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Received at : 12h 16m, 2/17/2004

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:eceived at : 12h 16m, 2/17/2004

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PreManufacture Notice an d

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A Public Commitment
P. 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 : B0410 9

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 Laboratory

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 .

Mei-Shu Zheng Jul. 02, 2004

StudyDirector Date

Biotoxtech Co., Ltd.

Jong-S ung Lee Jul. 02, 2004

Tasting Facility Management Date

Biotoxtech Co., Ltd.

Q W) x393
p. 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. Sponsor

Title ~

Address

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► 0394
p. 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 Kore a

7. Storage of record and data

1) Duration : 5 years aft er 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 ofstudy May. 03, 2004

Animal receipt(Prelimina ry test) May. 10, 200 4

Preliminary testdata May. 17, 2004 -- May. 23, 2004

Animals receipt(main test) May. 24, 2004

Group assignment May. 31, 2004

Beginning ofstudy 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

3,95
p. 95

BT Study Number: B04109

Report

9 . Responsible personne l

Study Director Mei-Shu Zheng

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 Kim

OF (} ~~396
p. 96

BT Study Number: B04109

Report

QUALITY ASSURANCE RECOR D

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- ► 0397
p. 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 mal e male ICR mize ( Individual, I~'=6) . . . . . . . . . . . . . . . 16

Appendix 3 . Result of prelimina ry test(NT =3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Appendix 4 . Certificate of an alysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. 18

(" [a 0398
p. 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>0399
P. 99

BT Study Number : B04109

Report

II. PURPOSE

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

Address

(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.)

00040A
p. 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 animals

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 acclimatio n

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