N,N'-methylenebis[methacrylamide]

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:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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

Test material information

Constituent 1

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

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

Results

Results 1

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

Results 2

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

Results 3

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.

Results 4

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 [µg/mL]

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 [µg/mL]	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. [μg/mL]	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. [μg/mL]	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. [μg/mL]	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.