2,4-Imidazolidinedione, 5-(2-methylpropylidene)-, (5Z)-

Skin sensitisation

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

Test material information

Constituent 1

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%)	500	Soluble	500	Soluble

(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)	20	29	44	66	99	148	222	333	500
Test Item	99.7	99.4	99.6	99.4	99.2	99.2	99.0	99.2	99.1

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 Item	Replicate#	Stimulation concentration (I)	Vehicle	Relative viability(II) (%)	Acceptance criteria (Pass/Fail)
Test Item	1	500 µM	DMSO 0.1%	99.3	Pass
	2			99.1
	3			99.7
Positive control, PPD	1	75 µM		91.5
	2			89.8
	3			92.7
Negative control, DMSO	1	0.10%	N/A	99.9
	2			99.2
	3			99.9
Unstimulated control (absolute viability, %)	1	N/A		96.7
	2			96.8
	3			97.2

(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 Item	Replicate#	RNA conc (ng/µL)	RIN	RNA QC (Pass/Fail)
Test Item	1	155	9.8	Pass
	2	131	10
	3	101	9.9
Positive control, PPD	1	100	10
	2	84	9.7
	3	75	9.8
Negative control, DMSO	1	130	9.9
	2	116	9.9
	3	128	10
Unstimulated control	1	151	10
	2	100	9.4
	3	82	9.6

 

ENDPOINT MEASUREMENT QUALITY CONTROL

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

Test/Reference Item	Replicate#	Imaging Quality	Binding Density	Limit of detection	Linearity	Enpoint measurement QA (Pass/Fail)
Test Item	1	0.98	0.43	6.6	0.998	Pass
	2	0.95	0.42	6.3	0.999
	3	0.98	0.47	5.4	0.998
Positive control, PPD	1	0.97	0.33	6.7	0.998
	2	0.96	0.33	7.5	0.995
	3	0.97	0.31	8.8	0.997
Negative control, DMSO	1	0.97	0.34	5.3	0.997
	2	0.98	0.44	5.6	0.998
	3	0.96	0.34	7.3	0.997
Unstimulated control	1	0.98	0.25	7.1	0.995
	2	0.97	0.42	5.6	0.997
	3	0.92	0.41	6.5	0.998

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 Item	Decision Value (DV)			Mean DV	GARDskin classification	Acceptance criteria(I) (Pass/Fail)
	Replicate #1	Replicate #2	Replicate #3
Test Item	1.09	-0.764	1.62	0.647	Sensitiser	Pass
Positive control, PPD	8.98	8.84	8.88	8.9	Sensitiser
Negative control, DMSO	-2.4	-3.25	-2.25	-2.63	Non-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.