Document 713Nb34pakBxMOQxop5L4dvNB

cir Chemical Manufacturers Association FILE COPY April 28,1997 James Swenberg, D.V.M., Ph.D. CB#7400, Room 357 Rosenau Hall Department of Environmental Sciences and Engineering School of Public Health The University of North Carolina Chapel Hill, NC 27599 RE: CMA Reference No. VCHC-MOLE-RES-SWENBERG Dear Dr. Swenberg: The Chemical Manufacturers Association Vinyl Chloride Health Committee has reviewed your proposal uProtocol for Molecular Dosimetry Studies on Vinyl Chloride" dated May 7, 1996 and the protocol entitled "Studies to Evaluate the Formation and Repair of DNA Adducts Induced in Adult and Neonatal CD Rats by Vinyl Chloride" dated February 17,1997 that was developed in conjunction with Huntingdon Life Sciences Laboratories of East Millstone, New Jersey. I am pleased to confirm our gift of $50,000 in support of the proposed investigation on vinyl chloride in 1997. Funds will be provided in two payments, A payment of $25,000 will be made upon execution of this letter. A second payment of $25,000 will be made on July 15, 1997. We request that this gift not be applied to your indirect costs. In making this gift, we ask the following: 1. We expect the outcome of the research program to be published in one or more peer-reviewed publications. We request the opportunity to review drafts of any publications made by you or your colleagues related to the work. CMA opportunity to review and comment is for the purpose of clarifications, format or editorial comments, and not for the purpose of substituting CMA's position for that of the researchers. Publications should acknowledge CMA sponsorship of the work. 2. We would like 50 reprints of any published papers to be provided to CMA, for distribution to other researchers and government officials. 004612.001 Innovation, Technology and Responsible Care At Work l&ResMnstteCn' 1300 Wilson Blvd., Arlington, VA 22209 Telephone 703-741-5000 Fax 703-741-6000 1 f AnufcCamnftnnt Dr. James Swenberg April 28.1997 Page 2 2. We would like 50 reprints of any published papers to be provided to CMA, for distribution to other researchers and government officials. 3. We ask you to be aware of the potential reporting obligations for member companies of die Vinyl Chloride Health Committee may have under Section 8 (e) of TSCA regarding certain health or environmental risks that may be identified as a result of your work. CMA assumes no legal liability or financial obligations other than the payments indicated above. Ifyou are in agreement with the terms of the gift, please sign both copies of this letter, retain one for your files, and return the other to Wendy K. Sherman, Manager of the Vinyl Chloride Health Committee at CMA. On ail correspondence concerning this gift, please refer to CMA Reference No. VCHC-MOLE-RES-SWENBERG. If you have any questions, please contact Ms. Sherman at (703) 741-5639. / Accepted By: University ofNorth Carolina at Chapel Hill 004612.002 LAB OF MOL CARCINO Fax:919-966-6123 May 7 *96 .19:12 i i P.02/07 PROTOCOL FOR MOLECULAR DOSIMETRY STUDIES ON VINYL CHLORIDE James A. Swenberg, D.V.M., PhJ). University ofNorth Carolina May 7,1996 Listed below is a series of studies on the formation and repair ofDNA adducts induced by vinyl chloride (VC) that will provide a vastly improved understanding ofthe mechanisms of vinyl chloride carcinogenesis. These studies will provide information relevant to cancer risk assessments of vinyl chloride. The studies would be conducted in a manner that will complement the two generation reproductive and developmental toxicity studies that will be conducted at Huntingdon Life Sciences Laboratory. Dose-response Studies Presently, we have datashowingthatpreweanling rats are more susceptible than adults to VC carcinogenesis, that preweanlings develop 3-fold greater numbers of VC DNA adducts, and that preweanlings have 3-fold higher expression of CYP 2E1. The adduct and carcinogenesis data are all from "high" exposures of-500 ppm. No information exists on lower exposures. In addition, we have clearly demonstrated that DNA adducts identical to those formed by VC are formed endogenously in unexposed rats, mice and humans. We have developed ultrasensitive and highly specific assays for the DNA adducts ofVC. These include imnumnflffinity^P-pogtlnhleing methods for 1 ^-ethenodeoxyadenosine (EdA) and 3,N*-ethenodcoxycytidinc (EdC), and GC/MS methods for N^-ethenoguanine (EG). By adding animals to the reproductive and developmental toxicity studies, we will be able to assess the effect of exposure concentration on 1) the molecular dose of etheno adducts, the primary promutagemc DNA adducts ofVC; 2) determine ifthere are differences in the dose-response between adult and weanling rats; 3) examine the m^jor DNA repair pathways for VC DNA adducts; and 4) characterize the utility of using DNA adducts excreted in the urine as a biomarker of exposure. 1 : 004612.003 LPB OF MOL CflRCIwf Fax:919-966-6123 May 7 *96 19:12 l i i t 1I P.03/07 Molecular Dosimetry Studies in Nonpancnchvmal Cells fNPO versos Heoatocvtes Understanding why VC targets the endothelial cell for its carcinogenic effect is critical for proper risk assessment We have preliminary data on EG in vinyl fluoride-exposed rats (2500 ppm, 6 hr/day, 5 days/wk, 4 wks) which shows that the number ofadducts is ~2.5-fold higher jn NPC than hepatocytes, even though metabolism of VF to its electrophile is thought to only take place in hepatocytes. Since the NPC are exposed by diffusion ofVC or VF electrophiles from neighboring hepatocytes and therefore have lower exposures, this suggested that NPC were deficient in DNA repair activity for EG. To test this hypothesis, we have developed a RT-PCR method for quantitating methylpurine-DNA glycosylasc (MPG) in tissues and cells. Preliminary data have shown that NPC have <20% ofthe MPG activity present in hepatocytes. In addition, we have shown that NPC have 5-fold greater numbers ofabasic sites in their DNA. Abasic sites are formed when MPG excises a damaged base, such as EG, EA and EC. These data suggest that NPC are deficient in a second step in the DNA repair of VC adducts. We think that the most likely candidate is a deficiency in AP endonuclease. The difference between hepatocyte and NPC abasic sites was seen in rats exposed to VF, but not in controls. The combination oftwo defects in DNA repair in the target cells for VC carcinogenesis provides a strong scientific basis for the induction of hcmangiosarcomas by this important chemical. In view of the feet that the increase in abasic sites was only seen in highly exposed rats shows die need to understand the dose response relationship for this endpoint. By conducting 4 week exposures to 0,10,100, and 1000 ppm VC and measuring etheno adducts, MPG expression and abasic sites, we will be able to determine ifnonlinearities exist in the molecular dose ofVC, in MPG expression, and in the number ofabasic sites present in the DNA. Our hypothesis is that EG will show a supralincar dose response; that MPG will he similar in exposed and unexposed hepatocytes, but lower in both exposed and unexposed NPC; but that abasic sites will exhibit a sublincar dose response characteristic of saturation, of DNA repair in NPC, but not hepatocytes. 2 004612.004 LAB OF MOL CflRCINO Fax .*919-566-6123 May 7 '96 19:13 P.04/07 StudieaJMngXSCJrYC Our data showing endogenous formation ofetheno DNA adducts that are identical to those induced by VC have important implications. We had previously published a paper that concluded that the DNA adducts ofVC were highlypersistenL We nowbelieve that this was not correct, in that what we were calling persistent DNA adducts were actually the endogenously formed adducts at steady-state. By utilizing [I3CJ-VC, the formation and repair of VC-induced DNA adducts can be studied relative to those adducts formed endogenously. I believe that these studies will critically impact on any risk assessment dealing with low exposures, such as might be associated with accidental releases that reach the fenceline. We will examine the amount of [,3CJ-VC induced EG in rats exposed to 10,100, or 1000 ppm for 5 days (6 hrs/day). These same animals will be housed in metabolism cages for 3 days prior to exposure and during each day's post exposure holding period (days 1-4) so that urine can be collected. The urine will be analyzed for endogenous and induced DNA adducts that have been repaired by the MPG pathway using GC/MS. We will also develop immunoaffimty-LC/MS methods for all three etheno, bases, hi addition, it may be possible to identify and quantitate [`'CJ-VC metabolites in the urine using NMR. A second set of t13CJ-VC exposures is contemplated that would consist ofsingle exposures. These will be designed after we get data from the above study. Significance We have developed highly sensitive and specific assays for the promutagenic DNA adducts ofvinyl chloride over the past 10 years. By exposing additional animals in the Huntingdon study, we can use these methods to answer several questions related to improved risk assessment of VC. First, the molecular dosimetry data will be the only information of its type that addresses doseresponse. All other data have come from high exposures of500-600 ppm. Second, we will address the issue of differences between weanling and adult animals. While we already know that preweanling rats are more sensitive than adults to the formation ofDNA adducts and carcinogenesis at high concentrations, it is not known ifthis is true for low exposures. Since the only VC exposures that childrenencounter would be extremely low, the 10 ppm data are ofparticular importance. Just 3 004612.005 LflB OF MOL CfiRCINO Fax:919-966-6123 May 7 '96 19:13 ! I P.05/0? i it because prcweanling rats exposed to concentrations that saturate metabolism develop more adducts does not necessarily mean that young animals exposed to low concentrations will also develop more DNA adducts than adults. Our recent development ofan assay for MPG and the finding that NPC are deficient in this DNA repair pathway provides the first mechanistic data that explains why VC causes hemangiosarcomas. The additional finding of greatly increased numbers of abasic sites in the NPC DNA further highlights fire importance of cell-specific differences in DNA repair. Hie experiments outlined above will provide compelling data on the importance ofDNA repair in VC carcinogenesis. Finally, the use of [l3CJ-VC will allow us to differentiate between induced and endogenous DNA adduct formation. If large numbers ofinduced EG are formed, they may lead to increased numbers of endogenous adducts due to competition for DNA repair. On the other band, we will be able to determine the extent of increased DNA damage relative to endogenous adducts for all three exposure groups. For example, we might find that 10 ppm VC for 5 days only doubles the amount ofEG present endogenously. Such a finding should have great impact on low dose risk: assessments. We would then look at various single exposures to simulate fcnceline exposure scenarios to determine ifthere was any detectable increase in risk. Similar studies could be used to mimic occupational or environmental exposures to VC. Protocols Study I. Adult Rats Exposed to Vinyl Chloridefor 1 Week Route: Whole body exposure conducted during file two generation animal portion ofthe main study Exposure groups: 0,10,100, or 1000 ppm No. ofrats/group: 32 Total: 128 Exposure duration; 1 week (6 hr/day, 5 days) Spccies/strain: CD Rat Age: Approximately 12 weeks of age Sex: Males Sacrifice: 8 rats/ group at end of last exposure (non-perfo$ed), and 8 rats/ group at end of last exposure and at 3 and 7 days post-exposure (perfused) Urine collections: during Days 1-4 of exposure (collected during nonexposure times) 4 004612.006 LflB CF MOL CflRCINO Fax:919-966-6123 May 7 *96 19:14 P.06/07 Study It, Weanling Rats Exposed to Vinyl Chloridefor 1 Week Route: Whole body exposure conducted during the two generation animal portion ofthe nw^ study Exposure groups; 0,10,100, or 1000 ppm No. ofrats/group; 32 Total: 128 Exposure duration; 1 week (6 hr/day, 5 days/week) Species/strain: CD Rat Age: Approximately 21 days ofage Sex: Males Sacrifice; 8 rats/ group at end. of last exposure (non-perfhsed), and 8 rats/ group at end of last exposure and at 3 and 7 days post-exposure (perfused) Urine collections: during Days 1-4 of exposure (collected during nonexposure times) Study ni. Adult Rats Exposed to Vinyl Chloridefor 4 Weeks Route: Whole body exposure conducted during the two generation animal portion ofthe study Exposure groups: 0,10,100, or 1000 ppm No. of rats/group: 32 Total: 128 Exposure duration: 4 weeks (6 hr/day, 5 days/week) Species/strain: CD Rat Age: Approximately 12 weeks of age Sex: Males Sacrifice: 8 ratsi group at end of last exposure (non-perfused), and 8 rats/ group at end of last exposure and at 3 and 7 days post-exposure (perfused) Urine collections: during last week ofexposure (collected during nonexposure times) 5 004612.007 LAB OF MOL CflRCINO Fax:919-966-6123 May 7 *96 19:14 P.07/07 Study IK Adult Rats Exposed to ^CJ-Vinyl Chloridefor l Week Route: Nose only exposure Exposure groups: 10,100, or 1000 ppm No. of rats/group: 8 Total: 24 Exposure duration: 5 days (6 hr/day) Species/strain: CD Rat Age: Approximately 12 weeks ofage Sex: Males Sacrifice: 8 rats/ group at end of last exposure (perfused) Urine collections: 3 days prior to exposure and Days 1-4 during exposure (collected during nonexposure times) 6 004612.008 Report Title and Contract Nuaber Epldebtologlcal Study of VC Markers (VC 1.4* Chronic Vapor rnhititten Study with Vinyl Chloride fVC 2.1) VC Pathology Report (VC 2.0 Pathology Report on the Brilni Cron Rice, Hamsters, and Rata Rapoasd to VC In IBT Study (VC I.M) Validation of Strip Chart* at IRT (VC <.OB) Sunnary of the Studla* Conducted on the Phsraacoklnsttea/Metabollsn Ol VC tn Rata (VC 3.1) Bffacte of Maternally Inhaled Vlayl Chloride oa tmbryoaal i ratal Davalopnant 1* Mica* Rate and Rabbits (VC 3.2) Raaaarch Techniques and Methods Cor RetactIon and Prevention at Carcino genesis In Industrial Workers (fr, 1) (VC 6.11 Raaearch Techniques and Methods for Detection and Prevention >f Carclaoqeseals in Industrial Health Workers (Tr. 1) fvc 6,5) Research Techniques and Methods for Oatactlon and Prevention of Carcino genesis in Industrial Workers (VC (.7) VIHVL C'llLOHlOE Contractor TaherehawCooper. Aasoc. laduatrial BIO VEST tabs., Inc. Ecperlaental Pathology tab* Experimental Pathology tabs Contract foecutlon Bale-------- April 25. 75 reb. , 71 Feb. 2. 7* April 15, SO Bob West Assoc. The Dow Chesical Conpany The Dow Chesical Conpany April B, SO Hov. I, 76 Aaq, 19, 71 Valvarsity of toulavllla Dec. 12, 76 University of Louisville University of Louisville Peh. 27, 7B March 23, 79 Present Level ol fond tag 42,SOS 110,450 3,617 16,460 4,000 115,000 224,090 191,771 252,620 247,704 600Z19P00 Consent* fanunt Released Peb. 23, 7S llfl Ternteated JBr Released Nay 30, 10 II.B Released May 30, 80 S.B- 23 pp. II. 31 Released Sept, 2, 77 |l.78* S5.K* Released May 22, go 17 pp $1.7* Released May 22, St U pp. 61.10 In Progress yrr-i rc^a