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EC number: 807-645-4 | CAS number: 701920-77-6
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
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- Nanomaterial surface chemistry
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- Nanomaterial pour density
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- Nanomaterial catalytic activity
- Endpoint summary
- Stability
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- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study complies to OECD Test Guidance 476 and GLP, and is considered to be relevant, adequate and reliable.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- thymidine kinase TK+/-
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- -Type and identity of media:
Complete Culture Medium
RPMI 1640 medium supplemented with 10% horse serum, 100U/100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 1.5 µg/mL amphotericin B
Treatment Medium
RPMI 1640 medium supplemented with 5% horse serum (in case of short term exposure) or 7.5 % horse serum (in case of long term exposure), 100U/100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B
Selective Medium
RPMI 1640 medium supplemented with 20% horse serum, 100U/100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B, 5 µg/mL TFT
-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:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- Experiment I without and with metabolic activation: 0.2, 0.5, 1.0, 2.5, 5.0, 7.5, 9.0 and 10 mM
Experiment II without metabolic activation: 0.05, 0.1, 0.2, 0.5, 1.0, 2.5, 5.0 and 7.5 mM
Experiment II with metabolic activation: 0.1, 0.3, 0.7, 2.0, 4.0, 6.0, 8.0 and 10.0 mM - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO (1% DMSO v/v)
- Justification for choice of solvent/vehicle: As for this test system the use of AOO (4:1 (v/v) acetone/olive oil;) was technically not possible, another vehicle was used according to OECD Guideline. A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 10 mM. Based on the results of the solubility test DMSO was used as solvent (1% DMSO v/v). Different test item stock solutions were prepared and added to the cells. The solution was treated with ultrasound for 12 minutes at 37°C and diluted prior to treatment. The solvent was compatible with the survival of the cells and the S9 activity. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- 1% DMSO v/v)
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- without S9, 200 µg/mL and 300 µg/mL
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- without S9, 8 µg/mL and 10 µg/mL
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- with S9, 2.5 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
Experiment I without and with metabolic activation and Experiment II with metabolic activation (4h short-term exposure)
- Preincubation period: 4 h
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 days
- Cloning efficieny: 6 days
- Selection time (if incubation with a selection agent): 14 days
Experiment II without activation (24h long-term exposure)
- Preincubation period: 24 h
- Exposure duration: 24 h
- Expression time (cells in growth medium): 2 days
- Cloning efficieny: 6 days
- Selection time (if incubation with a selection agent): 14 days
SELECTION AGENT (mutation assays): TFT (Trifluorothymidine)
NUMBER OF REPLICATIONS:
Cloning efficiency: duplicate
Mutagenicity : ?
NUMBER OF CELLS EVALUATED: ?
DETERMINATION OF CYTOTOXICITY
- Method: other: measuring the colony-forming ability and the growth rate of cultures - Evaluation criteria:
- ACCEPTABILITY OF THE ASSAY
A mutation assay is considered acceptable if it meets the criteria mentioned in current international guidelines and the current recommendations of the IWGT:
-At least three out of four 96-well plates from the TFT resistance-testing portion of the experiment are scorable
-The cloning efficiency of the negative and/or solvent controls is in the range 65% - 120%
-The spontaneous mutant frequency in the negative and/or solvent controls is in the range 50 – 170 mutants per 106 cells
-The number of the negaztive/solvent controls shouls undergo 8-32 fold increase during a 2 day growth period (short-term treatment) or 32-180 fold increase during a 3 day growth period (long-term treatment)
-The clastogenic positive controls (MMS and B[a]P) have to produce an induced mutant frequency (total mutant frequency minus concurrent negative control mutant frequency) of at least 300 mutants per 106 cells with at least 40% of the colonies being small colonies or with an induiced small colony mutant frequency of at least 150 mutants per 106 cells. The RTG must be greater than 10%.
EVALUATION OF RESULTS
The test item is considered mutagenic if the following criteria are met:
-The induced mutant frequency meets or exceeds the Global Evaluation Factor (GEF) of 126 mutants per 106 cells
-A dose-dependent increase in mitant frequency is detected.
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥ 40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation of the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative. - Statistics:
- The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the solvent/negative controls were used as reference.
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: growth inhibition was observed in experiment I without and with metabolic activation and in experiment II without metabolic activation; no growth inhibition in experiment II with metabolic activation.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was noted in the experiments.
- The pH-value detected with the test item was within the physiological range.
RANGE-FINDING/SCREENING STUDIES:
Pre-Experiment for Toxicology:
Experiment I: 0.2, 0.5, 2.5, 5.0, 7.5 and 10 mM were tested without and with metabolic activation.
Experiment II : 0.5, 1.0, 2.5, 5.0, 7.5 and 10 mM were testes without metabolic activation.
COMPARISON WITH HISTORICAL CONTROL DATA:
BioService historical data for mutatnt frequancies were available (negative, solvent and positive controls).
All frequencies were within the historical control range, except the mutant frequencies for the solvednt of expderiment I with metabolic actiation, which were below the range of historical data.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Growth inhibition was observed in experiment I without and with metabolic activation and in experiment II without metabolic activation. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item bis[2-(2-butoxyethoxy)ethyl]butanedioate is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L1578Y cells. - Executive summary:
The test item bis[2-(2-butoxyethoxy)ethyl]butanedioate was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.
The selection of the concentrations used in the main experiments was based on data from the pre-experiments. In experiment I 10 mM (without and with metabolic activation) was selected as the highest concentrations. In experiment II 7.5 mM (without metabolic activation) and 10 mM (with metabolic activation) were selected as the highest concentrations. Experiment I without and with metabolic activation and Experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as a 24 h long-term exposure assay.
The test item was investigated at the following concentrations:
Experiment I without and with metabolic activation: 0.2, 0.5, 1.0, 2.5, 5.0, 7.5, 9.0 and 10 mM
Experiment II without metabolic activation: 0.05, 0.1, 0.2, 0.5, 1.0, 2.5, 5.0 and 7.5 mM; with metabolic activation: 0.1, 0.3, 0.7, 2.0, 4.0, 6.0, 8.0 and 10.0 mM
No precipitation of the test item was noted in the experiments. Growth inhibition was observed in experiment I without and with metabolic activation and in experiment II without metabolic activation. In experiment II with metabolic activation no growth inhibition was observed.
In experiment I without metabolic activation the relative total growth (RTG) was 60.6% for the highest concentration (10 mM) evaluated. The highest concentration evaluated with metabolic activation was 10 mM with a RTG of 64.3%. In experiment II without metabolic activation the relative total growth (RTG) was 11.3% for the highest concentration (7.5 mM) evaluated. The highest concentration evaluated with metabolic activation was 10 mM with a RTG of 110%.
In experiment I and II no biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation). The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories was not exceeded by the induced mutant frequency at any concentration. No dose-response relationship was observed.
Reference
Table 1: Summary: Experiment I and II, without metabolic activation
|
Test Group |
Conc. [mM] |
RCEa |
RTGb |
MFc |
IMFd |
GEFe |
Statistical |
Precipitate |
Exp I |
C1 |
0 |
105.3 |
112.1 |
67.2 |
/ |
/ |
/ |
- |
C2 |
81.8 |
89.2 |
/ |
/ |
/ |
- |
|||
S1 |
0 |
100.0 |
100.0 |
53.5 |
/ |
/ |
/ |
- |
|
S2 |
/ |
/ |
/ |
- |
|||||
3 |
0.2 |
87.2 |
82.0 |
77.9 |
24.4 |
- |
+ |
- |
|
4 |
0.5 |
90.1 |
92.6 |
|
0.0 |
- |
- |
- |
|
5 |
1.0 |
99.7 |
93.2 |
53.5 |
-1.1 |
- |
- |
- |
|
6 |
2.5 |
80.6 |
77.2 |
52.4 |
32.3 |
- |
+ |
- |
|
7 |
5.0 |
78.2 |
63.4 |
85.8 |
33.2 |
- |
+ |
- |
|
8 |
7.5 |
74.7 |
63.6 |
86.7 |
9.6 |
- |
- |
- |
|
9 |
9.0 |
93.1 |
70.1 |
63.1 |
-1.6 |
- |
- |
- |
|
10 |
10 |
80.6 |
60.6 |
51.9 |
42.2 |
- |
+ |
- |
|
EMS |
300 µg/mL |
84.4 |
70.2 |
95.7 |
574.4 |
+ |
+ |
- |
|
MMS |
10 µg/mL |
57.6 |
49.2 |
627.9 |
631.9 |
+ |
+ |
- |
|
|
|||||||||
Exp II |
C1 |
0 |
116.9 |
135.9 |
66.0 |
/ |
/ |
/ |
- |
C2 |
116.9 |
128.7 |
/ |
/ |
/ |
- |
|||
S1 |
0 |
100.0 |
100.0 |
93.1 |
/ |
/ |
/ |
- |
|
S2 |
/ |
/ |
/ |
- |
|||||
1 |
0.05 |
97.0 |
100.3 |
90.6 |
-2.5 |
- |
- |
- |
|
2 |
0.1 |
106.3 |
102.6 |
86.1 |
-7.1 |
- |
- |
- |
|
3 |
0.2 |
101.5 |
91.8 |
90.3 |
-2.8 |
- |
- |
- |
|
4 |
0.5 |
115.0 |
92.9 |
89.0 |
-4.2 |
- |
- |
- |
|
5 |
1.0 |
103.0 |
79.1 |
108.0 |
14.9 |
- |
- |
- |
|
6 |
2.5 |
111.4 |
67.1 |
90.0 |
-3.1 |
- |
- |
- |
|
7 |
5.0 |
107.9 |
31.9 |
84.8 |
-8.3 |
- |
- |
- |
|
8 |
7.5 |
80.3 |
11.3 |
150.0 |
56.9 |
- |
+ |
- |
|
EMS |
200 µg/mL |
51.5 |
27.2 |
2490.8 |
2397.7 |
+ |
+ |
- |
|
MMS |
8 µg/mL |
63.1 |
39.8 |
1179.4 |
1086.3 |
+ |
+ |
- |
C: Negative Controls
S: Solvent Controls (DMSO)
a: Relative Cloning Efficiency, RCE = [(CEdose group/ CEof corresponding controls) x 100]
Cloning Efficiency, CE = ((-LN(((96-(mean P1,P2)) / 96)) / 1.6) x 100)
b: Relative Total Growth, RTG = (RSG x RCE)/100
c: Mutant Frequency,
MF = {-In [negative cultures/total wells (selective medium)] / -In [negative cultures/total wells (non selective medium)]} x 800
d: Induced Mutant Frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls
e: Global Evaluation Factor, GEF (126); +: GEF exceeded, -: GEF not exceeded
f: Statistical significant increase in mutant frequency compared to solvent controls (Mann-Whitney test, p < 0.05). +: significant; - not significant
EMS: Ethylmethanesulfonate [200 and 300 µg/ml]
MMS: Methyl methanesulfonate [8 and 10 µg/ml]
Table 2: Summary: Experiment I and II, witth metabolic activation
|
Test Group |
Conc. [mM] |
RCEa |
RTGb |
MFc |
IMFd |
GEFe |
Statistical |
Precipitate |
Exp I |
C1 |
0 |
86.4 |
81.9 |
58.3 |
/ |
/ |
/ |
- |
C2 |
95.0 |
93.6 |
/ |
/ |
/ |
- |
|||
S1 |
0 |
100.0 |
100.0 |
55.1 |
/ |
/ |
/ |
- |
|
S2 |
/ |
/ |
/ |
- |
|||||
3 |
0.5 |
83.8 |
73.5 |
53.7 |
-1.4 |
- |
- |
- |
|
4 |
0.5 |
89.1 |
92.1 |
56.5 |
1.3 |
- |
- |
- |
|
5 |
1.0 |
90.5 |
79.3 |
33.5 |
-21.7 |
- |
- |
- |
|
6 |
2.5 |
95.0 |
89.4 |
56.0 |
0.9 |
- |
- |
- |
|
7 |
5.0 |
106.9 |
97.7 |
32.1 |
-23.0 |
- |
- |
- |
|
8 |
7.5 |
93.4 |
85.4 |
54.1 |
-1.1 |
- |
- |
- |
|
9 |
9.0 |
77.7 |
60.5 |
52.6 |
-2.5 |
- |
- |
- |
|
10 |
10 |
74.4 |
64.3 |
57.1 |
1.9 |
- |
- |
- |
|
B[a]P |
2.5 µg/mL |
72.2 |
35.7 |
494.9 |
439.7 |
+ |
+ |
- |
|
|
|||||||||
Exp II |
C1 |
0 |
105.9 |
92.6 |
84.1 |
/ |
/ |
/ |
- |
C2 |
102.7 |
98.7 |
/ |
/ |
/ |
- |
|||
S1 |
0 |
100.0 |
100.0 |
89.9 |
/ |
/ |
/ |
- |
|
S2 |
/ |
/ |
/ |
- |
|||||
3 |
0.1 |
93.7 |
95.3 |
80.1 |
-9.8 |
- |
- |
- |
|
4 |
0.3 |
109.4 |
117.7 |
79.6 |
-10.3 |
- |
- |
- |
|
5 |
0.7 |
95.1 |
93.8 |
98.6 |
8.7 |
- |
- |
- |
|
6 |
2.0 |
81.0 |
83.6 |
131.4 |
41.5 |
- |
- |
- |
|
7 |
4.0 |
104.3 |
104.5 |
87.9 |
-2.0 |
- |
- |
- |
|
8 |
6.0 |
113.0 |
118.9 |
78.4 |
-11.5 |
- |
- |
- |
|
9 |
8.0 |
93.7 |
91.8 |
94.9 |
5.0 |
- |
- |
- |
|
10 |
10 |
105.9 |
110.0 |
77.5 |
-12.4 |
- |
- |
- |
|
B[a]P |
2.5 µg/mL |
92.3 |
62.6 |
575.9 |
+ |
+ |
+ |
- |
C: Negative Controls
S: Solvent Controls (DMSO)
a: Relative Cloning Efficiency, RCE = [(CEdose group/ CEof corresponding controls) x 100]
Cloning Efficiency, CE = ((-LN(((96-(mean P1,P2)) / 96)) / 1.6) x 100)
b: Relative Total Growth, RTG = (RSG x RCE)/100
c: Mutant Frequency,
MF = {-In [negative cultures/total wells (selective medium)] / -In [negative cultures/total wells (non selective medium)]} x 800
d: Induced Mutant Frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls
e: Global Evaluation Factor, GEF (126); +: GEF exceeded, -: GEF not exceeded
f: Statistical significant increase in mutant frequency compared to solvent controls (Mann-Whitney test, p < 0.05). +: significant; - not significant
B[a]P: Benzo[a]pyrene [2.5 µg/mL]
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
A key study for mammalian gene mutation with bis[2-(2-butoxyethoxy)ethyl]butanedioate was conducted in the mouse lymphoma thymidine kinase assay using the cell line L5178Y (BioService, 2015d).The selection of the concentrations used in the main experiments was based on data from pre-experiments. In experiment I 10 mM (without and with metabolic activation) was selected as the highest concentrations. In experiment II 7.5 mM (without metabolic activation) and 10 mM (with metabolic activation) were selected as the highest concentrations. Experiment I without and with metabolic activation and Experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as a 24 h long-term exposure assay.
The test item was investigated at the following concentrations:
- Experiment I without and with metabolic activation: 0.2, 0.5, 1.0, 2.5, 5.0, 7.5, 9.0 and 10 mM
- Experiment II
without metabolic activation: 0.05, 0.1, 0.2, 0.5, 1.0, 2.5, 5.0 and 7.5 mM;
with metabolic activation: 0.1, 0.3, 0.7, 2.0, 4.0, 6.0, 8.0 and 10.0 mM.
No precipitation of the test item was noted in the experiments. Growth inhibition was observed in experiment I without and with metabolic activation and in experiment II without metabolic activation. In experiment II with metabolic activation no growth inhibition was observed.
In experiment I without metabolic activation the relative total growth (RTG) was 60.6% for the highest concentration (10 mM) evaluated. The highest concentration evaluated with metabolic activation was 10 mM with a RTG of 64.3%. In experiment II without metabolic activation the relative total growth (RTG) was 11.3% for the highest concentration (7.5 mM) evaluated. The highest concentration evaluated with metabolic activation was 10 mM with a RTG of 110%.
In experiment I and II no biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation). The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories was not exceeded by the induced mutant frequency at any concentration. No dose-response relationship was observed.
Justification for selection of genetic toxicity endpoint
Key study
Justification for classification or non-classification
Based on the results and according to the EC criteria for classification and labelling requirements for dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC) and CLP regulation (EC No. 1272/2008 of 16 December 2008), bis[2-(2-butoxyethoxy)ethyl]butanedioate does not have to be classified and has no obligatory labelling requirement for mammalian muagenicity.
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