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Key value for chemical safety assessment

Skin sensitisation

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Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017
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)
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes
Details on the study design:
Cell line used: HaCaT cells
obtained from Givaudan and maintained following protocols outlined in the supplier’s standard operating procedures and publications (Emter et al., 2010; Natsch et al., 2011). To determine sensitization potential, keratinocytes were incubated with each test chemical for about 48 hours at approximately 37ºC. At the end of incubation, luciferase induction and cell viability was determined.

Media:
The maintenance medium for the HaCaT cell line was prepared by supplementing Dulbecco’s Modified Eagle Medium (D-MEM) media (Gibco) with 9.1% fetal bovine serum (FBS) and either with (DMEM9.1(+)) or without (DMEM9.1(-)) geneticin (antibiotic; final concentration 500 µg/ml). Treatment medium consisted of D-MEM with 1% FBS and no geneticin (DMEM1(-)). All medium prepared was stored at approximately 4°C and used within 28 days.

Preparation of the controls and treatment solutions:
DMSO was used as the solvent for the test material and the control treatment. Cinnamic aldehyde (CA, CAS # 104-55-2) was used as the positive control. CA was prepared at a concentration of 6.4 mM in DMSO and further diluted to 64 µM, 32 µM, 16 µM, 8 µM, and 4 µM in culture medium. Stock solutions of the test material were prepared fresh in DMSO, at an initial concentration of 200 mM. All stock solutions were further serial diluted in DMSO to obtain a “100X master plate” consisting of each test material at twelve consecutive two-fold dilutions (ranging from 0.098 to 200 mM). These stocks were further diluted in the assay procedure as outlined below to result in the testing of a concentration range of 0.98 to 2000 µM in the final assay.

Luciferase and Cytotoxicity assays:
Frozen HaCaT cells (approximately -150ºC) were thawed in a water bath at approximately 37ºC, resuspended in DMEM9.1(-), and were pelleted by centrifugation at 125 g for 5 minutes at room temperature. The cell pellet was resuspended in DMEM9.1(-), seeded in a flask, and maintained at about 37oC with 5% CO2. After reaching 80-90% confluency, the HaCaT cells were washed twice with Dulbecco’s Phosphate-Buffered Saline (DPBS), trypsinized, and incubated at approximately 37ºC for about 7 minutes. Detached cells were resuspended in DMEM9.1(-) and centrifuged at 125 g for 5 minutes. The resulting pellet was resuspended and hereafter maintained in DMEM9.1(+).
Cell Seeding For Testing
• Cells at 80-90% confluency were washed twice with DPBS, harvested as described above, re-suspended in DMEM9.1(-), and the cell density was adjusted to approximately 80,000 cells/ml.
• 125 µl of the cell-suspension was distributed to each well in a 96-well plate (approximately10,000 cells/well).
• Each 96 well plate consisted of EPON™ Resin CS-337 at twelve different concentrations, six negative control wells containing 1% DMSO, five wells containing the positive control CA at five different concentrations, and one well which is blank containing no cells (one well each/plate).
• In each experiment, three parallel plates for luciferase assay (solid white plate) and two plates for cytotoxicity assessment (transparent plate) were prepared as above.
• The plated cells were grown for about 24 h at approximately 37ºC and 5% CO2.

Treatment Regimen
• Following the ~24 h incubation, the medium was aspirated and replaced with 150 µl of DMEM1(-).
• The 100X stock master plate was diluted 25-fold (10 µl of test chemical solution from master plate + 240 µl of DMEM1(-)) in a fresh 96 well plate (4X master plate).
• 50 µl of solution from the resulting 4X master plate was transferred to each replicate plate already containing the keratinocytes and 150 µl of DMEM1(-) (ranging from 0.98 to 2000 µM in culture medium).
• All plates were covered with sealing tape (STR-SEAL-PLT, EXCEL Sci, Omaha, Nebraska) and incubated at approximately 37ºC for an additional ~48 h.
Luciferase Measurements
• At the end of the 48 h incubation, the supernatant from the 96 well plates was aspirated, washed once with DPBS and cells in each well was incubated with 20 µl of passive lysis buffer (Promega Corp., Madison, Wisconsin) on an orbital shaker at room temperature for
20 min.
• The plates with the cell lysate were read (relative luminescence units; RLU) in the luminometer using the following program:
i. 50 µl of the luciferase substrate was added to each well
ii. waited for 1 second and integrated the luciferase activity for 2 sec
• Luciferase induction for the chemicals was calculated using the following approach:
i. (RLUFG) – (RLUBG) = BG corrected (RLU)
ii. (BG corrected (RLU) of each chemical containing well)/( average BG corrected (RLU) of six negative control wells) = Luciferase induction
RLUFG = Foreground Relative luciferase units
RLUBG = Background Relative luciferase units (no cells blank)
Cytotoxicity Assessment
• For the cell viability assay plates, the medium was aspirated and replaced with 200 µl of DPBS and 27 µl of Thiazolyl blue tetrazolium bromide (MTT) reagent (5 mg/ml in DPBS). The plate was covered with sealing tape and was incubated at approximately 37ºC for 4 hours.
• Following incubation, the supernatant was aspirated and 200 µl of DMSO was added to each well. Following thorough mixing by repeated pipetting, the cell lysate was transferred to a new clear 96 well plate and absorbance was quantified at 600 and 630 nm. Cell viability for the cells was calculated using the following method:
i. (Abs600) – (AbsBG) = BG corrected (Abs600)
ii. (Abs630) – (AbsBG) = BG corrected (Abs630)
iii. BG corrected (Abs600) - BG corrected (Abs630) = (Abs600 - 630)
iv. ((Abs600 - 630) of each chemical containing well)/( average (Abs600 - 630) of six negative control wells) *100 = % viability
Abs600 = Foreground absorbance measured at 600 nm
Abs630 = Foreground absorbance measured at 630 nm

ii. waited for 1 second and integrated the luciferase activity for 2 sec
• Luciferase induction for the chemicals was calculated using the following approach:
i. (RLUFG) – (RLUBG) = BG corrected (RLU)
ii. (BG corrected (RLU) of each chemical containing well)/( average BG corrected (RLU) of six negative control wells) = Luciferase induction
RLUFG = Foreground Relative luciferase units
RLUBG = Background Relative luciferase units (no cells blank)
Cytotoxicity Assessment
• For the cell viability assay plates, the medium was aspirated and replaced with 200 µl of DPBS and 27 µl of Thiazolyl blue tetrazolium bromide (MTT) reagent (5 mg/ml in DPBS). The plate was covered with sealing tape and was incubated at approximately 37ºC for 4 hours.
• Following incubation, the supernatant was aspirated and 200 µl of DMSO was added to each well. Following thorough mixing by repeated pipetting, the cell lysate was transferred to a new clear 96 well plate and absorbance was quantified at 600 and 630 nm. Cell viability for the cells was calculated using the following method:
i. (Abs600) – (AbsBG) = BG corrected (Abs600)
ii. (Abs630) – (AbsBG) = BG corrected (Abs630)
iii. BG corrected (Abs600) - BG corrected (Abs630) = (Abs600 - 630)
iv. ((Abs600 - 630) of each chemical containing well)/( average (Abs600 - 630) of six negative control wells) *100 = % viability
Abs600 = Foreground absorbance measured at 600 nm
Abs630 = Foreground absorbance measured at 630 nm
AbsBG = Background absorbance (of no cells blank)

Acceptance Criteria
Cinnamic aldehyde (CA, positive control) was considered positive when the gene induction by CA was above the threshold of 1.5-fold in at least one dose level and cell viability at that dose was greater than 70%.
Maximum luciferase induction (Imax) and EC 1.5 (test material concentration at which luciferase induction was greater than 1.5 fold) was calculated for CA. The assay was acceptable only if at least one of the two following criteria were fulfilled:
• Average luciferase induction in the two replicates for CA at 64 µM was between 2 and 8.
• The EC 1.5 was between 7.5 µM and 30 µM.
If only one criterion is fulfilled, the dose-response of CA was carefully checked to decide on acceptability (Natsch et al., 2011).
The average variability in the 6 solvent control wells of each of the two parallel test plate should be below 20%. If the variability was higher, the assay was deemed unreliable and the results were discarded.
The results for these acceptable criteria were reported along with the test results. Final interpretation of the assay acceptability was based on the above criteria and expert judgment.

Test material data reporting and interpretation:
For the test chemical, Imax, EC 1.5, and cell viability were calculated as described above. A chemical was reported as positive if:
• Luciferase induction (Imax) was greater than 1.5-fold and EC 1.5 is below 1000 µM.
• At EC 1.5, the cellular viability was above 70%.
• There was an apparent overall dose-response for luciferase induction.
Final interpretation of the test material results were based on the above criteria as well as expert judgment.



















Positive control results:
Results for positive control and the test material were evaluated relative to the criteria specified in the OECD TG 442D. In the three independent replicates, the positive control compound, cinnamic aldehyde, exhibited a dose-dependent increase in luciferase activity with EC 1.5 values of 21.93, 14.24, and 10.93 µM, respectively. The relative cell viability at EC 1.5 was greater than 70%. In replicate 1, 2, and 3, cinnamic aldehyde exhibited a maximum luciferase induction (Imax) of 2.41, 4.32, and 4.44-fold, relative to the vehicle control. In addition, the average variability in the solvent control wells was below the acceptable 20% in both replicates, thereby demonstrating appropriate assay responsiveness.
Key result
Run / experiment:
other: 1 2 and 3
Parameter:
other: EC 1.5 (micro molar)
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
2-(Bis(2-ethylhexyl)amino)ethanol was tested at twelve concentrations ranging from 1 to 2000 µM in three independent assay replicates.
In replicate 1, 2-(Bis(2-ethylhexyl)amino)ethanol exhibited a maximum luciferase induction (Imax) of 1.82-fold, relative to the vehicle control. The EC1.5 value in replicate 1 was 5.75 μM and cell viability at this concentration was greater than 70%. In replicate 2, 2-(Bis(2-ethylhexyl)amino)ethanol exhibited a maximum luciferase induction (Imax) of 1.45-fold, relative to the vehicle control (i.e. less than 1.5-fold) and cell viability at this concentration was greater than 70%. In replicate 3, 2-(Bis(2-ethylhexyl)amino)ethanol exhibited a maximum luciferase induction (Imax) of 1.71-fold, relative to the vehicle control, however cell viability at this concentration was less than 70%. Therefore, 2-(Bis(2-ethylhexyl)amino)ethanol did not meet the criteria for a potential skin sensitizer in two of three replicates.

2-(Bis(2-ethylhexyl)amino)ethanolinterpretation criteria

 

Results

Interpretation

 

Rep-1

Rep-2

Rep-3

Average

 

Imax (relative fold)

1.82

1.45

1.71

1.66

 

EC 1.5 (µM)

5.75

NA

6.61

6.16 *

Potential non-sensitizer

Cell viability at EC 1.5 (%)

>70%

>70%

<70%

 

 

* criteria for skin sensitising potential not met due to high cyctotoxicity in replicate 3 and no EC 1.5 in replicate 2

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study, 2-(Bis(2-ethylhexyl)amino)ethanol is predicted to be a potential non-sensitizer to skin.
Executive summary:

2-(Bis(2-ethylhexyl)amino)ethanolwas evaluated for skin sensitization potential in anin vitroKeratinoSens assay. In this study the KeratinoSens cells were exposed to a vehicle control (1% DMSO), positive control (cinnamic aldehyde) at five concentrations (4 – 64µM), and2-(Bis(2-ethylhexyl)amino)ethanolat 12 concentrations (0.98 – 2000µM). Following 48 hours of exposure, the cell viability and luciferase activity were measured in treated and control cells. The test material was considered a sensitizer if relative luciferase activity was greater than 1.5-fold (EC 1.5 at concentration < 1000µM) and cell viability at EC 1.5 was greater than 70%. The positive control treated cells exhibited luciferase induction within the designated parameters and met all requirements for a viable assay. The mean relative luciferase activity of2-(Bis(2-ethylhexyl)amino)ethanol was greater than 1.5-fold at 6.16µM, however cell viability at this concentration was less than 70%. Therefore, under the conditions of this study, 2-(Bis(2-ethylhexyl)amino)ethanol is predicted to be a potential non-sensitizer to skin.

Endpoint:
skin sensitisation
Remarks:
other: QSAR
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: QSAR prediction made using a valid model; substance is within the applicability domain for the model
Justification for type of information:
QSAR prediction: migrated from IUCLID 5.6
Principles of method if other than guideline:
Prediction of skin sensitising potential using OASIS Times v2.27.16.8

This substance was assessed for skin sensitising potential using the QSAR tool OASIS Times. Parent compound and metabolites were with the domain of the tool and all predicted to be negative for sensitising potential.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The substance (and metabolites) was in domain for the QSAR tool and was predicted not to be a sensitiser.
Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
GLP compliance:
yes
Type of study:
direct peptide reactivity assay (DPRA)
Details on the study design:
Test Article Solubility Test
A solubility test was performed, in accordance with the OECD TG 442C, for the test articles prior to incubations to determine an appropriate solvent that dissolved the test article at a 100 mM concentration.
Test Article Preparation
The test article and positive control were prepared immediately before use at a concentration of 100 mM in acetonitrile as determined from the solubility testing.
Buffer Solution Preparation
According to OECD TG 442C, a 100 mM phosphate buffer (pH=7.5 ± 0.1) was prepared with sodium phosphate dibasic and sodium phosphate monobasic for use with cysteine containing peptide and adjusted to the appropriate pH using either the monobasic or dibasic. For lysine-containing peptide, a 100 mM ammonium acetate buffer (pH= 10.2 ± 0.1) was prepared with ammonium acetate and adjusted to appropriate pH with ammonium hydroxide.
Peptide Preparation
Two custom synthetic peptides, one containing cysteine (cysteine peptide) and another containing lysine (lysine peptide), were used in this assay. Peptide samples were prepared and a single preparation of the peptide was used for all samples as described below.
Cysteine Peptide Stock Solution
Based on the amount of cysteine peptide needed in the study, the actual amount of cysteine peptide was weighed into a container and the appropriate amount of pH 7.5 phosphate buffer solution was added to form a 0.667 mM stock cysteine peptide solution. The following calculation was used to determine the correct amount of buffer needed based on the actual weight of the peptide:
mL pH 7.5 buffer = mg Cysteine Peptide/ 0.501 mg/mL

Lysine Peptide Stock Solution
Based on the amount of lysine peptide needed in the study, the actual amount of lysine peptide was weighed into a glass vial and the appropriate amount of pH 10.2 ammonium acetate buffer solution was added to the glass vial to form a
0.667 mM stock lysine peptide solution. The following calculation was used to determine the correct amount of buffer needed based on the actual weight of the peptide:
mL pH 10.2 buffer = mg Lysine Peptide/ 0.518 mg/mL
Dilution Buffer
A total of 48 mL of buffer (pH 7.5 for cysteine and 10.2 for lysine) was mixed with 12 mL of acetonitrile to create a 80:20 buffer:solvent solution.
Peptide Standard Preparation
Two sets of standards were prepared, one of each peptide, by two-fold serial dilution of the highest standard (0.534 mM peptide which was prepared by mixing 1600 µL of the previously prepared peptide solution of 0.667 mM with 400 µL of acetonitrile) via the appropriate dilution buffer prepared above. The final sets of serially diluted standards were prepared with the concentrations of 0.534, 0.267, 0.134, 0.067, 0.033, and
0.017 mM and were labeled as Standards 1, 2, 3, 4, 5, and 6, respectively.
Control Preparation
Solvent controls (blanks) were prepared and injected several times during the analysis to re-equilibrate the system between peptides. Reference controls and co-elution controls were also prepared.
Solvent Controls
Solvent controls, made with 50:50 methanol:ultrapure water, were injected in between peptide sequences for re-equilibration purposes.
Reference Controls
Reference controls A, B, and C were peptide solutions that lacked the test chemical or positive control, cinnamaldehyde, which was replaced by acetonitrile. The reference controls serve as indicators for peptide depletion in solvent over time. These were prepared, in triplicate, by mixing 375 µL of peptide stock solution (0.667 mM) with 125 µL of acetonitrile.

Co-elution Controls
Cysteine peptide co-elution controls were prepared by mixing 375 µL of buffer solution, 100 µL of acetonitrile and 25 µL of 100 mM test chemical solution (or positive control). Lysine peptide co-elution controls were prepared by mixing
375 µL of buffer solution and 125 µL of 100 mM test chemical solution (or positive control). These controls verified that co-elution of the test material did not occur.
Peptide & Test Chemical Reaction Mixture Preparation
The cysteine peptide-related final reaction mixtures were prepared by mixing 375 µL of buffer solution, 100 µL of acetonitrile, and 25 µL of 100 mM test chemical or positive control. The lysine peptide final reaction mixtures were prepared by mixing 375 µL of lysine peptide stock solution with 125 µL of 100 mM test chemical or positive control. Triplicate samples were prepared for each test article and positive control.
The final reaction mixtures were prepared as 0.5 mL incubations for the test articles, positive control, and reference controls in the pre-labeled autosampler vials. Triplicate samples were prepared for each test article and control.
Incubation Procedure
All reaction mixture samples, peptide standards, reference controls, and co-elution controls were incubated in the dark at 25°C for 24 hours.
HPLC Conditions
For the HPLC/UV analysis, as described in the OECD TG 442C, specified eluents and a HPLC system were used to separate the analytes. An Agilent HPLC/UV Analysis system (Agilent, Palo Alto, CA, USA) was used for both peptide sample sets as well as a Zorbax SB-C18 2.1 mm x 100 mm column (Agilent, Palo Alto, CA, USA). Mobile phases consisted of ultrapure water with 0.1% formic acid (FA; A) and acetonitrile with 0.1% FA (B). HPLC analysis was performed using a flow of 0.35 mL/min and a linear gradient from 10-25% A over a period of 10 minutes followed by a rapid increase to 90% A to remove any lingering impurities. Detection of analytes was achieved at 220 nm. Detailed conditions are described below:
Once the column was equilibrated the time frame of 24 ± 2 hours post-incubation, the sample vials were placed into designated locations in the autosampler and analyzed.

Sample Analysis
Two analysis sequences for each peptide-related incubation sample set were prepared. The first sequence consisted of the cysteine peptide-related calibration standards, triplicates of Reference Controls A, B, and C, reaction mixtures, and Co-elution controls. The second sequence consisted of the lysine peptide-related calibration standards, triplicates of Reference Controls A, B, and C, reaction mixtures, and Co-elution controls.
Data Analysis
The graphical analysis, peak areas, and concentrations, were obtained from the Agilent MassHunter software package. Standard curves of peak area versus concentration were generated for each set of peptide standards. The concentrations were calculated for each sample using peak area and the equation from the appropriate standard curve. Microsoft Excel 2010 was used to generate means, standard deviations (STD), and coefficients of variance (CV) across replicated and peptides.
The percent depletion was calculated for the test article sample and the positive control samples as shown below. The peptide depletion for the test article was calculated using the appropriate reference controls which, in this assay, are replicates B and C.

% Peptide Depletion=[ 1-(Test Article or Positive Control Peptide Concentration)/(Mean Peptide Concentration of Reference Controls B & C )]x 100

For predicting sensitization, the following table was adapted from the OECD TG 442C and used as the prediction model:
Text Table 1. Cysteine 1:10/Lysine 1:50 Prediction Model

Mean of Cysteine and Lysine % Depletion Reactivity Class DPRA Prediction
0% ≤ mean % depletion ≤ 6.38% No or minimal reactivity Negative; Non-sensitizer
6.38% < mean % depletion ≤ 22.62% Low reactivity Positive; Sensitizer
22.62 < mean % depletion ≤ 42.47% Moderate reactivity Positive; Sensitizer
42.47% < mean % depletion ≤ 100% High reactivity Positive; Sensitizer

Criteria for a Valid Test (OECD TG 442C)
To consider a DPRA valid, several criteria must be met:

Standard curves for each peptide must have an r2 value greater than 0.99.
Mean peptide concentration of reference control A (three replicates of acetonitrile and peptide with no test article and run with the standards) must fit within the concentration range of 0.50 ± 0.05 mM.
The positive control (100 mM cinnamaldehyde reacted with each peptide) must have a mean percent peptide depletion of 60.8-100.0% for cysteine and 40.2-69.4% for lysine among the three replicates.
Positive control replicates must have a STD < 14.9% for cysteine and < 11.6% for lysine.
Reference Control B must have a peak area or concentration CV < 15% for cysteine and lysine.
Reference control C must have a peak area or concentration CV < 15% for cysteine and lysine and a mean peptide concentration of 0.50 ± 0.05 mM.
The standard deviations for the three replicates of the test article must have a STD
< 14.9% for cysteine and < 11.6% for lysine.
There must be no overlap observed between the test article peak of the co-elution control and the peptide peak.
Positive control results:
Average percent depletion values for cysteine and lysine were 68.9% and 36.0%, respectively. It should be noted that the positive control lysine peptide depletion value was 36.0%, which was lower than the OECD guideline value (40.2-69.4%). However, in consideration of the chemical structure of the test material which does not contain any electrophilic group, this lower positive lysine depletion value did not appear to impact the study conclusion
Key result
Run / experiment:
other: mean of all runs
Parameter:
other: Mean cysteine depletion (%)
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Run / experiment:
other: mean of all runs
Parameter:
other: mean lysine depletion (%)
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Interpretation of results:
GHS criteria not met
Conclusions:
Overall, the current study results showed that the average percentage depletion values of cysteine and lysine peptide for this substance were 0.0% and 0.0%, respectively, with a mean percent depletion of 0.0%. Based on the test guideline (OECD 442C) and the mean peptide depletion of 0.0%, this substance is not considered to be a sensitizer in the DPRA.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

QSAR -

OASIS Times v2.27.20.1 was used to predict skin sensitising potential. The structure was in domain for the QSAR tool, and it was predicted not to be a skin sensitiser. Potential metabolites were also assessed and none of these were identified as potential sensitisers.

In vitro -

Keratinosense assay - the substance was assessed for skins ensitisng potential in the Keratinosense assay. It failed to induce luciferase activity to 1.5 fold or greater versus control at concentrations where cell viability was >60% and thus was predicted to be a nonsensitiser.

DPRA - The substance failed to cause a depletion in either cystein or lysine peptides indicating a lack of ability to directly bind to proteins.

Based on the lack of structural alerts for skin sensitising potential and the negative results from the 2 in vitro assays, the WoE supports the conclusion that this substance is a non-sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The QSAR assessment identified no structural alerts for skin sensitising potential and the Keratinosense and DPRA assays were negative, indicating that this substance is unlikely to be a skin sensitiser.