Sodium 2-(1-carboxylatoethoxy)-1-methyl-2-oxoethyl laurate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-03-22 to 2018-10-11

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 Guideline 442E (In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation)

Version / remarks:

Adopted 09 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

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:

Sodium 2-(1-carboxylatoethoxy)-1-methyl-2-oxoethyl laurate

EC Number:

236-942-6

EC Name:

Sodium 2-(1-carboxylatoethoxy)-1-methyl-2-oxoethyl laurate

Cas Number:

13557-75-0

Molecular formula:

C18H32O6.Na

IUPAC Name:

sodium 2-{[2-(dodecanoyloxy)propanoyl]oxy}propanoate

Test material form:

solid

Specific details on test material used for the study:

- Purity: technically pure

SOURCE OF TEST MATERIAL
- Source and lot/batch no. of test material: Esterlac SLL (Lot No. 1805900121)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature; protected from light
- Solubility and stability of the test substance in the solvent/vehicle: All test item solutions were freshly prepared immediately prior to use. The test item was soluble in tetrahydrofuran (THF) at a concentration of 500 mg/mL.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Sonication and warming to 50 °C for 10-20 minutes were performed to aid solubilisation. Stock solutions were prepared by diluting the highest soluble concentration seven times with a constant dilution factor of 1:2.

- Final dilution of a dissolved solid, stock liquid or gel: The working stock solutions were then prepared by diluting each stock solution 250 times with cell culture medium.
The working stock solutions were applied to the cells by adding equal volumes of each solution to prepared cells, resulting in a further 1:2 dilution of the working solutions. The solvent (THF) was present at a constant volume ratio of 0.2 % (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.

In vitro test system

Details on the study design:

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

CELL LINE:
The test was carried out using THP-1 cells (ATCC® TIB-202TM), an acute human monocytic leukemic cell line used as a surrogate for dendritic cells. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and subcultured at least 2 weeks before they were used in the in vitro h-CLAT test. Cells at passage number (<30) were used. Cells are routinely passaged every 2-3 days at a density of 0.1–0.2E+06 cells/mL.
Cells were cultured in 75 cm² culture flasks (Greiner) in cell culture medium consisting of Roswell Park Memorial Institute medium (RPMI-1640, Gibco Life Science; Cat. No.: 31870-025) supplemented with 10 % foetal bovine serum, 25 mM HEPES, L-glutamine, 0.05 mM 2-mercaptoethanol and 100 U/mL penicillin / 100 µg/mL streptomycin at 37 ± 1 °C and 5 % CO2.

PRE-EXPERIMENTS:
Reactivity Check of the Cells Stock
Prior to testing, the quality of freshly thawed cell batch was checked by monitoring the doubling time and checking the reactivity towards positive controls. For the reactivity check of the cell batch additional negative and positive controls were included. DNCB at a final concentration of 4 µg/mL and nickel sulphate (NiSO4) at a final concentration of 100 µg/mL served as positive controls while lactic acid (LA) at a final concentration of 1000 µg/mL served as negative control. Cells were accepted when both DNCB and NiSO4 produce a positive response and LA produces a negative response for the upregulation of CD86 and CD54.

Solvent Finding
Solubility of the test item was determined prior to the main experiment. The test item was dissolved in 0.9 % NaCl at a final concentration of 100 mg/mL. Test items not soluble in 0.9 % NaCl solution were dissolved in DMSO at a concentration of 500 mg/mL. If the test item was not soluble in DMSO, other solvents (e.g. THF) were used. It was taken care that the test chemical was dissolved or stably dispersed in the chosen solvent and that it did not interfere with the test design.

EXPERIMENTAL PROCEDURE:
Dose Finding Assay
Using the 500 mg/mL stock solution of the test item, eight working stock solutions were prepared by 2-fold serial dilutions using the appropriate solvent (THF). These working stock solutions were further diluted 250-fold with cell culture medium (working solutions). The working solutions were finally used for treatment by adding an equal volume of working solution to the volume of THP-1 cell suspension in a 96-well plate to achieve a further 2-fold dilution.
For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of 0.1–0.2E+06 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation and were re-suspended in fresh culture medium at a density of 2.0E+06 cells/mL. Then 500 µL of the cell suspension were seeded onto a 24 well flat-bottom plate (1.0E+06 cells/well).
The solvent controls and the test item working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2.
After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 × g). The supernatant was discarded and the remaining cells were washed twice with Dulbecco’s phosphate buffered saline (DPBS) containing 0.1% bovine serum albumin (BSA; i.e. FACS buffer). After washing, cells were re-suspended in 600 µL FACS buffer.
200 µL of the cell suspension were transferred into a FACS tube and stained with a propidium iodide (PI) solution at a final concentration of 0.625 µg/mL.
The PI uptake of the cells (an indicator of cytotoxicity) was analysed immediately after the staining procedure by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ > 650 nm. A total of 10,000 cells were acquired and cell viability was calculated for each test concentration.
The CV75 value, i.e. the concentration showing 75 % cell survival, was calculated by log-linear interpolation. The CV75 value was used to calculate and set the concentration range of the test item for the main experiments.

CD54 and CD86 Expression
The test item was dissolved using THF as determined in the pre-experiment. Based on the concentration of the CV75 value determined in the dose finding assay 8 concentrations of the test item were defined for the measurement of the surface marker expression, corresponding to 1.2*CV75; CV75; CV75/1.2; CV75/1.22; CV75/1.23; CV75/1.24; CV75/1.25; CV75/1.26. If the CV75 could not be determined due to insufficient cytotoxicity of the test item in the dose finding assay, the highest soluble concentration of the test item prepared with each solvent was used as starting dose.
The test item was diluted to the concentration corresponding to 500-fold of the 1.2 × CV75. Then, 1.2-fold serial dilutions were made using the corresponding solvent to obtain the 8 stock solutions to be tested. The stock solutions were further diluted 250-fold into the culture medium (working solutions). These working solutions were finally used for cell treatment with a further final 2-fold dilution factor.
For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of 0.1–0.2E+06 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation (125 × g) and were re-suspended in fresh culture medium at a density of 2.0E+06 cells/mL. Then 500 µL of the cell suspension were seeded onto a 24 well flat-bottom plate (1.0E+06 cells/well).
The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared on the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5 % CO2.
After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 × g). The following steps were carried out on ice with pre-cooled buffers and solutions. The supernatant was discarded and the remaining cells were washed twice with FACS buffer. After washing, cells were blocked using 600 µL of a FcR blocking buffer (FACS buffer containing 0.01 % (w/v) Globulin Cohn Fraction) and incubated at 4 °C for 15 min. After blocking, cells were split in three aliquots into a 96-well V-bottom plate. After centrifugation (approx. 250 × g), cells were incubated with 50 µL of FITC-labelled anti-CD86, FITC-labelled anti-CD54, or mouse FITC-labelled IgG1 (isotype) antibodies in the dark for 30 min at 4 °C. All antibodies were diluted in FACS buffer. After incubating and then washing with FACS buffer two times, cells were re-suspended in FACS buffer and PI solution was added. PI staining was done just prior to the measurement by adding PI solutions to each sample (final concentration of PI was 0.625 µg/mL).
The expression levels of CD86 and CD54 as well as cell viability (as determined by living cells with no PI uptake) were analysed by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ = 530 nm ± 15 nm for FITC and λ > 650 nm for PI. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 were calculated. The cell viability was also calculated.

DATA ANALYSIS
FACS data analysis was performed using the software BD FACS DIVA 6.0. Further data analysis such as calculations of the CV75, RFI, Effective Concentration 150 (EC150) and Effective Concentration 200 (EC200) values were performed using the software Microsoft Excel 2010 as appropriate. The mean values and standard deviations of the single replicates were determined using the respective Excel commands.

PREDICTION MODEL
For CD86/CD54 expression measurement, each test item was tested in at least two independent runs to derive a single prediction. Each independent run was performed on a different day or on the same day provided that for each run independent fresh stock solutions and working solutions of the test chemicals and antibody solutions were prepared and independently harvested cells were used.
Sensitising potential of the test item was predicted from the mean percentage expression of CD86 and CD54. Any test item tested by the h-CLAT was considered positive if any of the three scenarios is met:
- if the RFI of CD86 was equal to or greater than 150% at any tested dose at a cell viability ≥ 50 % in at least two independent runs
- if the RFI of CD54 was equal to or greater than 200% at any tested dose at a cell viability ≥ 50 % in at least two independent runs
- if the RFIs of both the CD86 and CD54 were equal to or are greater than 150 % and 200% respectively at any tested dose at a cell viability ≥ 50% in at least two independent runs
In case of not concordant results a third run should be conducted to make the final prediction. Otherwise the results were considered as inconclusive.
A negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 × CV75 is < 90 %. In contrast, a positive test outcome was accepted irrespective of cell viability of > 90 % at a concentration of 1.2 × CV75. If no CV75 could be derived negative test results can be accepted when the test item is tested at the highest soluble concentration (i.e. up to 5000 µg/mL for 0.9 % NaCl solution; up to 1000 µg/mL for DMSO or a different organic solvent) even if the cell viability is > 90 %.

Results and discussion

Positive control results:

The positive control (DNCB) led to an upregulation of CD54 and CD86 in both experiments. The thresholds of 150 % for CD86 (302 % experiment 1; 275 % experiment 2) and 200 % for CD54 (428 % experiment 1; 248 % experiment 2) were clearly exceeded.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: 1

Parameter:

other: CD86 upregulation

Remarks:

at highest tested concentration of 125.56 µg/mL

Value:

2 273

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

severe cytotoxicity

Results 2

Key result

Run / experiment:

other: 1

Parameter:

other: CD54 upregulation

Remarks:

at highest tested concentration of 125.56 µg/mL

Value:

3 499

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

severe cytotoxicity

Results 3

Key result

Run / experiment:

other: 2

Parameter:

other: CD86 upregulation

Remarks:

at highest tested concentration of 125.56 µg/mL

Value:

102

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

severe cytotoxicity

Results 4

Key result

Run / experiment:

other: 2

Parameter:

other: CD54 upregulation

Remarks:

at highest tested concentration of 125.56 µg/mL

Value:

94

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

severe cytotoxicity

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
For individual results see Tables 1 and 2 in box "Any other information on results incl. tables".

Any other information on results incl. tables

Summary of Results:

Severe cytotoxicity was observed for the cells treated with the test item. Relative cell viability at the highest test item concentration of 125.56 μg/mL was reduced to 7.6 % (CD86), 7.5 % (CD54) and 7.8 % (isotype IgG1 control) compared to the solvent control in the first experiment and to 13.4 % (CD86), 13.3% (CD54) and 12.4 % (isotype IgG1 control) compared to the solvent control in the second experiment.

In both experiments, the expression of the cell surface marker CD86 was upregulated above the threshold of 150 % relative to the solvent control at the highest test item concentration of 125.56 μg/mL (2273 % in the first experiment, 203 % in the second experiment). At this concentration the test item showed severe cytotoxicity. No further upregulation above the threshold of 150 % was observed for CD86 in the other tested concentrations.

In both experiments, the expression of the cell surface marker CD54 was upregulated above the threshold of 200 % relative to the solvent control at the highest test item concentration of 125.56 μg/mL (3499 % in the first experiment, 377 % in the second experiment). At this concentration the test item showed severe cytotoxicity. No further upregulation above the threshold of 200 % was observed for CD54 in the other tested concentrations.

Table1: CD54 and CD86 Expression Experiment 1

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			Mean Fluorescence Intensity (corrected for IgG1)		Relative Fluorescence Intensity (RFI) [%]^1		Cell marker to Isotype IgG1 [%]^2
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	94.7	95.4	95.2	2174	1124	610	1564	514	84	95	356	184
Solvent Control (0.2% THF)	-	94.8	94.4	94.9	2036.0	1190.0	637.0	1399	553	100	100	320	187
Solvent Control (0.2% DMSO)	-	94.0	94.3	94.1	2469	1137	597	1872	540	100	100	414	190
DNCB	4.00	77.2	76.8	77.1	6275	2930	617	5658	2313	302	428	1017	475
Sodium lauroyl lactylate	125.56	7.6	7.5	7.8	34370	21922	2574	31796	19348	2273	3499	1335	852
	104.63	14.0	14.5	14.2	2363	1698	795	1568	903	112	163	297	214
	87.19	85.3	84.1	84.3	2473	1316	695	1778	621	127	112	356	189
	72.66	90.9	91.4	89.7	2403	1248	705	1698	543	121	98	341	177
	60.55	92.6	92.7	91.5	2317	1168	709	1608	459	115	83	327	165
	50.46	93.5	93.1	93.2	2234	1220	695	1539	525	110	95	321	176
	42.05	93.5	94.3	93.6	2453	1216	700	1753	516	125	93	350	174
	35.04	93.7	94.1	93.3	2259	1179	677	1582	502	113	91	334	174

 

Table2:  CD54 and CD86 Expression Experiment 2

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			Mean Fluorescence Intensity (corrected for IgG1)		Relative Fluorescence Intensity (RFI) [%]^1		Cell marker to Isotype IgG1 [%]^2
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	92.0	93.2	92.6	3138	1369	616	2522	753	98	109	509	222
Solvent Control 2 (0.2% THF)	-	93.4	93.7	93.6	2849	1258	672	2177	586	100	100	424	187
Solvent Control 1 (0.2% DMSO)	-	93.5	93.2	93.4	3159	1280	590	2569	690	100	100	535	217
DNCB	4.0	76.9	77.2	76.9	7652	2300	586	7066	1714	275	248	1306	392
Sodium lauroyl lactylate	125.56	13.4	13.3	12.4	5733	3528	1319	4414	2209	203	377	435	267
	104.63	28.8	29.6	29.7	3071	1607	743	2328	864	107	147	413	216
	87.19	70.5	69.1	69.7	3288	1466	735	2553	731	117	125	447	199
	72.66	88.6	89.2	89.0	3024	1392	765	2259	627	104	107	395	182
	60.55	90.9	90.7	90.7	3250	1334	772	2478	562	114	96	421	173
	50.46	91.0	91.9	92.1	2983	1327	781	2202	546	101	93	382	170
	42.05	92.2	92.6	91.7	3033	1337	734	2299	603	106	103	413	182
	35.04	93.2	93.7	93.4	2984	1301	753	2231	548	102	94	396	173

 

RFI is relative to the respective solvent control (i.e. 0.2% DMSO for DNCB and medium control and 0.2% THF for the test item).

¹(MFI_{negative control, solvent control, positive control, test item}* 100) / MFI_{solvent control}(all corrected for IgG1)

²(MFI_{negative control, solvent control, positive control, test item}* MFI_{solvent control}) * 100

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study (performed in accordance with OECD 442E) under the given conditions the test item sodium lauroyl lactylate did not trigger an upregulation of the expression of the cell surface markers CD86 and CD54 in two independent experiment runs. Therefore, the test item is considered to be non-sensitiser in this test.

Executive summary:

In a skin sensitisation study conducted according to OECD 442E, the sensitisation potential of the test item sodium lauroyl lactylate (technically pure) in tetrahydrofuran was assessed on the basis of dendritic cell activation by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1.

Cells were incubated with the test item (8 concentrations; range 35.04–125.56 µg/mL) for 24 h at 37 °C. After exposure cells were labelled with fluorescent antibodies of cell surface markers CD54 and CD86 and the expression levels of CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

In both experiments, the expression of the cell surface marker CD86 was upregulated above the threshold of 150 % relative to the solvent control at the highest test item concentration of 125.56 µg/mL (2273 % in the first experiment, 203 % in the second experiment). At this concentration the test item showed severe cytotoxicity. No further upregulation above the threshold of 150 % was observed for CD86 in the other tested concentrations.

In both experiments, the expression of the cell surface marker CD54 was upregulated above the threshold of 200 % relative to the solvent control at the highest test item concentration of 125.56 µg/mL (3499 % in the first experiment, 377 % in the second experiment). At this concentration the test item showed severe cytotoxicity. No further upregulation above the threshold of 200 % was observed for CD54 in the other tested concentrations.

Therefore, sodium lauroyl lactylate is considered to be a non-sensitiser in this test.