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INBIFO Institut fur biologische Forschung • Koln 111BiFu DR•MEU• R. RYLANDER 29•JUL•82 c/o FABRIQUES DE TABAC REUNIES S•A• DRRS/RM RM9 (R) Al S W I T Z E R L A N D I N T E G R A T I N G REPDRT A o5oo/3o47 21-DAY SMOKE INHALATION STUDY WITH MAINSTREAM AND SIDESTREAM CIGARETTE SMOKE OF STANDARD REFERENCE CIGARETTE TYPE 2R1 ON RATS COPY NO.: INBIFO Institut fur biologische Forschung GmbH, Fuggerstraee 3, D-5000 K81n 90 Sitz der Gesellschalt: KSln HR 8 367, 29. Oktober 1959 5t.Kw et Tele(on: Porz (02203) 303-1, telelax: (02203) 303362, Telex: 8874 675 lnbi d lnstitutsleiter und GeschVsliihrer: Dr. med. Ulrioh Hecicenberg

1NB1FO Institut filr biologische Forschung • K6ln INTEGRATING REPORT A o5oo/3o47 RM9 (R) A2 PAGE o-1 CONTENTS PAGE 1 SUMMARY 1-1 1.1 Introduction 1-1 1.2 Generation and Administration of smoke 1-2 1.3 Results 1-5 1.4 Discussion, Conclusion 1-13 2 RESPONSIBILITY 2-1 3 CIGARETTES 3-1 TABLE A: TYPES OF SIDESTREAM USED FOR INHALATION 3-2 TABLE B: SPECIFICATIONS OF CIGARETTE TYPES 2R1 3-3 TABLE C: PHYSICAL PROPERTIES AND CHEMICAL COMPOSITION OF FILLER OF CIGARETTE TYPE 2R1 3-4 TABLE D: COMPOSITION OF SMOKE COMPONENTS OF CIGARETTE TYPE 2R1 3-6 4 STO RAGE OF MATERIALS AND RECORDS 4-1 This integrating report contains, including front page, 26 pages. 2s.xwa2

INBIFO lnstitut ftir biologische Forschung • K6ln INTEGRATING REPORT A o5oo/3o47 RM9 (R) A3 PAGE 1-1 , SUMMARY 1.1 Introduction In previous studies at INBIFO the biological activity of inhaled mainstream cigarette smoke on small laboratory animals was inve- stigated. In order to quantitate biological activity, systemic responses, metabolic responses, effects indicative for mutageni- city, histological responses of the respiratory tract as well as free lung cell responses were studied (Bioassay program). In addition to mainstream, which leaves the burning cigarette through the mouth end or filter during puffing, smoke is also released from the burning zone directly into the surrounding atmosphere without passing through the filler and the mouth end. Additional smoke diffuses through the cigarette paper. This smoke types are termed sidestream smoke (a). Since "open" smoking is performed, smoke leaks out of the mouth end during puff intervals. This fraction is not included into the side- or mainstream fraction (see FIGURE A, TYPES OF SIDESTREAM USED FOR INHALATION). Though qualitatively similar, the chemical composition and physical properties of sidestream is known to be quantitatively different from mainstream (b). This difference is also reflected in the different biological activity of mainstream and sidestream cigar- ette smoke condensate in the mouse skin painting model (c). (a) According to DIN 1o24o "Maschinelles Abrauchen von Zigaretten und Bestimmung des Rauchkondensats", Apr.78, sidestream smoke is defined as the whole amount of smoke, which leaves the cig- arette by another way than through the cigarette mouth end. (b) see De Bardeleben, M., An Overview of sidestream Smoke: its components, its analysis, some influencing factors, PM inter- office corresp. March 1981 (c) sidestream condensate: higher tumorigenic activity than main- stream condensate (Wynder, E.L. and Hoffman, D., Tobacco and tobacco smoke, Acad. press, N.Y., pp. 183 and 291, 1967 ) sa.Kws2

INBIFO lnstitut fGr biologische Forschung • KtSIn INTEGRATING REPORT A o5oo/3o47 RM9 (R) A4 PAGE 1-2 These differences are related to the deviating burning- and distil- lation processes during puffing and puff intervals. Sidestream smoke will generally contain a higher proportion of oxidative combustion products, but a lower proportion of distillation and pyrolysis products as mainstream smoke. For normal cigarette types the particle phase of sidestream contains smaller, highly charged particles (Corn, 1974) and its gas phase relatively more volatile components than mainstream. In the present 21-day inhalation study on rats the subacute toxici- ty of sidestream cigarette smoke has been compared to the subacute toxicity of mainstream smoke. In addition to parameters of general toxic effects, local histological changes of the epithelium in the respiratory tract and cytotoxical effects on free lung cells were analysed. As a prerequisite, the methods for generation, administration and for analytica3l monitoring of cigarette sidestream smoke had to be acquired. Cigarette type 2R1 was used to generate sidestream and mainstream smoke. As in previous inhalation studies with mainstream, the TPM concentration was used to relate administered "doses" of side- and mainstream smoke. 1.2 Generation and Administration of Smoke Mainstream smoke was generated as in previous studies using 1 automatic 3o-port positive pressure smoking machine. Parallel to mainstream smoke generation, the resulting sidestream smoke ("puffed sidestream smoke") was quantitatively collected by a circular hood inside the smoking machine. 28. Kwa2

INBIFO lnstitut ftir biologische Forschung • Mln INTEGRATING REPORT A o5oo/3o47 RM9 (R) A5 PAGE 1-3 In a 2nd smoking machine, the cigarettes were only lighted with 1 mainstream puff, and the sidestream smoke originating from the nonpuffed smoldering cigarettes ("nonpuffed sidestream smoke") was collected in the same way as far as the puffed sidestream smoke. By adjusting the sample flow rates of the sidestream smoke and the flow rate of the diluting air for the mainstream smoke, the TPM concentration of all 3 types of cigarette smoke was adjusted to be equal (see IB TABLE A, GROUPS, EXPOSURE AND DOSES). loo male rats with an initial mean body weight of 17o g were randomly allocated to 5 groups of 2o rats each. The cage control group was not treated and was kept in cages. The tube control group was sham exposed to a similar flow of fresh air as the dose groups. The mainstream group was exposed on 42o min/day for 21 consecu- tive days to o.5 : 21o diluted mainstream smoke resulting in a TPM concentration of o.17 mg/3. (a). The "puffed" sidestream group was simultaneously exposed to equal TPM concentrations of sidestream smoke, generated in parallel. The "nonpuffed" sidestream group was simultaneously exposed to equal TPM-concentrations of sidestream smoke from nonpuffed cigarettes. (a) This represents about 1/3 of the highest TPM concentration in previous 21-day inhalation studies. For the given cigarette type and exposure duration this "daily TPM dose" is limited by the relatively high CO concentration of the puffed sidestream smoke. 28. KW 82

INBIFO Inst9tut fiar bfologische Forschung • Kt51n INTEGRATING REPORT A o5oo/3o47 RM9 (R) A6 PAGE 1-4 I The rats of the 3 dose groups were kept in glass tubes, sealed with rubber stoppers at the back and ending in a stainless steal wire head basket at the front end which extended into the aerosol duct of the inhalation chamber (head only exposure). The rats of the tube control group were kept in Makrolon tubes. The flow of the aerosol (or air) inside the inhalation chambers was adjusted to be approx. o.35 m/s for all groups. This resulted in a constant pressure of about lo mbar inside the inhalation chamber relative to the outside. The mean temperature of the diluted smoke (or air) inside the inhalation chamber was: 23.3 degrees centigrade for the tube control group, 25.2 degrees centigrade for the mainstream group, 33.5 degrees centigrade for the puffed sidestream group and 33.9 degrees centigrade for the nonpuffed sidestream group. The remarkable decrease of body temperature (see 1.3 Results) during exposure and its possible effect on the body weight develop- ment of the rats was investigated by exchanging some of the Makro- lon tubes with glass tubes (tube control group only) and some of the glass tubes with temperature controlled glass tubes (nonpuffed sidestream group only). 28.KW82

INBIFO Inst9tut fiar biologische Forschung - K6in INTEGRATING REPORT A o5oo/3o47 RM9 (R) A7 PAGE 1-5 , 1.3 Results Diluted mainstream and sidestream smoke from puffed and nonpuffed cigarettes were sampled from the inhalation chambers. The following parameters were analyzed: total particulate matter, nicotine, carbon monoxide, carbon dioxide, nitric oxide, acetaldehyde, hydrogen cyanide and the hydrogen-ion concentration as well as the buffer capacity of an aqueous extract of the particle and the gas/vapor phase (see AC TABLE 1, SURVEY OF SMOKE ANALYSIS). Within the accuracy of these analyses, none of these parameters showed remarkable differences between the chemical composition of puffed and nonpuffed sidestream smoke. The concentration of TPM and nicotine as well as the buffer capa- city and the pH of the particle phase showed no remarkable differ- ence between the diluted main- and sidestream smoke. The following gas/vapor phase components were distinctly higher in diluted sidestream smoke than in diluted mainstream smoke: carbon monoxide (4 times) (a), carbon dioxide (3 to 4 times), nitric oxide (6 to 7 times) and acetaldehyde (2 to 3 times). Only the hydrogen cyanide concentration was higher in diluted mainstream than in sidestream smoke. The factor was 2. The gas/vapor phase of sidestream smoke was more alkaline (pH 8.7) and had a much stronger buffer capacity (26-fold) than the main- stream gas/vapor phase (b). (a) The higher CO concentration in sidestream smoke was in accord- ance with the higher HbCO concentration in the blood of the sidestream exposed rats, which limited the maximum inhaled daily smoke dose. (b) Both effects may mainly be due to the known higher ammonia concentration in sidestream smoke (which was not analyzed in this study). 2a.Kwa2

lNBIFO Institut fiir biologische Forschung • Min INTEGRATING REPORT A o5oo/3o47 RM9 (R) AB PAGE 1-6 i As commonly seen in cigarette inhalation studies of this type, the rats resisted to the daily loading into the exposure tubes and continued to struggle inside the tubes right after the beginning of the exposure. By and large, the rats of the sidestream groups reacted more vigorously than those of the mainstream group. All rats showed general signs of exhaustion after the end of the daily exposure. In contrast to the rats of the mainstream group, which recovered by the next morning, the rats of the sidestream groups continued to show shaggy fur and some pronounced respiratory symptoms characterized by whistling and rattling sounds. 1 rat of the mainstream group, 9 rats of the puffed and 11 of the nonpuffed sidestream group died "spontaneously". 2 rats of the puffed sidestream group and 1 rat of the nonpuffed sidestream group were killed in moribund state. Additional 12 rats of all groups died for reasons which were considered to be of primarily technical nature. The rats of the sham and the cage control groups increased their body weight during the .21 days of exposure steadily in a normail way to approx. 15o percent of the initial body weight. The body weight of the mainstream group increased to approx. 13o percent. The sidestream groups showed a decrease to approx. 8o percent of their initial body weight. The rats of the sham and cage control groups consumed approx. 24 grams powdered diet per day. 28.KW82

INBIFO Institut ftir biologische Forschung • Kt31n INTEGRATING REPORT A o5oo/3o47 RM9 (R) A9 PAGE 1-7 This food consumption is in the normal range and was approx. constant during the 21 days of exposure. The food consumption of all dose groups dropped sharply during the first days of inhalation. The food consumption of the mainstream group increased gradually after day 4 and reached a total mean of 92 percent of the con- Aumption oC the sham control group. The food consumption of the sidestream groups remained low, with a resulting total mean of 45 percent of the sham control group. The water intake showed a similar pattern as the food consumption: it was 39 g/day for the rats of the sham and the cage control group, and 113 percent for the mainstream group and 59 percent for the sidestream groups relative to the sham control. The mean body temperature at the end of each daily exposure was in the range of 36.5 and 37.4 degrees centigrade for the rats of the cage control and the sham-exposed group regardless of whether kept in glass or Makrolon tubes. The mean body temperature of the mainstream exposed rats was 36.5 and that of both sidestream groups 32.o degrees centigrade. The respiratory frequency of the dose groups at the end of each daily exposure showed a consistent decrease similar to the body temperature. The mean respiratory frequency was 1o7/min for the sham and cage control groups, 91/min for the mainstream and 86/min for both sidestream-exposed groups. 28.KW82

INBIFO Institut fiir biologische 1=orschung • K6In INTEGRATING REPORT A o5oo/3o47 RM9 (R) Alo \„,PAGE 1-8 Some rats of sham control group, which were exposed after day 14 to nonpuffed sidestream smoke in temperature controlled exposure tubes (a) showed a similar drop in body weight, food consumption and water consumption as the other rats of the sidestream groups at the beginning of the study. The increased tube temperature and hence the decreased heat loss of the rats, showed no influence on these parameters. The body temperature increased with higher environmental tempera- ture of the tubes, but the difference between body and tube tempe- rature, which can be considered as proportional to the metaboli- cally generated heat, decreased from approximately 5 degrees centigrade for the nonheated tubes to 1.5 degrees centigrade at 35 degrees centigrade tube temperature. The nasal cavity of 53 rats (b) and the larynx and lungs of 2o rats (c) killed at dissection af ter 21 days of exposure were examined histopathologically. The nasal cavities (d)- of the rats of the control group were without histopathological alterations. (a) The 4 temperature levels of the tubes compared were: nonheated (equilibrium with room temperature of 2o degrees centigrade), 30, 33 and 35 degrees centigrade (b) 27 rats of the control groups, 16 mainstream-exposed rats and 6 and 4 rats, exposed to puffed and nonpuffed sidestream smoke respectively. (c) 9 rats of the control groups, 4 mainstream-exposed rats and 3 and 4 rats, exposed to puffed and nonpuffed sidestream smoke respectively. (d) 2 dorsoven a1 sections, 1 at the incisive papilla and 1 between the 1st and 2nd palatal ridge were histopathologi- cally evaluat d. 2a.xwa2