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Diss Factsheets

Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2011-12-13 to 2012-01-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
There are no official national or international guidelines for the MUSST Assay, 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)
Remarks:
BASF SE
Type of study:
other: Dendritic Cell Activation Assay Myeloid U937 Skin Sensitization Test (MUSST)

Test material

Constituent 1
Reference substance name:
2-Propenoic acid, C18-26-alkyl esters
EC Number:
285-348-3
EC Name:
2-Propenoic acid, C18-26-alkyl esters
Cas Number:
85085-17-2

In vitro test system

Details on the study design:
Skin sensitisation (In vitro test system) - Details on study design:

CELL LINE
U937

CONTROLS
- Negative Control (NC): Lactic acid(LA - 200 µg/mL)
- Positive Control (PC): Ethylene diamine (EDA - 70 µg/mL)
- 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).

TEST SUBSTANCE PREPARATION
- The test substance preparation was performed on a weight per volume basis shortly before application by stirring and ultrasonic treatment. The test substance was dissolved in medium as a 2 x stock solution of the highest concentration (2000 µg/mL). Further concentrations were prepared as 2 x concentrations by serial dilution.
- Vehicle: culture medium (according to physiological conditions)
- The test substance preparations were prepared within 4 hours of performing the assay (preparation of test substance samples).

EXPERIMENTAL PROCEDURE
- Preparation of the cells: U973 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 0.5 x 10^6 cells/mL cell suspensions). 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 2 x concentrated test substance preparations to their final concentration and the cells to 0.25 x 10^6 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.

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Run / experiment:
other: 125 µg/mL, 1st Experiment
Parameter:
other: % PI negative cells
Value:
0.99
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: 250 µg/mL, 1st Experiment
Parameter:
other: % PI negative cells
Value:
1.19
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: 500 µg/mL, 1st Experiment
Parameter:
other: % PI negative cells
Value:
1.24
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: 1000 µg/mL, 1st Experiment
Parameter:
other: % PI negative cells
Value:
1.26
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: 2000 µg/mL, 1st Experiment
Parameter:
other: % PI negative cells
Value:
1.58
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: 125 µg/mL, 2nd Experiment
Parameter:
other: % PI negative cells
Value:
1.03
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: 250 µg/mL, 2nd Experiment
Parameter:
other: % PI negative cells
Value:
1.12
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: 500 µg/mL, 2nd Experiment
Parameter:
other: % PI negative cells
Value:
1.16
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: 1000 µg/mL, 2nd Experiment
Parameter:
other: % PI negative cells
Value:
1.19
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: 2000 µg/mL, 2nd Experiment
Parameter:
other: % PI negative cells
Value:
1.33
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

The test substance was soluble in culture medium. The dilutions of the test substance were solutions. After 48 hours no precipitates were noticed in the samples by visual inspection. In summary, after 48 hours of exposure to test substance Behenylacrylate (Acrylate 22 45%) CD 86 expression was induced in U937 cells at concentration between 500 and 2000 μg/mL affording at least 70% viability. From this it has to be concluded that test substance Behenylacrylate (Acrylate 22 45%) does induce dendritic cell activation.

Applicant's summary and conclusion

Interpretation of results:
other: induction of dendritic cell activation
Conclusions:
No prediction can be made for skin sensitization according to GHS criteria based on the results of this in vitro study alone.
Based on the observed results it was concluded that the test substance does induce dendritic cell activation in the MUSST under the test conditions chosen.
Executive summary:

The change in the expression of the cell membrane marker CD86 induced by the test substance was evaluated in the Myeloid U937 Skin Sensitization Test (MUSST). For this purpose the test substance was incubated with human pro-monocytic cell line U937 for ca. 48 hours at 37 °C and membrane marker expression measured by flow cytometry.

A solubility experiment was performed. The test substance was soluble in culture medium at a concentration of 2000 µg/mL.

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. A CV75 value (= estimated concentration that affords 75 % cell viability) could not be determined as no cytotoxicity was observed up to 2000 µg/mL under the chosen exposure conditions on U937 cells.

In the main test, test substance was used at five final concentrations. After 48 hour exposure U937 cells were stained with FITC labeled anti-human-CD86 antibody and propidium iodide and the fluorescence intensity was analyzed using flow cytometry. 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 non-cytotoxic (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. After 48 hours no precipitates were noticed in the samples by visual inspection.

In summary, after 48 hours of exposure to test substance CD86 expression was induced in U937 cells at concentration between 500 and 2000 µg/mL affording at least 70 % viability. From this it has to be concluded that test substance does induce dendritic cell activation.