Revision 1

Page 1 of 19 Study Title

Pri mary Eye Irritation in Rabbits

T EST G UIDELINES : U.S. EPA Health Effects Test Guidelines, OPPTS 870.2400 (1998) OECD Guidelines for the Testing of Chemicals, Test No. 405 (2002)

S TUDY A MENDED ON : June 30, 2009

A UTHOR : Jennifer Durando, B.S.

S TUDY C OMPLETED ON : March 12, 2009

P ERFORMING L ABORATORY :

L ABORATORY

P ROJECT ID:

W ORK R EQUEST N UMBER :

S ERVICE C ODE N UMBER :

S PONSOR :Primary Eye Irritation in Rabbits Revisio n 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 characterized by the sponsor 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

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### 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 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.

Page 3 of 19Primary Eye Irritation in Rabbits Revisio n 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.

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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 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 Eye Irritation in Rabbits Revi sion 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 26, 2009 / February 6, 2009Primary Eye Irritation in Rabbits Revi sion 1

Page 7 of 19 

### 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

............................................................................................................ 19Primary Eye Irritation in Rabbits Revi sion 1

Page 8 of 19 

### 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 substan ce was not a severe skin irritant or corrosive to the skin.Primary Eye Irritation in Rabbits Revi sion 1

Page 9 of 19 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 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 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 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 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 Revisio n 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 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 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 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 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 o r 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.Primary Eye Irritation in Rabbits Revi sion 1

Page 11 of 19 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 in to the conjunctival sac of the right ey e of each rabbit by pulling the lower lid away from the eyeba ll. The upper and lower lids were then gently held together for about on e sec ond before releasing to m inimize lo ss 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-intens ity 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 conjunctiv ae, 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 co rnea, 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 ev aluations 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 Revi sion 1

Page 12 of 19 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 sep arately from numerical scores obtained at the 24-, 48-, and 72-hour observations. The results were interpreted accord ing 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 fo llowing 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 fo llowing 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 revers ible 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.Primary Eye Irritation in Rabbits Revi sion 1

Page 13 of 19 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 ar e 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 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 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 gain ed body weight during the st udy. Apart f rom the eye irritation noted below, there were no ot he r signs of gross to xicity, adverse pharmacologic effects, or abnormal behavior.Primary Eye Irritation in Rabbits Revi sion 1

Page 14 of 19 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 stud y, classification is not required.Primary Eye Irritation in Rabbits Revi sion 1

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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 2545Primary Eye Irritation in Rabbits Revi sion 1

Page 16 of 19 

### 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 0 1 0 0 0 0 1 0 0 0 0 1 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.Primary Eye Irritation in Rabbits Revi sion 1

Page 17 of 19 

### TABLE 3: MEAN SCORES FOR INDIVIDUAL RABBITS

Rabbit Number Corneal Opacity a Iritis a Conjunctival Redness a Conjunctival Chemosis a

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 scoresPrimary Eye Irritation in Rabbits Revi sion 1

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### TABLE 4: SCALE FOR SC ORING OCULAR LESIONS 1

1. Cornea A. Opacity-degree of density (area most dense taken for reading) No Op acity ..................................................................................................................... .....................................................0 Scattered or diffuse area, deta ils of iris cl early visible..................................................................... ...................................1 2 Easily discernible translucent areas, details of iris s lightly ob scured..................................................................................2 2 Opalescent areas, no details of iris vi sible, size of pupil barely discernible................................................. .......................3 2 Opaque, iris invisi ble ..........................................................................................................................................................4 2
B. Area of cornea involved One quarter (or le ss) but not zero............................................................................................. ...........................................1 Greater than one quarter , but less th an half................................................................................... ......................................2 Greater than half, but le ss than thr ee quarters ................................................................................ .....................................3 Greater than three quarter s, 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 (s luggish reaction is pos itive)................................................................... ....................................1 2 No reaction to light, hemorrhage, gross destruction (any or all of these)...................................................... ......................2 2 3. C onjunctivae A. Redness (refers to palpebral and bulbar conjunctivae excluding cornea and iris) Vessels normal ................................................................................................................. ...................................................0 Vessels definitely in jected above normal....................................................................................... .....................................1 More diffuse, deeper crimson red, indi vidual vessels not easily dis cernible .................................................... ..................2 2 Diffuse beefy red.............................................................................................................. ...................................................3 2 B. Ch emosis No swe lling .................................................................................................................... .....................................................0 Any swelling above normal (inc ludes nictitati ng membrane)...................................................................... .......................1 Obvious swelling with par tial eversion of lids................................................................................. ...................................2 2 Swelling with lids ab out half-c losed ........................................................................................... ........................................3 2 Swelling with lids ab out half-closed to completely closed ...................................................................... ...........................4 2 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 ha irs, 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 membrane s. J. Pharmacol. Exp. Ther. 1944; 82:377-390.

2 These scores represent a positive response.Primary Eye Irritation in Rabbits Revi sion 1

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### APPENDIX A: CERTIFICATE OF ANALYSIS