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
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))
GLP compliance:
yes
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
The correlation of protein reactivity with skin sensitisation potential is well established. Haptenation i.e. covalent binding of low molecular weight substances (Haptens) to proteins present in skin is considered a prominent mechanism through which chemicals or their metabolites become antigenic. Therefore, information inferred from the DRPA assay is relevant for assessment for skin sensitisation potential of substances.

Test material

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

In chemico test system

Details on study design:
On the day of test sample preparation, appropriate amount of cysteine and lysine peptide was weighed and dissolved in phosphate buffer and ammonium acetate buffer respectively. The stock concentration of cysteine peptide prepared was 0.501 mg/mL (0.667 mM) and lysine peptide was 0.518 mg/mL (0.667 mM). The preparations were done immediately before use.

Test item and positive control solutions were prepared at 100 mM concentration and used in the test sample preparation for analysis. Test item, positive control and reference control was added to cysteine and lysine peptides and incubated in dark at 25°C for 24±2 hours. Post incubation, the test samples were analysed using HPLC to measure the peptide depletion.


HPLC ANALYSIS :
1. Agilent Zorbax SB-C18 2.1 mm X 100 mm X 3.5 micron (or alternate column: Phenomenex Luna C18 (2) 2.0 mm X 100 mm X 3 micron) column was installed in the HPLC system.
2. HPLC Analysis was performed using a flow 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 to remove other materials.
3. Equal volumes of each standard, sample and control were injected onto the column. Injection volume range of 10 µL. Absorbance is monitored at 220 mm.
4. Column was re-equilibrated under initial conditions.

DATA ANALYSIS:
The concentration of cysteine or lysine peptides 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.

Results and discussion

Positive control results:
Positive control cinnamaldehyde showed 74.96% mean cysteine depletion and 51.27% mean lysine peptide depletion.

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: % of cysteine peptide depletion
Value:
90.93
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Parameter:
other: % of lysine peptide depletion
Value:
5.34
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes

Any other information on results incl. tables

Table 1.    Mean and SD of percent peptide depletion for cysteine and lysine.

For Cysteine peptide

Sample ID

Peak area of cysteine peptide

% peptide depletion

Mean % Peptide Depletion

SD

Cinnamic aldehyde-replicate 1

954336.0

72.77

74.96

1.90

Cinnamic aldehyde-replicate 2

835304

76.17

Cinnamic aldehyde-replicate 3

843654

75.93

K010-01 replicate 1

309768.0

91.16

90.93

2.72

K010-01 replicate 2

226878.0

93.53

K010-01 replicate 3

417155

88.10

 

For Lysine peptide

Sample ID

Peak area of Lysine peptide

% peptide depletion

Mean % Peptide Depletion

SD

Cinnamic aldehyde-replicate 1

1257486

59.35

51.27

12.59

Cinnamic aldehyde-replicate 2

1308743

57.69

*Cinnamic aldehyde-replicate 3

1956255

36.76

K010-01 replicate 1

3287685

-6.29

5.34

10.09

K010-01 replicate 2

2727511

11.82

K010-01 replicate 3

2768916

10.49

K010-01:6-Methyl-2-Thiouracil, %: Percent, SD: Standard deviation,        

*: replicate 3 is an slight outlier

Table 2. Reactivity class classification of test item and positive control as per cysteine 1:10/lysine 1:50 prediction model.

Sample ID/ Code

Name of the chemical

Mean % of Cysteine Peptide Depletion

Mean % of Lysine Peptide Depletion

Mean % peptide depletion

Reactivity class 

DPRA prediction

CA

Cinnamic aldehyde

74.96

51.27

63.12

High Reactivity

Positive

K010-01

6-Methyl-2-Thiouracil

90.93

5.34

48.14

High Reactivity

Positive

Applicant's summary and conclusion

Interpretation of results:
other: Positive result. Can be used in assessing weight of evidence for skin sensitisation
Conclusions:
The mean of cysteine and lysine peptide depletion by the positive control (cinnamaldehyde) was 63.12% which shows that the positive control is a sensitizer with high reactivity confirming the sensitivity of the assay. The mean of cysteine and lysine peptide depletion by the test item [6-Methyl-2-Thiouracil] was 48.14% which shows that the test item has high reactivity on the skin sensitisation potential. Under the testing conditions, test item [6-Methyl-2-Thiouracil] was concluded as positive or sensitiser with high reactivity in Direct Peptide Reactivity Assay (DPRA).
Executive summary:

Skin sensitisation test by in chemico test method, Direct Peptide Reactivity Assay (DPRA) was carried out using the nucleophile containing synthetic cysteine and lysine peptides to evaluate the skin sensitisation potential of the test item, 6-Methyl-2-Thiouracil.

Solubility check was performed for the test item before test sample analysis. The test item, 6-Methyl-2-Thiouracil, was insoluble in Acetonitrile, acetonitrile: water (1:1), Isopropanol, Acetone, Acetone: Acetonitrile (1:1), 10% DMSO at 100 mM concentrations. Test item was found soluble in 50% DMSO (with 50% Acetonitrile) at 100 mM concentration and formed a homogenous solution. Hence, 50% DMSO (with 50% Acetonitrile) was selected as solvent.

On the day of test sample preparation, appropriate amount of cysteine and lysine peptide was weighed and dissolved in phosphate buffer and ammonium acetate buffer respectively. The stock concentration of cysteine peptide prepared was 0.501 mg/mL (0.667 mM) and lysine peptide was 0.518 mg/mL (0.667 mM). The preparations were done immediately before use.  

Test item and positive control solutions were prepared at 100 mM concentration and used in the test sample preparation for analysis. Test item, positive control and reference control was added to cysteine and lysine peptides and incubated in dark at 25°C for 24±2 hours. Post incubation, the test samples were analysed using HPLC as per section 9 to measure the peptide depletion.

Cysteine and lysine peptide percent depletion values are then calculated from peptide peak areas obtained from the HPLC analysis. The test item 6-Methyl-2-Thiouracil showed 90.93% mean cysteine peptide depletion and 5.34% mean lysine peptide depletion. Under the same conditions positive control cinnamaldehyde showed 74.96% mean cysteine depletion and 51.27% mean lysine peptide depletion.

To classify test item as sensitizers and non- sensitizers, the cysteine 1:10/lysine 1:50 prediction was used. The mean of cysteine and lysine peptide depletion by the positive control (cinnamaldehyde) was 63.12% which shows that the positive control is a sensitizer with high reactivity confirming the sensitivity of the assay. The mean of cysteine and lysine peptide depletion by the test item [6-Methyl-2-Thiouracil] was 48.14% which shows that the test item has high reactivity on the skin sensitisation potential. Under the testing conditions, test item [6-Methyl-2-Thiouracil] was concluded as positive or sensitiser with high reactivity in Direct Peptide Reactivity Assay (DPRA).