sample="quota" bates="1002968498" isource="pm" decade="1960" class="ui" date="19690000" C: 1 Carpenter 2 Charles Dontenwill, W. Research Institute of the Cigarette Industry, Hamburg - Germany EXPERIMENTAL INVESTIGATIONS ON THE EFFECT OF CHRONIC CIGARETTE SMOKE INHALATION ON SYRIAN GOLDEN HAMSTERS AND APPLICATION OF CIGARETTE SMOKE CONDENSATE ON MOUSE SKIN Ever since numerous other working groups, especially Wynder and Hoffman in the USA as well as Druckrey and Schmahl in Germany have been engaged in clarifying the question of the tumor inducing effect of cigarette smoke condensate, to enable an elimination of harmful substances. Repeatedly, it was tried to develop methods and models in order to study the effect of whole smoke by smoke exposure in order to obtain more knowledge about the effect of whole smoke or to obtain information of more conclusive evidence by means of better bioassays. At the conference in Lausanne (1969) and Gatlinburg (1970), and more in detail at the conference in Washington (1970) and New York (1970) we had reported on our up to that date existing results, the fundamental conception of which you know. 1. ) All our endeavours are bent on the search for a way to a "safer or less harmful cigarette" in terms of definition, often explained by Wynder and others. Up to now, the condensed smoke was tested predominatly on the skin and the subcutaneous tissue. *Lecture held at the National Cancer Institute, Bethesda/Maryland USA 2. ) In order to better evaluate the biological effect we have not only performed extensive animal experiments but we have also looked for new bioassays with a higher value of evidence. All our efforts to improve the smoke exposure experiments were explained by us in detail, just as the equipment and methods we designed and resulting experiences. 3. ) A special problem is the question of comparability between the epicutaneous test and the smoke exposure test. On one hand we are investigating the effect of condensate on the skin, and on the other hand the effect of fresh whole smoke in the respiratory tract, i.e. at a site where it also coan become effective in man. Problems of dosage, as you know, are playing a decisive role in comparative investigations; also decisive are questions of the organotrophic effect of certain carcinogens suspected or proved in smoke. The question, whether certain nitrosamines, showing no carcinogenic effect on the skin, play a sifnificant role in the total effect of smoke in the respiratory tract is of decisive importance, as we have often discussed. 4. ) The test methods which are also required for other inhalable substances by the Food and Drug Administration in USA or by the Ministry of Health in Germany are applicable for studying new tobacco additives as well as the effect of whole smoke. The optimal test method is an effective smoke exposure experiment, i.e. that in a great number of animals alteratoins in organs or in the tissue are detectable. Only in a smoke exposure experiment we can study, for instance, the effect of the vapor phase. Factors, such as CO and other components of the vapor phase which are not effective on the skin, can only in this way be tested as a whole. 5.) The fundamental question is: When can a test be considered practicable and of evidence? In New York (1970) we have answered this question as follows: A smoke exposure experiment is then effective when, with a maximum tolerable dose, in a sufficient number of animals malignant growth in the respiratory tract is induced, and this in a quantity which allows evaluation of significant differences within the experimental groups, for example differences between standard cigarettes and cigarettes from reconstituted tobacco sheets or cigarettes with sodium nitrate as additive or other cigarettes. Only, in this way, we may be able to approximately assess what we may consider "safer" or "less harmful". The results of our second long-term experiment with 8775 mice which is in press in the Journal of Cancer Research and Clinical Oncology may be summarized as follows (table 1): Of five used condensates from different reconstituted tobacco sheet cigarettes, two (= EG1, EG2 and ES) are showing a storng reduction of the tumor inducing effect on the mouse skin, calculated on the basis per gram of the administered substance. This reduction is lying below 50% compared with the activity of standard condensate. A condensate of one reconstituted tobacco sheet cigarette (EB) shows a stronger, and two condensates from reconstituted tobacco sheet cigarettes (EW and EA) show an equally strong activity compared to the condensate of the standard cigarette. In the following, we want to discuss the results of the smoke inhalation experiment. The question of reproducibility, even if animals from different colonies are used, can be definitely answered in the affirmative and is explained by the following fingings. The dose-response-relationship is evident in various findings: 1. ) In the survival time: You will see in this table (6) that in comparison to the controls, the mean survival time is distinctly reduced in those animals having received increased doses of smoke and in those animals having received an additional treatment of DMBA. 2. ) In the development of body weight: This table (7) demonstrates that the highest decrease of body weight is observed in the group having received the highest amount of somke. The loss in body weight is also evident in comparison to the controls. 3.) In alterations of the larynx: This will be discussed in the following: The number of animals used as well as the dose administered is sufficient for statistical evaluation; this is apparent from statistical evaluation of significance in the original paper given for publication. As optimal dose may be considered the dose II, i.e. 30 cigarettes twice daily or 10 min. smoke exposure twice daily. Owing to the marked difference of the two sexes as to mortality and incidence of certain diseases, the use of male and female animals was necessary. We have alredy mentioned some of the alterations provoked by chronic smoke exposure extended throughout the whole lifespan. They are 1.) the findings in the larynx, 2.) the inhibition of body weight development, 3.) the reduction of survival time and, moreover, the increase of Hb and of erytrhocytes should be mentioned, which may be explained as consequence of the high dose of smoke offered without intervals and as a result of strong increase of the CO-Es. This result is distinctly influenced by method, as already explained, and, therefore, does not permit any conclusions as to the smoking habits of man. The test gives us the following information: 1. ) In 800 control animals and in 160 animals treated with the vapor phase of the standard cigarettes, laryngeal leucoplakia of stage 1 is found relatively seldom, and stage 2 of laryngeal leucoplakia was observed only in one control animal. Similar observations were made in the group of animals treated with nitroszmines only; there, too, only stage 1 of laryngeal alterations was found. The group of 160 hamsters treated once with DMBA shows an exception. Besides 4 cases of stage 1 and 1 case of stage 2 of laryngeal leucoplakia, 3 casees of the papillomatous stage 4 were found. 2. ) The various stages of laryngeal alterations appear in an order of time, i.e. they are dependent on dose-time factors. This may be demonstrated by some examples (tables 9 and 10), giving also information about the time of appearance, e.g. of stages 5 and 6 which in group 1 appeared distinctly earlier than, for instance, in the comparable group 5. Here already, the more-than-additive effect of the smoke is apparent, i.e. the effect cannot be only explained as summation of the effect of dose II = 2 x 30 cigarettes and DMEA. 3. ) In general, animals which died early or animals having received cigarettees with lower activity have more frequently stages of lower degree, e.g. stages 2 - 4. Cigarettes with lower activity clearly show more rarely the stages of higher degree, that is stage 5 and carcinomas (stage 6). The following tables (11 and 12) distinctly demonstrate the difference of stages within the groups. In order to offer a clear overall picture, we have summarized the most important findings in this table (13). It is evident that a reduction of condensate by using NaNO3 as additive or reconstituted tobacco sheets or filters reduced alterations, especially the incidence of stage 5 and 6 (cancer) in the larynx. This is also concerned for the black cigarette showing a very low condensate yield due to the mixture used in this experiment. A significant reduction of the biological effect is demonstrated also under consideration of different numbers of puffs and period of smoke exposure. This becomes very impressive when expressed in terms of R = relative potency. Here, we believe to find a way towards a safer cigarette which should inspire and influence our future work. Which findings have been enhanced by smoke exposure? The incidence of the so-called smoke cells which, as already CONNING replied to OTTO in Lausanne, are no smoke cells. In smoke-exposed and also in other groups this type of cells is increased. In controls, the freqeuncy was, for instance, 9.9 - 15%, in smoke-exposed animals 28 - 70%. An increase of the so-called "adenomatoid lesions" could not be clearly proved in smoke-exposed animals. Which findings may be attributed to single treatment with DMEA only? This will be clearly demonstrated in some of the following tables: 1. ) The increase of pharyngeal tumors (table 19) 2. ) the increase of tumors in the oral cavity (table 20) 3. ) the tumors of the esophagus and stomach, especially of the papillomas (table 21) 4. ) the increase of liver tumors (table 22) 5. ) th early induction of tumors of the hemopoietic or lymphoreticular system (table 23) 6. ) the increase of ovary cysts in controls (= 4.6%) and in animals treated with DMBA (= 20 - 30%). Which findings are not in relationship with the treatment? There is no evident relationship between treatment and induction of tumors in the soft tissue and subcutaneous tissule of the skin (table 23), in the nasal cavity (table 25) and in the urinary bladder. Only one bladder carcinoma appeared in the group 18A. The animals of this group were treated with DENA. The incidence of adrenal tumors was age-dependent, and the distribution of sexes with regard to tumor types was very different (table 26). The incidence of lung thrombi, pneumonia and bronchitis was similar in all groups. Smoke-exposed animals did not exhibit lung emphysema more frequently (table 27), and this could be confirmed by two quantitative measuring methods: 1 - by densimetric measurements of the lung of old, young, smoke-exposed and control animals (table 28) 2 - by determination of the number of alveoli in smoke-exposed and control animals (table 29). Also the incidence of gastric ulcer was not increased in smoke-exposed animals (table 30). We have intensively been engaged in studying the incidence of heart diseases and cardiovascular diseases in smoke-exposed animals. The results show (table 31) that 1 - diseases of heart valves could not be demonstrated more frequently in somke-exposed animals 2 - inflammatory vascular diseases occurred more frequently in female that in male animals. They are, however, in no relationship to smoke-exposure. 3 - arteriosclerotic alterations could be demonstrated only once as calcification of the media. This finding is worth mentioning, as animals with severe amyloidosis of the kidney, i.e. stage III - the so-called amyloid cirrhosis of the kidney - showed also hypertension (table 32 and figure). Additionally, it should be pointed out, that, regarding the subject of arteriosclerosis, the chronic CO-Hb increase which was dependent on the method and which was expressed in a low increase of Hb and erythrocytes, obviously did not become effective in the vessels. Moreover, it should be pointed out that the entire biochemical findings (tables 33 and 34), especially insofar as the fat metabolism is concerned, did not show any pathological alterations. Finally, it should be added that extensive examinations of the testicles (tables 35 and 36) did not give any indication that smoke-exposure is a cause of the severe testicle atrophy of the hamster, which could often be found at an early age. Since it would take far too long to go into all other findings. we have mentioned those of greatest importance. In summary it can be said that the method designed gives findings which can be evaluated and reproduced. A number of findings which could be expected, such as the increase of gastric ulcer, vascular diseases, the influence of fat metabolism and lung emphysema or bladder tumors, did not occur. The findings following combined treatment with DMBA and nitrosamine give a number of informations and further suggestions. Finally, as a summary of this paper, I should like to express our opinion that, on the basis of further international collaboration in such a good way, the way to a "safer cigarette" can possibly be found soon. This is the purpose of all our scientific endeavours. In conclusion, I would like to express my thanks to my collaborators, Dr. Chevalier, Dr. Harke, Dr. Lafrenz and Mr. Reckzeh, who have essentially participated in the experiments. Moreover, I would like to express my thanks for your kind attention to my presentation. Bibliographical references may be obtained on request.