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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:
2017-10-11 to 2017-10-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
04 February 2015
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

In chemico test system

Details on study design:
TEST-SUBSTANCE PREPARATION
- Stock solution: 84.5 mM
- Vehicle: water
- Reason for the vehicle: The test substance is soluble in water

CONTROLS
- Reference controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run.
- Co-elution control: buffer and test substance without the peptide
- Positive control: Cinnamic aldehyde in acetonitrile

PEPTIDES
- Synthetic peptides:
-- Cysteine- (C-) containing peptide: Ac-RFAACAA
-- Lysine- (K-) containing peptide: Ac-RFAAKAA
- Stock solution:
-- C-containing peptide: 0.667 mM of peptide in pH 7.5 phosphate buffer
-- K-containing peptide: 0.667 mM of peptide in pH 10.2 ammonium acetate buffer

EXPERIMENTAL PROCEDURE
- Replicates: 3 for each peptide
- Determination remaining non-depleted peptide concentration: HPLC at 220nm: HPLC analysis started 22 to 26 hours after sample preparation and the analysis time was less than 30 hours.
- Calibration samples: samples of a known peptide concentration are measured in parallel

PREPARATIONS SAMPLES
- Calibration sample was prepared from the peptide stock solution in 20% acetonitrile in the respective buffer using serial concentration: 0.534, 0.267, 0.134, 0.067, 0.033, 0.017 or 0.000 mM peptide
- Test-substance samples: samples were incubated at 25°C +/- 2.5°C in the dark for 24 +/- 2 hours and visually investigated for any precipitate that may occur during the exposure period.
- Reference controls, co-elution controls as well as the positive control were set up in parallel (see table below)

MEASUREMENT PEPTIDE CONCENTRATIONS
- Method: HPLC/DAD Agilent 1200 series with Chemstation
Wavelength: 220 nm for quantification and 258 nm as indicator of co-elution
Column: Zorbax SB-C18, 100 mm x 2.1 mm, 3.5 µm Agilent
Pre-Column: Phenomenex AJO 4286, 4 x 3 mm
Colunm temperature: 30°C
Sample temperature: 25°C
Run time: 20 min
Injection volume: 10 µL
- Detector: UV detector

DATA EVALUATION
- Calculation of the peptide concentrations: peptide concentration (mM) = [peak area (mAU * s) - b] /m
with b: axis intercept and m: slope
- The percent peptide depletion (PPD) was calculated according to the following formula:
PPD = [ 1 – ( Peptide Peak Area in the Replicate Injection / Mean Peptide Peak Area in the Reference Control C)] * 100

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.

EVALUATION RESULTS
- Chemical reactivity was determined by mean peptide depletion [%] and was rated as
-- high: mean peptide depletion > 42.47
-- moderate: mean peptide depletion > 22.62 ≤ 42.47
-- low: mean peptide depletion > 6.38 ≤ 22.62
-- minimal: mean peptide depletion ≤ 6.38
High, moderate and low reactivity are evaluated as positive.
- In case the mean peptide depletion cannot be determined due to invalid K-peptide depletion the
evaluation is performed as follows:
-- high: mean peptide depletion > 98.24
-- moderate: mean peptide depletion > 23.09 ≤ 98.24
-- low: mean peptide depletion > 13.89 ≤ 23.09
-- minimal: mean peptide depletion ≤ 13.89
High, moderate and low reactivity are evaluated as positive.

Results and discussion

Positive control results:
Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed precipitation was regarded as insignificant.

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: mean depletion % of peptide
Run / experiment:
cysteine
Value:
100
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: mean depletion % of peptide
Run / experiment:
lysine
Value:
2.66
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: mean depletion % of peptide
Run / experiment:
lysine and cysteine
Value:
51.33
Vehicle controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The mean peptide depletion of the positive control was 75.35% (cysteine) and 56.98% (lysine)

Any other information on results incl. tables

Results of the Cysteine Peptide Depletion

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

809.6354

0.1341

74.93

75.35

0.40

0.53

794.7175

0.1317

75.39

783.8397

0.1299

75.72

Test Item

0.0000

0.0036

100.00

100.00

0.00

0.00

0.0000

0.0036

100.00

0.0000

0.0036

100.00

Results of the Lysine Peptide Depletion

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

1768.9691

0.2117

57.97

56.98

1.07

1.87

1804.7944

0.2160

57.12

1858.1759

0.2224

55.85

Test Item

4077.2800

0.4880

1.87

2.66

0.84

31.54

4007.8833

0.4797

3.54

4048.2310

0.4846

2.57

Categorization 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

51.33

High Reactivity

sensitiser

100.00

High Reactivity

sensitizer

Positive Control

66.16

High Reactivity

sensitizer

75.35

Moderate Reactivity

sensitizer

Applicant's summary and conclusion

Interpretation of results:
other:
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 high reactivity towards the cysteine peptide. Based on these results the test item might be considered to have a sensitising potential.
Executive summary:

In the current study the skin sensitisation effect of the test item was assessed in an in chemico assay according to OECD 442C and in compliance to GLP. 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 water, based on the results of the pre-experiments. A blue- violet solution was obtained.

Based on a molecular weight of 327.82 g/mol a 84.5 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 all experiments no turbidity, precipitation or phase separation was observed when diluted with the cysteine or lysine peptide solution respectively.

After the 24 h±2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any samples. Precipitation was observed for the samples of the positive control and co-elution of the positive control. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed precipitation was 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 (RC Cwater).

The 84.5 mM stock solution of the test item showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 51.33 %, which is > 6.38% cut-off. According to the evaluation criteria in the guideline, if a test chemical is tested in concentration < 100 mM, a positive result can still be used to support the identification of the test chemical as a skin sensitiser. Based on the prediction model 1 the test item can be considered as 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 66.16%.

In conclusion, in this study, under the given conditions the test item was shown 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.