K06-1279 

212 

The inspection result of following phase was reported to the study director and 

management based on the report of process-based inspection relevant to this study type 

and timeframe. --"-,~, 

Phase Date of Inspection 

Date Reported to Study 

Director and Management 

Examination of Solubility of the Test 

Substance 

October 10,2008 April 14, 2009 

Preparation and Management of Positive 

Control Substance 

November 26, 2008 April 14,2009 

Preparation of Medium and Reagent December 12, 2007 April 14,2009 

Management of Cells 

November 30, 2007 

and 

March 4, 2008 

April 14,2009 

Collection of Cells and Preparation of 

Specimens 

November 7, 2008 April 14, 2009 

I, the undersigned, hereby declare that this report provides an accurate description of the 

methods and procedures used in this study and that the reported results accurately reflect 

obtained raw data. 

l-lead, Quality Assurance Unit: Signed in original April 15, 2009 

Masakazu Yoshida 

iv 

145 
K06-1279 


TABLE OF CONTENTS 


Page 


TITLE .............................................. 3 


SPONSOR .............................................. 
 3 


TESTING FACiLITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
 3 


PURPOSE OF STUDY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 


TESTING MET! [00 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 


GLP COMPLIANCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 


PERIOD OF STUDY ........................................ 4 


STORAGE AND RETENTION PERIOD OF DATA . . . . . . . . . . . . . . . . . . . . 4 


RETENTION Of ORIGINAL DOCUMENTS . . . . . . . . . . . . . . . . . . . . . . . . 4 


STUDY DIRECTOR AND PERSONS CONCERNED WITH 


THE STUDY Ai'lD THE OPERATION ................................ 4 


APPROVAL BY AUTHOR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 


SUMMARY 	 6 


MATERIALS AND METHODS 

1. 	 TEST SUBSTANCE AND POSITIVE CONTROL SUBSTANCES 7 


2. 	 CELLS ................................ 9 


...... ..... .......... .... ....... 9
 3. 	 MEDIUM AND S9 MIX 

....... " . . .• . . • . . .• . . .• . . .. . . . • 10
 4. 	 CELL PRE-CULTURE 

5. 	 PREPARATION Of TEST SUBSTANCE SOLUTION 


AND POSITIVE CONTROL SUBSTANCE SOLUTIONS ............ 10 


6. TEST PROCEDURE 	 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 


7. 	 JUDGEMENT CRITERIA OF RESl;LTS .. . . . . . . . .. . . . . . . .. . . . . . 14 


8. 	 VAUDITY OF TEST . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 


FACTORS AFFECTED RELIABILITY OF TEST ........ , . . . . . . . . . . . .. 15


TEST RESULTS 

1. CELL GROWTH INHIBITION TEST . . . . . . . . . . . . . . . . . . . . . . . . 15 


2. CHROMOSOMAL ABERRATION TEST ........................ 15 


3. TYPICAL PHOTOS 	 . . . . . . . . . . . . . . . . . . . . . . . . 17 


DISCUSSION AND CONCLUSION ....... ........... .......... 	 17


REFERENCES . . . . . . • . . . . . . . . . . . . . . . . . . . . . 	 18

]­

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K06-1279 


TABLES, FIGURES AND PHOTOS 


Table 1 Results of cell growth inhibition test of 

Karenz MT (Mixture ofPEMB and PE3MB) ~ .. • • • * • • • '" • • • • .. .. 19 

Table 2 Results of chromosomal aberration test of 

Karenz MT (Mixture of PEMB and PE3MB) · ... . . . .. . .. .. . ~ .. " . ~ 20 

Table 3 Results of chromosomal aberration test 

(short-tenn treatment without S9 mjx) .... '" .. .. .. .. .. . . .. ~ ........ 2] 

Table 4 Results of chromosomal aberration test 

(short-tenn treatment with 89 mix) ·.. .. ~ ", ~ ... .. .,,-.. .. . . . 22 

Table 5 Results of chromosomal aberration test (continuous treatment) . ~ ~ . 23 

Fig. 1 Results of cell growth inhibition test of 

Karenz MT (Mixture of PEMB and PE3MB) · ................ 24 

Fig. 2 eel1 growth rate in chromosomal aberration test of 

Karenz MT (Mixture ofPEMB and PE3MB) ... .. ....... .... 25 

Fig. 3 Results of chromosomal aberration test in short-tenn treatments of 

Karenz MT (Mixture of PEMB and PE3MB) . . . . . . . . . . . . . . . . 26 

Fig. 4 Results of chromosomal aberration test in continuous treatment of 

Karenz MT (Mixture ofPEMB and PE3MB) 27 

Photo 1 Normal. cell 28 

Photo 2 Structural aberration induced by 

Karenz MT (Mixture of PEMB and PE3MB)' . . . . . . . . . . . . . . . . . .. 28 

Photo 3 Structural aberration induced by 

Karenz MT (Mixture of PEMB and PE3MB)' . . . . . . . . . . . . . . . . . .. 29 

. :2 

147 
K06-1279 


Study Code: K06-1279 

Test Substance Code: HR7645 

Sponsor Code: S-0170 

TITLE 

Chromosomal Aberration Tesl of Karenz MT (Mixture of PEMB and PE3MB) Using 

Cultured Mammalian Cells 

SPONSOR 

Showa Denko K.K. 

13-9, Shiba Daimon I-Chome, Minato-ku, Tokyo 105-8518, Japan 

TESTING FACILITY 

CERI Hita 

Chemicals Evaluation and Research Institute, Japan 

822, 3-chome, Ishii-machi, llita, Oita 877-0061, Japan 

PURPOSE OF STUDY 

The ability of the test substance to induce chromosomal aberrations was examined by 

using Chinese hamster lung fibroblasts (CHLlIU cells). 

TESTING METHOD 

This study was conducted in accordance with «III Mutagenicity Test: Chromosomal 

Abellation Test Using Cultured Mammalian Cells" prescribed in "Concerning Testing 

Methods Relating to the New Chemical Substances" on Japanese Test Guideline 

(Notification No. 1121002 of the Pharmaceutical and Food Safety Bureau, MHLW, No.2 

(November 13, 2(03) of the Manufacturing Industries Bureau, Tv[ ETI & No. 031121002 of 

the Environmental Health DepaI1ment, MOE (November 21, 2003)). 

GLP COMPLIANCE 

This study was conducted In compliance wiLh "Concerning Standard of the Testjng 

Facilities Conducting thc Test Relating to the New Chemical Substances" on Japanese 

GLP (Notification No. 1121003 of the Pharmaceutical and Food Safety Bureau, MHLW, 

No. 3 (November 17, 2003) of the Manufacturing Industries Bureau, METl & No. 

031121004 ofthc EnvirollmentalllealLh Department, MOE (Novemher 21,2(03). 

- 3 

148 
K06-1279 


PERIOD OF STUDY 

Commencement of Study: November 26, 2008 

Initiation of Experiment 

(Initiation of Cell Growth Inhibition Test): December 1, 2008 

Completion of Experiment 

(Completion of Observation of Specimens): March 17,2009 

Completion of Study: April 15,2009 

STORAGE AND RETENTION PERIOD OF DATA 

The raw data, protocol, protocol amendment, letter of test request, table of test substance 

information, final report, other record documents and specimens will be stored in the 

archive of this testing facility for 10 years after the date of receipt of the notification that 

they are applicable to Article 4, Paragraphs 1 or 2, Article 4-2, Paragraphs 2, 3 or 8, 

Article 5-4, Paragraph 2, Article 24, Paragraph 2 or Article 25-3, Paragraph 2 of the 

Japanese Chemical Substances Control Law No. 117 (1973). Thc sponsor will inform 

the datc of receipt of the notification to this testing facility. After termination of the 

retention period, any measures taken will bc done so with the approval ofthe sponsor. 

The specimens to which the quality will be deteriorated will be retained only for the 

period when the quality can be secured. The sponsor's consent will be obtained before 

abandonment. 

RETENTION Of ORIGINAL DOCUMENTS 

An original protocol, original protocol amendments and an original final report will be 

retained at the testing facility. The copies of their original that the study director will be 

recognized to be accurate copy will be sent to the sponsor. 

STUDY DIRECTOR AND PERSONS CONCERNED WITli TjlE STUDY AND THE 

OPERATION 

Study Director: Shozo Ogura 

Section 3, CERI Hita 

Persons Concerned with The Study and Their Operation: 

Renge Suzuki 

(Preparation oftest substance solution and cell treatment) 

Shozo Ogura 

(Microscopic observation of specimens) 

Junko Kawaguchi 

(Microscopic obscrvation of specimens) 

4 ­

149 
K06-1279 


Chisato Iwami 

(Microscopic observation of specimens) 

APPROVAL BY AUTHOR 

Study Director: Signed in original 

Shozo Ogura 

April] 5,2009 

.5 

150 
K06-1279 


SUMMARY 


The ability of Karenz MT (Mixture of PEMB and PE3MB) to induce chromosomal 

aberrations was investigated by using Chinese hamster lung fibroblasts (CHUIl] cells). 

Based on the result of cell growth inhibition test, the doses in the chromosomal aberration 

test were set at 6.91, 9.77, 13.8, 19.5,27.6,39.1 and 55.2 ~g/mL in short-term treatment 

without 89 mix, 7&.1, 110, 156, 221, 313 and 442 ~tg/mL in short-tenn treatment with S9 

mix and 4.8&, 6.91, 9.77,13.8,19.5 and 27.6 ~g/mL in 24 hours continuous treatment. 

In chromosomal aberration test, the observation doses for evaluation were selected at 19.5, 

27.6 and 39.1 pg/mL in short-term treatment without S9 mix, 156, 221 and 313 Ilg/mL in 

short-term treatment with S9 mix and 4.88, 6.91, 9.77, 13.8 and 19.5 Ilg/mL in 24 hours 

continuous treatment. In the observation, the frequencies of cells with structural aberrations 

and numerical aberration cells were scored. 

As a result of observation of specimens, the frequencies of numerical aberration cclls showed 

below 5% at all observation doses of the test substance in all treatment methods, therefore, 

numerical aberration was judged to be negative. The frequencies of cells with structural 

aberrations showed below 5% at all observation doses of the test substance in the short-ternl 

treatment without and with S9 mix, therefore, stmctural aberrations was judged to be 

negative in these treatments. However, the frequencies of cells with structural aberrations 

showed 4.5% at 4.88 p.g/mL and 11.5% at 6.91 p.glmL and the maximum frequency was over 

1 0% in 24 hours continuous treatment. The frequencies of cells with stmctural abelTations 

at 9.77, 13.8 and 19.5 ~g/mL of test substance were 8.5, 6.5 and 3.0%, respectively, and a 

dose related decrease was observed. Bccause the cell groVvth rates at 13.8 and 19.5 Ilg/mL 

of test substance was about 50% and the frequency of metaphases was low at 19.5 pg/mL of 

test substance, it was considered that thc the appearance of chromosomal aberration was 

inhibited by cytotoxicity at 9. 77 ~lg/l11L or more. The frequencies of cells with stmctural 

aberrations at 4.88 Ilg/mL was 4.5%. Then, it was evaluated that a dose related increase 

was observed between 4.88 and 6.91 p.g/mL of test substance. Therefore, structural 

abcrration was judged to be positive in 24 hours continuous treatment. 

On the other hand, the frequencies of cells with structural aberrations or numerical aberration 

cells in the ncgative control treated with dimethyl sulfoxide showed below 5%, and the 

fi'equencies of cells with structural abelTations in the positive controls treated with 

mitomycin C or cyclophosphamide monohydrate, showed morc than 20%, indicating the 

proper performance of the present study. 

it was concluded that Karenz MT (Mixture of PEMB and PE3MB) did not induce numerical 

aberration but it indllced structural aberration under the present test conditions . 

.- () 

151 
K06-1279 


MATERIALS AND METHODS 

1. TEST SUBSTANCE AND POSITIVE CONTROL SUBSTANCES 

.....}.·L ...T.~~~.~~!?~.t.~I!~.~ .(I.l!f?.1E?~~~~.1! X~~~i.q~.'! .~Y. !h~ .~p.?!!~?~L ............................................... . 


7 ­

152 
K06-1279 


8) 	 Storagc conditions 

Stored at room temperature (cabinet No. 1 in the test substance storage room, 

permissible rangc: 10-30°C) and to avoid contact with oxidizing agcnts. 

9) 	 Prccautions 

Gloves, a mask, a head cap, safety gJasses and a lab coat were worn. The test 

substance was used in the draft ehamber because of its characteristic odor. 

1.2 Positive Control Substances 

1) 	 Mitomycin C (MMC) 


Manufacturer: Kyowa I1akko Kirin Co., Ltd. 


Lot No.: 518AGJ 


Appearance: royal purple powder 


Content: 102% 


Grade: for injection 


2) 	 Cyclophosphamide monohydrate (CPA) 

Manufacturer: Wako Pure Chemical Industries. Ltd. 


Lot No.: WKL2245 


Appearance: white crystals or crystallinc powder 


Content: 98.9% 


Grade: for biochemistry 


3) 	 Storage conditions 

MMC was stored at room temperature (cabinet No.2 in the test substance storage 

room, permissible range: 10-30°C) and CPA in a cold dark place (refrigerator No. 

005 in the test substance storage room, permissiblc range: I-10°C). 

4) 	 Precautions 


Gloves, a mask. a head cap and a lab coat were worn. 


- 8 

153 
K06-1279 


2. CELLS 

2.1 	 Cell Line and Reason for Sclection 

Chinese hamster lung fibroblasts (CHLiIU cells) were supplied by Hcalth Science 

Research Resources l3ank, Japan Hcalth Sciences foundation on April 17, 2002. The 

modal number of chromosomes was 25 per cell. The doubling time was about 15 

hours. It was confirmed in tbe testing facility that the cells were mycoplasma free and 

the spontaneous frequencies of cclls with structural aberration and thc numerical 

aberration cells were below 5%. 

CHLlIU cells have been recommended the availability on in vitro chromosome 

aberration test prcscribed in "Concerning Testing Methods Relating to the New 

Chemical Substances" on Japanese Test Guideline. 

2.2 	 Storage 

Cells were suspended in medium lEagle's minimum essential medium (Nissui 

Pharmaceutical Co., Ltd.) and 10 vol% heat-inactivated newborn calf serum (NBCS, 

Sanko J\myaku Co., Ltd.)] including 10 vol% DMSO and were frozen in liquid 

nitrogen. 

2.3 	 Culture Condition 

Cells were cultured in a C02 incubator (MCO-18AIC, SANYO Electric Co.), which 

was sct at 37°C and 5% CO2 under humid condition. 

2.4 	 Subculture 

Cells were subcultured in 90-mm diameter Petri dishes (Nunc A/S) twice a week. 

Passage number of cells was at 14 for the cell gro\\1h inhibition test and 18 for the 

chromosomal aberration test after the receipt. 

3. MEDIUM AND S9 MIX 

3.1 	 Medium 

L-glutamine (final concentration: 0.292 giL) and sodium hydrogen carbonate (final 

concentration: approximately 1.85 gIL) were added to Eagle's minimum essential 

medium (Lot 1:\0. 588803, Nissui Phannaccutical Co., Ltd.) and basal medium (MEM) 

was prepared. This medium was then supplemented with ] 0 vol% heat-inactivated 

NBC8 (Lot No. A8C29136, Hyelone). 

3.2 	 S9 Mix 

1 ) 	 Rat liver S9 

S9 (Lot No. 08071104, manufactured on July 11, 2008, 89 protein content: 20.4 

mg/mL, Oriental Yeast Co., Ltd.), which was prepared from livers of 7-week-old 

male SD rats administered intraperitoneally a combination of phenobarbital (one 

time at 30 mglkg and three times at 60 mg/kg) and 5,6-benzoflavone (one time at 80 

mg/kg) was used. 89 was frozen and preserved in an ultra-deep freezer (MDF9 

154 
K06-1279 


U481;\ TR, SANYO Electric Co., Ltd., the tolerance temperature: below -80°C) 

until use. S9 was used within six months after the day of manufacturing. 

2) 	 Composition of S9 mix 

One milliliter of S9 mix consisted of 0.3 mL S9, 5 ).lmol MgCh, 33 /lOlol KCl, 5 

).lmol G-6-P, 4 ).lmol NADP and 4 ).lmol HEPES (pH 7.2) and S9 mix was preparcd 

just prior to use and was stored in ice tmtil usc. 

4. 	 CELl. PRE-CULTURE 

A 60-0101 diameter plastic dish (Asahi Glass Co., Ltd.) was used for ccll culture. Five 

milliliter of a cell suspension of 5x 103 cells/mL were seeded into a dish and were 

cultured continuously for 3 days. 

5. 	 PREPARATION OF TEST SUBSTANCE SOLUTION AND POSITIVE CONTROL 

SUBSTANCE SOLUTIONS 

5.1 Preparation of Test Substance Solution 

1) 	 Solvent 

DMSO (Lot No. WF032, 100.0% in purity, pme solvent for measuring ultraviolet 

absorption spectrum, DOJINDO Laboratories) was used. 

2) 	 Reason for selection of solvent 

The test substance was insoluble II1 distilled water (the infonnation from the 

sponsor) and was dissolved at 500 mg/mL in DMSO. The solution at 500 mg/mL 

prepared with DMSO was not indicated any change in color nor exothermic at room 

temperature within 2 hours after preparation. Therefore, DMSO was selected as a 

solvent. 

3) 	 Preparation method 

After the test substance was weighed, DMSO was added to the test substance to 

make an original solution using a laboratory mixer. The test substance solutions of 

100 times concentrations of the test substance in the medium were prepared with the 

solvent. It wa') prepared without correcting purity of the test substance because the 

purity of the test substance was above 95%. 

4) 	 Preparation time and storage 

The test substance solutions were prepared immediately before lise, stored at room 

temperature and used within 2 hours after preparation. 

5.2 Preparation of Positive Control Substance Solutions 

1) 	 Preparation method and storage 

MMC and CPA were dissolved in distilled water at 0.01 mg/mL and 1 mg/mL, 

respectively. The positive control substance solutions were frozen in an ultra-deep 

freezer (MDF-U481ATR, SANYO Electric Co., Ltd., the tolerance temperature: 

-	 10 ­

155 
K06-1279 


below -BO°C). 

2) 	 Preparation time and expiry in use 

The positive control substance solutions were thawed at the time of use and used 

within 2 hours. The stock solutions wcrc used within 6 months after preparation. 

6. TEST PROCEDURE 

6.1 Cell GroVvth Inhibition Test 

1) 	 Procedure 

For the short-term treatment without S9 mix, the medium was removed from a preculture, and the cells were treatcd for 6 hours in well-mixed mcdium containing 30 

ilL of the test substance solution or the solvent and 3 mL of the fresh medium. For 

the short-term treatment with S9 mix, the medium was removed from a pre-culture, 

and tht.: t.:ells were treated for 6 hours in well-mixed medium consisting of 0.5 mT. of 

S9 mix and 30 ilL of the test substance solution or the solvent and 2.5 mL of the 

fresh medium. After treatment, the medium was removed, and the cells were 

rinsed 3 timcs with 2 mL of Dulbecco's physiological phosphate buffered solution 

without Ca2

+ and Mg2+. Cells were then cultured for another 18 hours in 5 mL of 

fresh medium. 

For the continuous treatment, the mcdium was removed from a pre-culture, and the 

cells were treated for 24 hours with well-mixed medium containing 50 ilL of the test 

substance solution or the solvent and 5 mL ofthe fresh medium. 

In the short-tenn and the continuous treatments, 50 IJ.L of a 10 I1gimL demecolcine 

solution was added to each dish at 2 hours before the end of the culture. 

At the start and the end of the treatment and at the end ofthe culture, precipitation of 

the test substance, the color change of the medium and the corrosion of the dish 

were observed macroscopically. 

At the end ofthe culture, a cell suspension was preparcd to collect from each dish by 

a treatment with 2 mI. of 0.25 w/v% trypsin. After 200 ilL of the cell suspension 

was diluted with 10 mL of Cell Pack (Sysmex Corporation), the number of the cells 

was measured using a Microcell counter (CDA-500, Sysmex Corporation), and the 

cell growth rate and the 50% cell growth inhibition concentration (lC:,o) was 

calculated. TI1C ICso was obtained from a linear line drawn between 2 plots; the 

onc was the lowest dose with the cell growth rate of below 50% and the other was 

the next lower dose ofthis dose. 

Leftover cells were collected by a centrifugation at 1000 rpm (l85 x g) for 5 minutes 

and were treated hypotonically with 3 mL of 0.075 mollL KCI at 37°C for 15 

minutes. Following the hypotonic treatment, the cells were pre-fixed once with 

approximately 0.3 mL of a fixative solution (methanol : acetic acid = 3 : 1). and 

--- 11 ­

156 
--

6.2 

K06-1279 


were completely fixed twice with 3 mL of fixative solution. Then, the cell 

suspension was prepared with a fixative solution, two drops of the suspension were 

placed on a glass slide, and stained about J5 minutes with 2 vol% Giemsa solution 

in 1115 malll phosphate butTer solution (pfI6.8). One specimen was prepared per 

dose. 

2) 	 Dose levels 

In each treatment method, the highest dose was set at 5000 ~lg/ml, as the maximum 

dose in the case of no cytotoxicity on the guideline, and 19.5,39.1,78.1,156,313, 

625, 1250, 2500 and 5000 ~lglmL were set based on a geometric progression of 2, 

respectively. Duplicate dishes were used ror each dose. 

J) 	 Observation and scoring 

Specimens were observed to check the presence or absence of mitotic metaphases, 

and the frequency of the cells vv'ith chromosomal aberration was calculated by 

observed 50 metaphases per dose, at which the dose setting of chromosomal 

aberration test, was considered to be referred. 

(1) Structural aberration 

The number of cells with structural abelTations excluding gaps was recorded. 

Gaps were defined as an achromatic region smaller than the widlh of one chromatid. 

(2) Numerical aberration 


The number of cells showing triploid or more was scored. 


Chromosomal Aberration Test 


I) 	 Procedure 

Chromosomal aberration test was carried out using the same procedure as that of the 

cell growth inhibition test, with the following positive controls. Four specimens 

per dose (two speCImens per d'ISh) were prepanx I . 

Treatment method 

Without S9 mix 

Short -term treatment 

with S9 mix 

24 hours continuous treatment 

Suhstance 

MMC 


CPA 


MMC 


Dose 

0.1 1l.g/mL 

6 Jlg/mL 

0.05 Jlg/mL 

Tn the positive control, each dish was added with 30 ilL of a 0.01 mg/mL MMC 

solution and 18 ~lL of a I mglmL CPA solution for the short-term treatments 

without and with S9 mix, respectively, and 25 111. of a 0.01 mg/mL MMC solution 

for the continuous treatment. 

2) 	 Dose levels ofthe test substance 

The dose levels of the test substance wt!re set on the basis of results of the cell 

growth inhibition test. The setting doses and the reason for selection are shown 

below. 

Por the shorHerm treatments without and with 89 mix and 24 hours continuous 

157 

--

K06-1279 


treatment, the highest dose of thc test substance was selectcd the dose that was over 

ICso, hecause a cytotoxicity that the cell growth rate was below 50% was obtained in 

the cell growth inhibition test. lCso were calculated at 27 JlglmL in the shorHerm 

treatment without S9 mix, then the highest dose was selected at 55.2 ~lg/mL and the 

following 6 lower doses were set based on a geometric progression orf2. lC50 

were calcula1ed at 270 ~tgimL in the short-term trea1ment with S9 mix, then the 

highest dose was selected at 442 Ilg/mL and the following 5 lower doses were set 

based on a geometIic progression Of)2. IC50 were calculated at 14 ~LglmL in 24 

hours continuous treatment, then the highest dose was selected at 27.6 Jlg/mL and 

the following 5 lower doses were sct based on a geometric progression of.J2 . 

Treatment method I Setting doses of test substance 

Short-term 

treatment 

without S9 mix i 6.9L 9.77, 13.8, 19.5, 27.6, 39.1 and 55.2J1gfmL 

with S9 mix I 

78. L 110, 156, 22L 313 and 442 J1g1mL 

24 hours continuous treatment I 4.88,6.91,9.77,13.8, 19.5 and 27.6 ~lglmL 

Duplicate dishes wcre used for each dose. 

3) Observation 

(1) Dose for observation 

All spccimens of the negative and the positive controls set as the control groups 

were observed. The consecutive doses of three stages were selected for the 

observation dose of the test substance. The observed doses and the reason for 

scJ.cction arc shown below. 

In each treatment, because a cytotoxicity that the cell growth rate was below 50% 

was obtained, the highest dose for observation of specimens was selected at the 

lowest dose that the cell growth rate was below 50%. The lowest dose such that 

the cell growth rate was below 50% was 39.1 Ilg/mL in the short-tenll treatment 

without S9 mix, 313 ~lg/mL in the short-term treatment with S9 mix and 19.5 

Jlg/mL in 24 hours continuolls treatment. Therefore, these doses were selected as 

the highest dose and two consecutive doses shown in the following table were 

selected as the observation doses of the test substance. 

Treatment method Observation doses of test substance 

Short-tenl1 

treatment 

without S9 mix 19.5,27.6 and 39.1 )lg/mL 

with S9 mix 156, 221 and 313 jlg/mL 

24 hours continuous treatment 9.77, 13.8 and 19.5jlg/mL 

After the selection of the observation doses, slide numbers were randomly 

allocated to all specimens to be observed. AU specimens were observed in a 

blinded manner. 

As a result of the observation of specimens, the frequency of the cells with 

structural aberration was over 5% at the lowest dose for observation in 24 hours 

13 

158 
K06-1279 


continuous treatment. Therefore, in 24 hours continuous treatment, the 

additional observation of specimens at 4.88 and 6.91 J.lg/mL was conducted in 

order to evaluate the no-observed effect level that the frequency of aberrant cells 

become below 5%. The slide numbers were allocated to all specimens to be 

observed additionally. 

(2) Structural Aberration 

Two hundred metaphase cells per dose (50 cells per specimen) containing 25±2 

chromosomes were observed using a microscope. The total number of cells with 

structural aberrations and the number of aberrant cells in each aberration category 

were recorded. Gaps were defined as an achromatic region smaller than the 

width of one chromatid and were recorded separately from the structural 

aberrations. 

(3) Numerical Aberration 

The numbers of polyploid (cell with 38 or more chromosomes) cells among 200 

metaphase cells per dose (50 cells per specimen) were recorded. 

6.3 	 Confirmation lest 

In the chromosomal aberration test for 24 hours continuous treatment, the structural 

aberration was judged to be positive obviously, therefore, a confirmation test was not 

conducted. 

7. 	 JUDGEMENT CRITERIA Of RESULTS 

The tindings were judged to be positive when the frequencies of cells with structural 

aberrations showed 10% or more with a dose-related increase or the frequencies of cells 

with structural aberrations showed 5% or more both in the chromosomal aberration test 

and the confirmation tcst. The other cases were judged to be: negative. No statistical 

analyses were used. Thc frequency of numerical aberration cells was judged according 

to the same criteria as that of the structural aberration. D10 value indicating a 

concentration that chromosomal aberration was observed in 20% of cells was calculated 

in the treatment method that the frequency of cells with chromosomal aberrations were 

5% or more. 

8. 	 VALIDITY or TEST 

This study was regal"ded as valid as follows: 1) the frequencies of cells with chromosomal 

aberrations did not fluctuate markedly betwecn two culture dishcs, 2) the frequencies in 

the negative control showed below 5%, and 3) the frequencies of cells with structural 

aberrations excluding gaps in the positive controls showed 20% or more. 

14 	 -­ 159 
K06-1279 

FACTORS AFFECTED RELIABILITY OF TEST 

There were no factors, which might affect the reliability of the lest. 

TEST RESULTS 

I. 	CELL GROWTH INHIBITION TEST (Table 1 and Fig. 1) 

The ICso were calculated at 27 ~lg/mL in the short-term treatment without S9 mix, at 270 

I1g/mL in the short-telm treatment with S9 mix and at 14 l1g1mL in 24 hours continuous 

treatment. 

In the short-term treatment without S9 mix, precipitation of the test substance was 

observed at 78.1 I1gimL or more at the start of the treatment, at 156 I1g/mL or more at the 

end of the treatment and at 2500 I1g/mL or more at the end of the culture. In the shortterm treatment with S9 mix, precipitation of the test substance was observed at all doses 

at the start of the treatment, at 313 I1g/mL or more at the end of the treatment and at 2500 

I1g/mL or more at the end of the culture. In 24 hours continuous treatment, precipitation 

of the test substance was observed at 78.1 I1g/mL or more at the start of the treatment and 

at 156 I1g/mL or more at the end of the treatment. The color change of the medium and 

the eorrosion of the culture dish were not observed at any doses. 

As a result of the observation of specimens, the maximum frequencies of the cells with 

structural aberration were 10.0% in the short-term treatment without S9 mix and 24 hours 

continuous treatment. The frequencies of numerical aberration cells were below 5% at 

all observation doses ofthe test substance in each treatment. 

2. 	 CHROMOSOMAL ABERRATION TEST 

2.1 	 Short-tern1 Treatment (Tables 2, 3,4 and Figs. 2, 3) 

1) Without S9 mix 

(1) 	Cell growth rate and IC50 

The cell gwwth rates at 6.91,9.77, 13 .8, 19.5,27.6,39.1 and 55.2 ).1g/mL of the test 

substance were 78.9, 72.4, 66.2, 58.3, 50.3, 42.6 and 31.8%, respectively. The 

IC50 was calculated at 28 l1g1mL. 

(2) 	Precipitation of the test substance, color change of medium and corrosion of culture 

dish 

Precipitation of the test substance, color ehange of the medium and the corrosion of 

the culture dish were not observed at any doses. 

(3) Frequency of eells with structural aberrations 

The frequencies of cells with structural abenations were 1.5% in the negative 

contra! and 45.0% in the positive control. The frequencies of cells with structuml 

15 	 ­ 160 
K06-1279 


aberrations at 19.5,27.6 and 39.1 J.lg/mL of the test substance were 2.5, 2.0 and 

4.0%, respectively, and were below 5%, therefore, the results were judged to be 

negative. 

(4) Frequency of numerical aberration cells 

The frequencies of numerical aberration cells were below 5% at all doses including 

the negative and positive controls, therefore, the results were judged to be negative. 

2) With 89 mix 

(1) Cell growth rate and 1Cso 

The cell growth rates at 78.1, 110, 156, 221, 313 and 442 )..lg/mL of the test 

substance were 82.3, 81.3, 70.4,57.4,42.6 and 21.3%, respectively. The ICsowas 

calculated at 270 ~lglmL. 

(2) Precipitation of the test substance, color change of medium and corrosion of culture 

dish 

Precipitation of the test substance was observed at all doses at the start of the 

treatment and at 156 )..lg/mL or more at the end of the treatment, however, not 

observed at the end of the culture. The color change of the medium and the 

COiTosion of the culture dish were not observed at any doses. 

t)) Frequency of cells with structural aberrations 

The frequencies of cclls with structural aberrations were 1.0% in the negative 

control and 36.0% in the positive control. The frequencies of cells with structural 

aberrations at 156,221 and 313 )..lg/mL ofthe test substance were 3.5,1.5 and 3.0%, 

respectively, and were below 5%, therefore, the results were judged to be negative. 

(4) Frequency of numerical aberration cells 

The frequencies of numerical abenation cells were below 5% at all doses including 

the negativc and positive controls, therefore, the results were judged to be negative. 

2.2 Twenty Four Hours Continuous Treatment (Tables 2,5 and Figs. 2,4) 

1) Cell growth rate and IC50 

The cell growth rates at 4.88, 6.91, 9.77, 13.8, 19.5 and 27.6 )..lg/mL of the test 

substance were 84.6, 73.2, 65.3, 58.2, 48.2 and 34.4%, respectively. The 1Cso was 

calculated at 18 )..lg/mL. 

2) Precipitation of the test substance, color change of medium and corrosion of culture 

dish 

Precipitation of the test 'substance, color change of the medium and the corrosion of 

the culture dish were not observed at any doses. 

3) Frequency of cells with structural aberrations 

The frequencies of cells with structural aberrations were 2.5% in the negative 

control and 59.5% in the positive control. The frequencies of cells with structural 

aberrations at 4.88,6.91, 9.77,13.8 and 19.5 )..lg/mL were 4.5, 11.5,8.5,6.5 and 

- 16 ­

161 
K06-1279 

3.0%, respectively, and a dose related increase was observed in 4.&8 and 6.91 J.lg/mL 

and the maximum frequency was over 10%, therefore, the results were judgcd to be 

posItIve. The dose that the frequencies of cells with structural aberrations showed 

below 5% was 4.88 ~lglmL. 

4) Frequency of numerical aberration eells 

The frequencies of numerical aberration cells were below 5% at all doses including 

the negative and positive controls, therefore, the results were judged to be negative. 

5) 	 D:w value 


It was calculated at 0.014 mg/mL for structural aberration. 


3. 	 TYPICAL PHOTOS 

The normal cell was shown in photo 1 and cell with structural aberration induced by 

Karenz MT (Mixture ofPEMB and PE3MB) was shown in photos 2 and 3. 

DISCUSSION AND CONCLUSION 

In each treatment method in the chromosomal aberration test, the frequencies of cells with 

chromosomal aberrations did not fluctuate markedly between two culture dishes, and the 

frequencies of cells with aberrations were below 5% in the negative controls, and the 

frequencies of cells with structural aberrations excluding gaps were 20% or more in the 

positive controls, indicating that the present study was appropriately performed. 

As a result of the chromosomal aberration test, the frequencies of numerical aberration cells 

were below 5% at all doses of the test substance in the short-term treatments without and 

with S9 mix and 24 hours continuous treatment, there tore, numerical aherration was judged 

to be negative. The frequencies of cells with structural aberrations showed below 5% at all 

observation doses of the test substance in the short-term treatments without and with 89 mix, 

therefore, structural aberrations was judged to be negative. However, the frequencies 

showed 4.5% at 4.88 J.lg/mL and 11.5% at 6.91 J.Lg/mL and the maximum frequency of cells 

with structural aberrations was over 10% in 24 hours continuous treatment. The 

frequencies of ceJls with structural aberrations at 9.77, 13.8 and 19.5 J.lg/mL of test substance 

were 8.5, 6.5 and 3.0%, respectively, and a dose related decrease was observed. Because 

the cell growth rates at 13.8 and 19.5 f,!g/mL oftest substance was approximately 50% and a 

mitotic inhibition was occurred at 19.5 pg/mL of test substance, it was considered that the 

the appearance of aberrant cells was suppressed by cylotoxicity at 9.77 jlg/mL or more. The 

frequencies of cells with structural aberrations at 4.88 jlglmL was 4.5%. Then, it was 

evaluated that a dose related increase was observed in 4.88 and 6.91 ~lglmL oftest substance. 

Therefore, structural aberration was judged to be positive in 24 hours continuous treatment. 

Based on the above results, it was concluded that Karenz MT (Mixture of PEMR and 

--	 17 

162 
K06-1279 

PE3MB) did not induce numerical aberration but it induced structural aberration under the 

present test conditions. 

REFERENCES 

1) 	 Toshio Sofuni (cd.) (1999) Data book of chromosomal aberration test in vitro. Revised 

edition, 1998 (in Japanese). Life-science Information Center. 

2) 	 Japan Environmental Mutagenicity SocietylMammalian Mutagenicity Study Group (ed.) 

(1988) Atla., of chromosomal aberration induced with chemical substance (in Japanese). 

Asakura Publishing Co., Tokyo. 

--	 18 

163 
K06-1279 

Table I Results of cell growth inhibition test of Karenz MT (Mixture of PEMB and PE3MB) 

Precipitation ortest Frequency ofcells with Treatment Illdex of Dose - recovery S9 Cell growth substance in medium Hi aberrations (%}bl

Suhstance metil.phR~e 

(flg/mL) time mix rate (%) Treatment Treatn'ICnt Clillure <ell Siructnral Numerical 

(hour) aberration aberration slar1 clld end 

DMSO 0 6-18 100 abundant 4.0 0.0 

Karenz MT 195 6-18 55.2 abundant 6.0 0.0 

(Mixture of 

PEMR and 39.1 6-18 41.0 abundant 10.0 0.0 

PUMS) 78.1 6-18 17.7 + few meta n.o. n.o. 

156 6·18 5.7 + + no meta n.o. n.o. 

313 6-18 3.4 + + no meta n.o. n.o. 

625 6-18 9.8 j. + no meta n.o. n.o. 

1250 6-18 6.6 + + no meta n.o. n.o. 

2500 6·18 9.4 + + no meta n.o. n.o. 

SOOO 6-18 5.4 ~ no meta n.o. n.o. 

(Cso : 27/.lg/mL 

DMSO 0 6-18 + 100 abundant 0.0 0.0 

KarcnzMr 19.5 6-18 + 100.6 abundant n.o. n.o. (Mixture of 

PEMB and 39.1 6-18 93.7 abundant n.o. n.o. 

PlDMrl) 78.1 6-18 87.0 abundant 2.0 2.0 

156 6-18 71.6 + abundant 4.0 0.0 

313 6-18 41.6 + abundant 2.0 0.0 

625 6-18 + 10.2 + few meta n.o. n.o. 

1250 6-18 + 13.9 + + no meta n.o, n.o. 

2500 6-18 + 5.0 + + + no meta n.o. n.o. 

5000 6-18 + 3.8 + -t 1­ no meta n.o. n.o. 

ICso : 270 IlgirnL 

DMSO 0 24-0 100 0.0 2.0 

Karcnz MT 19.5 24-0 44.2 10.0 0.0 

(MixlurCHf 

PEMB and 39.1 24-0 25.0 few meta n.O. n.o. 

I'EJM[l) 78.1 24-0 15.7 no mela n.o. n.o. 

156 24-0 16.0 + + no meta n.o. 11.0. 

313 24-0 17.6 + -1 no meta 11.0. n.o. 

625 24-0 9.6 .j + no meta 11.0. n.o. 

1250 24-0 CiA + no meta n.o. n.o. 

2500 24-0 2.4 t + no meta 11.0. n.o. 

5000 24-0 2,2 + + no 1t1<.;ta n.o. n.O. 

IC\o: 14 ~lglmL 

DMS(): Dimethyl sulfoxide 

n.o.: not observed, abllndant: metaphase, existed abundantly, a fi:w: the li'cquency of metaphases was relatively low, 

lew meta: the fn:quency of metaphases was extremely lew, no l1leHl: metup]'ose5 were 1I0t observt.!d 

a) Precipitation ofthe lest substance: -. absence; +, presence. 

b) The rrequC'IICY of cells with dm'nlosomal aberrations was calculated by observating 50 metaphase, per dose. 

The highest dose was sel at 5000 flglrnL of the leSI substance, as the maximum dose in case of no C),10loxicil), on the guideline: 

the do!'", levels based on a geoll1etric progn;ssicHI of 2 w;.:rc s",lccl~d. 

19 164 
K06-1279 

Table 2 Results of chromosomal aberration test of Karenz MT (Mixture of PEMB and PE3MB) 

Preclpitalion onest F"equency of cells with TrcfJlmcnl 

substance in medium .) huj", or aberrations (%)b) Dose - recovery SY Cell gro\\1h Substance mettlphase 

(JlglmL) time mix rale (%} Treatment Trealme"! Culture edl Stnlctural Numerical 

(hoor) aberratIon aberration stan end end 

OMSO 0 6-18 100 abundant L5 0.0 

Kar(>nz MT 6.91 6-18 78.9 abundant n.tI. n.o. 

(Mi"tureof 

PEMB and 9.77 6-18 72.4 abundant n.o. n.o. 

PE3MB) 13.8 6-18 66.2 abundant n.o. n.o. 

19.5 6-18 58.3 abundant 2.5 0.5 

27.6 6-18 50.3 abundant 2.0 0.0 

39.1 6-18 42.6 abundant 4.0 0.0 

55.2 6-18 -,~ ,,-"'" ... ,"---~-' ,-..-._-."- ~-,-~-,~----

31.8 a few n.o. n.o. 

MMC 0.1 6-18 NO n.o. 45.0 1.0 

ICso : 28 ).tglmL 

DMSO 0 6-18 + 100 ahundant 1.0 0.0 

Karenz MT 

(Mixture of 

i'EMBand 

J>E3M8) 

78.1 

110 

156 

6-18 

6-18 

6-18 

+ 

+ 

82.3 

81.3 

70.4 

+ 

+ + 

abundant 

abundant 

abundant 

n.o. 

n.o. 

3.5 

n.o. 

n.o. 

0.5 

221 6-18 + 57.4 + + abundant 1.5 1.0 

313 6-18 + 42.6 + abundant 3.0 0.0 

CPA 

442 6-18 ""~-----.-~. 

6 6-18 

+ 

+ 

21.3 

ND 

\. + a few 

n.o. 

n.o. n.o. _._----­

36.0 0.5 

ICso : 270 ).tglmL 

OMSO 0 24-0 100 2.5 1.0 

Karen? MT 4.&8 24-0 84.6 4.5 0.0 

(Mixture of 

J>EMBand 6.91 24-0 73.2 abundant 11.5 0.0 

['E3MB) 9.77 24-0 65.3 abundant 8.5 0.5 

13.8 24-0 58.2 abundant 6.5 2.5 

19.5 24-0 48.2 a few 3.0 1.0 

27.6 24·0 34.4 few meta n.o. n.o. 

MMC 0.05 24-0 NO n.o. 59.5 0.0 

IC50 : 18 ).tg/mL D20: 0.014 mg/tnt 

DMSO: Dimethyl ~l!llbxidc. MMC: mitomycin C, CPA: cyclophosphamide monohydrale 

ND: fI('l d0tcctctl 

n.o.: not observed, abundullt: metnphases existed abundanlly, a few: the rrequency ofmctaphases lI'ns relatively low. 

fewmcta: the Ih:qucncy ofmclaphases was extremely few 

a) Prcci~lituli()11 ofthe lest slibslaIlC~: -, abscnce: +. presence, 

b) The frequcncy of cells with cnromo50mal anerraliol1, was cakulated by obs<',yuting 20() metaphases per dose. 

20 165 
Table 3 Res.ults of ('hfOrnOS()Ill(ti abcrratlOIl test \:;hon~le0111reatmeal without 59 mix) 


Nnl11e "fr.51 ~ut\,mlflj)e: K"ellz MT iMlxture ofPE:YIB and I'E3Ml:!) K06-1279 


Negative controf () 

6- 18 (DMSOi o tl 

I 0,0)' o ( o (00); 0 ( 0,01 "( 001 

6 18 

--"", ..... 

~ - 18 13.S 

0-1& 

6 18 2;,6 
 N 


(,. i8 JQ I 

(, is 55,~ 

43 o 'J 


6 IS 


"' " 

1n:aUl1ellt tin:es c()mpnscd trea11l1eoHlme and recovery-time:: 


TI!c m:l'raber ofaberrallt C'1!'11s at each dish \\'ere SIH)WII attl~ th:'l.t and $!;Cunu lines, The tola! number ofthem was shtn.\r at tbe lhird :1Ti~, 

Cell ~rowlll ralo 81 ~ach d"h "ere 5ilO\\11 311lie first and second liues The aver.'ge ofthem was shown at the thirtllhe, 


D05e 

i~g!mL) 

Number of celts w11h structmal chromotiP1n::J.1 aberrations {frcqlJency~/o) NllJnper of ct11s lvilh numcnral chmmosomn~ aberratioos 

8'Jml~ Tf~tment ------;--"_..

time (11) Number of Chromatid Chromatid 

.cells ob~rvedl br~ak exchange Others Polvpknds Others 

() - 18 

9.77 

Totalnumi>er of 

cells with 

jans 

DMSO Dimelhyl ".iltoxide 


~1MC: Mltomy.:in C 


Til; ,,,"CIIII.US at 6.91, ? '7. 13 8 and 55.l"i-1mL were nO! observed. 


~ o 

C» 'r' C» ..­

"-J 

\0 

IV 
Table 4 Rti31.i!ti of (;hromc~omal ;~be:mHioll t.:'Si (Short~lcm1lrca1ment \\.ith $9 mix) 


N.')\11t.' orte.S! 5!1Ds:t2lncf:" Kareni. M'T' (;V1l:":I~re ofPEMB !\,ld PEJ.MB) }(()6~ 12'79 


t'~umber of celhi \\It:l nuntcflc31 chrolHosomal ali;:rratlcns ~h;mbcf' of cells \\"ith s,:mctur.al chromc!:\omo! abe-mttic)!1.t: (trequCl)CY%) 21o,,, Cell Trcatnenl -----~-",.,.. ',' 

(pg/mL) 

Ntgative 

$9 VIi, 

,'"

fr¢(luenc../ J'I1)" 

i!!fO\\1h ratc------- ~ 

liml.'f,h) Chr01n31id Chr.lIllaUd Chromosome ~ Chromosome ., !\umhC'f 0: 


Olh"" ('Yo) cells obse:f\'ed P0:yplcid~ r:ln:'Jk ~change break exch<1n~e 

',)1) 

6 IS 

(, - 18 78,! t 

6 - i& 110 t 

;, - is 156 t 

6 - IS 2:: I t 

6· Iii .313 l' 

1--,)

t--.) 

6 - l~ 441 t 

POSlln!1! cOlltrol 38 

6 - 16 l(PA) 

6 

Treafment times. e()lHpri<;ed tn~:1IlmenHjl1l~ and recovtl')'~ttlne 

rile nlllllber0rabcn~m cd!!) i\1 (!,lch df:,h were ShO\Vll ~(th~~ tin.t :lila second lilles The tOfallHHl1bcr cfthem \\35 sllow ot the third ~ine 

~ty'owlh ralc.H CllCl1 dish were sho\\1\ ill the t1rfit and ~C!;OJ1(! lir.c:,<; The ovemge of them w2;,", Sh(\\1;1l flt I:U' thlrJ !ill\.! 

O'l~rs III the numb.:r nt'ccll$ \'tilt rfumel'k~ll chromosomal avaratlons iwJn:.ale elluOIeduplic.,'llion 

l)\lSO Dlme:l,,1 sulfox,de 

CPA' Cyciophv')pIHlmide. mCr10hydrate 

t: Ptccipitation of Ihe 1("sl slIb51i1n.::c was observed at all dD$t:~ lit Ihe !)tan ('If the tr..;:a1mcl1t and at 150 itg/mL or mm~.at the end of the treatment 

rII;: ,,)?e"cimcns nr 78 1. 1\ 0 and 442 IlgtmL W'i)ft 1101 ubserved. 

0) 

-..J 

I, 

Q 

~ 

o 

0,­

I 

t-.l 

-..J 

'-0 
Table 5 Results ofchro010SQmaf aberration test (continUOus trcanncnlj 

~a',1Z of lest substance Knrcnz MT IMIXillre of PEMB and PE3MB) K06·1279 

XUll1bcr of ce!ls: with omnericai chf(1mo~of'l1al aherrations l\umber of cells with structural chrOln"somnl aberralions (fre'l"ency%) Doso Number I" Cell \ffequency%} 'T rcat1n~nl 

\~Jg/mL) IIrn~ (h} Chrmna:id Chmmalid •. 0 I II 1 h ' ., POlvpk'lds I leTS Ct:" S \1,,'lU break ex\: ange cell,ohserv<<lj - nl1~rrallon' 

24. 

o 

24 - C 4.88 

14 ~ 6.91 

24 9.77 

24 - () IH 

1'1 a 19.5 

24 0 17.6 ,OX if~w metaphases) TOX I lew melaph",e,) 

34..i.l...._ 

(I 

:'4 - 0 (MMC) () 

O,(lS 

o 

() i 1),0)' (\ 0) OJ 

Tn:almenl tI1TH~$ compnsed tremr.lelU-lime and recovery-lime 

The nWT!ber 01 a:>eITant l~ils at cach dish Were shown at tho fits! and second Ilncs, The 10131 number of Ihem was ,how at the third line, 

Cdll~ro\\1:) ra:e :ll ~ach dish wert: Sh(Win at ihe first :!\nd st!cofH.llincs, The av(xage (.'Ifthem was shown;).t the th~rd 

DMSl)' Dllnethyl slIlfoXldc 

WvlC: Mi10mycin C 

few met;Jphnsl'':9' til;; frc(IUenCy ofmu(apha~l'::$ '\'a~ \!xtrcmclv fc\\' and it \-\,10\5 unable to ar'lulyz~ chrt)lntlsomcs 

9'

..... 

N 

-.J 

;C 

Q) 

(Xl 

8 
K06-1279 


100 

ICso : 27 llg/mL 

10 100 1000 10000 

Dose (pg/mL) 

Shorl-tenn treatment without S9 mix 

[DO 

50 

ICso : 270 llg/mL 

ot-~~~~~~~~~~~--~~~~ 

10 100 1000 10000 

Do~e (llg/mL) 

Short-term treatment with S9 mix 

100 

50 

o+---~~~~~+---~~~~~~~~~~~~ 

to 100 1000 10000 

Dose (Jlg/mL) 

24 hours continuous treatment 

Fig. 1 Results ofcell growth inhibition test of Karenz MT (Mixture of PEMB and PE3MB) 

- 24 ­ 169 

K06-1279 


100 

c 

~ 

,0 

., 

E 

~ e 50 bI> 

-;:; 

U 

100 

~ 

~ ,2! 

I:: 

l 50 bO 

-;:; 


U 


leso: 28 /lglmL 

10 100 

Dose (~glmL) 

Short-term treatment without 89 mix 

o+-----~~--~~~~~~~------~--~~~~~~~ 

10 100 1000 

Dose (~lglmL) 

Short-term treatment with S9 mix 

100 

,-.. 

~ 

~ 

C> 

~ 

~ so ~ 

-;:; 

w 

0 

JO 100 

Dose (!is/in L) 

24 hours continuous treatment 

Fig. 2 Cell growth rate in chromosomal aherralion test of Knrenl MT (Mixture of PEMB and PE3 MB) 

- 25 ­ 170 
K06-1279 


~ 

'" c:

0 

.~ 

... 

.J:; 

o:l 

<ii 

e 

0 

VI 

0 

E 

8 

.c 

<.) 

-S 

.~ 

~ 

"8.... 

0 

» 

u

c:

... 

:::> 

cr 

~ 

"'­

o m.unerical aberration 

• structural aberration 

20 

to 

o~--------==========~===---~-----------e 

30 

20 

10 

o 19.5 27.6 39.1 

Dose ().lg/mL) 

without S9 mix 

o numerical aberration 

• structural aberration 

o~=-----~========~~==~~==~===-~ 

o 156 221 313 

Dose (J,lg/mL) 

With 89 mix 

Fig. 3 	 Results of chromosomal aberration test in short-term treatments 


of Karenz MT (Mixture of PEMB and PE3MB) 


26 

171 
K06-1279 


30 

o numerical aberration 

• structural aberration 

020; 0.014 mg/mL (structural aberration) 

o~======~~~~==~~~--~--------~ o 4.88 6.91 9.77 

Dose (fJ.glmL) 

13.8 19.5 

24 hours treatment 

Fig.4 Results of chromosomal aberration test in continuous treatment 

of Karenz MT (Mixture of PEMB and PE3MB) 

27 .­ 172 

K06-1279 


Photo 1 Nonnal cell 

Negative control in 24 hours continuous treatment 

Photo 2 	 Structural aberration induced 

by Karenz MY (Mixture of PEMB and PE3MB) 

13.8 ,ug/mL in 24 hours continuous treatment 

a: chromatid break 

28 

173 

K06-1279 


Photo 3 	 Structural aberration induced 

by Karenz MT (Mixture ofPEMB and PE3MB) 

9.77 JlglmL in 24 hours continuous treatment 

b: chromatid exchange 

- 29 ­

174 

PAGE 1 OF 20 PAGES 

Mixture of PEMB and PE3MB: 

LOCAL LYMPH NODE ASSAY IN THE MOUSE 

PROJECT NUMBER: 0146/0200 

AUTHOR: A Pooles 

STUDY SPONSOR: TEST FACILITY: 

Showa Denko K.K. Harlan Laboratories Ltd 

13-9, Shiba Daimon 1-Chome Shard low Business Park 

Minato-ku Shard low 

TOKYO 105-8518 Derbyshire 

JAPAN DE722GD 

UK 

Telephone: +44 (0) 1332792896 

Facsimile: +44 (0) 1332799018 

0145'()200.doeJJS 

175 
PROJECT NUMBER: 014510200 PAGE 2 

QUALITY ASSURANCE REPORT 


This study type is classed as short-lenn. The standard test method for this study type 

("General Study Plan" in OECD tenninology) was reviewed for compliance once only on 

initial production. Inspection of the routine and repetitive procedures that constitute the 

study is carried out as a continuous process designed to encompass the major phases at 

or about the time this study was in progress. In addition, inspection of general facilities 

not specifically related to this sludy are done monthly or annually in accordance with QA 

Standard Procedure. 

This report has been audited by the Quality Assurance Unit, and is considered to be an 

accurate account of the data generated and of the procedures followed. 

In each case, the outcome 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 type and schedule of this study were as follows: 

12 November 2008 Standard Test Method Compliance Audit 

16 February 2009 Test Material Preparation 

23 February 2009 Test System Preparation 

19 February 2009 Animal Preparation 

16 February 2009 Dosing 

19 February 2009 Assessment of Response 

§ 19 March 2009 Draft Report Aud it 

§ Date of QA Signature Final Report Audit 

§ Evaluation specific to this study 

~.J-\,Vb~~ . 2 5 MAR 2009 ...,........ ~ ~ ..................... ~.~ ...... "........................ ~ ....... ~ .................. . DATE ................................................ . 


For the Quality Assurance Unit* 

*Authorlsed QA Signatures: 

Deputy Head of Department JM Crowther MIScT MROA 

Senior Audit Staff: G Wren ONC MROA 

176 

PROJECT NUMBER: 014510200 PAGE 3 

GLP COMPLIANCE STATEMENT 


The work described was performed in compliance with UK GLP 'standards (Schedule 1, 

Good Laboratory Practice Regulations 1999 (SI 199913106 as amended by 

SI200410994». These Regulations are in accordance with GLP standards published as 

OECD Principles on Good Laboratory Practice (revised 1997, ENVIMC/CHEM(98)17); 

and are in accordance with, and implement, the requirements of Directives 2004/9/EC 

and 2004/10/EC. 

This report fully and accurately reflects the procedures used and data generated . 

........A.:.. P<?9..~................................................ DATE: ...... ':2.S..\.9..19.3................:........ 
 A Pooles BA (Hons) 

Study Director 

This report may be presented in final form as a digital (pdt) document. Such documents are prepared by scanning the paper onginal, 

and are considered of equivalent integrity and authenticity to versions produced by optical photocopy. However, in all cases the 

hand-signed paper original, held in secure archives, Is the definitive document. 

177 
PROJECT NUMBER: 014510200 PAGE 4 

QUALITY ASSURANCE REPORT 

CONTENTS 


2 


GLP COMPLIANCE STATEMENT 3 


CONTENTS 4 


SUMMARY 5 


1. INTRODUCTION 6 


2. TEST MATERIAL 6 


2.1 Description, Identification and Storage Conditions 6 


2.2 Preparation of Test Material 7 


3. METHODS 7 


3.1 Animals and Animal Husbandry 7 


3.2 Procedure 8 


3.3 Interpretation of Results 10 


4. ARCHIVES 10 


5. RESULTS 11 


5.1 Preliminary Screening Test 11 


5.2 Main Test 11 


6. CONCLUSION 12 


Table 1 Clinical Observations, Bodyweight and Mortality Data-


Preliminary Screening Test 13 


Table 2 Disintegrations per Minute, Disintegrations per Minute/Node and 


Stimulation Index 14 


Table 3 Individual Clinical Observations and Mortality Data 15 


Table 4 Individual Bodyweights and Bodyweight Changes 16 


Appendix 1 Current Positive Control Study for the Local Lymph Node Assay 17 


Appendix 2 Summary of Positive Control Data for the Local Lymph Node Assay 18 


Appendix 3 Vehicle Determination Record 19 


Appendix 4 Statement of GLP Compliance in Accordance with Directive 2004/9/EC 20 


178 

PROJECT NUMBER: 014510200 	 PAGE 5 

Mixture of PEMB and PE3MB: 

LOCAL LYMPH NODE ASSAY IN THE MOUSE 

SUMMARY 

Introduction. A study was performed to assess the skin sensltisation potential of the 

test material in the CBAlCa strain mouse following topical application to the dorsal 

surface of the ear. The method was designed to meet the requirements of the following: 

• 	 OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local 

Lymph Node Assay" (adopted 24 April 2002) 

• 	 Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission 

Directive 2004n3/EC 

Methods. Following a preliminary screening test in which no clinical signs of toxiCity 

were noted at a concentration of 100%, this concentration was selected as the highest 

dose investigated in the main test of the Local Lymph Node Assay. Three groups. each 

of four animals, were treated with 50 ~I (25 '".11 per ear) of the undiluted test material or 

the test material as a solution in acetone/olive oil 4: 1 at concentrations of 50% or 

25% vlv. A further group of four animals was treated with acetone/olive oil 4:1 alone. 

Results. The Stimulation Index expressed as the mean radioactive incorporation for 

each treatment group divided by the mean radioactive incorporation of the vehicle control 

group are as follows: 

Concentration (% v/v) in 

acetone/olive oil 4:1 Stimulation Index Result 

25 22.52 Positive 

50 25.71 Positive 

100 23.11 Positive 

Conclusion. The test material was considered to be a sensitiser under the conditions of 

the test. 

179 

2.1 

PROJECT NUMBER: 014510200 	 PAGE 6 

Mixture of PEMB and PE3MB: 


LOCAL LYMPH NODE ASSAY IN THE MOUSE 


1. INTRODUCTION 

A study was perfonned to assess the skin sensitisation potential of the test material in 

the CBAlCa strain mouse following topical application to the dorsal surface of the ear. 

The method was designed to meet the requirements of the following: 

• 	 OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local 

Lymph Node Assay" (adopted 24 April 2002) 

• 	 Method 842 Skin Sensitisation (Local Lymph Node Assay) of Commission 

Directive 2004/73/EC 

The assay has undergone extensive inter-laboratory validation and has been shown to 

reliably detect test materials that are moderate to strong sensitisers. 

The strain of mouse used in these laboratories has been shown to produce satisfactory 

responses using known sensitisers and non-sensitisers during the in-house validation. 

The results of routine positive control studies are shown in Appendix 1 and Appendix 2. 

The results of the study are believed to be of value in predicting the sensitisation 

potential of the test material to man. 

The study was perfonned between 16 February 2009 and 10 March 2009. 

2. TEST MATERIAL 

Description, Identification and Storage Conditions 

Sponsor's identification Mixture of PEMB and PE3MB .. _--_._-_._-_ ... _---_ ...._._. Description 	 : : 

Batch number 	 . L ........................... : 


Date received 15 December 2008 

Storage conditions room temperature in the dark 

The integrity of supplied data relating to the identity. purity and stability of the test 

material is the responsibility of the Sponsor. 

180 
PROJECT NUMBER: 014510200 PAGE 7 

2.2 Preparation of Test Material 

For the purpose of the study, the test material was used undiluted and freshly prepared 

as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced the most 

suitable formulation at the required concentration. The concentrations used are given in 

the procedure section. The vehicle determination record is included as Appendix 3. 

Determination, by analysis, of the concentration, homogeneity and stability of the test 

material preparations was not appropriate because it was not specified in the Study Plan 

and is not a requirement of the Test Guideline. 

3. METHODS 

3.1 Animals and Animal Husbandry 

Female CBAJCa (CBAJCaOlaHsd) strain mice were supplied by Harlan Laboratories UK 

Limited, Bicester. Oxon, UK. On receipt the animals were randomly allocated to cages. 

The animals were nulliparous and non-pregnant After an acclimatisation period of at 

least five days the animals were selected at random and given a number unique within 

the study by indelible ink-marking on the tail and a number written on a cage card. At 

the start of the study the animals were in the weight range of 15 to 23 g, and were eight 

to twelve weeks old. 

The animals were individually housed in suspended solid-floor polypropylene cages 

furnished with softwood woodflakes. Free access to mains tap water and food (2014 

Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK) 

was allowed throughout the study. 

The temperature and relative humidity were controlled to remain within target ranges of 

19 to 25°C and 30 to 70%, respectively. Any occasional deviations from these targets 

were considered not to have affected the purpose or integrity o( the study. The rate of 

air exchange was approximately fifteen changes per hour and the lighting was controlled 

by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours 

darkness. 

181 

PROJECT NUMBER: 0145/0200 PAGES 

The animals were provided with environmental enrichment items which were considered 

not to contain any contaminant of a level that might have affected the purpose or integrity 

of the study. 

3.2 Procedure 

3.2.1 Preliminary Screening Test 

As no toxicological information was available regarding the systemic toxicity/irritancy 

potential of the test material, a preliminary screening test was performed using one 

mouse. The mouse was treated by daily application of 251-11 of the undiluted test 

material to the dorsal surface of each ear for three consecutive days (Days 1,2,3). The 

mouse was observed twice daily on Days 1,2 and 3 and once daily on Days 4, 5 and 6. 

Any signs of tOXicity or excessive local irritation noted during this period were recorded. 

The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6. 

3.2.2 Main Test 

3.2.2.1 Test Material Administration 

Groups of four mice were treated with the undiluted test material or the test material at 

concentrations of 50% or 25% v/v in acetone/olive oil 4:1. The preliminary screening test 

suggested that the test material would not produce systemic toxicity or excessive local 

irritation at the highest suitable concentration. The mice were treated by daily application 

of 25 1-11 of the appropriate concentration of the test material to the dorsal surface of each 

ear for three consecutive days (Days 1, 2, 3). The test material formulation was 

administered using an automatic micropipette and spread over the dorsal surface of the 

ear using the tip of the pipette. 

A further group of four mice received the vehicle alone in the same manner. 

182 

PROJECT NUMBER: 014510200 PAGE 9 

3.2.2.2 3H-Methyl Thymidine Administration 

Five days following the first topical application of the test material (Day 6) all mice were 

injected via the tail vein with 250 IJI of phosphate buffered saline (PBS) containing 

3H-methyl thymidine eHTdR: 80 IJCilml, specific activity 2.0 Ci/mmol, GE Healthcare UK 

Ltd) giving a total of 20 IJCi to each mouse. 

3.2.2.3 Observations 

Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and 

on a daily basis on Days 4.5 and 6. Any signs of toxicity or signs of ill health during the 

test were recorded. 

Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) 

and Day 6 (prior to tennination). 

3.2.2.4 Terminal Procedures 

Termination: Five hours following the administration of 3HTdR all mice were killed by 

carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice 

were excised and pooled for each experimental group. For each group 1 ml of PBS was 

added to the pooled lymph nodes. 

Preparation of Single Cell Suspension: A single cell suspension of pooled lymph 

node cells was prepared by gentle mechanical disaggregation through a 200-mesh 

stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 ml of 

PBS into a petri dish labelled with the project number and dose concentration. The 

lymph node cell suspension was transferred to a centrifuge tube. The petri dish was 

washed with an additional 5 ml of PBS to remove all remaining lymph node cens and 

these were added to the centrifuge tUbe. The pooled lymph node cells were pelleted at 

1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 ml of 

PBS and re-pelleted. To precipitate out the radioactive material, the pellet was 

resuspended in 3 ml of 5% Trichloroacetic acid (TCA). 

183 

3.3 

PROJECT NUMBER: 014510200 PAGE 10 

Determination of 3HTdR Incorporation: After approximately eighteen hours incubation 

at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm 

(approximately 450 g) for ten minutes, resuspended in 1 ml of TCA and transferred to 

10 ml of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by 

~-scintillation counting. The "Poly aTMII vials containing the samples and scintillation fluid 

were placed in the sample changer of the scintillator and left for approximately 

twenty minutes. The purpose of this period of time in darkness was to reduce the risk of 

luminescence, which has been shown to affect the reliability of the results. After 

approximately twenty minutes, the vials were shaken vigorously. The number of 

radioactive disintegrations per minute was then measured using the Beckman LS6500 

scintillation system (Beckman Instruments Inc, Fullerton, CA, USA). 

Interpretation of Results 

The proliferation response of lymph node cells was expressed as the number of 

radioactive disintegrations per minute per lymph node (disintegrations per minute/node) 

and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to 

that recorded for the control nodes (Stimulation Index). 

The test material will be regarded as a sensitiser if at least one concentration of the test 

material results in a threefold or greater increase in 3HTdR incorporation compared to 

control values. Any test material failing to produce a threefold or greater increase in 

3HTdR incorporation will be classified as a "non-sensitiser". 

4. ARCHIVES 

Unless instructed otherwise by the Sponsor, all original data and the final report will be 

retained in the Harlan Laboratories Ltd, Shardlow, UK archives for five years, after which 

instructions will be sought as to further retention or disposal. 

184 

PROJECT NUMBER: 014510200 PAGE 11 

5. RESULTS 

5.1 Preliminary Screening Test 


Clinical observations, bodyweight and mortality data are given in Table 1. 


No signs of systemic toxicity were noted. 


Based on this information the undiluted test material and the test material at 


concentrations of 50% and 25% v/v in acetone/olive oil 4:1 were selected for the main 

test. 

5.2 Main Test 

5.2.1 Estimation of the Proliferative Response of Lymph Node Cells 

The radioactive disintegrations per minute per lymph node and the stimulation index are 

given in Table 2. 

A stimUlation index of greater than 3 was recorded for the undiluted test material and the 

test material at concentrations of 50% and 25% v/v in acetone/olive oil 4:1. 

5.2.2 Clinical Observations and Mortality Data 

Individual clinical observations and mortality data for test and control animals are given 

in Table 3. 

There were no deaths. No signs of systemic toxicity were noted in the test or control 

animals during the test. 

185 

PROJECT NUMBER: 0146/0200 PAGE 12 

5.2.3 Bodyweight 

Individual bodyweights and bodyweight changes for test and control animals are given in 

Table 4. 

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to 

those observed in the corresponding control group animals over the same period. 

6. CONCLUSION 

The test material was considered to be a sensitiser under the conditions of the test. 

186 
PROJECT NUMBER: 014510200 	 PAGE 13 

Mixture of PEMB and PE3MB : LOCAL LYMPH NODE ASSAY IN THE MOUSE 

Table 1 	 Clinical Observations, Bodyweight and Mortality Data ­

Preliminary Screening Test 

Concentration Animal 

Number 

Bodyweight 

(g) 

Day Day 

1 6 

1 

Pre- Post 

Dose Dose 

2 

Pre- Post 

Dose Dose 

Day 

3 

Pre- Post 

Dose Dose 

4 5 6 

100% S-1 17 19 0 0 0 0 0 0 0 0 0 

o= No signs of systemic toxicity 

187 
PROJECT NUMBER: 014510200 	 PAGE 14 

Mixture of PEMB and PE3MB : LOCAL LYMPH NODE ASSAY IN THE MOUSE 

Table 2 	 Disintegrations per Minute, Disintegrations per Mlnute/Node and 

Stimulation Index 

Concentration 

(% v/v) in 

acetone/olive oil 

4:1 

dpm dpm/Node a Stimulation Index b Result 

Vehicle 6889.00 861.13 na na 

25 155117.80 19389.73 22.52 Positive 

50 177120.30 22140.04 25.71 Positive 

100 159191.00 19898.88 23.11 Positive 

dpm = Disintegrations per minute 

a = Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 

(total number of lymph nodes) 

b = Stimulation Index of 3.0 or greater indicates a positive result 

na = Not applicable 

188 

PROJECT NUMBER: 014510200 PAGE 15 

Mixture of PEMB and PE3MB : LOCAL LYMPH NODE ASSAY IN THE MOUSE 

Table 3 Individual Clinical Observations and Mortality Data 

Concentration 

(% v/v) in 

acetone/olive oil 

4:1 

Animal 

Number 

Day 1 Day 2 Day 3 Day 

4 

Day 

5 

Day 

Pre- 6

Dose 

Post 

Dose 

PreDose Post 

Dose 

PreDose 

Post 

Dose 

Vehicle 

1-1 0 0 0 0 0 0 0 0 0 

1-2 0 0 0 0 0 0 0 0 0 

1-3 0 0 0 0 0 0 0 0 0 

14 0 0 0 0 0 0 0 0 0 

25 

2-1 i 0 0 0 0 0 0 0 0 0 

2-2 0 0 0 0 0 0 0 0 0 

2-3 0 0 0 0 0 0 0 0 0 

24 0 0 0 0 0 0 0 0 0 

50 

3-1 0 0 0 0 0 0 0 0 0 

3-2 0 0 0 0 0 0 0 0 0 

3-3 0 0 0 0 0 0 0 0 0 

3-4 0 0 0 0 0 0 0 0 0 

100 

4-1 0 0 0 0 0 0 0 0 0 

4-2 0 0 0 0 0 0 0 0 0 

4-3 0 0 0 0 0 0 0 0 0 

4-4 0 0 0 0 0 0 0 0 0 

o= No signs of systemic toxicity 

189 
PROJECT NUMBER: 014510200 PAGE 16 

Mixture of PEMB and PE3MB : LOCAL LYMPH NODE ASSAY IN THE MOUSE 

Table 4 Individual Bodyweights and Bodyweight Changes 

Concentration Bodyweight (9) (% v/v) in Animal Number Bodyweight 

acetone/olive oil Day 1 Day 6 Change (9) 

4:1 

1-1 18 17 -1 

1-2 18 18 0 

Vehide i 

1-3 18 17 -1 

1-4 18 18 0 

2-1 20 19 -1 

2-2 18 18 0 

25 

2-3 19 19 0 

2-4 18 19 1 

3-1 17 17 0 

3-2 17 18 1 

50 

3-3 17 17 0 

3-4 17 17 0 

4-1 17 16 -1 

4-2 20 19 -1 

100 

4-3 20 19 -1 

4-4 17 17 0 

190 
PROJECT NUMBER: 014510200 	 PAGE 17 

Mixture of PEMB and PE3MB: LOCAL LYMPH NODE ASSAY IN THE MOUSE 

Appendix 1 Current Positive Control Study for the Local Lymph Node Assay 

Introduction. A study was perfonned to assess the sensitivity of the strain of mouse 

used at these laboratories to a known sensitiser. The method was designed to meet the 

requirements of the following: 

• 	 OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local 

Lymph Node Assay" (adopted 24 April 2002) 

• 	 Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission 

Directive 2004173/EC 

Test Material: a-Hexylcinnamaldehyde, Tech, 85% 

Project number: 0039/1057 

Study dates: 17 October 2008 to 23 October 2008 

Methods. One group of five animals was treated with 50 jJl (25 jJl per ear) of 

a-Hexylcinnamaldehyde, Tech, 85% as a solution in acetone/olive oil 4:1 at a 

concentration of 15% vlv. A further group of five animals was treated with acetone/olive 

oil 4: 1 alone. 

Results. The Stimulation Index expressed as the mean radioactive incorporation for the 

treatment group divided by the mean radioactive incorporation of the vehicle control 

group are as follows: 

Concentration % vlv in 

acetone/olive oil 4:1 Stimulation Index Result 

15 6.55 Positive 

Conclusion. a-Hexylcinnamaldehyde, Tech, 85% was considered to be a sensitiser 

under the conditions of the test. 

191 

Mixture of PEMB and PE3MB ; LOCAL LYMPH NODE ASSAY IN THE MOUSE 

Appendix 2 Summary of Positive Control Data for the Local Lymph Node Assay. 

Project 

Number Start Date Finish Date Test Material Concentration Vehicle Stimulation Indexa Classificationb 

0039/1017 06103/08 12103/08 a-Hexylcinnamaldehyde 5%, 10%, 25% v/v acetone 5.52, 5.05, 12.89 Positive 

0039/1018 07/03/08 13/03/08 a-Hexylcinnamaldehyde 5%, 10%, 25% vlv ethanol/distilled water 

7:3 4.70, 10.57,17.76 Positive i 

0039/1028 28/03/08 03/04/08 Phenylacetaldehyde (90%) 1%, 2.5%, 5% v/v propylene glycol 2.23,4.25, 23.51 Positive I 

0039/1029 10/04/08 16/04/08 2,4-0Initrobenzenesulfonic 

acid, sodium salt 1%,5%.10% wlw 1% pluronic L92 in distilled water 0.96,4.29,13.15 Positive 

003911032 11104108 17104/08 a-Hexylcinnamafdehyde 15% vlv acetone/olive oil 4: 1 10.91 Positive 

0039/1037 

0039/1038 

23/05/08 

05/06/08 

29/05/08 

11/06108 

a-Hexylcinnamaldehyde 

a-Hexylcinnamaldehyde 

5%,10%,25% v/v 

5%,10%,25% v/v 

liquid paraffin BP 

polyethylene glycol 300 

1.73, 2.32,4.58 

2.07.4.22,10.92 

Positive ----~ 

Positive 

0039/1057 17/10/08 23/10/08 a-Hexylcinnamaldehyde 15% v/v acetone/olive oil 4:1 6.55 Positive 

0039/1058 29/10/08 04111108 a-Hexylcinnamaldehyde 15% v/v dimethyl formamide 4.55 

---­

4.05 

Positive 

0039/1059 29/10/08 04111/08 a-Hexylcinnamaldehyde 15% v/v butanone 

-. 

Positive 

0039/1060 05/11/08 17112108 a-Hexylcinnamaldehyde 15% v/v dimethyl sulphoxlde 5.73 Positive 

0039/1061 05/11/08 11/11/08 a-Hexylcinnamaldehyde 15% vlv acetone 4.21 Positive 

0039/1062 05/11/08 11/11/08 a-Hexylcinnamaldehyde 15% vlv ethanol/distilled water 

7:3 9.49 

-

Positive 

(0 .= Standard Test Method 599 ('Individual' nOdes) I',) 

a= Ratio of test to control lymphocyte proliferation 

b= Stimulation index greater than 3.0 indicates a positive result 

PROJECT NUMBER: 014510200 PAGE 18 
PROJECT NUMBER: 014510200 PAGE 19 

Mixture of PEMB and PE3MB : LOCAL LYMPH NODE ASSAY IN THE MOUSE 

Appendix 3 Vehicle Determination Record 

Vehicle Concentration I 

Method of Description of Suitability* Preparation Formulation 

acetone/olive oil 50% 

0.5 ml test material Vortex mixer solution suitable for dosing i (4:1) -+- 0.5 ml vehicle 

'" = Suitable for dosing if formulation is a solution or fine homogenous suspension which can be 

administered via a micropipette 

193 
PROJECT NUMBER: 014510200 PAGE 20 

Appendix 4 Statement of GLP Compliance in Accordance with Directive 

2004/91EC 

,DH) Department 

\.:..: of Health 

THE DEPARTMENT OF :QEALTH OF THE GO'1ERNMENT 


OF THE UNITED KINGDOM 


GOOD LABORATORYPRACTICH 

STATEMENrOFCOMPLlAl\iCE 


IN ACCORDANCE WITH DIRECTIVE ZOO4J91EC 


TEST FACrJ.JTI! TES'fTY1'E 

Harlan Lllbontories Ltd. AualytkallClillic:a.l Chemistry 

Shardlow Bosille!s :Park Environmental Fate 

I.ondotl Road Envil'Oomontt.1 Toxicology 

Sliardlaw Mutagenicity 

Derby Pti)'llChom 

DEnlGD Toxicology 

DATEOF~ON 

19th August 1008 

A general inspection for compliance with the POnciplc;s of Good Laboratory Prnctice 

was camed out lit me llbove.test facility as part of the UK GLPCompiiance Programme. 

At the time of inspection no deviations were found of ~ufticienl magnitude to affect 

the validity of non.clinical studies perfol'Tiled at these facilities. 

Head, UKGLP Monitoring Aulilorily 

194 

PAGE 1 OF 17 PAGES 

Mixture of PEMB and PE3MB: 

ACUTE EYE IRRITATION IN THE RABBIT 

PROJECT NUMBER: 0145/0199 

AUTHOR: A Pooles 

STUDY SPONSOR: TEST FACILITY: 

Showa Denko K. K. Harlan Laboratories Ltd 

13-9. Shiba Daimon 1-Chome Shardlow Business Park 

Minato-ku Shardlow 

TOKYO 105-8518 Derbyshire 

JAPAN DE722GD 

UK 

Telephone: +44 (0) 1332 792896 

Facsimile: +44 (0) 1332799018 

0145·0199.doefJS 

195 
PROJECT NUMBER: 0145/0199 PAGE 2 

QUALITY ASSURANCE REPORT 


This study type is classed as short-term. The standard test method for this study type 

("General Study Plan" in DECO terminology) was reviewed for compliance once only on 

initial production. Inspection of the routine and repetitive procedures that constitute the 

study is carried out as a continuous process designed to encompass the major phases at 

or about the time this study was in progress. In addition, inspection of general facilities 

not specifically related to this study are done monthly or annually in accordance with QA 

Standard Procedure. 

This report has been audited by the Quality Assurance Unit, and is considered to be an 

accurate account of the data generated and of the procedures followed. 

In each case, the outcome 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 type and schedule of this study were as follows: 

13 November 2008 Standard Test Method Compliance Audit 

16 February 2009 Test Material Preparation 

16 February 2009 Animal Preparation 

16 February 2009 Dosing 

16 February 2009 Assessment of Response 

§ 09 March 2009 Draft Report Audit 

§ Date of QA Signature Final Report Audit 

§ Evaluation specific to this study 

~.N.Qb~ 1 8 MAR 2009 ............ "......................... ~~ ...... ~ ......."................ ~ ................... . DATE: ................................................


For the Quality Assurance Unit'" 

*Authorised QA Signatures: 

Deputy Head of Department: JM Crowther MJScT MRQA 

Senior Audit Staff: G Wren ONe MRQA 

196 

PROJECT NUMBER: 014510199 PAGE 3 

GLP COMPLIANCE STATEMENT 


The work described was performed in compliance with UK GLP standards (Schedule 1, 

Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by 

SI 2004/0994». These Regulations are in accordance with GLP standards published as 

DECO Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17); 

and are in accordance with, and implement, the requirements of Directives 2004/9/EC 

and 2004/10/EC. 

This report fully and accurately reflects the procedures used and data generated . 

.....................A...:.. p.C?~b,................................... DATE: ........ .!..1:.I..~J?9....................... 


A Pooles BA (Hons) 

Study Director 

This report may be presented in final form as a digital (pdf) document. Such documents are prepared by scanning the paper original, 

and are considered of equivalent Integrity and authenticity to versions produced by optical photocopy. However. in all cases the 

hand-signed paper Original. held in secure archives. is the definitive document 

197 
PROJECT NUMBER: 014510199 PAGE 4 

CONTENTS 

QUALITY ASSURANCE REPORT 2 


GLP COMPLIANCE STATEMENT 3 


CONTENTS 4 


SUMMARY 5 


1. INTRODUCTION 6 


2. TEST MATERIAL 6 


2.1 Description, Identification and Storage Conditions 6 


2.2 Preparation of Test Material 6 


2.3 Measurement of pH 7 


3. METHODS 7 


3.1 Animals and Animal Husbandry 7 


3.2 Procedure 8 


3.3 Interpretation of Results 9 


4. ARCHIVES 9 


5. RESULTS 10 


5.1 Ocular Reactions 10 


5.2 Bodyweight 10 


6. CONCLUSION 10 


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


Table 2 Individual Total Scores and Group Mean Scores for9cular Irritation 12 


Table 3 Individual Bodyweights and Bodyweight Changes 13 


Appendix 1 Initial Pain Reaction 14 


Appendix 2 Draize Scale for Scoring Ocular Irritation 15 


Appendix 3 Modified Kay and Calandra Interpretation of Eye Irritation Test 16 


Appendix 4 Statement of GLP Compliance in Accordance with Directive 2004/9/EC 17 


198 
PROJECT NUMBER: 014510199 	 PAGE 5 

Mixture of PEMB and PE3MB: 


ACUTE EYE IRRITATION IN THE RABBIT 


SUMMARY 


Introduction. The study was performed to assess the irritancy potential of the test 

material to the eye of the New Zealand White rabbit. The method was designed to meet 

the requirements ofthe following: 

• 	 OECD Guidelines for the Testing of Chemicals No. 405 "Acute Eye 

Irritation/Corrosion" (adopted 24 April 2002) 

• 	 Method B5 Acute Toxicity (Eye Irritation) of Commission Directive 2004173/EC 

Result A single application of the test material to the non-irrigated eye of three rabbits 

produced moderate conjunctival irritation. All treated eyes appeared normal at the 

48-hour observation. 

Conclusion. The test material produced a maximum group mean score of 8.0 and was 

classified as a minimal irritant (Class 3 on a 1 to 8 scale) to the rabbit eye according to a 

modified Kay and Calandra classification system. 

199 

PROJECT NUMBER: 014510199 	 PAGES 

Mixture of PEMB and PE3MB: 


ACUTE EYE IRRITATION IN THE RABBIT 


1. INTRODUCTION 

The study was performed to assess the irritancy potential of the test material following a 

single application to the rabbit eye. The method was designed to meet the requirements 

of the following: 

• 	 OECD Guidelines for the Testing of Chemicals No. 405 "Acute Eye 

Irritation/Corrosion" (adopted 24 April 2002) 

• 	 Method 85 Acute Toxicity (Eye Irritation) of Commission Directive 2004f73/EC 

The albino rabbit has been shown to be a suitable model for this type of study and is 

recommended in the test method. The results of the study are believed to be of value in 

predicting the likely eye irritancy potential of the test material to man. 

The study was performed between 02 February 2009 and 12 February 2009. 

2. TEST MATERIAL 

2.1 Description, Identification and -Storage Conditions 

Sponsor's identification Mixture of PEM8 and PE3MB ....-.. .. -~ -...---.-...-.. --~ Description .

: .: 


Batch number : : 


Date received "-1S-0ecembe'r200"Ef' 


Storage conditions room temperature in the dark 


The integrity of supplied data relating to the identity, purity and stability of the test 


material is the responsibility of the Sponsor. 


2.2 Preparation of Test Material 

For the purpose of the study the test material was used as supplied. 

The absorption of the test material was not determined. 

200 

3.1 

PROJECT NUMBER: 0145/0199 PAGE 7 

2.3 Measurement of pH 

The pH of the test material was detennined prior to commencement of the study and 

found to be as follows: 

Preparation pH Measurement 

immediately after 10 minutes after 20 minutes 

Undiluted as Supplied unobtainable not applicable not applicable 

90% vlv aqueous preparation approximately 4.3 approximately 4.1 4.3 ofthe test material 

3. METHODS 

Animals and Animal Husbandry . 

Three New Zealand White rabbits were supplied by Harlan Laboratories UK Limited, 

Bicester, Oxon, UK. At the start of the study the animals were in the weight range of 

2.53 to 2.95 kg and were twelve to twenty weeks old. After an acclimatisation period of 

at least five days each animal was given a number unique within the study which was 

written with a black indelible marker-pen on the inner surface of the ear and on the cage 

label. 

The animals were individually housed in suspended cages. Free access to mains 

drinking water and food (2030 Teklad Global Rabbit diet supplied by Harlan Teklad, 

Blackthorn, Bicester, Oxon, UK) was allowed throughout the study. The diet and 

drinking water were considered not to contain any contaminant of a level that might have 

affected the purpose or integrity of the study. 

The temperature and relative humidity were set to achieve limits of 17 to 23°C and 30 to 

70% respectively. Any occasional deviations from these targets were considered not to 

have affected the purpose or integrity of the study. The rate of air exchange was at least 

fifteen changes per hour and the lighting was controlled by 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 any contaminant of a level that might have affected the purpose or integrity 

of the study. 

201 
PROJECT NUMBER: 014510199 PAGES 

3.2 Procedure 

Immediately before the start of the test, both eyes of the provisionally selected test 

rabbits were examined for evidence of ocular irritation or defect with the aid of a light 

source from a standard ophthalmoscope. Only animals free of ocular damage were 

used. 

Initially, a single rabbit was treated. A volume of 0.1 ml of the test material was placed 

into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from 

the eyeball. The upper and lower eyelids were held together for about one second 

immediately after treatment, to prevent loss of the test material, and then released. The 

left eye remained untreated and was used for control purposes. Immediately after 

administration of the test material, an assessment of the initial pain reaction was made 

according to the six pOint scale shown in Appendix 1. 

After consideration of the ocular responses produced in the first treated animal, two 

additional animals were treated. Due to a technician error the initial pain reaction in one 

animal was not recorded. This deviation from the Standard Test Method was considered 

not to affect the purpose or integrity of the study. 

Assessment of ocular damagelirritation was made approximately 1 hour and 24, 48 and 

72 hours following treatment. according to the numerical evaluation given in Appendix 2, 

(from Draize J H (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures 

for Evaluating the Toxicity of Household Substances, National Academy of Sciences, 

Washington DC p,48 to 49). 

Any other ocular effects were also noted. Examination of the eye was facilitated by the 

use of the light source from a standard ophthalmoscope. 

Individual bodyweights were recorded on Day 0 (the day of dosing) and at the end of the 

observation period. 

202 

3.3 

PROJECT NUMBER: 014510199 PAGE 9 

Interpretation of Results 

The numerical values corresponding to each animal, tissue and observation time were 

recorded. The data relating to the conjunctivae were designated by the letters 

A (redness), B (chemosis) and C (discharge), those relating to the iris designated by the 

letter D and those relating to the cornea by the letters E (degree of opacity) and F (area 

of cornea involved). For each tissue the score was calculated as follows: 

Score for conjunctivae = (A + B + C) x 2 

Score for iris = Dx5 

Score for cornea = (E x F) x 5 

Using the numerical data obtained a modified version of the system described by 

Kay J H and Calandra J C (1962), J. Soc. Cosmet. Chem. 13,281-289 (see Appendix 3) 

was used to classify the ocular irritancy potential of the test material. This was achieved 

by adding together the scores for the cornea, iris and conjunctivae for each time point for 

each rabbit. The group means of the total scores for each observation were calculated. 

The highest of these group means (the maximum group mean score) together with the 

persistence of the reactions enabled classification of the eye irritancy potential of the test 

material. 

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

corrosive to the eye. 

4. ARCHIVES 

Unless instructed otherwise by the Sponsor, all original data and the final report will be 

retained in the Harlan Laboratories Ltd, Shard low, UK archives for five years, after which 

instructions will be sought as to further retention or disposal. 

203 
PROJECT NUMBER: 014510199 PAGE 10 

5. RESULTS 

5.1 Ocular Reactions 


Individual and group mean scores for ocular irritation are given in Table 1 and Table 2. 


No corneal or iridial effects were noted during the test. 


Moderate conjunctival irritation was noted in all treated eyes one hour after treatment 


with minimal conjunctival irritation noted at the 24-hour observation. 


All treated eyes appeared normal at the 48-hour observation. 


5.2 Bodyweight 

Individual bodyweights and bodyweight changes are given in Table 3. 

All animals showed expected gain in bodyweight during the study. 

6. CONCLUSION 

The test material produced a maximum group mean score of 8.0 and was classified as a 

minimal irritant (Class 3 on a 1 to 8 scale) to the rabbit eye according to a modified Kay 

and Calandra classification system. 

204 

. Mixture of PEMB and PE3MS : ACUTE EYE IRRITATION IN THE RABBIT 

Table 1 Individual Scores and Individual Total Scores for Ocular Irritation 

68210 Male 68234 Male 

Rabbit Number and Sex 

IPR=2 IPR=2 

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 0 0 0 0 0 0 0 0 --­

Score (Ox 5) 0 0 0 0 0 0 0 0 

CONJUNCTIVAE 

A= Redness 2 1 0 0 2 1 0 0 

B=Chemosis 1 0 0 0 1 0 0 0 

C =Discharge 1 0 0 0 1 0 0 0 

Score (A + B + C) x 2 8 2 0 0 8 2 0 0 

Total Score 8 2 0 0 8 2 0 0 - -­

68235 Male 

IPR=® 

1 24 48 72 

Hour Hours Hours Hours 

0 0 0 0 ! 

0 0 0 0 

0 0 0 0 

• 

0 0 0 0 

0 0 0 0 

I 

2 1 0 0 

1 0 0 0 

1 0 0 0 

8 2 0 0 

8 2 0 0 

r-,) 

o 

t11 IPR =Initial pain reaction 

® =Due to technician error the IPR was not recorded 

PROJECT NUMBER: 014510199 PAGE 11 
PROJECT NUMBER: 014510199 PAGE 12 

Mixture of PEMB and PE3MB : ACUTE EYE IRRITATION IN THE RABBIT 

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 

68210 Male 8 2 0 0 

68234 Male 8 2 0 0 

68235 Male 8 2 0 0 

Group Total 24 6 0 0 

Group Mean 

Score 8.0 2.0 J 0.0 0.0 

206 

PROJECT NUMBER: 014510199 PAGE 13 

Mixture of PEMB and PE3MB: ACUTE EYE IRRITATION IN THE RABBIT 

Table 3 Individual Bodyweights and Bodyweight Changes 

Rabbit Number 

and Sex 

Individual Bodyweight (kg) 

Bodyweight Change (kg) 

Day 0 Day 3 

68210 Male 2.95 3.05 0.10 

68234 Male 2.82 2.90 0.08 

68235 Male 2.53 2.61 0.08 

201 

PROJECT NUMBER: 014510199 PAGE 14 

Mixture of PEMB and PE3MB: ACUTE EYE IRRITATION IN TlIE RABBIT 

Appendix 1 Initial Pain Reaction 

When the material is instilled in the eye there may be an initial local pain reaction. The 

reaction will be graded as follows: 

Class Reaction by Animal Descriptive Rating 

0 No response No initial pain 

1 A few blinks only, normal within one or 

two minutes Practically no initial pain 

2 Rabbit blinks and tries to open eye, but 

reflex closes it Slight initial pain 

3 .Rabbit holds eye shut and puts pressure 

on lids, may rub eye with paw 

Moderate initial pain 

4 Rabbit holds eye shut vigorously, may 

squeal Severe initial pain 

5 

Rabbit holds eye shut vigorously, may 

squeal, claw at eye, jump and try to 

escape 

Very severe initial pain 

There is often no correlation between the initial pain and the subsequent eye irritation. 

208 

PROJECT NUMBER: 014510199 	 PAGE 15 

Mixture of PEMB and PE3MB : ACUTE EYE IRRITATION IN THE RABBIT 

Appendix 2 Draize Scale for Scoring Ocular Irritation 

1. CONJUNCTIVAE 

(A) 	 Redness (refers to palpebral and bulbar conjunctivae excluding cornea and iris) 

Vessels normal o 

Vessels definitely Injected above normal 1 

More diffuse, deeper crimson red, individual vessels not easily discemible 2. 

Diffuse beefy red 3 

(B) 	 Chemosis 

No swelling o 

Any swelling above normal (includes nictitating membrane) 1 

Obvious swelling with partial eversion of Ods 2 

Swelling with lids about half closed 3 

Swelling with lids half closed to completely closed 4 

(C) 	 Discharge 

No discharge 0 

Any amount different from normal (does not include small amounts observed in inner 1 

canthus of normal animals) 

Discharge with moistening of the lids and hairs just adjacent to lids 2. 

Discharge with moistening of the lids and hairs a considerable area around the eye 3 

THE TOTAL SCORE =(A + B + C) X 2 MAXIMUM TOTAL = 20 

2. IRIS 

(D) 	 Values 

Normal o 

Folds above normal, congestion, swelling, circumcorneal injection (any or all of these or 

combination of any thereof) jris still reacting to light (sluggish reaction is positive) 

No reaction to light. haemorrhage, gross destruction (any or all of these) . 2. 

THE TOTAL SCORE =D X 5 MAXIMUM TOTAL =10 

3. CORNEA 

(E) 	 Degree of Opacity (most dense area used) 

No opacity o 

Scattered or diffuse areas, details of iris clearly visible 1 

Easily discernible translucent areas, details of Iris slightly obscured 2 

Opalescent areas, no detaRs of iris visible, size of pupil barely discernible 3 

Opaque, iris not discernible through the opacity 4 

(F) 	 Area of Cornea Involved 

One quarter (or lass) but not zero 1 

Greater than one quarter but less than half 2 

Greater than half but less than three quarters 3 

Greater than three quarters, up to whole area 4 

THE TOTAL SCORE =(E X F) X 5 MAXIMUM TOTAL = 80 

MAXIMUM TOTAL SCORE POSSIBLE =110 

209 

PROJECT NUMBER: 014!5/0199 PAGE 16 

Mixture of PEMB and PE3MB : ACUTE EYE IRRITATION IN THE RABBIT 

Appendix 3 Modified Kay and Calandra Interpretation of Eye Irritation Test 

MAXIMUM 

MEAN 

SCORE 

PERSISTENCE OF SCORE DESCRIPTION RATING 

(AND CLASS) 

0.0 to 0.5 Group mean total at 24 hours = 0 

Group mean total score at 24 hours> 0 

Non-Irritant (1 ) 

Practically non-inilant (2) 

0.5 to 2.5 Group mean total score at 24 hOUlS ;: 0 

Group mean total score at 24 hours> 0 

PraClically non.Jrritant (2) 

Minimallrrllanl (3) 

2.5 to 15 Group mean total SCOl8 at 48 hours" 0 

Group mean total score at 48 hours> 0 

Mlnimallrrllanl (3) 

Mild Irritant (4) 

15 to 25 Group mean total SCOI8 at 72 hours .. 0 

Group mean total score at 72 hours > 0 

Mild mtant (4) 

Moderate Irritant (5) 

25 to 50 Group mean total score at 7 days 

20 or less 

Group mean tolal score al 7 days> 20 

More than half of the Individual total 

scores at 7 days 10 or less 

More than half of the individual total 

SCOret at 7 days> 10 but no individual 

totat score at 7 days> 30 

More than half of the individual total 

scores at 7 days > 10 and any 

individual Store at 7 days> 30 

Moderate Irrttant (5) 

Moderate initant (5) 

Severe irritant (6) 

Severe irritant (e) 

50 to 80 Group mean tolal score at 7 days 

40 or less 

Group mean tolal SCOI8 at 7 days> 40 

More than half of the individual total 

scores at 7 days 30 or less 

More than half of the individual total 

scores at 7 days > 30 but no individual 

total scores at 7 days> 60 

More than half of the individual tolal 

scores at 7 days > 30 and Individual 

total score at 7 days > 60 

Severe irrttant (6) 

Severe irritant (6) 

Very severe lnitant (7) 

Very severe lnitant (7) 

80 to 100 Group mean total Store at 7 days 

80 or less 

Group mean total at 7 days> 80 

More than half of the individual total 

scores at 7 days 60 or less 

More than half of the individual tolal 

scores at 7 days > 60 but no Indlllfdual 

total SCOI8 at 7 days> 100 

More than half of the Individual total 

scores at 7 days > 60 and Individual 

total score at 7 days> 100 

Very severe irritant (7) 

Very severe irritant (7) 

Extremely severe Irritant (8) 

Extremely severe Irritant (8) 

100 to 110 

Group mean tolal SCOI8 at 7 days 

80 or less 

Group mean tolal score at 7 days .. 80 

Very severe lnitan! (7) 

Extremely severe irrtant (8) 

210 

PROJECT NUMBER: 014510199 PAGE 17 

Appendix 4 Statement of GLP Compliance in Accordance with Directive 

2004l9/EC 

ID.;H). Department C of Health 

THE DEPARTMENT OFHEALm OF THE GOVERNMENT 


OF THE UNITED KINGDOM 


GOOD LABORATORY PRACTICE 

STATEMENT OFCOMPUANCE 


IN ACCORDANCE WITH DJREcnVE ~IEC 

tEST FAClLI'I"Y TEST TYPE 

Hnrlan LlIbol'lItorios Ltd. AnalylicallCUnieal Chemistry 

Shardlow Business Park EnvlronmentaJ Fate 

Londol'lRoud Environmental TOlCieology 

ShArdlow Mutagenicity 

Derby Phys/Cheru 

DE72lGD Toxicology 

PATE OFlNSP£C'I'ION 

19th August 2008 

A general inspection for co.mpliance with the Principles of Good Laboratory Practice 

was Cllniedout III the above lest facility as part of the UK GLP Compliance Progrnmme. 

At the time of inspection DO deviations were found ofsufficiem magniwde to affect 

the validity of nOll-clinical stQdies performed at these faciJi~es. 

Dr. AndrewJ. Gray 

Head, UK GLP Monitoring Autkorit)' 

211 

PAGE 1 OF 15 PAGES 

Mixture of PEMB and PE3MB: 

ACUTE DERMAL IRRITATION 

IN THE RABBIT 

PRO..IECT NUMBER: 014510198 . 

STUDY SPONSOR: 

showa Denko K.K. 

13-9. shiba Daimon 1-Chome 

Minato-ku 

TOKYO 105-8518 

JAPAN 

0145-0198.doclJS 

AUTHOR: APooles 

TEST FACILITY: 

Harlan Laboratories Ltd 

shardlow Business Park 

Shard low 

Derbyshire 

DE722GD 

UK 

Telephone: +44 (0) 1332 792896 

Facsimile: +44 (0) 1332 799018 

212 
· PROJECT ~UMBER: 014510198 PAGE 2 

QUALITY ASSURANCE REPORT 


This study type is classed as short-term. The standard test method for this study type 

("General Study Plan" in OECD terminology) was reviewed for compliance once only on 

initial production. Inspection of the routine and repetitive procedures that constitute the 

study is carried out as a continuous process designed to encompass the major phases at 

or about the time this study was in . progress. In addition, inspection of general facilities 

not specifically related to this study are done monthly or annually in accordance with QA 

Standard Procedure. 

This report has been audited by the QualitY Assurance Unit, and is considered to be an 

accurate account of the data generated and of the procedures followed. 

In each case, the outcome 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 type and schedule of this study were as follows: 

14 November 2008 Standard Test Method Compliance Audit 

13 January 2009 Test Material Preparation 

13 January 2009 Animal Preparation 

13 January 2009 Dosing 

13 January 2009 Assessment of Response 

§ 18 February 2009 Draft Report Audit 

§ Date of QA Signature Final Report Audit 

§ Evaluation specific to this study 

\ . 

25 MAR 2009 .. ~:..b.!~~................
DATE: ............................................... . 


For the Quality Assurance Unit* 

*Authorlsed QA Signatures: 

Deputy Head of Department JM Crowther MIScT MRQA 

Senior Audit Staff: G Wren ONC ~RQA 

213 

PROJECT NUMBER: 014510198 PAGES 

GLP COMPLIANCE STATEMENT 


The work described was performed in compliance with UK GLP standards (Schedule 1. 

Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by 

SI 2004/0994»). These Regulations are in accordance with GLP standards published as 

OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17); 

and are in accordance with, and implement, the reqUirements of Directives 200419IEC 

and 2004/10/EC. 

This report fully and accurately reflects the procedures used and data generated . 

............. A.:..PQQ.E>........................................... DATE: ...... ?:~J3...\9.,g .......................... 


A Pooles SA (Hons) 

Study Director 

This report may be presented In final fonn as a digital (pdf) document. Such documents are prepared by scanning the paper original, 

and are considered ofequivalent integrity and authenticity to versions produced by optical photocopy. However, in all cases the 

hand-signed paper original, held in secure archives, i$ the definitive document 

214 
PROJECT NUMBER: 014510198 PAGE 4 

CONTENTS 


QUALITY ASSURANCE REPORT 2 


GLP COMPLIANCE STATEMENT 3 


CONTENTS 4 


SUMMARY 5 


1. INTRODUCTION 6 


2. TEST MATERIAL 6 


2.1 Description, Identification and Storage Conditions 6 


2.2 Preparation of Test Material 7 


2.3 Measurement of pH 7 


3. METHODS 7 


3.1 Animals and Animal Husbandry 7 


3.2 Procedure 8 


3.3 Interpretation of Results 9 


4. ARCHIVES 10 


5. RESliLTS 11 


5.1 Skin Reactions 11 


5.2 80dyweight 11 


6. CONCLUSION 11 


Table 1 Individual Skin Reactions Following 3-Minute and 1-Hour Exposures 12 


Table 2 Individual Skin Reactions Following 4-Hour Exposure 13 


Table 2 Individual 80dyweights and Bodyweight Change 14 


Appendix 1 Statement of GLP Compliance in Accordance with Directive 2004/9/EC 15 


215 

PROJECT NUMBER: 0145/0198 	 PAGES 

Mixture of PEMB and PE3MB: 


ACUTE DERMAL IRRITATION IN THE RABBIT 


SUMMARY 

Introduction. The study was performed to assess the irritancy potential of the test 

material to the skin of the New Zealand White rabbit. The method was designed to meet 

the requirements of the following: 

• 	 OECD Guidelines for the Testing of Chemicals No. 404 "Acute Dermal 

Irritation/Corrosion" (adopted 24 April 2002) 

• 	 Method 84 Acute Toxicity (Skin Irritation) of Commission Directive 2004n3/EC 

Results. 3-minute and 1-hour semi-occluded applications of the test material to the 

intact skin of one rabbit produced no evidence of skin irritation. 

A single 4-hour, semi-occluded application of the test material to the intact skin of three 

rabbits produced no evidence of skin irritation. No corrosive effects were noted. 

Conclusion. The test material produced a primary irritation index of 0.0 and was 

classified as non-irritant to rabbit skin according to the Draize classification scheme. 

216 

2.1 

PROJECT NUMBER: 0145J0198 	 PAGE 6 

Mixture of PEMB and PE3MB: 


ACUTE DERMAL. IRRITATION IN THE RABBIT 


1. INTRODUCTION 

The study was performed to assess the irritancy potential of the test material following 

single, 3~minute, 1 and 4-hour, semi-occluded applications to the intact rabbit skin. The 

method was designed to meet the requirements of the following: 

• 	 OECD Guidelines for the Testing of Chemicals No. 404 "Acute Dermal 

Irritation/Corrosion" (adopted 24 April 2002) 

• 	 Method B4 Acute Toxicity (Skin Irritation) of Commission Directive 2004173/EC 

The albino rabbit has been shown to be a suitable model for this type of study and is 

recommended in the test method. The results of the study are believed to be of value in 

predicting the likely skin irritancy potential of the test material to man. 

The study was performed between 21 January 2009 and 30 January 2009. 

2. TEST MATERIAL. 

Descriptiont Identification and Storage Conditions 

Sponsor's identification Mixture of PEMB and PE3MB I ....... • ..... ~- ... --............. -----...... • .. '; 
 Description 	 , 	 . , , , 	 . : 	 ' Batch number :....................................................... ,..",: 

Date received 15 December 2008 

Storage conditions room temperature in the dark 

The integrity of supplied data relating to the identity, purity and stability of the test 

material is the responsibility of the Sponsor. 

217 

3.1 

PROJECT NUMBER: 014510198 PAGE 7 

2.2 Preparation of Test Material 

For the purpose of the study the test material was used as supplied. 

The absorption of the test material was not determined. 

2.3 Measurement of pH 

The pH of the test material was determined prior to commencement of the study and 

found to be as follows: 

Preparation 

immediately 

pH Measurement 

after 10 minutes after 20 minutes 

Undiluted as Supplied unobtainable not applicable not applicable I 

90% vlv aqueous preparation 

of the test material approximately 4.3 approximately 4.1 4.3 

3. METHODS 

Animals and Animal Husbandry 

Three New Zealand White rabbits were supplied by Harlan Laboratories UK Limited, 

Bicester, Oxon, UK. At the start of the study the animals were in the weight range of 2.0 

to 3.5 kg and were twelve to twenty weeks old. After an acclimatisation period of at least 

five days each animal was given a number unique within the study which was written 

with a black. indelible marker-pen on the inner surface of the ear and on the cage label. 

The animals were individually housed in suspended cages. Free access to mains 

drinking water and food (2030 Teklad Global Rabbit diet supplied by Harlan Teklad, 

Blackthom, Bicester, Oxon, UK) was allowed throughout the study. The diet and drinking 

water were considered not to contain any contaminant of a level that might have affected 

the purpose or integrity of the study. 

The temperature and relative humidity were set to achieve limits of 17 to 23°C and 30 to 

70% respectively. Any occasional deviations from these targets were considered not to 

have affected the purpose or integrity of the study. The rate of air exchange was at least 

218 

PROJECT NUMBER: 014510198 PAGES 

fifteen changes per hour and the lighting was controlled by 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 any contaminant of a level that might have affected the purpose or integrity 

of the study. 

3.2 Procedure 

On the day before the test each rabbit was clipped free of fur from the dorsallflank area 

using veterinary clippers. Only animals with a healthy intact epidennis by gross 

observation were selected for the study. 

One rabbit was initially treated. Three suitable sites were selected on the back of the 

rabbit. At each test site a quantity of 0.5 ml of the test material was introduced under a 

2.5 cm x 2.5 em cotton gauze patch and placed in position on the shorn skin. Each 

patch was secured in position with a strip of surgical adhesive tape. To prevent the 

animal interfering with the patches, the trunk of the rabbit was wrapped in an elasticated 

corset for the duration of the exposure period. 

One patch was removed at each of three time points: 3 minutes, 1 hour and 4 hours after 

application. Any residual test material was removed by gentle swabbing with cotton wool 

soaked in distilled water. 

After consideration of the skin reactions produced in the first animal, two additional 

animals were treated with 0.5 ml of test material. One patch was applied to the back of 

each rabbit, and was allowed to remain in contact with the skin for a period of four hours. 

Approximately one hour following the removal of the patches, and 24, 48 and 72 hours 

later, the test sites were examined for evidence of primary irritation and scored according 

to the follOwing scale: 

219 

3.3 

PROJECT NUMBER: 0145/0198 PAGE 9 

EVALUATION OF SKIN REACTIONS 


Erythema and Eschar Formation Value 

No erythema .................................................................................................... . o 

Very slight erythema (barely perceptible) ....................................................... .. 1 

Well-defined erythema ................................................................................... .. 2 

Moderate to severe erythema ......................................................................... . 3 

Severe erythema (beef redness) to eschar formation preventing grading of 

erythema ......................................................................................................... . 4 

Oedema Formation 

No oedema....................................................................................................... 0 

Very slight oedema (barely perceptible) ........................................................... 1 

Slight oedema (edges of area well-defined by definite raising) ........................ 2 

Moderate oedema (raised approximately 1 millimetre) .................................... 3 

Severe oedema (raised more than 1 millimetre and extending beyond the area 

of exposure) ..................................................................................................... 4 

Any other skin reactions. if present, were also recorded. 

Individual bodyweights were recorded on Day 0 (the day of dosing) and at the end of the 

observation period. 

Interpretation of Results 

Calculation of Primary Irritation Index and Grading of Irritancy Potential Using the 

Draize Scheme 

The scores for erythema and oedema at the 24 and 72-hour readings were totalled for 

the three test rabbits (12 values) and this total was divided by six to give the primary 

irritation index of the test material. The test material was classified according to the 

following scheme devised by Draize J H (1959) ItDerrnal Toxicity" In: Appraisal of the 

Safety of Chemicals in Foods, Drugs and Cosmetics. Association of Food and Drug 

Officials of the United States, Austin, Texas, pAS-59: 

220 

PROJECT NUMBER: 0145/0198 PAGE 10 

Primary Irritation Index Classification of Irritancy 

o Non-irritant 

> Oto2 Mild irritant 

> 2toS Moderate irritant 

> StoB Severe irritant 

If irreversible alteration of the dermal tissue is noted in any rabbit, as judged by the Study 

Director, which include ulceration and clear necrosis or signs of scar tissue, the test 

material is classified as corrosive to rabbit skin. Classification according to Draize may, 

therefore, not be applicable. 

4. ARCHIVES 

Unless instructed otherwise by the Sponsor, all original data and the final report will be 

retained in the Harlan Laboratories Ltd, Shardlow. UK archives for five years, after which 

instructions will be sought as to further retention or disposal. 

221 

PROJECT NUMBER: 014510198 PAGE 11 

5. RESULTS 

5.1 Skin Reactions 

5.1.1 3-Minute Exposure Period 

The individual scores for erythema/eschar and oedema are given in Table 1. 

No evidence of skin irritation was noted during the study. 

5.1.2 i-Hour Exposure. P~riod .. 

The individual scores for erythemaleschar and oedema are given in Table 1. 

No evidence of skin irritation was noted during the study. 

5.1.3 4~Hour Exposure Period 

The individual scores for erythema/eschar and oedema are given in Table 2. 

No evidence of skin irritation was noted during the study. 

5.2 Bodyweight 

Individual bodyweights and bodyweight changes are given in Table 3. 

All animals showed expected gain in bodyweight during the study. 

6. CONCLUSION 

The test material produced a primary irritation index of 0.0 and was classified as 

NON-IRRITANT to rabbit skin according to the Draize classification scheme. No 

corrosive effects were noted. 

222 

PROJECT NUMBER: 014510198 	 PAGE 12 

Mixture of PEMB and PE3MB : ACUTE DERMAL IRRITATION IN THE RABBIT 

Table 1 	 Individual Skin Reactions Following 3-Minute and 1-Hour 

Exposures 

Skin Reaction Observation Time 

Individual Scores - Rabbit Number and Sex 

68162 Male 

3-Minute Exposure 1-Hour Exposure 

Erythema/Eschar 

Formation 

1 Hour 

24 Hours 

48 Hours 

72 Hours 

0 

0 

0 

0 

0 

0 

0 

0 

Oedema Formation 

1 Hour 

24 Hours 

48 Hours 

72 Hours 

0 

0 

0 

0 

0 

0 

0 

0 

223 

PROJECT NUMBER: 014510198 PAGE 13 

Mixture of PEMB and PE3MB : ACUTE DERMAL IRRITATION IN THE RABBIT 

Table 2 Individual Skin Reactions Following 4-Hour Exposure 

Skin Reaction Observation Time 

Individual Scores ­ Rabbit Number and Sex 

Total 

68162 Male 68192 Male 68193 Male 

Erythema/Eschar 

Formation 

1 Hour 

24 Hours 

48 Hours 

72 Hours 

0 

0 

0 

0 

0 

0 

0 

0 

·0 

0 

0 

0 

(0) 

0 

( D) 

0 

Oedema 

Formation 

1 Hour 

24 Hours 

48 Hours 

72 Hours 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

(0) 

0 

( D) 

0 

Sum of 24 and 72-hour Readings (S) 0 

Primary Irritation Index (S/6) 016 =0.0 

Classification NON-IRRITANT 

( ) = Total values not used for calculation of primary irritation index 

224 

PROJECT NUMBER: 0145/0198 PAGE 14 

Mixture of PEMB and PE3MB : ACUTE DERMAL IRRITATION IN THE RABBIT '. . 

Table 3 Individual Bodyweights and Bodyweight Change 

Rabbit Number 

and Sex 

Individual Bodyweight (kg) Bodyweight Change (kg) 

Day 0 Day 3 

68162 Male 2.85 2.88 0.03 

68192 Male 2.35 2.43 0.08 

68193 Male 2.46 2.54 0.08 

225 

Bead. UK. GLPMoaitorlugA1Ithorlty 

PROJECT NUMBER: 014510198 PAGE 15

Appendix 1 Statement of GLP Compliance in Accordance with Directive 

2004l9IEC 

,DH) Department 

\:.: of Health 

THE D~~AltTMENT OF HEALTH OF THE GOveRNMENT 

OF THE UNITED KINGDOM 

SOODLABORATORY PRACfiCE 

STATEMENT OF COMPLIANCE 

iN ACCORDANCEwrrH DIRECTIVE lOO4I9I.BC 

TIlSTTYPE 

Harlan Labora~ries LId. Ailldyii\:ilJlClinical Chemistry 

Sliardlow BuslDess Park Eln'ironmental Fate 

LoDdou Road EuviroDllleatal Toxicology 

SlIanilow M_geniclty 

Derby PhyalChesn 

DEl12GD Tuieoloay 

19th August 2008 

A general.inspection for compliance with the Principles of Good Laboratory Practice 

was carried at the above test facility as part of the UK GLP Compliance ProgramIl1e. 

At the tirIX: of inspccti9n no deviations found of sufficient magnitude to affect 

the vali~ly of noo-clin.ical StpdiC5 perfonncd at these facilit'iel. 

Dr. Andrew J. Gray 

226 

PAGE 1 OF 17 PAGES harlan'" ... 

Mixture of PEMB and PE3MB: 

ACUTE DERMAL TOXICITY 

(LIMIT TEST) IN THE RAT 

PROJECT NUMBER: 0145/0195 

AUTHOR: APooles 

STUDY SPONSOR: TEST FACILITY: 

Showa Denko K.K. Harlan Laboratories Ltd 

13-9, Shlba Dalmon 1-Chome Shard low Business Park 

Minato-ku Shardlow 

TOKYO 105-6516 Derbyshire 

JAPAN DE722GD 

UK 

Telephone: +44 (0) 1332 792696 

Facsimile: +44 (0) 1332799016 

0145-0195.doclJS 

227 

PROJECT NUMBER: 014510195 PAGE 2 

QUALITY ASSURANCE REPORT 


This study type is classed as short-term. The standard test method for this study type 

("General Study Plan" in OECD terminology) was reviewed for compliance once only on 

initial production. Inspection of the routine and repetitive procedures that constitute the 

study is carried out as a continuous process designed to encompass the major phases at 

or about the time this study was in progress. In addition, inspection of general facilities 

not speCifically related to this study are done monthly or annually in accordance with QA 

Standard Procedure. 

This report has been audited by the Quality Assurance Unit, and is considered to be an 

accurate account of the data generated and of the procedures followed. 

In each case, the outcome 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 type and schedule of this study were as follows: 

09 December 2008 Standard Test Method Compliance Audit 

11 February 2009 Test Material Preparation 

11 February 2009 Animal Preparation 

11 February 2009 Dosing 

11 February 2009 Assessment of Response 

12 February 2009 Necropsy 

§ 09 March 2009 Draft Report Audit 

§ Date of QA Signature Final Report Audit 

§ Evaluation specific to this study 

.~.-b.!.~.~ . 2 0 MAR 2009 .........
m ............. 
DATE ................................................ . 


For the Quality Assurance Unit* 

*Authorised QA Signatures: 

Deputy Head of Department JM Crowther MIScT MRQA 

Senior Audit Staff: G Wren ONC MRQA 

228 

PROJECT NUMBER: 014510195 PAGE 3 

GLP COMPLIANCE STATEMENT 


The work described was performed in compliance with UK GLP standards (Schedule 1, 

Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by 

SI2004/0994». except that the stated version of the standard test method was not 

available to be followed. These Regulations are in accordance with GLP standards 

published as OECD Principles on Good Laboratory Practice (revised 1997. 

ENV/MC/CHEM(98)17); and are in accordance with, and implement, the requirements of 

Directives 2004/9IEC and 2004/101EC. 

This report fully and accurately reflects the procedures used and data generated. 

A.(Joo~ \~ l~ lo~ , ............. ~ ...... ,,~ ............................ ....................... ~ ................. . DATE: ................................................. .. 


A Pooles BA (Hons) 


Study Director 


This I8port may be presented in final fonn as a digital (pdf) document. Such documents al8 prepared by scanning the paper original, 

and al8 considered of equivalent Integrity and authenticity to versions produced by optical photocopy. However. In all cases the 

hand-signed paper original, held In secure archives. is the definitive document. 

229 
PROJECT NUMBER: 0145/0195 PAGE 4 

CONTENTS 


QUALITY ASSURANCE REPORT 2 


GLP COMPLIANCE STATEMENT 3 


CONTENTS 4 


SUMMARY 5 


1. INTRODUC"nON 6 


2. TEST MATERIAL 6 


2.1 Description, Identification and Storage Conditions 6 


2.2 Preparation of Test Material 7 


3. METHODS 7 


3.1 Animals and Animal Husbandry 7 


3.2 Procedure 8 


3.3 Evaluation of Data 9 


4. ARCHIVES 10 


5. RESULTS 11 


5.1 Mortality Data 11 


5.2 Clinical Observations 11 


5.3 Dermal Reactions 11 


5.4 Bodyweight 11 


5.5 Necropsy 11 


6. CONCLUSION 11 


Table 1 Individual Clinical Observations and Mortality Data 12 


Table 2 Individual Dermal Reactions - Males 13 


Table 3 Individual Dermal Reactions - Females 14 


Table 4 Individual Bodyweights and Weekly Bodyweight Changes 15 


Table 5 Individual Necropsy Findings 16 


Appendix 1 Statement of GLP Compliance in Accordance with Directive 2004/9/EC 17 


230 

PROJECT NUMBER: 0145/0195 	 PAGE 5 

Mixture of PEMB and PE3MB: 


ACUTE DERMAL TOXICITY (LIMIT TEST) IN THE RAT 


SUMMARY 


Introduction. The study was performed to assess the acute dermal toxicity of the test 

material in the Wistar strain rat. The method was designed to meet the requirements of 

the following: 

• 	 DECO Guidelines for the Testing of Chemicals No. 402 "Acute Dermal Toxicity" 

(adopted 24 February 1987) 

• Method 83 Acute Toxicity (Dermal) of Commission Directive 92169/EEC 

Method. A group of ten animals (fIVe males and five females) was given a single. 

24-hour, semi-occluded dermal application of the undiluted test material to intact skin at 

a dose level of 2000 mg/kg bodyweight. Clinical signs and bodyweight development 


were monitored during the study. All animals were subjected to gross necropsy. 


Mortality. There were no deaths. 


Clinical Observations. There were no signs of systemic toxicity. 


Dermallrotation. There were no Signs of dermal irritation. 


Bodyweight All animals showed expected gains in bodyweight over the study period. 


Necropsy_ No abnormalities were noted at necropsy. 


Conclusion. The acute dermal median lethal dose (LD50) of the test material in the 


Wistar strain rat was found to be greater than 2000 mg/kg bodyweight. 

231 

2.1 

PROJECT NUMBER: 0145/0195 	 PAGE 6 

Mixture of PEMB and PE3MB: 


ACUTE DERMAL TOXICITY (LIMIT TEST) IN THE RAT 


1. INTRODUCTION 

The study was performed to assess the acute dermal toxicity of the test material in the 

Wistar strain rat. The method was designed to meet the requirements of the following: 

• 	 DECO Guidelines for the Testing of Chemicals No. 402 "Acute Dermal Toxicity" 

(adopted 24 February 1987) 

• 	 Method B3 Acute Toxicity (Dermal) of Commission Directive 92169/EEC 

The rat was selected for this study as it is a readily available rodent species historically 

used in safety evaluation studies and is acceptable to appropriate regulatory authorities. 

The dermal route was selected as the most appropriate route of exposure and the results 

of the study are believed to be of value in predicting the likely toxicity of the test material 

to man. 

The study was performed between 04 February 2009 and 18 February 2009. 

2. TEST MATERIAL 

Description, Identification and Storage Conditions 

Sponsor's identification Mixture of PEMB and PE3MB ...-------...... ----~-- ..-.-. Description : 1 	 : 

Batch number 	 ..• ___ ._. ____ • __ ••• __ •• ________1 

Date received 	 15 December 2008 

Storage conditions room temperature in the dark 

The integrity of supplied data relating to the identity, purity and stability of the test 

material is the responsibility of the Sponsor. 

232 

PROJECT NUMBER: 0145/0195 PAGE 7 

2.2 Preparation of Test Material 

For the purpose of the study the test material was used as supplied. The specific gravity 

was determined and used to calculate the appropriate dose volume for the required dose 

level. 

The absorption of the test material was not determined. 

3. METHODS 

3.1 Animals and Animal Husbandry 

Five male and five female Wistar (HsdRccHan®n.l:WIST®TM) strain rats were supplied by 

Harlan Laboratories UK Limited, Bicester, Oxon, UK. On receipt the animals were 

randomly allocated to cages. The females were nulliparous and non-pregnant. After an 

acclimatisation period of at least fIVe days the animals were selected at random and 

given a number unique within the study by indelible ink-marking on the tail and a number 

written on a cage card. At the start of the study the animals weighed at least 2009, and 

were eight to twelve weeks of age. The weight variation did not exceed ±20% of the 

mean weight for each sex. 

The animals were housed in suspended solid-floor polypropylene cages furnished with 

woodflakes. The animals were housed individually during the 24-hour exposure period 

and in groups of fIVe, by sex, for the remainder of the study. Free access to mains 

drinking water and food (2014 Teklad Global Rodent diet supplied by Harlan Teklad, 

Blackthorn, Bicester, Oxon, UK) was allowed throughout the study. The diet. drinking 

water and bedding were routinely analysed and were considered not to contain any 

contaminants that could reasonably be expected to affect the purpose or integrity of the 

study. 

The temperature and relative humidity were set to achieve limits of 19 to 25°C and 30 to 

70% respectively. Any occasional deviations from these targets were considered not to 

have affected the purpose or integrity of the study. The rate of air exchange was at least 

fifteen changes per hour and the lighting was controlled by a time switch to give twelve 

hours continuous light (06:00 to 18:00) and twelve hours darkness. 

233 

PROJECT NUMBER: 014510195 PAGE 8 

The animals were provided with environmental enrichment items which were considered 

not to contain any contaminant of a level that might have affected the purpose or integrity 

of the study. 

3.2 Procedure 

On the day before treatment the back and flanks of each animal were clipped free of 

hair. 

Using available information on the toxicity of the test material, a single group of animals 

was treated as follows: 

Dose Level Dose Volume Number of Rats 

(mg/kg) Specific Gravity (mllkg) Male Female 

2000 1.194 1.68 5 5 

The calculated volume of test material, as received, was applied as evenly as possible to 

an area of shorn skin (approximately 10% of the total body surface area) using a 

graduated syringe. A piece of surgical gauze was placed over the treatment area and 

semi-occluded with a piece of self-adhesive bandage. The animals were caged 

individually for the 24-hour exposure period. Shortly after dosing the dressings were 

examined to ensure that they were securely in place. 

After the 24-hour contact period the bandage was carefully removed and the treated skin 

and surrounding hair wiped with cotton wool moistened with distilled water to remove any 

residual test material. The animals were returned to group housing for the remainder of 

the study period. 

The animals were observed for deaths or overt signs of toxicity ~, 1, 2 and 4 hours after 

dosing and subsequently once daily for fourteen days. 

After removal of the dressings and subsequently once daily for fourteen days, the test 

sites were examined for evidence of primary irritation and scored according to the 

following scale from Draize J H (1977) "Dermal and Eye Toxicity Tests" In: Principles and 

Procedures for Evaluating the Toxicity of Household Substances, National Academy of 

Sciences, Washington DC p.31: 

234 

3.3 

PRO.IECT NUMBER: 014510195 PAGE 9 

EVALUATION OF SKIN REACTIONS 


Erythema and Eschar Formation Value 

No erythema ................................................................................................... . o 

Very slight erythema (barely perceptible) ....................................................... .. 1 

Well-defined erythema .................................................................................... . 2 

Moderate to severe erythema ........................................................................ .. 3 

Severe erythema (beef redness) to slight eschar formation (injuries in depth) 4 

Oedema Formation 

No oedema ...................................................................................................... 0 

Very slight oedema {barely perceptible)........................................................... 1 

Slight oedema (edges of area well~defined by definite raising) ........................ 2 

Moderate oedema (raised approximately 1 millimetre) ..................... ...... ......... 3 

Severe oedema (raised more than 1 millimetre and extending beyond the area 

of exposure) .................................................................................. :.... ........ ...... 4 

Any other skin reactions, if present were also recorded. 

Individual bodyweights were recorded prior to application of the test material on Day 0 

and on Days 7 and 14. 

At the end of the study the animals were killed by cervical dislocation. All animals were 

subjected to gross necropsy. This consisted of an external examination and opening of 

the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities 

was recorded. No tissues were retained. 

Evaluation of Data 

Data evaluations included the relationship, if any, between the exposure of the animal to 

the test material and the incidence and severity of all abnormalities including behavioural 

and clinical observations, gross lesions, bodyweight changes, mortality and any other 

toxicological effects. 

Using the mortality data obtained, an estimate of the acute dermal median lethal dose 

(LDso) of the test material was made. 

235 
PROJECT NUMBER; 014510195 PAGE 10 

4. ARCHIVES 

Unless instructed otherwise by the Sponsor, all original data and the final report will be 

retained in the Harlan Laboratories Ltd, Shardlow, UK archives for five years, after which 

instructions will be sought as to further retention or disposal. 

236 

PROJECT NUMBER: 014510195 PAGE 11 

5. RESULTS 


Individual clinical observations and mortality data are given in Table 1. 


5.1 Mortality Data 


There were no deaths. 


5.2 Clinical Observations 


There were no signs of systemic toxicity. 


5.3 Dermal Reactions 

Individual dermal reactions are given in Table 2 and Table 3. 

There were no signs of dermal irritation. 

5.4 Bodyweight 

Individual bodyweights and weekly bodyweight changes are given in Table 4. 

All animals showed expected gains in bodyweight over the study period. 

5.5 Necropsy 

Individual necropsy findings are given in Table 5. 

No abnormalities were noted at necropsy. 

6. CONCLUSION 

The acute dermal median lethal dose (LOso) of the test material in the Wistar strain rat 

was found to be greater than 2000 mglkg bodyweight. 

237 

Mixture of PEMB and PE3MB: ACUTE DERMAL TOXICITY (LIMIT TEST) IN THE RAT 

Table 1 Individual Clinical Observations and Mortality Data 

Dose 

Level 

mg/kg 

Animal 

Number 

and Sex 

Effects Noted After Initiation of 

Exposure (Hours) Effects Noted After Initiation of Exposure (Days) 

% 1 2 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 

2000 

1-0 

Male 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

1-1 

Male 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

1-2 

Male 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

1-3 

Male 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

1-4 

Male 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

2-0 

Female 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

2-1 

Female 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 i 

2-2 

Female 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

2-3 

Female 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

2-4 

Female 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

r-;, 

w 

o= No signs of systemic toxicity 

PROJECT NUMBER: 0145/0195 PAGE 12 

00 
Mixture of PEMB and PE3MB: ACUTE DERMAL TOXICITY (LIMIT TEST) IN THE RAT 

Table 2 Individual Dermal Reactions w Males 

Dose Animal Effects Noted After Initiation of Exposure (Days) 

Level Number Observation 

mg/kg and Sex 1 2 3 4 5 6 7 8 9 10 

Erythema 0 0 0 0 0 0 0 0 0 0 

1-0 Oedema 0 0 0 0 0 0 0 0 0 0 Male 

Other 0 0 0 0 0 0 0 0 0 0 

Erythema 0 0 0 0 0 0 0 0 0 0 

1-1 Oedema 0 0 0 0 0 0 0 0 0 0 Male 

other 0 0 0 0 0 0 0 0 0 0 

Erythema 0 0 0 0 0 0 0 0 0 0 

2000 1-2 Oedema 0 0 0 0 0 0 0 0 0 0 Male 

Other 0 0 0 0 0 0 0 0 0 0 

Erythema 0 0 0 0 0 0 0 0 0 0 

1-3 Oedema 0 0 0 0 0 0 0 0 0 0 Male 

Other 0 0 0 0 0 0 0 0 0 0 

Erythema 0 0 0 0 0 0 0 0 0 0 

1-4 Oedema 0 0 0 0 0 0 0 0 0 0 Male 

Other 0 0 0 0 0 0 0 0 0 0 

11 12 13 14 

0 0 0 0 

0 0 0 0 

0 0 0 0 

0 0 0 0 

0 0 0 0 

0 0 0 0 

0 0 0 0 

0 0 0 0 

0 0 0 0 -­

0 0 0 0 

0 0 0 0 

0 0 0 0 

0 0 0 0 

0 0 0 0 

0 0 0 0 

N 

(.,.) 

c.o 

o=No reactions 

PROJECT NUMBER: 0145/0195 PAGE 13 
Mixture of PEMB and PE3MB: ACUTE DERMAL TOXICITY (LIMIT TEST) IN THE RAT 

Table 3 Individual Dermal Reactions - Females 

Dose Animal Effects Noted After Initiation of Exposure (Days) I 

1 

Level Number ObseNation ---­ ----­

mglkg and Sex 1 2 3 4 5 6 7 8 9 10 11 12 13 14 

Erythema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

2-0 Oedema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Female 

Other 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

Erythema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

2-1 Oedema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Female 

Other 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

Erythema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

2000 2-2 Oedema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Female 

Other 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

Erythema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

2-3 Oedema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Female 

Other 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

Erythema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

2-4 Oedema 0 0 -0 0 0 0 0 0 0 0 0 0 0 0 Female 

Other 0 0 0 0 0 0 0 0 0 0 0 0 0 0 -­

N 

~ 

o= No reactions 

PROJECT NUMBER: 014510195 PAGE 14 
Mixture of PEMB and PE3MB: ACUTE DERMAL TOXICITY (LIMIT TEST) IN THE RAT 

Table 4 Individual Bodyweights and Weekly Bodyweight Changes 

Dose Level 

mglkg 

Animal Number 

and Sex 

0 

Bodyweight (g) at Day 

7 14 

Bodyweight Change (g) During Week 

1 2 

1-0 Male 223 230 253 7 23 

1-1 Male 242 273 304 31 31 

1-2 Male 238 270 312 32 42 

1-3 Male 239 263 288 24 25 

2000 

1-4 Male 

2-0 Female 

236 

207 

260 

217 

289 

228 

24 

10 

29 

11 

2-1 Female 221 226 237 5 11 

2-2 Female 216 218 226 2 8 

2-3 Female 201 202 216 1 14 

2-4 Female 202 203 212 1 9 

'" -'=" 

PROJECT NUMBER: 014510195 PAGE 15 
Mixture of PEMB and PE3MB: ACUTE DERMAL TOXICITY (LIMIT TEST) IN THE RAT 

Table 5 Individual Necropsy Findings 

Dose Level 

mg/kg 

Animal Number 

and Sex Time of Death Macroscopic Observations 

1-0 

Male Killed Day 14 No abnormalities detected 

1-1 

Male Killed Day 14 No abnormalities detected 

1-2 

Male Killed Day 14 No abnormalities detected 

1-3 

Male Killed Day 14 No abnormalities detected 

2000 

1-4 

Male 

2-0 

Female 

Killed Day 14 

Killed Day 14 

No abnormalities detected 

No abnormalities detected 

2-1 

Female Killed Day 14 No abnormalities detected 

2-2 

Female Killed Day 14 No abnormalities detected 

2-3 

Female Killed Day 14 No abnormalities detected 

2-4 

Female Killed Day 14 No abnormalities detected 

i') 

.;.. 

i') 

PROJECT NUMBER: 0145/0195 PAGE 16 
PROJECT NUMBER: 014510195 PAGE 17 

Appendix 1 Statement of GlP Compliance in Accordance with Directive 

2004/9/EC 

, pH). Department 

~ of Health 

TIJE DEPARTMENT OF HEALTH OF THE GOVERNMENT 


OF THE UNITED KINGDOM 


COOD LABORATORY PRACTICE 

STATEMEtI.'T OFCO~!PLlANCE 

IN ACCORDANCE wrmDlRF.c1tvE2004J9IEC 


TEST FACILITY TIi:STTVPE 

Hllrllm Laboratories Ltd. AnalytieallCliuitai Cllemistry 

Sllardlow BU$joe8sPark Environmental Fate 

London Road En"ironmenfal Toxicology 

Shan:llow Mutagenicity 

Derby PbysiChem 

DE?22GD Toxicology 

DATE OFJNSPftCTION 

19th August Z008 

kgeneral inspection for complia.nce with the Principles of Good Laboratory Practice 

was carritld out at1m: above test faci lity as pan ofthe UK GLP Compliance Programme. 

At the time of inspection 110 deviati.ons were foulld of sufficient magniwde to affect 

the validil)! ofnon-cUnica] studies performedallhese facilities, 

Dr. Andrew J. Gray 

Head, UKGLP MonitoringAuthorlt~ 

243 

PAGE 1 OF 16 PAGES harlan~ .... 

Mixture of PEMB and PE3MB: 

ACUTE ORAL TOXICITY IN THE RAT 

- FIXED DOSE METHOD 

PROJECT NUMBER: 0145/0194 

AUTHOR: A Pooles 

STUDY SPONSOR: TEST FACIUTY: 

Showa Denko K.K. Harlan Laboratories Ltd 

13-9, Shiba Daimon 1-Chome Shardlow Business Park 

Minato-ku Shardlow 

TOKYO 105-8518 Derbyshire 

JAPAN DE722GD 

UK 

Telephone: +44 (0) 1332 792896 

Facsimile: +44 (O) 1332799018 

0145-Q194.dodJS 

244 

PROJEc'r NUMBER: 014510194 PAGE 2 

QUALITY ASSURANCE REPORT 


This study type is classed as short-term. The standard test method for this study type 

("General Study Plan" in DECO terminology) was reviewed for compliance once only on 

initial production. Inspection of the routine and repetitive procedures that constitute the 

study is carried out as a continuous process designed to encompass the major phases at 

or about the time this study was in progress. In addition, inspection of general facilities 

not specifically related to this study are done monthly or annually in accordance with QA 

Standard Procedure. 

This report has been audited by the Quality Assurance Unit, and is considered to be an 

accurate account of the data generated and of the procedures followed. 

In each case, the outcome 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 type and schedule of this study were as follows: 

12 November 2008 Standard Test Method Compliance Audit 

11 February 2009 Test Material Preparation 

11 February 2009 Animal Preparation 

11 February 2009 DOSing 

11 February 2009 Assessment of Response 

12 February 2009 Necropsy 

§ 09 March 2009 Draft Report Audit 

§ Date of QA Signature Final Report Audit 

§ Evaluation specific to this study 

~! . 1 8 MAR 2009 ..N:..~~....................
DATE ................................................ . 


For the Quality Assurance Unit* 

*Authorlsed QA Signatures: 

Deputy Head of Department JM Crowther MISeT MRQA 

Senior Audit Staff: . G Wren ONC MRQA 

245 

PROJECT NUMBER: 014510194 PAGE 3 

GLP COMPLIANCE STATEMENT 


The work described was performed in compliance with UK GLP standards (Schedule 1, 

Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by 

SI200410994». These Regulations are in accordance with GLP standards published as 

GECD Principles on Good Laboratory Practice (revised 1997, ENVIMC/CHEM(98)17); 

and are in accordance with, and implement, the requirements of Directives 2004/9/EC 

and 2004/10/EC. 

This report fully and accurately reflects the procedures used and data generated. 

_................A.:.J\~o.~.................. _________ ........... DATE: ........ ~.!:J~_.,~.~........................ 
 A Pooles BA (Hons) 

Study Director 

This report may be presented in final foil'll as 8 digital (pdf) document. Such documents are prepared by scanning the paper original, 

and are considered of equivalent Integrity and authenticity to versions produced by optical photocopy. However, In all cases the 

hand-signed paper original, held in secure archives, Is the definitive document 

246 

PROJECT NUMBER: 014510194 PAGE 4 

CONTENTS 


QUALITY ASSURANCE REPORT 2 


GLP COMPLIANCE STATEMENT 3 


CONTENTS 4 


SUMMARY 5 


1. INTRODUCTION 6 


2. TEST MATERIAL AND EXPERIMENTAL PREPARATION 6 


2.1 Description, Identification and Storage Conditions 6 


2.2 Experimental Preparation 6 


3. METHODS 7 


3.1 Animals and Animal Husbandry 7 


3.2 Procedure 7 


3.3 Evaluation of Data 8 


4. ARCHIVES 9 


5. RESULTS 10 


5.1 Mortality 10 


5.2 Clinical Observations 10 


5.3 Bodyweight 10 


5.4 Necropsy 10 


6. CONCLUSION 10 


Table 1 Individual Clinical Observations and Mortality Data 11 


Table 2 Individual Bodyweights and Bodyweight Changes 12 


Table 3 Individual Necropsy Findings 13 


Appendix 1 Flow Chart for the Sighting Test 14 


Appendix 2 Flow Chart for the Main Test 15 


Appendix 3 Statement of GLP Compliance in Accordance with Directive 2004/9/EC 16 


241 

PROJECT NUMBER: 014510194 	 PAGE 5 

Mixture of PEMB and PE3MB: 

ACUTE ORAL TOXICITY IN THE RAT 

- FIXED DOSE METHOD 

SUMMARY 

Introduction. The study was performed to assess the acute oral toxicity of the test 

material in the Wistar strain rat. The method was designed to meet the requirements of 

the following: 

• 	 OECD Guidelines for Testing of Chemicals No 420 "Acute Oral Toxicity - Fixed 

Dose Method" (adopted 17 December 2001) 

• 	 Method 61 bis Acute Toxicity (Oral) of Commission Directive 2004n3/EC 

Method. Following a sighting test at a dose level of 2000 mglkg, an additional four 

fasted female animals were given a single oral dose of test material at a dose level of 

2000 mglkg bodyweight. Clinical signs and bodyweight development were monitored 

during the study. All animals were subjected to gross necropsy. 

Mortality. There were no deaths. 

Clinical Observations. There were no signs of systemic toxicity. 

Bodyweight Animals showed expected gains in bodyweight except for one animal 

which showed bodyweight loss during the first week'with expected gain in bodyweight 

during the second week. 

Necropsy. No abnormalities were noted at necropsy. 

Conclusion. The acute oral median lethal dose (LDso) of the test material in the female 

Wistar strain rat was estimated to be greater than 2000 mglkg bodyweight (Globally 

Harmonised Classification System - Unclassified). 

248 

PROJECT NUMBER: 014510194 	 PAGE 6 

Mixture of PEMB and PE3MB: 

ACUTE ORAL TOXICITY IN THE RAT 


- FIXED DOSE METHOD 


1. INTRODUCTION 

The study was perfonned to assess the acute oral toxicity of the test material in the 

Wistar strain rat. The method was designed to meet the requirements of the following: 

• 	 OECD Guidelines for Testing of Chemicals No 420 "Acute Oral Toxicity - Fixed 

Dose Method" (adopted 17 December 2001) 

• 	 Method B1 bis Acute Toxicity (Oral) of Commission Directive 2004173/EC 

The test system was chosen because the rat has been shown to be a suitable model for 

this type of study and is recommended in the test method. The results of the study are 

believed to be of value in predicting the likely toxicity of the test material to man. 

The study was performed between 27 January 2009 and 19 February 2009. 

2. TEST MATERIAL AND EXPERIMENTAL PREPARATION 

2.1 Description. Identification and Storage Conditions 

Sponsors identification Mixture of PEMB and PE3MB ~ ... '" ............ '" ."'--_... _............., 


o 0 

, 

o 

.

: 

Description 	 o 

o 

0 

• 

Batch number , 	 . ...---------.-..... ---.---.-~ 

Date received 	 15 December 2008 

Storage conditions room temperature in the dark 

The integrity of supplied data relating to the identity. purity and stability of the test 

material is the responsibility of the Sponsor. 

2.2 Experimental Preparation 

For the purpose of the study the test material was used as supplied. The specific gravity 

was determined and used to calculate the appropriate dose volume for the required dose 

level. 

249 
PROJECT NUMBER: 014510194 PAGE 7 

3. METHODS 

3.1 Animals and Animal Husbandry 

Female Wistar (HsdRccHan®m:WIS~) strain rats were supplied by Harlan 

Laboratories UK Limited, Bicester, Oxon, UK. On receipt the animals were randomly 

allocated to cages. The females were nulliparous and non-pregnant. After an 

acclimatisation period of at least five days the animals were selected at random and 

given a number unique within the study by indelible ink-marking on the tail and a number 

written on a cage card. At the start of the study the animals were eight to twelve weeks 

of age. The bodyweight variation did not exceed ± 20% of the initial/mean bodyweight of 

any previously dosed animal(s). 

The animals were housed in groups of up to four in suspended solid-floor polypropylene 

cages furnished with woodflakes. With the exception of an overnight fast immediately 

before dosing and for approximately three to four hours after dosing, free access to 

mains drinking water and food (2014 Teklad Global Rodent diet supplied by Harlan 

Teklad, Blackthorn, Bicester, Oxon, UK) was allowed throughout the study. The diet, 

drinking water and bedding were routinely analysed and were considered not to contain 

any contaminants that would reasonably be expected to affect the purpose or integrity of 

the study. 

The temperature and relative humidity were set to achieve limits of 19 to 25°C and 30 to 

70% respectively. Any occasional deviations from these targets were considered not to 

have affected the purpose or integrity of the study. The rate of air exchange was at least 

fifteen changes per hour and the lighting was controlled by 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 any contaminant of a level that might have affected the purpose or integrity 

of the study. 

3.2 Procedure 

Using available information on the tOXicity of the test material, 2000 mglkg was chosen 

as the starting dose. 

Dose Level 

(mglkg) Specific Gravity Dose Volume 

(mllkg) 

Number of Rats 

Female 

2000 1.194 1.68 1 

250 

3.3 

PROJECT NUMBER: 014510194 PAGE 8 

In the absence of toxicity at a dose level of 2000 mglkg. an additional group of animals 

was treated as follows: 

Dose Level Dose Volume Number of Rats 

(mglkg) Specific Gravity (mVkg) Female 

2000 1.194 1.68 4 

A total of five animals were therefore treated at a dose level of 2000 mg/kg in the study. 

All animals were dosed once only by gavage using a metal cannula attached to a 

graduated syringe. The volume administered to each animal was calculated according 

to its fasted bodyweight at the time of dosing. 

Clinical observations were made %, 1, 2, and 4 hours after dosing and subsequently 

once daily for fourteen days. Morbidity and mortality checks were made twice daily. 

Individual bodyweights were recorded on Day 0 (the day of dosing) and on Days 7 and 

14. 

At the end of the observation period the animals were killed by cervical dislocation. All 

animals were subjected to gross necropsy. This consisted of an external examination 

and opening of the abdominal and thoracic cavities. The appearance of any 

macroscopic abnormalities was recorded. No tissues were retained. 

Evaluation of Data 

The test material will be classified according to Annex 3 of the DECO Guidelines for 

Testing of Chemicals No. 420 "Acute Oral Toxicity - Fixed Dose Method" (adopted 17 

December 2001) as shown in the Flow Chart in Appendix 2. 

Evaluation of data included identification of the number of animals that died during the 

study (or that were killed for humane reasons), and determination of the nature, severity, 

onset and duration of the toxic effects. If possible, the signs of evident toxicity were 

described. Evident toxicity refers to the toxic effects of sufficient severity that 

administration of the next higher dose level could result in development of severe signs 

of toxicity and probable mortality. Effects on bodyweights and abnormalities noted at 

necropsy were also identified. 

251 
PROJECT NUMBER: 014510194 PAGE 9 

Using the mortality data obtained, an estimate of the acute oral median lethal dose 

(LD50) of the test material was made. 

4. ARCHIVES 

Unless instructed otherwise by the Sponsor, all original data and the final report will be 

retained in the Harlan Laboratories Ltd, Shardlow, UK archives for five years, after which 

instructions will be sought as to further retention or disposal. 

252 

PROJECT NUMBER: 0145/0194 PAGE 10 

5. RESULTS 


Individual clinical observations and mortality data are given in Table 1. 


5.1 Mortality 


There were no deaths. 


5.2 Clinical Observations 


No signs of systemic toxicity were noted. 


5.3 Bodyweight 

Individual bodyweights and bodyweight changes are given in Table 2. 

Animals showed expected gains in bodyweight except for one animal which showed 

bodyweight loss during the first week with expected gain in bodyweight during the 

second week. 

5.4 Necropsy 

Individual necropsy findings are given in Table 3. 

No abnormalities were noted at necropsy. 

6. CONCLUSION 

The acute oral median lethal dose (LD50) of the test material in the female Wistar strain 

rat was estimated to be greater than 2000 mg/kg bodyweight (Globally Harmonised 

Classification System - Unclassified). 

253 

Mixture of PEMB and PE3MB: ACUTE ORAL TOXICITY IN THE RAT - FIXED DOSE METHOD 

Table 1 Individual Clinical Observations and Mortality Data 

Dose Animal Effects Noted After Dosing Effects Noted During Period After Dosing 

Level Number lHours) (Days) 

mglkg and Sex % 1 2 4 1 2 3 4 5 6 7 8 9 10 11 

1"'() 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Female 

2~O 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Female 

2000 2-1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Female 

2-2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Female 

2-3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Female 

12 13 14 

0 0 0 

0 0 0 

0 0 0 

0 0 0 

0 0 0 

I':l 

(J1 

.j:>. 

0:::: No signs of systemi9 to)Ciqity__ _ 

PROJECT NUMBER: 014510194 PAGE 11 
---------

Mixture of PEMB and PE3MB: ACUTE ORAL TOXICITY IN THE RAT - FIXED DOSE METHOD 

Table 2 Individual Bodyweights and Bodyweight Changes 

Dose Bodyweight (g) at Day Bodyweight Gain (g) During Week

Level Animal Number and Sex 

mg/kg 0 7 14 1 2

1-0 Female 192 211 221 19 10

2-0 Female 192 199 209 7 10

2000 2-1 Female 188 194 205 6 11

2-2 Female 174 189 204 15 15

2-3 Female 189 185 197 -4 12 -.....•.---­

N 

CJ'I 


CJ'I 


PROJECT NUMBER: 014510194 PAGE 12 
Mixture of PEMB and PE3MB: ACUTE ORAL TOXICITY IN THE RAT - FIXED DOSE METHOD 

Table 3 Individual Necropsy Findings 

Dose Level 

mg/kg 

Animal Number 

and Sex 

1-0 Female 

Time of Death 

Killed Day 14 

Macroscopic Observations 

No abnormalities detected 

2-0 Female Killed Day 14 No abnormalities detected 

2000 2-1 Female 

2-2 Female 

Killed Day 14 --­

Killed Day 14 

--­

No abnormalities detected 

No abnormalities detected 

2-3 Female Killed Day 14 No abnormalities detected 

N 

(11 

0) 

PROJECT NUMBER: 014510194 PAGE 13 
Mixture of PEMB and PE3MB: ACUTE ORAL TOXICITY IN THE RAT - FIXED DOSE METHOD 

Appendix 1 Flow Chart for the Sighting Test 

1~;;~fjJ9;P.'ii~~\1i.lli;1~ 

START 

~ 

• for outt;ome at 5 mgJkg there is an optional supplementary 

procedure to confirm the GHS classification 

... Death 

Evident toxicity N

c:.n • No deaths or evident toxicity -...J 

~ 

PROJECT NUMBER: 0145/0194 PAGE 14 
I') 

c.n 

00 

Outcome 

• .::2deaths 

• .:: 1with evident toxicity and/or s: 1 death 

• No deaths or evident toxicity 

..... 

., Animal welfare override 

Ifthis dose level caused death in the sighting 

animals will be tested. Go directly to outcome 

no further 

Mixture of PEMB and PE3MB: ACUTE ORAL TOXICITY IN THE RAT - FIXED DOSE METHOD 

Appendix 2 Flow Chart for the Main Test 

(~jr'j!'f,iL~Wi'a"1!iW!iiiMii~ 

START 

PROJECT NUMBER: 0145/0194 PAGE 15 
PROJECT NUMBER: 014510194 PAGE 16 

Appendix 3 Statement of GLP Compliance in Accordance with Directive 

2004l9IEC 

iDH)..Department 'Cot HE#31th 

THE DEPARTMENT OF HEACI1I OF TIlE GOVERNMENT 


OF THE UNITED KINGDOM 


GOOD LABORATORY PRACTICE 

STA'~fENT OFCOMPLIA.'IICE 


fl\f ACCORDANCE WITHDIRECllVE 20041!11F£ 


TEST FACIT.rI'Y TI'.5T1'VPE 

Hlll'lan Laboratories Ltd. Analyticul/Owcal Cbem:istry 

Shardlo" BuSll:les5 Park Environmcntllt Fllfe 

London Road Environmenfal Toxicology 

Sliardiow Mutagenicity 

Herby . PbyslCliem 

DE722GD Toxicology 

DATB OF INSPECTION 

19th·AUgust 2008 

A general inspectlon for compliance with the Principles of Oood LabomlOry Practice 

Wll$ eamed out at the above test facility as pllrt of the VKGLPComplillDCe Prognnnme. 

At me time of illspection no deviations were found of sufficient magnitude to affect 

the validity of non-cHnkai sllld~s performed atlbese fudlities. 

Dr. AmJrcw J. Gray 

Head. UK GLPMonltorlng Authority 

259 

v 


CERI 


Receipt No. 827-08-D-3387 

STUDY CODE: 811-0908 

FINAL REPORT 

TWENTY-EIGHT-DAY REPEATED-DOSE 


ORAL TOXICITY STUDY OF 


KarenzMT (Mixture ofPEMB aDd PE3MB) 


IN RATS 

March 2007 

Hita Laboratory 


Chemicals Evaluation and Research Institute 


Japan 


- i­ 260 
B11-0908 

STATEMENT 

I, the undersigned, hereby declare that this report provides correct English translation of the final 

report (Study Code B11-0908, issued on March 19,2009). 

Katsumi Miyata, M.S., J.A.C.L.C.T. Date 

Hita Laboratory 

Chemicals Evaluation and Research 1nstitute, Japan 

- ii ­ 261 
1311-0908 

GLP STATEMENT 

Hila Laboratory 


Chemicals Evaluation and Research Institute, Japan 


Sponsor: Showa Dcnko K.K. 

..!cT.!.!it""lc",-:____l"-'w!!..c""n~tyJ_-_"E""igl[;>l.!ht-12ID' Repeated-Dose Oral Toxicity Study of KarenzMT (MixturELQf 

PEMB and PEJMB) in Rats 

Study Code: B 11-0908 

I, the undersigned, hereby declare that this study was conducted in compliance with "Concerning 

Standard of the Testing Facilities Conducting the Test Relating to the New Chemical Substances" 

(Notification No. 1121003 of the Phannaceutica1 and Food Safety Bureau, MHLW, No. 3 

(2003.11.17) of the Manufacturing Industries Bureau, METI & No. OJ 1121004 of the Environmental 

Health Depanment, MOE (November 21, 2003)". 

And, I con filmed that this report accurately reflects the raw data obtained and that data of the study 

has reliability. 

Study Director: Signed in original March 19, 2009 

Katsumi Miyata, M.S., lA.C.L.C.T. 

- iii ­ 262 
\J 11-0908 

QUALITY ASSURANCE STATEMENT 

!lita Laboratory 


Chemicals Evaluation and Research Institute, Japan 


Sponsor: Showa Ocn]<J2J<.K. 

Titl~: Twenty-Eight-QaLE...~S'!ated-Dose Oral Toxicity Study of KarenzMT (Mixture of 

I'.EMB and PE3MB) in Rats 

Sjudy Code: B 11-0908 

This study was audited and inspected by Quality Assurance Section of Hita Laboratory, Chemicals 

Evaluation and Research Institute, Japan. The dates audited and/or inspected and the dates reported 

ofthese results to the study director and management are as follows. 

Items of Inspections 

and Audits 

Dates of Inspections 

and Audits 

Dales oflnspections and 

Audits Reports 

Protocol October 27, 2008 October 27, 2008 

I Assessment of response October 30, 2008 October 30,2008 

Preparation of test substance November 6, 2008 November 6, 2008 

Administration and clinical 

observation November 7,2008 November 10,2008 

Amendment to protocol November 10,2008 November 11, 2008 

Animal data January 27, 2009 January 27, 2009 

Detailed clinical observation and 

sensorimotor function January 27, 2009 January 27, 2009 

Amendment to protocol (2"") February 5, 2009 February 5, 2009 

Clinical chemistry data February 19, 2009 February 19, 2009 

Pathological data February 19, 2009 February 19; 2009 

Re~inspection of clinical chemistry 

data February 20,2009 February 20, 2009 

Documents of test substance and 

housing conditions March 10, 2009 March 10,2009 

Draft of final report March 11, 2009 March 11,2009 

Amendment to protocol (3'u) MarchI 1,2009 March II, 2009 

Re-inspection of draft final report March 16, 2009 MarcIl 16, 2009 

Draft offinal report (2=) March IS, 2009 March 18, 2009 

Re-inspection of draft final report 

(2nd) March 19,2009 March 19, 2009 

Final report March 19,2009 March J9, 2009 

Following items were reported to the study director and management on the basis of the audit of 

f:aCLTJty or aud'It resu I'ts In other studly, 

Items ofAudits Dates of Audits Dates of Audits Reports 

Preparation investigation June 4,2008 March 19, 2009 

Animal receipt October 7, 2008 March 19, 2009 

Quarantine and acclimatization October 7, 2008 March 19, 2009 

Animal care October 7,2008 March J9, 2009 

263 
BII-0908 

~n - and animal September 8, 2008 March 19, 2009 ation 


ight measurements 
 October 7,2008 March 19,2009 

Food intake measurements September 8, 2008 March 19, 2009 

Detailed clinical observation and October 6, 2008 March J9,2009 sensorimotor function 

Urine sampling October 6, 2008 March 19,2009 

Blood sampling October 7,2008 March 19, 2009 

Trimming, autopsy and organ October 7,2008 March 19,2009 weight measurements 

Hematology October 7,.2008 March 19, 2009 

Blood chemistry October 8, 2008 March 19,2009 

Urinalysis October 7,2008 March 19,2009 

October 17, 21 and 29, Pathological preparation March 19,2009 2008 


Microscopic examinations 
 October 20, 2008 March 19,2009 

I, the undersigned, hereby declare that this report provides an accurate description ofthe methods and 

procedures used in this study and that the reported results accurately reflect the raw data obtained. 

Section Chief. Quality Assurance: Signed in original March 19, 2009 

Ryuichiro Mizuguchi, B.S. 

-v- 264 
B 11-0908 

TABLE OF CONTENTS 

TITLE ............................................................................................................................ . 


SPONSOR ..................................................................................................................... . 


TESTING FACILITY ................................................................................................... . 


PURPOSE OF THE STUDY ......................................................................................... . 


TESTING 1\1ETHOD·· .... ··· ..· .. ·.. ·· .... ··....·· .. ·..····..· .... · .... ····· ...... · ..................................... . 


GLP COMPLIANCE .................................................................................................... .. 


PERIOD OF THE STUDY ............................................................................................. 2 


LOCA TION AND PERIOD FOR RETENTION OF RA W DATA AND 


SPECIMENS, .................................... ............................................................................. 2 


RETENTION OF THE ORIGINAL PROTOCOL AND FINAL REPORT .................. 2 


AUTHOR AND PERSONNEL CONCERNED WITH THE STUDy·.... ····· ....· .... ··· .. ·· 3 


AUTHOR APPROVAL·.. ·· ..· ...... · .... ·..··..· ........ ·..· .... ···· ........ · ..·.. · .... ·............................... 3 


SUMMARy·..· .... ·..· .... · ..·.. ··..· .... ···· ...... · .... ······ ...... · .... ····................................................. 4 


MATERIALS AND METHODS·· ..··.. ·..···· ...... · ..···..·····....·····.... ·· ..··....··· ..·..................... 6 


1. TEST SUBSTANCE ................................................. ..................................... 6 


2. ANIMALS ...................................................................................................... 7 


3. HOUSING CONDITIONS ............................................................................. 7 


4. GROUPING .................................................................................................... 8 


5. STABILITY OF THE TEST SUBSTANCE·.. ··.. ·..····..···..···.. ·· .. ···..· .............. 9 


6. PREPARA TION OF FORMULATIONS ...................................................... 9 


7. ADMINISTRA TION ...................................................................................... 10 


8. OBSERVA TIONS .......................................................................................... 10 


9. STATISTICAL ANALYSIS .......................................................................... 17 


ENVIRONMENTAL FACTORS THAT MIGHT HAVE AFFECTED 


TI IE RELIABILITY OF TIlE STUDY RESLJL TS ....................................................... 
 17 


RESULTS ..................................................................................................................... . 
 18 

I. CLINICAL SIGNS ........................................................................................ . 
 18 

2. DETAILED OBSERVATIONS ..................................................................... 
 19 

3. SENSORIMOTOR FUNCTION .................................................................... 
 19 

4. BODY WEIGHTS .......................................................................................... 
 20 

5. FOOD INTAKES .......................................................................................... . 
 20 

6. HEMATOLOGICAL EXAMINATIONS ...................................................... 
 20 

7. BLOOD CHEMICAL EXAMINATIONS .................................................... . 
 21 

8. URINALySES· ..·..····..··..·······.. ·· .. ····..·..· .......... ·· .... ···· .................................... . 21 


9. ORGAN WE1GIITS ...................................................................................... . 
 21 

- vi­ 265 

BII-0908 

10. NECROPSy····· ..· .... · ...... ···· .. ·· .. ····· ...... ··...... ·.. ·.. ········...................................... 22 


11. HISTOPATHOLOGICAL EXAMINATIONS .............................................. 23 


DISCUSSION ................................................................................................................. 23 


REFERENCE . ................................................................................................................ 26 


FIGURES 


Body weights· ....................... ......... ........... ................... .......... ......................... 27 


2 Food intakes .................................................................................................... 29 


TABLES 


Summary of clinical signs .............................................................................. 31 


2 Summary of detailed clinical observations ..................................................... 32 


3 Summary ofreflex .......................................................................................... 57 


4 Summary of grip strength ............................................................................... 60 


5 Summary of motor activity ..· ........ · ................................ · ..· ........ ··· ...... · .... ·...... 61 


6 Summary of body weights .............. ............... ................................................. 62 


7 Summary of food intakes ................................................................................ 64 


8 Summary ofhematological examinations ...................................................... 66 


9 Summary of blood chemical examinations .. ·..·..· .......... ··....................·..·..· .... · 68 


10 Summary of urinalyses ................................................................................... 70 


l] Summary of absolute organ weights ............................................................... 73 


12 Summary ofrelative organ weights ................................................................ 74 


13 Summary of macroscopic examinations ......................................... ................ 75 


14 Summary of histopathological examinations .................................................. 76 


ADDENDA 


Clinical signs ofindividual animals ..····· ...... · ...... · .... · ..· ........ · ...... · .......... · ..· .... · 80 


2 Detailed clinical observations of individual animals ...................................... 95 


3 Reflex of individual animals ........................................................................... J65 


4 Grip strength of individual animals ................................................................ 167 


5 Motor activity of individual animals .............................................................. 169 


6 Body weights of individual animals ............................................................... 172 


7 Food intakes of individual animals ................................................................. 175 


8 Hematological data of individual animals ...................................................... 178 


9 Blood chemical data of individual animals .................................................... 182 


10 Urinalytic data of individual animals ....................................... ....................... 186 


11 A bsolute organ weights ofindividual animals ......... ...................................... 192 


12 Relative organ weights of individual animals ................................................ 194 


13 Pathological findings of individual animals ............................................. .,.... 196 


APPENDIX 1 ............................................................ HISTOPA THOLOGICAL PHOTOS 


APPENDIX 2 .................................................................. PHYSICOCHEMICAL REPORT 


- vii­ 266 
B 11-0908 

Study Code: B 11-0908 

Test Substance Code: HR7645 

Sponsor Code: S-O 170 

TITLE 

Twenty-Eight-Day Repeated-Dose Oral Toxicity Study of KarenzMT (Mixture of PEMB and 

PE3l'vID) in Rats 

SPONSOR 

Showa Denko K.K. 

13-9, Shiba Daimon l-chome, Minato-ku, Tokyo 105-8518, Japan 

TESTING F ACILlTY 

Hila Laboralory 

Chemicals Evaluation and Research Institute, Japan 

822, 3-chome, Ishii-machi, Hita, Oila 877-0061, Japan 

PURPOSE OF THE STUDY 

The purpose of this study is to define the type, severity and reversibility of toxicological signs of the 

test substance by observing the functional and morphological changes in animals receiving repeated 

doses orally for 28 days. 

TESTING METHOD 

This study was conducted in accordance with "28-day Repeated Dose Toxicity Study in Mammalian 

Species" prescribed in "Concerning Testing Methods Relating to the New Chemical Substances" 

(Notification No. 1121002 of the Pharmaceutical and Food Safety Bureau, MHLW, No. 2 

(2003.11.13) of the Manufacturing Industries Bureau, METJ & No. 031121002 of the Environmental 

Health Department, MOE (November 21, 2003)". 

GLP COMPLIANCE 

This study was conducted in conformity "Concerning Standard of the Testing Facilities Conducting 

the Test Relating to the New Chemical Substances" (Notification No. I 12! 003 of the Pharmaceutical 

and Food Safety Bureau, MHLW, No.3 (2003.11.17) of the Manufacturing Industries Bureau, METI 

& No. 031121004 of the Environmental Health Department, MOE (November 21, 2003)". 

- 1­ 267 
B 1)-0908 

PERIOD OF THE STUDY 

Commencement ufthe Study: October 23, 2008 

Animal Receipt: October 28, 2008 

Initiation ofExamination (Initiation of Dosing): November 7, 2008 

Terminal Necropsy ofDosing Period: December 5,2008 

Initiation ofRecovery Period: December 5, 2008 

Terminal Necropsy of Recovery Period: December 19, 200S 

Termination of Examination (Termination of Histology): February 10, 2009 

Completion of the Study: March 19,2009 

LOCATION AND PERIOD FOR RETENTION OF RAW DATA AND SPECIMENS 

The raw data, protocol and amendment, study contract documents, test substance information, final 

report, other record documents and specimens will be stored in the archive of the Hita Laboratory of 

our organization, and samples of every lot of the test substance in the test substance storage room, for 

a period of 10 years from the date of receipt of the notification that they are applicable to Article 4, 

Paragraphs 1 or 2, Article 4-2, Paragraphs 2,3 or 8, Article 5-4, Paragraph 2, Article 24, Paragraph 2 

or Article Paragraph 2 of the Japanese Chemical Substances Control Law No. I 17 (1973). 

The sponsor will inform the Hita Laboratory of our organization of the date of receipt of the 

notification. After termination of the retention period, any measures taken will be done so with the 

approval of the sponsor. Samples and specimens that are liable to deteriorate markedly will be 

retained for 10 years after reeeipt of the notification or only for as long as the quality of the 

preparation pClmits evaluation WitJl the sponsor's consent. 

RETENTION OF THE ORIGINAL PROTOCOL AND FINAL REPORT 

The original protocol and amendments will be retained at Hita Laboratories. A copy of the 

document will be retained by the sponsor. 

- 2 ­ 268 
Bll--0908 

AUTHOR AND PERSONNEL CONCERNED WITH THE STUDY 

Study Director: 	 Katsumi Miyata, M.S., .l.A.C.L.C.T. 

(Planning and management of the study, evaluation of the 

results, report creation, and over all responsible for the technical 

conduct of the study) 

Study Staff: 	 Saori Takakura, J.L.A.T., J.A.C.L.C.T. 

Hiroshi Ohshima. D.V.M. 

(Quarantine, acclimation and housing management of animals, 

preparation and administration of the test substance, clinical 

observation, detailed clinical observation, sensorimotor 

Function, body weights and food intakes measurements, and 

responsible for the animal examination) 

Person in charge ofPathoJogic Examination: 

Satsuki Houshuyama, Ph.D., D.V.M. 

(Necropsy, collection of tissues, organ weight measurements, 

histopathological examinations, and responsible for the 

histopathology) 

Person in charge ofClinical Chemistry: 

Nobuhiko Higasihara, B.S. 

(Hematological and blood chemical examinations, urinalysis, 

and responsible for the biochemistry of the specimens) 

AUTHOR APPROVAL 

Study Director: Signed in original March 19, 2009 

Katsumi Miyata, M.S., J.A.C.L.C.T. 

General Toxicology Section 

Hita Laboratory 

·3· 269 
B 11-0908 

SUMMARY 

A 28-day repeated oral dose toxicity study of KarenzMT (Mixture of PEMB and PE3MH) was 

performed in groups of five male and five female Crl:CD(SD) rats at 5 weeks of age, The high dose 

was set at 1,000 mglkg/day, and altogether three doscs including 150 and 25 mg/kg/day were 

employed. Recovery groups were also set for the 1,000 mg/kg and vehicle control groups to 

investigate the reversibility ofthe effects, 

Effects ofthe test substance were observed in the liver and kidneys. 

In the liver, relative weights in males and females and absolute weight in females were increased in 

the 1,000 mg/kg groups. Enlargement of the liver was observed in females of the 150 and 1.000 

mg/kg groups and males of the 1,000 mglkg group. In the histopathological examinations, diffuse 

fatty change of the hepatocytes in males and fatty changes of the periportal hepatocytes in females 

were observed in the 1,000 mglkg groups. 111 the blood chemistry, increased total bilirubin in males 

and females, increased ALT in males and decreased cholinesterase in females were noted in the 1,000 

mglkg groups, 

As the effects to the kidney. relative weights in males and females and absolute weights in females 

were increased in the 1,000 mg/kg groups. Enlargement of the kidneys were observed in males and 

females of the 1,000 mglkg groups. Degeneration and necrosis of the tubular epithelium with the 

regenerative epithelium was observed in females of the ) ,000 mglkg group histopathologically. In 

the blood chemistry, BUN was increased in males of the 1,000 mg/kg group. White blood cells and 

epithelial cells in the urinary sediments were increased in males ofthe 1,000 mglkg group. 

Salivation was observed just after dosing in males and females of the 150 and 1,000 mglkg groups. 

Moistening and fur loss of the neck or mandible due to salivation were observed in males and females 

of the ] ,000 mglkg groups. Decreased spontaneous locomotion in males of the 150 and 1,000 

mglkg groups and females of the 1,000 mg/kg group, incomplete eyelids opening and plenty of food 

on the tray in males and females of the 1,000 mg/kg groups were observed. Lacrimation in males 

and staining lower abdomen in females were observed in the 1,000 mg/kg groups, 

Body weights were decreased in males oftlle 1,000 mglkg group after day 21 ofthe treatment 

Food consumption was decreased in males and females on day 3 and increased in females after day 

J5 in the 1,000 mg/kg groups. 

-4­ 270 
B 11-0908 

In the organ weights, absolute weights in males and females and relative weight in females in the 

thymus were decreased in the 1,000 mg/kg groups without histopathological lesion. 

In tile recovery groups, fur loss of the neck and mandible were observed by day II of recovery, and 

plenty of food on the tray was observed by day 3, and these were disappeared subsequently. 

Although liver weights were increased in females of the 1,000 mg/kg recovery group, the severity 

was decreased compared to those at the end of administration period. Kidney weights were also 

increased in males of the 1,000 mglkg recovery group; however, they showed recovery. No 

histopathological changes were observed in the liver and kidney at the end ofthe recovery period. 

No toxicological changes were noted in the sensorimotor functions and hematology. 

Based on these results, the NOAEL of Karen7.MT (Mixture of PEMB and PE3MB) under the 

conditions tested was considered to be 25 mg/kg/day due to decreased spontaneous locomotion in 

males and enlargement of the liver in females of the 150 and 1,000 mglkg groups. 

The NOEL was considered to be 25 mg/kglday due to salivation related to an irritation of the test 

substance observed in males and females of the 150 and 1,000 mg/kg groups. 

- 5 ­ 271 
Bll"()908 

MATERIALS AND METHODS 

1. TEST SUBSTANCE (Information provided by the sponsor) .* __ ...___ .__ ~~. ____ a ___ *_. __ ._._..__ ...__ ._ ..••_•• _••••• _.••• _.•_•••. _.... ___ ~ ____ .. ________ .____ .____ .. _. __ •• _•• _._ ••• ~ ••••••• _.I

. 

. ~.---*-------.- .. ~ ... -.~ .....------------------..-----.---.----_._.. ___ ._______ .. ~ ....._. _________ .. _______ .... -..... _----- ______ 1 

-6­ 272 
B11-0908 


... ~.- ...--.---.--------.--.--........----...--.--.--.-----_ ........_--_ ............----.--.........
 . 

1.8 	 Storage Conditions 

The test substance was stored at room temperature (19.6-22.4"C from obtaining of the test 

substance to the final preparation of the dosing formulations) in the test substance storage 

room. A void oxidizing agent. 

],9 	 Handling Precaution 

Glove. mask, cap, protective glasses and lab coat were put on. The test substance was treated 

in a draft, since the test substance has characteristic odor. 

2. 	 ANIMALS 

Crl:CD(SD) rats (SPF) of thirty three males and thirty three females were obtained from 

Charles River Japan Hino Breeding Center at four weeks old. Animals were acclimati7.ed for 

10 days including 7 days quarantine. No abnonnalities were noted in all animals during the 

quarantine and acclimation periods. The animals were allocated to groups to ensure 

homogeneity of mean body weights using body weight-stratified randomization on two days 

before the start of the administration. The animals not treated were excreted from the study 

and euthanized under ether anesthesia. At the onset of the treatment, the animals were five 

weeks old with body weight ranges of 155.2-173.9 g and 120.2-149.8 g for males and females, 

respectively. Animals were identified by means of a marker on the tail before grouping, and 

ear-tags after grouping. However, the detailed clinical examinations, reflex and grip strength 

measurements were performed ad blind tests. Racks and cages were identified by individual 

cards. 

3. 	 HOUSING CONDITIONS 

The barrier-system animal room was maintained at a stable temperature (21-25"C) and relative 

humidity (40-70%) with 10-15 air changes per hour and artificial light-dark cycle of 12-12 

hours (light on: 7:00 and light off: 19:00). The actual temperature and humidity were 

22.7-24.5°C and 49.3-62.3%, respectively. 

- 7 ­ 273 
Bl1-090!\ 

The rats were housed in hanging stainles!> steel cages with wire-mesh noor at three or five 

animals/cage (260 Wx380 Dx l80 H mm, TOKIWA KAGAKU KIKAl) for quarantine and 

acclimation, and at one animal/cage (l65 Wx300 Dxl50 II mm, TOKIWA KAGAKU KIKAI) 

for after grouping. Under trays wcrc changed once before grouping, and twice weekly and 

when plenty of food on the tray was observed after grouping. Feeders, cages and racks were 

changed once at the grouping, and once at the termination of the dosing period for the recovery 

group. The animals had free access to an Mf pelleted diet (Lot No. 080710 and 080916, 

Oriental Yeast) and chlorinated water from the Hita City supply via automatic watering system 

with sipper tubes. The diet and housing materials were auroclaved at 121°C for 30 min prior 

to usc. Analysis of the diet was performed in Japan Food Research laboratories, and the 

analytical data were provided by the manufac·turer. The tested parameters met the 

requirements in our laboratories according to the "Toxic Substances Control Act of US-EPA". 

Contaminants in drinking water were analyzed t"vice yearly in Gita Prefecture Pharmaceutical 

Association according to the water regulations of the "Noti fication No. 101 of Environmental 

Health Bureau. MHW" except for the taste and residual chlorine. In the examination of 

drinking water sampled in November 18, 2008, the concentration of chloric acid was 0.89 

mgfL and deviated from the quality criterion value, :SO.6 mg/L, described in ministerial 

ordinance for Ministry of Health, Labour and Welfare. However, it was confirmed that other 

data were within each criterion on water quality. 

4. GROUPING 

Range finding study of 7 days repeated oral treatment was performed at 0, 25, 250, 500 and 

1,000 mglkglday. As a result, blackish region of the mucosa in the forestomach in males of 

the 500 and 1,000 mglkg groups and females of the 1,000 mglkg group were observed. 

Relative kidney weights in males and relative liver and kidney weights in females were 

increased in the 1,000 mglkg group without a histopatholigicallesion. Accordingly, the high 

dose was set at 1,000 mglkg/day and lower doses of 150 and 25 mglkglday were set for the 

present study. A further group of fivc males and five females were set for the recovery 

groups to investigate the reversibility ofthe effects. 

Dose Volume Concentration No. of Animals (Animal No.) 

Group 

~mg/kgldal:) (mLlkgl ~w/v%) Male Female 

Vehicle control 0 10 ° 5( - 5 ) 5 ( 31 - 35 ) 

Vehicle control (recovery) 0 10 ° 5 ( 6 - 10 ) 5 ( 36 - 40 ) 

Low dose 25 10 0.25 5 ( 11 - 15 ) 5 ( 41 - 45 ) 

Intermediate dose 150 10 1.5 5 ( 16 - 20 ) 5 ( 46 - 50 ) 

High dose 1,000 10 10.0 5 ( 21 - 25 ) 5 ( 51 - 55 ) 

High dose (recovery) 1,000 10 10.0 5 ( 26 - 30 ) 5 ( 56 - 60 ) 

- 8 ­ 274 
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5. 	 STABILITY OF THE TEST SUBSTANCE (see attached physicochemical report) 

Stability of the test substance during the dosing period was con finned with infrared 

spectrophotometer in Hita laboratory. IR spectrum of the test substance within 4600 

cm· 1

-400 em'] was compared with that provided by the sponsor before dosing to determine the 

identity. The test substance was also analyzed to confirm the stability before and after the 

dosing period to confirm the stability. 

6. 	 PREPARATION OF FORMULATlONS 

6.1 	 Vehicle 

In preparation investigation of the dosing formulations, the test substance did not dissolved in 

purified water. In olive, although oily droplets of the test substance were observed, a 

homogeneous formulation was obtained by mixing. Therefore, olive oil (Lot No. 0460YS, 

Fujimi Phannaceutical) was selected as the vehicle. 

6.2 	 Preparation and Storage 

The test substance was accurately weighed in a glass beaker and mixed with olive oil to 

prepare 10.0 w/v% by stirring with a magnetic stirrer. The lower concentrations of 1.5 and 

0.25 w/v% were diluted from 10.0 w/v% with olive oil by stirring with a magnetic stirrer. 

The formulations including olive oil for the vehicle control were stored at the dark and cold 

place (2-9°C, refrigerator No.7 in the test substance preparation room). 

Concentration Test substance weights Volume 

Date 

(w/v%) and dilution methods \mL) 

November 6, 2008 10.0 40.00 g 400 

(Concentration 1.5 Diluted with 30 mL of 10.0 w/v% 200 

Conformation) 0.25 Diluted with 5 mL of 10.0 w/v% 200 

November 13,2008 10.0 	 50.00 g 500 

1.5 Diluted with 37.5 mL of 1 0.0 w/v% 250 

0.25 Diluted with 6.25 mL of 10.0 w/yOlo 250 

November 20, 2008 10.0 50.00 g 500 

1.5 Diluted with 37.5 mL of 10.0 w/v% 250 

0.25 Diluted with 6.25 mL of 10.0 w/v% 250 

November 27,2008 10.0 60.00 g 600 

1.5 Diluted with 45 mL of 10.0 w/v% 300 

0.25 Diluted with 7.5 filL of 1 0.0 w/v% 300 

·9­ 275 
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6.3 	 Homogeneity lind Stability Tests (see attached physicochemical report) 

The homogeneity and stability tests were performed in Hila Laboratory. In the homogeneity 

test, top, middle and bottom layers of 10.0 and 0.25 w/v% formulations were taken just arter 

preparation. These samples were quantitatively analyzed by a high performance liquid 

chromatography. In the stability test, the formulations for homogeneity samples were stored 

at the dark and cold place and analyzed after 3 and 9 days. The test substance in formulations 

was confirmed to be stable for 8 days. 

6.4 	 Concentration Conformation (see attached physicochemical report) 

Concentration of the test substance was confirmed in Hita Laboratory. The concentrations of 

10.0, 1.5 and 0.25 w/v% formulations were confilmed to be within 1 00±1O% of each nominal 

concentration at the first preparation of the dosing period. 

7. 	 ADMINISTRATION 

The formulations were mixed with a magnetic stirrer and repeatedly administered daily in the 

morning (8:55-11 :58) by oral gavage using a syringe (Terumo) connected to a Nelaton catheter 

(Terumo) for 28 days. Thereafter, a 14-day recovery period was set. 

8. 	 OBSERV A nONS 

Concerning the numbering of day and week, the day of the initiation of dosing was regarded as 

day I, and the week of initiation of dosing as w~ek 1. The day after the last dosing was 

regarded as day I (recovery) and the week of the initiation of recovery as week 1 (recovery). 

8.1 	 Clinical Signs 

During the dosing period, animals were observed three times a day, i.c., before dosing (in the 

morning), during and just after dosing, and in the afternoon, daily from day 1 to day 28. 

During the recovery period, observation was performed once daily. 

8.2 	 Detailed Clinical Observations 

The detailed examination in all animals was performed ad blind tests before dosing and once 

weekly thereafter. 

I) Removal from cage 

The case with which the animal was removed from the home cage was recorded. 

Ease ofremoval and vocalization. 

2) Handling observations 

In hand observations 

Muscle tone, subnormal temperature, piloerection, hair appearance (staining and 

unkempt hair), skin color and mucous (paleness, reddening and cyanosis), eyes 

(lacrimation, exophthalmos and pupillary size), salivation and secretion. 

" 10 ­ 276 
B 11-0908 

3) 	 Observation in arena 

Animals were placed in the standard arena and observed, and number of defecation and 

urination were re~'orded for 1 min. 

Posture, motor activity level, respiration, lid closure, gait characteristics, tremor, 

twitch, convulsion, stereotypes and abnormal behavior. 

8.3 	 Sensorimotor Function 

All animals were examined in week 4. Locomotor activity counts were measured in females 

of the vehicle and 1,000 mg/kg recovery groups in recovery week 2, since a statistically 

significant difference was noted during the dosing period in females ofthe 1,000 mg/kg group. 

I) Reflex 

Sensory reactivity to stimuli of different types was performed ad blind tests. 

(I) 	 A pproach contact/touch response 

A blunt probe was bought approximately 3 em from the animal's nose for 4 

seconds. 

(2) 	 Pinna response 

The animal's response to a sudden sound of finger snap was assessed. 

(3) 	 Pain response 

The animal's tail was pinched with a clothespin between one-third and base of the 

lail. 

(4) 	 Pupillary reflex 

Following darkness adaptation of the animal's eyes, pupil constriction in response to 

a bright beam of light was observed. 

(5) 	 Air righting renex 

The animal was held ventral surface uppermost approximately 30 cm height from 

the flat surface and released. 

2) 	 Grip strength 

Forelimbs and hindlimbs grip strengths were measured with automated grip strength meter 

(FGC-2, MATYS) ad blind tests. Two trials were performed, and the mean values were 

calculated. 

3) 	 Locnmotor activity counts 

Locomotor activity level of each animal was counted with activity monitoring system for 

rats (ACTIMO-IO, SH INTECHNO) by the number of crossing IR beam for 1 hour at 10 

min intervals. 

8.4 	 Body Weigbts 

Body weight was measured at allocation to groups, and on days 1,3,8, 12, 17,21,26 and 28 

during the dosing period and on days I, 5, 10 and 14 (recovery) during the recovery period. 

- 11 ­ 277 
811-0908 

[n addition, immediately before necropsy, body weights were mea~ured for calculation of the 

relative organ weights. 

8.5 	 .Food Intakes 

Food consumption was measured at allocation to groups, and on days I, 3, 8, IS, 22 and 28 

during the dosing period and on days 1,4, 8 and 14 during the recovery period. Mean food 

intakes per day were calculated from their remainders for each period. 

8.6 	 Hematological Examinations 

Blood or plasma samples obtained by blood sampling from the abdominal aorta under ether 

anesthesia after overnight fasting (16 to 20 hr) at completion of the dosing period (excluding 

the recovery groups) and at completion of the recovery period were determined for the 

following items. In addition, the blood smears stained with Wright staining were made. As 

an anticoagulant, 3.2% sodium citrate aqueous solution (Lot No. PEQ4430, Wako Pure 

Chemical Industries) was used for the determination of prothrombin time and activated partial 

thromboplastin time, and EDT A-2K (Lot No. 07039, Sysmex) for other measurements. 

Method 

Electrical resistance detection 

Electrical resistance detection 

Noncyanhemoglobin method 

RBCxMCV 

103 

Electrical resistance detection 

Hb x 103 

RBe 

Hb x 10" 

Ht 

Electrical resistance detection 

RNA staining 

Mab'TIetic sensor 

Magnetic sensor 

Flow cytometry technique 

Parameters 

I) Red blood cell count (RBC) 

2) White blood cell count (W8C) 

3) Hemoglobin cone. (Hb) 

4) Hematocrit value (Ht) 

5) Mean corpuscular volume (MCV) 

6) Mean corpuscular hemoglobin (MCH) 

7) Mean corpuscular hemoglobin conc. (MCHC) (g/dL) 

4 

(xlO /~L) 

(x102/~L) 

(g/dL) 

(%) 

(tL) 

(pg) 

8) Platelet count (Platelet) 

9) Reticulocyte count (Reticulo) 

10) Prothrombin time (PT) 

11) Activated partial thromboplastin time (APTT) 

12) Differentiation of leukocytes 

Neutrophils (Neutro) 


Eosinophils (Eosino) 


Basophils (Baso) 


Lymphocytes (Lymph) 


Monocytes (Mono) 


Large unstained cells (LUC) 


(x 104/IlL) 

(%) 

(sec) 

(sec) 

(%) 

- 12 ­ 278 
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I) - 8) CELL-DYN3500, Abbott Laboratories 


9),12) ADVIA 120, SIEMENS 


to), 11) KC-lOA, AMELUNG 


8.7 	 Blood Chemical Examinations 

Serum samples were separated from blood samples collected at the same times as those 

described in section 8.6, and the following items were determined in the obtained serum 

samples. 

Parameters Method 

\) Aspartate aminotransferase (AST) (lUlL) UV method (method based on JSCC) 

2) Alanine aminotransferase (AL T) (lUlL) UV method (method based on JSCC) 

3) Alkaline phosphatase (ALP) (lUlL) p-Nitrophenyl phosphate method 

4) Cholinesterase (ChE) OWL) Butyrylthiocholinc iodide method 

5) y-Glutamyl transpeptidase (y-GTP) (lUlL) L-y-glutamyl-3-carboxy-4-nitroanilide 

method 

6) Total cholesterol (ToCho) (mg/dL) COD'ADPS method 

7) Triglyceride (TG) (mg/dL) GPO'ADPS glycerol blocking method 

8) Glucose (mg/dL) Hexokinasc'G-6-PDH method 

9) Total protein (T-Protein) (gldL) Biuret method 

to) Albumin (gldL) Bromocresol green method 

Albumin II) AIG ratio 

T - Protein - Albumin 

12) Blood urea nitrogcn (BUN) (mg/dL) Urcase·GIDH method 

13) Creatinine (mg/dL) Creatininasc'F-DAOS method 

14) Total bilirubin (T-BiI) (mg/dL) Enzyme method 

15) Calcium (Ca) (mg/dL) OCPC method 

\6) Inorganic phosphorus (IP) (mg/dL) Fiske-Subbarow method 

17) Sodium (Na) (mEq/L) Crown-Ether membrane 

electrode method 

18) Potassium (K) (mEq/L) Crown-Ether membrane 

electrode method 

19) Chloride (CI) (mEq/L) Coulometric titration method 

1)-10), 12)-16) 7170 Automatic Analyzer, Hitachi 

17)-19) PVA-EXII, A & T 

- 13 ­ 279 
B I] -0908 

8.8 	 Urinalyses 

Urinalysis was performed once (day 28) during the dosing period (excluding the recovery 

groups) and once (day 14) during the recovery period. Urine samples (accumulated for 15-] 7 

hr) collected in individual metabolic cages (150 Wx200 D><263 H mm) were determined with 

drinking water ad lib. The urine sediments was stained and examined in males and females of 

the vehicle control and 25 and 150 mg/kg groups at the end orthe dosing period, and in males 

and females of the vehicle control and 1,000 mglkg recovery groups at the end ofthe recovery 

period, since WBC and epithelial cells were increased in males and females of the 1,000 mg/kg 

groups. 

Parameters Method 


1) Urine volume (mIL) Volumetric method 


2) Color Macroscopy 


3) Turbidity Macroscopy 


4) Urine osmotic pressure (Uosm) (mOsm/L) Freezing-point depression method 

5) pH Test paper 

6) Protein Test paper 

7) Glucose Test paper 

8) Occult blood Test paper 

9) Urinary sediments Stemheimer 

4) OM-6040, ARKRAY 


5)-8) Hema-Combistix, SIEMENS 


9) Biological microscope, BX41, OLYMPUS 


8.9 	 Pathological Examinations 

I) Necropsy 

All animals were subjected to the detailed gross necropsy including body surface, all 

orifices, cranial, subcutis, thoracic, abdominal and pelvic cavities, and the contents. 

2) Organ weights 

Liver, heal1, kidneys, testes, epididymides, ovaries, brain, spleen, thymus and adrenals 

were weighed in all animals. The relative organ weights were calculated based on the 

body weight at the time of necropsy. 

3) Histopathological examinations 

(1) 	 The following organs and tissues were taken. Trachea, lungs and urinary bladder 

were filled with 10% neutralized buffered formalin before taken. Stomach and 

intestines were filled and fixed with 10% neutralized buffered formalin and were wash 

with water. All organs/tissues were preserved in 10% neutralized buffered formalin. 

- 14 ­ 280 
1311-0908 

However, testes and epididymides were fixed in l3ouin's solution. 

Category Organs and Tissues 

Respiratory system Trachea, lungs 

Digestive system Stomach, intestine (duodenum to rectum, with Peyer's 

patches), liver 

Cardiovascular system Heart 

Urinary system Kidneys, urinary bladder 

Reproductive system Testes, epididymides, prostate, seminal vesicle, 

ovaries, uterus, vagina 

Nervous system Brain (c,erebrum, cerebellum and pons), spinal cord, 

sciatic nerve 

Hematopoietic and lymphatic Bone marrow (femur), axillar and mesenteric lymph 

systems nodes, spleen, thymus 

Endocrine system Pituitary gland, thyroids (with parathyroids), adl'enals 

Special sense organ Eye balls 

(2) 	 Skin was taken as a macroscopic region in one male (:--Jo. 22) of the 1,000 mg/kg 

group. 

(3) 	 Light microscopic examinations were perfonned for the organs and tissues of the 

following groups after embedding in paraffin, sectioning and hematoxylin and eosin 

(HE) staining. Decalcification was done for bone marrow (femur) with 10% formic 

acid fonnalin before cutting. In the table, "d'/ct.'· show groups which were examined 

and "-" shows examinations were not done. Parentheses show HE staining samples 

were prepared but not examined, since there were no changes attributable to the test 

substance in the 1,000 mglkg groups. 

Vehicle 	 1,000 Vehicle 	 25 150 1,000 control 	 mglkg Organ • tissue control 	 mgfkg mg/kg mglkg recovery 	 recovery group 	 group group group group 	 grol,fP 

Trachea 1:0 	 ('1: i) c+ vi 

Lungs eN (r'rJ 

Forestomach 

Glandular stomach 

Duodenum-ileum 

Cecum- rectum 

Liver*I 

281 
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,

Kidneys"" ,'1 

) i 


Urinary bladder r-~' ~~ 

Testes C 

1 
 , 

Epididymides"" ,-t 

Prostate 

/[ Seminal vesicles ~, 

Ovaries ( 

'[: 

Uterus (r' 

Vagina ,"

y 

Cerebrum, cerebellum, r:(') ~'" .J + '-' 

pons 

Spinal cord 

Sciatic nerve (')'(;' 

Bone marrow 

Axillar Jymph nodes Co")'? 

Mesenteric lymph nodes 

Spleen 

Thymus*5 d'~ (9) (~) (9) c!9 (2) 

Pituitary gland 

Thyroids d'S? CJ 

I 

Parathyroids 

Adrenals (Jq () ~'( 

Eye balls j'~{ eN 

'" I Since changes suspected to be effects of the test substance in the necropsy and organ weights were 

noted in males and females of the 1,000 mglkg groups, histopathological examinations for both 

sexes of all groups including the recovery groups were done. 

*2 Since changes suspected to be effects of the test substance in the organ weights were noted in 

females of the 1,000 mglkg recovery group, histopathological examinations for females of the 

recovery group were done. 

*J Since changes suspected to be effects of the test substance were observed in femaJes of the 1,000 

mg/kg group, histopathological examinations for females of all groups including the recovery 

groups were done. In males, since changes suspected to be effects of the test substance in the 

necropsy and organ weights were noted in the 1,000 mg/kg group, HE staining samples for males 

of all groups including the recovery groups were prepared. And since changes suspected to be 

effects of the test substance in the organ weights were noted in the 1,000 mglkg recovery group, 

histopathological examinations for the recovery groups were done. 

·16 ­ 282 
B 11-0908 

*4 Since changes suspected to be effects of the test sub:;tance in the organ weights were notcd in 

males of the 1.000 mg/kg group, histopathological examinations for males of the recovery groups 

were done, 

*5 Since changes suspected to be effects of the test substance in the organ weights were noted in 

females of the 1,000 mglkg group, HE staining sample:; were prepared in the recovery groups, 

(4) 	 Gross lesions in the jejunum (animal No, 16) and glandular stomach (animal No, 48) 

in the 150 mglkg group. and skin (animal No, 22) in the 1,000 mglkg group were 

examined histopathologically, 

(5) 	 Vacuolization in the hepatocytes was obscrved in HE stained samples in the 1,000 

mglkg groups and was suggestive of accumulation of lipid. Oil red 0 stain was 

perrormed using frozen section in one male (No. 24) and one female (No, 53) of the 

1,000 mg/kg groups. Oil red 0 positive substance was observed diffusely in male 

and in the centrilobular region in female. 

9. 	 STA TISTICAL ANALYSIS 

Data regarding body weights (excluding those at the time of necropsy), food intakes, 

hematological examinations, blood chemical examinations, urine volume, urine osmotic 

pressure, organ weights and FOB metrical da1a (grip strength and locomotor activity count) 

were analyzed using the Bartlett's test for homogeneity of variance, If the variances were 

homogeneous at a significance level of 5%, one way analysis of variance was performed. If 

there was a significant difference in this analysis, the difference between the vehicle control 

group and each of the treatment groups was analyzed by the Dunnett's test. If the variances 

were not homogeneous, the Kruskal-Wallis's test was used. If there was a significant 

difference in this analysis, the difference between the vehicle control group and each of the 

treatment group was analyzed by the nonparametrie Dunnett's test. 

FOB numerical data (defecation and urination) was analyzed using the Kruskal-Wallis's test. 

ENVIRONMENTAL FACTORS THAT MIGHT HA VE AFFECTED THE RELlA BTLITY OF 

THE STUDY RESULTS 

In the examination of drinking water sampled in November 18, 2008. the concentration of chloric 

acid was 0.89 mg/L and deviated from the quality criterion value, 50.6 mglL, described in ministerial 

ordinance for Ministry of Health, Labour and Welfare, It was reported that the NOAEL of ehloric 

acid was 30 mg/kg/day in the 90-day drinking water study in rats (Shokuhin Anzen Iinkai, 2007 (in 

Japanese) and McCauley et aI., 1995). Therefore, the required concentration correspond to the 

NOAEL was calculated at 180 mg/L for rats, if body weight of rat was 300 g and volume of drinking 

- 17­ 283 
B J 1-0908 

water per day was 50 mL. Since 180 mg/L was apprmdmately 200-fold from 0.89 mg/L and there 

was no change in clinical signs in thc vehicle control groups expected to be caused by the increased 

concentration of chloric acid, it was considered that this deviation did not affect the study results. 

RESULTS 

1. 	 CLINICAL SIGNS (Table 1, Addendum 1) 

1.1 	 During Dosing Period 

In males, salivation and decreased spontaneous locomotion were observed in all animals and 

two animals respectively in the 150 mg/kg group. In the 1,000 mg/kg group, salivation was 

observed in all animals, and moistening of the neck or mandible due to salivation in seven 

animals, fur loss in the neck or mandible in five animals, decreased spontaneous locomotion in 

all animals, incomplete eyelids opening and lacrimation in each one animal, and plenty of food 

on the tray in seven animals were observed. 

In females, salivation was observed in three animals of the ISO mg/kg group. Salivation and 

decreased spontaneous locomotion in all animals, incomplete eyelids opening, moistening of 

the neck or mandible and fur loss of the mandible in each four animals and staining lower 

abdomen and plenty of food on the tray in each eight animals were observed in the 1,000 

mglkg group. 

No abnormalities were noted in males and females of the 25 mg/kg groups. 

Salivation was observed just after dosing in almost males and females of the 150 and 1,000 

mg/kg groups. One male of the 1 SO mg/kg group and four females of the 1,000 mg/kg also 

salivated just before dosing sporadically. 

Moistening of tbe neck or mandible was sporadically observed in the afternoon from day 13 

of the dosing period. 

Fur loss was observed from day 11 ofthe dosing period. 

Incomplete eyelids opening was observed just after dosing form day 6. 

Lacrimation was observed just after dosing in the afternoon from day 19. 

Decreased spontaneous locomotion was observed just after dosing in almost animals of the 

150 mg/kg groups from day 16 and of the 1,000 mg/kg groups from day 4. Five males and 

• 18 ­ 284 
BII-0908 

one female showed decreased spontaneous locomotion in the afternoon in the 1,000 mg/kg 

groups. 

Staining lower abdomen was observed sporadically from day 4 of the dosing period, 

Plenty of food on the tray was observed continuously or sporadically from day 20. 

'1.2 	 During Recovery Period 

In males, fur loss of the neck or mandible was observed in one animal of the 1,000 mg/kg 

recovery group by day II of the recovery. Plenty of food on the tray was observed in one 

animal of the ],000 mglkg recovery gTOUp by day 3. 

In females of the 1,000 mg/kg recovery group, plenty of food on the tray was observed in one 

animal by day 3. 

2. 	 DETAILED CLIUNICAL OBSERVATIONS (Table 2, Addendum 2) 

2.1 	 During Dosing Period 

In males, lacrimation was observed in each one animal of the 1,000 mglkg group in week 3 and 

week 4. Stained fur was observed in four animals in week 4 in the I ,000 mg/kg group. 

In females, stained fur was observed in three, one, one and seven animals in weeks 1, 2, 3 and 

4, respectively. 

No abnormalities were noted in males and females ofthe 25 and 150 mglkg groups. 

2.2 	 During Recovery Period 

No abnormalities were noted in both sexes of the 1,000 mglkg recovery groups. 

3. 	 SENSORIMOTOR FUNCTION (Tables 3, 4 and 5, Addenda 3, 4 and 5) 

3.1 	 During Dosing Period 

No abnormalities were noted in all males. 

In females, locomotor activity counts were significantly increased in 30-40 min in the 1,000 

mg/kg group. No abnormalities were noted in the 25 and 150 mg/kg groups. 

3.2 	 During Recovery Period 

Males were not examined since no abnormalities were noted during the dosing period. 

- 19­ 285 
B 11-0908 

No abnormal locomotor activity counts were detected in lcmalcs of the 1,000 mg/kg recovery 

group, although locomotor activity count was increased during the c10sing period. 

4. 	 BODY WEIGHTS (Fig. 1 , Table 6, Addendum 6) 

4.1 	 During Dosing Period 

I n males, lower body weights were noted in the 1,000 mg/kg group on days 2 I. 26 and 28, 

compared to the control group. No abnormalities were noted in the 25 and 150 mg/kg groups. 

No statistically significant changes of body weight were noted in all treated females. 

4.2 	 During Recovery Period 

No statistically significant changes of body weights were noted in males ofthe recovery group. 

Statistically significant increases of body weights were noted in females of the 1,000 mg/kg 

recovery group on day 1 of the recovery. 

5. 	 FOOD INTAKES (Fig.2, Table 7, Addendum 7) 

5.1 	 During Dosing Period 

Food consumption was significantly decreased in males of the 1,000 mg/kg group on day 3. 

In females, a significant decrease was noted in the 1,000 mglkg group on day 3, and significant 

increases werc noted in the same group on days 15, 22 and 28. 

No significant changes were noted in males and females of the 25 and 150 mg/kg groups. 

5.2 	 During Recovery Period 

Significantly increased food consumption was noted in males of the 1,000 mg/kg recovery 

group on day 14. 

No significant changes were noted in females. 

6. 	 HEMATOLOGICAL EXAMINATIONS (Table 8, Addendum 8) 

6.1 	 At Termination of Dosing Period 

No statistically significant changes of hematological parameters were noted in males of all 

treatment groups. 

Decreased lymphocytes and increased monocytes were noted in females of the 1,000 mg/kg 

group. No abnormalities were noted in females of the 25 and 150 mg/kg groups. 

- 20­ 286 
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6.2 	 At Termination of Recovery Period 

No statistically significant changes were noted in both sexes. 

7. 	 BLOOD CHEMICAL EXAMINATIONS (Table 9, Addendum 9) 

7.1 	 At Termination of Dosing .Period 

ALT, albumin, A/G ratio, Bl3N and total bilirubin were significantly increased in males of the 

1,000 mg/kg group. 

Cholinesterase was significantly decreased in females of the 1,000 mg/kg group. Total 

bilirubin and inorganic phosphorus were significantly increased in females of the 1,000 mg/kg 

group. 

No statistically significant changes were noted in males and females of the 25 and 150 mglkg 

groups. 

7.2 	 At Termination of Recovery Period 

Although no abnormalities were noted at the end ofthe dosing period, significant decreases of 

glucose, creatinine and calcium and a significant increase of potassium were newly noted in 

males of the 1,000 mglkg recovery group. 

Inorganic phosphorus was significantly increased in females of the 1,000 mglkg recovery 

group. 

8. 	 URINALYSES (fable 10, Addendum 10) 

8.1 	 At Termination of Dosing Period 

WBC and epithelial cells were increased in the urine sediment in males and females of the 

1,000 mg/kg recovery groups. 

No abnormalities were noted in males and females of the 25 and 150 mg/kg groups. 

8.2 	 At Termination of Recovery .Period 

No abnormalities were noted in the 1,000 mg/kg recovery groups. 

9. 	 ORGAN WEIGHTS (Tables 11 and 12, Addenda 11 and 12) 

9.1 	 At Termination of Dosing Period 

In males, absolute thymus weight was significantly decreased in the 1,000 mg/kg group. 

Relative liver, kidney and testis weights were significantly increased in the 1,000 mg/kg group. 

- 21 ­ 287 
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[n females, absolute and relative liver and kidney weights were significantly increased in the 

1,000 mglkg group. Absolute and relative thymus weights were significantly decreased in the 

1,000 mg/kg group. 

No statistically significant changes were noted in males and females of the 25 and 150 mg/kg 

groups. 

9.2 	 At Termination of Recovery Period 

In males, relative kidney weights were significantly increased as shown at the end of the 

dosing period in the 1,000 mg/kg recovery group; however, the severity was decreased 

compared to those at end of the dosing period. Absolute epididymis and brain weights were 

newly significantly decreased in the 1,000 mglkg recovery group. No significant changes of 

I iver, testis or thymus weights were noted at the end of the recovery period. 

]n females, absolute and relative liver weights were significantly increased as shown at the end 

of the dosing period, however the severities were decreased. Significant increases of absolute 

and relative heart weights were newly noted in the 1,000 mg/kg recovery group. Kidneys and 

thymus did not show significant changes of organ weights at the end of the recovery period. 

10. 	 NECROPSY (Table 13, Addendum 13) 

10.1 	 At Termination of Dosing Period 

[n males, enlargement of the liver was observed in two animals of the 1,000 mg/kg group. 

Enlargement of the kidneys was observed in all animals of the J,000 mg/kg group. Although 

diverticulum in the jejunum was observed in one animal of the 150 mg/kg group, this was 

single occurrence. Pelvic dilatation of the kidney and fur loss were observed in each one 

animal ofthe 1,000 mgikg group. 

In females, enlargement of the liver was observed in one animal of the 150 mg/kg group and 

all animals of the 1,000 mgikg group. Enlargement of the kidney was observed in four 

animals of the ],000 mglkg group. Although, blackish region of the mucosa in the glandular 

stomach was observed in one animal of the 150 mglkg group, this sign showed no 

dose-relationship. 

10.2 	 At Termination of Recovery Period 

Liver and kidney lesions as shown at the end or the dosing period were not observed in all 

males and females at the end ofthe recovery period. 

- 22­ 288 
n11-0908 

11. 	 HISTOPATHOLOGICAL EXAMINATIONS (Table 14, Addendum 13) 

11.1 	 At Termination of DoSin2 Period 

rn males, diffuse fatty change of the hepatocytes in the liver was observed in one animal of the 

1,000 mglkg group. Following changes did not show dose·relationships; focal hypertrophy of 

the cortical cells in one male of the vehicle control group, focaillecrosis of the hepatocytes in 

one male of the 25 mglkg group and diverticulum in the jejunum in one male orthe 150 mg/kg 

group. Microgranuloma was observed in each one male of the 25, 150 and 1,000 mg/kg 

groups. Unilateral pelvic dilatation in the kidney in one male and scab formation on the skin 

in one male were observed in the 1,000 mg/kg group. No histopathological changes were 

observed in the thymus and testis. 

In females, fatty change of the periportal hepatocytes in the liver was observed in two animals 

and degeneration and necrosis of the tubular epithelium with the regenerative epithelium in the 

kidney in other two animals were observed in the 1,000 mg/kg group. No dose-related 

changes were observed as rollows, solitary cyst in the medullar of the kidney in one female of 

the 25 mglkg group and necrosis of the fundic mucosa in the glandular stomach in one female 

of the 150 mg/kg group. Focal inflammation in the lung in one female and microgranuloma 

in the liver in one female were observed in the 1,000 mglkg group. No histopathological 

changes were observed in the thymus. 

11.2 	 At Termination of Recovery Period 

Histopathological changes in liver as shown at the end of the dosing period disappeared in 

males. 

Histopathological changes in liver and kidney disappeared in females. 

DISCUSSION 

The effects of the test substance were obServed in the liver and kidney. 

As effects on the liver, relative weights in males and females and absolute weight in females were 

increased in the 1,000 mg/kg groups. Enlargement of the liver was observed in females of the 150 

and 1,000 mglkg groups and males of the 1,000 mglkg group. In the histopathological examinations, 

diffuse fatty change of the hepatocytes in males and fatty changes of the periportal hepatocytes in 

females were observed in the 1,000 mglkg groups. Although these changes were considered to be of 

effects to the lipid metabolism, the histopathological lesions were limited to few animals. In the 

blood chemistry, the changes related to the liver were observed; increased total bilirubin in males and 

females, increased AL T in males and decreased cholinesterase in females ofthe 1,000 mg/kg groups. 

- 23 • 289 
B 11-0908 

As effects on the kidney. relative weights in males and females and absolute weights in females were 

increased in the 1.000 mg/kg grours. Enlargement of the kidneys were observed in all males and 

four females or the 1,000 mg/kg groups; however, in the pathological examination, degeneration and 

necrosis of the tubular epithelium with the regenerative epithelium were observed in only two females 

of the 1,000 mg/kg group. In the urinalysis, WBC and epithelial cells in urine sediments were 

increased in males and females of the 1,000 mglkg groups, and BUN was increased in males of the 

1,000 mg/kg group in the blood chemistry. 

In the clinical signs, salivation was observed just after dosing in males and females of the I SO and 

1,000 mg/kg groups, however, was differed from that continuously obst'rved as the neurological signs. 

This sign was considered to be related to irritability of the test substance, since moistening and fur 

loss in the neck or mandible were observed, even though hyperplasia of the mucosa was not observed 

in the forestomach as seen in an irritable substance. Plenty of food on the tray observed in males 

and females of the 1,000 mg/kg group was considered to be probably related to increased food biting 

to reduce discomfort in the mouth, because the plenty of food on the tray was observed in the 

salivated animals. Salivation observed just before dosing was considered to be Pavlovian response. 

Decreased spontaneous locomotion in males of the 150 and 1,000 mg/kg groups and in females ofthe 

1,000 mg/kg group, and incomplete eyelids opening in males and females of the 1,000 mg/kg group, 

lacrimation in males and staining lower abdomen in females of the 1,000 mg/kg groups were 

observed. Decreased spontaneous locomotion was suspected to be neurological effects, since this 

sign was observed not only just after dosing but also in the afternoon. Lacrimation was also 

suspected to be neurological effects since this was observed in the afternoon, fcw hours later after 

dosing. Staining lower abdomen due to urination was considered to be effect to voiding. These 

clinical signs were not observed during the recovery period, except spilled food on the tray observed 

by early period ofthe recovery. Therefore, all of the clinical signs were considered to be reversible. 

In the detailed clinical observations, lacrimation and stained fur in males of the 1,000 mg/kg group 

and stained fur in females of the 1,000 mg/kg group were observed, as seen in the daily clinical 

observation. 

Body weight was decreased in males of the 1,000 mg/kg group from day 21 of the dosing period. 

There were no severe liver and kidney lesions and no abnom1al food consumption in the same period. 

Body weight gains in the high dose group were lower compared to those ofthe vehicle control group. 

The decreased body weight was considered to be treatment-related, although cause of the decrease 

was unclear. 

Food intakes were decreased in males and remales of the 1,000 mg/kg groups on day 3 of the dosing 

period. This was considered related to the test substance since it was observed in both sexes, 

- 24­ 290 
BII·0908 

however. body weights showed no abnormalities during the same period. Therefore. the decreased 

food intakes were considered not to be toxicologically significant. Increased food intakes observed 

in females was considered related to the increased food biting in the 1,000 mg./kg group after day 15 

of trea1ment. 

In the organ weights, absolute thymus weights in males and females, and relative thymus weight in 

females were decreased in the 1,000 mg/kg groups without histopathological changes. Although 

these changes were considered related to treatment since absolute and relative weights were 

decreased, they were not considcred to be toxicologically significant. Decreased relative testis 

weights in males of the 1.000 mglkg group were considered to bc secondary effects of the decreased 

body weights, since absolute weights were not affected and histopathological changes were not seen. 

In the sensorimotor function, locomotor activity counts were increased in females of the 1,000 mg/kg 

group. However, this was a single occurrence observed in 30-40 min and considered to be no 

treatment related. 

In the hematology, decreased lymphocytes and increased monocytes were not considered to be related 

to treatment since there were no abnormalities in the lymphatic system including the spleen. 

In the blood chemistry, increased albumin and A/G ration in males of the 1,000 mglkg group were 

considered to be secondary effects resulted from malnutrition. since body weight was lower. 

Increased inorganic phosphorus in females of the 1.000 mg/kg and 1,000 mg/kg recovery groups was 

considered to be a single occurrence, since no related abnormalities were noted in the kidney. 

In the histopathological exanlinations, microgranuloma in the liver in males of the 25, 150 and 1,000 

mg/kg groups and in females of the 1,000 mg/kg group, unilateral pelvic dilatation in the kidney and 

scab ft)rmation on the skin in males of the 1,000 mglkg group and focal int1ammation in the lung in 

females of the 1,000 mglkg group werc considered to be single occurrences since they wcre observed 

in each one animal, and the same signs are also observed in the vehicle control groups. 

In the recovery groups, fur loss of the neck or mandible was observed by day 11 of the recovery 

period and plenty of food on the tray was observed by day 3, and these were disappeared 

subsequently. Although, liver weights in females and kidney weights in males were increased in the 

1,000 mg/kg recovery groups, the severities were decreased compared to those at the end of the 

dosing period and the histopathological changes were not observed in these organs. 

All other changes observed only in the recovery groups were considered not to be toxicologically 

significant. Body weights were increased in remales of the 1,000 mglkg recovery group on day 1 of 

the recovery period without food intake changes, and these wcre considered to be a single occurrence. 

- 25­ 291 
B 11-0908 

3805showadenAithough food intakes were increased in males of the 1,000 mg/kg recovery group on 

day 14, there were no body weight changes related to this and it was considered to be no 

toxicologically significant. In the blood chemistry, decreased blood glucose, creatinine and calcium 

and increased potassium were noted in males of the 1,000 mglkg recovery group. However, the 

same changes were not noted at the end of the dosing period and there were no histopathological 

lesions related to them. Therefore, these were not suggestive of toxicological significant. In the 

organ weights, absolute epididymis and brain weights were decreased in males and absolute and 

relative heart weights were increased in females of the 1,000 mglkg recovery groups without 

histopathological changes. The same changes were not noted at the end of the dosing period. 

Therefore, they were not considered related to treatment. 

In conclusion, the 'No Observed Adverse Effect Level' (NOAEL) was considered to be 25 mglkg/day 

due to decrea<;ed spontaneous locomotion in males and enlargement of the liver in females of the 150 

and 1,000 mg/kg groups. 

The 'No Observed Effect Level' (NOEL) wa" considered to be 25 rng/kglday due to salivation related 

to irritable effects ofthe test substance in males and females ofthe 150 and 1,000 mglkg groups. 

REFERENCES 

Shokuhin anzen iinkai (2007) Seiryo Inryosui Hyokasho, Seiryo inryosui nt kakaru kagakubushitsu 

no shokuhin kenko eikyo hyoka ni tsuite, ensosan (in Japanese). 

McCauley PT, Robinson M, Daniel FB, Olson GR (J995) The effects of subehronic chlorate 

exposure in Sprague-Dawley rats. Drug and Chemical Toxicology 18 (2, 3): 185-199. 

- 26­ 292 
811-0908 

() : Vehicle control 

0: 25 mg/kg!day 

-2 3 g ta 17 21 26 28 1 5 10 J.4 (days) N 

(g 

(.) Fig. 1-1 	 Twenty-eight-day repeated-dose oral toxicity study in rats 

Body weights : Male 

( ,) 

450 

400 

350 

, 

N 

-.::.

, 

100 Administration 

~: 150 mg/kg/day 

<> : 1,000 mg!kg!day 

Recovery 
(g) 

250 

200 

I 

t>:> 

00 

I 

150 

100 

I'» 

~ 

~ rig. 

B11-0908 

o : Vehicle control 

0: 25 mg/kg/day ~: 150 mg/kg/day 

<> : 1,000 mg/kg/day 

Administration Recovery

k ~ 

-z 3 8 12 17 21 Z6 28 5 10 14 (days) 

1-2 Twenty-eight-day repeated-dose oral toxicity study in rats 

weights : Female 
--

811-0908 

'0 

30 

>. 

-c "" 20 

I 

N +-" 

<0 ... I 

"" 

10 

o 

~4_~ 

() : Vehicle control 

0: 25 mg/kg/day 

{:,.: 150 mg/kg/day 

o ; 1,000 mg/kg/day 

Administration 	 Recovery ~ ~ Ie: )j 

3 8 	 15 22 28 4 8 14 (days l ....., 

<0 

c:.n Fig. 2-1 	 Twenty-eight-day repeated-dose oral toxicity study in rats 

~ood intakes : Male 
--

--

40 

3ll 

>. 

... 20 '"CI 

I 

c.o 

0 '" ... I 

"" 

10 

o 

B11-0908 

_A-. 

::;:;;; 

o : Vehicle contro 1 

0: 25 mg/kg/day 

A: 150 rug/kg/day 

<> : 1,000 mg/kg/day 

Administration Recovery ~ ~ ~ )1 

N 3 8 15 22 28 .( 8 14 (days) (Q 

0) Fig. 2-2 Twenty-eight-day repeated-dose oral toxicity study in rats 

Food intakes : Female 
III 1-090& 

Table 1 Twenty-eight-day repeated-dose oral toxicity study in rats 

Summary of clinica.l signs 

Sex Signs Administration Period Recovery Period 

mglkglday ve VC 

(R) 2S ISO 1,000 1,000 (R) VC 1,000 

Male ta ta ta ta ta til ta til 

Sa) S 5 S S 5 S 5 

No abnormalities detected S 5 5 5 J 

Salivation 5 S 5 

Decreased spontaneous locomotion 2 5 5 

Incomplete eyelid opening 

Moistening 3 4 

Loss of hair 2 3 

Lacrimation 

.-"'....----.--~ 

Much food on tray .. 3 

Female ta la ta la ta ta ta ta 

5') 5 5 S 5 5 5 5 

No abnormalities detected 5 5 S 2 S 

Salivation 3 S S 

Decreased spontaneous locomotion 5 S 

Incomplete eyelid opening 3 

Moistening 3 

Loss of hair 3 

Staining lower abdomen 3 S 

Much food on tray 4 .. 1\) Number of animals examined. 


ve, Vehicle control; (R), Recovery 


ta, terminal autopsy. 


Salivation was observed just after dosing in males and females ofthe ISO and 1,000 mg/kg groups, and just before dosing in one male 


ofthe 150 mg/kg group and four males ofthe 1,000 mg/kg group. 


Incomplete eyelid opening was observed just after dosing. 


Moistening lind lacrimation were observed in the afternoon. 


Decreased spon!!Uleous locomotion was observed just after dosing in males of the 150 and 1,000 mg/kg groups and females ofthe 1,000 


mg/kg group, and in the afternoon in five males and one female of the 1,000 mg/kg goups. 


Staining lower abdomen was observed before dosing, during and just after dosing or in the afternoon. 


-31- 297 


4 
Table 2 Twcnty-eight-day repeated-dose oral toxicity study in rats BII-090& 

Summary ofdetailed clinieal observations (scoring scale for detailed clinical observations) 

REMOVAL FROM CAGE 

Ease of removal 

-2 

-I 

o 

+1 

+2 

Vocalization 

o 

+1 

+2 

HANDLING OBSERVATIONS 

Muscle tone 

-} 

o 

+1 

Suhnomlill temperature 

+ 

Piloerection 

+ 

Staining hair 

+ 

Unkempt hair 

+ 

Paleness 

+ 

Reddening 

+ 

Cyanosis 

+ 

Lacrimation 

+ 

No reaction 

Very easy 

Easy (slight rcsisllIJIcc) 

Difficult 

Very difficult 

None 

Vocalization during handling 

Continuous vocalization 

Decreased 

Nonnal 

Increased 

Absent 

Present 

Absent 

Present 

Absent 

Present 

Absent 

Present 

Absent 

Present 

Absent 

Present 

Absent 

Present 

Absent 

Present 

-32- 298 
Table 2 Twenty-eight-day repeated-dose oral toxicity study in rats Bl1-0908 

Summary ofdetailed clinical observations (scoring scale for detailed clinical observations) 

HANDLING OBSERVATIONS-continued 

Exophthalmos 

+ 

Pupi\lary size 

·1 

o 

+] 

Salivation 

+ 

Secretion 

+ 

OBSERVATIONS IN ARENA 

Posture 

o 

+1 

+2 

Motor activity 

-2 

-1 

o 

+1 

+2 

Respiration 

o 

+1 

+2 

+3 

Lid closure 

+ 

Gait 

S 

T 

P 

GO 

Absent 

Present 

Miosis 

Normal 

Mydriasis 

Absent 

Present 

Absent 

Present 

Normal 

Crouching position or hunchback position 

Prone position or lateral position 

Significantly decreased 

Decreased 

Normal 

Increased 

Significantly increased 

Normal 

Slightly insufficiency 

Moderately insufficiency 

Severely insufficiency 

Absent 

Present 

Normal 

Staggering gait 

Tip toe gait 

Shuffling (paralytic) gait 

Gait disturbance 

-33- 299 

Table 2 Twenty-eight-day repeated-dose oral toxicity study in rats BII-0908 

Summary of detailed clinical observations (scoring scale for detailed clinical observations) 

OBSERVATIONS IN ARENA.continued 

Tremor/twitch/convulsion 

o 

+1 

+2 

+3 

Stereotypic behavior 

C 

G 

S 

H 

Abnormal behavior 

S 

B 

C 

R 

W 

V 

8T 

T 

None 

Tremor 

Twitch or convulsion 

Systematic 10nic convulsion (opisthotonus or epistholonus etc.) 

None 

Circling 

Grooming 

Sniffing 

Head bobbing 

None 

Self-biting 

Backing 

Circling 

Rolling 

Writhing 

Vocalization 

Straub tail 

Tnil lashine behavior 

-34­ 300 
Table 2·1 Twenty-eight-day repeated-dose oral toxicity study in rats BII-0908 

Summary of detailed clinical observations 

Removal from cage 

St:x Period Exp. group Number of Ease of removal Vocal ization 

(mg!kglday) animals ·2 ·1 0 +] +2 0 +1 +2 

Vehicle control 10 0 0 10 0 0 10 0 0 

Predosing 

25 

150 

.5 

5 

0 

0 

0 

0 

.5 

5 

0 

0 

0 

0 

5 

:; 

0 

0 

0 

0 

. 1.000 10 0 0 10 0 0 9 I 0 

Vehicle control 10 0 0 10 0 0 10 0 0 

week I 

25 5 0 0 5 0 0 4 I 0 

150 .5 0 0 .5 0 0 5 0 0 

1.000 10 0 0 10 0 0 9 1 0 

Vehicle control 10 0 0 10 0 0 10 0 0 

week 2 

25 

150 

5 

.5 

0 

0 

0 

I 

.5 

4 

0 

0 

0 

0 

.5 

.5 

0 

0 

0 

0 

Male 1,000 

Vehicle control 

10 

10 

0 

0 

0 

0 

10 

]0 

0 

0 

0 

0 

9 

10 0 

0 

0 

week 3 

25 

150 

5 

5 

0 

0 

0 

0 

5 

:; 

0 

0 

0 

0 

.5 

5 

0 

0 

0 

{) 

1,000 ]0 0 0 10 0 0 9 0 

Vehicle control 10 0 0 10 0 0 9 0 

week 4 

25 .5 0 I 4 0 0 4 0 

150 .5 0 0 .5 0 0 4 0 

1,000 10 0 0 10 0 0 8 2 0 

Recovery Vehicle control .5 0 0 5 0 0 5 0 0 

weekI 1,000 .5 0 0 5 0 0 2 3 0 

Recovery Vehicle control 5 0 0 .5 0 0 4 I 0 

week 2 1,000 .5 0 0 .5 0 0 5 0 0 

-35­ 301 
Table 2-2 Twenty-eight-day repeated-dose oral toxicity study in rats BII-0908 

Summary of detailed cliniclll observations 

Removal from cage 

Sex Period Exp. group Number of Ease of removal Vocalization 

(mglkglday) animals -2 -1 0 +I +2 0 +1 +2 

Vehicle control 10 0 0 10 0 0 10 0 0 

Predosing 

25 

ISO 

.5 

5 

0 

0 

0 

0 

.5 

.5 

0 

0 

0 

0 

4 

4 

0 

0 

1,000 10 0 0 10 0 0 10 0 0 

Vehicle control JO 0 0 10 0 0 10 0 0 

week 1 

25 

150 

5 

5 

0 

0 

0 

0 

5 

5 

0 

0 

0 

0 

5 

4 

a 0 

0 

1,000 10 0 0 10 0 0 10 0 0 

Vehicle control 10 0 0 10 0 0 10 0 0 

week 2 

25 

150 

.5 

.5 

0 

0 

0 

0 

.5 

5 

0 

0 

0 

0 

5 

.5 

0 

0 

0 

0 

Female 1,000 

Vehicle control 

10 

10 

0 

0 

0 

0 

10 

10 

0 

0 

0 

0 

9 

10 

1 

0 

0 

0 

week 3 

25 

150 

5 

.5 

0 

0 0 

4 

S 

0 

0 

0 

0 

4 

4 

1 0 

0 

1,000 10 0 0 10 0 0 9 0 

Vehicle control 10 0 0 10 0 0 7 3 0 

week 4 

25 

150 

5 

.5 

0 

0 

0 

0 

5 

5 

0 

0 

0 

0 

3 

5 

2 

0 

0 

0 

1,000 10 0 I 9 0 0 8 2 0 

Recovery Vehicle control 5 0 0 5 0 0 S 0 0 

week 1 1,000 .5 0 0 5 0 0 .5 0 0 

Recovery Vehicle control 5 0 0 5 0 0 4 I 0 

week 2 1,000 5 0 0 5 0 0 4 0 

-36- 302 

Table 2-3 Twenty-eight-day repeated-dose oral toxicity study in rats D1I-0908 

Summ!!!l of detailed clinical observations 

Handling observations 

Sex Period Exp. group Numbcrof Muscle tOile Subnormal temperature Piloerection 

(mglkglday) animals -I 0 H + + 

Vehicle control 10 0 10 0 ]0 0 10 0 

25 5 0 5 0 5 0 .5 0 

Predosing 

150 5 0 5 0 5 0 5 0 

1,000 10 0 10 0 10 0 10 0 

Vehicle control 10 0 10 0 10 0 10 0 

week I 

25 

150 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

1,000 10 0 10 0 10 0 10 0 

Vehicle control 10 0 10 0 10 0 10 0 

week 2 

25 

150 

5 

.5 

0 

0 

5 

5 

0 

0 

.5 

.5 

0 

() 

5 

5 

0 

0 

Male 

1,000 

Vehicle control 

10 

10 

0 

0 

10 

10 

0 

0 

10 

10 

0 

0 

10 

10 

0 

0 

week 3 

25 

150 

5 

5 

0 

0 

.5 

5 

0 

0 

5 

5 

0 

0 

5 

.5 

0 

0 

1,000 10 0 10 0 10 0 10 0 

Vehi cle control 10 0 10 0 10 0 10 0 

week 4 

25 

150 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

1,000 10 0 10 0 10 0 10 0 

Recovery Vehicle control .5 0 S 0 5 0 .5 0 

week I 1,000 5 0 5 0 5 0 5 Q 

Recovery Vehicle control .5 0 5 0 5 0 5 0 

week 2 1,000 .5 0 5 0 5 0 5 0 

-37­ 303 
Table 2-4 Twenty-eight-day repeated-dose oral toxicity study in rats B 11-0908 

Summa!r of detailed clinical observations 

Handling observations 

Sex Period Exp. group Number of Muscle tone Subnormal temperature Piloerection 

(mglkglday) animals .J 0 +1 + + 

Vehicle control 10 0 10 0 10 0 10 0 

Predosing 

2S 

ISO 

5 

5 

0 

0 

5 

.5 

0 

0 

5 

5 

0 

0 

5 

S 

0 

0 

1,000 10 0 10 . 0 10 0 10 0 

Vehicle control 10 0 10 0 10 0 10 0 

week I 

25 

150 

.5 

.5 

0 

0 

5 

5 

0 

0 

5 

.5 

0 

0 

.5 

5 

0 

0 

1,000 10 0 \0 0 10 0 10 0 

Vehicle control 10 0 10 0 10 0 10 0 

week 2 

25 

150 

5 

5 

0 

0 

5 

.5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

Female 1,000 

Vehicle control 

10 

10 

0 

0 

10 

10 

0 

0 

10 

10 

0 

0 

10 

10 

0 

0 

week 3 

25 

150 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

1,000 10 0 10 0 10 0 10 0 

Vehicle control 10 0 10 0 10 0 10 0 

week 4 25 .5 0 5 0 5 0 .5 0 

150 .5 0 .5 0 S 0 5 0 

1,000 10 0 10 0 10 0 10 0 

Recovery Vehicle control .5 0 5 0 5 0 5 0 

week I 1,000 5 0 .5 0 .5 0 .5 0 

Recovery Vehicle control 5 0 5 0 .5 0 S 0 

week 2 1,000 S 0 S 0 .5 0 .5 0 

-38­ 304 
Table 2-5 Twenty-eight-day repeated-dose oralloxicity study in rats BII·0908 

Summ!:!l: of detailed clinical observations 

Handling observations 

Sex Period Exp. group Number of Staining hair Unkempt hair Paleness Reddening 

(mglkglday) animals + + + + 

Vehicle control 10 10 0 10 0 10 0 10 0 

Predosing 

25 

150 

5 

5 

5 

5 

0 

0 

5 

5 

0 

0 

5 0 

0 

5 

5 

0 

0 

1,000 10 10 0 10 0 10 0 10 0 

Vehicle control 10 10 0 10 0 10 0 10 0 

week I 

25 5 5 0 5 0 .5 0 5 0 

150 5 5 0 5 0 .5 0 5 0 

1,000 10 10 0 10 0 10 0 10 0 

Vehicle control 10 10 0 \0 0 10 0 10 0 

week 2 

25 

150 

5 

5 

5 

5 

0 

0 

5 

5 

0 

0 

.5 

5 

0 

0 

5 

5 

0 

0 

Male 1,000 

Vehicle control 

10 

10 

10 

10 

0 

0 

10 

10 

0 

0 

10 

10 

0 

0 

10 

)0 

0 

0 

week 3 

25 

150 

5 

!S 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

1,000 10 10 0 10 0 10 0 10 0 

Vehicle control 10 10 0 10 0 \0 0 10 0 

week 4 

25 5 5 0 5 0 5 0 5 0 

150 5 5 0 5 0 5 0 .5 0 

1,000 10 6 4 10 0 10 0 10 0 

Recovery Vehicle control 5 5 0 5 0 5 0 5 0 

week I 1,000 5 5 0 5 0 5 0 5 0 

Recovery Vehicle conlrol 5 5 0 5 0 5 0 5 0 

week 2 1,000 5 5 0 5 0 5 0 5 0 

-39­ 305 
Table 2-6 	 Twenty-eight-day repeated-dose oral toxicity study in rats B 11-0908 

Summ~ of detailed clinical observations 

Handling observalions 

Sex Period Exp. group Number of Staining hair Unkempt hair Paleness Reddening 

(mglkglday) animals + + + + 

Vehicle control 10 10 0 10 0 10 0 10 0 

Predosing 25 

150 

5 

5 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

1,000 10 10 0 10 (I 10 0 10 0 

Vehicle control 10 10 0 10 0 10 0 10 0 

week) 25 

150 

5 

5 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

1,000 10 7 3 10 0 ]0 0 10 0 

Vehicle control 10 10 0 10 0 10 0 10 0 

week 2 

25 

150 

5 

5 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

Female 1,000 

Vehicle control 

10 

10 

9 

10 

1 

0 

10 

10 

0 

0 

10 

10 

0 

0 

10 

10 

0 

0 

week 3 25 

150 

5 

5 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

1,000 10 9 I 10 0 10 0 10 0 

Vehicle control 10 10 0 10 0 10 0 10 0 

week 4 

25 5 5 0 5 0 5 0 5 0 

150 5 5 0 5 0 5 0 5 0 

1,000 10 3 7 10 0 10 0 10 0 

Recovery Vehicle control 5 5 0 5 0 5 0 5 0 

weeki 1,000 5 5 0 5 0 5 0 5 0 

Recovery Vehicle control 5 5 0 5 0 5 0 5 0 

week 2 1,000 5 5 0 5 0 5 0 5 0 

-40-	 306 

Table 2-7 Twenty-eight-day repealed-dose oral toxicity study in rats B 11-0908 

Surrun~ of detailed clinical observations 

Handling observations 

Sex Period Exp. group Number of Cyanosis Lacrimation Exophthalmos 

(mglkglday) animals + + + 

Vehicle control 10 10 0 10 0 10 0 

25 5 0 5 0 5 0 

Prcdosing 

150 5 5 0 5 0 5 0 

1,000 10 10 0 10 0 10 0 

Vehicle control 10 10 0 10 0 10 0 

week I 

25 

150 

5 

5 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

1,000­ 10 10 0 10 0 10 0 

Vehicle control 10 10 0 10 0 10 0 

week 2 

25 

150 

5 

5 

5 

5 

0 

0 

5 

5 

0 

0 5 

0 

0 

Male 

1,000 

Vehicle control 

10 

10 

10 

10 

0 

0 

10 

10 

0 

0 

10 

10 

0 

0 

week 3 

25 

150 

5 

5 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

1,000 10 10 0 9 10 0 

Vehicle control 10 10 0 10 0 10 0 

week 4 

25 

150 

5 

5 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

1,000 10 10 0 9 10 0 

Recovery Vehicle control 5 5 0 5 0 5 0 

week 1 1,000 5 5 0 5 0 5 0 

Recovery Vehicle control 5 5 0 5 0 5 0 

week 2 1,000 5 5 0 5 0 5 0 

-41- 307 

Table 2-8 Twenty-eigbt-day repeated-dose oral toxicity study in rats BIl·0908 

Summ~ of detailed clinical observations 

Handling observations 

Sex Period Exp. group Number of CYWlosis Lacrimation Exophthalmos 

(mglkglday) animals + + + 

Vehicle control 10 to 0 10 0 10 0 

25 5 5 0 .5 0 5 0 

Predosing 150 5 5 0 5 0 5 0 

1,000 10 10 0 10 0 10 0 

Vehicle control 10 10 0 10 0 10 0 

week I 

25 

150 

5 

.5 

5 

5 

0 

0 

5 

.5 

0 

0 

.5 

j 

0 

0 

1,000 10 10 0 10 0 10 0 

V chicle control 10 10 0 10 0 10 0 

week 2 

25 

150 

.5 

j 

5 

5 

0 

0 

.5 

.5 

0 

0 

5 

5 

0 

° 

Female 1,000 

Vehicle control 

10 

10 

10 

10 

0 

0 

10 

10 

0 

0 

10 

10 

0 

0 

week] 25 

150 

j 

5 

.5 

5 

0 

0 

.5 

5 

0 

0 

5 

5 

0 

0 

1,000 10 10 0 10 0 10 0 

Vehicle control 10 10 0 10 0 10 0 

week 4 

25 

150 

5 

:; 

5 

5 

0 

0 

.5 

.5 

0 

0 

.5 

5 

0 

0 

1,000 10 10 0 10 0 [0 0 

Recovery Vehicle control 5 .5 0 .5 0 5 0 

week 1 1,000 .5 .5 0 .5 0 .5 0 

Recovery Vehicle control 5 .5 0 5 0 5 0 

week 2 1,000 .5 .5 0 .5 0 .5 0 

-42­ 308 
Table 2·9 	 Twenty-eight-day repeated-dose oral toxicity study in rats BII-0908 

Summarv of detailed clinical observations , 

Handling observations 

Sex Period Exp. group Number of Pupillary size Salivation Secretion 

(mgtkglday) animals ·1 0 +1 + + 

Vehicle control 10 0 10 0 10 0 \0 0 

Predosing 

25 

150 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

1,000 10 0 10 0 10 0 10 0 

Vehicle control 10 0 10 0 10 0 10 0 

weeki 

25 

150 

.5 

5 

0 

0 

.5 

5 

0 

0 

5 

.5 

0 

0 

5 

5 

0 

0 

1,000 10 0 10 0 10 0 10 0 

Vehicle control 10 0 10 0 10 0 10 0 

week 2 

25 

150 

5 

5 

0 

0 

.5 

5 

0 

() 

:; 

.5 

0 

0 

.5 

5 

0 

0 

Male 1,000 

Vehicle control 

10 

10 

0 

0 

10 

10 

0 

0 

10 

10 

0 

0 

10 

10 

0 

0 

week 3 

25 

150 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

1,000 10 0 10 0 10 0 10 0 

Vehicle control 10 0 10 0 ]0 0 10 0 

week 4 

25 5 0 5 0 5 0 5 0 

150 .5 0 5 0 5 0 5 0 

1,000 10 0 10 0 10 0 ]0 0 

Recovery V chicle control 5 0 5 0 5 0 5 0 

week I 1,000 5 0 5 0 5 0 5 0 

Recovery Vehicle control 5 0 5 0 5 0 5 0 

week 2 1,000 5 0 5 0 5 0 5 0 

-43­ 309 
Table 2-10 Twenty-eighl-day repeated-dose oral toxicity study in rats DII-0908 

Summ!!;!l of detailed clinical observations 

Handling observations 

Sex Period Exp. group Number of Pupillary size Salivation Secretion 

(mg/kglday) animals -I 0 +1 + + 

Vehicle control 10 0 10 0 10 0 10 0 

25 5 0 5 0 5 0 5 0 

Predosing 

150 5 0 5 0 5 0 5 0 

1,000 10 0 10 0 10 0 10 0 

Vehicle control 10 0 10 0 10 0 10 0 

week I 

25 

150 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

1,000 10 0 10 0 10 0 10 0 

Vehicle control 10 0 10 0 10 0 10 0 

week 2 

25 

150 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

Female 1,000 

Vehicle control 

10 

10 

0 

0 

10 

10 

0 

0 

10 

10 

0 

0 

10 

10 

0 

0 

week 3 

25 

ISO 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

5 

5 

0 

0 

1,000 10 0 10 0 10 0 10 0 

Vehicle control 10 0 10 0 10 0 10 0 

week 4 

25 

150 

5 

5 

0 

0 

5 

5 

0 

0 

S 

5 

0 

0 

5 

5 

0 

0 

1,000 10 0 10 0 10 0 10 0 

Recovery Vehicle control 5 0 5 0 5 0 5 0 

week J 1,000 5 0 5 0 5 0 5 0 

Recovery Vehicle control S 0 5 0 5 0 5 0 

week 2 1,000 5 0 5 0 5 0 5 0 

-44- 310 
Table 2-11 Twenty-eight-day repeated-dGse oral toxicity study in rats 811·0908 

Summary of detailed clinical observations 

Observations in arena 

Sex Period Exp. group Number of PostlJre Motor activity 

(mglkglday) animals 0 +) +2 ·2 ·1 0 +1 +2 

Vehicle control 10 10 0 0 0 0 9 0 

Predosing 

25 

150 

5 

5 

.5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

S 

5 

0 

0 

0 

0 

1,000 10 10 0 0 0 0 ·10 0 0 

Vehicle control 10 10 0 0 0 7 2 0 

week) 

25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

° 

0 

0 

4 

4 

1 0 

0 

1,000 10 10 0 0 0 0 9 0 

Vehicle control 10 10 0 0 0 0 9 I 0 

week 2 

25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

5 

5 

0 

0 

0 

0 

Male 1,000 

Vehicle control 

]0 

10 

lO 

10 

0 

0 

0 

0 

0 

0 

0 

0 

8 

10 

2 

0 

0 

0 

week 3 

2S 

150 

S 

5 

5 

5 

0 

0 

() 

0 ° 0 

0 

° 

5 

5 

0 

0 

0 

0 

1.000 10 10 0 0 0 0 10 0 0 

Vehicle control 10 10 ° 0 0 9 0 0 

week 4 

25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 0 

4 

4 

0 

1 ° 0 

1,000 10 10 0 0 0 2 8 0 0 

Recovery Vehicle control 5 5 0 0 0 I 4 0 0 

weekI 1,000 5 5 0 0 0 0 5 0 0 

Recovery Vehicle control 5 5 0 0 0 4 0 0 

week 2 1,000 .5 5 0 0 0 0 5 0

° 

-45- 311 

Table 2·12 Twenty-eight.day repeated.dose oral toxicity study in rats Bll·0908 

Sununary of detailed clinical obscrvalionJ 

Observations in arena 

Sex. Period Exp. group Numbcrof Posture Molar activity 

(mg/kglday) animals 0 +1 +2 ·2 ·1 0 +1 +2 

Vehicle control 10 10 0 0 0 0 9 I 0 

Predosing 

25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

0 

0 5 

0 

0 

0 

0 

1,000 10 to 0 0 0 0 10 0 0 

Vehicle control 10 10 0 0 0 0 7 3 0 

week 1 

25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

5 

5 

0 

0 

0 

0 

1,000 10 10 0 0 0 0 8 2 0 

Vehicle control 10 10 0 0 0 0 10 0 0 

week 2 . 25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

4 

5 () 

0 

0 

Female 

1,000 

Vehicle control 

10 

10 

10 

10 

0 

0 

0 

0 

0 

0 

0 

0 

9 

10 

1 

0 

0 

0 

week 3 

25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

5 

5 

0 

0 

0 

0 

1,000 10 10 0 0 0 0 10 0 0 

Vehicle control 10 10 0 0 0 1 8 0 

weck4 

25 

150 

:5 

5 

:5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

:5 

:5 

0 

0 

0 

0 

1,000 10 10 0 0 0 6 3 0 

Recovery V chicle control :5 5 0 0 0 0 5 0 0 

week I 1,000 :5 5 0 0 0 0 :5 0 0 

Recovery Vehicle control :5 5 0 0 0 0 4 0 

week 2 1,000 5 5 0 0 0 0 :5 0 0 

-16- 312 
Table 2-13 Twenty-eight-day repealed-dose oral toxicity study in rats BIl-0908 

Summary of detailed clinical o\>servalions 

Observations in arena 

Sex .Period Exp. group Number of Respiration Lid closure 

(mglkg/dIlY) animals 0 +1 +2 +3 + 

Vehicle control 10 10 0 10 ° ° ° 
 25 5 5 0 0 5 0 

Predosing ° 
 150 5 5 5 0

° ° ° 
 1,000 IO 10 0 0 0 10 ° Vehicle control 10 IO 0 0 0 10 0 

week 1 

25 5 5 0 ° ° 5 0 

]50 .5 .5 0 0 0 5 0 

1,000 10 10 D 0 0 10 0 

Vehicle control 10 10 0 0 0 10 0 

week 2 

25 

150 

.'i 

.'i 

5 

5 

0 

0 

0 

0 

0 

0 

5 

5 

0 

0 

Male 1,000 

Vehicle control 

10 

10 

10 

10 

0 

0 

0 

0 

0 

0 

10 

10 

0 

0 

week 3 

25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

5 

5 ° 0 

1,000 10 10 0 0 0 10 0 

Vehicle control 10 10 0 0 0 10 0 

week 4 

25 5 .5 0 0 0 5 0 

150 5 5 0 0 0 5 0 

],000 10 10 0 0 0 10 0 

Recovery Vehicle control 5 5 0 0 0 5 0 

week I 1,000 5 5 0 0 0 5 0 

Recovery Vehicle control 5 5 0 5 0 

week! ° ° 1,000 5 5 0 0 5 0 ° 

-47- 313 

Table 2.14 Twenty-eight-day repeated-dose oral toxicity study in rats B 11-0908 

Summary of detailed clinical observations 

Observations in arena 

Sex Period Exp. group Number of Respiration Lid closure 

(mglkglday) animals 0 +1 +2 +3 + 

Vehicle control 10 10 0 0 0 10 0 

Predosing 

25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

5 

5 

0 

0 

],000 !O 10 0 0 0 10 0 

Vehicle control 10 10 0 0 0 10 () 

week I 

25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

() 

5 

5 

0 

0 

1,000 IO IO 0 0 0 10 0 

Vehicle control 10 10 0 0 0 10 0 

week 2 

25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

5 

5 

0 

0 

Female 1,000 

Vehicle control 

10 

10 

IO 

10 

0 

0 

0 

0 

0 

0 

10 

10 

0 

0 

week 3 

25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

5 

5 

0 

0 

1,000 10 10 0 0 0 IO 0 

Vehicle control 10 IO 0 0 0 10 0 

week 4 

25 5 5 0 0 0 5 0 

150 5 5 0 0 0 5 0 

1,000 10 10 0 0 0 10 0 

Recovery Vehicle control 5 5 0 0 0 5 0 

week 1 1,000 5 5 0 0 0 5 0 

Recovery Vehicle control 5 5 {) 0 0 5 0 

week 2 1,000 5 5 0 0 0 5 0 

-48­ 314 
Table 2·1 S Twenty·eight-day repeated·dose oral toxicity study in rats Bll·0908 

Summary of detailed clinical observations 

Observations in arena 

Sex. Period Exp. group Number of Gait 

(mglkglday) animals S T P GD 

Vehicle control 10 10 0 0 0 ° 


25 5 5 0 0 0 0 

Predosing ISO 5 S 0 0 0 0 

1,000 JO 10 0 0 0 0 

Vehicle control 10 10 0 0 0 0 

25 5 5 0 0 0 0 

week 1 

150 5 S 0 0 0 0 

1,000 10 10 0 0 0 0 

Vehicle control to 10 0 0 ° 0 

week 2 

25 

ISO 

5 

5 

5 

5 ° 0 

0 

0 

0 

0 

0 

0 

Male 1,000 

Vehicle control 

10 

10 

10 

10 

0 

0 

0 

0 

0 

0 

0 

0 

week 3 

25 

ISO 

S 

5 

5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

1,000 10 10 0 ° 0 0 

Vehicle control 10 10 0 0 0 0 

week 4 

25 

150 

5 

S 

5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

1,000 10 10 0 0 0 0 

Recovery Vehicle cootrol 5 5 0 0 0 0 

week 1 1,000 5 5 ° 0 0 0 

Recovery Vehicle control 5 5 0 0 0 0 

week 2 1,000 5 5 0 0 0 0 

-49­ 315 
Table 2-16 Twenty-eight-day repeated-dose oral toxicity study in rats 811-0908 

SUtnllUll'Y of detailed clinical observations 

Observations in arena 

Sex Period Exp. group Number of GIIit 

(mglkglday) animals S T P GD 

Vehicle control \0 10 0 0 0 0 

25 5 5 0 0 0 0 

Predosing 

150 5 5 0 0 0 0 

1,000 10 10 0 0 0 0 

Vehicle control 10 10 0 0 0 0 

week 1 

25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

1,000 10 10 0 0 a 0 

Vehicle control 10 }O 0 0 0 0 

week 2 

25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

Female 1,000 

Vehicle control 

10 

\0 

10 

10 

0 

0 

0 

0 

0 

0 

0 

0 

week 3 

25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

1,000 10 10 0 0 0 0 

Vehicle control 10 10 0 0 0 0 

week: 4 

25 

ISO 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

1,000 10 10 0 0 0 0 

Recovery Vehicle control 5 5 0 0 0 0 

week 1 1,000 5 5 0 0 0 0 

Recovery Vehicle control 5 5 0 0 0 0 

week 2 1,000 5 5 0 0 0 0 

-50- 316 

Table 2-17 Twenty-eight-day repeated-dose oral toxicity study in rats B 11-0908 

Summary of detailed clinical observations 

Observations in arena 

Sex Period Exp. group Number of Trcmorltwitch/convlllsion Defecation Urination 

(mglkglday) animals 0 +l +2 +3 (count/min)") (count/min)') 

Vehicle control 10 10 0 0 0 0.3 ±0.48 2.0 ±3.40 

25 5 5 0 0 0 0.0 ±O.OO 4.4 ±9.29 

Predosing 

150 5 0 0 0 0.4 ±0.89 4.2 ±6.69 

1,000 . 10 10 0 0 0 0.1 ±0.32 l.5 ±1.58 

V chicle control 10 10 0 0 0 0.0 ±O.OO 0.5 ±1.27 

week I 

25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

0.2 

0.0 

±0.45 

±O.OO 

0.4 

2.8 

±0.55 

±5.22 

1,000 10 10 0 0 0 0.0 ±O.OO 0.5 ±0.85 

Vehicle control 10 10 0 0 0 0.2 ±D.63 0.7 ±1.34 

week 2 

25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

0.2 

0.0 

±0.45 

±O.OO 

0.6 

1.6 

±1.34 

±2.07 

Male 

1,000 

Vehicle control 

IO 

10 

10 

10 

0 

0 

0 

0 

0 

0 

0.4 

0.3 

±0.70 

±0.95 

0.9 

0.2 

±1.29 

±0.42 

week 3 

25 

150 

5 

.5 

5 

5 

0 

0 

0 

0 

0 

0 

0.0 

0.4 

±O.OO 

±0.89 

0.0 

0.6 

±O.OO 

±0.89 

1,000 10 10 0 0 0 0.1 ±0.32 0.5 ±1.27 

Vehicle control 10 10 0 0 0 0.1 ±D.32 0.1 ±O.32 

week 4 

25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

0.2 

0.4 

±0.45 

±0.89 

0.0 

0.2 

±O.OO 

±0.45 

1,000 10 10 0 0 0 0.3 ±0.48 0.6 ±1.90 

Recovery Vehicle control 5 5 0 0 0 0.4 ±0.89 0.6 ±0.89 

week 1 1,000 5 .5 0 0 0 0.2 ±0.45 0.2 ±0.45 

Recovery Vehicle control 5 5 0 0 0 0.4 ±0.89 0.4 ±0.89 

week 2 1,000 5 5 0 0 0 0.0 ±O.OO 0.2 ±0.45 

a) Mean±S.D. 

... Significantly different from vehicle control at P<O.05 . 

.. Significantly different from vehicle control at P<O.Ol. 

-51- 317 

Table 2-18 Twenty-eight-day repeated-dose oral toxicity study in rats llll·0908 

Summary of detailed clinical observations 

Observations in arena 

Sex Period Exp. gTOUp Number of Tremor/twitch/convulsion Defecation Urination 

(mglkglday) animals 0 +1 +2 +3 (count/min)') (count/min)') 

Vehicle control 10 10 0 0 0 0.0 :1:0.00 2.0 ±1.89 

25 5 5 0 0 0 0.0 :1:0.00 4.4 :1:5.59 

Predosing 150 5 5 0 0 0 0.2 :1:0.45 2.2 :1:3.90 

1,000 10' 10 0 0 0 0.2 :1:0.42 1.4 :1:1.58 

Vehicle control 10 10 0 0 0 0.0 :1:0.00 0.6 ±0,84 

week I 

25 5 5 0 0 0 0.0 ±D,OO 1.8 :1:3 .03 

150 .5 5 0 0 0 0,0 ±O.OO 1.6 :1:3.58 

1,000 10 10 0 0 0 0.0 :1:0,00 0.7 ±1.06 

Vehicle control 10 10 0 0 0 0.0 :1:0.00 0.3 ±0,67 

week 2 

25 5 .5 0 0 0 0.0 :1:0,00 0.4 :1:0.55 

150 5 5 0 0 0 0.0 :1:0.00 0.4 :1:0.89 

Female 1,000 10 10 0 0 0 0.0 :1:0.00 0.5 :1:1.27 

Vehicle control 10 10 0 0 0 0.0 ±D.OO 0.5 ±1.08 

week) 

25 

150 

5 

5 

5 

.5 

0 

0 

0 

0 

0 

0 

0.0 :1:0.00 

0.0 ±D.oo 

0.0 

2.6 

:1:0.00 

±5.81 

1,000 10 10 0 0 0 0.0 :1:0.00 0.5 :1:1.27 

Vehicle control 10 10 0 0 0 0.0 ±O.OO 0.0 :1:0.00 

25 .5 5 0 0 0 0,0 ±D.OO 0.0 ±O,OO week 4 

150 5 .5 0 0 0 0.0 ±D,OO 1.2 :\'2.68 

1,000 10 10 I) 0 0 0.1 :1:0.32 0.3 :1:0.95 

Recovery Vehicle control 5 5 0 0 0 0.0 ±D.OO 0.8 :1:1.30 

week I 1,000 5 5 0 0 0 0.0 :1:0.00 0.0 ±O.OO 

Recovery Vehicle control 5 5 0 0 0 0.0 :1:0.00 0.0 :1:0.00 

week 2 1,000 5 .5 0 0 0 0.0 :1:0.00 0.0 :1:0,00 

a) 	 Mean*S.D. 

Significantly different from vehicle control at P<O.05. * 

•• 	Significantly different from vehicle control at P<O.Ol. 

-52-	 318 

Table 2-19 Twenty-eight-day repeated-dose oral toxicity study in rids BII-0908 

Summary of detailed clinical observations 

Observations in arena 

Sex Period Exp. group Number of Stcreotypic behavior 

(mglkgldIlY) animals C G S H 

Vehicle control 10 10 0 0 0 {} 

25 5 5 0 0 0 0 

Preclosing 

150 5 5 0 0 0 0 

1,000 10 ]0 '0 0 0 0 

Vehicle control 10 10 0 0 0 0 

week 1 

25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

1,000 10 10 0 0 0 0 

Vehicle control 10 10 0 0 0 0 

week 2 

25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

Male 1,000 

Vehicle control 

10 

10 

10 

\0 

0 

0 

0 

0 

{} 

0 

0 

0 

week 3 

25 

ISO 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

1,000 10 10 0 0 0 0 

Vehicle control 10 10 0 0 0 0 

week 4 

25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

1,000 10 10 0 0 0 0 

Recovery Vehicle control 5 5 0 0 0 0 

wee.k 1 1,000 5 5 0 0 0 0 

Recovery Vehicle control 5 5 0 0 0 0 

week 2 1,000 5 5 0 0 0 0 

-53- 319 
Table 2-20 Twenty-eight-day repeated-dose oral toxicity study in rats BII-0908 

Summary of detailed clinical observations 

Observations in arena 

Sex Period Exp. group Number of Stcreotypic behavior 

(mglkglday) animals C G S H 

Vehicle control 10 10 0 0 0 0 

Predosing 

25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

1,000 10 10 0 0 0 0 

Vehicle control 10 10 0 0 0 0 

week 1 

25 

150 

5 

:5 

5 

:5 

0 

0 

0 

0 

0 

0 

0 

0 

1,000 10 10 0 0 0 0 

Vehicte control to 10 0 0 0 0 

week 2 

25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

Female 1,000 

Vehicle control 

10 

10 

10 

10 

0 

0 

0 

0 

0 

0 

0 

0 

week 3 

25 

ISO 

:5 

:5 

5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

1,000 10 10 0 0 0 0 

Vehicle control 10 10 0 0 0 0 

week 4 

25 

ISO 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

1,000 10 10 0 0 0 0 

Recovery Vehicle control 5 :5 0 0 0 0 

week 1 1,000 5 5 0 0 0 0 

Recovery Vehicle control 5 5 0 0 0 0 

week 2 1,000 5 5 0 0 0 0 

-54- 320 

Table 2-21 Twenty-eight-day repeated-dose oral toxicity study in rats Bll-090S 

Summary of detailed clinical observations 

Observations in arena 

Sex Period Exp. group Number of Abnormal behavior 

(rnglkglday) animals S 8 C R W V ST T 

Vehicle control 10 10 0 0 0 0 0 0 0 0 

25 5 5 0 0 0 0 0 0 0 0 

Predosing 

150 5 5 0 0 0 0 0 0 0 0 

1,000 10 10 0 0 0 0 0 0 0 0 

Vehicle control 10 10 0 0 0 0 0 0 0 0 

weekI 

25 

150 

5 

5 

5 

.5 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

1,000 10 10 0 0 0 0 0 0 0 0 

Vehicle control 10 10 0 0 0 0 0 0 0 0 

week 2 

2.5 

1.50 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

Male 

1,000 

Vehicle control 

10 

10 

10 

10 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

week 3 

25 

150 

5 

.5 

5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

1,000 10 10 0 0 0 0 0 0 0 0 

Vehicle control 10 10 0 0 0 0 0 0 0 0 

week 4 

25 

150 

5 

5 

.5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

1,000 10 to 0 0 0 0 0 0 0 0 

Recovery Vehicle control .5 .5 0 0 0 0 0 0 0 0 

week I 1,000 .5 5 0 0 0 0 0 0 0 0 

Recovery Vehicle control 5 .s 0 0 0 0 0 0 0 0 

week 2 1,000 5 5 0 0 0 0 0 0 0 0 

-55­ 321 
Table 2-22 Twenty-eight-day repeated-dose oral toxicity study in rats BI1-0908 

Summary of detailed clinical observations 

Observations in arena 

Sex Period Exp. group Number of Abnormal behavior 

(mglkg/day) animals S B C R W V ST T 

Vehicle control 10 JO 0 0 0 () 0 0 0 0 

2S S 5 0 0 0 0 0 0 0 0 

Predosing 

150 5 S 0 0 0 0 0 0 0 0 

1,000 10 10 0 0 0 0 0 (} 0 0 

Vehicle control 10 10 0 0 0 0 0 0 0 0 

week I 

2S 5 . 5 0 0 0 0 0 0 0 0 

150 5 5 0 (} 0 0 0 0 0 0 

1,000 10 10 0 0 0 0 0 0 0 0 

Vehicle control 10 10 0 0 0 0 0 0 0 0 

week 2 

25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

Female 1,000 

Vehicle conlTol 

10 

10 

10 

10 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

week 3 

25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

1,000 10 10 0 0 0 0 0 0 0 0 

Vehicle control 10 10 0 0 0 0 0 0 0 0 

week 4 

25 

150 

5 

5 

5 

5 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

1,000 10 10 0 0 0 0 0 0 0 0 

Recovery Vehicle control 5 S 0 0 0 (} 0 0 0 0 

week 1 1,000 5 5 0 0 0 0 0 0 0 0 

Recovery Vehicle control 5 5 0 0 0 (} 0 0 0 0 

week 2 1,000 5 S 0 0 0 0 0 0 0 0 

-56- 322 

Table 3 Twenty-eignt-day repeated-dose oral toxicity study in rats B11-0908 

Summary of reflex (scoring scale for reflex) 

SENSORIMOTOR FUNCTION 

Approach contactltoucb response 

·1 

0 

+1 

Pinna response 

-1 

0 

+1 

Pain respollse (tail pin eh) 

-I 

0 

+1 

Pupillary reflex 

+ 

Air righting reflex 

+ 

No reaction 

Nonnal 

Hyper reaction 

No reaction 

Nonnal 

Hyper reaction 

No reaction 

Normal 

Hyper reaction 

Nonna! 

Abnonnal reaction 

Normal 

Abnormal reaction 

-57- 323 

Table 3-1 Twenty-tight·day repealed·dose oral toxicity study in rat.~ BIl·0908 

Summary of reflex 

Sensorimotor function 

Sex Period Exp. group 

(m~g!day) 

Number of 

animals 

Approach contact 

touch response 

Pinna response 

.1 0 +1 -1 0 +1 

Vehicle control 10 0 10 0 0 10 0 

Male week 4 

25 5 0 5 0 0 S 0 

150 5 0 5 0 0 5 0 

1,000 10 0 10 0 0 10 0 

Vehicle control 10 0 10 0 0 \0 0 

Female week 4 

25 5 0 5 0 0 5 0 

150 5 0 5 0 0 5 0 

1,000 10 0 10 0 0 10 0 

-58­ 324 
Table 3·2 Twenty-eight-day repeated-dose oral toxicity study in rats Bll·0908 

Swnmaty of reflex 

Sensorimotor function 

Sex Period Exp.group Number of Pain respon.'1e (tail pinch) Pupillaty reflex Air righting reflex 

(rnglkglday) animals -I 0 +1 + + 

Vehicle control 10 0 10 0 10 0 10 0 

Male week 4 

25 ,5 0 ,5 0 ,5 0 5 0 

150 5 0 5 0 5 0 5 0 

1,000 10 0 10 0 10 0 10 0 

Vehicle control 10 0 10 0 10 0 10 0 

Female week 4 

25 5 0 ,5 0 ,5 0 5 0 

150 5 0 5 0 5 0 5 0 

1,000 10 0 10 0 10 0 10 0 

-59­ 325 
Table 4 Twenty-eight-day repeated-dose oral toxicity study in rats BII-0908 

Summwy of grip strength 

Sex Period Exp. group Number of Forelimb Hindlimb 

(mglkglday) llIlimals (g) (g) 

Vehicle control 10 4]8 :1:28 426 :1:25 

25 5 417 ±25 462 ±13 

Male week 4 

150 5 428 :1:39 429 :1:49 

1,000 10 425 :1:37 -405 :1:29 

Vehicle control 10 372 ±26 378 ±23 

25 5 367 ±18 364 :1:23 

Female week 4 

150 5 375 ±16 377 ±30 

1,000 ]0 356 ::1:30 384 ±24 

Mean ±S.D. 

* Significantly different from vehicle control at P<O.05. 

** Significantly different from vehicle control at P<O.O 1. 

-60­ 326 
Table 5 Twcnty-eight-day repeated-dose oral toxicity study in rats 811-0908

Summary of motor activiljl 

Sex Period Exp. group Number of Interval (min) 

(mglkglday) animals 0-10 10-20 20-30 30-40 40-50 50-60 Total 

Vehicle control 10 174 107 79 57 46 61 525 

=94 ±46 ±50 ±26 ±46 ±52 ±198 

Male weck4 

25 5 188 

±79 

86 

±4O 

60 

±40 

61 

±53 

65 

±66 

60 

±39 

518 

±141 

150 S 126 

±72 

103 

±54 

68 

±58 

46 

±40 

67 

±42 

71 

±60 

481 

±255 

1,000 10 259 149 112 88 84 65 

±66 ±63 ±65 ±45 ±66 ±63 ±268 

Vehicle control 10 236 133 98 54 36 59 

±61 ±58 :!:51 ±38 ±46 ±60 ±203 

week 4 

25 5 261 

±32 

190 

±85 

150 

±73 

80 

±79 

89 

±41 

49 

±59 

820 

±223 

Female 

150 S 195 

±30 

121 

±33 

47 

±47 

40 

±34 

51 

±35 

10 

:i:9 

463 

±155 

1,000 10 290 

±48 

179 

±46 

115 

±66 

123 

±75 

• 83 

±72 

89 

±66 

879 

±213 

Vehicle control 142 75 40 18 39 31 345 

Recovery 

week 2 

±83 ±80 ±36 ±18 ±49 .:1:31 ±246 

1,000 S 229 

±38 

143 

±74 

79 

±99 

66 

±73 

69 

±21 

58 

±56 

644 

.:1:190 

Mean±S.D. 

• Significantly different from vehicle control at P<O.05 . 

.. Significantly different from vehicle control at P<O.OI. 

-61­ 327 

757 

617 
Table 6-1 Twenty-eight-day repeated-dose oral toxicity study in rats 811-0908 

Swuary of body lIeights(g) 

Sex Bxp.group NUliber of Adl.inistration period 

(fig/kg/day) anillals -2 1 3 8 12 17 21 26 28 (days) 

Vehicle 10 148.5 165.2 180.9 222.7 253.3 294.0 325.8 356.2 366.5 

control ± 4.4 6.5 ± 7.8 t 14.0 ± 18.8 ± 21.5 ± 23.7 ± 25.6 ± 25.2 

25 5 148.7 166.3 181.1 222.5 250.5 290.8 318.1 345.8 355.2 

Male ± 4.9 ± 5.5 ± 7.0 ± 11. 3 ± 13.7 ± 19.6 ± 21.2 ± 2B.5 ± 31.1 

150 5 148.7 164.4 177.9 216.5 245.4 285.0 312.0 342.9 350.9 

± 5.5 ± 6.3 ± 9.2 ± 16.8 ± 22.1 ± 33.1 ± 41. 7 ± 51.8 ± 55.7 

1,000 10 147.8 165.3 172.0 206.7 234.2 268.0 290.2* 313.2* 320.5* 

± 4.2 ± 4.9 ± 8.4 ± 11.2 ± 16.2 ± 20.4 ± 24.6 ± 27.2 ± 29.1 

Vehicle 10 123.8 134.6 142.9 162.1 174.0 191. 6 203.4 212.4 217.7 

control ± 6.8 ± 7.8 ± 9.6 ± 11.9 ± 12.4 ± 12.8 ± 14.2 ± 13.8 ± 14.9 

25 5 123.1 132.3 140.7 157.4 169.3 183.1 194.1 203.9 211.6 

lellole ± 5.2 ± 7.3 ± 7.2 ± 12.4 ± 13.7 ± 15.8 ± 16.7 ± 14.6 i 17.6 

150 5 122.9 134.3 141.3 165.9 177.3 195.9 206.2 217.5 222.1 

± 7.0 ± 8.5 ± 10.3 ± 17.5 ± 13.6 ± 11.7 ± 11. 3 ± 11. 8 ± 13.6 

1,000 10 124.0 134.5 142.5 165.1 181.9 203.0 214.7 225.0 229.6 

± 5.1 ± 7.2 ± 7.8 ± 8.8 ± 11.6 ± 13.8 ± 16.1 ! 16.1 ± 16.5 

I 

0') Mean ±S.D. 


l'.:> * Significantly different fro. vehicle control at P<O.05. 


I ** Significantly different frail vehicle control at P<O.Ol. 


W 

N 

CO 
Table 6-2 Twenty-eight-day repeated-dose oral toxicity study in rats 811-0908 

Suuary of body weights(g) 

Sex Exp.group Number of Recovery period 

(llg/kg/day) animals 1 5 10 14 (days) 

Kale 

Vehicle 

control 

5 363.7 

± 23.7 

386.2 

± 23.1 

410.9 

± 24.0 

426.8 

± 21.7 

1,000 5 327.6 

± 33.2 

346.5 

± 35.7 

372.9 

± 38.1 

396.0 

± 40.1 

FellAle 

Vehicle 

control 

5 213.4 

± 10.7 

222.7 

± 10.4 

227.2 

± 12.1 

233.6 

± 11.9 

1,000 5 228.1* 

± 10.0 

235.2 

± 11.4 

239.1 

± 8.4 

243.8 

± 9.4 

Hean ±S.D. * Significantly different froll vehicle control at P<O.05. ** Significantly different froa vehicle control at P<O.OI. 

I 

c;T) 

c.u 

I 

W 

N 

co 
Table 7-1 Twenty-eight-da¥ repeated-dose oral toxicity study in rats 811-0908 

Sumlary of fQod intakes(g/rat/day} 

Sex Exp.group NUilber of Atiainistration period 

(lII(/kg/daY) ani.is 1 3 8 15 22 28 (days) 

Vehicle 10 19.7 19.6 21.1 20.8 20.9 19.6 

control ± 1.5 :t 1.9 2.4 t 'l. 4 ± 2.1 ! 2.1 

25 5 20.4 19.5 21.1 20.7 19.9 1S.S 

Kale ± 1.3 ± 1.6 f 1.9 t 2.8 ± 2.6 ± 2.7 

. 150 5 20.5 19.5 21. 5 22.1 :U.8 20.7 

... 1.3 ± 1.6 2.7 ! 3.4 3.7 ± 4.6 

1,000 10 20.5 15.6** 19.0 21. 3 22.2 22.0 

t 0.9 ! 2.0 .t 2.2 2.6 ± 2.9 t 3.2 

Vehicle 10 16.0 14.7 14.3 13.0 13.0 12.3 

control 1.3 ± 1.7 ± 1.4 1.3 i 1.1 f 0.9 

25 I) 15.3 14.6 14.0 12.8 12.4 12.5 

re.le ! 1.9 ! 1.6 ! <l.1 t 1.8 1.5 1.5 

150 5 16.2 14.4 15.9 14.0 14.5 13.4 

! 1.0 t 1.1 ! 2.0 i 1.2 1.0 t 0.6 

1,000 10 18.0 12.7* 14.1 15.8u 16.8** 16.2** 

f 1.6 0.8 i 1.4 ! 1.6 

I 

a.o lI&an ±S.Il • .po.. * Significantly different from vehicle control at P<O.05. I ** Significantly different fro. vehicle control at P<O.OI. 

cu 

cu 

o 
Table 7-2 T1ienty-eight-day repeated-dose oral toxici t:y study in rah Bll-0908 

SUlIBry of food intakes(g/rat/day) 

Sex Exp.r;roup Humber of Recovery period 

(ag/kr;/day) animals 4 8 14 (days) 

Kale 

Vehicle 

control 

5 

i 

22.5 

1.7 

24.0 

1.5 t 

24.9 

1.0 

1,000 I) 

± 

22.7 

2.1 f 

24.1 

1.6 t 

26.7* 

1.3 

Vehicle 5 16.4 16.0 16 a 

Female control :t 1.1 ± 1.3 ! 1.1 

1,000 5 17.8 17.5 17.7 

1.1 ± 0.8 f 0.6 

Kean ±S.D. *,Significantly different froll vehicle control at P<O.05. ** Significantly different froll vebicle control at P<O.Ol. 

I 

O:l 

01 

I 

(..) 

(..) 
Table 8-1 T~enty-eight-day repeated-dose oral toxicity study in rats 

Su..ary of he.atologlcal exaainations 

811-0908 

Sex 

Rxp.group 

(lig/kg/day) 

!luBber of 

ani.ais 

!Be WBe lIb 

(xlO4! p.L) (x103/,uL) (g/dL) 

Ht 

(%) 

Hev 

(iL) 

MeH 

(pg) 

Melle 

(g/dL) 

I'latelet Reticulo 

(xJ04/,ut) (%) 

I'T 

(see) 

AI'TT 

(sec) 

Vehicle 

control 

5 749 

± 43 

124 

± 15 

15.0 

± 0.8 

44.1 

± 2.6 

59.S 

i 0.3 

20.1 

± 0.2 

33.6 

± 0.4 

102.6 

± 4.7 

2.6 

to.3 

18.0 

:: 1. 0 

28.7 

± 2.2 

25 5 742 lZO 14.7 43.8 59.0 19.9 33.6 100.1 2.4 16.0 28.8 

± 11 ± 23 ± 0.3 ± 0.8 ± 1. Z ± 0.4 ± 0.4 ± 3.2 to.3 ± 1.1 .± 2.9 

Male 

150 5 749 

± 31 

114 

± 37 

14.9 

± 0.2 

44.9 

± 0.9 

60.1 

± 1.8 

20.0 

± 0.7 

33.3 

± 0.3 

99.4 ± 8.9 

2.3 

±0.5 

·18.5 

± 3.6 

28.7 

± 3.2 

1,000 5 790 

+ 41 ReComy-------""-­

120 

i 27 

15.3 

± 0.7 

45.8 57.9 19.3 

...LLL-_U...:.L__ ± 0.8 

33.4 103.4 i O. 3 ___.....Lll.) Z.8 17.8 32.8 

to.6______.LLL_____LL1_____ 

Vehicle 

control 

5 791 

± 46 

115 

± 16 

15.2 

± 0.4 

44.7 

± 1.1 

56.5 

± 2.2 

19.3 

± 0.8 

34.1 

± 0.1 

109.9 

± 7.4 

2.4 

±0.3 

20.2­

± 4.0 

35.5 

± 2.2 

1,000 5 760 

± 30 

131 

± 12 

14.8 

± 0.4 

43.8 

t 1. 5 

57.S 

± 1. 5 

19 4 ± 0.4 33.7 

± 0.5 

10S.9 

I 7.7 

2.7 

to.5 

19.4 

! 5.2 

35.7 

± 3.3 

Vehicle 

control 

5 763 

± 20 

106 

± 4 

14.9 

± 0.2 

43.9 

± O.S 

57.5 

± 0.8 

19.6 

± 0.4 

34.0 

± 0.3 

106.8 

± 9.3 

1.8 

iO.2 

15.1 

± 0.9 

24.1\ 

± 2.9 

25 5 763 

± 13 

86 

± 22 

15.0 

± 0.5 

44.2 

± 1.4 

58.0 

± 0.9 

19.6 

± 0.4 

33.8 

± 0.1 

109.3 

± 10.9 

1.7 

±0.2 

14.8 

± 1. 2 

26.1 

± 1. 4 

I 

0') 

0') Fe.ale 

150 5 755 

± 20 

97 

± 27 

14.9 

± 0.5 

43.9 

± 1. 2 

5S.2 

± 1. 5 

19.8 

± 0.6 

34.0 

± 0.4 

107.8 

± 9.7 

La 

±0.2 

15.0 

± 0.5 

23.1 

± 2.0 

I 1,000 5 738 118 14 5 42.7 58.0 19.7 34.0 11:.2 2.0 14.8 &3.7 ___•___________._..L!1.____._L.l~_______..t_Q..:_~______L_!~.1 _____.l..!L.!L__...LQ.ce___LQ..:_~_______t__] .._~__l.Q.:j____L1_·2___.1 2 9 

ltecovery 

Vehic le 

control 

5 777 

± 24 

92 

11 

14.9 

± 0.5 

42 7 

! 1. 9 

54.9 

J. 1.8 

19.2 

± 0.6 

35.0 

± 0.5 

126.6 

± U.B 

2.1 

±0.4 

14.5 

! 0.3 

24.2 

!: 2.7 

1,000 5 784 

± 22 

76 

13 

14.9' 

± 0.2 

42.7 

± 0.5 

54.4 

± 1. 6 

19.0 

± O.B 

34.8 

t 0.3 

119.5 

± 1.4 

1.9 

to.1 

14.6 

± 0.4 

23.9 

i 2.2 

Mean ±S.ll. * Significantly different fro. vehicle control at P<O.OS. *. Significantly different fro. vehicle control at P<O.Ol. 

t.) 

W 

N 
Table 8-& Twenty-eight-day repeated-doss oral toxicity study in rats 811-0908 

Sumlary of hematological examinations 

Exp.group NUBber of DIfferentiation of leukocyte (~) 

Sex (llg/kg/day) 3ninls lfeutro Ensinn Baso Lymph MOllo LUC 

Vehicle 5 15.2 0.5 0.2 79.8 3.1 1.2 

control ± 3.7 ±O.l iO.O f 4.6 ±1. 0 ±0.3 

25 5 17 .6 0.5 0.1 71.9 2.5 1.3 ± 4.0 ±0.1 ±0.1 ± 3.3 f1.0 ±0.7 

150 5 20.0 0.7 0.1 74.9 3.1 1.2 

Hale ± 3.6 to.4 fO.l ± 3.9 :to.7 to.S 

1.000 5 Z2.5 O.S 0.2 11.7 4.0 0.9 _____________J_ 4.2 to. 2___-t2..:..!_____LUL____±l. 1 ___.J.9..:...L ___.._ Recovery Vehicle 5 15.5 0.6 0.2 19.4 3.0 1.4 

control ± 7.0 ±0.2 fO.1 ± 7.3 fO.8 fO.S 

1,000 5 13.8 0.7 0.1 81.8 2.4 1.2 

± 3.0 ±0.3 to.O ± 3.9 H.l ±0.4 

Vehicle 5 12.1 0.9 0.1 84.1 1.9 0.9 

control t 2.7 to.S ±O.O ± 2.8 11.0 ±0.2 

25 5 16.6 1.1 0.1 79.1 2.0 1.0 ± 3.5 ±0.4 ±O.O t 3.8 iO.6 to.3 

I 160 16.6 0.7 0.1 79.9 1.6 1.1 


-::/ Felale t 7.6 ±0.2 fO.O ± 7.6 ±0.3 10.4 
 J 1,000 5 11.5 1.4 0.1 75.9* 3.8* 1.3 

en 5 

----1.!!.:...L______tL.L________ -.l....!:.L..._ 1 . 1 fO.l ± 4.2 

ReCOvery 

Vehicle 5 14.8 1.4 0.2 79.7 2.8 1.2 

control t 4.6 fO.S to.l f 4.7 to.7 ±0.3 

1.000 5 .7 1.3 0.1 8Z.4 3.2 1.4 

.9 ±0.4 fO.O ± 4.2 ±O.7 to.l 

lIean <±S.D • 
 .. Siglificantly different frol vehicle control at P<O.05.
 ** Signlficantlr different from vehicle control at P<O.Ol. 


w 

w 

w 
Table 9-1 Twenty-eight-day repeated-dose oral toxicity study in rats BII-0908 

Sumaary of blood che.ieal examinations 

Kxp.group KUJlber of AST ALi ALP ChE -r-GTP T-Cho TG Glucose T-Protein Albumin A/G ratio 

Sex (.,;/kg/day) aniJlais (lUlL) (lUlL) (lUlL) (lUlL) (lUlL) (mg/dL) (lIg/dL) (lg/dL) (g/dL) (g/dL) 

Vehicle 

control 

5 63 

± 7 

18 

± 3 

559 

±l35 

64 

17 

0.8 

to.1 

52 

t 4 

89 

136 

167 

:t 10 

5.9 

to.2 

2.1.1 

to. 

0.89 

+0.04 

25 5 65 

± 6 

19 

± 5 

574 

t 66 

54 

14 

0.8 

to.l 

50 

± 5 

62 

il9 

160 

± 21 

5.8 

to.4 

2.7 

10.1 

0.89 

to.08 

Kale 

150 5 60 

± 7 

42 

± 3 

531 

1122 

54 ± 18 

O.S 

±0.2 

52 

± 4 

83 

±56 

162 

± 16 

5.7 

to 2 

2.8 

±0.1 

0.98 

fO.05 

1,000 

Recovery 

Vehicle 

control 

5 

5 

71 

± 7 

62 

± 6 

44** 

_t 6 

22 

± 4 

515 

tIO! 

334 

± 41 

61 

14 

51 

7 

0.8 

;1;0.1 

0.8 

±0.2 

56 

± 9 

51 

± 9 

44 

~36 

66 

±l8 

140 

~ 17 

152 

.t 10 

5.9 

;0.2 

6.0 

to.2 

3. On 1.06** tU____t~__ 

2.7 0.83 

to.l ±0.08 

1,000 5 69 

no 

35 

± 5 

311 

± 33 

49 

9 

0.7 

±0.1 

52 

± 6 

56 

U5 

13h 

± 14 

5.8 

±0.1 

2.6 

to .1 

0.84 

±0.06 

Vehicle 

control 

5 69 

± 6 

21 

± 5 

395 

±226 

255 

± 54 

0.9 

to.2 

63 

± .7 

27 

± B 

128 

± 10 

6.0 

±0.2 

3.0 

±O 1 

I 02 

±0.05 

I 

0':> 

00 

I 

fellale 

25 

150 

1,000 

Recovery-­

Vehicle 

control 

5 

5 

5 

5 

6a 

± 6 

56 

flO 

66 

U6 

19 

± 3 

18 

± 6 

39 

±I5 

272 268 0.8 49 2Z 117 5.9 3 0 L03 

± 31 ± 73 ±0.1 ± 6 ± 8 ± 13 to.2 ±O 1 ±0.09 

351 257 1.0 64 26 139 5.9 30 1. 04 

± 95 ± 75 ±0.3 ±I5 ± 2 ± 11 ±0.2 ±0.2 ±0.07 

328 125** 0.9 75 26 134 6.2 3.2 1. 05 ______HQ_L_____.J__~~______±O ct._____U _______L!___!__ J_______tQ.:J________tLL_____!.!LJ1______ 

219 460 0.8 68 36 122 6.3 3.0 0.92 

± 69 ±117 to. Z ±l3 t26 ± 16 to.2 ±0.2 ±O.08 

1,000 5 1.1 

.3 

81 

±11 

46 

±37 

131 

± 15 

6.1 

±0.3 

3.0 

±0.1 

0.94 

to.06 

lIean ±S.D. * Significantly different fro. vehicle 'control at P<0.05.

** Significantly different fro. vehicle control at P<O.Ol. 

(.) 

(.) 

~ 
Table 9-2 iKenty-eight-day repeated-dose oral toxicity study in rats 811-0908 

Su..ary of blood chemical examinations 

Exp.group Number of BUN Creatinine T-BU Ca IP lia K CI 


Sex (me/kif/day) aniaals (mg/dL) (mg/dL) (ag/dL) (lIg/dL) (mg/dL) (IlEq/L) (IIIEq/L) (JEq/L) 


Vehicle 

control 

5 8.6 

± 1.5 o 21 

±0.04 

0.06 

±0.02 

10.2 

± 0.2 

B.O 

±O.S 

144 

± 1 

4.1 

±O.2 

105.9 

± 0.9 

25 s 9.0 

± 1.8 

0.21 

±O.04 

0.06 

±0,02 

10.1 

± 0.2 

7.8 

to 6 1 

4.4 

to.3 106,8± 1,3 

lIale 

150 6 9.0 

1.6 

ZO 

Oll 

0.07 

±0,02 

10.1 

± 0,3 

8.2 

fl.1 

143 

± 1 

4.2 

fO.2 

105,5

± 0.9 

1,000 

Recovery 

Vehicle 

control 

I) 

5 

13,7*

± 4. i 

14.1 

± 1.7 

0.26 

iO. 04 

0.22 

±0.01 

0.10* to. 03 

0.06 

to.Ol 

10.0 9.2 143 4.3 107.1 ____L<Lg___...iQ..:_;!______L-l_____tLL______;__L.L_____ 

9.9 7.0 142 3.9 104.5 ± 0.2 to.3 ± 1 10.2 ± 1.2 

1,000 5 14.3 

! 1.9 

0.19** 

±0.01 

0.07 

±0.02 

9.6* 

± 0.1 

7.1 

fO.S 

143 

± 2 

4.2* 

±0.2 

104.9 

± 1.1 

Vehicle 

control 

5 10.2 

± 1.1 

0.23 

±O.01 

0.06 

to.02 

9.9 

0.1 

6.9 

to.5 

143 

± 1 

3.9 

±0.2 

107.9 

• 2.0 

25 5 11.4 0.24 0.04 9.7 6.8 143 3.8 109.4 

I 

0'> 

'" hllde 

150 5 

± 2.5 

10.2 

± 2.5 

±0.04 

0.21 

fO.03 

±0.01 

0.07 

±0.02 

± 0,3 

10.1 

i 0.4 

±0.5 

7,4 

±1.2 

± 1 

142 

± 1 

±0.2 

4.1 

to.3 

± 0.8 

107.2 

± 2.1 

I 1,000 5 11.1 0.26 0.09* 10,1 8,8u 141 

± 3.2 to.03 ±0.01 ± 0,3 fO.3 ± 6 fe-c-amy----------------------------------------------~-----------

4.0 

+0.3 •. 

104.5 

± 3.7 

Vehicle 

control 

5 17.0 

t 3.1 

0.28 

to.05 

0.09 

±0.03 

9.8 

0.1 

5.2 

±os 

142 

z 1 

3.7 

10,2 

106,4

± 1.5 

1,000 5 16.6 

± 3.5 

0.25 

±0.03 

0.09 

to.03 

9.7 

0.3 

6.1** 

fO,5 

142 

± 2 

4.0 

±0,5 

107. 1 

± Z. 5 

Hean ±S.D. , Significantly different from vehicle contrDl at P<0.05.

** Significantly different froll vehicle control at P<O.Ol. 

w 

w 

(J1 
-----------

Table 10-1 Twenty-eight-day repeated-dose oral toxicity study in rats 811-0908 

Sum.ary of urinalyses 

Exp.group }lumber of Urine volUllll Uosa 

Sex (llg/kg/day) animals (mL) (IIOsII/L) 

Hale 

Vehicle 

control 

25 

150 

1,000 

Recol'ery Vehicle 

control 

1,000 

5 13 

± 8 

718 

± 687 

5 4 1708 

2 ± 624 

5 5 1611 

3 ± 965 

.~ 15 

± 2 

567 

± 308 

5 9 

4 

997 

± 478 

5 14 751 

± 5 ± 401 

I 

-.:J 

0 

I 

Vehicle 5 5 1391 

control ± 4 ± 944 

25 5 10 845 

± 9 731 

150 5 5 965 

Female 2 292 

1,000 5 21 448 

t13 ± 325 

Recovery 

Vehicle 5 6 1302 

control ± 2 ± 678 

1,000 6 4 1513 

± 2 ± 518 

Hean ±S.D. * Significantly different fro. vehicle control at P<O.05. 

** Significantly different from vehicle control at P<O.OI. 

....._----------­

w 

w 

CJ) 
Tahle 10-2 Twenty--eight-day repellted-dose oral toxicity study in rats 811-0908 

Summary of urinalyses 

Exp.group Numbel' of Colo.- Turbidity pH Protein Glucose Occult blood 

Sex (mglkgfday) animals SY Y NT 5.0 6.0 6.5 7.0 ±1+2+ ± 

Vehicle 

control 

Z5 

5 

5 

3 

0 

2 

5 

5 

5 

.0 

0 

1 

J 

2 

2 

2 

0 

0 

0 

2 

0 

2 

2 

1 

3 

5 

5 

5 

5 

0 

0 

Male 

150 

1,000 

5 

5 4 

4 5 

5 0 

2 

3 

2 

2 

0 

0 

0 

0 

0 

2 

2 

3 

3 

0 

5 

5 

5 

5 

0 

0 

Recovery 

Vehicle 

control 

1,000 

5 

5 

3 

4 

2 5 

5 

0 

0 

0 

0 

3 2 

4 

0 

0 3 

4 

2 

0 

0 

5 

5 

4 

5 0 

Vehicle 5 2 3 5 2 0 3 0 0 2 2 5 5 0 

control 

25 5 3 2 5 0 2 2 2 5 5 0 

! 

-:t 150 5 4 5 0 2 3 0 0 0 4 5 5 0 ~ 

I Female 

1,000 5 5 0 5 0 2 2 2 2 0 5 5 0

-_....--.-­

Recovery 

Vehicle 5 4 5 0 " 0 0 3 5 5 0 

control 

1,000 5 4 5 0 0 4 0 3 Ii 5 0 

SY, Slightly yellow. 

y, Yellow. 

NT, No turbidity. 

(.,) 

(.) 

...... 
Table 10-3 Twenty-eignt-day repeated-dose oral toxicity study in rats B11-0908 

Summary ofurinalyses (Urinary sediment) 

Exp.group Number of Red blood cells·) White blood cells.l Epitnelial cellsa) Castsbl Crystalsc) 

Sex (mg/kg/day) 

Vehicle 

control 

25 

animals 

5 

I) 

0 

5 

5 

0 

5 

5 

1-5 

0 

0 

6-20 

0 

0 

0 

2 

3 

1-5 

3 

2 

6-20 

0 

0 

21-50

0 

0 

51-499 

0 

0 

0 

5 

5 

5 

2 

± 

0 

+ 

0 

2 

++ 

0 

0 

Male 

150 

1,000 

5 

5 

5 

5 

5 0 0 

3 

3 

0 

2 

0 

0 

3 

0 0 5 

5 

3 

5 

2 

0 

0 

0 

0 

0 

Recovery 

Vehicle 

control 

1,000 

5 

5 

5 

5 

4 

5 0 

0 

0 

3 

2 

2 

3 

0 

0 

0 

0 

0 

0 

5 

5 

3 

4 

0 2 

0 

0 

0 

Vehicle 5 5 5 0 0 0 5 0 0 0 5 1 0 0 

control 

25 5 5 3 2 0 2 3 0 0 0 5 5 0 0 0 

I 

-::. 

N 

I Female 

150 

1,000 

5 

5 

5 

5 

4 

3 0 

0 

2 

2 3 0 

2 

0 

0 

0 5 

5 

4 

5 

0 0 

0 0 0 

-_...._----­

Recovery 

Venlcle 5 5 2 3 0 4 0 0 0 5 1 0 0 

control 

1,000 5 5 4 0 0 5 0 0 0 5 2 2 0 

a) Number or cells/IO views (X 400). 

b) Number of cllsts/t8xIS mm 2

• 

c) Incidence ofcrysUlls/18x18 mm 2

• 

w 

w 

(X) 
Table 11 	 Tventy-eight-day repeated-dose oral toxicity study in fats Bll-0908 

Sunary of absolute organ weights 

Sex h:p.group Nuber of Liver Heart Kidney Testis Epididymis Ovary Brain Spleen ThYJllus Adrenal !lady weight a) 

(ag/kg/day) animals (g) (g) (g) (g) (g) (mg) (g) (g) (ag) (mg) (g) 

5 11.49 1.18 2.40 3.01 	 1. 97 0.65 608.8 50 1 353 8 

± 1. 21 ± 0.02 0.10 0.38 0.03 0.13 0.10 126.2 7.0 25.6 

25 5 10.69 1.13 2. '19 3.15 O.BO 1. 96 0.62 605.4 47.9 338.2 

± 0.91 0.08 ± 0.32 ± 0.17 ± 0.05 ± 0.03 0.06 89.1 3.2 30.0 

150 5 10.92 1.16 2.36 3.00 0.75 2.00 0 57 507.7 42.5 333.3 


Hale ± 1.76 t 0.15 t 0.30 ± 0.06 0.07 ± 0.07 011 168.2 60 52.1 


1,000 11.14 1.07 2.71 3.11 0.74 1.84 0.48 373.2* 41.9 286.1 
 _____ ._______________LL.Q1__L9..!.q!L______LU~____i.J~.:J§____L.Q.&~___________: _______.Ut&L___LJL\.~_____.L_'!.~.:_L____L __ii_c.L___ i._l~.·L____ _ Recovery 

Vehicle 10.96 1.34 2.82 3.27 1.10 2.13 0.68 529.6 53.6 399.8 

control ± 0.63 0.09 ± 0.14 ± 0.23 ± 0.08 0.09 0.08 90.S 4.0 24.6 

1,000 5 10.54 1.28 2.98 3.10 0.93** 1.99* 0.62 452.3 47.1 367.0 

± 1.66 0.15 ± 0.24 ± 0.19 ± 0.06 0.03 0.11 ± 119.9 8.8 36.3 

Vehicle Ii 6.41 0.81 1.56 76.1 1.82 0.42 520.2 58 8 212.3 

cOlltrol 0.57 0.10 0.11 11 a 0.11 0.06 + 53.5 4.3 16.4 

25 5 5.93 0.73 1. 45 70.9 1. 85 0.37 485.1 63.1 201. 6 

I 0.68 0.09 0.22 8.9 0.12 0.06 105 4 7.2 17. 4 

-J 

CO 

I Female 

150 5 7.15 

0.72 

0.82 

± 0.07 

1. 61 

0.12 

76.9 

7.0 

1. 86 

0.05 

0.43 

0.06 

431.6 

76.9 

686 

14.0 

210.1 

11.9 

000 5 9.30n 0.85 2,05*& 79.4 1.84 U.44 373.2* 60.8 213.0 :-:=-~~__---.LLIL----J:....Q..:.QI------.t-I!.~~-~----------.-:---- _______=_______.L.ILL___L.9..!.Q.L___t.-'LI!.1____L..lLL_____;.._.!.9_c.L_.__L1L_"-_____ Recover)' 

Vehicle II 5.92 o. 1. 55 76.9 1. 88 0.46 413.3 59.8 220.7 

control 0.G3 0, 0.09 ! B.6 0.06 0.02 i 57. 4 ! 6.1 10.4 

1,000 5 6.70* 

0.55 

0.93* 

0.10 

1. 76 

± 0.20 t 

63.4 

7.6 

1. 85 

0.05 

0.52 

0.06 1 

424.9 

54.0 ! 

66.4 

7.5 ! 

6 

11. 7 

Mean ±S.D. 


a) Statistical allalysis was not applied. 
 * liignificantly different fro. vehicle control at P<O.05. ** Significantly different from vehicle control at P<O.OI. 

(,,) 

(,,) 

co 
Table 12 Twenty-eight-day repeated-dose oral toxicity study in rats 81 H}908 

Suuary (If relative organ weights 

Sex Exp.group Nuber of Liver Heart Kidney Testis Epididyais Ovary Brain Spleen Thymus Adrenal Body weight .) 

(Ig/kg/day) animals (,/100g) (g/100g) (g/100g) (&/100g) (g/100g) (lIg/lOOg) (,/100g) (g/IOOg) (ag/IOOg) (mg/IOOg) (I) 

Vehicle 5 3.24 0.34 0.68 0.85 0.21 0.56 0.18 171.9 14.1 353.8 

control to.lS 10.03 ±0.04 ±0.07 !0.01 10.03 to.03 1 33.1 1 4 25.6 

25 5 3,17 

to.28 

0,34 

to.03 

0.73 

±0.03 

0.94 

±0.09 

0 24 

±0.01 

0.58 

!0.05 

0.19 

±O.OZ 

178.7 

± 16.7 ! 

14.3 

2.0 i 

338.2 

30.0 

150 5 3.28 0.35 0.71 0 92 0.23 0.61 0.17 151 6 12.8 333.3 

Male to 09 to.03 to.09 iO.13 ±0.04 ±0.08 fO.03 ! 42.7 1.4 52.1 

1.000 5 3.90** 0.37 0.95** 1.09** 0.26 0.65 0.17 130.4 14.6 2.86.1 __________________________tQ.,,1,L_____..tJ!!.!l.L______HL_Q.L__-..J.!L1L_____ l~_·_Q.L __________..::______1Q".QL ______ j9_&L______l.llJL___ L.Q.,_~ _____••L_~JLl_____ 

~-~ . Vehicle 5 2.74 0.34 0.70 0.82 0 28 0.53 0.17 132.0 13.5 399.8 

contl'ol iO.12 ±0.03 ±0.05 ±0.10 to.03 to.04 to.Ol ± 16.8 1.3 t 24.6 

1,000 5 2.86 0.35 0.81** 0.85 0.25 0.55 0.17 122.2 12.8 367.0 

±0.19 to.02 ±0.04 iO.09 10.02 ±0.06 to.02 ± 25.7 1.5 36.3 

Vehicle 

control 

5 

13 

0.38 

iO.02 

0.74 

±0.06 

36.2 

± 6.6 

0.85 

to.08 O.~O to.04 

245.0 

~ 17 S 

27.9 

3.4 

212.3 

16.4 

I 

....::l

..,. 

I Fe.ale 

25 

150 

5 

6 

2.94 

±0.21 

3.40 

±0.27 

0.36 

±0.04 

0.39 

±O.OZ 

0.72 

!0.06 

0.77 

±O.OS 

35.4 

5.4 

36 5 

1.8 

0.92 

to.03 

0.89 

to.03 

0.18 

iO.02 

0.20 

±0.03 

241.0 

± 48.9 

205.4 

t 33.4 

31.3 

1.7 

3Z.6 

6.1 

201.S 

17.4 

210 1 

11.9 

1,000 

Recovcry 

Vehicle 

control 

6 

5 

4.36:,,* 0.40 0.96** 37.7 0.67 0.21 175.7** Z8.7 213.0 !Q.:JJL.__.__.t.Q~QL___._H)_J11L_____._.__..::______________:____.____J ... Lp_______1Q"J1.L______J 0 J1.L._.___.LJjU._._.._~___L!______L_JLL__._. 

2.69 

10.06 

0.36 

±0.02 

0.70 

10.05 

34.8 

3.4 

0.85 

±0.06 

0.21 

iO.02 

187.9 

± 29.6 

27.1 

t 3.1 

220.7 

t 10.4 

1,000 5 2.93** 

±O .13 

0.41* 

to.03 

0.77 

to.08 :I. 

36.6 

3.9 

0.81 

to.06 

0.23 

±0.02 

185.7 

t 19.8 

29.1 

± 3.6 1: 

2Z8.6 

11 7 

Mean ±S.D. 


a) Statistical analysis was not applied.
 * Significantly different from vehicle control at P<0.06. '* Significantly different fro. vehicle control at P<O.OI. 

(,.) 

~ 
811-0908 

Table 13 Twcnty-eight4ay repeated-dose oral toxicity study in rats 

Summary ofmacroscopic examlnations~ _~__ 

Male female 

Findings 

Vehicle 

cootrol 

Vehicle 

control 

(Recovery) 

25 150 1000 

, 1,000 

(Recovery) 

Vehicle 

control 

Vehicle 

25 150 1,000 

1,000 

(Recovery) (mglk.glday) 

ta ta ta ta ta ta ta 18 ta ta 

5') 5 5 5 5 5 5 5 S S 5 5 

No abnormalilies detected 5 5 5 4 0 S 5 5 5 .3 0 S 

OlaDdWar stoJll8Cb 

Blackish region of mucosa 

Jejunum 

Diverticulum 

() 

0 

0 

0 

0 

0 

0 0 

0 

0 

0 

0 

0 

0 

0 

0 

0 0 

0 

0 

0 

0 

Liver 

I 

-..J 

01 

EnJarpneat 0 0 0 0 2 0 0 0 0 

IGdney 

Enlargement 0 0 0 0 5 0 0 0 0 0 

S 0 

4 0 .......................................................................................... "' .. __.............. _ ..__ .....a ............................................. '" ............................................ '" __ ........ _ ................................. - ...... - ....... _ ...... .......... "',. .......... 

Pelvic dilatation 0 0 0 0 0 0 0 0 0 0 0 

Skin 

Loss ofbair 0 0 0 0 0 (') (') 0 () 0 0 

ta, terminal autopsy. 

a) Number ofanimals examined. 

w

.:.. 
B 11-0908 

Table 14-1 	 Twenly-eight-day repeated-dose oral toxicity study in rats 

Summary ofhistopathological examination:; 

Mt.lc Female 

Findings Grade 

Vehicle 

control 

Vehicle 

control 

(Recovery) 

25 150 1,000 1,000 

(Recovery) 

Vehicle 

control 

Vehicle 

control 

(Recovery) 

25 150 1,000 1,000 Ikgld) (Recovery) (mg ay 

ta la ta ta ta ta ta ta ta ta !a la 

5a) S 5 5 5 5 5 5 5 5 5 5 

Trachea 

No abnonnalities detected 515b

) 515 515 515 

Lung 

No abnormalities detected 5/5 515 515 4/5 ...........--. _:-- ..............._ ••_ ..........__..... ., .................__....................., ..................... _ .. _ .......... _ ............................................................................. _ ..................... 'Ol • ..._ ..................................... 


Focal inflammation + DIS 	 0/5 0/5 115 

Forestomach 

No abnormalities detected 5/5 515 SIS 515 

I Glandular stomach -J 

m No abnormalities detected 515 	 5/5 515 011 5/5 

I ......................._..........................-.........'..... "'-.................-............................."'...................................................................... "'..........--_.............................................................................................. ... 
 Necrosis offundic mucosa -+ 0/5 	 015 015 iii Oi5 

Duodenum 

No abnormalities detected '5/5 5/5 S/5 515 

Jejunum 

No abnonnalities detected 515 011 5/5 SIS 515 ...................- ..................................,........................ _ ...................................................................................III _ ............ _ ..............._ ....-..................................._ ............................ ", ...... "' ................ - .. 


Diverticulum 0/5 Ifl 0/5 015 0/5 

Ileum 

No abnormalities detected 515 515 515 515 

Cecum 

No abnormalities detected 5/5 515 515 5/5 

Colon 

No abnonnalities detected 515 515 515 SIS 

Rectum 

No abnormalities detected 515 515 515 5/5 

C.J ta, terminal autopsy, ..c=. 

N 	 a) Number of animals autopsied, 

b) Number of anima,ls affi:tted I Number of animals examined. 

-, Not examined. 

+, slight. 
••••••••••••••••••••••• 

BII·0908 

Table: 14-2 Twc:nty-e:ight-day repeated-dose oral toxicity study in rats 

Summary of histopathological e.xaminations 

MahI Female 

Findings Grade 

Vehicle 

control 

Vehicle 

control 

(Re ) covery 

25 150 I 000 

• 1,000 (Recovery) 

Vehicle 

I contro 

Vehicle 

control 

(Recovery) 

25 150 1,000 1000 . ' (mglkg/day) (Recovery) 

ta ta fa Ia la ta Ia II la ta III 18 

51) S S 5 5 5 S S S S S S 

Uver 


No abnormalities detected 51Sb) 515 315 415 3/5 515 515 515 5/5 515 3/5 5/5 


Diffuse fatty change of + 015 015 DIS 015 115 0/5 0/5 015 0/5 0/5 0/5 015 

.~:~;~.................................................-'" .........................-_.............................................., 


Fatty change ofperiportal + 0/5 015 0/5 0/5 0/5 015 015 0/5 0/5 0/5 2/5 0/5 

.~~PJ5?:~~_•.•.............................................................................................._•.•.••...••••... 0 ••••••••••••••••••••_ 

Focal necrosis of hepalocyles + 0/5 0/5 115 0/5 0/5 0/5 0/5 015 015 0/5 0/5 0/5 ............. __...-_.. "" ..................-........................................................................................... -~-.- .......................................................--........................... __ ................... "' ............ _........ 
 I Microgranuloma + 0/5 0/5 1/5 liS 115 0/5 0/5 0/5 0/5 0/5 115 0/5 

-l 

-l Heart 

I No abnormalities detected SIS 51S 515 5/5 515 515 

Kidney 

No abnormalities detected 515 515 4/5 515 5/5 515 4/5 5/5 3/5 515 .................. ---...................................... _-.... __ .. __ ............ '"' .... ~ .............. _................................... _...... _- ................................................... _.. -............................... -----_ ............................. -................ -. ---_ ........... ~"' ... 


Degeneration and necrosis of 


tubular epithelium with + 0/5 015 0/5 0/5 0/5 0/5 0/5 0/5 215 0/5 


regenerative epithelium 
 ..................................................-........................-........................._-_._....--........................................................-...................................-........................................................... _....................................-.. 
 Pelvic dilatation, unilateral + 0/5 0/5 	 115 0/5 0/5 0/5 0/5 0/5 0/5 0/5 ....................__ ........................................... " ........ _ .... ......._..................._ ............................................................ _ .................... __ ................................................................................... "' •• eo 


Solitary cyst in medulla + 015 0/5 	 0/5 015 0/5 0/5 1/5 015 0/5 015 

Urinary bladder 

No abnormalities detected 5/5 5/5 51S 5/5 

Testis 

No abnormalities detected SI5 515 

Epididymis 

No abnormalities dt:tected 5/5 515 515 SIS 

18, terminal autopsy. w 

.j:>. 	 a) Number of animals autopsied . w 	 b) Number of animals affected I Number ofanimals examined. 

-, Not examined. 

+, slight 
011-0908 

Table 14-3 Twenty-eight-day repeated-dose oral toxicity study in rats 

Summary ofhistopatbological examinations 

Male Female 

findings Grade 

Vehicle 

control 

Vehicle 

control 

(Recovery) 

25 150 1,000 

1,000 

(Recovery) 

Vehicle 

cootrol 

Vehicle 

cootrol 

(Recovery) 

25 150 1,000 1,000 (Recovery) (mglkglday) 

ta fa ta ta ta ta ta la ta ta la ta 

5") 5 s s s s 5 5 !i 5 j 5 

Prostate 

No abnormalities detected 515b) 515 

SemiDaJ 'RiSicle 

No abnormalities detected 515 5/5 

Ovary 

No abnormalities detected 515 515 

Uterus 

I No abnormalities detected 515 5/5 

--:J 

00 

I 

Vagina 

No abnormalities detected 515 515 

Cerebrum 

No abnormalities detected 515 515 515 515 5/5 5/5 

Cerebellum 

No abnormalities detected 515 515 5/5 515 SIS 5/5 

Pons 

No abnonnalities detected 515 515 515 515 515 515 

Spinal cord 

No abnormalities detected 

Sciatic nerve 

5/5 515 515 SIS 

No abnormalities detected 515 5/5 515 515 

Bone marrow 

No abnormalities detected 5/5 515 515 515 

t.) 

:t 

ta, terminal autopsy. 

a) Number ofanimals autopsied. 

b) Number of animals affected I Number ofanimals c)(amined. 

-. Not e'l:amined.