Adenosine phosphate

Endpoint summary

Description of key information

A DEREK assessment, DPRA assay (OECD 442C) and KeratinoSens assay (OECD 442D) were performed.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

19 December 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE: DEREX NEXUS
2. MODEL (incl. version number): DEREK NEXUS 5.0.2.
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL: C([C@@H]1[C@@H](O)[C@@H](O)[C@@H](O1)N2C=NC=3C(N)=NC= NC2=3)OP(=O)(O)O
4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL: See the QMRF in the study document attached
5. APPLICABILITY DOMAIN (OECD principle 3)

- Descriptor domain:The scopes of the structure-activity relationships describing the skin sensitisation endpoint are defined by the developer to be the applicability domain for the model. Therefore, if a chemical activates an alert describing a structure-activity for skin sensitisation it can be considered to be within the applicability domain. The applicability of potency predictions may be judged, and modified, by the user based on the displayed data for nearest neighbours. If a compound does not activate an alert or reasoning rule in Derek, a result of ‘no alerts fired’ is presented to the user. This can be interpreted as a negative prediction or that the query compound is outside the domain of the model. Which of these is more appropriate may depend on the endpoint of interest. For the endpoint of skin sensitisation, which features multiple alerts believed to cover most of the mechanisms and chemical classes responsible for activity, ‘no alerts fired’ may be extrapolated to a negative prediction

- Structural fragment domain: DEREK NEXUS is applicable to organic structures including their salts, e.g. sodium, potassium. For skin sensitisation, which features 80 alerts believed to cover most of the mechanisms and chemical classes responsible for activity, ’no alerts fired’ may be extrapolated to a negative prediction. The present structure has no extraordinary features and is considered to fall within the applicability domain.

- Mechanism domain: as the prediction is ‘no alerts fired’ none of the mechanisms predicted in the 80 skin sensitisation alerts is applicable to this structure.

-Metabolic domain: no evident metabolism that might lead to skin sensitization is predicted for this structure

6. ADEQUACY OF THE RESULT
-Regulatory purpose: The present prediction may be used for preparing the REACH Registration Dossier on the substance for submission to ECHA as required by Regulation (EC) 1907/2006 and related amendments.
- Approach for regulatory interpretation of the model result: This result can be directly used within a weight-of-evidence approach to complete the endpoint skin sensitization.

Guideline:

other: REACH Guidance on QSARs R.6

Version / remarks:

Prediction on the potential for skin sensitization with the in silico model DEREK NEXUS, version 5.0.2.

Principles of method if other than guideline:

- Software tool(s) used including version: in silico model DEREK NEXUS version 5.0.2
- Knowledge Base: Derek KB 2015 2.0
- Model description: see field 'Justification for non-standard information', 'Attached justification'
- Justification of QSAR prediction: see field 'Justification for type of information', 'Attached justification'

GLP compliance:

no

Specific details on test material used for the study:

SMILES:C([C@@H]1[C@@H](O)[C@@H](O)[C@@H](O1)N2C=NC=3C(N)=NC= NC2=3)OP(=O)(O)O

Parameter:

other: alerts for skin sensitization

Remarks on result:

other: DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item. Adenosine 5’-monophosphate (AMP) is predicted to be not sensitizing to the skin.

Interpretation of results:

other: Non-Sensitizer

Conclusions:

DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item. Adenosine-5’-monophosphate (AMP) (CAS Nr. 61-19-8) is predicted to be not sensitizing to the skin.

Executive summary:

The objective of this study was to obtain a prediction on the potential for skin sensitization of Adenosine-5’-monophosphate (AMP) (CAS Nr. 61-19-8) with the in-silico model DEREK NEXUS. In this assessment version 5.0.2 of DEREK NEXUS was used.

DEREK NEXUS is a knowledge-based system that contains 80 alerts for skin sensitization based on the presence of molecular substructures. LHASA has inserted validation comments for the skin sensitization alerts:The DEREK NEXUS system has been designed for the qualitative prediction of the possible toxicity of chemicals. The predictions made by DEREK NEXUS are intended as an aid to toxicological assessment and, where appropriate, should be used in conjunction with other methods. “No alerts fired” may be extrapolated to a negative prediction.

DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item Adenosine-5’-monophosphate (AMP) (CAS Nr. 61-19-8)

Therefore, substance should not be classified according to DEREK NEXUS; however, this (Q)SAR prediction cannot be used as stand-alone for classification purposes or for covering the endpoint skin sensitization for registration under REACH.

The result is adequate to be used in a weight-of-evidence approach together within chemico/in vitro studies to complete the endpoint skin sensitization.

Reference 2

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

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)

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

Positive control results:

The mean depletion value for the positive control was 70.3% showing a high reactivity (Sensitizer)

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).

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

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.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

20 February 2017 - 9 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

2015

Deviations:

yes

Remarks:

yes In the second experiment, the average coefficient of variation of the luminescence reading for t he negative (solvent) control DMSO was 22.5%.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

- Test concentrations: final test concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95 and 0.977 µM (final concentration vehicle DMSO of 1%)
- All concentrations of the test item were tested in triplicate.
- Positive control: Ethylene dimethacrylate glycol, final concentration 7.81 to 250 µM (final concentration DMSO of 1%)
- Negative control: vehicle: 1% DMSO in exposure medium

- Test System A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switzerland).

- Cell culture:
Basic medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.
Manteinance Medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/ml).
Exposure medium: Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

- Environmental conditions:
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 60 – 93 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.9 – 37.3°C).

EXPERIMENTAL DESIGN
- Plating of cells
For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each test item, one plate was used for the luciferase activity measurements, and one parallel plate was used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was 21 in experiment 1 and 25 in experiment 2.

- Treatment of cells
The medium was removed and replaced with fresh culture medium (150 µL culture medium containing serum but without Geneticin) to which 50 µL of the 25-fold diluted test chemical and control substances were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours at 37±1.0 °C in the presence of 5% CO2. In total 2 experiments were performed.

- Luciferase activity measurement
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the luminometer to assess the quantity of luciferase (integration time one second).
- Cytotoxicity assessment
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.81 to 250 µM).
b) The EC1.5 should be between 5 and 125 µM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 µM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

INTERPRETATION
- Data analysis
The following parameters are calculated in the KeratinoSensTM test method:
• The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
• The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained
• The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.
In case the luciferase activity induction is larger than 1.5 fold, statistical significance is shown by using a two-tailed Student’s t-test, comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data. The lowest concentration with > 1.5 fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.

- Data interpretation
A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 µM (or < 200 µg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction

Positive control results:

• Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.40 and the EC1.5 24.4 µM.
• Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 6.17 and the EC1.5 15.8 µM.

Key result

Run / experiment:

other: 1

Parameter:

other: maximal average fold induction of luciferase activity (Imax)

Value:

21.9

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

Imax: 3.40

Key result

Run / experiment:

other: 2

Parameter:

other: maximal average fold induction of luciferase activity (Imax)

Value:

13.64

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

Imax: 13.62

Key result

Run / experiment:

other: 1

Parameter:

other: EC 1.5 (µM) (concentration for which induction of luciferase activity is above the 1.5 fold threshold)

Value:

22.8

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

EC1.5: 24.4 µM

Key result

Run / experiment:

other: 2

Parameter:

other: EC 1.5 (µM) (concentration for which induction of luciferase activity is above the 1.5 fold threshold)

Value:

25.8

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

EC1.5 15.8 µM

Other effects / acceptance of results:

Acceptance criteria:
• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
• The EC1.5 of the positive control was between 5 and 125 µM (24.4 µM and 15.8 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (3.40-fold and 6.17-fold in experiment 1 and 2, respectively).
• Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (9.6% and 22.5% in experiment 1 and 2, respectively).

Adenosine-5’-monophosphate (AMP) showed slight toxicity in experiment 1 (IC₃₀values of 1441µM, no IC₅₀value), and no toxicity in experiment 2 (no IC₃₀and IC₅₀value). A statistically significant (p<0.001, Student’s t test), dose-related induction of the luciferase activity (EC_1.5values of 22.8 µM and 25.8 µM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (I_max) was 21.9-fold and 13.6-fold in experiment 1 and 2 respectively. Adenosine-5’-monophosphate (AMP) is classified as positive in the KeratinoSens^TMassaysince positive results (>1.5-fold induction) were observed at test concentrations <1000 µM with a cell viability of >70% compared to the vehicle control.

Overview luminescence induction and cell viability of Adenosine-5’-monophosphate (AMP) in Experiment 1 and 2

Concentration (µM)	0.977	1.95	3.91	7.81	15.63	31.25	62.50	125	250	500	1000	2000
Exp 1 luminescence	1.20	1.03	1.32	1.44	1.30	1.74	1.76	2.18	5.40^***	21.90^***	11.35^***	6.82^***
Exp 1 viability (%)	117.3	120.9	92.1	97.7	92.9	98.8	131.2	139.0	77.4	101.0	70.3	69.6
Exp 2 luminescence	0.85	0.92	0.99	1.09	1.25	1.63	1.99	2.44	6.43^**	10.97^***	5.57^*	13.64^***
Exp 2 viability (%)	105.2	102.3	102.1	105.9	103.3	104.0	98.7	102.5	104.6	79.6	79.7	70.9

^*p<0.05,^**p<0.01,^***p<0.001 Students t test

  
Overview luminescence induction and cell viability positive control Ethylene dimethacrylate glycol in Experiment 1 and 2

Concentration (µM)	7.81	15.6	31.3	62.5	125	250
Exp 1 luminescence	0.99	1.36	1.61^***	1.77^***	2.54^***	3.40^***
Exp 1 viability (%)	90.7	106.2	98.8	127.9	90.2	104.7
Exp 2 luminescence	1.13	1.28	1.75^***	2.26^***	3.29^***	6.17^***
Exp 2 viability (%)	104.6	104.5	125.9	141.7	148.6	155.3

*** p<0.001 Students t test

        
Overview EC_1.5, I_max, IC₃₀and IC₅₀values

	EC1.5 (µM)	Imax	IC30(µM)	IC50(µM)
Test item Experiment 1	22.8	21.9	1441	NA
Test item Experiment 2	25.8	13.6	NA	NA
Pos Control Experiment 1	24.4	3.40	NA	NA
Pos Control Experiment 2	15.8	6.17	NA	NA

NA = Not applicable

Interpretation of results:

other: activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes

Remarks:

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

Conclusions:

In conclusion, the KeratinoSensTM assay is valid and Adenosine-5’-monophosphate (AMP) (CAS no 61-19-8) is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions used in this study.

Executive summary:

The objective of this study was to evaluate the ability of Adenosine-5’-monophosphate (AMP) (CAS No 61-19-8) to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay.

The study procedures described in this report were based on OECD Guideline TG 442D: In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method (adopted February, 2015).

The test item was a white crystalline powder. A correction factor of 1.054 was used to correct for the purity (94.9%). The test item was suspended or dissolved in dimethyl sulfoxide at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.977– 2000 µM (2-fold dilution series). Two independent experiments were performed.

Both experiments passed the acceptance criteria:

·        The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration. 

·        The EC_1.5of the positive control was between 5 and 125 µM (24.4 µM and 15.8 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (3.40-fold and 6.17-fold in experiment 1 and 2, respectively).

·        Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was 9.6% and 22.5% in experiment 1 and 2, respectively.

Overall it is concluded that the test conditions were adequate and that the test system functioned properly. 

Adenosine-5’-monophosphate (AMP) showed slight toxicity in experiment 1 (IC₃₀values of 1441µM, no IC₅₀value), and no toxicity in experiment 2 (no IC₃₀and IC₅₀value). A statistically significant (p<0.001, Student’s t test), dose-related induction of the luciferase activity (EC_1.5values of 22.8 µM and 25.8 µM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (I_max) was 21.9-fold and 13.6-fold in experiment 1 and 2 respectively. Adenosine-5’-monophosphate (AMP) is classified as positive in the KeratinoSens^TMassay since positive results (>1.5-fold induction) were observed at test concentrations of <1000 µM with a cell viability of >70% compared to the vehicle control.

In conclusion, the KeratinoSens^TMassay is valid and Adenosine-5’-monophosphate (AMP) (CAS No 61-19-8) is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions used in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item. Adenosine-5’-monophosphate (AMP) is predicted to be not sensitizing to the skin.

A valid DPRA assay was performed according to OECD 442C and GLP principles. The test item was dissolved in sodium hydroxide in water (125 mM) at 100 mM. A stability assessment demonstrated that both Cysteine and Lysine peptides were stable in sodium hydroxide in water (125 mM). There was no evidence of co-elution of AMP with either Cysteine or Lysine peptide. 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.

A valid Keratinosens assay was performed according to OECD 442D and GLP principles. A stock solution of the test item was prepared in dimethyl sulfoxide. In the first experiment, the stock solution of 200 mM was a clear solution, whereas in the second experiment it was a translucent suspension. Clear solutions were obtained at concentrations of 100 mM and below. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.977 – 2000 µM (2-fold dilution series). The test item formed a clear solution at each of the test concentrations. Two independent experiments were performed.

The test item showed slight toxicity in experiment 1 (IC30 value of 1441 µM and no IC50 value) and no toxicity in experiment 2. A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 22.8 and 25.8µM in experiment 1 and 2, resp.) was measured in both experiments. The maximum luciferase activity induction (Imax) was 21.9-fold and 13.6-fold in experiment 1 and 2, respectively. AMP is classified as positive in the KeratinoSens assay since positive results (>1.5-fold induction) were observed at test concentrations of <1000 µM with a cell viability of >70% compared to the vehicle control. However, this result is considered a false positive (see discussion on Justification for classification/no classification).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

AMP is an endogenous substance involved in many metabolic processes in the cell, for example as part of the energy system of any cell, including those in the skin. ATP and AMP are interconverted in the cell and highly regulated intra- and extracellularly. ATP may hydrolyze to AMP and pyrophosphate, and AMP can be regenerated to ATP via ADP. When lesion of the skin occurs, the skin is being exposed to extracellular substances and cell content following necrosis of cells. Consequently, AMP, an endogenous substance omnipresent in the body, is not considered to be a skin sensitizer.

 

The results of QSAR DEREK and DPRA assay (OECD 442C), predict as expected that AMP is not to a skin sensitizer.

The positive result obtained in the Keratinosens assay (OECD 442D) is considered to be a false positive. Upon contacting the Keratinosens assay developers, they confirm that false positive results have been observed for AMP. They suspect that some cellular ATP and AMP receptors (still unidentified) are triggered, which leads to intracellular (MAP) kinase activation which in turn leads to non-canonical Nrf2 activation.

 

Performance of an in vitro assay for key event 3 is considered not relevant. Extracellular ATP is known to up-regulate CD86 and CD54 in monocytic and dendritic cells via binding to a P2 receptor on the plasma membrane (Schnurr, 2000; Miyazawa, 2008). Interconversion of AMP to ATP could be expected as result of normal metabolic activity of the cells during the experiment. Therefore, performance of a h-CLAT or U-Sens is not considered appropriate as the result is expected to be false positive.

In conclusion, based on the above data, AMP (CAS 61-19-8) is not considered to be a skin sensitizer . AMP does not need to be tested further and does not need to be classified for sensitization according to Regulation 1272/2008 and amendments.

 

References:

Miyazawa, M etal, Role of TNF-a and extracellular ATP in THP-1 cell activation following allergen exposure. J Toxicol Sci 33:71-83 (2008); https://www.jstage.jst.go.jp/article/jts/33/1/33_1_71/_pdf

Schnurr, M etal, Extracellular ATP andTNF-a synergize in the activation and maturation of human dendritic cells.J Immunol 165: 4704-4709 (2000);http://www.jimmunol.org/content/165/8/4704.long