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. R1VltEGED AP1D CONFID~~T1A~ REDUCTION OF CARCINOGENICITY ON MOUSE SKIN f OF CIGARETTE SMOKE CONDENSATE WITH PALLADIUM CATALYST I §Life Sciences Division, Arthur D. Little, Inc., Cambridge, Mass. 02140; and tResearch Department Liggett Group, Inc., Durham, North Carolina 27702 ABSTRACT (0 The carcinogenicity of cigarette smoke condensate (CSC) toward mouse skin has been dramatically reduced by 79-100% through a com- bination of palladium metal catalyst with blends high in Burley tobacco content or with lower Burley tobacco blends supplemented by the addition of compensating amounts of a nitrate salt. This lowering of carcinogenicity is associated with decreases in the level of polycyclic aromatic hydrocarbons (PAH) in the smoke con- densate. Through the use of filters designed to complement this additive combination, the other measured components of the smoke stream are either lower than or equivalent to those of conventional best selling US cigarette brands. This is confirmed by lowered in vitro ciliatoxicity and cytotoxicity responses as well as in vivo sub- acute inhalation studies with rats.

REDUCTION OF CARCINOGENICITY ON MOUSE SKIN OF CIGARETTE SMOKE CONDENSATE WITH PALLADIUM CATALYST §Life Sciences Division, Arthur D. Little, Inc., Cambridge, Mass. 02140; and tResearch Department, Liggett Group, Inc., Durham, North Carolina 27702 4 In a separate report (Mold, 1978), we have described the experi- mentation which has led us to conclude that the polycylic aromatic hydrocarbons (PAH) in cigarette smoke are of major significance in its carcinogenicity toward mouse skin. The complexity of this mix- ture caused us to direct our further efforts toward a reduction in the yield of this total class of compounds rather than to attempt to further subdivide it. Important requirements of these studies were the availability of the procedure described in our previous report for rapidly ob- taining a concentrate of PAH's and the estimation of the quantity of these present by the Sebaceous Gland Suppression (SGS) test. A correlation of SGS activity and carcinogenic potency of pure PAH compounds has been noted (Bock, 1958) and, at the degree of purity obtained for our PAH concentrate from cigarette smoke condensate (CSC), we have also found a very good correlation to exist between the SGS ED50 and the IR absorbance, thus facilitating our studies. Materials and Methods ~ ~ ~ Preparation of Tobacco Samples Containing Additives. Solid, sW water-insoluble additives were ground finer than 100 mesh and added ~ W

, - 2 - to a casing mixture consisting of glycerine,propylene glycol, invert sugar, corn syrup, flavorant materials, and water. Water-soluble additives were added to the premixed casing solution in a minimum amount of water. The thoroughly-mixed casing/additive dispersion was sprayed onto an uncut Bright, Burley, Maryland, and Turkish (BBMT) tobacco blend at a rate equivalent to 14.14% casing and 1-10% additives. Reconstituted tobacco was added in some cases and the treated tobacco was then cut in a laboratory guillotine cutter at 32 cuts per inch and allowed to equilibrate at 60% RH and 68°F for a minimum of 48 hours. Preparation of Cigarettes Containing Additives. Soluble addi- tives were dissolved in water and added to a casing mixture of glycerin, propylene glycol, invert sugar, corn syrup, flavorant materials, and water. Insoluble additives were added to the casing mixture and pebble-milled for a minimum of 12 hours. The contin- uously stirred casing/additive mixture was applied to a blend of BBMT tobaccos in a casing cylinder. Reconstituted tobacco was added to the blend and the blend was cut and driedtwith cut tobacco stems and expanded tobacco added during the drying operation. In most cases, non-filter cigarettes were manufactured on factory production equipment. In certain instances, cigarettes were.manufactured with filters designed to yield the desired levels of smoke constituents. Several of the additives were incorporated in a reconstituted tobacco sheet to maximize catalytic availability. To prepare the treated reconstituted tobacco, the additives were dry-mixed with a blend of ground BBMT tobacco strips, a-cellulose, and sodium carboxymethylcellulose. A solution of water, propylene glycol, and glycerine was added to the dry blend and the combination wet-mixed until the appropriate consistency was achieved. The material was

then pressed into sheets on factory production equipment, cut into 4 inch squares and finally shredded using rotary cutters at a rate of 32 cuts per inch. The resulting product was used to manufacture cigarettes. Tobacco Pyrolysis Procedure. Approximately 160 g of cut tobacco, equilibrated at 60% RH and 68°F, was packed to a density in the range of commercial cigarettes in the apparatus shown in Figure 1. The tobacco was lit at the surface of the screen cylinder and the rate of combustion was controlled by pulling a vacuum through an exit tube located at the axis of the cylinder while simultaneously bleeding N2 into the tobacco mass through the two end screens. The flow conditions were selected to give a PAH composition similar to that observed for CSC. The pyrolysis condensate was collected in two glass traps, cooled in liquid air, connected in tandem at each end of the exit tube. After pyrolysis, the traps were washed with redistilled acetone and the yield of "dry" condensate determined by evaporation of an aliquot. Preparation of the PAH concentrate was carried out as described in our previous report for CSC (Mold, 1978), (Standard Procedure for Preparation of a PAH Concentrate from Cigarette Smoke Condensate). Evaluation for PAH content was performed as described previously, either by the SGS Test, or by measurement of total infrared absor- bance at 11.9-14.Ou (Fuson, 1956) or the ratio of absorbance at 3050 cm-' to that at 2960 cm-1 (Wiberley, 1961). When the esti- mation of activity was by SGS, the activity is reported as the ED50. When the measurement of infrared absorbance was used to predict tne biological activity, it is reported as the PED50. In order to place

d , 4 the PAH level, or activity, on the basis of its concentration in the pyrolysate or CSC, these results have been expressed in terms of the number of ED50's per gram of dry condensate (DC). Mass Spectrometric Analysis of PAH Concentrates. The pro- cedure used for these measurements was suggested by a publication by Biros (1970) and is essentially similar to that later described by Blumer (1975). Over the period of time during which samples were analyzed by this technique several different instrumental com- binations were used. The most recent utilized a DuPont 21-490 mass spectrometer in combination with a Finnegan-Incos Model 2300 Data System. An aliquot of an ether solution was placed in the probe glass capillary (1.5 mm ID x 19 mm) such that, after careful evaporation under vacuum, 10 ug of the PAH concentrate and 1.2 ug of o-tolidine (mol wt 212), used for internal standard, remained. The mass spec- trometer ionizing voltage was set at 12 e.v. and the data system was programmed to acquire 150 spectra at the rat e of 1 spectrum every 6 sec. Scanning was begun, and the sample in the solid sam- pling probe was introduced through the vacuum lock into the ion source. The temperature of the sampling probe was manually increased from 75°C to a final hold temperature of 290°C over a period of 7.5 minutes. After acquisition of the 150 scans, one hundred consecutive spectra were averaged by the data system beginning with the spectrum taken at the time the probe was first introduced into the ion source. The resultant averaged spectrum was normalized on the internal standard peak at m/e 212. Figure 3 presents this low

- 5 - voltage molecular ion summation for several samples of PAH concen- trates calculated as the percent of total ion intensity. Characterization of the PAH Concentrate by High Pressure Liquid Chromatography (HPLC) and Gas Liquid Chromatography(GLC). The presence of PAH's in the concentrate was demonstrated as follows. The concentrate was fractionated by gel permeation chromatography (GPC) (Severson, 1976). The GLC pattern published by Severson (1976) was essentially duplicated when the GPC fraction was subjected to similar conditions. The authors used GC-MS to identify most of the components as PAH'.s. Further verification was obtained by reverse phase HPLC analysis of the GPC fraction with detection at wave- lengths of 340, 308, 384, and 430 nm, allowing identification of pyrene, chrysene, benzo(a)pyrene (BaP) and anthanthrene by retention volume. The presence of BaP was additionally confirmed by injecting a portion of the GPC fraction into the GLC, collecting the BaP peak, and rechromatographing by reverse phase HPLC using 384 nm detection. A 30-40% recovery was obtained when 7,10-14C-BaP was treated in this manner. Evaluation of CSC for Carcinogenicity by Painting on Mouse Skin. Cigarettes were equilibrated at 60% RH and 68°F and smoked to 30mm butts on Liggett & Myers Model 4 smoking machines (Hackney, 1965). Thirtyfive-ml puffs of 2-sec duration were taken at one minute intervals. The smoke condensate was collected in two liquid air cooled traps, dissolved in acetone, and the acetone removed under reduced pressure at 40-50°C. The final "dry" condensate was 2 ~ dissolved in an appropriate volume of acetone to give 50 mg of ~ condensate per 100 mg of solution. W C1' N W I

- 6 - For each test group, 50 Swiss Ha/ICR female albino mice were used. Hair was clipped from the dorsal test area prior to each painting using an Oster Co. (Racine, Wis), Model A-2 small animal clipper with a size 40 blade. The animals were 9 weeks of age at the start of the experiment. Application of 100 mg of smoke con- densate solution was accomplished with a Grumbacker No. 6 camel hair brush. Test materials were applied five days per week for 80 weeks. (Insert Page 6a) Evaluation of Cigarette Smoke for Cytotoxicity and Ciliatoxicity. The procedures used were identical to those described in the report of the U.S. Dept. of HEW, Appendices A and C (Gori, 1976). Evaluation of Cigarette Smoke for Mutagenicity. .n (Described smoke preparation) The mutagenicity of CSC was examined using histidine auxotrophs of Salmonella typhimurium. The strains used in this study were obtained from B.N. Amesb, and are identified as TA-98, TA-100 and TA-1538. Their properties and the specific details of the assay are given by Ames et al. (1975). Briefly, the test organisms, microsomal preparations (S-9 fraction) where required, and the material to be assayed was mixed in a culture medium containing insufficient histidine to allow the bacteria to proliferate sufficiently to give colonies. After incubation for two days at 37°C, the clones of

- 6a - Analyses For Smoke Constituents of Cigarettes. Cigarettes used for these analyses were equilibrated for at least 24 hours at 60% RH and 68°F prior to smoking for analysis. The nicotine-free dry smoke (NFDS) and nicotine were determined by procedures described by Bates (1967) which are essentially identical to those used by the U.S. Federal Trade Commission (FTC) for reporting "tar" and nicotine values in commercial cigarettes. Carbon monoxide was determined as described by Collins (1973). Catechol was determined essentially as described by Guerin (1976). Acetaldehyde and acrolein were determined by the procedure described by Norman (1968). Hydrogen cyanide was determined as described by Collins (1973). Phenols were determined essentially by the colorimetric procedure described by Oakley (1964) modified to adapt to automation with Technicon modules. Formaldehyde was determined as described by Spincer (1971) modified to adapt to automation with Technicon modules. For the determination of nitric oxide (NO) in cigarette smoke, cigarettes were smoked on an 8-port syringe smoker, and each 8x35cc puff was exhausted into a polyethylene bag enclosed in a glass aspiration flask. A 12cm X 8mm OD tube containing silica gel was inserted ahead of the NO analyzer to remove aldehydes and water which otherwise interfere in the determination. The system was otherwise identical to that described by Collins (1973) with the exception that the syringes contained no oil. Within 15 seconds after a puff, the bag was exhausted through a non-dispersive IR ~ r 4 s

- 6b - .s analyzer (Beckman Model 215A) and the NO concentration calculated from calibration curves obtained with standard gases. The per puff values, including clearance puffs, were summed to give ciga- rette deliveries. Values obtained by this method agree with those by colorimetric and chemiluminescent determinations on the same cigarettes. Details of this procedure will be published at a later date. For the analysis crf nitrosonornicotine (NNN), cigarette smoke was collected in aqueous alkali, concentrated by extraction into benzene-chloroform (9:1) followed by back-extraction from chloroform into aqueous acid and re-extraction into chloroform at pH 5. Further separation of NNN was achieved by thin layer chromatography on silica gel and reverse phase high pressure liquid chromatography. Detection was by UV at 254 mu with isotope dilution to measure recovery. Details of this procedure will be published at a later date. Smoke was analyzed for N-nitrosodimethylamine (NDMA) using a method basically similar to that of Rhoades (1972). Smoke samples were collected in either sodium hydroxide or ascorbic acid solution to prevent artifactual formation and were cleaned up by distilla- tions from basic and acidic solutions and by distribution between solvent pairs prior to GLC analysis for NDMA which was accomplished with a dual column system and a Hall electrolytic conductivity detector. Recoveries of NDMA through the cleanup and recovery procedures were determined using a radioactive tracer technique O^ j employing NDMA-C14. W (Jt T) a 0 ,

- 7 - bacterial outgrowth were counted. Usually, three replicates per assay level were employed. Dimethylsulfoxide was used to dissolve the smoke condensates for ali.quoting. Evaluation of Filter Cigarette Prototypes for Subacute Toxi- city by Inhalation in Rats. The diagram in Figure 8 shows the essential features of the inhalation apparatus. Cigarettes were smoked on a Liggett & Myers Model 4 smoking machine (Hackney, 1965) modified to smoke 30 cigarettes with 35-m1 puffs of 2-second dura- tion at one minute intervals. All cigarettes utilized in the experi- ment were kept refrigerated and sealed until approximately 48 hours prior to use, when the cigarettes were humidified in a chamber con- taining a supersaturated solution of sodium bromide in sterile water. Ten puffs were taken on each cigarette. A peristaltic pump supplied the pressure differential for the puffing. Air to dilute the fresh smoke stream was supplied from a reservoir through a flow regulator which was adjusted to give the desired smoke dilu- tion. The inhalation exposure chamber was situated directly beneath the smoke-mixing chamber and consisted of a cylindrical manifold into which twenty cassettes, each designed to contain one rat, could be securely inserted. During the experiment, the animals' noses were pushed into the manifold where they were secured for exposure to the smoke stream by means of an adjustable plunger from the rear. All rats on test were examined twice daily for general appearance, clinical signs and survival. Body weight and (Z food intake of individual animals were monitored weekly. Water con- .~ ~ sumption was recorded daily. Animals were maintained throughout the V, . N . study on Purina Lab Meal and water ad libitum 0 . - i-+