Document XQkbqqRN3NBVB43BE2pjM8VG

A Selected Literature Review Concerning Possible Affects of Vinyl Chloride Monomer (VCM) On Reproduction Prepared For: Georgia Pacific Company International Square 1875 Eye Street, N.W. Washington, DC 20006 Prepared By: ToxicoConsultants, Inc. 3025 Sylvania Drive Raleigh, NC 27607 Edward 0. Gralla, VMD President February 1984 Summary and Conclusion The objective of this review was to examine the scientific literature concern ing vinyl chloride monomer's (VCM) toxic properties with regard to possible affects on the developing human fetus. This effort encompassed the following sources: Three published surveys of the reproductive histories of women who were living in a relatively close proximity to industrial plants engaged in the manufacture of polyvinyl chloride (PVC) or other products that involve vinyl chloride; two full animal teratology studies, and one preliminary report of a transplacental carcinogenesis study are also a part of this review. This review also delved into related areas such as general toxic properties and pharmacodynamics, as needed. The following conclusions are drawn from this exercise: Vinyl chloride has the attributes that suggest it could have fetal toxic prop erties, being toxic, carcinogenic, mutagenic and capable of crossing the mam malian placenta. However, there are no published studies which prove conclusively that VCM has ever caused fetal maldevelopment or carcinogenesis following intrauterine exposure. In short, neither the safety nor the hazardous risks to pregnant women have been unequivocally proven for this chemical. Introduction VCM has been shown to be a carcinogenic agent in a variety of species including man, and a mutagenic chemical in non-mammalian bioassay systems and mammalian cell tissue culture systems. Such an array of toxic properties raised concerns over the possibility that it might be teratogenic as well. In an attempt to answer this question, a search of the scientific literature was undertaken for relevant information that might clarify this situation. Sources of Information This review will set forth the findings from selected publications. It is not intended to be a full and complete review of vinyl chloride toxicology. A general survey of the literature was conducted which focused on reports deal ing with vinyl chloride studies in either humans or laboratory animals, that were concerned either with reproductive effects or ancillary, but important concerns, such as pharmacodynamic properties. The principle sources of infor mation are listed in the reference portion of this review. The toxic properties of vinyl chloride, including carcinogenicity, were first discovered in long term animal studies where it was learned that high exposures for long periods of time, e.g. 30,000 ppm for 12 months, induced tumors of the skin, lungs and bones in rats. Shortly thereafter, the occurrence of fatal liver angiosarcomas in workers manufacturing polyvinyl chloride resins was re ported (1). Animal Studies In 1977 the first of two full studies of VCM for possible teratogenic effects was conducted and published (2). Rats and rabbits were exposed to vinyl chlo ride gas at concentrations of 500 to 2,500 ppm. Mice received either 50 or 500 ppm of this agent. Administration was during critical and sensitive stages of gestation, days 6-15 for rats and mice, and 6-18 for rabbits. Pregnant mice exposed to the higher level had lower body weight gains during their exposure. This was accompanied by and possibly related to a significantly increased early fetal mortality seen as a higher level of resorptions. An exposure-limiting, maternal mortality appeared in the rabbits and mice, i.e., 14% for the former at 2,500 ppm and 17% for the latter at 500 ppm, respectively. Rats were more resistant with only one death out of 17 dams occurring at 2,500 ppm. The following year a second teratology study was published involving only rats exposed to 1,500 ppm during either the first, second or third trimester of preg nancy (3). A concurrent experiment examined transplacental crossing of inhaled vinyl chloride in female rats in their 18th day of pregnancy. Exposure was for 2.5 hours at levels of 2,000, 7,000 or 12,000 ppm. Fetal blood levels were from 63% to 70% of maternal levels indicating that vinyl chloride freely crosses the placenta. The exposure of pregnant rats to 1,500 ppm of VCM for three seg ments of gestation, i.e., days 1-9, 8-14, or 14-21, caused a slight but signifi cant increase in resorptions in the earlier stage of pregnancy. Otherwise, there were no adverse affects on fetal development reported. In a pilot study, Sprague-Dawley rats were exposed to 10,000 or 6,000 ppm of vinyl chloride 4 hours/day for 7 days from the 12th to the 18th days of preg nancy (4). The offspring were delivered normally and held for postpartum ob servations. Subcutaneous angiosarcomas developed in one offspring after 24 weeks of age from the 10,000 ppm level, and one in the 6,000 ppm group at 22 weeks. All the data from these studies was published seven years later in tabular form. This later report failed to mention dermal angiosarcomas but did indicate that nephroblastomas and zymbal gland carcinomas appeared in the offspring of exposed pregnant rats at a high incidence as well as an occasional epitheloma of the skin, benign tumors of the stomach, and nonspecific malignant mammary tumors (5). Human Studies Three complete studies of VCM's affect on human reproduction have appeared in the literature. Each one followed the same general format. That was to examine the hospital birth records of mother's who delivered while they were living in close proximity to an industrial plant engaged in producing or using VCM, and causing substantial amounts of vinyl chloride to be released into the atmosphere. Thus the search was for the effects of an assumed wind-borne teratogen. In West Virginia, Canada, and four separate locations in Ohio, clusters of birth defects were found to be located in communities that also contained one or more VCM plants (6, 7, 8). In no instance was a direct relationship proven to exist between abnormal births and VCM plants. None of the mothers had ever worked in the plants, while one study concentrated on families in which the father was employed at a VCM plant. A significantly increased level of birth defects in these later families was discovered (6). Moreover, in families with more than one child, the incidence of birth defects was higher in children born after the father began working at a plant. The investigators who conducted this study suggested that VCM may induce a transmissible mutagenic affect whereby defective genes are passed from the father to the offspring. A Canadian study examined temporal relationships over several seasons (8). They found that the incidence of birth defects dropped in the winter months which was when the cold temperatures caused VCM to precipitate from the atmos phere. VCM's mutagenic affects were reported to have been shown in the lymphocytes of a group of PVC workers, who were studied twice, before and after a 2.5 year interval (9). In the first study, a significantly higher frequency of chromo somal aberrations was found to exist in exposed workers over that of age and gender matched controls, 3,41% and 1.79% respectively. In the follow-up study, the exposed workers' rate of chromosome damage had dropped to that of controls. During the same time period, the air concentrations of VCM had been reduced from 80 to 1 ppm. Dose Response Effects and Pharmacodynamics These two characteristics must be taken together in considering vinyl chloride's toxicity profile, since it has been shown that the ultimate toxicant for vinyl chloride is a metabolite and not the parent compound. Moreover the metabolite is detoxified in rats by a conjugation with glutathione of the liver and sub sequently excreted (10). Hence, the body is protected against VCM-induced cancer as long as there are ample quantities of liver glutathione available to covert the vinyl chloride metabolite to its conjugate. This hypothesis has withstood the test of rigorous examination, which also indicated that a criti cal break point appears at the 100 ppm concentration. Exposures below that point cause a dose-related depletion of hepatic glutathione. Exposures above this level are incapable of causing further depletions probably because a maxi mum reduction has been achieved. Moreover, at about the same exposure concen trations (11), the amount of macromolecular binding of radiolabelled VCM io hepatic proteins is increased significantly (12). Discussion The published literature is lacking in specific articles that deal with the problems of hazards to women who are or have been working in the PVC industry. There may be too few females with sufficiently long work histories to support such a study. In fact, it may be impossible to ever conduct such an investi gation since the ambient work place levels of this chemical have been steadily reduced in response to improved work practices, in the wake of demands for greater safety which followed the discovery that this chemical is a potent human carcinogen. Prior to 1972, the TWA for VCM in the work place was 500 ppm. This was lowered to 200 ppm until 1974 at which time the 0SHA standard was set at 1 ppm. This requirement appears to afford a comfortable margin of safety in preventing cancer and, assuming that the above described principal applies to the developing fetus, then in utero development is protected as well. Alternatively, the investigations cited centered on women who lived in a general vicinity of VCM manufacturing plants and were assumed to have received an ex posure to this material. Little information is available on measured levels of VCM in these locations, and, since these same cities are highly industrial ized, the possibility of a multiple exposure to other chemicals at the same time is likely. A weakness in some of the human study data is that it was collected by question naire which required that the average, usually untrained woman, diagnose fetal death and early abortion. In many institutions, the diagnosis of abortion is considered proven only after a histopathological confirmation by an expert path ologist. Data from animal studies appears to be showing that a species-specific toxicity exists, and that the rat is considerably less sensitive than the mouse or rabbit. Of the three species studied, only the pregnant rat survived the top level of 2,500 ppm, while the mouse and rabbit showed significant mortality. This resis tance to VCM toxicity was further demonstrated in the second study, reported as a pilot investigation, where the VCM was studied only in the rat at 6,000 SGC 003634 and 10,000 ppm for a seven day exposure. It was in this study that the occur rence of congenital tumors were reported to have occurred. The evidence presented in the literature suggests that the teratogenicity of VCM is an unsettled question. The most striking finding that has been reported to date was the induction of tumors in the offspring of pregnant rats that were exposed in utero to high concentrations of vinyl chloride. While the data that supports this conclusion is far from overwhelming, it is the basis for at least one writer who reviewed vinyl chloride toxicity to proclaim that "vinyl chloride is unequivocally a transplacental carcinogen for the rat" (13). This statement may be accurate at extremely high concentrations, that are unlikely to occur under normal working conditions. Moreover, as was discussed, the mammalian body seems to have a protective mechanism against the toxic properties of vinyl chloride that takes the form of glutathione conjugation. Only when the exposure levels of this chemical are sufficiently high to deplete hepatic levels of glutathione can the carcinogenicity of vinyl chloride be expressed. Presum ably, the same system would buffer any teratologic potential. bbC 003685 SELECTED REFERENCES 1. Milby, T. H. Vinyl Chloride: An Information Resource. HEW Publication (NIH) 78-1559, 1978. 2. John, J. A., et al. The Effects of Maternally Inhaled Vinyl Chloride on Embryonal and Fetal Development in Mice, Rats and Rabbits. Toxicology and Applied Pharmacology 39:497-513, 1977. 3. Ungvary, G. Y. et al. Effects of Vinyl Chloride Exposure Alone and in Combination with Trypan Blue Applied Systematically During all Thirds of Pregnancy on the Fetuses of CFY Rats. Toxicology, 11:45-54, 1978. 4. Maltoni, C. N., Lefemine, G. Carcinogenicity Bioassays of Vinyl Chloride. I Research Plan and Earlier Results. Environmental Research 7:387-405, 1974. 5. Maltoni, C., et al. Carcinogenicity Bioassays of Vinyl Chloride Monomer: A Model of Risk Assessment on an Experimental Basis. Environmental Health Perspectives, 41:3-29, 1981. 6. Infante, P. F., et al. Carcinogenic, Mutagenic and Teratogenic Risks Associated with Vinyl Chloride. Mutation Research 41:131-142, 1976. 7. Edmonds, L. D., et al. Central Nervous System Malformations in Vinyl Chloride Monitoring Exposure: A Community Study. Teratology 17:137-142, 1978. 8. Theriault, G. et al. Evaluation of the Assocation Between Birth Defects and Exposure to Ambient Vinyl Chloride. Teratology 27:359-370, 1983. 9. Hansteen, I. L. et al. Fate of Vinyl Chloride in Man. A Cytogenetic Followup Study. Mutation Research, 51:271-278, 1978. 10. Gehring, P. J. et al. Resolution of Dose Response Toxicity Data for Chemi cals Requiring Metabolic Activation: Example, Vinyl Chloride. Toxicology and Applied Pharmacology, 44:581-591, 1978. 11 Gehring, P. J. et al. Risk of Angiosarcoma in Workers Exposed to Vinyl Chloride as Predicted from Studies in Rats. Toxicology and Applied Pharma cology, 49:15-21, 1979. 12. Watanabe, P. J. et al. Protein and Macromolecular Binding Following Ex posure to Vinyl Chloride. Toxicology and Applied Pharmacology, 44:571-579, 1978. 13. Rice, J. M. Prenatal Susceptibility to Carcinogenesis by Xenobiotic Sub stances Including Vinyl Chloride. Environmental Health Perspectives, 41:179-188, 1981. Environmental Health Perspectives Vol. it, pp. 179-188, 1981 Prenatal Susceptibility to Carcinogenesis by Xenobiotic Substances Including Vinyl Chloride by Jerry M. Rice* The carcinogenicity of vinyl chloride for experimental animals when administered transplacentally is reviewed in comparison with known transplacental carcinogens, including those that, like vinyl chloride, are dependent on enzyme-mediated metabolic conversion to a reactive intermediate in maternal or fetal tissues. Vinyl chloride is converted by mixed-function oxidases to the reactive metabolite chlorooxirane, the carcinogenicity of which is also reviewed. Vinyl chloride is unequivocally a transplacental carcinogen for the rat. No evidence exists, however, to support the hypothesis that exposure of male rats to vinyl chloride or any other carcinogen confers an increased risk of tumor development on their progeny. Many structural analogs of vinyl chloride, i.e., substituted ethylenes, are also carcinogenic for adult animals, and can with confidence likewise be predicted to be effective transplacental carcinogens. Introduction The carcinogenicity of vinyl chloride and its predilection for the hepatic blood vessels of both experimental animals and man is now well recog nized, and the substance is rightly regarded as a serious hazard in the workplace. Moreover, the demonstration by Maltoni (1) that vinyl chloride is not only carcinogenic for adult rodents but is a transplacental carcinogen for the rat as well, has raised concern over a possible risk of carcinogenesis in children born to mothers who had been employed in vinyl chloride manufacturing during their preg nancy. In the United States this has also led to questions of whether it is reasonable to single out women in the workplace as individuals especially at risk and whether to do so unfairly infringes upon their rights to equality in employment. It has been asked whether men may not equally be at risk in ^ terms of the potential of workplace exposure to vinyl chloride and related compounds to cause genetic damage in workers so exposed, and in this indirect way to contribute to an increased risk of cancer in their offspring. These are important questions, not all of which can be fully answered at the present time. Our laboratory has not been engaged in research on vinyl chloride and there fore can contribute no new data to what has been presented during the course of this conference. However, we have been engaged for many years in studying the phenomena of prenatal carcinogenesis by a variety of chemical carcinogens and in species as diverse as the mouse and .subhuman primates, and it is the purpose of this presentation to provide a context of current knowledge about the phenom ena of prenatal carcinogenesis within which the risk of prenatal exposure to this agent and related compounds can be evaluated. Mechanisms of Chemical Carcinogenesis When one tests a substance or a mixture of substances for carcinogenic activity in rodents, a positive result commonly takes one or more of the following three forms: (l)a higher incidence of tumors of one or more organ systems is observed 179 among treated animals in comparison with controls; if several different doses have been administered, test animals receiving higher dosage levels (except at very high, toxic levels) have a higher incidence of tumors than test animals receiving lower dosage levels; (2) a higher multiplicity of tumors occurs in one or more organ systems in treated versus control animals; if more than one dosage level is given to different groups of test animals, multiplici ty, like tumor incidence, also varies in rough proportion to dose, and animals that receive the highest nontoxic dosage regimen develop the high est multiplicity of tumors; (3) the latency for tumor development is shortened in test animals that develop tumors in comparison with control animals that develop the same tumors; this effect also may be proportional to dose, so that animals that receive the highest dosage of test compound develop tumors after the shortest period of time from the beginning of treatment. The results of a bioassay by itself can only assert that a substance indeed is carcinogenic, in the purely phenomenological sense of the term, by one or more of the above criteria. A bioassay says nothing about the mechanism by which carci nogenesis has been effected. This is a significant point, as evidence is steadily accumulating that not all of the extremely diverse agents capable of inducing tumors in animals or man act by the same mechanism. There exist a wide variety of chemicals that by these criteria have been shown to have the capacity to induce tumors in animals. These agents are extremely diverse in their chemical features, and include certain heavy metal cations; mineral fibers such as asbestos; and organic polymers, both soluble (iron-dextran; DEAE-dextran) and insoluble, such as plastic films. By far the greatest number of known substances carcinogenic for man or animals, however, are small organic molecules. For the overwhelming majority of these carcinogens there is a common mechanism of action. Agents belonging to this class either react chemically, or are trans formed in the course of metabolism to products that react chemically with intracellular nucleophiles, including nucleic acids, to form covalent bonds. Such reactions are irreversible, and proceed through the intermediate formation of an electropositive, or electrophilic, intermediate (2). Agents of this sort react with DNA to induce mutations and can be detected by their capacity to induce nonscheduled DNA repair synthesis in nondivkling cells; the term "genotoxic" is becoming accepted as a descriptive term for agents of this class, to which vinyl chloride belongs. Most genotoxic carcinogens are not themselves chemically reactive and require metabolic trans 180 formation to a chemically reactive metabolite or ultimate carcinogen. Although there exist a vari ety of mechanisms involving different enzyme sys tems by which such changes can be effected, the most common route, which is applicable to vinyl chloride, involves the mixed function oxidases {JThis is a class of cytochrome-containing enzymes, dependent on molecular oxygen and reduced nico tinamide-adenine dinucleotide phosphate (NADPH). which have very broad substrate specificities and are capable of catalyzing a variety of reactions, including the epoxidation of carbon-carbon double bonds, the oxidation of aromatic compounds through the formation of arene oxides, and the oxidative dealkylation of compounds such as nitrosamines by hydroxylation of carbon atoms alpha to a nitrogen or oxygen atom. The capacity of these enzyme systems to metabolize different classes of foreign substances, including chemical carcinogens, varies from individual to individual and from tissue to tissue, and is in part responsible for variation among individuals and from one organ system to another in susceptibility to different types of chem ical carcinogens. Transplacental Carcinogenesis in Experimental Animals To demonstrate transplacental carcinogenesis, experiments must be designed to eliminate the possibility of exposure to a chemical carcinogen by any route other than across the placenta. For rats and mice, in which species most such experiments have been carried out, this is usually accomplished by allowing the carcinogen-treated, timed pregnant female to deliver her young on top of a wire mesh screen so that the pups, as they are delivered, fall through the mesh into a cage below which is inhabited by a lactating female. With luck, the latter will gather up the newborns and care for them as her own, raising them to maturity without their having come postnatally into contact with their carcinogen-contaminated natural mother. For mice, such screens are generally made of 1/2 in. hardware cloth; for rats, poultry fencing generally proves adequate. General features of experimental transplacental carcinogenesis have been reviewed (4-6). The peri ods of susceptibility to different types of transpla cental toxic effects in rodents by chemical carcino gens are strictly related to stages of prenatal development. Exposure during the interval between conception and implantation of the blastocyst will either be without effect or will be embryocidal. Exposure of rats and mice to the same agent Environmental Health Perspectives GGC 0036S8 between approximately days 7 and 10, or between implantation of the blastocyst and development of the true placenta, when embryogenesis is occurring with great rapidity, may be lethal to the conceptus; at nonlethal doses there may be severe develop mental abnormalities. At lower dosage levels, in the range of exposures that are carcinogenic during later periods of development, tumors are generally not induced in offspring of animals exposed to carcinogens during this period even though their life expectancy is not significantly shorter than that of untreated offspring. After day 12, however, and usually with increasing efficiency thereafter until termination of pregnancy, exposure of a gravid female to a carcinogen will cause tumors to develop in her offspring. Tumors develop in different organ systems in different species in response to a given agent, but are generally at least in part morpholog ically and anatomically similar to those inducible by postnatal exposure to the same agent. A given agent may not affect a given tissue or organ similarly in all species, and the tumor spectrum seen as a consequence of prenatal exposure may vary markedly from one species to another. The most potent transplacental carcinogens are direct-acting alkylating agents, which, like methylnitrosourea (Fig. 1), decompose to reactive inter mediates without enzymatic catalysis. The next higher homolog of this compound, ethylnitrosourea (ENU), is extremely active transplacentally and has been extensively studied in both rodent and nonrodent species. When this compound is adminis tered as a single injection to a pregnant rat on one of the first 11 days of gestation, high doses result in devastating teratogenic effects or in death of the embryo, but not in tumorigenesis in surviving offspring. Beginning on day 12 and with increasing efficiency thereafter the offspring, which appear normal at birth if dosage is kept at a level below the acutely toxic range, subsequently develop tumors of the central and peripheral nervous system, with overt signs of disease developing 2 months to 2 years after birth (~). All three of the parameters previously mentioned as indices of carcinogenicity can be observed in experiments with ENU; the incidence of tumors and the multiplicity of tumors of the nervous system in treated offspring are both directly proportional to the dose administered to the mother, while latency is inversely proportional to dose. Despite the fact that ENU is a direct acting agent and is distributed nearly uniformly throughout the tissues of the rat fetus, tumors rarely appear in organ systems other than the nervous system. Occasionally, tumors of the kidney are seen, and an occasional offspring may develop leukemia. Epithelial tumors of the liver and the lung, for example, are virtually never encountered in rats transplacentally exposed to ENU. When one compares the dose-response relation ships for offspring of rats treated on day 15 of gestation with the response of adult rats to the same agent, it is found that the offspring are approximately 50-fold more susceptible than adults to the carcinogenic effects of this agent: the dose necessary to induce one or more neurogenic tumors in 50^ of exposed adult rats is on the order of 150 mg/kg, but the corresponding dose for transplacen tally exposed offspring is approximately 3 mg/kg (calculated on the basis of the mother's total body weight). If exactly the same sort of experiment is con ducted in the mouse, however, the results are qualitatively much different. Tumors of the ner vous system are relatively rare in mice following transplacental exposure to ENU. As in rats, how ever, transplacentally treated offspring exposed on or after day 12 of gestation develop tumors. In contrast to rats, mice exposed transplacentally to ENU develop epithelial tumors of the lung "and hepatocellular tumors of the liver in high incidence and multiplicity (<). In both species, the fetus is quantitatively more susceptible than the adult: susceptibility is greatest during the second half of the period of gestation, and increases as gestation proceeds towards parturition. Adult animals are less sensitive, by one to two decimal orders of magnitude, to the carcinogenic effects of ENU. This much higher prenatal-susceptibility is one of the principal reasons for concern that transplacen tal exposure to carcinogens in the mother's workplace or environment may be of significant risk for the human fetus. h*"h CH,-|V JNH 3C H *-. I O -ncom" r i x- Ich^n^n-ohI -- x-ch3 -n2 -OH* Figure 1. Decomposition of methylnitrosourea to yield a reactive alkylating diazonium hydroxide product. The initial step is pH-dependent, but does not require enzyme-mediated catalysis. October 1981 181 Recent studies carried out with ENU in our laboratories have shown, however, that two sig nificant differences from the patterns of response seen in rodents occur when transplacental carcino genesis experiments are carried out in a nonhuman primate. Different tissues are affected by the carcinogen, and the period of greatest prenatal susceptibility is early rather than late in gestation. Experiments on transplacental carcinogenesis in the Old World monkey, Erythrocebus /hitax, whose gestation period averages 170 day; have clearly shown that ENU is a transplacental carcinogen for this primate (9), but that it causes tumors princi pally of the vascular connective tissues and to a lesser extent the liver, kidney and brain, a pattern of response different from either the rat or the mouse. Tumor incidence is higher and latency is shorter in offspring than in pregnant or nonpreg nant treated adults; in that respect the pattern resembles that seen in both rodent species, but neither the rat or the mouse yields a spectrum of tumors predictive of the results of exposing mon keys to the same agent. In our experiments pregnant patas monkeys were exposed to ENU during either the first half or the second half of gestation, or throughout pregnancy. It has become strikingly apparent that animals exposed for the first time at day 30 are at much higher risk for tumor develop ment than animals given comparable exposure but beginning even 30 days later in gestation. This marks a significant departure from the patterns seen in rodents and suggests that the period of maximum intrinsic susceptibility to chemical car cinogens in other primates, including man, may well be during the first trimester of pregnancy and in that respect may resemble the period of greatest susceptibility to teratogens. Obviously, this includes the early fraction of gestation during which, at the time of possibly greatest vulnerability of her conceptus, a woman may not know for certain that she is pregnant and may therefore not be warned to take special precautions to prevent exposure to noxious agents. In other important respects, such as the greater susceptibility of the fetus, experience in the patas monkey is comparable to that in the rat and mouse and suggests that the phenomenon of high fetal susceptibility to carcinogens is a general one. The fact that the organ systems principally affected by ENU in this species are different from those of the rodent species further emphasizes that one should be extremely cautious in extrapolating from any other species to man in predicting the site of action of a chemical carcinogen, even a direct acting one. The one common pattern valid across species lines 182 in the matter of prenatal organ specificity is that the nervous system and the kidneys appear to be susceptible to at least some extent to this agent in all species tested, an observation which brings to mind the fact that tumors of the kidney and nervous system predominate among solid tumors of childhood- Transplacental Carcinogenesis by Metabolism-Dependent Carcinogens The vast majority of genotoxic chemical carcino gens are not direct-acting, but, as indicated pre viously, require metabolic conversion to a chemi cally reactive ultimate carcinogen in order to effect carcinogenesis. It has been shown that in rodents, the mixed function oxidase enzymes principally involved in activation of chemical carcinogens are present at low or virtually undetectable levels in fetal tissues until immediately prior to parturition, and even then are present at levels that are minus cule in comparison with those in adult tissues (10). The picture is complicated by the fact that these enzymes are inducible, and their levels in tissues such as the liver may be significantly altered by exposure to chemical agents that are substrates for these enzymes, including carcinogens such as methylcholanthrene (.i). The role of enzyme induction in modifying fetal susceptibility to transplacental carcin ogens has not yet been well studied and remains conjectural. However, the low levels of enzymes present in noninduced fetal rodent tissues result in extremely inefficient conversion of most substances to reactive ultimate carcinogenic metabolites. Thus, when the chemically reactive metabolite is very unstable, i.e., has an extremely short half-life under physiologic conditions, it cannot be effectively gen erated in maternal tissues and transported via mater nal and fetal bloodstreams to fetal tissues. Very short-lived reactive metabolites must be generated in situ in any fetal tissue in which carcinogenesis is to occur. Agents whose carcinogenicity is mediated by such metabolites are extremely poor transpla cental carcinogens. An example is dimethylnitrosamine (DMN; Fig. 2). DNM is metabolized by mixed function oxidases by the N-dealkylation mechanism, generating an intermediate methyl(hydroxymethyl)nitrosamine which is far too unstable to demonstrate even spectroscopically. It has never been synthesized. This is an excellent example of an agent which presumably must be formed by fetal enzymes. When the parent compound, DMN, was tested for trans placental carcinogenic activity it was found as expected to be much less efficient in offspring than in their mothers in the induction of tumors of the kidney (11). Environmental Health Perspectives i Fi GGC 003690 < N* = 0 MFO Nv -- o2 h3c' XH3 NAOPH /? to h3c' ch2 [ch3-n=h*oh] ch2o Figure 2. Metabolic activation of dimethylnitrosamine by a mixed-function oxidase* (MFO) to a reactive intermediate formally equivalent to that generated nonenzymatically from methvlnitrosourea as diagrammed in Figure 1. The enzyme catalyzed ,V-demethy!ation reaction requires molecular oxygen and NADI'II. and yields a |imdiict too unstable and short-lived to isolate or synthesize. From the example of dimethylnitrosamine, one might be tempted to infer that the transplacental route of exposure is insignificant for metabolismdependent carcinogens and, since this encompasses the vast majority of genotoxic carcinogens, is not a significant route of human exposure. Such a predic tion is probably wrong. Proximate and ultimate carcinogenic metabolites of many metabolism depen dent carcinogens are much more stable than methyl(hydroxymethyl)nitrosamine. An excellent example is afforded by the polynuclear aromatic hydrocar bons, especially 7,l2-dimethylbenz[a]anthraeene (DMBA, Fig. 3) which in common with many of the other carcinogenic substances belonging to this chem ical class can undergo metabolism to a variety of chemically reactive, arene oxide metabolites which are all ultimate carcinogens of varying potency. The most carcinogenic metabolites of this type of compound are the bay region diolepoxides {12), formed by three sequential metabolic steps as indi cated in Figure 3. The arene oxides, dihydrodiol, and diolepoxide (Fig. 3) have all been synthesized and are sufficiently stable for not only nynthom and characterization, but for direct testing of earcino- OH ON Fwvrf. 3. Some mixed-function oxida.se reactions of the poly nuclear aromatic hydrocarbon 7,12-dimethylbenz|a)anthracene (DMBA), yielding various mutagenic arene oxide products. These may subsequently be hydrolyzed by epoxide hydratase (EH) to dihydrodiols, which in turn may serve as substrates for MFO reactions that yield diolepoxides. Although carcino genic and very reactive, diolepoxides can be synthesized. October 1981 genic potency in experimental animals (1,1. 11,). Accordingly, it is quite possible that metabolites of this sort may be formed in maternal tissues and may succeed in traversing the maternal bloodstream and placenta to reach the fetus. When the parent hydrocarbon DMBA was tested for transplacental carcinogenicity in rats by using a foster nursing procedure (15), it was found to be extremely potent. The experiment was terminated at 52 weeks with only 20% of the offspring still surviving; large num bers of tumors were seen in the central and periph eral nervous systems, the kidneys, and blood ves sels, as well as in other sites. Thus, not only is this particular metabolism dependent agent a potent transplacental carcinogen, but it actually affects a broader spectrum of organ systems in the fetus of the rat than the extremely potent direct acting carcinogen ENU. Metabolism and Carcinogenicity of Vinyl Chloride The carcinogenicity of vinyl chloride for rats, mice, and hamsters over an extremely wide range of doses as well as its transplacental carcinogenic effects in rats, are presented by Maltoni (16); an earlier version of his data for rats (1) is summarized in Table 1. It can be seen that on inhalation of vinyl chloride, tumors are induced in dose-dependent fashion in adult rats in-the liver, nasal cavity, kidneys, Zymbal's gland, and most importantly and consistently, in the blood vessels, especially those of the liver. Transplacental exposure for a period of one week, between the 12th and 18th days of preg nancy, generated tumors in three of these tissues, the kidney, blood vessels, and Zymbal's gland, in the offspring (Table 2). Although no controls were included specifically in the transplacental study, the very large series of historical control animals care fully examined in that laboratory are convincing proof that the elevated incidence of tumors of these three tissues is real, and that vinyl chloride is ef fectively a transplacental carcinogen in the rat. It is noteworthy that the sites and kinds of tumors in- 183 GQc duced by vinyl chloride are different in part from those that resulted from transplacental exposure to any of the agents discussed previously, none of which affected the Zymbal's glands. The tissues and organs affected in offspring exposed transplacentally to vinyl chloride included some but not all those in which tumors developed in adults subjected to much more prolonged exposures (cf. Tables 1 and 2), and in the transplacental study, significant num bers of tumors were induced in the offspring but not in the mothers. Vinyl chloride is a metabolism-dependent carcin ogen, dependent for its carcinogenicity on mixed function oxidases (1?) which convert vinyl chloride to its epoxide derivative chloroethylene oxide (chlorooxirane, Fig. 4). The earcinogeneity of chloroethyl ene oxide and of its rearrangement product, chloro- acetaldehyde, were recently investigated by research ers at the International Agency for Research on Cancer in Lyon, France, who tested both com pounds by subcutaneous injection and by skin paint ing, the latter followed by phorbol ester promotion, in mice. Chloroethylene oxide proved to be an effective carcinogen, inducing both papillomas and carcinomas in the skin and giving rise to sarcomas on injection (Table 3). Its rearrangement product, chloroacetaldehyde, was toxic, but not demonstra bly carcinogenic (IS). It was noted by the IARC investigators is that the half-life of chloroethylene oxide for hydrolysis at 37 C is on the order of 0.9 min. This is sufficient time for chloroethylene oxide formed in maternal tissues to reach the fetus by way of the placenta, and provides strong sugges tive evidence that maternal metabolism contributes to transplacental carcinogenicity of vinyl chloride. To date, however, chloroethylene oxide has not itself been tested for transplacental earcinogeneity. Analogs of Vinyl Chloride It is important to note that vinyl chloride is by no means unique with respect to chemical structure, and that a wide variety of compounds are in current use in large volumes in industrial processes which differ from vinyl chloride only by further substitu tion of the vinyl chloride molecule. A partial list of such substances is given in Table 4. Furthermore Table 1. Tumors in Sprague-Dawtey rats after inhalation exposure to vinyl chloride, 4 hr daily and 5 days weekly for 52 weeks. Partial results after 135 weeks." Vinyl chloride, ppm Rats at risk (both sexes) Zymlial's gland carcinoma Nephro blastoma Rats with tumors Angiosarcoma Liver Other Liver cell tumors Nasal eavityb 30,000 10,000 6,000 2.500 500 250 50 None 60 35 I) 18 1 1 1 69 16 5 9 3 1 7 72 74 13 3 1 3 74 2 13 3 2 5 67 44 72 3 0 67 06 42 00 64 01 1i 00 68 0 t) 0 0 0 0 Data from Maltoni (1). ''Originally reported U) as neuroblastoma of the brain. Table 2. Tumors in female Sprague-Dawley rats exposed to vinyl chloride by inhalation 4 hr daily from day 12 through day 18 of gestation, and in their offspring," Generation Vinyl chloride concentration (ppm) Rats with tumors after 115 weeks Rats exposed Zymbal's gland Nephro Total Survivors carcinoma blastoma Angiosarcoma Liver Other Parents Offspring 10,000 30 30 i 0 0 1 6,000 30 30 0 0 0 0 10,000 54 12 3 1 0 2 6,000 32 8 1 0 0 2 "Data from Maltoni (1). 184 Environmental Health Perspectives GGC 003692 Cl o \_y Cl =j MFO VINYL NADPH CHLORIDE (carcinogenic) XT O CHLOROOXIRANE (carcinogenic) Ti/2(37*). CHLOROACETALDEHYDE (toxic i not carcinogenic) 0.9 min Covalent bonding to cellular nucleophiles including DNA Figure 4. Metabolism of vinyl chloride by mixed function oxidase? to chlorooxirane, a carcinogenic substance that is stable enough to synthesize. Table 3. Carcinogenicity of chloroethylenc oxide in adult mice.* 1 Effective number Route Tumor-bearing animals, K Duration of experiment, days 28 M 32 SC injections 24 F Control 30 M i 28 M 1 mg initiation (skin); TPA promotion < Control 28 M \ 'Data from Zajdela et al. (18). 15 (54) 12 (50) 0 (0) 20 CJ.'J), papillomas 5 (18), carcinomas 4 (15), papillomas 0, carcinomas 549 549 549 590 590 Table 4. Carcinogenicity in adult rodents of structural analogs of vinyl chloride reviewed in the IARC Monograph Series. Compound Structure Reference Rats Site of lesions Mice Vinyl chloride CHZ=CHC1 Vinylidene chloride Trichlorethylene Tetrachlorethylene Chloroprene CH2 = CC1z CHC1 = CCIZ CCI2-CC12 CH2=CH-CC1 = CH2 (22a) (22b) (2.1a) (2.1b) (22c) Angiosarcoma Angiosarcoma Zymbal's gland Lung CNS Mammary Kidney Similar to vinyl chloride, but incomplete in 1979 (Inadequate) , Lung, liver Negative Liver Inadequate - there is an even larger list of substances that resem ble vinyl chloride in that they are substituted deriv atives of ethylene, but differ in that they lack a chlorine atom. A very brief list of important deriva tives of this sort is given in Table 5. A much longer 4 list is given in the review by Posner and Falk (19). It is reasonable to postulate that metabolism via 4 reactive epoxides is one route of metabolism to be < expected for all these compounds, and mutagenic I October 1981 < i i i i metabolites of some of them have been demon strated (20, 21). If mutagenic epoxides are formed in significant quantities in maternal tissues and are chemically reactive, yet stable enough to reach the fetus, it is probable that they too would have some measure oftransplacental carcinogenic activity. Some of the substances listed in Tables 4 and 5 have been reviewed for risk of carcinogenicity to man in the IARC monograph series (22, 2J). Other compounds I I i Table 5. Compounds structurally similar to vinyl chloride that may have similar carcinogenic activity. 1 _______________________________________________ Site of lesion Compound Structure Reference Rats Mice < Vinyl bromide Styrene Acrylonitrile Acrylamide Ethyl acrylate CH, = CHBr Ch, = CHC(1H-, CH^CHCN CH.. = CHCONH., CtL = CHCOOC.H- Not vet reviewed (AM) UJe) Not yet reviewed Not yet reviewed _ Brain Forestomach Zymbal's gland Lung (7) 1 1 i t < i listed here have been tested in the United States by the National Cancer Institute-National Toxicology Program. As a significant number have been found to be carcinogenic in at least one rodent species, it is reasonable to regard the entire class with suspi cion as possible potential carcinogens and transpla cental carcinogens pending the acquisition of fur ther data. Effects of Transplacental Carcinogens on Subsequent Generations: Lack of Male Parental Risk for Prenatal Carcinogenesis In the context of our present knowledge, prena tal carcinogenesis is virtually synonymous with trans placental carcinogenesis. That is, there is very little evidence to indicate that exposure of either male or female parents to a chemical carcinogen prior to conception confers an increased risk of carcinogen esis on their offspring. It should be pointed out that carcinogenic risk is not the only risk, and that exposure of males to toxic agents may cause steril ity or reduce fertility. In rodents it has also been shown that dominant lethal mutations may be induced in this manner, the consequence of which is death of the conceptus Ui). These hazards however are different from carcinogenic risk for surviving off spring, which has not been show'n to be a conse quence of exposure of adult males or of adult females prior to conception. The experimental data which are relevant to this question generally concern what has been termed the "second generation effect" in prenatal carcino genesis, which has been reviewed by Tomatis (A5). The general design of experiments of this sort is as follows: The female of a parental generation, P, is given a chemical carcinogen during pregnancy. Male and female offspring of the treated female, consti- 186 tuting the Fi generation, are then at risk for transplacental carcinogenesis. On attaining sexual maturity, and before tumors resulting from transplacental exposure appear, F j males and females are mated to produce an F2 generation. Both parents of the F2 generation had been exposed to carcinogen during prenatal life, but at no time were the F2 animals exposed. In addition, males of the prenatally exposed F i generation were bred with untreated females to produce offspring designated F2M, whose male parents only had been subjected to chemical carcinogens. Likewise, the F, females were bred with untreated control males to produce a F2 F generation, of which only female parents had experienced exposure to chemical carcinogens. Similar experiments have been continued to the third and higher generations in attempts to demonstrate a higher incidence of tumors in comparison with untreated controls. Such experiments have been carried out w ith polynuclear aromatic hydrocarbons, direct acting nitrosourea carcinogens, and with ethyl carbamate, in three laboratories. They have consistently demonstrated that a small, often statistically in significant but nevertheless reproducible and per sistent excess risk of carcinogenesis is present in F2 and F2F generations. F2M offspring in general have shown no excess risk. The numbers of tumors observed in the second generations in experiments of this sort have invariably been extremely small, leaving much to be desired in terms of statistical significance. However, these studies constitute the only experimental evidence for a chemically induced enhanced susceptibility to cancer mediated by dam age to the germ cells. Present evidence indicates that if this effect is real, it is most significant for the female germ cells, which are undergoing rapid mitotic division during the final week of intrauterine devel opment in the female rat or mouse fetus. On the basis of present information, therefore, it is clear that prenatal carcinogenesis by chemicals is principally a direct effect of chemicals or their fetal or maternal metabolites upon fetal tissues, rather than upon the germ cells of the parents of either Environmental Health Perspectives * * * * * . . j j j * ' j j , j , t i t i 1 GGC 003694 sex; if an effect mediated via damage to parental germ cells exists, that damage with its attendant increased risk of carcinogenesis in the offspring appears greatest when the female parent is exposed to a carcinogen; and that the fetus is at risk on account of maternal exposures to potential carcino gens, not only because it is inevitably exposed to any agent that may find its way into the maternal bloodstream, but also because carcinogenic or toxic metabolites which may be even more dangerous than the environmental precursor may be gener ated by maternal tissues and transferred via the placenta to the fetus. The incidence of tumors that will develop in a given organ system following transplacental expo sure to a carcinogen is not necessarily the maxi mum incidence possible. Cocarcinogenie phenome na, in which exposure to a second, noncarcinogenie agent accelerates the development and increases the yield of tumors resulting from a previous carci nogenic exposure, are well demonstrated in trans placental carcinogenesis (26), especially in the mouse where they have been documented for the skin and for the liver. Potential tumor cells may remain quiescent for prolonged periods after exposure to the inducing agent, and in evaluating the significance of transplacental exposure one must bear in mind that subsequent, postnatal exposure to noncareinogenic promoting agents may act in a strongly syn ergistic fashion with prenatal exposure to a carcin ogen and may greatly increase the risk of carcino genesis. Finally, all agents known to be prenatal--that is, transplacental--carcinogens are also carcinogenic to some extent during postnatal life in one or more species. To some extent, this is a consequence of the manner in which the science of transplacental carcinogenesis has developed; known carcinogens have been selected for testing for transplacental effects. Nonetheless, the generalization holds that there is no known purely transplacental carcinogen. REFERENCES 1. Maltoni, C. Predictive value of carcinogenesis bioassays. Ann. N.Y, Acad. Sci. 271: 431-443 (1976). 2. Miller, E. C., and Miller, J. A. The metabolism of chemical carcinogens to reactive electrophiles and their possible mechanisms of action in carcinogenesis. In: Chemical Car cinogens, (ACS Monograph 173), C. E. Searle. ed.. Ameri can Chemical Society, Washington, D.C.. 1976, pp, 737-762. 3. Lu, A. Y. H.,and Levin, W. The resolution of the liver microsomal hydroxylation system. Biochem. Biophvs. Acta 344:205-240(1974). 4. Tomatis, L., and Mohr, U., Eds. Transplacental Carcino genesis (IARC Scientific Publications No. 4). International Agency for Research on Cancer, Lyon, 1973. 5. Rice, J. M.. Ed. Perinatal Carcinogenesis (NCI Monograph October 1981 51). C.S. Government Printing Office. Washington, D.C., 1979. 6. Rice, J. M. Carcinogenesis: a late effect of irreversible toxicdamage during development. Environ. Health Perspect. 18; 133-13911976). 7. Ivankovic, S. and Druckrey, H. Transplacentare Erzeugung maligner Tumoren des Nervensystems. 1. Athylnitrusohamstoff (ANH) an BD-IX Ratten. Z, Krebsforsch. 71: 320-360(1968). 8. Vesselinovitch, S. D., Koka, M.. Kao, K. V. N., Mihailovich, N.. and Rice, J. M. Prenatal carcinogenesis by ethvlnitrosourea in mice. Cancer Res. 37: 1822-1828 0977). 9. Rice, J. M., Palmer, A, E., London, W. T,, Sly, D- L. and Williams, G. M. Transplacental effects of ethylnitrosourea in the patas monkey. In: Tumours of Early Life in Man and Animals, L. Seven, Ed.. Perugia Quadrennial International Conferences on Cancer, Perugia, Italy, 1978. pp. 893-906. 10. Lucier. G. W., Lui, E. M. K.,and Lamartiniere, 0. A. Metabolic activation/deactivation reactions during prenatal development. Environ, Health Perspect. 29: 7-16 (1979). 11. Alexandrov, V. A. Blastomogenic effect of dimethylnitrosamine on pregnant rats and their offspring. Nature 218: 280-281 (1968). 12. Jerina, I). M.. Thakker, D. R., Yagi, H,, Levin, W., Wood. A. W., and Conney. A. H. Carcinogenicity of benzofa,'pvrene derivatives; the bav region theory. Pure Appl. Chem. 50: 1033-1044(1978). 13. Sukumaran. K. B., and Harvey. R. G. Synthesis of /mifs-3.4-dihyro.\y-3,4-dihydro-7,I2-dimethylbenz[a]anthracene, a highly carcinogenic metabolite of 7,12-dimethylbenz(alanthracene. J. Am. Chem. Soc. 101:1353-1354(1979). 14. Slaga, T. J., Huberman, E.. Digiovan, J.. Gleason, G.. and Harvey, R. Q. Importance of the bay region diol-epo.xide in 7,12-dimethylbenzfalanthracene skin tumor initiation and mutagenesis. Cancer Lett. 6: 213-220 (1979). 15. Rice. J. M., Josbi, S. R., Shenefelt, R. E., and Wenk. M. Transplacental carcinogenic activity of 7,12-dimethylbenz[alanthracene. In: Carcinogenesis, Vol. 3: Polynuclear Aro matic Hydrocarbons. P. W. Jones and R. I, Freudenthal, eds. Raven Press, New York, 1978, pp. 413-422. 16. Maltoni. C., Lefemine. G.. Ciliberti, A.. Cotti. G.. and Carretti, D. Carcinogenicity bioassays of vinyl chloride monomer: a model of risk assessment on an experimental basis. Environ. Health Perspect. 41; 3-30 (1981). 17. Salmon, A, G. Cytochrome P-450 and the metabolism of vinyl chloride. Cancer Lett. 2: 109-114 (1976). 18. Zajdela. F., Croisy, A., Barbin, A., Malaveille, C.. Tomatis, L., and Bartsch, H. Carcinogenicity of ehloroethylene oxide, an ultimate reactive metabolite of vinyl chloride, and bis(chloromethyl)ether after' subcutaneous administration and in initiation-promotion experiments in mice. Cancer Res. 40; 352-356 (1980). 19. Posner, H. S.. and FaJk, H. L, Haloetbylene-related com pounds of industrial, environmental, and medical significance. Environ. Health Perspect, 21: 293-326 (1977). 20. Bartsch, H. Malaveille, C.. Barbin, A., and Blanche. G. Mutagenic and alkylating metabolites of haloethylenes, chlorobutadienes and dichlorobutenes produced by rodent or human liver tissue. Arch. Toxicol. 41: 249-277 (1979). 21. Simmon. V. F.. and Baden. J. M. Mutagenic activity of vinyl compounds and derived epoxides. Mutat. Res. 78: 227-231 (1980). 22. IARC. IARC Monographs on the Evaluation of the Carci nogenic Risk of Chemicals to Humans. Vol. 19, Some Monomers. Plastics and Synthetic Elastowers. and Acrole in, International Agency for Research on Cancer. Lyon, 1979, (a) pp. 377-437: (b) pp. 452-453: (c) pp, 131-156: 187 \ (d) pp. 231-274; (e) pp. 73-133. 23. IARC. IARC Monographs on the Evaluation of the Carci nogenic Risk of Chemicals to Humans, Vol. 20, Some Halogenated Hydrocarbons, International Agency for Re search on Cancer, Lyon, 1979, (a) pp. 543-572; (b) pp. 491-514. 24. Health and Welfare Canada. The Testing of Chemicals for Carcinogenicity, Mutagenicity. Carcinogenicity. Health and Welfare Canada, Ottawa, 1975. 25. Tomatis, L. Prenatal exposure to chemical carcinogens and its effect on subsequent generations. Natl. Cancer Inst. Monogr. 51: 159-184 (1979). 26. Goerttler, K., and Lohrke, H. Diaplacental carcinogenesis; initiation with the carcinogens dimethylbenzanthracene (DMBA) and urethane during fetal life and postnatal promo tion with the phorbol ester TPA in a modified 2-stage Berenblum/Mottram experiment. Virchows'Arch. Pathol. A 372: 29-38 (1976). 188 Environmental Health Perspectives GGC 00369*