Isopentyl salicylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January - April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD guideline 442E h-CLAT

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

Isobutyl salicylate

EC Number:

201-729-9

EC Name:

Isobutyl salicylate

Cas Number:

87-19-4

Molecular formula:

C11H14O3

IUPAC Name:

Isobutyl salicylate

In vitro test system

Details on the study design:

TEST SYSTEM
- Cell line: THP-1 cells; human monocytic leukemia cell line
- Source: American Type Culture Collection Manassas, USA (ATCC, TIB-202)

TEST-SUBSTANCE PREPARATION
- Concentrations: Dose range finder: 7.81, 15.63, 31.25, 62.50, 125, 250, 500, 1000 µg/mL; Main experiment: 97.71, 81.42, 67.85, 56.54, 47.12, 39.27, 32.72, 27.27 µg/mL
- Stock: 2x concentration of the highest concentration stock solution
- Solvent: DMSO

CONTROLS
- Negative control (NC):Medium
- Solvent control (SC): 0.2% DMSO
- Isotype control: In order to help distinguish non-specific staining from specific antibody staining each test-substance concentration and control is additionally incubated with mouse IgG1
- Positive control: 1-chloro-2-4-dinitrobenzene (DNCB); 4.0 µg/mL

MEDIUM
- Culture medium: RPMI 1640: with L-glutamine, 25 mM HEPES (Gibco) + 10% FBS inactivated + 1% Penicillin/Streptomycin + 0.05 mM 2-Mercaptoethanol (Gibco)
- FACS Buffer: Phosphate Buffered Saline (DPBS) without Ca2+ / Mg2+ (Gibco) + 0.1% BSA (Sigma A7030)
- Blocking Solution: 0.01% Globulins Cohn fraction II,III with DPBS
- Reagent for cytotoxicity test: Propidium iodide (Sigma)

EXPERIMENTAL PROCEDURE
- Replicates: 1
- Experiments: 3
- Exposure period: 24 hours
- Preparation of cells: THP-1 cells were thawed and cultured in complete RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin and 0.05 mM 2 -mercaptoethanol (30 > passage >= 5)

ANALYSIS
- FACS: cell staining and flow cytometric analysis 24 hours after exposure

DATA EVALUATION
- CV75 calculation: Relative survival rate is calculated by linear extrapolation. This value is the substance concentration at which cell viability is 75% compared to the vehicle control.
- Relative cell viability: % relative cell viability = (number of living cells / total number of aquired cells) * 100
- Relative fluorescence intensity: RFI (%) = (MFI of chemical-treated cells - MFI of chemical-treated isotype control cells) / (MFI of solvent control cells - MFI of solvent isotype control cells) * 100
- Calculation of EC 150% and EC 200%: If applicable, the concentration resulting in a positive response (RFI of 150% (CD86) or 200% (CD54) and viability >50%) was calculated for each cell surface marker from each experiment. The calculation is performed by linear regression from the two concentrations directly above and below the EC 150% / EC 200% concentration.

ACCEPTANCE CRITERIA
- A tested concentration is not to be further evaluated when viability is less than 50%
- Cell viability of vehicle control cells must yield at least 90%
- In the positive control (DNCB), RFI (relative fluorescence intensity) values of both CD86 and CD54 should be exceed the positive criteria (RFI CD86 =150% and CD54 =200%) and cell viability should be =50%.
- For all vehicle control, the MFI (mean fluoresence intensity) ratio of both CD86 and CD54 to isotype controls should be =105%.
- A study is considered to be acceptable if the positive, negative and vehicle control data lies within the range of the historic data.

EVALUATION RESULTS
- Positive result: A test is considered to be positive when the dendritic cells are activated meaning that CD86 expression is increased =150% and/or CD54 expression increased =200% in relation to vehicle control in at least 2 independent experiments.
- Negative result: A test is considered to be negative when the criteria mentioned above are not met up to the maximum concentration (=5000 µg/mL for the vehicle culture medium or 2000 µg/mL for 0.2% DMSO in culture medium).

Results and discussion

Positive control results:

Positive controls achieved RFI values of both CD86 and CD54 over the positive criteria (CD86= 264% experiment 1; 336% experiment 2; 304% experiment 3) and 200% for CD54 (231% experiment 1; 288% experiment 2; 257% experiment 3).

In vitro / in chemico

Results

Key result

Run / experiment:

other: Please see 'Any other information on results incl. tables'.

Parameter:

other: RFI CD54 mean (%) and RFI CD54 mean (%)

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

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	-	94.7	94.7	94.3	2943	1205	613	2330	592	101	111	480	197
Solvent Control	0.20%	94.8	95.2	94.8	2988	1214	679	2309	535	100	100	440	179
DNCB	4.00	80.9	80.5	80.0	6868	2013	779	6089	1234	264	231	882	258
ISOBUTYL SALICYLATE	97.71	12.4	11.9	12.3	4427	3822	942	3485	2880	151	538	470	406
	81.43	57.3	58.4	57.4	3480	1781	726	2754	1055	119	197	479	245
	67.85	83.9	83.4	84.1	3116	1540	673	2443	867	106	162	463	229
	56.55	88.9	89.3	89.5	3066	1380	658	2408	722	104	135	466	210
	47.12	93.2	93.5	93.6	2778	1235	669	2109	566	91	106	415	185
	39.27	94.5	94.3	93.9	2801	1230	686	2115	544	92	102	408	179
	32.72	94.4	94.8	94.9	2996	1287	660	2336	627	101	117	454	195
	27.27	94.7	94.6	94.7	3063	1276	669	2394	607	104	113	458	191

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	-	94.7	94.8	93.9	4619	1554	646	3973	908	102	95	715	241
Solvent Control	0.20%	95.0	95.1	94.9	4571	1620	669	3902	951	100	100	683	242
DNCB	4.0	81.4	80.5	81.2	13735	3370	633	13102	2737	336	288	2170	532
ISOBUTYL SALICYLATE	97.71	13.2	12.8	12.4	7467	5420	935	6532	4485	167	472	799	580
	81.43	45.9	45.5	45.7	6028	3615	1213	4815	2402	123	253	497	298
	67.85	79.3	79.0	79.7	5291	3011	782	4509	2229	116	234	677	385
	56.55	90.0	90.8	89.7	4710	2761	874	3836	1887	98	198	539	316
	47.12	93.5	93.5	92.7	4543	2413	735	3808	1678	98	176	618	328
	39.27	93.4	92.5	92.8	4587	2304	746	3841	1558	98	164	615	309
	32.72	94.6	94.1	94.2	4335	2049	756	3579	1293	92	136	573	271
	27.27	95.1	95.0	94.9	4489	2013	790	3699	1223	95	129	568	255

CD54 and CD86 Expression Experiment 3

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	-	95.2	94.5	94.3	3732	1289	640	3092	649	88	87	583	201
Solvent Control	0.20%	94.7	94.5	94.5	4176	1395	649	3527	746	100	100	643	215
DNCB	4.0	81.9	81.1	82.4	11303	2502	583	10720	1919	304	257	1939	429
ISOBUTYL SALICYLATE	97.71	40.5	38.6	39.3	4938	2769	816	4122	1953	117	262	605	339
	81.43	64.4	64.9	64.8	4625	2417	806	3819	1611	108	216	574	300
	67.85	86.4	85.4	86.3	4465	2304	739	3726	1565	106	210	604	312
	56.55	93.0	91.8	93.0	4049	1925	742	3307	1183	94	159	546	259
	47.12	94.0	93.8	94.4	3792	1728	740	3052	988	87	132	512	234
	39.27	94.5	94.1	93.9	3707	1636	730	2977	906	84	121	508	224
	32.72	95.0	94.9	95.2	3538	1580	735	2803	845	79	113	481	215
	27.27	95.2	95.6	95.0	4119	1701	811	3308	890	94	119	508	210

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Based on the results, it is concluded that the test substance induces dendritic cell activation (CD54 expression increased =200% in at least one assay).

Executive summary:

The potential of test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h-CLAT). For this purpose the test substance was incubated with human pro-monocytic cell line THP-1 for ca. 24 hours at 37°C and membrane markers expression measured by flow cytometry. In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 10 concentrations of the test substance and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (=estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve.

 

In the main test after 24 hour exposure THP-1 cells were stained with FITC labelled anti-human-CD86/ anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analysed using flow cytometry. A total of 3 valid experiments were performed. The expression of the cell surface marker CD54 clearly exceeded the threshold at one concentration with a cell viability ≥50% in at least two independent runs.

The positive control (DNCB) led to an upregulation of CD54 and CD86 in all three experiments. The threshold of 150% for CD86 (264% experiment 1; 336% experiment 2; 304% experiment 3) and 200% for CD54 (231% experiment 1; 288% experiment 2; 257% experiment 3) were clearly exceeded.

 

In summary, it has to be concluded that the test substance induces dendritic cell activation.