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EC number: 801-773-4 | CAS number: 1550-44-3
- 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 bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 13 JUNE 2013 to 26 AUGUST 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 013
- Report date:
- 2013
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- 2,2-difluoroethyl acetate
- EC Number:
- 801-773-4
- Cas Number:
- 1550-44-3
- Molecular formula:
- C4H6F2O2
- IUPAC Name:
- 2,2-difluoroethyl acetate
- Test material form:
- liquid
- Remarks:
- clear and colorless.
Constituent 1
Method
- Target gene:
- S. typhimurium strains: histidine requirement for growth.
E. coli strain: tryptophan requirement for growth.
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- Source of S9: the S9 (lot No 3080, protein content 42.2 mg/mL) was purchased from Moltox (Boone, NC, USA) where it was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice. Upon receipt at the testing facility, the S9 was stored at -60°C or colder until used.
- Method of preparation of S9 mix / Concentration or volume of S9 mix / S9 in the final culture medium: the S9 mix was prepared immediately before its use and contained 10% S9, 5 mM glucose-6-phosphate, 4 mM ß-nicotinamide-adenine dinucleotide phosphate, 8 mM MgCl2 and 33 mM KCl in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture (Sham mix), containing 100 mM phosphate buffer at pH 7.4, was prepared immediately before its use. 0.5 mL of the Sham mix was used in the experiments with metabolic activation
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability):
* Each bulk preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and 2-aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.
* To confirm the sterility of the S9 and Sham mixes, a 0.5 mL aliquot of each was plated on selective agar. - Test concentrations with justification for top dose:
- - Initial test - experiment B1: 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate.
- Initial test - experiment B2: 50, 150, 500, 1500 and 5000 μg per plate.
- Confirmatory test - experiment B3: 50, 150, 500, 1500 and 5000 μg per plate.
- Justification for top dose: 5000 µg/plate (= 5 mg/plate) is the maximum test concentration recommended in OECD test guideline 471 for soluble non-cytotoxic substances.
See more details in the section "Any other information on results incl. tables" below. - Vehicle / solvent:
- - Solvent used: DMSO (lot No SHBC3749V, purity 99.92%).
- Justification for choice of solvent: a solubility test was conducted to determine the vehicle. The test was conducted using water and DMSO to determine the vehicle, selected in order of preference, that permitted preparation of the highest soluble or workable stock concentration up to 50 mg/mL for aqueous solvents and up to 500 mg/mL for organic solvents. DMSO was selected as the solvent of choice based on the solubility of the test material and compatibility with the target cells. The test material formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test conducted at the test facility.
Remark: All positive controls were diluted with DMSO except sodium azide, which was diluted with water.
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO.
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- sodium azide
- methylmethanesulfonate
- other:
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration:
1) Initial test: duplicate.
2) Confirmatory test: triplicate.
- Number of independent experiments:
1) Initial test: 2 consecutive experiments. The second experiment was carried with strain TA100 without S9 only due to an unacceptable vehicle control value in the first experiment. See more details in the results sections below.
2) Confirmatory test: 1 experiment.
METHOD OF TREATMENT/ EXPOSURE:
- Test material formulation: the plate-incorporation method was applied. The test substance was formulated in DMSO at the concentrations described in the field "Test concentrations with justification of top dose". The actual concentrations of the test substance in the test formulations were not determined. Therefore, the concentrations quoted in this report are nominal concentrations.
- Exposure conditions: on the day of its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCl (W/V), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 μM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, the water used was sterile and deionized. Bottom agar was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956) containing 1.5 % (W/V) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (W/V) agar and supplemented with 2.5% (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). One-half (0.5) mL of S9 or Sham mix, 100 μL of tester strain (cells seeded) and 50 μL of vehicle or test material dilution were added to 2.0 mL of molten selective top agar at 45±2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test material aliquot was replaced by a 50 μL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
The condition of the bacterial background lawn was evaluated for evidence of test material toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate.
METHODS FOR MEASUREMENTS OF GENOTOXICITY:
Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity. For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and reported. - Rationale for test conditions:
- Initial Test:
The initial assay (experiment B1) was used to establish the dose-range for the confirmatory assay and to provide a preliminary mutagenicity evaluation. Vehicle control, positive controls and eight dose levels of the test material were plated, two plates per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9. This initial test was repeated (experiment B2) with strain TA100 only due to an unacceptable vehicle control value in the first experiment. See more details in the results sections below.
Confirmatory Test:
The confirmatory mutagenicity assay was used to evaluate the mutagenic potential of the test material. Five dose levels of test material along with appropriate vehicle control and positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective agar in the presence and absence of Aroclor-induced rat liver S9. All dose levels of test material, vehicle control and positive controls were plated in triplicate. - Evaluation criteria:
- For the test material to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test material. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0 times the mean vehicle control value.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited.
A response was evaluated as negative, if it was neither positive nor equivocal. - Statistics:
- No statistical analysis was performed.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- STUDY RESULTS
- Signs of toxicity: Neither precipitate nor toxicity was observed in initial (experiments B1 and B2) and confirmatory (experiment B3) tests.
- Genotoxicity results:
* In Experiment B1 (Initial test, see Table 1 in the field "Any other information on results incl. tables"), no positive mutagenic responses were observed with any of the tester strains in the presence of S9 activation or with tester strains TA98, TA1535, TA1537 and WP2 uvrA in the absence of S9 activation. Due to an unacceptable vehicle control value, tester strain TA100 in the absence of S9 activation was not evaluated for mutagenicity but was retested in Experiment B2 based on the toxicity and precipitate profile observed.
* In Experiment B2 (Repeat of the Initial test, see Table 2 in the field "Any other information on results incl. tables"), no positive mutagenic response was observed with tester strain TA100 in the absence of S9 activation.
* In Experiment B3 (Confirmatory test, see Table 3 in the field "Any other information on results incl. tables"), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.
HISTORICAL CONTROL DATA
See Table 4 in the field "Any other information on results incl. tables".
Any other information on results incl. tables
Sterility results:
No contaminant colonies were observed on the sterility plates for the vehicle control, the test material dilutions or the S9 and Sham mixes.
Tester strain titer results:
Experiment | Tester Strain | ||||
TA98 | TA100 | TA1535 | TA1537 | WP2 uvrA | |
Titer Value (x 10E9 cells per mL) | |||||
B1 | 0.1 | 0.8 | 1.7 | 2.1 | 2.4 |
B2 | - | 0.8 | - | - | - |
B3 | 0.9 | 1.1 | 1.5 | 2.1 | 2.8 |
Tabulated results:
Table 1: Initial Test: experiment B1
| TA 98 | TA 100 | TA1535 | TA 1537 | WP2uvrA | |||||
| - S9 | + S9 | - S9 | + S9 | - S9 | + S9 | - S9 | + S9 | - S9 | + S9 |
Test material dose level (µg) per plate | Mean revertants per plate (standard deviation) | |||||||||
5000 | 22 (0) | 25 (4) | - | 97 (14) | 15 (7) | 14 (7) | 9 (1) | 8 (5) | 23 (1) | 29 (3) |
1500 | 16 (4) | 31 (3) | - | 69 (16) | 8 (2) | 18 (4) | 9 (1) | 4 (1) | 20 (4) | 21 (8) |
500 | 10 (1) | 31 (3) | - | 77 (4) | 14 (11) | 16 (6) | 8 (2) | 8 (2) | 16 (4) | 22 (7) |
150 | 22 (3) | 30 (6) | - | 89 (1) | 12 (2) | 15 (1) | 6 (3) | 7 (2) | 14 (1) | 24 (1) |
50 | 12 (1) | 20 (1) | - | 76 (1) | 11 (3) | 13 (2) | 7 (4) | 12 (2) | 12 (8) | 22 (10) |
15 | 10 (1) | 26 (2) | - | 75 (6) | 11 (1) | 12 (9) | 5 (2) | 9 (4) | 12 (8) | 28 (1) |
5.0 | 14 (5) | 34 (11) | - | 99 (16) | 9 (7) | 11 (1) | 8 (5) | 12 (4) | 28 (1) | 22 (6) |
1.5 | 17 (0) | 25 (2) | - | 88 (11) | 5 (1) | 15 (1) | 9 (0) | 12 (5) | 20 (1) | 20 (4) |
Negative control (DMSO) | 18 (4) | 20 (0) | - | 95 (17) | 10 (1) | 18 (0) | 7 (4) | 11 (0) | 18 (4) | 22 (2) |
Positive control | 161 (4) | 346 (57) | - | 374 (77) | 419 (23) | 97 (1) | 283 (64) | 47 (2) | 311 (6) | 264 (73) |
Table 2: Initial Test: experiment B2
| TA 100 |
| - S9 |
Test material dose level (µg) per plate | Mean revertants per plate (standard deviation) |
5000 | 95 (6) |
1500 | 96 (16) |
500 | 109 (34) |
150 | 107 (1) |
50 | 117 (17) |
Negative control (DMSO) | 98 (18) |
Positive control | 693 (8) |
Table 3: Confirmatory Test: experiment B3
| TA 98 | TA 100 | TA1535 | TA 1537 | WP2uvrA | |||||
| - S9 | + S9 | - S9 | + S9 | - S9 | + S9 | - S9 | + S9 | - S9 | + S9 |
Test material dose level (µg) per plate | Mean revertants per plate (standard deviation) | |||||||||
5000 | 10 (4) | 28 (4) | 100 (13) | 113 (20) | 12 (6) | 11 (7) | 8 (4) | 9 (2) | 22 (6) | 22 (8) |
1500 | 11 (4) | 25 (3) | 98 (24) | 117 (19) | 11 (6) | 18 (5) | 8 (2) | 6 (2) | 22 (2) | 28 (3) |
500 | 11 (3) | 22 (4) | 94 (12) | 107 (6) | 11 (2) | 16 (9) | 5 (3) | 8 (5) | 23 (10) | 23 (5) |
150 | 13 (3) | 31 (4) | 104 (3) | 115 (25) | 11 (3) | 18 (5) | 5 (4) | 10 (6) | 19 (5) | 22 (2) |
50 | 14 (4) | 24 (6) | 104 (7) | 119 (21) | 14 (1) | 19 (7) | 6 (3) | 8 (3) | 19 (7) | 23 (7) |
Negative control (DMSO) | 21 (9) | 28 (4) | 98 (5) | 104 (14) | 12 (2) | 19 (4) | 4 (1) | 10 (3) | 23 (4) | 30 (3) |
Positive control | 222 (74) | 488 (35) | 569 (30) | 531 (189) | 493 (44) | 111 (12) | 227 (44) | 32 (1) | 276 (24) | 277 (18) |
Historical control data:
Table 4: Historical negative and positive control values 2010 – 2012
Revertants per plate:
|
| - S9 | + S9 | ||||||
Strain | Control | Mean | SD | Min | Max | Mean | SD | Min | Max |
TA 98 | Negative | 21 | 10 | 3 | 64 | 26 | 10 | 4 | 61 |
positive | 266 | 168 | 51 | 1871 | 428 | 196 | 54 | 3355 | |
TA 100 | Negative | 102 | 21 | 47 | 251 | 115 | 23 | 41 | 247 |
positive | 645 | 160 | 232 | 1393 | 744 | 273 | 235 | 2301 | |
TA 1535 | Negative | 12 | 5 | 1 | 45 | 12 | 5 | 1 | 50 |
positive | 523 | 174 | 20 | 1593 | 114 | 110 | 20 | 1472 | |
TA 1537 | Negative | 7 | 4 | 0 | 29 | 8 | 4 | 0 | 28 |
positive | 503 | 384 | 17 | 3448 | 65 | 71 | 13 | 1272 | |
WP2 uvrA | Negative | 32 | 11 | 5 | 84 | 35 | 11 | 8 | 80 |
positive | 422 | 173 | 42 | 1796 | 223 | 88 | 44 | 969 |
SD = standard deviation; Min = minimum value; Max = maximum value;
Applicant's summary and conclusion
- Conclusions:
- Under the conditions of this study, 2,2-Difluoroethyl acetate did not cause mutagenic responses in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in Escherichia coli strain WP2 uvrA in either the presence or absence of metabolic activation.
- Executive summary:
The mutagenic potential of 2,2-Difluoroethyl acetate was investigated in bacteria in an in vitro study performed according to OECD test guideline 471 (Ames test) under GLP compliance.
The histidine-requiring Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and the tryptophan-requiring Escherichia coli strain WP2uvrA were tested in both the absence and presence of a liver fraction of Aroclor-induced rats for metabolic activation (S9-mix). The study was performed in two phases, using the plate incorporation method. The first phase (i.e. initial test with two subsequent experiments) was used to establish the dose range for the confirmatory test and to provide a preliminary mutagenicity evaluation. The second phase (i.e. confirmatory test) was used to evaluate and confirm the mutagenic potential of the test material. The following conditions were applied in the different experiments:
- Initial test (experiment B1):
- Tested strains: all.
- Tested concentrations: 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate.
- Metabolic activation: with and without.
- Initial test (experiment B2):
- Tested strains: Salmonella typhimurium strain TA100.
- Tested concentrations: 50, 150, 500, 1500 and 5000 μg per plate.
- Metabolic activation: without.
- Confirmatory test (experiment B3):
- Tested strains: all.
- Tested concentrations: 50, 150, 500, 1500 and 5000 μg per plate.
- Metabolic activation: with and without.
Negative controls (DMSO) and positive controls were run simultaneously. The plates were incubated at 37±2 °C for 48 to 72 hours. Subsequently, the revertant colonies were counted.
No positive mutagenic responses were observed at any concentration or with any tester strain in either the absence or presence of metabolic activation. No test substance precipitation or appreciable toxicity was observed. All validity criteria of the OECD test guideline 471 were met.
Under the conditions of this study, 2,2-Difluoroethyl acetate did not cause mutagenic responses in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA in either the presence or absence of Aroclor-induced rat liver S9.
- Initial test (experiment B1):
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