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FROM: F.R.``Perini SUBJECT: Candidates For Tobacco Nitrosation Prevention At Hogshead Levels Given that nitrosation prevention coupled with fermentation . tolerance may be useful, the following proposal is made. It aims at assessing the practicality of eliminating nitrosamine growth ( i.e., NNN) with concomitant nondisruption of the fermentation process. Questions dealing with the choice of additives, their concentrations and scale of operations had been addressed in a 1979 report (1): "Exact correspondence on a time scale cannot be expected due to the intensity of conditions in the fermentation chamber, especially the moisture level and the tumbling effect. These conditions have been adopted to enhance NNN formation during (laboratory) fermentation. Levels of additives are set much higher than would be desirable in Lexington and Louisville so as to quickly detect useful protection agents. Efficiency comparisons can readily be made among the additives themselves. A decision can then be made to apply from two to five of the most desirable additives to tobacco in hogsheads for a long term protection study." A practical test system~which would be intermediate towards hogshead application involves a number of wooden boxes, such as those in which some foreign tobacco is received. Their dimensions are approximately 4x2 1/2x2 1/2 ft, and they hold 440 lbs of pressed bright tobacco. Alternatively one might use hogsheads. In either case, the contents would be useable in blending later on~. We know from a 1980 memorandum (2), that NNN levels in green or unaged PF tobacco during the years 1973-78 ranged'from 1.5 to 4.4 ppm. The six year average was 2.7 ppm~, The PF2-1977 tobacco NNN level rose from 4.4 to 30.6 ppm after aging and hogshead forced fermentation in Louisville. It is noteworthy that the crucial time for NNN increase takes place at the very beginning of aging, as mentioned in a 1979 report (3): "The early, and possibly most active, stage in aging may occur in the first few days, even hours, after putting,up the tobacco at 28% moisture. The slight changes in Leaf Analysis data and NNN level occurring over an O eleven week period may,be real." ~ O A proper time for antinitrosation additive application is then~ O during tobacco prizing at the inception.of aging, if not even 0 earlier. en kD.

As cited in a 1981 overview (4), the two main classes of com- pounds under consideration for hogshead-scale nitrosationpre- vention are certain phenolic antioxidants and'some inorganic chemical reducers. The first class embraces hydroquinone (HQ) a) nitrosation prevention at a reasonable dose (,< 0.1%), b) fermentation tolerance (fermentation must go on as usual), c) aqueous carrier preferability due to economic reasons, and related compounds. These are the most effective protectants yet found~by us, but are not on the GRAS list.* Others, such as n-propyl gallate (PG) are less effective but are GRAS, as is the case with inoraanic salts such as sodium sulfite. One should take note that,currently, tobacco companies employ potassium or sodium sorbate and benzoate as preservatives to the extent of 0.25% (w/w) in chewing tobaccos. This number can give us some perspective in choosing reasonable levels for the proposed' protective additives. The factors to be considered are: 2 d) utmost importance of health and safety (a GRAS additive is preferred), -1 ( e) the taste of the final product must be acceptable. On the last point, the simplest and earliest test would consist of applying a reasonable level of additive to the existing chewing tobacco with normal casing. It is possible that (a) a be gleaned during long term (months to years) experimentation~. after application, but not after fermentation, and (b) even a persistent problem of this sort could be addressed by masking with other materials. Additive levels on the tobacco at the end of fermentation are a key measurement. This information can only borderline or unacceptable taste would be detected immediately *rlost additives on FDA's Generally Recognized as Safe (GRAS) list actually have been found to be safe by a special committee. Completing the first scientific review of all the 415 original ingredients on the 21-year-old list, a select panel from the Federation of American Societies for Experimental Biology has declared that 305 of the compounds are safe in their present use and for foreseeable future uses. Another 68 are safe at least at their present use levels. The panel gave conditional approval to the other ingredients (5). ` N 0 ~ 0 0

RECOMMENDED ADDITIVES The following materials are recommended for testing, criteria (a) - (e) above: (1) n_-Propyl Gallate (PG) OH NO~~~ oH v ~j -© CHZ Chr 2. C /y3 O Laboratory fermentation effectiveness,* 53% @ 1% (w/w) application level. Carrier (solubility), water (0.35 g/100 mL). Current status and usage, FDA-GPAS (LD50 rats 3800 mg/kg);`used' as a food antioxidant. Maximum use level, 100&ppm (0.1%', w/w) (6). ~Level recommended for testing, 1000 ppm (0.1%, w/w). (2) Tannic Acid (TA)~ Laboratory fermentation effectiveness, 33-38% @ 1% (w/w), application level. NOTE: Mutagenicity of tobacco tars on Salmonella is markedly decreased with tannic acid. Thus, tannic acid was sprayed onto shredded tobacco to 15% and the treateditobacco was made into cigarettes. The number of mutant colonies of Salmonella was 253 and 483 for tars from the tannic acid- treated cigarettes and those from the control, respectively (7)• Carrier (solubility), water (1.00 g/0.35 mL). *Percentage not formed relative to untreated~fermented control. 010600fi1

4 Current status and~usage FDA-GRAS(LD rats 6000 m /k ) clari-• , S g g ; fying agent in the brewing and'wine in8ustries and as a flavoring agent; medicinal (oral dose, 0.2-1.3 g). Maximum use level, 140 ppm (0.014%, w/w) in and hard candy (8). Level recommended for testing, (3) Sodium bisulfite (SBS) NaHSO3 effectiveness, 24% @ 0.5% (w/w) application level and 54% @'1g (w/w). :- ~ Laboratory fermentation is acidic). Carrier (solubility), water (freely soluble, 1.00 g/3.5 mL; solution Current status and usage, FDA-GRAS; antioxidant, chemical preser- vative. Maximum use level, 2000 ppm at start, 300 ppm at end imdried fruits, etc. (6). Level recommended for testing, 1000 ppm (0.1g', w/w). (4) Sodium sulfite (SS) Na2SO3 Laboratory.fermentation effectiveness, 33% @ 0.5% (w/w) application level. Carrier (solubility)'„ ' water (very soluble, 1.00 g/1.6 mL; solution is alkaline, pH ti 9). Current status and usage, FDA-GRAS; antioxidant, chemical preser- vative for meats unless recognized as vitamin B1 source, egg yolks, etc. Maximum use level, 20,00 ppm at start, 300 ppm at end, in molasses, dried'fruits, etc.; 350 ppm in finished wines (6). p Level recommended for testing, 1410 ppm ( 0 . 014%) . 1000 ppm (0.1%, w/w). • N

(5) Sodium metabisulfite Na2S205 Carrier (solubiiity), water (freely soluble; solution is acidic). Current status and usage, FDA-GRAS; antioxidant, c-hemical preser- vative. Maximum use level, 2000 ppm at start, 300 ppm~at end of process in molasses, dried fruits, etc.; 350 ppm in finished wines (6). Level recommended for w/w). (or pyrosulfite) (SMBS) Laboratory fermentation effectiveness, not quantitated, but this chemical behaves much like Na2S03. NOTE: A 1980 report cites: "T.C. Tso...found that bright- type tobacco treated'with 2 mmoles of sodium metabisulfite per kg during a homogenized leaf curing process had 1/3 less of the dimethylnitrosantine found after conventional curing. In the case of burley, 80% of the expected dimethylnitrosamine was prevented from forming" (9). The level used was 0.380~g per 1000 g of tobacco, or 380 ppm. r testing, 1000 ppm (0.1%, (6) 2,6-Di-l-butyl-4-methylphenol (Butylated hydroxytoluene (BHT)) OH Laboratory fermentation effectiveness, 91% @ 0.8% (w/w) application level (in FC-11). Carrier solubility, FC-11 (freon~, 1.00 g/mL) ; also ethanol, acetone, ether (&. 4 g/10 mL) ; insoluble in water.

6 Current status and usage, FDA-GRAS; antioxidant, chemical preser- vative. Maximum use level, 1000 ppm (0.1%, w/w) in chewing-gum base; 200 ppm in fats, oils, sausages, etc. (6). Level recommended for testing, 1000 ppm (0.1%, w/w). (7) Hydroquinone (p-hydroxyphenol) (HQ) Laboratory fermentation effectiveness, 65% @ 0.25% (w/w) application level and 93% @ 1% (w/w). ~ Carrier solubility, water (1.00 g/14 mL); also freely soluble in ethanol, ether. Current status and usage, reducing agent and antioxidant (not in the food'ind'ustry); LD 0 rats 370 mg/kg; relatively safe in low concentrations (10). Aarmful effects are likely to occur to workers who inhale the dust or vapors or whose skin and eyes come in contact with the dust or vapors for prolonged periods (11). HQ is oxidized to 1,4-benzoquinone (LD rats 130 mg/kg), which also displays excellent protection agai~st nitrosation ( i.e., 99% effectiveness @ 1% (w/w) application~level) (12). Maximum use level, unkno~n; hygienic standard of permissible exposure, as TLV, 2 mg/m . -Level recommended for testing, but without preliminary taste ` testing, 500 ppm. F.R. Perini /jw:I Xc: J.H. Bell a N F.J. Schultz ." O M 0 0 ~ 4h

REFERENCES 1. F.R. Perini, Lorillard Report Accession No. 1399 (Aug. 16, 1979). 2. F.R. Perini, Lorillard Memorandum, "NNN Concentration in Louisville Force Fermented PF 2-1977 Tobacco," (Aug. 22, 19 8'0 ) . 3. F.R. Perini and J.H. Bell, Lorillard' Report Accession No. 1400 (Aug. 22, 1979). 4. F.R. Perini, Lorillard Report Accession No. 1585 (Jan. 1981), . 5. T.E. Furia, ed., "Handbook of Food Additives," 2nd ed. Press, Cleveland', GH., C 1972. Chemical &__En5ir.eering News, 59, 17 (Jan. 12, 1981). CRC 7. "Tannic acid for decrealsing,mutagenicity of tobacco tars," (Japan Tobacco and Salt Public Corp.) (Japan). Jpn. Kokai Tokkyo Koho 81 15,681 (Cl. A24B3/12), Feb. 14, 1981, CA, 94:('21) : 171,237c (1981). 8. G.W. Irving, Jr., Food and Drug, Administration Repcrt No. PB-274669, "Evaluation of the Health!Aspects of Tannic Acid . as a Food ingredient," (1977). 9. F'.R. Perini, Lorillard Report Accession No. 1530 (Aug. 19, 1980;) • 10. M. Windholz, ed., "The Merck Index," 9th ed., Merck & Co., Inc., Rahway, N.J., 1976, p. 635. 11. W.B. Deichmann and M.L. Keplinger, in "Patty's Industrial Flyg,iene and Toxicology," 3rd rev. ed., Vol. 2A, G.D. Clayton and F.E. Clayton, eds., Wiley-Interscience, N.Y., N.Y., 1981, pp. 2589 ff. I 12. F.R. Perini, Lorillard Report Accession No;.. 1528 (Aug. 11, 1980').