Method for testing toxicity of test sample

Abstract

An object of the present disclosure is to provide a toxicity test system that does not use an animal and can determine the toxicity of rhododenol having a relatively weak white spot formation ability.SOLUTION: It has been found that a melanocyte state in which cytotoxicity is easily detected can be created by adding a melanization-inducing factor to a medium. By utilizing this finding, a culture toxicity test system containing melanocytes capable of detecting the toxicity of rhododenol was established.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to the technical field of methods for testing toxicity of test samples. [Background technology]

[0002] Skin pigmentation, such as age spots, melasma, and freckles, creates an aged appearance and is a major cosmetic problem, leading to the development of various skin-whitening ingredients. Rhododenol, developed as a skin-whitening ingredient, caused vitiligo in 2.4% of users, becoming a social issue. While rhododenol inhibits tyrosinase, it is believed that rhododenol metabolites produced by the action of tyrosinase are toxic to melanocytes, resulting in the formation of vitiligo (Non-Patent Document 1: Pigment Cell & Melanoma Research 27 (5). doi:10.1111 / pcmr.12269). Acquired vitiligo resulting from repeated application of skin-whitening ingredients is called chemical leukoderma. Chemical leukoderma is an acquired depigmentation caused by repeated exposure to certain drugs that damage epidermal melanocytes. It has been reported to have characteristics that differ from vitiligo vulgaris, such as occurring only at the site of drug application and the presence of residual melanocytes (Non-patent document 2: Bjerke, DL, et al., 2022. 131: p. 105157).

[0003] In the development of new whitening active ingredients, toxicity tests using animals were previously conducted as a means of eliminating the possibility of vitiligo. However, in recent years, there has been an international trend toward non-animal toxicity tests, such as restrictions on animal testing of cosmetics in the EU. Meanwhile, since the problems with rhododenol, the screening of quasi-drugs has become stricter, making it necessary to develop toxicity tests that can replace animal testing, and such methods have been proposed (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2024-35677 [Non-patent literature]

[0005] [Non-Patent Document 1] Pigment Cell & Melanoma Research 27 (5). doi:10.1111 / pcmr.12269 [Non-patent document 2] Bjerke, DL, et al., 2022. 131: p. 105157 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present disclosure is to provide a toxicity test system that does not use animals and that is capable of determining the toxicity of rhododenol, which has a relatively weak ability to form vitiligo. [Means for solving the problem]

[0007] As a result of extensive research, the inventors discovered that adding a melanization-inducing factor to culture medium can create a melanocyte state in which cytotoxicity can be easily detected. Utilizing this finding, they established a melanocyte-containing culture toxicity test system capable of detecting the toxicity of rhododenol. Thus, the present disclosure relates to the following: [1] A method for testing toxicity of a test sample, comprising: culturing a culture containing melanocytes in a medium containing a melanization-inducing factor; contacting and culturing a test sample; and A step of measuring the number of melanocytes or an index based on the number of melanocytes and determining the cytotoxicity of the test sample compared to a control. [2] The method according to item 1, wherein the culture is selected from the group consisting of an ex vivo skin culture, a three-dimensional skin model, and a cell culture. [3] The method according to Aspect 1 or 2, wherein the melanization-inducing factor is one selected from the group consisting of αMSH, SCF, ET-1, NGF, KGF, HGF, bFGF, NT-3, NRG-1, TGF-β, DKK1, sFRP, Sema7a, Sema4D, CCN (CYR61), and FAP-α, or any combination thereof. [4] The method according to any one of items 1 to 3, wherein the melanization-inducing factor is a combination of αMSH and SCF. [5] The method according to any one of items 1 to 4, wherein the measurement of the number of melanocytes or an index based on the number of melanocytes is carried out by a measurement method targeting the protein or expression level of c-KIT, MelanA, or TYPR1. [6] The method according to any one of items 1 to 5, wherein the measurement of the number of melanocytes is carried out by detecting the number of positive melanocytes using an anti-c-KIT antibody, an anti-MelanA antibody, or an anti-TYPR1 antibody. [7] The method according to any one of items 1 to 6, wherein the control is the number of melanocytes measured by contacting and culturing a control sample containing a predetermined concentration of a control substance instead of the test sample, or an index determined from the number of melanocytes. [8] The method according to any one of items 1 to 7, wherein when a sample containing a vitiligo-inducing substance is used as the control sample, the test sample is determined to have cytotoxicity if the number of melanocytes is reduced compared to the control. [9] The vitiligo inducer is rhododenol, hydroquinone, MBEH (monobenzyl ether of hydroquinone), 4-TBP (4-tert-butylphenol), 4-TBC (4-tert-butylcatechol Item 9. The method according to item 8, wherein the ketone is selected from the group consisting of RK (Raspberry ketone).

[10] The method according to any one of items 1 to 9, wherein the toxicity test is a test for determining the vitiligo-forming potential of a test sample for a transdermal administration formulation.

[11] The method according to any one of items 1 to 10, wherein the ex vivo skin culture comprises keratinocytes and melanocytes.

[12] The method according to any one of items 1 to 11, wherein the ex vivo skin culture is obtained by peeling the epidermis from an animal or human skin section. [Effects of the Invention]

[0008] By adding a melanization-inducing factor to the culture medium, it is possible to create a state in which melanocytes are susceptible to cytotoxicity, and by using a culture containing melanocytes, it is possible to provide a toxicity test system that can detect the toxicity of rhododenol without using animals. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 shows the results of melanocyte proliferation following application of various concentrations of rhododenol in medium without melanization-inducing factor, medium supplemented with αMSH, medium supplemented with SCF, or medium supplemented with αMSH and SCF. [Figure 2] Figure 2 shows the quantification of immunostaining results using antibodies against TYRP1 or Gp100 in cultures containing melanocytes cultured in medium with or without αMSH and SCF, to which various concentrations of rhododenol (RD) or 50% EtOH as a control were applied. [Figure 3] Figure 3 shows the quantification of immunostaining results using antibodies against TYRP1, cKIT, Gp100, or MelanA in melanocyte-containing cultures cultured in medium supplemented with αMSH and SCF, which were treated with various concentrations of rhododenol (RD), potassium 4-methoxysalicylate (4MSK), or 50% EtOH as a control. [Figure 4]Figure 4 shows the quantification of immunostaining results using antibodies against TYRP1, MelanA, or Gp100 in melanocyte-containing cultures cultured in medium supplemented with αMSH and SCF, which were treated with various concentrations of hydroquinone monobenzyl ether (MBEH) or 50% EtOH as a control. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present disclosure relates to a method for testing the toxicity of a test sample, more specifically, the method comprising: culturing a culture containing melanocytes in a medium containing a melanization-inducing factor; contacting and culturing a test sample; and A step of measuring the number of melanocytes or an index based on the number of melanocytes The present invention relates to a method for determining the cytotoxicity of a test sample by comparing it with a control, comprising culturing a melanocyte-containing culture in a medium containing a melanization-inducing factor, thereby rendering the melanocytes in the culture susceptible to the cytotoxic effects of the test sample (Figure 1). This enabled the toxicity of samples with relatively weak vitiligo-forming potential to be determined, and the sensitivity of melanocytes to rhododenol toxicity was amplified (Figure 2).

[0011] In the present disclosure, a toxicity test method for a test sample refers to a test that determines whether a test sample causes toxicity to the skin to which the test sample is applied. Typically, the test sample is evaluated in two stages, such as "toxic" or "non-toxic," but this is not limited thereto. If the test sample is "toxic," measures such as adjusting the concentration of the toxic component in the test sample or excluding the test sample from the development candidates are taken. In one embodiment, toxicity refers to the ability to form vitiligo, and whether or not the test sample has the ability to form vitiligo can be determined.

[0012] In one embodiment, when a vitiligo-inducing sample is used as a control, a test sample is determined to have cytotoxicity if the number of melanocytes is reduced compared to the vitiligo-inducing sample. In one embodiment, the toxicity testing method of the present disclosure is a test for determining the vitiligo-forming potential of a test sample for a transdermal administration formulation. In one embodiment, a control sample containing rhododenol, which is known to have a relatively weak vitiligo-forming potential, can be used as the control.

[0013] The test sample can be the ingredient itself, which is typically applied to the skin, or a sample with a specific formulation containing the ingredient. Such ingredients can be ingredients that have been investigated as whitening ingredients in particular. Examples of such ingredients include, but are not limited to, organic compounds, inorganic compounds, and mixtures thereof. Such ingredients may be naturally derived or synthetic, or may be ingredients contained in existing compound libraries, extract libraries, etc. Furthermore, not only existing ingredients but also ingredients to be developed in the future can be used as test ingredients. Typically, test samples are contained in transdermal formulations (e.g., skincare products such as lotions, serums, emulsions, and creams, whitening cosmetics, moisturizing cosmetics, and foundations) or quasi-drugs for the skin (e.g., medicated creams such as hand creams, whitening agents, etc.). Alternatively, transdermal formulations and quasi-drugs themselves may be used as test samples.

[0014] The step of culturing a melanocyte-containing culture in a medium containing a melanocyte-inducing factor may be carried out in any manner as long as the melanocyte-containing culture is exposed to the melanocyte-containing culture. The melanocyte-inducing factor or a diluted solution thereof may be added directly to the culture medium, or the medium may be replaced with a medium containing the melanocyte-inducing factor. The culture is carried out under conditions typically used for culturing human epidermal cells. For example, the cells are cultured in an incubator at 37°C in a humidified atmosphere of 5% CO2. The culture step in a medium containing a melanocyte-inducing factor is carried out for a period sufficient to sufficiently enhance the sensitivity of the melanocytes to cytotoxicity. This period can be set so that toxicity by a control sample can be detected. For example, the cells can be cultured for 1 to 10 days, particularly 3 to 6 days.

[0015] A melanin-inducing factor refers to a factor that has the effect of increasing melanin production by melanocytes. It is thought that melanin-inducing factors, in particular, increase tyrosinase activity in melanocytes and amplify the cytotoxicity sensitivity of melanocytes. In one embodiment, the melanocyte cytotoxicity is vitiligo. In one embodiment, the melanin-inducing factor amplifies the cytotoxicity sensitivity of melanocytes to rhododenol, thereby making it possible to determine the vitiligo-forming ability of rhododenol.

[0016] Examples of melanization-inducing factors that can be used include, but are not limited to, one selected from the group consisting of α-Melanocyte-stimulating hormone (αMSH), SCF (Stem cell factor), ET-1 (Endothelin-1), NGF (Nerve growth factor), KGF (Keratinocyte growth factor), HGF (Hepatocyte growth factor), bFGF (β-Fibroblast growth factor), NT-3 (Neurotrophin-3), NRG-1 (Neuregulin 1), TGF-β (Transforming growth factor β), DKK1 (Dickkopf-related protein 1), sFRP (Secreted Frizzled Related Protein), Sema7a (Semaphorin 7A), Sema4D (Semaphorin 4D), CCN (CYR61) (Cysteine-rich 61), and FAP-α (Fibroblast activation protein-α), or any combination thereof. From the perspective of melanin induction, combinations of αMSH and ET-1, SCF and KGF, and αMSH and SCF are preferred. By using a combination of αMSH and SCF as a melanin induction factor, the toxicity sensitivity of melanocytes can be detected at a control sample of rhododenol of 0.01% or more, preferably 0.1% or more, and more preferably 1% or more. There are no particular limitations on the upper limit of the control sample of rhododenol, but it can be, for example, 50% or less, preferably 20% or less, and more preferably 10% or less.

[0017] The melanization induction factor may be contained in the medium at 0.0001% to 10% depending on the components used. The melanization induction factor may be contained in the medium at 0.0001% or more, 0.001% or more, preferably 0.01% or more. The melanization induction factor may be contained in the medium at 10% or less, 1% or less, preferably 0.1% or less.

[0018] The melanocyte-containing culture may be any cell culture containing melanocytes, including cell cultures of melanocytes themselves, ex vivo skin cultures, and 3D skin culture models. Such cultures may contain cells other than melanocytes, such as keratinocytes and Langerhans cells. Ex vivo skin cultures may be obtained by peeling the epidermis from animal or human skin sections. The 3D skin culture model may be commercially available or may be prepared according to a commonly disclosed method.

[0019] The step of contacting and culturing a test sample may be carried out in any manner as long as the test sample and melanocyte-containing culture are in contact. The test sample may be applied to the melanocyte-containing culture, the test sample or a diluted solution thereof may be added directly to the culture medium, or the culture medium may be replaced with a test sample-containing culture medium. Culture is performed under typical conditions used for culturing human epidermal cells. For example, the cells are cultured in an incubator at 37°C in a humidified atmosphere of 5% CO2. The test sample may be applied once or multiple times. In the latter case, the test sample is typically applied over a predetermined period (e.g., 4 or 5 days) at predetermined intervals (e.g., 12 hours, 24 hours, or 2 days). The intervals may be constant or may vary. The amount applied each time may be the same or different. The test sample may be applied to the culture surface at a concentration of 0.01% to 50%. To achieve a concentration close to that used in actual human use, the test sample may be contained in the culture medium at 1% to 10%. Preferably, it may be applied at 2% or more, 3% or more, or 5% or more. Preferably, it may be applied at 10% or less, 5% or less, or 2% or less.

[0020] The number of melanocytes or an index based on the number of melanocytes may be determined by directly counting the number of melanocytes or by determining the protein amount or expression level of a factor specifically expressed in melanocytes. For example, the number of melanocytes may be determined by immunostaining a melanocyte-specific protein (e.g., c-KIT, MelanA, TYPR1, etc.) and quantifying its color or brightness with a secondary antibody. Factors specifically expressed in melanocytes may include c-KIT, MelanA, or TYPR1. Measurement methods targeting the protein or expression level of c-KIT, MelanA, or TYPR1 can be performed using techniques well known in the art, such as immunological techniques or quantitative PCR. The protein amount or expression level of these factors reflects the number of viable melanocytes and may also reflect cellular activity. The number of melanocytes may be measured by detecting the number of positive melanocytes using an anti-c-KIT antibody, anti-MelanA antibody, or anti-TYPR1 antibody. Use of these antibodies allows for a more accurate determination of the number of surviving melanocytes. Anti-c-KIT antibodies, anti-MelanA antibodies, and anti-TYPR1 antibodies are readily available commercially. An index based on the number of melanocytes may be calculated based on the number of detected positive melanocytes. Examples of such an index include, but are not limited to, the number of apoptotic melanocytes.

[0021] The control may be the number of melanocytes measured by contacting a control sample containing a predetermined concentration of a control substance instead of the test sample and culturing it at a different point, or an index set from the number of melanocytes. The control may be a control group obtained by previously conducting an experiment and setting a threshold based on the number of melanocytes in the control group or an index set from the number of melanocytes, or the culture and measurement steps may be performed in parallel.

[0022] If the number of melanocytes is reduced compared to the control sample, the test sample is determined to have cytotoxicity. When the number of apoptotic melanocytes is used as an index based on the number of melanocytes, if the number of apoptotic melanocytes increases, i.e., the number of positive melanocytes decreases, compared to the control sample, the test sample can be determined to have cytotoxicity. This allows us to determine that the test sample has at least higher toxicity than the control sample.

[0023] The control sample is not particularly limited and may be an organic compound, an inorganic compound, or a mixture thereof. The control sample may be derived from a natural product or may be synthetic. Furthermore, not only existing substances but also substances to be developed in the future can be used as control samples. Vitiligo inducers may be used as control samples, but are not limited to these. Examples of vitiligo inducers include, but are not limited to, rhododenol, hydroquinone, monobenzyl ether of hydroquinone (MBEH), 4-tert-butylphenol (4-TBP), 4-tert-butylcatechol (4-TBC), and raspberry ketone (RK).

[0024] The control sample may be contained in the medium at 0.01% to 50%. The control sample may be contained in the medium at 0.5% or more, 1% or more, preferably 2% or more. The control sample may be contained in the medium at 5% or less, 10% or less, preferably 20% or less. The strength of toxicity to be evaluated can be adjusted by appropriately selecting the control sample and its concentration.

[0025] Rhododenol, an example of a vitiligo-inducing substance, induces melanocyte cell death through a reaction with tyrosinase. Rhododenol reacts with tyrosinase and ROS are produced during its metabolism. Subsequently, melanocyte cell death is thought to be induced by the endoplasmic reticulum stress response or by the formation of a complex between rhododenol metabolites and tyrosinase, which becomes a melanocyte-specific autoantigen and induces melanocyte-specific cytotoxic lymphocytes. Therefore, the level of tyrosinase activity in melanocytes is thought to increase or decrease sensitivity to rhododenol-induced toxicity.

[0026] In addition to the steps described above, the method of the present disclosure may include any processing steps typically performed in toxicity testing methods for test samples, including, but not limited to, a washing step, an immunostaining step for counting cell numbers, and the like.

[0027] The method of the present disclosure may include a washing step to remove components contained in the medium from the previous step. Examples of washing solutions include, but are not limited to, buffer solutions such as PBS, TBS, and HEPES, medium, and water. The washing step may be performed before, but is not limited to, the step of culturing a melanocyte-containing culture in a medium containing a melanization-inducing factor, the step of contacting and culturing a test sample, or the step of measuring the number of melanocytes or an index based on the number of melanocytes.

[0028] The method of the present disclosure may include an immunostaining step for measuring the number of melanocytes. For example, the immunostaining step involves washing the culture, fixing it with formaldehyde, etc., washing it again, and then reacting it with an anti-c-KIT antibody, an anti-MelanA antibody, or an anti-TYPR1 antibody against a protein specifically expressed in melanocytes as a primary antibody, followed by reacting it with a labeled secondary antibody, and then observing it under a fluorescence microscope.

[0029] All documents mentioned herein are incorporated by reference in their entirety.

[0030] The examples of the present disclosure described below are for illustrative purposes only and do not limit the technical scope of the present disclosure. The technical scope of the present disclosure is limited only by the claims. The present disclosure may be modified, for example, by adding, deleting, or substituting components of the present disclosure, provided that the modifications do not depart from the spirit of the present disclosure. [Example]

[0031] Example 1: Measurement of melanocyte proliferation (1) Measurement of cell proliferation Normal human melanocytes (Kurashiki Boseki Co., Ltd.) were cultured for 24 hours in maintenance medium (supplied by Kurashiki Boseki Co., Ltd.). Culture conditions were 37°C in a humidified 5% CO2 atmosphere. The cells were cultured overnight (day 1) in medium without melanization-inducing factors, medium supplemented with 100 nM αMSH (supplied by Sigma-Aldrich), medium supplemented with 10 ng / ml SCF (supplied by Sigma-Aldrich), or medium supplemented with 100 nM αMSH and 10 ng / ml SCF. Rhododenol (0.0001%, 0.001%, 0.01%, or 0.1%) was applied to the culture surface (day 2). Cell proliferation was measured by Alamar Blue assay (Thermo Fisher Scientific) on day 6. The results are shown in Figure 1.

[0032] Example 2: Addition of melanin-inducing factor increases sensitivity of melanocytes to cytotoxicity (1) Cultivation of melanocyte-containing cultures Melanocyte-containing cultures were cultured for 1 day in medium supplemented with or without 100 nM αMSH and 10 ng / ml SCF. Subsequently, the cultures were cultured for 5 days in the presence of melanization-inducing factors, with 2%, 3%, or 5% rhododenol applied to the culture surface. Control cultures were cultured with 50% EtOH instead of rhododenol. (2) Immunostaining After culture, the epidermal sheets were fixed with 4% PFA, washed, and then incubated with anti-TYPR1 antibody (distributor: Santa Cruz Biotechnology) or anti-Gp100 antibody (distributor: Santa Cruz Biotechnology) for 48 hours at 4°C. After washing, the sheets were incubated with 0.5% goat-anti-mouse 564 or goat-anti-rabbit 488-conjugated secondary antibody (distributor: Thermo Fisher Scientific) and Hoechst (Thermo Fisher Scientific) for 48 hours. Then, the sheets were observed under a fluorescent microscope. The number of TYPR1- or Gp100-positive cells in a given area was quantified by brightness (Figure 2).

[0033] Example 3: Detection of cytotoxicity by rhododenol (1) Cultivation of melanocyte-containing cultures Melanocyte-containing cultures were cultured for 1 day in medium supplemented with 100 nM αMSH and 10 ng / ml SCF. Subsequently, while still containing the melanization-inducing factor, 2%, 3%, or 5% rhododenol or 2%, 3%, or 5% 4-methoxysalicylic acid potassium salt (4MSK) was applied to the culture surface and cultured for 5 days. Control cultures were cultured with 50% EtOH instead of rhododenol or 4MSK. (2) Immunostaining After culture, the epidermal sheets were fixed with 4% PFA, washed, and then incubated with anti-TYPR1 antibody (Santa Cruz Biotechnology), anti-c-KIT antibody (Abcam), anti-c-MelanA antibody (Santa Cruz Biotechnology), or anti-Gp100 antibody (Santa Cruz Biotechnology) for 48 hours at 4°C. After washing, the sheets were incubated with 0.5% goat-anti-mouse 564 or goat-anti-rabbit 488-conjugated secondary antibodies (Thermo Fisher Scientific) and Hoechst (Thermo Fisher Scientific) for 48 hours. Then, the sheets were observed under a fluorescent microscope. The number of TYPR1-, c-KIT-, MelanA-, or Gp100-positive cells in a given area was quantified by brightness (Figure 3).

[0034] Example 4: Detection of cytotoxicity by MBEH (1) Cultivation of melanocyte-containing cultures Melanocyte-containing cultures were cultured for 1 day in medium supplemented with 100 nM αMSH and 10 ng / ml SCF. Subsequently, while still containing the melanization-inducing factor, 1M or 3M MBEH was applied to the culture surface and cultured for 5 days. Control cultures were cultured in medium supplemented with 50% EtOH instead of MBEH. (2) Immunostaining After culture, the epidermal sheets were fixed with 4% PFA, washed, and then incubated with anti-TYPR1 antibody (Santa Cruz Biotechnology), anti-c-MelanA antibody (Santa Cruz Biotechnology), or anti-Gp100 antibody (Santa Cruz Biotechnology) for 48 hours at 4°C. After washing, the sheets were incubated with 0.5% goat-anti-mouse 564 or goat-anti-rabbit 488-conjugated secondary antibody (Thermo Fisher Scientific) and Hoechst (Thermo Fisher Scientific) for 48 hours. Then, the sheets were observed under a fluorescent microscope. The number of TYPR1-, MelanA-, or Gp100-positive cells in a given area was quantified by brightness (Figure 4).

Claims

1. A method for testing toxicity of a test sample, comprising: Cultivating a culture containing melanocytes in a medium containing a melanization-inducing factor; contacting and culturing a test sample; and A step of measuring the number of melanocytes or an index based on the number of melanocytes and determining the cytotoxicity of the test sample compared to a control.

2. 10. The method of claim 1, wherein the culture is selected from the group consisting of an ex vivo skin culture, a three-dimensional skin model, and a cell culture.

3. 3. The method according to claim 1, wherein the melanization-inducing factor is one selected from the group consisting of αMSH, SCF, ET-1, NGF, KGF, HGF, bFGF, NT-3, NRG-1, TGF-β, DKK1, sFRP, Sema7a, Sema4D, CCN (CYR61), and FAP-α, or any combination thereof.

4. 4. The method of claim 3, wherein the melanization-inducing factor is a combination of αMSH and SCF.

5. The method according to claim 1 or 2, wherein the measurement of the number of melanocytes or an index based on the number of melanocytes is carried out by a measurement method targeting the protein or expression level of c-KIT, MelanA, or TYPR1.

6. 3. The method according to claim 1, wherein the measurement of the number of melanocytes is carried out by detecting the number of positive melanocytes using an anti-c-KIT antibody, an anti-MelanA antibody, or an anti-TYPR1 antibody.

7. The method according to claim 1 or 2, wherein the control is the number of melanocytes measured by contacting and culturing a control sample containing a predetermined concentration of a control substance instead of the test sample, or an index set from the number of melanocytes.

8. The method according to claim 7, wherein when a sample containing a vitiligo-inducing substance is used as the control sample, the test sample is determined to have cytotoxicity if the number of melanocytes is reduced compared to the control.

9. The vitiligo inducer is rhododenol, hydroquinone, MBEH (monobenzyl ether of hydroquinone), 4-TBP (4-tert-butylphenol), 4-TBC (4-tert-butylcatechol), 9. The method of claim 8, wherein the ketone is selected from the group consisting of RK (Raspberry ketone).

10. The method according to claim 1 or 2, wherein the toxicity test is a test for determining the vitiligo-forming potential of a test sample for a transdermal administration formulation.

11. 3. The method of claim 2, wherein the ex vivo skin culture comprises keratinocytes and melanocytes.

12. The method of claim 11, wherein the ex vivo skin culture is obtained by peeling the epidermis from an animal or human skin section.

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