N,N'-methylenebis[methacrylamide]

Endpoint summary

Description of key information

Skin sensitising potential of N,N´-Methylenebis[methacrylamide] was examined in three guideline studies under GLP conditions.

- In the Direct Peptide Reactivity Assay (DPRA, OECD guideline 442C), no prediction could be made due to co-elution of test item with the cysteine peptide peak.

- The KeratinoSens^TM assay (OECD guideline 442D) showed that the test item did not induce luciferase activity at all tested concentrations.

- In the human cell line activation test (h-CLAT, OECD guideline 442E), the test item did not lead to an upregulation of CD54 and CD86 above the threshold values as described in the guideline.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-07-13 to 2017-07-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

adopted: February 04, 2015

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154

Version / remarks:

January 12, 2013

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

The test item was freshly prepared immediately prior to use, unless stability data demonstrate the acceptability of storage. The test item was pre-weighed into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared. The test item was not soluble in water but soluble in the other tested solvents. Since acetonitrile is the preferred solvent according to the guideline, acetonitrile was chosen as suitable vehicle.

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
The DPRA is supposed to address the molecular initiating event of the adverse outcome pathway (AOP), namely protein reactivity, by quantifying the reactivity of test chemicals towards synthetic model peptides containing either lysine or cysteine. The percentage depletion value of the cysteine and lysine peptide is used to categorize a substance in one of four reactivity classes to support discrimination between skin sensitisers and non-sensitisers.
The correlation of protein reactivity with skin sensitisation potential of a chemical is well established and represents the first and initial key event in the skin sensitisation process as defined by the AOP. It is therefore a crucial step for the sensitising potential of a chemical. This test may be used for the hazard identification of sensitising chemicals 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 DPRA 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”.

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 65.66 %. Precipitation was observed in the cysteine peptide solution (excluding the co-elution control of the positive control). Phase separation was observed in the lysine peptide solution (including the co-elution control of the positive control). Since the acceptance criteria the depletion range of the positive control was fulfilled, the observed precipitations and phase separation were regarded as insignificant. Thus, the positive control results are considered valid.

Key result

Run / experiment:

other: CYSTEINE PEPTIDE

Parameter:

other: % Depletion of Peptide

Value:

4.93

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Co-elution of test item with the cysteine peptide peak was observed.

Key result

Run / experiment:

other: LYSINE PEPTIDE

Parameter:

other: % Depletion of Peptide

Value:

2.84

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

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 (vehicle) control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Pre-Experiments

Solubility of the test item was determined prior to the main experiment. All test item solutions were freshly prepared immediately prior to use. The test item was soluble in acetonitrile. No turbidity, precipitation and phase separation was observed for the test item solutions. All test item preparations of the main experiment were prepared using acetonitrile.

Precipitation and Phase Separation

All test item solutions were freshly prepared immediately prior to use.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the positive control (excluding the co-elution control of the positive control). Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the samples of the positive control (including the co-elution control of the positive control). Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria the depletion range of the positive control was fulfilled, the observed precipitations and phase separation were regarded as insignificant.

Co-elution with the Peptide Peaks

Co-elution of the test item with the cysteine peptide peak was observed.

Results Calibration Curve

Table 6: Cysteine and Lysine Values of the Calibration Curve. Based on these results, linear regression was performed, and the following calibration curves were determined.

Cysteine Peptide Calibration Curve :y = 9090.22 x -99.48; R² = 0.9926.

Lysine Peptide Calibration Curve : y = 7923.41 x +6.83; R² = 1.000

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD1	4835.2368	0.5340	4231.4258	0.5340
STD2	2314.6648	0.2670	2129.2986	0.2670
STD3	782.4597	0.1335	1075.6375	0.1335
STD4	525.4504	0.0667	541.5288	0.0667
STD5	266.4071	0.0334	266.7184	0.0334
STD6	135.9798	0.0167	133.0853	0.0167
STD7	0.0000	0.0000	0.0000	0.0000

Results of the Cysteine Peptide Depletion

Table 7: Depletion of the Cysteine Peptide

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1381.2087	0.1629	71.03	71.19	0.17	0.24
	1364.7275	0.1611	71.37
	1374.1948	0.1621	71.18
Test Item	4542.8496	0.5107	4.71	4.93	0.27	5.43
	4518.3960	0.5080	5.23
	4536.8809	0.5100	4.84

Results of the Lysine Peptide Depletion

Table 8: Depletion of the Lysine Peptide

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1578.2926	0.1983	60.21	60.13	0.17	0.29
	1589.7759	0.1998	59.93
	1577.3129	0.1982	60.24
Test Item	3859.0159	0.4862	2.72	2.84	0.11	3.89
	3850.4365	0.4851	2.94
	3853.1375	0.4854	2.87

Categorization of the Test Item

Based on the results of the peptide depletion, categorization according to the prediction model might be performed. Since co-elution with the cysteine peptide was observed, no prediction can be made.

Table 9: Categorization of the Test Item

Prediction Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)			Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	3.88	Minimal Reactivity	--	4.93	Minimal Reactivity	--
Positive Control	65.66	High Reactivity	sensitizer	71.19	Moderate Reactivity	sensitizer

Acceptance Criteria

Table 10: Acceptance Criteria for Cysteine Peptide

Cysteine Peptide Run
Acceptance Criterion	Range	Value	pass/fail
coefficient of determination	R² > 0.99	0.9926	pass
mean peptide concentration of RC A	0.45 ≤ x ≤ 0.55 mM	0.5429	pass
mean peptide concentration of RC C (PC)	0.45 ≤ x ≤ 0.55 mM	0.5354	pass
mean peptide concentration of RC C (TI)	0.45 ≤ x ≤ 0.55 mM	0.5354	pass
CV of the peak area of RC B	< 15 %	1.98	pass
CV of the peak area of RC C (PC)	< 15 %	0.57	pass
CV of the peak area of RC C (TI)	< 15 %	0.57	pass
mean peptide depletion of the PC	60.8 % < x < 100 %	71.19	pass
SD of peptide depletion of the PC replicates	< 14.9 %	0.17	pass
SD of peptide depletion of the TI replicates	< 14.9 %	0.27	pass

Table 11: Acceptance Criteria for Lysine Peptide

Lysine Peptide Run
Acceptance Criterion	Range	Value	pass/fail
coefficient of determination	R² > 0.99	1.0000	pass
mean peptide concentration of RC A	0.45 ≤ x ≤ 0.55 mM	0.5021	pass
mean peptide concentration of RC C (PC)	0.45 ≤ x ≤ 0.55 mM	0.4998	pass
mean peptide concentration of RC C (TI)	0.45 ≤ x ≤ 0.55 mM	0.4998	pass
CV of the peak area of RC B	< 15 %	0.51	pass
CV of the peak area of RC C (PC)	< 15 %	0.28	pass
CV of the peak area of RC C (TI)	< 15 %	0.28	pass
mean peptide depletion of the PC	40.2 % < x < 69.0 %	60.13	pass
SD of peptide depletion of the PC replicates	< 11.6 %	0.17	pass
SD of peptide depletion of the TI replicates	< 11.6 %	0.11	pass

Historical data

Table 12: Historical Data Cysteine Peptide

Cysteine Peptide
	mean	SD	N
linearity of the calibration curve	0.9991	0.0005	22
mean peptide concentration of reference A [mM]	0.52	0.0193	22
mean peptide concentration of reference C [mM]	0.50	0.0164	22
CV of the peak area of control B [%]	2.10	1.43	22
CV of the peak area of control C [%]	1.60	1.25	22
mean peptide depletion of the PC [%]	74.67	2.15	22
SD of peptide depletion of the PC replicates [%]	0.84	0.52	22
SD of peptide depletion of the test items [%]	4.60	1.72	79

Table 13: Historical Data Lysine Peptide

Lysine Peptide
	mean	SD	N
linearity of the calibration curve	0.9998	0.0001	19
mean peptide concentration of reference A [mM]	0.49	0.0170	19
mean peptide concentration of reference C [mM]	0.49	0.0181	19
CV of the peak area of control B [%]	1.26	0.02	19
CV of the peak area of control C [%]	0.81	0.83	19
mean peptide depletion of the PC [%]	59.54	5.72	20
SD of peptide depletion of the PC replicates [%]	2.58	1.53	20
SD of peptide depletion of the test items [%]	1.02	0.74	63

 

Interpretation of results:

other: expert statement

Remarks:

Sensitizing potential of the test item can not be predicted and the test result must be considered as inconclusive.

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards the peptides. Due to co-elution of test item with the cysteine peptide peak, sensitizing potential of the test item could not be predicted and the test result must be considered as inconclusive.

Executive summary:

In an in chemico skin sensitisation study performed according to OECD test guideline 442C (Direct Peptide Reactivity Assay), the reactivity of N,N´-methylenebis[methacrylamide] was evaluated by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides.

A 100 mM stock solution of N,N´-methylenebis[methacrylamide] in acetonitrile was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently, samples were analysed by HPLC.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. Precipitation was observed for the samples of the positive control (excluding the co-elution control of the positive control).

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. Phase separation was observed for the samples of the positive control (including the co-elution control of the positive control).

Since the acceptance criteria the depletion range of the positive control was fulfilled, the observed precipitations and phase separation were regarded as insignificant. The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 65.66 %.

The 100 mM stock solution of N,N´-methylenebis[methacrylamide] showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38 %. Co-elution of test item with the cysteine peptide peak was observed.

In conclusion, in this study under the given conditions, N,N´-methylenebis[methacrylamide] showed minimal reactivity towards the peptides. Due to co-elution of test item with the cysteine peptide peak, sensitizing potential of the test item could not be predicted and the test result must be considered as inconclusive.

The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-08-21 to 2017-10-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

adopted: February 04, 2015

Qualifier:

according to guideline

Guideline:

other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155

Version / remarks:

July 1st, 2015

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

- Skin sensitisation (In vitro test system) ARE-Nrf-2 Luciferase Test Method (KeratinoSens™): The induction of the Keap1-Nrf2-ARE signalling pathway by small electrophilic substances such as skin sensitizers represents the second key event of the skin sensitisation process. This in vitro method is designed to predict and classify the skin sensitising potential of a chemical by assessment of its potential to induce the Keap1-Nrf2-ARE signalling pathway by quantifying the luciferase gene expression using luminescence detection.
- Details on study design / experimental procedure:
A cell suspension of 8.00E+04 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1.00E+04 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability.
After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5 % CO2. Thereafter, the assay medium was discarded and replaced by 150 µL test item exposure medium. 50 µL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item.
All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5 % CO2.
Luciferase activity
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS (Gibco Life Science; Lot No.: 1877596). Subsequently 20 µL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light.
Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1.000 ms before assessing the luciferase activity for 2.000 ms. This procedure was repeated for each individual well.
Cell viability
For the cell viability plate the medium was replaced with 200 µL test item exposure medium. 27 µL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5 % CO2. Afterwards the medium was removed and replaced by 200 µL 10 % SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5 % CO2 overnight (experiment 1 and 2). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.
For detailed information on materials, please see section “any other information on materials and methods incl. tables”.
Data Analysis
For each test item two independent repetitions using separately prepared test item solutions and independently harvested cells are necessary to derive a prediction. Each independent run consisted of three replicates for every concentration step of the test item and the positive control. In case of discordant results, a third independent run is performed. For the parameters calculated, please see figure 1 in section “illustration (picture/graph)”.
For every concentration showing > 1.5 fold luciferase activity induction, statistical significance (p < 0.05) was calculated using a two-tailed Student’s t-test comparing the luminescence values for the three replicated samples with the luminescence values in the solvent (negative) control wells.
The lowest concentration with > 1.5 fold luciferase activity induction was the value determining the EC1.5 value. It was checked in each case whether this value was below the IC30 value, indicating that there was less than 30 % reduction on cellular viability at the EC1.5 determining concentration.
Prediction Model:
The test item is considered positive in accordance with UN GHS “Category 1” if the following conditions were met in at least two independently prepared test repetitions:
- Imax is > 1.5 fold increased and statistically significant (p < 0.05) compared to the negative control
- cell viability is > 70 % at the lowest concentration with an induction of luciferase activity > 1.5
- EC1.5 value is < 1000 µM an apparent overall dose-response for luciferase induction
If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations < 1000 µM is considered as inconclusive.
Acceptance Criteria:
The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is < 20 % in each repetition.

Positive control results:

The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (3.92 in experiment 1; 4.55 in experiment 2). The calculated EC1.5 was between 7 and 34 µM (16.35 µM in experiment 1; 12.29 µM in experiment 2). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20 % (11.1 % in experiment 1; 12.7 % in experiment 2). Thus, the results fulfil the given criteria and the positive controls are considered valid. For details please see section “any other information on results incl. tables”.

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: max. luciferase activity (Imax) induction

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: No EC1.5 value could be calculated. The corresponding cell viability was 64.9 %. No sign of sensitisation.

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: max. luciferase activity (Imax) induction

Value:

1.35

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: No EC1.5 value could be calculated. The corresponding cell viability was 42.9 %. No sign of sensitisation.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not reported.
DEMONSTRATION OF TECHNICAL PROFICIENCY: the measured values 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

In the first experiment, a max luciferase activity (I_max) induction of 1.50 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 64.9 %. No significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5value could be calculated.

In the second experiment, a max luciferase activity (I_max) induction of 1.35 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 42.9 %. No significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5value could be calculated.

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Under the condition of this study the test item is therefore considered as non sensitiser.

The controls confirmed the validity of the study. The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (3.92 in experiment 1; 4.55 in experiment 2). The calculated EC_1.5 was between 7 and 34 µM (16.35 µM in experiment 1; 12.29 µM in experiment 2). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20 % (11.1 % in experiment 1; 12.7 % in experiment 2).

Table 1: Results of the Cytotoxicity Measurement

	Concentration [µM]	Cell viability [%]
		Experiment 1	Experiment 2	Mean	SD
Solvent control	-	100	100	100	0.0
Positive control	4.00	95.5	98.9	97.2	2.4
	8.00	104.9	102.4	103.7	1.8
	16.00	120.2	104.5	112.4	11.1
	32.00	119.6	112.8	116.2	4.8
	64.00	123.6	119.1	121.3	3.2
Test item	0.98	113.6	86.8	100.2	19.0
	1.95	90.3	82.7	86.5	5.4
	3.91	95.7	86.8	91.3	6.3
	7.81	100.8	88.0	94.4	9.1
	15.63	95.0	87.0	91.0	5.7
	31.25	82.3	85.1	83.7	2.0
	62.50	87.0	85.5	86.3	1.0
	125.00	79.2	78.4	78.8	0.6
	250.00	69.8	65.1	67.5	3.3
	500.00	63.9	63.6	63.7	0.2
	1000.00	64.6	50.7	57.7	9.8
	2000.00	64.9	42.9	53.9	15.5

 

Table 2: Induction of Luciferase Activity Experiment 1. * = significant induction according to Student’s t-test, p < 0.05.

Experiment 1	Concentration [µM]	Fold induction
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent control	-	1.00	1.00	1.00	1.00	0.0
Positive control	4.00	1.20	1.24	1.11	1.18	0.07
	8.00	1.26	1.36	1.26	1.30	0.06
	16.00	1.53	1.53	1.38	1.48	0.09
	32.00	2.81	2.22	2.12	2.38 *	0.37
	64.00	4.08	4.13	3.53	3.92 *	0.33
Test item	0.98	0.97	1.17	1.08	1.07	0.10
	1.95	1.00	1.02	0.98	1.00	0.02
	3.91	1.03	1.02	0.96	1.01	0.04
	7.81	0.97	1.07	1.01	1.02	0.05
	15.63	0.94	1.00	1.13	1.02	0.10
	31.25	1.02	1.05	1.01	1.02	0.02
	62.50	1.02	1.15	1.10	1.09	0.07
	125.00	1.08	1.16	1.07	1.10	0.05
	250.00	1.12	1.32	1.09	1.18	0.12
	500.00	1.23	1.15	1.06	1.15	0.09
	1000.00	1.27	1.13	1.19	1.20	0.07
	2000.00	1.52	1.64	1.33	1.50	0.16

 

Table 3: Induction of Luciferase Activity Experiment 2. * = significant induction according to Student’s t-test, p < 0.05.

Experiment 1	Concentration [µM]	Fold induction
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent control	-	1.00	1.00	1.00	1.00	0.0
Positive control	4.00	1.03	1.21	1.13	1.12	0.09
	8.00	1.28	1.38	1.30	1.32	0.05
	16.00	1.57	1.75	1.65	1.66 *	0.09
	32.00	2.43	2.71	2.42	2.52 *	0.16
	64.00	3.82	5.53	4.29	4.55 *	0.88
Test item	0.98	1.16	1.16	1.12	1.15	0.02
	1.95	1.02	1.19	1.02	1.08	0.10
	3.91	0.99	1.16	1.02	1.06	0.09
	7.81	1.04	1.21	0.98	1.08	0.12
	15.63	1.06	1.28	0.97	1.10	0.16
	31.25	1.03	1.17	1.10	1.10	0.07
	62.50	0.98	1.19	0.99	1.06	0.12
	125.00	1.04	1.24	0.98	1.09	0.14
	250.00	1.04	1.27	1.01	1.11	0.14
	500.00	1.11	1.31	0.93	1.12	0.19
	1000.00	1.11	1.36	1.08	1.18	0.16
	2000.00	1.17	1.56	1.31	1.35	0.20

 

Table 4: Induction of Luciferase Activity - Overall Induction. * = significant induction according to Student’s t-test, p < 0.05.

Overall induction	Concentration [µM]	Fold induction
		Experiment 1	Experiment 2	Mean	SD
Solvent control	-	1.00	1.00	1.00	0.00
Positive control	4.00	1.18	1.12	1.15	0.04
	8.00	1.30	1.32	1.31	0.02
	16.00	1.48	1.66	1.57 *	0.12
	32.00	2.38	2.52	2.45 *	0.10
	64.00	3.92	4.55	4.23 *	0.45
Test item	0.98	1.07	1.15	1.11	0.05
	1.95	1.00	1.08	1.04	0.05
	3.91	1.01	1.06	1.03	0.04
	7.81	1.02	1.08	1.05	0.04
	15.63	1.02	1.10	1.06	0.06
	31.25	1.02	1.10	1.06	0.05
	62.50	1.09	1.06	1.07	0.02
	125.00	1.10	1.09	1.09	0.01
	250.00	1.18	1.11	1.14	0.05
	500.00	1.15	1.12	1.13	0.02
	1000.00	1.20	1.18	1.19	0.01
	2000.00	1.50	1.35	1.42	0.11

 

Table 5: Additional Parameters; n.a. = not applicable

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5 [µM]	n/a	n/a	n/a	n/a
Imax	1.50	1.35	1.42	0.11
IC30 [µM]	247.61	204.36	225.99	30.59
IC50 [µM]	n/a	1093.87	n/a	n/a

 

Table 6: Acceptance criteria

Criterion	Range	Experiment 1	Pass/fail	Experiment 2	Pass/fail
CV Solvent Control	< 20 %	11.1	pass	12.7	pass
No. of positive control concentration steps with significant luciferase activity induction > 1.5	≥1	2.0	Pass	3.0	Pass
EC1.5 PC	7 < x < 34 µM	16.35	Pass	12.29	Pass
Induction PC at 64 µM	2 < x < 8	3.92	pass	4.55	pass

 

Table 7: Historical data

Acceptance criterion	Range	Mean	SD	n
CV Solvent Control	< 20 %	11.3	3.3	41
No. of positive control concentration steps with significant luciferase activity induction > 1.5	≥1	2.3	0.6	41
EC1.5 PC	7 < x < 34 µM	20.4	6.7	41
Induction PC at 64 µM	2 < x < 8	3.3	1.1	41

Interpretation of results:

other: Expert statement

Remarks:

Negative. No indication of sensitisation.

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non-sensitiser.

Executive summary:

In this guideline study according to OECD 442d (adopted: February 04, 2015), the skin sensitising potential of N,N'-Methylenebis[methacrylamide] was assessed via its potential to induce the Keap1-Nrf2-ARE signalling pathway followed by quantification of luciferase gene expression.

Cells were incubated with N,N'-Methylenebis[methacrylamide] for 48 h at 37 °C at the following concentrations [µM]: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 and 0 (solvent control). Afterwards, the test substance was removed, the cells were lysed and luminescence subsequently measured with a plate reader. Besides the luminescence the cell viability was measured using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay method.

The KeratinoSens^TM assay is considered to provide positive results if the following conditions are all met in two of two independent experimental repetitions:

- I_max is > 1.5 fold increased and statistically significant (p < 0.05) compared to the negative control

- cell viability is > 70 % at the lowest concentration with an induction of luciferase activity > 1.5

- EC_1.5 value is < 1000 µM

- there is an apparent overall dose-response for luciferase induction

In the first experiment, a max luciferase activity (I_max) induction of 1.50 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 64.9 %. No significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5value could be calculated. In the second experiment, a max luciferase activity (I_max) induction of 1.35 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 42.9 %. No significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5value could be calculated.

Thus, in two independent experiments, no dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

The controls confirmed the validity of the study. The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (3.92 in experiment 1; 4.55 in experiment 2). The calculated EC_1.5was between 7 and 34 µM (16.35 µM in experiment 1; 12.29 µM in experiment 2). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20 % (11.1 % in experiment 1; 12.7 % in experiment 2).

In this study under the given conditions, N,N'-Methylenebis[methacrylamide] did not induce the luciferase activity in the transgenic KeratinoSens^TM cell line in at least two independent experiment runs. Therefore, the test item can be considered as non-sensitiser. However, the data generated with this method may be not sufficient to conclude definitely on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Reference 3

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

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

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.

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”.

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.

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

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

The skin sensitising potential of N,N´-Methylenebis[methacrylamide] was examined in three OECD guideline studies, an in chemico study (Direct Peptide Reactivity Assay (DPRA), OECD 442C), an in vitro approach measuring activation of the ARE-Nrf2 pathway in human keratinocytes (KeratinoSens^TM, OECD 442D) and the in vitro human cell line activation test (h-CLAT) according to OECD guideline 442E.

Firstly, in the in chemico approach (Direct Peptide Reactivity Assay), the 100 mM stock solution of N,N´-methylenebis[methacrylamide] showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38 %. Co-elution of test item with the cysteine peptide peak was observed. In the framework of an IATA the test substance may be considered as non-sensitiser to skin in accordance with UN GHS “No Category” if the mean depletion of both peptides is below 6.38 %. However, due to the observed co-elution of test item with the cysteine peptide peak, the obtained negative result may reflect an underestimation of the reactivity. Therefore, a conclusion on the lack of reactivity could not be drawn with sufficient confidence and a prediction could not be made.

In an in vitro OECD guideline study using the KeratinoSens^TM model under the given conditions, N,N´-methylenebis[methacrylamide] did not induce the luciferase activity in the transgenic KeratinoSens^TM cell line in at least two independent experiment runs. Therefore, the test item can be considered as non-sensitiser according to this assay.

In the in vitro human cell line activation test (h-CLAT) according to OECD guideline 442E, the test item showed no upregulation of the cell surface markers CD54 and CD86 in at least two of three independent experiments when tested up to 80.0 µg/mL. No cytotoxicity was observed under the tested concentrations.

The Direct Peptide Reactivity Assay (DPRA), the KeratinoSens^TM model and the in vitro human cell line activation test (h-CLAT) and are validated test methods for the assessment of skin sensitisation which have not been developed as stand-alone test methods, but to be used in a Weight-of-Evidence approach. When used in an AOP-based IATA under consideration of ENV/JM/MONO(2016)29, the outcome of these studies targets key events along the defined toxicity pathway and the results enable a regulatory decision.

Taken together, N,N´-Methylenebis[methacrylamide] is considered to be a non-sensitiser.