WO2023038026A1 - Toxicity test method and toxicity test kit - Google Patents

Toxicity test method and toxicity test kit Download PDF

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WO2023038026A1
WO2023038026A1 PCT/JP2022/033427 JP2022033427W WO2023038026A1 WO 2023038026 A1 WO2023038026 A1 WO 2023038026A1 JP 2022033427 W JP2022033427 W JP 2022033427W WO 2023038026 A1 WO2023038026 A1 WO 2023038026A1
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test
toxicity
test substance
cells
value
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French (fr)
Japanese (ja)
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洋幸 津田
デビット ビー アレクサンダー
モハメッド マラード サリ ディナ
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公立大学法人名古屋市立大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

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  • the present invention relates to toxicity test methods and toxicity test kits.
  • This application claims priority based on Japanese Patent Application No. 2021-146197 filed on September 8, 2021, and the entire contents of the same Japanese application are incorporated by reference.
  • Patent Document 1 proposes an inhalation exposure test device. According to the inhalation exposure test apparatus of Patent Document 1, inhalation toxicity can be evaluated by housing a test substance and a test animal in the same space.
  • Poisonous and Deleterious Substances Control Law designates approximately 500 compounds as target substances. Inhalation exposure studies are required for these designated compounds. However, inhalation exposure studies require extensive dedicated facilities. In addition, high cost is required to operate the inhalation exposure test equipment. In addition, the Japan Bioassay Research Center is the only facility in Japan that can conduct inhalation exposure tests. Therefore, an inhalation exposure test for these specified compounds cannot be easily carried out. As a result, inhalation exposure tests are often replaced by oral administration tests and intraperitoneal administration tests. Furthermore, there are many chemical substances for which inhalation exposure tests have not yet been conducted.
  • the present invention provides a toxicity test method and a toxicity test kit for inexpensively and simply evaluating the inhalation toxicity of chemical substances.
  • a toxicity test method comprising; a: determining the usage amount ⁇ of the test substance at which the viability of the cell is within a predetermined range when cells are cultured using a culture solution containing the test substance and dye; b: converting the amount ⁇ used to a total dose ⁇ to the test animal based on the body weight of the test animal, and; c: the total dose ⁇ of the test substance was administered by spraying it into the trachea of the test animal, and from the survival rate of the test animal after administration of the total dose ⁇ of the test substance, the test substance determining the toxicity value ⁇ of [2]
  • the method of [1] wherein the test substance is sprayed into the trachea of the test animal and administered multiple times over a period of several hours to half a day.
  • [7] The method of any one of [1] to [6], wherein the pigment in a above is neutral red.
  • [8] The method of any one of [1] to [7], wherein the cells in a above are human lung-derived cells.
  • the toxicity test kit of [9], wherein the dye is neutral red.
  • the toxicity test kit of [9] or [10], wherein the cells are human lung-derived cells.
  • a toxicity test method and a toxicity test kit are provided that can inexpensively and simply evaluate the inhalation toxicity of chemical substances.
  • FIG. 1 is a photograph showing the results of the Neutral Red test in Experimental Example A.
  • FIG. 2 is an explanatory diagram of a method for determining the usage amount ⁇ by the Neutral Red test in Experimental Example A.
  • FIG. 3 is an explanatory diagram of a method for determining the total dose ⁇ in Experimental Example A.
  • FIG. 4 is an explanatory diagram of the results of obtaining the toxicity value ⁇ of glycidol.
  • FIG. 5 is an explanatory diagram of the acquisition results of the toxicity value ⁇ of N,N-dimethylformamide.
  • FIG. 6 is an explanatory diagram of the acquisition results of the toxicity value ⁇ of the acrylic acid polymer.
  • FIG. 1 is a photograph showing the results of the Neutral Red test in Experimental Example A.
  • FIG. 2 is an explanatory diagram of a method for determining the usage amount ⁇ by the Neutral Red test in Experimental Example A.
  • FIG. 3 is an explanatory diagram of a method for determining the total dose
  • FIG. 7 is an explanatory diagram of the acquisition result of the toxicity value ⁇ of acetylacetone.
  • FIG. 8 is a diagram comparing the toxicity value ⁇ with the LC50 value of the conventional inhalation exposure method.
  • FIG. 9 shows an outline of the TIPS test in Experimental Example B.
  • FIG. 10 is an explanatory diagram of the acquisition results of the toxicity value ⁇ of 1,4-dioxane.
  • FIG. 11 is an explanatory diagram of the acquisition results of the toxicity value ⁇ of glycidyl methacrylate.
  • FIG. 12 is an explanatory diagram of the acquisition result of the toxicity value ⁇ of acrolein.
  • FIG. 13 is an explanatory diagram of the acquisition result of the toxicity value ⁇ of xylene.
  • FIG. 14 is an explanatory diagram of the acquisition results of the toxicity value ⁇ of 1,2-dichloroethane.
  • FIG. 15 is an explanatory diagram of the acquisition results of the toxicity value ⁇ of quinoline.
  • FIG. 16 is an explanatory diagram of the acquisition results of the toxicity value ⁇ of t-butyl alcohol.
  • FIG. 17 is a diagram comparing the toxicity value ⁇ obtained in Experimental Example B with the LC50 value of the conventional inhalation exposure method.
  • toxicity is a concept including carcinogenicity, carcinogenicity, and mutagenicity. "-" indicating a numerical range means that the numerical values before and after it are included as lower and upper limits.
  • the toxicity test method of the present invention the toxicity of a test substance is tested by combining an in vitro preliminary test and an in vivo intratracheal administration test.
  • the toxicity test method of the present invention includes the following a, b, and c.
  • a Determine the amount ⁇ of the test substance to be used so that the viability of the cells is within a predetermined range when the cells are cultured using the culture medium containing the test substance and the dye.
  • b Convert the amount ⁇ used to the total dose ⁇ to the test animal based on the body weight of the test animal.
  • Toxicity value ⁇ of the test substance is calculated from the survival rate of the test animal after administering the total dose ⁇ of the test substance by spraying it into the trachea of the test animal and administering the total dose ⁇ of the test substance.
  • the usage amount ⁇ of the test substance is determined so that the viability of the cells is within a predetermined range when the cells are cultured using the culture solution containing the test substance and the dye. As long as the cells are alive, the dye is taken up into the cells. On the other hand, when a cell dies, no more dye is taken into the cell. Therefore, the viability of cells when cultured using a culture solution containing a test substance and a dye is reflected in the visual staining rate and degree of staining. Cell viability is affected by test substance toxicity. Therefore, the toxicity of the test substance can be preliminarily tested in vitro by culturing cells using a culture medium containing the test substance and the dye.
  • the cells are not particularly limited. Lung-derived cells are preferred in terms of evaluating inhalation toxicity to humans.
  • lung cells derived from various animals can be used. Examples include various animals such as mice, rats, dogs, cats, cows, horses, sheep and goats.
  • a dye is not particularly limited. It is appropriately selected according to the cell type. For example, hematoxylin, eosin, acridine orange, bismarck brown, carmine, coomassie blue, cresyl violet, crystal violet, DAPI (“2-(4-amidinophenyl)-1H-indole-6-carboxamidine”), ethidium bromide, acid fuchsine, Hoechst dye, iodine, malachite green, methyl green, methylene blue, neutral red, nile blue, nile red, osmium tetroxide, rhodamine, safranin.
  • dyes are not limited to these examples.
  • neutral red is preferred in that a test with good sensitivity and low cost has been established, such as the Neutral Red test.
  • the Neutral Red test is a cell viability assay based on neutral red uptake.
  • the composition of the culture solution is not particularly limited. Various additives and culture components can be used. Also, the culture time and temperature are not particularly limited. Culture conditions for these cells may be in accordance with general protocols. Culture conditions can be changed as appropriate depending on the type of cells, the content of the test substance, and the expected toxicity of the test substance.
  • a plurality of culture solutions with different test substance contents can be prepared.
  • cells are seeded in a plurality of petri dishes using culture media containing different test substances, and the cells in each petri dish are cultured under the same conditions.
  • a petri dish having a cell viability of 10 to 90%, 40 to 60%, preferably 45 to 55%, and more preferably 50% can be selected from a plurality of petri dishes.
  • the usage amount ⁇ can be obtained from the usage amount of the test substance in the selected petri dish during the culture.
  • the usage amount ⁇ is the usage amount of the test substance at which the viability of the cells is within a predetermined range when the cells are cultured.
  • the survival rate that results in a predetermined range is not particularly limited.
  • the survival rate may be, for example, 10-90%, 40-60%, 45-55%, 48-53%, 50% and the like.
  • the total dose ⁇ for intratracheal administration in c below is determined from the usage ⁇ . Specifically, the amount ⁇ used is converted into the total dose ⁇ to the test animal based on the body weight of the test animal.
  • Test animals are not particularly limited. For example, rats and mice are preferably used as in carcinogenesis tests. The test animal may be appropriately changed according to the test substance and purpose of the test.
  • the usage amount ⁇ obtained in a is converted into the total dose ⁇ for intratracheal administration in c below. Conversion is based on the body weight of the test animal. In addition to the body weight of the test animal, the age, age in weeks, sex, vital capacity, physical condition, activity level, respiratory rate per minute, etc. of the test animal are additionally taken into account to convert the dose ⁇ to the total dose ⁇ . good.
  • the total dose ⁇ can be calculated using the following formula. Calculation formula: (amount used ⁇ ) ⁇ (liquid density of test substance in a) / (body weight of test animal) The approximate value obtained by the above formula may be used as the total dose ⁇ as it is. In addition, to the approximate value obtained by the above calculation formula, the age, age in weeks, sex, vital capacity, physical condition, activity level, and respiratory rate per minute of the test animal are quantified and reflected as appropriate, so that the total dose ⁇ may be
  • a total dose ⁇ of the test substance is administered by intratracheal nebulization of the test animal.
  • the toxicity value ⁇ of the test substance is calculated from the survival rate of the test animal after administration of the total dose ⁇ of the test substance.
  • this toxicity value ⁇ is derived as a value indicating the toxicity of the test substance.
  • a relatively small value of the toxicity value ⁇ means a relatively high toxicity of the test substance.
  • a relatively large toxicity value ⁇ means that the toxicity of the test substance is relatively low.
  • TIPS Intra-Traceal Intra-pulmonary Spray
  • TIPS for example, the contents of the documents listed below can be incorporated herein.
  • the number of administrations of the test substance is not particularly limited. That is, when administering the test substance, the total dose ⁇ of the test substance may be administered in multiple doses or may be administered in one dose. For example, the total dose ⁇ of the test substance can be divided into multiple doses in order to facilitate determination of the toxicity value ⁇ by preventing annihilation of test animals.
  • the test substance can be administered multiple times by nebulization into the trachea of the test animal over a period of several hours to half a day.
  • the dosage of the test substance for multiple times may be the same or may be different as long as the effects of the invention are obtained.
  • the doses of the test substance are the same each time, that is, constant.
  • the intervals between each administration may be constant, or may be irregular as long as the effects of the invention are obtained. From the viewpoint of the reliability of the obtained toxicity value ⁇ , it is preferable that the administration interval is constant, that is, the interval is equal.
  • test animals are not particularly limited.
  • one dose group is typically 50 animals.
  • the number of animals may be appropriately changed according to the test substance and purpose of the test. For example, a plurality of groups of several animals to more than ten animals are prepared per group, and the animals are extracted so as to obtain at least the LD50 value.
  • the toxicity value ⁇ is the test substance administration at which the survival rate of the test animal becomes 40 to 60%, preferably 45 to 55%, more preferably 50% after the observation period has elapsed after the administration of the test substance. It can be calculated as a quantity. It is particularly preferred to derive the toxicity value ⁇ as the dose at which half of the test animals die, ie the LD50 value. Therefore, a relatively small value of the toxicity value ⁇ means a relatively high toxicity of the test substance. On the other hand, a relatively large value of toxicity value ⁇ means that the toxicity of the test substance is relatively low.
  • the observation period can be changed as appropriate depending on the purpose of the study.
  • the endpoint of the observation period should be chosen so that the toxicity value ⁇ can be derived from the 50% survival rate of the test animals.
  • the acute toxicity of the test substance can be assessed by observing the test animal without administration of the test substance for at least one week after the end of administration of the total dose ⁇ of the test substance.
  • the chronic toxicity (LD50 value) of the test substance is determined by observing the test animal without administering the test substance to the test animal for at least 2 weeks after the end of administration of the test substance at the total dose ⁇ . can be evaluated.
  • the observation period is preferably 2 weeks or longer, more preferably 4 weeks or longer.
  • carcinogenicity of the test substance is evaluated by observing the test animal without administration of the test substance for at least 12 months after the end of administration of the total dose ⁇ of the test substance.
  • the test period is defined as 24 to 30 months for rats and 18 to 24 months for mice and hamsters. It is
  • the total dose ⁇ is calculated from the preliminary test ⁇ , and the toxicity value ⁇ is obtained by administering TIPS.
  • This method does not require the use of expensive equipment.
  • the resulting toxicity value ⁇ is very close to that obtained by the conventional inhalation exposure method, which requires expensive equipment and operating costs. In other words, in this method, a practically satisfactory value can be obtained as the toxicity value ⁇ . Therefore, according to the toxicity test method of the present invention, the inhalation toxicity of chemical substances can be evaluated easily and inexpensively.
  • the toxicity test kit of the present invention is used for the toxicity test method of the present invention described above.
  • a toxicity test kit comprises a dye, cells, and a medium for culturing the cells. The details and preferred aspects of the dye, cells, and culture medium are the same as those already explained for the toxicity test method.
  • the toxicity test kit may further include at least one selected from the group consisting of a spray needle, a syringe and an instruction manual, in addition to the dye, cells, and culture medium for culturing the cells.
  • a spray needle a syringe and an instruction manual
  • the contents and techniques already explained for the toxicity test method can be applied.
  • Acetylacetone, ethylene glycol monoethyl acetate, acrylic acid polymer, and N,N-dimethylacetamide were prepared as test substances.
  • a test substance was added to the culture medium of lung cancer-derived A549 cells, and cultured for a short time of about 10 to 30 minutes. After that, the medium was replaced with a medium containing neutral red but no test substance.
  • a serum-free RPMI medium supplemented with 10% Bovine Calf Serum was used as the culture medium.
  • the dose ⁇ was converted to the total dose ⁇ based on the body weight and vital capacity of the rat.
  • a total dose ⁇ was determined so that about 10 doses were obtained from the amount ⁇ used.
  • FIG. 2 shows an example in which the test substance is acetylacetone.
  • the total dose ⁇ was between 12.5 mg/kg and 100 mg/kg.
  • FIG. 3 shows values obtained by converting the concentration (mg/L) of the test substance in the Neutral Red test into body weight/kg of rats administered intratracheal spray (TIPS administration).
  • Column “A” in FIG. 3 "Neutral Red test result (in Vitoro dose ⁇ L/ml)” corresponds to the amount ⁇ used.
  • the “H” column, “Total Dose of 4 TIPS/4 TIPS (mg/kg)” corresponds to the total dose ⁇ .
  • TIPS was administered in 4 divided doses.
  • the upper panel of FIG. 3 shows the conversion of the 90% viable dose in culture of A549 cells to the dose administered to rats.
  • the bottom row shows the conversion of the survival dose of 10% or less in the medium of A549 cells to the dose administered to rats.
  • toxicity value ⁇ is the LD50 value.
  • toxicity value ⁇ are shown in Figures 4-7. These are the LD50 values in the example of 5-7 dose groups with 8 animals per group. In Figures 4-7, fractions such as 8/8 indicate survival rates of rats. For example, "6/8" indicates that 6 out of 8 rats are alive (and so on).
  • the LD50 value, ie the toxicity value ⁇ was between 160 mg/kg and 320 mg/kg. As shown in FIG. 5, the LD50 value, ie toxicity value ⁇ , was between 1280 mg/kg and 2560 mg/kg for N,N-dimethylformamide. As shown in FIG. 6, the LD50 value, ie toxicity value ⁇ , was between 120 mg/kg and 240 mg/kg for the acrylic acid polymer. For acetylacetone as shown in Figure 7, the LD50 value, ie the toxicity value ⁇ , was between 400 mg/kg and 800 mg/kg.
  • the toxicity value ⁇ was compared with the LC50 value of the conventional inhalation exposure method (4-hour inhalation exposure).
  • TIPS LD50 mg/kg corresponds to the toxicity value ⁇ .
  • each value of TIPS LD50 is within 4 times the acute toxicity LC50 value. shown to fit.
  • the toxicity value ⁇ determined in this way, that is, TIPS LD50 is considered to be a valid value.
  • LD50 ⁇ 154.7 ⁇ L/mL of 1,4-dioxane obtained by the same method as in Experimental Example A was obtained as the usage amount ⁇ .
  • the dose used in the TIPS test was set between the minimum usage amount (75%) and the maximum usage amount (200%).
  • the dose ( ⁇ /1) for one TIPS was determined by the following formula.
  • the rat vital capacity was converted to vital capacity per kg.
  • TIPS was administered to 10- to 12-week-old rats at a dose of ⁇ /4 for a total of 4 times, 1 hour, 2 hours, and 3 hours after the initial administration. Thereafter, the rats were observed for a total of 8 days without administration of the test substance, and the toxicity value ⁇ was determined as the dose at which half of the rats died by the 8th day (Fig. 9).
  • the toxicity value ⁇ is the LD50 value.
  • LD50 values were obtained from graphs plotting the survival rate of test animals at each total dose. Values obtained from known inhalation exposures were approximated. The results are shown in FIG. As a result, the LD50 value, ie toxicity value ⁇ , was between 1280 mg/kg and 1600 mg/kg for 1,4-dioxane.
  • the International Agency for Research on Cancer (IARC) carcinogenicity classification for 1,4-dioxane is G2B. Substances classified as G2B are potentially carcinogenic to humans.
  • Toxicity value ⁇ was determined for the following test substances by the same method as for 1,4-dioxane described above. • Glycidyl methacrylate • Acrolein • Xylene • 1,2-dichloroethane • Quinoline • t-butyl alcohol The results are shown in Figures 11 to 16, respectively.
  • the LD50 value, ie the toxicity value ⁇ was between 240 mg/kg and 480 mg/kg.
  • the LD50 value, ie toxicity value ⁇ was between 1 mg/kg and 2 mg/kg.
  • the LD50 value, ie toxicity value ⁇ was between 100 mg/kg and 400 mg/kg.
  • the LD50 value, ie toxicity value ⁇ was between 240 mg/kg and 480 mg/kg.
  • FIG. 14 the LD50 value, ie toxicity value ⁇
  • quinoline had an LD50 value, that is, a toxicity value ⁇ of 80 mg/kg or more.
  • the LD50 value that is, the toxicity value ⁇
  • the LD50 value was 800 mg/kg or more for t-butyl alcohol.
  • the toxicity value ⁇ was compared with the LC50 value of the conventional inhalation exposure method (4-hour inhalation exposure).
  • TIPS LD50 mg/kg corresponds to the toxicity value ⁇ .
  • each value of TIPS LD50 is within 4 times the acute toxicity LC50 value. It was also shown in Experimental example B that it was accommodated.
  • the toxicity value ⁇ determined in this way, that is, TIPS LD50 is considered to be a valid value.
  • the present inventors have developed a test system that combines an in vitro Neutral Red test and an in vivo TIPS administration.
  • the total dose ⁇ is calculated from the preliminary test ⁇ , and the toxicity value ⁇ is determined by TIPS administration. Satisfactory data consistency was obtained when the toxicity value ⁇ was compared with the LC50 of conventional inhalation toxicity tests.
  • the method of the present invention is inexpensive because it does not require expensive operating costs for inhalation exposure test equipment.
  • the methods of the invention can be simple and inexpensive compared to inhalation exposure studies in expensive dedicated facilities. Therefore, the method of the present invention is useful as an alternative to conventional inhalation exposure tests.
  • a toxicity test method and a toxicity test kit are provided that can inexpensively and simply evaluate the inhalation toxicity of chemical substances.

Abstract

To provide a toxicity test method which enables cheaply and simply evaluating the inhalation toxicity of chemicals, and a toxicity test kit.　The toxicity test method in one embodiment involves a, b and c below.　a: Calculate the use amount α of a test substance from the survival rate of the cells when the cells have been cultured using a culture fluid that contains the test substance and a pigment.　b: Convert the use amount α into a test animal total dosage on the basis of the body weight of the test animal.　c: Dose the test animal with a total dosage β of the test substance by spraying in the trachea, and calculate the toxicity γ of the test substance from the survival rate of test animals after dosing with the total dosage β of the test substance.　The toxicity test kit in the present embodiment is provided with the aforementioned pigment, the aforementioned cells, and a culture fluid for culturing the cells.

Description

毒性試験方法、毒性試験キットToxicity Test Method, Toxicity Test Kit
　本発明は、毒性試験方法、毒性試験キットに関する。
　本願は２０２１年９月８日に出願した日本国特許出願２０２１－１４６１９７号に基づく優先権を主張し、同日本国出願の全内容を参照により援用する。 The present invention relates to toxicity test methods and toxicity test kits.
This application claims priority based on Japanese Patent Application No. 2021-146197 filed on September 8, 2021, and the entire contents of the same Japanese application are incorporated by reference.
　化学物質の吸入毒性の評価は重要である。空気中の化学物質は不可避的に肺から体内に取り込まれる。そのため、例えば、事業場における空気中の化学物質の安全性の評価および管理が必要となる。
　特許文献１では、吸入曝露試験装置が提案されている。特許文献１の吸入曝露試験装置によれば、試験物質および試験動物を同一の空間内に収容することで、吸入毒性を評価することができる。 Evaluation of the inhalation toxicity of chemicals is important. Airborne chemicals are inevitably taken into the body through the lungs. Therefore, for example, it is necessary to evaluate and manage the safety of chemical substances in the air at business sites.
Patent Document 1 proposes an inhalation exposure test device. According to the inhalation exposure test apparatus of Patent Document 1, inhalation toxicity can be evaluated by housing a test substance and a test animal in the same space.
特開２０１４－１０１２１号公報JP 2014-10121 A
　日本国の毒物及び劇物取締法（以下、「毒劇法」）においては、対象物質として約５００種の化合物が指定されている。これらの指定化合物には吸入曝露試験が必要である。しかし、吸入曝露試験は大規模な専用設備を要する。また、吸入曝露試験装置の稼働には高額な費用が要求される。加えて、吸入曝露試験を実施できる施設は、日本国内では日本バイオアッセイ研究センターのみである。よって、これら指定化合物の吸入曝露試験は簡便に実施できない。
　結果、吸入曝露試験が経口投与試験、腹腔内投与試験で代替されていることが多い。さらには、吸入曝露試験が未実施の化学物質も多く存在する。世界的にも、吸入曝露試験を実施できる施設は少ない。本発明者の知見によれば、米国、英国、独国の各国においても１施設ずつしかない。また、ＧＨＳ（Ｇｌｏｂａｌｌｙ　Ｈａｒｍｏｎｉｚｅｄ　Ｓｙｓｔｅｍ　ｏｆ　Ｃｌａｓｓｉｆｉｃａｔｉｏｎ　ａｎｄ　Ｌａｂｅｌｌｉｎｇ　ｏｆ　Ｃｈｅｍｉｃａｌｓ）に記載の化学物質でも吸入曝露試験が未実施の化合物も多い。 Japanese Poisonous and Deleterious Substances Control Law (hereinafter referred to as "Poisonous and Deleterious Substances Law") designates approximately 500 compounds as target substances. Inhalation exposure studies are required for these designated compounds. However, inhalation exposure studies require extensive dedicated facilities. In addition, high cost is required to operate the inhalation exposure test equipment. In addition, the Japan Bioassay Research Center is the only facility in Japan that can conduct inhalation exposure tests. Therefore, an inhalation exposure test for these specified compounds cannot be easily carried out.
As a result, inhalation exposure tests are often replaced by oral administration tests and intraperitoneal administration tests. Furthermore, there are many chemical substances for which inhalation exposure tests have not yet been conducted. There are few facilities in the world that can conduct inhalation exposure tests. According to the findings of the present inventor, there is only one facility each in the United States, the United Kingdom, and Germany. In addition, many of the chemical substances listed in GHS (Globally Harmonized System of Classification and Labeling of Chemicals) have not undergone an inhalation exposure test.
　以上の理由から、従来、化学物質の吸入毒性を充分に評価できているとは言い難い。
　本発明は、化学物質の吸入毒性を安価で簡便に評価できる毒性試験方法；および毒性試験キットを提供する。 For the above reasons, it is difficult to say that the inhalation toxicity of chemical substances has been sufficiently evaluated.
The present invention provides a toxicity test method and a toxicity test kit for inexpensively and simply evaluating the inhalation toxicity of chemical substances.
　本発明は下記の［１］～［１１］の態様を有する。
［１］毒性試験方法であって；
　ａ：試験物質および色素を含む培養液を用いて細胞を培養したときに前記細胞の生存率が所定の範囲となる前記試験物質の使用量αを求めること；
　ｂ：前記使用量αを試験動物の体重に基づいて、前記試験動物への総投与量βに換算すること、および；
　ｃ：総投与量βの前記試験物質を前記試験動物の気管内で噴霧して投与し、かつ、総投与量βの前記試験物質を投与した後の前記試験動物の生存率から、前記試験物質の毒性値γを求めること；を含む、方法。
［２］前記ｃでは、数時間から半日の期間をかけて、前記試験物質を前記試験動物の気管内で噴霧して複数回投与する、［１］の方法。
［３］前記ｃでは、総投与量βの前記試験物質の投与終了後少なくとも１週間以上、前記試験動物に前記試験物質を投与せずに前記試験動物を観察することで、前記試験物質の急性毒性を評価する、［１］または［２］の方法。
［４］前記ｃでは、総投与量βの前記試験物質の投与終了後少なくとも２週間以上、前記試験動物に前記試験物質を投与せずに前記試験動物を観察することで、前記試験物質の慢性毒性を評価する、［１］または［２］の方法。
［５］前記ｃでは、総投与量βの前記試験物質の投与終了後少なくとも１２か月以上、前記試験動物に前記試験物質を投与せずに前記試験動物を観察することで、前記試験物質のがん原性を評価する、［１］または［２］の方法。
［６］前記ａでは、前記細胞の生存率が１０～９０％となるときの前記試験物質の使用量から前記使用量αを求める、［１］～［５］のいずれかの方法。
［７］前記ａにおける前記色素が、ニュートラルレッドである、［１］～［６］のいずれかの方法。
［８］前記ａにおける前記細胞が、ヒトの肺由来の細胞である、［１］～［７］のいずれかの方法。
［９］［１］～［８］のいずれかの方法のための毒性試験キットであり；前記色素と、前記細胞と、前記細胞を培養するための培養液と、を備えた、毒性試験キット。
［１０］前記色素が、ニュートラルレッドである、［９］の毒性試験キット。
［１１］前記細胞が、ヒトの肺由来の細胞である、［９］または［１０］の毒性試験キット。 The present invention has the following aspects [1] to [11].
[1] A toxicity test method comprising;
a: determining the usage amount α of the test substance at which the viability of the cell is within a predetermined range when cells are cultured using a culture solution containing the test substance and dye;
b: converting the amount α used to a total dose β to the test animal based on the body weight of the test animal, and;
c: the total dose β of the test substance was administered by spraying it into the trachea of the test animal, and from the survival rate of the test animal after administration of the total dose β of the test substance, the test substance determining the toxicity value γ of
[2] In the above c, the method of [1], wherein the test substance is sprayed into the trachea of the test animal and administered multiple times over a period of several hours to half a day.
[3] In c above, acute The method of [1] or [2] for evaluating toxicity.
[4] In c above, chronic The method of [1] or [2] for evaluating toxicity.
[5] In the above c, by observing the test animal without administering the test substance to the test animal for at least 12 months after the end of administration of the test substance at the total dose β, The method of [1] or [2] for evaluating carcinogenicity.
[6] In the method a, the method according to any one of [1] to [5], wherein the usage amount α is obtained from the usage amount of the test substance when the survival rate of the cells is 10 to 90%.
[7] The method of any one of [1] to [6], wherein the pigment in a above is neutral red.
[8] The method of any one of [1] to [7], wherein the cells in a above are human lung-derived cells.
[9] A toxicity test kit for the method of any one of [1] to [8]; a toxicity test kit comprising the dye, the cells, and a culture medium for culturing the cells. .
[10] The toxicity test kit of [9], wherein the dye is neutral red.
[11] The toxicity test kit of [9] or [10], wherein the cells are human lung-derived cells.
　本発明によれば、化学物質の吸入毒性を安価で簡便に評価できる毒性試験方法；および毒性試験キットが提供される。 According to the present invention, a toxicity test method and a toxicity test kit are provided that can inexpensively and simply evaluate the inhalation toxicity of chemical substances.
図１は、実験例ＡにおけるＮｅｕｔｒａｌ　Ｒｅｄ試験の結果を示す写真である。FIG. 1 is a photograph showing the results of the Neutral Red test in Experimental Example A. FIG. 図２は、実験例ＡにおけるＮｅｕｔｒａｌ　Ｒｅｄ試験による使用量αの決定方法の説明図である。FIG. 2 is an explanatory diagram of a method for determining the usage amount α by the Neutral Red test in Experimental Example A. FIG. 図３は、実験例Ａにおける総投与量βの決定方法の説明図である。FIG. 3 is an explanatory diagram of a method for determining the total dose β in Experimental Example A. FIG. 図４は、グリシドールの毒性値γの取得結果の説明図である。FIG. 4 is an explanatory diagram of the results of obtaining the toxicity value γ of glycidol. 図５は、Ｎ，Ｎ－ジメチルホルムアミドの毒性値γの取得結果の説明図である。FIG. 5 is an explanatory diagram of the acquisition results of the toxicity value γ of N,N-dimethylformamide. 図６は、アクリル酸ポリマーの毒性値γの取得結果の説明図である。FIG. 6 is an explanatory diagram of the acquisition results of the toxicity value γ of the acrylic acid polymer. 図７は、アセチルアセトンの毒性値γの取得結果の説明図である。FIG. 7 is an explanatory diagram of the acquisition result of the toxicity value γ of acetylacetone. 図８は、毒性値γを従来の吸入暴露法のＬＣ５０値と比較した図である。FIG. 8 is a diagram comparing the toxicity value γ with the LC50 value of the conventional inhalation exposure method. 図９は、実験例ＢにおけるＴＩＰＳ試験の概要を示す。FIG. 9 shows an outline of the TIPS test in Experimental Example B. 図１０は、１，４－ジオキサンの毒性値γの取得結果の説明図である。FIG. 10 is an explanatory diagram of the acquisition results of the toxicity value γ of 1,4-dioxane. 図１１は、グリシジルメタクリレートの毒性値γの取得結果の説明図である。FIG. 11 is an explanatory diagram of the acquisition results of the toxicity value γ of glycidyl methacrylate. 図１２は、アクロレインの毒性値γの取得結果の説明図である。FIG. 12 is an explanatory diagram of the acquisition result of the toxicity value γ of acrolein. 図１３は、キシレンの毒性値γの取得結果の説明図である。FIG. 13 is an explanatory diagram of the acquisition result of the toxicity value γ of xylene. 図１４は、１，２－ジクロロエタンの毒性値γの取得結果の説明図である。FIG. 14 is an explanatory diagram of the acquisition results of the toxicity value γ of 1,2-dichloroethane. 図１５は、キノリンの毒性値γの取得結果の説明図である。FIG. 15 is an explanatory diagram of the acquisition results of the toxicity value γ of quinoline. 図１６は、ｔ－ブチルアルコールの毒性値γの取得結果の説明図である。FIG. 16 is an explanatory diagram of the acquisition results of the toxicity value γ of t-butyl alcohol. 図１７は、実験例Ｂで取得した毒性値γを従来の吸入暴露法のＬＣ５０値と比較した図である。FIG. 17 is a diagram comparing the toxicity value γ obtained in Experimental Example B with the LC50 value of the conventional inhalation exposure method.
　本明細書において「毒性」とは、発がん性、がん原生、変異原性を含む概念である。
　数値範囲を示す「～」は、その前後に記載された数値を下限値および上限値として含むことを意味する。 As used herein, "toxicity" is a concept including carcinogenicity, carcinogenicity, and mutagenicity.
"-" indicating a numerical range means that the numerical values before and after it are included as lower and upper limits.
［毒性試験方法］
　（概要）
　本発明の毒性試験方法では、ｉｎ　ｖｉｔｒｏにおける予備試験と、ｉｎ　ｖｉｖｏにおける気管内投与試験とを組み合わせることで、試験物質の毒性を試験する。具体的に本発明の毒性試験方法は、下記のａ、ｂ、ｃを含む。
　ａ：試験物質および色素を含む培養液を用いて細胞を培養したときに、細胞の生存率が所定の範囲となる試験物質の使用量αを求めること。
　ｂ：使用量αを試験動物の体重に基づいて、試験動物への総投与量βに換算すること。
　ｃ：総投与量βの試験物質を試験動物の気管内で噴霧して投与し、かつ、総投与量βの試験物質を投与した後の試験動物の生存率から、試験物質の毒性値γを求めること。 [Toxicity test method]
(overview)
In the toxicity test method of the present invention, the toxicity of a test substance is tested by combining an in vitro preliminary test and an in vivo intratracheal administration test. Specifically, the toxicity test method of the present invention includes the following a, b, and c.
a: Determine the amount α of the test substance to be used so that the viability of the cells is within a predetermined range when the cells are cultured using the culture medium containing the test substance and the dye.
b: Convert the amount α used to the total dose β to the test animal based on the body weight of the test animal.
c: Toxicity value γ of the test substance is calculated from the survival rate of the test animal after administering the total dose β of the test substance by spraying it into the trachea of the test animal and administering the total dose β of the test substance. Ask.
　（ａ：ｉｎ　ｖｉｔｒｏ試験）
　ａでは、試験物質および色素を含む培養液を用いて細胞を培養したときに、細胞の生存率が所定の範囲となる試験物質の使用量αを求める。細胞が生存している間は、色素が細胞内に取り込まれる。一方、細胞が死滅すると、色素がそれ以上細胞内に取り込まれなくなる。
　よって、試験物質および色素を含む培養液を用いて細胞を培養したときの細胞の生存率は、目視の染色率、染色の程度に反映される。細胞の生存率は試験物質の毒性に影響を受ける。そのため、試験物質および色素を含む培養液を用いて細胞を培養することで、試験物質の毒性を予備的にｉｎ　ｖｉｔｒｏで試験できる。 (a: in vitro test)
In a, the usage amount α of the test substance is determined so that the viability of the cells is within a predetermined range when the cells are cultured using the culture solution containing the test substance and the dye. As long as the cells are alive, the dye is taken up into the cells. On the other hand, when a cell dies, no more dye is taken into the cell.
Therefore, the viability of cells when cultured using a culture solution containing a test substance and a dye is reflected in the visual staining rate and degree of staining. Cell viability is affected by test substance toxicity. Therefore, the toxicity of the test substance can be preliminarily tested in vitro by culturing cells using a culture medium containing the test substance and the dye.
　細胞は特に限定されない。ヒトに対する吸入毒性を評価する点では、肺由来の細胞が好ましい。その他、種々の動物由来の肺細胞を使用できる。例えば、マウス、ラット、イヌ、ネコ、ウシ、ウマ、ヒツジ、ヤギ等の種々の動物が挙げられる。 The cells are not particularly limited. Lung-derived cells are preferred in terms of evaluating inhalation toxicity to humans. In addition, lung cells derived from various animals can be used. Examples include various animals such as mice, rats, dogs, cats, cows, horses, sheep and goats.
　色素は特に限定されない。細胞の種類に応じて適宜選択される。例えば、ヘマトキシリン、エオジン、アクリジンオレンジ、ビスマルクブラウン、カーミン、クーマシーブルー、クレシルバイオレット、クリスタルバイオレット、ＤＡＰＩ（「２－（４－アミジノフェニル）－１Ｈ－インドール－６－カルボキサミジン」）、エチジウムブロミド、酸性フクシン、ヘキスト染色剤、ヨウ素、マラカイトグリーン、メチルグリーン、メチレンブルー、ニュートラルレッド、ナイルブルー、ナイルレッド、四酸化オスミウム、ローダミン、サフラニンが挙げられる。ただし、色素はこれらの例示に限定されない。
　これらのなかでも、Ｎｅｕｔｒａｌ　Ｒｅｄ試験のように、感度が良好で費用が安い試験が確立されている点で、ニュートラルレッドが好ましい。Ｎｅｕｔｒａｌ　Ｒｅｄ試験は、ニュートラルレッドの取込みに基づく細胞の生存率のアッセイである。 A dye is not particularly limited. It is appropriately selected according to the cell type. For example, hematoxylin, eosin, acridine orange, bismarck brown, carmine, coomassie blue, cresyl violet, crystal violet, DAPI (“2-(4-amidinophenyl)-1H-indole-6-carboxamidine”), ethidium bromide, acid fuchsine, Hoechst dye, iodine, malachite green, methyl green, methylene blue, neutral red, nile blue, nile red, osmium tetroxide, rhodamine, safranin. However, dyes are not limited to these examples.
Among these, neutral red is preferred in that a test with good sensitivity and low cost has been established, such as the Neutral Red test. The Neutral Red test is a cell viability assay based on neutral red uptake.
　培養液の組成は特に限定されない。種々の添加物、培養成分を使用できる。また、培養時間、温度も特に限定されない。これらの細胞の培養条件は、一般的なプロトコルにしたがえばよい。培養条件は、細胞の種類、試験物質の含有量、試験物質の予想される毒性に応じて適宜変更できる。 The composition of the culture solution is not particularly limited. Various additives and culture components can be used. Also, the culture time and temperature are not particularly limited. Culture conditions for these cells may be in accordance with general protocols. Culture conditions can be changed as appropriate depending on the type of cells, the content of the test substance, and the expected toxicity of the test substance.
　一態様において、ａでは、試験物質の含有量が異なる培養液を複数用意できる。例えば、互いに試験物質の含有量が異なる培養液を用いて、複数のシャーレに細胞を播種して各シャーレの細胞を同一条件下で培養する。このとき、複数のシャーレのうちから、細胞の生存率が１０～９０％、４０～６０％、好ましくは４５～５５％、より好ましくは５０％となるシャーレを選択できる。選択したシャーレの試験物質の培養時の使用量から、使用量αを求めることができる。 In one aspect, in a, a plurality of culture solutions with different test substance contents can be prepared. For example, cells are seeded in a plurality of petri dishes using culture media containing different test substances, and the cells in each petri dish are cultured under the same conditions. At this time, a petri dish having a cell viability of 10 to 90%, 40 to 60%, preferably 45 to 55%, and more preferably 50% can be selected from a plurality of petri dishes. The usage amount α can be obtained from the usage amount of the test substance in the selected petri dish during the culture.
　使用量αは、細胞を培養したときに、細胞の生存率が所定の範囲となる試験物質の使用量である。このときに結果的に所定の範囲となる生存率は、特に限定されるものではない。該生存率としては、例えば、１０～９０％、４０～６０％、４５～５５％、４８～５３％、５０％等であり得る。 The usage amount α is the usage amount of the test substance at which the viability of the cells is within a predetermined range when the cells are cultured. At this time, the survival rate that results in a predetermined range is not particularly limited. The survival rate may be, for example, 10-90%, 40-60%, 45-55%, 48-53%, 50% and the like.
　（換算）
　ｂでは、後段のｃでの気管内投与のための総投与量βを使用量αから求める。具体的には、使用量αを試験動物の体重に基づいて、試験動物への総投与量βに換算する。
　試験動物は特に限定されない。例えば、がん原生試験のようにラット、マウスが好適に用いられる。試験動物は、試験物質、試験目的に応じて適宜変更すればよい。 (conversion)
In b, the total dose β for intratracheal administration in c below is determined from the usage α. Specifically, the amount α used is converted into the total dose β to the test animal based on the body weight of the test animal.
Test animals are not particularly limited. For example, rats and mice are preferably used as in carcinogenesis tests. The test animal may be appropriately changed according to the test substance and purpose of the test.
　ｂでは、ａで求めた使用量αを後段のｃでの気管内投与のための総投与量βに換算する。換算に際しては、試験動物の体重に基づいて行う。試験動物の体重に加えて、試験動物の年齢、週齢、性別、肺活量、体調、活動度、毎分呼吸量等を追加的に勘案して使用量αを総投与量βに換算してもよい。 In b, the usage amount α obtained in a is converted into the total dose β for intratracheal administration in c below. Conversion is based on the body weight of the test animal. In addition to the body weight of the test animal, the age, age in weeks, sex, vital capacity, physical condition, activity level, respiratory rate per minute, etc. of the test animal are additionally taken into account to convert the dose α to the total dose β. good.
　例えば、総投与量βは以下の算出式を用いて算出され得る。
　算出式：（使用量α）×（ａにおける試験物質の液体密度）／（試験動物の体重）
　前記算出式で得られた概算値はそのまま総投与量βとしてもよい。また、前記算出式で得られた概算値に、さらに、試験動物の年齢、週齢、性別、肺活量、体調、活動度、毎分呼吸量を適宜数値化して反映することで、総投与量βとしてもよい。 For example, the total dose β can be calculated using the following formula.
Calculation formula: (amount used α) × (liquid density of test substance in a) / (body weight of test animal)
The approximate value obtained by the above formula may be used as the total dose β as it is. In addition, to the approximate value obtained by the above calculation formula, the age, age in weeks, sex, vital capacity, physical condition, activity level, and respiratory rate per minute of the test animal are quantified and reflected as appropriate, so that the total dose β may be
　（ｃ：ｉｎ　ｖｉｖｏ試験）
　ｃでは、総投与量βの試験物質を試験動物の気管内で噴霧して投与する。そして、総投与量βの試験物質を投与した後の試験動物の生存率から、試験物質の毒性値γを求める。
　本発明の毒性試験方法においては、この毒性値γが試験物質の毒性を示す値として導き出される。毒性値γの値が相対的に小さいほど、試験物質の毒性が相対的に高いことを意味する。一方、毒性値γの値が相対的に大きいほど、試験物質の毒性が相対的に低いことを意味する。 (c: in vivo test)
In c, a total dose β of the test substance is administered by intratracheal nebulization of the test animal. Then, the toxicity value γ of the test substance is calculated from the survival rate of the test animal after administration of the total dose β of the test substance.
In the toxicity test method of the present invention, this toxicity value γ is derived as a value indicating the toxicity of the test substance. A relatively small value of the toxicity value γ means a relatively high toxicity of the test substance. On the other hand, a relatively large toxicity value γ means that the toxicity of the test substance is relatively low.
　試験動物の気管内での試験物質の噴霧投与は、本発明者が確立したＴＩＰＳ（Ｉｎｔｒａ－Ｔｒａｃｈｅａｌ　Ｉｎｔｒａ－ｐｕｌｍｏｎａｒｙ　Ｓｐｒａｙ）投与を使用することが好ましい。 It is preferable to use TIPS (Intra-Traceal Intra-pulmonary Spray) administration established by the present inventors for spray administration of the test substance into the trachea of the test animal.
　ＴＩＰＳに関しては、例えば、以下に列挙する文献の内容を本明細書に援用できる。
・Ｓｉｚｅ－ａｎｄｓｈａｐｅ－ｄｅｐｅｎｄｅｎｔ　ｐｌｅｕｒａｌ　ｔｒａｎｓｌｏｃａｔｉｏｎ，ｄｅｐｏｓｉｔｉｏｎ，ｆｉｂｒｏｇｅｎｅｓｉｓ　ａｎｄ　ｍｅｓｏｔｈｅｌｉａｌ　ｐｒｏｌｉｆｅｒａｔｉｏｎ　ｂｙ　ｍｕｌｔｉ－ｗａｌｌｅｄ　ｃａｒｂｏｎ　ｎａｎｏｔｕｂｅｓ．Ｃａｎｃｅｒ　Ｓｃｉｅｎｃｅ．１０５：７６３－７６９，２０１４
・Ｍｕｌｔｉｗａｌｌｅｄ　ｃａｒｂｏｎ　ｎａｎｏｔｕｂｅｓ　ｉｎｔｒａｔｒａｃｈｅａｌｌｙ　ｉｎｓｔｉｌｌｅｔｄ　ｉｎｔｏ　ｔｈｅ　ｒａｔ　ｌｕｎｇ　ｉｎｄｕｃｅ　ｄｅｖｅｌｏｐｍｅｎｔ　ｏｆ　ｐｌｅｕｒａｌ　ｍａｌｉｇｎａｎｔ　ｍｅｓｏｔｈｅｌｉｏｍａ　ａｎｄ　ｌｕｎｇ　ｔｕｍｏｒｓ．　Ｃａｎｃｅｒ　Ｓｃｉｅｎｃｅ，１０７（７）：９２４－９３５　２０１６．
・Ｐｅｒｓｉｓｔｅｎｔ　Ｐｌｅｕｒａｌ　Ｌｅｓｉｏｎｓ　ａｎｄ　Ｉｎｆｌａｍｍａｔｉｏｎ　ｂｙ　Ｐｕｌｍｏｎａｒｙ　Ｅｘｐｏｓｕｒｅ　ｏｆ　Ｍｕｌｔｉｗａｌｌｅｄ　Ｃａｒｂｏｎ　Ｎａｎｏｔｕｂｅｓ，Ｃｈｅｍ．Ｒｅｓ．Ｔｏｘｉｃｏｌ．，１５；３１（１０）：１０２５－１０３１．２０１８．
・Ｃｏｍｐａｒａｔｉｖｅ　ｐｕｌｍｏｎａｒｙ　ｔｏｘｉｃｉｔｙ　ｏｆ　ａ　ＤＷＣＮＴ　ａｎｄ　ＭＷＣＮＴ－７　ｉｎ　ｒａｔｓ，Ａｒｃｈ．Ｔｏｘｉｃｏｌ．，９３：４９－５９，２０１９
・Ｐｕｌｍｏｎａｒｙ　ａｎｄ　ｐｌｅｕｒａｌ　ｔｏｘｉｃｉｔｙ　ｏｆ　ｐｏｔａｓｓｉｕｍ　ｏｃｔａｔｉｔａｎａｔｅ　ｆｉｂｅｒｓ，　ｒｕｔｉｌｅ　ｔｉｔａｎｉｕｍ　ｄｉｏｘｉｄｅ　ｎａｎｏｐａｒｔｉｃｌｅｓ，ａｎｄ　ＭＷＣＮＴ－７　ｉｎ　ｍａｌｅ　Ｆｉｓｃｈｅｒ　３４４　ｒａｔｓ，Ａｒｃｈ．Ｔｏｘｉｃｏｌ．，９３（４）：９０９－９２０，２０１９
・Ｄｅｖｅｌｏｐｍｅｎｔ　ｏｆ　ｉｎｔｒａｔｒａｃｈｅａｌ　ｉｎｔｒａｐｕｌｍｏｎａｒｙ　ｏｆ　ｓｐｒａｉｎｇ　（ＴＩＰＳ）　ａｄｍｉｎｉｓｔｒａｔｉｏｎ　ａｓ　ａ　ｆｅａｓｉｂｌｅ　ａｓｓａｙ　ｍｅｔｈｏｄ　ｆｏｒ　ｔｅｓｔｉｎｇ　ｔｈｅ　ｔｏｘｉｃｉｔｙ　ａｎｄ　ｃａｒｃｉｎｏｇｅｎｉｃ　ｐｏｔｅｎｔｉａｌ　ｏｆ　ｍｕｌｔｉｗａｌｌ　ｃａｒｂｏｎ　ｎａｎｏｔｕｂｅｓ，ｉｎ　Ｖｉｖｏ　Ｉｎｈａｌａｔｉｏｎ　Ｔｏｘｉｃｉｔｙ　Ｓｃｒｅｅｎｉｎｇ　Ｍｅｔｈｏｄｓ　ｆｏｒ　Ｍａｎｕｆａｃｔｕｒｅｄ　Ｎａｎｏｍａｔｅｒｉａｌｓ，Ｉｎ　Ｃｕｒｒｅｎｔ　Ｔｏｐｉｃｓ　ｉｎ　Ｅｎｖｉｒｏｎｍｅｎｔａｌ　Ｈｅａｌｔｈ　ａｎｄ　Ｐｒｅｖｅｎｔｉｖｅ　Ｍｅｄｉｃｉｎｅ，　Ｓｐｒｉｎｇｅｒ　（Ｓｐｒｉｎｇｅｒ　Ｎａｔｕｒｅ　Ｓｉｎｇａｐｏｒｅ　Ｐｔｅ　Ｌｔｄ），ｐｐ１４５－１６３，２０１９，ＤＯＩ：１０．１００７／９７８－９８１－１３－８４３３－２
・ＭＷＣＮＴ－７　ａｄｍｉｎｉｓｔｅｒｅｄ　ｔｏ　ｔｈｅ　ｌｕｎｇ　ｂｙ　ｉｎｔｒａｔｒａｃｈｅａｌ　ｉｎｓｔｉｌｌａｔｉｏｎ　ｉｎｄｕｃｅｓ　ｄｅｖｅｌｏｐｍｅｎｔ　ｏｆ　ｐｌｅｕｒａｌ　ｍｅｓｏｔｈｅｌｉｏｍａ　ｉｎ　Ｆ３４４　ｒａｔｓ，Ｃａｎｃｅｒ　Ｓｃｉ．，１１０（８）：２４８５－２４９２，　２０１９
・Ｃａｒｃｉｎｏｇｅｎｉｃ　ｅｆｆｅｃｔ　ｏｆ　ｐｏｔａｓｓｉｕｍ　ｏｃｔａｔｉｔｎａｔｅ（ＰＯＴ）ｆｉｂｅｒｓ　ｉｎ　ｔｈｅ　ｌｕｎｇ　ａｎｄ　ｐｌｅｕｒａ　ｏｆ　ｍａｌｅ　Ｆｉｓｃｈｅｒ　３４４　ｒａｔｓ　ａｆｔｅｒ　ｉｎｔｒａｐｕｌｍｏｎａｒｙ　ａｄｍｉｎｉｓｔｒａｔｉｏｎ，Ｐａｒｔｉｃｌｅ　ａｎｄ　Ｆｉｂｒｅ　Ｔｏｘｉｃｏｌｏｇｙ，１６：ｈｔｔｐｓ：／／ｄｏｉ．ｏｒｇ／１０．１１８６／ｓ１２９８９－０１９－０３１６－２，２０１９，
・Ｐｌｅｕｒａｌ　ｔｒａｎｓｌｏｃａｔｉｏｎ　ａｎｄ　ｌｅｓｉｏｎｓ　ｂｙ　ｐｕｌｍｏｎａｒｙ　ｅｘｐｏｓｅｄ　ｍｕｌｔｉ－ｗａｌｌｅｄ　ｃａｒｂｏｎ　ｎａｎｏｔｕｂｅｓ，Ｊ．Ｔｏｘｉｃ．Ｐａｔｈｏｌ．，３３（３）：１４５－１５１，　２０２０
・Ｃｏｍｐａｒｅｔｉｖｅ　ｃａｒｃｉｎｏｇｅｎｉｃｉｔｙ　ｓｔｕｄｙ　ｏｆ　ａ　ｔｈｉｃｋ，　ｓｔｒａｉｇｈｔ－ｔｙｐｅ　ａｎｄ　ａ　ｔｈｉｎ，　ｔａｎｇｌｅｄ－ｔｙｐｅ　ｍｕｌｔｉ－ｗａｌｌｅｄ　ｂｙ　ｃａｒｂｏｎ　ｎａｎｏｔｕｂｅ　ａｄｍｉｎｉｓｔｅｒｅｄ　ｂｙ　ｉｎｔｒａ－ｔｒａｃｈｅａｌ　ｉｎｓｔｉｌｌａｔｉｏｎ　ｉｎ　ｔｈｅ　ｒａｔ．　　Ｐａｒｔｉｃｌｅ　ａｎｄ　Ｆｉｂｒｅ　Ｔｏｘｉｃｏｌｏｇｙ，１７：４８（２０２０）． Regarding TIPS, for example, the contents of the documents listed below can be incorporated herein.
- Size-and-shape-dependent pleural translocation, deposition, fibrogenesis and mesothelial proliferation by multi-walled carbon nanotubes. Cancer Science. 105:763-769, 2014
・Multiwalled carbon nanotubes intracheally instilled into the rat lung induce development of pleural alignment mesothelioma and lung tumors. Cancer Science, 107(7):924-935 2016.
• Persistent Pleural Lesion and Inflammation by Pulmonary Exposure of Multiwalled Carbon Nanotubes, Chem. Res. Toxicol. , 15;31(10):1025-1031.2018.
- Comparative pulmonary toxicity of a DWCNT and MWCNT-7 in rats, Arch. Toxicol. , 93:49-59, 2019
- Pulmonary and pleural toxicity of potassium octitatanate fibers, rutile titanium dioxide nanoparticle, and MWCNT-7 in male Fischer 344 rats, Arch. Toxicol. , 93(4):909-920, 2019
・Ｄｅｖｅｌｏｐｍｅｎｔ ｏｆ ｉｎｔｒａｔｒａｃｈｅａｌ ｉｎｔｒａｐｕｌｍｏｎａｒｙ ｏｆ ｓｐｒａｉｎｇ （ＴＩＰＳ） ａｄｍｉｎｉｓｔｒａｔｉｏｎ ａｓ ａ ｆｅａｓｉｂｌｅ ａｓｓａｙ ｍｅｔｈｏｄ ｆｏｒ ｔｅｓｔｉｎｇ ｔｈｅ ｔｏｘｉｃｉｔｙ ａｎｄ ｃａｒｃｉｎｏｇｅｎｉｃ ｐｏｔｅｎｔｉａｌ ｏｆ ｍｕｌｔｉｗａｌｌ ｃａｒｂｏｎ ｎａｎｏｔｕｂｅｓ，ｉｎ Ｖｉｖｏ Ｉｎｈａｌａｔｉｏｎ Ｔｏｘｉｃｉｔｙ Ｓｃｒｅｅｎｉｎｇ Ｍｅｔｈｏｄｓ ｆｏｒ Ｍａｎｕｆａｃｔｕｒｅｄ Ｎａｎｏｍａｔｅｒｉａｌｓ，Ｉｎ Ｃｕｒｒｅｎｔ Ｔｏｐｉｃｓ ｉｎ Ｅｎｖｉｒｏｎｍｅｎｔａｌ Ｈｅａｌｔｈ ａｎｄ Ｐｒｅｖｅｎｔｉｖｅ Ｍｅｄｉｃｉｎｅ， Ｓｐｒｉｎｇｅｒ （ Springer Nature Singapore Pte Ltd), pp145-163, 2019, DOI: 10.1007/978-981-13-8433-2
・MWCNT-7 administered to the lung by intratracheal installation induces development of pleural mesothelioma in F344 rats, Cancer Sci. , 110(8):2485-2492, 2019
・Carcinogenic effect of potassium octatinate (POT) fibers in the lung and pleura of male Fischer 344 rats after intrapulmonary administration, Particle and Fiber topic 1: org/10.1186/s12989-019-0316-2, 2019,
- Pleural translocation and lesions by plummonary exposed multi-walled carbon nanotubes, J. Am. Toxic. Pathol. , 33(3):145-151, 2020
・Comparative carcinogenicity study of a thick, straight-type and a thin, tangled-type multi-walled by carbon nanotube administrator by intra-tracheal installation in the. Particle and Fiber Toxicology, 17:48 (2020).
　試験物質の投与回数は特に限定されない。つまり、試験物質の投与に際しては、総投与量βの試験物質を複数回に分けて投与してもよく、１回で投与してもよい。例えば、試験動物の全滅を防止することで毒性値γを求めやすくするために、総投与量βの試験物質を複数回に分けて投与することができる。 The number of administrations of the test substance is not particularly limited. That is, when administering the test substance, the total dose β of the test substance may be administered in multiple doses or may be administered in one dose. For example, the total dose β of the test substance can be divided into multiple doses in order to facilitate determination of the toxicity value γ by preventing annihilation of test animals.
　ｃでは、数時間から半日の期間をかけて、試験物質を試験動物の気管内で噴霧して複数回投与することができる。複数回の試験物質の投与量は互いに同一でもよく、発明の効果が得られる範囲内であれば異なっていてもよい。
　ただし、得られる毒性値γの信頼度の点から、複数回の試験物質の各回の投与量は、互いに同一、つまり一定であることが好ましい。ｎ回に分けて噴霧投与を行うとき、ｎ回の試験物質の投与量が互いに同一であるなら、各回の試験物質の投与量はβ／ｎとなる。 In c, the test substance can be administered multiple times by nebulization into the trachea of the test animal over a period of several hours to half a day. The dosage of the test substance for multiple times may be the same or may be different as long as the effects of the invention are obtained.
However, from the viewpoint of the reliability of the obtained toxicity value γ, it is preferable that the doses of the test substance are the same each time, that is, constant. When nebulization is performed in n divided doses, if the doses of the test substance n times are the same, the dose of the test substance in each time is β/n.
　総投与量βの試験物質を複数回に分けて投与する場合、各回の投与間隔は一定でもよく、発明の効果が得られる範囲内であれば不規則でもよい。得られる毒性値γの信頼度の点から、投与間隔は一定、つまり等間隔とすることが好ましい。 When administering the total dose β of the test substance in multiple doses, the intervals between each administration may be constant, or may be irregular as long as the effects of the invention are obtained. From the viewpoint of the reliability of the obtained toxicity value γ, it is preferable that the administration interval is constant, that is, the interval is equal.
　試験動物の動物数は特に限定されない。例えば、例えば米国Ｎａｔｉｏｎａｌ　Ｔｏｘｉｃｏｌｏｇｙ　Ｐｒｏｇｒａｍ（ＮＴＰ）レポートにおけるがん原性試験では、１用量群の通例は５０匹である。動物数は、試験物質、試験目的に応じて適宜変更すればよい。例えば、１群あたり数匹から十数匹の動物群を複数用意し、そのうちから少なくともＬＤ５０値が得られるように抽出する。 The number of test animals is not particularly limited. For example, in carcinogenicity studies, eg, in the US National Toxicology Program (NTP) report, one dose group is typically 50 animals. The number of animals may be appropriately changed according to the test substance and purpose of the test. For example, a plurality of groups of several animals to more than ten animals are prepared per group, and the animals are extracted so as to obtain at least the LD50 value.
　毒性値γは、試験物質の投与終了後、観察期間が経過したときの試験動物の生存率が４０～６０％、好ましくは４５～５５％、より好ましくは５０％となるときの試験物質の投与量として算出できる。毒性値γは、半数の試験動物が死亡する投与量、すなわち、ＬＤ５０値として導出することが特に好ましい。したがって、毒性値γの値が相対的に小さいほど、試験物質の毒性が相対的に高いことを意味することになる。一方、毒性値γの値が相対的に大きいほど、試験物質の毒性が相対的に低いことを意味することになる。 The toxicity value γ is the test substance administration at which the survival rate of the test animal becomes 40 to 60%, preferably 45 to 55%, more preferably 50% after the observation period has elapsed after the administration of the test substance. It can be calculated as a quantity. It is particularly preferred to derive the toxicity value γ as the dose at which half of the test animals die, ie the LD50 value. Therefore, a relatively small value of the toxicity value γ means a relatively high toxicity of the test substance. On the other hand, a relatively large value of toxicity value γ means that the toxicity of the test substance is relatively low.
　観察期間は、試験目的に応じて適宜変更可能である。観察期間の終了点は、毒性値γを試験動物の５０％生存率から導出できるように選択すればよい。
　一態様においてｃでは、総投与量βの試験物質の投与終了後少なくとも１週間以上、試験動物に試験物質を投与せずに試験動物を観察することで、試験物質の急性毒性を評価できる。 The observation period can be changed as appropriate depending on the purpose of the study. The endpoint of the observation period should be chosen so that the toxicity value γ can be derived from the 50% survival rate of the test animals.
In one aspect c, the acute toxicity of the test substance can be assessed by observing the test animal without administration of the test substance for at least one week after the end of administration of the total dose β of the test substance.
　一態様においてｃでは、総投与量βの試験物質の投与終了後少なくとも２週間以上、試験動物に試験物質を投与せずに試験動物を観察することで、試験物質の慢性毒性（ＬＤ５０値）を評価できる。この場合、観察期間は２週間以上が好ましく、４週間以上がより好ましい。 In one aspect c, the chronic toxicity (LD50 value) of the test substance is determined by observing the test animal without administering the test substance to the test animal for at least 2 weeks after the end of administration of the test substance at the total dose β. can be evaluated. In this case, the observation period is preferably 2 weeks or longer, more preferably 4 weeks or longer.
　一態様においてｃでは、総投与量βの試験物質の投与終了後少なくとも１２か月以上、試験動物に試験物質を投与せずに試験動物を観察することで、試験物質のがん原性を評価できる。例えば、医薬品のがん原生試験に関するガイドライン（平成１１年１１月１日付）においては、ラットでは２４か月以上３０か月以内、マウスおよびハムスターでは１８か月以上２４か月以内と試験期間が定められている。 In one aspect c, carcinogenicity of the test substance is evaluated by observing the test animal without administration of the test substance for at least 12 months after the end of administration of the total dose β of the test substance. can. For example, in the Guidelines for Oncogenesis Tests for Pharmaceuticals (dated November 1, 1999), the test period is defined as 24 to 30 months for rats and 18 to 24 months for mice and hamsters. It is
　（作用機序）
　以上説明した本発明の毒性試験方法では、予備試験αから総投与量βを算出し、ＴＩＰＳ投与によって毒性値γを求める。この本法は高価な設備の使用を必要としない。また、得られる結果としての毒性値γは従来の高価な設備と稼働費用を要する吸入暴露法による結果と非常に近い値である。つまり、本法では実用上充分に満足できる値が毒性値γとして得られる。よって、本発明の毒性試験方法によれば、化学物質の吸入毒性を安価で簡便に評価できる。 (Mechanism of action)
In the toxicity test method of the present invention described above, the total dose β is calculated from the preliminary test α, and the toxicity value γ is obtained by administering TIPS. This method does not require the use of expensive equipment. Also, the resulting toxicity value γ is very close to that obtained by the conventional inhalation exposure method, which requires expensive equipment and operating costs. In other words, in this method, a practically satisfactory value can be obtained as the toxicity value γ. Therefore, according to the toxicity test method of the present invention, the inhalation toxicity of chemical substances can be evaluated easily and inexpensively.
［毒性試験キット］
　本発明の毒性試験キットは、上述の本発明の毒性試験方法のために使用される。
　毒性試験キットは、色素と、細胞と、該細胞を培養するための培養液とを備える。色素、細胞、培養液の詳細および好ましい態様は、既に毒性試験方法について説明した内容と同じである。 [Toxicity test kit]
The toxicity test kit of the present invention is used for the toxicity test method of the present invention described above.
A toxicity test kit comprises a dye, cells, and a medium for culturing the cells. The details and preferred aspects of the dye, cells, and culture medium are the same as those already explained for the toxicity test method.
　毒性試験キットは、色素、細胞、および該細胞を培養するための培養液に加えて、噴霧針、注射器および取扱説明書からなる群から選ばれる少なくとも１つをさらに備え得る。
　毒性試験キットの使用方法については、既に毒性試験方法について説明した内容および技法を適用できる。 The toxicity test kit may further include at least one selected from the group consisting of a spray needle, a syringe and an instruction manual, in addition to the dye, cells, and culture medium for culturing the cells.
For the method of using the toxicity test kit, the contents and techniques already explained for the toxicity test method can be applied.
［実験例Ａ］
　以下、いくつかの実験例を示して本発明をさらに詳細に説明するが、本発明は以下の記載に限定されない。 [Experimental example A]
Hereinafter, the present invention will be described in more detail by showing several experimental examples, but the present invention is not limited to the following description.
　（使用量αの取得）
　Ｎｅｕｔｒａｌ　Ｒｅｄ試験を用いることで、ヒトの肺がん由来のＡ５４９細胞の生存率から使用量αを求めた。Ｎｅｕｔｒａｌ　Ｒｅｄ試験について、以下の参考文献の記載を参考にした。
　参考文献：Ｒｅｐｅｔｔｏ，Ｇ．ｅｔ　ａｌ．，　Ｎｅｕｔｒａｌ　Ｒｅｄ　ｕｐｔａｋｅ　ａｓｓａｙ　ｆｏｒ　ｔｈｅ　ｅｓｔｉｍａｔｉｏｎ　ｏｆ　ｃｅｌｌ　ｖｉａｂｉｌｉｔｙ／ｃｙｔｏｔｏｘｉｃｉｔｙ．　Ｎａｔｕｒｅ　Ｐｒｏｔｏｃｏｌｓ，　７，　１１２５，　２０００． (Acquisition of usage amount α)
Using the Neutral Red test, the usage amount α was determined from the viability of human lung cancer-derived A549 cells. Regarding the Neutral Red test, the following references were referred to.
References: Repetto, G.; et al. , Neutral Red uptake assay for the estimation of cell viability/cytotoxicity. Nature Protocols, 7, 1125, 2000.
　試験物質として、アセチルアセトン、エチレングリコールモノエチルアセテート、アクリル酸ポリマー、Ｎ，Ｎ－ジメチルアセトアミドを準備した。肺がん由来のＡ５４９細胞の培養液に試験物質を加えて１０～３０分程度の短時間で培養した。その後試験物質を含まず、かつ、ニュートラルレッドを含む培養液に交換した。培養液としては、無血清のＲＰＭＩ培地に１０％　Ｂｏｖｉｎｅ　Ｃａｌｆ　Ｓｅｒｕｍを加えたものを用いた。次いで、Ａ５４９細胞のニュートラルレッドの取り込み率（＝生存率）を計測した（図１）。その後、Ａ５４９細胞のＬＤ５０％を示す培養液中の試験物質の投与量（ｍｇ／Ｌ）から使用量αを求めた。 Acetylacetone, ethylene glycol monoethyl acetate, acrylic acid polymer, and N,N-dimethylacetamide were prepared as test substances. A test substance was added to the culture medium of lung cancer-derived A549 cells, and cultured for a short time of about 10 to 30 minutes. After that, the medium was replaced with a medium containing neutral red but no test substance. A serum-free RPMI medium supplemented with 10% Bovine Calf Serum was used as the culture medium. Next, the neutral red uptake rate (=viability) of A549 cells was measured (Fig. 1). After that, the amount α used was determined from the dose (mg/L) of the test substance in the culture medium showing LD50% of A549 cells.
　（総投与量βへの換算）
　本例では、ラットの体重および肺活量に基づいて使用量αを総投与量βに換算した。使用量αから１０用量程度となるような総投与量βを求めた。図２に、試験物質がアセチルアセトンの例を示す。図２のアセチルアセトンの場合、総投与量βは１２．５ｍｇ／ｋｇと１００ｍｇ／ｋｇの間であった。 (Conversion to total dose β)
In this example, the dose α was converted to the total dose β based on the body weight and vital capacity of the rat. A total dose β was determined so that about 10 doses were obtained from the amount α used. FIG. 2 shows an example in which the test substance is acetylacetone. For acetylacetone in FIG. 2, the total dose β was between 12.5 mg/kg and 100 mg/kg.
　換算式、換算方法についてアクリル酸ポリマーの例を用いて図３を参照しながら説明する。
　図３は、Ｎｅｕｔｒａｌ　Ｒｅｄ試験における試験物質の濃度（ｍｇ／Ｌ）を気管内噴霧投与（ＴＩＰＳ投与）のラットの体重／ｋｇに換算した値を示す。
　図３中「Ａ」の列、「Ｎｅｕｔｒａｌ　Ｒｅｄ試験の結果（ｉｎ　Ｖｉｔｏｒｏ　用量μＬ／ｍｌ）」が、使用量αに対応する。また、「Ｈ」の列、「ＴＩＰＳの４回分の合計用量Ｄｏｓｅ／４ＴＩＰＳ（ｍｇ／ｋｇ）」が、総投与量βに対応する。実験例Ａでは４回に分けてＴＩＰＳ投与を行った。
　図３の上段は、Ａ５４９細胞の培地中の９０％生存用量のラット投与用量への換算を示す。下段はＡ５４９細胞の培地中の１０％以下の生存用量のラット投与用量への換算を示す。 A conversion formula and a conversion method will be described with reference to FIG. 3 using an example of an acrylic acid polymer.
FIG. 3 shows values obtained by converting the concentration (mg/L) of the test substance in the Neutral Red test into body weight/kg of rats administered intratracheal spray (TIPS administration).
Column "A" in FIG. 3, "Neutral Red test result (in Vitoro dose μL/ml)" corresponds to the amount α used. Also, the "H" column, "Total Dose of 4 TIPS/4 TIPS (mg/kg)" corresponds to the total dose β. In Experimental Example A, TIPS was administered in 4 divided doses.
The upper panel of FIG. 3 shows the conversion of the 90% viable dose in culture of A549 cells to the dose administered to rats. The bottom row shows the conversion of the survival dose of 10% or less in the medium of A549 cells to the dose administered to rats.
　（ＴＩＰＳ投与による毒性値γの取得）
　１群あたり５～１０匹のラットからなる用量群を５～１０群用意した。各群において、ラットにＴＩＰＳ投与を行った。
　１０～１２週齢のラットに対して、初回の投与から、１時間後、２時間後、３時間後の計４回にわたって、各回の投与量をβ／４としてＴＩＰＳ投与を実施した。その後、試験物質を投与せずに２週間観察し、投与終了後２週までに半数のラットが死亡する投与量として毒性値γを求めた。毒性値γは、ＬＤ５０値である。 (Acquisition of toxicity value γ by TIPS administration)
Five to ten dose groups of 5 to 10 rats per group were prepared. In each group, rats were administered TIPS.
TIPS was administered to 10- to 12-week-old rats four times in total, 1 hour, 2 hours, and 3 hours after the initial administration, with a dose of β/4 each time. Thereafter, the rats were observed for 2 weeks without administration of the test substance, and the toxicity value γ was determined as the dose at which half of the rats died within 2 weeks after the end of administration. The toxicity value γ is the LD50 value.
　毒性値γの結果を図４～７に示す。これらは、１群８匹で５～７用量群の例におけるＬＤ５０値である。図４～７中、８／８等の分数は、ラットの生存割合を示す。例えば「６／８」は、８匹中６匹のラットが生存していることを示す（以下同様）。 The results of toxicity value γ are shown in Figures 4-7. These are the LD50 values in the example of 5-7 dose groups with 8 animals per group. In Figures 4-7, fractions such as 8/8 indicate survival rates of rats. For example, "6/8" indicates that 6 out of 8 rats are alive (and so on).
　図４に示すようにグリシドールについて、ＬＤ５０値、すなわち、毒性値γは１６０ｍｇ／ｋｇと３２０ｍｇ／ｋｇの間であった。
　図５に示すようにＮ，Ｎ－ジメチルホルムアミドについて、ＬＤ５０値、すなわち、毒性値γは１２８０ｍｇ／ｋｇと２５６０ｍｇ／ｋｇの間であった。
　図６に示すようにアクリル酸ポリマーについて、ＬＤ５０値、すなわち、毒性値γは１２０ｍｇ／ｋｇと２４０ｍｇ／ｋｇの間であった。
　図７に示すようにアセチルアセトンについて、ＬＤ５０値、すなわち、毒性値γは４００ｍｇ／ｋｇと８００ｍｇ／ｋｇの間であった。 For glycidol as shown in Figure 4, the LD50 value, ie the toxicity value γ, was between 160 mg/kg and 320 mg/kg.
As shown in FIG. 5, the LD50 value, ie toxicity value γ, was between 1280 mg/kg and 2560 mg/kg for N,N-dimethylformamide.
As shown in FIG. 6, the LD50 value, ie toxicity value γ, was between 120 mg/kg and 240 mg/kg for the acrylic acid polymer.
For acetylacetone as shown in Figure 7, the LD50 value, ie the toxicity value γ, was between 400 mg/kg and 800 mg/kg.
　図８に示すように、毒性値γを従来の吸入暴露法（４時間吸入暴露）のＬＣ５０値と比較した。図８中、ＴＩＰＳ　ＬＤ５０ｍｇ／ｋｇが毒性値γに対応する。
　従来の４時間吸入暴露試験によって得られている急性毒性ＬＣ５０値と、本発明の毒性値γ、すなわち、ＴＩＰＳ　ＬＤ５０とを比較すると、ＴＩＰＳ　ＬＤ５０の各値は、急性毒性ＬＣ５０値の４倍以内に収まることが示された。このように求めた毒性値γ、すなわち、ＴＩＰＳ　ＬＤ５０は、妥当性のある数値であると考えられる。 As shown in FIG. 8, the toxicity value γ was compared with the LC50 value of the conventional inhalation exposure method (4-hour inhalation exposure). In FIG. 8, TIPS LD50 mg/kg corresponds to the toxicity value γ.
When comparing the acute toxicity LC50 value obtained by the conventional 4-hour inhalation exposure test and the toxicity value γ of the present invention, that is, TIPS LD50, each value of TIPS LD50 is within 4 times the acute toxicity LC50 value. shown to fit. The toxicity value γ determined in this way, that is, TIPS LD50, is considered to be a valid value.
［実験例Ｂ］
　（使用量αの取得）
　Ｎｅｕｔｒａｌ　Ｒｅｄ試験を用いることで、ヒトの肺がん由来のＡ５４９細胞の生存率から使用量αを求めた。Ｎｅｕｔｒａｌ　Ｒｅｄ試験については、実験例Ａの項にて説明した通りである。２ｍＬに分取したＲＰＭＩ培養液中に１，４－ジオキサンを２，４，８，１６，３２，６４，１２８，２５６，５１２，１０２４μＬそれぞれ加えた。それぞれの培養液中にＡ５４９肺がん細胞を入れた。１５分の培養後に培養液を除去した。その後、Ｎｅｕｔｒａｌ　Ｒｅｄ（０．０３３ｍｇ／ｍｌ）を加えた培養液にてＡ５４９細胞を３時間培養した。この結果に基づいて実験例Ａと同じ手法により得られた１，４－ジオキサンのＬＤ５０≒１５４．７μＬ／ｍＬを使用量αとして取得した。
　求めた１，４－ジオキサンの使用量α：１５４．７μＬ／ｍＬから、最低使用量（７５％）と最高使用量（２００％）の間でＴＩＰＳ試験に使用する用量を設定した。最低使用量は、１５４．７μＬ／ｍＬ×０．７５＝１１６μＬ／ｍＬと算出した。最高使用量は、１５４．７μＬ／ｍＬ×２＝３１０μＬ／ｍＬと算出した。 [Experimental example B]
(Acquisition of usage amount α)
Using the Neutral Red test, the usage amount α was determined from the viability of human lung cancer-derived A549 cells. The Neutral Red test is as described in Experimental Example A. 2, 4, 8, 16, 32, 64, 128, 256, 512 and 1024 μL of 1,4-dioxane were added to each 2 mL of RPMI culture medium. A549 lung cancer cells were placed in each culture medium. After 15 minutes of incubation, the medium was removed. After that, the A549 cells were cultured for 3 hours in a culture medium containing Neutral Red (0.033 mg/ml). Based on this result, LD50≈154.7 μL/mL of 1,4-dioxane obtained by the same method as in Experimental Example A was obtained as the usage amount α.
From the calculated amount α of 1,4-dioxane used: 154.7 μL/mL, the dose used in the TIPS test was set between the minimum usage amount (75%) and the maximum usage amount (200%). The minimum usage was calculated as 154.7 μL/mL×0.75=116 μL/mL. The maximum amount used was calculated as 154.7 μL/mL×2=310 μL/mL.
　（総投与量βへの換算）
　総投与量βを算出するために、まず、ＴＩＰＳ１回分の投与量（β／１）を以下の算出式で求めた。
　β／１＝（使用した１，４－ジオキサンの濃度：１．０３４ｍｇ／ｍＬ）×（１，４－ジオキサンの使用量α：１５４．７μＬ／ｍＬ）×（ラット肺活量：２μＬ／ｋｇ）＝３２０ｍｇ／ｋｇ。ここで、ラット肺活量は１ｋｇあたりの肺活量に変換した。
　４回投与するための総投与量βとして、β＝４×（β／１）、つまり、３２０ｍｇ／ｋｇ×４＝１２８０ｍｇ／ｋｇとして総投与量βを求めた。 (Conversion to total dose β)
In order to calculate the total dose β, first, the dose (β/1) for one TIPS was determined by the following formula.
β/1=(concentration of 1,4-dioxane used: 1.034 mg/mL)×(amount of 1,4-dioxane used α: 154.7 μL/mL)×(rat vital capacity: 2 μL/kg)=320 mg /kg. Here, the rat vital capacity was converted to vital capacity per kg.
As the total dose β for 4 administrations, the total dose β was calculated as β=4×(β/1), that is, 320 mg/kg×4=1280 mg/kg.
　（ＴＩＰＳ投与による毒性値γの取得）
　１群あたり７匹のラットからなる用量群を６群用意した。各群において、ラットにＴＩＰＳ投与を行った。各群の投与量は、ＴＩＰＳ試験の総投与量に対して７５％，１００％，１２５％，１５０％，１７５％，２００％の６点とした。つまり、総投与量を９６０ｍｇ／ｋｇ，１２８０ｍｇ／ｋｇ，１６００ｍｇ／ｋｇ，１９２０ｍｇ／ｋｇ，２２４０ｍｇ／ｋｇ，２５６０ｍｇ／ｋｇとする６群をそれぞれ用意した。 (Acquisition of toxicity value γ by TIPS administration)
There were 6 dose groups of 7 rats per group. In each group, rats were administered TIPS. The dosage of each group was set to 6 points of 75%, 100%, 125%, 150%, 175%, and 200% of the total dosage in the TIPS test. That is, six groups were prepared with total doses of 960 mg/kg, 1280 mg/kg, 1600 mg/kg, 1920 mg/kg, 2240 mg/kg and 2560 mg/kg.
　１０～１２週齢のラットに対して、初回の投与から、１時間後、２時間後、３時間後の計４回にわたって、各回の投与量をβ／４としてＴＩＰＳ投与を実施した。その後、試験物質を投与せずに合計８日間観察し、８日目までに半数のラットが死亡する投与量として毒性値γを求めた（図９）。毒性値γは、ＬＤ５０値である。 　TIPS was administered to 10- to 12-week-old rats at a dose of β/4 for a total of 4 times, 1 hour, 2 hours, and 3 hours after the initial administration. Thereafter, the rats were observed for a total of 8 days without administration of the test substance, and the toxicity value γ was determined as the dose at which half of the rats died by the 8th day (Fig. 9). The toxicity value γ is the LD50 value.
　各総投与量の試験動物の生存率をプロットしたグラフから、ＬＤ５０値を得た。既知の吸入暴露から得られた値と近似した。結果を図１０に示す。結果、１，４－ジオキサンについて、ＬＤ５０値、すなわち、毒性値γは１２８０ｍｇ／ｋｇと１６００ｍｇ／ｋｇの間にあった。
　１，４－ジオキサンの国際がん研究機関（ＩＡＲＣ）による発がん性の分類は、Ｇ２Ｂである。Ｇ２Ｂに分類された物質は、ヒトに対して発がん性がある可能性がある。 LD50 values were obtained from graphs plotting the survival rate of test animals at each total dose. Values obtained from known inhalation exposures were approximated. The results are shown in FIG. As a result, the LD50 value, ie toxicity value γ, was between 1280 mg/kg and 1600 mg/kg for 1,4-dioxane.
The International Agency for Research on Cancer (IARC) carcinogenicity classification for 1,4-dioxane is G2B. Substances classified as G2B are potentially carcinogenic to humans.
　以上説明した１，４－ジオキサンの場合と同じ手法によって、以下の試験物質についても毒性値γを求めた。
・グリシジルメタクリレート
・アクロレイン
・キシレン
・１，２－ジクロロエタン
・キノリン
・ｔ－ブチルアルコール
　結果を図１１－図１６にそれぞれ示す。 Toxicity value γ was determined for the following test substances by the same method as for 1,4-dioxane described above.
• Glycidyl methacrylate • Acrolein • Xylene • 1,2-dichloroethane • Quinoline • t-butyl alcohol The results are shown in Figures 11 to 16, respectively.
　図１１に示すようにグリシジルメタクリレートについて、ＬＤ５０値、すなわち、毒性値γは２４０ｍｇ／ｋｇと４８０ｍｇ／ｋｇの間であった。
　図１２に示すようにアクロレインについて、ＬＤ５０値、すなわち、毒性値γは１ｍｇ／ｋｇと２ｍｇ／ｋｇの間であった。
　図１３に示すようにキシレンについて、ＬＤ５０値、すなわち、毒性値γは１００ｍｇ／ｋｇと４００ｍｇ／ｋｇの間であった。
　図１４に示すように１，２－ジクロロエタンについて、ＬＤ５０値、すなわち、毒性値γは２４０ｍｇ／ｋｇと４８０ｍｇ／ｋｇの間であった。
　図１５に示すようにキノリンについて、ＬＤ５０値、すなわち、毒性値γは８０ｍｇ／ｋｇ以上にあった。
　図１６に示すようにｔ－ブチルアルコールについて、ＬＤ５０値、すなわち、毒性値γは８００ｍｇ／ｋｇ以上にあった。 For glycidyl methacrylate as shown in Figure 11, the LD50 value, ie the toxicity value γ, was between 240 mg/kg and 480 mg/kg.
As shown in Figure 12, for acrolein, the LD50 value, ie toxicity value γ, was between 1 mg/kg and 2 mg/kg.
As shown in Figure 13, for xylene, the LD50 value, ie toxicity value γ, was between 100 mg/kg and 400 mg/kg.
For 1,2-dichloroethane as shown in FIG. 14, the LD50 value, ie toxicity value γ, was between 240 mg/kg and 480 mg/kg.
As shown in FIG. 15, quinoline had an LD50 value, that is, a toxicity value γ of 80 mg/kg or more.
As shown in FIG. 16, the LD50 value, that is, the toxicity value γ, was 800 mg/kg or more for t-butyl alcohol.
　図１７に示すように、毒性値γを従来の吸入暴露法（４時間吸入暴露）のＬＣ５０値と比較した。図１７中、ＴＩＰＳ　ＬＤ５０ｍｇ／ｋｇが毒性値γに対応する。
　従来の４時間吸入暴露試験によって得られている急性毒性ＬＣ５０値と、本発明の毒性値γ、すなわち、ＴＩＰＳ　ＬＤ５０とを比較すると、ＴＩＰＳ　ＬＤ５０の各値は、急性毒性ＬＣ５０値の４倍以内に収まることが実験例Ｂにおいても示された。このように求めた毒性値γ、すなわち、ＴＩＰＳ　ＬＤ５０は、妥当性のある数値であると考えられる。 As shown in FIG. 17, the toxicity value γ was compared with the LC50 value of the conventional inhalation exposure method (4-hour inhalation exposure). In FIG. 17, TIPS LD50 mg/kg corresponds to the toxicity value γ.
When comparing the acute toxicity LC50 value obtained by the conventional 4-hour inhalation exposure test and the toxicity value γ of the present invention, that is, TIPS LD50, each value of TIPS LD50 is within 4 times the acute toxicity LC50 value. It was also shown in Experimental example B that it was accommodated. The toxicity value γ determined in this way, that is, TIPS LD50, is considered to be a valid value.
　以上説明したように本発明者はｉｎ　ｖｉｔｒｏにおけるＮｅｕｔｒａｌ　Ｒｅｄ試験とｉｎ　ｖｉｖｏにおけるＴＩＰＳ投与を組み合わせた試験系を開発した。この試験系では、予備試験αから総投与量βを算出し、ＴＩＰＳ投与によって毒性値γを求める。毒性値γを従来の吸入毒性試験のＬＣ５０と比較したところ、データの一致性において満足できる結果が得られた。
　本発明の方法は、高額な吸入曝露試験装置の稼働費用を必要としないため安価である。本発明の方法は、高価な専用施設における吸入曝露試験と比べて簡便かつ安価であり得る。よって、本発明の方法は、従来の吸入曝露試験の代替法として有用である。 As described above, the present inventors have developed a test system that combines an in vitro Neutral Red test and an in vivo TIPS administration. In this test system, the total dose β is calculated from the preliminary test α, and the toxicity value γ is determined by TIPS administration. Satisfactory data consistency was obtained when the toxicity value γ was compared with the LC50 of conventional inhalation toxicity tests.
The method of the present invention is inexpensive because it does not require expensive operating costs for inhalation exposure test equipment. The methods of the invention can be simple and inexpensive compared to inhalation exposure studies in expensive dedicated facilities. Therefore, the method of the present invention is useful as an alternative to conventional inhalation exposure tests.
　本発明によれば、化学物質の吸入毒性を安価で簡便に評価できる毒性試験方法；および毒性試験キットが提供される。 According to the present invention, a toxicity test method and a toxicity test kit are provided that can inexpensively and simply evaluate the inhalation toxicity of chemical substances.

Claims (11)

  1. 　毒性試験方法であって、
    　ａ：試験物質および色素を含む培養液を用いて細胞を培養したときに前記細胞の生存率が所定の範囲となる前記試験物質の使用量αを求めること、
    　ｂ：前記使用量αを試験動物の体重に基づいて、前記試験動物への総投与量βに換算すること、および、
    　ｃ：総投与量βの前記試験物質を前記試験動物の気管内で噴霧して投与し、かつ、総投与量βの前記試験物質を投与した後の前記試験動物の生存率から、前記試験物質の毒性値γを求めること、
    　を含む、方法。     A toxicity test method comprising:
    a: determining the usage amount α of the test substance at which the viability of the cell is within a predetermined range when cells are cultured using a culture medium containing the test substance and dye;
    b: converting the amount α used to a total dose β to the test animal based on the body weight of the test animal; and
    c: the total dose β of the test substance was administered by spraying it into the trachea of the test animal, and from the survival rate of the test animal after administration of the total dose β of the test substance, the test substance determining the toxicity value γ of
    A method, including
  2. 　前記ｃでは、数時間から半日の期間をかけて、前記試験物質を前記試験動物の気管内で噴霧して複数回投与する、請求項１に記載の方法。 The method according to claim 1, wherein in c, the test substance is sprayed into the trachea of the test animal and administered multiple times over a period of several hours to half a day.
  3. 　前記ｃでは、総投与量βの前記試験物質の投与終了後少なくとも１週間以上、前記試験動物に前記試験物質を投与せずに前記試験動物を観察することで、前記試験物質の急性毒性を評価する、請求項１または２に記載の方法。 In c above, acute toxicity of the test substance is evaluated by observing the test animal without administering the test substance to the test animal for at least one week after the end of administration of the test substance at the total dose β. 3. The method of claim 1 or 2, wherein
  4. 　前記ｃでは、総投与量βの前記試験物質の投与終了後少なくとも２週間以上、前記試験動物に前記試験物質を投与せずに前記試験動物を観察することで、前記試験物質の慢性毒性を評価する、請求項１または２に記載の方法。 In c above, the chronic toxicity of the test substance is evaluated by observing the test animal without administering the test substance to the test animal for at least two weeks after the end of administration of the test substance at the total dose β. 3. The method of claim 1 or 2, wherein
  5. 　前記ｃでは、総投与量βの前記試験物質の投与終了後少なくとも１２か月以上、前記試験動物に前記試験物質を投与せずに前記試験動物を観察することで、前記試験物質のがん原性を評価する、請求項１または２に記載の方法。 In the above c, by observing the test animal without administering the test substance to the test animal for at least 12 months after the end of administration of the test substance at the total dose β, the carcinogenicity of the test substance 3. The method according to claim 1 or 2, wherein sex is evaluated.
  6. 　前記ａでは、前記細胞の生存率が１０～９０％となるときの前記試験物質の使用量から前記使用量αを求める、請求項１または２に記載の方法。 The method according to claim 1 or 2, wherein in said a, said usage amount α is obtained from the usage amount of said test substance when said cell viability is 10 to 90%.
  7. 　前記ａにおける前記色素が、ニュートラルレッドである、請求項１または２に記載の方法。 The method according to claim 1 or 2, wherein said pigment in said a is neutral red.
  8. 　前記ａにおける前記細胞が、ヒトの肺由来の細胞である、請求項１または２に記載の方法。 The method according to claim 1 or 2, wherein the cells in said a are human lung-derived cells.
  9. 　請求項１または２に記載の方法のための毒性試験キットであり、
    　前記色素と、前記細胞と、前記細胞を培養するための培養液と、を備えた、毒性試験キット。     A toxicity test kit for the method of claim 1 or 2,
    A toxicity test kit comprising the dye, the cells, and a culture medium for culturing the cells.
  10. 　前記色素が、ニュートラルレッドである、請求項９に記載の毒性試験キット。 The toxicity test kit according to claim 9, wherein the dye is neutral red.
  11. 　前記細胞が、ヒトの肺由来の細胞である、請求項９に記載の毒性試験キット。 The toxicity test kit according to claim 9, wherein the cells are human lung-derived cells.
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