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EC number: 258-380-0 | CAS number: 53126-67-3
- 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 mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2012-02-07 to 2012-05-23
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 012
- Report date:
- 2012
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Test material
- Reference substance name:
- Dibutyl hydrogen phosphate
- EC Number:
- 203-509-8
- EC Name:
- Dibutyl hydrogen phosphate
- Cas Number:
- 107-66-4
- Molecular formula:
- C8H19O4P
- IUPAC Name:
- dibutyl hydrogen phosphate
- Test material form:
- liquid
Constituent 1
Method
- Target gene:
- HPRT locus
Species / strain
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Additional strain / cell type characteristics:
- other: the cells have a stable karyothype with a modal chromosome number of 22
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mixPhenobarbital/beta-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 were prepared from 8 – 12 weeks old male Wistar rats (Hsd Cpb: WU, weight approx. 220 – 320 g, Harlan Laboratories B.V., 5960 AD Horst, The Netherlands) induced by intraperitoneal applications of 80 mg/kg b.w. phenobarbital (Desitin, 22335 Hamburg, Germany) and by peroral administrations of 80 mg/kg b.w. -naphthoflavone (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) each, on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at –80 °C. Small numbers of the ampoules were kept at –20 °C for up to one week. Each batch of S9 mix was routinely tested with 2-aminoanthracene as well as benzo(a)pyrene. The protein concentration of the S9 preparation was 25.9 mg/mL (Lot. No.: 021211) in the pre-experiment, 22.5 mg/mL (Lot. No.: 020212) in experiment I, and 27.2 mg/mL (Lot. No.: 050412) in experiment II.
- Test concentrations with justification for top dose:
- 137.5, 275.0, 550.0, 1100.0 and 2200.0 µg/mL
A range finding pre-experiment was performed using a concentration range of 17.2 to 2200 μg/mL (≈10 mM) to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. No relevant toxic effect occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. No precipitation occurred up to the highest concentration with and without metabolic activation following 4 and 24 hours treatment. In the pre-experiment the two highest concentrations were adjusted with 2N NaOH. There was no relevant shift of osmolarity of the medium even at the maximum concentration of the test item. Based on the results of the pre-experiment, the maximum concentration of the main experiments was again, 2200 μg/mL or 10 mM. The lower concentrations were spaced by a factor of 2. - Vehicle / solvent:
- - solvent used: water
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- The treatment time was 4 hours in the first experiment with and without metabolic activation. In the second experiment the cells were exposed to the test item for 24 hours without metabolic activation and 4 hours with metabolic activation.
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments . 2
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5*10^2 cells
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 μl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps with "saline G". In the second experiment the cells were exposed to the test item for 24 hours in complete medium, supplemented with 10 % FBS, in the absence of metabolic activation.
- Harvest time after the end of treatment (sampling/recovery times): 7 days expression time, 8 days cultivation in medium containing 6-TG, afterwards staining of the colonies
FOR GENE MUTATION:
- Expression time: 7 days
- Selection time: 8 days in medium containing 6-thiogunanine (3 - 5x10^5 cells per culture flask)
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency
METHODS FOR MEASUREMENTS OF GENOTOXICIY
mutation frequency - Rationale for test conditions:
- according to OECD Guideline
- Evaluation criteria:
- A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concen-trations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration. - Statistics:
- A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Relevant cytotoxic effects were only noted at the maximum concentration of the first experiment without metabolic activation.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.
Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.
Any other information on results incl. tables
Table 1: Results of Experiment I, Culture I
|
Conc [µg/mL] |
S9-mix |
Rel. cloning efficiency I [%] |
Rel. cell density [%] |
Rel. cloning efficiency II [%] |
Mutant colonies/ 10^6 cells |
Induction factor |
solvent |
|
- |
100 |
100 |
100 |
8.9 |
1 |
EMS |
150 |
- |
89.5 |
106.9 |
102 |
116.8 |
13.1 |
Test item |
68.8 |
- |
100 |
Culture was not continued |
|||
Test item |
137.5 |
- |
102.6 |
95 |
77.8 |
28.6 |
3.2 |
Test item |
275 |
- |
100.1 |
85.2 |
102.3 |
8.1 |
0.9 |
Test item |
550 |
- |
94.5 |
75.9 |
122.2 |
17.1 |
1.9 |
Test item |
1100 |
- |
90.9 |
92 |
115.7 |
11.1 |
1.2 |
Test item |
2200 |
- |
60.8 |
18.6 |
99.1 |
0 |
0 |
solvent |
|
+ |
100 |
100 |
100 |
11.7 |
1 |
EMS |
150 |
+ |
55.3 |
83.9 |
101.7 |
615.9 |
52.8 |
Test item |
68.8 |
+ |
103.1 |
Culture was not continued |
|||
Test item |
137.5 |
+ |
99.6 |
79.8 |
100.6 |
12.1 |
1 |
Test item |
275 |
+ |
100.7 |
78.5 |
102.8 |
18.4 |
1.6 |
Test item |
550 |
+ |
100.5 |
76.1 |
107.4 |
10.6 |
0.9 |
Test item |
1100 |
+ |
103.7 |
78.6 |
98 |
31.3 |
2.7 |
Test item |
2200 |
+ |
102.8 |
76.3 |
112.8 |
21.9 |
1.9 |
Table 2: Results of Experiment I, Culture II
|
Conc [µg/mL] |
S9-mix |
Rel. cloning efficiency I [%] |
Rel. cell density [%] |
Rel. cloning efficiency II [%] |
Mutant colonies/ 10^6 cells |
Induction factor |
solvent |
|
- |
100 |
100 |
100 |
18.5 |
1 |
EMS |
150 |
- |
91.9 |
89 |
83.5 |
69.9 |
3.8 |
Test item |
68.8 |
- |
102.7 |
Culture was not continued |
|||
Test item |
137.5 |
- |
101.5 |
89 |
83.5 |
21.4 |
1.2 |
Test item |
275 |
- |
101.5 |
117.3 |
105.4 |
20 |
1.1 |
Test item |
550 |
- |
101.2 |
131.8 |
100.6 |
18.3 |
1 |
Test item |
1100 |
- |
100.1 |
113.2 |
113.7 |
11.9 |
0.6 |
Test item |
2200 |
- |
71.6 |
24.1 |
103.6 |
0 |
0 |
solvent |
|
+ |
100 |
100 |
100 |
10.1 |
1 |
DMBA |
150 |
+ |
52.8 |
127.4 |
79 |
826.2 |
82 |
Test item |
68.8 |
+ |
98.7 |
Culture was not continued |
|||
Test item |
137.5 |
+ |
100.8 |
101.4 |
89.3 |
6.8 |
0.7 |
Test item |
275 |
+ |
99.4 |
105.8 |
101.1 |
20.2 |
2 |
Test item |
550 |
+ |
99.5 |
103 |
88.7 |
24.3 |
2.4 |
Test item |
1100 |
+ |
102 |
122.1 |
91.9 |
19.3 |
1.9 |
Test item |
2200 |
+ |
98.9 |
114.1 |
89.9 |
14.8 |
1.5 |
Table 3: Results of Experiment II, Culture I
|
Conc [µg/mL] |
S9-mix |
Rel. cloning efficiency I [%] |
Rel. cell density [%] |
Rel. cloning efficiency II [%] |
Mutant colonies/ 10^6 cells |
Induction factor |
solvent |
|
- |
100 |
100 |
100 |
15 |
1 |
EMS |
150 |
- |
79.8 |
96.4 |
76.9 |
233.4 |
15.6 |
Test item |
68.8 |
- |
99.2 |
Culture was not continued |
|||
Test item |
137.5 |
- |
103.3 |
96 |
97.4 |
14.2 |
0.9 |
Test item |
275 |
- |
99.3 |
98.6 |
90.7 |
19.2 |
1.3 |
Test item |
550 |
- |
90.5 |
93.4 |
85.8 |
20.4 |
1.4 |
Test item |
1100 |
- |
85.1 |
84 |
93.2 |
18.7 |
1.3 |
Test item |
2200 |
- |
73.7 |
80.7 |
91.6 |
13.4 |
0.9 |
solvent |
|
+ |
100 |
100 |
100 |
21 |
1 |
DMBA |
150 |
+ |
44.5 |
141.4 |
94.2 |
549 |
26.1 |
Test item |
68.8 |
+ |
100.6 |
Culture was not continued |
|||
Test item |
137.5 |
+ |
100.1 |
160.1 |
93.9 |
13.7 |
0.7 |
Test item |
275 |
+ |
101.5 |
168.4 |
97.7 |
17.3 |
0.8 |
Test item |
550 |
+ |
100.8 |
128.1 |
94.6 |
20.2 |
1 |
Test item |
1100 |
+ |
99.9 |
98 |
93.1 |
16.5 |
0.8 |
Test item |
2200 |
+ |
103.1 |
160.4 |
100.8 |
15.2 |
0.7 |
Table 4: Results of Experiment II, Culture II
|
Conc [µg/mL] |
S9-mix |
Rel. cloning efficiency I [%] |
Rel. cell density [%] |
Rel. cloning efficiency II [%] |
Mutant colonies/ 10^6 cells |
Induction factor |
solvent |
|
- |
100 |
100 |
100 |
20 |
1 |
EMS |
150 |
- |
92 |
101.8 |
77.5 |
377.6 |
18.9 |
Test item |
68.8 |
- |
96.8 |
Culture was not continued |
|||
Test item |
137.5 |
- |
97.8 |
102.5 |
104.4 |
27.9 |
1.4 |
Test item |
275 |
- |
98.7 |
106.3 |
103.4 |
25 |
1.3 |
Test item |
550 |
- |
91.6 |
98.5 |
110.1 |
17.7 |
0.9 |
Test item |
1100 |
- |
86.1 |
88.9 |
98 |
17.7 |
0.9 |
Test item |
2200 |
- |
78.8 |
66.6 |
96.9 |
27.9 |
1.4 |
solvent |
|
+ |
100 |
100 |
100 |
32.9 |
1.0 |
DMBA |
150 |
+ |
43.8 |
81.8 |
91.8 |
570.6 |
17.3 |
Test item |
68.8 |
+ |
103.6 |
Culture was not continued |
|||
Test item |
137.5 |
+ |
100.1 |
100.4 |
91.7 |
23.3 |
0.7 |
Test item |
275 |
+ |
103.3 |
89.5 |
119.6 |
19.4 |
0.6 |
Test item |
550 |
+ |
100.9 |
104.2 |
113.7 |
17.6 |
0.5 |
Test item |
1100 |
+ |
104.4 |
61.2 |
94.8 |
22.3 |
0.7 |
Test item |
2200 |
+ |
105.6 |
109.5 |
95.6 |
29.9 |
0.9 |
Applicant's summary and conclusion
- Conclusions:
- The test item was negative in this HPRT assay.
- Executive summary:
The study was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD guideline 476. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration of 2200 µg/mL was equal to approximately 10 mM.
No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item was negative in this HPRT assay.
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