烟用香精香料对卷烟安全性的影响
|
摘要
卷烟是一种特殊食品,而烟用香精香料是这一特殊食品的添加剂,对于卷烟风格特征的形成具有不可或缺的作用,其配方更是卷烟工艺中的核心技术,烟用香精香料的安全性已备受消费者和烟草企业的关注。香精香料加入到卷烟后的毒性很值得研究和探讨。细胞毒性试验和遗传毒性试验作为有害物质危害性评价的重要手段被广泛应用。本研究采用CORESTA(国际烟草科学研究合作中心)推荐的三项体外毒理学评价方法MTT(四甲基偶氮唑盐比色法)细胞毒性试验,微核试验和AMES试验(鼠伤寒沙门氏菌回复突变实验)对卷烟中的香精香料及其拆分组分进行了评价。本论文的主要研究内容与结果如下：
1、香精香料及其拆分组分未添加至卷烟之前,随物质浓度的增大细胞的存活率均出现下降,表现出剂量效应,呈负相关。全香精细胞毒性明显高于全香料(p<0.01),全香料的10个拆分组分在最高所试剂量(50ml／L)未表现出细胞毒性,全香精的10个拆分组分细胞毒性存在差异,其中3＃、5＃、7＃和8＃(此编号为拆分组分的编号,具体成分为商业秘密,在此不给出)香精组分的细胞毒性较低,其他几种香精组分的细胞毒性相当,但所有香精拆分组分的细胞毒性均明显低于全香精(p<0.01)。
2、香精香料及其拆分组分添加至卷烟后,随CSC(卷烟烟气凝集物)浓度的增大细胞的存活率均出现下降,也表现出剂量效应,呈负相关。与对照烟比较,添加香精的卷烟,细胞毒性无明显增加,与全香精比较,10个香精拆分组分的细胞毒性无差别。与对照烟比较,添加香料的卷烟,细胞毒性也无明显增加,与全香料比较,10个香料拆分组分的细胞毒性无差别。
3、对香精香料及其拆分组分的卷烟烟气CSC进行微核分析,流式细胞仪检测结果显示经卷烟烟气染毒细胞微核率高于未染毒正常细胞,全香精5#、10＃拆分组分比其他组分微核率稍高,但是全香精的微核率与未添加香精空白对照烟无差异。全香料3＃、4＃、5＃拆分组分比其他组分微核率稍高,但是全香料的微核率明显低于未添加香料空白对照烟。提示某些拆分组分增加了卷烟的遗传毒性,提示在烟用香精香料配伍时可少用或者不用毒性较高的组分,保证其在卷烟应用时的安全性。
4、对香精香料及其拆分组分的卷烟烟气CSC进行致突变分析,CSC的加入量与突变菌落数呈极显著正相关,存在线性剂量关系,即随着CSC剂量的增加,其致突变性也相应增强。与空白对照烟比较,全香精、全香料及其拆分组分致突变性无显著差异。整个实验结果提示该香精香料在卷烟中的应用是安全的。
Tabacco essence and flavor, additives for the special foodstuff—Cigarette, are essential to form the cigarette style, and the prescription of them is the key technology in cigarette technics. The safety of the tabacco essence and flavor has raised much attention of the consumers and tobacco companies. The toxicity of the essence and flavor in tobacco is worth to research and discuss. The tests of Cytotoxicity and Genetic toxicity are widely used as important means to evaluate the dangers of the detrimental agents. This study evaluated the tabacco essence and flavor and it's splited components by Three in vitro toxicology methods(recommended by CORESTA)、MTT Cytotoxicity Test、Micronucleus Test and AMES Test.The results were as follows:
1、Before injection of essence and flavor and their splited components into the cigarettes, the cell survival rate declined as the detriments increased, which showed dose-dependent trend of the cell. The cytotoxicity of whole essence was apparently higher than the whole flavor(p<0.01). The ten splited components of whole flavor showed no cytotoxicity in the maximal concentration (50ml/L) of the test dose. The cytotoxicity of ten splited components of whole essence was different. The 3#、5#、7#、8# components had lower cytotoxicity and the rests had relatively same level of cytotoxicity. However, the cytotoxicity of all splited components of essence were obviously lower than the whole essence (p<0.01).
2、After injection of essence and flavor and their splited components into the cigarettes, the Cell survival rate also decreased when the dosage of CSC(cigarette somking condensates) increased, and showed a dose-dependent trend as well. Compared with blank cigarettes, the essence or flavor injected cigarettes had no obvious cytotoxicity increase and the cytotoxicity of the ten splited components of whole essence or flavor had no difference in comparation with whole essence or flavor separately.
3、The cigarette somking condensates(CSC) of essence and flavor and their splited components injected cigarettes were evaluated by Micronucleus Test. The Flow Cytometry showed that:the cell micronucleus rate of cells poisoned by CSC was higher than the unpoisoned ones. The Cell micronucleus rate of splited components of whole essence 5#、10# were a little higher than other components, but the cell micronucleus rate of whole essence had no difference with the blank cigarettes. The Cell micronucleus rate of splited components of whole flavor 3#、4#、5# were a little higher than other components, but the cell micronucleus rate of whole flavor was obvious lower than the blank cigarettes. The results shows that some splited components increased the descendiblity toxicity of cigarettes, therefore, we could use less or even no the higher toxicity components to ensure the safety of cigarettes.
4、The cigarette somking condensates(CSC) of essence and flavor and their splited components injected cigarettes were evaluated by the mutagenicity AMES test. The increase of the CSC resulted in a higher mutagenicity of the Revertant Clonies, which showed an apparent positive correlation and a linear does effect between them. The cigarette somking condensates of the whole essence and flavor and their splited components injected cigarettes had the same level of mutagenicity as blank cigarettes. All the experiment results demonstrated that it's safe to use essence and flavor in cigarettes.
1、香精香料及其拆分组分未添加至卷烟之前,随物质浓度的增大细胞的存活率均出现下降,表现出剂量效应,呈负相关。全香精细胞毒性明显高于全香料(p<0.01),全香料的10个拆分组分在最高所试剂量(50ml／L)未表现出细胞毒性,全香精的10个拆分组分细胞毒性存在差异,其中3＃、5＃、7＃和8＃(此编号为拆分组分的编号,具体成分为商业秘密,在此不给出)香精组分的细胞毒性较低,其他几种香精组分的细胞毒性相当,但所有香精拆分组分的细胞毒性均明显低于全香精(p<0.01)。
2、香精香料及其拆分组分添加至卷烟后,随CSC(卷烟烟气凝集物)浓度的增大细胞的存活率均出现下降,也表现出剂量效应,呈负相关。与对照烟比较,添加香精的卷烟,细胞毒性无明显增加,与全香精比较,10个香精拆分组分的细胞毒性无差别。与对照烟比较,添加香料的卷烟,细胞毒性也无明显增加,与全香料比较,10个香料拆分组分的细胞毒性无差别。
3、对香精香料及其拆分组分的卷烟烟气CSC进行微核分析,流式细胞仪检测结果显示经卷烟烟气染毒细胞微核率高于未染毒正常细胞,全香精5#、10＃拆分组分比其他组分微核率稍高,但是全香精的微核率与未添加香精空白对照烟无差异。全香料3＃、4＃、5＃拆分组分比其他组分微核率稍高,但是全香料的微核率明显低于未添加香料空白对照烟。提示某些拆分组分增加了卷烟的遗传毒性,提示在烟用香精香料配伍时可少用或者不用毒性较高的组分,保证其在卷烟应用时的安全性。
4、对香精香料及其拆分组分的卷烟烟气CSC进行致突变分析,CSC的加入量与突变菌落数呈极显著正相关,存在线性剂量关系,即随着CSC剂量的增加,其致突变性也相应增强。与空白对照烟比较,全香精、全香料及其拆分组分致突变性无显著差异。整个实验结果提示该香精香料在卷烟中的应用是安全的。
Tabacco essence and flavor, additives for the special foodstuff—Cigarette, are essential to form the cigarette style, and the prescription of them is the key technology in cigarette technics. The safety of the tabacco essence and flavor has raised much attention of the consumers and tobacco companies. The toxicity of the essence and flavor in tobacco is worth to research and discuss. The tests of Cytotoxicity and Genetic toxicity are widely used as important means to evaluate the dangers of the detrimental agents. This study evaluated the tabacco essence and flavor and it's splited components by Three in vitro toxicology methods(recommended by CORESTA)、MTT Cytotoxicity Test、Micronucleus Test and AMES Test.The results were as follows:
1、Before injection of essence and flavor and their splited components into the cigarettes, the cell survival rate declined as the detriments increased, which showed dose-dependent trend of the cell. The cytotoxicity of whole essence was apparently higher than the whole flavor(p<0.01). The ten splited components of whole flavor showed no cytotoxicity in the maximal concentration (50ml/L) of the test dose. The cytotoxicity of ten splited components of whole essence was different. The 3#、5#、7#、8# components had lower cytotoxicity and the rests had relatively same level of cytotoxicity. However, the cytotoxicity of all splited components of essence were obviously lower than the whole essence (p<0.01).
2、After injection of essence and flavor and their splited components into the cigarettes, the Cell survival rate also decreased when the dosage of CSC(cigarette somking condensates) increased, and showed a dose-dependent trend as well. Compared with blank cigarettes, the essence or flavor injected cigarettes had no obvious cytotoxicity increase and the cytotoxicity of the ten splited components of whole essence or flavor had no difference in comparation with whole essence or flavor separately.
3、The cigarette somking condensates(CSC) of essence and flavor and their splited components injected cigarettes were evaluated by Micronucleus Test. The Flow Cytometry showed that:the cell micronucleus rate of cells poisoned by CSC was higher than the unpoisoned ones. The Cell micronucleus rate of splited components of whole essence 5#、10# were a little higher than other components, but the cell micronucleus rate of whole essence had no difference with the blank cigarettes. The Cell micronucleus rate of splited components of whole flavor 3#、4#、5# were a little higher than other components, but the cell micronucleus rate of whole flavor was obvious lower than the blank cigarettes. The results shows that some splited components increased the descendiblity toxicity of cigarettes, therefore, we could use less or even no the higher toxicity components to ensure the safety of cigarettes.
4、The cigarette somking condensates(CSC) of essence and flavor and their splited components injected cigarettes were evaluated by the mutagenicity AMES test. The increase of the CSC resulted in a higher mutagenicity of the Revertant Clonies, which showed an apparent positive correlation and a linear does effect between them. The cigarette somking condensates of the whole essence and flavor and their splited components injected cigarettes had the same level of mutagenicity as blank cigarettes. All the experiment results demonstrated that it's safe to use essence and flavor in cigarettes.
引文
[1]张悠金,金闻博,烟用香料香精[M].合肥：中国科学技术大学出版社,1996
[2]董丽,刑均,吴采樱.香精香料的分析方法进展[J].分析科学学报2003,19(2)：189－193
[3]肖协忠等.烟草化学[M].北京：中国农业出版社,1997.
[4]刘海森,何唯平.香精香料基本原理及发展趋势[J].中国食品添加剂2003,5：6-10
[5]D.Layten Davis,MarkT.Nielsen.Tobacco Production,Chemistry and Technology [M]. Blackwell Selenee,1999:336
[6]汪秋安,罗俊霞.香料香精分析技术研究[J].中国调味品,2004,8：13－17
[7]DOOL ITTLE D J, LEE C K. Comparative studies on the genotoxic activity of mains-tream smoke condensatefrom cigarettes which burn or only heat tobacco [J]. Envir Mo-lecular Mutag,1990,15 (2):932105.
[8]DOOL ITTLE D J, LEE C K. Analysis of cytogenetic effects in bone2marrow cells of rats subchronically exposed to smoke from cigarettes which burn or only heat tobacco [J]. Mutat. Res.1990,240 (4):251-277.
[9]DOOL ITTLE D J, BOMB ICK D W. Evaluation of the genotoxic and cytotoxic potential of mainstream whole smoke and smoke condensate from a cigarette contai ning a novel carbon filter[J]. Fundam App 1 Toxicol,1997,39 (1):1127.
[10]BOMB ICK B R, MURL I H, AVALOS J T, et al. Chemical and biological studies of a new cigarette that primarily heats tobacco. Part 2. In vitro toxicology of mainstream smoke condensate [J]. Food Chem. Toxicol.1998,36 (3):1832190.
[11]R ICHARD R BAKER, GRAHAM SM ITH.烟草香味添加剂是否具有毒性的探讨[C]第57届烟草科学研究会议论文集,2003.
[12]PH IL IPMORR ISUSA RESEARCH CENTER. In vitro exposure to 1R4F reference cigarette smoke:Evaluation of developmental toxicity[J]. Toxicol.Sci,2003,75 (1): 1342147.
[13]PH IL IPMORR IS RESEARCH LABORATOR IES GMBH. Chemical composition, cytotoxicity and mutagenicity of smoke from US commercial and reference cigarettes smoked under two sets of machine smoking conditions[J]. Toxicology,2004,195 (1):31252.
[14]Clements, J. Study Design Considerations for in vitro Toxicity Assays on Ingredients 烟草制品及卷烟添加剂安全评价与低潜在性危害卷烟产品开发国际研讨会杭州,2004.10
[15]Massey, E. The Effect of Tobacco Ingredients on Smoke Chemistry and Toxicology: An Overview烟草制品及卷烟添加剂安全评价与低潜在性危害卷烟产品开发国际研讨会杭州,2004.10
[16]http://www.coresta.org/
[17]Doolittle, D.J., Massey, E.D., CORESTA Task Force On In Vitro Toxicity Testing of Tobacco Smoke烟草制品及卷烟添加剂安全评价与低潜在性危害卷烟产品开发国际研讨会杭州,2004.10
[18]朱茂祥.中式低危害卷烟的毒理学评价技术[C].中式卷烟理论内涵讨论论文汇编[C].北京：国家烟草专卖局科技教育司,2004.
[19]郭琳,朱茂祥,黄焕桂,等.复方C卷烟添加剂的安全性及其降低卷烟危害作用[J].中国烟草学报,2007,13(6)：7-12.
[20]吕功煊,聂聪,赵明月,等.应用含纳米贵金属催化材料降低卷烟烟气中CO技术研究[J].中国烟草学报,2003,9(3)：18-27.
[21]Baker R R,Jose R. Pereira da Silva,SmithG. The effect of tobacco ingredients on smoke chemistry. Part Ⅰ:Flavourings and additives[J]. Food and Chemical Toxicology.2004 42(supplement):3-37.
[22]David M D. Genotoxicity of tobacco smoke and tobacco smoke condensate:a review [J]. Mutation Research,2004,567 (2—3):447-474.
[23]高美伶,范家林,周建华,高氟茶浸泡液的细胞毒性及致突变性[J].环境与职业医学,2006,26(2)：166-174.
[24]卢斌斌,Ryan Meng,Lynne Wisse,焦延福.三种不同焦油卷烟烟气的细胞毒性比较[J].烟草科技,2007,12(245)38-41.
[25]赵嘉惠,张华屏,王春芳.MTT法在检测细胞增殖方面的探讨[J].山西医科大学学报,2007,38(3)262－263
[26]司徒镇强,吴军正.细胞培养[M].西安：世界图书出版公司,2007.
[27]Dariasz S, Sarah JS, Riehard H,et al. An improved MTT assay[J]. Immunol Methods,1993,157:203.
[28]Green LM, Reade JL,Ware CF. Rapid colorimetric assay for cell viability of cytotoxic and growth inhibitory lymphokines[J]. Immunol Methods,1984,70(2):257-268.
[29]Maj JG, Kankofer M. Activity of 72 kD and 92 kD matrix metal-loproteinases in placental tissues of cows with and without retained fetal membranes[J]. Placenta, 1997,18:683-687.
[30]赵斌,葛金芳,朱娟娟,黄晓晖,李俊,小议在MTT法测细胞增殖抑制率中IC50的计算方法[J].安徽医药,2007,11(9)834－836
[31]Schlede E,Genschow E, Spielmann H, et al. Oral acute toxic class method:a successful alternative to the oral LD50 test[M]. Regul Toxicol Pharmaco.l United States,2005:15-23.
[32]李寿祺,毒理学原理与方法(第2版)[M]1成都：四川大学出版社,2003,153－177
[33]曹佳,林真,余争平.微核试验[M].北京：军事医学科学出版社,2000：2642272.
[34]刘毓谷.卫生毒理学基础[M].北京：人民卫生出版社,1997：53－81.
[35]Kirsch-Volders M, Sofuni T, Aardema M, Albertini S, Eastmond D, Fenech M, Ishidate M Jr, Kirchner S, Lorge E, Morita T, Norppa H, Surralles J, Vanhauwaert A, Wakata A. Report from the in vitro micronucleus assay working group. Mutat. Res. 2003;540:153-163. [PubMed:14550499]
[36]Matsushima T, Hayashi M, Matsuoka A, Ishidate M Jr, Miura KF, Shimizu H, Morimoto K, Ogura H,Mure K, Koshi K, Sufuni T. Validation study of the in vitro micronucleus test in a Chinese hamster lung cell line (CHL/IU). Mutagenesis 1999; 14:569-580. [PubMed:10567032]
[37]Miller B, Potter-Locher F, Seelbach A, Stopper H, Utesch D, Madle S. Evaluation of the in vitro micronucleus test as an alternative to the in vitro chromosome aberration assay:position of the GUM working group on the in vitro micronucleus test. Mutat. Res.1998;410:81-116. [PubMed:9587425]
[38]Lorge E, Thybaud V, Aardema MJ, Oliver J, Wakata A, Lorenzon G, Marzin D. SFTG international collaborative study on in vitro micronucleus test. I. General conditions and overall conclusions of the study. Mutat.Res.2006;607:13-36. [PubMed: 16815079]
[39]Zhang Y,Holford TR,Leaderer B,et al. Hair-coloring product use and risk of non-Hodgkin's lymphoma:a population-based casecontrol study in connecticut. Am J Epidemiol,2004,159(2):148-154.
[40]杜立颖,冯仁青.流式细胞术[M].北京大学出版社2008,7
[41]Viaggi S, Braselmann H, Nusse M. Flow cytometric analysis of micronuclei in the CD2+ subpopulation of human lymphocytes enriched by magnetic separation. Int J Radiat Biol 1995;67:193-202. [PubMed:7884288]
[42]Nusse M, Marx K. Flow cytometric analysis of micronuclei in cell cultures and human lymphocytes:advantages and disadvantages. Mutat. Res.1997;392:109-115. [PubMed:9269335]
[43]Roman D, Locher F, Suter W, Cordier A, Bobadilla M. Evaluation of a new procedure for the flow cytometric analysis of in vitro, chemically induced micronuclei in V79 cells. Environ Molec Mutagen 1998;32:387-396. [PubMed:9882014]
[44]Avlasevich SL, Bryce SM, Cairns SE, Dertinger SD. In vitro micronucleus scoring by flow cytometry:differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability. Environ Molec Mutagen.2006;47:56-66. [PubMed:16180205]
[45]Steven M. Bryce, Jeffrey C. Bemis, Svetlana L. Avlasevich, and Stephen D. Dertinger In vitro micronucleus assay scored by flow cytometry provides a comprehensive evaluation of cytogenetic damage and cytotoxicity. Mutat. Res.2007 June 15; 630(1-2):78-91.
[46]Garriott ML, Phelps JB, Hoffman WP.2002. A protocol for the in vitro micronucleus test. I. Contributions to the development of a protocol suitable for regulatory submissions from an examination of 16 chemicals with different mechanisms of action and different levels of activity. Mutat. Res.517:123-134.
[47]Kirsch-Volders M, Sofuni T, Aardema M, Albertini S, Eastmond D, Fenech M, Ishidate M, Jr., Kirchner S, Lorge E, Morita T, Norppa H, Surralle's J, Vanhauwaert A, Wakata A.2003. Report from the in vitro micronucleus assay working group. Mutat. Res.540:153-163.
[48]OECD.2004. In vitro micronucleus test. OECD Guideline for the Testing of Chemicals, Draft Proposal for a New Guideline 487. Paris:OECD.Available at http://www.oecd.org/dataoecd/60/28/32106288.pdf.
[49]von der Hude W, Kalweit S, Engelhardt G, McKiernan S, Kasper P,Slacik-Erben R, Miltenburger HG, Honarvar N, Fahrig R, Gorlitz B, Albertini S, Kirchner S, Utesch D, Potter-Locher F, Stopper H, Madle S.2000. In vitro micronucleus assay with Chinese hamster V79 cells—results of a collaborative study with in situ exposure to 26 chemical substances. Mutat. Res.468:137-163
[50]Zhan L, Sakamoto H, Sakuraba M, Wu de S, Zhang LS, Suzuki T, Hayashi M, Honma M.2004. Genotoxicity of microcystin-LR in human lymphoblastoid TK6 cells. Mutat.Res.557:1-6.
[51]Ames, B.N., McCann, J., and Yamasaki, E.1975.Methods for detecting carcino-gens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res.31:347-364.
[52]Kristien Mortelmans,Errol Zeiger.The Ames Salmonella/microsome mutagenicity assay.Mutation Research,2000:29-60
[53]Kristien Mortelmans,Errol Zeiger The Ames Salmonella/microsome mutagenicity assay. Mutation Research 455 (2000) 29-60
[54]N.F. Cariello, W.W. Piegorsch, The Ames test:the two-fold rule revisited, Mutat. Res. 369(1996)23-31.
[55]P. Gee, C.H. Sommers, A.S. Melick, X.M. Gidrol, M.D.Todd, R.B. Burris, M.E. Nelson, R.C. Klemm, E. Zeiger,Comparison of responses of base-specific Salmonella tester strains with the traditional strains for identifying mutagens:the results of a validation study, Mutat. Res.412 (1998) 115-130.
[56]J. McCann, E. Choi, E. Yamasaki, B.N. Ames, Detection of carcinogens in the Salmonella/microsome test. Assay of 300 chemicals, Proc. Natl. Acad. Sci. U.S.A.72 (1975)5135-5139.
[57]E. Zeiger, B. Anderson, S. Haworth, T. Lawlor, K. Mortelmans, W. Speck, Salmonella mutagenicity tests:Ⅲ. Results from the testing of 255 chemicals, Environ. Mutagen. Suppl.9 (9) (1987) 1-110.
[58]E. Zeiger, B. Anderson, S. Haworth, T. Lawlor, K. Mortelmans, Salmonella mutagenicity tests:V. Results from the testing of 311 chemicals, Environ. Mol. Mutagen.19 (Suppl.21) (1992) 1-141.
[59]E. Zeiger, K. Mortelmans, The Salmonella (Ames) test for mutagenicity, D. Mahin E.D. Maines L.G. Costa D.J. Reed S. Sassa I.G Sipes (Eds.), Current Protocols in Toxicology(1999) 3.1.1-3.1.29 WileyNew York
[60]林翔云.调香术[M]化学工业出版社2001,7
[61]Baker R R,Jose R. Pereira da Silva,SmithG. The effect of tobacco ingredients on smoke chemistry. Part Ⅰ:Flavourings and additives[J]. Food and Chemical Toxicology.2004 42(supplement):3-37.
[62]David M D. Genotoxicity of tobacco smoke and tobacco smokecondensate:a review [J]. Mutation Research,2004,567 (2-3):447-474.
[2]董丽,刑均,吴采樱.香精香料的分析方法进展[J].分析科学学报2003,19(2)：189－193
[3]肖协忠等.烟草化学[M].北京：中国农业出版社,1997.
[4]刘海森,何唯平.香精香料基本原理及发展趋势[J].中国食品添加剂2003,5：6-10
[5]D.Layten Davis,MarkT.Nielsen.Tobacco Production,Chemistry and Technology [M]. Blackwell Selenee,1999:336
[6]汪秋安,罗俊霞.香料香精分析技术研究[J].中国调味品,2004,8：13－17
[7]DOOL ITTLE D J, LEE C K. Comparative studies on the genotoxic activity of mains-tream smoke condensatefrom cigarettes which burn or only heat tobacco [J]. Envir Mo-lecular Mutag,1990,15 (2):932105.
[8]DOOL ITTLE D J, LEE C K. Analysis of cytogenetic effects in bone2marrow cells of rats subchronically exposed to smoke from cigarettes which burn or only heat tobacco [J]. Mutat. Res.1990,240 (4):251-277.
[9]DOOL ITTLE D J, BOMB ICK D W. Evaluation of the genotoxic and cytotoxic potential of mainstream whole smoke and smoke condensate from a cigarette contai ning a novel carbon filter[J]. Fundam App 1 Toxicol,1997,39 (1):1127.
[10]BOMB ICK B R, MURL I H, AVALOS J T, et al. Chemical and biological studies of a new cigarette that primarily heats tobacco. Part 2. In vitro toxicology of mainstream smoke condensate [J]. Food Chem. Toxicol.1998,36 (3):1832190.
[11]R ICHARD R BAKER, GRAHAM SM ITH.烟草香味添加剂是否具有毒性的探讨[C]第57届烟草科学研究会议论文集,2003.
[12]PH IL IPMORR ISUSA RESEARCH CENTER. In vitro exposure to 1R4F reference cigarette smoke:Evaluation of developmental toxicity[J]. Toxicol.Sci,2003,75 (1): 1342147.
[13]PH IL IPMORR IS RESEARCH LABORATOR IES GMBH. Chemical composition, cytotoxicity and mutagenicity of smoke from US commercial and reference cigarettes smoked under two sets of machine smoking conditions[J]. Toxicology,2004,195 (1):31252.
[14]Clements, J. Study Design Considerations for in vitro Toxicity Assays on Ingredients 烟草制品及卷烟添加剂安全评价与低潜在性危害卷烟产品开发国际研讨会杭州,2004.10
[15]Massey, E. The Effect of Tobacco Ingredients on Smoke Chemistry and Toxicology: An Overview烟草制品及卷烟添加剂安全评价与低潜在性危害卷烟产品开发国际研讨会杭州,2004.10
[16]http://www.coresta.org/
[17]Doolittle, D.J., Massey, E.D., CORESTA Task Force On In Vitro Toxicity Testing of Tobacco Smoke烟草制品及卷烟添加剂安全评价与低潜在性危害卷烟产品开发国际研讨会杭州,2004.10
[18]朱茂祥.中式低危害卷烟的毒理学评价技术[C].中式卷烟理论内涵讨论论文汇编[C].北京：国家烟草专卖局科技教育司,2004.
[19]郭琳,朱茂祥,黄焕桂,等.复方C卷烟添加剂的安全性及其降低卷烟危害作用[J].中国烟草学报,2007,13(6)：7-12.
[20]吕功煊,聂聪,赵明月,等.应用含纳米贵金属催化材料降低卷烟烟气中CO技术研究[J].中国烟草学报,2003,9(3)：18-27.
[21]Baker R R,Jose R. Pereira da Silva,SmithG. The effect of tobacco ingredients on smoke chemistry. Part Ⅰ:Flavourings and additives[J]. Food and Chemical Toxicology.2004 42(supplement):3-37.
[22]David M D. Genotoxicity of tobacco smoke and tobacco smoke condensate:a review [J]. Mutation Research,2004,567 (2—3):447-474.
[23]高美伶,范家林,周建华,高氟茶浸泡液的细胞毒性及致突变性[J].环境与职业医学,2006,26(2)：166-174.
[24]卢斌斌,Ryan Meng,Lynne Wisse,焦延福.三种不同焦油卷烟烟气的细胞毒性比较[J].烟草科技,2007,12(245)38-41.
[25]赵嘉惠,张华屏,王春芳.MTT法在检测细胞增殖方面的探讨[J].山西医科大学学报,2007,38(3)262－263
[26]司徒镇强,吴军正.细胞培养[M].西安：世界图书出版公司,2007.
[27]Dariasz S, Sarah JS, Riehard H,et al. An improved MTT assay[J]. Immunol Methods,1993,157:203.
[28]Green LM, Reade JL,Ware CF. Rapid colorimetric assay for cell viability of cytotoxic and growth inhibitory lymphokines[J]. Immunol Methods,1984,70(2):257-268.
[29]Maj JG, Kankofer M. Activity of 72 kD and 92 kD matrix metal-loproteinases in placental tissues of cows with and without retained fetal membranes[J]. Placenta, 1997,18:683-687.
[30]赵斌,葛金芳,朱娟娟,黄晓晖,李俊,小议在MTT法测细胞增殖抑制率中IC50的计算方法[J].安徽医药,2007,11(9)834－836
[31]Schlede E,Genschow E, Spielmann H, et al. Oral acute toxic class method:a successful alternative to the oral LD50 test[M]. Regul Toxicol Pharmaco.l United States,2005:15-23.
[32]李寿祺,毒理学原理与方法(第2版)[M]1成都：四川大学出版社,2003,153－177
[33]曹佳,林真,余争平.微核试验[M].北京：军事医学科学出版社,2000：2642272.
[34]刘毓谷.卫生毒理学基础[M].北京：人民卫生出版社,1997：53－81.
[35]Kirsch-Volders M, Sofuni T, Aardema M, Albertini S, Eastmond D, Fenech M, Ishidate M Jr, Kirchner S, Lorge E, Morita T, Norppa H, Surralles J, Vanhauwaert A, Wakata A. Report from the in vitro micronucleus assay working group. Mutat. Res. 2003;540:153-163. [PubMed:14550499]
[36]Matsushima T, Hayashi M, Matsuoka A, Ishidate M Jr, Miura KF, Shimizu H, Morimoto K, Ogura H,Mure K, Koshi K, Sufuni T. Validation study of the in vitro micronucleus test in a Chinese hamster lung cell line (CHL/IU). Mutagenesis 1999; 14:569-580. [PubMed:10567032]
[37]Miller B, Potter-Locher F, Seelbach A, Stopper H, Utesch D, Madle S. Evaluation of the in vitro micronucleus test as an alternative to the in vitro chromosome aberration assay:position of the GUM working group on the in vitro micronucleus test. Mutat. Res.1998;410:81-116. [PubMed:9587425]
[38]Lorge E, Thybaud V, Aardema MJ, Oliver J, Wakata A, Lorenzon G, Marzin D. SFTG international collaborative study on in vitro micronucleus test. I. General conditions and overall conclusions of the study. Mutat.Res.2006;607:13-36. [PubMed: 16815079]
[39]Zhang Y,Holford TR,Leaderer B,et al. Hair-coloring product use and risk of non-Hodgkin's lymphoma:a population-based casecontrol study in connecticut. Am J Epidemiol,2004,159(2):148-154.
[40]杜立颖,冯仁青.流式细胞术[M].北京大学出版社2008,7
[41]Viaggi S, Braselmann H, Nusse M. Flow cytometric analysis of micronuclei in the CD2+ subpopulation of human lymphocytes enriched by magnetic separation. Int J Radiat Biol 1995;67:193-202. [PubMed:7884288]
[42]Nusse M, Marx K. Flow cytometric analysis of micronuclei in cell cultures and human lymphocytes:advantages and disadvantages. Mutat. Res.1997;392:109-115. [PubMed:9269335]
[43]Roman D, Locher F, Suter W, Cordier A, Bobadilla M. Evaluation of a new procedure for the flow cytometric analysis of in vitro, chemically induced micronuclei in V79 cells. Environ Molec Mutagen 1998;32:387-396. [PubMed:9882014]
[44]Avlasevich SL, Bryce SM, Cairns SE, Dertinger SD. In vitro micronucleus scoring by flow cytometry:differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability. Environ Molec Mutagen.2006;47:56-66. [PubMed:16180205]
[45]Steven M. Bryce, Jeffrey C. Bemis, Svetlana L. Avlasevich, and Stephen D. Dertinger In vitro micronucleus assay scored by flow cytometry provides a comprehensive evaluation of cytogenetic damage and cytotoxicity. Mutat. Res.2007 June 15; 630(1-2):78-91.
[46]Garriott ML, Phelps JB, Hoffman WP.2002. A protocol for the in vitro micronucleus test. I. Contributions to the development of a protocol suitable for regulatory submissions from an examination of 16 chemicals with different mechanisms of action and different levels of activity. Mutat. Res.517:123-134.
[47]Kirsch-Volders M, Sofuni T, Aardema M, Albertini S, Eastmond D, Fenech M, Ishidate M, Jr., Kirchner S, Lorge E, Morita T, Norppa H, Surralle's J, Vanhauwaert A, Wakata A.2003. Report from the in vitro micronucleus assay working group. Mutat. Res.540:153-163.
[48]OECD.2004. In vitro micronucleus test. OECD Guideline for the Testing of Chemicals, Draft Proposal for a New Guideline 487. Paris:OECD.Available at http://www.oecd.org/dataoecd/60/28/32106288.pdf.
[49]von der Hude W, Kalweit S, Engelhardt G, McKiernan S, Kasper P,Slacik-Erben R, Miltenburger HG, Honarvar N, Fahrig R, Gorlitz B, Albertini S, Kirchner S, Utesch D, Potter-Locher F, Stopper H, Madle S.2000. In vitro micronucleus assay with Chinese hamster V79 cells—results of a collaborative study with in situ exposure to 26 chemical substances. Mutat. Res.468:137-163
[50]Zhan L, Sakamoto H, Sakuraba M, Wu de S, Zhang LS, Suzuki T, Hayashi M, Honma M.2004. Genotoxicity of microcystin-LR in human lymphoblastoid TK6 cells. Mutat.Res.557:1-6.
[51]Ames, B.N., McCann, J., and Yamasaki, E.1975.Methods for detecting carcino-gens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res.31:347-364.
[52]Kristien Mortelmans,Errol Zeiger.The Ames Salmonella/microsome mutagenicity assay.Mutation Research,2000:29-60
[53]Kristien Mortelmans,Errol Zeiger The Ames Salmonella/microsome mutagenicity assay. Mutation Research 455 (2000) 29-60
[54]N.F. Cariello, W.W. Piegorsch, The Ames test:the two-fold rule revisited, Mutat. Res. 369(1996)23-31.
[55]P. Gee, C.H. Sommers, A.S. Melick, X.M. Gidrol, M.D.Todd, R.B. Burris, M.E. Nelson, R.C. Klemm, E. Zeiger,Comparison of responses of base-specific Salmonella tester strains with the traditional strains for identifying mutagens:the results of a validation study, Mutat. Res.412 (1998) 115-130.
[56]J. McCann, E. Choi, E. Yamasaki, B.N. Ames, Detection of carcinogens in the Salmonella/microsome test. Assay of 300 chemicals, Proc. Natl. Acad. Sci. U.S.A.72 (1975)5135-5139.
[57]E. Zeiger, B. Anderson, S. Haworth, T. Lawlor, K. Mortelmans, W. Speck, Salmonella mutagenicity tests:Ⅲ. Results from the testing of 255 chemicals, Environ. Mutagen. Suppl.9 (9) (1987) 1-110.
[58]E. Zeiger, B. Anderson, S. Haworth, T. Lawlor, K. Mortelmans, Salmonella mutagenicity tests:V. Results from the testing of 311 chemicals, Environ. Mol. Mutagen.19 (Suppl.21) (1992) 1-141.
[59]E. Zeiger, K. Mortelmans, The Salmonella (Ames) test for mutagenicity, D. Mahin E.D. Maines L.G. Costa D.J. Reed S. Sassa I.G Sipes (Eds.), Current Protocols in Toxicology(1999) 3.1.1-3.1.29 WileyNew York
[60]林翔云.调香术[M]化学工业出版社2001,7
[61]Baker R R,Jose R. Pereira da Silva,SmithG. The effect of tobacco ingredients on smoke chemistry. Part Ⅰ:Flavourings and additives[J]. Food and Chemical Toxicology.2004 42(supplement):3-37.
[62]David M D. Genotoxicity of tobacco smoke and tobacco smokecondensate:a review [J]. Mutation Research,2004,567 (2-3):447-474.