Sodium p-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzenesulphonate

Respiratory sensitisation

Administrative data

Endpoint:

respiratory sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

other information

Study period:

2022

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:

2022

Materials and methods

Principles of method if other than guideline:

Introduction
The Genomic Allergen Rapid Detection (GARD) methods are a series of assays for hazard assessment, hazard characterization and risk assessment of sensitizers. The assays evaluate exposure-induced transcriptional patterns of endpoint-specific genomic biomarker signatures in the SenzaCell™ cell line, following Test Item exposure, in order to provide classifications of Test Items.
GARD™air
The Genomic Allergen Rapid Detection™ assay for hazard assessment of respiratory sensitizers (GARD™airl provides binary hazard identification of respiratory sensitizers li.e. UN GHS Category 1/No classification!. The method evaluates the transcriptional patterns of an endpoint-specific genomic biomarker signature, referred to as the GARDair Genomic Prediction Signature (GPS). in
the SenzaCell™ cell line exposed to test chemicals.
An early proof-of-concept of the ability of the SenzaCell™ cell line to differentiate respiratory sensitizers from skin sensitizers and non-sensitizers was demonstrated in Forreryd et al., 2015, in which endpoint-specific genomic biomarkers were proposed following a genome-wide analysis of the SenzaCell™ cell line exposed to a panel of well-characterized respiratory sensitizers, skin
sensitizers and non-sensitizers. Following these findings, the proposed set of biomarkers was refined in work supported by the EU Programme Horizon 2020, leading to the establishment of the GARDair GPS and finalized protocols and data analysis pipeline, as outlined in this Study plan (Johansson et al., 20211.
The assay is proposed to monitor transcriptional changes in an in vitro model of DCs, induced specifically by respiratory sensitizers, related to the bridging of innate and adaptive immune functions and skewing towards Th2 type immune responses. The GARDair prediction model classifies test chemicals as respiratory sensitizers or respiratory non-sensitizers, by utilization of a fixed Support Vector Machine ISVM) model (Cortes and Vapnik, 19951. The model was trained using data collected
during assay establishment !Johansson et al., 2021I. The endpoint value of each GARDair
measurement is a Decision Value (DV). as derived from the SVM model output.
The GARDair assay was subjected to an inter-laboratory ring trial (Johansson et al., 2021, Forreryd et al. 20201. The assay was demonstrated to be functional, with an estimated accumulated predictive accuracy of 74 %, specificity of 95 % and sensitivity of 53 %. The reproducibility between labs was 79 %.
The method is as of to date not included in the DECO Test Guideline Programme, and the data originating from the ring trial has not been independently reviewed by validating bodies. For these reasons, GARDair is an experimental method intended for research-oriented purposes. Of note, while both estimates of reproducibility and sensitivity indicate that GARDair is not currently considered appropriate for stand-alone regulatory use, the high specificity and positive predictive value suggest that GARDair can advantageously be utilized within opt-out applications, e.g. within relative risk assessment or pipeline prioritization projects, as it constitutes a useful screening tool to identify hazardous candidate molecules, which can subsequently be removed from prioritization in order to prevent harm.

Test method
The Test Method used was GARDair. The method has been optimized and validated at the Test Facility and this Study followed internal Standard Operating Procedures ISOPsi which governed the work at the Test Facility.

The standard experimental procedures of the method were followed and are described in section
Standard GARD Assay Procedures. The following is to note:

In this Study a mixture was tested. The main component, which was the component of interest is present at 21.48 %. To calculate the maximum in well concentration of 500 µM the molecular weight and purity of the main component was used. The other components in the mixture (see appendix 3) were present during the stimulations and except water and Sodium Chloride, the components were present at a concentration below the maximum allowed concentration of 500 µM. The cell stimulations were performed in a water-based cellculture medium containing higher amounts of Sodium Chloride and the Test Item Sodium Chloride is neglectable.
Since the Test Item is water soluble it was dissolved directly in cellculture medium.

The standard acceptance criteria of the method are described in section Standard GARD Assay Acceptance Criteria.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium p-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzenesulphonate

EC Number:

223-989-2

EC Name:

Sodium p-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzenesulphonate

Cas Number:

4156-21-2

Molecular formula:

C9H6Cl2N4O3S.Na

IUPAC Name:

sodium 4-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzenesulfonate

Details on test material:

- Name of test material (as cited in study report): Active substance of Granofin Easy F-90 liq in aqueous solution
- Physical state: Solid, ivory colours
- Analytical purity: 69.8 %
- Purity test date:2011-07-21
- Lot/batch No.: ITA0056831
- Expiration date of the lot/batch: 2013-07-21
- Stability under test conditions: Not specified
- Storage condition of test material: 6 +/- 2 °C, protected from light, in original container

Results and discussion

Results:

The Test Item GRANOFIN EASY F 90 was soluble at the maximum in well-concentration of 500 µM of the main component, and no solubility issues were reported. The positive and negative control were correctly classified as respiratory sensitizer and non-sensitizer, respectively, in the GARDair assay. In addition, all specified acceptance criteria passed in this Study, resulting in an overall valid Study.

No amendments or deviations occurred the conduct of this Study.

The average decision value was -0.098 and based on this result the Test Item GRANOFIN EASY F 90 was not classified as a respiratory sensitizer in the GARDair assay when tested at an in-well concentration of 500 µM of the main component. Note the variance of the resulting decision values and that the sensitivity of the GARDair assay is 53 %.

Positive control results:

Sensitizer

Negative control results:

Non-sensitizer

Applicant's summary and conclusion

Interpretation of results:

other: Non sensitizer

Conclusions:

Non sensitizer

Executive summary:

The purpose of this Study was to assess the Test Item GRANOFIN EASY F 90 for respiratory sensitizing hazard properties using the GARDair assay under GLP compliance.

 

 

The GARDair assay was developed for hazard assessment of respiratory sensitizers, which can give rise to the adverse outcome of respiratory sensitization, an allergic hypersensitivity reaction of the respiratory tract. The GARDair assay captures the combined expression of human genes involved in the respiratory sensitization response. The GARDair biomarker prediction signature comprises gene transcripts of mechanistically relevant genes, induced specifically by respiratory sensitizers, related to the bridging of innate and adaptive immune functions and skewing towards T helper type 2 immune responses. The GARDair assay was subjected to an inter-laboratory ring trial [Johansson et al., 2021, Forreryd et al. 20201. The assay was demonstrated to be functional, with anestimated accumulated predictive accuracy of 74 %, specificity of 95 % and sensitivity of 53 %.

 

 

The Test Item GRANOFIN EASY F 90 was freely soluble in cell media at the maximum in well concentration (500 µM of the main component). For cytotoxicity assessment, cells were incubated with the Test Item in a range of 500 to 1 µM of the Test Item main component. under standard conditions. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis. No cytotoxicity was observed. Therefore, the maximum concentration was selected for downstream stimulations.

 

After cell stimulations with the selected Test Item concentration, RNA was isolated and endpoint measurements were performed using the GARDair GPS. All samples passed the standard acceptance criteria for the GARDair assay.

 

Based on the results in this study, the GRANOFIN EASY F 90 was not classified a respiratory sensitizer, when tested with GARDair at an in-well concentration of 500 µM of the main component.

 

No amendments to or deviations from the Study Plan occurred during the conduct of this Study.