1,1'-(benzene-1,3-diyldimethanediyl)bis(1H-pyrrole-2,5-dione)

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

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. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

1-({3-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl]phenyl}methyl)-2,5-dihydro-1H-pyrrole-2,5-dione

EC Number:

695-930-2

Cas Number:

13676-53-4

Molecular formula:

C16H12N2O4

IUPAC Name:

1-({3-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl]phenyl}methyl)-2,5-dihydro-1H-pyrrole-2,5-dione

Test material form:

solid: particulate/powder

In chemico test system

Details on the 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.

Results and discussion

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

In vitro / in chemico

Results

Key result

Run / experiment:

other: cysteine peptide

Parameter:

other: Mean Peptide Depletion prediction model 2 (Cysteine Peptide/Test item ratio: 1:10)

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

Any other information on results incl. tables

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%.

Applicant's summary and conclusion

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.