Tetraphosphorus trisulphide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 19th, 2017 to January 23rd, 2018 (included preliminary solubility test)

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:

2017

Report date:

2018

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

This skin sensitisation study was performed according to SENS-IS method. Even though the OECD Guideline of SENS-IS method is under validation, SENS-IS method is already included among the in vitro tests to investigated the skin sensitisation potential of a substance in ECHA guidance R7a (July 2017). Following the Adverse Outcome Pathway scheme (AOP), key events of the skin sensitisation are: 1) covalent binding of the electrophilic substance to skin proteins; 2) release of pro-inflammatory cytokines and induction of cyto-protective pathways in keratinocytes; 3) activation and maturation of dendritic cells, and their migration to the local lymph nodes; 4) presentation of the chemical allergen by the dendritic cells (allergen processed by the dendritic cell and displayed in its surface as an epitope) to naïve T-cells, which leads to their differentiation and proliferation into allergen specific memory T-cells. SENS-IS investigates the second key event (Keratinocyte response). SENS-IS has the potential to serve as a stand-alone method regarding the classification of test item as skin sensitiser.
The experiment was performed according to:
- Genes specifically modulated in sensitized skins allow the detection of sensitizers in a reconstructed human skin model. Development of the SENS-IS assay. Francoise Cottrez, Elodie Boitel, Claude Auriault, Pierre Aeby, Hervé Groux. Toxicology in vitro 29: 787-802, 2015.
- SENS-IS, a 3D reconstituted epidermis based model for quantifying chemical sensitization potency: reproductibility and predictivity results from an inter-laboratory study. Francoise Cottrez, Elodie Boitel, Jean-Claude Ourlin, Jean-Luc Peiffer, Isabelle Fabre, lmène-Sarah Henaoui, Bernard Mari, Ambre Vallauri, Agnes Paquet, Pascal Barbry, Claude Auriault, Pierre Aeby, Hervé Groux . Toxicology in Vitro 32: 248-260, 2016.
- OECD n°439: OECD guidelines for the testing of chemicals. In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, Adopted July 28th 2015.

GLP compliance:

yes

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

Tetraphosphorus trisulphide

EC Number:

215-245-0

EC Name:

Tetraphosphorus trisulphide

Cas Number:

1314-85-8

Molecular formula:

P4S3

IUPAC Name:

3,5,7-trithia-1,2,4,6-tetraphosphatricyclo[2.2.1.0²,⁶]heptane

Test material form:

solid: particulate/powder

In vitro test system

Details on the study design:

GENERAL PRINCIPLE:
The objective of this study was to evaluate the in vitro sensitization potential of the test item applied onto 3D human reconstructed epidermis (EpiSkinTM) using the SENS-IS assay, based on the quantitative analysis of specific gene biomarkers.
The expression profile of 61 genes divided in three sets were analysed: one set of 23 genes related to the irritation process and the two other sets of genes, named "SENS-IS" and "ARE", with 21 and 17 biomarkers respectively, involved in skin sensitization.
The expression level of these separate sets of genes was determined by qRT-PCR (quantitative real-time polymerase chain reaction) after a 15 min incubation period with the test item and a 6 hours post-incubation period at 37°C.
The fold expression of each gene were calculated as a ratio between the mRNA level of test item-treated epidermis versus control (vehicle treated) epidermis.

CONTROLS:
- sodium lauryl sulfate (SLS) 10 %: positive control for irritation;
- 2,4,6-Trinitrobenzenesulfonic acid (TNBS) 1M : positive control for sensitisation;
- dimethylsulfoxide (DMSO)

TEST SYSTEM:
The test system used in this study was the EpiSkinTM model, an in vitro reconstructed human epidermis from normal human keratinocytes cultured on a collagen matrix at the air-liquid interface. This model is histologically similar to the in vivo human epidermis, with a functional horny layer.
Some quality controls are performed by the provider of the model and must fulfill the following criteria: IC50 value of SLS by the MTT test ≥ 1.2 mg/ml
The two batches used in the study satisfied the requirement:
17-EPIS-045: 1.4 mg/ml
17-EPIS-047: 1.5 mg/ml

PRELIMINARY TEST: SOLUBILITY TEST
The test item was solubilized in phosphate buffered saline (PBS), olive oil (OO), dimethylsulfoxide (DMSO) and dipropylene glycol (DPG) at a concentration of 10 % and 50 % at room temperature, at 37 °C and at 60 °C, with the aid of an ultrasonic disruptor. The solubility of the test item was assessed by visual inspection of each preparation

MAIN TEST: SENS-IS assay
The test item (30 mg) was deposited on the epidermis surface and gently spread on the entire surface . After 15 minutes of exposure, the EpiskinTM was rinsed with PBS and then incubated at 37 °C for 6 hours.
After incubation, reconstructed epidermis was removed from the inserts with forceps and placed in a cryotube for freezing in liquid nitrogen. The epidermis was then transferred in a tube containing 1 ml of Qiazol reagent and 2 steel beads. Epidermis was homogenized using the TissueLyser II. After centrifugation, the supernatant was collected and stored at -20 °C until RNA extraction.
After addition of bromochloropropane, total RNA were purified using the miRNeasy extraction Kit according to the manufacturer's instructions. RNA quality was assessed by measuring 260/280 absorbance ratio. The mRNA was then reversed as cDNA using SuperScript III Reverse Transcriptase kit and RNase inhibitor.
After reverse transcription, quantitative gene expression was measured by qRT-PCR using a SYBRGreen® buffer and specific primers defined for the SENS-IS test. The housekeeping genes (Glucuronidase ß, ß2 microglobuline, and Nono « non-POU domain containing octamer-binding >>) were analyzed in parallel.


DATA ANALYSIS AND INTERPRETATION
Samples were analyzed by the LightCycler 480 software using the second derivative maximum method. This method allows the calculation of the crossing point (Cp) of each sample defined as the number of cycles from which the fluorescence signal enters the exponential phase of the reaction. This value is dependent of the amount of the mRNA present in the sample. For each sample, the mRNA content for each gene of interest was normalized to the mean mRNA content of the 3 house-keeping genes. For each gene, the fold increase expression over vehicle controls was calculated as followed:
AE = Expression level for the test item / Expression level for the vehicle controls*
* Mean of OO and PBS samples
The endpoint values are the number of positive genes (i.e., genes obtaining a AE > 1.25) in each group.

EVALUATION CRITERIA:
A test item is classified as irritant if at least 16/23 genes of the "IRRITATION" group are significantly over-expressed.
A test item is classified as sensitizer if at least 7/17 genes of the "ARE" group, and/or 7/21 genes in the "SENS-IS" group are significantly over-expressed.
Moreover, the results obtained with the different concentrations allow the classification of the test item according to the lowest concentration that gives a positive result (AE > 1.25). Thus, a test item is classified in:
- category 1A: strong to extreme skin sensitizer, when a positive result is obtained at concentrations of 0.1 and/or 1 %,
-category 1 B: weak to moderate sensitizer, when a positive result is obtained at concentrations of 10 and/or 50 %.
A test item is classified as a non-sensitizer when no positive result is observed for all tested concentrations (0.1, 1, 10, 50 and 100 %).

ACCEPTABILITY:
#ACCEPTANCE CRITERIA FOR THE EXPERIMENT:
- Negative sensitization control (DMSO): this control should induce the over-expression of 6 genes at most in the SENS-IS and ARE groups of genes.
- Positive irritation control and negative sensitization control (SLS at 5 %): this control should induce the over expression of at least 16 genes in the IRRITATION group of genes and 6 genes at most in the SENS-IS and ARE groups of genes.
-Positive sensitization control (TNBS at 1 M): this control should induce the over expression of at least 7 genes in the SENS-IS or ARE group of genes.
#ACCEPTANCE CRITERIA FOR THE SAMPLE:
- The Cp value of HSP90AA1 gene must be ≤ 21.
- If more than 20 genes are over-expressed in the "IRRITATION" set of genes for a given concentration, the result is classified as false positive to take into account non-specific genes up regulation that could be due to cell stress.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: experiment 1

Parameter:

other: ARE gene

Value:

15

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Test item was applied as solid due to its low solubility. No subcategorisation possible.

Results 2

Key result

Run / experiment:

other: experiment 2

Parameter:

other: ARE gene

Value:

15

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Test item was applied as solid due to its low solubility. No subcategorisation possible.

Other effects / acceptance of results:

RESULTS:
After application of crude powder onto the epidermis, the test item Tetraphosphorus trisulphide induced more than 6 genes in the "ARE" gene group and more than 15 genes in the IRRITATION gene group. Considering the number of over-expressed gene in "ARE" gene groups, the test gave positive results (more than 6 genes induced) after application of crude powder onto the epidermis. In conclusion, under the experimental conditions of this SENS-IS assay, the test item Tetraphosphorus trisulphide can to be classified as a sensitizer. Since the test item could not be tested at the classical concentrations of the assay (not soluble), it was not possible to evaluate the degree of sensitization in this assay. In addition, the test item appears irritant in this assay since more than 15 genes were induced in the IRRITATION gene group. Nevertheless, this result must be considered only as informative it was not confirmed by the results of the specific test for skin irritation OECD TG 439. Tetraphosphorus trisulphide was negative in the skin irritation test according to OECD TG 439 Reconstructed human epidermis model (cell viability: 106 %).

PRELIMINARY SOLUBILITY TEST
The test item was not soluble in any solvent (Phosphate buffered saline, Olive oil, DMSO, diproylene glycol) at room temperature and after heating (at 37 °C and 60 °C) and sonication neither at 50% w/v nor at 10 % w/v.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: DMSO at 100 % was classified as a non-sensitizer, the number of overexpressed genes in both the SENS-IS and ARE groups being below 7 (Experiment 1: SENS-IS over-expressed genes: 2 and ARE overexpressed genes: 1; Experiment 2: SENS-IS over-expressed genes: 0 and ARE overexpressed genes: 0).
- Acceptance criteria met for positive control:
#Positive control for sensitisation: TNBS at 1M was classified as sensitizer since more than 6 genes are overexpressed in at least one of the two groups of genes (SENS-IS or ARE) (Experiment 1: ARE overexpressed genes: 16; Experiment 2: ARE overexpressed genes: 14)
#Positive control for irritation: SLS at 5% was classified as irritant (number of over- expressed irritant genes > 15) and non-sensitizer, the number of overexpressed genes in both the SENS-IS and ARE groups being below 7 (Experiment 1: IRRITATION overexpressed genes:23, SENS-IS overexpressed genes: 5 and ARE overexpressed genes: 2; Experiment 1: IRRITATION overexpressed genes: 23, SENS-IS overexpressed genes: 2 and ARE overexpressed genes: 2)

Any other information on results incl. tables

Analysis of test item

Experiment 1:

	Number of over-expressed genes
Gene group	100% (not solubilized)*
IRRITATION	19
SENS-IS	6
ARE	15
Cp value - HSP90AA1	18.6
Conclusion
SENSITIZATION	POSITIVE

Experiment 2:

	Number of overexpressed genes
Gene group	100% (not solubilized)*
IRRITATION	17
SENS-IS	3
ARE	15
Cp value - HSP90AA1	18.2
Conclusion
SENSITIZATION	POSITIVE

Analysis of positive and negative controls

Experiment 1:

	Number of overexpressed genes
Gene group	SLS 5%	TNBS 1M	DMSO 100%
IRRITATION	23	7	2
SENS-IS	5	4	2
ARE	2	16	1
Conclusion
IRRITATION	POSITIVE	NEGATIVE	NEGATIVE
SENSITIZATION	NEGATIVE	POSITIVE	NEGATIVE

Experiment 2:

	Number of overexpressed genes
Gene group	SLS 5%	TNBS 1M	DMSO 100%
IRRITATION	23	3	2
SENS-IS	2	3	0
ARE	2	14	0
Conclusion
IRRITATION	POSITIVE	NEGATIVE	NEGATIVE
SENSITIZATION	NEGATIVE	POSITIVE	NEGATIVE

Applicant's summary and conclusion

Interpretation of results:

other: classified as skin sensitiser according to the CLP Regulation (EC n.1272/2008).

Conclusions:

The test item resulted to be positive (sensitiser) in SENS-IS test. No subcategorisation was possible because the test item was tested as solid due to its low solubility.

Executive summary:

The test item resulted to be positive (sensitiser) in SENS-IS test. No subcategorisation was possible because the test item was tested as solid due to its low solubility.

The potential of Tetraphosphorus trisulphide to induce the expression of specific irritation and sensitisation biomarkers in a 3D-reconstructed epidermis model was evaluated according to SENS-IS method.

The results obtained for the positive and negative controls were within acceptance criteria and validated the study. Considering the number of over-expressed gene in "ARE" gene groups, the test item " gave positive results (more than 6 genes induced) after application of crude powder onto the epidermis.

In conclusion, under the experimental conditions of this SENS-IS assay, the test item Tetraphosphorus trisulphide can to be classified as a sensitiser. Since the test item could not be tested at the classical concentrations of the assay (not soluble), it was not possible to evaluate the degree of sensitisation in this assay.

In addition, the test item appears irritant in this assay since more than 15 genes were induced in the IRRITATION gene group. The irritation result was only informative and it was not confirmed by the results of the specific test for skin irritation OECD TG 439. Tetraphosphorus trisulphide was negative in the skin irritation test according to OECD TG 439 Reconstructed human epidermis model (cell viability: 106 %).