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EC number: 217-576-6 | CAS number: 1892-29-1
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 09 March 2017 to 31 May 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Version / remarks:
- 2015
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- other: in vitro gene mutation in mammalian cells
Test material
- Reference substance name:
- 2,2'-dithiobisethanol
- EC Number:
- 217-576-6
- EC Name:
- 2,2'-dithiobisethanol
- Cas Number:
- 1892-29-1
- Molecular formula:
- C4H10O2S2
- IUPAC Name:
- 2-[(2-hydroxyethyl)disulfanyl]ethan-1-ol
Constituent 1
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: No assay of test item stability, nor its concentration and homogeneity in solvent were undertaken.
- Solubility and stability of the test substance in the solvent/vehicle: The test item was found to be soluble in culture medium at the maximum concentration of
50mg/mL based on solubility test.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not specified
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in culture medium
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable
Method
- Target gene:
- TK locus (TK+/−)
Species / strain
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: American Type Culture Collection, Rockville, Maryland
- Suitability of cells: Recommended in Test Guideline
- Cell cycle length, doubling time or proliferation index: not specified
- Sex, age and number of blood donors if applicable: not applicable
- Whether whole blood or separated lymphocytes were used if applicable: not applicable
- Number of passages if applicable: not applicable
- Methods for maintenance in cell culture if applicable: Permanent stocks of the L5178Y TK+/− cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use.
- Modal number of chromosomes: not specified
- Normal (negative control) cell cycle time: not specified
MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital – 5,6-Benzoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- Experiment 1, 3-hour treatment with metabolic activation: 385, 193, 96.3, 48.1, 24.1 and 12.0 μg/mL
Experiment 1, 3-hour treatment without metabolic activation: 385, 193, 96.3, 48.1, 24.1, 12.0 and 6.02 μg/mL
Experiment 2, 24-hour treatment without metabolic activation: 50.0, 40.0, 32.0, 25.6, 20.5, and 16.4 μg/mL
The top dose was selected based on toxicity observed in the preliminary toxicity assay. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: The solvent used in this study was RPMI Minimal medium A
- Justification for choice of solvent/vehicle: based on solubility test
Controlsopen allclose all
- Untreated negative controls:
- yes
- Remarks:
- complete medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- minimal medium
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- without metabolic activation
- Untreated negative controls:
- yes
- Remarks:
- complete medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- minimal medium
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): not specified
ACTIVATION: The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows (for each 10 mL): S9 tissue fraction 0.408mL, NADP (30 mM) 0.204mL, G-6-P (590 mM) 0.204mL, KCl (150 mM) 0.204mL, Complete medium (5%) 8.98mL
DURATION
- Preincubation period:
- Exposure duration: experiment 1 3 hours with and without metabolic activation; experiment 2 24 hours without metabolic activation
- Expression time (cells in growth medium): two days after treatment at 37°C in a 5% CO2 atmosphere
- Selection time (if incubation with a selection agent): one to two weeks incubated at 37°C in a 5% CO2 atmosphere
- Fixation time (start of exposure up to fixation or harvest of cells): not specified
SELECTION AGENT (mutation assays): trifluorothymidine
NUMBER OF REPLICATIONS: duplicate cultures apart from positive control
NUMBER OF CELLS EVALUATED: not specified
DETERMINATION OF CYTOTOXICITY
- Method: plating efficiency; relative total growth
- Any supplementary information relevant to cytotoxicity: none
OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no - Rationale for test conditions:
- A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined.
- Evaluation criteria:
- For a test item to be considered mutagenic in this assay, it is required that:
1. The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126×10−6) at one or more doses.
2. There is a significant dose-relationship as indicated by the linear trend analysis. - Statistics:
- Statistical analysis was performed according to UKEMS guidelines:
- Test for consistency between plates;
- Heterogeneity factors for replicate cultures;
- Test for overall consistency;
- Updated heterogeneity factors;
- Comparison of each treatment with the control;
-Test for linear trend
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Remarks:
- 3-hour treatment
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- severe toxicity was noted at the two highest dose levels reducing RS below 10%
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Remarks:
- 24-hour treatment
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- severe toxicity was noted at the two highest dose levels reducing RS below 10%
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Remarks:
- 3-hour treatment
- Metabolic activation:
- with
- Genotoxicity:
- not determined
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- severe toxicity was noted at the three highest dose levels
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: the test item solution did not have any obvious effect on the pH of the treatment medium.
- Effects of osmolality: the test item solution did not have any obvious effect on the osmolality of the treatment medium.
- Evaporation from medium: not specified
- Water solubility: not specified
- Precipitation: No precipitation of the test item was noted upon addition of the test item to the cultures or by the end of the treatment period.
- Definition of acceptable cells for analysis:
- Other confounding effects: not specified
RANGE-FINDING/SCREENING STUDIES: Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 1540µg/mL and at a wide range of lower dose levels: 770, 385, 193, 96.3, 48.1, 24.1, 12.0 and 6.02µg/mL. No precipitation of the test item was noted upon addition of the test item to the cultures or by the end of the treatment period.
NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
- Indication whether binucleate or mononucleate where appropriate:
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: within the ranges of positive control data
- Negative (solvent/vehicle) historical control data: within the ranges of negative control data
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RICC
- Other observations when applicable: NONE
Any other information on results incl. tables
Table 1: Experiment 1: 3 HOURS - WITHOUT S9, Cytotoxicity test
Dose-level (µg/mL) |
106cells / mL Cell count Post treatment factor |
106cells / mL Cell count Post treatment factor |
Total wells
|
Plate counts # |
Plate counts # |
Cloning efficiency |
Relative survival |
0.00 6.02 12.0 24.1 48.1 96.3 193 385 770 1540 |
0.56 0.55 0.58 0.42 0.38 0.36 0.28 0.25 0.26 0.23 |
1.00 0.98 1.04 0.75 0.68 0.64 0.50 0.45 0.46 0.41 |
192 192 192 192 192 192 192 192 192 192 |
70 71 66 68 67 45 47 25 8 0 |
72 73 62 72 65 46 45 28 9 0 |
0.84 0.87 0.69 0.82 0.73 0.40 0.41 0.20 0.06 0.00 |
100% 101% 85% 73% 59% 31% 24% 11% 3% 0% |
Table 2: Experiment 2: 24 HOURS - WITHOUT S9, Cytotoxicity test
Dose-level (µg/mL) |
106 cells / mL Cell count Post treatment factor |
106 cells / mL Cell count Post treatment factor |
Total wells
|
Plate counts #
|
Plate counts # |
Cloning efficiency |
Relative survival |
0.00 6.02 12.0 24.1 48.1 96.3 193 385 770 1540 |
0.53 0.58 0.45 0.47 0.14 0.03 0.03 0.01 0.00 0.00 |
1.00 1.09 0.85 0.89 0.26 0.06 0.06 0.02 0.00 0.00 |
192 192 192 192 192 0 0 0 0 0 |
81 77 80 64 39 £ £ £ £ £ |
79 80 78 68 35 £ £ £ £ £ |
1.12 1.06 1.08 0.73 0.30 £ £ £ £ £ |
100% 104% 82% 58% 7% 0% 0% 0% 0% 0% |
# = Wells with clones/plate - 1.6 cells in each well of two 96-well plates plated for survival £ = Insufficient number of cells recovered after treatment period
Table 3: Experiment 1: 3 HOURS - WITH S9, cytotoxicity test
Dose-level (µg/mL) |
106 cells / mL Cell |
106 cells / mL Cell |
Total wells |
Plate counts # |
Plate counts # |
Cloning efficiency |
Relative survival |
|
count Post treatment factor |
count Post treatment factor |
|
|
|
|
|
0.00 6.02 12.0 24.1 48.1 96.3 193 385 770 1540 |
0.40 0.40 0.39 0.41 0.36 0.37 0.35 0.39 0.36 0.38 |
1.00 1.00 0.98 1.03 0.90 0.93 0.88 0.98 0.90 0.95 |
192 192 192 192 192 192 192 192 192 192 |
76 80 79 70 60 44 37 16 1 0 |
77 77 75 71 55 49 37 12 1 0 |
1.00 1.06 1.01 0.83 0.57 0.41 0.30 0.10 0.01 0.00 |
100% 107% 99% 85% 52% 38% 27% 10% 1% 0% |
# = Wells with clones/plate - 1.6 cells in each well of two 96-well plates plated for survival £ = Insufficient number of cells recovered after treatment period
Table 4:
SUMMARY TABLE, MAIN ASSAY I - TREATMENT: 3 HOURS - WITH S9
Dose-level (µg/mL) |
RTG |
MF § |
P |
IMF |
Proportion |
Precipitation |
|
|
|
|
§ |
small colony mutants |
|
0.00 |
100% |
74.1 |
- |
- |
0.12 |
- |
12.0 |
39% |
70.2 |
NS |
- |
- |
- |
24.1 |
78% |
73.2 |
NS |
- |
- |
- |
48.1 |
37% |
98.2 |
NS |
24.03 |
- |
- |
96.3 |
18% |
182.3 |
** |
108.14 |
- |
- |
193 |
15% |
298.9 |
** |
224.8@ |
0.33 |
- |
385 |
6% |
698.9 |
$ |
624.8@ |
- |
- |
B(a)P 2.00 |
38% |
1140.4 |
- |
1066.2@ |
0.20 |
- |
Linear trend |
|
|
*** |
|
|
|
Table 5: SUMMARY TABLE, EXPERIMENT 1 - TREATMENT: 3 HOURS - WITHOUT S9
Dose-level (µg/mL) |
RTG |
MF § |
P |
IMF |
Proportion |
Precipitation |
|
|
|
|
§ |
small colony mutants |
|
0.00 |
100% |
65.6 |
- |
- |
0.19 |
- |
6.02 |
99% |
70.5 |
NS |
4.95 |
- |
- |
12.0 |
81% |
66.5 |
NS |
0.98 |
- |
- |
24.1 |
55% |
61.7 |
NS |
- |
- |
- |
48.1 |
37% |
73.1 |
NS |
7.50 |
- |
- |
96.3 |
19% |
100.0 |
NS |
34.43 |
- |
- |
193 |
7% |
317.7 |
$ |
252.1@ |
- |
- |
385 |
2% |
445.5 |
$ |
379.9@ |
- |
- |
Linear trend |
|
|
* |
|
|
|
Table 6: SUMMARY TABLE, EXPERIMENT 2 - TREATMENT: 24 HOURS - WITHOUT S9
Dose-level (µg/mL) |
RTG |
MF § |
P |
IMF |
Proportion |
Precipitation |
|
|
|
|
§ |
small colony mutants |
|
0.00 |
100% |
77.2 |
- |
- |
0.19 |
- |
16.4 |
61% |
96.2 |
NS |
19.03 |
- |
- |
20.5 |
56% |
91.2 |
NS |
14.03 |
- |
- |
25.6 |
43% |
69.3 |
NS |
- |
- |
- |
32.0 |
29% |
75.0 |
NS |
- |
- |
- |
40.0 |
3% |
134.5 |
$ |
57.28 |
- |
- |
50.0 |
0% |
125.1 |
$ |
47.84 |
- |
- |
MMS 5.00 |
53% |
628.1 |
- |
550.8@ |
0.29 |
- |
Linear trend |
|
|
NS |
|
|
|
§ |
= |
per 10 viable cells |
NS |
= |
Not statistically significant |
* |
= |
Statistically significant at p < 5% |
** |
= |
Statistically significant at p < 1% |
*** |
= |
Statistically significant at p < 0.1% |
+ |
= |
Opacity of treatment medium |
++ |
= |
Precipitation |
$ |
= |
Treatment excluded from test statistics due to excessive toxicity |
@ |
= |
Induced mutant frequency (IMF) > global evaluation factor (GEF = 126 x 10-6) |
Applicant's summary and conclusion
- Conclusions:
- Di-2-hydroxyethyl disulfide has been tested for mutagenicity in mouse lymphoma L5178Y cells according to OECD TG 490, and under GLP (RTC, 2017). No test-substance induced increase in the number of mutations was observed when treated without metabolic activation. However, a test substance induced increase in the induced mutant frequency (IMF) relative to the global evaluation frequency (GEF) was observed when treated with metabolic activation. This increase occurred only at the highest dose of 193 μg/mL, at which the RTG = 15%. At the next lower dose, 96.3 μg/mL, RTG value 18%, the IMF was lower than the GEF. Appropriate solvent, negative (treatment medium) and positive controls were included and gave expected results. It is concluded that the test substance does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in the absence of S9 metabolic activation. In the presence of S9 metabolic activation, under the conditions of the study, it is not possible to conclude whether the substance is negative or positive for mutagenicity to mammalian cells.
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