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:
24 June 2019 - 11 July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

In chemico test system

Details on study design:
Skin sensitisation (In chemico test system):
The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 ± 2 hours incubation with the test item at 25 ± 2.5°C.

Preparation of the cysteine or lysine-containing peptides:
Stock solutions of cysteine (Ac-RFAACAA-COOH) and lysine (Ac-RFAAKAA-COOH) containing synthetic peptides of purity higher than 95% were freshly prepared just before their incubation with the test item. The final concentration of the cysteine peptide was 0.666 mM in pH 7.5 phosphate buffer, whereas the final concentration of the lysine peptide was 0.668 mM in pH 10.2 ammonium acetate buffer

Preparation of the test item:
Solubility of the test item in an appropriate solvent was assessed before performing the assay. 156.06 mg test item was dissolved in 3 mL acetonitrile immediately before testing to prepare a 100 mM solution. The test item solution was then tested as such without any further dilution by incubating at 1:10 or 1:50 ratio with the cysteine peptides and lysine peptides, respectively.

Positive control, reference controls and co-elution control
Cinnamic aldehyde (CAS no. 14371-10-9) was used as positive control (PC) at a concentration of 100 mM in acetonitrile. In addition reference controls (i.e. samples containing only the peptide and added acetonitrile) were also included in the HPLC run sequence and these were used to verify the HPLC system suitability prior to the analysis (reference controls A) and the stability of the reference controls over time (reference control B). To verify that the solvent used to dissolve the test item does not impact the percent peptide depletion the reference control C was prepared by adding acetonitrile to the peptide solution. The reference control C was used to calculate the percent peptide depletion for the test item. In addition a co-elution control constituted by the test item alone for the test item analysed was included in the run sequence to detect possible co-elution of the test item with either the lysine or the cysteine peptide.

Incubation of the test item with the cysteine and lysine peptide solutions:
Cysteine and lysine peptide solutions were incubated in glass autosampler vials with the test item at 1:10 and 1:50 ratio, respectively. The reaction solution was left in the dark at 25 ± 2.5°C for 24 ± 2 hours before running the HPLC analysis. The test item assay was analysed in triplicate for both peptides. Samples were visually inspected prior to HPLC analysis. If a precipitate would be observed immediately upon addition of the test item solution to the peptide solution, due to low aqueous solubility of the test item, in this case one cannot be sure how much test item remained in the solution to react with the peptide. Therefore, in such a case, a positive result could still be used, but a negative result is uncertain and would be interpreted with due care. No precipitate or phase separation was observed.

Preparation of the HPLC standard calibration curve:
A standard calibration curve was generated for both the cysteine and the lysine peptides. Peptide standards were prepared in a solution of 20% acetonitrile : buffer using 100 mM sodium phosphate buffer (pH 7.5) for the cysteine peptide and 100 mM ammonium acetate buffer (pH 10.2) for the lysine peptide. Using serial dilution standards of the peptide stock solution (nominal concentrations: 0.666 mM of cysteine peptide in sodium phosphate or 0.666 mM lysine peptide in ammonium acetate), 6 calibration standards were prepared to cover the range from 0.534 to 0.0167 mM. A blank of the dilution buffer was also included in the standard calibration curve. Suitable calibration curves should have an r2 > 0.99.

Data evaluation
HPLC analysis for the cysteine and lysine peptides was performed on one day. All test item solutions were freshly prepared for both assays on one day. The analysis was timed to assure that the injection of the first sample (reference control C) started 22 to 26 hours after the test item had been mixed with the peptide solution. The HPLC run sequences were set up in order to keep the HPLC analysis time to less than 30 hours. The concentrations of cysteine or lysine peptide were photometrically determined at 220 nm in each sample by measuring the peak area (area under the curve, AUC) of the appropriate peaks and by calculating the concentration of peptide using the linear calibration curve derived from the standards.

Acceptance criteria
The following criteria must be met for a run to be considered valid:
a) The standard calibration curve should have an r2 > 0.99.
b) The mean percent peptide depletion value of the three replicates for the positive control cinnamic aldehyde should be between 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide and the maximum standard deviation (SD) for the positive control replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion.
c) The mean peptide concentration of reference controls A should be 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C in acetonitrile should be <15.0%.
If one or more of these criteria is not met, the run would have been repeated.

The following criteria must be met for a test item’s results to be considered valid:
a) The maximum standard deviation for the test item replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion.
b) The mean peptide concentration of the three reference controls C in the appropriate solvent should be 0.50 ± 0.05 mM.
If these criteria were not met, the data would have been rejected and the run have been repeated for that specific test item.

Prediction model:
The mean percent cysteine and percent lysine depletion value was calculated for each test item. Negative depletion was considered as “0” when calculating the mean. By using the 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 in the framework of an Integrated Approach to Testing and Assessment (IATA). Application of the prediction model for assigning a test item to a reactivity class (i.e. low, moderate and high reactivity) may perhaps prove useful to inform potency assessment within the framework of an IATA.

Results and discussion

Positive control results:
Treatment with the positive control item revealed a cysteine and lysine peptide depletion of 69.85% for cysteine and 56.79% for lysine peptide. These values are within the required range of 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide.

In vitro / in chemico

Results
Key result
Parameter:
other: mean peptide depletion in %
Run / experiment:
1st experiment
Value:
8.85
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- No precipitate in the reaction mixture at the end of the incubation time and no co-elution were observed.

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

Any other information on results incl. tables

TABLE 1   Mean of cy steine and lysine % depletion

Test item Art. 851009

Cysteine peptide

Sample

no.

Peak area

Actual
peptide
[mM]

Peak area
reference
control C

Peptide
depletion
[%]

Corrected
peptide
depletion
[%]

Replicate 1
Replicate 2
Replicate 3

45.5235
45.6717
45 7667

0.471
0.473
0.474

49.0051
48.9476
48 6365

6.83
6.53
6.29

6.83
6.53
6.29

Mean
SD
CV

45.661
0.133
0.003

0.473
0.001
0.003

48.8631
0.198
0.004

6.55

0.272

0.042

6.55
0.272
0.042

Lysine peptide

Sample

no.

Peak area

Actual
peptide
[mM]

Peak area
reference
control C

Peptide
depletion
[%]

Corrected
peptide
depletion

[%]

Replicate 1
Replicate 2
Replicate 3

35.2319
35.0659
35.0171

0.437
0.435
0.435

39.4984
39.4912
39.5254

10.82
11.24
11.36

10.82
11.24
11.36

Mean
SD
CV

35.105
0.113
0.003

0.436
0.001
0.003

39.5050

0.0180

0.0005

11.14
0.285
0.026

11.14

0.285
0.026

Acceptance criteria:

SD % depletion Cysteine < 14.9 met:
SD % depletion Lysine < 11.6 met:

YES
YES

Result:

Art. 851009

 

 

 

Mean
depletion
(Cysteine + Lysine)
8.85

Reactivity
Class
(Cysteine + Lysine)
LOW

Reactivity
Class
(Cysteine only)
NEGATIVE

SD = standard deviation
CV = coefficient of variation

TABLE 1  Mean of cysteine and lysine % depletion (continued)

Positive control: Cinnamic aldehyde, vehicle: acetonitrile

Cysteine peptide

 

 

 

 

 

 

 

 

 

Sample

Peak area

Actual

Peak area

Peptide

Corrected

no.

 

peptide

reference

depletion

peptide

 

 

[mM]

control C

[%]

depletion
[%]

Replicate 1

14.9565

0.154

49.0051

69.39

69.39

Replicate 2

14.5266

0.150

48.9476

70.27

70.27

Replicate 3

14.7173

0.152

48.6365

69.88

69.88

Mean

14.733

0.152

48.8631

69.85

69.85

SD

0.215

0.002

0.1983

0.441

0.441

CV

0.015

0.015

0.0041

0.006

0.006

Lysine peptide

Sample

Peak area

Actual

Peak area

Peptide

Corrected

no.

 

peptide

reference

depletion

peptide

 

 

[mM]

control C

[%]

depletion
[%]

Replicate 1

16.7054

0.206

39.4984

57.71

57.71

Replicate 2

17.1234

0.212

39.4912

56.66

56.66

Replicate 3

17.3847

0.215

39.5254

55.99

55.99

Mean

17.071

0.211

39.5050

56.79

56.79

SD

0.343

0.004

0.0180

0.867

0.867

CV

0.020

0.020

0.0005

0.015

0.015

Acceptance criteria:

60.8 < Mean % depletion Cysteine < 100

YES

 

 

40.2< Mean % depletion Lysine < 69.0

YES

 

 

SD % depletion Cysteine < 14.9

 

YES

 

 

SD % depletion

Lysine < 11.6

 

YES

SD = standard deviation
CV = coefficient of variation

 

Applicant's summary and conclusion

Interpretation of results:
other: peptide depletion positive
Conclusions:
In this study under the given conditions the test item is considered positive regarding peptide depletion and predicted to be a sensitiser (low reactivity). The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation Adverse Outcome Pathway (AOP).
Executive summary:

A study was conducted according to OECD TG 442C in order to evaluate the reactivity of the test item towards cysteine (Cys-) and lysine (Lys-) containing peptides. The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 ± 2 hours incubation with the test item at 25 ± 2.5°C. The test item was dissolved at a concentration of 100 mM in acetonitrile. Relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm.

Cinnamic aldehyde was used as positive control at a concentration of 100 mM in acetonitrile. Treatment with the positive control item revealed a cysteine and lysine peptide depletion of 69.85% for cysteine and 56.79% for lysine peptide. These values are within the required range of 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide.

Test item-treated samples revealed a cysteine peptide depletion of 6.55% and a lysine peptide depletion of 11.14% (mean peptide depletion of 8.85%) and, hence, were below 22.62% and above 6.38%. The test item is considered positive and predicted to be a sensitiser (low reactivity) in the Direct Peptide Reactivity Assay (DPRA).