Document GzrOJyJeErGbwp8XdvZRE045n

Chemical Manufacturers Association FILE COPE 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 "Protocol 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. Innovation, Technology and Responsible Care At Work 1300 Wilson Blvd., Arlington, VA 22209 Telephone 703-741-5000 * Fax 703-741-6000 ft Responsible Care' A Mmc Commitment CMA 120946 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 the 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. If you 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 all 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 of North Carolina at Chapel Hill CMA 120947 LJ|" l'1U_ CHKLif'tO 1 Q'v' 3i j flay . ao ia-** ...... LABORATORY OF MOLECULAR CARCINOGENESIS AND MUTAGENESIS THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL CAMPUS BOX 7400, ROSENAU HALL SOUTH COLUMBIA STREET CHAPEL HILL, NC 27599 PHONE 919-966-6142 FAX 919-966-6123 FACSIMILE TRANSMISSION To: Bob Venezia Address: CMA FAX number: 703-741-6091 From; James A. Swenberg, D.V.M., Ph.D. Date: May 7, 1996 Bob: Here is the- research- proposal/protocol. I made a few changes from our discussions in New, . Jersey. I sent a copy to Jim Knaak for his comments. He said that the issue of dynamic versus static exposures will be determined in the future. Dr. Rudy Jagcr has some dynamic exposure systems that will use even less than the 27 gm. As I do not have access to the needed information, I cannot go any farther today. I hope that this is what you need. If you have any questions, please give me a call tomarrow. CMA 120948 PROTOCOL FOR MOLECULAR DOSIMETRY STUDIES ON VINYL CHLORIDE James A. Swcnberg, D.V.M., Ph.D. University of North Carolina May 7,1996 Listed below is a scries of studies on the formation and repair of DNA 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 maimer 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 data showing thatpreweanling 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 immunoaffinity/J1P-postlablcing methods for 1 ;N*-ethenodeoxyadenosme. (EdA) and 3,N4-ethenodeoxycytidinc (EdC), and GC/MS methods for N2,3-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 promutagcnic DNA adducts ofVC; 2) determine ifthere are differences in the dose-response between adult and weanling rats; 3) examine the major 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. Molecular Dosimetry Studies in Nonparenehvmal Cells (NPC) versus Hepatncvtes Understanding why VC targets the endothelial cell for its carcinogenic effect is critical for proper risk assessment We have preliminary data on HG in vinyl fluoride-exposed rats (2500 ppm, 6 hr/day, 5 days/wk, 4 wks) which shows that the number of adducts is -2.5-fold higher in 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 of VC or VF electrophiles from neighboring hepatocytes and therefore have lower exposures, this suggested that NPC were deficient in DNA repair activity for G. To test this hypothesis, we have developed a RT-PCR method for quantitating methylpurine-DNA giycosylase (MPG) in tissues and cells. Preliminary data have shown that NPC have <20% of the MPG activity present in hepatocytes. In addition, we have shown that NPC have 5-fold greater numbers of abasic 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 arc 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 of two defects in DNA repair in the target cells for VC carcinogenesis provides a strong scientific basis for the induction of hemangiosarcomas by this important chemical. In view of the fact that the increase in abasic sites was only seen in highly exposed rats shows the 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 if nonlinearities exist in the molecular dose of VC, in MPG expression, and in the number of abasic sites present in the DNA. Our hypothesis is that EG will show a supralincar dose response; that MPG will be 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 CMA 120950 Uifc i_r I'ILl - I" JA 'ji J-i'DO_0 a i'loy ir r 'J J SimIig.Using.rjCz]^{ Our data showing endogenous formation of ethcno 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 highly persistent. We now believe 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 [`^CJ-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 ['^CJ-VC induced EG in rats exposed to 10, 100, or 1000 . pptn 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 GOMS. We whl also develop immunoafGnity-LC/MS methods for all three etheno. bases. In addition, it may be possible to identify and quantitate [`'CJ-VC metabolites in the urine using NMR. A second set of [I3CJ-VC exposures is contemplated that would consist of single 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 of vinyl chloride over the past 10 years. By exposing additional animals in the Huntingdon study, wc can use these methods to answer several questions related to improved risk assessment of VC. First, the molecular dosimehy data will be the only information of its type that addresses doseresponse. All other data have come from high exposures of 500-600 ppm. Second, we will address the issue of differences between weanling and adult animals. While we already know that preweanling rats arc 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 children encounter would be extremely low, the 10 ppm data are ofparticular importance. Just 3 CMA 120951 i-no .J- ,'ILu LHhuinC ra* 3j.n-30o-0i.-j lfidu 13 ` iw r. because prcwcanling 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 of an assay for MPG and the finding that NPC are deficient in this DNA repair pathway provides the first mechanistic dam that explains why VC causes hemangiosarcomas. The additional finding of greatly increased numbers of abasic sites in the NPC DNA further highlights the importance of cell-specific differences in DNA repair. The experiments outlined above will provide compelling data on the importance ofDNA repair in VC carcinogenesis. Finally, the use of ['^CJ-VC will allow us to differentiate between induced and endogenous DNA adduct formation. If large numbers of induced EG are formed, they may lead to increased numbers of endogenous adducts due to competition for DNA repair. On the other hand, we will be able to determine the exrent 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 fenceline exposure scenarios to determine if there 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 the two generation animal portion ofthe main study Exposure groups: 0,10,100, or 1000 ppm No. of rats/group: 32 Total: 128 Exposure duration: 1 week (6 hr/day, 5 days) Species/strain: CD Rat Age: Approximately 12 weeks of age Sex: Males Sacrifice: 8 rats/ 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 Days 1-4 of exposure (collected during nonexposure times) 4 CMA 120952 *r I Study II. Weanling Rats Exposed to Vinyl Chloridefor 1 Week Route: Whole body exposure conducted during the two generation animal portion ofthe main study Exposure groups: 0,10,100, or 1000 ppm No. of rats/group: 32 Total: 128 Exposure duration: 1 week (6 hr/day, 5 days/week) Species/strain: CD Rat Age: Approximately 21 days of age Sex: Males Sacrifice: 8 rats/ 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 Days 1-4 of exposure (collected during nonexposure times) Study III. Adult Rats Exposed to Vinyl Chloridefor 4 Weeks Route; Whole body exposure conducted during the two generation animal portion of the main 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 rats/ group al 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 of exposure (collected during nonexposure times) 5 CMA 120953 LMb UT IV1L1_ U-iKiilNlj rax-aia-ado-olio Ilay f SO 13 W i r . v* * \ iVwdy /K ^l^/zc/jr JZdtr Exposed to f3CJ-Vinyl Chloridefor I 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 of age Sex: Males Sacrifice: 8 rats/ group at end oflast exposure (perfused) Urine collections: 3 days prior to exposure and Days 1-4 during exposure (collected during nonexposure times) 6 CMA 120954