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Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 Feb - 01 Jun 2021
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:
2021
Report date:
2021

Materials and methods

Principles of method if other than guideline:
- Principle of test: The Genomic Allergen Rapid Detection™ assay for hazard assessment of skin sensitizers (GARD™skin) provides binary hazard identification of skin sensitizers (i.e., UN GHS Category 1/No category). The method evaluates the transcriptional patterns of an endpoint-specific genomic biomarker signature, referred to as the GARDskin Genomic Prediction Signature (GPS) in the SenzaCell™ cell line exposed to test chemicals.
- Short description of test conditions: The test system (SenzaCell™ cells) is exposed to a non-cytotoxic concentration of the test item and concurrent negative and positive controls for 24 h at 37 °C and 5% CO2. Cells are lysed after exposure, and RNA is isolated from the lysed cells. 100 ng of RNA is used as sample input in a hybridization assay with the endpoint specific GARDskin CodeSet. Each hybridized sample is prepared on cartridge using nCounter MAX Prep Station and individual transcripts of the endpoint specific biomarker signature are quantified using nCounter MAX Digital Analyzer
- Parameters analysed / observed: the quantified transcriptional levels of the endpoint specific biomarker signature are analysed with the GARD Data Analysis Application (GDAA) software in order to determine the Decision Value (DV), which indicates whether a test item has skin sensitising potential or not.
GLP compliance:
yes (incl. QA statement)
Type of study:
other: Genomin Allergen Rapid Detection™ assay for hazard assessment of skin sensitizers (GARD™skin)
Justification for non-LLNA method:
Not required for transported isolated intermediates <1000 t/a

Test material

Constituent 1
Chemical structure
Reference substance name:
(5Z)-5-(2-methylpropylidene)imidazolidine-2,4-dione
EC Number:
700-726-4
Cas Number:
1369499-44-4
Molecular formula:
C7H10N2O2
IUPAC Name:
(5Z)-5-(2-methylpropylidene)imidazolidine-2,4-dione
Test material form:
solid: particulate/powder

In vitro test system

Details of test system:
other: human myeloid leukemia-derived cell line SenzaCell™
Vehicle / solvent control:
DMSO
Negative control:
not applicable
Positive control:
other: p-Phenylenediamine (CAS No. 106-50-3)

Results and discussion

Positive control results:
The positive control substance p-Phenylenediamine (PDD) was tested at 75 µM, assigned a mean Decision Value (DV) of 8.9, and thus correctly identified as a skin sensitizer.

In vitro / in chemico

Results
Key result
Group:
test chemical
Run / experiment:
mean
Parameter:
other: mean Decision Value (DV)
Value:
0.647
Cell viability:
99.37% (mean of three replicates)
Vehicle controls validity:
valid
Remarks:
mean DV: -2.63; mean relative cell viability: 99.67%
Negative controls validity:
valid
Remarks:
mean DV: n.a.; mean absolute cell viability: 96.9%
Positive controls validity:
valid
Remarks:
mean DV: 8.9; mean relative cell viability: 91.33%
Remarks on result:
positive indication of skin sensitisation
Outcome of the prediction model:
positive [in vitro/in chemico]

Any other information on results incl. tables

SOLUBILITY ASSESSMENT


The Test ltem solubility was assessed and the Test ltem was found to be soluble at 500 µM in cell medium using DMSO as vehicle. No other vehicles were tested.



















Vehicle (max in-well concentration of vehicle)(I)Maximum assessed concentration in vehicle (mM)Solubility conclusion in vehicle(II)Solubility in medium (µM)Solubility conclusion in-well(II)
DMSO (0.5%)500Soluble500Soluble

(I) Maximum concentration with an accurate classification as non-sensitiser in GARDskin. Based on historical data and previous experience. Data not shown.


(II) As defined by ocular inspection.


PHENOTYPIC QUALITY CONTROL


To ensure that the cells are maintained in an inactivated state and to detect a potential phenotypic drift, a quality control of the cells was performed the day of cell stimulation. The cells passed the specified acceptance criteria in all cell stimulations performed in the Study.


CYTOTOXICITY ASSESSMENT


The Test ltem was assayed in the range 20-500 µM. The Test ltem did not induce cytotoxicity and the Test ltem in-well concentration chosen for Main stimulations was 500 µM.

































 Relative viability (%)
In-well concentration (µM)2029446699148222333500
Test Item99.799.499.699.499.299.299.099.299.1

MAIN STIMULATIONS


The relative viability of the Test and Reference ltems and absolute viability of the unstimulated control are presented below. All samples passed the acceptance criteria.








































































Test/Reference ItemReplicate#Stimulation concentration (I)VehicleRelative viability(II) (%)Acceptance criteria (Pass/Fail)
Test Item1500 µMDMSO 0.1%99.3Pass
299.1
399.7
Positive control, PPD175 µM91.5
289.8
392.7
Negative control, DMSO10.10%N/A99.9
299.2
399.9
Unstimulated control (absolute viability, %)1N/A96.7
296.8
397.2

(I) Concentration based on max. solubility and cytotoxicity


(II) Observed viability, calculated based on the equation in section Standard GARD Assay Procedure


 


RNA QUALITY CONTROL


The result from the RNA Quality Control is presented below. All samples passed the acceptance criteria.













































































Test/Reference ItemReplicate#RNA conc (ng/µL)RINRNA QC (Pass/Fail)
Test Item11559.8Pass
213110
31019.9
Positive control, PPD110010
2849.7
3759.8
Negative control, DMSO11309.9
21169.9
312810
Unstimulated control115110
21009.4
3829.6

 


ENDPOINT MEASUREMENT QUALITY CONTROL


The result from the Endpoint Measurement Quality Control is below. All samples passed the acceptance criteria.







































































































Test/Reference ItemReplicate#Imaging QualityBinding DensityLimit of detectionLinearityEnpoint measurement QA (Pass/Fail)
Test Item10.980.436.60.998Pass
20.950.426.30.999
30.980.475.40.998
Positive control, PPD10.970.336.70.998
20.960.337.50.995
30.970.318.80.997
Negative control, DMSO10.970.345.30.997
20.980.445.60.998
30.960.347.30.997
Unstimulated control10.980.257.10.995
20.970.425.60.997
30.920.416.50.998

CLASSIFICATION OF TEST AND REFERENCE ITEMS


Decision Values were generated for each replicate sample by the GARDskin prediction model using the GDAA. as described in section Standard GARD Assay Procedure.


Individual Decision Values and corresponding mean Decision Values used for GARDskin classifications of Test and Reference ltems are presented below. All samples passed the acceptance criteria.










































Test/Reference ItemDecision Value (DV)Mean DVGARDskin classificationAcceptance criteria(I) (Pass/Fail)
Replicate #1Replicate #2Replicate #3
Test Item1.09-0.7641.620.647SensitiserPass
Positive control, PPD8.988.848.888.9Sensitiser
Negative control, DMSO-2.4-3.25-2.25-2.63Non-sensitiser

(I) Reference Item acceptance criteria as described in section Standard GARD Assay Acceptance Criteria


 


STATISTICAL SIGNIFICANCE OF READOUT


The conclusions presented in this Study report are based on generated output from the GARD prediction model, which is based on a Support Vector Machine. The SVM is trained on a training dataset consisting of 120 samples with measurements of 200 variables, each of which have been shown to be statistically significantly regulated by sensitizers compared to non-sensitizers (p < 5x10-20, q < 2x10-6, multiple hypothesis correction using Benjamini-Hochberg).


 

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
For the GARDskin classification, the mean DV of three replicates samples for each Test and Reference Item were used. The mean DV generated for the Test Item was 0.647, and thus ≥0 (skin sensitiser). The positive and negative controls were correctly classified as skin sensitiser (mean DV: 8.9) and skin non-sensitiser (mean DV: -2.63), respectively, in the GARDskin assay.
All specified acceptance criteria passed in this study and the study was therefore valid.
In conclusion, based on the results of this study, the Test Item was classified as sensitiser in the GARDskin assay tested at an in-well concentration of 500 µM.