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Study Title 

 Primary Eye Irritation in Rabbits 

TEST GUIDELINES: U.S. EPA Health Effects Test Guidelines, OPPTS 870.2400 

(1998) 

OECD Guidelines for the Testing of Chemicals, Test No. 405 

(2002) 

STUDY AMENDED 

ON: 

 June 30, 2009 

AUTHOR: Jennifer Durando, B.S. 

STUDY COMPLETED 

ON: 

March 12, 2009 

PERFORMING 

LABORATORY: 

LABORATORY 

PROJECT ID: 

WORK REQUEST 

NUMBER: 

SERVICE CODE 

NUMBER: 

SPONSOR: 
Primary Eye 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 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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Primary Eye 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 3, 2009 Apr 23, 2008; Mar 3, 2009 

In-process inspection: 

48 hour scoring Jan 28, 2009 Mar 3, 2009 

Raw data audit Mar 3, 2009 Mar 3, 2009 

Draft report review Mar 3, 2009 Mar 3, 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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Primary Eye 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 Eye 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 Eye 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 26, 2009 / February 6, 2009 
Primary Eye Irritation in Rabbits Revision 1 

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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

PRIMARY EYE 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. DEVIATIONS 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 SCORES FOR OCULAR IRRITATION ................................................................................16

TABLE 3: MEAN SCORES FOR INDIVIDUAL RABBITS ..........................................................................................17

TABLE 4: SCALE FOR SCORING OCULAR LESIONS ............................................................................................18

APPENDIX A: CERTIFICATE OF ANALYSIS ............................................................................................................19
Primary Eye Irritation in Rabbits Revision 1 

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PRIMARY EYE 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 26, 2009 

EXPERIMENTAL COMPLETION DATE: February 6, 2009 

STUDY COMPLETION DATE: March 12, 2009 

NOTEBOOK NO.:

1. PURPOSE 

To provide information on the ey e irritation likely to arise fro m a sin gle instillation to 

2. SUMMARY 

A primary eye irritation test was conducted wi th rabbits to determ ine the potential for 

 to produce irrita tion from a single instillat ion via t he ocular route. A skin irritation 

study was previously conducted. The test substance was not a severe skin irritant or corrosive to 

the skin. 
Primary Eye Irritation in Rabbits Revision 1 

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At the request of the Sponsor, the study was conduc ted in a stepwise fashion. Initially, one-tenth 

of a m illiliter of the test substance was instilled in to the conjunctival sac of the right e ye of one 

healthy rabbit by pulling the lower lid away from the eyeball. The upper and lower lids were then 

gently held together for about one second before re leasing to minimize loss of the test substance. 

The left ey e remained untreated and se rved as a control. Ocular irritation was evaluated by the 

method of Draize et al. 1

 (see Table 4). Since there was no significant irritation observed in this 

animal, the test was completed on two additional animals, as described above. 

There was n o corneal opacity noted i n any treat ed ey e during t he study. One hour after test 

substance instillation, all three treated ey es e xhibited iritis and conjunctivit is. The over all 

incidence of irritation decreased with time. All animals were free of ocular irritation by 48 hours. 

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 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 instilled as received . Documentation of the methods of synthesis, 

fabrication, or derivation of the test substance is retained by the Sponsor.

Information on the test substance provided to by the 

Sponsor was (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 non-pregnant. 

3.B.3 Species/Strain: Rabbit/New Zealand albino. 

3.B.4 Age: Young adult. 

1

 Draize, J.H., Woodward, G. and Calvery, H.O. Methods for the study of irritation and toxicity of substances applied 

topically to the skin and mucous membranes. J. Pharmacol. Exp. Ther. 1944; 82:377-390. 
Primary Eye Irritation in Rabbits Revision 1 

Page 10 of 19 

3.B.5 Source: Received from Robinson Services, Inc. Clemmons, NC on January 14 and 

21, 2009. 

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 30-59%, 

respectively. 

4.A.3 Photoperiod: 12 hour light/dark cycle 

4.A.4 Acclimation Period: 12 or 14 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 a

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 

Prior to test initiation, bot h eyes of a group of an imals were examined using a white light source 

and a fluorescein dy e procedure. One drop of 2% ophthalmic fluorescein sodium was insti lled 

into both e yes of each rabbit. The e yes were ri nsed with phy siological s aline (0.9% NaCl) 

approximately 30 seconds after instillation of th e fluorescein and then evaluated for corneal 

damage using an ultraviolet light sour ce. Prio r to instillation, t he ey es were re-exam ined and 

scored for abnorm alities according to the "Scal e f or Scoring Ocular Lesions" (see Tab le 3). 

Three health y nai ve animals (not previousl y test ed) without pre-existing ocul ar irritation were 

selected for test. 

Initially, only one rabbit was placed on test. In the absence of significant irritation in this animal, 

the remaining two animals were tested to confirm the result. 
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B. Instillation 

Prior to instillation, two to three drops of ocular anesthetic (Tetracaine Hydrochloride Ophthalmic 

Solution, 0.5%) were placed into both the treated and control eye of each animal. One-tenth of a 

milliliter of the test substance was then instilled into the conjunctival sac of the right ey e of each 

rabbit by pulling the lower lid away from the eyeball. The upper and lower lids were then gently 

held together for about on e second before releasing to m inimize loss of the test substance. The 

other eye of each rabbit remained untreated with th e test substance and served a s a control. The 

rabbits were then returned to their designated cages. 

C. Ocular Scoring 

Ocular irritation was evaluated using a high-intensity white light ( Mag Lite) in accordance with 

Draize et al .

1

 (see T able 4) at 1, 24, 48, and 72 hours post-ins tillation. The fluorescein dy e 

evaluation procedure described in Section 5.A. w as used in the treated ey e at 24 hours to v erify 

the absence of corneal damage. Individual scores we re recorded for each animal. In addition t o 

observations of the cornea, iris and conjunctivae, any other observed lesions were noted. 

D. Classification of Eye Scores 

The observed ocular effects were classified as follows: 

European Economic Community (EEC)

 (Classification based on the mean scores of the cornea, iris, or conjunctiva averaged across 24, 48 

and 72 hrs for all animals) 

 R-36 Risk Phrase and “Xi” Symbol..................................Irritating to Eyes 

Assigned if, when applied to the e ye of the t est animal, the test substance causes significant 

ocular lesions which occur within 72 hours after exposure and which persist for at least 24 hours. 

Ocular lesions are significant if the individual an imal mean values of the 24-, 48-, and 72- hour 

evaluations in 2 or more animals comply with any of the following criteria: 

- corneal opacity equal to or greater than 2.0, but less than 3.0 

- iridial lesion equal to or greater than 1.0, but less than 2.0 

- conjunctival redness equal to or greater than 2.5 

- conjunctival chemosis equal to or greater than 2.0 

 R-41 Risk Phrase and “Xi” Symbol..................................Severe Damage to the Eyes 

Assigned if the test subst ance c auses severe ocul ar lesions whic h occur within 72 hours after 

exposure and which persist for at least 24 ho urs. Ocular lesion s are severe if the indivi dual 

animal mean value of the 24-, 48-, and 72-hour evaluations in 2 or more animals comply with any 

of the following criteria: 

- corneal opacity equal to or greater than 3.0 

- iridial lesion equal to 2.0 

 

1

 Draize, J.H., Woodward, G. and Calvery, H.O. Methods for the study of irritation and toxicity of substances 

applied topically to the skin and mucous membranes. J. Pharmacol. Exp. Ther. 1944; 82:377-390. 
Primary Eye Irritation in Rabbits Revision 1 

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also required if: 

 - ocular lesions are still present 21 days following treatment 

 - the test substance causes irreversible coloration of the eyes 

 (Reference: European Communities D irective on Classifi cation, Packaging and Labeling of 

Dangerous Substances (June 27, 1967), 67/548/EEC and subsequent amendments) 

Globally Harmonized System (GHS) 

Mean values for each lesion (corneal opacity, iris, conjunctival redness, conjunctival 

chemosis) were calculated for each animal separately from numerical scores obtained at the 

24-, 48-, and 72-hour observations. 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. 

Category 1 (irreversible effects on the eye) is a test substance that produces: 

- at least in one animal effects on the cornea, iris, or conjunctiva that 

are not expected to reverse or have not fully reversed within an 

observation period of normally 21 days, and/or 

- at least in 2 of 3 tested animals, a positive response of: 

corneal opacity ≥ 3 and/or 

iritis > 1.5 

- calculated as the mean scores following grading at 24, 48, and 72 

hours after instillation of the test substance 

Category 2 (reversible effects on the eye) (irritating to eyes) 

 Category 2A (irritating to eyes) is a test substance that produces:

- at least in 2 of 3 tested animals a positive response of: 

corneal opacity ≥ 1, and/or 

iritis ≥ 1, and/or 

conjunctival redness ≥ 2, and/or 

conjunctival chemosis ≥ 2 

- calculated as the mean scores following grading at 24, 48, and 72 

hours after instillation of the test substance, and 

- which fully reverses within an observation period of normally 21 

days 

Within this category an eye irritant is considered mildly irritating to eyes (Category 2B) when 

the effects listed above are fully reversible within 7 days of observation. 

E. Body Weights 

Individual body weights of the animals were reco rded prior t o test substance instillation (init ial) 

and again on the last day of individual scoring. 
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F. 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 gros s evaluation of ey e and fu r, eyes 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 870.2400 (1998) 

OECD Guidelines for the Testing of Chemicals, Test No. 405 (2002) 

8. QUALITY ASSURANCE 

The final report was audited for agreement with th e raw data records and for compliance with the 

protocol and 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. DEVIATIONS FROM FINAL PROTOCOL 

None. 

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 in Table 1. Individual eye irritation scores are presented in 

Table 2. A summary of mean scores is presented in Table 3. The Draize Pri mary Eye Irritation 

Scoring System is presented in Table 4. 

All animals a ppeared active and healthy and gained body weight during the st udy. Apart f rom 

the eye irritation noted below, there were no ot her signs of gross to xicity, adverse pharmacologic 

effects, or abnormal behavior. 
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There was n o corneal opacity noted i n any treat ed ey e during t he study. One hour after test 

substance instillation, all three treated ey es e xhibited iritis and conjunctivit is. The over all 

incidence of irritation decreased with time. All animals were free of ocular irritation by 48 hours. 

12. CONCLUSION 

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 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 

3401 F 2534 2550 

3402 F 2710 2740 

3403 F 2530 2545 
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TABLE 2: INDIVIDUAL SCORES FOR OCULAR IRRITATION 

Rabbit No.: 3401 (Female) Rabbit No.: 3402 (Female) Rabbit No.: 3403 (Female) 

Hours Hours 

1 24 48 72 1 24 48 72 1 24 48 72 

I. Cornea 

A. Opacity 0 01 0 0 0 01 0 0 0 01 0 0 

B. Area 4 4 4 4 4 4 4 4 4 4 4 4 

II. Iris 

A. Values 1 0 0 0 1 0 0 0 1 0 0 0 

III. Conjunctivae 

A. Redness 1 0 0 0 1 1 0 0 1 0 0 0 

B. Chemosis 0 0 0 0 0 0 0 0 0 0 0 0 

C. Discharge 1 0 0 0 1 1 0 0 1 0 0 0 

 

1

 2% ophthalmic fluorescein sodium used to verify the absence of corneal opacity. 
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TABLE 3: MEAN SCORES FOR INDIVIDUAL RABBITS 

Rabbit 

Number 

Corneal 

Opacitya Iritisa

Conjunctival 

Rednessa

Conjunctival 

Chemosisa

3401 0.00 0.00 0.00 0.00 

3402 0.00 0.00 0.33 0.00 

3403 0.00 0.00 0.00 0.00 

a Calculated from the 24-, 48-, and 72-hour scores 
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TABLE 4: SCALE FOR SCORING OCULAR LESIONS1

1. Cornea 

A. Opacity-degree of density (area most dense taken for reading) 

No Opacity ..........................................................................................................................................................................0 

Scattered or diffuse area, details of iris clearly visible........................................................................................................12

Easily discernible translucent areas, details of iris slightly obscured..................................................................................22

Opalescent areas, no details of iris visible, size of pupil barely discernible........................................................................32

Opaque, iris invisible ..........................................................................................................................................................42

B. Area of cornea involved 

One quarter (or less) but not zero........................................................................................................................................1 

Greater than one quarter, but less than half.........................................................................................................................2 

Greater than half, but less than three quarters .....................................................................................................................3 

Greater than three quarters, up to whole area......................................................................................................................4 

2. Iris 

A. Values 

Normal ................................................................................................................................................................................0 

Folds above normal, congestion, swelling, circumcorneal injection (any or all of these or combination of any thereof) 

iris still reacting to light (sluggish reaction is positive).......................................................................................................12

No reaction to light, hemorrhage, gross destruction (any or all of these)............................................................................22

3. C onjunctivae 

A. Redness (refers to palpebral and bulbar conjunctivae excluding cornea and iris) 

Vessels normal ....................................................................................................................................................................0 

Vessels definitely injected above normal............................................................................................................................1 

More diffuse, deeper crimson red, individual vessels not easily discernible ......................................................................22

Diffuse beefy red.................................................................................................................................................................32

B. Ch emosis 

No swelling .........................................................................................................................................................................0 

Any swelling above normal (includes nictitating membrane).............................................................................................1 

Obvious swelling with partial eversion of lids....................................................................................................................22

Swelling with lids about half-closed ...................................................................................................................................32

Swelling with lids about half-closed to completely closed .................................................................................................42

C. Disch arge 

No discharge .......................................................................................................................................................................0 

Any amount different from normal (does not include small amounts observed in inner canthus of normal animals)........1 

Discharge with moistening of the lids and hairs just adjacent to lids..................................................................................2 

Discharge with moistening of the lids and hairs, and considerable area around the eye.....................................................3 

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. 

2

 These scores represent a positive response. 
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APPENDIX A: CERTIFICATE OF ANALYSIS