(4-carboxybutyl)triphenylphosphonium bromide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Feb 2020 - 29 Apr 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The study is conducted according to the internal requirements from Senza Gen AB (DB-Alm Protocol GARD – Genomic Allergen Rapid Detection (GARDskin)).
The Genomic Allergen Rapid Detection (GARD™) is an in vitro assay designed to predict the ability of chemical substances to induce skin sensitization based on the analysis of the relative expression levels of a biomarker signature of 196 genes using a human myeloid leukemia cell line called SenzaCell. The GARD™ is based on chemical stimulation of the SenzaCell line, acting as an in vitro model of human Dendritic Cells (DCs). The readout of the assay is a transcriptional quantification of the genomic predictors, collectively termed the GARD™ Prediction Signature (GPS), using Nanostring nCounter technology.
The prediction of a substance to be either a sensitizer or non-sensitizer bases on a derived decision value (DV) from a Support Vector Machine (SVM) model. The algorithm of the machine uses known data sets from already done cell stimulations with known chemicals from the training set. With this data set a DV is created for each new sample. The mean of the DV is used for classification.
GARD™ is intended to be used as either a stand-alone assay, or as part on an Integrated Testing Strategy (ITS), for the assessment of chemical skin sensitizers.

GLP compliance:

yes (incl. QA statement)

Remarks:

The GLP compliance will not be effective for the sample analysis at SenzaGen AB as this part of the study will be performed under non-GXP at test site 1.

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

The GARD™ method mimics the immune system by using the human dendritic cells. It predicts the ability of chemical compounds to induce skin sensitization by measuring changes in the genomic profile of the cells after chemical treatment. The GARD™ addresses the third key event of the AOP, the activation of human dendritic cells. Genomics-based models offer reliable alternatives to animal testing.

Test material

Test material information

Constituent 1

Reference substance name:

(4-carboxybutyl)triphenylphosphonium bromide

EC Number:

241-782-5

EC Name:

(4-carboxybutyl)triphenylphosphonium bromide

Cas Number:

17814-85-6

Molecular formula:

C23H24BrO2P

IUPAC Name:

(4-carboxybutyl)triphenylphosphonium bromide

Test material form:

solid: crystalline

In vitro test system

Details on the study design:

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%). Vortex mixing was used to aid solubilisation.
After a suitable solvent was determined, the solubility of the test item solution was tested after a 1:100 dilution in semi-complete media.
The solvent was present at a constant volume ratio of 0.1% (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.

Controls
Medium controls, a negative 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 (two times).
Solvent Control: DMSO was used as a Solvent control. The final concentration of DMSO in cell culture medium was 0.1% (v/v).
Positive Control: p-Phenylenediamine (PPD; CAS No.: 106-50-3) at a final concentration of 50 μM was tested concurrently with the test item. PPD was dissolved in DMSO and diluted according to the procedure described for the test item further below under Main Stimulation, resulting in a final DMSO concentration of 0.1% (v/v).

Test System

FACS
FACS: BD Lyric
Software: BD FACSuite V1.0

Cell line
The test was carried out using human myeloid leukemia cell line Senza cells provided by SenzaGen AB. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and sub-cultured at least 2 weeks before they were used in the in vitro GARD™. Only cells at a low passage number (< 16) were used. Cells are routinely passaged every 3-4 days at a density of 0.2 x 10E06 cells/mL.
Cells are cultured in 175 cm² culture flasks in MEM/Alpha Modification with L-glut, Ribo- Deoxyribo supplemented with 20% fetal bovine serum (= semi-complete medium), and freshly added 40 ng/mL GM-CSF in a humidified incubator at 37 ± 1 °C and 5% CO2 (complete medium).

Dose groups
Medium Control: semi-complete medium
Solvent Control: 0.1% (v/v) for DMSO
Positive Control: 50 μM PPD
Test Item:
Input finder: 9 concentrations of the test item down to 1 mM stock solution (see Input Finder Assay below)
Main stimulation: 1 concentration, which leads to a relative cell viability of 90 ± 5% or above (see Main Stimulation below).

Pre-Experiments
Solvent Finding
Solubility of the test item was determined prior to the Input Finder assay. The test item was dissolved in 100% DMSO at a final concentration of 500 mM. If soluble, DMSO was used as the solvent. If the test item was not soluble in DMSO at 500 mM, the test item was dissolved at 500 mM in sterile water. If soluble, sterile water was used as the solvent. If the test item was not soluble in sterile water at 500 mM the highest soluble concentration was determined by diluting the solution from 500 mM to a minimal concentration of 1 mM in both solvents. Heating up to 37 °C and vortexing was applied to achieve complete dissolution.
After a solvent was determined, the solubility of the test item solution in semi-complete medium was tested by a 1:100 dilution. If precipitation, phase separation or turbidity is observed the test item was further diluted in the solvent and tested again in semi-complete medium at lower concentrations after a 1:100 dilution. This was done until no precipitation, phase separation or turbidity was observed in semi-complete medium.

Phenotypic Quality Control
At the same day, as the chemical stimulation was performed, the cells were quality controlled by a phenotypic analysis. 2 x 10E05 cells were transferred six times into FACS tubes. The cells were washed twice with Dulbecco’s phosphate buffered saline (DPBS) containing 0.5-1% bovine serum albumin (BSA; i.e. FACS buffer). After washing the cells were resuspended in 50 μL FACS buffer and stained with the following surface markers for 15 minutes at 2-8 °C in the dark: Isotype, CD86, CD54, HLA-DR, CD80, CD34, CD14, CD1α. Additionally, for cells were stained with propidium iodide for cell viability measurement.
After staining the cells were washed once with 1 mL FACS Buffer and the cell pellets were resuspended in 200 μL FACS buffer for analysis.
If the phenotypic quality control passes all criteria, the cells were used for stimulation.

Input Finder Assay:
Starting from the highest soluble concentration in the most applicable solvent and medium determined in the solvent finding experiment, a maximum of nine stock solutions (i.e. nine concentrations) down to 1 mM were prepared. (i.e. 500 mM – 400 mM – 300 mM – 200 mM – 100 mM – 50 mM – 10 mM – 5 mM – 1 mM). These stock solutions were further diluted 100-fold into semi-complete medium giving the stock B solutions. For testing, the highest concentration was 500 μM, even if this concentration is non-toxic. The stock B solutions were finally used for treatment by adding the test item to the SenzaCell suspension in complete medium in a 24-well plate to achieve a further 10- fold dilution.
For testing, SenzaCells were pre-cultured for 3 – 4 days in culture flasks at a cell density of 0.2 x 10E06 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation and were re-suspended in fresh semi-complete medium at a density of 2.22 x 10E05 cell/mL. Then 1.8 mL of cell suspension was seeded into a 24-well flat-bottom plate (4 x 10E05 cells/well).
The stock B solutions of the solvent control and the dilution range of the test item was mixed 1:10 (v/v) with the cell suspension prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37°C ± 1 °C and 5% ± 0.5% CO2.
The cells were stained to check the quality of the cells.
After 24 h ± 0.5 h of exposure, cells were transferred and distributed into two FACS tubes. FACS buffer was added and the cells were collected by centrifugation (approx. 300 x g). The supernatant was discarded and the remaining cells were washed once with FACS buffer. After washing, cells were re-suspended in 50 μL propidium iodide (PI) solution (1 μg/mL) in FACS buffer. Cells were stained ~ 10 minutes at 2-8°C in the dark. After staining cells were washed with FACS buffer again and re-suspended in 200 μL FACS buffer.
The PI uptake of the cells (as an indicator of 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.
The test item that induced cytotoxicity was used for the main stimulation at the concentration that induces 90% ± 5% relative viability (RV90). Test items that induced no or weak cytotoxicity (≥ 95.5% relative viability) were tested at 500 μM final concentration or at the highest soluble concentration.
If cytotoxic effects were determined, but the relative viability is not between the range of 90% ± 5% the input finder was repeated with another dilution range.
The determined concentration was used for the main stimulation experiment (= GARD input concentration).

Main Stimulation:
A stock solution of the positive control (50 mM) in DMSO and a stock solution of 1000 x the GARD input concentration of the test item in the solvent determined in the solvent finding pre-experiment were freshly prepared immediately before use.
The stock solution of the positive control, pure DMSO as a negative control, the stock solution of the test item and the solvent of the test item were diluted 100-fold into semi-complete medium (= stock B) to achieve the in-well concentration of 0.1% DMSO.
For testing, SenzaCells were pre-cultured for 3 – 4 days in culture flasks at a cell density of 0.2 x 10E06 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation and were re-suspended in fresh semi-complete medium at a density of 2.22 x 10E05 cell/mL. Then 3.6 mL of cell suspension were seeded into a 12 well plate-bottom plate (8 x 10E05 cells/well).
The stock B solutions of the positive control, the solvent control of the test item input concentration and the solvent of the test item were mixed 1:10 (v/v) with the cell suspension prepared in the 12-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37°C ± 1 °C and 5% ± 0.5% CO2.
The cells were stained to check the quality of the cells as described in chapter 10.6.2.
After 24 h ± 0.5 h of exposure, 1 mL of the cells were transferred three times into small reaction tubes and further 500 μL were transferred twice into two FACS tubes. The cells of the reaction tubes were collected by centrifugation (approx. 300 x g), re-suspended in 500 μL TRIzol®and frozen at < -20 °C. The cells of the FACS tubes were washed twice with FACS buffer. After washing, cells were re-suspended in 50 μL propidium iodide (PI) solution (1 μg/mL) in FACS buffer. Cells were stained for approx. 10 minutes at 2-8°C in the dark. After staining, cells were washed with FACS buffer again and were re-suspended in 200 μL FACS buffer.
The PI uptake of the cells (as an indicator of 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 were calculated for each sample.
If the cell viability passes the criteria, described in chapter 11.2 the RNA of the TRIzol®samples were collected as described as follows.

RNA Isolation:
The first tube of each TRIzol®sample triplicate was thawed slowly on ice. The samples were centrifuged to remove particulate debris and the supernatant was transferred into new tubes. An equal volume of 95% - 100% ethanol was added and the whole mixture was transferred on the column of a Zymo-SpinTM IIC Columns.
The flow through was discarded and the column was washed twice with 400 μL with the Direct-zolTM RNA PreWash. The flow through was discarded again. After the pre-washing step the column was washed with 700 μL RNA wash buffer. The flow through was discarded again and the column was placed in a new RNAse-free tube. The RNA was eluted with 25 μL RNAse-free water. After centrifugation the collected RNA-eluate was transferred onto the column again to elute remaining RNA. The RNA was stored at -80°C for shipment.

End-point Measurement

RNA Quality Control:
The RNA quality and quantity was measured with an Agilent 2100 BioAnalyser. If the RNA sample did not pass the quality control criteria the RNA had to be isolated from the last two TRIzol®samples and quality had to be measured again.
RNA samples which had passed the criteria underwent Nanostring endpoint measurement with a codeset designed for GARD skin analysis.

Nanostring Hybridisation:
All hybridization samples used a total RNA input of 100 ng.
All RNA samples were diluted to 20 ng/μL with RNA-free water: 5 μL of each diluted sample was used for hybridization.
For hybridization mix 8 μL of the master mix (provided in the Nanostring master kit), 5 μL RNA sample (20 ng/μL) and 2 μL of the capture code set were mixed. The mixture was incubated in a thermocycler at 65°C for 24 h ± 0.5 h.

Setting up the Nanostring Preparation and Measurement:
The nCounter Prepstation (Nanostring) was prepared as described in the manual. After hybridization the samples were removed from the thermocycler and centrifuged before opening. The samples were placed in the nCounter Prepstation, too.
The cartridges were analysed by an nCounter Digital Analyser (Nanostring).


Data analysis
Calculation relative viability:
The cell viability was measured with FACS. The FACS data analysis was performed using the software BD FACSSuite V1.0. Further data analysis like calculation of the GARD input concentration and calculation of the relative viability was performed using the software Microsoft Excel 2010. The relative viability percentage calculation is based on the following equation:
Rv= (Vs/Vc)*100
(Rv = Relative viability of the sample in %
Vs = Absolute viability of the sample in %
Vc = Absolute viability of the mean of the two unstimulated control samples in %)

Nanostring data analysis:
The Nanostring raw data files were analysed using the GARD Data Analysis Application provided from SenzaGen AB.
If the DV shows sensitisation potential, the RNA samples can be measured with a different codeset again to get information on the potency of the substance in order to categorize the test item into packaging group 1A and 1B according to EU-CLP.

Vehicle / solvent control:

DMSO

Negative control:

other: semi-complete medium

Positive control:

other: p-Phenylenediamine

Results and discussion

In vitro / in chemico

Results

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: mean Decision Value (DV)

Value:

-1.9

Cell viability:

mean relative cell viability: 100.9%

Vehicle controls validity:

valid

Remarks:

mean DV: -1.88; mean relative cell viability: 99.77%

Negative controls validity:

valid

Remarks:

mean relative cell viability: 100.0%

Positive controls validity:

valid

Remarks:

mean DV: 6.63; mean relative cell viability: 90.96%

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

The decision values for the test item are < 0 (-1.9). Therefore the test item has to be classified as a
non-sensitizer
The decision values of the positive control are > 0 (6.63) and for the negative control are < 0 (-1.88).
The controls confirmed the validity of the study for all experiments for cytotoxicity.

Any other information on results incl. tables

Solvent Finding

All test item solutions were freshly prepared immediately prior to use. The test item was soluble in DMSO at a concentration of 500 mM.

Input Finder

The Input Finder assay was performed using stock solutions with a concentration of 500 mM (applied concentration 500 μM) and cells harvested in passage 4.

Relative cell viability of the Input Finder Assay after 24 h Test Item exposure

The Input Finder Assay was performed once. The first Input Finder showed no toxicity.

Quality Control

The cells for the Input Finder Assay passed the Quality Control Criteria.

Main Stimulation

For determination of the sensitizing potential of the test substance three independent experiments were performed using separate thawed, cultivated and harvested cells at passage 6 (main stimulation 1 and 2) and passage 7 (main stimulation 3) for all three main stimulations. For each experiment separately weighted samples and preparations were used.

Quality Control

The cells for the Main Stimulation passed the Quality Control.

RNA Quality

For the Nanostring measurement all single TRIzol®RNA isolations fulfilled the acceptance criteria.

Nanostring measurement

The Nanostring measurement fulfilled all the acceptance criteria.

Data Analysis for GARD-Skin

The “rlf files” of the Nanostring measurement were transferred for analysis to the testing facility. The decision values were generated by analysing the rlf files with the GARD Data Analysis Application provided from SenzaGen AB. These decision values are reported in the following table.

Decision Values of the Test Item and the Controls

Negative Control		Positive Control		(Methoxymethyl)triphenylphosphonium chloride (1:1) (MMC) (10 μM)
Main Stimulation	Decision Values	Main Stimulation	Decision Values	Main Stimulation	Decision Values
1	-1.57	1	6.73	1	-1.91
2	-2.31	2	6.49	2	-2.21
3	-1.75	3	6.68	3	-1.59
Mean	-1.88	Mean	6.63	Mean	-1.90
Prediction
negative		positive		negative

 

Acceptance Criteria for toxicity

All the acceptance criteria are met for the BioAnalyser and Nanostring measurement.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item did not change the genomic profile of the cells for sensitization in at least three independent experiment runs. Therefore, the test item might be considered as non-sensitizer.
The data generated with this method may be not sufficient to conclude a sensitization potential of the test item and should be considered in the context of integrated approach such as IATA.