Document RZK8qZk8yY3zO9Ox76dYyK68

4&Z A/<Wo(ion Research. 4* (1977 ) 327-^J O Elsevier/Norlh-Hnlland Biomedical PresJ/-' JfUL 2 7ft 327 VINYL CHLORIDE MUTAGENESIS IN DROSOPHILA ME LASOC, ASTER F.G. VERBURGT and E. VOGEL Department of liudiatton Genetics and Chemical Mulaecnesis. State Lfnrrsity of Leiden. Syh ri/s Lahoratnru s. Leiden (The Netherlands} (Hc-C'i vi-ti Oc-mhor 26th. 1976) (Revision ii'rpivi'd J.mu.ivy 20th, 1977) (Accepted February 2nd. 1977) Summary In inhalation experiments, Drosophila males were exposed to vinyl chloride at concentrations of 200, 850, 10,000, 30,000, or 50,000 ppm for 2 days, and to 30 or 850 ppm for 17 days. VCM was mutagenic in the recessive-lethal test both after short-term and long-term exposures. The lowest effective concentra tion (LEC) was 850 ppm after 2 day exposure, and this value could be lowered to 30 ppm by prolonging the exposure time to 17 days. With the concentration levels tested, the mutation frequency increased with concentrations and reached a plateau at 10.000 ppm. This indicates a substrate saturation effect. In contrast with the recessive lethal assay, negative results were obtained when tests on dominant lethals, translocations, entire and partial sex-chromo some loss wore carried out with VCM at 30,000 ppm for 2 days. This finding of a false negative seems a logical consequence of the observed saturation effect, and strengthens the concept that there exist two effective concentrations for point mutations vs the induction of chromosome breakage events. Vinyl chloride monomer provides another example to support our view that chromo some breakage is not a reliable measure of mutagenic activity. Introduction A 1975 survey of the top fifty chemical products f 1) lists vinyl chloride monomer at position 23, thereby expressing its great economic importance. Over the past two years it lias become clear that VCM can cause late toxic effects in experimental animats and man. Several reports indicate that VCM is \ hhr, i n. lit. oti,l< >1,1.1 sin ic i I'lic-ulum . f*' DMT . 3,.,|.<liin,-tltv|. ] -< :-(> s rid I) tru/ ri.-l-iinm-Ui \ l-1 UJ-pvruJ vl-'v -> \ ide) Ina/citc; VCM. vmvl i-hluriilc munmiicr. '. I`\ i1 DMT, RSV 0016287 328 mutagenic in Salmonella /y/;/iimwrrin8s7{,2,3,13t16|, Escherichia coli [7], and Schisosaecharomyces pombe [11). rfflrTjtfwim etial. [5], Funes-Cravioto et al. [61, and Purchase et al. [15] have found chromosomal aberrations in workers exposed to VCM. A study of Infante et al. [81 on pregnancy among wives of workers exposed to vinyl chloride has indicated a significantly raised fetal loss, iii comparison with controls. Systemic carcinogenic activity of VCM was ob served in rats, mice and hamsters [ 14,191. All these findings have raised serious concern about the genetic and carcinogenic risks of VCM to man. Vinyl chloride as a chlorinated ethylene shows low reactivity, and its sys temic action suggests that the biological effects of VCM are dependent on its biotransformation. Tins is in line with the failure of VCM to exert mutagenic activity in microorganisms in the absence of active microsomal enzymes | 2.3.7.13,16]. Our interest in VC'M stems from the observation that Drosophila can execute the essential activation steps needed to convert such pro-carcinogens into electrophilic species |24). Its mode of action and its chemical properties made VCM a suitable material for further investigating the ability of the Drosophila system to detect different typos of environmental mutagens. Preliminary data from our group [20] and the work by Magnusson and Ramel [12] demonstrate that Drosophila is capable of activating VCM. In this study we report the mutagenicity of VCM in Drosophila at low and high concentrations, by comparing short-term vs long-term exposure, and by using different genetic end-points. It will be seen from the data that the detec tion of the mutagenic activity of VCM is dependent on the category of genetic damage used as a diagnostic criterion. Materials and methods Treatment procedure VCM, produced by Matheson Gas Products (supplied by Hock Loos, Amster dam), was contaminated with acetylene (1--2 ppm), water (1 ppm) and phenol (82 ppm). For exposure, groups of 50 males (2 days old) of our tester strain Berlin K were continuously exposed to VCM in 1-1 bottles for a period of 2--17 days. Each bottle contained about 10 g of food (normal food with killed yeast) and was air-tight sealed with a rubber screw-cap. A known quantity of pure VCM gas was injected with a disposable syringe through this cap into the bottle. A Packard gas chromatograph, model 417, with a stainless steel column (4 m, 3 mm^'filled with poropack Q, 200 mesh) was used to exactly determine the initial quantity of VCM injected into each bottle. The temperature of the injection port, the column and the flame ionization detector was 2005C. N2 with a flow of 1.5 1/h was used as the carrier gas. When low VCM concentrations were used, a known quantity of pure VCM gas was. injected into a 35-ml bottle. From the resulting air-VCM mixture a sample was transferred into a new 1-1 hottle, to obtain the desired concentration of VCM. The final concentration of VCM in the bottles was measured by gas chromatography analysis. This analysis show that the deviation in VCM content obtained by this simple device was less than lOTr, except for the lowest concentration of 30 ppm for which the error was larger, 30 ppm 10 ppm. r The sur days. At t enhanced, be detect) survical ra In these every 3 or Mutagenic: Sex-lim. Berlin h V(\M (30 itusc (In (/ to VCM <' analysis, u of 12 days Dominn The ind mature sp< [17]. To s (30,000 pi The flies u Since the homogemA slight. exposure (control) i ratio being males thoTile males and allowi nous will summarize Sc.x-chr, Males < (30,000 ip 99 for a p was scored Transit!, Since il focused oi storage e (30,000 pi by a proci RSV 0016288 [7], and vioto ot al. < in workers my wives of l fetal loss, ! M was ob liged serious and its sysulont on its mutagenic '1 I'nzymos ! irosophila arcinopens ;! projierties ility of the mutagens, i and Ramel ; at low and ure, and by it the detecy of genetic -i. Amsteri and phenol am Berlin K . 17 days, il yeast) and f pure VCM aottle. teel column iy determine ature of the - 2GCTC. N3 aeentrations >5-mI bottle, to a new 1-1 `titration of 1 <i is analysis vice was less ,-h the error 329 The survival rate was 957c for maierc^bsed to 200--50,000 ppm for 2 or 4 days. At the highest concentration of 5QaQflfr,ppm, the mortality rate was not enhanced, though the files seemed sIightfy*narcotized. No killing effect could be detected even in the long-term experiments of 17 days duration. The survival rate was equal (80--90%) for both the treated and the control groups. In these experiments the males were transferred to freshly prepared bottles every 3 or 4 days. Mutagenicity assays Sex-linked recessive lethals Berlin K males, 2 days old, were exposed to increasing concentrations of VCM (30--50.000 ppm), for 2 or 1 days and then individually mated to 3-/ i',asc (In (1) sc*"' xeKU + S. sc1 scK ir` U) virgins [ 10|. The mutagenic response to VCM of successive stages of spermatogenesis was tested by brood pattern analysis, with breeding intervals of 2 or 3 days each and total breeding period of 12 days. All suspected recessive lethals were verified by re-tests. Dominant lethals The induction of dominant lethals byVCM at 30,000 ppm was tested for m mature sperm and spermatids by the technique developed by Sankaranarayanan [ 17]. To sample mature sperm, 2-day-old Berlin K males were exposed to VCM (30,000 ppm) for 2 days and then mated with one 4-day-old Berlin K virgin. The flies were allowed to oviposit in egg-laying units [17] for two 24-h periods. Since the data for both the control subjects and the treated groups were homogeneous, the flics were assumed to be from one 4S-h brood. A slightly modified technique was used for the analysis of spermatids. After exposure of 2-day-old Berlin K males to 30,000 ppm for 2 days, exposed (control) males were mass-mated to 3- to 6-day-old Base virgins for 3 days, the ratio being 4 females : 1 male. This procedure was aimed at depleting from the maies those germ cells that were already spermatozoa at the time of treatment. The males were then re-collected, individually mated to one Berlin K female and allowed to oviposit for two 24-h periods. Since the material was homoge nous within the control and the treated groups, the date were again summarized to one 48-'n brood. Sex-ehromosomc loss Males of the genetic constitution A', v/j9s Y y* were exposed to VCM (30.000 ppm) for two days and then mated with 3-4 y ac In 49 v f rnal su(f) , 99 for a period of 3 days followed by a second 2-day brood. The [*', progeny was scored for entire sex-chromosome loss, and for partial loss (YL; Ys). Translocations Since the yield in recessive lethals was highest in spermatids, attention was focused on the induction of autosomal (2--3) translocations in this stage. In a storage experiment, 2-day-old Berlin K maies were exposed to VCM (30,000 ppm) for 2 days. Exposed sperm were again depleted from the males by a procedure similar that used in the dominant-lethal experiments. The males RSV 0016289 330 were then mated individually*'-/*j scSlL $c*H + dl -- 49, y scsl scR (Inscy); bw; st; virgins for 2 days. AHjigtS h, the males were discarded and the females allowed to lay eggs for a perfiBfcpi 5 consecutive 2- to 3-day broods, yielding for the last brood a total storag^period of 14 days. The F, males were individually back-crossed to females of the maternal genotype, and the F2 cultures were scored for the presence of autosomal translocations. Results and discussion Short-term experiments When inhaled by Drosophila males for 2 days, vinyl chloride monomer was dearly efficient between 850 and 50,000 ppm (Table 1). However, the incidence of recessive letlials was not a simple function of concentration: it increased from S50 to 10,000 ppm VCM and then remained constant at eoncentrauons above 10.000 ppm. The weighted mean mutation frequencies were 2.19 0-25% at 10,000 ppm, 2.22 * 0.24 at 30,000 ppm. and 2.30 0.26 at 50.000 ppm, indicating no further enhancement in the recessive lethal yield above 10.000 ppm VCM. Since VCM is believed to be a pre-carcinogen that must be oxidized to exert mutagenic activity, it is inferred that, above a certain concentration (which is between 850 and 10,000 ppm) the Drosophila enzymes are no longer capable of metabolizing additional substrate: this indicates that the detoxication (activation) mechanisms are saturated when too high doses are administered. This conclusion is also supported by the fact that an LD<0 for mortality of flies could not be determined for VCM. A similar "saturation effect" to that described for VCM has been observed for other pre-carcino gens -- PvODMT, PyDMT [22], cyclophosphamide, trofosfamide, and ifosfamide [23] -- all of which are well-known compounds that need biotransfor mation to become biologically active. (For review see ref. (20).) The vinyl chloride data thus provide another instructive example of the need to study the effects over a range of concentrations as well as of problems involved in any attempt at an extrapolation from higher to lower concentrations. With the mating procedures adopted, maximal sensitivity to the mutagenic action of VCM seemed to be in the second brood, about day 4 to day 5 after termination of treatment. In brood 2 the germ cell stages were sampled that were predominantly m the spermatid stage at tile time of treatment. Though the differences in recessive lethal yield between that brood and the first and third was only slight, this .tendency showed consistency through all experiments (series 4--8 in Table I), and the effect can be seen most clearly if the data, from series 6. 7 and 8 are pooled (Table II). The data aiso show a considerable decline in the recessive lethal yield in broods 4 and 5. Broods 3,4 and 5 all gave indications of a sterilizing effect. The mutational pattern described here for vinyl chloride coincides well with that observed for other pre-carcinogens, all indicating a typical, that is, a rather uniform, sensitivity pattern with a consistent peak activity in brood 2 or 3 [20,24]. Chandiey and Bateman [4| have indicated that gametes in the spermatid stage during treatment become available for fertilization on the third to fifth day, whereas spermatocytes become available from the sixth to ninth day, and it is precisely these stages that have a highly developed endoplasmic reculum (ER) [18]. In fact, in our j i : i ; i ! ' ' r i T A It I, F I INDUCTION VINYL OIL' ' Srnos Cm- (PI'I 1 2 3 4 !|; ~ :i" H i'liiiirri is, y scsl sc8 warded and the = 3-day broods, F, males were ;n\ and the F2 . monomer was However, the meentration: it instant at con'vqueneies were I 2.30 0.26 at ive lethal yield arcinogen that . above a certain -ophiia enzymes indicates that ) high doses are nat an LD*0 for ilar "saturation ner pre-carcinonide, and ifosfed biotransforu(>|.) The vinyl ed to study the .tvolved in any the mutagenic i to day 5 after re sampled that atment. Though nd the first and I'Ugh all experi>st clearly if tlie -how a consider 'd:, 3, 4 and 5 all i described here pre-carcinogens, pattern with a Bateman [4] tinient become ,s spermatocytes sely these stages |. In fact, in our TABLE l INDUCTION OK SKX-MNKKD RffCSS| VINYL CflLORIDK KOR 2 OR. 4 DAYS rilALS IN DROSOPHILA MALES EXPOSED TO Sr rics Cono-ntraiion (ppm) Exposure time (days) Brood ^jpapOTiift UntimiA Qftronmsomts listed Number of lethal* 0 1 0- 3 495 II 3-- r, 414 III 0-- 7 324 IV 7-10 238 V 10-12 t>od 1 -V 0--12 1990 2 30 2 I 0- 3 5`li> II 3- 5 rxj ill f> 7 r,Ri, IV 7 |0 i.n V lO 12 ddtt - l-V 0--12 2810 23 200 1 0- 3 404 11 3- 5 012 111 5-- 7 420 IV 7 -- 1 IJ 37(1 V 10-12 300 l-V 0-12 2172 0_ l 0.24 0.24 0-- 01 0.20 0.20 2 0.10 ' 0.07 1 0.17 ' 0.17 1 0.17 ` 0.17 o 1 0.23 0.23 2 0.32 ' 0.23 5 0.18 ' 0.06 0_ 00-- 1 0.27 ' 0.27 1 0.28 :: 0.28 2 0.00 ' 0.07 A k;>o 2 1 0- 3 780 1! 3- 5 530 Ill 3- 7 58 0 IV 7-10 324 V 10-12 304 3 0.3H ' 0.22 4 0.72 0.31, 2 0.34 0.24 1 0.31 0.31 0- l-V 0- 12 2550 10 0.30 '` 0.12 b 8 50 3 4 1 0- 3 1884 8 0.43 ' 0.15 II 3 - f> 1800 II! 5- ? 1420 IV 7 -10 1048 20 1.70 : 0.24 0 0.53 ' 0.21 2 0.10 0.13 V 1 0--12 1078 4 0.37 0.19 l-V 0-12 7200 43 0.50 ' 0.0!) 0 10.000 4 2 i II III IV V l-V 0- 3 3 - r, b- 7 7-10 10-12 0-12 107 l 110 1 020 228 207 3377 1 7 1.50 0.38 3 8 3.27 ' 0.52 If, 2.58 ' 0.54 0 3 1.01 !' 0.58 74 2.10 '' 0.25 7 30.000 a 2 3K 50.000 4 1 II III IV V l-V 1 I! Ill IV V t-v 0- 3 3- 5 5-- 7 7 -- 10 10-12 O--I 2 0- 3 3- r> 7 7 -10 10 -- 12 0-12 1 400 1040 572 255 527 3800 1470 1 140 287 170 300 330 1 34 2.31 ' 0.3!) 37 3.54 ' 0.57 1 1 1.92 ' 0.57 1 0.30 ' 0.3!) 3 0.57 ` 0.33 HO 2.22 ' 0.2 1 31 2.10 0.37 35 .3,(15 .' 0.51 2.14 ' n.`i i 1 0.50 0.55 4 1.33 0.55 78 2.31) ' 0.25 I',>nlt'<l fi.ltj from lo RSV 0016291 332 TABLE II ^SSlr.'TV THE WEIGHTED MEAN AVERAGES FROM 6 EXPERIMENTS * ON THE INDUCTION OK RECES SIVE LETHALS IN SUCCESSIVE STAGES OK SPERMATOGENESIS Brood Days after treament Cull stacks sampled '' Uami-tes tested Numlirr of lethals Prrrmt.iee lethals 1 11 in !V V 1V O-- .1 4 - T> 7 7 -11 0 m -i1 2 O1 SP. St St st. sr SC sc 40111 3353 1479 on 2 1 124 1 0.037 82 1 10 34 2 10 2.18 2 1)4 0.22 3.28 ` (1,11 2.30 :: 0.39 0.30 :l 0.21 0.89 11 0.28 2 24 1'014 .1 sri u-s ' f two \V> iriimilU t-.irh il h VCM a t lO.Oito. 1 10,000 .mil 50.000 1 M't" $j>. sporrn.; St, spe rm jIkIs, SC,. sprMttjtoevles; sn. sp<-rmat HCOtiU. experiments, spermatocytes were found particularly sensitive to killing by pre-carcinogens. The drop in the number of lcthals in broods 4 an 5, then, reflects the lower response to VCM of spermatogonia in whieht the ICR is only poorly developed. In the present study, it was considered useful to determine the ability of VCM to induce chromosome breakage. The results, presented in Table II, show that, in contrast with its substantial activity in the recessive lethal assay, VCM failed to produce dominant lethals to a noticeable extent in males that had been exposed to a high concentration of VCM (30,000 ppm) for 2 days. Although the size of the experiment was rather large, the survival rate in the treated groups did not differ significantly from that in controls. Moreover, one storage experiment for translocations was carried out, as a further check on the ability of VCM to break chromosomes in Drosophila. However, only 1 trans location was obtained amongst 1683 germ cells tested. In tests on the incidence of loss of X or V chromosomes or partial loss of tin* Y (Y1 : Ys> no significant increase over control frequencies was found, despite the large numbers of gametes tested (Table IV). These findings are entirelv in line with the negative results for dominant lethals and translocal ions. In the treated groups 6 mosairs were found, one of which was a double' mosaic (not included in Table IV). Since no mosaics were observed in the control groups, a slight mutagenic effectiveness by VCM cannot tie ruled out. What is obvious from these findings is that mutagenic effectiveness cannot be estab lished for VCM on grounds of chromosome breakage phenomena, though TABLE HI K AII. U UI Ol- VCM IN 1)1 (T DOMINANT LI JIlALS IN SPERM 1)111001) l) AND IN SPERMATIDS ( BROOD 1!) AFTER FNPOMfll l\ TO .10.000 I1 PM I- (111 2 DAYS Davs after Irodimcnl 0-2 0 -2 3-5 3 CmirriHr;i 1 i<>n Ippiti I O 30.000 1) 30,000 o CXt Nurtiln r of CCCV total 9889 8212 J 1.300 11 a u-lia til l (<. 8 1.0 1.9 82.9 1.0 82.8 11.1 81.8 - 2.8 Dkiiiiiuii lethals 'v 0 2.0 ; 2.9 0 1.2 - 5.1 f I TABLE r ERKQUh AND StO Brood RSV 0016292 KECF.S- dug by then, is only tlity of 1. show . . VCM .it had : Jays, m the t. one on the ' mans- nf the -pite rely in the mosaic * mtrol A nat is estabmough .MAT1DS 2.9 333 TABLE IV FKEQUKNCIES OK ENTIRE CHHOMOSOMF. LOSS LOSS IN SPERM (BROOD i) AND SPERMATIDS (BROOD 2) EXPOSED TO VCM OO.OOo'pFfclVVOR 2 DAYS Brnod 1 2 1 *2 Concentration Kopular (ppm) propcr>% i> 30.0DO o 30,000 O 30.000 7311 1 3.087 0 7 3.'. 8702 1 4.030, 22.t)89 Loss if X or V n T. 8 Oil Ra o.oi; 4 o.oo, 13 b O, I 5 12 0.00 . 0,02 21 0.00 0.02 Loss of yL n 3 0.04 2 0-01 -- 4 o.os .3 0.02 i 0.01 0 0.03 - 0.01 Loss iof YS n i o.m ia 0.01 _ 3 0.03 1 0.01 0.01 4 0.02 't 0.0 1 .*ir included i.m - included mutation induction provides evidence that there has been interaction with DNA under comparable experimental conditions. It has become clear over the past two years that there exists a significant difference between point muta tions and breakage effects, in that the concentrations needed to increase the amount of the former are consistently lower than those required for the production of breaks [ 21.23.14 ]. The "two effective levels", together with the peculiar saturation effect, explain the failure of VCM to register positive in the Drosophila assays on chromosome breakage. Failure of VCM to produce dominant lethals lias also been reported for mammalian test [15]. There was no significant increase in the number of early deaths per implantation after male exposure to such extraordinarily high levels as 3,000, 10,000 and 30.000 ppm. These findings have been interpreted as resulting front the inabil ity of active metabolites of VCM to reach the testis [15]. On the basis of our findings, a negative result in the dominant-lethal Lest cannot be construed as a decisive one for demonstrating the absence of mutagenic effects. Long-term experiments The lowest effective VCM concentration recorded in the short-term inhala tion studies was S50 ppm (2-days exposure). In a second series of experiments we attempted to evaluate the mutagenic activity of VCM on Drosophila males at lower concentrations comparable to those to which rats, mice and hamsters were exposed in tests on carcinogenicity. Frequencies of recessive lethals obtained after exposure of males to 30 and S50 ppm for 17 days are given in Table V. For VCM at 850 ppm the estimated mean mutation frequency was 1.02 i 0.167c, this being significantly higher than the frequencies found m the controls. The experiments with 30 ppm consisted of three treated series, two of which had concomitant controls (scries 1 and 2). The mutation frequencies seemed slightly enhanced in all three experiments with 30 ppm. Statistical analysis by the significance test of Kastenbaum and Bowman and the xJ test did not reveal any significant differences in the mutation rates between treated and control groups. The best result (on the verge of significance) was ob tained when the data from broods 1 and 2 were pooled and compared with RSV 0016293 CgMHttBrflaMifl 334 TABLE V ' __ INDUCTION OF SEX-LINKKD RECESSIVE LEVELS OF VCM FOR 17 DAYS ri&S? IN DROSOPHILA MALES EXPOSED TO LOW Senes Concentration VCM tppm) Brood Days alter treatment Number nf chromosomes tested Number nf Irthals found Percentage lethal* 0 604 2 II A- 7 476 0 III 7-10 527 2 I--III 0-10 1 697 4 0.29 i 0.20 - 0.38 ` 0.27 0.23 0.12 30 0-- 4 1163 3 II 4- 7 7GH 4 III 7-10 588 1 1 - III n -in 25111 K 0.26 0.15 0.52 0.26 0.17 0.17 II..12 0.1 1 20 I II III ! --Ul -0--4 4- 7 7-10 0-10 1001 724 605 2424 1 1 2 4 0.09 * O OM 0.1* i 0.14 0.33 r 0.23 0.17 0.08 2 30 I 11 III I --HI 0-- A 4- 7 7-JO 0--10 1205 605 103 2093 0 9 0.51 : 0.20 0.33 t 0.23 0.43 0.14 3 30 l 11 111 i-m 0- 4 4- 7 7-10 0--10 1 468 4*10 64 3 2001 3 4 1 8 0.20 0.12 0.82 0.4 1 0.1G (MG 0.3 I 0.1 1 3- 2 0 i n m i-m 0- 4 4- 7 7-]0 0 -- 10 1 785 1200 1 130 4121 .3 1 4 8 0.17 . 0.09 0 08 0 08 0.3 5 0.1 8 0.19 11.07 1 -3 30 - 4* 850 i n in l-IU i n m i-m 0- 4 4- 7 7-10 0-10 0- 4 4- 7 7-10 o--in 3726 1 803 1 124 7213 20.34 1 001 904 3939 13 10 2 28 r> 40 O 33 0.0*1 0.54 - 0.1 7 0.14 0.10 0.35 0.07 1.38 n,2G 0.00 * 0.22 0.77 0.29 1.02 0.16 Mean values from two experiments. the pooled data from the controls. However, since an increased mutation frequency was consistently found in all three treated series, the question of whether VCM at 30 ppm can induce mutations seems entirely a statistical problem, for with a larger materiel of about 20.000 gametes (instead of the 11,334 tested in our experiments) the differences would become statistically significant (25). In sum ments, ti> at the vci (ration th VCM in resolving Acknowle This in from the also reocr of Lcidoi excellent Sankaram Reference Amlersm 2 B-irbin. micr'sou and Qio|> 3 Qjrorh. Mini rill4 Oi.imlli' tritiati-d i r' Dur.il in ,i 6 1- uni s-O T Ciri-iui. 11 H Infant. , rfilur nii-, 9 K iislt'lili.. 1 f**t| lie lit 1 <i l.imldi s . 1`iil.t. 1.2' 1 1 I,, i p l-*n. U Cm,.-fli-rls n 1 2 Maioiii-.-.' U. < , 38 i 13 <t`7T.) a. M..In.m. i Puri'h.is. i nn I trlilif. li.iiunu:. ini l.ilmli. S.*i iil%.i r.u i iiiilurt-cl i (i`*r.7 I .Ill s, A it\ r Li'i RSV 0016294 JSF.D TO LOW ntae* lethals 0.27 0.12 0.15 0 26 0.17 0.11 0.09 0.14 u.23 O.OS O.20 0.23 0.14 0.12 0.41 0.16 0.1 1 (1.00 H.08 - 0.18 0.07 13 0.09 I - 0.17 ; 4 - 0.10 16 * 0.07 38 0.26 60 0.22 7 r 0.2(1 "2 1 O.JG aod mutation .e question of > a statistical instead of the ne statistically 33& In summary then, on the'basis^dBSJ^Sstnsistent effect in three large experi ments, the conclusion seems justified CH(fi/VCM still shows mutagenic activity at the very low concentration of 30 ppn%ffhi& constitutes a far lower concen tration than those of 625--200,000 ppm used to demonstrate mutagenicity of VCM in other assays [2,3,7,11,13], and this further documents the high resolving power of the Drosphila recessive-lethal test. Acknowledgement This investigation was supported by PHS Research Grant No. ESO 1 27-03 from the National Institute of Environmental Health Sciences (U.S.A.), and also received support from the contract between the EEC and the University of Leiden, contract No. C 30-74-1 ENV N. We thank Netty Pex for her excellent technical assistance. We are indebted to Prof. F.II. Sobols and Dr. K. Sankaranarayanan for critically reading the manuscript. References 1 Anderson. K. V.. Tup fihv eht-mira) prnclucis. Clicm. t'.iift. Nous, 5 (1975) 29 -- 33. 2 B.irbin. A.. II Brusil. A. Cmisv, I'. JacnmRi'nn, C. Malavrillc, U. Mnntcsano and 11. Bartsch. Livvr- i'iir i .'si't'M'-Tm-iii.Urcl forma turn >'f alkvlnlim: acents from vinyl bromine and vinvl chloride. Bioehrm. .md Dii'phw l!ci. C"i'ini.. 6 7 ( 1 !>7M 696--0033 B.irlsch. U.. C Marseille ami U MonU-sann, Human. ral and mouse liver-mediated lmitacemeits nf i in' I chloride in S' r v p/n rnur m in si Tams. Ini. .1. Cancer. 1 3 (1 975 I 4 29 --437 . 4 Ch.mdli'i . A.C. .md A .1 Bateman. TimiiiR of spcrmaloceiiesis in Drosophila nwlanotHixIrr usms tntiatcd 11 j v-in id mi*. Nalure . 4 8 1 2 (1 962) 299--300. 5 On r.ii m.m. A.. K Hirsrhhnrn jivl 1..I. Selikuff, Vinyl chloride exposure and human chromosome ahiTi atoms. Mul.ilnm Krs.. 31 (19*5) 163--168. 6 I- uues-Crj' |i>l". h .. R. I.ambert. .! Lmdst en. L. Flireilbore. A ,T. Nal.ir.oan and S. Osierulkar, Ch mums. 11 in- ,i bvrr.iluuis in nrkrrs e x posed in iinvl chin r id, .The Laurel. 22 (1976) 4 59--460. 7 (7 mm. 11.. G . Bouse. '/ . 1? adu .in. I). Reichert and D. Henschlrr. Mutanenicuv of chlorinated rh vleues as .1 hinrlnm <>( m.'luboirc oxu am form a l inn. Riorhem. Pli.mnae.. 24 (1975) 2013 -- 2017. 8 111 f .i 111, P.l .IK. WiiBoncr. A ..l. Me Mieiiad, R J Waxueilrr arid II. Kalk. Genetic Risks nf vim I chloride. The I. a nee l. 3 (1976) 734 -- 73 5. 9 k j Mi-iib.m hi. M A . .mil K .(). Dim in.in. Tabl< s for <1 derm mine the statistical significance of inul.itiou frequencies, .................... I(rs.. 9 (1 970) 527 -- 549 ID I.ni'Ui,'. 1)1.. and K.ll Or.41, Genetic vanalmns nf In nsuphila niriaiiucaslrr_ Carnegie Jus! Wish. I'iiM. 627. ( 1 >1681 472 DP 11 l.opnnin S' . I< U.i r.ib'. S U.i r> >nr eili. C. Bauer. G. Urn n /ell i. A. Cam me II mi, G. Ccrr icn.ini. C Cm m. (I Cer' jsi . C. 1,1-i'nrini. It . N u*n. Anna M. Ri'ssi. G. StrcLli and G. Turchi. Kvulu athn nf the criu*t ic rffecis induced b\ vinvl chloride nmiiiimiT (VCM) under mammalian metabolic acUvalmn studies in vitro jnd m vimi, Mutation lies.. 40 (19761 85 --96. 12 Maamiss"n. '. and C. Kamel, Millj genie ef(eels nf vinvl chloride in Drosophila rnrlanocaslcr. Mulalinn llis., 3 R (1 976) 1 1 5. 13 Malavrille. C.. 11. B.irtsch. A. Barbin. A M. Camus. R. Morilrsunn, MutaRenicitv of vim 1 chloride, rhlom^thi lenenyide, ehloruacel.driehvde and rhinro**thanol. Bioclicm. Bioplivs.. Res. Comm.. 63 (1975) 363-370. 14 M a Horn. C.. An example. Vinvl chloride, Am bio, 4 (197 5) 1 8--23. 15 Purchase. I.I'.H., C.K Richardson mil I). Anderson. Chromosomal and dominant lethal effects nf Mini chloride. The Lancet. II (1975) 410--411. 16 Kantiuc. lr.. A. Johansson. C. Kamel and C.A. Wachtmcisler. The niuiagenintv of vmvl chloride utter me lab oil c art i va lion. Am bio. 3 (1974) 194 -- 197. 17 Smikaranai.ivanan. K.. The effects nf imrngvn and oxvfcn treatments in the frrriueneii-s of N-r.iyinitueeti dominant lethals on the phvsinlogv of the sperm in Drosophila nialaimeaslcr, Mutation Res.. 4 (1967) 6 41 --667. 18 Tales. A.D., CviodifferenliHlion during spci iiMloRenesiS in Drosophila ntrlauuuaslrr. Thesis, (miser;, il\ of Lridcii. 1971. RSV 0016295 1 9 ViuU. A. Biemu a"'1 A. Caputn. OiviVpA^taespons** of rat kin. luncs and bones to vim I chlo ride . Caneer Res., 31 (1971) 51G --522. 20 Vo*el. F.. and F.H. Snbels. Tin* function of Drong)U*rtn Bcnru&tOMcolocv testing. in A. HolUcnricr (nd). 'CHrmic-iil niulaeens. Principles and melhofls"IorUwir detection". Vol, 4, Plenum Press. Nv York. 1976. pp 31--142 \'nit<'l, F. and B. Lcndi. Cunecnti.ition-irfecl studies ith MMS. TEB. 2. 4. 6-lriCI"Pl)MT, amt DKN on tin- lndiatinn of rinnim.mi and recessm- lelhals, fliroiiimmiif loss and translmaliuns in Drosophila sperm. Motulinn Research. 20 (1075) 3K.1 --(190. 22 Voti'l K-. Chnmichi' Knnsiiiutinn uml im'tacenc tVirkiuc. V|. indnktion dnimiiantrr und re/cssiv- Ri-sr hli'cltlsKotuinrlriHT l.el al in u t.i iinni'M durt'li Ar5'ldialkvliru/cm- bei Drosophila mcianoeos/rr. Mut.itnm FUs.. I I (1071 ) 397-410. 2.7 Voeel, K... Mul.iCrmr urlnilv "( cvclniih'isphamide. trofnsfamidi'. and ifnsfaiiiiiJe in Itroxuptnla rUincnxlrr. Specific induction of icecssive lelhals in the absence of delectable chromosome break- ace. Mutation Hes.. 17 n07.'>) 221-22B Vogel. F.. Some aspects nf the delection of potential mul.iEcnie aR.nts m Drosophila. Mutation Ur* . 20 (1 075) 24 1 --250. \\ u ruli'v, K.F., l' tii.if ,md \\ . (leech t old. Rt.ilixUc.d problems connected with 10 sox "linked iiccssur 1,.| |li4| in t i fluUi ft'. I. TOP use of tip* Kaslr nh.iu in - Uo\s m.n i lest, A rchp (ur ln`hi`1 , 48 (1975) 108 -- 173. ,!iriinrii: IIS I biit/ii. (Received Ji (Revision H-. Accepted I' Summary Chronn* evils crow compound cidos and t Sixty-th with dost" pounds kn urethane* ; In* enreimn nitroyiianu sodium nil ti's1 s, hut l Inltoduclii The del nu*nt is m rapid and assays in n iiianiinaliai also he ap| substances chances m; certain mu if the eomi bacterial a> Mil.',-. n /(`r.sr'urr/i, <>7 (l!>7!)) I T.'t I fU Kl .'\i i 1 Nii i r ' HirlNmJ HiomriliCill l`n JUL 12 879171 mi rr uiKN'K.rrv ok viNYL onr.QRiDK, viNvr.rnFNF rHT.onmr and nil dtOI'HRNR IN V70 CNINKSi; IIAMSTRII CRLLS I- DKKVON T. KLmOKl * 1 t Chemical Carcinogenesis, International Agency for Research on Cancer, 150 court -t Fi.oinn.- >J72 l.}on Cedex 2 H'raruc) ' i ' .,!.i JuiK- 11)78) i* \ !muii rKVK : 1 January 1979) ( < '-d l ii .i.muary 1 D7`.)) mu: mry I mutagenicity of vinyl chloride, vinylidone chloride (1,1-dichioroethylrn.-i aiul ri loprene 12-chloro-1.3-butadiene) was tested in V79 Chinese Ii.i 'T cells in the presence of i 15 000 X" liver supernatant from phenoiMri.i:..iH'-pr ' `.mated ruts and mice. Mutations in terms of 8-azaguanine and i-u .i en r.-sist..nee were induced in a do.-,e-relalod fashion Ly exposure to vapour ni' \ , I chloride in the presence of liver supernatant front phenobarbitone-pre* ... iii<(I rau. i .uiourj of vmviidcne chloride and chlormwvne induced a dosere'. i toxicity in tin* presence ot liver supernatant from phenobarbitone-pre't c i| ruts, i-ut these two compounds were not mutagenic in V79 Chinese n t cells under the present assay conditions. The results are discussed with t to tl;. metabolic activation of the compounds ami to the correlation ii h ' heir can-mogenu.ity in inanfgpd experimental animals. \ i-innated hydrocarbons, suv.li as VCM, VDC and chloropcene, are pro1 Inc.' I in Lip puanmies and an- widely used in the production of plastics and '.ic ruhiiers. Tijcre is uniMpiicocal evidence that VCM is carcinogenic to nun;. us well o to a number of other animal species (I'd- VDC, a structurally . '.o I compound and a copotym- r of VCM. is carcinoi ,-nic to mice and rats, wi, data . ' availul ile on its cun. mogenicity to man ( 5 j. Chloroprene has so i n imi been found to he carcinogenic in mice and rats, although some human I 'fiu ..ii i, 4: ni'iMrtiu rni ot r C.-ll U .scar<- h. [ nsliLu to .it Science, University of i. > <i. -Ui'riik.mi-a.ii. \1 iu.itii-ku. Tok - >> I < I n ( l.ipaii . 1 1 'ithui. A '/. \'. n ' 'aicuanini' r- u>i.> i C v f .. (<>i c<lf scr 3. 17 Uju < j u|un,.iljni fraclioi. . < U vinyt ilil rule iinoiio. 0.1 -aU-UU '`thyUoy-v. < U/\r, ouabain resistant <-, . i VL)C. vinyiniettc Chl-ri.t- 174 studies revealed an increased cancer incidence and a high incidence of chromo somal aberratio'|[|^^S!,!P5ntjheral Urmp^^ges of workers exposed to this sub stance [15]. AJljpfee of these chlonS^QsHiijjCaitobns have been reported to be mutagenic in microbial systems ri^^^l4^^~24,26,28.30,31 ]. The adverse biological effects of these co-many*Other chemical car cinogens, are attributable to the formation of reactive metabolites by micro somal mixed-function oxidases. In recent years, several short-term tests using mammalian cells have been developed: of these, the mutagenicity test in V79 Chinese hamster cells has been relatively well evaluated, using a series of direct-acting carcinogens and a number of those requiring metabolic activation, e.g. N-nitrosamines [8,19,20], polycyclic aromatic hydrocarbons ([13,17] and unpublished) and aflatoxins ([17,20] and unpublished). In mutagenicity assays of some ester derivatives of N-hydroxy-2-aminoTluorene [18] and of praziquantel, a new anti-schistosomal drug [3], this system was found to be useful, when incorporated into a battery of short-term tests, in increasing the predictive value of these tests for carcino genicity. We now report on the mutagenicity of VCM, VDC and chloroprene in V79 Chinese hamster cells in the presence of liver post-mitochondrial frac tion plus cofactors. The results are discussed in correlation with the carcino genicity of these substances in man and experimental animals. Materials and methods VCM (purity 99.9%) was generously provided by Rhone-Progii (Lyon, France); it was contaminated with ethanol (30 ppm), water (20 ppm), methyl chloride (<20 ppm) and non-volatile substances (<5*ppm). VDC, containing 0.3% 4*methoxyphenol as antioxidant, was obtained from Merck-Schuchardt (Darmstadt, Federal Republic of Germany). Chloroprene (2-chloro-l,3-butadiene; purity 99%) was provided by Distugil (Le Pont de Claix, France); it was contaminated with 0.8% 1-chloro-l ,3-butadiene, crotylchloride at <200 ppm and 2-chIorobutene-l and 2-chIorobutene-2 at 80 ppm. 8-Azaguanine (Pfaitz and Bauer, Flushing, NY, U.S.A.), ouabain (Sigma Chemical Co., St. Louis, MO, U.S.A.) and agar (special agar Noble, Difco Lab., Detroit, MI, U.S.A.) were purchased. Autociavable Eagle MEM was obtained from Flow Laboratories (Irvine, Ayrshire, Great Britain). FCS was purchased from Grand Island Biologi cal Co. (Grand Island, NY, U.S.A.). Mutagenicity in V79 Chinese hamster cells Mutations in terms of resistance to 8-azaguanine, a purine analogue, and to ouabain, a specific, inhibitor of Na*/K*-activated ATPase in cell membranes, were recorded iiM&f79 Chinese hamster cells. Experimental procedures were a modification of t^piassay system for nitrosamines reported previously [ 8,19]. V79 Chinese hamster cells were plated at a concentration of 1.5 X 106 cells/ 60 mm petri dish and were cultured overnight in Eagle MEM, supplemented with 10% FCS. They were then incubated at 37C for 5 h or more in 2.5 ml of the reaction mixture, either in liquid suspension or suspended in 0.3% agar. The reaction mixture consisted of 0.75 ml S15 post-mitochondrial fraction from the livers of BDVI male rats or OF-1 male mice, which had been treated with phenobart Sdrensen : MgCla ' 61 *imol glue MEM and vitamins f ensure sur agar was a The pei the vapou: the desicc levels corr the pressi which are volume of desiccator min at 37 atmosphei foil, was t trations o: mined prr riod, th ^ere was: control ce chemicals (glucose ' reason, cc plating efi those for ' The cel mutagenic 60 mm pt genesis, 2 respective tion drugj 1 mM oui later: for non-essen stained w frequene\ plating ef confirmee twice wit refer to t OUAr col which wt used for s RSV 0016298 I7f> phenobarbitone (1 mg/ml) in drinking water for 7 (lavs; 0.75 ml modified Sdrrusvn phosphate buffer (0.055 M, pll 7.4, containing b.9% NaCl and 1.0 mg M;CI: 0H:O per ml); and 0.75 ml phosphate*bufferel *uhno containing 12.5 ^mul glucose 0-phosphate, 2 ^mol NADP*, amino acids, vitamins from Eagle MEM and 0.25 ml FCS. (Components of culture medium, such as ammo acids, vitamins from Eagle MEM and FCS, were included in the reaction mixture to ensu e survival of the cells for long incubation periods.) For agar suspensions, agar was added to modified S0rensen phosphate buffer at 1.0%. The petri dishes were then placed in a desiccator (10--151) and exposed lo the . jpour of the chemicals being tested. For VCM, the gas was introduced into the desiccator, which had been evacuated, with the aid of a manometer, at level' corresponding to 5, 10, 20 and 30% in volume; after 20--30 min at 37<'C the nressure was adjusted to atmospheric level. For VDC and chloroprene, which are liquids at low temperatures, the volume calculated to give a known volume of vapour (on the basis of Avogadro's law) was frozen and placed in the deMccator. 'I'm-* desiccator was partially evacuated and incubated for 20--30 mu: ai 37( to allow the compound to vaporize; air was then introduced until atmospheric pressure was obtained. The desiccator, covered with aluminium foil, was then incubated in a water-bath at 37C for 5, 10 or 15 h. The coneenitMtt- ns of the compounds in aqueous phase under these conditions were deter mined previously, using gas-liquid chromatography [6,24]. After the exposure pern- the vapour was removed under vacuum and replaced by air; the cells were washed twice and incubated for 2--3 h in fresh culture medium. The co, Mol cells were treated in the same way, either simply in the absence of the chemicals or in reaction mixture from which the S15 fraction or cofactors iclucose 6-phosphaie and NADP*) had been omitted. For some unknown reason, controls for which cofactors were omitted repeatedly yielded a lower plating efficiency as compared with cells incubated in the complete mixture or tho>e for which the S15 fraction was omitted (see Table 2), The cells were then plated for the determination of the cytotoxicity and mutagenicity induced. CytotojTftty was determined by plating 100 cells/ t'h mm petri di$h (4 dishes foi^Kjh point) and cultured for7 days. FormutagmrMs, 2 X 104 and 10s cellsl&fmm dish were plated for AZAr and OUAr, respectively (S dishes for each pofnt). After an expression period of 48 h, selec tion drugs were added to give final concentrations of 20 ug/ml 8-azaguanine or 1 uiM ouabain. The media containing the drugs were changed once, 5--7 days later; for the 8-azaguumne medium, FCS was replaced by dialysed FCS and :um tosL-tuial amino acids were added to 0.1 raM, The cultures were fixed and stained with Giemsa at 12 days for AZ.V and at 14 days forOUAV Mutation frequency was calculated per 10s survivors, the number of cells plated and the plating efficiency being taken into account. Reproducibility of the results was confirmed by repeated experiments; 5 times with VCM, 4 times with VDC and twice with chloroprene. Means and standard errors in the figures and tables refer to the variability in a single experiment. The stability of the AZAr and OC.V colonies was demonstrated in 10 and 11 isolated colonies, respectively, which were cultured for more than one month in tl.c absence of the drug usoii for selection [8,19]. RSV 0016299 o[ VCM____ . .. ^ 1':. <h eompount additions for detecting the mutagenicity of 'sgere composed to 20% (v/v) VCM in air ". *0 or 15 h in the pr< 54?;om phenobarbitone-pre-treated ? n< . xt*-' cells were suspende<FHIfiFm*liquid (left column of Fig. 1) or in 0.3r? :f- .U- iright column of Fig. 1). Agar suspension was used, since VCM has been reported to induce mutations in Salmonella typhimurium only when tested in the agar-incorporation assay and not in the liquid-suspension assay (5]. Post- mitochondrial fractions from phenobarbitone-pre-treated rats or mice have boon shown to increase VCM-induced mutagenicity in 5. typhimurium over that obtained with fractions from untreated animals (5,24]. As seen in Fig. 1, the reaction mixture, in either suspension, was not by itself toxic to the V79 cells within 10-h incubation time; 15-h incubation in liquid Liauio AGAR 40 20 30 20 10 -3. INCUBATION TIME {h#u.) Pie. I. jR* courses of (he induction of c y to tonicity and of mutations h v exposure to 2(V- (v,'v) VCM *n sir 1 j*a reaction mixture containmc SIS liver fraction from f>iirii.l>.irLiiliiic-|irt-Lrf.Ucd rats plus coiactor*. in either liquid suspension (left column) or m 0 3% agar suspension (right column). Controls (C) were treated in the same way. but without VCM. Cvtotoniru v w.ts ilrernninrci from the platint i Ifirienr v and is repressed * percentage o( pin Unit ((>*,<nr v mrimlToh Mnl-il ton Irniuciir v is i-s mm-vx <1 n* llir mimlii'r <<f rrsistnnl colonics per 10* survivors. lukim: into > , < ..uni do- uiiinlii-r <>( cvtli plutcl un.t Itv pl.iluik' i llii iriif v, liars: KK. RSV 0016300 caused a dturease in plating efficiency of about 50~ . and 15-h incubation u. -U'p.'.-p.Nii'jn caused only slight toxicity. VCM induced cytotoxicity and nullaynf.ji .y vvhen added in liquid suspension or in suspension. The- Urnc eia rjes of mutation induction were much more gradual in agar suspension than in liquid incubation: with liquid incubation, the highest mutation frequencies \wtv observed at 5 h and were followed by a sharp decline: whereas with agar suspension, mutation frequencies reached almost a plateau after 5 h, suggesting that microsomal enzymes are stabilized in this suspension, as reported by M.davetlle et al. [25]. In both the agar and liquid incubations, the highest number of resistant colonies per 105 survivors was about 30 for AZAr and 3 for Of' \r, obtained at 5 h in liquid incubation and at 10--15 h in agar incubation. Consequently, incubation for 5 h in a liquid reaction mixture was chosen for the following experiments. VCM induced dose-related mutations and toxicity when given at concentra te .s of between 5 and 30% (v/v) in air (Fig. 2). With a concentration of 30' -. it-v.), mutation frequencies were 10--20 times that of the spontaneous back- L rf i CONCENTRATION OF VCM (imairj I-''*'-- VCM induced cy lu toxicity and mutagenicity m V79 cells t* a function of concentration rVCM in J" <T. v/v>. *. complete reaction mixtuiv containing SIS liver fraction rrom phenob&rtiitonc-pre-lrcalcd r. i j>lu* cofaetors. rt-acuon mixture containing no SIS. Cytotoxicity and mutation frequency arc , d a* described in ihv iegond to Mg. I. RSV 0016301 178 ground: 51 AZAr and 4 OUAr cofSip'oc-were obtained per 10! survivors. Neither toxicity nor mutation was in< the S15 fraction was omitted from the reaction mixture (Fig. 2). Mutagenicity of VDC The mutagenicity of VDC in V79 Chinese hamster cells was tested by exposing them to 2.0 and 10.0% of the vapour in air for 5 h in reaction mixture containing S15 fraction of liver from phenobarbitone-pre-treated rats and mice. S15 fraction from mice was used because VDC was mutagenic in S. typhimurium only when tested with mouse-liver homogenate and was almost inactive when tested with liver homogenate from rats [6], although VDC has been shown to be carcinogenic in both species {15]. These results are summarized in Table 1. A dose-related toxicity was induced when ceils were incubated in reaction mixture containing S15 fraction from rat liver plus cofactors, but VDC was not toxic with the S15 fraction from mouse liver; this suggests that a toxic metabolite(s) was formed by rat microsomal enzymes. However, no increase in the appearance of AZAr and OUAr colonies was observed after incubation in the presence of liver SI 5 fraction from either rats or mice. Mutagenicity of chioroprene V79 cells were exposed to 0.2, 1.0, 2.0 and 10% (v/v) chioroprene vapour in air for 5 h in reaction mixture containing S15 fraction of livers from phenobarbitone-pre-treated rats, plus cofactors. The results are summarized in Table 2. Chioroprene was toxic to the cells, in the absence of a metabolic activation system, at concentrations of more than 1%. This toxic effect could be a direct action of chioroprene or that of one of its enzymic (from the V79 cells) or non-enzymic breakdown products. An enhanced toxic response was observed when the cells were incubated with S15 fraction plus cofactors; this suggests TABLE l CYTOTOXICITY AND MUTAGENICITY OF VDC IN V79 CHINESE HAMSTER CELLS IN THE PRF.5FNCE OF POST-MITOCHONDRIAL FRACTION (SI5) OF LIVER FROM PHENOBARBITONE-PRETREATED RATS AND MICE Concentration in air <%> Incubation Cytotoxicity * Mulacemcity b A7.Ar OUAr 0 2.0 10.0 0 2.0 10.0 0 2.0 10.0 .v complete, with rat SIS T' complete, with mouse SI 5 minus SI 5 63.0 15.3 2.3 68.3 62.3 64.4 6 2.8 49.5 60.3 4.5 4-2 . 0 6.4 3.1 3.9 1.0 3.8 2.1 0.4 0 0 0.4 0 0.2 0.4 0.3 0.6 * Expressed as plating efficiency: the percentage of cells that formed colonies. b Mutation frequency is expressed as the number of resistant colonics per 10s survivors, the number of ceils piated and plating efficiency being taken into account. TABLE 2 CYTOTO IN THE I' BJTONE-f Concentra In ui (%) enzym mutat: presen Discus Tat chlorc graphs and, f been produ tested TABLI SVMM ROPK VCM VDC Chlorc Care RSV 0016302 TAIU.K i \ i c 11 >.\ n 11'\ \mi mi' i i nu:i rv nr 11u m oru knc in vti i iiim si. iiamstkk cti.i.s IN 1 III I'lll SIM I. Ill'' I'OaT-MI II ICIIUN IIIU A I. I ll ACTION (Slf>) 1)1 I I \ l I' IIOM I'MEN < 111 Alt nil UNI l-lu I l< h.\ ll l) H ATS J I'ondi'rt ration in air (' e | Incubation Cytotoxicity b Muiaiii'iiuily C A/.Ar OUA1 ii it * 10 30 HI 0 u 1) J ] '1 ' ll 10 II ' 'IrwAntc minus cofdCtors ii4.f. 3t,.H 1) 0 0 31.3 23.8 11.0 8.8 0 2.9 2. it 0 O 0 2.1 79 7.7 0 0 0.8 1.4 0 0 0 0.8 1.0 0 0 0 ih. . 11* uf in<> mdepcnd'-m ; vpcrinu'nt* arc cuinmnul: ehluroiircne was tested at 0, 0.2 and 1,0"e in T , n i .mil ul (I, v.O and li i.cl'i m k \ pi. II. Values for con Iritis <()%) are taken (rorn Ex pt. 1. although -miil.ii ul'ai v-i r ibtjiruil in k.xpt. 11. *' 1 \ i .i ui|i , (fi cn nc\ . tiie pei cent u ( cells that formed colonies. ^ Mui.ii, .a (ri cfuiiM. v is c x pi ess* d as the number of resistant Colonies per 10s survivors, the number of . IT ,jI ili'il ami ijunu effieiencv bemu tnken into account. enzymic formation of a toxic motabolite(s) from chloroprene. However, no inu! i'ions. in terms of AZAr and Ol'Ar, were induced by this compound m the j.r -'vm i' or absence of metabolic activation systems. I JisCUnMlon Table 3 summarizes the data on the carcinogenicity of VCM, VDC and chloroprene m man and experimental animals, taken from the IARC Mono* graphs 11 5 |, and juxtaposes these-with data on mutagenicity in S. typhimurium and. from the present study, wftlMhat in V7& Chinese hamster cells. VCM has been shown to be carcinogenic in man as well as in experimental animals: it produced Lumours at different sites, including angiosarcomas of the liver, when levied by tile oral route or inhalation exposure in rats, mice and hamsters. ' \m i 3 M'MMaKY of data on carcinogenicity and MUTAGENICITY OF VCM, VDC AND CHLO- '(! ! IO -t. i' 111111<iutul tvidoncL* of coroncicciiiCity a in Kvjcitfof mutagenicity in Man Animals S. fyphimurium V79 VCM VIK i tiT.i..i.niie * . _ + ; 7 < it. iiinui-iiicily .!ju arc taken from IA RO \1nr*oi'tj|>h Vol. 19 (1 r>|. Sec . \ for detaili. 180 Epidemiological studies'have shown that AttptSure to VCM results in an increased carcinogenic risk to man, involving liver, brain, lung and the lymphato-haemapoietic system. The data in experimental animals, also indicative of a carcinogenic effect of VDC, are inadequate to allow an evaluation of the carcinogenicity of VDC or chloroprene. Occupational exposures to chloroprene have, however, been reported to be associated with a variety of toxicological effects, including chromosomal aberrations among exposed workers. In one study, an excess of lung and skin cancers was related to occupational exposure to chloroprene. These observations raise the possibility that chloroprene is a human carcinogen. The present study confirms previous results on the mutagenicity of VCM in 5. typhimurium [1,5,10,24,26,28], Escherichia coii K12 [11], Schizosaccharomyces pombe (22], Saccharomyces cereuisiae [22] and Drosophila melanogaster [23,30]. This subject has been reviewed by Bartsch and Montesano [7], Bartsch et al. [4], Fishbein [9] and the IARC Monograph [15]. Recently, Styles [29] reported VCM*induced transformation in BHK-21 cells in the presence of a microsome preparation. Although VCM induced mutations in S. typhimurium in the absence of a metabolic activation system, a much higher mutagenic response was observed when a 9000 X g supernatant from rat liver was added [1,5,26]. Pre-treatment of rats with phenobarbitone increased the mutation rate over that with a fraction from untreated rats [5,24]. Mutations have also been observed in the presence of liver homogenates from mice [5,10, 22,24] and from human biopsies [5,24] as well as in the host-mediated assay in mice [22). These results, together with those from the present study, indicate enzymic formation of a mutagenic metabolite(s) from VCM. Among the possible metabolites that have been tested, chloroethylene oxide, an obligatory epoxide in metabolism by microsomal mixed-function oxidases, was the strongest mutagen in V79 Chinese hamster cells [12] as well as in microbial assays [14.21,24,27]; chloroacetaldehyde was mutagenic [5,12,21,24, 26,27], but chloroacetic acid was not mutagenic, in such assays [5,12,24,26, 27]. The above data strongly suggest that chloroethylene oxide is the metabo lite of VCM that is principally responsible for the various adverse biological effects of the parent compound. VDC and chloroprene may follow a metabolic pathway similar to that of VCM [2,16]. VDC and chloroprene have been shown to be mutagenic in microbial assays: VDC produced reverse mutations in S. typhimurium and in E. coli in the pres ence of a metabolic activation system [6,11 ]; chloroprene induced reverse muta tions in 5. typhimu&iim in the absence of metabolic activation, but an increased mutagenic response was observed when liver homogenate from mice was added to such assays [6]. In Drosophila, recessive lethal mutations were induced by feeding male flies with chloroprene [31], In V79 Chinese hamster cells, how ever, these two chemicals induced a dose-related toxicity in the presence of liver SI 5 fraction from phenobarbitone-pre-treated rats but were not mutagenic within the constraints of the present assay conditions. This suggests that there is at least enzymic formation of toxic metabolites from VDC and chloroprene by microsomal enzymes. Acknowledge The autho with gaseous for editorial the critical re NCI Contract References 1 Andrews. A. it vmvl chlondc 2 Barbin. A.. I; microsome-n. Biophys. Res 3 Bartsch. H.. Rainaidi. F. schistosomal 142. 4 Bartsch. H.. metabolism o: 5 Bartsch. H.. C vinvl chlondc 6 Bartsch. H.. i chloride and 7 Bartsch, H . 32 (1975)93 8 Drevon, C.. T line by uno Toxieolnei. h 9 Kishbcin. I. . lion lies . 32 ' 10 Garro. A.J.. ex tracts and t 11 Grcim. 11.. G carrinoi'cnici". Pharmacol.. 2 12 Huberman. Ivmvl chloride 639--1>4- 13 Huberman. c Kens, int. J. C 14 Hussain, S.. metabolites, i 15 International genic Risk of Lyon,1979 16 Jones. B K . act.. 20 (t97)i 17 Krahn. [).h. cells by polyc 18 Kuroki. T.. a Huort-nc in V 19 Kuroki. T., C cells bv vanoi 20 Langenbach. with liver can 21 Lopneno. N. Nieri. C. Lep vinyl chloride RSV 0016304 I .'It Acknowledgements Tin authors thank Mr. C. Malaveille for his guidance concerning treatment with gaseous compounds; Miss J. Mitchell for secretarial aid; Mrs. E. Heseltine for editorial assistance; and Drs. L. Tomatis, R. Montesano and H. Bartsch for the critical reading of this manuscript. This work was supported in part by LTS NCI Contract 1CP-55630 and CEC contract No. 190-77-1 ENVF. References 1 Andrews, A W.. F S. Zawistowski and C.R. Valentine. A comparison of the mutagenic properties of mu vl chloride a rut methyl chloride. Mutation Res.. 40 <1976) 273--276. 2 M.ii <n. a . |I. Until. A. Cruisy, P. Jacquignon. C. MaUveille, R. Montesano and H. Baruch. Liver h: i<T.)Snme-in<MJij' cd formation of alkylating agents from vinyl bromide and vinyl chloride. Biochrm. Eh.ohvs, Res. Commun., 67 (1975) 596--603. 3 II , h, H.. T. Kuroki. C. Malaveille, N. Loprieno, R. Barale, A. Abbondandolo, S. Bonatti. G. I'jmjkU. E. \'oC,,l and A. Davis, Absence of mutagenicity of praziquantel, a new. effective anti* - hivtiisumal ,lrun in bacteria, yeasts, insects and mammalian cells. Mutation Res., 58 (1978) 133 -- I1 l II n .cli. II . C Mjlavftlli', a. Barbin. H. Bresil. L. Tomatis and R. Montesano. Mutagenicity and i'iv' i 'nil sin iui)l r hloride and tela ted compounds. Environ. Health Persp., 17 (1976) 193--198. r, I! .> '.. II.. C. Malavnllr and R. Mnnicsjim. Human, rat and mouse liver mediated mutagenicity of Mi.*, i tiiltiri.il* in s iyphi.-'itiniim strains, int. J. Cancer. 15 <1975) 4 29--13 7. i D .rum ![.. C. MalavciU. R. Montesano and L. Tomatis. Tissue-mediated mutagenicity of vinylidine i and 2-ehlurobui.idivnc in Salmcnc '.In typhupurium. Nature <London 1, 255 (1975) 641--643. 7li. I.. If an-: It Miintrsar.'i, Mutagenic and carcinogenic effect* of vinyl chloride. Mutation Kes., 12 (1 975| 9 3 l l 4, .1 r>r- . 'ii. c . r Kuroki and R. Montesano. Microsome-medtated mutagenesis of a Chinese hamster cell lint- to various cnnmcals. in- D. Scott. B.A. Bridges and K.H. Sobeis (Ed.*.), Progress in Genetic I ..m. olocy . 1- I-.. i. r iN nrlli- Holland, Anutcrdjm. 1077. pp. 207--213. I oi..-in. [... I n du stria I mutagens and potential mutagens, l. (lalogenated aliphatic derivatives. Mutal:.,t 2 ( I <17G ) 267--308. l*r >i.rro. \..l . i n GuUenplan and P. MUvy, Vinyl chloride dependent mutagenesn: effects of liver i \- ,tls .mil fro radicals. Mutation Res.. 38 (1976) 61--88. 11 * 11, ii" II.. G Bouse. A. Rad wan. D. Reichert and D. Henschler. Mutagenicity in vitro and potential i-.mmogrniciiy of chlorinated echylencs a* a function of metabolic osuane formation. Biochem. Pb -TTi.icol . 24 (1975) 2013-2017. 12 II. -iijii, E., II. Bartsch and L. Sachs, flotation Induction in Chinese hamster V79 cells by two .nivl ililomli1 metabolites. ehloroethylene^&tidc and 2-chloroacetaldehyde. Int. J. Cancer. 16 (1975) Li J 1) ' 4 ! - *** ' id HiiA -rinan. F... and L. Sachs. Cell-mediated, mutagenesis of mammalian cells with chemical earcino..i ii, 111 .1 Cancer, 13 (1974) 326--333. ' I Ik '. in. 6.. ami S. OslL-rman*Golkar. Comment on the mutagenic effectiveness of vinyl chloride in. 'I Cli. m.-Riol. interact.. 12 (1976) 265 -267. 1-> l.ir.-in.ktiunal Agency for Research on Cancer, IARC Monograph on the Evaluation of the Carclno- t -ni< Risk of Chemicals to Man, Vol. 19. Some monomers, plastics and synthetic elastomers, acrolein, r V -n. 1979. Hi i.ai. *. II K . .iiuj D.E. Hathway. The biological fate of vtnylidene chloride in rats, Chem.-Biol. Intcrv < . -d lHi78) 27 -11. 7 .............. H I' . an.I C. IIcidi-lix-rgcr. Liver homogenate-mediated mutaRcm-sis in Chinese hamster V79 i-. IL liy polycyclic hydrocarbons jnd anatoxins, Mutation Res.. 46 (1977) 27 44. 16 Km.,i.i. r . ,md II. Bartsch, Mutagenicity of some N<- and O-acyl derivatives of /V-hydroxy-2-ammo.......... ' in V'?*i Chinese hamster cells. Cancer Lett., 6 11979) 67--72. 19 Kur.iki. f . C Urevon and R. Montesano. Microsome-mediated mutagenesis in V79 Chinese hamster c< Il m vnn.ius unrnsamines, Cancer Res.. 37 (1977) 1044--1050. in I :i..;, nbai h. R , H..I. Freed and F. HuWrman, Liver cell-mt'diatcd mutagenesis of mammalian cells ,.itli Id.t can-in..whs. Proc. Natl. Acad. Sci. (U.S.AA.75 <1978)2864-2867. 11 I.mj.. ii-tio, N . l<. Barjlv, 5. Baroncelli. H. Bartsch. G- Bronzctti, A. Camm, Hini, C. Coni, D. Frezza, R. Ni. . ( . Ltputmi, D. KoscUmi and A.M. R >si. Induction of gene mutation* and gene conversions by ) *P ) I V hloriilr n, in Polite* in v.MSt. Cancer Rev. 37 (1977) 253--257. 182 22 Lopricno. N.. R. Barale. S. Baroncetli. C. rHiiiT^Pgffwirrnt A. Cammellini. G. Cercignani. C. Coni. G. Gervaa. C. Leporini. R. Nieri, A.M. Rossi, G. ActUi.-Md G. Turchi. Evaluation of the genetic effecu induced by vinyl chloride monomer (VCMl^Mfrleiniinitiie metabolic activation: studies in vitro and in vivo. Mutation Ret., 40 (1976) 85--96. K 23 Magnusson, J.. and C. Ramel. Mutagenic effecu ol vinyl chloride in Drotophila mtlanotatur, Muta tion Res.. 38 (1976) 115. 24 Malaveille. C.. H. Bartsch. A. Bartoln, A.M. Camus and R- Montesano. Mutacenicity of vinyl chloride, chlorethylenc oxide, chloroaeetaldehyde and chloroethanol. Biochem. Biophys. Res. Comnuin,, 63 (1975) 363--370. 25 Malaveille. C.. G. Blanche and H. Baruch. Factors lor efficiency of the Salmonella/microsome mutagenicity assay, Chem. Biol. Interact., 17 <1977) 129--136. 26 McCann. J.. V. Simmon. D. Streitu/icser and B.N. Ames. Mutagenicity of chloroaeetaldehyde. a pos sible metabolic product of 1.2-dichloroethane (ethylene dichlonde), chlorocthanol (ethylene chlorohydrin), vinyl chiond^ and cyclophosphamide. Proc. Natl. Acad. Sci. (U.S.A.). 72 (1975) 3190--3193. 27 Rannug, U., R. Gothe and C.A. Wachtmeister, The mutagenicity of chloroethylene oxide, chloroacctaldchyde. 2-chloroethanol and chloroacetic acid, conceivable metabolites of vinyl chloride. Chem.-Biol. Interact.. 12 (1976) 251--263. 28 Kamiug. U., A. Johansson. C. Knrm-I and C.A. Wachlmcislcr. The mutacenicity of vinyl chloride after metabolic activation. Ambio. 3 (1974 l 194--197. 29 SlyW-s. J.A.. A method for detecting carcinogenic organic chemicals using mammalian cells in culture. Br. J. Cancer. 36 (1977) 558--563. 30 Verburgt, F.G.. and E. Vogel. Vinyl chloride mutagenesis in Drotophila melanogotler, Mutation Res.. 48 (1977) 327--336. 31 Vogel. E-. Mutagenicity of carcinogens in Drosophila as a (unction of genotype-controlled metabo lism. in: F.J. dc Serres. J.R. Fouls. J.U. Bend and R.M. Philpot (Eds ). In vitro Metabolic Activation in Mutagenesis Testing, Elsevier/North-HolUnd. Amsterdam. 1976. pp. 63--79. Mutalion Re. Eisevier/N Short Com CHEMICA typbimuri; RICHARD 1 Litron Labe (Received g (Revision tv (Accepted Previou their pho been synt 4-fluoro-S inactivatu amino ac azides ca: the mech (DNPA) test systi bacteria the mnt: frameshii tagenic c chemical which in Ames sti experirm ferent U DNPA it strains T Material The tained f testing for plat cedure concent RSV 0016306 snu 7V'i ` i cure /" i/ir 7WfM-.'mvr/>/mnif(. II (1075*1111 A ' Fk.-i n- Sfi.-nl ifii1 Pnhlivhiuc ('nM)fxmy. A nisi riibni llil IVinleil in Tin- N'HhrvI tmls Ill / a -C- K. POTENTIAL HALOGENATKD INDUSTRIAL CARCINOGENIC AN!); Ml't AGENIC CHEMICALS L HALOGENATED UN.SATURATED HYDItOCARRONS LAWRENCE F1SHBEIN HOTICid I do ..... aa be protected by copyright .\atinnal Center for Toxicological Research, Jefferson, .-!/? 72079 (U.S.A.I NOV o * 1970 (Received July 14th, 1978) ABSTRACT Tilt; halogcnatcd aliphatic hydrocarbons represent one of the mnsi important cate goiu-> of industrial chemicals from a consideration of use categories. production volume. environmental and toxicological cousideralions, and hence most importantly, potential population risk. 7'Ih> major unsaturated hydrocarbons reviewed. prnnariK in terms <>f their synthesis? utility, stability, distribution and levels of exposure as well as their reactivity, enreino' jtiMiii-ily. imil.-igcnicily and metabolism, include' vmvl chloride, vinylidoiio chloride. trichloroethylene, perchloroethylene, chluroprcne, (rails-1.4-tlichlorohntene. hexachlnro butadiene ana ajlyl cnloridi. ' " HALOGENATED UNSATURATED HYDROCARBONS The major objective of this .series of reviews is to consider a number of the most industrially significant halngenaled compounds: (a) unsaluralcd hydro carbons; (b) saturated hydrocarbons; {<) alkanes, alloinols and others: (dl aryl derivatives; and fe) polyaromatic derivatives reported to be carcinogenic and or mutagenic in order to better assess the nature rtf the preseni potential risk Additionally, germane aspects (when1 known) of their synthesis, prim arily in terms of the nature of the possible hazardous trace impurities, production volumes and use patterns (U.S.A., Western Europe and Japan), chemical and biological reactivity and stability, environmental occurrence, national permissible worker exposure levels (TLV's and MAC's) have been included for eohesiveness of treatment and hopefully for enhanced utility for a broader spectrum of scientists and public health officials. In recent years, there has been recognized concern over the env.ronmental and toxicological effects of a spectrum of halogenatcd hydrocarbons, prim arily the organochlorine insecticides and related derivatives. II) \ in vl rhinrutr \ i ".s I chli'rule (chi*u i ><! hy Iciic. rl h v lour im Hu >chli ri< ; chi* m * u I heno; \ ( \ I; c'il: CllCU >s used in mormons quantities primarily (cn. !M> *.>7'- ) for tin production of homo-polymer (for PVC production) anti co-polymer resins 112 (c.gM Saran and other plastics). Lessor quantities of vinyl chloride arc used in the production of l,l,l-lriehloroelhan<* (methyl chloroform), as an additive in specialty coatings and as a component of certain propellant mixtures. It is important to note the VCM production processes because of the halocarbon precursors and intermediates as well as the nature of the potential carcino genic and mutagenic trace impurities. VCM monomer production processes employ one of the following: (1) the acetylene plus hydrogen reaction; (2) the direct chlorination of ethylene and dehvdrochlorination: and (3) the balanced direct and oxychlorinalion of ethylene and dehydrochlorination. The overall processes differ primarily in '.he manner in which the inter mediate ethylene dichloride is produced. The hulk of VCM is produced by process (3) above [1--3| (over 95T of VCM produced m the C.S. was made from ethylene) |3|. A typical commercial product can contain the following impurities in mg/kg {2 ] : unsnluruled hydrocarbons, 10; acetaldehyde. 2: diehloro compounds, 16: water, 15; HC1. 2: non-volatiles. 200; iron, 0.1; phenol as a stabilizer, 25--50: and trace amounts of organic impurities including: acetylene, 1,3-butadiene, methyl chloride, vjnylidene chloride and vinyl* acetate |1], VCM is generally supplied .is a liquid under pressure and currently mosL VCM is not inhibited for shipping | 3 ] . As will he discussed in moredclaillaler, ethylene dichioride is mutagenic in Drosophila [1,51 in .S', lyphnnunion T.\ 1530. TA 1535 and T.\ 100 tester strains (without metabolic activation) [G.7] and in K. coil (l)\A polymerase deficient po\ A" strain [8). The growth patterns of VCM per si' as well ns that of its pnmary end-product polyvinylchloride (TVC) plastic resin have been well docu mented [1--3,8--12]. The total world production of VCM in 1071 was 7.059 million kg and in various areas was estimated us follows (in millions of kg) [ 2] : Western Europe (2,-197): Eastern Europe ($17): U.S. < 1.969): .Japan (1,275); and other areas (-199). VCM production in the U.S.A. in 197 1 exceeded 2.6 billion kg (about one-third Western World's output) with tire annual growth rate expected to exceed 1GV per year through the 19$0's [ 1 ]. (In 1976. nine U.S. companies reported the production of 2,580 million kg of VCM [9).) Countries producing VCM, listed in decreasing order of estimated annual production in recent years and the numbers of producers in each year are as follows: Japan (15), the Federal Republic of Germany (5), Italy (3), France (-11. Belgium (3). the United Kingdom (4). The Netherlands (2). Brazil (3). Spain (3), Turkey (3). Taiwan (2). Argentina (2). Sweden (1), Mexico (1). U.S.S.R. (2), South Korea (1), Finland (1), Czechoslovakia (1). Venezuela (1), Egypt (1), Thailand (1), Rumania (1), Chile (1), Greece (1) and Australia (1) [2, 13). About 9f/T tr the 2,27 1 million kg of VCM consumed in the U.S.A. in 1976 was used for the product ion of vinyl chloride homojmlymer and copolymer resins [3], '['lie remainder was ibid (essentially by one company internally) as a co-tnonomer with vinyhdene chloride in the production of resins, and in the production of inclhyichloiolonn (3 ]. The consumption patterns of vinyl chloride monomer in Western Europe and Japan are used in idditive res. It is carbon earcinorocesses ion; (2) i3) the mation. c intcrired hv is made Mowing \2;di. phenol luding: 1 vinylire and :tagoiuc ['A 100 <DNA primary 1 doeu'71 was lions of c. Japan .n 1974 vith the 0's [ij. llion kg 1 annual ir are as . France izil 13), tco (i >. nezuela 1) and .S.A, in ;ier and mpany I ion of impi ion pan are 113 believed to be similar to that in the U.S.A. [31. The world production of PVC in 1975 is estimated to be 9--10 million tons. The major market for PVC resins is in the production of plastic pip** and conduit. Other important uses include: in floor coverings, consumer goods, electrical.applications, and in transportation [31. The total world-wide employment in the VCM and PVC industries is over 70,000 workers. Those employed in industries using PVC as a basic element are believed to number in the millions [111. PVC is produced (in U.S.) via 4 major processes (in % total production) as follows f 11: (1) suspension polymerization, 78: (2) emulsion polymeri/.at ion, 12; (3) bulk polvmerizaLion, 0; and (1) solution, 4. The current U.S. Occupational Safety ami Health Administration tOSUA) health standards for exposure to air contaminants requires that an employee's exposure to vinyl chloride does not exceed an 8-h time weighted average of 1 ppin in the work-place air in any S-li workshift of a lt)-h work week. During any work shift, an employee's exposure may not exceed a ceiling concentration limit of 5 ppm averaged over any period of 15 min or less [31. IARC [31 has summarized the work environment hygiene standards for exposure to vinyl chloride as reported by Bertram [14J in terms of time weighted averages for 8-hour (8-h) and 15-minute (15-min) (n'ceiling con centration) time period unless otherwise stated for the following countries: Belgium -- 200 ppm max; Canada -- 10 ppm (8-h) and 25 ppm (15-min); Finland -- 5 ppm (8-hr) and 10 ppm (10-min period); France- no limits; Federal Republic of Germany -- 5 ppm max. technical standard, with the average concentration not exceeding 15 ppm over the period of 1 h; United Kingdom -- 25 ppm (8-h) and 50 ppm (15-min); Italy -- 50 ppm (S-h) (this is expected to change to 25 ppm (8-h) and 15 ppm (15-min)); Japan -- expected to be 10 ppm; The Netherlands -- 10 ppm (8-h); Norway -- 1 ppm (8-h) and 5 ppm (15-min); Sweden -- 1 ppm (8-h and 5 ppm (15-min); Switzerland -- 100 ppm (this is expected to change to 10 ppm (8-h)) and U.S.S.R. -- 12 ppm. The hazard of vinyl chloride was originally believed to primarily concern workers employed in the conversion of VCM to PVC who may receive a particularly high exposure of VCM in certain operations (o.g.. cleaning of [>olymerizalion kettles) or a long-term exnosure to relatively Ion' con centrations in air of VCM at different factory sites. Much larger populations are now believed to be potentially at risk including: (1) producers of VCM; (2) people living in close proximity to VCM- or PVC-producing industries; (3) users of VCM as propellant in aerosol sprays; (4) persons in contact with resins made from VCM; (5) consumers of food and beverage products containing leachable amounts of unreacted VCM from PVC packaged materi als; and (6) ingestion of water containing unreacted VCM leached from PVC pipes. Gaseous vinyl chloride is emitted at hol.li vinyl chloride and PVC resin plants and is dist rihnl>i 1 into the al mnsplmi'e sum mnilinc Hi*1 emissions source in patterns that depend on (he .minimi of'vinyl chloride released Ihe nature of the plant area from which it is released and the meteorological RSV 0016309 114 conditions [11. It is estimated by the U.S. Environmental Protection Agenry that the tola! vinyl chloride escaping In the atmosphere in the I'.S.A. exceeded 100 million kg per year prior to 1075 [11. V;- yt chloride loss rroin the average VC plant is estimated to he about 0.45kg'10O kg of VCM produced [1[. Based on limited data, ambient concentration* of vinyl chloride exceeded 1 ppm (2500 pg/m3) !e>s than 111'- of llte lime in re*tdenlial areas located in the vicinity of plants producing \C or PVC. The maximum concentration of vinyl chloride found in ambient atr was 33.0 ppm (84,480/jg/m3)at adistance of 0.5 km from the center of the plant [1]. The average concentration of VCM in air around the PVC plants prior to 1975 was 17 ppb [15]. VCM has been determined in the air at levels of 3.1--1250 ppb in the Houston, Texas area (where an estimated 10'; of the U.S. production capacity of PVC is located) [16]. The U.S. Environmental Protection Agency recently proposed new rules to reduce the.national emission standard for vinyl chloride from iO to 5 ppm in order to effect a reduction of vinyl chloride emissions by one-half within 3 years of Hie actual rulemaking. Bast'd on new average-sized plains, libs would result in hourly emissions of 5.1 kg from an ethylene dichloride vinyl chloride plant instead of 10.3 kg; 9 kg from a dispersion PVC plant instead of 17.5 kg and 13.5 kg from a suspension PVC resin plant instead of 16 kg [17f. Early occupational exposure studies have revealed a wide range of VCM concentrations dependent on the manufacturing processes involved [1<S|. The air concentration of VCM in a polymerization reaction prior to vent ilation has been reported |18| to he 7800 mg/m3 (3000 ppm) anil range from 1560--2600 mg/m3 (600--1000 ppm) in a polymerization reactor after washing [ 19 ]. Concentrations of VCM in the working atmospheres in some plants producing PVC have been reported in the ranges of 100--800 mg/m3 MO312 ppm) with peaks up to 87,300 mg/m3 (34,000 ppm) [20] . Additional concentrations of vinyl chloride have been reported for work place air including: (1) 0.15 to 0.35 ppm in air in throe English cable factories [21 ]; (2) greater than 75 ppm in air in a Yugoslav ian PVC manufaeturing plant [22]; and (3) greater than 113.6 mg/m3 in air in a Russian synthetic leather plant [23J. In 1974, Heath [24] estimated that 20,000 U.S. workers in the past and to 1974 had been exposed to VCM in manufacturing plants. Baretta [25] in 1969 reported that on a time-weighted average, the concentrations of VCM to which coagulator operators are exposed ranges from 130--650 mg/m3 (50--250 ppm). A more recent survey by NIOS1I of 3 VCM plants reported that the time-weighted-average exposure to VCM ranged from 0.07--26.46 ppm [3, 26]. The concentration of residual VCM monomer in PVC powder that is fabricated into final products is also an important determinant <3 VCM m the ppm range. The ml rapped i niiceiil i at ion is dependent upon I lie pr< id lielion process and can range from 0.1 lo 5.8 thousand ppm, which can be liberated during fabrication, particularly when heated [1|. PVC leaving Agency L'.S.A. nss from <>f VCM o/ vinyl in resi- VC. The air was lie plant nls prior lewis of - of the w rules n r> ppm If within nts, this le--vinyl instead of 16 kg of VCM d [IS), to vent 'd range tor after e plants n3 flo or work er cable nanufac- Russian past and i [ 251 in of VCM 0 mg/m3 reported 7-26 .*46 r that is VCM in protluc1 can be leaving 115 certain plants may contain 200--400 ppm VCM, on delivery to the customer, the level of VCM is about 250 ppm. and after processing, levels of 0.5 -- 20 ppm are reached, depending on the method of fabrication [27). How ever, new processing methods developed since 1971, leave as little as 1 to 2 ppm residual VCM in VCM resins (2S|. Residual VCM in commercial food grade resins have been reduced by special techniques to 115 ppb Iwt wt) for resin and less than 0.048 ppb (wt./wtl for compound anti sheet PVC |29| . VCM has been detected in effluents discharged by chemical anti latex manufacturing plants and in raw water in the IJ.R.A. [30J. The l:.S. Environmental Protection Agency estimated in 197-1 that about 12.3 kg'day of VCM were discharged in the wastewater effluent from two VCM plants in the Lone Beach, California area 13 j. Vinyl chloride has been found in municipal wafer supplies in the U S.A. )1, 14) in representative samples of the nation's community drinking water supplies that ehloriuale their water and represent a wide variety of raw wafer sources, treatment techniques and geographical locations. The highest con centration of vinyl chloride detected in finished drinking water in ttie U.S.A. was 10.0/jg/l (31). The sources of the vinyl chloride found in the Miami. Florida and Philadelphia water supplies (5.6 and 0.27 pg/1, respectively) have not been iclenti find j 11. Available results indicate that migration of vinyl chloride from rigid I'YC water pipes does occur, and that it is a linear function of the residual vinyl chloride level in the pipe itself [ 1J. Only limited data are available on vinyl chloride emissions from the incineration of plastics. The quantities of vinyl chloride and combustion products varied as a function of temperature as well as with the type of plastics and their polymers [ 1). It is believed that vinyl chloride should disappear significantly in its transport over long distances, however, in the immediate vicinity of emission sources, vinyl chloride can be considered a stable pollutant [1). While no mechanism is presently known for the removal of vinyl chloride from the air at night, biological sinks such as microbial removal in soil may be of significance in depletion of vinyl chloride over a long time period. However, such sinks would not be expected to be important in terms of urban scale transport of vinyl chloride [1 ]. In studies performed in a laboratory model ecosystem, it was reported that vinyl chloride, despite its lipophilicity, is so volatile (vapor pressure in 2660 mm at 25 C) that it does not bioaccumulate or transfer appreciably through food chains, at least at ordinary temperatures [32]. Vinyl chloride lias been found in a relatively small number of products packaged in PVC containers (e.g., in edible oils in a concentration of 0.05 to 2 ppm [33] and in butter and margarine [34]). The U.S. Treasury Department in 1973 banned the use of PVC for the packaging of alcoholic beverages as a result of FDA reports indicating the presence of VCM at levels up to 20 rng/kg in some alcoholic beverages packaged in this material [351. Vinyl choride has been found in domeslie and foreign cigarettes and liLlle cigars in concentrations of 15--17 nanograms/eigarette | 36 j. RSV 0016311 I llfi New automobile interiors have been found to contain vinyl chloride in concentrations of 0.4 to 1.2 ppm [3, 37 |. Experimental as well as human evidence of tin- carcinogenicity of vmyl chloride has been reviewed by Uartsch and Monlesano |10| ami I ARC 12. 3(. The results to date show that vmyl chloride adminUh-rnl by in halation is carcinogenic in rats, mice and hamster, producing angiosarcoma of the liver as well as of other tissues in all three species. In addition in rats, vinyl chloride induces tumors of the Zymhal glands and of the skin, nephro blastomas, hepatomas and neuroblastomas, in mice tumors of tin1 lung and skin were also observed [38-48]. The lowest reported effective close winch is carcinogenic in rats and mice is .70 ppm [ 10. I !-- IS [. Twu angiosarcomas of the subcutaneous tissue and a carcinoma of the Z\ mbal glands were observed in the offspring of mothers exposed during pregnancy to inhalation of vinyl chloride at 10,000 and 5000 ppm [3.10J, It is noteworthy that these experiments not only indicate the carcinogenicity of vinyl chlondr but also demonstrate that the type of tumor induced, angiosarcoma of the liver, was the same as that observed in workers exposed to VC'M |2. 3. 10] The relationship between exposure to V('M and development of angio sarcoma of the liver was first reported by Creech and .Johnson [4P| who cited three cases among workers employed in a single vinyl polymerization facility in Lhe IRS.A. These men had cleaned reactor vessels as part of their employment. AL present, a total of 17 cases of angiosarcoma of the liver have been reported among workers exposed to VCM in the U.S.A. Extensive world wide epidemiological studies to date have indicated about 50 cast's of angiosarcomas of the liver associated with VCM exposure among workers employed in the muiuilacture of 1'VC i esins (Table 11 [ 2, 3. 10. 12, IP 32 |. Most of the tumors were observed in workers employed in the cleaning of autoclaves where- the concentration of VCM is high (e.g., ranging from 500 tO 2000 or more ppm in the air). It should be noted however that angio sarcomas of the liver were also found in people exposed to much lower concentrations of VCM (e.g.. in a worker in the production of I'VC bags who had been exposed to VCM for a total of 3 years and in an accountant employed for 10 years iu a vinyl fabric factory) [10|. It has also been suggested that at least two other types of cancer, carcinoma of the brain and of the lung, appear with increased frequency in workers exposed to VCM [10, 54. 55], Infante et al. [52] cited a significant excess of mortality from cancer of the lung and brain in addition to cancer of the liver, among workers occupationally exposed to VCM. The risk of dying from cancer of the lymphatic and hematopoietic system also appears to increase with an in crease in latency. A study of cancer mortality among populations residing proximate to VCM polymerization facilities also demonstrated an increased risk of dying from CNS and lymphatic cancer [52]. However, it was tailed by Infante el. al. | 52 | that although these findings raise cause for concern about out-plan! emissions of VCM. without, further sludv these rancors cannot be unequivocally interpreted as being related to out-planl exposure Lo VCM. lyl chloride in nicity of vinyl 0] and IAHC listered by in; angiosarcoma ddition in rats, i* skin, nephro f the lung and .ive dose which angiosarcomas al glands w.-re y to inhalation Aeworthy that yl chloride but ma of the liver, 3.10). merit of angioison [49) who polymerization as part of their ma of the liver .S.A. Kxtensive iout 50 cases of imong workers 0, 12, 49-521. the cleaning of iging from 500 ver that angioto much lower n of J'VC bags i an accountant t has also been >f the brain and vposed to VCM from cancer of imong workers cancer of the ase with an in flations residing ed an increased *r, it was noted ist* for eonoern these cancers plant expo-nm* TAJ1LK l CASKS UK ANGIOSARCOMA UK THE LIVKi: AMONG UOKKKK.S EXPOSED TO VCM OR PVC[10j Country Number Age at of diagnosis cases Years from first exposure lo Total exposure in ye.iis Occupation L'.S.A. 15 46(36-61)" 201 12-30)" 17(4 --30 l" Polymerization WOI kers L`.K. 2 71 26 20 Poly meri/.alion WOI k 1 !> .17 s 3,5 Pul v iin-i i/.at miii workers Norway 1 56 oo 21 Polymerizal un win kers Sweden 1 43 10 lb I'l 1 > Ull'l l/.ll lOll W< 11 1. I'l s Federal f{<-|iul)lii- r> 10 14 1 2 l'i >1 vi in-i i/al ion VV'I lll.l'IS ill l riTlll.lliy 3H 1 2 12 I'olv tneii/.al inn workers 44 17 17 Poly merizal ion workers 19 17 1 1 Poly merizal ion workers 43 13 1 2 Poly m<-rt/.ai ion vvoi k ei s t- raiK-c 2 43 10 19 I'olv ineri/.at mil W Ol Ll'o 54 15 12 Polymerization vvoi Uns Italy > 43 !5 6 Tol v Min i/,il uni workers 55 21 Polymerization Rumania lb 27 11 workers5.5 Polymerization workers Czechoslovakia 2 37 14 14 Polymerization 45 15 vvoi keis 15 Poly men/.ation Canada 9 no data available workers Polymerization Yugoslavia 4 no data available workers Polymerization Japan i no data available workers Poly imrization workers L'.K. 1 55 24 1 1 Pouring PVC oil Sweden 1 61 27 onto 1 abnc base 23 VCM production Federal Republic i>f Germany Italy 1 43 1 ;tu 14 worker 14 Kilting pesticide cans with VCM as prupellanl 3 Pi i< 1 M'l ii >n o 1 * - - ' .... , - - . ... . ,, _ ... __ PVC bags Average with, m parenthesis, minimal ;iticj maximal values. This cum- was d iag nosed as licpalocarcmoma associal nl with choking ii lsarcoin.i. I IK Tin* risk of < aueer mortality among residents -15 years of age an<l older in three communities having vinyl ehloride polymerization facilities \v;k studied hy Infante |T11J. Among males the dead; rale !.<m ('NS Timmy was higher than that for the state as a whole. No aleukemia or from lymphoma was found. mortality from leukemia ami A four- to five-fold excess of cancer oi the liver and pancreas was found among 771 workers employed in a Swedish vinyl chloride.'PVC plain since its start in the early 1940's [57 j. Although the risk of cancer nf ihe brain and of the lung was increased among workers exposed to vinyl chloride, u was reported hy Hyren el a!. | 57 | not lo he statistically significant. The cancer mortality among 75bl males who were at some lime between 1910 and 197 1 exposed to vinyl chloride in the manufacture ol l'\ C in the United Kingdom wui investigated by Fox and ('oilier |58|, Wliilr mi cso-ss mortality from liver cancer was reported for each group of workers whether exposure was thought lo lie high, medium or Imv was reported, no iwulenee was found lo support an excess of mortality from cancer other than of the liver. Relatively few' persons had long term exposure to VCM and e\cn m cases in which men had completed 20 years of employment, the follow-up period was considered too short to permit an evaluation of the carcinogenic effect of vinyl chloride (3,58 |. While no excess mortality from lung cancer was demonstrated among individuals currently employed 15 years after initial occupational exposure to vinyl chloride, a 56rr excess of lung cancer was observed among those individuals who had terminated employment prior to 15 years since initial VCM exposure. Among this later group, the excess lung cancer mortality was found for each duration of exposure [3. 59). An excess of deaths due to cancel' of four organ systems (e.g., brain and CNS, rcspiraLory system, hepatic system and lymphatic and hematopoietic systems) was found hy Waxweiller el al. |(H)j among 1294 individual* who had 5 or more years of employment and 10 years since onset of emplnvmeut in departments or jobs directly related to VCM exposure at mu* of four VCM/PVC production plants in the C.S.A. The cancer risk among a cohort <>( males who had at least one veai'of occupational exposure Lo VCM was studied hy Tabers haw and CalTey 155) When compared lo the U.S. white male population, excess of cancer of the digestive system {primarily angiosarcoma of the liver), respiratory system, brain and of unknown sites, as welt as Lymphomas was observed in those members of the study cohort with the greatest estimated exposure1 Lo vinyl chloride. Vital statistics was undetermined for 15N of the study cohort and only 50'r of the study cohort had 15 or more years since onset of exposure to VCM [31. A number of cytogenic studies have revealed high incidences of chromosome aberrations among workers occupationally exposed lo vinyl chloride in I lie U ,,S. A . | <> I <5.3 | , 1 lie I in u ed Kingdom | < > I | . Sh < den | ro | delgimu |dd|. Norway |<i7| and Hungary |li8|. In most eases. these aher rations included "unstable" chromosome changes (e.g.. fragments, dicentrics and rings) and simple aberrations (e.g.. breaks and gaps). i\' and older in s was studied uir wus higher leukemia and as was found `C plant since r of the brain yl chloride. it am. time between of I'YC in the ' liilf an iwi-r.ss 'rkers whether d. no evidence er than of tlie 'l and even in lho follow-up e carcinogenic iraU'd among otial exposure I .imong those -rs since initial mortality was eg.. brain and hematopoietic divuluals who f employment t one of four 'l one year of l r.al'fey 1 no | . cancer of the alory system, rved m those -isure in vinyl dy cohort. and i't of exposure incidences oT >osed to vinyl Sweden [93). 's, I li' .< ill ,i r ills, i lieen I vies 119 Infante et al. [691 reported a significant excess of fetal deaths in wives whose husbands were exposed lo vinyl chloride, e.g., 15.fi'-'. 23 fetal deaths in 139 pregnancies as compared to 8.8'6 (24 out of 273) in the age adjn..ied control group. The. mutagenicity of vinyl chloride' has heen reviewed try Itartseh and Muntesano [10J, Burlsch et al. [70], Fishbein [71] and lAKC |3|. Vinyl chloride vapor induced reverse mutations of the base-pair sub stitution type in S. typhimurium G ift, TA 1530, TA 1535 and TA 100 in the presence of 9000 g supernatant from rat liver [72--77], mouse liver [72, 75. 76] and human biopsies [72, 75]. Although vinyl chloride induced mutations in the absence of a metabolic activation system, a much higher mutagenic effect was noted when a 9000 g supernatant from liver was added [72.7 1.77]. Whereas vinyl chloride in aqueous solution was not mutagenic in the Salmonella test system [72,_73], it produced reverse mutations in L'. cali K-12 [78], gene conversions in S. cereiisiae in the presence of 9000 g supernatant from mouse liver. Vinyl chloride also induced forward mutations in .S'. pombe in the host-mediated assay in mice [79. 84]. Vinyl chloride as a vapor or in elhanolic solution was non-mulagenic in Xeurnspora crassu in the presence or absence of metabolic derivation [81 | . While vinyl chloride was mutagenic in inhalation experiments in Dros ophila niclanoguslar [82, 83|, it was non-mulagenic in the tests for domi nant lethals, translocations and sex-chromosome loss 183]. Anderson et al. [84] reported no dominant lethals in male CD-I mice after exposure by inhalation to 3000, 10,000 and 30,000 ppm vinyl chloride Cor 6 h/day for 5 days. Forward mutations (in terms of 8-azaguanine and oubain resistance) were induced in V79 Chinese hamster cells following their exposure to vinyl chloride vapor in the presence of 15,000 g supernatant from rat liver [85]. The mutagenicity of possible metabolites of vinyl chloride (e.g, chloroethylme oxide, ehloroacetaldehyde, chloroetlianol and ehloroaeetic acid) has been extensively studied. Chloroethylene oxide was reported to be the strongest mutagen among those tested in S. typhimurium G46. TA 1530 and TA 1535 strains [72, 73. 75|, E. colt [86], S. cerei'isiae [79] ,,S\ pomhe [791. and V-79 Chinese hamster cells [87 J. ehloroacetaldehyde was mutagenic in .S', typhimurium [72--751 and V-79 Chinese hamster cells 187). Chloroetlianol was weakly mutagenic in ,S. typhinwrium [72--75, 88], while ehloroaeetic acid was not mutagenic in S. typhimurium [72--75]. Klmore et al. [891 screened without exogenous activation seven potential metabolites of vinyl chloride in their pure forms as well as the related cpichlorohydrin in tester strains of limilliis and Sitlmonrlla. ('hlorooxirnue Ichloroethylene oxide), ehloroacetaldehyde, ehloroacetaldehyde monomer hydrate, ehloroacetaldehyde dimer hydrate, chloroaeclaldehyde trimer and cpichlorohydrin produced significant unit ngenir arl ivil.y in .S' /v/i/iminiiiini shams scnsilixc Jo b.isr pair mu t nJ ii <u. A recombination repair deficienl slram of It. tmhtihs was mhihiled in growth by these compounds, whereas RSV 0016315 120 excision repair deficient and wild type strains of /?. sublilts were relatively unaffected. Vinyl chloride at a concentration of 0.01'' M |723 ppmi in nutrient broth was negative in both the Salmonella and Hacillus eultures. (High concentrations of VCM (20% v/v in air -- 200,000 ppm) produced mutagenic action in previous assays with Salmonella tester strains {72, 73].) Chloroethanol and chloroacetic acid were non-mutagenie at 1 niM con centrations in the above mutagenicity assays of Elmore ot al. [89 |. Among the compounds tested by Elmore et al. [89 j, chloruacetaldehyde anti chlorooxirane were the most mutagenic with the lowest toxic side-effects. !lenri\ it was suggested that they may be the active carcinogenic derivatives of vmyl chloride 189). However, chloroacelaldehyde monomer hydrate \va> <on sidered a more realistic choice as the uUinwl*' carcinogen than the mono mer compound which reacts immediately with water. The lower mutagenic activity of chlorooxirane compart'd to chloroacelaldehyde monomer hydrate may "reflect the unstable nature of chlorooxirane as an cv-chloroether [73] . One mode of action of chlorooxirane is a rearrangement to chloroacetaldehyde (90] via the NIH shift [91). Another is a homolytie ring cleavage to yield a stabilized diradical intermediate C'ICH-C! IjO with both being cap able of reacting with DNA.to account for the mutagenicity of chlorooxirane (73). Hartsch [92| recently reported on a collaborative study involving the biological activity of vinyl chloride, three of its identified mammalian metabolites (chloroethylene oxide, chloroacetaldehyde and chloroacetic acid) and a putative metabolic intermediate (2-chloroethano)) which were assayed in several prescreening tests for carcinogens, t'.g., (a) assay for electrophilic reactivity; (b) tissue-mediated mutagenicity test* with S. lyphtmurium or with (c) 5. pombe and S. cerevisiae\ fd) host-mediated assav in mice with S', pombe; (e) in vitro mutagenicity in Chinese hamster Y79 cells using ouabain or 8-nzuguanine resistance; (f) induction of DNA repair syn thesis ("unscheduled" incorporation of ['ll) thymidine) in cultured human fibroblasts; and (g) tests for recessive Lethal mutations in Drosophila mclanogaster. Chloroetliylene oxide, which was the most active compound in the assay systems (a) to (f), induced local tumors in mice upon repeated subcutaneous administration. It was slated by Bartsch 192) that these results strongly support the hypothesis that chloroethylene oxide is one of the principal mutagenic and/or carcinogenic intermediates formed from vinyl chloride by mammalian metabolism. Negative results were reported by Mattern et al. [93] in their attempts to use the mutagenicity testing of urine from 20 workers in vinyl chloride producing plants in Holland, and vinyl chloride-treated rats using the Sal monella strains TA 100 in the former case and strains TA 1535. TA 98 and TA 100 in the latter case.- Urine samples were assayed in the presem-e or absence of liver homogenates from Aroelor I2rv!-trcate<l rats, with or without/i-glucuronidase in the plates. Whether a different treatim iil of (hr urine samples would reveal mutagenic aelivity, or whether no mutagenic mrlahol ites of vinyl chloride are excreted in the urine remains to be determined 193]. Its wore relatively ppm) in milrienl cultures. (High 'tluced mutagenic 73].) he at 1 mM eona). |89). Among hyde and chioro-cffects. Ik-nee, it rivatives oT vinyl tydvale was eon i Ilian (In' humidlowcr mutagenic monomer hydrate hhoroether [73]. : to chloroaeotaln: ring cleavage In i both being eapr>f chlorous iraiu- nly involving the ified mammalian and chloroacet ic mol) which wore g.. la) assay for sts with S. typhilediated assay in namster V79 cells DNA repair synn cultured human osophiia melanocompound in the e upon repeated that these results :de is one of the >rmed from vinyl their attempts to in vinyl chloride ats using the Sul1535. TA 9S and i the presence or als. with or withincut of Hit- urine utapeme metnbol o be determined 121 While the predominant risk has hern at the level of occupation health duo to the exposures in vinyl chloride and PVC plants, it is also of importance to determine whether other products in the process could also be carcin .genic and/or mutagenic. The synthesis of vinyl chloride either from acetylene or ethylene or from a mixture of these substances, results in a tar-like by product called EDC-tar. This name originates from one of the main com ponents, ethylene dichloride for 1.2-dichlorootham*) 101. 95], F.DC-tnr. however, is a complex mixture, consisting chiefly of chlorinated aliphatic hydrocarbons [94] (e.g., approximately 33% ethylene dichloride; 1,1.2trichloroethane and about 0.06rr vinyl chloride monomer). The composition of EDC-tar varies not only from factory to factory, hut also from time to lime from llu* same factory 196). RerenL mutagenicity studies of a Swedish sample of KI)('-t;ir utilizing Salnn>nella/n\nmmixlinn microsome test (TA 1535) showed that this sample contained direct as well as indirect mutagenic compounds 196). The mutagenic effect observed was not due to any significant extent to one of the main components (ethylene dichloride). The in vivo and in ritro metabolism of vinyl chloride lias been extensively studied resulting in a diversity of results and conclusions [10.75.78, 97 107|. The metabolism of vinyl chloride has recently been reviewed by l'flugge and Safe 1108| and IARC 13]. The major metabolic pathway for the formation of the major urinary metabolites (chloroacelate; S-carhoxy methyl cysteine; S-( 2-hydroxy ethyl )cysteine; N-acetyi-S-{ 2-hydroxy ethyl ) cysteine and thiodiglycollate) is shown in Fig. 1 [108]. Vinyl chloride is metabolized by the liver mixed function oxidase system (MFOS) (predom inantly through the cytochrome P-450 system) (109--112) to the oxirano, chloroethylene oxide and chloroaectaldehyde) which then can cither bind directly or enzymatically via glutathione-epoxide-lransferase 1113) or other glutathione transferases [114) to form S-formyl methyl glutathione. Chloroaivinldehyde can also be directly oxidized to chloroaeeLaldehyde which is CH,-CHCI vinylchlonde \~C CH CiCH0,V' ':'::,> O ''X00H H 0 VH ## ' ehloroelhylene- chloroocet- cNoroccetate oxide 1 aldehyde * i J V G-S-CH-CHO --G-S-CH-C00H S-formyimethyfgiuicth'one S-car doxymethy1glutathione I cys-5-CK.-CH OH <--eys-S-CH.-CHO S-te-hydroxyethyip S-fiymylmethyi- cysteine cysteine I* cys-S-CH -C00H S-corboxymerhylcysteine I N-ocetyl-S-tz-hydroxyethyl)-cyotp>nr It-nh. . 1 *C0 * rto'.vWievive detected m vitro HOOV-VM h-CM ihioUrglycoll-jU' Kic. 1 Major metabolic pathway of vjnylchloridc fur lho. formation of Uh- major urinary nirliibolites. RSV 0016317 122 chd--ch2--c--c --`C-jOcho-- o c.rcc o 'h Cl ^ cceve H-C--C-H ---c- o CH \ iuttT'f------ iJ'.C', Col qV/o^io'e----- .maw* *---- - (,ifr*o'a` =. cjq t *; seccr'O''me ''fu'in. --o>o'oocetQie D!r c ocia eye* --,,4C02 - _fc<-- c*-; ce 3 ri"citrcg>i: i- j lie^yae '"c-cs < : -iUc j-~<sr> , r--rfhfie',''e v* *o----....... -t't rf Fig. MeLtUnlism nf vinyichlnmh* via <:til<M<i.iccnUd`l\yti>' then either excreted or hound to CiSI l to form carboxymethyl glutathione which can also he formed from S-formylmethyl glutathione. The generation of ,4C02 following the administration of 1 40vinyl chloride in inhalation (105|, oral [106j, intragastric [101.1021 UIU* i'Uraperitonal [101,1021 W;1S postulated by IMngge and Safe [10S| to occur via two possible pathways: (11 addition/transfer of a ehioroaeetyl group to f'oA followed by metabolism in the TCA cycle (Fig. 2) or (21 the formation of glycollate followed by its oxidation to glyoxylate (Fig. 3) after winch it enters the C2 andCi pools. The main eliminative route for ,4C*-viny! chloride after oral. i.v. or i.p. administration to rats is pulmonary: both unchanged vinyl chloride and !4CO> are excreted by that route and other 14 ('-metabolites via the kidneys. AFtcr intragastric administration, pulmonary output of unchanged vinyl chloride is proportional to the logarithm of reciprocal dose. Pulmonary excretion of unchanged vinyl chloride after oral dosing is complete with 3 -- 1 h; but pulmonary elimination of C02 and excretion of metabolites occupies 3 days. In comparison, 99% of a small i.v. dose is excreted unchanged within 1 h of injection; S0% after 2 min. The rate of elimination of single oral doses of 1 4C-vinvI chloride is uninfluenced by up to 60 days chronic dosing with the unlahelcd substance [ 101,102 J. A scheme for the metabolism of vinyl chloride is rats (Fig. It was proposed by Onvn and IlaUnvay [102J in which the urinary metabolites S-{2-ehloropt.hyl)cy.stoine (a) and N-acctyl-S-(2-chloToethyl)cystei!ie (b> are formed only through direct, interaction of glutathione derived cysteine with \ inyl chloride per sc. / There i.s a strong .supposition that in rats ehluvoelhy lene oxide I Fig, 5) | 101) is formed from vinyl ehloride ami transformed jspnnl anronsly sooirete i*ce, cnooswccnne hyl glutathione ii of 1 4C-vinyl 102] and intra>8] to occur via vl group to CoA ie formation of i after which it o' '.v. or i.p. A ride and boliuis via the t of unchanged lose. Pulmonary iplete with 3--4 bolites occupies changed within single oral doses mic dosing with is (Fig. 4) was cry metabolites ysteine (b) are d cysteine with thy lone oxide I spontaneously f rotw 0H H-C--- C-H OH OH i-coroelhone-|,2dol mc ch2ohcho -- glycoiaideftyde c^ohc: glyccdote lormyCoA CH0CCS-* glyoxy*^ - CO. N^-'or rnyi'enabyOiGlotole f-- (yf- fOrm/'EtrohyC'-otolQie I -lormoie:. I * CO2-- Ci'flC etc Cyoe* N 'NIOrt>e'l'yi<n-f'>ii,'ttohytJ'Oli>ale --* 'rcthyfc.'mieirotiydioloiaio -- fiemocyVoirie -- --- tnifirvaninc *ditocif><l as netob<Vi*c "i o-Odl^-'-iylnofliCCySl'.-n'! --- '----S-aaenO'.y:rneTvr"-V! Fig. 3. Metabolism of vinyJehloride via chloruethanoi. into chloroacetaldehyde. There is supporting evidence for vinyl chloride epoxidation in vitro [10,111,115]. \2> Vinylidene chloride Vinylidene chloride (1,1-dichloroethylene; asym. dichloroethylene; l,l-*dichloroethenu; DCE; VDC; Cll2 =:CCl2 ) is produced commercially in the U.S.A. and Japan by the dehydrochlorination of 1.1.1-trichloroethane HSC62<HC02H nh2 Fig. 4. Scheme for the metabolism of vinyl chloride in raus j ]02) /sites RSV 0016319 jC-CHCI --* M3C-CH------ CICH5CHO--* ^ CICMjCOOM 'OSH * C.o CHCHjSCHjCOjH NH COjH CHCHjSCHjCOjH (i| S|CH3C02H|j (j) Fig. 5. Scheme for the biogenesis of S-containing vinyl chloride mclubolUes | 10 1 1 (methyl chloroform) or 1,1,2-trichloroethane, both of which are derived from ethylene dichloride (116.1171. Specific impurities in vinylidene chloride monomer depend upon u~ method of manufacture and isolation. A typical commercial grade of vinyl idene chloride, 99.7% by weight as prepared by dehydrochlorination of 1,1,2-trichloroethane with lime or caustic [ 117j contains the following impurities (in ppm): vinyl chloride. 850; cis-1.2-dichloroolhylone. 500. rans-l,2-dichloroethy\ene, 1500: 1.1-dichloroethane, 10. Phenol at levels ot 0.6--0.8% or 200 ppm or monomrlhyl ether or hydroquinone (MKMfi) nr<added to prevent polymerization during shipment and storage. A typical analysis of vinylidene chloride monomer (unstalulixed) largely produced in the U.S.A. (118) includes as impurities (in ppm): vinyl chloride. 28; vinyl bromide; <rus-l,2-riirhlororl.hvlene, 1 000; and cts-1,2-<licl\lororihvlene, `110. It was estimated that two U.S. companies at three sites (('>ulf (`nadi in 1976 produced a combined total of 70 million kg of vinylidene chloride and 1RSV 0016320 .oolites (101). uch are derived pend upon its grade of vinylchlorination of the following ethylene. 500; mol at levels of in? (MEMO) are rage. A typical ly produced in oride. 28: vinvi ililmoi'llu'U'ni', ((lull' ConM 1 in no chloride and I 25 that one of these companies manufactured an additional 50 million kg of vinylidene chloride for captive use ,*is an unisolated intermediate in the production of 1,1,1-lrichloroclhanc [119|. It was postulated that in the U.S.A., the growth rate during the 1974--1979 period in the production of vinylidene-chloride will be hasod on 1.1,2-trichloroethane which is used as a feedstock in one vinylidene chloride production method. It was also estimated that the consumption of 1,1,1-trichloroethane used in a second vinylidene chloride will not change over this time period [120]. In 1976, three Japanese companies produced a combined total of 28.2 million kg of vinylidene chloride 11191. No information is available concern ing the production of vinylidene chloride elsewhere. Excluding the amount of vinylidene chloride used as an unisolated inter mediate in the production of 1,1,1-trichloroethane, more than 90T of the vinylidene chloride produced in the U.S.A. and Japan is used in tile pro duction of copolymers of high vinylidene chloride content, the other major monomer usually being vinylidene chloride (1191. The remaining 10'< or less of vinylidene chloride produced is used for I he manufacture oT modacrylic fibers which are largely based on acrylonitrile with small amounts of vinyl idene chloride and other monomers [119]. A number of copolymers of vinylidene chloride (in latex, fiber, film and resin forms) are referred to as "Saran" and have wide utility for film wraps for food- Saran production is estimated at about 150 million pounds per year [120 J. The copolymers presently of commercial interest in the' U.S.A. include: vinylidene chloride-vinyl chloride (Saran B): vinylidene chiorido-alkylncrylate (Saran C) and vinylidene chloride-acrylonitrile (Saran F> [119]. t\S. production of vinylidene chloride copolymers in 1977 1121] has been estimated at 68 million kg while in 1976, four Japanese companies produced aii estimated 31.9 million kg of vinylidene chloride-vinyl chloride copoly mers. 2.7 million kg of which was fibers and the remainder was latex and film 11191. The number of workers engaged in the production of vinylidene chloride monomer per sc in the II.S.A. (compared to vinyl chloride monomer) appears to be small e.g,, 75 and 12 -15 at two major vinylidene chloride production facilities [122]. Vinylidene chloride is polymerized to plastic resins at 12 facilities and the resins arc then fabricated into plastics at 60 to 7,i plants thoughout the country [120]. Estimates of the number of workers engaged in the preparation of polymers and co-polymers of vinylidene chloride and vinyl chloride (e.g., the preparation of Saran wrap) are not available, nor are data available at present on workers exposed to only \ mylidone chloride during their working lifetimes. the American Conference of Governmental Industrial Hygienists (ACGUD ti-iommcnds that an employee's exposure to vinylidene chloride does not Meed an eight-hour time* weighted average of 10 ppm ( 10 iug/m'l in ihr workplaee air m any eight-hour work shift of a lorly-bnur work week. Ihiring any lifn'cn.mimite period. Mu' Al'Glll proposes an absolute coiling -Mieentralion limit of 29 ppm (80nig/m3) provided the daily threshold RSV 0016321 limit value fin terms of 8-hour time* weighted average values) is not-exceeded [1231. There is a paucity of data concerning worker Nposure to vinylidene chloride. Worker exposure has generally not been monitored in (he past according to EPA [120|. Tests have shown that 20.000 ppm cun cosily inreached in the proximity of a spill in some cases past worker exposure to vinylidene chloride may have exceeded those of vinyl chloride which were measured at 300--1000 ppm before OS11A limits were imposed. Workers involved in manufacturing facilities using vinylidene chloride in polymerization processes such as the production of PYC have been repotted to be pnposed to vinylidene chloride in amounts of Joss than 5 ppm and most frequently to trace levels 1124,125). Levels of 2 ppm of vinylidene chloride have also been detected as a contaminant of submarine and spacecraft atmospheres [119,126]. A substantial amount of vinylidene chloride appears In ho vented In the atmosphere during its production, polymerization and fabrication. Emission* of vinylidene chloride in the U.S.A. in 197-1 have he<-n estimated nt I n million kg from monomer synthesis operations. 1'his was reduced to 277 thousand kg by new control technology in late 1975. Vinylidene chloride losses from polymer synthesis operations and polymer fabrication operations were 308 thousand kg and 13.8 thousand kg, respectively [127] . To estimate the population at risk due to vinylidene chloride emissions. EPA [120] assumed that the populations of the cities and countries which the production and use of vinylidene chloride exist are at risk. Using a total U.S. population figure of 212 million, (lie population at risk due to Ihesr producers and major users of vinylidene chloride (>$1000 annually or >1000 lbs annually) was estimated to be about 4.79: to the U.S. population or approximately 10 million people. Other sources of exposure to the population include the other user facilities located throughout the country and those involved in transportation. Based on solubility data, it is estimated by EPA that alt vinylidene chloride in wastewater is released to the atmos phere and thus probably little exposure occurs through the use of water 1120]. Vinylidene chloride has been detected in effluent discharged by chemical and latex manufacturing plants in the U.S.A. [119] and in effluent dis charged from chemical manufacturing plants in the Netherlands at a concen tration of 32 pg/I [128]. Vinylidene chloride has also been identified in Hie U.S.A. in well, river, and raw water [129]. It has also been found in finished drinking water in the U.S.A. where the highest reported concentration was 0.1 Mg/1 [130). Vinylidene chloride has also been found as an impurity in vinyl chloride monomer [131], and trichloroethylene [132] and at a level of 0.0119 in commercial chloroprene [133]. As much as 259. of the vinylidene chloride used in any given Karan production run has been estimated to be disposed of in landfill (primarily in polymerized form) although Ihore are no estimates of the levels of unreaeted monomer [120]. RSV 0016322 1 not exceeded M vinylidene >n the past can easily be r exposure to :e which were ve chloride in been reported 'f>m and most done chloride nd spacecraft vented to tlu* on. Emissions mated at 1.5 I need to 277 dene chloride on operations ute emissions, unlries which . Using a total due to these * annually or S. population >sure to the the country it is estimated to the atniususe of water d bv chemical i effluent disi.s at a concenentified in the nd in finished entration was vinyl chloride of 0.0117?- in given Saran I (primarily in ' of unreacted Commercial household and industrial Saran wrap have been malyz'*d for residual vinylidenr chloride monomer. Six rolls of household film had monomer concentrations ranging from 0.5 to 10.1 ppm with an nveingr ;.i 8.8 ppm with no significant differences found in samples taken from the beginning (outside) or the end (inside) of each roll. Level? of monomer ranging from 10.8 to 26.2 ppm were found in the industrial film with levels increasing from the beginning to the end of the roll [ 119,1311. Although the widespread use of vinylidene polymers as food wraps could result in the release of unreacted monomer into the food chain (121.135). and vinylidene chloride copolymers containing a minimum of 85';- vinyl* idene chloride have been approved for use with irradiated foods |t30|. information is scant as to the migration of imreacU-d monomers from Ihc^e sources cither into food or via disposal of the polymeric material pe<- sc. < hie report stales that no more than 10 ppm of imrcnded vinylidenr rlilnridr is contained in Dow's product Saran Wrap and that within detectable limits, no more than 10 ppb could get into food, even under severe condition-, of vise U371. An investigation of the cancer risk among a cohort of 138 workers exposed to vinylidiue chloride (where vinyl chloride was not used as a copolymer) revealed no findings statistically related or individually attri buted to vinylidene chloride exposure [12-1] . Fifty-five people had less than 15 years since first exposure and only 5 deaths were observed: \>,,1 workers were lost Ln follow up but were considered alive in the analysis l I 2 I | . The health effects of vinylidene chloride have been reviewed by II.dec j!38| and IAHC 1119) and the U.S, Environmental Protection \grncy l139|. Aspects of the reported carcinogenicity of vinylidene chloride appear conflicting and indicate sex, species, and strain specifieiLy. Twenty-four of 150 Swiss male mice exposed to 25 ppm of vinylidene chloride in air for 1 hours daily. 4--5 weeks for 52 weeks, developed adenocarcinoma of ihc kidney (compared to 1 out of 150 females). No such tumors occurred m mice exposed to 10 ppm for 52 weeks or in controls (110.! 41 |, nor in HALB/C, C56BL or CjII mice or Sprague-Dawley rats and hamsters similarly exposed to vinylidene chloride [142]. An increased incidence of mammary fibroadenomas and carcinomas was reported in female Sprague-Dawley rats exposed to 10, 25, 50, 100 and 150 ppm vinylidene chloride in air for 4 h/dav, 4--5 days a week for 52 weeks and observed for up to 82 weeks. No dose-response relation was bumd and in one rat treated with 100 ppm, vinylidene chloride, one 2ymb.il eland carcinoma was observed [141). Viola [143] reported (hat male and female Wistar rats exposed to 100 ppm of vinylidene chloride by inhalation developed abdominal lymphomas and subcutaneous fibromas. I vvo-year studies at Dow Chemical Co., involving both vinylidene chloride administered in Lhe drinking waler (60, 100 and 120 ppm) and repealed inhalation (10 or 10 ppm 6 h/dav: 5 days/wcek; after 5 weeks. 75 ppm tor up to 18 months) to male and female Sprague-Dawley rats have been arrietl mil |145,1-1G| and indicated no dose-related clinical diffcri-nces or RSV 0016323 i cumulative mortality differences or findings of neoplasia. Reproduction studies with vinyiidene chloride administered to Spraguo-Dawley rats Ininhalation or ingestion in the drinking water- ..bowed tin' compound to neither a teratogen or mutagen or one adversely affecting reproductivity [145]. The vinyiidene chloride l99.5'7) tested in Dow studies contained trace amounts (ppm) of the following impurities: vinyl bromide. A: vinyl chloride, 3--50; trans-l ,2-dichloroethylene, 138--1300: cik-1.2-dichloroethylene, 0.013--0.16%; 1,1,1-triehloroethane, 0.03: and 1.1.2-trichloroethane [145]. Winston et al. [147] and Lee [148] reported the only tumor in (.'D-rats exposed to 55 ppm vinyiidene chloride for 9 months was a subcutaneous hemangiosarcoma of the skin in one of the rats tested, llemangiosareoma of the liver and lung in CD rats exposed for 9--12 months to 250 or 1000 ppm of vinyiidene chloride have been reported. In addition, there were no lesions in the testes or accessory organs indicative of a treatment related effect on reproductive performance [66]. in a preliminary report by Maltoni et al. [141] of an ongoing study involving Sprague-Dawley rats administered 5, 10 or 20 mg'kg body weight, vinyiidene chloride by stomach tube once daily, 4--5 days/week for 52 weeks, 1 carcinoma of the Zymbal gland was observed in a rat treated with a 10 mg/kg dose. At the time of reporting, the rats had been observed for 93 weeks after the start of treatment. In most recent studies at I ARC [119], vinyiidene chloride when given by the oral route, induced malignant and benign liver tumors in C57R1 mice <>T both sexes and gastric tumors in female mice [1191 and or oral cavity tumors and of meningiomas in male BDIV rats. Maltoni et al. [141 ] reported in a study still in progress that no tumors had occurred at 74 weeks among a group of 30 male and 30 female Chinese hamsters, 28 weeks of age exposed to 25 ppm vinyiidene chloride in air for 4 h/day, 4--5 days/week for 52 weeks. Vinyiidene chloride in air (2% and 20%) produced reverse mutations in S. typhimurium TA 1530 and TA 100 in (he presence of 9000 g supernatant from mouse and rat liver, lung and kidney [149], The lower mutagenic response observed with a concentration of 20% vinyiidene chloride may have resulted from an inhibitory action of vinyiidene chloride and/or its metaboiite(s) on the microsomal enzymes responsible for its metabolic activation. It was postulated by Bartsch et al. [149j that 1,1-dichloroethylenc* oxide (in analogy with chloroethylene oxide the suggested primary metabolite of vinyl chloride) may be a primary reactive metabolite of vinyiidene chloride. It is also considered possible that partial dechlorination of vinyiidene chloride by microsomal enzymes results in vinyl chloride and its metabolic products [149]. Vinyiidene chloride in solution induced reverse mutations in l'. co/i K12 in the present r of 9000 g snpcrimlanl Irom nmnsr liver |7K| Vinyiidene rhlornle was noi millagenie in Ihe dominant lethal lest in male CD-I mice exposed by inhalation to 10, 30, 50 [150] and 55 [151] ppm for 6 h/day for 5 days/week. JRSV 0016324 . Reproduction Hawley rats hy .impound to be i; reproductivity ' udies contained romide, 4; vinyl cts-l,2-dichloro1,1.2-trichloro- umor in CD-rats a subcutaneous angiosarcoma of 50 or 1000 ppm ` were no lesions related effect tin \ ongoing study kg body weight, ivs/week For 52 at treated with a i observed for 93 le wlu'n given by n C57B1 mice of cl or oral cavity it no tumors had female Chinese hloride in air for e mutations in S. 00 g supernatant lower mutagenic iiloridc may have and/or its metaibolic activation, ihylene oxide (in >tabolite of vinyl ne chloride. It is dene chloride by tabolic products is in E. coli K12 I. `thal test in male 5 11511 ppm for ] 29 H,C = CCIj - H5C-CC( -- CKHjC-c/ -- OO CSH CICMjCOOH i* -cII -cI hcm5* sch*,1c=o ON------------------- * f \ COOH 1 AC y .C.CH.CHjtCHjCOOH I ONH I J HOOCCHCH jSCHjCOOH NHj J HOOC-CMCH jSCHjCOOH OM I S|CH }<OON)j l HSCHjCOOH ^SCH^COOHjj Fig. 6. Proposed mammalian metabolism i.f vinylidenc chloride { ir>2 | No chromosomal aberrations have been found in Sprague-Daw ley rats exposed to 75 ppm vinylidene chloride 6 h -dav, 5 dayS'Wivk for 2f> weeks |1IT>]. Biolransformalion mechanisms have been proposer! for vinylidi-mchloride by Haley (138| and I lathway [1521 (Fig. fi) analogous to (hat of vinyl ehoride involving the initial Formation of a chloroelhylene oxide; r.g., 1.1 -diehloroelhylenc oxide. Of the compounds shown in Fig. f. chloroarel ic acid. Lhiodiglycollic acid, thioglycollic acid, dithioglycollie arid and lactam compounds have already been isolated from (he urine of vinylidene chloride tn-ated animals [152]. (3) Trichloroethylene trichloroethylene (l-chlorO'2,2-dichloroethylene; 1.1.2-triehloroethylene: trichloroothene; acetylene trichloride; TCF: ClClI=Cn2 may be produced from acetylene or ethylene. Although the acetylene process (involving chlorination to 1,1.2,2-ti'trachloroethane, which is then dehvdroohlnrinatrd) has been the dominant method in the past, only 8% of the reported U.S. capacity relied on this process. Trichloroethylene is produced primarily by die chlorination and dehydrochlorinalion of ethylene dichloride. U.S. pro duction of trichloroethylene in 1974 amounted to approximately 193 million kg [153,154]. This represents a 30% decrease from the record annual production of 1970 [154], and was due primarily to legislation restricting the use and emissions of trichloroethylene and to the closing of three acetylene-based and one ethylene-based plants. Five U.S. companies pro duced 98 million kg of trichloroethylene during the first 9 months of 1975 [ 155]. In Japan in 1974, four companies produced 90 million kg. compart'd b> 112 million kg in 1970 [153). It was forecast that the world market for trichloroethylene during 1975 would he about G80 million kg [15C>|. Approximately 90% of trichloroethylene consumed in (he U.S.A. (315 million pounds in 197 l) is for vapor degreasing and cold cleaning of fabricated metal parts. Because of its implications in smog production in the 130 U.S.A. and resultant legislation restricted its use, it is expected that during the next five years consumption of trichloroethylene for metal cleaning.will decline at an average rate of 3% and tie most pitiably replaced hy 1.1.1trichloroethane and perchloroethylene [ 157]. Six percent (25 million pounds) of tin? trichloroethylene production is used as a chain terminator for polyvinyl chloride production 1153,1511. Additional areas of utility include: as an extract in food processing to.g.. for decaffeinated coffee), as a chemical intermediate: as a solvent in the textile industry and research laboratories; as an ingredient in printing inks lacquers, varnishes and adhesives, and in the dry-cleaning of fabrics. A .pharmaceutical grade of trichloroethylene is used as a general anesthetic in surgical, dental and obstetrical procedures. Largely because of its solvent properties, trichloroethylene is incorporated in a number of consumer products (o.g., cleansers for automobiles, buffing solution, spot remover, rug cleaner, disinfectant and deodorant) [ 158 |. The" threshold limit value for trichloroethylene in the IJ.S.A. is 0.535 mg/m3 (100 ppm). OS11A is currently in the process of approving the Criteria Document for trichloroethylene which recommends that a TLV expressed as a time-weighted average exposure for an 8-h workday continue at 100 ppm and that the presenl ceiling be reduced from 1.07 tng.'l (200 ppm) to 0.80 mg/1 (150 ppm) [159,160], The maximum allowable concentration in the U.S.S.R. is 10 mg/m3 .mil the MAC in several European countries has been set at 0.273 mg/1 (50 ppm I or even lower [ 161 ]. The number of U.S. workers exposed to trichloroethylene lias been estimated to be about 283 thousand [158] (Table 2). Levels of 1076--43,000 mg/m3 (200- 8000 ppm) of trichloroethylene have been reported in a small U.S. factory (1G21. Emissions of commercial organic solvent vapors into the atmosphere have been increasing dramatically in the last decade [1631 . The loss of trichloro ethylene and perchloroethylene to the global environment in 1973 was estimated to be each over 1 million tons [163]. Trichloroethylene emissions can occur principally from three sources, production, transportation and consumption. Estimated emissions from trichloroethylene production are 57.0 lbs emitted/ton produced [ 154.16 1 ]. The quantity of trichloroethylene discharged from domestic transport is very difficult to evaluate but it is believed that emissions occur almost inevitably from loading and transfer operations as well as accidental spills [165]. The major sources of emissions resulting from trichloroethylene con sumption can be attributed to its use as a solvent in open top vapor degreasers [154]. The average emission rate for an open top vapor degreaser in the U.S.A. is 110 tons/year [166]. Assuming that 55?0 of the vapor degreasing operations in 1974 used trichloroethylene, the total national emissions would have been approximately 121.000 tons or roughly 70'/ of the lolal amount of trichloroethylene used in metal cleaning operations [154.167|. Cold cleaners are another type of metal degreaser which ran contribute to trichloroethylene emissions. The aver age emission rate lor a cold eleam i is RSV 0016326 -4 575- afa no . <3 - = CL rD 1 c p 3cn r"-t V c crt> Ofi n rP> -- G 2 Op>"C P CL -- t-- D C3 --J <cn? C.J 3' < v> ptn T5 3" nn o' y p c .D 5 x tr p < O' 2 p 2. tn w> cr cr <9 rp o 3 Xi 3 3 rn M P '< >y p!- T3< O o TOPH y*i O *-j o 3 'T 0? 2 < W3 y TO it IT*- --i 3 t. C ft> (P c h" O' X Up1 r> O yj > cr c 3 O --*v P fP <J\ TO CL 3C. o' P C3tVL P cac. d. cr rr>* O TaO Ps &o v t w. r~ r: r > ~ o C> s 6 m = a ',r5:-u3-s2* _=;. 7- :-c.=gS.-3sci^tigaC.?-2-2Stoirn2e. if! &5 55 _3- 25 2so1j_..0?=o. ^ * O- i ? 3 ?c 3i = ss =i "s- s 2 S3 s X S?-LS2S?=3d3O C. -2 S. 4 9- 5, 3 re. C3 oQ -^K c -r C. JZ o 3 ? 75 JS < 2 t ' it O oCjiL ftj o 3 5 3 5= 'TC C V- T <* Pw i3'sC`5r3-eic' 3ic5; 52iCS"=..u3*9-tfi-gS31aa<ivxr.>e-?2_^--. 3-t CC- _ rt w S^ =o * I T K IC H L O R O E T llY U iN li BY INDUSTRY to CWD mOto' , "-- CO fO C 3 O' -j -4 t '-- CD tC O' to i- tO -J -I 'Ll O2' ro W!l OW MW ov IOt WO' N cn ci -- x. >- cn -o cOi o-->--c-r.-5i--(O*o V od C8 W -> X <J) O W - -4 O -t C W W p O' i. Vj -- ---- to o. to It to "-jci cl "to -l O' X M - V. ISc-j cp to ce -- O' -> ~. (?) <0t-S O' O' O' M M P C N 1C JO ill to *-- Cto O-o' C-ol t--o 'r!3<j wVJT. "a z s ct=r 5 C y. to 3J C<fl Oo --it. p> CO no >4 132 articles [168], and between 150 and 250 ppm in degreasing rooms per se* [169). Concentrations of vapor in a dial assembly workshop in a Japanese factory ranged from below 135 mg/trr (25 ptunl to over 538 mg. m3 (100 ppm) [169]. Concentrations of anesthetics (including trichloroethylene) in operating rooms to which surgeons and nurses were exposed varied from 1.6-~554 mg/in'3 (0.3 to 103 ppm) ]170|. It is estimated that about 5000 medical, dental, and hospital personnel are routinely exposed In trichloro ethylene [ 158 j. Typical concentrations of trichloroethylene at 3 locations in 5 U.S. states in 1974 ranged from 970 ng/nr' (180 ppt) in urban areas to Ic'S than llQng/m3 (20 ppl> in rural areas J171). )a*ss than 30ng'm' (5ppt) were found in air samples in rural I'ullman, Washington. from December. ] 97 1 to February, 1975 [172). Trichloroethylene lias been recently delected in air over 3 New Jersey industrialized cities [173|. Concentrations of trichloro ethylene in air samples taken at 5 land stations ranged from 2 28 tig, m 1 (0.5 5 pptl at 11 sea stations from 1 -22 ng/nv1 (0.2 $ ppt) anti over tin* imrl heast Atlant ic (>ee:m from 5 1 1 ng'nr' ( 1.2 ppt ) (17 11 . Slrghlly enhanced levels of trichloroethylene following ehlovjualum of water at vwnge treatment plants have her-n found in the U.S.A. 1 175,1701 . Trichloroethylene has been found in the organic constituent* of Mississippi ltiw-r wnler (before and after treatment) and in (he organic constituents of commercial deionized charcoal filtered water [ 177 |. Trichloroethylene con centrations of 54 kg/day of 1.2 ug/l in average raw wastewater flow have resulted from a doeaffviuatton process used in the manufacture of soluble (instant) coffee in California [17S|. Trichloroethylene has been found in foodstuffs such as dairy products, meats, oils and fats, beverages and fruits and vegetables in levels ranging from 0.02 Mg/kg in wine to 60 pg/kg in packaged tea [163]. Trace levels of trichloroethylene have also been found in edible oils after extraction [ 179]. The use of trichloroethylene for caffeine has recently hhen discontinued in the U.S.A. and the FDA in 1977 announced Lhat it intends to ban the vise of trichloroethylene in foods, drugs anil cosmetics. In the latter category, it had been used as a topical anesthetic* in some cosmetics j 180,181J. The National Cancer Insitute (NCI) in the (I.S.A. has recently issued a ``state of concern" alert, warning producers, users, and regulatory agencies that trichloroethylene administered by gastric intubation to H6(*3F mice induced predominantly hepatocellular carcinomas with some metnsases to the lungs, e.g., .70 of 98 I 30. f >'7) of the mice given (he low dose (! 200 mg/kg and 90(J mg/kg for male and female respectively) and 41 or 95 ( 13.2'-) of the mice given the higher dose (2400 mg/kg and 1800 mg/kg fur male and female, respectively). Only one of 40 (2.5'rl control mice developed these carcinomas j 158,182]. No hep.iioeellnlar carcinomas were observed in both sexes of Osborne Mendel rats administered trichloroethylene at levels of 1.0 or 0.5 g kg hv gastric intubation 5 times weekly for an unspecific period j 158 |. N<t liver lesions or hepatomas were found in NbC mice given oral doses by g rooms per se > in a Japanese >T 538 mg/m3 ) in operating varied from at about 5000 d to trichloro- m 5 U.S. states s to less Ilian <5 ppU wm-o mher, 107 t to detected in air >s of trichloron 2- 28 ng/nv1 ) and over the hlorinatiun of .A. [ 17 5,1 7 6 | . of Mississippi onsiituenls of oethvlene con iter flow have lure of soluble lairv products, ranging from .ace levels of raction [179}. lisconlinued in ban lhe use of ategory, it had entiy issued a latory agencies > B6C3F mice metasases to e (1200 me kg 95 (13.29') uf I for male and eveloped these s of OKfinnii' i>> 0.5 g/kg i>v '1 n oral doses by gavage or 0.1 ml of a 40% solution of trichloroethylene in oil twice weekly for an unspecified period [183]. Henschler et al. [184) recently postulated that the carcinogenic effect >f the technical sample of trichloroethylene used in the bumssay experiments [185] is most probably predominantly, if not exclusively, due to the epoxides [186] which are added to some but by no means to all brands of trichloroethylene. Technical grade trichloroethylene contains several impuri ties which must be stabilized for use as a degreasing agent, by antioxidants [153,184) (e.g., amines at levels of 0.001 to 0.2%) or combinations of epoxides and esters (0.2 to 27C total) [153]. Gas chromatographic -- mass spiH-trometrie analysis of the trichloroethylene sample performed by Henschler el al. 1181] indicated a proportion of identified contaminants amounting to 0.65% including strong monofimclional. alkylating agents of known mutagenicity and/or carcinogenicity (e.g., epiehlorohydrin and 1,2 epoxybuUme). Trichloroethylene (3.3 mMT in the presence of a metabolic activating microsomal system induced reverse mutations in E. coli strain K12 |78|. It has also been shown to induce frameshifl as well as base substitution imitation in A', ee/vei-suie strain W185-'4C in the presence of mice liver homogenate [ 187 j . Trichloroethylene exhibited dose-dependent mutagenicity at concentra tions of 0.01, 0.1 and 1 mg/ml when tested in S. lypiumiirnnn strains TA 1535 and 1538 without metabolic activation [188J. In tin* hostmediated assay (with female ICR mice! using strains TA 1950. TA 1951 and TA 1952, there was a significant increase in the number of rev rtnnls al trichloroethylene and perehloroethylene doses on the level of M)50 and 1 -'LDS 0. Cytogenetic analysis of mice bone marrow cells performed after single and repeated i.p. applications (5 applications in one-day intervals) after 0, 21 and 18 h following Liu* last application showed no significant increase in chromo somal aberrations following treatment by either trichloroethylene or per ehloroethylene 1188|. No adverse effects on embryonal or fetal development were observed billowing exposure of pregnant mice and rats to levels of 0.34 mg/1 (65 ppni) or 1.605 mg/l <300 ppm) of trichloroethylene [1891 The metabolism of trichloroethylene has been reviewed by IA11C [153]. Kelley [190] Henschler [191], Bonse and Henschler [192] and Van Houron |193|. There are three* major metabolic transformations: (1) oxidation lo chloral hydrate which takes place in the microsomal fraction of liver cells; (21 reduction to trichloroethanoi and trichloroethylene is metabolized to Lrii hloroethanol and trichloracetic acid in rats [194] and dogs [195]. The eiidier suggestion of Powell [196] in 1945 that formation of these metab olites implies rearrangement of the transient trichloroethylene oxide inter mediate into chloral has been confirmed by a number of findings including: ill llii* identification of ehlnrut as a trichloroethylene metabolite in ritn> 1197,198) and (2) by study of the rearrangement of the oxides belonging to a writ's of chlorinated ethylenrs 1191,192). Tim formation of triehlorn ethylene oxide (2,2,3-trirhloro-oxirnnei from trichluroethyIme m aerobi. incubates of liver micronios and NADIMI was c- ifirmed by spec!rat imestiga lion of tiie cytochrome IM50 complex [lily j. Epoxides are now recognized as obligatory intermediates in the metab olism of olei'tns by hepatic micro-omal mixed function oxidases [200). The metabolism of trichloroethylene through the oxirane includes diffemu rear Live molecular species. The participation of radical intermediates during opening of the oxirane ring and chlorine migration is possible (192) . Trichlompropane oxide lias recently been reported to augment the micro somal dependent covalent binding of trichloroethylene ]o |)\ \ or protein. 1o a greater degree in male Hum female mice. \ vti. l;iti<mi heuv.-n -ach binding and Iner Lumor induction Ity Lra him nrthvlenr in IU i( 1 mice i lm t not in ()>home-Mendel rats) was suggested ) 19.`j.20 1.2U2 ) . I he ivrr\rrsiblr binding of 1 '('-labeled trichloroethylene to mice hepatic protein in t iro and in utro has also recently been reported [203]. < 1I VWri.-Wdoiorf/n Iriu" Teirachloroei.hylene I perchlornribylen**; C!,C (T'U i is prepared pri marily via two processes [203|: (l) I he ! fuels, method whereby direct (T.lorinaimn of ethylene yields 70'.' pevhlurneihylrn<\ 2(1'' carbon tetra chloride anti 10'^ other chlorinated products and (2) hydrocarbons >iich as methane, ethane or propane are simukenously chlorinated and pyroly/ed Lo yield ewer 95'; perchloroethylene plus C( '14 and 1 KT The workl-producLion of perchloroeUivlem* in 1972 was 680 million kg: its growth rate estimated at 7^/year with a total world production estimated at 1100 million kg for 19R0 [204 1. The consumption pattern for perchloroethylene in Lhe U.S.A. in 197 1 is estimated to have been as follows: textile anil dry cleaning industries, G9Cf; metal cleaning. lOU; chemical intermediate <e.g.. preparation of trichloroacetii acid in some fluorocarbon* i, 12'anil miscellaneous 3U. (Vrchloroethylene is usecj as a solvent m tlie manufacture of rubber solutions, paint removers, printing inks, and solvent soaps, as a solvent for fat.x, oils, silicones and sulfur and as a heat-lvansfer medium [2031Some 500,000 workers currently are at risk of exposure to pen hloro- ethylenc according to NIOSII. It was also noted that over 20.000 drycleaning establishments and a large number of other industries manufacture or use perchloroethylene )205|. The current TLV for perchloroethylene in the U.S.A. is OTOmgon' (100 ppm). OSHA is attempting to reduce this standard to 50 ppm (206) . Work environment hygiene standards (all in terms of 8-hour lime weighted averages! for perchloroethylene reported by Winell |2U71 tire as follows (mg,in1!: Federal Republic of (armany. 070. the (lerman Democratic I! e public. 300; S wed mi, 20 lb ('/eela>*h" akia. 250. The M \ ( fur peirliloro elh\ lene m die U .S.S.K. is 10 mg/m ' . Depending mi its source strength, melonrolngieal dilution. sunlight intens ity, and the presence of oLber trace c< >n( ituenl s, peivhlmoethylene or its mi of trichloroIrne in aerobic i`etral investiga- in the metabises [200]. I he > different reacipdiates during [192]. netu the microN.\ or protein, i between surli .'3F1 mice (but Che irreversible lein in vivo and prepared pr i /hen-by dip" t carbon tetraarbons such as d pyrolyzed to >80 million kg; tion estimated > for perchlorojllows: textile il intermediate ms), 12^; and h> manufacture mt soaps. ai a insfer medium to pen hloror 20.000 dry 's manufacture is 070 meon3 ppm [200 |. t ime weighted are us follows in IVtno<r:it ic for | irrcl ilc `i i Miilicht intensc `I hy lene i >r il s predominant product, phosgene, accordingly may or may not be observed in nonurbau areas [ 171J. It was estimated that an ambient concentration of 10 ppb perchloroethylone observed in New York City should lead to the formation of 12 ppb phosgene [171] (TT.A'= 100 ppb). In 197). at S locations, in 5 industrial stales, the concentrations of perchlonhylene ranged from 1.2 ppb in the urban area to less than 0.02 ppb in rural areas. Perchloroethylene was measured at concentrations exceeding 0.0(3 ppb at least 507f of the time at all locations [171J. rearson and McConnell [20S| reported that city atmospheres contain from less than 0.1 to up to 10 ppb (<0.68--08 pg/m3) of perchloroelhylene. The air in the center nt Munich was found to In* contaminated with 0.8,8 ppb (0/jg/nv') >f perehlmoethylene in 1975, while the suburbs of Munich had an ambient concen tration of 0.59 ppb 14 pg/m31 [209]. Populations living in urban atmospheres polluted by 6 /jg/'m3 of perchloroelhylene were calculated to breathe in daily levels of about 90 pg [210]. Persons consuming three liters of tap water contaminated by 2S.3 ppb of the solvent as described by Giger and Molnar [211], would ingest 85 ng pprchloroethyjene/day (210 [. Chlorination at sewage treatment plants has resulLed in slightly < nhaiuvd levels of perchlorui-lhylcne in water [212,176].. Similarly to trichloro ethylene, perchloroelhylene (5^/1) lias been found in the organic con stituents of Mississippi Itiver water and in the organic consULaenis of commercial deionized charcoal-filtered water [177]. Pearson and McConnell [208] reported the concentration of perehloroethylene in animals to range from 0.5 Ui 50 ppb. The levels in tissues of animals at the upper end of food chains were elevated 100 times the environmental concentrations at most. This was considered a w**nk bioaccumulation potential as compared to other halogen compounds. 'Die accumulation coefficient in the quotient from the uptake rate and clearance. Welny et al. [213] found an accumulation ratio of perchloroelhylene of 39.6 5.5. The respective value of hexachlorobenzene (HCB) is 7880. Perchloroethylene has very recently reported to be carcinogenic in NCI studies, producing liver hepatocellular tumors in B6C3F1 hybrid male and Ik-male mice when tested at MTD and I'/MTI) dose levels in corn oil solution by gavage [214,415]. No carcinogenic activity was observed in analogously treated Oshorne-Mcndel rats of both sexes [214 ) . Perchloroethylene has not been found to be carcinogenic in inhalation -indies with rabbits, mice [216], rats, guinea pigs and monkeys. IVrchloro tliylene showed dose-dependent mutagenicity at concentrations of 0.01,0.1 .mil 1 mg/ml in S. typfumurum? TA 100 without metabolic activation [ 1<S8 |. iVrchloroethylene (0.6 mM) as well as the cis- and fro/js-isomers of 1.2.hchloroethylene were found to be non-mutageuic when tested in the metaUdi/.ing in vitro system with E. coli K12 [78,217]. The mutagenicity of Miiyl chloride, vinyiidene chloride, t riehloroelhylene, in Hie alrne lest -S'b'm was ati ril nil.ed In Iheir iml.edlv Inrming unstable nxnanrs, whereas b ilneai li*ns such as perehlornelhvlene ami eis and .'oms 1.2 la hlnrnelhyleiu' which form much more stable oxiranes were non-nuiluc-me I 7 K. 1 92,2 I 7 l . Trichioroethyleneoxide in HjO Cl fo) H c d--^ ct Cl (o) ,H in vivo V a '-^ci r---------- - - - - - - CHa2-*ooci * 3;*icooce*^tFe Cl 0XN_. *.H *. ,c c> ci - ci CCIj-CHO ---CCI.-C00H i CCIj-CHpOH Chloroethyleneoxide CL>-L-H HN--^ H a CHjCI -- CHG1-- a--s : weetfecrr*ycc H H Fig. 7. Rearrangements of Lrichioi'oelhyleneoxide and chlnr|li ide Figure 7 illustrates the metabolic pathways of tetrachloroethylcm* as proposed by Hortso and llenschler [192). The metabolic formation ol trichloroacetic acid can he explained by the primary formation of the oxirane and subsequent rearrangement to trichloroacetvl chloride anti it'subsequent hydrolysis. In a recent review, Ilenseher [271] contrasted the metabolism and mum genicity of chlorinated olefins. In this class of molecules, the electron withdrawal effect dominates over the mesomoric donator effect of tieinvolved carbon atom hence deereasim: the election density in the douhh bond which in turn results in a chemical stabilization against electrophilic attack [192]. It is optimal in pcrehloroethylene as has been demonstrated by the reactivity of chlorinated ethylenes with ozone [218]. As has been previously cited [108,192,217], the chlorinated ethvlenes may undergo a variety of reactions, e.g., (1) reaction with nucleophilic cellular macromolecules under alkylation; (2) conjugation wiih low molec ular nucleophiles (mainly glutathione) both enzymatically and noti-cnzymatically; (3) hydrolysis to diols. with anti without the catalytic action of enzymes such as epoxide hydrase; and i l) intramolecular rearrangement. Tinlatter reaction according to llonschler [217] represents a deactivation mm-h anism and is of considerable importance for the potential of acute toxicity as well as of carcinogenicity and mutagenicity of the different members of the series of chlorinated ethylenes. (5) Ch loroprene Chloroprene (2-chlorobutadiene; 2-chioro-l.3-butadiene: beta-chloro- prene; Cl 12 ~C--CU = Cli2) is the monomer for neoprene, the specialty rub l'\ her. It is pn-pau-d by two major vuub-s: ll i the ihmrn/.alion ol ao-tyU-uo to mouovinylaeetylenr ami atklilinn of hydrogen chloride ami (2) the chlori nation of butadiene Lo a mixture of dichlorobutenes, from which 2.1-dichloro-l-butcne is isolated and then is sui>jecled to dehydrnchlorinatinn RSV 0016332 1 H neoN idi'. oethylene as formation ol' ation of the oride and its and mulathe electron ffect of the >n the double electrophilic lemonstrated ied ethylones mu loophihc h low mokrnd iion-i'ii/.y. lie action of .Igl'lUL'lU. Che ivatinn moch;te toxicity as embers of the beta-chloro>peeialty rub- acrl ylrn>- In .11 the chlori- vliich 3.1-diichlormatiim 137 {219, 220). The latter method is believed to be the basis of the current U.S. production of ehloroprene. In this procedure, butadiene is Tirst reacted with chlorine to yield a mixture of dichlorobutene isomers from which the' 3.4-dichlorobutene-l isomer is isolated and then reacted with caustic to form ehloroprene. 1, t-l)irhlornbulen<'-2, the other isomer, can either be isomerized to 3,4-diehlorobutene-l for additional ehloroprene production or it can be utilized for in the production of adiponitrile [2201. A typical specification for ehloroprene made from butadiene is as follows: ehloroprene, 98.59o min., 1-chlorobutadiene, 1.07r max., aldehydes (as acetaldehydes), 0.2?r max., 3,4-dichlorohutene-l. 0.01rr max., dimers, 0.01'V max., peroxides, 1 ppm max., and no detectable amount of vinyl acetylene (221J. In 1976, two U.S. companies produced an estimated 164 million kg of ehloroprene, while throe Japanese companies produced a total of 80 million kg (70'i of ehloroprene is based on acetylene and JU'.r on butadiene). The total Western European production in 1977 amounted to an estimated 100 million kg of ehloroprene. The total world production of ehloroprene in 1977 is estimated to have been 300 million kg [220]. Chloroprene is extremely reactive, e.g., it can polymerize spontaneously at room temperatures, the process being catalyzed by light, peroxides, and other free radical initiators. It can also react with oxygen to form polymeric peroxides and because of its instability, flammability and toxicity, eldoroprene has no end product uses. It is used exclusively and without isolation in the production of neoprene elastomers [222 [. Neoprene as obtained by emulsion polymerization of ehloroprene consists mainly of traus-polychloroprene. There are two main classes, the sulfur modified type and the non-sulfur modified type, indicating the differences in polymerization techniques [222, 223). About 100 million kg of neoprene were consumed in the IJ.S.A. in 1976 with the following consumption pattern: the production of industrial and automotive rubber goods, 63';; wire and coble applications, 13';; eon-miction applications, 10adhesives applications, 8% and miscellaneous m-s.6' ; [2201. An estimated 2500 workers are currently exposed to chloroprene in the i s.A. 1222, 224). I he TLV for chloroprene in the U.S.A. is 25 ppm (90 mg/m1). In August, ' NIOSH recommended that occupational exposure to chloroprene he l initt-d to a maximum concentration of 1 ppm (3.6 mg/nrM in air deter mined ns a ceiling for a 15-min. period during a 40-h work week [ 225 j . Work environment hygiene standards (all in terms of 8-hour time weighted nrr.igcs) for chloroprene reported by Winetl [207] are as follows (in mem3): Federal Republic of Germany, 90; the German Democratic Re public, 10; Sweden, 90 and Czeckostovakia, 50. The MAC of chloroprene in du' 1 '.S.S.R. is 2 mg/m3 . ( hlornprenc has been detected as an impurity in commercial vinyl ehlor> !< m Italy (22li |. Japan [227 j and in acrylonitrile in the U.S.S.It. 1228). * hloroprene concentrations of 14.5--53.4 mg/m3 have been reported in RSV 0016333 i :tK the air inside a Russian neoprene rubber plant; 0.2- 1.57 mg/nr1 500 met- i from the plant and 0.12-0.38 mg-m3 7000 meters from the plant j 2`JO! . I another neoprene rubber plant in the tin* chknoprene comer tration in air in the immediate vicinity was 28.-45 mg m1: 0.727 mg in' 5m meters from the plant, and 0.199 mg nr 7000 meters away (230). Worker in a Russian shoe factory have hem reported to be often exposed t. chloroprene concentrations of 20--25 mg nv' |231(. Chloroprene which has been used since 1930 in the manufacture " synthetic rubber lias recently been suggested to be responsible fur tin increased incidence of skin ancl lung eancer in exposed workers in tin U.S.S.R. 1232.233}. During the period 1956--1970. epidemiological Mudie- of industrial workers in the Yerevan region re\ealed 137 c;w`s of skin oinri'i among approximately 25.000 workers over 25 years of ace. Kach of ih- 25,000 workers was classified as belonging to one of tile following group* (I) Never worked in industrial plants: ill) persons working in non-cheinira1 industries; (HI) persons working in chemical industries but not exposed p. chloroprene or its derivatives: (1 \*> persons working in industries using chloroprene derivatives: and (V) persons with extended work experience in chloroprene production. The incidence of skin cancer within these group* v. order from (l) to (VI revealed a striking gradient, e.g.. 0.12';, 0.40' -. O.fift' . 1.60% and 3.0%. The study also indicated a gradient m the average age of tin skin cancer cases and the average duration of employment, with worker*' exposed to chloroprene or the derivatives showing the lowest values. The chloroprene workers who developed skin cancer had an average age of 59.6 years and an average duration of employment of 9.5 years [2331 During the same period. 87 cases of lung cancer were identified among 19,979 workers in the same region. The groups exposed to chloroprene or it* derivatives had the highest incidence of lung cancer (1.16%). These workers' average age was 44.5 years with an average duration of employment of 8.7 years. Of the 34 cases of lung cancer in this group. 18 were among persons having a direct and prolonged exposure to chloroprene monomer, the re maining 16 involved individuals exposed to chloroprene latexes [232}. The frequency ratio of primary lung cancer occurrence among the comparison or control groups compared to the chloroprene groups was: 2.67 times lower in workers with chemicals unrelated to chloroprene; 6.3 times lower in workers in non-chemical industries and 17.5 times lower in workers in cultural and civic institutions. The limitations of the above two studies (232,233) have been recently cited by 1ARC (220) and include: failure to distinguish prevalent from incident cast's, to document completeness of case ascertainment among the exposure group, to adjust for effect of age and sex, to measure the extent of exposure, to control for the potential confounding effect of smoking and to furnish pathology information on cell type (particularly important in the study of reported skin cancer). A study oT ennerr inortnIiH among (wo cohorts of males engaged in Hie production and pol> men/alum of chluniprenc in Liu* U.S.A. was recently RSV 0016334 m3 500 meters Plant [229J. In *prem? coneen'27 mg/nv 500 1230). Workers exposed to nannfacture of 'nsjJ>!t> for the workers in the "logical siudics ' of ^kin cancer Kadi of the (lowing groups: 0 non-chemicaJ not exposed to ndustries using experience in these groups in 0.4b%, 0.66:;, *'rage age of the with workers est values. The -`ge age of 59 q -3J. entified among oroprene or its "hese workers' loyment of 8.7 among persons nomer. the re\es 12321. The ' comparison or 1 times lower in >wer in workers in cultural and '' been recently prevalent from :>ent among the re the extent of smoking and to iportant in the ongapt'd in was recently i no reported l)y Pell [234). One cohort consisted of 270 men first exposed between 1931--1948 arid the other of 1576 men first exposed between 1942--1957. The number of lung cancer deaths in each cohort (3 in the fir.>t and 16 in the second) were about the same as was expected on the basis of U.S. or company wide rates. However, the? risk of digestive cancer (19 versus 13.3) and of lymphatic and hematopoietic cancer (7 versus 4.5) were slightly elevated when contrasted with company wide experience. Among maintenance mechanics in the study/coliort of 1576, there were 8 lung cancer cases 14 living and 4 deceased) which accounted for approx imately 37% of the lung cancers found in the total study cohort. In < entrust, only 17ri of the total cohort was composed of maintenance mechanics. U must be noLed that these mechanics whose tasks include the general maintenance in the reactor areas, installation of equipment and the replace ment of leaking pipe fittings would be expected to have a potential for exposure (perhaps to high levels) to cliloroprene (220), A number of limit ations to the above Pell study [234] were enumeratred [220j. These include: no data were presented on any potential confounding variables such as smoking history and other occupational exposure: no specific exposure information bused on chemical measurements was provided: no dala pre sented on cell type analysis of the malignancies; and methodological short comings of combining of workers engaged in cliloroprene monomer produc tion and those in polymerization. There may also have been selective removal of high risk, high exposure workers from the cohort examined, in that retirees, disabled workers and former chloroprene individuals exposed in job categories not ucticcly involving cliloroprene were not included in the inception cohort. Additionally, the major limitations in interpreting this study were cited by IARC [220] to be: (a) the period of follow-up for the cohort is still quite incomplete for an adequate latent period and thus there may eventually be demonstration of statistically significant excesses and (b) the power of this study is limited due to the small number of person-years of exposure. Mo carcinogenic effects of chloroprene have been noted to date in animal Mmlies [220) involving oral [235J and intratraehael [235] administration to rats and dermal application [235,236] and subcutaneous injection |23o, 236] of chloroprene to mice and rats. A number of additional studies -ire in progress to investigate the carcinogenicity by oral administrations to r.its and by inhalation exposure in rats and hamsters [237--239). Kxposure of S. typhimurium TA 100 and TA 1530 strain to 0.5-8^ of vhloroprene vapor in air in the absence of any metabolic activation system 1 xused a linear increasing mutagenic response (reaching 3 limes Hie spon taneous mutations rate at a concentration of 8rr) [149], Kxposure to a hiclier concentration (20/r) caused a strong toxicity in the bacteria. This ntilagenic and/or toxic effect could be caused by a direct action of chloroI'rcne or more likely, by one of its en/.ymie (bacteria) or non enzymic l*r< akcl(nvn products. Up to a 3-fold increased mutagenic, response was found "hen a fortified 9000 g liver supernatant from either pheiiuharhilnne (vented 1 I 140 or untreated mice was added to such assays supporting an enzymic for mation of mutagenic metabolite(s) from chloroprene [149] (probably an oxirane (epoxide) in analogy with vinyl chloride, vinylidene chloride, and trichloroethylene) [10,149]. Liver supernatant from some human biopsies also enhanced mutagenicity of chloroprene [10], Treatment of male Drosophila for 3 days with 5.7 mM and 11.4 mM chloroprene resulted in an increase of X-linked recessive lethal mutations from 0.8 0.04% in the control to 0.58 0.3% and 1.0 0.4%, respectively [240]. Vapor of chloroprene (0.04--1.0 ppm) [241--244] induced dominant lethal mutations in sperm and chromosome aberrations in bone marrow cells of rats and of mice exposed to 1.83--3.5 mg/m3 [243]. Mixtures of chloro prene and methyl methacrylate [24,246] chloroprene, dodecylmercaptan and ammonia [245] also induced chromosomal aberrations in bone-marrow cells of rats. An increase of chromosomal aberrations have been reported in cultured peripheral lymphocytes from workers occupationally exposed to chloro prene [243, 244,'247] or chloroprene and methyl methacrylate [245]. For example, Katosova [247} noted a significant rise in the number of chromo some aberrations in blood cultures of workers exposed to an average chloro prene concentration of 18 ppm for 2 to more than 10 years. In addition, decreases in motility and number of sperm were noted in exposed male workers as well as a three-fold excess of miscarriages in the wives of chloroprene workers have also been noted [224]. Testicular atrophy and reduction in the numbers and mobility of sperm in rats with non-atrophoid testicles have been noted to an exposure level of chloroprene down to 0.06 ppm [241,244], while spermatogenesis in C57B1/6 mice was affected after 2 months exposure to 0.32 ppm chloro prene [243,248]. Sanotiskii [243] reported that the threshold for chronic effects of chloro prene on animals based upon the indicators of general systemic effect is 1.69 0.987 mg/m3, about the same as the maximum permissible concen tration (MPC)in the U.S.S.R. formerly adopted. The threshold concentration based on specific indicators (e.g., embryotropic, gonadotropic and mutagenic effects) was 0.15 0.0059 mg/m3 (e.g., one order of magnitude below the former MPC). Neither embryotoxic nor teratological effects have been noted after exposure of pregnant rats to 90.5 mg/m3 (25 ppm) of chloroprene 4h/day from day 1 until day 12 and day 3 until day 20 of gestation [249]. The biotransformation of chloroprene has been postulated by Haley [250] to occur in an analogous fashion to vinyl chloride and vinylidene chloride, e.g., via the formation of the epoxide by the action of mixed function oxidases. The intermediate epoxide would give rise to the aldehyde or combine with glutathione and subsequently form a mercapturic acid derivative. The known oxidation of chloroprene in positions 1 and 2 as well as the decreased tissueSH content would appear to lend some support to this postulated biotrans formation. .... ar; -;r RSV 0016336 an enzymic for>9] (probably an I'ltf chloride, am) human biopsis iM and 11.1 m.M lethal mutations OA't. respectively iduced dominant 'one marrow cells Aliirn-; ol chlovo^odecylmercupUin s in bone-marrow ortcd in cultured posed to ch.lorowlale 1245]. For imiicr of chromom aw'rage r;Vitr>r* i ears. In addition, in exposed male in the wires of bility of sperm in exposure level of `rmatogenesis in ).32 ppm chloro- effeets of chloroystemic effect is rmissible concern 'Id concentration >ic and mutagenic aitude below the have been noted ) of chloroprene gestation (219). y Haley (250 ) to ene chloride, e.g., unction oxidases, or combine with alive. The 1; nown -Uikued blot rails- 141 (6) Trans-l,4-dichlorobutenv Cl |I 7Vuus-l,4'dichlorobutene (l,4-dich!oro-2-butene; 1 iC`--C=c--c--11) isVm- 11 II 11 Cl ployed in the U.S.A. mainly as an intermediate in the manufacture of hexamethylenediamine and chloroprene. Hexamethylenediamine is further used as a chemical intermediate in the production of nylon 66 and 612 polyamide resins, while chloroprene is used in the production of polvchloroprene rubber. While the U.S. production of hexamethylenediamine and polychloroprene rubber in 1975 was 340 and 143.9 million kg respectively. the percentage originally derived from trans-l.l-diehlorobuU'ne is not known 1251). 7>a/j.s-l,4-dichlorobuleno has been shown to be weakly carcinogenic by subcutaneous and intraperitoneal administration in ICR/HA Swiss mice but not carcinogenic in mice via skin application (252]. 7Viws-l,4-dichlorobutene produced mutations in S. typfwmunwiM TA 100 strains (2531 with the mutagenic effect enhanced hy liver microsomal fractions from mouse or humans. It has also been reporLeil mutagenic in K. culi (251) and S', ctwi'isiac j255). It has been suggested that trs-l,4-dichlorobulene-2 could conceivably be metabolized to an epoxide intermediate which is analogous in structure to open-chain fl-chloroethers (252 ]. l" ) Hexachlorobutadivne Hexachlorobutadiene (1,1,2,3,4,4-hexachlorobutadione; C1 Cl HCBD; per- chlorobutadiene; Cl2-- C=C--C=CC1-.) is normally obtained in commercial quantities as a by-product in some chlorinated hydrocarbon processes (e.g., perchloroethylene production). It is found in the tarry wastes (HEX wastes) along with hexachlorobenzene, hexachloroethane and other chlorinated by products [256]. In 1974, although no HCBI) was produced in the U.R.A., 200.000 to 500,000 pounds were reported imported in the same year [256]. 1 bo production of perchloroethylene, trichloroethylene, carbon tetra chloride and chlorine in the U.S.A. in 1972 produced I1CBD (thousands of pounds) as follows: 8670; 3000; 2790; and 70 (255). The production of perchloroethylene, trichloroethylene, and carbon tetrachloride accounts lor '.'HT of the HCBD in the U.S.A. [256]. Approximately 10 million pounds of HCBI) and 5 milion pounds of Iwxachlorobenzene (HCB) are generated annually as hex waste in the U.S.A. [2571. I he largest use for HCBD in the U.S.A. is for the recovery of "snift" or < lili>riiif'-eoMlaining gas in chlorine plants. HCBI) is also used us a chemical i` immediate to produce lubricants, as a solvent and in heat transfer and hydraulic fluids. RSV 0016337 J 142 I HCBD (analogous to HCB) is highly resistant to chemical, biological and the physical degradation and hence is a stable environmental pollutant. the } HCBD in the ppb range has been found in water, soil, selected aquatic usee i organisms [258] and food [257,259] (fish, eggs, milk, vegetables) samples usee taken along the lower Mississippi River in Louisiana. usef There is a paucity of carcinogenicity and mutagenicity information on hexachlorobutadiene. In one limited study, no tumors were found in rats after 6 months administration of HCBD at levels of 2--7 mg/kg in the diet [260]. Recent chronic toxicological studies at Dow Chemical Co. in the U.S.A. [261,262] suggest the possibility of a threshold level for HCBD. For example, in a study where male and female Sprague-Dawley rats were maintained on-diets containing 20, 2.0, 0.2 mg/kg/day of HCBD for two years, the lowest dosage caused no observed adverse effects. Ingestion of the intermediate dose level of 2.0 mg/kg/day caused some degree of toxicity, affecting primarily the kidney in which increased renal tubular epithelial hyperplasia as well as an increase of urinary excretion of coproporphyrin was be t cope uyat u hil OtlK A allyl and .-.titi as tl T abo noted. Ingestion of the highest dose level (20 mg/kg/day) resulted in renal No tubular adenomas and adenocarcinomas, some of which metastasized to the N lung [261]. nos* A review of health data on Dow employees working in areas where HCBD has been found, revealed no abnormalities which could be attributed to the chemical. It was also noted that wastes containing the material are being recycled or incinerated in specially designed facilities [262]. li> Fed tu a mil: 'I HCBD has been found mutagenic (with and without activation) in Salmonella typhimurium TA 1535 and TA 100 [263]. Schwetz et al.[264] described the results of a reproduction study in Sprague-Dawley fed diets containing 0.2, 2.0, or 20 mg/kg/day for 90 days prior to mating, 15 days during mating, and subsequently throughout gestation and lactation. Signs of toxicity among the adult rats were observed at the two higher dose levels and included decreased weight gain and food consumption as well as alterations in kidney structure. There was no effect on pregnancy or neonatal survival and development. No toxic effects were observed among the adults at a dose level of 0.2 mg/kg/day or among the neonates at dose levels of 0.2 or 2.0 mg/kg/day [264]. 1 In eon bet' lion (50 (80 '1 chic guii to s 8. Allyl chloride Allyl chloride (3-chloro-l-propene; 3-chloro-propylene; chlorallylene; CH2=CHCH1C1) is the most important of all commercial allyl compounds. It is reactive, both as an organic halide and as an olefin. In contrast to the vinyl halides which are characteristically inert in either SN 1 or Sn 2 nucleo philic substitution reactions, allyl halides are very reactive, much more than corresponding saturated compounds in both Sn 1 and SN 2 reactions. The double bond facilitates breaking the bond to the functional gTOup in dis placement and substitution reactions. In addition, the allyl group when introduced into other molecules, is usually reactive hence permitting many syntheses of potential commercial interest [265]. Although other reactions for the preparation of allyl chloride are known. I wel chit inac vol; vap mn' mit gen ablact fro RSV 0016338 biological and ntanr. 'fleeted aquatic tables) samples information on <* found in rats 1 mg'kg in (.be ai Co. in the for llcpn. Por u'li*y nits were IlCBD for two digestion of the roe of toxicity, bnlar epithelial 'porphyrin was '`suited in renal nstavized to Hie as whore HCIU> Unbilled to the terial are being activation) in >vetz et al.[2(i4 | >awley fed diets lating. 15 days tation. Signs of ' dose levels and 'll as alterations eonata] survival adults at a dose Is of 0.2 or 2.0 : chlorallylene; llyl compounds. ' contrast to the 1 or SN 2 nucleomuch more than ' reactions. The .il group *n ibs(vl crimp w ben cmiUting many ride are known. l \:\ the high-temperature substitutive chlorination of propylene is believed to be the only route used commercially at present [265). Allyl chloride is mainly used as a monomer in the production of various plastics ami resins that nv used per nc or incorporated into surface routing* adhesives, ole. \ number of useful specialty resins are derived from allyl esters and polvesi.-rs that may be made directly from allyl chloride. Resin uses also include a number of copolymers and inter-polymers of allyl chloride with acrylonitrile, vmyliclene cyanide, styrene and diallyl esters developed to provide special properties, while allyl chloride also serves as a catalyst or modifier in the pmdui-iion of other resins. A number of commercially important compounds are math' dived k from allyl ehlonde, e.g.. "Iirsl-generation derivatives", glycerol, rpi'-hlorohvdriu ami allyl alcohol. Medicinal derivatives of allyl chloride include Phi sub stituted barbiturates, and mercury diuretics derived from allvlamine, ;is well the anesthetic, cyclopropane {265). The production of allyl chloride in the l FS.A. in 197d was ed hunted !<* be about .300 million pounds most of it produced lv two maun fact i iivrs j yji>i > J . \'> data are available on (.be amounts of allyl chloride produced elsewhere. NIOSU estimates that approximately 5000 workers are pulentially e \ posed In allyl chloride during its manufaeiure <,r use. N'lOSII i vis si i1 >uuU ed to OSIIA a criteria document recommending adherence to the preseni Federal standard of 1 ppm of allyl chloride as a lime-weigbled average for up i" ;i 10-h workday. K)-h work week and proposed the addition ni a 3 ppm iling concentration for any 15-min period [2G6j. 1 lie MAC (267) for allyl chloride in the IJ.S.S.U. is 3 mg m . Data are sparse concerning environmental exposure levels of allyl chloride. In one study of an allyl chloride production facility in the ITS.S.lt.. the 'memUration of allyl chloride in air of production-working areas varied h'-tween 6.-1--140 mg/m3. Workers in this facility showed early renal ftmer:,,n impairment, higher glomercular fillraLion of creatinine |10rd and urea '>0A) considerable hypernitremia, moderate kaliemia, higher blood Cl" 1 Ml' 1 and dysproteinemia [267). I here is a paucity of information regarding the chronic effects of nllvl kl'M'ide. In a limited study, allyl chloride cl id not induce tumors in rats, : ani>a pigs and rabbits exposed to 3 ppm of the agent by inhalation fur up t-1 d\ months [268]. In the standard Salmonella mutagenicity assay in which the bacteria as '11 as the test agent are incorporated in the agar overlay |26d|. allyl Monde did not exhibit any significant mutagenic activity 127U). This initial iiiiuty of allyl chloride under the above conditions can be ascribed to its '.aiility. When steps are taken to minimize dissipation of allyl chloride i'ers mto the atmosphere (e.g., via impregnation of filter discs with the test vihnal then placing them on the surface of the agar plates containing the r'lurganisins, and the plates then sealed in separnle plnslie bags) mula "iif art ivi t y o] allyl ehlm ide \ 0. | . \ and Ml pi/pi a I e ) was I lien < leuu i at ' I.- fin S ixphimunmn T.\ 100 and T \ 1 5X5. imL mb T.\ I bF.S, Mul .ip.eiuc '.aity was not significantly enhanced by micro,some preparations derived m Ui' livers of rul.s induced wiLh Aroelor [261)). lienee these findings RSV 0016339 144 suggest that allyl chloride acts as a direct acting base-substitution mutagen since it affects only those strains* TA 1535 and to some extent TA 100 capable of detecting such mutations [270]. Allyl chloride (at 10 p1) is mutagenic in the E. coli DNA polymerase deficient (E. coli pol A*/pol A") disc test [271,272] procedure, prefer entially inhibiting the growth of the poi A" strain which is indicative of DNA modifying activity [270]. Allyl chloride (at levels of 18.4, 24.5 and 30.7 x 10*5M) induces gene conversion in S. cerevisiae D4 [270]. Aspects of the biotransformation of allyl halides have been described by Kaye et al. [273]. Allyl mercapturic acid, its sulphoxide, and 3-hydroxypropylmercapturic acid were identified as urinary metabolites following subcutaneous administration of allyl chloride, bromide and iodide to rats [273]. S-allyl glutathione and S-ally1-2-cysteine were also detected in the bile of a rat dosed with allyl chloride. The study of Kaye et al. [272] did not establish with certainty the pathway or pathways whereby allyl halides give rise to the formation of 3-hydroxypropylmercapturic acid. There are a number of pathways by which this may occur because the allyl halide can undergo reaction either at the double bond or at the site of the halogen atom. Although it is not known whether metabolic formation of a 3-halogenopropanol (HOCH2CH2CH2X) from an allyl halide can occur, if such a reaction did take place it would probably be followed by the formation of 3-hydroxypropylmercapturic acid since the excretion of this mercapturic acid has been reported following the administration of 3-chloropropanoi to rats [274]. The conversion of allyl chloride to S-allyl glutathione and the conversion of the latter compound to allyl mercapturic acid was demonstrated by the work of Kaye et al. [272]. Additional potentially possible metabolic pathways for allyl chloride could involve the epoxidation of the double bond to form epichlorohydrin (e.g., CH2=CHCH3C1 CH2--CHCH2Cl) a known carcinogen and mutagen. The sub- sequent oxidation products of epichlorohydrin are glycidol (H2C--CH-- V CH2OH) and glycidaldehyde (H2C--CHCHO) both are mutagenic and the V latter is carcinogenic as well. The second metabolic pathway involves conversion to allyl alcohol, then acrolein, then acrylic acid, viz., H2C=CH3CH2Cl - H2C=CHCH2OH *- CH2=CHCHO - CH2 = CHCOOH. Acrolein (vinyl aldehyde) is mutagenic in Drosophila and S. typhimurium (strains TA 1538 and TA 98) [275, 276] and non-mutagenic in the dominant lethal assay in ICR/HA Swiss mice when tested at 1.5 and 2 2 mg/kg [277]. REFERENCES 1 Environmental Protection Agency, Scientific and technical assessment report on vinyl chloride and polyvinyl chloride. Office of Research and Development, EPA, Washington, DC, June, 1975. i 91 t \ 10 H _\ 11 C J< 12 L ri: t;< D w l4 C T. 15 U. Po (1`16 S. Te 17 U.! U,.1 IS \v. os: 19 C. km 20 V. . pro (19 21 I..A vn> 22 T. t proci 23 A. M Vop. 24 C. W sarco Acad 25 E. 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S| ><-ri i * >/*lioi * >>i i,-. > i. ni.ili...! I... del riM i rning ell I* o opr cue in ;ur. toy N.init . tin ( I !1 > 11) li I . A. A. Buyanov and (I. A. Svischchev, Pn.'ection of the .n mo-phere hom shoe 232 factory industrial emissions, Zv. Vyssii. Ucheb. Zaved. Tekhnol l.egk. Prom, (1973) US. E. A. Khaehatryan, Lung cancer morbidity among people working with chloi'o prenc. V* *ji Oncol , I S ( I *. 17 11 .X 233 l\ A K liaehal i v.i n, Tile ode > f .................. Itti tit.il t, ni , m ] | J'i ol /.i ho I IS | 19,31 .3 |. in 11 * pr*>i*--- <( -km net pl.e.m RSV 0016350 iouikI and <1 inking igress Preliminary .ci- >( Tox.c Sub licieni to measure n. Sci. Technol., 5 iiocnrbmis. NIEHS ' Chloride and He lene Carcinogenic. i bhnopl hyi !ii> and nl . 22 f 1 Pfi.v, 379 ms - a comj3.ii i-on ;e with chlorinated ds.). Encyclopedia New York . 1 964, Chemicals *.<> Man. , in press. icne, Hydrocarbon million on ihioru- ent, Vol. 5, Barnes carcinogenesis and , New York. Sept. 2 11977) (4 c determination of "ii of high-boiling Kagaku Zasshi. 7 1 rylmutrile, Zavod. mekler, and A. S ' synthetic /ubber ion of white mis. aisakanvan. G A. metric method lor -phere from shoe4. Li'gk. Prom . 3 iking with chloro- of \|, 1 M 111-. ,|>1 |M11 155 234 S. Pell, Mortality of workers exposed to cliUvroprcne, Presumed nl Uonlrrencc of American Occupational Health, Boston, April 26 < 1977), J. Occup. Med.. 20 (1978)21, 235 V. N. Zil'fynii. B S. Fichidzhy an. 0. K. Garibyan and A. M. Poyosova. Expenmnninl study of chloroprene for carcinogenicity, Vtip. Onkol., 23 (1977) 61 236 V. N. ZilTyim. Results of testing chloroprene for carcinogenicity, J. Lxp. Clin. ,Mrd Akad. Sci. Armen. SSR, 15 ( 1975) 5-1. 237 Toxicology Information Programme, A long-term inhalation study of born* chloroprene to rats, Tox-Tips, 3 ( 1976) -I. 238 Toxicology Informal ion Prog ram me, Chrome inhalation of bi*ia-i:iilo.o|io`in' in hamsters, Tox-Tips. 1 3 f 1977 ) 31. 239 IARC, Information bulletin on the Survey of Chemicals Being Tested for Carnnogeniciiv, [nturnationai Agency for Research on Cancer, No. 16, Lyon. 1976,p. 13. 240 E. Vogel, Mutagenicity of CMfcinoucn.s in Drom>phila as a Inni'lion >l genotype controlled metabolism, in F. .1. deSerres. ). R. Fouls, J. R. Bend ami R M Pliilp<it (Eds.), Elsevier North Holland Biomedical Press, Amsterdam, 1976 241 R. M. Davtyan, Toxicological characteristics of the action of chlomprenc on the reproductive function of male rats, Toksikol. Cog. Prod. Neflrklicim. Prozvod. Vses-Konf. Dokl., 2nd. 1971 (Publ. 1972) 95--97; Cliem. Absir., 80 (1971) 91720A. 242 R. M. Davtyan, V. N. Fumenko and G. P. Andreeva, Effect of chlomprenc on the generative function of mammals Imales)). Chcm. Absir., 79 (1973) fin. 243 1. V. Sanotxkii, Aspects of toxicology of chloroprene: Immediate and long-term effects, Environ. Health Persp., 17 (197 6) Hf>. 244 2. A. Volkova, Establishing the value of the MPEL of chloroprene tn the air of a work area. Gig. Trud. Prof. Zabol.. 3 ( 1976) 3 I. 245 S. B. Bagramjam and E. A. Babajan, Cytogenetic study of the mutagenic activity of chemical substances isolated from Nairit latexes MKA and LNT-1. Biol. Zb. Arm., 27 (1974) 102. 246 S. B. Bagramjam, A. S. Pogosyan, E. A. Babajan, R. D. Ovanesjnn and S. M. Charjan. Mutagenic effect of small concentrations of volatile substances, emitted from polychioroprene latexes LNT-I and MKH, during their combined uptake by the animals. Biol. Zh. Arm., 29 (1976) 9b. 247 L. D. Katosovu, Cylogenic analysis of peripheral blood of workers engaged in the production of chloroprene. Gig. Tr. Prof. Zabol., 10 (1973) 30. 248 I. V. Sunotskii. Correlations between the gonadolvpes, embrvolropic and blasto mogenic types of effect of chemical compounds, Mater. Nauch Ses.ii Posvy. Sozdan. SSR Tbilisi. (1972) 117. 249 R. Culik, D. P. Kelly and J. J. Clary. (J-chloroprene (2-chlombutadiene-1,3) Embivotoxic and teratogenic studies in rats, Toxicol. Appl. Pharmacol., 37 (1976) 172. 250 T. J. Haley. Chloroprene (2-chloro, 1-3-hutadiene). What is the evidence for its carcinogenicity, Toxicol. Annu, (1977) in press. 2a 1 U. S. International Trade Commission, Synthetic Organic Chemicals United States Production And Sales of Elastomers, 1975 Preliminary, June, Washington. DC, U.S. Government Printing Office, 1976, p. 2. 252 B. L. Van Duuren, B. M. Goldschmidt and 1. Sicdman, Carcinogenic activity of di- and tri-functional Oochloroethers and of l,4-dichiorobulene-2 in ICR/HA Swiss mice. Cancer Res., 35 (1975) 2553. .'Ml H. Bartsch, C. Mal.iveille, A. Burbin, G. Pbmclic nnd R. Mojilcsano, Alkylating and mutagenic metabolites of halogenated oleins produced by human and animal tissues, Proc. Am. Assoc. Cancer lies., (1976) 17, Toronto. May 4 K. Pr>c. 67th Ann. MitIiiii; of Am. Assoc. C;m-rr Ki'si-noh. J l I' II. Muk.ii anil l ll.itvivlnk, 'flu* inn I .igcnit'il v of somi' h.ilm-l In-rs .nut halo ketones, Mill,ill,Ml II.-s., *JJ ( 22N. I RSV 0016351 irr> 25f> 256 N. Loprieno, Mutagenicity assays using \ easts with carcinogenic compound-. See ond Meeting of Scientific Committee of the Carlo Ecba Foundation. Dec 1'.'. I*'75 pp. 129-140. EPA, Survey of Industrial Processing Data Task l-hexiichloinbeniene .nul Ih\.t chlorobutodiene pollution from chlorocarbon processes, Environmental Pmiiocuoi* Agency. Washington, DC, June 1976. 257 M. P. Yurawecz, P. A. Dreifuss and L. R. Kamps. Determination of hexaidiloio-1 .3 butadiene in spinach, eggs, fish, and milk by electron capture g.i-liqind ehroni.ito graphy, J. Ass. Offic. Anal. Chetn., 59 ( 1970) 552. 258 259 260 A. C. Laska, C. K. Uarlell and J. L. LasiMrr, Distribution of hcxachN'I'obeiizem1 and hexachlorohutadiene in water, soil and selected aquatic organism- alone l lie lower Mississippi River, Louisiana. Bull. Environ. Contain. Toxicol . | ,*i i juTt.i V,.', G. Yip, Survey for hrxachlnro-1 ,3-hutailicno in fish. ecus, milk and saci.ddi--. .1 Ass Ot'l it- Anal. Chi'in.. 59 ( M)7r) a`9 !'.(>. Mnr/.akaev, Action exerted hy low lie xachlnrolmtad iene d> on I hi' ,iei i \ u \ <l the crniral ni'ivoiis system anil morphologic.d changes in annual- . p,,i-oiiei! Gig. Tr. Prof. Zabo|. I t ( 1967) 25 2H; C'hem Ab.-tr . 67 ( I 0 67 ) I n t<> \ 261 R. J. Kocilia, D. G. Keyes, G. C. Jersey. J. J. Ballard el al.. Ke-uh - u a i wo \ ear chronic toxicity study with hexachlorohutadiene fHCBD) in rats. Ab-iraci of 16th Annua) Meeting of Sociotv of Toxicology. Toronto, Canaria. March 2* 50 1977. 262 Anon, Dow Tests ol carcinogen indicate safe exposure levels, ('In-m. I'eol (i.-ioli.' ( 1976) 1. 265 V, Simmon, Mutagenic halogen.u ed hydrocarbon-. Presented at Meeting ni Sinn lural Parameters Associated with Carcinogenesis. Annapolis. MU, Aug '!l Sept 2 1977. 264 265 266 I). A. Schwetz, K. A. Smith, C. G Humiston. J. F Qunst and R. J Kociha Re-uli<jf a reproduction study in rats led diets containing hcxnch loi i >h m . r j n n. Josuol Appl. Pharmacol., 52 (1977 ) 387. B. H. Pilorz, Ally I chloride, in: Kirk-Othnicr Encyclopedia of Chemical J ci imnlngv , 2nd ed, VnJ. 5, Wilcy-Interscionce. New York, 1961, pp. 205 2 5 5 Anon, Adherence to l ppm stamlnad for allyl chloride exposure recommended. Occup. Health Safety Letter, Oct l ( 1976) 6. 267 268 G. A. Alizade, F. G. Guselinov. L. P. Agamovn, R. S. Guseinova and F. A. Aleskerov, Functional state of the kidneys of workers in contact with allyl chloride. Azerb Med. Zh..53(1976) 54,Chem. Absir., 86(1977) 194353M T. R. Torkelson, M. A. Wolf. F. Oyen and V. K. Rowe, Vapor toxicity id allvl chloride as determined in laboratory animals, Am Ind. Hyg. Assoc. J. I'll i 1959) 217. 269 B. N. Ames, J. McCann and E. Yamasaki. Method* for detecting carcinogens and mutagens with the .So/nioncffa/mammalian-nucrosume mutagenicity te-i. Mutation Res., 31 (1975) 347. 270 E. C. McCoy, L. Burrows and H. S. Rnsenkranz, Genetic activity of alhl rhlonde. Mutation Res., 57 (1978) 1 1. 271 H. S. Rosenkranz, B. Gutter and W T. Speck, Mutagenicity and DN A-nmdify mg activity. A comparison of two microbial assays, Mutation Res .41 ( 1976'6 I . 272 H. S. Rosenkranz, W. T. Speck, and B. Gutter. Microbial assay procedme* Experi ence with two systems, in: F. J. deSerres. J. R. Fouls. J. R. Bend and ' Pliilpol (Eds.). In Vitro Metabolic Activation m Mutagenesis Testing. Fl-mict North Holland, Amsterdam, 1976 pp. 337 363 273 C. M. Kaye, J. J. Clapp, and L. Young, The metabolic formation of mcn apture acids from allyl halide*, Xenohiolica, 2 (1972) 129. 274 C. M. Kaye, PJi.IV Thesis, University of Dmdon, London, England. 197 I 275 I A. Kapnporl, Mutations muler the lofluiunr of nnsu I in al ci) .dilrhv ill--. Dol.l Ak.id. Nank SSKR, f. I { I97S) 'i 13. 276 M. Bignami. G. Carilunionc, P. Cmiilw, V A. Orlali, G. Morpmgn and A ('.urre -genic compounds. Sec- -idation, Dec 12, 1975, inrobenzene and hexa\ironmental Protection -i ion of hexachioru-1.3tre gasiiquid chromato- hexachlorobenzene and anisms along the lower . 15(1976) 535. milk and vegetables, J. 'ie doses on the activity n animals so poisoned. '67) 31040 A. Results of a two-year 'J in rats. Abstract of Canada, March 27-.30, . Chem. Eco)., October d at Meeting of SlrucMD, Aug. 3 I Sept. 2, -1 R. J. Kociba, Results aiiobuladienv, Toxicol. Chemical Tedmologv, 215. posnre recommended. .'useinova and F. A. art with allvl chloride. ;.vlm apor toxicity of allyl Assoc. J.. 20 (J 959) cling carcinogens and lenicity test. Mutation ivity of allyl chloride. and DNA-modifying . 4 1 ( 19761 151 iv proceduit's Expcri Bend and .1. Phifpol sting, Elsevier/North nation of mercapture land, 197 1. 'led aldehydes. |1ok 1. purge and A. Carere, 157 Relationship between chemical structure and mutagenic activity in Mime pesticides: the use of S. Typ/tmturtum ami A. A'ididuns, Mutation Ftes.v 46 (1977) 2 13. 277 278 279 280 2M 282 283 284 285 286 S. S. Epstein. E. Arnold, J. Andrea. W. Hass and Y. Bishop, Detection of clicmtcal mutagens by the dominant lethal assay in the mouse, Toxicol. Appl. Pharmocol . 23 (1972)288. R. Spirtas and R. Kaminski. Angiosarcoma of the liver in vinyl chloride/polyvinyl chloride workers, J. Occup. Med., 20 ( 1978) 427. I. Fleig anti A. M. Thiess, Mutagenicity of vinyl chloride. External chromosome studies on persons with and without VC illness and on VC exposed animals, J. Occup. Med.. 20 (1978) 557. P C. Watannbe, J. A. Zempel, D. C. Pegg and P. J. Gehring, Hepatic macro* molecular binding following exposure to vinyl chloride. Toxicol Appl Pharmacol.. \1(1978)57t. Y Suzuki. Pulmonary tumors induced in mice by vinyl chloride monomer. Km iron Res., 16 (1978) 285. J. Higginson, The role of the pathologist in environmental medicine and public health. A review. Am. J. Pathol., 86 I I 977) 4G0. R. B. LeLllanc. Flume resistant fibers. Fiber Producers, April (19771pp. 10, 12. 61. G. M. Sassu, F. Zilio-Grandi and A. Conte, Gas-chromatographic determination of impurities in vinyl chloride, J. Chromatogr., 34 (I 968) 394. M. Kurosaki, S. Taima, T. Hatla and A. Nakamura, Identification of high boiling materials as by-products in vinyl chloride manufacture, Kogyo Kngaku Zasshi, 7 1 (1968)488. Anon. Vinyl bromide: Possible carcinogenicity indicated in IS-mmuh study sub mitted to e'PA. Chem. Reg. Replr.. 2 |22 ) (1978) 961. 2S7 288 289 290 H. Barisch, C. Malavcille, A. Harbin, G. Plnnehr anti R. Monicsaun, Alkylating and mutagenic emlaboliles of halogenaled olef-ns produced by human and antm.d tissues, Proc. Am. Assoc. Cancer Res.. 17 (1976) 17. A. Rarbin, G. Plunche, A. Croisy, C. Mnlaveille and H. Harisch. Detection of electrophilic metabolites of haloeenalcd oiefins with W4-iimobenzyl)pynriine (NBP) or with S. lyphimurium. Mutation Res.. 53 (1978) 150. C. C. Lee, J. C. Bhandari. J. M. Winston, \V. B. House, R. L. Dixon anti J. S. Woods, Carcinogenicity of vinyl chloride and vinylidcne chloride, J. Toxicol., Environ. Health. 4 (1978) 15. D. E. Halhwny, Comparative mammalian metabolism of vinyl chloride and vmvlidrne chloride in relation lo oncogenic potential, Environ. Health. Pcrspecl.. 21. (1977) 55. 291 292 293 294 29,-> 296 ; R. K. -Jones and 0. E. Hathaway, Differences between mice anti rats in the met a hol ism of vinylidene chloride, Hr. J. Cancer, (1978) in press, M. -1. McKenna, J. A. Zempel, E 0. Madrid, W H. Braun and P. J. Gehring. Metab olism and pharmacokinetic profile of vinylidcne chloride in rats following oral administration,Toxicol. Appl. Pharmacol , 45 (1978) 821. R. J. Jaeger, R. Connnlly and S. D. Murphy, Effect of 18 hr fast and glutathione depletion on 1,1 -dichioroethylene-mdticed hepaLotoxicily and lethality in rats. Exp. Mol. Pathol., 20 ( 197 I) 187. K. J. Jaeger, M. -I. Trabulus and S. D. Murphy, Biochemical effects of 1,1 -Dtrhloroethylene in rats: Dissociation of its hepntoloxicity from a lipopi'roxnlalive mechan ism, Toxicol. Appl. Pharmacol., 24 (1973) 457. H. Konielzko, W. Haberlandt, H. Herrbronner. G. Reill and H. Weichardt, Cytogcnelischc Unlersuchungen an Trichloral hylen-arbciteran, Arch. Toxicol., 40(1978) 201. M. Iked a, Metaholism of triehlornellivlene and I etrarhloroct hylone m human subt-'Hs, Knviri <ii I Ira II li IVrs|<-rl . 21 | 1977) 2:'M i' A u'Ui'ii. K . \ mh'rsson. (\ I logs I <-d I, It. I b ilnilii-1 g. I Molina and A de\ ei di-i . A cohort siiiity on Inchlovoclliylenr I'spoMirc and canci-r morlably, <1. Occup Med., 20 ( 1978) 194. I l RSV 0016353 158 298 299 300 301 302 G. Bronzetti, E. Zeiger and D. Frezza, Genetic activity of trichloroethylene in yeast, J. Environ. Pathol. Toxicol., 1 (1978) 411. Anon, Trichloroethylene is not a teratogen, Chem. Eng. News, Sept. 12 (1977) 20. R. Culik, D. P. Kelly and J. J. Clary, Inhalation studies to evaluate the teratogenic ; and embryotoxic potential of chloroprene (2-chlorobutadiene-l ,3), Toxicol. Appl. Pharmacol., 44 (1978) 81. < L. S. Salnikova, Embryotoxic effect of volatile substances from Nairit latexes, Toksikol. Nov. Prom. Khim. Veshchetsv., 11 (1968) 106. L. S. Salnikova and V. N. Fomenko, Comparative characteristic of the embryotoxic effect of chloroprene in relation to mode of action associated with various routes of entry into the organism. Gig. Tr. Prof. Zabol., 7 (1975) 30. j Note added in proof ` \ The present update of the NIOSH register provided details on 64 cases of angiosarcoma (world-wide) among vinyl chloride polymerization workers reported as of October, 1977 (278). Of this total, 23 cases of angiosarcoma have now been reported in the U.S.A., 10 in Canada, 9 in the Federal s Republic of Germany, 8 in France, 3 in Sweden, 2 each in the U.K., Italy, Czechoslovakia and Yugoslavia and 1 each in Belgium, Japan and Norway."The 23 cases identified among'U.S. polymerization workers represents approxi- ' mately 10% of all liver angiosarcomas identified in the U.S.A. since 1964 [278]. The ages at diagnosis for the 64 cases ranged from 37 to 71 years : with a median of 49 years, while the latency periods (years from first j exposure to diagnosis) ranged from 9 to 38 years with a median of 21 years. ' The total years exposed ranged from 4 to 31 years with a median of 18 years [278]. j Fleig (278] reported an increased incidence of chromosomal aberrations in lymphocyte cultures from workers occupationally exposed to both esti mated and monitored concentrations (5000 ppm and 600 mg/kg) of vinyl chloride only in those subjects showing symptoms of VC illness or in an angiosarcoma case. In 200 metaphases from exposed subjects, the rate of ; aberrant metaphases including gaps and isogaps was 16.6% compared to a control group with a rate of 5.5%. The frequency of structural anomalies, inclusive and exclusive of gaps was also increased compared to controls in bone marrow cells of Chinese hamsters exposed to inhalation of 2500 and 5000 ppm VC for 6 h/day at 24 h intervals over 5 days, or after i.p. injection , of 300 and 600 mg VC/kg/day for 5 days. j Watanabe et al. [280] studied the covalent binding to hepatic macro- ; molecules in rats exposed to ,4C-labeled vinyl chloride to determine if ! VC-induced carcinogenesis may be related to electrophilic alkylation of ! macromolecules in vivo. At exposures of VC > 50 ppm, the amount of 14C ; bound to macromolecuies in the liver correlated with induction of hepatic angiosarcoma, there was no detectable binding of radioactivity to either ; DNA or RNA in the liver. The hepatic glutathione content was depressed only at exposure concentrations > 100 ppm. j Recent studies by Suzuki 1281] on the induction of pulmonary tumors in ? CD1 Charles River white strain male mice suggests that the mouse lung may be an extremely sensitive indicator of the oncogenicity of vinyl chloride. The \ juilmo: cancer' type II various mustarm mic .imong Vin> eo-mor fibers, child rei applica comme disclosi submit: months the liv*. bromid effect : period, malign; that sk hromid mice d initiato carcino any sig (PMA a Expt gaseous enhanc* pretrea epoxidprene) air thrc metabo 115]. The idene c more n hemang of VC hepatic did not 55 pprr or subt than m chlovoethyUn* in yeast. us. Sept. 1 2 11977) 20. evaluate the iC'atogenic ene-1,3), Toxicol. App). vs from Nairi! I ilexes, ">t ic uf the embryo toxic "(I with various .omes of lotails on 64 <.asr5 of vmerization workers asps of angiosuvoniu ia, 9 in the Federal h in the L.K.. Italy, >an and Norway. The ' represents approxie U.S.A. since 1964 from 37 to 71 years Is (years from first i median of 21 years, a median of 13 years nosomai aberrations xposed to both esti>00 mg/kg) of vinyl VC illness or m an subjects, the rate of '>.67r compared to a tructural anomalies, pared to controls in alation of 2500 and t after i.p. injection <* to hepatic macroide to determine if pliiiic alkylation of . the amount of 1 4 C nduetion of hepatic lioactivity to either ntent was depressed `Ultnonarv I amors in the mouse bmp m:tv 1 vinyl rhlonde. The 159 pulmonary tumors corresponded to ` alveologenic tumors or alveologenic cancer'*, it was suggested that the neoplastic colls were transformed front type II alveolar epithelium via its hyperplastic form. It was also noted that various carcinogens such as polycyclic aromatic hydrocarbons, nitrogen mustard, and chromate compounds are known u> induce alveologenic lumnrs in mice and to also be associated with excess bronchcngenic carcinoma among workers exposed to the carcinogens (282). Vinyl bromide (bromoethene) is used in small amounts ns a flame retardant co-monomer with acrylonitrile and other vinyl monomers in modaevylic fibers. These fibers are used in fabrics and fabric blends with polyesters for children's sleepwear and other apparel, home furnishings and in industrial applications (283). Vinyl bromide bus also been delected as an impurity in commercial vinyl chloride (284, 285). Vinyl bromide has been recently disclosed to bo a possible carcinogen in a preliminary 18-month study submitted to the I'.S. Environmental 1`roln lion Agency }28(".|. Aflcr 18 months of a 24-munLh chronic inha'alion sLudy, primary angiosarcomas of ihe liver were found aL sacrifice in malt' and female rats exposed to vinyl bromide at 50, 250 and 1250 ppm. There also appeared to be carcinogenic effect in Zvmbal's gland in rats exposed to 250 and 1250 ppm during this period. In addition, two primary lung neoplasms were found as well as some malignant mammary neoplasms in female rais. Van Duuren (19o[ reported that skin application of vinyl bromide in acetone solution and polyvinylbromide in commercial aqueous latex suspension using female ICR/lla Swiss mice did not result in any skin tumors in either group When tested as initiators with phorbol myristnlo acetate (PMAi as promoter (in two stage carcinogenesis) the tost group (vinylbromide followed by I'MA) did not show any significant increase in tumors induced compared with the control group (I'MA alone). Exposure of S. lyphimurium TA 1530 |287) or TA 100 [288, 289) to gaseous mixtures of vinylbromide in air caused mutagenic effects which were enhanced by the addition of liver supernatant fractions from phenobarbitone pretreated mice or from human liver biopsies (287). In addition, the epoxide formation from vinyl bromide (as weil as vinyl chloride and chloroprene) was suggested by passing a gaseous mixture of the lest compound and air through a mouse liver microsomal system and trapping volatile alkylating metabolites by reaction with excess 4-(4-nitrobenyl)pyridine (NBP) |3, 115). The species sensitivity and specificity to both vinyl chloride and vinylidene chloride has been further elaborated by Lee et al. (289) . Rats were more resistant than mice to the carcinogenic effects of VC or VDC. Hepatic hrmangio sarcomas were observed in rats exposed to 250 or 1000 ppm r>f VC starting in the ninth month. In contrast to mice, many of the rats with hepatic hemangiosarcomas also developed hemangiosarcomas in the lung; VC did not cause any oilier tumors in I he rat. A small number r >T ral s exposed to af> pprn of VDC developed hemangiosarcomas in llic meseiilcric lymplniode nr subcutaneous lissue (289). Kals were also found lo be more resistant hum mice to Liie acute or uthei chronii effects of VC or VUC [46, 289). RSV 0016355 160 Comparative studies suggest clues in the differences between rats and mice in the processing of vinylidene chloride (152, 290, 291]. In mice, for example, the production of thiodiglycollic acid is considerably reduced and the formation of the N-acetyl-S-cysteinylacetyl metabolite is increased. The higher beta-thionase activity in mice than in rats accounts for the greater conversion of thioglycollic acid into dithioglycollic acid via thioglycollic acid in the former species. In mice, the metabolic pathway from chloroacetic acid to thiodiglycollic acid seems to be readily saturable, possibly on account of an inadequacy in the reaction catalyzed by glutathione S-acyl transferase [290], Hence, under these circumstances, detoxification of 1,1-dichloroethylene oxide by glutathione S-epoxide transferase and the modification of DNA by 1,1-dichloroethylene oxide or chloroacetyl chloride would be expected to be more significant in mice than in rats (152, 290, 291]. According to Hathaway [291], vinylidene chloride is an agent of low, perhaps very low, oncogenic potential, which can be damaging only in a special set of biological circumstances. McKenna et al. [292] recently elaborated the metabolism and pharmaco kinetic profile of vinylidene chloride in rats following oral administration. The fate of VCD following oral administration to rats is dependent upon both the dose administered and the nutritional status of the animal. The diminished ability of fasted animals to metabolize the high dose (50 mg/kg) of VDC correlated well with the previously reported enhancement of VDCinduced hepatoxicity in fasted animals [293, 294]. Both the hepatotoxic response to vinylidene chloride, and the extent of its detoxification appear to be dependent on the concentration of glutathione (GSH) in the liver. When hepatic GSH is depleted (e.g., in fasted animals or at higher doses of VDC) a toxic response to VDC is elicited. After a single oral dose of 1 mg/kg of 14C-VDC, 78% of the dose was metabolized and excreted in urine and feces as non-voltile metabolites of VDC the remainder was exhaled as ,4C02 (21%) and unchanged 1 4C-VDC (1--3%). After a single 50 mg/kg dose of 1 4C-VDC, excretion of the parent compound via the lungs was 19 and 29% of the dose in fed and fasted rats, respectively. Two of the major urinary metabolites of 14C-VDC were identified as S-(2-hydroxyethyl)N-acetylcysteine and thiodiglycolic acid. The identification of these metab olites substantiates the hypothesis that detoxification of VDC occurs mainly via conjugation with glutathione. While GSH may act as a site of detoxification, other tissue nucleophiles may be sites of attack or toxicity. This may arise via covalent binding of the reactive metabolite of VDC to macromolecules in the liver cells, subsequently causing liver injury (292]. A cytogenic investigation on 28 individuals employed in degreasing oper ations with trichloroethylene has been recently reported by Konietzko et al. (295], On the basis of chromosome studies on lymphocyte cultures, 9 of these cases showed pathological rates of hypodiploid cells, but otherwise normal karyotypes. In these 9 cases, the trichloroethylene load, but not the exposure periods in the working place, was significantly higher than in the control group. In a study of the metabolism of trichloroethylene (TCE) and tetrachloroethylene in human subjects, Ikeda [296] reported a linear correlation between the T total trichloro equation: Y * trichloro comp related to TCE the linear rela ethylene expos consequently t chloroethylene half-lives for T The respiraton and tetrachloro Axelson et comprising 516 estimated thro\ any excess can cohort with tr supposed to be and observed exposure (548 authors cautior no means be i common malig that "there is p Trichloroeth somal activatio and mitotic gei yeast. When a pension tests, c were noted at s TCE induced 1 covered from : [298]. Preliminary suggests that T changes in ger inhaled air con behavior reacti offspring. In tl centration ofTin the offspring Additional r of 25 ppm chic bility and are l vi th previous st embryotoxic, t< low the existi 1 ppm [301, 3C t RSV 0016356 ween rats and mice !)1 ]. In mice, for rably reduced and e is increased. The its for the greater a thioglycollic acid n chloroacetic acid ibiy on account of S-acyl transferase n of 1,1-dichlorohe modificiition of hloride would be [152, 290. 291] . an agent of low, (imaging only in a ism and pharmacoral administration, is dependent upon f the animal. The :h dose (50 mg/kg) ancement of VDCh the hepatotoxie (oxification appear ) in the liver. When or doses of VDC) a ose of 1 mg/kg of in urine and feces exhaled as 14CO: 50 mg/kg dose of gs was 19 and 29% the major urinary -(2-hydroxy ethyl)m of these metabi of VDC occurs iy act as a site of attack or toxicity, abolite of VDC to t injury [292]. in degreasing oper'iy Konietzko et al. oyte cultures, 9 of oils, but otherwise o load, but not the higher than in the and tetraehloroiinear correlation 101 between the TCE concentration in the work environments and the level of total trichloro compounds in the urine of the workers, as expressed by the equation: V = 7.25=* +5.5 where V is TDK in air (ppm) and .V is-1 ilal trichloro compounds in urine (mg/1). Trichloroethanol level is also linearly related to TCE concentration, while trichloroacetic acid level deviates from the linear relation when the TCE level exceeds 50 ppm. For letrachloroethylene exposure, both trichloroethanol and trichloroacetic acid levels, and consequently the total trichloro compound level, reach a plateau at tetrachloroolhylene level well below 100 ppm. The mean urinary biological half-lives for TCE and tetniehioroelhylono arc 41 ami 1 1 1 hrs respectively. The rospimlorv half-life is shorter than thr urinary half-life for both `IT'D ami tetrachloroethy lone [288]. Axelson et al. [297] recently described a fairly small cohort study comprising 518 men with rather low levels of trichloroethylene exposure as estimated through trichloroacetic acid in the urine. This study did not reveal any excess cancer mortality? Requiring 10 years of latency time, the sub* cohort with trichloroacetic acid in urine above 100 mg/1 (e.g.. exposure supposed to be above 30 ppm) showed a close agreement between expected and observed number of cancer cases as did the subcohorl with lower exposure (5 18 and 3643 person-years of observation, respectively). The authors cautioned that the cancer risk to man from trichloroethylene can by ni> means be ruled out from this study, particularly with regard to on -innum malignancies such as liver cancer. However, they also concluded that "there is probably no serious cancer hazard at low exposures" [297 | . Trichloroethylene was toxic but not genetically active without micro-"nuil activation when tested for its ability to induce troth poim mutation i ul mitotic gene conversion in diploid strain of S. cc/criswc (strain U7) of yrast. When a mouse liver 10,000 kg supernatant was included in the sus pension tests, dose-related increases in both mutation and gene conversion -w re noted at survival levels of greater than 50%. In the host-mediated assay. ICE induced both point mutation and gene conversion in D-l when re""ered from the liver and kidneys after both acute and subacute dosing 1298]. Preliminary data submitted to OSHA and NIOS1I on two rat studies -cggi-sts that TCE does not cause abnormalities in animal embryos or genetic - hangi's in germinal cells [299]. In the first study, pregnant female rats inhaled air containing 300 ppm of TCE for 6 h/day. There were no deaths or 'havior reactions among the pregnant rats and no abnormalities in their "ff.pring. In the second study, male rats were exposed to the same con alration of TCE for 9 months, then mated. No genetic changes were found ci the offspring [299]. Additional recent studies by Culik ol ui. [300 ] suggest that concentrations -f 25 ppm chloroprene do not adversely affect male rat reproduction enpa` Uty and are not embryotoxie or teratogenic. These results are at variance i :h previous studies from the U.S.S.R. which reported that chloroprene was -iduy'otoxic, teratogenic and mutagenic at atmospheric concentrations bel w `lie existing U.S.S.R. maximum allowable concentration (MAC) of 1 ppm |301, 302J. RSV 0016357 1* 1 ) 16 '28. >965. 34871958) (195U U, 522 cV 4/1 * en'3 i-Tit Toxicology, 13 (1979) 25--28 EJsevier/North-Holland Scientific Publishers Ltd. fOrtGSt fkls oftterlal ^ v-v Troyrleht 13? SEQUENCE INHALATION TOXICITY STUDY OF [YL CHLORIDE) IN RATS. I. GROWTH, MORTALITY, HAEMATO LOGY, CLINICAL CHEMISTRY AND ORGAN WEIGHTS* << V.J. FERON* *, A. KRUYSSE and H.P. TIL Centre/ Institute for jVi/tritton and Food Research TNO, Zeist (The Netherlands) (Received April 30th. 1979) (Revision received June 19th, 1979) (Accepted June 20th, 1979) SUMMARY Wistar rats were exposed to atmospheres containing 0 (control) or 5000 ppm vinyl chloride monomer (VCM), 7 h/day, 5 days/week, for a period of 52 weeks. After -4, 13, 26 and 52 weeks each time 10 rats/sex/group were killed and subjected to extensive examinations. The present paper deals with growth, mortality, haematology, clinical chemistry and organ weights. Slight growth retardation throughout the experimental period and high mortality in the second half of the study were observed in VCM-exposed animals. Some of the haematological parameters and biochemical blood parameters were slightly influenced by VCM after an experimental period of 52 weeks only. Blood clotting time was generally slightly shorter in VCMexposed rats than in controls. There were minor indications of increased potassium contents of the blood serum in VCM-exposed animals during the first half of the test period. The kidneys were adversely affected by VCM as appeared from increased blood urea nitrogen levels and relative kidney weights. After 52 weeks increased weights of heart and spleen, and slight signs of anaemia were noticed in VCM-exposed rats. The present study did not produce obviously suitable parameters for early diagnosing "VCM-disease" in man. * The study was sponsored by a group of co-operating European industries, including s*Terband Kunststofferzeugende Industrie e.V. (F.R.G.), Shell Nederland Chemie, Dutch State Mines, Akzo Zout Chemie Nederland B.V. and Dow Chemical Europe S.A. Abbreviations: BSP, bromsulfophthalein; VCM, vinyl chloride monomer; UGOT, urine glutarmc-oxalacetic transaminase. ** Present address Dr V.J. Feron. Department of Toxicology, Central Institute for Nutrition and Food Research TNO, P.O. Box 360, 3700 A Zeist. Netherlands. This is an abrideed paper Copies of lhe full paper are available from Hie Editor on request, which should be accompanied by 85.00, or equivalent, to cover reproduction and postage 25 INTRODUCTION Industrial exposure to vinyl chloride monomer (VCM) has been associated with several disorders such as acro-osteolysis, non-neoplastic liver lesions, angiosarcoma of the liver and other types of tumours at various sites [2,8, 16,171. Moreover, experimental exposure to VCM by inhalation has been found to result in degenerative and neoplastic changes of the liver and other organs in several species [1,10,14,18,191. From the point of industrial safety it is important to recognize VCMeffects in plant workers as early as possible [3,4,5,9,11,12,13,151. There fore, experiments aimed at detecting the earliest changes attributable to VCM-exposure as well as at studying their further development and con sequences, were deemed desirable. Against this background a 1-year inhal ation study with a serial killing design was conducted in rats repeatedly exposed to VCM at a level as high as 5000 ppm. The results of this invest igation are presented in 3 papers of which this is the first. It deals with the design of the study, growth, mortality, haematology, clinical chemistry and organ weights. The 2 other papers describe the morphological changes found in several organs [6,71. MATERIALS AND METHODS VCM was obtained from Akzo Zout Chemie, Rotterdam. The Nether lands, in stainless steel pressure bottles of about 50 litres. 124 male and 124 female, newly weaned, rats (Cpb ; WU: Wistar random^ were evenly distributed according to sex and body weight over 2 inhalation chambers. In one of the chambers the rats were exposed to 5000 ppm VCM 7 h/day, 5 days/week, for a period of 52 weeks; in the other chamber the controls were housed, where they were exposed to fresh air. The institute's stock diet and tap water were constantly available to the animals. Individual body weights were frequently recorded, and at weeks 4, 13, 26 and 52 haematological examinations, serum and urine analyses and organ function tests were conducted. Haematological examinations involved determinations of haemoglobin concentrations, packed cell volume, blood clotting time and counts of erythrocytes, thrombocytes and of total and ^differential leucocytes. Serum analyses involved measurements of total protein, albumin, globulins, a,-feto-protein and the activity of glutamicoxalacetic and glutamic-pyruvic transaminases, alkaline phosphatase and lactic dehydrogenase and of electrolytes, viz. sodium, potassium, calcium, magnesium and chlorine. Measurements were made of fasting blood glucose and blood urea nitrogen some days prior to each autopsy. Urine examin ations included volume, specific gravity, uric add, activity of glutamicoxalacetic transaminase, pi 1. protein, glucose, kclones. occult blood and microscopic constituents. Liver function tests involved brninsulfophthalem (BSP) and sleeping time. The kidney function wiu memun-d by the phenolred excretion tost. 20 'd \s, ,8, en her M*re- to onhaldly estthe and ;lges ther- lom) ation ppm mber o the 13, s and olved blood d and total tamice and. 'Icium, :lucose <amin^tarmc-d and thalein )henol- After 4, 13, 26 and 52 weeks, 10 rats/sex/group were killed and autopsied, and the weights of heart, kidneys, liver, spleen, brain, gonads, thymus, pituitary, thyroid, adrenals and lungs were recorded. RESULTS In week 33 the first rat died (a male of the test group). Thereafter, mortal ity among VCM-exposed rats gradually increased, and in week 52 only 9 males and 10 females of the test group were still alive. The controls remained in good shape, except for one lethargic animal which was killed in week 50. Mean body weights in the test group were significantly lower than those of the controls throughout the experimental period. Haemoglobin content, packed cell volume and the number of erythrocytes were slightly decreased and the total number of leucocytes were somewhat increased in VCM-exposed males and females at week 52. Blood clotting time in test animals was lower than in controls at ail intervals examined. Blood urea nitrogen levels were slightly increased in the test group in females at week 26 and in males and females at week 52. Albumin and 7-globulin levels were slightly decreased in the test group in both sexes at week 52 only. Alpha-feto-protein levels were slightly increased in the test group after 26 and 52 weeks, the differences with the controls being statis tically significant after 52 weeks only. A markedly elevated level of this protein indicating the presence of a a,-feto-protein-producing tumour, was not found in any of the animals. The potassium content of the blood serum was somewhat higher in test animals than in controls during the first half of the experimental period. Specific gravity values and uric acid levels were decreased, whereas the volume of the urine produced and UGOT-vaiues were increased in both males and females of the test group at week 52 only. However, the UGOTvalues are within the normal range and well below those considered pathol ogical, BSP-retention was decreased in the test group at weeks 13, 26 and 52, but slightly increased in females at week 4. In the test group, the relative weights of the liver and kidneys were nearly always significantly increased in both sexes at weeks 13, 26 and 52 and those of the spleen at weeks 26 and 52. The relative weights of the heart and lungs were slightly increased in the test group at week 52. DISCUSSION* An attempt was made to indicate the earliest VCM-related changes detect ed in the present study. Comparison of toxicity data on VCM from the liter ature with those obtained in the study described here, and being aware of * The reader is referred to the unabridged paper for a detailed discussion on all VCMeffecU observed. 27 RSV 0016360 the fact that "a statistically significant difference between the control and test group" is not always synonymous with "a VCM-effeet", the following changes might represent early VCM-effects, i.e. growth retardation, short ened blood dotting time and increased potassium content of the blood serum. From the results published in a subsequent paper [7] it appears that another early VCM-effect is the occurrence of swollen and malformed mitochondria in hepatocytes. VCM-effects appearing at a later stage were increased urea nitrogen content of the blood and enlargement of kidneys, liver and spleen. Foci of cellular alterations in the liver and increased amount of smooth endoplasmic reticulum in the parenchymal cells of the liver were also found at this later stage [7J. Other VCM-effects became visible only at the final stage when tumours had already emerged in many VCM-exposed animals. Because of the nature or the scantiness of the early VCM-effects observed in the present itudy, it seems unlikely that any of the parameters affected by VCM at an early stage are obviously suitable as tools for a timely diagnosis of "VCM-disease" in humans. On the other hand, these and other obser vations made in the experiment described here may indicate the direction in which such an "early diagnostic tool" should be looked for. ACKNOWLEDGEMENT The authors thank Mr. J. Cats burg and Mr. F. Hendriksma for competent technical assistance, and Dr. A.P. de Groot for critically reviewing the manuscript. REFERENCES A.V. Basalaev, A.N. Vazin and A.G. Kotchetkov. Gig. Tr. Prof. Zabol., 16 (1972) 24, P.D. 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