Methylthiouracil

Skin sensitisation

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

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

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

Test material information

Constituent 1

Reference substance name:

Methylthiouracil

EC Number:

200-252-3

EC Name:

Methylthiouracil

Cas Number:

56-04-2

Molecular formula:

C5H6N2OS

IUPAC Name:

methylthiouracil

Test material form:

solid: crystalline

In chemico test system

Details on the 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

Results

Results 1

Key result

Parameter:

cysteine depletion

Value:

90.93 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Parameter:

cysteine depletion

Value:

5.34 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

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).