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Study Title 

Primary Skin Irritation in Rabbits 

TEST GUIDELINES: U.S. EPA Health Effects Test Guidelines, OPPTS 870.2500 

(1998) 

OECD Guidelines for Testing of Chemicals, Test No. 404 (2002)

STUDY AMENDED 

ON: 

 June 30, 2009 

AUTHOR: 

STUDY COMPLETED 

ON: 

March 19, 2009 

PERFORMING 

LABORATORY: 

LABORATORY 

PROJECT ID: 

WORK REQUEST 

NUMBER: 

SERVICE CODE 

NUMBER: 

SPONSOR: 
Primary Skin Irritation in Rabbits 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 ch aracterized by the S ponsor 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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Primary Skin Irritation in Rabbits Revision 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, 20081; 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. 

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Primary Skin Irritation in Rabbits Revision 1 

CERTIFICATION 

I, the undersigned, declare that the methods, results and data contained in this report faithfully reflect the 

procedures used and raw data collected during the study. 

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Primary Skin Irritation in Rabbits Revision 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. 

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Primary Skin Irritation in Rabbits Revision 1 

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

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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.............................................................................................................19
Primary Skin Irritation in Rabbits Revision 1 

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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 conducted 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 dermal 
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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 study, 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 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: 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. 
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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 size 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 during 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 contaminants 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. Since 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. 
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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 stem1

 (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 produces full thickness destruction of the skin tissue 

on at least 1 animal during the skin irritation test. 

Risk phrases will be assigned in accordance 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 for 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 erythema 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 Revision 1 

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- Erythema and eschar formation 2.0 or more 

- Edema 2.0 or more 

All scores at each of the reading times (24, 48, and 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, hyperplasia, and scaling; or 

(3) In some cases where there is pronounced 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 recorded prior to test substance ap plication (initial) 

and again on the last day of individual scoring. 
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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 gross 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 are 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 records 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 calculated in accordance with EEC is presented in Table 

3. The Draize Primary Skin Irritation Scoring System is presented in Table 4. 
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All animals appeared active and healthy and gained body weight during the study. There were no 

signs of gross toxicity, adverse pharmacologic 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 study, classification is not required. 
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TABLE 1: INDIVIDUAL BODY WEIGHTS 

Body Weight (g) Animal No. Sex

Initial Terminal 

3501 F 2284 2536 

3502 F 2059 2225 

3503 F 2320 2431 
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TABLE 2: INDIVIDUAL SKIN 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 0/0 
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TABLE 3: SUMMARY OF MEAN SCORES FOR DERMAL RESPONSES

RABBIT NUMBER ERYTHEMAa EDEMAa

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). 
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TABLE 4: PRIMARY SKIN IRRITATION 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 approximately 1 millimeter).......................................................... 3 

Severe edema (raised more than 1 millimeter and extending beyond the area of exposure) 4 
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APPENDIX A: CERTIFICATE OF ANALYSIS