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Topical Report NCI/S&HP/ORNL #48 SITE VISIT I TO ENVIRO CONTROL INC. INHALATION LABORATORIES Temple Hills, Maryland, May 23-25, 1977 8-17-77 R. A. Jenkins, R. B. Quincy, and B. E. Gill 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) Charge No. 3390-0224 Intended for informal communication with project management only. Confidential until published'or released by author.

SITE VISIT I TO ECI R. A. Jenkins, R. B. Quincy, and B. E. Gill Summary. A first working site visit was made to ECI Inhalation Laboratories to observe and document the chronic exposure there. The BNW exposure devices in use were in poor working condition. This was assessed to be due to weak- nesses in the basic machine-smoke delivery system design, rather than in- attention or lack of maintenance on the part of ECI staff. Data gathered on the visit indicated that the apparent smoke delivery efficiency of the machines, under samplinq conditions, was good. The smoke doses offered the animals, on a per cigarette basis, are comparable to other beagle studies. Detailed chemical analysis of the particulate phase of the smoke of the Code 32 variant indicates that none of the visualized components are being selectively enriched or depleted upon passage through the exposure devices. Introduction. A first working site visit was made to Enviro Control Inc. Inhalation Laboratories (ECI) on May 23-25, 1977. The primary purpose of this visit was to "shake down" monitoring procedures for this particular site, identify future monitoring needs, collect some smoke samples for preliminary dose definition, and observe the beagle inhalation exposure which is being conducted there. The staff was very courteous and helpful, and because of this, we were able to obtain more than 130 smoke samples. These samples were used to provide some measure of the smoke dose offered the animals and a tentative picture of the chemical profile of the particulate phase of the smoke of at least one variant. Experimental. Briefly, the inhalation studies at ECI are directed toward exposing four groups of male beagles to the smoke of four experimental ciga- rette variants. For cigarette Codes 11, 13, SEB IV (04, 14, 29, 32), and 67, there are 48, 47, 46, and 60 dogs, respectively, in each exposure group, in addition to a total of 115 sham and control animals. There are 18 expo- sure wards at ECI, three for each variant plus six for housing the sham and control dogs. The wards being used to.expose the Code 11, 13, and 32 groups each contained six exposure•devices, whereas the three wards for the Code 67 animals each contained 10 exposure devices. (Code 32 will be used to denote all of the SEB-IV Variants.) At the time of our visit, the exposures employing

Codes 11, 13, and 32 cigarettes generally used the BNW exposure device, with an uncuffed cannula, whereas the Code 67 exposures were being conducted with ADL-II exposure systems which utilized a cuffed cannula. The exhalation valve, necessary for a cuffed cannula system, is mounted on the head of the cannula by means of a plastic "T-tube." Each animal is exposed to twelve cigarettes per day, seven days a week. The animals are given three cigarettes consecutively before taking them off the expo- sure system. Because of the large number of exposure devices in this study (84), it was impossible to sample each device on any one visit. Measurements were made on approximately half of the machines in service at the time of the visit. The frequency and duration of puffing are measured with a stop watch. While this is a somewhat crude procedure for measuring small time intervals, it is sufficient for detecting large excursions from specified operating parameters. Puff volumes are measured with a soap bubble meter. Cigarette static burn rates are determined by lighting a cigarette, taking one puff, turning off the machine, and measuring the amount of time required to burn to a 23 mm butt length. To collect smoke particulates generated by the experimental variants, a Cambridge filter pad is installed between the cigarette and exposure device. For cannula exit samples, a filter pad is installed on the end of the cannula, which is, in turn, attached to a Harvard respirator (ventilator) set to withdraw air through the cannula at a rate of 16 strokes per minute, 100 ml per stroke. Smoke samples obtained at ECI were returned to ORNL for analysis. Nicotine is determined by extracting the filter pad with ethanol and analyzing an aliquot of the extract via gas chromatography on a Casterwax stationary phase. 3-phenyl-5-methyl-1, 2,4-oxadiazole (PMO) is analyzed via gas chromatography on a Carbowax- poly-phenyl ether stationary phase. Other analyses (referred to as a multi-component, multi-aliquot procedure, and discussed later) are per- formed by extracting the filter pad with pyridine, subjecting small aliquots of the extract to a tri-methyi silylating procedure and analyzing these derivatized aliquots under va'rying gas chromatographic conditions. The multi-component, multi-aliquot procedure permits a much more detailed chemical description of the particulate phase of the smoke, as several components correlate highly with the presence of entire classes of smoke constituents.

t General Observations. General observations concerning the operation of the exposure devices and environmental conditions in the exposure wards have been discussed previously in Topical Report #43. Results and Conclusions. Table I lists the measured puffing parameters for selected BNW and ADL-II exposure devices in service at the time of the visit. The puffing period and frequency for both exposure systems are within specified limits. Puff volumes for the BNW exposure devices were slightly larger than the specified value (35 ml), but only by a few percent. When judged solely in terms of measured puffing parameters, the performance of both BNW and ADL-II machines was good. However, the mechanical reliability of the BF1W system is very poor. There was frequent binding of the moving parts, and most of the solenoid by-pass valves were occluded with condensed smoke. This has been discussed in detail previously, and, at the time of the visit, ECI was planning on changing over to the sole use of ADL-II exposure systems. We also observed several of the ADL-II systems sticking, some to the point where they had to be "puffed°@ manually. Careful attention should be paid to the lubricant on the large o-ring of the piston of the ADL-II. After use, these o-rings will frequently expand, and cause the piston to sieze up within the cylinder. Table II compares the static burn rate of Code 32 cigarettes smoked at ECI with that of those smoked under analytical conditions at ORNL. There are no significant differences in the burn rates among wards, but the ciga- rettes burn a little more slowly on-site than under analytical conditions- This is probably due to the higher relative humidity (75%-80% RH at 23°C) and low ventilation of air flow rate on-site. The total particulate matter (TPM) and nicotine content of the smoke generated by some selected exposure devices at ECI is tabulated for indi- vidual samples on Table III. The data indicates that the machine-variant combinations are producing a fairly consistent amount of TPM and nicotine. Table IV compares the average input TPM and nicotine values with those of cigarettes sampled from the same batch and smoked under analytical conditions at ORNL. The TPM values under both conditions are virtually identical, which is the ideal situation. Nowever,•the analytical nicotine values are higher, especially in the case of the Code 13 variant, than those generated on-site. We have analyzed Code 13 cigarettes from other chronic exposures, ?;sV+A -

and have never observed such a high (by a factor of two) nicotine content. We suspect that this is due to having sampled a non-representative lot of cigarettes at ECI. Significant tray-to-tray variations in cigarette tar and nicotine content have been observed in other chronic inhalation studies. Every effort should be made to identify batches of cigarettes which deviate substantially from the average prior to their use in a chronic exposure, in order to avoid exposing animals to widely varying smoke doses. Table V tabulates the individual TPM and nicotine values of the smoke reaching the cannula exit of both the BNW and RDL-II exposure systems. Within a given code, the values are relatively consistent, suggesting that, if the animal inhales all the smoke which is made available at the cannula exit, the smoke dose will not vary appreciably as the animals are shifted from machine to machine. Table VI compares the average cannula exit results with input TFM and nicotine values for each variant (from Table IV). Several things warrant comment. First, nicotine is generally believed to be a some- what better indicator of the amount of smoke arriving at the cannula exit because the volumes of air required to insure complete withdrawal of the smoke from the stand tube (ti1.6 liters/minute) may evaporate some of the more volatile constituents from the filter pad. Thus an artifactually low TPM weight may be ovserved for output samples. Secondly, the same approximate relative nicotine ratios of the smoke of the Code 13, 11, and 32 variants (1:3:9) is maintained at the cannula exit. For all of the variants, the average output nicotine values are lower than the input values. However, these differences are not statistically significant, except in the case of the Code 13 variant. The high efficiency of smoke delivery to the cannula under sampling conditions indicates that few smoke particulates are likely to condense in the delivery system as long as the animals withdraw the smoke at a reasonable rate. Thus, the animals are probably offered a smoke which has a ratio of gas phase to particulate phase constituents very similar to that of °normal" smoke. Of course, under exposure conditions, the animals being exposed with the BNW devices were not withdrawing all of the smoke from the smoke delivery system. This is most likely due to the use of the uncuffed cannula in conjunction with these devices. The uncuffed cannula, permits animals to bypass the machine inhalation channel, and thus inhale less than the full dose of smoke. The smoke which remains is free to condense 00 ~ W ~ .~ t~7 FA

within the smoke generation chamber or in the narrow orifices of the solenoid by-pass valve, which is supposed to permit un-inhaled smoke to be expelled from the system prior to taking the next puff. Based on the observation that most of the by-pass tubes and solenoids became quickly occluded with condensed smoke, we concluded that a large number of animals exposed on the BNW devices were not receiving a full smoke dose. Table VII compares the average PMO contents of Code 67 smoke generated analytically with that generated by the ADL-II exposure devices and that arriving at the cannula exit. The apparent low delivery efficiency of PMO may be an artifact of sampling, due to the higher air flow across the filter pad during cannula exit sampling. A very large cigarette-to-cigarette variation was noted in both the analytical and input smoke samples. We were alerted to this possibility by Dr. Buhl at ECI. Ultimately, the most important chemical parameter relating to the actual exposures is the smoke dose which the animals receive. For chronic exposures, given the limitations of present techniques, our best estimate of dose is the amount of smoke offered the animal. For the ADL-II-cuffed cannula system, the dose offered should be a good estimate of the amount of smoke inhaled by the animal. Table VIII lists our estimates of the amount of smoke offered the animals, per variant, on a weekly basis. For Codes 13 and 32, the cannula exit values of TPM and nicotine, per cigarette, are very similar to another P1CI-sponsored inhalation bioassay. However, the weekly dose at ECI is significantly higher on a weekly basis, because the number of cigarette exposures per week is about 40% greater. While each animal group is being offered a slightly different amount of TPM, the differences vary from a mean value by less than ± 15%. In an effort to more exactly define the chemical composition of the smoke offered the animals, we have initiated a more detailed chemical characterization of particulate phase samples acquired on-site. Table IX compares chemical profiles of smoke samples taken at the machine input and cannula exit for the Code 32 variant. There appears to be very little difference in the relative amounts of the visualized constituents of the input and output samples, sugoesting that gross changes in the chemical composition of the smoke praticulates do not occur as the smoke passes through the exposure system. The slight depletion of phenol in the output samples is probably an artifact of the sampling procedure, since phenol is relatively.

volatile. In addition, the Code 32 smoke generated at ECI on the BNW exposure system is chemically very similar to that generated by an ADL-II system in another inhalation bioassay (See Topical Report #36-A).

Table I Measured Puffing Parameters for Selected BNW and ADL-II Exposure Devices in Service at ECI on May 23-24, 1977 L Ward No. Cigarette Code Exposure Machine Device No. Puff Period (sec) Puff Duration (sec) Puff Volume (ml) 10 11 gNW 022 30.1 2.2 36.5 001 31.1 2.2 37.8 065 32.0 2.1 36.9 043 30.1 2.3 37.5 23 31.0 2.3 38.7 16 11 BNW 025 31.0 2.1 38.9 019 31.0 2.2 37.2 048 31.0 2.3 37.0 032 30.5 2.2 36.5 29 34.0 2.3 35.8 Average Puff Volume for Selected Code 11 Machines 37.3 ± 1.0 ml 7 13 aNW 057 31.0 2.0 35.0 060 30.7 2.0 36.7 010 * * * 059 * * * 004 * * * 18 13 BNW 085 30.5 2.1 35.8 073 30.5 2.2 32.8 055 30.5 2.1 36.0 035 29.5 2.2 37.7 014 * * * Average Puff Volume for Selected Code 13 Machines 35.7 ± 1.7 ml 11 32 BNW 06 30.3 2.1 35.8 038 31.0 2.1 36.5 03 30.5 2.0 36.5 002 31.0 2.1 36.0 017 31.0 2.6 36.5 12 32 BNW 030 31.0 2.1 36.3 101 25.0 2.0 35.8 021 27.0 2.0 36.7 031 31.0 2.1 37.0 046 '31.0 2.1 36.8 . Average Puff Volume for Selected Code 32 Machines 36.4 ± 0.4 ml ~ 4 *Machine inoperative at time of measurement. W ~ ~ W 1

Table I (cont'd) Ward Cigarette Exposure Machine Puff Period Puff Duration Puff Volume No. Code Device No. (sec) (sec) (ml) 1 67 ADL-II 62 29.0 2.1 33.6 38 31.0 2.0 35.5 72 30.5 2.0 35.1 13 30.5 2.1 35.6 84 31.0 2.0 35.0 6 67 ADL-II 40 31.0 2.0 35.2 41 32.0 2.1 35.3 83 30..0 2.1 34.1 24 30.0 1.9 35.3 55 30.0 2.0 34.7 Average Puff Volume for Selected Code 67 Machines 34.9 ± 0.6 ml

Table II Comparison of Code 32 Static Burn Rates: Exposure Conditions at ECI vs Analytical Smoking Conditions at ORNL Site Burn Rate, mm/mina Burn Rate, mg/minb ECI, Ward 4 4.22 ± 0.08 60.7 ± 1.9 ECI, Ward 6 4.27 ± 0.21 60.5 ± 1.9 ECI, Ward 12 4.30 ± 0.12 62.C ± 2.5 ECI, Ward 13 4.36 ± 0.33 62.1 ± 2.5 ORNL 4.82 ± 0.10 67.6 ± 2.5 aBurn rate given in terms of actual length of cigarette burned. bBurn rate given in terms of actual weight of cigarette burned.