Ethanol, 2,2'-oxybis-, reaction products with 3-(triethoxysilyl)-1-propanamine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-04-09 to 2018-05-08

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: human Cell Line Activation Test (h-CLAT)” adopted 29 July 2016

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st, 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

In vitro testing strategy

Test material

Test material information

Constituent 1

Reference substance name:

Ethanol, 2,​2'-​[1,​2-​ethanediylbis(oxy)​]​bis-​, reaction products with 3-​(triethoxysilyl)​-​1-​propanamine

EC Number:

813-152-5

Cas Number:

152261-44-4

Molecular formula:

Unspecified

IUPAC Name:

Ethanol, 2,​2'-​[1,​2-​ethanediylbis(oxy)​]​bis-​, reaction products with 3-​(triethoxysilyl)​-​1-​propanamine

Test material form:

liquid

In vitro test system

Details on the study design:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

Results and discussion

Positive control results:

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (252% experiment 1; 303% experiment 2) and 200% for CD54 (201% experiment 1; 226% experiment 2) were clearly exceeded.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

116

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 11979.17 µg/mL

Results 2

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

122

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration:14375.00 µg/mL

Results 3

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

119

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 17250.00 µg/mL

Results 4

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

113

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 17250.00µg/mL

Other effects / acceptance of results:

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 the medium and DMSO control, is >105%.

The test met the acceptance criteria.

Any other information on results incl. tables

In all experiments no precipitation or turbidity of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 79.4% (CD86), 79.4% (CD54) and 79.5% (isotype IgG1 control) in the first experiment and to 87.5% (CD86), 86.1% (CD54) and 86.8% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Therefore, the test item is considered to be no skin sensitiser.

The controls confirmed the validity of the study for all experiments.

Results of the Cell Batch Activation Test (Batch 19)

Sample	Concentration [µg/mL]	CD86			CD54			Activated	Pass /Fail
		Cell Viability [%]	RFI	Threshold OECD TG 442E	Cell Viability [%]	RFI	Threshold OECD TG 442E	yes/no
DNCB	4 µg/mL	81.1	347	>150	79.7	269	>200	yes	pass
NiSO4	100 µg/mL	82.1	347	>150	82.5	391	>200	yes	pass
LA	1000 µg/mL	96.7	89	≤150	96.4	109	≤200	no	pass

 

Results of the Cell Batch Activation Test (Batch 20)

Sample	Concentration [µg/mL]	CD86			CD54			Activated	Pass /Fail
		Cell Viability [%]	RFI	Threshold OECD TG 442E	Cell Viability [%]	RFI	Threshold OECD TG 442E	yes/no
DNCB	4 µg/mL	89.2	266	>150	88.4	206	>200	yes	pass
NiSO4	100 µg/mL	82.3	220	>150	80.6	283	>200	yes	pass
LA	1000 µg/mL	96.2	79	≤150	96.9	101	≤200	no	pass

Results of the Dose Finding Assay

Sample		Experiment 1		Experiment 2
		Concentration applied [µg/mL]	Cell Viability [%]	Concentration applied [µg/mL]	Cell Viability [%]
Medium Control	--	--	93.10	--	95.70
Solvent Control	NaCl	--	92.60	--	94.70
Ethanol, 2,2’[1,2-ethanediylbis(oxy)] bis-, reaction products with 3-(triethoxysilyl)-1-propanamine	C8	26.95	95.10	134.77	94.90
	C7	53.91	95.40	269.53	95.40
	C6	107.81	95.40	539.06	94.80
	C5	215.63	95.30	1078.13	95.00
	C4	431.25	95.00	2156.25	95.40
	C3	862.50	95.00	4312.50	94.60
	C2	1725.00	95.00	8625.00	92.20
	C1	3450.00	93.30	17250.00	84.90
Calculated CV75 [µg/mL]		No CV75		No CV75
Mean CV75 [µg/mL]		No CV75
SD CV 75 [µg/mL]		No SD

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	-	93.6	93.2	94.0	5539	1543	723	4816	820	100	100	766	213
Solvent Control	0.20%	92.7	92.2	92.8	5225	1464	712	4513	752	94	92	734	206
DNCB	4.00	83.8	84.4	84.5	12159	2295	786	11373	1509	252	201	1547	292
Ethanol, 2,2’[1,2-ethanediylbis(oxy)] bis-, reaction products with 3-(triethoxysilyl)-1-propanamine	17250	79.4	79.4	79.5	6232	1753	820	5412	933	112	114	760	214
	14375.00	82.0	81.4	81.7	6213	1893	896	5317	997	110	122	693	211
	11979.17	84.6	84.6	84.1	6359	1619	787	5572	832	116	101	808	206
	9982.64	87.5	88.3	87.9	5801	1697	779	5022	918	104	112	745	218
	8318.87	89.6	90.3	89.3	5688	1620	767	4921	853	102	104	742	211
	6932.39	91.5	91.7	92.0	5302	1523	781	4521	742	94	90	679	195
	5776.99	91.5	91.1	90.9	5600	1518	788	4812	730	100	89	711	193
	4814.16	91.3	92.2	91.7	5326	1491	779	4547	712	94	87	684	191

 

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	CD86	CD54
Medium Control	-	94.6	94.3	94.1	5423	1785	731	4692	1054	100	100	742	244
Solvent Control	0.20%	94.7	95.1	94.5	5772	1783	704	5068	1079	108	102	820	253
DNCB	4.0	81.4	82.1	82.3	16087	3161	726	15361	2435	303	226	2216	435
Ethanol, 2,2’[1,2-ethanediylbis(oxy)] bis-, reaction products with 3-(triethoxysilyl)-1-propanamine	17250.00	87.5	86.1	86.8	6383	1991	801	5582	1190	119	113	797	249
	14375.00	87.2	88.3	88.4	5982	1863	999	4983	864	106	82	599	186
	11979.17	89.6	89.6	89.8	5988	1944	970	5018	974	107	92	617	200
	9982.64	91.0	90.9	90.9	6024	1830	813	5211	1017	111	96	741	225
	8318.87	92.5	92.2	92.4	5436	1761	819	4617	942	98	89	664	215
	6932.39	92.7	92.3	92.8	5946	1769	807	5139	962	110	91	737	219
	5776.99	93.9	93.4	93.9	5103	1761	799	4304	962	92	91	639	220
	4814.16	93.5	93.6	94.1	5217	1671	825	4392	846	94	80	632	203

Acceptance Criteria

Acceptance Criterion	Range	Experiment 1			pass/fail	Experiment 2			pass/fail
cell viability solvent controls [%]	>90	92.2	-	94.0	pass	94.1	-	95.1	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	252			pass	303			pass
RFI of positive control of CD54	≥200	201			pass	226			pass
RFI of solvent control of CD86	<150	94			pass	108			pass
RFI of solvent control of CD54	<200	92			pass	102			pass
MFI ratio IgG1/CD86 for medium control [%]	>105	766			pass	742			pass
MFI ratio IgG1/CD86 for DMSO control [%]	>105	734			pass	820			pass
MFI ratio IgG1/CD54 for medium control [%]	>105	213			pass	244			pass
MFI ratio IgG1/CD54 for DMSO control [%]	>105	206			pass	253			pass

 

Historical Data

Criterion	mean	SD	N
cell viability solvent controls [%]	97.0	1.3	672
number of test doses with viability >50%	-	-	1786
RFI of positive control of CD86	401.0	146.8	112
RFI of positive control of CD54	576.6	312.0	112
RFI of solvent control of CD86	115.0	15.1	112
RFI of solvent control of CD54	118.8	25.5	112
MFI ratio IgG1/CD86 for medium control [%]	202.4	50.0	112
MFI ratio IgG1/CD86 for DMSO control [%]	221.6	58.5	112
MFI ratio IgG1/CD54 for medium control [%]	141.0	24.7	112
MFI ratio IgG1/CD54 for DMSO control [%]	147.7	25.6	112

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item did not upregulate the expression of the cell surface markers in at least two independent experiment runs. Therefore the test item might be considered as non-sensitiser.

Executive summary:

In the present study the test item was dissolved in 0.9% NaCl. Due to a lack of cytotoxicity, no CV75 could be derived. Therefore the main experiment was performed covering the following concentration steps:

5000.00, 4166.67, 3472.22, 2893.52, 2411.27, 2009.39, 1674.49, 1395.41µg/mL

This corresponds to the following weights, since a correction factor of 3.45 was applied to correct for the active component of the test item: 17250, 14375, 11979.17, 9982.64, 8318.87, 6932.39, 5776.99, 4814.16 µg/mL.

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 79.4% (CD86), 79.4% (CD54) and 79.5% (isotype IgG1 control) in the first experiment and to 87.5% (CD86), 86.1% (CD54) and 86.8% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments.

Therefore, the test item is considered to be no skin sensitiser.

The positive control (DNCB) led to an upregulation of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (252% experiment 1; 303% experiment 2) and 200% for CD54 (201% experiment 1; 226% experiment 2) were clearly exceeded.