2-phenoxyethanol; phosphoric acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-03-14 to 2018-04-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 442E

Version / remarks:

adopted 2017

Deviations:

no

GLP compliance:

yes

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

h-CLAT is an in vitro method proposed to address the third key event (dendritic cell activation) of the skin sensitization adverse outcome pathway (AOP) by quantifying changes in the expression of cell surface markers associated with the process of activation of dendritic cells (i.e. CD54 and CD86) in the human leukemia cell line THP-1, following exposure to sensitizers. The measured expression levels of CD54 and CD86 cell surface markers are then used for supporting the discrimination between skin sensitizers and non-sensitizers. The correlation of upregulation of immunologically relevant cell surface markers with the skin sensitising potential of a chemical has been reported and represents the third key event of the skin sensitisation process as described by the AOP for skin sensitisation. This method, which measures the markers of DC activation, using the DC-like cell line THP-1 is considered relevant for the assessment of the skin sensitisation potential of chemicals.

Test material

Test material information

Constituent 1

Reference substance name:

2-phenoxyethanol; phosphoric acid

EC Number:

609-691-9

Cas Number:

39464-70-5

Molecular formula:

(C2-H4-O)mult-C6-H6-O.x-H3-O4-P

IUPAC Name:

2-phenoxyethanol; phosphoric acid

Specific details on test material used for the study:

- Purity: 100%
- Appearance: clear yellow liquid
- Storage: controlled room temperature

In vitro test system

Details on the study design:

Skin sensitisation (In vitro test system)
- Details on study design:

Solubilization of the test substance and solvent selection (Day 1):
As the test substance was soluble both in physiological solution (PS) and in DMSO, pre-feasibility test and Dose finding assay were performed with test substance dissolved both in PS and DMSO.
The assay was carried out in duplicate, starting from two different cultures (suspension A and B) and using 2 different sets of test substance dilutions (First preparation and second preparation) and 2 different solvents (PS and DMSO).

Solubilization of the test substance in physiological solution (PS):
A test substance solution 500 mg/mL was prepared by adding up to 1 ml of physiological solution to about 500 mg (499.8 mg for First preparation and 500.2 mg for Second preparation) of the test substance in a volumetric flask. 100 μL of this solution were diluted 1:50 with 4.900 mL of culture medium to produce the working solution 10,000 μg/mL (C1). Then, 7 dilutions (labeled from C2 to C8) were prepared with the constant dilution factor 1:2 in culture medium.
Solubilization of the test substance in DMSO:
A test substance solution 500 mg/mL was prepared by adding up to 1 ml of DMSO to about 500 mg (499.9 mg for First preparation and 500.4 mg for Second preparation) of the test substance in a volumetric flask. 20 μL of this solution were diluted 1:250 with 4.980 mL of culture medium to produce the working solution 2,000 μg/mL (C1). Then, 7 dilutions (labeled from C2 to C8) were prepared with the constant dilution factor 1:2 in culture medium: C1 in DMSO First (or second) preparation (2,000 μg/mL)

CELL LINE:
Cell line: THP-1
Supplier’s name and cat. n°: ATCC TIB-202
Monocytes from peripheral blood
Culture conditions: 37 °C ± 1 °C, 5 % CO2, 90 % ± 10 % relative humidity
Culture Medium: RPMI enriched with 10 % heat-inactivated Fetal Bovine Serum (FBS), 2 mM L-Glutamine, 0.05 mM 2-mercaptoethanol, 1 % penicillin/streptomycin solution (NRI 226 VIT/2018; 280 VIT/2018; 296 VIT/2018).
The cells are stored as frozen stocks in liquid nitrogen.
A new batch of THP-1 cell stored in liquid nitrogen was thawed at least 2 weeks prior to start with the test. After thawing, the batch of cells that was employed in the assay was monitored for the absence of mycoplasma applying the procedure described in SOP 80. Cells can be propagated up to 2 months after thawing.

TEST METHOD AND EXPERIMENTAL DESIGN:
Procedure for Pre-feasibility test and Dose finding (Day 1)
2 different cultures were collected by centrifugation at 250 g for 5 minutes at room temperature and re-suspended in fresh culture medium. For each culture, a 5 mL cell suspension (suspension A and suspension B) at a density of 2 x 10^6 cells/mL were prepared. 80 μL of suspension A were added to 36 wells of a 96-well flat-bottom plate (1.6 x 10^5 cells/well) and 80 μL of suspension B were added to 18 wells of the same plate. Afterwards, 80 μL of the corresponding test substance concentration (C1-C8) or 80 μL of solvent control were added to the corresponding wells of the same plate. The plate was covered with sealing tape and incubated for 24 hours at 37 °C and 5 % CO2. In the meanwhile FACS buffer (D-PBS with 0.1 % (w/v) BSA) was prepared and stored in the fridge until the next day.

Procedure for Pre-feasibility test and Dose finding (Day 2):
At the end of the incubation period cells were moved from each well to 96 well plate V bottom in the same position. Cells were centrifuged at 250 g for 5 min at 4 °C and resuspended in 200 μL of ice-cold FACS buffer. This washing step was repeated twice.
In the meanwhile, FACS buffer containing PI at a final concentration of 0.625 μg/mL was prepared starting from a 1 mg/mL PI stock solution as follows: 6.25 μL Propidium Iodide (PI) in 10 mL of FACS buffer. At the end of the second washing step, cells were resuspended in 200 μL of FACS buffer or in 200 μL of FACS buffer + PI.
Unstained cells were used for pre-feasibility test to verify that the test substance does not interfere with the Propidium iodide fluorescent signal due to intrinsic fluorescence. Cells stained with PI were used for dose finding assay for the determination of CV75 value. During the whole study all solutions (FACS buffer and PI solutions) were stored on ice or in the fridge till use.

EXPERIMENTAL PROCEDURE:
THP-1 cells were exposed to the test chemical for 24 hours and changes in CD54 and CD86 expression levels were measured by flow cytometry following cell staining with fluorescein isothiocyanate (FITC)-labeled antibodies. In parallel, cytotoxicity measurement was conducted to assess whether up-regulation of surface marker expression occurs at sub-cytotoxic concentrations.
The test was divided in 4 phases:
1) Reactivity check of the cells which were thawed for the assay, performed by treating cells for 24 hours with 2 positive controls, DNCB and NiSO4 6H2O and the negative control lactic acid.
2) Pre-feasibility test, to verify that the test substance did not interfere with the Propidium Iodide fluorescent signal due to intrinsic fluorescence.
3) Dose finding assay, to calculate the CV75 needed for determining the concentrations of test substance to be used in the CD54/CD86 expression experiment; in this phase cells were exposed for 24 hours to 8 different concentrations of test substance. Viability was measured after Propidium Iodide (PI) staining as the ration between number of living cells and total number of acquired cells.
4) CD54/CD86 expression assay, in which cells were incubated for 24 hours with 8 doses of test substance. As it was not possible to calculate the CV75 value, due to insufficient cytotoxicity of the test substance, 5000 μg/mL concentration was used as the first concentration for the CD54/CD86 expression assay, the highest concentration of the test chemical stably dissolved in physiological solution. Then 7 serial dilutions with a 1.2-fold dilution factor were used.
Expression levels of CD54 and CD86 were analyzed by flow cytometry and the relative fluorescence intensity (RFI) was calculated in order to define the test substance as positive (potential sensitizer) or negative (non-sensitizer). The test substance was assayed in 2 independent runs, performed in two different days. A third run was not necessary.

CULTURING OF CELL LINE
Culturing of the cell line was carried out following the procedure described in SOP 80 and as below detailed.
Thawing: At least 2 weeks before starting the test, one cryovial containing the cell line was removed from the liquid nitrogen tank and was thawed in a water bath at 37 °C. The vial was removed from the water bath and the entire volume was transferred into a sterile tube containing 9 mL of culture medium and spinned for 5 minutes at 250 g. Supernatant was discarded and cell pellet was resuspended in fresh culture medium. Finally, cells were transferred into flasks containing culture medium and incubated at 37 °C ± 1 °C, 5 % CO2.
Maintenance of cell cultures: Cells were maintained in suspension at a cell density from 0.1 to about 0.8 x 106 cells/mL using culture medium and incubated at 37 °C and 5 % CO2 under gentle agitation (60-100 rpm). The cell density did not exceed 1 x 106 cells/mL. Every 2-3 days cells were counted on a Burker chamber, centrifuged, resuspended in fresh medium and passaged into new flasks at the concentration of 0.2 x 106 cells/mL.
Cells can be propagated up to two months after thawing but not in excess of 30 passages.
For cell counting throughout the whole assay, the Burker chamber was employed. Cell counting was performed as by internal procedure. In detail, 20 μL of the cell suspension to be counted was mixed with 20 μL of trypan blue (1:2 dilution), an agent used to discriminate within dead and live cells. 20 μL of the mixture was gently applied at the interface between Burker chamber and coverslip. Only live cells were counted as the dead cells were discriminated because of the blue colour due to the trypan blue. The cells contained in the inner of the 12 little squares of each diagonal were counted together with those cells touching the upper and the right side of each square. A total of 4 diagonals (2 per each squared reticulate) were counted and hence 48 little squares. The number of cells per mL (CI) were then determined applying the following formula:
cells in 1 mL (CI) = (N/48) x 250 x 2 x 1000
where:
N is the number of cells counted in 48 squares;
48 are the total little squares counted;
250 represents the volume of each square of the Burker chamber;
2 is the dilution factor due to the addition of trypan blue;
1000 is the conversion factor from μl to ml.
To calculate the total number of cells, the number of cells/ml was multiplied by the total number of ml.
Cell count was expressed with 3 decimal digits. The approximation was done by excess, i.e. values were rounded up when the last digit was ≥ 5, they were rounded down when the last digit was < 5.
Cells were also controlled for mycoplasma absence following the procedure described in SOP 80.
The cells appeared free of mycoplasma, therefore they were employed in the assay.
Monitoring of doubling time: One week after thawing, the doubling time of the thawed batch was controlled. For measurement of doubling time cells were seeded at the density of 0.2 x 106 cells/mlL and incubated at 37 °C and 5 % CO2. The density of the cell suspension was measured at 24, 48 and 72 hours after seeding.

REACTIVITY CHECK
Two weeks after thawing, the reactivity check was performed on the cell batch that was employed in the assay. This step was needed to establish the sensitivity of the cells to the assay, i.e. if after incubation with 2 known sensitizers, the levels of CD54/CD86 receptors increases and if after exposure to 1 known non-sensitizer, CD54/CD86 levels remain unaltered. Only cells which passed the reactivity check are to be used in the assay. Cell reactivity was assessed as described below.
48 hours before the assay, cells were seeded so that the day of the test they were at least 10 x 10^6 total cells.

PREPARATION of THREE CONTROL SUBSTANCES (Day 1)
The day of the assay, solutions of the controls were prepared and were stored in the dark.
DNCB and NiSO4 6H2O were used as positive controls, lactic acid was used as negative control. For DNCB, a 2 mg/mL stock solution was prepared in DMSO by adding up to 5 mL of DMSO to 10.3 mg of DNCB in a volumetric flask. 10 μL of this solution was diluted 1:250 with 2.49 mL of culture medium to obtain the solution 8 μg/mL.
For NiSO4 6H2O, a 10 mg/mL stock solution was prepared in physiological solution by adding up to 2 mL of physiological solution to 20.4 mg of NiSO4 6H2O in a volumetric flask. 50 μL of this solution were diluted 1:50 with 2.45 mL of culture medium to obtain the solution 200 μg/mL.
For LA, a 100 mg/mL stock solution was prepared in physiological solution by adding up to 2 mL of physiological solution to 199.6 mg of LA in a volumetric flask. 50 μL of this solution were diluted 1:50 with 2.45 mL of culture medium to obtain the solution 2000 μg/mL.
Notably, the 3 control substances were prepared double concentrated since they were subjected to a further 1:2 dilution once mixed with the cells for the assay.
As solvent controls were employed:
- NC: culture medium
- NC+PS: culture medium with physiological solution (50 μL of physiological solution in 2.45 mL of culture medium)
- NC+DMSO: culture medium with DMSO (10 μL of DMSO in 2.49 mL of culture medium)

PROCEDURE FOR THE REACTIVITY TEST (DAY 1)
Cells were collected by centrifugation at 250 g for 5 minutes at room temperature and re-suspended in fresh culture medium at a density of 2 x 10^6 cells/mL. 80 μL of cell suspension were added to 18 well of a 96-well flat-bottom plate (1.6 x 10^5 cells/well). Afterwards, 80 μL of control solutions were added to the corresponding wells of the same plate. Therefore, due to the mixing with the cells, the final concentration of DNCB was 4 μg/mL, the one of NiSO4 6H2O 100 μg/mL and LA was 1000 μg/mL.
The plate was covered with sealing tape and incubated for 24 hours at 37 °C and 5 % CO2.
In the meanwhile FACS buffer (D-PBS with 0.1 % (w/v) BSA) and 0.1 % blocking buffer (0.1 % globulin solution in
FACS buffer) were prepared and stored in fridge until the next day.

PROCEDURE FOR THE REACTIVITY TEST (DAY 2)
The 0.01 % blocking buffer was prepared by diluting 1 mL of 0.1 % blocking buffer with 9 mL of FACS buffer (dilution 1:10). The solution was stored on ice.
At the end of the incubation period cells were moved from each well to 96 well plate V bottom in the same position.
Cells were centrifuged at 250 g for 5 min at 4 °C and resuspended in 200 μL of ice-cold FACS buffer.
This washing step were repeated twice. At the end of the second washing step, the cell pellet were resuspended in 100 μL of 0.01 % blocking buffer and incubated for 15 minutes in the fridge.
Pre-mixed antibody solutions (master mix) were prepared as described in table 5. Notably, the volume of master mix was prepared according to the number of samples stained with the same antibody plus three sample in excess, in order to cover the eventual loss of solution due to pipetting. After blocking cells were centrifuged at 250 g for 5 min at 4 °C, supernatant was discarded and cell pellet was resuspended with 30 μL of the corresponding antibody solution. Cells were then incubated in the fridge for 30 minutes in the dark.
After staining cells were centrifuged at 250 g for 5 min at 4 °C, the supernatant was discarded and cell pellet was resuspended with 200 μL of FACS buffer. This washing was repeated twice. In the meanwhile, FACS buffer containing PI at a final concentration of 0.625 μg/mL was prepared starting from a 1 mg/mL PI stock solution as follows: 6.25 μL Propidium Iodide (PI) in 10 mL of FACS buffer.
At the end of the second washing step, cells were re-suspended in 200 μL of FACS buffer + PI.
During the whole study all solutions (FACS buffer, blocking buffer, antibody and PI solutions) were stored on ice or in the fridge till use.

ACQUISITION
Expression of cell surface antigen was analysed by flow cytometry. The Excitation and Emission wavelength of CytoFLEX (Beckman Coulter) are shown below:
Excitation wavelength: 488 nm
Emission wavelength: for FITC 525/40 nm; for PI 610/20 nm
At least 10000 cells including dead cells were acquired. For flow cytometry analysis the QC settings obtained by reading of “Daily QC fluorospheres” were used. The gain configuration of the instrument was reported only in the raw data.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: 1

Parameter:

other: max. CD86 upregulation

Value:

105.58

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Run / experiment:

other: 1

Parameter:

other: max. CD54 upregulation

Value:

124.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Run / experiment:

other: 2

Parameter:

other: max. CD86 upregulation

Value:

67.45

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 4

Key result

Run / experiment:

other: 2

Parameter:

other: max. CD54 upregulation

Value:

73.38

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

In the CD54/CD86 expression assay, RFI of CD54 is < 200 % and RFI CD86 is < 150 % at any tested dose, therefore it was not possible to calculate the EC200 and EC150 values and all data met the acceptance criteria. For detailed results please refer to Table 1 in box "Any other information on results incl. tables".

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, 98.89 % (> 90 %)
- Acceptance criteria met for positive control: Yes, 98.68 % (> 90 %)

Any other information on results incl. tables

Table 1: Results of CD54/ CD86 expression assay

	Test dose in well (µg/ml)	RFI CD54 (%)		RFI CD86 (%)		Viability (%)
		Run 1	Run 2	Run 1	Run 2	Run 1	Run 2
Test substance C1	5000.00	85.34	38.07	89.24	35.84	95.71	96.43
Test substance C2	4166.67	121.36	39.53	101.70	36.07	96.32	97.65
Test substance C3	3472.23	124.70	40.44	90.72	29.31	96.55	98.07
Test substance C4	2893.53	108.97	44.13	103.79	32.93	96.93	98.31
Test substance C5	2411.28	105.11	46.62	92.16	39.22	97.13	98.46
Test substance C6	2009.40	108.62	53.24	85.17	56.67	97.26	98.58
Test substance C7	1674.50	104.59	53.21	79.36	56.61	97.71	98.67
Test substance C8	1395.42	100.24	73.38	105.58	67.45	97.86	98.84
DNCB	4.00	860.78	268.33	328.59	392.53	85.28	85.79
NC	/	/	/	/	/	98.35	98.91
NC+PS	/	81.71	93.01	84.60	94.68	98.38	98.88
NC+DMSO	/	67.63	102.20	65.27	85.66	98.20	98.52

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In the present study, Ethoxylated phenol phosphate is to be considered negative (non-sensitizer) up to the concentration 5000 μg/mL. A third run was not necessary, since the first 2 runs produced concordant results.

Executive summary:

In a skin sensitisation study conducted according to OECD 442E with Ethoxylated phenol phosphate (100% purity), the sensitisation potential of the test item was assessed on the basis of the activation of dendritic cells using the in vitro human cell line activation test (h-CLAT). Cells were incubated with the test item for 24 h at 37 °C and later checked for cell viability and expression of CD86 and CD54 cell surface markers. Sensitisation was scored by measuring cell viability and checking the expression of both cell surface markers. CD54 and CD86 were not upregulated above the threshold of 200% (CD54) and 150% (CD86) in both experiments upto the highest concentration of 5000 µg/mL. Based on these results, the test item is not considered to be a skin sensitizer under the UN GHS criteria.