Calcium glycerophosphate

Skin sensitisation

Administrative data

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:

18 October - 16 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Calcium glycerophosphate

EC Number:

248-328-5

EC Name:

Calcium glycerophosphate

Cas Number:

27214-00-2

Molecular formula:

C3H9O6P.Ca

IUPAC Name:

calcium glycerophosphate

Test material form:

solid: particulate/powder

Details on test material:

Batch: CGP0416003

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: Calcium glycerophosphate
Trade Name: Vivcal-G
Batch: CGP0416003
CAS No.: 27214-00-2
EC No.: 248-328-5
Purity: 91.7%* (w/w) (calculated)
Partition coefficient
(n-octanol/water):
log Pow: < -1.7
Water solubility: 25 g/L (20 °C)
Appearance: White powder
Expiry Date: 29 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.

In vitro test system

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).

Results and discussion

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

In vitro / in chemico

Results

Key result

Run / experiment:

other: all

Parameter:

other: cell activation

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable because of methodological limitations

Remarks:

The test item Calcium glycerophosphate with a log Pow of < -1.7 activated THP-1 cells under the test conditions of this study. However, due to the observed precipitations after the treatment of the cells in all three h-CLAT runs and no clear observed dose response, a false positive activation of the cells cannot be excluded. Therefore the results of the test item is considered inconclusive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Any other information on results incl. tables

 Resultsof the Dose Finding Assay (XTT Test)

Results of the first XTT test for Test Item Calcium 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	0.733	0.023	0.235	0.499	88.73
Solvent Control	-	no	0.784	0.036	0.222	0.562	100.00
Test Item	39.1	no	0.730	0.013	0.222	0.508	90.41
	78.1	no	0.767	0.012	0.230	0.537	95.64
	156.3	no	0.750	0.016	0.230	0.520	92.49
	312.5	no	0.865	0.024	0.320	0.545	97.02
	625^P	no	1.017	0.038	0.395	0.622	110.62
	1250^P	no	1.117	0.038	0.483	0.634	112.75
	2500^P	yes	1.396	0.065	0.572	0.824	146.60
	5000^P	yes	1.139	0.025	0.539	0.601	106.91

^P    Precipitation (results are excluded from the evaluation, shaded grey)

*            mean absorbance (absolute) of 7 wells

**          relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium control was 112.70%.

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 Calcium 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	0.627	0.032	0.256	0.372	101.45
Solvent Control	-	no	0.621	0.015	0.255	0.366	100.00
Test Item	39.1	no	0.630	0.022	0.253	0.378	103.14
	78.1	no	0.620	0.014	0.258	0.362	98.97
	156.3	no	0.637	0.014	0.259	0.378	103.22
	312.5^P	no	0.717	0.031	0.338	0.379	103.58
	625^P	no	0.853	0.034	0.427	0.426	116.24
	1250^P	no	1.071	0.029	0.566	0.505	137.85
	2500^P	no	1.118	0.033	0.604	0.514	140.39
	5000^P	no	1.091	0.062	0.617	0.474	129.42

^P    Precipitation (results are excluded from the evaluation, shaded grey)

*            mean absorbance (absolute) of 7 wells

**          relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium control was 98.57%.

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 Calcium 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	440.7*	744.9*	63.1
	3.0	759.3*	859.5*	52.5
Test Item	174	146.2	71.2	106.8
	209	163.5	74.5	107.4
	251	173.1	85.6	105.2
	301	151.9	106.6	89.5
	362	157.7	97.9	86.0
	434^P	167.3	105.3	86.2
	521^P	155.8	114.4	85.5
	625^P	301.9*	151.4*	76.8

^P    Precipitation (results are excluded from the evaluation, shaded grey)

*   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 Calcium 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	174	104.2	111.0	100.1
	209	109.3	111.0	97.3
	251	104.2	119.3	98.0
	301	192.4	189.4*	79.2
	362^P	193.2	168.8*	77.6
	434^P	205.9*	173.9*	74.7
	521^P	233.1*	185.8*	73.1
	625^P	349.2*	336.7*	68.9

^P    Precipitation (results are excluded from the evaluation, shaded grey)

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

 

 

Results of the third h-CLAT run for the Test Item Calcium 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	145	142.3	96.0	99.2
	174	167.9	110.4	98.4
	209	133.3	106.9	98.9
	251	110.3	148.0	95.5
	301	141.0	188.1*	91.1
	362^P	166.7	193.1*	90.1
	434^P	202.6*	185.1*	85.1
	521^P	262.8*	246.5*	78.0

^P    Precipitation (results are excluded from the evaluation, shaded grey)

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

 

The test item concentrations for the third h-CLAT run (main experiment) were adjusted, due to precipitations observed in four test item concentrations after the treatment of the cells in the second h-CLAT run.

 

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

The test item Calcium glycerophosphate with a log Pow of < -1.7 activated THP-1 cells under the test conditions of this study. However, due to the observed precipitations after the treatment of the cells in all three h-CLAT runs and no clear observed dose response, a false positive activation of the cells cannot be excluded. Therefore the results of the test item is considered inconclusive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of Calcium glycerophosphate stable 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 Calcium glycerophosphate was previously determined by two XTT tests.

Cytotoxic effects were observed by microscopic evaluation but not by photometric evaluation following incubation with the test item starting with the concentration of 2500 µg/mL up to the highest tested concentration (5000 µg/mL) in the first XTT test. No cytotoxic effects were observed following incubation with the test item up to the highest tested concentration (5000 µg/mL) in the second XTT test. A CV75 value could not be calculated, due to the lack of cytotoxicity by photometric evaluation and observed precipitations in both XTT tests. Thereforea test item concentration of625 µg/mL (the first concentration with precipitations in the first XTT test and the second concentration with precipitations in the second XTT test)wasused for the first and second h-CLAT runs.

The following concentrations of the test item (dissolved inculture medium)were tested in the main experiment (h-CLAT run):

174, 209, 251, 301, 362, 434, 521 and 625 µg/mL in the first and second main experiment

and

145, 174, 209, 251, 301, 362, 434 and 521 µg/mL in the third experiment.

The test item with a log Pow of < -1.7 was tested in 3 independent runs. The treatment time of the cells in the third main experiment was 24 hours + 35 minutes instead of 24 hours ± 30 minutes. The three highest test item concentrations of the first run and the four highest test item concentrations of the second h-CLAT run showed precipitations after the treatment of the cells. Therefore, the test item concentrations for the third run were adjusted. Due to the precipitations observed in all three runs, the test item concentrations with precipitations were excluded from the evaluation. Under this consideration the RFI of CD86 was greater than 150% in one concentration of 2 out of 3 independent runs. Therefore the h-CLAT prediction is considered positive for the test item in this h-CLAT. However, a false positive activation of the cells cannot be excluded since no clear dose response was observed. Furthermore, due to the observed precipitations after the treatment of the cells in all three h-CLAT runs, it cannot be excluded that small non-observed precipitates were also present in the test item concentrations with a RFI of CD86 > 150%. Therefore the h-CLAT prediction is considered inconclusive 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.