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Registration Dossier
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EC number: 256-974-4 | CAS number: 51115-67-4
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 7 August 1986 till 8 September 1986
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Remarks:
- OECD TG 471
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 1 986
- Report date:
- 1986
Materials and methods
Test guideline
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- with and without pre-incubation
- Deviations:
- not specified
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- 2-isopropyl-N,2,3-trimethylbutyramide
- EC Number:
- 256-974-4
- EC Name:
- 2-isopropyl-N,2,3-trimethylbutyramide
- Cas Number:
- 51115-67-4
- Molecular formula:
- C10H21NO
- IUPAC Name:
- 2-isopropyl-N,2,3-trimethylbutyramide
- Test material form:
- solid: particulate/powder
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source: PPF/U.K.
- Sample Number: S15100-T01
- Test item: N 2,3-Trimethyl-2-isopropyl butanamide (WS23),
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Stock solutions of DMSO - 50 mg/ml.
Method
- Target gene:
- TA 1535 hisG46, rfa-, uvrB-
TA 1537 hisC3076, rfa-, uvrB-
TA 1538 hisD3052, rfa-, uvrB-, pKM101
TA 100 hisG46, rfa-, uvrB-, pKM101
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- other: histidine-dependent bacteria strains
- Species / strain / cell type:
- S. typhimurium TA 1538
- Additional strain / cell type characteristics:
- other: histidine-dependent bacteria strains
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 derived from Aroclor-induced rat liver
- Test concentrations with justification for top dose:
- Number of dose levels and concentration (mg/plate): 1.58, 2.5, 5, 7,5 and 10 (concentrations spaced at log10 intervals).
The concentrations of WS-23, which were used in the mutation assay were determined from a dose range finding toxicity assay using 5 strains of S. typhimurium. The toxicity assay was performed with 5 concentrations of WS-23 spaced at log10 intervals. - Vehicle / solvent:
- Vehicle: DMSO.
WS23 was found to be readily soluble in DMSO.
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- other: 2-aminoanthracene
- Remarks:
- Positive controls: 2-aminoanthracene - stock solution in DMSO Sodium azide - stock solutions in distilled water and filter sterilised 2-nitrofluorene - stock solution in DMSO 9-aminoacridine - stock solution in DMSO
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
Bacteria, test material and metabolising system were incorporated into the top agar of the Petri dishes. The strains were tested routinely for sensitivity to crystal violet, ultraviolet light and, where applicable, resistance to ampicillin.
Negative controls were treated only with the solvent - DMSO.
The petri dishes were incubated for 2 to 3 days, as appropriate, and the number of revertants was determined for each treatment.
Cultures of bacteria of known concentrations were diluted using PBS to about 5x10E3 cells/ml. 0.1 ml of diluted bacteria was added to 2 ml molten top agar followed by 0.1 ml test solution or solvent and 0.5 ml S9 mix or cofactor mix. The top agar was allowed to solidify at room temperature before inversion of the plates for incubation at 37 degrees C. Three plates were prepared for each concentration tested.
After 2-3 days, the plates were removed from the incubator and the number of colonies on each plate were determined. - Rationale for test conditions:
- Certain auxotrophic histidine requiring strains of Salmonella exist which readily undergo reversion to histidine independence under the action of chemical mutagens. The test consists of treating histidine-dependent bacteria with the test material plating out on selective medium (i.e. medium not containing histidine), incubating for 2-3 days and finally counting the mutant colonies.
- Evaluation criteria:
- The average number of mutant colonies per plate is compared with the average number of spontaneous revertants in the control. A dose-related increase in the number of colonies which reaches at least a doubling of the control values is usually considered to be a positive response.
- Statistics:
- Thee number of bacterial colonies on each plate were determined using Artek 880 automatic colony counter. The number of colonies of bacteria which grew following each treatment is expressed as a percentage of the number of colonies which grew on control plates. The mean number of colonies and standard deviation for each set of plates were calculated, as were the percentage survival including 95% confidence limits.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 7,5 and 10 mg/plate with metabolic activation
- Vehicle controls validity:
- valid
- Remarks:
- DMSO
- Untreated negative controls validity:
- other: untreated cells
- Positive controls validity:
- valid
- Remarks:
- 2-aminoanthracene, sodium azide
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 10 mg/plate without and 7,5 and 10 mg/plate with metabolic activation
- Vehicle controls validity:
- valid
- Remarks:
- DMSO
- Untreated negative controls validity:
- other: untreated cells
- Positive controls validity:
- valid
- Remarks:
- 2-aminoanthracene, 9-aminoacridine
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 7,5 and 10 mg/plate with metabolic activation
- Vehicle controls validity:
- valid
- Remarks:
- DMSO
- Untreated negative controls validity:
- other: untreated cells
- Positive controls validity:
- valid
- Remarks:
- 2-aminoanthracene, sodium azide
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 7,5 and 10 mg/plate with metabolic activation
- Vehicle controls validity:
- valid
- Remarks:
- DMSO
- Untreated negative controls validity:
- other: untreated cells
- Positive controls validity:
- valid
- Remarks:
- 2-aminoanthracene, 2-nitrofluorene
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 7,5 and 10 mg/plate with metabolic activation
- Vehicle controls validity:
- valid
- Remarks:
- DMSO
- Untreated negative controls validity:
- other: untreated cells
- Positive controls validity:
- valid
- Remarks:
- 2-aminoanthracene, 2-nitrofluorene
- Remarks on result:
- other: negative
Any other information on results incl. tables
Table 1: Mutagenicity Assay Results Test 1 – TA1537 strain |
||||||
Strain |
+/- pre-incubation |
Dose level (µg/ml) |
% S9 |
Mean Revertant Colony Counts |
S.D. |
Individual Revertant Colony Counts |
TA1537 |
- |
0 |
5 |
2.7 |
1.5 |
3, 4, 1 |
1580 |
7.7 |
1.5 |
8, 9, 6 * |
|||
2500 |
8.5 |
0.7 |
C, 9, 8 * |
|||
5000 |
6 |
2.6 |
7, 3, 8 * |
|||
7500 |
4 |
1.7 |
2, 5, 5 |
|||
10000 |
3.7 |
2.1 |
3, 2, 6 |
|||
- |
0 |
10 |
8.3 |
4 |
4, 9, 12 |
|
1580 |
7.3 |
0.6 |
8, 7, 7 |
|||
2500 |
5.7 |
1.2 |
7, 5, 5 |
|||
5000 |
5.7 |
0.6 |
6, 6, 5 |
|||
7500 |
5.7 |
2.5 |
3, 6, 8 |
|||
10000 |
4.3 |
2.9 |
6, 6, 1 |
|||
- |
0 |
20 |
4 |
0 |
4, 4, 4 |
|
1580 |
6 |
3.5 |
2, 8, 8 |
|||
2500 |
3.3 |
1.2 |
2, 4, 4 |
|||
5000 |
3.3 |
1.5 |
2, 3, 5 |
|||
7500 |
4 |
2.6 |
3, 2, 7 |
|||
10000 |
1.7 |
1.2 |
1, 3, 1 |
|||
Untreated |
0 |
9.7 |
4.9 |
12, 4, 13 |
||
+ |
0 |
5 |
4.3 |
2.9 |
1, 6, 6 |
|
1580 |
5.3 |
3.5 |
5, 9, 2 |
|||
2500 |
6.3 |
2.1 |
8, 4, 7 |
|||
5000 |
5.3 |
2.9 |
2, 7, 7 |
|||
7500 |
1.7 |
1.2 |
1, 1, 3 |
|||
10000 |
2.7 |
1.5 |
3, 1, 4 |
|||
+ |
0 |
10 |
6.3 |
0.6 |
6, 6, 7 |
|
1580 |
6 |
3 |
6, 9, 3 |
|||
2500 |
3.3 |
2.5 |
6, 3, 1 |
|||
5000 |
4 |
1 |
5, 4, 3 |
|||
7500 |
0.3 T |
0.6 |
0, 0, 1 |
|||
10000 |
0.3 T |
0.6 |
1, 0, 0 |
|||
+ |
0 |
20 |
6 |
2.6 |
4, 9, 5 |
|
1580 |
4.7 |
1.5 |
5, 6, 3 |
|||
2500 |
5.7 |
1.5 |
4, 6, 7 |
|||
5000 |
1 T |
1 |
2, 1, 0 |
|||
7500 |
0 T |
|
|
|||
10000 |
0 |
|
|
|||
Untreated |
0 |
6.7 |
1.5 |
5, 7, 8 |
||
Positive control |
+/- pre-incubation |
Concentration (µg/plate) |
% S9 |
Mean Revertant Colony Counts |
S.D. |
Individual Revertant Colony Counts |
DMSO |
-
|
|
10 |
8.3 |
4 |
4, 9, 12 |
2AA |
2 |
|
554 |
56 |
497, 556, 609 |
|
DMSO |
+ |
|
10 |
6.3 |
0.6 |
6, 6, 7 |
2AA |
2 |
|
446.3 |
21.6 |
431, 437, 471 |
|
* = doubling of negative control values S.D. = standard deviation T = toxic dose level |
+ = with pre-incubation - = without pre-incubation 2AA = 2-aminoanthracene administered with DMSO as a vehicle |
|||||
Table 2: Mutagenicity Assay Results Test 1 – TA1538 Strain |
||||||
Strain |
+/- pre-incubation |
Dose level (µg/ml) |
% S9 |
Mean Revertant Colony Counts |
S.D. |
Individual Revertant Colony Counts |
TA1538 |
- |
0 |
5 |
19.3 |
6.5 |
24, 28, 16 |
1580 |
19.7 |
5 |
24, 17, 19 |
|||
2500 |
16.3 |
2.1 |
28, 19, 16 |
|||
5000 |
19.7 |
5.7 |
23, 16, 25 |
|||
7500 |
16 |
2.6 |
25, 24, ND |
|||
10000 |
12.7 |
6.4 |
23, 26, 16 |
|||
- |
0 |
10 |
22.7 |
6.1 |
24, 28, 16 |
|
1580 |
20 |
3.6 |
24, 17, 19 |
|||
2500 |
21 |
6.2 |
28, 19, 16 |
|||
5000 |
21.3 |
4.7 |
23, 16, 25 |
|||
7500 |
24.5 |
0.7 |
25, 24, ND |
|||
10000 |
21.7 |
5.1 |
23, 26, 16 |
|||
- |
0 |
20 |
23.7 |
5.7 |
19, 22, 30 |
|
1580 |
|
24 |
5.8 |
C, 22, 26 |
||
2500 |
|
19 |
3 |
22, 16, 19 |
||
5000 |
|
21 |
2.6 |
20, 19, 24 |
||
7500 |
|
22.7 |
5.1 |
27, 17, 24 |
||
10000 |
|
21.3 |
2.1 |
23, 19, 22 |
||
Untreated |
0 |
11.3 |
2.1 |
12, 13, 9 |
||
+ |
0 |
5 |
16 |
1 |
15, 17, 16 |
|
1580 |
24.7 |
2.1 |
23, 27, 24 |
|||
2500 |
20 |
4.6 |
25, 16, 19 |
|||
5000 |
13 |
2.6 |
16, 11, 12 |
|||
7500 |
6 T |
6.9 |
14, 2, 2 |
|||
10000 |
0 T |
|
|
|||
+ |
0 |
10 |
17.3 |
4.5 |
13, 17, 22 |
|
1580 |
|
21.7 |
5.5 |
16, 27, 22 |
||
2500 |
|
24 |
9 |
33, 24, 15 |
||
5000 |
|
10 |
1.7 |
9, 9, 12 |
||
7500 |
|
0 T |
|
|
||
10000 |
|
0.3 T |
0.6 |
0, 0, 1 |
||
+ |
0 |
20 |
21.3 |
302 |
25, 19, 20 |
|
1580 |
|
21.3 |
5 |
16, 26, 22 |
||
2500 |
|
24.7 |
308 |
22, 23, 29 |
||
5000 |
|
18 |
5.3 |
24, 14, 16 |
||
7500 |
|
12.3 T |
5.1 |
11, 8, 18 |
||
10000 |
|
0 T |
|
|
||
Untreated |
0 |
19.3 |
5.1 |
15, 18, 25 |
||
Positive control |
+/- pre-incubation |
Concentration (µg/plate) |
% S9 |
Mean Revertant Colony Counts |
S.D. |
Individual Revertant Colony Counts |
DMSO |
- |
|
10 |
22.7 |
6.1 |
24, 28, 16 |
2AA |
|
2 |
|
1785.3 |
33.8 |
1750, 1817, 1789 |
DMSO |
+ |
|
10 |
17.3 |
4.5 |
13, 17, 22 |
2AA |
|
2 |
|
642 |
30.8 |
607, 665, 654 |
S.D. = standard deviation T = toxic dose level |
+ = with pre-incubation - = without pre-incubation |
|||||
2AA = 2-aminoanthracene administered with DMSO as a vehicle |
||||||
Table 3: Mutagenicity Assay Results Test 1 – TA98 strain |
||||||
Strain |
+/- pre-incubation |
Dose level (µg/ml) |
% S9 |
Mean Revertant Colony Counts |
S.D. |
Individual Revertant Colony Counts |
TA98 |
- |
0 |
5 |
33 |
4.4 |
38, 30, 31 |
1580 |
27.7 |
7.1 |
20, 34, 29 |
|||
2500 |
26 |
10.1 |
15, 38, 35 |
|||
5000 |
26.7 |
11 |
26, 38, 16 |
|||
7500 |
27.7 |
6.4 |
35, 25, 23 |
|||
10000 |
24.3 |
2.5 |
27, 22, 24 |
|||
|
- |
0 |
10 |
38.7 |
3.1 |
38, 42, 36 |
1580 |
27.7 |
3.2 |
24, 30, 29 |
|||
2500 |
34.3 |
6.7 |
31, 42, 30 |
|||
5000 |
28 |
3.5 |
30, 24, 30 |
|||
7500 |
29.3 |
9 |
38, 30, 20 |
|||
10000 |
29 |
5.6 |
28, 35, 24 |
|||
|
- |
0 |
20 |
31 |
3 |
34, 28, 31 |
1580 |
33 |
4 |
33, 37, 29 |
|||
2500 |
32 |
4.6 |
33, 27, 36 |
|||
5000 |
28.7 |
7.6 |
27, 37, 22 |
|||
7500 |
28.3 |
5.5 |
34, 28, 23 |
|||
10000 |
36 |
0 |
36, 36, 36 |
|||
Untreated |
0 |
34.3 |
6.4 |
39, 27, 37 |
||
|
+ |
0 |
5 |
32 |
4.4 |
34, 35, 27 |
1580 |
29.3 |
7.8 |
27, 38, 23 |
|||
2500 |
29.7 |
3.5 |
26, 33, 30 |
|||
5000 |
26 |
7 |
18, 29, 31 |
|||
7500 |
18.3 |
2.1 |
16, 20, 19 |
|||
10000 |
15.3 |
5.7 |
9, 20, 17 |
|||
|
+ |
0 |
10 |
33 |
5.6 |
34, 27, 38 |
1580 |
31 |
3.5 |
29, 29, 35 |
|||
2500 |
31.7 |
4 |
28, 31, 36 |
|||
5000 |
25 |
2.6 |
28, 23, 24 |
|||
7500 |
5 T |
5 |
5, 0, 10 |
|||
10000 |
5.7 T |
9 |
16, 1, 0 |
|||
|
+ |
0 |
20 |
37.7 |
2.5 |
35, 40, 38 |
1580 |
36.5 |
2.1 |
38, 35, ND |
|||
2500 |
35.3 |
2.3 |
34, 38, 34 |
|||
5000 |
25 |
2.6 |
27, 22, 26 |
|||
7500 |
6.3 T |
7.6 |
3, 1, 15 |
|||
10000 |
0.7 T |
1.2 |
0, 2, 0 |
|||
Untreated |
0 |
29.7 |
13.3 |
45, 22, 22 |
||
Positive control |
+/- pre-incubation |
Concentration (µg/plate) |
% S9 |
Mean Revertant Colony Counts |
S.D. |
Individual Revertant Colony Counts |
DMSO |
- |
|
10 |
38.7 |
3.1 |
38, 42, 36 |
2AA |
|
2 |
|
1986.3 |
179.6 |
2096, 2084, 1779 |
DMSO |
+ |
|
10 |
33 |
5.6 |
34, 27, 38 |
2AA |
|
2 |
|
2101 |
178.3 |
2285, 2089, 1929 |
S.D. = standard deviation T = toxic dose level |
S.D. = standard deviation T = toxic dose level |
|||||
2-AA = 2-aminoanthracene administered with DMSO as a vehicle |
||||||
|
||||||
Table 4: Mutagenicity Assay Results Test 1 – TA1535 strain |
||||||
Strain |
+/- pre-incubation |
Dose level (µg/ml) |
% S9 |
Mean Revertant Colony Counts |
S.D. |
Individual Revertant Colony Counts |
TA1535 |
- |
0 |
5 |
9 |
4 |
9, 5, 11 |
1580 |
6.7 |
3.8 |
5, 11, 4 |
|||
2500 |
12.7 |
1.5 |
13, 14, 11 |
|||
5000 |
7 |
2.6 |
4, 9, 8 |
|||
7500 |
7.7 |
4.6 |
5, 5, 13 |
|||
10000 |
6.3 |
1.5 |
5, 6, 8 |
|||
- |
0 |
10 |
8 |
2.6 |
6, 7, 11 |
|
1580 |
7 |
2 |
9, 7, 5 |
|||
2500 |
8 |
3 |
11, 5, 8 |
|||
5000 |
9.7 |
1.2 |
9, 9, 11 |
|||
7500 |
6.7 |
3.8 |
4, 5, 11 |
|||
10000 |
3.7 |
1.2 |
5, 3, 3 |
|||
- |
0 |
20 |
10.7 |
1.5 |
9, 12, 11 |
|
1580 |
4.7 |
2.1 |
7, 3, 4 |
|||
2500 |
5 |
1.7 |
6, 3, 6 |
|||
5000 |
7.3 |
3.2 |
6, 11, 5 |
|||
7500 |
6.3 |
4.9 |
4, 12, 3 |
|||
10000 |
8 |
5.3 |
4, 14, 6 |
|||
Untreated |
0 |
6.3 |
2.5 |
6, 4, 9 |
||
+ |
0 |
5 |
7.3 |
1.2 |
8, 8, 6 |
|
1580 |
9.3 |
3.8 |
5, 12, 11 |
|||
2500 |
7.3 |
2.1 |
9, 8, 5 |
|||
5000 |
8.3 |
3.2 |
12, 7, 6 |
|||
7500 |
3 T |
1.7 |
1, 4, 4 |
|||
10000 |
3.3 T |
1.5 |
2, 3, 5 |
|||
+ |
0 |
10 |
10.3 |
2.1 |
8, 11, 12 |
|
1580 |
13.3 |
2.5 |
13, 11, 16 |
|||
2500 |
8 |
1 |
9, 7, 8 |
|||
5000 |
5.3 T |
1.5 |
7, 4, 5 |
|||
7500 |
3.7 T |
3.8 |
8, 1, 2 |
|||
10000 |
2.3 T |
1.5 |
4, 2, 1 |
|||
+ |
0 |
20 |
6.7 |
0.6 |
6, 7, 7 |
|
1580 |
8.7 |
3.8 |
7, 6, 13 |
|||
2500 |
6 |
1 |
7, 6, 5 |
|||
5000 |
8 |
3.6 |
12, 5, 7 |
|||
7500 |
2 T |
3.5 |
0, 6, 0 |
|||
10000 |
3.3 T |
0.6 |
3, 4, 3 |
|||
Untreated |
0 |
3.7 |
2.1 |
2, 3, 6 |
||
Positive control |
+/- pre-incubation |
Concentration (µg/plate) |
% S9 |
Mean Revertant Colony Counts |
S.D. |
Individual Revertant Colony Counts |
DMSO |
- |
|
10 |
38.7 |
3.1 |
38, 42, 36 |
2AA |
|
2 |
|
1986.3 |
179.6 |
2096, 2084, 1779 |
DMSO |
+ |
|
10 |
33 |
5.6 |
34, 27, 38 |
2AA |
|
2 |
|
2101 |
178.3 |
2285, 2089, 1929 |
S.D. = standard deviation T = toxic dose level |
S.D. = standard deviation T = toxic dose level |
|||||
2-AA = 2-aminoanthracene administered with DMSO as a vehicle |
||||||
Table 5: Mutagenicity Assay Results Test 1 – TA100 strain |
||||||
Strain |
+/- pre-incubation |
Dose level (µg/ml) |
% S9 |
Mean Revertant Colony Counts |
S.D. |
Individual Revertant Colony Counts |
TA100 |
- |
0 |
5 |
105.3 |
15.5 |
94, 123, 99 |
1580 |
98 |
17.3 |
108, 108, 78 |
|||
2500 |
94.3 |
20.5 |
118, 82, 83 |
|||
5000 |
83.7 |
1.1 |
85, 83, 83 |
|||
7500 |
90 |
2.6 |
89, 88, 93 |
|||
10000 |
82.7 |
12.6 |
96, 71, 81 |
|||
|
- |
0 |
10 |
105.3 |
15.7 |
100, 93, 123 |
1580 |
114.3 |
13.2 |
117, 100, 126 |
|||
2500 |
121.7 |
6.7 |
120, 116, 129 |
|||
5000 |
103 |
3 |
106, 100, 103 |
|||
7500 |
107.7 |
29.4 |
121, 128, 74 |
|||
10000 |
101.3 |
4 |
99, 106, 99 |
|||
|
- |
0 |
20 |
122.3 |
4.2 |
119, 121, 127 |
1580 |
120.3 |
18 |
139, 119, 103 |
|||
2500 |
103.7 |
4 |
108, 103, 100 |
|||
5000 |
102.3 |
14.5 |
119, 95, 93 |
|||
7500 |
111.3 |
10.4 |
108, 103, 123 |
|||
10000 |
127.7 |
13.4 |
143, 122, 118 |
|||
Untreated |
0 |
55.3 |
12.7 |
49, 47, 70 |
||
|
+ |
0 |
5 |
109.7 |
16.4 |
116, 91, 122 |
1580 |
126.7 |
4.2 |
128, 130, 122 |
|||
2500 |
127.3 |
2.9 |
129, 129, 124 |
|||
5000 |
103.3 |
2.1 |
101, 105, 104 |
|||
7500 |
83 |
10.6 |
95, 75, 79 |
|||
10000 |
0 T |
|
|
|||
|
+ |
0 |
10 |
117.3 |
20.1 |
123, 134, 95 |
1580 |
121 |
20.5 |
120, 101, 142 |
|||
2500 |
122.7 |
4.1 |
127, 119, 122 |
|||
5000 |
94.7 |
13.8 |
79, 105, 100 |
|||
7500 |
63.3 |
24.5 |
35, 78, 77 |
|||
10000 |
52 T |
20.3 |
70, 56, 30 |
|||
|
+ |
0 |
20 |
130 |
8.9 |
123, 140, 127 |
1580 |
123.7 |
9.9 |
117, 119, 135 |
|||
2500 |
114.3 |
10.8 |
119, 122, 102 |
|||
5000 |
94.7 |
5.5 |
95, 89, 100 |
|||
7500 |
76 |
14.8 |
69, 66, 93 |
|||
10000 |
0.3 T |
0.6 |
1, 0, 0 |
|||
Untreated |
0 |
115.7 |
8.5 |
122, 106, 119 |
||
Positive control |
+/- pre-incubation |
Concentration (µg/plate) |
% S9 |
Mean Revertant Colony Counts |
S.D. |
Individual Revertant Colony Counts |
DMSO |
-
|
|
10 |
38.7 |
3.1 |
38, 42, 36 |
2AA |
|
2 |
|
1986.3 |
179.6 |
2096, 2084, 1779 |
DMSO |
+ |
|
10 |
33 |
5.6 |
34, 27, 38 |
2AA |
|
2 |
|
2101 |
178.3 |
2285, 2089, 1929 |
S.D. = standard deviation T = toxic dose level |
S.D. = standard deviation T = toxic dose level |
|||||
2AA = 2-aminoanthracene administered with DMSO as a vehicle |
||||||
Table 6: Mutagenicity Assay Results Test 2 – TA1537 strain |
||||||
Strain |
+/- pre-incubation |
Dose level (µg/ml) |
% S9 |
Mean Revertant Colony Counts |
S.D. |
Individual Revertant Colony Counts |
TA1537 |
- |
0 |
5 |
6.3 |
2.5 |
9, 4, 6 |
1580 |
6 |
1 |
6, 5, 7 |
|||
2500 |
9.3 |
4.6 |
12, 4, 12 |
|||
5000 |
5 |
2 |
3, 7, 5 |
|||
7500 |
8 |
1 |
7, 9, 8 |
|||
10000 |
2 T |
1 |
3, 1, 2 |
|||
- |
0 |
10 |
7.7 |
1.2 |
7, 9, 7 |
|
1580 |
7 |
1 |
8, 7, 6 |
|||
2500 |
6.3 |
1.2 |
7, 7, 5 |
|||
5000 |
6 |
3.6 |
7, 2, 9 |
|||
7500 |
5.3 |
1.5 |
5, 7, 4 |
|||
10000 |
2.7 |
1.5 |
3, 1, 4 |
|||
- |
0 |
20 |
12 |
5.6 |
7, 11, 18 |
|
1580 |
9.3 |
3.2 |
8, 13, 7 |
|||
2500 |
5.7 |
1.2 |
5, 7, 5 |
|||
5000 |
7.7 |
1.5 |
8, 9, 6 |
|||
7500 |
6 |
2 |
8, 6, 4 |
|||
10000 |
6.7 |
1.5 |
7, 8, 5 |
|||
Untreated |
0 |
6.3 |
1.2 |
7, 5, 7 |
||
+ |
0 |
5 |
5.3 |
2.3 |
8, 4, 4 |
|
1580 |
7 |
1.7 |
5, 8, 8 |
|||
2500 |
8.3 |
2.5 |
11, 8, 6 |
|||
5000 |
6.3 |
2.9 |
3, 8, 8 |
|||
7500 |
1.3 T |
1.5 |
1, 3, 0 |
|||
10000 |
3.3 T |
3.2 |
1, 7, 2 |
|||
+ |
0 |
10 |
6.3 |
2.1 |
8, 4, 7 |
|
1580 |
4.3 |
1.2 |
5, 5, 3 |
|||
2500 |
5.3 |
2.9 |
2, 7, 7 |
|||
5000 |
7 |
1 |
6, 7, 8 |
|||
7500 |
4.7 |
1.5 |
6, 5, 3 |
|||
10000 |
2.3 T |
1.2 |
1, 3, 3 |
|||
+ |
0 |
20 |
10 |
4.6 |
5, 14, 11 |
|
1580 |
9.3 |
2.3 |
8, 8, 12 |
|||
2500 |
8.3 |
3.2 |
7, 12, 6 |
|||
5000 |
1 T |
1.7 |
3, 0, 0 |
|||
7500 |
3 T |
3 |
3, 0, 6 |
|||
10000 |
1.7 T |
1.2 |
3, 1, 1 |
|||
Untreated |
0 |
7.7 |
2.9 |
6, 6, 11 |
||
Positive control |
+/- pre-incubation |
Concentration (µg/plate) |
% S9 |
Mean Revertant Colony Counts |
S.D. |
Individual Revertant Colony Counts |
DMSO |
-
|
|
10 |
7.7 |
1.2 |
7, 9, 7 |
2AA |
2 |
|
499 |
18.1 |
480, 516, 501 |
|
DMSO |
+ |
|
10 |
6.3 |
2.1 |
8, 4, 7 |
2AA |
2 |
|
415 |
27.8 |
401, 447, 397 |
|
S.D. = standard deviation T = toxic dose level |
+ = with pre-incubation - = without pre-incubation |
|||||
2AA = 2-aminoanthracene administered with DMSO as a vehicle |
||||||
Table 7: Mutagenicity Assay Results Test 2 – TA1538 Strain |
||||||
Strain |
+/- pre-incubation |
Dose level (µg/ml) |
% S9 |
Mean Revertant Colony Counts |
S.D. |
Individual Revertant Colony Counts |
TA1538 |
- |
0 |
5 |
32.3 |
7.4 |
38, 35, 24 |
1580 |
21.7 |
1.5 |
23, 20, 22 |
|||
2500 |
28 |
2.6 |
29, 25, 30 |
|||
5000 |
27.3 |
7 |
28, 20, 34 |
|||
7500 |
24.3 |
4.7 |
26, 19, 28 |
|||
10000 |
26 |
9.5 |
25, 17, 36 |
|||
- |
0 |
10 |
28.3 |
12.1 |
17, 27, 41 |
|
1580 |
24 |
4.6 |
19, 25, 28 |
|||
2500 |
24.7 |
6.7 |
17, 28, 29 |
|||
5000 |
30 |
3 |
33, 27, 30 |
|||
7500 |
27.3 |
6.7 |
23, 35, 24 |
|||
10000 |
29.7 |
4.7 |
35, 26, 28 |
|||
- |
0 |
20 |
23.3 |
6.7 |
25, 29, 16 |
|
1580 |
|
21.3 |
5.1 |
27, 20, 17 |
||
2500 |
|
26 |
8.5 |
17, 27, 34 |
||
5000 |
|
25 |
12.2 |
39, 19, 17 |
||
7500 |
|
25.3 |
3.5 |
29, 22, 25 |
||
10000 |
|
24.3 |
3.8 |
27, 20, 26 |
||
Untreated |
0 |
15.7 |
2.5 |
13, 18, 16 |
||
+ |
0 |
5 |
22.7 |
5.5 |
29, 20, 19 |
|
1580 |
25.7 |
7 |
33, 19, 25 |
|||
2500 |
23 |
9.2 |
13, 25, 31 |
|||
5000 |
23.3 |
4.2 |
20, 28, 22 |
|||
7500 |
20.7 |
4.2 |
16, 24, 22 |
|||
10000 |
18.3 |
1.2 |
19, 17, 19 |
|||
+ |
0 |
|
32 |
3.6 |
35, 28, 33 |
|
1580 |
|
28.7 |
4 |
25, 28, 33 |
||
2500 |
10 |
27.7 |
5 |
27, 23, 33 |
||
5000 |
|
24.7 |
4.9 |
28, 19, 27 |
||
7500 |
|
21.7 |
4.7 |
20, 18, 27 |
||
10000 |
|
14 T |
13.1 |
26, 16, 0 |
||
+ |
0 |
|
25 |
3 |
22, 28, 25 |
|
1580 |
|
28.7 |
5.5 |
25, 35, 26 |
||
2500 |
20 |
27.7 |
4.2 |
29, 23, 31 |
||
5000 |
|
23.3 |
4.2 |
22, 20, 28 |
||
7500 |
|
2 T |
2.6 |
5, 0, 1 |
||
10000 |
|
0 T |
|
|
||
Untreated |
0 |
26.3 |
9.1 |
16, 33, 30 |
||
Positive control |
+/- pre-incubation |
Concentration (µg/plate) |
% S9 |
Mean Revertant Colony Counts |
S.D. |
Individual Revertant Colony Counts |
DMSO |
- |
|
10 |
28.3 |
12.1 |
17, 27, 41 |
2AA |
|
2 |
|
1930.7 |
73.7 |
1929, 1858, 2005 |
DMSO |
+ |
|
10 |
32 |
3.6 |
35, 28, 33 |
2AA |
|
2 |
|
2176.7 |
98.3 |
2063, 2235, 2232 |
S.D. = standard deviation T = toxic dose level |
+ = with pre-incubation - = without pre-incubation |
|||||
2AA = 2-aminoanthracene administered with DMSO as a vehicle |
||||||
Table 8: Mutagenicity Assay Results Test 2 – TA98 strain |
||||||
Strain |
+/- pre-incubation |
Dose level (µg/ml) |
% S9 |
Mean Revertant Colony Counts |
S.D. |
Individual Revertant Colony Counts |
TA98 |
- |
0 |
5 |
36.7 |
8.6 |
46, 29, 35 |
1580 |
25.3 |
4.7 |
29, 20, 27 |
|||
2500 |
30.7 |
2.1 |
33, 30, 29 |
|||
5000 |
32.7 |
4.5 |
28, 33, 37 |
|||
7500 |
32.3 |
5 |
27, 33, 37 |
|||
10000 |
31 |
11.5 |
44, 27, 22 |
|||
|
- |
0 |
10 |
38.7 |
2.1 |
37, 38, 41 |
1580 |
43.3 |
5.9 |
39, 41, 50 |
|||
2500 |
33.7 |
5.8 |
27, 37, 37 |
|||
5000 |
36.7 |
7.4 |
31, 34, 45 |
|||
7500 |
29 |
5.6 |
30, 23, 34 |
|||
10000 |
39.3 |
3.8 |
42, 35, 41 |
|||
|
- |
0 |
20 |
44.7 |
11.1 |
33, 46, 55 |
1580 |
37 |
5.3 |
41, 39, 31 |
|||
2500 |
42 |
7.2 |
44, 48, 34 |
|||
5000 |
41.3 |
7.1 |
49, 35, 40 |
|||
7500 |
41 |
5.2 |
38, 47, 38 |
|||
10000 |
34.3 |
5.7 |
28, 36, 39 |
|||
Untreated |
0 |
42.3 |
7.5 |
35, 50, 42 |
||
|
+ |
0 |
5 |
37.7 |
11 |
27, 37, 49 |
1580 |
36.7 |
4 |
41, 36, 33 |
|||
2500 |
35 |
5.3 |
33, 41, 31 |
|||
5000 |
26.3 |
2.9 |
28, 28, 23 |
|||
7500 |
28 |
3 |
31, 28, 25 |
|||
10000 |
14.7 |
10.8 |
27, 7, 10 |
|||
|
+ |
0 |
10 |
34.7 |
4 |
37, 37, 30 |
1580 |
31.3 |
3.8 |
34, 27, 33 |
|||
2500 |
35.3 |
2.5 |
35, 38, 33 |
|||
5000 |
26.3 |
9 |
35, 17, 27 |
|||
7500 |
8.7 T |
8.1 |
10, 16, 0 |
|||
10000 |
2.7 T |
3.8 |
0, 1, 7 |
|||
|
+ |
0 |
20 |
34.3 |
0.6 |
35, 34, 34 |
1580 |
37.7 |
1.2 |
39, 37, 37 |
|||
2500 |
36.3 |
3.1 |
33, 39, 37 |
|||
5000 |
23.7 |
1.2 |
23, 23, 25 |
|||
7500 |
21.3 |
4 |
22, 25, 17 |
|||
10000 |
0 T |
|
|
|||
Untreated |
0 |
42.7 |
3.8 |
47, 40, 41 |
||
Positive control |
+/- pre-incubation |
Concentration (µg/plate) |
% S9 |
Mean Revertant Colony Counts |
S.D. |
Individual Revertant Colony Counts |
DMSO |
- |
|
10 |
38.7 |
2.1 |
37, 38, 41 |
2AA |
|
2 |
|
1645 |
54.8 |
1704, 1635, 1596 |
DMSO |
+ |
|
10 |
34.7 |
4 |
37, 37, 30 |
2AA |
|
2 |
|
1599.3 |
77.9 |
1511, 1629, 1658 |
S.D. = standard deviation T = toxic dose level |
+ = with pre-incubation - = without pre-incubation |
|||||
2AA = 2-aminoanthracene administered with DMSO as a vehicle |
||||||
Table 9: Mutagenicity Assay Results Test 2 – TA1535 strain |
||||||
Strain |
+/- pre-incubation |
Dose level (µg/ml) |
% S9 |
Mean Revertant Colony Counts |
S.D. |
Individual Revertant Colony Counts |
TA1535 |
- |
0 |
5 |
8.3 |
4 |
4, 12, 9 |
1580 |
11 |
5.3 |
17, 9, 7 |
|||
2500 |
11 |
2.6 |
8, 12, 13 |
|||
5000 |
9.7 |
2.1 |
9, 8, 12 |
|||
7500 |
10 |
2.6 |
9, 8, 13 |
|||
10000 |
6.3 |
0.6 |
6, 6, 7 |
|||
- |
0 |
10 |
8.7 |
3.8 |
6, 7, 13 |
|
1580 |
7 |
0 |
7, 7, 7 |
|||
2500 |
8.7 |
6.7 |
3, 7, 16 |
|||
5000 |
8.7 |
4 |
11, 4, 11 |
|||
7500 |
5.3 |
1.5 |
7, 5, 4 |
|||
10000 |
7.3 |
2.1 |
9, 8, 5 |
|||
- |
0 |
20 |
14.3 |
2.5 |
14, 17, 12 |
|
1580 |
8.7 |
2.1 |
7, 8, 11 |
|||
2500 |
9.3 |
3.5 |
9, 6, 13 |
|||
5000 |
11.3 |
2.1 |
9, 13, 12 |
|||
7500 |
7.7 |
4.2 |
9, 3, 11 |
|||
10000 |
9 |
2 |
11, 9, 7 |
|||
Untreated |
0 |
9.3 |
1.5 |
9, 8, 11 |
||
+ |
0 |
5 |
7.7 |
2.1 |
6, 10, 7 |
|
1580 |
12.7 |
2.9 |
16, 11, 11 |
|||
2500 |
6.3 |
1.5 |
5, 6, 8 |
|||
5000 |
7.3 |
1.2 |
8, 8, 6 |
|||
7500 |
6.7 |
1.2 |
6, 6, 8 |
|||
10000 |
1.7 T |
2.9 |
0, 5, 0 |
|||
+ |
0 |
10 |
6.3 |
1.2 |
7, 5, 7 |
|
1580 |
11 |
5 |
11, 16, 6 |
|||
2500 |
8 |
3.5 |
12, 6, 6 |
|||
5000 |
7 |
2 |
9, 7, 5 |
|||
7500 |
2 T |
2 |
4, 0, 2 |
|||
10000 |
1.7 T |
1.2 |
1, 1, 3 |
|||
+ |
0 |
20 |
11.3 |
3.8 |
7, 13, 14 |
|
1580 |
8.3 |
2.5 |
6, 11, 8 |
|||
2500 |
10 |
2.6 |
8, 13, 9 |
|||
5000 |
10.7 |
3.2 |
7, 12, 13 |
|||
7500 |
1.3 T |
2.3 |
0, 0, 4 |
|||
10000 |
3.7 T |
3.2 |
0, 5, 6 |
|||
Untreated |
0 |
11 |
2.6 |
8, 13, 12 |
||
Positive control |
+/- pre-incubation |
Concentration (µg/plate) |
% S9 |
Mean Revertant Colony Counts |
S.D. |
Individual Revertant Colony Counts |
DMSO |
- |
|
10 |
8.7 |
3.8 |
6, 7, 13 |
2AA |
|
2 |
|
75.7 |
10.4 |
64, 84, 79 |
DMSO |
+ |
|
10 |
6.3 |
1.2 |
7, 5, 7 |
2AA |
|
2 |
|
137.3 |
11.8 |
130, 131, 151 |
S.D. = standard deviation T = toxic dose level |
+ = with pre-incubation - = without pre-incubation |
|||||
2AA = 2-aminoanthracene administered with DMSO as a vehicle |
||||||
Table 10: Mutagenicity Assay Results Test 2 – TA100 strain |
||||||
Strain |
+/- pre-incubation |
Dose level (µg/ml) |
% S9 |
Mean Revertant Colony Counts |
S.D. |
Individual Revertant Colony Counts |
TA100 |
- |
0 |
5 |
130.3 |
3.1 |
131, 133, 127 |
1580 |
140 |
14.1 |
142, 153, 125 |
|||
2500 |
126.7 |
14.2 |
119, 143, 118 |
|||
5000 |
111.7 |
11.9 |
125, 108, 102 |
|||
7500 |
111 |
15.1 |
106, 99, 128 |
|||
10000 |
112.7 |
10.7 |
125, 107, 106 |
|||
|
- |
0 |
10 |
139 |
6.6 |
132, 140, 145 |
1580 |
130 |
10.6 |
118, 138, 134 |
|||
2500 |
121.7 |
18.1 |
105, 141, 119 |
|||
5000 |
111.3 |
10.3 |
114, 120, 100 |
|||
7500 |
123.7 |
9.1 |
114, 132, 125 |
|||
10000 |
116.7 |
3.8 |
121, 114, 115 |
|||
|
- |
0 |
20 |
131.3 |
6 |
132, 137, 125 |
1580 |
131.3 |
23.9 |
148, 104, 142 |
|||
2500 |
120.7 |
16.7 |
102, 134, 126 |
|||
5000 |
131.3 |
10.3 |
140, 134, 120 |
|||
7500 |
133.3 |
4.5 |
133, 138, 129 |
|||
10000 |
117.3 |
15.5 |
117, 133, 102 |
|||
Untreated |
0 |
145.3 |
11.8 |
138, 159, 139 |
||
|
+ |
0 |
5 |
118.7 |
10.7 |
121, 107, 128 |
1580 |
130 |
21.6 |
106, 148, 136 |
|||
2500 |
129 |
15 |
114, 144, 129 |
|||
5000 |
93.3 |
14.6 |
95, 78, 107 |
|||
7500 |
82.3 |
2.5 |
82, 85, 80 |
|||
10000 |
46.7 T |
34 |
60, 8, 72 |
|||
|
+ |
0 |
10 |
109.7 |
8.4 |
115, 100, 114 |
1580 |
129 |
9 |
129, 138, 120 |
|||
2500 |
124 |
19.3 |
145, 120, 107 |
|||
5000 |
94 |
21.7 |
81, 119, 82 |
|||
7500 |
62.7 T |
19.6 |
41, 79, 68 |
|||
10000 |
26 T |
22.7 |
36, 42, 0 |
|||
|
+ |
0 |
20 |
130 |
10.1 |
139, 132, 119 |
1580 |
138 |
6.6 |
137, 145, 132 |
|||
2500 |
122 |
1.8 |
120, 123, 123 |
|||
5000 |
105.3 |
24.5 |
77, 119, 120 |
|||
7500 |
51.3 T |
40.3 |
71, 5, 78 |
|||
10000 |
21.7 T |
31.7 |
0, 58, 7 |
|||
Untreated |
0 |
139.7 |
18.6 |
138, 122, 159 |
||
Positive control |
+/- pre-incubation |
Concentration (µg/plate) |
% S9 |
Mean Revertant Colony Counts |
S.D. |
Individual Revertant Colony Counts |
DMSO |
- |
|
10 |
139 |
6.6 |
132, 140, 145 |
2AA |
|
2 |
|
1850 |
125.7 |
1728, 1979, 1843 |
DMSO |
+ |
|
10 |
34.7 |
4 |
37, 37, 30 |
2AA |
|
2 |
|
1599.3 |
77.9 |
1511, 1629, 1658 |
S.D. = standard deviation T = toxic dose level |
+ = with pre-incubation - = without pre-incubation |
|||||
2AA = 2-aminoanthracene administered with DMSO as a vehicle |
||||||
Applicant's summary and conclusion
- Conclusions:
- Based on this testing, it is concluded, that under conditions used in the assay, WS-23 was not mutagenic towards five strains of S. typhimurium.
- Executive summary:
The objective of this study was to investigate the potential of WS-23 to be a bacterial mutagen.
For this reason, a standard Plate Incorporation Assay (Ames test) was performed twice on WS-23 using five concentrations of WS-23 spaced at log10 intervals. The concentrations of the test material used per Petri dish were, as follows: 1.58, 2.5, 5, 7.5 and 10 mg/plate. The assay was performed with three levels of S9 (5, 10 and 20 %) with and without pre-incubation.
Three plates were prepared for each concentration of material tested. Similar numbers of plates were also prepared for appropriate positive and negative (solvent, DMSO) controls. Untreated controls were also included. The plates were incubated at 37 degrees C for either 2 or 3 days. Colonies were counted on an Artek 880 counter.
A reduction in the number of revertants and thinning or complete absence of background lawn were observed on several plates treated with the higher concentration (10 mg/plate) of WS 23. The toxicity was dependent on the concentration of S9 used and was more evident with pre-incubation (observed toxicity at 7.5 and 10 mg/plate).
No increase in the number of the revertant colonies occurred with any of the five strains of bacteria at test concentrations of WS-23 up to 10 mg/plate at any of the three levels of S9 mix, either with of without pre-incubation.
In one test only, with TA 1537 in the presence of 5% S9, there was an increase in the number of revertants three concentrations of twice the spontaneous control value. These increases were neither dose related nor reproducible. The spontaneous revertant values on the control plates were unusually low in this test and the observed increase was therefore considered spurious.
In the absence of any evidence of mutagenic potential it is concluded that WS-23 is not mutagenic towards five strains of S. typhimurium up to 5 mg/plate.
Interpretation of the data described in this study suggests that WS-23 is unlikely to present a genotoxic hazard.
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