Alcohols, C10-16, ethoxylated, sulfosuccinates, disodium salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 Dec 2017 - 08 Mar 2018

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, No. 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

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158

Version / remarks:

July 1st, 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP-Landesleitstelle Bayern, Bayerrisches Landesamt für Gesundheit und Lebensmittelsicherheit

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

Alcohols, C10-16, ethoxylated, sulfosuccinates, disodium salts

EC Number:

500-232-7

EC Name:

Alcohols, C10-16, ethoxylated, sulfosuccinates, disodium salts

Cas Number:

68815-56-5

Molecular formula:

not applicable to UVCB

IUPAC Name:

Alcohols, C10-16, ethoxylated, sulfosuccinates, disodium salts

In vitro test system

Details on the study design:

- Details of the test procedure used: For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks and then harvested, afterwards the cell suspension were seeded into a 24 well flat-bottom plate at a final density of 1 x 10E6 cells/well.
The solvent controls, the positive control and the 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 exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x 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 x g), cells were stained with 50 µL of FITC-labelled anti-CD86, anti-CD54, or mouse IgG1 (isotype) antibodies in the dark for 30 min. All antibodies were diluted in FACS buffer at an appropriate manner. After washing with FACS buffer two times, cells were re-suspended in FACS buffer and PI solution (propidium iodide) was added. PI staining was done just prior to the measurement by adding PI solutions to each sample (final concentration of PI was 0.625µg/mL). Finally the expression levels of CD86 and CD54 as well as cell viability 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.

- Cell line used: THP-1 cells (ATCC® TIB-202TM).

- Doses of test chemical and control substances used:
Test Item:
Dose finding assay 1 and 2: 1000, 500.0, 250.0, 125.0, 62.50, 31.25, 15.63 and 7.81 µg/mL
Main experiment 1 and 2: 21.09, 17.58, 14.65, 12.21, 10.17, 8.48, 7.06, 5.89 µg/mL
Medium Control: cell culture medium
Solvent Control: cell culture medium
Positive Control: 4 µg/mL DNCB (2, 4-dinitrochlorobenzene)

- Duration and temperature of exposure: 24 h ± 0.5 h at 37 °C± 1 °C and 5% CO2.

- Cell reactivity check and Dose finding assay: Prior to testing, the quality of freshly thawed cell batch was checked by monitoring the doubling time and checking the reactivity towards positive controls. DNCB (4 µg/mL) and nickel sulphate (100 µg/mL) served as positive control while lactic acid (1000 µg/mL) as negative control. Cells were accepted when both positive controls produce a positive response for CD86 and CD54, and lactic acid produces a negative response for CD86 and CD54.
In dose finding assay, 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 (with the final density of 1 x 106 cells/well). The 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. The CV75 value (75% cell survival) was calculated and used to calculate the concentration range of the test item for the main experiment.

- Number of tissue replicates used per test chemical and controls: Two repeated Experiments respectively with one sample per treatment: test substance, positive and negative controls.

- Acceptance criteria:
The test meets acceptance criteria if:
• the cell viability of the solvent controls is >90%,
• the cell viability of at least four tested doses of the test item in each run is >50%,
• the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
• the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
• the MFI ratio of CD86 and CD54 to isotype IgG1 control for both, the medium and DMSO control, is >105%.

- Evaluation criteria:
Test chemical tested by the h-CLAT was considered positive 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
- or 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
- 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

A negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is <90%. But a negative result for test items with a log Kow >3.5 should be considered as inconclusive.

Results and discussion

Positive control results:

The positive control (DNCB) led to an upregulation of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (326% experiment 1; 309% experiment 2) and 200% for CD54 (572% experiment 1; 309% experiment 2) were clearly exceeded.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: CD86/Experiment 1

Parameter:

other: RFI (relative fluorescence intensity)

Value:

122

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

The expression of the cell surface marker CD86 was upregulated above the threshold of 150 starting from a concentration of 21.09 µg/mL to 12.20 µg/mL

Results 2

Key result

Run / experiment:

other: CD54/Experiment 1

Parameter:

other: RFI (relative fluorescence intensity)

Value:

83

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

The expression of CD54 was not upregulated above the threshold of 200%

Results 3

Key result

Run / experiment:

other: CD86/Experiment 2

Parameter:

other: RFI (relative fluorescence intensity)

Value:

116

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

The expression of CD86 was upregulated above the threshold of 150 starting from a concentration of 21.09 µg/mL to 17.58 µg/mL

Results 4

Key result

Run / experiment:

other: CD54/Experiment 2

Parameter:

other: RFI (relative fluorescence intensity)

Value:

101

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

The expression of CD54 was not upregulated above the threshold of 200%

Other effects / acceptance of results:

Slight cytotoxic effects were observed for the cells treated with the test item in the highest concentrations. Relative cell viability at the highest test item concentration was reduced to 76.2% (CD86), 75.4% (CD54) and 74.9% (isotype IgG1 control) in the first experiment and to 78.5% (CD86), 78.0% (CD54) and 77.8% (isotype IgG1 control) in the second experiment.
The expression of the cell surface marker CD86 was upregulated above the threshold of 150% starting from a concentration of 21.09 µg/mL to 12.20 µg/mL in the first experiment and of 21.09 µg/mL to 17.58 µg/mL in the second experiment. In contrast, the expression of the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Since one of the cell surface markers (CD86) clearly exceeded the threshold in two independent experiments the test item is considered to be a skin sensitiser.
All the controls confirmed the validity of the study. The positive control (DNCB) led to an upregulation of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (332% experiment 1; 272% experiment 2) and 200% for CD54 (304% experiment 1; 544% experiment 2) were clearly exceeded.

Any other information on results incl. tables

Table 1: CD54 and CD86 Expression Experiment 1

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	97.1	96.3	96.0	2863	1435	657	2206	778	100	100	436	218
Solvent Control	0.20%	96.6	94.9	95.7	3156	1228	668	2488	560	113	72	472	184
DNCB	4.00	81.3	80 .8	80.6	8942	2395	693	8249	1702	332	304	1290	346
NEO-HITENOL LM-20 (Alcohols, C10-16, ethoxylated, sulfosuccinates, disodium salts)	21.09	76.2	75.4	74.9	5612	1631	750	4862	881	220	113	748	217
	17.58	87.7	88.4	87.7	4980	1521	714	4266	807	193	104	697	213
	14.65	91.1	91.2	91.6	4390	1492	726	3664	766	166	98	605	206
	12.20	93.9	93.4	93.2	4160	1494	737	3423	757	155	97	564	203
	10.17	94.3	94.2	94.1	3990	1591	786	3204	805	145	103	508	202
	8.48	94.7	95.0	94.8	3698	1515	871	2827	644	128	83	425	174
	7.06	95.0	94.9	94.7	3692	1453	774	2918	679	132	87	477	188
	5.89	95.4	95.6	95.0	3398	1507	713	2685	794	122	102	477	211

Table 2: CD54 and CD86 Expression Experiment 2

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	IgG Isotype	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	C86	CD54
Medium Control	-	96.9	96.8	96.5	2594	1130	618	1976	512	100	100	420	183
Solvent Control	0.20%	96.6	95.8	96.6	2483	1056	619	1864	437	94	85	401	171
DNCB	4.0	84.1	84.5	83.4	5810	3109	732	5078	2377	272	544	794	425
NEO-HITENOL LM-20 (Alcohols, C10-16, ethoxylated, sulfosuccinates, disodium salts)	21.09	78.5	78.0	77.8	4943	1222	615	4328	607	219	119	804	199
	17.58	92.7	92.9	92.5	3601	1166	578	3023	588	153	115	623	202
	14.65	94.1	94.4	94.4	3209	1191	600	2609	591	132	115	535	199
	12.20	95.4	95.3	95.4	3301	1168	636	2665	532	135	104	519	184
	10.17	95.6	95.4	95.9	3395	1162	584	2811	578	142	113	581	199
	8.48	96.1	96.2	96.1	3309	1146	611	2698	535	137	104	542	188
	7.06	95.6	96.1	95.7	2893	1123	604	2289	519	116	101	479	186
	5.89	94.7	95.5	95.6	3007	1103	584	2423	519	123	101	515	189

Table3: Acceptance Criteria

Acceptance Criterion	Range	Experiment 1			pass/fail	Experiment 2			pass/fail
cell viability solvent controls [%]	>90	94.9	-	97 .1	pass	95.8	-	96.9	pass
number of test dosed with viability >50% CD86	≥4	8			pass	8			pass
number of test dosed with viability >50% CD54	≥4	8			Pass	8			pass
number of test dosed with viability >50% IgG1	≥4	8			pass	8			pass
RFI of positive control of CD86	≥150	332			pass	272			pass
RFI of positive control of CD54	≥200	304			pass	544			pass
RFI of solvent control of CD86	<150	113			pass	94			pass
RFI of solvent control of CD54	<200	72			pass	85			pass
MFI ratio IgG1/CD86 for medium control [%]	>105	436			pass	420			pass
MFI ratio IgG1/CD86 for DMSO control [%]	>105	472			pass	401			pass
MFI ratio IgG1/CD54 for medium control [%]	>105	218			pass	183			pass
MFI ratio IgG1/CD54 for DMSO control [%]	>105	184			pass	171			pass

Applicant's summary and conclusion

Interpretation of results:

other: skin sensitising based on the key event “activation of dendritic cells”

Conclusions:

In this study under the given conditions the test item did upregulate the cell surface marker CD86 in at least two independent experiment runs. Therefore, based on the observed results it was concluded that the test substance induces dendritic cell activation. The result alone does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required. Based on the results of all three key events the test substance has to be classified as skin sensitiser.