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EC number: 271-663-3 | CAS number: 68603-55-4
- 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:
- 2 018
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Amines, C12-14-alkyl, C6-10-alkyl phosphates
- EC Number:
- 271-663-3
- EC Name:
- Amines, C12-14-alkyl, C6-10-alkyl phosphates
- Cas Number:
- 68603-55-4
- Molecular formula:
- not available (UVCB)
- IUPAC Name:
- Amines, C12-14-alkyl, C6-10-alkyl phosphates
- Test material form:
- liquid: viscous
Constituent 1
Method
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 97
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- S. typhimurium TA 98
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- S. typhimurium TA 100
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- S. typhimurium TA 102
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- S. typhimurium TA 1535
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 Mix
- Test concentrations with justification for top dose:
- In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 mL/L in demineralized water, dimethyl sulfoxide (DMSO) and acetone. Acetone was chosen as vehicle, because the test item was sufficiently soluble in this solvent, and it does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations. On the day of the start of the experiment 1a, a stock solution containing 50 mL/L of the test item in acetone was prepared. The test item 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: 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 2a, a stock solution containing 5 mL/L of the test item in acetone was prepared. The following nominal concentrations were prepared for the experiment 1b: 0.5 µL/plate, 0.15 µL/plate, 0.05 µL/plate, 0.015 µL/plate, 0.005 µL/plate and 0.0015 µL/plate. The following nominal concentrations were prepared for the experiment 2a: 0.5 µL/plate, 0.25 µL/plate, 0.125 µL/plate, 0.063 µL/plate, 0.031 µL/plate, 0.016 µL/plate and 0.08 µL/plate. On the day of the start of the experiment 2b, a stock solution containing 1.25 mL/L of the test item in acetone was prepared. The following nominal concentrations were prepared for the experiment 2b for the bacteria strains TA98, TA102 and TA1535: 0.125 µL/plate, 0.063 µL/plate, 0.031 µL/plate, 0.016 µL/plate, 0.008 µL/plate and 0.004 µL/plate. The following nominal concentrations were prepared for the experiment 2b for the bacte-ria strains TA97a and TA100: 0.016 µL/plate, 0.008 µL/plate, 0.004 µL/plate, 0.002 µL/plate, 0.001 µL/plate and 0.0005 µL/plate.
- Vehicle / solvent:
- Acetone
Controls
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- 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:
- Mutation present in strain
Name Category Effect TA97a TA98 TA100 TA102 TA1535
hisD6610 frame shift histidine deficiency x
hisD3052 frame shift histidine deficiency x
hisG46 base pair substitution histidine deficiency x x
hisG428 base pair substitution histidine deficiency x
uvrB deletion UV sensitivity, biotin deficiency x x x x
rfa deletion lipopolysaccharide side chain x x x x x
pKM101 plasmid ampicillin resistance x x x x
pAQ1 plasmid tetracycline re-sistance x
All Salmonella typhimurium strains were obtained from TRINOVA BioChem GmbH (batch: TA97a: 4997D, TA98: 5011D, TA100: 4996D, TA102: 4982D, TA1535: 5012D) 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. - Evaluation criteria:
- The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard devia-tions 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 item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of re-vertant 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:
- S. typhimurium TA 97
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- The test item showed signs of toxicity towards all the bacteria strains in both the absence and presence of metabolic activation (details see in other information on results)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- The test item showed signs of toxicity towards all the bacteria strains in both the absence and presence of metabolic activation (details see in other information on results)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- 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:
- The test item showed signs of toxicity towards all the bacteria strains in both the absence and presence of metabolic activation (details see in other information on results)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- 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:
- The test item showed signs of toxicity towards all the bacteria strains in both the absence and presence of metabolic activation (details see in other information on results)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- The test item showed signs of toxicity towards all the bacteria strains in both the absence and presence of metabolic activation (details see in other information on results)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
Any other information on results incl. tables
The detailed data of all the experiments are listed in the annex (Exp. 1a see chapter 12, page 35ff., Exp. 1b see chapter 13, page40ff., Exp. 2a see chapter14, page 45, Exp. 2b see chapter 15, page 50).
Confirmation of genotype is performedfor each batch of lyophilized bacteria before stock culture preparation. The last performance showed no abnormalities .
1.1 Experiment 1a
1.1.1 Confirmation of the Criteria and Validity
All strains met the criterion of at least 109bacteria/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 (historical data of the laboratory see chapter 17, page 57).All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
1.1.2 Solubility and Toxicity
In this experiment, the test itemshowed no precipitates on the plates in all tested concentrations.
At the three highest concentrations (5, 1.5 and 0.5 µL/plate) no bacteria growth and no bacterial lawn was observed. The test itemshowed signs of toxicity towards all the bacteria strains in both the absence and presence of metabolic activation in these concentrations.
At the two lower concentrations, the bacterial background lawn was not reduced and no decrease of the spontaneous revertants was observed.
1.1.3 Mutagenicity
No significant 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.
Based on the toxicity results, a repetition of this experiment with lower concentrations was performed.
1.1.4 Survey of the Findings
The mean revertant values of the three replicates are presented in the following table.
Table8.1‑a Mean Revertants Experiment 1a
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
||||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
Demin. water |
Mean |
73 |
81 |
26 |
25 |
77 |
93 |
308 |
353 |
19 |
23 |
sd |
7.6 |
11.3 |
4.7 |
6.2 |
7.5 |
7.0 |
45.1 |
4.6 |
1.0 |
4.2 |
|
DMSO |
Mean |
67 |
85 |
26 |
29 |
72 |
83 |
305 |
292 |
19 |
21 |
sd |
3.6 |
13.0 |
3.6 |
6.5 |
7.8 |
9.5 |
45.5 |
56.7 |
3.8 |
4.7 |
|
Acetone |
Mean |
76 |
83 |
28 |
30 |
80 |
93 |
344 |
329 |
19 |
21 |
sd |
17.2 |
20.7 |
4.7 |
5.6 |
10.2 |
11.8 |
47.2 |
84.3 |
3.5 |
3.0 |
|
Positive |
Mean |
520 |
609 |
407 |
86 |
324 |
1001 |
1005 |
1363 |
224 |
247 |
sd |
84.3 |
136.8 |
56.0 |
6.8 |
55.6 |
0.0 |
334.9 |
224.2 |
49.2 |
48.1 |
|
f(I) |
7.76 |
7.16 |
15.65 |
2.97 |
4.21 |
12.06 |
3.30 |
4.67 |
11.79 |
11.76 |
|
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 |
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 |
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 |
|
0.15 µL/plate |
Mean |
69 |
59 |
20 |
31 |
73 |
82 |
255 |
227 |
22 |
16 |
sd |
2.9 |
7.2 |
1.0 |
1.7 |
7.5 |
14.4 |
82.4 |
44.1 |
5.0 |
4.7 |
|
f(I) |
0.91 |
0.71 |
0.71 |
1.03 |
0.91 |
0.88 |
0.74 |
0.69 |
1.16 |
0.76 |
|
0.05 µL/plate |
Mean |
74 |
81 |
27 |
29 |
83 |
79 |
277 |
308 |
14 |
18 |
sd |
10.3 |
12.0 |
5.2 |
4.6 |
8.0 |
11.3 |
73.4 |
39.4 |
3.6 |
1.0 |
|
f(I) |
0.97 |
0.98 |
0.96 |
0.97 |
1.04 |
0.85 |
0.81 |
0.94 |
0.74 |
0.86 |
f(I) = increase factor, calculation see chapter7.4, page23
* Different positive controls were used, see chapter6.3, page13
1001 colonies per plate means the bacteria growth was too strong for counting
1.2 Experiment 1b
1.2.1 Confirmation of the Criteria and Validity
All strains met the criterion of at least 109bacteria/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 (historical data of the laboratory see chapter17, page57).All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
1.2.2 Solubility and Toxicity
In this experiment, the test itemshowed no precipitates on the plates in all tested concentrations.
At the highest concentration (0.5 µL/plate), no bacteria growth was observed in the following bacteria strains: TA97a, TA98 and TA100. Towards the bacteria strains TA102 and TA1535 a clear decrease in the spontaneous revertants was observed.
The bacterial background lawn was visible in all concentrations.
1.2.3 Mutagenicity
No significant 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 item is stated as not mutagenic under the test conditions.
1.2.4 Survey of the Findings
The mean revertant values of the three replicates are presented in the following table.
Table8.2‑a Mean Revertants Experiment 1b
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
||||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
Demin. water |
Mean |
79 |
100 |
32 |
34 |
97 |
98 |
311 |
285 |
12 |
16 |
sd |
12.6 |
17.6 |
3.1 |
2.5 |
5.1 |
14.2 |
28.1 |
78.0 |
4.4 |
1.5 |
|
DMSO |
Mean |
89 |
84 |
36 |
31 |
93 |
107 |
290 |
308 |
12 |
18 |
sd |
15.0 |
0.6 |
4.6 |
1.5 |
8.5 |
9.9 |
67.7 |
45.1 |
2.9 |
1.5 |
|
Acetone |
Mean |
79 |
81 |
32 |
32 |
99 |
105 |
432 |
363 |
20 |
15 |
sd |
5.7 |
15.2 |
4.0 |
2.3 |
11.4 |
6.1 |
82.7 |
48.1 |
7.5 |
2.9 |
|
Positive |
Mean |
605 |
747 |
608 |
155 |
291 |
1001 |
1496 |
817 |
413 |
201 |
sd |
101.7 |
61.1 |
89.1 |
63.6 |
33.5 |
0.0 |
72.0 |
86.9 |
177.1 |
18.5 |
|
f(I) |
6.80 |
8.89 |
16.89 |
5.00 |
3.00 |
9.36 |
5.16 |
2.65 |
34.42 |
11.17 |
|
0.5 µL/plate |
Mean |
0 |
0 |
0 |
0 |
0 |
0 |
68 |
57 |
3 |
2 |
sd |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
25.5 |
4.2 |
0.6 |
1.2 |
|
f(I) |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.16 |
0.16 |
0.15 |
0.13 |
|
0.15 µL/plate |
Mean |
67 |
80 |
29 |
30 |
80 |
90 |
241 |
209 |
13 |
12 |
sd |
8.0 |
12.5 |
7.8 |
2.0 |
12.8 |
11.9 |
68.4 |
35.9 |
1.0 |
3.8 |
|
f(I) |
0.85 |
0.99 |
0.91 |
0.94 |
0.81 |
0.86 |
0.56 |
0.58 |
0.65 |
0.80 |
|
0.05 µL/plate |
Mean |
84 |
82 |
35 |
28 |
72 |
96 |
235 |
311 |
18 |
16 |
sd |
0.6 |
15.9 |
1.5 |
6.2 |
8.0 |
6.7 |
15.1 |
47.7 |
2.3 |
2.9 |
|
f(I) |
1.06 |
1.01 |
1.09 |
0.88 |
0.73 |
0.91 |
0.54 |
0.86 |
0.90 |
1.07 |
|
0.015 µL/plate |
Mean |
76 |
83 |
25 |
32 |
88 |
110 |
265 |
292 |
14 |
17 |
sd |
10.4 |
11.1 |
4.7 |
6.5 |
6.0 |
12.0 |
74.2 |
6.9 |
2.5 |
1.7 |
|
f(I) |
0.96 |
1.02 |
0.78 |
1.00 |
0.89 |
1.05 |
0.61 |
0.80 |
0.70 |
1.13 |
|
0.005 µL/plate |
Mean |
100 |
96 |
33 |
32 |
99 |
99 |
249 |
273 |
13 |
14 |
sd |
22.1 |
26.5 |
10.1 |
3.6 |
14.7 |
24.1 |
12.9 |
19.7 |
3.1 |
2.1 |
|
f(I) |
1.27 |
1.19 |
1.03 |
1.00 |
1.00 |
0.94 |
0.58 |
0.75 |
0.65 |
0.93 |
|
0.0015 µL/plate |
Mean |
71 |
66 |
35 |
25 |
95 |
101 |
233 |
239 |
13 |
11 |
sd |
4.7 |
11.0 |
5.2 |
5.5 |
11.7 |
18.0 |
48.1 |
38.9 |
1.7 |
2.1 |
|
f(I) |
0.90 |
0.81 |
1.09 |
0.78 |
0.96 |
0.96 |
0.54 |
0.66 |
0.65 |
0.73 |
f(I) = increase factor, calculation see chapter7.4, page23
* Different positive controls were used, see chapter6.3, page13
1001 colonies per plate means the bacteria growth was too strong for counting
1.3 Experiment 2a
1.3.1 Confirmation of the Criteria and Validity
All strains met the criterion of at least 109bacteria/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 (historical data of the laboratory see chapter17, page57).All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
1.3.2 Solubility and Toxicity
In this experiment, the test itemshowed no precipitates on the plates in all tested concentrations.
At the three highest concentrations (0.5, 0.25 and 0.125 µL/plate) no bacteria growth and no bacterial lawn was visible. The test itemshowed signs of toxicity towards all bacteria strains in both the absence and presence of metabolic activation in these concentrations.
Towards the bacteria strains TA97a and TA100, signs of toxicity were also observed in the concentrations 0.063, 0.031 and 0.016 µL/plate.
1.3.3 Mutagenicity
No significant 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.
Due to the toxicity results, the experiment was repeated.
1.3.4 Survey of the Findings
The mean revertant values of the three replicates are presented in the following table.
Table8.3‑a Mean Revertants Experiment 2a
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
||||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
Demin. water |
Mean |
74 |
81 |
36 |
33 |
84 |
100 |
280 |
341 |
21 |
11 |
sd |
5.6 |
5.0 |
3.1 |
2.1 |
11.1 |
19.1 |
34.6 |
124.7 |
7.0 |
1.0 |
|
DMSO |
Mean |
76 |
78 |
33 |
35 |
84 |
92 |
283 |
343 |
12 |
12 |
sd |
16.0 |
13.6 |
4.2 |
3.6 |
8.5 |
13.9 |
14.0 |
37.2 |
2.0 |
2.6 |
|
Acetone |
Mean |
80 |
85 |
38 |
33 |
83 |
77 |
301 |
349 |
15 |
13 |
sd |
1.0 |
9.0 |
15.0 |
7.2 |
9.8 |
5.9 |
20.1 |
42.8 |
3.2 |
1.7 |
|
Positive |
Mean |
295 |
495 |
129 |
128 |
269 |
361 |
661 |
720 |
272 |
99 |
sd |
37.8 |
177.6 |
23.4 |
24.3 |
83.9 |
2.3 |
59.4 |
14.4 |
90.1 |
12.9 |
|
f(I) |
3.88 |
6.35 |
3.91 |
3.66 |
3.20 |
3.92 |
2.34 |
2.10 |
12.95 |
8.25 |
|
0.5 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 |
|
0.25 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 |
|
0.125 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 |
|
0.063 µL/plate |
Mean |
2 |
6 |
37 |
29 |
14 |
9 |
280 |
239 |
13 |
14 |
sd |
1.2 |
0.0 |
5.9 |
7.9 |
3.8 |
1.5 |
28.0 |
37.0 |
1.2 |
3.2 |
|
f(I) |
0.03 |
0.07 |
0.97 |
0.88 |
0.17 |
0.12 |
0.93 |
0.68 |
0.87 |
1.08 |
|
0.031 µL/plate |
Mean |
21 |
32 |
36 |
32 |
23 |
45 |
277 |
297 |
13 |
14 |
sd |
2.1 |
13.0 |
7.6 |
5.0 |
15.5 |
3.6 |
51.4 |
52.8 |
0.0 |
3.5 |
|
f(I) |
0.26 |
0.38 |
0.95 |
0.97 |
0.28 |
0.58 |
0.92 |
0.85 |
0.87 |
1.08 |
|
0.0016 µL/plate |
Mean |
10 |
36 |
31 |
32 |
21 |
39 |
264 |
285 |
15 |
17 |
sd |
1.2 |
7.2 |
7.1 |
7.8 |
7.2 |
9.9 |
52.9 |
30.6 |
3.5 |
2.1 |
|
f(I) |
0.13 |
0.42 |
0.82 |
0.97 |
0.25 |
0.51 |
0.88 |
0.82 |
1.00 |
1.31 |
|
0.008 µL/plate |
Mean |
65 |
83 |
36 |
36 |
82 |
83 |
265 |
248 |
14 |
16 |
sd |
6.0 |
7.6 |
4.5 |
4.2 |
8.2 |
9.8 |
30.6 |
74.1 |
3.2 |
1.7 |
|
f(I) |
0.81 |
0.98 |
0.95 |
1.09 |
0.99 |
1.08 |
0.88 |
0.71 |
0.93 |
1.23 |
f(I) = increase factor, calculation see chapter7.4, page23
* Different positive controls were used, see chapter6.3, page13
1.4 Experiment 2b
1.4.1 Confirmation of the Criteria and Validity
All strains met the criterion of at least 109bacteria/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 (historical data of the laboratory see chapter17, page57).All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
1.4.2 Solubility and Toxicity
In this experiment, the test itemshowed no precipitates on the plates in all tested concentrations.
Signs of toxicity were observed in the following concentrations towards the respective bacteria strains:
· TA97a: 0.016 µL/plate (a decrease in the spontaneous revertants with and without S9 mix)
· TA98: 0.125 µL/plate (in the treatment without S9 mix no bacteria growth was observed, in the treatment with S9 mix a decrease in the spontaneous revertants was observed)
· TA100: 0.016 µL/plate (a decrease in the spontaneous revertants with and without S9 mix)
· TA102: 0.125 µL/plate (a decrease in the spontaneous revertants with and without S9 mix)
· TA1535: 0.125 µL/plate (no bacteria growth was observed in the treatment with and without S9 mix)
1.4.3 Mutagenicity
No significant 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 item is stated as not mutagenic under the test conditions.
1.4.4 Survey of the Findings
The mean revertant values of the three replicates are presented in the following table.
Table8.4‑a Mean Revertants Experiment 2b
Strain |
TA98 |
TA102 |
TA1535 |
||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
Demin. water |
Mean |
31 |
35 |
269 |
244 |
26 |
22 |
sd |
5.1 |
4.7 |
12.2 |
4.0 |
4.0 |
3.5 |
|
DMSO |
Mean |
35 |
33 |
307 |
311 |
21 |
21 |
sd |
3.5 |
6.9 |
46.9 |
6.1 |
3.5 |
1.5 |
|
Acetone |
Mean |
35 |
40 |
253 |
264 |
23 |
25 |
sd |
5.5 |
3.1 |
10.1 |
42.3 |
3.2 |
3.5 |
|
Positive |
Mean |
441 |
104 |
1320 |
1312 |
331 |
272 |
sd |
18.9 |
18.0 |
36.7 |
42.3 |
53.3 |
32.0 |
|
f(I) |
12.60 |
3.15 |
4.30 |
4.22 |
12.73 |
12.95 |
|
0.125 µL/plate |
Mean |
0 |
3 |
90 |
58 |
0 |
0 |
sd |
0.0 |
2.9 |
24.0 |
5.3 |
0.0 |
0.0 |
|
f(I) |
0.00 |
0.08 |
0.36 |
0.22 |
0.00 |
0.00 |
|
0.063 µL/plate |
Mean |
28 |
31 |
264 |
223 |
20 |
20 |
sd |
4.4 |
6.0 |
0.0 |
20.1 |
6.0 |
2.5 |
|
f(I) |
0.80 |
0.78 |
1.04 |
0.84 |
0.87 |
0.80 |
|
0.031 µL/plate |
Mean |
28 |
34 |
240 |
277 |
20 |
20 |
sd |
8.9 |
6.0 |
22.3 |
55.5 |
3.2 |
2.6 |
|
f(I) |
0.80 |
0.85 |
0.95 |
1.05 |
0.87 |
0.80 |
|
0.016 µL/plate |
Mean |
37 |
33 |
225 |
252 |
19 |
19 |
sd |
3.1 |
3.5 |
34.0 |
34.9 |
5.7 |
3.6 |
|
f(I) |
1.06 |
0.83 |
0.89 |
0.95 |
0.83 |
0.76 |
|
0.008 µL/plate |
Mean |
34 |
32 |
244 |
265 |
21 |
17 |
sd |
2.1 |
4.6 |
4.0 |
18.5 |
4.4 |
0.6 |
|
f(I) |
0.97 |
0.80 |
0.96 |
1.00 |
0.91 |
0.68 |
|
0.004 µL/plate |
Mean |
35 |
32 |
223 |
264 |
18 |
22 |
sd |
2.1 |
5.1 |
18.9 |
28.0 |
2.6 |
3.1 |
|
f(I) |
1.00 |
0.80 |
0.88 |
1.00 |
0.78 |
0.88 |
f(I) = increase factor, calculation see chapter7.4, page23
* Different positive controls were used, see chapter6.3, page13
1001 colonies per plate means the bacteria growth was too strong for counting
Table8.4‑b Mean Revertants Experiment 2b
Strain |
TA97a |
TA100 |
|||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
|
Demin. water |
Mean |
83 |
81 |
115 |
116 |
sd |
14.4 |
8.1 |
7.5 |
8.4 |
|
DMSO |
Mean |
76 |
90 |
86 |
92 |
sd |
11.6 |
10.6 |
8.4 |
12.5 |
|
Acetone |
Mean |
85 |
78 |
93 |
105 |
sd |
7.2 |
14.2 |
12.2 |
14.3 |
|
Positive |
Mean |
331 |
313 |
531 |
1001 |
sd |
40.1 |
34.0 |
45.5 |
0.0 |
|
f(I) |
4.36 |
3.48 |
4.62 |
10.88 |
|
0.016 µL/plate |
Mean |
16 |
23 |
33 |
31 |
sd |
0.0 |
7.4 |
3.8 |
5.5 |
|
f(I) |
0.19 |
0.29 |
0.35 |
0.30 |
|
0.008 µL/plate |
Mean |
65 |
85 |
79 |
96 |
sd |
1.2 |
21.9 |
4.6 |
25.5 |
|
f(I) |
0.76 |
1.09 |
0.85 |
0.91 |
|
0.004 µL/plate |
Mean |
75 |
80 |
93 |
114 |
sd |
16.7 |
5.3 |
15.0 |
11.1 |
|
f(I) |
0.88 |
1.03 |
1.00 |
1.09 |
|
0.002µL/plate |
Mean |
76 |
79 |
81 |
107 |
sd |
8.4 |
11.4 |
5.5 |
9.5 |
|
f(I) |
0.89 |
1.01 |
0.87 |
1.02 |
|
0.001 µL/plate |
Mean |
71 |
100 |
83 |
110 |
sd |
14.7 |
16.8 |
6.4 |
17.9 |
|
f(I) |
0.84 |
1.28 |
0.89 |
1.05 |
|
0.0005µL/plate |
Mean |
74 |
106 |
102 |
114 |
sd |
7.2 |
15.9 |
22.8 |
8.7 |
|
f(I) |
0.87 |
1.36 |
1.10 |
1.09 |
f(I) = increase factor, calculation see chapter7.4, page23
* Different positive controls were used, see chapter6.3, page13
1001 colonies per plate means the bacteria growth was too strong for counting
1.5 Mutagenicity of Test Item
The test item Deophos 228showed 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 thatDeophos 228is not mutagenic in theSalmonella typhimuriumtest strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.
Applicant's summary and conclusion
- Conclusions:
- In all experiments, no precipitation of the test item Deophos 228 was observed at any of the tested concentrations up to 5 µL/plate. The test item showed toxicity towards all bacteria strains in all experiments. 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 (historical data of the laboratory chapter 17, page 57) 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.
- Executive summary:
Four valid experiments were performed. The study procedures described in this report were based on the most recent OECD and EC guidelines. The test item Deophos 228 was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535). The test was performed in four experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.
Experiment 1a:
In this experiment,the test item (dissolved in acetone) was tested up to concentrations of 5 µL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.
The test item showed no precipitates on the plates at any of the concentrations.
At the three highest concentrations (5, 1.5 and 0.5 µL/plate) no bacteria growth and no bacterial lawn was observed. The test itemshowed signs of toxicity towards all bacteria strains in both the absence and presence of metabolic activation in these concentrations.
At the lower two concentrations, the bacterial background lawn was not reduced and no decrease of the spontaneous revertants was observed.
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.
Experiment 1b:
Based on the toxicity results of the experiment 1a,the test item was tested up to concentrations of 0.5 µL/plate in the absence and presence of S9-mix in all bacteria strains using the plate incorporation method.
The test item showed no precipitates on the plates at any of the concentrations.
At the highest concentration (0.5 µL/plate), no bacteria growth was observed in the following bacteria strains: TA97a, TA98 and TA100. Towards the bacteria strains TA102 and TA1535 a clear decrease in the spontaneous revertants was observed.
The bacterial background lawn was observed in all concentrations.
The results of this experiments showed that the test item 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 item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.
Experiment 2a:
In this experiment, the test item (dissolved in acetone) was tested up to concentrations of 0.5 µL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the pre-incubation method.
The test item showed no precipitates on the plates at any of the concentrations.
At the three highest concentrations (0.5, 0.25 and 0.125 µL/plate) no bacteria growth and no bacterial lawn was visible. The test itemshowed signs of toxicity towards all bacteria strains in both the absence and presence of metabolic activation in these concentrations.
Towards the bacteria strains TA97a and TA100, signs of toxicity were observed in the concentrations 0.063, 0.031 and 0.016 µL/plate, too.
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.
Experiment 2b:
In this experiment, the test item was tested up to concentrations of 0.125 µL/plate in the absence and presence of S9-mix in the bacteria strains TA98, TA102 and TA1535 resp. up to concentrations of 0.016 µL/plate in the absence and presence of S9-mix in the bacteria strains TA97a and TA100.
The test item showed no precipitates on the plates at any of the concentrations.
Signs of toxicity were observed in the following concentrations towards the respective bacteria strains:
· TA97a: 0.016 µL/plate (a decrease in the spontaneous revertants with and without S9 mix)
· TA98: 0.125 µL/plate (in the treatment without S9 mix no bacteria growth was observed, in the treatment with S9 mix a decrease in the spontaneous revertants was observed)
· TA100: 0.016 µL/plate (a decrease in the spontaneous revertants with and without S9 mix)
· TA102: 0.125 µL/plate (a decrease in the spontaneous revertants with and without S9 mix)
· TA1535: 0.125 µL/plate (no bacteria growth was observed in the treatment with and without S9 mix)
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 results of this study it is concluded that Deophos 228 is not mutagenic in the Salmonella typhimuriumstrains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.
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