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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
Study period:
2017-06-07 to 2017-09-01
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:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
yes
Remarks:
These deviations did not influence the quality or integrity of the present study.
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
In accordance with Annex VII of REACH regulation, new REACH requirements for skin sensitisation entered into force on 11 October 2016

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: solid yellow powder

In chemico test system

Details on study design:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was pre-weighed into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared.


PREPARATION OF PEPTIDES
- 18.12 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (33.52 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
- 21.3 mg lysine peptide with an amino acid sequence of Ac RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (39.76 mL) to reach a concentration of 0.667 mM.
All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.

PREPARATION OF CONTROLS
- Reference controls (RC):
- Reference control A (undiluted) was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run
- Reference control B (undiluted) was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run
- Reference control C (undiluted) was set up for the test item and the positive control. RC C for the test item was prepared using the respective solvent used to solubilize the test item. RC C for the positive control was prepared using acetonitrile. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples.
- Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution.. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.
- Positive control: Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetronitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.

DOSE GROUPS
- Reference Control C: undiluted
- Test item: 100 mM stock solution
- Positive control: 100 mM stock solution

PRELIMINARY EXPERIMENT
Solubiluty of the test item was determined prior to the main experiment and was tested at the highest final concentration applied in the study (100mM). The test item was dissolved in dimethylformamide (DMF)

MAIN TEST
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide).
The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis.

Results and discussion

Positive control results:
Mean cysteine peptide depletion: 70.9% and mean lysine peptide depletion: 62.34%

In vitro / in chemico

Results
Key result
Parameter:
other: Mean cysteine peptide depletion (%)
Remarks:
Prediction Model 2
Run / experiment:
1
Value:
80.95
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

1. Pre-experiments:

Solubility of the test item was determined. The test item was not soluble in acetonitrile but completely soluble in DMF. No turbidity, precipitation and phase separation was observed for the test item solutions. All test item preparations of the main experiment were prepares using DMF.

2. Precipitation and phase separations:

All test item solutions were freshly prepared immediately prior to use. No precipitation, turbidity or phase separation was observed for the samples of the test item. Precipitation was observed for the samples of the positive control, however, since the acceptance criteria for the depletion of the positive contrtol were fulfilled, the observed precipitation and turbidity was regarded as insignificant.

3. Co-elution with the peptide peaks:

Co-elution of the test item with the lysine peptide peak was observed. Therefore prediction model 2 was applied.

4. Results Calibration Curve: Cysteine and Lysine values of the Calibration curve:

Sample Cysteine peptide Lysine peptide
Peak Area 220 nm Peptide Concentration mM Peak Area 220 nm Peptide concentration mM
STD1 4946.3618 0.5340 4453.7617 0.5340
STD2 2473.8633 0.2670 2214.0920 0.2670
STD3 1198.9833 0.1335 1103.6776 0.1335
STD4 613.2764 0.0667 542.1525 0.0667
STD5 313.3556 0.0334 261.3623 0.0334
STD6 155.0795 0.0167 131.8409 0.0167
STD7 0.0000 0.0000 0.0000 0.0000

Cysteine Peptide Calibration Curve: y = 9265.42x-5.69 ; R² = 0.9999

Lysine Peptide Calibration Curve: y = 8353.22x-10.69 ; R² = 1.0000

5. Results of Cysteine Peptide Depletion:

Sample Peak Area 220 nm Peptide concentration (mM) Peptide Depletion (%) Mean Peptide Depletion (%) SD of Peptide Depletion (%) CV of Peptide Depletion (%)
Positive Control 1367.6532 0.1482 70.48 70.90 0.44 0.62
1350.3103 0.1464 70.86
1327.2146 0.1439 71.36
Test Item 1035.8650 0.1124 77.63 80.95 3.57 4.41
903.7498 0.0982 80.48
707.2005 0.0769 84.73

6. Results of Lysine Peptide Depletion:

Sample Peak Area 220 nm Peptide concentration (mM) Peptide Depletion (%) Mean Peptide Depletion (%) SD of Peptide Depletion (%) CV of Peptide Depletion (%)
Positive Control 1562.8215 0.1884 62.85 62.34 0.45 0.72
1598.4821 0.1926 62.00
1591.5383 0.1918 62.17
Test Item 4383.3223 0.5267 0.00 1.04 0.96 91.63
4222.1846 0.5067 1.25
4195.4971 0.5035 1.88

7. Categorization of the Test item

Co-elution of test item with the lysine peptide peak was observed. Therefore, sensitizing potential of the test item was predicted from the mean peptide depletion of the cysteine peptide using prediction model 2:

Predicition Model

Prediction Model 1
(Cysteine 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

41.00

Moderate Reactivity

sensitiser

80.95

Moderate Reactivity

sensitiser

Positive Control

66.62

High Reactivity

sensitiser

70.90

Moderate Reactivity

sensitiser

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Remarks:
The study alone cannot be used for the classification purpose but in a WoE approach of minimally 2 in vitro assessment representing the key events in AOP
Conclusions:
In a DPRA study, the test item showed moderate reactivity towards the cysteine peptide. Based on these results the test item might be considered to have a sensitising potential (PPD 80.95%).

Executive summary:

In this in chemico study Direct Peptide Reactive Assay (DPRA), the skin sensitisation potential of the test item Mordand Red 11 was assessed according to the OECD 442C and under GLP without signiticant deviations.

Following a pre-experiment to determine the solubility of the test item in different solvent (dissolved in DMF), the test item (in triplicate) was incubated at a concentration of 100 mM (24.022 mg/mL) with the peptides containing either cystiene or lysine for 24 +/- 2h at 25 +/- 2.5 °C. After the incubation periode, the samples were analysed by HPLC in order to quantifyed the percentage of the mean peptide depletion.

Co-elution of test item with the lysine peptide peak was observed. Sensitizing potential of the test item was hence predicted from the mean peptide depletion of cysteine peptide alone by comparing the peptide concentration of the test item treated samples to the corresponding reference control C. (RC C). The mean depletion of the cysteine peptide was > 13.89% (i.e. 80.95%). Based on the prediction model 2 the test item can be considered as skin sensitiser (moderate).

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of positive control on both peptides was 66.62%.

In conlusion, in this study under the given conditions the test item showed moderate reactivity towards the cysteine peptide. Therefore, the test item is considered to have skin sensitising potential.The data generated with this method may be not sufficient to conclude on the skin sensitisation potential of chemicals and should be considered in a weight of evidence approach in which minimally 2 key events of the adverse outcome pathway (AOP) are tested.