1,1-Bis(acryloyloxymethyl)ethyl isocyanate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation, other

Remarks:

The available in vitro data meets the data requirements for this endpoint, as the 2 available in vitro tests allow classification and risk assessment according to 8 .3 of Annex 7 of Regulation (EC) No. 1907/2006.

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

06 - 27 Oct 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

Jul 2016

Deviations:

yes

Remarks:

cytotoxicity measurement and estimation of the CV75 value of the dose finding assay was performed by XTT test, measuring the standard absorbance (OD) of a XTT cleavage product

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

2-isocyanato-2-methyl-3-(prop-2-enoyloxy)propyl prop-2-enoate

Cas Number:

886577-76-0

Molecular formula:

C11H13NO5

IUPAC Name:

2-isocyanato-2-methyl-3-(prop-2-enoyloxy)propyl prop-2-enoate

Test material form:

liquid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: At room temperature

In vitro test system

Details on the study design:

TEST CELL LINE- Source: ATCC, #TIB-202; Stocks of the THP-1 cell line were stored in liquid nitrogen in the cell bank of Envigo CRS GmbH, Germany- Passage number: 11 - 14 (XTT test); 17 - 18 (h-CLAT)CELL CULTURE CONDITIONS- Type and identity of media: RPMI-1640 supplemented with 10% (v/v) FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate and 1% (v/v) L-glutamine- Temperature (°C): 37 ± 1.5- CO2 (%): 5.0 ± 0.5The following information relates to details on XTT:The XTT test is a method to investigate the cytotoxicity of a test substance. The test is based on the cleavage of the yellow tetrazolium salt, sodium 3'-(1-phenylaminocarbonyl)-(3,4 - tetrazolium)-bis-(4–methoxy-6-nitro)-benzenesulfonic acid hydrate (XTT), to form an orange water soluble formazan dye by dehydrogenase activity in active mitochondria. Two independent cytotoxicity experiments were performed with different cell cultures and on different days to obtain a reliable concentration showing 75% cell viability (CV75).CONTROLSNegative Control- Substance: culture mediumVehicle control- Substance: dimethyl sulfoxide (DMSO)- Concentration: 0.2%EXPOSURE CONDITIONS- Exposure duration: 24 ± 1 hTEST CONCENTRATIONS- Justification for top dose: The test substance was soluble in DMSO up to and including 125 μg/mL.0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5 and125 μg/mLNUMBER OF REPLICATIONS: 3 samples per dose groups were tested in two independent experimentsMEASUREMENT- Device: microplate reader (Versamax Molecular Devices)- Wavelength: 450 nm- Reference wavelength: 690 nmEVALUATION CRITERIAA decrease in number of living cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample. This decrease directly correlates to the amount of orange formazan formed, as monitored by the absorbance. The relative absorbance as compared with the solvent control is calculated using the formula:Relative absorbance [%] = 100 x [(mean absorbance test substance) - (absorbance blank)] / [(mean absorbance vehicle control) - (absorbance blank)]To calculate the concentration of test substance needed to reduce the relative absorbance to 75% of the vehicle control (CV75) the following formula is used:CV75 = Conc. > 75 - ([(Conc. > 75) - (Conc. < 75)] x [(% > 75) - 75] / [(% > 75) - (% < 75)])a) Conc. > 75: max. measured concentration with the % of vehicle control > 75%b) Conc. < 75: min. measured concentration with the % of vehicle control < 75%c) % > 75: relative absorbance at a) in %d) % < 75: relative absorbance at b) in %ACCEPTANCE CRITERIAThe XTT test is considered to be acceptable if it meets the following criteria:- mean absorbance of the negative control is ≥ 0.5- mean viability of the vehicle control is ≥ 70%The following information relates to details on h-CLAT:The h-CLAT method is an in vitro method which quantifies changes of cell surface marker expression (CD86 and CD54) on a human cell line, THP-1 cells, following 24 h exposure to the test substance. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorescein isothiocyanate (FITC)-labelled antibodies. The relative fluorescence intensity of surface markers compared with solvent control is calculated and used in the prediction model to discriminate between sensitisers and non-sensitisers.CONTROLSNegative Control- Substance: culture mediumVehicle control- Substance: DMSO- Concentration: 0.2%Positive control- Substance: 2,4-dinitrochlorobenzene in DMSO- Concentration: 2 and 3 μg/mLEXPOSURE CONDITIONS: The cells treated with the test substance and with the control, respectively, were pooled, equally distributed into three samples tubes and washed with FACS buffer. Thereafter, the cells were centrifuged, re-suspended and blocked with blocking solution on ice at 2 - 8 °C for approximately 15 min. After that, the cells were washed once again and stained with FITC-labelled anti CD-86, CD54 antibody or mouse IgG1 (isotype control).- Exposure duration: 24 ± 1 hTEST CONCENTRATIONS- Concentrations: 3.8, 4.5, 5.4, 6.5, 7.8, 9.3, 11.2 and 13.4 μg/mL- Justification for top dose: The highest concentration used was 1.2 × Mean CV75 of both XTT tests. The calculated mean CV75 value of both XTT tests was 11.2 μg/mL. NUMBER OF REPLICATIONS: 3 samples per dose groups were tested in two independent experimentsSTAINING- Antibodies: FITC anti-CD54; FITC anti-CD86; FITC mouse IgG1 (isotype) - Temperature: 2 - 8 °C- Duration: 30 ± 5 minMEASUREMENT- Device: FACSCalibur (Becton Dick)EVALUATION CRITERIAThe relative fluorescence intensity (RFI) is used as an indicator of CD86 and CD54 expression, and is calculated as follows for each concentration:RFI (%) = [(MFI of test substance treated cells) - (MFI of test substance isotype control cells) / (MFI of vehicle control cells) - (MFI of vehicle isotype control cells)] x 100MFI: geometric mean fluorescence intensity (GeoMean)The cell viability is calculated as follows for each concentration:Cell viability (%) = (Mean cytotox of vehicle control cells) / (Mean cytotox of test substance treated cells) x 100Where the Mean cytotox is the mean of GeoMean (7-AAD) isotype control, GeoMean (7-AAD) CD54 and GeoMean (7-AAD) CD86.The test substance is tested in 2 independent runs. If the RFI of CD86 is ≥ 150% or if the RFI of CD54 is ≥ 200% in both independent run data, the test substance is considered to be a sensitizer. Otherwise it is considered to be a non-sensitizer. In case of different results in both runs, a third run has to be performed. If the RFI of CD86 is ≥ 150% at any dose in at least 2 of 3 independent run data, or if the RFI of CD54 is ≥ 200% in at least 2 of 3 independent run data, the test substance is considered to be a sensitizer. Otherwise it is considered to be a non-sensitizer.ACCEPTANCE CRITERIAThe study is considered as valid, if the following criteria are met:- Cell viability of negative control is adjusted to 100% and the cell viability of the vehicle control should be more than 90% in comparison to the negative control.- In the positive control, RFI values of both CD86 and CD54 should exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50%.- In the vehicle control, RFI values compared with the negative control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).- For negative and vehicle controls, the MFI ratio of CD86 and CD54 to isotype control should be > 105%. - For the test substance resulting in negative outcome, the cell viability at the 1.2 × CV75 value should be less than 90%. If the cell viability at the 1.2 × CV75 value is more than 90% for a positive tested test substance, the data will be acceptable. If 5 mg/mL in saline, 1 mg/mL in DMSO or the highest soluble dose will be used as the maximal test concentration instead of CV75-based dose, the data for test substance are accepted independent by the cell viability.- The cell viability of at least 4 doses in each experiment should be ≥50%.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: 24 h incubation

Parameter:

other: relative fluorescence intensity of CD54 (%)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: 24 h incubation

Parameter:

other: relative fluorescence intensity of CD86 (%)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:XTT testCytotoxic effects were observed following incubation with the tested concentrations ≥ 15.63 µg/mL). The viability of the cells was reduced to 50.17 and 31.55%, respectively, in both XTT tests. The calculated mean CV75 value of both XTT tests was 11.2 µg/mL. ACCEPTANCE OF RESULTS:- Acceptance criteria met for vehicle control: yes, the RFI values of the solvent control compared with the medium control was 119.6% (first run) and 113.3% (second run) for CD54 and 87.6% (first run) and 116.9% (second run) for CD86, and thus fulfilled the acceptance criteria. The MFI ratio was determined in both runs and was 151.4% and 159.4%, respectively, for Medium CD54, 220.2% and 176.2%, respectively, for Medium CD86, 169.2% and 170.8%, respectively, for MDSO CD54 and 218.4% and 193.8%, respectively, for DMSO CD86.- Acceptance criteria met for positive control: The RFI values of the positive control (2 and 3 µg/mL, respectively) for CD86 (230.1 and 294.5%, respectively) and CD54 (230.1 and 391.4%, respectively) exceeded the positive criteria (CD86 >150% and CD54 >200%) and the cell viability was >50%. Please refer to Table 3 and 4 under "Any other information on results incl. tables).

Any other information on results incl. tables

Table 1: Results of the first XTT test

	Concentration [µg/mL]	Mean absorbance *	Blank	Absorbance in % of vehicle control **
Negative control	Cell culture medium	0.673± 0.037	0.185	102.83
Vehicle control	DMSO (0.2%)	0.669 ± 0.051	0.195	100.00
Test substance	0.98	0.679 ± 0.023	0.194	102.18
	1.95	0.710 ± 0.036	0.195	108.40
	3.91	0.758 ± 0.037	0.193	119.09
	7.81	0.805 ± 0.049	0.195	128.42
	15.62	0.467 ± 0.017	0.229	50.17
	31.25	0.331 ± 0.017	0.126	43.33
	62.5	0.223 ± 0.015	0.184	8.24
	125	0.190 ± 0.010	0.179	2.17

*: mean absorbance (absolute) of 7 wells

** relative absorbance

Table 2: Results of the second XTT test

	Concentration [µg/mL]	Mean absorbance *	Blank	Absorbance in % of vehicle control **
Negative control	Cell culture medium	0.939± 0.027	0.193	106.65
Vehicle control	DMSO (0.2%)	0.887 ± 0.033	0.187	100.00
Test substance	0.98	1.035 ± 0.032	0.201	119.31
	1.95	0.970 ± 0.038	0.194	110.88
	3.91	0.976 ± 0.030	0.192	112.06
	7.81	0.788 ± 0.027	0.193	84.99
	15.62	0.416 ± 0.026	0.195	31.55
	31.25	0.421 ± 0.030	0.176	35.04
	62.5	0.201 ± 0.011	0.174	3.86
	125	0.196 ± 0.007	0.172	3.54

*: mean absorbance (absolute) of 7 wells

** relative absorbance

Table 3: Results of the first h-CLAT run

	Concentration [µg/mL]	Antibody	RFI [%]	Cell viability [%]
Negative control	Cell culture medium	CD 54	100.0	100.0
		CD86	100.0
Vehicle control	DMSO (0.2%)	CD 54	100.0	100.0
		CD86	100.0
Positive control	DNBC (2.0 µg/mL)	CD 54	221.1 *	70.5
		CD86	230.1 *
	DNBC (3.0 µg/mL)	CD 54	391.4 *	69.3
		CD86	294.5 *
Test substance	3.8	CD 54	168.8	86.7
		CD86	162.6 *
	4.5	CD 54	219.5 *	88.6
		CD86	207.3 *
	5.4	CD 54	267.2 *	80.2
		CD86	219.2 *
	6.5	CD 54	232.8 *	68.1
		CD86	235.2 *
	7.8	CD 54	246.9 *	70.3
		CD86	220.5 *
	9.3	CD 54	222.7 *	68.2
		CD86	228.8 *
	11.2	CD 54	235.9 *	49.2
		CD86	302.7 *
	13.4	CD 54	203.1 *	37.4
		CD86	320.1 *

*: RFI value of CD86 or CD54 exceeded the criteria for a positive response (CD86 ≥ 150% and CD54 ≥ 200%).

Table 4: Results of the second h-CLAT run

	Concentration [µg/mL]	Antibody	RFI [%]	Cell viability [%]
Negative control	Cell culture medium	CD 54	100.0	100.0
		CD86	100.0
Vehicle control	DMSO (0.2%)	CD 54	100.0	100.0
		CD86	100.0
Positive control	DNBC (2.0 µg/mL)	CD 54	294.1 *	74.7
		CD86	460.0 *
	DNBC (3.0 µg/mL)	CD 54	380.1 *	76.4
		CD86	483.9 *
Test substance	3.8	CD 54	147.1	88.0
		CD86	145.6
	4.5	CD 54	139.7	92.8
		CD86	132.2
	5.4	CD 54	152.9	89.8
		CD86	168.9 *
	6.5	CD 54	164.7	84.8
		CD86	138.3
	7.8	CD 54	210.3 *	73.3
		CD86	191.7 *
	9.3	CD 54	246.3 *	71.1
		CD86	236.7 *
	11.2	CD 54	245.6 *	66.9
		CD86	260.0 *
	13.4	CD 54	246.3 *	44.6
		CD86	350.0 *

*: RFI value of CD86 or CD54 exceeded the criteria for a positive response (CD86 ≥ 150% and CD54 ≥ 200%).

Applicant's summary and conclusion

Interpretation of results:

other: positive according to OECD 442E

Remarks:

the results of this study as a stand-alone study are not suitable for classification according to CLP/EU GHS criteria; the results may be used for classification purposes in a weight of evidence approach.

Conclusions:

Under the conditions of the Human Cell Line Activation Test the test substance increased the expression of CD54 and CD86 associated with the process of activation of monocytes and dendritic cells. Based on this result, the h-CLAT prediction is considered positive for skin sensitisation.