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Topical Report NCI/S&HP/ORNL #55 INTERCOMPARISON OF TOBACCO SMOKE DOSE BEAGLE DOG INHALATION BIOASSAYS 11-17-77 R. A. Jenkins Tobacco Smoke Research Program Bio/Organic Analysis Section Analytical Chemistry Division Oak Ridge National Laboratory Oak Ridge, Tennessee 37830 Interagency Agreement (ERDA-NIH/NCI) No. 40-485-74, Part II Internal (ORNL) Contract Charge No. 3390-0224 Intended for informal cornnunication with project management only. Confidential until published or released by author. r Cx) I W Q Q ~ ~ ....

Intercomparison of Tobacco Smoke °'Dose°" Beagle Dog Inhalation Bioassays R. A. Jenkins In response to several inquiries concerning unanticipated deaths in the Code 67 exposure group at Borriston Research Laboratories, we have prepared a status report on estimates of smoke dose in the three NCI- sponsored inhalation studies which utilize the beagle as an animal model. Table I compares the estimated mean weekly amounts of smoke particulates offered at the cannula for each cigarette code. (In the interest of clarity, standard deviation values have been omitted. These generally amount to ± 10-20,10.) In the case of the Hazleton and VAH studies, the figures represent averages of estimated amounts of smoke over several monitoring visits. For the BNW-BRL study, the values are from one site visit only. This visit was made in May, 1977, just prior to the change from the BNW machine - uncuffed cannula system for Codes 11, 13, and 32 to the ADL-II - cuffed cannula sys- tem (see below). Note that the values listed are given as the amount of smoke particulates offered for inhalation. These are determined by collecting the total partic- ulate matter (TPM) output of the exposure device on filter pads at the down- stream end of the cannula. A large-animal ventilator (respirator) is used to draw smoke from the system in a pulsed manner, which somewhat mimics the breathing pattern of the dog. The true "dose" (that quantity retained by the animal) will be lower than the quantity offered for inhalation. Furthermore, the values listed in Table I do not reflect levels of gas phase constituents, which may or may not be related to the level of smoke particulates. For example, at the VAH study, carbon monoxide levels as determined at the cannula exit show that the gas phase dose offered the animals is substantially higher than would be extrapolated from the amount of nicotine reaching the cannula. Thus the values in Table I should be taken as a maximum possible dose of total particulate matter and nicotine. The implications of the values on •Table I can be explained in terms of some of the differences amohg•the bioassays, which are delineated in Table II. For example, Table I indicates that the weekly offered amounts of smoke par- ticulates in the Hazleton and VAH studies are similar. Since the animals at VAH receive the particulates from 72 cigarettes per week smoked at 3 puffs per minute, one might expect a significantly higher offered dose than the

Hazleton aninrals, which are exposed to 60 cigarettes per week smoked at 2 puffs per minute. However, the VAH study employed the ADL-I exposure device, which was subject to high levels of internal smoke particulate deposition. Our monitoring work showed that only 50-60% of the smoke particulates which an AaL-I generates under chronic exposure conditions ever reaches the can- nula exit. It is this internal smoke deposition which is responsible for lowering the offered particulate dose down to a level which is comparable to that at Hazleton. The °offered°' amounts of smoke particulates in the BNW-BRL study appear to be a factor of two greater than those in the Hazleton experiment. This is primarily a result of the greater number of cigarettes smoked per week (84 vs. 60) in the BNW-BRL experiment. Also, on the one monitoring visit made to date to BRL, environmental conditions (air flow, temperature, rela- tive humidity) caused a"wetter°' particulate matter to be produced. Nicotine is probably a somewhat better indicator of active smoke constituents arriving at the cannula than TPM. In comparing the offered nicotine doses in Table I for the Code 13 and 32 cigarettes in the BRL and Hazleton studies, only a 50% greater level is observed at BRL. Most of this difference can be accounted for by the 40% greater number of cigarettes used in the BNW-BRL exposure. Because of the differences in exposure systems, smoking protocols, cigarette variants, etc., the three bioassays are not strictly comparable. Many of these factors can ultimately affect the amount of smoke offered or retained. Probably the most important factor in determining that fraction of offered smoke which is actually inhaled is the cannula design. The pri- mary advantage of the uncuffed tracheal cannula is that it permits smoke to be diluted with air in the trachea, in much the same way as the smoke is drawn into human lungs. However, since this type of cannula fits very loosely inside the trachea, there is no way of insuring that the smoke which the exposure device delivers to the stand tube upstream of the cannula will actually be inhaled. The advantages of the cuffed cannula are two-fold. First, the cuff seals off the upper part of the trachea, insuring that virtually all of the smoke reaching the cannula will be inhaled. Secondly, since the animal exhales smoke through 'an exhalation channel attached to the cannula, and not through the Upper respiratory tract, the animal is much less visibly irritated during the smoking procedure.

Of course, the animal on either system does not retain all of the particulate matter which is inhaled. Thus, without some sort of dosimetry information, any estimate of the retained dose from the level of smoke de- livered to the cannula exit amounts to an educated guess. A preliminary 14C-dotriacontane deposition study conducted in November, 1973, at VAFH using an ADL-I, uncuffed cannula system suggested that the animals may retain as much as 60% of the particulates which can be collected at the cannula. However, there is reason to believe that this value is probably lower. ORNL and Borriston Research Laboratories are presently engaged in a collaborative beagle dosimetry experiment. One of the objectives of the experiment is to determine relative smoke retention with the two cannula systems. If it is assumed that animals retain -,90% of offered smoke particulates with a cuffed cannula, and 50% of the offered particulates with an uncuffed cannula, then Table I translates to Table III, a comparison of the estimated retained smoke dose in the three bioassays. The data on Table III warrants several comments. First, because the VAH dogs are exposed with an uncuffed cannula, which does not force them to inhale deeply all of the offered particulate matter, they retain,only about half of the smoke particulates that the Hazleton animals retain when exposed with a cuffed cannula. Secondly, for the first ti330 days of full dose exposure (until June, 1977), the Code 11, 13, and 32 exposure groups in the BNW-BRL study were exposed with an uncuffed cannula. This caused them to retain about the same amount of smoke particulates per week as the I-Eazleton animals (exposed with a cuffed cannula), even though the values on Table I indicate that the BRL dogs were offered considerably more smoke per week, because of a greater number of offered cigarettes. In contrast, the Code 67 exposure group has received only -150 days of full dose exposure. However, all of that exposure has been with an ADL-II, cuffed cannula system. Thus, while all of the exposure gorups in the BNW-BRL study were offered about the same amount of smoke, the Code 67 group was probably retaining considerably more of the parficulate matter than the other groups. Since June, 1977, the Code ll', 13, and 32 exposure groups were switched to a cuffed cannula system. Thus, they are now probably retaining considerably more smoke. Preliminary data from a November, 1977, monitoring visit (in which all of the BNW-BRL exposure groups were exposed with ADL-II, cuffed cannula systems) suggest that the amount of retained smoke particulates is

2.9, 3.1, 3.4 and 3.9 grams 7PM per weekk for the Code 11, 13, 32, and 67 exposure groups, respectively. In other words, assuming that the BNW ex- posure device performed, in terms of smoke generation, as well at BNW as it performed at Borriston (which is comparable to the ADL-II when properly maintained) during our May, 1977, site visit, and that incomplete inhalation occurred at BNW as was observed at Borriston with an uncuffed cannula, it is highly probable that the Code 11, 13, and 32 exposure groups received a smaller smoke dose for the first n,330 days of exposure (200 days at BNW plus the first 130 days at Borriston) than they are currently receiving or than the Code 67 dogs have ever received. Using the.figures in this report as a guide, dogs exposed to the Code 11, 13, and 32 variants retained approxi- mately 2 grams of TPM (109 mg nicotine for Code 32) per week for "'330 days and then 3-4 grams of TPM (185 mg nicotine for Code 32) per week thereafter. On the other hand, dogs exposed to the Code 67 cigarette have retained 3-4 grams of TPM per week since the beginning of chronic exposures. The long term exposure at a lower dose for the Code 11, 13, and 32 variants may represent what amounts to an additional period of acclimation to smoke ex- posure for these animal groups. If the higher levels of exposure approxi- mate the maximum tolerable dose (MTD), early deaths might be expected for the Code 67 group, and one would predict an increase in fatalities in the other groups following conversion to the cuffed cannula system. If retention (with a cuffed cannula system) of smoke from 84 cigarettes per week does represent MTD, then early deaths may be anticipated also in the cuffed cannula exposure groups of the Borriston-ORNL collaborative dosimetry experiment.

TABLE I Estimated Weekly Amount of Offered Smoke Particulates NCI Beagle Bioassays CIGARETTE CODE SITE 11 13 32 67 79 90 HN LN Total Particulate Matter (TPM), 9/week BNW-BRL1 3.8 3.6 4.1 4.6 .Hazleton2 1.8 2.0 1.8 1.7 '},rAH 3 - 1.6 1.7 Nicotine, mg/week BP,W-BRL1 78 23 217 205 Hazleton2 15 140 129 125- VAH3 150 55 IBNW: Battelle Pacific Northwest Laboratories, Richland, WA - first 200 days of chronic exposure BRL: Borriston Research Laboratories, Temple Hills, MD 2Hazleton: Hazleton Laboratories America, Reston, VA 3VAH: Veteran"s Administration Hospital, East Orange, NJ 994=4+68 •1i1

TABLE II Comparison of NCI Beagle Inhalation Bioassays Site Smoking Machine Cannula BNW-BRL BNl1/ADL-II* Uncuffed/cuffed* Haz1eton ADL-II Cuffed VAH ADL-I Uncuffed Puffs/min 2 2 3 Variants in use 11, 13, 32, 67 13, 32, 79, 90 HN, LN Cigarettes/day 12 10 12 Days/week 7 6 6 Exposure Sequence . 3 + 3 in a.m., 3 + 3 i n p. m. 5 in a.m., 5 in p.m., 6 in a.m., 6 in p.m., (chain) (chain) *For first u330 days of exposure, animals smoking Codes 11, 13, and 32 were exposed with a BNW-uncuffed cannuia system. Since June, 1977, these animals have been exposed with an ADL-II, cuffed cannula system. Code 67 dogs have received -,150 days full-dose exposure, all on the ADL-II, cuffed cannula. Z+94Z'j:469 -

TABLE III Estimated Weekly Retained Smoke Particulate Dose* NCI Beagle Bioassays CIGARETTE CODE SITE 11 13 32 67 79 90 HN LN Total Particulate Matter (_TPM), g/week BNW-BRL 1.9 1.8 2.1 4.1 -Hazleton 1.6 1.8 1.7 1.5 8 0 0 9 VAH . . Nicotine, mg/week BNW-BRL 39 12 109 185 Hazleton 14 126 116 113 VAH 75 28 *Assumes %50°d retention of offered particulates with uncuffed cannula and ti90% retention with cuffed cannula. 694=4fi8