# PAGE

I OF 6 PAGES 

# STUDY SPONSOR:

CBI 

# STUDY TYPE: TEST ITEM: STUDY NUMBER: 

# INTRODUCTION A study was performed

to assess the irritation
of the te s t item to the eye of the New Zealand White rabbit (General Study Plan 560-17)

. This study was designed

to be compatible with the procedures indicated
by the following internationally accepted guidelines and recommendations: • OECD Guidelines

for the Testing

of Chemicals
No. 405 "Acute Eye Irritation/Corrosion

" (adopted
02 October 2012) • Method B5 

# Acute Toxicity (Eye Irritation

) of Commission Regulation (EC) No

. 440

/ 2008 

# Initial considerations Available information indicated that the test item had the potential

to produce severe effects
in a rabbit eye and to confirm this initial

a sse
s s m e nt, a Rabbit

E nucl

e ated Eye Test was performed prior to the in vivo 

# test. The results are given in the Appendix and indicated that the test item was unlikely

to cau

s e severe ocular irritancy. The ne g ative

c o ntrol data was shared withjCBI jCBI



# I

**~--J** 

# Experimental Starting

Date

: Experimental Completion Date

:

2 4 June 2014 17 July 2014 Test item characterisation
data are the responsibility

of the Sponsor. All raw data will be retained
in Harlan Laboratories
Ltd.

, Shardlow

, UK archives

. 

# METHOD A single application

of 0

. 1 mL of the test item was administered

to the non-irrigated
eye of two rabbits. Ocular reactions were recorded

1, 24

, 48 and 72 hour

s after admini

s tration.Report

Page2 

### RESULTS Individual and group mean scores for ocular irritation are given in Table

1 and Table
2. A sing le application

of the test item to the non-irrigated
eye of two rabbits produced iridial inflammation and/or moderate conjunctiva

l irritation. Both treated eyes appeared normal

at the 72-Hour observation. 

### CONCLUSION The test item produced

a maximum group mean score of 10.5 and was classifi

e d as
a mild irritant (Class

4 on
a 1 to 8 sca

l e) to the rabbit eye according

to a modified Kay and Calandra classification system

. This study was conducted

in a facility operating

to Good Laboratory Practice within the UK national GLP monitoring programme

, but the study report has not been audited by the QA Unit. No formal claim of GLP compliance

is made for this study. This report

is an accurate record of the study and its outcome

. 

# A
. Poo5 A Pool es Study Director

# I

**~1** Report 

# Page3


# TABLES
Table 1 Individual Scores and Individual Total Scores for Ocular Irritation
74465 Female 74508 Fema le Rabbit Number and Sex IPR=O IPR=O Time After Treatment 1 

# 24 

# 48 72 1 24 

# 48 72 

# Hour Hours Hours Hours Hour Hours Hours Hours CORNEA E = Degree of Opacity 0 

# 0 0 0 0 0 0 

# 0 F =Area of Cornea Involved 0 

# 0 0 0 0 0 0 

# 0 Score ( E x F) x 5 

# 0 0 0 0 0 0 

# 0 0 

# IRIS D I 0 0 0 

# 0 0 0 

# 0 Score (D x 5) 5 0 0 0 

# 0 0 0 

# 0 CONJUNCTIVAE A=Redness 

# 2 1 

# I 

# 0 2 I I 

# 0 

# B = Chemosis I 1 

# 0 

# 0 1 

# I 

# 0 0 

# C =Discharge 1 

# 0 

# 0 0 I 0 

# 0 0 

# Score (A+ B +C) x 2 

# 8 4 

# 2 0 8 4 2 0 

# Total Score 13 

# 4 

# 2 0 8 4 

# 2 0



# IPR = 

# Initial pain reaction

# Report

Page4 

# Table

2 Individual Total Scores and Group Mean Scores for Ocular Irritation Rabbit Number

Individual Total Scores At: and Sex

1 Hour

24 Hours

48 Hours

72 Hours 74465 Female

13



# 4

2

0 74508 Female

8



# 4

2

0 Group Total

21

8



# 4

0 Group Mean Score
10.5

4.0

2

. 0

0.0ReportPage5APPENDIXRabbitEnucleatedEyeTestIntroductionTheocularirritancypotentialofthetestitemwasassessedusingtheRabbitEnucleatedEyeTest.Thismethodinvolvedtheapplicationofthetestitemontothecorneaoftheenucleatedeye.TheRabbitEnucleatedEyeTestisusedasafirststageintheassessmentofocularirritancypotential.Anegativeocularirritancypotentialmayrequirefurtherinvestigationusinganinvivoocularirritationtest.Thetesthasundergonevalidationandhasbeenshowntoreliablydetectsubstancesthatarenegligible,ormoderatetosevereocularirritants.MethodsFiveenucleatedeyes,obtainedfromtheNewZealandWhitestrainofrabbit,weremaintainedatatemperatureof32±1.5°Cwithinthesuperfusionapparatus.0.1mLwasappliedontothecorneaofeachofthreeenucleatedeyes.Thedirecteffectofthetestitemonthecorneawasassessedbyevaluationofcornealthickness,cornealopacity,alterationofcornealepitheliumandfluoresceinuptake,throughoutthedurationofthetest.Thedataforallendpointswasassessedandanestimateofocularirritancypotentialmade.Afurthertwoenucleatedeyesremaineduntreatedforcontrolpurposes.ThenegativecontroldatawassharedwithICBIIResultsCornealOpacityObservationPeriod(minutespostdosing)60120180240CldyAreaCldyAreaCldyAreaCldyArea00000000TestEyes0000000000000000Control00000000Eyes*00000000Cldy=Cornealcloudiness*=ControldatasharedwithHarlanLaboratoriesLtd.Studynumber41401640Report

Page

6 

### Corneal Epithelium Condition Observation Period (minutes post dosing) 60

120

180

240 Normal Normal Normal Normal Test Eyes Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Control Eyes• Normal Normal

Normal Normal 

### Fluorescein Uptake (240 minutes post dosing) Test Eyes
Control Eyes* Int

0

0

0

0

0 Area

0

0

0

0

0 

### Corneal Swelling (%) 

### (minutes post dosing) 60



### 120

240 Test Eyes

a

12.1

8 .3

2.2 Control Eyes*b

10.3

8

. 8



### 6.3 Conclusion Following assessment

of the data for all endpoints the test item was considered unlikely

to have the potential

to cause severe ocular irritancy

in vivo. 

# . .. Int = a= b = Control data

s hared withiCBI intensity
of fluore

s cein **u!::'lptr:;'lak~e~-------------"** for each time point the corneal swelling recorded

is the mean of three eyes for each time point the corneal swelling recorded

i s the mean of two eyes