Tetramethylcyclotetrasiloxane substituted dipropylether bisphenol A

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2020 to March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (Tetramethylcyclotetrasiloxane propylether) prop-1-enylether bisphenol A and Di(tetramethylcyclotetrasiloxane propylether) bisphenol A

Cas Number:

203874-34-4

Molecular formula:

(OSiHxCH3)4-CH2-CH2 -CH2- O-C6H4-C(CH3)2-C6H4-0-CH2-CH2-CH2-(SiHxCH3O)4

IUPAC Name:

Reaction mass of (Tetramethylcyclotetrasiloxane propylether) prop-1-enylether bisphenol A and Di(tetramethylcyclotetrasiloxane propylether) bisphenol A

Test material form:

liquid

Details on test material:

Appearance: Pale yellow liquid

In vitro test system

Details of test system:

THP-1 cell line [442E]

Details on the study design:

* Dose Finding Assay:
The test article working solutions or solvent controls were mixed 1:1 (v/v) with the cell suspensions in the 96-well plates. The plates were sealed and then incubated for 24±0.5 hours (incubator set to 37ºC, 5% CO2).
After the 24-hour incubation period, all cells from the 96-well flat-bottomed plate were transferred into a 96-well round-bottomed plate. The cells were washed at least twice in 200 µL of phosphate buffered saline containing 0.1% bovine serum albumin (FACS buffer) and re-suspended in 190 µL of FACS buffer. 10 µL of propidiumiodide solution (PI) was added just before FACS analysis (final concentration of PI = 0.625 µg/mL). PI uptake was analysed using flow cytometry with the acquisition channel FL-3. A total of 10,000 viable cells were acquired.

* CD86/CD54 Expression Measurement:
Two independent runs (experiments) were needed to drive a prediction. Each independent run was performed on the same day provided that for each run:
a) Independent fresh stock solutions and working solutions of the test article and antibody solutions were prepared and
b) Independently harvested cells were used (i.e. cells were collected from different culture flasks).

On the days of testing, cells harvested from the flasks were resuspended with fresh culture medium at 2 x 10E6 cells/mL. The cells were then distributed into a 24-well plate (500 µL/1 x 10E6 cells per well).

The test article working solution or solvent control was mixed 1:1 (v/v) with the cell suspensions in the 24-well plates. The plates were sealed and then incubated for 24±0.5 hours (incubator set to 37ºC, 5% CO2).

After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation (approximately 250 g, 5 minutes) and washed twice with 1 mL of FACS buffer. After washing, the cells were blocked with 600 µL of blocking solution (FACS buffer containing 0.01% (w/v) globulin) on ice for 15 minutes.

After blocking, the cells were split into three aliquots of 180 µL into a 96-well plate and centrifuged (approximately 250 g, 3 minutes).

After centrifugation, the cells were stained with 50 µL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies on ice for 30 minutes.

The stained cells were washed three times with an excess of FACS buffer, re-suspended in FACS buffer and 12.5 µg/mL PI solution was added (to give a final PI concentration of 0.625 µg/mL).

The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

Vehicle / solvent control:

DMSO

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Results and discussion

Positive control results:

For the positive control, RFI values were =150% for CD86 and =200% for CD54, and cell viability was >50% in each independent run.

In vitro / in chemico

Results

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

EC150, CD86 [442E]

Value:

314.14 µg/mL

Cell viability:

>50%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

In both experiments, the RFI values for CD86 were >150% (with cell viability >50%) at multiple concentrations and the RFI values for CD54 were <200% at all concentrations.
The EC150 value for CD86 calculated by linear regression of endpoint assay data was
314.14 µg/mL. No EC200 value was calculated for CD54 as this marker was negative in both experiments.

All assay acceptance criteria were met. The cell viabilities of medium and solvent/vehicle control were higher than 90% in each independent run. In the solvent control, RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI =150% and CD54 RFI =200%).

For both medium and solvent/vehicle controls, the MFI ratio of both CD86 and CD54 to isotype control was >105% on all occasions. For the positive control, RFI values were 150% for CD86 and 200% for CD54, and cell viability was >50% in each independent run.

For the test article, the cell viability was more than 50% in all tested concentrations in each independent run.

Any other information on results incl. tables

Dose Finding Assay: Cell Viability

Viability (%) at Concentration (μg/mL)
Run	7.81	15.63	31.25	62.5	125	250	500	1000
1	99.1	98.6	99	98.8	98.7	98.1	98.3	98.1
2	98.8	98.6	98.8	98.9	99	98.2	98.3	97.9

Expression Assay: MFI and Cell Viability Values

Experiment 1

Concentration (µg/mL)	MFI (Geo Mean)			Corrected MFI		Viability
	CD86	CD54	Isotype	CD86	CD54	IgG	CD86	CD54
279.08	1425	575	481	944	94	97.5	96.6	97.0
334.90	1567	561	480	1087	81	96.4	96.0	96.2
401.88	1420	544	469	951	75	96.3	95.7	96.0
482.25	1224	557	463	761	94	97.7	96.5	95.6
578.70	1244	520	423	821	97	99.1	96.8	96.8
694.44	1453	573	485	968	88	96.7	95.1	95.4
833.33	1346	589	493	853	96	96.4	95.3	95.4
1000.00	1197	587	497	700	90	96.9	95.8	96.8
Culture medium	922	555	467	455	88	98.9	98.8	98.5
DMSO 0.2%	940	498	418	522	80	99.6	99.1	99.3
DNCB 4 μg/mL	2475	1687	580	1895	1107	84.9	80.9	82.3

Experiment 2

Concentration (µg/mL)	MFI (Geo Mean)			Corrected MFI		Viability
	CD86	CD54	Isotype	CD86	CD54	IgG	CD86	CD54
279.08	1139	591	502	637	89	95.9	96.2	94.9
334.90	1350	568	471	879	97	95.6	95.1	94.8
401.88	1380	587	505	875	82	93.1	95.1	93.5
482.25	1254	568	489	765	79	94.1	94.5	93.8
578.70	1350	583	511	839	72	94.5	93.6	93.8
694.44	1354	603	529	825	74	92.6	93.3	92.5
833.33	1331	605	525	806	80	95.4	92.5	94.2
1000.00	1207	604	510	697	94	97.2	95.5	94.7
Culture medium	913	539	457	456	82	98.3	98.4	98.1
DMSO 0.2%	981	555	455	526	100	98.7	98.5	98.3
DNCB 4 μg/mL	2173	1115	569	1604	546	84.9	85.1	85.5

The relative fluorescence intensity (RFI) values:

Concentration (μg/mL)	RFI (CD86)		RFI (CD54)
	Exp1	Exp2	Exp1	Exp2
279.08	181	121	118	89
334.90	208	167	101	97
401.88	182	166	94	82
482.25	146	145	118	79
578.70	157	160	121	72
694.44	185	157	110	74
833.33	163	153	120	80
1000.00	134	133	113	94

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

The data can only be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

Conclusions:

The results of an in vitro skin sensitization test, performed according to OECD/EC guidelines and under GLP principles indicate that the test item has skin sensitizing properties.

Executive summary:

An in vitro skin sensitisation study (human Cell Line Activation Test) was conducted to investigate the potential of the test item to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1 by quantifying changes in the expression of cell surface markers (CD86 and CD54). For the dose finding assay, the test item was formulated in dimethyl sulphoxide (DMSO) at a concentration of 500 mg/mL giving a maximum test concentration of 1000 μg/mL. No reduction in viability was observed.

For the expression measurements, test concentrations in a range from 279.08 to 1000 μg/mL (after dilution in medium) were used. Aliquots of 500 μL of each of the working solutions were mixed 1:1 with cell suspensions at 1 x 10⁶ cells per well. After blocking, the cells were stained with FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies on ice for 30 minutes. The stained cells were washed, re-suspended in FACS buffer and propidium iodide solution was added. The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

In the two experiments performed, the RFI values for CD86 were >150% (with cell viability >50%) at multiple concentrations and the RFI values for CD54 were <200% at all concentrations. The EC150 value for CD86 was calculated to be 314.14 μg/mL. All acceptance criteria of the h-CLAT assay parameters were met in each experiment.

As a result, the substance was considered to be positive in the human Cell Line Activation Test.