Revision 1

Page 1 of 19 Study Title
Pri mary Skin Irritation in Rabbits

T EST G UIDELINES : U.S. EPA Health Effects Test Guidelines, OPPTS 870.2500 (1998) OECD Guidelines for Testing of Chemicals, Test No. 404 (2002)
S TUDY A MENDED ON : June 30, 2009

A UTHOR :
S TUDY C OMPLETED ON : March 19, 2009

P ERFORMING L ABORATORY :

L ABORATORY

P ROJECT ID:
W ORK R EQUEST N UMBER :
S ERVICE C ODE N UMBER :
S PONSOR :Primary Skin Irritation in Rabbits Re vision 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 curre nt OECD Good Laboratory Practices, except for the item documented below. The item listed do es not impact the validity of the study.
The test substance was ch aracterized by the S ponsor pr ior 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.

Page 2 of 19Primary Skin Irritation in Rabbits Re vision 1



### QUALITY ASSURANCE STATEMENT

The Quality Assurance Unit has reviewed this final study report to assure the report accur ately describes the methods 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 Apr 23, 2008 1 ; Mar 4, 2009 Apr 23, 2008; Mar 4, 2009 In-process inspection: 24 hour scoring Jan 22, 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 19, 2009 Mar 19, 2009

1 The protocol used for this study was reviewed by the Quality Assurance group on this date.
Page 3 of 19Primary Skin Irritation in Rabbits Re vision 1



### CERTIFICATION

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

Page 4 of 19Primary Skin Irritation in Rabbits Re vision 1



### 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 i n Ap pendix A of this report. The Stud y Information page was updated to reflect the revise d com position. The raw data noteboo k 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.

Page 5 of 19Primary Skin Irritation in Rabbits Re vision 1

Page 6 of 19 

### STUDY INFORMATION

Substance Tested: 

### •

•
Nu mber:
Co mposition:

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 21, 2009 / January 30, 2009Primary Skin Irritation in Rabbits Re vision 1

Page 7 of 19 

### TABLE OF CONTENTS
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT

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

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

............................................................................................................................... .............................. 4
STUDY INFORMATION

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

............................................................................................................................... .................. 7
PRIMARY SKIN IRRITATION IN RABBITS

.................................................................................................. 8
1. PURPOSE

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

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

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

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

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

............................................................................................................................... ... 13
7. TEST GUIDELINES

............................................................................................................................... .. 13
8. QUALITY ASSURANCE

.......................................................................................................................... 13
9. DEVIATION FROM FINAL PROTOCOL

................................................................................................. 13
10. FINAL REPORT AND RECORDS RETENTION

..................................................................................... 13
11. RESULTS

............................................................................................................................... ................. 13
12. CONCLUSION

............................................................................................................................... .......... 14
TABLE 1: INDIVIDUAL BODY WEIGHTS

................................................................................................................... 15
TABLE 2: INDIVIDUAL SKIN IRRITATION SCORES

................................................................................................ 16
TABLE 3: SUMMARY OF MEAN SCORES FOR DERMAL RESPONSES ..............................................................17
TABLE 4: PRIMARY SKIN IRRITATION SCORING SYSTEM

.................................................................................. 18
APPENDIX A: CERTIFICATE OF ANALYSIS

............................................................................................................. 19Primary Skin Irritation in Rabbits Re vision 1

Page 8 of 19 

### PRIMARY SKIN IRRITATION IN RABBITS

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 21, 2009
EXPERIMENTAL COMPLETION DATE: January 30, 2009
STUDY COMPLETION DATE: March 19, 2009
NOTEBOOK NO.:

1. PURPOSE To provide i nformation o n the skin irritation lik ely to arise fro m a single topical exposure to

2. SUMMARY A prim ary skin irritation test was conducted wi th rabbits to determ ine the potential for to produce irritation after a single topical application. At the request of the S ponsor, the study was c onducted in a stepwise manner using a single patch applied initially to one rabbit for 4-hours. Five-t enths of a milliliter of the test substance was applied to the skin of one healthy rabb it for 4- hours. The dose site was evaluated for dermalPrimary Skin Irritation in Rabbits Re vision 1

Page 9 of 19 irritation by the method of Draize et al. 1 (see Table 4) immediately following patch removal, and at 30-60 m inutes and 24, 48, and 72 hours after patch rem oval. Since there was no dermal irritation observed in this animal, the test was co mpleted on two additional animals, as described above. There was no dermal irritation noted for any treated dose site during the study. In accordance with the provisions of D irective 67/ 548/EEC, classification by the dermal route i s not required based on the results of this study.
According to the Globally Harmonized System (GHS) of classification and labeling of chem icals and under the conditions of this stud y, classification is not required.

3. MATERIALS A. Test Substance The test sub stance, identifi ed as was received on January 8 , 200 9 and was further identified with Reference Nu mber The test su bstance was stored at room temperature. The sample was applied as r eceived. Documentation of the methods of synthesis, fabrication, or derivation of the test substance is retained by the Sponsor.
The following inform ation related to the characterization of the test substanc e was provided b y 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 rep ort, unless otherwise specified by the Sponsor. After th is time period all remaining test substance will be properly disposed. Records of samp le disposition are maintained by
B. Animals

3.B.1 Number of Animals: 3 3.B.2 Sex: Female, nulliparous and nonpregnant 3.B.3 Species/Strain: Rabbit/New Zealand albino. 3.B.4 Age: Young adult. 3.B.5 Source: Received from Robinson Services, Inc. Clemmons, NC on January 14 and 21, 2009.

1 D raize, J.H., Woodward, G. an d Cal very, H.O. Methods f or th e st udy o f irr itation and t oxicity o f su bstances applied topically to the skin and mucous membranes. J. Pharmacol. Exp. Ther. 1944; 82:377-390.Primary Skin Irritation in Rabbits Re vision 1

Page 10 of 19 4. METHODS A. Husbandry 4.A.1 Housing: The animals were singly housed in s uspended stainless steel caging w ith mesh floors which conform to the si ze recommendations in the most recent Guide for the Care and Use of Laboratory Animals DHEW (NIH) . Enrichment (e.g. toy) was placed in eac h cage. Litter pa per was placed beneath the cage and was changed at least three times per week. 4.A.2 Animal Room Temperature and Re lative Humidity Ranges: 19-21 ° C and 24-60%, respectively. Lo w humidity was n oted duri ng the study. Po rtable humidifiers were used to increase the humidity levels during this time. 4.A.3 Photoperiod: 12 hour light/dark cycle 4.A.4 Acclimation Period: 6 or 7 days 4.A.5 Food: Pelleted Purina Rabbit Chow #5326 4.A.6 Water: Filtered tap water w as supplied ad libitum by an autom atic water dispensing system. 4.A.7 Contaminants: There were no known contamin ants reasonably expected to be found in the food or water at leve ls 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 and identification and sex of the animal. 4.B.2 Animal: A number was allocated to each rabbit on receipt and a stainless steel ear tag bearing this number was attached to the animal. This number, together with a sequential animal number assigned to study constituted unique identification. 5. PROCEDURE A. Preparation and Selection of Animals Approximately 24 h ours p rior to application, a group of animals was prepared b y clippin g the dorsal area and the trunk. On the day of d osing, but prior to application, t he anim als were examined for health and t he sk in checked for any abnormalities. Three heal thy nai ve ani mals (not previously tested) without pre-existing skin irritation were selected for test. Initially, only one rabbit was placed on test. Sin ce there was no dermal irritation observed in this animal, the test was completed with two additional rabbits.

B. Application of Test Substance Five-tenths of a milliliter of the test su bstance was applied to one 6-cm 2 intact dose site on each animal and covered with a 1-inch x 1-inch, 4- ply gauze pad. The pad and entire trunk of each animal were then wrapped with semi-occlusive 3- inch Micropore tape to avoid dislocation of the pad. Elizabethan collars were plac ed on each rabb it and the y were returned to their designate d cages. No other substances were tested on these rabbits.Primary Skin Irritation in Rabbits Re vision 1

Page 11 of 19 After 4 hours of exp osure to the test su bstance, the pads and coll ars were removed and the test sites were gently cleansed of any residual test substance.

C. Evaluation of Test Sites/Classification Schemes Individual dose site s w ere scor ed ac cording to the Draiz e scoring sy stem 1 (see T able 4) immediately following pat ch rem oval and at a pproximately 30-60 minutes and 24, 48, an d 72 hours after patch removal. European Economic Community (EEC) Mean values for each lesion (erythema and edema) were calculated for each animal separately from numerical scores obtained at the 24-, 48-, and 72-hour observations. The results were interpreted according to 67/548/EEC relative to the general classification and labeling requirements for dangerous substances.
Corrosive
The test substance will be considered to be "CORROSIVE" and will require the symbol "C" and the indication of danger "CORROSIVE" if it produ ces full thickness destructi on of the skin tissue on at least 1 animal during the skin irritation test. Risk phrases will be assigned in accord ance with the following criteria.
R35 "CAUSES SEVERE BURNS" If, when applied to healthy intact animal skin, full thickness destruction of skin tissue occurs as a result of up to 3 minutes exposure or if this result can be predicted.
R34 "CAUSES BURNS" If, when applied to healthy intact animal skin, full thickness destruction of skin tissue occurs as a result of up to 4 hours exposure or if this can be predicted.
Irritant The test substance will be classified as "IRRITANT" and will require the symbol "Xi" and the indication of danger "IRRITANT" in accordance with the criteria given below. In addition, the following risk (R) phrase will be assigned to substances, if appropriate, according to the criteria indicated below:
R38 "IRRITATING TO SKIN" If, when applied to healthy intact animal skin fo r up to 4 hours, significant inflammation is caused and is present 24 hours or more after the end of the exposure period. Inflammation is significant if the mean values of the scores for either er ythema and eschar formation or edema formation corresponds to one or more of the following mean values calculated for each animal separately and has been observed in 2 or more animals:

1 D raize, J.H., Woodward, G. an d Cal very, H.O. Methods f or th e st udy o f irr itation and t oxicity o f su bstances applied topically to the skin and mucous membranes. J. Pharmacol. Exp. Ther. 1944; 82:377-390.Primary Skin Irritation in Rabbits Re vision 1

Page 12 of 19 - Erythema and eschar formation 2.0 or more - Edema 2.0 or more All scores at each of the reading times (24, 48, a nd 72 hours) for an effect should be used in calculating the respective mean values. An R38 "IRRITATING TO SKIN" phrase should also be assigned if: The infla mmation persists in at least 2 ani mals at t he end of the observation time. Partic ular effects such as hyperplasia, scaling, discoloration, fissures, scabs, and alopecia should be t aken into account. Globally Harmonized System (GHS) Mean values for each l esion (ery thema and ede ma) were cal culated for ea ch a nimal s eparately from nu merical score s obtained at the 24-, 48-, and 72-h our o bservations. The results were interpreted according to United Nations Economic Commission for Europe, Globally Harmonized System of Classification and Labeling of Chemicals (GHS), Part 3 - Health Hazards. Skin Corrosive Category and Subcategories Corrosive Corrosive subcategories Corrosive in ≥ 1 of 3 animals (Category 1) Exposure Observation 1A ≤ 3 minutes ≤ 1 hour 1B > 3 minutes ≤ 1 hour ≤ 14 days corrosive

1C > 1 hour ≤ 4 hours ≤ 14 days Skin Irritation Categories Categories Criteria Irritant (Category 2) (1) Mean value of ≥ 2.3 < 4.0 for erythema/eschar or for edema in at least 2 of 3 tested animals from scores at 24, 48, and 72 hours after patch removal, or if reactions are delayed, from scores on 3 consecutive days after the onset of skin reactions; or (2) Inflammation that persists to the end of the observation period normally 14 days in at least 2 animals, particularly taking into account alopecia (limited area), hyperkeratosis, hyperp lasia, and scaling; or (3) In some cases where there is pron ounced variability of response among animals, with very definite positive effects related to chemical exposure in a single animal but less than the criteria above.

Mild Irritant (Category 3) Mean value of ≥ 1.5 < 2.3 for erythema/eschar or for edema from scores in at least 2 of 3 tested animals from scores at 24, 48, and 72 hours or, if reactions are delayed, from scores on 3 consecutive days after the onset of skin reactions (when not included in the irritant category above). (Reference: United Nations Econom ic Commission for Europe, Globally Harmonized System of Classification and Labeling of Chemicals (GHS), Part 3 – Health Hazards).

D. Body Weights Individual body weights of the ani mals were record ed prior to test substance ap plication (initial) and again on the last day of individual scoring.Primary Skin Irritation in Rabbits Re vision 1

Page 13 of 19 E. Clinical Observations The animals were observed for signs of gross toxi city and behavioral changes at least once daily during the test period. Observations included gro ss evaluation of skin and fur, e yes and mucous membranes, respiratory, circulatory, autonomic and central n ervous s ystems, so matomotor activity and behavior pattern. Particu lar atte ntion was directed to observation of tremors, convulsions, salivation, diarrhea, and coma.
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 ar e based on the following testing guidelines:
U.S. EPA Health Effects Test Guidelines, OPPTS 870.2500 (1998) OECD Guidelines for Testing of Chemicals, Test No. 404 (2002)

8. QUALITY ASSURANCE The final report was audited for agreement with th e raw data records and for compliance with the protocol and s Standard Operating Procedures. Dates of inspections and audits performed during the study, and the dates of reporting of the inspection and audit findings to the Study Director and Facility Management are presen ted in the Quality Assurance Statement.

9. DEVIATION FROM FINAL PROTOCOL The protocol requires if the pH of the test subs tance was not supplied b y the Sponsor it is to be determined by the testing laboratory prior to st udy initiation. Due to a scheduling error the pH was not determined. This deviation had no impact on the outcome of the study.

10. FINAL REPORT AND RECORDS RETENTION A copy of t he signed report, copies of all raw data generated at and the original signed protocol, will be maintained in the archives. will maintain these reco rds for a period of at least five years.
Laboratory-specific or site-specific raw data, su ch as personnel files and equipment record s 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 are presented i n Table 1. Individual skin irritation scores are presented in Table 2. A summary of mean score s calcula ted in accordance with EEC is presented in Table 3. The Draize Primary Skin Irritation Scoring System is presented in Table 4.Primary Skin Irritation in Rabbits Re vision 1

Page 14 of 19 All animals appeared active and healthy and gained body weight during the study. There were no signs of gross toxicity, adverse pharmac ologic effects, or abnormal behavior. There was no dermal irritation noted for any treated dose site during the study.

12. CONCLUSION In accordance with the provisions of D irective 67/ 548/EEC, classification by the dermal route i s not required based on the results of this study.
According to the Globally Harmonized System (GHS) of classification and labeling of chem icals and under the conditions of this stud y, classification is not required.Primary Skin Irritation in Rabbits Re vision 1

Page 15 of 19 

### TABLE 1: INDIVIDUAL BODY WEIGHTS

Body Weight (g) Animal No. Sex Initial Terminal 3501 F 2284 2536
3502 F 2059 2225
3503 F 2320 2431Primary Skin Irritation in Rabbits Re vision 1

Page 16 of 19 

### TABLE 2: INDIVIDUAL SK IN IRRITATION SCORES

ERYTHEMA/EDEMA

Time After Patch Removal Animal No. Sex Immediately after patch

removal 30-60 mins 24 hrs 48 hrs 72 hrs

3501 F 0/0 0/0 0/0 0/0 0/0 3502 F 0/0 0/0 0/0 0/0 0/0 3503 F 0/0 0/0 0/0 0/0 0/0Primary Skin Irritation in Rabbits Re vision 1

Page 17 of 19 

### TABLE 3: SUMMARY OF MEAN SCORES FOR DERMAL RESPONSES

RABBIT NUMBER ERYTHEMA a EDEMA a 3501 0.00 0.00 3502 0.00 0.00 3503 0.00 0.00 a Calculated from the 24-, 48-, and 72-hour dermal responses (EEC).Primary Skin Irritation in Rabbits Re vision 1

Page 18 of 19 

### TABLE 4: PRIMARY SKIN IR RITATION SCORING SYSTEM

Evaluation of Skin Reactions Value

Erythema and eschar formation:
No erythema.................................................................................................................... ...... 0
Very slight erythema (barely perceptible) ............................................................................ 1
Well-defined erythema.......................................................................................................... 2
Moderate to severe erythema ................................................................................................ 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) ..................... 4

Edema formation:

No edema ....................................................................................................................... ....... 0
Very slight edema (barely perceptible)................................................................................. 1
Slight edema (edges of area well defined by definite raising).............................................. 2
Moderate edema (raised appr oximately 1 millimeter).......................................................... 3 Severe edema (raised more than 1 millimeter and extending beyond the area of exposure) 4Primary Skin Irritation in Rabbits Re vision 1

Page 19 of 19 

### APPENDIX A: CERTIFICATE OF ANALYSIS