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Registration Dossier
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EC number: 801-773-4 | CAS number: 1550-44-3
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
- Gene mutation (bacterial reverse mutation assay / Ames test): 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.
- Gene mutation (mammalian cell gene mutation test using the thymidine kinase gene / Mouse Lymphoma Assay, MLA): 2,2-Difluoroethyl acetate showed no evidence of mutagenicity in L5178Y (TK+/- -3.7.2C) mouse lymphoma cells in both the absence and presence of metabolic activation.
- Cytogenicity (mammalian chromosome aberration test): 2,2-Difluoroethyl acetate was not found to induce structural or numerical chromosome aberrations in human peripheral blood lymphocytes in both the absence and presence of metabolic activation.
Link to relevant study records
- 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
- 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
- Target gene:
- S. typhimurium strains: histidine requirement for growth.
E. coli strain: tryptophan requirement for growth. - 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. - 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.
- 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". - 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):
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- FROM 14 MARCH 2019 to 20 FEBRUARY 2020.
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Version / remarks:
- 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- TK: Thimidine Kinase gene.
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: Mouse lymphoma L5178Y (TK +/- -3.7.2C) cell line from ATCC CRL-9518.
- Absence of Mycoplasma contamination: confirmed in the study.
- Number of passages: 8.
- Methods for maintenance in cell culture:
- Periodically ‘cleansed’ of spontaneous mutants: the cell cultures were cleansed of pre-existing mutant cells.
MEDIA USED
- Type and composition of media: the culture medium was RPMI1640 and 3 types of RPMI 1640 medium (0, 10, 20) were prepared by adding horse serum and other ingredients as follows:
Horse serum: 0%, 10% and 20%.
Penicillin-Streptomycin: 1%.
Sodium pyruvate: 2%.
RPMI10 was used during cell daily culture and cell treatment, RMPI0 was used during cell treatment and RPMI20 was used during cell cloning culture in 96-microwell plate.
- CO2 concentration: L5178Y cells were maintained with 4.9 ~5.0% CO2.
- Humidity: L5178Y cells were maintained in a humidified atmosphere.
- Temperature: L5178Y cells were maintained at 35°C~37.3°C. - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- Source of S9: the S9 was prepared from the livers of rats induced with the enzyme-inducing agent Aroclor 1254; male Sprague Dawley rats were treated by a single intraperitoneal injection of Aroclor 1254 at the dose of 500 mg/kg 5 days prior to the S9 preparation. The livers of the rats were taken out under asepsis conditions, homogenized in a 0.15 ml/L KCl solution (1g liver: 3 mL KCl solution) and centrifuged at 11 000 rpm for 10 minutes with Allegra 64R Auto Freeze Centrifuge. The supernatant of S9 was stored in liquid nitrogen.
- Method of preparation of S9 mix: a 10% S9 mix was prepared immediately before being used and placed in a mixture of water and ice.
- Concentration or volume of S9 mix and S9 in the final culture medium: The composition (as volume) of S9 mix was as follows:
* Phosphate buffer (0.2 mol/L) : 35000 µL.
* MgCl2 (0.4 mol/L) - KCl (1.65 mol/L) solution: 1400 µL.
* G-6-P-Na2 solution (140 mg/mL): 700 µL.
* NADP-Na2 solution (300 mg/mL): 700 µL.
* S9: 7000 µL
* Sterile distilled water: 25200 µL.
The final concentration of S9 fraction in the culture medium was 2% (v/v) (i.e. 20% S9 mix).
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Before being used, the S9 was tested for its sterility, its protein content (not higher than 40 mg/mL) and its ability to activate known mutagens in the Ames test. The S9 used in this test was prepared on March 12, 2018. The results of the test indicated that each index of S9 was qualified, which met the test requirements. - Test concentrations with justification for top dose:
- - Test concentrations: 10, 4, 1.6, 0.64, 0.256 and 0.1024 mM (corresponding to 1240.9, 496.4, 198.5, 79.4, 31.8 and 12.7 µg/ml, respectively). These same concentrations were applied in the 3 exposure conditions:
* 3-hour exposure with metabolic activation,
* 3-hour exposure without metabolic activation,
* 24-hour exposure with metabolic activation.
- Justification for top dose: No precipitate and limited cytotoxicity, i.e. RSG% (relative suspension growth) well above 20% in most of the cases, were observed during the preliminary test (performed at 3.2, 16, 80, 400, 2000 µg/mL). Therefore, the highest concentration tested was 10 mM as recommended in the OECD Guideline 490. - Vehicle / solvent:
- - Solvent used: Dimethyl sulfoxide (DMSO) was used for test material preparation and solvent control.
- Justification for choice of solvent: approximately 0.5 g of test material did not dissolve in water, but dissolved in DMSO thoroughly. Consequently DMSO was chosen as solvent.
- Percentage of solvent in the final culture medium: DMSO did not exceed 1% (v/v) in the final treatment medium as recommended in OECD test guideline 490. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate.
- Number of independent experiments: 1 mutagenicity test (preceded by a preliminary experiment for evaluation of solubility and cytotoxicity).
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: all cell cultures contained 20 mL of a solution of 3x10E5 cells/mL. Consequently all cultures contained 6x10E6 cells.
- Exposure to the test substance: The test material was weighed (0.99359 g) and formulated in DMSO to a volume of 4 mL as the test solution with the highest stock concentration of 2M (248.18 mg/mL). Under asepsis conditions, the other stock solutions were prepared by dilution. The final test material concentrations were obtained by diluting 5µL of the appropriate stock solution in DMSO into 1 mL culture medium. All prepared test material solutions were applied to the test system just after being formulated. The test material solutions or control solutions were added to the corresponding 20 mL of cell cultures.
- Duration of treatment: The mixtures in the treatment conditions of 3-hour exposure with and without S9 were shaken for 3-4 hours and the mixtures in the treatment conditions of 24-hour exposure without S9 were kept unshaken for nearly 24 hours.
- Handling of cells at the end of treatment: After the treatment, for the cells in the treatment conditions of 3-hour exposure with and without S9, the cultures were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, the cells were washed with HBSS solution twice and resuspended in 20 mL RPMI10 to be cultured for nearly 24 hours. For the cells in the treatment conditions of 24-hour exposure without S9, after resuspending the cells in 20 mL RPMI10 following the same conditions as above, the cell count was determined, the density was adjusted to 3x10E5 cells/mL, and culture was continued for approximately 22 hours.
- Expression time: The mutant expression period began after washing the cells and putting them into the culture incubators. The cells were cultured in incubators for 2 days to allow mutant expression. On day 1 of the expression period, the cell density was determined and diluted to 3x10E5 cells/mL with RPMI10. Cell cultures with less than 3x10E5 cells/mL were incubated without density adjustment. On day 2 of the expression period, the cells were counted again.
- Selection time: after 11 to 12 days.
- Method used: microwell plates.
- Selective agent used: trifluorothymidine (TFT, Batch No BCBW1167, supplied by Sigma Aldrich) at a final concentration of 3 µg/mL in cell cultures. The cell exposure to TFT lasted 12 days for the cells previously treated for 3 hours with the test material and 11 days for the cells previously treated for 24 hours with the test material.
- Number of cells seeded and method to enumerate numbers of viable cells: 1.6 cells per well for viability plates (PE = plating efficiency).
- Number of cells seeded and method to enumerate numbers of mutants cells: 2000 cells per well for selection plates (TFT).
- Criteria for small (slow growing) and large (fast growing) colonies:
* Large colony:
Size: the diameter was not less than a quarter of diameter of the well;
Morphology: thin and dispersive.
* Small colony:
Size: the diameter was less than a quarter of diameter of the well;
Morphology: compact and nubbly.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Plating efficiency (PE) of the cells after expression was determined and the relative total growth was calculated to evaluate cytotoxicity:
PE% = -ln(EW/TW)/1.6.
Where
EW: number of the wells without cell colony.
TW: total number of wells being counted.
1.6: number of cells plated per well.
SG (Suspension Growth) = SG1 x SG2
Where
SG1 = Cell density on Day 1 of expression period / Cell density at the beginning of cell treatment.
For the 24-hour treatment condition, the formula is adjusted as:
SG1 = (Cell density after cell treatment / Cell density at the beginning of cell treatment) x (Cell density on Day 1 of expression period / Adjusted cell density after cell treatment).
SG2 = Cell density on Day 2 of expression period / Adjusted cell density on Day 1.
RPE% (Relative Plating Efficiency) = [PE (treated) / PE (solvent control)] x 100.
RSG% (Relative Suspension Growth) = [SG (treated) / SG (solvent control)] x 100.
RTG% (Relative Total Growth) = RSG x RPE x 100.
METHODS FOR MEASUREMENTS OF GENOTOXICITY
The mutant frequency (MF) and the induced mutant frequency (IMF, i.e. increase in MF above solvent control) of each culture, and the IMF of small colonies (IMFsc) of the highest concentration and all controls were determined after the selection with TFT:
MF (Mutant Frequency) = [- ln(EW/TW)/N] / PE.
Where
EW: number of wells without mutant cell colony (EW = TW — CW).
TW: total number of wells being counted.
CW: wells with viable mutant colony.
N: number of cells plated per well (2000 cells in that case).
PE: expressed in decimal value in the formula (and not in %).
Mutant frequency of the small colony (MFsc) was calculated as the formula of MF, but EW was the number of the wells without small colony (EWsc = TW —CW+LW).
Where
LW: number of wells with large colony.
IMF (Induced Mutant Frequency) = MF (treated) – MF (solvent control).
Induced mutant frequency of the small colony (IMFsc) was calculated using the formula of IMF with the substitution of MF by MFsc.
OTHER MEASUREMENTS
The potential occurrence of test material precipitation was observed by naked eyes at the beginning and end of the treatment. - Evaluation criteria:
- When IMF(s) in one or several dose levels are more than the GEF* of 126x10E-6 and the increase is concentration-related and/or replicated, the result is evaluated as positive.
The result is evaluated as negative, if none of the above criteria is met.
In case the criteria above are not met at the same time (i.e. IMF value and concentration-dependence), the repeated test should be performed using modified experimental conditions and the biological relevance of the result should be considered. If the result is still not clear, the result is concluded to be equivocal.
*GEF = Global Evaluation Factor, established for the MLA making use of the version of microwell plates as average background mutation frequency of negative controls. - Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- See more details in section "Any other information on results incl. tables".
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- PRELIMINARY TEST
The results of the preliminary test showed that no test material precipitation occurred at any of the tested dose levels. The cell count results showed that the test material produced dose-related cytotoxicity. In the 3-hour treatments in the absence and presence of S9-mix, the relative suspension growths were 68% and 49%, respectively, at the test material concentration of 2000 µg/mL compared to the relative suspension growth of the solvent control. In the 24-hour treatment in the absence of S9-mix, the relative suspension growth was 28% at the test material concentration of 2000 µg/mL compared to the relative suspension growth of the solvent control. The detailed results are in Table 1 in the field "Any other information on results incl. tables".
STUDY RESULTS
- Test-specific confounding factors:
* Precipitation and time of the determination: In the cell cultures under the three treatment conditions, no test material precipitation was observed at all test concentrations, either at the beginning or the end of treatment.
- Concurrent vehicle negative and positive control data: The results of the solvent and positive controls were in accordance with historical data (see in Table 3 in the field "Any other information on results incl. tables"). Consequently the sensitivity of the assay and the efficacy of the S9 mix were validated.
- Cytotoxicity results: Cytotoxicity was assessed through the determination of the relative total growth (RTG), the key parameter representing overall cell proliferation and survival across all steps of the test. RTG includes the relative suspension growth during treatment (RSG: test culture vs. solvent control) and during the incubation time for mutant expression along with the relative plating/cloning efficiency (RPE: test culture vs. solvent control) at the time of mutant selection.
The test material produced dose-related cytotoxicity under the three treatment conditions (see table 4 below). In the 3-hour treatments in the absence and presence of S9-mix, the RTG values were 56.97% and 51.54% at the highest test material concentration of 10 mM, respectively. In the 24-hour treatment in the absence of S9-mix, the RTG value was 27.92% at the highest test material concentration of 10 mM.
The RTG values calculated for the positive controls were 42.58% (MMS at 5 µg/mL for the 3-hour treatment without S9), 48.31% (CP at 3 µg/mL for the 3-hour treatment with S9) and 31.83% (MMS at 5 µg/mL for the 24-hour treatment without S9).
In all groups under all treatment conditions, the RTG values were thus well above the values of 20 to 10% between which the OECD 490 test guideline recommends to interpret the results with care.
- Genotoxicity results: The mutant frequency data showed that the IMF of all cultures exposed to the test material for 3 hours and 24 hours in the absence and presence of S9 were less than the global evaluation factor (GEF) of 126x10-6, the generally accepted average background mutant level for the MLA assay. The result of the study is thus considered as negative. Because no positive response was observed in cultures exposed to the test material, results obtained for small and large colonies were not discussed.
HISTORICAL CONTROL DATA
See Table 3 in the field "Any other information on results incl. tables". - Conclusions:
- Under the conditions of this study, 2,2-Difluoroethyl acetate showed no evidence of mutagenicity in L5178Y (TK+/- -3.7.2C) mouse lymphoma cells in both the absence and presence of metabolic activation.
- Executive summary:
The mutagenic potential of 2,2-Difluoroethyl acetate was investigated in mammalian cells in an in vitro study performed according to OECD test guideline 490 (Mouse Lymphoma Assay, MLA) under GLP compliance.
L5178Y (TK+/--3.7.2C) mouse lymphoma cells were tested in both the absence and presence of a liver fraction of Aroclor-induced rats for metabolic activation (S9-mix). Six test material concentrations were tested using dimethylsulphoxide (DMSO) as solvent: 0.1024, 0.256, 0.64, 1.6, 4 and 10 mM. Negative controls (DMSO) and positive controls were run simultaneously. The cells were treated for 3 and 24 hours in the absence of metabolic activation and for 3 hours in the presence of metabolic activation. The cells were then subcultured for two days to allow mutant phenotypic expression. Mutant frequency was then determined by seeding known numbers of cells in medium containing the selective agent 5-trifluorothymidine (TFT) to detect mutant colonies, and in medium without TFT to determine the cloning efficiency (viability). After 11 to 12 days of incubation, colonies were counted. Mutant frequency was calculated based on the number of mutant colonies corrected by the cloning efficiency at the time of mutant selection.
The induced mutant frequencies were consistently less than the global evaluation factor (GEF) of 126x10-6 in any condition, meaning that the results of the study were negative. No test material precipitation was observed. As reflected by the Relative Total Growth (RTG), test material related cytotoxicity was recorded, but the RTG values were well above the values of 20 to 10 % between which the OECD test guideline 490 recommends to interpret the results with care. All validity criteria of OECD test guideline 490 were met.
Under the conditions of this study, 2,2-Difluoroethyl acetate showed no evidence of mutagenicity in L5178Y (TK+/--3.7.2C) mouse lymphoma cells in both the absence and presence of metabolic activation.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- FROM 7 AUGUST 2015 TO 05 JANUARY 2016.
- Reliability:
- 1 (reliable without restriction)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
- Version / remarks:
- 2014.
- Deviations:
- not specified
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- other: Human peripheral blood lymphocytes (HPBL).
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: blood was drawn from a healthy man without previous chemotherapy or radiotherapy and without recent (within the last 6 months) viral disease or X-ray exposure. The blood was collected by site medical personnel at the Stine-Haskell Site Medical Facility (USA).
- Suitability of cells: Human peripheral blood lymphocytes, exposed to test substances in vitro, are routinely used to detect clastogenic activity.
- Sex, age and number of blood donors: 1 male donor, 35-year old.
- Whether whole blood or separated lymphocytes were used: whole blood.
- Mitogen used for lymphocytes: phytohemagglutinin-M (PHA-M).
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: 0.5 mL of heparinized blood was inoculated to 4.5 mL of complete medium (RPMI 1640 medium containing approximately 15% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units penicillin/mL, and 100 μg streptomycin/mL) supplemented with 1-2% phytohemagglutinin-M (PHA-M). Cultures were incubated at 37°C ± 2°C in a humidified atmosphere of 5 ±2% CO2 in air. - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- Source of S9 : Liver homogenate (S9), prepared from male Sprague-Dawley rats induced with Aroclor 1254, was purchased commercially (Moltox, Inc., Boone, North Carolina, U.S.A.) and stored frozen at approximately -80°C until used.
- Method of preparation of S9 mix / Concentration or volume of S9 mix / S9 in the final culture medium: immediately prior to use, the S9 liver homogenate was thawed and mixed with a cofactor pool. The stock S9 protein concentration (Moltox, lot 3497) was 40.7 mg/mL in both the preliminary toxicity and main chromosome aberration assay. The final concentration of the cofactors and S9 in the metabolic activation system (S9 mixture) was 4 mM nicotinamide adenine dinucleotide phosphate (NADP), 5 mM glucose-6-phosphate, 8 mM MgCl2, 33 mM KCl, 100 mM sodium phosphate buffer, pH 7.4, and 10% S9. The S9 mixture was prepared immediately before use and kept on ice until diluted 1:4 (v/v) in complete RPMI.
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): the metabolic activity of the S9 was demonstrated by the response of the cyclophosphamide treated cultures. - Test concentrations with justification for top dose:
- - Test concentrations: For all test conditions (with and without metabolic activation and with different treatment durations), the test material concentrations were 0 (negative control), 100, 250, 500, 750 and 1240 µg/mL.
- Justification for top dose: These concentrations were based on the results of a preliminary toxicity test during which the cells were exposed to the test material concentrations of 5, 10, 25, 50, 100, 250, 500, 750 and 1240 µg/mL (one culture per concentration) for:
* 4-hour, with metabolic activation,
* 4-hour, without metabolic activation,
* 22-hour, without metabolic activation.
Neither cytotoxicity nor precipitation was observed. The highest concentration of 1240 µg/mL was thus retained for the definitive chromosome aberration assay. It corresponded to the highest test concentration recommended in the OECD test guideline 473 (equivalent to 10 mM considering a molecular weight of 124.09 g/mol). - Vehicle / solvent:
- - Solvent used: dimethyl sulfoxide (DMSO).
- Justification for choice of solvent: a visual solubility determination was conducted to determine the maximum soluble concentration or workable suspension up to a maximum of 20 mg/mL for aqueous vehicles and 200 mg/mL for organic vehicles. Vehicles compatible with this test system, in order of preference, included, but were not limited to sterile water, dimethyl sulfoxide (DMSO), ethanol, or acetone. The vehicle of choice for this study was DMSO, which permitted preparation of the highest workable/soluble stock concentration.
- Percentage of solvent in the final culture medium: under the conditions of this test system, the final concentration of DMSO in the treatment medium did not exceed 1% of the treatment medium as recommended in OECD test guideline 473. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO.
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Remarks:
- Positive control substances were dissolved in sterile water. Two test concentrations of each positive control substance were used to ensure a valid assay; however only one concentration of each positive control was included in the cytogenetic analysis.
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate cultures.
- Number of independent experiments: one main chromosome aberration test (preceded by a preliminary toxicity test).
METHOD OF TREATMENT/ EXPOSURE AND HARVEST:
- Test material formulation: Serial stock concentrations of 124, 75, 50, 25 and 10 mg/mL were prepared in DMSO. An aliquot (50 µL) of these solutions was added to the test cultures (total volume of 5 mL) to reach the target concentrations of 1240, 750, 500, 250 and 100 µg/mL.
- Preincubation period (Cell initiation): the HPBL cultures were initiated in labeled sterile, 15 mL centrifuge tubes by inoculating 0.5 mL of heparinized blood to 4.5 mL of complete medium (RPMI 1640 medium containing approximately 15% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units penicillin/mL, and 100 μg streptomycin/mL) supplemented with 1-2% phytohemagglutinin-M (PHA-M). Cultures were incubated at 37°C ± 2°C in a humidified atmosphere of 5 ±2% CO2 in air. Approximately 48 hours after culture initiation, the whole blood cultures were centrifuged and the culture medium was discarded and replaced with treatment medium (with or without 10% S9 mixture) such that addition of the test material volume (50 μL) resulted in a total volume of 5 mL.
- Exposure conditions and duration: duplicate cultures were administered an aliquot of the test substance (5 concentrations were tested), the vehicle control and 2 positive control substance concentrations for each test condition. The treatment medium was RPMI 1640 medium for all testing conditions. The cells were treated for approximately 4 and 22 hours in the non-activated test condition, and for approximately 4 hours in the S9-activated test condition. For the 4-hour condition only, after completion of the exposure period, the cells were collected by centrifugation, and the treatment medium replaced with complete RPMI 1640 culture medium and incubated until cell harvest. All the incubations were conducted at 37 ± 2°C in a humidified atmosphere of 5 ± 2% CO2 in air.
- Spindle inhibitor and harvest time: the cells were arrested in metaphase at approximately 19 hours after treatment initiation by adding Colcemid® to the cultures at a 0.1 μg/mL final concentration in the culture. Approximately 22 hours after treatment initiation the cells were harvested by centrifugation and the medium removed.
- Methods of slide preparation and staining technique used: the cells were treated with 0.075M KCl hypotonic buffer, fixed in methanol/glacial acetic acid (3:1 v/v) and stored frozen. To prepare the slides, the cells were collected by centrifugation and resuspended in fixative. The slides were prepared by applying an aliquot of the fixed cells onto clean microscope slides and air-drying them. The slides were stained by Giemsa and permanently mounted.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI). MI, based on the percentage of cells in metaphase per at least 1000 cells scored per concentration level (at least 500 from each duplicate culture), was determined prior to coding the slides. Precipitation was evaluated with the naked eye at the end of the treatment of the cell cultures with the test material. After selection of the slides for cytogenetic analyses, the slides were coded and scored blind to control for bias.
METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Number of cells spread and analysed per concentration: for each test condition, cytogenetic analyses were conducted for at least 3 test material concentrations (here, 500, 750 and 1240 µg/mL), the vehicle control and a positive control. Metaphase cells were selected for scoring based on good chromosome morphology and staining characteristics. Only metaphase cells with 46 centromeres were analysed for structural aberrations. At least 300 metaphases per concentration level (150 from each duplicate culture), when available, were analysed for structural aberrations. The number of metaphases evaluated per duplicate culture may be less if 10 or more aberrant cells were observed among the first 50 cells scored.
- Criteria for scoring chromosome aberrations: Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures. Chromosome-type aberrations include chromosome breaks and exchange figures. Pulverized chromosome(s) and cell(s) and severely damaged cells (i.e. cells with ≥ 10 aberrations per cell) were recorded and included in the analyses. The XY coordinates for the microscope stage were recorded for cells with structural aberrations. Numerical aberrations were recorded as well.
OTHER MEASUREMENTS
The pH of the treatment medium was evaluated both at the beginning and end of the treatment period by visual determination using the pH-sensitive colour indicator present in the treatment medium. Precipitation was evaluated both at the beginning and the end of the treatment period by visual determination. In addition, osmolality and pH measurements were taken from the vehicle control, lowest precipitating test material concentration, and the highest soluble test material concentration in the culture media with and without 10% S9-mixture. - Evaluation criteria:
- The following conditions were used as a guide to determine a positive response:
- A statistically significant increase (p < 0.05, Fisher’s exact test) in the percentage of cells with structural aberrations was seen in one or more treatment groups relative to the vehicle control response.
- The observed increased frequencies were accompanied by a concentration-related increase when evaluated by the trend test.
- Any of the results were outside the 95% control limit distribution of the historical negative control data.
- Note: statistically significant values that did not exceed the historical control range for the negative/vehicle control may be judged as not being biologically significant.
The following condition was used as a guide to determine an equivocal response:
- Results observed in any of the assays resulted in statistically significant elevations in structural chromosome aberrations at more than one test concentration level, except the highest dose, without demonstrating a dose-responsive trend.
The test material was judged negative if the following condition was met:
- There was no statistically significant increase in the percentage of cells with structural aberrations in any treatment group relative to the vehicle control group.
- There was no concentration-related increase when evaluated with an appropriate trend test.
- All results were within the 95% control limit of the distribution of the historical negative control database. - Statistics:
- Statistical analysis was used as a guide to determine whether or not the test material induced a positive response.
Interpretation of the statistical analysis also relied on additional considerations including the magnitude of the observed test material response relative to the vehicle control response and the presence of a dose-responsive trend. Statistical analysis consisted of a Fisher’s exact test (with Bonferroni-Holm Adjustment) to compare the percentage of cells with structural or numerical aberrations (or the percentage of cells with more than one aberration, if required) in the test material treated groups with the vehicle control response. A Cochran-Armitage test for dose responsiveness was conducted only on values within a test condition only if statistically significant values, based on the Fisher’s exact test, were found. At the discretion of the study director, statistical analyses could be conducted on the percentage of cells with numerical aberrations as well. - Key result
- Species / strain:
- other: Human peripheral blood lymphocytes (HPBL).
- 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:
- PRELIMINARY TOXICITY ASSAY RESULTS
- Precipitation and time of the determination: no test substance precipitation was observed at the beginning or end of treatment for any test condition.
- Cytotoxicity results: substantial toxicity (i.e., 55 ± 5% mitotic reduction in relation to the vehicle control) was not observed in any test condition.
CHROMOSOME ABERRATION ASSAY RESULTS
- Test-specific confounding factors:
* Data on pH: see Table 2 in the field "Any other information on results incl. tables".
* Data on osmolality: see Table 2 in the field "Any other information on results incl. tables". The observed changes in osmolality were ≤ 20% and were, therefore, not considered significant.
* Precipitation and time of the determination: no test substance precipitation was observed at the beginning or end of treatment for any test condition.
- Cytotoxicity results: substantial toxicity (i.e., 55 ± 5% mitotic reduction in relation to the vehicle control) was not observed in any test condition:
* 4-hour, without metabolic activation: The mitotic index for the highest test concentration evaluated microscopically for chromosome aberrations, 1240 μg/mL, was 9.0%, compared with 11.0% for the vehicle control. This represents an 18.2% mitotic inhibition in relation to the vehicle control.
* 4-hour, with metabolic activation: The mitotic index for the highest test concentration evaluated microscopically for chromosome aberrations, 1240 μg/mL, was 7.5%, compared with 9.9% for the vehicle control. This represents a 24.2% mitotic inhibition in relation to the vehicle control.
* 22-hour, without metabolic activation: The mitotic index for the highest test concentration evaluated microscopically for chromosome aberrations, 1240 μg/mL was 7.7%, compared with 9.5% for the vehicle control. This represents a 19.0% mitotic inhibition in relation to the vehicle control.
- Genotoxicity results:
* 4-hour, without metabolic activation: The concentrations selected for microscopic analysis of chromosome aberrations were 500, 750 and 1240 μg/mL and were selected based on the highest concentration tested. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased above that of the vehicle control at any concentration (p ≥ 0.05, Fisher's exact test). The percentage of cells with structurally damaged chromosomes in the MMC (positive control) treatment group (27.0%) was statistically significant (p < 0.05, Fisher’s exact test).
* 4-hour, with metabolic activation: The concentrations selected for microscopic analysis of chromosome aberrations were 500, 750 and 1240 μg/mL and were selected based on the highest concentration tested. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased above that of the vehicle control at any concentration (p ≥ 0.05, Fisher's exact test). The percentage of cells with structurally damaged chromosomes in the CP (positive control) treatment group (23.0%) was statistically significant (p < 0.05, Fisher’s exact test).
* 22-hour, without metabolic activation: The concentrations selected for microscopic analysis of chromosome aberrations were 500, 750 and 1240 μg/mL and were selected based on the highest concentration tested. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased above that of the vehicle control at any concentration (p ≥ 0.05, Fisher's exact test). The percentage of cells with structurally damaged chromosomes in the MMC (positive control) treatment group (13.0%) was statistically significant (p < 0.05, Fisher’s exact test).
HISTORICAL CONTROL DATA
See Table 4 in the field below "any other information on results incl. tables". - Conclusions:
- Under the conditions of this study, 2,2-Difluoroethyl acetate was not found to induce structural or numerical chromosome aberrations in human peripheral blood lymphocytes in both the absence and presence of metabolic activation.
- Executive summary:
The cytogenic potential of 2,2-Difluoroethyl acetate was investigated in human peripheral blood lymphocytes in an in vitro study performed according to OECD test guideline 473 (chromosome aberration test) under GLP compliance.
Human peripheral blood lymphocytes (HPBL) were tested in both the absence and presence of a Iiver fraction of Aroclor-induced rats for metabolic activation (S9-mix). Five test material concentrations were tested using dimethylsulphoxide (DMSO) as solvent: 100, 250, 500, 750 and 1240 μg/mL (i.e. the limit dose of 10 mM). Negative controls (DMSO) and positive controls were run simultaneously. The cells were treated for 4 and 22 hours in the absence of metabolic activation and for 4 hours in the presence of metabolic activation. The cells were arrested in the metaphase stage of mitosis at approximately 19 hours after treatment initiation by the addition of colcemid. Approximately 22 hours after treatment initiation, the cells were harvested and slides were prepared for microscopic examinations. As a measure of cytotoxicity, the mitotic index was determined. Subsequently, the cultures of selected concentrations of the test material (selected based on mitotic index analysis), together with the negative and positive control cultures, were analysed for the induction of structural and numerical chromosomal aberrations.
The percentage of cells with structural and numerical aberrations in the test material treated groups was not significantly increased above that of the solvent control at any concentration. No test material precipitation or substantial toxicity was observed. All validity criteria of the OECD test guideline 473 were met.
Under the conditions of this study, 2,2-Difluoroethyl acetate was not found to induce structural or numerical chromosome aberrations in human peripheral blood lymphocytes in both the absence and presence of metabolic activation.
Referenceopen allclose all
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;
Table 1: preliminary test results
Treatment condition | Doses (µg/mL) | Precipitation | RSG% | |
At the beginning of treatment | A the end of treatment | |||
3 hours – S9 | 3.2 | None | None | 80 |
16 | None | None | 95 | |
80 | None | None | 90 | |
400 | None | None | 73 | |
2000 | None | None | 68 | |
3 hours + S9 | 3.2 | None | None | 98 |
16 | None | None | 106 | |
80 | None | None | 93 | |
400 | None | None | 65 | |
2000 | None | None | 49 | |
24 hours – S9 | 3.2 | None | None | 95 |
16 | None | None | 85 | |
80 | None | None | 73 | |
400 | None | None | 75 | |
2000 | None | None | 28 | |
3 hours – S9: exposure for 3 hours without metabolic activation.
3 hours + S9: exposure for 3 hours with metabolic activation.
24 hours – S9: exposure for 24 hours without metabolic activation.
Table 2: validity results
Criteria | Required | Observed | ||
3-hour –S9 | 3-hour + S9 | 24-hour –S9 | ||
Solvent control (DMSO) | ||||
Plating Efficiency (PE, %) | 65 - 120 | 101.33 | 93.54 | 104.7 |
Mutant Frequency (MF, x10-6) | 50 - 170 | 120.17 | 123.12 | 102.21 |
Suspension Growth (SG), 3-hour treatment | 8 - 32 | 9.84 | 9.36 | - |
Suspension Growth (SG), 24-hour treatment | 32-180 | - | - | 68.83 |
Positive controls (MMS at 5 µg/mL without S9, CP at 3 µg/mL with S9) | ||||
Relative Total Growth (RTG, %) | > 10 | 42.58 | 48.31 | 31.83 |
Induced Mutant Frequency (IMF, x10-6) and | > 300 | 609.26 | 639.15 | 615.99 |
Induced Mutant Frequency for
| > 40% IMF | 69.97% IMF | 82.69% IMF | 72.04% IMF |
OR Induced Mutant Frequency for small colonies (IMFsc, x10-6) | > 150 | 426.27 | 528.50 | 443.78 |
(MMS= methymethanesulfonate; CP = cyclophosphamide)
Table 3: Historical negative and positive control data from 19 studies (2018)
Year | Treatment | Control (µg/mL) | Mutant frequencies ( x10-6) | ||||
Mean | SD | Min | Max | 95% control | |||
2018 (18) | 3-hour with S9 | Solvent | 108.20 | 21.28 | 86.92 | 129.47 | 65.65 ~150.75 |
CP (3.0) | 728.21 | 122.73 | 605.48 | 850.94 | 482.75~973.68 | ||
2018 (19) | 3-hour without S9 | Solvent | 115.02 | 21.58 | 93.44 | 136.59 | 71.87~158.17 |
MMS (5.0) | 719.29 | 147.48 | 571.81 | 866.78 | 424.33~1014.26 | ||
2018 (8) | 24-hour without S9 | Solvent | 134.06 | 16.08 | 117.98 | 150.13 | 101.90~166.21 |
MMS (5.0) | 831.84 | 123.48 | 708.36 | 955.32 | 584.88~1078.80 | ||
Table 4: Survival, cytotoxicity results
TC* | Test conc. ** | PE (%) | RPE (% ) | SG | RSG (%) | RTG (%) |
3-hour – S9 | DMSO | 101.3 | 100.00 | 9.84 | 100.00 | 100.00 |
0.1024 | 89.72 | 88.54 | 9.83 | 99.92 | 88.47 | |
0.256 | 98.83 | 97.53 | 10.24 | 104.13 | 101.56 | |
0.64 | 105.0 | 103.63 | 9.79 | 99.56 | 103.18 | |
1.60 | 95.27 | 94.02 | 7.95 | 80.86 | 76.03 | |
4 | 93.54 | 92.31 | 6.94 | 70.57 | 65.14 | |
10 | 86.70 | 85.56 | 6.55 | 66.59 | 56.97 | |
MMS (5.0) | 69.66 | 68.74 | 6.09 | 61.94 | 42.58 | |
3-hour + S9 | DMSO | 93.54 | 100.00 | 9.36 | 100.00 | 100.00 |
0.1024 | 86.70 | 92.69 | 9.13 | 97.47 | 90.35 | |
0.256 | 92.34 | 98.72 | 8.34 | 89.12 | 87.98 | |
0.64 | 89.13 | 95.29 | 8.38 | 89.51 | 85.29 | |
1.60 | 85.47 | 91.38 | 8.37 | 89.44 | 81.73 | |
4 | 79.32 | 84.81 | 6.92 | 73.90 | 62.67 | |
10 | 66.86 | 71.48 | 6.75 | 72.11 | 51.54 | |
CP (3.0) | 66.65 | 71.26 | 6.35 | 67.80 | 48.31 | |
24-hour – S9 | DMSO | 104.7 | 100.00 | 68.83 | 100.00 | 100.00 |
0.1024 | 105.0 | 100.28 | 60.28 | 87.57 | 87.81 | |
0.256 | 104.0 | 99.36 | 57.99 | 84.24 | 83.70 | |
0.64 | 106.6 | 101.81 | 45.84 | 66.60 | 67.81 | |
1.60 | 93.13 | 88.93 | 40.66 | 59.06 | 52.53 | |
4 | 53.95 | 51.51 | 38.27 | 55.60 | 28.64 | |
10 | 70.68 | 67.49 | 28.48 | 41.37 | 27.92 | |
MMS (5.0) | 56.32 | 53.78 | 40.74 | 59.18 | 31.83 |
* TC: test conditions.
** mM for test material and µg/mL for positive controls.
Table 5: Mutagenicity results
TC* | Test conc. ** | MF (x10-6) | IMF (x10-6) | MFsc (x10-6) | IMFsc (x10-6) |
3-hour – S9 | DMSO | 120.17 | 0.00 | 10.39 | 0.00 |
0.1024 | 130.19 | 10.02 | - | - | |
0.256 | 118.19 | -1.98 | - | - | |
0.64 | 132.04 | 11.88 | - | - | |
1.60 | 149.16 | 28.99 | - | - | |
4 | 144.58 | 24.41 | - | - | |
10 | 157.96 | 37.79 | 22.98 | 12.59 | |
MMS (5.0) | 729.43 | 609.26 | 436.66 | 426.27 | |
3-hour + S9 | DMSO | 123.12 | 0.00 | 11.25 | 0.00 |
0.1024 | 146.23 | 23.10 | - | - | |
0.256 | 144.61 | 21.48 | - | - | |
0.64 | 157.51 | 34.38 | - | - | |
1.60 | 162.23 | 39.10 | - | - | |
4 | 190.15 | 67.02 | - | - | |
10 | 215.15 | 92.03 | 23.74 | 12.49 | |
CP (3.0) | 762.27 | 639.15 | 539.75 | 528.50 | |
24-hour – S9 | DMSO | 102.21 | 0.00 | 22.92 | 0.00 |
0.1024 | 125.54 | 23.33 | - | - | |
0.256 | 165.71 | 63.50 | - | - | |
0.64 | 191.99 | 89.78 | - | - | |
1.60 | 161.96 | 59.75 | - | - | |
4 | 216.53 | 114.32 | - | - | |
10 | 181.74 | 79.53 | 33.96 | 11.04 | |
MMS (5.0) | 718.20 | 615.99 | 466.70 | 443.78 |
-: not measured, not evaluable.
* test conditions.
** mM for test material and µg/mL for positive controls.
Table 1: Preliminary toxicity assay results
Treatment dose (µg/mL) | 4-hour | 22-hour | ||||
- S9 | + S9 | - S9 | ||||
Mitotic Index (MI) | % change | Mitotic Index (MI) | % change | Mitotic Index (MI) | % change | |
0 (DMSO) | 12.1 | NA | 8.1 | NA | 12.4 | NA |
5 | 11.1 | -8.3 | 7.9 | -2.5 | 13.4 | 8.1 |
10 | 10.7 | -11.6 | 5.5 | -32.1 | 8.2 | -33.9 |
25 | 10.3 | -14.9 | 7.6 | -6.2 | 11.1 | -10.5 |
50 | 11.1 | -8.3 | 6.1 | -24.7 | 11.0 | -11.3 |
100 | 11.1 | -8.3 | 5.6 | -30.9 | 11.4 | -8.1 |
250 | 11.9 | -1.7 | 5.9 | -27.2 | 9.3 | -25.0 |
500 | 11.5 | -5.0 | 9.1 | 12.3 | 8.2 | -33.9 |
750 | 10.6 | -12.4 | 6.2 | -23.5 | 10.3 | -16.9 |
1240 | 11.2 | -7.4 | 4.9 | -39.5 | 11.6 | -6.5 |
Table 2: pH and osmolality during the chromosome aberration assay
Treatment condition | Test material concentration (µg/mL) | pH | Osmolality (mOsm/kg) |
Without metabolic activation | 0 (DMSO) | 8.02 | 647 |
750 | 7.86 | 627 | |
1240 | 7.86 | 651 | |
With metabolic activation (S9) | 0 (DMSO) | 7.58 | 672 |
750 | 6.86 | 701 | |
1240 | 6.24 | 702 |
Table 3: Chromosome aberration assay results (summary)
Test material dose (µg/mL) | S9 | ha | Mitotic index (%) | Cells scored | Aberrations per cell | Cells with aberrationsb | |||
Numerical | Structural | ||||||||
Numerical | Structural | Mean | SD | % | % | ||||
0 | -S9 | 4 | 11.0 | 300 | 300 | 0.010 | 0.005 | 0.0 | 1.0 |
500 | -S9 | 4 | 8.7 | 300 | 300 | 0.007 | 0.000 | 0.0 | 0.7 |
750 | -S9 | 4 | 10.0 | 300 | 300 | 0.010 | 0.005 | 0.0 | 1.0 |
1240 | -S9 | 4 | 9.0 | 300 | 300 | 0.010 | 0.005 | 0.0 | 1.0 |
MMCc 0.4 | -S9 | 4 | 5.8 | 100 | 100 | 0.300 | 0.028 | 0.0 | 27.0e |
0 | +S9 | 4 | 9.9 | 300 | 300 | 0.007 | 0.000 | 0.0 | 0.7 |
500 | +S9 | 4 | 8.5 | 300 | 300 | 0.027 | 0.009 | 0.0 | 2.7 |
750 | +S9 | 4 | 8.7 | 300 | 300 | 0.003 | 0.005 | 0.3 | 0.3 |
1240 | +S9 | 4 | 7.5 | 300 | 300 | 0.007 | 0.000 | 0.0 | 0.7 |
CPd 10 | +S9 | 4 | 4.8 | 100 | 100 | 0.280 | 0.028 | 0.0 | 23.0e |
0 | -S9 | 22 | 9.5 | 300 | 300 | 0.013 | 0.000 | 0.0 | 1.3 |
500 | -S9 | 22 | 8.8 | 300 | 300 | 0.007 | 0.000 | 0.0 | 0.7 |
750 | -S9 | 22 | 8.2 | 300 | 300 | 0.020 | 0.009 | 0.0 | 1.7 |
1240 | -S9 | 22 | 7.7 | 300 | 300 | 0.027 | 0.019 | 0.0 | 2.3 |
MMC 0.2 | -S9 | 22 | 3.6 | 200 | 200 | 0.165 | 0.184 | 0.0 | 13.0e |
a treatment duration.
b excluding cells with only gaps.
c MMC: mitomycin C.
d CP: cyclophosphamide.
e Statistically significant difference from control at p < 0.05 by Fisher's test.
Table 4: Historical control data:
Vehicle control 95% control limits | ||||
| % Structural aberrations | % Numerical aberrations | ||
Non activated test system | 0-2.8 | 0-0.4 | ||
Activated test system | 0-3.0 | 0-1.0 | ||
Positive controls | ||||
| Non activated test system Mitomycin C (MMC) | Activated test system Cyclophosphamide (CP) | ||
Structural | Numerical | Structural | Numerical | |
% Aberrations | ||||
Mean | 23.2 | 0.1 | 21.6 | 0.2 |
Standard deviation | 13.7 | 0.2 | 13.3 | 0.45 |
range | 7-60 | 0-1 | 7-52 | 0-4 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
- Cytogenicity (mammalian erythrocyte micronucleus test during a 28-day repeated dose toxicity study by oral route): 2,2-Difluoroethyl acetate showed no evidence of in vivo cytogenicity with no statistically significant or biologically relevant increase in micronucleated reticulocytes in rats.
- Cytogenicity (mammalian erythrocyte micronucleus test during a 28-day repeated dose toxicity study by inhalation route): 2,2-Difluoroethyl acetate showed no evidence of in vivo cytogenicity with no statistically significant or biologically relevant increase in micronucleated reticulocytes in rats.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- This genotoxicity evaluation was combined with a 28-day repeated-dose toxicity study by gavage. Only the information relevant for the micronucleus evaluation were described here (for the ‘classical’ repeated-dose toxicity study, see IU section 7.5.1).
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- FROM 29 AUGUST 2014 TO 5 MAY 2015.
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Remarks:
- The present micronucleus evaluation was done during a 28-day repeated dose toxicity study by oral route.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- 1997
- Deviations:
- not specified
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian erythrocyte micronucleus test
- Species:
- rat
- Strain:
- other: Crl:CD(SD).
- Details on species / strain selection:
- Rats have historically been used in safety evaluation studies for oral toxicity testing. The Crl:CD(SD) rat was selected based on consistently acceptable health status and on extensive experience with this strain at the testing facility.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories International, Inc., Raleigh, North Carolina, U.S.A.
- Females: nulliparous and non pregnant.
- Age at study initiation: 7 weeks old.
- Weight at study initiation: the males mean body weight varied between 224.9 and 228.4 g and the females mean body weight varied between 171.6 and 176.7 g within all dose groups. The weight variation of selected animals did not exceed ± 20% of the mean weight for each sex.
- Assigned to test groups randomly: yes, the animals were distributed by computerized, stratified randomization into study groups.
- Fasting period before study: not specified.
- Housing: animals were housed in groups in solid-bottom caging with bedding and appropriate species-specific enrichment.
- Diet: all animals were fed PMI® Nutrition International LLC Certified Rodent LabDiet® 5002 ad libitum.
- Water: all animals were provided tap water ad libitum.
- Acclimation period: 6 days. The animals were released from quarantine based on normal observations for body weights and clinical signs.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC.
- Humidity (%): 30-70%.
- Photoperiod: Animal rooms were artificially illuminated (fluorescent light) on an approximate 12-hour light/dark cycle.
IN-LIFE DATES: From 16 September 2014 to 14 October 2014. - Route of administration:
- oral: gavage
- Vehicle:
- - Solvent used: corn oil.
- Justification for choice of solvent: the chosen solvent was among those recommended in the guideline and it was shown through sampling and analysis that the test material at 10 to 100 mg/mL in the vehicle and stored at room temperature for up to approximately 15 days was stable.
- Concentration of test material in vehicle: 0, 10, 30 and 100 mg test material/mL of vehicle.
- Amount of vehicle (gavage): 10 mL/kg bw. - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test material was dissolved in corn oil. Neither the amount nor nature of any contaminants in the vehicle was expected to affect the integrity or validity of this study. Dose formulations were prepared without a correction for the sponsor reported purity. Dosing formulations of the test material were prepared and used within the established range of stability and stored at room temperature until used. - Duration of treatment / exposure:
- 29 days.
- Frequency of treatment:
- Animals were dosed daily at approximately the same time (± 2 hours) by intragastric intubation at a dose volume of 10 mL/kg body weight for 29 days. The amount of test material each animal received was based on the most recently collected body weight and the formulation concentration. Control animals were dosed with corn oil at a volume of 10 mL/kg of body weight.
- Post exposure period:
- Post exposure period is only relevant for the repeated-dose toxicity study described in details under IUCLID section 7.5.1. For micronucleus evaluation, assessments were performed on Day 4 and on the day of sacrifice (Day 30) and a post exposure period is therefore not applicable.
- Dose / conc.:
- 1 000 mg/kg bw/day
- Dose / conc.:
- 300 mg/kg bw/day
- Dose / conc.:
- 100 mg/kg bw/day
- Dose / conc.:
- 0 mg/kg bw/day
- Remarks:
- Vehicle alone.
- No. of animals per sex per dose:
- - 10 animals/sex/dose in the control and 1000 mg/kg/day test groups,
- 5 animals/sex/dose in the 100 and 300 mg/kg/day test groups. - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- No positive control was used in this specific study. The study report presents the historical control data (see in section "Any other information on results incl. tables") including positive control data, demonstrating proficiency of the testing facility in the conduct of the test, as requested by the guideline.
- Tissues and cell types examined:
- The frequency of micronuclei was analysed in the peripheral blood by analysing reticulocytes.
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION
Samples from each exposure level and the negative control were analyzed at both collection time points.
TREATMENT AND SAMPLING TIMES
Peripheral blood samples were collected by sublingual bleeding from 5 animals per sex per group. There were 2 blood collections, a bleed on day 4 and on the day of sacrifice prior to the scheduled sacrifice.
DETAILS OF SLIDE PREPARATION
Approximately 60-120 μL of blood was collected from each animal directly into a labelled microcentrifuge tube containing 350 μL of anticoagulant/diluent (anticoagulant) found in the In Vivo MicroFlow Plus® Rat Micronucleus assay kit (Litron Laboratories, Rochester, New York). The tubes were capped and inverted several times to mix the blood with anticoagulant. The blood/anticoagulant mixture was stored at room temperature for up to 6 hours or refrigerated for up to 24 hours before fixing. The blood samples were fixed in duplicate (approximately 180 μL of blood/anticoagulant mixture each) in 2 mL ultra-cold methanol and stored below -75ºC until processed.
METHOD OF ANALYSIS:
The micronucleus evaluation was conducted by flow cytometry. Whenever feasible, at least 20000 reticulocytes were analyzed per blood sample. The samples were analyzed and evaluated using the In Vivo MicroFlow Plus® Rat Micronucleus assay kit. - Evaluation criteria:
- - Toxicity was evaluated by the frequency of immature erythrocytes (%RETs) among the total erythrocytes (RETs plus NCEs).
- The frequency of micronucleated reticulocytes (%MN-RETs) was used as a measure of induction of aneugenic or clastogenic alterations by the test material.
Where:
RET: reticulocyte
NCE: normochromatic erythrocytes
MN-RET: micronucleated reticulocyte - Statistics:
- Micronucleus data was evaluated using scientific judgment taking into account both statistical and biological significance. The individual animal was considered the experimental unit. For each treatment group, the mean and standard deviation of % RETs and % MN-RETs was calculated. Data was transformed prior to analysis using an arcsine square root or Freeman-Tukey function. This transformation was appropriate for proportions since the distribution of the transformed data more closely approximates a normal distribution than does the nontransformed proportion. All micronucleus data analyses was one-tailed and conducted at a significance level of 5%. See also Table 1 in "Any other information on materials and methods incl. tables".
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Remarks:
- The vehicle (negative) control group exhibited a response consistent with the %MN-RETs historical control data.
- Negative controls validity:
- valid
- Remarks:
- The negative control was the vehicle administered alone, see above.
- Positive controls validity:
- not applicable
- Additional information on results:
- - Induction of micronuclei: No statistically significant or biologically relevant increases in the micronucleated reticulocyte frequency (% MN-RET) were observed in any evaluated test substance treated group of male or female animals at either time point.
- Appropriateness of dose levels and route: A reduction in %RETs among the total erythrocytes was observed indicating that the test substance reached the target cells. In male rats statistically significant decreases were observed at 300 and 1000 mg/kg bw/day at day 4. In female rats, although not statistically significant, a dose related decrease in %RETs was also observed at day 4. However, no decrease in %RET was observed at day 30 in either male or female animals. - Conclusions:
- Under the conditions of this study, 2,2-Difluoroethyl acetate showed no evidence of in vivo cytogenicity with no statistically significant or biologically relevant increase in micronucleated reticulocytes in rats.
- Executive summary:
The cytogenic potential of 2,2-Difluoroethyl acetate was investigated in the context of a 28-day repeated-dose toxicity study by gavage. This study was performed according to OECD test guidelines 407 (for the repeated-dose toxicity study) and 474 (for the micronucleus evaluation) under GLP compliance.
Four groups of male and female Crl:CD(SD) rats were administered 0, 100, 300, and 1000 mg/kg bw/day test material by gavage for 29 days consecutively (10 animals per sex in the control and 1000 mg/kg/day test groups and 5 animals per sex in the 100 and 300 mg/kg/day test groups).
Peripheral blood samples were collected from 5 animals per sex on days 4 and 30 for micronucleus evaluation by flow cytometry. The frequency of micronucleated reticulocytes (%MN RETs) was used as a measure of induction of aneugenic or clastogenic alterations by the test material. Samples from the negative control and each exposure level were analysed at both collection time points.
No statistically significant or biologically relevant increases in the micronucleated reticulocyte (RET) frequency were observed in bone marrow in any test material treated group of male or female rats at either time point. The negative control group exhibited %MN-RETs values consistent with the historical control data.
Under the conditions of this study, 2,2-Difluoroethyl acetate showed no evidence of in vivo genotoxicity with no biologically relevant increase in micronucleated reticulocytes.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- This genotoxicity evaluation was combined with a 28-day repeated-dose toxicity study by inhalation. Only the data relevant for the micronucleus evaluation were described here (for the ‘classical’ repeated-dose toxicity study, see IU section 7.5.2).
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- FROM 28 APRIL 2014 to 1 SEPTEMBER 2015.
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Remarks:
- The present micronucleus evaluation was done during a 28-day repeated dose toxicity study by inhalation route.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- 1997.
- Deviations:
- not specified
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian erythrocyte micronucleus test
- Species:
- rat
- Strain:
- other: Crl:CD(SD).
- Details on species / strain selection:
- Rats have historically been used in safety evaluation studies for inhalation toxicity testing. The Crl:CD(SD) rat was selected based on consistently acceptable health status and on extensive experience with this strain at the testing facility.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories International, Inc., Raleigh, North Carolina, U.S.A.
- Females: nulliparous and non pregnant.
- Age at study initiation: approximately 8 weeks old.
- Weight at study initiation: male rats weighed between 248 and 308 grams; female rats weighed between 176 and 226 grams. The weight variation of selected rats did not exceed ± 20% of the mean weight for each sex.
- Assigned to test groups randomly: yes, the rats were distributed by computerized, stratified randomization into study groups, so that there were no statistically significant differences among group body weight means within a sex.
- Fasting period before study: not specified.
- Housing: except during exposure, animals were housed in pairs (sexes separated) in solid-bottom caging with Enrich-o'Cobs™ as bedding and enrichment.
- Diet: except during exposure, PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 was available ad libitum.
- Water: except during exposure, tap water was available ad libitum.
- Acclimation period: at least 6 days. The animals were released from quarantine based on normal observations for body weights and clinical signs.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26°C.
- Humidity (%): 30-70%.
- Photoperiod: Animal rooms were artificially illuminated (fluorescent light) on an approximate 12-hour light/dark cycle.
IN-LIFE DATES: From 20 May 2014 to 15 July 2014. - Route of administration:
- inhalation: vapour
- Vehicle:
- - Vehicle used: air.
- Justification for choice of vehicle: chamber atmospheres were generated by flash evaporation of the test material in air with a round-bottom evaporation flask.
- Concentration of test material in vehicle: chamber concentrations of the test material were controlled by varying the test material feed rate to the heated flasks. - Details on exposure:
- TYPE OF INHALATION EXPOSURE
Whole body.
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: exposure chambers were constructed of stainless steel and glass (NYU style) with a nominal internal volume of 350 L. A tangential feed inside the chamber promoted uniform chamber distribution of the test atmosphere. The chamber volume was chosen so that the total body volume of the test animals did not exceed 5% of the chamber volume.
- Method of holding animals in test chamber: during exposure, animals were individually placed in stainless steel, wire-mesh cages and exposed, whole-body, inside the exposure chamber.
- Source and rate of air: Houseline generation air, metered into the flask by a Brooks model 5850E mass flow controller, carried the vaporized test substance into a supply air stream leading to the exposure chamber.
- System of generating test material vapor: chamber atmospheres were generated by flash evaporation of the test material in air with a round-bottom evaporation flask. The test material was metered into the evaporation flask with a Harvard Apparatus model 22 syringe infusion pump. The evaporation flasks were placed in Unimantle heaters that were heated up to 175°C for the 100 and 750 ppm chambers and 195°C for the 1500 ppm chamber to vaporize the test material. Heat tapes set at approximately 95°C were used to wrap the glass connection tubes from the evaporation flasks to the chamber inlets for the 1500 ppm chamber. Houseline generation air, metered into the flask by a Brooks model 5850E mass flow controller, carried the vaporized test material into a supply air stream leading to the exposure chamber. Chamber concentrations of test material were controlled by varying the test material feed rate to the heated flasks.
- Temperature, humidity in air chamber: chamber temperature was targeted at 19-25°C and recorded approximately once/hour during each exposure. Chamber relative humidity was targeted at 30-70% and recorded approximately once/hour during each exposure. Temperature and humidity were measured with a VWR dial-type thermometer/hygrometer.
- Air flow rate and air change rate: chamber airflow was set at the beginning of each exposure to achieve at least 10 air changes per hour and monitored continually with a Brooks model 5850E mass flow controller. Airflows were recorded initially, approximately once/hour and whenever changes were made during each exposure. Chamber oxygen concentration was targeted to be at least 19%, measured with a Teledyne Analytical Instruments model GB300 oxygen analyser and recorded once during each exposure.
- Treatment of exhaust air: test atmospheres were exhausted into the laboratory’s exhaust stack.
TEST ATMOSPHERE
- Brief description of analytical method used: during each exposure, the atmospheric concentration of the test material was determined by gas chromatography (GC) at approximately 12 times per day in the test chambers. The control chamber was monitored at least once per day. Samples of chamber atmosphere were directly injected into an Agilent Technologies model 6890N GC equipped with a pneumatically operated gas sample valve and a flame ionization detector. All samples were chromatographed using an oven temperature rate of 80°C on a 30-meter J&W Scientific Inc. DB-5 fused silica glass column. The atmospheric concentration of the test material was determined from a standard curve derived from vapor standards. Standards were prepared prior to each exposure by injecting known volumes of the liquid test material into Tedlar® bags containing known volumes of air. Sample results (injection time, date, valve position and measured concentration) were recorded by CITADS. Upon completion of the exposures, GC sample results were transferred to the Camile Inhalation Reporting and Analysis System (CIRAS), which collated sample calculations.
- Samples taken from breathing zone: yes. Prior to the start of the exposure phase, the distribution of the test material was determined in the high-concentration chamber. Vapor samples were collected from the center of the chamber and 8 separate locations inside the exposure chamber and averaged. Individual samples from the 8 separate locations in the chamber were compared to the overall average for determination of homogeneous distribution of test material in the exposure chamber. - Duration of treatment / exposure:
- 4 weeks.
- Frequency of treatment:
- Each group of animals was exposed for 6 hours/day, 5 days/week over a 4-week period (weekends and holidays excluded) for a total of 20 exposures.
- Post exposure period:
- Post exposure period is only relevant for the repeated-dose toxicity study described in details under IUCLID section 7.5.2. For micronucleus evaluation, assessments were performed on Days 3 and 29 and a post exposure period is therefore not applicable.
- Dose / conc.:
- 1 500 ppm
- Remarks:
- Equivalent to 7.6 mg/L
Conversion from ppm to mg/L:
Test concentrations (mg/m3) = Test concentrations (ppm) x Molecular weight (g/mol) / 24.5 (L/mol)
Where:
Test concentrations (ppm) = 1500
Molecular weight (g/mol) = 124.09
24.5 L/mol = gas constant at 25 °C and 1013.25 hPa
Test concentrations (mg/m3) = 1500 x 124.09 / 24.5
Test concentrations (mg/m3) = 7597
Test concentrations (mg/L) = 7.6 - Dose / conc.:
- 750 ppm
- Remarks:
- Equivalent to 3.8 mg/L
Conversion from ppm to mg/L:
Test concentrations (mg/m3) = Test concentrations (ppm) x Molecular weight (g/mol) / 24.5 (L/mol)
Where:
Test concentrations (ppm) = 750
Molecular weight (g/mol) = 124.09
24.5 L/mol = gas constant at 25 °C and 1013.25 hPa
Test concentrations (mg/m3) = 750 x 124.09 / 24.5
Test concentrations (mg/m3) = 3799
Test concentrations (mg/L) = 3.8 - Dose / conc.:
- 100 ppm
- Remarks:
- Equivalent to 0.51 mg/L
Conversion from ppm to mg/L:
Test concentrations (mg/m3) = Test concentrations (ppm) x Molecular weight (g/mol) / 24.5 (L/mol)
Where:
Test concentrations (ppm) = 100
Molecular weight (g/mol) = 124.09
24.5 L/mol = gas constant at 25 °C and 1013.25 hPa
Test concentrations (mg/m3) = 100 x 124.09 / 24.5
Test concentrations (mg/m3) = 506
Test concentrations (mg/L) = 0.51 - Dose / conc.:
- 0 ppm
- Remarks:
- (Air control only).
- No. of animals per sex per dose:
- 20 animals/sex/dose.
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- No positive control was used in this specific study. The study report presents the historical control data (see in section "Any other information on results incl. tables") including positive control data, demonstrating proficiency of the testing facility in the conduct of the test, as requested by the guideline.
- Tissues and cell types examined:
- The frequency of micronuclei was analysed in the peripheral blood by analysing reticulocytes.
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION
Samples from each exposure level and the negative control were analyzed at both collection time points.
TREATMENT AND SAMPLING TIMES
Peripheral blood samples were collected by tail vein from 5 animals per sex per group. There were 2 blood collections, a bleed after the third exposure (on Day 3) and following the animals’ final exposure (on Day 29). Blood collections occurred within 4 hours after the third and final exposure.
DETAILS OF SLIDE PREPARATION
Approximately 60-120 μL of blood was collected from each animal directly into a labelled microcentrifuge tube containing 350 μL of anticoagulant/diluent (anticoagulant) found in the In Vivo MicroFlow Plus® Rat Micronucleus assay kit (Litron Laboratories, Rochester, New York). The tubes were capped and inverted several times to mix the blood with anticoagulant. The blood/anticoagulant mixture were stored either at room temperature or refrigerated for up to 2 hours before fixing. The blood samples were fixed in duplicate (approximately 180 μL of blood/anticoagulant mixture each) in 2 mL ultra-cold methanol and stored below -75ºC until processed.
METHOD OF ANALYSIS
The micronucleus evaluation was conducted by flow cytometry. Whenever feasible, at least 20000 reticulocytes were analyzed per blood sample. The samples were analyzed and evaluated using the In Vivo MicroFlow Plus® Rat Micronucleus assay kit. - Evaluation criteria:
- - Toxicity was evaluated by the frequency of immature erythrocytes (%RETs) among the total erythrocytes (RETs plus NCEs).
- The frequency of micronucleated reticulocytes (%MN RETs) was used as a measure of induction of aneugenic or clastogenic alterations by the test material.
Where:
RET: reticulocyte
NCE: normochromatic erythrocytes
MN-RET: micronucleated reticulocyte - Statistics:
- Micronucleus data was evaluated using scientific judgment taking into account both statistical and biological significance. The individual animal was considered the experimental unit. For each treatment group, the mean and standard deviation of % RETs and % MN-RETs was calculated. Data was transformed prior to analysis using an arcsine square root or Freeman-Tukey function. This transformation was appropriate for proportions since the distribution of the transformed data more closely approximates a normal distribution than does the nontransformed proportion. All micronucleus data analyses was one-tailed and conducted at a significance level of 5%. See also Table 1 in "Any other information on materials and methods incl. tables".
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Remarks:
- The vehicle (negative) control group exhibited a response consistent with the %MN-RETs historical control data.
- Negative controls validity:
- valid
- Remarks:
- The negative control was the vehicle administered alone (air), see above.
- Positive controls validity:
- not applicable
- Additional information on results:
- - Induction of micronuclei: No statistically significant or biologically relevant increases in the micronucleated reticulocyte frequency (% MN-RET) were observed in any evaluated male or female exposure group at either time point.
- Appropriateness of dose levels and route: A reduction in the frequency of immature erythrocytes (%RETs) among the total erythrocytes was observed in both male and female rats indicating that the test substance reached the target cells. In male rats statistically significant decreases were observed at all exposure levels on test day 3 and in the 750 and 1500 ppm exposure groups on test day 29. In female rats statistically significant decreases were observed in the 750 and 1500 ppm exposure groups on test day 3 only. - Conclusions:
- Under the conditions of this study, 2,2-Difluoroethyl acetate showed no evidence of in vivo cytogenicity with no statistically significant or biologically relevant increase in micronucleated reticulocytes in rats.
- Executive summary:
The cytogenic potential of 2,2-Difluoroethyl acetate was investigated in the context of a 28-day repeated-dose toxicity study by inhalation. This study was performed according to OECD test guidelines 412 (for the repeated-dose toxicity study) and 474 (for the micronucleus evaluation) under GLP compliance.
Four groups of male and female Crl:CD(SD) albino rats (20 animals per sex per group) were exposed whole body, 6 hours per day, 5 days a week to vapour concentrations of 0 (air control), 100 ± 0.63, 750 ± 1.3 and 1500 ± 2.9 ppm test material over a 4-week period for a total of 20 exposures. Test atmospheres were generated by flash evaporation of the test material in air. Peripheral blood samples were collected from 5 animals per sex per exposure group on days 3 and 29 for micronucleus evaluation by flow cytometry. The frequency of micronucleated reticulocytes (%MN RETs) was used as a measure of induction of aneugenic or clastogenic alterations by the test material. Samples from the negative control and each exposure level were analysed at both collection time points.
No statistically significant or biologically relevant increases in the micronucleated reticulocyte (RET) frequency were observed in bone marrow in any test material treated group of male or female rats at either time point. The negative control group exhibited %MN-RETs values consistent with the historical control data.
Under the conditions of this study, 2,2-Difluoroethyl acetateshowed no evidence of in vivo genotoxicity with no biologically relevant increase in micronucleated reticulocytes.
Referenceopen allclose all
Dose formulation analysis:
The formulation analyses showed that the test material was at the targeted concentrations and stable when stored at room temperature for up to 15 days. See more details in the IUCLID section 7.5.1.
Historical control data:
Table 2: Historical Control Data
Negative control animalsa,b
Parameter | % RET- Males | % RET - Females | % MN-RET- Males | % MN-RET - Females |
Mean | 1.64 | 1.41 | 0.11 | 0.11 |
Standard deviation | 0.90 | 0.67 | 0.05 | 0.05 |
Range | 0.59 – 5.55 | 0.34 – 4.14 | 0.03 – 0.31 | 0.02 – 0.37 |
Positive control animalsa,c
Parameter | % RET- Males | % RET - Females | % MN-RET- Males | % MN-RET - Females |
Mean | 1.26 | 0.83 | 1.03 | 1.03 |
Standard deviation | 0.87 | 0.52 | 0.34 | 0.55 |
Range | 0.25 – 3.96 | 0.07 – 2.38 | 0.27 – 1.85 | 0.31 – 3.12 |
a Historical control data was compiled from studies conducted at DuPont Haskell Global Centers since 2009.
b Negative controls include all vehicles, routes of administration and sample times.
c The positive control is cyclophosphamide, administered by oral gavage. Blood was collected 48 hours after a single administration.
Tabulated results:
Table 3: Micronucleus Evaluation for Male Rats
Dose group | Group 1 0 mg/kg bw/day | Group 2 100 mg/kg bw/day | Group 3 300 mg/kg bw/day | Group 4 1000 mg/kg bw/day |
RET % : Mean SD (a) | ||||
Day 4 | 4.79 | 4.02 | 3.84* | 2.79* |
0.63 (5) | 0.87 (5) | 0.23 (5) | 0.48 (5) | |
Day 30 | 1.61 | 1.30 | 1.47 | 1.53 |
0.29 (5) | 0.37 (5) | 0.40 (5) | 0.88 (5) | |
MN-RET % : Mean SD (a) | ||||
Day 4 | 0.11 | 0.08 | 0.08 | 0.09 |
0.03 (5) | 0.03 (5) | 0.03 (5) | 0.04 (5) | |
Day 30 | 0.07 | 0.08 | 0.07 | 0.07 |
0.02 (5) | 0.05 (5) | 0.02 (5) | 0.03 (5) | |
(a) Standard deviation (number of values included in calculation).
* Statistically significant difference from control at p < 0.05 by Dunnett/Tamhane-Dunnett test.
Table 4: Micronucleus Evaluation for Female Rats
Dose group | Group 1 0 mg/kg bw/day | Group 2 100 mg/kg bw/day | Group 3 300 mg/kg bw/day | Group 4 1000 mg/kg bw/day |
RET % : Mean SD (a) | ||||
Day 4 | 2.00 | 2.17 | 1.76 | 1.52 |
0.26 (5) | 0.57 (5) | 0.60 (5) | 0.38 (5) | |
Day 30 | 0.97 | 0.94 | 0.72 | 0.90 |
0.23 (5) | 0.30 (5) | 0.24 (5) | 0.14 (5) | |
MN-RET % : Mean SD (a) | ||||
Day 4 | 0.08 | 0.09 | 0.07 | 0.10 |
0.04 (5) | 0.04 (5) | 0.04 (5) | 0.03 (5) | |
Day 30 | 0.06 | 0.07 | 0.07 | 0.05 |
0.02 (5) | 0.03 (5) | 0.03 (5) | 0.03 (5) | |
(a) Standard deviation (Number of values included in calculation).
Inhalation concentrations
The chamber concentrations and environmental conditions were considered adequate for the conduct of the study. See more details in the IUCLID section 7.5.2.
Historical control data:
Table 2: Historical Control Data
Negative control animalsa,b
Parameter | % RET- Males | % RET - Females | % MN-RET- Males | % MN-RET - Females |
Mean | 1.64 | 1.41 | 0.11 | 0.11 |
Standard deviation | 0.90 | 0.67 | 0.05 | 0.05 |
Range | 0.59 – 5.55 | 0.34 – 4.14 | 0.03 – 0.31 | 0.02 – 0.37 |
Positive control animalsa,c
Parameter | % RET- Males | % RET - Females | % MN-RET- Males | % MN-RET - Females |
Mean | 1.26 | 0.83 | 1.03 | 1.03 |
Standard deviation | 0.87 | 0.52 | 0.34 | 0.55 |
Range | 0.25 – 3.96 | 0.07 – 2.38 | 0.27 – 1.85 | 0.31 – 3.12 |
a Historical control data was compiled from studies conducted at DuPont Haskell Global Centers since 2009.
b Negative controls include all vehicles, routes of administration, and sample times.
c The positive control is cyclophosphamide, administered by oral gavage. Blood was collected 48 hours after a single administration.
Tabulated results:
Table 3: Micronucleus Evaluation for Male Rats
Dose group | Group 1 0 ppm | Group 2 100 ppm | Group 3 750 ppm | Group 4 1500 ppm |
RET % : Mean SD (a) | ||||
Day 3 | 4.61 | 3.57* | 3.02* | 1.88* |
0.40 (5) | 0.59 (5) | 0.47 (5) | 0.74 (5) | |
Day 29 | 1.27 | 1.05 | 0.76* | 0.76* |
0.35 (5) | 0.24 (5) | 0.11 (5) | 0.21 (5) | |
MN-RET % : Mean SD (a) | ||||
Day3 | 0.13 | 0.08 | 0.08 | 0.08 |
0.05 (5) | 0.03 (5) | 0.03 (5) | 0.03 (5) | |
Day 29 | 0.08 | 0.06 | 0.05 | 0.07 |
0.02 (5) | 0.03 (5) | 0.02 (5) | 0.03 (5) | |
(a) Standard deviation (Number of values included in calculation).
* Statistically significant difference from control at p < 0.05 by Dunnett/Tamhane-Dunnett test.
Table 4: Micronucleus Evaluation for Female Rats
Dose group | Group 1 0 ppm | Group 2 100 ppm | Group 3 750 ppm | Group 4 1500 ppm |
RET % : Mean SD (a) | ||||
Day 3 | 1.24 | 1.19 | 0.76* | 0.53 * |
0.31 (5) | 0.15 (5) | 0.14 (5) | 0.12 (5) | |
Day 29 | 0.89 | 0.95 | 0.78 | 0.70 |
0.21 (5) | 0.29 (5) | 0.20 (5) | 0.16 (5) | |
MN-RET % : Mean SD (a) | ||||
Day 3 | 0.05 | 0.05 | 0.07 | 0.08 |
0.01 (5) | 0.02 (5) | 0.02 (5) | 0.03 (5) | |
Day 29 | 0.06 | 0.06 | 0.08 | 0.07 |
0.01 (5) | 0.02 (5) | 0.03 (5) | 0.02 (5) | |
(a) Standard deviation (Number of values included in calculation).
* Statistically significant difference from control at p < 0.05 by Dunnett/Tamhane-Dunnett test.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Genetic toxicity in vitro: Three experimental in vitro studies performed under GLP compliance and in accordance with OECD test guidelines 471 (bacterial reverse mutation assay / Ames test), 490 (mammalian cell gene mutation test using the thymidine kinase gene / Mouse Lymphoma Assay, MLA) and 473 (mammalian chromosome aberration test) are available. All were flagged as key studies and assigned a Klimisch score of 1.
Genetic toxicity in vivo: Two in vivo mammalian erythrocyte micronucleus tests were performed under GLP compliance and in accordance with OECD test guideline 474 in the context of 28-day repeated-dose toxicity studies by gavage and by inhalation. Both were flagged as key studies and assigned a Klimisch score of 1.
Justification for classification or non-classification
The three in vitro and the two in vivo tests all provided negative results. Consequently, in accordance with criteria of the CLP Regulation, 2,2-Difluoroethyl acetate does not need to be classified for genotoxicity.
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