Document YGwZ11n8bqwj5eRG0gVMOXBQ0

r FINAL DRAFT RcC'iVj.J 11/7/7.1 Contract No, Cl 76-0401 Toxicologic Investigation of Vinyl Chloride with Relevance to Non-*worker Populations: With Emphasis on Transplancental Carcinogenesis end Co-factors UU&tA * Binghan/Macchois, Ph.D, Principal Investigator University of Cincinnati College of Medicine Cincinnati, Ohio 45219 aayraona k. su^y.irui, h,d. Director, Capartaant of Environmental Health University of Cincinnati College of Medicine Cincinnati, Ohio 45219 Robert S. Daniels, ii.D. Interim Dean Univerlsty of Cincinnati College of Medleina Cincinnati, Ohio 45219 qr Robert T. Binhasacr, Ph.D. Assistant Dean, College of Medicine 1 APPENDIX F UCC 094846 Toxicologic Investigation of Vinyl Chloride vith Relevance to Nen-vorker Populations: vith ccphnsis on transplacental carcinogenesis and co-factors. Principal Investigator: E. Bingham Matthcis, Ph.Ji. tnrhnround * Vinyl chloride monomer <\'CM) is & chemical of increasing industrial and cnviroiwsnul importance, VCM hn* many uses, o,g. the production of poly vinyl chloride (PVC) rosin, as a co-polymer in saran and other plastics, as a solvent, as a propellant for pesticides and hair-sprays, as a refrigerant, and, in the past, as an anaesthetic. Despite the apparent usefulness of this chesical, 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 experiments vith various levels of VCM perforat'd by Mai toni and in Italy and by iotas t laboratories, Northbrook, Illinois have demonstreted that simi lar rare neoplastic lotions can be induced in experimental nnlstals, A great need exists for data on toxicity, persistence and for the assessment of risks associated with low level concentrations of VCM, inol^.nr." those levels that are likely to exist beyond the fence line of the :.:atw;'.".of urine plant. Materials loss in PVC production processes hns been round to be approx imately 6?,, but this varies vith typo 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 looses occur as air emissions and as components of water effluent and solid wastes, A recent report indicates that a vaan, living downwind fron fi pvc plant and having r.o occupational exposure to this substance, developed hepatic anftiosarcoina. Russian scientists have detomined that exposure to VCM rr.y be very widespread. It has been found, in a study performed in a newly constructed UCC 094847 building for child care, that VCM is continuously released freo PVC flooring for nt least two tor.ths after installation, they have recommended excessive Ventilation of a newly constructed building be nandatory before occupancy. Iho 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 segment? of the population, in addition to tho industrial workers, way 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. Xt has been reported that the enaymes responsible for ethanol metab olism may be involved in the metabolise of VCM (Cehrlng, 1974) since the effects of other chlorinated hydrocarbons, euch as trichlorenthylenc. and chloral hydrate, which are biologically transformed by alcoholic dehydrogenase are potentiated by ethyl alcohol, it appears thc comparable effects ray oceur with VCM, Xbc*c investigations r.ay 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 vinylidcnc chloride. Since both of theac co-polymers have similar properties and structures, there is a need to investigate what effects this combination nay have on the bio logical outcome. Purpose The purpose of this study is threefold: UCC 094848 a) To investigate the effects of VCM on the developing fetus through inhala tion exposure in an effort to determine the risk of developing ontogenle lesions via the iransplaccntal route and to determine the effects of inhalation exposure to VCX on the neonate. b) To dotevriine Aether certain dietary factors such ae vitamin C deficiency _ or ethanol consu^tion ray influence the biological response to VCM. c) To develop cell culture techniques useful for the. rapid screening of potential C oncogenic agents, such as VCM and co-factors* Iheec techniques will be uscc to provide the meant for studying mechanisms of the actiou of similar agents and to serve as an indicator of dose-effect responses. Scoop of"Berk A* Inhalation exposure of pregnant cntoals to level; of VOX tu determine the potential transplacental oncogenic and other bio)os-.leal effects. Sprague-Pawley rats will he obtained from Charlee River Laboratories cither rlinod-pregnant or they will be bred In our un.b-al facilities, In the first experiment, twenty pregnant tats will he cy.pot.cu to 20,ODD ppm vinyl chloride, or as agreed upon with the contract officer, in an inhalation r-h&rbur from the 10th day of fcstatlon to the tire of delivery. After hijih, litters of five young will rennin with each Mother, Additional you.-.y `will he uscri licet! and cleared by the Schultz-Doveon csethod to visualise 1!k shell on for osr.ec.uF chouses. Hie mothers and their surviving young will be fcopt is-, box cages until vanning 0 at four veths of age. Then the. mothers and their young will be fed Furins Lab Chow and maintained in rooms controlled for tcr.pwtirurc, humidity and light cycle. The 100 surviving young will be examined for congenital r.alfornations end then observed for signs of tumors and other diseases, particularly acroosteolysis. X-ray excninacion will he done in selected cases at various tines. Observation!; nay have to he continued for one to-two years. After weaning of the first batch of young from exposed nothcr.*, another batch of twenty pregnant fcstales will ho subjected to a similar vinyl chloride treatment. In this experiment, ten Mothers with their litters of five UCC 094849 young each will be handled like the anil talc 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 oi mothers and offspring will be treated like`the first end 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 n room with controlled lightipg cycle, temperature, and humidity, and fed liquid diets gd libitum (16 hours/day). Hie liquid diets (General biochemical, Chagrin Falls, Ohio) will be purchased in dry form and reconstituted as needed, Ihe diets will consist of a control diet (without ethanol) and the same diet supplemented with 5* ethanol Appropriate controls will be used* In addition to rats fad an ethanol supplemented diet and exposed to VCM, guinea pigs on vitamin C deficient diets and their controls viVj be used. Ihc protocol involving guinea pigs will serve a dual purposei a second species susceptibility to VCM and the co-factor response to vitamin t' All exposures will be maintained for 12 months, 5 dsya/wvob, 4 houra/day to the VCM, or os agreed upon by the project officer. In addition, rats will be exposed to VCM and vinylidcne chloride at levels agreed upon with the contract officer. Ibe exposure chambers will bo located in a large room which has been previously used for human inhalation exposures. This roo?, is very suitable for the proposed testing since it is sealed to insure against leakego. Hie main chamber has a double door entry. Ibis chassbnr in a chamber arrangement will UCC 094850 sub;tr.r.3K|'Viftrlc pressures to insure against leakage. Air flow in the outer Chamber is in a ct-iling to floor direction with a complete air change every four minutes. Makeup air for the exposure chamber and negative pressure room Will N> prcfilrered through absolute filter*. Periodic sampling of both cham bers using Sipin pumps and activated charcoal tubes will be nadfc. lo addition, appropriate protection of personnel will be provided, Including mandatory wearing of full face gas masks vith 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 ch&nber and the negative pressure roor. will be removed from the nir using a fabricated activated charcoal filter. Spent activated charcoal will be removed from the filter by por&onnel, 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 a.-.aunt of exhaled VCM from exposed animals and rh tiws at which they can be moved to houcing quarters after exposure. In r.he event the exhaled air concentrations arc. unacceptable, the animals will ba housed in suitable quarters, which are isolated from all personnel. 1* 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 method* could ba used for the analysis of VCM including a halide merer, long pathlength (Brooks-Koran) IP. spectrophotometer or a gas chromatograph with a flame ionization detector. The GC is the method preferred, facilities and personnel experience in analytical UCC 094851 procedures are available for this project* The first year will be entirely devoted to Betting up, calibration and exposing the pregnant aninals from Part A, bo additional animals {cn various dietary regimens) will be exposed during the first year* The second yep.r will be devoted to inhalation exposure at one or two different levels of VO!, c.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 ns shown in the following table. Suitable control animals will also be used. Diet Dumber of Anir-ale 1) Vitamin C deficient AO X 2 2) Vitorrdn C sufficient/ guinea 3) Vitamin A deficient || Pig* AO X 2 40 A) Vitamin C sufficient,. A0 VCM Exposure! (ppr) 50, 500 50, 500 Done Done 5) Control diet with n 57, Cthanol j 6) Control diet 1 n Control diet with 1 rats 5% Ethanol f 8) Control diet 1 A0 X 2 A0 X 2 A0 A0 50, 500 50, 500 Done Done 9) Control diet AO VCM + Vinylidctie Cl. During the third year animal exposures and observations will be continued, oe based on previous data. The animal# must be kept under obser vation for one to two years, C. Tissue and blood samples from, tho exposed animals (part B) will be oollcictcd and transported to ETT.L, KERC - Cincinnati for clinical chemistry and pathological determinations with special emphasis on electrorcaicroscopy. UCC 094852 The number of animals to be used will be determined as agreed upon by the project officer, Hie current figures represent ten animals fro:* each group for the sampling. This portion of tho work will begin the second year or where appropriate the first year as agreed upon with the project ofiicer, D, Tissue CulLurc Lab - Proposed for Second Year and Beyond The malignant potential of clinical carcinogens has been demonetrated 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 ceil sysien; 2. mouse cell lines; and 3* the 3X3 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 A'C:*; will be added to the cuKruas in the gas phase, in a mixture of % CO^ in air. The design of the chamber and the concentrations of VC.*: will be determined in con sultation with the contractor. It should be possible to obtain a dose respoma* curve in this system with concentrations between 5 - 20,000 ppm Vf:M. The celir will be exposed for seven days, allowed to grow for an additional 8-10 day:, in control medium and then fixed and stained. Determination of cytotoxicity and examination for transformed ccIIe is dona in the same dieh. Cytotoxicity is determined as a percentage o cells exposed to the agent ogeinet control. The host mediated assay is of particular interest to this protect because VCfl is a gas and this is probably the route of entrance into the ani vil or man. This project is also interested in the effect of this agent on the embryo. In Lhis assay the pregnant hartster or rat will he 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 form t ion as above. These techniques have proved quite satisfactory for the study of a Ucc 094853 ti-.Li-.k'/jwiHL OtiU i*W4t* l*L. 4-Jli? UVl-h **14*>*-*< ^ *44 but because of the unusual nature of this carcinogen scver.il other techniques Vill be used in consultation with the contractor. Endothelial cells ore avail able for exposure to VOS ns veil as Inman fibroblastic cells* These vill be used In teste Bfoilar to those outlined shove. A somvhet different, but very Interesting approach Lo this studyf vill be .the observation of hyperplastic or prcaalignant changes of tissues in organ culture, lhese otudleB vill be elxailar to Lhosc previously reported by Dirksen and Palekar.on rat trachea. Eat blood vessels and email pieces of rot liver will be grown in organ culture in an exposure chnoher with an atmospheric mixture of VC'I, CO^ and air or 0^, The tissue vill bo placed on rayon rafts and floated on nedium in organ cul ture dishes. The tissues will be fixed at weekly interval', for five weeks and provided to the contractor for potbologieal examination. Tnese changes coy be similar to changes in the rots exposed to VC" in the inhalation checkers. Periodic reports vill be submitted to the project officer on the......... statue of the experiments. racimifsThe deportment possesses extensive nnirial quarters used for both short and long range toxicologic end rxtcbolic studies on t`,rpcrimontal animale. A special area war. recently constructed permitting the maintenance.of anirsls 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 cost laboratory animals. Large air UCC 094854 v the carcinogenic rccponse of skin to various carcinogens (pure, complex *r.tx' cocorclnof.cns, etc.), inhalation studies using coal tar we currently unden Five stainless steel, rectangular exposure chamber* (Young and Bet Co., Cincinnati) arc operational. Each has a cone shape cop and batter) and *3 capacity of lit ft. (340L). In addition, a larger chamber ( 26 ft. ) of th ':-.4 * j" :j r.J acme design te available, These chcnbsre are normally operated at airflows t permit one exchange of air per,minute, Eacl) chamber can accomodate 60 rats rats capacity - large chamber) or 90 hamsters or 9 rabbits in individually h stainless steel holding cages. After the primary air supply is drawn through an absolute filter t cont&sinent Is added and introduced into the chamber, which permits very uni dispersion as deremitted by tests in the laboratory, Tnc chambers are opera under eubcrr-osphuyic pressures and the exhaust air is paused through either absolute filter, charcoal filter, or a water spray to remove the contaminant exhausting tc the atmosphere. In addition, four other types of ehar.ber* are. used, One hexagonal Stainless steel chamber with a capacity of 703 liters is operated in a rrinnei Similar to thrt described above. Four it liter capacity sesihe-mispherical g; chambers are also employed. This particular type ir, useful for range finding , tlgation as well as for chronic exposures. Each chamber can accomodate up tc % 10 rata. Three chamber rec-as, each 12.5 ft.'long X 7.5 ft, wide X 9 ft. high A with a volume o hjO ft. (24,700L), can singularly accomodate two large cage rocks for 120 individually housed rata* Air.is circulated to these chambers m rate of 150 ft, par minute to each chamber by caens of two blowers (site C fuaerricoft Blower Co.) with absolute filters positioned on both the inlet and e ducts. These chambers arc also kept under a slight substnosphcric pressure. Contaminant generation equipment for inhalation chamber's include#: 1) Dust goneretion * 7 Kright Dust Fccdorc (l, Adams Ltd,. London, England) '4 'X equipped with cyclones and 15 dost tuber. A supply of replacement parts :rn is maintained. UCC 094855 2) Aerosol gentracing equipment including Aspirating equipment'such as that used for coal tar inhalation. 3) Fines - Various technique* as used for the generation of metal fires* 4) Vapors - generated by modified Crctnb;rg-S.-ith Implngors. 5) Caseous exposure using various regulator valves and orifaces. All chambers have-indicating t.*.onoTMr.terc (Hsgnchelic) to icoa&urc subataosphcric prosr-urefl. Air flow is monitored with cither stainless steel orIfaces or rotometers, t Other air flow calibration and monitoring equipment Includes: 9 Rockwell dry te6t voters (175 ft, 3/hr, capacity); one calibrated nastcr 140 ft. 3 test cetera; one vet test meter (3 literc/revolution) for calibration of low flow rate rotoxfeters. Routine sampling of the contaminant, etc. is porforusd hourly. Soopling equipment routinely used includes! 12 1I3A midget bubblers, Greenbcrg-Srith le-piii" gors with fritted <J1ec; 7 Anderson cascade lupoctort for particle site distribution; nd various filter holder samplers for gravimetric and/or analytical procedures. In Addition the industrial hygiene and analytical departments have a fairly complete And varied Hoc of supplementary sampling equipment that is readily available, thirteen gast piep <S*103ll, 4-0522, 1-0822) of various air flow capabilities And many other standby pumps are used routinely. I UCC 094856  I-v,:-- hcrpvcin, K., U'7C. Pure hydrocarbons, liquified pvopcHants and vic.yl chloride * 6& aerosol yirpt Hants and mixture*, lUfswilk-i'firfurdrogen Uundsrhan R:20-22, Dirt-can, B, J>,, V. A, Cook, V,', K, Hhiitl.ouse, II, J, Munition And 7. DUeheck, 1971, Occupational iierosUolynls l* An rpidc.r.-.iologiciil study, Arch. Environ. Health 22:61-73. Dlrkstn, E. ho ter find 7. Timothy Crocker. ISltrntitmetural alterations produced by polycyclic areiirttie hydrocarbons find rat Lrachcitl epithslius in organic culture. Canter Research 2:i*`06-923, May 1966. Gabor, B,, 15, Utce-Rcdu, end 1. Hftnta. 1962, Certain blochtr.icd indexes of the blood 11. workers exposed to toxic substances (benrene, chloreb-r.cer.c, vinyl chloride). Frro. Toksikul. i, Klinika Prof. ZtboUvf.nii Klim bliol 1962:221-223. Ces&ner, P, K. 1973. Effect of trirhoroetlienol and of chloral hydrate on the in vivo rate of disappearance of ethanol in rice. Arch, lnt, Phatv.acodyn, 202:392-401, Kal'fcfinovich, T. L. 196B. Sanitary chemical characteristics of polyvinyl-chloride floor coatings, i!yg, .Ssnicatior. 33:274-260, Kaplan, H, N, C. Jain, Y,, f>. Forney, and A, R. kidiads, 1969, Chlorrtlhydrate- cth&nol into .fictions in the mouse and deg, fox. Appl, i':.air.acol. 14:127-137, Kogan, A, K,, V. H, Tugcrinova. 1959. 'ihe blaster.'genic effects of polyvinyl chloride. Voprosy Ottkologi b\ 540-545. Pol char, L. 1'., Kuschnar and S, baskin, Ihc effect of 3-r ethylchol.r,;thronn on rut trachea ir; organ culture. Canter Kcseavch, 29; 20`,;b-2H>4, Or. taler 196b. Sellers, E. It,, IS, bang, J. Kock-Wesc, E. be Blanc, rind !i. K&lam. 1972, Inter action of chloral, hydrate and ethanol in nan, Clin. Pham, *.nd IViersp. 1,3:27-49, ficyth, H, F, find C, S, h>*l, 1966. Chronic oral toxicity to rats of a vinyl chloride - vinyl fleet tie cepolywsT, box, Appl, Pherr jirol. : 5=01 -604, Sokol, W. K,, Y, Aclong, fj-.d C, ft. Refill. 1573, Mott-vrapfore estrera, A new cyndronft? JAliA 226:639-641. Springer, E. 1965. Animal ewerirents on the toxicity of sow vinyl chloride - vinylidcne chloride nixed polymeratv* end tvo stobiliters. JJtcch fdg Kyi-, ihre Grengebeste U:442; bull, of Hyp,. 40:1355!. Suciu, I.. I, Drc-Jnaa, and K, Vnleskai. 1963. Investigation of the diseases produced by vinyl chloride. Ikd. intern' (Bacharesc) ,15/.967-976, Torkclsori, T, n., F., and Rove, V. K. 1961* The toxicity of vinyl chloride ss rirtrrrmod by repeated exposure of laboratory Animals. Amer. lnduat. Hygiene Ass, J, 22:354-301. I Tauchim, T. end Surd, T, 1967. Thermal decomposition products of pelYfvlnvll chloride, j. APp. Chem. U:364. ' 1 yv>in>A; Viola, P.'L. 1970. Pathology of vinyl chloride, lied, 3*sb. 61:174-160* JOv 12:156, 1571. " Viola, P, L,, A. Bipotti, and A, Caputo. 1971. Oncogenic response -f rat chin, UCC 094857 i/::3 pSTr*: ; 4%. 4 fe;-sa .............. t ... v ......................... / , . lungs*and bones to""vinyl chloride. Cancer Rc.p, ^1:516-522, *' 'Kohl, H, C. and K* $* Goodhort* ....tat-end VefcJ gor, Vhll, p .. ***.' *. is6S. Kodcrn nutrition In health and dleecse >ti>tii ***> ***** ' *'** fc. * ''vi;in*" , : . \",y- ] ;&>v ';;J . IV:? jfU*. ; * ~ -jf srj..- Hi IfpX ''.;if vi41.v* .-- v t ./a ws-sr-W C'il . ?^.i .'3 ;f -i Lt; ." /i^c .nv: *'* * - k.* *uU' >-t* y*. !+K'?t+i ./.t.u\u UCC 094858