Fatty acids, coco, esters with 3,3'-oxybis[1,2-propanediol]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-08-01 to 2016-11-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Draft Proposal for a new test guideline, “In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)”

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

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, coco, esters with 3,3'-oxybis[1,2-propanediol]

EC Number:

285-089-6

EC Name:

Fatty acids, coco, esters with 3,3'-oxybis[1,2-propanediol]

Cas Number:

85029-63-6

Molecular formula:

not applicable, mixture

IUPAC Name:

Fatty acids, coco, esters with 3,3'-oxybis[1,2-propanediol]

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 (345% experiment 1; 347% experiment 2) and 200%
for CD54 (496% experiment 1; 491% 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:

153

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

208

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD86 [%]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The 150% threshold was not exceeded at any concentration of the test item.

Results 4

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

204

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

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 mets the acceptance criteria.

Any other information on results incl. tables

Table1:  Results of the Cell Batch Activation Test

Sample	Concentration [µg/mL]	CD86		CD54		Activated
		Cell Viability [%]	RFI	Cell Viability [%]	RFI	yes/no
DNCB	4 µg/mL	85.8	393	85.6	408	Yes
NiSO4	100 µg/mL	90.4	272	90.4	332	Yes
LA	1000 µg/mL	98.1	56	98.5	56	No

The positive controls DNCB and NiSO₄led to upregulation of the cell surface markers CD54 and CD86. The negative control LA did not induce an upregulation of CD54 and CD86.

The cell batch was accepted for further testing.

Table2:  Results of the Dose Finding Assay

Sample	Experiment 1		Experiment 2		Experiment 3
	Concentration applied [µg/mL]	Cell Viability [%]	Concentration applied [µg/mL]	Cell Viability [%]	Concentration applied [µg/mL]	Cell Viability [%]
Medium Control	0	96.50	0.00	97.40	0.00	96.00
DMSO Control	0.00	96.10	0.00	97.00	0.00	98.90
Hostastat FE 20 LIQ	7.81	96.10	7.81	97.90	279.08	98.50
	15.63	96.50	15.63	97.60	334.90	98.30
	31.25	96.30	31.25	97.30	401.88	98.20
	62.50	95.70	62.50	97.30	482.25	98.40
	125.00	96.00	125.00	97.30	578.70	98.30
	250.00	96.00	250.00	96.90	694.44	98.10
	500.00	95.00	500.00	95.40	833.33	97.30
	1000.00	59.70	1000.00	81.80	1000.00	95.80
Calculated CV75 [µg/mL]	740.50		No CV75		No CV75
Mean CV75 [µg/mL]	740.50
SD CV 75 [µg/mL]	No SD

 

Table3:  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	-	97.2	97.4	97.4	1341	863	751	590	112	83	54	179	115
DMSO Control	0.20%	97.3	97.4	97.4	1457	954	745	712	209	100	100	196	128
DNCB	4.00	83.7	84.4	85.3	3237	1819	783	2454	1036	345	496	413	232
Hostastat FE 20 LIQ	1000.00	93.7	93.6	93.5	1747	1470	909	838	561	118	268	192	162
	833.33	94.5	93.9	94.7	1862	1395	872	990	523	139	250	214	160
	694.44	95.8	95.5	95.0	1901	1357	858	1043	499	146	239	222	158
	578.70	95.4	95.7	96.1	1965	1310	875	1090	435	153	208	225	150
	482.25	96.7	97.0	96.9	1534	1214	807	727	407	102	195	190	150
	401.88	96.2	96.5	97.0	1691	1153	816	875	337	123	161	207	141
	334.90	97.1	97.4	97.6	1640	1118	799	841	319	118	153	205	140
	279.08	95.1	95.4	95.3	2070	1475	1118	952	357	134	171	185	132

 

Table4: 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	-	98.3	98.2	97.7	1351	885	713	638	172	85	106	189	124
DMSO Control	0.20%	98.0	97.9	98.2	1437	848	685	752	163	100	100	210	124
DNCB	4.0	88.3	89.0	87.9	3363	1554	753	2610	801	347	491	447	206
Hostastat FE 20 LIQ	1000.00	93.0	93.0	93.6	1863	1518	915	948	603	126	370	204	166
	833.33	95.8	96.0	95.9	1837	1255	835	1002	420	133	258	220	150
	694.44	97.0	97.4	96.9	1646	1155	850	796	305	106	187	194	136
	578.70	97.2	97.7	97.4	1821	1124	819	1002	305	133	187	222	137
	482.25	98.0	97.9	97.4	1663	1152	819	844	333	112	204	203	141
	401.88	98.0	98.0	97.6	1596	1049	809	787	240	105	147	197	130
	334.90	98.1	97.8	97.8	1518	1056	753	765	303	102	186	202	140
	279.08	98.0	97.6	97.9	1642	1022	777	865	245	115	150	211	132

 

Table5:  Acceptance Criteria

Acceptance Criterion	Range	Experiment 1			pass/fail	Experiment 2			pass/fail
cell viability solvent controls [%]	>90	97.2	-	97.4	pass	97.7	-	98.3	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	345			pass	347			pass
RFI of positive control of CD54	≥200	496			pass	491			pass
RFI of solvent control of CD86	<150	121			pass	118			pass
RFI of solvent control of CD54	<200	187			pass	95			pass
MFI ratio IgG1/CD86 for medium control [%]	>105	179			pass	189			pass
MFI ratio IgG1/CD86 for DMSO control [%]	>105	196			pass	210			pass
MFI ratio IgG1/CD54 for medium control [%]	>105	115			pass	124			pass
MFI ratio IgG1/CD54 for DMSO control [%]	>105	128			pass	124			pass

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In this study under the given conditions the test item did upregulate the cell surface marker in at least two independent experiment runs. Therefore, the test item is considered to be a skin sensitiser in accordance with UN GHS category 1.

Executive summary:

In the present study the test item was dissolved in DMSO. A CV75 of 740.5 µg/mL was derived in the dose finding assay. Based on the CV75, the main experiment was performed covering the following concentration steps:

1000.00, 833.33, 694.44, 578.70, 482.25, 401.88, 334.90 and 279.08 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 93.7% (CD86), 93.6% (CD54) and 93.5% (isotype IgG1 control)in the first experiment and to 93.0% (CD86), 93.0% (CD54) and 93.6% (isotype IgG1 control)in the second experiment.

The upregulation above the threshold of 150% of the cell surface marker CD86 was observed in the first experiment only at a concentration of 578.70 µg/mL (153%). In the second experiment no upregulation above the threshold of 150% of the cell surface marker CD86 was observed. In the first experiment the expression of the cell surface marker CD54 was upregulated starting with 208% at a concentration of 578.70 µg/mL up to 268 % at the highest tested concentration (1000.00 µg/mL). In the second experiment the expression of the cell surface marker CD54 was upregulated to 204% at a concentration of 482.25 µg/mL, and 258% at a concentration of 833.33 µg/mL, up to 370% at the highest tested concentration (1000.00 µg/mL). The upregulation of the cell surface markers CD86 and CD54 were observed for both independent experiments at test item concentrations exhibiting a cell viability >50%. Since the expression of the cell surface marker CD86 exceeded the threshold in one experiment, and the cell surface marker CD54 clearly exceeded the threshold in both independent experiment the test item is considered to be a sensitiser.

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (345% experiment 1; 347% experiment 2) and 200% for CD54 (496% experiment 1; 491% experiment 2) were clearly exceeded.