Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: Genomic Allergen Rapid Detection (GARDTM)
Version / remarks:
currently under ECVAM validation.
Principles of method if other than guideline:
please see documentation attached regarding GARD skin test.
It is a new test currently under validation with ECVAM/OECD.
Genomic Allergen Rapid Detection (GARDTM) is an in vitro assay designed to predict the ability of chemical substances to induce skin sensitisation 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 GARDTM 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 GARDTM Prediction Signature (GPS), using Nanostring nCounter technology.
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of dendritic cells

Test material

Constituent 1
Reference substance name:
Docosan-1-ol, icosan-1-ol, octadecan-1-ol and their reaction products with betaine and ethanesulphonic acid
IUPAC Name:
Docosan-1-ol, icosan-1-ol, octadecan-1-ol and their reaction products with betaine and ethanesulphonic acid
Test material form:
solid: bulk

In vitro test system

Details of test system:
other: Human myeloid leukemia-derived cell line SenzaCellTM (acting as an in vitro model of a human Dendritic Cell)
Details on the study design:
TEST SYSTEM AND EQUIPMENT
The test system and equipment which was used in the study are detailed below:

Test system : SenzaCells : SenzaGen
Flow cytometry : FACSverse : BD
RNA isolation : Zymo-spinTM IIC Columns (Cat R5052) : ZymoResearch
RNA Quantification and Quality Control:
-2100 Bioanalyzer : Agilent
-2100 Expert Software : Agilent
-Agilent RNA 6000 Nano Kit : Agilent
Endpoint Measurement
-CodeSet: GPS200_v2 :Nanostring
-nCounter Prepstation : Nanostring
-nCounter Digital Analyzer : Nanostring
-nCounter Master kit : Nanostring
-SimpliAmp Thermal Cycler : Applied Biosystems
GARD Data Analysis Application: GDAA v2.2.1 : SenzaGen


The test system consists of the human myeloid leukemia-derived cell line SenzaCellTM acting as an in vitro model of a human Dendritic Cell (DC), and maintained in a-MEM medium (GE-Healthcare Life sciences, Hycline) supplemented with 20% (volume/volume) fetal calf serrum ((Life Technologies, Carlsbad, CA) and 40ng/ml recombinant human Granulocyte macrophage colony stimulating factor (rhGM-CSF, Miltenyi Biotec, Germany). During cell expansion, subculture with fresh medium was performed every 2-3 days to a concentration of 0.2x10^6 cells/ml. Working stocks of culturess were grown for a maximum of 16 passages or two months after thawing. Cells were seeded for stimulation directly following a cell split, i.e. test substance stimulations were scheduled to coincide with routine cell culture maintenance. The cell stimulations were initiated when a stable cell culture was established ie when at least a dupllication of the cells between cell passages was seen, and depending on the purpose of the cell stimulation, at specific cell passage ranges:
-for GARD imput finder, cells at passage number 4-16 are used
-for GARD main stimulation, cells at passage number 6-12 are used.
To ensure that the senzacells were maintained in an inactivated state, and detection of a potential phenotypic drift, the cells were stained with a panel of biomarkers, CD54, CD86, HLA-DR, CD34, CD80; CD14 and CD1a.


TEST ITEM SOLUBILITY AND CYTOTOXICITY ASSESSMENT
The test item was tested for solubility in the full range of vehicles compatible with the GARD assay.
After extensive solubility testing it was found that the test item was not soluble in a-MEM medium but could be added as a dispersion in a-MEM medium to the cells as follows. The test item was added to isopropanol and heated to its melting point, 70°C +/- 5°C, under these conditions the test item was fully dissolved in isopropanolol. The dissolved, hot test item was then added to equilly hot a-MEM medium. The mixture was immediately vortexed vigorously and cooled to 37°C +/- 2°C in a cell incubator and then added to the cells together with 20% FBS and 40ng/ml rh-GMCSF. The test item was, efter addition to medium, dispersed in the solution.

As a test or reference item may have a cytotoxic effect on the cells, this effect was monitored. If the test item was poorly dissolved in cell media, the maximum soluble concentration was assessed. The test item was titrated to concentrations ranging from 1µM to the maximum soluble concentration in cell media. For a freely soluble test item, 500µM was set as the upper limit of the titration range.
For cytotoxic assessment, senzacells were seeded in a 24 well plate followed by addition of different concentrations of the test or reference item, concentration range determined in the solubililty assessment.
The final cell concentration was 0.2x10^6 cells/ml in 2 ml. After incubation for 24h +/- 0.5h at 37°C +/-1°C andd 5%+/-0.5% CO2, harvested cells were stained with the viability marker propidium iodide (PI) (BD Bioscience USA) and analysed using flow cytometry. PI-negative cells were defined as viable, and the relative viability (Rv) of cells stimulated with each concentration in the titration range was calculated as : Rv = (Vs/Vc)x100
Vs = viability of the sample (test item) in %
Vc= mean viability of the two unstimulated control samples in %.

For a cytotoxic test or reference item, the concentration yielding 90% relative viability (Rv90) was used for the GARD assay, the reason being that this concentration demonstrates bioavailability of the test or reference item used for stimulation, while not impairing immunological responses. For a non-cytotoxic test or reference item, a concentration of 500µM was used if possible. For a non-cytotoxic test or reference item that is insoluble at 500µM in cell media, the highest soluble concentration was used. Whichever of these three criteria were met, only one concentration was used for gene expression analysis. The concentration to be used for any given chemical is termed the 'GARD input concentration'.

GARD MAIN STIMULATION
In the main stimulation ,test and reference items werer assayed in bibological replicates(n>=3), using different senzacell cultures. These cell stimulations were performed in the same way as in the previous step, with the exception that the final amount of cells/well was 0.2x10^6 clls/ml in 4 ml cell medium. After incubation for 24h +/60.5h at 37°C +/-1°C and 5%+/-0.5% CO2, the cells were lysed in TRIzol reagent (life technologies) and stored at -20°C +/-5°C. In parallel, the cells were PI stained and analysed by flow cytometry to verify the expected relative viability.

ISOLATION, QUANTIFICATION AND QUALITY CONTROL OF RNA
RNA was isolated according to manufacturer's instructions (Zymo-Research) for cells lysed with TRIzol reagent and a 2%l RNA sample aliquit was taken from each RNA sample for RNA quantification and quality control.
For RNA quantification, the RNA smaple aliquots were thawed on ice, maximum 12 samples at a time, and assayed with the Bioanalyzed 2100. For each completed RNA analysis, a software generated PDF report was saved together with raw XAD files. The PDF report contains software calculateed values for total RNA content and RNA Integrity Number (RIN) for each analyzed sample. All RNA samples were checked against the Quality control criteria and passed samples were further used for the endpoint measurements.

ENDPOINT MEASUREMENTS
a total of 100ng of RNA was used as sample input in a hybridization assay with the endpoint specific GARDskin CodeSet (NanoString Technologies, Seattle, WA). Each hybridized sample was prepared on cartridge using nCounter Prep Station and individual transcripts of the endpooint specific biomarker signature was quantified using nanostring digital analyzed (nCounterMAX, NanoString Technologies, Seattle, WA).

RCC raw data files were exported from the Nanostring n Counter and quality controlled, according toe the acceptance criteeria in section 9, prior to using the GARD data analysis application for classification of skin sensitizers. Data files that did not pass the acceptance criteria were removed from the classification analysis.

DATA ANALYSIS AND CLASSIFICATION
For assessment of skin sensitization, a support vector machine previously modelled on a training data set corresponding to samples used for assay development was used. For a comprehensibe overview of the training data set and methods, see Forreryd et al., 2016. Batch variations between the training data set and the test data set was elminated using the batch adjustment by reference alignment (BARA) method, using unstimulated cells as reference control. Each sample in the test set was assifned a Decision Value (DV) based on its transcriptional levels of the enedpoint specific biomarker signature.

In the GARDskin assay, a positive mean Decision Value >=0 (n>=2) will classify the test item as a sensitizer, whereas a negative mean Decision Value ill classify the test item as a non-sensitizer. Both positive and negative reference items should be accurately classified as sensitizer and non-sensitizer, respectively, for the classification to be valid.
Vehicle / solvent control:
other: isopropanolol
Negative control:
other: solvent control: isopropanolol
Positive control:
other: PPD in DMSO

Results and discussion

Positive control results:
quality criteria: pass

In vitro / in chemico

Results
Key result
Run / experiment:
mean
Parameter:
other: Mean decision value
Value:
-0.99
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
All the acceptance criteria are met for the BioAnalyser and Nanostring measurement.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Executive summary:

The sensitization potential of the test substance was performed according to the GARD assay (It is a new test currently under validation with ECVAM/OECD). Genomic Allergen Rapid Detection (GARDTM) is an in vitro assay designed to predict the ability of chemical substances to induce skin sensitisation 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 GARDTM 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 GARDTM Prediction Signature (GPS), using Nanostring nCounter technology.)


 


In this study under the given conditions the test item (at a concentration of 500µM using isopropanol at 0.25% as vehicule) did not change the genomic profile of the cells for sensitisation in at least three independent experiment runs. Therefore, the test item might be considered as non-sensitiser.


 


Please note that other tests are under investigation because the OECD tests proposed by the AOP in sensitization have proved to be unfeasible on the substance:
-DPRA not feasible on UVCB
-Keratinosens: preliminary solubility tests in the study were carried out and did not lead to obtaining a stable mixture in solvents, not meeting the conditions required for the study.