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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:
weight of evidence
Study period:
28 July 2017 - 14 December 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013
Version / remarks:
2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

In chemico test system

Details on study design:
Skin sensitisation (In chemico test system)

Synthetic peptides used:
- cysteine peptide with an amino acid sequence of Ac-RFAACAA, JPT Peptide Technologies GmbH; > 95%; Lot. No.: 260515HS_DWW1115
- lysine peptide with an amino acid sequence of Ac-RFAAKAA, JPT Peptide Technologies GmbH; > 95%; Lot. No.: 120514HSDW W0517

Controls used:
- Positive control: Cinnamic aldehyde 100 mM in acetonitrile
- Co-elution control: test item or positive control without cysteine or lysine peptide
- Reference controls (RCs): cysteine or lysine peptide in acetonitrile with and without test item; Reference control A 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 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 was set up for the test item and the positive control. RC C for the positive control was prepared using acetonitrile. RC C for the test item was prepared using the respective solvent used to solubilise the test item. 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.

Test substance preparation:
- The test substance was prepared as a 100 mM preparation in acetonitrile.

Peptide stock solution preparation:
- 21.94 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (40.59 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
- 18.33 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 (34.8 mL) to reach a concentration of 0.667 mM.

Experimental procedure:
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. Reference controls, co-elution controls as well as the positive control were set up in parallel. Test item solutions were inspected on a visual basis for the formation of precipitates, turbidity and phase separation prior and after HPLC analysis, if a precipitate or phase separation was observed after the reaction period and prior to the HPLC analysis, samples might have been centrifuged at low speed (100 -400x g) to force precipitates to the bottom of the vial. After the incubation period of 24 ± 2 h the test item was analysed in triplicate for both peptides via HPLC.

HPLC conditions:
Peptide depletion was monitored by HPLC coupled with an UV detector at A = 220 nm using a reversed-phase HPLC column (Zorbax SB-C-18 2.1 mm x 100 mm x 3.5 micron) as preferred column. The entire system was equilibrated at 30 °C with 50% phase A (0.1% ( v/v) trifluoroacetic acid in water) and 50% phase B (0.085% ( v/v) trifluoroacetic acid in acetonitrile) for at least 2 hours before running the analysis sequence. The HPLC analysis was performed using a flow rate of 0.35 mL/min and a linear gradient from 10% to 25% acetonitrile over 10 minutes, followed by a rapid increase to 90% acetonitrile. The column was re-equilibrated under initial conditions for 7 minutes between injections. Equal volumes of each standard, sample and control were injected. HPLC analysis for the cysteine and lysine peptide was performed concurrently (if two HPLC systems were available) or on separate days. If analysis was conducted on separate days all test chemical solutions were freshly prepared for both assays on each day.
The analysis was timed to assure that the injection of the first sample started 22 to 26 hours after the test chemical was mixed with the peptide solution. The HPLC run sequence was set up in order to keep the HPLC analysis time less than 30 hours.

Calculation and data evaluation:
The concentration of the cysteine and lysine peptide was determined in each sample from absorbance at A = 220 nm, measuring the area of the appropriated peaks (peak area (PA)) and calculating the concentration of peptide using the linear calibration curves derived from the standard solutions.
PPD = (1- (Peptide Peak Area in the Replicate Injection / Mean Peptide Peak Area in Reference Control C)) * 100
Sensitising potential of the test item is predicted from the mean cysteine and lysine PPD value. The test item is considered positive to be a skin sensitiser in accordance with UN GHS "Category 1", if the mean depletion of both peptides exceeds the threshold of the respective prediction model. Negative depletion is considered as "0" when calculating the mean. Sensitizing potential might not be predictable if the test item was incubated using a concentration differently from 100 mM.
By using the prediction model 1 (cysteine 1:10 / lysine 1:50 prediction model) the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers.
By using the prediction model 2 (cysteine 1:10 prediction model) the threshold of 13.89% peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers.

Acceptance criteria:
The run meets the acceptance criteria if:
- the standard calibration curve has a r2 > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.
The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Results and discussion

Positive control results:
The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 65.19%.

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: combined peptide depletion [%]
Run / experiment:
mean depletion of both peptide depletions
Value:
0.11
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Parameter:
other: mean peptide depletion [%]
Run / experiment:
cysteine run
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Parameter:
other: mean peptide depletion [%]
Run / experiment:
lysine run
Value:
0.21
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: Precipitation and phase seperation was observed for the positive control samples. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed precipitations and phase separation were regarded as insignificant.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Table 3: Depletion of the Cysteine Peptide

Cysteine Peptide

Sample

Peak Area
at 220 nm

Peptide Conc. [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

1381.2087

0.1629

71.03

71.19

0.17

0.24

1364.7275

0.1611

71.37

1374.1948

0.1621

71.18

Test Item

4804.2617

0.5395

0.00

0.00

0.00

n/a

4819.4985

0.5411

0.00

4785.0088

0.5373

0.00

n/a not applicable

Table 4: Depletion of the Lysine Peptide

Lysine Peptide

Sample

Peak Area
at 220 nm

Peptide Conc. [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

1633.4226

0.2075

58.76

59.19

0.48

0.81

1595.8407

0.2028

59.71

1619.8287

0.2058

59.10

Test Item

3877.1357

0.4921

0.27

0.21

0.17

81.66

3886.7849

0.4933

0.02

3873.7730

0.4917

0.35

Table 5: Categorisation of the Test Item

Prediction Model

Prediction Model 1
(Cysteine Peptide 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

0.11

Minimal Reactivity

no sensitizer

0.00

Minimal Reactivity

no sensitizer

Positive Control

65.19

High Reactivity

sensitizer

71.19

Moderate Reactivity

sensitizer

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item showed minimal reactivity towards the peptides. The test item might be considered as “non-sensitiser”.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
Executive summary:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

In the present study the test item was dissolved in distilled water based on the results of the pre-experiments. Based on a molecular weight of 132.07 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the positive control (excluding the co-elution control of the positive control). Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the samples of the positive control (including co-elution control of the positive control). Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control was fulfilled, the observed precipitations and phase separation were regarded as insignificant.

No co-elution of test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C.

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was 6.38% (0.11%). Based on the prediction model 1 the test item can be considered as non-sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 65.19%.