4-hydroxybutyl acrylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Jul 2012 to 09 Aug 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: In Vitro Skin Sensitisation: U937 Skin Sensitisation Test

Version / remarks:

20 July 2016

Principles of method if other than guideline:

There are no official national or international guidelines for the MUSST Assay; however, the study is performed according to the methods described in the following publications:

Python F, Goebel C, Aeby P. (2007) Assessment of the U937 cell line for the detection of contact allergens. Toxicol Appl. Pharmacol. 220(2), 113-24.

Bauch C, Kolle SN, Fabian E, Pachel C, Ramirez T, Wiench B, Wruck CJ, van Ravenzwaay B, Landsiedel R. (2011) Intralaboratory validation of four in vitro assays for the prediction of the skin sensitizing potential of chemicals. Toxicology in Vitro 25, 1162 – 1168.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

4-hydroxybutyl acrylate

EC Number:

219-606-3

EC Name:

4-hydroxybutyl acrylate

Cas Number:

2478-10-6

Molecular formula:

C7H12O3

IUPAC Name:

4-hydroxybutyl acrylate

Test material form:

not specified

Details on test material:

- Name of test material: 4-hydroxybutyl acrylate
- Lot/batch No.: 010232EDA0
- Expiration date of the lot/batch: 30 Nov 2012
- Stability under test conditions: at least until 30 Nov 2012

Specific details on test material used for the study:

- Name of test material: 4-hydroxybutyl acrylate
- Lot/batch No.: 010232EDA0
- Expiration date of the lot/batch: 30 Nov 2012
- Stability under test conditions: at least until 30 Nov 2012

In vitro test system

Details on the study design:

Concentrations: 3.26, 6.53, 13.05, 26.10, 52.20 µg/mL

PRE-TEST
In order to determine the concentrations suitable for the main experiment a pre-test (experimental conduct in accordance with GLP but without a GLP status) was performed. Cells were exposed to 9 concentrations of the test substance (0.5 μg/mL up to 2000 μg/mL) and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA.

MAIN-TEST
Test substance preparation
The test-substance preparation was performed on a weight per volume basis shortly before application by stirring.

Test-substance preparation: The test substance was dissolved in medium as a 2x stock solution of the highest concentration. Further concentrations were prepared as 2x concentrations by serial dilution.
Vehicle: Culture medium
Reason for the vehicle: Culture medium was used according to the physiological conditions.
Form of application: Solutions in culture medium

The test substance preparations were prepared within 4 hours of performing the assay (preparation of test-substance samples).

CONTROLS
Negative control (NC): Lactic acid (LA – 200 μg/mL), CAS no.: 50-21-5
Positive control (PC): Ethylene diamine (EDA – 70 μg/mL), CAS no.: 97-90-5
Vehicle control: Culture medium Isotype control: In order to help distinguish non-specific (“background”) staining from specific antibody staining each test-substance concentration and control is additionally incubated with IgG1 FITC (CD86)

PREPARATION OF CELLS
U937 cells from the working cell bank were thawed and cultured in suspension using complete RPMI 1640 medium with 25 mM HEPES buffer and 2 mM L-glutamine supplemented with 10% fetal bovine serum and 100 U/mL penicillin and 100 μg/mL streptomycin under standard culture conditions (37°C, ca. 5% CO2, ≥ 90% humidity) until for 5 passages but not longer than passage 13 prior to testing. For substance incubation, cells seeded in 96-well microtiter plates (100 μL of 0.5x10^6 cells/mL). As a rule, two independent experiments were performed. In each experiment, duplicates of each treatment were tested.

TEST SUBSTANCE APPLICATION
Treatment was performed by adding 100 μL of test-substance preparation to the cells, thus diluting the 2x concentrated test-substance preparations to their final concentration and the cells to 0.25x10^6 cells/mL. After application the plates were sealed with semi-permeable plate sealers in order to prevent evaporation of the test substance. The plates were placed into the incubator under standard culture conditions for the exposure period of 48 hours.

VISUAL INSPECTIONS
A visual inspection for test-substance precipitates was performed for each test-substance concentration prior to application. In addition, each well was inspected under a microscope after the exposure period of 48 hours in order to detect irregularities in cell morphology or test-substance precipitates.

CELL STAINING AND FLOW CYTOMETRIC ANALYSIS
After visual inspection the cells were transferred into V-shaped 96-well microtiter plates and centrifuged. Supernatants were discarded. The cells were washed once with PBS with 5% FBS at 4°C. Cells were resuspended in 100 μL PBS (with 5% FBS) and labeled for 30 min at 4°C in the dark with 5 μL IgG-FITC (isotype control) or 5 μL anti-CD86-FITC antibody. Following incubation, cells were washed twice with PBS (with 5% FBS) and once with PBS and were then resuspended in 200 μL PBS. For cell viability analysis, cells were stained with PI (1.25 μg/mL final concentration in PBS) for 5 min at 4°C in darkness. Fluorescence intensity was analyzed using flow cytometry.

DATA EVALUATION
Analysis of the membrane markers was performed in 10,000 viable cells, determined by PI staining. Concentrations affording viability less than 70% were not considered for further assessment of dendritic cell activation. For data analysis, the CXP software (Beckman Coulter) was used. Data evaluation was performed with percentage of CD86 positive cells among the viable cells. An isotype control was used to quantify and remove non-specific antibody binding. The CD86 result was expressed as fold induction of CD86 expression compared to the respective vehicle control.

ACCEPTANCE CRITERIA
A tested concentration is not to be further evaluated when relative viability is less than 70%. A study is considered acceptable if the positive and negative and vehicle control data lie within the range of the historical data. The cell viability of untreated cells must yield at least 90%. The expression marker CD86 of the vehicle control cells should lie between 8% – 20%.

EVALUATION OF RESULTS
A test substance is predicted to activate dendritic cells when CD86 cell surface expression exceeds the threshold of 1.2 in relation to vehicle control in at least two independent experiments.

Results and discussion

In vitro / in chemico

Results

Key result

Run / experiment:

other: 2 experiments (concentration in μg/mL)

Parameter:

other: CD86

Value:

3.26 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Raw data main experiments

Concentration µg/mL	1st Experiment		2nd Experiment
	CD 86 induction	Rel. viability	CD 86 induction	Rel. viability
VC	1.00	100.0	1.00	100.0
3.26	1.29	99.9	1.45	99.6
6.53	1.54	99.4	1.97	99.0
13.05	1.59	95.5	2.13	88.6
26.10	0.65	73.3	0.74	53.3
52.20	0.33	59.9	0.44	21.0

Control data

Concentration µg/mL	1st Experiment		2nd Experiment
	CD 86 induction	Rel. viability	CD 86 induction	Rel. viability
VC	1.00	100.0	1.00	100.0
LA 200 µg/mL	1.09	100.0	1.02	99.9
EDA 70 µg/mL	2.09	92.0	2.21	80.6

Pre-test

The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve to be 26.10 μg/mL.

Main tests

A test substance was predicted to have a dendritic cell activating potential when the marker expression exceeded the threshold of 1.2 with respect to vehicle treated cells (VC) at any tested sufficiently noncytotoxic (cell viability ≥ 70%) concentration in at least two independent experiments.

The MUSST showed the following results:

The test substance was soluble in culture medium.

The dilutions of the test substance were solutions.

In summary, after 48 hours of exposure to test substance 4 -hydroxybutyl acrylate CD86 expression was induced in U937 cells at concentration between 3.26 and 13.05 μg/mL affording at least 70% viability. From this it has to be concluded that test substance 4 -hydroxybutyl acrylate does induce dendritic cell activation.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

in combination with DPRA and LuSens in chemico/vitro tests