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EC number: 287-820-4 | CAS number: 85586-18-1
Toxicological information
Genetic toxicity: in vitro
Currently viewing:
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:
- 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: This study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criteria were met.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 011
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- UK GLP Monitoring authority
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Fatty acids, C18-unsatd., reaction products with acrylic acid and polyethylenepolyamines
- EC Number:
- 287-820-4
- EC Name:
- Fatty acids, C18-unsatd., reaction products with acrylic acid and polyethylenepolyamines
- Cas Number:
- 85586-18-1
- Molecular formula:
- Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)
- IUPAC Name:
- (Z,9Z)-N-(2-{2-[(8Z)-heptadec-8-en-1-yl]-4,5-dihydro-1H-imidazol-1-yl}ethyl)octadec-9-enimidic acid; 3-[(2-carboxyethyl)({2-[3-(2-carboxyethyl)-2-[(8Z)-heptadec-8-en-1-yl]imidazolidin-1-yl]ethyl})amino]propanoic acid; 3-{2-[(8Z)-heptadec-8-en-1-yl]-3-{2-[(E)-[(9Z)-1-hydroxyoctadec-9-en-1-ylidene]amino]ethyl}imidazolidin-1-yl}propanoic acid
Constituent 1
Method
- Target gene:
- Histidine and Tryptophan synthesis genes
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 prepared from the livers of male rats treated with 80/100 mg Phenobarbitone/B-naphthoflavone
- Test concentrations with justification for top dose:
- Preliminary test: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate for Salmonella strains TA100, TA1535, TA98 (presence of S9) and E. Coli strain WP2uvrA- (Absence and presence of S9)
Definitive test: 5, 15, 50, 150, 500, 1500, 5000ug/plate for Salmonella strains TA100, TA1535, TA98 (absence of S9) and TA1537 (Absence and presence of S9) - Vehicle / solvent:
- Sterile distilled water
Controlsopen allclose all
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- 2 ug/plate for WP2uvrA- without S9
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- 3 ug/plate for TA100 without S9
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- 5 ug/plate for TA1535 without S9
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- 80 ug/plate for TA1537 without S9
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- 0.2 ug/plate for TA98 without S9
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other:
- Remarks:
- 2-Aminoanthracene 1ug/plate for TA100 with S9
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other:
- Remarks:
- 2-Aminoanthracene 2ug/plate for TA1535 and TA1537 with S9
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other:
- Remarks:
- 2-Aminoanthracene 10ug/plate for WP2uvrA- with S9
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- 5 ug/plate for TA98
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- no
- Positive control substance:
- other:
- Remarks:
- Water
- Details on test system and experimental conditions:
- Summary
Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using both the Ames plate incorporation and pre-incubation methods at up to 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 ranged between 5 and 5000 μg/plate, depending on bacterial strain type and presence or absence of S9-mix. The experiment was repeated on a separate day (pre-incubation method) using a similar dose range to Experiment 1, fresh cultures of the bacterial strains and fresh test material 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 material.
Tester strains
The following strains were used:
Salmonella typhimurium TA1535, TA1537, TA98 and TA100
Escherichia coli WP2uvrA
The four strains of Salmonella used in the test were obtained either from the University of California, Berkeley, on culture discs, on 04 August 1995 or from Syngenta CTL, Alderley Edge, as frozen vials, on 20 March 2007. E. coli strain WP2uvrA- was obtained from the British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987. All of the strains were stored at approximately ¯196°C in a Statebourne liquid nitrogen freezer, model SXR 34. Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate (5). In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 757012 03/14) and incubated at 37°C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates
Preparation of Test and Control Materials
The test material was fully miscible in sterile distilled water at 50 mg/ml in solubility checks performed in-house. Sterile distilled water was therefore selected as the vehicle. The test material was accurately weighed and approximate half-log dilutions prepared in sterile distilled water by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated impurity content (ethylene glycol at 38.5%) of the test material. Analysis for concentration, homogeneity and stability of the test material formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Vehicle and positive controls were used in parallel with the test material. A solvent treatment group was used as the vehicle control and the positive control materials used in the series of plates without S9-mix were as follows:
N-ethyl-N'-nitro-N-nitrosoguanidine (without S9) abbreviated to ENNG
9-Aminoacridine (without S9) abbreviated to 9AA
4-Nitroquinoline-1-oxide (without S9) abbreviated to 4NQO
2-Aminoanthracene (with S9) abbreviated to 2AA
Benzo(a)pyrene (with S9) abbreviated to BP
Preliminary Toxicity test
In order to select appropriate dose levels for use in the main test, a preliminary test wascarried out to determine the toxicity of the test material. 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 material 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 material formulation and a vehicle control (sterile distilled water) were tested. In addition, 0.1 ml of the maximum concentration of the test material 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 material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn
Mutation test - Experiment 1
Up to seven concentrations of the test material were assayed in triplicate against each tester strain, using the direct plate incorporation method. In the initial test, the toxicity of the test material yielded results that differed slightly from the preliminary toxicity test and consequently, there was an insufficient number of non-toxic dose levels for the Salmonella strains (predominantly in the absence of S9-mix). Therefore, part of the experiment was repeated using additional dose levels and expanded dose range. Dose ranges were allocated as follows:
Salmonella strains TA100, TA1535, TA98 (presence of S9-mix) and E.coli strain WP2uvrA- (absence and presence of S9-mix): 50, 150, 500, 1500, 5000 μg/plate.
Salmonella strains TA100, TA1535, TA98 (absence of S9-mix) and TA1537 (absence and presence of S9-mix): 5, 15, 50, 150, 500, 1500, 5000 μg/plate.
Additional dose levels and an expanded dose range were selected (where applicable) in order to achieve both four non-toxic dose levels and the toxiclimit of the test material.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test material formulation, vehicle 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 material 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 Domino colony counter
Mutation test - Experiment 2
The second experiment was performed using fresh bacterial cultures, test material and control solutions. The test material dose range was amended slightly, following the results of Experiment 1, as follows:
Salmonella strains TA100, TA1535, TA98 (presence of S9-mix): 5, 15, 50, 150, 500, 1500, 5000 μg/plate.
E.coli strain WP2uvrA- (absence and presence of S9-mix): 15, 50, 150, 500, 1500, 5000 μg/plate.
Salmonella strains TA100, TA1535, TA98 (absence of S9-mix) and TA1537 (absence and presence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 μg/plate
Additional dose levels and an expanded dose range were again selected in order to achieve both four non-toxic dose levels, the toxic limit of the test material and to account for the change in test methodology.
The test material formulations and vehicle control were dosed using the pre incubation method as follows: 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 0.1 ml of the vehicle or test material 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 material both with and without S9-mix.
The positive and untreated controls were dosed using the standard plate incorporation method previously described..
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter - Evaluation criteria:
- The reverse mutation assay may be considered valid if the following criteria are met:
1. All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls within historic norms for the laboratory
2. The appropriate characteristics for each tester strain have been confirmed, eg rfa cellwall mutation and pKM101 plasmid R-factor etc.
3. All tester strain cultures should be in the range of 1 to 9.9 x 109 bacteria per ml.
4. Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9 mix.
4. There should be a minimum of four non-toxic test material dose levels.
5. There should be no evidence of excessive contamination.
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material 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 a definitive judgement about the test material activity. Results of this type will be reported as equivocal. - Statistics:
- Standard deviation only
Results and discussion
Test resultsopen allclose all
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- From 500 ug/plate absence of S9 and from 1500 ug/plate with S9
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- From 500 ug/plate absence of S9 and from 1500 ug/plate with S9
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- From 500 ug/plate absence of S9 and from 1500 ug/plate with S9
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- From 500 ug/plate absence of S9 and from 1500 ug/plate with S9
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
Any other information on results incl. tables
Preliminary Toxicity test
| S9 | Strain | Dose ug/plate | ||||||||||
| 0 | 0.15 | 0.5 | 1.5 | 5 | 15 | 50 | 150 | 500 | 1500 | 5000 | ||
| - | TA100 | 93 | 115 | 120 | 110 | 109 | 134 | 118 | 108 | 140 | 129 | 154 |
| + | TA100 | 99 | 70 | 91 | 89 | 87 | 151 | 144 | 130 | 115 | 154 | 135 |
| - | WP2uvrA- | 42 | 31 | 23 | 27 | 34 | 23 | 37 | 29 | 34 | 19 | 30 |
| + | WP2uvrA- | 42 | 51 | 48 | 41 | 40 | 44 | 49 | 43 | 44 | 36 | 23 |
Experiment 1
| Experiment | One | ||||||||||
| S9 Mix | Concn (ug/plate) |
Strain | |||||||||
| TA100 | TA1535 | WP2uvrA | TA98 | TA1537 | |||||||
| Count | Mean (SD) | Count | Mean (SD) | Count | Mean (SD) | Count | Mean (SD) | Count | Mean (SD) | ||
| - | 0 | 126 | 111 (13.1) |
11 | 14 (3.8) |
47 | 45 (3.8) |
20 | 20 (1.5) |
12 | 14 (4.4) |
| 107 | 18 | 41 | 18 | 11 | |||||||
| 101 | 12 | 48 | 21 | 19 | |||||||
| - | 5 | 115 | 103 (10.7) |
18 | 17 (2.6) |
NT | NT | 27 | 22 (5.5) |
7 | 10 (3.6) |
| 94 | 14 | 16 | 9 | ||||||||
| 101 | 19 | 22 | 14 | ||||||||
| - | 15 | 110 | 104 (7.8) |
10 | 12 (1.5) |
NT | NT | 19 | 20 (1.5) |
11 | 10 (1) |
| 106 | 13 | 20 | 10 | ||||||||
| 95 | 12 | 22 | 9 | ||||||||
| - | 50 | 87 | 97 (21.5) |
11 | 10 (1.5) |
42 | 44 (2) |
21 | 21 (3.5) |
7 | 10 (4.6) |
| 83 | 8 | 46 | 25 | 7 | |||||||
| 122 | 10 | 44 | 18 | 15 | |||||||
| - | 150 | 75 | 94 (16.8) |
12 | 14 (2.1) |
45 | 43 (4) |
19 | 24 (4.2) |
7 | 8 (1) |
| 107 | 13 | 45 | 25 | 8 | |||||||
| 100 | 16 | 38 | 27 | 9 | |||||||
| - | 500 | 90 S | 85 (5) |
5 S | 7 (2.5) |
42 | 47 (4.6) |
17 S | 17 (4.5) |
4 S | 4 (2.5) |
| 86 S | 10 S | 51 | 21 S | 2 S | |||||||
| 80 S | 7 S | 48 | 12 S | 7 S | |||||||
| - | 1500 | 54 S | 47 (5.9) |
7 S | 11 (3.6) |
40 | 42 (4) |
3 S | 2 (1) |
1 V | 2 (1.2) |
| 45 S | 14 S | 47 | 1 S | 3 V | |||||||
| 43 S | 12 S | 40 | 2 S | 1 V | |||||||
| - | 5000 | 27 S | 38 (9.9) |
6 V | 7 (2.1) |
41 | 41 (3) |
4 S | 4 (1) |
0 V | 0 (0) |
| 45 S | 9 V | 44 | 3 S | 0 V | |||||||
| 43 S | 5 V | 38 | 5 S | 0 V | |||||||
| - | Name | ENNG | ENNG | ENNG | 4NQO | 9AA | |||||
| Concn (ug/plate) |
3 | 5 | 2 | 0.2 | 80 | ||||||
| Results | 553 | 469 (73.9) |
198 | 190 (14.7) |
285 | 280 (9.5) |
152 | 154 |
1154 | 1078 (66.1) |
|
| 438 | 173 | 286 | 140 | 1038 | |||||||
| 415 | 199 | 269 | 169 | 1041 | |||||||
Where S = Sparse bacterial background lawn, V= Very sparse bacterial background lawn
| Experiment | One | ||||||||||
| S9 Mix | Concn (ug/plate) |
Strain | |||||||||
| TA100 | TA1535 | WP2uvrA | TA98 | TA1537 | |||||||
| Count | Mean (SD) | Count | Mean (SD) | Count | Mean (SD) | Count | Mean (SD) | Count | Mean (SD) | ||
| + | 0 | 97 | 99 (7.6) |
9 | 13 (3.8) |
48 | 48 (3.5) |
29 | 32 (3.6) |
15 | 12 (3.1) |
| 92 | 15 | 44 | 36 | 11 | |||||||
| 107 | 16 | 51 | 31 | 9 | |||||||
| + | 5 | NT | NT | NT | NT | NT | NT | NT | NT | 12 | 13 (3.1) |
| 16 | |||||||||||
| 10 | |||||||||||
| + | 15 | NT | NT | NT | NT | NT | NT | NT | NT | 12 | 13 (4.6) |
| 18 | |||||||||||
| 9 | |||||||||||
| + | 50 | 108 | 95 (13) |
13 | 12 (1.2) |
35 | 42 (6.2) |
31 | 31 (4.5) |
12 | 11 (1.2) |
| 96 | 11 | 47 | 27 | 10 | |||||||
| 82 | 13 | 44 | 36 | 10 | |||||||
| + | 150 | 89 | 96 (6.5) |
13 | 13 (1.5) |
46 | 49 |
31 | 30 (3.1) |
12 | 11 (3.2) |
| 96 | 15 | 49 | 27 | 13 | |||||||
| 102 | 12 | 51 | 36 | 7 | |||||||
| + | 500 | 93 | 86 (6.6) |
12 | 13 (1.2) |
59 | 47 (11.1) |
41 | 37 (4.6) |
9 | 11 (2.1) |
| 85 | 14 | 37 | 38 | 13 | |||||||
| 80 | 12 | 45 | 32 | 12 | |||||||
| + | 1500 | 55 | 67 (10.6) |
16 | 13 (2.5) |
43 | 42 (4.6) |
31 | 25 (5.3) |
3 S | 3 (1) |
| 75 | 11 | 37 | 23 | 4 S | |||||||
| 71 | 13 | 46 | 21 | 2 S | |||||||
| + | 5000 | 40 S | 40 (11.5) |
8 S | 10 (3.8) |
48 | 48 (3) |
11 S | 8 (4.2) |
3 S | 3 (1.5) |
| 52 S | 7 S | 51 | 9 S | 1 S | |||||||
| 29 S | 14 S | 45 | 3 S | 4 S | |||||||
| - | Name | 2AA | 2AA | 2AA | BP | 2AA | |||||
| Concn (ug/plate) |
1 | 2 | 10 | 5 | 2 | ||||||
| Results | 1656 | 1207 (431.7) |
236 | 236 (13) |
223 | 235 (13.9) |
229 | 238 (27.6) |
246 | 246 (9.5) |
|
| 1169 | 223 | 250 | 216 | 236 | |||||||
| 795 | 249 | 231 | 269 | 255 | |||||||
Where S = Sparse bacterial background lawn
Experiment 2
| Experiment | Two | ||||||||||
| S9 Mix | Concn (ug/plate) |
Strain | |||||||||
| TA100 | TA1535 | WP2uvrA | TA98 | TA1537 | |||||||
| Count | Mean (SD) | Count | Mean (SD) | Count | Mean (SD) | Count | Mean (SD) | Count | Mean (SD) | ||
| - | 0 | 130 | 117 (11.6) |
19 | 17 (3.5) |
32 | 31 (2.1) |
18 | 21 (3.1) |
11 | 14 (3.8) |
| 109 | 13 | 29 | 22 | 12 | |||||||
| 111 | 19 | 33 | 24 | 18 | |||||||
| - | 1.5 | 128 | 117 (13.6) |
19 | 18 (1.7) |
NT | NT | 18 | 18 (0.6) |
12 | 13 (2.6) |
| 102 | 16 | 18 | 16 | ||||||||
| 122 | 19 | 19 | 11 | ||||||||
| - | 5 | 107 | 114 (7) |
21 | 15 (6) |
NT | NT | 20 | 21 (1) |
15 | 13 |
| 114 | 9 | 22 | 13 | ||||||||
| 121 | 16 | 21 | 10 | ||||||||
| - | 15 | 111 | 101 (8.5) |
14 | 17 (3.1) |
36 | 33 (5.2) |
21 | 21 (4.5) |
12 | 14 (1.5) |
| 98 | 16 | 27 | 25 | 14 | |||||||
| 95 | 20 | 36 | 16 | 15 | |||||||
| - | 50 | 95 | 88 (5.9) |
16 | 18 (2.9) |
29 | 29 (2.5) |
20 | 21 (1) |
9 | 12 (2.6) |
| 84 | 16 | 31 | 21 | 13 | |||||||
| 86 | 21 | 26 | 22 | 14 | |||||||
| - | 150 | 101 | 109 (7.2) |
18 | 17 (3.6) |
31 | 30 (2.3) |
20 | 19 (3.6) |
9 | 11 (2.5) |
| 114 | 13 | 27 | 15 | 11 | |||||||
| 113 | 20 | 31 | 22 | 14 | |||||||
| - | 500 | 98 S | 99 (0.6) |
16 | 15 (1.2) |
24 | 26 (1.7) |
20 | 16 (3.5) |
8 S | 6 (1.7) |
| 99 S | 14 | 27 | 16 | 5 S | |||||||
| 99 S | 16 | 27 | 13 | 5 S | |||||||
| - | 1500 | 47 S | 55 (9.1) |
4 S | 4 (0.6) |
23 | 26 (2.1) |
4 S | 5 |
2 S | 3 (2.1) |
| 54 S | 5 S | 27 | 6 S | 5 S | |||||||
| 65 S | 4 S | 29 | 4 S | 1 S | |||||||
| - | 5000 | NT | NT | NT | NT | 29 | 33 (4) |
NT | NT | NT | NT |
| 37 | |||||||||||
| 32 | |||||||||||
| - | Name | ENNG | ENNG | ENNG | 4NQO | 9AA | |||||
| Concn (ug/plate) |
3 | 5 | 2 | 0.2 | 80 | ||||||
| Results | 503 | 497 (13.7) |
64 | 162 (85.3) |
262 | 283 (22.6) |
114 | 116 (5.3) |
2297 | 2530 (315.3) |
|
| 481 | 216 | 280 | 122 | 3889 | |||||||
| 506 | 207 | 307 | 112 | 2405 | |||||||
Where S = Sparse bacterial background lawn
| Experiment | Two | ||||||||||
| S9 Mix | Concn (ug/plate) |
Strain | |||||||||
| TA100 | TA1535 | WP2uvrA | TA98 | TA1537 | |||||||
| Count | Mean (SD) | Count | Mean (SD) | Count | Mean (SD) | Count | Mean (SD) | Count | Mean (SD) | ||
| + | 0 | 117 | 109 (6.8) |
18 | 18 (0.6) |
36 | 36 (4) |
26 | 27 (1.7) |
13 | 12 (2.1) |
| 107 | 19 | 32 | 26 | 10 | |||||||
| 104 | 18 | 40 | 29 | 14 | |||||||
| + | 1.5 | NT | NT | NT | NT | NT | NT | NT | NT | 14 | 14 (0.6) |
| 14 | |||||||||||
| 15 | |||||||||||
| + | 5 | 114 | 102 (12) |
18 | 16 (2.1) |
NT | NT | 31 | 27 (3.6) |
12 | 12 (0.6) |
| 90 | 15 | 24 | 12 | ||||||||
| 102 | 14 | 26 | 13 | ||||||||
| + | 15 | 118 | 112 (5.1) |
15 | 14 (1.2) |
38 | 39 (2.1) |
28 | 25 (3.5) |
11 | 12 (2.6) |
| 111 | 13 | 37 | 21 | 10 | |||||||
| 108 | 15 | 41 | 25 | 15 | |||||||
| + | 50 | 103 | 112 (10.7) |
21 | 19 (1.5) |
23 | 34 (10.2) |
25 | 25 (2) |
11 | 12 (1.5) |
| 124 | 19 | 36 | 27 | 14 | |||||||
| 110 | 18 | 43 | 23 | 12 | |||||||
| + | 150 | 125 | 112 (11.4) |
21 | 18 (3.1) |
38 | 32 (5.6) |
30 | 27 (3.5) |
12 | 12 (1.5) |
| 107 | 15 | 31 | 23 | 11 | |||||||
| 104 | 19 | 27 | 27 | 14 | |||||||
| + | 500 | 99 | 112 (12.1) |
14 | 13 (2.6) |
37 | 35 (3.2) |
23 | 25 (4.4) |
14 | 13 (1.2) |
| 123 | 15 | 31 | 22 | 12 | |||||||
| 113 | 10 | 36 | 30 | 12 | |||||||
| + | 1500 | 97 S | 108 (9.8) |
14 | 18 (4) |
42 | 36 (5.5) |
14 | 15 (2.1) |
5 S | 8 (3) |
| 114 S | 18 | 33 | 17 | 8 S | |||||||
| 114 S | 22 | 32 | 13 | 11 S | |||||||
| + | 5000 | 57 S | 54 (3) |
13 S | 12 (0.6) |
31 | 32 (1.5) |
4 S | 4 (1.5) |
NT | NT |
| 54 S | 12 S | 32 | 6 S | ||||||||
| 51 S | 12 S | 34 | 3 S | ||||||||
| + | Name | 2AA | 2AA | 2AA | BP | 2AA | |||||
| Concn (ug/plate) |
1 | 2 | 10 | 5 | 2 | ||||||
| Results | 629 | 801 (157.7) |
288 | 292 (15.4) |
143 | 130 (11.9) |
180 | 189 (18.8) |
100 | 95 (5.5) |
|
| 939 | 309 | 120 | 177 | 89 | |||||||
| 834 | 279 | 126 | 211 | 95 | |||||||
Where S = Sparse bacterial background lawn
Note History profile for vehichle (Distilled water) and positive controls for 2008 and 2009 are available and indicate that the results reported here for these controls are within historic norms for the laboratory.
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results:
negative with metabolic activation
negative without metabolic activation
The test material (PR-4758) was considered non-mutagenic under the conditions of the assay. The sensitivity of the assay and the efficacy of the S9-mix were validated as the vehicle (sterile distilled water) 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. - Executive summary:
At the request of Nalco Limited UK, Harlan laboratories performed a Bacterial reverse mutation assay on PR-4758. The study was conducted in accordance with Good laboratory practise.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 harmonized guidelines.
Salmonella typhimuriumstrains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using both the Ames plate incorporation and pre-incubation methods at up to 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 ranged between 5 and 5000 μg/plate, depending on bacterial strain type and presence or absence of S9-mix.
The experiment was repeated on a separate day (pre-incubation method) using a similar dose range to Experiment 1, fresh cultures of the bacterial strains and fresh test material 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 material.
The vehicle (sterile distilled water) 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 material caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains, initially from 500 and 1500 μg/plate, in the absence and presence of S9-mix, respectively. No toxicity was noted for Escherichia coli strain WP2uvrA- at any test material dose level. The sensitivity of the tester strains to the toxicity of the test material varied slightly between strain type, exposures with or without S9-mix and experiment number. The test material was, therefore, either 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 material 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 material, either with or without metabolic activation or exposure method. PR-4758 was considered non-mutagenic under the conditions of the assay.
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