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EC number: 701-357-1 | CAS number: -
- 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
- Study period:
- From October 9, 2018 to November 15, 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
- Report date:
- 2019
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- Adopted on 21 July 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- Adopted 30 May 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
Reference
- Name:
- Unnamed
- Type:
- Constituent
- Test material form:
- liquid
Method
Species / strain
- Species / strain / cell type:
- other: Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver homogenate metabolizing system (4% liver S9 in standard co-factors).
- Test concentrations with justification for top dose:
- Plate incorporation method
Experiment 1a: 0.05, 0.15, 0.5, 1.5 and 5 µL/plate, triplicate against each tester strain
Experiment 1b: 0.005, 0.015, 0.05, 0.15, 0.5, 1.5 µL/plate, triplicate against each tester strain
Pre-incubation method
Experiment 2: 0.006, 0.012, 0.023, 0.047, 0.094, 0.188, 0.375, 0.75 and 1.5 µL/plate
The maximum dose level of the test substance in the first experiment was selected as the maximum recommended dose level of 5 µL/plate. Both in test 1a and 1b no precipitation was observed in all the tested concentration. The test substance showed signs of toxicity towards all five bacterial strains in both absence and presence of metabolic activation in the highest concentration (5 µL/plate) and in the next lower concentration (1.5 µL/plate). The bacterial background lawn was not reduced at any of the concentrations in both the experiments. Based on the results of the experiment 1a and 1b, the test substance was tested up to concentrations of 1.5 µL/plate in the absence and presence of metabolic activation in all bacteria strain using the pre-incubation method. The test substance showed signs of toxicity (no bacteria growth and no bacterial background lawn) with 0.375, 0.75 and 1.5 µL/plate concentrations for all five tester strains, whereas TA100 and TA1535 showed signs of toxicity as decrease in the number of revertants with concentration of 0.188 µL/plate. - Vehicle / solvent:
- In a non-GLP pre-test, the solubility of the test substance was tested in a concentration of 50 mL/L in de-mineralized water, dimethyl sulfoxide (DMSO) and acetone. The liquid test substance is sufficiently soluble in DMSO, only. Based on the non-GLP pre-test, DMSO was chosen as vehicle, because the test substance was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO and Demineralised water
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- benzo(a)pyrene
- other: 4-Nitro-1,2-phenylene Diamine, 2-Amino-Anthracene
- Details on test system and experimental conditions:
- Test system
All Salmonella typhimurium strains were obtained from TRINOVA BioChem GmbH (batch: TA97a: 5033D, TA98: 5136D, TA100: 5141D, TA102: 5145D, TA1535: 5138D) and were stored as lyophilizates in the refrigerator at 2-8°C. The lyophilizates were used to prepare permanent cultures which were filled into vials and stored at < -75°C. Eight hours before the start of each experiment, an aliquot of a permanent culture per strain to be used was taken from the deep freezer to inoculate a culture vessel containing nutrient broth. After incubation overnight for eight hours at 37 ± 1°C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.
Preparation
In a non-GLP pre-test, the solubility of the test substance was tested in a concentration of 50 mL/L in de-mineralized water, dimethyl sulfoxide (DMSO) and acetone. The liquid test substance is sufficiently soluble in DMSO, only. Based on the non-GLP pre-test, DMSO was chosen as vehicle, because the test substance was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of sponta-neous revertants in the tested concentrations. On the day of the start of the experiment, a stock solution containing 50 mL/L of the test substance in DMSO was prepared. The test substance solution was not sterile filtrated before use. The stock solution was used to prepare the geometric series of the concentrations to be tested.
The following nominal concentrations were prepared for the experiment 1a for all bacteria strains: 5 µL/plate, 1.5 µL/plate, 0.5 µL/plate, 0.15 µL/plate and 0.05 µL/plate
On the day of the start of the experiment 1b and 2, a stock solution containing 15 µL/L of the test substance in DMSO was prepared. The following nominal concentrations were prepared for the experiment 1b for all bacteria strains: 5 µL/plate, 1.5 µL/plate, 0.5 µL/plate, 0.15 µL/plate, 0.05 µL/plate and 0.015 µL/plate
The following nominal concentrations were prepared for the experiment 2 for all bacteria strains: 1.5 µL/plate, 0.75 µL/plate, 0.38 µL/plate, 0.19 µL/plate, 0.09 µL/plate, 0.05 µL/plate, 0.03 µL/plate, 0.02 µL/plate and 0.01 µL/plate
Solvent Controls
The following substances were used as solvent controls:
Dimethylsulfoxide (DMSO), batch: 187256959, for the positive controls nitrophenylendiamine, benzo-a-pyrene and 2-amino-anthracene and batch: 218270919 for the test substance. Demineralised water, batch: 20180614 for the positive control sodium azide
Positive Controls
All mutagenic substances were stored as ready to use solution in the test facility in the deep freezer (-20 ± 5 °C). The respective positive control solution was thawed on the day of each experiment.
The following mutagenic substances were used as positive controls in all experiments:
A) 4-Nitro-1,2-phenylene diamine, C6H7N3O2; CAS-No.: 99-56-9
Concentration per plate: 20 µg
Solvent: DMSO
Strains: TA97a, TA98 and TA102
Metabolic activation: none
B) Sodium azide, NaN3; CAS-No.: 26628-22-8
Concentration per plate: 1 µg
Solvent: H2O
Strains: TA100 and TA1535
Metabolic activation: none
C) 2-Amino-anthracene, C14H11N; CAS-No.: 613-13-8
Concentration per plate: 1 µg
Solvent: DMSO
Strains: TA97a, TA100, TA102 and TA1535.
Metabolic activation: S9
D) Benzo-a-pyrene, C20H12; CAS-No.: 50-32-8
Concentration per plate: 20 µg
Solvent: DMSO
Strain: TA98
Metabolic activation: S9
Note: each batch of S9 is characterized with a mutagen that requires metabolic activation by microsomal enzymes (e.g., benzo(a)pyrene). - Evaluation criteria:
- The colonies were counted visually and the numbers were recorded. A validated spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control. The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test substance solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants less mean spontaneous revertants) was given. A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- other: Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535
- Remarks:
- Experiment 1a
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Highest concentration (5 µL/plate; no bacteria growth) and in the next lower concentration (1.5 µL/plate, decrease in the num-ber of revertants)
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- other: Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535
- Remarks:
- Experiment 1b
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Highest concentration (1.5 µL/plate; no bacteria growth)
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- other: Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535
- Remarks:
- Experiment 2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- ≥0.19 µL/plate (decrease in the number of revertants, no bacteria growth and no bacterial background lawn)
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
Any other information on results incl. tables
Results
Confirmation of genotype is performedfor each batch of lyophilized bacteria before stock culture preparation. The last performance showed no abnormalities.
A) Experiment 1a
Confirmation of the Criteria and Validity
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
Solubility and Toxicity
In this experiment, the test substance showed no precipitates on the plates in all tested concentrations. The test substance showed signs of toxicity towards all five bacteria strains in both the absence and presence of metabolic activation in the highest concentration (5 µL/plate; no bacteria growth) and in the next lower concentration (1.5 µL/plate, decrease in the number of revertants).The bacterial background lawn was not reduced at any of the concentrations.
Mutagenicity
No increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found. Therefore, the test substance is stated as not mutagenic under the conditions of this experiment. To verify this result, a further experiment was performed with lower concentrations.
Survey of the Findings
The mean revertant values of the three replicates are presented in the following table.
Table: Mean Revertants Experiment 1a
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
||||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
Demin. water |
Mean |
98 |
83 |
50 |
53 |
96 |
107 |
435 |
440 |
9 |
9 |
sd |
15.7 |
7.5 |
4.6 |
2.3 |
12.5 |
12.1 |
37.8 |
27.7 |
2.1 |
0.0 |
|
DMSO |
Mean |
91 |
96 |
48 |
45 |
90 |
95 |
445 |
437 |
10 |
9 |
sd |
13.3 |
6.0 |
2.1 |
1.2 |
8.7 |
12.2 |
25.7 |
24.1 |
3.0 |
1.7 |
|
Positive |
Mean |
456 |
484 |
349 |
417 |
1251 |
1261 |
1061 |
1064 |
349 |
109 |
sd |
79.7 |
144.4 |
75.6 |
6.1 |
48.2 |
72.6 |
115.5 |
92.3 |
46.2 |
23.4 |
|
f(I) |
5.01 |
5.04 |
7.27 |
9.27 |
13.03 |
13.27 |
2.38 |
2.43 |
38.78 |
12.11 |
|
5 µL/plate |
Mean |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
sd |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
|
f(I) |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
1.5 µL/plate |
Mean |
2 |
2 |
9 |
10 |
1 |
4 |
3 |
6 |
1 |
3 |
sd |
1.2 |
1.2 |
1.0 |
0.6 |
0.6 |
4.2 |
1.7 |
3.8 |
0.0 |
0.6 |
|
f(I) |
0.02 |
0.02 |
0.19 |
0.22 |
0.01 |
0.04 |
0.01 |
0.01 |
0.10 |
0.33 |
|
0.5 µL/plate |
Mean |
85 |
87 |
36 |
39 |
31 |
15 |
63 |
241 |
10 |
12 |
sd |
7.2 |
6.0 |
4.7 |
4.5 |
7.0 |
6.0 |
11.1 |
22.7 |
0.0 |
1.2 |
|
f(I) |
0.93 |
0.91 |
0.75 |
0.87 |
0.34 |
0.16 |
0.14 |
0.55 |
1.00 |
1.33 |
|
0.15 µL/plate |
Mean |
91 |
84 |
42 |
43 |
75 |
92 |
387 |
433 |
11 |
11 |
sd |
10.0 |
11.5 |
1.5 |
1.5 |
4.9 |
5.3 |
67.1 |
24.4 |
0.6 |
1.5 |
|
f(I) |
1.00 |
0.88 |
0.88 |
0.96 |
0.83 |
0.97 |
0.87 |
0.99 |
1.10 |
1.22 |
|
0.05 µL/plate |
Mean |
98 |
102 |
39 |
42 |
101 |
103 |
352 |
456 |
11 |
8 |
sd |
11.0 |
4.0 |
0.6 |
1.2 |
17.2 |
24.3 |
32.7 |
78.7 |
2.6 |
0.6 |
|
f(I) |
1.08 |
1.06 |
0.81 |
0.93 |
1.12 |
1.08 |
0.79 |
1.04 |
1.10 |
0.89 |
f(I) = increase factor
* Different positive controls were used
B) Experiment 1b
Confirmation of the Criteria and Validity
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
Solubility and Toxicity
In this experiment, the test substance showed no precipitates on the plates in all tested concentrations. The test substance showed signs of toxicity towards all five bacteria strains in both the absence and presence of metabolic activation in the highest concentration (1.5 µL/plate; no bacteria growth).The bacterial background lawn was not reduced at any of the concentrations.
Mutagenicity
No increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found. Therefore, the test substance is stated as not mutagenic under the conditions of this experiment.
Survey of the Findings
The mean revertant values of the three replicates are presented in the following table.
Table: Mean Revertants Experiment 1b
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
||||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
Demin. water |
Mean |
77 |
117 |
45 |
44 |
90 |
93 |
239 |
245 |
12 |
13 |
sd |
4.0 |
7.6 |
9.0 |
2.6 |
11.0 |
9.0 |
20.5 |
52.8 |
3.5 |
3.6 |
|
DMSO |
Mean |
87 |
108 |
44 |
41 |
94 |
109 |
207 |
203 |
11 |
12 |
sd |
7.4 |
22.5 |
10.5 |
4.5 |
12.5 |
9.9 |
43.1 |
10.1 |
1.2 |
2.9 |
|
Positive |
Mean |
621 |
497 |
305 |
483 |
717 |
941 |
1291 |
1349 |
145 |
313 |
sd |
128.6 |
55.2 |
2.3 |
120.1 |
57.2 |
219.4 |
101.3 |
74.3 |
37.2 |
68.9 |
|
f(I) |
7.14 |
4.60 |
6.93 |
11.78 |
7.97 |
8.63 |
6.24 |
6.65 |
12.08 |
26.08 |
|
1.5 µL/plate |
Mean |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
sd |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
|
f(I) |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
0.5 µL/plate |
Mean |
70 |
92 |
36 |
40 |
88 |
84 |
285 |
301 |
10 |
11 |
sd |
10.3 |
17.1 |
3.6 |
5.0 |
2.6 |
6.4 |
74.3 |
19.7 |
1.0 |
1.5 |
|
f(I) |
0.80 |
0.85 |
0.82 |
0.98 |
0.94 |
0.77 |
1.38 |
1.48 |
0.91 |
0.92 |
|
0.15 µL/plate |
Mean |
92 |
86 |
45 |
43 |
81 |
98 |
237 |
253 |
10 |
10 |
sd |
15.9 |
8.1 |
2.1 |
10.1 |
0.6 |
19.3 |
61.1 |
27.2 |
1.5 |
0.6 |
|
f(I) |
1.06 |
0.80 |
1.02 |
1.05 |
0.86 |
0.90 |
1.14 |
1.25 |
0.91 |
0.83 |
|
0.05 µL/plate |
Mean |
83 |
95 |
40 |
39 |
87 |
114 |
228 |
279 |
11 |
11 |
sd |
1.5 |
16.1 |
3.0 |
8.6 |
8.1 |
7.2 |
10.6 |
55.5 |
1.5 |
1.2 |
|
f(I) |
0.95 |
0.88 |
0.91 |
0.95 |
0.93 |
1.05 |
1.10 |
1.37 |
1.00 |
0.92 |
|
0.015 µL/plate |
Mean |
81 |
113 |
42 |
46 |
82 |
91 |
251 |
299 |
10 |
10 |
sd |
12.0 |
14.7 |
8.7 |
7.6 |
5.6 |
8.7 |
22.0 |
23.4 |
1.5 |
1.7 |
|
f(I) |
0.93 |
1.05 |
0.95 |
1.12 |
0.87 |
0.83 |
1.21 |
1.47 |
0.91 |
0.83 |
|
0.005 µL/plate |
Mean |
86 |
118 |
43 |
43 |
109 |
112 |
276 |
272 |
11 |
10 |
sd |
5.3 |
6.0 |
8.9 |
4.0 |
6.1 |
13.8 |
44.5 |
27.7 |
1.2 |
0.6 |
|
f(I) |
0.99 |
1.09 |
0.98 |
1.05 |
1.16 |
1.03 |
1.33 |
1.34 |
1.00 |
0.83 |
f(I) = increase factor
* Different positive controls were used
C) Experiment 2
Confirmation of the Criteria and Validity
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
Solubility and Toxicity
In this experiment, the test substanceshowed no precipitates on the plates in all tested concentrations.
The following signs of toxicity were observed towards the bacteria strains:
- Strain TA97a: 1.5 µL/plate, 0.75 µL/plate, 0.38 µL/plate (no bacteria growth and no bacterial background lawn)
- Strain TA98: 1.5 µL/plate, 0.75 µL/plate, 0.38 µL/plate (no bacteria growth and no bacterial background lawn)
- Strain TA100: 1.5 µL/plate, 0.75 µL/plate, 0.38 µL/plate (no bacteria growth and no bacterial background lawn), 0.19 µL/plate (decreasein the number of revertants)
- Strain TA102: 1.5 µL/plate, 0.75 µL/plate (no bacteria growth and no bacterial background lawn), 0.38 µL/plate (decreasein the number of revertants)
- Strain TA1535: 1.5 µL/plate, 0.75 µL/plate, 0.38 µL/plate (no bacteria growth and no bacterial background lawn), 0.19 µL/plate (decreasein the number of revertants)
Mutagenicity
No increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found. Therefore, the test substance is stated as not mutagenic under the conditions of this experiment.
Survey of the Findings
The mean revertant values of the three replicates are presented in the following table.
Table: Mean Revertants Experiment 2
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
||||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
Demin. water |
Mean |
82 |
110 |
42 |
41 |
99 |
113 |
245 |
235 |
12 |
11 |
sd |
10.1 |
6.9 |
1.2 |
10.0 |
5.0 |
6.4 |
34.0 |
8.3 |
1.7 |
2.6 |
|
DMSO |
Mean |
75 |
105 |
43 |
45 |
91 |
91 |
229 |
235 |
12 |
12 |
sd |
8.7 |
12.9 |
9.7 |
2.1 |
14.2 |
8.1 |
66.6 |
53.7 |
1.5 |
3.1 |
|
Positive |
Mean |
513 |
456 |
379 |
456 |
552 |
1205 |
1277 |
1389 |
291 |
251 |
sd |
40.5 |
113.4 |
53.3 |
36.7 |
138.8 |
212.3 |
120.4 |
44.1 |
32.3 |
28.1 |
|
f(I) |
6.84 |
4.34 |
8.81 |
10.13 |
5.58 |
13.24 |
5.58 |
5.91 |
24.25 |
20.92 |
|
1.5 µL/plate |
Mean |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
sd |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
|
f(I) |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
0.75 µL/plate |
Mean |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
sd |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
|
f(I) |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
0.38 µL/plate |
Mean |
0 |
0 |
0 |
0 |
0 |
0 |
14 |
54 |
0 |
0 |
sd |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
2.6 |
3.2 |
0.0 |
0.0 |
|
f(I) |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.06 |
0.23 |
0.00 |
0.00 |
|
0.19 µL/plate |
Mean |
79 |
80 |
33 |
40 |
9 |
3 |
211 |
219 |
3 |
3 |
sd |
12.6 |
7.1 |
5.5 |
1.5 |
2.1 |
3.5 |
16.2 |
16.2 |
0.0 |
0.6 |
|
f(I) |
1.05 |
0.76 |
0.77 |
0.89 |
0.10 |
0.03 |
0.92 |
0.93 |
0.25 |
0.25 |
|
0.09 µL/plate |
Mean |
83 |
85 |
39 |
39 |
81 |
83 |
248 |
291 |
10 |
8 |
sd |
9.5 |
9.6 |
2.1 |
3.6 |
10.5 |
11.3 |
73.0 |
54.0 |
0.0 |
1.5 |
|
f(I) |
1.11 |
0.81 |
0.91 |
0.87 |
0.89 |
0.91 |
1.08 |
1.24 |
0.83 |
0.67 |
|
0.05 µL/plate |
Mean |
100 |
85 |
38 |
34 |
78 |
106 |
214 |
271 |
10 |
13 |
sd |
6.4 |
3.1 |
5.5 |
3.1 |
3.8 |
17.1 |
8.7 |
51.1 |
1.0 |
1.5 |
|
f(I) |
1.33 |
0.81 |
0.88 |
0.76 |
0.86 |
1.16 |
0.93 |
1.15 |
0.83 |
1.08 |
|
0.03 µL/plate |
Mean |
85 |
95 |
44 |
37 |
93 |
78 |
238 |
265 |
12 |
14 |
sd |
6.1 |
16.5 |
2.5 |
5.3 |
12.0 |
5.5 |
17.4 |
39.7 |
2.9 |
0.6 |
|
f(I) |
1.13 |
0.90 |
1.02 |
0.82 |
1.02 |
0.86 |
1.04 |
1.13 |
1.00 |
1.17 |
|
0.02 µL/plate |
Mean |
84 |
73 |
41 |
41 |
91 |
89 |
283 |
267 |
9 |
11 |
sd |
9.6 |
1.5 |
10.5 |
14.2 |
7.0 |
8.3 |
10.3 |
49.7 |
1.7 |
2.9 |
|
f(I) |
1.12 |
0.70 |
0.95 |
0.91 |
1.00 |
0.98 |
1.24 |
1.14 |
0.75 |
0.92 |
|
0.01 µL/plate |
Mean |
76 |
122 |
41 |
49 |
94 |
107 |
259 |
263 |
14 |
12 |
sd |
11.6 |
7.2 |
3.0 |
2.9 |
11.1 |
17.0 |
21.4 |
16.0 |
2.6 |
1.0 |
|
f(I) |
1.01 |
1.16 |
0.95 |
1.09 |
1.03 |
1.18 |
1.13 |
1.12 |
1.17 |
1.00 |
f(I) = increase factor
* Different positive controls were used
Mutagenicity of Test substance
The test substanceshowed no increase in the number of revertants in all bacteria strains in all experiments. All negative and all strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that test substance is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.
Acceptability of Study, Discussion
In all experiments, no precipitation of the test substance was observed at any of the tested concentrations up to 5 µL/plate. The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value of 109 bacteria/mL. All of the means of all replicates of the spontaneous revertants (in negative and solvent controls) were within the range of the historical data of the test facility. All numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory and were increased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens. Since all criteria for acceptability have been met, the study is considered valid.
Applicant's summary and conclusion
- Conclusions:
- Under ths study conditions, the test substance was determined to be non-mutagenic with and without metabolic activation in the Ames test.
- Executive summary:
An in vitro study was conducted to determine the mutagenicity potential of the test substance, C12 -14 HEDMAC (39.6% active) in a bacterial reverse mutation test, according to the OECD Guideline 471 and EU Method B13/B14, in compliance with GLP. Using five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535), the test was performed in three experiments in the presence and absence of metabolic activation. In the experiment 1a, the test substance (5, 1.5, 0.5, 0.15, 0.05 µL/plate dissolved in DMSO) was tested using the plate incorporation method. The test substance showed no precipitates on the plates at any of the concentrations. The test substance showed signs of toxicity towards all five bacteria strains in both the absence and presence of metabolic activation in the highest concentration (5 µL/plate; no bacteria growth) and in the next lower concentration (1.5 µL/plate, decrease in the number of revertants). The bacterial background lawn was not reduced at any of the concentrations. The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation. Based on the toxicity results of the experiment 1a, the test substance (1.5, 0.5, 0.15, 0.05, 0.015, 0.005 µL/plate) was tested up to concentrations of 1.5 µL/plate using the plate incorporation method. This experiment was performed to verify the toxicity results of the experiment 1a with lower concentrations. The test substance showed no precipitates on the plates at any of the concentrations. The test substance showed signs of toxicity towards all five bacteria strains in both the absence and presence of metabolic activation in the highest concentration (1.5 µL/plate; no bacteria growth). The bacterial background lawn was not reduced at any of the concentrations. The results of this experiment showed that the test substance caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test substance did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation. Based on the results of the experiment 1a and 1b, the test substance (1.5, 0.75, 0.38, 0.19, 0.09, 0.05, 0.02, 0.01, 0.006 µL/plate) was tested up to concentrations of 1.5 µL/plate using the pre-incubation method. The test substance showed no precipitates on the plates at any of the concentrations. The following signs of toxicity were observed towards the bacteria strains: Strain TA97a: 1.5 µL/plate, 0.75 µL/plate, 0.38 µL/plate (no bacteria growth and no bacterial background lawn); Strain TA98: 1.5 µL/plate, 0.75 µL/plate, 0.38 µL/plate (no bacteria growth and no bacterial background lawn); Strain TA100: 1.5 µL/plate, 0.75 µL/plate, 0.38 µL/plate (no bacteria growth and no bacterial background lawn), 0.19 µL/plate (decrease in the number of revertants); Strain TA102: 1.5 µL/plate, 0.75 µL/plate (no bacteria growth and no bacterial background lawn), 0.38 µL/plate (decrease in the number of revertants); Strain TA1535: 1.5 µL/plate, 0.75 µL/plate, 0.38 µL/plate (no bacteria growth and no bacterial background lawn), 0.19 µL/plate (decrease in the number of revertants). Vehicle and positive control studies were considered valid. The results of this experiment showed that the test substance caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test substance did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation. Under the study conditions, the test substance was determined to be non-mutagenic with and without metabolic activation in the Ames test (Andres, 2019).
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