CN102156188A - Neutral erythrocyte toxicity testing method for biological evaluation of cigarette tipping paper - Google Patents
Neutral erythrocyte toxicity testing method for biological evaluation of cigarette tipping paper Download PDFInfo
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- CN102156188A CN102156188A CN2011100353531A CN201110035353A CN102156188A CN 102156188 A CN102156188 A CN 102156188A CN 2011100353531 A CN2011100353531 A CN 2011100353531A CN 201110035353 A CN201110035353 A CN 201110035353A CN 102156188 A CN102156188 A CN 102156188A
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Abstract
The invention relates to a neutral erythrocyte toxicity testing method for biological evaluation of a piece of cigarette tipping paper and belongs to the technical field of tobaccos. The method concretely comprises the following steps of: taking artificial saliva as tipping paper leach liquor; measuring and taking the tipping paper with a certain area, shearing to a strip with a length and a width of 1cm and 6cm and placing in a 50-250ml conical flask with a ground glass stopper for later use; pouring the leach liquor into the conical flask according to a proportion of the tipping paper area to the leach liquor of 24-36cm<2>/ml and putting back the stopper; placing the conical flask in a water-bath vibrator at a temperature of 36-38 DEG C, vibrating and leaching for 5-180min and preparing a sample stock solution; carrying out a neutral erythrocyte toxicity test, wherein doses of the sample stock solution per ml of nutrient solution successively are 1mg, 5mg, 10mg, 25mg, 50mg, 100mg, 200mg, 300mg, 400mg, 500mg; and evaluating the security of the tipping paper according to the result of the neutral erythrocyte toxicity test. The method provided by the invention has the advantages that the actual situation of the contact between the tipping paper and a mouth is effectively simulated to prepare a sample; the dose range of the sample stock solution for the neutral erythrocyte toxicity test is suitable; and the method is practical, simple and convenient.
Description
Technical field:
The present invention relates to a kind of dimethyl diaminophenazine chloride cell toxicity test method that is used for the cigarette tipping paper biological assessment, belong to technical field of tobacco.
Background technology:
Though cigarette tipping paper is not participated in burning, but directly contact with the consumer oral cavity.At present, the concern of cigarette tipping paper is mainly concentrated on standard [1～2] to its physical index, microbiological indicator, heavy metal and the index of quality.Along with deepening continuously of lowering harm and decreasing coking, the new cigarette tipping paper constantly occurs, and need carry out the cigarette tipping paper safety research in a deep going way, thereby provides basic data and theory support for cigarette material supervision.
The sample preparation methods that is used for the cigarette tipping paper biological assessment, though not the core of safety evaluatio work, but an initial step of all analyzing and testing work.Accurately simulation actual exposed condition prepares key factor and basic premise that the migration sample of cigarette tipping paper in artificial saliva is influence follow-up safety evaluatio objectivity, accuracy and reliability.
Up to now, do not see the actual state that the simulation cigarette tipping paper contacts with the oral cavity at home and abroad, carry out the open report of the dimethyl diaminophenazine chloride cell toxicity test method of tipping paper biological assessment.
List of references:
[1] People's Republic of China's tobacco business standard YC 170-2002 " body paper for tobacco-use tipping paper ".
[2] People's Republic of China's tobacco business standard YC 171-2002 " cigarette tipping paper ".
[3] Song Yubing, Zong Yongli, Ma Ji, Li Peng, Zhang Jie, Peng Shuhai. a kind of method of measuring nonpolar or low-polar flavor component release rate in the smoke-free tobacco product. number of patent application 200910227274.3.
[4] State Standard of the People's Republic of China GB/T 16886.15-2003/ISO 10993-15:2000 " medicine equipment biological assessment the 15th part: the qualitative and quantitative of Metal and Alloy catabolite ".
[5] Mao Youan, Li Jun, Gu Junpin, Liu Wei, Zhong Kejun, Wei ten thousand it. the device that the simulation human oral cavity absorbs main flume in the smoking process. number of patent application 200910226704.X.
[6] LI XUEMEI, Ceng Xiaoying, Cui Yang, Yang Yekun, person are section flame green grass or young crops, Geng Yongqin, Wang Mingfeng. the method for a kind of whole smoke trapping and pH pH-value determination pH thereof. and number of patent application 201010123588.1.
Summary of the invention:
The object of the present invention is to provide a kind of easy, practical dimethyl diaminophenazine chloride cell toxicity test method that is used for the cigarette tipping paper biological assessment.
The dimethyl diaminophenazine chloride cell toxicity test method that is used for the cigarette tipping paper biological assessment, the concrete steps of dimethyl diaminophenazine chloride cell toxicity test method that it is characterized in that being used for the cigarette tipping paper biological assessment are as follows:
A. adopt the leaching liquor of the artificial saliva of State Standard of the People's Republic of China GB/T 16886.15-2003/ISO 10993-15:2000 " medicine equipment biological assessment the 15th part: the qualitative and quantitative of Metal and Alloy catabolite " regulation as cigarette tipping paper;
B. measuring certain area (as: measures cigarette tipping paper 600cm * 6cm, is equivalent to the tipping paper consumption of 500 cigarette; Measure 300cm * 6cm, be equivalent to the tipping paper consumption of 250 cigarette; Measure cigarette tipping paper 240cm * 6cm, be equivalent to the tipping paper consumption of 200 cigarette; Measure cigarette tipping paper 120cm * 6cm, be equivalent to the tipping paper consumption of 100 cigarette; Measure 60cm * 6cm, be equivalent to the tipping paper consumption of 50 cigarette) cigarette tipping paper, be cut into the strip of 1cm * 6cm, place 50～250mL conical flask standby with grinding port plug;
C. be 24～36cm according to tipping paper area/leaching liquor ratio
2/ mL injects leaching liquor in the conical flask that tipping paper is housed, tipping paper is built stopper after all immersing;
D. simulate the actual exposed condition, it is 37 ± 1 ℃ that extraction temperature is set;
E. this conical flask is placed water-bath oscillator concussion lixiviate 5～180min of 37 ± 1 ℃, obtain sample stoste;
F. the sample stoste that the e step is obtained is carried out the dimethyl diaminophenazine chloride cell toxicity test, well-grown Chinese hamster ovary line is inoculated in the 96 porocyte culture plates every hole 1 * 10 that is:
4Individual cell, placing condition setting is 37 ℃, 5%CO
2Hatch 24h in the incubator; Discard the nutrient solution in the culture plate, add the sample stoste of various dose, in every mL nutrient culture media the dosage of given the test agent stoste be followed successively by 1,5,10,25,50,100,200,300,500mg, continue to hatch 24h, it is parallel that each group dosage is done 6 holes; Discard the solution in the culture plate, add neutral red solution by 100 μ g/mL, every hole 200 μ L place incubator to hatch 3h; Discard the solution in the culture plate, add 1% (v/v) formalin, every hole 200 μ L fix 1～2min; Discard formalin, add 1% (v/v) acid ethanol solution, acid ethanol solution is a glacial acetic acid: absolute ethyl alcohol: water=1: 50: 49, and every hole 200 μ L place concussion 10min in the microplate oscillator; Measure every hole absorption value of 540nm wavelength on microplate reader, test repeats twice;
G. according to the security of dimethyl diaminophenazine chloride cell toxicity test evaluation of result cigarette tipping paper.
The invention has the advantages that: the actual state that tipping paper contacts with the oral cavity in the time of effectively simulating smoking cigarette prepares sample, and the selected sample stoste dosage range that is used for the dimethyl diaminophenazine chloride cell toxicity test is suitable, method practicality, easy.
Description of drawings:
Accompanying drawing is the dimethyl diaminophenazine chloride cell toxicity test method and technology route map that is used for the cigarette tipping paper biological assessment.
Embodiment:
Below in conjunction with specific embodiment the present invention is done detailed description, but do not limit the present invention.
Example one is used for the evaluation of HH cigarette tipping paper
1 experimental section
1.1 key instrument and reagent
Series II CO
2Incubator (ThermoForma company); Inverted microscope (Olympus company); Microplate reader (Bio-Rad company); 96 porocyte culture plates (Corning company); Tissue Culture Flask (Corning company).
DMEM nutrient culture media (Dulbecco ' s modification of Eagle ' s medium, Solarbio company); Hyclone (Clark company); Phosphate buffer (CMF-PBS, Solarbio company); Trypsase (Solarbio company); Dimethyl diaminophenazine chloride dyestuff (Sigma company); Penicillin-streptomysin (Solarbio company); Formaldehyde; Ethanol; Glacial acetic acid; It is pure that organic solvent is analysis.
The preparation of cell culture medium is the penicillin-streptomysin that adds 10% (v/v) hyclone and 100 units/mL in the DMEM nutrient culture media.Neutral red solution is that concentration is 100 μ g/mL with the preparation of DMEM nutrient culture media.Acid ethanol solution is by volume, glacial acetic acid: absolute ethyl alcohol: water=preparation in 1: 50: 49.
1.2 subject cell
Chinese hamster ovary line (Chinese hamster ovary cell line, Chinese hamster ovary celI) is available from Kunming animal institute of Chinese Academy of Sciences cell bank.
1.3 specimen preparation
With reference to the artificial saliva preparation method of State Standard of the People's Republic of China GB/T 16886.15-2003/ISO 10993-15:2000 " medicine equipment biological assessment the 15th part: the qualitative and quantitative of Metal and Alloy catabolite " regulation, the leaching liquor of the cigarette tipping paper that preparation contacts with the oral cavity.
Measure HH cigarette tipping paper 600cm * 6cm (the tipping paper consumption that is equivalent to 500 cigarette), be cut into about 1cm * 6cm fine strip shape, place the 250mL conical flask of tool grinding port plug.Inject the artificial saliva 100mL for preparing in the conical flask that the HH cigarette tipping paper is housed, cause the tipping paper slip to immerse all that (ratio of tipping paper area/leaching liquor is 36cm
2/ mL is equivalent to the tipping paper with 5 cigarette of 1mL leaching liquor lixiviate), build stopper.Simulation actual exposed condition, it is 37 ± 1 ℃ that extraction temperature is set; According to the soak time of fading of regulation among People's Republic of China's tobacco business standard " body paper for tobacco-use tipping paper " (YC 170-2002) and " cigarette tipping paper " (YC 171-2002), it is 2h that extraction time is set.This conical flask is placed 37 ± 1 ℃ water-bath oscillator concussion lixiviate 2h (concussion speed 200r/min), preparation tipping paper sample stoste.
1.4 test method
During test, well-grown Chinese hamster ovary line is inoculated in the 96 porocyte culture plates every hole 1 * 10
4Individual cell, place incubator (37 ± 1 ℃, 5%CO
2) in hatch 24h; Discard the solution in the culture plate, add the sample stoste of various dose, in every mL cell culture medium the dosage of given the test agent stoste be 1,5,10,25,50,100,200,300 successively, 500mg, continue to hatch 24h, it is parallel that each group dosage is done 6 holes; Discard the solution in the culture plate, add neutral red solution, every hole 200 μ L place incubator to hatch 3h; Discard the solution in the culture plate, add 1% (v/v) formalin, every hole 200 μ L fix 1～2min; Discard formalin, add 1% (v/v) acid ethanol solution, every hole 200 μ L place concussion 10min in the microplate oscillator; On microplate reader, measure every hole absorption value of 540nm wavelength.Test repeats twice.
2, result
Known that by table 1 HH cigarette tipping paper sample stoste has slight inhibiting effect to Chinese hamster ovary celI, along with sample dose increases from 1mg/mL to 500mg/mL, its cell inhibitory rate also increases thereupon, is significant dose-response relationship.
Table 1HH cigarette tipping paper sample stoste dimethyl diaminophenazine chloride cell toxicity test result
* the solvent of given the test agent is a water, does not therefore do solvent control.
Example two is used for the evaluation of HD cigarette tipping paper
Substantially with example one.Difference is:
Measure HD cigarette tipping paper 300cm * 6cm (the tipping paper consumption that is equivalent to 250 cigarette), be cut into about 1cm * 6cm fine strip shape, place the 250mL conical flask of tool grinding port plug.Inject the artificial saliva 150mL for preparing in the conical flask that the HD cigarette tipping paper is housed, cause the tipping paper slip to immerse all that (ratio of tipping paper area/leaching liquor is 24cm
2/ mL is equivalent to the tipping paper with 3.3 cigarette of 1mL leaching liquor lixiviate), build stopper.Simulation actual exposed condition, it is 37 ± 1 ℃ that extraction temperature is set, and this conical flask is placed 37 ℃ water-bath oscillator concussion lixiviate 1h (concussion speed 200r/min), preparation HD cigarette tipping paper sample stoste.
Known that by table 2 HD cigarette tipping paper sample stoste has slight inhibiting effect to Chinese hamster ovary celI, along with sample dose increases from 1mg/mL to 500mg/mL, its cell inhibitory rate also increases thereupon, is significant dose-response relationship.
Table 2HD cigarette tipping paper sample stoste dimethyl diaminophenazine chloride cell toxicity test result
* the solvent of given the test agent is a water, does not therefore do solvent control.
Example three is used for the evaluation of YY cigarette tipping paper
Substantially with example one.Difference is:
Measure YY cigarette tipping paper 120cm * 6cm (the tipping paper consumption that is equivalent to 100 cigarette), be cut into about 1cm * 6cm fine strip shape, place the 50mL conical flask of tool grinding port plug.Inject the artificial saliva 20mL for preparing in the conical flask that the YY cigarette tipping paper is housed, cause the tipping paper slip to immerse all that (ratio of tipping paper area/leaching liquor is 36cm
2/ mL is equivalent to the tipping paper with 5 cigarette of 1mL leaching liquor lixiviate), build stopper.Simulation actual exposed condition, it is 37 ± 1 ℃ that extraction temperature is set, and this conical flask is placed 37 ± 1 ℃ water-bath oscillator concussion lixiviate 180min (concussion speed 200r/min), preparation tipping paper sample stoste.
Known that by table 3 YY cigarette tipping paper sample stoste has slight inhibiting effect to Chinese hamster ovary celI, along with sample dose increases from 1mg/mL to 500mg/mL, its cell inhibitory rate also increases thereupon, is significant dose-response relationship.
Table 3YY cigarette tipping paper sample stoste dimethyl diaminophenazine chloride cell toxicity test result
* the solvent of given the test agent is a water, does not therefore do solvent control.
The example four-function is estimated in the RS cigarette tipping paper
Substantially with example one.Difference is:
Measure RS cigarette tipping paper 240cm * 6cm (the tipping paper consumption that is equivalent to 200 cigarette), be cut into about 1cm * 6cm fine strip shape, place the 100mL conical flask of tool grinding port plug.Inject the artificial saliva 40mL for preparing in the conical flask that the RS cigarette tipping paper is housed, cause the tipping paper slip to immerse all that (ratio of tipping paper area/leaching liquor is 36cm
2/ mL is equivalent to the tipping paper with 5 cigarette of 1mL leaching liquor lixiviate), build stopper.Simulation actual exposed condition, it is 37 ± 1 ℃ that extraction temperature is set, and this conical flask is placed 37 ± 1 ℃ water-bath oscillator concussion lixiviate 5min (concussion speed 200r/min), preparation RS cigarette tipping paper sample stoste.
Known that by table 4 RS cigarette tipping paper sample stoste has slight inhibiting effect to Chinese hamster ovary celI, along with sample dose increases from 1mg/mL to 500mg/mL, its cell inhibitory rate also increases thereupon, is significant dose-response relationship.
Table 4RS cigarette tipping paper sample stoste dimethyl diaminophenazine chloride cell toxicity test result
* the solvent of given the test agent is a water, does not therefore do solvent control.
Claims (1)
1. dimethyl diaminophenazine chloride cell toxicity test method that is used for the cigarette tipping paper biological assessment, the concrete steps of dimethyl diaminophenazine chloride cell toxicity test method that it is characterized in that being used for the cigarette tipping paper biological assessment are as follows:
A. adopt artificial saliva commonly used in the research of medicine equipment biological assessment leaching liquor as cigarette tipping paper;
B. measure the cigarette tipping paper of certain area, be cut into the strip of 1cm * 6cm, place 50～250mL conical flask standby with grinding port plug;
C. be 24～36cm according to tipping paper area/leaching liquor ratio
2/ mL injects leaching liquor in the conical flask that tipping paper is housed, tipping paper is built stopper after all immersing;
D. simulate the actual exposed condition, it is 37 ± 1 ℃ that extraction temperature is set;
E. this conical flask is placed water-bath oscillator concussion lixiviate 5～180min of 37 ± 1 ℃, obtain sample stoste;
F. the sample stoste that the e step is obtained is carried out the dimethyl diaminophenazine chloride cell toxicity test, well-grown Chinese hamster ovary line is inoculated in the 96 porocyte culture plates every hole 1 * 10 that is:
4Individual cell, placing condition setting is 37 ℃, 5%CO
2Hatch 24h in the incubator; Discard the nutrient solution in the culture plate, add the sample stoste of various dose, in every mL nutrient culture media the dosage of given the test agent stoste be followed successively by 1,5,10,25,50,100,200,300,500mg, continue to hatch 24h, it is parallel that each group dosage is done 6 holes; Discard the solution in the culture plate, add neutral red solution by 100 μ g/mL, every hole 200 μ L place incubator to hatch 3h; Discard the solution in the culture plate, add 1% (v/v) formalin, every hole 200 μ L fix 1～2min; Discard formalin, add 1% acid ethanol solution (v/v), acid ethanol solution is a glacial acetic acid: absolute ethyl alcohol: water=1: 50: 49, and every hole 200 μ L place concussion 10min in the microplate oscillator; Measure every hole absorption value of 540nm wavelength on microplate reader, test repeats twice;
G. according to the security of dimethyl diaminophenazine chloride cell toxicity test evaluation of result cigarette tipping paper.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104198249A (en) * | 2014-09-05 | 2014-12-10 | 云南中烟工业有限责任公司 | Simulating extraction method of cigarette filter extracting solution |
| CN104293876A (en) * | 2014-09-23 | 2015-01-21 | 云南中烟工业有限责任公司 | Cigarette filter tip water extract cytotoxicity test positive control setting method |
| CN114184441A (en) * | 2021-10-19 | 2022-03-15 | 威凯检测技术有限公司 | Preparation device and method of animal-derived medical instrument biological safety evaluation experiment leaching liquor |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104198249A (en) * | 2014-09-05 | 2014-12-10 | 云南中烟工业有限责任公司 | Simulating extraction method of cigarette filter extracting solution |
| CN104293876A (en) * | 2014-09-23 | 2015-01-21 | 云南中烟工业有限责任公司 | Cigarette filter tip water extract cytotoxicity test positive control setting method |
| CN114184441A (en) * | 2021-10-19 | 2022-03-15 | 威凯检测技术有限公司 | Preparation device and method of animal-derived medical instrument biological safety evaluation experiment leaching liquor |
| CN114184441B (en) * | 2021-10-19 | 2024-04-05 | 威凯检测技术有限公司 | Preparation device and method of animal-derived medical instrument biosafety evaluation experiment leaching solution |
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Application publication date: 20110817 |