Registration Dossier
Registration Dossier
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EC number: 236-492-0 | CAS number: 13402-02-3
Endpoint summary
Administrative data
Description of key information
Read across has been done to a dataset of two mixtures of long-chain acrylates: Behenylacrylate (Acrylate 22 45%, mixture of C18-C22) and 2-Propenoic acid, C16-18-alkyl esters (mixture of C16-C18 acrylates).
Sensitization involves a number of key steps in order to take place, and
can be described in terms of an adverse outcome pathway (AOP). These
include reactivity with skin proteins (peptide (cysteine and lysine)
reactivity), activation of skin cells (keratinocyte activation) and
immune cells (dendritic cell activation). The results of these studies
are used in a predefined evaluation scheme ("2 out of 3"-approach) to
determine hazard classification as a sensitizer by weight-of-evidence
approach.
C16-18:
The sensitizating potential was tested in in vitro studies (Direct
Peptide Reactivity Assay (DPRA), ARE Reporter Assay-LuSens and Myeloid
U937 Skin Sensitization Test (MUSST)). The test item did reveal
sensitising properties.
C18-22:
The sensitizating potential was tested in in vitro studies (Myeloid U937
Skin Sensitization Test (MUSST), ARE Reporter Assay-LuSens and Direct
Peptide Reactivity Assay (DPRA)). The test item did not reveal any
sensitising properties.
Conclusion:
Based on the in vitro results and as a worst case assumption, the test
item hexadecyl acrylate (C16) is considerd to be a skin sensitiser.
Key value for chemical safety assessment
Skin sensitisation
Link to relevant study records
- 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
- 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)
- 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. - 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
- 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.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2011-10-24 to 2012-02-08
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- There are no official national or international guidelines for the DPRA Test, the study is performed according to the methods described in the following publications: Gerberick GF, Vassallo JD, Bailey RE, Chaney JG, Morrall SW, Lepoittevin JP. Development of a Peptide Reactivity Assay for Screening Contact Allergens. Toxicological Sciences 81,332-343, 2004.Gerberick GF, Vassallo JD, Bailey RE, Chaney JG, Morrall SW, Lepoittevin JP.
Development of a Peptide Reactivity Assay for Screening Contact Allergens. Toxicological Sciences 81,332-343, 2004. Bauch C, Kolle SN, Fabian E, Pachel C, Ramirez T, Wiench B, Wruck CJ, van Ravenzwaay B, Landsiedel R. Intralaboratory validation of four in vitro assays for the prediction of the skin sensitizing potential of chemicals. Toxicology in Vitro 25, 1162 – 1168, 2011.
Maxwell G, Aeby P, Ashikaga T, Bessou-Touya S, Diembeck W, Gerberick F, Kern P, Marrec-Fairley M, Ovigne JM, Sakaguchi H, Schroeder K, Tailhardat M, Teissier S, Winkler P. Skin sensitisation: the Colipa strategy for developing and evaluating nonanimal test methods for risk assessment. ALTEX 28(1): 50-5, 2011. - GLP compliance:
- yes (incl. QA statement)
- Remarks:
- BASF SE
- Type of study:
- other: Direct Peptide Reactivity Assay (DPRA)
- Details on the study design:
- Skin sensitisation (In vitro test system) - Details on study design:
CONTROLS
- Negative Control (NC): vehicle control = acetone (for the test substance) and acetonitrile (for the PC)
- Positive Control (PC): p-Benzoquinone, puriss.
TEST SUBSTANCE PREPARATION
- The test substance was prepared within 4 hours of performing the assay (preparation of samples). The test substance was prepared as a 100 mM suspension in acetone. As the test substance was a suspension in acetone the preparation was stirred with a magnetic stirrer until and during sample preparation.
- Vehicle: acetone (The test substance was not soluble in one of the vehicles used for the assay. In acetone a homogeneous suspension was achieved).
EXPERIMENTAL PROCEDURE
- The test substance was formulated in a suitable vehicle. Per run three samples of the test substance were incubated with each peptide. Additionally triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. In addition peptide standards of known concentration, prepared from the respective peptide stock solution used for test substance incubation, were measured in parallel with the same analysis method.
- Test substance solubility: Prior to the assay the solubility of the test substance was tested. A suitable non-reactive, water-miscible solvent which dissolves the test substance completely should be used. The preferred solvent was acetonitrile. When not soluble in acetonitrile, deionized water, methanol, propanol, isopropanol, acetone or mixtures of these solvents were tried. As a last possibility mixtures of DMSO with acetonitrile up to a ratio of 1 : 1 and EDGE were tried. The test substance was soluble in a concentration of 100 mM in EDGE, only. However, EDGE could not be used due to instability of the C-peptide in the vehicle. No homogeneous preparations were obtained in a concentration of 100 mM in the following vehicles: acetonitrile, water, methanol and DMSO : ACN (1 : 1).
- Preparation of peptide stock solutions: Peptide stock solutions in a concentration of 0.667 mM of peptide were prepared in pH 7.5 phosphate buffer (C-containing peptide) or pH 10.2 ammonium acetate buffer (K-containing peptide). The peptide stock solution was used for preparing the peptide standard and the test substance and control samples.
- Preparation of the test substance samples: The samples were prepared in triplicates for each peptide. The test substance was incubated with the C-containing peptide in a ratio of 1 : 10 (0.5 mM peptide, 5 mM test substance) and with the K-containing peptide in a ratio of 1 : 50 (0.5 mM peptide, 25 mM test substance). The samples were prepared in suitable tubes, capped tightly and incubated at room temperature in the dark for 24 +/- 2 hours. Prior to HPLC analysis the samples were visually investigated for any precipitate that may have formed during the exposure period. Samples which were visually turbid or displayed precipitates were centrifuged or filtrated prior to injection into the HPLC in order to remove any unsolved particles. The HPLC analysis of the batch of samples started about 24 hours after sample preparation and the analysis time itself did not exceed 30 hours.
- Preparation of the vehicle controls: Vehicle controls were prepared in triplicates in the same way as the test substance samples described above with the vehicle instead of the test substance.
- Preparation of the co-elution control: The co-elution control was prepared in the same way as the test substance samples described above but without the peptide. Instead the respective peptide buffer was used. The samples were analyzed directly after preparation. Samples which were visually turbid or displayed precipitates were centrifuged or filtrated prior to injection into the HPLC in order to remove any unsolved particles. - Run / experiment:
- mean
- Parameter:
- other: cysteine-peptide depletion [%]
- Value:
- 0.3
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Run / experiment:
- mean
- Parameter:
- other: lysine-peptide depletion [%]
- Value:
- 0
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Run / experiment:
- mean
- Parameter:
- other: mean peptide depletion [%]
- Value:
- 0.2
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- When mixed with the peptide stock solution the samples became cloudy directly after preparation. Additionally the test substance precipitated in all samples. Thus all samples were centrifuged prior to HPLC analysis.
No co-elution of the test substance and peptides occured. - Interpretation of results:
- other: minimal chemical reactivity
- 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 and applying the prediction model proposed in Gerberick et al. (2007) it was concluded that the test substance shows a minimal chemical reactivity in the DPRA under the test conditions chosen. - Executive summary:
The reactivity of the test substance towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose the test substance was incubated with synthetic peptides for ca. 24 hours at room temperature and the remaining non-depleted peptide concentration was determined thereafter by high performance liquid chromatography with gradient elution and UV-detection at 220 nm.
The test substance was formulated at a 100 mM concentration in acetone. One (K-peptide) or two (C-peptide) test runs were performed. Per test run three samples of the test substance were incubated with each peptide in ratios of 1 : 10 (for C-peptide) or 1 : 50 (for K-peptide). Additionally triplicates of the concurrent vehicle control (= NC) were incubated with the peptides.
Additionally, a co-elution control was performed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide.
The following results were obtained in the DPRA:
The test substance was not soluble in one of the vehicles used for the assay. Thus a homogeneous suspension in acetone was used for sample preparation.
When mixed with the peptide stock solution the samples became cloudy directly after preparation. Additionally the test substance precipitated in all samples. Thus all samples were centrifuged prior to HPLC analysis.
In a first test run the mean C-peptide depletion, caused by the test substance was determined to be 18 %. However, due to high standard deviation and irregularities during centrifugation of the samples the test run was considered to be invalid and was repeated.
The mean C-peptide depletion, caused by the test substance was determined to be 0.3 % in the second test run. The mean K-peptide depletion, caused by the test substance was determined to be 1.9 %.
Negative depletions were considered to be "zero" for calculation of the mean peptide depletion, which was thus calculated to be 0.2 %.
No co-elution of test substance and peptides was noticed.
Based on the observed results and applying the prediction model proposed in Gerberick et al. (2007) it was concluded that the test substance shows a minimal chemical reactivity in the DPRA under the test conditions chosen.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- Oct 2012 - Jan 2013
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- There are no official national or international guidelines for the LuSens Assay; however, the study is performed according to the methods described in the following publication:
Bauch C, Kolle SN, Ramirez T, Eltze T, Fabian E, Mehling A, Teubner W, van Ravenzwaay B, Landsiedel R, (2012), Putting the parts together: Combining in vitro methods to test for skin sensitizing potentials, Regul Toxicol Pharmacol, 63(3):489-504. - GLP compliance:
- yes (incl. QA statement)
- Remarks:
- (from the competent authority) Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz)
- Type of study:
- other: ARE Reporter Assay - LuSens
- Details on the study design:
- Skin sensitisation (In vitro test system) - Details on study design:
CONTROLS
- Negative Control (NC): DL-Lactic acid (LA 450 µg/mL)
- Positive Control (PC): Ethylene glycol dimethacrylate (EGDMA 15 µg/mL)
- Vehicle Control: Culture medium 3
- Blank Control: Medium without cells
- Basal Control: Medium with cells
TEST SUBSTANCE PREPARATION
- The test substance preparation was prepared on a weight per volume basis shortly before application by stirring treatment. The test substance was dissolved in culture medium 3 as a 2 x stock solution of the highest concentration. Further concentrations were prepared as 2 x concentrations by 1 : 1.2 serial dilution.
- Vehicle: Culture medium 3 (good homogeneity of the preparation)
- Form of application: Emulsion in culture medium at 1157 µg/mL and higher.
- The test substance preparations were prepared within 4 hours of performing the assay (preparation of test substance samples).
EXPERIMENTAL PROCEDURE
- Preparation of the cells: LuSens cells from the working cell bank were thawed and cultured using culture medium 1, under standard culture conditions (37 °C, ca. 5 % CO2, >= 90 % humidity) for at least 2 weeks at passage > 5 but not longer than 15 passages prior to testing. Before the substance incubation, cells were seeded in 96-well microtiter plates (120 µL of 0.83 x 10^5 cells/mL cell suspensions), using culture medium 2 for incubation for 24 hours. As a rule, two independent experiments were performed. In each experiment, three replicates of each treatment were tested. If contradictory results are obtained in the first and second experiment, a third experiment will be usually conducted.
- Test substance preparation and application of MTT and Luciferase Assay: The substance was prepared up to a 2 x stock concentration in culture medium 3. Based on the stock concentration, a 1 : 1.2 master plate with culture medium 3 was prepared. After cell adaption for 24 hours cell culture medium was aspirated and replaced with 100 µL culture medium 3 and 100 µL of each dilution of the 2 x master plate was added to each well. 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. For the luciferase assay a white plate (luminescence compatible plate) was used. In addition a clear plate was treated in parallel for the determination of cell viability.
- 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.
- Luciferase assay: After visual inspection of the cells, the supernatant was aspirated from the white assay plate and discarded. The cells were washed twice with 300 µL PBS (with Ca2+ / Mg2+), 100 µL Steady-Glo-Mix and 100 µL PBS (without Ca2+ / Mg2+) per well were added and cells were shaken with a plate shaker for 10 min at room temperature in darkness. After the incubation the luminescence was measured in the luminometer.
- Cell viability assay MTT: Cell culture medium was aspirated from all wells and 180 µL medium 3 was added. Briefly, a 5 mg/mL thiazolyl blue tetrazolium bromide (MTT) stock solution was prepared in PBS (without Ca2+ / Mg2+). 20 µL of MTT solution was added to each well of the 96-well microtiter plate and incubated for further 2 hours after sealing the plates in the incubator. For analysis, medium was aspirated and cells were lysed by adding 100 µL of lysis solution (99.6 mL DMSO; 10 g sodium dodecyl sulfate, SDS; and 0.4 mL glacial acetic acid). Absorbance was measured at 570 nm with reference wavelength at 590 nm and 690 nm using the Sunrise Absorbance Reader. - Run / experiment:
- other: 804 µg/mL, 1st Experiment
- Parameter:
- other: induction of luciferase activity
- Value:
- 1.01
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 965 µg/mL, 1st Experiment
- Parameter:
- other: induction of luciferase activity
- Value:
- 1.36
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 1157 µg/mL, 1st Experiment
- Parameter:
- other: induction of luciferase activity
- Value:
- 1.36
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 1389 µg/mL, 1st Experiment
- Parameter:
- other: induction of luciferase activity
- Value:
- 1.33
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 1667 µg/mL, 1st Experiment
- Parameter:
- other: induction of luciferase activity
- Value:
- 1.08
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 2000 µg/mL, 1st Experiment
- Parameter:
- other:
- Value:
- 1.01
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 804 µg/mL, 2nd Experiment
- Parameter:
- other: induction of luciferase activity
- Value:
- 0.96
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 965 µg/mL. 2nd Experiment
- Parameter:
- other: induction of luciferase activity
- Value:
- 0.89
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 1157 µg/mL, 2nd Experiment
- Parameter:
- other: induction of luciferase activity
- Value:
- 0.96
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 1389 µg/mL, 2nd Experiment
- Parameter:
- other: induction of luciferase activity
- Value:
- 1.05
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 1667 µg/mL, 2nd Experiment
- Parameter:
- other: induction of luciferase activity
- Value:
- 0.93
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 2000 µg/mL, 2nd Experiment
- Parameter:
- other: induction of luciferase activity
- Value:
- 0.89
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 804 µg/mL, 3rd Experiment
- Parameter:
- other: induction of luciferase activity
- Value:
- 1.01
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 965 µg/mL, 3rd Experiment
- Parameter:
- other: induction of luciferase activity
- Value:
- 0.92
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 1157 µg/mL, 3rd Experiment
- Parameter:
- other: induction of luciferase activity
- Value:
- 0.97
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 1389 µg/mL, 3rd Experiment
- Parameter:
- other: induction of luciferase activity
- Value:
- 0.88
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 1667 µg/mL, 3rd Experiment
- Parameter:
- other: induction of luciferase activity
- Value:
- 0.83
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 2000 µg/mL, 3rd Experiment
- Parameter:
- other: induction of luciferase activity
- Value:
- 0.83
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Interpretation of results:
- other: no induction of luciferase activity
- 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 not induce luciferase activity in LuSens cells under the test conditions chosen. - Executive summary:
The keratinocyte activating potential of the test substance was evaluated in the LuSens assay. For this purpose the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37 °C and incubation was measured in a luminometer.
The test substance was a emulsion in culture medium at a concentration of 1000 µg/mL (2 x stock solution) onward.
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 MTT assay. No decrease in cell viability below 70 % was observed.
In the main test, test substance was used at six final concentrations. After 48 hour exposure luciferase activity was measured in a luminometer. A test substance was concluded to exhibit a keratinocyte activating potential when the luciferase activity exceeds 1.5 fold induction with respect to the vehicle control and at concentrations that did not reduce a viability below 70 %. In parallel a MTT assay was performed to assess cytotoxicity of the test substance.
The LuSens showed the following results:
The test substance was a emulsion in culture medium at 1157 µg/mL onward. The dilutions of the test substance were emulsions at 1157 µg/mL onward. After 48 hours no precipitates were noticed.
In summary, after 48 hours of exposure to test substance luciferase activity in LuSens cells was not induced at concentration between 804 and 2000 µg/mL affording at least 70 % viability. From this it has to be concluded that the test substance has no keratinocyte activating potential.
- Endpoint:
- skin sensitisation, other
- Remarks:
- Expert Statement
- Type of information:
- other: Expert Statement
- Adequacy of study:
- weight of evidence
- Study period:
- 2013-02-20
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The combination of the individual test methods into the test battery evaluation is performed as described in the following publication:
-Bauch C Knolle SN, Ramirez T, Eltze T, Fabian E, Mehling A, Teubner W, van Ravenzwaay B, Landsiedel R., 2012. Putting the parts together: combining in vitro methods to test for skin sensitizing potentials. Regul Toxicol Pharmacol. 63: 489-504. - GLP compliance:
- yes
- Remarks:
- Expert Statement (All studies were conducted in accordance with GLP.)
- Type of study:
- other: Expert Statement
- Details on test animals and environmental conditions:
- The data used for the evaluation of the in vitro sensitization potential of the test item were generated in separate in vitro sensitization studies:
- Direct Peptide Reacitivity Assay (DPRA)
- Keratinocyyte Activation Assay- LuSens
- Dendritic Cell Line Acitivation Assay Myeloid U937 Skin Sensitization Test (MUSST) - Run / experiment:
- other: DPRA
- Parameter:
- other: peptide depletion [%]
- Value:
- 0.2
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: LuSens
- Parameter:
- other: luciferase activity induction
- Value:
- 1.5
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: MUSST
- Parameter:
- other: CD86 expression induction
- Value:
- 1.2
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- Based on the results and applying the evaluation criteria the test substance is predicted not to be a skin sensitizer.
- Executive summary:
A combination of several in vitro methods addressing key steps of the adverse outcome pathway (AOP) for skin sensitization as defined by OECD, has been conducted to assess the skin sensitizing potential of the test substance,
- protein reactivity (DPRA),
- activation of keratinocytes (LuSens), and
- activation of dendritic cells (MUSST).
In this report, the results of the individual studies are summarized and evaluated to predict the presence or absence of skin sensitizing potential of the test substance.
The combination of test methods and the evaluation of their results has been evaluated and published by Bauch et al., 2012. Based on the performance standards of the OECD test guideline No. 429 (Local Lymph Node Assay, LLNA, OECD 2010), the evaluation based on the DPRA, LuSens and MUSST methods yields an overall accuracy of 95 % compared to results in humans (for comparison: for the same data set the LLNA yielded an overall accuracy of 86 %). A skin sensitizing (quantitative) potency assessment using the reported results was not validated at the time of writing of this report.
Based on the results and applying the evaluation critera, the test substance is predicted not to be a skin sensitizer.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2012-04-23 to 2012-05-09
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- There are no official national or international guidelines for the DPRA Test; however, the study is performed according to the methods described in the following publications:
- Gerberick GF, Vassallo JD, Bailey RE, Chaney JG, Morrall SW, Lepoittevin JP. Development of a Peptide Reactivity Assay for Screening Contact Allergens. Toxicological Sciences 81,332-343, 2004.
- Gerberick GF, Vassallo JD, Foertsch LM, Price BB, Chaney JG, Lepoittenvin JP. Quantificationn of Chemical Peptide Reactivity for Screening Contact Allergens: A Classification Tree Model Approach. Toxicological Sciences 97(2), 417-427, 2007.
- Bauch C, Kolle SN, Fabian E, Pachel C, Ramirez T, Wiench B, Wruck CJ, van Ravenzwaay B, Landsiedel R. Intralaboratory validation of four in vitro assays for the prediction of the skin sensitizing potential of chemicals. Toxicology in Vitro 25, 1162 – 1168, 2011.
- Maxwell G, Aeby P, Ashikaga T, Bessou-Touya S, Diembeck W, Gerberick F, Kern P, Marrec-Fairley M, Ovigne JM, Sakaguchi H, Schroeder K, Tailhardat M, Teissier S, Winkler P. Skin sensitisation: the Colipa strategy for developing and evaluating nonanimal test methods for risk assessment. ALTEX 28(1): 50-5, 2011. - GLP compliance:
- yes (incl. QA statement)
- Remarks:
- BASF SE
- Type of study:
- other: Direct Peptide Reactivity Assay (DPRA)
- Details on the study design:
- Skin sensitisation (In vitro test system) - Details on study design:
CONTROLS
- Negative Control (NC): vehicle control = isopropanol; acetonirile was performed as an additional vehicle control for the PC EGDMA
- Positive Control (PC): Ethylene glycol dimethacrylate (EGDMA, prepared as a 50 mM solution in isopropanol and in acetonitrile), p-Benzoquinone (puriss., prepared as a 100 mM solution in isopropanol); as EGDMA may result in different peptide depletions when formulated in the different vehicles the standard vehicle acetonitrile was used additionally to the vehicle for test substance formulation
TEST SUBSTANCE PREPARATION
- The test substance solutions were prepared within 4 hours of performing the assay (preparation of samples). The test substance was prepared as a 100 mM solution in isopropanol. After short shaking the test substance was soluble in the vehicle.
- Vehicle: isopropanol
EXPERIMENTAL PROCEDURE
- The test substance was solved in a suitable vehicle. Three samples of the test substance were incubated with each peptide. Additionally triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. In addition calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test substance incubation, were measured in parallel with the same analytical method.
- Test substance solubility: Prior to the assay the solubility of the test substance was tested. A suitable non-reactive, water-miscible solvent which dissolves the test substance completely should be used. The preffered solvent was acetonitrile. When not soluble in acetonitrile, deionized water, methanol, propanol, isopropanol, acetone or mixtures of these solvents were tried.
- Preparation of peptide stock solutions: Peptide stock solutions in a concentration of 0.667 mM of peptide were prepared in pH 7.5 phosphate buffer (C-containing peptide) or pH 10.2 ammonium acetate buffer (K-containing peptide). The peptide stock solution was used for preparing the calibration samples and the test substance and control samples.
- Preparation of the test substance samples: The samples were prepared in triplicates for each peptide. The test substance was incubated with the C-containing peptide in a ratio of 1 : 10 (0.5 mM peptide, 5 mM test substance) and with the K-containing peptide in a ratio of 1 : 50 (0.5 mM peptide, 25 mM test substance). The samples were prepared in suitable tubes, capped tightly and incubated at room temperature in the dark for 24 +/- 2 hours. Prior to HPLC analysis the samples were visually investigated for any precipitate that may have formed during the exposure period. As the samples were visually turbid or displayed precipitates they were centrifuged (samples of the K-peptide) or centrifuged and filtrated (samples of the C-peptide) prior to injection into the HPLC in order to remove any unsolved particles. The HPLC analysis of the batch of samples started about 24 hours after sample preparation and the analysis time itself did not exceed 30 hours.
- Preparation of the vehicle controls: Several isopropanol controls were prepared in triplicates in the same way as the test substance samples described above but with isopropanol instead of the test substance: One set (set A) was analyzed together with the calibration samples without incubation and serves as a performance control. Another three sets (two sets B and set C) were prepared and incubated with the samples. Sets B were placed at the very start and ending of the sample list and serves as stability control of the peptide over the analysis time. Set C was analyzed with the samples and serves for calculation of the peptide depletion of any chemical formulated in isopropanol. As one set of the PC EGDMA was formulated in acetonitrile a respective set of vehicle controls (set C) was analyzed with the samples and was used for calculation of peptide depletion.
- Preparation of the co-elution control: The co-elution control was prepared in the same way as the test substance samples described above but without the peptide. Instead the respective peptide buffer was used. The samples were analyzed together with the calibration samples. As the samples were visually turbid or displayed precipitates they were centrifuged prior to injection into the HPLC in order to remove any unsolved particles. - Run / experiment:
- mean
- Parameter:
- other: cysteine-peptide depletion [%]
- Value:
- 25.5
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Run / experiment:
- mean
- Parameter:
- other: lysine-peptide depletion [%]
- Value:
- 0
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Run / experiment:
- mean
- Parameter:
- other: mean peptide depletion [%]
- Value:
- 12.8
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Other effects / acceptance of results:
- When mixed with the peptide stock solutions the samples became cloudy directly after preparation. After 24 hours precipitates were noticed in the samples with the C-peptide, additionally.
Thus all samples were centrifuged prior to HPLC analysis. The samples containing the C-peptide were additionally filtered as a separation could not be achieved by centrifugation.
No co-elution of the test substance and peptides occured as demonstrated by the consistent values of the area ratios 220/258. - Interpretation of results:
- other: low chemical reactivity
- 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 and applying the prediction model proposed in Gerberick et al. (2007) it was concluded that the test substance shows a low chemical reactivity in the DPRA under the test conditions chosen. - Executive summary:
The reactivity of the test substance towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose the test substance was incubated with synthetic peptides for ca. 24 hours at room temperature and the remaining non-depleted peptide concentrations were determined thereafter by high performance liquid chromatography with gradient elution and UV-detection at 220 nm.
The test substance was dissolved at a 100 mM concentration in isopropanol. Three samples of the test substance were incubated with each peptide in ratios of 1 : 10 (for C-peptide) or 1 : 50 (for K-peptide). Additionally triplicates of the concurrent vehicle control (= NC) were incubated with the peptides.
Additionally, a co-elution control was performed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover the samples were additionally analyzed by measuring UV absorbance at 258 nm and the area ratio 220 / 258 was calculated as a measure of peak purity.
The following results were obtained in the DPRA:
The test substance was soluble in isopropanol. However when mixed with the peptide stock solutions the samples became cloudy directly after preparation. After 24 hours precipitates were noticed in the samples with the C-peptide, additionally.
Thus all samples were centrifuged prior to HPLC analysis. The samples containing the C-peptide were additionally filtered as a separation could not be achieved by centrifugation.
The mean C-peptide depletion, caused by the test substance was determined to be 25.5 %. The mean K-peptide depletion, caused by the test substance was determined to be - 0.2 %.
Negative depletions were considered to be "zero" for calculation of the mean peptide depletion, which was thus calculated to be 12.8 %.
No co-elution of test substance and peptides was noticed.
Based on the observed results and applying the prediction model proposed in Gerberick et al. (2007) it was concluded that the test substance shows a low chemical reactivity in the DPRA under the test conditions chosen.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2012-05-07 to 2012-06-29
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- 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)
- Remarks:
- BASF AG
- Type of study:
- other: In vitro sensitization: Dendritic Cell Line Activation Assay; Myeloid U 937 Skin Sensitization Test (MUSST)
- Details on the study design:
- Skin sensitisation (In vitro test system) - Details on study design:
CELL LINE
- U937
ANTIBODIES
- FITC Mouse anti-human CD86
- IgG1 FITC CD86
VIABILITY MARKER
- Propidium iodide
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. Further concentrations were prepared as 2 x concentrations by serial dilution.
- Vehicle: Culture medium (according to the physiological conditions)
- Form of application: Emulsion in culture medium 285.6 µg/mL onward
- 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. If contradictory results were obtained in the first and second experiments, a third expereiment was conducted.
- 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. - Run / experiment:
- other: 0.5 µg/mL
- Parameter:
- other: % PI negtive cells
- Value:
- 99.47
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 1 µg/mL
- Parameter:
- other: % PI negative cells
- Value:
- 99.44
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 5 µg/mL
- Parameter:
- other: % PI negative cells
- Value:
- 99.32
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 10 µg/mL
- Parameter:
- other: % PI negative cells
- Value:
- 99.33
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 50 µg/mL
- Parameter:
- other: % PI negative cells
- Value:
- 99.2
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 100 µg/mL
- Parameter:
- other: % PI negative cells
- Value:
- 99.21
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 500 µg/mL
- Parameter:
- other: % PI negative cells
- Value:
- 81.65
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 1000 µg/mL
- Parameter:
- other: % PI negative cells
- Value:
- 31.63
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: precipitation of test substance after 48 h incubation
- Run / experiment:
- other: 2000 µg/mL
- Parameter:
- other: % PI negative cells
- Value:
- 21.2
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: precipitation of test substance after 48 h incubation
- Interpretation of results:
- other: no 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 not induce dendritic cell activation in the MUSST under the test conditions chosen. - Executive summary:
The potential of the test substance to induce the cell membrane marker CD86 expression 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.
The test substance was an emulsion in culture medium at a concentration 50 µg/mL onward. Precipiation of test substance after 48 hours occured at 1000 µg/mL onward.
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. The CV75 value (= estimated concentration that affords 75 % cell viability) was determined by linear regression from the concentration response curve to be 571.2 µg/mL.
In the main test, test substance was used at six final concentrations determined with regard to the CV75 value: CV75x2, CV75, CV75/2, CV75/4, CV75/8, CV75/16. After 48 hour exposure U937 cells were stained with FITC labeled anti-human-CD86 antibody and propidium iodode 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 not soluble at Topdose in any of the vehicles used for the assay. Thus a homogeneous emulsion in culture medium was used for sample preparation. Up to 142.8 µg/mL the dilutions were solutions. At 285.6 µg/mL onward the dilutions of the test substance were emulsions.
In summary, after 48 hours of exposure to test substance CD86 expression was not induced in U937 cells at concentration affording at least 70 % viability. From this it has to be concluded that test substance does not induce dendritic cell activation.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2012-09-05 to 2012-12-07
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- There are no official national or international guidelines for the LuSens Assay; however, the study is performed according to the methods described in the following publication:
Bauch C, Kolle SN, Ramirez T, Eltze T, Fabian E, Mehling A, Teubner W, van Ravenzwaay B, Landsiedel R, (2012), Putting the parts together: Combining in vitro methods to test for skin sensitizing potentials, Regul Toxicol Pharmacol, 63(3):489-504. - GLP compliance:
- yes (incl. QA statement)
- Remarks:
- BASF SE
- Type of study:
- other: in vitro LuSens
- Details on the study design:
- Skin sensitisation (In vitro test system) - Details on study design:
CELL LINE
- LuSens, transgenic keratinocyte cell line derived from HaCaT cells
CONTROLS
- Negative Control (NC): DL-Lactic acid (LA 450 µg/mL)
- Positive Control (PC): Ethylene glycol dimethacrylate (EGDMA 15 µg/mL)
- Vehicle Control: 1 % DMSO in culture medium
- Blank Control: Medium without cells
- Basal Control: Medium with cells
TEST SUBSTANCE PREPARATION
- The test substance preparation was prepared on a weight per volume basis shortly before application by stirring treatment. The test substance was dissolved in DMSO as a 100 x stock solution (= 200 mg/mL) of the highest concentration. Further concentrations were prepared as 100 x concentrations by 1 : 1.2 dilution and thereafter be further diluted (1 : 25) in medium to obtain 4 x concentrations (final DMSO concentration in the test medium = 1 %).
- Vehicle: DMSO (good homogeneity of the preparation)
- Form of application: Emulsion in DMSO and culture medium at 804 µg/mL onward.
- The test substance preparations were prepared within 4 hours of performing the assay (preparation of test substance samples).
EXPERIMENTAL PROCEDURE
- Preparation of the cells: LuSens cells from the working cell bank were thawed and cultured using culture medium 1, under standard culture conditions (37 °C, ca. 5 % CO2, >= 90 % humidity) for at least 2 weeks at passage > 5 but not longer than 15 passages prior to testing. For substance incubation, cells were seeded in 96-well microtiter plates (120 µL of 0.83 x 10^5 cells/mL cell suspensions), using culture medium 2 for incubation for 24 hours. As a rule, two independent experiments were performed. In each experiment, three replicates of each treatment were tested.
- Test susbtance preparation and application of MTT and Luciferase Assay: The substance was prepred up to a stock concentration of 2000 µg/mL in DMSO. The final DMSO concentration in the test was adjusted to 1 %. Based on the stock concentration, a 100 x master plate with DMSO was prepared. Based on the 100 x DMSO master plate a 4 x master plate with medium 3 was prepared. After cell adaption for 24 hours cell culture medium was aspirated and replaced with 150 µL medium 3 and 50 µL of each dilution of the 4 x master plate was added to each well. 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. For the luciferase assay a white plate (luminescence compatible plate) was used. In addition a clear plate was treated in parallel for the determination of cell viability.
- 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. - Run / experiment:
- other: 804 µg/mL, 1st Experiment
- Parameter:
- other: Luciferase activity (fold induction)
- Value:
- 6.96
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Run / experiment:
- other: 965 µg/mL, 1st Experiment
- Parameter:
- other: Luciferase activity (fold induction)
- Value:
- 6.54
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Run / experiment:
- other: 1157 µg/mL, 1st Experiment
- Parameter:
- other: Luciferase activity (fold induction)
- Value:
- 8.64
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Run / experiment:
- other: 1389 µg/mL, 1st Experiment
- Parameter:
- other: Luciferase activity (fold induction)
- Value:
- 7.51
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Run / experiment:
- other: 1667 µg/mL, 1st Experiment
- Parameter:
- other: Luciferase activity (fold induction)
- Value:
- 5.94
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Run / experiment:
- other: 2000 µg/mL, 1st Experiment
- Parameter:
- other: Luciferase activity (fold induction)
- Value:
- 9.76
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Run / experiment:
- other: 804 µg/mL, 2nd Experiment
- Parameter:
- other: Luciferase activity (fold induction)
- Value:
- 7.34
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Run / experiment:
- other: 965 µg/mL, 2nd Experiment
- Parameter:
- other: Luciferase activity (fold induction)
- Value:
- 7.46
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Run / experiment:
- other: 1157 µg/mL, 2nd Experiment
- Parameter:
- other: Luciferase activity (fold induction)
- Value:
- 9.19
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Run / experiment:
- other: 1389 µg/mL, 2nd Experiment
- Parameter:
- other: Luciferase activity (fold induction)
- Value:
- 5.32
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Run / experiment:
- other: 1667 µg/mL, 2nd Experiment
- Parameter:
- other: Luciferase activity (fold induction)
- Value:
- 5.65
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Run / experiment:
- other: 2000 µg/mL, 2nd Experiment
- Parameter:
- other: Luciferase activity (fold induction)
- Value:
- 9.42
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Interpretation of results:
- other: induction of luciferase activity
- 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 subsance does induce luciferase activity in LuSens cells under the test conditions chosen. - Executive summary:
The keratinocyte activating potential of the test substance was evaluated in the LuSens assay. For this purpose the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37 °C and incubation was measured in a luminometer.
The test substance was a homomgenous emulsion in 1 % DMSO at a concentration of 2000 µg/mL (100 x stock solution).
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 (7.8 µg/mL up to 2000 µg/mL) and cytotoxicity was determined thereafter by MTT assay. No decrease in cell viability below 70 % was observed.
In the main test, test substance was used at six final concentrations. After 48 hour exposure luciferase activity was measured in a luminometer. A test substance was concluded to exhibit a keratinocyte activating potential when the luciferase activity exceeds 1.5 fold induction with respect to the vehicle control and at concentrations that did not reduce a viability below 70 %. In parallel a MTT assay was performed to assess cytotoxicity of the test substance.
The LuSens showed the following results:
The test substance was not soluble in any of the vehicles used for the assay. Thus a homogeneous emulsion in 1 % DMSO was used for sample preparation.
The dilutions of the test substance were emulsions. However, after 48 hours no precipitates were noticed in the samples; the test substance preparation appeared as homogeneous emulsion.
In summary, after 48 hours of exposure to test substance luciferase activity in LuSens cells was induced at concentration between 804 and 2000 µg/mL affording at least 70 % viability. From this it has to be concluded that test substance has an keratinocyte activating potential.
- Endpoint:
- skin sensitisation, other
- Remarks:
- Expert Statement
- Type of information:
- other: Expert Statement
- Adequacy of study:
- weight of evidence
- Study period:
- 2013-02-20
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The combination of the individual test methods into the test battery evaluation is performed as described in the following publication:
-Bauch C Knolle SN, Ramirez T, Eltze T, Fabian E, Mehling A, Teubner W, van Ravenzwaay B, Landsiedel R., 2012. Putting the parts together: combining in vitro methods to test for skin sensitizing potentials. Regul Toxicol Pharmacol. 63: 489-504. - GLP compliance:
- yes
- Remarks:
- Expert Statement (All studies were conducted in accordance with GLP.)
- Type of study:
- other: Expert Statement
- Details on test animals and environmental conditions:
- The data used for the evaluation of the in vitro sensitization potential of the test item were generated in separate in vitro sensitization studies:
- Direct Peptide Reacitivity Assay (DPRA)
- Keratinocyyte Activation Assay- LuSens
- Dendritic Cell Line Acitivation Assay Myeloid U937 Skin Sensitization Test (MUSST) - Run / experiment:
- other: DPRA
- Parameter:
- other: peptide depletion [%]
- Value:
- 12.8
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Run / experiment:
- other: LuSens
- Parameter:
- other: luciferase activity induction
- Value:
- 1.5
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Run / experiment:
- other: MUSST
- Parameter:
- other: CD86 expression induction
- Value:
- 1.2
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Interpretation of results:
- Category 1 (skin sensitising) based on GHS criteria
- Conclusions:
- Based on the results and applying the evaluation criteria the test substance is predicted to be a skin sensitizer.
- Executive summary:
A combination of several in vitro methods addressing key steps of the adverse outcome pathway (AOP) for skin sensitization as defined by OECD, has been conducted to assess the skin sensitizing potential of the test substance,
- protein reactivity (DPRA),
- activation of keratinocytes (LuSens), and
- activation of dendritic cells (MUSST).
In this report, the results of the individual studies are summarized and evaluated to predict the presence or absence of skin sensitizing potential of the test substance.
The combination of test methods and the evaluation of their results has been evaluated and published by Bauch et al., 2012. Based on the performance standards of the OECD test guideline No. 429 (Local Lymph Node Assay, LLNA, OECD 2010), the evaluation based on the DPRA, LuSens and MUSST methods yields an overall accuracy of 95 % compared to results in humans (for comparison: for the same data set the LLNA yielded an overall accuracy of 86 %). A skin sensitizing (quantitative) potency assessment using the reported results was not validated at the time of writing this report.
Based on the results and applying the evaluation criteria the test substance is predicted to be a skin sensitizer.
Referenceopen allclose all
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.
The test substance was not soluble in one of the vehicles used for the assay. Thus a homogeneous suspension in acetone was used for sample preparation. When mixed with the peptide stock solution the samples became cloudy directly after preparation. Additionally the test substance precipitated in all samples. Thus all samples were centrifuged prior to HPLC analysis. In a first test run the mean C-peptide depletion, caused by the test substance was determined to be 18%. However, due to high standard deviation (17.4) and irregularities during centrifugation of the samples the test run was considered to be invalid and was repeated. The mean C-peptide depletion, caused by the test substance was determined to be 0.3% in the second test run. The mean K-peptide depletion, caused by the test substance was determined to be -1.9%. Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 0.2%. No co-elution of test substance and peptides was noticed.
Behenylacrylate (Acrylate 22 45%) shows a minimal chemical reactivity in the DPRA under the test conditions chosen.
for deteils see "Overall remarks, attachments"
The test substance was soluble in isopropanol. However when mixed with the peptide stock solutions the samples became cloudy directly after preparation. After 24 hours precipitates were noticed in the samples with the C-peptide, additionally. Thus all samples were centrifuged prior to HPLC analysis. The samples containing the C-peptide were additionally filtered as a separation could not be achieved by centrifugation.
The mean C-peptide depletion, caused by the test substance was determined to be 25.5%.
The mean K-peptide depletion, caused by the test substance was determined to be -0.2%.
Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 12.8%.
No co-elution of test substance and peptides was noticed.
Based on the observed results and applying the prediction model proposed in Gerberick et. al (2007), it was concluded that the test substance shows a low chemical reactivity in the DPRA under the test conditions chosen.
The test substance was not soluble at Topdose in any of the vehicles used for the assay. Thus a homogeneous emulsion in culture medium was used for sample preparation.
Up to 142.8 μg/mL the dilutions were solutions. At 285.6 μg/mL onward the dilutions of the test substance were emulsions.
In summary, after 48 hours of exposure to the test substance CD 86 expression was not induced in U937 cells at concentration affording at least 70% viability. From this it has to be concluded that the test substance does not induce dendritic cell activation.
The test substance was not soluble in any of the vehicles used for the assay. Thus a homogeneous emulsion in 1 % DMSO was used for sample preparation. The dilutions of the test substance were emulsions. However, after 48 hours no precipitates were noticed in the samples; the test substance preparation appeared as homogenous emulsion.
In summary, after 48 hours of exposure to the test substance luciferase activity in LuSens cells was induced at concentration between 804 and 2000 μg/mL affording at least 70% viability. From this it has to be concluded that the test substance has an keratinocyte activating potential.
for deteils see "Overall remarks, attachments"
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (sensitising)
- Additional information:
Read across to C18 -22
The skin sensitizing potential of the substance Behenylacrylate (Acrylate 22 45 %) was investigated in three in vitro tests:
MUSST:
The change in the expression of the cell membrane marker CD 86 induced by Behenylacrylate (Acrylate 22 45%,mixture of CAS no. 4813-57-4, 48076-38-6, 18299-85-9) 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-CD 86 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 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. 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.
DPRA:
The reactivity of Behenylacrylate (Acrylate 22 45%) towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose the test substance was incubated with synthetic peptides for ca. 24 hours at room temperature and the remaining non-depleted peptide concentration was determined thereafter by high performance liquid chromatography with gradient elution and UV-detection at 220 nm. The test substance was formulated at a 100 mM concentration in acetone. One (K-peptide) or two (C-peptide) test runs were performed. Per test run three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-peptide) or 1:50 (for K-peptide). Additionally triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. Additionally, a co-elution control was performed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. The following results were obtained in the DPRA: The test substance was not soluble in one of the vehicles used for the assay. Thus a homogeneous suspension in acetone was used for sample preparation. When mixed with the peptide stock solution the samples became cloudy directly after preparation. Additionally the test substance precipitated in all samples. Thus all samples were centrifuged prior to HPLC analysis. In a first test run the mean C-peptide depletion, caused by the test substance was determined to be 18%. However, due to high standard deviation (17.4) and irregularities during centrifugation of the samples (for details see section 4.1) the test run was considered to be invalid and was repeated. The mean C-peptide depletion, caused by the test substance was determined to be 0.3% in the second test run. The mean K-peptide depletion, caused by the test substance was determined to be -1.9%. Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 0.2%. No co-elution of test substance and peptides was noticed.
LuSens:
The keratinocyte activating potential of Behenylacrylate was evaluated in the LuSens assay. For this purpose the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37 °C and incubation was measured in a luminometer.
The test substance was a emulsion in culture medium at a concentration of 1000 µg/mL (2 x stock solution) onward.
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 MTT assay. No decrease in cell viability below 70 % was observed.
In the main test, test substance was used at six final concentrations. After 48 hour exposure luciferase activity was measured in a luminometer. A test substance was concluded to exhibit a keratinocyte activating potential when the luciferase activity exceeds 1.5 fold induction with respect to the vehicle control and at concentrations that did not reduce a viability below 70 %. In parallel a MTT assay was performed to assess cytotoxicity of the test substance.
The LuSens showed the following results:
The test substance was a emulsion in culture medium at 1157 µg/mL onward. The dilutions of the test substance were emulsions at 1157 µg/mL onward. After 48 hours no precipitates were noticed.
In summary, after 48 hours of exposure to test substance luciferase activity in LuSens cells was not induced at concentration between 804 and 2000 µg/mL affording at least 70 % viability. From this it has to be concluded that the test substance has no keratinocyte activating potential.
Summary Report:
A combination of serval in vitro methods addressing key steps of the adverse outcome pathway (AOP) leading to skin sensitization: peptide reactivity (DPRA), keratinocyte activation (LuSens or KeratinoSens) and dendritic cell activation (MUSST or h-CLAT). The combination of the test methods and the evaluation of their results has been evaluated and published by Bauch et al. 2012. Based on the prformance standards of the OECD test guideline no. 429 (local lymph Node Assay, LLNA), the evaluation based o the DPRA, LuSens and MUSST methods yields an overall accuracy of 95 % compared to results in humans (for comparision: for the same data set the LLNA yielded an overall accuracy of 86%).Behenylacrylate (Acrylate 22 45%) has been assessed in vitro methods addressing major steps of the sensitization process. The results of the invidual studies are summarized in the table below:
Test method
Test result
Test Evaluation
Direct peptide Reactivity Assay (DPRA)
0.2% mean peptide depletion (0.3% cysteine peptide depletion- 1.9% lysine peptide depletion)
Negative
Keratinocyte Activation Assay-LuSens
In at least two independent experiments no ARE-dependent luciferase acitivity induction above 1.5-fold was observed.
Negative
Dendritic Cell Line Activation Assay Myeloid U937 Skin Sensitization Test (MUSST)
In at least two independent experiments an induction of the expression of CD 86 above 1.2-fold was observed at sufficiently non-cytotoxic (cell viability >= 70%) concentration
Positive
The read across substance Behenylacrylate (Acrylate 22 45 %), mixture of CAS no. 4813-57-4, 48076-38-6, 18299-85-9 is predicted not to be a skin sensitizer.
Read across to C16 -18
DPRA:
The reactivity of 2-Propenoic acid, C16-18-alkyl esters towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose the test substance was incubated with synthetic peptides for ca. 24 hours at room temperature and the remaining non-depleted peptide concentrations were determined thereafter by high performance liquid chromatography with gradient elution and UV-detection at 220 nm. The test substance was dissolved at a 100 mM concentration in isopropanol. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-peptide) or 1:50 (for K-peptide). Additionally triplicates of the concurrent vehicle control (= NC) were incubated with the peptides.
Additionally, a co-elution control was performed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover the samples were additionally analyzed by measuring UV absorbance at 258 nm and the area ratio 220 / 258 was calculated as a measure of peak purity.
The following results were obtained in the DPRA:
The test substance was soluble in isopropanol. However when mixed with the peptide stock solutions the samples became cloudy directly after preparation. After 24 hours precipitates were noticed in the samples with the C-peptide, additionally. Thus all samples were centrifuged prior to HPLC analysis. The samples containing the C-peptide were additionally filtered as a separation could not be achieved by centrifugation.
The mean C-peptide depletion, caused by the test substance was determined to be 25.5%.
The mean K-peptide depletion, caused by the test substance was determined to be -0.2%.
Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 12.8%.
No co-elution of test substance and peptides was noticed.
Based on the observed results and applying the prediction model proposed in Gerberick et. al (2007), it was concluded that the test substance shows a low chemical reactivity in the DPRA under the test conditions chosen.
ARE Reporter Assay- LuSens:
The keratinocyte activating potential of the test substance was evaluated in the LuSens assay. For this purpose the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and incubation was measured in a luminometer. The test substance was a homogenous emulsion in 1% DMSO at a concentration of 2000 μg/mL (100x stock solution).
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 (7.8 μg/mL up to 2000 μg/mL) and cytotoxicity was determined thereafter by MTT assay. No decrease in cell viability below 70% was observed.
In the main test, test substance was used at six final concentrations. After 48 hour exposure luciferase activity was measured in a luminometer. A test substance was concluded to exhibit a keratinocyte activating potential when the luciferase activity exceeds 1.5 fold induction with respect to the vehicle control and at concentrations that did not reduce a viability below 70%. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. The LuSens showed the following results:
The test substance was not soluble in any of the vehicles used for the assay. Thus a homogeneous emulsion in 1 % DMSO was used for sample preparation. The dilutions of the test substance were emulsions. However, after 48 hours no precipitates were noticed in the samples; the test substance preparation appeared as homogenous emulsion.
In summary, after 48 hours of exposure to test substance luciferase activity in LuSens cells was induced at concentration between 804 and 2000 μg/mL affording at least 70% viability. From this it has to be concluded that the test substance has an keratinocyte activating potential.
MUSST:
The potential of the test substance to induced the cell membrane marker CD 86 expression 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. The test substance was an emulsion in culture medium at a concentration 50 μg/mL onward. Precipitaton of test substance after 48 hours occurred at 1000 μg/mL onward.
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. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve to be 571.2 μg/mL. In the main test, test substance was used at six final concentrations determined with regard to the CV75 value: CV75x2, CV75, CV75/2, CV75/4, CV75/8, CV75/16. After 48 hour exposure U937 cells were stained with FITC labeled anti-human-CD 86 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 not soluble at Topdose in any of the vehicles used for the assay. Thus a homogeneous emulsion in culture medium was used for sample preparation.Up to 142.8 μg/mL the dilutions were solutions. At 285.6 μg/mL onward the dilutions of the test substance were emulsions. In summary, after 48 hours of exposure to the test substance CD86 expression was not induced in U937 cells at concentration affording at least 70% viability. From this it has to be concluded that the test substance does not induce dendritic cell activation.
Summary Report:
A combination of several in vitro methods addressing key steps of the adverse outcome pathway (AOP) for skin sensitization as defined by OECD, has been conducted to assess the skin sensitizing potential of the test item: peptide reactivity (DPRA), keratinocyte activation (LuSens) and dendritic cell activation (MUSST). In this report, the results of the individual studies are summarized and evaluated to predict the presence or absence of the skin sensitizing potential of the test item.
The combination of the test methods and the evaluation of their results has been evaluated and published by Bauch et al. 2012. Based on the performance standards of the OECD test guideline no. 429 (local lymph Node Assay, LLNA, OECD 2010), the evaluation based on the DPRA, LuSens and MUSST methods yields an overall accuracy of 95 % compared to results in humans (for comparision: for the same data set the LLNA yielded an overall accuracy of 86%). A skin sensitizing (quantitative) potency assessment using the reported results was not validated at the time of writing of this report.
The results of the invidual studies are summarized in the table below:
Test method
Test result
Test Evaluation
Direct peptide Reactivity Assay (DPRA)
12.8% mean peptide depletion (25.5% cysteine peptide depletion- 0.2% lysine peptide depletion)
Positive
Keratinocyte Activation Assay-LuSens
In at least two independent experiments no ARE-dependent luciferase acitivity induction above 1.5-fold at the substance concentrations that did not reduce cell viability below 70% was observed.
Positive
Dendritic Cell Line Activation Assay Myeloid U937 Skin Sensitization Test (MUSST)
In at least two independent experiments an induction of the expression of CD 86 above 1.2-fold was observed.
Negative
In conclusion, the tested analogous substance 2-Propenoic acid, C16-18-alkyl ester is predicted to be a skin sensitizer.
Overall conclusion
Read across has been done to a dataset generated for a mixture of C18-C22 acrylates and to a dataset generated for a mixture of C16 -C18 acrylates. The substance to be registered (hexadecyl acrylate, C16) was part of one of those mixtures. Whilst the latter mixture revealed skin sensitising properties in respective tests, the mixture of C18 -C22 acrylates did not. This demonstrates and shows that the bioavailability and reactivity of the acrylates decrease concurrently with an increasing tail length. Thus, due to the fact that the test item hexadecyl acrylate (C16) represents a borderline substance in the group of long-chain acrylates, and for precautionary reasons 2-Propenoic acid, hexadecyl ester is considerd to be a skin sensitizer.
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008 (GHS Category 1, H317), as amended for the tenth time in Regulation (EC) No. 2017/776. A subcategorization for potency is not possible from the existing experimental data.
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