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REPORT ON MUTAGENICITY TEST RESULTS USING MICROORGANISMS
1. General Information Nomenclature of novel chemical substance (by IUPAC nomenclature) Other name
Structure or empirical formula (outline of preparation method when not certain)
Purity of novel chemical substance used in test
Impurity name and content CAS No. Molecular weight Melting point
Boiling point
34455-29-3 570.37
-
Stability
Solubility in solvent, etc.
solvent water
DMSO
acetone
Other(*3)
[Notes] Hydrol}
Deity: unknown
Lot No. of novel chemical substance
used in test
ca ii
Vapor pressure
Distribution coefficient
-- -
Appearance at room temperature
Brown viscous solid
Unknown Solubility <50 mg/mL*2 <50 mg/mL*2
--
*3
Stability in solvent
--
--
-
*2 Measured on site at Hita Works *3 Ethanol soluble (solubility unknown) *4 50-mg/mL suspension showed no discoloration, heat emission, etc., up to 2 h after preparation, thus judged stable (confirmed on site at Hita Works)._________________
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[Note: no pages 2 and 3]
6. Minimum-glucose agar plate medium
Self-made or acquired
1. Self-made Acquired (producer: Oriental Kobo Kogyo K.K.)
Production date
November 8, 2002
Lot No. for acquired specimen
ANI740KR
Name of agar used, manufacturer, Lot No.
-
7. Testing method
(1) Testing method and reason for its selection Testing m ethod selected
Reasons for selection in the case of "Other"
Preincubation method
3. Other (
)
-
2. Plate method
(2) Testing conditions
Bacterial suspension
Test substance solution
Composition Na phosphate buffer solution (in direct method) S9 mix (in metabolism activation method)
Top agar
Other (-)
Preincubation
Temperature Time
Incubation
Temperature Time
0.1 mL 0.1 mL 0.5 mL 0.5 mL 2.0 mL
--
37 0.5C 20 min
37 0.5C 48 h
8. Colony counting method Counting method Manual
Correction
None
Instrumental 2. Yes (correction method: area and number drop)
9. Test results
(1) Test results are shown in a separate table
(2) Evaluation of results Evaluation (circled)
Positive
<_ Negativi
Reasons for evaluation Regardless of the presence or absence of S9 mix, against all bacteria, the number of back-
mutation colonies was less than 2 times the negative control, thus mutagenicity was judged to be negative.
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(3) Notes_____________________________________________________________________ _ * Dosage setting Dosage setting experiment-1 With a maximum of 5000 pg/mL, an overall 6-step dosage was set with a nominal dilution ratio of 4. Dosage setting experiment-2 As a result of dosage setting experiment-1, regardless of the S9 mix, growth inhibition was realized against all test bacteria. More than 4 doses for the effective dosage showing no growth inhibition could not be obtained with TA100, TA1535, and TA1537 in the absence of the S9 mix. For the dosage-setting experiment evaluation and dosage setting of this test with the effective dosage established, with a maximum dosage of 313 pg/plate showing growth inhibition, 6-step dosage was set at a nominal dilution ratio of 4. Main test From the dosage-setting experiment results, with the maximum dosage showing growth inhibition, in the absence of the S9 mix, the 313-pg/plate for TA100, TA1535, and TA1537, and the 1250-pg/plate for WP2 uvrA and TA98, the 6-step dosage was set with a nominal dilution ratio of 2. In the presence of the S9 mix, the total [overall] 6-step dosage was set with a nominal dilution ratio of 2 at the most with the 1250-pg/plate for TA100, TA1535, and TA1537 and the 5000-pg/plate for WP2 uvrA and TA98. Growth inhibition was observed at a dosage above 156 pg/plate for TA100, above 313 pg/plate for TA1535 and TA1537, and above 1250 pg/plate for WP2 uvrA and TA98; in the absence of S9 mix, and above 1250 pg/plate for TA100, TA1535, and TA1537 and 5000 pg/plate for WP2 uvrA and TA98 in the presence of S9 mix. No precipitation of the test substance occurred. Number of back-mutation colonies of the negative control and positive control was within the historical data range. No staining in test system occurred, indicating that the testing was a proper procedure._________________________________________________________________
10. Others
Testing facilities
Principal tester Test No.
Test duration
Name
Address Post and name
Experience
Hita Works, Kagakubusshitsu Heikakenkyukiko
3-822 Ishii, Hita, Oita 877-0061
S 0973(24)7211
FAX: 0973(23)9800
Testing Department 3
Shozo Ogura
21 years
KOI-2869
2/4/03 to 3/5/03
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Test results (dosage setting experiment-1)
Test substance name
T est duration
M etabolism Test substance
activation
dosage
(pg/plate)
- S9 mix
Negative control
4.88
19.5
78.1
313
1250
+ S9 mix
5000
Negative control
4.88
19.5
78.1
313
1250
Positive control
W ithout S9 mix
With S9 mix
5000
Name Dosage (gg/plate)
C o lon ies/p late
Name Dosage (pg/plate)
Colonies/plate
2/4/03 to 2/6/03 Back mutation (N o. o f colonies/plate)
Base pair substitution
TA100
TA1535
tit 99 9B (103)
7 13
4 (8)
109 11 100 (105) 16 (14)
97 14 95 (96) 11 (13)
101 105
88* 79*
81* 74*
59* 75*
104 120
15 (103)
3* (84) 3*
s* 08) S*
2* (67) _____0*. 90 8 (105) 10
03)
(3)
(6)
- ....(P 10 (9)
89 5 .97 (93) 16 (11)
97 13 112 (105) 14 (14)
99 17
107 (103)
9 (13)
107 108 (108)
11 8. (10)
101* 106* (104)
16* 12* (14)
83* 68*
AF-2
0.01
06)
2* 5*
fatti
0.5
(4)
257 261
2AA
1
(259)
278 304
2AA
2
(291)
395 142 307 (351) 120 (131)
WLuvrA
35 23 27 (28)
19 28 (24)
25 25 (25)
25 30 (28)
25 26 (26)
21 24* (23)
29* 18* (24)
32 30 37 (33)
26 21 (24)
38 28 (33)
31 25 (28)
30 30 (30)
25 27
28* 25*
AF-2
0.01
(26) (27)
184 174
2AA
10
(179)
742 797 (770)
Frame shift
TA98
15 21
22 17
13 23
18 20
17 12
. 22* IB *
15* 16*
40 31
22 (19)
(20)
(18)
(19)
(15)
(20)
(16) 25 (32)
27 (30)
23 32 (28)
25 24 (25)
35 29 (32)
31 34
15* 15*
AF-2
(33) (16)
0.1
510 532 (521)
2AA
0.5
476 482 (479)
TA1537
63 5 (5)
8 4 (6)
5 2 (4)
4 6 (5)
2* 1* (2)
3* 4* (4)
3* 2* (3)
-.16 17 14 (16)
18 19 (19)
15 21 (18)
17 18 (18) !
13 19 (16)
10* 8* (9)
3* 4* (4) ICR-191
0.5
1000 1054 (1027)
2AA
2
1Q1 156 (175)
[Notes] (): average value of number of colonies of each plate *: growth inhibition of bacteria was observed. ' AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide NaN3: sodium azide ICR-191: 2-methoxy-6-chloro-9-[3-(2-chIoroethyl)aminopropylamino]acridine*2HCl 2AA: 2-aminoanthracene
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Test results (dosage setting experiment-2)
Test substance name'
Test duration
M etabolism activation
Test substance dosage
(pg/plate)
Negative control
0.305
1.22
- S9 mix
4.88
19.5
78.1
Positive control
313
W ithout S9 mix
Name Dosage (rg/plate)
C o lon ies/p late
2/7/03 to 2/10/03 Back mutation (No. o f colonies/plate)
Base pair substitution
Frame shift
TA100
TA153S
TA1537
96 102
12
95 (98)
6
6 6 12
(8) 5
(8)
110 106 (108)
13 10
(12)
6 4
(5)
89 9 96 (93) 10 (10)
5 8
(7)
95 86 113 100 106 99 95* 82*
AF-2 0.01
(91) (107) (103)
(89)
5 12
8 6 7 12 5* 6*
Nalfe
0 .5
(9) (7) (10) (6)
8 2 7 7
7 9
4* 5* ICR-191
0.5
(5) (7) (8) (5)
244 351
228
(236)
354
(353)
893 852
(873)
[Notes] (): average value of number of colonies of each plate *: growth inhibition of bacteria was observed. AF-2: 2-(2-furyl)-3 -(5-nitro-2-furyl)acrylamide NaNs: sodium azide ICR-191: 2-methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine*2HCl
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Test results (main test)
Test substance name!
T est duration
M etabolism activation
Test substance dosage
(Hg/plate)
Negative control
9.77 19.5
39.1
- S9 mix
78.1 156 313 625
1250
Negative control
39.1
+ S9 mix
78.1 156 313 625
1250
2500
Without S9 mix
With S9 mix
5000
N am e D osage (vtg/piate)
C o lo n ies/p la te
N am e Dosage (gg/plate)
C olon ies/p late
Positive control
2/13/03 to 2/18/03 Back mutation (N o. o f colonies/plate)
Base pair substitution
TA100
TAI53S
murrA
107 115
11
107 (110)
11
111 13 103 (107) ______ .
114 119 (117)
7 8
110 7
127 (119) _____ 6
110 9 98 (104) _____ 8
107* 7 73*....... (90) - *
102* 104* (103)
6* 3*
----
9 (10)
(U ) (8)
(7) (8) (8) (5)
--
119 118 106 126
124 117
123 133
104 129 90 108 95* 100*
--
114 (117) (116)
(121) (128) (117) (99) (98)
--
9 11 12 6
11 3
12 6 9 9 15 9 8* 7*
9 no) (9) (7) (9) (9) (12) (8)
--
AF-2 0.01
300 287 (294)
2AA 1 861 682 (772)
--
NaNi
0.5
275 280
2AA 2
175 177
(278) (176)
28 28
--
29 (28)
--
27 23 30 26 23 24
24 23 18 21 30* 22* 29 33
--
(25)
(28)
(24)
(24)
(20)
(26) 29 (30)
--
32 29 (31)
35 27 (31)
36 33 (35)
33 27 (30)
35 40 (38)
34* 33*
AF-2
0.01
(34)
162 200
2AA
10
(181)
534 509 (522)
Fram e shift
TA98 21 19
--
27 (22)
--
16 19 18 19 16 23
10 17 23 21 13* 14* 41 46
--
(18)
(19)
(20)
(14)
(22)
(14) 45 (44)
--'
36 39 (38)
35 ' 27 (31)
43 35 (39)
32 39 (36)
31 42 (37)
5* 15* (10)
AF-2 0.1
613 572 (593)
2AA 0.5
481 493 (487)
TA1537
7 7
7 3
8 s 10 8
7 9
5 6
6* 2*
5 (8 (s: rr. (9' (S'. (6! (4)
--
13 15
17 IB
26 19
16 14
22 24
25 16
19* 21*
19 (15) (18) (23)
(15) (23) (21) (20)
--
ICR-191. 0.5
825 596 (761)
2AA 2
263 293 (278)
[Notes] (): average value of number of colonies of each plate *: growth inhibition of bacteria was observed. AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide NaNs: sodium azide ICR-191: 2-methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine*2HCl 2AA: 2-aminoanthracene
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Dosage-Reaction Curve (dosage setting experiment-1)
No. of back-mutation colonies/plate
O : TA100 : TA1S3S O : W luvrA A : TA98
7
Figure 1: Dosage-reaction curve without S9 mix
No. of back-mutation
O : TA100 : TA153S
*V>
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Dosage-Reaction Curve (dosage setting experiment-2)
No. of back-mutation colonies/plate
8
Figure 3: Dosage-reaction curve without S9 mix
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No. of back-mutation colonies/plate
150 _
120 ..
90 . .
60 . .
Dosage-Reaction Curve (main test)
9
o : TA100 : TA1S3S O : tfP2uvrA A : TA98 V : TA1537
0 9.77 19. S 39.1 78.1 156 313 625 1250
Test substance dosage (pg/plate) Figure 4: Dosage-reaction curve without S9 mix
No. of back-mutation colonies/plate
o : TA100 : TA1535 O : %?2uvrA A : TA98
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10
Kiatsu No. 90 February 22, 2001
Jiro Hiraishi Chairman, Board of Directors Kagakubusshitsu Heikakenkyukiko
Labor Standards Bureau Ministry of Health, Labor and Welfare
Safety-Health Law GLP Feasibility Confirmation of Test Facilities
This is notification of the following judgments of the title matter requested on March 24, 2000.
Description
1 Name of test facilities for feasibility confirmation Hita Works, Kagakubusshitsu Heikakenkyukiko
2 Title of test requiring feasibility confirmation Mutagenicity test using microorganisms and chromosomal aberration test using mammalian cultured cells
3 Inspection date July 24, 2000 to July 25, 2000
4 Judgment Pass
5 Effective duration of feasibility confirmation July 26, 2000 to July 25, 2003
6 Notes None
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STATEMENT
Test Requester: Test Title: Test Code No.:
Dupont K.K. Mutagenicity test o KOI-2869
Hita Works Kagakubusshitsu Heikakenkyukiko
smg microorganisms
The test described above was carried out in accordance with Standards for Facilities for Toxicity Test (1988 Ministry of Labor Bulletin No. 76, partially amended on March 29, 2000), Test Facilities Specified in Ministry Order Article 4: Defining Tests for New Chemical Substances and Toxicity Test of Designated Chemical Substances (March 31,1984, Kanhogyo No. 39, Yakuhatsu No. 229, 59 Kikyoku No. 85, amended on March 1, 2000), and OECD Principles of Good Laboratory Practice (November 26,1997).
Yoshifumi Fujino Manager
March 5,2003
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STATEMENT
I Hita Works
Kagakubusshitsu Heikakenkyukiko
Test Requester:
Dupont K.K.
Test Title:
Mutagenicity test ol
ising microorganisms
Test Code No.:
KO1-2869
The test described above was carried out in accordance with Standards for Facilities for Toxicity Test (1988 Ministry of Labor Bulletin No. 76, partially amended on March 29, 2000), Test Facilities Specified in Ministry Order Article 4: Defining Tests for New Chemical Substances and Toxicity Test of Designated Chemical Substances (March 31,1984, Kanhogyo No. 39, Yakuhatsu No. 229, 59 Kikyoku No. 85, amended on March 1, 2000), and OECD Principles of Good Laboratory Practice (November 26,1997).
The final report precisely reflects raw data, and it is to confirm the test data as being effective.
Shozo Ogira Principal Tester March 5, 2003
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RELIABILITY WARRANTY
Hita Works Kagakubusshitsu Heikakenkyukiko
Test Requester
Dupont K.K.
Test Title:
Mutagenicity test o:
sing microorganisms
Test Code No.:
KO1-2869
This test has been audited or inspected by the QA Division of Hita Works, Kagakubusshitsu Heikakenkyukiko. Supervision or inspection dates and reporting dates to the principal tester and management are given below.
Auditing or inspection items
Test plan documented Test substance preparation and test start Amendment of test plan document Amendment of test plan document (No. 2) Recording and report draft Final report draft review Final report
Auditing or inspection date
2/5/2003 2/13/2003 2/17/2003 2/19/2003 2/25/2003 3/5/2003 3/5/2003
Auditing or inspection result report date 2/6/2003 2/14/2003 2/17/2003 2/19/2003 2/25/2003 3/5/2003 3/5/2003
The test described above was carried out in accordance with Standards for Facilities for Toxicity Test (September 1,1988 Ministry of Labor Bulletin No. 76, partially amended on March 29, 2000), Test Facilities Specified in Ministry Order Article 4: Defining Tests for New Chemical Substances and Toxicity Test of Designated Chemical Substances (March 31,1984, Kanhogyo No. 39, Yakuhatsu No. 229, 59 Kikyoku No. 85, amended on March 1, 2000), and OECD Principles of Good Laboratory Practice (November 26,1997) and in accordance with test methods defined in Toxicity Test Standards (Ministry of Labor Bulletin No. 77, September 1, 1988; Ministry of Labor Bulletin No. 67, June 2, 1997), Specific Procedure of Mutagenicity Test Using Microorganisms (February 8, 1999), Testing Methods for New Chemical Substances,
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partial amendment (Kanhogyo No. 700, Yakuhatsu No. 1039, 61 Kikyoku No. 1014, December 5,1986; Kanhoan No. 287, Eisei No. 127,1997-10-31 Kikyoku No. 2, October 31, 1997).
This report accurately describes the testing method and procedure; the reported results accurately reflect raw data of the tests.
March 5,2003 Keiji Shiraishi Reliability Warranty Officer
Yoshifumi Fujino Manager
March 5,2003
Language Services Unit Phoenix Translations January 13, 2004
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