Phosphocreatine, sodium salt

Endpoint summary

Description of key information

-       QSAR DEREK: No alert for skin sensitization

-       DPRA (OECD 442C): mean peptide depletion: 1%, minimal reactivity, Non sensitizer

-       Keratinosens^TM(OECD 442D): Negative (No cytotoxicity, Imax: 0.96 and 0.97 fold induction), Non sensitizer

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:

12 September 2017

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: N(CC(O)=O)(C(=N)NP([O-])([O-])=O)C.[Na+].[Na+]
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 Phosphocreatine, sodium salt (CAS No. 922-32-7) 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: N(CC(O)=O)(C(=N)NP([O-])([O-])=O)C.[Na+].[Na+]

Key result

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. Phosphocreatine, sodium salt 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. Phosphocreatine, sodium salt (CAS No. 922-32-7) 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 Phosphocreatine, sodium salt (CAS No. 922-32-7) 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 Phosphocreatine, sodium salt (CAS No. 922-32-7).

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:

08 August - 20 October 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:

no

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:
- Cysteine: Catalogue No. SP-CAT-002, Batch No. 2605151H5_DWW1115, purity 97.47%
- Lysine: Catalogue No. 110716-2, Batch No. P161108-LC107617, purity 98.14%

BUFFERS USED:
- Phosphate buffer: ca 100 mM, pH 7.49
- Ammonium acetate buffer: ca 100 mM, pH 10.22

SOLUBILITY ASSESSMENT:
- ultrapure water was selected as the most suitable solvent for the test material

PREPARATION PEPTIDE STOCK SOLUTIONS:
- CYSTEINE: stock solution of 0.665 mM in phosphate buffer
- LYSINE: stock solution of 0.667 mM in ammonium acetate buffer

CYSTEINE PEPTIDE ASSAY:
-PREPARATION: test item was dissolved in ultrapure water and mixed by inversion and vortex until fully in solution. The concentration of the test solution corrected for purity, was 100 mM (100% of target, 100 mM). Cinnamic aldehyde was dissolved in acetonitrile with a concentration of 100 mM (100% of target, 100 mM). All test item and control solutions 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 peptide stock (750 µL) and three replicates containing ultrapure water (50 µL)!, acetonitrile (200 µL) and 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 1:10 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 ultrapure water and mixed by inversion and vortex until fully in solution. The concentration of the test solution corrected for purity, was 100.25 mM (100.25% of target, 100 mM). Cinnamic aldehyde was dissolved in acetonitrile with concentration of 100 mM (100 % of target, 100 mM). All tets item and control solutions were prepared immediately prior to use.
- PREPARATION OF THE STANDARD CURVE: Dilution buffer was prepared by mixing ammonium acetate buffer (pH XX, 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 79.9% showing a high reactivity (Sensitizer)

Key result

Run / experiment:

other: DPRA cysteine and lysine prediction model

Parameter:

other: % Peptide Depletion (mean value)

Value:

1

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 0.9999. This met the acceptance criteria for r2 which was >0.990.
The mean peptide concentration of Reference Control A was 0.484 mM ± 0.002 mM (mean ± SD). These samples met the acceptance criteria of 0.5 mM ± 0.05 mM.

The calculated peptide concentration in the Reference Control C samples was 0.469 mM ± 0.005 mM (acetonitrile), and 0.466 mM ± 0.009 mM (ultrapure water). These samples met the acceptance criteria of 0.5 mM ± 0.05 mM. In addition, for the six Reference Control B and three Reference Control C in acetonitrile, the coefficient of variation (CV) was 2.2%. This met the acceptance criteria of <15.0%.

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 a peptide depletion value of 96.7 ± 0.1% (mean ± SD). The standard deviation of replicate positive control samples achieved the acceptance criteria of <14.9% (0.1%, actual).

Finally, the standard deviation of replicate test item samples was 0.6% for Phosphocreatine, sodium salt (acceptance criteria: <14.9%). Positive control and test item peptide peak areas and percent peptide depletion data for Cysteine binding.


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 0.497 mM ± 0.002 mM (mean ± SD). These samples met the acceptance criteria of 0.5 mM ¿ 0.05 mM.

The calculated peptide concentration in the Reference Control C samples was 0.499 mM ± 0.007 mM (acetonitrile) and 0.482 mM ± 0.009 mM (ultrapure water). These samples met the acceptance criteria of 0.5 mM ± 0.05 mM. In addition, for the six Reference Control B and three Reference Control C samples in acetonitrile, the CV was 2.5% (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 63.1% ± 2.2% (mean ± SD).

Finally, the standard deviation of replicate test item samples was 1.4% for Phosphocreatine, sodium salt (acceptance criteria: <11.6%). Positive control and test item peptide peak areas and percent peptide depletion data for Lysine binding.

PROTOCOL DEVIATIONS
The study was performed in accordance with the protocol and protocol amendment 1 with no deviations.

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).

Peptide depletion data for the cysteine and lysine assays

Test Item	% Peptide Depletion Cysteine (Mean  ± SD)	% Peptide Depletion Lysine (Mean ± SD)	Mean of Cysteine and Lysine	DPRA Classification (Cysteine and Lysine Prediction Model)
Phosphocreatine, sodium salt	2.0±0.6	0.0 ±1.4	1.0	Minimal Reactivity (Non-Sensitizer)
Positive control (cinnamic aldehyde)	96.7 ± 0.1	63.1 ± 2 .2	79.9	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

Conclusions:

In conclusion, according to the DPRA cysteine and lysine prediction model, Phosphocreatine, sodium salt (CAS No. 922-32-7) 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 Phosphocreatine, sodium salt (CAS No. 922-32-7) 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 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

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 nucelophilic reaction centres and phenylalanine to facilitate detection by HPLC analysis.

The solubility of the test item was assessed and ultrapure water selected as the most suitable solvent for use in both peptide assays. The test item and peptides were combined and incubated together for ca 24 h at ambient temperature. The test item was prepared at a concentration of 100 mM. 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.

The results obtained are presented in the following table:

Test Item	% Peptide Depletion (Mean±SD)		Mean Peptide depletion (%)	DPRA Prediction
	Cysteine	Lysine
Phosphocreatine, sodium salt	2.0 ± 0.6	0.0 ± 1.4	1.0	Minimal Reactivity (Non Sensitiser)
Cinnamic Aldehyde (Positive Control)	96.7 ± 0.1	63.1 ± 2.2	79.9	High Reactivity (Sensitiser)

Negative depletion has been corrected to 0.00

All acceptance criteria were fulfilled. There was no evidence of co-elution of the test item with either Cysteine or Lysine peptide. Peptide depletion was calculated as 0.0% and 2.0% in Lysine and Cysteine Assays, respectively, resulting in a mean peptide depletion of 1.0%. This value places Phosphocreatine, sodium salt in the minimal reactivity classification resulting in a DPRA prediction of Non-Sensitiser

In conclusion, according to the DPRA cysteine and lysine prediction model, Phosphocreatine, sodium salt (CAS No. 922-32-7) 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:

3-16 October 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:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

- Test concentrations:Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated in the dosed plates were as follows: 2000 µM, 1000 µM, 500 µM, 250 µM, 125 µM, 62.5 µM, 31.3 µM, 15.6 µM, 7.8 µM, 3.9 µM, 1.95 µM and 0.98 µM.
- All concentrations of the test item were tested in triplicate.
- Positive control :Positive control stock solution (cinnamic aldehyde, 6.4 mM stock in DMSO): 64 µM, 32 µM, 16 µM, 8 µM and 4 µM.
- Negative control: vehicle: 1% Ultrapure water 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). KeratinoSens™ cells were supplied by Charles River, Den Bosch who previously obtained them from Givaudan, Switzerland. Stocks of cells were prepared according to the methods specified in the European Union Reference Laboratory for Alternatives to Animal Testing (EURL-ECVAM) protocols.

- Environmental conditions:
Unless otherwise stated, all incubations of cells were performed in a humidified incubator set to maintain a temperature of 37°C, and an atmosphere of 5% CO2 in air.

- Cell Thawing:
The cells were removed from liquid nitrogen storage and warmed in a ca 37ºC water bath until thawed. The cells were resuspended in KeratinoSens™ Medium 2 (10 mL) and collected by centrifugation at ca 125 g for 5 min. The cell pellet was resuspended in KeratinoSens™ Medium 2 (10 mL) and seeded into two tissue culture dishes, with the final volume of cells and media being 10 mL/dish.

-Culture maintenance:
Cells were maintained in KeratinoSens™ Medium 1. Cells were passaged when ca 80 90% confluent; if it was required to passage cells of lower or higher confluence, the passage ratio was adjusted accordingly. Cells were washed twice with Dulbecco’s phosphate buffered saline (DPBS) containing EDTA (0.05%, w/v; 5 mL), then Trypsin/EDTA (1 mL) was added and dishes were returned to the incubator. After cells had detached (ca 10 min), they were resuspended in KeratinoSens™ Medium 1 (10 mL) and reseeded into fresh dishes using an appropriate split ratio and fresh KeratinoSens™ Medium 1 to bring the total dish volume up to 10 mL.



EXPEIMENTAL DESIGN
- Test system setup
In the week prior to testing, cells were split and grown for 4 days in tissue culture dishes.

For the 2 experiments performed, KeratinoSens™ cells were at passage numbers p19 (test run 1) and p21 (test run 2).

The cells were washed twice with DPBS containing EDTA (0.05%, v/v; 5 mL), then Trypsin/EDTA (1 mL) was added and plates returned to the incubator. After cells had detached (ca 10 min), they were resuspended, counted and adjusted to a density of 80,000 cells/mL in KeratinoSens™ Medium 2.

The cells were then seeded into 96 well plates, 125 µL per well, except for one empty well (blank). Four parallel plates were prepared: three white 96 well plates and one transparent 96 well plate. Plates were then incubated for ca 24 h.

Following incubation and prior to dosing, the medium was removed and replaced with KeratinoSens™ Medium 3 (150 µL).

- Preparation of master plates
On each testing occasion, a stock solution of Phosphocreatine, sodium salt was prepared on the day of use. Phosphocreatine, sodium salt was dissolved in ultrapure water (filter sterilised though a 0.45 µm filter) at a concentration of ca 200mM, taking purity into account. Actual concentrations prepared were 99.98% and 100.15% of target, in test run 1 and test run 2, respectively.

Positive control stock solution (cinnamic aldehyde, 6.4 mM stock in DMSO) was also prepared on the day of use, taking purity into account. Actual concentrations were 100.15% and 101.8% of target, in test run 1 and test run 2, respectively. DMSO was the vehicle control.

The 100x solvent plate was then prepared using these stocks, following a pre-defined plate plan. DMSO vehicle controls (6 wells) and cinnamic aldehyde positive controls (5 concentrations) were included on every plate. Serial dilutions of the test item in ultrapure water (12 concentrations) and positive control in DMSO (5 concentrations) stock solutions were then prepared in the 100x plate (1:1, v/v).

Into a fresh plate corresponding to the 100x plate layout, KeratinoSens™ Medium 3 (240 µL/well) was added to wells that would receive cinnamic aldehyde or DMSO from the 100x plate and, KeratinoSens™ Medium 3 (230 µL/well) was added to wells that would receive the test items from the 100x plate. Test item or controls (10 µL) were transferred from the 100x plate to the 4x plate followed by DMSO (10 µL) to the test item wells, according to the pre-defined plate plan.


Each independent repetition was performed on a different day, with fresh stock solutions of test items and independently harvested cells.

Aliquots (50 µL) of each dosing solution from the 4x plate were transferred to the pre prepared replicate assay plates (Section 8.3, three white plates and one clear plate) and mixed by gentle aspiration. The blank wells were not dosed. All plates were sealed with sealing tape to avoid evaporation of volatile compounds and to avoid cross contamination between wells. The plates were then incubated for 48 h +/- 2 h.


- Luciferase assay
Following 48 h +/- 2 h incubation, dosing solutions were aspirated from the white assay plates and discarded. The cells were then washed once with DPBS (300 µL). KeratinoSens™ Medium 3 (100 µL, ambient temperature) was then added to each well, including the blank well. ONE-Glo™ (Promega) reagent mix was allowed to thaw and equilibrate to room temperature. The ONE-Glo™ reagent mix (100 µL) was then added to each well. The plates were incubated at room temperature, protected from light, for 6 min, before analysis with a luminometer

- Cytotoxicity assessment
For the cell viability assay, the solutions in the clear plate were aspirated and replaced with MTT solution (0.59 mg/mL in KeratinoSens™ Medium 3, 227 µL per well, including the blank well) following the 48 h +/- 2 h incubation. The plate was sealed with sealing tape and then incubated for 4 h +/- 30 min. After 4 h +/- 30 min incubation, the MTT solution was removed and sodium dodecyl sulphate solution (SDS, 10%, w/v, 200 µL per well) was added. The plate was sealed with sealing tape and placed protected from light in the incubator. After weekend incubation to dissolve the cells, plates were placed on an orbital shaker for 10 min to homogenise the solutions, then the optical absorption at 600 nm was determined for each well.

ACCEPTABILITY CRITERIA
• The luciferase induction obtained with the positive control, cinnamic aldehyde, should be statistically significant above the threshold of 1.5 in at least one of the tested (non cytotoxic) concentrations.
• The EC1.5 value for the positive control should be within two standard deviations of the historical mean of the testing facility (2.86 to 26.68)
• The average luciferase induction in the three replicates for cinnamic aldehyde at 64 µM (highest dose tested) should be between 2 and 8. If the latter criterion is not fulfilled, tests may still be accepted if there is a clear dose response, with increasing luciferase induction at increasing concentrations of cinnamic aldehyde.
• The average coefficient of variation of the luminescence reading for the vehicle control, DMSO, should be <20% in each experiment (18 wells). If the variability is higher, and is due to a single value, this result can be rejected, otherwise, the results should be discarded and the test repeated.

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.

- Skin Sensitising Prediction Model
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 luciferase induction is >1.5-fold and statistically significant compared to the vehicle control.
2. The EC1.5 value is <1000 µM (<200 µg/mL for test chemicals with no defined MW) in all 3 repetitions or in at least 2 repetitions.
3. At the lowest concentration with a gene induction above 1.5-fold (i.e. at the EC1.5 determining value), the cell viability is >70%.
4. There is an apparent overall dose-response for luciferase induction, which is similar between the repetitions.
For each test item, concordant data from 2 independent repetitions (performed on different days) is required to assign sensitising potential.

Positive control results:

• Experiment 1: The positive control ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.30 and the EC1.5 27.6 µM.
• Experiment 2: The positive control ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.09 and the EC1.5 36.8 µM.

Key result

Run / experiment:

other: 1

Parameter:

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

Value:

0.96

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

Imax: 2.30

Key result

Run / experiment:

other: 2

Parameter:

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

Value:

0.97

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

Imax: 2.09

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)

Vehicle controls validity:

valid

Remarks:

NA

Positive controls validity:

valid

Remarks:

EC 1.5: 27.6

Remarks on result:

not determinable

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)

Vehicle controls validity:

valid

Remarks:

NA

Positive controls validity:

valid

Remarks:

EC 1.5: 36.8

Remarks on result:

not determinable

Other effects / acceptance of results:

All acceptance criteria were met for each run.
Gene induction in the positive control was significant in at least one concentration for run 1 and run 2, with a p-value of 0.01 and <0.01, respectively. The EC1.5 values for the positive control were 27.6 µM and 36.8 µM for Run 1 and Run 2, respectively. These were greater than two standard deviations above the historical mean (2.84-26.28 µM), and were accepted. The average luciferase induction in the three replicates for cinnamic aldehyde at 64 µM (highest dose tested) for Run 1 and Run 2 was 2.30 and 2.09, respectively. This was within the acceptance criteria of 2-8. The average coefficient of variation of the luminescence reading for the vehicle control, DMSO, was 12.41% and 15.10%, for Run 1 and Run 2 respectively.

Cell Viability:
Tested concentrations the positive control (cinnamic aldehyde) did not cause a reduction in cell viability. Two of the tested concentrations of the test item caused a reduction in viability of ca 50%, however these were anomalous results, likely due to a processing error as the concentrations affected were the lowest tested (0.98, 1.95 µM). Therefore it was not possible to calculate an IC30.

Table 1          Run 1 Gene Induction and Cell Viability Results

Test Item	IC30 (µM)	IC50 (µM)	EC1.5 (µM)	Imax	Dose response for luciferase	Significant at p<0.05	Result accepted
Phosphocreatine, sodium salt	N/A	N/A	N/A	0.96	N/A	N/A	Yes
Positive Control – Cinnamic Aldehyde	N/A	N/A	27.6	2.30	Yes	Yes	Yes

Coefficient of variation for this test was 12.41%. This run satisfied all acceptance criteria and therefore passed.

N/A: Not applicable – unable to calculate values as viability was over 70% and there was no luciferase induction.

Table 2          Run 2 Gene Induction and Cell Viability Results

Test Item	IC30 (µM)	IC50 (µM)	EC1.5 (µM)	Imax	Dose response for luciferase	Significant at p<0.05	Result accepted
Phosphocreatine, sodium salt	N/A	N/A	N/A	0.97	N/A	N/A	Yes
Positive Control – Cinnamic Aldehyde	N/A	N/A	36.8	2.09	Yes	Yes	Yes

Coefficient of variation for this test was 15.10%. This run satisfied all acceptance criteria and therefore passed.

N/A: Not applicable – unable to calculate values as viability was over 70% and there was no luciferase induction.

Table 3          Summary of Data

Test Item	IC30(µM) values		IC30(µM) Geometric Mean	EC1.5(µM) Geometric Mean	Imax Average	KeratinoSens^™Classification
Phosphocreatine, sodium salt	N/A	N/A	N/A	N/A	N/A	Non-Sensitiser
Positive Control – Cinnamic Aldehyde	N/A	N/A	N/A	31.9	2.2	Sensitiser

N/A: Not applicable – unable to calculate values as viability was over 70% and there was no luciferase induction.

 

Interpretation of results:

other: Test item did not induce activation of the ARE-dependant pathway in keratinocytes

Remarks:

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

Conclusions:

In conclusion Phosphocreatine, sodium salt (CAS No.: 992-32-7) did not induce activation of the ARE-dependant pathway in keratinocytes under experimental conditions described in this report and therefore can be considered a non-sensitiser.

Executive summary:

Chemical exposure to humans often occurs via the skin and compounds able to pass through the skin can cause disease such as sensitisation. A skin sensitiser is a substance that will lead to an allergic response following skin contact.

The KeratinoSens™ assay detects up-regulation of the Keap1/Nrf2 antioxidant response element (ARE) in KeratinoSens™ cells, a human keratinocyte cell line (HaCaT) stably transfected with a reporter construct containing an ARE coupled to luciferase.

Following exposure to Phosphocreatine, sodium salt (CAS No.: 992-32-7) at non-cytotoxic concentrations, the luciferase activity of cells was measured. An increase in luciferase activity indicates upregulation of the reporter gene through activation of the Keap1/Nrf2/ARE signalling pathway, indicating skin sensitising potential.

KeratinoSens™ cells were exposed to Phosphocreatine, sodium salt (in filter sterilised ultrapure water) at 12 concentrations in 96 well plates for 48 h. Luciferase activity was then determined by addition of ONE-Glo™ reagent (Promega) followed by measurement of luminescence. Cell viability was determined using an MTT assay. A compound would be classed as a skin sensitiser where an increase in luminescence was seen at non-cytotoxic

concentrations.

In two independent repetitions of the test which both met all the acceptance criteria (with the exception that the EC1.5 for the positive control was not within 2 standard deviations of the historical mean), Phosphocreatine, sodium salt did not induce luciferase activity or cause cytotoxicity at any concentration (no EC1.5 value or IC30/50). The maximum luciferase

induction value (Imax) was 0.96-fold for Run 1 and 0.97-fold for Run 2. Therefore Phosphocreatine, sodium salt can be classified as a non-sensitiser according to the UN GHS classification system since negative results (¿1.5-fold induction) were observed at all test concentrations.

In conclusion Phosphocreatine, sodium salt (CAS No.: 992-32-7) did not induce activation of the ARE-dependant pathway in keratinocytes under experimental conditions described in this report and therefore can be considered a non-sensitiser.

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. phosphocreatine, sodium salt (CAS 922-32-7) 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 acetonitrile at 100 mM. There was no evidence of co-elution of the test item with either Cysteine or Lysine peptide. Peptide depletion was calculated as 0.0% and 2.0% in Lysine and Cysteine Assays, respectively, resulting in a mean peptide depletion of 1.0%. This value places Phosphocreatine, sodium salt in the Minimal Reactivity Class resulting in a DPRA prediction of non-sensitiser.

A valid Keratinosens assay was performed according to OECD 442D and GLP principles. The test item was dissolved in water at 200 mM. From this stock 11 spike solutions were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 µM (2-fold dilution series). No precipitate was observed at any dose level tested. Two independent experiments were performed.

The test item showed no toxicity (no IC30 value calculated). No biologically relevant, dose-related induction of the luciferase activity was measured in both experiments. The maximum luciferase activity induction (Imax) was 0.96-fold and 0.97-fold in experiment 1 and 2, respectively.

Phosphocreatine, sodium salt is classified as negative in the KeratinoSens assay since negative results (<1.5-fold induction) were observed.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Considering the negative outcome of the DEREK assessment, the DPRA and KeratinoSens assay, phosphocreatine, sodium salt is not considered to be skin sensitizer. Performance of an in vitro assay assessing key event 3 (activation of dendritic cells) is not expected to give additional information as irrespective of the outcome, phosphocreatine, sodium salt is not considered to be skin sensitizer based on the tests performed.

Moreover, phosphocreatine is an endogenous substance, involved in many energetic processes in the cell, and serves as a rapidly mobilizable reserve of high-energy phosphates. When lesions of the skin occur, the skin is being exposed to cell contents, including phosphocreatine, following necrosis of cells. Consequently, phosphocreatine is not considered to be a skin sensitizer.

 

Based on the above data, phosphocreatine, sodium salt (CAS 922 -32 -7) is not considered to be skin sensitizer. The test item is not classified for skin sensitization according to Regulation 1272/2008 and amendments.