JP7590751B2 - Method for exposing poorly water-soluble test substances to suspension culture cells - Google Patents
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Description
本発明は、浮遊系培養細胞への難水溶性被験物質の曝露方法に関する。 The present invention relates to a method for exposing suspension cultured cells to a poorly water-soluble test substance.
培養細胞へ被験物質を曝露する場合、被験物質を培地へ直接添加するか、あるいは被験物質を水や生理食塩水等の水溶性溶媒に溶解させて培地へ添加する方法がある。また、被験物質が難水溶性の場合、被験物質をジメチルスルホキシド（ＤＭＳＯ）やエタノール等の有機溶媒に溶解させ、培地へ添加する方法がある。しかしながら、これらの有機溶媒は、培地中の濃度が高くなると細胞毒性を呈するため、予め最終濃度の１００倍から１０００倍になるように被験物質を有機溶媒に溶解し、培地の総量に対して０．１重量％から１．０重量％になるように添加する必要がある。そのため、被験物質の有機溶媒への溶解性が低い場合、最終濃度を高く設定できない場合がある。 When exposing cultured cells to a test substance, the test substance can be added directly to the medium, or the test substance can be dissolved in a water-soluble solvent such as water or physiological saline and then added to the medium. If the test substance is poorly water-soluble, the test substance can be dissolved in an organic solvent such as dimethyl sulfoxide (DMSO) or ethanol and then added to the medium. However, these organic solvents become cytotoxic when their concentration in the medium becomes high, so the test substance must be dissolved in the organic solvent in advance to 100 to 1000 times the final concentration, and then added to the medium at 0.1 to 1.0% by weight relative to the total volume of the medium. Therefore, if the test substance has low solubility in organic solvents, it may not be possible to set the final concentration high.
また、被験物質が水にも有機溶媒にも溶解しない場合は、被験物質を培地に分散させる方法がある。しかしながら、被験物質が培地の上部に浮遊したり、培地の下部に沈んだりする場合、被験物質が細胞と接触しているのかどうかはっきりしない場合がある。特に、浮遊系培養細胞を用いた場合は、被験物質と細胞が共に培地中で一定の位置を保つことができないため、被験物質と細胞が接触しているのかどうか猶更はっきりしない。そのため、浮遊系培養細胞を用いて難水溶性被験物質の安全性評価を行う際、本来は陽性であるものが陰性と判定される場合があり（非特許文献１）、改良が望まれていた。 In addition, when the test substance is not soluble in either water or organic solvent, there is a method of dispersing the test substance in a culture medium. However, when the test substance floats on the top of the culture medium or sinks to the bottom of the culture medium, it may not be clear whether the test substance is in contact with the cells. In particular, when suspension culture cells are used, the test substance and the cells cannot maintain a fixed position in the culture medium, making it even more unclear whether the test substance and the cells are in contact. Therefore, when safety evaluation of poorly water-soluble test substances is performed using suspension culture cells, there are cases where what should be positive is judged to be negative (Non-Patent Document 1), and improvements were desired.
難水溶性被験物質を培養細胞へ曝露する方法としては、これまでに、１分子内に正電荷と負電荷の両方を有する双性イオンを含む培地用添加剤を用いる方法（特許文献１）、可逆的にゲル化可能な生体適合性ゲルに細胞を包埋し、細胞を含むゲルと難水溶性の被験物質を接触させる方法（特許文献２）、温度感受性ハイドロゲル培地に難水溶性被験物質を浸潤させることにより、該被験物質と培地中のヒト単核球細胞を曝露させる方法（特許文献３）、液体パラフィンに難水溶性被験物質を溶解させ、細胞と短時間接触させる方法（非特許文献２）等が提案されてきた。しかしながら、特許文献１の手法は、双性イオンが細胞へ影響することによって評価結果に影響を及ぼす場合があり、特許文献２及び３の手法は、難水溶性被験物質が、培地のゲル化に影響を及ぼし、評価結果に影響を及ぼす場合があった。また、非特許文献２の手法では、液体パラフィンに溶解できない被験物質を適切に評価することができない。 As methods for exposing poorly water-soluble test substances to cultured cells, there have been proposed a method using a medium additive containing a zwitterion having both positive and negative charges in one molecule (Patent Document 1), a method of embedding cells in a reversibly gelling biocompatible gel and contacting the gel containing the cells with a poorly water-soluble test substance (Patent Document 2), a method of exposing human mononuclear cells in a temperature-sensitive hydrogel medium to a poorly water-soluble test substance by infiltrating the poorly water-soluble test substance into the medium (Patent Document 3), and a method of dissolving a poorly water-soluble test substance in liquid paraffin and contacting it with cells for a short period of time (Non-Patent Document 2). However, the method of Patent Document 1 may affect the evaluation results by the effect of the zwitterion on the cells, and the methods of Patent Documents 2 and 3 may affect the evaluation results by the poorly water-soluble test substance affecting the gelation of the medium. In addition, the method of Non-Patent Document 2 cannot properly evaluate test substances that cannot be dissolved in liquid paraffin.
本発明は、浮遊系培養細胞への難水溶性被験物質の曝露方法を提供することを課題とする。 The objective of the present invention is to provide a method for exposing poorly water-soluble test substances to suspension culture cells.
即ち、本発明は、浮遊系培養細胞への難水溶性被験物質の曝露方法であって、（Ａ）難水溶性被験物質、（Ｂ）２５℃で液状の炭化水素及び（Ｃ）２５℃で固型状且つ７０℃で液状の炭化水素を７０℃以上に加熱溶解させた後に冷却して固型物を得た後、該固型物の上から浮遊系培養細胞の懸濁液を適用し、一定時間放置することを特徴とする、浮遊系培養細胞への難水溶性被験物質の曝露方法を提供するものである。 In other words, the present invention provides a method for exposing suspension cultured cells to a poorly water-soluble test substance, which comprises heating and dissolving (A) the poorly water-soluble test substance, (B) a hydrocarbon that is liquid at 25°C, and (C) a hydrocarbon that is solid at 25°C and liquid at 70°C to 70°C or higher, cooling the mixture to obtain a solid, applying a suspension of suspension cultured cells onto the solid, and allowing the mixture to stand for a certain period of time.
また、本発明は、（Ａ）難水溶性被験物質のオクタノール／水分配係数が３．５以上である浮遊系培養細胞への難水溶性被験物質の曝露方法を提供するものである。 The present invention also provides (A) a method for exposing a poorly water-soluble test substance to suspension culture cells, the test substance having an octanol/water partition coefficient of 3.5 or more.
また、本発明は、（Ｂ）２５℃で液状の炭化水素がスクワランである浮遊系培養細胞への難水溶性被験物質の曝露方法を提供するものである。 The present invention also provides (B) a method for exposing a poorly water-soluble test substance to suspension cultured cells, in which the hydrocarbon that is liquid at 25°C is squalane.
また、本発明は、（Ｃ）２５℃で固型状且つ７０℃で液状の炭化水素が４８～６９℃に融点を有するパラフィンワックスである浮遊系培養細胞への難水溶性被験物質の曝露方法を提供するものである。 The present invention also provides (C) a method for exposing a poorly water-soluble test substance to suspension culture cells, the method being paraffin wax, a hydrocarbon that is solid at 25°C and liquid at 70°C and has a melting point of 48 to 69°C.
また、本発明は、（Ｃ）２５℃で固型状且つ７０℃で液状の炭化水素の含有量が、（Ａ）＋（Ｂ）＋（Ｃ）の総量に対して５０重量％以上である浮遊系培養細胞への難水溶性被験物質の曝露方法を提供するものである。 The present invention also provides a method for exposing suspension cultured cells to a poorly water-soluble test substance, in which the content of (C) a hydrocarbon that is solid at 25°C and liquid at 70°C is 50% by weight or more relative to the total amount of (A) + (B) + (C).
また、本発明は、上記曝露方法を用いた難水溶性皮膚感作性物質のｉｎ ｖｉｔｒｏ評価法であり、浮遊系培養細胞の一種であるヒト単球性白血病由来（ＴＨＰ－１）細胞上に発現される細胞表面マーカーＣＤ８６及びＣＤ５４の発現量を指標にした、難水溶性皮膚感作性物質のｉｎ ｖｉｔｒｏ評価法を提供するものである。 The present invention also provides an in vitro evaluation method for poorly water-soluble skin sensitizers using the above-mentioned exposure method, in which the expression levels of cell surface markers CD86 and CD54 expressed on human monocytic leukemia-derived (THP-1) cells, a type of suspension cultured cell, are used as indicators.
本発明により、これまで困難であった浮遊系培養細胞への難水溶性被験物質の曝露が可能となり、難水溶性皮膚感作性物質のｉｎ ｖｉｔｒｏ評価を適切に実施することが可能となる。 The present invention makes it possible to expose poorly water-soluble test substances to suspension culture cells, which was previously difficult, and enables appropriate in vitro evaluation of poorly water-soluble skin sensitizing substances.
本発明における浮遊系培養細胞とは、血液、髄液、リンパ球等の体液中に浮遊して存在している細胞を指し、例えば白血球、骨髄細胞、骨髄幹細胞、造血幹細胞、間葉系細胞等を指す。具体的には、ヒト単球性白血病由来（ＴＨＰ－１）細胞、ヒトＴ細胞性白血病由来（Ｊｕｒｋａｔ）細胞、ヒトリンパ芽球由来（ＴＫ６）細胞、ヒト前骨髄性白血病由来（ＨＬ－６０）細胞等が挙げられる。 In the present invention, the term "suspension culture cells" refers to cells that exist in suspension in bodily fluids such as blood, cerebrospinal fluid, and lymphocytes, and includes, for example, leukocytes, bone marrow cells, bone marrow stem cells, hematopoietic stem cells, and mesenchymal cells. Specific examples include human monocytic leukemia-derived (THP-1) cells, human T-cell leukemia-derived (Jurkat) cells, human lymphoblast-derived (TK6) cells, and human promyelocytic leukemia-derived (HL-60) cells.
本発明における（Ａ）難水溶性被験物質とは、水にもＤＭＳＯやエタノール等の有機溶媒にも溶解しない被験物質を指す。本発明は、難水溶性被験物質のオクタノール／水分配係数が３．５以上の場合、特に有用であり、例えば、サリチル酸ヘキシル、Ｎ，Ｎ－ジブチルアニリン、１－クロロメチルピレン、クロトリマゾール、安息香酸ベンジル等が挙げられる。尚、オクタノール／水分配係数とは、ある化学物質のｎ－オクタノールおよび水への分配度の比（ｎ－オクタノール相及び水相中における濃度の比）であり、例えばＯＥＣＤガイドライン１０７に規定されるフラスコ振とう法、ＯＥＣＤガイドライン１１７に規定されるＨＰＬＣ法、あるいは量子化学計算や分子動力学シミュレーション等により算出することができる。 In the present invention, the (A) poorly water-soluble test substance refers to a test substance that is not soluble in water or in organic solvents such as DMSO or ethanol. The present invention is particularly useful when the poorly water-soluble test substance has an octanol/water partition coefficient of 3.5 or more, such as hexyl salicylate, N,N-dibutylaniline, 1-chloromethylpyrene, clotrimazole, and benzyl benzoate. The octanol/water partition coefficient is the ratio of the partition degree of a certain chemical substance between n-octanol and water (the ratio of concentrations in the n-octanol phase and the aqueous phase), and can be calculated, for example, by the shake flask method defined in OECD Guideline 107, the HPLC method defined in OECD Guideline 117, or by quantum chemical calculations, molecular dynamics simulations, etc.
本発明における（Ｂ）２５℃で液状の炭化水素とは、例えばスクワラン、ミネラルオイル、水添ポリイソブテン等が挙げられる。特に、非鉱物油であるスクワランが好ましく用いられる。 In the present invention, (B) the hydrocarbon that is liquid at 25°C includes, for example, squalane, mineral oil, hydrogenated polyisobutene, etc. In particular, squalane, which is a non-mineral oil, is preferably used.
本発明における（Ｃ）２５℃で固型状且つ７０℃で液状の炭化水素とは、例えばパラフィンワックス、マイクロクリスタリンワックス等が挙げられる。特に、ハンドリングの点から４８～６９℃に融点を有するパラフィンワックスが好ましく用いられ、例えば、１１５°（融点約４８℃）、１３０°（融点約５６℃）、１５５°（融点約６９℃）等を使用することができる。 In the present invention, (C) the hydrocarbon that is solid at 25°C and liquid at 70°C includes, for example, paraffin wax, microcrystalline wax, etc. In particular, from the viewpoint of handling, paraffin wax having a melting point of 48 to 69°C is preferably used, for example, 115° (melting point about 48°C), 130° (melting point about 56°C), 155° (melting point about 69°C), etc. can be used.
本発明における固型物とは、（Ａ）難水溶性被験物質、（Ｂ）２５℃で液状の炭化水素及び（Ｃ）２５℃で固型状且つ７０℃で液状の炭化水素を７０℃以上で加熱することにより均一に溶解させた後に冷却することにより得られるものであり、（Ａ）、（Ｂ）及び（Ｃ）を混合させる順番は特に限定されない。例えば、予め（Ａ）難水溶性被験物質と（Ｂ）２５℃で液状の炭化水素を均一に溶解させた後、（Ｃ）２５℃で固型状且つ７０℃で液状の炭化水素と混合し、７０℃以上で加熱することにより均一に溶解させた後に冷却することにより固型物を得ることができる。固型物を調製するための容器は特に限定されず、例えば、細胞培養用ディッシュ、マイクロプレート、マイクロチューブ等を使用することができる。 The solid substance in the present invention is obtained by heating (A) a poorly water-soluble test substance, (B) a hydrocarbon that is liquid at 25°C, and (C) a hydrocarbon that is solid at 25°C and liquid at 70°C to a temperature of 70°C or higher to dissolve uniformly, and then cooling. The order in which (A), (B), and (C) are mixed is not particularly limited. For example, (A) a poorly water-soluble test substance and (B) a hydrocarbon that is liquid at 25°C are first uniformly dissolved, and then (C) a hydrocarbon that is solid at 25°C and liquid at 70°C is mixed with the hydrocarbon, and the mixture is heated to a temperature of 70°C or higher to dissolve uniformly, followed by cooling to obtain a solid substance. The container for preparing the solid substance is not particularly limited, and for example, a cell culture dish, a microplate, a microtube, etc. can be used.
本発明における浮遊系培養細胞の懸濁液を調製する際に使用する溶液は、細胞を懸濁することができれば特に限定されない。例えば、培地、生理食塩水、リン酸緩衝生理食塩水（ＰＢＳ）等を使用することができる。浮遊系培養細胞へ難水溶性被験物質を長時間曝露する場合は、細胞の生育への影響を最小限にするため、培地を使用するのが好ましい。 The solution used in preparing the suspension of suspension cultured cells in the present invention is not particularly limited as long as it can suspend the cells. For example, culture medium, saline, phosphate-buffered saline (PBS), etc. can be used. When exposing suspension cultured cells to a poorly water-soluble test substance for a long period of time, it is preferable to use culture medium in order to minimize the effect on cell growth.
本発明における浮遊系培養細胞の懸濁液を適用する方法は、特に限定されない。例えば、パスツールピペット、マイクロピペット等を用いて、固型物の上から浮遊系培養細胞の懸濁液を適用する。その後、ＣＯ２インキュベーター等に入れ、一定時間放置することにより、自重により浮遊系培養細胞が懸濁液の下部に沈み、固型物の上面と接触する。これにより、固型物中に含まれる難水溶性被験物質と浮遊系培養細胞を効率よく接触させることが可能となる。 The method of applying the suspension of suspension cultured cells in the present invention is not particularly limited. For example, a Pasteur pipette, a micropipette, or the like is used to apply the suspension of suspension cultured cells from above the solid object. After that, the suspension cultured cells are placed in a CO2 incubator or the like and left for a certain period of time, whereby the suspension cultured cells sink to the bottom of the suspension due to their own weight and come into contact with the upper surface of the solid object. This allows the suspension cultured cells to be efficiently contacted with the poorly water-soluble test substance contained in the solid object.
本発明における一定時間とは、難水溶性被験物質と浮遊系培養細胞を効率よく接触させるために十分な時間のことを指し、特に限定されないが、例えば６時間、２４時間、４８時間等が挙げられる。 In the present invention, a certain period of time refers to a period of time sufficient to efficiently bring the poorly water-soluble test substance into contact with the suspension cultured cells, and is not particularly limited, but examples thereof include 6 hours, 24 hours, 48 hours, etc.
本発明における（Ｃ）２５℃で固型状且つ７０℃で液状の炭化水素の含有量は特に限定されないが、冷却後の操作上の点から（Ａ）＋（Ｂ）＋（Ｃ）の総量に対して５０重量％以上であることが好ましい。 In the present invention, the content of (C) the hydrocarbon that is solid at 25°C and liquid at 70°C is not particularly limited, but from the viewpoint of operation after cooling, it is preferable that it be 50% by weight or more based on the total amount of (A) + (B) + (C).
本発明における浮遊系培養細胞への難水溶性被験物質の曝露方法を用いることにより、被験物質の細胞毒性、遺伝子発現あるいはタンパク発現を指標にした各種有効性あるいは安全性の評価に応用することができる。 By using the method of exposing poorly water-soluble test substances to suspension culture cells in the present invention, it is possible to apply it to various evaluations of efficacy or safety using the cytotoxicity, gene expression, or protein expression of the test substance as an indicator.
本発明における難水溶性皮膚感作性物質のｉｎ ｖｉｔｒｏ評価法とは、浮遊系培養細胞の一種であるヒト単球性白血病由来（ＴＨＰ－１）細胞上に発現される細胞表面マーカーＣＤ８６及びＣＤ５４の発現量の変化を指標にして評価するものである。ＣＤ８６及びＣＤ５４の発現量の測定方法は特に限定されないが、例えば抗ヒトＣＤ８６及びＣＤ５４抗体を用いて細胞を染色し、フローサイトメーターを用いて測定する方法等が挙げられる。評価方法としては、例えば難水溶性被験物質処理群におけるＣＤ８６及びＣＤ５４の平均蛍光強度を、難水溶性被験物質未処理群の平均蛍光強度で除して相対蛍光強度を算出し、評価する方法等が挙げられる。 The in vitro evaluation method of poorly water-soluble skin sensitizers in the present invention uses as an index the change in the expression levels of cell surface markers CD86 and CD54 expressed on human monocytic leukemia-derived (THP-1) cells, a type of suspension cultured cell. The method for measuring the expression levels of CD86 and CD54 is not particularly limited, but examples include a method in which cells are stained with anti-human CD86 and CD54 antibodies and measured using a flow cytometer. Examples of evaluation methods include a method in which the average fluorescence intensity of CD86 and CD54 in a group treated with a poorly water-soluble test substance is divided by the average fluorescence intensity in a group not treated with the poorly water-soluble test substance to calculate the relative fluorescence intensity, and evaluation is performed.
以下に実施例を挙げて本発明を具体的に説明するが、本発明の技術的範囲がこれらに限定されるものではない。 The present invention will be specifically explained below with reference to examples, but the technical scope of the present invention is not limited to these examples.
１．固型物の調製
難水溶性被験物質であるサリチル酸ヘキシル（Ｆｌｕｋａ社、オクタノール／水分配係数５．０６）及びＮ，Ｎ－ジブチルアニリン（富士フィルム和光純薬社、オクタノール／水分配係数５．１２）をそれぞれスクワラン（シュガースクワラン、日光ケミカルズ社）に溶解し、難水溶性被験物質のスクワラン溶液を調製した。３００ｍｇのスクワラン溶液及び３００ｍｇのパラフィンワックス（１５５°パラフィン、日本精蝋社）を１．５ｍＬのマイクロチューブへ入れ、７２℃にて６０分間加熱溶解した。その後、室温まで冷却することにより固型物を得た。
1. Preparation of solids Hexyl salicylate (Fluka, octanol/water partition coefficient 5.06) and N,N-dibutylaniline (Fujifilm Wako Pure Chemicals, octanol/water partition coefficient 5.12), poorly water-soluble test substances, were dissolved in squalane (sugar squalane, Nikko Chemicals) to prepare squalane solutions of the poorly water-soluble test substances. 300 mg of the squalane solution and 300 mg of paraffin wax (155° paraffin, Nippon Seiro) were placed in a 1.5 mL microtube and heated to 72° C. for 60 minutes to dissolve. The mixture was then cooled to room temperature to obtain a solid.
２．浮遊系培養細胞への難水溶性被験物質の曝露
ＴＨＰ－１細胞を１０％ウシ胎児血清（ＦＢＳ）、０．０５ｍＭ ２－メルカプトエタノール、１００単位／ｍＬペニシリン、１００μｇ／ｍＬストレプトマイシンを添加したＲＰＭＩ－１６４０培地を用いて培養し、細胞を回収した後、２．０×１０６個／ｍＬになるように新しい培地で再懸濁し、細胞懸濁液を得た。５００μＬの細胞懸濁液を、マイクロピペットを用いて固型物の上に適用した後、３７℃、５％ＣＯ２環境下で４８時間培養した。
2. Exposure of poorly water-soluble test substances to suspension culture cells THP-1 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 0.05 mM 2-mercaptoethanol, 100 units/mL penicillin, and 100 μg/mL streptomycin, and the cells were collected and then resuspended in fresh medium to a concentration of 2.0 × 10 6 cells/mL to obtain a cell suspension. 500 μL of the cell suspension was applied onto a solid object using a micropipette, and then cultured at 37°C in a 5% CO 2 environment for 48 hours.
３．難水溶性皮膚感作性物質のｉｎ ｖｉｔｒｏ評価
上記手法にて難水溶性被験物質を曝露した後のＴＨＰ－１細胞を回収し、染色用緩衝液で細胞を洗浄した。その後、ブロッキング溶液を加えて４℃にて１５分間インキュベートした後、ＦＩＴＣ標識抗ＣＤ８６抗体、抗ＣＤ５４抗体及びマウスＩｇＧ抗体で細胞を４℃にて３０分間染色した。ＣＤ８６及びＣＤ５４の発現量は、フローサイトメーターを用いて測定した。即ち、抗ＣＤ８６抗体及び抗ＣＤ５４抗体で処理された細胞の平均蛍光強度と、マウスＩｇＧ抗体で処理された細胞の平均蛍光強度の差を算出し、被験物質無しの場合の平均蛍光強度を１００とした場合の相対蛍光強度を算出した。
3. In vitro evaluation of poorly water-soluble skin sensitizers: THP-1 cells were collected after exposure to poorly water-soluble test substances by the above method, and the cells were washed with a staining buffer. After that, a blocking solution was added and incubated at 4°C for 15 minutes, and the cells were stained with FITC-labeled anti-CD86 antibody, anti-CD54 antibody, and mouse IgG antibody at 4°C for 30 minutes. The expression levels of CD86 and CD54 were measured using a flow cytometer. That is, the difference between the average fluorescence intensity of cells treated with anti-CD86 antibody and anti-CD54 antibody and the average fluorescence intensity of cells treated with mouse IgG antibody was calculated, and the relative fluorescence intensity was calculated when the average fluorescence intensity in the absence of the test substance was set to 100.
＜判定基準＞
ＣＤ８４の相対蛍光強度が１５０％以上又はＣＤ５４の相対蛍光強度が２００％以上の場合を陽性、その他の場合を陰性と判定した。
<Criteria>
Cases in which the relative fluorescence intensity of CD84 was 150% or more or the relative fluorescence intensity of CD54 was 200% or more were judged to be positive, and other cases were judged to be negative.
被験物質の濃度（重量％）は、（Ａ）の含有量を（Ａ）＋（Ｂ）＋（Ｃ）の含有量で除して算出した。
The concentration (wt %) of the test substance was calculated by dividing the content of (A) by the content of (A)+(B)+(C).
表１に示したように、サリチル酸ヘキシルの２０重量％、２４重量％及びＮ，Ｎ－ジブチルアニリンの１２．５重量％及び１５重量％において、ＣＤ５４の相対蛍光強度が２００％以上となった。また、Ｎ，Ｎ－ジブチルアニリンの１０重量％において、ＣＤ８６の相対蛍光強度が１５０％以上となった。このことから、サリチル酸ヘキシル及びＮ，Ｎ－ジブチルアニリンは、共に陽性と判定された。 As shown in Table 1, the relative fluorescence intensity of CD54 was 200% or more at 20% and 24% by weight of hexyl salicylate and 12.5% and 15% by weight of N,N-dibutylaniline. In addition, the relative fluorescence intensity of CD86 was 150% or more at 10% by weight of N,N-dibutylaniline. From these results, both hexyl salicylate and N,N-dibutylaniline were determined to be positive.
１）Ｔａｋｅｎｏｕｃｈｉ Ｏ ｅｔ ａｌ、Ｊ． Ｔｏｘｉｃｏｌ． Ｓｃｉ．、３８（４）、５９９－６０９、２０１３
1) Takenouchi O et al, J. Toxicol. Sci. , 38(4), 599-609, 2013
表２に、従来の手法と本発明の手法との判定結果を示した。ｉｎ ｖｉｖｏ評価法の一種である局所リンパ節アッセイ（ＬＬＮＡ）では、サリチル酸ヘキシル及びＮ，Ｎ－ジブチルアニリン共に陽性と判定された。一方、ｉｎ ｖｉｔｒｏ評価法の一種であるｈ－ＣＬＡＴでは、サリチル酸ヘキシルは陽性と判定されたのに対し、Ｎ，Ｎ－ジブチルアニリンは陰性と判定され、偽陰性となった。このことから、本発明の手法を用いることにより、難水溶性皮膚感作性物質のｉｎ ｖｉｔｒｏ評価を適切に実施できることが明らかとなった。 Table 2 shows the results of the evaluation using the conventional method and the method of the present invention. In the local lymph node assay (LLNA), which is a type of in vivo evaluation method, both hexyl salicylate and N,N-dibutylaniline were evaluated as positive. On the other hand, in the h-CLAT, which is a type of in vitro evaluation method, hexyl salicylate was evaluated as positive, while N,N-dibutylaniline was evaluated as negative, resulting in a false negative. From this, it became clear that the method of the present invention can be used to appropriately perform in vitro evaluation of poorly water-soluble skin sensitizing substances.
本発明によれば、難水溶性被験物質の細胞毒性、遺伝子発現あるいはタンパク発現を指標にした各種有効性あるいは安全性の評価、特に難水溶性皮膚感作性物質のｉｎ ｖｉｔｒｏ評価を適切に実施することができ、動物実験代替試験法への貢献も高いことから、化粧品だけでなく、医薬品等にも応用可能である。
According to the present invention, it is possible to appropriately perform various evaluations of efficacy or safety using the cytotoxicity, gene expression or protein expression of poorly water-soluble test substances as indicators, in particular in vitro evaluation of poorly water-soluble skin sensitizing substances, and since this invention greatly contributes to alternative testing methods to animal testing, it can be applied not only to cosmetics but also to pharmaceuticals, etc.
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