Registration Dossier
Registration Dossier
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EC number: 700-655-9 | CAS number: 857288-56-3
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: OECD guideline and EU method. GLP study.
- 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)
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Experiment 1: 5, 15, 50, 150, 500, 1500, 5000 μg/plate
Experiment 2: Salmonella strains: 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.
E.coli strain WP2uvrA: 5, 15, 50, 150, 500, 1500, 5000 μg/plate. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- Without metabolic activation: TA100, TA1535, E.coli WP2uvrA
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- Without metabolic activation: TA98
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- Without metabolic activation: TA1537
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- With metabolic activation: TA100, TA1535, TA1537, E. coli WP2uvrA
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- With metabolic activation: TA98
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
Experiment 1: Plate incorporation method: Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the vehicle, test item formulation or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix. All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a colony counter.
Experiment 2: Pre-incubation method: Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 ml of S9-mix or phosphate buffer and 0.05 ml of the vehicle or test item formulation and incubated for 20 minutes at 37°C with shaking at approximately 130 rpm prior to the addition of 2 ml of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix. All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a colony counter.
NUMBER OF REPLICATIONS: Triplicates
DETERMINATION OF CYTOTOXICITY
- Method: In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test item. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate. The test was performed by mixing 0.1 ml of bacterial culture (TA100 or WP2uvrA), 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of test item formulation and 0.5 ml of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten concentrations of the test item formulation and a vehicle control (dimethyl sulphoxide) were tested. In addition, 0.1 ml of the maximum concentration of the test item and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile nutrient agar plate in order to assess the sterility of the test item. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a colony counter and examined for effects on the growth of the bacterial background lawn. - 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 (De Serres and Shelby (1979)).
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. - 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:
- cytotoxicity
- Remarks:
- (all tester strains, initially from 500 μg/plate in the absence and presence of S9-mix).
- Vehicle 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:
- cytotoxicity
- Remarks:
- (all tester strains, initially from 500 μg/plate in the absence and presence of S9-mix).
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
RANGE-FINDING/SCREENING STUDIES: The test item was toxic to TA100 at and above 1500 μg/plate (absence and presence of S9-mix) and to WP2uvrA at 5000 μg/plate in the absence of S9-mix.
ADDITIONAL INFORMATION ON CYTOTOXICITY: In the first experiment, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 500 μg/plate in the absence and presence of S9-mix. In the second experiment (pre-incubation method) the test item induced a slightly stronger toxic response with weakened bacterial background lawns noted to all of the tester strains initially from 500 μg/plate in both the absence and presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. - Conclusions:
- Interpretation of results (migrated information):
negative
The test item was considered to be non-mutagenic under the conditions of this test. - Executive summary:
The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines. Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the first experiment was determined in a preliminary toxicity assay and was 5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using an amended dose range Between 1.5 and 5000 μg/plate), fresh cultures of the bacterial strains and fresh test item formulations. Additional dose levels and an expanded dose range were selected in both experiments in order to achieve both four non-toxic dose levels and the toxic limit of the test item. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant
colonies generally within the normal range. All of the positive controls 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. In the first experiment, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 500 μg/plate in both the absence and presence of S9-mix. In the second experiment (pre-incubation method)
the test item induced a slightly stronger toxic response with weakened bacterial background lawns noted to all of the tester strains initially from 500 μg/plate in both the absence and presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. The test item was tested up to the maximum recommended dose level of 5000 μg/plate or the toxic limit, depending on bacterial strain type and experiment number. 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. The test item was considered to be non-mutagenic under the conditions of this test.
Reference
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.
Table 1: Test Results: Experiment 1 – Without Metabolic Activation
Test Period |
From: 21 June 2012 |
To: 24 June 2012 |
||||||||||||||||
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) |
106 104 76 |
(95) 16.8# |
15 31 22 |
(23) 8.0 |
43 43 41 |
(42) 1.2 |
20 23 20 |
(21) 1.7 |
16 17 13 |
(15) 2.1 |
||||||||
5 μg |
104 104 88 |
(99) 9.2 |
16 23 17 |
(19) 3.8 |
56 43 52 |
(50) 6.7 |
15 12 13 |
(13) 1.5 |
12 12 15 |
(13) 1.7 |
||||||||
15 μg |
81 98 79 |
(86) 10.4 |
17 20 15 |
(17) 2.5 |
40 49 44 |
(44) 4.5 |
9 15 19 |
(14) 5.0 |
12 5 11 |
(9) 3.8 |
||||||||
50 μg |
94 95 110 |
(100) 9.0 |
15 12 20 |
(16) 4.0 |
44 45 36 |
(42) 4.9 |
23 19 24 |
(22) 2.6 |
15 15 15 |
(15) 0.0 |
||||||||
150 μg |
74 76 111 |
(87) 20.8 |
21 16 17 |
(18) 2.6 |
41 43 29 |
(38) 7.6 |
11 19 20 |
(17) 4.9 |
12 12 8 |
(11) 2.3 |
||||||||
500 μg |
116 88 107 |
(104) 14.3 |
17 15 21 |
(18) 3.1 |
37 28 28 |
(31) 5.2 |
12 28 23 |
(21) 8.2 |
11 S 12 S 12 S |
(12) 0.6 |
||||||||
1500 μg |
9 V 13 V 19 V |
(14) 5.0 |
9 S 15 S 13 S |
(12) 3.1 |
49 S 41 S 49 S |
(46) 4.6 |
23 S 12 S 19 S |
(18) 5.6 |
7 S 12 S 15 S |
(11) 4.0 |
||||||||
5000 μg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
17 S 29 S 20 S |
(22) 6.2 |
0 V 0 V 0 V |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
||||||||
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 |
||||||||||||||
326 330 330 |
(329) 2.3 |
167 139 221 |
(176) 41.7 |
547 413 472 |
(477) 67.2 |
119 116 122 |
(119) 3.0 |
266 400 428 |
(365) 86.6 |
|||||||||
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
S Sparse bacterial background lawn
T Toxic, no bacterial background lawn
V Very weak bacterial background lawn
# Standard deviation
Table 2 Test Results: Experiment 1 – With Metabolic Activation
Test Period |
From: 21 June 2012 |
To: 24 June 2012 |
||||||||||||||||
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) |
98 87 78 |
(88) 10.0# |
12 8 16 |
(12) 4.0 |
41 43 36 |
(40) 3.6 |
23 24 29 |
(25) 3.2 |
13 11 13 |
(12) 1.2 |
||||||||
5 μg |
75 118 95 |
(96) 21.5 |
4 8 11 |
(8) 3.5 |
44 39 47 |
(43) 4.0 |
25 29 29 |
(28) 2.3 |
12 16 13 |
(14) 2.1 |
||||||||
15 μg |
106 99 107 |
(104) 4.4 |
12 8 9 |
(10) 2.1 |
43 51 37 |
(44) 7.0 |
28 24 28 |
(27) 2.3 |
19 13 15 |
(16) 3.1 |
||||||||
50 μg |
71 99 114 |
(95) 21.8 |
15 5 12 |
(11) 5.1 |
45 44 35 |
(41) 5.5 |
16 25 20 |
(20) 4.5 |
13 12 12 |
(12) 0.6 |
||||||||
150 μg |
100 86 88 |
(91) 7.6 |
12 8 9 |
(10) 2.1 |
35 48 28 |
(37) 10.1 |
24 15 31 |
(23) 8.0 |
12 13 8 |
(11) 2.6 |
||||||||
500 μg |
84 95 86 |
(88) 5.9 |
9 16 7 |
(11) 4.7 |
51 51 53 |
(52) 1.2 |
33 27 27 |
(29) 3.5 |
13 S 13 S 12 S |
(13) 0.6 |
||||||||
1500 μg |
27 S 29 S 29 S |
(28) 1.2 |
7 S 11 S 13 S |
(10) 3.1 |
33 S 41 S 35 S |
(36) 4.2 |
23 S 29 S 25 S |
(26) 3.1 |
11 S 5 S 4 S |
(7) 3.8 |
||||||||
5000 μg |
0 T 0 T 0 T |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
37 S 44 S 55 S |
(45) 9.1 |
0 V 0 V 0 V |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
||||||||
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 |
||||||||||||||
1660 1624 1629 |
(1638) 19.5 |
265 261 281 |
(269) 10.6 |
663 480 517 |
(553) 96.8 |
210 180 180 |
(190) 17.3 |
194 184 225 |
(201) 21.4 |
|||||||||
2AA 2-Aminoanthracene
BP Benzo(a)pyrene
S Sparse bacterial background lawn
T Toxic, no bacterial background lawn
V Very weak bacterial background lawn
# Standard deviation
Table 3 Test Results: Experiment 2 – Without Metabolic Activation
Test Period |
From: 06 July 2012 |
To: 09 July 2012 |
||||||||||||||||
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) |
106 110 61 |
(92) 27.2# |
12 24 2 7 |
(21) 7.9 |
29 37 27 |
(31) 5.3 |
23 24 16 |
(21) 4.4 |
9 9 13 |
(10) 2.3 |
||||||||
1.5 μg |
84 83 83 |
(83) 0.6 |
16 17 16 |
(16) 0.6 |
N/T |
19 20 16 |
(18) 2.1 |
4 8 13 |
(8) 4.5 |
|||||||||
5 μg |
83 80 67 |
(77) 8.5 |
31 13 21 |
(22) 9.0 |
40 27 25 |
(31) 8.1 |
31 9 23 |
(21) 11.1 |
7 17 17 |
(14) 5.8 |
||||||||
15 μg |
98 82 60 |
(80) 19.1 |
15 21 21 |
(19) 3.5 |
16 39 16 |
(24) 13.3 |
15 27 20 |
(21) 6.0 |
8 11 23 |
(14) 7.9 |
||||||||
50 μg |
67 75 95 |
(79) 14.4 |
28 27 27 |
(27) 0.6 |
49 25 25 |
(33) 13.9 |
20 21 12 |
(18) 4.9 |
4 11 11 |
(9) 4.0 |
||||||||
150 μg |
87 79 83 |
(83) 4.0 |
24 17 27 |
(23) 5.1 |
44 29 29 |
(34) 8.7 |
23 13 19 |
(18) 5.0 |
9 9 7 |
(8) 1.2 |
||||||||
500 μg |
63 S 123 S 74 S |
(87) 31.9 |
17 S 21 S 15 S |
(18) 3.1 |
35 32 43 |
(37) 5.7 |
24 S 20 S 29 S |
(24) 4.5 |
15 S 4 S 7 S |
(9) 5.7 |
||||||||
1500 μg |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
21 S 20 S 28 S |
(23) 4.4 |
12 S 11 S 28 S |
(17) 9.5 |
0 V 0 V 0 V |
(0) 0.0 |
||||||||
5000 μg |
N/T |
N/T |
0 V 0 V 0 V |
(0) 0.0 |
N/T |
N/T |
||||||||||||
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 |
||||||||||||||
520 436 488 |
(481) 42.4 |
183 187 216 |
(195) 18.0 |
508 509 532 |
(516) 13.6 |
100 112 106 |
(106) 6.0 |
345 408 372 |
(375) 31.6 |
|||||||||
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
N/T Not tested at this dose level
S Sparse bacterial background lawn
V Very weak bacterial background lawn
# Standard deviation
Table 4 Test Results: Experiment 2 – With Metabolic Activation
Test Period |
From: 06 July 2012 |
To: 09 July 2012 |
|
||||||||||||||||
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) |
82 69 100 |
(84) 15.6# |
9 13 19 |
(14) 5.0 |
47 35 44 |
(42) 6.2 |
19 35 40 |
(31) 11.0 |
8 20 13 |
(14) 6.0 |
|
||||||||
1.5 μg |
100 83 108 |
(97) 12.8 |
16 13 23 |
(17) 5.1 |
N/T |
25 33 27 |
(28) 4.2 |
19 24 23 |
(22) 2.6 |
|
|||||||||
5 μg |
100 82 80 |
(87) 11.0 |
20 11 20 |
(17) 5.2 |
24 56 49 |
(43) 16.8 |
40 31 32 |
(34) 4.9 |
17 9 24 |
(17) 7.5 |
|||||||||
15 μg |
82 87 84 |
(84) 2.5 |
23 16 7 |
(15) 8.0 |
37 49 51 |
(46) 7.6 |
27 23 23 |
(24) 2.3 |
12 15 11 |
(13) 2.1 |
|||||||||
50 μg |
100 75 72 |
(82) 15.4 |
13 17 11 |
(14) 3.1 |
39 56 39 |
(45) 9.8 |
23 32 27 |
(27) 4.5 |
13 12 20 |
(15) 4.4 |
|||||||||
150 μg |
72 71 71 |
(71) 0.6 |
11 20 1 |
(11) 9.5 |
44 40 29 |
(38) 7.8 |
35 39 39 |
(38) 2.3 |
15 16 5 |
(12) 6.1 |
|||||||||
500 μg |
79 S 48 S 60 S |
(62) 15.6 |
1 S 7 S 11 S |
(6) 5.0 |
29 45 19 |
(31) 13.1 |
20 24 36 |
(27) 8.3 |
11 12 19 |
(14) 4.4 |
|||||||||
1500 μg |
35 V 28 V 24 V |
(29) 5.6 |
0 V 0 V 0 V |
(0) 0.0 |
19 S 19 S 17 S |
(18) 1.2 |
13 S 20 S 17 S |
(17) 3.5 |
0 V 0 V 0 V |
(0) 0.0 |
|||||||||
5000 μg |
N/T |
N/T |
7 S 13 S 19 S |
(13) 6.0 |
N/T |
N/T |
|
||||||||||||
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 |
|
||||||||||||||
1425 1421 1958 |
(1601) 308.9 |
262 234 274 |
(257) 20.5 |
412 472 337 |
(407) 67.6 |
223 188 188 |
(200) 20.2 |
369 267 390 |
(342) 65.8 |
|
|||||||||
2AA 2-Aminoanthracene
BP Benzo(a)pyrene
N/T Not tested at this dose level
S Sparse bacterial background lawn
V Very weak bacterial background lawn
# Standard deviation
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Genetic toxicity in vitro:
Key study: OECD 471 and EU method B.13/14. GLP study.
The test item was considered to be non-mutagenic under the conditions of this test.
Justification for selection of genetic toxicity endpoint
Only one study available.
Justification for classification or non-classification
Based on the available data, the substance is not classified.
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