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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 June 2017 - 04 July 2017
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:
04 February 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Nitrosodiphenylamine
EC Number:
201-663-0
EC Name:
Nitrosodiphenylamine
Cas Number:
86-30-6
Molecular formula:
C12H10N2O
IUPAC Name:
N-nitroso-N-phenylaniline
Test material form:
solid: pellets

In chemico test system

Details on the study design:
The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm.
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 (in the appropriate solvent).

Cysteine peptide (batch 111016HS_MHeW0117, supplier JPT Peptide Technologies GmbH)
Lysine peptide (batch 220114HSDWW0117, supplier JPT Peptide Technologies GmbH)

Vehicle used: Based on solubility results, the retained vehicle was acetonitrile (without sonication step).

Positive control: cinnamaldehyde (CAS No. 104-55-2), batch No. MKBV4784V, supplied by Sigma Aldrich. Its molecular weight was 132.16 g/mol and the purity of the batch used was 98.9%. The positive control was pre-weighed and stored under appropriate conditions until ready to perform testing. It was dissolved in acetonitrile at 100 mM. The formulation was a colorless limpid solution and was used just after its preparation.

Criteria: Interpretation of results
The run was considered valid if the following criteria were fully met:
- the calibration curve should have a coefficient of determination (r2) >= 0.99,
- the mean peptide concentrations of the reference control A samples should be within ± 10% of the nominal concentration,
- the cinnamaldehyde depletion control samples should meet the following acceptance criteria:
- for the cysteine peptide, the mean percent depletion value should be between 60.8 and 100% with a SD < 14.9%,
- for the lysine peptide, the mean percent depletion value should be between 40.2 and 69.0% with a SD < 11.6%,
- the CV of the mean peptide peak area of the nine reference control B and C samples in acetonitrile must be < 15.0%.
The test item’s results were considered valid if the following criteria were fully met:
- the mean peptide concentrations of the reference control C samples prepared in the appropriate solvent should be within ± 10% of the nominal concentration,
- the maximum SD for the test item replicates should be < 14.9% for the percent cysteine depletion value and < 11.6% for the percent lysine depletion value.

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Parameter:
other: % depletion
Remarks:
lysine peptide
Value:
0.02
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Parameter:
other: % depletion
Remarks:
cysteine peptide
Value:
91.34
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Parameter:
other: % mean depletion
Remarks:
Cysteine + Lysine
Value:
45.68
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: The mean of cysteine and lysine %depletion = 45.68%. Accordingly, the test item was considered have a high peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test item may have potential to cause skin sensitization.
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

Analysis of the chromatograms of the co-elution samples (Figures 1 and 4) indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide using the formula described in § Data analysis and calculation:
- for the cysteine peptide, the mean depletion value was 91.34%,
- for the lysine peptide, the mean depletion value was 0.02%.

It is to be noted that since precipitate and micelles were observed at the end of the incubation with the lysine peptide, the corresponding peptide depletion may be underestimated. Therefore, the mean depletion value of 0.02% cannot be drawn with sufficient confidence.

The mean of the percent cysteine and percent lysine depletions was equal to 45.68%. Accordingly, the test item was considered have a high peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test item may have potential to cause skin sensitization.

Conclusion: Under the experimental conditions of this study, the test item N-Nitrosodiphenylamine was considered to have a high peptide reactivity. The test item is considered positive in the DPRA assay.

Applicant's summary and conclusion

Interpretation of results:
other: positive in the DPRA assay (high peptide reactivity)
Conclusions:
Under the experimental conditions of this study, the test item N-Nitrosodiphenylamine was considered to have a high peptide reactivity. The test item is considered positive in the DPRA assay.
Executive summary:

The objective of this study was to evaluate the reactivity of the test item to synthetic cysteine and lysine peptides. This test is part of a tiered strategy for skin sensitization assessment.

The design of this study was based on the OECD guideline No. 442C and the study was performed in compliance with CiToxLAB France standard operating procedures and with the OECD Principles of Good Laboratory Practice.

 

Methods

The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides.The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violetdetection at 220 nm.

Peptide reactivity was reported as percent depletion basedon the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent).

 

Results

The test item was dissolved at 100 mM inacetonitrile.

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

 

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide:

.            for the cysteine peptide, the mean depletion value was 91.34%,

.            for the lysine peptide, the mean depletion value was 0.02%.

 

The mean of the percent cysteine and percent lysine depletions was equal to 45.68%. Accordingly, the test item was considered to have a high peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test item may have potential to cause skin sensitization.

 

Conclusion

Under the experimental conditions of this study, the test item N-Nitrosodiphenylamine was considered to have a high peptide reactivity. The test item is considered positive in the DPRA assay.