Magnesium glycerophosphate

Endpoint summary

Description of key information

hCLAT

The test item Magnesium glycerophosphate with a log Pow of < -1.7 did not activate THP-1 cells up to a concentration of 5000 µg/mL under the test conditions of this study. Therefore the test item is considered negative for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

DPRA

Solutions of Magnesium glycerophosphate were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides. The overall mean result of 1.98% depletion places Magnesium glycerophosphate in the reactivity class of “minimal” and hence it is predicted by DPRA to be a non-skin sensitizer.

Keratinosens

The human skin sensitisation potential of Magnesium Glycerophosphate was assessed using the validatedin vitromethod, the KeratinoSens^TMassay, adapted to animal product-free conditions by XCellR8, and validated in-house to determine keratinocyte activation.

After 48h exposure of cells with 12 concentrations of Magnesium Glycerophosphate, Luciferase measurements and MTT viability testing were performed.

Magnesium Glycerophosphate was classified as Positive according to the KeratinoSens prediction model.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Remarks:

Human Cell Line Activation Test (h-CLAT)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))

Deviations:

no

GLP compliance:

yes

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

The test item was supplied by or on behalf of the Sponsor including the following information:
Identification: Magnesium glycerophosphate
Trade Name: Magna-C
Batch: INVG003917
CAS No.: 927-20-8
EC No.: 213-149-3
Purity: 96.5% (w/w) (calculated)*
Partition coefficient
(n-octanol/water):
log Pow: < -1.7
Water solubility: 79 g/L (20 °C)
Appearance: White powder
Expiry Date: 10 June 2021
Storage Conditions: At room temperature, light protected, protected from moisture
Stability in Solvent: Stable in water (not quantified)
Purpose of Use: Industrial chemical
* Dose calculation was not adjusted to purity.

Details on the study design:

TEST ITEM PREPARATION
On the day of the experiment (immediately prior to start) Magnesium glycerophosphate was stable suspended/dispersed in culture medium.
The maximum concentration of test item was a stable suspension/dispersion of 100 mg/mL in culture medium, as tested by a solubility test.
For the XTT test (dose finding assay) eight concentrations of the test item were analysed. For this, dilutions were prepared by 1:2 serial dilutions from 5000 µg/mL culture medium.

TEST SYSTEM AND SUPPORTING INFORMATION
Reasons for the Choice of THP-1 Cells
THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202. THP-1 cells are used as surrogate for human myeloid dendritic cells and show enhanced CD86 and/or CD54 expression when treated with sensitisers.

THP-1 Cell Cultures
Stocks of the THP-1 cell line are stored in liquid nitrogen in the cell bank of Envigo CRS GmbH (aliquots of cells in freezing medium at 1  106 to 2  106 cells/mL) allowing the repeated use of the same cell culture batch in experiments. Therefore, the parameters of the experiments remain similar, because of the reproducible characteristics of the cells. Thawed stock cultures are propagated at 37 ± 1.5 °C in plastic flasks. The cells are sub-cultured twice weekly. The cell density should not exceed 1  106 cells/mL. The THP-1 cell suspension is incubated at 37 ± 1.5 °C and 5.0 ± 0.5 % carbon dioxide atmosphere. Cells can be used up to two months after thawing (passage number should not exceed 30).
The passage numbers of the used THP-1 cells were 20 and 8 in the XTT assay and 10 and 15 in the h CLAT for runs 1 and 2, respectively.

Culture Medium
RPMI-1640 supplemented with 10 % FBS (v/v), 0.05 mM 2 mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate, 1% (v/v) L-glutamine and appropriate antibiotics (100 U/mL of penicillin and 100 µg/mL of streptomycin) is used to culture the cells during the assay. Medium with supplements has to be stored at 2 – 8 °C and used within one month. The culture medium has to be warmed to room temperature just before use.

Preparation and Seeding of THP-1 Cells
On the day of the cytotoxicity experiment (XTT) directly before the application of the test item, solvent and medium control, a volume of 100 µL with a cell density of 0.9 - 1  106 THP-1 cells/mL was seeded in each well of a 96-well flat bottom plate.
For the main experiment (h-CLAT) 0.9 - 1  106 cells/well in a volume of 500 µL were seeded in a 24-well plate before the treatment. 

Experimental Design and Procedures of XTT
Dose Finding Assay (XTT Test)
The test item concentrations investigated in the main experiment (h-CLAT) were determined with two XTT tests.
The XTT test is based on the cleavage of the yellow tetrazolium salt XTT [= (sodium 3'-(1-phenylaminocarbonyl) - (3,4 - tetrazolium) – bis - (4 – methoxy – 6 - nitro) - benzenesulfonic acid hydrate)] to form an orange water soluble formazan dye by dehydrogenase activity in active mitochondria. This method was first described 1988 by SCUDIERO et al. and improved in subsequent years by several other investigators.
Two independent cytotoxicity experiments were performed with different cell cultures to obtain a reliable CV75. The CV75 could not be determined therefore the highest stable suspended/dispersed test item concentration was used to calculate the dose-range for the main experiment (h-CLAT).

XTT Labelling Mixture
The XTT labelling mixture consists of two components, a XTT buffer solution and the substrate solution. Both components were mixed right before application at a ratio of 1:100.

Treatment
After the cell seeding, 100 µL of the test item dilutions, the medium and solvent controls, respectively, were added to the cells. All dose groups were tested in 7 replicates for each XTT test. At the end of the incubation period of 24 ± 0.5 hours, the cell cultures were microscopically evaluated for morphological alterations.

XTT Labelling and Measurement
At the end of the incubation period, 50 µL of the XTT labelling mixture were added to each well. The cells were incubated and subsequently transferred to a microplate reader (Versamax® Molecular Devices). The absorbance was measured at 450 nm (reference wavelength 690 nm). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

Evaluation of the XTT results
A decrease in number of living cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample. This decrease directly correlates to the amount of orange formazan formed, as monitored by the absorbance. The relative absorbance (= viability in [%]) as compared to the solvent control is calculated.

Calculation of the h-CLAT Test Item Concentrations
Two independent cytotoxicity experiments were performed with different cell cultures to obtain a reliable CV75.
Since the CV75 could not be determined, a stock solution of the highest stable suspended/dispersed test item concentration was prepared and seven further concentrations of the test item were prepared by serial 1:1.2 dilution.

Acceptability of the Assay
The XTT test is considered to be acceptable if it meets the following criteria:
•mean absorbance of the medium control is ≥ 0.5
•mean viability of the solvent control is ≥ 90% in comparison to the medium control


Experimental Design and Procedures of h-CLAT
The test item was tested in two independent runs.

Treatment of the Cells
For the test item exposure the highest concentration of the XTT test was used instead of 1.2 × CV75, since no CV75 could be determined. Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment.
Each volume (500 µL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours.
Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).

Staining of the Cells
The triplicates of each test item-treated and not test item-treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250  g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 µL of blocking solution at 2 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 µL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control).
All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis procedures.
The cells with the different antibodies or the IgG1 were mixed and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).

Sample Preparation for Measurement
After staining with the antibodies, the cells were washed twice (2 8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 µL of a 7-AAD solution were added.

Flow Cytometry Acquisition
Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions.
The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry using the software Cellquest Pro 6.0. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7 AAD fluorescence signal.
Preparation of the acquisition
The following acquisition plots were prepared:
•2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
•Histogram plot of each channel (FL-1 and FL-3, respectively)
The voltage of FSC and SSC was set with untreated cells to appropriate levels. FSC and SSC are not needed for the analysis, but the FSC/SSC plot was checked to make sure that a single population appears without contamination or excessive debris. The FL-1 and FL-3 voltage were set and compensate to appropriate position. The FL-1 voltage was set using the FITC labelled-mouse IgG1 medium-treated cells tube, as such that the MFI of control cells was set in the range between 1.0 and 4.0 (Geo Mean) and in the range between 3.0 and 4.0 (Geo Mean) with the FITC labelled CD54 medium-treated cells (FACSCalibur, Becton Dickinson GmbH).
The maintenance of the flow cytometer was in accordance with the manufacturer’s instructions. The process of washing was conducted very carefully since insoluble chemicals could flow into the flow line.
Acquisition
Dead cells were determined by staining with 7-AAD. Gating by FSC (forward scatter) and SSC (side scatter) was not done. A total of 10,000 living cells were analysed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The other tubes were acquired without changing the settings of the cytometer. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was not calculated, if the cell viability was less than 50% (due to diffuse labelling of cytoplasmic structures that could be generated due to cell membrane destruction).

Data Analysis and Interpretation
Flow Cytometry Analysis
The RFI is used as an indicator of CD86 and CD54 expression, and is calculated as follows for each concentration of every chemical.
The cell viability of the h-CLAT experiment is calculated for each concentration of every chemical.

Acceptance Criteria
The following acceptance criteria should be met when using the h-CLAT method:
•Cell viability of medium control is adjusted to 100% and the cell viability of the DMSO control should be more than 90% in comparison to the medium control.
•In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
•For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
•In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50% in at least one concentration of the two tested positive control concentrations.
•For the test chemical, the cell viability should be more than 50% in at least four tested concentrations in each run.
Negative results are acceptable only for test items exhibiting a cell viability of < 90% at the highest concentration tested (i.e. 1.2 × CV75). If the cell viability at 1.2 × CV75 is ≥ 90% the negative result should be discarded. In such case it is recommended to try to refine the dose selection by repeating the CV75 determination. It should be noted that when 5000 μg/mL in saline (or medium or other solvents/vehicles), 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test chemical, a negative result is acceptable even if the cell viability > 90% (OECD 442E guideline).

Positive control results:

Concurrent controls with DNCB ((2,4-dinitrochlorobenzene, CAS No.: 97-00-7) final concentration: 2 and 3 µg/mL, Purity ≥ 99%) in DMSO were used

Key result

Parameter:

other: RFI (%) CD 86 Antibody

Value:

92.9

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Parameter:

other: RFI (%) CD 54 Antibody

Value:

103.8

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: all

Parameter:

other: cell viability

Value:

93.7

Vehicle controls validity:

valid

Positive controls validity:

valid

Resultsof the Dose Finding Assay (XTT Test)

Results of the first XTT test for Test Item Magnesium glycerophosphate

 

		Microscopic Evaluation	Photometric Evaluation
Test Group	Concen-tration [µg/mL]	Cytotoxicity	Mean Ab-sorbance*	Standard-Deviation	Chem. Blank	Mean Ab-sorbance – Chemical Blank	Absorbance in % of Solvent Control**
Medium Control	-	no	0.743	0.026	0.251	0.491	107.42
Solvent Control	-	no	0.712	0.012	0.255	0.457	100.00
Test Item	39.1	no	0.730	0.016	0.247	0.484	105.75
	78.1	no	0.801	0.026	0.249	0.552	120.71
	156.3	no	0.793	0.019	0.248	0.546	119.32
	312.5	no	0.835	0.015	0.244	0.591	129.14
	625	no	0.809	0.021	0.246	0.563	123.04
	1250	no	0.818	0.025	0.239	0.580	126.77
	2500	no	0.700	0.036	0.219	0.481	105.15
	5000	no	0.686	0.035	0.209	0.478	104.44

*            mean absorbance (absolute) of 7 wells

**          relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium control was 93.09%.

Due to the lack of cytotoxicity in the XTT test, a CV75 value could not be calculated.

Results of the second XTT test for Test Item Magnesium glycerophosphate

 

		Microscopic Evaluation	Photometric Evaluation
Test Group	Concen-tration [µg/mL]	Cytotoxicity	Mean Ab-sorbance*	Standard-Deviation	Chem. Blank	Mean Ab-sorbance – Chemical Blank	Absorbance in % of Solvent Control**
Medium Control	-	no	0.553	0.036	0.216	0.337	91.78
Solvent Control	-	no	0.588	0.084	0.220	0.367	100.00
Test Item	39.1	no	0.605	0.089	0.218	0.387	105.22
	78.1	no	0.587	0.059	0.233	0.354	96.33
	156.3	no	0.593	0.014	0.229	0.363	98.90
	312.5	no	0.583	0.013	0.229	0.354	96.39
	625	no	0.584	0.021	0.222	0.362	98.48
	1250	no	0.591	0.020	0.233	0.358	97.39
	2500	no	0.561	0.010	0.198	0.363	98.85
	5000	no	0.566	0.011	0.193	0.373	101.66

*            mean absorbance (absolute) of 7 wells

**          relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium control was 108.95%.

Due to the lack of cytotoxicity in the XTT test, a CV75 value could not be calculated.

     Results of the h-CLAT Test

Results of the first h-CLAT run for the Test Item Magnesium glycerophosphate

	Concentration (µg/mL)	RFI (%) CD 54 Antibody	RFI (%) CD 86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	100.0
DMSO Control	-	100.0	100.0	100.0
Positive Control (DNCB)	2.0	236.2*	427.5*	72.4
	3.0	590.4*	562.0*	60.0
Test Item	1395	92.4	107.8	90.8
	1674	97.5	100.0	91.3
	2009	102.5	113.8	93.3
	2411	102.5	110.1	92.4
	2894	139.8	95.0	94.0
	3472	111.0	102.8	93.3
	4167	132.2	109.2	92.7
	5000	125.4	116.1	92.6

*   RFI value of CD86 or CD54 fulfilled the positive criteria (CD86≥150% and CD54≥200%).

 

Results of the second h-CLAT run for the Test Item Magnesium glycerophosphate

	Concentration (µg/mL)	RFI (%) CD 54 Antibody	RFI (%) CD 86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	100.0
DMSO Control	-	100.0	100.0	100.0
Positive Control (DNCB)	2.0	322.0*	863.5*	68.1
	3.0	581.7*	814.6*	61.2
Test Item	1395	146.2	133.7	100.2
	1674	147.4	125.7	99.5
	2009	138.5	130.2	97.3
	2411	111.5	92.6	93.7
	2894	103.8	108.9	95.6
	3472	125.6	108.4	98.6
	4167	150.0	111.9	95.8
	5000	189.7	117.8	94.9

*   RFI value of CD86 or CD54 exceeded the positive criteria (CD86≥150% and CD54≥200%).

 

The individual raw data are included in Annex 1.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item Magnesium glycerophosphate with a log Pow of < -1.7 did not activate THP-1 cells up to a concentration of 5000 µg/mL under the test conditions of this study. Therefore the test item is considered negative for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

This in vitroHuman Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) ofMagnesium glycerophosphatestable suspended/dispersed in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h‑CLAT)of Magnesiumglycerophosphate was previously determined by two XTT tests.

Cytotoxic effects were not observed following incubation with the test item up to the highest tested concentration (5000 µg/mL). Due to the lack of cytotoxicity, a CV75 value could not be calculated. Therefore the OECD 442E guideline recommended maximal to be tested test item concentration (5000 µg/mL) was used for the h-CLAT runs.

The following concentrations of the test item (stable suspended/dispersed in culture medium) were tested in themain experiment (h-CLAT): 1395, 1674, 2009, 2411, 2894, 3472, 4167 and 5000 µg/mL

The test item with a log Pow of < -1.7 was tested in 2 independent runs. The RFI of CD86 and CD54 was not equal or greater than 150% and 200%, respectively at any dose in both runs. Therefore the h-CLAT prediction is considered negative for the tested test item in this h-CLAT.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria(CD54 ≥ 200% and CD86 ≥ 150%)and the cell viability was >50%. For details see Annex 2.

Further results of the testing battery (including e.g. DPRA, ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential are not available. Therefore,consideration of the test method results within the context of an IATA (Integrated Approaches to Testing and Assessment) is not possible.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: DPRA

Specific details on test material used for the study:

Magnesium glycerophosphate

CAS No. 927-20-8
Lot No. INVG003917
Purity 96.5% w/w

Molecular weight
194.36 g/mol

Appearance: White powder

Expiry/retest date
29 June 2021

Storage conditions
Room temperature, dark (15ºc to 25ºC)

Details on the study design:

Incubation
The appearance of the Magnesium glycerophosphate and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to injection of the samples as part of analytical run. Before initiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis
The concentration of both the Cysteine and Lysine peptides in the presence of Magnesium glycerophosphate and the associated positive controls was quantified by HPLC using UV detection as detailed in the chromatographic section.

Positive control results:

Positive control chemical: Cinnamic Aldehyde

Cysteine Peptide Depletion: 71.4 % (mean of 3 measurements)
Lysine Peptide Depletion: 56.7 % (mean of 3 measurements)

Key result

Run / experiment:

other: all 3

Parameter:

other: Cysteine Peptide Depletion

Remarks:

Mean of 3 runs

Value:

10.3

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: all

Parameter:

other: Lysine Peptide Depletion

Remarks:

mean of 3 runs

Value:

-0.345

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Cysteine Peptide Depletion

Sample	Peak area (μV.sec)	Peptide concentration1 (μg/mL)	Peptide Depletion (%)	Mean Depletion (%)	SD (%)
Positive control	221250	104.22	71.6
	223039	105.07	71.4	71.4	0.22
	224624	105.83	71.2
Magnesium glycerophosphate	651354	310.30	17.03
	744467	354.91	5.163	10.3	6.09
	716423	341.47	8.743

Lysine Peptide Depletion

Sample	Peak area (μV.sec)	Peptide concentration1 (μg/mL)	Peptide Depletion (%)	Mean Depletion (%)	SD (%)
Positive control	344921	163.70	57.8
	353280	167.68	56.8	56.7	1.09
	362791	172.21	55.6
Magnesium glycerophosphate	808415	384.50	-0.209
	801982	381.43	0.589	-0.345	1.01
	818134	389.13	-1.41

Interpretation of results:

GHS criteria not met

Conclusions:

Solutions of Magnesium glycerophosphate were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides. The overall mean result of 1.98% depletion places Magnesium glycerophosphate in the reactivity class of “minimal” and hence it is predicted by DPRA to be a non-skin sensitizer.

Executive summary:

The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of Magnesium glycerophosphate.

Solutions of Magnesium glycerophosphate were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides. The mean result of 1.98% depletion places Magnesium glycerophosphate in the reactivity class of “minimal” and therefore it is predicted by DPRA to be a non-skin sensitizer.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

The study was conducted between 18 April 2018 and 21 September 2018.

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)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

Skin sensitisers have been reported to induce genes that are regulated by the antioxidant response element (ARE). The KeratinoSensTM test is a method for which validation studies have been completed followed by an independent peer review conducted by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM). The KeratinoSensTM test method was considered scientifically valid to be used as part of an IATA (Integrated Approach to Testing and Assessment), to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling. The method cannot be used on its own, neither to sub-categorise skin sensitisers into subcategories 1A and 1B as defined by the UN GHS, for authorities implementing these two optional subcategories, nor to predict potency for safety assessment decisions. However, depending on the regulatory framework a positive result may be used on its own to classify a chemical into UN GHS category 1.

Specific details on test material used for the study:

Test Item Name: Magnesium Glycerophosphate (Magna C)
Supplier batch/lot number: INVG003917
Purity: 96.5%
Expiry Date: 10 June 2021
Physical state: White powder
Storage Conditions: Room temperature (20 ± 5°C), in the dark, in closed bottle

Details on the study design:

Test Item
Solvent: 1% DMSO in cell culture medium
Administration method: In cell culture medium
Concentrations tested (µM): 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.813, 3.906, 1.953, 0.977 µM
Analyses for achieved concentration and stability of test item formulations were not conducted as part of this study. Based on the information supplied by the sponsor, the test item was stable, and test item formulations were prepared and used on the day of dosing. This is not considered to affect the validity of the study.

Negative Control
Reference Item name: DMSO
Purity: ≥99.7%
Concentration to be tested : 1%
Administration method: In cell culture medium
Expiry Date: Rep1 and Rep2: 04APR23; Rep 3: 27APR23
Storage Conditions: Room Temperature

Positive Control
Reference Item name: Cinnamic Aldehyde
Purity: ≥99%
Concentrations tested: 8, 16, 32, 64 and 128 µM
Administration method: In cell culture medium
Solvent: 1% DMSO in cell culture medium
Expiry Date: JUL 22
Storage Conditions: 4°C

Method of administration of test item
Per plate, a single application of 12 concentrations of the test item was applied in cell culture medium (dilution factor of 2) with a final concentration of 1% DMSO. The top concentration was previously determined by solubility testing.

Method of administration of reference items
Per plate, a single application of 5 concentrations of the positive control was applied in cell culture medium (dilution factor of 2) with a final concentration of 1% DMSO and a single application of culture medium with 1% DMSO was applied as the negative control (6 wells per plate). One well per plate was left empty (no cells).

Exposure times of test items and reference items
Cells were incubated with the test or reference item for 48 ± 2h prior to endpoint measurements.

Number of repetitions
Three repetitions (runs) were performed. Each repetition consisted of 3 x 96-well plates for luminescence (n=9 overall) and 2 x 96-well plates for MTT (n=6 overall). The validity of each repetition was assessed following the acceptance criteria described.

Study Design
Details of materials, reagents and equipment used are recorded in the study data.
Overview
Preliminary testing: Determination of the top concentration by solubility testing
Day 1: Cell seeding (3 x 96-well plates for Luminescence; 2 x 96-well plate for MTT); 10,000 cells per well, passage number 18.
Day 2: 24 hours after seeding, the test and control items were applied and plates were incubated at 37 °C, 5% CO2, ≥ 95% relative humidity for 48 ± 2 hours.
Day 4: Evaluation of luciferase activity by luminescence (3 plates) and cell viability by MTT testing (2 plates)

Data Analysis
XCellR8 Form F0056: “KeratinoSens data processing” v.1 was used to analyse data. This form is a Microsoft Excel workbook, validated in-house (May 2018) containing formulae to process the raw data as described in SOP L0057.
The following parameters were calculated using the KeratinoSensTM test method:
• The maximal average fold induction of luciferase activity (IMAX) value observed at any concentration of the test item and positive control.
• The EC1.5 value representing the concentration at which the induction of luciferase activity was above the 1.5-fold threshold (i.e. 50% enhanced luciferase activity).
• For each concentration showing > 1.5-fold luciferase activity induction, statistical significance is calculated (e.g. by 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). 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.
• The percentage of viability as compared to the Negative control.

Assay Acceptance Criteria
Test results are acceptable if:
• The positive control (cinnamic aldehyde) produces positive results, i.e. the luciferase gene induction produced by this control is above the threshold of 1.5 in at least one of the tested concentrations and this induction is statistically significant compared to the solvent (negative) control (p<0.05).
• The lMAX and the EC1.5 for cinnamic aldehyde is calculated and meet either or both of the following targets:
o Average induction in the three replicates for cinnamic aldehyde at 32 µM is within the XCellR8 historical range (currently 1.6 and 3)
o EC1.5 value for cinnamic aldehyde is within the XCellR8 historical range (currently 6 µM and 39 µM).
Note: At least one of these criteria must be met, otherwise the run is discarded unless there is sufficient reason not to do this as determined by the Study Director.
If only one criterion is met, it is recommended to check the dose-response curve of cinnamic aldehyde in order to decide on acceptability.
• CV% of blank values < 20%

Interpretation of Results and Skin Sensitisation Prediction model
A test item is considered positive using the KeratinoSens prediction model if the following conditions are met in 2 of 3 repetitions:
• 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).
• 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). Test items that only induce the gene activity at cytotoxic levels are not rated positive, as in the case for some non-sensitising skin irritants.
• The EC1.5 value is < 1000 µM or < 200 µg/mL for test items with no defined MW.
• There is an apparent overall dose-response for luciferase induction (or a biphasic response).

Positive control results:

See "Any Other Information on Results"

Run / experiment:

other: 1

Parameter:

other: EC1.5

Remarks:

in µM

Value:

39.882

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: 2

Parameter:

other: EC1.5

Remarks:

in µM

Value:

34.843

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: 3

Parameter:

other: EC1.5

Remarks:

in µM

Value:

30.68

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

See "Any Other Information on Results"

Solubility Assessment

The test concentrations of Magnesium Glycerophosphate (Magna C) used in the KeratinoSens^TM method were selected on the basis of solubility test carried out during the study:

Solubility of the test item in cell culture medium was confirmed up to 2 mM in 1% DMSO. Subsequent dilution in cell culture medium, which gave a top concentration of 2000 µM.

Determination of the skin sensitisation potential of Magnesium Glycerophosphate (Magna C)

Rep 1	Test item concentration (µM)
	0.977	1.953	3.906	7.813	15.625	31.250	62.5	125	250	500	1000	2000
Mean fold induction	1.114	0.986	1.053	1.107	1.184	1.342	1.914	2.624	3.694	5.618	20.488	0.081
Viability %	99.313	93.843	93.568	89.894	80.940	96.504	98.215	103.958	87.213	76.318	50.238	2.956
T-test	2.14E-01	4.97E-01	3.29E-01	2.23E-01	1.19E-01	2.51E-02	1.01E-05	2.85E-10	4.70E-15	1.10E-26	1.27E-55	1.10E-03
SD	0.198	0.106	0.130	0.109	0.132	0.117	0.241	0.562	1.102	0.734	1.242	0.021
IMAX	20.488 at 1000 µM
EC1.5	39.882 µM
IC30	621.127 µM
IC50	1005.034 µM

Rep 2	Test item concentration (µM)
	0.977	1.953	3.906	7.813	15.625	31.250	62.5	125	250	500	1000	2000
Mean fold induction	1.151	1.056	1.021	1.117	1.173	1.447	1.908	2.276	3.913	4.448	10.013	1.492
Viability %	107.850	81.920	87.832	89.498	88.975	85.800	79.862	95.694	86.528	88.222	76.845	2.187
T-test	1.61E-01	3.22E-01	4.05E-01	2.06E-01	1.32E-01	7.62E-03	9.34E-06	2.07E-08	3.53E-18	1.83E-21	1.63E-38	1.33E-02
SD	0.217	0.112	0.124	0.081	0.144	0.170	0.082	0.125	0.701	0.635	1.152	0.961
IMAX	10.013 at 1000 µM
EC1.5	34.843 µM
IC30	1091.685 µM
IC50	1359. 573 µM

Rep 3	Test item concentration (µM)
	0.977	1.953	3.906	7.813	15.625	31.250	62.5	125	250	500	1000	2000
Mean fold induction	1.014	1.025	1.047	1.086	1.236	1.510	1.905	3.221	3.899	5.878	19.384	0.068
Viability %	121.397	109.646	110.021	106.946	94.394	100.087	94.102	105.229	100.353	89.885	70.533	2.067
T-test	4.22E-01	3.97E-01	3.44E-01	2.68E-01	7.94E-02	3.68E-03	1.21E-05	2.01E-13	5.17E-18	6.35E-23	2.22E-33	9.22E-04
SD	0.106	0.186	0.230	0.302	0.333	0.258	0.264	0.827	0.721	1.542	4.708	0.037
IMAX	19.384 at 1000 µM
EC1.5	30.680 µM
IC30	1007.785 µM
IC50	1299.901 µM

Determination criteria for the skin sensitisation potential ofthe test item
	REP1	REP2	REP3
Does at least one concentration ofTest Iteminduce luciferase activity>1.5-fold:	Yes	Yes	Yes
Does the first concentration inducing luciferase activity above 1.5, have a viability above 70%:	Yes	Yes	Yes
Is p value < 0.05 for at least one concentration that yielded>1.5-fold induction with viability above 70%	Yes	Yes	Yes
Does EC1.5value occur at a concentration <1000µM (or <200µg/ml)	Yes	Yes	Yes
Does the test item induce the luciferase in a dose-dependent manner	Yes	Yes	Yes
Classification	Positive	Positive	Positive

Assay Acceptance Criteria (Mean of 3 repetitions)

Criteria		Result	PASS or FAIL
1	Positive Control (PC) (Cinnamic aldehyde) induction>1.5-fold in at least one concentration	Yes	PASS
2	Average induction of PC at 32µM is [1.6-3.0]	Yes (2.938)	PASS
3	EC1.5value is [6-39µM]	Yes (13.569)	PASS
4	CV% of blank values < 20%	Yes (15.736)	PASS

Interpretation of results:

other: Substance was classified as a Positive using the KeratinoSens prediction model.

Remarks:

Criteria used: EU CLP

Conclusions:

In this study, Magnesium Glycerophosphate (Magna C) was classified as a Positive using the KeratinoSens prediction model.

Executive summary:

The human skin sensitisation potential of Magnesium Glycerophosphate (Magna C) was assessed using the validated in vitro method, the KeratinoSens^TM assay, adapted to animal product-free conditions by XCellR8, and validated in-house to determine keratinocyte activation.

After 48h exposure of cells with 12 concentrations of Magnesium Glycerophosphate (Magna C), Luciferase measurements and MTT viability testing were performed.

Magnesium Glycerophosphate (Magna C) was classified as Positive according to the KeratinoSens prediction model.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

no classification

The test item did not activate human dentritic cells in an hCLAT study.

The test item did not bind to target peptides in an DPRA study.

The test item did not activate luciferase in a Keratinosens study.