# Patent

## Title
Method for evaluating the sensitizing potential of a test compound

## Abstract

The invention relates to a method for evaluating the sensitizing potential of a test compound, and to a kit for implementing said method.

## Background
The present invention relates to a method for evaluating the sensitizing potential of a test compound and a kit for implementing said method.
The perfume, cosmetics and pharmacy industries must remain competitive and effective and continue to regularly offer new products, with the constraint of complying with the human and environmental safety standards attached to their use. Contact allergy is one of the major risks associated with the use of such products.
Cutaneous contact allergy (or atopic dermatitis) is a major public health problem in humans. It represents a serious and restrictive environmental immunotoxic event, whose effects must be anticipated when marketing products that could induce it. Skin sensitization and, consequently, the associated allergic manifestation, is the result first of an interaction of an allergenic molecule with specialized epidermal cells, antigen-presenting cells (Langerhans cells, dendritic cells) and then, second, their presentation by these cells to CD4+ and CD8+ effector T cells. These T cells are the basis of the allergic and inflammatory reaction. However, the allergens, especially those that can be present in a fragrance, are small molecules that cannot be recognized directly. To be recognized, they must be associated beforehand with self proteins. Thus, it is the newly-formed heterodimeric complex in the skin that will finally be presented to T cells in the proximal lymph nodes. Accordingly, the ability of a chemical molecule (fragrance compound or cosmetic ingredient) to be associated with a protein of the user of this product is a prerequisite for the induction of the consecutive pathological skin reaction. This pathological skin reaction could be irritation, sensitization, or in the majority of cases, both irritation and sensitization.
Irritation is a reversible inflammatory reaction in living tissues by chemical action at the contact site. This is recognized by edema consecutive to the influx of fluid to the tissues, redness, heat and/or pain. In response to a chemical attack, keratinocytes of the epidermis and fibroblasts of the dermis are stimulated and release cytokines IL1, TNF alpha, IL6 and IL8, as well as mediators such as prostaglandins (PGE2) into the skin which will initiate the inflammatory response.
The delayed and immediate hypersensitivities that are the basis of sensitization involve the concept of “memory” which emphasizes their irreversible nature, unlike irritation. In this case as well, the mechanisms occur in two phases:

    
    
        the first, called sensitization phase, during which the antigen/allergen is presented to the immune system and, in particular, to the T cells that record the molecular signal and regulate, via the cytokines produced, the other cell populations involved (B cells, T CD8, endothelial cells, macrophages, mast cells keratinocytes, etc.);
        the second phase, called the effector phase, during which various skin cell populations will act via the chemical mediators responsible for pathologic disorders. In the case of immediate hypersensitivity, anaphylactic antibodies such as IgE are generated and bind mast cells and basophils, leading to the release of histamine, the main vector for allergic manifestations. In the case of delayed hypersensitivity, it is cytotoxic T cells (TCD8) that are responsible for skin lesions by destroying keratinocytes.
    
    


Thus, although from a histological point of view, sensitizing and irritant contact dermatitis are very similar, the immunological consequences analyzed at the cellular level are not necessarily similar. Consequently, it is important to have reliable methodologies for distinguishing them. An original predictive approach is even more necessary since currently no clear correlation has been demonstrated between a given molecular structure and allergenicity in the broadest sense of term.
So far, animals have been used to identify skin sensitizing molecules and the LLNA (local lymph node assay), based on the induced proliferation of lymph node lymphocytes after contact with the sensitizer, has been developed. This test was adopted as “Testing guideline 429” by the Organization for Economic Cooperation and Development (OECD) and is still considered as the standard test for determination of a sensitizing chemical agent.
The new European restrictions now require the use of methods that do not use animals and it is therefore vital to develop alternative methods for determining if a new composition or new product is likely to represent a risk for humans due to its sensitizing properties.
The Inventors have shown that in vivo recognition of a substance does not occur at the draining lymph node, as is generally accepted, but rather at the tissue where the substance comes into contact with the body, the skin in the present case. This would explain why some substances are sensitizing in one tissue and not in another, as is often observed. It therefore appears that it is the reaction in skin tissue that sends the message to the allergen-presenting cells: the dendritic cells will then transmit it to the T cells in the draining lymph node.
It is generally accepted that skin models are not sufficient to analyze sensitizing responses and it is necessary to have dendritic cells (EP 0857971).
The Inventors have now demonstrated that skin constitutes a sufficient model to show specific biomarkers for sensitization and/or irritation in humans and in mice, and that it is not necessary to add other types of cells if the identified genes are analyzed at a specific time. The EPISKIN model can thus be the standard tissue for evaluating the sensitizing nature of a test compound.
Moreover, the Inventors have shown that it was not sufficient to show an overexpression of only one of said biomarkers to conclude the sensitizing potential of a test compound, and that only studying at least six specific markers for sensitization would allow drawing conclusions about the sensitizing potential of a test compound.
Thus, the present invention relates to a method for evaluating the sensitizing potential of a test compound, comprising the steps of:
a) contacting a test compound with a biological sample;
b) determining the expression level of at least six genes chosen in the group consisting of: BRAK (CXC chemokine ligand 14), CTSS (cathepsin S), DAPK2 (death-associated protein kinase 2), FABP4 (fatty acid binding protein 4), HSP27 (heat shock 27 kDa protein), IL18 (Interleukin-18), HSP90 (heat shock 90 kDa protein), IL1R2 (interleukin-1 receptor type II), TPSAB1 (tryptase alpha/beta 1), CXCR1 (interleukin 8 receptor, alpha), DEFB1 (defensin, beta 1), DHFR (dihydrofolate reductase), EHF (ets homologous factor), IVL (involucrin) KRT4 (keratin 4), MELANA (melan-A), NGAL (gelatinase-associated lipocalin), PDZK1IP1 (PDZK1 interacting protein interaction 1), PI3 (peptidase inhibitor 3), PSME2 (proteasome activator subunit 2), SERPINB3 (serpin peptidase inhibitor member 3), AKR1B10 (aldo-keto reductase family 1, member B10), AKR1C1 (aldo-keto reductase family 1, member C1), AKR1C2 (aldo-keto reductase family 1, member C2), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), FTH1P (ferritin, heavy polypeptide 1), FTL (ferritin, light polypeptide 1), G6PD (glucose-6-phosphate dehydrogenase), GCLM (glutamate-cysteine ligase modifier subunit), NQO2 (NAD(P)H dehydrogenase, quinone 2), SLC7A11 (solute carrier family 7, Member 11), TXNRD1 (thioredoxin reductase 1), UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1), UGT1A9 (UDP glucuronosyltransferase 1 family, polypeptide A9, YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide), CD36 (CD36 molecule), CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide 1), GCLC (glutamate-cysteine ligase, catalytic subunit), HMOX1 (heme oxygenase 1), NQ01 (NAD(P)H dehydrogenase, quinone 1) and S100A8 (S100 calcium binding protein A8).
Preferably, said at least six genes are chosen in the group consisting of: BRAK (CXC chemokine ligand 14), CTSS (cathepsin S), DAPK2 (death-associated protein kinase 2), FABP4 (fatty acid binding protein 4), HSP27 (heat shock 27 kDa protein), IL18 (IL-18), HSP90 (heat shock 90 kDa protein), IL1R2 (interleukin-1 receptor type II), TPSAB1 (tryptase alpha/beta 1), CXCR1 (interleukin 8 receptor, alpha), DEFB1 (defensin, beta 1), DHFR (dihydrofolate reductase), EHF (ets homologous factor), IVL (involucrin) KRT4 (keratin 4), MELANA (melan-A), NGAL (gelatinase associated lipocalin), PDZK1IP1 (PDZK1 interacting protein interaction 1), P13 (peptidase inhibitor 3), PSME2 (proteasome activator subunit 2), SERPINB3 (serpin peptidase inhibitor member 3), AKR1B10 (aldo-keto reductase family 1, member B10), AKR1C1 (aldo-keto reductase family 1, member C1), AKR1C2 (aldo-keto reductase family 1, member C2), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), FTH1P (ferritin, heavy polypeptide 1), FTL (ferritin, light polypeptide 1), G6PD (glucose-6-phosphate dehydrogenase), GCLM (glutamate-cysteine ligase modifier subunit), NQO2 (NAD(P)H dehydrogenase, quinone 2), SLC7A11 (solute carrier family 7, Member 11), TXNRD1 (thioredoxin reductase 1), UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1), UGT1A9 (UDP glucuronosyltransferase 1 family, polypeptide A9) and YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide),
more preferentially still chosen in the group consisting of: BRAK (CXC chemokine ligand 14), CTSS (cathepsin S), DAPK2 (death-associated protein kinase 2), FABP4 (fatty acid binding protein 4), HSP27 (heat shock 27 kDa protein), IL18 (IL-18), HSP90 (heat shock 90 kDa protein), IL1R2 (interleukin-1 receptor type II), TPSAB1 (tryptase alpha/beta 1), CXCR1 (interleukin 8 receptor, alpha), DEFB1 (defensin, beta 1), DHFR (dihydrofolate reductase), EHF (ets homologous factor), IVL (involucrin) KRT4 (keratin 4), MELANA (melan-A), NGAL (gelatinase associated lipocalin), PDZK1IP1 (PDZK1 interacting protein interaction 1), P13 (peptidase inhibitor 3 and SERPINB3 (serpin peptidase inhibitor member 3),
and even more preferentially still chosen in the group consisting of: BRAK (CXC chemokine ligand 14), CTSS (cathepsin S), DAPK2 (death-associated protein kinase 2), FABP4 (fatty acid binding protein 4), HSP27 (heat shock 27 kDa protein) IL18 (IL-18), HSP90 (heat shock 90 kDa protein), IL1R2 (interleukin-1 receptor type II), TPSAB1 (tryptase alpha/beta 1), or in the group made up of: CXCR1 (interleukin 8 receptor, alpha), DEFB1 (defensin, beta 1), DHFR (dihydrofolate reductase), EHF (ets homologous factor), IVL (involucrin) KRT4 (keratin 4), MELANA (melan-A), NGAL (lipocalin 2), PDZK1IP1 (PDZK1 interacting protein interaction 1), PI3 (peptidase inhibitor 3, PSME2 (proteasome activator subunit 2) and SERPINB3 (serpin peptidase inhibitor member 3)
Preferably, the method according to the present invention can also comprise another step c) for determining the sensitizing potential of a test compound.
Preferentially, said step c) can consist of a step of selecting said compound as presenting a sensitizing potential if the expression level of at least 6 of said genes is above a threshold value.
Preferably, the method according to the present invention is an in vitro method. As used here, the term “biological sample” refers to any solid or liquid sample from a subject.
Preferably, said biological sample is a skin sample.
In a particularly preferred manner, the skin sample is a skin sample reconstructed in vitro, such as, for example, the EpiSkin (EPISKIN, Lyon France), EpiDermTM (MATEK Corporation, Ashland, Mass.) or SkinEthicTM RHE (SKINETHIC, Nice, France) model. Preferably, said skin sample reconstructed in vitro also comprises a keratin layer.
Even more preferably, the skin sample does not comprise other types of additional cells, and more preferentially no additional Langerhans cells.
The test compound can be a compound of various type, structure and origin, especially a biological compound, chemical compound, synthetic, etc.
The test compound can be any product present in the isolated form or mixed with other products. The test compound can be defined in terms of structure and/or composition or can be defined functionally. The test compound can be, for example, an isolated and structurally defined product, an isolated product of undefined structure, a mixture of known and characterized products or a composition comprising one or more products. One or more compounds can be tested in this way, mixed or separately.
Such compositions can be, for example, samples of a cosmetic or dermatological product.
Preferably, said test compound is suitable for use on the skin and may be used in a cosmetic or dermatological composition.
Preferentially, said method allows assessing the sensitizing potential of a test compound in humans, comprising a step b) of determining the expression level of at least six genes such as defined in Table 1, and preferably chosen in the group made up of: CXCR1 (interleukin 8 receptor, alpha), DEFB1 (defensin, beta 1), DHFR (dihydrofolate reductase), EHF (ets homologous factor), IVL (involucrin) KRT4 (keratin 4), MELANA (melan-A), NGAL (lipocalin 2), PDZK1IP1 (PDZK1 interacting protein interaction 1), P13 (peptidase inhibitor 3), PSME2 (proteasome activator subunit 2), and SERPINB3 (serpin peptidase inhibitor member 3).
“Sensitizing potential” means the risk for the test compound to provoke an immunological reaction when contacted with a mammal, preferably a human. Thus, sensitizing potential can be considered as the risk of developing a contact allergy to the test compound.
“Irritant potential” means the risk for the test compound to provoke a reversible inflammation of living tissue by chemical action at the contact site.
Preferably, the method according to the present invention allows evaluating whether the test compound is likely to induce a contact allergy or atopic dermatitis.
The present invention is particularly suited to identifying a large number of compounds. This simple and effective screening can be done in a very brief period of time. In particular, the methods described may be partially automated, thus allowing effective and simultaneous screening of diverse and numerous compounds, either in mixed or separate form.
Preferably, in the method according to the present invention, the expression level of each of said genes is determined by measuring the expression level of the polypeptides encoded by said gene or a fragment thereof, or by determining the expression level of the mRNA from said gene or a fragment thereof.
In one particularly preferred embodiment, the expression level of each of said at least six genes is determined by analysis of the expression of mRNA transcripts or mRNA precursors, such as a native RNA, of said gene. Said analysis can be done by preparing mRNA/cDNA from cells of a patient's biological sample, and hybridizing the mRNA/cDNA with a reference polynucleotide. The prepared mRNA/cDNA may be used in analysis by hybridization or amplification that includes, without being limiting, Southern and Northern analysis, PCR (“polymerase chain reaction”), such as quantitative PCR (Taqman) and the use of probes (“probe arrays”) such as GeneChip® DNA matrices® (AFFYMETRIX).
Advantageously, the analysis of the mRNA expression transcript of each of said at least six genes involves a nucleic acid amplification process, such as, for example, RT-PCR (experimental method described in U.S. Pat. No. 4,683,202), ligase chain reaction (BARANY, Proc. Natl. Acad. Sci. USA, vol. 88, p: 189-193, 1991), self sustained sequence replication (GUATELLI et al., Proc. Natl. Acad. Sci. USA, vol. 87, p: 1874-1878, 1990) and transcriptional amplification system. (KWOH et al., Proc. Natl. Acad. Sci. USA, vol. 86, p: 1173-1177, 1989), “Q-Beta Replicase” (LIZARDI et al., Biol. Technology, vol. 6, p: 1197, 1988), “rolling circle replication” (U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by a step of detecting the amplified molecules by techniques well known to the skilled person. These detection modes are particularly useful for detecting nucleic acid molecules in very small quantities. Thus, according to a preferred embodiment, the method according to the present invention comprises an additional step of amplifying the mRNA or cDNA of each of said six genes, the complementary sequence thereof or a fragment thereof.
Such as used here, amplification primers are defined as being a pair of nucleic acid molecules that can respectively pair with the 3′ and 5′ regions of a gene in a specific manner (positive and negative strands or vice versa) and encompassing a short region of said gene. Generally, amplification primers have a length of 10 to 30 nucleotides and allow amplifying a region of a length comprised between 50 and 200 nucleotides. Advantageously the primers used in the present invention are those listed in Table 1.
In another particularly preferred embodiment, the expression level of each of said at least six genes is determined by determining the expression level of the polypeptide encoded by said gene or a fragment thereof. Said analysis can be done by using an antibody (for example, a radiolabeled antibody, an antibody labeled with a chromophore, a fluorophore or an enzyme) an antibody derivative (for example, an antibody conjugated to a substrate or to a protein, or a ligand of a protein of a ligand/protein pair (for example biotin-streptavidin)) or an antibody fragment (for example, a single chain antibody, a hypervariable domain of an isolated antibody, etc.) which specifically binds the polypeptide encoded by said gene.
Said analyses can be done by many techniques known to the skilled person including, without being limiting, immunological tests based on the use of enzymatic activity (“enzyme immunoassay” EIA), immunological tests based on the use of radioactive isotopes (RIA), western blot analysis and ELISA (“enzyme linked immunosorbent assay”) tests.
In the sense of the present invention, “polypeptide” means a sequence comprising at least two amino acids, and the terms “polypeptide”, “peptide” and “protein” may be used interchangeably.
In the sense of the present invention, “mRNA or cDNA fragment” means a sequence of at least 50 nucleic acids, for example at least 100 or 150 nucleic acids, preferably at least 200 nucleic acids, for example at least 250 or 350 nucleic acids, and in a particularly preferred manner, a polypeptide [sic; nucleic acid sequence] of at least 400 nucleic acids.
In the sense of the present invention, “polypeptide fragment” means a sequence of at least 50 amino acids, for example at least 100 or 150 amino acids, preferably at least 200 amino acids, for example at least 250 or 350 amino acids, and in a particularly preferred manner, a polypeptide of at least 400 amino acids.
Preferably, the method according to the present invention also comprises a step of comparing the expression level of each of said at least six genes with a reference value. This reference value can serve as a positive and/or negative control.
A positive control can be conducted, for example, by comparing the expression level of said at least one gene in the presence of the test compound with the expression level of said at least one gene in the presence of a compound known to be sensitizing.
As an example of a compound known to be sensitizing, the following can be named: 2,4,6-trinitrobenzene sulfonic acid, p-phenylenediamine, dinitrochlorobenzene, benzaldehyde, resorcinol, tetramethylthiuram disulfide, oxazolone, chloroatranol, diphenylcyclopropenone, potassium dichromate, cinnamaldehyde, 2-bromo-2-(bromomethyl) glutaronitrile, glyoxal, saccharin, formaldehyde, trimellitic anhydride, methylchloroisothiazolinone, benzyl benzoate, alpha-hexyl cinnamaldehyde, eugenol, 2-mercaptobenzothiazole, isoeugenol, diphenylcyclopropenone (DCPP), lauryl gallate (LG), 3-3-dimethylaminopropylamine (3-DMAPA), cinnamaldehyde (CA), citral (Cal), 1,4-hydroquinone (HQ), glutaraldehyde (GA), 1,2-benzisothiazolin-3-one (Ben), phenylacetaldehyde (PA) and lilial (Li), preferably diphenylcyclopropenone, lauryl gallate, 1,4-hydroquinone and glutaraldehyde, and particularly preferably 1,4-hydroquinone.
Alternatively, in the present invention, a sensitizing compound can be used as a positive control, such as “fragrance mix”.
A negative control can be conducted in the absence of the test compound or in the presence of a compound known to be non-sensitizing, such as, for example, olive oil, glycerol, cetyltrimethylammonium bromide (CTAB) and dipropylene glycol.
In the scope of the present invention, we will conclude that a test compound has a sensitizing potential if an overexpression of said gene is observed with regard to its expression level in the absence of said test compound.
“Overexpression” means a significantly higher level of said gene compared to its normal expression level. Preferably, overexpression means an expression level in a biological sample that is greater by at least 20% than the normal expression level of said gene, preferably greater by at least 50% than the normal expression level of said gene, and more particularly preferably greater by at least 90% than the normal expression level of said gene.
“Expression level in the absence of said test compound” or “normal level” is the expression level of said gene in a control sample potentially corresponding to the biological sample of a patient who does not present sensitization or, preferably, to the mean of the expression level of said gene in different control samples.
Preferably, step b) of said method for evaluating sensitizing potential comprises measuring the expression of at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24 and more preferentially, at least 25 genes chosen in the group made up of the genes such as defined in Table 1.
Thus, one can conclude that a test compound has a sensitizing potential if there is an overexpression of at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24 and more preferentially, at least 25 genes chosen in the group made up of genes such as defined in Table 1.
In a particularly preferred manner, step b) of said method for evaluating sensitizing potential comprises determining the expression of the following group of genes: AKR1B10 (aldo-keto reductase family 1, member B10), AKR1C1 (aldo-keto reductase family 1, member C1), AKR1C2 (aldo-keto reductase family 1, member C2), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), FTH1P (ferritin, heavy polypeptide 1), FTL (ferritin, light polypeptide 1), G6PD (glucose-6-phosphate dehydrogenase), GCLM (glutamate-cysteine ligase modifier subunit), NQO2 (NAD(P)H dehydrogenase, quinone 2), SLC7A11 (solute carrier family 7, Member 11), TXNRD1 (thioredoxin reductase 1), UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1), UGT1A9 (UDP glucuronosyltransferase 1 family, polypeptide A9, YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide), CD36 (CD36 molecule), CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide 1), GCLC (glutamate-cysteine ligase, catalytic subunit), HMOX1 (heme oxygenase 1), NQ01 (NAD(P)H dehydrogenase, quinone 1) PSME2 (proteasome activator subunit 2), and S100A8 (S100 calcium binding protein A8), preferably chosen in the group consisting of: AKR1B10 (aldo-keto reductase family 1, member B10), AKR1C1 (aldo-keto reductase family 1, member C1), AKR1C2 (aldo-keto reductase family 1, member C2), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), FTH1P (ferritin, heavy polypeptide 1), FTL (ferritin, light polypeptide 1), G6PD (glucose-6-phosphate dehydrogenase), GCLM (glutamate-cysteine ligase modifier subunit), NQO2 (NAD(P)H dehydrogenase, quinone 2), SLC7A11 (solute carrier family 7, Member 11), TXNRD1 (thioredoxin reductase 1), UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1), UGT1A9 (UDP glucuronosyltransferase 1 family, polypeptide A9, PSME2 (proteasome activator subunit 2), and YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide).
In this particularly preferred embodiment, the test compound is determined to have sensitizing potential if at least 11 of said genes are overexpressed compared to a reference value.
In a particularly preferred manner, step b) of said method for evaluating sensitizing potential comprises determining the expression of at least one of the following group of genes: BRAK (CXC chemokine ligand 14), CTSS (cathepsin S), DAPK2 (death-associated protein kinase 2), FABP4 (fatty acid binding protein 4), HSP27 (heat shock 27 kDa protein), IL18 (IL-18), HSP90 (heat shock 90 kDa protein), IL1R2 (interleukin-1 receptor type II), TPSAB1 (tryptase alpha/beta 1), CXCR1 (interleukin 8 receptor, alpha), DEFB1 (defensin, beta 1), DHFR (dihydrofolate reductase), EHF (ets homologous factor), IVL (involucrin) KRT4 (keratin 4), MELANA (melan-A), NGAL (lipocalin 2), PDZK1IP1 (PDZK1 interacting protein interaction 1), PI3 (peptidase inhibitor 3) and SERPINB3 (serpin peptidase inhibitor member 3),
and even more preferentially, the group of genes BRAK (CXC chemokine ligand 14), CTSS (cathepsin S), DAPK2 (death-associated protein kinase 2), FABP4 (fatty acid binding protein 4), HSP27 (heat shock 27 kDa protein), IL18 (IL-18), IL1R2 (interleukin-1 receptor type II), HSP90 (heat shock 90 kDa protein), and TPSAB1 (tryptase alpha/beta 1), or the group of genes CXCR1 (interleukin 8 receptor, alpha), DEFB1 (defensin, beta 1), DHFR (dihydrofolate reductase), EHF (ets homologous factor), IVL (involucrin) KRT4 (keratin 4), MELANA (melan-A), NGAL (lipocalin 2), PDZK1IP1 (PDZK1 interacting protein interaction 1), PI3 (peptidase inhibitor 3) and SERPINB3 (serpin peptidase inhibitor member 3).
In this particularly preferred embodiment, the test compound is determined to have sensitizing potential if at least 7 of said genes, preferably at least 8 of said genes are overexpressed compared to a reference value.
In one particular embodiment, step b) of said method for evaluating the sensitizing potential of a test compound comprises a step α) of determining the expression level of at least 10, preferably at least 11, 12, 13, 14, 15, 16 and still more preferentially of the following group of genes: AKR1B10 (aldo-keto reductase family 1, member B10), AKR1C1 (aldo-keto reductase family 1, member C1), AKR1C2 (aldo-keto reductase family 1, member C2), CTGF (connective tissue growth factor), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), FTH1P (ferritin, heavy polypeptide 1), FTL (ferritin, light polypeptide 1), G6PD (glucose-6-phosphate dehydrogenase), GCLM (glutamate-cysteine ligase modifier subunit), IER3 (immediate early response 3), NQO2 (NAD(P)H dehydrogenase, quinone 2), SLC7A11 (solute carrier family 7, Member 11), TXNRD1 (thioredoxin reductase 1), UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1), UGT1A9 (UDP glucuronosyltransferase 1 family, polypeptide A9), YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide), CD36 (CD36 molecule), CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide 1), GCLC (glutamate-cysteine ligase, catalytic subunit), HMOX1 (heme oxygenase 1), NQ01 (NAD(P)H dehydrogenase, quinone 1) and S100A8 (S100 calcium binding protein A8).
and optionally a step β) of measuring the expression level of at least 10, preferably 11, 12, 13, 14, 15, 16, 17, 18, 19 and more preferentially of the following group of genes: BRAK (CXC chemokine ligand 14), CTSS (cathepsin S), DAPK2 (death-associated protein kinase 2), FABP4 (fatty acid binding protein 4), HSP27 (heat shock 27 kDa protein), IL18 (IL-18), IL1R2 (interleukin-1 receptor type II), TPSAB1 (tryptase alpha/beta 1), HSP90 (heat shock 90 kDa protein), CXCR1 (interleukin 8 receptor, alpha), DEFB1 (defensin, beta 1), DHFR (dihydrofolate reductase), EHF (ets homologous factor), IVL (involucrin) KRT4 (keratin 4), MELANA (melan-A), NGAL (lipocalin 2), PDZK1IP1 (PDZK1 interacting protein interaction 1), P13 (peptidase inhibitor 3), PSME2 (proteasome activator subunit 2) and SERPINB3 (serpin peptidase inhibitor member 3), and
a step c) of determining the sensitizing potential of a test compound wherein the compound is determined to be sensitizing if:

    
    
        the expression level of at least 7 genes measured in step α) is greater than a threshold value; and/or
        the expression level of at least 7 genes measured in step β) is greater than a limit value.
    
    


Preferably, step b) is done between 2 and 24 hours after step a), still more preferably between 4 and 18 hours after step a), preferentially between 5 and 7 hours after step a) and most preferably of all, 6 hours after step a).
Another aspect of the invention relates to a method for evaluating the sensitizing power of a test compound, comprising the following steps:
1) obtaining at least one test compound dilution, and
2) determining the sensitizing potential of said test compound at said at least one dilution by a method such as defined according to any one of the preceding claims.
“Sensitizing power” means the ability of a given compound to induce a sensitization reaction according to the concentration of said compound. Sensitizing power is dependent on the quantity of substance necessary to induce sensitization. Thus, the lower the sensitizing quantity necessary to induce a sensitizing response, the stronger the sensitizer is, and vice versa, the higher the sensitizing quantity necessary to induce a sensitizing response, the weaker the sensitizer is.
It is thus possible to perform a quantitative analysis of the sensitizing potential of a compound.
Preferably, said test compound is subject to successive dilutions. Thus, steps 1) and 2) will be done for each of the dilutions.
Preferably, said successive dilutions will allow determining the maximum dilution at which said test compound retains sensitizing potential.
Said method for evaluating the sensitizing power of a test compound can also comprise a step of evaluating the sensitizing power of the test compound.
Thus, the more a product retains a sensitizing potential after successive dilutions, the more powerful a sensitizer the test compound is.
Moreover, the sensitizing power of a test compound will also be a function of the irritant potential of said test compound. Thus, the fact that a test compound has an irritant potential increases the sensitizing power of said test compound.
Consequently, in one preferred embodiment, said method also comprises a step of determining the irritant potential of the test compound.
The irritant potential of a test compound can be evaluated by using, for example, the method described in French patent 1051638.
Thus, one can conclude that a product is extremely sensitizing (Extreme E), if:

    
    
        it has a sensitizing potential at a dilution of 1/1000, and/or
        it has a sensitizing potential at a dilution of 1/100, but does not have a sensitizing potential at 1/1000, and has an irritant potential.
    
    


Thus, one can conclude that a product is strongly sensitizing (Strong S), if:

    
    
        it has a sensitizing potential at a dilution of 1/100, but does not have a sensitizing potential at 1/1000, or
        it has a sensitizing potential at a dilution of 1/10, but does not have a sensitizing potential at 1/100 and has an irritant potential.
    
    


Thus, one can conclude that a product is moderately sensitizing (Moderate M), if:

    
    
        it has a sensitizing potential at a dilution of 1/10, but does not have a sensitizing potential at 1/100, or
        it has a sensitizing potential at a dilution of 1/2, but does not have a sensitizing potential at dilutions below 1/2 and has an irritant potential.
    
    


Thus, one can conclude that a product is weakly sensitizing (weak W) if it does not have a sensitizing potential at dilutions below 1/2.
According to another aspect, the present invention relates to the use of at least one, preferably at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 or at least 8 genes chosen in the group consisting of: BRAK (CXC chemokine ligand 14), CTSS (cathepsin S), DAPK2 (death-associated protein kinase 2), FABP4 (fatty acid binding protein 4), HSP27 (heat shock 27 kDa protein), IL18 (IL-18), HSP90 (heat shock 90 kDa protein), IL1R2 (interleukin-1 receptor type II), TPSAB1 (tryptase alpha/beta 1), CXCR1 (interleukin 8 receptor, alpha), DEFB1 (defensin, beta 1), DHFR (dihydrofolate reductase), EHF (ets homologous factor), IVL (involucrin) KRT4 (keratin 4), MELANA (melan-A), NGAL (gelatinase associated lipocalin), PDZK1IP1 (PDZK1 interacting protein interaction 1), PI3 (peptidase inhibitor 3), PSME2 (proteasome activator subunit 2), SERPINB3 (serpin peptidase inhibitor member 3), AKR1B10 (aldo-keto reductase family 1, member B10), AKR1C1 (aldo-keto reductase family 1, member C1), AKR1C2 (aldo-keto reductase family 1, member C2), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), FTH1P (ferritin, heavy polypeptide 1), FTL (ferritin, light polypeptide 1), G6PD (glucose-6-phosphate dehydrogenase), GCLM (glutamate-cysteine ligase modifier subunit), NQO2 (NAD(P)H dehydrogenase, quinone 2), SLC7A11 (solute carrier family 7, Member 11), TXNRD1 (thioredoxin reductase 1), UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1), UGT1A9 (UDP glucuronosyltransferase 1 family, polypeptide A9), and YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) for the in vitro evaluation of the sensitizing potential of a test compound.
The present invention also relates to a kit for the implementation of a method for in vitro evaluation of the sensitizing potential of a test compound, comprising means for determining the expression level of at least six genes selected from the group consisting of BRAK (CXC chemokine ligand 14), CTSS (cathepsin S), DAPK2 (death-associated protein kinase 2), FABP4 (fatty acid binding protein 4), HSP27 (heat shock 27 kDa protein), IL18 (IL-18), HSP90 (heat shock 90 kDa protein), IL1R2 (interleukin-1 receptor type II), TPSAB1 (tryptase alpha/beta 1), CXCR1 (interleukin 8 receptor, alpha), DEFB1 (defensin, beta 1), DHFR (dihydrofolate reductase), EHF (ets homologous factor), IVL (involucrin) KRT4 (keratin 4), MELANA (melan-A), NGAL (lipocalin 2), PDZK1IP1 (PDZK1 interacting protein interaction 1), PI3 (peptidase inhibitor 3), PSME2 (proteasome activator subunit 2), SERPINB3 (serpin peptidase inhibitor member 3), AKR1B10 (aldo-keto reductase family 1, member B10), AKR1C1 (aldo-keto reductase family 1, member C1), AKR1C2 (aldo-keto reductase family 1, member C2), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), FTH1P (ferritin, heavy polypeptide 1), FTL (ferritin, light polypeptide 1), G6PD (glucose-6-phosphate dehydrogenase), GCLM (glutamate-cysteine ligase modifier subunit), NQO2 (NAD(P)H dehydrogenase, quinone 2), SLC7A11 (solute carrier family 7, Member 11), TXNRD1 (thioredoxin reductase 1), UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1), UGT1A9 (UDP glucuronosyltransferase 1 family, polypeptide A9), YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide), CD36 (CD36 molecule), CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide 1), GCLC (glutamate-cysteine ligase, catalytic subunit), HMOX1 (heme oxygenase 1), NQ01 (NAD(P)H dehydrogenase, quinone 1) and S100A8 (S100 calcium binding protein A8).
Preferably, said kit will comprise at least six primer pairs each amplifying at least one gene chosen from the group consisting of: BRAK (CXC chemokine ligand 14), CTSS (cathepsin S), DAPK2 (death-associated protein kinase 2), FABP4 (fatty acid binding protein 4), HSP27 (heat shock 27 kDa protein), IL18 (IL-18), IL1R2 (interleukin-1 receptor type II), HSP90 (heat shock 90 kDa protein), TPSAB1 (tryptase alpha/beta 1), CXCR1 (interleukin 8 receptor, alpha), DEFB1 (defensin, beta 1), DHFR (dihydrofolate reductase), EHF (ets homologous factor), IVL (involucrin) KRT4 (keratin 4), MELANA (melan-A), NGAL (gelatinase associated lipocalin), PDZK1IP1 (PDZK1 interacting protein interaction 1), P13 (peptidase inhibitor 3), PSME2 (proteasome activator subunit 2), SERPINB3 (serpin peptidase inhibitor member 3), AKR1B10 (aldo-keto reductase family 1, member B10), AKR1C1 (aldo-keto reductase family 1, member C1), AKR1C2 (aldo-keto reductase family 1, member C2), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), FTH1P (ferritin, heavy polypeptide 1), FTL (ferritin, light polypeptide 1), G6PD (glucose-6-phosphate dehydrogenase), GCLM (glutamate-cysteine ligase modifier subunit), NQO2 (NAD(P)H dehydrogenase, quinone 2), SLC7A11 (solute carrier family 7, Member 11), TXNRD1 (thioredoxin reductase 1), UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1), UGT1A9 (UDP glucuronosyltransferase 1 family, polypeptide A9), YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide), CD36 (CD36 molecule), CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide 1), GCLC (glutamate-cysteine ligase, catalytic subunit), HMOX1 (heme oxygenase 1), NQ01 (NAD(P)H dehydrogenase, quinone 1) and S100A8 (S100 calcium binding protein A8).
In a particularly preferred manner, said at least one primer pair is chosen in Table 1.


EXAMPLES
1) Demonstration of Biomarkers According to the Skinethic Protocol
Skin pieces of 1.07 cm2 were purchased from EP1SKIN. Various substances were applied either in the liquid form (30 μL at different concentrations) or in the solid form (30 μL PBS or olive oil+30 μg or fewer to assess different powder concentrations) on the skin pieces. After an incubation of 15 min at room temperature, the skin pieces were washed with PBS (25 mL) and incubated 3 h, 6 h or 18 h at 37° C. in a CO2 incubator. After incubation, the skin pieces were sampled with a punch and separated from the collagen support. They were then directly placed in a “Tri Reagent” solution (Ambion) (1 ml) and immediately dissociated mechanically.
cDNA Preparation
The tissues were placed in a “Tri Reagent” solution (Ambion) and mechanically crushed. The RNA was prepared according to the protocol described by the supplier with isopropanol precipitation. To prepare the cDNA, the total RNAs were pretreated with DNAse to remove genomic DNA contaminants. One to 5 μg of total RNA was used for the treatment, with RNAse-free DNAse, RNAsin (1 μL) and random primers (3 μg). The superscript III RT (1.5 mL at 200 U/μL) was then added. The cDNAs were then tested by RT-PCR.
Quantitative PCR
Real time quantitative PCR was performed using the SYBR Green technology of Roche LC480 cyclers. The primers were designed to cover the intron-exon junctions to prevent any traces of genomic DNA amplification. Amplification gives amplicons between 100 and 150 bp. All the primer pairs were qualified by digestion with restriction enzymes and analyzed by electrophoresis. PCR was performed on 10 μL by using the Sybr green 2×PCR mix from Roche in PCR plates from Roche. Target gene expression was measured after RNA normalization by means of four housekeeping genes, and the values are expressed by using the CT method, and expressed in additional expression rates with regard to a theoretical zero (User Bulletin no. 2, Applied Biosystems, December 1997).
Results
For this analysis, an application of 15 min followed by washing and a post-incubation of 6 h were done before biopsy and analysis of the gene transcription. The results are shown in Table 1.







TABLE 1







expression level of genes specific for sensitivity in the EPISKIN model


















Expression
Expression








level in
level in








the
the pre-








absence of
sence of




Abbrevi-
Gene name

Accession
sensiting
sensiting
Sens
Antisense


ation
in French
English name
number
agent
agent
primer
primer





BRAK
CXC 
chemokine
NM_004887.4
0.653688171
1.8
AAGTACCCGCAC
TTGGTGCTCTGCAGC



chimiokine
(C-X-C motif)



TGCGAGGAGAAG
TTGGGGTGC



ligand 14
ligand 14



(SEQ ID NO: 1)
(SEQ ID NO: 2)


 


HSP90
Protéine 
Heat shock
NM_005348.3
1.054321721
1.75
TCTGCCTCTGGT
TTTCCGAAGACGTTC



de choc 
90kDa protein



GATGAGATGGTT
CACAAAGGC



thermique




(SEQ ID
(SEQ ID 



de90kDa




NO: 81)
NO: 82)


 


CTSS
cathepsine 
cathepsin S
NM_004079.3
0.51868364
2.296020251
TCATACGATCTG
TGGGAACTCTCAGGG



S




GGCATGAACCAC
AACTCATCA








(SEQ ID NO: 3)
(SEQ ID NO: 4)


 


DAPK2
protéine 
death-
NM_014326.3
1.012589915
1.454643548
TTTCCTTCAGCA
TGTCACTCTCACAGT



kinase
associated



TCGTGTCCCTGT
TCCTCAGGT



associée à
protein kinase



(SEQ ID NO: 5)
(SEQ ID NO: 6)



la mort 2
2







 


FABP4
Proteine 
fatty acid
NM_001442.2
0.844425344
2.920748581
ACTGGGCCAGGA
TTTCTGCACATGTAC



de 
binding



ATTTGACGAAGT
CAGGACACC



fixation 
protein 4,



(SEQ ID NO: 7)
(SEQ ID NO: 8)



des acides
adipocyte








gras 4








 


HSP27
Protéine 
heat shock
NM_001540.3
0.868543127
1.612936908
TGCTTCACGCGG
CTGGGATGGTGATCT



de choc 
27kDa protein



AAATACACGCT
CGTTGGACT



thermique
1



(SEQ ID NO: 9)
(SEQ ID 



de27kDa





NO: 10)


 


IL18
Inter-
Interleukin 18
NM_001562.2
1.190956418
2.309301387
TCATTGACCAAG
AGCCATACCTCTAGG



leukine 18
(interferon-



GAAATCGGCCTC
CTGGCTAT




gamma-inducing



(SEQ ID
(SEQ ID 




factor)



NO: 11)
NO: 12)


 


IL1R2
Recepteur
interleukin 1
NM_173343.1
1.280621228
1.9
GCCAGCCTTGCA
TTGCGGGTATGAGAT



type II de
receptor,



GGAGGACTCTG
GAACGGCAG



l'inter-
type II



(SEQ ID
(SEQ ID 



leukine 1




NO: 13)
NO: 14)


 


TPSAB1
tryptase
tryptase
NM_003294.3
0.048878149
33.92776528
ACGGCCCATACT
CAGCAGCTGGTCCTG



alpha/beta
alpha/beta 1



GGATGCACTTCT
GTAGTAGA



1




(SEQ ID
(SEQ ID 








NO: 15)
NO: 16)


 


CXCR1
Recepteur
interleukin 8
NM_000634.2
1.442894074
3.492178966
TATGAATCTGTCC
ACCTCATAGCAAACT



alpha de
receptor,



CTGCCCTTC
GGACTGGAA



l'inter-
alpha



(SEQ ID
(SEQ ID 



leukin 8




NO: 17)
NO: 18)


 


DEFB1
defensine,
defensin,
NM_005218.3
0.667424597
2.431463704
TTCCTGAAATCCT
AGGCCTGTGAGAAA



beta 1
beta 1



GGGTGTTGCCT
GTTACCACCT








(SEQ ID
(SEQ ID 








NO: 19)
NO: 20)


 


DHFR
dihydro-
dihydrofolate
NM_000791.3
1.033017625
2.6
CTCATTTTCTTTC
TGCCACCAACTATCC



folate
reductase



CAGAAGTCTAG
AGACCATGT



reductase




(SEQ ID
(SEQ ID 








NO: 21)
NO: 22)


 


EHF
Facteur
ets homologous
NM_012153.3
0.827499527
2.815783657
TTGGCTCTCTCAT
AGGTATGACACTGTG



homologue 
factor



GTCCTTGGCTT
GTAGGTGCT



ets




(SEQ ID
(SEQ ID 








NO: 23)
NO: 24)


 


IVL
involucrine
involucrin
NM_005547.2
0.943876521
1.859505427
TGCCCACAAAGG
TCTGGACACTGCGGG








GAGAAGTATTGC
TGGTTATTT








(SEQ ID
(SEQ ID 








NO: 25)
NO: 26)


 


KRT4
keratine 
keratin 4
NM_002272.2
0.925148858
1.35742071
TCACATATGTCCC
TGCCGGGTGTTGGAG



4




TTCCCAGTCCA
AAGTAGTTT








(SEQ ID
(SEQ ID 








NO: 27)
NO: 28)


 


MELANA
melane-A
melan-A
NM_005511.1
0.763327598
2.7
TCTTACTGCTCAT
TGAAGAGACACTTTG








CGGCTGTTGGT
CTGTCCCGA








(SEQ ID
(SEQ ID 








NO: 29)
NO: 30)


 


NGAL
Gelatinase 
lipocalin 2
NM_005564.3
1.069357565
1.8
GTGAGCACCAAC
AGTTCCGAAGTCAGC



des




TACAACCAGCAT
TCCTTGGTT



neutro-




(SEQ ID
(SEQ ID 



philes




NO: 31)
NO: 32)


 


PDZK11P1
Protéine
PDZK1
NM_005764.3
0.705479404
2.3
CAATCGCCTTTGC
ACCAGGACTCCATCT



d'inter-
interacting



AGTCAACCACT
GCCTTGTTT



action
protein 1



(SEQ ID
(SEQ ID 



PDZK1




NO: 33)
NO: 34)


 


PI3
Inhibiteur
peptidase
NM_002638.3
1.19471567
2.5
TCTTGATCGTGGT
GACTGGCTCTTGCGC



de 
inhibitor 3,



GGTGTTCCTCA
TTTGACTTT



peptidase
skin-derived



(SEQ ID
(SEQ ID 








NO: 35)
NO: 36)


 


PSME2
Sous unité
proteasome
NM_002818.2
1.066067422
1.9
TCCCTCAATGTG
TCTCATTCCCAGGGA



activa- 
activator



GCTGACTTGACT
GAAATCCAC



trice du 
subunit 2



(SEQ ID
(SEQ ID 



proteasome
(PA28 beta)



NO: 37)
NO: 38)



2








 


SERPINB3
Membre 3
serpin
NM_006919.2
0.462228737
4.366336795
ACTCCTGGGTGG
ACTCCTGGGTGGAA



des Inhi- 
peptidase



AAAGTCAAACGA
AGTCAAACGA



biteurs de
inhibitor,



(SEQ ID
(SEQ ID 



la serpine
clade B



NO: 39)
NO: 40)



peptidase
(ovalbumin),









member 3







 


AKR1B10
Membre B10
aldo-keto
NM_020299.4
1.219844195
1.9
AAGATGATAAAGG
AGCTTCTCGATCTGG



de la 
reductase



TAATGCCATCG
AAGTGGCTG



famille
family 1,



(SEQ ID
(SEQ ID 



des aldo-
member B10



NO: 41)
NO: 42)



keto
(aldose








reductases
reductase)







 


AKR1C1
Membre C1
aldo-keto
NM_001353.5
1.587681403
3.432182344
GCAGAGGTTCCTA
ACCTGCTCCTCATTA



de la 
reductase



AAAGTAAAGCTTT
TTGTATAAATGA



famille
family 1,



A
(SEQ ID 



des aldo-
member C1



(SEQ ID
NO: 44)



keto




NO: 43)




reductases








 


AKR1C2
Membre C2
aldo-keto
NM_001354.4
0.686185206
3.101161617
CATTGCATGAGGT
CTTAGCTGTAGCTTA



de la 
reductase



CTGCCAGAAGG
CTGAAGTCG



famille 
family 1,



(SEQ ID
(SEQ ID 



des aldo-
member C2



NO: 45)
NO: 46)



keto









reductases








 


CYP1B1
cytochrome
cytochrome
NM_000104.3
0.751299588
17.37117803
ATCAACAAGGACC
TCATTTGGGTTGGCC



P450, 
P450,



TGACCAGCAGA
CTGAAATCG



famille
family 1,



(SEQ ID
(SEQ ID 



1, sous
subfamily B,



NO : 47)
NO: 48)



famille B,
polypeptide 1








polypep-









tide 1








 


FTH1P
Polypeptide
ferritin,
NM_002032.2
1.032878604
1.942318842
CTTTGACCGCGAT
TCAGTTTCTCAGCAT



lourd 
heavy



GATGTGGCTTT
GTTCCCTCT



de la
polypeptide 1



(SEQ ID
(SEQ ID 



ferritine,




NO: 49)
NO: 50)



1








 


FTL
Polypeptide
ferritin,
NM_000146.3
0.681444874
2.940199851
TTGGATCTTCATG
AGTCGTGCTTGAGAG



léger de la
light



CCCTGGGTTCT
TGAGCCTTT



ferritine,
polypeptide



(SEQ ID
(SEQ ID 



1




NO: 51)
NO: 52)


 


G6PD
glucose-6-
glucose-6-
NM_000402.3
0.581030262
1.7
GAACCTCATGGTG
TGAGGATAACGCAG



phosphate
phosphate



CTGAGATTTGC
GCGATGTTGT



dehydro-
dehydrogenase



(SEQ ID
(SEQ ID 



genase




NO: 53)
NO: 54)


 


GCLM
Sous unité
glutamate-
NM_002061.2
0.874803304
5.083406994
ATGGCCTGTTCAG
TCCCAGTAAGGCTGT



modifica-
cysteine



TCCTTGGAGTT
AAATGCTCC



trice 
ligase,



(SEQ ID
(SEQ ID 



de la
modifier



NO: 55)
NO: 56)



glutamate-
subunit








cysteine









ligase








 


NQO2
NAD(P)H
NAD(P)H
NM_000904.3
1.170224823
2.8
CACGAAGACAGGA
CGGATGCAATTTCAG



dehydro-
dehydrogenase,



GTCAATGGAGA
GAGCAAAGC



genase des 
quinone 2



(SEQ ID
(SEQ ID 



quinone s2




NO: 57)
NO: 58)


 


SLC7A11
Membre 11
solute carrier
NM_014331.3
0.502347786
5.088930603
TATCCCTGGCATT
TGCCCACAGCTGTAA



de la 
family 7,



TGGACGCTACA
TGAGCTTGA



famille
(cationic



(SEQ ID
(SEQ ID 



7 des  
amino acid



NO: 59)
NO: 60)



porteurs
transporter,








de
y+ system)








molécules
member 11








solubles








 


TXNRD1
thiore-
thioredoxin
NM_182729.1
0.725407592
3.70757292
GGTCCTCACAGGA
TGCCCAAGTAACGTG



doxine 
reductase 1



TTAAGGCAACA
GTCTTTCAC



reductase




(SEQ ID
(SEQ ID 



1




NO: 61)
NO: 62)


 


UGT1A1
Polypeptide
UDP glucurono-
NM_000463.2
0.527392251
1.408908304
ACAGAACTTTCTG
AGCCAGACAGATGC



A1 de la
syltransferase



TGCGACGTGGT
AGAGCTCAAT



famille 
1 family,



(SEQ ID
(SEQ ID 



des UDP 
polypeptide



NO: 63)
NO: 64)



glucurono-
A1








syltrans-









ferase 1








 


UGT1A9
Polypeptide
UDP glucurono-
NM_021027.2
0.568212175
1.402794916
GCTTTGCCGAGGC
ATGAGTTTCTCCACC



A9 de la
syltransferase



AGGGAAGCTAC
ACCGACCTC



famille 
1 family,



(SEQ ID
(SEQ ID 



des UDP 
polypeptide



NO: 65)
NO: 66)



glucurono-
A9








syltrans-









ferase 1








 


YWHAZ
Polypeptide
tyrosine 3-
NM_001135702.1
1.154023086
3.1
ACAGCAGATGGCT
TGCTCTCTGCTTGTG



zeta de la
monooxygenase/



CGAGAATACAG
AAGCATTGG



proteine
tryptophan 5-



(SEQ ID
(SEQ ID 



d'activa-
monooxygenase



NO: 67)
NO: 68)



tion 
activation








de tyrosine
protein, zeta








3-monooxy- 
polypeptide








genase/









tryptophan









5-monooxy-









genase









polypeptide








 


CD36
CD36 
CD36 molecule
NM_001127444.1
0.640243459
5.375531509
ACAGATGCAGCCT
GGGATTCCTTTCAGA



molecule
(thrombospondin



CATTTCCACCT
TTAACGTCGG




receptor)



(SEQ ID
(SEQ ID 








NO: 69)
NO: 70)


CYP1A1
cytochrome
cytochrome
NM_000499.3
0.549457486
3.763056606
AGTGGCAGATCAA
ACACCTTGTCGATAG



P450, 
P450, family



CCATGACCAGA
CACCATCAG



famille
1, subfamily



(SEQ ID
(SEQ ID 



1, sous
A, polypeptide



NO: 71)
NO: 72)



famille A,
1








poly-









peptide 1








 


GCLC
Sous unité
glutamate-
NM_001498.2
0.905634705
2.728685624
AATGGGCAATTGC
AAAGGGAGATGCAGC



catalitique
cysteine



TGTCTCCAGGT
ACTCAAAGC



de la
ligase,



(SEQ ID
(SEQ ID 



glutamate-
catalytic



NO: 73)
NO: 74)



cysteine
subunit








ligase








 


HMOX1
heme oxy-
heme oxygenase
NM_002133.1
0.76137536
1.461320376
GGGCCAGCAACAA
TCGCCACCAGAAAGC



genase 1
(decycling) 1



AGTGCAAGATT
TGAGTGTAA








(SEQ ID
(SEQ ID 








NO: 75)
NO: 76)


 


NQO1
NAD(P)H
NAD(P)H
NM_001025434.1
0.740574662
1.948391073
AGTGGCTCCATGT
TTCTCCAGGCGTTTC



dehydro-
dehydrogenase,



ACTCTCTGCAA
TTCCATCCT



genase,
quinone 1



(SEQ ID
(SEQ ID 



des 




NO: 77)
NO: 78)



quinones 1








 


S100A8
Protéine
S100 calcium
NM_002964.3
0.970410479
1.890390064
GGGATGACCTGAA
TGTTGATATCCAACT



de fixation
binding



GAAATTGCTA
CTTTGAACCA



du calcium
protein A8



(SEQ ID
(SEQ ID 



S100 A8




NO: 79)
NO: 80)









Moreover, 35 substances classified according to their characteristics (non-irritant (non -IRR), non-sensitizer (NS), irritant (IRR) or sensitizing by using the official classification of Extreme (E), Strong (S), Moderate (M) and Weak (W) were tested at different doses (Table 2(a) and Table 2(b)). For analysis, the dose permitting the maximum response without inducing too much tissue destruction (corrosion) was selected.












Class
Name
CAS#







S
Hydroquinone
123-31-9


S
2-aminophenol
95-55-6


E
4-Nitrobenzylbromide (electrophile)
100-11-8


S
2,4,6-Trinitrobenzene sulfonic acid
2508-19-2


S
p-Phenylenediamine
106-50-3


M
Citral
5392-40-5


S
Dinitrochlorobenzene
97-00-7


M
Benzaldehyde
100-52-7


W
Resorcinol
108-46-3


M
Benzisothiazolin-3-one (Benzisothiazolinone)
2634-33-5


M
Tetramethylthiuram disulfide
137-26-8


S
Oxazolone
15646-46-5


S
Chloroatranol
57074-21-2


E
Diphenylcyclopropenone
886-38-4


S
Potassium Dichromate
7778-50-9


M
cinnamaldehyde
104-55-2


S
2-Bromo-2-(bromomethyl)glutaronitrile
35691-65-7


M
Glyoxal
107-22-2


M
Saccharine
6485-34-3


S
Formaldehyde
50-00-0


M
Trimellitic anhydride
552-30-7


E
Methylchloroisothiazolinone
26172-55-4


M
Phenyl Acetaldehyde
122-78-1


W
Benzyl benzoate
120-51-4


W
Lilial
80-5406


W
alpha-Hexyl cinnamaldehyde
101-86-0


W
Eugenol
97-53-0


M
2-mercaptobenzothiazole
149-30-4


S
Glutaraldehyde
111-30-8


M
Isoeugenol
97-54-1


NS
Dipropylene Glycol
25265-71-8


NS
Glycerol
56-81-5


NS
Cetyl trimethylammonium bromide
57-09-0


NS
Limonen
5989-54-8


IRR
Lactic acid (cys pos)
598-82-3


IRR
Sodium lauryl sulfate
151-21-3









Three groups of biomarkers were tested on the samples, those specific for irritation, those represented by the ARE gene family (genes under the control of ARE “antioxidant responsive element promoters”, i.e., AKR1B10, AKR1C1, AKR1C2, CYP1B1, FTH1P, FTL, G6PD, GCLM, NQO2, SLC7A11, TXNRD1, UGT1A1, UGT1A9, YWHAZ, CD36, CYP1A1, GCLC, HMOX1, NQO1, PSME2 and S100A8 genes) and another group of genes specific for sensitization, notably for sensitizing substances that do not induce ARE genes.
Irritants for which the expression of the IL-8 gene is 50 times greater than the control and that induce a non-specific expression of ARE genes were retested at lower doses.
The results are presented in Tables 2 and 3, with a code according to the degree of overexpression of the genes. If the expression is greater than 1.3 compared to the control, a grade of 11s given; if this expression is lower, the grade is 0. Note that, preferably, if the number of ARE genes is greater than 11, one can conclude that the substance is certainly sensitizing. For substances that do not strongly induce ARE genes, the other group of sensitization genes is analyzed.
Observe that, preferably, if at least 8 of these genes are overexpressed, then the substance is clearly sensitizing.
Preferably, said test is done both on the group of ARE genes and the group of “non-ARE” genes.







TABLE 2













 








 








 















In order to be able to evaluate the sensitizing power of the compounds, a selection of compounds was subjected to successive dilutions in order to be tested again for their sensitizing potential, as shown in Table 3.


























DAPK2
CTSS
EHF
NGAL
S100A8
DEFB1
P13
CXCR1
IL1R2
FABP4
IVL
CD36
HSP27





methyl-chloro-


isothiazolinone


MCIT-50%
1.0
2.0
5.8
2.2
1.6
3.0
2.2
1.6
5.1
1.5
5.0
0.8
1.3


NUMBER OF GENES
0
1
1
1
1
1
1
1
1
1
1
0
1


MCIT-10%
0.8
1.1
3.7
2.2
1.5
2.6
2.1
1.0
3.2
1.4
3.7
0.7
1.1


NUMBER OF GENES
0
0
1
1
1
1
1
0
1
1
1
0
0


MCIT-1%
0.8
1.3
1.8
1.2
1.2
1.7
1.6
1.2
1.7
1.3
2.2
0.8
0.8


NUMBER OF GENES
0
0
1
0
0
1
1
0
1
1
1
0
0


MCIT-0.1%
0.6
0.7
1.1
2.2
1.0
1.1
1.9
1.4
1.7
1.0
1.3
1.1
1.3


NUMBER OF GENES
0
0
0
1
0
0
1
1
1
0
0
0
0


diphenyl cyclopropenone


DPCP-50%
1.2
0.9
2.1
0.6
1.1
1.4
1.2
1.5
1.2
0.9
1.4
1.0
1.1


NUMBER OF GENES
0
0
1
0
0
1
0
1
0
0
1
0
0


DPCP-10%
1.2
0.9
1.2
1.2
1.1
1.1
1.1
1.2
0.9
1.0
1.3
1.0
1.0


NUMBER OF GENES
0
0
0
0
0
0
0
0
0
0
0
0
0


DPCP-1%
1.2
1.0
1.2
1.0
1.2
1.3
1.1
1.5
1.4
0.8
1.3
1.3
1.0


NUMBER OF GENES
0
0
0
0
0
0
0
1
1
0
0
0
0


DPCP-0.1%
1.3
1.6
0.8
1.1
1.0
1.2
1.1
2.3
1.1
1.1
1.3
0.8
0.9


NUMBER OF GENES
0
1
0
0
0
0
0
1
0
0
0
0
0































TOTAL






KRT4
BRAK
SERPINB3
MELANA
HSP90
PDZK1iP1
IL18
DHFR
SENS
GCLC
NQO1
FTL





methyl-chloro-


isothiazolinone


MCIT-50%
1.1
0.3
3.2
0.4
3.5
0.8
0.9
0.6

16.5
4.6
17.0


NUMBER OF
0
0
1
0
1
0
0
0
13
1
1
1


GENES


MCIT-10%
1.6
0.3
2.7
0.6
2.0
0.9
0.7
0.7

14.8
6.8
11.0


NUMBER OF
1
0
1
0
1
0
0
0
11
1
1
1


GENES


MCIT-1%
1.2
0.5
1.7
0.7
1.3
0.9
0.8
0.8

5.3
5.1
4.4


NUMBER OF
0
0
1
0
1
0
0
0
8
1
1
1


GENES


MCIT-0.1%
2.4
1.5
1.6
0.3
1.4
1.3
1.3
1.2

1.7
1.8
1.2


NUMBER OF
1
1
1
0
1
1
0
0
9
1
1
0


GENES


diphenyl


cyclopropenone


DPCP-50%
0.5
0.3
1.2
1.2
2.7
0.8
0.9
0.9

7.2
6.2
4.9


NUMBER OF
0
0
0
0
1
0
0
0
5
1
1
1


GENES


DPCP-10%
0.7
0.7
1.0
1.5
1.2
1.0
0.8
0.9

5.0
4.3
3.2


NUMBER OF
0
0
0
1
0
0
0
0
4
1
1
1


GENES


DPCP-1%
1.0
0.7
1.3
0.4
1.2
1.2
1.2
0.8

1.7
2.0
1.5


NUMBER OF
0
0
0
0
0
0
0
0
2
1
1
1


GENES


DPCP-0.1%
1.0
1.2
0.9
0.8
1.0
0.9
0.6
0.9

1.0
1.8
1.4


NUMBER OF
0
0
0
0
0
0
0
0
2
0
1
1


GENES





















FTH1P
AKR1B10
AKR1C2
AKR1C1
CYP1B1
CYP1A1
TXNRD1
HMOX1
UGT1A1
UGT1A9





methyl-chloro-


isothiazolinone


MCIT-50%
5.7
1.0
10.6
24.8
177.9
3855.0
45.1
22.4
1.3
1.7


NUMBER OF
1
0
1
1
1
1
1
1
1
1


GENES


MCIT-10%
3.8
1.3
9.6
27.0
49.5
852.8
29.7
14.3
1.4
1.2


NUMBER OF
1
1
1
1
1
1
1
1
1
0


GENES


MCIT-1%
2.0
1.8
6.6
15.6
35.0
217.3
10.9
3.1
2.1
1.5


NUMBER OF
1
1
1
1
1
1
1
1
1
1


GENES


MCIT-0.1%
1.1
1.4
1.5
1.3
49.0
115.5
1.5
1.5
2.1
1.0


NUMBER OF
0
0
1
0
1
1
1
1
1
0


GENES


diphenyl


cyclopropenone


DPCP-50%
2.5
1.9
7.3
19.3
32.5
13.9
23.5
10.0
3.4
1.9


NUMBER OF
1
1
1
1
1
1
1
1
1
1


GENES


DPCP-10%
2.3
2.0
5.0
9.8
68.6
87.6
10.0
2.2
4.6
3.1


NUMBER OF
1
1
1
1
1
1
1
1
1
1


GENES


DPCP-1%
1.3
1.2
2.1
3.6
40.5
45.7
3.7
1.6
2.5
1.8


NUMBER OF
0
0
1
1
1
1
1
1
1
1


GENES


DPCP-0.1%
1.2
1.1
1.6
0.9
2.6
9.2
1.6
0.9
2.1
1.1


NUMBER OF
0
0
1
0
1
1
1
0
1
0


GENES




















NQO2
SLC7A11
GCLM
AHR
TOTAL ARE
SENS >1.3
ARE >1.3





methyl-chloro-


isothiazolinone


MCIT-50%
2.0
29.1
21.8
1.0


NUMBER OF
1
1
1
0
15
13
15
Sensitizing
EXTREME


GENES


MCIT-10%
1.8
26.5
19.1
1.3


NUMBER OF
1
1
1
1
16
11
16
Sensitizing


GENES


MCIT-1%
1.6
12.8
11.3
1.1


NUMBER OF
1
1
1
0
16
8
16
Sensitizing


GENES


MCIT-0.1%
1.4
2.3
1.5
1.6


NUMBER OF
0
1
1
1
11
9
11
Sensitizing


GENES


diphenyl


cyclopropenone


DPCP-50%
1.5
24.3
13.8
1.4


NUMBER OF
1
1
1
1
17
5
17
Sensitizing
EXTREME


GENES


DPCP-10%
1.7
13.5
7.3
0.8


NUMBER OF
1
1
1
0
16
4
16
Sensitizing


GENES


DPCP-1%
1.4
2.8
2.4
0.6


NUMBER OF
1
1
1
0
14
2
14
Sensitizing


GENES


DPCP-0.1%
1.1
1.7
1.3
0.9


NUMBER OF
0
1
0
0
8
2
8
Sensitizing


GENES
























DAPK2
CTSS
EHF
NGAL
S100A8
DEFB1
P13
CXCR1
IL1R2
FABP4
IVL
CD36
HSP27





2-aminophenol


2-aminophenol-
1.0
1.3
1.7
1.8
1.3
1.9
1.5
2.3
2.0
1.1
1.8
0.7
1.0


50%


NUMBER OF GENES
0
1
1
1
1
1
1
1
1
0
1
0
0


2-aminophenol-
1.0
1.2
1.5
1.9
1.2
1.8
1.4
1.5
1.8
1.1
1.8
0.5
1.1


10%


NUMBER OF GENES
0
0
1
1
0
1
1
1
1
0
1
0
0


2-aminophenol-
0.9
1.4
1.4
1.5
1.2
1.4
1.2
2.0
1.3
0.7
1.3
0.6
0.8


1%


NUMBER OF GENES
0
1
1
1
0
1
0
1
0
0
1
0
0


2-aminophenol-
1.0
1.4
1.2
1.1
1.4
1.1
1.3
1.0
1.0
0.9
0.9
0.9
0.8


0.1%


NUMBER OF GENES
0
1
0
0
1
0
0
0
0
0
0
0
0


Hydroquinone


hydroquinone-
1.0
1.6
1.5
1.3
1.4
2.0
1.7
2.9
1.5
1.4
2.6
0.8
1.3


50%


NUMBER OF GENES
0
1
1
0
1
1
1
1
1
1
1
0
0


hydroquinone-
1.2
0.8
1.2
2.0
1.4
1.5
1.4
2.2
1.4
1.2
1.8
0.7
1.0


10%


NUMBER OF GENES
0
0
0
1
0
1
0
1
0
0
1
0
0


hydroquinone-1%
1.2
1.2
1.1
1.7
1.4
1.1
1.2
2.8
1.2
1.1
1.5
0.7
0.9


NUMBER OF GENES
0
0
0
1
1
0
0
1
0
0
1
0
0


hydroquinone-
1.1
0.7
1.2
1.5
1.1
1.7
1.1
2.9
1.3
1.1
1.6
0.7
0.8


0.1%


NUMBER OF GENES
0
0
0
1
0
1
0
1
0
0
1
0
0






























TOTAL





KRT4
BRAK
SERPINB3
MELANA
HSP90
PDZK1iP1
IL18
DHFR
SENS
GCLC
NQO1





2-aminophenol


2-aminophenol-
1.7
0.3
1.4
0.2
1.5
1.0
0.9
0.9

4.0
3.3


50%


NUMBER OF GENES
1
0
1
0
1
0
0
0
12
1
1


2-aminophenol-
1.7
0.3
1.2
0.2
1.5
1.0
0.9
0.9

2.3
2.5


10%


NUMBER OF GENES
1
0
0
0
1
0
0
0
9
1
1


2-aminophenol-
1.1
0.5
1.2
0.5
1.2
1.0
0.8
0.9

2.2
1.1


1%


NUMBER OF GENES
0
0
0
0
0
0
0
0
6
1
0


2-aminophenol-
1.7
0.8
1.3
1.1
1.0
1.0
1.1
1.2

1.1
0.9


0.1%


NUMBER OF GENES
1
0
1
0
0
0
0
0
4
0
0


Hydroquinone


hydroquinone-
1.1
0.8
1.1
1.6
1.8
1.0
0.9
1.1

2.4
2.8


50%


NUMBER OF GENES
0
0
0
1
1
0
0
0
11
1
1


hydroquinone-
1.2
0.8
1.1
0.9
1.2
1.0
0.9
0.7

1.5
1.5


10%


NUMBER OF GENES
0
0
0
0
0
0
0
0
4
1
1


hydroquinone-1%
1.5
0.8
1.0
0.7
1.1
1.0
0.8
0.8

1.3
1.3


NUMBER OF GENES
1
0
0
0
0
0
0
0
5
0
0


hydroquinone-
2.0
0.7
1.1
0.7
1.3
1.2
0.8
0.4

1.3
1.2


0.1%


NUMBER OF GENES
1
0
0
0
0
0
0
0
5
0
0





















FTL
FTH1P
AKR1B10
AKR1C2
AKR1C1
CYP1B
CYP1A1
TXNRD1
HMOX1
UGT1A1





2-aminophenol


2-aminophenol-
3.5
2.0
1.3
3.3
8.6
37.7
66.5
6.4
1.9
2.1


50%


NUMBER OF GENES
1
1
1
1
1
1
1
1
1
1


2-aminophenol-
2.5
1.5
1.1
3.4
9.3
45.1
122.0
7.6
1.7
1.8


10%


NUMBER OF GENES
1
1
0
1
1
1
1
1
1
1


2-aminophenol-
2.1
1.2
0.9
1.2
4.3
15.6
14.6
4.8
1.4
1.6


1%


NUMBER OF GENES
1
0
0
0
1
1
1
1
1
1


2-aminophenol-
1.0
0.9
0.8
1.1
1.2
5.3
7.1
3.0
1.1
0.9


0.1%


NUMBER OF GENES
0
0
0
0
0
1
1
1
0
0


Hydroquinone


hydroquinone-
3.5
2.1
1.1
3.7
3.5
49.5
57.8
7.9
3.5
1.9


50%


NUMBER OF GENES
1
1
0
1
1
1
1
1
1
1


hydroquinone-
1.9
1.4
1.0
1.8
2.6
17.1
33.2
2.6
1.2
1.5


10%


NUMBER OF GENES
1
1
0
1
1
1
1
1
0
1


hydroquinone-1%
1.5
1.3
1.0
1.3
2.0
7.1
15.4
2.1
1.0
1.4


NUMBER OF GENES
1
0
0
0
1
1
1
1
0
0


hydroquinone-
1.5
1.2
0.9
0.7
1.2
1.8
8.0
2.3
1.1
1.7


0.1%


NUMBER OF GENES
1
0
0
0
0
1
1
1
0
1


























TOTAL







UGT1A9
NQO2
SLC7A11
GCLM
AHR
ARE
SENS >1.3
ARE >1.3





2-aminophenol


2-aminophenol-
0.4
1.3
6.0
4.7
1.3


50%


NUMBER OF GENES
0
0
1
1
0
14
12
14
Sensitizing
STRONG


2-aminophenol-
1.2
1.4
3.8
4.6
0.7


10%


NUMBER OF GENES
0
1
1
1
0
14
9
14
Sensitizing


2-aminophenol-
1.0
1.1
3.2
3.5
0.6


1%


NUMBER OF GENES
0
0
1
1
0
10
6
10
Sensitizing


2-aminophenol-
1.1
1.0
2.1
2.1
1.0


0.1%


NUMBER OF GENES
0
0
1
1
0
5
4
5
Non-











sensitizing


Hydroquinone


hydroquinone-
1.7
2.0
5.4
3.8
0.9


50%


NUMBER OF GENES
1
1
1
1
0
15
11
15
Sensitizing
STRONG


hydroquinone-
1.3
1.5
1.6
2.0
0.6


10%


NUMBER OF GENES
0
1
1
1
0
13
4
13
Sensitizing


hydroquinone-1%
1.5
1.4
1.5
1.8
0.6


NUMBER OF GENES
1
1
1
1
0
9
5
9
Sensitizing


hydroquinone-
0.8
0.9
1.3
0.7
0.8


0.1%


NUMBER OF GENES
0
0
0
0
0
5
2
5
Non-











sensitizing
























DAPK2
CTSS
EHF
NGAL
S100A8
DEFB1
P13
CXCR1
IL1R2
FABP4
IVL
CD36
HSP27





Isoeugenol


isoeugenol-50%
1.4
1.4
2.0
2.7
1.5
2.1
1.7
2.6
1.7
1.8
2.8
0.6
1.2


NUMBER OF GENES
1
1
1
1
1
1
1
1
1
1
1
0
0


isoeugenol-10%
0.9
1.5
1.4
2.1
1.3
1.5
1.4
1.6
1.4
1.2
1.9
0.6
0.9


NUMBER OF GENES
0
1
1
1
1
1
1
1
1
0
1
0
0


isoeugenol-1%
0.9
0.8
1.2
1.1
1.0
1.1
0.9
1.1
1.4
0.9
1.4
1.3
0.8


NUMBER OF GENES
0
0
0
0
0
0
0
0
1
0
1
1
0


isoeugenol-0.1%
1.1
0.9
1.5
1.1
1.1
1.0
1.1
0.7
1.2
0.8
1.3
0.9
1.0


NUMBER OF GENES
0
0
1
0
0
0
0
0
0
0
1
0
0


Glyoxal


Glyoxal-50%
0.9
2.7
0.9
0.6
0.9
1.6
1.4
2.7
2.0
0.8
1.5
1.1
0.8


NUMBER OF GENES
0
1
0
0
0
1
1
1
1
0
1
0
0


Glyoxal-10%
0.9
2.2
0.9
1.3
0.9
1.6
1.3
1.8
2.2
0.8
1.6
1.3
0.8


NUMBER OF GENES
0
1
0
1
0
1
1
1
1
0
1
1
0


Glyoxal-1%
0.9
1.3
0.9
0.6
0.9
1.4
1.4
1.6
1.4
0.8
1.7
1.1
0.8


NUMBER OF GENES
0
1
0
0
0
1
1
1
1
0
1
0
0


Glyoxal-0.1%
0.9
1.0
0.8
0.7
0.7
0.8
0.8
1.1
0.9
0.4
0.7
1.1
0.7


NUMBER OF GENES
0
0
0
0
0
0
0
0
0
0
0
0
0






























TOTAL





KRT4
BRAK
SERPINB3
MELANA
HSP90
PDZK1iP1
IL18
DHFR
SENS
GCLC
NQO1





Isoeugenol


isoeugenol-50%
1.2
0.6
1.5
0.4
1.7
1.1
0.8
0.9

1.3
1.8


NUMBER OF GENES
0
0
1
0
1
0
0
0
13
1
1


isoeugenol-10%
1.0
0.8
1.1
0.6
1.3
1.0
0.7
0.9

0.8
1.0


NUMBER OF GENES
0
0
0
0
1
0
0
0
10
0
0


isoeugenol-1%
0.8
0.9
1.2
1.4
1.3
1.1
1.1
0.9

0.9
1.0


NUMBER OF GENES
0
0
0
1
1
0
0
0
5
0
0


isoeugenol-0.1%
1.2
0.6
1.2
0.9
0.9
1.0
0.6
0.8

0.8
1.1


NUMBER OF GENES
0
0
0
0
0
0
0
0
2
0
0


Glyoxal


Glyoxal-
0.6
0.5
1.7
1.1
2.0
1.0
1.0
0.9

0.9
0.9


50%


NUMBER
0
0
1
0
1
0
0
0
8
0
0


OF GENES


Glyoxal-
1.4
0.5
1.3
1.1
1.5
1.4
1.3
0.9

1.1
1.2


10%


NUMBER
1
0
1
0
1
1
1
0
13
0
0


OF GENES


Glyoxal-
0.6
0.5
1.1
1.1
1.1
1.0
1.0
0.9

1.1
1.1


1%


NUMBER
0
0
0
0
0
0
0
0
6
0
0


OF GENES


Glyoxal-
0.9
0.6
1.0
0.5
1.0
0.9
0.8
0.9

1.0
1.0


0.1%


NUMBER
0
0
0
0
0
0
0
0
0
0
0


OF GENES





















FTL
FTH1P
AKR1B10
AKR1C2
AKR1C1
CYP1B1
CYP1A1
TXNRD1
HMOX1
UGT1A1





Isoeugenol


isoeugenol-50%
1.5
1.0
1.0
1.3
2.2
39.1
87.1
2.6
1.4
1.2


NUMBER OF GENES
1
0
0
1
1
1
1
1
1
0


isoeugenol-10%
0.9
0.9
0.5
0.7
1.1
39.8
510.5
1.5
1.2
1.2


NUMBER OF GENES
0
0
0
0
0
1
1
1
0
0


isoeugenol-1%
1.0
0.9
0.7
0.9
1.2
25.3
219.2
1.3
1.2
1.1


NUMBER OF GENES
0
0
0
0
0
1
1
1
0
0


isoeugenol-0.1%
1.0
0.8
0.9
1.0
1.1
12.2
11.9
1.1
0.9
1.0


NUMBER OF GENES
0
0
0
0
0
1
1
0
0
0


Glyoxal


Glyoxal-
0.9
0.9
0.8
0.9
0.8
3.3
1.1
1.4
1.4
1.4


50%


NUMBER
0
0
0
0
0
1
0
1
1
1


OF GENES


Glyoxal-
1.1
1.1
0.7
1.2
1.0
0.8
0.0
1.3
1.0
1.0


10%


NUMBER
0
0
0
0
0
0
0
1
0
0


OF GENES


Glyoxal-
1.1
1.0
1.1
1.0
1.0
1.1
0.9
0.9
0.9
1.2


1%


NUMBER
0
0
0
0
0
0
0
0
0
0


OF GENES


Glyoxal-
1.0
0.9
0.6
0.8
0.8
0.6
0.6
1.0
1.0
0.9


0.1%


NUMBER
0
0
0
0
0
0
0
0
0
0


OF GENES


























TOTAL

ARE





UGT1A9
NQO2
SLC7A11
GCLM
AHR
ARE
SENS >1.3
>1.3





Isoeugenol


isoeugenol-50%
1.2
1.1
2.2
2.0
1.0


NUMBER OF GENES
0
0
1
1
0
11
13
11
Sensitizing
MODERATE


isoeugenol-10%
1.6
0.9
1.1
1.5
0.9


NUMBER OF GENES
1
0
0
1
0
5
10
5
Sensitizing


isoeugenol-1%
1.0
1.0
1.1
1.2
0.9


NUMBER OF GENES
0
0
0
0
0
3
5
3
Sensitizing


isoeugenol-0.1%
0.8
1.0
1.1
1.1
0.7


NUMBER OF GENES
0
0
0
0
0
2
2
2
Sensitizing


Glyoxal


Glyoxal-
1.3
0.9
1.0
1.5
0.9


50%


NUMBER
1
0
0
1
0
6
8
6
Sensitizing
MODERATE


OF GENES


Glyoxal-
0.2
1.0
1.9
1.7
1.0


10%


NUMBER
0
0
1
1
0
3
13
3
Sensitizing


OF GENES


Glyoxal-
0.6
1.0
1.4
0.9
1.1


1%


NUMBER
0
0
1
0
0
1
6
1
Sensitizing


OF GENES


Glyoxal-
0.3
0.8
1.0
0.9
0.9


0.1%


NUMBER
0
0
0
0
0
0
0
0
Sensitizing


OF GENES
























DAPK2
CTSS
EHF
NGAL
S100A8
DEFB1
P13
CXCR1
IL1R2
FABP4
IVL
CD36
HSP27





Eugenol


Eugenol-50%
1.5
0.1
1.2
1.3
1.9
3.0
1.4
2.2
1.3
1.4
2.6
2.3
5.4


NUMBER OF GENES
1
0
0
1
1
1
1
1
1
1
1
1
1


Eugenol-10%
1.3
1.0
1.3
0.7
1.0
1.1
0.9
0.8
0.8
0.6
0.9
1.7
1.1


NUMBER OF GENES
1
0
1
0
0
0
0
0
0
0
0
1
0


alpha hexyl


cinnamaldehyde


HCA-50%
0.8
0.8
1.3
1.1
1.1
1.1
1.0
1.2
1.1
1.2
1.4
1.9
1.1


NUMBER OF GENES
0
0
1
0
0
0
0
0
0
0
1
1
0


HCA-10%
1.0
1.0
1.0
0.8
1.0
0.9
0.9
0.7
0.8
0.8
0.9
2.2
1.0


NUMBER OF GENES
0
0
0
0
0
0
0
0
0
0
0
0
0


ipropylene


glycol


DPG-50%
1.3
1.2
0.9
1.0
1.2
1.2
1.0
2.2
1.2
1.2
1.1
1.1
1.1


NUMBER OF GENES
1
0
0
0
0
0
0
1
0
0
0
0
0


DPG-10%
1.4
0.7
1.0
0.6
1.1
0.9
1.0
0.5
1.0
0.8
0.9
1.1
1.2




















KRT4
BRAK
SERPINB3
MELANA
HSP90
PDZK1iP1
IL18
DHFR
TOTAL SENS





Eugenol


Eugenol-50%
4.6
0.4
1.4
1.0
9.1
1.7
0.7
0.6


NUMBER OF GENES
1
0
1
0
1
1
0
0
15


Eugenol-10%
0.8
0.7
1.2
1.9
1.1
0.9
1.1
0.8


NUMBER OF GENES
0
0
0
1
0
0
0
0
4


alpha hexyl


cinnamaldehyde


HCA-50%
1.0
0.8
1.2
1.3
1.7
1.1
1.0
0.9


NUMBER OF GENES
0
0
0
1
1
0
0
0
5


HCA-10%
0.7
1.0
1.5
2.0
1.6
1.1
1.1
0.8


NUMBER OF GENES
0
0
0
1
1
0
0
0
2


ipropylene


glycol


DPG-50%
1.3
1.1
1.1
0.6
1.0
1.1
1.2
1.2


NUMBER OF GENES
1
0
0
0
0
0
0
0
3


DPG-10%
1.1
0.8
1.2
0.6
0.9
1.0
1.1
1.1






















GCLC
NQO1
FTL
FTH1P
AKR1B10
AKR1C2
AKR1C1
CYP1B1
CYP1A1
TXNRD1
HMOX1





Eugenol


Eugenol-50%
3.2
0.7
4.2
2.3
1.0
0.7
0.7
0.9
2.3
3.0
2.4


NBRE GENES
1
1
0
1
1
0
0
0
0
1
1


Eugenol-10%
1.3
1.1
1.1
1.1
0.9
1.1
1.3
0.6
0.5
1.1
1.3


NBRE GENES
1
0
0
0
0
0
1
0
0
0
1


alpha hexyl


cinnamaldehyde


HCA-50%
0.9
1.5
1.7
1.0
1.1
1.2
1.1
1.7
12.3
1.5
1.0


NBRE GENES
0
1
1
0
0
1
0
1
1
1
0


HCA-10%
1.0
1.2
1.1
1.1
1.1
1.1
0.9
0.8
0.3
0.9
1.1


NBRE GENES
0
0
0
0
0
0
0
0
0
0
0


Dipropylene


glycol


DPG-50%
1.0
1.2
1.2
1.2
1.1
1.4
1.2
1.6
1.5
1.2
1.1


NBRE GENES
0
0
0
0
0
1
0
1
1
0
0


DPG-10%
1.1
1.0
1.0
1.1
1.0
0.9
1.3
0.4
0.2
1.1
1.1


NBRE GENES
0
0
0
0
0
1
0
0
0
0
0




























TOTAL

ARE





UGT1A1
UGT1A9
NQO2
SLC7A11
GCLM
AHR
ARE
SENS >1.3
>1.3





Eugenol


Eugenol-50%
0.5
25.0
1.3
2.6
1.1
1.3


NBRE GENES
1
1
1
1
0
1
10
15
10
Sensitizing
WEAK


Eugenol-10%
1.0
0.7
1.0
1.2
1.1
0.8


NBRE GENES
1
0
0
0
0
0
3
4
3
Non-sensitizing


alpha hexyl


cinnamaldehyde


HCA-50%
0.8
1.6
1.2
1.3
1.2
1.0


NBRE GENES
0
1
0
1
0
0
8
5
8
Sensitizing
WEAK


HCA-10%
0.7
0.7
1.1
0.8
0.8
0.8


NBRE GENES
0
0
0
0
0
0
0
2
0
Non-sensitizing


Dipropylene


glycol


DPG-50%
1.3
1.2
1.3
0.9
1.0
0.9


NBRE GENES
0
0
1
0
0
0
4
3
4
Non-sensitizing


DPG-10%
1.2
0.4
1.0
1.1
1.0
0.9


NBRE GENES
0
0
0
0
0
0
1
1
1
























DAPK2
CTSS
EHF
NGAL
S100A8
DEFB1
P13
CXCR1
IL1R2
FABP4
IVL
CD36
HSP27





Acide


Lactique


Lactic acid
1.1
1.4
0.9
1.0
1.1
1.1
0.9
0.8
0.8
0.8
0.9
0.9
0.8


50%


NUMBER OF GENES
0
1
0
0
0
0
0
0
0
0
0
0
0


Lactic acid
1.1
1.1
0.9
1.0
1.0
1.0
0.8
0.7
1.0
0.9
0.9
1.1
0.9


10%


NUMBER OF GENES
0
0
0
0
0
0
0
0
0
0
0
0
0


Lactic acid
1.2
1.0
0.9
0.9
0.8
0.9
0.8
0.7
1.0
0.7
1.0
1.1
1.0


1%


NUMBER OF GENES
0
0
0
0
0
0
0
0
0
0
0
0
0


SLS


SLS-50%
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0


(Corrosive)


NUMBER OF GENES
0
0
0
0
0
0
0
0
0
0
0
0
0


SLS-10%
1.2
1.4
0.4
0.9
1.2
0.8
0.4
1.0
0.8
1.1
1.0
1.1
0.3


NUMBER OF GENES
0
1
0
0
0
0
0
0
0
0
0
0
0


SLS-1%
0.9
1.4
1.2
1.1
0.9
1.1
1.1
0.6
0.7
1.2
1.1
0.6
1.1


NUMBER OF GENES
0
1
0
0
0
0
0
0
0
0
0
0
0




















KRT4
BRAK
SERPINB3
MELANA
HSP90
PDZK1iP1
IL18
DHFR
TOTAL SENS





Acide


Lactique


Lactic acid
0.8
0.7
1.2
0.6
0.9
1.0
0.8
0.9


50%


NUMBER OF GENES
0
0
0
0
0
0
0
0
1


Lactic acid
0.8
1.0
1.1
0.8
0.9
1.0
1.0
1.0


10%


NUMBER OF GENES
0
0
0
0
0
0
0
0
0


Lactic acid
0.9
0.8
0.9
0.7
1.0
1.0
0.9
0.9


1%


NUMBER OF GENES
0
0
0
0
0
0
0
0
0


SLS


SLS-50%
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0


(Corrosive)


NUMBER OF GENES
0
0
0
0
0
0
0
0
0


SLS-10%
0.7
1.4
0.7
2.5
0.7
0.6
1.0
1.2


NUMBER OF GENES
0
1
0
1
0
0
0
0
3


SLS-1%
1.1
1.3
1.1
1.6
0.6
0.7
0.5
0.8


NUMBER OF GENES
0
0
0
1
0
0
0
0
2






















GCLC
NQO1
FTL
FTH1P
AKR1B10
AKR1C2
AKR1C1
CYP1B1
CYP1A1
TXNRD1
HMOX1





Acide


Lactique


Ac Lactique-
0.9
1.0
1.1
0.9
1.0
0.8
1.2
1.0
1.1
1.0
1.0


50%


NUMBER OF
0
0
0
0
0
0
0
0
0
0
0


GENES


Ac Lactique-
0.9
1.0
1.0
0.9
1.0
1.0
1.2
1.3
1.8
1.2
1.1


10%


NUMBER OF
0
0
0
0
0
0
0
0
1
0
0


GENES


Ac Lactique-
1.0
0.9
1.2
1.1
0.7
1.0
1.0
1.1
0.6
1.0
0.9


1%


NUMBER OF
0
0
0
0
0
0
0
0
0
0
0


GENES


SLS


SLS-50%
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0


(Corrosif)


NUMBER OF
0
0
0
0
0
0
0
0
0
0
0


GENES


SLS-10%
1.7
1.1
1.6
1.3
1.1
1.2
1.6
0.3
0.2
1.2
0.5


NUMBER OF
1
0
1
0
0
0
1
0
0
0
0


GENES













SLS-1%
1.3
1.1
1.2
1.0
1.0
1.1
1.0
0.6
1.0
0.9
0.4


NUMBER OF
0
0
0
0
0
0
0
0
0
0
0


GENES





















UGT1A1
UGT1A9
NQO2
SLC7A11
GCLM
AHR
TOTAL ARE
SENS >1.3
ARE >1.3





Acide


Lactique


Ac Lactique-
1.3
0.8
1.2
1.0
1.1
0.8


50%


NUMBER OF
0
0
0
0
0
0
0
1
0
Non-sensitizing


GENES


Ac Lactique-
1.3
0.7
1.1
1.2
1.0
0.5


10%


NUMBER OF
0
0
0
0
0
0
1
0
1


GENES


Ac Lactique-
0.8
0.4
0.9
1.0
0.8
0.9


1%


NUMBER OF
0
0
0
0
0
0
0
0
0


GENES


SLS


SLS-50%
0.0
0.0
0.0
0.0
0.0
0.0


(Corrosif)


NUMBER OF
0
0
0
0
0
0
0
0
0


GENES


SLS-10%
1.0
1.4
1.0
5.2
1.0
1.4


NUMBER OF
0
0
0
1
0
1
5
3
5
Non-


GENES









sensitizing


SLS-1%
0.5
0.6
1.1
2.9
1.0
1.2


NUMBER OF
0
0
0
1
0
0
1
2
1


GENES

## Claims

1. A method for evaluating the sensitizing potential of a test compound, comprising the steps of:
a) contacting a test compound with a biological sample, wherein the biological sample is a skin sample reconstructed in vitro, and said skin sample comprises no Langerhans cells;
b) determining in the contacted skin sample the expression level of at least six genes chosen in the group consisting of: BRAK (CXC chemokine ligand 14), CTSS (cathepsin S), DAPK2 (death-associated protein kinase 2), FABP4 (fatty acid binding protein 4), HSP27 (heat shock 27 kDa protein), IL18 (IL-18), HSP90 (heat shock 90 kDa protein), IL1R2 (interleukin-1 receptor type II), TPSAB1 (tryptase alpha/beta 1), CXCR1 (interleukin 8 receptor, alpha), DEFB1 (defensin, beta 1), DHFR (dihydrofolate reductase), EHF (ets homologous factor), IVL (involucrin) KRT4 (keratin 4), MELANA (melan-A), NGAL (lipocalin 2), PDZK1 IP1 (PDZK1 interacting protein interaction 1), PI3 (peptidase inhibitor 3), PSME2 (proteasome activator subunit 2), SERPINB3 (serpin peptidase inhibitor member 3), AKR1B10 (aldo-keto reductase family 1, member B10), AKR1C1 (aldo-keto reductase family 1, member C1), AKR1C2 (aldo-keto reductase family 1, member C2), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), FTH1P (ferritin, heavy polypeptide 1), FTL (ferritin, light polypeptide 1), G6PD (glucose-6-phosphate dehydrogenase), GCLM (glutamate-cysteine ligase modifier subunit), NQO2 (NAD(P)H dehydrogenase, quinone 2), SLC7A 11 (solute carrier family 7, Member 11), TXNRD1 (thioredoxin reductase 1), UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1), UGT1A9 (UDP glucuronosyltransferase 1 family, polypeptide A9), YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide), CD36 (CD36 molecule), CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide 1), GCLC (glutamate-cysteine ligase, catalytic subunit), HMOX1 (heme oxygenase 1), NQO1 (NAD(P)H dehydrogenase, quinone 1) and S100A8 (S 100 calcium binding protein A8),
in which the expression level of each of said at least six genes is determined by measuring the expression level of the polypeptide encoded by said at least six genes or a fragment thereof, or by determining the expression level of the mRNA from said at least six genes or a fragment thereof; and

c) comparing the expression level of each of said at least six genes with a reference value,
wherein overexpression of the at least six genes indicates that the test compound has a sensitizing potential.

2. The evaluation method according to claim 1, comprising an additional step of amplifying the mRNA or cDNA of each of said six genes, the complementary sequence thereof or a fragment thereof.

3. The method according to claim 1, in which step b) comprises measuring the expression of at least 7 genes selected from the group consisting of: BRAK (CXC chemokine ligand 14), CTSS (cathepsin S), DAPK2 (death-associated protein kinase 2), FABP4 (fatty acid binding protein 4), HSP27 (heat shock 27 kDa protein), IL18 (IL-18), HSP90 (heat shock 90 kDa protein), IL1R2 (interleukin-1 receptor type II), TPSAB1 (tryptase alpha/beta 1), CXCR1 (interleukin 8 receptor, alpha), DEFB1 (defensin, beta 1), DHFR (dihydrofolate reductase), EHF (ets homologous factor), IVL (involucrin) KRT4 (keratin 4), MELANA (melan-A), NGAL (lipocalin 2), PDZK1IP1 (PDZK1 interacting protein interaction 1), PI3 (peptidase inhibitor 3), PSME2 (proteasome activator subunit 2), SERPINB3 (serpin peptidase inhibitor member 3), AKR1B10 (aldo-keto reductase family 1, member B 10), AKR1C1 (aldo-keto reductase family 1, member C1), AKR1C2 (aldo-keto reductase family 1, member C2), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), FTH1P (ferritin, heavy polypeptide 1), FTL (ferritin, light polypeptide 1), G6PD (glucose-6-phosphate dehydrogenase), GCLM (glutamate-cysteine ligase modifier subunit), NQO2 (NAD(P)H dehydrogenase, quinone 2), SLC7A11 (solute carrier family 7, Member 11), TXNRD1 (thioredoxin reductase 1), UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1), UGT1A9 (UDP glucuronosyltransferase 1 family, polypeptide A9), YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide), CD36 (CD36 molecule), CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide 1), GCLC (glutamate-cysteine ligase, catalytic subunit), HMOX1 (heme oxygenase 1), NQO1 (NAD(P)H dehydrogenase, quinone 1) and S100A8 (S100 calcium binding protein A8).

4. The method according to claim 1, in which step b) is performed between 2 and 24 hours after step a).

5. The method according to claim 4, in which step b) is performed between 4 and 18 hours after step a).

6. The method according to claim 4, in which step b) is performed between 5 and 7 hours after step a).

7. The method according to claim 4, in which step b) is performed 6 hours after step a).

8. A method for evaluating the sensitizing power of a test compound, comprising the following steps:
1) obtaining at least one test compound dilution, and
2) determining the sensitizing potential of said test compound at said at least one dilution by a method such as defined according to claim 1.

9. The method according to claim 1, in which step b) comprises measuring the expression of at least 8 genes selected from the group consisting of: BRAK (CXC chemokine ligand 14), CTSS (cathepsin S), DAPK2 (death-associated protein kinase 2), FABP4 (fatty acid binding protein 4), HSP27 (heat shock 27 kDa protein), IL18 (IL-18), HSP90 (heat shock 90 kDa protein), IL1R2 (interleukin-1 receptor type II), TPSAB1 (tryptase alpha/beta 1), CXCR1 (interleukin 8 receptor, alpha), DEFB1 (defensin, beta 1), DHFR (dihydrofolate reductase), EHF (ets homologous factor), IVL (involucrin) KRT4 (keratin 4), MELANA (melan-A), NGAL (lipocalin 2), PDZK1IP1 (PDZK1 interacting protein interaction 1), PI3 (peptidase inhibitor 3), PSME2 (proteasome activator subunit 2), SERPINB3 (serpin peptidase inhibitor member 3), AKR1B10 (aldo-keto reductase family 1, member B10), AKR1C1 (aldo-keto reductase family 1, member C1), AKR1C2 (aldo-keto reductase family 1, member C2), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), FTH1P (ferritin, heavy polypeptide 1), FTL (ferritin, light polypeptide 1), G6PD (glucose-6-phosphate dehydrogenase), GCLM (glutamate-cysteine ligase modifier subunit), NQO2 (NAD(P)H dehydrogenase, quinone 2), SLC7A11 (solute carrier family 7, Member 11), TXNRD1 (thioredoxin reductase 1), UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1), UGT1A9 (UDP glucuronosyltransferase 1 family, polypeptide A9), YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide), CD36 (CD36 molecule), CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide 1), GCLC (glutamate-cysteine ligase, catalytic subunit), HMOX1 (heme oxygenase 1), NQO1 (NAD(P)H dehydrogenase, quinone 1) and S100A8 (S100 calcium binding protein A8).

10. The method according to claim 1, in which step b) comprises measuring the expression of at least 9 genes selected from the group consisting of: BRAK (CXC chemokine ligand 14), CTSS (cathepsin S), DAPK2 (death-associated protein kinase 2), FABP4 (fatty acid binding protein 4), HSP27 (heat shock 27 kDa protein), IL18 (IL-18), HSP90 (heat shock 90 kDa protein), IL1R2 (interleukin-1 receptor type II), TPSAB1 (tryptase alpha/beta 1), CXCR1 (interleukin 8 receptor, alpha), DEFB 1 (defensin, beta 1), DHFR (dihydrofolate reductase), EHF (ets homologous factor), IVL (involucrin) KRT4 (keratin 4), MELANA (melan-A), NGAL (lipocalin 2), PDZK1IP1 (PDZK1 interacting protein interaction 1), PI3 (peptidase inhibitor 3), PSME2 (proteasome activator subunit 2), SERPINB3 (serpin peptidase inhibitor member 3), AKR1B10 (aldo-keto reductase family 1, member B 10), AKR1C1 (aldo-keto reductase family 1, member C1), AKR1C2 (aldo-keto reductase family 1, member C2), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), FTH1P (ferritin, heavy polypeptide 1), FTL (ferritin, light polypeptide 1), G6PD (glucose-6-phosphate dehydrogenase), GCLM (glutamate-cysteine ligase modifier subunit), NQO2 (NAD(P)H dehydrogenase, quinone 2), SLC7A11 (solute carrier family 7, Member 11), TXNRD1 (thioredoxin reductase 1), UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1), UGT1A9 (UDP glucuronosyltransferase 1 family, polypeptide A9), YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide), CD36 (CD36 molecule), CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide 1), GCLC (glutamate-cysteine ligase, catalytic subunit), HMOX1 (heme oxygenase 1), NQO1 (NAD(P)H dehydrogenase, quinone 1) and S100A8 (S100 calcium binding protein A8).

11. The method according to claim 1, in which step b) comprises measuring the expression of at least 10 genes selected from the group consisting of: BRAK (CXC chemokine ligand 14), CTSS (cathepsin S), DAPK2 (death-associated protein kinase 2), FABP4 (fatty acid binding protein 4), HSP27 (heat shock 27 kDa protein), IL18 (IL-18), HSP90 (heat shock 90 kDa protein), IL1R2 (interleukin-1 receptor type II), TPSAB1 (tryptase alpha/beta 1), CXCR1 (interleukin 8 receptor, alpha), DEFB1 (defensin, beta 1), DHFR (dihydrofolate reductase), EHF (ets homologous factor), IVL (involucrin) KRT4 (keratin 4), MELANA (melan-A), NGAL (lipocalin 2), PDZK1IP1 (PDZK1 interacting protein interaction 1), PI3 (peptidase inhibitor 3), PSME2 (proteasome activator subunit 2), SERPINB3 (serpin peptidase inhibitor member 3), AKR1B10 (aldo-keto reductase family 1, member B 10), AKR1C1 (aldo-keto reductase family 1, member C1), AKR1C2 (aldo-keto reductase family 1, member C2), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), FTH1P (ferritin, heavy polypeptide 1), FTL (ferritin, light polypeptide 1), G6PD (glucose-6-phosphate dehydrogenase), GCLM (glutamate-cysteine ligase modifier subunit), NQO2 (NAD(P)H dehydrogenase, quinone 2), SLC7A11 (solute carrier family 7, Member 11), TXNRD1 (thioredoxin reductase 1), UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1), UGT1A9 (UDP glucuronosyltransferase 1 family, polypeptide A9), YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide), CD36 (CD36 molecule), CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide 1), GCLC (glutamate-cysteine ligase, catalytic subunit), HMOX1 (heme oxygenase 1), NQO1 (NAD(P)H dehydrogenase, quinone 1) and S100A8 (S100 calcium binding protein A8).

12. The method according to claim 1, in which step b) comprises measuring the expression of at least 11 genes selected from the group consisting of: BRAK (CXC chemokine ligand 14), CTSS (cathepsin S), DAPK2 (death-associated protein kinase 2), FABP4 (fatty acid binding protein 4), HSP27 (heat shock 27 kDa protein), IL18 (IL-18), HSP90 (heat shock 90 kDa protein), IL1R2 (interleukin-1 receptor type II), TPSAB1 (tryptase alpha/beta 1), CXCR1 (interleukin 8 receptor, alpha), DEFB 1 (defensin, beta 1), DHFR (dihydrofolate reductase), EHF (ets homologous factor), IVL (involucrin) KRT4 (keratin 4), MELANA (melan-A), NGAL (lipocalin 2), PDZK1IP1 (PDZK1 interacting protein interaction 1), PI3 (peptidase inhibitor 3), PSME2 (proteasome activator subunit 2), SERPINB3 (serpin peptidase inhibitor member 3), AKR1B10 (aldo-keto reductase family 1, member B 10), AKR1C1 (aldo-keto reductase family 1, member C1), AKR1C2 (aldo-keto reductase family 1, member C2), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), FTH1P (ferritin, heavy polypeptide 1), FTL (ferritin, light polypeptide 1), G6PD (glucose-6-phosphate dehydrogenase), GCLM (glutamate-cysteine ligase modifier subunit), NQO2 (NAD(P)H dehydrogenase, quinone 2), SLC7A11 (solute carrier family 7, Member 11), TXNRD1 (thioredoxin reductase 1), UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1), UGT1A9 (UDP glucuronosyltransferase 1 family, polypeptide A9), YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide), CD36 (CD36 molecule), CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide 1), GCLC (glutamate-cysteine ligase, catalytic subunit), HMOX1 (heme oxygenase 1), NQO1 (NAD(P)H dehydrogenase, quinone 1) and S100A8 (S100 calcium binding protein A8).

13. The method according to claim 1, in which step b) comprises measuring the expression of at least 12 genes selected from the group consisting of: BRAK (CXC chemokine ligand 14), CTSS (cathepsin S), DAPK2 (death-associated protein kinase 2), FABP4 (fatty acid binding protein 4), HSP27 (heat shock 27 kDa protein), IL18 (IL-18), HSP90 (heat shock 90 kDa protein), IL1R2 (interleukin-1 receptor type II), TPSAB1 (tryptase alpha/beta 1), CXCR1 (interleukin 8 receptor, alpha), DEFB1 (defensin, beta 1), DHFR (dihydrofolate reductase), EHF (ets homologous factor), IVL (involucrin) KRT4 (keratin 4), MELANA (melan-A), NGAL (lipocalin 2), PDZK1IP1 (PDZK1 interacting protein interaction 1), PI3 (peptidase inhibitor 3), PSME2 (proteasome activator subunit 2), SERPINB3 (serpin peptidase inhibitor member 3), AKR1B10 (aldo-keto reductase family 1, member B10), AKR1C1 (aldo-keto reductase family 1, member C1), AKR1C2 (aldo-keto reductase family 1, member C2), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), FTH1P (ferritin, heavy polypeptide 1), FTL (ferritin, light polypeptide 1), G6PD (glucose-6-phosphate dehydrogenase), GCLM (glutamate-cysteine ligase modifier subunit), NQO2 (NAD(P)H dehydrogenase, quinone 2), SLC7A11 (solute carrier family 7, Member 11), TXNRD1 (thioredoxin reductase 1), UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1), UGT1A9 (UDP glucuronosyltransferase 1 family, polypeptide A9), YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide), CD36 (CD36 molecule), CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide 1), GCLC (glutamate-cysteine ligase, catalytic subunit), HMOX1 (heme oxygenase 1), NQO1 (NAD(P)H dehydrogenase, quinone 1) and S100A8 (S100 calcium binding protein A8).

14. The method according to claim 1, in which step b) comprises measuring the expression of at least 13 genes selected from the group consisting of: BRAK (CXC chemokine ligand 14), CTSS (cathepsin S), DAPK2 (death-associated protein kinase 2), FABP4 (fatty acid binding protein 4), HSP27 (heat shock 27 kDa protein), IL18 (IL-18), HSP90 (heat shock 90 kDa protein), IL1R2 (interleukin-1 receptor type II), TPSAB1 (tryptase alpha/beta 1), CXCR1 (interleukin 8 receptor, alpha), DEFB1 (defensin, beta 1), DHFR (dihydrofolate reductase), EHF (ets homologous factor), IVL (involucrin) KRT4 (keratin 4), MELANA (melan-A), NGAL (lipocalin 2), PDZK1IP1 (PDZK1 interacting protein interaction 1), PI3 (peptidase inhibitor 3), PSME2 (proteasome activator subunit 2), SERPINB3 (serpin peptidase inhibitor member 3), AKR1B10 (aldo-keto reductase family 1, member B10), AKR1C1 (aldo-keto reductase family 1, member C1), AKR1C2 (aldo-keto reductase family 1, member C2), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), FTH1P (ferritin, heavy polypeptide 1), FTL (ferritin, light polypeptide 1), G6PD (glucose-6-phosphate dehydrogenase), GCLM (glutamate-cysteine ligase modifier subunit), NQO2 (NAD(P)H dehydrogenase, quinone 2), SLC7A11 (solute carrier family 7, Member 11), TX-NRD1 (thioredoxin reductase 1), UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1), UGT1A9 (UDP glucuronosyltransferase 1 family, polypeptide A9), YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide), CD36 (CD36 molecule), CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide 1), GCLC (glutamate-cysteine ligase, catalytic subunit), HMOX1 (heme oxygenase 1), NQO1 (NAD(P)H dehydrogenase, quinone 1) and S100A8 (S100 calcium binding protein A8).