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Topical Report NCIJS&I-[PjORNL #64 SITE VISIT V TO NAZLETON LABORATORIES Reston, Virginia, November 5-6, 1977 2-9-78 R. A. Jenkins, B. E. Gill, R. B. Quincy, and D. D Pair Tobacco Smoke Research Program Bio/Organic Analysis Section Analtyical Chemistry Division Oak Ridge National Laboratory Oak Ridge, Tennessee 37830 V Interagency Agreement (ERDA-NIH/NCI) No. 40-485-74, Part II Internal (ORNL) Contract Charge Number 3390-0224 ® Intended for informal communication with project management only. ~ Confidential until published or released by author. ~ C,b N GD

SITE VISIT V TO HAZLETON LABORATORY R. A. Jenkins, B. E. Gi11,-P.. B. Quincy, and D. D. Pair Summaa. Data gathered on the fifth working site visit to the chronic tobacco smoke exposure at Hazleton Laboratory indicate that the exposures continue to be very consistent. Offered smoke doses for most exposure groups are about the same as measured on previous visits. By controling humidity, temperature and air flow in the exposure wards, environmental factors have been essentially eliminated as potential sources of dose variability in this bioassay. The carbon monoxide and carbon dioxide contents of the smoke reaching the cannula exit suggest that the Code 90 and 79 exposure groups are being offered the targeted level of gas phase constituents. A survey of the resistance-to-draw (RTD) of a number of Code 90 ciqarettes indicate that animals in that exposure group probably have been ex eriencing a wide variation in offered carbon monoxide dose. Introduction. A fifth site visit was made to Hazleton Laboratory (HL) on November 5-6, 1977, to observe and document the chronic exposure of beagles to tobacco smoke. As has been the case on previous visits, our primary purpose was to collect data to enable us to better estimate the dose which the animals receive and to determine the extent to which certain smoking factors can contribute to the variability of that dose. Both exposure and experimental details have been previously discussed, and can be found in reports covering previous site visits (Topical Reports NCI/S&HP/ORNL #24, 29, 33, 36A, 46). Ex eriny ~ental. The experimental approach and methods used on this site visit were similar to those of previous visits. Both gas and particulate phase smoke samples were taken to estimate the smoke dose which the ani- mals are offered. Smoke samples generated by the exposure systems were compared with analytical results to assess the importance of cigarette variability on smoke dose.• Detailed chemical analyses were performed on the smoke of one variant to detern~ine the extent to which the smoke chemically interacts with the exposure system. Mechanical reliability of the smoking devices were judged.

Table V tabulates individually the TPM and nicotine content of the smoke reaching the cannula exit of selected exposure systems. Again, there appears to be somewhat less scatter in the outputs of the Series IV variants. This scatter is probably attributable to cigarette-to-cigarette variability (e.g. Note the duplicate outputs for Machine #20 smoking the Code 90 cigarette), which means that the animals will not necessarily ex- perience a drastic change in offered smoke dose as they are shifted from machine to machine. Table VI compares the average TPM and nicotine values of smoke reaching the cannula with those of smoke at the ADL/II input. Clearly, the ADLJII, cuffed-cannula systems exhibited very high efficiency in making smoke particulates which are generated by the system available for inhalation. That the ADL/II's smoking the Code 90 variant are greater than "1001 efficient" at delivering smoke to the cannula is probably a result of the high degree of variability of smoke delivery of the Code 90 cigarette. As has been stated so frequently, the most important single chemical parameter of the bioassay is the smoke dose which the animals receive. At present, our best estimate of this dose is the amount of smoke offered the animals at the cannula exit. In an exposure such as Hazleton's in which a cuffed cannula is utilized, we believe that the dose offered approximates well the amount of smoke inhaled. Actual retention of the smoke or its components is likely to be a highly individual matter which can easily be affected by the volume and rate of respiration during the smoke exposure. Table VII lists our estimates of the average amount of smoke offered each variant group on a weekly basis. There appear to be some differences in offered nicotine doses among the Code 90, 79, and SEB IV exposure groups, but these differences are not statistically significant at the 95% confidence level. Except for the Code 90 exposure group, nicotine doses for each group are statistically indistinguishable from those observed on the last visit. The Code 90 group, as estimated from this visit, was offered a 28% larger nicotine dose. For the purpose of more exact7y defining the chemical composition ~ of the smoke offered the animals, we have been conducting detailed chem- ical characterizations of some of the smoke particulate phase samples ~ acquired at the bioassay laboratory. In addition, comparison of smoke ~ .~ samples before and after passage through the exposure system help deter- ~ m mine the extent to which, if any, certain constituents or classes of ~ O

constituents selectively interact with the system. Table VIII compares relative constituent concentrations in TPM samples taken at the machine input and cannula exit for the Code 13 variant. With the exception of the phenol and the cresols, the relative concentrations are the same in both input and output samples, suggesting that little selective depletion occurs as the smoke passes through the exposure system. The relative concentrations of the phenol and cresols are much lower in the cannula exit smoke. We have observed this phenomenon at this and other bioassay sites for phenol, but not to this extent for the cresols. We believe this to be an artifact of the cannula.exit sampling procedures in which a relatively high air flow through the cannula is used to insure quanti- tative removal of smoke from the exposure system. The relatively volatile phenol and cresols may be partially evaporated out of the particulate matter collected on the filter pad during sampling. Table IX compares the average smoke deliveries to the cannula exit of systems smoking the Code 90 and 79 variants with deliveries of ciga- rettes from the same batch smoked under analytical conditions. For the sake of comparison, analytical results for Codes 04 and 13 are included. For the Code 90 variant, the amount of carbon monoxide in the cannula exit smoke was about twice the amount measured on the previous visit. This suggests that animals being exposed to the Code 90 cigarettes may have been receiving a much larger amount of CO in November than they were in May (Site Visit IV). The amount of CO and C02 in the offered smoke was nearly equal to that of smoke generated analytically, suggesting that the Code 90 group is being exposed to the "targeted dose" of CO/CO2. However, because of the high degree of variability in the Code 90 smoke delivery, this similarity could be just coincidental. Note that for the Code 90, the CO:CO2 ratio in the smoke is much lower than that of the other cigarettes. This is indicative of a lower effective puff volume through the fire cone, and is a result of the air dilution holes on the Code 90 "filter" tip. For the Code 79 variants at HL,`the per cigarette CO delivery is 25% lower than the targeted level (analytical smoking results). However, the CO:nicotine ratio of the smoke is the same under both exposure and analytical conditions. This indicates that the lowered amount of CO is just a result of less whole smoke being produced and delivered to

the cannula. Thus, the whole smoke dose which the Code 79 exposure group is offered should be the same composi_tion as would have been anticipated from analytical smoking. That is, there is probably no significant loss of either particulate or gas phase constituents to the exposure system. One of the themes of the topical reports covering the Hazleton site visits has been the high degree of variability of the smoke from Code 90 cigarettes. The Code 90 is a blended cigarette', wrapped with high porosity paper, and tipped with a hollow filter tip, to which have been added two rings of air dilution holes. The air dilution holes act to lower the resistance-to-draw of the cigarette because they permit a significant fraction of the 35-ml puff volume to be drawn through the holes, around the fire cone of the cigarette. It has also been claimed that the holes permit faster diffusion of the gas phase constituents emanating from the tobacco rod out of the mainstream smoke. At any rate, most cigarettes with air dilution holes tend to have lower gas phase deliveries than cigarettes without them. On several occasions, we have observed what we believed to be large variations in the sizes of the holes on the air dilution tips of the Code 90 cigarettes. Typically, we have also found that relative standard deviations of the TPM and nicotine contents of Code 90 smoke (input or cannula exit) were greater than those of the smoke of other cigarettes. In the Topical Report covering Site Visit IV to Hazleton (~46, Table IX), we discussed how the whole smoke output of the Code 90 cigarette can be drastically affected by changes in resistance-to-draw of the cigarette. Essentially, a"1ow" RTD (ti20 mm H20) Code 90 cigarette will produce about 30% less particulates and 50% less gas phase than a Code 90 with a relative- ly high RTD (-u40 mm H20). Figure 1 is a histogram of the resistance-to-draw values of a relatively small number (N = 159) of Code 90 cigarettes returned to ORfdL from recent site visits. While the number of cigarettes sampled is too small to draw any definitive conclusions, the histogram does suggest a bimodal distribution of RTD values. If such is the case, it can seriously affect interpretation of data in the bioassay results, especially if an attempt is made to correlate carboxyhemoglobin levels with smoke dose. Within a relatively small variation in TPM delivery, carbon monoxide con- tents of the smoke may vary widely. Thus, different levels of COHb may be observed, depending on the resistance-to-draw of the cigarettes. This, of course, may be masked by even greater animal-to-animal differences in whole smoke inhalation and retention.

Results and Discussion. Table I lists puff durations and volumes of the ADL/II exposure systems in service at the time of our visit. (Note that all averages in this and all other tables are means ± one standard devi- ation.) Throughout this study, the mechanical performance and reliability of these systems has been very good. Findings on this visit followed that pattern. Specifically, the ADL/II's smoking Code 90 cigarettes were .observed to have slightly higher than specified puff volumes, while machines smoking the other codes had slightly lower than the specified 35 ml per puff. The overall average puff volume was 34.4 ± 1.6 ml, which is very close to the specified value. Table II compares static burn rates of Code 04 cigarettes smoked in the exposure wards at Hazleton with those determined under analytical conditions at ORNL. There is very little difference between burn rates determined at both sites. Over the past year, Hazleton has brought environmental conditions to where they almost mimic those of analytical smoking. Relative humidity was measured to be 63% at 22°C, which is near perfect. Combined with a somewhat lowered ventilating air flow in the exposure wards, Nazleton has essentially eliminated environmental conditions as a potential source of dose variability in the chronic exposure. The total particulate matter (TPM) and nicotine content of the smoke generated by the variants as smoked on the ADL/II exposure systems are tabulated individually on Table III. The average machine input values are virtually identical to those determined on the previous visit. The NCI Series III variants (Codes 79 and 90) exhibit somewhat greater scatter in the nicotine deliveries than do the Series IV variants (Codes 13 and 04), but the sample is really too small to draw any conclusions. Table IV compares the average smoke deliveries from cigarettes smoked on-site with those from the same batch returned to ORNL and smoked under analytical conditions. All codes exhibited about 11-21% less TPM generation on-site, and Codes 90, 79, and 04 delivered significantly less nicotine than under analytical conditions. We are not sure of the reason for this phenomenon, since puff volumes of the exposure 'systems were measured to be only a few percent low, and enviromnental conditions should not contribute to a lowered smoke delivery. We do not believe that this lowered delivery represents a serious difficulty. In general, the amount of nicotine generated by the variants on-site was very similar to that determined on the previous visit.

Figure 1 Resistance-to-Draw Histogram Code 90 Cigarettes (Total number of cigarettes in sample = 159) 15._ ,# o f 10_ Cigarettes y J~~ ! I IE' E € ~L11.L~.. ~ 20 30 44 5 0 Resistance to Draw (RTD, in mm H20) VZt34Usg

Table I F Measured Puffing Parameters for_ Selected ADL/II Smoking Machines in Service at Hazleton on November 5-6, 1977 Room No. Cigarette Machine Code No. Puff Duration (sec) Puff Volume (ml) 1 90 34 2.0 36.0 6 2.0 36.0 1 2.2 34.5 20 2.2 37.0 23 2.0 38.0 3 2.0 37.0 9 2.1 37.0 30 2.2 37.0 Average Puff Volume 36.6 ± 1.1 1 79 31 2.0 33.0 14 2.1 34.0 36 2.0 33.0 19 2.1 34.0 39 2.0 34.0 28 2.0 32.0 25 2.2 35.5 10 2.2 36.5 Average Puff Volume 34.0 ± 1.4 2 SEB IV 2 2.0 33.0 (04) 18 2.0 33.0 15 2.0 34.0 8 2.0 34.0 7 2.0 33.5 4 2.0 32.0 13 2.0 33.0 21 2.1 34.0 Average Puff Volume 33.3 ± 0.7 2 13 32 1.9 34.0 5 2.0 34.0 37 2.0 34.5 17 2.0 34.0 22 2.0 34.0 29 1.9 33.0 -• 2.1 34.0 16 _ 2.1 33.0 Average Puff,Volume 33.8 ± 0.5

Table II Comparison of Code-04 Static Burn Rate: Exposure Conditions at HL on November 5-6, 1977 vs. Analytical Smoking Conditions at ORNL Conditions Burn Ratea mm/min Burn Rateb mq/min HL, Ward #1, on Code 90 Machines 5.06 ± 0.29 72.4 ± 2.7 HL, Ward #1, on Code 79 Machines 5.04 ± 0.23 70.8 ± 2.2 HL, Ward #2, on Code 04 Machines 4.64 ± 0.31 66.5 ± 3.5 ORNL, Analytical Smoking Conditions 4.75 ± 0.24 67.0 ± 3.2 aBurn rate given in terms of actual length of cigarette burned. bBurn rate given in terms of actual weight of cigarette burned.

Table III Total Particulate Matter (TPM) and Nicotine Delivery of Cigarettes Smoked on Selected ADL-II Smoking Machines in Service on November 5-6, 1977 Cigarette Code Machine Number TPM, mg/cig Nicotine, mg/cig Nicotine:TPM Ratio 90 34 49.6 2.39 .0482 34 35.9 2.11 .0588 6 34.6 1.86 .0538 6 40.6 2.22 .0547 1 27.6 1.61 .0583 1 35.6 1.29 .0362 20 40.7 1.90 .0467 20 35.9 1.40 .0390 Average 37.6 ± 6.3 1.85 ± 0.39 .0495 ± .0085 79 31 41.2 1.79 .0434 14 42.7 2.25 .0527 36 46.0 2.36 .0513 19 42.8 2.32 .0542 Average 43.2 ± 2.0 2.18 ± 0.26 .0504 ± .0048 SEB IV 2 50.6 2.75 .0543 (04) 18 50.5 2.71 .0537 .;~~. 15 46.5 2.75 .0591 8 49.3 3.00 .0609 Average 49.2 ± 1.9 2.80 ± 0.13 .0570 ± .0035 13 32 38.8 .22 .0057 32 43.1 .23 .0053 5 41.0 .22 .0054 5 41.5 .22 .0053 37 42.0 .21 .0050 37 36.2 .19 .0052 17 35.4 .20 .0056 17 37.9 .21 .0055 Average 39.5 ± 2.8 0.21 ± 0.01 .0054 ± .0002 v i