1,1-diethoxyethane

Endpoint summary

Description of key information

h-CLAT: The test item induces dendritic cell activation.

LuSens: The test item does not activate keratinocytes.

DPRA: The test item shows low chemical reactivity towards Cys and Lys containing peptides under the test conditions chosen.

In conclusion, the test item is not considered to be skin sensitising.

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:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Experimental procedure of the DPRA:
The test substance was dissolved 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 at a concentration of 100 mM was tested. A suitable non-reactive, water-miscible solvent which dissolves the test substance completely (no visible precipitation or cloudiness of the test-substance preparation) should be used. The preferred solvent was acetonitrile. When not soluble in acetonitrile solutions in water, isopropanol, acetone, propanol, methanol or mixtures of these solvents were tried.

Preparation of peptide stock solutions:
Peptide stock solutions in a concentration of 0.667 mM 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 according to the pipetting scheme given below. The samples were prepared in suitable tubes, capped tightly and incubated at 25 °C ± 2.5 °C in the dark for 24 ± 2 hours. Visual inspection for solubility was performed directly after sample preparation and prior to HPLC analysis. Unsolved samples were centrifuged and/or filtrated prior to injection into the HPLC in order to remove any unsolved particles. The HLPC 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 vehicle controls were prepared in triplicates in the same way as the test-substance samples described above but with the vehicle (acetonitrile) instead of the test substance: One set (set A) was analysed 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 serve as stability control of the peptide over the analysis time. Set C was analysed with the samples and serves for calculation of the peptide depletion of any chemical formulated in the vehicle.

Preparation of the co-elution control:
One sample per peptide was prepared in the same way as the test-substance samples described above but without the peptides. Instead the respective peptide buffer was used. The samples were analysed together with the calibration samples. Samples which were visually turbid or display precipitates were centrifuged and/or filtrated prior to injection into the HPLC in order to remove any unsolved particles.

Synthetic peptides: (DPRA)
Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol)
Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)
The peptides are custom material (Supplier: GenScript, Piscataway, NJ, USA and RS Synthesis, Louisville KY, USA) containing phenylalanine to aid in detection and either cysteine or lysine as the reactive centre.

Controls for the DPRA
Negative control (NC): vehicle control = acetonitrile
Positive control (PC): Ethylene glycol dimethacrylate (EGDMA; CAS-no. 97-90-5), prepared as a 50 mM solution in acetonitrile.
Co-elution control (SK): Sample prepared of the respective peptide buffer and the test substance but without peptide.

Key result

Parameter:

other: mean peptide depletion (%)

Value:

16.81

Remarks on result:

other: show low chemical reactivity

The test substance was dissolved in acetonitrile at a concentration of 100 mM. The samples of the test substance with the peptides were solutions at the time of preparation. Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides.

No co-elution of test substance and peptides was present.

The mean C-peptide depletion, caused by the test substance was determined to be 31.92 %.

The mean K-peptide depletion, caused by the test substance was determined to be 1.70 %.

Thus, the mean peptide depletion was calculated to be 16.81 %.

Conclusions:

Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that 1,1-diethoxyethane shows low chemical reactivity in the DPRA under the test conditions chosen.

Executive summary:

A study according to OECD TG 442C - Direct Peptide Reactivity Assay (DPRA) was conducted under GLP to determine the reactivity of the test item towards model synthetic peptides containing either cysteine or lysine. The test substance was dissolved in acetonitrile as a 100 mM preparation. 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. Peptide stock solutions in a concentration of 0.667 mM were prepared in pH 7.5 phosphate buffer (C-containing peptide - Ac-RFAACAA-COOH) or pH 10.2 ammonium acetate buffer (K-containing peptide - Ac-RFAAKAA-COOH). The peptide stock solution was used for preparing the calibration samples and the test substance and control samples. Negative control (NC), positive control (PC) - Ethylene glycol dimethacrylate (EGDMA) and co-elution control (SK) ran parallel to check the validity of the test. The samples of the test substance with the peptides were solutions at the time of preparation. Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides. No co-elution of test substance and peptides was present. The mean C-peptide depletion, caused by the test substance was determined to be 31.92 %. The mean K-peptide depletion, caused by the test substance was determined to be 1.70 %. Thus, the mean peptide depletion was calculated to be 16.81 %. Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test item shows low chemical reactivity in the DPRA under the test conditions chosen.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442 E (In (Vitro skin Sensitization: human Cell Line Activation Test (h-CLAT))

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Details on the study design:

Experimental procedure of the h-CLAT:
Preparation of the cells:
THP-1 cells from the working cell bank were thawed and cultured in suspension using complete RPMI 1640 medium supplemented with 10 % fetal bovine serum (heat inactivated), 100 U/mL penicillin, 100 μg/mL streptomycin and 0.05 mM 2-mercaptoethanol under standard culture conditions (37 °C, ca. 5 % CO2, ≥ 90 % relative humidity) until for 5 passages but not longer than passage 30 prior to testing. Prior to use of the cells for a study, a reactivity check was performed with each new-thawed cells, as proposed in the OECD test guideline, using Nickel(II)sulfate hexahydrate, lactic acid and 1- chloro-2,4-dinitrobenzene in order to demonstrate qualification of the cells for the assay. For substance incubation, cells were seeded in 24-well plates (500 μL of 2.0 x 10^6 cells/mL cell suspensions). Two independent, valid experiments were performed. In each experiment, duplicates of each test-substance concentration were tested.

Test-substance application:
Treatment was performed by adding 500 μL of test-substance preparation to the cells, thus diluting the 2x concentrated test-substance preparations to their final concentration and the cells to 1.0 x 10^6 cells/mL. After test-substance application the plates were sealed with semipermeable 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 24 ± 0.5 hours.

Visual inspections:
Each test-substance concentration was visually inspected directly after application and after the exposure period of 24 ± 0.5 hours in order to detect test-substance precipitates.

Cell staining and flow cytometric analysis:
After visual inspection the cells were transferred into safe-lock tubes, collected by centrifugation and washed twice with 1 mL buffer. Cells were incubated with 600 μL of 0.01 % Globulins Chon fraction II,III at 4 °C for 15 min to block FC receptors (FcR). After FcR blocking, cells of each treatment condition were divided into 3 aliquots (approximately 0.3 x 10^6 cells/180 μL/group) in 96-well microtiter plates. Cells were centrifuged, supernatant was discarded and 50 μL working antibody solution was added to each pellet.
Cell staining was performed at 4 °C for ca. 30 min in the dark. After staining the cells were washed twice with 200 μL buffer and finally re-suspended in 200 μL buffer. Before analysis in flow cytometer the cells were stained with 5 μL of PI (50 μg/mL diluted in buffer) to yield a final concentration of 1.22 μg/mL PI (Propidium iodide).

Controls for the h-CLAT
Negative control (NC): Lactic acid (LA, CAS no.: 50-21-5), 1000 μg/mL in culture medium
Positive control (PC): 1-chloro-2,4-dinitrobenzene (DNCB, CAS no.: 97-00-7), 4.0 μg/mL in 0.2 % DMSO in culture medium
Vehicle control (VC): 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 mouse IgG1.

h-CLAT:
Cell line: THP-1 cells
The human monocytic leukemia cell line was obtained from “American Type Culture Collection, Manassas, USA” (ATCC, TIB-202).

Key result

Remarks on result:

other: induces dendritic cell activation

At concentrations used in the main experiment the test substance was soluble in culture medium (2 x stock preparations and final concentrations). After 24 hours, the highest concentration was unsolved, only.

The EC150 % (the concentration resulting in a RFI of 150 %) for CD86 was calculated to be 2293 μg/mL (experiment 2). The EC200 % (the concentration resulting in a RFI of 200 %) for CD54 was calculated to be 1911 μg/mL (experiment 2).

Calculation of an EC150 %/EC200 % was not applicable for the 3rd experiment as fold inductions above 150 %/200 % were obtained in the lowest tested concentrations, respectively. In summary, after 24 hours of exposure to test substance CD86 and CD54 expression was induced in THP-1 cells affording at least 50 % viability in at least two independent experiments. From this it has to be concluded that test substance induces dendritic cell activation.

Conclusions:

From this it has to be concluded that the test substance induces dendritic cell activation.

Executive summary:

In an OECD 442E study the potential of the test item to induce dendritic cell activation was tested according to the in vitro human Cell Line Activation Test (h-CLAT). THP-1 cells were prepared and used in two independent, valid experiments. In each experiment, duplicates of each test-substance concentration were tested. Treatment was performed by adding 500 μL of test-substance preparation to the cells, thus diluting the 2x concentrated test-substance preparations to their final concentration and the cells to 1.0 x 10^6 cells/mL. After test-substance application the plates were sealed with semipermeable 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 24 ± 0.5 hours. Each test-substance concentration was visually inspected directly after application and after the exposure period in order to detect test-substance precipitates. After visual inspection the cells were transferred, washed, centrifuged and incubated to block FC receptors (FcR). After FcR blocking, cells of each treatment condition were divided into 3 aliquots in 96-well microtiter plates. Cells were centrifuged, supernatant was discarded and 50 μL working antibody solution was added to each pellet. Cell staining was performed at 4 °C for ca. 30 min in the dark. After staining the cells were washed twice with 200 μL buffer and finally re-suspended in 200 μL buffer. Before analysis in flow cytometer the cells were stained with 5 μL of PI (50 μg/mL diluted in buffer) to yield a final concentration of 1.22 μg/mL PI. Concurently, 4 types of controls were performed - negative, positive, isotype and vehicle, in order to determine the validity of the test method. The EC150 % (the concentration resulting in a RFI of 150 %) for CD86 was calculated to be 2293 μg/mL (experiment 2). The EC200 % (the concentration resulting in a RFI of 200 %) for CD54 was calculated to be 1911 μg/mL (experiment 2). Calculation of an EC150 %/EC200 % was not applicable for the 3rd experiment as fold inductions above 150 %/200 % were obtained in the lowest tested concentrations, respectively. In summary, after 24 hours of exposure to test substance CD86 and CD54 expression was induced in THP-1 cells affording at least 50 % viability in at least two independent experiments. From this it has to be concluded that test substance induces dendritic cell activation.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Experimental procedure of the LuSens
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 % relative humidity) for at least passage ≥5 but not longer than 15 passages prior to testing. Before substance incubation, cells were seeded in 96-well microtiter plates (120 μL of 0.83 x 10^5 cells/mL cell suspension), using culture medium 2 for incubation for ca. 24 hours. Three independent, valid experiments were performed. In each experiment, three replicates of each test-substance concentration were tested.

Test-substance application for MTT and luciferase assay:
After cell adaption for ca. 24 hours, culture medium 2 was aspirated and replaced with 150 μL culture medium 3. Each preparation of the dilution plate was then applied in a ratio of 1:4 (50 μL) to the cells (final DMSO concentration in the test medium = 1%).After test-substance 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 ± 1 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:
Each test-substance concentration was visually inspected directly after application and after the exposure period of 48 ± 1 hours in order to detect 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+). Subsequently 200 μL of One-Glo-preparation (= 100 μL One-Glo- Mix and 100 μL PBS (without Ca2+/Mg2+)) per well was added and cells were shaken on a plate shaker for ca. 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. Thereafter 200 μL of a 0.5 mg/mL thiazolyl blue tetrazolium bromide (MTT) solution (prepared 1:10 from a 5 mg/mL (MTT) stock solution in PBS (without Ca2+/Mg2+) and culture medium 3) was added to each well of the 96-well microtiter plate and incubated for at least further 2 hours 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 690 nm using a spectral-photometer.

Cell line: LuSens
Human transgenic keratinocyte cell line derived from HaCaT cells, prepared in collaboration with Christoph J. Wruck, RWTH Aachen, Germany.

Controls for the LuSens:
Negative control (NC): DL-Lactic acid (LA, CAS no.: 50-21-5), 5000 μM (= 450 μg/mL) in 1 % DMSO in culture medium 3
Positive control (PC): Ethylene glycol dimethacrylate (EGDMA, CAS no.: 97-90-5), 90.8 μM (= 18 μg/mL) in 1 % DMSO in culture medium 3
Vehicle control (VC): 1 % DMSO in culture medium 3
Blank control: culture medium 3 without cells
Basal control: culture medium 3 with cells

Key result

Remarks on result:

other: does not have a keratinocyte activating potential

In the main test luciferase activity was measured after 48-hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 3 valid experiments were performed. One further experiment was conducted but was invalid for not meeting acceptance criteria and is not included in the report. The following results were observed: The test substance was soluble in 4 % DMSO in culture medium 3 (4 x stock preparations) and in 1 % DMSO in culture medium 3 (final concentrations). No precipitates were noticed in any concentration after 48 hours.

Calculation of an EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was not applicable due to the overall negative result of the study.

In summary, after 48 hours of exposure to test substance, luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70 % viability in at least two independent experiments. From this it has to be concluded that test substance does not have a keratinocyte activating potential.

Conclusions:

Based on the observed results and applying the evaluation criteria, the test item does not activate keratinocytes.

Executive summary:

A study according to OECD 442D was perfomed to determine if the test substance activates keratinocytes following the ARE-Nrf2 Luciferase Test (LuSens) method. LuSens cells were cultured using culture medium 1, under standard culture conditions (37 °C, ca. 5 % CO2, ≥ 90 % relative humidity) for at least passage ≥5 but not longer than 15 passages prior to testing. Before substance incubation, cells were seeded in 96-well microtiter plates, using culture medium 2 for incubation for ca. 24 hours. Three independent, valid experiments were performed. In each experiment, three replicates of each test-substance concentration were tested. After cell adaption for ca. 24 hours culture medium 2 was aspirated and replaced with 150 μL culture medium 3. Each preparation of the dilution plate was then applied in a ratio of 1:4 (50 μL) to the cells (final DMSO concentration in the test medium = 1 %). After test-substance 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 ± 1 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. Each test-substance concentration was visually inspected directly after application and after the exposure period of 48 ± 1 hours in order to detect test-substance precipitates. 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+). Subsequently 200 μL of One-Glo-preparation (= 100 μL One-Glo-Mix and 100 μL PBS (without Ca2+/Mg2+)) per well were added and cells were shaken on a plate shaker for ca. 10 min at room temperature in darkness. After the incubation the luminescence was measured in the luminometer.

Cell viability assay was determined by aspiration of the cell culture medium from all wells. Thereafter, 200 μL of a 0.5 mg/mL thiazolyl blue tetrazolium bromide (MTT) solution (prepared 1:10 from a 5 mg/mL (MTT) stock solution in PBS (without Ca2+/Mg2+) and culture medium 3) was added to each well of the 96-well microtiter plate and incubated for at least further 2 hours 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 690 nm using a spectral-photometer. In parallel, a negative control (NC), a positive control (PC), a vehicle control (VC), one blank control and one casal control were performed. The test substance was soluble in 4 % DMSO in culture medium 3 (4 x stock preparations) and in 1 % DMSO in culture medium 3 (final concentrations). No precipitates were noticed in any concentration after 48 hours. Calculation of an EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was not applicable due to the overall negative result of the study.

In summary, after 48 hours of exposure to test substance, luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70 % viability in at least two independent experiments. From this it has to be concluded that test substance does not have a keratinocyte activating potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

h-CLAT

In an OECD 442E study (reference 7.4.1-1) the potential of the test item to induce dendritic cell activation was tested according to the in vitro human Cell Line Activation Test (h-CLAT). THP-1 cells were prepared and used in two independent, valid experiments. In each experiment, duplicates of each test-substance concentration were tested. Treatment was performed by adding 500 μL of test-substance preparation to the cells, thus diluting the 2x concentrated test-substance preparations to their final concentration and the cells to 1.0 x 10^6 cells/mL. After test-substance application the plates were sealed with semipermeable 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 24 ± 0.5 hours. Each test-substance concentration was visually inspected directly after application and after the exposure period in order to detect test-substance precipitates. After visual inspection the cells were transferred, washed, centrifuged and incubated to block FC receptors (FcR). After FcR blocking, cells of each treatment condition were divided into 3 aliquots in 96-well microtiter plates. Cells were centrifuged, supernatant was discarded and 50 μL working antibody solution was added to each pellet. Cell staining was performed at 4 °C for ca. 30 min in the dark. After staining the cells were washed twice with 200 μL buffer and finally re-suspended in 200 μL buffer. Before analysis in flow cytometer the cells were stained with 5 μL of Propidium iodide (50 μg/mL diluted in buffer) to yield a final concentration of 1.22 μg/mL PI. Concurently, 4 types of controls were performed - negative, positive, isotype and vehicle, in order to determine the validity of the test method. The EC150 % (the concentration resulting in a RFI of 150 %) for CD86 was calculated to be 2293 μg/mL (experiment 2). The EC200 % (the concentration resulting in a RFI of 200 %) for CD54 was calculated to be 1911 μg/mL (experiment 2). Calculation of an EC150 %/EC200 % was not applicable for the 3rd experiment as fold inductions above 150 %/200 % were obtained in the lowest tested concentrations, respectively. In summary, after 24 hours of exposure to test substance CD86 and CD54 expression was induced in THP-1 cells affording at least 50 % viability in at least two independent experiments. From this it has to be concluded that test substance induces dendritic cell activation.

LuSens

A study (reference 7.4.1-2) according to OECD 442D was performed to determine if the test substance activates keratinocytes following the ARE-Nrf2 Luciferase Test (LuSens) method. LuSens cells were cultured using culture medium 1, under standard culture conditions (37 °C, ca. 5 % CO2, ≥ 90 % relative humidity) for at least passage ≥ 5 but not longer than 15 passages prior to testing. Before substance incubation, cells were seeded in 96-well microtiter plates, using culture medium 2 for incubation for ca. 24 hours. Three independent, valid experiments were performed. In each experiment, three replicates of each test-substance concentration were tested. After cell adaption for ca. 24 hours culture medium 2 was aspirated and replaced with 150 μL culture medium 3. Each preparation of the dilution plate was then applied in a ratio of 1:4 (50 μL) to the cells (final DMSO concentration in the test medium = 1 %). After test-substance 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 ± 1 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. Each test-substance concentration was visually inspected directly after application and after the exposure period of 48 ± 1 hours in order to detect test-substance precipitates. 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+). Subsequently 200 μL of One-Glo-preparation (= 100 μL One-Glo-Mix and 100 μL PBS (without Ca2+/Mg2+)) per well were added and cells were shaken on a plate shaker for ca. 10 min at room temperature in darkness. After the incubation the luminescence was measured in the luminometer.

Cell viability assay was determined by aspiration of the cell culture medium from all wells. Thereafter, 200 μL of a 0.5 mg/mL thiazolyl blue tetrazolium bromide (MTT) solution (prepared 1:10 from a 5 mg/mL (MTT) stock solution in PBS (without Ca2+/Mg2+) and culture medium 3) was added to each well of the 96-well microtiter plate and incubated for at least further 2 hours 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 690 nm using a spectral-photometer. In parallel, a negative control (NC), a positive control (PC), a vehicle control (VC), one blank control and one casal control were performed. The test substance was soluble in 4 % DMSO in culture medium 3 (4 x stock preparations) and in 1 % DMSO in culture medium 3 (final concentrations). No precipitates were noticed in any concentration after 48 hours. Calculation of an EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was not applicable due to the overall negative result of the study.

In summary, after 48 hours of exposure to test substance, luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70 % viability in at least two independent experiments. From this it has to be concluded that test substance does not have a keratinocyte activating potential.

DPRA

A study (reference 7.4.1-3) according to OECD TG 442C - Direct Peptide Reactivity Assay (DPRA) was conducted under GLP to determine the reactivity of the test item towards model synthetic peptides containing either cysteine or lysine.The test substance was dissolved in acetonitrile as a 100 mM preparation. 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. Peptide stock solutions in a concentration of 0.667 mM were prepared in pH 7.5 phosphate buffer (C-containing peptide - Ac-RFAACAA-COOH) or pH 10.2 ammonium acetate buffer (K-containing peptide - Ac-RFAAKAA-COOH). The peptide stock solution was used for preparing the calibration samples and the test substance and control samples. Negative control (NC), positive control (PC) - Ethylene glycol dimethacrylate (EGDMA) and co-elution control (SK) ran parallel to check the validity of the test. The samples of the test substance with the peptides were solutions at the time of preparation. Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides. No co-elution of test substance and peptides was present. The mean C-peptide depletion, caused by the test substance was determined to be 31.92 %. The mean K-peptide depletion, caused by the test substance was determined to be 1.70 %. Thus, the mean peptide depletion was calculated to be 16.81 %. Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test item shows low chemical reactivity in the DPRA under the test conditions chosen.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. According to Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776, the test item does not have to be classified as skin sensitiser.