l!f:!IH-10234 

English version of the final report 

Final Report 

An Acute Dermal Irritation/Corrosion Study of in Rabbits 

Study No. H-10234 

February 5, 2011 

Nippon Experimental Medical Research Institute Co., Ltd. 

1967·11 Arima, Shibukawa·shi, Gunma, Japan 
I!1!J H-10234 

STATEMENT OF COMPLIANCE 

Title : An Acute Dermal Irritation/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: J lA. ne il . 2o I 2.-

Takashi Sato, M.S. 

Study Director (translator) 

Nippon Experimental Medical Research Institute Co., Ltd. 
PREPARATION OF THE FINAL REPORT 

Title: An Acute Dermal Irritation/Corrosion Study of 

Study No.: H-10234 

in Rabbits 

I!L!J H -10234 

1/5 

The study listed above was performed in compliance with the Organization for 

Economic Co-operation and Development (OECD) Principles of Good 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 this report. 

Study director: Takashi Sato, M.S. (Impression of the seal) 

Date : February 5, 2011 

Nippon Experimental Medical Research Institute Co., Ltd. 
l!'f!l H-10234 

2/5 

OUTLINE OF THE STUDY CONDUCT 

Title: An Acute 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 

Experimental Medical Research Institute Co., Ltd. (Approval No. 2006013). 

5. Sponsor 

Name: 

Address: 

6. Contract laboratory 

Name: 

Address: 

Nippon Experimental Medical Research Institute Co., Ltd. 

1967·11 Arima, Shibukawa-shi, Gunma, Japan 

7. Testing facility 

Name: 

Address: 

Shibukawa Laboratories 

Nippon Experimental Medical Research Institute Co., Ltd.6. 

1967·11 Arima, Shibukawa·shi, Gunma, Japan 
8. Storage of records and materials 

(1) Storage period 

The items are stored for 10 years after completion of the study. 

(2) Storage items and storing location 

~H-10234 

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The study protocol and its amendment (original), documents relating to 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 of the storing institute 

1967 ·11 Arima, Shibukawa ·shi, Gunma, Japan 

9. Schedule of the study 

Study initiation 

Animal arrival 

End of the quarantine period 

Start of the experiment 

First administration 

Observation 

Completion of the experiment 

Submission of the draft final report 

Preparation of the final report 

Completion of the 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 

:February 5, 2011 
10. Study personnel and work responsibility 

l!i!'J H -10234 

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Study director, protocol preparation, work supervising/management and preparation 

of the final report : Takas hi Sa to Note) 

Clinical observation and body weight measurement during the quarantine/ 

acclimation period : Masaaki Shirai, Yoko Wakana, 

Animal health assessment 

Preparation of the test substance 

Control of the test substance 

Moeko Masuyama, Takashi Sato 

: Yoko Wakana 

: Takashi Sato 

: Takashi Sato 

Administration, removal, washing, clinical observation and body weight 

measurement : Masaaki Shirai, Yoko Wakana, 

Takashi Sato 

Observation of treatment sites : Yoko Wakana, Takashi Sa to 

Note) Affiliation: Safety Research Department, Nippon Experimental Medical Research 

Institute Co., Ltd. 

Address: 1967·11 Arima, Shibukawa·shi, Gunma, Japan 
l!i!J H -10234 

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UNPREDICTED EVENTS CONSIDERED TO HAVE AFFECTED THE 

RELIABILITY OF THE STUDY AND DEVIATION FROM THE PROTOCOL 

Title: An Acute Dermal Irritation/Corrosion Study of 

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. on January 7, 2011, recording 

the lowest value of 29.9% and 22.6%, respectively. The deviations occurred due to 

vapor supply disruption caused by an error of the maintainer shutting the vapor valve 

of the boiler. 

2. Deviation from the protocol 

There were no relevant items. 

3. Effects on the reliability of the study 

The deviations of the humidity were considered to have no effect on the reliability of 

the study, because no abnormal finding was observed in general conditions of animals 

after the humidity returned to the normal range. 
f!l!'JH-10234 

CONTENTS 

page 

·PREPARATION OF THE FINAL REPORT · .. ·-- .. · .. · · · · · .. ., .. · · · · · · .. · ·" · · 115 

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

· UNPREDICTED EVENTS CONSIDERED TO HAVE AFFECTED THE 

RELIABILITY OF THE STUDY AND DEVIATION FROM THE PROTOCOL · · · · 5/5 

I. Summary ......................................................................................................................... 1 

II. Purpose of study ............................................................................................................. 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 of rabbits in acute dermal irritation/corrosion study of 

3 Body weights of rabbits in acute dermal irritation/corrosion study of 

Appendix 

1 Individual irritation scores in acute dermal irritation/corrosion study of 

in rabbits 
l!l!J H -10234 

I. Summary 

was investigated for acute dermal irritation/corrosion m male Japanese 

White rabbits. 

Three application sites were prepared in the clipped dorsal skin of one rabbit. A 0.5g 

of the 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 site just after removal of the patch, the animal 

was subjected to occlusive exposure for 1hr. As a result, corrosion was noted just after 

removal; therefore, no more observation was performed. 

Based on the above results, it was concluded that 

under the conditions of this study. 

II. Purpose of study 

was a corrosive substance 

As part of safety assessment, 

irritation/corrosion in rabbits. 

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) Molecular formula 

(5) Molecular weight 

(6) CAS No. 

(7) Lot No. 

(8) Purity 

(9) Property at ambient temperature 

(10) Name of impurities and the concentration 

:Unknown, 0.5% 

(11) Storing conditions : Light shielding at room temperature 

Note 1: Characteristics and the stability were based on information (Non-GLP) from 

the sponsor 

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l!f!J H -10234 

(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 of the 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) Handling ofthe residual test substance 

Name 

Address 

:Residual test substance is returned to the department given 

below after the end of the 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 electronic balance (E12000S, Sartorius Co., Ltd.) on the first and last days, 

2 
I!E!J H -10234 

observed for clinical signs once every day, and assessed for health conditions on the 

last day of the 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 

was removed with an electric hair-clipper (2.0- and 0.5-mm edges, DAlTO ELECTRIC 

MACHINE INDUSTRY CO., LTD.) and pre-treatment skin conditions were examined. 

Based on the results, 3 healthy animals with good skin conditions were selected for 

the study, and the other animal which had island skins was reserved as one of pooled 

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 of the test 

substance, type of the 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/acclimation period 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: 18.4-22.4 oC, Room No.9: 19.6-20.1 oC ), humidity of 

50±20% (actual values: 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 

Pellet diet for experimental animals CR-3 (Lot No. R3070 and, R3080 Clea Japan, 

Inc., 1-2-7 Higashiyama, Meguro·ku, Tokyo, Japan) was provided ad libitum via 

3 
~H-10234 

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) Drinking water 

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 entrusted to Envil:onmental Technical Co., Ltd. (I709·I Kaneko·cho, 

Takasaki·shi, Gunma, Japan) based on the "Ministerial Ordinance Concerning 

Water Quality Standards (MHLW Ordinance No. IOI, 2003)", "Partial Amendment 

of Ministerial Ordinance Concerning Water Quality Standards (MHLW Ordinance 

No. I7 4, 2008)", and "Partial Amendment of the Ordinance for Enforcement of the 

Water Law (MHLW Ordinance No.I75, 2008)". Based on the results, the conformity 

to the above-mentioned water quality standards was confirmed. 

5. Number of animals, dose level and animal numbers 

(I) Number of animals, dose level and animal numbers 

Number of animals Dose level Animal No. 

I 0.5g/treatment site I 

(2)Justification for selection of dose level and number of animals. 

The dose level and number of animals were according to the OECD Guideline. 

6. Administration 

(I) Route of administration 

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 elastic bandage cut in 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 
I!'C!J H -10234 

CD First administration (Figure 1) 

Three application sites (A to c: Figure 1) were prepared on 1 animal (Animal 

No.1). Firstly, a patch of the test substance was applied to Site A (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 of removing each patch, the test substance 

remaining on the application sites was removed by gently wiping with absorbent 

cotton (J.P., KAWAMOTO CORPORATION) moistened with water for 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 

® Administration for confirmation 

Since corrosion was observed at observation in the first administration, further 

administration for confirmation was not performed. 

(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 dermal irritation/corrosion 

Since corrosion was observed at observation in the first administration, scoring 

was not performed. 

5 
l!i!] H -10234 

(2) Evaluation criteria for dermal reactions 

CD Erythema and eschar formation score 

No erythema ..................................................................................................... O 

Very slight erythema (barely perceptible) ..................................................... 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 of area 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 irritation intensity 

P.C.I. < 0.5 

0.5 ~ P.C.I. ~ 3 

3 < P.C.I. ~ 5 

5 < P.C.I. ~ 8 

Non irritant 

Slightly irritant 

Moderately irritant 

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 of the residual animals 

After the end of observation, the used animals were reserved as pooled animals. 

6 
l!'f!1 H-10234 

8. Statistical analysis 

The mean values were calculated for the scores and values obtained in this study; 

however statistical analyses were not performed. 

IV. Results 

1. Evaluation of dermal irritation/corrosion (Tablel, Appendix 1) 

In the 3min·treatment site in the first administration, very slight erythema was 

noted at observation just after removal ofthe patch; 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 of exposure, as well as corrosion, 

were noted at observation just after removal of the patch,. 

2. Clinical observation (Table 2) 

No abnormal finding was observed during the observation period. 

3. Body weight measurement (Table 3) 

The body weight at the time of administration was 2.13kg. 

V. Discussion and conclusion 

was investigated for acute dermal irritation/corrosion in male Japanese 

White rabbits. 

Three application sites were prepared in the clipped dorsal skin of one rabbit. A 0.5g 

of the 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 of this study. 

7 
I!T!J H -10234 

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. 

8 
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 Irritation Index. 

1) : Time after removal of the closed patch 

2) :Association Francaise de Normalization 

M . 1) ean scormg 

24 hr. 48 hr. 

) * ( * ) * ( * 

) * ( * ) * ( * 

) * ( * ) * ( * 

* : Not scored as corrosion was noted just after removal of the closed patch. 

ND: Not detected 

72 hr. 

) * ( * ) 

) * ( * ) 

) * ( * ) 

in rabbits 

P.C.I. Classification Z) 

ND Corrosive 

Study No. H-10234 
Table 2 Clinical signs of rabbits in acute dermal irritation/corrosion study of 

Observation period (day) 

Animal number 

0 

1 

day 0: Initiation day - :No abnormality 

Study No. H-10234 
Table 3 Body weights of rabbits in acute dermal irritation/corrosion study of 

Observation period (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 1 hr. Test substance number 

Individual irritation score 1) ~--···"~~-~---------. ·----·----- -- ---·------~-----···-··~-------- . """_" ____ -·------~-~------- ·----------·-·- ---- --~~--.. ------··----·--··-

24 hr. 48 hr. 72 hr. 

Erythema * * * * 

1 Edema * * * * 

Other remark * * * * 

Erythema * 

* Total score Edema 

* * * 

* * * 

Mean total score * * * * 

1J : Time after removal of the closed patch. 

* : Not scored as corrosion was noted just after removal of the closed patch. 

Study No. H-10234 
l!T!J H-10234 

QUALITY ASSURANCE STATEMENT 

Title :An Acute Irritation/Corrosion Study of 

Study No. : H-10234 

Inspection items 

Study protocol 

Amendment to the protocol (1) 

Animal receipt and body weight 

measurement at delivery 

Quarantine/acclimatization, animal health 

assessment and grouping 

Test substance weighing and 

administration (3min/1hr applications in 

the first administration) 

Observation of skin irritation/corrosion 

Performed 

Dec. 28, 2010 

Jan. 6, 2011 

Jan. 6, 2011 

Jan. 11, 2011 

Jan. 12, 2011 

(3min/1hr applications m the fin Jan. 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 

Final report 

(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 operating procedures 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 

obtained by the conduct of the study. 

Quality Assurance Unit Manager : Miki Wakabayashi <Impression of seal> 

Date: February 5, 2011 

Nippon Experimental Medical Research Institute Co., Ltd