Revision 1 

Study Title 

Acute Oral Toxicity – Fixed Dose Procedure in Rats 

TEST GUIDELINES: U.S. EPA Health Effects Test Guidelines, OPPTS 870.1100 

(2002) 

OECD Guidelines for the Testing of Chemicals, Test No. 420 

(2001)

STUDY AMENDED 

ON: 

 June 30, 2009 

AUTHOR: 

STUDY COMPLETED 

ON: 

March 12, 2009 

PERFORMING 

LABORATORY: 

LABORATORY 

PROJECT ID: 

WORK REQUEST 

NUMBER: 

SERVICE CODE 

NUMBER: 

SPONSOR: 

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Acute Oral Toxicity – Fixed Dose Procedure Revision 1 

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 current 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, 20081; 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 and 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: •

•

Number:

Composition: 

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.............................................................................................................................. 12

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 Revision 1 

 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 pharmacologic effects, or abnorm al behavior. No 

gross abnormalities were noted for any of the animals 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 study, 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, nulliparous 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 contaminants 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 the 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 pharmacologic effects, or abnorm al behavior. No 

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gross abnormalities were noted for any of the animals 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 study, classification is not required. 

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TABLE 1: INDIVIDUAL BODY WEIGHTS AND DOSES 

Body Weight (g) Dose1

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