Registration Dossier
Registration Dossier
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EC number: 836-437-6 | CAS number: 577978-76-8
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames Test:
The mutagenicity of the test substance was judged positive because the number of revertant colonies was increased two times or more than that of negative control, and the numbers increased with reproducibility in TA1535, WP2 uvrA and TA98 with and without S9 mix. Consequently, GK-5016-2 was considered to have mutagenic potential.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 04 June 2002 to 03 July 2002
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- No further details specified in the study report
- Target gene:
- Histidine and Tryprophan
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- Not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- 1) Rat liver S9
An S9 (Lot No. 02050902, manufactured on May 9, 2002, Oriental Yeast Co., Ltd.) prepared from the liver of rats (body weight 206. 7±8.4 g) administered phenobarbital and 5,6-benzoflavone was used. The S9 was cryopreserved in an ultra-deep freezer (MDF-291, SANYO Electric Co., Inc.) until use.
2) Composition of S9 mix
A S9 mix was prepared using a cofactor for S9 mix (Lot No. 999202, Oriental Yeast Co., Ltd.) immediately before use.
One mL of the S9 mix consisted of 8 µmol MgC12, 33 µmol KCl, 5 µmol G-6-P, 4 µmol NADPH, 4 µmol NADH, 100 µmol sodium-phosphate buffer (pH 7.4) and 0.1 mL of S9. - Test concentrations with justification for top dose:
- Dose-range-finding test-1
A total of 6 doses was set, 5,000 µg/plate was the highest dose and 5 lower doses diluted with a geometric progression of 4.
Dose-range-finding test-2
In the dose-range-finding test-1, growth inhibition were observed in all the tester strains. Four or more doses without growth inhibition (effective dose) were not obtained in all the tester strains without S9 mix and in TAlO0 and TA1535 with S9 mix. Therefore, dose-range-finding test-2 was carried out due to secure for four or more effective dose in a main test. Highest doses in the second test were selected at 78.1 µg/plate without S9 mix, at 313 µg/plate with S9 mix, and added at each 5 lower doses diluted with a geometric progression of 4. - Vehicle / solvent:
- DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, 2-Aminoanthracene, 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino] acridine-2H Cl
- Details on test system and experimental conditions:
- The tests were performed using the pre-incubation method with and without S9 mix. Three plates were used for the negative control group and two plates for the test substance treatment and positive control groups. The test code number, name of tester strain, presence or absence of S9 mix and dose level were noted on the plate.
Procedures
fter 0. lmL of the test substance solution or positive control 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 test tube, and the mixture was shaken for 20 minutes at 37±0.5°C. Two mL of the soft agar was then added to the test tube, and the mixture was poured onto a minimal glucose agar plate. The number of reve1iant colonies was counted after incubation at 37±0.5°C for 48 hours.
Sterility
Each of the test substance solution of the highest dose and S9 mix was mixed with 2 mL of the soft agar and poured onto a minimal glucose agar plate. The plate was incubated at 37±0.5°C for 48 hours to investigate a bacterial contamination.
Main test
n the dose-range-finding tests, the growth inhibition and increase of the revertant colonies were observed, so highest doses were selected at 39.1 µg/plate in TAlO0 and TA1535, at 78.1 µg/plate in WP2 uvrA and TA98, at 19.5 µg/plate in TA1537 in the without S9 mix, and added at each 5 or 6 lower doses diluted with a geometric progression of 2. In the with S9 mix, highest doses were selected at 625 µg/plate in TAl00 and TA1535, at 1,250 µg/plate in WP2 uvrA, TA98 and TA 153 7, and added 6 lower doses diluted with a geometric progression of 2. - Rationale for test conditions:
- Not specified
- Evaluation criteria:
- The test substance was judged positive when the number of revertant colonies was increased two times or more that of the negative control in a dose-dependent manner, and also when reproducibility of the test results was determined. In all the other cases, it was judged negative. Statistical treatment was not carried out.
In the case of a positive result, specific activities were calculated for dose levels of which the number of revertant colonies was twice or more than that in the negative - Statistics:
- Statistical treatment was not carried out.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- DOSE-RANGE-FINDING TEST-1
Growth inhibition was observed in all the tester strains, so there were not four or more effective doses in all the tester strains without S9 mix and in TAlO0 and TA1535 with S9 mix. Correctly, evaluation of the test results were not completed. In WP2 uvrA and TA98 with S9 mix, the numbers of revertant colonies were increased two times or more that of the each negative control. And dose-related increase was showed in WP2 uvrA.
DOSE-RANGE-FINDING TEST-2
In TA1535, WP2 uvrA and TA98 without S9 mix and TA1535 with S9 mix, the numbers of revertant colonies were increased two times or more that of the negative control, and dose-related increase were shown in WP2 uvrA and TA98.
The numbers of revertant colonies were less than two times that of the negative control in the other test strains.
Growth inhibition was observed at 19.5 µg/plate or more in TAIO0, TA1535, TA98 and TA1537 and at 78.1 µg/plate in WP2 uvrA without S9 mix. Growth inhibition was observed at 313 µg/plate in TAl00 and TA1535 with S9 mix.
The precipitate of the test substance was not observed.
MAINTEST
The numbers of revertant colonies were increased in TA1535, WP2 uvrA and TA98 with and without S9 mix, and shown dose-related increased in WP2 uvrA and TA98. The numbers of revertant colonies were less than two times that of the negative control in TAIO0 and TA1537 with and without S9 mix.
Growth inhibition was observed at 19.5 µg/plate or more in TAIO0, TA1535 and TA98, at 39.1 µg/plate or more in WP2 uvrA and at 9.77 µg/plate or more in TA1537 without S9 mix. Growth inhibition was observed at 313 µg/plate or more in TAl00 and TA1535 and at 625 µg/plate or more in WP2 uvrA, TA98 and TA1537 with S9 mix. The precipitate of the test substance was observed at 625 µg/plate or more with S9 mix.
Mutagenicity of the test substance was judged positive because the numbers of revertant colonies were reproducible increased two times or more that of negative control in TA1535, WP2 uvrA and TA98 with and without S9 mix. And in WP2 uvrA and TA98 with and without S9 mix, the numbers of revertant colonies were increased two times or more that of negative control and dose-related increase was shown. The specific activity was 13,730 Revertants/mg. In the other test strains, the number of revertant colonies was not increased.
The numbers of revertant colonies of the positive and negative controls were within the range of the historical data in our laboratory. It was also confirmed that the test system was free from bacterial contamination, indicating that this study was appropriately performed.
Based on the above results, GK-5016-2 was considered to have mutagenic potential under the conditions in the present study. - Conclusions:
- GK-5016-2 was considered to have mutagenic potential under the conditions in the present study.
- Executive summary:
The ability of GK-5016-2 to induce mutations was investigated in Salmonella typhimurium strains TAlO0, TA1535, TA98 and TA1537 and Escherichia coli strain WP2 uvrA using a pre-incubation method with and without metabolic activation system (S9 mix).
As a result, the mutagenicity of the test substance was judged positive because the number of revertant colonies was increased two times or more that of negative control and the numbers increased with reproducibility in TA1535, WP2 uvrA and TA98 with and without S9 mix. Consequently, GK-5016-2 was considered to have mutagenic potential.
Reference
Dose-range-finding test-I
Test substance: GK-5016-2
Test dates |
From June 7 2002 to June 10 2002 |
|||||
With (+) or without (-) S9 mix |
Test substance dose (μg/plate) |
Number of revertants per plate |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA 1535 |
WP2 uvrA |
TA 98 |
TA 1537 |
||
-S9 mix
|
Negative control |
97 84 (94) 102 |
7 10 (8) 8 |
29 25 (29) 34 |
20 28 (24) 24 |
8 4 13 (8) |
4.88 |
147 131 (139) |
21 19 (20) |
109 74 (92) |
46 40 (43) |
7 5 (6) |
|
19.5 |
137* 152* (145) |
9* 22* (16) |
131 128 (130) |
86* 89* (88) |
2* 3* (3) |
|
78.1 |
36* 38* (37) |
6* 6* (6) |
77* 66* (72) |
42* 42* (42) |
1* 0* (1) |
|
+313 |
0* 0* (0) |
0* 0* (0) |
28* 33* (31) |
0* 0* (0) |
0* 0* (0) |
|
+1250 |
0* 0* (0) |
0* 0* (0) |
0* 0* (0) |
0* 0* (0) |
0* 0* (0) |
|
+5000 |
0* 0* (0) |
0* 0* (0) |
0* 0* (0) |
0* 0* (0) |
0* 0* (0) |
|
+S9 mix
|
Negative control |
108 123 (120) 129 |
8 11 5 (9) |
28 26 22 (25) |
28 37 23 (29) |
16 20 18 (18) |
4.88 |
110 93 (102) |
21 8 (15) |
43 42 (43) |
33 26 (30) |
22 21 (22) |
|
19.5 |
130 130 (130) |
11 5 (8) |
31 27 (29) |
33 51 (42) |
23 12 (18) |
|
78.1 |
127 137 (132) |
18 16 (17) |
72 49 (61) |
37 34 (36) |
16 23 (20) |
|
313 |
107* 91* (99) |
12* 11* (12) |
165 114 (140) |
59 66 (63) |
20 17 (19) |
|
+1250 |
0* 0* (0) |
0* 0* (0) |
14* 15* (15) |
0* 1* (1) |
0* 0* (0) |
|
+5000 |
0* 0* (0) |
0* 0* (0) |
14* 11* (13) |
0* 0* (0) |
0* 0* (0) |
|
Positive control -S9 mix |
Chemical |
AF-2 |
NaN3 |
AF-2 |
AF-2 |
ICR-191 |
Dose(μg/plate) |
0.01 |
05 |
0.01 |
01 |
05 |
|
Number of revertants/plate |
236 223 (230) |
433 409 (421) |
187 159 (173) |
414 424 (419) |
358 420 (389) |
|
Positive control +S9 mix |
Chemical |
2AA |
2AA |
2AA |
2AA |
2AA |
Dose(μg/plate) |
1 |
2 |
10 |
0.5 |
2 |
|
Number of revertants/plate |
838 805 (822) |
199 191 (195) |
580 677 (629) |
425 462 (444) |
416 457 (437) |
|
[Notes] Parenthesis shows the mean of each plate.
+: Precipitate of test substance.
*: Observed bacterial growth inhibition.
· AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide
• NaN3 : Sodium azide
· ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino ]acridine · 2HCI
· 2AA: 2-Aminoanthracene
Dose-range-finding test-2
Test substance: GK-5016-2
Test dates |
From June 7 2002 to June 10 2002 |
|||||
With (+) or without (-) S9 mix |
Test substance dose (μg/plate) |
Number of revertants per plate |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA 1535 |
WP2 uvrA |
TA 98 |
TA 1537 |
||
-S9 mix
|
Negative control |
97 87 (91) 91 |
10 7 (8) 8 |
32 32 (29) 24 |
18 29 (22) 20 |
9 5 9 (8) |
0.0763 |
88 87 (88) |
11 9 (10) |
39 36 (38) |
29 15 (22) |
8 6 (7) |
|
0.305 |
111 93 (102) |
12 9 (11) |
37 28 (33) |
16 21 (19) |
7 3 (5) |
|
1.22 |
122 115 (119) |
7 8 (8) |
44 64 (72) |
29 20 (25) |
5 1 (3) |
|
4.88 |
141 133 (137) |
23 14 (19) |
89 102 (96) |
32 37 (35) |
6 5 (6) |
|
19.5 |
105* 111* (108) |
12* 11* (12) |
139 130 (135) |
70* 83* (77) |
3* 5* (4) |
|
7801 |
21* 18* (20) |
2* 2* (2) |
67* 57* (62) |
44* 38* (41) |
2* 0* (1) |
|
+S9 mix
|
Negative control |
105 111 119 (112) |
5 12 5 (7) |
-- |
--
|
-- |
0.305 |
97 122 (110) |
9 6 (8) |
-- |
-- |
-- |
|
1.22 |
92 100 (96) |
7 7 (7) |
-- |
-- |
-- |
|
4.88 |
118 94 (106) |
3 6 (5) |
-- |
-- |
-- |
|
19.5 |
106 132 (119) |
8 12 (10) |
-- |
-- |
-- |
|
78.1 |
147 153 (150) |
17 13 (15) |
-- |
-- |
-- |
|
313 |
85* 106* (96) |
23* 13* (18) |
-- |
-- |
-- |
|
Positive control -S9 mix |
Chemical |
AF-2 |
NaN3 |
AF-2 |
AF-2 |
ICR-191 |
Dose(μg/plate) |
0.01 |
05 |
0.01 |
01 |
05 |
|
Number of revertants/plate |
284 263 (274) |
407 448 (428) |
183 181 (182) |
315 350 (333) |
302 292 (297) |
|
Positive control +S9 mix |
Chemical |
2AA |
2AA |
2AA |
2AA |
2AA |
Dose(μg/plate) |
1 |
2 |
10 |
0.5 |
2 |
|
Number of revertants/plate |
973 824 (899) |
210 216 (213) |
-- |
-- |
-- |
|
[Notes] Parenthesis shows the mean of each plate.
*: Observed bacterial growth inhibition.
• AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide
· NaN3 : Sodium azide
• ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino ]acridine · 2HC1
· 2AA: 2-Aminoanthracene
Main test
Test substance: GK-5016-2
Test dates |
From June 7 2002 to June 10 2002 |
|||||
With (+) or without (-) S9 mix |
Test substance dose (μg/plate) |
Number of revertants per plate |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA 1535 |
WP2 uvrA |
TA 98 |
TA 1537 |
||
-S9 mix
|
Negative control |
114 96 (111) 124 |
12 10 (10) 7 |
24 26 (29) 38 |
26 15 (20) 18 |
7 7 3 (6) |
0.610 |
88 87 (88) |
7 12 (10) |
41 23 (32) |
-- |
9 4 (7) |
|
1.22 |
117 113 (115) |
11 12 (12) |
30 36 (33) |
19 30 (25) |
8 2 (5) |
|
2.44 |
150 133 (142) |
14 13 (14) |
51 53 (52) |
28 30 (29) |
8 6 (7) |
|
4.88 |
145 121 (133) |
23 12 (18) |
66 71 (69) |
41 36 (39) |
5 6 (6) |
|
9.77 |
156 158 (157) |
19 24 (22) |
95 88 (92) |
71 54 (63) |
9* 4* (7) |
|
19.5 |
160 148 (154) |
11* 6* (9) |
130 131 (131) |
86* 92* (89) |
3* 9* (6) |
|
39.1 |
53* 51* (52) |
5* 6* (6) |
123* 111* (117) |
82* 85* (84) |
-- |
|
78.1 |
-- |
-- |
99* 98* (99) |
42* 50* (46) |
-- |
|
+S9 mix
|
Negative control |
113 113 124 (117) |
6 11 5 (7) |
36 29 32 (32) |
30 28 30 (29) |
21 16 16 (18) |
9.77 |
132 125 (129) |
10 6 (8) |
-- |
-- |
-- |
|
19.5 |
145 138 (142) |
7 11 (9) |
24 39 (32) |
45 25 (35) |
10 15 (13) |
|
39.1 |
135 155 (145) |
12 11 (12) |
19 42 (31) |
38 34 (36) |
15 18 (17) |
|
78.1 |
173 156 (165) |
19 13 (16) |
39 47 (43) |
34 25 (30) |
19 19 (19) |
|
156 |
198 180 (189) |
17 17 (17) |
80 79 (80) |
47 45 (46) |
18 21 (20) |
|
313 |
85* 94* (90) |
14* 11* (13) |
159 146 (153) |
63 71 (67) |
20 12 (16) |
|
+625 |
54* 58* (56) |
9* 7* (8) |
41* 49* (45) |
14* 18* (16) |
7* 4* (6) |
|
+1250 |
-- |
-- |
21* 20* (21) |
8* 4* (6) |
3* 6* (5) |
|
Positive control -S9 mix |
Chemical |
AF-2 |
NaN3 |
AF-2 |
AF-2 |
ICR-191 |
Dose(μg/plate) |
0.01 |
05 |
0.01 |
01 |
05 |
|
Number of revertants/plate |
320 313 (317) |
400 367 (384) |
190 164 (177) |
436 425 (431) |
378 287 (333) |
|
Positive control +S9 mix |
Chemical |
2AA |
2AA |
2AA |
2AA |
2AA |
Dose(μg/plate) |
1 |
2 |
10 |
0.5 |
2 |
|
Number of revertants/plate |
1343 1287 (1315) |
215 206 (211) |
523 604 (564) |
478 469 (474) |
504 473 (489) |
|
[Notes] Parenthesis shows the mean of each plate.
+: Precipitate of test substance.
*: Observed bacterial growth inhibition.
· AF-2: 2-(2-Furyl)-3-(5-nitro-2-fmyl)acrylamide
· NaN3: Sodium azide
· ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino ]acridine · 2HCI
· 2AA: 2-Aminoanthracene
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for classification or non-classification
Test item not classified for genetic toxicity as no further study data is available to scientifically justify classification
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
