JP7808465B2 - Organic compounds, reagents, and analytical methods - Google Patents

Description

The present invention relates to an organic compound for measuring trace amounts of chemical substances, a reagent containing the organic compound, and an analytical method using the organic compound or the reagent.

It is important that products that come into contact with humans, such as pharmaceuticals and cosmetics, do not cause allergic reactions under actual conditions of use. Therefore, during product development, it is necessary to evaluate the risk of skin sensitization of the ingredients used. Skin sensitization has been evaluated using animal tests such as the Guinea Pig Maximization Test, Buehler Test, and Local Lymph Node Assay.

In recent years, alternative tests have been developed to evaluate the skin sensitization potential of chemicals in vitro without using animals, and skin sensitization tests using cysteine peptides, lysine peptides (Non-Patent Document 1) and amino acid derivatives have been proposed (Patent Documents 1 and 2).

Japanese Patent Application Laid-Open No. 2014-37995 JP 2011-59102 A

G.F.Gerberick et al., Quantification of Chemical Peptide Reaction for Screening Contact Allergens: A Classification Tree Model Approach,Toxicol. Sci., 97(2), 417-427:2007.

From the perspective of animal welfare, animal testing should be avoided, and it is necessary to realize skin sensitization assessment using alternative methods that do not use animals. While the above-mentioned alternative tests are good methods that can measure the skin sensitization potential of the main components contained in test substances in a simple and rapid manner, there are concerns that they may not properly evaluate the risk of skin sensitization caused by trace impurities. In skin sensitization tests using cysteine peptides, lysine peptides (Non-Patent Document 1) and amino acid derivatives (Patent Documents 1 and 2), it was found that there were problems with the detection sensitivity of the resulting trace amounts of bound substances, even when highly sensitive mass spectrometry was used.

The object of the present invention is to provide an organic compound for easily, quickly, and accurately measuring trace amounts of chemical substances (test substances), a reagent containing the organic compound, and an analytical method using the organic compound or the reagent.

As a result of intensive research conducted by the inventors to solve the above-mentioned problems, they discovered that trace amounts of chemical substances can be analyzed easily, selectively, and with high sensitivity by labeling chemical substances such as skin sensitizers with a labeling reagent having a specific structure and then subjecting the labeled chemical substances to analysis, leading to the completion of the present invention.

That is, the present invention relates to an organic compound represented by the following chemical formula (1).
(In the formula, X is a thiol group or an amino group, R ¹ , R ² , R ³ , R ⁴ , and R ⁵ each independently represent a hydrocarbon group, A is a functional group containing a single bond between a heteroatom and a carbon atom, and Y ⁻ is an anion.)

The present invention also relates to a reagent containing the organic compound.

Furthermore, the present invention relates to an analytical method in which the organic compound is reacted with a chemical substance to obtain a reaction product, and then the reaction product is separated and detected.

The organic compound of the present invention has a functional group (thiol group or amino group) that is highly reactive to trace amounts of chemicals (such as skin sensitizers), a functional group (quaternary ammonium group) that enables highly sensitive detection of the reactant, and a functional group (functional group containing a single bond between a heteroatom and a carbon atom) that enables highly selective detection of the reactant. Therefore, trace amounts of chemicals can be measured simply, quickly, and accurately using general-purpose analytical methods and analytical equipment. The organic compound, reagent, and analytical method of the present invention can be used, for example, to evaluate the skin sensitization potential of trace amounts of chemicals and to quantitatively measure trace amounts of chemicals.

Chromatograms of the reaction products of TMAS, Cys-P, or NAC with the test substance.

The present invention is described in detail below.

<Organic compounds>
The organic compound of the present invention is represented by the following chemical formula (1).
(In the formula, X is a thiol group or an amino group, R ¹ , R ² , R ³ , R ⁴ , and R ⁵ each independently represent a hydrocarbon group, A is a functional group containing a single bond between a heteroatom and a carbon atom, and Y ⁻ is an anion.)

X is a thiol group or an amino group, a functional group that is reactive with chemicals such as skin sensitizers (e.g., α,β-unsaturated ketones, acid anhydrides, and aldehydes).

^R1 , ^R2 , ^R3 , ^R4 , and ^R5 are each independently a hydrocarbon group, and examples of the hydrocarbon group include a linear or branched aliphatic saturated or unsaturated hydrocarbon group, an alicyclic saturated or unsaturated hydrocarbon group (including a bridged ring or a fused ring), an aromatic hydrocarbon group, and an organic group in which two or more of these are bonded together. The hydrocarbon group may have various substituents as long as they do not impede the effects of the present invention.

From the viewpoint of facilitating detection by an analytical device, ^R1 is preferably a hydrocarbon group having no substituent, more preferably a linear or branched saturated aliphatic hydrocarbon group, and even more preferably a linear saturated aliphatic hydrocarbon group.

From the viewpoint of facilitating detection by an analytical device, ^R2 is preferably a hydrocarbon group having no substituent, more preferably an aromatic hydrocarbon group, and even more preferably a phenylene group.

The number of carbon atoms in the hydrocarbon groups in R ¹ and R ² (not including the number of carbon atoms in the substituents) is not particularly limited, but from the viewpoint of facilitating detection by an analytical device, in the case of a linear aliphatic saturated hydrocarbon group, it is 1 or more, preferably 2 or more, and preferably 12 or less, more preferably 8 or less; in the case of a linear unsaturated hydrocarbon group, it is 2 or more, preferably 3 or more, and preferably 12 or less, more preferably 8 or less; in the case of a branched aliphatic saturated or unsaturated hydrocarbon group, it is 3 or more, preferably 4 or more, and preferably 12 or less, more preferably 8 or less; and in the case of an alicyclic saturated or unsaturated hydrocarbon group or an aromatic hydrocarbon group, it is preferably 6 or more, and preferably 14 or less, more preferably 10 or less.

From the viewpoint of facilitating detection by an analytical device, ^R3 , ^R4 , and ^R5 are preferably unsubstituted hydrocarbon groups, more preferably linear or branched saturated aliphatic hydrocarbon groups, and even more preferably linear saturated aliphatic hydrocarbon groups.

The number of carbon atoms in the hydrocarbon groups in ^R3 , ^R4 , and ^R5 (not including the number of carbon atoms in substituents) is not particularly limited, but from the viewpoint of facilitating detection by an analytical device, in the case of a linear aliphatic saturated hydrocarbon group, it is 1 or more, preferably 2 or more, and preferably 12 or less, more preferably 6 or less; in the case of a linear unsaturated hydrocarbon group, it is 2 or more, preferably 3 or more, and preferably 12 or less, more preferably 6 or less; in the case of a branched aliphatic saturated or unsaturated hydrocarbon group, it is 3 or more, preferably 4 or more, and preferably 12 or less, more preferably 6 or less; and in the case of an alicyclic saturated or unsaturated hydrocarbon group or an aromatic hydrocarbon group, it is preferably 6 or more, and preferably 14 or less, more preferably 10 or less.

A is a functional group containing a single bond between a heteroatom and a carbon atom, and the single bond is cleaved by heating or other means. Introducing this functional group makes it possible to detect fragment ions derived from the organic compound, enabling highly selective and sensitive analysis. Examples of such functional groups include amide bonds, ester bonds, ether bonds, urethane bonds, and urea bonds. From the perspective of highly selective detection of reactants, the functional group is preferably an amide bond.

Y ⁻ is an anion, and examples thereof include a halogen anion, ClO ₄ ⁻ , BF ₄ ⁻ , PF ₆ ⁻ , CH ₃ COO ⁻ , CF ₃ COO ⁻ , and CH ₃ (C ₆ H ₄ )SO ₃ ⁻ .

Specific examples of the organic compound represented by the above chemical formula (1) include the following compounds.

The organic compound represented by the above chemical formula (1) can be easily synthesized using known raw materials and reaction reagents in accordance with commonly used chemical reactions. For example, the above TMAS and TMAA can be synthesized by the following chemical reaction using the following raw materials and reaction reagents. Detailed methods for synthesizing TMAS and TMAA are shown in the examples. Those skilled in the art will be able to easily synthesize organic compounds represented by the above chemical formula (1) other than the above TMAS and TMAA in accordance with commonly used chemical reactions using appropriate raw materials and reaction reagents.

In the above reaction scheme, DMT-MM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) is the condensing agent, TCEP (tris(2-carboxyethyl)phosphine) is the reducing agent, and TFA is trifluoroacetic acid.

In the above reaction scheme, DMT-MM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) is a condensing agent, Boc is a t-butoxycarbonyl group, and TFA is trifluoroacetic acid.

The organic compound of the present invention can be used, for example, to evaluate the skin sensitization of trace amounts of chemical substances (such as skin sensitizers) and to quantitatively measure trace amounts of chemical substances. Furthermore, from the perspective of trace analysis, the organic compound of the present invention can be used preferably as a labeling reagent for detecting trace amounts of chemical substances (such as skin sensitizers), more preferably as a labeling reagent for mass spectrometry, and even more preferably as a labeling reagent for mass spectrometry of skin sensitizers.

<Reagents>
The reagent of the present invention may consist solely of the organic compound, or may contain one or more additives in addition to the organic compound as the measurement agent. Examples of additives include pH adjusters and stabilizers. The reagent of the present invention may also be prepared by dissolving the organic compound and, if necessary, the additives in water, an aqueous buffer solution, an organic solvent, or a mixture of these solvents. The reagent of the present invention may be in the form of a solution, liquid, or solid (powder, granules, lyophilized product, tablet, etc.).

The reagent of the present invention can be used, for example, to evaluate the skin sensitization of trace amounts of chemical substances (such as skin sensitizers) and to quantitatively measure trace amounts of chemical substances. Furthermore, from the perspective of trace analysis, the reagent of the present invention can be used preferably as a labeling reagent for detecting trace amounts of chemical substances (such as skin sensitizers), more preferably as a labeling reagent for mass spectrometry, and even more preferably as a labeling reagent for mass spectrometry of skin sensitizers.

<Analysis method>
The analytical method of the present invention involves reacting the organic compound with a chemical substance (test substance) to obtain a reaction product, and then separating and detecting the reaction product. The analytical method of the present invention will be described below with specific examples.

The chemical substance (test substance) is not particularly limited as long as it is reactive with or reacts with the thiol or amino group of the organic compound, but is preferably a sensitizing substance, more preferably a skin sensitizing substance.

The reagent containing the organic compound is used, for example, as an aqueous buffer solution containing an organic acid salt such as ammonium acetate or an inorganic salt such as a phosphate, or as a mixed solvent buffer solution in which the organic compound is dissolved in water or a mixed solvent of these with an organic solvent. The concentration of the organic compound in the aqueous buffer solution or mixed solvent buffer solution is not particularly limited, but is, for example, about 0.01 μM to 1 M, typically about 0.1 mM to 500 mM.

The chemical substance (test substance) is dissolved in an organic solvent such as methanol, ethanol, acetonitrile, or acetone, or a mixture thereof, to a concentration of, for example, about 0.01 μM to 1 M, typically about 0.1 mM to 500 mM.

The aqueous buffer solution or mixed solvent buffer solution containing the reagent is then mixed with a test substance solution containing the test substance so that the molar concentration ratio of the organic compound to the test substance is, for example, 1:100 to 10:1, and the organic compound is allowed to react with the test substance to obtain a reaction product. The reaction can be carried out by stirring or leaving the mixture to stand, typically for 1 minute to 2 days, while keeping the mixture at a temperature ranging from about 4°C to 60°C.

The resulting reaction products are then separated and detected. There are no particular limitations on the method for separating the reaction products, but chromatography is preferred from the perspective of excellent separation and detectability. Examples of chromatography include supercritical chromatography, ion chromatography, liquid chromatography, gas chromatography, and thin-layer chromatography. From the perspective of excellent detectability, supercritical chromatography, ion chromatography, or liquid chromatography is preferred, and liquid chromatography is more preferred.

Examples of liquid chromatography include normal phase chromatography, reverse phase chromatography, size exclusion chromatography, and ion exchange chromatography. From the viewpoint of superior detectability, reverse phase chromatography or ion exchange chromatography is preferred, and reverse phase chromatography is more preferred.

The method for detecting the reaction product is not particularly limited, and examples include spectroscopic analysis using ultraviolet light, visible light, or infrared light, mass spectrometry, fluorescence analysis, differential refractive index analysis, electrical conductivity analysis, and evaporative light scattering analysis. From the perspective of microanalysis, mass spectrometry is preferred.

The mass spectrometry method is not particularly limited as long as it is an analytical method that utilizes mass analysis, and examples include conventional mass spectrum acquisition methods; selected ion monitoring methods; and tandem mass spectrometry methods (MS/MS methods) such as data-dependent acquisition methods, data-independent acquisition methods, precursor ion scanning methods, selected reaction monitoring methods, and constant neutral scanning methods. From the perspective of trace analysis, tandem mass spectrometry methods (MS/MS methods) are preferred.

Examples of MS/MS methods include selected reaction monitoring, precursor ion scanning, and constant neutral loss scanning. Methods developed in the future can also be used as long as they are analytical methods included in the MS/MS method.

According to the present invention, test substances such as skin sensitizers can be analyzed easily, sensitively, and accurately by using specific labeling reagents to enhance selectivity. In particular, test substances can be quantitatively analyzed using mass spectrometry such as MS/MS. Furthermore, multiple test substances can also be measured simultaneously.

The analytical method of the present invention is extremely useful because it can be widely used in industry, particularly in the fields of cosmetics, daily necessities, pharmaceuticals, and analytical equipment.

The present invention will now be described in detail with reference to examples.

Synthesis Example 1
Synthesis of TMAS (4-TriMethylAmmoniobenzoyl 2-Sulfanylethylamine)
4-Aminobenzoic acid (200 mg, 1.46 mmol) was placed in a 30 mL screw tube and dissolved in DMSO (3 mL). The resulting solution was ice-cooled, and iodomethane (726 μL, 8.0 eq.) was added dropwise with stirring. After the addition was complete, the mixture was returned to room temperature (25°C) and left overnight (approximately 20 hours). The resulting reaction solution was ice-cooled, and 0.5 M aqueous KOH was added to terminate the reaction and hydrolyze the mixture. After 20 minutes, the mixture was neutralized with hydrochloric acid and washed with ethyl acetate to obtain an aqueous solution containing potassium 4-trimethylammonium benzoate.
A portion of the recovered aqueous layer (equivalent to 0.1 mmol) was removed and placed in a 20 mL screw tube. MeOH (2 mL) was added, followed by the addition of cystamine dihydrochloride (11.3 mg, 0.05 mmol) and DMT-MM (42.0 mg, 0.15 mmol). The mixture was allowed to react overnight (approximately 20 hours) at room temperature (25°C). An appropriate amount of TCEP was added to the resulting reaction solution to reduce the disulfide bond. The insoluble matter was filtered and concentrated, and the mixture was purified by HPLC (0.1% TFA aqueous solution was used as the eluent for preparative purification of the target product). The resulting fraction was lyophilized to obtain the target product, TMAS. The ¹ H NMR data for the resulting TMAS were as follows:
¹ H NMR (D ₂ O): δ7.89 (m, 4H), 3.49 (s, 9H), 2.80 (t, 2H), 2.62 (t, 2H)

Synthesis Example 2
Synthesis of TMAA (4-TrimethylAmmoniobenzoyl 6-Aminohexylamine) An aqueous solution containing potassium 4-trimethylammonium benzoate was obtained in the same manner as in Synthesis Example 1.
A portion of the recovered aqueous layer (equivalent to 0.1 mmol) was removed and placed in a 20 mL screw tube. MeOH (2 mL) was added, followed by N-Boc-1,6-diaminohexane (26.0 mg, 0.12 mmol) and DMT-MM (42.0 mg, 0.15 mmol), and the mixture was allowed to react overnight (approximately 20 hours) at room temperature (25°C). The resulting reaction solution was concentrated to dryness, and 10% aqueous TFA solution (2 mL) was added to deprotect the Boc group. Insoluble matter was filtered and concentrated, followed by purification by HPLC. The resulting fraction was lyophilized to obtain the target product, TMAA. The ¹ H NMR data for the resulting TMAA were as follows:
¹ H NMR (D ₂ O): δ7.96 (m, 4H), 3.66 (s, 9H), 3.40 (t, 2H), 2.98 (t, 2H), 1.63 (br, 4H), 1.42 (br, 2H)

The following drugs were used in the following test examples.
(Labeling Reagent)
TMAS: Compound obtained in Synthesis Example 1 TMAA: Compound obtained in Synthesis Example 2 Cysteine peptide (hereinafter also referred to as Cys-P): Compound sold by RS Synthesis NAC: Obtained as an ADRA kit from Fujifilm Wako Pure Chemical Industries (sensitizing compound, skin sensitization: strong)
Benzoyl peroxide (molecular weight 242.23) obtained from Tokyo Chemical Industry Co., Ltd. Diphenylcyclopropenone (molecular weight 206.24) obtained from Fujifilm Wako Pure Chemical Industries, Ltd. CMI/MI (5-chloro-2-methyl-4-isothiazolin-3-one/2-methyl-4-isothiazolin-3-one) (skin sensitization: strong) (CMI molecular weight 149 .59, MI molecular weight 115.15) Obtained from Fluorochem. Cinnamaldehyde (molecular weight 132.16) Obtained from Fujifilm Wako Pure Chemical Industries. Trimellitic anhydride (molecular weight 192.13) Obtained from Fujifilm Wako Pure Chemical Industries. p-Benzoquinone (molecular weight 108.09) Obtained from Fujifilm Wako Pure Chemical Industries. Formaldehyde (molecular weight 30.03) Formaldehyde liquid Obtained from Fujifilm Wako Pure Chemical Industries, Ltd.; Oxazolone 4-ethoxymethylene-2-phenyloxazolin-5-one (molecular weight 217.22) obtained from Fujifilm Wako Pure Chemical Industries, Ltd.; Palmitoyl chloride (molecular weight 274.87) obtained from Tokyo Chemical Industry Co., Ltd. The above skin sensitization is an evaluation result by the LLNA test (a test using mice) described in the aforementioned Non-Patent Document 1 (GF Gerberick et al., Toxicol. Sci., 97(2), 417-427:2007).

Test Example 1: Evaluation of detection sensitivity of reaction products with sensitizing compounds (1) TMAS (present invention)
[Preparation of TMAS solution]
12.5 mg of TMAS (molecular weight 337.38) was weighed out and dissolved in 100 mM phosphate (sodium) buffer (pH = 8.0), and the volume was adjusted to 100 mL (125 mg/L solution).

(2) Cys-P (Comparative Example)
[Preparation of Cys-P solution]
12.5 mg of the cysteine peptide (Ac-Arg-Phe-Ala-Ala-Cys-Ala-Ala-COOH: hereinafter referred to as Cys-P) (molecular weight: 750.87) described in Non-Patent Document 1 (GF Gerberick et al., Toxicol. Sci., 97(2), 417-427:2007) was weighed out and dissolved in 100 mM phosphate (sodium) buffer (pH = 8.0), and the volume was adjusted to 100 mL (125 mg/L solution).

(3) NAC (Comparative Example)
[Preparation of NAC solution]
To a reagent bottle containing 14.5 μg of N-(2-(1-naphthalyl)acetyl)-L-cysteine (hereinafter also referred to as NAC) (molecular weight: 289.34) described in Patent Document 2 (JP 2011-59102 A), 1 mL of 100 mM phosphate (sodium) buffer (pH = 8.0) was added and dissolved (14.5 mg/L solution).

[Method for preparing reaction solution]
600 μL of a 125 mg/L solution of TMAS, Cys-P, or NAC and 150 μL of a 50 μg/L or 5000 μg/L acetonitrile solution of the test substance (cinnamaldehyde) were added to a 1 mL vial and stirred (water/solvent = 80/20). This mixture was reacted at room temperature for 24 hours and then subjected to HPLC under the conditions described below.

[HPLC measurement conditions]
Apparatus: Agilent 1290 Series (manufactured by Agilent), TripleTOF 6600 (manufactured by Sciex)
Column: Kinetex polar C18 (2.1 mm x 150 mm, 2.6 μm)
Column temperature: 40°C
Flow rate: 0.15ml/min.
Detection: ESI (+)
Detection method: High Resolution Multiple Reaction Monitoring
Measurement mass range: MS (depending on reaction product), MS/MS (m/z 100-1000)
Eluent A: Ultrapure water (0.1% formic acid)
Eluent B: acetonitrile (0.1% formic acid)
Elution conditions: gradient as follows
Injection volume: 2μl
Analysis time: 30 minutes

[Measurement results]
The chromatograms are shown in Figure 1. Comparing the S/N ratios of the test substance at 50 μg/L, TMAS was approximately 60, while Cys-P and NAC were not detected. Comparing the S/N ratios of the test substance at 5000 μg/L, TMAS was approximately 6000, Cys-P was approximately 20, and NAC was approximately 110, demonstrating that TMAS can be detected with sensitivity several tens to several hundreds times higher than that of Cys-P or NAC.

Test Example 2: Evaluation of reactivity with sensitizing compounds and detection sensitivity of reaction products
[Test substance]
Using the compounds shown in Table 3 as sensitizing compounds, skin sensitization was measured according to the method described below.

(1) About TMAS (the present invention)
[Preparation of TMAS solution]
A TMAS solution was prepared in the same manner as in Test Example 1.

[Method for preparing reaction solution]
A reaction solution was prepared in the same manner as in Test Example 1 above.

[HPLC measurement conditions]
Apparatus: Agilent 1290 Series (manufactured by Agilent), TripleTOF 6600 (manufactured by Sciex)
Column: Kinetex polar C18 (2.1 mm x 150 mm, 2.6 μm)
Column temperature: 40°C
Flow rate: 0.25ml/min.
Detection: ESI (+) m / z 70-1300
Detection Method: Information Dependent Acquisition
Measurement mass range: TOF MS (m/z 70-1300), MS/MS (m/z 70-1300)
Eluent A: Ultrapure water (0.1% formic acid)
Eluent B: acetonitrile (0.1% formic acid)
Elution conditions: gradient as follows

The S/N ratio was calculated, and the concentration (μg/L) was determined with an S/N ratio of 3 as the detection limit. The results are shown in Table 3.

Test Example 3: Evaluation of reactivity with sensitizing compounds and detection sensitivity of reaction products
[Test substance]
Using the compounds shown in Table 4 as sensitizing compounds, skin sensitization was measured according to the method described below.
(2) TMAA (the present invention)
[Preparation of TMAA solution]
12.5 mg of TMAA (molecular weight 505.46) was weighed out and dissolved in 100 mM phosphate (sodium) buffer (pH = 10.2), and the volume was adjusted to 100 mL (125 mg/L solution).

[Method for preparing reaction solution]
A reaction solution was prepared in the same manner as in Test Example 1 above.

[HPLC measurement conditions]
Same as Test Example 1.

The S/N ratio was calculated, and the concentration (μg/L) was determined using an S/N ratio of 3 as the detection limit. The results are shown in Table 4.

The organic compounds, reagents, and analytical methods of the present invention are suitable for use in evaluating the skin sensitization potential of trace amounts of chemical substances and for quantitatively measuring trace amounts of chemical substances.

Claims (10)

An organic compound represented by the following chemical formula (1):
(In the formula, X is a thiol group or an amino group, ^R1 is a linear aliphatic hydrocarbon group having from 1 to 12 carbon atoms, ^R2 is an aromatic hydrocarbon group having from 6 to 10 carbon atoms, ^R3 , ^R4 , and ^R5 are each independently a linear aliphatic hydrocarbon group having from 1 to 6 _carbon atoms, A is an amide bond, and ^Y- is an anion selected from _a halogen anion, _ClO4- ^{, BF4- ,} _PF6- , _CF3COO- ^, and _CH3 ( _C6H4 ) _SO3- . ⁾ The organic compound according to claim 1 , wherein R ² is a phenylene group. 2. The organic compound according to claim 1, wherein the compound is a salt of 4-TriMethylAmmoniobenzoyl 2-Sulfanylethylamine and an anion, or a salt of 4-TriMethylAmmoniobenzoyl 6-Aminohexylamine and an anion . A reagent containing the organic compound described in any one of claims 1 to 3. An analytical method comprising reacting an organic compound according to any one of claims 1 to 3 with a chemical substance to obtain a reaction product, and then separating and detecting the reaction product. The analytical method described in claim 5, wherein the chemical substance is reactive with a thiol group or an amino group possessed by the organic compound. The analytical method described in claim 5 or 6, wherein the chemical reacts with a thiol group or an amino group of the organic compound. The analytical method according to any one of claims 5 to 7, wherein the separation method is chromatography. The analytical method according to any one of claims 5 to 8, wherein the detection method is mass spectrometry. The analytical method according to any one of claims 5 to 9, wherein the chemical substance is a sensitizing substance.