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Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: SENS-IS assay (Cottrez et al., 2015)
Version / remarks:
1. Genes specifically modulated in sensitized skins allow the detection of sensitizers in a reconstructed human skin model. Development of the SENS-IS assay. Franc;oise Cottrez, Elodie Boitel, Claude Auriault, Pierre Aeby, Herve Groux. Toxicology in vitro 29: 787-802, 2015.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: SENS-IS assay (Cottrez et al., 2016)
Version / remarks:
2. SENS-IS, a 3D reconstituted epidermis based model for quantifying chemical sensitization potency: reproductibility and predictivity results from an inter-laboratory study. Franc;oise Cottrez, Elodie Boitel, Jean-Ciaude Ourlin, Jean-Luc Peiffer, Isabelle Fabre, lmeme-Sarah Henaoui, Bernard Mari, Ambre Vallauri, Agnes Paquet, Pascal Barbry, Claude Auriault, Pierre Aeby, Herve Groux. Toxicology in Vitro 32: 248-260, 2016.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Reconstructed Human Epidermis Test System
Version / remarks:
OECD no439: OECD guidelines for the testing of chemicals. ln Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, Adopted July 28th 2015.
Deviations:
not applicable
Principles of method if other than guideline:
The SENS-IS test protocol for the in vitro detection of sensitizers is based on a reconstructed human skin model (Episkin) as the test system and on the analysis of the expression of a large panel of genes.
Briefly, the test item is applied on the skin model at 4 different concentrations (50%, 10%; 1% and 0.1%) in the appropriate vehicle. After exposure, gene expression of two groups of genes is measured: One group (REDOX group = ARE group) includes a selection of 17 genes that have an antioxidant responsive element in their promoter and monitor the redox protective signals induced through the interaction of sensitizers binding to cysteine amino acids of the Keap1-NRF2 complex (Uruno and Motohashi, 2011). The second group (SENS-IS group) includes a selection of 21 genes involved in inflammation, danger signals and cell migration to address the complex cascade of events leading to activation of DCs by a sensitizing chemical. Finally, the prediction model (PM), based on the number of over-expressed genes makes a decision according to the following rules: if at a given test concentration a compound induces at least 7 genes in either the REDOX or SENS-IS group of genes, it is classified as a sensitizer.
GLP compliance:
yes (incl. QA statement)
Type of study:
other: The SENS-IS test protocol (Cottrez, 2015) describes a new approach based on a reconstructed human skin model (Episkin) as the test system and on the analysis of the expression of a large panel of genes relevant to the considered biological processes.
Justification for non-LLNA method:
The SENS-IS assay is an innovative in vitro system for the detection of sensitizers.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
batch number C-015073-001

In vitro test system

Details on the study design:
1) Preliminary solubility tests (phosphat buffere salin, olive oil, dimethylsulfoxide)
2) Main test: SENS-IS assay
The test item (30µl) was deposited on the reconsituted human epidermis (RHE) surface and gentyl spread on the entire surface. After 15 minutes of exposure, the RHE was rinsed with PBS and then incubated at 37°C for 6 hours. After incubation RHE was removed from the inserts with forceps and frozen in liquid nitrogen. The RHE was prepared for lysis and RNA extration by means of milling it with steel beads in Qiazol reagent. Total RNA quality was assessed by means of 260/280 absobance ratio. After reverse transciption, gene expression was measured by qRT-PCR using SYBRGreen buffer and specific primers defined for the SENS-IS test. Three housekeeping genes (Glucoronidase ß, ß2 microglobuline, and non-POU domaine containing octamer-binding) were analysed in parallel.
The expression profile of 61 genes divided in three sets were analysed: one set of 23 genes (HAS1, IL-8, IL-1 A, PLAUR, IL-7R, JUN, TNFA,
RELT, IER3, IL-4R, ICAM2, CCL20, COX2, MMP10, MKP1, GM-CSF, IL-23, IL-24, ICAM1, CXCL1, MMP3, MMP9, EGR1) related to the irritation process and two other sets of genes,named "SENS-IS"-set and "ARE"-set, with 21 and 17 biomarkers respectively, involved in skin sensitisation (according to literatures Cottrez F et al. (2015); Toxicology in vitro 29; 787-802; Cottrez F et al. (2016); Toxicology in vitro 32; 248-260.
3) Data analysis and interpretation
Samples were analysed semiquantitatively by light cycler software using the socalled second derivative maximum point where the increasing fluorescence signal enters the exponential phase of the amplification reaction. For each sample the specific mRNA content of interest was normalized to the mean mRNA content of the housekeeping genes.For each gene the x-fold increase expression over the vehicle controls was calculated: delta E.
4) Validation of the study
The following criteria must be met for an experiment to be considered valid.
- Negative sensitisation 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 1M): this control should induce the over expression of at least 7 genes in the SENS-IS or ARE group of genes.
The following criteria must be met for sample result tobe considered valid:
- The Cp value of HSP90AA1 gene must be <= 21.
- lf more than 20 genes are overexpressed 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.
5) Criteria for test item classification (SENS-IS assay)
A test item is classified as irritant if at least 16/23 genes of the "IRRITATION" group are significantly overexpressed.
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 overexpressed.
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 (L.\E > 1.25). Thus, a test item is classified in:
category 1A: streng 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 1 0 and/or 50%.
A test item is classified as a non-sensitizer when negative results are observed at 100% and at all other tested concentrations.
At least two independent experiments (repetitions) are performed in order to obtain two concordant conclusions. lf three repetitions should be performed for a given concentration, a majority of positive results (2 out of 3) must be obtained so that the final outcome is positive, otherwise the final outcome is negative.

Results and discussion

Positive control results:
SLS at 5% was classified as irritant (number of overexpressed irritant genes > 15) and non-sensitizer, the number of overexpressed genes in both the SENS-IS and ARE groups being below 7.
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).
DMS0 at 100% was classified as a non-sensitizer, the number of overexpressed genes in both the SENS-IS and ARE groups being below 7.

In vitro / in chemico

Resultsopen allclose all
Run / experiment:
other: experiment 1: not interpretable
Parameter:
other: 50% test item in DMSO
Remarks:
not interpretable
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: gene expression not interpretable due to tissue damage; cp value for HSP90AA1 > 21
Key result
Run / experiment:
other: experiment 1: irritation 17; SENS-IS: 3, ARE: 4
Parameter:
other: 10% test item in DMSO
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: cp value for HSP90AA1 = 17.6
Key result
Run / experiment:
other: experiment 1: irritation 15; SENS-IS: 3, ARE: 3
Parameter:
other: 1% test item in DMSO
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: cp value HSP90AA1 = 16.2
Key result
Run / experiment:
other: experiment 1: irritation 03; SENS-IS: 2, ARE: 0
Parameter:
other: 0.1% test item i DMSO
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: cp value HSP90AA1 = 16.8
Run / experiment:
other: experiment 2: not interpretable
Parameter:
other: 25% test item in DMSO
Remarks:
not interpretable
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: gene expression not interpretable due to tissue damage; cp value HSP90AA1 = 27.6
Key result
Run / experiment:
other: experiment 2: irritation 17; SENS-IS: 4, ARE: 4
Parameter:
other: 10% test item in DMSO
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: cp vale HSP90AA1 = 17.5

Any other information on results incl. tables

ln the first experiment, the test item induced less than 7 genes in the "SENS-IS" and "ARE" gene groups when tested at 0.1, 1 and 10% (v/v) in DMSO. When the test compound was tested at 50% (v/v),

the Cp value for HSP90AA1 was above 21 , traducing a degradation of the tissue by the test item. Therefore, the results obtained at this concentration can't be taken into consideration.

ln the second experiment, the test item induced less than 7 genes in the "SENS-IS" and "ARE" gene groups when tested at 10% (v/v) in DMSO. When the test compound was tested at 25% (v/v), the Cp value for HSP90AA1 was above 21, traducing a degradation of the tissue by the test item. Therefore, the results obtained at this concentration can't be taken into consideration.

Applicant's summary and conclusion

Interpretation of results:
other: negative for skin sensitisation
Conclusions:
The results obtained for the positive and negative controls were within acceptance criteria defined in the study plan.
Considering the number of over-expressed genes in the "SENS-IS" and "ARE" gene groups, the test item gave negative result (less than 7 genes induced) when it was tested diluted at 0.1, 1, and 10% (v/v) in DMSO. When the test item was tested diluted at 25 or 50% (v/v) in DMSO, the Cp value for HSP90AA1 was too high and the results can't be taken into consideration.
In conclusion, under the experimental conditions of this SENS-IS assay, the test item is a non-sensitiser when applied at 0.1, 1 and 10% onto the epidermis. No result could be obtained with higher concentrations due to the cytotoxic potential of the test compound.