### K06-1279 

### 212
The inspection result of following phase was reported to the study director and management based on the report ofprocess-based inspection relevant to this study type and timeframe.**--"-,~,** 

### Phase Date ofInspection Date Reported to Study Director and Management Examination of Solubility of the Test Substance October 10,2008 April 14, 2009 Preparation and Management ofPositive Control Substance November 26, 2008 April 14,2009 Preparation ofMedium and Reagent December 12, 2007 April 14,2009 Management ofCells 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 ofthe 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 OFCONTENTS Page TITLE
.............................................. 

### 3 SPONSOR
.............................................. 3 TESTING FACiLITY
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 PURPOSE OF STUDY
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 TESTING MET! 

### [00
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 GLP COMPLIANCE
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

### 3 PERIOD OF STUDY
........................................ 4 

### STORAGEANDRETENTION PERIOD OF DATA
. . . . . . . . . . . . . . . . . . . . 

### 4 RETENTION Of ORIGINALDOCUMENTS
. . . . . . . . . . . . . . . . . . . . . . . . 

### 4 STUDY DIRECTORAND PERSONS CONCERNED WITH THESTUDYAi'lD THE OPERATION
................................ 

### 4 APPROVALBYAUTHOR
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
SUMMARY 

### 6
MATERIALS AND METHODS 

### 1. TESTSUBSTANCEAND POSITIVE CONTROL SUBSTANCES 7 2. CELLS 

### ................................ 9

...... ..... .......... .... ....... 9 

### 3. MEDIUMAND S9MIX

....... " . . .• . . • . . .• . . .• . . .. . . . • 10 

### 4. CELL PRE-CULTURE 5. PREPARATION OfTESTSUBSTANCE SOLUTION AND POSITIVE CONTROL SUBSTANCE SOLUTIONS
............ 10 

### 6. TESTPROCEDURE
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 7. JUDGEMENTCRITERIA OF RESl;LTS
.. . . . . . . . .. . . . . . . .. . . . . . 14 

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



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

### 1. CELLGROWTH INHIBITION TEST
. . . . . . . . . . . . . . . . . . . . . . . . 15 

### 2. CHROMOSOMALABERRATION TEST
........................ 15 3. TYPICAL PHOTOS
. . . . . . . . . . . . . . . . . . . . . . . . 17

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



### . . . . . . • . . . . . . . . . . . . . . . . . . . . . 18 REFERENCES ]­ 

### 146

### K06-1279

TABLES, FIGURES 

### AND PHOTOS Table 1 Results ofcell growth inhibition test of KarenzMT (Mixture ofPEMB and PE3MB)**~** .. • • • * • • • '" • • • • .. .. 

### 19 Table 2 Results ofchromosomal aberration test of KarenzMT(Mixture ofPEMB and PE3MB) 

### ·

## . .. 

## . . ... 

### . .. .. 

### .**~** .. " 

## .**~** 

### 20 Table 3 Results ofchromosomal aberrationtest (short-tenntreatment without S9 mjx) .... '" .. .. .. 

### .. .. 

# . ...**~** ........ 

### 2] Table 

### 4 Results ofchromosomal aberrationtest (short-tenntreatmentwith 89 

### mix) 

## · .. ..**~** ",**~** 

## . .. .. 

# . ,,-.. .. 

# . . . 22 Table 5 

### Results ofchromosomal aberration test (continuous treatment) 

### .**~ ~**  

## . 23 Fig. 

### 1 Results ofcell growth inhibition test of Karenz MT (Mixture ofPEMB and PE3MB) 

### ·

## ................ 24 Fig. 

### 2 eel1 growthrate in chromosomal aberration test of KarenzMT (Mixture ofPEMB and PE3MB)
... .. ....... .... 25 Fig. 

### 3 Results ofchromosomal aberration test in short-tenn treatments of Karenz MT(Mixture ofPEMB and PE3MB) . . . . . . . . . . . . . . . . 26 Fig. 4 

### Results ofchromosomal aberrationtest in continuoustreatment of KarenzMT (Mixture ofPEMB andPE3MB) 27 Photo 1 Normal. cell 28 Photo 2 Structural aberration induced by KarenzMT(Mixture ofPEMB and PE3MB)'
. . . . . . . . . . . . . . . . . .. 28 Photo 3 Structural aberration induced by KarenzMT (Mixture ofPEMB 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 ShowaDenko 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).
TESTINGMETHOD 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 ofthe Pharmaceutical and Food Safety Bureau, MHLW, No.2 (November 13, 2(03) ofthe 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 ofthe Pharmaceutical and Food Safety Bureau, MHLW, No. 3 (November 17, 2003) of the Manufacturing Industries Bureau, METl & 

### No. 031121004 ofthc EnvirollmentalllealLh Department,MOE (Novemher21,2(03).

- 3 148

### K06-1279

PERIOD OF STUDY Commencement ofStudy: November 26, 2008 Initiation ofExperiment (Initiation ofCell Growth Inhibition Test): December 1, 2008 Completion ofExperiment (Completion ofObservation ofSpecimens): March 17,2009 Completion ofStudy: April 

### 15,2009
STORAGEAND RETENTION PERIOD OFDATA The raw data, protocol, protocol amendment, letter oftest request, table oftest substance information, final report, other record documents and specimens will be stored 

### in the archive ofthis testing facility for 10 

### years after the date ofreceipt ofthe 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 ofreceipt of the notification to this 

### testing facility. After termination of the retention period, anymeasures taken will bc done so withthe 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 ORIGINALDOCUMENTS An original protocol, original protocol amendments and an original final report 

### will be retained at the testing facility. The copies oftheir original that the studydirector 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 andcell treatment) Shozo Ogura (Microscopic observation ofspecimens) Junko Kawaguchi (Microscopic obscrvation ofspecimens)

4 ­ 

### 149

### K06-1279

Chisato Iwami (Microscopic observation ofspecimens)
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 24hours 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 ofcells with structural aberrations andnumerical aberrationcells were scored. As a result ofobservation ofspecimens, the frequencies ofnumerical aberration cclls showed below 5% at all observation doses ofthe test substance in all treatment methods, therefore, numerical aberration wasjudged to be negative. The frequencies of cells with structural aberrations showed below 5% at all observation doses ofthe 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 10% in 24 hours continuous treatment. The frequencies ofcells with stmctural abelTations at 9.77, 13.8 and 19.5**~g/mL** 

### oftest 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 oftest substance was about 50% and the frequency ofmetaphases 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 wasjudgedto be positive in 24 hours continuous treatment. On the other hand, the frequencies ofcells with structural aberrations ornumerical 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 ofthe present study. it was concluded that Karenz MT (Mixture ofPEMB 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, permissiblerangc: 10-30°C) and to avoid contactwithoxidizing 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 ehamberbecause ofits characteristic odor. 1.2 Positive Control Substances 

### 1) MitomycinC (MMC) Manufacturer: Kyowa I1akko Kirin Co., Ltd. LotNo.: 518AGJ Appearance: royal purplepowder Content: 

### 102% Grade: for injection 2) Cyclophosphamidemonohydrate (CPA) Manufacturer: Wako Pure Chemical Industries. Ltd. LotNo.: WKL2245 Appearance: white crystals orcrystallincpowder 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 inthe 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" onJapanese 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 

### underhumid condition. 2.4 Subculture Cells were subcultured in 90-mm diameter Petri dishes (Nunc 

### A/S) twice a week. Passage number ofcells 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 wasused within six months afterthe day ofmanufacturing. 2) Composition ofS9 mix One milliliter of S9 mix consisted of0.3 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 justpriorto 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 ofTest 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 ofsolvent 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 ofthe test substance in the medium were prepared with the solvent. It wa') 

### prepared without correcting purity ofthe test substance because the purity ofthe 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 afterpreparation. 5.2 Preparation ofPositive 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 afterpreparation.
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 ofthe test substance solution or the solvent and 3 mL ofthe 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 of0.5 

### mT. of S9 mix and 30 ilL ofthe 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 ofDulbecco'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 ofthe test substance solution orthe solvent and 5 mL ofthe fresh medium. In the short-tenn and the continuous treatments, 50 IJ.L ofa 10 I1gimL demecolcine solution was added to eachdish at 2hours before the end ofthe culture. Atthe startand the end ofthetreatment and atthe 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 preparcdto collect from each dishby a treatment with 2 mI. of0.25 

### w/v% trypsin. After 200 ilL ofthe cell suspension was diluted with 10 mL ofCell Pack (Sysmex Corporation), the number ofthe 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 ofbelow 50% and the other was the next lowerdose ofthis dose. Leftover cells were collected 

### by a centrifugation at 1000 rpm (l85x

### 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 ofthe suspension were placed on a glass slide, and stained about J

### 5 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 ofno 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 of2, respectively. Duplicate dishes were used ror each dose. 

### J) Observation and scoring Specimens were observed to check the presence or absence ofmitotic 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 smallerthan the widlh ofone chromatid. (2) Numerical aberration The number ofcells showing triploid ormore was scored. Chromosomal Aberration Test I) Procedure Chromosomal aberration test was carried out using the same procedure as that ofthe cell growth inhibition test, with the following positive controls. Four specimens perdose (two speCImens 

### per d' IS

### h) were prepanx . I Treatmentmethod 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 ofa 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. ofa 0.01 mg/mL MMC solution forthe 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 ofthc 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. 

### lCsowere 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 5lower doses were sctbased on a geometric progression of.J2
. Treatment method 

# I Setting doses oftest substance Short-term treatment without S9 mix i 

### 6.9L 9.77, 13.8, 19.5, 27.6, 39.1 

### and55.2J1gfmL with S9 mix I 

### 78.L 110, 156, 22L 313 

### and442J1g1mL 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 ofthe 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 observationdoses ofthe test substance. Treatment method Observation doses oftest substance Short-tenl1 treatment without S9 mix 19.5,27.6and 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 ofaberrant 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 ofcells with structural aberrations and the number ofaberrant 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 ofpolyploid (cell with 38 or more chromosomes) cells among 200 metaphase cells perdose (50 cellsper specimen) wererecorded. 6.3 Confirmation lest In the chromosomal aberration test for 24 hours continuous treatment, the structural aberration wasjudged to be positive obviously, therefore, a confirmation test was not conducted.


### 7. JUDGEMENT CRITERIA OfRESULTS The tindings werejudged to be positive when the frequencies of cells with structural aberrations showed 10% or more with a dose-related increase or the frequencies ofcells with structural aberrations showed 5% or more both in the chromosomal aberration test and the confirmation tcst. The other cases werejudged to be: negative. No statistical analyses were used. Thc frequency ofnumerical aberration cells wasjudged according to the same criteria as that of the structural aberration. D10 

### value indicating a concentration that chromosomal aberration was observed in 20% ofcells was calculated in the treatment method that the frequency ofcells with chromosomal aberrations were 5%ormore.
8. VALIDITY

# or TEST This study wasregal"ded as valid asfollows: 1) the frequencies ofcells 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 ofcells with structural aberrations excluding gaps in the positive controls showed 20%ormore.

14 -­ 

### 159

### K06-1279

FACTORS AFFECTED RELIABILITY OF TEST
There were no factors, which might affect the reliability ofthe lest.
TESTRESULTS
I. 

### CELL GROWTH INHIBITION TEST (Table 1and 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 ormore at the start ofthe treatment, at 156 I1g/mL or more at the end ofthe treatment and at 2500 I1g/mL or more atthe end ofthe culture. In the shortterm treatment with S9 mix, precipitation ofthe test substance was observed at all doses at the start ofthe treatment, at 313 I1g/mL or more at the end ofthe treatment and at 2500 I1g/mL or more at the end ofthe culture. In 24 hours continuous treatment, precipitation ofthe test substance was observed at 78.1 I1g/mL 

### or more at the start ofthe treatment and at 156 I1g/mL 

### or more at the end ofthe treatment. The color change ofthe medium and the eorrosion ofthe culture dish were not observed at any doses. As a result ofthe observation of specimens, the maximum frequencies ofthe cells with structural aberration were 10.0%in the short-term treatment without S9 mix and 24 hours continuous treatment. The frequencies ofnumerical aberration cells were below 5% at all observation doses ofthe test substance ineach 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 ofthe 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 ofthe test substance, color change ofmedium and corrosion ofculture dish Precipitation ofthe test substance, color ehange ofthe medium and the corrosion of the culture dish were not observed at any doses. (3) Frequency ofeells 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 ofcells 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 ofnumerical aberration cells The frequencies ofnumerical aberration cells were below 5% at all doses including the negative andpositive controls, therefore, the results werejudged to be negative. 2) 

### With 89 

### mix

(1) 

### Cell growthrate 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 calculatedat270**~lglmL.** 

### (2) 

### Precipitation ofthe test substance, color change ofmedium and corrosion ofculture 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 ofthe culture dish were not observed at any doses. t)) 

### Frequency ofcellswith 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 ofcells 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 werejudgedto be negative. (4) 

### Frequency ofnumerical aberration cells The frequencies ofnumerical abenationcells were below 5% at all doses including the negativc andpositive controls, therefore, the results werejudged to be negative. 2.2 Twenty FourHours Continuous Treatment (Tables 2,5 and Figs. 2,4) 1) 

### Cell growthrate 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 1Csowas calculated at 18 )..lg/mL. 2) 

### Precipitation ofthe test substance, color change ofmedium and corrosion 

### ofculture dish Precipitation ofthe test 'substance, colorchange ofthe medium and the corrosion of the culture dish were not observed at any doses. 3) Frequency ofcells 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 ofcells 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 adose related increase was observed in 4.&8 and 6.91 J.lg/mL and the maximum frequency was over 10%, therefore, the results werejudgcd to be posItIve. The dose that the frequencies ofcells with structural aberrations showed below 5% was 4.88**~lglmL.** 

### 4) Frequency ofnumerical aberration eells The frequencies ofnumerical aberration cells were below 5% at all doses including the negative and positive controls, therefore, the results werejudged 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 ofcells 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 appropriatelyperformed. As a result ofthe chromosomal aberration test, the frequencies ofnumerical aberration cells were below 5% at all doses ofthe test substance in the short-term treatments without and with S9 mix and 24 hours continuous treatment, theretore, numerical aherration wasjudged to be negative. The frequencies ofcells with structural aberrations showed below 5% at all observation doses ofthe 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 ofcells with structural aberrations was over 10% in 24 hours continuous treatment. The frequencies ofceJls with structural aberrations at 9.77, 13.8 and 19.5 J.lg/mL oftest 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 oftest substance, it was considered that the the appearance ofaberrant 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 wasjudgedto 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 ofchromosomal aberration test 

### in vitro. Revised edition, 1998 (inJapanese). Life-science Information Center. 2) Japan Environmental Mutagenicity SocietylMammalian Mutagenicity Study Group (ed.) (1988) Atla., ofchromosomal aberration induced 

### with chemical substance (in Japanese). Asakura Publishing Co., Tokyo.

-- 18 163

### K06-1279

Table I Results ofcell growth inhibition test ofKarenzMT (Mixture ofPEMB 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 ofmetaphases was relatively low, lewmeta: the fn:quency ofmetaphases was extremely lew, no l1leHl: metup]'ose5 were 1I0tobservt.!d a) Precipitation ofthelest substance: -. absence; +, presence. b) The rrequC'IICY ofcells with dm'nlosomal aberrations was calculated by observating 50 metaphase, per dose. The highest dose was sel at 5000 flglrnL ofthe leSI substance, as the maximum dose in case ofno C),10loxicil), on the guideline: the do!'", levels based on ageoll1etric progn;ssicHI of2w;.:rc**s",lccl~d.**



### 19 164

### K06-1279

Table 2 Results ofchromosomal aberration testofKarenzMT(Mixture ofPEMB and PE3MB) Preclpitalion onest F"equency ofcells 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 ofcells with cnromo50mal anerraliol1, was cakulated by obs<',yuting 20() metaphasesper dose.



### 20 165Table 3 Res.ults of('hfOrnOS()Ill(ti abcrratlOIl test**\:;hon~le0111reatmeal** without 59 mix) Nnl11e "fr.51**~ut\,mlflj)e:** K"ellzMTiMlxture 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! numberofthem was shtn.\r at tbe lhird**:1Ti~,** Cell**~rowlll** ralo 81**~ach** d"h "ere5ilO\\11 311lie first and second liues Theaver.'ge ofthem was shown at the thirtllhe, D05e**i~g!mL)** Numberofcelts w11h structmal chromotiP1n::J.1 aberrations**{frcqlJency~/o)** NllJnper ofct11s 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.USat 6.91, ? '7. 13 8 and 55.l"i-1mL were nO! observed.**~** o C» 

### 'r' ..­ C» "-J \0 IVTable 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** ofcelhi \\It:l nuntcflc31 chrolHosomal ali;:rratlcns**~h;mbcf'** ofcells \\"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'f0: 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 ofthem 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 ofIhe 1("sl slIb51i1n.::c was observed at all**dD$t:~** lit Ihe !)tan ('Ifthe tr..;:a1mcl1t and at 150 itg/mL or**mm~.at** the end ofthe 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 '-0Table 5 Results ofchro010SQmaf aberration test (continUOus trcanncnlj**~a',1Z** oflest substance Knrcnz MT IMIXillre ofPEMB and PE3MB) K06·1279 XUll1bcr ofce!ls: with omnericai**chf(1mo~of'l1al** aherrations l\umberofcells 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 Ilew melaph",e,) 34..i.l...._ (I :'4 - 0 (MMC) () O,(lS 

### o () i 1),0)' (\ 0) OJ Tn:almenl**tI1TH~$** compnsed tremr.lelU-lime and recovery-lime The nWT!ber01 a:>eITant**l~ils** at cach dish Were shown at tho fits! and second Ilncs, The 10131 number ofIhem was ,how at the third line,**Cdll~ro\\1:)** ra:e :ll**~ach** dish wert: Sh(Win at ihe first :!\nd st!cofH.llincs, The av(xage (.'Ifthem was shown;).t the**th~rd** DMSl)' Dllnethyl slIlfoXldc WvlC: Mi10mycin C few met;Jphnsl'':9' til;; frc(IUenCy**ofmu(apha~l'::$ '\'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-tenntreatment 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 continuoustreatment Fig. 1 Results ofcell growth inhibition test ofKarenz MT(Mixture ofPEMB 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-termtreatment with S9mix

100

,-..**~ ~**  C>**~**

### ~ 

## so**~** -;:; 

### w

0 JO 100 Dose (!is/inL) 24 hours continuoustreatment Fig. 2 Cell growth rate in chromosomal aherralion test ofKnrenl MT (Mixture ofPEMB and PE3MB)



### - 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 S9mix



### o numerical aberration • structural aberration

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

## o 156 221 313 Dose (J,lg/mL) 

### With 89 

### mix Fig. 3 Results ofchromosomal aberration test 

### in short-termtreatments ofKarenz MT(Mixture ofPEMB 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 hourstreatment Fig.4 Results ofchromosomal aberration test in continuous treatment ofKarenz MT(Mixture ofPEMB and PE3MB)



### 27 .­ 172

### K06-1279



### Photo 1 Nonnal 

## cell Negative control in24hours continuoustreatment

Photo 2 Structural aberration induced 

### by Karenz

### MY (Mixture ofPEMB and PE3MB)

13.8 ,ug/mL in 24 hourscontinuous treatment a: chromatid break 

## 28 173

### K06-1279

Photo 3 Structural aberration induced by Karenz MT (MixtureofPEMB and PE3MB) 9.77 JlglmL in 24 hours continuoustreatment b: chromatid exchange

- 29 ­ 

### 174PAGE 1 OF 20 PAGES



### Mixture ofPEMB and PE3MB: LOCAL LYMPH NODEASSAY IN THE MOUSE PROJECT NUMBER: 

### 0146/0200

AUTHOR: A Pooles

STUDYSPONSOR: 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) 1332792896 Facsimile: +44 (0) 1332799018 0145'()200.doeJJS

175PROJECT 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 ofthe routine and repetitive procedures that constitute the study is carried outas a continuous process designed to encompassthe major phases at or about the time this study was in progress. In addition, inspection of general facilities not specifically related tothis 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

## ofthe data generated and ofthe proceduresfollowed.
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 appropriatetothetype and schedule ofthis study were asfollows: 12 November2008 Standard Test Method Compliance Audit 16 February 2009 Test Material Preparation 23 February 2009 Test System Preparation 19 February2009 Animal Preparation 16 February2009 Dosing 19 February2009 Assessment of Response 

### § 19 March 2009 Draft ReportAudit 

## § Date ofQA

### Signature Final ReportAudit 

## § Evaluation specificto this study

# ~.J-\,Vb~~ . 2 5 MAR 2009



# ... ,

# ........**~**

# ..................... 

# ...... "

## ........................ 

# ....... 

# ................... DATE

.................................................**~ ~.~** **~ ~**  

### Forthe QualityAssurance Unit*

*Authorlsed QA

### Signatures: 

### Deputy Head of Department JM Crowther MIScT MROA SeniorAudit

### Staff: GWren ONC MROA



### 176PROJECT 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 reportfully 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 adigital (pdt) document. Such documents are prepared byscanning the paper onginal,

and are considered ofequivalent integrityand authenticityto versions produced by optical photocopy. However, in all cases the hand-signed paperoriginal, held in secure archives, Isthe definitive document.



### 177PROJECT NUMBER: 014510200 PAGE 4



### QUALITYASSURANCE 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 ofTest 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 Studyforthe Local Lymph Node Assay 17 Appendix 2 Summary of Positive Control Data forthe 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

178PROJECT NUMBER: 014510200 PAGE 5 

### Mixture of PEMB and PE3MB: LOCAL LYMPH NODEASSAY 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 ofthe ear. The method was designed to meet the requirements ofthe following:

• OECD Guideline for the Testing 

### of Chemicals No. 429 "Skin Sensitisation: Local Lymph NodeAssay" (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 ofthe Local Lymph Node Assay. Three groups. each offour 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 offour 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 bythe mean radioactive incorporation ofthe vehicle control group are asfollows: 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 underthe 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 ofthe test material in the CBAlCa strain mouse following topical application to the dorsal surface of the ear. The method was designed to meetthe requirements ofthefollowing:

• OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph NodeAssay" (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 detecttest materials that are moderateto 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 ofthe test material to man. The studywas 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 December2008 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 ofthe Sponsor.

180PROJECT NUMBER: 014510200 PAGE 7 

## 2.2 Preparation ofTest Material 

### For the purpose ofthe 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 atthe 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 ofthe 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 ofthe study the animals were in the weight range of 15to 23 g, and were eight totwelve 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 throughoutthe 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 approximatelyfifteen changes per hourand the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

181PROJECT NUMBER: 0145/0200 PAGES 

### The animals were provided with environmental enrichment items which were considered notto contain any contaminant ofa level that might have affected the purpose or integrity ofthe 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 tothe dorsal surface ofeach earforthree 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 oftOXicity or excessive local irritation noted during this period were recorded. The bodyweightwas recorded on Day 

### 1 (priorto dosing) and on Day 

### 6. 3.2.2 Main 

### Test 3.2.2.1 TestMaterialAdministration Groups offour mice were treated with the undiluted test material orthe test material at concentrations of50% or25% 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 atthe highest suitable concentration. The mice were treated by daily application of25 1-11 ofthe appropriate concentration ofthe test material to the dorsal surface ofeach ear for three consecutive days (Days 

### 1, 2, 3). The test material formulation was administered using an automatic micropipette and spread overthe dorsal surface ofthe ear using thetip ofthe pipette. A further group offour mice received the vehicle alone in the same manner.

182PROJECT NUMBER: 014510200 PAGE9


## 3.2.2.2 3H-Methyl ThymidineAdministration Five days following the first topical application ofthe 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 atotal of20 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 oftoxicity or signs of ill health during the testwere recorded. Bodyweights: The bodyweight ofeach mouse was recorded on Day 1 (prior to dosing) and Day 6 (priorto tennination). 

## 3.2.2.4 TerminalProcedures 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) 

### forten minutes. The pelletwas resuspended in 10 ml of PBS 

### and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 

## 3 ml of5% Trichloroacetic acid (TCA).

183

## 3.3 PROJECT NUMBER: 014510200 PAGE 10 

### Determination of3HTdR 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 ofthis period oftime 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 forthe control nodes (Stimulation Index). The test material 

### will 

### be regarded as a sensitiser 

### ifat 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 forfive years, afterwhich instructions will be sought as tofurther retention or disposal.

184PROJECT NUMBER: 014510200 PAGE 11


### 5. RESULTS 

## 5.1 Preliminary ScreeningTest Clinical observations, bodyweight and mortality data are given in Table 1. No signs ofsystemic 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 ofthe Proliferative Response ofLymph Node Cells The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 2. A stimUlation index ofgreaterthan 3 was recorded forthe undiluted test material and the test material at concentrations of50% 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.

185PROJECT NUMBER: 0146/0200 PAGE 12


## 5.2.3 Bodyweight 

### Individual bodyweights and bodyweight changes for test and control animals are given in Table4. Bodyweight changes ofthe test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals overthesame period. 

## 6. CONCLUSION 

### The test material was considered to be a sensitiser underthe conditions ofthe test.

186PROJECT NUMBER: 014510200 PAGE 13 

### MixtureofPEMB and PE3MB : LOCAL LYMPH NODE ASSAY IN THE MOUSE 

### Table 1 Clinical Observations, Bodyweightand 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 ofsystemictoxicity



### 187PROJECT NUMBER: 014510200 PAGE 14 Mixture ofPEMB 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 bydividing the disintegrations per minutevalue by8 (total numberoflymph nodes) b

# = Stimulation Indexof3.0 orgreater indicates a positive result na 

### = Notapplicable



### 188PROJECT 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 

### 6 Pre- 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 ofsystemictoxicity

189PROJECT NUMBER: 014510200 PAGE 16 MixtureofPEMB and PE3MB : LOCAL LYMPH NODE ASSAY IN THE MOUSE 

### Table 4 Individual Bodyweights and BodyweightChanges 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

190PROJECT NUMBER: 014510200 PAGE 17 Mixtureof PEMB and PE3MB: LOCAL LYMPH NODE ASSAY IN THE MOUSE 

### Appendix 1 Current Positive Control Studyforthe Local Lymph Node Assay Introduction. A studywas perfonned to assessthe sensitivity ofthe strain ofmouse used atthese laboratories to a known sensitiser. The method was designed to meetthe requirements ofthefollowing:

• OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph NodeAssay" (adopted 24April 2002)

• Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Directive 2004173/EC
Test Material: a-Hexylcinnamaldehyde, Tech, 85% Project number: 0039/1057 Studydates: 17 October2008to23 October2008 

### Methods. One group offive animals was treated with 50 jJl (25 jJl perear) of a-Hexylcinnamaldehyde, Tech, 85% as a solution in acetone/olive oil 4:1 ata concentration of 15%vlv. A further group offive animals was treated with acetone/olive oil 4:1 alone. 

### Results. The Stimulation Index expressed as the mean radioactive incorporation forthe treatment groupdivided bythe mean radioactive incorporation ofthevehicle control group are asfollows: Concentration % vlv in

acetone/olive oil 4:1 Stimulation Index Result 15 

### 6.55 Positive 

### Conclusion. a-Hexylcinnamaldehyde, Tech, 85% was considered to bea sensitiser underthe conditions ofthe test.



### 191Mixture of PEMB and PE3MB ; LOCAL LYMPH NODE ASSAY IN THE MOUSE 

### Appendix 2 Summary of Positive Control Data forthe 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 oftest to control lymphocyte proliferation b= 

### Stimulation indexgreaterthan 

### 3.0 indicates a positive result PROJECT NUMBER: 014510200 PAGE 18PROJECT NUMBER: 014510200 PAGE 19 

### Mixture ofPEMB and PE3MB : LOCAL LYMPH NODE ASSAY IN THE MOUSE 

### Appendix3 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 suitablefordosing i (4:1) -+- 

### 0.5 ml vehicle



# '"= Suitable fordosing ifformulation is asolution orfine homogenous suspension which can be administered via a micropipette

193PROJECT NUMBER: 014510200 PAGE 20 

### Appendix4 StatementofGLP Compliance in Accordancewith Directive 2004/91EC



# ,DH) Department \.:..: ofHealth

THEDEPARTMENTOF

### :QEALTH OFTHEGO'1ERNMENT 

### OFTHEUNITED KINGDOM GOODLABORATORYPRACTICH STATEMENrOFCOMPLlAl\iCE INACCORDANCEWITHDIRECTIVEZOO4J91EC

TESTFACrJ.JTI! TES'fTY1'E
HarlanLllbontories Ltd. AualytkallClillic:a.l Chemistry Shardlow Bosille!s:Park EnvironmentalFate I.ondotl Road Envil'Oomontt.1Toxicology Sliardlaw Mutagenicity Derby Pti)'llChom DEnlGD Toxicology**DATEOF~ON** 19thAugust 1008

Ageneral inspection forcompliance with the POnciplc;s ofGoodLaboratory Prnctice wascamedout litmellbove

### .testfacilityaspartoftheUKGLPCompiianceProgramme. At the time ofinspection no deviations were found of**~ufticienl** magnitude to affect the validity ofnon.clinical studies perfol'Tiled atthese facilities.

Head,UKGLPMonitoringAulilorily



### 194PAGE 1 OF 17 PAGES



### Mixture ofPEMB 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

195PROJECT 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 ofthe routine and repetitive procedures that constitute the study is carried outas a continuous process designed to encompassthe 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 QualityAssurance Unit, and is considered to be an accurate account ofthe data generated and ofthe 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 thetype and schedule ofthis studywere asfollows: 13 November2008 Standard Test Method Compliance Audit 16 February 2009 Test Material Preparation 16 February 2009 Animal Preparation 16 February 2009 Dosing 16 February 2009 Assessmentof Response 

### § 09 March 2009 Draft ReportAudit 

### § Date ofQA

### Signature Final ReportAudit 

## § Evaluation specific to this study

# ~.N.Qb~ 1 8 MAR 2009



# ............ "

# ......................... ......**~**

# ....... "

# ................**~**

## .................... DATE:

................................................**~~** 

### Forthe QualityAssurance Unit'"

*Authorised QA 

### Signatures: Deputy Head ofDepartment: JM Crowther MJScT MRQA SeniorAudit Staff: G Wren 

### ONe MRQA



### 196PROJECT 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 adigital (pdf) document. Such documents are prepared by scanning the paperoriginal,

and are considered ofequivalent Integrity and authenticityto versions produced by optical photocopy. However. in all cases the hand-signed paper Original. held in secure archives. is the definitive document



### 197PROJECT NUMBER: 014510199 PAGE 4


### CONTENTS QUALITYASSURANCE 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 ofTest Material 

### 6 2.3 Measurement ofpH 

## 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 Scoresfor Ocular Irritation 11 Table 2 Individual Total Scores and Group Mean Scoresfor9cular Irritation 12 Table 3 Individual Bodyweights and Bodyweight Changes 13 Appendix 1 Initial Pain Reaction 

## 14 Appendix 2 Draize Scalefor Scoring Ocular Irritation 15 Appendix 3 Modified Kay and Calandra Interpretation of Eye Irritation Test 16 Appendix 4 StatementofGLP Compliance in Accordance with Directive 2004/9/EC 17

198PROJECT NUMBER: 014510199 PAGE 5 

### MixtureofPEMB 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 ofthe New Zealand White rabbit. The method was designed to meet the requirements ofthefollowing:

• OECD Guidelines for the Testing of Chemicals No. 405 "Acute Eye Irritation/Corrosion" (adopted 24April 2002)

• Method B5 AcuteToxicity (Eye Irritation) ofCommission Directive 2004173/EC 

### Result A single application ofthe test material to the non-irrigated eye 

## ofthree 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 1to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

199PROJECT NUMBER: 014510199 PAGES


### Mixture 

### ofPEMB 

### and PE3MB: 

### ACUTE EYE IRRITATION IN THE RABBIT 1. INTRODUCTION The study was performed to assess the irritancy potential ofthe test material following a single application to the rabbit eye. The method was designed to meet the requirements ofthefollowing:

• OECD Guidelines for the Testing of Chemicals No. 405 "Acute Eye Irritation/Corrosion" (adopted 24 April 2002)

• Method 85Acute Toxicity (Eye Irritation) ofCommission Directive 2004f73/EC The albino rabbit has been shown to be a suitable model for this type of study and is recommended in thetest method. The results ofthe study are believed to be ofvalue in predicting the likely eye irritancy potential ofthe test material to man. The studywas performed between 02 February 2009 and 12 February 2009. 

### 2. TEST MATERIAL 2.1 Description, Identification and-Storage Conditions Sponsor's identification Mixture ofPEM8 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 ofthe Sponsor. 

### 2.2 Preparation ofTest Material Forthe purpose ofthe studythetest materialwas used as supplied. The absorption ofthe test material was not determined.

200

### 3.1 PROJECT NUMBER: 0145/0199 PAGE 7 

### 2.3 Measurement

## ofpH 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 after20 minutes Undiluted as Supplied unobtainable notapplicable not applicable 90% vlv aqueous preparation approximately 4.3 approximately4.1 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 ofthe 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 waterwere considered notto contain any contaminant ofa level that might have affected the purpose orintegrity ofthe study. Thetemperature and relative humiditywere setto 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 ofthe study. The rate ofairexchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00to 18:00) and twelve hours darkness. The animals were provided with environmental enrichment items which were considered notto contain anycontaminant ofa levelthat might have affected the purpose or integrity ofthe study.

201PROJECT 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 ofthe test material was placed into the conjunctival sac ofthe 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 aftertreatment, to prevent loss ofthe test material, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration ofthe test material, an assessment ofthe initial pain reaction was made according tothe six pOint scaleshown in Appendix 1. After consideration of the ocular responses produced in the first treated animal, two additional animals were treated. Dueto a technician errorthe initial pain reaction in one animal was not recorded. This deviation from the Standard Test Method was considered notto affect the purpose or integrityofthe 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 ofthe eye was facilitated by the use ofthe lightsourcefrom a standard ophthalmoscope. Individual bodyweights were recorded on Day 0 (the day ofdosing) and at the end ofthe observation period.

202

### 3.3 PROJECT NUMBER: 014510199 PAGE 9 

### Interpretation ofResults 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 bythe letters E (degree ofopacity) and F (area ofcornea involved). For each tissue the scorewas calculated asfollows: Scorefor conjunctivae = 

### (A 

### + B 

### + C) 

### x2 Score for iris = Dx5 Scorefor cornea 

# = (E

## xF)

## x5 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 classifythe ocular irritancy potential ofthe test material. This was achieved by adding together the scores forthe cornea, iris and conjunctivaeforeach time pointfor each rabbit. The group means ofthe total scores for each observation were calculated. The highest ofthese group means (the maximum group mean score) together with the persistence ofthe reactions enabled classification ofthe eye irritancy potential ofthe test material.
Ifevidence 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, Shardlow, UK archives forfive years, afterwhich instructionswill be soughtastofurther retention ordisposal.

203PROJECT 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 effectswere 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 ofPEMB and PE3MS : ACUTE EYE IRRITATION IN THE RABBIT 

### Table 1 Individual Scoresand IndividualTotal Scores forOcular Irritation 68210 Male 68234 Male Rabbit Numberand Sex IPR=2 IPR=2

Time AfterTreatment 1 24 48 

### 72 1 24 48 72 Hour Hours Hours Hours Hour Hours Hours Hours CORNEA E = Degree ofOpacity 0 0 

### 0 0 0 0 0 0 F = Area ofCornea Involved 0 0 0 0 0 0 

### 0 0 Score (Ex F) x 5 0 0 0 

### 0 0 0 

### 0 0 IRIS D 

### 0 0 0 0 0 0 0 0

--­ Score (Ox5) 

### 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 totechnician errorthe IPR was not recorded PROJECT NUMBER: 014510199 PAGE 11PROJECT NUMBER: 014510199 PAGE 12 

### Mixtureof 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



### 206PROJECT NUMBER: 014510199 PAGE 13 

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

### Table 3 Individual Bodyweights and BodyweightChanges
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



### 201PROJECT 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 

### begraded as follows: 

### Class Reaction byAnimal 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 shutvigorously, may squeal Severe initial pain 

## 5 Rabbit holds eye shutvigorously, 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.



### 208PROJECT NUMBER: 014510199 PAGE 15 Mixture

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

### Appendix2 Draize Scale forScoring 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, deepercrimson 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 ofOds 2 Swelling with lids about halfclosed 3 Swelling with lids halfclosed to completely closed 4 

### (C) Discharge No discharge 0 Any amount different from normal (does not include small amounts observed in inner 1 canthus ofnormal animals) Discharge with moistening ofthe lids and hairsjustadjacentto lids 

### 2. Discharge with moistening ofthe 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 ofanythereof) jris still reacting to light (sluggish reaction is positive) No reaction to light. haemorrhage, gross destruction (any orall ofthese)

. 2. 

### THE TOTAL SCORE

# =D X 

### 5 MAXIMUM TOTAL

# =10 3. CORNEA (E) Degree ofOpacity(mostdensearea used) No opacity 

## o Scattered or diffuse areas, details ofiris clearly visible 1 Easily discernibletranslucent areas, detailsofIris slightly obscured 2 Opalescent areas, no detaRs ofirisvisible, size ofpupil barely discernible 3 Opaque, iris not discernible through the opacity 4 

### (F) Area ofCornea Involved One quarter (or lass) but notzero 1 Greaterthanone quarterbutlessthan half 2 Greaterthan halfbut lessthanthree quarters 3 Greaterthanthree quarters, uptowhole area 4 

### THE TOTAL SCORE 

## =(E X 

### F) X

### 5 MAXIMUM TOTAL 

### =80 MAXIMUM TOTAL SCORE POSSIBLE

# =110



### 209PROJECT NUMBER: 014!5/0199 PAGE 16 Mixtureof PEMB and PE3MB : ACUTE EYE IRRITATION INTHE RABBIT 

### Appendix3 Modified Kay and Calandra Interpretation of Eye Irritation Test

MAXIMUM

MEAN SCORE PERSISTENCE OF SCORE DESCRIPTION RATING (AND CLASS)
0.0to 0.5 Group mean total at24 hours = 0 Group mean total score at24 hours> 0 Non-Irritant (1) Practically non-inilant (2) 0.5to 2.5 Group meantotal score at24 hOUlS ;:0 Group meantotal score at24 hours> 0 PraClically non.Jrritant (2) Minimallrrllanl (3) 2.5to 15 Group meantotal SCOl8at48 hours"0 Group mean total score at48 hours> 0 Mlnimallrrllanl (3) Mild Irritant (4) 15to 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 at7days 20 or less

Group meantolalscoreal7days>20 More than half of the Individual total scores at 7days 10orless More than half of the individual total SCOret at 7 days> 10 but no individual totat score at7days> 30 More than half of the individual total scores at 7 days > 10 and any individual Store at7days> 30 Moderate Irrttant (5)

Moderate initant (5)

Severe irritant (6) Severe irritant (e)

50 to 80 Group meantolal score at7days 40orless

Group mean tolal SCOI8 at 7days> 40 More than half of the individual total scores at 7days 30 orless More than half of the individual total scores at 7 days > 30 but no individual total scores at7days> 60 More than half of the individual tolal scores at 7 days > 30 and Individual total score at7days >60 Severe irrttant (6)

Severe irritant (6)
Very severe lnitant (7) Very severe lnitant (7)

80 to 100 Group mean total Storeat 7days 80 orless

Group meantotal at7days>80 More than half of the individual total scores at7days 60 orless More than half of the individual tolal scores at 7 days >60 but no Indlllfdual total SCOI8 at7days> 100 More than half of the Individual total scores at 7 days > 60 and Individual total score at 7days> 100 Very severe irritant (7)
Verysevere irritant (7)
Extremely severe Irritant (8)
Extremelysevere Irritant (8) 100to 110 Group meantolal SCOI8at 7days 80 or less Group mean tolalscore at 7days

.. 80 Verysevere lnitan! (7) Extremelysevere irrtant (8)



### 210PROJECT NUMBER: 014510199 PAGE 17 

### Appendix4 StatementofGLP Compliance in Accordance

## with Directive 2004l9/EC


# ID. ;H). 

## Department 

# C ofHealth

THEDEPARTMENTOFHEALmOFTHE

### GOVERNMENT OFTHEUNITEDKINGDOM GOODLABORATORYPRACTICE
STATEMENTOFCOMPUANCE INACCORDANCEWITHDJREcnVE**~IEC**
tESTFAClLI'I"Y TESTTYPE Hnrlan LlIbol'lItorios Ltd. AnalylicallCUniealChemistry ShardlowBusiness Park EnvlronmentaJFate Londol'lRoud EnvironmentalTOlCieology ShArdlow Mutagenicity Derby Phys/Cheru DE72lGD Toxicology

PATEOFlNSP£C'I'ION 19thAugust 2008

Ageneral inspection for co.mpliance 

### with the Principles ofGood Laboratory Practice was CllniedoutIII the above lestfacility aspartoftheUKGLPComplianceProgrnmme. At the time ofinspection DO deviations werefound ofsufficiem magniwde to affect the validityofnOll-clinical stQdies performedatthese**faciJi~es.**

Dr.AndrewJ.Gray Head, UKGLPMonitoringAutkorit)'



### 211PAGE 1 OF 15 PAGES



### MixtureofPEMB 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 Shardlow Derbyshire DE722GD 

# UK Telephone: +44 (0) 1332 792896 Facsimile: +44 (0) 1332 799018

212· PROJECT**~UMBER:** 014510198 PAGE 2


### QUALITYASSURANCE 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 ofthe 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 tothis 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 ofthe data generated and ofthe proceduresfollowed. 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 appropriatetothetype and schedule ofthis studywere as follows: 14 November2008 Standard Test Method ComplianceAudit 13 January2009 Test Material Preparation 13January2009 Animal Preparation 13January2009 Dosing 13 January2009 Assessment of Response 

### § 18 February2009 Draft Report Audit 

### § Date ofQA 

### Signature Final ReportAudit 

### § Evaluation specificto this study \


### . 

# 25 MAR 

# 2009 

# ..**~:** 

# ..**b.!~~** 

# ................ DATE:

................................................ Forthe QualityAssurance Unit*

*Authorlsed QA Signatures: Deputy Head ofDepartment JM Crowther MIScT MRQA SeniorAudit 

### Staff: GWren ONC**~RQA**



### 213PROJECT 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 reportfully 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 adigital (pdf) document. Such documents are prepared by scanningthe paper original,

and are considered ofequivalent integrityand authenticitytoversions produced byoptical photocopy. However, in all cases the hand-signed paperoriginal, held in secure archives, i$ the definitive document



### 214PROJECT NUMBER: 014510198 PAGE 4


### CONTENTS QUALITYASSURANCE REPORT 

### 2 GLPCOMPLIANCE STATEMENT 

## 3 CONTENTS 

### 4 SUMMARY 

## 5 1. INTRODUCTION 6 2. TEST MATERIAL 

### 6 2.1 Description, Identification and Storage Conditions 

### 6 2.2 Preparation ofTest 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 ofGLP Compliance in Accordance with Directive 

### 2004/9/EC 15

215PROJECT NUMBER: 0145/0198 PAGES



### Mixture ofPEMB and PE3MB: 

### ACUTE DERMAL IRRITATION IN THE RABBIT SUMMARY 

### Introduction. The study was performed to assess the irritancy potential of the test material tothe skin ofthe New Zealand White rabbit. The method was designed to meet the requirements ofthefollowing:

• OECD Guidelines for the Testing of Chemicals No. 404 "Acute Dermal Irritation/Corrosion" (adopted 24April 2002)

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

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

## ofone rabbit produced no evidence ofskin irritation. A single 4-hour, semi-occluded application 

## ofthe test material tothe intact skin ofthree rabbits produced no evidence ofskin irritation. No corrosive effectswere noted. 

### Conclusion. The test material produced a primary irritation index of 0.0 and was classified as non-irritantto rabbit skin according to the Draize classification scheme.

216

### 2.1 PROJECT NUMBER: 0145J0198 PAGE 6


### Mixture ofPEMB 

### 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 methodwas designed to meetthe requirements ofthe following:

• OECD Guidelines for the Testing of Chemicals No. 404 "Acute Dermal Irritation/Corrosion" (adopted 24April 2002)

• Method B4 Acute Toxicity (Skin Irritation) ofCommission 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 ofthe study are believed to be ofvalue in predicting the likely skin irritancy potential ofthe test material to man. The studywas performed between 21 January 2009 and 30 January 2009.
2. TEST MATERIAL. 

### Description t 

### Identification and Storage Conditions Sponsor's identification Mixture of PEMB and PE3MB

I

....... • .....**~-** ... -- ............. ----- ...... • .. '; , 

# . Description , , , 

# . : ' 

### Batch number 

### : .......................................................,..",: 

### Date received 15 December2008 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 ofthe Sponsor.

217

### 3.1 PROJECT NUMBER: 014510198 PAGE 7



### 2.2 Preparation ofTest Material Forthe purpose ofthe studythetest material was used as supplied. The absorption ofthe test material was not determined. 

### 2.3 Measurement ofpH 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 after20 minutes Undiluted as Supplied unobtainable notapplicable not applicable 

## I 90% vlv aqueous preparation ofthe test material approximately4.3 approximately4.1 4.3 

### 3. METHODS Animals and Animal Husbandry Three New Zealand White rabbits were supplied by Harlan Laboratories UK Limited, Bicester, Oxon, UK. Atthe start ofthe studythe animals were in the weight range of2.0 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 innersurface ofthe earand 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 dietand drinking waterwere considered notto contain any contaminant ofa level that might have affected the purpose or integrity ofthe study. The temperature and relative humiditywere 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 ofthe study. The rate ofair exchange was at least

218PROJECT 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 notto contain any contaminant ofa level that might have affected the purpose or integrity ofthe study. 

## 3.2 Procedure On the day before the test each rabbit was clipped free offur 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 of0.5 ml ofthe 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 ofthe rabbit was wrapped in an elasticated corset for the duration ofthe exposure period. One patch was removed at each ofthree time points: 3 minutes, 1 hourand 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 offour hours. Approximately one hourfollowing the removal ofthe patches, and 24, 48 and 72 hours later, the test sites were examined for evidence ofprimary irritation and scored according tothefollOwing 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 Moderateto severe erythema

.......................................................................... 

## 3 Severe erythema (beef redness) to eschar formation preventing grading of erythema

.......................................................................................................... 

## 4 Oedema Formation No oedema....................................................................................................... 0 Very slightoedema (barely perceptible)

........................................................... 1 Slight oedema (edges ofarea 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 ofexposure)

..................................................................................................... 4 Any otherskin reactions. if present, were also recorded. Individual bodyweights were recorded on Day 0 (the day ofdosing) and atthe end ofthe observation period. 

### Interpretation ofResults 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 ofthe United States, Austin, Texas, pAS-59:

220PROJECT NUMBER: 0145/0198 PAGE 10 

### Primary Irritation Index Classification of Irritancy 

# o Non-irritant > Oto2 Mild irritant 

### > 2toS Moderate irritant 

### > StoB Severe irritant Ifirreversible alteration ofthe dermaltissue is noted in any rabbit, asjudged bythe 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 forfive years, afterwhich instructionswill 

### be sought astofurther retention ordisposal.



### 221PROJECT 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 ofskin irritation was noted during the study. 

## 5.1.2 i-Hour Exposure.**P~riod** 

### .. The individual scoresforerythemaleschar and oedema are given in Table 1. Noevidence ofskin irritation was noted during thestudy. 

## 5.1.3**4~Hour** 

### Exposure Period The individual scoresfor erythema/eschar and oedema are given in Table 2. No evidence ofskin 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 effectswere noted.



## 222PROJECT NUMBER: 014510198 PAGE 12 

### Mixture ofPEMB 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 Numberand 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



### 223PROJECT NUMBER: 014510198 PAGE 13 

### MixtureofPEMB and PE3MB : ACUTE DERMAL IRRITATION IN THE RABBIT 

### Table 2 Individual Skin Reactions Following 4-Hour Exposure

Skin Reaction Observation Time Individual Scores
­ Rabbit Numberand 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 of24 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



### 224PROJECT NUMBER: 0145/0198 PAGE 14 

### Mixture 

### ofPEMB and PE3MB :ACUTE DERMAL IRRITATION IN THE RABBIT '. 

### . Table 3 Individual Bodyweights and BodyweightChange Rabbit Number and Sex Individual Bodyweight (kg) BodyweightChange (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



## 225Bead.UK.GLPMoaitorlugA1Ithorlty PAGE 15 PROJECT NUMBER: 014510198 

### Appendix 1 Statement of GLP Compliance in Accordance with Directive 2004l9IEC



# ,DH) Department \:.: ofHealth

THE**D~~AltTMENT** 

### OFHEALTH OFTHEGOveRNMENT OF

## THE UNITEDKINGDOM SOODLABORATORYPRACfiCE STATEMENTOFCOMPLIANCE 

### iNACCORDANCEwrrHDIRECTIVElOO4I9I.BC

TIlSTTYPE Harlan**Labora~ries** LId. Ailldyii\:ilJlClinicalChemistry Sliardlow BuslDessPark Eln'ironmentalFate LoDdou Road EuviroDllleatalToxicology SlIanilow M_geniclty Derby PhyalChesn 

### DEl12GD Tuieoloay

19thAugust 2008 Ageneral.inspection forcompliance with the Principles of

### Good Laboratory Practice wascarried attheabovetestfacility aspartoftheUKGLPComplianceProgramIl1e. Atthe tirIX: ofinspccti9n no deviations found ofsufficientmagnitude to affect the**vali~ly**ofnoo-clin.ical StpdiC5perfonncd atthesefacilit'iel.

Dr.AndrewJ.Gray



### 226

### PAGE 1 OF 17 PAGES 

# harlan'" 

# ...

Mixture ofPEMB and PE3MB:

ACUTE DERMALTOXICITY

(LIMITTEST) 

### 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 Shardlow Business Park Minato-ku Shardlow TOKYO 105-6516 Derbyshire JAPAN DE722GD UK Telephone: +44 (0) 1332 792696 Facsimile: +44 (0) 1332799016 0145-0195.doclJS



## 227PROJECT NUMBER: 014510195 PAGE 2


### QUALITYASSURANCE 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 ofthe routine and repetitive procedures that constitute the study is carried out as a continuous process designed to encompassthe 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 ofthedata generated and ofthe proceduresfollowed.
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 thetype and schedule ofthis studywere asfollows: 09 December2008 Standard Test Method ComplianceAudit 11 February 2009 Test Material Preparation 11 February 2009 Animal Preparation 11 February 2009 Dosing 11 February 2009 Assessment of Response 12 February2009 Necropsy 

## § 09 March 2009 Draft ReportAudit 

## § Date of

### QA Signature Final ReportAudit 

## § Evaluation specificto this study 

# . 2 0MAR 

# 2009

# .~.-b.!.~.~ ......... m ........... .. 

### DATE

................................................. Forthe QualityAssurance Unit*



## *Authorised QA Signatures: Deputy Head of Department 

### JM Crowther MIScT MRQA SeniorAudit Staff: GWren ONC MRQA


## 228PROJECT 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 reportfully 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 paperoriginal,

and al8 considered ofequivalent Integrity and authenticityto versions produced by optical photocopy. However. In all cases the hand-signed paperoriginal, held In secure archives. is the definitive document.



## 229PROJECT NUMBER: 0145/0195 PAGE 4


### CONTENTS QUALITYASSURANCE 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 ofTest 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 Table4 Individual Bodyweights and Weekly Bodyweight Changes 15 Table 5 Individual Necropsy Findings 16 Appendix 1 StatementofGLP Compliance in Accordance with Directive 2004/9/EC 17

230PROJECT NUMBER:0145/0195 PAGE 5



### Mixture ofPEMB and PE3MB: ACUTE DERMALTOXICITY (LIMIT TEST) IN THE RAT SUMMARY 

### Introduction. The study was performed to assess the acute dermal toxicity ofthe test material in the Wistar strain rat. The method was designed to meet the requirements of thefollowing:

• DECO Guidelines for the Testing of Chemicals No. 402 "Acute Dermal Toxicity" (adopted 24 February 1987)

• Method 

## 83AcuteToxicity (Dermal) ofCommission Directive 92169/EEC 

### Method. A group of ten animals (fIVe males and five females) was given a single. 24-hour, semi-occluded dermal application ofthe 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 animalswere subjected to gross necropsy. 

### Mortality. Therewere no deaths. 

### Clinical Observations. Therewere no signs ofsystemictoxicity. 

### Dermallrotation. There were no Signs ofdermal irritation. 

### Bodyweight All animals showed expected gains in bodyweight overthe study period. 

### Necropsy_ No abnormalitieswere noted at necropsy. 

### Conclusion. The acute dermal median lethal dose (LD50) of the test material in the Wistar strain ratwas found to begreaterthan 2000 mg/kg bodyweight.

231

### 2.1 PROJECT NUMBER: 0145/0195 PAGE 6


### Mixture ofPEMB 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 Wistarstrain rat. The method was designed to meetthe requirements ofthe following:

• DECO Guidelines for the Testing of Chemicals No. 402 "Acute Dermal Toxicity" (adopted 24 February 1987)
• Method B3 Acute Toxicity (Dermal) ofCommission 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 acceptableto appropriate regulatory authorities. The dermal route was selected as the most appropriate route ofexposure and the results ofthe study are believed to be ofvalue in predicting the likely toxicity ofthe test material to man. The studywas performed between 04 February2009 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 December2008 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 ofthe Sponsor.



## 232PROJECT NUMBER: 0145/0195 PAGE 7 

### 2.2 Preparation ofTest Material Forthe purpose ofthe studythe test material was used as supplied. The specific gravity was determined and used to calculate the appropriate dose volume forthe required dose level. The absorption ofthe test material was notdetermined. 

### 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 ofthe study the animals weighed at least 2009, and were eight to twelve weeks of age. The weight variation did not exceed 

## ±20% of the meanweight 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 ofthe 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 ofthe study. The rate ofairexchangewas at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00to 18:00) and twelve hours darkness.



## 233PROJECT NUMBER: 014510195 PAGE 8 

### The animals were provided with environmental enrichment items which were considered notto contain any contaminant ofa level that might have affected the purpose or integrity ofthe 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 ofthe test material, a single group of animals was treated asfollows: Dose Level DoseVolume Numberof Rats

(mg/kg) 

### Specific Gravity (mllkg) Male Female



### 2000 1.194 1.68 

### 5 5


### The calculated volume oftest 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 thattheywere securely in place. Afterthe 24-hourcontact period the bandage was carefully removed and the treated skin and surrounding hairwiped 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 oftoxicity**~,** 

### 1, 2 and 4 hours after dosing and subsequently once dailyfor fourteen days. After removal ofthe 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 ToxicityTests" 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 Veryslight erythema (barely perceptible)

......................................................... 

### 1 Well-defined erythema

..................................................................................... 

## 2 Moderate to severe erythema

.......................................................................... 

## 3 Severe erythema (beef redness) to slight escharformation (injuries in depth) 

### 4 

### Oedema Formation No oedema

...................................................................................................... 0 Very slight oedema {barely perceptible)........................................................... 1 Slight oedema (edges ofarea**well~defined** 

### bydefinite raising)

........................ 2 Moderate oedema (raised approximately 1 millimetre)

.................................... 3 Severe oedema (raised morethan 1 millimetre and extending beyond the area ofexposure)

..................................................................................:.................. 4 Any otherskin reactions, ifpresentwere 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 ofthe study the animals were killed by cervical dislocation. All animals were subjected to gross necropsy. This consisted ofan external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. No tissueswere retained. 

### Evaluation of Data Data evaluations included the relationship, ifany, between the exposure ofthe animal to thetest material and the incidence and severity ofall 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) ofthetest material was made.



### 235PROJECT 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 forfive years, after which instructions will be sought as tofurther retention ordisposal.



## 236PROJECT 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 Therewere no signs ofsystemictoxicity.


### 5.3 Dermal Reactions Individual dermal reactions are given in Table 2 and Table 3. There were no signs ofdermal irritation. 

## 5.4 Bodyweight Individual bodyweights and weekly bodyweight changes are given in Table 4.
All animals showed expected gains in bodyweight overthe study period. 

### 5.5 Necropsy Individual necropsyfindings are given in Table 5. Noabnormalitieswere noted at necropsy. 

### 6. CONCLUSION The acute dermal median lethal dose 

### (LOso) ofthe test material in the Wistar strain rat wasfound to be greaterthan 2000 mglkg bodyweight.



### 237Mixture of PEMB and PE3MB: ACUTE DERMALTOXICITY (LIMITTEST) 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 ofsystemic toxicity PROJECT NUMBER: 0145/0195 PAGE 12 00

### MixtureofPEMB and PE3MB: ACUTE DERMAL TOXICITY (LIMITTEST) 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 ofPEMB and PE3MB: ACUTE DERMALTOXICITY (LIMITTEST) IN THE RAT 

### Table 3 Individual Dermal Reactions
- Females
Dose 

### Animal Effects Noted After Initiation ofExposure (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 14Mixture of PEMB and PE3MB: ACUTE DERMALTOXICITY (LIMITTEST) IN THE RAT 

### Table 4 Individual Bodyweights and Weekly BodyweightChanges
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 15Mixture of PEMB and PE3MB: ACUTE DERMALTOXICITY (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 16PROJECT NUMBER: 014510195 PAGE 17 

### Appendix 1 Statement of GlP Compliance in Accordance with Directive 2004/9/EC



## , pH). Department

## ~ ofHealth

TIJEDEPARTMENTOFHEALTHOFTHEGOVERNMENT OFTHEUNITEDKINGDOM COODLABORATORY

### PRACTICE
STATEMEtI.'T**OFCO~!PLlANCE** INACCORDANCE

## wrm DlRF.c1tvE2004J9IEC

TESTFACILITY TIi:STTVPE
Hllrllm LaboratoriesLtd. AnalytieallCliuitai Cllemistry SllardlowBU$joe8sPark EnvironmentalFate London Road En"ironmenfalToxicology Shan:llow Mutagenicity Derby PbysiChem DE?22GD Toxicology

DATE OFJNSPftCTION 19th August Z008 kgeneral inspection for complia.nce with the Principles ofGood Laboratory Practice wascarritldout at

## 1m:above testfacilityaspanoftheUKGLPComplianceProgramme. At the time ofinspection 110 deviati.ons were foulld ofsufficient magniwde to affect the validil)! ofnon-cUnica] studiesperformedallhesefacilities,

Dr.AndrewJ.Gray Head,UKGLP**MonitoringAuthorlt~**



### 243PAGE 1 OF 16 PAGES

# harlan~ 

# ....

MixtureofPEMB and PE3MB: ACUTE ORALTOXICITY 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



### 244PROJEc'r NUMBER: 014510194 PAGE 2


### QUALITYASSURANCE 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 ofthe 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 tothis study are done monthly orannually in accordance with QA Standard Procedure. This report has been audited bythe Quality Assurance Unit, and is considered to 

## be an accurate account ofthe data generated and ofthe 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 appropriateto thetype and schedule ofthis study were asfollows: 12 November2008 Standard Test MethodComplianceAudit 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 ReportAudit 

### § Date of QA Signature Final ReportAudit 

### § Evaluation specific to this study 

# . 1 8 MAR 2009

# ~! ..

# N:
..**~~** 

# ....................DATE

................................................. Forthe QualityAssurance Unit*

*Authorlsed QA

### Signatures: Deputy Head ofDepartment JM Crowther MISeT MRQA SeniorAuditStaff: . G Wren ONC MRQA



### 245PROJECT NUMBER: 014510194 PAGE 3


### GLPCOMPLIANCE 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 reportfully and accurately reflects the procedures used and data generated.



# _ ................**A.:.J\~o.~** 

# .................._________........... DATE:

........

# ~.!:J~_.,~.~

# ........................ A Pooles BA (Hons) Study Director

This report maybepresented infinal foil'll as 8 digital (pdf) document. Such documents are prepared byscanningthe paperoriginal,

and are considered ofequivalent Integrity and authenticityto versions produced byoptical photocopy. However, In allcasesthe hand-signed paper original, held in secure archives, Isthe definitive document



### 246PROJECT 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 ofData 

### 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 Chartforthe Sighting Test 14 Appendix 2 Flow Chartforthe Main Test 15 Appendix 3 Statement ofGLP Compliance in Accordancewith Directive 2004/9/EC 16



## 241PROJECT NUMBER: 014510194 PAGE 5



### Mixture of PEMB and PE3MB: ACUTE ORALTOXICITY 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 thefollowing:

• OECD Guidelines for Testing of Chemicals No 420 "Acute Oral Toxicity
- Fixed Dose Method" (adopted 17 December 2001)

• Method 61 

### bisAcute Toxicity (Oral) ofCommission 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 nodeaths. 

### Clinical Observations. There were nosigns ofsystemic 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 duringthe second week. 

### Necropsy. No abnormalities were noted at necropsy. 

### Conclusion. The acute oral median lethal dose (LDso) ofthetest material in the female Wistar strain rat was estimated to be greater than 2000 mglkg bodyweight (Globally Harmonised Classification System
- Unclassified).

248PROJECT NUMBER: 014510194 PAGE 6



### Mixture ofPEMB and PE3MB: ACUTE ORALTOXICITY 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 meetthe requirements ofthe following:
• OECD Guidelines for Testing of Chemicals No 420 "Acute Oral Toxicity
- Fixed Dose Method" (adopted 17 December2001)
• Method B1 

### bisAcute Toxicity (Oral) ofCommission Directive 

### 2004173/EC The test system was chosen because the rat has been shown to be a suitable model for this type ofstudy and is recommended in the test method. The results ofthe study are believed to be ofvalue in predicting the likely toxicity ofthetest material to man. The studywas performed between 27 January2009 and 19 February 2009. 

## 2. TEST MATERIALAND 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 December2008 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 ofthe Sponsor. 

## 2.2 Experimental Preparation Forthe purpose 

## ofthe study the test material was used as supplied. The specific gravity was determined and used to calculate the appropriate dose volume forthe required dose level.

249PROJECT 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. Atthe start ofthe study the animals were eight to twelve weeks ofage. The bodyweight variation did not exceed 

## ±20% 

## ofthe 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 setto 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 ofthe study. The rate ofair 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 notto contain any contaminant ofa levelthat might have affected the purpose or integrity ofthe study. 

## 3.2 Procedure Using available information on the tOXicity 

## ofthe test material, 2000 mglkg was chosen asthe starting dose. Dose Level

(mglkg) 

### Specific Gravity Dose Volume (mllkg) 

### Numberof 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 of2000 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 offive animals were therefore treated at a dose level of2000 

### 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 itsfasted bodyweight atthe time ofdosing.
Clinical observations were made 

## %, 1, 2, and 4 hours after dosing and subsequently once dailyfor 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. Atthe end ofthe 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. Notissues were retained. 

### Evaluation ofData 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 December2001) as shown in the Flow Chart in Appendix2. Evaluation of data included identification ofthe number of animals that died during the study (or thatwere 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 ofthe next higher dose level could result in development of severe signs of toxicity and probable mortality. Effects on bodyweights and abnormalities noted at necropsywere also identified.

251PROJECT NUMBER: 014510194 PAGE 9 

### Using the mortality data obtained, an estimate of the acute oral median lethal dose (LD50) ofthetest materialwas 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 forfive years, afterwhich instructionswill be sought as to further retention ordisposal.



## 252PROJECT 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 ofsystemictoxicity 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) ofthe 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 ofPEMB and PE3MB: ACUTE ORALTOXICITY 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 ofsystemi9to)Ciqity

___ PROJECT NUMBER: 014510194 PAGE 11--------- 

### MixtureofPEMB and PE3MB: ACUTE ORAL TOXICITY IN THE RAT
- FIXED DOSE METHOD 

### Table 2 Individual Bodyweightsand BodyweightChanges Bodyweight (g) at Day Bodyweight Gain (g) During Week Dose Level Animal Numberand Sex mg/kg 2 0 

### 7 14 1 192 1-0 Female 211 10 221 19 2-0 Female 7 10 192 199 209 2000 

### 11 2-1 Female 

### 188 194 205 

### 6 2-2 Female 189 204 

### 15 15 174 12 2-3 Female 189 

### 185 197 

### -4

- .....•.---­

N CJ'I CJ'I PROJECT NUMBER: 014510194 PAGE 12

### Mixture ofPEMB and PE3MB: ACUTE ORALTOXICITY 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 Chartforthe Sighting Test**1~;;~fjJ9;P.'ii~~\1i.lli;1~** START**~** • for outt;ome at 5mgJkg there is an optional supplementary procedure to confirm the GHS classification ... Death Evidenttoxicity N c:.n • No deaths orevidenttoxicity -...J

### ~

PROJECT NUMBER: 0145/0194 PAGE 14I') 

### c.n 00 Outcome
• .::2deaths • .:: 1 with evident toxicity and/or

### s: 1death • No deaths orevidenttoxicity 

# .....

., Animal welfare override Ifthis dose level caused death in the sighting animalswill betested. Godirectlyto outcome no further MixtureofPEMB and PE3MB: ACUTE ORALTOXICITY INTHE RAT
- FIXED DOSE METHOD 

### Appendix 2 Flow Chartforthe Main Test**(~jr'j!'f,iL~Wi'a"1!iW!iiiMii~** START

PROJECT NUMBER: 0145/0194 PAGE 15PROJECT NUMBER: 014510194 PAGE 16 

### Appendix 3 StatementofGLP 

### Compliance in Accordance 

### with Directive 2004l9IEC 

# iDH).
.Department 

# 'CotHE#31th

THEDEPARTMENTOFHEACI1I

## OFTIlEGOVERNMENT OFTHEUNITED KINGDOM GOOD

### LABORATORYPRACTICE**STA'~fENT** OFCOMPLIA.'IICE fl\fACCORDANCEWITHDIRECllVE20041!11F£

TESTFACIT.rI'Y TI'.5T1'VPE Hlll'lan LaboratoriesLtd. Analyticul/OwcalCbem:istry Shardlo"BuSll:les5 Park EnvironmcntlltFllfe LondonRoad EnvironmenfalToxicology Sliardiow Mutagenicity Herby
. PbyslCliem DE722GD Toxicology

DATBOFINSPECTION 19th·AUgust 2008 Ageneral inspectlon for compliance with the Principles ofOood LabomlOry Practice Wll$eamedoutatthe above test facility as pllrtofthe

### VKGLPComplillDCePrognnnme. At

### me time ofillspection no deviations were found ofsufficient magnitude to affect thevalidityofnon-cHnkai**sllld~s**performed atlbesefudlities.

Dr.AmJrcwJ.Gray Head.UK GLPMonltorlngAuthority



### 259

# v 

# CERI
ReceiptNo. 827-08-D-3387

STUDYCODE: 811-0908



## FINAL REPORT

TWENTY-EIGHT-DAY REPEATED-DOSE ORALTOXICITY STUDY OF KarenzMT (MixtureofPEMB aDd 

### PE3MB) INRATS

March 2007

HitaLaboratory ChemicalsEvaluation and Research Institute Japan



### - i­ 

### 260B11-0908

STATEMENT

I, the undersigned, hereby declare that this report provides correct English translation ofthe final report (Study Code B11-0908, issued onMarch 19,2009).

Katsumi Miyata, M.S., J.A.C.L.C.T. Date 

### HitaLaboratory 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: 

### B11-0908

I, the undersigned, hereby declare that this study was conducted in compliance with "Concerning Standard ofthe 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) ofthe Manufacturing Industries Bureau, METI & 

### No. OJ 

### 1121004 of

### the Environmental Health Depanment, MOE (November 21, 2003)". And, I confilmed that this report accurately reflects the raw data obtained and that data ofthe 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

QUALITYASSURANCE STATEMENT
!litaLaboratory 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: B11-0908

This study was audited and inspected by Quality Assurance Section ofHita Laboratory, Chemicals Evaluation and Research Institute, Japan. The dates audited and/or inspected and the dates reported ofthese results to the study directorand management are as follows. Items ofInspections and Audits Dates ofInspections and Audits Dales oflnspections and Audits Reports Protocol October 27, 2008 October27, 2008 

## I Assessment ofresponse October 30, 2008 October 30,2008

Preparation oftest substance November 6, 2008 November 6, 2008 Administration and clinical observation November7,2008 November 10,2008 Amendment to protocol November 10,2008 November 11, 2008 Animal data January 27, 2009 January 27, 2009 Detailed clinical observation and sensorimotorfunction January 27, 2009 January 27, 2009 Amendmentto 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** ofclinical chemistry data February 20,2009 February 20, 2009 Documents of test substance and housingconditions March 10, 2009 March 10,2009 Draft offinal report March 11, 2009 March 11,2009 Amendment to protocol (3'u) MarchI 1,2009 March II, 2009 Re-inspection ofdraft 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: aCL



## T

Jty oraud' It resu ts 

### I' In ot

### herstu

### d ly, Items ofAudits Dates ofAudits Dates ofAudits Reports Preparation investigation June 4,2008 March 19, 2009 Animal receipt October7, 2008 March 19, 2009 Quarantine and acclimatization October7, 2008 March 19, 2009 Animal care October7,2008 March 

### J9, 2009



### 263BII-0908

### ~n - and animal September 8, 2008 March 19, 2009 ation ight measurements October7,2008 March 19,2009 

### Food intake measurements September 8, 2008 March 19, 2009 Detailed clinical observation 

### and October6, 2008 March J9,2009 sensorimotor function Urine sampling October 6, 2008 March 19,2009 Blood sampling October7,2008 March 19, 2009 Trimming, autopsy 

### and organ October7,2008 March 19,2009 weight measurements Hematology October7,.2008 March 19, 2009 Blood chemistry October 8, 2008 March 19,2009 Urinalysis October7,2008 March 19,2009 October 17, 21 and 29, Pathological preparation March 19,2009 2008 Microscopic examinations October20, 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

### B11-0908

TABLE 

### OF CONTENTS
TITLE
............................................................................................................................. SPONSOR
...................................................................................................................... TESTING FACILITY
.................................................................................................... PURPOSE OFTHE STUDY
.......................................................................................... TESTING 1\1ETHOD··
....···..·..·..··....··....·· ..·..····..·....·....·····......·...................................... GLP COMPLIANCE
...................................................................................................... PERIOD OFTHE STUDY
............................................................................................. 2 LOCATION ANDPERIOD FOR RETENTION OF RAW DATA AND SPECIMENS,
................................................................................................................. 2 RETENTION OFTHEORIGINAL PROTOCOL AND FINAL REPORT
.................. 2 AUTHORAND PERSONNELCONCERNED WITH THE STUDy·
....·····....·....···..·· 3 AUTHORAPPROVAL·.. ·· ..·......·....·..··..·........·..·....····........·..·..·....·............................... 3 SUMMARy·
..·....·..·....·..·.. ··..·....····......·....······......·....····................................................. 4 MATERIALS AND METHODS··
..··..·..····......·..···..·····....·····....··..··....···..·..................... 6 1. TEST SUBSTANCE
...................................................................................... 6 2. ANIMALS
...................................................................................................... 7 3. HOUSING CONDITIONS
............................................................................. 7 4. GROUPING
.................................................................................................... 8 5. STABILITY OFTHETEST SUBSTANCE·.. ··..·..····..···..···.. ··..···..·.............. 9 6. PREPARATION OFFORMULATIONS
...................................................... 9 7. ADMINISTRATION
...................................................................................... 10 8. OBSERVATIONS
.......................................................................................... 10 9. STATISTICAL ANALYSIS
.......................................................................... 17 ENVIRONMENTAL FACTORS THAT MIGHTHAVE AFFECTED TIIE RELIABILITY OF TIlE STUDY RESLJLTS
....................................................... 17 RESULTS
...................................................................................................................... 

### 18 I. CLINICAL SIGNS
......................................................................................... 

### 18 2. DETAILED OBSERVATIONS
..................................................................... 19 3. SENSORIMOTORFUNCTION
.................................................................... 

### 19 4. BODY WEIGHTS
.......................................................................................... 

### 20 5. FOOD INTAKES
........................................................................................... 

### 20 6. HEMATOLOGICAL EXAMINATIONS
...................................................... 

### 20 7. BLOODCHEMICALEXAMINATIONS
..................................................... 21 8. URINALySES·
..·..····..··..·······..··..····..·..·..........··....····..................................... 21 9. ORGAN WE1GIITS
....................................................................................... 

### 21

- vi­ 

### 265BII-0908

10. NECROPSy·····
..·....·......····.. ··..·····......··......·..·..········...................................... 22 11. HISTOPATHOLOGICAL EXAMINATIONS
.............................................. 23 DISCUSSION
................................................................................................................. 23 REFERENCE
................................................................................................................. 26 FIGURES Body weights·
................................................................................................. 27 2 Food intakes
.................................................................................................... 29 TABLESSummary ofclinical signs
.............................................................................. 31 2 Summary ofdetailed clinical observations
..................................................... 32 3 Summary ofreflex
.......................................................................................... 57 4 Summary ofgrip strength
............................................................................... 60 5 Summary ofmotor activity
..·........·................................·..·........···......·....·...... 61 6 Summary ofbody weights
.............................................................................. 62 7 Summary offood intakes
................................................................................ 64 8 Summary ofhematological examinations
...................................................... 66 9 Summary ofblood chemical examinations
..·..·..·..........··....................·..·..·....· 68 10 Summary ofurinalyses
................................................................................... 70 l] Summary ofabsolute organ weights
............................................................... 73 12 Summary ofrelative organ weights
................................................................ 74 13 Summary ofmacroscopic examinations
......................................................... 75 14 Summary ofhistopathological examinations
.................................................. 76 ADDENDA Clinical signs ofindividual animals
..·····......·......·....·..·........·......·..........·..·....· 80 2 Detailed clinical observations ofindividual animals
...................................... 95 3 Reflexofindividual animals
........................................................................... 

### J65 4 Grip strength ofindividual animals
................................................................ 167 5 Motoractivity ofindividual animals
.............................................................. 169 6 Body weights ofindividual animals
............................................................... 172 7 Food intakes ofindividual animals
................................................................. 175 8 Hematological dataofindividual animals
...................................................... 178 9 Blood chemical data ofindividual animals
.................................................... 182 10 Urinalytic data ofindividual animals
.............................................................. 186 11 Absolute organ weights ofindividual animals
............................................... 192 12 Relative organ weights ofindividual animals
................................................ 194 13 Pathological findings ofindividual animals
..............................................,.... 196 APPENDIX 1
............................................................ HISTOPATHOLOGICALPHOTOS APPENDIX 2
.................................................................. PHYSICOCHEMICAL REPORT



### - vii­ 

### 266

### B11-0908

Study Code: B11-0908 Test Substance Code: HR7645 SponsorCode: 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


### TESTINGFACILlTY HilaLaboralory Chemicals Evaluation and Research Institute, Japan 822, 3-chome, Ishii-machi, Hita, Oila 877-0061, Japan 

### PURPOSE OFTHE STUDY The purpose ofthis study is to define the type, severity and reversibility oftoxicological signs ofthe test substance by observing the functional and morphological changes in animals receiving repeated doses orally for 28 days.


### TESTINGMETHOD 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) ofthe Manufacturing Industries Bureau, METJ & 

### No. 031121002 ofthe Environmental Health Department, MOE (November21, 2003)".


### GLP COMPLIANCE This study was conducted in conformity "Concerning Standard ofthe Testing Facilities Conducting the Test Relating to the NewChemical Substances" (Notification No. I 12!003 ofthe Pharmaceutical and Food Safety Bureau, MHLW, No.3 (2003.11.17) ofthe Manufacturing Industries Bureau, METI & 

### No. 031121004 ofthe Environmental Health Department, MOE (November 21, 2003)".



### - 1­ 

### 267B1)-0908



### PERIOD OFTHE STUDY Commencementufthe Study: October 23, 2008 Animal Receipt: October 28, 2008 Initiation ofExamination (Initiation ofDosing): November7, 2008 Terminal Necropsy ofDosingPeriod: December5,2008 Initiation ofRecovery Period: December 5, 2008 Terminal Necropsy ofRecovery Period: December 19, 200S Termination ofExamination (Termination ofHistology): February 10, 2009 Completion ofthe Study: March 19,2009


### LOCATIONAND PERIOD FORRETENTION OFRAW 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 ofthe Hita Laboratory of
our organization, and samples ofevery lotofthe test substance in the test substance storage room, for
a period of 10 years from the date ofreceipt ofthe 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 ofthe 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 OFTHE ORIGINALPROTOCOLAND FINALREPORT The original protocol and amendments 

### will be retained at Hita Laboratories. A copy of the document

### will be retained bythe sponsor.

- 2 ­ 

### 268

### Bll--0908



### AUTHORAND 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 conductofthe study) StudyStaff: 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 ofthe specimens)


### AUTHORAPPROVAL Study Director: Signed in original March 19, 2009 Katsumi Miyata, M.S., J.A.C.L.C.T. GeneralToxicology Section Hita Laboratory



### ·3· 

### 269B11-0908

SUMMARY
A 28-day repeated oral dose toxicity study of KarenzMT (Mixture of PEMB and PE3MH) was performed in groups offive male and five female Crl:CD(SD) rats at 5 weeks ofage, 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 ofthetest substancewere 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 ofthe liver was observed in females ofthe 150 and 1.000 mg/kg groups and males ofthe 1,000 mglkg group. In the histopathological examinations, diffuse fatty change ofthe hepatocytes in males and fatty changes ofthe 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 mglkggroups,
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 ofthe kidneys were observed in males and females ofthe 1,000 mglkg groups. Degeneration and necrosis ofthe tubular epithelium with the regenerative epithelium was observed in females ofthe ),000 mglkg group histopathologically. 

### In the blood chemistry, BUN 

### was increased in males ofthe 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 observedjust after dosing in males and females ofthe 150 and 1,000 mglkg groups. Moistening and fur loss ofthe neck ormandible 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 ofthe 1,000 mg/kg group, incomplete eyelids opening and plenty offood
on the tray in males and females ofthe 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 weredecreased 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/kggroups.

-4­ 

### 270

### B11-0908

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 ofthe neck and mandible were observed by day II ofrecovery, and plenty of food on the tray was observed by day 3, and these were disappeared subsequently. Although liver weights were increased in females ofthe 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 oftherecovery 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 enlargementofthe liver in females ofthe 

### 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 ofthe 150 and 1,000 mg/kg groups.

- 5 ­ 

### 271Bll"()908



### MATERIALSAND METHODS
1. TESTSUBSTANCE (Information provided by

### the sponsor) 

# .*

__...___.__**~~.** 

# ____ a ___

### *_.

__._._..__...__._..••

_ ••

_ •••••

_ .•••

_ .•

_ •••.

_ ....___**~**

### ____..________.____.____.._.__••

_ ••_._••• •••••••

## _.I**~**


# .

.**~ -.~** **~.---*-------.-** 

## .. ... .....------------------..-----.---.----_._..___._______..**~**

## ....._._________.._______....-....._-----______ 1 -6­ 

### 272

### B 11-0908



## ..**~.-** 

## ...--.---.--------.--.--

## ........

# ----...--.--.--.-----_........

### _--_............----.--

### ......... 

# . .

1.8 

### StorageConditions 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. Avoid oxidizingagent. 

### ],9 HandlingPrecaution Glove. mask, cap, protective glasses and lab coat were put on. Thetest 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 ofmean body weights using body weight-stratified randomization on two days before the start ofthe administration. The animals not treated were excreted from the study and euthanized under ether anesthesia. At the onset ofthe 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 ofa 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 ­ 

## 273Bl1-090!\

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 offood on the tray was observed after grouping. Feeders, cages and racks were changed once at the grouping, and once at the termination ofthe 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 ofUS-EPA". Contaminants in drinking water were analyzed t"vice yearly in Gita Prefecture Pharmaceutical Association according to the water regulations ofthe "Notification 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 ofHealth, Labour and Welfare. However, it was confirmed that other data were within each criterion on waterquality.


### 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 ofthe 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 groupsto investigate the reversibility ofthe effects. Dose Volume Concentration No. ofAnimals (AnimalNo.) Group**~mg/kgldal:)** (mLlkgl**~w/v%)** Male Female Vehicle control 0 10 

# ° 5( 

# - 5 ) 

### 5 ( 31

- 35 ) Vehiclecontrol (recovery) 0 10 

# ° 5 ( 6 

# - 10 ) 5 ( 36

- 40 ) Lowdose 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 ­ 

### 2741311-0908



### 5. STABILITY OFTHETEST SUBSTANCE (see attached physicochemical report) Stability of the test substance during the dosing period was confinned 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 OFFORMULATlONS 6.1 Vehicle In preparation investigation ofthe 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 thevehicle.


### 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, refrigeratorNo.7 in the testsubstancepreparation 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 10.0 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 of10.0 w/v% 300



### ·9­ 

### 275

### BII-0908

6.3 Homogeneity lind StabilityTests (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 takenjust 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 ofthe 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% ofeach nominal concentration at the first preparation ofthe 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-dayrecovery period was set.


### 8. OBSERV AnONS Concerning the numbering ofday and week, the day ofthe initiation ofdosing 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 ofthe initiation ofrecovery 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. Duringthe 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, exophthalmosand pupillary size), salivation and secretion.
" 10 ­ 

### 276B11-0908

3) Observation in arena Animals were placed in the standard arena and observed, and number ofdefecation and urination were**re~'orded** for 1min. 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 ofdifferent types was performed ad blind tests. (I) Approach contact/touchresponse A blunt probe was bought approximately 3 em from the animal's nose for 4 seconds. (2) Pinna response The animal'sresponse to a sudden sound offinger snap was assessed. (3) Pain response The animal's tail was pinched with a clothespin between one-third and base ofthe 

### lail. (4) Pupillary reflex Following darkness adaptation ofthe animal'seyes, pupil constriction in responseto a bright beam oflight was observed. (5) Airrighting 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) Locnmotoractivity counts Locomotor activity level ofeach animal was counted with activity monitoring system for rats (ACTIMO-IO, SHINTECHNO) by the number ofcrossing IR beam for 1 hour at 10 min intervals.


### 8.4 BodyWeigbts 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.

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[n addition, immediately before necropsy, body weights were**mea~ured** for calculation ofthe 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 ofthe 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 ofprothrombin time and activated partial thromboplastin time, and EDTA-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) Plateletcount (Platelet) 9) Reticulocyte count (Reticulo) 10) Prothrombin time (PT) 11) Activated partial thromboplastin time (APTT) 12) Differentiation ofleukocytes Neutrophils (Neutro) Eosinophils (Eosino) Basophils (Baso) Lymphocytes (Lymph) Monocytes (Mono) Large unstained cells (LUC) (x 104/IlL) (%) (sec) (sec)

(%)

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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 (ALT) (lUlL) UV method (method based on JSCC) 3) Alkaline phosphatase (ALP) (lUlL) p-Nitrophenyl phosphate method 4) Cholinesterase (ChE) OWL) Butyrylthiocholinciodide 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 blockingmethod 8) Glucose (mg/dL) Hexokinasc'G-6-PDH method 9) Total protein (T-Protein) (gldL) Biuret method to) Albumin (gldL) Bromocresolgreen 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-Subbarowmethod 17) Sodium (Na) (mEq/L) Crown-Ethermembrane electrode method 18) Potassium (K) (mEq/L) Crown-Ethermembrane electrodemethod 19) Chloride (CI) (mEq/L) Coulometrictitration method

1)-10), 12)-16) 7170AutomaticAnalyzer, Hitachi 17)-19) PVA-EXII, A & T

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### 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 Hmm) were determined with drinking water ad lib. Theurine 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 ofthe 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 ofthe 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 Testpaper 6) Protein Test paper 7) Glucose Test paper 

### 8) Occultblood Testpaper 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 pelviccavities, andthe 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 ofnecropsy. 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.

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However, testes and epididymides were fixed in l3ouin's solution. Category Organs and Tissues Respiratory system Trachea, lungs Digestive system Stomach, intestine (duodenum torectum, 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 inthe 1,000 mglkggroups. 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 1:0 ('1: i) Trachea c+ vi Lungs 

## eN (r'r J Forestomach Glandular stomach Duodenum-ileum Cecum- rectum Liver

## *I



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### BII-0908

, 

### 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 AxillarJymph 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 ofthe 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 ofall groups includingthe recovery groups were done. *2 Since changes suspected to be effects ofthe 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 weredone. 

## *J 

### Since changes suspected to be effects ofthe test substance were observed in femaJes ofthe 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 ofthe 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 ofthe test substance in the organ weights were noted in the 1,000 mglkg recovery group, histopathological examinationsfor the recovery groups were done.



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### *4 Since changes suspected to be effects of the test sub:;tance in the organ weights were notcd in males ofthe 1.000 mg/kg group, histopathological examinations for males 

### ofthe recovery groups were done, 

### *5 Since changes suspected to be effects ofthe test substance in the organ weights were noted in females ofthe 1,000 mglkg group, HE stainingsample:; were prepared 

### in the recovery groups, (4) Gross lesions in thejejunum (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) ofthe 1,000 mg/kg groups. Oil red 

### 0 positive substance was observed diffusely in male and in the centrilobularregion in female.


### 9. STATISTICALANALYSIS 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, Ifthe variances were homogeneous at a significance level of 5%, one way analysis ofvariance was performed. If there was a significant difference in this analysis, the difference between the vehicle control group and each ofthe treatment groups was analyzed by the Dunnett's test. Ifthe 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 ofthe 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 MIGHTHAV

### EAFFECTEDTHERELlABTLITY OF THE STUDY RESULTS

In the examination ofdrinking 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 ofHealth, Labour and Welfare, It was reported that the NOAEL ofehloric 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, ifbody weight ofrat was 300g and volume ofdrinking

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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 ofchloric acid, it was considered that this deviation did not affect the study results.


### RESULTS
1. CLINICALSIGNS (Table 1, Addendum 1) 1.1 DuringDosingPeriod 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 offood
on the tray in seven animals were observed.

In females, salivation was observed in three animals ofthe 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 mglkggroup.
No abnormalities were noted in males and females ofthe 25 mg/kggroups.


### Salivation was observedjust after dosing in almost males and females ofthe 150 and 1,000 mg/kg groups. One male ofthe 1 SO mg/kg group and four females ofthe 1,000 mg/kg also salivatedjust before dosing sporadically. 

### Moistening of

### tbe neckor mandible was sporadically observed in the afternoon from day 13 ofthedosing period.


### Furloss was observed from day 11 ofthedosing period. 

### Incomplete eyelids opening was observedjustafter dosing form day 6.


### Lacrimation was observedjust after dosingin the afternoon from day 19.


### Decreased spontaneous locomotion was observedjust after dosing in almost animals ofthe 150 mg/kg groups from day 16 and ofthe 1,000 mg/kg groups from day 4. Five males and

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### 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 ofthedosing period, Plentyoffood 

### on thetraywas observed continuously or sporadically from day 20.
'1.2 

### DuringRecoveryPeriod 

### In males, fur loss ofthe neck or mandible was observed in one animal ofthe 1,000 mg/kg recovery group by day 

### II of

### the recovery. Plenty offood on the tray was observed 

### in one animal ofthe ],000mglkg recovery gTOUp 

### by day 3.


### In females 

### ofthe 1,000 mg/kg recovery group, plenty offood on the tray was observed in one animal by day 

### 3.
2. DETAILEDCLIUNICALOBSERVATIONS (Table

### 2, Addendum 

### 2) 2.1 DuringDosing 

### Period In males, lacrimation was observed in each one animal ofthe 1,000 mglkg group in week 3 and week4. Stainedfur was observed in four animals in week 4 in the I,000 mg/kggroup.
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 DuringRecoveryPeriod Noabnormalities were noted in both sexes ofthe 1,000 mglkg recovery groups.
3. SENSORIMOTORFUNCTION (Tables 3, 4 and 5, Addenda 3, 4 and 5) 

### 3.1 DuringDosing 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/kggroup. Noabnormalities were noted in the 25 and 150 mg/kg groups.
3.2 DuringRecoveryPeriod Males were not examined since no abnormalities were noted during the dosing period.



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### B11-0908

No abnormal locomotor activity counts were detected in lcmalcs ofthe 1,000 mg/kg recovery group, although locomotor activity count was increased duringthe 

### c10sing period.


### 4. BODYWEIGHTS (Fig.1 ,Table 6, Addendum 6) 4.1 DuringDosing Period I n males, lower body weights were noted in the 1,000 mg/kg group on days 2I. 

### 26 and 28, compared to the control group. No abnormalities were noted in the 25 and 150 mg/kg groups.
No statistically significant changes ofbody weight were noted in all treated females.


### 4.2 During Recovery Period No statistically significant changes ofbody weights were noted in males ofthe recovery group. Statistically significant increases ofbody weights were noted in females ofthe 1,000 mg/kg recovery group on day 1ofthe recovery.


### 5. FOOD INTAKES (Fig.2,Table 7, Addendum7) 5.1 DuringDosing Period Food consumption was significantly decreased in males ofthe 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 ofthe 25 and 150 mg/kg groups.


### 5.2 DuringRecoveryPeriod Significantly increased food consumption was noted in males ofthe 1,000 mg/kg recovery group on day 14.
No significant changes were noted in females.


### 6. HEMATOLOGICALEXAMINATIONS (Table 8, Addendum 8) 6.1 AtTermination ofDosing Period No statistically significant changes ofhematological parameters were noted in males of all treatment groups.
Decreased lymphocytes and increased monocytes were noted in females ofthe 1,000 mg/kg group. No abnormalities were noted in females ofthe 25 and 150 mg/kg groups.

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### 286BII-0908

6.2 AtTermination ofRecovery Period No statistically significantchanges were noted in both sexes.
7. BLOOD CHEMICALEXAMINATIONS (Table 9, Addendum9) 7.1 AtTerminationofDosing.Period 

### ALT, albumin, A/G ratio, 

### Bl3N and total bilirubin were significantly increased in males ofthe 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 ofthe 1,000 mg/kg group.


### No statistically significant changes were noted in males and females ofthe 25 and 150 mglkg groups.
7.2 AtTermination ofRecovery Period Although no abnormalities were noted atthe end ofthe dosing period, significant decreases of glucose, creatinine and calcium and a significant increase of potassium were newly noted in males ofthe 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 AtTermination ofDosingPeriod WBC and epithelial cells were increased in the urine sediment in males and females ofthe 1,000 mg/kgrecovery groups.
No abnormalities were noted in males and females ofthe 25 and 150 mg/kggroups.
8.2 AtTerminationofRecovery .Period No abnormalities were noted inthe 1,000 mg/kg recovery groups.
9. ORGANWEIGHTS (Tables 11 and 12, Addenda 11 and 12) 

### 9.1 AtTerminationofDosingPeriod In males, absolute thymus weight was significantly decreased in the 1,000 mg/kg group. Relative liver, kidney and testis weights were significantly increased inthe 1,000mg/kg group.

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### 287Bll-0908

[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 ofthe 25 and 150 mg/kg groups.


### 9.2 At TerminationofRecovery 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 ofthe dosing period. Absolute epididymis and brain weights were newly significantly decreased in the 1,000 mglkg recovery group. No significant changes of Iiver, testis orthymus weights were noted atthe end ofthe recovery period.
]n females, absolute and relative liver weights were significantly increased as shown atthe end ofthe dosing period, however the severities were decreased. Significant increases ofabsolute and relative heart weights were newly noted in the 1,000 mg/kg recovery group. Kidneys and thymus did not show significant changes oforgan weights at the end ofthe recoveryperiod.


### 10. NECROPSY (Table 13, Addendum 13) 10.1 AtTermination ofDosing Period [n males, enlargement of the liver was observed in two animals ofthe 1,000 mg/kg group. Enlargement ofthe kidneys was observed in all animals ofthe J,000 mg/kg group. Although diverticulum in the jejunum was observed in one animal ofthe 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 mgikggroup.
In females, enlargement ofthe liver was observed in one animal ofthe 150 mg/kg group and all animals of the 1,000 mgikg group. Enlargement of the kidney was observed in four animals ofthe ],000 mglkg group. Although, blackish region ofthe 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 ofRecoveryPeriod Liver and kidney lesions as shown at the end orthe dosing period were not observed in all males and females at the end ofthe recovery period.



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# n11-0908

11. HISTOPATHOLOGICALEXAMINATIONS (Table 14, Addendum 13) 11.1 AtTermination ofDoSin2 Period 

# rn males, diffuse fatty change ofthe hepatocytes in the liver was observed in one animal ofthe 1,000 mglkg group. Following changes did not show dose·relationships; focal hypertrophy of the cortical cells in one male ofthe vehicle control group, focaillecrosis ofthe hepatocytes in one male ofthe 25 mglkg group and diverticulum in thejejunum 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 ofthe periportal hepatocytes in the liver was observed in two animals and degeneration and necrosis ofthe 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 ofthe kidney in one female of the 25 mglkg group and necrosis ofthe fundic mucosa in the glandular stomach in one female ofthe 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 AtTermination ofRecovery 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 ofthe 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 ofthe liver was observed in females ofthe 150 and 1,000 mglkg groups and males ofthe 1,000mglkg group. In the histopathological examinations, diffuse fatty change of the hepatocytes in males and fatty changes ofthe periportal hepatocytes in females were observed in the 1,000 mglkg groups. Althoughthese 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 ALT in males and decreased cholinesterase in females ofthe 1,000 mg/kg groups.



### - 23 • 

### 289B11-0908

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 ofthe kidneys were observed in all males and four females orthe 1,000 mg/kg groups; however, in the pathological examination, degeneration and necrosis ofthe 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 ofthe 1,000 mglkg groups, and BUN was increased in males ofthe 

### 1,000 mg/kg group in the blood chemistry.

In the clinical signs, salivation was observedjust after dosing in males and females ofthe ISO 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 ofthe test substance, since moistening and fur loss in the neck ormandible were observed, even though hyperplasia ofthe mucosa was not observed in the forestomach as seen in an irritable substance. Plenty offood on the tray observed in males and females ofthe 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 observedjust before dosing was considered to be Pavlovian response. Decreased spontaneous locomotion in males ofthe 150 and 1,000 mg/kg groups and in females ofthe 1,000 mg/kg group, and incomplete eyelids opening in males and females ofthe 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 byearlyperiod oftherecovery. Therefore, all ofthe clinical signs were considered to 

### be reversible.

In the detailed clinical observations, lacrimation and stained fur in males ofthe 1,000 mg/kg group and stained fur in females ofthe 1,000 mg/kg group were observed, as seen in the daily clinical observation. Body weight was decreased in males ofthe 1,000 mg/kg group from day 21 ofthe 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 lowercompared tothose ofthe vehicle control group. The decreased body weight was considered to be treatment-related, although cause ofthe decrease was unclear.
Food intakes were decreased in males and remales ofthe 1,000 mg/kg groups on day 3 ofthe dosing period. This was considered related to the test substance since it was observed in both sexes,



### - 24­ 

### 290BII·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 oftrea1ment.
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 ofthe 1.000 mglkg group were considered to bc secondary effects ofthe 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 ofthe 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 notconsidered 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 ofthe 1,000 mglkg group were considered to be secondary effects resulted from malnutrition. since body weight was lower. Increased inorganic phosphorus in females ofthe 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 ofthe 25, 150 and 1,000 mg/kg groups and in females ofthe 1,000 mg/kg group, unilateral pelvic dilatation in the kidney and scab ft)rmation on the skin in males ofthe 1,000 mglkg group and focal int1ammation in the lung in females ofthe 1,000 mglkg group werc considered to be single occurrences sincethey wcre observed in each one animal, and the same signs are also observed in the vehicle control groups.
In the recovery groups, fur loss ofthe 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 dosingperiod 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 ofthe 1,000 mglkg recovery group on day 1of the recovery period without food intake changes, and these wcre considered to be a single occurrence.



### - 25­ 291

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3805showadenAithough food intakes were increased in males ofthe 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 oftoxicological 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 ofthe liver in females ofthe 150 and 1,000 mg/kg groups.
The 'NoObserved Effect Level' (NOEL) wa" considered to be 25 rng/kglday due to salivation related to irritable effects ofthetest 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 hyokani tsuite, ensosan (in Japanese). McCauley PT, Robinson M, Daniel FB, Olson GR (J995) The effects of subehronic chlorate exposure in Sprague-Dawleyrats. Drugand 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 

## Recovery Administration 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 contro1 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 : FemaleIII 1-090&

Table 1 Twenty-eight-dayrepeated-dose oral toxicity study in rats Summaryofclinica.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 ofhair 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 Noabnormalities detected 5 5 S 2 S Salivation 3 S S Decreased spontaneouslocomotion 5 S Incomplete eyelid opening 3 Moistening 3 Loss ofhair 3 Staininglowerabdomen 3 S Much food on tray 4 

# .. 1\) Numberofanimals examined. 

### ve, Vehiclecontrol; (R), Recovery ta, terminal autopsy. Salivation was observedjustafter dosing in males and females ofthe ISO and 1,000 mg/kg groups, andjustbefore dosing in one male ofthe 150 mg/kg group and four males ofthe 1,000 mg/kg group. Incomplete eyelid opening was observedjust after dosing. Moistening lind lacrimation were observed in the afternoon. Decreased spon!!Uleous locomotionwasobservedjustafter dosing in malesofthe 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 ofthe 1,000mg/kggoups. Staininglower abdomen was observed before dosing, during andjust afterdosing orinthe afternoon.



## -31- 297 4Table 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 ofremoval -2 -I 

### o +1 +2 Vocalization 

## o +1 +2
HANDLING OBSERVATIONS Muscle tone -} 

### o +1 Suhnomlilltemperature

+ Piloerection

+ Staininghair

+ Unkempthair

+ Paleness

+ Reddening

+ Cyanosis

+ Lacrimation

+ No reaction Very easy Easy (slightrcsisllIJIcc) Difficult Very difficult
None Vocalization duringhandling Continuous vocalization

Decreased Nonnal Increased
Absent Present
Absent Present
Absent Present
Absent Present
Absent Present
Absent Present
Absent Present
Absent Present



### -32- 298Table 2 Twenty-eight-day repeated-dose oral toxicity study in rats Bl1-0908 Summary ofdetailed clinical observations (scoring scalefor 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 Crouchingposition orhunchback position Proneposition orlateral position Significantlydecreased Decreased Normal Increased Significantly increased
Normal Slightlyinsufficiency Moderately insufficiency Severely insufficiency
Absent Present
Normal Staggeringgait Tiptoe gait Shuffling(paralytic) gait Gait disturbance



## -33- 299Table2 Twenty-eight-day repeated-dose oral toxicity study in rats BII-0908 Summary ofdetailed clinical observations (scoring scalefor detailed clinical observations)
OBSERVATIONS IN ARENA.continued Tremor/twitch/convulsion 

### o +1 +2 +3 Stereotypicbehavior
C G S H Abnormal behavior
S B C R W V 8T T None Tremor Twitch orconvulsion Systematic 10nicconvulsion (opisthotonus orepistholonus etc.)
None Circling Grooming Sniffing Head bobbing
None Self-biting Backing Circling Rolling Writhing Vocalization Straubtail Tnil lashinebehavior



## -34­ 300Table 2·1 Twenty-eight-day repeated-dose oral toxicity study in rats BII-0908 Summary ofdetailed clinical observations Removal from cage St:x Period Exp. group Number of Easeofremoval Vocalization (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 Vehiclecontrol .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 301Table 2-2 Twenty-eight-day repeated-dose oral toxicity study in rats BII-0908 Summary ofdetailed cliniclll observations Removal from cage Sex Period Exp. group Number of Ease ofremoval 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 Vehiclecontrol 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 Vehiclecontrol 10 0 0 10 0 0 7 3 0 week4 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 week2 1,000 5 0 0 5 0 0 4 0



## -36- 302Table 2-3 Twenty-eight-dayrepeated-dose oral toxicity study in rats D1I-0908 Summ!!!lofdetailed clinical observations Handling observations Sex Period Exp. group Numbcrof MuscletOile Subnormal temperature Piloerection (mglkglday) animals -I 0 H + + Vehiclecontrol 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 Vehiclecontrol 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 Vehicle 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 week2 1,000 .5 0 5 0 5 0 5 0



### -37­ 303Table 2-4 Twenty-eight-day repeated-dose oral toxicity study in rats B11-0908 Summa!rofdetailed clinical observations Handlingobservations Sex Period Exp. group Numberof 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 Vehiclecontrol 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 Vehiclecontrol 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 week4 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 Vehiclecontrol .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

week2 1,000 S 0 S 0 .5 0 .5 0



## -38­ 304Table 2-5 Twenty-eight-day repeated-dose oralloxicity study in rats BII·0908 Summ!:!l: ofdetailed clinical observations Handling observations Sex Period Exp. group Numberof Staininghair Unkempthair 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 week4 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 305Table 2-6 Twenty-eight-day repeated-dose oral toxicity study in rats B11-0908**Summ~** ofdetailed clinical observations Handling observalions Sex Period Exp. group Number of Staining hair Unkempthair 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 Vehiclecontrol 5 5 0 5 0 5 0 5 0 weeki 1,000 5 5 0 5 0 5 0 5 0 Recovery Vehiclecontrol 5 5 0 5 0 5 0 5 0 week 2 1,000 5 5 0 5 0 5 0 5 0



## -40- 306Table 2-7 Twenty-eight-dayrepealed-dose oral toxicity study in rats B11-0908**Surrun~** ofdetailed 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 week2 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 week4 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- 307Table 2-8 Twenty-eigbt-dayrepeated-dose oral toxicity study in rats BIl·0908**Summ~** ofdetailedclinical observations Handling observations Sex Period Exp. group Numberof 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 Vchicle 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 Vehiclecontrol 10 10 0 10 0 10 0 week4 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 Vehiclecontrol 5 .5 0 5 0 5 0 week2 1,000 .5 .5 0 .5 0 .5 0



## -42­ 308Table 2·9 Twenty-eight-day repeated-dose oral toxicity study in rats BII-0908 Summarv ofdetailed 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 Vehiclecontrol 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 week4 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 Vchicle 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 309Table 2-10Twenty-eighl-dayrepeated-dose oral toxicity study in rats DII-0908 Summ!!;!l ofdetailed clinical observations Handlingobservations Sex Period Exp. group Numberof 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 Vehiclecontrol 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 week4 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

week2 1,000 5 0 5 0 5 0 5 0



## -44- 310Table 2-11 Twenty-eight-day repeated-dGse oral toxicity study in rats 811·0908 Summary ofdetailed 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 Vehiclecontrol ]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 week4 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 Vehiclecontrol 5 5 0 0 0 I 4 0 0

weekI 1,000 5 5 0 0 0 0 5 0 0 Recovery Vehiclecontrol 5 5 0 0 0 4 0 0

week 2 1,000 .5 5 0 0 0 0 5 0 

# °

-45- 311Table 2·12 Twenty-eight.day repeated.dose oral toxicity study in rats Bll·0908 Sununaryofdetailed clinical obscrvalionJ Observations in arena Sex. Period Exp. group Numbcrof Posture Molaractivity (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 week2 . 25 150 5 5 5 5 0 0 0 0 0 0 0 0 4 5 () 0 0 Female 1,000 Vehiclecontrol 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 Vchicle control :5 5 0 0 0 0 5 0 0 week I 1,000 :5 5 0 0 0 0 :5 0 0 Recovery Vehiclecontrol :5 5 0 0 0 0 4 0 week2 1,000 5 5 0 0 0 0 :5 0 0



## -16- 312Table 2-13 Twenty-eight-day repealed-dose oral toxicity study in rats BIl-0908 Summary ofdetailed 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 week2 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- 313Table2.14 Twenty-eight-day repeated-dose oral toxicity study in rats B11-0908 Summary ofdetailed 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 Vehiclecontrol 10 IO 0 0 0 10 0 week4 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 Vehiclecontrol 5 5 {) 0 0 5 0 week 2 1,000 5 5 0 0 0 5 0



## -48­ 314Table2·1STwenty·eight-day repeated·dose oral toxicity study in rats Bll·0908 Summary ofdetailed clinical observations Observations in arena Sex. Period Exp. group Number of Gait (mglkglday) animals S T P 

### GD Vehiclecontrol 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 Vehiclecontrol to 10 0 0 

# ° 0 week 2 25 

### ISO 5 5 5 5 

# ° 0 0 0 0 0 0 0 Male 1,000 Vehiclecontrol 10 10 10 10 0 0 0 0 0 0 0 0 week3 25 ISO S 5 5 5 0 0 0 0 0 0 0 0 1,000 10 10 0 

# ° 0 0 Vehiclecontrol 10 10 0 0 0 0 week4 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 315Table2-16Twenty-eight-dayrepeated-dose oral toxicity study in rats 811-0908 SUtnllUll'Y ofdetailed clinical observations Observations in arena Sex Period Exp. group Numberof 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 week2 25 150 5 5 5 5 0 0 0 0 0 0 0 0 Female 1,000 Vehiclecontrol 10 \0 10 10 0 0 0 0 0 0 0 0 week3 25 150 5 5 5 5 0 0 0 0 0 0 0 0 1,000 10 10 0 0 0 0 Vehiclecontrol 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- 316Table 2-17Twenty-eight-day repeated-dose oral toxicity study in rats B 11-0908 Summary ofdetailed 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 Vchicle 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 atP<O.05
.

.. Significantly differentfrom vehicle control at P<O.Ol.



## -51- 317Table 2-18 Twenty-eight-day repeated-dose oral toxicity study in rats llll·0908 Summary ofdetailed 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 week4 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 Vehiclecontrol 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 differentfrom vehicle control atP<O.05. 

### * ••Significantly differentfrom vehicle control atP<O.Ol.



## -52- 318Table2-19Twenty-eight-dayrepeated-dose oral toxicity study in rids BII-0908 Summaryofdetailed clinicalobservations Observations in arena Sex Period Exp. group Number of Stcreotypic behavior (mglkgldIlY) animals C G S H Vehiclecontrol 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 week4 25 150 5 5 5 5 0 0 0 0 0 0 0 0 1,000 10 10 0 0 0 0 Recovery Vehiclecontrol 5 5 0 0 0 0

wee.k 1 1,000 5 5 0 0 0 0 Recovery Vehiclecontrol 5 5 0 0 0 0 week2 1,000 5 5 0 0 0 0



## -53- 319Table 2-20Twenty-eight-day repeated-dose oral toxicity study in rats BII-0908 Summary ofdetailed clinical observations Observations in arena Sex Period Exp. group Numberof Stcreotypic behavior (mglkglday) animals C G S H Vehiclecontrol 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 Vehiclecontrol 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 Vehictecontrol to 10 0 0 0 0 week2 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 Vehiclecontrol 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 Vehiclecontrol 5 :5 0 0 0 0 week 1 1,000 5 5 0 0 0 0 Recovery Vehiclecontrol 5 5 0 0 0 0 week 2 1,000 5 5 0 0 0 0



## -54- 320Table 2-21 Twenty-eight-day repeated-dose oral toxicity study in rats Bll-090S Summary ofdetailed clinical observations Observations in arena Sex Period Exp. group Number of Abnormal behavior (rnglkglday) animals S 8 C R W V ST T Vehiclecontrol 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 Vehiclecontrol 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 Vehiclecontrol 10 10 10 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 week3 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 week4 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 Vehiclecontrol .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 321Table2-22Twenty-eight-dayrepeated-dose oral toxicity study in rats BI1-0908 Summary ofdetailed clinical observations Observationsin arena Sex Period Exp. group Numberof Abnormalbehavior (mglkg/day) animals S B C R W V ST T Vehiclecontrol 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 VehicleconlTol 10 10 10 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 week3 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 Vehiclecontrol 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 Vehiclecontrol 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 week2 1,000 5 

### S 0 0 0 0 0 0 0 0



## -56- 322Table 3 Twenty-eignt-day repeated-dose oral toxicity study inrats B11-0908 Summary ofreflex (scoring scaleforreflex)
SENSORIMOTORFUNCTION Approach contactltoucb response ·1 0 +1 Pinnaresponse -1 0 +1 Pain respollse (tail pineh) -I 0 +1 Pupillary reflex + Airrighting reflex + No reaction Nonnal Hyperreaction
No reaction Nonnal Hyperreaction
No reaction Normal Hyperreaction
Nonna! Abnonnal reaction
Normal Abnormal reaction



## -57- 323Table 3-1 Twenty-tight·dayrepealed·dose oral toxicity study in**rat.~** BIl·0908 Summaryofreflex Sensorimotorfunction Sex Period Exp. group**(m~g!day)** Numberof animals Approach contact touch response Pinnaresponse
.1 0 +1 -1 0 +1 Vehicle control 10 0 10 0 0 10 0 Male week4 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 week4 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 324Table3·2 Twenty-eight-dayrepeated-dose oral toxicity studyin rats Bll·0908 Swnmaty ofreflex Sensorimotor function Sex Period Exp.group Numberof Pain respon.'1e (tail pinch) Pupillaty reflex Airrightingreflex (rnglkglday) animals -I 0 +1 + + Vehicle control 10 0 10 0 10 0 10 0 Male week4 

### 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 week4 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 325Table4 Twenty-eight-dayrepeated-dose oral toxicity study in rats BII-0908 Summwy of grip strength Sex Period Exp. group Numberof Forelimb Hindlimb (mglkglday) llIlimals (g) (g) Vehiclecontrol 10 4]8 :1:28 426 :1:25 25 5 417 ±25 462 ±13 Male week4 150 5 428 :1:39 429 :1:49 1,000 10 425 :1:37 -405 :1:29 Vehiclecontrol 10 372 ±26 378 ±23 25 5 367 ±18 364 :1:23 Female week4 150 5 375 ±16 377 ±30 1,000 ]0 356 ::1:30 384 ±24 Mean ±S.D. 

## *Significantly different fromvehicle control at P<O.05. 

## ** Significantly different from vehicle control atP<O.O 1.



## -60­ 326811-0908 Table 5 Twcnty-eight-day repeated-dose oraltoxicity study in rats Summary ofmotor activiljl Sex Period Exp. group Numberof 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 week2 ±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 differentfrom vehicle control atP<O.05
.

..Significantlydifferentfrom vehiclecontrol atP<O.OI.



## -61­ 327 757

617811-0908 Table 6-1 Twenty-eight-day repeated-dose oral toxicity study in rats 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 CO811-0908 Table 6-2 Twenty-eight-day repeated-dose oral toxicity study in rats 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 co811-0908 Table 7-1 Twenty-eight-da¥ repeated-dose oral toxicity study in rats 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 

### oTable 7-2 T1ienty-eight-day repeated-dose oral toxicit: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 Vehicle 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 N811-0908 Table 8-& Twenty-eight-day repeated-doss oral toxicity study in rats 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 

### wTable 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.

(.) (.)**~**811-0908 Table 9-2 iKenty-eight-day repeated-dose oral toxicity study in rats 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 0I 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)811-0908 Tahle 10-2 Twenty--eight-day repellted-dose oral toxicity study in rats Summary ofurinalyses Exp.group Numbel' of Colo.- Turbidity pH Protein Glucose Occult blood Sex (mglkgf day) 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, Slightlyyellow. y, Yellow. NT, No turbidity.

(.,) (.)



### ......Table 10-3 Twenty-eignt-dayrepeated-dose oral toxicity study in rats B11-0908 Summary ofurinalyses (Urinary sediment) Exp.group Number of Redblood cells·) White bloodcells.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

## orcells/IO views (X400). b) Number ofcllsts/t8xIS mm2• c) Incidence ofcrysUlls/18x18 mm2•



### 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.

(,,) (,,) coTable 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

_____ __________..::

______1Q".QL j9_&L

______l.llJL

___ _____••**L_~JLl l~_·_Q.L**  

### ______**L.Q.,_~** 

### _____**~-~** . 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-eight4ayrepeated-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 Noabnormalilies detected 5 5 5 4 0 S 5 5 5 .3 0 S OlaDdWarstoJll8Cb

Blackishregion ofmucosa 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 

### .:..B11-0908
Table 14-1 Twenly-eight-dayrepeated-dose oral toxicity studyin rats Summaryofhistopathological 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 Glandularstomach -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) Numberofanimals autopsied, b)Numberofanima,ls affi:tted INumber ofanimals examined. -,Notexamined. +, slight.••••••••••••••••••••••• BII·0908
Table: 14-2 Twc:nty-e:ight-day repeated-dose oral toxicity study in rats Summaryofhistopathological 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 changeofperiportal + 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 ofhepalocyles + 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 tubularepithelium 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 Solitarycyst 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) Numberofanimals autopsied
. 

### w b)Numberofanimals affected INumberofanimals examined.
-,Notexamined. +, slight011-0908
Table 14-3 Twenty-eight-day repeated-dose oral toxicity studyin rats Summaryofhistopatbological 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 abnormalitiesdetected 515 515 5/5 515 SIS 5/5 

### Pons

No abnonnalities detected 515 515 515 515 515 515 Spinal cord

No abnormalities detected Sciaticnerve 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)Numberofanimals autopsied. b)Numberofanimals affected INumber ofanimals c)(amined. 

# -. Not e'l:amined.