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EC number: 219-102-3 | CAS number: 2359-15-1
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
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- Density
- Particle size distribution (Granulometry)
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- Additional physico-chemical information
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- Endpoint summary
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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
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- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
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- Repeated dose toxicity
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- Specific investigations
- Exposure related observations in humans
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- Additional toxicological data
Skin sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017-07-13 to 2017-11-14
- 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: human Cell Line Activation Test (h-CLAT)"
- Version / remarks:
- adopted 29 July 2016
- 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)
- Type of study:
- activation of dendritic cells
Test material
- Reference substance name:
- N,N'-methylenebis[methacrylamide]
- EC Number:
- 219-102-3
- EC Name:
- N,N'-methylenebis[methacrylamide]
- Cas Number:
- 2359-15-1
- Molecular formula:
- C9H14N2O2
- IUPAC Name:
- N,N'-methylenebis(2-methylacrylamide)
- Test material form:
- solid
Constituent 1
- Specific details on test material used for the study:
- The test item was freshly prepared immediately prior to use. The test item was soluble in dimethyl sulfoxide (DMSO) at a concentration of 40 mg/mL. Sonication and warming to 37 °C was used to aid solubilisation. Stock solutions were prepared by diluting the highest soluble concentration seven times with a constant dilution factor of 1:2. The working stock solutions were prepared by diluting each stock solution 250 times with cell culture medium. The working stock solutions were applied to the cells by adding equal volumes of each solution to prepared cells, resulting in a further 1:2 dilution of the working solutions. The solvent was present at a constant volume ratio of 0.2 % (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.
In vitro test system
- Details on the study design:
- The h-CLAT is supposed to address the third key event of the skin sensitisation process as defined by the adverse outcome pathway (AOP), the activation of dendritic cells (DC) typically accompanied by expression of specific cell surface markers, chemokines and cytokines. The h-CLAT quantifies the expression of the two surface markers CD86 and CD54 which are considered to be associated with the process of DC activation by using the human monocytic leukemia cell line THP-1 as a surrogate. The expression level of CD86 and CD54 following exposure to test chemicals are used for supporting the discrimination between sensitisers and non-sensitisers. The correlation of upregulation of immunological relevant cell surface markers with the skin sensitising potential of a chemical has been reported is considered relevant for the assessment of the skin sensitisation potential of chemicals.
This test may be used for supporting the discrimination between skin sensitisers and non-sensitisers in accordance with UN GHS "Category 1". It does not allow the classification of chemicals to the subcategories 1A and 1B as defined by UN GHS nor predict potency for safety assessment decisions. Therefore, all substances giving a positive result in the h-CLAT will be classified into "UN GHS Category 1".
For detailed information on experimental procedure, materials, methods, controls, prediction model and acceptance criteria please see section “any other details on materials and methods incl. tables”.
Results and discussion
- Positive control results:
- The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in all experiments. The threshold of 150% for CD86 (359% experiment 1; 390% experiment 2; 360 experiment 3) and 200% for CD54 (263% experiment 1; 389% experiment 2; 256 experiment 3) were clearly exceeded.
In vitro / in chemico
Resultsopen allclose all
- Key result
- Run / experiment:
- other: 1, 2 and 3
- Parameter:
- other: CD54 expression (%)
- Value:
- 200
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Run / experiment:
- other: 1 and 3
- Parameter:
- other: CD86 expression (%)
- Value:
- 150
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 2
- Parameter:
- other: CD86 expression (%)
- Value:
- 156
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- other: Upregulation above the threshold of 150% was observed at a concentration of 32.15 µg/mL. No further upregulation of the cell surface marker CD86 above the threshold was observed in the tested concentration range.
- Run / experiment:
- other: determination of cytotoxicity
- Parameter:
- other: CV75 (µg/mL)
- Value:
- 80
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- other: No cytotoxic effects observed.
- Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: not reported
DEMONSTRATION OF TECHNICAL PROFICIENCY: the results of the controls are well within the historical range.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
Any other information on results incl. tables
Reactivity Check of the Cell Stock
Doubling time of the cells was monitored and found to be 46.49 h which is within the doubling time range specified by the manufacturer (35 - 50 h).
Doubling time of the cells was monitored and found to be 46.50 h (batch 16 used for dose finding assay and main experiments 1 and 2) and 48.7 (batch 17 used for main experiment 3) which is within the doubling time range specified by the manufacturer (35 - 50 h).
Table 2: Results of the Cell Batch Activation Test (batch 16)
Sample |
Concentration |
CD86 |
CD54 |
Activated |
Pass /Fail |
||||||||
Cell Viability [%] |
RFI |
Threshold OECD TG 442E |
Cell Viability [%] |
RFI |
Threshold OECD TG 442E |
yes/no |
|
|
|||||
DNCB |
4 µg/mL |
86.9 |
374 |
>150 |
86.7 |
282 |
>200 |
Yes |
pass |
|
|||
NiSO4 |
100 µg/mL |
88.8 |
226 |
>150 |
86.9 |
250 |
>200 |
Yes |
pass |
|
|||
LA |
1000 µg/mL |
95.8 |
76 |
£150 |
95.4 |
93 |
£200 |
No |
pass |
|
|||
Table 3: Results of the Cell Batch Activation Test (batch 17)
Sample |
Concentration |
CD86 |
CD54 |
Activated |
Pass /Fail |
||||
Cell Viability [%] |
RFI |
Threshold OECD TG 442E |
Cell Viability [%] |
RFI |
Threshold OECD TG 442E |
yes/no |
|||
DNCB |
4 µg/mL |
86.2 |
282 |
>150 |
86.5 |
256 |
>200 |
yes |
pass |
NiSO4 |
100 µg/mL |
79.6 |
229 |
>150 |
80.2 |
573 |
>200 |
yes |
pass |
LA |
1000 µg/mL |
96.1 |
84 |
£150 |
96.1 |
96 |
£200 |
no |
pass |
The positive controls DNCB and NiSO4led to upregulation of the cell surface markers CD54 and CD86. The negative control LA did not induce an upregulation of CD54 and CD86. The cell batch was accepted for further testing.
Solvent Finding
All test item solutions were freshly prepared immediately prior to use. The test item was soluble in DMSO at a concentration of 40 mg/mL.
Dose Finding Assay
The dose finding assay was performed using stock solutions with a concentration of 80µg/mL. Since no cytotoxicity was observed no CV75 could be determined. The main experiment was performed covering a concentration range from 80.00 – 22.33µg/mL (40.00 – 11.16 mg/mL stock solution).
Table 4: Results of the Dose Finding Assay
Sample |
Concentration applied [µg/ml] |
Cell Viability [%] |
Medium Control |
0.00 |
95.10 |
Solvent Control |
0.00 |
95.10 |
N,N´- Methylene bis (methacrylamide) |
0.63 |
95.40 |
1.25 |
95.30 |
|
2.50 |
95.40 |
|
5.00 |
95.70 |
|
10.00 |
95.70 |
|
20.00 |
95.00 |
|
40.00 |
94.80 |
|
80.00 |
95.30 |
|
Calculated CV75 [µg/mL] |
No CV75 |
Results CD54 and CD86 Expression
For determination of the cell surface markers CD54 and CD86 three independent experiments were performed using separate cultivated cells at passage 18 (dose finding assay), passage 27 (first experiment), passage 30 (second experiment) and passage 15 (third experiment). For each experiment separately weighted samples and preparations were used.
Table 5: 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 |
- |
97.7 |
96.6 |
96.6 |
2467 |
1277 |
723 |
1744 |
554 |
116 |
89 |
341 |
177 |
Solvent Control |
0.20 % |
96.5 |
96.8 |
96.6 |
2220 |
1342 |
716 |
1504 |
626 |
100 |
100 |
310 |
187 |
DNCB |
4.00 |
86.9 |
85.6 |
86.3 |
6045 |
2297 |
649 |
5396 |
1648 |
359 |
263 |
931 |
354 |
N, N´- Methylene bis (methacrylamide) |
80 |
97.0 |
96.9 |
96.7 |
2371 |
1299 |
786 |
1585 |
513 |
105 |
82 |
302 |
165 |
66.67 |
96.3 |
96.6 |
96.5 |
2492 |
1257 |
684 |
1808 |
573 |
120 |
92 |
364 |
184 |
|
55.56 |
95.6 |
95.8 |
95.5 |
2530 |
1298 |
685 |
1845 |
613 |
123 |
98 |
369 |
189 |
|
46.30 |
96.4 |
97.1 |
96.6 |
2399 |
1392 |
692 |
1707 |
700 |
114 |
112 |
347 |
201 |
|
38.58 |
96.8 |
96.5 |
96.8 |
2372 |
1291 |
669 |
1703 |
622 |
113 |
99 |
355 |
193 |
|
32.15 |
96.2 |
96.7 |
96.4 |
2465 |
1269 |
692 |
1773 |
577 |
118 |
92 |
356 |
183 |
|
26.79 |
95.8 |
97.1 |
96.6 |
2202 |
1222 |
703 |
1499 |
519 |
100 |
83 |
313 |
174 |
|
22.33 |
96.4 |
96.8 |
96.0 |
2224 |
1471 |
732 |
1492 |
739 |
99 |
118 |
304 |
201 |
Table 6: 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 |
Isotype IgG1 |
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
CD86 |
CD54 |
C86 |
CD54 |
||
Medium Control |
- |
95.8 |
95.6 |
95.4 |
1278 |
843 |
654 |
624 |
189 |
73 |
58 |
195 |
129 |
Solvent Control |
0.20 % |
95.8 |
95.9 |
95.6 |
1433 |
906 |
582 |
851 |
324 |
100 |
100 |
246 |
156 |
DNCB |
4.0 |
79.2 |
78.6 |
78.5 |
3968 |
1910 |
651 |
3317 |
1259 |
390 |
389 |
610 |
293 |
N, N´- Methylene bis (methacrylamide) |
80.00 |
95.7 |
95.7 |
95.3 |
1676 |
1029 |
637 |
1039 |
392 |
122 |
121 |
263 |
162 |
66.67 |
95.9 |
95.4 |
95.3 |
1703 |
1075 |
642 |
1061 |
433 |
125 |
134 |
265 |
167 |
|
55.56 |
95.9 |
95.8 |
95.7 |
1593 |
1031 |
687 |
906 |
344 |
106 |
106 |
232 |
150 |
|
46.30 |
95.6 |
95.8 |
95.9 |
1782 |
1027 |
652 |
1130 |
375 |
133 |
116 |
273 |
158 |
|
38.58 |
96.0 |
95.6 |
95.9 |
1630 |
1039 |
643 |
987 |
396 |
116 |
122 |
253 |
162 |
|
32.15 |
95.3 |
95.7 |
95.8 |
1973 |
1049 |
649 |
1324 |
400 |
156 |
123 |
304 |
162 |
|
26.79 |
95.8 |
95.4 |
96.0 |
1643 |
1033 |
636 |
1007 |
397 |
118 |
123 |
258 |
162 |
|
22.33 |
95.8 |
96.6 |
96.2 |
1758 |
994 |
600 |
1158 |
394 |
136 |
122 |
293 |
166 |
Table 7: CD54 and CD86 Expression Experiment 3
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 |
C86 |
CD54 |
||
Medium Control |
- |
96.7 |
95.9 |
96.1 |
1451 |
1126 |
644 |
807 |
482 |
83 |
87 |
225 |
175 |
Solvent Control |
0.20 % |
95.2 |
95.1 |
94.3 |
1609 |
1192 |
639 |
970 |
553 |
100 |
100 |
252 |
187 |
DNCB |
4.0 |
85.6 |
84.8 |
84.4 |
4107 |
2025 |
612 |
3495 |
1413 |
360 |
256 |
671 |
331 |
N, N´- Methylene bis (methacrylamide) |
80.0 |
95.7 |
96.2 |
95.9 |
1578 |
1161 |
667 |
911 |
494 |
94 |
89 |
237 |
174 |
66.67 |
95.8 |
95.6 |
95.3 |
1728 |
1239 |
673 |
1055 |
566 |
109 |
102 |
257 |
184 |
|
55.56 |
95.0 |
95.0 |
94.6 |
1705 |
1153 |
650 |
1055 |
503 |
109 |
91 |
262 |
177 |
|
46.30 |
95.0 |
95.6 |
93.9 |
1603 |
1116 |
657 |
946 |
459 |
98 |
83 |
244 |
170 |
|
38.58 |
96.5 |
96.3 |
95.7 |
1538 |
1210 |
666 |
872 |
544 |
90 |
98 |
231 |
182 |
|
32.15 |
95.4 |
95.3 |
95.7 |
1646 |
1165 |
664 |
982 |
501 |
101 |
91 |
248 |
175 |
|
26.79 |
96.2 |
96.2 |
95.6 |
1581 |
1166 |
667 |
914 |
499 |
94 |
90 |
237 |
175 |
|
22.33 |
95.3 |
95.2 |
95.8 |
1552 |
1182 |
681 |
871 |
501 |
90 |
91 |
228 |
174 |
Table 8: Acceptance criteria
Acceptance criterion |
range |
Experiment 1 |
pass/fail |
Experiment 2 |
pass/fail |
Experiment 3 |
pass/fail |
||||||
cell viability medium and solvent control [%] |
>90 |
96.5 |
- |
97.7 |
pass |
95.4 |
- |
95.9 |
pass |
94.3 |
- |
96.7 |
pass |
number of test dosed with viability >50% CD86 |
≥4 |
8 |
pass |
8 |
pass |
8 |
pass |
||||||
number of test dosed with viability >50% CD54 |
≥4 |
8 |
pass |
8 |
pass |
8 |
pass |
||||||
number of test dosed with viability >50% IgG1 |
≥4 |
8 |
pass |
8 |
pass |
8 |
pass |
||||||
RFI of positive control of CD86 |
≥150 |
359 |
pass |
390 |
pass |
360 |
pass |
||||||
RFI of positive control of CD54 |
≥200 |
263 |
pass |
389 |
pass |
256 |
pass |
||||||
RFI of solvent control of CD86 |
<150 |
86 |
pass |
136 |
pass |
120 |
pass |
||||||
RFI of solvent control of CD54 |
<200 |
113 |
pass |
171 |
pass |
115 |
pass |
||||||
MFI ratio IgG1/CD86 for medium control [%] |
>105 |
341 |
pass |
195 |
pass |
225 |
pass |
||||||
MFI ratio IgG1/CD86 for solvent control [%] |
>105 |
310 |
pass |
246 |
pass |
252 |
pass |
||||||
MFI ratio IgG1/CD54 for medium control [%] |
>105 |
177 |
pass |
129 |
pass |
175 |
pass |
||||||
MFI ratio IgG1/CD54 for solvent control [%] |
>105 |
187 |
pass |
156 |
pass |
187 |
pass |
||||||
Table 9: Historical data
Criterion |
mean |
SD |
N |
cell viability solvent controls [%] |
97.4 |
1.2 |
462 |
number of test doses with viability >50 % |
- |
- |
1060 |
RFI of positive control of CD86 |
417.5 |
170.7 |
77 |
RFI of positive control of CD54 |
660.0 |
319.7 |
77 |
RFI of solvent control of CD86 |
116.9 |
14.6 |
77 |
RFI of solvent control of CD54 |
124.6 |
26.9 |
77 |
MFI ratio IgG1/CD86 for medium control [%] |
193.3 |
48.6 |
77 |
MFI ratio IgG1/CD86 for DMSO control [%] |
213.5 |
60.5 |
77 |
MFI ratio IgG1/CD54 for medium control [%] |
130.1 |
16.6 |
77 |
Applicant's summary and conclusion
- Interpretation of results:
- other: Expert statement
- Remarks:
- Negative. No indication of sensitisation.
- Conclusions:
- In this in vitro assay, the test item showed no upregulation in at least two of three independent experiments and thus, the test item is considered to be no skin sensitiser. No cytotoxic effects were observed for the cells treated with the test item.
- Executive summary:
In an in vitro human cell line activation test (h-CLAT) according to OECD guideline 442E, the sensitising potential of N,N´-Methylenebis[methacrylamide] by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1 was investigated.
N,N´-Methylenebis[methacrylamide] was dissolved in DMSO. Cells were incubated with the test item for 24 h at 37 °C. After exposure, cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.
The main experiment was performed covering the following concentration steps: 80.0, 66.67, 55.56, 46.30, 38.58, 32.15, 26.79 and 22.33 µg/mL
Relative cell viability at the highest test item concentration was reduced to no less than 95.7 % for CD86 and 95.7 % for CD54. Due to a lack of cytotoxicity at the given concentrations, no CV75 could be derived.
The expression of cell surface marker CD54 was not upregulated above the threshold of 200 % in any of the experiments.
In the second experiment the expression of the cell surface marker CD86 was upregulated to 156 % only at a concentration of 32.15µg/mL. In the first and third experiment the expression of the cell surface marker CD86 was not upregulated above the threshold of 150 %.
The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in all experiments. The threshold of 150 % for CD86 and 200 % for CD54 was clearly exceeded in all experiments.
The controls confirmed the validity of the study. The viability of the solvent control was > 90 %. The number of tested test item concentrations with cell viability > 50 % was ≥ 4 (8 in all three experiments). The RFI for CD86 and CD54 of cells treated with the solvent DMSO was ≤ 150 % and ≤ 200 %. The MFI ratio of the medium control and isotype IgG1 control was ≥ 105 % for CD86 and CD54. The MFI ratio of the solvent control (DMSO) and isotype IgG1 control was ≥ 105 % for CD86 and CD54.
Since the test item showed no upregulation in two of three independent experiments, the test item is considered to be no skin sensitiser.
The data generated with this test should be considered in the context of an integrated approach such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.
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