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s/ro * MUTAGENICITY OF WASTE PRODUCTS FROM VINYL CHLORIDE INDUSTRIES U. Rannug, C. Ramel Environmental Toxicology Unit, Wallenberg Laboratory, Univeisiiy of Stockholm, Sim.kliolm. Sweden The byproduct from vmvl chloride production, I DC-tar, n o complex m\:ure oh am/v short-chained chlorinated aliphatic hydrocarbons. This mixture has been tested tor mutagenicity by means of Ames' b>e\mone\\.i/nianimaltan miernstnne method. same most of the components m the tar ate poorly soluble m water, three auenls lane ttsed as soli Tnfs or rntulsl/ler ethanol, ItMstl, and I s\ een SO. I he results tsilh all these agents showed that I.UL-tar contains dncct as well as indirect mutagenic tore st tuents. It could he concluded that the mutagenic effect observed in the test could not he due to any significant es tent to one of the main components, ethylene dkhtoride 1.2-dichlorncthane). This substance showed a weak mutagenic effect, but only at higher concentrations than could be available in the highest concentration tested of the tar, Although the microsomal system enhanced the mutagenicity both of the EOC-tar and of i.2-dicldoroethune, this enhancement was dependent on NADPH in the case of tDC-tar but independent of NADPH with t,2-dich/oroethane. The Salmon ell a/momma/ion microsomc method Seems to be a suitable tool tor both mutagenicity screening of complex chemical mixtures and identification of mutagenic constituents in such mixtures. INTRODUCTION The carcinogenic effects of vinyl chloride on humans and on laboratory animals have recently been reviewed (Haley, 1975). The mutagenic effect of this substance is also well documented (Rannug et al., 1974, 1976; Bartsch et al., 1975; Malavcille et al., 1975; Lopfieno el al., 1976) and has been reviewed by Bartsch and Montcsano (1975). These harmlul properties This mscMigation was supported by grants Irom the Swedish Hoard for fcihmc.it Develop ment and Ihc National Swedish Lnvnonmcni I'roieclinn Hoard A preliminary report of this work was presented at the fifth annual nicetinR of ihc European Environmental Mutagen Society, Fircn/c, Italy, 1975. The author-* arc grateful to Drs S jensen, l , Renberg, and (., A. W.ichimnstcr, W.ihcnuerg Laboratory, for `clpfui discussions and criticism, arid to Mrs. A, Medcnstedt and Miss A. Su-.JvaH for their jcchn-c.: assisiancc. Wo also gratefully acknowledge chemical analyses performed tor tin*, investigation lw 11. I.msson, Wallenberg lahoi.iuny, Requests a'lirinis should be sent to U, Rannug, Wallenberg Laboratory, Lilia T'cs^ali, 5-104 05 Stockholm 50, Sweden. ;oi9 journal of Toxi eulogy and Environmental Health, 21019-1029, 1977 Copyright 1977 hy Hemisphere Publishing Corporation 1020 U. KANNLKj AND C. KAMLL together with its enormous annual production worldwide has nude vm>l chloride a serious environmental problem. The situation has been most obvious at the level of occupational health, since the predominant risk has been exposures in vinyl chloride and PVC plants. The circumstances mentioned above therefore make the manu facturing processes of vinyl chloride of interest. Since the final product is mutagenic as well as carcinogenic, it is of importance to know whether other products formed in the processes could have the same properties. When vinyl chloride is synthesized either from acetylene or ethylene or from a mixture of these substances, a tarlikc by-product is formed, called EDC-tar. This name lor the by-pioduct oiigmatcs fiom one of the main components, ethylene bichloride or l ,2-dichlorocthanc (Jensen eta!., 1970, 1975). EDC-tar is, however, a complex mixture, mainly consisting of chlor inated, aliphatic hydrocarbons; a detailed chemical analysis of the con stituents has recently been done (jensen ct al., 1975), Another factor that renders EDC-tar important Trom an environmental point of view is the fact that until recently it has been dumped in large quantities in the North Sea (Jensen ct al., 1970). Reports dealing with toxicity, accumulation, and excretion of EDCtar among marine invertebrates and fishes have been published (Jernelov et al., 1972; Braaten et al., 1972; Rosenberg et al., 1975; Jensen et al., 1975). Toxic effects on the bacterium Escherichia coli have also been studied (Hagstrom and Normark, 1974). So far, however, no mutagenicity testing of EDC-tar has been reported. The mutagenicity test adopted in the present study is the Saimonellalmammalian microsome method (Ames ct al., 1973, 1975), This method has proved to be a very useful tool in mutagenicity testing and in carcinogenicity screening. A recent study of 300 carcinogens and noncarcinogens of a wide variety of chemical types has revealed a high correlation between carcinogenicity and mutagenicity. It was shown that 90% of the carcinogens were mutagenic in the test (McCann ct al., 1975a; McCann and Ames, 1976). The screening of chemicals for mutagenicity and carcinogenicity has mostly been performed with defined chemicals. When dealing with chemi cals in the evironment, however, one often has to consider complex mix tures rather than identified substances. The Salmonella microsomal test system has been used successfully for detection of the mutagenic activity of cigarette smoke condensates (Kicr ct al., 1974). One of the purposes of the present study was to use EDC-tar as a model for the procedure of mutagenicity screening of complex industrial waste products. EDC-tar is suitable in this respect because it exhibits some of the characteristics and problems often encountered in this connection--it is highly com plex, with many constituents poorly soluble in water. Because of the latter problem, a comparative investigation was performed with different solvents. MUTAGENICITY OF WASTE PRODUCTS FROM VINYL CHLORIDE INDUSTRIES 1021 In order to identify separate components responsible lor a mutagenic effect of such complex mixtures, the results with the whole mixture should be followed up by successive testing of ("ructions of the complex down to single compounds. This is being done at this laboratory in cooperation with S. jensen in the division of analytical chemistry. The aim of investigation of EDC-tar at this stage, however, has been to gain some experience in using the 5u//jfonc7/u/mammalian microsomc test for screening complex mixtures rather than identifying separate mutagenic constituents that may occur. As one of the main components is 1,2-dichlorocthanc (approximately 35%) this compound has been studied in some detail separately. This chemical is in itself a very impor tant industrial product also used as an additive in gasoline and has shown weak mutagenic effect m uthei iincsiigaiionx (Uuenbeig el al., 1(374; Brem cl al., 1974; McCann ct al., 1975b). MATERIAL AND METHODS Test Compounds 1 he EDC-tar used was obtained from Kenu Not'd, Stenungsund, Sweden. The content of vinyl chloride monomer in the tar was approxi mately 0,06% according to GC-analysis carried out by the division of analytical chemistry at Wallenberg Laboratory. Ethylene dichloride (1,2dichloroethane) was obtained from BDH Chemicals Ltd. Methylmcthane sulfonate (MMS) was from Ega-Chemie KG and 2-aminoanthraccne from Aldrich. Solvents Ethanol, 95%; dimcthylsulfoxide (DMSO), spectrophotometric grade (Merck); and polyoxyethylene sorbitan mono-oleate (Tween 80) (Hopkin & Williams) were used for EDC-tar, but only DMSO was used for 1,2dichloroethane. Bacterial Strain and Metabolizing System Salmonella typhimunum strain TA 1535 has been used earlier by our group (Rannug et al., 1974, 1976) and was originally kindly provided by B. Ames. For a detailed description of the strain, see Ames ct al. (1973, 1975). The 9,000 x y liver fraction was prepared from male rats (strain R bred at our laboratory) maintained on normal did. The animals were starved 16-20 hr before they were sacrificed. The livers were washed and minced in 0.15 M KCI and then homogenized in three volumes of 0.15 M KCI (3 ml/g wet liver). No drug was used to induce liver enzymes for these experiments. The microsomal system consisted of three-tenths of the 9,000 x y supernatant and seven-tenths of an NADPH generating system SL 1019^7 10i2 U. KANNUG AND C. KAMEL. | NADP, glucose fi-phosphaic, MgCI2, KCI, and phosphate bufler(pl l 7.4)]. In the series without NADP a corresponding volume of phosphate buffer was used (see Rannug ct al., 1974). Test Procedure EDC-tar was added to ethanol, DMSO, or Tween 80 to give the con centrations indicated in figures and tables per 0.1 ml. In the case of Tween 80 the tar was emulsified in 10% Tween 80 (v/v in 0.9% saline). It should be mentioned, however, that with ethanol the highest concentration of the tar did not dissolve completely. Portions (0.1 ml) of the solutions or emulsions were added together with 0.5 ml of the miirosont.il system or phosphate butter and 0.1 ml of hactciia to 2 ml soli agar (0,6% agar in 0.9% NaCI) and poured onto the plates. The bacterial suspension was either an undiluted overnight culture in complete medium (Antibiotic medium 3) or an appropriate dilution in 0.9% NaCI of the same culture. In the first case the soft agar was poured onto minimal medium (Vogel and .Bonner, 1956) with supplements according to Ames (1971). Five plates were used per concentration and control. When a diluted hucteiiul suspension was added, Lhrec plates with complete medium were used instead. The latter plates give the number of surviving cells and the plates with minimal medium the number of mutants after 24 and 48 hr incuba tion, respectively, in the dark at 37C. The numbers given in figures and tables are thus mean values of three plates for survival measures and five plates for mutations. For positive controls MMS was used in experiments without a micro somal system and 2-aminoanthracene was used when the metabolizing system was included. RESULTS The toxic and mutagenic effects of EDC-tar on S. typhimurium TA 1535 are summarized in Table 1. Tested directly without a microsomal system, EDC-tar exhibits both a toxic and a mutagenic effect in all three solvents. With ethanol and DMSO survival is reduced to about 40% with the highest concentration of EDC-tar tested (900 pg per plate). The number of mutants per plate rises to about five times the control value. When the tar is emulsified in 10% Tween 80 it docs not cause the same reduction in survival. Only 30% of the bacteria were killed with the highest concentration in this ease. The corresponding increase in number of mutants per plate was more than tenfold. When EDC-tar is tested in the presence of the microsomal system the results pualitatively leseiuble those obtained without a metabolizing system; that is, a toxic as well as a mutagenic ellect is pioduced. Also, in this senes the toxic ellect with Tween 80 differs liom that with the otlici two solvents. Under these conditions, however, a slionger_ioxk action SL 101938 \ML 1)1. ilfcr conveen >uld II of is or n or >t in WJS Hilic I ure. ogel Five e-rial used Fites uba- hree ero ding TA `trial 'iree with The due. uine the iber the 'em; this .her lion MUTAGENICITY OF WASTE PRODUCTS FROM VINYL CHLORIDE INDUSTRIES 1023 TABLE 1. I' f fee t of f'DC-ljf in Different Solvents on S iJhnnttcfla typhirrwriufn f A f S 3 S {ll.iscp.nr Substitution) with ,uul without Microsomal S>s!vm Solvent Dose plate) Without microsomal sysiem Survival (%i No. of mutants per plate i SE With marosotn.il system Survival (%) No, of mutants per pUlc SL 0 100 100 85 Ethanol 300 95 000 69 900 42 10.4 * 1.33 16.2 t 1.98 27.0 i 3.22 46.8 * 2.08 55.4 t 4.65 100 15.0 s 1.41 104 29.6 i 2.36 101 76.4 _* 2.50 77 124.2 t 4.48 46 1 12.4 t 14.98 DMSO 0 100 100 94 300 80 600 58 900 40 13.6 '161 19 2 * 2.89 18.6 t 1.69 52.0 i 4.83 63.8 t 1.65 1 till 17,' 1 t.` 98 31.6 ` 3.31 95 78.0 i 6.46 69 119.6 i 5.21 52 137.0 i 3.81 I wetn 80 0 100 300 600 900 100 94 87 79 69 7.4 > 0.9J 14.6 , 1,08 29.6 < 1.86 53.0 *. 2.47 83.2 t 3.76 9S IS,2 ' 1.02 89 90,2 * 6 22 74 1 12.4 ' 1.38 53 1 16.6 t 6,38 20 116.8 * 4.84 occurs with Tween 80. Only 20% of the bacteria survive 900 pg EDC-tar with Tween 80, while ethanol and DMSO both give approximately 50% survival. The effect of the liver microsomes on the survival with EDC-tar is not the same with all the solvents. With ethanol and DMSO the addi tion of liver microsomes seems to decrease the toxic effect by the tar, while the opposite is true with Tween 80. From Table 1 it can be seen that the mutagenic effect of EDC-tar is greatly enhanced by the microsomal system. This holds true for all three solvents. The lowest concentration of EDC-tar tested (100 pg per plate) differs in all cases significantly from the corresponding controls (p < 0.01). The enhancement of the mutagenic effect of EDC-tar caused by the microsomal system is also illustrated in Fig. 1, where the effect of EDC-tar in a normal metabolizing system with all necessary co factors is compared with the corresponding effects in a microsomal system where NADP has been omitted. EDC-tar causes little or no killing in the incomplete metabolizing system, but if the system is made complete by adding NADP the situation changes. In the latter case the number of surviving cells decreases strongly with increasing EDC-tar concentration, resulting in approximately 30% survival for the highest concentration in the experiment shown in Fig, 1. Further more it is evident that the NADPH-dcpcndcnt functions in the metabo lizing system arc necessary for the high mutagenic effect. In this SL IOI939 1024 U. RANNUG AND C. RAMEL portico!,ir experiment the maximum number of mounts, within the con centration range, was approximately 200 per plate. The main component, 1,2-dichloroethane, has been tested in the con centration range 5-45 ^amol per plate (Fig. 2). This substance, however, docs not show the same pattern as the entire EDC-tar in this mutagenicity test. Irrespective of whether 1,2-dichlorocthanc is tested in the presence of a complete or an incomplete microsomal system-that is, with or without NADP or in a system where the liver fraction has been replaced by a KCI solution--the survival curves largely have the same slope (Fig. 2). The highest concentration, 45 /amol per plate, reduces the survival approximately 30% compared with the corresponding control. To attain a high mutagenic effect, on the other hand, the presence of the liver fraction is necessary, although the NADP addition has no influence on MfiUKT 1 ! "'it. ,mt| nml,ii;enn. i fleets nt ||)( |,,ron Ivphimnt mm IA I S IS Pdir subsli(ui'iin) m the presenee ol ,t miuosumal system with () nr without ( > NADI*. solvent used :n this espeiintent w.o DMSO. }hc SL 101940 MUTAGENICITY OF WASTE PRODUCTS FROM VINYL CHLORIDE INDUSTRIES 1025 FIGURE 2, Toxic and mutagenic effects of 1,2-dichloroethane (ethylene dichlondc) on Salmonella lyphimurium TA 1535 (base-pair substitution) in the presence of a microsomal system with () or without (o) NADP or in a system with NADP but without the 9,000 X g liver fraction (*). the mutagenicity. Under these circumstances the highest concentration resulted in approximately 125 mutants per plate. In the absence of liver fraction the corresponding number was 25 mutants per plate. DISCUSSION The by-product from vinyl chloride production, EDC-tar, is a com plex mixture of mainly short-chained chlorinated aliphatic hydrocarbons (Jensen ct al., 1970). From the present results it is evident that this mix ture includes direct as well as indirect mutagenic constituents. Since most of the components arc poorly soluble in water, three agents were used as solvents oi emulsifier: ethanol, DMSO, and I'wcen 80. Qualitatively SL 101941 1026 U. RANNUG AND C, RAMEL no difference was found between the solvents used; that is, the mutagenic effect was apparent in all series. Quantitatively, however, some differences in both survival and mutagenicity, were obtained. These differences between the solvents can have many causes, for instance different effects on the permeability of the bacterial cell wall or various interactions with the microsomal system (see Rannug et al., 1975). Concerning the effects of the individual solvents on the mutagenicity of EDC-tar, it is evident from the results both with and without liver microsomcs that ethanol and DMSO are quite similar. Tween 80, on the other hand, deviates from the other two agents. This is particularly true with the microsomal system present. Ethanol and DMSO give an expected dose-effect relation, while with Tween SO the mutagenicity vaiies only slightly (between 90 and 117 mutants per plate) from the lowest to the highest dose of the tar, indicating that a plateau has been reached around the lowest dose. This plateau may occur because Tween 80 causes more severe damage to the metabolizing system, resulting in stronger inhibition, at least with the concentrations used in these experiments (Rannug el al., 1975). Tween 80 deviates from the other solvents in another respect also. The effect of the lowest dose with liver microsomcs is clcaily higher than with ethanol or DMSO. Apparently Tween 80 affects the availability of the test compound to the action of the microsomal enzyme system. It should, however, be emphasized that independently of the solvents used, an induction of mutations could be detected. When it comes to interpreting the mutagenic constituents it is clear that one or several directly acting mutagens are present. The enhance ment of mutagenicity with liver microsomes furthermore points to the' fact that one or several constituents are converted into mutagens by the liver microsomal fraction. This biotransformation can occur cither through the microsomal enzymes of the mixed function oxygenase category or by other enzymatic or noncnzymatic processes. Biotransformation through the mixed function oxygenase enzymes can be inferred from the fact that it is NADPH-dependent, To analyze the process through which chemicals arc activated into mutagens by the liver microsomal system, tests should therefore be performed both with and without NADP in the NADPH-generating system. The usefulness of this NADP control was evident in the present investigation. The enhancement in mutagenicity found with total EDC-tar was shown to be NADPHdependent (Fig. 1). The corresponding enhancement of the muta genicity of 1,2-dichlorocthane by the microsomal system, on the other hand, turned out to be independent of NADPH. Further conclusions can also be drawn from these results. Although 1,2-dichloroethane is one of the main components of EDC-tar, constituting approximately 35% of the total (Jensen et al., 1975), its contribulion to the mutageniiity ofthe i.n must be miller limited. Appioximalely 3 /zmol ol I,dii IiIoioi lb,me r, |iie .eu| m the lughr.l < onumlialioii of I.UC-!ai, SL 10X942 MUTAGENICITY OF WASTE PRODUCTS FROM VINYL CIILORIOE INDUSTRIES 1027 but the lowest concentration of 1,2-dichlorocthanc tested, 5 mmol per plate, is not significantly different from the corresponding control value under any of the conditions shown in Fig. 2. From the data presented in Table 1 and Fig. 1 it is evident that the mutagenic effect of EDC-tar with out NADP is as low as, or even lower than, the corresponding value without a microsomal system. This is a further indication that the contribution of 1,2-dichlorocthanc to the mutagenicity of total EDC-tar is negligible. The behavior of 1,2-dichlorocthane in the mutagenicity test will, however, be further discussed in another context (Rannug and Ramcl, in preparation). Of oilier compounds that could he lespousihle foi the muiauenii.il y of LIX'-lar, vinyl ihloiidc itscll should also he consideied. As mentioned above, EDC-tar contains about 0.06% of the vinyl chloride monomer. Now, according to Bartsch ct al. (1975) the lowest dose of vinyl chloride tested in Salmonella was 0.2% in air, which cot responded to 4 X 10 ~5 M in the substrate. "I his is a ten times higher concentration of vinyl chloride than in our experiment with the highest dose of LDC-lar, and judging from the data presented by Bartsch ct al. vinyl chloride in this concentration can not be expected to give a measurable increase of mutations. It can therefore be concluded that the contribution of vinyl chloride to the mutagenicity of EDC-tar is negligible. The mutagenic effects discussed in this paper were detected in the concentration range 100-900 Mg per plate. In terms of parts per 106 (ppm) it would equal 40-300 in the top agar layer or 4-40 if calculated on the whole agar medium content of a plate. Concentrations of EDCtar at which other biological effects arise have been reported by several authors, Jernelov ct al. (1972) found an increased frequency of cmitotic cells in Allium cepa at concentrations from 1-50 ppm. Acute toxicity in sonic marine animals gave LCS0 values ranging from 2-20 ppm (see Jensen et al., 1975). Effects on l4C fixation of phytoplankton were found at even lower concentrations. Rosenberg et al. (1975) have shown, however, that there can be differences in acute toxicity between Swedish and Norwegian EDC-tar, In their experiments the Swedish tar was approximately nine times more toxic (48 hr LC50) to shrimps (Crangon crungon). They also point out that the composition of EDC-tar varies not only from factory to factory but also from time to time from the same factory. These circumstances together with different ways of exposing the test organisms to the EDC-tar-that is, momentary or successive exposures--give rise to discrepant results con cerning LCjo as well as other biological parameters. Considering the cause of the toxicity of EDC-tar, Hagstrom and Normark (1974) conclude Irom their work on E. coli that the killing effect is associated with a decreased stability ol the cytoplasmic mem brane, and they ihoicloic suggest an interaction with the membrane. I liese liinlings seem to lie m good agieement with the stiong tendency of EDC-tar to adhere to particles noted by )erncldv ct al. 11972). 1028 U. KANNUG AND C. KAMEL Anolhcr conclusion tlr.iwn by H.i^stiom ,imJ Norm.uk (1974) ,is well .is Rosenberg et ul. (1975) is lh.it neither of the two m.iin components 1,2dichloroethanc or 1,1,2-trichlorocthane could be responsible for the biological effects caused by EDC-tar. This is in good agreement with the results reported here, which show that 1,2-dichlorocthanc gives a different mutagenicity pattern when tested as a pure chemical than does total EDC-tar. The appearance of both 1,2-dichlorocthane and 1,1,2-trichloroethane in the mutagenicity test with Salmonella and microsomal system is, however, different from that of EDC-tar (Rannug and Ramcl, in preparation). In conclusion, it should be emphasized that the ,Wmo/;('//i;/mumnulian miciosomc method seems to be a suitable tool for both muta genicity screening of complex chemical mixtures and identification of mutagenic constituents in such mixtures. It may be pointed out that the limited detoxification action in the indicator organism, Salmonella, is an advantage in this connection. It makes it possible to manipulate experimentally with the liver microsomal system in order to elucidate the process of metabolic conversion of the chemicals to mutagens. REFERENCES Ames, 8. N, 1971. The detection of chemical mutagens with enteric bacteria. In Chemical mutagens: Principles and methods for their detection, vol. 1, ed. A. Hoilaender, pp. 267-281. New York: Plenum. Ames, 8. N., Ourston, W. E., Yamasaki, E. and Lee, F. D. 1973. Carcinogens are mutagens: A simple lest system combining liver homogenates for activation and bacteria for detection. Proc. Nath Acad, Sci. US.A, 70:2281-2283. Ames, B. N., McCann, J. and Yamasaki, E. 1975. Methods for detecting carcinogens and mutagens with the .Su/mone/Zu/mammahan-microsome mutagenicity test. Mutat. Res. 31:347-364. Bartsch, II. and Montcsano, K. 1973. Mutagenic and carcinogenic effects of vinyl chloride. Mutat. Res, 32:9 3-1 14. Bartsch, H., Malaveille, C. and Montesano, R. 1975. Human, rat and mouse liver-mediated muta genicity of vinyl chloride in Salmonella typhimurium strains. Int. /. Cancer 15:429-437, Braaten, B., Mdllcrud, E. E, and Solemdal, P. 1972. The influence of some byproducts from vinylchloridc production on fertilization, development and larval survival on plaice, cod and herring eggs. Aquaculture 1:81-96. Brcm, H,, Stein, A, B. and Roscnkranz, H. S. 1974. The mutagenicity and DNA-modifying effect of haloalkancs. Cancer Res. 34:2576-2579. Ehrcnberg, L., Ostcrman-Golkar, S., Singh, D. and Lundqvist, U. 1974. On the reaction kinetics and mutagenic activity of methylating and d-halogenoclhylaling gasoline additives. Radial. Bot. 15:185-194. Hagstrom, A. and Normark, S. )974. Toxic effect and action of chlorinated by-products from vinyl chloride production on Escherichia coli K 12. Ambio 3:77-79. Haley, T. I. 1975. Vinyl chloride: How many unknown problems? j. Toxicol. Environ. Health 1:47-73. (ensen, S., (crnclov, A., Lange, R. and Palmork, K. M. 1970. In t AO technical conference on marine pollution, TIR:MI'/70 E 88. Rome: I AO. Jensen, S., Lange, R,, lierge, G., Palmork. K. H. and Renherg, L, 1975, On the chemistry of LI)Ct.tr and Us luiilogic.il significance. Prm. R. Soc. I out/. (Ilioij 189:3 13- M(i, si 1-01944 MUTAGENICITY OF WASTE PRODUCTS FROM VINYL CHLORIDE INDUSTRIES 1029 Jernclov, A., Rosenberg, R. and lensen, 5. 1972. Biological effects and physical properties in the marine environment of aliphatic chlorinated by-products from vinyl chloride production, Water Res. 6:1 181-1191. Kier, L. D., Yamasaki, E. and Ames, B. N. 1974. Detection of mutagenic activity in cigarette smoke condensates. Proc. Natl. Acad. Sci. U.S.A. 71:4159-4163. Lopricno, N., Baralc, R., Baroncclli, S., Bauer, C., Bronzctti, G., Cammcllini, A., Cercignani, G., Corsi, C., Gervasi, G., Lcporini, C., Nicri, R., Rossi, A. M., Strctti, G. and Turchi, G. 1976. Evaluation of the genetic effects induced by vinyl chloride monomer (VCM) under mammalian metabolic activation: Studies in vitro and in vivo. Mutat. Res. 40:85-96. Malavcille, C,, Bartsch, H,, Barbin, A., Camus, A. M., Montcsano, R., Croisy, A. and jacquignon, P, 1975. Mutagenicity of vinyl chloride, chlorocthylencoxidc, chloroacctaldchydc and chlorocthanol. Biochcm. Riaphys. Res. Cattunun. 63:363-370. McCann, and Ames. II, N. 1076, Detection ol carcinogens .is mutagens m the s,//ruorn7/u/miciosoioe test: Assay of J00 chemicals. Discussion. Proc. Nall. Acad. Sci. U.S.A, 73:950-954. McCann, Choi, E., Yamasaki, . and Ames, B. N. 1975a. Detection of carcinogens as mutagens in the Salmonella/microsome test; Assay of 300 chemicals. Proc. Natl. Acad. Sci. U.S.A, 72:5135-5139. McCann, J., Simmon. V., Streitwieser, D. and Ames, B, N. 1975b. Mutagenicity of chloroacetaldehyde, a possible metabolic product of 1,2-dichlorocthanc (ethylene dichloridc), chlorocthanol (ethylene chlorohydrin), vinyl chloride, and cyclophosphamide, Proc. Natl. Acuil. Sci. U.S.A. 72:3190-3193. Rannug, U., lohansson, A,, Ramcl, C. and Wachtmeister, C, A. 1974. I he mutagenicity ot vinyl chloride after metabolic activation. Ambio 3:194-197. Rannug, U., Hedenstedt, A., Arrhenius, E. and Zetlcrqvist, M*A, 1975, Influence of solvents on the metabolizing system used for the detection of mutagenic intermediates. iUPHAR satellite symposium on active intermediates: Formation, toxicity and inactivation, Turttu, Finland, abstracts, p. 43. Rannug, U., Gothc, R. and Wachtmeister, C. A. 1976. The mutagenicity of chlorocthylene oxide, chloroaceuldehyde, 2-chloroethanol and chloroacetic acid, conceivable metabolites of vinyl chloride. Chem, Biol. Interact. 12:251-263, Rosenberg, R., Grahn, O. and Johansson, L. 1975. Toxic effects of aliphatic chlorinated by-products from vinyl chloride production on marine animals. Water Res. 9:607-612. Vogel, H. J. and Bonner, D. M. 1956. Acctylornithinase of Escherichia coli: Partial purification and some properties.;. Biol. Chem. 218:97-106. Received /uly 6, 1976 Accepted October i 7, 1976 SL 101945