l!f:!IH-10234

English version ofthe final report



### Final Report AnAcute Dermal Irritation/Corrosion Study of inRabbits Study No. H-10234

February 5, 2011
Nippon ExperimentalMedical Research Institute Co., Ltd. 1967·11Arima, Shibukawa·shi, Gunma, Japan

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### STATEMENT OF COMPLIANCE

Title : AnAcute DermalIrritation/Corrosion Study of in Rabbits Study No. H-10234

I, the undersigned, hereby declare that this report is the English version of the original report that has been written in Japanese language. Further, I declare that the data are exactly reflected in this report and similar to that of the original (Japanese) report.

Date: 

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## 2o I2.- Takashi Sato, M.S. StudyDirector (translator) Nippon ExperimentalMedical Research Institute Co., Ltd.

### PREPARATION OF THE FINAL REPORT Title:AnAcute DermalIrritation/Corrosion Study of Study No.: H-10234 in Rabbits 

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The study listed above was performed in compliance with the Organization for Economic Co-operation and Development (OECD) Principles ofGood Laboratory Practice (as revised in 1997), ENV /MC/CHEM(98)17. This study was conducted according to the methods described herein, and the data are accurately reflected in thisreport.

Study director: Takashi Sato, M.S. (Impression ofthe seal) Date : February 5, 2011 NipponExperimentalMedical Research Institute Co., Ltd.

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### OUTLINE OF THE STUDY CONDUCT Title:AnAcute Dermal Irritation/Corrosion Study of Study No. :H-10234 1. Purpose As part of the safety assessment, irritation/corrosion in rabbits. 2. GLP compliance in Rabbits

was investigated for dermal

Organisation for Economic Co-operation and Development(OECD)Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98)17 3. Guidelines referred This study was conducted by reference to the OECD Guideline for the Testing of Chemicals #404 (adopted on April 24, 2002) (hereinafter referred to as "OECD Guideline"). 4. Animal welfare This study was approved by the Animal Experimental Committee of Nippon ExperimentalMedical Research Institute Co., Ltd. (Approval No. 2006013). 5. Sponsor Name: Address: 6. Contract laboratory Name: Address: Nippon ExperimentalMedical Research Institute Co., Ltd. 1967·11Arima, Shibukawa-shi, Gunma, Japan 7. Testingfacility Name: Address: Shibukawa Laboratories Nippon ExperimentalMedical Research Institute Co., Ltd.6. 1967·11Arima, Shibukawa·shi, Gunma, Japan8. Storage ofrecords and materials (1) Storageperiod The items are stored for 10 years after completion ofthe study. (2) Storage items and storinglocation

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The studyprotocol and its amendment (original), documents relatingto the study, raw data obtained in the course of the study, final report (original) and other materials are stored in Shibukawa Laboratories, Nippon Experimental Medical Research Institute Co., Ltd. (3) Address ofthe storinginstitute 1967·11Arima, Shibukawa·shi, Gunma, Japan 9. Schedule ofthe study Study initiation Animal arrival End ofthe quarantine period Startofthe experiment First administration Observation Completion ofthe experiment Submission ofthe draft final report Preparation ofthe final report Completion ofthe study :December 28, 2010 :January 6, 2011 :January 11, 2011 :January 12, 2011 :January 12, 2011 :January 12, 2011 :January 12, 2011 :January 24, 2011 :February 5, 2011 :February5, 2011

### 10. Studypersonnel and workresponsibility 

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Study director, protocol preparation, work supervising/management and preparation ofthe final report :Takas hi Sa to Note) 

### Clinical observation and body weight measurement during the quarantine/ acclimationperiod :Masaaki Shirai,Yoko Wakana, Animal health assessment Preparation ofthe test substance Control ofthe test substance Moeko Masuyama, Takashi Sato :Yoko Wakana :Takashi Sato :Takashi Sato Administration, removal, washing, clinical observation andbody weight measurement :Masaaki Shirai,Yoko Wakana, Takashi Sato Observation oftreatment sites :Yoko Wakana, Takashi Sa to Note) Affiliation: Safety Research Department, Nippon Experimental Medical Research Institute Co., Ltd. Address: 1967·11Arima, Shibukawa·shi, Gunma, Japan

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### UNPREDICTED EVENTS CONSIDERED TO HAVE AFFECTED THE RELIABILITY OF THE STUDYAND DEVIATION FROM THE PROTOCOL Title:AnAcute DermalIrritation/Corrosion Studyof Study No.: H-10234 1. Unpredicted events in Rabbits

The humidity in Animal Room 8 deviated from the lower limit between 9:54:40 and 9:54:47 a.m., and between 9:54:56 a.m. and 10:16=28 a.m. onJanuary 7, 2011, recording the lowest value of 29.9% and 22.6%, respectively. The deviations occurred due to vaporsupply disruption caused by an error ofthe maintainer shutting the vapor valve ofthe boiler. 2. Deviation from the protocol There were no relevant items. 3. Effects on the reliability ofthe study The deviations ofthe humiditywere consideredto have no effect on the reliabilityof the study, because no abnormal finding was observed in general conditions ofanimals after the humidityreturned to the normal range.

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### CONTENTS page ·PREPARATION OF THE FINALREPORT · .. ·-- .. · .. · · · · · .. ., .. · · · · · · .. · ·" · · 115

· OUTLINE OF THE STUDY CONDUCT · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 2/5

· UNPREDICTED EVENTS CONSIDERED TO HAVE AFFECTED THE

RELIABILITY OF THE STUDYAND DEVIATION FROMTHE PROTOCOL · · · · 5/5 I. Summary......................................................................................................................... 1 II. Purpose ofstudy ............................................................................................................. 1 III. Materials and methods................................................................................................... 1 IV. Results.............................................................................................................................? 

### V. Discussion and conclusion..............................................................................................7 VI. Reference.........................................................................................................................8 Tables 1 Mean irritation scores in acute dermal irritation/corrosion study of m rabbits 2 Clinical signs ofrabbits in acute dermalirritation/corrosion studyof 3 Body weights ofrabbits in acute dermalirritation/corrosion studyof Appendix 1 Individualirritation scores in acute dermalirritation/corrosion studyof in rabbits

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### I. Summary
was investigated for acute dermal irritation/corrosion m male Japanese White rabbits. Three application siteswereprepared inthe clipped dorsal skin ofone rabbit.A 0.5g ofthe test substance was spread on each of 2.5 x 2.5-cm lint patches, and occlusively applied to each application site. First, a patch was removed after application for 3min, and the topical site was observed for the presence or absence of corrosion. Since no corrosion was noted in the application sitejust after removal ofthe patch, the animal was subjected to occlusive exposure for 1hr. As a result, corrosion was notedjust after removal; therefore, no more observation wasperformed. Based on the above results, it was concluded that under the conditions ofthis study. 

### II. Purpose ofstudy was a corrosive substance

As part of safety assessment, irritation/corrosion inrabbits. was investigated for acute dermal



### III.Materials and methods 1. Test substance Note 1)

(1) Name (abbr. name) (2) Chemical name (3) Rational formula (4) Molecularformula (5) Molecularweight (6) CAS No. (7) Lot No. (8) Purity (9) Property at ambient temperature (10) Name ofimpurities and the concentration :Unknown, 0.5% (11) Storingconditions :Light shielding at room temperature Note 1: Characteristics and the stabilitywere based on information (Non-GLP) from the sponsor 

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(12) Expiration date :May 27, 2011 (13) Solubility : Soluble in water, dimethyl sulfoxide (DMSO) and acetone (14) Identity (SOP 13-01-05) (GLP)

(15) Source Name Address : In the study (Study No. H·10233) using the same test substance, before the start of experiment, the infrared absorption spectrum ofthe test substance was measured with an infrared spectrophotometer (Fourier transform infrared spectrophotometer, FT-720, HORIBA, Ltd) (December 28, 2010). Comparing with the infrared absorption spectrum of the lot provided from the sponsor, confirmed the identity of the test substance based on the similarity of main peak shapes.

(16) Handlingofthe residual test substance

Name Address :Residual test substance is returned to the department given below after the end ofthe relevant experiments.

2. Preparation ofthe test substance The test substance was not diluted and used as supplied. 3. Experimental animals Rabbits were chosen according to the OECD Guideline, especially Japanese White rabbits because they were generally used in acute dermal irritation/corrosion studies. Four 10-week old male Japanese White rabbits (Kbs:JW, Healthy) (body weight range: 1.90-1.96kg) produced by Kitayama Labes Co., Ltd. (3052·1 Arai, Ina·shi, Nagano, Japan) were purchased from Oriental Bioservice Kanto, Inc. (3-15·15Azuma, Tsukuba·shi, Ibaraki, Japan). The animals were quarantined during the 6 days following their arrival. During this period, the animals were weighed with a Sartorius electronicbalance (E12000S, Sartorius Co., Ltd.) on the first andlast days, 

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observed for clinical signs once every day, and assessed for health conditions on the last day ofthe period. No abnormal finding was observed throughout the quarantine period, indicating good health conditions. Animals except for the animal used for the first administration were continuously acclimated to housing conditions after the quarantine period, and observed for general conditions. At approximately 24hr before the first administration, the animals' dorsal hair wasremovedwith an electrichair-clipper (2.0- and 0.5-mmedges, DAlTO ELECTRIC MACHINE INDUSTRY CO., LTD.) andpre-treatment skinconditions were examined. Based on the results, 3 healthy animals with good skin conditions were selected for the study, and the other animal which hadisland skins was reserved as one ofpooled animals. Since the experiment was terminated at first administration, used was only one animal. The animal's age at the time of administration was 10 weeks and the body weight was 2.13kg. Cages were identified with color labels (showing the Study no., name ofthe test substance, type ofthe study, sex and temporary animal no. or animal no., etc), and animals with animal nos. written inside of the auricle (left auricle during the quarantine/acclimationperiod and right auricle after grouping) with oil ink. 4. Housing and environmental conditions

(1) Environmental conditions The animals were individually housed in automatic water-washing aluminum cages for rabbits (350W x 480D x 350H mm) in a conventional system animal room (Room No.8 during the quarantine period, Room No.9 after the quarantine period), where the environmental conditions were maintained to a temperature of 20±3°C (actual values: Room No.8: 18.4-22.4oC, Room No.9: 19.6-20.1oC), humidity of 50±20% (actualvalues: Room No.8: 22.6-65.1%, Room No.9: 49.5-58.3%), ventilation frequency of 10 times or more per hour (all-fresh-air system), and lighting of 12hr per day (from TOO a.m. to 7:00p.m., 150-300 lux).

Cages and feeders were sterilized with 500-fold aqueous dilution of sodium hypochlorite prior to use, and changed for new ones at the time of changing the animal room. The animal room was cleaned every day after work and the floor was sterilized by wiping with 200-fold aqueous dilution of benzethonium chloride (Hyamine solution; Daiichi Sankyo Co., Ltd.). (2) Feed Pelletdiet for experimental animals CR-3 (Lot No. R3070 and, R3080 CleaJapan, Inc., 1-2-7 Higashiyama, Meguro·ku, Tokyo, Japan) was provided 

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feeders. Feed analysis for impurities and contaminants in the lot used in the study was entrusted by the maker to Tokyo Kenbikyo·in foundation (44-I Nihonbashi Hakozaki-cho, Chuo·ku, Tokyo, Japan). Based on the results (obtained as a copy), the conformity to the standards specified by Nippon Experimental Medical Research Institute Co., Ltd. was confirmed before feeding. (3) Drinkingwater Shibukawa·city tap water was provided 

### ad libitum via an automatic watering system. Water analysis in the samples periodically collected at sites specified by this facility was entrustedtoEnvil:onmentalTechnical Co., Ltd. (I709·I Kaneko·cho, Takasaki·shi, Gunma, Japan) based on the "Ministerial Ordinance Concerning Water Quality Standards (MHLW Ordinance No. IOI, 2003)", "PartialAmendment ofMinisterial Ordinance Concerning Water Quality Standards (MHLW Ordinance No. I74, 2008)", and "PartialAmendment ofthe Ordinance for Enforcement ofthe Water Law (MHLW Ordinance No.I75, 2008)". Based on the results, the conformity to the above-mentioned water quality standardswas confirmed. 5. Number ofanimals, dose level and animal numbers

(I) Number ofanimals, dose level and animal numbers Number ofanimals Dose level Animal No. I 0.5g/treatment site I (2)Justificationfor selection ofdose level and number ofanimals. The dose level and number ofanimals were according to the OECD Guideline. 6. Administration

(I) Route ofadministration Percutaneous route (occlusive patch) (2) Administration method Hair-clipped areas (approximately 2.5 x 2.5 em, approximately 6 cm2 each) ofthe dorsal region served as application sites. A 0.5g of the test substance was evenly spread on a 2.5 x 2.5-cm lint patch (OSATO EISEl ZAIRYO SEIZOSHO, 

### Y.K.) and applied to each application site to contact to the skin. The lint patch was occluded with adhesive elasticbandage cutin 3.5 x 3.5cm (Silky Tex, ALCARE CO., LTD.) and fixed with Silky Tex and surgical tape (NICHIBAN CO., LTD.) wrapped around the trunk. 4

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## CD First administration (Figure 1) Three application sites (A to 

## c: Figure 1) were prepared on 1 animal (Animal No.1). Firstly, a patch ofthe test substance was applied to SiteA (not wrapped with dressing around the trunk). The patch was removed after an exposure period of 3min and the topical site was observed for corrosion. Because of the absence of corrosion, another patch was applied to Site B. The patch was removed after an exposure period of lhr and the topical site was observed for corrosion. Because of the presence of corrosion in Site B, the experiment was terminated and further process was not performed. At the time ofremoving each patch, the test substance remaining on the application sites was removed by gently wiping with absorbent cotton (J.P., KAWAMOTO CORPORATION) moistened with waterfor injection (J.P., Lot No.00610D, Fuso Pharmaceutical Industries, Ltd.). Head 

### [A] A: 3min o:Application site 

## [[] B: lhr 

### [QJ C:4hr Tail Figure 1. Application sites and duration 

## ® Administrationfor confirmation Since corrosion was observed at observation in the first administration, further administration for confirmation was notperformed. (3) Rationale for administration route and method The administration route and method were m accordance with the OECD Guideline. 7. Observation and measurement

(1) Observation and scoring for dermalirritation/corrosion Since corrosion was observed at observation in the first administration, scoring was notperformed.



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(2) Evaluation criteria for dermalreactions



## CD Erythema and escharformation score No erythema.....................................................................................................O Very slight erythema (barelyperceptible) .....................................................1 Well defined erythema ....................................................................................2 Moderate to severe erythema .........................................................................3 Severe erythema (beet redness) to slight eschar formation preventing grading erythema........................................................................4 Maximum score: 4 

## ® Edema formation score No edema..........................................................................................................O Very slight edema (barely perceptible) ..........................................................1 Slight edema (edges ofarea well defined by definite raising)......................2 Moderate edema (raised approximately 1 mm).............................................3 Severe edema (raised more than 1 mm and extending beyond area of exposure) ..........................................................................................................4 Maximum score: 4 (3) Calculation ofP.C.I. Since corrosion was observed m the first administration, P.C.I was not calculated. (4) A.F.N.O.R. standards for dermal irritationintensity P.C.I. < 0.5 0.5**~** P.C.I.**~** 3 3 < P.C.I.**~** 5 5 < P.C.I.**~** 8 Non irritant Slightlyirritant Moderatelyirritant Severely irritant (5) Clinical observation and body weight measurement One treated animal was observed for clinical signs, and weighed on the day of administration (Day 0) with a Sartorius electronic balance (E 12000S, Sartorius Co., Ltd.). (6) Handling ofthe residual animals After the end ofobservation, the used animals were reserved as pooled animals.



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8. Statisticalanalysis The meanvalueswere calculatedfor the scores and values obtainedinthis study; however statistical analyses were notperformed. 

### IV. Results 1. Evaluation ofdermal irritation/corrosion (Tablel,Appendix 1) In the 3min·treatment site in the first administration, very slight erythema was noted at observationjustafterremoval ofthepatch; however, no necrosis or corrosive reaction was observed. In the lhr-treatment site, well defined erythema, and edema raised lmm or more and extending beyond the area ofexposure, as well as corrosion, were noted at observationjust afterremoval ofthe patch,. 2. Clinical observation (Table 2) No abnormal finding was observed during the observationperiod. 3. Body weight measurement (Table 3) The body weight atthe time ofadministration was 2.13kg. 

### V. Discussion and conclusion
was investigated for acute dermal irritation/corrosion in male Japanese White rabbits. Three application sites were prepared inthe clipped dorsal skin ofone rabbit.A 0.5g ofthe test substance was spread on each of 2.5 x 2.5-cm lint patches, and occlusively applied to each application site. First, a patch was removed after application for 3min, and the topical site was observed for the presence or absence of corrosion. Since no corrosion was observed in the application site just after removal of the patch, the animal was subjected to occlusive exposure for lhr. Just after removal, corrosion was observed adding to well defined erythema and severe edema; therefore, no more application was performed. Based on the above results, it was concluded that was a corrosive substance under the conditions ofthis study.



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### VI. Reference 1) Guillot, J.P. et al.: Chemicals, ocular and cutaneous local tolerance, "cosmetic", A.F.N.O.R. and O.E.C.D. protocols, 2.2 Test for the evaluation of the cutaneous, Irritation and/or corrosivity in the rabbit, 20-25, 1982.



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### Table 1 Mean irritation scores in acute dermal irritation/corrosion study of
Test substance Lesion Number of

animals 1 hr. Erythema 

### * ( * 1 Edema 

### * ( * Total 

### * ( * ( ) :mean P.C.I.: Primary Cutaneous IrritationIndex. 1) :Time after removal ofthe closed patch 2) :AssociationFrancaise de Normalization M . 1) ean scormg 24 hr. 48 hr. ) 

### * ( * ) 

### * ( * ) 

### * ( * ) 

### * ( * ) 

### * ( * ) 

### * ( *

* :Not scored as corrosion was notedjustafter removal ofthe closed patch. ND: Not detected 72 hr. ) 

### * ( * ) ) 

### * ( * ) ) 

### * ( * ) 

### in rabbits
P.C.I. Classification Z)

ND Corrosive

Study No. H-10234

### Table 2 Clinical signs ofrabbits in acute dermal irritation/corrosion study of Observationperiod (day) Animal number 0

1 day 0: Initiation day

- :No abnormality Study No. H-10234

### Table 3 Body weights ofrabbits in acute dermal irritation/corrosion study of Observationperiod (day) Animal number 0

1 2.13

Gain Day 0: Initiation day Unit: kg Study No. H-10234

### Appendix 1 Individual irritation scores in acute dermal irritation/corrosion study of in rabbits Animal Lesion 1hr. Test substance number Individualirritation score 1)**~--···"~~-~---------.**·----·----- --**---·------~-----···-··~--------** . """_" ____**-·------~-~-------** ·----------·-·- ----**--~~--** ..------··----·--··- 

### 24 hr. 48 hr. 72 hr. Erythema 

### * * 

### * * 1 Edema 

### * * 

### * * Other remark 

### * * * * * Erythema 

### * Total score Edema 

### * * 

### * * * * Mean total score 

### * * * * 1J : 

### Time after removal ofthe closed patch.



### * :Not scored as corrosion was notedjust after removal ofthe closed patch. Study No. H-10234

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### QUALITYASSURANCE STATEMENT Title :AnAcute Irritation/Corrosion Study of Study No. :H-10234 Inspectionitems Studyprotocol Amendmentto the protocol (1) Animalreceipt and body weight measurement at delivery Quarantine/acclimatization, animal health assessment and grouping Test substance weighing and administration (3min/1hr applicationsin the first administration) Observation ofskinirritation/corrosion Performed Dec. 28, 2010 Jan. 6, 2011 Jan. 6, 2011 Jan. 11, 2011 Jan. 12, 2011
(3min/1hr applications m the finJan. 12, 2011 administration) in Rabbits
Reported to Study director Management Dec. 28, 2010 Dec. 28, 2010 Jan. 6, 2011 Jan. 6, 2011 Jan. 6, 2011 Jan. 6, 2011 Jan. 11, 2011 Jan. 11, 2011 Jan. 12, 2011 Jan. 12, 2011

Jan. 12,2011 Jan. 12,2011 Raw data Finalreport

(draft) (re·inspection) (final) Jan. 24·25, 2011 Jan. 25, 2011 Jan. 25, 2011 Jan. 24·25, 2011 Jan. 25, 2011 Jan. 25, 2011 Jan. 31, 2011 Feb. 5, 2011 Jan. 31,2011 Jan. 31,2011 Feb.5,2011 Feb.5,2011 Based on the above inspections, I have certified that this study was conducted in accordance with the GLP, protocol and standard operatingprocedures prescribed by Nippon Experimental Medical Research Institute Co., Ltd., and that this report precisely describes the methods and procedures employed in this study, and accurately reflects the raw data obtainedby the conduct ofthe study. QualityAssurance Unit Manager : MikiWakabayashi <Impression ofseal> Date: February 5, 2011 Nippon ExperimentalMedical Research Institute Co., Ltd