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EC number: 500-232-7 | CAS number: 68815-56-5 1 - 2.5 moles ethoxylated
- 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
Skin sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 28 Dec 2017 - 08 Mar 2018
- 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:
- other: OECD Guidelines for Testing of Chemicals, No. 442E: In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation”
- Version / remarks:
- adopted 09 October 2017
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158
- Version / remarks:
- July 1st, 2015
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- GLP-Landesleitstelle Bayern, Bayerrisches Landesamt für Gesundheit und Lebensmittelsicherheit
- Type of study:
- activation of dendritic cells
Test material
- Reference substance name:
- Alcohols, C10-16, ethoxylated, sulfosuccinates, disodium salts
- EC Number:
- 500-232-7
- EC Name:
- Alcohols, C10-16, ethoxylated, sulfosuccinates, disodium salts
- Cas Number:
- 68815-56-5
- Molecular formula:
- not applicable to UVCB
- IUPAC Name:
- Alcohols, C10-16, ethoxylated, sulfosuccinates, disodium salts
Constituent 1
In vitro test system
- Details on the study design:
- - Details of the test procedure used: For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks and then harvested, afterwards the cell suspension were seeded into a 24 well flat-bottom plate at a final density of 1 x 10E6 cells/well.
The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2. After exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The following steps were carried out on ice with pre-cooled buffers and solutions. The supernatant was discarded and the remaining cells were washed twice with FACS buffer. After washing, cells were blocked using 600 µL of a FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction) and incubated at 4 °C for 15 min. After blocking, cells were split in three aliquots into a 96-well V-bottom plate. After centrifugation (approx. 250 x g), cells were stained with 50 µL of FITC-labelled anti-CD86, anti-CD54, or mouse IgG1 (isotype) antibodies in the dark for 30 min. All antibodies were diluted in FACS buffer at an appropriate manner. After washing with FACS buffer two times, cells were re-suspended in FACS buffer and PI solution (propidium iodide) was added. PI staining was done just prior to the measurement by adding PI solutions to each sample (final concentration of PI was 0.625µg/mL). Finally the expression levels of CD86 and CD54 as well as cell viability were analysed by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ = 530 nm ± 15 nm for FITC and λ > 650 nm for PI.
- Cell line used: THP-1 cells (ATCC® TIB-202TM).
- Doses of test chemical and control substances used:
Test Item:
Dose finding assay 1 and 2: 1000, 500.0, 250.0, 125.0, 62.50, 31.25, 15.63 and 7.81 µg/mL
Main experiment 1 and 2: 21.09, 17.58, 14.65, 12.21, 10.17, 8.48, 7.06, 5.89 µg/mL
Medium Control: cell culture medium
Solvent Control: cell culture medium
Positive Control: 4 µg/mL DNCB (2, 4-dinitrochlorobenzene)
- Duration and temperature of exposure: 24 h ± 0.5 h at 37 °C± 1 °C and 5% CO2.
- Cell reactivity check and Dose finding assay: Prior to testing, the quality of freshly thawed cell batch was checked by monitoring the doubling time and checking the reactivity towards positive controls. DNCB (4 µg/mL) and nickel sulphate (100 µg/mL) served as positive control while lactic acid (1000 µg/mL) as negative control. Cells were accepted when both positive controls produce a positive response for CD86 and CD54, and lactic acid produces a negative response for CD86 and CD54.
In dose finding assay, The solvent controls and the test item working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate (with the final density of 1 x 106 cells/well). The cytotoxicity was analysed immediately after the staining procedure by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ > 650 nm. The CV75 value (75% cell survival) was calculated and used to calculate the concentration range of the test item for the main experiment.
- Number of tissue replicates used per test chemical and controls: Two repeated Experiments respectively with one sample per treatment: test substance, positive and negative controls.
- Acceptance criteria:
The test meets acceptance criteria if:
• the cell viability of the solvent controls is >90%,
• the cell viability of at least four tested doses of the test item in each run is >50%,
• the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
• the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
• the MFI ratio of CD86 and CD54 to isotype IgG1 control for both, the medium and DMSO control, is >105%.
- Evaluation criteria:
Test chemical tested by the h-CLAT was considered positive if
- the RFI of CD86 was equal to or greater than 150% at any tested dose at a cell viability ≥ 50% in at least two independent runs
- or the RFI of CD54 was equal to or greater than 200% at any tested dose at a cell viability ≥ 50% in at least two independent runs
- the RFIs of both the CD86 and CD54 were equal to or are greater than 150% and 200% respectively at any tested dose at a cell viability ≥ 50% in at least two independent runs
A negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is <90%. But a negative result for test items with a log Kow >3.5 should be considered as inconclusive.
Results and discussion
- Positive control results:
- The positive control (DNCB) led to an upregulation of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (326% experiment 1; 309% experiment 2) and 200% for CD54 (572% experiment 1; 309% experiment 2) were clearly exceeded.
In vitro / in chemico
Resultsopen allclose all
- Key result
- Run / experiment:
- other: CD86/Experiment 1
- Parameter:
- other: RFI (relative fluorescence intensity)
- Value:
- 122
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Remarks:
- The expression of the cell surface marker CD86 was upregulated above the threshold of 150 starting from a concentration of 21.09 µg/mL to 12.20 µg/mL
- Key result
- Run / experiment:
- other: CD54/Experiment 1
- Parameter:
- other: RFI (relative fluorescence intensity)
- Value:
- 83
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other:
- Remarks:
- The expression of CD54 was not upregulated above the threshold of 200%
- Key result
- Run / experiment:
- other: CD86/Experiment 2
- Parameter:
- other: RFI (relative fluorescence intensity)
- Value:
- 116
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Remarks:
- The expression of CD86 was upregulated above the threshold of 150 starting from a concentration of 21.09 µg/mL to 17.58 µg/mL
- Key result
- Run / experiment:
- other: CD54/Experiment 2
- Parameter:
- other: RFI (relative fluorescence intensity)
- Value:
- 101
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other:
- Remarks:
- The expression of CD54 was not upregulated above the threshold of 200%
- Other effects / acceptance of results:
- Slight cytotoxic effects were observed for the cells treated with the test item in the highest concentrations. Relative cell viability at the highest test item concentration was reduced to 76.2% (CD86), 75.4% (CD54) and 74.9% (isotype IgG1 control) in the first experiment and to 78.5% (CD86), 78.0% (CD54) and 77.8% (isotype IgG1 control) in the second experiment.
The expression of the cell surface marker CD86 was upregulated above the threshold of 150% starting from a concentration of 21.09 µg/mL to 12.20 µg/mL in the first experiment and of 21.09 µg/mL to 17.58 µg/mL in the second experiment. In contrast, the expression of the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Since one of the cell surface markers (CD86) clearly exceeded the threshold in two independent experiments the test item is considered to be a skin sensitiser.
All the controls confirmed the validity of the study. The positive control (DNCB) led to an upregulation of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (332% experiment 1; 272% experiment 2) and 200% for CD54 (304% experiment 1; 544% experiment 2) were clearly exceeded.
Any other information on results incl. tables
Table 1: CD54 and CD86 Expression Experiment 1
Sample |
Conc. |
Cell Viability [%] |
Mean Fluorescence Intensity |
corrected Mean Fluorescence Intensity |
Relative Flourescence Intensity (RFI) |
Ratio Isotype IgG1 to [%] |
|||||||
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
CD86 |
CD54 |
CD86 |
CD54 |
||
Medium Control |
- |
97.1 |
96.3 |
96.0 |
2863 |
1435 |
657 |
2206 |
778 |
100 |
100 |
436 |
218 |
Solvent Control |
0.20% |
96.6 |
94.9 |
95.7 |
3156 |
1228 |
668 |
2488 |
560 |
113 |
72 |
472 |
184 |
DNCB |
4.00 |
81.3 |
80 .8 |
80.6 |
8942 |
2395 |
693 |
8249 |
1702 |
332 |
304 |
1290 |
346 |
NEO-HITENOL LM-20 (Alcohols, C10-16, ethoxylated, sulfosuccinates, disodium salts) |
21.09 |
76.2 |
75.4 |
74.9 |
5612 |
1631 |
750 |
4862 |
881 |
220 |
113 |
748 |
217 |
17.58 |
87.7 |
88.4 |
87.7 |
4980 |
1521 |
714 |
4266 |
807 |
193 |
104 |
697 |
213 |
|
14.65 |
91.1 |
91.2 |
91.6 |
4390 |
1492 |
726 |
3664 |
766 |
166 |
98 |
605 |
206 |
|
12.20 |
93.9 |
93.4 |
93.2 |
4160 |
1494 |
737 |
3423 |
757 |
155 |
97 |
564 |
203 |
|
10.17 |
94.3 |
94.2 |
94.1 |
3990 |
1591 |
786 |
3204 |
805 |
145 |
103 |
508 |
202 |
|
8.48 |
94.7 |
95.0 |
94.8 |
3698 |
1515 |
871 |
2827 |
644 |
128 |
83 |
425 |
174 |
|
7.06 |
95.0 |
94.9 |
94.7 |
3692 |
1453 |
774 |
2918 |
679 |
132 |
87 |
477 |
188 |
|
5.89 |
95.4 |
95.6 |
95.0 |
3398 |
1507 |
713 |
2685 |
794 |
122 |
102 |
477 |
211 |
Table 2: CD54 and CD86 Expression Experiment 2
Sample |
Conc. |
Cell Viability [%] |
Mean Fluorescence Intensity |
corrected Mean Fluorescence Intensity |
Relative Flourescence Intensity (RFI) |
Ratio Isotype IgG1 to [%] |
|||||||
CD86 |
CD54 |
IgG Isotype |
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
CD86 |
CD54 |
C86 |
CD54 |
||
Medium Control |
- |
96.9 |
96.8 |
96.5 |
2594 |
1130 |
618 |
1976 |
512 |
100 |
100 |
420 |
183 |
Solvent Control |
0.20% |
96.6 |
95.8 |
96.6 |
2483 |
1056 |
619 |
1864 |
437 |
94 |
85 |
401 |
171 |
DNCB |
4.0 |
84.1 |
84.5 |
83.4 |
5810 |
3109 |
732 |
5078 |
2377 |
272 |
544 |
794 |
425 |
NEO-HITENOL LM-20 (Alcohols, C10-16, ethoxylated, sulfosuccinates, disodium salts) |
21.09 |
78.5 |
78.0 |
77.8 |
4943 |
1222 |
615 |
4328 |
607 |
219 |
119 |
804 |
199 |
17.58 |
92.7 |
92.9 |
92.5 |
3601 |
1166 |
578 |
3023 |
588 |
153 |
115 |
623 |
202 |
|
14.65 |
94.1 |
94.4 |
94.4 |
3209 |
1191 |
600 |
2609 |
591 |
132 |
115 |
535 |
199 |
|
12.20 |
95.4 |
95.3 |
95.4 |
3301 |
1168 |
636 |
2665 |
532 |
135 |
104 |
519 |
184 |
|
10.17 |
95.6 |
95.4 |
95.9 |
3395 |
1162 |
584 |
2811 |
578 |
142 |
113 |
581 |
199 |
|
8.48 |
96.1 |
96.2 |
96.1 |
3309 |
1146 |
611 |
2698 |
535 |
137 |
104 |
542 |
188 |
|
7.06 |
95.6 |
96.1 |
95.7 |
2893 |
1123 |
604 |
2289 |
519 |
116 |
101 |
479 |
186 |
|
5.89 |
94.7 |
95.5 |
95.6 |
3007 |
1103 |
584 |
2423 |
519 |
123 |
101 |
515 |
189 |
Table3: Acceptance Criteria
Acceptance Criterion |
Range |
Experiment 1 |
pass/fail |
Experiment 2 |
pass/fail |
||||
cell viability solvent controls [%] |
>90 |
94.9 |
- |
97 .1 |
pass |
95.8 |
- |
96.9 |
pass |
number of test dosed with viability >50% CD86 |
≥4 |
8 |
pass |
8 |
pass |
||||
number of test dosed with viability >50% CD54 |
≥4 |
8 |
Pass |
8 |
pass |
||||
number of test dosed with viability >50% IgG1 |
≥4 |
8 |
pass |
8 |
pass |
||||
RFI of positive control of CD86 |
≥150 |
332 |
pass |
272 |
pass |
||||
RFI of positive control of CD54 |
≥200 |
304 |
pass |
544 |
pass |
||||
RFI of solvent control of CD86 |
<150 |
113 |
pass |
94 |
pass |
||||
RFI of solvent control of CD54 |
<200 |
72 |
pass |
85 |
pass |
||||
MFI ratio IgG1/CD86 for medium control [%] |
>105 |
436 |
pass |
420 |
pass |
||||
MFI ratio IgG1/CD86 for DMSO control [%] |
>105 |
472 |
pass |
401 |
pass |
||||
MFI ratio IgG1/CD54 for medium control [%] |
>105 |
218 |
pass |
183 |
pass |
||||
MFI ratio IgG1/CD54 for DMSO control [%] |
>105 |
184 |
pass |
171 |
pass |
Applicant's summary and conclusion
- Interpretation of results:
- other: skin sensitising based on the key event “activation of dendritic cells”
- Conclusions:
- In this study under the given conditions the test item did upregulate the cell surface marker CD86 in at least two independent experiment runs. Therefore, based on the observed results it was concluded that the test substance induces dendritic cell activation. The result alone does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required. Based on the results of all three key events the test substance has to be classified as skin sensitiser.
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