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EC number: 219-102-3 | CAS number: 2359-15-1
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- Short-term toxicity to fish
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Administrative data
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 KeratinoSensTM 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
- 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 - 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.
- 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 - 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 KeratinoSensTM assay is considered to provide positive results if the following conditions are all met in two of two independent experimental 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
- there is an apparent overall dose-response for luciferase induction
In the first experiment, a max luciferase activity (Imax) 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 EC1.5value could be calculated. In the second experiment, a max luciferase activity (Imax) 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 EC1.5value 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 EC1.5was 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 KeratinoSensTM 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.
- 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 - 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.
Referenceopen allclose all
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 |
Peptide Concentration [mM] |
Peak Area |
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 |
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 |
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 |
Prediction Model 2 |
||||
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 |
In the first experiment, a max luciferase activity (Imax) 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 EC1.5value could be calculated.
In the second experiment, a max luciferase activity (Imax) 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 EC1.5value 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 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).
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 |
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 |
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 (KeratinoSensTM, 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 KeratinoSensTM model under the given conditions, N,N´-methylenebis[methacrylamide] did not induce the luciferase activity in the transgenic KeratinoSensTM 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 KeratinoSensTM 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.
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