Adenosine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

20 - 24 October 2017

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))

Version / remarks:

2015

Deviations:

yes

Remarks:

Due to the nature of the test item and its incompatibility with any solvents known to be suitable for cysteine testing, this DPRA study was terminated prior to its completion.

GLP compliance:

no

Remarks:

Due to the nature of the test item and its incompatibility with any solvents known to be suitable for cysteine testing, this DPRA study was terminated prior to its completion.

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Adenosine

EC Number:

200-389-9

EC Name:

Adenosine

Cas Number:

58-61-7

Molecular formula:

C10H13N5O4

IUPAC Name:

adenosine

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

Test material form:

solid: particulate/powder

Details on test material:

Batch No.: 10004539
Storage at room temperature approx. 20°C

In chemico test system

Details on the study design:

SUMMARY: DPRA measures the reaction of the test item with synthetic peptides containing cysteine (Ac-RFAACAA-COOH) or lysine (Ac-RFAAKAA-COOH). The custom peptides contained cysteine or lysine as the nucleophilic reaction centres and phenylalanine to facilitate HPLC detection. Test item and peptide were combined and incubated together for 24 h at room temperature. Following this incubation, the concentration of free (i.e. unreacted) peptide remaining was measured by HPLC immediately prior to the lysine peptide assay.


EXPERIMENTAL PROCEDURES

PEPTIDES:
Source: RS Synthesis
Batch:
- Lysine: Catalogue No. 110716-2, Batch No. P161108-LC107617, purity 98.14%

BUFFERS USED:
- Ammonium acetate buffer: ca 100 mM, pH 10.2

SOLUBILITY ASSESSMENT:
- NMP:DMSO (1:1, v/v) was selected as the most suitable solvent for the test material

PREPARATION PEPTIDE STOCK SOLUTIONS:
- LYSINE: stock solution of 0.667 mM in ammonium acetate buffer

LYSINE PEPTIDE ASSAY:
-PREPARATION: Adenosine was dissolved in NMP:DMSO (1:1, v/v) at a final concentration of 101 mM (101% of target). Cinnamic aldehyde was dissolved in acetonitrile at a final concentration of 101 mM (101% of target). All solutions were prepared immediately prior to use.
- PREPARATION OF THE STANDARD CURVE: Dilution buffer was prepared by mixing ammonium acetate buffer (pH 10.2, 8 mL) with acetonitrile (2 mL). Standard 1 (STD1-Lys) was prepared by mixing lysine peptide stock solution (1600 µL) with acetonitrile (400 µL). Serial dilutions (1:1, v/v) were prepared from this to make a standard curve (STD1-Lys to STD6-Lys). An additional sample containing only dilution buffer was included as a blank (0 mM) standard. The standard curve was analyzed by HPLC.
- REFERENCE CONTROL:Acetonitrile (250 µL) was mixed with lysine peptide stock solution (750 µL) Three replicates of this were produced for Reference Control A, which were used to determine the accuracy of the lysine peptide calibration curve.. Six replicates of Reference Control B were prepared as described for Reference Control A. Three replicates were analysed at the beginning of the testing run, and three at the end of the testing run, to demonstrate peptide stability over the analysis time. Two groups of Reference Control C samples were prepared containing either acetonitrile (250 µL) and lysine peptide stock (750 µL), or NMP:DMSO (1:1, v/v, 250 µL) and lysine peptide stock (750 µL). Three replicates of each were distributed throughout the run. These samples were included in the assay run together with the samples containing each test item to verify that the solvent does not impact upon peptide stability during the assay, and to calculate percentage peptide depletion.
- PEPTIDE ASSAY METHOD: TThe lysine assay contained a 1:50 molar ratio of peptide to test item. Cinnamic aldehyde (positive control) or test item (250 µL) were mixed with lysine peptide stock (750 µL). The vials were mixed by inversion. The co-elution control was prepared by mixing together ammonium acetate buffer (750 µL) and test item (250 µL). All vials were stored in the dark at ambient temperature for 24-36 h until analysed by HPLC.

CHROMATOGRAPHIC AND DETECTOR PARAMETERS
- Column: Phenomenex Luna C18 (2) (2 x 100 mm, 3 µm)
- Run Time: 20 min
- Mobile Phase Conditions: Mobile Phase A: trifluoracetic acid (0.1%, v/v) in Milli-Q H2O
Mobile Phase B: trifluoracetic acid (0.085%, v/v) in acetonitrile
- Flow Rate: 0.35 mL/min
- Column Temperature: 30°C
- Auto Sampler Temperature: Room temperature
- Injection Volume: 7 µL
- UV Wavelength: 220 nm
- HPLC Gradient: see below

CALCULATIONS:
The concentration of peptide remaining in each sample following incubation was calculated from integrated peak area, with reference to the peptide standard curve. Percent peptide depletion was calculated from the following formula:
Peptide Depletion (%) = 1 – ( Peak Area (Sample) / Mean Peak Area (Reference Control C)) x 100

Results and discussion

Positive control results:

The % lysine depletion value for the positive control was 48.6%

In vitro / in chemico

Results

Run / experiment:

other: DPRA cysteine and lysine prediction model

Parameter:

other: % Lysisne Peptide Depletion

Value:

-1.2

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

Cinnamic aldehyde

Remarks on result:

not determinable

Other effects / acceptance of results:

System Suitability for the Lysine Assay:
The calibration linearity, r2, for the lysine standard curve was 0.9998. This met the acceptance criteria for r2 which was >0.990.
The mean peptide concentration of Reference Control A was 0.501 ± 0.009 mM (mean ± SD).
The calculated peptide concentration in the Reference Control C samples in acetonitrile was 0.499 ± 0.005 mM. The calculated peptide concentration in the Reference Control C samples in NMP:DMSO was 0.494 ± 0.007 mM. These controls met the acceptance criteria (0.5 ± 0.05 mM).
For the six Reference Control B and three Reference Control C samples in acetonitrile, the coefficient of variation (CV) was 1.1% (acceptance criteria for CV was <15%).
The mean percentage peptide depletion value of the three replicates for cinnamic aldehyde fell within the lower bound and upper bound values of 40.2% and 69.0% for lysine, with the SD <11.6%. The actual percentage peptide depletion value reported for cinnamic aldehyde was 48.6% ± 2.4% (mean ± SD).
Finally, the standard deviation of replicate test item samples was <11.6% for the test item (actual SD was 1.2%).
The time between sample preparation and the last injection of a sequence was not greater than 48 h.

Guideline deviations:
Due to the nature of the test item and its incompatibility with any solvents known to be suitable for cysteine testing, this DPRA study was terminated prior to its completion.

Adenosine binding with the lysine peptide resulted in -1.2% ± 1.2% peptide depletion and no co-elution of the test item with the lysine peptide was observed. Due to the nature of the test item and its incompatibility with any solvents known to be suitable for cysteine testing, this DPRA study was terminated prior to its completion.

Any other information on results incl. tables

Test Item	% Peptide Depletion Lysine (Mean ± SD)
Adenosine	-1.2 ± 1.2
Positive control (cinnamic aldehyde)	48.6 ± 2.4

Classification cannot be determined by the lysine assay alone and, therefore, reactivity of Adenosine (CAS No. 58-61-7) was inconclusive.

Applicant's summary and conclusion

Interpretation of results:

other: Classification cannot be determined by the lysine assay alone and was inconclusive.

Conclusions:

In conclusion, the reactivity of Adenosine (CAS No. 58-61-7) was inconclusive due to the incompatibility of the test item.

Executive summary:

Skin sensitisation is a type IV (delayed) hypersensitivity reaction that results from the interaction of a sensitising agent with host proteins to form an immunogenic complex.

Small molecules that can interact with proteins in this way are referred to as haptens, and are generally not immunogenic in isolation.  Hapten-modified proteins are recognised as foreign by antigen presenting cells, leading to T-cell activation and localised inflammation at the site of all subsequent exposures to the hapten.

Most skin sensitising agents are electrophiles, i.e. will accept an electron pair from a nucleophile to form a covalent bond.  The amino acids, cysteine and lysine, are thought to be the nucleophiles most frequently modified in proteins during sensitisation, and the ability of small molecules to react with these amino acids forms the basis of the Direct Peptide Reactivity Assay (DPRA).

The objective of this study was to assess the peptide binding capabilities of Adenosine (CAS No. 58-61-7) using synthetic cysteine and lysine peptides and to classify the test item to one of the four reactivity classes leading to a DPRA prediction according to the prediction model.

Due to the nature of the test item and its incompatibility with any solvents known to be suitable for cysteine testing, this DPRA study was terminated prior to its completion.

The solubility of the test item was assessed and 1 methyl-2-pyrrolidinone (NMP): dimethyl sulfoxide (DMSO), (1:1, v/v) chosen as the appropriate solvent.  Test item and lysine peptide were combined and incubated together for ca 24-36 h at room temperature.  Following this incubation, the concentration of free (i.e. unreacted) peptide remaining was measured by HPLC.  The percent lysine peptide depletion values calculated for the test item are presented in the table below.  All acceptance criteria for the lysine assay were met, however, classification cannot be determined by the lysine assay alone and, therefore, reactivity of Adenosine (CAS No. 58-61-7) was inconclusive.

 

Test Item	% Lysine Peptide Depletion (Mean ± SD)
Adenosine	-1.2 ± 1.2
Cinnamic Aldehyde (Positive Control)	48.6 ± 2.4

In conclusion, the reactivity of Adenosine (CAS No. 58-61-7) was inconclusive due to the incompatibility of the test item.