The cigarette ingredients cocoa powder, glycerol, and saccharose were investigated regarding their potential effect on the resulting mainstream smoke, i.e., smoke chemistry (Hoffmann analytes), mammalian cell cytotoxicity (Neutral Red Uptake assay), and bacterial mutagenicity (Ames assay). Each ingredient was added at three concentrations to the tobacco of a 6 mg and 10 mg ‘tar’ yield experimental American blend filter cigarette (obtained under ISO/FTC smoking regime). The lowest application concentration was equivalent to the normal approximate use level of the ingredients; the highest application level was up to 5-fold higher. The resulting data were compared with the respective control cigarettes without addition of the ingredients. The addition of cocoa powder did not lead to any consistent effects on the measured mainstream smoke analytes. Neither the in vitro cytotoxicity nor the in vitro mutagenicity was affected by cocoa addition. The addition of glycerol resulted in a decrease in the delivery of several smoke constituents (generally around 20%), e.g. aldehydes, phenolics, and N-nitrosamines. Water in the particulate phase (TPM) was distinctly increased (up to +150%). The cytotoxicity of the TPM was decreased (approx. !15%). Mutagenicity was not affected. Saccharose addition consistently increased formaldehyde delivery in smoke by up to 40% and decreased tobacco-specific N-nitrosamines by up to approximately 20%. The increase in formaldehyde is discussed in the context of the human smoker. The cytotoxicity was not affected by the addition of saccharose, while the mutagenicity of the TPM was decreased in tester strain TA98 with metabolic activation (!15%). The results are in agreement with currently available literature. Some investigations summarized in this publication are novel and have not yet been reported in the literature. Based on the total evidence, it can be concluded that the three ingredients added at their current use levels do not increase the inherent toxicity of the cigarette smoke.
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1. Brooks, J. E.: The Mighty Leaf. Tobacco through the centuries; Alvin Redman, London and Sydney, 1953.
2. Rodgman, A.: Some Studies of the Effects of Additives on Cigarette Mainstream Smoke Properties. II. Casing Materials and Humectants; Beitr. Tabakforsch. Int. 20 (2002) 279–299.
3. Fisher, P.: Cigarette manufacture, 11A, Tobacco blending; in: Tobacco Production, Chemistry and Technology, edited by D. L. Davis and M. T. Nielsen, Blackwell Science Inc., Malden, MA., 1999, pp. 346–352.
4. Fagerson, I. S.: Thermal degradation of carbohydrates. A review; J. Agric. Food Chem. 17 (1969) 747–750.
5. Phillpotts, D. F., D. Spincer, and D. T. Westcott: The Effect of the Natural Sugar Content of Tobacco Upon the Acetaldehyde Concentration Found in Cigarette Smoke; Beitr. Tabakforsch. Int. 8 (1975) 7–10.
6. Coleman III, W. M. and T. A. Perfetti: The Roles of Amino Acids and Sugars in the Production of Volatile Materials in Microwave Heated Tobacco Dust Suspensions; Beitr. Tabakforsch. Int. 17 (1997) 75–95.
7. Seeman, J. I., M. Dixon, and H. J. Haussmann: Acetaldehyde in Mainstream Tobacco Smoke: Formation and Occurrence in Smoke and Bioavailability in the Smoker; Chem. Res. Toxicol. 15 (2002) 1331– 1350.
8. Rustemeier, K., R. Stabbert, H. -J. Haussmann, E. Roemer, and E. Carmines:. Evaluation of the potential effects of ingredients added to cigarettes. Part 2: Chemical composition of mainstream smoke; Food Chem. Toxicol. 40 (2002) 93–104.
9. Sanders, E. B., A. I. Goldsmith, and J. I. Seeman: A model that distinguishes the pyrolysis of D-Glucose, D-Fructose, and sucrose from that of cellulose. Application to the understanding of cigarette smoke formation; J. Anal. Appl. Pyrolysis 66 (2003) 29–50.
10. Seeman, J. I., S. W. Laffoon, and A. J. Kassman: Evalu-ation of Relationships Between Mainstream Smoke Acetaldehyde and “Ttar” and Carbon Monoxide Yields in Tobacco Smoke and Reducing Sugars in Tobacco Published by Intorp et al. in this issue on page 139. Blends of U. S. Commercial Cigarettes; Inhal. Toxicol. 15 (2003) 373-395.
11. Baker, R. R. and L. J. Bishop: The pyrolysis of tobacco ingredients; J. Anal. Appl. Pyrolysis 71 (2004) 223-311.
12. Baker, R. R., S. Coburn, C. Liu, and J. Tetteh: The pyrolysis of saccharide tobacco ingredients: a TGA-FTIR investigation; J. Anal. Appl. Pyrolysis 74 (2005) 171-180.
13. Baker, R. R., J. R. Pereira da Silva, and G. Smith: The effect of tobacco ingredients on smoke chemistry. Part I: Flavourings and additives; Food Chem. Toxicol. 42S1 (2004) 3-37.
14. Baker, R. R.: The generation of formaldehyde in cigarettes - Overview and recent experiments; Food Chem. Toxicol. 44 (2006) 1799-1822.
15. Baker, R. R., S. Coburn, and C. Liu: The pyrolytic formation of formaldehyde from sugars and tobacco; J. Anal. Appl. Pyrolysis 77 (2006) 12-21.
16. Sato, S., T. Ohka, M. Nagao, K. Tsuji, and T. Kosuge: Reduction in mutagenicity of cigarette smoke conden-sate by added sugars; Mutat. Res. 60 (1979) 155-161.
17. Heck, J. D., CL. Gaworski, N. Rajendran, and R. L. Morrissey: Toxicologic evaluation of humectants added to cigarette tobacco: 13-week smoke inhalation study of glycerin and propylene glycol in Fischer 344 rats; Inhal. Toxicol. 14 (2002) 1135-52.
18. Roemer, E., FJ. Tewes, TJ. Meisgen, D. J. Veltel, and E. Carmines: Evaluation of the potential effects of ingredients added to cigarettes. Part 3: In vitro genotoxicity and cytotoxicity; Food Chem. Toxicol. 40 (2002) 105-111.
19. Vanscheeuwijck, P. M., A. Teredesai, P. M. Terpstra, J. Verbeeck, P. Kuhl, B. Gerstenberg, S. Gebel, and E. Carmines: Evaluation of the potential effects of ingredients added to cigarettes. Part 4: Subchronic inhalation toxicity; Food Chem. Toxicol. 40 (2002) 113-131.
20. Baker, R. R., E. D. Massey, and G. Smith: An overview of the effects of tobacco ingredients on smoke chemistry and toxicity; Food Chem. Toxicol. 42S (2004) S53-S83.
21. Renne, R. A., H. Yoshimura, K. Yoshino, G. Lulham, S. Minamisawa, A. Tribukait, D. D. Dietz, K. M. Lee, and R. B. Westerberg: Effects of Flavoring and Casing Ingredients on the Toxicity of Mainstream Cigarette Smoke in Rats; Inhal Toxicol. 18 (2006) 685-706.
22. Stavanja, M. S., P. H Ayres, D. R. Meckley, E. R. Bom-bick, M. F. Borgerding, M. J. Morton, CD. Garner, D. H. Pence, and J. E. Swauger: Safety assessment of high fructose corn syrup (HFCS) as an ingredient added to cigarette tobacco; Exp. Toxicol. Pathol. 57 (2006) 267-281.
23. ISO, International Organization for Standardization: ISO/IEC 17025:2005, General requirements for the competence of testing and calibration laboratories. Geneva, ISO (2000).
24. ISO, International Organization for Standardization: ISO 3308:2000, Routine analytical cigarette-smoking machine - Definitions and standard conditions. Geneva, ISO (2000).
25. Health Canada: Health Canada Tobacco Reporting Regulations Part 3, Emissions from designated tobacco products. Canada Gazette, Part II, 134 (2000) (15, Part 3). Test methodologies with their designated test numbers have been posted by Health Canada in the internet at the site http://www.hc-sc.gc.ca/hc-ps/tobac-tabac/legislation/reg/indust/method/index-eng.php. (Accessed Sep. 25, 2010).
26. CPSC, U. S. Consumer Products Safety Commission in Consultation with the US Department of Health and Human Services, Toxicity Testing Plan 5. DHHS, Washington, DC 20207 (1993) pp D1-D38.
27. Bundesgesetzblatt, Deutschland (Federal Register, Germany): Grundsätze der Guten Laborpraxis (GLP), Neufassung des Chemikaliengesetzes vom 20. Juni 2002 (Principles of Good Laboratory Practice (GLP), revised version of the Chemical Acts of 20 June 2002). Bundesgesetzblatt I 40 (2002) pp. 2090-2130.
28. Babich, H. and E. Borenfreund: Cytotoxic and mor-phological effects of phenylpropanolamine, caffeine, nicotine, and some of their metabolites studied In Vitro; Toxicol. In Vitro 6 (1992) 493-502.
29. Borenfreund, E. and J. A. Puerner: Toxicity determined in vitro by morphological alterations and neutral red absorption; Toxicol. Lett. 24 (1985) 119-124.
30. Maron, D. M. and B. N. Ames: Reviewed methods for the Salmonella mutagenicity test; Mutat. Res. 113 (1983) 173-215.
31. OECD, Organization for Economic Cooperation and Development, 1997. Guideline 471, bacterial reverse mutation test. In: OECD Guidelines for Testing of Chemicals. OECD, Paris, France.
32. Guzelian, P. S., M. S. Victoroff, N. C Halmes, R. C. James, and CP. Guzelian: Evidence-based toxicology: a comprehensive framework for causation; Hum. Exp. Toxicol. 24 (2005) 161-201.
33. Hoffmann, S. and T. Hartung: Towards an evidence based toxicology; Human Exp. Toxicol. 25 (2006) 497-513.
34. European Community: Directive 2001/37/EC of the European Parliament and of the council of 5 June 2001 on the approximation of the laws, regulations and administrative provisions of the Member States con-cerning the manufacture, presentation and sale of tobacco products. Accessible at http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2001:194:0026:0034:EN:PDF (Accessed Sep. 25, 2010).
35. DIN, Deutsches Institut für Normung: The Toxicologi-cal Evaluation of Additives for Tobacco Products - A Guide, 1st Edition, Technical Report 133. Beuth Verlag, Berlin, 2004.
36. Rabinoff, M., N. Caskey, A. Rissling, and C. Park: Pharmacological and Chemical Effects of Cigarette Additives; Am. J. Public Health 97 (2007) 1981-1991.
37. Craig, W. J. and T. T. Nguyen: Caffeine and Theobro-mine Levels in Cocoa and Carob Products; J. Food Sci. 49 (1984) 302-303.
38. Stavric, B.: Methylxanthines: toxicity to humans. 3. Theobromine, paraxanthine and the combined effects of methylxanthines; Food Chem. Toxicol. 26 (1988) 725-733.
39. Rossner, S.: Chocolate and nutrition - divine food, fattening junk or nutritious supplementation; Manu-fact. Confectioner 79 (1999) 57-60.
40. Bates, C, M. Jarvis, and G. Connolly, 1999: Tobacco additives. Cigarette engineering and nicotine addiction. Action on Smoking and Health.
41. Rambali, B., I. van Andel, E. Schenk, G. Wolterink, G. van de Werken, H. Stevenson, and W. Vleeming: RIVM report 650270002/2002, The contribution of cocoa additive to cigarette smoking addiction; RIVM, Bilthoven, Netherlands, 2003.
42. Simons, F. E. R, A. B. Becker, K. J. Simons, and CA. Gillespie: The bronchodilator effect and pharmaco-kinetics of theobromine in young patients with asthma; J. Allergy Clin. Immunol. 76 (1985) 703-707.
43. IARC, International agency for Research on Cancer: IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Volume 51. Coffee, Tea, Mate, Methylxanthines and Methylglyoxal; IARC, Lyon, France, 1991, pp. 421-441.
44. Greger, G.: Testing of lung function after theophylline inhalation (author's transl.); Z. Erkr. Atmungsorgane 157 (1981) 270-275.
45. IITRI, Illinois Institute of Technology Research Institute: Chemical analysis of mainstream smoke from cigarettes containing target levels of 0, 10,000, 20,000 or 40,000 ppm of cocoa. Final Report, IITRI Project Number 8739-117-002; IITRI, Chicago, 2003.
46. IITRI, Illinois Institute of Technology Research Institute: Cytotoxicity assay (neutral red uptake) of smoke fractions from cigarettes containing target levels of 0, 10,000, 20,000 or 40,000 ppm of cocoa. Final Report, IITRI Project Number 8739-117-004; IITRI, Chicago, 2003.
47. IITRI, Illinois Institute of Technology Research Institute: In vitro mutagenicity of mainstream smoke from cigarettes containing target levels of 10,000, 20,000 or 40,000 ppm cocoa in Salmonella/microsome assay. Final Report, IITRI Project Number 8739-117-003; IITRI, Chicago, 2003.
48. IITRI, Illinois Institute of Technology Research Institute: 90-day rat inhalation toxicity study of the mainstream smoke from cigarettes containing target levels of 0, 10,000, 20,000 or 40,000 ppm of cocoa. Volumes I to III. Final Report, IITRI Project Number 8739-117-001; IITRI, Chicago, 2006.
49. NCI, National Cancer Institute: Smoking and Health Program, Report No. 3, Towards less hazardous cigarettes: the third set of cigarettes, G. B. Gori, Editor. DHEW, Washington, 1977.
50. Roemer, E. and U. Hackenberg: Mouse skin bioassay of smoke condensates from cigarettes containing different levels of cocoa; Food Addit. Contam. 7 (1990) 563-569.
51. Gaworski, CL., J. D. Heck, M. B. Bennett, and M. L. Wenk: Toxicologic evaluation of flavor ingredients added to cigarette tobacco: skin painting bioassay of cigarette smoke condensate in SENCAR mice; Toxi-cology 139 (1999) 1-17.
52. Lee, P. N, B. A. Forey, J. S. Fry, J. S. Hamling, J. F. Hamling, E. B. Sanders, and R. A. Carchman: Does use of flue-cured rather than blended cigarettes affect international variation in mortality from lung cancer and COPD?; Inhal. Toxicol. 21 (2009) 404-30.
53. Hine C. H., H. H. Anderson, H. D. Moon, M. K. Dunlap, and M. S. Morse: Comparative toxicity of synthetic and natural glycerin; Arch Ind. Hyg. Occup. Med. 7 (1953) 282-291.
54. Doolittle, D. J., D. A. Lee, and CK. Lee: The genotoxic activity of glycerol in an in vitro test battery; Food Chem. Toxicol. 26 (1988) 631-635.
55. Renne, R. A.: 2-Week and 13-Week Inhalation Studies of Aerosolized Glycerol in Rats; Inhal. Toxicol. 4 (1992) 95-111.
56. Mauderly, J. L.: Respiration of F-344 rats in nose-only inhalation exposure tubes; J. Appl. Toxicol. 6 (1986) 25-30.
57. Laurene, A. H., R. H. Cundiff, and G. H. Greene: De-termination of glycerol and propylene glycol in cigarette smoke; Tob. Sci. 9 (1965) 1-4.
58. Liu, C: Glycerol Transfer in Cigarette Mainstream Smoke; Beitr. Tabakforsch. Int. 21 (2004) 111-116.
59. Carmines, E. L. and CL. Gaworski: Toxicological evaluation of glycerin as a cigarette ingredient; Food Chem. Toxicol. 43 (2005) 1521-1539.
60. IITRI, Illinois Institute of Technology Research Institute: 90-day rat inhalation toxicity study of the mainstream smoke from cigarettes containing target levels of 0, 50,000, 100,000 or 150,000 ppm of glycerin. Volumes I to III. Final Report, IITRI Project Number 8739-114-001, Chicago, 2004.
61. NIOSH, National Institute of Occupational Safety and Health: Pocket Guide to Chemical Hazards, DHHS (NIOSH) Publication No. 2005-149, NIOSH, Atlanta 2005.
62. ACGIH, American Conference of Governmental Industrial Hygienists: TLVs® and BEIs®: Threshold Limit Values for Chemical Substances and Physical Agents, ACGIH, Cincinnati 2002.
63. MAK Kommission (Commission for maximum concentration at the workplace): Gesundheitsschäd-liche Arbeitsstoffe, Toxikologisch-arbeitsmedizinische Begründungen von MAK-Werten, Mitteilung 1-44, Formaldehyd, Wiley-VCH, Weinheim, 2000.
64. IARC, International Agency on Research on Cancer: IARC Monographs on the Evalauation of Carcinogenic Risks to Humans, Vol. 88, Formaldehyde, 2-Butoxy-ethanol and 1-tert-Butoxypropan-2-ol, IARC, Lyon, 2006.
65. G. Jaccard, Philip Morris International, R&D, Market Map Study on 202 cigarette brands from 10 countries, 2008, Internal Report, not published.
66. Scherer, G., J. Engl, M. Urban, G. Gilch, D. Janket, and K. Riedel, K.: Relationship between machine-derived smoke yields and biomarkers in cigarette smokers in Germany; Regul. Toxicol. Pharmacol. 47 (2007) 171-183.
67. OSHA, Occupational Safety & Health Administration: Occupational Safety and Health Standard 1919. 1048, Formaldehyde, OSHA, Washington, 2008.
68. CIIT, Chemical Industry Institute of Toxicology: Formaldehyde: Hazard Characterization and Dose-response Assessment for Carcinogenicity by the Route of Inhalation (revised edition); CIIT, Research Triangle Park, NC, 1999.
69. Conolly R. B., J. S. Kimbell, D. Janszen, P. M. Schlosser, D. Kalisak, J. Preston, and F. J. Miller: Human Respiratory Tract Cancer Risks of Inhaled Formaldehyde: Dose-response Predictions Derived from Biologically-motivated Computational Modeling of a Combined Rodent and Human Dataset; Toxicol. Sci. 82 (2004) 279–296.
70. Crump K. S., C. Chen, J. F. Fox, C. Van Landingham C, and R. Subramaniam: Sensitivity Analysis of Biolo-gically Motivated Model for Formaldehyde-induced Respiratory Cancer in Humans; Ann. Occup. Hyg. 52 (2008) 481–495.
71. Schulte, A., U. Bernauer, S. Madle, H. Mielke, U. Herbst, H. -B. Richter-Reichhelm, K. -E. Appel, and U. Gundert-Remy: Assessment of the Carcinogenicity of Formaldehyde [CAS No. 50-00-0]. Bericht zur Bewer-tung der Karzinogenität von Formaldehyd, Bundes-institut fuer Risikobewertung, Berlin. 2006.
72. IITRI, Illinois Institute of Technology Research Institute: Cytotoxicity assay (neutral red uptake) of smoke fractions from cigarettes containing target levels of 0, 36,000, 72,000 or 100,000 ppm of sucrose. Final Report, IITRI Project Number 8739-106-004, Chicago, 2002.
73. IITRI, Illinois Institute of Technology Research Institute: 90-day rat inhalation toxicity study of the mainstream smoke from cigarettes containing target levels of 0, 36,000, 72,000 or 100,000 ppm of sucrose. Final Report, IITRI Project Number 8739-106-001, Chicago, 2003.
74. Gaworski, CL., M. M. Dozier, J. D. Heck, J. M. Ger-hart, N. Rajendran, R. M. David, L. H. Brennecke, and R. Morrissey: Toxicologic evaluation of flavor ingre-dients added to cigarette tobacco: 13 week inhalation exposures in rats; Inhal. Toxicol. 10 (1998) 357-381.
75. European Community: Directive 2001/37/EC of the European Parliament and of the council of 5 June 2001on the approximation of the laws, regulations and administrative provisions of the Member States con-cerning the manufacture, presentation and sale of tobacco products. Accessible at http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L: 2001:194:0026:0034:EN:PDF (Accessed Sep. 25, 2010).