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# 00U388p . 88 Received at : 12h 16m, 2/17/200 4

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# Respons

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# Public CommitmentP. 91 BIOTOXTECH 

### FINAL REPORT In vivo Micronucleus test of J

STUDY No. : B04109 CERTIFICATE OF TRANSLATIO N This report is translated from Korean into English as an original contents, therefore, is not assigned on the relevant pages . This report is partially modified the Korean report for an expression of exact meaning but it did not influence on the oriainal copy. Translated by Date

Mei=sflu Zhen,- Biotoxtech Co . . Ltd .p. 92 BT Study Number : B04109

Report STUDY DIRECTOR AUTHENTICATION AND GLP COMPLIANCE STATEMEN T Study Title In vivo Micronucleus test cf



# 1 Study No. : B04109 The Section 10, Genetic toxicity test, in Chapter 4 of Annex 5 in the TCCA-Good Laborator y Practice Standards and Test Guidelines, [ NIER (National Institute of Environmental Research) notification No . 1998-41, December 23, 1998] . OECD Guidelines for the Testing of Chemicals 474, Mammalian Erythrocyte Micronucleus Test (Adopted 21 st July 1997 ) The study was performed under the responsibility of the Study Director. This report was Written based on the raw data obtained through the study . M ei-Shu Zheng Jul . 02, 2004 Study Director Date Biotoxtech Co., Ltd. Jong-S ung Lee Jul . 02, 2004 Tasting Facility Management Date Biotoxtech Co ., Ltd. 

# Q W) x393p. 93 BT Study Number: B04109

Report OUTLINE OF STUD Y Study Title: In vivo Micronucleus test of' . ~ Study No . : B04109 1 . Purpos e The objective of this study was to determine a mutagenic potential o f In the micronucleus test system using a bone marrow in mice . 2 . GLP TCCA (Toxic Chemical Control Act)-Good Laboratory Practice described in Annex 1 of TCCA-Good Laboratory Practice Standards and Test Guidelines [NIER (National Institute of Environmental Research)Notification Na . 1998-41, December 23, 1998 ] Some unstated matters in the test plan in terms of GLP regulation should be observed by the Standard Operation Process (SOP) of Biotoxtech co ., Li:d . 3 . Guideline The Section 10, Genetic toxicity test, in Chapter 4 of Annex 5 in the TCCA-Good Laboratory Practice Standards and Test Guidelines, [NIER (National Institute of Environmental Research) notification No . 1998-41, December 23, 19981 . OECD Guidelines for the Testing of Chemicals 471, Bacterial Reverse Mutation Test (Adopted Jul 21, 1997 ) 4 . Sponso r Title ~ Addres s 5. Test managemen t Title Biotoxtech Co ., Ltd . Address 58-1Block Ochang Scientific Industrial Complex, Ochano-im1eon,

Cheongwon-gun, Chungcheongbuk-do, 363-883 Korea y 

# ot►0394p. 94 BT Study Number

: B04109 Report 6 . Test facility Name Biotoxtech Co ., Ltd. Address 58-1Block Ochang Scientific Industrial Complex, Ochang-myeon,

Cheongwon-gun, ChunQchcongbuk-do, 363-883 Korea 7. Storage of record and data 1) Duration : 5 years after the completion of Study 2) Record and data : Protocol, raw data, final report (draft and original), references, documents related with the test (documents of GLP and all correspondences) . 3) Storage place and addres s Place Archives of Biotoxtech Co ., Ltd . Address ~ 58-1Block Ochang Scientific Industrial Complex, Ochang-myeon,

Cheongwon-gun, Chungcheongbuk- do, 363-883 Kore a 8. Schedule

Initiation of study May . 03, 2004 Animal receipt(Preliminary test) May. 10, 200 4 Preliminary test data May . 17, 2004 -- May. 23, 2004 Animals receipt(main test) May . 24, 2004 Group assignment May . 31, 2004 Beginning of study May . 31, 2004 Storage and formulation of test item May . 31, 2004 - Jun . 01, 2004 Slide preparation Jun . 02, 2004 Observation of experiments Jun . 17, 2004 - Jun . 18, 2004 End of experiments Jun . 18, 2004 Presentation of draft final report Jun . 24, 2004 Presentation of final report Jul . 02, 2004 Completion of study Jul . 02 . 2004 3, 

## 95p. 95 BT Study Number: B04109

Report 9 . Responsible personnel Study Director Mei-Shu Zhen g Preparation of protocol Mei-Shu Zheng, Su-Hyon Kim Evaluation of healthy conditions of animal-r Kap-Ho Ki m Management of test substance Eun-Ae Ki m Application of test item Mei-Shu Zheng, Mi-Young Lee Arrangement of raw data & evaluation Mei-Shu Zheng, MI-Young Lee Presentation of final report Mei-Shu Zheng , Su-Hyon Ki m



# OF (} ~~396p. 96 BT Study Number

: B04109 Report 

## QUALITY ASSURANCE RECORD AND AUTHENTICATION STATEMENT Study Title : In vivo Micronucleus test o f J in mice Study No . : B04109 This study was subject to audit by the independent Quality Assurance Unit of BIOTOXTECH as indicated below . The findings of each audit were reported to the Study Director and Management as prescribed by Standard Operating Procedures . The report audit was designed to confirm that as far as can be reasonably established the methods described and results incorporated in the report accurately r .-flect the raw data produced during the study. Audit phase and each date reported to responsible personnel were as indicated below and these were based upon audit records . Audit type Audit phase Audit date To Study To Management Director Study based Protocol May . 04, 2004 May . 04, 2004 May . 04, 2004 audit Protocol Amendment May . 25, 2004 May . 27, 2004 May . 27, 2004

Protocol Amendment Jun . 25, 2004 Jun . 25, 2004 Jun . 25, 2004 Animal receipt May . 24, 2004 May . 27, 2004 May . 27, 2004 Group assignment May . 31, 2004 Jun . 02, 2004 Jun . 02, 2004 Storage and Formulation May . 31, 2004 Jun . 02, 2004 Jun . 02, 2004 of test item Administration May . 31, 2004 Jun . 02, 2004 Jun . 02, 2004 Slide preparation Jun . 02, 2004 Jun . 03, 2004 Jun . 03, 2004 Observation Jun . 17, 2004 Jun . 19, 2004 Jun . 19, 2004 Record & Raw data Jun . 28, 2004 Jun . 30, 2004 :un . 30, 2004 Record & Raw data Jul . 02, 2004 Jul . 02, 2004 Jul . 02, 2004 Report (Draft) Jun . 28, 2004 Jun . 30, 2004 Jun . 30, 2004 Report Jul . 02, 2004 Jul . 02, 2004 Jul . 02, 2004 Young, Hyun Lee Jul . 02 . 2004 Director, Quality Assurance Unit Dat,2 Biotoxtech Co ., Ltd. 

# M-►0397p. 97 BT Study Number: B04109

Report CONTENTS Page TITLE PAGE STUDY DIRECTOR AUTHENTICATION AND GLP COMPLIANCE STATEMENT OUTLINE OF STUS Y QUALITY ASSURANCE RECORD AND AUTHENTICATION STATEMENT 1 . SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I II. PURPOSE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 III. MATERIAL AND METHOD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 . Test item . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2. Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 3 .- The test Animal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 4. Environmental condition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 5 . Analysis of contaminant in diet and water ••• • . .••• ••• ••• ••• ••• ••• ••• ••• ••• ••• ••• ••• ••• . . . . . . 4 6. Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 7 . Preparation and analysis of test item • .•••• •• . ••••••••••••••••••••••••••••••••••• .• . . . . . . . . 5 8 . Administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 9 . Test group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 10 . Observation, measu'rement and examination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 11 . Collection of bone marrow .•••••••••• .•••••••••••••••••••• . .• . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 12. Collection of the bone marrow and preparation of micronucleus specimens • • . . ••••• 6 13 . Observation, measurement and examination •••• . .• .•• .•••••••••••••••••••• .•••••••• . .••• 7 14. Statistics analysis . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 IV. RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 V. DISCUSSIONS AND CONCLUSIONS ••• .•••••••••••• .••••••••••••••••••••• . . . .• . . . . . . . . . . . 9 VI.REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Table 1 . Change of body weight (Group Summary) •••••• . .••••••••••••••• .••••••••• 12 Table 2. In vivo Micronucleus test of ~



### I in male ICR mice (Group Summary) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 APPENDICES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Appendix 1 . Change of body weight (Individual, N=6) •••••••••••••• .• . .••••••••• 15 Appendix 2 . In vivo Micronucleus test Offin male male ICR mize ( Individual, I~'=6) . . . . . . . . . . . . . . . 16 

### Appendix 3 . Result of prelimina

### ry test(

### NT=3) 

### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 

### Appendix 4 . 

### Certificate of a

### nalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 18 

# (" [a0398p. 98 BT Study Number: B04109 Report 1 . SUMMARY 

### This study was designed to determine the mutagenic potential of ~ Jin the micronucleus test using bone marrow cells in mice . In the main study, the nominal concentrations of the test item such as 2000, 1000, and 500 mg/kg for the highest test dose, middle dose and lowest dose, respectively, were administered once by gavages except the positive control to 6 male mice per group in accordance with the preliminary test . The negative control and positive control were 0 .5°o MC 1500 (10 ml/kg) and MMC (2 mg/kg), respectively . Bone'marrow cells were collected and fixed from the sacrificed mice in 24 hours from treatment . And the Giemsa solution was used for dying to observe the ratio of polychlomatic erythrocytes . There were no significant difference in appearance rate of micronucleated polychrornatic erythrocytes (IMNPCEs) in polychromatic erythrocytes (PCEs) between treatment and control groups . In addition, PCE/(P(-'E+NCE) ratio did not show sign of cytotoxicity . In the positive control group, micronucleated polychromatic erythrocytes (MNPCEs) was increased significantly in contrast with that of the negative control group . In conclusion, Ddid not induce microriucleated polychromatic erythrocytes (MNPCEs) in t :ie bone marrow cells in mice .


## 0t>0399P. 99 BT Study Number: B04109

Report II . PURPOS E The objective of this study was to determine a mutaQenic potential o f in the micronucleus assay system using bone marrow cells in mice . y III . MATERIAL AND METHO D 1 . Test item (1) Name ] L (2) Lot No . L JLot No . : 0-6182 ) (3) Appearance Orange solid (4) Purity 100 % (5) Attraction Hydrophobic, Lipophilic (6) Stability Stabl ~ (6) Storage Conditions Cold storage (7) Handling & notice Non e (8) Supplier

Name Addres s (9) Residue treatment After the study, the residue of the test item will be discarded . (10) Certificate of analysis Provided by the sponsor . 2 . Control (1) Negative contro l 0 .5% methylcellulose 1500(MC1500, Lot No . : TCR1966, Wako) (2) Positive contro l MMC ~ Mitomycin C (Lot No . : 122K2509, Sigma, USA ) (3) MMC was dissolved in Water for injection (Lot No . : AA~V3AS, Choonowae Pharma Corp .) 

## 00040Ap. 100 BT Studjy Number: B04109

Report 3-'The test anima l (1) Species, family Mouse, ICR [sam : Tac Ha(ICR)] (2) Supplier Name Samtako Bio Korea Inc . Address 77-1 Seorang-dong, Osan-si, Gyeonggi-do, Korea (3) Producer Name Samtalco Bio Korea Inc . Address 77-1 Seoran-dong, Osan-si, Gyeonggi-do, Korea (4) Justification of test system The ICR mouse were selected because it was widely used at toxicity study and available toxicological background data have been accumulated . (5) Number and sex of selected animal s 1) Preliminary test and Collection of bone marrow : male, 30 2) Main test : male, 35 (6) Labeling When animals were received, to identify between animals, their tails were labeled using an oil base pen in accordance with the method of identification (SOP/SGE/110 and140) . (7) Quarantine and acclimation Animals will be quarantined and acclimatized for 7 days and general signs will be observed once a day . When received and completed quarantine & acclimatization of animals, they were weighed using a scale (BP3100S, Sartorius, Germany) to select suitable healthy animals for the test. (8) Classification After the quarantine and acclimatization, 15, and 30 animals for the preliminary, micronucleus induction, and main test, respectively, and normal increase in body weight ; and without abnormal `;eneral signs will be selected . When classified, mean weight and mean standard deviation were considered . (9) Arrangement of remaining animal s After the classif

►cation, remaining animals were euthanized . (10) Identification of individual animal 

# ~i~ ► ~~401