PAGE 1 OF 16PAGES 

SafePharm 

Laboratories 

EMULGEN B-66: 

ACUTE EYE IRRITATION IN THE RABBIT 

SPL PROJECT NUMBER: 140/1100 

STUDY SPONSOR: 

Kao Corporation 

Division of Product Safety 

Safe匂I& Environmenta1 Research Center 

2606,Akab組 e,Ichikairnachi 

Haga 

TOCHIGI 321-3497 

JAPAN 

140.1100.docIJS 

AUTHOR: A Sanders 

TEST FACILITY: 

Safephann Laboratories Limited 

P.O. Box No. 45 

DERBY 

DE12BT 

U.K 

Tel巴:phone:(01332) 792896 

Facsimile: (01332) 799018 
SPL PROJECT NUMBER: 140/1100 PAGE2 

QUALITY ASSURANCE REPORT 

This studyザpeis classedぉ short-term.官les旬nd紅 dtest method for this study type (“General 

Study Plan"血 OECDterr凶nology)was reviewed for compliance once only on initial production. 

Inspection of the routine and repetitive procedures也a1conぬ 旬tethe study is carried out as a 

con出 uousprocess designed to encompぉs也emajorphぉ esat or about the也nethis study was in 

progress. 

百註sreport hぉ beenaudited by Safepharm Quality Assurance U凶t,and is consider官dto be an 

accurate account of世leda臼 generatedand ofthe proced町巴sfollowed. 

In each cぉ e,也.eoutcome of QA evaluation is reported to the Study Director and Management on 

the day of evaluation. Audits of study documentation, and process inspections appropriate to the 

句!peand scheduIe of this託udywere as follows: 

15 August2001 

19 March 2002 

07 March 2002 

05 March 2002 

Standard Test Method Compliance Audit 

Test Material Preparation 

13, 20 March 2002 

S 08 April 2002 

Animal Preparation 

Dosing 

S Date of QA Signature 

Assessment of Response 

Draft Report Audit 

Final Report Audit 

S Evaluation specific to白isstudy 

For Safepharm Quality 

以ム

Assurance Unit* 

• Authorised QA Signatures: 

DATE: 

Head ofDep紅 白lent: JR Pateman CBiol MIBiol DipRQA FRQA 

JMCrow也erMIScT MRQA 

26 APR 2002 

Deputy Head ofDepar町lent

Senior Audit S阻ff: JV Johnson BSc MRQA; G Wren ONC MRQA; R Hurst MRQA 
SPL PROJECT NUMBER: 140/1 100 PAGE3 

GLPCOMPLlANCESTATEMENT 

百lework described wぉ performedin compliance with UK GLP standards (Schedule 1, Good 

Laboratory Practice RegulatiollS 1999 (SI 1999/3106)).百leseRegulatiollS紅'ein accordance with 

GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, 

ENV IMC/CHEM(98) 17); and紅 ein accordance wi由, and implement, the req凶rementsof 

Directives 87/18AEC (ぉ amendedby Directive 1999111厄C)and 88/320厄EC(as amended by 

Directive 1999/12厄C).

These intemational standards are a∞eptable to the Regulatory agencies of也efollowing 

countries: Australia, Aus位ia, B巴Igium,Canada, the Czech Republic, Denmark, Finland, France, 

Germany, Greece, Hungary, Iceland, Ireland, Israel, Italy, Japan, Republic of Korea, 

Luxembo町 g,Mexico, The Netherlands, New Zealand, Norway, Poland, Portugal, Slovenia, 

Sp垣見 Sweden,Switzerland, Turkey, the United Kingdom, and the U凶t巴dStates of America 

ηlIs reportぬllyand accurately reflects也eproced出 回 usedand data generated. 

'""'-..,ミ?と~一一 DATE: .........2.~..A P.R .. Z~Q2 A Sanders 

Study Director 
SPL PROJECT NUMBER: 140/1100 PAGE4 

CONTENTS 

QUALITY ASSURANCE REPORT 

GLP COMPLIANCE STATEMENT 

CONTENTS 

SUMMARY 

1. INTRODUCTION 

3. 

TEST MATERIAL 

2.1 Description, Identification and Storage Conditions 

2.2 Preparation ofTest MateriaI 

2.3 Measurement of pH 

METHODS 

3.1 AnimaIs and Animal Husbandry 

3.2 Procedure 

3.3 Interpretation ofRc巴sults

ARCHIVES 

9La'34aTF3rokoroζり勺

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2. 

4. 

5. RESULTS 10 

6. CONCLUSION 10 

Table 1 IndividuaI Scores and IndividuaI Total Scores for Ocular Irritation 11 

Table 2 IndividuaI To凶 ScoresAnd Group Mean Scores For Ocular Irritation 12 

Appendix 1 InitiaI Pain Reaction 13 

Appendix 2 Draize ScaIe for Scoring Ocular Irritation 14 

Appendix 3 Modified Kay and CaIandra Interpretation ofEye Irritation Test 15 

Appendix 4 Statement of GLP Compliance in Accordance with Directive 88/320厄EC 16 
SPL PROJECT NUMBER: 140/1100 PAGE5 

EMULGENB・66:

ACUTE EYE IRRITATION 11可THERABBIT

SUMMARY 

lnlroduction. The study wぉ performedto assess the irritancy potentiaI of the test material to出E

eye of由巴 NewZeaIand White rabbit. The method was designed to meet the requirements ofthe 

follow泊g

• OECD Guidelines for the Testing of ChemicaIs No. 405“Acute Eye IrritationlCoITosion" 

(adopted 24 February 1987) 

• Commission Directive 92/69厄ECMe血odB5 Acute Toxicity (Eye Irritation) 

Result. A single application of the test materiaI to也enon-irrigated eye ofせrreerabbits 

produced moderate conjunctivaI i凶 匂.tion.All eyes appe眠 dnormaI at the 48・hourobservation. 

Conclusion.百letest material produced a maximum group mean score of 10.0 and was classified 

as a minimaI irritant (Class 3 on a 1 to 8 scaIe) to the rabbit eye according to a modified Kay and 

CaIandra classification system. 

The test materiaI did not meet也巴 criteriafor classification as irritant according to EU labelling 

regulations Commission Directive 93/21AEC. 
SPL PROJECT NUMBER: 14011100 PAGE6 

EMULGENB・66:

ACUTEEYEll主RlTATIONIN THE RABBIT 

1. INTRODUCTION 

The study was performed to拙 巴ss也e凶 回cypotential of也etest material following a single 

application to the rabbit eye. The method was designed to meet the requirements of出efollowing: 

• OECD Guidelinωfor the Testing of Chemicals No. 405“Acute Eye IrritationlCoITosion" 

(adopted 24 February 1987) 

• Commission Directive 92/69AEC Method B5 Acute Toxicity (Eye Irritation) 

百lealbino rabbit has been shown to be a suitable model for也1S句rpeof study and is 

recommended in吐1巴 testme白od.The results of the study紅 'eb巴lievedto be of value in predicting 

也elik巴Iyeye註討旬且cypotential of the test material to m釦 .

百lestudy was performed between 04 March 2002 and 10 March 2002. 

2. TEST MATERlAL 

2.1 Description, Identification and Storage Conditions 

Sponsor's identification 

Description 

Batch number 

Date received 

Storage conditions 

EMULGEN B-66 

liめtbrown slightly turbid viscous liquid 

2376 

31 December 2001 

room temperature in the dark 

Data relating to也eidentity, purity釦 dstability of the test material l由 也eresponsibili旬。f也E

Sponsor. 

2.2 Preparation ofTest Material 

For the purpose ofthe study the test material wぉ usedぉ supplied

The absorption of the test material was not determined. 
l , 

SPL PROJECT NUMBER: 140/1100 PAGE7 

2.3 Measurement of pH 

百lepH of a 90% v/v aqueous prep制 tionofせletest material was determined戸orto 

commencement ofthe study and found to be approximately 8.5. 

3. METHODS 

3.1 Animals and Animal Husbandry 

τbree New Zealand White rabbits were supplied by David Percival Ltd, Moston, Sandbach, 

Cheshire, UK.. At也es泊rtofthe study the animals were in the weight r佃.geof 2.0 to 3.5 kg and 

were twelve to sixteen weeks old. After an acclimatisation period of at least five days each 

animal was given a number u且iquewi也inthe study which wぉ wri世enwi仕1a black indelible 

marker-pen on the inner surface of也ee紅 andon也ecage label. 

τ1:te創出nalswere individually housed in suspended metal cages. Free access to mains drinking 

water and food (Certified Rabbit Diet (Code 5322) supplied by PMl Nutrition lnternational, 

No仕inghanl,UK.) Wぉ allowedthroughout the study. The diet and drinking water were 

considered not to contain any contaminant of a level也atrnight have affected也epurpose or 

integrity of也estudy. 

The tempera旬reand relative humidity were set to achieve 1並litsof 17 to 230C飢 d30 to 70% 

respectively. Any 0∞asional deviations企omthese t紅 getswere considered not to have affected 

白 purposeor integrity of the study. The rate of air exchange was at least fifteen changes per 

hourand也eligh出 gwぉ con甘olledby a time switch to give twelve hours continuous light (06:00 

to 18:00) and twelve hours darkness. 

The animals were provided with environmental enrichment items which were considered not to 

contain釦 ycontanlinant of a 1巴vel白紙 rnighthave affected the purpose or integrity of the 山 dy.

3.2 Procedure 

Immediately before吐lestart of the test, both eyes of也eprovisionally selected test rabbits were 

exalllIned for evidence of ocular irritation or defect with the aid of a light so町 cefrom a standard 

ophthalrnoscope. Dnly animals企eeof ocular danlage were used. 

lnitially, a single rabbit was treated. A volume of 0.1 lnl of也etest material was placed into由E

conjunctival sac of the right eye, formed by gently p叫lingthe lower lid away from批 eyeball.

百leupper and lower eyelids wer巴 heldtogether for about one second inunediately after仕切回ent,
r-

~ 

SPL PROJECT NUMBER: 140/1100 PAGE8 

to prevent loss ofせletest material, 釦d也enreleased 百1eleft eye remained un出 atedand was 

used for control purposes. Immediately after adminis回 tionof吐1etest mat巴rial, 組 assessmentof 

the initial pain reaction was made according to也esix point scale shown in Appendix 1. 

After consideration of也eocu1ar responses produced in the first仕eatedaninlal, two additional 

祖 国alswere困 ated.

Assessment of oc叫 紅 damage/irri旬.tionwas made approximately 1 ho町 組d24, 48 and 72 hours 

followi時 間atment,according to也enumerical evaluation given in Appendix 2, (企omDraizeJH 

(1977) "Dermal姐 dEye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity 

ofHousehold Substances, National Academy ofSciences, Washington DC p.48 to 49) 

Anyotheroc叫紅巳ffectswere also noted. Exan由1ationof也eeye was facilitated by the use of the 

light source企omastand紅 dophthalmoscope. 

3.3 Interpretation of ResuIts 

The numerical values corresponding to each ru由nal,tissue and observation time were recorded. 

The data rela出 gto吐1e∞ 勾unctivaewere designated by the letters A (redness), B (chemosis)飢 d

C (disch紅 ge),也oserelating to the iris designated by the 1出 erD and those relating to血ecornea 

by也巴 lettersE (degree of opacity) and F (訂'eaof cornea involved). For each tissue the score w,ぉ

calculatedぉ follows:

Score for coniunctivae 

Score for iris 

Score for cornea 

(A+B + C)x 2 

Dx5 

(E xF) x 5 

Using the numerical data obtained a modified version of the system described by Kay J H and 

CalandraJ C, J. Soc. Cosmet. Chem., 1962 13 281-289 (see Appendix 3) was used to classi今也E

ocu1紅紅rit組 cypotential of也etest material. This was achieved by adding together也escores for 

也ecornea, iris and conjunctivae for each time point for each rabbit. The group means of the total 

scores for each observation were calcu1ated. 百1ehighest of出巴segroup means (the maximum 

group mean score) toge出巴rwith the persistence of the reactions enabled classification of the eye 

1m阻ncypotential ofthe test material. 

If evidence of irreversible ocu1ar damage is noted, the test material will be classified as corrosive 

to the eye. 
「

、、一

SPL PROJECT NUMBER: 140/1100 PAGE9 

4. ARCHIVES 

Unless ins住uctedotherwise by the Sponsor, all original da包 釦d由efinal report will be retained 

in the Safepharm archives for five ye紅 s,after which instructions will be sought as to fur血er

retention or disposal. 
SPL PROJECT NUMBER: 140!1l00 PAGE 10 

5. RESULTS 

Individual and group mean scores for ocular irritation紅 egiven in Table 1 and Table 2. 

No corneal or iridial effects were noted during the study. 

Moderate conjunctival討itationwas noted in all回 atedeyes one hoぽ after仕切回entwi白

minimal coniunctival re也lessat the 24-hour observation. 

All treated eyes appeared normal at白e48-hour observation. 

6. CONCLUSION 

百letest material produced a maximum group mean score of 10.0 and was classified as a 

M別勘仏LIRRlTANT (CLASS 3 ON A 1 TO 8 SCALE) to也erabbit eye according to a 

modified Kay and Calandra classification system. 

立letest material did not meet the criteria for classification as註ri1antaccording to EU labelling 

regu1ations Commission Directive 93/21AEC. 
Table 1 

EMULGEN B-66: ACUTE EYE IRRITATION IN THE RABBIT 

Individual Scores and Individual Total Scores for Ocular Irritation 

IPR=2 IPR=2 

Rabbit Number and Sex 

165 Male 109 Male 

Time After Treatment 24 48 72 24 48 72 

Hour Hours Hours Hours Hour Hours Hou悶 Hours 

CORNEA 

E = Degree of Opacity 。 。 。 。 。 。 。 。

F = Area of Cornea Involved 。 。 。 。 。 。 。 。

Score (E x F) x 5 。 。 。 。 。 。 。 。

IRIS 

D 。 。 。 。 。 。 。 。

Score (D x 5) 。 。 。 。 。 。 。 。

CONJUNCTIVAE 

A = Redness 2 。 。 2 。 。

B = Chemosis 。 。 。 。 。 。

C = Discharge 2 。 。 。 2 。 。 。

Score (A + B + C) x 2 10 2 。 。 10 2 。 。

Total Score 10 2 。 。 10 2 。 。

IPR = Initial pain reaction 

SPL PROJECT NUMBER: 140/1100 

IPR=2 

III Male 

24 48 72 

Hour Hours Houl's Hours 

。 。 。 。

。 。 。 。

。 。 。 。

。 。 。 。

。 。 。 。

2 。 。

。 。 。

2 。 。 。

10 2 。 。

10 2 。 。

PAGE 11 
SPL PROJECT NUMBER: 140/1100 PAGE 12 

EMULGEN B-66 : ACUTE EYE IRRIT A TION IJ可THERABBIT

Table2 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 

165 Male 10 2 。 。

109 Male 10 2 。 。

111 Male 10 2 。 。

Group Total 30 6 。 。

Group Mean Score 10.0 2.0 。。 。.0
SPL PROJECT NUMBER: 140/1100 PAGE I3 

EMULGENB・66:ACUTE EYE IRRITATION IN THE RABBIT 

Appendix 1 Initial Pain Reaction 

When the material is instiIled in吐leeye白eremay be組 凶tiallocalpain reaction.百lereaction 

will be graded as foIlows: 

Class Reaction by Animal Descriptive Rating 

。 No response No initial pain 

A few blinks 0凶y,normal wi血inoneortwo Practically no泊itialpain mmutes 

2 Rabbit blinks and往iesto open eye, but Slight initial pain reflex closes it 

3 Rabbit holds eye shut and puts press町 'eon Moderate initial pain lids, may rub eye wi也 paw

4 Rabbit holds eye shut vigorously, may squeal Severe initial pain 

5 R且bbitholds eye shut vigorously, may Ve巧rsevere initial pain squeal, claw at eye, jump and try to escape 

There is often no correlation between the initial pain and the subsequent eye irritation. 
SPL PROJECT NUMBER: 140!llOO PAGE 14 

EMULGEN B-66: ACUTE EYE IRRITATION IN THE RABBIT 

Appendix2 Draize Scale for Scoring Ocular Irritation 

1. CONJUNCTIV AE 

0123 

Redness (refers to palpebral and bulbar conjunctivae excluding cornea四 diris) 

Vessels normal 

Vessels definitely i町ectedabove normal 

More diffuse, deeper crimson red, individual vessels not easily discemible 

Diffuse beeぢred

(A) 

nu'且今,&吋‘JA

値T

Chemosis 

No swelling 

Any swelling above nonnal (inc1ud田 nic凶 加gmembrane) 

Obvious swelling with parti剖 eversionof lids 

Swelling with lids abOUI half closed 

Swellingwi血 lidshalf closed 10 completely closed 

。)

Discharge 

No discharge 

Any阻 ountdifferent from normal (does oot include small町noun.匝 observedin inner c血血usof 

normal animals) 

Discharge with moistening ofthe lids皿 dhai目 justadjacent to lids 

Discharge wi也 moisteningof曲elids四 dh司1Sa considerable町田町'Ound出eeye

THE TOTAL SCORE = (A + B + c) x 2 MAXIMUM TOTAL = 20 

。

2 

3 

(c) 

2. IRIS 

。

2 

Values 

Normal 

Folds above norma1. congestion, swelIing, circumco四 国1injection (any or all ofthese or combination 

of any thereof) iris still rea凶 ngto light (sluggish reaction is positive) 

No reaction to light, haemorrhage, gross destruction (四yor all of曲 目e)

THE TOTAL SCORE = D x 5 

(D) 

MAXIMUM TOTAL = 10 AU'Iヲ白司3SAT

CORNEA 

Degree of Opacity (most dense area used) 

No opacity 

Scattered or diffuse町 eas,details of iris clearly visible 

E白 ilydiscemible回 nslucentareas, details ofiris slightly obscured 

Opalescent areas, no details ofiris visible, size ofpupil barely discemible 

Opaque. iris not discemible血rough由eopacity 

Area of Cornea Involved 

(E) 

3. 

-1234 

MAXIMUM TOTAL = 80 

σy 

One quarter (or less) but not zero 

Greater白田 onequarter but less th四 half

Greater than halfbut less出回 threequarters 

Greater th四 threequarters, up to whole area 

THE TOTAL SCORE = (E x F) x 5 

MAXIMUM TOTAL SCORE POSSffiLE = 110 

、
SPL PROJECT NUMBER: 140/1100 PAGE 15 

EMULGEN B-66: ACUTE EYE IRRITATION IN THE RABBIT 

Appendix3 Modified Kay and Calandra Interpretation of Eye Irritation Test 

MAX町四JM DESCRIP百 ONRAτlNG MEAN PERSISTENCE OF SCORE 

SCORE (ANDCLASS) 

0.0 toO.5 Groupm田 nto阻 Iscore at 24 hours = 0 Non-Iπltant (1) 

Groupm担 nto阻 1score at 24 hOll回 >0 Pr富山田lIynon-irritant (2) 

0.5 t02.5 Groupm田 ntO回1score at 24 hou目 -0 hMircutm回al11yu mn imtant (2) 

Groupm田 nlO凶 E回 reat 24 hours > 0 1π1包nt (3) 

2.5 to 15 Groupm国 n阿 国Iscore at 48 hours = 0 Minimal irri飽nt (3) 

Groupm国 nto阻 Iscore at 48 hours > 0 Mild irri回 t (4) 

15t025 Groupm阻 nto也1sco悶 at72ho四 百 -0 Mild irri凶 nt (4) 

Groupm田 nto阻 Iscore at 72 hour宮 >0 Moderate町 出nt (5) 

Mo陪白血 halfof曲eindividual同 国11sco陀 S Moderate町出血 (5) at 7 days 10 or less 

Groupm田 nto飽1s∞re at 7 days More血 叩 ha!foftheindividual to凶 scores

25to 50 200fl at 7 days > 10 but no individual to凶 s∞re Mode汀ateirrt泊 目 (5) 田 S at 7 day!l > 30 

Moreth四 ha1foftheindividual to凶 scor,田

at 7 days > 10血 dany individual sco陪 at7 Severe irri也nt (6) 

days> 30 

Groupm田 ntO也Is回 目 at7 days >20 Severe irritant (6) 

Moreth叩 halfoftheindividual 10凶 500毘 S Severe iπi阻nt (6) a17 days 30 or les市

Groupm田 nto凶 scoreal 7 da'戸 Morelh血 halfoftheindividual to凶 scores

50to 80 40 or less at 7 days > 30 bUl no individual total scores Severe irri回 t (6) 

at 7 days >60 

More出 叩 halfoftheindividual to阻 1s四 回S

al 7 days > 30 and individual total score at Very severe irritant (7) 

7 days >60 

Groupm田 nto回 1score at 7 days > 40 Very severe irri也nt (7) 

More th血 haJfoftheindividual to組Jsco陀 S Very severe irritant (7) at 7 days 60 or 1田 S

Groupm田 ntotal score at 7 days Moreth叩 halfofthe individual to凶 scores

80tol00 at 7 days > 60 but no individual to阻 1score Very severe i而 阻nt (7) 800rl田 S at 7 days > 100 

More白 血 halfofthe individual total scores 

at 7 days > 60 and individual to凶 s∞reat Extremely severe irritant (8) 

7 days > 100 

Groupm田 nto回1score at 7 days > 80 Extremely severe irritant (8) 

Grollpm田 nto也Jscore at 7 days Very severe irri包nt (7) 

100 to 110 80 or less 

Groupm国 nto凶 s∞reat7 days >80 Extre江nelysevere irri旬nt (8) 
SPL PROJECT NUMBER: 140/1100 PAGE 16 

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El¥侃JLGENB・66:

ACUTE EYE IRRITATION IN THE RABBIT 

SPL PROJECT NUMBER: 140/1100 

1 verifシ 也at也1S1S an exact copy of the original report which is located in也eArchives of 

Safephann Laboratories Ltd., Derby, UK. 

¥ 

A Sanders 

Stud y Director 

DATE: ...........?J.A P.H...?~~? ・.,.