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I. MONITORING OF THE CHRONIC INHALATION EXPOSURES R. A. Jenkins, B. E. Gill, R. B. Quincy, D. D. Pair Magnitude of Exposure. One of the primary purposes in monitoring the bioassays is to determine the amount of smoke which the animals retain. Since it is not possible to determine this value quantitatively for each animal in the bioassay, it is necessary to estimate the amount of smoke inhaled or re- tained, based on measurements of machine smoke delivery, apparent exposure chamber depletion, etc. The first procedure in making this estimation is the determination of the maximum possible amount of smoke inhaled. For dogs, we take this maximum possible amount to be the amount of smoke made available for inhalation at the exit of the tracheal cannula. For the rat bioassays, the maximum possible amount inhaled is taken as the product of the fraction of smoke particulates depleted from the exposure chamber with animals present and the amount of smoke actually getting into the chamber. Monitoring proto- cols for the pigeon studies are still being formulated, but we have taken the maximum possible amount of smoke inhaled to be the amount of smoke (or smoke constituent) depleted in the exposure channels after passage across the beaks of the animals. Our determination of the maximum possible amount of nicotine inhaled for the seven bioassays with which we have interacted are compared on Table 11-4. These values are considered to be a good single estimate to describe the maximum possible inhaled smoke over the entire course of the bioassay. For the sake of simplicity, standard deviations have been eliminated from the Table. Generally, these are about ± 10-15% over the duration of the exposure. A few comments are in order. The dogs exposed to the Code HN cigarette at VAH are offered about the same amount of nicotine as the animals in three of the exposure groups in the Hazieton study. Rats in the Code 27 and 32 ~ ~ G.! ~ CD

TABLE 11-4 Maximum Possible Quantity* of Inhaled Nicotine NCI Inhalation Bioassays (mg Nicotine Offered per Animal per Week) Cigarette Code Site (Animal) 11 13 16 23 27 32 67 79 90 HN LN Battelle (Rat)a 0.6 5.7 8.2 Beth Israel (Pigeon)c --- 8.3 Borriston (Dog)b 67 21 207 195 Hazleton (Dog)b 14 144 130 127 Hazleton Cofactor (Dog)b 5.4 64 ORNL '(Rat)a 1.6 VA Hospital (Dor)b 150 55 *f+faximum Possible Quantities of Inhaled Smoke are defined as aFor Rats: Product of the fraction of particulates depleted while the animals are exposed and the amount of particulates present inside the exposure chamber, bFor Dogs: Amount of smoke made available at cannula exit cFor Pigeons: Average amount of smoke depleted in exposure channel divided by the number of birds per channel. 0494U68

109 groups may have inhaled more nicotine than their counterparts in the ORNL bioassay. Also, it would appear that the pigeons at Beth Israel could inhale about the same level of nicotine as the Code 32 rats at BNW. And, at Borriston, there appears to be a 1:3:10 nicotine dose ratio in the Code 13, 11, and 32 exposure groups. Without particulate deposition experiments, such as those underway as a collaborative effort between ORNL and Borriston Research Laboratories, it is impossible to determine the actual quantity of particulates retained by the animal. Based on some preliminary dosimetry experiments conducted several years ago at VAH, we estimate that beagles retain about 50% of the particulates offered for inhalation with an uncuffed cannula. We estimate that as much of 90% of the.offered particulates may be retained with a cuffed cannula. Given these fractional retentions, we have made estimates for the levels of retained nicotine in the four bioassays for which we have the most complete data. For rats, the retained dose should be considered as a maximum possible whole body dose, since significant levels of particulates will deposit on the head and fur of the animal. These estimated retentions are compared on Table 11-5. Note that these do not necessarily reflect the dose level over the entire course of the study. (Within the past year the Code 11, 13, and 32 exposure groups at BRL have been switched to cuffed cannulas.) Estimated retained doses have been corrected for estimated mean body weights. Note that, due to differences in cannula design, the Code HN dogs probably do not retain as much nicotine as the three groups of dogs at Hazleton. Interestingly, on a body weight normalized basis, the Code 32 rats at BN4W appear to retain propoftionately more nicotine than do the dogs being' exposed to the same cigarette at BRL or Hazleton.

TABLE 11-5 Current Best Estimatea Maximum Possible Weekly Retained Nicot~ne Dose Nicotine (mg/Week/kg Body Weight ) Cigarette Code Site (Animal) 11 13 27 32 67 79 90 HN LN Battelle (Rat)c 3 26 40 Borriston (Dog) 6 2 18 17 Hazleton (Dog) 1.3 13 12 11 VA Hospital (Dog) 8 3 aAssumes 90% retention of offered nicotine with cuffed cannula, 50% retention with uncuffed cannula. bAssumes 200 g Fisher Rat, 10 kg Beagle Dog. cShould be taken as a whole body dose, since a significant fraction of the particulates can deposit on the fur of the animal. z49=6e

111 Variabili~y of Expasu.re. The quantity of smoke offered or retained by the animal will vary over the course of the bioassay. While it is difficult to manipulate retention variation due to the animal, it is important to understand what factors external to the animal can give rise to variability in offered dose, so that these factors can be controlled. Also, it is im- portant to document the extent of potential variation, in order to allow for this when interpreting results of the bioassay. For example, variation in the estimated retained dose of particulate matter over the 24-month duration of the Hazleton bioassay is presented in Figure II-1. This variation was found to be due to both environmental fac- tors in the exposure wards and cigarette-to-cigarette variability. However, it is important to note that most of the dose levels lie within a cluster of values around 1.3-2.0 grams of total particulate matter per week. Variation in estimated retained nicotine dose over the last two years of the dog study at VAH is presented in Figure II-2. During the last 16 months of the bioassay, the dose levels retained a relatively parallel course, sug- gesting good dose group differentiation in the two animal populations. Most of the variability observed was due to a problem with machine maintenance which affected the exposure systems smoking the Code HN cigarette to a greater extent. This had the effect of collapsing the dose group distinction, and thus could be important to consider if, for example, measurements of cardiovascular physi- ology had been made between the 24th and 28th months of the study. Changes in retained dose as a result of changes in cannula design in the Oorriston study are depicted on Figure II-3. It is estimated that when the Code 11, 13, and 32 groups wer`e switched from an uncuffed to a cuffed cannula', the dose of particulates retained increased substantially. The particulate dose of the Code 67 group remained about the same, since that group had been exposed with a cuffed cannula from the initiation of the bioassay.

FIGURE II-1 ORNL-DWG 78-9043 VARIATIG~y OF ESTIMATED RETAINED PARTICULATE MATTER DOSE* HAZLETON LABORATORIES (90 % CONFIDENCE INTERVAL) TOTAL PARTICULATE MATTER (TPM) ' (g/week) *ASSUMES 90 % RETENTION OF OFFERED DOSE WITH CUFFED CANNULA vf.+94U68

FIGURE 11-2 ORNL-DWG 78-9011 VARIATION OF ESTIMATED RETAINED NICOTINE DOSE~ VETERAN'S ADMINISTRATION HOSPITAL (90 % CONFIDENCE INTERVAL) 150 100 NICOTINE ( mg/week) 50 0 20 24 28 32 36 TIME (months) 40 44 wASSUMES 50% RETENTION OF OFFERED DOSE WITH UNCUFFED CANNULA 9494C468

114 FIGURE II-3 ORNL-DWG 78 -907 5 VARIATI®N OF ESTIMATED RETAINED PARTICULATE MATTER DOSE* BORRISTON RESEARCH LABORATORIES (90 % CONFIDENCE INTERVAL ) I 4.0 3.0 RETAINED TPM DOSE Code r 32 (g/week) 2.0 I 45 20 TIME (month) *ASSUMES 50 ®!o RETENTION OF OFFERED DOSE WITH UNCUFFEC CANNULA, 90% RETENTION WITH CUFFED CANNULA

115 Chemical Composition of Smoke Dose. Since the chemistry of the smoke which the animals are offered is not necessarily the same as that which might have been predicted from analytical smoking (See Part I of this report), it is important to define the chemical composition of the smoke actually offered the animals at the bioassay laboratory. For example, one of the prime purposes of inhalation bioassay is to insult the target organs with cigarette smoke constituents which would not be present in a condensate skin painting experiment. The most obvious example of this is the class of gas phase constituents. Over the past year, we have begun to measure the concentrations of CO and C02 in exposure atmospheres to assess overall expo- sure levels of gas phase constituents. Briefly, the smoke is withdrawn through the cannula into a large gas sampling bag, a sample of the contents of which are returned to ORNL for gas chromatographic analysis. On Table 11-6 are compared the ADL/I cannula exit deliveries of CO, CO,, total par- ticulate matter (TPM), and nicotine on-site, with the amounts of constituents generated by smoking the Code LN cigarettes under analytical conditions but at the exposure laboratory puffing rate. Since CO and C02 (arid many other organic gas phase constituents) are not easily condensible, amounts similar to those which would have been predicted from analytical smoking reach the cannula. But only half of the predicted level of TPM and nicotine reach the cannula. (The remainder appear to condense within the exposure system.) Thus, the smoke which the animals were being offered at the time of these measurements was a substantially different "type`° of smoke (particulates to gas phase ratio) than might have been predicted. Throughout the course of'the first Hazleton study, we have observed a much higher degree of variability in the delivery of smoke constituents from the Code 90 variants than from others in the bioassay. (The Code 90 is an

TABLE 11-6 Comparison of Cigarette Deliveries* August 1977, Code LN Constituent Analytical, ORNL Cannuia Exit, On-Site TPM, mg 64.9 ± 4.0 36.9 ± 3.3 Nicotine, mg 1.63 ± 0.17 0.81 ± 0.18 Carbon Monoxide, ml 24.G ± 1.2 22.2 ± 1.5 Carbon Dioxide, ml 51.7 ± 4.0 51.6 ± 4.1 *Cigarettes smoked at 3 puffs per minute. . 8494UFB