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B051350

Appendices

Appendix 1 Clinical Sign (Dosing period) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Appendix 2 Clinical Sign (Recovery period) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 7

Appendix 3 Detailed Clinical Observations (Week -1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Appendix 4 Detailed Clinical Observations (Week 1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29

Appendix 5 Detailed Clinic al Observations (Week 2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37

Appendix 6 Detailed Clinical Observations (Week 3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .45

Appendix 7 Detailed Clinical Observations (Week 4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53

Appendix 8 Function Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .61

Appendix 9 Motor Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69

Appendix 10 Body Weight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .77

Appendix 11 Food Consumption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .85

Appendix 12 Hematology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .93

Appendix 13 Blood Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

Appendix 14 Urinalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .117

Appendix 15 Organ Weight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .125

Appendix 16 Relative Organ Weight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 3

Appendix 17 Pathologic al Findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14 1

Final page 158

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~ B051350

Photographs 1-4

~--

(

3C:4, 0,
p. 174

,~. .

~- .-

Photograph 1 .

Control group, Male, Animal No. 10102 (Scheduled sacrifice Week 5)

Liver: No remarkable change . (HE, x 200)

:

Photograph 2 . MTDID 1000 mg/kg group, Male, Animal No . 10402 (Scheduled sacrifice Week 5)

Liver: Hypertrophy, hepatocyte, centrilobular, mild in grade . Single cell necrosis,

hepatocyte, minimal in grade. (HE, x 200)

3~~ -4-
p. 175

' a •

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Photograph 3 .

Control group, Male, Animal No . 10102 (Scheduled sacrifice Week 5)

Liver: No remarkable change . (HE, x 200)

°-

Photograph 4 .

MTDID 1000 mg/kg group, Male, Animal No . 10402 (Scheduled sacrifice Week 5)

Liver: Vacuolation, hepatocyte, perilobular, minimal in grade . (HE, x 200)

~ do -~-
p. 176

B051350

Quality Assurance Statement

Sponsor: Sumitomo 3M Limited

Title: A 28-Day Repeated Dose Oral Toxicity Study of MTD ID3663 in Rats

Study No.: B051350

This study was carried out in accordance with the following standard . I hereby certify

that this final report faithfully describes the methods and results in this study . This

inspection and reporting are as follows.

Standards Concerning Testing Facilities Implementing Studies Related to New Chemical Substances

(November 21, 2003, Pharmaceutical and Food affairs No . 1121003, Pharmaceutical and Food Safety

Bureau, Ministry of Health, Labor, and Welfare, November 17, 2003, Manufacturing Industries Bureau,

No. 3, Ministry of Economy, Trade, and Industry, No . 031121004, Environmental Policy Bureau,

Ministry of the Environment)

Reporting Date

Phase Inspector Inspection Date to the to the Management

Study Director

Protocol

Draft Chen Fujun Feb. 17, 2006 Feb. 17, 2006 Feb. 17, 2006

(Reinspection) (Chen Fujun) (Feb . 21, 2006) (Feb. 21,-2006) (Feb. 21, 2006)

Final Chen Fujun (Feb. 21, 2006) (Feb. 21, 2006) (Feb. 21, 2006)

MiTOX protocol Chen Fujun Feb. 24, 2006 Feb. 24, 2006 Feb. 24, 2006

Protocol amendment 1

Draft 1 Chen Fujun March 6, 2006 March 6, 2006 March 6, 2006

(Reinspection) (Chen Fujun) March 7, 2006 March 7, 2006 March 7, 2006

Final 1 Chen Fujun March 7, 2006 March 7, 2006 March 7, 2006

Study procedure

Animal receipt Mayumi Watanabe March 1, 2006 March 1, 2006 March 1, 2006

Stability analysis of Chen Fujun March 8, 2006 March 8, 2006 March 8, 2006 the test substance

Grouping Chen Fujun March 8, 2006 March 8, 2006 March 8, 2006

~ Preparation of the

dosing suspensions Chen Fujun March 9, 2006 March 9, 2006 March 9, 2006

Concentration analysis Chen Fujun March 9, 2006 March 9, 2006 March 9, 2006 of dosing suspensions

Measurement of body

weight and food Chen Fujun March 10, 2006 March 10, 2006 March 10, 2006

consumption

Administration Chen Fujun March 10, 2006 March 10, 2006 March 10, 2006

Detailed clinical Chen Fujun March 14, 2006 March 14, 2006 March 14, 2006 observations

Necropsy, measurement Chen Fujun April 7, 2006 April 7, 2006 April 7, 2006 of organ weights

Histopathology Chen Fujun May 17, 2006 May 17, 2006 May 17, 2006 (Microscopic examination)

Raw data and final report

Draft Chen FJuly 24, 2006 July 27 2006 July Fujun - July 27, 2006 ~ y~, 2006
p. 177

B051350

(Reinspection) (Chen Fujun) July 28, 2006 July 28, 2006 July 28, 2006

Final Chen Fujun August 1, 2006 August 1, 2006 August 1, 2006

Quality Assurance Manager: Signed and sealed Date: August 1, 200 6

Kazuo Morotome

Quality assurance unit of Kashima Laboratory

Mitsubishi Chemical Safety Institute Ltd.

~--

~

C~
p. 178

ST-131 Final Report 6-7-2005

SANITIZED

Final Report

Acute Inhalation Toxicity Study ofc

(ToxDocs Study Number 05-003, MTDID 3663)

Study Director : Paul Lieder, Ph.D., DABT.

Senior Toxicology Specialist

3M Corporate Toxicology and Regulatory Services, 220-2E-02,

St. Paul, MN 5514 4

Study Toxicologist:

;

Testing Facility: 3M Strategic Toxicology Laboratory, 3M Center, 270-3S-05,

St. Paul, MN 55144

Sponsor:

Experimental Start Date (in-life) : 12-16-2004

Experimental Termination Date (end-life): 12-23-2004

CONFIDENTIALITY STATEMENT

This report contains the unpublished results of research sponsored by 3M. These results may not

be published, either wholly or in part, or reviewed or quoted in any other publication without the

pr ior authorization of 3M .

'-A
p. 179

ST-131 Final Report ,~

O

~~

E

D 6-7-2005-

Study Summary

The purpose of this study (Tox Docs Study Number 05-003) was to evaluate the acute inhalation

toxicity of [ ~ in male Sprague Dawley rats. Four rats were exposed, whole body, to the

test material at a concentration of 2000 ppm for 4liours. All animals appeared normal during the

exposure and during the post exposure observation phase . Another group of 4 rats were used as

control rats and they were not exposed to any test atmosphere . Gross necropsy was conducted 7-

days post exposure for all the rats (due to holiday schedule) . No abnormalities were found.

The 4-hour LC50 for is greater than 2000 ppm

Study Obiective

The objective of this study was to evaluate the acute inhalation toxicity of L `1 in rats . Animals were observed throughout the study for clinical signs of toxicity . Gross necropsy was

performed at the termination of the study . -

Experimental Procedures

Test Material:

The test material was identified as [ ~I "MTDID 3663) . Chemical characterization

of the material was performed by 19F/1H-NMR and is attached to this report as Appendix

1 . The purity fort was 97.4%.

Species Selection:

The Animal Usage Application protocol.(AUA 2004-0405) requires the use of a small

rodent species to optimize use of test material . Male Sprague Dawley rats supplied by

Harlan were selected as they are commonly_ used in acute inhalation toxicity testing to

generate data for human health risk assessment.

Animal Husbandry:

Housing - After exposure, the rats were group housed in standard solid bottomed cages

through out the study.

Diet/Water - Harlan Teklad LM-485 Mouse/Rat Sterilizable Diet, supplied by Harlan

Teklad, Madison, WI, and tap water were provided to all animals ad libitum throughout

the study, except during the exposure period.

Environment - Environmental controls for the animal room were set to maintain a

temperature of 72 ± 3°F, humidity of 30-70%, a minimum of 10 exchanges of room air

per hour, and a 12 hour light/dark cycle .

Observation - Each animal was observed daily for morbidity/mortality.

~-A o L}

-~-~-
p. 180

ST-131 Final Report 6-7-2005 SANITIZED

Body Weights - Each animal, was weighed immediately prior to initial dosing, daily

thereafter, and immediately prior to euthanasia .

Exposure chamber - 40-liter plexiglas chamber specially designed to observe animal

exposed to volatile fluorochemicals.

Exposure

L ~

2000 ppm exposure :

Four rats were exposed, whole body, to an atmosphere of approximately 2000 ppm (v/v)

of the test material for 4 hours. The atmosphere was generated by adding 0 .735 mL of

the test material to a closed 40-liter test chamber containing the animals . To maintain

proper oxygen and carbon dioxide levels, the test atmosphere was regenerated every one

hour and twenty minutes until the termination of the exposure .

Control :

Four rats were used as control for this study . They were not exposed to any test

atmosphere .

Results & Discussion

Four animals rat were exposed, whole body, to the test material at a concentration of 2000 ppm

for 4 hours. All animals appeared normal during the exposure and during the post exposure

observation phase. No urine was collected and gross necropsy was conducted 7-days post

exposure for all the rats. No abnormalities were found.

The body weight data prior to and after the exposure are listed in Table 1 for all the control and

treatment rats. All animals gained weight and there was no mortality.

Conclusions

The 4-hour inhalation LC50 for in rats is greater than 2000 ppm.

~o~ ~
p.181

ST-131 Final Report SANITIZED 6-7-2005

Signatures

Report Prepared By:

Date

Reviewed By:

Paul Lieder, Ph

Senior Toxicology

.D., DABT

Specialis

. Date

t

Study Director

IA © ~° -4-
p. 182

ST-131 Final Repo rt SANITIZED 6-7-2005

List of Tables:

Table 1 : Body Weights Data

List ofAppenilix:

Appendix 1 :1- ~ Characterization Report

LA ~~ .~-
p. 183

ST-131 Final Report 6-7-2005 - SANITIZED

Table 1 : Body Weights Data

Dose Tail Day 0 Day 4 Day 7

Animal # Group Marking BW BW BW

4R5438 BlackI' 194 227' 247

C

4R5439 000 ppm~ lack II 185 211 230

4R5440 Red 1 191 225 245

4R5441 Red lI 197 230 248

4R5442 Blue I 195 225 245

4R5443 control Blue II 196 221 244

4R5444 Blue III 192 219 239

4R5445 Blue IV 190 210 232

1- A ©% ,~,
p. 184

SANITIZED

P~ yo~ LA
p. 185

ST-135 Final Report 7-7-2005

SAN ITIZED

Final Report

5-Day Inhalation Toxicity Study ofL in Rats

(ToxDocs Study Number 05-027, MTDID 3663)

Study Director : Paul Lieder, Ph.D., DABT.

Senior Toxicology Specialist

3M Corporate Toxicology and Regulatory Services, 220-2E-02,

St. Paul, MN 55144

Study Toxicologist:

Testing Facility: 3M Strategic Toxicology Laboratory, 3M Center, 270-3S-05,

St. Paul, MN 55144

Sponsor :

CONFIDENTIALITY STATEMENT

This report contains the, unpublished results of research sponsored by 3M. These results may not

be published, either wholly or in part, or reviewed or quoted in any other publication without the

prior authorization of 3M.

~~\ ~_
p. 186

ST-135 Final Repo rt SANITIZED 7-7-2005

Study Summary

The purpose of this study was to evaluate the 5-day inhalation toxicity of [ 3(ToxDocs

Study Number 05-027, MTDID 3663) in rats. Four male Sprague Dawley rats were exposed,

whole body, to 2000 ppm of the test material for 6 hours for 5 consecutive days. Observations

were made continually during the study . Animal behavior was normal during the first three days

of exposure but occurrences of facial muscle twitching were noted on the fourth and fifth days of

exposure. All animals were active after removal from the chamber at the end of exposure ; and

all animals exhibited normal eating and drinking patterns. Urine samples were collected

everyday after the exposure and also on day 4 and day 7 post last exposure . Necropsy was

conducted on day 7 post last exposure and serum samples were collected . NMR analysis

performed on collected urine samples revealed 2 fluorinated acid metabolites and inorganic

fluoride. No clear connection could be established between these results and any biological

activity. No serum chemistry was performed . The facial twitching observed in this study may

be the result of neurotoxicity. If so, the mechanism of neurotoxicity action is presently unknown

but not likely due to chemical narcosis .

Study Obiective

The purpose of this study was to evaluate the 5-day inhalation toxicity of in rats

. Animals were observed throughout the study for clinical signs of toxicity and necropsy was

performed 7 days after the last exposure .

Experimental Procedures

Test Material :

The test material was supplied in a plastic bottle by 3M SMMD . The material was

identified as -3ToxDocs

Study Number 05-027, MTDID 3663) . Chemical characterization of the material was

performed by 19F/1H-NMR and is attached to this report as Appendix I. The

corresponding purity for was 97 .8%.

Species Selection :

The Animal Usage Application protocol (AUA 2004-0405) requires the use of a small

rodent species to optimize use of test material. Male Sprague Dawley rats supplied by

Harlan were selected as they are commonly used in acute inhalation toxicity testing to

generate data for human health risk assessment .

Animal Husbandry:

Housing - After exposure, the rats were placed in metabolism cages and urine was

collected every 18 hours for 5 days post exposure, and again on day 4 and day 7 after the

5'' exposure. When not in metabolism cages, the rats were group housed in standard

solid bottomed cages through out the study .

-~--
p. 187

ST-135 Final Report 7-7-2005

Diet/Water - Harlan Teklad LM-485 Mouse/Rat Sterilizable Diet, supplied by Harlan

Teklad, Madison, WI, and tap water were provided to all animals ad libitum throughout

the study, except during the exposure period.

Environment - Environmental controls for the animal room were set to maintain a

temperature of 72 ± 3°F, humidity of 30-70%, a minimum of 10 exchanges of room air

per hour, and a 12 hour light/dark cycle .

Observation - Each animal was observed daily when practical for morbidity/mortality .

Body Weights - Each animal was weighed immediately prior to initial dosing and daily

thereafter when practical and immediately prior to euthanasia.

Exposure chamber - 40-liter plexiglas chamber specially designed to observe animal

exposed to volatile fluorochemicals.

Exposure

2000 ppm exposure :

Four rats were exposed, whole body, to an atmosphere of approximately 2000 ppm (v/v)

of the test material for 6 hours for 5 days. The atmosphere was generated by adding

0.735 mL (corresponding to 0.08 liter of vapor) of the test material to a closed 40-liter

test chamber containing the animals. To maintain 02 and CO2 levels, every one and half

hours, the test chamber was flushed with fresh air over a 10-minute period to ensure

adequate oxygen level (static renewal) and test atmosphere was then regenerated by

adding 0.735 mL of the test material.

Results & Discussion

Four rats were exposed whole body to the test material daily at a concentration of 2000 ppm for

6 hours. Observations were made continually during the study .

First exposure :

Animal behavior was normal throughout the first exposure .

Second exposure :

Animal behavior was normal throughout the second exposure .

Third exposure :

Animal behavior was normal throughout the third exposure .

V

~ .~ __
p. 188

ST-135 Final Repo rt SANITIZED 7-7-2005

Fourth exposure :

During fourth exposure, occurrences of facial muscle twitching were noted about one

hour into the exposure . Such occurrences were noted throughout the rest of the exposure

but no seizure activity was observed. All the rats were responsive to sounds throughout

the exposure . At the end of exposure, all the rats were immediately removed from the

exposure chamber. They immediately resumed normal eating and drinking patterns after

they were returned to their cage s

Fifth exposure:

During the fifth exposure, occurrences of facial muscle twitching were also noted

throughout the exposure but to a lesser extent . No seizure activity was observed . Similar

to previous exposures, all the rats immediately resumed their normal eating and drinking

patterns after they were returned to their cages .

Urine samples were collected everyday after the exposure and also on day 4 and day 7 post last

exposure. The body weight data prior and after the exposure are listed in Table 1 for both

control and treatment rats. All animals gained weights and there was no mortality. Gross

necropsy was conducted 7 days after the last exposure and serum samples were collected . The

t -~l -treated rats had increased liver weights. No abnormality or other significant findings

was found during the necropsy . No serum chemistry was performed. NMR analysis (see

Appendix 2) performed on collected urine samples revealed 2 fluorinated acid metabolites and

inorganic fluoride. No clear connection could be established between these results and any

biological activity.

Conclusions

The facial muscle twitching observed in this study may be the result of neurological effect. If so,

the mechanism of neurotoxicity action is presently unknown but not likely due to chemical

narcosis.

1A

4
p. 189

ST-135 Final Report 7-7-2005

SANITIZED

Signatures

Report Prepared By :

Date

Reviewed By:

Paul Lieder, Ph

Senior Toxicology

.D., DABT. Date

Specialist

Study Director

'-A k5

,~-~--
P. 190

ST-135 Final Repo rt SANITIZED 7-7-2005

List of Tables:

Table 1 : Body Weights Data

List ofAppendices:

Appendix 1 : ~Characterization Report

Appendix 2 : Urine NMR Analysis

'-A

---6 - -
p. 191

ST-135 Final Report SANITIZED

7-7-2005

Table 1 : Body Weights, Liver Weights, Kidney Weights, and Testes Weights Data

Liver Kidneys Testes

Animal Dose Tail Day 0 Day 1 Day 2 Day 3 Day 4 Day 7 Day 11 Weight Weight Weight

# Group Marking BW BW BW BW (g) W BW (g) BW (g) (g) (g) (g)

4R3147 Black I 243 250 250 261 267 289 310 13 .7 2.4 3.6

4R3148 control Black II 206 209 210 217 220 236 245 10 2.3 3.0

-

4R3149 Black III 226 229 234 238 245 258 278 11 .6 2.0 3.3

4R3150 Black IV 205 206 208 212 212 222 237 9.6 2.0 3.0

4R3256 Blue I 235 228 239 241 247 265 281 12.2 2.0 3. 3

4R3257 Blue II 207 209 209 212 213 238 248 12.5 1.5 3.1 2000 ppm

4R3258 Blue III 229 233 236 241 246 261 273 13.8 2.4 3.3

4R3259 Blue IV 236 236 239 244 247 270 288 14.6 2.0 3.5

`t

-- ~---
p. 192

SANITIZED

PP 'A\% - 4DL. Lk
p. 193

Statement of Translator

Title: Bacterial Reverse Mutation Test of MTDID3663

Study No. : B051346

This study was conducted in General Testing Research Institute of Japan Oilstuff

Inspectors' Corporation . The original study report was written in Japanese . I hereby

~ declare that this report faithfully reflects the original study report as accurately as

possible.

SANITIZED

~~.5
p. 194

B051346

FINAL REPORT

Bacterial Reverse Mutation Test of MTDID3663

(Study No.: B051346)

June 8, 2006

GENERAL TESING RESEARCH INSTITUTE,

JAPAN OILSTUFF INSPECTORS' CORPORATION

--I--

`~2-~
p. 195

B051346

1 . GLP Compliance Statement

Title: Bacterial Reverse Mutation Test of MTDID3663

Study No. : B051346

This study was conducted in compliance with the following Good Laboratory Practice

Standards.

Standard Concerning Testing Facilities Implementing studies Related to New

Chemical Substances, (Pharmaceutical and Food affairs No . 1121003, November 17,

2003, No.3, Ministry of Health, Labor, and Welfare, Ministry of Economy, Trade,

and Industry, No.031121004, 2003 )

Notification for Standard of Testing Facilities Based on Rule for Industrial Safety and

Health, Article 34-3-2

(Notification No 76, Ministry of Labor, September 1, 1988 )

A Part Revision of Notification for Standard of Testing Facilities Based on Rule for

Industrial Safety and Health, Article 34(3)- 2

(Notification No 13, Ministry of Labor, March 29, 2000)

Management : Date: June 8, 2006 Eiichi Machigaki sealed

General Testing Research Institute of

Japan Oilstuff Inspectors' Corporation

Study director : Date: June 8, 2006 Masato Nakamura seale d

General Testing Research Institute of

Japan Oilstuff Inspectors' Corporation

-2-

A
p. 196

B051346

2. Table of Contents

1 . GLP Compliance Statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2 . Table of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3. Study Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3.1 Title . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3 .2 Study No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . 6

3 .3 Purpose . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3 .4 Guideline . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 6

3.5 GLP. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3.6 Sponsor. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

3.6.1 Sponsor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

3 .6.2 Organization under contract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

~ 3.7 Testing facility. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . . . . . . . . . . . . . . .. ~ . . . . . . . . . . . . . .. . . . . . . . ., . 7

3.8 Study director. . . .. . . . .. . . . . .. . . . .. . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 7

3.9 Study contributors . . . . . . . . .. . .. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . .. . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . 7

3.10 Study schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 7

3 .11 Retention . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . 8

3 .12 Retention materials. . . . . . . . . . . . . . . . . .. . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

4. Study Director Signature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

5 . Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

6. Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1

6.1 Test substance . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .11

6.1 .1 Name and characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

6.1 .2 Storage conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

6.1 .3 Handling precautions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

6.2 Control substances. . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .12

6.2.1 Negative control substance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

6.2.2 Positive control substance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

6.3 Tester strains [1 1

,

[21 . . . . .. . . . . . . . .. . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . .13

~. 6.3 .1 Tester strains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

6.3 .2 Reason for selection of the tester strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

6.3.3 Storage of the test strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

6.3 .4 Genetic properties of the test strain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

6.3 .5 Bacterial suspensions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 4

6.4 Media. . . . . . .. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .15

6.4.1 Preparation of liquid growth medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

6.4.2 Preparation of top agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

6.4.3 Minimal glucose agar plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5

6.5 S9 mix. . . . . . . . . . . . . . . . . . . . . . . .. . .. . . . : . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . .16

6.5 .1 S9. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

6. 5 .2 Cofactor mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

6.5.3 S9 mix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

--3-

1A
p. 197

B051346

6.6 Preparation of the test substance and positive control solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

6.6.1 Preparation of the test substance solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

6.6.2 Preparation of positive control solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 7

6.7 Doses of the test substance and positive controls. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . 18

6.7.1 Doses of the test substance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

6.7.2 Doses of the positive controls. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 8

6.8 Reverse mutation test PI . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .19

6.8.1 Selection of the test method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

6. 8 .2 Pre-incubation method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

6. 8 .3 Sterility test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

6. 8 .4 Observation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

6.8 .5 Measurement of colony number. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

6.8.6 Number of plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

6.8.7 Expression of data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

t 6.8.8 Acceptable criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

6.8 .9 Judgment of test results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

6.8 .10 Confirmation of the reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

7. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

7.1 Dose-finding test (Table 1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 21

7.2 Main test (Table 2 and Figure 1-5) . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . 21

7.3 Sterility test . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. .. . . . . . . . . . . . . . . . . . . . . . . . 21

8 . Discussion and Conclusion.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

9. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

10. Study Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

10.1 Environmental conditions that many have had an effect on the reliability of the study

results. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

10.2 Deviations from the protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 23

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Tables

Table 1 Result of the Dose-finding Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Table 2 Result of the Main Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Figures

Figure 1-5 Dose-response curve (Main Test ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Appendix

Appendix 1 Acceptable ranges of negative (solvent) and positive control values 31

Quality Assurance Statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

(Final page : 32)

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3. Study Outline

3.1 Title

Bacterial Reverse Mutation test of MTDID3663

3.2 Study No.

B051346

3.3 Purpose

To assess the mutagenicity of MTDID3663 in a reverse mutation assay using Salmonella

typhimurium and Escherichia coli.

3.4 Guideline

(1) Testing Method Concerning Designated Chemical

~ (November 21, 2003, Pharmaceutical and Food affairs, No.1121002,

Pharmaceutical and Food Safety Bureau, Ministry of Health, Labor, and Welfare,

November 13, 2003, Manufacturing Industries Bureau, No .2, Ministry of Economy,

Trade, and Industry, No .031121002, Environmental Policy Bureau, Ministry of the

Environment)

(2) Notification for Standard of Established by Minister of Labor Based on Rule for

Industrial Safety and Health, Article 57(3)- 1

(Notification No 77, Ministry of Labor, September 1, 1988 )

(3) Notification for Standard of Established by Minister of Labor Based on Rule for

Industrial Safety and Health, Article 57(3)- 1

(Notification No 67, Ministry of Labor, June 2, 1997)

3.5 GLP

(1) Standard Concerning Testing Facilities Implementing studies Related to New

Chemical Substances, (Pharmaceutical and Food Safety Bureau, Manufacturing

Industries Bureau, Environmental Policy Bureau, Ministry of the Environment,

( Pharmaceutical and Food- affairs No. 1121003, November 17, 2003, No .3,

November 21, 2003, Ministry of Health, Labor, and Welfare, Ministry of Economy,

Trade, and Industry, No.031121004)

(2) Notification for Standard of Testing Facilities Based on Rule for Industrial Safety

and Health, Article 34-3-2

(Notification No 76, Ministry of Labor, September 1, 1988 )

(3) A Part Revision of Notification for Standard of Testing Facilities Based on Rule for

Industrial Safety and Health, Article 34(3)-2

(Notification No 13, Ministry of Labor, March 29, 2000)

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3.6 Sponsor

3.6.1 Sponsor

Sumitomo 3M Limited

8-8, 3-Chome, Minamihashimoto, Sagamihara-shi, Kanagawa, Japan

3.6.2 Organization under contract

Mitsubishi Chemical Safety Institute Ltd .

1-30, Shiba 2-Chome, Minato-ku, Tokyo, Japan

3.7 Testing facility

General Testing Research Institute, Japan Oilstuff Inspector' Corporation

10-4, 1-Chome, Mikagetsukamachi, Higashinada-ku, Kobe-shi, Hyogo, Japan

3.8 Study director

Masato Nakamura

General Testing Research Institute, Japan Oilstuff Inspector' Corporation

3.9 Study contributors

Osamu Iwaihara, Emi Sekito, Aiko Seo

3.10Study schedul e

Initiation of the study: April 5, 2006

Dose-finding test: April 17, 2006 - April 20, 2006

Main test: April 25, 2006 - April 28, 2006

Completion of the study: June 8, 2006

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3.11Retention

The materials shown in the following section will be retained in the archives of the

Analytical Technical Laboratory. The test substance will be retained for whichever is

shorter, 10 years after receiving the notification according to the Japanese Industrial

Chemicals (Section 4-1, 4-2, 4(2)-2, 4(2)-3, 4(2)-8, 5(4)-2, 24-2, or 25(3)-2, No .117,

1973), 10 years after the notification according to the Japanese Industrial Safety and

Health Law (Section 57(3)-1), or the period that allows for stable storage without

deterioration in quality. The other materials will be retained for the whichever is longer,

10 years after receiving the notification to the Japanese Industrial Chemicals or 10 years

after the notification according to the Japanese Industrial Safety and Health Law .

Further retention will be determined in consultation with the sponsor.

~ . 3.12Retention materials

(1) Protocol

(2) Test substance

(3) Raw data

(4) Test substance and control substance records

(5) Test strain records

(6) Study result records

(7) Correspondence documents

(8) Final report

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4. Study Director Signatur e

Title: Bacterial Reverse Mutation Test of MTDID3663

Study No. : B051346

Study director : Date: June 8, 2006 Masato Nakamura sealed

General Testing Research Institute of

Japan Oilstuff Inspectors' Corporation

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5. Summary

Mutagenicity of the test substance, MTDID3663, was assessed in a bacterial reverse

mutation test using five strains, Salmonella typhimurium TA100, TA1535, TA98, and

TA1537 and Escherichia coli WP2 uvrA-. The test was conducted by the pre-incubation

method with and without S9 mix .

A dose-finding test was performed at 5000 μg/plate as the maximum dose using 8 doses

with ratio of 4. According to the results, the number of revertant colonies treated with

the test substance was less than twice of the negative control (solvent) in any tester strain

with or without S9 mix. Microbial growth inhibition and precipitation of the test

substance were not observed in any tester strain or in any dose with or without S9 mix .

A main test was performed at 5000 μg/plate as the maximum dose using 5 doses with

ratio of 2 based on the results of the dose-finding test . According to the results, the

number of revertant colonies treated with the test substance was less than twice of the

negative control in any tester strain with or without S9 mix. Microbial growth

inhibition and precipitation of the test substance were not observed in any tester strain or

in any dose with or without S9 mix .

From the results described above, it was concluded that MTDID3663 was not mutagenic

under the conditions employed in this study .

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6. Materials and Methods

6.1 Test substanc e

6.1.1 Name and characteristics

F

SANITIZED

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6.1.2 Storage conditions

Refrigeration, Shielded from light in an air tight container

6.1.3 Handling precaution s

Use safety equipment (rubber gloves, protective goggles, and a safety mask)

6.2 Control substances

6.2.1 Negative control substanc e

6.2.1 .1 Name and manufacturer and other related informatio n

Name (Abb.) Manufacturer LotNo. Grade Purity(%)

dimethyl sulfoxide Wako Pure Chemical EWH5027 for biochem

. min.99.5 (DMSO) Industries Ltd

.2.1.2 Reason for selection of the negative control substanc . 6 e

The solubility of the test substance is less than 230ppm(wt) in water, and the test

substance is soluble in DMSO and acetone in preliminary information . In the solvent

selection test, the test substance was not dissolved or suspended in distilled water at

50mg/mL, but was dissolved in DMSO at 50mg/mL and in acetone at 100mg/mL.

Increases in temperature, discoloration and foaming were not observed when the test

substance was mixed with DMSO . According to these results, DMSO was selected as the

solvent for the test substance in this study .

6.2.2 Positive control substance

6.2.2.1 Name, manufacturer and other related information

Name (Abb.) Manufacturer LotNo. Purity(%)

2-(2-Fury)-3-(5-nitro-2-furyl) Wako Pure Chemical SEL1402 99

.0 acrylamide (AF-2) Industries Ltd.

Sodium azide (NaN3) Wako Pure Chemical DWM4758 99

C Industries Ltd .8

.

9-Aminoacridine hydrate (9-AA) Aldrich Chemica l

Company 03024JR 98.8

N-Ethyl-N'-nitro-N-nitrosoguanidine Aldrich Chemical 12219AS 97

(ENNG) Company

2-Aminoanthracene (2-AA) Wako Pure Chemical ELJ6826 97

.4 Industries Ltd .

6.2.2.2 Reason for selection of the positive control substance

These positive control substances are widely used in reverse mutation assays using

bacteria and are recommended in the applied guidelines .

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6.3 Tester strains [ l] ' [21

6.3.1 Testerstrains

Stains Receivedfrom (Date)

Salmonella typhimurium

Japan Bioassay Research Center

TA100, TA1535, TA98, TA1537 (May 13, 1998)

Escherichia coli Japan Bioassay Research Center

WP2 uvrA- (May 13, 1998 )

6.3 .2 Reason for selection of the tester strain s

These strains are widely used in reverse mutation assays using bacteria and

( recommended in the applied guidelines.

6.3.3 Storage of the test strains

6.3.3.1 Composition

Bacterial suspension (8 .0 mL) cultured at 37 °C for 8 hours in nutrient broth were added

to 0.7mL DMSO (Wako Pure Chemical Industries Ltd .).

6.3.3.2 Storag e

Divided into freezing tubes (0 .4 mL) and frozen

6.3.3.3 Storage conditions

Below-80'C (Sanyo Ultralow freezer; MDF-192AT)

6.3.4 Genetic properties of the test strain

6.3.4.1 Genetic properties

S~~ Amino acid (2) Membrane Drug

requirement (l UV sensitivity mutation (

3

)

resistance (4)

TA100 his (base-pair change) A uvrB rfa +(pKM101)

TA1535 his (base-pair change) A uvrB rfa -

TA98 his (frame-shift) A uvrB rfa +(pKM101)

TA1537 his (frame-shift) A uvrB rfa -

WP2 uvrA- trp (base-pair change) A uvrA Wild type -

(1) his, histidine requirement; and trp , tryptophan requirement

(2) A uvrA and A uvrB represent deletion of uvrA or uvrB gene (coding for DNA

excision repair system), and the strains with each mutation have UV sensitivity.

(3) rfa denotes the deep rough character of cell wall, and the strains with this mutation

have crystal violet sensitivity.

(4) +(pKV1101) is the ampicillin resistance factor (R-factor), and the strains with this

plasmid have ampicillin resistance .

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6.3.4.2 Confirmation of genetic propertie s

The genetic properties of the test strains had already been checked with regard to the

following phenotypic characteristics. Strains having the required properties were used for

this study.

(1) Amino acid requirement

(2) W sensitivity

(3) Membrane mutation

(4) Drug resistance

6.3.5 Bacterial suspensions

6.3.5.1 Culture condition

Temperature : 37°C (kept at 7°C until culture)

Time: 8 hours

Method: Reciprocal shaking (shaking frequency: 90 times/minute)

Vessel : L-type test tube (volume : 22 mL)

Medium: Liquid growth medium (10 mL)

Stain and inoculate volume : A frozen stock suspension of the test strain was thawed and

0.02 mL was inoculated .

6.3.5.2 Measurement of cell number in bacterial suspension

After pre-culture, bacterial density of each strain was measured by a digital photometer

(Tietech Co., Ltd. Mini photo 518R), and then the bacterial suspension for counting the

number of cells was prepared by the stepwise dilution method. The suspension was

overlaid to a minimal glucose agar plate and incubated at 37°C for 48 hours. The cell

number was calculated by counting the number of colonies on the plate . The bacterial

suspension for the reverse mutation test was stored at room temperature .

Cell number of each bacterial suspension is shown in the following table ;

Strain Base-pair chan e type Frame-shift type

TA100 TA1535 . .WP2 uvrA- TA98 TA1537

Cell number pose-finding test 3.86 4.14 6.03 3.38 2.30

(x 10 9/mL) Main test 3.74 4.04 6.19 3.20 3.04

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B051346

6.4 Media

6.4.1 Preparation of liquid growth mediu m

Nutrient Broth No.2 (Oxoid Ltd., Lot No. 349915, 2.5 g) was dissolved in 100 mL of

purified water. The medium was autoclaved for 15 minutes at 121 °C and stored in a cold

place.

6.4.2 Preparation of top agar

6.4.2.1 Preparation of soft agar

Bacto-agar (Becton Dickinson and Company, Lot No. 5089012, 0.6 g) and sodium

chloride (Wako Pure Chemical Industries Ltd., 0.5 g) were added to 100 mL of purified

water. This mixture was autoclaved for 15 minutes at 121°C and then was stored at 45°C

until use .

6.4.2.2 Preparation of top agar

Each of the following each amino acid solution was added to the soft agar at a volume

ratio of 1 to 10 just before use . The top agar was kept at 45°C until use .

Salmonella typhimurium: 0.5 mmoUL D-biotin and 0.5 mmoUL L-histidine solution

mixture

Escherichia coli: 0.5 mmoUL L-tryptophan solution

6.4.3 Minimal glucose agar plate

6.4.3.1 Name

TESMEDIA AN MEDIUM

6.4.3.2 Manufacturer

Oriental Yeast Co ., Ltd.

6.4.3.3 No.

ANI170BV

~ 6.4.3.4 Used agar

Ina agar BA-30A (Ina Food Industry Co., Ltd, Lot No.511115)

6.4.3.5 Preparation date

February 16, 2006

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6.5 S9 mix

6.5.1 S9

6.5.1.1 Manufacturer

Kikkoman Corporation

6.5.1.2 Lot No.

RAA-539

6.5.1.3 Preparation date

March 10, 2006

6.5.1.4 S9 preparation

The S9 was prepared from the livers of 7-week old male Sprague-Dawley rats (body

weight: 215 to 272 g) treated with phenobarbital (PB, 4 times at 24-hour intervals at i .p.

doses of 30, 60, 60 and 60 mg/kg, respectively) and 5, 6-benzoflavone (once at a single

i.p. dose of 80 mg/kg on the same day of the third PB injection) .

6.5.1.5 Protein content

25 .63 mg/mL

6.5.1 .6 Storage conditions

Below-80 °C (Sanyo Ultralow freezer; MDF-192AT )

6.5.1 .7 Expiration date

September 10, 2006 (6 months after preparation)

6.5.2 Cofactor mix

6.5.2.1 Name

Cofactor-I

6.5.2.2 Manufacturer

Oriental Yeast Co ., Ltd.

6.5.2.3 Lot No.

999601

6.5.2.4 Cofactor preparation

Cofactor-I was dissolved in 9 mL of sterile purified water and filtrated with membrane

filter (pore size: 0.45 μm) to give the cofactor mix . The cofactor mix was freshly

prepared just prior to use .

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B051346

6.5.3 S9 mix

One milliliter of S9 was added to 9 mL of the cofactor mix to give the proper ratio for the

S9 mix. The S9 mix was freshly prepared just prior to use in each test and was kept in an

ice bath until use. The ingredients and their contents per milliliter of the S9 mix

S9 0

reparation were as follows.

.1mL

Magnesium chloride 8 μmol

Potassium chloride 33 μmol

D -Glucose 6 -phosphate 5 μmol

(3-NADPH 4 μmol

P-NADH 4 μmol

Sodium phosphate buffer (pH 7 .4) 100 μmol

{ 6.6 Preparation of the test substance and positive control solutions

6.6.1 Preparation of the test substance solutions

6.6.1.1 Preparation

(1) The test substance was weighed and dissolved in DMSO with vortex mixing to

make a 50mg/mL solution.

(2) A portion of this solution was diluted stepwise with DMSO to make test substance

solutions of each concentration

(3) All procedures, including those for weighing the test substance, dilution,

dispensation and treatment of the solutions, were performed under a yellow light at

room temperature .

6.6.1.2 Storage time and temperatur e

The storage period and temperature after preparation of the test substance solutions to the

time of use were as follows;

Dose-finding test: 2 hours and 20 minutes at room temperature (26°C)

Main test: 1 hour and 55 minutes at room temperature (25°C)

6.6.2 Preparation of positive control solution s

6.6.2.1 Preparation

Positive control solutions were prepared by the following method;

(1) NaN3 was dissolved in DW (Wako Pure Chemical Industries Ltd .) and AF-2, 9-AA,

ENNG and 2-AA were dissolved in DMSO(Dojndo Laboratories) .

(2) The above preparations were diluted with each solvent for preparing stock solutions .

6.6.2.2 Usage of the positive control solution s

The positive control solutions were prepared and stored as stock solutions, the stock

solutions of positive control were thawed and used for the study before use .

2-AA stock solution was diluted more with DMSO at tested concentration .

The remainders of thawed solution for usage in the assay was not reuse d

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6.6.2.3 Storage of the positive control solutions

Divided into freezing tubes (1 .OmL) and frozen

6.6.2.4 Storage conditions

Below-20'C (Sanyo MPR-411FS) (AF-2, NaN3, ENNG, and 9-AA)

Below-80'C (Sanyo Ultralow freezer; MDF-192ATI (2-AA)

6.7 Doses of the test substance and positive controls

6.7.1 Doses of the test substance

6.7.1.1 Dose-finding test

According to the test guidelines, the dose-finding test was performed at doses in the

following table.

Strains Dose(μ late)

( With and without S9 mix

TA100, TA1535, TA98, 0.305, 1 .22, 4.88, 19.5, 78.1, 313, 1250, 5000

TA1537, WP2 uvrA6.7.1 .2 Main test

According to the results of the dose-finding test, revertant colonies did not incre ase and

microbial growth inhibition was not observed in any tester strains with or without S9 mix .

Based on these results, the main test was performed at doses in the following table.

Str

ains Dose μg/ late)

Without S9 mix With S9 mix

TA100, TA1535, TA98, 313, 625, 1250, 2500, 5000 313, 625, 1250, 2500, 5000 TA1537, WP2 uvrA6.7.2 Doses of the positive controls

6.7.2.1 Names and doses of the positive controls

Strains Names and doses (μ late)

~. - Without S9 mix With S9 mix

TA100 AF-2 0.01 2-AA 1.0

TA1535 NaN3 0.5 2-AA 2.0

TA98 AF-2 0.1 2-AA 0.5

TA1537 9-AA 80 2-AA 2.0

WP2 uvrA- ENNG 2 2-AA 10

6.7.2.2 Reason for dose selection of the positive control

The positive controls are known to induce increasing revertant colonies of the tester

strains at these doses .

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B051346

6.8 Reverse mutation test 131

6.8 .1 Selection of the test metho d

This study was preformed using the pre-incubation method with and without S9 mix .

6.8.2 Pre-incubation method

(1) For each treatment, 0 .1 mL of the test substance solution, negative (solvent) control

or positive control solution was added into a sterilized test tube .

(2) For assays without S9 mix, 0 .5 mL of 0.lmol/L sodium phosphate buffer (pH 7 .4)

was mixed and 0 .1 mL of the bacterial suspension was added to this mixture .

(3) For assays with S9 mix, 0 .5 mL of S9 mix was mixed in place of 0 .lmol/L sodium

phosphate buffer.

(4) The mixture was incubated with gentle shaking (Shaking frequency : 120

times/minute) for 20 minutes at 37°C (pre-incubation) .

(5) After pre-incubation, 2 mL of the molten top agar was added to this mixture and

~ then poured onto a minimal glucose agar plate.

(6) After the overlaid agar had solidified, the plates were incubated for 48 hours at

37°C.

6.8.3 Sterility tes t

In both the tests, bacterial contamination was observed using one plate for each of the

highest dose of the test substance solution and the S9 mix .

(1) 0.1 mL of the highest dose of the test substance solution or 0.5 mL of S9 mix was

mixed with 2 mL of the molten top agar.

(2) The mixture was poured onto a minimal glucose agar plate.

(3) After the overlaid agar had solidified, the plates were incubated for 48 hours at 37°C

and then checked for bacterial contamination with the unaided eye .

6.8.4 Observation

Microbial growth inhibition : Background lawn of the bacterial cells that have amino-acid

requirement was observed by a stereoscopic microscope (40x Nikon, SMZ-10), and

{ microbial growth inhibition was judged by the relationship between the test substance

treated plates and the non-treated plate .

Precipitation: Precipitation was judged by observation of the plate surface by unaided

eyes.

6.8.5 Measurement of colony number.

Revertant colonies were measured using an automatic colony counter (Toyo Sokki Ltd .,

CA-7) or manual counting. Revertant colonies in positive control treatment groups, in

TA100 of test substance treatment groups, and in the test substance treatment groups in

which many revertant colonies were observed were measured using an automatic colony

counter and the others measured by manual counting . Correction of the measuring area

was employed using instrumental analysis .

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6.8.6 Number of plate

Dose-finding test: 2 plates/dose

Main test: 2 plates/dose

6.8.7 Expression of data

The mean of the measured number of revertant colonies was calculated for the negative

(solvent) control, the positive control and the test substance treatments . The mean was

expressed by rounding to the first decimal place .

6.8.8 Acceptable criteria

The test was accepted as valid if all the following criteria were satisfied .

(1) The negative (solvent) control values (mean) and the positive control values (mean)

are within the proper ranges calculated based on the historical data at our laboratory.

(2) The positive control values (mean) show clear positive responses in the respective

test strains, as evidenced by the number of revertant colonies being greater than

2-fold of the respective negative (solvent) control value.

(3) There are more than 4 doses showing no microbial growth inhibition and more than

5 doses applicable to the evaluation.

(4) The result of the sterility test indicates that there is no bacterial contamination .

(5) There are no plates that became invalid for measurement due to contamination or

other unexpected situations.

6.8.9 Judgment of test results

Test substance was judged to have mutagenicity (positive) when the test substance

induced a dose-dependent increase in the number of the revertant colonies (mean) to a

level equal to or greater than 2-fold of the negative (solvent) control value (mean value)

in any one of the tester strains with or without S9 mix, and when the dose-dependent

increase was reproducible. Other results were judged to be negative. When the test

substance was positive, mutation activity (number of revertant colonies/mg) was

calculated. No statistical analysis was performed with the test results in this study.

6.8.10 Confirmation of the reproducibility

The reproducibility of the test result was confirmed in the dose-fmding and main tests .

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7. Results

7.1 Dose-finding test (Table 1)

Number of revertant colonies: The number of revertant colonies in the test

substance-treated groups was less than twice that in

the corresponding negative (solvent) control in all

test strains with and without S9 mix .

Microbial growth inhibition: Microbial growth inhibition was not observed in any test

strains with or without S9 mix.

Precipitation : Precipitation of the test substance was not observed in any dose with or

without S9 mix.

~ 7.2 Main test (Table 2 and Figure 1-5)

Number of revertant colonies: The number of revertant colonies in the test

substance-treated groups was less than twice that in

the corresponding negative (solvent) control in all

test strains with and without S9 mix.

Microbial growth inhibition: Microbial growth inhibition was not observed in any test

strains with or without S9 mix.

Precipitation: Precipitation of the test substance was not observed in any dose with or

without S9 mix.

7.3 Sterility test

Bacterial or fungous contamination was not observed in plates of the highest dose of the

test substance solution or S9 mix in either two tests .

~

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8. Discussion and Conclusion

In the dose-finding and main tests, the number of revertant colonies induced by the test

substance was less than twice of that of the corresponding negative control value for all

test strains with and without S9 mix. Furthermore, both tests indicated that they were

performed accurately as the tests satisfied acceptable the specified criteria :

From the results described above, it was concluded that MTDID3663 was not mutagenic

under the conditions employed in this study .

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