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BIO-MEDICAL. RESEARCH DOCUMENT DESCRIPTION FORM 68 69 76 Duplicate in all cards:-> I'rP I 00003571 year as-1961- File number [Right justify fNumeric only] Author(s), as Last Name FS (No Punctuation) and coden for journal as JAMA preceeded by one blank space 1 20 21 _____ 40 4160 61 62 ' 77 78 Sub-Index Code Title of Report; end with space-hyphen-hyphen-space. Follow with Index Terms, separated from each other with comma-space. Avoid other punctuation; Brief Summary 12 10 31 32 61 62 0000357 Toxicologic Investigation of Vinyl Chloride with Relevance to Non-worker Populations: with emphasis on transplacental carcinogenesis and co-factors. R&S 112106 Principal Investigator: E. Bingham Mattheis, Ph.D. S73 "S'??'f'' -57?! Background ' Vinyl chloride monomer (VCM) is a chemical of increasing industrial and environmental importance. VCM has many uses, e.g. the production of poly- , / '/ ` vinyl chloride (PVC) resin, as a co-polymer in saran and other plastics, as a solvent, as a propellant for pesticides and hair-sprays, as a refrigerant, and-; ??. An the past, as an anaesthetic. Despite the apparent usefulness of this chemical, recent findings implicate VCM as the agent responsible for induction of a rare liver cancer, angiosarcoma, among workers employed in the production of PVC. Toxicological Ljt 7c^ - experiments with various levels of VCM performed by Maltoni and in Italy and by Biotest Laboratories, Northbrook, Illinois have demonstrated that simi lar rare neoplastic lesions can be induced in experimental animals. A great need exists for data on toxicity, persistence and for the assessment of risks associated with low level concentrations of VCM, including those levels that are likely to exist beyond the fence line of the manufacturing plant. Materials loss in PVC production processes has been found to be approx imately 6%, but this varies with type of process, the age of the plant, the level of technology employed and the manufacturing processes. However, there is no doubt that substantial amounts of VCM have been discharged into the envir onment during PVC production processes. These PVC and VCM losses occur as air emissions and as components of water effluent and solid wastes. A recent report indicates that a woman, living downwind from a PVC V, /. So - plant and having no occupational exposure to this substance, developed hepatic angiosarcoma. Russian scientists have determined that exposure to VCM may be very widespread. It has been found, in a study performed in a newly constructed building for child care, that VCM is continuously released from PVC flooring for at least two months after installation. They have recommended excessive ventilation of a newly constructed building be mandatory before occupancy. Hie use of vinyl chloride as a propellent in aerosol containers has raised a concern for individuals exposed in the general population who may have been exposed before removal of this propellent from the market. It appears, therefore, that segments of the population, in addition to the industrial workers, may be at risk from exposure to VCM. The risk of such exposures to the various elements of the population must be determined, in particular the more sensitive groups such as the developing fetus and new born, or that segment which experiences dietary deficiencies. It has been reported that the enzymes responsible for ethanol metab olism may be involved in the metabolism of VCM (Gehring, 1974) since the effects of other chlorinated hydrocarbons, such as trichlore^thylene and chloral hydrate, which are biologically transformed by alcoholic dehydrogenase are potentiated by ethyl alcohol, it appears that comparable effects may occur with VCM. These investigations may help to determine a mechanism of action for this carcinogenic agent as well as for other related potentially carcinogenic chlorinated hydro carbons . There is no information available on the carcinogenic effects of VCM given together with common co-polymeric agents such as vinylidene chloride. Since both of these co-polymers have similar properties and structures, there is a need to investigate what effects this combination may have on the bio logical outcome. Purpose The purpose of this study is threefold: a) To investigate the effects of VCM on the developing fetus through inhala tion exposure in an effort to determine the risk of developing oncogenic lesions via the transplacental route and to determine the effects of inhalation exposure R&S 112107 to VCM on the neonate. b) To determine whether certain dietary factors such as vitamin C deficiency or ethanol consumption may influence the biological response to VCM. c) To develop cell culture techniques useful for the rapid screening of potential oncogenic agents, such as VCM and co-factors. Ihese techniques will be used to provide the means for studying mechanisms of the action of similar agents, and to serve as an indicator of dose-effect responses. Scope of"Work A. Inhalation exposure of pregnant animals to levels of VCM to determine the potential transplacental oncogenic and other biological effects. Sprague-Dawley rats will be obtained from Charles River Laboratories either timed-pregnant or they will be bred in our animal facilities. In the first experiment, twenty pregnant rats will be exposed to 20,000 ppm vinyl chloride, or as agreed upon with the contract officer, in an inhalation chamber from the 10th day of ,Jestation to the time of delivery. After birth, litters of five young will remain with each mother. Additional young will be sacrificed and cleared by the Schultz-Dawson method to visualize the skelton for osseous changes. The mothers and their surviving young will be kept in box cages until weaning at four weeks of age. Uien the mothers and their young will be fed Purina Lab Chow and maintained in rooms controlled for temperature, humidity and light cycle. The 100 surviving young will be examined for congenital malformations and then observed for signs of tumors and other diseases, particularly acroosteolysis. X-ray examination will be done in selected cases at various times. Observations may have to be continued for one to two years. After weaning of the first batch of young from exposed mothers, another batch of twenty pregnant females will be subjected to a similar vinyl chloride treatment. In this experiment, ten mothers with their litters of five j i ; I ji R&S 112108 R&S 112109 young each will be handled like the animals described above. Another group of ten mothers and their young will be kept in the vinyl chloride chamber until after weaning. In this way, 50 neonates will be exposed to test their suscep tibility to vinyl chloride. This second batch of mothers and offspring will be treated like the first and observed for oncologic and other morbid effects. Control mothers will be subjected to chamber treatment with only absolute filter air. B. Interaction effects of VCM with other agents. It is proposed that male rats will be subjected to various dietary manipulations and exposed to one or two levels of VCM (as agreed upon by the contract officer). The animals will be housed in a room with controlled lighting cycle, temperature, and humidity, and fed liquid diets ad libitum (16 hours/day). Hie liquid diets (General Biochemical, Chagrin Falls, Ohio) will be purchased in dry form and reconstituted as needed. Hie diets will consist of a control diet (without ethanol) and the same diet supplemented with 57. ethanol Appropriate controls will be used. In addition to rats fed an ethanol supplemented diet and exposed to VCM, guinea pigs on vitamin C deficient diets and their controls will be used. The protocol involving guinea pigs will serve a dual purpose: a second species susceptibility to VCM and the- co-factor response to vitamin C. All exposures will be maintained for 12 months, 5 days/week, 4 hours/day to the VCM, or as agreed upon by the project officer. In addition, rats will be exposed to VCM and vinylidene chloride at levels agreed upon with the contract officer. The exposure chambers will be located in a large room which has been previously used for human inhalation exposures. Ihis room is very suitable for the proposed testing since it is sealed to insure against leakage. The main chamber has a double door entry. This chamber in a chamber arrangement will Insure safety of all laboratory personnel. Both chambers will, be kept under subatmospheric pressures to insure against leakage. Air flow in the outer chamber is in a ceiling to floor direction with a complete air change every four minutes. Makeup air for the exposure chamber and negative pressure room will be prefiltered through absolute filters. Periodic sampling of both cham bers using Sipin pumps and activated charcoal tubes'will be made. In addition, appropriate protection of personnel will be provided, including mandatory wearing of full face gas masks with outside air, supplied by the umbilical cord arrangement, emergency full face organic vapor canister type gas masks, and portable air supplies with rechargeable type cylinders. Periodic personnel monitoring will be made using Sipin pumps and charcoal tubes. All VCM from the exposure chamber and the negative pressure room will be removed from the air using a fabricated activated charcoal filter. r ^ R&S 112110 Spent activated charcoal will be removed from the filter by personnel, appro priately protected, and placed into heavy plastic bags. These bags will be incinerated. A similar chamber for control exposure will be used. Preliminary experiments will determine the amount of exhaled VCM from exposed animals and the time at which they can be moved to housing quarters after exposure. In the event the exhaled air concentrations are unacceptable, the animals will be housed in suitable quarters, which are isolated from all personnel. Either the charcoal tube method or gas tight syringes will be used to collect the samples. An analytical gas chromatographic method to be used for determining VCM levels in the exposure chambers was developed by our in-house analytical department. However, a number of methods could be used for the analysis of VCM including a halide meter, long pathlength (Brooks-Moran) IR spectrophotometer or a gas chromatograph with a flame ionization detector. The GC is the method preferred. Facilities and personnel experience in analytical r# procedures are available for this project. The first year will be entirely devoted to setting up, calibration and exposing the pregnant animals from Part A. No additional animals (on various dietary regimens) will be exposed during the first year. The second year will be devoted to inhalation exposure at one or two different levels of VCM, e.g., 50 and 500 ppm as agreed upon with the contract officer. Animals will be maintained on the proper deficiency or supplemented diets coupled with various exposure regimens as shown in the following table. Suitable control animals will also be used. Diet Number of Animals VCM Exposure (ppm) 1) Vitamin C deficient 2) Vitamin C sufficient/ l guinea 3) Vitamin A deficient J pigs 40 X 2 40 X 2 40 50, 500 50, 500 None 4) Vitamin C sufficient./ 40 None 5) Control diet with \ 5% Ethanol 40 X 2 50, 500 6) Control diet 40 X 2 50, 500 7) Control diet with ^ 57. Ethanol / rats 40 None 8) Control diet 9) Control diet I J 40 40 None VCM + Vinylidene Cl. R&S 112111 During the third year animal exposures and observations will be continued, as based on previous data. The animals must be kept under obser vation for one to two years. C. Tissue and blood samples from the exposed animals (part B) will be collected and transported to ETRL, NERC - Cincinnati for clinical chemistry ,, and pathological determinations with special emphasis on electronmicroscopy. The number of animals to be used will be determined as agreed upon by the project officer. The current figures represent ten animals from each group for the sampling. This portion of the work will begin the second year or where appropriate the first year as agreed upon with the project officer. D. Tissue Culture Lab - Proposed for Second Year and Beyond The malignant potential of chemical carcinogens has been demonstrated by in vitro cell culture methods. This technique should also be applicable to the vinyl chloride monomer. Three in vitro carcinogenic assay systems are in general use: 1. the hamster embryo cell system; 2. mouse cell lines; and 3. the 3T3 cell line. This proposed work for a rapid screening system will be developed using the hamster embryo cell system without a feeder layer. Twelve to 14 day old fetuses are sacrificed and their cells grown in culture. The VCM will be added to the cultrues in the gas phase in a mixture of 5% CO^ in air. The design of the chamber and the concentrations of VCM will be determined in con sultation with the contractor. It should be possible to obtain a dose response curve in this system with concentrations between 5 - 20,000 ppm VCM. The cells will be exposed for seven days, allowed to grow for an additional 8-10 days in control medium and then fixed and stained. Determination of cytotoxicity and examination for transformed cells is done in the same dish. Cytotoxicity is determined as a percentage of cells exposed to the agent against control. The host mediated assay is of particular interest to this project because VCM is a gas and this is probably the route of entrance into the animal or man. This project is also interested in the effect of this agent on the embryo. In this assay the pregnant hamster or rat will be exposed to VCM in an inhalation chamber 48 hours before the fetuses are obtained from the mother and sacrificed. The fetal cells are then cultured and observed for trans formation as above. These techniques have proved quite satisfactory for the study of a R&S 112112  number of carcinogenic agents and may be the most satisfactory in this case, ^lt because of the unusual nature of this carcinogen several other techniques vill be used in consultation with the contractor. Endothelial cells are avail able for exposure to VCM as well as human fibroblastic cells. These will be used in tests similar to those outlined above. A somewhat different, but very interesting approach to this study, will be the observation of hyperplastic r premalignant changes of tissues in organ culture. These studies will be similar to those previously reported by Dirksen and Palekar.on rat trachea. Eat blood vessels and small pieces of rat liver will be grown in organ culture in an exposure chamber with an atmospheric mixture of VCM, C0_ and air or 0 . The tissue will be placed on rayon rafts and floated on medium in organ cul ture dishes. The tissues will be fixed at weekly intervals for five weeks and provided to the contractor for pathological examination. These changes may be ^jnilax to changes in the rats exposed to VCM in the inhalation chambers. Periodic reports will be submitted to the project officer on the status of the experiments. Facilities The department possesses extensive animal quarters used for both short and long range toxicologic and metabolic studies on experimental animals. A special area was recently constructed permitting the maintenance.of animals on rigidly controlled diets, including diets deficient in trace metals, etc. The main area contains more than 10,000 square feet*of animal rooms for acute and chronic experiments for most laboratory animals. Large air R&S 112113 R&S 112114 conditioned animal rooms containing cabinet sinks with hot and cold running water are available. Racks of cages for dogs, rabbits, rats, mice, guinea pigs, and hamsters are available in sufficient quantity for research efforts. Special facilities are available for monkeys. Special rooms and equipment are also available for the housing of animals used in metabolic studies involving the lungs, in which air, passed through absolute filters, is circulated under positive pressure (laminar air flow). Facilities for washing and sterilizing, the racks and cages and for maintaining cleanliness and sanitation in the animal rooms are utilized by a staff trained in animal care and under the supervision of an experienced D.V.M., Dr. Steele Mattingly. Pathology laboratory under the direction of Dr. Klaus Steramer, has a w 11 developed facility and is fully equipped for chemical pathology and histopathology. As part of the teaching and research activities of the Department of Environmental Health, an epidemiology and biostatistics group has been active for many years. It is under the direction of Dr. Ralph Buncher. Data manage ment can be handled through the Medical Computing Center which has as its major equipment an IBM 360 Model 20 which connects directly with an IBM 360 Mod 65 time-sharing computer located in the College of Engineering on the main campus, close to the Medical Center. The Department has extensive facilities for the study of pulmonary toxicology. Among areas of active interest are the effects of exposure to Pb, Cd, Ni and other metals in the atmosphere and the physiology of pulmonary macrophages. Other active interests include investigations covering pulmonary metabolism of carcinogenic substances and other environmental* agents using the isolated perfused lung preparation of various animal species. In addition to R&S 112115 the carcinogenic response of skin to various carcinogens (pure, complex mixtures, cocarcinogens, etc.), inhalation studies using coal tar are currently underway. Five stainless steel, rectangular exposure chambers (Young and Bertke Co., Cincinnati) are operational. Each has a cone shape top and bottom and a capacity of 12 ft. 3 (340L). In addition, a larger chamber ( 24 ft. 3 ) of the same design is available. These chambers are normally operated at airflows which permit one exchange of air per minute. Each chamber can accomodate 60 rats (120 rats capacity - large chamber) or 90 hamsters or 9 rabbits in individually housed stainless steel holding cages. After the primary air supply is drawn through an absolute filter the contaminant is added and introduced into the chamber, which permits very uniform dispersion as determined by tests in the laboratory. The chambers are operated under subatmospheric pressures and the exhaust air is passed through either an absolute filter, charcoal filter, or a water spray to remove the contaminant before exhausting to the atmosphere. In addition, four other types of chambers are used. One hexagonal stainless steel chamber with a capacity of 700 liters is operated in a manner similar to. that described above. Four 32 liter capacity semihemispherical glass chambers are also employed. This particular type is useful for rangefinding inves tigation as well as for chronic exposures. Each chamber can accomodate up to 10 rats.' Three chamber rooms, each 12.5 ft. long X 7.5 ft. wide X 9 ft. high 3 with a volume of 850 ft. (24,700L), can singularly accomodate two large cage racks for 120 individually housed rats. Air is circulated to these chambers at 3 a rate of 150 ft. per minute to each chamber by means of two blowers (size C American Blower Co.) with absolute filters positioned on both the inlet and exit ducts. These chambers are also kept under a slight substmospheric pressure* Contaminant generation equipment for inhalation chambers includes: 1) Dust generation - 7 Wright Dust Feeders (L. Adams Ltd.,`London, England) equipped with cyclones and 15 dust tubes. A supply of replacement parts is maintained 2) Aerosol generating equipment including aspirating equipment such as that used for coal tar inhalation. 3) Fumes - various techniques as used for the generation of metal fumes. 4) Vapors - generated by modified Greenberg-Smith Impingers. 5) Gaseous exposure using various regulator valves and orifaces. All chambers have indicating monometers (Magnehelic) to measure subatmospheric pressures. Air flow is monitored with either stainless steel orifaces or rotometers. Other air flow calibration and monitoring equipment includes: 9 R ckwell dry test meters (175 ft. 3/hr. capacity); one calibrated master 140 ft. 3 test meters; one wet test meter (3 liters/revolution) for calibration of low flow rate rotometers. Routine sampling of the contaminant, etc. is performed hourly. Sampling equipment routinely used includes: 12 MSA midget bubblers, Greenberg-Smith Impin gers with fritted disc; 7 Anderson cascade Impactors for particle size distribution and various filter holder samplers for gravimetric and/or analytical procedures. In addition the industrial hygiene and analytical departments have a fairly complete and varied line of supplementary sampling equipment that is readily available. Thirteen gast pumps (S-10311, 4-0522, 1-0822) of various air flow capabilities and many other standby pumps are used routinely. % I R&S 112116 Bergwein, K., 1970. Pure hydrocarbons, liquified propellants and vinyl chloride as aerosol propellancs and mixtures. Kosmetik-Parfumdrogen Rundschau 8^:20-22. Dinman, B. D., W. A. Cook, W. M. Whitehouse, H. J. Magnuson and T. Ditcheck, 1971. Occupational acrosteolysis I. An epidemiological study. Arch. Environ. Health 22:61-73. Dirksen, E. Roter and T, Timothy Crocker. Ultrastructural alterations produced by polycyclic aromatic hydrocarbons and rat tracheal epithelium in organic culture. Cancer Research 28:906-923, May 1968. Gabor, S., M. Lecca-Radu, and 1. Manta. 1962. Certain biochemical indexes of the blood in workers exposed to toxic substances (benzene, chlorobenzene, vinyl chloride). Prom. Toksikol. i. Klinika Prof. Zabolevanii Klim. Etiol 1962:221-223. Gessner, P. K. 1973. Effect of trichoroethanol and of chloral hydrate on the in vivo rate of disappearance of ethanol in mice. Arch. Int. Pharmacodyn. 202:392-401. Kal'manovich, F. L. 1968. Sanitary chemical characteristics of polyvinyl-chloride floor coatings. Hyg. Sanitation 33:274-280. Kaplan, H. L., N. C. Jain, R. B. Forney, and A. B. Richards. 1969. Chloralhydrateethanol interactions in the mouse and dog. Tox. Appl. Pharmacol. 14:127-137. Kogan, A. K., V. N. Tugarinova. 1959. The blastomagenic effects of polyvinyl chloride. Voprosy Onkologi 5:540-545. Palekar, L. M. Kuschner and S. Laskin. The effect of 3-methylcholanthrene on rat trachea in organ culture. Cancer Research. 2!3:2098-2104. October 1968. Sellers, E. M., M. Lang, J. Kock-Wese, E. Le Blanc, and H. Kalant. 1972. Inter action of chloral hydrate and ethanol in man. Clin. Pharm. and Therap. 13:37-49. Smyth, H. F. and C. S. Weil. 1966. Chronic oral toxicity to rats of a vinyl chloride - vinyl acetate copolymer. Tox. Appl. Pharmacol. 9^:501-504. Sokol, W. N., Y. Aelong, and G. N. Beall. 1973. Meat-wrappers asthma. A new syndrone? JAMA 226:639-641. Springer, E. 1965. Animal experiments on the toxicity of some vinyl chloride vinylidene chloride mixed polymerates and two stabilizers. Ztach fdg Hyg. ihre Grengebeste 11_:442; Bull, of Hyg. 40:1358. Suciu, I., I. Drejman, and M. Valaskai. 1963. Investigation of the diseases 'produced by vinyl chloride. Med. intern; (Bacharest) 15:967-978. Torkelson, T. R., Oyen, F., and Rowe, V. K. 1961. The toxicity of vinyl chloride as determined by repeated exposure of laboratory animals, Amer. Indust. Hygiene.Ass. J. 22:354-361. Tsuchiza, T. and Sumi, T. 1967. Thermal decomposition products of poly(vinyl) chloride. J. App. Chem. 17:364. Viola, P. L. 1970. Pathology of vinyl chloride. Med. Lab.* 61:174-180; J0M 13:156, 1971. Viola, P. L., A. Bigotti, and A. Caputo. 1971. Oncogenic response of rat skin. L V lZ ll SSU R&S 112118 lungs, and bones to vinyl chloride. Cancer Res. 31:516-522. Wohl, M, G. and R. S. Goodhart. 1968. Modern nutrition in health and disease Lea and Febiger, Phil. \