Complexation products of bismuth(3+) neodecanoate with propane-1,2-diol, propoxylated and 2,2',2'',2'''-ethylenedinitrilotetraethanol

Endpoint summary

Description of key information

Test item is to be classified as Skin Sensitizer, Cat. 1: based on the following:

- Under the experimental conditions of an OECD 442D test, test item may be classified as potential skin sensitizer.

- The test item with a log Pow of app. -0.2 activated THP-1 cells under the test conditions of a study conducted according to OECD guideline 442E. Therefore, the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

- Test according to OECD 442C was not conducted because test item is an UVCB.

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:

key study

Study period:

26 March 2018 - 18 January 2019

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)

Version / remarks:

dated 4 February 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

dated 27 April 2017

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

OECD 442D is one of the in-vitro skin sensitization test method now recommended for REACh registration.

Details on the study design:

STUDY OBJECTIVE:
A skin sensitizer refers to a substance that will lead to an allergic response following skin contact.
One of the biological key events takes place in the keratinocytes and includes inflammatory responses as well as gene expression associated with specific cell signaling pathways such as the antioxidant/electrophile response element (ARE) dependent pathways.
The genes under the ARE control, including AKR1C2 gene identified as a target gene for detecting skin sensitizers in keratinocytes, are induced by the protein Nrf2 via Keap1.
The test consists of evaluating the activation of AKR1C2 in transformed keratinocytes (KeratinoSens™), by monitoring the induction of the luciferase gene fused to AKR1C2. The luciferase produced by the cells complexes with luciferin which, in the presence of ATP, produces light measured in relative light units (RLU).
After contact between a potentially sensitizing element with a KeratinoSens™ monolayer, the induction of the luciferase is quantified. In parallel, the cytotoxicity is measured, in order to exclude a false positive generated by skin irritation.
Since activation of the Keap1-Nrf2-ARE pathway addresses only the second key event of the skin sensitization AOP, information from test methods based on the activation of this pathway is unlikely to be sufficient when used on its own to conclude on the skin sensitization potential of chemicals.
Therefore data generated should be considered in the context of integrated approaches.
This study was carried out according to the OECD Guideline 442D dated February, 04th, 2015 and the ECVAM DB-ALM protocol 155: KeratinoSensTM.

SERIES DEFINITION:
- Test item: The test item was tested at 12 concentrations according to a geometric progression of ratio 2 from 0.98 µM to 2000 µM.
- Reference item:
Negative control: 6 wells of solvent control (1% DMSO in treatment medium) on each culture plate.
Positive control: 5 concentrations of cinnamaldehyde on each culture plate. The concentration varies from 4 to 64 µM according to a geometric progression of ratio 2.
- Blanks: 1 well by culture plate is left without cell and was filled with negative control.
The study was composed of two independent repetitions. For each repetition the test item and the reference items were replicated on three independent plates for the measurement of induction and two plates for the measurement of cytotoxicity. Each repetition was performed on a different day with fresh stock solution.

REFERENCE ITEMS:
- Positive control: Cinnamaldehyde (SIGMA ALDRICH Ref W228613):
- Negative control: Treatment culture medium, 1% DMSO, 1% Non-heat inactivated foetal calf serum. The negative control is prepared just prior the test and used within the day.

TEST SYSTEM:
Cells: KeratinoSens™ (Givaudan) maintained according to the current working instruction.
Cells are cultured in maintenance medium at 37°C, 5% CO2.
Cells are exempt of mycoplasma. Assessment of mycoplasma was performed according to the current working instruction.
Cells were used at passage 17 in repetition 1 and passage 19 in repetition 2.
 
MEDIA AND REAGEANTS:
- Maintenance medium: DMEM 1 g/l glucose, 9.1% non-heat inactivated foetal calf serum, 0.05% geneticin - stored at 5°C ± 3°C
- Seeding medium: DMEM 1 g/l glucose, 9.1% non-heat inactivated foetal calf serum - stored at 5°C ± 3°C
- Treatment medium: DMEM 1 g/l glucose, 1% non-heat inactivated foetal calf serum - stored at 5°C ± 3°C
- Trypsin (0.5 g/l) - EDTA (0.2 g/l) - stored at -20°C ± 5°C
- Diluent for the test item: Sterile water - stored at room temperature 20°C ± 5°C
- Diluent for the positive control: DMSO (1% maximum final concentration) - stored at room temperature 20°C ± 5°C
- Luciferase substrate: Bright Glo™ Luciferase Assay System (Promega) - stored at -80°C after reconstitution
- Cell washing solution: Dulbecco’s PBS Ca2+ and Mg2+ free with 0.05% EDTA - stored at 5°C ± 3°C
- Dulbecco’s PBS Ca2+ and Mg2+ free - stored at room temperature 20°C ± 5°C
- Staining solution: 5 mg/ml MTT* (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) solution in PBS - prepared extemporaneously and used within the day
- Desorption solution: 10% SDS in water - stored at room temperature 20°C ± 5°C
* Note: MTT powder is stored at 5°C ± 3°C

EQUIPMENT AND CONSUMABLES:
- Luminometer: GloMax™ (Promega)
- MULTISKAN EX plate reader (Thermo life sciences) - reading range 0 - 3.5 units of Absorbance -linearity range 0 - 2.200 units of Absorbance
- White cell culture 96-well plates for luminescence reading
- Transparent cell culture 96-well plates for absorbance reading
- Plastic adhesive foils
- Conventional cell culture laboratory equipment.

TEST PROTOCOL:
- Cells seeding (first day)
The cells were trypsinized according to the current working instruction IL 09.
After removal the culture medium from the culture flask, the cell layer was rinsed with PBS 0.05% EDTA, Ca2 + and Mg² + free to remove all traces of serum. The solution of trypsin-EDTA was added and left for a few minutes at 37°C, 5% CO2 until the detachment of cells. The action of trypsin was stopped by addition of maintenance medium.
Cell concentration was determined on Malassez cell. Cells suspension was adjusted to a density of 8.104 cells/ml in seeding medium.
125 µl of the cell suspension at 8.104 cells/ml (i.e. 104 cells per well) were distributed in three white cell culture plates (96 wells) for the induction measurement and two transparent cell culture plates (96 wells) to assess the cytotoxicity. In order to ensure a good homogeneity of seeding, cells suspension was regularly mixed all along the seeding.
Note: the H12 wells were left without cells for the measurement of blanks.

- Preparation of test item and positive control dilutions (second day)
Given the high MW of the test item, the stock solution was prepared at 79.2 mM* (4X) in treatment medium, 4% DMSO instead of 100X.
*During the experimentation, the purity of the test item was considered at 10.1 %; taking into account the 100% purity, the concentration of the stock solution was 79.2 mM.
The positive control stock solution was prepared at 200 mM in DMSO, then diluted to the concentration of 6.4 mM in DMSO.

A 100-fold concentrated dilutions series was prepared in 96-well plate:
Positive control
100 µl of DMSO were distributed in row G from columns 7 to 10. 200 µl of the 6.4 mM stock solution were placed in column 11 then the series dilutions were prepared by transferring 100 µl from the column 11 to the column 10 and so on until the column 7. Dilutions were mixed by repeated pipetting, at least 3 times, between each concentration.
Negative control
100 µl of DMSO were distributed in row G columns 1 to 6 and 12 and in the well H12.

Preparation of the 4 X dilution plate
Test item: The test item was placed in one of the rows B to F.
100 µl of treatment culture medium 4% DMSO were distributed from columns 1 to 11. 200 µl of the 79.2 mM stock solution were placed in column 12 then the series dilutions were prepared by transferring 100 µl from the column 12 to the column 11 and so on until the column 1. Dilutions were mixed by repeated pipetting, at least 3 times, between each concentration.
Positive and negative control
The 100 X DMSO plate was diluted 25 fold with treatment medium in the 4X plate.

- Contact between the cells and the test and reference items (second day):
In the 5 seeded plates, the medium was aspirated and replaced with 150 µl of treatment medium. Then the 4 X plate was replicated 5 times: 50 µl from the 4 X plate were placed in each of the three white plates and in the two transparent plates. The plates (1 X) were covered with an adhesive plastic foil to prevent evaporation and incubated for 48 hours ± 1 hour (37°C, 5% CO2).

- Luciferase activity (day 4):
After 48 hours, the homogeneity of the test item dilutions are checked, no precipitation was observed. The medium was aspirated and each well was gently washed once with 200 µl of PBS. Then 100 µl of luciferase substrate (luciferin + ATP + lysing agent) were then added in each well. The plates were incubated at least 15 minutes at room temperature to ensure cells lysis.
The plates were placed in the luminometer then the luciferase activity was measured with an integration time of 2 seconds.

- Cell viability assessment with MTT method (day 4):
After 48 hours, the medium was aspirated and each well was gently washed once with 200 µl of PBS. Then, 225 µl of staining solution diluted at 0.6 mg/ml in treatment medium (from the 5 mg/ml stock solution) were distributed in each well. The plates were covered with an adhesive plastic foil and incubated for 4 hours ± 30 minutes (37°C, 5% CO2).
After this contact time, the staining solution was eliminated and the cells were treated with 200 µl of 10% SDS one night in the dark (37°C, 5% CO2). After a 10 minutes homogenization, the absorbances were measured at 540 nm.

RESULTS AND INTERPRETATION:
Two parameters are measured, the luciferase induction and the cytotoxicity.
The test item is identified as potential skin sensitizer if the 4 following conditions are met in 2 of 2 or in 2 of 3 repetitions. Otherwise the KeratinosensTM prediction is considered as negative:
• the Imax is strictly 1.5 fold higher of the basal luciferase activity* statistically significantly to the value obtained for the negative control (as determined by a two-tailed, unpaired Student’s t-test on the raw RLU values),
* If the Imax is exactly equal to 1.5, the test item is rated as negative and no EC1.5 value is calculated
• the EC1.5 value is strictly below 1000 µM,
• at the lowest concentration with a gene induction above 1.5, the cell viability must be strictly above 70% (i.e. EC1.5 < IC70),
• there is an apparent overall dose-response for luciferase induction, which is similar between the repetitions.
In rare cases, the test items which induce the gene activity at a concentration very close to the cytotoxic levels, are positive in some repetitions at non-cytotoxic levels, and in other repetitions only at cytotoxic levels. In this case, the test item must be tested again with a narrower range using a dilution factor of 4/3 instead of 2.
Test items that only induce the gene activity at cytotoxic levels are not rated as positive, as it is the case for some non-sensitizing skin irritants.
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, then the result of that repetition should be considered inconclusive and further testing may be required. In addition, in case of a test item with poor solubility tested at a concentration lower than 1000 µM, a negative result obtained should also be considered as inconclusive.
Negative results should be interpreted with caution as substances with an exclusive reactivity towards lysine-residues can be detected as negative by the test method. Furthermore, because of the limited metabolic capability of the cell line used and because of the experimental conditions, pro-haptens (i.e.chemicals requiring enzymatic activation for example via P450 enzymes) and pre-haptens (i.e. chemicals activated by auto-oxidation) in particular with a slow oxidation rate may also provide negative results.
Test chemicals that do not act as a sensitizer but are nevertheless chemical stressors may lead on the other hand to false positive results. Furthermore, highly cytotoxic test chemicals cannot always be reliably assessed.
Finally, test chemicals that interfere with the luciferase enzyme can confound the activity of luciferase in cell-based assays causing either apparent inhibition or increased luminescence.

Positive control results:

Rep 1:
4 µM: 1.26 - 8 µM: 1.46 - 16 µM: 1.94 - 32 µM: 2.40 - 64 µM: 4.13 - EC1.5 = 8.74 and Imax = 4.13
Rep 2:
4 µM: 1.14 - 8 µM: 1.24 - 16 µM: 1.43 - 32 µM: 1.67 - 64µM: 3.95 - EC1.5=20.67 and Imax = 3.95
Mean:
4 µM: 1.20 - 8 µM: 1.35 - 16 µM: 1.69 - 32 µM: 2.03 - 64µM: 4.04 - EC1.5=13.44 (geometric mean) and Imax = 4.0

Key result

Run / experiment:

other: induction

Parameter:

other: EC1.5

Remarks:

expressed in mM

Value:

0.01

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: viability

Parameter:

other: Imax

Remarks:

mean value

Value:

145.79

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: induction

Parameter:

other: IC70

Remarks:

geometric mean expressed in mM

Value:

0.11

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

All validity criteria are met, study is considered valid.

The test item dilutions were checked by visual inspection after 48 hours at 37°C, no undissolved residues or precipitate or phase separation remained.

Interference of the test item with the detection of luminescence was not checked.

	VIABILITY	INDUCTION
	IC70 (mM)	Imax	Linear EC 1.5 (mM)	EC 1.5 Lin/lLog (mM)
Rep 1	0.07	56.36	< 0.010	< 0.010
Rep 2	0.19	235.22	< 0.010	< 0.010
Mean		145.79
Geometric mean	0.11

      

Interpretation of results:

other: May be classified as a potential skin sensitizer

Remarks:

The test method KeratinoSensTM is considered scientifically valid to be used as part of an integrated approaches to testing and assessment, to support the identification of the sensitization potential of test item for hazard classification and labeling purposes.

Conclusions:

Under the retained experimental conditions of this OECD 442D test, test item may be classified as potential skin sensitizer.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 August 2018 - 6 February 2019

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

other: OECD 442E; In Vitro Sensitisation Assays addressing the Key Event on activiation of dendritic cells on the Adverse Outcome Pathway for Skin Activiation. Annex I: In Vitro Skin Sensitisation: human Cell Line Activation Test (hCLAT)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dated 14 September 2015

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

OECD 442E is one of the in-vitro skin sensitization test method now recommended for REACh registration.

Details on the study design:

MATERIALS AND TEST METHODS:
- Controls for Citotoxicity and h-CLAT
* Medium Control and Solvent Control for the Test Item
Name: Culture medium
* Positive Control (h-CLAT)
Name: DNCB (2,4-dinitrochlorobenzene, CAS No.: 97-00-7) final concentration: 2 and 3 µg/mL, Purity ≥ 99%)
Solvent: DMSO
* Solvent Control for the Positive Control (h-CLAT)
Name: DMSO (Dimethyl sulfoxide, CAS No. 67-68-5) in culture medium, final concentration 0.2%, Purity ≥ 99%

TEST ITEM PREPARATION:
On the day of the experiment (prior to start) test item was dissolved in culture medium.
The maximum concentration of test item was 5000 µg/mL in culture medium, as tested by a solubility test.
For the cytotoxicity test (dose finding assay) eight concentrations of the test item were analysed. For this, dilutions were prepared by 1:2 serial dilutions from 5000 µg/mL in culture medium. The test item formulation was heated up to 37 °C for five minutes to prepare the test item.

TEST SYSTEM AND SUPPORTING INFORMATION:
- Reasons for the Choice of THP-1 Cells
THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202. THP-1 cells are used as surrogate for human myeloid dendritic cells and show enhanced CD86 and/or CD54 expression when treated with sensitisers.

- THP-1 Cell Cultures
Stocks of the THP-1 cell line are stored in liquid nitrogen in the cell bank of Envigo CRS GmbH (aliquots of cells in freezing medium at 1 x 10E6 to 2 x 10E6 cells/mL) allowing the repeated use of the same cell culture batch in experiments. Therefore, the parameters of the experiments remain similar, because of the reproducible characteristics of the cells. Thawed stock cultures are propagated at 37 ± 1.5 °C in plastic flasks. The cells are sub-cultured twice weekly. The cell density should not exceed 1  106 cells/mL. The THP-1 cell suspension is incubated at 37 ± 1.5 °C and 5.0 ± 0.5 % carbon dioxide atmosphere. Cells can be used up to two months after thawing (passage number should not exceed 30).
The passage numbers of the used THP-1 cells were 25 in both cytotoxicity assays and 27 and 28 in the h CLAT runs 1 and 2, respectively.

- Culture Medium
RPMI-1640 Medium, GlutaMAXTM Supplement including 25 mM HEPES, supplemented with 10 % FBS (v/v), 0.05 mM 2 mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate and appropriate antibiotics (100 U/mL of penicillin and 100 µg/mL of streptomycin) is used to culture the cells during the assay. Medium with supplements has to be stored at 2 – 8 °C and used within one month. The culture medium has to be warmed to room temperature just before use.

- Preparation and Seeding of THP-1 Cells
On the day of the cytotoxicity experiment directly before the application of the test item, solvent and medium control, a volume of 500 µL with a cell density of 1.8 - 2 x 10E6 THP-1 cells/mL was seeded in each well of a 24-well flat bottom plate.
For the main experiment (h-CLAT) 0.9 - 1 x 10E6 cells/well in a volume of 500 µL were seeded in a 24-well plate before the treatment.

- Experimental Design and Procedures of the Cytotoxicity Test
* Dose Finding Assay (Flow cytometer)
The test item concentrations investigated in the main experiment (h-CLAT) were determined with two cytotoxicity tests. The tests were performed with independent cell cultures (cells are collected from different culture flasks). The test item was prepared separately for each run.
* Treatment of the Cells
The test item dilutions were prepared freshly before each experiment.
Each volume (500 µL) of the dilutions of the test item, culture medium and solvent control (e.g. 0.2% (v/v) DMSO in culture medium) was added to the cells. Only culture medium was tested as solvent control. The treated THP-1 cells were incubated for 24 ± 0.5 hours. All dose groups were tested in one replicate for each cytotoxicity test. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations.
Each concentration of the test item, culture medium and solvent control were prepared for the 7-AAD staining.
* Staining of the Cells
Each test item-treated and not test item treated cells were collected in sample tubes centrifuged (approx. 250 x g, 5 min), washed twice (2 8 °C) with approx. 600 µL FACS buffer (PBS with 0.1% (w/v) BSA) and re-suspended in a final volume of 1 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 µL of a 7-AAD solution were added in each sample tube.
* Flow Cytometry Acquisition (Cytotoxicity Test)
The following acquisition plots were prepared:
2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
Histogram plot of the FL-3 channel
The voltage of FSC and SSC was set to appropriate levels. FSC and SSC are not needed for the analysis, but the FSC/SSC plot should be checked to make sure that a single population appears without contamination or excessive debris. The FL-3 voltage was set and compensate to appropriate position (FACSCalibur, Becton Dickinson GmbH).
The cell viability was measured by gating-out dead cells stained with 7-AAD. A total of 10,000 living cells were analysed.
The maintenance of the flow cytometer was in accordance with the manufacturer’s instructions. The process of washing was conducted very carefully since insoluble chemicals could flow in the flow line.
* Flow Cytometry Analysis (Cytotoxicity Test)
The cell viability is shown by the cytometry analysis program (% total) or is calculated according to the following equation:
Cell Viability [%]= (Number of living cells)/(Number of acquired cells) ×100
The CV75 value, a concentration showing 75% of THP-1 cell survival (25% cytotoxicity), is calculated by log-linear interpolation using the following equation:
Log CV75 = ((75-c)×Log (b)- (75-a) ×Log (d) )/(a-c )
Where:
a is the minimum value of cell viability over 75%
c is the maximum value of cell viability below 75%
b and d are the concentrations showing the value of cell viability a and c respectively
* Acceptability of the Cytotoxicity Assay
The cytotoxicity test is considered to be acceptable if it meets the following criteria:
The cell viability of the medium and solvent control (if the test item is solved in DMSO) should be more than 90%.
* Calculation of the Test Doses for the Main Experiment (h-CLAT)
The mean of two CV75 values was used to determine the dose-range for the main experiment (h-CLAT).
Eight final concentrations (µg/mL) were used for the test item in the main experiment (h CLAT). The highest concentration used was 1.2 × CV75 and seven further concentrations of the test item were prepared by serial 1:1.2 dilution.

- Experimental design and Procedures of h-CLAT
The test item was tested in two independent runs. The h-CLAT runs were performed on different days.
* Treatment of the Cells
For the test item exposure the highest dose solution calculated from the cytotoxicity assay was prepared corresponding to 1.2 × CV75. Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment.
Each volume (500 µL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations.
Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).
* Staining of the Cells
The triplicates of each test item-treated and not test item-treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 x g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 µL of blocking solution at 2 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 µL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control).
All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis procedures.
The cells with the different antibodies or the IgG1 were mixed and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).
* Sample Preparation for Measurement
After staining with the antibodies, the cells were washed twice (2 8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 µL of a 7-AAD solution were added.
* Flow Cytometry Acquisition
Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH, software FACSComp 6.0), the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions.
The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry using the software Cellquest Pro 6.0. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7 AAD fluorescence signal.
Preparation of the acquisition
The following acquisition plots were prepared:
2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
Histogram plot of each channel (FL-1 and FL-3, respectively)
The voltage of FSC and SSC was set with untreated cells to appropriate levels. FSC and SSC are not needed for the analysis, but the FSC/SSC plot was checked to make sure that a single population appears without contamination or excessive debris. The FL-1 and FL-3 voltage were set and compensate to appropriate position. The FL-1 voltage was set using the FITC labelled-mouse IgG1 medium-treated cells tube, as such that the MFI of control cells was set in the range between 1.0 and 4.0 (Geo Mean) and in the range between 3.0 and 4.0 (Geo Mean) with the FITC labelled CD54 medium-treated cells (FACSCalibur, Becton Dickinson GmbH).
The cell viability was detected by setting an R1-gate (dead cells are gated-out by staining with 7-AAD). Therefore, the R1 gate was set approximately at the middle position between the peak of the negative fraction and the positive fraction in the FL-3 histogram using DNCB-treated cells. The negative fraction corresponds to the living cells and was kept for the subsequent analyses. For each controls and all test item concentrations, the cell viability was recorded from the isotype control cell tube (stained with FITC labelled-mouse IgG1), the CD54 and CD86 cell tube. Where only the isotype control cells were used for the cell viability evaluation.
The maintenance of the flow cytometer was in accordance with the manufacturer’s instructions. The process of washing was conducted very carefully since insoluble chemicals could flow into the flow line.
Acquisition
Dead cells were gated-out by staining with 7-AAD. Gating by FSC (forward scatter) and SSC (side scatter) was not done. A total of 10,000 living cells were analysed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The other tubes were acquired without changing the settings of the cytometer. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was calculated, but excluded from the evaluation, if the cell viability was less than 50% (due to diffuse labelling of cytoplasmic structures that could be generated due to cell membrane destruction).
* Data Analysis and Interpretation
Flow Cytometry Analysis: The RFI is used as an indicator of CD86 and CD54 expression.
The cell viability from the isotype control cells, CD54 and CD 86 cells is calculated according to the following equation:
Cell Viability [%]= (Number of living cells)/(Number of acquired cells) ×100
Where only the isotype control cells (which are stained with mouse IgG1 (isotype) antibodies) are used for the cell viability evaluation.
* Acceptance Criteria
The following acceptance criteria should be met when using the h-CLAT method:
Cell viability of medium control and DMSO control should be more than 90%.
In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50% in at least one concentration of the two tested positive control concentrations.
For the test chemical, the cell viability should be more than 50% in at least four tested concentrations in each run.
Negative results are acceptable only for test items exhibiting a cell viability of < 90% at the highest concentration tested (i.e. 1.2 × CV75). If the cell viability at 1.2 × CV75 is ≥ 90% the negative result should be discarded. In such case it is recommended to try to refine the dose selection by repeating the CV75 determination. It should be noted that when 5000 μg/mL in saline (or medium or other solvents/vehicles), 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test chemical, a negative result is acceptable even if the cell viability > 90% (OECD 442E guideline).
* Prediction model
For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs (OECD 442E guideline):
− The RFI of CD86 is ≥ 150% at any tested concentration (with cell viability ≥ 50%);
− The RFI of CD54 is ≥ 200% at any tested concentration (with cell viability ≥ 50%).
Otherwise, the h-CLAT prediction is considered NEGATIVE (see chapter 5.6.7.1).
Based on the above, if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered POSITIVE and a third run does not need to be conducted. Similarly, if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE without the need for a third run. If, however, the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred to as N), the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered POSITIVE. An h-CLAT prediction should be considered in the framework of an IATA (OECD 442E guideline).

Positive control results:

In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.
Results: 2 µg/mL DNCB (CD 54): 247.0%
2 µg/mL DNCB (CD 86): 259.0%
3 µg/mL DNCB (CD 54): 361.2%
3 µg/mL DNCB (CD 86): 325.9%

Run / experiment:

other: cytotoxicity test

Parameter:

other: CV75

Remarks:

mean value expressed in µg/mL

Value:

393.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: See Remarks

Remarks:

Cytotoxic effects were observed following incubation with the test item starting with a concentration of 625 µg/mL up to the highest tested concentration (5000 µg/mL) in both cytotoxicity tests.

Key result

Run / experiment:

other: h-CLAT test

Parameter:

other: RFI of CD 86

Remarks:

expressed as %

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

RFI of CD86 was > 150 for the following concentrations: 132, 190, 228, 273, 328, 393 and 472 µg/mL (first run) and 273, 328, 393, 472 µg/mL (second run).

Key result

Run / experiment:

other: h-CLAT test

Parameter:

other: RFI of CD54

Remarks:

expressed as %

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

RFI of CD54 was > 200 for the following concentrations: 158, 190, 228, 273, 328, 393 and 472 µg/mL (first run) and 273, 328, 393, 472 µg/mL (second run).

Other effects / acceptance of results:

The test item with a log Pow of app. -0.2 was tested in 2 independent runs. No precipitations were observed in the wells of all test item treated cells in both h-CLAT runs after treatment. The RFI of CD86 and CD54 was equal or greater than 150% and 200%, respectively, in at least one concentration of both runs. Therefore, the h-CLAT prediction is considered positive for the tested test item in this h-CLAT.
It is possible to calculate the EC200 value for CD54 for both runs and the EC150 value for CD86 of the second run. However, it is not possible to calculate the EC150 for CD86 of the first run, since the lowest tested test item concentration of the first h-CLAT run showed a RFI value > 150%. Furthermore, only two instead of three h-CLAT runs were conducted in this study, therefore a more precisely value of the EC150 and EC200 could not be calculated as recommended in the OECD 442E Guideline. For this reason no EC200 and EC150 value was calculated.
In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was > 50%. Except the CD54 RFI and CD86 RFI value of the positive control (2.0 µg/mL DNCB) in the second h-CLAT run did not exceed the positive criterion (CD54 ≥ 200%, CD86 ≥ 150%). However, this is considered to be acceptable since the respective RFI values of the positive control (3.0 µg/mL DNCB) in the second h-CLAT run exceeded the positive criteria.

Interpretation of results:

other: May be classified as a potential skin sensitizer

Remarks:

This human cell line activation test can be used as part of a testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.

Conclusions:

The test item with a log Pow of app. -0.2 activated THP-1 cells under the test conditions of this study conducted according to OECD guideline 442E. Therefore, the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available