### TRANSLATION STATEMENT

Study Title: Bacterial Reverse Mutation Test of Study Number: SR09242

I, the undersigned, hereby certify that this is a true and accurate translation from the original Japanese final report into English, which was reviewed properly.

Safety Research Institute for Chemical Compounds Co., Ltd.
Translated by: 

## /lf -lO (d.- 1 

### Koko Nagaoka, Study Director Date

### Study Title:
Study Number: FINAL REPORT 

### (Translation)

Bacterial Reverse Mutation Test of

SR09242

Safety Research Institute for Chemical Compounds Co., Ltd.

-1/38-

### STATEMENT Study Title: Bacterial Reverse Mutation Test of Study Number: SR09242 I. This study was conducted in compliance with GLP standards "On the Standard for the Test Facility Conducting Tests Concerning New Chemical Substances, etc.," Notification No. 1121003 ofthe Pharmaceutical and Food Safety Bureau (PFSB), Ministry ofHealth, Labour and Welfare, Japan (MHLW), November 21, 2003; No. 3 ofthe Manufacturing Industries Bureau, Ministry ofEconomy, Trade and Industry, Japan (METI), November 17, 2003; and No. 031121004 ofthe Environmental Policy Bureau, Ministry ofthe Environment, Japan (MOE); and "Partial Amendment of 'On the Standard for the Test Facility Conducting Tests Concerning New Chemical Substances, etc.'," Notification No. 0704001 ofthe PFSB, MHLW, July 4, 2008; No.2 ofthe Manufacturing Industries Bureau, METI, June 30, 2008; and No. 080704001 ofthe Environmental Policy Bureau, MOE. The test method used in this study was based on "On the Test Method Concerning New Chemical Substances, etc." (Notification No. 1121002 ofthe PFSB, MHLW, November 21, 2003; No.2 ofthe Manufacturing Industries Bureau, METI, November 13, 2003; and No. 031121002 ofthe Environmental Policy Bureau, MOE); and "Partial Amendment of 'On the Test Method Concerning New Chemical Substances, etc.'" (Notification No. 1120001 ofthe PFSB, MHLW, November 20, 2006; No. 2 ofthe Manufacturing Industries Bureau, METI, November 13, 2006; and No. 061120001 ofthe Environmental Policy Bureau, MOE) 2. This study was conducted in compliance with the study protocol, and no environmental factors that could have affected the reliability ofthe study were found.
Safety Research Institute for Chemical Compounds Co., Ltd.

Name and seal affixed in the original August 10, 2010 Koko Nagaoka, Study Director Date

-2-1/2 

### QUALITY ASSURANCE STATEMENT Study Title: Bacterial Reverse Mutation Test of Study Number: SR09242 This study was inspected by the Quality Assurance Unit ofSafety Research Institute for Chemical Compounds Co., Ltd. as follows: Phase ofstudy inspection Date ofinspection Date ofreport to Date ofreport to Study Director Management Study protocol April 23,2010 April 23, 2010 April 23, 2010 Study protocol amendment (No. I) June 4, 2010 June 4, 2010 June 7, 2010 Study protocol amendment (No. 2) June 29, 2010 June 29, 2010 June 29, 2010 Receipt, labeling and storage of April23, 20IO April 23,2010 April23, 2010 test substance Preparation oftest substance May 19,2010 May I9, 2010 May 19,2010 Conduct ofstudy May 19,2010 May I9, 2010 May 19,2010 Observation and colony counting May 21,2010 May 21,2010 May 21,2010 Raw data June 23,2010 June 23, 20I0 June 23, 2010 Final report (draft): Tables and June 23, 20 I0 June23,2010 June 23,2010 Figures Final report (draft): Text June 23, 20 I0 June 23,2010 June23, 2010 Final report August IO, 2010 August IO, 2010 August 10, 2010 1. This study was conducted in compliance with "On the Standard for the Test Facility Conducting Tests Concerning New Chemical Substances, etc.," Notification No. 1121003 ofthe Pharmaceutical and Food Safety Bureau (PFSB), Ministry ofHealth, Labour and Welfare, Japan (MHLW), November 21, 2003; No.3 ofthe Manufacturing Industries Bureau, Ministry ofEconomy, Trade and Industry, Japan (METI), November 17, 2003; No. 031121004 ofthe Environmental Policy Bureau, Ministry ofthe Environment, Japan (MOE); "Partial Amendment of'On the Standard for the Test Facility Conducting Tests Concerning New Chemical Substances, etc.'," Notification No. 0704001 ofthe PFSB, MHLW, July 4, 2008; No.2 ofthe Manufacturing Industries Bureau, METI, June 30, 2008; and No. 080704001 ofthe Environmental Policy Bureau, MOE; "On the Test Method Concerning New Chemical Substances, etc." (Notification No. 1121002 ofthe PFSB, MHLW, November 21, 2003; No.2 ofthe Manufacturing Industries Bureau, METI, November 13, 2003; and No. 031121002 ofthe Environmental Policy Bureau, MOE); and "Partial Amendment of 'On the Test Method Concerning New Chemical Substances, etc."' (Notification No. 1120001 ofthe PFSB, MHLW, November 20, 2006; No.2 ofthe Manufacturing Industries Bureau, METJ, November 13, 2006; and No. 061120001 ofthe Environmental Policy Bureau, MOE).



### -3-

### SR09242 2/2

2. 

### The Quality Assurance Unit has reviewed the final report and determined the following: this study was conducted in compliance with the study protocol, the methods and procedures ofthis study were accurately described in this report, and the results presented in this report accurately reflect the raw data generated during this study. Safety Research Institute for Chemical Compounds Co., Ltd.

Name and seal affixed in the original August I0, 2010 Taku Katano, QA Representative Date

-4-

### SR09242 CONTENTS 

### Page Title page ······· ······················· ··· ······················ ····· ········· ···· ··· ·········· ····· 1 

### STATEMENT······ ··· · ··· ······ · ······ ··· ······ ···· ······ ··· · ·· ····· · ···· · ········ · · · ······ · · ···· 2 QUALITY ASSURANCE STATEMENT······ ········· ······ · ·· ·· ············· · · ··· ········· 3 CONTENTS· ·· · ··· ··············································· · ·········· ·· · ·· ·· ········ · · · · ····5 Study title, study number, purpose ofthe study, Good Laboratory Practice

standards and test guidelines, and study sponsor ··· ············ ············· ····· ···· ······ 7 Test facility, Study Director, study personnel, and testing period ··········· ····· ·········· 8 SUMMARY····· · ·· ······················· · ················ ······· ·· ·· ·········· ·· · ·· ··········· ···9 INTRODUCTION···· · ········ ·· ········ · ··· ···· · · ······· ····· ·········· · · ·· ······ · ···· ··· ··· ·· · I0 MATERIALS AND METHODS ································· ············ ················· 10 RESULTS· ·· ··· · · · · ·········· ················· ··· ······· ····· · ·· ······ · ·························· 18 DISCUSSION· · · ·· · ·· ·· · ··· ·· ························ · ··· · ···· ·· ··········· · ·············· ·· ··· ·· 19 ENVIRONMENTAL FACTORS THAT MIGHT HAVE AFFECTED THE RELIABILITY OF THE TEST RESULTS ·· ·· · ······ ········ · ·· ·· · · ········· ·· ···· 20 STORAGE OF DOCUMENTS · ····· · ············· ·· · ·· · ··· ··· ·· ··· ··· ·· ········· ············· 20 NAME AND SEAL OF STUDY DIRECTOR ·············································· 20 Tables Table I

Table 2

Table 3

Table 4

Table 5 Reverse mutation test of in Salmonella typhimurium 

### TA 1 

### 00, TA 1535, TA98, TA1537 and Escherichia coli WP2uvrA 

### without metabolic activation (dose-finding test) (SR09242) ····· ··· ········· ······ 21 Reverse mutation test of in Salmonella typhimurium 

### TA100, TA1535, TA98, TA1537 and Escherichia coli WP2uvrA 

### with metabolic activation (dose-finding test) (SR09242) ··· · ·· · · · · ··· ··· ···· ··· 22 Reverse mutation test of in Salmonella typhimurium 

### TA100, TAl 535, TA98, TA1537 and Escherichia coli WP2uvrA 

### without metabolic activation (main test) (SR09242) ···················· ·· ···· ··· ··· 23 Reverse mutation test of in Salmonella typhimurium 

### TA 100, TA1535, TA98, TA1537 and Escherichia coli WP2uvrA 

### with metabolic activation (main test) (SR09242) ·························· ······ 24 Reverse mutation test of in Salmonella typhimurium 

### TA98 without metabolic activation (confirmatory test) (SR09242) ·· ······ ··· · 25

- 5 -

### SR09242 Figures Figure 1-1 Reverse mutation test of in Salmonella typhimurium TA100 without metabolic activation (dose-response curves) (SR09242) ···· ··· 26 Figure 1-2 Reverse mutation test of in Salmonella typhimurium TA1 00 with metabolic activation (dose-response curves) (SR09242) ··· ········ 27 Figure 2-1 Reverse mutation test of in Salmonella typhimurium TA1535 without metabolic activation (dose-response curves) (SR09242) ······ · 28 Figure 2-2 Reverse mutation test of in Salmonella typhimurium TA1535 with metabolic activation (dose-response curves) (SR09242) ··· ····· ··· 29 Figure 3-1 Reverse mutation test of in Escherichia coli WP2uvrA without metabolic activation (dose-response curves) (SR09242) ··· · ······· ···· ·· 30 Figure 3-2 Reverse mutation test of in Escherichia coli WP2uvrA with metabolic activation (dose-response curves) (SR09242) ···· ······· ······ 31 Figure 4

- 1 Reverse mutation test of in Salmonella typhimurium TA98 without metabolic activation (dose-response curves) (SR09242) ······· 32 Figure 4

-2 Reverse mutation test of in Salmonella typhimurium TA98 with metabolic activation (dose-response curves) (SR09242) ··········· 33 Figure 5-1 Reverse mutation test of in Salmonella typhimurium TAl 537 without metabolic activation (dose-response curves) (SR09242) ····· ·· 34 Figure 5-2 Reverse mutation test of in Salmonella typhimurium TA1537 with metabolic activation (dose-response curves) (SR09242) ··· ········ 35 Appendices Appendix 1-1 Analysis table (January 14, 2010) · ··· ·· · ··· ··· ··· ·· ·· · · ·········· ···· · · 36 Appendix 1-2 Analysis table (August 1, 2010) ····· · · · · · · · ··· ···· ····· ·· ···· ·· · · · ··· ·· 37 Appendix 2 Historical control data for reverse mutation test······· ······ · ·· ·· ·· ·· 38



### - 6-SR09242 Study title: Bacterial reverse mutation test of Study number: SR09242 Purpose ofthe study: was investigated for the potential to induce genetic mutation in bacteria using 

### Salmonella typhimurium and 

### Escherichia coli. Good Laboratory Practice standards and test guidelines Good Laboratory Practice (GLP) standards:

Test guidelines:

Study sponsor Name: Address: Study monitor: "On the Standard for the Test Facility Conducting Tests Concerning New Chemical Substances, etc.," Notification No. 1121 003 ofthe Pharmaceutical and Food Safety Bureau (PFSB), Ministry ofHealth, Labour and Welfare, Japan (MHLW), November 21, 2003; No.3 ofthe Manufacturing Industries Bureau, Ministry ofEconomy, Trade and Industry, Japan (METI), November 17, 2003; and No. 031121004 ofthe Environmental Policy Bureau, Ministry ofthe Environment, Japan (MOE) "Partial Amendment of 'On the Standard for the Test Facility Conducting Tests Concerning New Chemical Substances, etc.'," Notification No. 0704001 ofthe PFSB, MHLW, July 4, 2008; No. 2 ofthe Manufacturing Industries Bureau, METI, June 30, 2008; and No. 080704001 ofthe Environmental Policy Bureau, MOE "On the Test Method Concerning New Chemical Substances, etc." (Notification No. 1121 002 ofthe PFSB, MHL W, November 21, 2003; No.2 ofthe Manufacturing Industries Bureau, METI, November 13, 2003; and No. 031121002 ofthe Environmental Policy Bureau, MOE) "Partial Amendment of 'On the Test Method Concerning New Chemical Substances, etc."' (Notification No. 1120001 ofthe PFSB, MHLW, November 20, 2006; No.2 ofthe Manufacturing Industries Bureau, METI, November 13, 2006; and No. 061120001 ofthe Environmental Policy Bureau, MOE)



### - 7-

### SR09242

Test facility Name: Address: Management: Study Director Safety Research Institute for Chemical Compounds Co., Ltd. 363-24 Shin-ei, Kiyota-ku, Sapporo 004-0839, Japan Masao Kiguchi
Name: Koko Nagaoka Company and division: Safety Research Division, Safety Research Institute for Chemical Compounds Co., Ltd. Study personnel Test substance management: Shiho Kodama, Misako Ohkubo, Kohtaro Kawamura, and Takashi Sakakibara Test operation: Koko Nagaoka, Takashi Sakakibara, and Wataru Shimatani Testing period Study initiation: Receipt oftest substance: Start oftest operation: Dose-finding test Initiation ofsubculture: Treatment with test substance: Colony counting: Main test Initiation ofsubculture: April23, 2010 January 18,2010 May 19,2010 May 18,2010 May 19,2010 May 21, 2010 June 1, 2010 Treatment with test substance: June 2, 201 0 Colony counting: June 4, 2010 Confirmatory test Initiation ofsubculture: Treatment with test substance: Colony counting: Experiment completion: Study completion: June 8, 2010 June 9, 2010 June 11,2010 June 11, 2010 August 10,2010



### - 8-SR09242 

## SUMMARY The potential of to induce genetic mutation in bacteria was evaluated by a reverse mutation test using 

### Salmonella typhimurium TAIOO, TA1535, TA98, TA1537 and 

### Escherichia coli WP2uvrA. The test was conducted by the pre-incubation method, in the non-activated assay (in the absence ofthe S9 mix metabolic activation system) and activated assay (in the presence ofthe S9 mix metabolic activation system). In the dose-finding test, the highest dose ofthe test substance was set at 5000 flg/plate, and a total of7 doses (5 to 5000 flg/plate) were set in approximately threefold dilution series (6-step serial dilution) for each test strain in both the non-activated and activated assays. In the main test, the highest dose ofthe test substance was set at 5000 flg/plate, and a total of7 doses (78.1 to 5000 flg/plate) were set in a twofold dilution series (6-step serial dilution) for each test strain in both the non-activated and activated assays. In addition, a confirmatory test was conducted using S. 

### typhimurium TA98 by the non-activated assay at the same doses as in the main test. The results ofthe dose-finding test were as follows: In the non-activated assay using S. 

### typhimurium TA98, the average numbers ofrevertants at 150 to 1500 Jlg/plate increased and was twice or more than in the negative control group, which was not dose-related. The average number ofrevertants in the test substance groups was less than twice that in the negative control group in the other test strains both the non-activated and activated assays, and no dose-related increase in the number ofrevertants was noted. No inhibition ofcell growth or precipitation ofthe test substance was detected in any test strains, either the non-activated and activated assays. The results ofthe main and confirmatory tests were as follows: the average number of revertants in the test substance groups were less than twice that in the negative control group in each test strain both the non-activated and activated assays, and no dose-related increase in the number ofrevertants was noted. No inhibition ofcell growth or precipitation ofthe test substance was detected in any test strains, under all test conditions used in these tests. In the dose-finding, main, and confirmatory tests, all the average numbers ofrevertants in the negative control group ofall test strains were within the control range based on the historical control data ofthe test facility. The average number ofrevertants in the positive control group clearly increased and was twice or more that in the negative control group. These results confirmed that each test strain had appropriate sensitivity to the mutagens. On the basis ofthese results, the increase in the numbers ofrevertants inS. 

### typhimurium TA98 in the non-activated assay in the dose-finding test was not dose-related, which showed no reproducibility; therefore, this was not considered to be positive reaction. In conclusion, is not mutagenic to the bacteria under the conditions ofthis test.



### - 9-

### SR09242 INTRODUCTION 

### The potential of to induce genetic mutation in bacteria was evaluated by a reverse mutation test using Salmonella typhimurium TA100, TA1535 , TA98, TA1537 and Escherichia coli WP2uvrA. 

### The test was conducted by the pre-incubation method, in the non-activated assay (in the absence ofthe S9 mix metabolic activation system) and activated assay (in the presence ofthe S9 mix metabolic activation system)
.

1. 

### Test substance 

### Name: Abbreviation: CAS No. : MATERIALS AND METHODS



### Molecular formula: Structural formula :

Molecular weight: Physicochemical properties: LotNo. : Supplier: Amount obtained:

Stability:

Storage conditions : Study sponsor 2 containers (NET 1000 g and 1090 g, shared with related tests) Obtained as an aqueous solution (clear and colorless liquid) containing solid content 24.0%, impurities 0.85% (to the solid content), and Cl ion 35 ppm (Appendix 1-1). Mter completion oftest operation, the analysis results of the test substance were obtained and stability was confirmed (Appendix 1-2)
. Containers should be sealed to avoid moisture absorption
. Direct sunlight is strictly prohibited (protected from light), and attention should be paid for water and high humidity

- 10-

### Storage location:

Caution in handling: SR09242 (measured temperature: 2 to 8 °C). Test substance storage room (January 18 [date received] to April29, 2010) and mutagenicity test room (April29 to June 9, 20I0 [date oftreatment with the test substance in the confirmatory test]) Sufficient ventilation should be provided in the workplace. Proper protection equipment such as protection glasses and gloves are used
. Generating dust should be avoided. Remaining test substance: After completion ofall test operation including that in the related tests, the remaining test substance was returned to the supplier. 

### 2. Preparation oftest substance Preparation method: The test substance was dissolved in and diluted with Japanese Pharmacopoeia water for injection (Lot No. 8L88, Otsuka Pharmaceutical Factory, Inc.) to the prescribed concentrations. Calculations for the preparation was made on the basis that the purity ofthe test substance was 24.0% by the following procedure: For the dose-finding test, the 50 mg/mL preparation (value calculated from the purity: 208.33 mg/mL) was serially diluted with Japanese Pharmacopoeia water for injection in approximately threefold dilution series to prepare 15, 5, 1.5, 0.5, 0.15 and 0.05 mg/mL preparations
. For the main and confirmatory tests, the 50 mg/mL preparation (value calculated from the purity: 208.33 mg/mL) was serially diluted with the vehicle in a twofold dilution series to prepare 25, 12.5, 6.25, 3.13, 1.56, and 0.781 mg/mL preparations. Rationale for selection ofvehicle: Because the test substance is soluble in water. Preparation frequency: The preparation was prepared before use, and used within 2.0 h in the dose-finding test, within 2.1 h in the main test, and within 0.7 h in the confirmatory test. Caution in preparation: Prepared in a clean bench using protection glasses, protection mask, masks, gloves, and white lab coat to avoid inhalation and contact with eyes, skin and clothes. Confirming for stability ofpreparations: Preparations showed no visible reactivity (such as discoloration, generation ofheat, and foaming) at preparation oftest substance in the dose-finding, main, and confirmatory tests.

- 11

-SR09242 Disposal ofthe remaining preparations: Remaining preparations were collected as industrial waste to be incinerated. 

### 3. Negative control substance Japanese Pharmacopoeia water for injection (Lot No. 8L88; Otsuka Pharmaceutical Factory, Inc.), which was the vehicle used to prepare the test substance, was used as the negative control substance without any further preparation. 

### 4. Positive control substances and their preparation The known mutagens shown in the table below were used as the positive control substances. These positive control substances were stored in a cool and dark place (set at 2 to 8 °C). Positive control substances were prepared to the concentrations shown in the table below without correction for the content. The preparations were aliquoted and cryopreserved at -20°C or below, and used within 1
.8 h after thawing. The preparations were used within 9 months after the day ofpreparation (expiration: one year after preparation). Remaining preparations ofthe positive control substance were collected as the industrial waste to be incinerated. Positive control substance Concentration Preparation vehicle 2-(2-Furyl)-3-(5-nitro-2-furyl) 0.1 and Dimethyl sulfoxide

acrylamide (content: 98.3%) Lot No. WF032 Lot No. SDJ4376 I 11g/mL Dojindo Laboratories Wako Pure Chemical Industries, Ltd. Sodium azide (purity: 99
.8%) Japanese Pharmacopoeia water for Lot No. SDH6348 5 11g/mL injection Wako Pure Chemical Industries, Ltd
. Lot No. 8L88 Otsuka Pharmaceutical Factory, Inc. 9-Aminoacridine hydrochloride Dimethyl sulfoxide

hydrate (purity: 99.9%) 800 11g/mL Lot No. WF032 Lot No. S32398-347 Dojindo Laboratories Sigma-Aldrich Corporation 2-Aminoanthracene (content: 97.2%) 5, 10, 20 and Dimethyl sulfoxide Lot No. TSP5974 100 11g/mL Lot No. FW032 Wako Pure Chemical Industries, Ltd. Dojindo Laboratories 

### 5. Bacterial test strains The test strains used in this test were 

### Salmonella typhimurium (expressed as 

### "S.typhimurium" hereinafter) TAlOO, TA1535, TA98, and TA 1537 and 

### Escherichia coli (expressed as 

### "E.coli" hereinafter) 

### WP2uvrA. These test strains were obtained from National Institute ofHygienic Sciences (current National Institute ofHealth Sciences) on October 18, 1991. The test strains were selected because they were widely accepted as the bacteria suitable for investigating the genotoxic potential of chemicals. To 8 mL ofthe culture ofeach test strain, 

### 0.7 mL ofdimethyl sulfoxide (Lot No.

- 12-SR09242 BTl59; Dojindo Laboratories) was added. The culture was aliquoted and cryopreserved at -80°C or below. Using an aliquot ofthe culture ofeach test strain, characteristics ofthe test strain (amino acid-requirement, deep rough character 

### [rfa], UV sensitivity, and drug resistance), and the number ofrevertants in the negative and positive control substance groups were checked, and the test strains in which these characteristics were normal were used in the test. 

### 6. Culture medium 6.1. Culture medium for subculture As the culture medium for subculture, nutrient broth medium (Oxoid Nutrient Broth No.2; Lot No. 464616; Oxoid Ltd.) was prepared with Japanese Pharmacopoeia water for injection (Lot No. 8L88; Otsuka Pharmaceutical Factory, Inc.) to yield a concentration of25 giL. To the culture media ofS. 

### typhimurium TA98 and TA 100, ampicillin sodium (Lot No. M8B0619; Nacalai Tesque, Inc.) was added to yield a concentration of25 flg/mL before use. 6.2. Culture medium for the test (minimum glucose agar medium: expressed as "a plate" hereinafter) The components in 1000 mL ofthe minimum glucose agar medium (Vitalmedia AMT-0; Lot No.DZLB2Q01; manufactured on February 26, 201 0; Kyokuto Pharmaceutical Industrial Co., Ltd.) used as the culture medium for the test are shown in the following table. Components in 1000 mL ofculture medium for the test Magnesium sulfate heptahydrate Citric acid monohydrate Potassium phosphate dibasic Ammonium phosphate monobasic Sodium hydroxide Glucose Agar powder (Oxoid AgarNo.1; Lot No. 1 073337-02) 6.3. Top agar 0.2 g 2.0 g 10.0 g

1.92 g 0.66 g 20.0 g 15.0 g

Soft agar (shown as [A] in the table below) and amino acid solution (shown as [B] in the table below) with the components shown in the table below were prepared with Japanese Pharmacopoeia water for injection (Lot Nos. 8L88 and 9K88; Otsuka Pharmaceutical Factory, Inc.). Before use, (A) and (B) were mixed in a volume ratio of 10:1. Amino acid solution ofL-histidine and D-biotin was applied to 

### S.typhimurium, and that ofL-tryptophan was applied to 

### E. 

### coli. These top agars were kept at 47°C until use.



### - 13-SR09242 Components oftop agar (A) Soft agar BactoTM Agar (Lot No. 9131 109; Becton, Dickinson and Company) 0.6% Sodium chloride (Lot No. I06P5602; Kanto Chemical Co., Ltd.) 0.5% (B) Amino acid solution Solution ofL-histidine and D-biotin 0.5 mmoi/L each (L-histidine; LotNo. WKK2889; Wako Pure Chemical Industries, Ltd.) (D-biotin; Lot Nos. PEN6828 and CDJ4769; Wako Pure Chemical Industries, Ltd.) or L-tryptophan solution 0.5 mmol/L (L-tryptophan; LotNo. PEP6208; Wako Pure Chemical Industries, Ltd.) 7. S9 

### mix S9 mix was prepared with S9 (Lot No. RAA-609; manufactured on February 5, 2010; Kikkoman Corporation), Cofactor for S9 mix (Cofactor-I; Lot No. 999903; Oriental Yeast, Co., Ltd.) and Japanese Pharmacopoeia water for injection (Lot No. 8L88; Otsuka Pharmaceutical Factory, Inc.) before use. S9 was cryopreserved at -80°C or below after purchase, and used within 4 months from the date ofmanufacture (expiration: 6 months after manufacture). The S9 was prepared from liver homogenates from Slc:SD rats (male, 7 weeks ofage) to which phenobarbital and 5,6-benzoflabone had been intraperitoneally administered for enzyme induction. The components in 1 mL ofS9 mix are shown in the following table:

S9 Magnesium chloride Potassium chloride Glucose-6-phosphate Components in 1 mL ofS9 mix

Nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) Nicotinamide adenine dinucleotide, reduced form (NADH) Sodium-phosphate buffer (pH 7.4) 8. Test 

### groups 8.1. Dose-finding test 0.1 mL 8 f.LIDOI 33 f.Lmo)

5 f.Lmo) 4 f.Lmo) 4 f.Lmo) 100 f.LIDOI

The test was conducted by the non-activated assay (in the absence ofmetabolic activation system S9 mix) and by the activated assay (in the presence ofmetabolic activation system S9 mix) for each test strain. For both ofthe non-activated and activated assays, the highest dose ofthe test substance was set at 5000 !J.g/plate (value calculated from the purity: 20833 !J.g/plate), and a total of

### 7 doses (5 to 5000 !J.g/plate: values calculated from the purity: 20.83 to 20833 !J.g/plate) were set in approximately threefold dilution series (6-step serial dilution). These test groups are shown in the following table:

- 14-Test series

Non-activated assay

Activated assay Test group

Test substance group

Test substance group

8.2 Main test and confirmatory test Dose (Jlg/plate) 

### 5 15 50 150 500 1500 5000 5 15 50 150 500 1500 5000 SR09242 Number ofplates 

### 2 2 2 2 2 2 2 2 2 2 2 2 2 2
These tests were conducted by the non-activated and activated assays for each test strain. As the results ofdose-finding test, though the number ofrevertants increased inS. 

### typhimurium TA98 at 150 to 1500 11g/plate in the non-activated assay, inhibition of cell growth or precipitation ofthe test substance was not detected in any test strains in both the non-activated and activated assays; therefore, the highest concentration in the main test was set at 5000 11g/plate (value calculated from the purity: 20833 flg/plate), and a total of7 doses (78.1 to 5000 11g/plate: values calculated from the purity: 325 to 20833 11g/plate) were set in twofold dilution series (6-step serial dilution). A confirmatory test was conducted in S. 

### typhimurium TA98 at the same doses as in the main test, in the non-activated assay. These test groups in the main and confirmatory tests are shown in the following table: Test series

Non-activated assay

Activated assay Test group

Test substance group

Test substance group

- 15

- Dose (Jlg/plate) Number ofplates 

### 78.1 2 156 2 313 

### 2 625 2 1250 2 2500 2 5000 2 78.1 2 !56 2 313 

### 2 625 2 1250 2 2500 2 5000 2SR09242 8.3. Negative and positive control groups A negative control group (Japanese Phannacopoeia water for injection) and a positive control group shown in the following table were set for each test strain in the dose-finding, main, and confirmatory tests. Test strains Positive control substance (dose: f!g/plate) Non-activated assay Activated assay S. typhimurium TAIOO AF-2 (0.01) 2-AA (1) S. typhimurium TA1535 NaN3 (0.5) 2-AA (2) E. coli WP2uvrA AF-2 (0.01) 2-AA (10) S. typhimurium TA98 AF-2 (0.1) 2-AA (0.5) S. typhimurium TA1537 9-AA (80) 2-AA (2) AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide NaN3 : sodium azide 2-AA: 2-aminoanthracene 9-AA: 9-aminoacridine hydrochloride hydrate
8.4. Number and identification ofplates Each test strain was tested using 3 plates in the negative control group, 2 plates per dose in the test substance groups, and 2 plates in the positive control group. For identification, a label indicating the study number and the test group was attached to each plate. 9. Test 

### method 9

### .1. Subculture oftest strains Each thawed test strain suspension (12 )lL) was inoculated into an L-shaped tube (capacity approximately 40 mL) containing 12 mL ofthe culture medium for subculture (nutrient broth medium). The mixture in the L-shaped tube was cooled with ice until the start ofincubation, for 7.5 h in the dose-finding test, 7.4 h in the main test, and 7.0 h in the confirmatory test. Then, the mixture was incubated for 10 h in a shaking water bath (Personal- II·EX, Taitec Corporation) set at 37°C, 40 mm in stroke, and at a rate 100 times/min. At the termination ofincubation, OD66onm ofthe culture obtained was measured using a spectrophotometer (mini photo 518, Taitec Corporation) to calculate the number ofviable cells by the correlation equation between the number ofviable cells and OD660nm· A culture in which the number ofviable cells was above 1 

### x I 09cells/mL was used in the test. The numbers ofviable cells (calculated value) in the culture ofeach test strain were as follows: Test strain S. typhimurium TA I 00 S. typhimurium TA 1535 E. coli WP2uvrA S. typhimurium T A98 S. typhimurium TA1537 Number ofviable cells (calculated value) (x I09 cells/mL) Dose-finding test Main test Confirmatory test 2.60 2.52 3.20 3.20 4.42 4.29 2.82 2.82 2.63 1.37 1.34

- 16-

### SR09242 9.2. Treatment with the test and control preparations The pre-incubation method was used for treatment with the test and control preparations. To a polyethylene tube (capacity of5 mL) with a cap containing 0. I mL ofthe test or control preparation, 0.5 mL of0.1 mol/L sodium-phosphate buffer (pH 7.4) was added and mixed for the non-activated assay, and 0.5 mL ofS9 mix was added and mixed for the activated assay. To these mixtures, 0.1 mL ofeach culture was added, which were incubated (pre-incubation) for 20 min in a shaking water bath (Personal-11 ·EX, Taitec Corporation) set at 37°C, 40 mm in stroke, and at a rate 100 times/min. After the termination ofpre-incubation, 2 mL 

### oftop agar containing 0.05 mmol/L L-histidine and 0.05 mmol/L D-biotin was added to the mixtures of

### S.typhimurium, and 2 mL oftop agar containing 0.05 mmol/L L-tryptophan was added to the mixtures of

### E
.coli, which were mixed and spread over each plate. After the top agar was solidified on a flat place, the plates were placed in an incubator (MIR-262, SANYO Electric Co., Ltd.) set at 37°C and incubated for 49 h. In each ofthe dose-finding, main, and confirmatory tests, a sterility test was performed to check bacterial contamination in the test preparation ofthe highest concentration and in the S9 mix used in the test. 9.3. Observation The plates in the negative control group, test substance groups and positive control group ofeach test strain were observed with a stereoscopic microscope (SZ6045TR, Olympus Optical Co., Ltd.) for inhibition ofcell growth, and the plates in the test substance groups were macroscopically checked for precipitation ofthe test substance. Then, revertants in each plate in the negative control group, test substance groups, and positive control group ofeach test strain were counted using a colony analyzer (CA-11D, System Science Co., Ltd.). Evaluation ofinhibition ofcell growth was performed according to the following criteria, and an evaluation result scored 1 or more was considered to be inhibition ofcell growth. 0: No inhibition ofcell growth Small background colonies (visible with a magnification approximately x 

### 50) are observed all over the plate, and no difference is noted when compared with those in the negative control group. 

### 1: Slight inhibition ofcell growth

Density ofbackground colonies decreases and the size ofeach colony increases when compared with those in the negative control group. 2: Moderate inhibition ofcell growth

Protruding and large revertants and small-plane background colonies are observed on the same plate.

- 17-

### SR09242 3: Severe inhibition ofcell growth

Background colonies grow to the sizes equivalent to those ofthe revertants, and they are not distinguishable. 4: No survivors are observed. 9.4. Calculation ofobservation results The average number ofrevertants was calculated for the negative control group, test substance groups (for each dose), and positive control group ofeach test strain. 10. Evaluation oftest results I 

### 0.1
. Confirmation ofsensitivity oftest strains A test strain wasjudged to have appropriate sensitivity when the average number of revertants in the negative control group ofthe test strain was within the range of control values based on the historical control data ofthe test facility, and the value in the positive control group ofthe test strain was twice or more that in the negative control group. I 

### 0.2. Evaluation criteria oftest results The test substance wasjudged to be positive for potential to induce genetic mutation when the average number ofrevertants in the test substance groups was twice or more that in the negative control group, and reproducibility in the dose-related increase in the number ofrevertants was demonstrated, in at least one test strain. No statistical analyses were applied for evaluation ofthe test results. I 

### 0.3. Calculation ofspecific activity in mutagenicity As positive results were not obtained in the test substance groups, specific activity in mutagenicity was not calculated in this test. RESULTS The results ofthe dose-finding test are shown in Tables I 

### and 2, those in the main test in Tables 3 and 4, and those in the confirmatory test in Table 5. The dose-response curves obtained from the doses of the test substance and the numbers of revertants in the dose-finding, main, and confirmatory tests are shown in Figures 1-1 to 5-2. The results of the dose-finding test (5 to 5000 1.1.g/plate) were as follows: In S. 

### typhimurium 

### TA98 in the non-activated assay, the average numbers ofrevertants at 150 to 1500 11g/plate increased and was twice or more than in the negative control group, which was not dose-related. The average number of revertants in the test substance groups was less than twice that in the negative control group in the other test strains both the non-activated and activated assays, and no dose-related increase in the number of revertants was noted. No inhibition of cell growth or precipitation of the test substance was detected in any test strains, either the non-activated and activated assays. The results ofthe main test (78.1 to 5000 1.1.g/plate) were as follows: the average number

- 18-

### SR09242 of revertants in the test substance groups were less than twice that in the negative control group in each test strain both the non-activated and activated assays, and no dose-related increase in the number of revertants was noted. No inhibition of cell growth or precipitation ofthe test substance was detected in any test strains, either the non-activated and activated assays. As the increase in the number ofrevertants in S. 

### typhimurium TA98 in the non-activated assay was not reproducible between the dose-finding test and the main test, a confirmatory test was conducted by the non-activated assay inS. typhimurium TA98 at the same doses as in the main test to assess reproducibility. As the results, the average number ofrevertants in the test substance groups was less than twice that in the negative control group, and no dose-related increase was noted, and no inhibition ofcell growth or precipitation ofthe test substance was detected. In the dose-finding, main, and confirmatory tests, all the average numbers ofrevertants in the negative control group of all test strains were within the control range based on the historical control data (Appendix 2) of the test facility. The average number of revertants in the positive control group of each test strain clearly increased, and was twice or more that in the negative control group in all test strains. In the sterility tests in the dose-finding, main, and confirmatory tests, no bacterial contamination was detected in the test preparation ofthe highest dose or in the S9 mix. DISCUSSION The potential of to induce genetic mutation in bacteria was evaluated by a reverse mutation test using Salmonella typhimurium TA 100, TA 1535, TA98, TAI537 and Escherichia coli WP2uvrA. In the dose-finding test, the highest dose ofthe test substance was set at 5000 flg/plate, and a total of7 doses were set in approximately threefold dilution series (6-step serial dilution) for each test strain in both the non-activated and activated assays. Based on the results of the dose-finding test, the highest dose of the test substance in the main test was set at 5000 f.ig/plate, and a total of 7 doses were set in a twofold dilution series (6-step serial dilution) for each test strain in both the non-activated and activated assays. In addition, a confirmatory test was conducted in S typhimurium TA98 in the non-activated assay at the same doses as in the main test. The results were as follows: In the non-activated assay using S typhimurium TA98 in the dose-finding test, the average numbers ofrevertants at the intermediate to high doses increased and was twice or more than in the negative control group, which was not dose-related. The increase in the number of revertants was not detected in S. typhimurium TA98 in the non-activated assay in the main or confirmatory test. Thus, the increase in the numbers ofrevertants in S typhimurium TA98 in the non-activated assay in the dose-finding test was not dose-related, and showed no reproducibility; therefore, this was not considered to be positive reaction. The increase in the number ofrevertants

- 19-SR09242 in the dose-finding test was considered to be incidental change due to the low values in the negative control group, which were at the lowest level ofthe historical control data ofthe test facility. In the other 4 test strains, the average number ofrevertants in the test substance groups was less than twice that in the negative control group of all test strains in the activated and non-activated assays, and no dose-related increase in the number ofrevertants was noted. In the dose-finding, main, and confirmatory tests, all the average numbers ofrevertants in the negative control group of all test strains were within the control range based on the historical control data of the test facility. The average number of revertants in the positive control group clearly increased and was twice or more that in the negative control group. These results confirmed that each test strain had appropriate sensitivity to the mutagens. In conclusion, is not mutagenic to the bacteria under the conditions ofthis test.**E~ONMENTALFACTORSTHATN.UGHTHAVEAFFECTEDTHE** 

### RELIABILITY OF THE TEST RESULTS No environmental factors that might have affected the reliability ofthe test results were found. 

### STORAGE OF DOCUMENTS The following documents will be stored in the archives of Safety Research Institute for Chemical Compounds Co., Ltd. for 10 years after the study completion, and their storage thereafter will be decided by an agreement with the study sponsor. 1.1
. Study protocol and study protocol amendment 1
.2. Raw data and other documents 1.3. Final report 

### NAME AND SEAL OF STUDY DIRECTOR Name and seal affixed in the original August 10, 2010 Koko Nagaoka, Study Director Date

-20-Table I Reverse mutation test of in Salmonella typhimurium TAIOO, TAI535, TA98, TA1537 and Escherichia coli WP2uvr A without metabolic activation (dose-finding test) (SR09242)
Revertants per plate Compound Concentration (Mean) (Jlg/plate) S9 (-) TAlOO TAI535 WP2uvrA TA98 TA1537
Control a I16 ' 111 12 ' 13 12 ' 11 12 8 12 9 110 10 19 12 11 (112) ( 12) ( 14) ( 11) ( II) 5 96 ' I27 13 ' 9 17 ' 17 23 ' I9 5 ' 12 (II2) ( 11) ( I7) ( 21) ( 9) 15 128 ' 119 10 , IO 16 , I2 I8 , 19 5 7 (I24) ( 10) ( 14) ( 19) ( 6) 50 I26 ' 110 13 8 16 , 13 21 , 16 5 8 (118) ( 11) ( 15) ( 19) ( 7) 150 II0 , 114 13 , 7 15 , 15 27 , 2I 9 ' 11 (II2) ( 10) ( 15) ( 24) ( 10) 500 120 , 110 7 5 23 , 16 22 ' 25 12 , 14 (115) ( 6) ( 20) ( 24) ( 13) 1500 116 ' 118 7 , 12 13 , 14 27 ' 21 9 , 10 (II7) ( 1 0) ( I4) ( 24) ( 10) 5000 I06 ' 115 9 8 23 , 24 16 , 21 6 7 (111) ( 9) ( 24) ( 19) ( 7)
AF-2 NaN3 AF-2 AF-2 9

-AA Positive Concentration control (Jlg/plate) 0.01 0.5 0.01 0.1 80 Rev./plate 557 '622 267 '369 54 , 76 275 , 287 187 , 151

(Mean) (590) (318) ( 65) (281) (169)

a: Water for injection (Japanese pharmacopoeia) AF-2 : 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide NaN3 : Sodium azide 9-AA : 9-Aminoacridine hydrochloride hydrate Rev. : Revertants

- 21

-Table 2 Reverse mutation test of in Salmonella typhimurium TAIOO, TA1535, TA98, TA1537 and Escherichia coli WP2uvr A with metabolic activation (dose-finding test) (SR09242)
Revertants per plate Compound Concentration (Mean) (IJ.glplate) S9(+) TAlOO TA1535 WP2uvrA TA98 TA1537
Control a 144 ' 162 12 ' 14 27 ' 19 30 ' 43 7 ' 13 119 5 26 26 12

(142) ( 10) ( 24) ( 33) ( II)
5 171 ' !56 9 ' 10 15 ' 13 31 ' 35 17 ' 21 (164) ( 10) ( 14) ( 33) ( 19) 15 133 ' 158 12 ' 11 13 10 39 ' 49 15 ' 17 (146) ( 12) ( 12) ( 44) ( 16) 50 177 ' 138 13 ' 13 18 ' 18 36 ' 31 8 ' 14 (158) ( 13) ( 18) ( 34) ( 11) !50 141 ' 156 8 ' 10 13 ' 13 43 ' 29 13 ' 9 (149) ( 9) ( 13) ( 36) ( II)
500 130 ' 143 10 6 18 ' 12 24 ' 32 15 ' 16 (137) ( 8) ( 15) ( 28) ( 16) 1500 143 ' 153 10 7 14 ' 14 24 ' 37 5 ' 16 (148) ( 9) ( 14) ( 31) ( 11) 5000 136 ' 131 7 ' 13 21 ' 27 36 ' 31 15 ' 9 (134) ( 10) ( 24) ( 34) ( 12)
2-AA 2-AA 2-AA 2-AA 2-AA Positive Concentration control (IJ.g/plate) 2 10 0.5 2 Rev./plate 1395 ' 1321 603 '650 1161 ' 1244 143 ' 141 386 '345

(Mean) (1358) ( 627) (1203) ( 142) ( 366)

a: Water for injection (Japanese pharmacopoeia) 2-AA : 2-Aminoanthracene Rev. : Revertants

-22-Table 3 Reverse mutation test of in Salmonella typhimurium TAIOO, TA1535, TA98, TA1537 and Escherichia coli WP2uvr A without metabolic activation (main test) (SR09242)
Revertants per plate Compound Concentration (Mean) (f.lg/plate) S9 (-) TA100 TAI535 WP2uvrA TA98 TA1537
Control a 140 ' 129 7 7 I8 ' 13 I2 ' 19 9 ' 11 108 10 13 I5 I5

(126) ( 8) ( 15) ( I5) ( 12)
78
. 1 123 ' 121 6 ' II 16 ' 22 13 ' 17 9 ' 10 (122) ( 9) ( 19) ( 15) ( I0)
156 132 ' 102 13 ' 11 13 ' 18 17 ' 25 6 7 (117) ( 12) ( 16) ( 21) ( 7) 313 131 ' 140 15 ' 8 12 ' 11 16 ' 18 12 ' 11 (136) ( 12) ( 12) ( I7) ( 12) 625 132 ' 127 II 2 17 , 8 17 , 6 8 6 (130) ( 7) ( 13) ( I2) ( 7) 1250 114 , 123 8 , 13 11 , 18 16 , 15 9 8 (119) ( I 1) ( 15) ( 16) ( 9)
2500 128 , I23 3 8 12 9 17 , 18 9 9 (126) ( 6) ( 11) ( 18) ( 9)
5000 112 , 141 4 3 20 , 12 14 , 23 9 , 11 (127) ( 4) ( 16) ( 19) ( 10)
AF-2 NaN, AF-2 AF-2 9-AA Positive Concentration control (f.!g/plate) 0
.01 0.5 0.01 0.1 80 Rev./plate 599 , 618 245 , 265 70 , 60 266 , 305 193 ' 334

(Mean) (609) (255) ( 65) (286) (264)

a : Water for injection (Japanese pharmacopoeia) AF-2 : 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide NaN3 : Sodium azide 9-AA : 9

-Aminoacridine hydrochloride hydrate Rev.: Revertants



### -23-Table4 Reversemutationtestof iin Salmonellatyphimurium TAIOO, TA1535, TA98, TA1537 and Escherichia coli WP2uvr A with metabolic activation (main test) (SR09242)
Revertants per plate Compound Concentration (Mean) (11g/plate) S9 (+) TAIOO TA1535 WP2uvrA TA98 TA1537
Control a I34 ' I71 8 ' 9 15 ' 20 35 ' 27 I3 ' 8 I59 I6 18 35 IO (155) ( I1) ( I 8) ( 32) ( 10)
78
. I I34 ' I67 12 ' 13 12 ' 23 20 ' 22 13 ' 6 (151) ( 13) ( 18) ( 21) ( IO)
156 I64 ' 158 6 ' 14 6 ' I9 20 ' 34 13 ' 10 (I6I) ( IO) ( I3) ( 27) ( I2)
313 I46 ' I9I 9 ' I 1 25 ' 2I 20 ' 36 7 ' I2 (169) ( IO) ( 23) ( 28) ( IO)
625 I67 ' I4I I2 ' II I4 ' I8 26 ' 32 9 ' 15 (154) ( 12) ( 16) ( 29) ( 12)
1250 I45 ' 137 12 ' 8 I4 ' I8 31 ' 25 I2 6 (141) ( 10) ( 16) ( 28) ( 9)
2500 124 ' 149 8 ' I2 12 ' 18 29 ' 22 6 ' IO (137) ( 10) ( 15) ( 26) ( 8)
5000 I25 ' 160 9 8 13 ' I6 18 ' 31 7 ' 14 (143) ( 9) ( I5) ( 25) ( 11)
2-AA 2-AA 2-AA 2-AA 2

-AA Positive Concentration control (11g/plate) 2 IO 0.5 2 Rev./plate 1481 ' 1447 545 '613 I280 ' I365 152 '159 415 '471

(Mean) (1464) ( 579) (1323) ( 156) ( 443)

a: Water for injection (Japanese pharmacopoeia) 2-AA : 2-Arninoanthracene Rev.: Revertants

-24-Table 5 Reverse mutation test of in Salmonella typhimurium TA98 without metabolic activation (confirmatory test) (SR09242)
Compound

Control a

Positive control Concentration (J.Lg/plate)

78.1

156

313

625

1250

2500

5000

Concentration

(J.Lg/plate) Rev./plate

(Mean)
a : Water for injection (Japanese pharmacopoeia) AF-2 : 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide Rev.: Revertants



### - 25- Revertants per plate (Mean) S9(-) TA98
26 , I8 I4

( I9) I 8 , I 7

( I8) 20 , 18

( 19) 17 , 27

( 22) I6 , 22

( I9)
22 , 18

( 20) 15 , 19

( 17) 12 , 16

( 14)
AF-2
0.1 320 , 350 (335)200


### %l 150 

### 0. '-< Q) 0.. 

### "' 

## § 100 t:: Q) >

### ~ 50

0

200


### %l 150 

### 0. '-< Q) 0.. 

### "' 100 ...... 

### § t:: Q) > Q) p:::; 50

0 0

0 

### [ T A I00, S9 (-), Dose-finding test)

5 15 50 150 500 1500 5000 Concentration of (J.lg/plate)



### [ TAIOO, S9 (-),Main test)

78.1 156 313 625 1250 2500 5000 Concentration of (J.lg/plate)


### Figure 1-1 Reverse mutation test of 

### in 

### Salmonella typhimurium TA100 without metabolic activation (dose-response curves) (SR09242)

-26-[ TAIOO, S9(+), Dose-finding test] 250
200 <!) "'iti 

### 0.. a> 150 0.. {/)

### ~ t:: 100 <!) ;> <!)**~** 50
0 0 5 15 50 150 500 1500 5000 Concentration of (f.Lg/plate)

[ TAIOO, S9(+), Main test] 250

<!) 200 "'iti 

### 0..



### .... 

### & 150 {/) 

### ..... § t:: 100 <!) ;> <!)**~** 50
0 0 78.1 156 313 625 1250 2500 5000 Concentration of (f.Lg/plate)


### Figure 1-2 Reverse mutation test of in 

### Salmonella typhimurium TAlOO with metabolic activation (dose-response curves) (SR09242)

-27-[ TA1535, S9 (-),Dose-finding test] 20

Q) 15**~** 

### 0..

..... Q) 0.. 

### "' 10 

## § t:: Q) ;;. Q)**~** 5

0 0 5 15 50 150 500 1500 5000 Concentration of (flg/p1ate)

[ TA1535, S9(-), Main test] 20

Q) 15**~** 

### 0..

..... Q) 0.. 

### "' 10 -<;::**~** t:: Q) ;;. Q)**~** 5

0 0 78.1 156 313 625 1250 2500 5000 Concentration of (f!g/plate)


### Figure 2-1 Reverse mutation test of in 

### Salmonella typhimurium 

### TA1535 without metabolic activation (dose-response curves) (SR09242)

-28-[ TA1535, S9 (+),Dose-finding test] 20

(!) 15**~** 

### 0..

..... (!) 0.. 

### "' 10 

## .... § t (!) ;;. (!)**~** 5

0 0 5 15 50 150 500 1500 5000 Concentration of (Jlg/plate)

[ TA1535, S9(+), Main test] 20

(!) 15 

### ] 0..

..... (!) 0.. 

### "' 10 

## .... § t (!) ;;. (!)**~** 5

0 0 78.1 156 313 625 1250 2500 5000 Concentration of (Jlg/plate)


### Figure 2-2 Reverse mutation test of 

### in Salmonella typhimurium 

### TA1535 with metabolic activation (dose-response curves) (SR09242)

-29-( WP2uvr A, S9 (-) , Dose-finding test

## J 30

Q) 1d 20 

### 0..

..... Q) 0.. rn 

### 1:: Clj t:: Q) 10 ;> Q)**~**
0 0 5 15 50 150 500 1500 Concentration of (Jlg/plate)

( WP2uvr A, S9 (-) , Main test

## J 30

Q) 1d 20 

### 0..

..... Q) 0.. rn +->**~** Clj t:: Q) 10 ;> Q)**~**

0 0 78.1 156 313 625 1250 2500 Concentration of (Jlg/plate)


### Figure 3-1 Reverse mutation test of in 

### Escherichia coli 

### WP2uvrA without metabolic activation (dose-response curves) (SR09242)

- 30- 5000

5000( WP2uvr A, S9 (+) , Dose-finding test

### J 40

Q) 30 d 

### 0..

..... Q) 0.. 

### "' 20

...... § t: Q) ;> Q)**~** 10

0 0 5 15 50 150 500 1500 Concentration of (J.lg/plate)

( WP2uvr A, S9 (+) , Main test

### J 40

Q) 30 d 

### 0..

..... Q) 0.. 20 

### "'

...... § t: Q) ;> Q) 10**~**
0 0 78.1 156 313 625 1250 2500 Concentration of (J.lg/plate)


### Figure 3-2 Reverse mutation test of in Escherichia coli 

### WP2uvrA with metabolic activation (dose-response curves) (SR09242)

- 31

- 5000

5000[ TA98, S9(-), Main test 

## J 40

Q) 30 "'id 

### 0..

..... Q) 0.. U) 

## § 20 t:: Q) ;> Q) p::; 10

0 0 78
. 1 156 313 625 1250 2500 5000 Concentration of**(~J.g/plate)**
[ TA98, S9 (-), Confirmatory test 

## J 40

Q) 30 "'id 

### 0..

..... Q) 0.. U) 20 

### ..... @ t:: Q) ;> Q) p::; 10

0 0 78.1 156 313 625 1250 2500 5000 Concentration of**(~J.g/plate)** 

### Figure 4-1 Reverse mutation test of in 

### Salmonella typhimurium TA98 without metabolic activation (dose-response curves) (SR09242)

- 32-[ TA98, S9 (+),Dose-finding test] 

### 60

Q) 

## .... ro 40 

### 0. 1-< Q) 0.. r.n

### ~ t:: 20 Q) ;> Q)**~**
0 0 5 15 50 150 500 1500 5000 Concentration of (!J.g/plate)

[ TA98, S9(+), Main test] 

### 60

Q)**~** 40 

### 0.

..... Q) 0.. r.n

......**~** ro t:: Q) 20 ;> Q)**~**
0 0 78
. 1 156 313 625 1250 2500 5000 Concentration of J.g/plate)


### Figure 4-2 Reverse mutation test of 

### in Salmonella typhimurium 

### TA98 with metabolic activation (dose-response curves) (SR09242)

-33-[ TA1537, S9(-), Dose-finding test] 20

Q)**~** 15 

### 0..



### .... Q) 0.. <I) 

## § 10 t:: Q) :> Q)**~** 5

0 0 5 15 50 150 500 1500 5000 Concentration of (Jlg/plate)

[ TA1537, S9(-), Main test]

0 78
. 1 156 313 625 1250 2500 5000 Concentration of (Jlg/plate)


### Figure 5-1 Reverse mutation test of 

### in Salmonella typhimurium TA1537 without metabolic activation (dose-response curves) (SR09242)

- 34-[ TA1537, S9 (+),Dose-finding test] 30

Q)**~** 

### 0.. 20

..... Q) 0.. Cll

### ~ t:: Q) > Q) 10**~**

0 0 5 15 50 150 500 1500 5000 Concentration of (Jlg/plate)

[ TA1537, S9(+), Main test] 30

Q) 

### ] 0.. 20

..... Q) 0.. Cll

### ~ t:: Q) > 10 Q)**~**

0 0 78.1 156 313 625 1250 2500 5000 Concentration of (Jlg/plate)


### Figure 5-2 Reverse mutation test of in 

### Salmonella typhimurium 

### TA1537 with metabolic activation (dose-response curves) (SR09242)

-35-

### Appendix I- I 

### SR09242 January 14, 2010


### Analysis table

1. Name ofarticle

2. Analysis values Items Analysis values Notes



### -36-

### Appendix 1-2 SR09242 August I, 2010


### Analysis table

I. Name ofarticle

2. Analysis values



### - 37-Appendix 2 Historical control data for reverse mutation test

Strain Metabolic activation S9 (-) TAIOO S9(+) No. ofdata n = 47 n = 95 Mean±S
.D. 105 ±II 131 ± 13 Maximum 128 169 Minimum 75 I00 X-R-Rs' 73 -137 96-166

No. ofdata n = 704 n = 741 Mean±S.D. 123 ± 19 136 ± 18 Maximum 248 219 Minimum 75 82 X-R-Rs' 86**~** 160 96**~** 176

Strain Metabolic activation Compound Concentration

{[.Lg/plate) TAIOO S9 (-) S9(+ ) AF-2 2-AA 0
.01 Control data (Date :**2009 6~2010** **4)** TAI535 WP2uvrA S9(-) S9 (+) S9 (-) S9 (+)

n = 39 n = 39 n = 38 n = 38 10 ± 4 II ± 3 19 ± 5 20 ± 4

24 22 30 32 5 6 9 12 3-35 7-33 Reference: Control data (Date: 1992.2-2010
.4)

n = 600 n = 594 n = 585 n = 583 10 ± 2 I I ± 3 18 ± 4 21 ± 4

24 39 44 45 5 6 7 II 5-15**6~16** 7-29 10-32 Positive control data (Date : 2009
.6-2010.4) TA1535 WP2uvrA S9(-) S9(+ ) S9(-) S9(+ ) 2-AA AF-2 2-AA 0.5 2 0.01 10 SR09242 (201 0
. 5)

TA98 TAI537

. S9(-) S9 (+) S9(-) S9(+)

n = 40 n =39 n = 38 n = 38 18 ± 4 31 ± 5 17 ± 6 18 ± 7

27 40 29 33 12 21 5 8 7-29 15-47 0-36 2-34

n = 706 n = 693 n = 600 n = 596 17 ± 4 29 ± 5 12 ± 4 16 ± 5

39 45 29 39
7 10 4 2 6**~28** 16**~42 4~20**  5**~27**

TA98 TA1537 S9(-) S9(+) S9(-) S9(+) AF-2 2-AA 9-AA 2-AA 0.1 0.5 80 2 No. ofdata n = 47 n = 95 n = 39 n = 39 n = 38 n = 38 n = 40 n = 39 n = 38 n = 38 Mean± S.D. 644 ± 92 1643 ± 307 300 ± 47 546 ± 61 81 ± 17 1214 ± 127 348 ± 75 209 ± 60 222 ± 52 469 ± 108 Maximum 882 2291 482 650 123 1424 510 317 356 695 Minimum 505 1006 214 346 53 851 184 122 89 263**490~798** 1034-2252**191~409** 386-706**60~102** 924-1504 226-470 148-270 68-376 192-746 Reference :Positive control data(Date: S9(-);**1998.4~2010.4,** S9(+); 1992.2-2010.4) No.ofdata n=527 n=624 n=495 n=542 n=494 n=533 n=529 n=559 n=498 n=541 Mean± S.D
. 788 ± 155 1299 ± 341 263 ±51 303 ± 106 124 ± 46 880 ± 224 360 ± 87 269 ± 68 273 ± 71 273 ± 109 Maximum 1352 2501 482 686 454 1765 594 607 640 791 Minimum 505 227 132 I05 53 309 180 I I 6 89 73 X-R-Rs •**543~1033** 796-1802 165-361 178-428 76-172 598-1162 211-509 155-383 116-430 137-409

a X-R-Rs =X±2
.66Rs

X=Mean Rs =Mean of (Xi -Xi-1) 

### -38-