Registration Dossier
Registration Dossier
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EC number: 940-433-0 | CAS number: -
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- The experimental phase started on 24 February 2014 and ended on 10 March 2014
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Meets the criteria for classification as Reliable without restriction according to Klimisch et al (1997).
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 014
- Report date:
- 2014
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- No analysis was carried out, however, as the test item was formulated within four hours of it being applied to the test system; it is assumed that the formulation was stable for this duration.
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- lithium hydrogen 7-{(E)-[2-amino-4-(2-naphthyl)-1,3-thiazol-5-yl]diazenyl}naphthalene-1,3,5-trisulfonate (2:1:1)
- EC Number:
- 940-433-0
- Molecular formula:
- C23H14Li2N4O9S4
- IUPAC Name:
- lithium hydrogen 7-{(E)-[2-amino-4-(2-naphthyl)-1,3-thiazol-5-yl]diazenyl}naphthalene-1,3,5-trisulfonate (2:1:1)
- Test material form:
- solid: particulate/powder
- Remarks:
- migrated information: powder
Constituent 1
Method
- Target gene:
- Histidine for Salmonella
Tryptophan for E. Coli
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- Not Applicable
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- not applicable
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbitone/betanaphthoflavone induced rat liver S9.
- Test concentrations with justification for top dose:
- Experiment one: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: 50, 150, 500, 1500 and 5000 µg/plate
Test concentrations relate to active substance as formulation concentrations were adjusted to allow for the stated water and impurity content (13.9% w/w) of the test item. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water at 50 mg/mL and acetone at 100 mg/mL but was fully soluble in dimethyl sulphoxide at 50 mg/mL in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.
Controlsopen allclose all
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rate of TA100
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 1 ug/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rate of TA1535
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 2-ug/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rate of TA1537
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 2ug/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rate of TA98
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: benzo(a)pyrene: 5ug/plate
- Remarks:
- With S9
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rate of WP2uvrA
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 10 ug/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rate of TA100
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 ug/plate
- Remarks:
- Without S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rate of TA1535
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 ug/plate
- Remarks:
- Without S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rate of TA1537
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 9-aminoacridine:80 ug/plate
- Remarks:
- Without S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rate of TA98
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-Nitroquinoline-1-oxide: 0.2 ug/plate
- Remarks:
- Without S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rate WP2uvrA
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 ug/plate
- Remarks:
- Without S9 mix
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1 and pre-incubation for Experiment 2.
DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable
NUMBER OF REPLICATIONS: Triplicate plating.
DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for reductions in bacterial lawn in both experiment 1 and 2. - Evaluation criteria:
- Acceptance Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks.
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
All tester strain cultures should be in the range of 0.9 to 9 x 10^9 bacteria per mL.
Positive control chemicals must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
Evaluation Criteria
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response.
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal. - Statistics:
- None
Results and discussion
Test resultsopen allclose all
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- (Tested up to maximum recommended dose of 5000 ug/plate)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- (Tested up to maximum recommended dose of 5000 ug/plate)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
Any other information on results incl. tables
Test Results for Each Experiment are as follows:
Codes:
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
I Intense test item induced coloration
# Standard deviation
BP Benzo(a)pyrene
2AA 2-Aminoanthracene
Test Results: Experiment 1 – Without Metabolic Activation
Test Period |
From: 28 February 2014 |
To: 03 March 2014 |
||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (DMSO) |
67 87 65 |
(73) 12.2# |
19 8 8 |
(12) 6.4 |
27 21 15 |
(21) 6.0 |
17 16 17 |
(17) 0.6 |
9 7 21 |
(12) 7.6 |
||
1.5 µg |
65 83 61 |
(70) 11.7 |
16 16 15 |
(16) 0.6 |
20 29 23 |
(24) 4.6 |
17 15 23 |
(18) 4.2 |
8 4 9 |
(7) 2.6 |
||
5 µg |
71 64 61 |
(65) 5.1 |
11 11 12 |
(11) 0.6 |
28 23 15 |
(22) 6.6 |
13 13 9 |
(12) 2.3 |
12 4 7 |
(8) 4.0 |
||
15 µg |
83 71 74 |
(76) 6.2 |
9 8 12 |
(10) 2.1 |
25 23 27 |
(25) 2.0 |
13 8 16 |
(12) 4.0 |
8 5 13 |
(9) 4.0 |
||
50 µg |
83 79 72 |
(78) 5.6 |
11 11 11 |
(11) 0.0 |
15 21 29 |
(22) 7.0 |
19 16 11 |
(15) 4.0 |
13 5 8 |
(9) 4.0 |
||
150 µg |
72 75 74 |
(74) 1.5 |
11 15 9 |
(12) 3.1 |
17 21 24 |
(21) 3.5 |
15 15 9 |
(13) 3.5 |
13 5 5 |
(8) 4.6 |
||
500 µg |
106 I 79 I 74 I |
(86) 17.2 |
12 I 13 I 11 I |
(12) 1.0 |
22 I 10 I 14 I |
(15) 6.1 |
9 I 20 I 18 I |
(16) 5.9 |
5 I 12 I 5 I |
(7) 4.0 |
||
1500 µg |
69 I 70 I 65 I |
(68) 2.6 |
10 I 8 I 11 I |
(10) 1.5 |
16 I 16 I 14 I |
(15) 1.2 |
19 I 21 I 13 I |
(18) 4.2 |
6 I 9 I 13 I |
(9) 3.5 |
||
5000 µg |
58 I 56 I 60 I |
(58) 2.0 |
8 I 9 I 8 I |
(8) 0.6 |
21 I 17 I 16 I |
(18) 2.6 |
13 I 9 I 17 I |
(13) 4.0 |
6 I 5 I 10 I |
(7) 2.6 |
||
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
||||||
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
||||||||
337 291 361 |
(330) 35.6 |
114 103 107 |
(108) 5.6 |
718 510 679 |
(636) 110.6 |
179 120 151 |
(150) 29.5 |
1149 706 1405 |
(1087) 353.6 |
|||
Test Results: Experiment 1 – With Metabolic Activation
Test Period |
From: 28 February 2014 |
To: 03 March 2014 |
||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (DMSO) |
83 64 94 |
(80) 15.2# |
9 11 11 |
(10) 1.2 |
37 27 25 |
(30) 6.4 |
17 27 21 |
(22) 5.0 |
9 12 4 |
(8) 4.0 |
||
1.5 µg |
68 84 74 |
(75) 8.1 |
11 11 8 |
(10) 1.7 |
20 28 35 |
(28) 7.5 |
20 16 20 |
(19) 2.3 |
5 4 7 |
(5) 1.5 |
||
5 µg |
65 79 65 |
(70) 8.1 |
9 11 9 |
(10) 1.2 |
37 29 31 |
(32) 4.2 |
20 17 19 |
(19) 1.5 |
9 8 7 |
(8) 1.0 |
||
15 µg |
65 68 68 |
(67) 1.7 |
13 12 11 |
(12) 1.0 |
27 20 27 |
(25) 4.0 |
13 15 17 |
(15) 2.0 |
4 5 4 |
(4) 0.6 |
||
50 µg |
64 91 64 |
(73) 15.6 |
11 13 9 |
(11) 2.0 |
31 19 25 |
(25) 6.0 |
23 25 17 |
(22) 4.2 |
4 4 5 |
(4) 0.6 |
||
150 µg |
68 74 68 |
(70) 3.5 |
11 11 11 |
(11) 0.0 |
25 17 19 |
(20) 4.2 |
28 23 27 |
(26) 2.6 |
8 13 9 |
(10) 2.6 |
||
500 µg |
79 I 67 I 69 I |
(72) 6.4 |
14 I 11 I 11 I |
(12) 1.7 |
21 I 13 I 26 I |
(20) 6.6 |
24 I 29 I 26 I |
(26) 2.5 |
11 I 8 I 4 I |
(8) 3.5 |
||
1500 µg |
84 I 70 I 64 I |
(73) 10.3 |
14 I 11 I 10 I |
(12) 2.1 |
16 I 19 I 14 I |
(16) 2.5 |
14 I 13 I 20 I |
(16) 3.8 |
6 I 8 I 6 I |
(7) 1.2 |
||
5000 µg |
64 I 57 I 70 I |
(64) 6.5 |
10 I 8 I 8 I |
(9) 1.2 |
22 I 16 I 29 I |
(22) 6.5 |
21 I 18 I 18 I |
(19) 1.7 |
7 I 6 I 6 I |
(6) 0.6 |
||
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
BP |
2AA |
||||||
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
||||||||
531 528 532 |
(530) 2.1 |
120 210 158 |
(163) 45.2 |
678 744 716 |
(713) 33.1 |
155 140 176 |
(157) 18.1 |
134 135 159 |
(143) 14.2 |
|||
Test Results: Experiment 2 – Without Metabolic Activation
Test Period |
From: 06 March 2014 |
To: 09 March 2014 |
||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (DMSO) |
94 84 76 |
(85) 9.0# |
13 11 16 |
(13) 2.5 |
32 36 21 |
(30) 7.8 |
13 23 23 |
(20) 5.8 |
11 9 21 |
(14) 6.4 |
||
50 µg |
90 98 79 |
(89) 9.5 |
11 11 9 |
(10) 1.2 |
33 21 27 |
(27) 6.0 |
11 11 17 |
(13) 3.5 |
8 23 3 |
(11) 10.4 |
||
150 µg |
92 86 102 |
(93) 8.1 |
9 17 11 |
(12) 4.2 |
35 28 15 |
(26) 10.1 |
25 23 24 |
(24) 1.0 |
15 11 9 |
(12) 3.1 |
||
500 µg |
89 I 85 I 86 I |
(87) 2.1 |
16 I 10 I 20 I |
(15) 5.0 |
27 I 24 I 20 I |
(24) 3.5 |
27 I 18 I 22 I |
(22) 4.5 |
13 I 8 I 4 I |
(8) 4.5 |
||
1500 µg |
94 I 86 I 81 I |
(87) 6.6 |
14 I 16 I 19 I |
(16) 2.5 |
18 I 23 I 18 I |
(20) 2.9 |
17 I 23 I 21 I |
(20) 3.1 |
10 I 12 I 6 I |
(9) 3.1 |
||
5000 µg |
64 I 63 I 69 I |
(65) 3.2 |
14 I 16 I 19 I |
(16) 2.5 |
17 I 21 I 21 I |
(20) 2.3 |
22 I 21 I 15 I |
(19) 3.8 |
6 I 11 I 9 I |
(9) 2.5 |
||
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
||||||
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
||||||||
1201 981 893 |
(1025) 158.6 |
168 176 162 |
(169) 7.0 |
531 615 620 |
(589) 50.0 |
180 188 229 |
(199) 26.3 |
498 326 687 |
(504) 180.6 |
|||
Test Results: Experiment 2 – With Metabolic Activation
Test Period |
From: 06 March 2014 |
To: 09 March 2014 |
||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (DMSO) |
78 83 103 |
(88) 13.2# |
15 16 12 |
(14) 2.1 |
27 28 32 |
(29) 2.6 |
9 24 27 |
(20) 9.6 |
9 19 12 |
(13) 5.1 |
||
50 µg |
83 83 80 |
(82) 1.7 |
9 8 17 |
(11) 4.9 |
24 33 28 |
(28) 4.5 |
27 21 20 |
(23) 3.8 |
13 17 16 |
(15) 2.1 |
||
150 µg |
90 91 90 |
(90) 0.6 |
8 11 18 |
(12) 5.1 |
25 33 24 |
(27) 4.9 |
29 15 20 |
(21) 7.1 |
8 5 13 |
(9) 4.0 |
||
500 µg |
83 I 67 I 81 I |
(77) 8.7 |
11 I 14 I 18 I |
(14) 3.5 |
26 I 28 I 35 I |
(30) 4.7 |
28 I 21 I 28 I |
(26) 4.0 |
15 I 7 I 14 I |
(12) 4.4 |
||
1500 µg |
85 I 95 I 93 I |
(91) 5.3 |
12 I 9 I 8 I |
(10) 2.1 |
25 I 28 I 32 I |
(28) 3.5 |
29 I 26 I 24 I |
(26) 2.5 |
11 I 12 I 16 I |
(13) 2.6 |
||
5000 µg |
74 I 86 I 64 I |
(75) 11.0 |
10 I 9 I 8 I |
(9) 1.0 |
27 I 26 I 29 I |
(27) 1.5 |
19 I 18 I 28 I |
(22) 5.5 |
10 I 8 I 6 I |
(8) 2.0 |
||
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
BP |
2AA |
||||||
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
||||||||
870 807 945 |
(874) 69.1 |
241 211 230 |
(227) 15.2 |
274 255 225 |
(251) 24.7 |
111 138 130 |
(126) 13.9 |
294 249 287 |
(277) 24.2 |
|||
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
negative
The test item was considered to be non-mutagenic under the conditions of this test. - Executive summary:
Introduction
The test item was tested using a protocol designed to be compatible with OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test" and Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008.
Methods
Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre‑incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for the plate incorporation experiment was 1.5 to 5000 µg/plate and for the pre-incubation experiment 50 to 5000 µg/plate.
Results
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.
Conclusion
The test item was considered to be non-mutagenic under the conditions of this test.
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