JP7016533B2 - Test method for vitiligo toxicity and melasma toxicity - Google Patents

Description

The present invention relates to a technique for evaluating vitiligo toxicity and melasma toxicity. More specifically, the present invention relates to a test method for vitiligo toxicity and melasma toxicity using experimental animals. This application claims priority based on Japanese Patent Application No. 2016-104698 filed on May 25, 2016, and the entire contents of the patent application are incorporated by reference.

In recent years, the damage caused by "white spots" caused by the use of whitening agents containing 2% rhododenol has become a major social problem. Vitiligo is a disease associated with mottled whitening of the skin (depigmentation) (Non-Patent Document 1). It has also been reported that about 38% of patients with vitiligo who used the whitening agent develop "melanosis" accompanied by pigmentation (Non-Patent Document 2). It has been reported that the onset time is 1 to 2 months for early people and 3 to 5 months in many cases, and it can be said that one of the characteristics is that there are large individual differences in susceptibility. Rhododenol is a reduced form of raspberry ketone that has already been reported to be vitiligo toxic, but it was commercialized as a whitening agent because it cleared the safety standards for several types of animals set by the Ministry of Health, Labor and Welfare. Nevertheless, considering the background of this vitiligo problem, the sensitivity required for the current safety test is not sufficient. Furthermore, the Ministry of Health, Labor and Welfare has reported that 19 chemical substances contained in cosmetics, etc. other than rhododenol are suspected to have vitiligo-induced toxicity, and there is concern that the damage caused by whitening agents will further increase in the future.

The discovery of toxicity of whitening agents is a major problem that threatens corporate life, and countermeasures are urgently needed. In addition to cosmetics, many cases of vitiligo damage have been reported to people who handle chemical substances as a profession in factories, which is also an industrial hygiene problem. Furthermore, from the aspect of food safety due to the increase in imported foods, the demand from cosmetics, food and pharmaceutical manufacturers for safety tests using animal models is extremely high.

A technique using a hairy guinea pig has been reported as being related to the evaluation of vitiligo toxicity (Non-Patent Document 3). In this technique, vitiligo is induced by external application of 30% rhododenol in the skin whose pigment production is enhanced by artificial stimuli such as shaving and ultraviolet rays. However, vitiligo toxicity has not been evaluated at low concentrations (eg 2%) of rhododenol, and even 10% rhododenol has not been able to induce vitiligo. In addition, it is an evaluation technique that limits the chemical substance to rhododenol, and has not evaluated the toxicity of melasma. On the other hand, an evaluation method using hairless hk14-SCF Tg mice has also been reported (Non-Patent Document 4), but it is a vitiligo toxicity evaluation technique limited to 30% rhododenol external use, and uses a low concentration (for example, 2%) of rhododenol. Vitiligo toxicity has not been evaluated. In addition, it is a technique for evaluating vitiligo toxicity in which the chemical substance is limited to rhododenol, and the toxicity of melanosis has not been evaluated.

Pigment Cell Melanoma Res, 27,754-763,2014 Journal of the Japan Skin Association: 124 (11), 2095-2109, 2014 J Toxicol Sci, 39,615-23,2014 J Dermatol Science 81: 35-43, 2016 J Invest Dermatol. 127 (5): 1244-9,2007 J Biol Chem. 14; 281, 10365-73, 2006 Toxicology in Vitro 32; 339-346, 2016 Pigment Cell Melanoma Res. 29 (5): 541-9,2016

In response to the above social needs, it is desired to develop more sensitive evaluation technology in order to prevent the recurrence of health hazards such as vitiligo caused by chemical substances. However, at present, there is no technique capable of highly sensitively evaluating melasma toxicity / melasma toxicity caused by chemical substances. Therefore, the main object of the present invention is to provide a highly practical evaluation method capable of evaluating vitiligo toxicity and / or melasma toxicity with high sensitivity.

The present inventors have repeated studies to solve the above problems. As a result, we succeeded in establishing a highly sensitive and versatile evaluation system using mice with certain characteristics. Since the evaluation system does not require secondary stimuli such as shaving and ultraviolet irradiation, it is possible to centrally evaluate only the toxicity of chemical substances, and highly reliable evaluation results can be obtained. One of the notable points is that when a specific mouse (hairless RET mouse) is used, vitiligo toxicity and melasma toxicity can be evaluated at the same time. Further studies have also succeeded in constructing a cell-based evaluation system (in vitro test method) capable of simultaneously evaluating vitiligo toxicity, melasma toxicity, or both.
The inventions completed by the contributions of the present inventors are shown below.
[1] A test method for vitiligo toxicity, which comprises the following steps (1) and (2):
(1) A step of applying the test substance to the tail of a hairless mouse having melanin-producing ability,
(2) A step of evaluating the presence / absence and / or degree of induction of vitiligo in the tail of the hairless mouse, and determining the vitiligo toxicity of the test substance based on the evaluation result.
[2] The test method according to [1], wherein the hairless mouse is a hairless mouse having a normal melanin-producing ability.
[3] The test method according to [1], wherein the genetic background of the hairless mouse is C57BL / 6.
[4] The hairless mouse was obtained by re-mating a hairless mouse having a genetic background of C57BL / 6 to a RET / ^o -transgenic mouse in which the activated RET gene was introduced into a heterozygous form. The test method according to [3], which is a mouse or a hairless RET mouse obtained by mating the mouse with a brother and sister.
[5] The hairless mouse having a genetic background of C57BL / 6 is a mouse obtained by backcrossing a C57BL / 6 mouse to a Hos: HRM hairless mouse, or a mouse obtained by backcrossing the mouse. , [4].
[6] A test method for melasma toxicity, which comprises the following steps (1) and (2):
(1) A hairless RET mouse obtained by returning a hairless mouse having a genetic background of C57BL / 6 to a RET / ^o -transgenic mouse into which the activated RET gene has been introduced into a heterozygous form, or the mouse. The step of applying the test substance to the skin of hairless RET mice (excluding the skin of the tail) obtained by mating brothers and sisters.
(2) A step of evaluating the presence / absence and / or degree of induction of melasma in the skin of the hairless RET mouse, and determining the toxicity of the test substance to melasma based on the evaluation result.
[7] The test method according to [6], wherein the skin is the skin of the back, abdomen or head.
[8] Simultaneous test method for vitiligo toxicity and melasma toxicity, including the following steps (1) to (3):
(1) A hairless RET mouse obtained by returning a hairless mouse having a genetic background of C57BL / 6 to a RET / ^o -transgenic mouse into which the activated RET gene has been introduced into a heterozygous form, or the mouse. The step of applying the test substance to the tail and skin (excluding the skin of the tail) of a hairless RET mouse obtained by mating brothers and sisters.
(2) A step of evaluating the presence / absence and / or degree of induction of vitiligo in the tail of the hairless RET mouse, and determining the vitiligo toxicity of the test substance based on the evaluation result.
(3) A step of evaluating the presence / absence and / or degree of induction of melasma in the skin of the hairless RET mouse, and determining the toxicity of the test substance to melasma based on the evaluation result.
[9] The test method according to [8], wherein the skin is the skin of the back, abdomen or head.
[10] The test method according to any one of [4] and [6] to [9], wherein the activated RET gene is an RFP-RET gene generated by a hybrid of a c-RET gene and an RFP gene. ..
[11] The test method according to any one of [4] and [6] to [10], wherein the activated RET gene is linked to the metallothionein I promoter.
[12] The RET / ^o -transgenic mouse is 192 line RET / ^o -transgenic mouse, 242 line RET / ^o -transgenic mouse, 304 line RET / ^o -transgenic mouse, or 304 / B6 line RET /. ^o -The test method according to any one of [4], [6] to [11], which is a transgenic mouse.
[13] Mice susceptible to vitiligo toxicity and melasma toxicity, consisting of the following mice (i) or (ii):
(I) A hairless RET mouse obtained by backcrossing a hairless mouse having a genetic background of C57BL / 6 to a RET / ^o -transgenic mouse in which the activated RET gene has been introduced into a heteromorphic form.
(Ii) A hairless RET mouse obtained by mating the mice of (i) with brothers and sisters.
[14] The susceptible mouse according to [13], wherein the activated RET gene is an RFP-RET gene generated by a hybrid of a c-RET gene and an RFP gene.
[15] The susceptible mouse according to [13] or [14], wherein the activated RET gene is linked to the metallothionein I promoter.
[16] The RET / ^o -transgenic mouse is 192 line RET / ^o -transgenic mouse, 242 line RET / ^o -transgenic mouse, 304 line RET / ^o -transgenic mouse, or 304 / B6 line RET /. ^o -The susceptible mouse according to any one of [13] to [15], which is a transgenic mouse.
[17] An in vitro test method for evaluating vitiligo toxicity of a test substance using melanocytes collected from the tail of a hairless mouse capable of producing melanin or passage cells thereof.
[18] A hairless RET mouse obtained by re-mating a hairless mouse having a genetic background of C57BL / 6 to a RET / ^o -transgenic mouse into which the activated RET gene has been introduced into a heteromorphic form, or the mouse. It is characterized in that the melanosis toxicity of the test substance is evaluated using melanosite or its passage cells collected from the skin (excluding the tail skin) of hairless RET mice obtained by mating brothers and sisters. In vitro test method.
[19] An in vitro test method for evaluating vitiligo toxicity and / or melasma toxicity, which comprises the following steps (a) to (d):
(A) A hairless RET mouse obtained by returning a hairless mouse having a genetic background of C57BL / 6 to a RET / ^o -transgenic mouse into which the activated RET gene has been introduced into a heterozygous form, or the mouse. A step of preparing a sample containing the skin melanosite of a hairless RET mouse obtained by mating brothers and sisters and dividing it into a test group and a control group.
(B) The step of culturing the test group in the presence of the test substance,
(C) For the test group after step (b) and the control group cultured under the same conditions except that the test substance was absent, c-Met, RFP-RET, c-Ret, Enothelin receptor B ( Ednrb), c-kit, Mc1r, Mitf-m, Tyrosinase, Tyrosinase-realted protein-2, TGF-beta type receptor 2 (Tgfr2), Fibroblast growth factor (Fgfr), Bcl2, p53, survivin, CCAAT-enhancer-binding protein homologous protein (CHOP), Glutamate cysteine ligase (GCLC), NF-E2 related factor2 (Nrf2), Heat shock protein (HSP) 70, Foxp1, Foxp3, CCR6, Bach2, NATHT leucine-rich-repeat protein 1 (Nalp) , A step of detecting the expression or activity of one or more marker molecules selected from the group consisting of Cytotoxic T-lymphocyte antigen-4 (Ctla4) and Granzyme B (GZMB).
(D) A step of comparing the detection results between the test group and the control group, and determining the vitiligo toxicity and / or melasma toxicity of the test substance based on the comparison results.
[20] An in vitro test method for evaluating vitiligo toxicity and / or melasma toxicity, which comprises the following steps (A) to (D):
(A) A step of preparing a melanocyte cell line derived from a RET / ^o -transgenic mouse into which an activated RET gene has been introduced into a heterozygous form and dividing it into a test group and a control group.
(B) The step of culturing the test group in the presence of the test substance,
(C) For the test group after step (B) and the control group cultured under the same conditions except that the test substance was absent, c-Met, RFP-RET, c-Ret, Enothelin receptor B ( Ednrb), c-kit, Mc1r, Mitf-m, Tyrosinase, Tyrosinase-realted protein-2, TGF-beta type receptor 2 (Tgfr2), Fibroblast growth factor (Fgfr), Bcl2, p53, survivin, CCAAT-enhancer-binding protein homologous protein (CHOP), Glutamate cysteine ligase (GCLC), NF-E2 related factor2 (Nrf2), Heat shock protein (HSP) 70, Foxp1, Foxp3, CCR6, Bach2, NATHT leucine-rich-repeat protein 1 (Nalp) , A step of detecting the expression or activity of one or more marker molecules selected from the group consisting of Cytotoxic T-lymphocyte antigen-4 (Ctla4) and Granzyme B (GZMB).
(D) A step of comparing the detection results between the test group and the control group, and determining the vitiligo toxicity and / or melasma toxicity of the test substance based on the comparison results.
[21] A method for screening a drug for preventing or treating vitiligo, which comprises the following steps (a) to (c):
(A) A step of inducing vitiligo on the tail of a hairless mouse capable of producing melanin,
(B) The step of administering the test substance to the hairless mouse,
(C) A step of determining the prophylactic or therapeutic effect of the test substance.
[22] A method for screening a drug for preventing or treating melasma, which comprises the following steps (A) to (C):
(A) A hairless RET mouse obtained by re-mating a hairless mouse having a genetic background of C57BL / 6 to a RET / ^o -transgenic mouse in which the activated RET gene has been introduced into a heterozygous form, or the mouse. A step of inducing melanosis on the skin (excluding the tail skin) of a hairless RET mouse obtained by mating brothers and sisters.
(B) The step of administering the test substance to the hairless RET mouse,
(C) A step of determining the preventive or therapeutic effect of the test substance.

Mice used for evaluation of vitiligo / melasma toxicity. 8-week-old hairless wild-type mice (C57BL / 6 background) (A: back skin on the left, tail skin on the right) and hairless RET mice (242 strains) used to evaluate vitiligo toxicity and melasma toxicity. C57BL / 6 background) (B: back skin on the left, tail skin on the right) shows typical macroscopic findings. L ^* values (n = 5) (mean ± SD) in the back skin of hairless wild-type mice and hairless RET mice are shown relative values when the L ^* value of hairless wild-type mice is 1. (C). ** p <0.01 (according to the Mann-Whitney U test). Vitiligo and melasma induced by application of 2% rhododenol and 2% raspberry ketone. Hairless wild-type mice (C57BL / 6 background) and hairless RET mice (242 strains) (C57BL / 6 background) with solvent (50% ethanol) and 2% rhododenol or 2% raspberry ketone externally applied for 4 months. The relative values (n = 5) (mean ± SD) of the L ^* values of the dorsal skin and the gross findings (A) are shown when the L ^* value of the hairless wild-type mice externally applied to the solvent is 1. (B), typical macroscopic findings of the tail skin (C), and relative values (n = 5) (mean ± SD) (D) of the skin color (blackness) of the tail skin are shown. * p <0.05, ** p <0.01 (according to the corresponding Student's t-test). Histological analysis of the back skin with external application of 2% rhododenol and 2% raspberry ketone. Representative of the back skin of hairless wild-type mice (C57BL / 6 background) and hairless RET mice (242 strains) (C57BL / 6 background) to which the solvent (50% ethanol) and 2% rhododenol or 2% raspberry ketone were externally applied for 4 months. Fontana Masson stained image (A), epidermal melanin density (n = 5) (mean ± SD) (B), representative histological image showing the distribution of Dopachrometautomerase (Dct) positive cells (melanocytes), epidermis (C), epidermis ( The number of Dct-positive cells (n = 5) (mean ± SD) (D) per 500 μm) is shown. The arrowhead of A shows melanin stained with Fontana masson, and the arrowhead of C shows Dct-positive cells. In the original figure, the blue fluorescence shows the nucleus stained with Dapi. The scale bar is 50 μm. * p <0.05, ** p <0.01 (according to Tukey-Kramer test). Histological analysis of tail skin with external application of 2% rhododenol and 2% raspberry ketone. Representative of tail skin of hairless wild-type mice (C57BL / 6 background) and hairless RET mice (242 strains) (C57BL / 6 background) to which solvent (50% ethanol) and 2% rhododenol or 2% raspberry ketone were externally applied for 4 months. Fontana Masson stained image (A), epidermal melanin density (n = 5) (mean ± SD) (B), representative histological image showing the distribution of Dopachrometautomerase (Dct) positive cells (melanocytes), epidermis (C), epidermis ( The number of Dct-positive cells (n = 5) (mean ± SD) (D) per 500 μm) is shown. The arrowhead of A shows melanin stained with Fontana masson, and the arrowhead of C shows Dct-positive cells. In the original figure, the blue fluorescence shows the nucleus stained with Dapi. The scale bar is 50 μm. ** p <0.01 (according to Tukey-Kramer test). Evaluation of vitiligo toxicity and melasma toxicity using cell lines isolated from RET-mice. Changes in RFP-RET mRNA expression (RFP-RET / Hprt) (n = 3, mean ± SD) (A) corrected by the mRNA expression level of Hypoxanthine phosphoribosyltransferase (Hprt), and changes in c-Met mRNA expression (c-). Met / Hprt) (n = 3, mean ± SD) (B) is shown. The relative values are shown when the RFP-RET / Hprt or c-Met / Hprt values are set to 1 when rhododenol is not exposed. (*, P <0.05, according to Mann-Whitney U-test).

1. 1. Test Method for Vitiligo Toxicity The present invention relates to a toxicity test using mice. In the first aspect, a test method for vitiligo toxicity is provided. Vitiligo toxicity refers to the action or effect of inducing vitiligo. Vitiligo is a pathological condition caused by the decrease or disappearance of melanocytes, which are pigment cells distributed in the basal layer of the skin. As is clear from the object and the mode of use of the present invention, the vitiligo in the present invention does not include congenital vitiligo (albinism) and corresponds to so-called vitiligo vulgaris. In the vitiligo toxicity test method of the present invention, the following steps (1) and (2) are performed.
(1) Step of applying the test substance to the tail of a hairless mouse having melanin-producing ability (2) The presence or absence and / or degree of induction of vitiligo in the tail of the hairless mouse is evaluated, and the vitiligo toxicity of the test substance is evaluated based on the evaluation result. Step to determine

In step (1), a specific mouse, that is, a hairless mouse having a melanin-producing ability is prepared, and the test substance is applied to the tail thereof. "Hairless mice capable of producing melanin" have melanin in the epidermis and show the genotype of hairless (hairless) individuals. This feature is important in that it is necessary for the induction of vitiligo and allows easy and objective evaluation. Examples of hairless mice capable of producing melanin include Hos: HRM, Hos: HRM-2 and HR / DeF1 (all available from Hoshino Test Animal Breeding Center). As a hairless mouse capable of producing melanin, molecules involved in the regulation of melanin production, specifically Microphthalmia-associated transcription factor, Tyrosinase, Tyrosinase related protein-1, Tyrosinase related protein 2, Pmel 17, alpha-Melanocyte-stimulating hormone, Hairless mice in which molecules such as Stem cell factor, c-Kit, Endothelin, and Endothelin receptor are functioning and have normal melanin-producing ability should be used. Preferably, hairless mice with a genetic background of C57BL / 6 are used. For example, by returning and mating a C57BL / 6 mouse (for example, a C57BL / 6 mouse provided by Japan Charles River or Japan Claire can be used) to the above-mentioned hairless mouse, a genetic background that can be used in the present invention can be obtained. C57BL / 6 hairless mice (mice that have not undergone genetic modification by introducing the RET gene and may be referred to as "hairless wild-type mice" to distinguish them from the following hairless RET mice) can be obtained. It is advisable to repeat backcrossing using C57BL / 6 mice so that the genetic background is preferably 95.0% or more, more preferably 99.9% or more.

In one embodiment, a hairless RET mouse obtained by backcrossing a RET / ^o -transgenic mouse into which an activated RET gene has been introduced into a heterozygous gene with a hairless mouse having a genetic background of C57BL / 6, or the said. Hairless RET mice obtained by mating mice with brothers and sisters are used as "hairless mice having melanin-producing ability". When hairless RET mice are used, the evaluation system assumes a type of human who develops vitiligo over a relatively long period of time (see Examples for details). On the other hand, when a mouse other than the hairless RET mouse (that is, the above-mentioned hairless wild-type mouse) is used, a more sensitive evaluation system can be realized. Therefore, if these two aspects are used in combination, toxicity evaluation for humans with different susceptibility becomes possible.

As described above, the hairless RET mouse is a method of backcrossing a hairless mouse having a genetic background of C57BL / 6 to a RET / ^o -transgenic mouse in which the activated RET gene is heterozygously introduced (creation method 1). ), Or the mouse obtained by the production method 1 can be obtained by the method of mating brothers and sisters (production method 2). According to these methods, a hairless mouse having a genotype of hr / hr; Ret / ^O and a genetic background of C57BL / 6 can be obtained. A RET / ^o -transgenic mouse in which an activated RET gene is introduced into a heterogeneous form typically uses the RFP-RET gene generated by a hybrid of the c-RET gene and the RFP gene as the activated RET gene. It is a mouse possessed by a hetero. It is preferred that the activated RET gene is linked to the metallothionein I promoter (MT). Specific examples of RET / ^o -transgenic mice are 192 strains RET / ^o -transgenic mice, 242 strains RET / ^o -transgenic mice, 304 strains RET / ^o -transgenic mice (Kato et al., Oncogene, 18 ( 3): 837-42,1999; Iwamoto et al., EMBO J, 10 (11), 3167-3175, 1991), and 304 strain RET / ^o -transgenic mice were mated with C57BL / 6 mice. The obtained 304 / B6 strain RET / ^o -transgenic mouse (Kato et al, .Oncogene, 17, 1885-1888, 1998). Preferably, 242 lines are used. Frozen fertilized eggs and frozen sperm of RET trasgenic mice are stored in the Life Resources Research and Support Center (CARD) of Kumamoto University and the RIKEN BioResource Center (Tsukuba) of RIKEN.

In production method 1, hairless mice with a genetic background of C57BL / 6 are backcrossed to RET / ^o -transgenic mice as described above. As the hairless mouse having a genetic background of C57BL / 6, the above-mentioned hairless wild-type mouse can be used. In a preferred embodiment, a mouse obtained by backcrossing a C57BL / 6 mouse to a Hos: HRM hairless mouse or a mouse obtained by mating the mouse with a brother and sister is referred to as a "hairless mouse having a genetic background of C57BL / 6". Adopt as.

In the production method 2, the mice obtained by the production method 1 are mated with each other. The mice thus obtained have the advantage that the uniformity of the genetic background is enhanced and the influence of individual differences can be reduced, so that stable toxicity evaluation becomes possible. Brother-sister mating may be performed, for example, 10 times or more, preferably 20 times or more (specifically, 20 to 30 times).

The test substance is applied to the tail of the "hairless mouse having melanin-producing ability" prepared as described above. That is, in the present invention, the tail is adopted as the application site of the test substance based on the finding that the induction of vitiligo was specifically observed in the tail. The method of applying the test substance is not particularly limited. For example, the test substance itself or a composition containing the test substance (solution, ointment, cream, cosmetics, etc.) is applied or dropped on the skin of the tail, or injected into the tail. Prick tests and scratch tests are also possible. It is also possible to apply the test substance by putting or immersing the tail in the test substance or a solution containing it.

Any substance that requires an assessment of vitiligo toxicity, i.e., any substance that may exhibit vitiligo toxicity, can be the test substance. The structure, molecular size, existence state (for example, other substances may be mixed or a complex may be formed with other substances) of the test substance are not particularly limited. Therefore, various organic compounds or inorganic compounds, or various compositions containing them can be test substances. The test substance may be of natural or synthetic origin. Considering that the toxicity of white spots caused by chemical substances may be enhanced by physical irritation (for example, light rays) (for example, photosensitivity white spot keratoderma), physical irritation should be added when applying the test substance. You may do it. Further, not only existing substances but also substances to be developed in the future can be used as test substances. Typically, for example, cosmetics (for example, basic cosmetics such as lotion, beauty liquid, milky lotion, cream, whitening cosmetics, moisturizing cosmetics, foundations, lipsticks, shampoos, soaps, hairdressing agents, etc.), pharmaceuticals for skin. External products (for example, medicated creams such as hand creams, whitening agents, deodorant agents, gray hair dyes, hair coloring agents, hair growth agents, hair growth agents, etc.), pharmaceuticals (external agents, internal preparations (for example, siazide diuretics, muscle tension) Main components, sub-ingredients, additives or base materials such as therapeutic agents), drip agents, injectable agents, etc.) are suitable test substances. Further, the cosmetic substance itself, the quasi-drug itself, the drug itself and the like may be used as the test substance. Various substances (for example, chemical substances, foods, substances derived from animals and plants) that can be exposed to workers and surrounding residents in industrial processes such as chemical factories and raw material processing industries may be used as test substances.

The test substance is applied once or multiple times (eg, 2 to 360 times). In the latter case, typically, there are predetermined intervals (eg, 6 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks) and a predetermined period (eg, 1 week, 2 weeks). It will be applied over 3 weeks, 4 weeks, January, February, March, April, May, June). The interval here may be constant or may vary in the middle. Moreover, the applied amount of each time may be the same or different.

In step (2), the presence or absence or degree of induction of vitiligo in the tail of the hairless mouse treated in step (1), or both of them are evaluated. Then, the vitiligo toxicity of the test substance is determined based on the evaluation result. Typically, when vitiligo is induced, it will be determined that the test substance has vitiligo toxicity. However, instead of such a definitive judgment, it is decided to give a judgment result such as "the test substance is likely to have vitiligo toxicity" or "the test substance may have vitiligo toxicity". May be good. If vitiligo is not induced, a determination result such as "the test substance does not have vitiligo toxicity" or "the test substance is unlikely to have vitiligo toxicity" is obtained. When determining based on the degree of vitiligo induction, for example, multiple levels of toxicity corresponding to the degree of vitiligo induction (time until vitiligo is induced, size (area) of induced vitiligo, color tone of vitiligo, etc.) Is set, it is confirmed which toxicity level the evaluation result corresponds to, and the toxicity level of the test substance is judged or determined. The presence or absence or degree of vitiligo induction is evaluated by the time until vitiligo is induced, the size (area) of the induced vitiligo, the color tone of vitiligo, the skin color of the test substance application site, and the amount of skin melanin at the test substance application site. It can be performed according to evaluation items such as the amount of skin melanocytes at the site of application of the test substance, tyrosinase activity, and the expression level of melanin-related molecules in melanocytes and surrounding cells. The time until vitiligo is induced, the size (area) of the induced vitiligo, the color tone of the vitiligo, and the skin color of the test substance application site can be evaluated visually, but a color difference meter and image analysis software ( For example, it may be evaluated using WinROOF image analysis software (Mitani Shoji Co., Ltd.). When evaluating the color tone and skin color of white spots with a color difference meter, it is advisable to adopt the international color standard L ^* a ^* b ^* color system. According to the L ^* value (brightness) of the color difference meter, it is possible to more objectively evaluate the color tone and skin color of vitiligo. It is also possible to evaluate by a formula system such as L * C * h or HSL.

In order to improve the accuracy and reliability of the evaluation, two or more evaluation items may be used together. Examples of combined use of endpoints include (i) time to induce vitiligo, (i) time to induce vitiligo, and (ii) time to induce vitiligo. , Combination of two endpoints of vitiligo color tone, (iii) Combination of three endpoints of time until vitiligo is induced, size (area) of induced vitiligo, and vitiligo color tone. can.

Preferably, a hairless mouse (test group) to which the test substance is applied and a hairless mouse (control group) having the same conditions except that the test substance is not applied are prepared. Then, the test group and the control group are compared with respect to the adopted evaluation items, and the judgment of step (2) is performed based on the result. By comparing the test group and the control group in this way, the toxicity of the test substance can be easily determined with high reliability. The number of individuals included in the test group and the control group is not particularly limited. In general, the larger the number of individuals used, the more reliable the results can be obtained, but handling a large number of individuals at the same time involves difficulties in securing and operating (including breeding) the individuals to be used. Therefore, the number of individuals included in each group is, for example, 1 to 50, preferably 2 to 30, and more preferably 5 to 20.

2. 2. Test Method for Melasma Toxicity The second aspect of the present invention provides a test method for melasma toxicity. Melasma is a condition in which the skin changes from dark brown to purple-brown due to pigmentation. In the melasma toxicity test method of the present invention, the following steps (1) and (2) are performed. In the following, the details of each step will be described, but for the terms and requirements common to the first aspect (white spot toxicity test method), the explanation in the first aspect will be referred to, and duplicate explanations will be omitted.
(1) A hairless RET mouse obtained by re-mating a hairless mouse having a genetic background of C57BL / 6 to a RET / ^o -transgenic mouse in which the activated RET gene has been introduced into a heteromorphic form, or the mouse. Steps of applying the test substance to the skin of a hairless RET mouse (excluding the skin of the tail) obtained by mating brothers and sisters (2) Presence / absence and / or degree of induction of melanosis in the skin of the hairless RET mouse. And determine the toxicity of the test substance to melanosis based on the evaluation results.

In step (1), a hairless RET mouse obtained by re-mating a hairless mouse having a genetic background of C57BL / 6 to a RET / ^o -transgenic mouse in which the activated RET gene has been introduced into a heterozygous form, or A hairless RET mouse obtained by mating the mouse with a brother and sister (that is, a hairless mouse having a genotype of hr / hr; Ret / ^O and a genetic background of C57BL / 6) is prepared, and the test substance is applied to the skin. .. The test substance is applied to the skin of a part other than the tail, but preferably the skin of the back, abdomen or head is the application part of the test substance. The method of applying the test substance is not particularly limited. For example, the test substance itself or a composition containing the test substance (solution, ointment, cream, cosmetics, etc.) is applied or dropped on the skin (other than the tail) of a mouse, or injected into the skin (other than the tail). Prick tests and scratch tests are also possible.

Any substance that requires an assessment of melasma toxicity, i.e., any substance that may exhibit melasma toxicity, can be the test substance. Therefore, various substances can be adopted as in the case of the first aspect (see the description of the test substance in the first aspect for details). Further, as in the case of the first aspect, physical stimulation may be applied together with the administration of the test substance. The number of times the test substance is applied and the application schedule are also in accordance with the first aspect.

In the step (2) following the step (1), the presence or absence or degree of induction of melasma in the skin (other than the tail) of the hairless RET mouse treated in the step (1), or both of them are evaluated. Then, the toxicity of the test substance to melasma is determined based on the evaluation result. As a matter of course, the evaluation and judgment will be made at the site corresponding to the application site of the test substance. That is, when the test substance is applied to the back of a mouse, the "back" becomes the evaluation site. Similarly, when the test substance is applied to the abdomen, the "belly" is used, and when the test substance is applied to the head, the "head" is the evaluation site. Typically, when melasma is induced, it will be determined that the test substance has melasma toxicity. However, instead of such a definitive judgment, a judgment result such as "the test substance is likely to have melasma toxicity" or "the test substance may have melasma toxicity" is obtained. You may decide to put it out. If melasma is not induced, a determination result such as "the test substance does not have melasma toxicity" or "the test substance is unlikely to have melasma toxicity" is given. When judging based on the degree of induction of melasma, for example, the degree of induction of melasma (time until the induction of melasma, the size (area) of the induced pigmentation, the color tone of the pigmentation, etc. ), A plurality of toxicity levels are set, it is confirmed which toxicity level the evaluation result corresponds to, and the toxicity level of the test substance is determined or determined. The presence or absence or degree of melasma induction is evaluated by the time until melasma is induced, the size (area) of the induced pigmentation, the color tone of the pigmentation, the skin color of the test substance application site, and the test substance application. It can be performed according to evaluation items such as the amount of skin melanin at the site, the amount of skin melanocytes at the site where the test substance is applied, tyrosinase activity, and the expression level of melanin-related molecules in melanocytes and surrounding cells. The time until melasma is induced, the size (area) of the induced pigmentation, the color tone of the pigmentation, and the skin color of the test substance application site can be visually evaluated. Image analysis software (for example, WinROOF image analysis software (Mitani Shoji Co., Ltd.)) may be used for evaluation. When evaluating the color tone and skin color of white spots with a color difference meter, it is advisable to adopt the international color standard L ^* a ^* b ^* color system. According to the L ^* value (brightness) of the color difference meter, it is possible to more objectively evaluate the color tone and skin color of pigmentation.

In order to improve the accuracy and reliability of the evaluation, two or more evaluation items may be used together. Examples of combined endpoints include (i) the time to induce melasma and the combined size (area) of induced pigmentation, and (ii) induced melasma. Combined use of two endpoints of time to hyperpigmentation and color tone of pigmentation, (iii) time to induce melasma, size (area) of induced pigmentation, and color tone of pigmentation. The combined use of the three evaluation items can be mentioned.

As in the case of the first aspect, in the melasma toxicity test method, it is preferable to prepare a test group and a control group, and evaluate and judge the toxicity of the test substance based on these comparisons.

3. 3. Mice useful for simultaneous evaluation of vitiligo toxicity and rhododenol toxicity and test methods using them As shown in the examples below, hairless RET mice (ie, genotype hr / hr; Ret / ^O and genetic background) In C57BL / 6 hairless mice), application of low concentrations of rhododenol and raspberry ketone induced melasma in the tail skin and melasma in the back skin. That is, the hairless RET mouse has a unique and useful property that it is highly susceptible to vitiligo toxicity and melasma toxicity, and its utility value is high. Therefore, as a further aspect, the present invention provides a mouse susceptible to vitiligo toxicity and melasma toxicity, which comprises the following mice (i) or (ii). Since the mice of (i) and (ii) are the same as the mice used in the first aspect and the second aspect, the overlapping description will be omitted. In the following, matters specific to this aspect will be explained.
(I) Hairless RET mouse (ii) obtained by re-mating a hairless mouse with a genetic background of C57BL / 6 to a RET / ^o -transgenic mouse in which the activated RET gene has been introduced into a heterozygous form. i) Hairless RET mouse obtained by mating the mouse of brother and sister

The mouse of (ii) has a higher uniformity of genetic background than the mouse of (i) and can reduce the influence of individual differences, so that it has an advantage that stable toxicity evaluation becomes possible, which is particularly preferable. It can be said that. Brother-sister mating may be performed, for example, 10 times or more, preferably 20 times or more (specifically, 20 to 30 times).

Specific examples of RET / ^o -transgenic mice are 192 strains RET / ^o -transgenic mice, 242 strains RET / ^o -transgenic mice, 304 strains RET / ^o -transgenic mice, and 304 strains RET / ^o -transgenic. 304 / B6 strain RET / ^o -transgenic mice obtained by mating C57BL / 6 mice with mice, preferably 242 strains are used.

Hairless RET mice are useful for assessing vitiligo toxicity (first phase) and melasma toxicity (second phase), but this aspect is highly sensitive to both melasma toxicity and melasma toxicity. Taking advantage of the characteristics shown, we provide an extremely unique test method for simultaneously evaluating and determining vitiligo toxicity and melasma toxicity. In the test method (simultaneous test method for vitiligo toxicity and melasma toxicity), the following steps (1) to (3) are performed. In the following, the details of each step will be described, but for the terms and requirements common to the first aspect (white spot toxicity test method), the explanation in the first aspect will be referred to, and duplicate explanations will be omitted.
(1) A hairless RET mouse obtained by re-mating a hairless mouse having a genetic background of C57BL / 6 to a RET / ^o -transgenic mouse in which the activated RET gene has been introduced into a heteromorphic form, or the mouse. Steps of applying the test substance to the tail of a hairless RET mouse obtained by mating brothers and sisters and the skin (excluding the skin of the tail) (2) Presence / absence and / or degree of white spots on the tail of the hairless RET mouse. (3) The presence or absence and / or degree of induction of melanosis in the skin of the hairless RET mouse of the hairless RET mouse is evaluated, and the test substance of the test substance is evaluated based on the evaluation result. Steps to determine proto-onset toxicity

In step (1), a hairless RET mouse is prepared, and the test substance is applied to the tail and the skin (excluding the skin of the tail). That is, the test substance is applied to roughly two places. The application to each site is performed without a substantial time difference (that is, substantially at the same time) or at predetermined time intervals.

A substance that requires evaluation of white spot toxicity is applied to the tail as the first test substance, and another substance that requires evaluation of melanosis toxicity is applied to the skin (other than the tail) as the second test substance. Although it is possible to evaluate the toxicity of each test substance at the same time, if the same test substance is applied to both the tail and the skin (other than the tail), the toxicity of the white spot and the toxicity of melanosis of the substance are to be evaluated. Can be evaluated at the same time. In this case, the substance that requires evaluation of vitiligo toxicity, melasma toxicity, or both of them is used as the test substance. Various substances can be adopted as in the case of the first phase / the second phase (for details, refer to the description of the test substance in the first phase / the second phase). The number of times the test substance is applied and the application schedule are also the same as in the first phase / second phase.

In step (2), the presence or absence or degree of induction of vitiligo in the tail of the hairless RET mouse treated in step (1), or both of them are evaluated. Then, the vitiligo toxicity of the test substance is determined based on the evaluation result. On the other hand, in step (3), the presence or absence or degree of induction of melasma in the skin (other than the tail) of the hairless RET mouse treated in step (1), or both of them are evaluated. Then, the toxicity of the test substance to melasma is determined based on the evaluation result. Either step (2) or (3) may be performed first. Further, steps (2) and (3) may be performed in parallel. The details of the evaluation and judgment in step (2) (evaluation items and judgment results, etc.) and the details of the evaluation and judgment in step (3) (evaluation items, judgment results, etc.) are the first phase and the second phase, respectively. Same as. In addition, in each step, two or more evaluation items may be used together in order to improve the accuracy and reliability of the evaluation, and a test group and a control group are prepared, and the toxicity of the test substance is based on these comparisons. It is the same as the above-mentioned aspect that it is preferable to evaluate and judge.

4. In vitro test system This aspect relates to cell-based test methods. Specifically, a test method for evaluating vitiligo toxicity of a test substance using melanocytes collected from the tail of a hairless mouse used in the first aspect or subculture cells thereof (including established cells), and the first. Evaluation of vitiligo toxicity of the test substance using melanocytes collected from the skin of hairless RET mice used in the second aspect (excluding the skin of the tail) or passage cells thereof (including established cells) Test methods are provided. For evaluation of leukoplakia toxicity or melanocytic toxicity, for example, in addition to cell physiological function tests such as melanin expression level, melanocyte proliferation ability, melanocyte survival rate, and melanocyte migration ability, molecules that affect these cell physiological functions. Evaluation items such as expression or activity level of (tyrosinase, αMSH, endoserine, endoserin receptor, SCF, c-Kit, POMC, GM-CSF, Mitf, etc.) can be utilized.

As a more specific in vitro test system, a test method for evaluating vitiligo toxicity, melasma toxicity, or both of them is provided, which comprises the following steps (a) to (d).
(A) Hairless RET mouse obtained by returning and mating a hairless mouse having a genetic background of C57BL / 6 to a RET / ^o -transgenic mouse in which the activated RET gene has been introduced into a heteromorphic form, or the mouse thereof. A step of preparing a sample containing skin melanosite of a hairless RET mouse obtained by mating brothers and sisters and dividing it into a test group and a control group (b) A step of culturing the test group in the presence of a test substance (c) Step (b) ) C-Met, RFP-RET, c-Ret, Enothelin receptor B (Ednrb), c-kit for the later test group and the control group cultured under the same conditions except that the test substance was absent. , Mc1r, Mitf-m, Tyrosinase, Tyrosinase-realted protein-2, TGF-beta type receptor 2 (Tgfr2), Fibroblast growth factor (Fgfr), Bcl2, p53, survivin, CCAAT-enhancer-binding protein homologous protein (CHOP) , Glutamate cysteine ligase (GCLC), NF-E2 related factor2 (Nrf2), Heat shock protein (HSP) 70, Foxp1, Foxp3, CCR6, Bach2, NACHT leucine-rich-repeat protein 1 (Nalp), Cytotoxic T-lymphocyte gene -4 Steps to detect the expression or activity of one or more marker molecules selected from the group consisting of (Ctla4) and Granzyme B (GZMB) (d) Compare the detection results between the test group and the control group, and compare the results. Steps to determine white spot toxicity and / or scab toxicity of the test substance based on

In step (a), a hairless RET mouse (that is, a RET / ^o -transgenic mouse in which an activated RET gene has been introduced into a heteromorphic gene) is crossed back with a hairless mouse having a genetic background of C57BL / 6. Prepare a sample containing the skin melanosite of the obtained hairless RET mouse or the hairless RET mouse obtained by mating the mouse with brothers and sisters. Since the hairless RET mouse is as described above (1. column of test method for vitiligo toxicity), detailed description thereof will be omitted. As the sample here, cells (skin melanosite alone or in combination with other cells constituting the skin tissue) or tissue pieces (skin tissue, epidermis tissue, or dermis tissue) can be used. In the former case, cell culture (single culture or co-culture of melanocytes) is performed, and in the latter case, organ culture is performed. The sample used in step (a) can be collected or isolated from the back skin or tail skin of hairless RET mice by a known method. The following is an example of the procedure for isolating and culturing (primary culture) of skin melanocyte cells of hairless RET mice.

Use the back skin or tail skin of 0.5-day-old hairless RET mice. The isolated back or tail skin is washed with calcium-magnesium-free phosphate buffer, soaked in 0.25% trypsin solution and allowed to stand overnight at 2 ° C. Peel off the epidermis and dermis with tweezers. Only the peeled epidermis sheet is treated with 0.02% EDTA solution at 37 ° C. for 10 minutes, and the cells are dissected by pipetting. Remove the remaining keratin sheet with tweezers and centrifuge to obtain cell pellets. After suspending the cell pellet in Ham's F-10 medium, incubate in melanocyte selective medium at 37 ° C. and 5% CO ₂ (medium composition is shown below). By changing the medium composition, pure cultures of "undifferentiated melanocytes", "differentiated melanocytes", "proliferative undifferentiated melanocytes", and "proliferative differentiated melanocytes" can be obtained (Hirobe T. Melanocyte stimulating hormone induces the differentiation of mouse). epidermal melanocytes in serum-free culture. J Cell Physiol. 152 (2): 337-45, 1992 .; Hirobe T. Endothelins are involved in regulating the proliferation and differentiation of mouse epidermal melanocytes in serum-free primary culture. J Investig Dermatol Symp Proc. 6 (1): 25-31, 2001.). Any melanocyte-based cell may be used, but more commonly used melanocytes (differentiated melanocytes or proliferative differentiated melanocytes) are preferably used. Among the melanocytes, proliferative differentiated melanocytes are suitable for evaluation of many test substances, and can be said to be particularly preferable. As the skin melanocyte-based cells used as a sample, cells after subculture may be used.

<Type and composition of medium>
Medium A: Undifferentiated melanocyte medium
Ham's F-10 Medium Insulin (10 μg / ml)
Bovine serum albumin (1 mg / ml)
Ethanolamine (1 μM)
Phosphoethanolamine (1 μM)
Sodium selenite (10 nM)
Penicillin G (100 U)
Streptomycin sulfate (100 μg / ml)
Gentamycin sulfate (50 μg / ml)
Amphotericin B (0.25 μg / ml)

Medium B: Differentiated melanocyte medium A medium A supplemented with α-melanocyte stimulating hormone (α-MSH) (100 nM).

Medium C: Proliferative Differentiated Melanocyte Medium Medium A supplemented with Dibutyryl adenosine 3-cyclic monophosphate (DBcAMP) (0.5 mM)

Medium D: Proliferative undifferentiated melanocyte medium Medium C with basic fibroblast growth factor (bFGF) (2.5 ng / ml) added.

The prepared sample is divided into a test group and a control group, and each is cultured under predetermined conditions (step (b)). The test group is cultured in the presence of the test substance. In order to culture in the presence of the test substance, for example, the sample may be cultured and the test substance may be added to the culture solution or replaced with the culture solution to which the test substance has been added. Immediately after the start of culturing, the test substance may be added or replaced with a culture solution containing the test substance. Further, the culture solution to which the test substance is added in advance may be used so that the “state in which the test substance is present in the culture solution” is formed at the same time as the start of the culture. As in the case of the first aspect / the second aspect, various substances can be adopted as the test substance (for details, refer to the description of the test substance in the first aspect / the second aspect).

The culture time in the presence of the test substance is not particularly limited, but is, for example, 0.5 minutes to 5 days, preferably 30 minutes to 5 hours. The optimum culture time can be determined by a preliminary experiment.

For matters not mentioned in the present specification (medium, culture temperature, etc.), general culture conditions may be followed. The culture conditions may be determined with reference to past reports and books, or through preliminary experiments. The culture temperature is usually 37 ° C.

In step (c), c-Met, RFP-RET, c-Ret, Enothelin receptor B (Ednrb), c-kit, Mc1r, Mitf-m, Tyrosinase, Tyrosinase-realted protein-2 for the test group and the control group. , TGF-beta type receptor 2 (Tgfr2), Fibroblast growth factor (Fgfr), Bcl2, p53, survivalin, CCAAT-enhancer-binding protein homologous protein (CHOP), Glutamate cysteine ligase (GCLC), NF-E2 related factor2 (Nrf2) ), Heat shock protein (HSP) 70, Foxp1, Foxp3, CCR6, Bach2, NACHT leucine-rich-repeat protein 1 (Nalp), Cytotoxic T-lymphocyte antigen-4 (Ctla4) and Granzyme B (GZMB) Detects the expression or activity of one or more selected marker molecules. Preferably, c-Met and / or RFP-RET are used. A detection method suitable for the marker molecule to be adopted is adopted. For example, transcriptional activity (luciferase activity evaluation method, etc.), gene expression level (real-time PCR, etc.), protein expression level (Western blot, ELISA, immunohistochemistry, etc.), activation level such as phosphorylation (Western blot, ELISA, immunohistochemistry, etc.) (Tissue chemistry, etc.) is detected by a conventional method. If it is a secreted protein, the protein in the culture supernatant can be detected / quantified by ELISA or the like.

In step (d), the detection results are compared between the test group and the control group, and the vitiligo toxicity and / or melasma toxicity of the test substance is determined based on the comparison results. That is, in the present invention, the detection results of the test group and the target group are used to determine vitiligo toxicity, melasma toxicity, or both at the same time.

Among the marker molecules listed above, RFP-RET, c-Met, c-Ret, Ednrb, c-Kit, Mc1r, Mitf-m, Tyrosinase, TRP2, Tgfbeta-2, Fgfr, Bcl2, p53, survivalin, GCGL. , Nrf2, HSP70, Foxp1, Focp3, Bach2, Nalp, Ctla4 show a positive correlation between their expression level / activity and melanosis toxicity. Therefore, an increase in the expression level / activity of these marker molecules is an indicator of melasma toxicity, and typically, when the expression level / activity of the marker molecule is higher in the test group than in the control group. It will be determined that the test substance has melasma toxicity. On the other hand, CHOP, CCR6, and GZMB show a negative correlation between their expression level / activity and melasma toxicity. Therefore, a decrease in the expression level / activity of these marker molecules is an indicator of melasma toxicity, and typically, when the expression level / activity of the marker molecule is lower in the test group than in the control group. It will be determined that the test substance has melasma toxicity. The degree (level) of melasma toxicity may be evaluated based on the expression level / degree (level) of activity. When a plurality of test substances are used, the melasma toxicity of each test substance can be comparatively evaluated based on the degree of increase or decrease in the expression or activity of the marker molecule.

Among the marker molecules listed above, CHOP, CCR6, and GZMB show a positive correlation between their expression level / activity and vitiligo toxicity. Therefore, an increase in the expression level / activity of these marker molecules is an index of vitiligo toxicity, and typically, when the expression level / activity of the marker molecule is higher in the test group than in the control group, "test substance". Has vitiligo toxicity. " On the other hand, RFP-RET, c-Met, c-Ret, Ednrb, c-Kit, Mc1r, Mitf-m, Tyrosinase, TRP2, Tgfbeta-2, Fgfr, Bcl2, p53, survivalin, GCGL, Nrf2, HSP70, Foxp1, Focp3, Bach2, Nalp, and Ctla4 show a negative correlation between their expression level / activity and white spot toxicity. Therefore, a decrease in the expression level / activity of these marker molecules is an indicator of vitiligo toxicity, and typically, when the expression level / activity of the marker molecule is lower in the test group than in the control group, "test". The substance has vitiligo toxicity. " The degree of vitiligo toxicity (level) may be evaluated based on the expression level / degree of activity (level). When a plurality of test substances are used, the vitiligo toxicity of each test substance can be comparatively evaluated based on the degree of increase or decrease in the expression or activity of the marker molecule.

Preferably, c-Met is adopted as an index of vitiligo toxicity, and RFP-RET is adopted as an index of melasma toxicity. In one of the most preferable embodiments, these two indexes are used in combination to simultaneously determine and evaluate vitiligo toxicity and melasma toxicity.

As a further aspect of the in vitro test system, a melanosite cell line derived from a RET / ^o -transgenic mouse (hereinafter referred to as "RET-mouse") into which an activated RET gene has been introduced into a heterotype was used. A test method is provided. It can be said that this test method is simpler than the above test method in that it uses a melanocyte cell line that is easy to prepare in advance. The RET / ^o -transgenic mice into which the activated RET gene has been introduced into the heterozygous form are as described above (1. Column of test method for white spot toxicity), and detailed description thereof will be omitted. Mouse melanocyte cell lines can be established, for example, from cells isolated from skin tissue containing MET mouse melanocyte cell lines. More specifically, a method of organ-culturing skin tissue containing melanocyte-based cells collected from mice and repeating subculture of cells migrating outside the organ, trypsin of skin tissue containing melanocyte-based cells collected from mice, etc. A method of repeating subculture of cells dissociated by the above enzyme, a method of inducing immortalization using a viral gene such as Simian virus 40 (SV40) T antigen, and expression of telomea reverse transcription protein (TERT). A melanocyte cell line derived from RET-mouse can be established by an operation such as a method for inducing immortalization.

In this embodiment, the following steps (A)-(D) are typically performed to assess the vitiligo toxicity and / or melasma toxicity of the test substance.
(A) Prepare a melanosite cell line derived from a RET / ^o -transgenic mouse into which the activated RET gene has been introduced into a heterozygous form, and divide it into a test group and a control group. (B) The test group is a test substance. Steps for culturing in the presence (C) For the test group after step (B) and the control group cultivated under the same conditions except that the test substance was absent, c-Met, RFP-RET, c- Ret, Enothelin receptor B (Ednrb), c-kit, Mc1r, Mitf-m, Tyrosinase, Tyrosinase-realted protein-2, TGF-beta type receptor 2 (Tgfr2), Fibroblast growth factor (Fgfr), Bcl2, p53, survivin , CCAAT-enhancer-binding protein homologous protein (CHOP), Glutamate cysteine ligase (GCLC), NF-E2 related factor2 (Nrf2), Heat shock protein (HSP) 70, Foxp1, Foxp3, CCR6, Bach2, NATHT leucine-rich- Step to detect expression or activity of one or more marker molecules selected from the group consisting of repeat protein 1 (Nalp), Cytotoxic T-lymphocyte antigen-4 (Ctla4) and Granzyme B (GZMB) (D) Control with test group A step of comparing the detection results between the groups and determining the white spot toxicity and / or melanosis toxicity of the test substance based on the comparison results.

In step (A), a melanocyte cell line derived from RET-mouse is prepared and divided into a test group and a control group. Subsequently, each is cultured under predetermined conditions (step (B)). The culturing method, culturing time, etc. are the same as in step (b) of the above test method using skin melanocytes of hairless RET mice. Since steps (C) and (D) are the same as steps (c) and (d) of the above test method using skin melanocytes of hairless RET mice, the description thereof will be omitted.

5. A method for screening a drug for preventing or treating melasma or melasma A further aspect of the present invention provides a method for screening a drug for preventing or treating melasma or melasma. The first aspect of this aspect is a method for screening a drug for preventing or treating vitiligo, which comprises the following steps (a) to (c).
(A) Step of inducing vitiligo on the tail of a hairless mouse having melanin-producing ability (b) Step of administering a test substance to the hairless mouse (c) Step of determining a preventive effect or a therapeutic effect of the test substance

The term "drugs for preventing or treating vitiligo" is used not only for drugs used for the purpose of improving the symptoms of patients who have vitiligo, but also for those who may develop vitiligo. It is also used as a term that includes drugs used in (including the purpose of preventing recurrence). As described above, the drug obtained by utilizing the screening method of the present invention can be used for the purpose of prevention or treatment of vitiligo.

In step (a), a hairless mouse having a melanin-producing ability is prepared, and its tail is subjected to vitiligo-inducing treatment. Since the hairless mouse having the ability to produce melanin is the same as the first aspect, the description thereof will be omitted. The vitiligo-inducing treatment is carried out, for example, by application (application, dripping, injection, etc.) of a substance toxic to vitiligo (eg, rhododenol, raspberry ketone).

Examples of the method for administering the test substance in step (b) include application or dropping to the skin of the tail, oral administration, intravenous administration, intraarterial, subcutaneous, muscle, or intraperitoneal injection. The test substances include organic compounds of various molecular sizes (nucleic acids, peptides, proteins, lipids (simple lipids, complex lipids (phosphoglycerides, sphingolipids, glycosylglycerides, celebrosides, etc.), prostaglandins, isoprenoids, terpenes, steroids, etc.)). ) Or an inorganic compound can be used. The test substance may be of natural or synthetic origin. In the latter case, for example, an efficient screening system can be constructed by using a method of combinatorial synthesis. Nucleic acid / protein, cell extract containing lipid, culture supernatant and the like may be used as the test substance. Two or more kinds of test substances may be used in combination.

The administration timing (administration timing) of the test substance is not particularly limited, and the test substance is administered before the vitiligo-inducing treatment in step (a), simultaneously with the vitiligo-inducing treatment, or after the vitiligo-inducing treatment. The number of administrations is not particularly limited, and single or multiple administrations are performed.

In step (c), the therapeutic or preventive effect of the test substance is determined. A test substance determined to have a preventive or therapeutic effect is a promising drug candidate. The therapeutic or preventive effect can be determined by the presence or absence or degree of induction of vitiligo, or by evaluating both of them. Since the evaluation index and the evaluation method are the same as those in the first aspect, detailed description thereof will be omitted.

The second aspect of this aspect is a method for screening a drug for preventing or treating melasma, which comprises the following steps (A) to (C).
(A) A hairless RET mouse obtained by back-mating a hairless mouse having a genetic background of C57BL / 6 to a RET / ^o -transgenic mouse into which the activated RET gene has been introduced into a heterozygous form, or the mouse. (B) Step of administering the test substance to the hairless RET mouse (C) Test substance Steps to determine the preventive or therapeutic effect of

The term "melasma preventive or therapeutic drug" is used not only for drugs used for the purpose of improving the symptoms of patients with melasma, but also for the risk of developing melasma. It is used as a term that includes drugs that are used prophylactically (including the purpose of preventing recurrence) for people. As described above, the drug obtained by utilizing the screening method of the present invention can be used for the purpose of prevention or treatment of melasma.

In step (A), a hairless RET mouse obtained by re-mating a hairless mouse having a genetic background of C57BL / 6 to a RET / ^o -transgenic mouse in which the activated RET gene has been introduced into a heterozygous form. Alternatively, prepare a hairless RET mouse obtained by mating the mouse with a brother and sister (that is, a hairless mouse having a genotype of hr / hr; Ret / ^O and a genetic background of C57BL / 6), and its skin (for example, back, belly). Or the head) is treated to induce melanosis. Since the hairless RET mouse is the same as the second aspect, the description thereof will be omitted. The melasma-inducing treatment is carried out, for example, by application (application, dripping, injection, etc.) of a substance toxic to melasma (eg, rhododenol, raspberry ketone).

The method of administration of the test substance in step (B) (however, in the case of administration by application, dropping, etc., the site to be subjected to the melasma-inducing treatment is the application site), the administration time, the substance that can be adopted as the test substance, etc. Is the same as the first aspect.

In step (C), the therapeutic or preventive effect of the test substance is determined. A test substance determined to have a preventive or therapeutic effect is a promising drug candidate. The therapeutic or prophylactic effect can be determined by the presence or absence or degree of induction of melasma, or by evaluating both of them. Since the evaluation index and the evaluation method are the same as those in the second aspect, detailed description thereof will be omitted.

In the screening method of the present invention (first aspect and second aspect), a model animal group (test group) to which the test substance is administered and a model animal group under the same conditions except that the test substance is not administered are preferably used. (Control group) is prepared. Then, the test group and the control group are compared with respect to the adopted index, and a judgment (step (c), step (C)) is made based on the result. As a result of comparison, for example, when the onset of melasma (in the case of the first aspect) or melasma (in the case of the second aspect) is observed only in the control group, the test group has white spots (first aspect) than the control group. Therapeutic or preventive effect was observed in the test group, such as when the symptoms of melasma (in the case of the second aspect) did not progress or became serious, or when the test group showed a significant improvement in the symptoms. If so, it can be determined that the test substance is a promising candidate for a drug for preventing or treating vitiligo (in the case of the first aspect) or a drug for preventing or treating melasma (in the case of the second aspect). By comparing the group to which the test substance is administered (test group) and the group to which the test substance is not administered (control group) in this way, the effectiveness of the test substance can be easily determined with high reliability.

The number of individuals included in the test group and the control group is not particularly limited. Generally, the larger the number of individuals used, the higher the reliability of the results, but handling a large number of individuals at the same time involves difficulties in securing and operating (including breeding) the individuals to be used. Therefore, for example, the number of individuals included in each group is set to 1 to 50, preferably 2 to 30, and more preferably 5 to 20.

If the compound selected by the screening method of the present invention has sufficient efficacy against vitiligo (in the case of the first aspect) or melasma (in the case of the second aspect), the compound can be used as it is as a drug. It can be used as an active ingredient. On the other hand, if it does not have a sufficient medicinal effect, it is modified by chemical modification to enhance its medicinal effect, and then a prophylactic or therapeutic drug for vitiligo (in the case of the first aspect) or prevention or melanosis. The compound can be used as the active ingredient of the therapeutic agent (in the case of the second aspect). Of course, even if it has a sufficient medicinal effect, the same modification may be made for the purpose of further increasing the medicinal effect.

Matters not specifically mentioned in this specification (conditions, operation methods, etc.) may be subjected to conventional methods, such as Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York), Current protocols in molecular biology (edited by Frederick). M. Ausubel et al., 1987), Current protocols in Immunology, John Wiley & Sons Inc, etc. can be referred to.

A. Construction of toxicity evaluation system using hairless mice 1. As hairless hairless mice that do not require shaving, a hairless wild-type mouse (FIG. 1A) and a hairless RET mouse (FIG. 1B) were prepared. The production method of both mice is as follows.

(1) Preparation method of hairless wild-type mouse Hairless wild-type mouse (Hos: HRM: CBA / J x C57BL / 6J Jms background) that can produce melanin with hairless (hr / hr) purchased from Hoshino Test Animal Breeding Center) A hairless wild-type mouse (C57BL / 6 background) was prepared by back-crossing C57BL / 6 mice 7 times, and then bred by 10 or more sibling matings.

(2) Method for producing hairless RET mice Previously, the research group of the present inventors had 192 lines (once mated with BCF1 mice and then once with C57BL / 6) and 242 lines (once mated with BCF1 mice). After that, we developed RET / ^o -transgenic mice of 304 strains (1 mating with C57BL / 6 and then 6 backcrosses with C57BL / 6) (Iwamoto et al., 1991; Kato et al., 1998). Hairless RET mice (C57BL / 6 background) were prepared by backcrossing these 3 strains of RET / ^o -transgenic mice 7 times with the hairless wild-type mice (C57BL / 6 background) prepared in (1). Made. After that, hairless RET mice bred by sibling mating 10 times or more were used in the following experiments.
(3) Characteristics of each mouse Of the mice produced by the above method, 192 strains and 304 strains develop tumors, so 242 strains of hairless RET mice are preferably used. FIG. 1 shows the back (left) and tail (right) of a hairless wild-type mouse (A) and a 242 line hairless RET mouse (B). In the back skin of hairless wild-type mice, blackness of the skin is hardly confirmed with the naked eye. On the other hand, in hairless RET mice, the entire back skin is black. The L * value, which indicates skin brightness (whiteness) measured by a color difference meter (Konica Minolta CR400), was shown to be statistically significantly smaller in hairless RET mice than in hairless wild-type mice (). FIG. 1C). Regarding the skin of the tail, a certain blackness can be confirmed even in the hairless wild-type mouse, but the skin color of the hairless RET mouse is darker (FIGS. 1A and 1B).

The hairless RET mice (C57BL / 6 background) prepared by the above method are 242 hairless RET mice (CBA / J) prepared by mating once with the previously reported hairless RET mice (Hos: HRM mice). x C57BL / 6J Jms background. See Japanese Patent Application Laid-Open No. 2013-106554) is another strain with a different genetic background. The difference between the two strains appears as a difference in the darkness of the skin, and the above method is used. The prepared hairless RET mice have weak skin blackness. This feature affects the sensitivity in the evaluation of melanosis toxicity (it works favorably).

2. 2. External study using low concentrations of rhododenol and raspberry ketone 2% dissolved in 70% ethanol on the back and tail skin of hairless wild-type mice (C57BL / 6 background) and hairless RET mice (C57BL / 6 background) Rhododenol or 2% raspberry ketone was applied externally once daily and 5 times a week (test group). Only the solvent (70% ethanol) was externally applied to the control group (control). The external dose was 9.0 μl / cm ² .

(1) Evaluation by visual inspection, color difference meter and image analysis (1-1) Evaluation result of back skin
About 3 months after the first external application of 2% rhododenol or 2% raspberry ketone, melasma was induced in the back skin of hairless RET mice (Fig. 2A). On the other hand, in the back skin of hairless wild-type mice, no change in skin color was observed in any skin to which 2% rhododenol or 2% raspberry ketone was applied externally (Fig. 2A). From the results of the objective evaluation based on the L ^* value measured by the color difference meter, the level of the L ^* value was higher in the back skin of the hairless RET mouse to which 2% rhododenol or 2% raspberry ketone was externally applied than in the back skin to which the solvent was externally applied. It was shown to be significantly lower (skin is darker) (Fig. 2B). On the other hand, in hairless wild-type mice, there was no significant difference in L ^* values between the skin to which the solvent was externally applied and the skin to which 2% rhododenol or 2% raspberry ketone was applied externally (Fig. 2B).

(1-2) Evaluation results of tail skin About 1 month after the start of external application, depigmentation was observed in the tail skin of hairless wild-type mice by external application of 2% rhododenol or 2% raspberry ketone (Fig. 2C). Depigmentation could not be observed macroscopically on the back skin of the tail of hairless RET mice 1 month after external application, but about 3 months after the start of external application, it was removed by external application of 2% rhododenol or 2% raspberry ketone. A dye was observed (Fig. 2C). Since it was difficult to measure the tail skin with a color difference meter due to the structural restrictions of the device, an objective evaluation was performed by quantifying the blackness of the skin with image analysis software (WinROOF) after taking a photograph. As a result, it was shown that the darkness of the tail skin to which 2% rhododenol or 2% raspberry ketone was externally applied was significantly lower than that of the tail skin to which the solvent was externally applied in both the hairless wild-type mouse and the hairless RET mouse. (Fig. 2D).

(2) Evaluation by pathological analysis Next, pathology to confirm whether melanosis and depigmentation induced by 2% rhododenol or 2% raspberry ketone are similar to the pathology of human melanosis / melasma. Analysis was performed (Figs. 3 and 4). Decreased / disappeared melanin pigments and decreased / disappeared melanocyte counts have been reported in human vitiligo. Little information is available at this time regarding melanin and melanocytes in rhododenol- and raspberry ketone-induced melasma, but in melasma, an increase in melanin pigment is generally confirmed, and the number of melanocytes remains unchanged. Increasing cases have been reported. In order to detect skin melanin, skin paraffin sections were prepared and stained with Fontana Masson. In addition, in order to detect melanocytes, immunohistochemistry using an antibody against Dopachromet automerase (Dct), which is a melanocyte-specific antigen, was performed.

(2-1) Results of evaluation of back skin In both histological observation and objective evaluation by WinROOF, the back skin of hairless wild-type mice to which 2% rhododenol or 2% raspberry ketone was externally applied and the back skin to which a solvent was externally applied were used. No significant difference in melanin distribution and melanin density was observed between them (Fig. 3A, B). On the other hand, in the back skin of hairless RET mice in which melasma was observed, histological observation and objective evaluation by WinROOF showed that the melanin density was significantly higher than that of the back skin to which the solvent was externally applied (Fig.). 3A, B). In addition, in hairless wild-type mice, no change in the number of melanocytes due to external application of 2% rhododenol or 2% raspberry ketone was observed (Fig. 3C, D), but in hairless RET mice, skin with external application of 2% rhododenol or 2% raspberry ketone was observed. A clear increase in the number of melanocytes (about twice the number of melanocytes in the skin to which the solvent was applied externally) was observed in (Fig. 3C, D).

(2-2) Evaluation result of tail skin
Histology that the melanin density in the tail skin of hairless wild-type mice and hairless RET mice confirmed to be depigmented by external application of 2% rhododenol or 2% raspberry ketone was significantly lower than that of the tail skin to which the solvent was applied externally. It was clarified by scientific observation and objective evaluation by WinROOF (Figs. 4A and 4B). In addition, histological observations and objective evaluation by WinROOF showed that the number of melanocytes in the tail skin externally applied with 2% rhododenol or 2% raspberry ketone was significantly smaller than the number of melanocytes in the tail skin externally applied with a solvent. (FIGS. 4C, D).

(3) Discussion-It has become possible to evaluate vitiligo toxicity and melasma toxicity using low-concentration chemical substances contained in actual whitening agents. Hairless wild-type mice can be evaluated for vitiligo toxicity in the tail skin, and hairless RET mice can be evaluated for vitiligo toxicity in the tail skin and melanosis toxicity in the back skin at the same time.
・ Since vitiligo was induced in hairless wild-type mice by an external application period of about 1 month and in hairless RET mice by an external application period of about 3 months, the test using hairless wild-type mice assumed a more sensitive human. It is especially useful as a vitiligo toxicity evaluation system. On the other hand, the test using hairless RET mice can be said to be particularly useful as a vitiligo toxicity evaluation system assuming a type of human who develops vitiligo over a relatively long period of time. If hairless wild-type mice and hairless RET mice are evaluated in combination, toxicity evaluation can be performed in humans with different susceptibility.
-The evaluation system using hairless wild-type mice and hairless RET mice does not require secondary stimuli such as shaving and UV irradiation, so it is possible to centrally evaluate the toxicity of chemical substances and it is highly reliable. Evaluation results are obtained.
-Hairless wild-type mice and hairless RET mice were able to evaluate not only rhododenol but also raspberry ketone vitiligo and melasma toxicity. This fact indicates that an evaluation system using a hairless wild-type mouse or a hairless RET mouse has excellent versatility and can be used for toxicity evaluation of a wide variety of chemical substances.

B. Construction of in vitro toxicity evaluation system With the aim of establishing an evaluation system that can evaluate vitiligo toxicity and melasma toxicity more easily and in a short time, melanosis due to exposure to rhododenol using cell lines isolated and established from RET-mice. Changes in the expression of related genes and vitiligo-related genes were investigated. It has been reported that the RFP-RET gene has been introduced into RET-mice, which causes melasma (Non-Patent Document 5). Therefore, using the cell line isolated from this model, the toxicity of melasma was evaluated using the RFP-RET gene expression as an index. On the other hand, for the evaluation of vitiligo toxicity, we focused on the c-Met gene. c-Met is a receptor tyrosine kinase and is a representative molecule involved in the proliferation and function of human melanocytes (Non-Patent Document 6).

1. 1. Method
Melanocyte cell lines isolated from RET-mice were seeded on 6-well plates at a density of 1.0 x 10 ⁵ / well. Rhododenol was dissolved in dimethyl sulfoxide (DMSO) to a concentration 1000 times the final concentration, and 1/1000 of them were added to the culture medium (0.1% DMSO). After exposing the melanocyte cell line to rhododenol for 5 hours, the mRNA expression of RFP-RET, c-Met, and Hprt was analyzed by quantitative PCR.

2. 2. result
The addition of 0.2% rhododenol increased the mRNA expression level of RFP-RET about 2-fold. In addition, the mRNA expression level of c-Met decreased to about 30% by adding 0.00002% rhododenol (Fig. 5).

3. 3. Discussion (1) Since exposure to rhododenol increased the mRNA expression level of RFP-RET, which is a melasma marker, and decreased the mRNA expression level of c-Met, which is a survival marker of melanocytes, this test method should be used. It was shown that the toxicity of rhododenol-induced melasma and melasma toxicity can be evaluated at the same time.
(2) The prior art (Non-Patent Documents 1, 7, and 8) relating to the evaluation of toxicity by rhododenol using an in vitro test system is a test limited to the evaluation of vitiligo toxicity. In contrast, according to this evaluation system, vitiligo toxicity and melasma toxicity can be evaluated at the same time.
(3) In humans, rhododenol not only exhibits vitiligo toxicity, but it has been reported that about 38% of patients develop melasma with pigmentation (Non-Patent Document 2). Since this evaluation method can evaluate not only vitiligo toxicity but also melasma toxicity, it is possible to evaluate toxicity more suitable for human cases.
(4) As in the case of humans, 2% rhododenol induced vitiligo toxicity and melasma toxicity in hairless wild-type mice and hairless RET mice, but in this evaluation system, about 1/10000 (c-Met is used as an index). It was possible to evaluate the toxicity with the sensitivity of about 1/10 (toxicity of melanosis using RFP-RET as an index). Compared with the past reports (Non-Patent Document 8) that evaluated vitiligo toxicity most sensitively in the in vitro test system, this evaluation system is about 50 times more sensitive.
(5) In the past in vitro test system, evaluation was performed after 24-hour or 48-hour exposure (Non-Patent Documents 1, 7, and 8), but in this evaluation system, toxicity can be evaluated even by exposure for only 5 hours. be.

5. summary
By using the strain cells established from RET-mice, it became possible to evaluate vitiligo toxicity and melasma toxicity at the same time with high sensitivity and in a short time. By constructing an in vitro evaluation system, more test substances can be evaluated in a short period of time. In addition, it is possible to respond to various needs such as in vitro evaluation only, in vivo evaluation only, or both, and it is more reliable by comprehensively evaluating the results of in vitro evaluation and in vivo evaluation. Highly toxic evaluation is possible.

According to the test method of the present invention, vitiligo toxicity / melasma toxicity can be evaluated with high sensitivity. The test method of the present invention is highly versatile and can be used for toxicity evaluation of various substances. For example, the present invention is useful as a means for confirming safety in the development stage of cosmetics, pharmaceuticals and the like.

The present invention is not limited to the description of the embodiments and examples of the above invention. Various modifications are also included in the present invention to the extent that those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of the papers, published patent gazettes, patent gazettes, etc. specified in this specification shall be cited by reference in their entirety.

Claims (20)

A test method for vitiligo toxicity, which comprises the following steps (1) and (2):
(1) A step of applying the test substance to the tail of a hairless mouse having melanin-producing ability,
(2) A step of evaluating the presence / absence and / or degree of induction of vitiligo in the tail of the hairless mouse, and determining the vitiligo toxicity of the test substance based on the evaluation result. The test method according to claim 1, wherein the hairless mouse is a hairless mouse having a normal melanin-producing ability. The test method according to claim 1, wherein the genetic background of the hairless mouse is C57BL / 6. The hairless mouse is a hairless RET mouse obtained by back-mating a hairless mouse having a genetic background of C57BL / 6 to a RET / ^o -transgenic mouse in which an activated RET gene has been introduced into a heterozygous form. The test method according to claim 3, which is a hairless RET mouse obtained by mating the mouse with a brother and sister. Claimed that the hairless mouse having a genetic background of C57BL / 6 is a mouse obtained by backcrossing a C57BL / 6 mouse to a Hos: HRM hairless mouse, or a mouse obtained by mating the mouse with a brother and sister. The test method according to 4. Test method for melasma toxicity, including the following steps (1) and (2):
(1) A hairless RET mouse obtained by returning a hairless mouse having a genetic background of C57BL / 6 to a RET / ^o -transgenic mouse into which the activated RET gene has been introduced into a heterozygous form, or the mouse. The step of applying the test substance to the skin of hairless RET mice (excluding the skin of the tail) obtained by mating brothers and sisters.
(2) A step of evaluating the presence / absence and / or degree of induction of melasma in the skin of the hairless RET mouse, and determining the toxicity of the test substance to melasma based on the evaluation result. The test method according to claim 6, wherein the skin is the skin of the back, abdomen or head. Simultaneous test method for vitiligo toxicity and melasma toxicity, including the following steps (1) to (3):
(1) A hairless RET mouse obtained by returning a hairless mouse having a genetic background of C57BL / 6 to a RET / ^o -transgenic mouse into which the activated RET gene has been introduced into a heterozygous form, or the mouse. The step of applying the test substance to the tail and skin (excluding the skin of the tail) of a hairless RET mouse obtained by mating brothers and sisters.
(2) A step of evaluating the presence / absence and / or degree of induction of vitiligo in the tail of the hairless RET mouse, and determining the vitiligo toxicity of the test substance based on the evaluation result.
(3) A step of evaluating the presence / absence and / or degree of induction of melasma in the skin of the hairless RET mouse, and determining the toxicity of the test substance to melasma based on the evaluation result. The test method according to claim 8, wherein the skin is the skin of the back, abdomen or head. The test method according to any one of claims 4, 6 to 9, wherein the activated RET gene is an RFP-RET gene generated by a hybrid of a c-RET gene and an RFP gene. The test method according to any one of claims 4, 6 to 10, wherein the activated RET gene is linked to the metallothionein I promoter. The RET / ^o -transgenic mouse is 192 line RET / ^o -transgenic mouse, 242 line RET / ^o -transgenic mouse, 304 line RET / ^o -transgenic mouse, or 304 / B6 line RET / ^o -trans. The test method according to any one of claims 4 and 6 to 11, which is a genetic mouse. Mice susceptible to vitiligo and melasma toxicity: consisting of the following (i) or (ii) mice:
(I) A hairless RET mouse obtained by backcrossing a hairless mouse having a genetic background of C57BL / 6 to a RET / ^o -transgenic mouse in which the activated RET gene has been introduced into a heteromorphic form.
(Ii) A hairless RET mouse obtained by mating the mice of (i) with brothers and sisters, wherein the RET / ^o -transgenic mouse is a 192 line RET / ^o -transgenic mouse or a 242 line RET / ^o- . Transgenic mouse,
Sensitive mouse. The susceptible mouse according to claim 13, wherein the activated RET gene is an RFP-RET gene generated by a hybrid of a c-RET gene and an RFP gene. The susceptible mouse according to claim 13 or 14, wherein the activated RET gene is linked to the metallothionein I promoter. A hairless RET mouse obtained by returning a hairless mouse having a genetic background of C57BL / 6 to a RET / ^o -transgenic mouse into which the activated RET gene has been introduced into a heteromorphic gene, or a brother-sister mating of the mouse. An in vitro study characterized by evaluating the melanosis toxicity of a test substance using melanosite or its passage cells collected from the skin (excluding the tail skin) of the hairless RET mouse thus obtained. Method. An in vitro test method for assessing vitiligo toxicity and / or melasma toxicity, comprising the following steps (a)-(d):
(A) A hairless RET mouse obtained by returning a hairless mouse having a genetic background of C57BL / 6 to a RET / ^o -transgenic mouse into which the activated RET gene has been introduced into a heterozygous form, or the mouse. A step of preparing a sample containing the skin melanosite of a hairless RET mouse obtained by mating brothers and sisters and dividing it into a test group and a control group.
(B) The step of culturing the test group in the presence of the test substance,
(C) For the test group after step (b) and the control group cultured under the same conditions except that the test substance was absent, c-Met, RFP-RET, c-Ret, Enothelin receptor B ( Ednrb), c-kit, Mc1r, Mitf-m, Tyrosinase, Tyrosinase-realted protein-2, TGF-beta type receptor 2 (Tgfr2), Fibroblast growth factor (Fgfr), Bcl2, p53, survivin, CCAAT-enhancer-binding protein homologous protein (CHOP), Glutamate cysteine ligase (GCLC), NF-E2 related factor 2 (Nrf2), Heat shock protein (HSP) 70, Foxp1, Foxp3, CCR6, Bach2, NATHT leucine-rich-repeat protein 1 (Nalp) ), Steps to detect the expression or activity of one or more marker molecules selected from the group consisting of Cytotoxic T-lymphocyte antigen-4 (Ctla4) and Granzyme B (GZMB),
(D) A step of comparing the detection results between the test group and the control group, and determining the vitiligo toxicity and / or melasma toxicity of the test substance based on the comparison results. In vitro test methods for assessing vitiligo toxicity and / or melasma toxicity, comprising the following steps (A)-(D):
(A) A step of preparing a melanocyte cell line derived from a RET / ^o -transgenic mouse into which an activated RET gene has been introduced into a heterozygous form and dividing it into a test group and a control group.
(B) The step of culturing the test group in the presence of the test substance,
(C) For the test group after step (B) and the control group cultured under the same conditions except that the test substance was absent, c-Met, RFP-RET, c-Ret, Enothelin receptor B ( Ednrb), c-kit, Mc1r, Mitf-m, Tyrosinase, Tyrosinase-realted protein-2, TGF-beta type receptor 2 (Tgfr2), Fibroblast growth factor (Fgfr), Bcl2, p53, survivin, CCAAT-enhancer-binding protein homologous protein (CHOP), Glutamate cysteine ligase (GCLC), NF-E2 related factor 2 (Nrf2), Heat shock protein (HSP) 70, Foxp1, Foxp3, CCR6, Bach2, NATHT leucine-rich-repeat protein 1 (Nalp) ), Steps to detect the expression or activity of one or more marker molecules selected from the group consisting of Cytotoxic T-lymphocyte antigen-4 (Ctla4) and Granzyme B (GZMB),
(D) A step of comparing the detection results between the test group and the control group, and determining the vitiligo toxicity and / or melasma toxicity of the test substance based on the comparison results. A method for screening a drug for preventing or treating vitiligo, which comprises the following steps (a) to (c):
(A) A step of inducing vitiligo on the tail of a hairless mouse capable of producing melanin,
(B) The step of administering the test substance to the hairless mouse,
(C) A step of determining the prophylactic or therapeutic effect of the test substance. A method for screening a drug for preventing or treating melasma, which comprises the following steps (A) to (C):
(A) A hairless RET mouse obtained by re-mating a hairless mouse having a genetic background of C57BL / 6 to a RET / ^o -transgenic mouse in which the activated RET gene has been introduced into a heterozygous form, or the mouse. A step of inducing melanosis on the skin (excluding the tail skin) of a hairless RET mouse obtained by mating brothers and sisters.
(B) The step of administering the test substance to the hairless RET mouse,
(C) A step of determining the preventive or therapeutic effect of the test substance.

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