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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)

Test material

1
Chemical structure
Reference substance name:
N,N'-(methylenedi-p-phenylene)bis(aziridine-1-carboxamide)
EC Number:
231-034-6
EC Name:
N,N'-(methylenedi-p-phenylene)bis(aziridine-1-carboxamide)
Cas Number:
7417-99-4
Molecular formula:
C19H20N4O2
IUPAC Name:
N,N'-(methylenedi-p-phenylene)bis(aziridine-1-carboxamide)
Test material form:
solid

In chemico test system

Details of test system:
cysteine peptide, (Ac-RFAACAA-COOH)
lysine peptide (Ac-RFAAKAACOOH)
Details on the study design:
PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions:
Stock solutions of Cysteine (Ac-RFAACAA-COOH) and Lysine (Ac-RFAAKAA-COOH) were freshly prepared just before their incubation with the test item. The final concentration of the Cysteine peptide was 0.667 mM in pH 7.5 phosphate buffer whereas the final concentration of the Lysine peptide was 0.667 mM in pH 10.2 ammonium acetate buffer.
- Preparation of the test chemical solutions:
Test item was weighed into clean and sterile vials. Test item was dissolved in 3.0 mL of the 1:1 DMSO: acetonitrile to prepare a 100 mM solution immediately prior to use. The weight of test item to be added in the vial was determined based on the molecular weight (MW) and purity.
- Preparation of the positive controls, reference controls and co-elution controls:
Cinnamic aldehyde was used as positive control (PC) at a concentration of 100 mM in acetonitrile.
Reference control A was prepared with acetonitrile and used to verify the accuracy of the calibration curve for peptide quantification.
Preparation of reference control A (0.5 mM): 750 µL Cysteine/Lysine peptide (0.667 mM) + 250 µL of acetonitrile
Reference control B was prepared with acetonitrile and its replicates were injected in the beginning and in the end of the experimental run to verify the stability of the peptide over the analysis time.
Preparation of reference control B: Same as preparation for reference control A (but run before and after 24 ± 2 hours incubation at 25 ± 2.5 °C in dark).
Reference control C was prepared with the solvent used to solubilize the test items, i.e., 1:1 mixture of DMSO:Acetonitrile and included in every assay run together with the samples. They were used to verify that the solvent does not impact the Percent Peptide Depletion. An appropriate Reference Controls C for each test item was used to calculate Percent Peptide Depletion.
Preparation of reference control C: 750 µL of Cysteine peptide stock + 200 µL of acetonitrile + 50 µL 1:1 mixture of DMSO:Acetonitrile (for Cysteine). 750 µL of Lysine peptide stock + 250 µL of 1:1 mixture of DMSO:Acetonitrile (for Lysine).
Co-elution control was prepared without peptide, to verify whether the test item absorbs at 220 nm and has a similar retention time as a peptide and/or interfere with the data analysis.
Co-elution Control preparation: 750 µL of phosphate buffer (pH 7.5) + 200 µL of acetonitrile + 50 µL test item (Cysteine). 750 µL of ammonium acetate buffer (pH 10.2) + 250 µL of test item (Lysine).

INCUBATION
- Incubation conditions: Cysteine and Lysine peptide solutions were incubated in glass auto sampler vials with the test item at 1:10 and 1:50 ratio, respectively. The reaction solution was left in the dark at 25 ± 2.5 ºC for 24 ± 2 hours before running the HPLC analysis. Test item was analysed in triplicate for both peptides.
- Precipitation noted: The test item was found to be insoluble in acetonitrile, Milli-Q water, isopropanol, acetone, 1:1 acetone: acetonitrile, and 1:9 dimethyl sulfoxide (DMSO): acetonitrile and found to be soluble in 1:1 DMSO: acetonitrile at the concentration of 100 mM. Precipitation was not observed at 100 mM.

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys: A standard calibration curve was generated on each day of HPLC analysis and value of r2 obtained was 0.99987 for Cysteine and 0.99909 for Lysine containing peptides. This showed that system was suitable for assay.

DATA EVALUATION
- Cys and Lys peptide detection wavelength: 220 nm



Vehicle / solvent:
mix DMSO:acetonitrile
Positive control:
cinnamic aldehyde

Results and discussion

Positive control results:
Values of the mean percent peptide depletion value of positive control cinnamic aldehyde was 76% for cysteine peptide and 45% for Lysine peptide.

In vitro / in chemico

Resultsopen allclose all
Key result
Group:
test chemical
Run / experiment:
mean
Parameter:
mean lysine depletion
Value:
1 %
At concentration:
100 mM
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Group:
test chemical
Run / experiment:
mean
Parameter:
mean cystein depletion
Value:
-122 %
At concentration:
100 mM
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
not determinable
Remarks:
(co-elution was observed with cysteine peptide)
Key result
Group:
test chemical
Run / experiment:
mean
Parameter:
other: Mean of lysine and cysteine peptides
Remarks on result:
not determinable
Remarks:
(Mean percent depletion was not calculated since co-elution was observed with cysteine peptide)
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, the 10 proficiency substances recommended in the OECD guideline 442C were performed, obtaining values that fall within the respective reference range for 8 out of the 10 for each peptide.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for calibration curve: yes, coefficient r2 was higher than 0.99 for lysine and cysteine.
- Acceptance criteria met for reference control A: yes, the mean concentration of peptide was 0.50± 0.05 mM
- Acceptance criteria met for reference control B: yes, the CV (coefficient of variation) of the controls B was 1.60% for Cysteine peptide and 3.78% for Lysine peptide which are lower than 15%.
- Acceptance criteria met for reference control C: yes, the mean concentration of peptide was 0.39 mM ((might be due to solvent). The CV (coefficient of variation) of the controls C was 0.66% for Cysteine peptide and 0.71% for Lysine peptide which are lower than 15%.
- Acceptance criteria met for positive control: The depletion mean rate was 76% for cysteine peptide and 45% for lysine peptide which are between 60.8% and 100% for the cysteine and between 40.2% and 69.4% for the lysine.

Any other information on results incl. tables

Mean percent depletion for the test item was not calculated since co-elution was observed with cysteine peptide.

Applicant's summary and conclusion

Interpretation of results:
other: DPRA test result is considered in an overall weight of evidence assessment, i.e. as part of an integrated approach to testing and assessment (IATA) in accordance with OECD guideline 442C.
Conclusions:
The test item cannot be classified as either “sensitizer” or “non-sensitizer” in the DPRA test since its elution was occurred at the same time as that of elution of Cysteine peptide.

Executive summary:

A DPRA skin sensitization test was performed for the test substance as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442C, under GLP conditions. The test item was prepared at 100 mM in 1:1 DMSO: acetonitrile and Cinnamaldehyde (positive control) was prepared at 100 mM in acetonitrile. Reference controls A, B and C were prepared with acetonitrile and 1:1 DMSO: acetonitrile (control C) in order to check the HPLC system suitability, the stability of peptide over time and the influence of the solvent on the peptide depletion respectively. Peptide solutions were prepared at 0.667 mM in phosphate buffer for cysteine and ammonium acetate buffer for lysine. Test item and positive control were incubated for 24 hours ± 2 hours at 25°C±2.5°C with the peptides solutions at 1:10 and 1:50 ratio for cysteine and lysine respectively, before HPLC analysis. Each sample was tested in triplicate for both peptides. All validity criteria were fulfilled. Percent peptide depletion values of the test substance for Cysteine and Lysine were -122% (which shows significant co-elution) and 1%, respectively. Mean percent depletion was not calculated since co-elution was observed with cysteine peptide. Thus, the test substance cannot be classified as either “sensitizer” or “non-sensitizer” and hence the result of DPRA test is considered as “inconclusive”.