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 Okazakibashi Building, 5-19, 2-chome, Nishihonmachi, Nishi-ku, Osaka

Tel. (06)6531-1881 (pilot number)
LIFE SCIENCE LABORATORY®

5-19, 2-chome, Nishihonmachi, 

Nishi-ku, Osaka

TeI.06-6531-1881 (pilot number)

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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, 2001 
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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 (NaN3) 

8. Names of vehicle control substances 

The solvents (vehicles) used to dissolve the test substance (1 wt% solution used as 
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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 the 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.

Storage 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 used

Salmonella typhimurium TA100

TA98

TA1535

TA1537

Escherichia coli WP2uvrA

Viable cell count ( 109/mL) at completion of preincubation [converted from 

turbidity (absorbance at 660 nm)]

Dose setting study

Main study
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2) Origin and management of bacterial strains

Salmonella strains 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 the 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) Positive 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.
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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 

strain

S9 mix (−) system S9 mix (+) system

Name Dose level

(μg/plate) Name Dose level(μg/plate)

Ingredient 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)
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5) Minimum glucose agar 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 sodium 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 

were 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.
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1) Dose 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 incubation, 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 tested 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 phosphate 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 compared 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.
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14. Results 

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 strains 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 setting 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 except 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/plate 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 (−) system 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 the 
LIFE SCIENCE LABORATORY®

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Nishi-ku, Osaka

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archive of Control Headquarters, LIFE SCIENCE LABORATORY (Room 126, 2nd 

floor, Okazakibashi 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. References

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 Yamasaki 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, Ministry of Health and Welfare, Jiho, Inc., Tokyo (1991).

End of text
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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. 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 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, NaN3: sodium azide

Study period June 25 to 28, 2001

Presence/

absence of 

metabolic 

activation

Dose level of the 

test substance 

(μg/plate)

Number of reverse mutant (No. of colonies/plate)

Base-pair substitution type Frame shift type

Positive 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 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, NaN3: sodium azide

Study period July 2 to 5, 2001

Presence/

absence of 

metabolic 

activation

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 

control

Positive 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
Number of reverse mutant 

colonies/plate

S9 mix (−) system

S9 mix (+) system

S9 mix (−) system

S9 mix (+) system

S9 mix (−) system

S9 mix (+) system

Number of reverse mutant 

colonies/plate

Number of reverse mutant 

colonies/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 WP2uvrA
Number of reverse mutant colonies/plate Number 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 Stability 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 temperature, 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” section.

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 Fermentation, 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 June 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 Administration method i.p

Age (weeks) 7 weeks

Treatment period and 

Body weight dose level (g/kg) 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

MgCl2 8 mol NADH 4 mol

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 suspension, 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) Viable 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

 109/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. Self-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 suspension 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 are 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.
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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)