### TRANSLATION STATEMENT

Study Title: Chromosomal Aberration Test of [ n Cultured Mammalian Cells Study Number: SR09243

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:

Kohtaro Kawamura, Study Director DateStudy Title:

Study Number: FINAL REPORT (Translation)

Chromosomal Aberration Test of Cells

SR09243 

### in Cultured Mammalian

Safety Research Institute for Chemical Compounds Co., Ltd.



### -1126-STATEMENT Study Title: Chromosomal Aberration Test of in Cultured Mammalian Cells Study Number: SR09243 1. 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, 201 0 Kohtaro Kawamura, Study Director Date

- 2-QUALITY ASSURANCE STATEMENT Study Title: Chromosomal Aberration Test of in Cultured Mammalian Cells Study Number: SR09243 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 April23, 20IO April23, 20IO April23, 2010 Study protocol amendment (No. I) May 17,2010 May I7, 20IO May I7, 20IO Study protocol amendment (No.2) June 29, 20IO June 29, 20IO June 29, 2010 Receipt, labeling and storage oftest April23, 20IO April23, 20IO April 23, 20I 0 substance Preparation oftest substance May I7, 2010 May I7, 20IO May I7, 20IO Conduct ofstudy May I7, 20IO May I7, 20IO May I7, 20IO Preparation ofspecimens May 20, 20IO May 20, 20IO May 20, 20IO Observation June 2, 2010 June 2, 20IO June2, 20IO Raw data June 25, 20IO June 25, 20IO June 25, 20IO Final report (draft): Tables and Figures June 25, 20 I0 June25, 20IO June25, 20IO Final report (draft): Text June 25, 20 I0 June25, 20IO June 25, 20IO Final report August I0, 20 I0 August 10, 2010 August 10, 2010 I. 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, METI, November 13, 2006; and No. 061120001 ofthe Environmental Policy Bureau, MOE). 2. The Quality Assurance Unit has reviewed the fmal 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 10,2010 Taku Katano, QA Representative Date

- 3 -

### SR09243 CONTENTS 

### Page Title page· ·· ·· ······ ··· ··· ·· ··· ·· ···· ··· ··· ··· ······ ······ ··· ······· ···· ··· ··· ··· ··· ··· ······ ····· · 1 STATEMENT · ···· ·· · · ····· · ············· ········ ······· ·· · · · ·· · · · · ··· · ·· · ·· ·· ·· · · · ·········· · ·· · 2 QUALITY ASSURANCE STATEMENT · ···· ····· · ·· · ·· · · ·· ·· ····· · · · ···· · ·· · ·· ··· ·· ····· ·· 3 CONTENTS·· · · · · · · ···· ······· · ··· ·· ···· ··········· ·· ···· · ···· ·· · ·· · ······ · ······· ·· ·· ······ ····· 4 Study title, study number, purpose ofthe study, Good Laboratory Practice

standards and test guidelines, and study sponsor·· ········· ··· ·· · ····· ··· ··· ··· ·· ···· ······ 5 Test facility, Study Director, study personnel, and testing period · · · ·· ·· ······ · ·· · ···· · ·· · · 6 SUMMARY ·· · ·· ··· · ·· · · · ·· ···· ·· ··· ··· ·· ···· ·· ··· · ············· · ···· · · ··· ·· ·· · · · · · ···· ···· · ····· · ? INTRODUCTION ·· ··· ··· ·· ···· ··· ··· ·· ··· ········ ······ ···· ····· ···· ·· ···· ····· ·· ···· ······ ···· 8 MATERIALS AND METHODS ···· · ····· ····· ·· ·· · ·· ··· ··· ·· · ··· ··· ·· · · ··· ···· ······ ···· · ···· 8 RESULTS ······· ···· · ·· ·· · ····· ··· ······· ·· ····················· · ······ · ··· ·· ··· · ·· · ······ · ······17 DISCUSSION ·· ···· ·· ··· ··· ····· ··· ······ ··· ······ ······ ··· ······· ···· ··· ··· ··· ···· ····· ····· ··· 18 ENVIRONMENTA L FACTORS THAT MlGHT HAVE AFFECTED THE RELIABILITY OF THE TEST RESULTS·· · ···· · ···· · ····· · ··· · ··· · ·· ·· ·········· 18 STORAGE OF DOCUMENTS AND MATERIALS ····· ····· ···· ·· ······ ··· · ········ ··· ··18 NAME AND SEAL OF STUDY DIRECTOR · ·········· ······ ··· ·· ······ ··· ·· ···· ······ ···18 Tables and Figure Table 1 Effects of on growth rate ofCHLIIU with or without metabolic activation (preliminary test) (SR09243) ···· · ····· ··· ··· ·· ·· · · ··· ···19 Figure 2 Effects of on growth rate ofCHL/IU with or without metabolic activation (preliminary test) (SR09243) ·· ···· ·· ··· ·· ···· ····· ··· 20 Table 2 Effects of on growth rate ofCHL/IU with or without metabolic activation (chromosomal aberration test) (SR09243) ······· · ·· 21 Table 3-1 Results ofthe chromosomal aberration test of (6 hours treatment without metabolic activation) (SR09243) ·· ··· ····· ··· 22 Table 3-2 Results ofthe chromosomal aberration test of (6 hours treatment with metabolic activation) (SR09243) ·· ··· ······ ······ 23 Table 3-3 Results ofthe chromosomal aberration test of (24 hours treatment without metabolic activation) (SR09243) ····· ······ · 24 Appendices Appendix 1-1 Analysis table (January 14, 2010)·· ········ ···· · ······ · · · ···· ···· ·· ··· ··25 Appendix 1-2 Analysis table (August I, 2010)· · ··· ······ ··· ··· ····· ···· ·· ·· · ··· · ·· ··· ·26

-4-

### Study title: Chromosomal Aberration Test of Cells SR09243 in Cultured Mammalian Study number: SR09243 Purpose ofthe study: The clastogenic potential of was evaluated in vitro 

### using Chinese hamster lung (CHL/IU) cells. 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. 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) "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) "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)



### - 5-SR09243

Test facility Name: Address: Safety Research Institute for Chemical Compounds Co., Ltd. 363-24 Shin-ei, Kiyota-ku, Sapporo 004-0839, Japan Management: Study Director Name: Masao Kiguchi
Kohtaro Kawamura Company and division: Safety Research Division, Safety Research Institute for Chemical Compounds Co., Ltd. Study personnel Test substance management:

Test operation: Testing period Study initiation: Shiho Kodama (responsible person), Misako Ohkubo, and Kohtaro Kawamura Ikuko Endo, Kohtaro Kawamura, and Wataru Shimatani

Receipt oftest substance: April 23, 20 I0 January 18, 2010 April29, 2010 Start ofexperiment: Preliminary test Initiation oftreatment with the test substance: April29, 2010 Fixation and staining: April30, 2010 Main test Initiation oftreatment with test substance: May I7, 20IO Preparation ofspecimens: May 18, 20 I0 Experiment completion (completion ofobservation): June 4, 20IO Study completion: August 10, 20 10



### -6-SR09243 SUMMARY The clastogenic potential of was evaluated in vitro using Chinese hamster lung (CHL/IU) cells. The test was performed in three test series: short-term treatment with and without metabolic activation and continuous treatment for 24 h.

In the preliminary test (cell-growth inhibition test: 14.6 to 1870 J..Lg/mL [equivalent to 10 mM]), cell growth was slightly inhibited at the high dose after continuous treatment for 24 h, and no effects were observed in cell growth after short term treatment with or without metabolic activation. Precipitation ofthe test substance or effects ofthe test substance treatment on the culture pH were not observed in any test series. Based on the results ofthe preliminary test, the highest dose ofeach test series was set at 1870 J..Lg/mL, and lower doses were set with a twofold dilution series (3 or 4 doses in total) in the main test (chromosomal aberration test). As the results, the incidences of structural and numerical aberrations ofchromosomes were less than 5% at all doses of all test series, which showed negative results. No precipitation ofthe test substance or effects ofthe test substance treatment on the culture pH were observed in any test series, which were the same as in the preliminary test. In the positive control group, the incidence ofstructural aberrations ofchromosomes was clearly positive in all test series, demonstrating that the cell line used in this study had sufficient sensitivity.

These results indicate that is not clastogenic in cultured mammalian cells under the conditions used in the present test.



### -7-

### SR09243 INTRODUCTION 

### To evaluate the clastogenic potential of 

### in vitro, a chromosomal aberration test was performed using Chinese hamster lung (CHL/IU) cells.

1. 

### Test substance 

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



### Reference Number in Gazetted List in Japan :

Rational formula (structural formula) :

Molecular weight: Physicochemical properties : Lot No. : Supplier: Amount obtained:

Stability:

Storage conditions : Storage location: Storage period : Caution in handling : Study sponsor 2 containers (NET 1000 g and 1090 g, shared with related tests) Obtained as an aqueous solution (clear liquid) containing solid content 24.0%, impurities 0.85% (to the solid content), and Cl ion 35 ppm (Appendix 1

- 1)

. Deliquescent, stable in water After completion oftest operation, the analysis results of the test substance were obtained and stability was confirmed (Appendix 1-2)

. Containers were sealed and stored in a refrigerator (measured temperature: 2 to 8 °C)

. The test substance storage room (refrigeration room) and the refrigerator in the mutagenicity test room January 18 (receipt) to May 17, 2010 (date offinal use) Handled in a clean bench wearing gloves, protection

- 8 -SR09243 glasses, masks, etc. Remaining test substance: After completion oftest operation including that in the related tests, the remaining test substance was returned to the supplier. 

### 2. Preparation oftest substance The test substance is soluble in water, and was dissolved in and diluted with Japanese Pharmacopoeia water for injection (Lot No. 8L88, Otsuka Pharmaceutical Factory, Inc.) to the prescribed concentrations before use. The prepared concentration was calculated on the basis that the purity ofthe test substance was 24.0%. For the preliminary test, an 18.7 mg/mL preparation was prepared, which was serially diluted in a twofold dilution series to prepare 9.35, 4.68, 2.34, 1.17, 0.584, 0.292, and 0.146 mg/mL preparations. For the main test, an 18.7 mg/mL preparation was prepared, which was serially diluted in a twofold dilution series to prepare 9.35, 4.68, and 2

.34 mg/mL preparations. Regarding the stability, the preparations showed no visible reactivity (such as discoloration, generation ofheat, and foaming) to the vehicle at preparation oftest substance in either the preliminary or main test. The test preparations were used within 0.8 h after preparation in both the preliminary and main tests. The test solutions were prepared in a clean bench using protection glasses, masks, gloves, white lab coat, etc
. to avoid inhalation and contact with eyes, skin and clothes. Remaining preparations were collected as industrial waste to be incinerated. 

### 3. Negative control substance Japanese Pharmacopoeia water for injection (Lot No

. 8L88; expiration date, December 2013; Otsuka Pharmaceutical Factory, Inc.) was used as the negative control substance, without any further preparation. The negative control substance was added at a concentration of 10% (val) to the culture medium in each plate. 

### 4. Positive control substances Mitomycin C (Lot No. 498AFJ; expiration date, October 2010; Kyowa Hakko Kogyo Co., Ltd.) was used as the positive control substance for treatment without metabolic activation

. The compound was stored at room temperature and dissolved in Japanese Pharmacopoeia water for injection (Lot No. 7K81 , Otsuka Pharmaceutical Factory, Inc

.) to obtain concentrations of5 and 10 jlg/mL. One vial ofmitomycin C contained 2 mg (potency) ofJapanese Pharmacopoeia mitomycin C; hence, calculations for preparation were made on the basis that 1 mg (potency) ofmitomycin C was equivalent to 1 mg ofmitomycin C.

-9-

### SR09243 3,4-benzopyrene (Lot No. 8JB8G; expiration date, July 2014 [for 5 years after purchase]; Tokyo Chemical Industry Co., Ltd.) was used as the positive control substance for treatment with metabolic activation. The compound was stored in a cool place (2 to 8 °C) and dissolved in dimethyl sulfoxide (Lot No. TA026, Dojindo Laboratories) to obtain a concentration of 1 

### mg/mL. The content of3,4-benzopyrene in the product purchased was 98.2%. The positive control preparations were aliquoted into test tubes and cryopreserved at -20°C or below. The preparations were used within 9 months after preparation (expiration date: one year after preparation), and stored preparations were used within 0.5 h after being thawed. Each preparation was added at a concentration of 1% (vol) to the culture medium in each plate. 5. Cellline 

### The cell line was CHL/IU, obtained at a passage number of 14 from Dainippon Pharmaceutical Co., Ltd., on May 17, 2005. These cells were derived from the lung ofa female newborn Chinese hamster, with 25 chromosomes (mode) (2n 

### = 22) and a cell doubling time of 13.3 h (measured value). This cell line was selected in consideration ofthe growth rate, chromosomal stability during subcultivation, ease ofobservation ofchromosome specimens, and sensitivity to known mutagens. Cells cryopreserved together with those used in the test were checked for mycoplasma contamination using fluorescent staining, and the results were negative for contamination. A cell suspension was prepared at a density of 1 x 106 

### cells/mL using culture medium containing 10% (vol) dimethyl sulfoxide and aliquoted into ampoules (1 

### mL/ampoule), which were then successively frozen and stored in liquid nitrogen. After being thawed, the cells were seeded in a 75-cm2 

### flask and incubated in a C02 

### incubator (MC0-175, Sanyo Electric Co., Ltd.; or MC0-18AIC [UV], Sanyo Electric Biomedical Co., Ltd.) at 5.0% C02 

### and 37.0°C. The cells were subcultured every three or four days. Cells with a passage number of 18 (preliminary test) or 17 (main test) were used in the test. 6. Culture medium 

### A culture medium was prepared as follows using Eagle MEM. First, 9.4 g ofEagle MEM (Code 05900, Lot Nos. 62791211 and 628001, containing kanamycin and phenol red, Nissui Pharmaceutical Co., Ltd.) was dissolved in Japanese Pharmacopoeia water for injection (Lot No. 8L88, Otsuka Pharmaceutical Factory, Inc.) to obtain a volume of 1 L. The solution was autoclaved for sterilization, then cooled to room temperature and mixed with a sterilized 7.5% sodium bicarbonate solution (special grade, Lot No. 905X1946, Kanto Chemical Co., Ltd.) to adjust the pH to 7.2 to 7.4. The solution was then mixed with a filter-sterilized L-glutamine solution (special grade, L-glutamine, Lot No. PEH6211, Wako Pure Chemical Industries, Ltd.) to obtain a concentration of0.292 giL. Fetal bovine serum

- 10-SR09243 (Lot No. 366272, GIBCO) was added to the mixture to obtain a fmal concentration of I0%. The fetal bovine serum was inactivated at 56°C for 30 min before use. All procedures following autoclaving were performed aseptically. 

### 7. S9 mix

S9 After purchase from Kikkoman Corporation, S9 mix (Lot No. CAM-605, manufactured on November 20, 2009) was cryopreserved at -80°C or below and used within 6 months after the production date (expiration date: 6 months after production). To prepare S9 mix, 2.45 mL ofcofactor mix was added to 1.05 mL ofS9 prepared using liver homogenate from Slc:SD rats (males, 7 weeks ofage) intraperitoneally administered with phenobarbital and 5,6-benzoflavone to induce production of drug-metabolizing enzymes. The composition ofS9 mix is presented in the following table: Composition of S9 mix in 1 mL (Kikkoman Corporation, Lot No. RAA-605, protein content in S9: 26.23 mg/mL) 0.3 mL MgCh (Wako Pure Chemical Industries, Ltd., Lot No. SDN0075) (Wako Pure Chemical Industries, Ltd., Lot No. CDP6106) (Oriental Yeast Co., Ltd., Lot No. 118904) 5 !liDO] KCI G-6-P NADP (Oriental Yeast Co., Ltd., Lot No. 045909) 33 llmol 5 !liDO] 4 !liDO] 4 !liDO] 0.1 mL HEPES buffer Distilled water 

### 8. Test methods (Dojindo Laboratories, Lot No. PE026)

8.1. Preliminary test (cell-growth inhibition test) 8.1.1. Test groups The preliminary test was performed in three test series: short-term treatment without metabolic activation, short-term treatment with metabolic activation, and continuous treatment for 24 h. The highest dose ofthe test substance was set at 1870 J.Lg/mL, equivalent to 10 mM (molecular weight: 187.07) according to the test guideline, and 7 lower doses were selected in a twofold dilution series (8 doses in total: 1870, 935, 468, 234, 117, 58.4, 29.2, and 14.6 11g/mL). In addition, a negative control group was included in each test series. Two plates were used for each group, each marked with an identification number. 8.1.2. Cell seeding Five milliliters ofcell suspension was seeded in a 60-mm plate and incubated in a C02 incubator at 5.0% C02 and 37.0°C. The cell density in the suspension was 0.4 x 104 cells/mL for short-term treatment without metabolic activation and for continuous treatment for 24 h, and 0.6 x 10 4 cells/mL for short-term treatment

- 11-with metabolic activation. 8

. 1.3

.Treatment with test solutions a) Short-term treatment without metabolic activation SR09243

On the third day from cell seeding, the culture medium in the plate was removed. After the test solution was mixed with fresh medium in a ratio of300 flL to 2.7 mL in a test tube, 3 mL ofthe mixture was added to the plate and incubated for 6 h. After completion ofincubation, the fluid in the plate was removed, and the remaining cells were rinsed with Ca 2+- and Mg2+ -free Dulbecco's phosphate buffer. Then, 5 mL offresh medium was added to the plate and incubated for an additional 18 h. b) Short-term treatment with metabolic activation On the third day from cell seeding, the culture medium in the plate was removed. After the mixture ofS9 mix and fresh medium was mixed with the test solution in a ratio of0.5 mL:2

.2 mL :300 flL in a test tube (final concentration ofS9: approximately 5% [vol]), 3 mL ofthe mixture was added to the plate and incubated for 6 h. After completion ofincubation, the fluid in the plate was removed, and the remaining cells were rinsed with Ca2+
- and Mg2+ -free Dulbecco 's phosphate buffer. Then, 5 mL offresh medium was added to the plate and incubated for an additional 18 h. c) Continuous treatment for 24 h

On the third day from cell seeding, the culture medium in the plate was removed

. After the test solution was mixed with fresh medium in a ratio of500 flL to 4.5 mL in a test tube, 5 mL ofthe mixture was added to the plate and incubated for an additional 24 h. 8.1.4. Observation for precipitation ofthe test substance At the start and end oftreatment with the test solution, the plate was macroscopically observed for precipitation ofthe test substance. 8.1.5. Observation for effects ofthe test substance on the culture pH At the start and end oftreatment with the test solution, the culture medium in the plate was macroscopically observed for color change. When no color change was found, the test substance was considered to have no effect on the culture pH. 8.1.6. Measurement ofthe cell growth rate and calculation ofthe concentration causing 50% inhibition ofcell growth (ICso) After completion ofincubation, the fluid in the plate was removed, and the remaining cells were rinsed with Ca2+
- and Mg2+-free Dulbecco's phosphate buffer. The cells were then fixed in 10% formalin for approximately 10 to 15 min and stained with 0.1% (w/v) crystal violet for approximately 10 to 15 min. After staining, the plate was washed with tap water in a water bath and air

-dried. The

- 12-SR09243 cell growth rate was measured for each plate using a monolayer culture cell densitometer (MONOCELLATER II, Toyo Sokki Co., Ltd.), with the growth rate in the negative control group defined as 100%. As a decrease in growth rate by 50% or more was not detected in the test substance groups, the concentration causing 50% inhibition ofcell growth (IC50) was not calculated. 8.2

. Main test (chromosomal aberration test) 8.2.1. Test groups In the preliminary test, cell growth was not affected by short-term treatment with or without metabolic activation, and was slightly inhibited at high concentration ofcontinuous treatment for 24 h. Accordingly, 1870 

### J.LglmL was selected as the highest dose ofeach test series, and lower doses were set with a twofold dilution series to select 3 doses in total for short-term treatment with and without metabolic activation, and 4 doses in total for continuous treatment for 24 h. Four plates were used for each group except the positive control group, including 2 plates for the satellite group to observe the effect ofthe test substance on cell growth. Two plates were used for the positive control group. Each plate was marked with an identification number. 8.2.2. Cell seeding The method described in "8. Test methods, 8.1. Preliminary test, 8.1.2. Cell seeding" was used

. 8.2.3. Treatment with test solutions a) Short-term treatment without metabolic activation The method described in "8. Test methods, 8.1. Preliminary test, 8.1.3. Treatment with test solutions, a) Short-term treatment without metabolic activation" was used. b) Short-term treatment with metabolic activation The method described in "8

. Test methods, 8.1. Preliminary test, 8.1.3. Treatment with test solutions, b) Short-term treatment with metabolic activation" was used. c) Continuous treatment for 24 h The method described in "8. Test methods, 8.1. Preliminary test, 8.1.3. Treatment with test solutions, c) Continuous treatment for 24 h" was used. 8.2.4. Observation for precipitation ofthe test substance The method described in "8. Test methods, 8.1. Preliminary test, 8.1.4. Observation forprecipitation of the test substance" was used. 8.2.5. Observation for effects ofthe test substance on the culture pH The method described in "8. Test methods, 8.1. Preliminary test, 8.1.5. Observation for effects of the test substance on the cultureplf' was used.



### - 13-

### SR09243 8.2.6. Measurement ofthe cell growth rate The method described in "8. Test methods, 8.1

. Preliminary test, 8.1

. 6. Measurement of the cell growth rate and calculation of the concentration causing 50% inhibition of cellgrowth (JC 50

)" 

### was used. The IC50 

### was not calculated. 8.2.7. Preparation ofchromosome specimens Approximately 2 h before the end ofincubation, colcemid (Lot No. 571750, GIBCO) was added to each plate at a final concentration of0.2 J.Lg/mL. At the end ofincubation, the fluid in each plate was collected into respective centrifuge tubes as follows: The plate was first treated with 0.02% EDTA (0.5M EDTA: Lot No. 1390894, GIBC0)-0.25% trypsin (2.5% trypsin: Lot Nos. 633330 and 690264, GIBCO) to detach the cells. The resultant cell suspension was then collected into a centrifuge tube and centrifuged at 1,000 rpm for 5 min. After the supernatant was removed, 0.075 moVL potassium chloride (Lot No. 81 OX1990, Kanto Chemical Co., Ltd.) was added to the cells, and the mixture was allowed to stand for 30 min at ordinary temperature with gentle pipetting while the cells swelled. After iced Carnoy's fixative (mixture ofmethanol and acetic acid in a ratio of3:1. Methanol, Lot No. 11 ON1127, Kanto Chemical Co., Ltd.; acetic acid, Lot No. KWF0791, Wako Pure Chemical Industries, Ltd.) was added to fix the cells, the mixture was centrifuged at 1,000 rpm for 5 min. After removing the supernatant, Carnoy's fixative was added to the cells. After these procedures were repeated three times, the cell suspension was dropped onto a glass slide, which was then allowed to air-dry at least overnight. Two chromosome specimens were prepared from each plate. The slide was stained with 2% Giemsa solution (Giemsa solution, Lot No. KN999, Wako Pure Chemical Industries, Ltd.; instant phosphate buffer (pH 7.2), Lot No. A941, Mitsubishi Chemical Medience Corporation) for 20 min. After being washed with water and air-dried, the slide was sealed with a mounting agent (Malinol: Lot No. 0701201, Muto Pure Chemicals Co., Ltd.). 8.2.8. Observation ofchromosome specimens Prior to observation ofspecimens, one slide was selected per plate for each dose and blind-coded. Using a microscope (BX51 TF, Olympus Corporation) with a total magnification of600, 100 metaphase chromosomes were observed in each slide. Chromosomal aberrations were assessed according to the following classification systems. Cells with 25 ± 

### 2 chromosomes were observed for structural aberrations.

a) Structural aberration Chromatid break (ctb) Distinctive discontinuity (break) in the chromatid; either a discontinuity wider than the chromatid or a break offthe major axis ofthe chromatid

- 14-

### SR09243

• Chromatid exchange (cte) At least two breaks in the chromatid exchanged with each other (combined)
• Chromosome break (csb) Break at the same position in a pair ofchromatids and judged according to the criteria for chromatid break
• Chromosome exchange (cse) Exchange at the same position in a pair of chromatids toward the same direction
• Others Other structural aberrations include fragmentation (defined as breaks or gaps affecting almost all metaphase chromosomes with no exchange-type aberration) b) Gap Unstained area (no stain) in chromatids or chromosomes with a width Jess than that ofthe chromatid c) Numerical aberration
• Polyploid (poly) Triploid, tetraploid, etc., the chromosomal constitution of a cell containing multiples ofthe normal number ofchromosomes (25 ± 

### 2)
• Others Other numerical aberrations include endoreduplication (defined as double chromosomes not separated but arranged in parallel), being distinguished from polyploid. 8.2.9.Calculation ofobservation results The number ofcells with aberrations described below was counted for each plate, and the total sum was calculated for each test group. In addition, the incidence (%) was calculated for the total number ofcells with structural aberrations (a cell with more than one structural aberration was counted as one) as well as the total number ofcells with numerical aberrations

. The incidence(%) was calculated as the percentage ofthe number ofcells with aberrations relative to the total number ofcells observed (number ofmetaphase chromosomes).

a) Structural aberrations
• ctb: number ofcells with chromatid breaks • cte: number ofcells with chromatid exchanges • csb: number ofcells with chromosome breaks • cse: number ofcells with chromosome exchanges • others: number ofcells with other structural aberrations • total: number ofcells with any structural aberrations



### - 15-SR09243 b) Gap gap: number ofcells with gaps c) Numerical aberration

• poly: number ofpolyploid cells • others: number ofcells with other numerical aberrations • total: number ofcells with any numerical aberrations 9. Evaluation oftest results The results were determined to be positive when the total incidence(%) ofstructural or numerical aberrations was I0% or more in a dose-dependent manner, or when reproducibility was shown in the results of5% or more in the confrrmatory test. All other results were determined to be negative. Statistical analysis was not used. As an incidence ofaberrations of5% or more was not detected in any test series, a D20 value (the concentration at which 20% ofthe cells show aberrations) was not calculated.



### - 16-SR09243 RESULTS 1. 

### Preliminary test The results ofthe cell growth rates are shown in Table 1 and Figure 1, and the results ofprecipitation ofthe test substance and effects on the culture pH are shown in Table 1. No effects were noted in cell growth after short-term treatment with or without metabolic activation. Cell growth was slightly inhibited at doses of935 and 1870 jlg/mL after continuous treatment for 24 h, and the cell growth rates were 64.5% and 65.5%, respectively. No precipitation ofthe test substance was noted in any test series. No effects ofthe test substance were noted in the culture pH in any test series. 

### 2. Main test The results ofthe cell growth rates, precipitation ofthe test substance, and effects on the culture pH in the main test are shown in Table 2, and the results ofclastogenicity evaluation are shown in Tables 3-1 to 3-3. In the evaluation ofeffects ofthe test substance on cell growth in the satellite group, which was performed in parallel with the evaluation ofclastogenicity, cell growth was slightly inhibited at 1870 jlg/mL only after continuous treatment for 24 h, and the cell growth rate was 63.0%. No precipitation ofthe test substance was noted in any test series. No effects ofthe test substance were noted in the culture pH in any test series. Both the incidences ofstructural and numerous aberrations were less than 5% after short-term treatment without metabolic activation (doses tested: 468 to 1870 jlg/mL), short-term treatment with metabolic activation (doses tested: 468 to 1870 jlg/mL), and continuous treatment for 24 h (doses tested: 234 to 1870 jlg/mL). The incidences ofstructural aberrations in the positive control group were 64.0% after short-term treatment without metabolic activation, 39.0% after short-term treatment with metabolic activation, and 64.0% after continuous treatment for 24 h.

- 17-The clastogenic potential of (CHLIIU) cells. SR09243 DISCUSSION
was evaluated in vitro using Chinese hamster lung

Based on the results ofthe preliminary test (cell-growth inhibition test), the highest dose was set at I0 mM, which is the upper limit concentration defined in the test guideline, for each test series, and totally 3 or 4 doses were set for the main test (chromosomal aberration test). In the main test, both the incidences ofstructural and numerous aberrations were less than 5% at all doses ofall test series, which showed negative results. In the positive control group, the incidence ofstructural aberrations ofchromosomes was clearly positive in all test series, demonstrating that the cell line used in this study had sufficient sensitivity. These results indicate that is not clastogenic in cultured mammalian cells under the conditions used in the present test. ENVIRONMENTAL FACTORS THAT MIGHT HAVE AFFECTED THE RELIABILITY OF THE TEST RESULTS No environmental factors that might have affected the reliability ofthe test results were found. STORAGE OF DOCUMENTS AND MATERIALS I. Archive facilities and documents and materials to be stored

The following documents and materials will be stored in the archives ofSafety Research Institute for Chemical Compounds Co., Ltd.:

1. Study protocol and study protocol amendments 2. Raw data and other documents 3. Final report 4. Specimens 2. Storage period The above-mentioned documents and materials will be stored for I0 years after the study completion. Their storage thereafter will be decided on agreement with the study sponsor. NAME AND SEAL OF STUDY DIRECTOR Name and seal affixed in the original August I0, 201 0 Kohtaro Kawamura, Study Director Date



### - I8-Table I Effects of on growth rate ofCHL/fU with or without metabolic activation (preliminary test) (SR09243)
Growth rate (% to the control) S9-

Group Concentration 6-18 h (f!g/mL) (Mean)
Control a 100 , 100 (100.0)
14.6 97 , 98 ( 97.5)
29.2 99 , 99 ( 99.0)
58.4 92 , 86 ( 89.0)
117 85 , 98 ( 91.5)
234 95 , 90 ( 92.5)
468 91 , 92 ( 91.5)
935 83 , 86 ( 84.5)
1870 78 , 82 ( 80.0)
ICso (f!g/mL)

a: Water for injection (Japanese pharmacopoeia) Precipitation and change ofpH in culture medium were not observed. The figure in parentheses represents mean value oftwo plates. -:Blank

- 19- S9+ S9- 6-18 h 24-0 h (Mean) (Mean)
100 , 100 100 , 100

(100.0) (100.0)
91 , 96 98 , 92 ( 93

.5) ( 95.0)
92 , 96 97 , 91 ( 94.0) ( 94.0)
97 , 90 99 , 87 ( 93.5) ( 93.0)
87 , 92 88 , 84 ( 89.5) ( 86.0)
97 , 92 94 , 91 ( 94.5) ( 92.5)
92 , 94 88 , 77 ( 93.0) ( 82.5)
89 , 96 70 , 59 ( 92

.5) ( 64.5)
88 , 106 78 , 53 ( 97.0) ( 65.5)

### 120

Q 

### 80 0

..... ...... s:: 0 u 0 ...c:

...... 0 

### 60 ...... ?J( '--' 0**~** -o-S9- 6-18 h ..... ...c:

### ~ 40 0 ---+-- S9+ 6-18 h

..... c:J
-J- S9- 24-0 h 

### 20

0 0 

### 14.6 29.2 58.4 117 

### 234 468 935 1870

Concentration of (f.lg/mL)

Figure 1 Effects of on growth rate ofCHL/IU with or without metabolic activation (preliminary test) (SR09243)
Each point represents mean value (n=2).



### - 20-Table 2 Ef fects of on growth rate ofCHL/IU with or without metabolic activation (chromosomal aberration test) (SR09243)
Growth rate (% to the control) S9-

Group Concentration 6-18 h (J.Lg/mL) (Mean)
Control a 100 , 100 (1 00.0)
234

468 100 , 102 (101.0)
935 99 , 106 (102.5)
1870 92 , 90 ( 91.0)

a : Water for injection (Japanese pharmacopoeia) Precipitation and change ofpH in culture medium were not observed. The figure in parentheses represents mean value oftwo plates

.

- :Blank

- 21

- S9+ 6-18 h (Mean)
100 , 100

(100.0)

97 , 104 (1 00.5)
105 , 104

(1 04

.5)
108 , 105

(1 06.5) S9- 24

-0 h (Mean)
100 , 100

(1 00.0)
92 , 115 (103.5)
96 , 101 ( 98.5)
78 , 85 ( 81.5)
62 , 64 ( 63.0)N N Table 3-1 Results ofthe chromosomal aberration test of (6 hours treatment without metabolic activation) (SR09243)

Time schedule • Concentration Growth Number of Structural aberrations (hours) S9 Group ()lg/mL) rate(%) metaphase observed ctb cte csb cse others total (%)
100 0 1 0 0 0 1 Control b - 100

.0 100 0 0 0 0 0 0 200 0 1 0 0 0 1 ( 0.5) 100 0 0 0 0 0 0 468 101.0 100 0 0 0 0 0 0 200 0 0 0 0 0 0 ( 0

.0) 100 0 0 0 0 0 0 6-18
- 935 102.5 100 0 0 0 0 0 0 200 0 0 0 0 0 0 ( 0

.0) 100 0 0 0 0 0 0 1870 91.0 100 0 0 0 0 0 0 200 0 0 0 0 0 0 ( 0.0)

# ~ 100 12 57 0 2 0 66 Mitomycin C 0.1 100 13 55 0 1 0 62 200 25 112 0 3 

## I 0 128 (64

.0) ctb, chromatid break cte, chromatid exchange csb, chromosome break cse, chromosome exchange poly, polyploid a : Time schedule ; treatment time-recovery time b : Water for injection (Japanese pharmacopoeia) c :Judgment was made according to the total(%) of structural aberrations and numerical aberrations; -,negative +, positive Numerical aberrations Gap Judgment c poly others total (%)
0 0 0 0 0 1 0 1

- 0 1 0 1 ( 0

.5) 0 0 0 0 0 0 0 0 0 0 0 0 ( 0.0) 0 0 0 0 0 1 0 1

- 0 1 0 1 ( 0.5) 0 0 0 0 0 0 0 0 0 0 0 0 ( 0.0) 0 0 0 0 1 0 0 0 + 1 0 0 0 ( 0.0)N w Table 3-2 Results ofthe chromosomal aberration test of (6 hours treatment with metabolic activation) (SR09243)

Time schedule • Concentration Growth Number of Structural aberrations (hours) S9 Group**(~-Lg/mL)** rate(%) metaphase observed ctb cte csb cse others total (%)
100 0 0 0 0 0 0 Control b - 100

.0 100 0 0 0 0 0 0 200 0 0 0 0 0 0 ( 0

.0) 100 0 0 0 0 0 0 468 100.5 100 0 0 0 0 0 0 200 0 0 0 0 0 0 ( 0

.0) 100 0 0 0 0 0 0 6-18 + 935 104

.5 100 0 0 0 0 0 0 200 0 0 0 0 0 0 ( 0

.0) 100 1 I 0 0 0 1 1870 106.5 100 0 0 0 0 0 0 200 I 1 0 0 0 1 ( 0

.5)

# ~ 100 7 32 0 0 0 37 3,4-Benzopyrene 10 100 12 33 2 0 0 41 200 19 65 2 0 0 78 (39

.0) ctb, chromatid break cte, chromatid exchange csb, chromosome break cse, chromosome exchange poly, polyploid a : Time schedule ; treatment time-recovery time b : Water for injection (Japanese pharmacopoeia) c : Judgment was made according to the total (%) of structural aberrations and numerical aberrations ; - , negative +, positive Numerical aberrations Gap Judgment c poly others total (%)
0 0 0 0 0 0 0 0

- 0 0 0 0 ( 0

.0) 0 0 0 0 0 0 0 0 0 0 0 0 ( 0.0) 0 0 0 0 0 0 0 0

- 0 0 0 0 ( 0

.0) 0 0 0 0 0 1 0 1 0 1 0 1 ( 0

.5) 0 0 0 0 0 0 0 0 + 0 0 0 0 ( 0.0)N**-~'>-** Table 3-3 Results ofthe chromosomal aberration test of (24 hours treatment without metabolic activation) (SR09243)

Time schedule • Concentration Growth Number of Structural aberrations (hours) S9 Group (llg!mL) rate(%) metaphase observed ctb cte csb cse others total (%)
100 0 0 0 0 0 0 Control b - 100.0 100 0 0 0 0 0 0 200 0 0 0 0 0 0 ( 0

.0) 100 0 0 0 0 0 0 234 103

.5 100 1 0 0 0 0 1 200 1 0 0 0 0 1 ( 0.5) 100 0 0 0 0 0 0 468 98.5 100 0 0 0 0 0 0 24-0 200 0 0 0 0 0 0 ( 0.0)



### - 100 0 0 0 0 0 0 935 81.5 100 0 0 0 0 0 0 200 0 0 0 0 0 0 ( 0

.0) 100 0 1 0 0 0 1 1870 63.0 100 0 1 0 0 0 1 200 0 2 0 0 0 2 ( 1.0)

# ~ 100 10 59 0 1 0 67 Mitomycin C 0

.05 100 6 58 0 0 0 61 200 16 117 0 1 0 128 (64

.0) ctb, chromatid break cte, chromatid exchange csb, chromosome break cse, chromosome exchange poly, polyploid a : Time schedule ; treatment time-recovery time b : Water for injection (Japanese pharmacopoeia) c :Judgment was made according to the total(%) of structural aberrations and numerical aberrations;

- , negative +, positive Numerical aberrations Gap Judgment c poly others total (%)
0 0 0 0 0 0 0 0 

### - 0 0 0 0 ( 0

.0) 0 0 0 0 0 1 0 1 0 1 0 1 ( 0.5) 0 0 0 0 0 1 0 1 0 1 0 1 ( 0

.5)

- 0 2 0 2 0 0 0 0 0 2 0 2 ( 1.0) 0 0 1 1 0 0 1 1 0 0 2 2 ( 1.0) 0 0 0 0 0 0 0 0 + 0 0 0 0 ( 0.0)Appendix 1-1

1. 

### Name ofarticle

2. Analysis values Items Analysis table



### Analysis values

-25- SR09243 

### January 14, 2010

NotesAppendix 1-2

1. Name ofarticle

2. Analysis values Items Analysis table

Analysis values

-26- SR09243 August I, 2010

Notes