Revision 1

Study Title Acute Oral Toxicity – Fixed Dose Procedure in Rats

T EST G UIDELINES : U.S. EPA Health Effects Test Guidelines, OPPTS 870.1100 (2002) OECD Guidelines for the Testing of Chemicals, Test No. 420 (2001)

S TUDY A MENDED ON : June 30, 2009

A UTHOR :
S TUDY C OMPLETED ON : March 12, 2009

P ERFORMING L ABORATORY :

L ABORATORY

P ROJECT ID:

W ORK R EQUEST N UMBER :
S ERVICE C ODE N UMBER :
S PONSOR :

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### GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT This study was conducted in compliance with U. S. EPA TSCA (40 CFR part 792) Good Laboratory Practice Standards, which are compatible with cu rrent OECD Good Laboratory Practices, except for the item documented below. The item listed does not impact the validity of the study. The test substance was characterized by the sponsor prior to the initiation of this study. Although the characterization was not performed under Good Laboratory Practice Standards, the accuracy of the data is considered sufficient for the purposes of this study.

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### QUALITY ASSURANCE STATEMENT

The Quality Assurance Unit has reviewed this final study report to assure the report accurately describes the m ethods and standard operating procedures, and that the reported results accurately reflect the raw data of the study.

QA activities for this study:

QA Activity Date Conducted Date Findings Reported To Study Director And Management Protocol review Aug 21, 2008 1 ; Mar 4, 2009 Aug 21, 2008; Mar 4, 2009 In-process inspection: Day 7 body weight for Animal #3101 Feb 4, 2009 Mar 4, 2009

Raw data audit Mar 4, 2009 Mar 4, 2009 Draft report review Mar 4, 2009 Mar 4, 2009 Final report review Mar 12, 2009 Mar 12, 2009

1 The protocol used for this study was reviewed by the Quality Assurance group on this date.

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### CERTIFICATION

I, the undersigned, declare that the m ethods, results a nd data contained in this report faithfully reflect the procedures used and raw data collected during the study.

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### AMENDMENTS TO FINAL REPORT

Report No.:

Amendment No.: 1
Report Sections : Study Information page, Appendix A and Notebook pages The Certificate of Analy sis was updated and can be found in Appendix A of this report. The Study Information page was updated to reflect the revi sed com position. The raw data notebook pages were updated to include this new information.

Reason: These changes were made at the request of the Sponsor.

Amendment No.: 2
Report Sections : All references to the Study Director Due to his unavailability as the Study Director of this study.

Reason: This change was required in order to finalize this amended report.

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### STUDY INFORMATION

Substance Tested: 

### •

•
Num ber:

Com position:

Purity: >99% (wt.%) Physical Characteristics: Colorless liquid Stability: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed. Study Initiated/Completed: January 13, 2009 / (see report cover page)

Experimental Start/Termination: January 28, 2009 / February 13, 2009

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### TABLE OF CONTENTS

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT

....................................................................... 2
QUALITY ASSURANCE STATEMENT

.................................................................................................................. 3
CERTIFICATION

............................................................................................................................... .......................4
AMENDMENTS TO FINAL REPORT

..................................................................................................................... 5
STUDY INFORMATION

............................................................................................................................... ............6
TABLE OF CONTENTS

............................................................................................................................... ............7
ACUTE ORAL TOXICITY – FIXED DOSE PROCEDURE IN RATS .....................................................8
1. PURPOSE

............................................................................................................................... ............... 8
2. SUMMARY

............................................................................................................................... .............. 8
3. MATERIALS

............................................................................................................................... ............ 9
4. METHODS

............................................................................................................................... ............. 10
5. PROCEDURE

............................................................................................................................... ........ 10
6. STUDY CONDUCT

............................................................................................................................... 12
7. TEST GUIDELINES

.............................................................................................................................. 1 2
8. QUALITY ASSURANCE

....................................................................................................................... 12
9. DEVIATIONS FROM FINAL PROTOCOL

............................................................................................ 12
10. FINAL REPORT AND RECORDS TO BE MAINTAINED

..................................................................... 12
11. RESULTS

............................................................................................................................... .............. 12
12. CONCLUSION

............................................................................................................................... ....... 13
TABLE 1: INDIVIDUAL BODY WEIGHTS AND DOSES

.................................................................................... 14
TABLE 2: INDIVIDUAL CLINICAL OBSERVATIONS

......................................................................................... 15
TABLE 3: INDIVIDUAL NECROPSY OBSERVATIONS

..................................................................................... 16
APPENDIX A: CERTIFICATE OF ANALYSIS

...................................................................................................... 17

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### ACUTE ORAL TOXICITY – FIXED DOSE PROCEDURE IN RATS

PROTOCOL NO.:

AGENCY: EPA (TSCA)

STUDY NUMBER:

SPONSOR:

SPONSOR REPRESENTATIVE:

SPONSOR STUDY MONITOR:

TEST SUBSTANCE IDENTIFICATION:

DATE RECEIVED: January 8, 2009

REFERENCE NO.:

STUDY INITIATION DATE: January 13, 2009

EXPERIMENTAL INITIATION DATE: January 28, 2009

EXPERIMENTAL COMPLETION DATE: February 13, 2009

STUDY COMPLETION DATE: March 12, 2009

NOTEBOOK NO.:

1. PURPOSE To provide information on health hazards likel y to arise from a short-term exposure to by the oral route.
2. SUMMARY An acute oral toxicity test (Fixed Dose Proce dure) was conducted with rats to determine the potential for to produce toxicity from a single dose via the oral route.

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An initial limit dose of 5,000 m g/kg was administered to one healthy female rat by oral gavage. Due to the absence of mortality in this animal, four additional fem ales received a dose level of 5,000 m g/kg, sim ultaneously. Fem ales were select ed for the test because they are frequently more sensitive to the toxicity of test com pounds than m ales. All anim als were observed for mortality, signs of gross toxicity, and behavioral changes at least once daily for 14 day s after dosing. Body weights were recorded prior to adm inistration and again on Day s 7 and 14 (termination) following dosing. Necropsies were performed on all animals at terminal sacrifice. All anim als survived, gained body weight, and appeared active and healthy during the study. There were no signs of gross toxicity , adverse pha rmacologic effects, or abnorm al behavior. No gross abnormalities were noted for any of the anim als when necropsied at the conclusion of the 14-day observation period. Under the conditions of this study, the acute oral LD 50 of the test substance is greater than 5,000 mg/kg of body weight in female rats. In accordance with the provisions of Directive 67/548/EEC, classification is not required based on the results of this study. According to the Globally Harmonized System (GHS) of classification and labeling of chemicals and under the conditions of this st udy, classification is not required.
3. MATERIALS A. Test Substance The test substance, identified as w as received on January 8, 2009 and was further identified with Reference Number The test substance was stored at room temperature. The sam ple was adm inistered as received. Docum entation of the methods of synthesis, fabrication, or derivation of the test substance is retained by the Sponsor. The following inform ation related to the character ization of the test substance was provided by the Sponsor (see Appendix A): Physical description: Colorless liquid Solubility: Not provided. Stability: The test substance was expected to be stable for the duration of testing Expiration Date: Not applicable
The test substance will be retained for at least 3 months following submission of the final report, unless otherwise specified by the Sponsor. After this time period all remaining test substance will be properly disposed. Records of sample disposition are maintained by

B. Animals

3.B.1 Number of Animals: 5 3.B.2 Sex: Female, nullipar ous and non-pregnant.

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3.B.3 Species/Strain: Rat/Sprague-Dawley derived, albino. 3.B.4 Age/Body weight: Young adult (9-10 weeks)/181-203 grams at experimental start. 3.B.5 Source: Received from Ace Anim als, Inc., Boy ertown, PA on January 13 and 20, 2009.
4. METHODS

A. Husbandry

4.A.1 Housing: The anim als were singly housed in suspended stainless steel caging with mesh floors which conform to the size recom mendations in the most recent Guide for the Care and Use of Laboratory Animals DHEW (NIH) . Enrichm ent (e.g. nylabone) was placed in each cage. Litte r paper was placed beneath the cage and was changed at least three times per week. 4.A.2 Animal Room Temperature and Relative Hum idity Ranges: 19-21oC and 20-58%, respectively. The hum idity was below th e targeted lower lim it of 30% during the study. A portable hum idifier was used to increase the hum idity levels during this time. 4.A.3 Photoperiod: 12-hour light/dark cycle 4.A.4 Acclimation Period: 10 or 15 days 4.A.5 Food: Purina Rodent Chow #5012 4.A.6 Water: Filtered tap water was supplied ad libitum by an automatic water dispensing system. 4.A.7 Contaminants: There were no known contam inants reasonably expected to be found in the food or water at levels which would have interfered with the results of this study. Analyses of the food and water are conducted regularly and the records are kept on file at

B. Identification

4.B.1 Cage: Each cage was identified with a cage card indicating at least the study number, dose level, identification and sex of the animal. 4.B.2 Animal: A num ber was allocated to each rat on receipt and a stainless steel ear tag bearing this number was attached to the rat. This number, together with a sequential animal number assigned to study number constituted unique identification.

5. PROCEDURE

A. Selection of Animals Prior to each dosing, experimentally naive rats were fasted overnight by removing the feed from their cages. During the fasting period, the rats we re examined for health and weighed (initial). Five healthy naive female rats were selected for test.

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B. Dose Calculations Individual doses were calculated based on the initial body weights, taking into account the specific gravity (determined by of the test substance.

C. Dosing The test substance was adm inistered using a stai nless steel ball-tipped gavage needle attached to an appropriate sy ringe. Following adm inistration, each animal was returned to its designated cage. Feed was replaced approximately 3-4 hours after dosing.
Individual animals were dosed as follows: Limit Test
Dosing Sequence Animal No. Dose Level (mg/kg) Short-Term Outcome Long-Term Outcome
1 3101 S S 2* 3102 S S 3* 3103 S S 4* 3104 S S 5* 3105 5,000

S S * - Animals were dosed simultaneously S – Survival Short-term result = animal response within 48 hours of dosing Long-term result = animal response at the end of the 14-day observation period

D. Body Weights Individual body weights of the anim als were recorded prior to test substance administration (initial) and again on Days 7 and 14 (termination) following dosing.

E. Clinical Observations The animals were observed for m ortality, signs of gross toxicity, and behavioral changes within 30 minutes and during the first several hours post-dosing and at least once daily thereafter for 14 days after dosing. Observations included gross evaluation of skin and fur, ey es and m ucous membranes, respiratory , circulatory , autonom ic and central nervous systems, somatomotor activity and behavior pattern. Pa rticular attention was directed to observation of trem ors, convulsions, salivation, diarrhea, and coma.

F. Necropsy

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All rats were euthanized via CO 2 inhalation at the end of the 14-day observation period. Gross necropsies were perform ed on all anim als. Tissu es and organs of the thoracic and abdom inal cavities were examined.

6. STUDY CONDUCT This study was conducted at The primary scientist for this study was

7. TEST GUIDELINES The procedures as described in the protocol are based on the following testing guidelines: U.S. EPA Health Effects Test Guidelines, OPPTS 870.1100 (2002) OECD Guidelines for the Testing of Chemicals, Test No. 420 (2001)

8. QUALITY ASSURANCE The final report was audited for agreem ent with th e raw data records and for com pliance with the protocol, s Standard Operating Procedures and appropriate Good Laboratory Practice Standards. Dates of in spections and audits performed during the study and the dates of reporting of the inspection and a udit findings to the Study Director and Facility Management are presented in the Quality Assurance Statement.

9. DEVIATIONS FROM FINAL PROTOCOL None.

10. FINAL REPORT AND RECORDS TO BE MAINTAINED A copy of the signed report, the protocol and all raw data generated at and the original signed protocol, will be maintained in the archives. will maintain these records for a period of at least five y ears. After this time the Sponsor will be offered the opportun ity to take possession of the records or request continued archiving at
Laboratory-specific or site-specific raw data, su ch as personnel files and equipm ent records will be retained by the facility where the work was done. The original signed final report and raw data and a copy of the protocol will be retained at

11. RESULTS Individual body weights and doses are presented in Table 1. Individual clinical and necropsy observations are presented in Tables 2 and 3, respectively.
All anim als survived, gained body weight, and appeared active and healthy during the study. There were no signs of gross toxicity , adverse pha rmacologic effects, or abnorm al behavior. No

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gross abnormalities were noted for any of the anim als when necropsied at the conclusion of the 14-day observation period.

12. CONCLUSION Under the conditions of this study , the acute oral LD 50 of is greater than 5,000 milligrams per kilogram of body weight in female rats. In accordance with the provisions of Directive 67/548/EEC, classification is not required based on the results of this study. According to the Globally Harmonized System (GHS) of classification and labeling of chemicals and under the conditions of this st udy, classification is not required.

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### TABLE 1: INDIVIDUAL BODY WEIGHTS AND DOSES

Body Weight (g) Dose 1 Animal No. Sex Dose Level (mg/kg) Initial Day 7 Day 14 mL
3101 F 200 244 261 0.62 3102 F 196 216 258 0.60 3103 F 181 198 246 0.56 3104 F 203 221 266 0.63 3105 F 5,000

194 224 258 0.60

1 The test substance was administered as received. Specific Gravity – 1.623 g/mL.

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### TABLE 2: INDIVIDUAL CLINICAL OBSERVATIONS

Animal Day of Number Findings Occurrence

3101 - 3105 Active and healthy 0-14

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### TABLE 3: INDIVIDUAL NECROPSY OBSERVATIONS

Animal Number Tissue Findings

3101 - 3105 All tissues and organs No gross abnormalities

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### APPENDIX A: CERTIFICATE OF ANALYSIS