Batilol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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

Test material information

Constituent 1

Reference substance name:

Batilol

EC Number:

208-874-7

EC Name:

Batilol

Cas Number:

544-62-7

Molecular formula:

C21H44O3

IUPAC Name:

3-(octadecyloxy)propane-1,2-diol

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Batch No.: 8462
Purity: approx. 99 %
Physical State: solid (powder)
Colour: white to pale yellow
Stability: stable
Storage Conditions: room temperature, protected from light
Expiry Date: 10.07.2021
Safety Precautions: The routine hygienic procedures will be sufficient to assure personnel health and safety.

In vitro test system

Details on the study design:

Preparation of the test item:
the test item is dissolved in either dimethyl sulfoxide (DMSO), water or in a different suitable solvent

Test system:
FACS:
FACS: BD Canto II
Software: BD FACS DIVA 6.0
Voltage Settings: FSC: 129 V, SSC: 435 V, FITC: 290 V, PE/PI: 315 V
Threshold value of FSC: 5000
Compensation: PI – 21.09 % FITC, FITC – 1.41 % PI

Cell line:
Human myeloid leukemia cell line Senza cells, provided by SenzaGen AB.
Cells tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and sub-cultured at least 2 weeks before the test.
Only cells at a low passage number (<16) were used.
Cells are cultured in 175cm2 culture flasks (Greiner) in MEM/Alpha Modification with L-glut, Ribo-Deoxyribo (Thermo Scientific; Cat. No.: SH30265.01) supplemented with 20% fetal bovine serum (FBS, Gibco Life Science, Cat No.: 10270-106) (= semi-complete medium), and freshly added 40 ng/mL GM-CSF (Miltenyi Biotec, Cat No.: 130-093-868) 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 or a different solvent for the test item and the positive control
Positive Control: 75 μM p-Phenylenediamine (CAS : 106-50-3)
Test Item:
Input finder: 9 concentrations of the test item down to 1 mM stock solution
Main stimulation: 1 concentration, which will lead to a cell viability of 90 ± 5%

Experimental procedure:
pre-test:
Nine concentrations (stock solutions) 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.
Highest concentration tested: 500µM.

Main test:
A stock solution of the positive control (75 mM) in DMSO and a stock solution 1000-fold higher as 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 at least 72 h - 96 h in culture flasks at a cell density of 0.2 x 10^6 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation and were re-suspended in fresh semi-complete medium at a density of 2.22 x 10^5 cell/mL. Then 3.6 mL of cell suspension were seeded into a 12 well plate-bottom plate (2 x 10^5 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.

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 will be 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 will be calculated for each sample according to the following equation:
Rv = (Vs/Vc)X100
where: 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 %

RNA isolation:
The first tube of each TRIzol sample triplicates 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 (ZymoResearch Cat.R5052).
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 was transferred on the column again to elute the whole RNA. The RNA was stored at -80 °C for shipment.


Endpoint Measurement (at Test Side 1)

1/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 (see section 11.3) 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 undergo Nanostring endpoint measurement with an appropriate code set for GARD skin.

2/ Nanostring Hybridisation
All hybridization samples were use a total RNA concentration of 100 ng.
All RNA samples were diluted to 20 ng/μL with RNA-free water to use 5 μL of each 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 whole approach was incubated in a thermocycler at 65 °C for 24 h ± 0.5 h.

3/ 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 spinned down before opening. The samples were placed in the nCounter Prepstation, too. The preparation of the nanostring cartridges took around 3 hours.
The cartridges were analysed by an nCounter Digital Analyser (Nanostring).

Results and discussion

In vitro / in chemico

Results

Key result

Run / experiment:

mean

Parameter:

other: Mean decision value

Value:

-1.2

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.

Any other information on results incl. tables

Prediction for skin sensitization:

The prediction is defined as described below:

-      If the mean decision value (DV) of biological replicate samples is ≥0, the substance is classified as a sensitizer

If the mean decision value (DV) of biological replicate samples is <0, the substance is classified as anon-sensitizer

Results table:

Negative Control		Positive Control		BATILOL
Decision Values	Main Stimulation	Decision Values	Main Stimulation	Decision Values
-1.57	1	10.17	1	-0.97
-1.56	2	9.37	2	-1.35
-1.99	3	9.1	3	-1.28
-1.71	Mean	9.55	Mean	-1.20
Predicition
negative		positive		negative

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 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.