Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Method equivalent or similar to guideline, non-GLP. Well documented study in accordance with scientific principles and sufficient for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
Remarks:
Additional investigations for dimerisation potential were conducted.
GLP compliance:
no
Remarks:
Method equivalent or similar to guideline, non-GLP. Well documented study in accordance with scientific principles and sufficient for assessment.
Type of study:
direct peptide binding assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Liquid
- Storage condition of test material: In refrigerator (4°C)

In chemico test system

Details on study design:
Skin sensitisation (In chemico test system) - Details on study design:
- The study was conducted in accordance with the OECD TG 422C – In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)
- The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442C. The results of the testing on the proficiency chemicals at the test facility is described in a separate test report (full references provided in the full study report): ‘Direct peptide reactivity assay (DPRA) for skin sensitization testing: Proficiency testing at the [name redacted] testing facility’.
- Acetonitrile (i.e. the standard solvent of the SOP) was used for preparation of test solutions. The test was run according to the final validated SOP published by ECVAM (DB-ALM protocol 154). Reference: ECVAM, Direct peptide reactivity assay (DPRA) for skin sensitization testing DB-ALM database (2014).
- HPLC-UV and LC-MS (Dimerization investigation) methodology are reported in the full study report.
- Acceptability criteria:
(i) Cys-peptide depletion standard deviation should be < 14.9% (Test item: Actual = 0.8%)
(ii) Lys-peptide depletion standard deviation < 11.6%. (Test item: Actual = 0.1%)
(iii) The co-elution controls indicated no co-elution with an UV-absorbing component.
(iv) Calibration curves for the HPLC-UV analysis were as follows: (i) Cysteine r2 = 0.9922 and (ii) Lysine r2 = 1.0000
(v) Mean peptide concentrations in reference item replicate control C: was 0.5 +/- 0.05 mM (actual = 0.50 to 0.50 mM in acetonitrile)
(vi) CoV in reference items B and C in acetonitrile: 6.42% (Cys peptide) and 0.34% (Lys peptide)

(vii) Positive Control: cinnamic aldehyde:
- Cysteine peptide: Should be between 60.8% and 100%
Actual = 65.4% (SD = 1.3%) and CoV = 2.0%
Cys-peptide depletion standard deviation should be < 14.9% (PC : Actual = 1.3%)
CoV should be < 15.0% (PC : Actual = 2.0%)
- Lysine peptide Should be between 40.2% and 69.0%
Actual = 43.5% (SD = 1.7%) and CoV = 3.9%
Lys-peptide depletion standard deviation < 11.6%. (Test item: Actual = 1.7%)
CoV should be < 15.0% (PC : Actual = 3.9%)
All acceptance criteria were fulfilled for the PC
(viii) Mean peptide concentrations in reference item replicate control A: was 0.5 +/- 0.05 mM (actual = 0.49 to 0.51 mM in acetonitrile)

(ix) Stability of vehicle control: A control experiment according to the DB-ALM protocol 154 to test whether Acetonitrile has no effect on peptide stability was performed. A 0.5mM Cysteine solution was incubated for 24 h in Phosphate buffer / acetonitrile, and subsequently injected every 3 h for 48 h. The CV of these samples was found to be 3.4% (DB-ALM protocol requirement: < 15%).

All acceptability criteria were met.

- Peptide dimerization: were monitored by LC-MS allowing quantification of the peptide dimers. Correct identification of the peptide dimer molecular formula was possible based on calculated and observed exact mass and comparison to synthetic references.
Reference: OECD TG 442C paragraph 23: 23. There may be test test items which could promote the oxidation of the cysteine peptide. The peak of the dimerised cysteine peptide may be visually monitored. If dimerization appears to have occurred, this should be noted as percent peptide depletion may be over-estimated leading to false positive predictions and/or assignment to a higher reactivity class.

- Synthetic peptides:
Cysteine- containing peptide: Ac-RFAACAA-COOH (MW=750.1) – full details on source provided in full study report.
Lysine-containing peptide: Ac-RFAAKAA-COOH (MW=775.4) – full details on source provided in full study report.
Cysteine-dimer peptide: (MW=1499.2) – full details on source provided in full study report.

Controls:
Positive control (PC): Cinnamic aldehyde (CAS 104-55-2; > 99% food-grade purity)
Negative control (NC): Vehicle = acetonitrile

Test item preparation:
The test substance was prepared as a 100 mM stock solution in acetonitrile prior to incubation with synthetic peptides. The test item was freely soluble in acetonitrile at 100 mM. Three replicates of the test item were incubated with each peptide. Additionally, triplicates of the concurrent vehicle control (NC) were incubated with the peptides. The peptide depletion of test item incubated samples was compared to the peptide depletion of the NC samples and expressed as relative peptide depletion. For the test item the mean peptide depletion as average of Cys and Lys peptide depletion is calculated and used for evaluation of the test item reactivity.

Dimerisation investigation:
In parallel, test item, PC and NC samples were 100-fold diluted and analysed with high resolution LC-MS were conducted. In this parallel analysis, peptide depletion, dimer formation and adduct formation were separately investigated, both for the test item and the control cinnamic aldehyde

Evaluation of results:
Test item reactivity was determined by mean peptide depletion and was rated as high, moderate, low, or minimal:
Mean peptide depletion [%] Reactivity
> 42.47 high reactivity
> 22.62 < 42.47 moderate reactivity
> 6.38 < 22.62 low reactivity
< 6.38 minimal reactivity

Dimerization investigation:
With completed LC-MS analysis, formation of peptide dimer and possible adducts is screened in addition to peptide depletion. If depletion of the Cys-peptide is observed AND the following two conditions are met:
a) Significantly enhanced formation of peptide Dimer or other peptide oxidation products such as the sulfoxide in test samples as compared to control reference samples
b) Lack of formation of a direct peptide adduct
Then there is no evidence for a direct reactivity, and observed peptide depletion can be explained by the chemical-catalysed peptide oxidation rather than direct reactivity. In this case, the scientifically most appropriate conclusion is that the test does not indicate reactivity of the test chemical.

However, if both peptide oxidation and adduct formation occur in parallel, then certainly chemicals are still rated as positive and peptide reactive.

This prediction model is not part of the validated protocol, but it takes into account a well described shortcoming of the DPRA. Since it does not form part of the validated protocol, this additional analysis should be used in a weight-of-evidence based analysis of the sensitization potential of a test chemical.

Results and discussion

Positive control results:
- All PC acceptability criteria were met.
- PC CYS-peptide depletion (mean): 64.4% (high reactivity)
- PC LYS-peptide depletion (mean): 43.5% (moderate reactivity)
- The positive control has a theoretical Michael acceptor structural alert for reactivity with a cysteine containing peptide, and the predicted direct peptide adduct is shown (within the full study report) with a theoretical exact mass of 882.4058. Therefore the [M+H]+ ion of 883.4131 was searched for by LC-MS. A new peak eluting at 9.34 min was observed and identified as [M+H]+ ion. The MS analysis indicated that the measured exact mass at this retention time is completely in line with the predicted mass which confirms that this peak is a direct adduct between the positive control and the test peptide, confirming that the positive control has direct peptide reactivity.

In vitro / in chemico

Resultsopen allclose all
Parameter:
other: Cys-peptide depletion
Run / experiment:
Mean (%)
Value:
29.9
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: Applicant indicates: NO or MINIMAL REACTIVITY CLASS
Remarks:
See 'any other information on results incl. tables' for dimerization investigations
Parameter:
other: Lys-peptide depletion
Run / experiment:
Mean (%)
Value:
0.1
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: None reported.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442C. The results of the testing on the proficiency chemicals at the test facility is described in a separate test report (full references provided in the full study report): ‘Direct peptide reactivity assay (DPRA) for skin sensitization testing: Proficiency testing at the [name redacted] testing facility’.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: All criteria met.
- Acceptance criteria met for positive control: All criteria met.
- Acceptance criteria met for variability between replicate measurements: All criteria met.
- Range of historical values if different from the ones specified in the test guideline: Not applicable.

Acceptability criteria: All acceptability criteria were met.
(i) Cys-peptide depletion standard deviation should be < 14.9% (Test item: Actual = 0.8%)
(ii) Lys-peptide depletion standard deviation < 11.6%. (Test item: Actual = 0.1%)
(iii) The co-elution controls indicated no co-elution with an UV-absorbing component.
(iv) Calibration curves for the HPLC-UV analysis were as follows: (i) Cysteine r2 = 0.9922 and (ii) Lysine r2 = 1.0000
(v) Mean peptide concentrations in reference item replicate control C: was 0.5 +/- 0.05 mM (actual = 0.50 to 0.50 mM in acetonitrile)
(vi) CoV in reference items B and C in acetonitrile: 6.42% (Cys peptide) and 0.34% (Lys peptide)

(vii) Positive Control: cinnamic aldehyde:
- Cysteine peptide: Should be between 60.8% and 100%
Actual = 65.4% (SD = 1.3%) and CoV = 2.0%
Cys-peptide depletion standard deviation should be < 14.9% (PC : Actual = 1.3%)
CoV should be < 15.0% (PC : Actual = 2.0%)
- Lysine peptide Should be between 40.2% and 69.0%
Actual = 43.5% (SD = 1.7%) and CoV = 3.9%
Lys-peptide depletion standard deviation < 11.6%. (Test item: Actual = 1.7%)
CoV should be < 15.0% (PC : Actual = 3.9%)
All acceptance criteria were fulfilled for the PC
(viii) Mean peptide concentrations in reference item replicate control A: was 0.5 +/- 0.05 mM (actual = 0.49 to 0.51 mM in acetonitrile)

(ix) Stability of vehicle control: A control experiment according to the DB-ALM protocol 154 to test whether Acetonitrile has no effect on peptide stability was performed. A 0.5mM Cysteine solution was incubated for 24 h in Phosphate buffer / acetonitrile, and subsequently injected every 3 h for 48 h. The CV of these samples was found to be 3.4% (DB-ALM protocol requirement: < 15%).

Any other information on results incl. tables

Dimerisation investigation:

1. Peptide adduct investigation:

LC-MS allowed quantification of the parent peptide as [M+H]+ion eluting at a retention time of 7.16 min. Correct identification of the parent peptide molecular formula was possible based on calculated and observed exact mass.This peak was integrated for all samples and used to calculated peptide depletion as done for HPLC-UV analysis. The test item gave 28.4% depletion of the Cys-peptide when measured with the LC-MS method. This value is close to the value (29.9%) reported using HPLC-UV, and thus peptide quantification with this alternative method is reliable (also consistent with the positive control results), and if using the depletion only, this alternative analysis method leads to identical conclusions.

2. Peptide dimerization LC-MS

LC-MS allowed quantification of the peptide dimer, which is observed as the double charged [M+2H]2+ ion eluting at a retention time of 8.17 minutes. Correct identification of the peptide dimer molecular formula was possible based on calculated and observed exact mass and comparison to synthetic reference. For each sample, this peak was integrated and quantified based on a calibration curve. This peak was enhanced in all three replicate samples of the test substance. Compared to Reference C, the parent peptide was reduced by 0.14 mM in the test samples, while the dimer peak in the test samples compared to the control samples was enhanced by 0.077mM corresponding to 0.15 mM test peptide. Quantitatively, the formed dimer can thus fully account for the peptide loss.

3. Expert Judgement assessment:

The test item does not have a typical structural alert for peptide adduct formation. Theoretically, the only presumed mode of action would be an acyl transfer, whereby the lactone is transformed to a thioester, although this process is energetically normally not favoured. Therefore the [M+H]+ ion of 1003.5695 was searched for by LC-MS. No new peak with this mass was observed. The test item did not clearly form a direct acyl transfer peptide adduct or similar direct adducts with the theoretically calculated MW 1002.6.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study, the test item is not considered to be potentially sensitising to the skin.
Executive summary:

The study was performed to the validated OECD TG 442C in chemico Direct peptide reactivity Assay (DPRA). The testing laboratory has previously demonstrated technical proficiency requirements associated with the assay. All test acceptability criteria were met. The test item was dissolved in acetonitrile and mixed with a Cysteine- and a Lysine-containing peptide according to the standard operating procedure of the DPRA. One study with three replicates was conducted. After 24 h incubation time, peptide depletion induced by the test item was determined by HPLC-UV. In parallel, the same samples were 100-fold diluted and analysed with high resolution LC-MS. In this parallel analysis, peptide depletion, dimer formation and adduct formation were separately investigated, both for the test item and the control cinnamic aldehyde.When using HPLC-UV analysis only, the test item led to 29.9.1% depletion of the Cys-peptide and 0.1% depletion of the Lys-peptide. In extension to the assay LC-MS investigations of Cys-peptide oxidation / dimerization was conducted. When using HPLC-UV analysis only, the test item led to 29.9% depletion of the Cys-peptide. When samples were analysed with high resolution LC-MS, the same amount of peptide depletion was observed. However, no direct adduct was formed in samples with the test item. At the same time the test item triggered significantly enhanced peptide dimerization, and the observed peptide depletion can be attributed to peptide oxidation / dimer formation rather than adduct formation / direct reactivity. No indication for a peptide adduct was found in this analysis.For the positive control (Cinnamic aldehyde), direct adduct formation was verified indicating direct reactivity for the positive control. Thus, based on this more sophisticated and detailed analysis, the test item is not directly peptide reactive, but can trigger peptide dimerization which is not considered itself as a molecular initiating event in skin sensitization.