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EC number: 232-601-0 | CAS number: 9001-37-0
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 006
- Report date:
- 2006
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- The study used selective agar plates containing fructose instead of glucose.
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Active enzyme protein of Glucose oxidase (EC no. 232-601-0, CAS no. 9001-37-0, EC name: Glucose oxidase, Enzyme Class no. 1.1.3.4)
- Molecular formula:
- Not applicable, see remarks.
- IUPAC Name:
- Active enzyme protein of Glucose oxidase (EC no. 232-601-0, CAS no. 9001-37-0, EC name: Glucose oxidase, Enzyme Class no. 1.1.3.4)
- Reference substance name:
- Protein as a constituent of enzyme deriving from the fermentation or extraction process
- Molecular formula:
- Not available. See remarks.
- IUPAC Name:
- Protein as a constituent of enzyme deriving from the fermentation or extraction process
- Reference substance name:
- Carbohydrates constituent of enzyme deriving from the fermentation or extraction process
- Molecular formula:
- Not available. See remarks.
- IUPAC Name:
- Carbohydrates constituent of enzyme deriving from the fermentation or extraction process
- Reference substance name:
- Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process
- Molecular formula:
- Not available. See remarks.
- IUPAC Name:
- Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process
- Reference substance name:
- Lipids as a constituent of enzyme deriving from the fermentation or extraction process
- Molecular formula:
- Not available. See remarks.
- IUPAC Name:
- Lipids as a constituent of enzyme deriving from the fermentation or extraction process
- Test material form:
- liquid
- Details on test material:
- - Substance type: UVCB
- Name of test material (as cited in study report): Glucose Oxidase
- Lot/batch No.: R-GOx-04004
- Physical state: Clear brown liquid
Constituent 1
Constituent 2
Constituent 3
Constituent 4
Constituent 5
Method
- Target gene:
- Operon for synthesis of amino acid histidine
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- S-9 mix prepared from Aroclor®-1254-induced rat livers
- Test concentrations with justification for top dose:
- 50, 160, 500, 1600, and 5000 µg/plate. The highest dose level tested is the maximum required by the OECD Guideline 471 for materials of low toxicity.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: saline (0.9% NaCl)
Controls
- Untreated negative controls:
- yes
- Remarks:
- sterile saline solution
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- cumene hydroperoxide
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Treat and plate method: Bacterial cultures were centrifuged at 1700 g for 10 minutes, the supernatant was discarded and the bacteria were resuspended in one third of the original volume of fresh broth. Test tubes were prepared containing the test substance solution, vehicle, or positive control solution, S-9 mix or 0.02 M phosphate buffer pH 7.4 and concentrated bacterial suspension. After incubation at 37°C for 30 minutes with gentle shaking, nutrient broth was added to each test tube and the incubation was continued for 3 hours. After incubation, the bacteria were sedimented by centrifugation, the supernatant removed and the bacteria resuspended in buffer. After another centrifugation and removal of supernatant, the bacteria were resuspended in buffer and top agar was added. The contents of each tube were mixed with a vortex mixer and spread on minimal fructose agar plates.
- Cell density at seeding (if applicable): 10E9 bacteria/mL
DURATION
- Preincubation period: 30 minutes at 30°C, nutrient broth added, incubation continued for 3 hours
- Exposure duration: plates were incubated for 72 hours at 37°C
NUMBER OF REPLICATIONS: 2 tests with 3 plates at each test point - Rationale for test conditions:
- In preliminary tests with the plate incorporation method, it was observed that the histidine in the test substance was interfering with the test system, preventing adequate assessment of the test substance for mutagenic activity. Therefore, the treat and plate method was carried out to avoid the problem.
- Evaluation criteria:
- The tests were considered valid if the following criteria were met:
- negative and positive control data were consistent with the historical control data for the laboratory
- the positive control data showed marked increases over the concurrent negative control values
- the evaluation of the data was not restricted by loss of plates (e.g., through contamination)
The test item would have been considered to have shown evidence for mutagenic activity in the study if all the following were met:
- increases in the number of revertant colonies were observed at one or more test points
- the mean number of revertant colonies at the test point showing the largest increase was more than twice the corresponding negative control value
- there was a credible scientific explanation for the observed dose-response relationship that involved a mutagenic effect of the test item
- the increases were reproducible between replicate plates and were observed in both main tests
- the increases were statistically significant
- increases were not directly related to increased growth of the non-revertant bacteria
The test substance would be considered to have shown no evidence of mutagenic activity if no dose-related increased in the numbers of revertant colonies which exceeded 1.5 times the negative control value were observed at any test point. The test item would also be considered to have shown no evidence of mutagenic activity if moderately larger increases had been observed that were not reproducible, not statistically significant, or which were sporadic (without a scientifically valid explanation for the dose-response relationship that involved a mutagenic effect of the test substance). Increases between 1.5 and 2-fold greater than the negative control values, which met the other criteria for a positive result, would provide some evidence for a weak effect of the test substance, but would be considered to be clear evidence of mutagenic activity. - Statistics:
- The numbers of revertant colonies at each treatment test point in the main tests were compared to the corresponding negative control values using the Analysis of Variance test. When this test showed statistically significant differences in the data, Dunnett's test was used to determine the statistical significance of the increases and decreases in the numbers of revertant colonies for each set of triplicate plates. The statistical analysis was performed with SAS® procedures (version 8.2) described in SAS/STAT® User's Guide, SAS OnlineDoc®, 1999, SAS Institute Inc., Cary, North Carolina 27513, USA.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- ≥500 µg (without S-9); 5000 µg (with S-9)
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- ≥160 µg (without S-9); ≥500 µg (with S-9)
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 5000 µg (without S-9)
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- ≥1600 µg (without S-9)
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- In the preliminary test with the treat and plate method, the test substance was toxic to the test bacteria at dose levels of 500, 1600, and 5000 µg/plate without S-9 mix and at 5000 µg/plate with S-9 mix: reduced growth of the background lawn of non-revertant bacteria and reductions in the number of revertant colonies.
The results of the two main tests showed the test substance was toxic to most of the tester strains at the higher does levels, causing reduced growth of background lawn and reductions in the number of revertant colonies. These effects were generally observed over a wider range of dose levels for treatments without S-9 than for treatments with S-9 mix. The amount of toxicity showed some variation between the tester strains.
No biologically significant increases in the number of revertant colonies were observed in any tester strain after treatment with the test substance at any dose level, either in the absence or presence of S-9 mix. A small, but statistically significant increase was observed in strain TA 1535 in the first main test after treatment with the test substance at 5000 µg/plate without S-9 mix. This increase does not meet the criteria for a mutagenic effect because it was too small and it was not reproduced in the second test.
Any other information on results incl. tables
Main Test Strain TA102 |
||||||
Dose |
Number of Revertant Colonies / Plate |
|||||
Plate 1 |
Plate 2 |
Plate 3 |
Mean |
SD |
Ratio |
|
Strain TA102, Without S-9 Mix, Main Test 1 |
||||||
Control |
436 |
432 |
472 |
446.7 |
22.0 |
|
5000 µg |
440T |
420T |
496T |
452.0 |
39.4 |
1.01 |
1600 µg |
416T |
400T |
576T |
464.0 |
97.3 |
1.04 |
500 µg |
416 |
368 |
500 |
428.0 |
66.8 |
0.96 |
160 µg |
424 |
404 |
400 |
409.3 |
12.9 |
0.92 |
50 µg |
380 |
420 |
324 |
374.7 |
48.2 |
0.84 |
Cumene hydroperoxide 100 µg |
800 |
1152 |
1040 |
997.3 |
179.8 |
2.23 |
Strain TA102, Without S-9 Mix, Main Test 2 |
||||||
Control |
648 |
488 |
432 |
522.7 |
112.1 |
|
5000 µg |
440T |
440T |
304T |
394.7 |
78.5 |
0.76 |
1600 µg |
344T |
536T |
528T |
469.3 |
108.6 |
0.90 |
500 µg |
520 |
472 |
480 |
490.7 |
25.7 |
0.94 |
160 µg |
352 |
480 |
408 |
413.3 |
64.2 |
0.79 |
50 µg |
376 |
432 |
448 |
418.7 |
37.8 |
0.80 |
Cumene hydroperoxide 100 µg |
1600 |
1408 |
1536 |
1514.7 |
97.8 |
2.90 |
Strain TA102, With S-9 Mix, Main Test 1 |
||||||
Control |
408 |
189 |
400 |
332.3 |
124.2 |
|
5000 µg |
352 |
304 |
424 |
360.0 |
60.4 |
1.08 |
1600 µg |
476 |
396 |
424 |
432.0 |
40.6 |
1.30 |
500 µg |
400 |
304 |
576 |
426.7 |
137.9 |
1.28 |
160 µg |
384 |
432 |
400 |
405.3 |
24.4 |
1.22 |
50 µg |
432 |
372 |
448 |
417.3 |
40.1 |
1.26 |
2-Aminoanthracene 4 µg |
1088 |
992 |
358 |
812.7 |
396.7 |
2.45 |
Strain TA102, With S-9 Mix, Main Test 2 |
||||||
Control |
320 |
360 |
456 |
378.7 |
69.9 |
|
5000 µg |
544 |
400 |
392 |
445.3 |
85.5 |
1.18 |
1600 µg |
376 |
440 |
408 |
408.0 |
32.0 |
1.08 |
500 µg |
360 |
376 |
448 |
394.7 |
46.9 |
1.04 |
160 µg |
344 |
440 |
448 |
410.7 |
57.9 |
1.08 |
50 µg |
384 |
384 |
352 |
373.3 |
18.5 |
0.99 |
2-Aminoanthracene 4 µg |
1344 |
1216 |
2496 |
1685.3 |
705.0 |
4.45 |
S.D. = Standard deviation RATIO =Mean number of revertants on treated plates/mean number of revertants on vehicle control plates * = Statistically significant at 5% level ** = Statistically significant at 1% level Otherwise, not statistically significant at 5% level (The positive controls were not included in the statistical analysis) T = Toxicity: reduced growth of background lawn of non-revertant bacteria |
Main Test Strain TA100 |
||||||
Dose |
Number of Revertant Colonies / Plate |
|||||
Plate 1 |
Plate 2 |
Plate 3 |
Mean |
SD |
Ratio |
|
Strain TA100, Without S-9 Mix, Main Test 1 |
||||||
Control |
218 |
198 |
272 |
229.3 |
38.3 |
|
5000 µg |
226 |
184 |
193 |
201.0 |
22.1 |
0.88 |
1600 µg |
214 |
201 |
209 |
208.0 |
6.6 |
0.91 |
500 µg |
173 |
178 |
200 |
183.7 |
14.4 |
0.80 |
160 µg |
206 |
204 |
212 |
207.3 |
4.2 |
0.90 |
50 µg |
241 |
218 |
188 |
215.7 |
26.6 |
0.94 |
Sodium azide 1 µg |
1301 |
1220 |
1208 |
1243.0 |
50.6 |
5.42 |
Strain TA100, Without S-9 Mix, Main Test 2 |
||||||
Control |
182 |
214 |
200 |
198.7 |
16.0 |
|
5000 µg |
180T |
159T |
120T |
153.0 |
30.4 |
0.77 |
1600 µg |
160 |
154 |
72 |
128.7 |
49.2 |
0.65 |
500 µg |
137 |
126 |
97 |
120.0 |
20.7 |
0.60 |
160 µg |
136 |
175 |
131 |
147.3 |
24.1 |
0.74 |
50 µg |
209 |
188 |
146 |
181.0 |
32.1 |
0.91 |
Sodium azide 1 µg |
1007 |
1173 |
1138 |
1106.0 |
87.5 |
5.57 |
Strain TA100, With S-9 Mix, Main Test 1 |
||||||
Control |
226 |
238 |
245 |
236.3 |
9.6 |
|
5000 µg |
198 |
210 |
221 |
209.7 |
11.5 |
0.89 |
1600 µg |
221 |
246 |
264 |
243.7 |
21.6 |
1.03 |
500 µg |
206 |
222 |
246 |
224.7 |
20.1 |
0.95 |
160 µg |
194 |
199 |
185 |
192.7 |
7.1 |
0.82 |
50 µg |
233 |
149 |
209 |
197.0 |
43.3 |
0.83 |
2-Aminoanthracene 2 µg |
1379 |
1612 |
1467 |
1486.0 |
117.7 |
6.29 |
Strain TA100, With S-9 Mix, Main Test 2 |
||||||
Control |
300 |
238 |
175 |
237.7 |
62.5 |
|
5000 µg |
184 |
169 |
167 |
173.3 |
9.3 |
0.73 |
1600 µg |
179 |
141 |
185 |
168.3 |
23.9 |
0.71 |
500 µg |
172 |
200 |
168 |
180.0 |
17.4 |
0.76 |
160 µg |
182 |
172 |
186 |
180.0 |
7.2 |
0.76 |
50 µg |
217 |
213 |
165 |
198.3 |
28.9 |
0.83 |
2-Aminoanthracene 2 µg |
2013 |
1820 |
2323 |
2052.0 |
253.8 |
8.63 |
S.D. = Standard deviation RATIO =Mean number of revertants on treated plates/mean number of revertants on vehicle control plates * = Statistically significant at 5% level ** = Statistically significant at 1% level Otherwise, not statistically significant at 5% level (The positive controls were not included in the statistical analysis) T = Toxicity: reduced growth of background lawn of non-revertant bacteria |
Main Test Strain TA98 |
||||||
Dose |
Number of Revertant Colonies / Plate |
|||||
Plate 1 |
Plate 2 |
Plate 3 |
Mean |
SD |
Ratio |
|
Strain TA98, Without S-9 Mix, Main Test 1 |
||||||
Control |
29 |
28 |
36 |
31.0 |
4.4 |
|
5000 µg |
12T |
23T |
24T |
19.7* |
6.7 |
0.63 |
1600 µg |
19T |
27T |
22T |
22.7 |
4.0 |
0.73 |
500 µg |
31 |
30 |
22 |
27.7 |
4.9 |
0.89 |
160 µg |
17 |
18 |
15 |
16.7** |
1.5 |
0.54 |
50 µg |
38 |
32 |
31 |
33.7 |
3.8 |
1.09 |
2-Nitrofluorene 1 µg |
121 |
144 |
127 |
130.7 |
11.9 |
4.22 |
Strain TA98, Without S-9 Mix, Main Test 2 |
||||||
Control |
31 |
23 |
25 |
26.3 |
4.2 |
|
5000 µg |
23T |
20T |
29T |
24.0 |
4.6 |
0.91 |
1600 µg |
29T |
32T |
31T |
30.7 |
1 .5 |
1.16 |
500 µg |
28T |
22T |
25T |
25.0 |
3.0 |
0.95 |
160 µg |
16 |
16 |
29 |
20.3 |
7.5 |
0.77 |
50 µg |
25 |
29 |
23 |
25.7 |
3.1 |
0.97 |
2-Nitrofluorene 1 µg |
140 |
104 |
104 |
116.0 |
20.8 |
4.41 |
Strain TA98, With S-9 Mix, Main Test 1 |
||||||
Control |
40 |
34 |
26 |
33.3 |
7.0 |
|
5000 µg |
20T |
23T |
37T |
26.7 |
9.1 |
0.80 |
1600 µg |
24 |
20 |
24 |
22.7 |
2.3 |
0.68 |
500 µg |
22 |
27 |
31 |
26.7 |
4.5 |
0.80 |
160 µg |
27 |
31 |
27 |
28.3 |
2.3 |
0.85 |
50 µg |
30 |
25 |
36 |
30.3 |
5.5 |
0.91 |
2-Aminoanthracene 2 µg |
600 |
644 |
584 |
609.3 |
31.1 |
18.28 |
Strain TA98, With S-9 Mix, Main Test 2 |
||||||
Control |
40 |
46 |
37 |
41.0 |
4.6 |
|
5000 µg |
40T |
34T |
33T |
35.7 |
3.8 |
0.87 |
1600 µg |
36 |
36 |
43 |
38.3 |
4.0 |
0.93 |
500 µg |
33 |
30 |
30 |
31.0 |
1.7 |
0.76 |
160 µg |
31 |
39 |
26 |
32.0 |
6.6 |
0.78 |
50 µg |
31 |
34 |
34 |
33.0 |
1.7 |
0.80 |
2-Aminoanthracene 2 µg |
752 |
896 |
704 |
784.0 |
99.9 |
19.12 |
S.D. = Standard deviation RATIO =Mean number of revertants on treated plates/mean number of revertants on vehicle control plates * = Statistically significant at 5% level ** = Statistically significant at 1% level Otherwise, not statistically significant at 5% level (The positive controls were not included in the statistical analysis) T = Toxicity: reduced growth of background lawn of non-revertant bacteria |
Main Test Strain TA1537 |
||||||
Dose |
Number of Revertant Colonies / Plate |
|||||
Plate 1 |
Plate 2 |
Plate 3 |
Mean |
SD |
Ratio |
|
Strain TA1537, Without S-9 Mix, Main Test 1 |
||||||
Control |
15 |
14 |
4 |
11.0 |
6.1 |
|
5000 µg |
11T |
8T |
9T |
9.3 |
1.5 |
0.85 |
1600 µg |
13T |
9T |
11T |
11.0 |
2.0 |
1.00 |
500 µg |
9T |
8T |
8T |
8.3 |
0.6 |
0.76 |
160 µg |
11T |
8T |
10T |
9.7 |
1.5 |
0.88 |
50 µg |
10 |
11 |
9 |
10.0 |
1.0 |
0.91 |
9-Aminoacridine 80 µg |
868 |
384 |
728 |
660.0 |
249.1 |
60.00 |
Strain TA1537, Without S-9 Mix, Main Test 2 |
||||||
Control |
17 |
28 |
31 |
25.3 |
7.4 |
|
5000 µg |
7T |
5T |
14T |
8.7** |
4.7 |
0.34 |
1600 µg |
9T |
10T |
9T |
9.3** |
0.6 |
0.37 |
500 µg |
6T |
18T |
10T |
11.3* |
6.1 |
0.45 |
160 µg |
11 |
13 |
18 |
14.0 |
3.6 |
0.55 |
50 µg |
11 |
13 |
20 |
14.7 |
4.7 |
0.58 |
9-Aminoacridine 80 µg |
1115 |
1196 |
528 |
946.3 |
364.5 |
37.36 |
Strain TA1537, With S-9 Mix, Main Test 1 |
||||||
Control |
14 |
8 |
11 |
11.0 |
3.0 |
|
5000 µg |
13T |
14T |
9T |
12.0 |
2.6 |
1.09 |
1600 µg |
9T |
8T |
5T |
7.3 |
2.1 |
0.67 |
500 µg |
4T |
6T |
10T |
6.7 |
3.1 |
0.61 |
160 µg |
8 |
11 |
14 |
11.0 |
3.0 |
1.00 |
50 µg |
10 |
12 |
15 |
12.3 |
2.5 |
1.12 |
2-Aminoanthracene 2 µg |
312 |
304 |
288 |
301.3 |
12.2 |
27.39 |
Strain TA1537, With S-9 Mix, Main Test 2 |
||||||
Control |
8 |
19 |
17 |
14.7 |
5.9 |
|
5000 µg |
8T |
10T |
13T |
10.3 |
2.5 |
0.70 |
1600 µg |
8T |
13T |
8T |
9.7 |
2.9 |
0.66 |
500 µg |
18 |
17 |
11 |
15.3 |
3.8 |
1.05 |
160 µg |
18 |
23 |
25 |
22.0 |
3.6 |
1.50 |
50 µg |
10 |
15 |
18 |
14.3 |
4.0 |
0.98 |
2-Aminoanthracene 2 µg |
103 |
93 |
108 |
101.3 |
7.6 |
6.91 |
S.D. = Standard deviation RATIO =Mean number of revertants on treated plates/mean number of revertants on vehicle control plates * = Statistically significant at 5% level ** = Statistically significant at 1% level Otherwise, not statistically significant at 5% level (The positive controls were not included in the statistical analysis) T = Toxicity: reduced growth of background lawn of non-revertant bacteria |
Main Test Strain TA1535 |
||||||
Dose |
Number of Revertant Colonies / Plate |
|||||
Plate 1 |
Plate 2 |
Plate 3 |
Mean |
SD |
Ratio |
|
Strain TA1535, Without S-9 Mix, Main Test 1 |
||||||
Control |
16 |
23 |
21 |
20.0 |
3.6 |
|
5000 µg |
36 |
33 |
30 |
33.0** |
3.0 |
1.65 |
1600 µg |
27 |
26 |
27 |
26.7 |
0.6 |
1.33 |
500 µg |
29 |
18 |
24 |
23.7 |
5.5 |
1.18 |
160 µg |
25 |
20 |
25 |
23.3 |
2.9 |
1.17 |
50 µg |
22 |
15 |
18 |
18.3 |
3.5 |
0.92 |
Sodium azide 1 µg |
500 |
352 |
448 |
433.3 |
75.1 |
21.67 |
Strain TA1535, Without S-9 Mix, Main Test 2 |
||||||
Control |
15 |
19 |
25 |
19.7 |
5.0 |
|
5000 µg |
11 |
14 |
15 |
13.3 |
2.1 |
0.68 |
1600 µg |
19 |
25 |
21 |
21.7 |
3.1 |
1.10 |
500 µg |
13 |
16 |
24 |
17.7 |
5.7 |
0.90 |
160 µg |
17 |
11 |
13 |
13.7 |
3.1 |
0.69 |
50 µg |
21 |
20 |
15 |
18.7 |
3.2 |
0.95 |
Sodium azide 1 µg |
400 |
408 |
396 |
401.3 |
6.1 |
20.41 |
Strain TA1535, With S-9 Mix, Main Test 1 |
||||||
Control |
24 |
34 |
34 |
30.7 |
5.8 |
|
5000 µg |
31 |
25 |
31 |
29.0 |
3.5 |
0.95 |
1600 µg |
31 |
20 |
27 |
26.0 |
5.6 |
0.85 |
500 µg |
31 |
25 |
31 |
29.0 |
3.5 |
0.95 |
160 µg |
23 |
29 |
18 |
23.3 |
5.5 |
0.76 |
50 µg |
26 |
28 |
16 |
23.3 |
6.4 |
0.76 |
2-Aminoanthracene 42 µg |
147 |
176 |
164 |
162.3 |
14.6 |
5.29 |
Strain TA1535, With S-9 Mix, Main Test 2 |
||||||
Control |
24 |
22 |
25 |
23.7 |
1 .5 |
|
5000 µg |
19 |
18 |
19 |
18.7 |
0.6 |
0.79 |
1600 µg |
16 |
22 |
26 |
21.3 |
5.0 |
0.90 |
500 µg |
20 |
25 |
18 |
21.0 |
3.6 |
0.89 |
160 µg |
18 |
20 |
20 |
19.3 |
1 .2 |
0.82 |
50 µg |
27 |
22 |
20 |
23.0 |
3.6 |
0.97 |
2-Aminoanthracene 2 µg |
184 |
187 |
210 |
193.7 |
14.2 |
8.18 |
S.D. = Standard deviation RATIO =Mean number of revertants on treated plates/mean number of revertants on vehicle control plates * = Statistically significant at 5% level ** = Statistically significant at 1% level Otherwise, not statistically significant at 5% level (The positive controls were not included in the statistical analysis) T = Toxicity: reduced growth of background lawn of non-revertant bacteria |
Applicant's summary and conclusion
- Conclusions:
- The test substance has not shown evidence of mutagenic activity with or without S-9 activation.
- Executive summary:
The test substance was tested in the Ames Test using Salmonella typhimurium strains TA 102, TA 100, TA 98, TA 1537, and TA 1535. The test was performed in accordance with OECD Guideline 471. The study was performed using selective agar plates containing fructose instead of glucose (the usual sugar in this type of plate) because glucose is the substrate for the enzymatic activity of this test substance.
A preliminary study was done using the plate incorporation method, but the histidine in the test substance interfered with the test system. A second preliminary study was done with strain TA 98 and the independent two main tests were carried out with the treat and plate method to avoid this problem. The bacteria were treated with solutions of the test substance prepared in sterile saline solution (0.9% NaCl) at five dose levels in the range 50 to 5000 µg/plate. The higest dose is equivalent to 1.87 mg active enzyme protein/ kg bw or 9.71 enzyme concentrate dry matter/kg bw.
All of the dose levels in this report are expressed in terms of the total protein content of the test substance (stated by the Sponsor to be 36.24 mg/mL in the solution supplied). Negative control plates were treated by the addition of sterile saline solution (200 µL/plate). The treatments were performed both with and without a metabolic activation system (S-9 mix). Triplicate plates were prepared at each test point.
A dose related amount of insoluble material was observed on the plates in some parts of the study. The test substance was toxic to most of the tester strains at the higher dose levels, causing reduced growth of the background lawn and reductions in the number of revertant colonies. These effects were generally observed over a wider range of dose levels for treatments without S-9 mix than for treatments with S-9 mix. The amount of toxicity showed variation between tester strains. No biologically significant increases in the number of revertant colonies were observed in any tester strain after treatment with the test substance at any dose level, either in absence or presence of S-9 mix. Results obtained with the negative and positive controls demonstrated the sensitivity of the tests and efficacy of the S-9 mix metabolic activation system. Based on the results obtained in this study, it is concluded that the test substance has not shown any evidence of mutagenic activity.
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