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Mutation Research, 33 (1975) 79--86 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

MUTAGENICITY R E S E A R C H AND TESTING IN SWEDEN C. RAMEL

Environmental Toxicology Unit, Wallenberg Laboratory, University of Stockholm (Sweden) (Received June 18th, 1975) (Accepted August 25th, 1975)

Summary A survey is given of Swedish legislation for control of chemicals in the environment. Although no direct legal requirements for mutagenicity testing of chemicals exist at present in Sweden, such requirements can be enforced within the existing laws. Testing and research in chemical mutagenicity are especially performed at the Environmental Toxicology Unit of the Wallenberg laboratory, University of Stockholm. An outline is given of the organization of the unit, which is based on an interdisciplinary cooperation, among divisions of organic and analytical chemistry, cellular toxicology, and genetics. As examples of projects under joint investigation results on polychlorinated biphenyl (PCB) and on vinyl chloride are briefly described.

Introduction The way legislation in Sweden has met the growing concern about genetic risks of chemicals in the environment is very similar to the situation in most other countries -- essentially nothing has been done a b o u t it. There are no legal requirements for mutagenicity testing of any groups of chemicals in Sweden. This may sound disappointing for those who have been working towards a better understanding of the genetic hazards encountered by environmental chemicals. However, the situation is neither that simple nor that dark. Although one may look in vain for any direct legal support of mutagenicity testing in Swedish official regulations, the whole legislation concerning chemicals in the environment has recently been changed rather thoroughly. The genetic risks of chemicals have received a comparatively wide public recognition, and they are referred to in many official connections. In governmental commissions set up for various specific issues such as herbicides, pesticides, nitrite, NTA and heavy metals, geneticists have regularly been included or at least consulted. Abbreviation: PCB, polychlorinated biphenyl(s).

80 The legal aspects of mutagenicity testing in Sweden can hardly be d e a l t with as a separate item, but t h e y must be considered in connection with the legislation for administrative control of chemicals in the environment. It may therefore be appropriate to give a short survey of the development in Sweden within this general area of legislation. Legislative control o f chemicals As I suppose is the case in other parts of the world, the reaction of administrative authorities, leading to amended legislation for the control of chemicals in the environment, has not come by itself, but as a consequence of particular issues which have focused the attention on specific problems. In Sweden particularly four such issues have played a decisive role with respect to the legislation: mercury, thalidomide, PCB and vinyl chloride. Each of these issues has contributed to a stricter regulation of chemicals. The influence of the mercury pollution in Sweden in this respect has been dealt with previously [16]; the thalidomide problem had an influence similar to m a n y countries. Some words might, however, be said about PCB. PCB as a pollutant in the environment was first identified in 1966 by Dr. S. Jensen. The pollution with PCB was considered alarming both from an ecological and a medical point of view, but it turned out that there was no legal way of solving the situation. PCB was released essentially indirectly from the industries and it could not be classified as a pesticide. In order to solve the problem, a special law had to be passed -- the PCB act of 1972, which regulated the use of PCB. However, that made it clear that the legislation in general was not adequate to meet the chemical pollution problems of modern time. Therefore, a governmental "Environmental Control Commission" was appointed in 1971 with the mission of preparing new legislation for the whole area of environmental pollution. That work resulted in a new law in 1973: " T h e Act on Products Hazardous to Health and to the Environment" [12] which gave the authorities very wide powers to intervene against any product that is, or can be suspected to be, dangerous to h u m a n beings and the environment. The main principles of the law are twofold. First, anyone who manifactures, sells or imports chemical products is responsible for preventing ill effects from the products. The burden of any uncertainty as to the hazard of a product, therefore, does not fall on the public but on those who manufacture and sell it. Second, the authorities are given powers to intervene even on the mere suspicion that a chemical product may be harmful, which means that they do not have to wait until damage has been done. An important point in the law is the fact that the manufacturer or seller must furnish the authorities with all information on the composition and other properties of the product, which is considered necessary for assessing the danger it presents to public health or to the-environment. As most of the legal acts of this kind in Sweden, the law itself only furnishes a framework for the actual regulations and to a great extent it delegates responsibility to administrative authorities who decide the practical applications. It therefore is up to the authorities to decide what tests are required to evaluate the hazards of chemical products. The responsibility for these questions has been given

81 primarily to a new authority created for this purpose, the Products Control Board. The Act on chemical products does not cover some groups of chemicals; namely, foodstuffs, pharmaceuticals, animal feeds, radioactive material and explosives, which are regulated by other laws. In no case is mutagenicity testing imposed directly. However, for pharmaceuticals, the Department of Drugs at the National Board of Health and Welfare, has started to require such tests for special drugs. They also have some laboratory facilities themselves where mutagenicity tests on mice are performed on a limited scale. Some guidelines for mutagenicity testing have been worked out b y the department. When it comes to foodstuffs, the Minister of Agriculture has made a special addition to the act on food products, in which he emphasizes that the evaluation of foodstuffs and food additives should include not only acute toxic effects, b u t also suspected genetic and other long-term effects. The latest issue, which has shaken b o t h the authorities and the general public, is the carcinogenic effect of vinyl chloride in man, which has been reported in several countries since 1973. Cases of angiosarcoma of the liver from an occupational exposition of vinyl chloride have occurred also in Sweden. Work at our and other laboratories has shown that the carcinogenic effect of vinyl chloride could have been detected b y simple mutagenicity tests. As a consequence, a working group has been set up by the National Board of Occupational Safety and Health in collaboration with our laboratory, in order to investigate potential carcinogens in industries primarily with Ames' test system on Salmonella. Within this project our laboratory is also engaged in the development of a method to measure the alkylation in vivo of people exposed to alkylating agents like vinyl chloride [ 15]. In summary it may be pointed o u t that Swedish legislation gives a rather farreaching p o w e r to the authorities to introduce, for instance, compulsory testing for mutagenicity within the existing laws, b u t that so far this has not been realized. One problem in this connection, in spite of the new Product Control Board which is supposed to handle most of the problems, is that there are many authorities involved and there is by no means a united opinion about toxicological testing and the need of mutagenicity screening. Another problem is the fact that a more general mutagenicity screening o f chemicals is hardly possible without furnishing the administrative authorities with a b o d y that has sufficient knowledge for the evaluation of test results. A scientific reference group for mutagenicity, like the one which has been set up in England, as reported by Dr. B. Kilbey in these Proceedings, seems to be highly appropriate. Hopefully, the example will be followed in Sweden. Concerning the industrial manufacturing side, it may be added that the interest for mutagenicity has increased very much the last year, mostly as a consequence of the vinyl chloride problem. The establishment of control laboratory facilities for mutagenicity screening of industrial products has also been discussed.

Research and testing at the university level At the university level, mutagenicity testing and development of screening methods are carried out at several places in Sweden. Of particular importance is

82 the Wallenberg Laboratory at the University of Stockholm, where a special unit, the Environmental Toxicology Unit, has been formed for the purpose of studying long-term toxicological actions of chemicals in the environment, in particular mutagenic and carcinogenic effects. The organization of the unit differs from ordinary o r t h o d o x university institutes in one important respect -- it is based on a direct interdisciplinary cooperation between chemical and biological divisions. The philosophy behind this organization is the obvious need for a joint effort between different scientific fields in order to meet the complexity of the problems concerning the hazards of chemicals in the environment. The Environmental Toxicology Unit was, as a matter of fact; organized under the influence of our experience from the investigation of the mercury problems in the 1960's when a rather unique cooperation between different disciplines grew up. It seems that the academic structure in general at the universities needs a reevaluation for certain areas of research because of the interdisciplinary nature of the problems. Environmental toxicology constitutes a good example of such a research area. From that point of view it may be of some interest to give a short account of the actual organization of the activities at our laboratory and also to give examples of research projects under joint investigation there. Wallenberg Laboratory, University o f Stockholm The Environmental Toxicology Unit comprises two chemical divisions, one in organic chemistry and one in analytical chemistry. There are furthermore two biological divisions, one in cellular toxicology and one in genetics. The Wallenberg laboratory also includes the departments of radiation biology, radiation chemistry and immunology. The radiobiology group is particularly engaged in the biochemistry and genetical effects of alkylation and the radiation chemistry group is working with repair mechanisms of DNA. Both have a close collaboration with the Environmental Toxicology Unit. Joint projects in the Environmental Toxicology Unit make it possible to follow certain lines of investigation among the chemical and biological divisions. Thus the analytical chemical division (which also constitutes the analytical laboratory for the National Environment Protection Board} has functioned as a detector of potentially hazardous compounds in the environment. It collects information on persistent compounds released to the aquatic environment. Compounds which have been traced through monitoring of samples from the environment can be synthesized b y the organic chemical division, b o t h for further chemical work and also for biological investigations. The biological testing in the division for cellular toxicology involves the fate and action of the chemicals on cellular organelles. In particular the enzymatic detoxication process in the liver microsomes is studied. This obviously is of direct relevance from a genetic point of view considering the large group of mutagens, which only act after metabolic activation in the liver. The genetic tests used or under development include the following: (1) Single-strand breaks and repair of DNA, (2) Back mutations in Salmonella with liver microsomes, (3) Host mediated assay,

83 (4) Genetic changes in Drosophila, (5) Mammalian bone marrow cells (micronucleus and metaphase analyses), (6) Hamster fibroblasts (point mutations, chromosomal aberrations, and neoplastic transformation}, and (7) Transformation o f human diploid fibroblast in vitro. PCB

As an example of the course of investigation in collaborative studies of chemicals, PCB may be mentioned. It was discovered as an environmental pollutant ten years ago b y Dr. Jensen, while he was analyzing the levels of chlorinated pesticides in wild animals and human fat tissues [7]. The gas chromatograms contained numerous peaks with retention times that did not correspond to chlorinated pesticides. The peaks were identified as PCB after a considerable a m o u n t of analytical detective work. The further analytical work involved an investigation of the extent o f environmental contamination in different ecosystems b y PCB and as is well known now, PCB turned out to be a b o u t as much of an environmental problem as DDT [8]. However, it was also of importance to characterize the various PCB peaks in the gas chromatograms, representing molecules which differed in the extent and position of the chlorinations. For that purpose a synthesis of about 90 of about 100 possible PCB molecules was performed by the organic chemical division [9,21]. The access to defined PCB molecules enabled the comparison of the PCB content in various samples of animals and humans in order to trace the origin and fate of PCB. It was found that the number of different PCB molecules decreased when moving upward in the ecosystems [6]. That observation indicated that in spite of the long biological life of PCB metabolism of at least some of the PCB molecules occurs. This metabolism was studied in some detail. In particular, the lowchlorinated c o m p o u n d s decreased most, b u t also some of the high-chlorinated c o m p o u n d s were metabolized. The metabolism varied clearly between different animals as revealed b y the analyses of herrings, guillemots and seals in the aquatic ecosystem. The metabolism primarily involved a hydrolysis of the PCB molecules and over 20 hydrolyzed metabolites were identified in seals and guillemots. Since the discovery of PCB as an environmental contaminant, the biological effects have been extensively studied in many laboratories. Concerning mutagenic effects of PCB, there is, to m y knowledge, no conclusive evidence. Tests by us on Drosophila with PCB of mixed degrees of chlorination did n o t indicate any chromosome-breaking effects [14]. However, PCB may have an indirect bearing on mutagenicity and carcinogenicity because it induces the enzymatic detoxication enzymes in the liver microsomes. The division of cellular toxicology has started investigations on the effect on the liver microsomal enzymes by various PCB molecules and also the metabolic hydroxylation of the PCB molecules themselves. For this work a liver perfusion system has been developed in which the metabolic activity of whole livers is studied in vitro. In the genetics division we are trying to develop methods to take advantage of this liver perfusion system in order to expose cells and bacteria to the liver perfusate and measure mutagenic effects of metabolites.

84

Vinyl chloride The investigations of PCB started at the analytical chemistry end through the identification of the c o m p o u n d s in samples from the environment. This led to chemical syntheses and biological investigations. However, a reverse order of events can also occur, when the biological and toxicological data fumish the starting point for a series of investigations involving chemical analyses and syntheses. An example of this is vinyl chloride, which we started to investigate after the reports a year and a half ago that it had caused angiosarcoma of the liver in workers who had been exposed to it [3]. The carcinogenic effect of vinyl chloride was at the same time also revealed b y Maltoni's large experiments on rats and mice [12]. Our experiments with vinyl chloride [ 18] have primarily been performed on back-mutations at the histidine-locus in Salmonella in the presence of liver microsomes according to Ames' test system. The bacteria were exposed to vinyl chloride gas in concentrations from 2% to 20% and with various exposure times. Several experiments were performed which showed a mutagenic effect by vinyl chloride b u t only together with liver microsomes. The results also showed that the presence of NADP was necessary for the mutagenic effect, which indicates that the liver microsomes actively metabolize vinyl chloride to a mutagenic compound. Mutations were induced only in the strain TA 1535, which responds to base substitutions, b u t not to the frame-shift strains. That indicated an alkylating action by the vinyl chloride metabolite(s). It may be added that no effect of vinyl chloride was obtained on mice with cytogenic tests and host mediated assay after exposures of I h to doses of up to 10% vinyl chloride. On the other hand 3 h exposure of male Drosophila to 20% vinyl chloride gas gave a significant increase of recessive lethals in F 1 and F 2 [11]. An important question in connection with the mutagenic effect of vinyl chloride obviously is the identity of the active mutagenic and carcinogenic metabolite or metabolites. This question was handed over at the laboratory to the chemists and physiologists to solve. Ehrenberg, Wachtmeister and their coworkers therefore set up experiments to identify the metabolites produced in the rat liver from vinyl chloride [5]. In order to facilitate the identification of the metabolites they used a thiol, 3,4-dichlorobenzenethiol, as a trapping agent. That c o m p o u n d is acid enough to form a sufficient concentration of negatively charged ions to function as a reactive nucleophilic agent. The presence of the dichlorophenyl group makes it easy to detect the reaction produets by combined gas chromatography--mass spectrometry technique. On the basis of these chemical analyses and other chemical considerations, four possible mutagenie derivatives of vinyl chloride could be identified; chloroethyleneoxide, chloroacetaldehyde, 2-chloroethanol and chloroacetic acid. All four compounds were then tested on Salmonella without liver mierosomes [17]. Chloroethyleneoxide and chloroacetaldehyde were synthesized for that purpose b y Wachtmeister's group in the organic chemistry division. Only chloroacetic acid turned out to be non-mutagenic in the tests. Of the other compounds 2-ehloroethanol had been reported earlier as a mutagen by Rosenkranz [20] b u t it only acted in such high doses in our experiments that it is

85 very unlikely to be responsible for the mutagenic effect of vinyl chloride. Chloroethyleneoxide was found to be b y far the most potent mutagen, about 450 times more effective than chloroacetaldehyde and 10 000--15 000 times more effective than ethyleneoxide, which was used as a positive control. Although it is n o t possible to determine the role of chloroacetaldehyde and chloroethyleneoxide in the mutagenicity of vinyl chloride, there are good reasons to believe that chloroethyleneoxide is the most important factor because of its pronounced reactivity and strong mutagenicity. The results indicate that the mutagenic effect of vinyl chloride primarily depends on an epoxidation by liver microsomes. Such an epoxidation is well known to occur with carcinogenic aromatic hydrocarbons and other compounds. The mutagenic effects of vinyl chloride and its metabolites have been studied also b y Loprieno in Italy [10] and Bartsch and Montesano at IARC in Lyon [1]. Their results are in accordance with ours. Ames and McCann have shown that vinyl chloride acts as a direct mutagen in their new strain TA 100 [13]. It should finally be mentioned that a joint study between Dr. Ehrenberg and Dr. Natarajan at the Wallenberg laboratory and Dr. Lindstens' group at Karolinska Institute in Stockholm performed an investigation on workers exposed to vinyl chloride. They found a significant increase of chromosomal aberrations in the exposed group as compared to the controls [4]. The vinyl chloride story draws one's attention to the chemicals in polymer industries where there m a y be more mutagenicity problems of a similar nature. The waste products from the polyvinyl chloride industries, the so called ethylenedichloride tars, have been d u m p e d in large amounts in the North Sea in Europe. Dr. Jensen at our laboratory has been engaged in analyses of these products [2]. In cooperation with his group we have started n~utagenicity studies of that tar and we have found that it contains b o t h compounds that are mutagenic directly on Salmonella and on those that need activation by liver microsomes [19]. By a succession of chemical fractionation and mutagenicity analyses, we hope to be able to identify the mutagenic factors present in that waste p r o d u c t from the polyvinyl chloride industry. References 1 Bartsch, H., C. Malaveille and R. Montesano, Human, rat and mouse liver-mediated mut a ge ni c i t y of vinyl chloride in Salmonella t y p h i m u r i u m strains, Int. J. Cancer, 15 (1975) 429--437. 2 Berge, G., S. Jensen, R. Lange, K.H. Palmork and L. Renberg, On the chemistry of EDC-t~Lr and its biological significance in the sea, Proe. R. Soc. Lond. B, (1975). 3 Creech, J.M., and M.N. Johnson, Angiosarcoma of liver in the m a n u h c t u r e of pol yvi nyl chloride, J. Oce. Med., 16 (1974) 150--151. 4 Funes-Cravioto, F., B. Lambert, J. Lindsten, L. Ehrenberg, A.T. Natarajan and S. Osterman-Golkar, C h r o m o s o m e aberrations in workers exposed to vinyl chloride, Lancet, 1 (1975) 459. 5 ODthe, R., C.J. CaUeman, L. Ehrenberg and C.A. Wachtmeister, Trapping with 3,4-dichlorobenzenethiol of reactive m e t a b o l i t e s formed in vitro from the carcinogen vinyl chloride, Ambio, 3 (1974) 234--236. 6 Jansson, B., S. Jensen, M. Olsson, L. Renberg, G. Sundstr~m and R. Vaz, Identification by GC-MS of p h e n o l i c metabolites of PCB and p,p'-DDE isolated from baltic guillemot and seal, Ambio, 4 (1975) 93--97. 7 Jensen, S , Chlorinated b i p h e n y l s in nature, No~lisk Bioeid-Information, 7 (1966). 8 Jensen, S., The PCB Story, Ambio, 1 (1972) 45---53. 9 Jensen, S., and G. Sundstrdm, Structures and levels of mos t chlorobiphenyls in two technical PCB p r o d u c t s and in h u m a n adipose tissue, Ambio 3 (1974) 70--81. 10 Loprieno, N., R. Barale, S. Baroncelli, C. Bauer, G. Bronzetti, A. Cammelini, G. Cercignani, C. Corsi, G. Gervasi, C. Leporini, R. Nieri, A.M. Rossi, G. Stretti and G. Turchi, Evaluation o f the genetic effects by vinyl e h l o ~ d e m o n o m e r (VCM) under the influence of liver microsomes, Submitted.

86 11 Magnusson, J., and C. Ramel, Mutagenic effect of vinyl chloride on Drosophila melanogaster, T o be pubHshed. 12 Maltoni, C., and G. Lefemine, Careinogenicity bio-assays of vinyl chloride, I. Research plan and early results, Environ. Res., 7 (1974) 387--405. 13 M c C a n n , J., V. S i m m o n , D. Streitwieser and B. Ames, Mutagenicity of chloroacetaldehyde, a possible metabolic product of 1,2-dichloroethane (ethylene dichloride), chloroethanol (ethylene chlorohydrin), vinyl chloride and cyclophosphamide, Proc. Natl. Acad. Sci. (U.S.), in press. 14 Nflsson, B., and C. Ramel, Genetic tests o n Drosophila melanogaster with polychlorinated biphenyls (PCB), Hereditas, 77 (1974) 319--322. 15 Osterman-Golkar, S., L. Ehrenberg, D. Segerbiick and I. H~11str/Sm, Evaluation of genetic risks of alkylating agents. If. Haemoglobin as a dose monitor, Mutation Res., in press. 16 Ramel, C., The mercury problem -- a trigger for environmental pollution control, Mutation Res., 26 (1974) 341--348. 17 Rannug, U., R. G~Jthe and C.A. Wachtmeister, The mut a ge ni c i t y of ehloroethylene oxide, ehloroacetaldehyde, If. Chloroethano] and chloroacetic acid, conceivable me t a bol i t e s of vinyl chloride, Chem. Biol. Interact., in press. 18 Rannug, U., A. Johansson, C. Ramel and C.A. Wachtmeister, The mut a ge ni c i t y of vinyl chloride after metabolic activation, Ambio, 3 (1974) 194--197. 19 Rannug, U., and C. Ramel, The mutagenicity of waste products from vinyl chloride industry. Subm i t t e d to Europ. Envir. Mut. SDe. 5th Annual Meeting, Florence, Oct. 19--22, 1975. 20 Rosenk~anz, H.S., and T.J. Wlodkowski, Mutagenicity of ethylene chlorohydrin. A degradation p r o d u c t present in foodstuffs exposed to ethylene oxide, J. Agr. Food Chem., 22 (1974) 407---409. 21 Sundstx~m, G., and C.A. Wachtmeister. Synthesis of 14C-labelied and uniabelled PCB c ompounds , PCB Conference II. Natl. Swedish Env. Prot. Board Publ., 1973: 4E, 73--86. 22 The Act on Products Hazardous to Health and to the Envi ronme nt ; The New Legislation with a C o m m e n t o r y , Royal Ministry for Foreign Affairs, Royal Ministry of Agriculture, Allm~inna F~rlaget, G~teborg, 1972.

Mutagenicity research and testing in Sweden.

A survey is given of Swedish legislation for control of chemicals in the environment. Although no direct legal requirements for mutagenicity testing o...
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