Adenosine phosphate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

31 March - 7 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

Time between sample preparation and the last injection exceed 35h(ca 36 and 38 h). Reference control C didn't meet the acceptance criteria. There was no impact on study integrity

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Adenosine phosphate

EC Number:

200-500-0

EC Name:

Adenosine phosphate

Cas Number:

61-19-8

Molecular formula:

C10H14N5O7P

IUPAC Name:

adenosine phosphate

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: crystalline

Remarks:

crystalline powder

Details on test material:

Batch No: 11679700
Storage: at room temperature, protected from light

In vitro 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:
- Cys: No. P170419LC180433
- Lys: No. P161108-LC107617
Purity:
-Cys: 96.47%
-Lys: 98.14%

BUFFERS USED:
- Phosphate buffer: ca 100 mM, pH 7.49
- Ammonium acetate buffer: ca 100 mM, pH 10.16

SOLUBILITY ASSESSMENT:
- Sodium hydroxide in water (125 mM) was selected as the most suitable solvent for the test material

STABILITY ASSESSMENT:
-The stability of each peptide was assessed in various solvents by mixing cysteine peptide (0.667 mM, 0.501 mg/mL in phosphate buffer, Section 8.1, 750 µL) with acetonitrile (50 µL) and each of the various solvents (200 µL) or mixing lysine peptide (0.518 mg/mL in ammonium buffer, 750 µL) with each of the various solvents (250 µL). Each sample was mixed by vortex, incubated for ca 24 h at ambient temperature and analysed by HPLC. Sodium phosphate dibasic heptahydrate, NMP:DMSO (1:1, v/v), ammonium acetate buffer and sodium hydroxide in water (125 mM) were investigated as potential solvents. Sodium hydroxide in water (125 mM) was selected as the appropriate solvent.


PREPARATION PEPTIDE STOCK SOLUTIONS:
- CYSTEINE: stock solution of 0.501 mg/mL (0.667 mM) in phosphate buffer
- LYSINE: stock solution of 0.518 mg/mL (0.667 mM) in ammonium acetate buffer

CYSTEINE PEPTIDE ASSAY:
-PREPARATION: Test item was dissolved in sodium hydroxide in water (125 mM, 3.04 mL) and mixed by inversion and vortex until fully in solution. The concentration of the test solution corrected for purity, was 34.7 mg/mL, 100 mM (100% of target, 100 mM). Cinnamic aldehyde was dissolved in acetonitrile with concentration of 13.2 mg/mL (100.1 % of target, 100 mM). All test item and control solutions/suspensions were prepared immediately prior to use.
-PREPARATION OF THE SANDARD CURVE: Dilution buffer was prepared by mixing phosphate buffer (pH 7.49, 8 mL) with acetonitrile (2 mL). Standard 1 (STD1) was prepared by mixing peptide stock solution (1600 µL) with acetonitrile (400 µL). Serial dilutions (1:1, v/v) were prepared from this to make a standard curve (from 0.534 to 0.0167 mM). An additional sample containing only dilution buffer was included as a blank (0 mM) standard. The standard curve was analysed by HPLC immediately prior to the cysteine peptide assay.
-REFERENCE CONTROL: Acetonitrile (250 µL) was mixed with peptide stock solution (750 µL). Three replicates of this were produced for Reference Control A. Reference Control B was prepared as described for Reference Control A. Three replicates were analyzed at the beginning of the testing run, and three at the end of the testing run, to demonstrate peptide stability over the analysis time. Reference Control C samples were prepared containing the solvent that the test item was dissolved in: three replicates containing acetonitrile (250 µL) and cysteine peptide stock (750 µL) and three replicates containing sodium hydroxide in water (125 mM, 50 µL), acetonitrile (200 µL) and cysteine peptide stock (750 µL). These samples were included in every assay run together with the samples containing test item. They are used to verify that the solvent does not impact upon peptide stability during the assay, and to calculate percentage peptide depletion.
- PEPTIDE ASSAY METHOD: The assay contained a 1:50 molar ratio of peptide to test item. Positive control or test item (50 µL) was mixed with acetonitrile (200 µL) and the peptide solution (750 µL). The vials were mixed by vortex. Co-elution controls were prepared by mixing together acetonitrile (200 µL), phosphate buffer (750 µL) and test item (50 µL). All test items and positive control samples were prepared in triplicate. All vials were stored in the dark at ambient temperature for ca 24 h until analyzed by HPLC.

LYSINE PEPTIDE ASSAY:
-PREPARATION: Test item was dissolved in sodium hydroxide solution (125 mM, 2.994 mL) and mixed by inversion and vortex until fully in solution. The concentration of the test solution corrected for purity was 34.7 mg/mL (100 mM, 100% of target). Cinnamic aldehyde was dissolved in acetonitrile with concentration of 13.2 mg/mL(100 % of target, 100 mM). All test item and control solutions/suspensions were prepared immediately prior to use.
- PREPARATION OF THE STANDARD CURVE: Dilution buffer was prepared by mixing ammonium acetate buffer (pH 10.6, 8 mL) with acetonitrile (2 mL). Standard 1 (STD1) was prepared by mixing peptide stock solution (1600 µL) with acetonitrile (400 µL). Serial dilutions (1:1, v/v) were prepared from this to make a standard curve (from 0.534 to 0.0167 mM). An additional sample containing only dilution buffer was included as a blank (0 mM) standard. The standard curve was analyzed by HPLC.
- REFERENCE CONTROL: like for cysteine
- PEPTIDE ASSAY METHOD: The assay contained a 1:50 molar ratio of peptide to test item. Cinnamic aldehyde or test item (250 µL) were mixed with peptide solution (750 µL). The vials were mixed by inversion and vortex. Co-elution controls were 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 ca 24 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 mean depletion value for the positive control was 70.3% showing a high reactivity (Sensitizer)

In vitro / in chemico

Results

Key result

Run / experiment:

other: DPRA cysteine and lysine prediction model

Parameter:

other: % Peptide Depletion (mean value)

Value:

0.65

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

Cinnamic aldehyde

Remarks on result:

other:

Remarks:

Minimal reactivity (Non-sensitizer)

Other effects / acceptance of results:

No co-elution of the test item with either peptide was observed.

SYSTEM SUITABILITY FOR THE CYSTEINE ASSAY
The calibration linearity, r2, for the cysteine standard curve was 0.9995. This met the acceptance criteria for r2 which was >0.990.

The mean peptide concentration of Reference Control A was 0.4919 mM ± 0.004 mM (mean ± SD).

The calculated peptide concentration in the Reference Control C samples was 0.4848 mM ± 0.004 mM (acetonitrile), and 0.4353 mM ± 0.008 mM (sodium hydroxide in water, 125 mM). Reference Control C (acetonitrile) met the acceptance criteria of 0.5 mM ± 0.05 mM. In addition, for the six Reference Control B and the three Reference Control C samples in acetonitrile, the coefficient of variation (CV) was 2.2% (acceptance criteria for CV was <15%).
The calculated peptide concentration in the Reference Control C (NaOH in water, 125 mM) samples was 0.4353 mM ± 0.008 mM. This did not meet the acceptance criteria (0.5 mM ± 0.05 mM). The data was consistent across all three replicates. All other Reference Control C samples had an acceptable peptide concentration, therefore this has been identified as a solvent effect (an alkaline solution) which was not identified during stability testing. The physico-chemical properties of the test item appear to have mitigated this effect, and therefore restored the stability of the peptide, therefore, these samples were not affected. Due to the slightly low peak area for Reference Control C (NaOH), the percent peptide depletion for the test item samples was calculated to be negative. However, their peak areas are comparable to the other Reference Control C samples, suggesting that the peptide is not being bound. In one test item sample the depletion is calculated as <-10%. However, there was no evidence of coelution of test item and peptide. Even if a worst case result were to be used (i.e. depletion calculated using the ideal Reference Control C values of ca 1550, the prediction would not change: there was no peptide depletion and the test item was not a sensitiser. Therefore, the results have been accepted, and there is no impact on study integrity.

The mean percentage peptide depletion value of the three replicates for cinnamic aldehyde fell within the lower bound and upper bound values of 60.8% and 100.0% for cysteine, with an SD of <14.9%, with a peptide depletion value of 83.8 ± 0.4% (mean ± SD).

Finally, the standard deviation of replicate test item samples was <14.9% for AMP (actual SD was 1.2%).

SYSTEM SUITABILITY FOR THE LYSINE ASSAY
The calibration linearity, r2, for the lysine standard curve was 0.9999. This met the acceptance criteria for r2 which was >0.990.
The mean peptide concentration of Reference Control A was 0.4770 mM ± 0.004 mM (mean ± SD).
The calculated peptide concentration in the Reference Control C samples was 0.4849 mM ± 0.002 mM (acetonitrile) and 0.5029 mM ± 0.001 mM (Sodium hydroxide in water, 125 mM). These samples met the acceptance criteria of 0.5 mM ± 0.05 mM. In addition, for the six Reference Control B and the three Reference Control C samples in acetonitrile, the coefficient of variation (CV) was 0.5% (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 56.7% ± 0.8% (mean ± SD).
Finally, the standard deviation of replicate test item samples was <14.9% for AMP (actual SD was 0.6%).

PROTOCOL DEVIATIONS
The study was performed in accordance with the protocol with the following deviations:
1) The protocol states the acceptance criteria for the test. The calculated peptide concentration in the Reference Control C (NaOH in water, 125 mM) samples was 0.4353 mM ± 0.008 mM. This did not meet the acceptance criteria (0.5 mM ± 0.05 mM). The data was consistent across all three replicates. All other Reference Control C samples had an acceptable peptide concentration, this was identified as a solvent effect (in alkaline solution) which was not identified during stability testing. The physico-chemical properties of the test item appear to have mitigated this effect, and therefore restored the stability of the peptide. Therefore, these samples were not affected. Due to the slightly low peak area for Reference Control C (NaOH), the percent peptide depletion for the test item samples was calculated to be negative. However, their peak areas are comparable to the other Reference Control C samples, suggesting that the peptide is not being bound. Even if a worst case result were to be used (i.e. depletion calculated using the ideal Reference Control C values of ca 1550, the prediction would not change. There is no peptide depletion and the test item is not a sensitiser. Therefore, the results have been accepted, and there is no impact on study integrity.
2) The protocol states that samples will be assessed visually prior to HPLC analysis however there is no record that this was done for the stability samples or lysine assay samples. There is no evidence of any precipitate forming and reference control B samples (analysed at the start and end of the run) had an acceptable CV. Therefore there is no impact on the study integrity.
3) The protocol states that the time between sample preparation and the last injection should not exceed 35 h. However in both the cysteine and the lysine runs this time was exceeded. There was ca 38 h between lysine sample preparation and the last injection and just under 36 h between cysteine sample preparation and the last injection. However the samples remained stable over this time, as shown by the CV of reference control B samples, and the peak areas, which had not changed. Therefore there was no impact on study integrity.

DEMONSTRATION OF TECHNICAL PROFICIENCY
Prior to use, Charles River Laboratories demonstrated technical proficiency in the DPRA test, using the panel of proficiency chemicals listed in OECD 442C (Toner, F, 2015).

Any other information on results incl. tables

Test Item	% Peptide Depletion Cysteine (Mean  ± SD)	% Peptide Depletion Lysine (Mean ± SD)	Mean of Cysteine and Lysine	DPRA Classification (Cysteine and Lysine Prediction Model)
AMP	0.0±1.2	1.3 ± 0.6	0.65	Minimal Reactivity (Non-Sensitizer)
Cinnamic Aldehyde (Positive Control)	83.8 ± 0.4	56.7 ± 0.8	70.3	High Reactivity (Sensitizer)

Using the cysteine and lysine prediction model (see Table below) the test material was categorised as minimally reactive and a non-sensitiser.

Mean depletion values (Cys Lys)	Mean Depletion values (cys only)	Reactivity classification	DPRA Prediction
<6.38 %	<13.89%	Minimal	Non Sensitizer
6.38 -22.62%	13.89 -23.09%	Low	Sensitizer
22.62 -42.47%	23.09%-98.24%	Moderate	Sensitizer
>42.47	>98.24%	High	Sensitizer

Applicant's summary and conclusion

Interpretation of results:

other: minimally reactive: non-sensitizer

Remarks:

Study will be used for classificatin in combination with other studies (Weight of Evidence)

Conclusions:

In conclusion, according to the DPRA cysteine and lysine prediction model, AMP (CAS no. 61-19-8) was classified as minimally reactive and was, therefore, a non-sensitiser.

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 capability of Adenosine-5’-monophosphate (AMP, CAS no. 61-19-8) 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 following prediction model.

Mean depletion values (Cys Lys)	Mean Depletion values (cys only)	Reactivity classification	DPRA Prediction
<6.38 %	<13.89%	Minimal	Non Sensitizer
6.38 -22.62%	13.89 -23.09%	Low	Sensitizer
22.62 -42.47%	23.09%-98.24%	Moderate	Sensitizer
>42.47	>98.24%	High	Sensitizer

This method of classification has been adopted in the Classification, Labelling and Packaging (CLP) regulation.

The reaction of the test item with synthetic peptides containing cysteine (Ac-RFAACAA-COOH) or lysine (Ac-RFAAKAA-COOH) was performed.  The custom peptides contained cysteine or lysine as the nucleophilic reaction centres and phenylalanine to facilitate detection by HPLC analysis.

The solubility of the test item was assessed and sodium hydroxide in water (125 mM) was selected as the appropriate solvent.  A stability assessment determined that both Cysteine and Lysine peptides were stable in sodium hydroxide in water (125 mM).  The test item was prepared at a concentration of 100 mM.  The test item and peptides were combined and incubated together for ca 24 h at room temperature.  Following this incubation, the concentration of free (i.e. unreacted) peptide remaining was measured by HPLC.  From the results obtained, a reactivity class was assigned and a DPRA prediction was made according to the above criteria.  Both peptide assays were successfully run with all acceptance criteria being met in the lysine assay.  One of the acceptance criteria was not met in the Cysteine assay, however, the assay was accepted as this did not affect the prediction.

The results obtained are presented in the following table:

Test Item	% Peptide Depletion Cysteine (Mean  ± SD)	% Peptide Depletion Lysine (Mean ± SD)	Mean % peptide depletion	DPRA Classification (Cysteine and Lysine Prediction Model)
AMP	0.0±1.2	1.3 ± 0.6	0.65	Minimal Reactivity (Non-Sensitizer)
Cinnamic Aldehyde (Positive Control)	83.8 ± 0.4	56.7 ± 0.8	70.3	High Reactivity (Sensitizer)

Peptide depletion was calculated as 0.0% and 1.3% in Cysteine and Lysine Assays, respectively, resulting in a mean peptide depletion of 0.65%.  This value places AMP in the Minimal Reactivity Classification resulting in a DPRA prediction of Non-Sensitiser.

In conclusion, according to the DPRA cysteine and lysine prediction model, AMP (CAS no. 61-91-8) was classified as minimally reactive and was, therefore, a non-sensitiser.