LIFE SCIENCE LABORATORY ® 5

- 19, 2

- chome, Nishihonmachi, Nishi

- ku, Osaka TeI.06

- 6531

- 1881 (pilot number)



### Study code No.: 01-VII-0601



# Final report

Bacterial reverse mutation study of

July 19, 2001

Yukio Yanagimoto, M.D., LlFE SCIENCE LABORATORY

New Okazaki bashi Building, 5

- 19, 2

- chome, Nishihonmach i, Nishi

- ku, Osaka
Tel. (06)6531

- 1881 (pilot number)LIFE SCIENCE LABORATORY ® 5

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- chome, Nishihonmachi, Nishi

- ku, Osaka TeI.06

- 6531

- 1881 (pilot number)



### - 1 - 

### Title: Bacterial reverse mutation study of

Summary: As a result of the bacterial reverse mutation study of none of the

bacterial strains showed the number of reverse mutant colonies twice or more that in the vehicle control although growth inhibition was noted in some of the strains at the highest dose level of 5000 μg/ plate in dose setting and main studies. Therefore, it was concluded that was not mutagenic.

1. Study objective Mutagenic property of was investigated as a part of safety evaluation. The main study was conducted based on SOP/REV/000 of this laboratory in accordance with the OECD Guidelines for the Testing of Chemicals: Toxicity
Studies.

2. Sponsor
Name
Location

3. Contract research organization
Name LIFE SCIENCE LABORATORY
Location 5 -19, 2 -chome, Nishihonmachi, Nishi-ku, Osaka

4. Testing facility Name Chihayaakasaka Research Institute (First Laboratory),

LIFE SCIENCE LABORATORY Location 1082 Aza-chihaya, Chihayaakasaka-mura, Minamikawachi-gun,

Osaka Prefecture

5. Study period Study initiation date June 11, 2001 Study completion date July 19, 2001LIFE SCIENCE LABORATORY ® 5

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### 6. Test substance
1 ) Name 2) Abbreviation 3) Lot No. 4) Structural formula

3) Purity 4) Description 5) Solubility 6) Stability Stabe under ordinary handling conditions. 7) Storage conditions To be stored in a sealed container in a dark and cool place avoiding moisture. 8) Precautions for handling Direct contact with the eyes, skin and clothes should
be avoided. 9) Supplier
Name
Location

7. Name and abbreviation of the positive control substance 1) N-Ethyl- N' -nitro-N-nitrosoguanidine (ENNG) 2) 2 -Nitrofluorene (2 -NF) 3) 9 -Aminoacridine hydrochioride (9

- AA) 4) 2 -Aminoanthracene (2

- AA) 5) Sodium azide (NaN 3 

### )

8. Names of vehicle control substances The solvents (vehicles) used to dissolve the test substance (1 wt% solution used asLIFE SCIENCE LABORATORY ® 5

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### the stock solution) and the positive control substance
Test substance: Purified water Positive control substance: Dimethyl sulfoxide (DMSO)

9. Reagents
S9 S9 manufactured at Oriental Yeast Co., Ltd. according to the following procedure was purchased on June 20, 2001, and used (lot No. 01032313). Method of preparation of S9 Phenobarbital (PB) was administered intraperitoneally in male SD rats (7

- week

- old, weighing 213.8 ± 8.3 g) at 30 mg/kg on th e first day and at 60 mg/kg for the following 3 consecutive days. An intraperitoneal administration of 5,6

- benzoflavone (BF) at 80 mg/kg was given 2 days later. The liver was extracted 4 days later and S9 was prepared according to the usual method.
Stora ge conditions S9 was stored at −80°C until use after purchase. Nutrient broth (Difco Laboratories, lot No. 123820JD) Agar (Difco Laboratories, lot No. 0286000)
Others Commercially available special grade reagents

10. Study system
1 ) Bacterial strains us ed Salmonella typhimurium TA100
TA98
TA1535
TA1537
Escherichia coli WP2uvrA Viable cell count (  10 9 

### /mL) at completion of preincubation [converted from turbidity (absorbance at 660 nm)]

Dose setting study Main studyLIFE SCIENCE LABORATORY ® 5

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### 2) Origin and management of bacterial strains
Salmonella strain s and E. coli WP2uvrA were portioned from the Institute for Fermentation, Osaka, on June 13, 2000. These bacterial strains are stored under the storage conditions (0.8 mL of bacterial suspension mixed with 0.07 mL of DMSO and stored at −80°C) designated at this institute because their properties are liable to change. They are maintained strictly to show stable mutagenicities by subculturing within 1 year and testing for bacterial properties every 3 months.

11. Preparation of test materials
1 ) Test substance The test substance solutions of the following concentrations were prepared using purified water. Dose setting study
In t he S9 mix (−) and (+) systems; 48.8, 195, 781, 3125, 12500 and 50000 μg/mL Main study (decided from the results of the dose setting study) In the S9 mix (−) and (+) systems; 1563, 3125, 6250, 12500, 25000 and 50000 μg/mL 2) P ositive control substance
DMSO solutions of the concentrations specified for respective bacterial strains according to the SOP of this institute were prepared and tested at the final dose levels shown in the following table.LIFE SCIENCE LABORATORY ® 5

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### 3) S9 mix S9 mix with the following composition was prepared aseptically just before use.

4) Nutrient broth culture solution
Nutrient broth 0.8 g
Sodium chloride 0.5 g
Purified water 100 mL This culture solution was dispensed in 10

- mL aliquots in L

- shaped tubes and
autoclaved. Bacterial strai n S9 mix (−) system S9 mix (+) system

Name Dose level

(μg/plate) Name Dose level (μg/plate)

Ingredie nt Amount in 1 mL of S9 mix

S9 0.4M magnesium chloride solution 1.65M potassium chloride solution 1M glucose

- 6

- phosphate solution 0.1M NADPH solution 0.1M NADH solution 0.2M sodium phosphate buffer Purified water Residual amount (to make 1 mL)LIFE SCIENCE LABORATORY ® 5

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### 5) Minimum glucose ag ar plate medium Vogel

- Bonner minimal media E (a stock solution of 10

- time concentration) with the following composition was prepared first. Magnesium sulfate heptahydrate 2 g Citric acid monohydrate 20 g Anhydrous dipotassium phosphate 100 g
Ammonium so dium hydrogenphosphate tetrahydrate 35 g These were dissolved in purified water in this order and made 1000 mL (pH 7)

. Then, this solution was used to prepare the minimum glucose agar plate medium with the following composition. Vogel

- Bonner minimal media E (stock solution of 10

- time concentration)
80mL
Glucose 16 g/100 mL purified water Agar 12 g/620 mL purified water These were autoclaved respectively and, after cooling to about 60°C, mixed and dispensed in 30

- mL aliquots in gamma

- ray sterilized plates ( 90 mm in diameter

, Ina Optica, Co., Ltd.) , and solidified. 6) Overlay agar An aqueous solution containing 0.6% agar and 0.5% sodium chloride was prepared, autoclaved and maintained at about 45°C

. For Salmonella strains, 10 volumes this agar solution and 1 volume each of solutions containing 0.5 mM L

- histidine and 5 mM D

- biotin, respectively, sterilized by filtration were mixed. For Escherichia coli , 10 volumes of the agar
solution and 1 volume of 0.5 mL L

- tryptophan solution sterilized by filtration

wer e mixed. These were maintained at about 45° C until use to prevent solidification. The amino acid solutions used were sterilized using the vacuum filtration device for sterilization ( 0.22 μm in pore diameter , Iwaki Glass Industries Co., Ltd.).

12. Study method The study was performed by the preincubation method. Two plates were used for each test, and each dose level.LIFE SCIENCE LABORATORY ® 5

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### 1) D ose setting study and main study In the dose setting study, bacterial suspensions of respective strains dispensed and
stored frozen were thawed and 20 μL each was inoculated in 10 mL of nutrient broth culture solution and incubated by shaking (65 reciprocations/minute) at
37°C in a dark room for 12 hours . A 0.1

- mL aliquot of this culture solution, 0.5
mL of S9 mix (0.5 mL of 0.1M sodium phosphate buffer, pH 7.4, when S9 mix was not used) and 0.1 mL of 48.8, 195, 781, 3125

, 12500 or 50000 μg/mL test
substance solution (4.88

, 19.5

, 78.1

, 312.5

, 1250 or 5000 μg/ plate) were measured in a sterilized test tube and preincubated at 37°C for 20 minutes

. Then, 2 mL of the overlay agar containing amino acid was added, mixed to spread over the minimum glucose agar plate medium, and incubated at 37° C for
48 hours . After incuba tion, reverse mutant colonies were counted macroscopically and bacterial growth inhibition was observed under a stereomicroscope. The main study was conducted in the same manner as in the dose setting study. Based on the results of the dose setting study, 6 concentrations (156.3

, 312.5

, 625

, 1250

, 2500 and 5000 μg/ plate) were provided with 50000 μg/mL (= 5000 μg/ plate) as the highest concentration and common ratio of 2. A vehicle control and a positive control were provided for each bacterial strain
and te sted in the same manner. 2) Sterility tests for S9 mix and the test substance solution On a plate of normal agar plate medium (20 mL) prepared using the normal agar plate medium (Eiken Chemical Co., Ltd., pH 7.2), 0.1 mL each of S9 mix, 0.1M sodium phospha te buffer, 2 types of overlay agar and the test substance solution of each concentration were spread and incubated at 37°C for 48 hours.

13. Evaluation criteria for the study results When the mean value of 2 plates obtained with the test substance is comp ared to the
data obtained with the vehicle control and the positive control, the result is judged as positive if the number of reverse mutant colonies is twice or more that of the vehicle
control and the result is dose

- dependence and reproducible.LIFE SCIENCE LABORATORY ® 5

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### 14. Re sults The result of the dose setting study is shown in Table 1. The result of the main study is shown in Table 2 and Fig. 1-2. The number of reverse mutant colonies in the positive control substance was twice
or more that in the vehicle control for all st rains both in the S9 mix (−) system and S9 mix (+) system in the dose setting study (Table 1) and the main study (Table 2) demonstrating mutagenicity. In the sterility test, microbial contamination was not detected in any of the test
solutions.
The dose se tting study was conducted at 6 dose levels with 5000 μg/plate at the highest dose level and common ratio of 4 for both S9 mix (−) and S9 mix (+) systems. As a result (Table 1), growth inhibition was observed at 5000 μg/plate for
all bacterial strains exce pt for TA98 in the S9 mix (−) system and for TA100, WP2uvrA and TA1537 in the S9 mix (+) system. However, the number of reverse
mutant colonies was not twice or more that in the vehicle control. The main study was conducted at 6 dose levels with 5000 μg/p late at the highest dose level and common ratio of 2 for both S9 mix (−) and S9 mix (+) systems based on the results of the dose setting study. As a result (Table 2), growth inhibition was observed at 5000 μg/plate for TA100 and TA1537 in the S9 mix (−) s ystem and for TA100, WP2uvrA and TA1537 in the S9 mix (+) system. However, the
number of reverse mutant colonies did not increase twice or more that of the vehicle
control for all bacterial strains.

15. Conclusion The bacterial reverse mutation study of did not show the number of reverse mutant colonies twice or more that in the vehicle control even at the highest dose level of 5000 μg/plate in the dose setting study and the main study with or without
metabolic activation. Growth inhibition was noted in some of the bacterial strains. Based on the above results, it was concluded that was not mutagenic.

16. Archiving The study protocol, source data, records and the final report shall be stored at theLIFE SCIENCE LABORATORY ® 5

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### archive of Control Headquarters, LIFE SCIENC E LABORATORY (Room 126, 2nd floor, Okazaki bashi Building, 9

- 11, 2

- chome, Utsubohonmachi, Nishi

- ku, Osaka) for 10 years from completion of the study.

17. Study personnel Study director Hitoshi Yamamoto Investigators Yutaka Kageyama, Kazusa Uchino

18. Re ferences 1) Ames, B.N., Durston, W.E., Yamasaki, E., and Lee, F.D.: Proc. Nat. Acad. Sci. USA 70, 2281

- 2285 (1973) 2) McCann, J., Spingarn, N.E., Kobari, J., and Ames, B.N.: Proc. Nat. Acad. Sci. USA 72, 979

- 983 (1975) 3) Ames, B.N., McCann, J. and Yamasak i E.: Mutation Res. 31, 347

- 364 (1975) 4) Yahagi, T., Degawa, M., Seino, Y., Matsushima, T., Nagao, M., Sugimura, T. and Hashimoto, Y.: Cancer Lett. 1, 91

- 96 (1975) 5) de Serres, F.J. and Shelby, M.D.: Science 203, 563

- 565 (1979) 6) Brusick, D.J., Simmon, V.F., Rosenkranz, H.S., Ray, V.A. and Stafford, R.S.: Mutation Res. 76, 169

- 190 (1980) 7) OECD Guideline for Testing of Chemicals: Toxicity studies, supervised by Office for Environmental Chemicals Safety, Planning Section, Environmental Health Bureau, Mi nistry of Health and Welfare, Jiho, Inc., Tokyo (1991).

End of textLIFE SCIENCE LABORATORY ® 5

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### - 10

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### LIFE SCIENCE LABORATORY

Study director Hitoshi Yamamoto

July 19, 2001

### Table 1 Reverse mutation study of in bacteria (dose setting study)

[Notes] 1. M ark numerical value with * on the right if growth inhibition of bacteria was observed.

2. Enter the mean colony count in each plate in ( ). 3. Show the number of reverse mutants by measured value and mean value in the ascending order of the dose levels of the test substance. 4. Mark the dose level when precipitation was observed on a plate. 5. ENNG: N

- ethyl

- N'

- nitro

- N

- nitrosoguanidine, 2

- NF: 2

- nitrofluorene 9

- AA: 9

- aminoacridine hydrochloride, 2

- AA: 2

- aminoanthracene, NaN 3 : sodium azide Study period June 25 to 28, 2001 Presence/ absence of metabolic activation Dos e level of the

test substance (μg/plate) Number of reverse mutant (No. of colonies/plate)

Base

- pair substitution type Frame shift typePositive control Not requiring S9 mix

Requiring S9 mix Name

Dose level (μg/plate)

No. of colonies/plate

Name Dose level (μg/plate)

No. of colonies/plate Vehicle control

Vehicle control

### Table 2 Reverse mutation study of in bacteria (Main study)

[Notes] 1. Mark numerical value with * on the right if growth inhibition of bacteria was observed.

2. Enter the mean colony count in each plate in ( ). 3. Show the number of rever se mutants by measured value and mean value in the ascending order of the dose levels of the test substance. 4. Mark the dose level when precipitation was observed on a plate. 5. ENNG: N

- ethyl

- N'

- nitro

- N

- nitrosoguanidine, 2

- NF: 2

- nitrofluorene 9

- AA: 9

- ami noacridine hydrochloride, 2

- AA: 2

- aminoanthracene, NaN 3 : sodium azide Study period July 2 to 5, 2001 Presence/ absence of metabolic activat ion Dose level of the

test substance (μg/plate) Number of reverse mutant (No. of colonies/plate)

Base

- pair substitution type Frame shift type

Vehicle control

Vehicle controlPositive control Not requiring S9 mix

Requiring S9 m ix Name

Dose level (μg/plate)

No. of colonies/plate

Name Dose level (μg/plate)

No. of colonies/plateNumber of reverse mutantcolonies/plate S9 mix (−) system S9 mix (+) system

S9 mix (−) system S9 mix (+) system

S9 mix (−) system S9 mix (+) systemNumber of reverse mutantcolonies/plateNumber of reverse mutantcolonies/plate

Dose level of the test substance (μg/plate) Fig. 1 Dose

- response curves in the reverse mutation study (main study) of using base

- pair

substitution mutant strains S. typhimurium TA100, TA1537 and E. coli WP2uvrANumber of reverse mutant colonies/plateNumber of reverse mutant colonies/plate S9 mix (−) system S9 mix (+) system

S9 mix (−) system S9 mix (+) system

Dose level of the test substance (μg/plate) Fig. 2 Dose

- response curves in the reverse mutation study (main study) of using frame

- shift

mutant strains S. typhimurium TA98 and TA1537

### ( 1 )



## Report on the results of the bacterial reverse mutation study

1. General matters
Name of the new chemical substance (by IUPAC nomenclature)

Abbreviation

Chemical formula

Purity of the new chemical substance used in the study Lot No. of the new chemical substance used in the study

Name and concentration of impurity –

CAS No. Vapor pressure

– Molecular weight Partition coefficient

– Melting point

– Description at room temperature An aqueous solution Boiling point

– Stability Stable under ordinary handling

Solubility, etc. in solvents Solvent Solubility S tability in the solvent Solvent Solubility Stability in the solvent

Water DMSO

– – Acetone

– – Ethanol

– –

[Notes] Describe the physical and chemical properties as much as possible as reference data. 1. Enter stabilities against temper ature, light, etc. in the “Stability” section. 2. Give the vapor pressure of the test substance in “Vapor pressure” section. 3. Give the partition coefficient, measurement temperature and the solvent used in the measurement in “Partition coefficient” secti on. 4. Give solubility of the test substance and its stability in the solvent in “Solubility, etc. in solvent” section.

### ( 2 )



### 2. Bacterial strains used in the study
Name of bacterial strain Supplier Date of receipt Salmonella typhimurium TA100 Institute for Fe rmentation, Osaka June 13, 1998 Salmonella typhimurium TA1537 Institute for Fermentation, Osaka June 13, 1998 Salmonella typhimurium TA1535 Institute for Fermentation, Osaka June 13, 1998 Salmonella typhimurium TA98 Institute for Fermentation, Osaka Jun e 13, 1998

Escherichia coli WP2uvrA Institute for Fermentation, Osaka June 13, 1998



### 3. S9Mix (1) Method of obtaining S9 (circle the number applicable and enter necessary matters)
Self

- made or purchased 1. Self

- made 2. Purchased (manufacturer: Oriental Yeast Co., Ltd.)

Date of manufacture March 23, 2001 Lot No. if purchased 01032313
Storage temperature

- 80  C



### (2) Method of preparation of S9

Animals used Inducer

Species/strain Rat, SD Name Phenobarbital (PB) 5,6

- benzoflavone (BF)

Sex Male Administr ation method i.p Age (weeks) 7 weeks Treatment period and dose level (g/kg) Body weight 213.8 ± 8.3 g



### (3) Composition of S9 mix Ingredient Amount in 1 mL of S9 mix Ingredient Amount in 1 mL of S9 mix

S9 0.1 mL NADPH 4  mol MgCl 2 8  mol NADH 4  mo l

KCl 33  mol Sodium phosphate buffer 100  mol

Glucose

- 6

- phosphate 5  mol Others ( )

### ( 3 )

4. 

### Preparation of the test substance solution (circle where applicable for the description of the test substance solution and presence/absence of purity conversion)

Solvent used Name Manufacturer Lot No. Grade Purity (%)

Purified
water – – – –

Reason for selection of the solvent Widely used in general

Description of the test substance solution

Method of suspen sion, etc. for a poorly

- soluble test substance

Storage period and temperature from preparation of the solution to its use 0 hour 15 minutes 23°C

Presence/absence of purity conversion Present Absent



### 5. Preincubation conditions, etc. (1) Conditions

Nutrient broth Name Manufacturer Lot No.

Nutrient Broth Difco Laboratories 123820JD Preincubation period 12 hours 0 minutes

Culture container (shape, volume) L

- shaped test tube, 15 mL

Volume of culture solution 10 mL Inoculum dose 20  L



### (2) Viabl e cell count, etc. at completion of preincubation

Name of bacterial strain Base

- pair substitution type Frame shift type
TA100 TA1535 WP2uvrA TA98 TA1537

Viable cell
count

 10 9 /mL Dose

- setting study 1.12 1.94 1.73 1.44 1.27

Main study 1.12 1.87 1.83 1 .41 1.24

Measurement method (circle one) 1 . Conversion from OD 2. Stepwise dilution method

3. Other ( )

### ( 4 )



### 6. Minimum glucose agar medium ( circle the number applicable and enter necessary matters)

Self

- made or purchased 1. Sel f

- made 2. Purchased (manufacturer)

Date of manufacture Manufactured on June 22 and 29, 2001 Lot No. if purchased

– Name, manufacturer, lot No. etc. of agar used Difco Bacto Agar, Difco Laboratories Lot No. 0286000



### 7. Study method (circle the number applicable and enter necessary matters) (1) Study method and reason for its selection

Study method selected 1. Preincubation method 2. Plate method 3. Other ( )

Reason for selection in case of “Other”



### (2) Study conditions

Composition Bacterial su spension 0.1 mL
Test substance solution 0.1 mL Na phosphate buffer (in case of the direct method) 0.5 mL

S9 mix (in case of the metabolic activation method) 0.5 mL
Overlay agar 2.0 mL Other ( )

Preincubation Temperature 37  C
Time 20 minutes

Incubation Temperature 37  C
Time 48 hours



### 8. Method of colony counting Counting method 1. Manual counting 2. Instrumental counting
Presence/absence of correction 1. No 2. Yes (method of correction: )

### ( 5 )



### 9. Study results (1) The study results ar e shown in the attached table. (2) Judgment of the results Judgment (circle one) Positive Negative

Reasons for the judgment The test substance did not increase the reverse mutant colony count twice or more that in the vehicle

control in the dose

- setting study and the main study although growth inhibition was noted in some of the strains at the highest dose level of 5000 μg/ plate. Based on these findings, the test substance was judged to be not mutagenic.

(Attach Appendix 2 Specific activity if the result is judged to be positive.



### (3) Reference matters

[Note] Enter the opinions, etc. of the study director on the result in the section of
“Reference matters”.

### ( 6 )



### 10. Others

Testing facility Name First Laboratory, Chihayaakasaka Research Institute, LIFE SCIENCE LABORATORY

Location 1082 Aza

- chihaya, Chihayaasasaka

- mura,

Minamikawachi

- gun, Osaka Prefecture Tel. (0721) 74

- 0316 Fax. (0721)74

- 0343

Study director Name and

position Hitoshi Yamamoto, senior researcher

Duration of experience 8 years

Study No. 01

- VII

- 0601

Study period June 11 to July 19, 2001

[Notes] 1. Fill in this form by transferring from the final report. 2. Enter the same study number as in the final report.LIFE SCIENCE LABORATORY ® 5

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### Form (i ) Certificate of reliability

1. Title: Bacterial reverse mutation study of

2 . Study code No. : 01-VII-0601 3. Name and location of the sponsor:

4. Study initiation date: June 11, 2001 5. Study completion date: July 19, 2001 6. Name and location of the testing facility: Contract research organization:

LIFE SCIENCE LABORATORY
5 -19, 2 -chome, Nishihonmachi, Nishi-ku, Osaka Test facility: Second Laboratory, Chihayaakasaka Research Institute,

LIFE SCIENCE LABORATORY 1082 Aza-chihaya, Chihayaakasaka-mura, Minamikawachi-gun

, Osaka Prefecture This study was conducted in accordance with the GLP standards . I have confirmed that the final report describes accurately the method and procedures used in the study and the results reflect the source data correctly. The study was inspected and the result was reported to the facility manager and the study
director as follows. Date of inspection Date of report Study protocol June 11, 2001 June 11, 2001 State of study conduct June 25 , 2001 June 25 , 2001 State of study conduct

State of study conduct Final report July 11 , 2001 July 11 , 2001



### July 11, 2001 Reliability Assurance Division, LIFE SCIENCE LABORATORY Quality Assurance Manager Shigehiro Fujii Person in charge of reliability assurance Shigehiro Fujii

# Statement

I hereby certify that the following study was conducted in accordance with the OECD Guidelines for the Testing of Chemicals: Toxicity Studies (Nos. 471 and 472).

Notice

Study code No.: 01-VII-0601

Title of the final report: Bacterial reverse mutation study of

July 19, 2001

LIFE SCIENCE LABORATORY

Shigeru Morita

(Facility Manager)