Reaction product of 2,2'-oxydiethanol and 2-hydroxyethyl acrylate and 2-hydroxyethyl methacrylate and hexan-6-olide and trimethylhexa-1,6-diyl diisocyanate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

30 Jan - 09 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (in vitro Skin Sensitisation: human Cell Line Activation Test)

Version / remarks:

adopted 25 Jun 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

OGYEI National Institute of Pharmacy and Nutrition, Budapest, Hungary

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

7,7,9(or 7,9,9)-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diyl bismethacrylate

EC Number:

276-957-5

EC Name:

7,7,9(or 7,9,9)-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diyl bismethacrylate

Cas Number:

72869-86-4

Molecular formula:

C23H38N2O8

IUPAC Name:

Reaction mass of 7,7,9-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diylbismethacrylate and 7,9,9-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diylbismethacrylate

In vitro test system

Details on the study design:

TEST METHOD:
The in vitro human Cell Line Activation Test (h-CLAT) is an alternative testing method for the evaluation of the skin sensitisation potential of a test compound. It quantifies phenotypic changes, such as cell surface marker expression in cell lines following 24 h treatment with chemicals. The human leukemia cell line THP-1 is used as surrogate for human myeloic dendritic cells, which show enhanced CD86 and CD54 surface protein expression when treated with sensitisers.

TESTS SUBSTANCE PREPARATION:
The test item was dissolved in dimethyl sulfoxide (DMSO).

CONCENTRATIONS:
Pre-experimental dose-finding study:
first run: 8, 16, 31, 63, 125, 251, 501 and 1003 µg/mL
second run: 18, 21, 25, 30, 37, 44, 53 and 63 µg/mL
Main experiment (h-CLAT): based on the results obtained in the pre-experimental dose-finding study: 15, 18, 22, 26, 31, 38, 45 and and 54 µg/mL.

VEHICLE CONTROL: 0.2% DMSO in Roswell Park Memorial Institute (RPMI) medium
POSITIVE CONTROL CV75: 1-chloro-2,4-dinetrobenzene (DNCB) prepared as 4.2 µg/mL in DMSO
POSITIVE CONTROL CD54 and CD86 expression: Nickel Sulphate prepared as 100 µg/mL in RPMI medium

TEST CELL LINE: THP-1 cells
- Source: ATCC (LGC Standards GmbH, Germany Office), #TIB-202
- Passage number 5 to 8

CELL CULTURE CONDITIONS:
- Type and identity of media: Complete RPMI-1640 culture medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, 2.05 mM L-glutamine and 0.05 mM 2-mercaptoethanol
- Temperature (°C): 37 ± 1.5
- CO2 (%): 5 ± 0.5
- Seeding h-CLAT test: 0.9-1 x 10E6 cells per well in 24-well format, total volume 550 µL

EXPOSURE CONDITIONS:
- Method of application: in medium
- Exposure duration: 24 ± 0.5 h

NUMBER OF REPLICATES: Each concentration was tested in three independent runs

DETERMINATION OF CYTOTOXICITY:
- Method: Propidium iodide uptake, 24 ± 0.5 h exposure with test item
- Detection: Flow cytometry, Apogee Flow Cytometer
- Determination of cell viability: calculation of the CV75, which corresponds to the concentration needed to reduce the relative absorbance to 75% of the solvent control.

DETERMINATION OF FLUORESCENCE:
- Flow cytometry, Apogee Flow Cytometer
- Antibodies: fluorescein isothiocyanate labelled CD86 and CD54

Results and discussion

Positive control results:

Relative fluorescence intensities first experiment:
4.2 µg/mL: CD54 = 514%, CD86 =520%

Relative fluorescence intensities, second experiment:
4.2 µg/mL: CD54 = 489%, CD86 = 378%

Relative fluorescence intensities, third experiment:
4.2 µg/mL: CD54 = 781%, CD86 = 570%

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: 24 h incubation

Parameter:

other: RFI in % for CD54 in µg/mL

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: in 2/3 independent runs

Results 2

Key result

Run / experiment:

other: 24 h incubation

Parameter:

other: RFI in % for CD54 in µg/mL

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: in 3/3 independent runs

Remarks:

cell viability was < 50% in the third run

Other effects / acceptance of results:

OTHER EFFECTS:
Cell viability at the highest dose in the third run was < 50%, therefore the corresponding RFI value was not considered valid and was excluded from the prediction. All other cell viabilities complied with the acceptance criteria.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, solvent control RFI values do not exceed the positive criteria CD86 ≥ 150% and CD54 ≥ 200% and cell viability is > 50%
- Acceptance criteria met for positive control: yes, RFI values CD86 ≥ 150% and CD54 ≥ 200% and cell viability is > 50%

Any other information on results incl. tables

Table 2: Results of the hCLAT test, first experiment

Compound	Concentration [µg/mL]	RFI CD86	RFI CD54	Cell viability [%]
control		100	100	91.6
DMSO	0.20%	59	96	93.5
DNCB	4.2	520	514	74.2
Test item	54	104	39	22.4
	45	149	112	44.4
	38	88	164	74.8
	31	121	117	82.1
	26	132	127	87.1
	22	88	130	88.5
	18	148	201	89.8
	15	161	102	92.3

Table 3: Results of the hCLAT test, second experiment

Compound	Concentration [µg/mL]	RFI CD86	RFI CD54	Cell viability [%]
control		100	100	92.1
DMSO	0.20%	121	88	92.9
DNCB	4.2	379	489	76.2
Test item	54	59	90	20.3
	45	94	100	27.8
	38	97	163	70.5
	31	89	268	81.8
	26	105	228	84.0
	22	63	174	88.7
	18	95	161	85.8
	15	52	125	89.9

Table 4: Results of the hCLAT test, third experiment

Compound	Concentration [µg/mL]	RFI CD86	RFI CD54	Cell viability [%]
control		100	100	91.4
DMSO	0.20%	97	119	93.4
DNCB	4.2	570	781	58.9
Test item	54	114	154	10.0
	45	199	347	39.3
	38	227	334	66.0
	31	200	387	85.2
	26	208	260	84.4
	22	183	400	83.0
	18	214	207	86.2
	15	142	101	90.6

Applicant's summary and conclusion

Interpretation of results:

other: positive for activation of dendritic cells

Conclusions:

The data generated with this method may not be sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of an integrated approach such as integrated approaches to testing and assessment (IATA).