volume V | INTERNATIONAL COURT OF JUSTICE

Document Number

17572

Parent Document Number

17564

Document File

17572.pdf

INTERNATIONAL COURT OF JUSTICE
____________________________________________

CASE CONCERNING
AERIAL HERBICIDE SPRAYING

(ECUADOR v. COLOMBIA)

REJOINDER OF THE

REPUBLIC OF COLOMBIA

VOLUME V

ANNEXES 56 - 59

1 FEBRUARY 2012 LIST OF ANNEXES

VOLUME V

Annex 56 United States Environmental Protection Agency (EPA),
Memorandum of 13 May 2003, Technical Review of the six acute
toxicity studies on the spray mixture for Eradication of Illicit Crops in
Colombia. .................................................... 1

Annex 56-A: Six Acute Toxicity Studies with Spray-Charlie, SLI Study
N° 3596.16, 20 Feb. 2003. ................................... 21

Annex 56-B: Six Acute Toxicity Studies with Spray-Alpha, SLI Study
N° 3596.3, 3 Sep. 2002. .................................... 221

Annex 56-C: Six Acute Toxicity Studies with Spray-Bravo, SLI Study
N° 3596.10, 4 Sep. 2002. ................................... 445

Annex 57 Letter by Ms Rebecca L. Puskas to the United States Environmental
ProtectionAgency, 11 Nov. 2008. ................................ 673

Annex 58 Embassy of the United States ofAmerica, List ofAerial Eradication
Verification Missions since 1997

Appendix: Implementation of the verification protocol January –

July 1998, carried out October 18-23, 1998. ........................ 679

Annex 59 United States Department of State, Bureau for International
Narcotics Matters, Herbicide Selection for Coca Eradication,
May 1984. .................................................. 711

iiiiv Annex 56

UnitedStateSenvironmentalProtectionagency (ePa),

m emorandUm of13 may 2003, echnicalreview of
the Six acUte toxicity StUdieS on the SPray mixtUre for
e radication illicic roPS icolombia

(United States Embassy in Bogotá, 2011)

12 Annex 56

3Annex 56

4 Annex 56

5Annex 56

6 Annex 56

7Annex 56

8 Annex 56

9Annex 56

10 Annex 56

11Annex 56

12 Annex 56

13Annex 56

14 Annex 56

15Annex 56

16 Annex 56

17Annex 56

18 Annex 56

1920 Annex 56-A

SixA cutet oxicitS tudieS witS prA-c hArli, Sli StudyN° 3596.16,

20 febrUary 2003

(United States Embassy in Bogotá, 2011)

2122 Annex 56-A

    

   

 

 





    

  

  

 

  
      
   
  

  



 


   
    
  

   

23Annex 56-A

    

      

             
             



  

 

24 Annex 56-A

25Annex 56-A

26 Annex 56-A

    

   

      

  

   

    

     

  

  

    

  

     

   

       

 

 

  

 

27Annex 56-A

    

     



   
   
     



   

28 Annex 56-A

    

 

          
               
             
           
           

          

          
           
          

             
         

            
         

29Annex 56-A

    

 

           
            

            
              
           

          
          
          
           

             
            
      

   

  

           

    
     
     



  
    

   
    
          
            

           
         
            

          
           

30 Annex 56-A

    

  

             
            
             
            
           

       

   

            
    

     

             
            
       

    

    

          
          

         
            
          
           
            

 

         
       
           

           
           
           

 

            
           
             

31Annex 56-A

    

           
      
       

          
            
           
            

 

          

           
           
          
          

            
        

 

          
          
            

         
  

  

           
           

          
          
          
            

       

  

 

             
             
             
 

32 Annex 56-A

    

       
    
    

  
       

           

         

  

            
          
           



  

            
           

  

          

          
        

  

      

     

           
       
             

            
         

33Annex 56-A

    

   

            

           
           
           

         
  

      

            
               
    

 

 

   

      

  

   

         
          

    

   

   

           

34 Annex 56-A

35Annex 56-A

    

 

            
  

36 Annex 56-A

(15)

---- ---- ----

STUDYL/NO.:U359E616ALESNT-O--MA-LE#------O-BAH6F6TIO-CEUS4NEDTA-AEDURSSI--HOFENLAL--USINT-REO--SK-M-
13SIAL-A---6N--PYE--A-------
-E-P---P1---=----------------

37Annex 56-A

(16)

---- ---- ----

STUDLY/NO.:S35E9616MALES--FEM-ALE#------6OB2671A--ED6LEDUE-SASOFTSAA-OEUSEDOO--LHANAS----------1--Y-
F
S
4--Y------7--PP---------------

38 Annex 56-A

(17)

---- ---- ----

STUDNYLNO.:US359MALE-S--ANIMAL#-5-----4M3846Y1---53TUDY--59-375--04-143A-45-EATH-75DAY)---5--------------------------
-------------------------------------
---------------------------------------------------------------------------------------------

39Annex 56-A

(18)

---- ---- ----

STUDY/N,O.:S359FEMALESANIM-AL#-----213171N-.NOF-S-----8---1---8---1----1-DE19---4DA29)-----------------------------
-------------------------------------
---------------------------------------------------------------------------------------------

40 Annex 56-A

(19)

1 ---- ---- ----

STUINL/A, US DE1MALES-----OAL0-A6/K-DOA23---30-YY-C-02646-SE-30-DEC----OESLUE--3--TNEN---N--ENILII-L
-TOPSRM---
L.XTTS-
---CM--
-----ON----A---

41Annex 56-A

(20)

2 ---- ---- ----

STINL/A, US DEFEMALES--IMA00-A6/KA-OFT----E--D-0-1--4-3----14T-1TISI-I-AT--OHIN--I-TH--TLII--IL-L
-RMAL----MITS------

42 Annex 56-A

    

 

  

43Annex 56-A

    

  

       



     

     

      

     

       

       

    

       

       

     

       

        

   

44 Annex 56-A

AN ACUTE DERMAL TOXICITY STUDY
IN RATS WITH SPRAY--CHARLIE

FINAL REPORT

OPPTS Guideline

870.1200

Author

Kimberly L. Bonnette, M.S., LATG

Study Completed on

February 20, 2003

Performing Laboratory

Springborn Laboratories (SLI),
a division of Charles River Company, Inc.

640 North Elizabeth Street
Spencerville, Ohio 45887

SLI Study No.

3596.17

Submitted to

INL/A
U.S. Department of State
2201 C St. NW SA-4
Washington, DC 20520

Page 1 of 29

45Annex 56-A

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SLI Study No. 3596.17

1. STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS
No claim of confidentiality is made for any information contained in this study on

the basis of its falling within the scope of FIFRA §10(d)(1)(A), (B), or (C).

Company:

Company Agent: Date

Title Signature

46 Annex 56-A

47Annex 56-A

48 Annex 56-A

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SLI Study No. 3596.17

4. TABLE OF CONTENTS

1. STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS......................... 2

2. COMPLIANCE STATEMENT....................................................................... 3

3. QUALITY ASSURANCE STATEMENT ....................................................... 4

4. TABLE OF CONTENTS............................................................................... 5

5. LIST OF TABLES AND APPENDICES........................................................ 6

6. SUMMARY................................................................................................... 7

7. INTRODUCTION.......................................................................................... 8

8. MATERIALS AND METHODS..................................................................... 8

9. EXPERIMENTAL PROCEDURES ............................................................. 11

10. DATA ACQUISITION AND ELECTRONIC RECORDS ........................... 12

11. ANALYSIS OF DATA........................................................................
....... 12

12. MAINTENANCE OF RAW DATA AND RECORDS................................. 13

13. RESULTS................................................................................................. 13

14. CONCLUSION ........................................................................
................. 14

15. REPORT REVIEW ........................................................................
........... 14

16. REFERENCES........................................................................
................. 15

49Annex 56-A

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SLI Study No. 3596.17

5. LIST OF TABLES AND APPENDICES

Tables

1. Individual Clinical Observations ..........................................................16
2. Individual Body Weights......................................................................19
3. Individual Gross Necropsy Observations ............................................21

Appendices

A. Macroscopic Dermal Grading System................................................23

B. SLI Personnel Responsibilities...........................................................28

50 Annex 56-A

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SLI Study No. 3596.17

6. SUMMARY

The single-dose dermal toxicity of Spray--Charlie was evaluated in Sprague
Dawley rats. A limit test was performed in which one group of five male and five
female rats received a single dermal administration of the test article at a dose of
5000 mg/kg body weight. Following dosing, the limit test rats were observed daily
and weighed weekly. A gross necropsy examination was performed on all

animals at the time of scheduled euthanasia (day 14).

No mortality occurred during the limit test. Clinical abnormalities observed during
the study included transient incidences of dark material around the facial area
and decreased defecation. Dermal irritation was noted at the site of test article

application. Body weight loss was noted in one male and two females during the
study day 7 to 14 body weight interval. Body weight gain was noted for all other
animals during the test period. No significant gross internal findings were
observed at necropsy on study day 14.

Under the conditions of this test, the acute dermal LD50 of Spray--Charlie was
estimated to be greater than 5000 mg/kg in the rat.

51Annex 56-A

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SLI Study No. 3596.17

7. INTRODUCTION

This study was performed to assess the short-term toxicity of Spray--Charlie in
Sprague Dawley rats when administered by a single dermal dose. This study

was intended to provide information on the potential health hazards of the test
article with respect to dermal exposure. Data from this study may serve as a
basis for classification and/or labeling of the test article. This study was

performed in accordance with the US EPA, Health Effects Test Guidelines,
OPPTS 870.1200, Acute Dermal Toxicity, August 1998. This study was
performed at Springborn Laboratories (SLI), 553 North Broadway, Spencerville,
Ohio. The protocol was signed by the Study Director on October 9, 2002 (GLP

initiation date). The in-life phase of the study was initiated with test article
administration on December 19, 2002 (day 0), and concluded with necropsy on
January 2, 2003.

8. MATERIALS AND METHODS

8.1. Test Article

The test article was received from the Sponsor and identified as follows:

Assigned Physical Receipt Expiration
Sponsor’s ID SLI ID Description Date Date
Spray—Charlie a S02.003.3596 Amber 12/09/02 None
liquid provided
b
Ingredients:
Herbicide: GLY-41 None
Lot No.: Manufactured 10/20/02 provided

Surfactant: Cosmo Flux-411F None
Lot No.: Manufactured 11/20/02 provided
aSample pooled at SLI from five different mixes of Spray--Charlie (top/middle/bottom).
b
Ingredients used in the five Spray--Charlie mixes that were prepared by the Sponsor.

The test article was stored at room temperature. The Sponsor was responsible
for any necessary evaluations related to identity, strength, purity, composition,

stability and method of synthesis of the test material according to
40 CFR 160.105 and 40 CFR 792.105. Springborn Laboratories, Inc. analyzed
the test article for the glyphosate (a.e.) which is presented in

SLI Study No. 3596.15.

52 Annex 56-A

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SLI Study No. 3596.17

8.2. Retention Sample

An approximate 1 mL retention sample of each test article mixture sample
(top/middle/bottom, maintained separately for a total of fifteen 1 mL samples)
was taken and stored at SLI at room temperature. In addition, a 10 mL retention
sample of the pooled test article samples (from the 5 test article mixtures) was
collected and stored at SLI at room temperature. These samples serve as the

retention samples for all studies conducted with this material.

8.3. Test Article Disposition

The test article was returned to the Sponsor following completion of all studies
with the test article.

8.4. Method of Test Article Preparation

The test articles were pooled and administered as received from the Sponsor
and dispensed fresh on the day of dosing. The test articles were stirred
continuously during dosing. The density of the test article was determined to be
1.08 g/mL.

8.5. Animals and Animal Husbandry

8.5.1. Description, Identification and Housing
Adult, Hsd: Sprague Dawley® SD® rats were received from Harlan Sprague

Dawley, Inc., Indianapolis, IN. Upon receipt, metal ear tags displaying unique
identification numbers were used to individually identify the animals. Cage cards
displaying at least the study number, animal number and sex were affixed to
each cage. The animals were housed individually in suspended stainless steel
cages. All housing and care were based on the standards recommended by the

Guide for the Care and Use of Laboratory Animals [1].

8.5.2. Environment
The animal room temperature and relative humidity ranges were 71-74°F
(22-23°C) and 40-53%, respectively. Environmental control equipment was

monitored and adjusted as necessary to minimize fluctuations in the animal room
environment. Light timers were set to maintain a 12-hour light/12-hour dark cycle
and room ventilation was set to produce 10-15 air changes/hour. The animal
room temperature and relative humidity were recorded a minimum of once daily.

8.5.3. Food

PMI Certified Rodent Chow #5002 (Purina Mills, Inc.) was provided ad libitum to
the animals throughout the study. The lot number and expiration date of each

53Annex 56-A

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SLI Study No. 3596.17

batch of diet used during the study were recorded. The feed was analyzed and
certified by the supplier for nutritional components and environmental
contaminants. Dietary limitations for various environmental contaminants,

including heavy metals, pesticides, polychlorinated biphenyls and total aflatoxin
are set by the manufacturer. Within these limits, contaminants which may have
been present were not expected to compromise the purpose of this study.
Results of the dietary analyses (Certificates of Analysis) were provided by the

manufacturer for each lot of diet. These are maintained by SLI.

8.5.4. Water

Municipal tap water treated by reverse osmosis was available ad libitum
throughout the study. The purified water was supplied by an automatic watering
system. Monitoring of the drinking water for contaminants is conducted by SLI
and the records are available for inspection. Within generally accepted limits,

contaminants which may have been present were not expected to compromise
the purpose of this study. The water meets the standards specified under the
EPA National Drinking Water Regulations (40 CFR Part 141).

8.5.5. Acclimation

Upon receipt, the animals were removed randomly from the shipping cartons,
examined by qualified personnel, identified with metal ear tags and then

acclimated to the laboratory conditions for a minimum of five days. The animals
were observed daily for overt physical or behavioral abnormalities, general
health/moribundity and mortality.

8.5.6. Animal Selection

The animals chosen for study use were randomly selected from healthy stock

animals using a computerized random numbers table to avoid potential bias. All
animals received a detailed pretest observation prior to dosing. Only healthy
animals were chosen for study use. Females were nulliparous and nonpregnant.
The male animals were approximately 9 weeks of age and weighed 265-290 g

prior to dosing. The female animals were approximately 9 weeks of age and
weighed 189-207 g prior to dosing.

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SLI Study No. 3596.17

9. EXPERIMENTAL PROCEDURES

9.1. Preliminary Procedures

On day -1, the fur was removed from the dorsal trunk area of the animals chosen
for the limit test using an animal clipper. The clipped area was approximately

10% of the animal’s body surface area (BSA). The region included the scapula
(shoulder) to the wing of the ilium (hipbone) and half way down the flank on each
side of the animal. Care was taken to avoid abrading the skin during the clipping

procedure.

9.2. Dosing

On the following day (day 0), the test article was administered dermally to
approximately 10% of the body surface area. The four corners of this area were
delineated in the clipped area with an indelible marker. The test article was then

spread evenly over the delineated test area and held in contact with the skin with
an appropriately sized 4-ply porous gauze dressing backed with a plastic wrap
which was placed over the gauze dressing (occlusive binding). Removal and

ingestion of the test article was prevented by placing an elastic wrap over the
trunk and test area. The elastic wrap was further secured with a tape harness on

the cranial end of the trunk and then secured with adhesive tape around the trunk
at the caudal end.

The test article was administered at the following level:

Dose Level Dose Volume Concentration No. of Animals
Male Female
(mg/kg) (mL/ka) (%)b
a 5000 4.63 100 5 5
Adjusted based on a density of 1.08 g/mL.
Pooled test article.

Individual doses were calculated based on the animal’s day 0 body weight. After

an approximate 24-hour exposure period, the binding materials were removed
and the corners of the test site were re-delineated using a marker. Residual test
article was removed using gauze moistened with deionized water followed by dry

gauze.

9.3. Dermal Observations

The test animals were examined for erythema and edema following patch
removal and the responses scored on study day 1 and daily thereafter

55Annex 56-A

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SLI Study No. 3596.17

(days 2-14) according to the Macroscopic Dermal Grading System provided in
Appendix A which is based on Draize [2]. The dermal test sites were reclipped
as necessary to allow clear visualization of the skin.

9.4. Clinical Observations

The animals were observed for clinical abnormalities two times on study day 0
(postdose) and daily thereafter (days 1-14). A mortality check was performed
twice daily, in the morning and afternoon.

9.5. Body Weights

Individual body weights were obtained for the animals prior to dosing on day 0
and on days 7 and 14.

9.6. Gross Necropsy
All animals were euthanized by carbon dioxide inhalation at study termination

(day 14) and necropsied. Body cavities (cranial, thoracic, abdominal and pelvic)
were opened and examined. No tissues were retained.

9.7. Protocol Deviations
On study day 1, edema was inadvertently not recorded for Animal No. A6709.

This occurrence was considered to have had no adverse effect on the outcome
of this study.

10. DATA ACQUISITION AND ELECTRONIC RECORDS

Electronic data were recorded on a Compaq Alpha Server DS10 utilizing the
Toxicology Analysis System Customized, Acute Toxicology Module, Version
1.0.0 or higher. The SLI study number assigned to this study is 3596.17. The
computer study number used to collect data for the study phases was 359617.

The tables within the report will display the applicable computer number.

11. ANALYSIS OF DATA

Data from the study were analyzed and an LD50 value estimated as follows:

< 50% Mortality: LD50 was estimated as greater than the administered dose.
= 50% Mortality: LD50 was estimated as equal to the administered dose.
> 50% Mortality: LD50 was estimated as less than the administered dose.

56 Annex 56-A

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SLI Study No. 3596.17

Body weight means and standard deviations were calculated separately for
males and females.

12. MAINTENANCE OF RAW DATA AND RECORDS

All original raw data, the final report and electronic records were transferred to
the SLI archives for a period of 10 years. The Sponsor will be contacted prior to
final disposition of these items.

13. RESULTS

13.1. Mortality

Individual Data: Table 1

No mortality occurred during the limit test.

13.2. Clinical/Dermal Observations

Individual Data: Table 1

Clinical abnormalities observed during the study included transient incidences of
dark material around the facial area and decreased defecation. Dermal irritation

was noted at the site of test article application.

13.3. Body Weight Data

Individual Data: Table 2

Body weight loss was noted in one male and two females during the study day 7

to 14 body weight interval. Body weight gain was noted for all other animals
during the test period.

13.4. Gross Necropsy
Individual Data: Table 3

No significant gross internal findings were observed at necropsy on study day 14.

57Annex 56-A

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SLI Study No. 3596.17

16. REFERENCES

1. Guide for the Care and Use of Laboratory Animals, DHHS Publication No.
(NIH) 96-03, 1996.

2.Draize, J.H., Appraisal of the Safety of Chemicals in Foods, Drugs and
Cosmetics, The Association of Food and Drug Officials of the United States,

49-51, 1959.

59Annex 56-A

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---- ---- ----

STUDYL/NO.:U3596D17ALESMENT--MAL-E#------OB-SHEFTERYAS-ETHEG0AIAI-REDRAAATE--UAIHKRKUOH-NDDLAL)-U--RRIRER---PS
DND
-
-8-PTM-----------

60 Annex 56-A

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---- ---- ----

STUDLY/NO.:.3.59617LESME-N---MALE#----0--OBSER-DUATRKR--TH0AIAI--AROUNDN--YE(S)-----------------D----
F
ST-P-YP
-P--7---P----1----P------P-14--R----------------

61Annex 56-A

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---- ---- ----

STUDY/N,O.:.359617MRALESN-FEMALE#-------OBSERVATI-RADATETMA---THEUMDNHEW-SRYARAETSA--ADAL)0-EUADEG-REODU-A5-A
P
PI---OU--P2P---------

62 Annex 56-A

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---- ---- ----

STUDLY/NO.:.S35MALES-TA-NIMAL#--86---9M9703Y0O--39-UDY--01-144A-95-EATH-05DAY)---5----------------------------------------
-------------------------------------
---------------------------------------------------------------------------------------------

63Annex 56-A

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---- ---- ----

STIUDY/NO.:.S35FEMALE-ANI-MAL#------151620Y0--040TU1Y---27--911-D2-----0DAY6--------------------------------------------
-------------------------------------
--------------------------------------I--N--L--/--A--,----U--.--S--.--M

64 Annex 56-A

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1 ---- ---- ----

STINL/A, U.S.MDAL-ES----5000------4A6696-ST---036--0-N-B--NJA----N---RESL-U-SS---TORR-N-N----TSIVI--M---
-L--
------------
-
----------------

65Annex 56-A

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2 ---- ---- ----

STINL/A, U.S.FDE-MALES--500-------96710--S5--1037-0-N-O--AJAN---ON---UESL-E-SU--SNOS-N-N----NTSN-T-MI---
LI-
ITS
----

66 Annex 56-A

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SLI Study No. 3596.17

APPENDIX A

Macroscopic Dermal Grading System

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SLI Study No. 3596.17

MACROSCOPIC DERMAL GRADING SYSTEM

ERYTHEMA AND EDEMA OBSERVATIONS

OBSERVATION DEFINITION CODE

Erythema – Grade 0 No erythema 0

Erythema – Grade 1 Very slight erythema (barely perceptible) 1

Erythema – Grade 2 Well-defined erythema 2

Erythema – Grade 3 Moderate to severe erythema 3

Erythema – Grade 4 Severe erythema (beet redness) 4

Maximized Grade 4 Notable dermal lesions (see below) M – 4
(see below)

Edema – Grade 0 No edema 0

Edema – Grade 1 Very slight edema (barely perceptible) 1

Slight edema (edges of area well defined by definite
Edema – Grade 2 raising) 2

Edema – Grade 3 Moderate edema (raised approximately 1 millimeter) 3

Edema – Grade 4 Severe edema (raised more than 1 millimeter and 4
extends beyond the area of exposure)

NOTE: Each animal was assigned an erythema and edema score. The most severely affected
area within the test site was graded. If eschar, blanching, ulceration and/or necrosis greater

than grade 1 was observed, then the “Maximized Grade 4" was assigned to the test site in
place of the erythema score and the type of notable dermal lesion(s) (e.g., eschar - grade 2,
blanching - grade 3, ulceration - grade 4, etc.) was noted. The presence of any other dermal
changes (e.g., desquamation, fissuring, eschar exfoliation, etc.) was also recorded.

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SLI Study No. 3596.17

MACROSCOPIC DERMAL GRADING SYSTEM

NOTABLE DERMAL LESIONS

OBSERVATION CODE DEFINITION

Eschar – Grade 1 ES-1 Focal and/or pinpoint areas up to 10% of test site.

Eschar – Grade 2 ES-2 > 10% < 25% of test site.

Eschar – Grade 3 ES-3 > 25% < 50% of test site.

Eschar – Grade 4 ES-4 > 50% of test site.

Blanching – Grade 1 BLA-1 Focal and/or pinpoint areas up to 10% of test site.

Blanching – Grade 2 BLA-2 > 10% < 25% of test site.

Blanching – Grade 3 BLA-3 > 25% < 50% of test site.

Blanching – Grade 4 BLA-4 > 50% of test site.

Ulceration – Grade 1 U-1 Focal and/or pinpoint areas up to 10% of test site.

Ulceration – Grade 2 U-2 > 10% < 25% of test site.

Ulceration – Grade 3 U-3 > 25% < 50% of test site.

Ulceration – Grade 4 U-4 > 50% of test site.

Necrosis – Grade 1 NEC-1 Focal and/or pinpoint areas up to 10% of test site
(color) (Note color of necrosis).

Necrosis – Grade 2 NEC-2 > 10% < 25% of test site (Note color of necrosis).
(color)
NEC-3
Necrosis – Grade 3 > 25% < 50% of test site (Note color of necrosis).
(color)
NEC-4
Necrosis – Grade 4 > 50% of test site (Note color of necrosis).
(color)

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SLI Study No. 3596.17

MACROSCOPIC DERMAL GRADING SYSTEM

ADDITIONAL DERMAL FINDINGS

OBSERVATION DEFINITION CODE

Characterized by scaling or flaking of
Desquamation dermal tissue with or without denuded DES
areas.

Characterized by cracking of the skin with
or without moist exudate. Fissuring should
Fissuring be checked prior to removing the animal FIS
from the cage and manipulating the test

site.
The process by which areas of eschar
Eschar Exfoliation EXF
flake off the test site.
Skin located at test site appears to be
TSS
Test Site Staining discolored, possibly due to test article (color)
(note color of staining).

The erythema extends beyond the test
Erythema Extends Beyond site. Note: A study director should be
the Test Site contacted for erythema extending beyond ERB
the test site.

Characterized by pale area(s) (almost a --
burn-like appearance) in the test site.
However, erythema may still be observed

through the pale area. Note: This
observation may affect the overall
erythema score of the test site. This
observation may progress to other
Superficial Lightening
observations resulting in notable dermal
lesions, but SL itself will not be considered
a notable dermal lesion that will result in a
dermal score to be maximized since it

does not result in any in-depth injury. To
be coded using an area designation (see
below).

Superficial Lightening - Focal and/or pinpoint areas up to 10% of
Grade 1 the test site SL-1

Superficial Lightening - > 10% < 25% of test site SL-2
Grade 2

Superficial Lightening - > 25% < 50% of test site SL-3
Grade 3
Superficial Lightening - > 50% of test site
SL-4
Grade 4

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SLI Study No. 3596.17

MACROSCOPIC DERMAL GRADING SYSTEM

ADDITIONAL FINDINGS

OBSERVATION DEFINITION CODE

Noticeable irritation outside of test site
Dermal Irritation - Outside oprobably due to the binding tape material. IT
the Test Site This notation will only be made for
reactions greater than what are normally

observed from tape removal which do not
interfere with the scoring of the test site.

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SLI Study No. 3596.17

APPENDIX B

SLI Personnel Responsibilities

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SLI Study No. 3596.17

SLI PERSONNEL RESPONSIBILITIES

Kimberly L. Bonnette, M.S., LATG Study Director/Director, Acute Toxicology

Dawn D. Rodabaugh, B.S. Alternate Contact/Toxicologist

Malcolm Blair, Ph.D. Managing Director Emeritus

Joseph C. Siglin, Ph.D., DABT General Manager

Rusty E. Rush, M.S., LAT, DABT Director, Neurotoxicity and Transgenics

Jason W. Smedley, B.S. Assistant Toxicologist

Pamela S. Smith, ALAT Study Supervisor, Acute Toxicology

Christina L. Zehender, B.S. Primary Technician/Acute Technician I

Delores P. Knippen Supervisor, Pharmacy

Steven H. Magness, B.S., LATG Senior Supervisor, Pathology

Anita M. Bosau, RQAP-GLP Senior Director, Compliance Assurance

Cheryl Bellamy Senior Supervisor, Report Writing

Deanna M. Talerico, RQAP-GLP Senior Supervisor, Quality Assurance

J. Dale Thurman, D.V.M., M.S., Senior Director, Pathology
DACVP

Kathy M. Gasser Archivist

73Annex 56-A

AN ACUTE NOSE-ONLY INHALATION TOXICITY
STUDY IN RATS WITH SPRAY--CHARLIE

FINAL REPORT

OPPTS Guidelines

870.1300

Author

Kimberly L. Bonnette, M.S., LATG

Study Completed on

March 14, 2003

Performing Laboratory

Springborn Laboratories (SLI),
a division of Charles River Laboratories, Inc.
640 North Elizabeth Street
Spencerville, Ohio 45887

SLI Study No.

3596.18

Submitted to

INL/A

U.S. Department of State
2201 C St. NW SA-4
Washington, DC 20520

Page 1 of 52

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SLI Study No. 3596.18

1. STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

No claim of confidentiality is made for any information contained in this study on
the basis of its falling within the scope of FIFRA §10(d)(1)(A), (B), or (C).

Company:

Company Agent: ____________________ Date

Title Signature

75Annex 56-A

76 Annex 56-A

77Annex 56-A

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SLI Study No. 3596.18

4. TABLE OF CONTENTS

1. STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS......................... 2

2. COMPLIANCE STATEMENT....................................................................... 3

3. QUALITY ASSURANCE STATEMENT ....................................................... 4

4. TABLE OF CONTENTS........................................................................
....... 5

5. LIST OF TABLES AND APPENDICES........................................................ 6

6. SUMMARY........................................................................
........................... 7

7. INTRODUCTION........................................................................
.................. 8

8. MATERIALS AND METHODS..................................................................... 8

9. EXPERIMENTAL PROCEDURES ............................................................. 11

10. ANALYSIS OF DATA........................................................................
....... 14

11. MAINTENANCE OF RAW DATA AND RECORDS................................. 14

12. RESULTS........................................................................
......................... 14

13. CONCLUSION ........................................................................
................. 16

14. REPORT REVIEW ........................................................................
........... 16

15. REFERENCE ........................................................................
................... 17

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SLI Study No. 3596.18

5. LIST OF TABLES AND APPENDICES

Tables

1. Summary of Aerosol Generation and Chamber Environmental Data......18
2. Individual Clinical Observations ..............................................................19
3. Individual Body Weights..........................................................................23

4. Individual Gross Necropsy Observations................................................25

Figure

1. Multi-Stage 10L Nose-Only Inhalation Chamber......................................27

Appendices

A. Preliminary Aerosol Generation Trials......................................................28

B. Analytical Chemistry Report.....................................................................36

C. Individual Aerosol Generation and Chamber Environmental Data...........43

D. SLI Personnel Responsibilities.................................................................51

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SLI Study No. 3596.18

6. SUMMARY

The four-hour nose-only inhalation toxicity of Spray--Charlie was evaluated in

Sprague Dawley rats. A limit test was performed in which a group of five male
and five female rats received a four-hour nose-only inhalation exposure to a time-
weighted average aerosol concentration (analytically determined) of 2.60 mg/L.

The mass median aerodynamic diameter and geometric standard deviation of the
sampled particles were 2.9 µ + 2.17. The percentage of particles < 4.0 µ was
determined to be 66%. Following the exposure, the limit test rats were observed
daily and weighed weekly. A gross necropsy examination was performed on all

limit test animals at the time of scheduled euthanasia (day 14).

No mortality occurred during the study. The most notable clinical abnormalities

observed during the study included breathing abnormalities, no/decreased
defecation, urine staining, rough haircoat, dark material around the facial area
and decreased food consumption. Body weight loss was noted in two males and

one female during the day 0 to 7 body weight interval. Body weight gain was
noted for all other animals during the test period. At study termination, the
animals had exceeded/maintained their initial body weight. No gross internal

findings were observed at necropsy on study day 14.

Under the conditions of this test, the acute inhalation LC50 of Spray--Charlie was

estimated to be greater than 2.60 mg/L in the rat (which was well above the EPA-
required 2.00 mg/L).

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SLI Study No. 3596.18

7. INTRODUCTION

This study was performed to assess the short-term toxicity of Spray--Charlie in

Sprague Dawley rats when administered by a four-hour nose-only inhalation
exposure. This study was intended to provide information on the potential health
hazards of the test article with respect to inhalation exposure. Data from this

study may serve as a basis for classification and/or labeling of the test article.
This study was conducted in accordance with the US EPA, Health Effects Test
Guidelines, OPPTS 870.1300, Acute Inhalation Toxicity, August, 1998. This

study was performed at Springborn Laboratories (SLI), 553 North Broadway,
Spencerville, Ohio. The protocol was signed by the Study Director on

October 9, 2002 (GLP initiation date). The in-life phase of the study was initiated
with test article administration on January 14, 2003 (day 0) and concluded with
terminal euthanasia on January 28, 2003.

8. MATERIALS AND METHODS

8.1. Test Article

The test article was received from the Sponsor and identified as follows:

Assigned Physical Receipt Expiration
Spansor’s ID SLI ID Description Date Date
Spray--Charlie S02.003.3596 Amber 12/09/02 None
liquid provided
b
Ingredients:
Herbicide: GLY-41 None
Lot No.: Manufactured 10/20/02 provided

Surfactant: Cosmo Flux-411F None

a Lot No.: Manufactured 11/29/02 provided
bSample pooled at SLI from five different mixes of Spray--Charlie (top/middle/bottom).
Ingredients used in the five Spray--Charlie mixes that were prepared by the Sponsor.

The test article was stored at room temperature. The Sponsor was responsible
for any necessary evaluations related to identity, strength, purity, composition,
stability and method of synthesis of the test material according to

40 CFR 160.105 and 40 CFR 792.105. Springborn Laboratories analyzed the
test article for the glyphosate (a.e.) which is presented in

SLI Study No. 3596.15.

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SLI Study No. 3596.18

8.2. Retention Sample

retention samples for all studies conducted with this material.

8.3. Test Article Disposition

The test article was returned to the Sponsor following completion of all studies
with the test article.

8.4. Method of Test Article Preparation

The test articles were pooled and administered as received from the Sponsor
and dispensed fresh on the day of dosing. The pooled test article was stirred
approximately 10 minutes prior to dispensation and stirred continuously during
dosing.

8.5. Animals and Animal Husbandry

8.5.1. Description, Identification and Housing
Young adult, Hsd: Sprague Dawley® SD® rats were received from Harlan

Sprague Dawley, Inc., Indianapolis, IN. Upon receipt, metal ear tags displaying
unique identification numbers were used to individually identify the animals.
Cage cards displaying at least the study number, animal number and sex were
affixed to each cage. The animals were housed individually in suspended
stainless steel cages. All housing and care were based on the standards

recommended by the Guide for the Care and Use of Laboratory Animals [1].

8.5.2. Environment
The animal room temperature and relative humidity ranges were 64-68°F
(18-20°C) and 37-55%, respectively. Environmental control equipment was

8.5.3. Food

PMI Certified Rodent Chow #5002 (Purina Mills, Inc.) was provided ad libitum to
the animals throughout the study (except during the time that the animals were

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SLI Study No. 3596.18

acclimated to the exposure tubes and maintained in the inhalation room for the
exposure procedure). The lot number and expiration date of each batch of diet
used during the study were recorded. The feed was analyzed and certified by the
supplier for nutritional components and environmental contaminants. Dietary

limitations for various environmental contaminants, including heavy metals,
pesticides, polychlorinated biphenyls and total aflatoxin are set by the
manufacturer. Within these limits, contaminants which may have been present
were not expected to compromise the purpose of this study. Results of the
dietary analyses (Certificates of Analysis) are provided by the manufacturer for

each lot of diet. These are maintained by SLI.

8.5.4. Water
Municipal tap water treated by reverse osmosis was available ad libitum
throughout the study (except during the time that the animals were acclimated to

the exposure tubes and maintained in the inhalation room for the exposure
procedure). The purified water was supplied by an automatic watering system.
Monitoring of the drinking water for contaminants is conducted by SLI and the
records are available for inspection. Within generally accepted limits,

8.5.5. Acclimation

Upon receipt, the animals were removed randomly from the shipping cartons,
examined by qualified personnel, identified with metal ear tags and then
acclimated to the laboratory conditions for a minimum of five days. The animals
were observed daily for overt physical or behavioral abnormalities, general
health/moribundity and mortality.

8.5.6. Animal Selection

The animals chosen for study use were randomly selected from healthy stock
animals using a computerized random numbers table to avoid potential bias. All
animals received a detailed pretest observation prior to dosing. Only healthy

animals were chosen for study use. Females were nulliparous and nonpregnant.
The male animals were approximately 9 weeks of age and weighed 248-275 g on
the day of exposure. The female animals were approximately 9 weeks of age
and weighed 201-212 g on the day of exposure.

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SLI Study No. 3596.18

9. EXPERIMENTAL PROCEDURES

9.1. Preliminary Procedures

9.1.1. Test Article Volatility Determination

The volatility of the test article relative to a distilled water standard was
determined prior to experimental initiation. This procedure was performed in
order to determine if the test article had sufficiently low volatility to allow for an

accurate gravimetric determination of the aerosol concentration. A known
quantity of the test article was placed on a preweighed filter disk and was allowed
to evaporate for a total of ten minutes. The test article weight was determined
each minute and the amount of evaporation of the test article was then

determined. The results of this volatility trial indicated that the test article
evaporation rate (0.82 mg/minute) was only slightly higher than the SLI
determined distilled water evaporation rate (0.55 mg/minute); therefore was
considered to not be volatile.

9.1.2. Preliminary Aerosol Generation Trials

Prior to experimental initiation, preliminary aerosol generation trials were
conducted. These trials were performed in order to determine the most efficient
means of generating an aerosol of the appropriate concentration while utilizing

equipment that would reduce the aerodynamic particle size. Data obtained
during the preliminary aerosol generation trials are presented in Appendix A.

9.2. Limit Test

9.2.1. Aerosol Generation Equipment

The test aerosol was generated with a Pistol Spraying System and a Master Flex
Pump and Pump Heads 77200-60 and 7523-30. Conditioned high pressure

external air was used in generating the test atmosphere. The aerosol was blown
through a 5L Elutriator, the Multi-Stage 10L nose-only inhalation chamber and
then vented from the chamber to an air treatment system which consisted of a
prefilter, a HEPA filter, a charcoal bed and a water scrubbing tower (see
Figure 1).

9.2.2. Dosing

On day 0, the animals chosen for the limit test were weighed, placed in a nose-
only exposure tube and allowed to acclimate to the exposure tube for at least

1 hour. Animals that appeared to have been acclimated to the exposure tube
(i.e., minimal struggling and no inversion) were considered to be acceptable,

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SLI Study No. 3596.18

removed from the exposure tube and returned to their cages until initiation of the

aerosol exposure. Animals that did not appear to acclimate to the exposure tube
were not acceptable, removed from the exposure tube and returned to their
cages.

The acceptable animals were then placed in exposure tubes, the tubes inserted
into the Multi-Stage 10L nose-only inhalation chamber and the test article
aerosolized at the following level:

Exposure Level No. of Animals
(mg/L) Male Female

2.60 5 5

The aerosol exposure consisted of a 3-minute T99 equilibration period, a
240-minute exposure period and a 3-minute de-equilibration period equal to the
T99 equilibration period. After each aerosol exposure, animals were removed
from the exposure tubes and residual test article was removed from the animal's

exterior surfaces (where practical) by wiping the haircoat with a towel. The
animals were then returned to ad libitum feed and water. The following
parameters were measured during the exposure.

9.2.2.1. Chamber Air Flow

Air flow readings were recorded at the initiation of the T99 equilibration period, at
approximate 30-minute intervals during the aerosol exposure and at the
conclusion of the de-equilibration period.

9.2.2.2. Aerosol Concentration

The aerosol concentration was measured at the beginning of the aerosol
exposure (after equilibration), at approximate 30-minute intervals during the

aerosol exposure and at the conclusion of the aerosol exposure (before de-
equilibration). The concentration of the test article aerosol was collected in the
inhalation chamber by gravimetric technique. A 5 L sample of the aerosol was
drawn from the breathing zone of the chamber through a preweighed glass fiber

filter. The change in weight of the filter (mg) was then determined and this value
was divided by the volume of chamber atmosphere sampled (L) to yield the
gravimetric concentration (mg/L). The average time-weighted gravimetric
concentration of the test atmosphere was then calculated for the exposure. For

the analytical concentration, the gravimetrically obtained samples were analyzed
by Springborn Laboratories for the glyphosate component, a non-volatile
component of the test article. These analyses were performed in order to
determine the analytical (actual) concentrations of the aerosol in the chamber for

each sampling period. The average time weighted analytical concentration of the

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SLI Study No. 3596.18

test atmosphere was then calculated for the exposure. Chemistry methods and
results are detailed in the Analytical Chemistry Report (Appendix B).

9.2.2.3. Chamber Temperature and Humidity

The chamber temperature and humidity were measured electronically and
recorded at approximate 30-minute intervals during the aerosol exposure using a
Vaisala HMI 41 Thermometer.

9.2.2.4. Aerosol Aerodynamic Particle-Size Distribution

The aerosol aerodynamic particle-size distribution was determined three times
during the aerosol exposure using the ITP 7 Stage Cascade Impactor. Each
stage of the impactor was fitted with a preweighed glass fiber filter. Five liters
per minute of the chamber air were drawn through the impactor and the change
in weight of each filter was then determined and recorded. The mean particle-

size distribution was subsequently determined using an Excel computer
adaptation of the manual method. The Mass Median Aerodynamic Diameter,
Geometric Standard Deviation and percentage of particles < 4.0 µ were then
determined. At least one hour passed between each aerosol particle-size

analysis.
9.2.2.5. Chamber Oxygen

Chamber oxygen content was measured and recorded at approximate 30-minute
intervals during the aerosol exposure using a GC-501 Oxygen Sensor.

9.2.3. Clinical Observations

The limit test animals were observed for clinical abnormalities during each
aerosol exposure, two times on study day 0 (post-exposure) and daily thereafter
(days 1-14). A general health/mortality check was performed twice daily (in the

morning and in the afternoon).

9.2.4. Body Weights
Individual body weights were obtained for the limit test animals prior to dosing on

day 0 and on days 7 and 14.
9.2.5. Gross Necropsy

All limit test animals were euthanized by carbon dioxide inhalation at study
termination (day 14) and necropsied. Body cavities (cranial, thoracic, abdominal

and pelvic) were opened and examined. No tissues were retained.

86 Annex 56-A

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SLI Study No. 3596.18

9.3. Protocol Deviations

The temperature of the animal room [64-68°F (18-20°C)] exceeded the preferred
range [66-77°F (19-25°C)] during this study. This occurrence was considered to

have had no adverse effect on the outcome of this study.

10. ANALYSIS OF DATA

Data from the limit tests were analyzed and an LC50 value estimated as follows:

< 50% Mortality: LC50 was estimated as greater than the administered dose.
= 50% Mortality: LC50 was estimated as equal to the administered dose.
> 50% Mortality: LC50 was estimated as less than the administered dose.

Body weight means and standard deviations were calculated separately for
males and females. The aerodynamic particle-size distribution of the test article
aerosol was plotted using an Excel computer adaptation of the three cycle
logarithmic probability paper as per the ITP Cascade Impactor instruction

manual. The Mass Median Aerodynamic Diameter, Geometric Standard
Deviation and particles < 4.0 µ were determined based on the plotted distribution.

11. MAINTENANCE OF RAW DATA AND RECORDS

All original raw data, the final report and electronic encoded records were
transferred to the SLI archives for a period of 10 years. The Sponsor will be
contacted prior to final disposition of these items.

12. RESULTS

12.1. Aerosol Generation and Chamber Environmental Data

12.1.1. Aerosol Generation Data

Individual Data: Table 1

The average time-weighted analytical concentration for the aerosol exposure was

determined to be 2.60 mg/L. The mass median aerodynamic diameter and
geometric standard deviation of the sampled particles were 2.9µ ± 2.17. The
percentage of particles < 4.0 µ was determined to be 66%.

87Annex 56-A

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SLI Study No. 3596.18

12.1.2. Chamber Environmental Data
Individual Data: Table 1

Chamber temperature and relative humidity for the aerosol exposure ranged from
68.3-70.7°F and 68.3-69.3%, respectively. Oxygen content was maintained at
20.9% throughout the exposure.

12.2. Limit Test Data

12.2.1. Mortality

Individual Data: Table 2

No mortality occurred during the study.

12.2.2. Clinical Observations

Individual Data: Table 2

No positive findings were noted at the time of observation during the 4-hour

exposure period. The most notable clinical abnormalities observed during the
study included breathing abnormalities, no/decreased defecation, urine staining,
rough haircoat, dark material around the facial area and decreased food
consumption.

12.2.3. Body Weight Data

Individual Data: Table 3

Body weight loss was noted in two males and one female during the day 0 to 7

body weight interval. Body weight gain was noted for all other animals during the
test period. At study termination, the animals had exceeded/maintained their
initial body weight.

12.2.4. Gross Necropsy

Individual Data: Table 4

No gross internal findings were observed at necropsy on study day 14.

88 Annex 56-A

89Annex 56-A

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SLI Study No. 3596.18

15. REFERENCE

1. Guide for the Care and Use of Laboratory Animals, DHHS Publication No.
(NIH) 96-03, 1996.

90 Annex 56-A

(18)

PAGE 1

10 5 24 3 3 66
2.60 95.24240 70.3060 2.9±2.17 20.9
68.37.03.-769.3

EXPOSURE LEVEL (MG/L)

TABLE 1

CHAMBER ENVIRONMENTAL DATA

AN ACUTE NOSE-ONLY INHALATION TOXICITY STUDY IN RATS

4.0 µ (%):
≤

STINL/A, U.S. DEPARTMENT OF STATEOTREIQLWLUQSUMMARY OF AEROSOL GENERATION ANDCDLGYSNSNGNE(%N:(%N):(MG/L):

91Annex 56-A

(19)

---- ---- ----

STUDYSNO.:PA3MALES--O----MALE#-------OBSERVATI--U0RCES68RD-LETCHEDUDSSASGREHANTCERI-LO----1--RNOSTDTAA-
FLSTUDUOC--EPE(S)-11R-----------P

92 Annex 56-A

(20)

---- ---- ----

STUDY.NDO.:A3-9LES---2--MAL--#------O-HSNRVAGIO-EBNBRES-E-GHKRKC---RIALALO-GHNDNNSUM-OTHON------
-
PAY--5F
S
P
Y--P-PP--P---LP--P----1R--------------------

93Annex 56-A

(21)

---- ---- ----

STUUDY.NO.:A3TFEMALES--FEMALE-#------OBS--ULSSUFOINUG-AHIHOT-U-G--EETNEREA1---ERERD---D0--CMAMU
--A
-AROUOCOU-2--S)PPNP---11-------------P

94 Annex 56-A

(22)

---- ---- ----

STUDY.NDO.:AFEM--8ES-F-EMALE#------SOBSERVATI--EBNBRESCES6-MTTETATSEDU-SSNRNIN-RIA-IDANAD--E2--LALAFOO-H--YEOSOPT--RNP-P-------V3-P--
-P--REP-----------

95Annex 56-A

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---- ---- ----

STUDYSNOD.:P359MMALES--ANI-MAL#----30386EA-3Y0OF---9--48---9--535-D---T7-(D-AY)-------------------------------------------
-------------------------------------
---------------------------------------------------------------------------------------------

96 Annex 56-A

(24)

---- ---- ----

STUDSY.NO.:AR35FEMAL-EA-NIMAL#--4858-5ME-60Y0OF--12U2Y---35-47AT-52-7T2-4-DAY)-------------------------------------------
-------------------------------------
---------------------------------------------------------------------------------------------

97Annex 56-A

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1 ---- ---- ----

STU.S. DEPARTMMALES----TMA0-MA6L-A-OFT----A--J-8-3--4-2----14T-1TISI-I-AT--LHIN--I-TH--TLII--IL-L
-IMAL----MITS------

98 Annex 56-A

(26)

2 ---- ---- ----

STU.S. DEPARTMFEM-ALEA-2.60-M--D---6850---TUD--3860---03S--AAL0-A------ILWG---ES---TMAP-M-OR---NVNT---S---SMIT--
-------
-
-------------------

99Annex 56-A

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100 Annex 56-A

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SLI Study No. 3596.18

APPENDIX A

Preliminary Aerosol Generation Trials

101Annex 56-A

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SLI Study No. 3596.18

1. PRELIMINARY AEROSOL GENERATION TRIALS
Prior to experimental initiation, preliminary aerosol generation trials were

conducted. These trials were performed to determine the appropriate means of
generating the aerosol exposure atmosphere of the test article at the targeted
gravimetric/analytical concentration of (2.00 mg/L, initially) and the aerodynamic
particle size (1-4 microns Mass Median Aerodynamic Diameter). The type of
equipment used during each trial procedure is presented in the table that follows.

It was determined that since the gravimetric concentration was proportional to the
analytical concentration it could be used as a “real time” estimate for the actual
analytical concentration thus allowing for changes during the exposure. The
results of the trials indicated that the equipment utilized during Trials # 1-7

produced an analytical concentration greater than 2.00 mg/L utilizing a pump
speed of 1.2 mL/minute or greater. In addition, the aerodynamic particle size
distribution was determined using the ITP 7 Stage Cascade Impactor during
Trial # 2 and was acceptable (3.0 + 1.78 µ ). Therefore, this equipment design

was used for the study exposure.

Note: The ability to generate a target gravimetric concentration of > 0.5 mg/L
(Trials # 8-10) were also explored. These trials revealed that the gravimetric
concentrations were also proportional to the analytical concentration at lower

concentrations. The trials provide an indication of the settings necessary to
achieve the target analytical concentration and that the gravimetric
concentrations could be used as a “real time” estimate of the analytical
concentration at lower concentrations in case additional levels would have been
required.

102 Annex 56-A

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PAGE 1

MAXIMUM ATTAINABLE
CONCENTRATIONS (MG/L)

GRAVIMETRIC ANALYTICAL

TEST
ARCONCEN-
TRATION (%)

30 100 2.40 100 -- 2.52 30 1040.829 2.54 4.688
INPAIR (PSI)

TRIAL TABLE 1

2.50 gravimetric concentration for Trials 1-3.

EQUIPMENT USED

4.50 mg/L analytical and >

5LMEl7tiprryi4ggmysEputrStap4lyirsumistI7-intrliiHLi/yludumb-gisNlAeradlui723-in0gndzzle

1 One Mult2i-Stage OLnNMseli-3tygehamLOerse-ultiltagem10LrNose-Only Chamber

STINL/A, U.S. DEPARTMENT OF STATE PRELIMINARY AEROSOL GENERATION TRIALSeting ≥

103Annex 56-A

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PAGE 2

MAXIMUM ATTAINABLE
CONCENTRATIONS (MG/L)

GRAVIMETRIC ANALYTICAL

TEST
ARTCONCEN-
TRATION (%)

30 100 1.600 100 -- 1.360 100 -- 1.50 3.169
INAIR (PSI)

TRIAL TABLE 1

1.50 mg/L gravimetric concentration for Trials 4-6.

EQUIPMENT USED

3.00 mg/L analytical and >

5LMEl7sirtr0x4ggaugsatst,Psp50Hgimyltdtdtnirliiozi/yludtmb-gisilderadlui723-in0gndzzle

4 One Multi-Stage OLnNoMselintygehamLberMselintygehamLberose-Only Chamber

STUINL/A, U.S. DEPARTMENT OF STATE PRELIMINARY AEROSOL GENERATION TRIALSng ≥

104 Annex 56-A

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PAGE 3

MAXIMUM ATTAINABLE
CONCENTRATIONS (MG/L)

GRAVIMETRIC ANALYTICAL

TEST
ARCONCEN-
TRATION (%)

30 100 1.00 100 2.940 0.8360 100 -- 0.52 1.202
INPAIR (PSI)

TRIAL TABLE 1

1.50 gravimetric concentration for Trial 7.

EQUIPMENT USED

3.≥0 mg/L analytical and >d gravimetric concentration for Trials 8-9.

5LMEl7tiprryi4ggmysEpu7tntil1lPei/yuidub,-nslzeAeadyu7tMb3-sgszHAerdlui723i-in0nodzzle

7 One Mult8i-Stage OLnNMseli-ntygehamLbeMseli-nygCehamLberose-Only Chamber

STINL/A, U.S. DEPARTMENT OF STATE PRELIMINARY AEROSOL GENERATION TRIALSng ≥ng

105Annex 56-A

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PAGE 4

MAXIMUM ATTAINABLE
CONCENTRATIONS (MG/L)

GRAVIMETRIC ANALYTICAL

TEST
ARCONCEN-
TRATION (%)

30 100 0.460 100 1.311 1.3300 100 -- 0.64 --
INPAIR (PSI)

TRIAL TABLE 1

EQUIPMENT USED

1.00 mg/L analytical and gravimetric concentration for Trials 10-12.

5LEa7stitrryi4gu5ysEpu7tntrl1lPei/yuEdus2-nrlNnAea/yu7dub3-nsszlAeradlui723-in0gndzzle

10 One Multi-Stage OLnNoMse--SnygehaOLbNeMselintygeha0Lberose-Only Chamber

STINL/A, U.S. DEPARTMENT OF STATE PRELIMINARY AEROSOL GENERATION TRIALSng ≥

106 Annex 56-A

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PAGE 5

MAXIMUM ATTAINABLE
CONCENTRATIONS (MG/L)

GRAVIMETRIC ANALYTICAL

TEST
ARTCONCEN-
TRATION (%)

30 100 0.72 --
INAIR (PSI)

TRIAL TABLE 1

EQUIPMENT USED

1.00 mg/L gravimetric concentration for Trial 13.

5LMEl7tir0r104ggaysnptub,ngpszHAeidlud523iin0gnodzzle

13 One Multi-Stage 10L Nose-Only Chamber

STINL/A, U.S. DEPARTMENT OF STATE Note: TargPRELIMINARY AEROSOL GENERATION TRIALS

107Annex 56-A

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SLI Study No. 3596.18

AN ACUTE NOSE-ONLY INHALATION TOXICITY STUDY IN RATS

AERODYNAMIC PARTICLE SIZE DATA
TRIAL 2

Effective

Cutoff Filter Weights (mg) Difference Cumulative
Stage Diameter Pre-sample Post-sample Weights % of Total % <ECD

1 10.00 103.2 103.3 0.1 1.4 98.6
2 6.11 102.9 103.7 0.8 11.4 87.1

3 3.70 103.6 105.0 1.4 20.0 67.1

4 2.22 103.4 106.1 2.7 38.6 28.6
5 1.39 103.1 104.5 1.4 20.0 8.6

6 0.79 103.5 104.0 0.5 7.1 1.4
7 0.50 103.8 103.9 0.1 1.4 0.0

Filter - 103.6 103.6 0.0 0.0
Total of Difference Weights: 7.0

Mass Median Aerodynamic Diameter = 3.0 microns

Geometric Standard Deviation = 1.78
Percentage ≤ 4.0 microns = 70 %

108 Annex 56-A

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SLI Study No. 3596.18

APPENDIX B

Analytical Chemistry Report

109Annex 56-A

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SLI Study No. 3596.18

1. SPRAY--CHARLIE ANALYSIS

The analytical method for the analysis of the glyphosate component of Spray--
Charlie was validated prior to the analytical chamber concentration analyses
performed at Springborn Laboratories, Inc. This method was utilized to
determine the inhalation chamber concentration during the Acute Nose-Only

Inhalation Toxicity Study.

1.1. Experimental System

1.1.1. HPLC System

Pump: Waters 600E System Controller
Injector: Waters WISP 717
Detector: Waters 2487
Data System: HP 3396B Integrator

Precolumn Phenomenex, SecurityGuard, C18, 4.0 x 3.0 mm ID
Column: Phenomenex, Spherex, C18, 5µ, 250 x 4.6 mm ID
Mobile Phase: A: 0.05 M HCO N2 , 4H 3.6/5% Acetonitrile
B1:00HPLACcetonitrile
Gradient: 100% A, hold for 6 minutes; linear change to 25% A/75%

B over 1 minute; hold for 5 minutes; linear change to
100% A over 1 minute; hold at 100% A for 15 minutes
Injection Volume: 10 µL
Flow Rate: 1.0 mL/min

Detection: 500nm; 0.4000 AUFS

1.1.2. Apparatus
Balance: Mettler AG 245, accuracy of 0.0001 gram

Glassware: Assorted volumetric glassware
Filters: Gelman, glass fiber, Whatman Puradisc 25PP, 0.45µm;
0.2 µ Nylon-66 filter
Shaker: Labline, Multi-Wrist Shaker

Oven: Boeke l,odel 7905
Pipet: Mettler-Toled1o00-1000 µL, 500 – 5000 µL
pH Meter Corning 320

110 Annex 56-A

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SLI Study No. 3596.18

1.1.3. Solutions and Reagents

1.1.3.1. Reagents

Water, Fisher, HPLC Grade, Lot # 023349
Acetonitrile, J.T. Baker, HPLC Grade, Lot # M15811
NBD-Chloride, Aldrich, Lot # 10926TO
Hydrochloric Acid, A.C.S. Grade, Lot # 012161

Potassium Tetraborate Tetrahydrate, Aldrich, Lot # 15325DI
Ammonium Formate, Fisher, Certified Grade, Lot # 990125
Formic Acid, Fisher, Laboratory Grade, Lot # 003630
Methanol, Fisher, HPLC Grade, Lot # 023883

1.1.3.2. Solutions

0.37M Borate Solution: Prepared by dissolving approximately 11.44 g of
potassium tetraborate tetrahydrate in 100 mL of HPLC grade water. The
resulting solution was mixed thoroughly and was stable for 6 months post-
preparation at room temperature.

1.2 N HCl: Prepared by diluting 10 mL of HCl in 90 mL of HPLC grade water.
The resulting solution was mixed thoroughly and was stable for 6 months post-
preparation at room temperature.

25 mM NBD-Cl: Prepared by dissolving approximately 2.5 g of NBD-Cl in
500 mL of HPLC grade methanol. The resulting solution was mixed thoroughly
and was stable for 6 months post-preparation at room temperature.

Mobile Phase A: Prepared by dissolving approximately 1.57 g of ammonium
formate in 950 mL of HPLC grade water. The pH of the resulting solution was
adjusted to approximately 3.6 with formic acid. Then, 50 mL of HPLC grade
acetonitrile was added. The resulting solution was mixed thoroughly, filtered
through a 0.2 µ m Nylon-66 filter, and degassed by helium sparging prior to use.

Different volumes were also prepared using the same ratio of components.

Mobile Phase B: 100% HPLC grade acetonitrile used as received.

Diluent: 100% HPLC grade water used as received.

Stock Standard Solution (Trial Work): Prepared by dissolving 116.8 mg of Spray-
-Charlie in a 25 mL flask with diluent.

111Annex 56-A

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SLI Study No. 3596.18

Standard Solutions (Trial Work) : Prepared by serially diluting the stock standard
solution with diluent. The final concentrations of the solutions were in the range
of approximately 0.47 to 3.3 mg/mL. These solutions were then filtered through
Whatman Puradisc 25PP 0.45 µm filters and diluted with HPLC water at a ratio of

1:10 prior to the derivatization.

Stock Standard Solution (Exposure #1): Prepared by dissolving 100.2 mg of
Spray--Charlie in a 25 mL flask with diluent.

Standard Solutions (Exposure #1) : Prepared by serially diluting the stock
standard solution with diluent. The final concentrations of the solutions were in
the range of approximately 0.4 to 1.6 mg/mL. These solutions were then filtered
through Whatman Puradisc 25PP 0.45 µm filters and diluted with HPLC water at
a ratio of 1:10 prior to the derivatization.

Chamber Concentration Solutions: Prepared by placing the weighed glass
fiber filter used for gravimetric concentration determination in a capped container
with 10 mL of diluent. The solutions were then agitated mechanically for

15 minutes and filtered through Whatman Puradisc 25PP 0.45 µ m filters. The
sample solutions were then diluted at a ratio of 1:10 with HPLC water prior to
derivatization.

Precolumn Derivatization: In order to analyze the glyphosate component, a

precolumn derivatization was performed by adding 1.2 mL of the appropriate
control, standard, or sample solution to a labeled scintillation vial. Both 0.8 mL of
the borate solution and 2.4 mL of the NBD-Cl solution were added to each vial.
The vials were then capped and shaken by hand prior to being heated in an oven
at 80° C for 30 minutes. After removal from the oven, the vials were allowed to

cool for 10 minutes followed by the addition of 0.9 mL of the HCl solution. After
the vials were again shaken by hand, they were allowed to stand for 10 minutes
in order for incipient precipitation to occur. These solutions were then transferred
to injection vials.

1.2. Analytical Procedures

1.2.1. Standard Curve Analysis
The peak areas of the glyphosate component of each standard were determined,
measured, and plotted as a function of concentration to generate a standard

curve. The actual values used for the calculations are shown in Chemistry
Tables 1 and 2.

112 Annex 56-A

113Annex 56-A

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SLI Study No. 3596.18

Chemistry Table 1

Standard Curve and Sample Analysis Values for Trial Work

Theoretical Conc. Analytical Chamber
Sample No. (mg/L) Peak Area Conc. (mg/L)
Std 1 0.9344 31125 NA

Std 2 2.804 97258 NA
Std 3 4.672 170507 NA
Std 4 6.540 249444 NA

Trial # 2 NA 179632 4.829
Trial # 3 NA 174130 4.688
Trial # 6 NA 114911 3.169
Trial # 6 NA 105992 2.940

Trial # 9 NA 38278 1.202
Trial # 10 NA 42531 1.311
NA – Not Applicable

Correlation coefficient = 0.9992

114 Annex 56-A

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SLI Study No. 3596.18

Chemistry Table 2

Standard Curve and Sample Analysis Values for Exposure #1

Theoretical Conc. Analytical Chamber

Sample No. (mg/L) Peak Area Conc. (mg/L)
Std 1 0.8016 25636 NA
Std 2 1.603 51542 NA
Std 3 2.404 70695 NA

Std 4 3.206 98772 NA
# 1 NA 81029 2.654
# 2 NA 62864 2.044

# 3 NA 85271 2.797
# 4 NA 87625 2.876
# 5 NA 79437 2.601
# 6 NA 80738 2.645

# 7 NA 80393 2.633
# 8 NA 77142 2.524
# 9 NA 82645 2.709

NA – Not Applicable
Correlation coefficient = 0.998

115Annex 56-A

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SLI Study No. 3596.18

APPENDIX C

Individual Aerosol Generation and
Chamber Environmental Data

116 Annex 56-A

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SLI Study No. 3596.18

2.60 mg/L Exposure Level

117Annex 56-A

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SLI Study No. 3596.18

AN ACUTE NOSE-ONLY INHALATION TOXICITY STUDY IN RATS
CHAMBER ENVIRONMENTAL DATA
EXPOSURE: 2.60 MG/L

TIME TEMPERATURE RELATIVE HUMIDITY OXYGEN CONTENT
(MIN.) (°F) (%) (%)
0 69.4 69.3 20.9

30 68.3 68.7 20.9
60 69.3 68.8 20.9
90 69.7 68.4 20.9
120 69.8 68.6 20.9

150 70.3 68.3 20.9
180 70.2 68.5 20.9
210 70.6 69.0 20.9
240 70.7 68.9 20.9

118 Annex 56-A

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SLI Study No. 3596.18

AN ACUTE NOSE-ONLY INHALATION TOXICITY STUDY IN RATS

TIME WEIGHTED ANALYTICAL CONCENTRATION
ANALYTICAL EXPOSURE: 2.60 MG/L

Mean Time
Aerosol Concentration Interval Weighted

Sample Sample Concentration Per Interval Length Concentration
No. Time (min.) (mg/L) (mg/L) (min.) Per Interval
1 0 2.65

2.35 30.00 70.35
2 30 2.04
2.42 30.00 72.60

3 60 2.80
2.84 30.00 85.20

4 90 2.88
2.74 30.00 82.20
5 120 2.60

2.63 30.00 78.75
6 150 2.65
2.64 30.00 79.20

7 180 2.63
2.58 30.00 77.25
8 210 2.52

2.62 30.00 78.45
9 240 2.71
TOTAL 240.00 624.00

TIME WEIGHTED MEAN ANALYTICAL CONCENTRATION (MG/L) 2.60

119Annex 56-A

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SLI Study No. 3596.18

AN ACUTE NOSE-ONLY INHALATION TOXICITY STUDY IN RATS
AERODYNAMIC PARTICLE SIZE DATA

SAMPLE NO. A
ANALYTICAL EXPOSURE: 2.60 MG/L

Effective
Cutoff Filter Weights (mg) Difference Cumulative

Stage Diameter Pre-sample Post-sample Weights % of Total % <ECD

1 10.00 102.0 102.2 0.2 8.0 92.0
2 6.11 102.2 102.4 0.2 8.0 84.0

3 3.70 102.1 102.5 0.4 16.0 68.0
4 2.22 102.7 103.7 1.0 40.0 28.0

5 1.39 103.5 103.9 0.4 16.0 12.0

6 0.79 103.7 103.9 0.2 8.0 4.0
7 0.50 103.3 103.4 0.1 4.0 0.0

Filter - 102.7 102.7 0.0 0.0
Total of Difference Weights: 2.5

Mass Median Aerodynamic Diameter = 3.1 microns

Geometric Standard Deviation = 2.10
Percentage ≤ 4.0 microns = 63 %

120 Annex 56-A

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SLI Study No. 3596.18

AN ACUTE NOSE-ONLY INHALATION TOXICITY STUDY IN RATS
AERODYNAMIC PARTICLE SIZE DATA

SAMPLE NO.: B
ANALYTICAL EXPOSURE: 2.60 MG/ML

Effective
Cutoff Filter Weights (mg) Difference Cumulative

Stage Diameter Pre-sample Post-sample Weights % of Total % <ECD

1 10.00 101.7 102.2 0.5 10.4 89.6
2 6.11 103.7 104.0 0.3 6.2 83.3

3 3.70 101.9 102.8 0.9 18.7 64.6
4 2.22 103.0 104.4 1.4 29.2 35.4

5 1.39 102.3 103.1 0.8 16.7 18.8

6 0.79 102.0 102.2 0.2 4.2 14.6
7 0.50 102.1 102.7 0.6 12.5 2.1

Filter - 102.3 102.4 0.1 2.1
Total of Difference Weights: 4.8

Mass Median Aerodynamic Diameter = 2.8 microns

Geometric Standard Deviation = 2.47
Percentage ≤ 4.0 microns = 65 %

121Annex 56-A

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SLI Study No. 3596.18

AN ACUTE NOSE-ONLY INHALATION TOXICITY STUDY IN RATS
AERODYNAMIC PARTICLE SIZE DATA

SAMPLE NO.: C
ANALYTICAL EXPOSURE: 2.60 MG/L

Effective
Cutoff Filter Weights (mg) Difference Cumulative

Stage Diameter Pre-sample Post-sample Weights % of Total % <ECD

1 10.00 102.7 102.9 0.2 4.4 95.6
2 6.11 103.4 103.7 0.3 6.7 88.9

3 3.70 103.2 103.9 0.7 15.6 73.3
4 2.22 102.8 104.3 1.5 33.3 40.0

5 1.39 102.7 103.8 1.1 24.4 15.6

6 0.79 102.9 103.5 0.6 13.3 2.2
7 0.50 103.0 103.1 0.1 2.2 0.0

Filter - 103.6 103.6 0.0 0.0
Total of Difference Weights: 4.5

Mass Median Aerodynamic Diameter = 2.8 microns

Geometric Standard Deviation = 1.95
Percentage ≤ 4.0 microns = 71 %

122 Annex 56-A

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SLI Study No. 3596.18

AN ACUTE NOSE-ONLY INHALATION TOXICITY STUDY IN RATS

AERODYNAMIC PARTICLE SIZE DATA

ANALYTICAL EXPOSURE: 2.60 MG/L

Effective Cutoff Cumulative % less than indicated size

Stage Diameter Sample A Sample B Sample C
1 10.00 92.0 89.6 95.6

2 6.11 84.0 83.3 88.9
3 3.70 68.0 64.6 73.3

4 2.22 28.0 35.4 40.0
5 1.39 12.0 18.8 15.6

6 0.79 4.0 14.6 2.2

7 0.50 0.0 2.1 0.0
Mean

Mass Median Aerodynamic Diameter 3.1 2.8 2.8 2.9
Geometric Standard Deviation 2.10 2.47 1.95 2.17

Percentage ≤ 4.0 microns 63 65 71 66

123Annex 56-A

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SLI Study No. 3596.18

APPENDIX D

SLI Personnel Responsibilities

124 Annex 56-A

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SLI Study No. 3596.18

SLI PERSONNEL RESPONSIBILITIES

Kimberly L. Bonnette, M.S., LATG Study Director/Director, Acute

Toxicology

Dawn D. Rodabaugh, B.S. Alternate Contact/Toxicologist

Malcolm Blair, Ph.D. Managing Director Emeritus

Rusty E. Rush, M.S., LAT, DABT Director, Neurotoxicity and Transgenics

Joseph C. Siglin, Ph.D., DABT General Manager

Jason W. Smedley, B.S. Assistant Toxicologist

Pamela S. Smith, ALAT Study Supervisor, Acute Toxicology

Kevin V. Weitzel, A.S. Primary Technician/Inhalation Team
Leader

Delores P. Knippen Supervisor, Pharmacy

Steven H. Magness, B.S., LATG Senior Supervisor, Pathology

Anita M. Bosau, RQAP-GLP Senior Director, Compliance Assurance

Cheryl A. Bellamy Senior Supervisor, Report Writing

Deanna M. Talerico, RQAP-GLP Senior Supervisor, Quality Assurance

J. Dale Thurman, D.V.M., M.S., DACVP Senior Director, Pathology

Kathy M. Gasser Archivist

125Annex 56-A

   

    
•  • •

 

 





    

  

  

 

  
      
   

  

  



 



   
    
  

   

126 Annex 56-A

    

      
             

             



  

 

127Annex 56-A

128 Annex 56-A

129Annex 56-A

    

   

  

   

       

 

 

  

  

130 Annex 56-A

    

     



   
   



   

   

     

    

   

131Annex 56-A

    

 

        
           

          
          
           

          
         

 

          
          ±   
             

  

 

 "      
           
          
         

             
           
          



 

             
            
            
    

132 Annex 56-A

    

 

          
        
          

            
             
            

         
        

         
             
           

          
  

            
             
           

          
           

   

  

           

   

     
          
            

133Annex 56-A

    

           

  

           
          

              
             
            
        

   

     
            

       

    

    

          
          
          
            
          

            
         

 
          E

°       
           
           
           

          

134 Annex 56-A

    

 

            
             
             
        
      

        
           
           
           
          

 

          
          
            
        

 

  
           

          
           
        
             
            

          

135Annex 56-A

    

  

  

            
           

          
  

  

  

             
             

 

                

           
      

           
            
       

 

              
              
            



     
       

      
  
   
  

              

            


136 Annex 56-A

    

 

            
             

           



     
     
     

      


  

   

         

           
            
   

  

            

        
       

  

         
            

 

  

           
              
        

  

         
         
    

137Annex 56-A

    

  

         
        
        
            

   

            
           

 ≥          ±      
            
         
≥             

  

      

 

   
     

            
           
     

  

   

          
          ±   

             
  

138 Annex 56-A

139Annex 56-A

    

 

           
   

140 Annex 56-A



  ±

 



    ±

 


                          



    ± 

 




           



      
    ±
           


           
 

    


       
    

141Annex 56-A



 

  
          ±       ±  

 



 

                     
 



  

      



 




           

142 Annex 56-A



 

 
          





 

   
           
 



  

      



 



  

          

143Annex 56-A

    

 

  

144 Annex 56-A

    

   

          

         
           

           
            

       

     

               
           

           
 

 

           
              

             

 

            
           

   

    
      
         
  
        
       

       

 
  
     

            

            
            

  

145Annex 56-A

    

         
           
            
   

  

            
          
    

  

         
          
     

  

          
    

  

         
         
   

 
            

           
     

146 Annex 56-A







 

   







  




  


               
                            

   


  





          

147Annex 56-A

    

 

  

148 Annex 56-A

    

  

   

  

      

       

           

       

          

        
 

         

        
     

          

       
          

                

                
                
        

149Annex 56-A

    

  

  

  

              

           

           

         

              

           

           

         

              

           

           

         

           
   
   

              


               


             


150 Annex 56-A

    

  

  

  

        
    

        
       
          
 

             

         
            

          
       
        

      
       
        
        

          
       
         
          

         
     
  
             

          
 
  
         

        
 
        

           
             
        
     

151Annex 56-A

    

 

   

152 Annex 56-A



 

 

  


 

   

      



  

    

153Annex 56-A



  

 

        
 

  

 
 

   

      

 
 

 

  
  
                      

154 Annex 56-A

    

 

   

155Annex 56-A

    

  

    
 """
   

 
          

"      
             
           
           

            
           
       

  
           

 

 
    
     
     
   

          
          

         

           
         

           
    

156 Annex 56-A

    

   

        
           
          
           
            

            
            
              
  

              
              
                
             
            

        
           
            
           
         

                
      

             

             
                
             
          
            

      

         
           
              

           
         
         

157Annex 56-A

    

      E °  
         
            

 
   

            
              
          
          ±   

             
  

             
            

             ±
             
     

 

    "    
             

         
"          

 

           
            

158 Annex 56-A

    

     
      
± ± ± ±  ± ±

        






 

   

  
      
±
        

 

   


 

 

      
        
± ± ±
        


   

 
 
   

 

"       
          
± ±

            

      

    
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

   



      
    ± ± ± ± ± ± ± ± ± ± ± ± ±



 
 

 
  
             

159Annex 56-A

    

 
± ± ± ± ± ± ± ± ± ±
 

          




  
  ± ± ± ± ± ± ± ± ± ± ± ±

        

 

± ± ± ± ± ± ± ±
 

            



 

  
   ± ± ± ± ± ± ± ±
  



      

 
      
 




           

160 Annex 56-A

    

 

 

          




 
 

          

 

 

          



 

  
  
  



      

 
       
 



         

161Annex 56-A

    

 

  
   

162 Annex 56-A

SLI Study No. 3596.21
(38)

163Annex 56-A

    

 

  

164 Annex 56-A

    

  

       



     

     

      

     

       

       

    

       

      


     

       

       


   

165Annex 56-A

    
   

 

 





    

  

  

 

  

  



 


   
    
  

   

166 Annex 56-A

    



  

 

167Annex 56-A

168 Annex 56-A

169Annex 56-A

    

   

   

       

 

  

  

170 Annex 56-A

    

     


    



   

    

   

171Annex 56-A

    

 

          
              
              

            
             


              
            

            
          
              

        

            
       

172 Annex 56-A

    

 

           

          
           
             

             
            
          

         
         
             

          
          
  

   

  

           

    
     
     



  
    

   
     
          
            

           
         
           

          
            
 

173Annex 56-A

    

  
           

          
              
             
            
        

   

             
    

     

           
            
  

    

    

         
         
          

            
          
            
         

 

         
       
           
           

           
           

 
            

            

174 Annex 56-A

    

             
        
      
        
           

           
           
          

 

          
           
           
          
          

 
          

          
            
         
  

  

           
          
           

            
  

  

  

              
           
          

         
           

175Annex 56-A

    

         
          

         
            
             


 

            
  

   
    
     
  

              

            
            
             
       

  

            
                 

            
         
          
              

          
             
           
          

  

       
           



176 Annex 56-A

    

  

             

  
          

         
   

  
          

       
           
      

   

           
           
             

            
            
           
             
       

      
           

              
       

177Annex 56-A

178 Annex 56-A

    

 
            

  

            
           
 

            
       

179Annex 56-A



 

 

 

  






 

 



 

  
  
  

   

           



         

                       

                      
                   
                    
         

180 Annex 56-A

    

 

  

181Annex 56-A

    

  

    
     

 

    
              

  
         

            

       

    

       

 

    

       

        

        

        

           

 

 

 

        
              

        

            

         

    

182 Annex 56-A

    

  

  
         

 

   

          

         

     

  
   

 

  

         

       

      

       

  

 

  
            

   
            

              

             

    

183Annex 56-A

    

  

 

  

            
   
              

  
              
   
            

 

  

  

             
   

            
 
              
     

                
           
       

        

               
   
           
    

             


184 Annex 56-A

    

  

   

 

  
          
          

 
     

  
         

  
          

  

 

    

       

      

       

         

185Annex 56-A

    

 

  

186 Annex 56-A

    

  

     
         

     
  
     

     

  
     

      

  
      

      
  

      

          

           
       
           
  
   
      

          

               
   
           
   
   
       

           

                
      
        
       

       

       
  
       

187Annex 56-A

    

 

  

188 Annex 56-A

    

  

       



     

     

      

     

       

       

    

       

       

     

       

        

   

189Annex 56-A

    

   

 

 





    

  

  

 

  
      

   
  

  



 


   

    
  

   

190 Annex 56-A

    

      



  

 

191Annex 56-A

192 Annex 56-A

193Annex 56-A

    

   

 

 

194 Annex 56-A

    

     



    



    

   

   

195Annex 56-A

    

 

          
              

              
             
          

           
          
            

              
           
    

            
              

  

196 Annex 56-A

    

 
           

           
           
             

             
            
         

          
         
             

          
          
  

   

  

           

    
     

     


  
    

          
            

197Annex 56-A

    

  

           
          
              
             
            

        

   

     

           
            
  

    

    

         
         

          
            
          
            
         

 

          
        
          

            
          
          

 

            
            
             

198 Annex 56-A

    

        
      
        
           

           
           
          

 

 

          
          
            
         
  

  

        
             
            


  

  

               
              
         

199Annex 56-A

    

 

                
             

    
     
           

  

            
              

              
           
            
            

            
             
         
     

  

           
              
         

       

  
       

           


  

         
   

200 Annex 56-A

201Annex 56-A

202 Annex 56-A

    

 

        

          

203Annex 56-A



 

  

 

   
  

           
   
      

  
     

         

  
            

204 Annex 56-A

    

 

   

205Annex 56-A

    

   

   

  

      

         

      

        

        

          
 

      

         

        
     

          

       
          

             
             

                
                
                
         

206 Annex 56-A

    

   

  

  

              

           

           

         

           

           

         

              

           

           

         

               
   


               


               


             

207Annex 56-A

    

   

  

  

        
    
        

         
        
 
          
  

            
         
        
 
           
    
      
       

        
        
       
          

         
          
         
     

              
 
  
         

          
 
  
       

        
           
        
            

      

208 Annex 56-A

    

 

  

209Annex 56-A

    

  

    


 

    

    

    

210 Annex 56-A

    

 

  

211Annex 56-A

    

  

       



     

     

      

     

      
 

    

       

       

     

       

       


   

212 Annex 56-A

   

    
  

 



    

  

  

   

  

 

  

  



 


   
    

  

   

213Annex 56-A

214 Annex 56-A

        

  

             

    

      
              
             

    
     
   

     
   

       
   
     

   
     
   

       
   
     

   
     
   

       
   
     

   
     
   

       
   
     

   
     
   

       
  

215Annex 56-A

Report Amendment No. 1

AN ACUTE DERMAL TOXICITY STUDY
IN RATS WITH SPRAY--CHARLIE

AMENDED FINAL REPORT

OPPTS Guideline

870.1200

Author

Kimberly L. Bonnette, M.S., LATG

Original Study Completion Date

February 20, 2003

Amended Study Completion Date

March 17, 2003

Performing Laboratory

Springborn Laboratories (SLI),
a division of Charles River Company, Inc.
640 North Elizabeth Street
Spencerville, Ohio 45887

SLI Study No.

3596.17

Submitted to

INL/A
U.S. Department of State

2201 C St. NW SA-4
Washington, DC 20520

Page 1 of 29

216 Annex 56-A

217Annex 56-A

SLI Study No. 3596.17 (8) Report Amendment No. 1

7. INTRODUCTION

This study was performed to assess the short-term toxicity of Spray--Charlie in

Sprague Dawley rats when administered by a single dermal dose. This study
was intended to provide information on the potential health hazards of the test
article with respect to dermal exposure. Data from this study may serve as a

basis for classification and/or labeling of the test article. This study was
performed in accordance with the US EPA, Health Effects Test Guidelines,
OPPTS 870.1200, Acute Dermal Toxicity, August 1998. This study was
performed at Springborn Laboratories (SLI), 553 North Broadway, Spencerville,

Ohio. The protocol was signed by the Study Director on October 9, 2002 (GLP
initiation date). The in-life phase of the study was initiated with test article
administration on December 19, 2002 (day 0), and concluded with necropsy on

January 2, 2003.

8. MATERIALS AND METHODS

8.1. Test Article

The test article was received from the Sponsor and identified as follows:

Assigned Physical Receipt Expiration
Spoasor’s ID SLI ID Description Date Date
Spray—Charlie S02.003.3596 Amber 12/09/02 None
liquid provided
b
Ingredients:
Herbicide: GLY-41 None
Lot No.: Manufactured 10/20/02 provided

Surfactant: Cosmo Flux-411F None
Lot No.: Manufactured 11/29/02 provided
aSample pooled at SLI from five different mixes of Spray--Charlie (top/middle/bottom).
bIngredients used in the five Spray--Charlie mixes that were prepared by the Sponsor.

40 CFR 160.105 and 40 CFR 792.105. Springborn Laboratories, analyzed the
test article for the glyphosate (a.e.) which is presented in
SLI Study No. 3596.15.

218 Annex 56-A

       

     

 

   

        

   



     



  

         

        

  



     



  

        

219Annex 56-A

220 Annex 56-B

SixA cutet oxicitS tudieS witS prA-A lphA, Sli tudy N° 3596.3,
3 SePtember 2002

(United States Embassy in Bogotá, 2011)

221222 Annex 56-B

AN ACUTE DERMAL TOXICITY STUDY IN RATS

WITH SPRAY--ALPHA

FINAL REPORT

OPPTS Guideline

870.1200

Author

Kimberly L. Bonnette, M.S., LATG

Study Completed on

September 3, 2002

Performing Laboratory

Springborn Laboratories, Inc. (SLI)
Ohio Research Center

640 North Elizabeth Street
Spencerville, Ohio 45887

SLI Study No.

3596.3

Submitted to

INL/A
U.S. Department of State

2201 C St. NW SA-4
Washington, DC 20520

Page 1 of 30

223Annex 56-B

SLI Study No. 3596.3 (2)

1. STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS
No claim of confidentiality is made for any information contained in this study on

the basis of its falling within the scope of FIFRA §10(d)(1)(A), (B), or (C).

Company:

Company Agent: Date Date

Title Signature

224 Annex 56-B

225Annex 56-B

226 Annex 56-B

SLI Study No. 3596.3 (5)

4. TABLE OF CONTENTS

1. STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS..........................2 ..

2. COMPLIANCE STATEMENT.........................................................................3 .......

3. QUALITY ASSURANCE STATEMENT..........................................................4 .....

4. TABLE OF CONTENTS.......................................................................................

5. LIST OF TABLES AND APPENDICES..........................................................6 .....

6. SUMMARY ......................................................................................................7.........

7. INTRODUCTION .............................................................................................

8. MATERIALS AND METHODS........................................................................8 ......

9. EXPERIMENTAL PROCEDURES.....................................................................10

10. ANALYSIS OF DATA.........................................................................................12

11. MAINTENANCE OF RAW DATA AND RECORDS......................................12

12. RESULTS.............................................................................................................13

13. CONCLUSION.....................................................................................................14

14. REPORT REVIEW..............................................................................................14

15. REFERENCES.....................................................................................................15

227Annex 56-B

SLI Study No. 3596.3 (6)

5. LIST OF TABLES AND APPENDICES

Tables

1. Individual Clinical Observations......................................................................16
2. Individual Body Weights..........................................................0......................2
3. Individual Gross Necropsy Observations......................................................22

Appendices

A. Macroscopic Dermal Grading System..........................................................24

B. SLI Personnel Responsibilities..................................................9..................2

228 Annex 56-B

SLI Study No. 3596.3 (7)

6. SUMMARY

The single -dose dermal toxicity of Spray --Alpha was evaluated in Sprague
Dawley rats. A limit test was performed in which one group of five male and five
female rats received a single dermal administration of the test article at a dose of
5000 mg/kg body weight. Following dosing, the limit test rats were observ
ed daily
and weighed weekly. A gross necropsy examination was performed on all

animals at the time of scheduled euthanasia (day 14).

No mortality occurred during the limit test. Clinical abnormalities observed
during the study included dark material around the facial area and red ocular
discharge. Minor/transient dermal irritation was noted at the site of test article

application. Body weight loss was noted in two males and two females dur
ing
the study day 0 to 7 body weight interval which is routinely observed in this study
type due to experimental manipulation. Body weight gain was noted for all other
animals during the test period. All animals exceeded their initial body
weight by
study termination (day 14). No significant gross internal findings were observed

at necropsy on study day 14.

Under the conditions of this test, the acute dermal LD50 of Spray --Alpha was
estimated to be greater than 5000 mg/kg in the rat.

229Annex 56-B

SLI Study No. 3596.3 (8)

7. INTRODUCTION

This study was performed to assess the short -term toxicity of Spray --Alpha in
Sprague Dawley rats when admini stered by a single dermal dose. This study

was intended to provide information on the potential health hazards of th
e test
article with respect to dermal exposure. Data from this study may serve as a
basis for classification and/or labeling of the test a rticle. This study was

performed in accordance with the US EPA, Health Effects Test Guidelines,
OPPTS 870.1200, Acute Dermal Toxicity, August 1998. This study was
performed at Springborn Laboratories, Inc., 553 North Broadway, Spencerville,
Ohio. The p rotocol was signed by the Study Director on April 30, 2002 (GLP

initiation date). The in- life phase of the study was initiated with test article
administration on June 25, 2002 (day 0), and concluded with necropsy on
July 9, 2002.

8. MATERIALS AND METHODS

8.1. Test Article

The test article was received from the Sponsor and identified as follows
:

Assigned Physical Receipt Expiration
Sponsor’s ID SLI ID Description Date Date
Spray—Alpha a S02.001.3596 Light amber 05/13/02 None
liquid provided
b
Ingredients
Herbicide:Fuete-SL None
Lot No.: 02- 01-02 Provided

Surfactant: Cosmo Flux -411F 10/2003
Lot No.: 244301
Sample pooled at SLI from five different mixes of Spray --Alpha (top/middle/bottom).
Ingredients used in the five Spray--Alpha mixes that were prepared by the Sponsor.

The test article was stored at room temperature. The Sponsor was responsible
for any necessary evaluations related to identity, strength, purity, composition,

stability and method of synthesis of the test material according to 40 CFR
160.105 and 40 CFR 792.105. Springborn Laboratories, Inc., analyzed the
test
article for the glyphosate (a.e.) which is presented in SLI Study No.
3596.1.

230 Annex 56-B

SLI Study No. 3596.3 (9)

8.2. Retention Sample

An approximate 1 mL retention sample of each test article mixture
(top/middle/bottom, maintained separately for a total of fifteen 1 mL
mples)
was taken and stored at SLI at room temperature. In addition, a 10 mL retention
sample of the pooled test article samples (from the 5 test article mix tures) was
collected and stored at SLI at room temperature. These samples serve as the

retention samples for all studies conducted with this material.

8.3. Test Article Disposition

The test article was returned to the Sponsor following completion of all studies
with the test article.

8.4. Method of Test Article Preparation

The test article was administered as received from the Sponsor and dispe
nsed
fresh on the day of dosing. The density of the test article was determi
ned to be
1.08 g/mL.

8.5. Animals and Animal Husbandry

8.5.1. Description, Identification and Housing

Adult, Hsd: Sprague Dawley® SD® rats were received from Harlan Sprague
Dawley, Inc., Indianapolis, IN. Upon receipt, metal ear tags displaying unique

identification numbers were used to individually identify the animals. Cage cards
displaying at least the study number, animal number and sex were affixed to
each cage. The animals were housed individually in suspended stainless steel
cages. All housing and care were based on the standards recommended by
the
Guide for the Care and Use of Laboratory Animals [1].

8.5.2. Environment

The animal room temperature and relative humidity ranges were 70- 75°F
(21-24°C) and 37 -57%, respectively. Environmental control equipment was
monitored and adjusted as necessary to minimize fluctuations in the animal room

environment. Light timers were set to maintain a 12-hour light/12-hour dark cycle
and room ventilation was set to produce 10- 15 air changes/hour. The animal
room temperature and relative humidity were recorded a minimum of once daily.

8.5.3. Food

PMI Certified Rodent Chow #5002 (Purina Mills, Inc.) was provided ad libitum to
the animals throughout the study. The lot number and expiration date of
each
batch of diet used during the study were recorded. The feed was analyzed
and

231Annex 56-B

SLI Study No. 3596.3 (10)

certified by the supplier for nutritional components and environmental
contaminants. Dietary limitations for various environmental contaminants
,
including heavy metals, pesticides, polychlorinated biphenyls and total
aflatoxin
are set by the manufacturer. Wi thin these limits, contaminants which may have

been present were not expected to compromise the purpose of this study.
Results of the dietary analyses (Certificates of Analysis) were provide
d by the
manufacturer for each lot of diet. These are maintained by SLI.

8.5.4. Water

Municipal tap water treated by reverse osmosis was available ad libitum
throughout the study. The purified water was supplied by an automatic w
atering

system. Monitoring of the drinking water for contaminants is conducted
by SLI
and the records are available for inspection. Within generally accepted limits,
contaminants which may have been present were not expected to compromise
the purpose of this study. The water meets the standards specified under the

EPA National Drinking Water Regulations (40 CFR Part 141).

8.5.5. Acclimation
Upon receipt, the animals were removed randomly from the shipping cartons,

examined by qualified personnel, identified with metal ear tags and then
acclimated to the laboratory conditions for a minimum of five days. Th e animals
were observed daily for overt physical or behavioral abnormalities, gener
al

health/moribundity and mortality.

8.5.6. Animal Selection
The animals chosen for study use were randomly selected from healthy stoc
k

animals using a computerized ( Alpha DS-10 AcuTox) random numbers table to
avoid potential bias. All animals received a detailed pretest observatio
n prior to
dosing. Only healthy animals were chosen for study use. Females were
nulliparous and nonpregnant. The male animals were approximately 11 weeks of

age and weighed 335- 374 g prior to dosing. The female animals were
approximately 11 weeks of age and weighed 226-249 g prior to dosing.

9. EXPERIMENTAL PROCEDURES

9.1. Preliminary Procedures

On day -1, the fur was removed from the dorsal trunk area of the animals chosen
for the limit test using an animal clipper. The clipped area was approxi
mately
10% of the animal’s body surface area (BSA). The region included the scapula

(shoulder) to the wing of the ilium (hipbone) and half way down the flank on each

232 Annex 56-B

SLI Study No. 3596.3 (11)

side of the animal. Care was taken to avoid abrading the skin during the clipping

procedure.

9.2. Dosing

On the following day (day 0), the test article was administered dermal
ly to
approximately 10% of the body surface area. The four corners of this are
a were
delineated in the clipped area with an indelible marker. The test article was then

spread evenly over the delineated test area and held in contact with the
skin with
an appropriately sized 4 -ply porous gauze dressing backed with a plastic wrap
which was placed over the gauze dressing (occlusive binding). Removal and

ingestion of the test article was prevented by placing an elastic wrap over the
trunk and test area. The elastic wrap was further secured with a tape harness on
the cranial end of the trunk and then secured with adhesive tape around the trunk

at the caudal end.

The test article was administered at the following level:

Dose Level Dose Volume Concentration No. of Animals
(mg/kg) (mL/kg) (%) Male Female
a b
a 5000 4.63 100 5 5
bAdjusted based on a density of 1.08 g/mL.
Pooled test article.

Individual doses were calculated based on the animal’s day 0 body weig
ht. After

an approximate 24- hour exposure period, the binding materials were removed.
Residual test article was removed using gauze moistened with deionized water
followed by dry gauze.

9.3. Dermal Observations

The test animals were examined for erythema and edema following patch

removal and the responses scored on study day 1 and daily thereafter (days 2 -
14) according to the Macroscopic D ermal Grading System provided in Appendix
A which is based on Draize [2]. The dermal test sites were reclipped as

necessary to allow clear visualization of the skin.

9.4. Clinical Observations

The animals were observed for clinical abnormalities a minimum of two times on
study day 0 (postdose) and daily thereafter (days 1- 14). A mortality check was
performed twice daily, in the morning and afternoon.

233Annex 56-B

SLI Study No. 3596.3 (12)

9.5. Body Weights

Individual body weights were obtained for the animals prior to dosing on day 0
and on days 7 and 14.

9.6. Gross Necropsy
All animals were euthanized by carbon dioxide inhalation at study termin
ation

(day 14) and necropsied. Body cavities (cranial, thoracic, abdominal
and pelvic)
were opened and examined. No tissues were retained.

9.7. Protocol Deviations
No protocol deviations occurred during this study.

10. ANALYSIS OF DATA

Data from the study were analyzed and an LD50 value estimated as follows:

Body weight means and standard deviations were calculated separately for

males and females.

11. MAINTENANCE OF RAW DATA AND RECORDS

All original raw data, the final report and magnetically encoded records were
transferred to the SLI archives for a period of 10 years. The Sponsor will be
contacted prior to final disposition of these items.

234 Annex 56-B

SLI Study No. 3596.3 (13)

12. RESULTS

12.1. Mortality

Individual Data: Table 1

No mortality occurred during the limit test.

12.2. Clinical/Dermal Observations

Individual Data: Table 1

Clinical abnormalities observed during the study included dark material
round
the facial area and red ocular discharge. Minor/transient dermal irritation was

noted at the site of test article application.

12.3. Body Weight Data

Individual Data: Table 2

Body weight loss was noted in two males and two females during the study day 0
to 7 body weight interval which is routinely observed in this study type
due to
experimental manipulation. Body weight gain was noted for all other animals
during the test period. All animals exceeded their initial body weight by study
termination (day 14).

12.4. Gross Necropsy

Individual Data: Table 3

No significant gross internal findings were observed at necropsy on study day 14.

Blood clots observed in one animal at necropsy were thought to have been
caused by a possible accidental injury prior to euthanasia.

235Annex 56-B

236 Annex 56-B

SLI Study No. 3596.3 (15)

15. REFERENCES

1. Guide for the Care and Use of Laboratory Animals, DHHS Publicat ion No.
(NIH) 96-03, 1996.

2. Draize, J.H., Appraisal of the Safety of Chemicals in Foods, Drugs and
Cosmetics, The Association of Food and Drug Officials of the United States,

49-51, 1959.

237Annex 56-B

(16)

---- ---- ----

STUDYL/NO.:U3596D.3ALESMENT--MA-LE#-A-5-2-OBSERVATITD53DRTATA-SIDMYORCREDTG0ASACTEULTGEM-RNUKCDSCE-2-ALBPCUDE5-TM
P
--R8--1-PPP-1SS-------

238 Annex 56-B

(17)

---- ---- ----

STUDLY/NO.:.S3596.A3LESMENT--M-ALE#-A-5-3-OBSC-EEATI--EAAAR-K-DELHI--OUA-O-EYE-NOSO------------0---DAY2-
F
ST-
--E-----
L------P----1--G-P-1-PB14-------------------

239Annex 56-B

(18)

---- ---- ----

STUDY/ANO.:.3596E.3MALESNT-FEMA-LE#-A-5-3-OBSERVATITD53DAT-DA-SIDMYORAR-STG0ASAIEURAAAMA-D-STRARR--2-ADAP(-ED5-RM
P
--S8-LHAP-PU1D--------

240 Annex 56-B

(19)

---- ---- ----

STUNDY/N,O.:.S3596.-3MALMESNTFEM-ALE#---------ES-IA-ATDTONSIAILAA-A-UNDN-N-SEU-TH------------------------R----
P---------F-----7---I------0---B----T-----4------------------------------------------------------------

241Annex 56-B

(20)

---- ---- ----

STUIDYL/NO.:U3S59MA3LES--ANI-MAL#-2---0204050N-Y0----7-4D--33-815--73--0-9TH-45DAY)--------------------------------------------
-------------------------------------
---------------------------------------------------------------------------------------------

242 Annex 56-B

(21)

---- ---- ----

STUNDY/NO.U:.359FEMAL-E-ANIMALA#----4043ME-43-0--4--26---8--18-47A-93DEAT---1DA-Y)-------------------------------------------
-------------------------------------
---------------------------------------------------------------------------------------------

243Annex 56-B

(22)

1 ---- ---- ----

STUDINL/A, U.S.MD ALES---EI-5000--5297G30A5----9-YYUA530--0-149--4T-1TIL-AL-MLUESSU-,LNINTH-IALSLRM-OOVNSMI-AS
L----LITS----E-------
-T

244 Annex 56-B

(23)

2 ---- ---- ----

STINL/A, U.S.FD.EMAL-EAN--500-0-533KG3FA0----9-YYUA534--0-149--4TI-TIV--LT-HUGS-UE-TAOT--I--D
OW-A--LDTN-TS--L-L
IMITS---------
-
-----------------FAT---E--

245Annex 56-B

SLI Study No. 3596.3 (24)

APPENDIX A

Macroscopic Dermal Grading System

246 Annex 56-B

SLI Study No. 3596.3 (25)

MACROSCOPIC DE RMAL GRADING SYSTEM

ERYTHEMA AND EDEMA OBSERVATIONS

OBSERVATION DEFINITION CODE

Erythema – Grade 0 No erythema 0

Erythema – Grade 1 Very slight erythema (barely perceptible) 1

Erythema – Grade 2 Well-defined erythema 2

Erythema – Grade 3 Moderate to severe erythema 3

Erythema – Grade 4 Severe erythema (beet redness) 4

Maximized Grade 4 Notable dermal lesions (see below) M – 4
(see below)

Edema – Grade 0 No edema 0

Edema – Grade 1 Very slight edema (barely perceptible) 1

Slight edema (edges of area well defined by definite
Edema – Grade 2 raising) 2

Edema – Grade 3 Moderate edema (raised approximately 1 millimeter) 3

Edema – Grade 4 Severe edema (raised more than 1 millimeter and 4
extends beyond the area of exposure)

NOTE: Each animal was assigned an erythema and edema score. The most severely affected
area within the test site was graded. If eschar, blanching, ulceration
and/or necrosis greater

than grade 1 was observed, then the “Maximized Grade 4" was assigned
to the test site in
place of the erythema score and the type of notable dermal lesion(s) (e.g., es- grade 2,
blanching - grade 3, ulceration - grade 4, etc.) was noted. The presence of any other dermal
changes (e.g., desquamation, fissuring, eschar exfoliation, etc.) wrecorded.

247Annex 56-B

SLI Study No. 3596.3 (26)

MACROSCOPIC DERMAL GRADING SYSTEM

NOTABLE DERMAL LESIONS

OBSERVATION CODE DEFINITION

Eschar – Grade 1 ES-1 Focal and/or pinpoint areas up to 10% of test site.

Eschar – Grade 2 ES-2 > 10% < 25% of test site.

Eschar – Grade 3 ES-3 > 25% < 50% of test site.

Eschar – Grade 4 ES-4 > 50% of test site.

Blanching – Grade 1 BLA-1 Focal and/or pinpoint areas up to 10% of test site.

Blanching – Grade 2 BLA-2 > 10% < 25% of test site.

Blanching – Grade 3 BLA-3 > 25% < 50% of test site.

Blanching – Grade 4 BLA-4 > 50% of test site.

Ulceration – Grade 1 U-1 Focal and/or pinpoint areas up to 10% of test site.

Ulceration – Grade 2 U-2 > 10% < 25% of test site.

Ulceration – Grade 3 U-3 > 25% < 50% of test site.

Ulceration – Grade 4 U-4 > 50% of test site.

NEC-1 Focal and/or pinpoint areas up to 10% of test site
Necrosis – Grade 1
(color) (Note color of necrosis).
NEC-2
Necrosis – Grade 2 > 10% < 25% of test site (Note color of necrosis).
(color)
NEC-3
Necrosis – Grade 3 (color) > 25% < 50% of test site (Note color of necrosis).

NEC-4
Necrosis – Grade 4 (color) > 50% of test site (Note color of necrosis).

248 Annex 56-B

SLI Study No. 3596.3 (27)

MACROSCOPIC DERMAL GRADING SYSTEM

ADDITIONAL DERMAL FINDINGS

OBSERVATION DEFINITION CODE

Characterized by scaling or flaki ng of
Desquamation dermal tissue with or without denuded DES
areas.

Characterized by cracking of the skin with
or without moist exudate. Fissuring should
Fissuring be checked prior to removing the animal FIS
from the cage and manipulating the test

The erythema extends beyond the test
Erythema Extends Beyond site. Note: A study director should be
the Test Site contacted for erythema extending beyond ERB
the test site.

Characterized by pale area(s) (almost a --
burn-like appearance) in the test site.
However, erythema may still be observed

does not result in any in- depth injury. To
be coded using an area designation (see
below).

Superficial Lightening - Focal and/or pinpoint areas up to 10% of
Grade 1 the test site SL-1

Superficial Lightening - > 10% < 25% of test site SL-2
Grade 2

Superficial Lightening - > 25% < 50% of test site SL-3
Grade 3
Superficial Lightening - > 50% of test site
SL-4
Grade 4

249Annex 56-B

SLI Study No. 3596.3 (28)

MACROSCOPIC DERMAL GRADING SYSTEM

ADDITIONAL FINDINGS

OBSERVATION DEFINITION CODE
Noticeable irritation outside of test site
Dermal Irritation - Outside probably due to the binding tape material. IT
the Test Site This notation will only be made for
reactions greater than what are normally

observed from tape removal which do not
interfere with the scoring of the test site.

250 Annex 56-B

SLI Study No. 3596.3 (29)

APPENDIX B

SLI Personnel Responsibilities

251Annex 56-B

SLI Study No. 3596.3 (30)

SLI PERSONNEL RESPONSIBILITIES

Kimberly L. Bonnette, M.S., LATG Study Director/Director, Acute Toxicology

Dawn D. Rodabaugh, B.S. Alternate Contact/Associate Toxicologist

Robert C. Springborn, Ph.D. Chairman, President and CEO

Malcolm Blair, Ph.D. Senior Vice President, Managing Director
Emeritus

Joseph C. Siglin, Ph.D., DABT Vice President, Managing Director

Jason W. Smedley, B.S. Assistant Toxicologist

Pamela S. Smith, ALAT Supervisor of Acute Toxicology

Kathy A. Pugh, ALAT Primary Technician/Team Leader

Delores P. Knippen Supervisor of Pharmacy

Steven H. Magness, B.S., LATG Senior Supervisor of Gross and Fetal
Pathology

Anita M. Bosau, RQAP-GLP Senior Director, Compliance Assurance

Deanna M. Talerico, RQAP-GLP Senior Supervisor of Quality Assurance

J. Dale Thurman, D.V.M., M.S., DACVP Senior Director, Pathology

Kathy M. Gasser Supervisor of Archives

252 Annex 56-B

AN ACUTE NOSE-ONLY INHALATION TOXICITY STUDY IN RATS
WITH SPRAY--ALPHA

FINAL REPORT

OPPTS Guidelines

870.1300

Author

Kimberly L. Bonnette, M.S., LAGT

Study Completed on

September 3, 2002

Performing Laboratory

Springborn Laboratories, Inc. (SLI)
Ohio Research Center
640 North Elizabeth Street

Spencerville, Ohio 45887

SLI Study No.

3596.4

Submitted to

INL/A
U.S. Department of State
2201 C St. NW SA-4
Washington, DC 20520

Page 1 of 48

253Annex 56-B

AN ACUTE NOSE-ONLY INHALATION TOXICITY STUDY IN RATS
WITH SPRAY--ALPHA

FINAL REPORT

OPPTS Guidelines

870.1300

Author

Kimberly L. Bonnette, M.S., LAGT

Study Completed on

September 3, 2002

Performing Laboratory

Springborn Laboratories, Inc. (SLI)
Ohio Research Center
640 North Elizabeth Street

Spencerville, Ohio 45887

SLI Study No.

3596.4

Submitted to

INL/A
U.S. Department of State
2201 C St. NW SA-4
Washington, DC 20520

Page 1 of 48

254 Annex 56-B

(2)

SLI Study No. 3596.4

1. STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

No claim of confidentiality is made for any information contained in this study on
the basis of its falling within the scope of FIFRA §10(d)(1)(A), (B), or (C).

Company:

Company Agent: ___________________ Date

Title Signature

255Annex 56-B

256 Annex 56-B

257Annex 56-B

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SLI Study No. 3596.4

4. TABLE OF CONTENTS

1. STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS..........................2 ..

2. COMPLIANCE STATEMENT.........................................................................3 .......

3. QUALITY ASSURANCE STATEMENT..........................................................4 .....

4. TABLE OF CONTENTS......................................................................................

5. LIST OF TABLES AND APPENDICES..........................................................6 .....

6. SUMMARY ......................................................................................................7.........

7. INTRODUCTION ............................................................................................8

8. MATERIALS AND METHODS........................................................................8 ......

9. EXPERIMENTAL PROCEDURES.....................................................................11

10. ANALYSIS OF DATA.........................................................................................14

11. MAINTENANCE OF RAW DATA AND RECORDS......................................14

12. RESULTS.............................................................................................................14

13. CONCLUSION.....................................................................................................16

14. REPORT REVIEW..............................................................................................16

15. REFERENCE.......................................................................................................17

258 Annex 56-B

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SLI Study No. 3596.4

5. LIST OF TABLES AND APPENDICES

Tables

1. Summary of Aerosol Generation and Chamber Environmental Data............18
2. Individual Clinical Observations......................................................................1
3. Individual Body Weights..................................................................................2

4. Individual Gross Necropsy Observations..........................................................

Figure

1. Multistage 10L Nose-Only Inhalation Chamber...............................................2 ...

Appendices

A. Preliminary Aerosol Generation Trials..............................................................

B. Analytical Chemistry Report............................................................................3

C. Individual Aerosol Generation and Chamber Environmental Data..................39

D. SLI Personnel Responsibilities.........................................................................4

259Annex 56-B

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SLI Study No. 3596.4

6. SUMMARY

The four -hour nose- only inhalation toxicity of Spray --Alpha was evaluated in

Sprague Dawley rats. A limit test was performed in which a group of five
male
and five female rats received a four-hour nose-only inhalation exposure to a time-
weighted average aerosol concentration ( analytically determined) of 3.27 mg/L.

Following the exposure, the limit test rats were observed daily and weighed
weekly. A gross necropsy examination was performed on all limit test animals at
the time of scheduled euthanasia (day 14).

No mortality occurred during this study. The most notable clinical abnormalities
observed during the study included decreased/no defecation, soft stools,
feces
small in size, rough coat, breathing abnormalities, decreased food consu
mption

and dark material around the facial area. Body weight loss was noted for
one
male and one female during the study day 0-7 body weight interval. Body weight
gain was noted for all other animals during the test period. All animals
exceeded

their initial body weight by study termination (day 14). No significant gross
internal findings were observed at necropsy on study day 14.

Under the conditions of this test, the acute inhalation LC50 of Spray --Alpha was
estimated to be greater than 3.27 mg/L in the rat.

260 Annex 56-B

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SLI Study No. 3596.4

7. INTRODUCTION

This study was performed to assess the short -term toxicity of S pray--Alpha in
Sprague Dawley rats when administered by a four- hour nose- only inhalation

exposure. This study was intended to provide information on the potentia
l health
hazards of the test article with respect to inhalation exposure. Data from this
study may serve as a basis for classification and/or labeling of the test article.

This study was conducted in accordance with the US EPA, Health Effects Te
st
Guidelines, OPPTS 870.1300, Acute Inhalation Toxicity, August 1998. This
study was performed at Spri ngborn Laboratories, Inc., 553 North Broadway,

Spencerville, Ohio. The protocol was signed by the Study Director on
April 30, 2002 (GLP initiation date). The in -life phase of the study was initiated

with test article administration on June 6, 2002 (day 0) and concluded with
terminal euthanasia on June 20, 2002.

8. MATERIALS AND METHODS

8.1. Test Article

The test article was received from the Sponsor and identified as follows
:

Assigned Physical Receipt Expiration
Sponsor’s ID SLI ID Description Date Date
Spray--Alphaa S02.001.3596 Light amber 05/13/02 None

liquid provided
Ingredientsb
Herbicide:Fuete-SL None
Lot No.: 02- 01-02 Provided

Surfactant: Cosmo Flux -411F 10/2003
a Lot No.: 244301
Sample pooled at SLI fr om five different mixes of Spray --Alpha (top/middle/bottom).
bIngredients used in the five Spray --Alpha mixes that were prepared by the Sponsor.

The test article was stored at room temperature. The Sponsor was respons
ible
for any necessary evaluations re lated to identity, strength, purity, composition,
stability and method of synthesis of the test material according to 40 C
FR

160.105 and 40 CFR 792.105. Springborn Laboratories, Inc., analyzed the
test
article for the glyphosate (a.e.) which is presented in SLI Study No. 3596.1.

261Annex 56-B

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SLI Study No. 3596.4

8.2. Retention Sample

An approximate 1 mL retention sample of each test article mixture
(top/middle/bottom, maintained separately for a total of fifteen 1 mL s
mples)
was taken and stored at SLI at room temperature. In addition, a 10 m L retention
sample of the pooled test article samples (from the 5 test article mixtures) was
collected and stored at SLI at room temperature. These samples serve as the

retention samples for all studies conducted with this material.

8.3. Test Article Disposition

The remaining test article was returned to the Sponsor following completion of all
studies with the test article.

8.4. Method of Test Article Preparation

The test article was utilized as received from the Sponsor and dispensed
fresh
on the day of dosing. The test article was stirred continuously during exposure.

8.5. Animals and Animal Husbandry

8.5.1. Description, Identification and Housing

Young adult, Hsd: Sprague Dawley® SD® rats were received from Harlan
Sprague Dawley, Inc., Indianapolis, IN. Upon receipt, met al ear tags displaying
unique identification numbers were used to individually identify the animals.

Cage cards displaying at least the study number, animal number and sex were
affixed to each cage. The animals were housed individually in suspended
stainless steel cages. All housing and care were based on the standards
recommended by the Guide for the Care and Use of Laboratory Animals [1].

8.5.2. Environment

The animal room temperature and relative humidity ranges were 71- 74°F (22-
23°C) and 35 -61%, respectivel y. Environmental control equipment was
monitored and adjusted as necessary to minimize fluctuations in the animal room
environment. Light timers were set to maintain a 12-hour light/12-hour dark cycle

and room ventilation was set to produce 10- 15 air cha nges/hour. The animal
room temperature and relative humidity were recorded a minimum of once d
aily.

262 Annex 56-B

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SLI Study No. 3596.4

8.5.3. Food
PMI Certified Rodent Chow #5002 (Purina Mills, Inc.) was provided ad l
ibitum to
the animals throughout the study (except during the time that the ani mals were

acclimated to the exposure tubes and maintained in the inhalation room for the
exposure procedure). The lot number and expiration date of each batch o
f diet
used during the study were recorded. The feed was analyzed and certified
by the
supplier for nutritional components and environmental contaminants. Dietary

limitations for various environmental contaminants, including heavy metals,
pesticides, polychlorinated biphenyls and total aflatoxin are set by the
manufacturer. Within these limits, co ntaminants which may have been present
were not expected to compromise the purpose of this study. Results of th
e
dietary analyses (Certificates of Analysis) are provided by the manufa
cturer for

each lot of diet. These are maintained by SLI.
8.5.4. Water

Municipal tap water treated by reverse osmosis was available ad libitum
throughout the study (except during the time that the animals were accli
mated to

the exposure tubes and maintained in the inhalation room for the exposure
procedure). The purified water was supplied by an automatic watering system.
Monitoring of the drinking water for contaminants is conducted by SLI and
the
records are available for inspection. Within generally accepted limits,
contaminants which may have been present were not expected to compromise

the purpose of this study. The water meets the standards specified under the
EPA National Drinking Water Regulations (40 CFR Part 141).

8.5.5. Acclimation
Upon receipt, the animals were removed randomly from the shipping cartons,

examined by qualified personnel, identified with metal ear tags and then
acclimated to the laboratory conditions for a minimum of five days. The
animals
were observed daily for overt physical or behavioral abnormalities, gener
al
health/moribundity and mortality.

8.5.6. Animal Selection

The animals chosen for study use were randomly selected from healthy stoc
k
animals using a computerized (Alpha DS -10 AcuTox) random numbers table to
avoid potential bias. All animals received a detailed pretest observati
n prior to
dosing. Only healt hy animals were chosen for study use. Females were

nulliparous and nonpregnant. The male animals were approximately 10 weeks
of
age and weighed 248- 293 g on the day of exposure. The female animals were
approximately 10 weeks of age and weighed 170-190 g on the day of exposure.

263Annex 56-B

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SLI Study No. 3596.4

9. EXPERIMENTAL PROCEDURES

9.1. Preliminary Procedures

9.1.1. Test Article Volatility Determination

accurate gravimetric determination of the aerosol concentration. A know
n
quantity of the test article was placed on a preweighed filter disk and
was allowed
to evaporate for a total of ten minutes. The test article weight was deter
mined
each minute and the amount of evaporation of the test article was then

determined. The results of this volatility trial indicated that the test
article
evaporation rate (0.45 mg/minute) was comparable to the SLI determined
distilled water evaporation rate (0.55 mg/minute); therefore, was consi
dered to
not be volatile.

9.1.2. Preliminary Aerosol Generation Trials

Prior to experimental initiation, preliminary aerosol generation trial s were
conducted. These trials were performed in order to determine the most e
fficient
means of generating an aerosol of the appropriate concentration while utilizing

equipment that would reduce the aerodynamic particle size. Data obtained
during the preliminary aerosol generation trials are presented in Appendix A.

9.2. Limit Test

9.2.1. Aerosol Generation Equipment

The test aerosol was generated with a Pistol Spraying System. Conditione
d high
pressure external air was used in generating the test atmosphere. The a erosol

was blown through the 5L Elutriator, the nose-only inhalation chamber and then
vented from the chamber to an air treatment system which consisted of a
prefilter, a HEPA filter, a charcoal bed and a water scrubbing tower (s
ee
Figure 1).

264 Annex 56-B

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SLI Study No. 3596.4

9.2.2. Dosing

On day 0, the animals chosen for the limit test were weighed, placed in a nose -
only exposure tube and allowed to acclimate to the exposure tube for at l
east 1
hour. Animals that appeared to have been acclimated to the exposure tube (i.e.,

minimal struggling and no inversion) were considered to be acceptable and
removed from the exposure tube and returned to their cages until initiation of the
aerosol exposure. Animals that did not appear to acclimate to the exposure tube
were not acceptable and were removed f rom the exposure tube and returned to

their cages.

The acceptable animals were then placed in exposure tubes and the tubes
inserted into the Multistage 10L nose-only inhalation chamber and the test article

aerosolized at the following level:

Exposure Level No. of Animals

(mg/L) Male Female
3.27 5 5

The aerosol exposure consisted of a 3 -minute T99 equilibration period, a 240-
minute exposure period and a 3-minute de-equilibration period equal to the T99

equilibration period. After each aerosol exp osure, animals were removed from
the exposure tubes and residual test article was removed from the animal's
exterior surfaces (where practical) by wiping the haircoat with a towel . The
animals were then returned to ad libitum feed and water. The followin g

parameters were measured during the exposure.

9.2.2.1. Chamber Air Flow
Air flow readings were recorded at the initiation of the T99 equilibration period, at

approximate 30- minute intervals during the aerosol exposure and at the
conclusion of the de-equilibration period.

9.2.2.2. Aerosol Concentration

The aerosol concentration was measured at the beginning of the aerosol
exposure (after equilibration), at approximate 30- minute intervals during the
aerosol exposure and at the conclusion of the aerosol exposure (before de-
equilibration). The concentration of the test article aerosol was coll
ected in the

inhalation chamber by gravimetric technique. A 5 L sample of the aerosol was
drawn from the breathing zone of the chamber through a preweighed glass
fiber
filter. The change in weight of the filter (mg) was then determined and this value
was divided by the volume of chamber atmosphere sampled (L) to yield the

gravimetric concentration (mg/L). The average time- weighted gravimetric

265Annex 56-B

(13)

SLI Study No. 3596.4

concentration of the test atmosphere was t hen calculated for the exposure. For
the analytical concentration, the gravimetrically obtained samples were analyzed
by Springborn Laboratories, Inc. for the glyphosate component, a non -volatile
component of the test article. These analyses were perform ed in order to

determine the analytical (actual) concentrations of the aerosol in the
chamber for
each sampling period. The average time weighted analytical concentration
of the
test atmosphere was then calculated for the exposure. Chemistry methods and
results are detailed in the Analytical Chemistry Report (Appendix B).

9.2.2.3. Chamber Temperature and Humidity

The chamber temperature and humidity were measured electronically and
recorded at approximate 30-minute intervals during the aerosol exposure.

9.2.2.4. Aerosol Aerodynamic Particle-Size Distribution

The aerosol aerodynamic particle -size distribution was determined three times
during the aerosol exposure using the ITP 7 Stage Cascade Impactor . Each
stage of the impactor was fitted with a preweighed glass fiber fil ter. Five liters
per minute of the chamber air were drawn through the impactor and the cha
nge

in weight of each filter was then determined and recorded. The mean particle -
size distribution was subsequently plotted using an Excel computer adaptation of
the manual method. The Mass Median Aerodynamic Diameter, Geometric
Standard Deviation and percentage of particles < 4.0 µ were then determined. At
least one hour passed between each aerosol particle-size analysis.

9.2.2.5. Chamber Oxygen

Chamber oxygen content was measured and recorded at approximate 30- minute
intervals during the aerosol exposure.

9.2.3. Clinical Observations

The limit test animals were observed for clinical abnormalities during each
aerosol exposure, two times on study day 0 (post -exposure) and daily thereafter
(days 1-14). A general health/mortality check was performed twice daily (in t
e
morning and in the afternoon).

9.2.4. Body Weights

Individual body weights were obtained for the limit test animals prior to dosing on
day 0 and on days 7 and 14.

9.2.5. Gross Necropsy

All limit test animals were euthanized by carbon dioxide inhalation at st
udy
termination (day 14) and necropsied. Body cavities (cranial, thoracic, abdominal
and pelvic) were opened and examined. No tissues were retained.

266 Annex 56-B

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SLI Study No. 3596.4

9.3. Protocol Deviations

No protocol deviations occurred during this study.

10. ANALYSIS OF DATA

Data from the limit tests were analyzed and an LC50 value estimated as follows:

Body weight means and standard deviations were calculated separately for
males and females. The aerodynamic particle -size distribution of the test article
aerosol was plotted using an Excel computer adaptation of the three cycle
logarithmic probability paper as per the ITP Cascade Impactor instruction

manual. The Mass Median Aerodynamic Diameter, Geometric Standard
Deviation and particles < 4.0 µ was determined based on the plotted distribution.

11. MAINTENANCE OF RAW DATA AND RECORDS

12. RESULTS

12.1. Aerosol Generation and Chamber Environmental Data

12.1.1. Aerosol Generation Data

Individual Data: Table 1

The average time-weighted analytical concentration for the aerosol exposure was

determined to be 3.27 mg/L. The mass median aerodynamic diameter and
geometric standard deviation of the sampled particles were 2.6 µ ± 1.8. The
percentage of particles  4.0 µ was determined to be 77%.

267Annex 56-B

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SLI Study No. 3596.4

12.1.2. Chamber Environmental Data
Individual Data: Table 1

Chamber temperature and relative humidity for the aerosol exposure ranged from
72.6-73.7°F and 65.7 -69.3%, respectively. Oxygen content was mainta ined at
20.9% throughout the exposure.

12.2. Limit Test Data

12.2.1. Mortality

Individual Data: Table 2

No mortality occurred during this study.

12.2.2. Clinical Observations

Individual Data: Table 2

The most notable clinical abnormalities observed during the study include
d
decreased/no defecation, soft stools, feces small in size, decreased food
consumption and rough coat. Clinical abnormalities also observed during the
study included transient incidences of breathing abnormalities and dark m
aterial
around the facial area, which were findings consistent with dosing an inhalation

study. No positive findings were noted at the time of observation during the 4 -
hour exposure period.

In addition, the dose level actually conducted was significantly higher (3.27
mg/kg) than the required dose level (2.0 mg/L) and did not result in any

mortality.
12.2.3. Body Weight Data

Individual Data: Table 3

Body weight loss was noted for one male and one female during the study d
ay 0-
7 body weight interval. Body weight gain was noted for all other animals during
the test period. All animals exceeded their initial body weight by stud
y
termination (day 14).

268 Annex 56-B

269Annex 56-B

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SLI Study No. 3596.4

15. REFERENCE

1. Guide for the Care and Use of Laboratory Animals, DHHS Publication No.
(NIH) 96-03, 1996.

270 Annex 56-B

(18)

PAGE 1

10 5 24 3 3 2.6 77 -73.-769.3
3.27 95.24 240 113.687 ± 1.8 20.9
72.665.7

OSURE LEVEL (MG/L)

EXP

TABLE 1

ONLY INHALATION TOXICITY STUDY IN RATS

CHAMBER ENVIRONMENTAL DATA

SUMMARY OF AEROSOL GENERATION AND

AN ACUTE NOSE-

: 4.0 µ (%):

-WEIGHTED MEAN ANALYTICAL CONCENTRATION (MG/L):

SLI STUDY NO.: 3596CHACMHELBMANVTET99EEPOISBRAELMRE(EIN)PDEMIT.)TIN):SNE-GHUMOIDITGNACNGET%N): (%):A

271Annex 56-B

(19)

---- ---- ----

STUIDYLNO.:US3596.4ALMESNT-O--MALE-#-A-5-2O-BSHRVATI-25EESOAA-EDENAFAD-EDEBRSOP-AREGHOFE-RGD-E-SO-ALME
-3F-:APEA-L--NG-
-8---P-PP-1PR------------

272 Annex 56-B

(20)

---- ---- ----

STIUDY/ANO.:S3D59P6.4LMESNT-----MAL-E#---4---COBAEIG57TDORCS--DUEE-DEDE-THE--ASI--G--------------------0---D----
-P
PT4--R-----------8--
------1----2------14-------------L-----

273Annex 56-B

(21)

---- ---- ----

STUDY/ANO.:S3596A.4MALES-OFFEMA-LE#-A-5-2-OBSERVATIGESTEDEAES-SMCRNGHOS-GLEDEE-BRNOSOPMAM--SIHIFEYA2-EEEBYELS5-
A
S
ZE-8-P------1-T------------

274 Annex 56-B

(22)

---- ---- ----

STUDLY/NO.:S3D596.4MALE-S-F-EMALE#-A-5-2-OBSEHEDENGBESTEDEBRETA-AMEAHELORAED-EUD-EOMMAA-INNALOOD-TUND
EM-
4S)YP-P-P--------9--------1--
-----P--P--P------------

275Annex 56-B

(23)

---- ---- ----

STUIDYL/NO.:US359M-ALES-EA-NIMAL-#----5352ME-57-0--F--86--65--64140A12-DEA--6-(D-AY)--------------------------------------------
-------------------------------------
---------------------------------------------------------------------------------------------

276 Annex 56-B

(24)

---- ---- ----

STUNDY/NO.U:S35EFEMAL-E-ANIMALA#----8482ME-82-0--7--90---8--94-48A-31DEAT---1DA-Y)-------------------------------------------
-------------------------------------
---------------------------------------------------------------------------------------------

277Annex 56-B

(25)

1 ---- ---- ----

STUDINL/A, US DEMA-LES----IOAL27-MG-4D------S3-2-0---J--257---R-NI0UN----M:AE----AALIA-I-O;E,E-O-ENTRAH--
L
L5M--MITSAS-ETER,-T-ORT
O-
-

278 Annex 56-B

(26)

2 ---- ---- ----

STINL/A, US DE6FEMAL-EAN-I3.2-7-527L52FA4---20-YY2282---0-N-J--4TI-TIV--LT-AWG--UE-SNO---I--IIO--A--MNO--TS--LI-
ITS-----
---
-
-------------------FATCHE--

279Annex 56-B

(27)

280 Annex 56-B

(28)

SLI Study No. 3596.4

APPENDIX A

Preliminary Aerosol Generation Trials

281Annex 56-B

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SLI Study No. 3596.4

1. PRELIMINARY AEROSOL GENERATION TRIALS
Prior to experimental initiation, preliminary aerosol generation trials
were

conducted. These procedures were performed in order to determine the most
efficient means of generating an aerosol of the test article. The type of
equipment used during each aerosol trial procedure is presented in Trial Table 1.
In each trial, attempts were made to generate the highest concentration of the
test article while utilizing equipment that would minimize the aerodynami
c particle

size of the aerosol.

The results indicated that the equipment design/pump speed utilized during
Trial #7 produced an analytical aerosol concentration ≥ 2.00 mg/L. Using the

equipment design determined by the aerosol generation trials, the aeroso
l
aerodynamic particle -size distribution was then determined utilizing the ITP 7
Stage Cascade Impactor . The aerodynamic particle size was acceptable.
Therefore, this equipment design was used for the LC50 study exposure.

282 Annex 56-B

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0.50
PAGE 1 -- >
3.71 4.132 1.20

ANALYTICAL

2.10 2.02 0.82 0.50
MAXIMUM ATTAINABLE
CONGRAVIMETRIC (MG/L)

TEST 100 100 100 100
ARCONCEN
TRATION (%)

30 30 30 30
(PSI) 1.00 mg/L gravimetric concentration for Trial 3 and
INPUT AIR >

-2;

-0 -0 -0 -0

TRIAL TABLE 1

30 and 77200 30 and 77200 30 and 77200 30 and 77200
- 3 - -

PRELIMINARY AEROSOL GENERATION TRIALS

-nly Chamber On-y Chamber Only-Chamber Only C-amber
tol Air/Fluid Mixing Nozzle
EQUIPMENT USED

2.00 mg/L gravimetric concentration for Trials 1
DEPARTMENT OF STATE ≥

OneLMElltitaorlOneuMoaetrarorlOp5ueaultttrFleOpnumMpltrt52rlxpPHmepdsnd523ump Heads 7523ixing Nozzle

NO.1 2 3 4
TRIAL
SLCLIENT: INL/A, US.4 Notmg/L gravimetric concentration for Trial 4.

283Annex 56-B

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PAGE 2
-- --
1.16 1.50 mg/L
>
ANALYTICAL

0.50 0.84 1.48
MAXIMUM ATTAINABLE
CONCENTRATIONS (MG/L)
GRAVIMETRIC

TEST 100 100 100
ARTCONCEN
TRATION (%)

(PSI) 30 30

INPUT AIR
1.00 mg/L gravimetric concentration for Trial 6 and
>
60 60 60
- - -
00
TRIAL TABLE 1

-0 and 77200 -0 and 77200 -0 and 772

PRELIMINARY AEROSOL GENERATION TRIALS

Only Chamber Only Chamber Only Chamber
- - -

EQUIPMENT USED
0L Nose

r Flex Pump and Pump Heads≥7523mg/L gravimetric concentration for Trial 5;

On5eLEulttitorlOPnumMpltrtaormOpnHNMalttirFerx0PLumopand Pump Heads 7523Mixing Nozzle

NO.5 6 7
TRIAL
SLICLIENT: INL/A, US DEPARTMENT OF STATE Nogravimetric concentration for Trial 7.

284 Annex 56-B

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SLI Study No. 3596.4

APPENDIX B

Analytical Chemistry Report

285Annex 56-B

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SLI Study No. 3596.4

1. SPRAY--ALPHA ANALYSIS

The analytical method for the analysis of the glyphosate component of Sp
ray --Alpha
was validated prior to the analytical chamber concentration analyses performed at
Springborn Laboratories, Inc. This method was utilized to determine the inhalation
chamber concentration during the Acute Nose-Only Inhalation Toxicity Study.

1.1. Experimental System

1.1.1. HPLC System

HPLC Model: Waters
Pump: Waters 600E
Injector: Waters WISP 717
Detector: Waters 2487

Data System: H-P 3396B Integrator
Precolumn: Phenomenex, SecurityGuard, C18, 4.0 x 3.0 mm ID
Column: Phenomenex, Spherex, C18, 5µ, 250 x 4.6 mm ID
Temperature: Ambient

Detection: 500 nm, 0.4000 AUFS
Mobile Phase: A: 0.05 M HCO N2 , 4H 3.6/5% ACN; B: 100% ACN
Gradient: 100% A hold for 6 minutes; linear change to 25% A/75% B over 1
minute; hold for 5 minutes; linear change to 100% A over 1 minute; ho
ld
at 100% A for 15 minutes.

Flow Rate: 1.0 mL/min
Injection Volume: 10 L

1.1.2. Apparatus

Balance: Mettler AG 245, accuracy of 0.0001 gram
Glassware: Assorted volumetric glassware
Filters: Gelman, glass fiber; Millipore 0.2µ Nylon-66; Whatman Puradisc 25PP
0.45µm

Shaker: Labline, Multi-Wrist Shaker
Oven: Boekel Model 107905

286 Annex 56-B

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SLI Study No. 3596.4

1.1.3. Solutions and Reagents

1.1.3.1. Reagents

Water, Fisher, HPLC Grade, Lot # 024948
Acetonitrile, Fisher, HPLC Grade, Lot # 011777
Methanol, Fisher, HPLC Grade, Lot # 011803
NBD Chloride, Aldrich, 98%, Lot #12214L1

Hydrochloric Acid, Fisher, ACS Grade, Lot # 012161
Potassium Tetraborate Tetrahydrate:, Aldrich, 99%, Lot # 15325D1
Formic Acid, Fisher, Laboratory Grade, Lot # 003630
Ammonium Formate, Fisher, Lot # 990125

1.1.3.2 Solutions

0.37 M Borate Solution: Prepared by dissolving approximately 11 .44 g of potassium
tetraborate tetrahydrate in 100 mL of water. The resulting solution was stable for 6
months under ambient storage conditions.

1.2 N HCl: Prepared by dissolving 10 mL of HCl in 90 mL of water. The resulting
solution was stable for 6 months under ambient storage conditions.

25 mM NBD- Cl: Prepared by dissolving approximately 2.5 g of NBD- Cl in 500 mL of

methanol. The resulting solution was stable for 6 months under ambient s
torage
conditions.

Mobile Phase A: Prepared by dissolving approximately 1.58 g of ammonium formate in
950 mL of water. The pH was adjusted to approximately 3.6 with formic a
cid. Added 50

ml of acetonitrile. The resulting solution was mixed thoroughly, fil
d through a 0.2 µ
Nylon-66 filter and degassed by helium sparging prior to use.

Mobile Phase B: Acetonitrile used 100% as received.

Diluent: All standards and samples were diluted in water.

Stock Standard Solution (Trial - mg/L): Prepared by dissolving 101.9 mg of the
Spray--Alpha formulation in a 25 mL flask with diluent.

Stock Standard Solution (Exposure #1- mg/L): Prepared by dissolving 236.0 mg of
Spray--Alpha formulation in a 25 mL flask with diluent.

287Annex 56-B

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SLI Study No. 3596.4

Standard Solutions : Prepared by serially diluting the stock standard solution with water.
The final concentrations of the solutions were in the range of approximately 0.4 to 2.9
mg/mL (trial) and 0.9 to 4.7 mg/mL (Exposure # 1). These solutions w
ere then further
diluted in diluent at a ratio of 1:10 and filtered through Whatman Puradi
sc 25PP 0.45µ m

filters prior to derivatization.

Chamber Concentration Solutions: Prepared by placing the weighed glass fiber filter
used for gravimetric concentration determination in a capped container with 10 mL of
diluent. The solutions were then agitated mechanically for 5 minutes further diluted in

diluent at a ratio of 1:10 and filtered through Whatman Puradisc 25PP 0.
45 µm filters
prior to derivatization.

Derivatization Procedure: In order to analyze the glyphosate component, a precolumn
derivatization was performed by adding 1.2 mL of the appropriate control, standard, or

sample solution to a labeled scintillation vial. Both 0.8 mL of the bor
ate solution and
2.4 mL of the NBD- Cl solution were added to each vial. The vials were then capped
and shaken by han d prior to being heated in an oven at 80° C for 30 minutes. After
removal from the oven, the vials were allowed to cool for 10 minutes foll
owed by the

addition of 0.9 mL of the HCl solution. After the vials were again shaken by hand, they
were allowed to stand for 10 minutes in order for incipient precipitation to occur. These
solutions were then transferred to injection vials.

1.2. Analytical Procedures

1.2.1. Standard Curve Analysis

The peak area of the glyphosate acid component of each standard were dete
rmined,
measured, combined, and plotted as a function of concentration to generat
e a standard
curve. The actual values used for the calculations are shown in Chemistry Tables 1

and 2.
1.2.2. Sample Analysis

The peak areas of the glyphosate acid component of each sample were measured and
combined and then the concentration was determined by linear fit to the standard curve.

The actual values used for the calculations are shown in Chemistry Table
s 1 and 2.

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SLI Study No. 3596.4

1.3. Results and Conclusions

1.3.1. Analytical Chamber Concentration

The actual sample results of the trial work are shown in Chemistry Table 1. The
ctual
sample results of the analytical chamber analysis are shown in Chemistry Table 2.

Date
M. Gardner Clemons, B.A.
Manager of Analytical Chemistry

And Pharmacy.

289Annex 56-B

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SLI Study No. 3596.4

Chemistry Table 1

Standard Curve and Sample Analysis Values for Trial Work

Theoretical Conc. Analytical Chamber

Sample No. (mg/L) Peak Area Conc. (mg/L)
Std 1 0.8152 25090 NA
Std 2 2.446 77738 NA
Std 3 4.076 131263 NA

Std 4 5.706 182542 NA
Trial # 1 NA 118551 3.707
Trial # 2 NA 132259 4.132
Trial # 4 NA 37811 1.204

Trial #5 NA 36312 1.158
* Correlation coefficient = 0.99997

290 Annex 56-B

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SLI Study No. 3596.4

Chemistry Table 2

Standard Curve and Sample Analysis Values for Exposure #1

Theoretical Conc. Analytical
Sample No. (mg/L) Peak Area Chamber Conc.

(mg/L)
Std 1 1.888 47622 NA
Std 2 3.776 114022 NA
Std 3 5.664 169206 NA

Std 4 7.552 225528 NA
Std 5 9.440 251583 NA
# 1 NA 111887 3.857
# 2 NA 107931 3.714

# 3 NA 90648 3.085
# 4 NA 93185 3.178
# 5 NA 92333 3.147

# 6 NA 89526 3.045
# 7 NA 94131 3.212
# 8 NA 97391 3.330
# 9 NA 91642 3.121

#10 NA 102623 3.521
#11 NA 100109 3.429
* Correlation coefficient = 0.991

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SLI Study No. 3596.4

APPENDIX C

Individual Aerosol Generation and
Chamber Environmental Data

292 Annex 56-B

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SLI Study No. 3596.4

3.27 mg/L Exposure Level

293Annex 56-B

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SLI Study No. 3596.4

AN ACUTE NOSE-ONLY INHALATION TOXICITY STUDY IN RATS
CHAMBER ENVIRONMENTAL DATA
EXPOSURE: 3.27 MG/L

TIME TEMPERATURE RELATIVE HUMIDITY OXYGEN CONTENT
(MIN.) (°F) (%) (%)
0 72.6 69.3 20.9
30 72.8 65.7 20.9

60 72.7 67.6 20.9
90 72.9 68.0 20.9
120 73.4 66.7 20.9

150 73.1 67.5 20.9
180 73.5 67.6 20.9
210 73.5 67.7 20.9
240 73.7 67.3 20.9

294 Annex 56-B

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SLI Study No. 3596.4

AN ACUTE NOSE-ONLY INHALATION TOXICITY STUDY IN RATS

TIME WEIGHTED ANALYTICAL CONCENTRATION
ANALYTICAL EXPOSURE: 3.27 MG/L

Mean Time

Aerosol Concentration Interval Weighted
Sample Sample Concentration Per Interval Length Concentration

No. Time (min.) (mg/L) (mg/L) (min.) Per Interval
1 0 3.86
3.79 14.00 52.99

2 14 3.71
3.40 7.00 23.80

3 21 3.09
3.14 9.00 28.22

4 30 3.18
3.17 30.00 94.95
5 60 3.15

3.10 30.00 93.00
6 90 3.05

3.13 30.00 93.90
7 120 3.21
3.27 30.00 98.10

8 150 3.33
3.23 30.00 96.75

9 180 3.12
3.32 30.00 99.60
10 210 3.52

3.47 30.00 104.10
11 240 3.42

TOTAL 240.00 785.41
TIME WEIGHTED MEAN ANALYTICAL CONCENTRATION (MG/L) 3.27

295Annex 56-B

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SLI Study No. 3596.4

AN ACUTE NOSE-ONLY INHALATION TOXICITY STUDY IN RATS

AERODYNAMIC PARTICLE SIZE DATA

SAMPLE NO.: A
ANALYTICAL EXPOSURE: 3.27 MG/L

Effective

Cutoff Filter Weights (mg) Difference Cumulative
Stage Diameter Pre-sample Post-sample Weights % of Total % <ECD

1 10.00 102.4 102.4 0.0 0.0 100.0
2 6.11 102.7 102.9 0.2 5.4 94.6

3 3.70 102.7 103.3 0.6 16.2 78.4

4 2.22 103.2 104.7 1.5 40.5 37.8
5 1.39 103.6 104.6 1.0 27.0 10.8

6 0.79 104.4 104.8 0.4 10.8 0.0

7 0.50 103.4 103.4 0.0 0.0 0.0
Filter - 102.6 102.6 0.0 0.0

Total of Difference Weights: 3.7

Mass Median Aerodynamic Diameter = 2.6 microns

Geometric Standard Deviation = 1.67
Percentage ≤4.0 microns = 80 %

296 Annex 56-B

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SLI Study No. 3596.4

AN ACUTE NOSE-ONLY INHALATION TOXICITY STUDY IN RATS

AERODYNAMIC PARTICLE SIZE DATA

SAMPLE NO.: B
ANALYTICAL EXPOSURE: 3.27 MG/L

Effective

Cutoff Filter Weights (mg) Difference Cumulative
Stage Diameter Pre-sample Post-sample Weights % of Total % <ECD

1 10.00 103.0 103.0 0.0 0.0 100.0
2 6.11 103.5 103.9 0.4 8.7 91.3

3 3.70 103.1 104.0 0.9 19.6 71.7

4 2.22 103.8 105.5 1.7 37.0 34.8
5 1.39 103.3 104.4 1.1 23.9 10.9

6 0.79 103.5 103.8 0.3 6.5 4.3

7 0.50 102.7 102.8 0.1 2.2 2.2
Filter - 103.1 103.2 0.1 2.2

Total of Difference Weights: 4.6

Mass Median Aerodynamic Diameter = 2.6 microns

Geometric Standard Deviation = 2.00
Percentage ≤4.0 microns = 74 %

297Annex 56-B

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SLI Study No. 3596.4

AN ACUTE NOSE-ONLY INHALATION TOXICITY STUDY IN RATS

AERODYNAMIC PARTICLE SIZE DATA

SAMPLE NO.: C
ANALYTICAL EXPOSURE: 3.27 MG/L

Effective

Cutoff Filter Weights (mg) Difference Cumulative
Stage Diameter Pre-sample Post-sample Weights % of Total % <ECD

1 10.00 103.4 103.4 0.0 0.0 100.0
2 6.11 103.5 103.8 0.3 7.5 92.5

3 3.70 103.3 104.1 0.8 20.0 72.5

4 2.22 103.7 105.1 1.4 35.0 37.5
5 1.39 103.2 104.1 0.9 22.5 15.0

6 0.79 103.4 103.9 0.5 12.5 2.5

7 0.50 103.3 103.4 0.1 2.5 0.0
Filter - 104.2 104.2 0.0 0.0

Total of Difference Weights: 4.0

Mass Median Aerodynamic Diameter = 2.6 microns

Geometric Standard Deviation = 1.82
Percentage ≤ 4.0 microns = 76 %

298 Annex 56-B

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SLI Study No. 3596.4

AN ACUTE NOSE-ONLY INHALATION TOXICITY STUDY IN RATS

AERODYNAMIC PARTICLE SIZE DATA

ANALYTICAL EXPOSURE: 3.27 MG/L

Effective Cutoff Cumulative % less than indicated size

Stage Diameter Sample A Sample B Sample C
1 10.00 100.0 100.0 100.0

2 6.11 94.6 91.3 92.5
3 3.70 78.4 71.7 72.5

4 2.22 37.8 34.8 37.5

5 1.39 10.8 10.9 15.0
6 0.79 0.0 4.3 2.5

7 0.50 0.0 2.2 0.0

Mean
Mass Median Aerodynamic Diameter 2.6 2.6 2.6 2.6

Geometric Standard Deviation 1.67 2.00 1.82 1.83
Percentage ≤ 4.0 microns 80 74 76 77

299Annex 56-B

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SLI Study No. 3596.4

APPENDIX D

SLI Personnel Responsibilities

300 Annex 56-B

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SLI Study No. 3596.4

SLI PERSONNEL RESPONSIBILITIES

Kimberly L. Bonnette, M.S., LAGT Study Director/Director, Acute

Toxicologist

Dawn D. Rodabaugh, B.S. Alternate Contact/Associate
Toxicologist

Robert C. Springborn, Ph.D. Chairman, President and CEO

Malcolm Blair, Ph.D. Senior Vice President, Managing
Director Emeritus

Joseph C. Siglin, Ph.D., DABT Vice President, Managing Director

M. Gardner Clemons, B.A. Manager of Analytical Chemistry and
Pharmacy

Pamela S. Smith, ALAT Supervisor of Acute Toxicology

Kevin V. Weitzel, A.S. Primary Technician/Inhalation Team
Leader

Delores P. Knippen Supervisor of Pharmacy

Steven H. Magness, B.S., LATG Senior Supervisor of Gross and Fetal
Pathology

Anita M. Bosau, RQAP-GLP Senior Director, Compliance Assurance

Deanna M. Talerico, RQAP-GLP Senior Supervisor of Quality Assurance

J. Dale Thurman, D.V.M., M.S., DACVP Senior Director, Pathology

Kathy M. Gasser Supervisor of Archives

301Annex 56-B

A DERMAL SENSITIZATION STUDY IN GUINEA PIGS WITH SPRAY--ALPHA

•MODIFIED BUEHLER DESIGN•

FINAL REPORT

OPPTS Guidelines

870.2600

Author

Kimberly L. Bonnette, M.S., LATG

Study Completed on

September 3, 2002

Performing Laboratory

Springborn Laboratories, Inc. (SLI)
Ohio Research Center

640 North Elizabeth Street
Spencerville, Ohio 45887

SLI Study No.

3596.7

Submitted to

INL/A
U.S. Department of State
2201 C St. NW SA-4
Washington, DC 20520

Page 1 of 41

302 Annex 56-B

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SLI Study No. 3596.7

1. STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

No claim of confidentiality is made for any information contained in this study on
the basis of its falling within the scope of FIFRA §10(d)(1)(A), (B), or (C).

Company:

Company Agent: Date:

Title Signature

303Annex 56-B

304 Annex 56-B

305Annex 56-B

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SLI Study No. 3596.7

4. TABLE OF CONTENTS

1. STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS..........................2 ..

2. COMPLIANCE STATEMENT.........................................................................3 .......

3. QUALITY ASSURANCE STATEMENT..........................................................4 .....

4. TABLE OF CONTENTS........................................................................................

5. LIST OF TABLES AND APPENDICES..........................................................6 .....

6. SUMMARY ......................................................................................................7.........

7. INTRODUCTION ..............................................................................................

8. MATERIALS AND METHODS........................................................................8 ......

9. EXPERIMENTAL PROCEDURES.....................................................................11

10. ANALYSIS OF DATA.........................................................................................13

11. MAINTENANCE OF RAW DATA AND RECORDS......................................13

12. RESULTS.............................................................................................................13

13. CONCLUSION.....................................................................................................14

14. REPORT REVIEW..............................................................................................14

15. REFERENCES.....................................................................................................15

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SLI Study No. 3596.7

5. LIST OF TABLES AND APPENDICES

Tables

1. Individual Induction Data (Spray--Alpha) .............................................1 ...
2. Individual Challenge Data (Spray--Alpha)............................................1 ..7

Appendices

A. Topical Range-Finding Study...............................................................1....

B. Dermal Grading System...........................................................................

C. Individual Body Weight Data....................................................................

D. HCA Historical Control Data.................................................................3...

E. SLI Personnel Responsibilities............................................................4....

307Annex 56-B

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SLI Study No. 3596.7

6. SUMMARY

The dermal sensitization potential of Spray --Alpha was evaluated in Hartley -
derived albino guinea pigs. Ten male and ten female guinea pigs were topically

treated with 100% Spray --Alpha, once per week, for three consecutive weeks.
Following an approximate two -week rest period, a challenge was performed
whereby the twenty test and ten previously untreated (naiv e) challenge control

guinea pigs were topically treated with 100% Spray --Alpha. Challenge
responses in the test animals were compared with those of the challenge c
ontrol
animals.

6.1. Spray--Alpha

Following challenge with 100% Spray --Alpha, dermal reactions in the test and
challenge control animals were limited to scores of 0. Group mean dermal

scores were noted to be the same in the test animals as compared with the
challenge control animals.

6.2. HCA

Using -Hexylcinnamaldehyde (HCA) as a positive control, Springborn
Laboratories, Inc., Spencerville, Ohio, has completed a study during the
past six
months which provided historical control data for contact sensitization
to this

agent utilizing the test system described herein (Modified Buehler Desig
n).
Following induction at 5% w/v HCA in ethanol and challenge at levels of 2.5%
and 1% w/v HCA in acetone, a contact sensitization response was observed,

thereby demonstrating the susceptibility of the test system to this sens
itizing
agent.

Based on the results of this study, Spray--Alpha is not considered to be a contact
sensitizer in guinea pigs. The results of the HCA historical control study
demonstrated that a valid test was performed and indicated that the test
design
would detect potential contact sensitizers.

308 Annex 56-B

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SLI Study No. 3596.7

7. INTRODUCTION

This study was performed to assess the dermal sensitization potential (
delayed
contact hypersensitivity) of Spray --Alpha in Hartley -derived albino guinea pigs

when administered by multiple topical applications. This study was inten
ded to
provide information on the potential health hazards of the test article wit
h respect
to dermal exposure. Data from this study may serve as a basis for class
ification

and/or labeling of the test article. This study was performed in accordance with
the US EPA, Health Effects Test Guidelines, OPPTS 870.2600, Skin
Sensitization, August 1998. This study was performed at Springborn

Laboratories, Inc., 553 North Broadway, Spencerville, Ohio. The protocol
was
signed by the Study Director on April 30, 2002 (GLP in itiation date). The in- life

phase of the main sensitization study was initiated with test article adm
inistration
on June 13, 2002 (day 0), and concluded with final scoring on July 12, 2002.

Prior to initiation of the main sensitization study, a topical range-finding study was
conducted in guinea pigs to aid in the selection of dosage levels. The in- life
phase of the range- finding study was initiated with test article administration on

June 10, 2002, and concluded on June 12, 2002. The experimental metho ds
and results of the range-finding study are included in Appendix A.

8. MATERIALS AND METHODS

8.1. Test Article

The test article was received from the Sponsor and identified as follows
:

Sponsor’s Assigned Physical Receipt Expiration
ID SLI ID Description Date Date
a
Spray—Alpha S02.001.3596 Light amber 05/13/02 None
liquid Provided
Ingredients
Herbicide: Fuete-SL None

Lot No.: 02-01-02 Provided

Surfactant: Cosmo Flux -411F 10/2003
Lot No.: 244301
Sample pooled at SLI from five different mixes o f Spray--Alpha (top/middle/bottom).
bIngredients used in the five Spray --Alpha mixes that were prepared by the Sponsor.

The test article was stored at room temperature. The Sponsor was responsible
for any necessary evaluations related to identity, stre ngth, purity, composition,

309Annex 56-B

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SLI Study No. 3596.7

stability and method of synthesis of the test material according to 40 C
FR
160.105, 40 CFR 792.105. Springborn Laboratories, Inc., analyzed the test
article for the glyphosate (a.e.) which is presented in SLI Study No.
3596.1.

8.2. Retention Sample

An approximate 1 mL retention sample of each test article mixture sample
(top/middle/bottom, maintained separately for a total of fifteen 1 mL s
mples)
was taken and stored at SLI at room temperature. In addition, a 10 mL retention
sample of the pooled test article samples (from the 5 test article mixtures) w
as

collected and stored at SLI at room temperature. These samples serve as the
retention samples for all studies conducted with this material.

8.3. Test Article Disposition
The remaining test article was returned to the Sponsor following completion of all

studies with the test article.

8.4. Method of Test Article Preparation

The test article was utilized at 100% (induction and challenge). The test article
was dispensed fresh on each day of dosing.

8.5. Animals and Animal Husbandry

8.5.1. Description, Identification and Housing

Young adult, Hartley-derived albino guinea pigs were received from Hilltop Lab
Animals, Inc., Scottdale, PA. Upon receipt, plastic ear tags displaying
unique

identification numbers were used to individually identify the animals. Cage cards
displaying at least the study number, animal number and sex were affixed to
each cage. The animals were housed individually in suspended stainless steel
cages. All housing and care were based on th e standards recommended by the
Guide for the Care and Use of Laboratory Animals [1].

8.5.2. Environment

The animal room temperature and relative humidity ranges were 63- 74F (17-
23°C) and 48- 82%, respectively. Environmental control equipment was

monitored and adjusted as necessary to minimize fluctuations in the animal room
environment. Light timers were set to maintain a 12-hour light/12-hour dark cycle
and room ventilat ion was set to produce 10- 15 air changes/hour. The room
temperature and relative humidity were recorded a minimum of once daily.

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SLI Study No. 3596.7

8.5.3. Food

PMI Certified Guinea Pig Chow #5026 (Purina Mills, Inc.) was provided ad libitum
to the animals throughout the study. The lot number and expiration date of each
batch of diet used during the study were recorded. The feed was analyzed and
certified by the supplier for nutritional components and environmental
contaminants. Dietary limitations for various environmental contaminants,

including heavy metals, pesticides, polychlorinated biphenyls and total
aflatoxin
are set by the manufacturer. Within these limits, contaminants which may have
been present were not expected to compromise the purpose of this study.
Results of the dietary analyses (Certificates of Analysis) are provided by the
manufacturer for each lot of diet. These are maintained by SLI.

8.5.4. Water

Municipal tap water treated by reverse osmosis was available ad libitum
throughout the study. The purified water was su pplied by an automatic watering
system. Monitoring of the drinking water for contaminants is conducted
by SLI

and the records are available for inspection. Within generally accepted
limits,
contaminants which may have been present were not expected to co mpromise
the purpose of this study. The water meets the standards specified under the
EPA National Drinking Water Regulations (40 CFR Part 141).

8.5.5. Acclimation

Upon receipt, the animals were removed randomly from the shipping cartons,
examined by qualified personnel, identified with plastic ear tags and then
acclimated to the laboratory conditions for a minimum of five days. The
animals
were observed daily for overt physical or behavioral abnormalities, gener
al

health/moribundity and mortality.

8.5.6. Animal Selection
The animals chosen for study use were arbitrarily selected from healthy s
tock

animals to avoid potential bias. All animals received a detailed pretest
observation prior to dosing. Only healthy animals were chosen for study use.
Females were nulliparous and nonpregnant. The male animals were
approximately 7 weeks of age and weighed 375- 458 g on the day prior to
Induction 1 dosing. The female animals were approximately 8 weeks of age and

weighed 346-389 g on the day prior to Induction 1 dosing.

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SLI Study No. 3596.7

9. EXPERIMENTAL PROCEDURES

9.1. Study Design

This study consisted of a topical range- finding group, a test group and a
challenge control group [2]. A rechallenge control group was maintained on this

study; however, the rechallenge procedure was not required since the challenge
results were definitive.

9.2. Sensitization Study

9.2.1. Preliminary Procedures

On the day prior to each dose administration, the guinea pigs had the hair
removed with a small animal clipper. Care was taken to avoid abrading
the skin.

9.2.2. Dosing

A dose of 0.3 mL of the test article was placed on a 25 mm Hilltop chamber

backed by adhesive tape (occlusive patch). The chambers were then app
lied to
the clipped surface as quickly as possible.

Following chamber application, the trunk of the animal was wrapped with
elastic
wrap which was secured with adhesive tape to prevent removal of the chamb
er
and the animal was returned to its cage.

9.2.2.1. Induction

On the day prior to the first induction dose administration (day -1), all test and
control animals were weighed and the hair was removed from the left side of the
test animals. On the day following clipping (day 0), chambers were applied as

follows:

Induction Concentration Test Site No. of Animals
Group Material No. (%) No. Male Female
Test Spray-- 1 100 a 1 10 10
a
Alpha 2 100 a 1
3 100 1
aPooled test article.

The induction procedure was repeated on study day 7 and on study day 14 so
that a total of three consecutive induction exposures were made to the t
est

animals.

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SLI Study No. 3596.7

9.2.2.2. Challenge

On the day prior to challenge dose administration, the test and challenge control

animals were weighed and the hair was removed from the right side of the
animals. On the day following clipping ( day 27), chambers were applied as
follows:

Concentration Test Site No. of Animals
Group Material (%) No. Male Female
a
Test Spray--Alpha 100 2 10 10
a
Challenge Control Spray--Alpha 100 2 5 5
a
Pooled test article.

9.2.3. Test Article Removal

Approximately six hours after chamber application, the binding materials
were

removed. The test sites were wiped with gauze moistened in deionized wa
ter,
followed by dry gauze, to remove test article residue. The animals were
then
returned to their cages.

9.2.4. Dermal Observations

The test sites were graded for irritation at approximate ly 24 and 48 hours

following chamber application (induction) or chamber removal (challenge) using
the Dermal Grading System presented in Appendix B.

9.2.5. Clinical Observations

Any unusual observations and mortality were recorded. The animals were

observed for general health/mortality twice daily, once in the morning and once in
the afternoon.

9.2.6. Body Weights

Individual body weights were obtained for all sensitization study animals on the
day prior to the first induction (day -1) and for the appropriate test and cha llenge

control animals on the day prior to challenge dosing.

9.2.7. Scheduled Euthanasia

All sensitization study animals were euthanized by carbon dioxide inhalation
following each animal's final scoring interval. Gross necropsy examinati
ons were

not required for these animals.

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SLI Study No. 3596.7

9.3. Protocol Deviations

The animal room temperature and relative humidity ranges [63- 74F (17-23°C)
and 48-82%] exceeded the preferred ranges [63- 73°F (17-23°C) and 30- 70%,
respectively] but were corrected on the same day. These occurrences were
considered to have had no adverse effect on the outcome of this study.

10. ANALYSIS OF DATA
The sensitization potential of the test article was based on the dermal responses

observed on the test and control animals at challenge. Generally, dermal scores
of 1 in the test animals with scores of 0 to  noted in the controls are considered
indicative of se nsitization. Dermal scores of 1 in both the test and control
animals are generally considered equivocal unless a higher dermal response ( 
grade 2) is noted in the test animals. Group mean dermal scores were

calculated for challenge.

11. MAINTENANCE OF RAW DATA AND RECORDS

All original raw data, the final report and magnetically encoded records
were
transferred to the SLI archives for a period of 10 years. The Sponsor will be
contacted prior to final disposition of these items.

12. RESULTS

12.1. Topical Range-Finding Study
Individual Topical Range-Finding Data: Appendix A

The results of the range-finding study indicated that a test article concentration of
100% was considered appropriate for induction and challenge since it was the
highest possible concentration which was nonirritating.

12.2. Sensitization Study

Individual Data: Tables 1-2

Following challenge with 100% Spray --Alpha, dermal reactions in the test and
challenge control animals were limited to scores of 0. Group mean dermal
scores were noted to be the same in the test animals as compared with the

challenge control animals.

314 Annex 56-B

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SLI Study No. 3596.7

15. REFERENCES

1. Guide for the Care and Use of Laboratory Animals, DHHS Publication No.
(NIH) 96-03, 1996.

2. E. V. Buehler, Delayed Contact Hypersensitivity in the Guinea Pig, Arch.
Dermat., 91 :171-177, 1965.

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PAGE 1 48 Hr0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

100%

24 Hr0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Induction 3 Dermal Scores

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
48 Hr

100%

) 24 Hr0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Induction 2 Dermal Scores

ALPHA

TABLE 1

(PRAY--
48 Hr0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

INDIVIDUAL INDUCTION DATA
Dermal Scores
100%

A DERMAL SENSITIZATION STUDY IN GUINEA PIGS

24 Hr0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Induction 1

Sex
G8270/F1/F2/F3/F4/F5/F6/F7/F8/F9/F
Animal No./1G81G45M46M4748149/50/51/52/M

GroupTest

Pooled test article.
Noae: See Appendix B for definition of codes.
SLICLIENT: INL/A, U.S. DEPARTMENT OF STATE

317Annex 56-B

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PAGE 1

48 Hr
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.0

100%

Dermal Scores

ALPHA)
24 H0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.0
TABLE 2

(SPRAY--

AL SENSITIZATION STUDY IN GUINEA PIGS
INDIVIDUAL CHALLENGE DATA

A DERM

Sex 72/F
Mean
Animal No./8G1851G8148/M8181/11M2/M/12F282/F2F282/F2F2/7F9/F

Groupest

PoAlemtelsfurcleut of binding at the time of patch removal.
SLICLIENT: INL/A, U.S. DEPARTMENT OF STATE Natbs: See Appendix B for definition of codes.

318 Annex 56-B

(18)

PAGE 2

48 0 0 0 0 0 0 0 0 0 0.0

100%

Dermal Scores

ALPHA)
IT IT
24 0 0 0 0 0 0 0 0 0 0 0.0
TABLE 2

(SPRAY--

DIVIDUAL CHALLENGE DATA
IN

A DERMAL SENSITIZATION STUDY IN GUINEA PIGS

Sex Mean
G81G8G8G181M1/7/M282/2/F284/F
Animal No./

Group

Challenge Control
SLICLIENT: INL/A, U.S. DEPARTMENT OF STATE Notes: See Appendix B for definition of codes.

319Annex 56-B

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SLI Study No. 3596.7

APPENDIX A

Topical Range-Finding Study

320 Annex 56-B

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SLI Study No. 3596.7

1. TOPICAL RANGE-FINDING STUDY

This appendix provides the experimental procedures and results of a topi
cal
range-finding study in guinea pigs with Spray --Alpha. The procedures for animal

husbandry were similar to those described for the main sensitization stud
y
animals. The male animals were approximately 7 weeks of age and weighed

405-458 g; the female animals were approximately 11 weeks of age and weighed
480-481 g on the day prior to dosing.

1.1. Method of Test Article Preparation

The test article was utilized at 100% and at 75%, 50% and 25% w/v in deionized
for the range-finding study. The test article was prepared and dispensed fresh

on the day of dosing. The dosing preparations were stirred continuously
during
dosing.

1.2. Dosing

On the day prior to dose administration, four topical range -finding guinea pigs
were weighed and the hair removed from the right and left side o f the animals

with a small animal clipper. Care was taken to avoid abrading the skin during

clipping procedures.

On the following day, four concentrations of the test article were prepar
ed and
each concentration was applied to the clipped area of each topical range-finding

animal as indicated below:

Concentration Test Site Amount a
Group Material (%b No. Applied Patch Design
Topical Spray-- 100 1 0.3 mL 25 mm Hilltop Chamber
Range- Alpha 75b, c 2 0.3 mL 25 mm Hilltop Chamber
Finding
50b, c 3 0.3 mL 25 mm Hilltop Chamber
b, c
25 4 0.3 mL 25 mm Hilltop Chamber
a
bOcclusive patch.
cPooled test article
The vehicle was deionized water.

The chambers were applied to the clipped surface as quickly as possible.
The
trunk of the animal was wrapped with elastic wrap which was secured with

adhesive tape to prevent removal of the chambers and the animal was retur
ned
to its cage.

321Annex 56-B

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SLI Study No. 3596.7

Approximately six hours after chamber application, the binding materials
were
removed. The test sites were then wiped with gauze moistened in deionized
water, followed by dry gauze, to remove test article residue and the animals
returned to their cages.

1.3. Dermal Observations

The test sites of the topical range- finding animals were graded for irritation at
approximately 24 and 48 ho urs following chamber application using the Dermal
Grading System in Appendix B.

1.4. Clinical Observations

Any unusual observations and mortality were recorded. The topical range- finding
animals were observed for general health/mortality twice daily, once in t
he
morning and once in the afternoon.

1.5. Body Weights

Individual body weights were obtained for the topical range- finding animals on
the day prior to dosing.

1.6. Scheduled Euthanasia

Following the 48- hour scoring interval, all topical range- finding animals were
euthanized by carbon dioxide inhalation. Gross necropsy examinations were not
required for these animals.

1.7. Results
The results of the range-finding study indicated that a test article concentration of

100% was considered appropriate for induction and challe nge since it was the
highest possible concentration which was nonirritating.

322 Annex 56-B

(22)

48 Hr
PAGE 1 a,b 0 0 0 0

25%

24 Hr
0 0 0 0

48 Hr

a,b 0 0 0 0

50%

24 Hr
0 0 0 0

Finding Dermal Scores
- 48 Hr
a,b 0 0 0 0

Ra75%
FINDING DATA
- ALPHA) 24 Hr
0 0 0 0

(SPRAY--

48 Hr
a 0 0 0 0
TOPICAL RANGE
100%

24 Hr
A DERMAL SENSITIZATION STUDY IN GUINEA PIGS

458 405 481 480
G7961/MG7969/MG7449/FG7477/F
Animal No./Sex
Body Weight (g)

-inding
Group

Range
SLI LITNT: IOL./:59.6..7DEPARTMENT OF STATE a PbolNote: See Appendix B for definition of codes.

323Annex 56-B

(23)

SLI Study No. 3596.7

APPENDIX B

Dermal Grading System

324 Annex 56-B

(24)

SLI Study No. 3596.7

DERMAL GRADING SYSTEM

ERYTHEMA AND EDEMA OBSERVATIONS

OBSERVATION DEFINITION CODE

Erythema – Grade 0 No reaction 0

Erythema – Grade ± Slight patchy erythema ±

Erythema – Grade 1 Slight, but confluent or moderate patchy erythema 1

Erythema – Grade 2 Moderate, confluent erythema 2

Erythema – Grade 3 Severe erythema with or without edema 3

M – 3
Maximized Grade 3 Notable dermal lesions
(see below)

Edema – Grade 1 Very slight edema (barely perceptible) ED-1

Edema – Grade 2 Slight edema (edges of area well defined by definite ED-2
raising)

Edema – Grade 3 Moderate edema (raised approximately 1 millimeter) ED-3
Severe edema (raised more than 1 millimeter and
Edema – Grade 4 ED-4
extends beyond the area of exposure)

An erythema code was assigned to each test site. An edema code was assigned on
ly if edema
was present at the test site. If notable dermal lesion(s) (> grade 1
) were present, then the
“Maximized Grade 3” was assigned to the test site in place of the
erythemre and the type
ES-2
of the notable dermal lesion(s) was noted (e.g., M -3

325Annex 56-B

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SLI Study No. 3596.7

DERMAL GRADING SYSTEM

NOTABLE DERMAL LESIONS

OBSERVATION CODE DEFINITION

Eschar – Grade 1 ES-1 Focal and/or pinpoint areas up to 10% of test site.

Eschar – Grade 2 ES-2 > 10% < 25% of test site.

Eschar – Grade 3 ES-3 > 25% < 50% of test site.

Eschar – Grade 4 ES-4 > 50% of test site.

Blanching – Grade 1 BLA-1 Focal and/or pinpoint areas up to 10% of test site.

Blanching – Grade 2 BLA-2 > 10% < 25% of test site.

Blanching – Grade 3 BLA-3 > 25% < 50% of test site.

Blanching – Grade 4 BLA-4 > 50% of test site.

Ulceration – Grade 1 U-1 Focal and/or pinpoint areas up to 10% of test site.

Ulceration – Grade 2 U-2 > 10% < 25% of test site.

Ulceration – Grade 3 U-3 > 25% < 50% of test site.

Ulceration – Grade 4 U-4 > 50% of test site.

NEC-1 Focal and/or pinpoint areas up to 10% of test site (note
Necrosis – Grade 1
(color) color of necrosis).
NEC-2
Necrosis – Grade 2 > 10% < 25% of test site (Note color of necrosis).
(color)

Necrosis – Grade 3 NEC-3 > 25% < 50% of test site (Note color of necrosis).
(color)

Necrosis – Grade 4 NEC-4 > 50% of test site (Note color of necrosis).
(color)

326 Annex 56-B

(26)

SLI Study No. 3596.7

DERMAL GRADING SYSTEM

ADDITIONAL DERMAL FINDINGS

OBSERVATION DEFINITION CODE

Characterized by scaling or flaking of dermal tissue or
Desquamation without denuded areas. DES

Characterized by cracking of the skin with or without
moist exudate. Fissuring should be checked prior to
Fissuring removing the animal from the cage and manipulating the FIS
test site.

Eschar Exfoliation The process by which areas of eschar flake off the test EXF
site.
Skin located at test site appears to be discolored, TSS
Test Site Staining possibly due to test article (note color of staining). (color)

Erythema Extends The erythema extends beyond the test site. Note: A
study director should be contacted for erythema ERB
Beyond the Test Site extending beyond the test site.

Characterized by pale area(s) (almost a burn -like
appearance) in the test site. However, erythema may
still be observed through the pale area. Note: This
observation may affect the overall erythema score of the

Superficial Lightening test site. This observation may progress to other --
observations resulting in notable dermal lesions, but SL
itself will not be considered a notable dermal lesion that
will result in a dermal score to be maximized since it

does not result in any in- depth injury. To be coded
using an area designation (see below).
Superficial Lightening -
Grade 1 Focal and/or pinpoint areas up to 10% of the test site SL-1

Superficial Lightening - > 10% < 25% of test site SL-2
Grade 2
Superficial Lightening -
Grade 3 > 25% < 50% of test site SL-3

Superficial Lightening - > 50% of test site SL-4
Grade 4
Noticeable irritation outside of test site probably due to

Dermal Irritation - the binding tape material. This notation will only be
Outside of the Test Site made for reactions greater than what are normally IT
observed from tape removal which do not interfere with
the scoring of the test site.

327Annex 56-B

(27)

SLI Study No. 3596.7

APPENDIX C

Individual Body Weight Data

328 Annex 56-B

(28)

PAGE 1

661 58659770636556757625545449860515125655564508
Day 26

1
-
434 37541944414584437399373835038343553838387366
Day

(SPRAY—ALPHA)

INDIVIDUAL BODY WEIGHT DATA

A DERMAL SENSITIZATION STUDY IN GUINEA PIGS

G8143G8G81G8G8G81G8G8G8152/M0282F282/82F282/82F2/F9/F
Animal No./Sex

Test
Group

SLI LITNT: IOL./:5.6..7DEPARTMENT OF STATE

329Annex 56-B

(29)

PAGE 2

69078576175834852497581511 -- -- ------ -- -- -- -- --
Day 26

hallenge were conclusive.

Body Weights

37898384363913536356358378 4314304153936363436536376
(SPRAY—ALPHA)

INDIVIDUAL BODY WEIGHT DATA

A DERMAL SENSITIZATION STUDY IN GUINEA PIGS
x

G8153/M1/5G861M7/M/8GF8/F83FG8158/M/M0/M/M/M82F282/F2F9/F

Animal No./Se

GroupControl Control
Challenge Rechallenge
A rechallenge control group was maintained on this study, but was not ut
ilized since the results from c
SLI LITNT: IOL./:59.6..7DEPARTMENT OF STATE a

330 Annex 56-B

(30)

SLI Study No. 3596.7

APPENDIX D

HCA Historical Control Data

331Annex 56-B

(31)

SLI Study No. 3596.7

SPRINGBORN LABORATORIES, INC.

MODIFIED BUEHLER HISTORICAL CONTROL DATA
USING -HEXYLCINNAMALDEHYDE
(SLI Study No. 999.171)

1. OBJECTIVE
This study was performed to assess the dermal sensitizat ion potential of -

Hexylcinnamaldehyde (HCA) when administered by multiple topical applications.
This study may be used to provide information on the ability of the test
system to
detect potential contact sensitizers and to update the historical posit
e control of
the testing facility. The protocol was signed by the Study Director on

February 6, 2002 (GLP initiation date). The in -life phase of the study was
initiated with test article administration on March 13, 2002, and conclu
ed with
final scoring on April 12, 2002.

2. TEST ARTICLE

The test article was received from the manufacturer, TCI America, and id
entified

as follows:

SLI Assigned
Supplier’s Assigned Physical Receipt Expiration
ID SLI ID Description Date Date
HCA S01.008.N Clear yellow 08/21/01 08/21/03
Lot No.: GF01 liquid

The bulk compound was stored desiccated, protected from light, at room
temperature. The manufacturer provided a Certificate of Analysis for the test

article which is presented as Attachment 1 of this Appendix.

The HCA was mixed with ethanol or acetone to produce the appropriate
concentrations for dose administration. For the sensitization study, the
test

article concentrations utilized were 5% w/v in ethanol (induction) and 1% and
2.5% w/v in acetone (challenge).

332 Annex 56-B

(32)

SLI Study No. 3596.7

3. EXPERIMENTAL PROCEDURES [1]

Young adult Hartley-derived albino guinea pigs were received on March 7, 2002,
from Hilltop Lab Animals, Inc., Scottdale, PA. The guinea pigs were uniquely
identified by ear tag, individually housed in suspended stainless steel cages and
received Purina Certified Guinea Pig Chow #5026 and water purified by rev
erse
osmosis ad libitum. The animals were acclimated for a minimum of 5 days
prior

to experimental initiation. The male guinea pigs were approximately 7 weeks of
age and weighed 370-463 g; the female guinea pigs were approximately 8 weeks
of age and weighed 336-396 g on the day prior to Induction I dosing.

On the day prior to the first induction dose administration (day -1), the hair was

removed from the left side of the twenty test animals. On the following day, 0.3
mL of 5% w/v HCA in ethanol was placed on a Hilltop chamber and applied to the
clipped area of each animal=s back. The trunk of each animal was wrapped with
elastic wrap which was secured with adhesive tape to prev ent removal of the
chamber. Approximately six hours after chamber application, the binding

materials were removed. The test sites were wiped with gauze moistened w
ith
deionized water, followed by dry gauze, to remove test article residue. The test
sites were graded for irritation at approximately 24 and 48 hours following
chamber application using the Dermal Grading System. The induction proce
dure

was repeated on study day 7 and on study day 14 so that a total of three
induction exposures were made to the animals.

On the day prior to challenge dose administration, the hair was removed from the
right side of the twenty test and ten challenge control animals. On the
following

day (day 28), 0.3 mL of 1% and 2.5% w/v HCA in acetone was placed on a 25
mm Hi lltop chamber and applied to the clipped area of each animal =s back.
Wrapping, unwrapping and rinsing procedures were the same as those utili
zed
for the induction phase. The test sites were graded for irritation at approximately
24 and 48 hours following chamber removal.

Any unusual observations and/or mortality were recorded. Body weights we
re
recorded for the test, challenge control and rechallenge control animals on the
day prior to first induction (day -1) and for the test and challenge control an imals
on the day prior to challenge dosing. All sensitization study animals w
ere

euthanized by carbon dioxide inhalation following each animal's final sco
ring
interval. Gross necropsy examinations were not required for these animal
s.

Note: The temperature and relative humidity of the animal room [64- 75°F (18-
24°C)] exceeded the preferred ranges [63- 73°F (17-23°C) and 30- 70%] during

333Annex 56-B

(33)

SLI Study No. 3596.7

this study. These occurrences were considered to have had no adverse eff
ect
on the outcome of this study.

4. RESULTS

Individual Data: Tables 1-2

Following challenge with 2.5% w/v HCA in acetone, dermal scores of 1 were

noted in 8/20 test animals at the 24- hour scoring interval. At the 48-hour scoring
interval, dermal scores of 1 were noted in 4/20 test animals. Dermal re
ctions in
the remaining test and challenge control animals were limited to scores of 0 to .
Group mean dermal scores were noted to be higher in the test animals as

compared with the challenge control animals.

Following challenge with 1% w/v HCA in acetone, dermal scores of 1 were n
oted
in 5/20 test animals at the 24- hour scoring interval. At the 48- hour scoring
interval, dermal scores of 1 were noted in 2/20 test animals. Dermal re
ctions in
the remaining test and challenge control animals were limited to scores
f 0 to  .

Group mean dermal scores were noted to be higher in the test anima ls as
compared with the challenge control animals.

5. CONCLUSION

The results of this -Hexylcinnamaldehyde positive control study demonstrated
that a valid test was performed and indicated that the test design would
detect
potential contact sensitizers. Based on the results of this study, -

Hexylcinnamaldehyde is considered to be a contact sensitizer in guinea p
igs.

6. REFERENCE

1. E.V. Buehler, Occlusive Patch Method for Skin Sensitization in Guinea Pigs:
The Buehler Method, Fd. Chem. Toxic., Vol. 32, No. 2, pp. 97-101, 1994.

334 Annex 56-B

(34)
SLI Study No. 3596.7

PAGE 1

- (BK), DES

-2, DES
48-1, DES -1, DE-1, DES -1,-1S, DE-1, DES -1, -1S-1S, DES
-1, DES -1, DES
-1, BLA-1,EBC1,A-, ESS -2, ES
-2, B-L-1S, 3L-1, B3 3 -1, -1, BL-1A,-1S, 3LADE -1, BL-2A, BL-2A, BL-1A, SL-1, DES
ED ED ED - ED - - ED ED ED ED ED - DESDESED ED ED ED ED
2 2 2 M 2 M M 2 2 1 1 1 M ± ± 2 2 2 2 1

a
5%

-1, DES

Induction 2 Dermal Scores
-1, DES -2 (BK), BLA3, DES -1, DES DES

24 Hr SL -1, DES-1, DES -1, DES SL-1, -1, SL-1, ES -1, -1S-4, -1SESDIT
-2, BLA-2,E-2C, BLA-2, SL-1, DES
-2, B-L-1S, BL-2, BLAD -2, -2, BL-2A,-2S, BL-2A, BL-1A, DES, IT-2, BL-2A, BL-2A, SL-2, SL-2, DES
- - 3-
E2 2 2 M 2 M M 2 2 2 2 2 2 1D ED ED ED ED ED D± 2 2 2 2D ED ED 2

-1, DES-1, DES -1, DES

TABLE 1
48 Hr -1, DES -1, BLA-1, BLA -1, BLA
B± ±1,ED± 1SE,B±A1,±-1±,DD±DS± 1D BL±-1S±SD±SDE±DE± 0S ED±DES±ES± 0

-EXYLCINNaMALDEHYDE)
IND(IVIDUAL INDUCTION DATA
5%

-1, DES -4 -1, D-E-1S, DE-1,-1S, DES -1, DES
A DERMAL SENSITIZATION STUDY IN GUINEA PIGS
-1, B-L1A, DE-1S, D-E1S, ,SI-1S, BL1, D-E1TS, IT-1, DES,-1, BL-1A, DES, IT
InductioED EDerED EDcoED ED ED ED ED ED ED ED DES,TIDDES,DES,T ED DESIT
1 1 ± 2 ± 1 1 1 1 2 ± 1 ± 1 ± ± 1 1 ± 0

Sex
G578G57G88/859/90/951M92/953M94/955/96/M54F95/956F97/988F99/009F01/029/03/F
Animal No./

Groupst

a ThNotes: See Appendix B for definition of codes. BK = black.

SLSTUDY NO.: 999.171OL

335Annex 56-B

SLI Study No. 3596.7 (35)

PAGE 2

-2 -1 -1

-1 -1, SL-1, SL -1, SL
-1 -4 -1 -2, DES -1 -4 -2 -4 -4

-1 -2, BL-1A, SL-1, SL--2S, SL-1, BL--1,-2, SL-1-1 -2, SL-1, SL-1, SL-1, SL-1, BLA
48 H2 2 2 2 2 2 2 2 2 2 1 1 2 1D ED ED ED ED E± 2 2 2 2 1ED ED

Induction 3 Dermal Scores

TABLE 1

-1 -1, DES
-4, DES-1, DES-2, L2, DESDES -1, DES, ITSL, DES-4S-4S, DES

-EXYLCINNAM24 HrYDE)BL--2, SL--2S, SL-2S, SL-2, S-2-1S-2S, SL-1, DE--2, SL-2, SL-2, SL-1, BLA
IND(IVIDUAL INDUCTION DATA2 2 2 2 2 2 1 1 2 1D ED ED ED ED E± 2 2 2 2 1ED ED

A DERMAL SENSITIZATION STUDY IN GUINEA PIGS

Sex
G57G57G887859/90/951M92/937M94/957/96/M48955/96/957F98/999F00/019F02/03/F
Animal No./

Group
Test aTheNote: See Appendix B for definition of codes.

SLISTUDY NO.: 999.171L

336 Annex 56-B

SLI Study No. 3596.7
(36)

IT ± 0 1 IT 0 ± ± 0 ± 0 ± ±IT 0IT 0 0 0 0
± ± 0 1 0.3
PAGE 1
48 Hr

IT IT IT IT IT IT
1 ± ± 1 ± ± ± 1 ± ± ± 1 ± ± 0 1 ± ± ± ± 0.6

24 Hr

Dermal Scores
± ± 0 1 1 0 ± 1 ± ± 0 ± ± 0 ± 1 0 0 ± ± 0.5

48 Hr

2.5%
TABLE 2
IT -1 IT IT
1 ± ±E1 1 ± ± 1 1 ± ± 1 1 ± ± 1 ± ± ± ± 0.7

-EXYLCINNAMALDEHYDE)
INDIVIDUAL CHALLENGE DATA

A DERMAL SENSITIZATION STUDY IN GUINEA PIGS See Appendix B for definition of codes.

Sex Mean
Animal No./5G5790/M G5794G5G5796/M895/F897/F899/F9G5G5903/F

Test
Group For the purpose of calculation, ± = 0.5.

SLISTUDY NO.: 999.171L aTheNotes:le was acetone.

337Annex 56-B

SLI Study No. 3596.7 (37)

PAGE 2
IT IT
48 Hr0 ± 0 0 0 0 0 0 0 0.1

a
%
1

I0 0T ±T0I0 I0 0 0IT0I0 0.1
24 Hr

Dermal Scores

48 Hr0 0 0 0 0 0 0 0I0 0.0

2.5%
TABLE 2

HEXYLCINNAMALDEHYDE)
 24 Hr0 0 0I0 I0 0 0 0 I0 0.0
INDIVIDUAL CHALLENGE DATA

See Appendix B for definition of codes.
A DERMAL SENSITIZATION STUDY IN GUINEA PIGS

Sex Mean
G5797/M G5801/M04/F G5908/FF
Animal No./

Group For the purpose of calculation, ± = 0.5.

SLSTUDY NO.: 999.171OLlenge aTheNotes:le was acetone.

338 Annex 56-B

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SLI Study No. 3596.7

ATTACHMENT 1

Certificate of Analysis
(Provided by the Manufacturer)

339Annex 56-B

SLIIStudyNoo.359467777
Doow Study No.0211090 (39)

340 Annex 56-B

(40)

SLI Study No. 3596.7

APPENDIX E

SLI Personnel Responsibilities

341Annex 56-B

(41)

SLI Study No. 3596.7

SLI PERSONNEL RESPONSIBILITIES

Kimberly L. Bonnette, M.S., LATG Study Director/Director, Acute

Toxicology

Dawn D. Rodabaugh, B.S. Alternate Contact/Associate
Toxicologist

Robert C. Springborn, Ph.D. Chairman, President and CEO

Malcolm Blair, Ph.D. Senior Vice President, Managing
Director Emeritus

Joseph C. Siglin, Ph.D., DABT Vice President, Managing Director

Jason W. Smedley, B.S. Assistant Toxicologist

Pamela S. Smith, ALAT Primary Technician/Supervisor of
Acute Toxicology

Delores P. Knippen Supervisor of Pharmacy

Steven H. Magness, B.S., LATG Senior Supervisor of Gross and Fetal

Pathology

Anita M. Bosau, RQAP-GLP Senior Director, Compliance
Assurance

Deanna M. Talerico, RQAP-GLP Senior Supervisor of Quality Assurance

J. Dale Thurman, D.V.M., M.S., Senior Director, Pathology
DACVP

Kathy M. Gasser Supervisor of Archives

342 Annex 56-B

A PRIMARY EYE IRRITATION STUDY IN RABBITS

WITH SPRAY--ALPHA

FINAL REPORT

OPPTS Guideline

870.2400

Author

Kimberly L. Bonnette, M.S., LATG

Study Completed on

August 28, 2002

Performing Laboratory

Springborn Laboratories, Inc. (SLI)
Ohio Research Center

640 North Elizabeth Street
Spencerville, Ohio 45887

SLI Study No.

3596.5

Submitted to

INL/A
U.S. Department of State
2201 C St. NW SA-4

Washington, DC 20520

Page 1 of 25

343Annex 56-B

SLI Study No. 3596.5 (2)

1. STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS
No claim of confidentiality is made for any information contained in this study on

the basis of its falling within the scope of FIFRA §10(d)(1)(A), (B), or (C).

Company:

Company Agent: Date:

Title Signature

344 Annex 56-B

345Annex 56-B

346 Annex 56-B

SLI Study No. 3596.5 (5)