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 of Health, Labour and Welfare, Japan (MHLW), November 21, 2003; No. 

3 of the Manufacturing Industries Bureau, Ministry ofEconomy, Trade and 

Industry, Japan (METI), November 17, 2003; and No. 031121004 of the 

Environmental Policy Bureau, Ministry of the 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 of the 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 of the Manufacturing Industries Bureau, METI, 

November 13, 2003; and No. 031121002 of the Environmental Policy Bureau, 

MOE); and "Partial Amendment of 'On the Test Method Concerning New 

Chemical Substances, etc.'" (Notification No. 1120001 of the 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 of the 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 of Safety Research Institute for 

Chemical Compounds Co., Ltd. as follows: 

Phase of study inspection Date of inspection Date of report to Date of report 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 of test substance May 19,2010 May I9, 2010 May 19,2010 

Conduct of study 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, 20 I 0 June 23, 2010 

Final report (draft): Tables and June 23, 20 I 0 June23,2010 June 23,2010 Figures 

Final report (draft): Text June 23, 20 I 0 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 of the 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 of the 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 of the 

Manufacturing Industries Bureau, METI, June 30, 2008; and No. 080704001 of the 

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 of the 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 of the Manufacturing Industries Bureau, METJ, November 13, 2006; and 

No. 061120001 of the 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 of this 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 I 0, 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 of the 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···· ········· ·· ········ ···· ···· ········· ····· ·········· ···· ······ ····· ··· ··· ·· · I 0 

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 of the 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 of the Pharmaceutical and Food Safety Bureau 

(PFSB), Ministry ofHealth, Labour and Welfare, Japan 

(MHLW), November 21, 2003; No.3 of the Manufacturing 

Industries Bureau, Ministry ofEconomy, Trade and Industry, 

Japan (METI), November 17, 2003; and No. 031121004 of the 

Environmental Policy Bureau, Ministry of the 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 of the 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 of the 

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 of subculture: 

Treatment with test substance: 

Colony counting: 

Main test 

Initiation of subculture: 

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 of subculture: 

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 of the S9 mix metabolic activation system) and 

activated assay (in the presence of the S9 mix metabolic activation system). 

In the dose-finding test, the highest dose of the 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 of the 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 of the dose-finding test were as follows: In the non-activated assay using S. 

typhimurium TA98, the average numbers of revertants 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 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 of the 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 of revertants was noted. No inhibition of cell growth or 

precipitation of the 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 of revertants 

in the negative control group of all test strains were within the control range based on 

the historical control data ofthe 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. 

On the basis ofthese results, the increase in the numbers of revertants 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 of this 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 WP2uvr A. The test was conducted by the pre-incubation method, in 

the non-activated assay (in the absence of the S9 mix metabolic activation system) and 

activated assay (in the presence of the S9 mix metabolic activation system). 

1. Test substance 

Name: 

Abbreviation: 

CAS No. : 

MATERIALS AND METHODS 

Molecular formula: 

Structural formula : 

Molecular weight: 

Physicochemical properties: 

Lot No. : 

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, 20 I 0 [date of treatment 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 of all test operation including that in the 

related tests, the remaining test substance was returned to 

the supplier. 

2. Preparation of test 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 of the 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 of vehicle: 

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 of preparations: 

Preparations showed no visible reactivity (such as 

discoloration, generation ofheat, and foaming) at 

preparation of test substance in the dose-finding, main, 

and confirmatory tests. 

- 11 -
SR09242 

Disposal of the 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 of preparation (expiration: one 

year after preparation). Remaining preparations of the 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 of the culture of each test strain, 0.7 mL of dimethyl sulfoxide (Lot No. 

- 12-
SR09242 

BTl 59; Dojindo Laboratories) was added. The culture was aliquoted and 

cryopreserved at -80°C or below. Using an aliquot of the culture of each test strain, 

characteristics ofthe test strain (amino acid-requirement, deep rough character [rfa], 

UV sensitivity, and drug resistance), and the number of revertants 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 of 25 giL. To the culture media of S. typhimurium TA98 and TA 100, 

ampicillin sodium (Lot No. M8B0619; Nacalai Tesque, Inc.) was added to yield a 

concentration of 25 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 of the 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 of culture medium for the test 

Magnesium sulfate heptahydrate 

Citric acid monohydrate 

Potassium phosphate dibasic 

Ammonium phosphate monobasic 

Sodium hydroxide 

Glucose 

Agar powder (Oxoid Agar No.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 4 7°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. I 06P5602; Kanto Chemical Co., Ltd.) 0.5% 

(B) Amino acid solution 

Solution ofL-histidine and D-biotin 0.5 mmoi/L each 

(L-histidine; Lot No. 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; Lot No. 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 of manufacture (expiration: 6 months after manufacture). The S9 was 

prepared from liver homogenates from Slc:SD rats (male, 7 weeks of age) to which 

phenobarbital and 5,6-benzoflabone had been intraperitoneally administered for 

enzyme induction. The components in 1 mL of S9 mix are shown in the following 

table: 

S9 

Magnesium chloride 

Potassium chloride 

Glucose-6-phosphate 

Components in 1 mL of S9 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 of metabolic 

activation system S9 mix) and by the activated assay (in the presence of metabolic 

activation system S9 mix) for each test strain. 

For both of the non-activated and activated assays, the highest dose of the 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 of plates 

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 of dose-finding test, though the number of revertants increased inS. 

typhimurium TA98 at 150 to 1500 11g/plate in the non-activated assay, inhibition of 

cell growth or precipitation of the 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 of plates 

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 2 
SR09242 

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 of plates 

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 of the culture medium for 

subculture (nutrient broth medium). The mixture in the L-shaped tube was cooled 

with ice until the start of incubation, 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 of incubation, 

OD66onm of the 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 of viable cells and OD660nm· A culture in 

which the number of viable cells was above 1 x I 09cells/mL was used in the test. 

The numbers ofviable cells (calculated value) in the culture of each 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 of viable cells (calculated value) (x I 09 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 of 5 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 of each 

culture was added, which were incubated (pre-incubation) for 20 min in a shaking 

water bath (Personal-11 ·EX, Taitec Corporation) set at 3 7°C, 40 mm in stroke, and 

at a rate 100 times/min. After the termination of pre-incubation, 2 mL of top 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 of top 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 of the dose-finding, main, and confirmatory tests, a sterility test was 

performed to check bacterial contamination in the test preparation of the 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 of each test strain were observed with a stereoscopic microscope (SZ6045TR, 

Olympus Optical Co., Ltd.) for inhibition of cell growth, and the plates in the test 

substance groups were macroscopically checked for precipitation of the test 

substance. Then, revertants in each plate in the negative control group, test 

substance groups, and positive control group of each test strain were counted using 

a colony analyzer (CA-11D, System Science Co., Ltd.). 

Evaluation of inhibition of cell growth was performed according to the following 

criteria, and an evaluation result scored 1 or more was considered to be inhibition 

of cell growth. 

0: No inhibition of cell 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 of cell growth 

Density of background colonies decreases and the size of each colony increases 

when compared with those in the negative control group. 

2: Moderate inhibition of cell growth 

Protruding and large revertants and small-plane background colonies are 

observed on the same plate. 

- 17-
SR09242 

3: Severe inhibition of cell growth 

Background colonies grow to the sizes equivalent to those of the revertants, and 

they are not distinguishable. 

4: No survivors are observed. 

9.4. Calculation of observation results 

The average number of revertants was calculated for the negative control group, 

test substance groups (for each dose), and positive control group of each test strain. 

10. Evaluation of test results 

I 0.1 . Confirmation of sensitivity of test strains 

A test strain was judged to have appropriate sensitivity when the average number of 

revertants in the negative control group of the test strain was within the range of 

control values based on the historical control data of the test facility, and the value 

in the positive control group of the test strain was twice or more that in the negative 

control group. 

I 0.2. Evaluation criteria of test results 

The test substance was judged to be positive for potential to induce genetic 

mutation when the average number of revertants 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 of revertants was demonstrated, in at least one 

test strain. No statistical analyses were applied for evaluation of the test results. 

I 0.3. Calculation of specific 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 of the 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 of the 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 of the test substance was detected in any test strains, either the 

non-activated and activated assays. 

As the increase in the number of revertants 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 of revertants 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 of cell growth 

or precipitation of the test substance was detected. 

In the dose-finding, main, and confirmatory tests, all the average numbers of revertants 

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 of the 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 of the test substance was set at 5000 flg/plate, 

and a total of 7 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 of revertants 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 of revertants 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 of revertants 

- 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 of the historical control data 

of the test facility. 

In the other 4 test strains, the average number of revertants 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 of revertants was 

noted. 

In the dose-finding, main, and confirmatory tests, all the average numbers of revertants 

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 of this test. 

E~ONMENTALFACTORSTHATN.UGHTHAVEAFFECTEDTHE 

RELIABILITY OF THE TEST RESULTS 

No environmental factors that might have affected the reliability of the 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 II 0 , 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) ( 1 0) ( 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) ( I 0) 

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) ( I 7) ( 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 

I 59 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 T A98 

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 

( I 8) 

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 I 00, 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 

0.. 20 ..... 

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 

0.. 20 ..... 

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 

WP2uvr A 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 

WP2uvr A 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) 

[ T A98, 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) 

~ 

0. 40 ..... 

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 of article 

2. Analysis values 

Items Analysis values Notes 

-36-
Appendix 1-2 SR09242 

August I, 2010 

Analysis table 

I. Name of article 

2. Analysis values 

- 37-
Appendix 2 

Historical control data for reverse mutation test 

Strain 

Metabolic 

activation S9 (-) 

TAIOO 

S9(+) 

No. of data n = 47 n = 95 

Mean±S.D. 105 ±II 131 ± 13 

Maximum 128 169 

Minimum 75 I 00 

X-R-Rs' 73 -137 96-166 

No. of data 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 I I 

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. of data 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 I 05 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-