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MUTAGENICITY OF WASTE PRODUCTS FROM VINYL CHLORIDE INDUSTRIES U. Rannug, C. Ramcl Environmental Toxicology Unit, Wallenberg Laboratory, Univmity of Stockholm, Stockholm, Swollen The by-product from vinyl chloride production, I DC-tar, is a complex mature of -uin/y short-chained chlorinated aliphatic hydrocarbons. This mixture has been tested tor mutaacnicity by means of Ames' Salmonclla/niurnroo/ion microsome method. C/ilie most of the components in the tar ate poorly soluble in water, three darn!. in7r used us solvents or enudsitiei: ethanol, DM'stt, and I ween HO, /he lesufts ssith ail these uqents showed that I.UC-tur contains direct as well as indirect mutagerm tonsttuents. It could be concluded that the mutagenic effect observed in the test could not he due to any significant extent to one of the main components, ethylene dichloride 1,2-dichloroethane). 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 HDC-tar and of t.J-dichlorocthanc, this enhancement was dependent on NADPH in the case of tDC-tar but independent of NADPH with 1,2-dichloroethane. The. S>s\moncUa/mammalian microsome 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). Th*c mutagenic effect of this substance is also well documented (Rannug ct al., 1974, 1976; Bartsch ct al., 1975; Malavcille et al., 1975; Lopricno el al., 1976) and has been reviewed by Bartsch and Montesano (1975). These harmlul properties This investigation was supported by grants from lho Swedish Hoard for Icilinical Develop ment and tile National Swedish Environment Protection Board. A preliminary report of this work was presented at the fifth annual meeting of the European Environmental Mutagen Society, Eircn/c, Italy, I97S. The authors arc grateful to Drs. S. Jensen, t. Kenherg, and I.. A. W.ichlineister, Walicnbcig Laboratorv, for cclpful discussions and criticism, and to Mrs. A. IIcdcnstcdl and Miss A. Sur.dvall for their (echo-cassistance. We also gralclully acknowledge chemical analyses pcrlornicd lor this investigation bs Dr. It, lanssim, Wallenberg Labuiatory. Requests tor reprints should be sent to U. Rannug, Wjllenbcrg Laboratory, Lilia frcstali, S-l04 05 Stockholm 50, Sweden. ;oi9 journal of Toxicology and Environmental Health, 2:1019*1029, 1977 Copyright O 1977 by Hemisphere Publishing Corporation 1020 U. KANNUG AND C. KAMEL together wiill its enormous annual production worldwide has made vinyl 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 tarlike by-product is formed, called EDC-tar, This name lor the by-pioduct oiiginalcs fiom one of the main components, ethylene dichloridc or 1,2-dichloroelhanc (Jensen ctal., 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 from an environmental point of view is the fact that until recently it has been dumped in large quantities in the North Sea (Jensen ct at., 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 Salmonella/mammalian microsome method (Ames ctal., 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. SL 041697 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 fractions 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 Salmonella/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-dichlorocthane (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 in other investigations (Lhienbeig et al., 1974; Brem et al., 1974; McCann et al., 1975b). MATERIAL AND METHODS Test Compounds 'I he EDC-tar used was obtained Irom Kema 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 (1,2dichloroethanc) was obtained from BDH Chemicals Ltd. Methylmethane sulfonate (MMS) was from Ega-Chemie KG and 2-aminoanlhraccne from Aldrich. Solvents Ethanol, 95%; dimcthylsulfoxidc (DMSO), spcctrophotomctric 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 typhimurium 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 et al, (1973, 1975). The 9,000 x g liver fraction was prepared from male rats (strain R bred at our laboratory) maintained on normal diet. 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 g supernatant and seven-tenths of an NAUPI-I generating system 1022 U. KANNUG AND C. KAMEL |NADP, glucose 6-phosphale, MgCl2, KCI, and phosphate buffer (pH 7.4) |. In the scries 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 microsomal system or phosphate buffer and 0.1 ml of bacteria to 2 ml soli agar (0.b% 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 ease 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 hatleiial suspension was added, three 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 arc 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 limes 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 LDC-tar is tested in the presence of the microsomal system the icsulls qualitatively resemble those obtained without a metabolizing system; that is, a toxic as well as a mutagenic olivet is pioduceil. Also, in this senes the toxic ellect with Tween 80 diflcrs born that with the othei two solvents. Under these conditions, however, a stronger-toxic action \MEL )l. ilfcr conveen i h ild is or n or n in lOtIC i ure. ogel Five used lates uba- liree cro|/ing TA >mal 'lice with The due. .ime the iber the cm; i his :her lion MUTAGENICITY OF WASTE PRODUCTS FROM VINYL CHLORIDE INDUSTRIES 1023 TABLE 1. Effect of niC-lar in Different Solvents on Siilmnurllu typhimurium TAI535 (ILnc p.ni Sulislilnlion) will! ,nul without Microsotndl System Solvent Dose M plate) Without microsomal system Suivival (%) No. of mutants per plate + SE With microsomal system Survival (%) No. of mutants per plate * SI. Ethanol 0 100 100 85 300 95 GOO 69 900 42 10.4 - 1.33 16.2 t 1.98 27.0 t 3.22 46.8 * 2.08 55.4 * 4.65 100 15.0 * 1.41 104 29.6 + 2.36 101 76.4 * 2.50 77 124.2 * 4.48 46 1 12.4 t 14.98 DMSO 0 100 100 94 300 80 600 58 900 40 12.0 1.0 t 19.2 * 2.89 38.6 1.69 52.0 t 4.83 63.8 + 1.65 100 17.2 l.o" 98 31.6 *. 3.31 95 78.0 6.46 69 119.6 5.21 52 137.0 3.81 1 0 100 7.4 0.93 95 15.2 ' 1.02 i 100 94 14.6 t. 1.08 89 90.2 > 6.22 ' I ween 80 300 87 29.6 > 1.86 71 1 12.1 4 IS 600 79 53.0 t 2.47 53 1 16.6 t 6.38 900 69 83.2 3.76 20 116.8 t 4.84 occurs with Tween 80. Only 20% of the bacteria survive 900 Mg 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 Mg pc 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 NADPM-dependent functions in the metabo lizing system arc necessary for the high mutagenic effect. In this 1024 U. KANNUG AND C. RAMEL p.irlicul.ir experiment the maximum number of mutants, within the con centration range, was approximately 200 per plate. The main component, 1,2-dichlorocthane, has been tested in the con centration range 5-45 prnol per plate (Fig. 2). This substance, however, does 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 pmol 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 FIGUKr. 1 lnMt .mil mut.icrnii rllriG ul I IX. i.ir ml Stilmtmcllti tyt'himni ium IA ISIS (b.ivr l>,nr sulisiiit:;!iiii) m (Ik- iiruM-ncc "I .1 mkriiMinijI sysictn willi () nr wilhoiu ( ) NADI'. Hie sulvmU used ;n this iwpciiment w.is DMSU. SL 041701 l.L MUTAGENICITY OF WASTE PRODUCTS FROM VINYL CHLORIDE INDUSTRIES 102S n- n- er, >y 'CC or ed 'Sal tin or on FIGURE 2, Toxic jnd mutagenic effects of 1,2-dichloroethanc (ethylene dichloridc) on Salmonella ryphimurium 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 (his mix JSC- ture includes direct as well as indirect mutagenic constituents. Since most I lie of the components arc poorly soluble in water, three agents were used as solvents or emulsifier: ethanol, DMSO, and I ween 80. Qualitatively SL 041702 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 et al., 1975). Tween 80 deviates from the other solvents in another respect also. The effect of the lowest dose with liver microsomcs is cleatly 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 arc 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 either 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-dichlorocthanc 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 maincomponents of EDC-tar, constituting approximately 35% of the total (Jensen et al., 1975), its contribution to the muta genicity of the i.ii must Ik- rather limited. Appioximaloly 3 pmol ol I111< liloim ili.iiii r. |iie ,i`iil m Hit* luglir.t < oiiccnli.diun ol I.DC-lar, SI* 041703 MUTAGENICITY OF WASTE PRODUCTS FROM VINYL CHLORIDE 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-dichloroethanc 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 conicxt (Rannugand Ramcl, in preparation). Of oilier compounds that could be lesponsible for the mutagenicity of ElX-lai, vinyl chloiide itsell should also he considered. 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 corresponded to 4 X 10M in the substrate. 1 his is a ten times higher concentration of vinyl chloride than in our experiment with the highest dose of LDC-lar, and fudging I rum 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 jug 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, jcrnelov ct al. (1972) found an increased frequency of cmitotic cells in Allium cepa at concentrations from 1-50 ppm. Acute toxicity in some marine animals gave LCSo values ranging from 2-20 ppm (see Jensen el al., 1975). Effects on l4C fixation of phytoplankton were found at even lower concentrations. Rosenberg ct 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 crangon). 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 LC5,, as well as other biological parameters. Considering the cause of the toxicity of EDC-tar, Hagstrom and Normark (1974) conclude from their work on C. coli that the killing effect is associated with a decreased stability ol the cytoplasmic mem brane, and they theielote suggest an inleiaction with the membrane. I hese findings seem to he in good agieemenl with the stiong tendency of EDC-tar to adhere to particles noted by |ernclov ct al. (1972). SL ow n03A 1028 U. RANNUG AND C. KAMEL Another conclusion ilr.iwn by It.lustrum .ind Norm.uk (1974) .is well as Rosenberg cl al. (1975) is that neither of the two main components 1,2dichlorocthanc or 1,1,2-trichlorocthanc 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-dichloroethanc gives a different mutagenicity pattern when tested as a pure chemical than does total EDC-tar. The appearance of both 1,2-dichlorocthanc and 1,1,2-trichlorocthane 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 Salnuuh'llafmMW malian microsomc method seems to be a suitable tool lor 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, B, N, 1971. The detection of chemical mutagens with enteric bacteria, in Chemical mutagens: Principles and methods for their detection, vol. 1, ed. A. Hollacnder, pp. 267-281. New York: Plenum, Ames, B. N., Durston, W. E., Yamasaki, E. and Lee, F. 0. 1973, Carcinogens are mutagens: A simple test system combining liver homogenates for activation and bacteria for detection. Prac. Natl. Acad. Sci. t/.S./t. 70:2281-2285. Ames, B. N,, McCann, |, and Yamasaki, E. 1975. Methods for detecting carcinogens and mutagens with the -Su//ur;e//w/mjmmalian-microsomc mutagenicity test. Mutat. Res. 31:347-364, Barlsch, II. anil Montesano, R. 1975. Mutagenic and carcinogenic effects of vinyl chloride. Mutat. Res. 32:43-1 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,, Mollcrud, E, E. and Solemdal, P. 1972. The influence of some byproducts from vinylchloride production on fertilization, development and larval survival on plaice, cod and herring eggs. Aquaculture 1:81-96. Brem, H., Stein, A. B. and Rosenkrana, H. S. 1974. The mutagenicity and DNA-modifying effect of haloalkanes. Cancer Res. 34:2576-2579. Ehrenberg, L,, Osterman-Golkar, 5., Singh, D. and Lundqvist, U. 1974. On the reaction kinetics and mutagenic activity of methylating and d-halogcnoethylating gasoline additives. Radial. Bot. 15:185-194. Hagstrom, A, and Normark, S, 1974. Toxic effect and action of chlorinated by-products from vinyl chloride production on Escherichia coli K 12, Ambio 3:77-79. Haley, T. ). 1975. Vinyl chloride: How many unknown problems? /. Toxicol. Environ. Health 1 :47-7 3. Icnscn, S., Icrnclov, A., Lange, R. 3nd Palmork, K. 11. 1970. In i'AO technical conference on marine pollution, l'IR:MP/7<) E 88. Rome: I AO. Icnscn, S,, Lange, R., Ilcrge, G., Palmork, K. II. and Renberg, I.. 1975. On the chemistry of I.DCl.ir and its biological significance, I'rnc. R. Sor, I ond. (Iliol.) 189:3.13- 146. MUTAGENICITY OF WASTE PRODUCTS FROM VINYL CHLORIDE INDUSTRIES 1029 Jernclov, A., Rosenberg, R. and lensen, S. 1972. Biological effects and physical properties in the marine environment of aliphatic chlorinated by-products from vinyl chloride production. Water Res. 6:1181*1191. Kier, L. D., Yamasaki, E. and Ames, B. N. 1974, Detection of mutagenic activity in cigarette smoke condensates. Proc. Nall. Acad. Sci. U.S.A. 71:4159-41G3. Lopricno, N., Baralc, R., Baroncclli, S., Bauer, C., Bronzetti, G., Cammcllini, A., Cercignani, G., Corsi, C., Gervasi, G., Leporini, C., Nicri, R., Rossi, A. M.p Stretti, 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, Malavcillc, C., Bartsch, H., Barbin, A., Camus, A. M., Montesano, R., Croisy, A. and lacquignon, P. * 1975. Mutagenicity of vinyl chloride, chlorocthylcncoxidc, chloroacctaldehydc and chloroethanol. Biochem. Biophys. Res. Common. 63:363-370. McCann, ), and Ames, II. N. 1976. Detection ol carcinogens as mulagt'ns in the s.i/nioiii /Ai/imciosonutest: Assay of 300 chemicals. Discussion. Proc. Natl. Acad. Sci. U.S.A, 73:950-954. McCann, Choi, E., Yamasaki, E, and Ames, B. N. 1975a. Detection of carcinogens as mutagens in the SalmonellaImictosome test: Assay of 300 chemicals. Proc. Natl. Acad. Sci. U.S.A. 72:5135-5139. McCann, Simmon, V., Streitwieser, D. and Ames, B. N. 1975b. Mutagenicity of chloroacetaldchyde, a possible metabolic product of 1,2-dichlorocthanc (ethylene dichlnridc), chlnmcthanol (ethylene chlorohydrin), vinyl chloride, and cyclophosphamide. Proc. Nall. Atad. Sci, l .S.A. 72:3190-3193. Rannug, U., loliansson, A., Kamel, C. and Waclilmcislcr, C. A. 1974. I lie mutagenicity ol vinyl chloride after metabolic activation. Ambio 3:194-197. Rannug, U., Hedenstedt, A., Arrhenius, E. and Zetterqvist, 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, Turku, Finland, abstracts, p. 43. Rannug, U., Gothc, R, and Wachtmeistcr, C. A. 1976. The mutagenicity of chloroclhylene oxide, chloroacctaldehyde, 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 coll: Partial purification and some properties. /. Biol. Chem. 218:97-106. Received July 6. 1976 Accepted October 17, 1976 041705