Document 4ap6KnNQVgm60pmjj16Ob2rnG
STUDY CODE : KOl-1815
ARROZZ
Receipt No. T96-2503 Report No, T-4663
FINAL REPORT
BACTERIAL REVERSE MUTATION TEST OF u-1
October, 1996
Hita Research Laboratories
Chemical Biotesting Center
Chemicals Inspection & Testing Institute
Japan
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j JilO SSfT
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QUALITY ASSURANCE STATEMENT
Hita Research Laboratories, Chemical Biotesting Center Chemicals Inspection & Testing Institute, Japan Sponsor: SUMITOMO 3M LIMITED Title:_______ Bacterial reverse mutation test of v -1 Study code: KO1-1815
This report was audited by the Quality Assurance Section. I, the undersigned, hereby declare that this report reflects the original Japanese report.
(Date) (Signature) Section Chief, Quality Assurance
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I, the undersigned, hereby declare that this report provides a correct English translation of the Final Report. (Study code No. KOl-1815 issued on October 30, 1996)
<da,e>
n , /9?e
(signature) Shozo Ogura
Hita Research Laboratories Chemical Biotesting Center Chemicals Inspection & Testing Institute, Japan
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GLP STATEMENT
Hita Research Laboratories, Chemical Biotesting Center Chemicals Inspection & Testing Institute, Japan
Sponsor:___________ SUMITOMO 3M LIMITED Title:_______________Bacterial reverse mutation test of v -1 Study Code No.: K01-1815
I, the undersigned, hereby declare that this study was conducted in compliances with "Standards to be observed by Testing Institutions for Toxicity Investigations" (Japan's MOL, No.76, September 1, 1988).
Management:
Signed in original Shigetaka Yamane, Ph. D.
October 30, 1996
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QUALITY ASSURANCE STATF.MF.NT
Hita Research Laboratories, Chemical Biotesting Center Chemicals Inspection & Testing Institute, Japan
Sponsor:___________ SUMITOMO 3M LIMITED Title:_______________ Bacterial reverse mutation test of u -1 Study Code No.: KOl-1815
This study was audited by the Quality Assurance Section and the study procedures were inspected on the following dates.
Dates of Inspections
Dates of Reports to
Dates of Reports to
and Audits
Study Director
Management
September 12, 1996
September 13, 1996
September 17, 1996
October 1, 1996
October 1, 1996
October 1, 1996
October 30, 1996
October 30, 1996
October 30, 1996
I, the undersigned, hereby declare that this report provides an accurate description of the
methods and procedures used in this study and that the reported results accurately reflect
the raw data obtained.
Section Chief, Quality Assurance:
Signed in original Keiji Shiraishi, B.S.
October 30, 1996
000203
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Study code:
KOl-1815
Test substance code: HR3291
Sponsor code:
S-030
TITLE Bacterial reverse mutation test of v -1
SPONSOR SUMITOMO 3M LIMITED 8-8, Minami-Hashimoto 3-chome Sagamihara-shi, Kanagawa, 229 Japan
TESTING FACILITY Hita Research Laboratories, Chemical Biotesting Center Chemicals Inspection & Testing Institute, Japan 822, 3-chome, Ishii-machi, Hita, Oita 877, Japan
PURPOSE OF STUDY The purpose of this study was to determine the mutagenic potential of the test substance using Salmonella typhimurium and Escherichia coli.
TESTING METHOD This study was conducted in accordance with the following guidelines: "Standards for Toxicity Investigations" (Japan's MOL, No.77, September 1, 1988).
GLP COMPLIANCE This study was carried out in compliance with the following GLP requirement: "Standards to be observed by Testing Institutions for Toxicity Investigations" (Japan's MOL, No.76, September 1, 1988).
PERIOD OF STUDY Commencement of test: Dose finding test: Completion of observation: Presentation of final report:
September 17, 1996 September 25, 1996 October 14, 1996 October 30, 1996
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LOCATION AND PERIOD FOR RETENTION OF RAW DATA Data and test substance are retained in the archives and the test substance storage room of Hita Research Laboratories for 10 years following the date of the notification specified under Item 1 of Article 57-2 of Industrial Safety & Health Law, respectively.
After termination of the retention period, any measures taken are done so with the approval of the sponsor.
PERSON CONCERNED WITH STUDY
Study Director:
Signed in original
October 30, 1996
Shozo Ogura
Hita Research Laboratories
Mutagenicity Section
Study Staff: Person in charge of Storage:
Tsunehiko Inai, B.S. Shizuka Kouda
ANY UNEXPECTED SITUATIONS AND DEVIATIONS FROM PROTOCOL There were no unexpected situations and deviations from protocol which might have affected the test results.
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CONTENTS
SUMMARY
...................................................
1
MATERIALS AND METHODS
1. TEST SUBSTANCE AND POSITIVE CONTROLS
.......................
2. BACTERIAL STRAINS
3. MEDIUM AND S9 MIX
4. PRE-CULTURES
5. PREPARATION OF TEST SUBSTANCE AND POSITIVE
CONTROLS
6. METHODS
7. MICROSCOPIC OBSERVATION AND COLONY COUNTING .......
8. INTERPRETATION OF RESULTS ...................................................
2 4 5 6
6 7 8 8
RESULTS
8
CONCLUSION
9
REFERENCES
9
APPENDIX TABLES AND FIGURES .................................................... 1-6
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SUMMARY
The reverse mutation test of v -1 was performed on Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 and a.Escherichia coli strain WP2 uvrA using the pre incubation method with and without metabolic activation. The results showed that the numbers of their revertant colonies for all strains in groups which were treated with the test substance were less than twice that of each negative control with and without S9 Mix. The numbers of the revertant colonies in the negative control and the positive controls were within the background data in our laboratories. Based upon the above results, v -1 was judged to have no reverse mutagenic potential under the present test conditions.
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MATERIALS AND METHODS
1. TEST SUBSTANCE AND POSITIVE CONTROLS 1.1 Test substance (Information provided by the sponsor)
1) Name Potassium salt of N-ethyl-N-perfluorobutylsulfonylglycine Other name: u - l
CAS No.: 67584-51-4
2) Lot No.
Lot 1
3) Supplier
SUMITOMO 3M LIMITED
4) Structural formula or rational formula (Outline of manufacturing method, in case
both were unknown)
C4F9SO2NCH2COOK
C2H 5
(molecular formula CSH7F9KNO4S)
5) Purity
97.3 w/w%
6) Impurities KC1 2.7 w/w%
7) Physicochemical properties
Appearance at ordinary temperature: light gray powder
Molecular weight:
423.30
Stability:
stable
Melting point:
Boiling point:
Vapor pressure:
Partition coefficient:
Solubility:
Degree of solubility:
Water: ^5w /v% *
DMSO: ^5w /v% *
Acetone: < 10 w/v%*
Others: --
* Examined in our laboratories
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8) Storage conditions
room temperature
9) Care on handling
Gloves, a mask, a head cap and a lab coat were worn when handling.
1.2 Positive controls
1) 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2)
Manufacturer: Wako Pure Chemical Industries, Ltd.
Lot No.:
LEN0571
Properties:
reddish-orange crystalline powder
Purity:
99.5%
Grade:
special grade
2) Sodium azide (NaNs)
Manufacturer: Wako Pure Chemical Industries, Ltd.
Lot No.:
DLP2438
Properties.
white crystalline
Purity:
99.4%
Grade:
special grade
3) 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine 2HCI
(ICR-191)
Manufacturer: Polysciences, Inc.
Lot No.:
412795
Properties:
yellow crystalline powder
Purity:
Grade:
--
4) 2-Aminoanthracene (2AA)
Manufacturer: Wako Pure Chemical Industries, Ltd.
Lot No. .
DLR7869
Properties:
yellowish-green-brown powder
Purity:
95.7%
Grade:
--
5) Storage conditions
A cold and dark place
6) Care on handling
Gloves, a mask, a head cap and a lab coat were worn when handling.
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2. BACTERIAL STRAINS 2.1 Strains selected
Salmonella typhimurium strains TAIOO, TA98, TA1535 and TA1537 were obtained from Dr. B.N. Ames, University of California, U.S.A. ,on June 20, 1990.
A Escherichia coli strain WP2 uvrA was obtained from Japan Bioassay Laboratories, on April 6, 1995.
S. typhimurium strains TA100, TA1535 and a E. coli strain WP2 uvrA were used
for the detection of base-pair substitution mutation, while S. typhimurium strains
TA98 and TA1537 were for the detection of frameshift mutation.
2.2 Storage
The test strains were stored as frozen stock cultures (0.045 ml of dimethyl
sulfoxide (DMSO)*/0.5 ml of broth culture) at - 8 0 't (ultra-deep freezer MDF-
291, Sanyo).
* Purity ^ 99.0%, Lot No. CF103, Dojindo Laboratories
2.3 Characterization of strains
1) Characteristics of strains________________________________________
Mutation on Mutation on Membrane R-factor
Strains
synthesis of excision mutation (pKMIOl)
___________
amino acid repair_______ (LPS)______________
Salmonella typhimurium
TA1535
hisG46
A uvrB
rfa
-
TA1537
hisC3076 AuvrB rfa -
TA98
hisD3052 A uvrB rfa +
TAIOO
hisG46
AuvrB
rfa
+
Escherichia coli
WP2 uvrA
trp
AuvrA
+
-
The amino acid requirement for growth was demonstrated by using histidine for S. typhimurium strains and tryptophan for E. coli strain. The presence o f Rfactor, membrane mutation and mutation on the ability to repair DNA lesions were confirmed by ampicillin resistance, sensitivity to crystal violet and UV sensitivity, respectively.
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Date of characterization Salmonella typhimurium
Escherichia coli
TA1535 TA1537 TA98 TA100 WP2 uvrA
July 18, 1996 June 7, 1996 June 7, 1996 March 6, 1996 April 18, 1996
MEDIUM AND S9 MIX
Medium
Minimal glucose agar plate (prepared in our Laboratories)
The medium was prepared as follows, and poured 30 ml into a petri dish.
Components
Amount included in one litre
20 x Vogel-Bonner E
50 ml
40 w/v% Glucose
50 ml
Agar
15 g
(1) Agar:
Bacto-Agar (Lot No. 71892AJB or 90800JA,
Difco Laboratories)
(2) Manufacturing date: dose finding test on September 11, 1996
main test on October 3, 1996
2) Soft agar
The solution containing 0.5 mM histidine and 0.5 mM biotin for S. typhimurium
strains or 0.5 mM tryptophan for E. coli strain was added to the soft agar solution containing 0.6 w/v% agar (Bacto-Agar, Lot No. 71892AJB, Difco
Laboratories) and 0.5 w/v% NaCl in a ratio of 1 : 10.
3.2 S9 Mix
1) Rat liver S9 (Kikkoman Co., Ltd.)
Induction method:
SD male rats, 7-week-old (203-254 g), were
intraperitoneally administrated phnobarbital (30 mg/kg
x 1 time, 60 mg/kg x 3 times) and 5,6-benzoflavone (80 mg/kg x i time).
Lot No.: Storage:
RAA-350 (manufactured on August 23, 1996, purchased on September 4, 1996) -80C (ultra-deep freezer MDF-291, Sanyo)
2) Cofactor for S9 Mix (Oriental Yeast Industries, Ltd.)
Lot No.: Storage:
999602 -20C (bio-freezer GS-2603, Nippon Freezer Ltd.)
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3) Composition of S9 Mix
One ml of S9 Mix contained 8 gmol MgCl2, 33 pmol KC1, 5 pmol G-6-P, 4 pmol NADPH, 4pmol NADH, 100 pmol of 0.2 M sodium-phosphate buffer (pH 7.4) and 0.1 ml S9.
4. PRE-CULTURES From the stock cultures, 20 pi of the bacterial suspension was inoculated to L-tube containing 10 ml nutrient broth No.2 (Lot No. 194 56443, OXOED Ltd.) and the bacterial culture was incubated at 37 0.5C for 8 h with shaking at 50 times/min by the Monod shaker (MONOSIN- II A, Taitec Co., Ltd.) The viable cell counts calculated from the values which were determined at 660 nm by spectrophotometry (Novaspec, LKB Japan) at the end of incubation are shown below,
No. of Dose viable cells finding test (x l0 5*9/ml) Main test
TA100 2.1 2.1
TA1535 WP2 uvrA 2.1 4.4 2.1 4.0
TA98 2.4 2.3
TA1537 2.1 2.0
5. PREPARATION OF TEST SUBSTANCE AND POSITIVE CONTROLS 5.1 Test substance
1) Preparation The test substance was dissolved in distilled water (distilled water for injection, Lot No. K6B74, Otsuka Pharmaceutical Factory) to make 5 w/v% concentration and diluted with the same solvent to give appropriate concentrations.
2) Stability of the test solution No denaturation of the test solution was observed for the color and the exothermic reaction until 2 hours after preparation.
3) Preparation time Prepared immediately before use and used within 0.5 h at room temperature.
5.2 Positive controls 1) Preparation NaN3 was dissolved in distilled water (Lot No. K6B74). AF-2, ICR-191 and 2AA were dissolved in DMSO (Lot No. CD069).
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2) Preparation time and storage condition Prepared on every 3 months and stored at -80C (ultra-deep freezer MDF-291, Sanyo).
6. METHODS The test was carried out for S. typhimurium strains TA1535, TA1537, TA98, TA100 and a E. coli strain WP2 uvrA using the pre-incubation method both with and without metabolic activation system. The plating was done in triplicate for the negative control and in duplicate for the test substance and positive controls.
6.1 Procedures After 0.1 ml of the test substance solution, 0.5 ml of 0.1 M sodium phosphate buffer (pH 7.4) or S9 Mix, and 0.1 ml of the bacterial culture were added to a tube, the mixtures were incubated for 20 min at 37 0.5C. Two ml of the soft agar was then added to each tube and poured onto a minimal glucose agar plate.
After incubation for 48 h at 37 0.5C, the number of revertant colonies were counted.
As the sterility test, each 0.1 ml of each bacterial suspension, test substance solution, S9 Mix or 0.1 M sodium phosphate buffer (pH 7.4) were smeared on a minimal glucose agar plate and incubated at 37 0.5C for 48 h, and then checked the bacterial contamination. Distilled water was used as a negative control, and the following positive controls were used for each bacterial strains.
S9 Mix (-) S9 Mix (+)
TA100 AF-2 0.01 2AA
1
TA1535 NaN3 0.5 2AA 2
WP2 uvrA AF-2 0.01 2AA 10
TA98 AF-2 0.1 2AA 0.5
TA1537 ICR-191
1 2AA
2
(pg/plate) 6.2 Dose selection
1) Dose finding test The test was carried out at the highest dose of 5,000 pg/plate and 6 doses of 1,000, 500, 100, 50, 10 and 5 pg/plate. As a result, growth inhibition was observed at 5,000 pg/plate both with and without S9 Mix.
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2) Main test Based on the results of the dose finding test, a main test was performed at the highest dose of 5,000 pg/plate and 5 lower doses diluted with a geometric progression of 2.
7. MICROSCOPIC OBSERVATION AND COLONY COUNTING 7.1 Microscopic observation
The state of revertant colonies (size and number of colonies), deposition of the test substance and the growth inhibition were examined with a stereo microscope. 7.2 Colony counting The number of colonies were counted with a manual counter or a colony analyzer (CA-7 or CA-9, Toyo-sokki Co., Ltd). Correction for counting errors was made for measurements with the colony analyzer. Each plate was measured three times, and the average of these three measurements was adopted as the number of revertant colonies on the plate. The average for each dose was calculated from the values of the plates used. Decimals of the average figures were rounded off.
8. INTERPRETATION OF RESULTS The test substance was judged to be positive, when the number of revertant colonies was twice or more of the negative control, and when the dose-relationship and the reproducibility were obtained. Any statistical procedures were not used.
RESULTS
The numbers of their revertant colonies for all strains in groups which were treated with the test substance were less than twice that of each negative control with and without S9 Mix.
The positive controls showed the distinct increase of revertant colonies, and the positive controls and the negative control were within a range of the background data in our laboratories.
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The growth inhibition was observed at more than 2,500 pg/plate both with and without S9 Mix. There were no fluctuations which affected the test results since the sterility test confirmed the absence of any micro-organisms.
CONCLUSION In conclusion, v -1 was judged to have no reverse mutagenic potential under the present test conditions.
REFERENCES 1. Ministry of Labor (1991) Guidebook on Mutagenicity Tests using Micro
organisms, New Edition (in Japanese) published by Japan Industrial Safety and Health Association, 2. Green M.H.L. and W.J. Muriel (1976) Mutagen testing using Trp+ reversion in Escherichia coli, Mutation Res., 38: 3-32. 3. Maron, D M ., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113: 173-215.
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Dose finding test Test substance: v --1
K01-1815
1STCOPYAVAILABLE
With(+) or without(-)
S9 Mix
Test substance
Number of revenants (number of colonies/plate)
concentration Base-pair substitution type
Frameshift type
(w/plate)
nceognattriovle TA 100 90 (101 101)
TA 1535
13
(10 12)
W 2uvrA
33
(41 37)
TA 98
30
(33 31)
TA 1537
9
6 ( 7)
S 9 Mix
111 14 37 31
7
S11315 m <114>
< ) 25 29> I t 9)
S10 (26 < 113' a < 121
< 42> n t 23) t T)
IS11M>50 " < 13> 32 ( 36) S t > 1 t ID
11( - )
100 116 < 107> < 14>
34> 36 t 34) i ! ( 10)
500 1000 5000
nceognattriovle
(13 12)
(114) 15 12>M 100> (
<38 <S 34)
S<S ` 78*>
4*) S < 27*)
(n o 85 95)
(10
15 12)
(41
40 38)
25 t 34)
l t 27)
(S t l3*>
34 36 34)
! t 9)
.j < 9 S t 7*)
(23
18 19)
S 9 Mix (+)
5
194> 1"> S(34) 1?<16>10 M<> K(13> 34<32> 24)50 M(<96)> "1<(10> (37> s' 27> '16>100 'S 98> "( <34> 2'44>500 ' ' '3,i "<1000 12*( i5< 27*> 24*>5000
91 12 96 12
t n o 12
1111*)
102*1
32 39 t 38)
40 t 38)
39 12) 35
fl10) 39)
" 1 32 t 32) 8,1 S t
33
39 35) St
16
23 t
23 t 22) 13>
S t 20) .S `
Positive control not requiring
S9 Mix
Name Concentration
(ug/plate) Number of colonies/plate
AF-2
N&Ns
I0 .0 1
0.5
m < 381> 385 < 384)
AF-2 0.01
188 t 4 )
AF-2 0.1
ICR-191 1
S t 527> m m )
Positive control
requiring S9 Mix
Name 2AA
Concentration
(ug/plate)
1
Number of
colonies/plate w <m)
2AA 2AA 2AA 2 10 0 .5
i S < 146 644 t 647> 274 t 289)
2AA 2
m t 158>
Notes Parenthesis shows the mean of each plate.
* : Observed bacterial growth inhibition.
AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylam ide
NaN3: Sodium azide
000216
ICR-191: 2-Kethoxy-6-chloro-9-(3-(2-chloroethyl)-aminopropylamino) acridine-2HC1 . 9AA 9-Ami
Main test
KOI--1815
Test substance: v - 1
With(+) or without(-)
S9 Mix
Test substance concentration
(fig/plate)
negative control
Number of revertants (number of colonies/plate)
Base-pair substitution type
Prameshift type
TA 100
113 112 ( 110) 106
TA 1535
18 10 ( 15) 17
WP2 uvrA
39 36 ( 37) 36
TA 98
28 25 ( 30) 38
TA 1537
13 10 ( 14) 19
156 ( 109) 13 < 13> 33 i 30> 34 i 31> l3
S9 M ix (-)
313 m < 116^ is< " ) 33 t 29> S t 32) 22 i 14> 625 m ( 117) H ' 32 31> S t 34> 1 1 2i>
1250 103 < 104> 12 < 12> 50 43> S ( 30) " ( 23)
2500 S t 87*> S i 8*) 30*i 33*> S t 22*> S t 6*>
5000 negative control
S < 84*^
108 117 ( 115) 120
9
9 ( ID 14
S < 35*)
38 29 ( 34) 34
24*t 21*>
42 46 ( 42) 39
f t 3*>
24 28 ( 26) 26
156 IS < ll0> 8 ( 7> 38 t 44> 40 t 38> ?5 2
S9 M ix
313 80 < 93> 10 < " > 42 47> l i 41> S 29>
(+ ) 625 115 < 112) " ( 12) 36 36> 3} ( 34) 26 t 26>
1250 m < 1111 *? ( 10) S ( 46> # < 431 l 29^
2500 S S t 121*> S i 7*> 38*i 36*) S t 34*) S t u *)
5000 111083**( in*)` f t 2*) S i 37*> S t 24*> S t 4*>
Positive control not requiring
S9 Mix
Name Concentration
(ug/plate) Number of colonies/plate
AF-2
0.01
356 / 337 < 347>
NaNa 0.5
295 312)
AF-2 AF-2 ICR-191
0.01 0.1
1
w < 123> m t 463> 2 <>
Positive control requiring S9 Mix
Name
Concentration
(lig/plate) Number of colonies/plate
2AA 1
874 < 839>
2AA 2
119613 (1 177)
Notes Parenthesis shows the Dean of each plate.
* : Observed bacterial growth inhibition.
AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide
2AA 2AA 2AA
10 0.5
2
56? i 572> 255 t 266> p ( 1651
NaN3: Sodium azide
ICR-191: 2-Methoxy-6-chloro-9-(3-(2-chloroethyl)-aminopropylamino) acridine-2HCl
2AA: 2-Aminoanthracene
0002iV
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Revertant colonies per plate
Revertant colonies per plate
K01-1815
test
fin d in g
Dose
000219
Revertant colonies per plate
Revertant colonies per plate
K01-1815
test
fin d in g
Dose
000220
Revertant colonies per plate
Revertant colonies per plate
Main test
K01-1815
000221
Revertant colonies per plate
Revertant colonies per plate
test
M ain
K01-1815
