Chem.-BioJ. Interactions, 12 (1976) 251-263 @ ElsevierScientific Publishing Company, Amsterdam

- Printed in Tae Netherlands

THE M~AGENI~~TY OF ~LGRG~LENE OXIDE, CHLOROACET~DB~DE, 2~HLORD~H~OL AND CH~ROACETIC ACID, CONCEIVABLE METABOLITES OF VINYL CHLORIDE ULF RANNUO, ROLF C&THE and CARL AXEL WAC~~EI&~R

~nvi~nme~taJ Toxicotogy Unit, WaJJe~be~ ~aboruto~, U~iue~ity of Stockholm, S-104 05 Stockholm (Sweden] (Received April 28th. 1975) (Revision received August 14th. 1975) (Accepted August 28th. 1975)

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SUMMARY

Previous investigations have shown that the carcinogen vinyl chloride causes base-pair substi~tion in the bacterium ~l~o~e~fa typ~~murium. The ability of four conceivable metabolites - chloroethylene oxide, chloroacetaldehyde, 2chloroethanol and chloroacetic acid - to cause base-pair substitution directly in Salmonella typhimurium TA1535 has been compared. The main comparison was performed at initial concentrations from 0.1 to 1.5 mM. In this region, however, a mu~genic effect was observed only with chlor~~ylene oxide and chloroace~dehyde, the former being approximately 20 times more effective than the aldehyde when compared on a molar basis. 2-Chloroethanol and chloroacetic acid were studied also at higher concentration (1 mM-1 M), and a weak mutagenic response was found with 1 M 2chloroethanol solution. With chforoacetic acid no enhancement of the mu~tion f~~uency could be detected. Chloroethylene oxide was found to be approximately 450 times more effective as a mutagen than chloroacetaldehyde when the comparison is based on exposure doses, defined as the time-dependent concentrations of the compounds in the treatment solutions, integrated between the times of onset and ~rminatlon of ~atrn~nt. agilely, chloroe~ylene oxide was 10 OOO15 000 times more effective as a mutagen than ethylene oxide, used as a positive control,

The carcinogenic effects of vinyl chloride (I) first shown by Viola et al. [ 1,2] were later confirmed through the extended work by Maltoni and Lefemine [3,43, In addition to these results in laboratory experiments, cases of 251

the rare malignant disease angiosarcoma of the liver have been reported among workers who have been exposed to vinyl chloride in plastic industry WI. Recent investigations in this laboratory using the test method worked out by Ames [ 71 clearly demonstrate a mutagenic effect of vinyl chloride after metabolic activation [ES]. From the results it could be concluded that vinyl chloride causes point mutations. By using different strains of Salmonella typhimurium it could be shown that the point mutations were due to basepair substitution, indicating that vinyl chloride could give rise to metabolites capable of alkylating DNA. These results were later confirmed by Bartsch et al. 191 and Yalaveille et al. [lo]. In addition they also reported a mutagenic effect of vinyl chloride without activation by liver microsomes with prolonged treatments (3-48 h). The most plausible primary metabolite was presumed to be chloroethylene oxide, known as a highly reactive, easily hydrolysable compound. Van Duuren [12] likewise discussed the possible mlechanism of carcinogenic action of vinyl chloride and emphasised chloroethylene oxide as being the probable reactive metabolite. This assumption received some support from experiments in this laboratory with 3,4-dichlorobenzenethiol as a trapping agent for reactive metabolites formed in vitro from vinyl chloride in the presence of a liver microsomal system [ 111. It was concluded from the identification of S-( 3,4-dichlorophenyl)-mercaptoacetaldehyde as a reaction product that either or both of the isomer compounds, chloroethylene oxide (II) and chloroacetaldehyde (III) were formed under the conditions used. A derivative of chloroacetic acid (IV), obviously a secondary metabolite, was also identified. A fourth compound, 2-chloroethanol (V), has been proposed as a possible primary metabolite of vinyl chloride [13j. CIHC=

CH, I

GIHC-CH, ‘d II

CICH,CHO III

CICH,COOH IV

CICH,CH,OH V

w,-,CHz 0 VI

In the present work, the mutagenicity of these four conceivable metabo&es (II-V) has been tested directly on Salmonella. In order to get a measure of the relative mutagenicity of the metabolites, the well known mutagen, ethylene oxide (VI) [14,15] was used as a positive control. In the previous investigation, the mutagenicity test on vinyl chloride after metabolic activation was limited to the fairly high concentrations of 11 and

20% (v/v) in the air. In the present work an experiment chloride in the air has therefore been included. MATERIAL

with 2% (v/v) vinyl

AND METHODS

Chemicals 24hloroethanol, Merck, Zur Synthcse, distilled (44” /20 mm Hg); chloroacetic acid, Baker Analyzed Reagent, Mp 61-63”; chloroacetaldehyde, was prepared as described by Natterer [16] from chloroacetaldehyde dimetbyl acetal (Merck, Zur Synthese) by treatment with concentrated sulphuric acid at 0" , to give a crystalline product, Mp 89-90” after repeated crystallisations from ethanol. The product was identified as 2,4,6-tris-chloromethyl-1,3,5trioxane by NMR [17] and molecular weight determination (Rast: M..W. 232) and by its mass spectrum, with parent ion at m/e 233 (M’-1,3 Cl) with an intensity of 1.7% of base peak (m/e 79,l Cl). Chloroacetaldehyde was prepared immediately before use by heating the crystallisate to ca. 150”) and allowing the chloroacetaldehyde gradually formed to distil through a Vigreux column, Bp 87-89”. IR spectrum of the aldehyde (CCL ) showed a band of high intensity at 1742 cm-’ [ 183, and bands of moderate intensity at 2720, 2830 and 2930 cm-‘. The NMR-spectrum (CC&, Varian A-60 apparatus: tetramethylsilane as internal standard) showed a doublet at STMs 3.95 ppm (2 H, J = 2 cps) and a triplet at 6 9.57 ppm (1 H, J = 2 cps) [19]. Chloroethylene oxide was prepared through a modification of a method described by Gross and Freiberg [20] . Ethylene oxide (Fluka, 44 g) was aefluxed in a Pyrex glass reacthn bottle (A in Fig. 1) equipped with a condenser (C), cooled by a dry iczacetone mixture. The evaporating ethylene oxide, containing hydrogen chloride as formed in the photoreaction, was freed from acid by means of an adsorption tube (B) containing sodium hydroxide pellets (40 g), protected from direct contact with the effluent from the condenser. The reaction bottle was irradiated with UV-light from a Hanau mercury lamp. Chlorine from a flask, kept at -30”) was passed into the bottle by means of a stream of nitrogen, which was adjusted so as to prevent an appreciable green coloration of the reaction solution due to excess chlorine. After 4 h when all chlorine was consumed (35 g), irradiation was stopped, the sodium hydroxide tube was removed ;md the reaction mixture was distilled at reduced pressure, using the condenser (C) as a fractionating column cooled by means of ice-water, The appropriate fraction was redistilled through a Vigreux column to give pure chloroethylene oxide (0.84 g, 25”/ 100 mm Hg). NMR-spectrum (CCL ) showed multiplets at 6~~s 2.88 ppm (2 H) and at d 5.05 ppm (1 H) in fair agreement with the ABX-spectrum described for the compound [21]. No signals from ethylene oxide (S 2.55 ppm) nor from chloroacetaldehyde, were observed in a 10% solution. The IR spectrum (10% Ccl, ) showed a strong band at 910 cm-’ [ 211, and bands of moderate intensity at 1010 cm” and at 1330 cm-*, No absorption at 870

253

Fig. 1. Apparatus used for UV-light induced chlorination of ethylene oxide: A. Pyrex reaction flask. B. Absorption tube witlh sodium hydroxide pellets. C. Reflux condenser cooled with dry ice-acetone mixture.

cm-’ (strong band in ethylene oxide spectrum) nor at 1742 cm” was observed. The pure chloroethylene oxide could be stored for several weeks at -78”. At room temperature, however, a rearrangement to chloroacetaldehyde occurs [20] . In hexane this reaction was found to be moderately slow, with a half life of chloroethylene oxide of several days (IR), while the half life of the compound in acetonitrile at room temperature was shown to be of the order of hours (IR, WMR). The pure chloroethylene oxide was highly sensitive to moisture, which in addition to hydrolysils also induced polymerisation. To minimise these reactions during the preparation of the final bacterial test solutions containing chloroethylene oxide, the inititi solutions and dilutions were made in 99.5% ethanol at 0”. In case of rapidly performed operations, hydrolysis and ethanolysis should be practically negligible under these conditions.

254

Bat terial strain The Salmonella typhimurium strain TA1535, kindly provided by B. Ames, was the base-pair substitution strain used in our previous investigation on vinyl chloride [8] . This strain reverts to histidine prototrophy by alkylating agents - as described by Ames et al. [ 221. Test procedure The bacteria were grown overnight in complete medium (Difco, Antibiolic medium 3), washed twice and resuspended in 0.1 M phosphate buffer (pH 7.4) to original or double density. The suspension with double cell density was divided into aliquots of 2 ml in test tubes. Water solutions (2 ml) of the substances, chloroacetaldehyde, chloroacetic acid, and 2-chloroethanol respectively were added to the test tubes to give final volumes of 4 ml with the concentrations indicated in the tables and in the figures. In case of chloroacetaldehyde, the sample was dissolved in 50 ~1 of ethanol prior to the initial dilution with water, in order to prevent a partial po’lymerisation of the aldehyde. Due to the instability of chloroethylene oxide this compound was dissolved in cold ethanol (0”). The same procedure was used for ethylene oxide. The solutions and dilutions in cold ethancl were made as quickly as possible and the treatment was started when 100 id of these solutions were added to aliquots of 4 ml of the Salmonella suspension. The solutions of the other compounds were also prepared immediately before each experiment. All treatments were carried out at the same cell density, approx. 3 lo9 cells per ml, at 25” for 1 h and terminated by centrifugation and resuspending in fresh buffer. All necessary dilutions of the bacteria before plating were ma.de in 0.9% saline. Platings with the soft agar technique, incubations and scoring the number of colonies were made in the same manner as described earlier [8] . The experiments with 2% vinyl chloride, apart from time of treatment (3 h), were performed in exactly the same way as the main experiments in the previous investigation [ 81 . l

RESUX ‘I’S

The toxic and mutagenic effects on Salmonella typhimurium ??A1535 in the comparative study of the four conceivable metaboiites chloroethylene oxide, chloroacetaldehyde, 2-chloroethanol and chloroacetic acid are snown in Fig. 2 and Table I. The same concentration range in terms of initial molar concentration (0.1-1.5 mM) was used for the substances. A slightly diminished range (0.15-0.75 mM) was, however, chosen for chloroethylene oxide due to its high mutagenic effect. In case of this very unstable substance (see DISCUSSION) the number of concentrations studied were increased to get a more reliable picture of the relation between mutagenic effect and initial molar concentration in the buffer suspension. As can be seen from Fig. 2, chloroethylene oxide was the

255

I

Ethylene

acid

oxide

Chloroacetic

2Chloroethanol

Chioroacetaidehyde 3.5 3.7 2.9 2.7

0 0.1 0.5

3.4 3.6 3.9 4.9 3.4 3.9 3.6 3.9 2.9 3.0 2.8 2.3 3.0 2.6

0.5 1.5

0 0.1 0.5 1.5

0 0.96 4.77 9.55 47.70 95.50

0 0.1

1.5

2.8 3.0 2.5 2.2 2.1 1.1

--I_-8.6

16.2 12.4 15.0 14.4

16.2 13.2 11.4 13.0

8.0 10.4 20.8 49.6

126.8 177.6 210.0 246.4 202.8

6.8 100 103.9 7.4 97.2 13.4 79.0 17.4 102.5 42.8 39.7 106.2 .-,__.._ ..-_~

100 113.6 109.8 115.1

100 105.9 113.7 144.1

100 107.3 83.2 77.6

75.0 40.6

90.6 79.3

100 109.4

2.36 2.47 4.79 7.65 14.46 41.16

4.74 3.19 4.00 3.66

4.74 3.65 2.94 2.64

2.30 2.78 7.18 18.35

3.10 41.65 70.76 95.53 118.63 179.47

t t 2 + f t

* t f 2

*** *** *** *** ***

0.55 0.51 0.27 0.69 0.69 3.30

0.71 0.73 0.70 0.57

** *** *** ***

0.44 0.42 0.63 *** 1.51 *** r 0.71 2 0.44 -’ 0.47 t 0.21

t f ? f

!. 0.44 ?. 2.98 t 2.35 f. 2.64 ? 3.71 t 9.44

- -Number of mutants per 10H surviving cells S.E. --1-

--

--

--

TREATED FOR 1 h AT 25’ (pH 7.4) WITH CHLOROAND CHLOROACETEC ACID AN3 ZTHYLENE OXIDE

* 9.01 < p < 0.05; ** 0.001 < p < 0.01; *** p < 0.001‘ _ _-_-,--. --------. Number of viable Survivai Number of mutants cells per plate - lOa per plate (%I Mean value of 3 plates Mean value of 5 plates -_-~. -.--_-m--

levels:

0 0.15 0.30 0.45 0.60 0.75

Initial concentration (mM)

with &test. Significance

oxide

analyses

Chioroethylene

Substance

Statistical

SALMONELLA TYP~Z~~~ZU~ TA 1535 (BASE-PAIR SUBSTITUTION} ETHYLENE OXIDE, CHLOROACETALDEHYDE, 2CHLQROETHANOL

TABLE

Fig. 2. Effect on survival and mutation frequency of Salmonella typhimurium TA1535 (base-pair substitution) treated for 1 h at 25’ (pH 7.4) with chloroethylene oxide (o), chloroacetaldehyde (m), 2chloroethanol (a) and chloroacetic acid (v).

only compound showing a definite toxic effect combined with a strong mutagenicity in this low concentration range. In the highest concentration it produced approx. 180 mutants per 10’ surviving cells which was 60 times higher than the corresponding control. Chloroacetddehyde also showed a mutagenic effect in this concentration range and caused 18 revertants per 10” surviving cells at the highest concentration. The spontaneous frequency was 2.3 revertants per 10” surviving cells. When compared on a molar basis, chloroethylene oxide is approx. 20 times more effective as a mutagen than chloroacetaldehyde. 2Chloroethanol and chloroacetic acid showed no effects up to 1.5 mM and were therefore tested at higher concentrations (1 mM-1 M) (Table II). 2-Chloroethanol was only faintly toxic and 1 M solution produced a weak mutagenic response. Chloroacetic acid finally caused a drop in the survival of the bacteria already below 0.5 M and at the concentration 0.5 M the survival was less than 0.01%. In Fig. 3 the effects of chloroethylene oxide and ethylene oxide are compared. A significant increase of mutants was first detected around 5 mM for

257

TABLE III

SALMONELLA

TYPHIMURIUM TA 1535 (BASE-PAIR SUBSTITUTION) TREATED FOR 3 h AT 25O IN AN ATMOSPHERE OF AIR CONTAINING 2% (v/v) GASEOUS VZNYL CHLORIDE (VC) St&stical

analyses, see Table I. Number of living cells lo+ Mean value of 3 plates

Number of mutants per 10s surviving cells +_S.E.

3.7

10.0 f 0.9 *** 3.6 r 0.3

l

VC + microsomal VC + buffer

system

Air + microsomal Air + buffer

system

3.3 3.4 3.2

3.6 f 0.6 3.2 + 0.6

mutation frequency, twice the control value, only at the highest concentration tested (1 M). The question arises whether the mutagenicity of 2-chloroethanol alone would be theoretically sufficient to account for the mutagenicity of vinyl chloride after metabolic activation. It must be pointed out that a comparison between the experiments with vinyl chloride and those with 2chloroethanol must be done with caution because the treatment conditions were far from identical. A rough calculation reveals that only if all vinyl chloride in the experiments with 2% gas in the air (10.3 mmoles vinyl chloride) is conve,rted to 2chloroethanol will the final concentration in the medium (350 ml) of this compound be sufficiently high to give a mutagenic effect, approaching the observed frequencies. However, the lipophilic gas vinyl chloride is of low solubility (cf. Bartsch et al. [9] and Malaveille et al. 1101) in the aqueous medium and most problably only a small fraction of the gas will be metabolised during the time of treatment. Therefore it is highly unlikely that 2ch!oroethanol is alone responsible for the mutagenicity found with vinyl chloride in the present test system. The two remaining compounds, chloroacetaldehyde and chloroethylene oxide, both give a mutagenic effect in the low concentration range. The aldehyde, however, is far less effective than chloroethylene oxide even if the short life time of the latter is not taken into account. Therefore, the difference between the two compounds will be far more apparent if a comparison is based on exposure doses, defined as the time-dependent concentrations of the compounds in the treatment solution, integrated between the times of onset and termination of treatment [15]. A rough estimate of the mutation frequency per dose unit in the low doserange using preliminary values of the rate constants at 25” for reactions of chloroethylene oxide with simple nucleophiles [ 25 ] to calculate the exposure doses, indicates that this compound induces mutations approx. 450 times more effectively than chloroacetaldehyde. Furthermore, with dosages giving approx. 80% survival, chloroethylene oxide gives a mutation frequen-

260

cy which is 6 times higher than that produced by chloroacetaldehyde. Chloroethylene oxide obviously exhibits a better capacity for producing mutants without killing the bacteria. It may be of interest in this connection to point out that neither in our experiments nor in those by Bartsch et al. could any toxic effects of vinyl chloride be detected. It should be mentioned, however, that t1.e killing effect found by us with chloroacetaldehyde may not be totally relenmt for the vinyl chloride experiments with liver microsomes, because Bartsch et al. found that the toxic effect of chloroacetaldehyde was reduced by the presence of liver fractions. The mutagenic effect as well as the reduction in toxicity were, however, found in experiments where the survival on the whole was below 10%. The results from the present investigation clearly show a mutagenic effect of chloroacetaldehyde at higher survival, and combined with a reduction of the toxic effect it is not excluded that this metabolite could play a part in the vinyl chloride mutagenicity. It is, however, likely that chloroethylene oxide is the active metabolite of major importance, because this substance has the most pronounced property of producing a high number of mutants at low doses without causing a toxic effect. The comparative study on the mutagenicity as a function of exposure doses of the two compounds chloroethylene oxide and ethylene oxide in Salmonella typhimurium TA1535 performed in the present investigation reveals a large difference in effectiveness. The mutation frequency induced per dose (mM - h) by chloroethylene oxide is 10 000-15 000 times higher than the corresponding value of ethylene oxide. Ross [26] has pointed out the close correlation between biological activities of alkylating agents on the one hand and their reaction mechanisms and reactivities towards simple nucleophiles on the other. Other authors [27,28] have shown that within groups of related alkylating agents a great part of the biological reaction pattern may be directly related to the absolute reaction rates. A comparison based on preliminary rate constants at 25” for reactions with the appropriate nucleophiles [ 251 gives a ratio of approx. 4000 between the rates for chloroethylene oxide and ethylene oxide. Within a factor of 2-3 this ratio, although preliminary, coincides with the ratio between mutagen effectivity of the two compounds referred to above, in reasonable agreement with theoretical expectations. It might therefore be concluded that chloroethylene oxide acts mainly as a monofunctional alkylating agent. Similar comparisons involving chloroacetaldehyde indicate this compound to be far more active as a mu% gen than expected from its reactivity as an alkylating agent. A possible interpretation of this fact is given in an addendum to this paper [25] . ACKNOWLEDGEMENTS The authors are grateful to Dr. B.N. Ames, University of California, Berkeley, for supplying the Salmonella typhimurium strain and to Dr. C. Ramel and Dr. L. Ehrenberg for helpful discussions and criticism,. The technical assistance of Miss I. Bamford and Mrs. A. I-Iedenstedt is gratefully acknowledged.

261

This investigation has been supported by grants from the Swedish Board for Technical Development and from the Swedish Work Env~onment Fund. REFERENCES

s 9 10 11 12 13 14 1s 16 17 18 19 20 21

P.L. Viola, Cancerogenic effect of vinyl chloride, X Intern. Cancer Congr., Houston, 1970, Ah&r. vol. 29. P.L. Viola,. A. Bigotti and A. Caputo, Gncogenic response of rat skin, lungs and bones to vinyl chloride, Cancer Res., 31 (1971) 516. C. Maltoni and 0, Lefemine, Carcinogenicity bioassays of vinyl chloride, I. Research plan and early results, Environ. Res., 7 (1974) 387. C. Maltoni, The value of predictive experimental bioassays in occupational and envie ronmental earcinogeneses. An example: vinyl chloride, Ambio, 4 {1975) 18. J.L. Creech and M.N. Johnson, Angi~~rcoma of liver in the manufacture of poiyvinyl chloride, J. Occup. Med., 16 (1974) 150. B. Holmberg and G. Molina, The industrial toxicology of vinyl chloride - A review, Work-Environm. Health, 11 (1974) 138. B.N. Ames, W.E. Durston, E. Yamasaki and F.D. Lee, Carcinogens are mutagens: A simple test system combining liver homogenates for activation and bacteria for detection, Proe. Natl. Acad. Sci. USA, 70 (1973) 2281. U. Rannug, A. Johansson, C. Ramel and CA. Wachtmeister, The mutagenicity of vinyl chloride after metabolic activation, Ambio, 3 (1974) 194. H. Bar&h, C. Malaveille and R. Montesano, Human, rat and mouse liver-mediated mutagenicity of vinyl chloride in Salmonella typhimurium strains, Int. J. Cancer, 15 (1975) 429. C. Malaveille, H. Bartsch, A. Barbin, A.M. Camus and R. Montesano, Mutagenicity of vinyl chloride, ChIoroethylen~xide, ~hIoroacet~dehyde and chloroethanol, Biothem. Biophys. Res. Commun., 63 (1975) 363. R. G&he, C.J. Calleman, L. Ehrenberg and C.A. Wachtmeister, Trapping with 3,4dichlorobenzenethiol of reactive metabolites formed in vitro from the carcinogen vinyl chloride, Ambio, 3 (1974) 234. B.L. van Duuren, On the possible mechanism of carcinogenic action of vinyl chloride, Ann. N.Y. Acad. Sci., 246 j1975) 258. R.E. Hefner Jr., P.G. Watanabe and P.J. Gehcing, Preliminary studies of the fate of inhaled vinyl chloride monomer (VCM) in rats, Ann. N.Y. Acad. Sci., 246 (1975) 135. G. K&mark and M. Westergaard, Further studies on chemically induced reversions at the adenine locus of Neurospora, Hereditas, 39 (1953) 209. L. Ehren-erg, K.D. Hiesche, S. Osterman-Golkar and I, Wennberg, Evaluation of genetic risks of alkylating agents: tissue doses in the mouse from air contaminated with ethylene oxide, Mutation Res., 24 (1974) 83. K. Natterer, Uber Monochloraldehyd, Monatah. Chemie, 3,457. J.L. Jungnickel and C.A. Reilly, Nuclear magnetic resonance studies on geometrical isomers of paraldehyde and other substituted trioxanes, J. Mol. Spectr., 16 (1965) 135. L.J. Be&my and R.L. Williams, Infrared spectra and polar effects, part IX. Field effects in ~-h~ogenated aldehydes and acid chiorid~?s, J. Chem. Sot., (1958) 3465. G J. Karabatsos and D.J. Fenoglio, Structural studies by nuclear magnetic resonance, XX. Conformational analysis of chloroacetaldehgde and bromoacetaldehyde, J. Amer. Chem. Sot. 91(1969) 1124. H. Gross and J. Freibere.-. Zur Existenz von Chloroiithylenoxid, J. Prakt. Chem., 311 (1969) 506. C. Wailing and P.S. Fredrieks, Positive halogen compounds, IV. Radical reactions of chlorine and t-butyl hypoehlorite with some small ring compounds, J. Amer. Chem. SOC., 84 (1962) 3326.

22 B.N. Ames, F.D. Lee and W.E. Dutston, An improved bacterial test system for the detection and cl~ification of mutagens and carcinogens, Proc. Nat]. Acad. Sci. USA, 70 (1973) 782. 23 H.S. Rosenkranz and T.J. Wlodkowski, Mutagenicity of ethylene cblorohydrin. A degradation product present in food stuffs exposed to ethylene oxide, J. Agr, Food Chem., 22 (1974) 407. 24 1. Grigorescu and Gh. TobH, Clorura de vinil, Aspecte de toxicologic industrial& Rev. Chim. Rom., 17 (1966) 499. 25 S. Hussain and S. Osterrnan-Golkar, Chem, -Biol. interact., submitted. 26 W.C.J. Ross, Biolo~icai A~kylating Agents, Butterw~rth, London, 1962. 27 S, Gsterman-Golkar, L. Ehrenberg and C.A. Wachtmeister, Reaction kinetics and biological action in barley of monofunctional methanesulfonie esters, Radiation Botany, 10 (1970) 303. 28 L. Ehrenberg, S. OstermanColkar, D Singh and U. Lundqvist, On the reaction kinetics and mutagenic activity of methylating and ~-haiogenoethylating gasoline additives, Radiation Botany, 15 (1974) 185.

263

The mutagenicity of chloroethylene oxide, chloroacetaldehyde, 2-chloroethanol and chloroacetic acid, conceivable metabolites of vinyl chloride.

Chem.-BioJ. Interactions, 12 (1976) 251-263 @ ElsevierScientific Publishing Company, Amsterdam - Printed in Tae Netherlands THE M~AGENI~~TY OF ~LGRG...
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