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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:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Qualifier:
according to
Guideline:
other: EU Method B.59 In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
The OECD TG 442 C may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitizers and non-sensitizers for the purpose of hazard classification and labelling.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.

PREPARATION OF TEST MATERIAL PRIOR TO TESTING
The test substance was prepared as a 100 mM preparation in methanol (considering a molecular weight of 426.99 g/mol and a purity/contents of 88.1%). After short stirring, the test substance was soluble in the vehicle.

In chemico test system

Details on study design:
TEST SYSTEM
- Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol)
- Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)
- The peptides are custom material (Supplier: GenScript, Piscataway, NJ, USA and RS Synthesis, Louisville KY, USA) containing phenylalanine to aid in detection and either cysteine or lysine as the reactive center.

SELECTION OF CONCENTRATIONS
- The C-containing peptide was incubated with the test substance in a ratio of 1:10 (0.5 mM peptide, 5 mM test substance).
- The K-containing peptide in a ratio of 1:50 (0.5 mM peptide, 25 mM test substance).

CONTROLS:
- Negative control (NC): vehicle control = methanol
- Positive control (PC): Ethylene glycol dimethacrylate (EGDMA; CAS-no. 97-90-5), prepared as 50 mM solution in methanol.
- Co-elution control: Sample prepared of the respective peptide buffer and the test substance but without peptide.

TEST SUBSTANCE PREPARATION:
- Reason for the vehicle : methanol was used since the test substance was soluble in this vehicle.
- Prior to the assay the solubility of the test substance at a concentration of 100 mM was tested. The preferred solvent was acetonitrile.

PREPARATION OF PEPTIDE STOCK SOLUTIONS AND CALIBRATION SAMPLES:
- Peptide stock solutions in a concentration of 0.667 mM were prepared in pH 7.5 phosphate buffer (C-containing peptide) or pH 10.2 ammonium acetate buffer (K-containing peptide). The peptide stock solution was used for preparing the calibration samples and the test-substance and control samples.
- Calibration samples were prepared from the peptide stock solutions in 20% methanol in the respective buffer (= dilution buffer) using serial dilution

EXPERIMENTAL PROCEDURE:
- Three samples of the test substance were incubated with each peptide. Triplicates of the concurrent vehicle control (= NC) were also incubated with the peptides.
- The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm.
- Calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, were measured in parallel with the same analytical method

DATA EVALUATION
- Integrated peak areas were transferred electronically into EXCEL data spreadsheets to carry out the necessary calculations.
- In the cases where proper integration and calculation of peptide depletion was not possible due to co-elution, the result for the respective peptide is reported as interference.
- For evaluation of peptide depletions peak areas at 220 nm are used.

Calculation of peptide concentrations:
- the peptide concentration of the samples is calculated with the respective calibration curve using linear regression (b= axis intercept; m=slope):
Peptide concentration [mM] = (peak area at 220 nm [mAU x s] - b)/ m

Calculation of peptide depletion:
Peptide depletion of sample = [1 - [(peptide concentration of sample (mM))/(mean peptide concentration of NC (mM))] x 100 (%)

- Mean peptide depletion of each of the two peptides is calculated as the mean value of the three samples conducted for each peptide and test substance (C-containing and K-containing peptide depletion; example calculation for C-containing peptide):
C-containing peptide depletion of a test substance [%] = mean [C-containing peptide depletion of samples 1-3] (%)

Limitations of the evaluation by insolubility and gravimetric procedure:
For test substances that are not completely soluble by visual observation in the sample preparations containing the peptides immediately after preparation or after 24 hours, or when a gravimetric procedure is applied (with the exception of application of the undiluted test substance (liquids) or the maximal soluble test-substance concentration (solids)), the result may be under-predictive due to limited availablity of the test substance. In this case mean peptide reactivity ≤ 6.38% (cysteine 1:10 / lysine 1:50 prediction model) or ≤ 13.89% (cysteine 1:10 prediction model) is interpreted as “inconclusive”. However, a mean peptide depletion > 6.38% or > 13.89% is considered as “positive”.

Results and discussion

Positive control results:
Ethylene glycol dimethacrylate (EGDMA; CAS-no. 97-90-5), prepared as 50 mM emulsion in de-ionized water was used as the positive control. It caused a mean C-peptide depletion of 55.20% and a mean K-peptide depletion of 12.90%.

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: Mean C-peptide depletion (%)
Run / experiment:
First test run
Value:
ca. 12.34
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Minimal or no reactivity (≤ 13.89) according to the cysteine 1:10 prediction model.
Key result
Parameter:
other: Mean C-peptide depletion (%)
Run / experiment:
Second test run
Value:
ca. 8.33
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Minimal or no reactivity (≤ 13.89) according to the cysteine 1:10 prediction model.
Key result
Parameter:
other: Mean K-peptide depletion (%)
Value:
ca. -15.26
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Limited solubility of test substance with the K-containing peptide, calculation of mean peptide depletion is not appliable and the cysteine 1:1 prediction model is used for evaluation.
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY
Acceptance criteria:
- The standard calibration curve should have an r² >0.99.
- The negative control (vehicle control) samples of sets A and C should be 0.50 mM +/- 0.05 mM.
- The CV of the nine vehicle controls B and C should be < 15%.
- Since the mean peptide depletion for each peptide is determined from the mean of three single samples, the variability between these samples should be acceptably low (SD < 14.9% for % cysteine depletion and < 11.6% for % lysine depletion).  
-  In addition the positive control should cause depletion of both peptides comparable to historic data

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes, but a mean lysine peptide depletion of 6.85 was obtained which is below the historic range. However it should be noted that a very limited number of historical data is available for the vehicle, methanol, and that for other vehicles, this value is not uncommon.
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: Please refer to the historical control data tables in the 'Attached background material'

Any other information on results incl. tables

Cysteine-peptide vehicle controls

1st test run

The mean peptide concentration of the three samples of set A was calculated to be 0.504 mM with a SD of 0.002 mM demonstrating good performance. The mean peptide concentration of the three samples of set B, analysed at the beginning of the sample list, was calculated to be 0.487 mM with a SD of 0.001 mM. The other three samples of set B, analysed at the end of the sample list had a mean peptide concentration of 0.468 mM with a SD of 0.018 mM. The CV of the 9 vehicle control samples of sets B and C was calculated to be 2.9%. Thus the peptide was considered stable over the time of analysis.

The mean area ratio 220 nm/258 nm of the 9 vehicle control samples of sets B and C was calculated to be 30.7. The area ratio 220/258 nm of the test-substance samples correspond to 96.6 -99.8% of the mean of the vehicle controls, demonstrating the absence of any interference.

 

2nd test run

The mean peptide concentration of the three samples of set A was calculated to be 0.504 mM with a SD of 0.002 mM demonstrating good performance. The mean peptide concentration of the three samples of set B, analysed at the beginning of the sample list, was calculated to be 0.487 mM with a SD of 0.001 mM. The other three samples of set B, analysed at the end of the sample list had a mean peptide concentration of 0.468 mM with a SD of 0.018 mM.The CV of the 9 vehicle control samples of sets B and C was calculated to be 2.9%. Thus the peptide was considered stable over the time of analysis.

The mean area ratio 220 nm/258 nm of the 9 vehicle control samples of sets B and C was calculated to be 30.1. The area ratio 220/258 nm of the test-substance samples correspond to 93.5 -98.7% of the mean of the vehicle controls, demonstrating the absence of any interference.

Lysine-peptide vehicle controls

The mean peptide concentration of the three samples of set A was calculated to be 0.481 mM with a SD of 0.002 mM, demonstrating good performance. The mean peptide concentration of set B, analysed at the beginning of the sample list, was calculated to be 0.549 mM with a SD of 0.060 mM. The other three samples of set B, analysed at the end of the sample list had a mean peptide concentration of 0.527 mM with a SD of 0.009 mM. The CV of the 9 vehicle control samples of sets B and C was calculated to be 6.7. Thus the peptide was considered stable over the time of analysis.

The mean area ratio 220/258 nm of the 9 vehicle control samples of sets B and C was calculated to be 32.7. Therefore the area ratio 220 nm/258 nm of the test substance samples correspond to 93.4 -97.0% if the mean of the vehicle controls, demonstrating the absence of interference.

Co-elution

No co-elution of the test substance and peptides occurred as demonstrated by the consistent values of the area ratios 220 nm/258 nm.

Calibration curves

Cysteine-containing peptide:

- 1st valid test run: slope = 962.0, axis intercept = -6.583, correlation = 0.99994

- 2nd valid test run: slope = 894.7, axis intercept = 0.966, correlation = 0.99994

Lysine-containing peptide:

- slope = 645.1, axis intercept = -0.722, correlation = 0.99997

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
negative prediction of skin sensitisation in DPRA assay
Conclusions:
Based on the observed results and applying the cysteine 1:10 prediction model, it was concluded that the test substance, Basic Orange 22 shows minimal or no chemical reactivity in the DPRA under the test conditions in accordance with the OECD Guideline 442C. Basic Orange 22 has been observed not to be peptide reactive and is predicted not be a skin sensitiser.
Executive summary:

The skin sensitising potential of the test substance, Basic Orange 22, was determined via the in chemico skin sensitisation method - Direct Peptide Reactivity Assay (DPRA). The study was conducted following the OECD Guideline for Testing of Chemicals TG. 442C, adopted 04 February 2015  (“In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)”) and the B.59 In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA); Official Journal of the European Union, No. L 112/1.

 

Main Assay

The test substance, Basic Orange 22 was dissolved in methanol at a 100 mM concentration considering a purity/contents of 88.1% and molecular weight of 426.99 g/mol. The C-containing peptide was incubated with the test substance in a ratio of 1:10 (0.5 mM peptide, 5 mM test substance) and the K-containing peptide in a ratio of 1:50 (0.5 mM peptide, 25 mM test substance). In total, three samples of the test substance were incubated with each peptide. Triplicates of the concurrent vehicle control were incubated with the peptides. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. The calibration samples of known peptide concentration, prepared from the respective peptide stock solution were used for test-substance incubation and measured in parallel with the same analytical method.

 

The mean peptide concentration of the cysteine-peptide and lysine-peptide vehicle controls in methanol demonstrated good performance and the coefficient of variation for the 9 vehicle control samples of the cysteine-peptide (2.9% in the first valid test run and 4.0% in the second valid test run) and lysine-peptide vehicle controls (6.7%), showed that the peptides remained stable over the time of analysis. There was no co-elution of the test substance and peptides occurred as demonstrated by the consistent values of the area ratios 220 nm/258 nm.

Due to insolubility of the test substance in the K-peptide samples, the calculation of mean cysteine- and mean lysine-peptide depletion is not applicable and instead the cysteine 1:10 prediction model is used for evaluation. The mean C-peptide depletion in the first test run was 12.34% and in the second test run 8.33%. Based on the cysteine 1:10 prediction model, since the depletion values were ≤ 13.89%, this indicates minimal or no reactivity.

The distinct negative value of K-peptide depletion indicates an artifical effect, such as interference, however this is not supported by the co-elution control or the area ration 220/258 nm.

The acceptance criteria required for acceptance of results in the test were satisfied according to OECD Test Guideline 422C. The positive control caused depletion of both peptides, comparable to historic data. However, a mean lysine peptide depletion of 6.85 was obtained for the positive control, which is below the historic range. Nonetheless, it should be noted that there is a very limited number of historic data available for the vehicle methanol and that this value is not uncommon for other vehicles.

 

Conclusion

Based on the observed results and applying the cysteine 1:10 prediction model, it was concluded that the test substance, Basic Orange 22 shows minimal or no chemical reactivity in the DPRA under the test conditions chosen. Basic Orange 22 has not been observed to be peptide-reactive and is not predicted to be a skin sensitiser.