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EC number: 224-632-3 | CAS number: 4432-31-9
- 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:
- 2017-08-01 to 2017-09-22
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- July 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- May 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Test material
- Reference substance name:
- 2-(4-morpholinyl)ethansulfonic acid hydrate
- Cas Number:
- 1266615-59-1
- Molecular formula:
- C6H13NO4S * xH2O
- IUPAC Name:
- 2-(4-morpholinyl)ethansulfonic acid hydrate
- Test material form:
- solid: particulate/powder
Constituent 1
Method
- Target gene:
- HPRT
Species / strain
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: CLS (Eppelheim, Germany)
- Suitability of cells: analysis of modal chromosome number
- Cell cycle length, doubling time or proliferation index: cell cycle time was determined in experiment and within the normal range; doubling time: 10-12 h
- Modal number of chromosomes: 22
MEDIA USED
- Type and identity of media including CO2 concentration : DMEM (from Biochrom AG) was used as a basis for complete culture medium with medium DMEM with 5% HS (5% horse serum, 1% Penicillin/Streptomycin, 1% phytohaemagglutinin solution) and selection medium (5% horse serum, 1% Penicillin/Streptomycin, 2 µg/mL 6-thioguanine (1 mg/mL))
- 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:
- Type and composition of metabolic activation system
- source of S9 : S9 (liver enzyme mixture used for the test with metabolic activation) was obtained from a specialized company (Trinova Biochem GmbH, Gießen), produced from the livers of male Sprague-Dawley rats
which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally.
- concentration or volume of S9 mix and S9 in the final culture medium: The final concentration of the S9 fraction during treatment is 0.4 %. - Test concentrations with justification for top dose:
- 0.31, 0.63, 1.25, 2.5, 5, 10 mM
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMEM
- Justification for choice of solvent/vehicle: no effects on cell viability and good solubility of test substance
Controlsopen allclose all
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Remarks:
- with metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- without metabolic activation
- 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:
- Cell density at seeding: 1000000/dish (10 cm diameter)
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Experiment 1: 4h (with and without metabolic activation)
Experiment 2: 24h (without metabolic activation)
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): at least 168 h
- Selection time: 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14-15 days
- Selection agent: 6-thioguanine
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency - Rationale for test conditions:
- The conditions of experiment 1 are according to the recommendations of the OECD TG 476.
The second experiment was performed to confirm the findings of experiment 1. - Evaluation criteria:
- Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- the increase is concentration-related when evaluated with an appropriate trend test,
- any of the results are outside the distribution of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system. - Statistics:
- Chi-square test with a significance level of 5%
Results and discussion
Test results
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
-
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Water solubility: the substance was highly soluble
- Precipitation: no
- Other confounding effects: none
RANGE-FINDING/SCREENING STUDIES:
- cytotoxicity pre-test: highest recommended concentration (10 mM) not cytotoxic, determined by cloning efficiency) and not precipitating after 4h exposure with and without metabolic activation
STUDY RESULTS
- Results from cytotoxicity measurements: see attached results
- Genotoxicity results: see attached results
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data (number of mutant colonies per 1000000 cells):
With S9 (4h treatment): DMBA: range: 100 - 461; mean: 2431; sd: 72.2
Without S9 (4h treatment): EMS: range: 71 - 207; mean: 127; sd: 33.4
Without S9 (24h treatment): EMS: range: 35 - 517; mean: 237; sd: 97.0
- Negative (solvent/vehicle) historical control data(number of mutant colonies per 1000000 cells):
With S9 (4h treatment): DMEM: range: 4 - 35; mean: 17; sd: 9.3
With S9 (4h treatment): DMSO: range: 2 - 39; mean: 15; sd: 9.2
Without S9 (4h treatment): DMEM: range: 2- 32; mean: 15; sd: 9.0
Without S9 (24h treatment): DMEM: range: 4 - 27; mean: 12; sd: 6.6
Any other information on results incl. tables
Table 1: Mutagenicity in Experiment I with Metabolic Activation
|
Conc. |
Culture |
Cells seeded |
Number of colonies per dish |
CE mut |
MF |
MF |
Mean |
||||
|
[mM] |
|
total |
I |
II |
III |
IV |
V |
|
|
Per 10E6 cells |
|
Solvent Control Test Item |
- |
A |
2503264 |
2 |
8 |
4 |
5 |
1 |
0.00001 |
0.00002 |
20 |
18 |
- |
B |
2500521 |
5 |
3 |
3 |
0 |
4 |
0.00001 |
0.00002 |
15 |
||
Solvent Control DMBA |
|
A |
2497798 |
12 |
4 |
6 |
8 |
6 |
0.00001 |
0.00003 |
34 |
25 |
- |
B |
2498400 |
0 |
1 |
3 |
5 |
4 |
0.00001 |
0.00002 |
17 |
||
Positive Control (DMBA) |
1.5 µg/mL |
A |
2499294 |
34 |
36 |
36 |
37 |
37 |
0.00007 |
0.00020 |
196 |
239 |
B |
2502737 |
47 |
65 |
51 |
43 |
50 |
0.00010 |
0.00028 |
282 |
|||
Test item |
10 |
A |
2499351 |
4 |
4 |
3 |
4 |
3 |
0.00001 |
0.00002 |
19 |
26 |
10 |
B |
2499539 |
9 |
10 |
5 |
3 |
7 |
0.00001 |
0.00003 |
34 |
||
Test item |
5 |
A |
2496838 |
2 |
3 |
4 |
2 |
4 |
0.00001 |
0.00002 |
18 |
12 |
5 |
B |
2501022 |
0 |
2 |
2 |
1 |
1 |
0.00000 |
0.00001 |
6 |
||
Test Item |
2.5 |
A |
2500169 |
3 |
6 |
3 |
4 |
7 |
0.00001 |
0.00003 |
26 |
21 |
2.5 |
B |
2495657 |
2 |
3 |
2 |
5 |
2 |
0.00001 |
0.00002 |
16 |
||
Test Item |
1.25 |
A |
2501957 |
1 |
0 |
1 |
1 |
1 |
0.00000 |
0.00000 |
4 |
9 |
1.25 |
B |
2501145 |
4 |
4 |
2 |
1 |
2 |
0.00001 |
0.00001 |
14 |
||
Test Item |
0.63 |
A |
2500115 |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
0.63 |
B |
2500176 |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
||
Test item |
0.31 |
A |
2498270 |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
0.31 |
B |
2498015 |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a = not analysed because the OECD 476 guideline requires only 4 concentrations
Table 2: Mutagenicity in Experiment I without Metabolic Activation
|
Conc. |
Culture |
Cells seeded |
Number of colonies per dish |
CE mut |
MF |
MF |
Mean |
||||
|
[mM] |
|
total |
I |
II |
III |
IV |
V |
|
|
Per 10E6 cells |
|
Solvent Control Test Item |
- |
A |
2497286 |
4 |
4 |
5 |
4 |
7 |
0.00001 |
0.00002 |
23 |
26 |
- |
B |
2498413 |
5 |
7 |
6 |
5 |
5 |
0.00001 |
0.00003 |
30 |
||
Solvent Control DMBA |
|
A |
2501274 |
0 |
6 |
2 |
2 |
5 |
0.00001 |
0.00002 |
1 |
11 |
- |
B |
2496879 |
0 |
3 |
1 |
0 |
2 |
0.00000 |
0.00001 |
6 |
||
Positive Control (DMBA) |
300 µg/mL |
A |
2496689 |
33 |
29 |
27 |
31 |
25 |
0.00006 |
0.00014 |
137 |
145 |
B |
2500960 |
26 |
23 |
24 |
31 |
23 |
0.00005 |
0.00015 |
153 |
|||
Test item |
10 |
A |
2497166 |
6 |
8 |
9 |
8 |
7 |
0.00002 |
0.00004 |
35 |
23 |
10 |
B |
2496330 |
2 |
3 |
3 |
1 |
1 |
0.00000 |
0.00001 |
10 |
||
Test item |
5 |
A |
2498978 |
3 |
5 |
4 |
1 |
7 |
0.00001 |
0.00002 |
21 |
21 |
5 |
B |
2497623 |
1 |
3 |
6 |
6 |
4 |
0.00001 |
0.00002 |
20 |
||
Test Item |
2.5 |
A |
2500349 |
3 |
5 |
2 |
3 |
3 |
0.00001 |
0.00002 |
16 |
20 |
2.5 |
B |
2503188 |
9 |
6 |
5 |
3 |
1 |
0.00001 |
0.00002 |
24 |
||
Test Item |
1.25 |
A |
2499500 |
4 |
5 |
4 |
2 |
5 |
0.00001 |
0.00002 |
21 |
21 |
1.25 |
B |
2502812 |
3 |
3 |
3 |
6 |
5 |
0.00001 |
0.00002 |
21 |
||
Test Item |
0.63 |
A |
2500478 |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
0.63 |
B |
2503155 |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
||
Test item |
0.31 |
A |
2497970 |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
0.31 |
B |
2499536 |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
Table 3: Mutagenicity in Experiment II without Metabolic Activation
|
Conc. |
Culture |
Cells seeded |
Number of colonies per dish |
CE mut |
MF |
MF |
Mean |
||||
|
[mM] |
|
total |
I |
II |
III |
IV |
V |
|
|
Per 106 cells |
|
Solvent Control Test Item |
- |
A |
2500933 |
0 |
0 |
3 |
1 |
1 |
0.00000 |
0.00001 |
7 |
11 |
- |
B |
2496490 |
1 |
3 |
2 |
2 |
1 |
0.00000 |
0.00001 |
14 |
||
Solvent Control DMBA |
|
A |
2497077 |
4 |
3 |
0 |
2 |
4 |
0.00001 |
0.00002 |
21 |
25 |
- |
B |
2501950 |
3 |
1 |
3 |
5 |
3 |
0.00001 |
0.00003 |
29 |
||
Positive Control (DMBA) |
300 µg/mL |
A |
2497340 |
22 |
25 |
23 |
21 |
24 |
0.00005 |
0.00027 |
270 |
280 |
B |
2503958 |
22 |
38 |
30 |
35 |
40 |
0.00007 |
0.00029 |
289 |
|||
Test item |
10 |
A |
2501018 |
1 |
2 |
0 |
2 |
0 |
0.00000 |
0.00001 |
7 |
12 |
10 |
B |
2498673 |
3 |
3 |
5 |
1 |
1 |
0.00001 |
0.00002 |
17 |
||
Test item |
5 |
A |
2505206 |
1 |
3 |
2 |
1 |
3 |
0.00000 |
0.00001 |
9 |
18 |
5 |
B |
2496744 |
5 |
3 |
3 |
1 |
2 |
0.00001 |
0.00003 |
27 |
||
Test Item |
2.5 |
A |
2499913 |
2 |
6 |
3 |
6 |
1 |
0.00001 |
0.00003 |
30 |
31 |
2.5 |
B |
2499050 |
3 |
4 |
6 |
0 |
4 |
0.00001 |
0.00003 |
32 |
||
Test Item |
1.25 |
A |
2501820 |
1 |
1 |
1 |
0 |
0 |
0.00000 |
0.00000 |
5 |
19 |
1.25 |
B |
2499000 |
8 |
1 |
3 |
2 |
4 |
0.00001 |
0.00003 |
34 |
||
Test Item |
0.63 |
A |
2498840 |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
0.63 |
B |
2503972 |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
||
Test item |
0.31 |
A |
2497800 |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
0.31 |
B |
2498438 |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a = not analysed because the OECD 476 guideline requires only 4 concentrations
Table 4: Regression Parameters
Treatment |
Correlation coefficient |r| |
p-value |
Exp. I with metabolic activation |
0.718 |
0.282 |
Exp. I without metabolic activation |
0.911 |
0.089 |
Exp. II without metabolic activation |
0.305 |
0.695 |
Table 5: Statistical Significance, pooled replicates (arithmetic means)
Treatment |
p-Values |
||
experiment I |
experiment II |
||
with S9 |
without S9 |
without S9 |
|
Positive control DMBA |
< 0.01 |
- |
- |
Positive control EMS |
- |
< 0.01 |
< 0.01 |
Test item 10 mM |
0.209 |
n/c |
0.487 |
Test item 5 mM |
n/c |
n/c |
0.182 |
Test item 2.5 mM |
0.435 |
n/c |
0.002* |
Test item 1.25 mM |
n/c |
n/c |
0.139 |
n.c.: not calculated because the MF is lower than or equal to the solvent control
* = statistically significantly increased in comparison to the solvent control.
Table 6: Statistical Significance, individual cultures (Comparison of values of replicate A with corresponding solvent control replicate of A and comparison of values of replicate B with corresponding solvent control replicate of B)
Treatment |
Culture |
p-Values |
||
experiment I |
experiment II |
|||
with S9 |
without S9 |
without S9 |
||
Positive control DMBA |
A |
< 0.01 |
- |
- |
B |
< 0.01 |
- |
- |
|
Positive control EMS |
A |
- |
< 0.01 |
< 0.01 |
B |
- |
< 0.01 |
< 0.01 |
|
Test item 10 mM |
A |
n/c |
0.113 |
n/c |
B |
0.006* |
n/c |
0.420 |
|
Test item 5 mM |
A |
n/c |
n/c |
0.430 |
B |
n/c |
n/c |
0.042* |
|
Test item 2.5 mM |
A |
0.313 |
n/c |
< 0.000* |
B |
0.490 |
n/c |
0.007* |
|
Test item 1.25 mM |
A |
n/c |
n/c |
n/c |
B |
n/c |
n/c |
0.003* |
n/c: not calculated because the MF is lower than or equal to the solvent control
* = statistically significantly increased in comparison to the solvent control.
Table 7: Historical Data of the Solvent Controls
|
Number of mutant colonies per 10E6 cells |
||
4 h treatment / with metabolic activation |
|||
|
Solvent control |
Positive control (DMBA) |
|
|
DMEM |
DMSO |
|
Mean value |
16 |
15 |
238 |
Standard deviation |
8.8 |
9.6 |
74.6 |
Range |
4 - 35 |
2 - 44 |
96 - 461 |
95 % confindence interval |
0* - 34 |
0* - 34 |
89 - 388 |
Number of experiments |
18 |
60 |
41 |
Study no.: 17050301G865 |
18 |
25 |
239 |
4 h treatment / with metabolic activation |
|||
|
Solvent control |
Positive control (DMBA) |
|
|
DMEM |
||
Mean value |
16 |
136 |
|
Standard deviation |
10.5 |
97.7 |
|
Range |
2 - 48 |
71 - 294 |
|
95 % confindence interval |
0* - 37 |
46 – 227 |
|
Number of experiments |
52 |
39 |
|
Study no.: 17050301G865 |
26, 11 |
145 |
|
24 h treatment / with metabolic activation |
|||
|
Solvent control |
Positive control (DMBA) |
|
|
DMEM |
||
Mean value |
12 |
232 |
|
Standard deviation |
6.6 |
97.7 |
|
Range |
4 - 27 |
35 – 517 |
|
95 % confindence interval |
0* - 25 |
36 – 427 |
|
Number of experiments |
40 |
34 |
|
Study no.: 17050301G865 |
11, 25 |
280 |
Applicant's summary and conclusion
- Conclusions:
- In conclusion, it can be stated that under the experimental conditions of this study the test substance did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation. Therefore, the test item is considered to be non-mutagenic under the conditions of the HPRT assay.
- Executive summary:
A study according OECD TG 476 was performed to investigate the potential of the substance to induce mutations at the hypoxanthineguanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster cells (V79).
The assay comprised a pre-test and two independent experiments (experiment I and II). The pre-test was done to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the main experiments were determined.
The first main experiment (experiment I) was performed with and without metabolic activation (liver S9 mix from male rats, treated with Aroclor 1254) and a treatment period of 4 h. The second experiment (experiment II) was performed with a treatment period of 24 hours without metabolic activation. The highest nominal concentration (10 mM) applied was chosen based on the good solubility of the test item in organic solvents and aqueous media and on the absence of cytotoxicity. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed enough sensitivity of the testing procedure and the activity of the metabolic activation system.
No substantial and reproducible dose-dependent increase in mutant colony numbers was observed in both experiments up to the maximal concentration (10 mM) of the test item.
In conclusion, it can be stated that under the experimental conditions of this study the test item did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation.
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