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EC number: 250-796-0 | CAS number: 31774-90-0
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Description of key information
Based on the OECD GUIDANCE DOCUMENT ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION Series on Testing & Assessment No. 256 (ENV/JM/MONO(2016)29 and ENV/JM/MONO(2016)29/ANN1) an Adverse Outcome Pathway-based "2 out of 3" integrated testing strategy approach to skin hazard identification (BASF) was chosen. This defined approach describes an integrated testing strategy (ITS) for the identification of the skin sensitisation hazard of a substance primarily for the purposes of classification and labelling without the use of animal testing. The combination of test methods used covers the first three key events (KEs) of the adverse outcome pathway (AOP) leading to skin sensitisation as formally described by the OECD: KE 1: protein binding (e.g. via the direct peptide reactivity assay (DPRA); OECD TG 442C); KE 2: keratinocyte activation (e.g. via the KeratinoSensTM or LuSens assay; OECD TG 442D); and dendritic cell activation [e.g. via the human cell line activation test (h-CLAT); OECD TG 442E. The prediction model entails that two concordant results obtained from methods addressing different steps of first three KEs of the AOP, determine the final classification. Performance and classifications derived from the “2 out of 3 - Sens ITS” of 213 substances were compared to both high quality animal and human data. Depending on the combination of tests used, the “2 out of 3 - Sens ITS” prediction model generally achieved accuracies slightly exceeding those of the murine local lymph node assay (LLNA) when compared to human data. These results compellingly verify the applicability of this easy to understand integrated testing approach (ITS) for a wide range of chemicals.[Ref. CASE STUDY I of ENV/JM/MONO(2016)29/ANN1, Dr. Robert Landsiedel, BASF SE] The test item does neither activate the Nrf2-Keap1-ARE toxicity pathway (key event 2 of skin sensitization AOP) nor induce incresed number of cell surface markers CD54 and CD86 (key 3 of the skin sensitization AOP). In line with the "2 out of 3" integrated testing strategy approach to skin hazard identification presented classification of the test item as skin sensitizer is not justified.
Key value for chemical safety assessment
Skin sensitisation
Link to relevant study records
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- In vitro study in accordance with Testing and assessment strategy for evaluating the skin sensitisation potential of substances of Chapter R.7a: Endpoint specific guidance Version 6.0 – July 2017.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of keratinocytes
- Specific details on test material used for the study:
- Name: Ethyltris(2-hydroxyethyl)ammonium ethyl sulphate
Product Description: Triethanolamine DES Quat
CAS No.: 31774-90-0
Physical state: colourless to yellow viscous liquid at 20 °C
Batch No.: PFS-755-175
Re-certification date of batch: 21 April 2018
Purity: 100 % (UVCB, water content 0.33 % (w/w))
pH, 5% in water 7.85
Acid Value , mg KOH/g 38.45
Moisture, % 0.33
Total Amine, mg/g 33.10
Viscosity,cps, #4@60,25C 1580
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light - Details on the study design:
- The KeratinoSens assay is supposed to address the second key event of the skin sensitisation process as defined by the adverse outcome pathway (AOP), the induction of cyto-protective signalling pathways in keratinocytes in response to electrophiles and oxidative stress. The KeratinoSens assay addresses the effect on the antioxidant response element (ARE)-dependent pathway in the KeratinoSens cell line by measuring the induction of an ARE dependent gene product, the luciferase gene. The luciferase gene induction following exposure to test chemicals is measured in cell lysates by luminescence detection, allowing the discrimination between sensitisers and non-sensitisers.
Preparation of the Test Item
All test item solutions were freshly prepared immediately prior to use. The test item was dissolved in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity ≥99%). A stock solution of 200 mM was prepared by pre-weighing the test material into a glass vial. Vortex mixing was used to aid solubilisation.Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent. These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.
Cell line
The test was carried out using the transgenic cell line KeratinoSens (Givaudan, Switzerland), a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and were used for routine testing. Only cells at a low passage number <25 (P 3 experiment 1 and 2) were used. Cells were cultured in 75 cm2 culture flasks (Greiner) in maintenance medium at 37 +- 1°C and 5% CO2. For test material exposure, cells were cultured in medium.
Composition of Media
Maintenance Medium
Dulbecco’s Modified Eagle Medium with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with the following components:
- 10% fetal bovine calf serum
- 1% geneticin (final concentration: 500 µg/mL)
Assay Medium
Dulbecco’s Modified Eagle Medium with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with the following components:
- 10% fetal bovine calf serum
Test Item Exposure Medium
Dulbecco’s Modified Eagle Medium with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with the following components:
- 1% fetal bovine calf serum
Experimental Procedure
A cell suspension of 8 × 10^4 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1 × 10^4 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability. After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 µL test item exposure medium. 50 µL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item. All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.
Luciferase activity
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 µL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light. Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1.000 ms before assessing the luciferase activity for 2.000 ms. This procedure was repeated for each individual well.
Cell viability
For the cell viability plate the medium was replaced with 200 µL test item exposure medium. 27 µL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 200 µL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 overnight (experiment 1 and 2). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.
Calculation of EC1.5, IC50 and IC30, Cell Viability and Maximal Induction of the Luciferase Activity was performed according to OECD 442 d guideline.
Prediction Model
The test item is considered positive in accordance with UN GHS “Category 1” if the following conditions were met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is <1000 µM
- an apparent overall dose-response for luciferase induction
If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 µM is considered as inconclusive.
Acceptance Criteria
The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition. - Positive control results:
- Controls
A blank, a negative control and a positive control were set up in parallel in order to confirm the validity of the test.
Blank
A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.
Negative Control
DMSO at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.
Positive Control
Cinnamic aldehyde (CA, (2E)-3-phenylprop-2-enal; CAS 104-55-2; >98%) was used as positive control. CA was dissolved in DMSO at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 µM – 64 µM. The final concentration of the solvent DMSO was 1% (v/v) for all wells. - Key result
- Run / experiment:
- other: 1
- Parameter:
- other: EC1.5
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- not determinable
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: EC1.5
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- not determinable
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: Imax
- Value:
- 1.03
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: Imax
- Value:
- 1.4
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Run / experiment:
- other: mean of experiment 1 and 2
- Parameter:
- other: Imax
- Value:
- 1.22
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: Experiment 1 & 2
- Parameter:
- other: IC30
- Remarks:
- [µM]
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- not determinable
- Run / experiment:
- other: Experiment 1 & 2
- Parameter:
- other: IC50
- Remarks:
- [µM]
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- not determinable
- Run / experiment:
- other: Positive control 1
- Parameter:
- other: EC1.5 PC
- Value:
- 23.29
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Run / experiment:
- other: Positive control 2
- Parameter:
- other: EC1.5 PC
- Value:
- 8.67
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
- Executive summary:
In this KeratinoSenss tudy under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
Conclusion:
Based on the OECD GUIDANCE DOCUMENT ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION Series on Testing & Assessment No. 256 (ENV/JM/MONO(2016)29 and ENV/JM/MONO(2016)29/ANN1) an Adverse Outcome Pathway-based "2 out of 3" integrated testing strategy approach to skin hazard identification (BASF) was chosen. This defined approach describes an integrated testing strategy (ITS) for the identification of the skin sensitisation hazard of a substance primarily for the purposes of classification and labelling without the use of animal testing. The combination of test methods used covers the first three key events (KEs) of the adverse outcome pathway (AOP) leading to skin sensitisation as formally described by the OECD: KE 1: protein binding (e.g. via the direct peptide reactivity assay (DPRA); OECD TG 442C); KE 2: keratinocyte activation (e.g. via the KeratinoSensTM or LuSens assay; OECD TG 442D); and dendritic cell activation [e.g. via the human cell line activation test (h-CLAT); OECD TG 442E. The prediction model entails that two concordant results obtained from methods addressing different steps of first three KEs of the AOP, determine the final classification. Performance and classifications derived from the “2 out of 3 - Sens ITS” of 213 substances were compared to both high quality animal and human data. Depending on the combination of tests used, the “2 out of 3 - Sens ITS” prediction model generally achieved accuracies slightly exceeding those of the murine local lymph node assay (LLNA) when compared to human data. These results compellingly verify the applicability of this easy to understand integrated testing approach (ITS) for a wide range of chemicals.[Ref. CASE STUDY I of ENV/JM/MONO(2016)29/ANN1, Dr. Robert Landsiedel, BASF SE] The test item does neither activate the Nrf2-Keap1-ARE toxicity pathway (key event 2 of skin sensitization AOP) nor induce incresed number of cell surface markers CD54 and CD86 (key 3 of the skin sensitization AOP). In line with the "2 out of 3" integrated testing strategy approach to skin hazard identification presented classification of the test item as skin sensitizer is not justified.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- In vitro study in accordance with testing and assessment strategy for evaluating the skin sensitisation potential of substances of Chapter R.7a: Endpoint specific guidance Version 6.0 – July 2017.
- Qualifier:
- according to guideline
- Guideline:
- other: OECD 442E guideline: human cell line activation test (h-CLAT)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of dendritic cells
- Specific details on test material used for the study:
- Name: Ethyltris(2-hydroxyethyl)ammonium ethyl sulphate
Product Description: Triethanolamine DES Quat
CAS No.: 31774-90-0
Physical state: colourless to yellow viscous liquid at 20 °C
Batch No.: PFS-755-175
Re-certification date of batch: 21 April 2018
Purity: 100 % (UVCB, water content 0.33 % (w/w))
pH, 5% in water 7.85
Acid Value , mg KOH/g 38.45
Moisture, % 0.33
Total Amine, mg/g 33.10
Viscosity,cps, #4@60,25C 1580
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light - Details on the study design:
- Test method
This in vitro method is designed to predict and classify the skin sensitising potential of a chemical or multi-constituent substances or mixtures by assessment of its potential to upregulate cell surface markers using fluorescence-activated cell sorting (FACS).
Preparation of the Test Item
The test item was freshly prepared immediately prior to use. The test item was dissolved in 0.9% NaCl solution at a concentration of 500 mg/mL. Stock solutions were prepared by diluting the highest soluble concentration seven times with a constant dilution factor of 1:2. The working stock solutions were prepared by diluting each stock solution 50 times with cell culture medium. The working stock solutions were applied to the cells by adding equal volumes of each solution to prepared cells, resulting in a further 1:2 dilution of the working solutions. The solvent (0.9% NaCl solution) was present at a constant volume ratio of 1% (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.
Controls
A medium control, a solvent control, and a positive control were set up in parallel in order to confirm the validity of the test.
Medium Control
A medium control was included in the test.
Solvent Controls
Solvent controls were included in the test. The solvent controls were set up depending on the appropriate solvent previously determined. Since the test item was solubilized in 0.9% NaCl, the medium control served as solvent control. Since the positive control was solubilized in DMSO, a DMSO control was included and served as solved control for the positive control. The solvent controls were diluted according to the procedure described for the test item, resulting in a final concentration of 1% (v/v) for 0.9% NaCl and 0.2% (v/v) for DMSO.
Positive Control
2,4-dinitrochlorobenzene (DNCB) at a final concentration of 4 µg/mL (alternatively at the concentration of the CV75) was tested concurrently with the test item. DNCB was dissolved in DMSO and diluted according to the procedure described for the test item, resulting in a final DMSO concentration of 0.2% (v/v).
Cell line
The test was carried out using THP-1 cells (ATCC TIB-202TM), an acute human monocytic leukemic cell line used as a surrogate for DC. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and subcultured at least 2 weeks before they were used in the in vitro h-CLAT test. Cells at passage number (<30) were used. Cells are routinely passaged every 2-3 days at a density of 0.1 – 0.2 x 10^6 cells/mL. Cells were cultured in 75 cm2 culture flasks (Greiner) in Roswell Park Memorial Institute medium (RPMI-1640) supplemented with 10% fetal bovine serum, 25 mM HEPES, L-glutamine, 0.05 mM 2-mercaptoethanol and 100 U/ml penicillin/ 100 µg/mL streptomycin at 37 +- 1°C and 5% CO2.
Reactivity Check of the Cells Stock
Prior to testing, the quality of freshly thawed cell batch was checked by monitoring the doubling time and checking the reactivity towards positive controls. For the reactivity check of the cell batch additional negative and positive controls were included. DNCB at a final concentration of 4 µg/mL and nickel sulphate at a final concentration of 100 µg/mL served as positive control while lactic acid at a final concentration of 1000 µg/mL served as negative control. Cells were accepted when both, DNCB and nickel sulphate produce a positive response for CD86 and CD54, and lactic acid produces a negative response for CD86 and CD54.
Solvent Finding
Solubility of the test item was determined prior to the main experiment. The test item was dissolved in 0.9% NaCl at a final concentration of 100 mg/mL. Test items not soluble in 0.9% NaCl solution were dissolved in DMSO at a concentration of 500 mg/mL. If the test item was not soluble in DMSO, other solvents (e.g. THF) were used. It was taken care that the test chemical is dissolved or stably dispersed in the chosen solvent and that it does not interfere with the test design. If the test item was not soluble in DMSO or a different organic solvent at 500 mg/mL, the highest soluble concentration was tested by diluting the solution from 500 mg/mL with a constant factor of 1:2 up to a minimal concentration of 1 mg/mL.
Dose Finding Assay
Starting from 100 mg/mL (in 0.9% NaCl) solutions of the test chemicals, eight stock solutions (eight concentrations) were prepared, by 2-fold serial dilutions using the corresponding solvent. These stock solutions were further diluted 50-fold (0.9% NaCl) or 250-fold (DMSO) into culture medium (working solutions). Since 0.9% NaCl was used as solvent and the top concentration of 1000 µg/mL was non-toxic, the maximum concentration was re-determined by performing a new cytotoxicity test, up to a final concentration of 5000 µg/mL. The working solutions were finally used for treatment by adding an equal volume of working solution to the volume of THP-1 cell suspension in a 96-well plate to achieve a further 2-fold dilution
For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of
0.1 – 0.2 x 10^6 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation and were re-suspended in fresh culture medium at a density of 2 x 106 cells/mL. Then, 500 µL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 10^6 cells/well).
The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at
37 °C ± 1 °C and 5% CO2. After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The supernatant was discarded and the remaining cells were washed twice with Dulbecco’s phosphate buffered saline (DPBS) containing 0.1% bovine serum albumin (BSA; i.e. FACS buffer). After washing, cells were re-suspended in 600 µL FACS buffer. 200 µL of the cell suspension were transferred into a FACS tube and stained by using propidium iodide (PI) solution at a final concentration of 0.625 µg/mL. The PI uptake of the cells and therefore cytotoxicity was analysed immediately after the staining procedure by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ > 650 nm. A total of 10,000 living (PI negative) cells were acquired and cell viability was calculated for each test concentration. - Positive control results:
- The positive controls DNCB and NiSO4 led to upregulation of the cell surface markers CD54 and CD86. The negative control LA did not induce an upregulation of CD54 and CD86. The cell batches were accepted for further testing.
- Key result
- Run / experiment:
- other: Experiment 1 - CD86 - 5000 µg/ml
- Parameter:
- other: Relative Flourescence Intensity (RFI)
- Value:
- 129
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks:
- DNCB
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Run / experiment:
- other: Experiment 1 - CD54 - 5000 µg/ml
- Parameter:
- other: Relative Flourescence Intensity (RFI)
- Value:
- 113
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks:
- DNCB
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Run / experiment:
- other: Experiment 2 - CD86 - 5000 µg/ml
- Parameter:
- other: Relative Flourescence Intensity (RFI)
- Value:
- 109
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks:
- DNCB
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Run / experiment:
- other: Experiment 2 - CD54 - 5000 µg/ml
- Parameter:
- other: Relative Flourescence Intensity (RFI)
- Value:
- 102
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks:
- DNCB
- Remarks on result:
- no indication of skin sensitisation
- Other effects / acceptance of results:
- Acceptance criteria:
The test meets acceptance criteria if:
- the cell viability of the solvent controls is >90%,
- the cell viability of at least four tested doses of the test item in each run is >50%,
- the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
- the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
- the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%. - Interpretation of results:
- GHS criteria not met
- Conclusions:
- In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experiment runs. Therefore the test item is considered to be no skin sensitiser. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
- Executive summary:
The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers. In the present study N-Ethyl-2-hydroxy-N,N-bis(2-hydroxyethyl)ethanaminium ethyl sulfate was dissolved in 0.9% NaCl. For the dose finding assay stock solutions with concentrations ranging from 500 mg/mL to 0.78 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis. Due to a lack of cytotoxicity, no CV75 could be derived. Therefore the main experiment was performed covering the following concentration steps: 5000; 4166.67; 3472.22; 2411.27; 2009.39; 1674.49; 1395.41 µg/mL. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.
No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 94.6% (CD86), 93.9% (CD54) and 94.3% (isotype IgG1 control) in the first experiment and to 95.8% (CD86), 95.9% (CD54) and 95.3% (isotype IgG1 control) in the second experiment. The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Therefore, the test item is considered to be no skin sensitiser. The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (504% experiment 1; 528% experiment 2) and 200% for CD54 (484% experiment 1; 441% experiment 2) were clearly exceeded.
Conclusion:
Based on the OECD GUIDANCE DOCUMENT ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION Series on Testing & Assessment No. 256 (ENV/JM/MONO(2016)29 and ENV/JM/MONO(2016)29/ANN1) an Adverse Outcome Pathway-based "2 out of 3" integrated testing strategy approach to skin hazard identification (BASF) was chosen. This defined approach describes an integrated testing strategy (ITS) for the identification of the skin sensitisation hazard of a substance primarily for the purposes of classification and labelling without the use of animal testing. The combination of test methods used covers the first three key events (KEs) of the adverse outcome pathway (AOP) leading to skin sensitisation as formally described by the OECD: KE 1: protein binding (e.g. via the direct peptide reactivity assay (DPRA); OECD TG 442C); KE 2: keratinocyte activation (e.g. via the KeratinoSensTM or LuSens assay; OECD TG 442D); and dendritic cell activation [e.g. via the human cell line activation test (h-CLAT); OECD TG 442E. The prediction model entails that two concordant results obtained from methods addressing different steps of first three KEs of the AOP, determine the final classification. Performance and classifications derived from the “2 out of 3 - Sens ITS” of 213 substances were compared to both high quality animal and human data. Depending on the combination of tests used, the “2 out of 3 - Sens ITS” prediction model generally achieved accuracies slightly exceeding those of the murine local lymph node assay (LLNA) when compared to human data. These results compellingly verify the applicability of this easy to understand integrated testing approach (ITS) for a wide range of chemicals.[Ref. CASE STUDY I of ENV/JM/MONO(2016)29/ANN1, Dr. Robert Landsiedel, BASF SE] The test item does neither activate the Nrf2-Keap1-ARE toxicity pathway (key event 2 of skin sensitization AOP) nor induce incresed number of cell surface markers CD54 and CD86 (key 3 of the skin sensitization AOP). In line with the "2 out of 3" integrated testing strategy approach to skin hazard identification presented classification of the test item as skin sensitizer is not justified.
Referenceopen allclose all
Results
The in vitro KeratinoSens assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers. In the present study N-Ethyl-2-hydroxy-N,N-bis(2-hydroxyethyl)ethanaminium ethyl sulfate was dissolved in DMSO. Based on a molecular weight of 303.37 g/mol a stock solution of 200 mM was prepared.
Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM. Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.In the first experiment, a max luciferase activity (Imax) induction of 1.03 was determined at a test item concentration of 1.95 µM. The corresponding cell viability was 90.5%.No significant luciferase induction >1.5 was found in the tested concentration range.Therefore, no EC1.5 value could be calculated. In the second experiment, a max luciferase activity (Imax) induction of 1.40 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 112.3%. No significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.
Under the condition of this study the test item is therefore considered as non sensitiser. The controls confirmed the validity of the study. The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (2.55 in experiment 1; 8.59 in experiment 2). The calculated EC1.5 was between 7 and 34 µM (24.29 µM in experiment 1; 8.67 µM in experiment 2). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20% (9.6% in experiment 1; 10.4% in experiment 2).
Table 1: Results of the cytotoxicity measurement
Concentration [µM] | Cell Viability [%] | ||||
Experiment 1 | Experiment 2 | Mean | SD | ||
Solvent control | - |
100 | 100 | 100 | - |
Positive control | 4.00 8.00 16.00 32.00 64.00 |
100.7 104.3 106.8 118.1 122.0 |
99.7 101.3 109.4 119.4 132.6 |
100.2 102.8 108.1 118.7 127.3 |
0.7 2.1 1.8 0.9 7.5 |
Test item |
0.98 1.95 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500.00 1000.00 2000.00 |
100.2 90.5 98.2 88.7 96.0 91.7 105.8 97.3 90.2 104.9 91.2 94.9 |
106.8 97.8 101.8 97.1 106.6 102.6 98.7 102.0 103.9 108.1 97.6 112.3 |
103.5 94.2 100.0 92.9 101.3 97.2 102.2 99.7 97.0 106.5 94.4 103.6 |
4.7 5.1 2.6 6.0 7.5 7.7 5.0 3.3 9.6 2.3 4.6 12.3 |
Table 2: Induction of Luciferase Activity Experiment 1
Experiment 1 | Concentration [µM] | Fold induction | Significance | ||||
Rep. 1 | Rep. 2 | Rep. 3 | Mean | SD | |||
Solvent control | - | 1.00 | 1.00 | 1.00 | 1.00 | 0.00 | |
Positive control | 4.00 8.00 16.00 32.00 64.00 |
1.08 1.18 1.34 1.58 2.39 |
1.13 1.14 1.30 1.61 2.60 |
1.21 1.25 1.40 1.73 2.66 |
1.14 1.19 1.35 1.64 2.55 |
0.07 0.06 0.05 0.08 0.14 |
* * |
Test item | 0.98 1.95 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500.00 1000.00 2000.00 |
1.10 1.00 1.00 0.98 0.98 0.95 0.98 1.04 0.99 0.92 0.85 0.75 |
0.93 1.04 1.07 0.93 0.99 0.95 1.00 0.95 1.01 0.92 0.90 0.94 |
1.03 1.05 0.97 1.03 1.09 0.97 1.00 1.05 0.99 0.91 0.89 0.81 |
1.02 1.03 1.01 0.98 1.02 0.96 0.99 1.01 1.00 0.92 0.88 0.83 |
0.08 0.02 0.05 0.05 0.06 0.01 0.01 0.06 0.01 0.01 0.03 0.09 |
* = significant induction according to Student’s t-test, p<0.05
Table 3: Induction of Luciferase Activity Experiment 2
Experiment 2 | Concentration [µM] | Fold induction | Significance | ||||
Rep. 1 | Rep. 2 | Rep. 3 | Mean | SD | |||
Solvent control | - | 1.00 | 1.00 | 1.00 | 1.00 | 0.00 | |
Positive control | 4.00 8.00 16.00 32.00 64.00 |
1.18 1.37 1.76 3.11 6.71 |
1.42 1.41 2.08 3.79 9.59 |
1.01 1.59 2.05 3.43 9.47 |
1.20 1.46 1.96 3.44 8.59 |
0.20 0.12 0.18 0.34 1.63 |
* * * |
Test item | 0.98 1.95 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500.00 1000.00 2000.00 |
1.16 0.95 0.93 1.01 0.90 1.08 0.92 0.97 0.88 0.97 0.96 1.48 |
0.87 0.74 1.03 1.03 1.11 1.11 1.16 1.06 1.07 1.18 1.02 1.58 |
0.94 0.87 1.29 0.85 1.13 1.06 1.17 1.12 1.18 1.04 1.07 1.14 |
0.99 0.85 1.08 0.97 1.05 1.08 1.09 1.05 1.05 1.06 1.02 1.40 |
0.15 0.10 0.19 0.10 0.13 0.02 0.14 0.07 0.15 0.11 0.05 0.23 |
* = significant induction according to Student’s t-test, p<0.05
Table 4: Induction of Luciferase Activity – Overall Induction
Overall Induction | Concentration [µM] | Fold Induction | Significance | |||
Experiment 1 | Experiment 2 | Mean | SD | |||
Solvent control | - | 1.00 | 1.00 | 1.00 | 0.00 | |
Positive control | 4.00 8.00 16.00 32.00 64.00 |
1.14 1.19 1.35 1.64 2.55 |
1.20 1.46 1.96 3.44 8.59 |
1.17 1.32 1.66 2.54 5.57 |
0.04 0.19 0.44 1.27 4.27 |
|
Test item | 0.98 1.95 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500.00 1000.00 2000.00 |
1.02 1.03 1.01 0.98 1.02 0.96 0.99 1.01 1.00 0.92 0.88 0.83 |
0.99 0.85 1.08 0.97 1.05 1.08 1.09 1.05 1.05 1.06 1.02 1.40 |
1.01 0.94 1.05 0.97 1.03 1.02 1.04 1.03 1.02 0.99 0.95 1.12 |
0.02 0.12 0.05 0.01 0.02 0.09 0.07 0.02 0.03 0.10 0.10 0.40 |
|
In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
Results
Dose Finding Assay
The first dose finding assay was performed using a highest final concentration of 1000 µg/mL and the second dose finding assay was performed using a highest final concentration of 5000 µg/mL.
Table 1: Results of the Dose Finding Assay
Sample | Experiment 1 | Experiment 2 | ||
Concentration applied [µg/ml] | Cell viability [%] | Concentration applied [µg/ml] | Cell viability [%] | |
Medium control | 0.00 | 95.30 | 0.00 | 95.30 |
Solvent control | 0.00 | 95.30 | 0.00 | 95.30 |
Test item | 7.81 15.63 31.25 62.50 125.00 250.00 500.00 1000.00 |
95.60 95.30 95.30 95.50 95.40 95.10 95.60 95.00 |
39.06 78.13 156.25 312.50 625.00 1250.00 2500.00 5000.00 |
95.30 95.10 95.70 95.40 95.60 95.80 95.40 95.30 |
Calculated CV75 [µg/mL] | No CV75 | No CV75 | ||
Mean CV75 [µg/mL] | - | |||
SD CV75 [µg/mL] | - |
Results CD54 and CD86 Expression
For determination of the cell surface markers CD54 and CD86 two independent experiments were performed using separate cultivated cells at passage 21 (first experiment) and 23 (second experiment). For each experiment separately weighted samples and preparations were used.
Discussion
The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitiser and non-sensitisers.
Prior to the main study the cell batch was checked for its reactivity towards known positive and negative controls and was found to be acceptable for further testing.
In the present study the test item was dissolved in 0.9% NaCl. For the dose finding assay stock solutions with concentrations ranging from 500 mg/mL to 0.78 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.
Due to a lack of cytotoxicity, no CV75 could be derived.
Therefore the main experiment was performed covering the following concentration steps: 5000; 4166.67; 3472.22; 2411.27; 2009.39; 1674.49; 1395.41 µg/mL
Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining. No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 94.6% (CD86), 93.9% (CD54) and 94.3% (isotype IgG1 control) in the first experiment and to 95.8% (CD86), 95.9% (CD54) and 95.3% (isotype IgG1 control) in the second experiment. The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Therefore, the test item is considered to be no skin sensitiser. The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (504% experiment 1; 528% experiment 2) and 200% for CD54 (484% experiment 1; 441% experiment 2) were clearly exceeded. The controls confirmed the validity of the study. The viability of the solvent control was > 90% (94.4-96.3% experiment 1; 95.4-96.6% experiment 2). The number of tested test item concentrations with cell viability > 50% was ≥ 4 (8 experiments 1 and 2). The RFI for CD86 and CD54 of cells treated with the solvent DMSO was ≤ 150% (93% experiment 1; 94% experiment 2) and ≤ 200% (120% experiment 1; 76% experiment 2). The MFI ratio of the medium control and isotype IgG1control was ≥ 105% for CD86 (246% experiment 1; 243% experiment 2) and CD54 (161% experiment 1; 182% experiment 2). The MFI ratio of the solvent control (DMSO) and isotype IgG1 control was ≥ 105% for CD86 (242% experiment 1; 216% experiment 2) and CD54 (177% experiment 1; 153% experiment 2).
The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.
Table 2: CD54 and CD86 Expression Experiment 1
Sample | Conc. [µg/mL] | Cell Viability [%] | Mean Fluorescence Intensity | corrected Mean Fluorescence Intensity | Relative Flourescence Intensity (RFI) | Ratio Isotype IgG1 to [%] | |||||||
CD86 | CD54 | Isotype IgG1 | CD86 | CD54 | Isotype IgG1 | CD86 | CD54 | CD86 | CD54 | CD86 | CD54 | ||
Medium Control | 96.3 | 95.3 | 94.4 | 1942 | 1272 | 791 | 1151 | 481 | 100 | 100 | 246 | 161 | |
Solvent Control | 0.2 | 95.5 | 94.7 | 95.2 | 1817 | 1329 | 752 | 1065 | 577 | 93 | 120 | 242 | 177 |
DNCB (positive control) | 4.0 | 82.6 | 83.6 | 80.8 | 6080 | 3503 | 711 | 5369 | 2792 | 504 | 484 | 855 | 493 |
Test item | 5000 4166.67 3472.22 2893.52 2411.27 2009.39 1674.49 1395.41 |
94.6 94.1 94.4 94.1 96.0 95.3 95.1 95.2 |
93.9 94.8 94.3 94.5 95.1 94.5 95.4 94.3 |
94.3 94.5 93.0 94.2 95.5 94.4 94.7 92.3 |
2244 2036 2027 1781 2277 1902 1956 1903 |
1307 1304 1235 1268 1297 1300 1395 1396 |
765 733 712 723 829 875 805 622 |
1479 1303 1315 1058 1448 1027 1151 1281 |
542 571 523 545 468 425 590 774 |
129 113 114 92 126 89 100 111 |
113 119 109 113 97 88 123 161 |
293 278 285 246 275 217 243 306 |
171 178 173 175 156 149 173 224 |
Table 3: CD54 and CD86 Expression Experiment 2
Conc. [µg/mL] | Cell Viability [%] | Mean Fluorescence Intensity | corrected Mean Fluorescence Intensity | Relative Flourescence Intensity (RFI) | Ratio Isotype IgG1 to [%] | ||||||||
CD86 | CD54 | Isotype IgG1 | CD86 | CD54 | Isotype IgG1 | CD86 | CD54 | CD86 | CD54 | CD86 | CD54 | ||
Medium Control | 96.6 | 96.2 | 96.2 | 1633 | 1218 | 671 | 962 | 547 | 100 | 100 | 243 | 181 | |
Solvent Control | 0.2 | 96.5 | 96.6 | 95.4 | 1693 | 1199 | 784 | 909 | 415 | 94 | 76 | 216 | 153 |
DNCB (positive control) | 4.0 | 80.3 | 80.1 | 81.8 | 5480 | 2514 | 685 | 4795 | 1829 | 528 | 441 | 800 | 367 |
Test item | 5000 4166.67 3472.22 2893.52 2411.27 2009.39 1674.49 1395.41 |
95.8 95.3 95.8 96.7 96.7 96.7 96.4 96.8 |
95.9 94.6 96.1 96.6 96.3 96.2 95.5 96.5 |
95.3 95.0 96.2 96.2 96.4 95.6 96.1 96.4 |
1827 1993 1845 2039 1874 1992 1862 1916 |
1333 1286 1262 1258 1290 1284 1233 1124 |
774 772 708 703 721 700 727 696 |
1053 1221 1137 1336 1153 1292 1135 1220 |
559 514 554 555 569 584 506 428 |
109 127 118 139 120 134 118 127 |
102 94 101 101 104 107 93 78 |
236 258 261 290 260 285 256 275 |
172 167 178 179 179 183 170 161 |
Table 4: Acceptance Criteria
Acceptance Criterion | Range | Experiment 1 | pass/fail | Experiment 2 | pass/fail |
cell viability solvent controls [%] number of test dosed with viability >50% CD86 number of test dosed with viability >50% CD54 number of test dosed with viability >50% IgG1 RFI of positive control of CD86 RFI of positive control of CD54 RFI of solvent control of CD86 RFI of solvent control of CD54 MFI ratio IgG1/CD86 for medium control [%] MFI ratio IgG1/CD86 for DMSO control [%] MFI ratio IgG1/CD54 for medium control [%] MFI ratio IgG1/CD54 for DMSO control [%] |
>90 ≥4 ≥4 ≥4 ≥150 ≥200 <150 <200 >105 >105 >105 >105 |
94.4 - 96.3 8 8 8 504 484 93 120 246 242 161 177 |
pass pass pass pass pass pass pass pass pass pass pass pass |
95.4 - 96.6 8 8 8 528 441 94 76 243 216 182 153 |
pass pass pass pass pass pass pass pass pass pass pass pass |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not sensitising)
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
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