Document KJXbveRL8jje74X12yeyeKVeK

Verh. Deutsch, Ges. Arbeitsmed. 13.(1978) Special issue from the annual report for 1978 Possibilities and limitations of biological monitoring. Industrial Hvciene nroblens of the services trades. Industrial Medicine colloouium Report from the 18th annual meeting of the German Society for Industrial Medicineijointly with the Union of the tech nical professional societies. Frankfurt am Main-Hbchst May 24 through 27 1978. Pharmacokinetics and Carcinogenesis of Vinyl Chloride. Industrial Medicine risk evaluation. A Butcher, H.M.Bolt**, J. Filser*, H.U. Goergens, R.J. Laib* and W. Bolt, Institute and Polyclinic for Industrial and Social Medicine from the University Of Cologne. * Toxicology Institute from the University of Tlibingen The alkylation of nucleic acids by the vinyl chloride-meta bolite and the ensuing damage to the base pairing mechanism are the likely explanations for the carcinogenesis of vinyl chloride((1,2,16,17); compare Laib,Bolt,Butcher.Kappus,3oIt, Verh .Dtsch.Ges.Arbaitsmed, 17 year, page 243-250,1977 ) The pharmacokinetics of vinyl chloride on rats was al ready evaluated by our work team (4). Figure 1 presents the experimental devices and the pharmaco kinetic models used. Vinyl chloride is rapidly picked up from the atmosphere through the lungs (figure 2). There is an equilibrium be*w tween the vinyl chloride concentration in the exposure at mosphere and the organism which is expressed by the equili brium constant _ v= amount of VC per of body weight amount of VC per mi or air In the body the vinyl chloride is eliminated via metabolic processes. In order to evaluate the risk involved in the inhalation of vinyl chloride one needs to determinethe rate of vinyl chloride pick up and the rate rate of its metabolic elimination. Figure 1: Pharmaco-kinetics of vinyl chloride by exposure in a closed system. Model by Bolt and coll(4) This model includes the metabolism of vinyl chi ride. The constants ware determined with expe rimental animals exposed to vinyl chloride ini tial concentrations below the 250 ppm level. SL 041730 (2) Figure 2: Pick up of gaseous vinyl chloride by rats which were previously treated with an inhibitor for the metabolism of vinyl chloride (6-nitro-l,2,3benzothiadiazol, see Bolt and coll. (4)), The level of vinyl chloride which is attained at equilibrium between the vinyl chloride in the gas phase and in the experimental animals, is repre sented as 100%. Comparative research of the vinyl chloride metabolism in ex perimental animals. The extraordinary importance of the pharmaco-kinetics of vinyl chloride in humans led us to further experimental investiga tions on various animal species in order to improve the calcu lation of the vinyl chloride metabolism in humans-and arrive to a better evaluation of the self-experiment. The following animals were employed in the investigation: 11 male white mice (Swiss mice) 380 g. altogether, 54 to 56 days old; 4 mongolic RennmaUse (meriones unguiculatus), altorether 240 g;male rab bit, 3.2 kg. ( The animlas came from Ivanovas,Kissleg,AllgHu) SL 041731 <3) Vinyl chloride was kindly supplied bv the Dynamit Nobel AC, Troisdorf (Degree of purity according to data supplied by the firm, 99.995%) The vinyl chloride concentration in the exposure atmosphere was determined by gas chromatography (Model no. 1440-1, Varian, Darmstadt). The samples were obtained with a disposa ble syringe and was injected into the gas chromatograph through a gas sampling valve provided with a 5 ml. sample loop. The column temperature was 150C(steel column 3m long 1/81', Poropak Q, 50-89 mesh), the temperature at the detec tor was 260C. The hydrogen gas flow was 30 ml/min, 300 ml/min of synthetic air and 60 ml/min of nitrogen. The retention time for vinyl chloride was 2.15 min. The addition of vinyl chloride was performed by way of a dosification sample loop through a septum in the exposure container. /Simirlarly to early experiments with rats, the corresponding initial concentration ranged about 50 ppm. The length of each experiment varied between 2.5 and 4 hours. Table 1 shows the results of the experimental studies on the various animal species. The half-life time and the corres ponding constant were determined for the "Invasion"(intoxi cation ?) for the initial time of the distribution of vinyl chloride in the organism until the establishment of the equi librium. The elimination constant, the half-life time and the clerance were determined for the mtabolic processes of vinyl chloride. The clearance of vinyl chloride based on the body weight was first used in a comparison of the metabolizing rates of vinyl chloride for the different species. A further discussion together with the experimental results with humans, follows. Investigation on the metabolism of vinyl chloride in humans. Vinyl chloride is a carcinogenic material which will be used in the future for economical reasons. Therefore, it is the obligation o`f the Industrial Medicine to reduce the risk,on the involved worker, to a minimum, especially using early diag nostic methods for the identification of the IlHmangio endothe- lum of the liver which is characteristic of tumors produced by vinyl chloride. The vinyl chloride limit concentration in the technique is the average yearly value for a work invol ving 8 hours per day and 40 hours per week, and the vinyl chlo ride concentrations allowed per day may increase as much as 3 times(using an hour as an average value). This leads to the main question of how to evaluate sporadic short-lived peak concentrations. Therefore, it will be of great significance that the technical limiting concentration, expressed as yearly average, allowed for sensibly higher short lived concentrations The previously obtained data on experimental animals cannot be applied to humans unless they undergo previous treatment. SL 041732 (4) On the other hand, we cannot overlook the need for an exact eva luation of the risk involved. Thus, the following studies of the metabolism of vinyl chloride among humans in selfdetermina tions were required. On the basis of the preceding studies on experimental animals the approximate order of magnitude was calculated for the expected for the vinyl chloride metabolism for humans. For these purposes one developed investigation devices which could provide extensive information on the dis tribution and rate of mtabolism of vinyl chloride in humans, without surpassing the limits of technical concentration and using the shortest possible exposure times. The studies were carried out according tot the rules and regulations of the pro fessional association for safety measures to protect the health in an environment containing vinyl chloride. They called for short-lived or incidental exposures to vinyl chloride which will "fail to produce effect". When there is an incident at a working place where there is a high exposure level to vinyl chloride, it may be expected to occur over the years in larger amounts. Research Methods for vinvl chloride-metabolites in humans. The studies were carried out in both, open and closed expo sure systems. The closed system consisted in a "spirograph" with post addition of oxygen to achieve horiaontal stabiliza tion. (Type 98,Dargatz, Hamburg). The volume of the system was determined using a standard gas. An initial concentration of 10 pp!p of vinyl chloride resulted after the addition of a given defined amount of vinyl chloride. After the preceding test with an empty probe, the inhalative exposure began. The volume of the spirograph system was kept constant using the oxygen post addition! A small air probe was removed from the spiro graph at every minute and its concentration in vinyl chloride was determined by the same method used in the studies with experiemental animals: gas chromatography, ( The condition of the measurements vary very little: Steel column 6m, 1/8", filled with 100/0 SE 30 out of Ehromosorb WAW/DMCS,100/120 mesh, column temperature 40C.Nitrogen 30ml/min synthetic air 300 ml/nin and hydrogen 30 ml/min, retention time for vinyl chloride 2.65 min). For the inhalation studies in the open system an apparatus was deviced to allow , for a constant inspiratoryt vinyl chloride concentration, the simultaneous measurement of the expiratory vinyl chloride concentration and the volume breathed per minute. Based on the system of the classic Respirations-Gasuhr (Maxplanck-Institut, Dortmund) the in- and expiratory parts of the apparatus were separated by a mouth piece valve. The inspira tor'/ apparatus consisted of a glass lined inlet and mixing part SL 041733 for synthetic air and for a mixture of air and vinyl chloride. It also consisted of a gas bag (Linde, Unterschleissheim), and a respiratory bag with a capacity of 3 1,(Dr&ger,LUbeck). Syn thetic air was supplied into the apparatus by a gas pump in a previously established quantities and in a continuous fashion. At the same tine, the air-vinyl chloride mixture, contained in a gas bag, was added to the system using a previously calibra ted pump ( Auer-toximeter,Auer-Gesellschaft.Berlin). The mix ture of air and vinyl chloride was obtained by evacuating the gas bag and then filling through a spirograph and additional ir'.z.r introduction of vinyl chloride. The overall ratio of the mix ture gave an inpiratory vinyl chloride concentration of about 2.55 ppm relative to the air. Attached to the expiratory part of the mouthpiece valve*there was a three necked flask (2 1. capacity) which functioned as mixing chamber. The size of the mixing chamber proved to be quite appropiate, because it gua ranteed an extensive and thorough mixing of the expiratory air and allowed for a a rapid evaluation of concentration changes. The expiratory volume was registerd by a gas meter, and finally exhausted into a hood. The inhalation air samples were obtained from the mouth piece, the samples from the expi ration gas were obtained from the mixing chamber, through a septum. The evaluation of the vinyl chlo.rlde concentration was carried out in the following manner. Kinetics of the distribution and metabolization of vinyl chloride in humans. There is a decrease in the vinyl chloride concentration in the spirograph at the beginning of the inhalation, due to the fact that the mixture of the vinyl chloride-containing air with the air remaining in the lungs of the subjectunder study. Similar ly to the test for the functioningoof lungs by the analysis of foreign gases, one is assuming that this thorough mixing step, lasts from two to 3 minutes in both subjects under research. Occurring at the same time but taking longer time, there is the phase of equilibration between the vinyl chloride in the exposure atmosphere and the vinyl chloride concentration in the organism of the subject. The metabolic process of vinyl chlo ride throughout the organism is also taking place. When one compares the present study with the previous research on expe rimental animals, the ratio between the volume of the exposure system and the body volume undergo a marked change.* The body volume is now, about 10 times the volume of the exposure sys- tem. Figures 3 and 4 show the decrease in the vinyl chloride con centration in the spirogranhs from persons A and 3. After 1 minute the vinyl chloride concentration in the spirograph,for person A, is already 537. and it is 567. for person B, relative to the initial concentration. SL 041734 6- After 5 minutes the decrease brought the levels down to 217, and 157o of the initial concentration. If one plots the vinyl chloride concentration ina logarithmic scale and uses the li near scale for the time of exposure ( figures 3 and 4), the curve describing the decrease in the vinyl chloride concentra tion in the spirograph system becomes a straight line after 15 minutes, for person A,and after 19 minutes for person B. The ensuing decrease in concentration takes place according to a first order kinetics. The decrease of the concentration with time is compatible with the assumption of an enzymatic metabolic process. Similarly to the previous studies on ex perimental animals regarding the pharmaco kinetics of vinyl chloride with or without inhibition of the oxidative metabo lite, one can state that the decrease on the vinyl chloride concentration, as indicated above, can be attributed Co^a me tabolic process of vinyl chloride throughout the organism be low the saturation point. The half life for the rate of the metabolic process of vinyl chloride in the selected system amounted to 18 min ( o.3 hr) for person A and 20.5 min (0.34 hr) for person 3. Fig.3.- Decrease in the vinyl chloride concentration in a closed spirograph system by inhalative vinyl chloride pick-up, for person A. SL 041735 -7- In order to achieve a better comparison, the clearance for both persons was calculated (see table 1). When the straight line describing the decrease in vinyl chloride concentration, in figures 3 and 4, is extrapolated to time 0, one obtains^ the vinyl chloride concentration that would exist in the spi rograph by momentary attainment of equilibrium, (between the vinyl chloride in the exposure atm.osphare and the vinyl chlo ride in the organism). Using this concentration, it was pos sible to calculate a total distribution volume of 57.6 with person A. After drawing off 7 1. from the volume of the spirograph and approximately 1 residual volume of the lungs, the claculation gave a distribution volume of 49.6 1 for per son A. The vinyl chloride clearance-for person A amounted 1.83 1 x min"^ and 1.74 1 x h* x kg" , respectively. Figure 4: Decrease in the Vinyl chloride concentration in a closed spirograph system by innalative vinyl chloride pick-up in person B. SL 041736 -8- For person B, the total distribution volume ainounted to 92.5 1 and the corresponding distribution volume 8 A. 5 1. The vinyl chloride clearance amounted to 3.36 1 x min-' and 2.A3 1 x h"^ x kg-*-, respectively. In agreement with the requirements of the pharmaco kinetic model for vinyl chloride, the ''equilibrium constants" were calculated. They amounted to 0.62 for person A and 1.1 for person B. For the initial phase of the vinyl chloride pick up, the calculated half-life was 1 minute and 38 seconds for person A and one minute and AO seconds for person B. During the inhalation of a constant vinyl chloride concentra tion in an open system (figures 5 and 6),a constant expiratory vinyl chloride concentration was obtained after about 5 minutes for person A and after 7 minutes for person B. Already after 1 minute, the expiratory concentration attained 3/A of the va lue measured later, when the expiratory equilibirium is attained. After the initial distribution of vinyl chloride in the orga nism, a constant percent of the vinyl chloride present in the inhalation is eliminated through the organism. The figures are, 267o for person A and 28% for person B. The vinyl chlo ride clearance amounted to 2.A 1/min for person A and 2.5 1/min for person B. On the basis of the preceding data on experi mental animal^,one can deduce that the constant difference be tween the inspiratory and expiratory vinyl chloride concentra tion, which exists after the first distribution phase of the elimination process, can be attributed to metabolic changes. Figure 5.- Inspiratory and expiratory vinyl chloride concen tration in person A..Inhalative vinyl chloride exposure in a open system. SL 041737 -9- Besides, figures 5 and 6 show that the inhalative pick-up of vinyl chloride at the beginning, and the elinination of vinyl chloride at the end of the exposure, take place in a very ra pid fashion. After one minute exoosure to vinyl chloride, 74 to 70% of the expiratory vinyl chloride concentration was reach ed. A minute ofter the end of the vinvl chloride exnosure, and breathing the room air, only 15 to 22 X of the previous expi ratory vinyl chloride concentration was detectable.in the expi ratory breathing gases. Discussion of the pharmaco kinetics of vinyl chloride in humans and in experimental animals. The basic results obtained in the studies on the metabolization in various animal species and among humans are comp iled in table 1. The data on rats is obtained from our previous studies, already published (3,4). The studies on the various animal species and on humans were performed in closed exposure system using a definite initial concentration of vinyl chloride In the last column of the table appear.' the research results with humans using the inspiration of a constant amount of vi nyl chloride. The selected vinyl chloride concentrations cor respond to actual situations in the working place for PVC po lymerization and are within the limits of the presently enfor ced limit concentration for vinyl chloride. Figure 6.- Inspirator1/ and expiratory vinyl chloride concentra tion for person B. Inhalative vinyl chloride expo sure in an open system. . y- timcnmi Mitt* i | I .* t i.i * . **m ,-T; SL 041738 -10- The initial phase of the distribution of vinyl chloride in the organism is defined by the establishment of a physical equilibrium between the vinyl chloride concentration in the or ganism, referred to the body weight, and the vinyl chloride concentration in the exposure atmosphere. Said "equilibrium constant" amounted to 2.86 in rats, 0.91 in rabbits, 0.62 in person A and 1.1 in person B, The dif ferences between the various animal species and various per sons can be attributed to a different content in fatty tissue. As we have previously shown (6),after the physical equilibrium is established, the concentration of vinyl chloride is ten times higher in the fatty tissue than in blood or in other organs. When studying comparable persons, not suffering from overweight, and after the establishment of the physical equi librium, one can assume as standard value the figure of 807., that is, 807. of the vinyl chloride concentration in the air, will be found in the organism, expressed relative to the body weight. Both in animals as well as in humans, the establish ment of the physical equilibrium takes only a few minutes. In the studies in closed systems with rats and rabbits,the half-life value for the rate of "invasion" amounted to approxi mately 3 minutes and for men about 1.5 to 2 minutes. This means that in humans, 2 minutes after the"exposure the orga nism has, already, one half of the expected vinyl chloride concentration that would result when the equilibrium is reach ed. Accordingly, the distribution phase, for constant inspira tory vinyl chloride concentration, was completed after 5 minutes for person A and after 7 minutes for person B. Studies on rats have shown (3,4) that the saturation point for the elimination of vinyl chloride from an exposure atmosphere is about 250 ppm with healthy animals. It is assumed that there is a saturation of the interested enzymatic systems (13). With isolated, perfused rats livers, on the contrary, no satu ration point was found for the enzymatic system.(14). An un equivocal interpretation for the divergence of these findings cannot be given at the present time. The present studies were carried out for air-vinyl chloride concentrations in the neigh borhood,or better within the range of 0.25 to 0.50 ppm. It is then assumed that the elimination of vinyl chloride from the exposure atmosphere took place below the saturation point of the enzymatic system relative to vinyl chloride. The metabolization rate of vinyl chloride showed different values for the individual animal species and for humans. The values for experimental animals and for humans, can also be compared using different research devices, via the calculation of the clearance for vinyl chloride per kg of body weight. The greatest clearance for vinyl chloride was shown by mice with 25.6 1 x h"-*- x kh--*- followed by the desert mouse with half that amount. SL 041739 -11- The clearance value for vinyl chloride for rats amounted to 8.4 1 xh"*- x kg"*-, for rabbits it was 2.74 of the same units. Among humans there was good agreement between the data obtained from various study devices used with both persons. The mean value of two studies on both persons gave a clearance for vinyl chloride of 2.02 1 x h"l x kg"l. This means that vinyl chloride metabolizes the slowest in humans. The vinyl chloride clearance for rats, in which most of the studies on the pharmaco kinetics of vinyl chloride were performed, is about 4.3 times the value for humans. On this regard one must consider,that the liver in rats (male Wistar-rats) exhibits a content in enzyme cytochrom P-450 which is twice as high as in humans. Based on the studies of induction and inhibition of the vinyl chloride metabolite in rats, it is possible to deduct that vinyl chloride is metabolized through the cytochrome P-450 system. The toxicity of vinyl chloride is due to the poisoning produ ced by its metabolic transformation in the organism. Also, the amount of metabolized vinyl chloride depends upon the amount contained in the dosis. Therefore, it is convenient to run the exposure to vinyl chloride at very low concentration over long periods of time. According to Henschler (14) this approach has a better chance for an enzymatic detoxication,inactivation by coupling onto glutathion, than that consisting of intermitent exposure to short and strongly increased vinyl chloride concen trations. The safety provisions for the protection of health in a vinyl chloride environment, as allowed by the professions as sociations of the chemical industry,- in force since 3uly 1st, 1977, do establish that an automatic alarm should be sounded/at the latest, 5 minutes after a vinyl chloride concentration of 40 ppm has been surpassed. The people concerned, for example, must immediately leave the autoclave room or carry protective respi ratory devices. This regulation is aDparently originated from the premise that the pick up of vinyl chloride by the organism and its subsequent distribution require a longer time, however, the studies that we carried on the pharmaco kinetics of vinyl chlo ride in humans show that, after 5 minutes, the distribution phase of the inhalated vinyl chloride is virtually completed. The re lease of a warning for the concerned individual, five minutes after the appearance of a high vinyl chloride concentration, comes from the assumption the assumption that peak concentrations are dangerous, a concept maintained for a long time. On the basis of new findings from studies with humans,it is recommended to limit to one minute the effective time for a peak vinyl chloride concentration. For the daily practice it means that , immedia tely after the recording of a higher than normal vinyl chloride . concentration in a measuring device, a warning must be given to the co-workers because,... SL 0417*0 t -12- Table I.- Studies on the phamaco kinetics of vinyl chloride on experimental animals and on humans.- Columns Rats Rabbits Mice desert mice / persons : Rows: Exposure system Initial concentration of vinyl chloride in ppm End concentration of vinyl chloride in ppm Distribution phase: Duration (min) Half-life(min) Equilibrium constant K Metabolization: Half-life ( min) Elimination constant k23(h"l) Clearance: 1 x min~^ 1 x h"l 1 x h"l x kg~l mean value ' ' t1 11 S'JsTfN" PC'SO* PcglQ* p*$ftM PcpiON Ratic* ICamhchcn f'AUiE h>u5 o,; kf i.i & e,j kf Aa `j kV n kV A fcEiT a 6*.a jFoimcssvsrcri 1 l > V-A*f AtC*ii0"2MTMf* VC*fc"30*IINTIUTl0* <-) l .. "i* J HiU"f*Ts:tiT t * l GlitMClCH?S*GkSTATC K 10.3 1.3 56.5 50 25 " 15 3.2 2,as 30 3.6 0.31 10.7 10,2 7,0 7.0 OMlkti $rT* 50 10 10 7.5 2.5 2 20 0,5 0.25 7.5 2.5 30 15 15 13 5 7 1,6 1.7 0.62 1.1 fTUOUftiUA | t.lrtlhAYlQN*XOHSTA*t[ _-*4 67.6 277 0.61 . 0.15 u 1 6,3 1,78 a k,*`i S.4 2.76 44 163 0.365 - 0.23 9.73 25.6 2.M6 12.68 1 16 70.5 , *" 2.31 2.03 1.63 3.36 2.6 7-25 105.5 201.7 144 135 1,76 2.63 2.23 1.63 2.16 161 2.67 SL 041741 J ...J {15] Kappas. H., It. M. So/:. .4. Buchter. IV. Bolt: Rat liver microsomes catalyse covalent binding of'`C-vinyl chloride to macrotnolecuics. Nature 257, 134-135 (1975). z ; 7:- ; - ' .. * ; ' [16] A\tpptts. II.. It. M. Boh, A. Bitchier. IV. Boll: Liver microsomal uptake of . "C-vinyl chloride and transformation to protein alkylating metabolites in vitro, Toxicol. Appl. Pharmacol. 37, 461-471 (1976). * hiV:!- 'I- /: I .[17] Lath. R. J.. It. M. Boh: Alkylation of RNA by vinyl chloride metabolites in vitro , !J. and in vivo: Formation of l-N`-ethenoadenosine. Toxicology, im Druck. t ' . {IS] Leibman. K. C.: Current literature: Metabolism of vinyl chloride. Drug Mctab. - * Disposition 5,93-94 (1977). ' '' -- [19] Muller. G..M. Spassotrski. D. Hensehler: Metabolism of trichloroethylene in man. . II. Pharmacokinetics of metabolites. Arch. Toxicol. 32, 2S3-295 (1974). -1 [20] Korpoth, A'.. G. Muller. V. Witting. D. Gottsch-tk, J. Gottsehclk: Untersuchungen ; . jv? ' fiber den StoiTwechsel des Vinylchlorid und fiber Wirkungcn der Vinylchlorid- . ; `; inhalation auf Regulationsmechanismen des LcberstoiTvvcchsels. Verh. Dtsch. _v.;y `; Ccs. Arbeitsmcd. 16. 187-194 (1976), (A. W. Gcntner Verlag, Stuttgart). ' -- [21] Potter. It. R.: Articles of general interest: Vinyl chloride Fd. Cosmct. Toxicol. 14, . 347-349 (pan I), 493-501 (part 11) (1976). . ' V "';' [22] Remolds. E. S.. U. T. Moslen, S. Szabo, R. J. Jaeger, S. D. Murphy:' :\i. - ! Hcpatotoxicity of vinyl chloride and 1,1-dichIoroethylcne. Am J. Pathol. 81,219- : 2 >. 236 (1975). ;. ; V' -. > ?.- ' ; [23] lan Dtturen. B. L.: On the possible mechanism of carcinogenic action of vinyl chloride. Ann. N. Y. Acad. Sci. 246, 258-267 (1975). ;v ' [24] Watanahe. P. G.. G. R. McGowan. P. J. Geitrittg: Fate of ,4C-vinyI chloride after single oral administration in rats. Toxicol. Appl. Pharmacol. 36, 339-352 (1976). ' v. j/ . ..,[25] Watancbe. P. G..J. D. Young. P. J. Gehrittg: The importance of non-linear (dose- V;'.`t ,, ; . 7 : ` dependent) pharmacokinetics in hazard assessment. J. Env. Pathol. Tox. 1, in :.V 7 .5 : `press. . " ' ' j"'.' , .-`-e. [26] IViiltey. J. R.: Pharmacodynamics and uptake of vinyl chloride monomer , administered by various routes to rats, J. Toxicol. Env. Hlth. 1, 381-394 (1976). ' , . ":S. ... .; .` - - . ^ \;r \;-yV- :Z- ' /. . , - . ' -;r; ", References of the author: Dr. A Buchter M.D., Dr in industrial medicine Institute and polyclinic, for Industrial-;an,d..c.iHt^ri Social medicine of thej University "of .Cologne'* .. Joseph-Stelzman Str;' ?.,5000,Kdln(Cologne)41. ' .V--. ' " :* .. 4 . ; 'K j 1 ,. t "j ,i.' *" ' ' -*** - * * . t v` ! 1 ' 7 '\`7* " " *> ' V 7. i - ' Amchrift tier Verfitsser: ' - --- > Dr. r.ted. A. Buchter, Aret fur Arbeitsmedizin, lnstitut und Poliklinik fur Arbcits- ; und Sozialmedizin dcr Univcrsitat zu Kbln, Joscph-Stelzmann-Str. 9^ 5000 Kdln 41 ,.\i 124 , -z-j-- - -'x- . -I r5*fjT,T'*V- -.`-7 j * - j;*,.. J' ,' . a :: .-,-.; r,'j- 7 . } ... * *4 Ii v -1; - 7,.: v 2;.* L r- - : ___ ,,r* J ; j ^ SL 041742