Test method for detecting cytotoxicity of buccal tobacco products

A test method and cytotoxicity technology, applied in the direction of biochemical equipment and methods, microbiological determination/inspection, etc., can solve the problems that cannot meet the needs of cytotoxicity detection of snus products, and achieve the effect of accurate and effective detection

Active Publication Date: 2015-07-22
CHINA TOBACCO YUNNAN IND
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Problems solved by technology

The use of snus products is different from that of traditional cigarette inhalation. The snus products are used directly by mouth, and their dissolved substances will enter the digestive system with saliva. During use, no smoke is produced, and the exposure route is significantly different from traditional cigarettes. For cigarettes, changes in the test object lead to differences in sample pretreatment methods, test cells, and test doses. The cytotoxicity test used to detect the cytotoxicity of traditional cigarettes cannot meet the needs of the cytotoxicity test of snus products, and the current research at home and abroad Institutions and tobacco companies have not formulated a standard method for the cytotoxicity test of snus products [3-5]. Therefore, it is necessary to establish a cytotoxicity test method suitable for snus products according to the characteristics of snus products to ensure product safety sex

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  • Test method for detecting cytotoxicity of buccal tobacco products
  • Test method for detecting cytotoxicity of buccal tobacco products
  • Test method for detecting cytotoxicity of buccal tobacco products

Examples

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Embodiment 1

[0040] It is used for the detection of cytotoxicity of Camel brand mint flavored Snus snus products.

[0041] (1) Sample pretreatment method: Accurately weigh 1.0g of Camel brand mint-flavored Snus snus product, add 30mL of commercially available serum-free medium, and place it in a constant temperature water bath at 37°C for 24h for extraction. After extraction, use qualitative filter paper for primary filtration to remove slag, and then use 0.45 μm organic filter membrane to filter and sterilize to obtain the test solution;

[0042] (2) Preparation of single cell suspension: Human oral mucosal fibroblast hOMF cells were incubated at 37°C, 5% CO 2 , 95% RH (relative air humidity) incubator, observe the confluence and morphology of the cultured cells through an inverted microscope, remove the medium when the cell confluence rate is 90%, wash twice with phosphate buffer, discard the washing solution ; Add 1-2 mL of trypsin solution with a concentration of 0.25% (w / v) to incuba...

Embodiment 2

[0061] It is used for the detection of cytotoxicity of Camel brand mint flavored Snus snus products.

[0062] (1) Sample pretreatment method: Accurately weigh 1.0g of Camel brand mint-flavored Snus snus product, add 30mL of commercially available serum-free medium, and place it in a constant temperature water bath at 37°C for 24h for extraction. After extraction, use qualitative filter paper for primary filtration to remove slag, and then use 0.45 μm organic filter membrane to filter and sterilize to obtain the test solution;

[0063] (2) Preparation of single cell suspension: Human oral mucosal fibroblast hOMF cells were incubated at 37°C, 5% CO 2 , 95% RH (relative air humidity) incubator, observe the confluence and morphology of the cultured cells through an inverted microscope, remove the medium when the cell confluence rate is 80%, wash twice with phosphate buffer, and discard the washing solution ; Add 1-2 mL of trypsin solution with a concentration of 0.25% (w / v) to in...

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Abstract

The invention provides a test method for detecting cytotoxicity of buccal tobacco products. The test method comprises the following steps: sample pretreatment, single-cell suspension preparation, cell concentration calculation, cell inoculation, test substance addition, test substance incubation, dye incubation, light absorption value measurement, cell inhibition rate calculation and cell half lethal dose calculation. According to the test method provided by the invention, the exposure means and action manners of the buccal tobacco products are comprehensively surveyed, a sample pretreatment method of the buccal tobacco products is established, human oral mucosa fibroblasts hoMF are selected as the cells for cytotoxicity test of the buccal tobacco products according to the acting target cells, the sample detection dosage is determined and a cytotoxicity test method suitable for the buccal tobacco products is formed.

Description

technical field [0001] The invention relates to a test method for detecting the cytotoxicity of snus products, and belongs to the technical field of biological safety evaluation of tobacco and tobacco products. Background technique [0002] The relative growth rate (RGR) of cells is a key indicator of cytotoxicity evaluation, and it is often used in the classification of cytotoxicity in the development of new drugs and the safety evaluation of medical devices [1，2] . The neutral red method and the thiazolium blue method (MTT) are simple, fast, and sensitive, and they are currently two widely used toxicological detection methods for cytotoxicity. In 2002, CORESTA, the International Tobacco Scientific Research Cooperation Center, established a working group for in vitro toxicological testing of cigarette smoke. Potential cytotoxicity is detected, and this detection method has been widely adopted by domestic and foreign tobacco companies. [0003] Increasingly stringent toba...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02
Inventor 管莹夭建华李雪梅米其利朱洲海高茜陈建华杨叶昆
Owner CHINA TOBACCO YUNNAN IND
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