of 27

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### A3

- 7 I. MATERIALS AND METHODS :

A. MATERIALS :

The following test substances which appear in the table below were evaluated in this study. Purity

and stability verification for the test substances were confirmed by the respective suppliers by

Certificates of Analysis or GLP analysis. All test substance formulations were stirred continuously

throughout the preparation sampling and dose administration procedures allowing for uniform

suspens ions or solutions. Dose volumes for each animal were based on the technical grade material

and were not adjusted for purity.



### 1. Test m aterial Pyrethrins :

Description: Clear, viscous, yellowish

- orange liquid

Lot/b atch #: 2000/4.2

Purity: 53.755% a.i.



### Test material: S

- bioallethrin

Description: Clear

, yellow liquid

Lot/b atch #: 9N0357B

Purity: 95.9% a.i.



### Test m aterial Permethrin Technical :

Description: Clear , light brown liquid

Lot/b atch #: PL00

- 0507

Purity: Not given



### Test m aterial Deltamethrin :

Description: Fine, off

- white powder

Lot/b atch #: ABDL0026

Purity: >99.6% a.i.

CAS # of TGAI: 52918

- 63

- 5

Structure:



### Test m aterial Beta

- cyfluthrin :

Description: Fine, off

- white powder

Lot/b atch #: AS024

Purity: 96.8% a.i.

CAS # of TGAI: 68359

- 37

- 5

Structure:



### Test m aterial Tefluthrin :

Description: Very light brown solid

Lot/b atch #: Y01059/143/002

Purity: 92.4% a.i.



### Test m aterial Cypermethrin Technical :

Description: Clear , brown, viscous liquidAcute Neurotoxicity Study (rats) ( 2006 ) / Page 8 of 27

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### A3

- 8 Lot/b atch #: PL00

- 0508

Purity: Not given



### Test m aterial Lambda

- cyhalothrin :

Description: Y ellow solid

Lot/b atch #: Y02637/321/001

Purity: 87.7% a.i.



### Test m aterial Fenpropathrin Technical :

Description: Yellow to orange fine powder with dark solids

Lot/b atch #: AS 459K

Purity: 90.8% a.i.



### Test m aterial Esfenvalerate Technical :

Description: Waxy, light brown solid

Lot/b atch #: YB656

- 84

Purity: 98.58% a.i.



### Test m aterial Bifenthrin Technical :

Description: Light yellow sol id

Lot/b atch #: PL00

- 0509

Purity: Not given



### Test m aterial Resmethrin :

Description: Brown solid containing dark brown solids

Lot/b atch #: 99N0803B

Purity: 91.9% a.i.



### 2. Vehicle and/or positive control :

Mazola ® 

### corn oil (Bestfoods, Englewood Cliffs, New Jersey) was used as the vehicle and

negative control. A positive control was not used in this study.

3. Test animals :

Species: Rat (males only)

Strain: Crl: CD ® (SD)IGS BR

Age/weight at dosing: 6 weeks; 167

- 279 g

Source: Charles River Laboratories, Inc., Raleigh, NC

Housing: Individually in wire

- mesh cages suspended above cageboard

Diet: Certified Rodent LabDiet ® 5002, PMI Nutrition International, Inc. ad libitum

Water: Reverse

- osmosis treated municipal water, ad libitum

Environmental conditions: Temperature:

Humidity:

Air changes:

Photoperiod: 19.6

- 23.2 **Ε** C

40.7

- 65.6 %

Not given

12 hrs dark/ 12 hrs light

Acclimation period: Minimum of 14 days



### B. STUDY DESIGN :

1. In life dates : Start: May 7, 2002 ; End: September 28, 2002

2. Animal assignment and treatment : Animals were assigned to the test groups noted in

Table 1 by a computerized random sort program based on body weight such that i ndividualAcute Neurotoxicity Study (rats) ( 2006 ) / Page 9 of 27

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### A3

- 9 body weig ht was within ± 20% of the mean. R ats were given a single dose by gavage in corn

oil at a volume of 5 mL/kg followed by FOB testing at the time of peak effect. Dose levels

and time to peak effect were chosen based on a range

- finding study ( MRID 47050504; see

Appendix). Following the FOB, s urvivors were sacrificed and discarded without necropsy.

The study was conducted in blocks of one to three test articles; each block had a concurrent

control group. Each control and treated group consisted of 10 males.

TABLE 1. Study design for male rats treated with a pyrethroid prior to FOB testing

Test article Dose Levels

(mg/kg) Number of

males Time of peak effect (hr)

Vehicle 0 10 /time 2, 4, 8

β

- Cyfluthrin 12.5, 25, 45 10/dose 2

S

- bioallethrin 150, 200, 300 10/dose 2

Fenpropathrin 15, 30 10/dose 2

λ

- Cyhalothrin 10, 20 10/dose 4

Cypermethrin 65, 100, 150 10/dose 4

Permethrin 200, 250 10/dose 4

Resmethrin 350, 500, 750 10/dose 4

Deltamethrin 12.5, 25, 35 10/dose 4

Pyrethrins 400, 800 10/dose 4

Tefluthrin 10, 15 10/dose 8

Esfenvalerate 15, 25, 50 10/dose 8

Bifenthrin 40, 50 10/dose 8

Data from text table p. 12, MRID 47050505.



### 3. T est Substance p reparation and a nalysis : Dose formulations were prepared as

weight/volume mixtures. The appropriate amount of the test article was ground using a

mortar and pestle, if necessary, and weighed into a storage container. A sufficient amount of

vehicle was added and the formulation mixed overnight using a magnetic stirrer. All

formulations were stirred continuously throughout dosing. Dose calculations were not

adjusted for purity. Samples of each dosing formulation were collected on the day of

preparation, but were not analyzed for concentration, stability, or homogeneity.

Results

Homogeneity a nalysis: not performed :

Stability an alysis: not performed

Concentration a nalysis: not performed

4. Statistics : Data are presented as mean ± SD (standard deviation) and the number of animals

used to calculate the mean. Standard statistical an alyses typically performed on FOB data were

not conducted. The study notes that due to the complexity of the data produced, the sponsor

analyzed the data set using Principle Component Analysis and Factor Analysis. Some plots from

that analysis are extracte d here. 

### This analysis is reported separately and was not available for

the current review, therefore, EPA has not performed a formal review of the PCA data (i.e.,Acute Neurotoxicity Study (rats) ( 2006 ) / Page 10 of 27

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### A3

- 10 repeated the analysis) but believes that the procedures conducted are a reasonable approach to

evaluating these data. (See Breckenridge, C., and Holden, L., 2007. Evidence for separate

mechanisms of action for Type I and Type II pyrethroid insecticides, PW G study number PWG

TOX

— 2007

- 001).



### C. METHODS / OBSERVATIONS :

1. Mortality and c linical o bservations : Animals were observed twice daily for mortality and

morbidity. Detailed clinical observations were recorded daily except on the day of the FOB.

2. Body weight : Animals were weighed at randomization and prior to dose administration.

Body weight was also recorded on the day the FOB was performed and prior to sacrifice.

3. Food consumption : Fo od consumption was not measured.

4. Cholinesterase d eter mination : Cholinesterase activity was not determined

.

5. Neurobehavioral a ssessment :

a. Functional Observational Battery (FOB ): The special FOB was conducted at the time of

peak effect for each test article. Testing was performed without the technician’s knowledge

of dose group assignment and inter

- observer reliability was established to verify consistency

among the technicians by verification of training in the laboratory with standard pyrethroids.

The FOB was performed in a sound

- attenuated room equipped with a white noise generator

set to operate at 70 ± 10 dB, while home cage observations were performed in the animal

room. Study technicians were given special training to distinguish classical symptoms of

pyrethroid intoxication. Duration of the open fie ld observation was two minutes.

Detailed scoring criteria were included for each parameter. The standard FOB parameters appear

in Table 2. The data from the enhanced FOB were divided into 56 numerical analysis variables

summarized in Table 3 and the corr elations between pairs of the four final factors are shown in

Figure 1 in the results section. The FOB was modified to include additional details (e.g., the

coarseness of tremor) to distinguish findings particularly associated with pyrethroid intoxication.

For instance, tremors were graded during home cage observations on a scale of coarseness:

slight, moderate and marked, and extreme. Duration of the open field observation was two

minutes. Detailed scoring criteria were included for each parameter. The CHECKED (X)

parameters were examined

.Acute Neurotoxicity Study (rats) ( 2006 ) / Page 11 of 27

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### A3

- 11 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/ ge n eral activity level*

X Tremors* X Piloerection* X Convulsions*

Abnormal Movements* X Fur appearance X Tremors*

X Palpebral closure* X Palpebral closure* X Abnormal movements*

X F eces consistency X Respiratory rate+ X Urination / defecation*

X Red/crusty deposits* X Grooming

SENSORY OBSERVATIONS X Mucous membranes /eye /skin

color X Gait abnormalities / posture*

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

X Touch response+ X Muscle tone* X Bizarre / stereotypic behavi o r*

X Startle response* X Backing

X Pain response* PHYSIOLOGICAL

OBSERVATIONS X Time to first step

X Pupil response* X Body weight*

X Eye blink response X Body temperature+ NEUROMUSCULAR

OBSERVATIONS

X Forelimb extension X Hind limb extensor strength

X Hind limb extension OTHER OBSERVATIONS X Forelimb grip strength*

X Air righting reflex+ X Catalepsy X Hind limb grip strength*

X Olfactory orientation X Landing foot splay*

X Rotarod performance

* R equired parameters; +Recommend e d parameters



### b. Locomotor a ctivity : Locomotor a ctivity was not ev aluated.

6. Sacrifice and p athology : Animals found dead were subjected to a complete gross necropsy.

All surviving animals were sacrificed by carbon dioxide inhalation after completion of the

FOB and disc arded without examination.

7. Positive c ontrols : Positive control data were not included. The reported stated that the

technicians were trained to reliably identify all clinical signs of neurotoxicity typically

expected for pyrethroids and that inter

- obs erver reliability had been established. Reference

was made to two validation studies, WIL

- 409006 and WIL

- 409007 conducted in 2001. It is

assumed that these studies as well as other positive control data from the testing facility have

been submitted to EP A and are considered adequate.

II. RESULTS :

A. OBSERVATIONS :

1. Clinical signs : Treatment

- related clinical signs were only observed in a few groups. At 25

and 35 mg deltamethrin/kg, dried yellow matting on the ventral abdominal area was seen in 4

and 6 animals, respectively; dried brown material at the base of the tail was seen in 5 and 5

animals, respectively; and dried red material around the eyes and nose was seen in 2 and 2

animals, respectively. Tremors were observed in one animal administered 55 mg

bifenthrin/mg.Acute Neurotoxicity Study (rats) ( 2006 ) / Page 12 of 27

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- 12 2. Mortality : A total of five rats were found dead during the study, one each in the 45 mg β

cyfluthrin/kg, 750 mg resmethrin/kg, and 55 mg bifenthrin/kg groups and two in the 300 mg

S

- bioallethrin group.

B. BODY WEIGHT AND BODY WEIGHT GAIN :

Selected body weight data are shown in Table 3. Only the groups which either lost weight or

did not gain weight between dosing (day 0) and sacrifice (day 1) are shown with their

concurrent control group. Dose

- related weight loss was found in the mid

- and high

- dose β

cyfluthrin groups, both λ

- cyhalothrin groups, the low

- , mid

- , and high

- dose cypermethrin

groups, the low

- , mid

- , and high

- dose deltamethrin groups, and both bifenthrin groups. No

change in bod y weight, compared with a slight gain in the controls, was seen in the high

- dose

permethrin group, the mid

- and high

- dose resmethrin groups, both tefluthrin groups, and the

mid

- and high

- dose esfenvalerate groups.

TABLE 3. Selected b ody weight data (g ± SD )

Treatment group Day 0 (prior to dosing) Day 1 (post

- FOB)

Vehicle 216 ±11.6 218 ±12.6

β

- Cyfluthrin, 25 mg/kg 215 ± 15.2 206 ±20.0

β

- Cyfluthrin, 45 mg/kg 216 ±13.9 205 ±15.5

Vehicle 219 ±17.9 223 ±17.5

λ

- Cyhalothrin, 10 mg/kg 217 ±23.4 210 ±25.2

λ

- Cyhalothrin, 20 mg/kg 219 ±11.6 200 ±11.0

Cypermethrin, 100 mg/kg 220 ±11.8 218 ±12.5

Cypermethrin, 150 mg/kg 222 ±16.8 215 ±16.8

Permethrin, 250 mg/kg 217 ±18.9 216 ±20.6

Vehicle 243 ±14.0 247 ±14.7

Resmethrin, 500 mg/kg 238 ±18.2 232 ±24.8

Resmethrin, 750 mg/kg 241 ±13.6 240 ±13.1

Deltamethrin, 25 mg/kg 242 ±16.3 226 ±25.9

Deltamethrin, 35 mg/kg 241 ±18.8 223 ±14.4

Vehicle 233 ±12.7 238 ±11.6

Tefluthrin, 10 mg/kg 230 ±13.2 229 ±10.9

Tefluthrin, 15 mg/kg 230 ±18.5 226 ±22.9

Esfenvalerate, 25 mg/kg 229 ±16.0 227 ±16.9

Esfenvalerate, 50 mg/kg 232 ±16.1 226 ±17.4

Bifenthrin, 40 mg/kg 232 ±14. 7 225 ±14.6

Bifenthrin, 55 mg/kg 231 ±18.6 218 ±18.8

Vehicle 197 ±10.4 201 ±10.7

Deltamethrin, 12.5 mg/kg 197 ±11.9 194 ±9.8

Vehicle 210 ±14.8 211 ±15.0

Cypermethrin, 65 mg/kg 209 ±15.5 207 ±14.3

Data taken from Table 3, pp. 113

- 120, MRID 47050505.Acute Neurotoxicity Study (rats) ( 2006 ) / Page 13 of 27

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- 13 C. FOOD CONSUMPTION :

Food consumption was not measured.

D. CHOLINESTERASE ACTIVITIES :

Cholinesterase activity was not measured.

E. NEUROBEHAVIORAL RESULTS :

1. FOB f indings :

Home cage observations : Findings during home cage observation s are shown in Table 4. β

Cyfluthrin resulted in an increased number of animals with abnormal posture and drooping

eyelids at the mid

- and high

- dose; four animals of the low

- dose group were observed with

clonic convulsions but only one each in the mid

- and high

- dose groups. Treatment with S

bioallethrin caused an increase in the incidences of abnormal posture, clonic convulsions, and

tremors in all three dose groups. Slightly inc reased incidences of abnormal posture and

tremors were seen in both fenpropathrin treated groups. λ

- Cyhalothrin resulted in a dose

related increased number of animals with abnormal posture and drooping eyelids in both

treated groups and clonic convulsions at the high

- dose. The mid

- and high

- dose

cypermethrin groups had a slight increase in the incidences of abnormal posture, clonic

convulsions, and drooping eyelids. Both groups treated with permethrin showed an increase

in clonic convulsions and tremors. Administration of resmethrin resulted in an increased

incidence of abnormal posture at the mid

- and high

- dose, and clonic convulsions and tremors

at all three doses. With deltamethrin, incidences of abnormal posture and clonic convulsions

at the mid

- an d high

- doses, and biting and drooping eyelids at the high dose were observed.

All animals treated with the highest dose of pyrethrins showed clonic convulsions. Slight

increases in the incidence of abnormal posture, clonic convulsions, and tremors were f ound

in both the high

- dose tefluthrin and high

- dose esfenvalerate groups. Both groups treated with

bifenthrin showed marked dose

- related increased incidences of abnormal posture, clonic

convulsions, and tremors.Acute Neurotoxicity Study (rats) ( 2006 ) / Page 14 of 27

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- 14 TABLE 4. Findings during home cage observa tion (no. animals showing sign /no. animals )

Treatment group Abnormal

posture Clonic

convulsions a Tremors b Biting Drooping

eyelids

Vehicle 0/10 0/10 0/10 0/10 0/10

β

- Cyfluthrin, 25 mg/kg 5/10 1/10 1/10 0/10 4/10

β

- Cyfluthrin, 45 mg/kg 7/10 1/10 1/10 1/10 2/10

S

- Bioallethrin, 200 mg/kg 2/10 6/10 5/10 0/10 0/10

S

- Bioallethrin, 300 mg/kg 3/10 6/10 6/10 0/10 0/10

Fenpropathrin, 15 mg/kg 2/10 0/10 2/10 1/10 0/10

Fenpropathrin, 30 mg/kg 1/10 1/10 3/10 0/10 0/10

Vehicle 7/10 0/10 0/10 0/10 6/10

β

- Cyfluthrin, 12. 5 mg/kg 2/10 4/10 0/10 2/10 0/10

S

- Bioallethrin, 15 0 mg/kg 3/10 2/10 3/10 0/10 2/10

Vehicle 1/10 0/10 0/10 0/10 1/10

λ

- Cyhalothrin, 10 mg/kg 3/10 1/10 1/10 0/10 2/10

λ

- Cyhalothrin, 20 mg/kg 6/10 3/10 1/10 1/10 4/10

Cypermethrin, 100 mg/kg 2/10 3/10 1/10 1/10 2/10

Cypermethrin, 150 mg/kg 4/10 3/10 1/10 0/10 2/10

Permethrin, 200 mg/kg 1/10 3/10 4/10 0/10 0/10

Permethrin, 250 mg/kg 2/10 6/10 9/10 1/10 0/10

Vehicle 3/10 0/10 0/10 0/10 5/10

Cypermethrin, 65 mg/kg 1/10 0/10 0/10 0/10 1/10

Vehicle 0/10 0/10 0/10 0/10 0/10

Resmethrin, 500 mg/kg 5/10 7/10 7/10 0/10 1/10

Resmethrin, 750 mg/kg 2/10 7/10 9/10 0/10 1/10

Deltamethrin, 25 mg/kg 5/10 2/10 0/10 0/10 0/10

Deltamethrin, 35 mg/kg 9/10 4/10 1/10 3/10 4/10

Pyrethrins, 400 mg/kg 0/10 1/10 1/10 0/10 0/10

Pyrethrins, 800 mg/kg 1/10 1/10 2/10 0/10 0/10

Vehicle 1/10 1/10 0/10 1/10 1/10

Resmethrin, 350 mg/kg 1/10 3/10 4/10 0/10 1/10

Deltamethrin, 12.5 mg/kg 3/10 0/10 0/10 0/10 2/10

Vehicle 0/10 0/10 0/10 0/10 0/10

Tefluthrin, 10 mg/kg 1/10 1/10 2/10 0/10 0/10

Tefluthrin, 15 mg/kg 3/10 2/10 3/10 0/10 1/10

Esfenvalerate, 25 mg/kg 1/10 0/10 0/10 0/10 0/10

Esfenvalerate, 50 mg/kg 2/10 3/10 2/10 1/10 0/10

Bifenthrin, 40 mg/kg 3/10 5/10 6/10 0/10 0/10

Bifenthrin, 55 mg/kg 4/10 9/10 10/10 0/10 1/10

Vehicle 3/10 0/10 0/10 1/10 1/10

Esfenvalerate, 15 mg/kg 0/10 0/10 0/10 0/10 0/10

Data taken from Table 4, pp. 121

- 134, MRID 47050505.

a Includes lying on side or curled up, alert, sitting with head held low, rearing, and prostration.

b Includes twitches of back, head, or body, jerking movements of limbs, whole

- body convulsions, and repetitive

chewing movements.Acute Neurotoxicity Study (rats) ( 2006 ) / Page 15 of 27

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- 15 Handling observations : Observations recorded while the animals were being handled are

shown in Table 5. β

- Cyfluthrin resulted in an increased number of animals with salivation

and wet or stained fur at the mid

- and high

- dose ; two high

- dose animals also exhibited

lacrimation . Treatment with S

- bioallethrin caused an i ncrease in the incidences of resistance

to removal and resistance to handling in all three groups. Slightly increase d incidences of

resistance to removal and salivation were seen in both fenpropathrin treated groups ; the low

dose group also had three anim als with resistance to handling but none in the high

- dose

group

. λ

- Cyhalothrin resulted in a dose

- related increased number of animals with resistance

to removal and handling, salivation, and wet or stained fur in both treated groups . The mid

and high

- do se cypermethrin groups had increase d incidences of resista nce to removal and

handling (high

- dose) , salivation, wet or stained fur , and lacrimation

. Both groups treated

with permethrin showed an increase in resistance to removal. The only clear effect from

a dministration of resmethrin was an increased incidence of red deposits around the nose in

the mid

- and high

- dose groups . With deltamethrin , incidences of resistance to removal and

handling were increased in all three dose group s, and salivation, wet or stained fur, and red

deposits around the nose in the mid

- and high

- dose groups were increased

. Both groups

treated with pyrethrins had increased incidences of resistance to removal and handling. The

high

- dose tefluthrin group had an increased inciden ce of resistance to removal from the cage.

I ncreases in the incidence of resistance to handling and removal, and salivation were noted in

the mid

- and high

- dose esfenvalerate groups and wet or stained fur was seen in the high

- dose

group. Both groups trea te d with bifenthrin showed dose

- related increased incidences of

resistance to removal and handling, salivation, and wet or stained fur.Acute Neurotoxicity Study (rats) ( 2006 ) / Page 16 of 27

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- 16 TABLE 5

. Handling observation s (no. animals showing sign/no. animals)

Treatment group Resistance to

removal Resistance to

handling Salivation Fur

wetness/staining Red deposit

– nose

Vehicle 1 /10 3 /10 0/10 0/10 0/10

β

- Cyfluthrin, 25 mg/kg 0/ 10 0 /10 4 /10 2 /10 0 /10

β

- Cyfluthrin, 45 mg/kg 1 /10 1 /10 10 /10 5 /10 0 /10

S

- Bioallethrin, 200 mg/kg 3 /10 2 /10 2 /10 0/10 0/10

S

- Bioallethrin, 300 mg/kg 2 /10 4 /10 1 /10 0/10 0/10

Fenpropathrin, 15 mg/kg 4 /10 3 /10 2 /10 0 /10 0/10

Fenpropathrin, 30 mg/kg 2 /10 0 /10 2 /10 0/10 0/10

Vehicle 1 /10 2 /10 0/10 0/10 0 /10

β

- Cyfluthrin, 12. 5 mg/kg 2/10 3 /10 0/10 0 /10 0/10

S

- Bioallethrin, 15 0 mg/kg 5 /10 3 /10 0 /10 0/10 1 /10

Vehicle 2/10 0/10 1 /10 0/10 0 /10

λ

- Cyhalothrin, 10 mg/kg 6/10 2 /10 4 /10 4 /10 0 /10

λ

- Cyhalothrin, 20 mg/kg 4/10 5 /10 1 0 /10 9 /10 0 /10

Cypermethrin, 100 mg/kg 5/10 0 /10 2 /10 6 /10 0 /10

Cypermethrin, 150 mg/kg 6/10 2 /10 4 /10 8 /10 0 /10

Permethrin, 200 mg/kg 3/10 0 /10 0 /10 1 /10 0/10

Permethrin, 250 mg/kg 9/10 0 /10 1 /10 1/10 0/10

Vehicle 1 /10 1 /10 0/10 0/10 0 /10

Cypermethrin, 65 mg/kg 2 /10 2 /10 1 /10 0/10 0 /10

Vehicle 2 /10 2 /10 0/10 0/10 0/10

Resmethrin, 500 mg/kg 2 /10 5 /10 1 /10 0/10 3 /10

Resmethrin, 750 mg/kg 2/10 3 /10 0 /10 1 /10 3 /10

Deltamethrin, 25 mg/kg 6 /10 2/10 5 /10 6 /10 2 /10

Deltamethrin, 35 mg/kg 6 /10 3 /10 1 0 /10 10 /10 2 /10

Pyrethrins, 400 mg/kg 2 /10 4 /10 0 /10 1 /10 0/10

Pyrethrins, 800 mg/kg 4 /10 4 /10 1 /10 1 /10 2 /10

Vehicle 1/10 3 /10 0/10 0 /10 0 /10

Resmethrin, 350 mg/kg 1/10 1 /10 0 /10 0/10 0 /10

Deltamethrin, 12.5 mg/kg 3/10 2 /10 1 /10 0/10 0 /10

Vehicle 1 /10 4 /10 1 /10 0/10 1 /10

Tefluthrin, 10 mg/kg 2 /10 1/10 0 /10 1 /10 0/10

Tefluthrin, 15 mg/kg 5 /10 2/10 0 /10 2 /10 0 /10

Esfenvalerate, 25 mg/kg 4 /10 2 /10 4 /10 0/10 0/10

Esfenvalerate, 50 mg/kg 4 /10 4 /10 5 /10 5 /10 1 /10

Bifenthrin, 40 mg/kg 4 /10 4 /10 4 /10 3 /10 4 /10

Bifenthrin, 55 mg/kg 5 /10 4 /10 2 /10 6 /10 1/10

Vehicle 0 /10 3 /10 0/10 0 /10 0 /10

Esfenvalerate, 15 mg/kg 0/10 1 /10 1 /10 0/10 0/10

Data taken from Table 6 , pp. 1 49

- 1 72 , MRID 47050505.Acute Neurotoxicity Study (rats) ( 2006 ) / Page 17 of 27

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- 17 Open field observations : Findings during the open field observation are shown in Table 6. β

Cyfluthrin resulted in an increased number of animals with abnormal gait at all three dose

levels and clonic convulsions, low arousal and stereotypic behavior at the mid

- and high

- dose

groups. Treatment with S

- bioallethrin caused an increase in the incid ences of abnormal gait,

clonic convulsions, and tremors in all three dose groups. Increased incidences of abnormal

gait, clonic convulsions, tremors, and stereotypic behaviors were seen in both fenpropathrin

treated groups, but were more pronounced at the high dose. λ

- Cyhalothrin resulted in a dose

related increased number of animals with abnormal gait and stereotypic behavior in both

treated groups and low arousal at the high

- dose. All three cypermethrin groups had increased

incidences of abnormal gait and low arousal and t he mid

- and high

- dose groups had a slight

increase in the incidences of stereotypic behaviors. Both groups treated with permethrin

showed an increase in abnormal gait, clonic convulsions, and tremors; two high

- dose animals

also displa yed stereotypic behavior. Administration of resmethrin resulted in an increased

incidence of abnormal gait, clonic convulsions

, and tremors at all three doses. With

deltamethrin, incidences of abnormal gait were increased in all three groups and clonic

c onvulsions , low arousal, and stereotypic behavior were increased at the mid

- and high

doses. Animals treated with the highest dose of pyrethrins showed tremors, and both treated

groups had a low incidence of animals with abnormal gait and clonic convulsio ns. Both

groups treated with tefluthrin had increase d in the incidence s of abnormal gait , clonic

convulsions, and tremors and the high

- dose group had two animals with stereotypic behavior.

Treatment with e sfenvalerate resulted in abnormal gait at all thr ee doses, and clonic

convulsions, tremors, and stereotypic behaviors in the mid

- and high

- dose groups ;

additionally three high

- dose animals had low arousal . Both groups treated with bifenthrin

showed marked dose

- related increase d incidences of abnormal ga it , clonic convulsions,

tremors , and stereotypic behavior; two high

- dose animals had low arousal

.Acute Neurotoxicity Study (rats) ( 2006 ) / Page 18 of 27

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- 18 TABLE 6

. Open field observation s (no. animals showing sign/no. animals)

Treatment group Abnormal

gait Clonic

convulsions a Tremors b Low arousal Bizarre

behavior

(stereotypic)

Vehicle 1 /10 0 /10 0/10 0/10 0/10

β

- Cyfluthrin, 25 mg/kg 7/ 10 5 /10 0 /10 2 /10 4 /10

β

- Cyfluthrin, 45 mg/kg 8 /10 2 /10 0 /10 5 /10 4 /10

S

- Bioallethrin, 200 mg/kg 5 /10 5 /10 6 /10 1 /10 0/10

S

- Bioallethrin, 300 mg/kg 6 /10 7 /10 7 /10 0/10 0/10

Fenpropathrin, 15 mg/kg 1 /10 2 /10 1 /10 0 /10 2 /10

Fenpropathrin, 30 mg/kg 4 /10 6 /10 4 /10 0/10 2 /10

Vehicle 1/10 0 /10 0/10 0/10 0/10

β

- Cyfluthrin, 12. 5 mg/kg 4 /10 0 /10 2 /10 0/10 0/10

S

- Bioallethrin, 15 0 mg/kg 4 /10 3/10 4 /10 0/10 0 /10

Vehicle 0 /10 0/10 0 /10 0/10 0/10

λ

- Cyhalothrin, 10 mg/kg 5 /10 0 /10 0 /10 1 /10 6 /10

λ

- Cyhalothrin, 20 mg/kg 10 /10 0 /10 0/10 8 /10 5 /10

Cypermethrin, 100 mg/kg 8 /10 0/10 1 /10 4 /10 2 /10

Cypermethrin, 150 mg/kg 10 /10 1 /10 1 /10 4 /10 2 /10

Permethrin, 200 mg/kg 2 /10 3 /10 4 /10 1 /10 0/10

Permethrin, 250 mg/kg 6 /10 4 /10 6 /10 0 /10 2 /10

Vehicl e 1/10 1/10 1 /10 0/10 0/10

Cypermethrin, 65 mg/kg 6 /10 0 /10 1/10 2 /10 0/10

Vehicle 0 /10 0 /10 0/10 0/10 0/10

Resmethrin, 500 mg/kg 7 /10 9 /10 9 /10 0/10 0 /10

Resmethrin, 750 mg/kg 4 /10 5 /10 10 /10 0 /10 0 /10

Deltamethrin, 25 mg/kg 9 /10 1 /10 0 /10 4 /10 3 /10

Deltamethrin, 35 mg/kg 10 /10 6 /10 0/10 7 /10 4 /10

Pyrethrins, 400 mg/kg 3 /10 2 /10 1 /10 0 /10 0/10

Pyrethrins, 800 mg/kg 5 /10 2 /10 4 /10 1/10 0 /10

Vehicle 0 /10 0 /10 0/10 1 /10 0/10

Resmethrin, 350 mg/kg 7 /10 3 /10 9 /10 0/10 0/10

Deltamethrin, 12.5 mg/kg 5 /10 0 /10 0 /10 0/10 1 /10

Vehicle 0 /10 0 /10 0 /10 0/10 0 /10

Tefluthrin, 10 mg/kg 6 /10 4 /10 3 /10 2 /10 0/10

Tefluthrin, 15 mg/kg 5/10 5 /10 3 /10 4 /10 2 /10

Esfenvalerate, 25 mg/kg 3 /10 6 /10 2 /10 0 /10 2 /10

Esfenvalerate, 50 mg/kg 9 /10 6 /10 2 /10 3 /10 3 /10

Bifenthrin, 40 mg/kg 10 /10 10 /10 9 /10 0 /10 7 /10

Bifenthrin, 55 mg/kg 8 /10 10 /10 10 /10 2 /10 8 /10

Vehicle 0/10 0 /10 0/10 0/10 0/10

Esfenvalerate, 15 mg/kg 4 /10 1/10 0 /10 0/10 0/10

Data taken from Table 8 , pp. 1 9 7

- 2 16 , MRID 47050505.

a In c l udes walkin g on tiptoes, dragging body, hind limbs splayed, hunched body, ataxia, and exaggerated hind limb

flexion.

b Includes twitches of back, head, or body, jerking movements of limbs, whole

- body convulsions, and repetitive

chewing movements.Acute Neurotoxicity Study (rats) ( 2006 ) / Page 19 of 27

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- 19 Count data : Count data collected during open field observation are shown in Table 7. The

main effect was a decrease in the mean number of rears for many treated groups although the

data were highly variable. The mean number of rears was slightly decreased in animals

treated with β

- cyfluthrin, fenpropathrin (high

- dose), λ

- cyhalothrin, cypermethrin, permethrin,

resmethrin, deltamethrin, tefluthrin, esfenva lerate (mid

- and high

- dose), and bifenthrin (high

dose). The high dose of deltamethrin also resulted in a marked increase in the time to first

step. Grooming data were highly variable such that no dose

- related trends could be

discerned.Acute Neurotoxicity Study (rats) ( 2006 ) / Page 20 of 27

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- 20 TABLE 7

. Count data from open field observation s ( mean ± S.D. )

Treatment group Time to first step

(s) Rearing Backing Grooming

Vehicle 0. 4 ± 0.07 4.8 ± 3.43 0.0 1.1 ± 1.60

β

- Cyfluthrin, 25 mg/kg 0.5 ± 0.44 2.8 ± 3.29 0.0 0.9 ± 1.20

β

- Cyfluthrin, 45 mg/kg 0.4 ± 0.09 1.7 ± 1.42 0.0 0.4 ± 0.97

S

- Bioallethrin, 200

mg/kg 0.4 ± 0.21 3.6 ± 3.95 0.0 1.7 ± 2.98

S

- Bioallethrin, 300

mg/kg 0.4 ± 0.14 4.1 ± 4.25 0.0 0.2 ± 0.42

Fenpropathrin, 15 mg/kg 0.3 ± 0.04 5.7 ± 2.21 0.0 2.7 ± 2.58

Fenpropathrin, 30 mg/kg 0.4 ± 0.15 3.3 ± 3.02 0.0 1.8 ± 1.55

Vehicle 0.4 ± 0.23 7.3 ± 1.64 0.0 0.1 ± 0.32

β

- Cyfluthrin, 12. 5 mg/kg 0.4 ± 0.09 5.8 ± 2.35 0.0 0.0

S

- Bioallethrin, 15 0

mg/kg 0.5 ± 0.19 6.9 ± 3.03 0.0 0.3 ± 0.67

Vehicle 0.4 ± 0.15 5.3 ± 2.21 0.0 0.6 ± 0.84

λ

- Cyhalothrin, 10 mg/kg 0.4 ± 0.18 3.8 ± 2.97 0.0 0.7 ± 0.95

λ

- Cyhalothrin, 20 mg/kg 0.5 ± 0.14 0.2 ± 0.42 0.0 0.0 ± 0.00

Cypermethrin, 100 mg/kg 0.4 ± 0.09 2.6 ± 2.37 0.0 0.5 ± 0.85

Cypermethrin, 150 mg/kg 0.5 ± 0.16 1.0 ± 1.89 0.0 0.0 ± 0.00

Permethrin, 200 mg/kg 0.4 ± 0.11 2.9 ± 2.85 0.0 1.1 ± 1.60

Permethrin, 250 mg/kg 0.4 ± 0.22 3.0 ± 2.98 0.0 1.0 ± 0.94

Vehicle 0.5 ± 0.21 6.8 ± 2.57 0.0 0.3 ± 0.48

Cypermethrin, 65 mg/kg 0.8 ± 1.07 4.0 ± 2.11 0.0 0.8 ± 0.92

Vehicle 0.4 ± 0.05 5.5 ± 4.30 0.0 0.2 ± 0.42

Resmethrin, 500 mg/kg 0.5 ± 0.14 3.1 ± 3.25 0.1 ± 0.32 0.5 ± 1.08

Resmethrin, 750 mg/kg 0.7 ± 0.39 3.2 ± 2.78 0.0 0.4 ± 0.7 0

Deltamethrin, 25 mg/kg 0.5 ± 0.23 2.7 ± 3.33 0.1 ± 0.32 0. 4 ± 0.97

Deltamethrin, 35 mg/kg 2.1 ± 3.86 0.2 ± 0.42 0.0 0.1 ± 0.32

Pyrethrins, 400 mg/kg 0.4 ± 0.16 5.3 ± 3.27 0.0 0.5 ± 0.53

Pyrethrins, 800 mg/kg 0.5 ± 0.17 5.9 ± 2.92 0.0 0.3 ± 0.48

Vehicle 0.4 ± 0.05 6.7 ± 2.95 0.0 0.1 ± 0.32

Resmethrin, 350 mg/kg 0.5 ± 0.20 3.6 ± 3.03 0.0 0.5 ± 0.71

Deltamethrin, 12.5 mg/kg 0.4 ± 0.16 4.8 ± 3.65 0.0 0.7 ± 0.95

Vehicle 0.4 ± 0.17 6.6 ± 2.07 0.0 0.9 ± 1.10

Tefluthrin, 10 mg/kg 0.4 ± 0.16 3.7 ± 2.58 0.0 0.5 ± 1.27

Tefluthrin, 15 mg/kg 0.6 ± 0.50 1.3 ± 1.25 0.0 0.8 ± 1.03

Esfenvalerate, 25 mg/kg 0.4 ± 0.09 4.2 ± 2.86 0.0 1.7 ± 2.45

Esfenvalerate, 50 mg/kg 0.5 ± 0.19 2.7 ± 2.95 0.0 0.6 ± 0.84

Bifenthrin, 40 mg/kg 0.6 ± 0.56 6.0 ± 5.23 0.0 1.1 ± 1.10

Bifenthrin, 55 mg/kg 0.9 ± 0.79 3.2 ± 3.33 0.0 1.8 ± 1.55

Vehicle 0.4 ± 0.08 5.6 ± 2.27 0.0 0.0

Esfenvalerate, 15 mg/kg 0.6 ± 0.28 6.2 ± 2.49 0.0 0.6 ± 0.84

Data taken from Table 8 , pp. 1 97

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- 21 Sensory observations : Several findings in the sensory observations showed either an increase

or decrease to stimuli. T reat ment with β

- cyfluthrin resulted in an increase in the number of

animals with a more energetic response to tail pinch in all three groups and a decrease in the

air righting reflex (slightly uncoordinated) in the mid

- and high

- dose groups. S

- bioallethrin

resulted in a more energetic startle response in all three treatment groups, and a decreased air

righting reflex in the high

- dose group. Both fenpropathrin groups showed an increase in the

number of animals with a more energetic response to tail pinch. At the high dose of λ

cyhalothrin, an increased number of animals showed no reaction to startle response, touch

response, approach response, olfactory orientation, decreased hind limb extension, and

greatly reduced air righting reflex. In co ntrast, the high dose λ

- cyhalothrin group had an

increased number of animals with a more energetic reaction to tail pinch. The high dose of

cypermethrin resulted in more energetic reaction to startle response and tail pinch but a

decrease in olfactory ori entation; both mid

- and high

- dose cypermethrin groups had a

decrease in number of animals with hind limb extension and normal air righting reflex. At

the high

- dose of permethrin, an increased number of animals showed more energetic

approach and startle re sponses.

An increase in the number of animals with a more energetic startle response was the only

effect observed at all three doses of resmethrin. Treatment with deltamethrin resulted in an

increased number of animals with more energetic touch and start le responses at the high dose

and tail pinch response at all three doses as well as no hind limb extension at the high dose

and greatly diminished air righting reflex at the mid

- and high

- dose groups. Both groups

administered pyrethrins had a slight incre ase in the number of animals with a more energetic

startle response. Both groups treated with bifenthrin had an increase in the number of

animals with a more energetic startle response and an uncoordinated air righting reflex.

No consistent dose

- or trea tment

- related changes in sensory observations were seen

following treatment with tefluthrin or esfenvalerate.

Neuromuscular evaluations : Findings from neuromuscular measurements are shown in Table

8

. Hind limb weakness was found in two animals in each o f the mid

- and high

- dose β

cyfluthrin groups and corresponded with reduced mean hind limb grip strength for these

animals. Four animals in the high

- dose λ

- cyhalothrin group showed hind limb weakness and

mean fore

- and hind limb grip strengths and landing footsplay were decreased in this group.

Two animals in the mid

- dose and seven in the high

- dose deltamethrin groups showed hind

limb weakness and mean fore

- and hind limb grip strengths were decreased in both groups

and landing footsplay was reduced in the high

- dose group. Reduced hind limb grip strength

was also found in the high

- dose S

- bioallethrin and fenpropathrin groups. Fore

- and hind

limb grip strengths were decreased in the high

- dose cypermethrin group. Forelimb grip

s trength was reduced in the high

- dose esfenvalerate and both bifenthrin groups.

Rotarod performance time was reduced for the high

- dose λ

- cyhalothrin, the mid

- and high

dose cypermethrin, the mid

- and high

- dose resmethrin, and all three deltamethrin groups.Acute Neurotoxicity Study (rats) ( 2006 ) / Page 22 of 27

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- 22 TABLE 8

. Neuromuscular observation s

Treatment group Hind limb

weakness Forelimb grip

strength (g) a Hind limb

grip strength

(g) Rotarod

performance (s) Hind limb

footsplay (mm)

Vehicle 0/10 448.2 ± 71.65 175.1 ± 41.97 50.4 ± 47.76 65.2 ± 12.15

β

- Cyfluthrin, 25 mg/kg 2/10 327 .5 ± 73.62 146.6 ± 24.92 52.0 ± 49.58 55.1 ± 18.73

β

- Cyfluthrin, 45 mg/kg 2/10 335.1 ± 120.73 149.3 ± 33.88 94.1 ± 45.69 60.5 ±21.52

S

- Bioallethrin, 200

mg/kg 0/10 406.6 ± 81.27 162.6 ± 61.81 78.6 ± 36.60 64.4 ±13.53

S

- Bioallethrin, 300

mg/kg 0/10 330.8 ± 70.29 133.4 ± 31.27 48.0 ± 49.50 49.1 ± 20.87

Fenpropathrin, 15 mg/kg 0/10 378.3 ± 109.09 156.6 ± 36.51 86.3 ± 48.77 60.2 ± 16.30

Fenpropathrin, 30 mg/kg 0/10 418.3 ± 86.78 141.8 ± 40.50 93.0 ± 39.71 64.1 ± 19.48

Vehicle 0/10 490.1 ± 86.73 264.1 ± 36.39 87.1 ± 46.69 61.9 ± 10.75

β

- Cyfluthrin, 12. 5 mg/kg 0/10 478.3 ± 111.93 252.5 ± 30.40 99.9 ± 33.66 75.5 ± 13.53

S

- Bioallethrin, 15 0

mg/kg 0/10 436.7 ± 88.84 270.8 ± 55.47 48.4 ± 42.46 64.7 ± 9.62

Vehicle 0/10 510.9 ±72.28 235.1 ± 40.56 95.3 ± 43.54 71.1 ± 8.97

λ

- Cyhalothrin, 10 mg/kg 0/10 513.3 ± 156.62 195.9 ± 51.85 109.8 ± 29.84 67.4 ± 14.00

λ

- Cyhalothrin, 20 mg/kg 4/10 341.8 ± 154.44 153.2 ± 50.70 53.2 ± 4 5 .34 51.0 ± 21.85

Cypermethrin, 100 mg/kg 1/10 463.4 ± 80.29 189.2 ± 46.82 79.9 ± 51.61 78.2 ± 11.68

Cypermethrin, 150 mg/kg 0/10 374.9 ± 107.41 156.6 ± 56.78 38.5 ± 43.81 66.4 ± 18.45

Permethrin, 200 mg/kg 0/10 423.2 ± 136.26 204.3 ± 52.20 94.6 ± 33.84 76.5 ± 13.18

Permethrin, 250 mg/kg 0/10 426.0 ± 86.69 180.7 ± 45.38 108.7 ± 35.73 66.8 ± 12.16

Vehicle 0/10 492.4 ± 91.62 279.1 ± 53.55 86.8 ± 43.93 65.3 ± 13.63

Cypermethrin, 65 mg/kg 0/10 505.8 ± 50.62 285.0 ± 63.34 82.4 ± 39.25 69.0 ± 5.75

Vehicle 0/10 518.4 ± 102.52 240.0 ± 60.47 85.9 ± 42.45 72.6 ± 12.38

Resmethrin, 500 mg/kg 0/10 410.1 ± 113.58 199.1 ± 40.73 55.7 ± 39.06 69.9 ± 12.86

Resmethrin, 750 mg/kg 0/10 517.4 ± 102.30 202.4 ±68.83 63.3 ± 41.04 64.0 ± 15.03

Deltamethrin, 25 mg/kg 2/10 325.1 ± 166.21 148.2 ± 57.40 73.3 ± 52.54 59.2 ± 21.83

Deltamethrin, 35 mg/kg 7/10 175.8 ± 130.48 85.7 ± 34.63 30.3 ± 49.97 45.9 ± 22.68

Pyrethrins, 400 mg/kg 0/10 543.5 ± 103.75 260.0 ± 48.46 93.7 ± 40.90 79.0 ± 12.62

Pyrethrins, 800 mg/kg 0/10 439.1 ± 99.20 177.4 ± 43.80 109.7 ± 25.22 66.0 ± 15.74

Vehicle 0/10 4 83.4 ± 74.73 265.7 ± 38.61 106.2 ± 30.16 72.0 ± 10.65

Resmethrin, 350 mg/kg 0/10 406.7 ± 74.40 244.2 ± 31.15 82.3 ± 45.49 70.2 ± 14.40

Deltamethrin, 12.5 mg/kg 0/10 430.9 ± 96.20 214.2 ± 55.73 76.7 ± 55.98 57.3 ± 14.19

Vehicle 0/10 536.6 ± 118.13 238.2 ± 47.48 69.8 ± 50.43 66.6 ± 13.91

Tefluthrin, 10 mg/kg 0/10 450.7 ± 136.26 221.6 ± 69.58 109.0 ± 34.88 59.9 ± 17.25

Tefluthrin, 15 mg/kg 0/10 417.4 ± 151.71 188.3 ± 66.19 83.5 ± 50.75 65.1 ± 12.69

Esfenvalerate, 25 mg/kg 1/10 410.9 ± 115.24 214.0 ±82.86 108.5 ± 35.36 53.8 ± 19.93

Esfenvalerate, 50 mg/kg 0/10 379.0 ± 79.71 184.4 ± 34.82 64.0 ± 44.06 54.8 ± 22.65

Bifenthrin, 40 mg/kg 0/10 300.8 ± 102.10 170.1 ± 61.20 77.3 ± 50.29 53.6 ± 11.58

Bifenthrin, 55 mg/kg 1/10 311.6 ± 71.25 177.6 ± 58.86 50.2 ± 52.27 46.9 ± 12.04

Vehicle 0/10 456.6 ± 95.80 264.3 ± 50.40 112.8 ± 15.83 69.3 ± 13.34

Esfenvalerate, 15 mg/kg 0/10 385.1 ± 79.42 236.0 ± 42.15 88.3 ± 38.62 59.8 ± 19.54

Data taken from Table 12 , pp. 1 69

- 276 , MRID 47050505.

a No. animals showing some weakness/No. testedAcute Neurotoxicity Study (rats) ( 2006 ) / Page 23 of 27

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- 23 Physiological measurements : Mean body temperature was decreased in the mid

- and high

dose β

- cyfluthrin, high

- dose λ

- cyhalothrin, high

- dose cypermethrin, and mid

- and high

- dose

deltamethrin groups. An increase in mean catalepsy time was measured in th e high

- dose

deltamethrin group. The other test articles did not affect body temperature or catalepsy time.

2. Motor activity : Motor activity measurements were not done.

F. SACRIFICE AND PATHOLOGY :

1. Gross pathology : The high

- dose bifenthrin animal that was found dead had dark red areas in

the glandular portion of the stomach. No gross lesions were seen in the other animals that

died on study. The surviving animals were sacrificed and discarded without examination.

2. Brain weight : Brain weight was not recorded.

3. Neuropathology : No neuropathology investigations were done.

III. DISCUSSION AND CONCLUSIONS :

PCA INFORMATION:



### Principal Component Analysis (PCA) and Factor Analysis (FA) are techniques which are

powerful statistical tools suited for the present dataset. The PCA approach is able to

simultaneously analyze 56 separate neurobehavioral findings across the 38 control an d treatment

groups that comprise this study in an objective manner. PCA also avoids the limitations of visual

or subjective examination. PCA can demonstrate associations or correlations empirically, based

on mathematical models. Such associations provide a deeper and more rigorous understanding of

possible patterns of pyrethroid toxicity syndromes.

In the PWG analysis, the different behavioral resp onse patterns of pyrethroids were defined by

four principal components, which demonstrate a dose response relationship with one or more

pyrethroids. Based on this analysis, individual chemicals were arrayed by a group of highly

correlated (p>0.5) behaviors, which constituted principle com ponent factors. The non

- cyano

pyrethroids tended to array themselves in a dose

- response manner primarily along two of the

principal components that were described by the following behavioral effects: tremors,

myoclonus, elevated temperature, exaggerated re sponse to sensory stimuli, difficulty handling,

head flicking, jerking movements, pulsating eyes (spontaneous nystagmus). The cyano

pyrethroids arrayed themselves in a dose

- related manner, primarily along two different principal

components described by the se behavioral effects: excessive salivation, impaired mobility, lower

temperature, non

- reactivity to sensory stimuli, lacrimation, writhing, neuromuscular weakness,

abnormal posture.

Table 10 defines the four primary patterns of variation in the FOB data that were attributed to

pyrethroid effects. For ease of comparison, the principle components that best described theAcute Neurotoxicity Study (rats) ( 2006 ) / Page 24 of 27

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- 24 Type I pyrethroids were designated Ta and Tb, while those for Type II pyrethroids were labeled

CSa and CSb.

Extracted from p. 107 in PWG Study Number: TOX

- 2007

- 01



### A. INVESTIGATOR ’ S CONCLUSIONS :

The study author concluded that the FOB findings in this study were consistent with the

known effects of pyrethroid compounds including tremors , body dragging, ataxia, profuse

salivation, clonic convulsions, abnormal hind limb function, and writhing

. Findings in the

pyrethrins and tefluthrin groups were limited to effects on CNS and neuromuscular

parameters. The remaining compounds also affecte d autonomic endpoints. The author also

mentioned that two patterns of clinical signs could be statistically distinguished by the

principle component analysis and factor analysis of the FOB data. These patterns were

defined as Type I (or T) syndrome, char acterized by the occurrence of tremors, and the Type

II (or CS) syndrome, associated with choreoathetosis and salivation.

B. REVIEWER COMMENTS :

A modified FOB was used to evaluate the effects of twelve pyrethroid compounds in rats. The

reviewer agrees with the study author in that the results are consistent with this class of chemical.

Doses and time to peak effect were appropriately chosen for each compound based on the results

of the range

- finding study. Desp ite similarity of effects, a wide range of potency was evident in

the doses used and severity of findings for the individual compounds. Deaths of five animals are

considered treatment

- related as they occurred at the highest dose administered for four of t he

compounds.

A NOAEL/LOAEL could not be established for most compounds as effects were observed at all

doses tested. However

, it was not the intent of the study to establish a NOAEL or to clearly

define the dose

- response for each compound. It is unfort unate that the statistical analyses

discussed by the study author w ere not available to the current reviewer.Acute Neurotoxicity Study (rats) ( 2006 ) / Page 25 of 27

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- 25 The results from the present study are consistent with the early reports of differences in

behavioral response patterns associated with Type I an d Type II pyrethroids, which were

described by Vershoyle and Aldridge (1980), Casida et al. (1983) and Soderlund et al. ( 2002).

The results from this study indicate that some pyrethroids clearly exhibit the historic

classification symptoms of the T and CS syndromes while others do so less obviously.

Use of the statistical technique of Principal Component Analysis (PCA) fur ther helped interpret

the study findings. Based on a review of the FOB data, analyzed by PCA, and other in vivo and

in vitro published data, two common mechanism groups are proposed by the Agency. Type I

would include pyrethrins, bifenthrin, resmethrin, pe rmethrin, S

- bioallethrin and tefluthrin. Type

II would include cypermethrin, deltamethrin, β

- cyfluthrin and λ

- cyhalothrin, while fenpropathrin

and esfenvalerate exhibited features of both groups.

C. STUDY DEFICIENCIES :

No deficiencies were noted in the conduct of this non

- guideline study that would prevent

interpretation of the results.Acute Neurotoxicity Study (rats) ( 2006 ) / Page 26 of 27

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- 26 APPENDIX: Range

– Finding Study

- Functional Observational Battery for Pyrethroids

TEST MATERIAL (PURITY): See Appendix Table below.

CITATION: Nemec, N.D. (2005) A Range

- Finding Acute Functional Observational Battery

Study in Rats. WIL Research Laboratories, LLC, 1407 George Road, Ashland,

OH. November 3, 2005. MRID 47050504. Unpublished.

SUMMARY: Groups of 5 male Crl : CD ® 

### (SD)IGS BR rats received one of 12 pyrethrin

compounds at each dose by oral gavage in order to determine dose levels and to establish times

of peak effect of 12 pyrethroid compounds. Testing of the chemicals was staggered with several

chemicals tested at a time along with a concurrent vehi cle control group. The vehicle control

was Mazola ® 

### corn oil. The dose volume was 5 mL/kg. Following completion of the initial

dosing, additional dosage levels of β

- cyfluthrin, deltamethrin, pyrethrins and tefluthrin were

evaluated to more fully characte rize the neurotoxicity or time of peak effect. Rats were treated

on day 0 and sacrificed on day 1. All animals were observed for clinical signs and mortality

prior to dosing and on study days 0 and 1. Animals were weighed on study days

- 3,

- 1, 0, and 1.

Neurotoxic effects and time of peak effect were evaluated during a modified functional

observational battery (FOB). The FOB was conducted at 3

- 4 specific times (2, 4, 8, or 12 hours,

depending on the specific compound) on the day of test material admini stration. A complete

necropsy was performed on all animals found dead or euthanized in extremis (via carbon dioxide

inhalation); surviving animals were euthanized and discarded without examination following the

FOB evaluation. For each chemical, dose lev el and time of peak effect during the FOB is

provided in the Appendix Table. Time of peak effect was generally from 2

- 8 hours post

- dose.

Only the predominant clinical signs are summarized in the Table. Not all animals in a group

displayed all signs. Sm all group mean body weight losses were noted at the higher dose levels

for several chemicals. Losses were also noted in several concurrent control groups.

Macroscopic examination revealed red discoloration or multiple dark red areas in the lungs of

one a nimal in each of the high

- dose group s of deltamethrin, λ

- cyhalothrin, and bifenthrin. Other

observations were incidental to treatment.

Compliance: Signed an d dated GLP , Data Confidentiality, and Flagging statements were

provided.

### A3

- 27 Appendix Table. Mortality, Time of Peak Effect , and Clinical Signs in Male Rats Administered 1 of 12 Pyr e throid Compounds

(Clinical signs and time of peak effects observed during Functional Observational Battery)

Chemical (purity) Doses tested (mg/kg) Mortality by dose

(no of animals/no of

animals tested) Dose/Time of

Peak Effect

after dosing Clinical signs

Pyrethrins (53.8%) 150, 250, 350, 375, 400 No mortality 400, 800: 4 hours Impaired mobility, altered gait,

convulsions, tremors, stereotypic

behavior, difficulty in removal from cage

Deltamethrin (>99.6%) 20, 30, 35, 40, 50 30: 5/5

40: 5/5

50: 5/5 25, 35: 4 hours Salivation, whole

- body convulsions,

splayed hind limbs , writhing

S

- Bioallerethrin (95.9%) 150, 250, 300 No mortality 200, 300: 2 hours Tremors, body dragging, ataxia

β

- Cyfluthrin (96.8%) 5, 25, 45, 60, 75 60: 1/5

75: 5/5 25, 45: 2 hours Salivation, whole

- body convulsions,

splayed hind limbs , writhing

Tefluthrin (92.4%) 5, 10, 15, 20 20: 2/5 10, 15: 8 hours Tremors, gait abnormalities, whole

- body

convulsions

Cypermethrin (not provided) 100, 150, 200 100: 1/5

200: 2/5 100, 150: 4 hours Salivation, whole

- body convulsions,

splayed hind limbs , writhing

Permethrin (not provided) 200, 250, 300 300: 2/5 200, 250: 4 hours Tremors, body dragging, ataxia

λ

- Cyhalothrin (87.7%) 10, 20, 35 35: 1/5 10, 20: 4 hours Salivation, whole

- body convulsions,

splayed hind limbs , writhing

Fenpropathrin (90.8%) 10, 20, 30 No mortality 15, 30: 2 hours Difficulty in handling, tremors, clonic

convulsions, low arousal

Esfenvalerate (98.6%) 5, 25, 50 No mortality 25, 50: 8 hours Difficulty in handling, salivation, slight

tremors, altered gait/mobility, clonic

convulsions

Bifenthrin (not provided) 40, 55, 70 70: 1/5 40, 55: 8 hours Vocalization, salivation, alterations in

gait, impaired mobility, ataxia

Resmethrin (91.1%) 500, 750, 900 500: 1/5

900: 1/5 500, 750: 4 hours Tremors, clonic convulsions, salivation,

impaired mobility

Dose calculations not adjusted for purity.