[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 May - 21 Jun 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:

December 2015

Deviations:

yes

Remarks:

cytotoxicity measurement and estimation of the CV75 value was performed by XTT test instead of flow cytometry

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. 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:

1,3-dimethylbutyl (E)-but-2-enoate

Molecular formula:

C10H18O2

IUPAC Name:

1,3-dimethylbutyl (E)-but-2-enoate

In vitro test system

Details on the study design:

TEST CELL LINE
- Source: American Type Culture Collection
- Passage number: 24 (XTT test); 5 - 7 (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) pyruvate and 1% (v/v) L-glutamine
- Temperature (°C): 37 ± 1.5
- CO2 (%): 5.0 ± 0.5

The following information relate 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).

CONTROLS
Negative Control
- Substance: culture medium
Vehicle control
- Substance: dimethyl sulfoxide (DMSO)
- Concentration: 0.2

EXPOSURE CONDITIONS
- Exposure duration: 24 ± 1 h

TEST CONCENTRATIONS
- Justification for top dose: The test substance was soluble in DMSO up to and including 500 µg/mL.
3.91, 7.81, 15.63, 31.25, 62.5, 125, 250 and 500 µg/mL

NUMBER OF REPLICATIONS: 4 samples per dose group were tested in two independent experiments

MEASUREMENT
- Device: microplate reader (Versamax Molecular Devices)
- Wavelength: 450 nm
- Reference wavelength: 690 nm

EVALUATION CRITERIA
A 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 to 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 CRITERIA
The 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 relate 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 to solvent control are calculated and used in the prediction model to support the discrimination between sensitisers and non-sensitisers.

CONTROLS
Negative Control
- Substance: culture medium
Vehicle control
- Substance: DMSO
- Concentration: 0.2%
Positive control
- Substance: 2,4-dinitrochlorobenzene in DMSO
- Concentration: 2 and 3 µg/mL

EXPOSURE CONDITIONS
- Exposure duration: 24 ± 1 h

TEST CONCENTRATIONS
- Justification for top dose: Since a CV75 value could not be determined the highest soluble test substance concentration was multiplied with 1.2 (600 µg/mL)
167.4, 200.9, 241.1, 289.4, 347.2, 416.7, 500 and 600 µg/mL

NUMBER OF REPLICATIONS: at least in triplicates for the different stainings in two independent experiments

STAINING
- Antibodies: FITC anti-CD54 (Clone: Fun-1); FITC anti-CD86 (Clone: 6.5B5); FITC mouse IgG1 (isotype control)
- Temperature: 2 - 8 °C
- Duration: 30 ± 5 min

MEASUREMENT
- Device: FACSCalibur (Becton Dickinson)

EVALUATION CRITERIA
The 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 100
MFI: 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 100
Where 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 CRITERIA
The 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 to 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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Phase separation: Due to phase separation observed in the two highest concentrations of the first experiment and the highest concentration of the second experiment, these results were excluded from the evaluation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
XTT test
Cytotoxic effects were not observed following incubation with the test substance up to the highest soluble concentration (500 µg/mL). The viability of the cells spaced within a range of 102.67 to 130.87% in the first experiment and within a range of 89.08 to 111.77% in the second experiment (threshold of cytotoxicity: < 75%). Due to the lack of cytotoxicity, a CV75 value could not be calculated.
h-CLAT
The cell viability was not negatively affected (viability > 75%) in both independent experiments.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: In the vehicle control, RFI values compared to the negative control of both CD86 and CD54 did not exceed the positive criteria (CD86 ≥150% and CD54 ≥ 200%).
- Acceptance criteria met for positive control: The RFI values of the positive control for CD86 and CD54 exceeded the positive criteria (CD86 >150% and CD54 >200%) and the cell viability was >50%.

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**
Medium control	-	0.973 ± 0.031	0.311	110.48
Vehicle control	-	0.922 ± 0.055	0.323	100.00
Test substance	3.91	1.000 ± 0.076	0.308	115.48
	7.81	0.924 ± 0.067	0.312	102.12
	15.63	0.924 ± 0.074	0.309	102.67
	31.25	0.956 ± 0.030	0.303	108.91
	62.5	0.968 ± 0.055	0.310	109.84
	125	1.044 ± 0.096	0.316	121.58
	250	1.068 ± 0.088	0.315	125.74
	500	1.101 ± 0.062	0.317	130.87

*: mean absorbance (absolute) of 4 wells

**: relative absorbance

 

Table 2: Results of the second XTT test

	Concentration [µg/mL]	Mean absorbance*	Blank	Absorbance in % of vehicle control**
Medium control	-	1.011 ± 0.092	0.309	120.38
Vehicle control	-	0.884 ± 0.046	0.301	100.00
Test substance	3.91	0.878 ± 0.037	0.309	97.45
	7.81	0.834 ± 0.074	0.314	89.08
	15.63	0.859 ± 0.053	0.307	94.58
	31.25	0.979 ± 0.069	0.306	115.28
	62.5	0.892 ± 0.070	0.310	99.85
	125	0.964 ± 0.092	0.312	111.77
	250	0.960 ± 0.059	0.316	110.33
	500	0.879 ± 0.064	0.308	97.91

*: mean absorbance (absolute) of 4 wells

**: relative absorbance

 

Table 3. Results of the first h-CLAT run

	Concentration [µg/mL]	Antibody / ISO	MFI GeoMean (FITC)	MFI - ISO	Csto(Geo) GeoMean (7-AAD)	Mean Cyto	RFI (%)	Viability (%)
Medium control	-	ISO	2.33	-	2.07	2.0	-	100.0
		CD54	3.01	0.68	1.88		100.0
		CD86	4.34	2.01	1.90		100.0
Vehicle control	-	ISO	2.35	-	1.88	1.9	-	100.0
		CD54	3.24	0.89	1.81		100.0
		CD86	4.00	1.65	1.91		100.0
Positive control	2	ISO	2.18	-	3.13	2.5	-	74.4
		CD54	3.39	1.21	2.20		136.0
		CD86	5.65	3.47	2.20		210.3
	3	ISO	2.67	-	3.38	2.4	-	77.3
		CD54	8.64	5.97	2.11		670.8
		CD86	11.07	8.40	1.75		509.1
Test substance	167.4	ISO	2.23	-	2.59	2.2	-	85.2
		CD54	3.93	1.70	1.98		191.0
		CD86	4.60	2.37	2.00		143.6
	200.9	ISO	2.32	-	2.09	1.9	-	99.3
		CD54	3.92	1.60	1.69		179.8
		CD86	4.26	1.94	1.86		117.6
	241.1	ISO	2.21	-	2.13	1.9	-	99.5
		CD54	4.12	1.91	1.85		214.6
		CD86	4.90	2.69	1.65		163.0
	289.4	ISO	2.20	-	3.06	2.5	-	75.7
		CD54	3.92	1.72	2.51		193.3
		CD86	5.37	3.17	1.83		192.1
	347.2	ISO	2.35	-	2.60	2.4	-	78.1
		CD54	3.64	1.29	2.13		144.9
		CD86	4.05	1.70	2.44		103.0
	416.7	ISO	2.27	-	2.35	2.3	-	80.3
		CD54	3.93	1.66	2.11		186.5
		CD86	4.03	1.76	2.51		106.7
	500^PS	ISO	2.27	-	2.34	2.1	-	90.2
		CD54	3.92	1.65	1.91		185.4
		CD86	4.53	2.26	1.96		137.0
	600^PS	ISO	2.27	-	2.11	2.1	-	90.5
		CD54	4.00	1.73	2.27		194.4
		CD86	4.47	2.20	1.81		133.3

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

PS: phase separation

 

Table 4. Results of the second h-CLAT run

	Concentration [µg/mL]	Antibody / ISO	MFI GeoMean (FITC)	MFI - ISO	Csto(Geo) GeoMean (7-AAD)	Mean Cyto	RFI (%)	Viability (%)
Medium control	-	ISO	2.18	-	1.73	1.8	-	100.0
		CD54	3.01	0.83	1.73		100.0
		CD86	3.07	0.89	1.80		100.0
Vehicle control	-	ISO	2.13	-	1.76	1.6	-	100.0
		CD54	3.14	1.01	1.61		100.0
		CD86	3.42	1.29	1.49		100.0
Positive control	2	ISO	2.13	-	2.38	1.8	-	91.0
		CD54	6.33	4.20	1.65		415.8
		CD86	9.61	7.48	1.31		579.8
	3	ISO	2.13	-	2.49	1.7	-	97.2
		CD54	10.94	8.81	1.10		872.3
		CD86	10.45	8.32	1.41		645.0
Test substance	167.4	ISO	1.95	-	2.36	2.1	-	78.9
		CD54	4.46	2.51	2.05		248.5
		CD86	4.45	2.50	1.75		193.8
	200.9	ISO	1.99	-	2.02	1.8	-	87.9
		CD54	4.48	2.49	1.89		246.5
		CD86	4.25	2.26	1.62		175.2
	241.1	ISO	1.92	-	2.28	1.9	-	83.2
		CD54	4.44	2.52	1.88		249.5
		CD86	4.52	2.60	1.68		201.6
	289.4	ISO	1.93	-	2.47	2.1	-	77.9
		CD54	4.41	2.48	1.88		245.5
		CD86	3.79	1.86	1.89		144.2
	347.2	ISO	1.98	-	2.49	2.1	-	76.4
		CD54	4.03	2.05	1.90		203.0
		CD86	3.91	1.93	1.97		149.6
	416.7	ISO	1.98	-	2.18	1.9	-	86.2
		CD54	4.37	2.39	1.64		236.6
		CD86	3.89	1.91	1.82		148.1
	500	ISO	1.99	-	1.97	1.8	-	88.2
		CD54	4.37	2.38	1.77		235.6
		CD86	3.94	1.95	1.77		151.2
	600^PS	ISO	1.90	-	2.29	2.0	-	80.2
		CD54	4.33	2.43	1.87		240.6
		CD86	4.02	2.12	1.90		164.3

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

PS: phase separation

Applicant's summary and conclusion

Interpretation of results:

other: positive according to OECD 442E

Conclusions:

Under the conditions of the Human Cell Line Activation Test the test substance increased the expression of both cell surface markers, CD86 and CD54, 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.

Executive summary:

Dendritic cell response of the test substance was determinded by a Human Cell Line Activation Test according to OECD 442E in compliance with GLP. The test substance increased the expression of both cell surface markers, CD86 and CD54, 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.