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 Date 
Study 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 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 of Economy, 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 of the 

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. 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 of the 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 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 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 of study May I7, 20IO May I7, 20IO May I7, 20IO 

Preparation of specimens 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 I 0 June25, 20IO June25, 20IO 

Final report (draft): Text June 25, 20 I 0 June25, 20IO June 25, 20IO 

Final report August I 0, 20 I 0 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 of Economy, 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 of the PFSB, MHLW, July 4, 2008; No.2 of the 

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 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 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 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 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 of the 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 

ENVIRONMENTAL 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 of CHL/IU with or without 

metabolic activation (chromosomal aberration test) (SR09243) · · · · · · · · · · 21 

Table 3-1 Results of the chromosomal aberration test of 

(6 hours treatment without metabolic activation) (SR09243) · · · · · · · · · · · · · 22 

Table 3-2 Results of the chromosomal aberration test of 

(6 hours treatment with metabolic activation) (SR09243) · · · · · · · · · · · · · · · · · 23 

Table 3-3 Results of the 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 of the 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 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. 1121002 ofthe PFSB, MHLW, 

November 21, 2003; No. 2 of the 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 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) 

- 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 I 0 

January 18, 2010 

Start of experiment: April29, 2010 

Preliminary test 

Initiation of treatment with the test substance: 

April29, 2010 

Fixation and staining: April30, 2010 

Main test 

Initiation of treatment with test substance: 

May I7, 20IO 

Preparation of specimens: May 18, 20 I 0 

Experiment completion (completion of observation): 

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 of the test 

substance treatment on the culture pH were not observed in any test series. 

Based on the results of the preliminary test, the highest dose of each 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 of chromosomes were less than 5% at all doses of 

all test series, which showed negative results. No precipitation of the test substance or 

effects of the 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 of structural aberrations of chromosomes 

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 of test 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 of final use) 

Handled in a clean bench wearing gloves, protection 

- 8 -
SR09243 

glasses, masks, etc. 

Remaining test substance: After completion of test operation including that in the 

related tests, the remaining test substance was returned to 

the supplier. 

2. Preparation of test 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 of the 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 of mitomycin C contained 

2 mg (potency) of Japanese Pharmacopoeia mitomycin C; hence, calculations for 

preparation were made on the basis that 1 mg (potency) of mitomycin C was 

equivalent to 1 mg of mitomycin 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 of 3,4-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 

of a 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 of the growth rate, chromosomal stability during subcultivation, ease 

of observation of chromosome 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 I 0%. 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 of cofactor mix was added to 1.05 mL of S9 prepared 

using liver homogenate from Slc:SD rats (males, 7 weeks of age) 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 of cell 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 104 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 of the mixture was added to the plate and 

incubated for 6 h. After completion of incubation, 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 of fresh 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 of S9 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 of the mixture was added 

to the plate and incubated for 6 h. After completion of incubation, 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 of fresh 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 of the mixture was added to the plate and 

incubated for an additional 24 h. 

8.1.4. Observation for precipitation of the test substance 

At the start and end oftreatment with the test solution, the plate was 

macroscopically observed for precipitation of the test substance. 

8.1.5. Observation for effects of the test substance on the culture pH 

At the start and end of treatment 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 of the cell growth rate and calculation ofthe concentration causing 

50% inhibition of cell growth (ICso) 

After completion of incubation, 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 of cell 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 

of continuous treatment for 24 h. Accordingly, 1870 J.LglmL was selected as the 

highest dose of each 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 of the 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 of the test substance 

The method described in "8. Test methods, 8.1. Preliminary test, 8.1.4. 

Observation for precipitation of the test substance" was used. 

8.2.5. Observation for effects of the 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 culture plf' was used. 

- 13-
SR09243 

8.2.6. Measurement of the 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 cell growth (JC50)" was used. The IC50 was not calculated. 

8.2. 7. Preparation of chromosome specimens 

Approximately 2 h before the end of incubation, colcemid (Lot No. 571750, 

GIBCO) was added to each plate at a final concentration of0.2 J.Lg/mL. At the end 

of incubation, 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 of chromosome specimens 

Prior to observation of specimens, one slide was selected per plate for each dose 

and blind-coded. 

Using a microscope (BX51 TF, Olympus Corporation) with a total magnification 

of 600, 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 off the major axis of the 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 of chromatids 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 of the normal number of chromosomes (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 of observation results 

The number of cells 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 of cells with structural aberrations (a cell 

with more than one structural aberration was counted as one) as well as the total 

number of cells with numerical aberrations. The incidence(%) was calculated as 

the percentage ofthe number of cells with aberrations relative to the total number 

of cells observed (number of metaphase chromosomes). 

a) Structural aberrations 

• ctb: number of cells with chromatid breaks 

• cte: number of cells with chromatid exchanges 

• csb: number of cells with chromosome breaks 

• cse: number of cells with chromosome exchanges 

• others: number of cells with other structural aberrations 

• total: number of cells with any structural aberrations 

- 15-
SR09243 

b) Gap 

gap: number of cells with gaps 

c) Numerical aberration 

• poly: number of polyploid cells 

• others: number of cells with other numerical aberrations 

• total: number of cells with any numerical aberrations 

9. Evaluation of test results 

The results were determined to be positive when the total incidence(%) of structural 

or numerical aberrations was I 0% or more in a dose-dependent manner, or when 

reproducibility was shown in the results of 5% or more in the confrrmatory test. All 

other results were determined to be negative. Statistical analysis was not used. 

As an incidence of aberrations of 5% 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 of the cell growth rates are shown in Table 1 and Figure 1, and the results 

of precipitation 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 of the test substance was noted in any test series. 

No effects of the test substance were noted in the culture pH in any test series. 

2. Main test 

The results of the cell growth rates, precipitation of the test substance, and effects on 

the culture pH in the main test are shown in Table 2, and the results of clastogenicity 

evaluation are shown in Tables 3-1 to 3-3. 

In the evaluation of effects of the test substance on cell growth in the satellite group, 

which was performed in parallel with the evaluation of clastogenicity, 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 of the test substance was noted in any test series. 

No effects of the test substance were noted in the culture pH in any test series. 

Both the incidences of structural 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 of structural 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 of the preliminary test (cell-growth inhibition test), the highest dose 

was set at I 0 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 of structural and numerous aberrations were less 

than 5% at all doses of all test series, which showed negative results. 

In the positive control group, the incidence of structural aberrations of chromosomes 

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 of the 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 of Safety 

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 I 0 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 I 0, 201 0 

Kohtaro Kawamura, Study Director Date 

- I8-
Table I Effects of on growth rate of CHL/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 of pH in culture medium were not observed. 

The figure in parentheses represents mean value of two 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 

0 80 ..... 

...... 

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 of CHL/IU with or without 

metabolic activation (preliminary test) (SR09243) 

Each point represents mean value (n=2). 

- 20-
Table 2 Effects 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 of pH in culture medium were not observed. 

The figure in parentheses represents mean value of two 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 of the 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 of the 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 of the 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 of article 

2. Analysis values 

Items 

Analysis table 

Analysis values 

-25-

SR09243 

January 14, 2010 

Notes 
Appendix 1-2 

1. Name of article 

2. Analysis values 

Items 

Analysis table 

Analysis values 

-26-

SR09243 

August I, 2010 

Notes