Registration Dossier

Administrative data

Description of key information

- positive in the DPRA, study conducted according to OECD TG 442 C, GLP

- positive in the KeratinoSens assaysensitizer, study conducted according to OECD TG 442 E, GLP

- positive in the h-CLAT, study conducted according to OECD TG 442 D, GLP

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-04-03 to 2017-08-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
adopted February 04, 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Details on study design:
Skin sensitisation (In chemico test system)
- Details on study design:
The in chemico direct peptide reactivity assay enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine. The test item was dissolved in acetonitrile at a concentration of 100 mM and the solutions were tested by incubating the samples with the peptides containing either lysine or cysteine (0.667 mM each in the stock solution and incubated in a 1:50 and 1:10 ration, respectively) for 24 ± 2 h at 25 ± 2.5 °C. Subsequently the samples were analysed by HPLC. Cinnamic aldehyd was used as a positive control, The test item solution without peptides was also used as a co-elution control. Three reference control samples were used to determine the accuracy of the calibration curve (acetonitrile; Control A), the stability of the respective peptide over analysis time (Control B) and to verify the impact of the solvent on PPD (acetonitrile with test item or positive control; Control C).
If co-elution of the test item occurrs with the peptide peak, the peak cannot be integrated correctly and the calculation of the PPD is not possible.

Cysteine peptide: AC-RFAACAA-COOH
Lysine peptide: AC-RFAAKAA-COOH
Number of replicates: triplicate; co-elution control samples: two samples per peptide, i.e. one for the test item solvent and one for the positive control solvent
- Co-elution control with cysteine peptide: 50 μL of test item or the positive control formulation was incubated with 750 μL of cysteine peptide dilution buffer (without cysteine peptide) and 200 μL of acetonitrile.
- Co-elution control with lysine peptide: 250 μL of test item or the positive control formulation was incubated with 750 μL of peptide stock solution (without lysine or cysteine peptide).

- Reference control A and B samples: 250 µL Acetonitrile were added to 750 µL of peptide solution (cysteine or lysine).
- Reference control C prepared with cysteine peptide: 750 µL peptide stock solution were incubated with 200 µL Acetonitrile and 50 µL Test item solvent.
- Reference control C prepared with lysine peptide: 750 µL peptide stock solution were incubated with 250 µL Test item solvent.
- Reactivity of cinnamaldehyde with cysteine peptide: 50 μL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 μL of cysteine peptide solution and 200 μL of acetonitrile.
- Reactivity of cinnamaldehyde with lysine peptide: 250 μL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 μL of lysine peptide solution.
- Reactivity of test item with cysteine peptide: 50 μL of test item formulation was incubated with 750 μL of cysteine peptide solution and 200 μL of acetonitrile.
- Reactivity of test item with lysine peptide: 250 μL of test item formulation was incubated with 750 μL of lysine peptide solution.

All Solutions were inspected for precipitation, turbity or phase separation prior to HPLC analysis.
If a precipitate or phase separation was observed after the reaction period and prior to the HPLC analysis, samples might have been centrifuged at
low speed (100 - 400x g) to force precipitates to the bottom of the vial. After the incubation period of 24 ± 2 h the test item was analysed in triplicate for both peptides using the following HPLC procedure.

The concentration of the cysteine and lysine peptide was determined in each sample from the absorbance at λ= 220 nm, measuring the area of the appropriated peaks and calculating the concentration of peptide using the linear calibration curves derived from the standard solutions. The percent peptide depletion ((PPD) was calculated. The test item is considered positive to be a sensitiser in accordance with UN GHS "Category 1", if the mean depletion of both peptides exceeds the threshold of the respective prediction model.
Positive control results:
Positive control:
Prediction model 2 (Cysteine Peptide/Test item Ratio:1:10)
Mean Peptide Depletion [%]: 70.26
SD of Peptide Depletion [%]: 0.23
Key result
Parameter:
other: Mean Peptide Depletion prediction model 2 (Cysteine Peptide/Test item ratio: 1:10)
Run / experiment:
cysteine peptide
Value:
96.64
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
Mean depletion 70.26 %
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible effects on test system: Determination of precipitation, turbity and phase separation for each replicate after the 24 h incubation time, Precipitation was observed for the test item samples and for the positive control in the dilution with the cysteine peptide, Slight turbity was observed for the test item samples and phase separation was observed for the positive and the co-elution control in the dilution with the lysine peptide. Samples were not centrifuged before HPLC.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed precipitation and turbity were regarded as insignificant.

ACCEPTANCE OF RESULTS:

- Acceptance criteria met for positive control:
yes, mean depletion of the positive control: 60.8% < x < 100% for cysteine, Value: 70.26%; 40.2% < x < 69%, Value: 59.36%
- Acceptance criteria met for variability between replicate measurements:
yes, CV < 15%, Value: 1.43 for cysteine and 0.39 for lysine

Categorization of 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

n.a.

n.a.

n.a.

96.64

Moderate reactivity

sensitizer

Positive control

64.81

High reactivity

sensitizer

70.26

Moderate recativity

sensitizer

In the present study m-Xylylenebismaleimide was given into acetonitrile, based on the results of the pre-experiments. The test item was completely soluble and the resulting solution was used for further testing.

For the 100 mM 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 precipitation was observed for the samples of the test item and samples of the positive control (excluding the co-elution control). Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM 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, slight turbidity was observed for the samples of the test item. Phase separation was observed for the samples of the positive control and the respective co-elution control. Samples were not centrifuged prior to HPLC analysis. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed precipitation and turbidity were regarded as insignificant. Co-elution of test item with the lysine peptide peak was observed. The sensitising potential of the test item was predicted from the mean peptide depletion of the cysteine peptide by comparing the peptide concentration of the test item treated samples to the corresponding reference control C. The 100 mM stock solution of the test item showed moderate reactivity towards the synthetic peptides. The mean depletion of both peptides was > 13.89% (96.64%). Based on the prediction model 2 the test item can be considered as sensitiser. Since a minor co-elution in the range of the cysteine peptide peak was observed, the obtained values for the peak areas are not considered to reflect the actual remaining cysteine peptide content and the true peptide depletion is suspected to be higher.

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 70.26%.

Interpretation of results:
other: positive in the DPRA
Conclusions:
In the present study conducted according to OECD guideline 442C, the sensitising potential of m-Xylylenebismaleimide was determined using the in chemico DPRA Test. This test is used to determine the reactivity of the test item with distinct cysteine and lysine peptides. Covalent binding of the test item results in a peptide depletion which can be measured by HPLC. Under the present test conditions m-Xylylenebismaleimide caused a peptide depletion with the cytsteine peptides and was therefore considered to be positive in the DPRA.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-04-21 to 2017-11-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
adopted February 04, 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Details on study design:
Skin sensitisation (In vitro test system) ARE-Nrf-2 Luciferase Test Method (KeratinoSens™)
- Details on study design:
Cells were cultured in 75 cm² culture flasks (Greiner) in maintenance medium at 37 ± 1°C and 5% CO2. 1E04 cells/well were seeded in either white (luciferase-measurement) or transparent (MTT-assay) 96-well plates for 24 h ± 1 h in assay medium at 37°C ± 1°C and 5% CO2. After 24 h the medium was discarded and replaced by 150 µL test item exposure medium. The following concentration range of the test item was tested in the assay: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM and for the positive control (Cinnamic aldehyde; CAS 104-55-2; > 98%; Sigma; Lot No.: MKBS2662V) the following concentrations were measured: 4.0, 8.0, 16.0, 32.0 and 64.0 µM. The assay was performed in three replicates in two independent experiments.
All plates were sealed and incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.

- Luciferase measurement:
After the incubation period (48 h ± 1 h) the medium was aspirated and the cells were washed once with DPBS.
Experiment 1:
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 for each well.
Experiment 2:
For experiment 2 after addition of 20 µL of passive lysis buffer into each well, the plate was incubated for approximately 2h at room temperature in the absence of light. Per well 50 µL of the luciferase substrate were pipetted using a multichannel pipette. Subsequently
pates were placed in the plate reader for luminescence measurement. The plate reader waited for 1.000 ms before assessing the luciferase activity for 2.000 ms for each 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% CO 2 overnight (experiment 2) or over the weekend (experiment 1). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.

- 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 EC 1.5 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.

- Controls:
A blank, a negative control and a positive control were set up in parallel in order to confirm the validity of the test.
Blank
A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.
Negative Control
DMSO (AppliChem; Lot No.: 000978834) at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.

Positive Control
Cinnamic aldehyde (CA, (2E)-3-phenylprop-2-enal; CAS 104-55-2; >98%; Sigma; Lot No.: MKBS2662V) was used as positive control. CA was dissolved in DMSO (AppliChem; Lot No.: 0000978834) at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 µM – 64 µM. The final concentration of the solvent DMSO was 1% (v/v) for all wells.

- 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
- EC 1.5 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.
Positive control results:
The positive control (cinnamic aldehyde) values are considered to be valid because they are within the acceptability range demanded by the OECD test guideline 442d and the determined historical data (see also Table 1).
Key result
Parameter:
other: EC1.5
Remarks:
µM
Run / experiment:
Experiment 1
Value:
4.09
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: EC1.5
Remarks:
µM
Run / experiment:
Experiment 2
Value:
2.13
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

Table 2: Cell viability

 

Concentration [µM]

Cell viability [%]

Experiment 1

Experiment 2

Mean

N

Solvent control

 -

100.0

100.0

100.0

0.0

Positive control

4.0

92.5

112.8

102.7

14.4

8.0

99.0

112.9

106.0

9.9

16.0

93.9

116.2

105.0

15.8

32.0

91.4

119.4

105.4

19.8

64.0

85.0

129.8

107.4

31.6

Test item

0.98

106.2

126.3

116.2

14.2

1.95

127.9

117.4

122.7

7.4

3.91

150.0

148.3

149.2

1.3

7.81

151.9

87.8

119.8

45.3

15.63

70.8

5.5

38.1

46.2

31.25

11.3

-0.1

5.6

8.1

62.50

0.4

-0.1

0.2

0.3

125.00

0.3

-0.1

0.1

0.3

250.00

0.3

0.0

0.2

0.2

500.00

0.3

0.1

0.2

0.1

1000.00

0.3

0.3

0.3

0.0

2000.00

0.0

0.7

0.3

0.5

Table 3: Induction of Luciferase Activity Experiment 1

Experiment 1

Concentration [µM] 

Fold Induction

Significance

Rep. 1

Rep. 2

Rep. 3

Mean

SD

Solvent Control

-

1

1

1

1

0

 

Positive Control

4

1.07

1.21

1.03

1.1

0.1

 

8

1.2

1.31

1.5

1.34

0.15

 

16

1.31

1.34

1.39

1.35

0.04

 

32

1.43

1.98

2.32

1.91

0.45

*

64

2.71

4.17

2.9

3.26

0.79

*

Test Item

0.98

1.66

0.88

0.99

1.18

0.42

 

1.95

1.46

1.19

0.85

1.17

0.3

 

3.91

1.4

1.45

1.36

1.4

0.05

 

7.81

2.78

4.09

3.63

3.5

0.67

 

15.63

2.68

4.05

3.5

3.41

0.69

 

31.25

0.23

0.94

0.54

0.57

0.36

 

62.5

0

0

0

0

0

*

125

0

0

0

0

0

 

250

0

0

0

0

0

 

500

0

0

0

0

0

 

1000

0

0

0

0

0

 

2000

0

0

0

0

0

 

Table 4: Induction of Luciferase Activity Experiment 2

Experiment 2

Concentration [µM]

Fold induction

Significance

 

Rep. 1

 Rep. 2

 Rep. 3

Mean

 SD

 

Solvent Control

-

1

1

1

1

0

 

Positive Control

4

1.2

1.16

1.28

1.21

0.06

 

8

1.19

1.43

1.2

1.27

0.14

 

16

1.42

1.36

1.41

1.4

0.04

 

32

1.37

1.52

1.47

1.45

0.08

 

64

2.71

3.39

2.95

3.02

0.34

*

Test Item

0.98

1.2

1.11

1.19

1.17

0.05

 

1.95

1.41

1.16

1.61

1.39

0.22

 

3.91

2.88

2.09

2.68

2.55

0.41

*

7.81

8.25

6.82

6.99

7.35

0.78

*

15.63

0.37

2.39

0.34

1.03

1.18

 

31.25

0.00

0.00

0.00

0.00

0.00

 

62.5

0.00

0.00

0.00

0.00

0.00

 

125

0.00

0.00

0.00

0.00

0.00

 

250

0.00

0.00

0.00

0.00

0.00

 

500

0.00

0.00

0.00

0.00

0.00

 

1000

0.00

0.00

0.00

0.00

0.00

 

2000

0.00

0.00

0.00

0.00

0.00

 

Table 5: Induction of Luciferase Activity – Overall Induction

 

Concentration [µM] 

Fold Induction

 

Significance

Experiment 1

 Experiment 2

Mean

SD

 

Solvent Control

-

1.00

1.00

1.00

0.00

 

Positive Control 

4

1.10

1.21

1.16

0.08

 

8

1.34

1.27

1.31

0.05

 

16

1.35

1.40

1.37

0.04

 

32

1.91

1.45

1.68

0.32

 

Test Item

64

3.26

3.02

3.14

0.17

*

0.98

1.18

1.17

1.17

0.01

 

1.95

1.17

1.39

1.28

0.16

 

3.91

1.40

2.55

1.98

0.81

 

7.81

3.50

7.35

5.43

2.72

 

15.63

3.41

1.03

2.22

1.68

 

31.25

0.57

0.00

0.29

0.40

 

62.5

0.00

0.00

0.00

0.00

 

125

0.00

0.00

0.00

0.00

 

250

0.00

0.00

0.00

0.00

 

500

0.00

0.00

0.00

0.00

 

1000

0.00

0.00

0.00

0.00

 

2000

0.00

0.00

0.00

0.00

 

Table 6: Acceptance Criteria

Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

CV Solvent Control

< 20%

17.8

pass

10.6

pass

No. of positive control concentration steps with significant luciferase activity induction >1.5

≥ 1

2

pass

1

pass

EC1.5 PC

7 < x < 34 µM

20.35

pass

32.99

pass

Induction PC at 64 µM

2.00 < x < 8.00

3.26

pass

3.02

pass


Interpretation of results:
other: positive in the KeratinoSens Assay
Conclusions:
In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered positive in the KeratinoSens Assay.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-04-03 to 2017-10-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD guideline 442E (In vitro Skin sensitisation: human Cell Line Activation Test (hCLAT))
Version / remarks:
adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of dendritic cells
Details on study design:
Skin sensitisation (In vitro test system)
- Details on study design:
- Preliminary Test:
Solvent finding: Insolubility of the test item in 0.9% NaCl and cell culture medium at a concentration of 100 mg/mL. Insolubility in DMSO at 500 mg/mL, stepwise dilution. Solubility was achieved in DMSO at 281.25 mg/mL.
Dose Finding: Starting from 281.25 mg/mL (in DMSO) solutions of the test chemicals, eight stock solutions (eight concentrations, 281.25, 140.63, 70.31, 35.16, 17.56, 8.79, 4.39, and 2.20 µg/mL ) were prepared, by 2-fold serial dilutions using the corresponding solvent. These stock solutions were further diluted 250-fold (DMSO) into culture medium (working solutions). The working solutions were finally used for treatment by adding an equal volume of working solution to
the volume of THP-1 cell suspension to achieve a further 2-fold dilution. Cell were seeded at a density of 1E06 cells/mL and incubated with the test item for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2. After the incubation period the cells were centrifuged and washed with Dulbecco´s phosphate buffered saline containing 0.1% bovine serum albumin, provided with propidium iodide and analysed for viability by measurement of PI uptake using FACS analysis. The CV75 value, i.e. the concentration showing 75% cell survival, was calculated by log-linear
interpolation. The CV75 value was used to calculate the concentration range of the test item for the main experiment.

CD54 and CD86 Expression
The test item was dissolved using DMSO as determined in the pre-experiment. Based on the
concentration of the pre-determined CV75 value 8 concentrations of the test item were defined for
the measurement of the surface marker expression, corresponding to 1.2*CV75; CV75; CV75/1.2; CV75/1.2²; CV75/1.2³; CV75/1.2E+4; CV75/1.2E+5; CV75/1.2E+6. If the CV75 could not be determined due to insufficient cytotoxicity of the test item in the dose finding assay, the highest soluble concentration
of the test item prepared with each solvent was used as starting dose.
The test item was diluted to the concentration corresponding to 500-fold (for DMSO) of the 1.2 × CV75. Then, 1.2-fold serial dilutions were made using the corresponding solvent to obtain the 8 stock solutions to be tested. The stock solutions were further diluted 250-fold (for DMSO) into the culture medium (working solutions). These working solutions were finally used for cell treatment with a further final 2-fold dilution factor. Alternative concentrations were used upon justification (poor solubility and cytotoxicity). For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of
0.1 – 0.2E+6 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation (125 x g) and were re-suspended in fresh culture medium at a density of 2E+6cells/mL. Then, 500 µL of the cell suspension were seeded into a 24 well flat-bottom plate (1E+6 cells/well).
The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell
suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO 2. After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The following steps were carried out on ice with pre-cooled buffers and solutions. The supernatant was discarded and the remaining cells were washed twice with FACS buffer. After washing, cells were blocked using 600 µL of a FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction) and incubated at 4 °C for 15 min. After blocking, cells were split in three aliquots into a 96-well V-bottom plate. After centrifugation (approx. 250 x g), cells were stained with 50 µL of FITC-labelled anti-CD86 (BD, art. no. 555657, Lot No.: 6036848 ), anti-CD54 (BioLegend, art. no.: 353108, Lot No.: B214036) or mouse IgG1 (isotype, BioLegend, art.
no.: 400110, Lot No.:B206037) antibodies in the dark for 30 min. All antibodies were diluted in FACS buffer at an appropriate manner. After washing with FACS buffer two times, cells were re-suspended in FACS buffer and PI solution was added. PI staining was done just prior to the
measurement by adding PI solutions to each sample (final concentration of PI was 0.625 µg/mL). The expression levels of CD86 and CD54 as well as cell viability were analysed by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ = 530 nm ± 15 nm for FITC and λ > 650 nm for PI. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 were calculated

- Controls:
1. Solvent Control: 0.2% DMSO (v/v) in cell culture medium, 2. Positive Control: 4 µg/mL DNCB
- Prediction Model:
The correlation of upregulation of immunological relevant cell surface markers with the skin sensitising potential of a chemical has been reported and represents the third key event in the skin sensitisation process as described by the AOP. This method that measures the markers of DC activation, based on DC-like cell line THP-1 is considered relevant for the assessment of the skin sensitisation potential of chemicals. The expression level of CD86 and CD54 following exposure to test chemicals are used for supporting the discrimination between sensitisers and non-sensitisers. Sensitising potential of the test item was predicted from the mean percentage expression of CD86 and CD54. Any test chemical tested by the h-CLAT was considered positive if the RFI of CD86 was equal to or greater than 150% at any tested dose at a cell viability ≥ 50% in at least two independent runs or if the RFI of CD54 was equal to or greater than 200% at any tested dose at a cell viability ≥ 50% in at least two independent runs or if the RFIs of both the CD86 and CD54 were equal to or are greater than 150% and 200% respectively at any tested dose at a cell viability ≥ 50% in at least two independent runs. In case of not concordant results a third run should be conducted to make the final prediction. Otherwise the results were considered as inconclusive. A negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is <90%. In contrast, a positive test outcome was accepted irrespective of cell viabilities >90% at a concentration of 1.2 x CV75. If no CV75 could be derived negative test results can be accepted when the test item is tested at the highest soluble concentration (5000 µg/mL for 0.9% NaCl solution; 1000 µg/mL for DMSO or a different organic solvent) even if the cell viability is >90%.

- Acceptance criteria: The test meets acceptance criteria if:
- the cell viability of the solvent controls is >90%,
- the cell viability of at least four tested doses of the test item in each run is >50%,
- the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
- the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
- the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%.

Positive control results:
The positive controls DNCB and NiSO4 led to upregulation of the cell surface markers CD54 and CD86.

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold values of 150% for CD86 (390% experiment 1; 344% experiment 2) and 200% for CD54 (417% experiment 1; 410% experiment 2) were clearly exceeded.
Key result
Parameter:
other: RFI of CD 86
Remarks:
at 2.90, 3.48 and 4.18 µg/mL
Run / experiment:
#1
Value:
> 150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: RFI of CD54
Remarks:
at any tested concentration with cell viability ≥ 50%
Run / experiment:
#1
Value:
< 200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: RFI of CD 86
Remarks:
at 2.90, 3.48 and 4.18 µg/mL
Run / experiment:
#2
Value:
> 150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: RFI of CD54
Remarks:
at all tested concentrations with a cell viability > 50%
Run / experiment:
#2
Value:
< 200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
Reactivity Check of the Cell Stock
Doubling time of the cells was monitored and found to be 43.9 h which is within the doubling time range specified by the manufacturer (35 - 50 h). The positive controls DNCB and NiSO 4 led 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.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, cell viability of the solvent controls was >90% in both experiments (Exp. 1: 97.1 - 97.7; Exp. 2: 95.0 - 95.8), The the RFI values of the solvent control was not ≥150% for CD86 (Exp. 1: 102; Exp. 2: 107) and not ≥200% for CD54 (Exp. 1: 97; Exp. 2: 99)
- Acceptance criteria met for positive control: yes, the RFI values of the positive control (DNCB) is ≥150% for CD86 (Exp. 1: 390; Exp. 2: 344) and ≥200% for CD54 (Exp. 1: 417; Exp. 2: 410) at a cell viability of >50%
- Acceptance criteria met for variability between replicate measurements: not applicable
- Range of historical values if different from the ones specified in the test guideline: see table

- test chemicals exhibiting a cell viability of less than 90% at the highest concentration tested
- the cell viability was more than 50% in at least four tested concentrations in each run

Table 1: Results of the Cell Batch Activation Tes

Sample

Concentration [µg/mL]

CD86

 

CD54

 

Activated

Cell Viability [%]

 

RFI

Cell Viability [%]

 

RFI

yes/no

DNCB

4 µg/mL

91.6

 

376

91.4

 

476

Yes

NiSO4

100 µg/mL

89.8

 

239

89.9

 

506

Yes

LA

1000 µg/mL

96.9

 

75

97.1

 

80

No

Table 2: Results of the Dose Finding Assay

Sample

Experiment 1

Experiment 2

Concentration applied

[µg/mL]

Cell Viability [%]

Concentration applied

[µg/mL]

Cell Viability [%]

Medium Control

0.00

97.80 

0.00 

97.40 

DMSO Control 

0.00

98.00 

0.00 

97.50 

Compimide®MXBI (m-

Xylylenebismaleimide)

4.39

8.79

17.58

35.16

70.31

82.30 

31.50 

24.10 

26.90 

26.50 

4.39 

8.79 

17.58 

35.16 

70.31 

85.00 

19.50 

4.40 

5.40 

8.30 

 

140.63

17.20 

140.63 

9.60 

 

281.25

15.80 

281.25 

11.00 

 

562.50

10.80 

562.50 

4.50 

Calculated CV75 [µg/mL]

4.85

4.89

Mean CV75 [µg/mL]

4.87

SD CV 75 [µg/mL]

0.02

Table 3: CD54 and CD86 Expression Experiment 1

Sample

Conc. [μg/mL]

Cell Viability [%] 

Mean Fluorescence Intensity 

corrected Mean Fluorescence Intensity 

Relative Fluorescence Intensity (RFI)

Ratio Isotype IgG1 to [%]

CD86 CD54 Isotype IgG1

CD86

CD54 Isotype IgG1

CD86

CD54

CD86

CD54

CD86

CD54

Medium Control

-

97.5

97.1

97.2

970

251

444

526

251

98

103

218

157

DMSO Control 

0.20%

97.7

97.3

97.1

967

244

432

535

244

100

100

224

156

DNCB

4

86.2

86.2

86.4

2608

1018

522

2086

1018

390

417

500

295

Compimide® MXBI (m-Xylylenebis maleimide)

10.39

2.8

2.9

2.9

1830

529

983

847

529

158

217

186

154

8.66

14.1

13.4

14

1623

459

696

927

459

173

188

233

166

7.22

34

34.5

34.6

1544

307

645

899

307

168

126

239

148

6.01

70.6

71.3

72.5

1097

150

567

530

150

99

61

193

126

5.01

75.7

74.2

73.8

1316

231

535

781

231

146

95

246

143

4.18

72.1

71.6

70

1397

404

541

856

404

160

166

258

175

3.48

82.3

83

82.1

1831

369

543

1288

369

241

151

337

168

2.9

89.3

89.9

89.4

1968

420

553

1415

420

264

172

356

176

Table 4: 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 IgG

CD86

 CD54

Isotype IgG

CD86

CD54

CD86

CD54

C86

CD54

Medium Control

-

95.7

95.6

95.8

1088

684

394

694

290

93

101

276

174

DMSO Control

0.20%

95.6

95

95

1166

707

420

746

287

100

100

278

168

DNCB

4

87.6

87.8

87.1

3032

1643

466

2566

1177

344

410

651

353

Compimide®

MXBI (m-

Xylylenebis maleimide) 

10.39

8.2

2.2

1.6

5075

3444

1288

3787

2156

508

751

394

267

8.66

13.5

9.1

8.8

2357

1300

706

1651

594

221

207

334

184

7.22

27.8

23.9

23.6

2328

1214

639

1689

575

226

200

364

190

6.01

53.9

52.8

52.9

1945

1020

567

1378

453

185

158

343

180

5.01

74.7

73.2

73.8

1417

790

502

915

288

123

100

282

157

4.18

74.2

75.1

74.6

1653

834

486

1167

348

156

121

340

172

3.48

79.3

78.7

77.3

1776

865

488

1288

377

173

131

364

177

2.9

87.7

88.6

88.6

1777

886

483

1294

403

173

140

368

183

Table 5: Acceptance Criteria

Acceptance Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

cell viability solvent controls [%]

number of test dosed with viability >50% CD86

>90

97.1 - 97.7

pass

95.0 - 95.8

pass

≥4

5

pass

5

pass

number of test dosed with viability >50% CD54

≥4

5

pass

5

pass

number of test dosed with viability >50% IgG1

≥4

5

pass

5

pass

RFI of positive control of CD86

≥150

390 

pass

344 

pass

RFI of positive control of CD54

≥200

417 

pass

410 

pass

RFI of solvent control of CD86

<150

102

pass

107

pass

RFI of solvent control of CD54

<200

97

pass

99

pass

MFI ratio IgG1/CD86 for medium control [%]

>105

218

pass

276

pass

MFI ratio IgG1/CD86 for DMSO control [%]

>105

224

pass

278

pass

MFI ratio IgG1/CD54 for medium control [%]

>105

157

pass

174

pass

MFI ratio IgG1/CD54 for DMSO control [%]

>105

156

pass

168

pass

Table 6: 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: positive in the h-CLAT
Conclusions:
In this study conducted according to OECD guideline 442E (2016) the test item induced upregulation of one cell surface marker (CD86) in two independent experiments. Therefore, the test item is considered to be positive in the h-CLAT.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

- DPRA:

In an in chemico skin sensitisation assay conducted according to OECD guideline 442C, adopted February 04, 2015,the reactivity of m-Xylylenemaleimide was evaluated by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides.

The test item was dissolved at 100 mM in acetonitrile. The positive control was cinnamaldehyde, and for each peptide, the analytical batch included co-elution control samples and three reference control samples.

Reactivity (%depletion) was determined following 24-hour contact between test item and peptide in acetonitrile at the ratios 1:10 cysteine:test item and 1:50 lysine:test item by HPLC with UV-detection.

Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples. The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied and thus demonstrated the validity of the study.

Analysis of the chromatograms of the co-elution samples indicated that co-elution occured with the lysine and marginally with the cysteine peptides. As a result, the mean percent depletion values were calculated for the cysteine peptide. For the cysteine peptide, the mean depletion value was 96.64%.

Since the mean of the percent cysteine depletions was above 42.47%, the test item was considered to have a high peptide reactivity. Therefore, the DPRA prediction was considered positive and m-Xylylenemaleimide may cause skin sensitisation.

- hCLAT:

In a study conducted according to OECD test guideline 442E (adopted 29 July 2016) THP-1 cells were incubated with m-Xylylenemaleimide dissolved in DMSO.

Based on the CV75, the main experiment was performed covering the following concentration steps:10.39, 8.66, 7.22, 6.01, 5.01, 4.18, 3.48, 2.90 µg/mL.

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.

Severe cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 2.8% (CD86), 2.9% (CD54) and 2.9% (isotype IgG1 control) in the first experiment and to 8.2% (CD86), 2.2% (CD54) and 1.6% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was upregulated above the threshold of 150% in experiment 1 at 7.22 µg/mL and in experiment 2 at 6.01 µg/mL. The expression of cell surface marker CD54 was upregulated above the threshold of 200% in experiment 1 at the highest dose (10.39 µg/mL) and in experiment 2 at 7.22 µg/mL.

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (417% experiment 1; 344% experiment 2) and 200% for CD54 (500% experiment 1; 410% experiment 2) were clearly exceeded. Thus, based on the present results the test item would be considered to exhibit sensitising potential.The data generated with this test should be considered in the context of integrated approached 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.

- KeratinoSens

In a study conducted according to OECD test guideline 442d (adopted February 04, 2015) transgenic keratinocytes constitutively expressing an ARE-reporter gene were incubated with m-Xylylenebismaleimide at concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 and 0 µM (solvent control) for 48 h at 37°C. Afterwards the test substance containing medium was removed and the cells lysed and luminescence subsequently measured with a plate reader for 2.000 ms. Beside the luminescence the cell viability was measured using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium  bromide) assay method. The KeratinoSens assay is considered to provide positive results if the following conditions are all met in two of two independent experimental repetitions:

- the  Imax   is  higher  than  (>)  1.5  fold  and  statistically  significant  different  as  compared  to  the solvent (negative) control (as determined by a two-tailed, unpaired Student’s T-test);

- the  cellular  viability  is  higher  than  (>)  70%  at  the  lowest  concentration  with  induction  of luciferase activity above 1.5 fold (i.e. at the EC 1.5  determining concentration);

- the EC 1.5  value is less than (<) 1000 µM (or < 200  µg/mL for test chemicals with no defined MW);

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

In the first experiment, a max luciferase activity (Imax) induction of 3.50 was determined at a test item concentration of 7.81 μM. The corresponding cell viability was 151.9%.

At lower concentrations no further  significant  luciferase  induction  >1.5  was  found.  The  calculated  EC 1.5  was  < 1000 µM (4.09 µM).

In the second experiment, a max luciferase activity (Imax) induction of 7.35 was determined at a test item concentration of 7.81 μM. The corresponding cell viability was 87.8%.

The  lowest  tested concentration  with  a  significant  luciferase  induction  >1.5  (2.55)  was  found  to  be  3.91  µM.  The corresponding cell viability was >70% (148.3%). The calculated EC1.5 was < 1000 µM (2.13 µM).

A 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 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 (3.26 experiment 1; 3.02 experiment 2). The calculated EC1.5 was between 7 and 34 μM (20.35 experiment 1; 32.99 experiment 2). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20% (7.8% experiment 1; 10.6% experiment 2).

In this study under the given conditions the test item induced the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item is considered to be a 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.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the reliable and relevant data m-Xylylenemaleimide needs to be classified as "sensitizer" "UN GHS Category 1" according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labeling of Chemicals (GHS).