Icosyl acrylate

Endpoint summary

Description of key information

A combination of several in vitro studies addressing key steps of the adverse outcome pathway (AOP) for skin sensitization as defined by OECD has been conducted with mixtures of long-chain acrylate esters (see table 1).

 

Table 1. Data from skin sensitization testing

Ester	Method	Species	Results	Reference
2-Propenoic acid, C12-14-alkyl esters [84238-60-8]	DPRA LuSens MUSST	in vitro	Sensitising	BASF SE 2013
2-Propenoic acid, C16-18-alkyl esters [90530-21-5]	DPRA LuSens MUSST	In vitro	sensitising	BASF SE 2013
2-Propenoic acid, C18-22-alkyl esters [85085-17-2]	DPRA LuSens MUSST	In vitro	not sensitising	BASF SE 2013

 

Conclusion

In conclusion, the long-chain alkyl acrylate esters (C12 – C22) are expected to be absorbed through the skin to a very low extend. Whilst the mixtures 2-Propenoic acid, C12-14-alkyl esters and 2-Propenoic acid, C16-18-alkyl esters revealed skin sensitizing properties in respective tests, the mixture 2-Propenoic acid, C18-22-alkyl esters did not. This demonstrates that the bioavailability and reactivity of the acrylates decrease concurrently with an increasing tail length. Thus, for precautionary reasons the long-chain acrylates are considered to be skin sensitizing.

 

The studies using the source substances were considered to be reliable and suitable to fulfil the REACH information requirements of Annex VII, section 8.3.

 

The variable part of the category approach is the length or configuration of the side chain of the parent ester and the alcohol metabolite, as well as their impacts on physico-chemical properties and subsequent properties. Despite these variations, the available data support the similarity in skin sensitisation potency for all the acrylate esters within the category, since data is availablefor the shortest and longest chain lengths present in the category. Skin sensitisation potency of acrylate esters involves reaction with tissue nucleophiles via Michael addition on the electrophilic C of the a,β-unsaturated carboxyl group (Freidig et al., 1999; Greim et al., 1995; McCarthy et al., 1994; cited in Borak et al., 2011). The prototype for such reactions is conjugation with GSH, which occurs spontaneously and enzymatically, leading to formation of thioethers and mercapturic acids. Increased urinary excretion of thioethers and depletion of hepatocyte GSH have been documented following in vivo and in vitro exposures to acrylate esters (Delbressine et al., 1981; Elovaara et al., 1983; cited in Borak et al., 2011). In skin sensitisation studies, a key early step in the process leading to sensitisation is the formation of covalent adducts with a carrier protein, thereby forming an antigenic hapten-protein complex (Natsch and Emter, 2008; Roberts et al., 2008; Roberts and Aptula, 2008; Smith and Hotchkiss, 2001; cited in Borak et al., 2011). Overall, the read across approach is applied with a high level of confidence.

 

A confirmatory skin sensitization tests with octadecyl acrylate (CAS 4813-57-4) is proposed to strengthen the read across.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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.

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.

Reference 2

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.

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.

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.

Reference 3

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.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Principles of method if other than guideline:

In vitro Sensitization: Direct Peptice Reactivity Assay (DPRA)

GLP compliance:

yes

Remarks:

non-GLP, but study conducted under similar Quality assurance system.

Type of study:

other: Direct Peptice Reactivity Assay (DPRA)

Details on the study design:

The test substance was solved at a 100 mM concentration in acetonitrile. 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. The peptide depletion of test-substance incubated samples was compared to the peptide depletion of the NC samples and expressed as relative peptide depletion. For the test substance the mean peptide depletion as average of C- and K-peptide depletion is calculated and used for evaluation of the chemical reactivity.

The study is performed according to the methods described in the following publications:
Bauch C, Kolle SN, Ramirez-Hernandez T, Eltze T, Fabian E, Teubner W, Mehling A, van Ravenzwaay B, Landsiedel R. Putting the parts together: Combining in vitro methods to test for skin sensitizing. Regulatory Toxicology and Pharmacology 63(3), 489-504, 2012. 2 Gerberick GF, Vassallo JD, Foertsch LM, Price BB, Chaney JG, Lepoittevin JP. Quantification of Chemical Peptide Reactivity for Screening Contact Allergens: A Classification Tree Model Approach. Toxicological Sciences 97(2), 417-427, 2007.

Run / experiment:

other: Cysteine-Peptide

Parameter:

other: mean peptide depletion [%]

Value:

30.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: moderate reactivity

Run / experiment:

other: Lysine-Peptide

Parameter:

other: mean peptide depletion [%]

Value:

2.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: minimal reactivity

Run / experiment:

other: mean of both depletions

Parameter:

other: mean peptide depletion [%]

Value:

16.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: low reactivity

The test substance was solved in acetonitrile. However, when mixed with the peptide stock solutions the samples became opaque. Visual observation after the 24-hour incubation time revealed precipitates in all samples. Based on the observed results and applying the prediction model proposed in Geberick et. al (2007) it was concluded that Laurylacrylate 1214 shows low chemical reactivity in the DPRA under the conditons chosen.

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.

Reference 5

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

In vitro sensitization: Dendritic Cell Line Activation Assay; Myeloid U 937 Skin Sensitization Test (MUSST)

GLP compliance:

yes

Remarks:

non-GLP, but study conducted under similar Quality assurance system.

Type of study:

other: In vitro sensitization: Dendritic Cell Line Activation Assay; Myeloid U 937 Skin Sensitization Test (MUSST)

Details on the study design:

- Cell line U937
- Vehicle: culture medium
- The myeloid U937 skin sensitization test is a dendritic cell activation test to predict skin sensitizing potential. The test is performed using the human pro-monocytic cell line U937 as surrogate fer dendritic cells. As readout, the change in the expression of the cell membrane marker GD 86 measured by Iow cytometry after 48 hours of test substance exposure is determined. A test substance is predicted to activate dendritic cells when GD86 cell surface expression exceeds the threshold in relation to vehicle control in at least two independent experiments.
- Positive control substance: Ethylene diamine (EDA – 70 μg/mL)
- The cytotoxicity of the test substance was evaluated by flow cytometry using propidium iodide staining after 48 hours exposure. For the purpose the CV75 value was derived from the concentration response curve. The CV75 is the estimated concentration that affords 75% cell viability and was determined to be 1297.8 µg/mL for Laurylacrylate 1214 under the chosen exposure conditions on U937 cells. In the main test, test substance was assessed at six final concentrations 2595.6 µg/mL, 1297.8 µg/mL, 648.9 µg/mL, 350.0 µg/mL and 324.0 µg/mL to be 162.2 µg/mL for Laurylacrylate 1214 under the chosen exposure conditions on U937 cells.
After 48 hours of 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 two experiments.
The strong sensitizer ethylene diamine (EDA, 70 µg/mL) was used as positive and lactic acid (LA, 200 µg/mL) as non-sensitizing negative control.

Run / experiment:

other: 162.2 µg/mL, 1st Experiment

Parameter:

other: CD86 fold induction

Value:

1.12

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 324.5 µg/mL, 1st Experiment

Parameter:

other: CD86 fold induction

Value:

1.23

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 350.0 µg/mL, 1st Experiment

Parameter:

other: CD86 fold induction

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not measured/tested

Run / experiment:

other: 648.9 µg/mL, 1st Experiment

Parameter:

other: CD86 fold induction

Value:

1.32

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 1297.8 µg/mL, 1st Experiment

Parameter:

other: CD86 fold induction

Value:

1.49

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 2595.6 µg/mL, 1st Experiment

Parameter:

other: CD86 fold induction

Value:

1.52

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 162.2 µg/mL, 2nd Experiment

Parameter:

other: CD86 fold induction

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: 324.5 µg/mL, 2nd Experiment

Parameter:

other: CD86 fold induction

Value:

1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 350.0 µg/mL, 2nd Experiment

Parameter:

other: CD86 fold induction

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not measured/tested

Run / experiment:

other: 648.9 µg/mL, 2nd Experiment

Parameter:

other: CD86 fold induction

Value:

1.26

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 1297.8 µg/mL, 2nd Experiment

Parameter:

other: CD86 fold induction

Value:

0.54

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 2595.6 µg/mL, 2nd Experiment

Parameter:

other: CD86 fold induction

Value:

0.91

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 162.2 µg/mL, 3rd Experiment

Parameter:

other: CD86 fold induction

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not measured/tested

Run / experiment:

other: 324.5 µg/mL, 3rd Experiment

Parameter:

other: CD86 fold induction

Value:

0.94

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 350.0 µg/mL, 3rd Experiment

Parameter:

other: CD86 fold induction

Value:

0.98

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 648.9 µg/mL, 3rd Experiment

Parameter:

other: CD86 fold induction

Value:

1.26

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 1297.8 µg/mL, 3rd Experiment

Parameter:

other: CD86 fold induction

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not measured/tested

Run / experiment:

other: 2595.6 µg/mL, 3rd Experiment

Parameter:

other: CD86 fold induction

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not measured/tested

In summary, after 48 hours of exposure to test substance Laurylacrylate 1214 CD 86 expression was not induced in U937 cells at concentration between 162.2 to 2595.6 µg/mL affording at least 70% viability. From this it has to be concluded that test substance Laurylacrylate 1214 does not induce dendritic cell activation.

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.
In summary, after 48 hours of exposure to the test substance CD86 expression was not induced in U937 cells at concentration between 162.2 to 2595.6 µg/mL affording at least 70 % viability in more than one experiment. From this it has to be concluded that the test substance does not induce dendritic cell activation.

Reference 6

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

In vitro Test LuSens (Bauch et al. 2012)

GLP compliance:

yes

Remarks:

non-GLP, but study conducted under similar Quality assurance system.

Type of study:

other: in vitro LuSens

Details on the study design:

The cell line LuSens was treated with 6 test substance concentrations for 48 hours in at least two independent experiments with each 3 replicates. Cells were lysed and luciferase induction was evaluated by measuring luminescence signal after substrate addition (Steady Glo®, Promega). ln parallel a MTT assay was performed to assess cytotoxicity of the test substance. A test substance was considered to have an ARE induction potential if the fold induction of luciferase activity was > 1.5 and viability determined in the MTT assay was > 70% at any test concentration.

MTT assay:
The assay is based on the tetrazolium salt MTT. ln active mitochondria the tetrazolium ring of yellow MTT is cleaved by various dehydrogenase enzymes and a purple formazan is formed. Gell culture medium was aspirated from all wells and 180 ~L 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 will be 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 lysed by adding 100 ~L of Iysis solution (99.6 mL DMSO; 10 g sodium dodecyl sulfate, SOS; and 0.4 ml glacial acetic acid). Absorbance was
measured at 570 nm with reference wavelength at 590 nm 690 nm using the Sunrise TM Absorbance Reader.

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 weil were added and cells shaked with a plate shaker for 10 min at room temperature in darkness. After the incubation the luminescence was measured in a luminometer.

Run / experiment:

other: 6.82 µg/mL, 1st Experiment

Parameter:

other: luciferase activity fold induction

Value:

4.29

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 8.18 µg/mL, 1st Experiment

Parameter:

other: luciferase activity fold induction

Value:

4.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 9.81 µg/mL, 1st Experiment

Parameter:

other: luciferase activity fold induction

Value:

4.71

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 11.78 µg/mL, 1st Experiment

Parameter:

other: luciferase activity fold induction

Value:

4.47

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 14.13 µg/mL, 1st Experiment

Parameter:

other: luciferase activity fold induction

Value:

4.01

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 16.96 µg/mL, 1st Experiment

Parameter:

other: luciferase activity fold induction

Value:

2.81

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 6.82 µg/mL, 2nd Experiment

Parameter:

other: luciferase activity fold induction

Value:

8.09

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 8.18 µg/mL, 2nd Experiment

Parameter:

other: luciferase activity fold induction

Value:

9.58

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 9.81 µg/mL, 2nd Experiment

Parameter:

other: luciferase activity fold induction

Value:

8.25

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 11.78 µg/mL, 2nd Experiment

Parameter:

other: luciferase activity fold induction

Value:

6.11

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 14.13 µg/mL, 2nd Experiment

Parameter:

other: luciferase activity fold induction

Value:

4.59

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 16.96 µg/mL, 2nd Experiment

Parameter:

other: luciferase activity fold induction

Value:

4.57

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

ln summary, after 48 hours of exposure to test substance Laurylacrylate 1214 luciferase activity in LuSens cells was induced at concentration between 6.82 and 9.81 µg/mL affording at least 70% viability. From this it has to be concluded that test substance Laurylacrylate 1214 has an keratinocyte activating potential.

Interpretation of results:

other: keratinocyte activating potential

Conclusions:

No prediction can be made for skin sensitization according to GHS criteria based on the results of this in vitro study alone.
In summary, after 48 hours of exposure to the test substance luciferase activity in LuSens cells was induced at concentration between 6.82 and 9.81 µg/mL affording at least 70 % viability. From this it has to be concluded that the test substance has an keratinocyte activating potential.

Reference 7

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.

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.

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.

Reference 8

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

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.

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.

Reference 9

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

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.

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 conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

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 with 2-Propenoic acid, C12-14-alkyl esters to assess the skin sensitizing potential of this mixture.

 

The substance 2-Propenoic acid, C12-14-alkyl esters was tested in the Direct Peptide Reactivity Assay (DPRA). The test substance was solved at a 100 mM concentration in acetonitrile. 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. The peptide depletion of test substance incubated samples was compared to the peptide depletion of the NC samples and expressed as relative peptide depletion. For the test substance the mean peptide depletion as average of C- and K-peptide depletion is calculated and used for evaluation of the chemical reactivity. The test substance was solved in acetonitrile. However, when mixed with the peptide stock solutions the samples became opaque. Visual observation after the 24-hour incubation time revealed precipitates in all samples. Based on the observed results and applying the prediction model proposed in Geberick et al. (2007) it was concluded that the test substance shows low chemical reactivity in the DPRA under the conditions chosen (BASF SE, 2013).

The test substance was also tested in the LuSens assay. A luciferase reporter cell line (LuSens cells) based on the activation of the antioxidant response element that can be used to assess the keratinocyte activating potential of a substance. The LuSens assay is an in vitro method for the identification of keratinocyte activating substances using the genetically modified keratinocytes. It employs the reporter gene for luciferase under the control of an ARE and hence monitors Nrf-2 transcription factor activity. The endpoint measurement is the upregulation of the luciferase activity after 48 hours incubation with the test substace. After 48 hours of exposure to the test substance luciferase activity in LuSens cells was induced at concentrations between 6.82 and 9.81 µg/mL affording at least 70 % viability. From this it has to be concluded that the test substance has a keratinocyte activating potential (BASF SE, 2013).

 

The substance 2-Propenoic acid, C12-14-alkyl esters was tested in the myeloid U937 skin sensitization test. The test is performed using the human pro-monocytic cell line U937 as surrogate for dendritic cells. As readout, the change in the expression of the cell membrane marker CD86 measured by flow cytometry after 48 hours of test substance exposure is determined. A test substance is predicted to activate dendritic cells when CD86 cell surface expression exceeds the threshold in relation to vehicle control in at least two independent experiments. After 48 hours of exposure to the test substance CD86 expression was not induced in U937 cells at concentrations between 162.2 to 2595.6 µg/mL affording at least 70 % viability in more than one experiment. From this it has to be concluded that the test substance does not induce dendritic cell activation (BASF SE, 2013).

 

Based on the positive result in the DPRA as well as in the LuSens assay, the mixture 2-Propenoic acid, C12-14-alkyl esters is predicted to be a skin sensitizer in thein vitromethods addressing major steps of the sensitization process (BASF SE, 2013).

 

A combination of severalin vitromethods addressing key steps of the adverse outcome pathway (AOP) for skin sensitization as defined by OECD has been conducted with 2-Propenoic acid, C16-18-alkyl esters to assess the skin sensitizing potential of this mixture.

 

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 HPLC 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 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 (BASF SE, 2013).

 

The keratinocyte activating potential of 2-Propenoic acid, C16-18-alkyl esters 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 homogeneous 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 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 hours 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 concentrations between 804 and 2000 µg/mL affording at least 70 % viability. From this it has to be concluded that test substance has a keratinocyte activating potential (BASF SE, 2013).

 

The potential of the mixture 2-Propenoic acid, C16-18-alkyl esters 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. Precipitation 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 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 hours 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 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 the test substance does not induce dendritic cell activation (BASF SE, 2013).

 

Based on the positive result in the DPRA as well as in the LuSens assay, the mixture 2-Propenoic acid, C16-18-alkyl esters is predicted to be a skin sensitizer in thein vitromethods addressing major steps of the sensitization process (BASF SE, 2013).

 

A combination of severalin vitromethods addressing key steps of the adverse outcome pathway (AOP) for skin sensitization as defined by OECD has been conducted with another mixture of long-chain alkyl esters to assess its skin sensitizing potential.

 

The reactivity of 2-Propenoic acid, C18-22-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 concentration was determined thereafter by HPLC 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 (BASF SE, 2013).

 

The keratinocyte activating potential of the 2-Propenoic acid, C18-22-alkyl esters 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 an 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 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 hours 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 an 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 concentrations between 804 and 2000 µg/mL affording at least 70 % viability. From this it has to be concluded that the mixture 2-Propenoic acid, C18-22-alkyl esters has no keratinocyte activating potential (BASF SE, 2013).

 

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 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 hours 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 the test substance CD86 expression was induced in U937 cells at concentrations between 500 and 2000 µg/mL affording at least 70 % viability. From this it has to be concluded that 2-Propenoic acid, C18-22-alkyl esters does induce dendritic cell activation (BASF SE, 2013).

 

Based on the negative result in the DPRA as well as in the LuSens assay, the mixture 2-Propenoic acid, C18-22-alkyl esters is predicted not to be a skin sensitizer in the in vitro methods addressing major steps of the sensitization process (BASF SE, 2013).

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.