Document o9o74Ka4gLqy97r3XgXEGQOGD

To cc$ : K. Beutel : L. Rinzema C. Loefgren Rotterdam, 11th June 1982 Horgen Horgen Horgen R&S 008323 Re : VINYL CHLORIDE TOX STUDIES In addition to my telex of April 26 1982 and the Shell letter of May 11 (CMCF/1) I am coming back to the above subject. Our 1980 committment to pay 50.000 Dfl. to Shell as Dow's contribution to the project has not yet resulted in an invoice, but I think this will come soon. The progress of the Shell studies has been slower than expected. The main reason was that the major adduct, 7-(2'oxoethyl)guanine did not produce detectable levels of antibodies in rabbits. Therefore another detection method had to be developed. In this method an unknown quantity of unlabeled adduct is spiked with 14 C-labeled adduct. A subsequent reduction of the adducts in volves the incorporation of tritiated sodium borohydride. 3 14 The purified reduced material is counted for H and C. The ratio 3H/ 14 C will then give the amount of adduct in the sample. The method involved in identifying adducts will be at our dis posal if we continue over and beyond the 50.000 Dfl. to partici pate in this project. In the particular case of vinyl chloride the method will enable the detection Of the amount of DNA adducts, if any, at levels that are not tumorigenic in rats, with a sensitivity of less then 7 1 adduct in 10 DNA molecules. If at no-tumor levels there are no DNA adducts either, we can make a significant step ahead in dealing with a possible no effect level for -carcinogens. 2 2 In the case of vinyl chloride this would be important for the food packaging materials made of vinyl chloride. But beyond vinyl chloride these methods could be useful for other foodpackaging materials. I realise that a request for sponsoring may come at a bad time for a product that has no great interest for Dow Chemical Europe. Nevertheless I request that you as product steward discuss this proposal with the appropriate product business team. Our collea gues in the USA may be willing to consider to take over from us this additional sponsoring, because of more business interest and the methodology involved. I therefore recommend that you also contact Bemy Schwetz and the USA product steward for vinyl chloride. reviewing the proposal one should consider that the other 3 sponsors, AKZO / DSM and Shell have equally bad times in the plastic business and some moral obligation in these circumstances can not be denied. The appropriate amount for additional sponsoring would be 50 75.000 Df1. or 20 to 30 thousand dollars. I would appreciate an answer from you before August 1. H.G. Verschuuren Enel. 1. telex of April 26 2. proposal by Shell R&s 008324 R&S 008325 APPENDIX 1 2nd Interim Report Molecular Dosimetry Studies with Vinyl Chloride Monomer XIie objectives of the study are stated in the Protocol, dated lAth October, 1980, and were re-stated in the 1st Interim Report, dated 16th June, 1981. Tills report addresses the points, listed on page 5 of the Protocol, covering the procedure to determine the quantitative relationship between Vinyl Chloride exposure and DNA dose in rats in the CIVO study. Progress to date is summarised below. 1) Synthesis of deoxyrlboslde - vinyl chloride adducts The major adduct, 7-(2'-oxoethyl)guanine, and the two minor ones, 1,N^-ethenodeoxyadenosine and 3,N^-ethenodeoxycytidine, have been chemically synthesised and structurally characterised. 2) Development of an hplc method to separate the adducts Conditions have been developed using reversed-phase hplc under which the three adducts are resolved from each other and from the four natural deoxyribosides present in a DNA hydrolysate. 3) A high dose pilot study using ^C-labelled vinyl chloride This was not carried out for two reasons: a) -^C-Vinyl chloride of high specific activity is extremely expensive to produce, and its stability is not guaranteed. b) At the start of this study, Professor Bolt from the University of Mainz published the results of an experiment very similar to the one which we were planning, which gave us the results we required. 4) Development of methods to assay DNA - vinyl chloride adducts in tissues of exposed animals The main criteria for such assays are sensitivity and specificity, i Tlie level of DNA alkylation which can be detected following administration of a l^C-labelled carcinogen to a mammal is of the order of 1 adduct in 10? nucleotides, which gives an idea of the sensitivity required for the assays under development. The analytical method chosen to develop assays for the three DNA adducts of vinyl chloride is radioimmunoassay. The principle of this technique and the steps involved in its development are outlined in the 1st Interim Report. Since the DNA adducts are small molecules, they must first be bound to a carrier macromolecule in order to stimulate antibody production when Injected into animals. The two minor adducts were both successfully bound to bovine serum albumin., a carrier protein, by application of a andard method. A modification of the standard method had to be developed bind the reduced (stable) form of the major adduct to a carrier protein. This was successfully achieved, and c njugates of the reduced major adduct R&S 008326 * 2- - tu two carrier proteins, bovine serum albumin and keyhoLe limpet haemocyaiiln, and to a third carrier, activated CH-Sepharose 4B, were obtained. The chree adducts were also prepared in tritium-labelled form as a requirement for the radioimmunoassay procedures. The conjugates of the two minor adducts, and the three conjugates of the reduced major adduct were injected into rabbits in order to produce antibodies. Antibodies against the two minor adducts were produced in substantial quantities in a matter of weeks. These antibodies were characterised for sensitivity, avidity of binding to substrate and crossreactivity in a radioimmunoassay procedure. A modification of the radioimmunoassays for the two minor adducts, called ultrasensitive enzymatic radioimmunoassay was investigated, but didn't result in any increase in sensitivity. The levels of sensitivity achieved with , radioimmunoassays for these two minor adducts are such that In 10 mg DNA isolated from the tissues of exposed animals we can expect to detect one molecule of ethenodeoxyadenosine in 3 x 10 nucleotides and one molecule of ethenodeoxycytidine in 1.2 x 10^ nucleotides. The sensitivities of both these assays can possibly be increased by 1-2 orders of magnitidue using ^25i_iabenej substrates which we have recently synthesised. Two recently-published studies have described experiments in which ^C-labelled vinyl chloride was administered to rats! and mice^. In the rat study ther of the two minor adducts was detected in liver DNA. The limit of sit*ivlty for this study can be calculated at one molecule of adduct in 1.5 x 10? nucleotides. In the mouse study tentative identification of ethenodeoxyadenosine was made in liver DNA at a level of-one molecule in 2.4 x 10 nucleotides. Injection of the three conjugates of the reduced major adduct into rabbits did not produce detectable levels of antibodies directed againsc this adduct. This result was completely unexpected. A possible resson"is that on injection into rabbits, the slightly basic physiological conditions caused a chemical modification of the adduct. If this were * .he case then any antibodies raised would have been produced in response to the chemically modified adduct. It was thus apparent that the radioimmunoassay technique could not be applied to the major adduct and that an alternative assay would have to be developed, leading inevitably to a delay in the progress of the study. The assay currently being developed is based on a radiolabelled derivatisation procedure. The sample, containing an unknown amount of the adduct is 'spiked' with a known amount of ^C-labelled adduct of high specific activity. The mixture of labelled and non-labelled adduct is reduced with tritlated sodium borohydride, a reaction which will Introduce tritium into both species. The reduced material is purified to constant specific activity and counted for 3h and 14C. The 3h/14C ratio will give t^i^amounc of adduct in the sample. In theory this method will enable us t^petect one adduct in 1.5 x 10? nucleotides. In the rat study! mentioned above 7-(2'-oxoethyl>guanine was detected in liver DNA at a level of approximately 1 adduct in 7.5 x 10^ nucleotides. It is anticipated that a further 2--3 months of effort will be required to develop this assay to - 3a stage where it can be applied to samples from the C1V0 study. 5) Collection, of samples from the CIVO study Samples of liver, lung, brain, kidney and testis taken after six weeks, nine months and eighteen months from rats in all exposure groups in the CIVO study have been collected. These are currently being stored at -80C in Shell Toxicology Laboratory pending assay. References 1. R.J. Laib et al, Chem.-Blol. Interactions, y7_t 219 (1981). 2. K. Bergman, Arch. Toxicol. 49, 117 (1982). STL, 22nd April, 1982 R&S 008327 APPENDIX 2 Proposal for Project Finalisation Ub jeticJvtjs/Justification The aims of the studies described in this proposal are to bring the current project to an expeditious and satisfactory conclusion in respect of the principal objectives of the project. These objectives are as follows:- 1. To determine the quantitative relationships between exposure dose of vinyl chloride and primary DNA damage (amounts of DNA adducts) in the target (liver) and in selected non-target tissue(s) of rats. 2. To determine the quantitative relationships between primary DNA damage and carcinogenic response in rats (second CIVO/TNO study). The results of these studies would directly address Important issues such as the question of thresholds in respect of the induction, of primary DNA damage and also in respect of the development of neoplasia as a response to this DNA damage* These studies (including comparisons of DNA alkylation in liver and a non-target tlssue(s)) may also yield important insights into the mechanism of action of vinyl chloride in rat liver, e.g. the results may signify whether or not factors other than DNA alkylacion are operative in determining the response of rat liver to this ^^mical. Experimental Programme The proposed programme comprises three discrete studies to be conducted in the following sequence:-- 1. Completion of Method Development The development phase of the project will be completed by providing a method for the assay of the major DNA adduct (7-(2'-oxoethyl)guanine). The method is currently being developed and is based on double-radlolabelled Jerivatisation procedures. It is anticipated that 0.3 dmy will be required for the completion of the assay. (Immunochemical assays for the two minor DNA adducts (see 2nd Interim Report) have been developed). This aspect of the work will be reported to the sponsors during August, 1982. 2. Pilot Study The implementation of this study is dependent upon the satisfactory completion of item 1. Prior to commlting the valuable CIVO/TNO samples to analysis, it is important to gauge whether or not the amounts of DNA adducts in the livers of the CIVO/TNO rats are likely to be below the threshold for detection using the methods developed/described in item 1, a^re. Therefore, a pilot study Is proposed in which vinyl chloride will b^wlmlnis. ered at a dose of 1.7 mg Kg"* body weight to a group of five ma^ rats for five days by oral intubation. (The need or desirability of conducting analogous studies at lower or higher exposures will be reviewed at the appropriate time). Following exposure, the rats will be killed, livers excised and liver DNA isolated. The liver DNA from the exposed R&S 008328 R&S 008329 _0- diumala will be assayed for Che three adduces. An estimated 0.1 dmy will be required for this aspect. 1. Application of Methods to the CIV0/TN0 Samples following successful application of the three assays to the pilot study, the C1V0 samples will be assayed. The first tissues to be examined will be livers from the high dose animals for all three exposure tiroes. Assuming that detectable levels of adducts are found, then liver samples from the other two dose groups will be assayed. The feasibility of assaying other (non-target) tissues for DNA adducts will depend on the levels found in Che liver. Accurate estimates of effort are dependent upon information to be gleaned from item 1. However, it is anticipated that all liver samples and a representative number of non-target tissues from the CIVO study can be assayed for the three DNA adducts using 0.6 dmy effort. It is therefore estimated that the project can be concluded In 1982 in respect of the principal objectives with the expenditure of 1.0 dmy of effort. STL, 22nd April, 1982 R&S 008330 Toxicoloeical studies with Vinvlchloride When it became known that vinvlchloride (VC) was the susnected agent to cause liver cancers (angiosarcoma's) not only in experimental animals but also in humans occuoationally exnosed to VC by inhalation, limits were introduced to safeguard health. These included limits for food nackaging materials and foodstuffs themselves although VC had not bee. tested orallv. A feeding studv was then carried out at CIVO-TNO to attemnt to establish a no toxic effect level (NTEL) for regulatory nurooses. The study was initiated by the German Plastics Producers Industry federation (VKE) who invited other than German Comnanies to co-snonsor. AKZO, DOW, DSM and Shell joined. This study failed to yield a NTEL and the VICE had. to commit themselves to a second study to avoid the introduction of very stringent regulations on PVC by German authorities. CXVO-TNO again was contracted for the second study and VICE invited the same sponsors to narticinate. This second study, however, would be with much lower dose levels and the number of animals could not realistically be increased to take account of the statistical requirements. This, taken together with the fact that a "no effect level" for a carcinogen was not accented in nrincinle by certain scientific bodies, oromoted us to nronose a DNA alkylation dosimetry study to establish the non-lineairitv of the dose-response relation i.e. the existence of a practical no effect level. This study would at the same time be of much wider interest in that it would help the case against the "one hit theory". The proposal was supported by AKZO, DOW and DSM but not by VKE who only intended to fulfill the commitment towards their regulatory authorities. It was agreed that two snonsor grouns would be formed to conduct the studies independently. The results would be exchanged on the basis that costs would be similar. Since the start of the dosimetry study two interim reports have been uroduced. Progress was not as easy as envisaged and costs are higher than originally estimated. For those reasons it was decided to review the situation and discuss it with the sponsors. The reasons in favour of nroceeding with the study even though the costs will be substantially higher are as follows: 1. The work has advanced to a stage that the methods can soon be applied to obtain the actual results (two thirds). 2. The subject is of high scientific interest in that it complements other work at renuted toxicology research institutes. It should yield new insights on finalisation. 3. The results should be of commercial value: Industry will be in a much better position to negotiate acceotable levels of residual VC in PVC for food packaging with regulatory authorities. R&S 008331 4. The work is complementary to the CIVO-TNO lifetime animal feed studies. Exchange will give us access to the results of the CIVOTNO work. 5. The study will have interesting side benefits. If required it will be possible to develoo a biological monitoring method for workers exnosed to VC. 6. On a more general basis it is our Companies' contribution to the nool of knowledge on VC toxicology to which the first important innut came from the soonsors of the Maltoni studies. (Italian, French, Belgian and UK PVC Producers.) SICM, 6th May, 1982 Molecular dosimetry studies with vinylchloride Costs Original project cost estimate STL reported actual costs 1980 STL reported actual costs 1981 CIV0-TN0 costs 1981 84000 38000 66000 Total to end 1981 Estimated costs to complete the programme 50000 Total for project 4.1 4.62 5.04 4.77 N.fls. 345000 175600 332600 62800 571000 238500 809500 SICM, 6th May, 1982 R&S 008332