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EC number: 203-940-1 | CAS number: 112-15-2
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Gene mutation in bacteria - Negative
Gene mutation in mammalian cells - Negative
Cytogenicity - Negative
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 September 04, 2020 to December 02, 2020
- 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:
- 1997-07-21, corrected 2020-06-26
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source of test material: Production lot
- Lot/batch number of test material: P230220120
- Expiration date of the lot/batch: 2023-04-29
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Cool, well ventilated place
- Stability under storage conditions: Stable
- Stability under test conditions: Stable - Target gene:
- his operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9 : Mitochondrial fraction from liver of male Sprague-Dawley rats treated with Aroclor 1254
- method of preparation of S9 mix: S9 fraction buffered and supplemented with the essential co-factors β-NADP and glucose-6-phosphate to form the S9 mix.
- concentration or volume of S9 mix and S9 in the final culture medium: 5% v/v S9 mix for plate incorporation assay, 10% v/v S9 mix for pre-incubation assay
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Yes, by supplier. - Test concentrations with justification for top dose:
- Plate incorporation assay: 0.0015 - 5 µLg/plate with and without metabolic activation
Pre-incubation assay: 0.1563 - 5 µLg/plate with and without metabolic activation - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Distilled water
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- Strain TA1537 in the absence of S9
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- Strains TA1535 and TA 100 in the absence of S9
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- Remarks:
- Strain TA98 in the absence of S9
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- Strain TA102 in the absence of S9
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene
- Remarks:
- All strains in the presence of S9
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Experiment 1: in medium; in agar (plate incorporation); Experiment 2: preincubation
DURATION
- Preincubation period: 30 min at 30 °C (only experiment 2)
- Exposure duration: 96 hours at 37 °C
NUMBER OF REPLICATIONS: triplicates per concentration in 2 independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: number of revertants, reduction of background lawn
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate/triplicate
- Number of independent experiments: 2
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): ca 2 x 10E+9 CFU/mL
- Test substance added in medium; in agar (plate incorporation) (1st assay) and pre-incubation (2nd assay)
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 minutes
- Exposure duration/duration of treatment: 48 hours
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition (thinning of background lawn) and reduction in number of colonies
METHODS FOR MEASUREMENTS OF GENOTOXICIY
Counting of numbers of revertant colonies - Rationale for test conditions:
- As prescribed by test guidelines
- Evaluation criteria:
- The following criteria were used to determine a valid assay:
Tester Strain Integrity - All tester strain cultures exhibited sensitivity to crystal violet, demonstrating presence of the rfa wall mutation. All tester strains demonstrated a requirement for biotin except strain TA102 which is biotin independent. All tester strains showed sensitivity to UV exposure except TA102 which is wild type. All the tester strains demonstrated a requirement for histidine for their growth. Tester strains TA98, TA100 and TA102 exhibited resistance to ampicillin, demonstrating presence of the pKM101 plasmid. Tester strains TA102 exhibited resistance to tetracycline, demonstrating presence of the pAQ1 plasmid.
The following criteria were used to determine a positive result:
A result was considered positive if a concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain, with or without metabolic activation, was seen. - Statistics:
- Mean values and standard deviations were calculated. Simple linear regression analysis was performed fto assess the dose dependent nature of any increase in revertant colonies.
- 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
- Positive controls validity:
- valid
- 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
- Positive controls validity:
- valid
- 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
- Positive controls validity:
- valid
- 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
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 102
- 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
- Positive controls validity:
- valid
- Additional information on results:
- ADDITIONAL INFORMATION ON CYTOTOXICITY:TEST-SPECIFIC CONFOUNDING FACTORS
- Possibility of evaporation from medium: No
- Water solubility: Suitable for the assay
- Precipitation: No precipitation occurred up to the limit concentration of 5 µL/plate
STUDY RESULTS
- Concurrent vehicle negative and positive control data : Valid
For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : None observed
Ames test:
- Signs of toxicity : Not observed
- Mean number of revertant colonies per plate and standard deviation : See tabulated data
- Positive historical control data: Test data fell within the historical control range
- Negative (solvent/vehicle) historical control data: Test data fell wihin the historical control range - Conclusions:
- The tested substance is non-mutagenic to any strain of Salmonella typhimurium, viz., TA1537, TA1535, TA98, TA100 and TA102, when tested under the specified conditions.
- Executive summary:
The mutagenic activity of 2 -(2 -ethoxyethoxy)ethyl acetate has been evaluated using the bacterial reverse mutation test, on five histidine deficient mutant tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100, and TA102) using methods described by OECD test guidelines.
The substance did not induce any significant increase in the number of revertants, in trials with and without S9 mix, in any tester strain. Values for the negative control were within the historical control ranges of the laboratory. Positive controls showed an increase in the number of revertant colonies, demonstrating the efficiency of the test system.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From August 17, 2020 to February 11, 2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
- Version / remarks:
- 2016-07-29
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source of test material: Production lot
- Lot/batch number of test material: P230220120
- Expiration date of the lot/batch: 2023-04-29
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Cool, well ventilated place
- Stability under storage conditions: Stable
- Stability under test conditions: Stable - Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Minimum Essential Medium, Eagle α-Modification with/without nucleosides
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Additional strain / cell type characteristics:
- other: CHO-K1
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction (Aroclor 1254 induced male SD rat liver ).
- Test concentrations with justification for top dose:
- Concentrations of 0.125, 0.25, 0.5, 1 and 2 µL/mL investigated, the highest concentration examined being that recommended by the OECD test guideline
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: aqueous solvent (water)
- Justification for choice of solvent/vehicle: Selected solvent is compatible with the test system - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- +S9
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- -S9
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: single
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 2E+5/mL
- Test substance added: in medium
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment: 8 days
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7-9 days
- Selection time (if incubation with a selective agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 16 days
- Selective agent: 6-thioguanine, 5 µg/mL, 8 days cell exposure.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2E+5 cells/mL seeded. Enumeration of cells by counting following staining
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency - Rationale for test conditions:
- In accordance with OECD test guideline
- Evaluation criteria:
- The test item was considered to be positive if:
- At least one of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control.
- The increase was concentration-related when evaluated with an appropriate trend test.
- Any of the results were outside the distribution of the historical negative control data (e.g. Poisson based 95% control limit)
The test item was considered negative if:
- None of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control.
- There was no concentration-related increase when evaluated with an appropriate trend test.
- All results were inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limit). - Statistics:
- Kastenbaum MA and Bowman KO: Tables for determining the statistical significance of mutation frequencies. Mutation Research 9 527-549 1970.
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: good
- Precipitation: no
- Other confounding effects: no
RANGE-FINDING/SCREENING STUDIES: yes - concentrations of 0.063, 0.125, 0.25, 0.5, 1 and 2 µL/mL
COMPARISON WITH HISTORICAL CONTROL DATA: yes
ADDITIONAL INFORMATION ON CYTOTOXICITY : N/A - Conclusions:
- 2 -(2 -ethoxyethoxy) ethyl acetate is considered to be non mutagenic in the CHO/HGPRT forward mutation assay
- Executive summary:
The potential for 2 -(2 -ethoxyethoxy) ethyl acetate to cause gene mutations in a mammalian cell line has been investigated using methods in accordance with OECD test guideline 476.
The results from the study demonstrate that 2 -(2 -ethoxyethoxy) ethyl acetate does not have potential to induce gene mutations at the hprt locus of CHO-K1 cells either in the absence or presence of a metabolic activation system
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From August 17, 2020 to March 01, 2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- 2016-07-29
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source of test material: Production lot
- Lot/batch number of test material: P230220120
- Expiration date of the lot/batch: 2023-04-29
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Cool, well ventilated place
- Stability under storage conditions: Stable
- Stability under test conditions: Stable - Species / strain / cell type:
- lymphocytes:
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: Human blood lymphocytes
- Sex, age and number of blood donors: Single male 28 years of age
- Whether whole blood or separated lymphocytes were used: Whole blood
- Whether blood from different donors were pooled or not: n/a
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI-1640 medium, 37 deg C, 5% CO2 - Cytokinesis block (if used):
- Cytochalasin B
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9: Mitochondrial fraction from liver of male Sprague-Dawley rats treated with Aroclor 1254
- method of preparation of S9 mix: S9 fraction buffered and supplemented with the essential co-factors β-NADP and glucose-6-phosphate to form the S9 mix.
- concentration or volume of S9 mix and S9 in the final culture medium: 2% v/v
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Yes, by supplier. - Test concentrations with justification for top dose:
- 0.0 (control), 0.125, 0.25, 0.5, 1.0 and 2 µL/mL
Highest tested concentration as recommended by test guideline - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: aqueous solvent (water)
- Justification for choice of solvent/vehicle: Selected solvent is compatible with the test system - Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- vinblastine
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium: yes
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Short-term treatment - 4 hours; Long-term treatment - 24 hours
- Harvest time after the end of treatment (sampling/recovery times): 24 hours
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: Identity of cytokinesis blocking substance - cytochalasin B; Concentration - 6 µg/mL; Duration and period of cell exposure - 24 hours
- Methods of slide preparation and staining technique used including the stain used: Slides were prepared from fixed cell suspension placed drop by drop onto pre-cleaned, ice-chilled slides and then dried over a slide warmer. Dried slides were stained with 5% Giemsa solution for 30 minutes
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 2 x replicates, 1000 cells/replicate
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
- Criteria for selecting cells suitable for scoring Micronuclei - The cytokinesis-blocked binucleated cells that were selected for scoring (for MN frequency) had the following characteristics:
• The cells were binucleated.
• The two nuclei in a binucleated cell had intact nuclear membranes and situated within the same cytoplasmic boundary.
• The two nuclei in a binucleated cell were approximately equal in size, staining pattern and staining intensity.
• The two nuclei within a BN cell whenever attached by a nucleoplasmic bridge, it was no wider than 1/4th of the nuclear diameter.
• The two main nuclei in a BN cell might touch but ideally they were not overlapping each other.
• A cell with two overlapping nuclei were scored only if the nuclear boundaries of each nucleus were distinguishable.
• The cytoplasmic boundary or membrane of a binucleated cell was intact and clearly distinguishable from the cytoplasmic boundary of adjacent cells.
- Criteria for scoring micronuclei (MNi): Micronuclei which had the following characteristics were considered for evaluation:
• The diameter of MNi in human lymphocytes usually varies between 1/16th and 1/3rd of the mean diameter of the main nuclei, which corresponds to 1/256th and 1/9th of the area of one of the main nuclei in a BN cell, respectively.
• Micronuclei (MNi) were non-refractile and therefore were readily distinguished from artifacts such as staining particles.
• Micronuclei (MNi) were not linked or connected to the main nuclei.
• Micronuclei (MNi) might touch but were not overlapping with the main nuclei and the micronuclear boundary was distinguishable from the nuclear boundary.
• Micronuclei (MNi) usually had the same staining intensity as the main nuclei but occasionally staining might be more intense.
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): As no increase in Frequency of micronuclei was seen, characterization of micronuclei for whole or fragmented chromosomes by kinetochore antibody binding was not required/applicable
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: replication index
- Rationale for test conditions:
- Test condiitions were those described by the relevant OECD test guideline
- Evaluation criteria:
- Assay considered negative if the following criteria were met:
- None of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control.
- There was no concentration-related increase when evaluated with an appropriate trend test (e.g., Kraskal-wallis).
- All results were inside the distribution of the historical control data. - Statistics:
- Data on micronuclei containing binucleated cells were subjected to Shapiro-Wilk’s test for normality and Bartlett’s test to assess homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. Where the data did not meet suitable homogeneity of variance, Student's t-test was performed to determine the level of significance between Negative control, three selected test concentrations (selected based on the replicative index data) and positive controls.
- Key result
- Species / strain:
- mammalian cell line, other: human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: 7.24 - 7.35
- Data on osmolality: 296 - 316 mOsm/kg H2O
- Possibility of evaporation from medium: No
- Water solubility: Soluble in selected vehicle (water) at 200 µL/mL
- Precipitation and time of the determination: None observed
- Definition of acceptable cells for analysis: Frequency of viable mono-nucleated, bi- nucleated and multinucleated cells was measured to determine cytostatic effects and the rate of mitotic division. Cell types having the following characteristics were considered for evaluation:
• Mono-nucleated, bi- nucleated and multinucleated cells should be with an intact cytoplasm and normal nucleus morphology containing one, two and three or more nuclei, respectively.
• These cells may or may not contain one or more micronuclei.
- Other confounding effects: None
RANGE-FINDING/SCREENING STUDIES:
Preliminary cytotoxicity assay at 6 concentrations. Cytostasis observed in the absence of the metabolic activation was 1.87%, 2.29%, 4.46%, 6.30%, 10.84% and 18.65% at concentrations of 0.0625, 0.125, 0.25, 0.5, 1, and 2 µL/mL, respectively and in the presence of metabolic activation was 0.34%, 2.50%, 4.84%, 7.95%, 5.73% and 11.91% at 0.0625, 0.125, 0.25, 0.5, 1, and 2 µL/mL, respectively
STUDY RESULTS
- Concurrent vehicle negative and positive control data
The number of binucleated cells with micronuclei in the negative control cultures were within the published range (Fenech, M., 2007: Cytokinesis-block micronucleus cytome assay, Nature Protocols, Vol. 2/5, pp. 1084-1104). The positive control, cyclophosphamide, produced a significant increase in the frequency of micronuclei containing binucleated cells (4.736% compared with a negative control value of 0.349%) following short term exposure in the presence of metabolic activation. The positive control, vinblastine, produced a significant increase (mean value of positive control was 3.977% MNBN against a negative control mean value of 0.342% MNBN negative control of 0.342) in the frequency of micronuclei containing binucleated cells following continuous exposure in the absence of metabolic activation.
For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : There was no concentration-related increase when evaluated with an appropriate trend test
- Statistical analysis; p-value if any : No statistically significant difference between treated cells and the negative control was apparent
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
Without metabolic activation - range 0.29 - 5.57; mean 3.80; standard deviation 0.90; 95% control limits 0.00 - 7.70; number of data 36
With metabolic activation - range 2.37 - 6.74; mean 3.91; standard deviation 0.75; 95% control limits 0.00 - 7.86; number of data 36
- Negative (solvent/vehicle) historical control data:
Without metabolic activation - range 0.10 - 0.60; mean 0.36; standard deviation 0.14; 95% control limits 0.00 - 1.56; number of data 36
With metabolic activation - range 0.10 - 0.60; mean 0.35; standard deviation 0.13; 95% control limits 0.00 - 1.53; number of data 36 - Conclusions:
- 2-(2 -ethoxyethoxy) ethyl acetate does not induce micronuclei and does not have clastogenic or aneugenic potential in isolated human peripheral blood lymphocytes both in the absence and presence of a metabolic activation system.
- Executive summary:
The clastogenic and aneugenic potential of EDGA 2 -(2 -ethoxyethoxy)ethyl acetate has been investigated in human peripheral blood lymphocytes that have undergone cell division after exposure to the substance in the absence and presence of metabolic activation. Methods used were in accordance with OECD test guideline 487.
The results from the study demonstrate that2 -(2 -ethoxyethoxy) ethyl acetate does not induce micronuclei and does not have clastogenic or aneugenic potential in isolated human peripheral blood lymphocytes both in the absence and presence of a metabolic activation system.
Referenceopen allclose all
Plate incorporation assay - Mean count of revertant colonies
Concentration (µL/plate) |
His+Revertant Colonies/Plate (Absence of Metabolic Activation) |
|||||||||
TA1537 |
TA1535 |
TA98 |
TA100 |
TA102 |
||||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
NC (DW) |
7.00 |
2.83 |
14.00 |
2.83 |
19.00 |
0.00 |
122.00 |
5.66 |
206.00 |
2.83 |
0.0015 |
7.00 |
1.41 |
12.50 |
2.12 |
21.50 |
3.54 |
128.00 |
7.07 |
249.00 |
39.60 |
0.005 |
7.50 |
0.71 |
13.50 |
0.71 |
21.00 |
4.24 |
111.00 |
8.49 |
231.00 |
0.00 |
0.015 |
5.00 |
1.41 |
14.00 |
2.83 |
20.50 |
3.54 |
136.50 |
36.06 |
228.50 |
20.51 |
0.05 |
6.50 |
3.54 |
15.50 |
2.12 |
23.00 |
2.83 |
128.00 |
9.90 |
222.50 |
12.02 |
0.15 |
7.00 |
2.83 |
16.00 |
1.41 |
20.00 |
2.83 |
140.50 |
6.36 |
228.00 |
4.24 |
0.5 |
7.50 |
3.54 |
13.50 |
0.71 |
15.00 |
11.31 |
120.00 |
4.24 |
235.00 |
7.07 |
1.5 |
9.00 |
1.41 |
13.00 |
5.66 |
18.00 |
0.00 |
138.00 |
8.49 |
220.00 |
5.66 |
5 |
6.50 |
0.71 |
13.50 |
0.71 |
14.50 |
7.78 |
126.50 |
2.12 |
219.00 |
19.80 |
PC |
173.00 |
63.64 |
307.00 |
55.15 |
460.00 |
29.70 |
682.50 |
55.86 |
927.50 |
101.12 |
Key: SD = Standard Deviation, NC = Negative Control, DW =Distilled Water, PC = Positive Control {TA1537 = 9-Aminoacridine Hydrochloride Monohydrate (75 µg/plate), TA1535 = Sodium Azide (0.5 µg/plate), TA98 = 2-Nitrofluorene (7.5 µg/plate), TA100 = Sodium Azide (5 µg/plate), TA102 = Mitomycin-C (0.5 µg/plate), 2-Aa = 2-Aminoanthracene (5 µg/plate for TA100)},- = Not Applicable.
Plate incorporation assay - Mean count of revertant colonies
Concentration (µL/plate) |
His+Revertant Colonies/Plate [Presence of Metabolic Activation (5% v/v S9 mix)] |
|||||||||
TA1537 |
TA1535 |
TA98 |
TA100 |
TA102 |
||||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
NC (DW) |
8.50 |
0.71 |
16.50 |
4.95 |
25.00 |
4.24 |
137.50 |
3.54 |
225.50 |
21.92 |
0.0015 |
6.50 |
0.71 |
11.00 |
2.83 |
22.50 |
2.12 |
131.50 |
10.61 |
230.00 |
9.90 |
0.005 |
11.50 |
4.95 |
18.00 |
7.07 |
19.00 |
7.07 |
135.00 |
45.25 |
205.50 |
9.19 |
0.015 |
10.50 |
3.54 |
19.00 |
1.41 |
19.50 |
6.36 |
121.50 |
28.99 |
227.50 |
6.36 |
0.05 |
11.00 |
0.00 |
17.00 |
7.07 |
20.00 |
1.41 |
161.50 |
19.09 |
209.50 |
2.12 |
0.15 |
9.50 |
7.78 |
13.50 |
9.19 |
18.00 |
5.66 |
125.00 |
29.70 |
243.50 |
2.12 |
0.5 |
8.00 |
0.00 |
12.50 |
2.12 |
20.00 |
2.83 |
118.00 |
9.90 |
235.50 |
23.33 |
1.5 |
8.50 |
4.95 |
11.00 |
4.24 |
21.00 |
4.24 |
133.00 |
5.66 |
234.50 |
19.09 |
5 |
9.00 |
4.24 |
12.00 |
4.24 |
21.50 |
4.95 |
107.00 |
4.24 |
244.50 |
24.75 |
PC |
242.50 |
3.54 |
270.00 |
12.73 |
676.50 |
94.05 |
688.50 |
78.49 |
947.50 |
99.70 |
Key: SD = Standard Deviation, NC = Negative Control, DW =Distilled Water, PC = Positive control {2Aa = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100)}.
Pre-incubation assay - Mean count of revertant colonies
Concentration (µL/plate) |
His+Revertant Colonies/Plate [Absence of Metabolic Activation] |
|||||||||
TA1537 |
TA1535 |
TA98 |
TA100 |
TA102 |
||||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
NC (DW) |
8.33 |
3.21 |
16.00 |
1.73 |
23.33 |
1.53 |
125.00 |
17.69 |
235.33 |
17.10 |
0.15625 |
8.67 |
1.15 |
15.33 |
3.06 |
26.33 |
3.06 |
134.33 |
13.05 |
234.33 |
7.37 |
0.3125 |
8.33 |
3.21 |
15.33 |
2.08 |
27.33 |
4.51 |
137.33 |
21.03 |
230.00 |
24.43 |
0.625 |
8.67 |
2.08 |
16.67 |
2.08 |
25.33 |
2.52 |
129.00 |
10.82 |
236.00 |
12.49 |
1.25 |
8.33 |
2.08 |
15.00 |
1.73 |
26.67 |
2.89 |
137.33 |
6.11 |
233.67 |
9.71 |
2.5 |
9.00 |
1.73 |
16.67 |
2.08 |
24.33 |
1.53 |
130.33 |
15.95 |
229.67 |
11.24 |
5 |
8.33 |
1.53 |
15.33 |
1.53 |
26.00 |
3.61 |
135.33 |
11.06 |
241.33 |
11.37 |
PC |
188.33 |
16.01 |
282.00 |
11.36 |
570.67 |
72.60 |
695.33 |
17.79 |
1009.33 |
69.51 |
2Aa |
- |
- |
- |
- |
- |
- |
122.33 |
11.06 |
- |
- |
Key: SD = Standard Deviation, NC = Negative Control, DW =Distilled Water,PC = Positive Control {TA1537 = 9-Aminoacridine Hydrochloride Monohydrate (75 µg/plate), TA1535 = Sodium Azide (0.5 µg/plate), TA98 = 2-Nitrofluorene (7.5 µg/plate), TA100 = Sodium Azide (5 µg/plate), TA102 = Mitomycin-C (0.5 µg/plate)}, 2-Aa = 2-Aminoanthracene (5 µg/plate for TA100),- = Not Applicable.
Pre-incubation assay - Mean count of revertant colonies
Concentration (µL/plate) |
His+Revertant Colonies/Plate [Presence of Metabolic Activation (10% v/v S9 mix)] |
|||||||||
TA1537 |
TA1535 |
TA98 |
TA100 |
TA102 |
||||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
NC (DW) |
9.33 |
2.52 |
16.00 |
2.00 |
27.33 |
2.52 |
127.00 |
8.00 |
228.00 |
20.95 |
0.15625 |
9.00 |
1.73 |
15.00 |
1.00 |
24.67 |
3.79 |
127.33 |
15.37 |
232.67 |
8.08 |
0.3125 |
7.00 |
2.65 |
15.33 |
3.06 |
27.33 |
1.53 |
133.67 |
9.61 |
236.67 |
12.42 |
0.625 |
7.67 |
0.58 |
16.00 |
2.65 |
26.67 |
2.52 |
130.67 |
10.02 |
238.00 |
16.09 |
1.25 |
9.33 |
3.06 |
14.67 |
2.08 |
25.33 |
4.04 |
127.67 |
10.50 |
230.33 |
9.71 |
2.5 |
7.67 |
2.08 |
17.00 |
1.00 |
27.33 |
3.79 |
137.67 |
12.22 |
228.67 |
19.43 |
5 |
8.00 |
1.00 |
15.67 |
2.52 |
25.33 |
2.08 |
134.00 |
13.23 |
237.00 |
14.00 |
PC |
170.33 |
16.77 |
283.33 |
26.95 |
508.00 |
42.53 |
551.00 |
32.51 |
1126.67 |
163.52 |
Key: SD = Standard Deviation, NC = Negative Control,DW =Distilled Water,PC = Positive Control{2Aa = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100)}.
Summary of Absolute and Relative Cloning Efficiency at selection
Group (µL/mL) |
Absence of Metabolic Activation |
Presence of Metabolic Activation (2% v/v S9 mix) |
||||
Mean ACE |
RCE |
RCE (%) |
Mean ACE |
RCE |
RCE (%) |
|
NC (DW) |
0.6419 |
1 |
100 |
0.6536 |
1.0000 |
100.00 |
T1 (0.125) |
0.6339 |
0.9876 |
98.76 |
0.5730 |
0.8767 |
87.67 |
T2 (0.25) |
0.6441 |
1.0034 |
100.34 |
0.5858 |
0.8963 |
89.63 |
T3 (0.5) |
0.6607 |
1.0294 |
102.94 |
0.6383 |
0.9767 |
97.67 |
T4 (1) |
0.6140 |
0.9566 |
95.66 |
0.5822 |
0.8908 |
89.08 |
T5 (2) |
0.6123 |
0.9539 |
95.39 |
0.6052 |
0.9259 |
92.59 |
PC |
0.6166 |
0.9607 |
96.07 |
0.5765 |
0.8821 |
88.21 |
Key: NC = Negative control, DW = Distilled water, ACE = Absolute cloning efficiency, RCE = Relative cloning efficiency, T = Treatment concentration, PC = Positive control [0.4 µL Ethyl Methanesulfonate/mL in absence of metabolic activation and 6 µg Benzo (a) pyrene/mL in presence of metabolic activation]
Mutant Frequency per 1 x 106Clonable Cells
Group (µL/mL) |
Absence of Metabolic Activation |
Presence of Metabolic Activation (2% v/v S9 mix) |
|||||||||
R |
ACE |
N° CP |
N° CC |
N° MC |
MF |
ACE |
N° CP |
N° CC |
N° MC |
MF |
|
NC (DW) |
1 |
0.6064 |
2439660 |
1479410 |
31 |
20.95 |
0.6750 |
2439360 |
1646568 |
34 |
20.65 |
2 |
0.6773 |
2440140 |
1652707 |
30 |
18.15 |
0.6321 |
2439360 |
1541919 |
29 |
18.81 |
|
T1 (0.125) |
1 |
0.6071 |
2439360 |
1480935 |
26 |
17.56 |
0.5821 |
2440320 |
1420510 |
30 |
21.12 |
2 |
0.6607 |
2439360 |
1611685 |
31 |
19.23 |
0.5638 |
2439360 |
1375311 |
28 |
20.36 |
|
T2 (0.25) |
1 |
0.6321 |
2439564 |
1542048 |
32 |
20.75 |
0.6179 |
2439360 |
1507281 |
29 |
19.24 |
2 |
0.6560 |
2439684 |
1600433 |
28 |
17.50 |
0.5536 |
2439564 |
1350543 |
27 |
19.99 |
|
T3 (0. 5) |
1 |
0.6750 |
2439600 |
1646730 |
29 |
17.61 |
0.6064 |
2439480 |
1479301 |
28 |
18.93 |
2 |
0.6464 |
2439564 |
1576934 |
30 |
19.02 |
0.6702 |
2439660 |
1635060 |
31 |
18.96 |
|
T4 (1) |
1 |
0.5851 |
2439480 |
1427340 |
32 |
22.42 |
0.5643 |
2439840 |
1376802 |
29 |
21.06 |
2 |
0.6429 |
2440200 |
1568805 |
22 |
14.02 |
0.6000 |
2439996 |
1463998 |
20 |
13.66 |
|
T5 (2) |
1 |
0.5709 |
2440140 |
1393076 |
32 |
22.97 |
0.6429 |
2439720 |
1568496 |
33 |
21.04 |
2 |
0.6536 |
2440236 |
1594938 |
32 |
20.06 |
0.5674 |
2439360 |
1384093 |
27 |
19.51 |
|
PC |
1 |
0.5857 |
2440104 |
1429169 |
557 |
389.74 |
0.5816 |
2440524 |
1419409 |
511 |
360.01 |
2 |
0.6475 |
2439840 |
1579796 |
468 |
296.24 |
0.5714 |
2440476 |
1394488 |
473 |
339.19 |
|
Key: NC = Negative control, DW = Distilled water, ACE = Absolute cloning efficiency, N° CP = N° of cells plated, N° CC = N° of clonable cells, N° MC = N° of mutant colonies, MF = Mutant frequency per 106clonable cells, T = Treatment concentration, PC = Positive control [0.4 µL Ethyl methanesulfonate/mL in absence of metabolic activation and 6 µg Benzo(a)pyrene/mL in presence of metabolic activation]
Summary of Cytostasis
Short-term treatment
Concentration |
Absence of Metabolic Activation |
Presence of Metabolic Activation |
||
% RI (Mean) |
Cytostasis |
% RI (Mean) |
Cytostasis |
|
NC (Distilled water) # |
100 |
0 |
100 |
0 |
0.125 µL/mL |
110.10 |
-10.10 |
102.54 |
-2.54 |
0.25 µL/mL |
90.05 |
9.95 |
101.28 |
-1.28 |
0.5 µL/mL # |
93.12 |
6.88 |
99.93 |
0.07 |
1 µL/mL # |
92.62 |
7.38 |
109.58 |
-9.58 |
2 µL/mL # |
104.03 |
-4.03 |
96.21 |
3.79 |
PC# |
- |
- |
79.74 |
20.26 |
Key: NC = Negative control, T = Treatment group, PC = Positive control (Cyclophosphamide @ 30 µg/mL in the presence of metabolic activation), # = Concentrations selected for scoring of binucleated cells containing micronuclei, RI = Replicative index.
Long-term treatment
Absence of Metabolic Activation |
|||
Concentration |
% RI (Mean) |
Cytostasis |
|
NC (Distilled water) # |
100 |
0 |
|
0.125 µL/mL |
99.33 |
0.67 |
|
0.25 µL/mL |
103.65 |
-3.65 |
|
0.5 µL/mL # |
102.02 |
-2.02 |
|
1 µL/mL # |
105.38 |
-5.38 |
|
2 µL/mL # |
97.16 |
2.84 |
|
PC# |
96.06 |
3.94 |
|
Key: NC = Negative control, T = Treatment group, PC = Positive control (Vinblastine @ 0.008 µg/mL in the absence of metabolic activation), # = Concentrations selected for scoring of binucleated cells containing micronuclei, RI = Replicative index.
Individual Observation of Slides for Frequency of Micronucleated Binucleated Cells
Short-term treatment
Concentration |
R |
Absence of Metabolic Activation |
Presence of Metabolic Activation |
||||
Total number of BN cells scored |
Number of MNBN |
Percent MNBN |
Total number of BN cells scored |
Number of MNBN |
% MNBN |
||
NC (Distilled water)
|
1 |
1004 |
5 |
0.498 |
1005 |
5 |
0.498 |
2 |
1002 |
3 |
0.299 |
1002 |
2 |
0.200 |
|
T3 (0.5 µL/mL) |
1 |
1003 |
4 |
0.399 |
1003 |
4 |
0.399 |
2 |
1007 |
2 |
0.199 |
1006 |
2 |
0.199 |
|
T4 (1 µL/mL) |
1 |
1002 |
4 |
0.399 |
1005 |
3 |
0.299 |
2 |
1003 |
3 |
0.299 |
1004 |
2 |
0.199 |
|
T5 (2 µL/mL) |
1 |
1002 |
3 |
0.299 |
1004 |
4 |
0.398 |
2 |
1001 |
5 |
0.500 |
1002 |
3 |
0.299 |
|
PC |
1 |
NA |
1002 |
47 |
4.691 |
||
2 |
1004 |
48 |
4.781 |
||||
Long-term treatment
Concentration |
R |
Total Number of BN Cells Scored |
Number of MNBN |
% MNBN |
NC (Distilled water)
|
1 |
1020 |
4 |
0.392 |
2 |
1029 |
3 |
0.292 |
|
T3 (0.5 µL/mL) |
1 |
1000 |
4 |
0.400 |
2 |
1002 |
2 |
0.200 |
|
T4 (1 µL/mL) |
1 |
1004 |
5 |
0.498 |
2 |
1102 |
3 |
0.272 |
|
T5 (2 µL/mL) |
1 |
1002 |
4 |
0.399 |
2 |
1005 |
2 |
0.199 |
|
PC |
1 |
1007 |
30 |
2.979 |
2 |
1005 |
50 |
4.975 |
Key: R = Replicate,T = Treatment group, NC = Negative control, BN cells = Binucleated cells, MNBN = Micronucleated binucleated cells, PC = Positive control (Cyclophosphamide 30 µg/mL in the presence of metabolic activation (short-term treatment) and Vinblastine 0.008 µg/mL in the absence of metabolic activation (long-term treatment)), NA = Not applicable.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Justification for classification or non-classification
Available data regarding the potential genotoxicity of 2 -(2 -ethoxyethoxy)ethyl acetate (CAS 112 -15 -2) do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and the data are therefore conclusive but not sufficient for classification.
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