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EC number: 433-060-5 | CAS number: 290822-07-0
- 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
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- Nanomaterial surface chemistry
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- Endpoint summary
- Stability
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- 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
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- Genetic toxicity
- Carcinogenicity
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- Specific investigations
- Exposure related observations in humans
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- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 011
- Report date:
- 2011
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Test material
- Details on test material:
- - Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance preparations.
- Storage stability: The stability of the test substance under storage conditions was guaranteed until 27 Sep 2014 as indicated by the sponsor and the sponsor holds this responsibility
- Physical state, appearance: Solid, white
- Storage conditions: Room temperature
Constituent 1
Method
- Target gene:
- HGPRT
Species / strain
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- - high proliferation rate (doubling time of about 12 - 16 hours)
- high plating efficiency (about 90%)
- karyotype with a modal number of 20 chromosomes
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix from phenobarbital and β-naphthoflavone induced rat liver
- Test concentrations with justification for top dose:
- 1st Experiment
without S9 mix (4-hour exposure period)
0; 3.13; 6.25; 12.5; 25.0; 50.0; 100.0; 200.0 μg/mL
with S9 mix (4-hour exposure period)
0; 3.13; 6.25; 12.5; 25.0; 50.0; 100.0; 200.0 μg/mL
2nd Experiment
without S9 mix
0; 12.5; 25.0; 50.0; 100.0; 200.0 μg/mL
with S9 mix
0; 18.8; 37.5; 75.0; 150.0; 200.0 μg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: tetrahydrofurane (THF)
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water and other commonly used vehicles (e.g. DMSO, ethanol, acetone), tetrahydrofurane (THF) was selected as the vehicle, which had been demonstrated to be suitable in the CHO/HPRT assay. The final concentration of the vehicle THF in the culture medium was 0.5% (v/v).
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- with and without S9 mix
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- other: methylcholanthrene (with metabolic activation, 20 µg/mL)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- pretreatment: During the week prior to treatment, spontaneous HPRT-deficient mutants were eliminated by pretreatment with "HAT" medium.
- Exposure duration: 20-24h
- Expression time (cells in growth medium): 4 and 24h --> washing and first cytotox determination, 1. passage day 5, days 7-9 2. passage into selection medium (TG medium) and second cytotox determination
- Fixation time (start of exposure up to fixation or harvest of cells): from day 16 drying, fixation, staining and counting of the selected colonies
SELECTION AGENT (mutation assays): 6-thioguanine
NUMBER OF REPLICATIONS: 2 independent experiments
NUMBER OF CELLS EVALUATED: number of colony per flask was counted and recorded
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
OTHER EXAMINATIONS:
pH, osmolarity, solubility, cel morphology - Evaluation criteria:
- A finding is assessed as positive if the following criteria are met:
• Increase of the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range (see Appendix 5).
• Evidence of reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship. Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 106 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significant increased above the concurrent negative control and is within our historical negative control data range. - Statistics:
- Due to the clearly negative findings, a statistical evaluation was not carried out.
Results and discussion
Test results
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES:
In the pretest for toxicity based on the solubility properties of the test substance in the most suitable vehicle 2500 μg/mL (approx. 5 mM) was used as top concentration both with and without S9 mix at 4-hour exposure time and without S9 mix at 24-hour exposure time. Higher concentrations could not be formulated in a suitable solvent. The pretest was performed following the method described for the main experiment. The cloning efficiency 1 (survival) was determined as toxicity indicator for dose selection and various parameters were checked for all or at least for some selected doses. In the pretest the parameters pH value and osmolarity were not influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. In culture medium test substance precipitation occurred at 78.1 μg/mL and above at the end of treatment in the absence and presence of S9 mix. After 4 and 24 hours treatment in the absence and presence of S9 mix no cytotoxicity indicated by reduced relative cloning efficiency of about or below 20% was observed.
Any other information on results incl. tables
In this study, no relevant increase in the number of mutant colonies was observed either without S9 mix or after the addition of a metabolizing system. In both experiments after 4 and 24 hours treatment with the test substance the values for the corrected mutation frequencies (MFcorr.: 0.00 – 9.68 per 106 cells) were close to the respective vehicle control values (MFcorr.: 0.50 – 6.92 per 106 cells) and clearly within the range of the historical negative control data (without S9 mix: MFcorr.: 0.00 – 15.95 per 106 cells; with S9 mix: MFcorr.: 0.00 – 15.68 per 106 cells). The positive control substances EMS (without S9 mix; 300 μg/mL) and MCA (with S9 mix; 20 μg/mL) induced clearly increased mutant frequencies as expected. The values of the corrected mutant frequencies (without S9 mix: MFcorr.: 108.87 – 381.88 per 106 cells; with S9 mix: MFcorr.: 48.01 – 72.64 per 106 cells) were clearly within the historical positive control data range (without S9 mix: MFcorr.: 48.83 – 1338.10 per 106 cells; with S9 mix: MFcorr.: 26.29 – 413.54 per 106 cells).
Table 5: Summary of results
Exp. |
Exposure period |
Test groups |
S9 mix |
Prec.* |
Genotoxicity** MFcorr. [per 106cells] |
Cytotoxicity*** |
|
CE1 [%] |
CE2 [%] |
||||||
1 |
4 h |
Vehicle control 3.13 µg/mL 6.25 µg/mL 12.50 µg/mL 25.00 µg/mL 50.00 µg/mL 100.00 µg/mL 200.00 µg/mL Positive control |
- - - - - - - - - |
- - - - - + + + - |
0.50 n.c. n.c. 2.12 0.00 1.19 4.44 2.56 108.87 |
100.0 100.9 94.7 89.9 94.9 91.1 93.8 96.2 96.2 |
100.0 n.c. n.c. 117.4 86.4 99.2 95.4 100.3 94.1 |
2 |
24 h |
Vehicle control 12.50 µg/mL 25.00 µg/mL 50.00 µg/mL 100.0 µg/mL 200.00 µg/mL Positive control |
- - - - - - - |
- - - + + + - |
1.58 5.84 1.58 2.02 1.54 2.90 381.88 |
100.0 107.9 101.0 91.4 88.6 92.3 71.7 |
100.0 85.9 80.2 79.6 82.3 95.7 70.5 |
1 |
4 h |
Vehicle control 3.13 µg/mL 6.25 µg/mL 12.50 µg/mL 25.00 µg/mL 50.00 µg/mL 100.00 µg/mL 200.00 µg/mL Positive control |
+ + + + + + + + + |
- - - - - + + + - |
3.46 n.c. n.c. 5.10 1.84 0.47 1.97 3.17 72.64 |
100.0 96.5 89.8 97.7 86.9 96.8 95.4 93.3 94.4 |
100.0 n.c. n.c. 92.6 93.7 96.9 96.7 90.6 91.2 |
2 |
4 h |
Vehicle control 18.80 µg/mL 37.50 µg/mL 75.00 µg/mL 150.00 µg/mL 200.00 µg/mL Positive control |
+ + + + + + + |
- - - + + + - |
6.92 1.44 0.00 0.35 9.68 4.45 48.01 |
100.0 107.9 99.5 110.7 108.4 117.2 124.3 |
100.0 98.2 102.0 103.9 95.9 79.5 88.7 |
* Precipitation in culture medium at the end of exposure period
** Mutant frequency MFcorr.: mutant colonies per 106 cells corrected with the CE2 value
*** Cloning efficiency related to the respective vehicle control
n.c. Culture was not continued since a minimum of only four analysable concentrations is required
Table 6: Cytotoxicity data - 1st Experiment without S9 mix; 4 -hour exposure period
Test groups |
Cell density (x 105/mL) At 1stsub-culture |
CE1(survival) (4 h after treatment; about 200 cells/flask seeded) |
CE2(viability) (at the end of the expression period; about 200 cells/flask seeded) |
|||||||
Cells Flask 1 |
Cells Flask 2 |
Cloning efficiency [%] |
Cells Flask 1 |
Cells Flask 2 |
Cloning efficiency [%] |
|||||
Abs. |
Rel. |
Abs. |
Rel. |
|||||||
Vehicle control* |
A |
3.76 |
109 |
125 |
66.3 |
100.0 |
142 |
137 |
63.1 |
100.0 |
B |
6.19 |
155 |
141 |
110 |
115 |
|||||
3.13 µg/mL |
A |
5.75 |
116 |
142 |
66.9 |
100.9 |
n.c. |
n.c. |
n.c. |
n.c. |
B |
6.13 |
132 |
145 |
n.c. |
n.c. |
|||||
6.25 µg/mL |
A |
5.52 |
115 |
110 |
62.8 |
94.7 |
n.c. |
n.c. |
n.c. |
n.c. |
B |
6.26 |
132 |
145 |
n.c. |
n.c. |
|||||
12.50 µg/mL |
A |
5.56 |
126 |
111 |
59.6 |
89.9 |
137 |
128 |
74.1 |
117.4 |
B |
6.46 |
136 |
103 |
138 |
189 |
|||||
25.00 µg/mL |
A |
5.35 |
114 |
112 |
62.9 |
94.9 |
135 |
95 |
54.5 |
86.4 |
B |
7.95 |
128 |
149 |
110 |
96 |
|||||
50.00 µg/mL |
A |
5.23 |
117 |
123 |
60.4 |
91.1 |
111 |
108 |
62.6 |
99.2 |
B |
6.94 |
124 |
119 |
149 |
132 |
|||||
100.00 µg/mL |
A |
6.43 |
109 |
131 |
62.2 |
93.8 |
119 |
109 |
60.2 |
95.4 |
B |
7.72 |
138 |
119 |
125 |
128 |
|||||
200.00 µg/mL |
A |
5.18 |
109 |
125 |
63.8 |
96.2 |
153 |
142 |
63.3 |
100.3 |
B |
6.86 |
134 |
142 |
114 |
97 |
|||||
300.00 µg/mL EMS |
A |
5.52 |
120 |
122 |
63.8 |
96.2 |
116 |
103 |
59.4 |
94.1 |
B |
6.51 |
133 |
135 |
150 |
106 |
*THF 0.5 % (v/v)
Table 7: Cytotoxicity data - 1st Experiment with S9 mix; 4 -hour exposure period
Test groups |
Cell density (x 105/mL) At 1stsub-culture |
CE1(survival) (4 h after treatment; about 200 cells/flask seeded) |
CE2(viability) (at the end of the expression period; about 200 cells/flask seeded) |
|||||||
Cells Flask 1 |
Cells Flask 2 |
Cloning efficiency [%] |
Cells Flask 1 |
Cells Flask 2 |
Cloning efficiency [%] |
|||||
Abs. |
Rel. |
Abs. |
Rel. |
|||||||
Vehicle control* |
A |
4.60 |
155 |
114 |
65.8 |
100.0 |
119 |
140 |
64.6 |
100.0 |
B |
5.34 |
135 |
122 |
127 |
130 |
|||||
3.13 µg/mL |
A |
5.34 |
150 |
90 |
63.5 |
96.5 |
n.c. |
n.c. |
n.c. |
n.c. |
B |
5.99 |
133 |
135 |
n.c. |
n.c. |
|||||
6.25 µg/mL |
A |
6.42 |
112 |
127 |
59.1 |
89.8 |
n.c. |
n.c. |
n.c. |
n.c. |
B |
6.31 |
123 |
110 |
n.c. |
n.c. |
|||||
12.50 µg/mL |
A |
6.50 |
133 |
153 |
64.3 |
97.7 |
137 |
105 |
59.8 |
92.6 |
B |
6.33 |
102 |
126 |
117 |
119 |
|||||
25.00 µg/mL |
A |
6.13 |
111 |
107 |
57.2 |
86.9 |
99 |
143 |
60.5 |
93.7 |
B |
6.00 |
122 |
117 |
103 |
139 |
|||||
50.00 µg/mL |
A |
6.69 |
136 |
119 |
63.7 |
96.8 |
128 |
109 |
62.6 |
96.9 |
B |
5.90 |
121 |
133 |
159 |
104 |
|||||
100.00 µg/mL |
A |
6.99 |
127 |
115 |
62.8 |
95.4 |
104 |
114 |
62.5 |
96.7 |
B |
6.89 |
109 |
151 |
136 |
146 |
|||||
200.00 µg/mL |
A |
7.06 |
128 |
116 |
61.4 |
93.3 |
75 |
111 |
58.5 |
90.6 |
B |
6.44 |
128 |
119 |
146 |
136 |
|||||
20.00 µg/mL MCA |
A |
6.80 |
129 |
110 |
62.1 |
94.4 |
98 |
139 |
58.9 |
91.2 |
B |
5.16 |
125 |
132 |
116 |
118 |
*THF 0.5 % (v/v)
Table 8: Cytotoxicity data - 2nd Experiment without S9 mix; 24 -hour exposure period
Test groups |
Cell density (x 105/mL) At 1stsub-culture |
CE1(survival) (24 h after treatment; about 200 cells/flask seeded) |
CE2(viability) (at the end of the expression period; about 200 cells/flask seeded) |
|||||||
Cells Flask 1 |
Cells Flask 2 |
Cloning efficiency [%] |
Cells Flask 1 |
Cells Flask 2 |
Cloning efficiency [%] |
|||||
Abs. |
Rel. |
Abs. |
Rel. |
|||||||
Vehicle control* |
A |
4.67 |
147 |
127 |
76.4 |
100.0 |
187 |
182 |
90.3 |
100.0 |
B |
7.38 |
156 |
181 |
177 |
176 |
|||||
12.5 µg/mL |
A |
6.46 |
170 |
177 |
82.4 |
107.9 |
194 |
181 |
77.6 |
85.9 |
B |
7.55 |
157 |
155 |
125 |
120 |
|||||
25.0 µg/mL |
A |
7.20 |
157 |
138 |
77.2 |
101.0 |
151 |
151 |
72.4 |
80.2 |
B |
7.61 |
182 |
140 |
127 |
150 |
|||||
50.0 µg/mL |
A |
6.57 |
134 |
145 |
69.8 |
91.4 |
161 |
139 |
71.9 |
79.6 |
B |
7.60 |
130 |
149 |
145 |
130 |
|||||
100.0 µg/mL |
A |
6.94 |
123 |
137 |
67.7 |
88.6 |
132 |
156 |
74.3 |
82.3 |
B |
7.94 |
148 |
133 |
163 |
143 |
|||||
200.0 µg/mL |
A |
7.51 |
135 |
151 |
70.5 |
92.3 |
193 |
194 |
86.4 |
95.7 |
B |
8.25 |
135 |
143 |
174 |
130 |
|||||
300.00 µg/mL EMS |
A |
5.65 |
129 |
106 |
54.8 |
71.7 |
125 |
120 |
63.7 |
70.5 |
B |
7.07 |
107 |
96 |
115 |
149 |
*THF 0.5 % (v/v)
Table 9: Cytotoxicity data - 2nd Experiment with S9 mix; 4 -hour exposure period
Test groups |
Cell density (x 105/mL) At 1stsub-culture |
CE1(survival) (4 h after treatment; about 200 cells/flask seeded) |
CE2(viability) (at the end of the expression period; about 200 cells/flask seeded) |
|||||||
Cells Flask 1 |
Cells Flask 2 |
Cloning efficiency [%] |
Cells Flask 1 |
Cells Flask 2 |
Cloning efficiency [%] |
|||||
Abs. |
Rel. |
Abs. |
Rel. |
|||||||
Vehicle control* |
A |
6.25 |
110 |
134 |
61.7 |
100.0 |
158 |
149 |
78.6 |
100.0 |
B |
5.90 |
126 |
123 |
163 |
158 |
|||||
18.8 µg/mL |
A |
6.59 |
135 |
132 |
66.6 |
107.9 |
170 |
150 |
77.2 |
98.2 |
B |
6.39 |
128 |
137 |
160 |
137 |
|||||
37.5 µg/mL |
A |
6.83 |
138 |
108 |
61.4 |
99.5 |
165 |
183 |
80.2 |
102.0 |
B |
6.31 |
122 |
123 |
141 |
152 |
|||||
75.0 µg/mL |
A |
6.02 |
113 |
124 |
68.3 |
110.7 |
162 |
166 |
81.7 |
103.9 |
B |
6.10 |
139 |
170 |
165 |
160 |
|||||
150.0 µg/mL |
A |
6.15 |
145 |
112 |
66.9 |
108.4 |
167 |
150 |
75.4 |
95.9 |
B |
5.19 |
126 |
152 |
147 |
140 |
|||||
200.0 µg/mL |
A |
6.30 |
144 |
149 |
72.3 |
117.2 |
127 |
123 |
62.5 |
79.5 |
B |
6.30 |
145 |
140 |
130 |
120 |
|||||
20.00 µg/mL MCA |
A |
6.86 |
128 |
155 |
76.7 |
124.3 |
155 |
137 |
69.7 |
88.7 |
B |
7.02 |
180 |
150 |
130 |
135 |
*THF 0.5 % (v/v)
Applicant's summary and conclusion
- Conclusions:
- Under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.
- Executive summary:
The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogeneous metabolic activation). Based on the solubility properties of the test substance and according to an initial range-finding cytotoxicity test for the determination of the experimental doses the following doses were tested and the doses in bold type were evaluated in this study:
1st Experiment
without S9 mix (4 -hour exposure period)
0; 3.13; 6.25; 12.5; 25.0; 50.0; 100.0; 200.0 µg/mL
with S9 mix (4 -hour exposure period)
0; 3.13; 6.25; 12.5; 25.0; 50.0; 100.0; 200.0 µg/mL
2nd Experiment
without S9 mix
0; 12.5; 25.0; 50.0; 100.0; 200.0 µg/mL
with S9 mix
0; 18.8; 37.5; 75.0; 150.0; 200.0 µg/mL
After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line.
Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations. In this study in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest applied concentration. On the basis from the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies both without S9 mix and / or after adding a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.
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