Document RaR4zKORzdpx95qEGqeDZd0n7

JPAmerican Petroleum lnsiiiute 1220 L Street, Northwest Washington, D.C. 20005 [i)ate: IFu-om: 'ii"o: ~e: January 25, 1993 Mary Paxton~_ <~~I~ Benzene Task Force Update The next meeting of the Benzene Task Force is scheduled for Tuesday, March 2, at API's Washington headquarters in Room 907. Please plan to be here by 9 a.m. for a full day's meeting. An agenda will follow. Please notify Roxie Landry (202-682-8330) by February 24 of whether you plan to attend or not. Attached are copies of the proposals submit-ted by Lovelace on benzene metabolism and dosimetry and on biomarkers of benzene exposure, which we will decide about at the March meeting. \ r- ----- - BP-3 35549 BP-00019204 Draft Proposal for the Study of BIOMARKERS OF EXPOSURE TO BENZENE Submitted to the American !Petroleum institute by the inhalation Toxicology Research linstitute IP. 0. Box 5890 Albuquerque, NM 87185 Operated by the Lovelace Biomedical and Environmental Research institute . for the U. S. Department of Energy under Contract No. DE-AC04-76EV01013 November 1992 ITRI Contact: C. H. Hobbs, DVM HTRI Assistant Director 505-845-1045 or R. F. Henderson, PhD Principal Investigator 505-845-1154 - --,..----- -- - ....... BP3 35550 BP-00019205 I. INTRODUCTION Benzene is a !mown hematotoxin and leukemogen in humans. 'fo determine the health risks associated with benzene exposure in a quantitative fashion, it is essential to determine dose/response relationships. The dose of greatest interest is the biologically effective dose, i.e., the dose that causes a change in the biological system that leads ao an adverse health ' effect. Hn the case of benzene, the mechanism by which benzene and/or its metabolites induces acute myelogenous leukemia in humans is not !mown. In the absence- of a mechanism, one cannot determine the true biologically effective dose, but the dose to the target tissue, the bone marrow can be detennined. One approach to quantitating dos~ is through the use of biological markers of exposure, as described by the National Research Council (1987). In the past, most toxicology studies were designed to monitor only the external exposure regimen and a developing a(Jverse health effect. Information from such studies may be deceiving, because the external dose may not-be linearly related to the internal body dose or the dose to the critical site of toxicity or most importantly, to the biologically effective dose. The ideal state of knowledge would be to know the quantitative relationships between the external exposure._ internal dose, dose to target tissue and biologically effective dose. U the kinetics of formation and removal of the specific markers is known, than mathematical models can be used to described the relationship between the various markers following different exposure regimens. Such a model will allow the use of readily available markers, such as those in blood or urine, to predict the level of less available markers of exposure in the target tissue, such as the bone marrow. - ---2 - - - - - - - - - - ...... BP3 35551 BP-00019206 II. BACKGROUND The !'rR.I has had extensive experience in measuring the dosimetry of benzene administered orally or by inhalation. We have determined the effect of species, route of exposure, and exposure concentration on the internal dose, and area under the curve for major metabolites in tissues of exposed rats and mice (Sabourin et eL, 1989). These studies indicated that the production of putative toxic metabolites (muconaldehye and hydroquinone) was favored at lower exposure concentrations. Limited studies in nonhuman primates suggested that hydroquinone production was also favored at low exposure concentrations in those species (Sabourin et al., 1992). Studies to determine the effect of repeated exposures to benzene on its metabolism showed little change from the results following single exposures (Sabourin, et al., 1990). Although area under the curve values were not obtained for &he bone marrow, the studies at KTJRI showed that bone marrow, the target tissue for benzene toxicity, contained metabolites that reflected those in the blood. Biological markers of exposure to benzene should be benzene specific (i.e., are produced only by benzene) and quantitatively relatable to prior exposures. Based on what is known about benzene metabolism (fig. 1), biological markers specific to benzene must be .related to either benzene itself or to the benzene epoxide that is the first step in metabolism of benzene. The benzene oxide can react with glutathione-to form phenyl mercapturic acids or it can act to phenylate nucleophilic sites on proteins or DNA The benzene oxide can also be further oxidized to form the ring-opened products, such as muconaldehyde or muconic acid. Benzene oxide can also be metabolized to phenolic products, the traditional biomarker of benzene exposure, but phenol products are not benzene specific. Phenol-is presenrin the environment and is a metabolic product of some aromatic amino acids. Urinary phenol was 3 BPJ 35552 BP-00019207 . I? BP-00019208 valid for exposures to 10 ppm benzene or above but, since the lowering of regulated exposures to 1 ppm, can no longer be used-due to the high background levels from nonbenzene sources. Besides being chemical specific, biological markers that are retained in the body with a long halflife are desirable because they can be used to integrate exposures over a longer time period. However, shorter halflived markers often occur in larger amounts and can be more easily detected soon after a single exposure. Good use can be made of a panel of biological markers of varying halflives. Biological markers that have a short halflife, such as a vapor in exhaled breath or metabolites in the urine, may be present in large amounts soon after the exposure, but may not be detectable at later times. Other markers, such as blood protein adducts or DNA adducts, may be formed in only trace amounts after a single exposure but are cleared more slowly and will accumulate with continued exposure. Thus one could distinguish between a recent single exposure, an ongoing exposure, and a past exposure with no recent exposures by the levels of the biological markers having different halflives (fig. 2). .m. SPECllFHC AIMS Of THIS PROPOSAL The purpose of the proposed work is to test the hypothesis that a panel of biomarkers of benzene exposures can be used to quantify prior exposures to benzene. This will be achieved by completing the following specific aims: 1. Develop a ~BPK model describing the time course relationships between several benzene specific biological markers of various halflives. The markers wHI be benzene in breath and blood, phenyl mercapturic acid, phenyl guanineand muconic acid in urine, phenyl cysteine adducts in blood proteins and 5 ,.,-----------. 8P3 35554 BP-00019209 ).. ~ !!!! ~ ~ ~ @, ~ r:&l ~ @= ; @, J ~ @ ~ 1!!!1 ~ .(!#)) ~ tQf: ~ (!#)) [Qf: liM ~ cg ~ g ~ d ~ ~ ~ @, fl&, 1\&fl (!# ~ d ~ ~ ~ liM ~ .@, IN 1\&fl ~ ~ ~ !=!!.. 0 @ IS 2J f!!RJ :. ~ c::::J @ c::::J c::::J c::1 [S WJ@ (S@ ~ <} c:} c:} c:} @ @ @ IS ((J IS c::::J c= @9 g083 <} <} '} 9 <{? ?? ?9 c} c} OC> ~ li0 (10) gg ~ =(9S c::::::J b @ @ c:} @!?) '} .-} @ '} <} g 0~ 0 ~ ; @e;::,I!2!Rl; @~ b~ ~ bW@Iil C> C:J b @ ~~ (10) @ ~ ~~b =I~S (lfiJ b 2J g, ~ 'BJ ~ c!5 @ ~ @li) g C:::l IS ~ w@ @ (9Ji) IS ~!S2J {5 = C!!iV @ b -@JS~ @g~ 000 ~ @ 2:S b ~ (Sij @ [Q = ===== @@@ ~~@ urou(iij gro/ ~ ~ II') II') on "...".,' ..., Q, .:Q BP-00019210 hydroquinone and muconic acid in bone marrow. The latter two markers are included so that we can relate the readily available markers in breath, urine and blood to target tissue metabolites. 2. Expose mice to benzene by different exposure regimens and-determine if the model al1ows us to distinguish between the animals that had been exposed recently, or sometime in the past or continuously. IV. EXPERIMENTAL APPROACJHJES A Developing the Model The experimental data that will be required for the -model are the amounts of the markers produced by a given amount of benzene exposure and the...rate at which the marker is removed. Much of this information is already known based on our past studies and the remainder are being developed under a separately funded project. An existing physiological model previously developed at ITRI for single exposure scenarios (Medinslcy et al., 1989) will be expanded to include the six biomarkers and will be modified fur multiple exposure scenarios. These-experimental data will be used in the development of the model describing the temporal, quantitative relationships between each of the markers and the benzene exposure levels. Exposures of mice by different regimens (see below) will-be used to validate the model and determine uncertainties of the model predictions. Simusolv (Dow Chemical Co., -1990) on a microvax computer will be-used. Statistical criteria provided in Simusolv will be used to test the goodness of fit and to select the be-st-fit pharmacokinetic model. --7 ,.- ------- --- BP3 35556 BP-00019211 lB. Exposure of mice by different regimens Once the model has been cbreloped, mice will be exposed to 1 or 10 ppm benzene once or repeatedly and the animals sacrificed either immediately after the exposures or one week or one month after the exposures. Based on our earlier wort (Sabourin et al., 1989), we know that either 1 or 10 ppm exposures are within the Hnear range for total metabolism of benzene as well as for metabolism by any of the pathways to individual metabolites. We will determine the levels of the six-biomarkers in the mice following the different exposure regimens and determine if the mode] is valid. To determine the relationship between the readily available biomarkers under study and the less available markers of dose in the target tissue (bone marrow), we will also measure markers of exposure in the marrow. This will allow us to use the model to predict target tissue doses based on the readily available markers of exposure. nf the model is not predictive of the results, we will experimentally determine the basis for the lack of validity and will correct the model. C. 1'ime Line The time line for the expected completion of these studies is shown in Figure 3. Although we know that the funding will have to be renewed on a yearly bas-is, we have projected studies over a three year period and are assuming we will need to expose mice twice to obtain the data we need for a valid model. V. EXPECfED RlESUL1'S These studies will determine if a panel of biomarkers with varying halflives can be used to characterize -prior expesures to benzene -in rats. r - - - - - --------~ BP3 35557 BP-00019212 Years Specific Aim 1. Develop PBPK model for biomarkers a. Model based on data in band b. Revisions based on new data 2. Mouse exposures and analysis of new data 3. Reports 1 2 Figure 3. Time JL..ine for Proposed Study 3 9 -------- BP3 35558 BP-00019213 VI!. fUTIJRE WORK A logical extension of this work would be to modify the PBJPK model for humans and test the validity of the human model in occupational workers with known exposures to benzene. Vll. B.EFJERENCJES Medinsky, M. A, l? .f. Sabourin, G, Lucier, JL. S. Birnbaum and R. JF. lH!enderson (1989) A physiological model for simulation of benzene metabolism by rats and mice. Toxicol. Appl. Pharmacol. 99: 193-206. National Research Council (1987) Biological mar~ers in environmental health research. Environ. Health l?erspect. 74:3-10. Sabourin, 1?. J., W. E. Bechtold, W: C. Griffith, L S. Birnbaum, G. Lucier and R. F. Henderson (1989) Effect of exposure concentration, exposure rate, and route of administration on metabolism of benzene by F344 rats and B6C3F1 mice. 'foxicol. Appl. l?harmacol. 99:421-444. Sabourin, P. J., l D. Sun, J. R. MacGregor, C. M. Wehr, L S. Birnbaum, G. W. Lucier and .R. F. Henderson (1990) Effect of repeated benzene inhalation exposures on benzene metabolism, binding to hemoglobin and induction of micronuclei. Toxicol. Appl. Pharmacal. 103:452-462. Sabourin, ]?; J., B. A, Muggenburg, R. C. Couch, D. Lefler, G. W. Lucier, L. S. Birnbaum and R. IF'. lfl!enderson (1992) Metabolism of [14C]benzene by cynomolgus monkeys and chimpanzees. To:xicol. Appl. Pharmacal. 114:277-284. 10 ~------- --- --- BP3 35559 BP-00019214 VJm. RESOURCE AND RESEARCH MANAGEMENT The institute!s resource management structure is-shown in Figure 4. ITJRI's Director, Dr. Joe L. Mauderly, serves also as President of LlBEJIU. Dr. Robert K. Jones is the Associate Director of JfTRI and Vice President-of LIBIERl Dr. Charles H. Hobbs, Dr. John F. Lechner and Dr. Bruce lB. Boecker act as Assistant Directors of ffl!U. Dr. Hobbs is responsible for management of the chemical toxicology projects at liTRI, Dr. Lechner is responsii>ie for molecular biology projects, and Dr. !Boecker is in charge of projects in radiation toxicology. These five members of the Directorate provide overall institutional resource management and each has responsibility for people, space and equipment included within their specific research groups and support service units. The research project described in this proposal will be under the overall supervision of Dr. C. H. Hobbs. !Each project also has a project coordinator or study director. In this case it will be Dr. R. F. Henderson. Since the Institute is operated under contract with the DOE, we must and do comply with all federal regulations relative to personnel activities, procurement,_property management, accounting standards, facility maintenance, security and health and safety procedures. We have de~'eloped and utilize a computerized management information system to account for all financial transactions and manpower data as well as for activities relating to property management and other support services act-ivities. Monthly printed reports on current period- and year-to-date transactions are provided to all-~ppropriate personnel for management_purposes. Finally, it should be noted that a year-end financial audit is conducted on t-he Institute's prior fiscal year financial transactions by U. S. Government auditors to assure compliance with federal regulations and to verify an appropriate indirect cost basis which is applicable to all sponsors for whom research has been conducted. In summary, we 11 ,..------------ BPJ 3 55 &o- BP-00019215 ~lNJ[HJ~fiA~r~[N] IF~@ llJJIRl ~ ~ u~~~'[L(Q)@V ~~~LEffi\[R1~[)={] (Q)!f@lDllDd;liDUDUlJ@J0 ~UIT'lUllr(U]lf D[NJ~uDuQJJu~ t Joe l. Mauderly Director I I I r~ Robs~ K. Jona~ Asso~lais Director rl Ch!Slfrts H. IHoillloo ~ ~ i8JNC IS. 18l!O$CCt'Sii' A9!!1Si81i11U lOirrsctou- Pl.slll!sUS!M lOllr~ou- I I~ John F. Lechner Asslsiani Director .I Aerosol Science ~ Group Exposure Section Chemistry & Biochemical Toxicology Gro~p 1- - Analytical Chemistry Section rl ~ ~ I Pathology Group . Clinical Pathology - Section Histopathology Section Necropsy Section -i Health Protection Unli Quality Assurance Unit Educational Program Internal Audiior I H Compuiar Unli Engineering & ~ Facilities Unli I HFinancial . Managemeni Unli ~ 4~ Human Resources Unit I H fl.llbrSJry Unli H Procurem~~tni Unli Pathophysiology Group ...... - Chronic Studies Section - Clinical Section ~ Propelty MmnSigement Unli I . Technlcei l- CommunlcaUons Unli H I 11Anlmmi Ctll~ l\Jn!R ~!om~ihsmmilcs Section H JAsrosoU l?ro@~SJM H II f-" Chemical 1roJ~Icoiomt t;=rogrtllm ~8ld!SJ~Ioi1l lOOill~RlSponS$ IP'ir~li'Slm H IChronic Eltposure ~ Rlta~Cl!oniUJcilo1e fDlooms~ l?mgrilm Pro!lii"Sli'l'il H IMolecular & Cellular ToJ~Icology Group H~hemlcal Can~er 1 Mechanisms P~ogr~m1 4Amdiatlon Canci1r 1 Mschmnlsms Pn)gram1 ~~ Comblns~ieltpO!!IUJV IPU'IIIl~I1'1Sllli'1l II i PaihCl!Jenesls Pr0!3Jrmm II l )BPJ 3556 ttl '"0 I 0 0 0...... \0 N...... 0\ feel that the Institute has the effective and experienced management team required to conduct both cost-effective and high-qJJality research for sponsors. IX. RESEAJRCH l?ROffiCf MANAGEMENT ANJD JPERSONNEL A multidisciplinary team of scientists has been chosen for work on this project. Table 1 lists the members of the research team. Curriculum vitae of all professional staff are included in the Appendix. The .KTRR scientists with primary responsibility for the management and conduct of these proposed studies are Dr. C. H. Hobbs and Dr. JR. JF. Henderson. In the context of the Good Laboratory Practice Regulations, Dr. Henderson will be the Study Director (Principal Investigator) for the studies-and as such she is responsible for overall conduct of the studies and will serve as the contact at KTRI for API for conducting the studies. Dr. Henderson is trained as a biochemical toxicologist and has extensive experience in the toxicokinetics of inhaled vapors. Dr. William Bechtold is a chemist with experience in analyzing for benzene metabolites. He will be in charge-of analyzing for benzene and its metabolites as biomarkers of benzene exposure. Dr. I-Yinn Chang is a biomathematician who has had training in the development of PBPK models and is familiar with the benzene PBPK model deyeloped at ITRX. Appropriate technical and other supporting personnel will be-assigned to the project to meet project objectives. The professional, technical and other supporting personnel will work as a team. Should the need arise f.or techitical:-expertise not available from personneLon the team, assistance of other members of the Institute staff will be enlisted. The Institute retains 13 BP3 35562 BP-00019217 Table 1 Summary of Scientific Personnel with Substantial Involvement in the Project Name JH[enderson, R. F. JBechtold, W. JE. Chang, I.-Y. Degree/Discipline Ph.D., Biochemical Toxicologist PhD., Chemist M.S., Biomathematician Role in St-udy Study Director (]Principal Investigator) Co-Investigator, Analysis of Metabolites Co-Investigator, Development of Model 14 ,....------ ----- -BP3 35563 BP-00019218 the authority to assign equivalent personnel or otherwise use institute personnel, within the limits of the authorized budget, to best achieve the project's objectives. X. REPORTS. At 6-mo intervals, a letter report on the progress of the project will be submitted to AlP! by LJBEJIU. The LJBJERI will immediately notify APJI of any private or governmental request for information on ongoing or completed research conducted under this Agreement, including any subpoena or other legal instrument requesting information. The Contractor will cooperate fully with any effort by APH to narrow the-scope of any such request, to obtain a protective order limiting the use or disclosure of information. XI. PUBUCATIONS Al?I and the JLBERI agree to -full public disclosure of scientific information deveioped through this Agreement. The LBERI shaJI not release such information-without prior API knowledge and review. The scientific conclusions and professional judgments arising out of performance of the project will be the responsibility of the LBERI. The LBERI, however, will not publish or otherwise release data, conclusions, or manuscripts in a citable or quotable fonn without prior API knowledge and-opportunity to comment: API opportunity to comment is for the purpose of clarification and format or editorial comments, but not for the purpose of substituting API's opinion for that of the LlBERl 15 ,----- ---- - - -- - - - - BP3 35564 BP-00019219 XIII. INSTITUTIONAL RESOURCES A General The Inhalation Toxicology Research Institute (ITRI) has been operated by the Lovelace organization for the U. S. Department of Energy (DOE) or its predecessor agencies since the early 1960's. lit is currently operated under a DOE five-year, cost-reimbursable, no-fee contract by the Lovelace Biomedical and Environmental Research Institute (LBlERX), a subsidiary of 1he Lovelace Medical Foundation (lLMf). LlBERI's only mission is to operate XTIU for DOE and to conduct research for IDOE and other sponsors where appropriate. LBERI has no financial assets other than those it is responsible for managing for the United States government or its other sponsors. rnu facilities are totally government owned and are located on Kirtland Air -Force Base East in Albuquerque, New Mexico. These facilities contain approximately 271,000 ~ of space for laboratories, animal breeding and maintenance activities, offices and essential support services. Since first occupied in 1963, new additions or modifications have been added on nearly an annual basis to meet the ever-changing nature of the Institute's research program. These facilities represent one of the most modem and complete laboratories for the conduct of inhalation toxicology research that are available anywhere in the world. Pertinent details are provided below. B. Facilities The Institute's government-owned facilities consist of approximately 271,000 ft2 of space. They include a central laboratory, office and small animal and primate maintenance complex, dog kennels on either side of the central laboratory and specialized facilities for machine shop, electronics shop, analytical chemistry, rodent quarantine and 16 BP3 35565 BP-00019220 rodent breeding, primate quarantine, receiving and warehouse, health protection and waste handling located more peripheral to the central complex. From the standpoint of this proposal, the following facilities are considered to be of principal importance: 1. Modem analytical chemistry laboratories containing stateoQf-the-art instruments. The instrumentation includes liquid scintillation eounters, spectrofluorometers, high-pressure liquid chromatographs with auto injectors-and auto sample collectors, UV and VJIS spectrophotometers, gas chromatograph/mass spectrometers with a computer data system, gas chromatographs, a laboratory data system for quanUtation of GC and HPLC data, infrared spectromete~ with a computerized data system, and a m~clear magnetic resonance spectrometer. 2. Laboratories for safely developing and testing generation procedures for inhalation exposure environments using either radiolabeled or unlabeled materials, including potential carcinogens. These laboratories are equi_2Ped with instruments for physicochemical characterization of the exposure environments. 3. Hazardous materiai storage and preparation area designed and constructed for-storage of hazardous materials in a controlled access area. The facility has storage capabilities adjacent to a_preparation area. This area provides for storage of radioactive materials in a shielded vault and storage of nonradioactive materials in a cabinet-or refrigerator. The preparation room is equipped with a hot cell and three hoods for 17 BP3 35566 BP-00019221 I . preparing aerosol generator solutions and other operations related to preparing materials for use in inhalation exposures. 4. Inhalation exposure facilities specifically designed to allow controlled access to an area fully equipped for acute inhalation exposures. !Each of two exposure complexes within the facility has its own control room, two exposure rooms and a preparation room. Associated equipment in the facility inc1udes an ultrasonic unit for decontamination of exposure apparatus and an electron microscope. A variety of laboratory animals have been exposed nose only in the facility, ranging in size from mice to dogs. Materials aerosolized for animal exposures have included chemical carcinogens, a variety of radioactive compounds and vapors. Exposures have included respiratory monitoring by whole body plethysmography in some caSes and the facility-is equipped to make these respiratory measurements for small laboratory animals, dogs and monkeys. 5. Quarantine facilities are available for isolation and stabilization of animals received from eommercial suppliers. lRigid standard operating procedures are followed during the quarantine period while the health status of newly acquired animals is--evaluated. 6. A barrier-sustained, rodent housing facility is operated on a clean-dirty corridor system. All personnel must shower before entering the facility and must w.ear clean laboratory-supplied clothing, face mask and gloves. All materials entering the facility are sanitized (i.e., cages, lids, caps, 18 BPJ 35567 BP-00019222 personnel clothing). !Feed is pasteurized by suppliers and all bedding is sterilized prior to use. All ilemuhat cannot be entered-through the tunnel washer or autoclaved are chemically sterilized. The animal rooms are maintained at 20 to 23C and at 30 to 50% relative humidity under a 12 h light and dark cycle. ITRI is a registered research facility, in compliance with Section 6 of JP.L. 89-544 {as amended), the Laboratory Animal Welfare Act, and has -been fully accredited by the American Association for Accreditation of Laboratory Animal Care since 1970. The last accreditation inspection was conducted in June, 1988. Animals are maintained in accordance with the recommendations in Nlli Publication No. 85-23, entitled 11Guide for the Care and Use of Laboratory Animals11 and in compliance with the Laboratory Animal Welfare Act. 7. A cellular toxicology facility is specifically designed and staffed to test the effects of chemical toxicants, carcinogens and radiation oneukaryotic and prokaryotic cells in culture. Equally important-are the capabilities in this facility to determine the in vitro functional capacities of cells exposed to toxicants in vivo. A FACSTAR PLUS @ectonDickinson) flow cytometer is available. 8. A comprehensive program for hazardous waste handling and disposal. Radioactive and hazardous wastes generated at the Institute are collected, treated, packaged and shipped in accordance with all EPA, 19 BP-00019223 DOT and DOE regulations, and in a manner that minimizes hazard to personnel or Jhe environment. Hazardous chemical wastes are collected and handled in a manner consistent with the requirements of the Resource ConseJVation and Recovery Act (RCRA). As with radioactive wastes, all hazardous chemical wastes are shipped to EPA-approved disposal sites for final disposal. 9. lin-house computer capability consisting of a network of Digital Equipment Corporation VAX series computers. Kt curr~ntly includes a VAX llnBO, three MicraVAX XR computers and five PDP-H series computers. The latter are required for real-time data acquisition. Extensive use is also made of personal computers for automated data collection, scientific calculations, word processing and graphics. These computer resources pro:vide facilities for collection, storage and processing of both scie~tific-and business or management data for the total Institute. 10. Emergency electrical generation capability is provided to all essential areas of the facilities. This capability includes two diesel-powered 750 KW and two 100 KW generators. They receive routine testing on a weekly basis and require only six seconds-for fuJI activation. All of the facilities described above are-incorporated into this emergency power grid. 20 ------ BPJ 35569 BP-00019224 11. A complete in-house shop capability including a machine shop, e)el;tronics shop and carpenter shop. The--personnel in each shop area continue to provide the necessary experience and skills for the initial design and fabrication or subsequent modification of prototype-exposure chambers, characterization devices and other instruments required at ITRK. 12. An excellent-research library which provides all services_required to support our research program. These include current subscription to 300 journals, over 9,500 books and approximately 19,000 documents. Local and regional interlibrary loan services are available through the University of New Mexico and a consortium of New Mexico Medical Libraries. On-line literature search services are maintained with over 100 data bases through 4 major systems. These services will be essential for providing available literature on specifiC chemicals which ITRH is requested to study. Although this by no means describes the Institute's total facility and equipment resources, it does describe those which would be of major use in the conduct of the proposed research and demonstrates thatITRI has all critical resources necessary to meet the needs of this research proposal. further, the essential resources will be available on a noninterference basis relative to our commitments to DOE and other federal~gencies currently supporting research at ITRI. 21 BP3 35570 BP-00019225 XHI. QUALITY ASSURANCE The research described in this proposal is explofcitory in nature and will be conducted in the spirit of the Good Laboratory Practice (GlP) regulations. Specific quality requirements will be addressed as integral parts of project-specific protocol(s) and standard operating ! . procedures that might be needed. The approval of the ffJR.H Quality Assurance Unit isrequired on all protocols and standard operating procedures at the HTRL General requirements fur research conducted in the spirit of GlPs an rm.H are that- study personnel will have documented training and experience; that work will be done following I'IR!-approved protocols and applicable K'lrlRJ-approved standard operating procedures; that study personnel follow good record!ceeping procedures and verify the accuracy of data collected; that personnel maintain calibration and maintenance documentation for equipment and instruments; and that study personnel ensure test chemicals, reagents, and specimens integrity throughout coJiection, analysis' and storage processes. It has been our experience that research such as this is difficult to conduct within strict GLP guidelines due to the many adjustments that occur in the research plan as protocols are developed and conducted. API staff, representatives of sponsoring companies, and cGnsultants retained-by API, will have reasonable access to LIBERI personnel and facilities engaged in the work covered, by this Agreement. API may designate one or more consultants who may-observe or audit the conduct of the work by this proposal. The LBERI shall cooperate fully with such observations and audits. 22 .- - --- ---------- BP3 35571 BP-00019226 All records, new data or other documentation relating to the studies covered by this Agreement shall be retained by LIBERI according to GLP regulations for a period of at least ten (10) years from the date of conclusion-of the studies. The materials retained pursuant to the above paragraph shall be stored in an archive which ensures that they will be maintained in a safe and secure manner and allows for their expeditious retrieval when needed. Material retained in ahe archive shall be indexed by test substance, date of study, test system and nature of study. In the event that the Contractor or its archiving facility discontinue operat-ions or for other reasons are not able to archive the material, all raw data, specimens and other documentation pertaining to the testing covered by this Agreement shall be-transferred to such other facility as Al?I may direct. XIV. HEALTH JPROTECf!ON All applicable !TIU health and safety requirement will be adhered to. These cover general laboratory practices. Specific safety requirements will be included as an integral part of the protocols and standard operating procedures required for the studies. The approval of the ITRI Health Protection Operations Unit is required on all protocols and standard operating procedures at the ITRl XV. CARE AND USE OF LABORATORY ANXMALS The use of-laboratory animals in well-designed biomedical research studies has resulted in significant advances in health care and prevention of disease in humans. Animals are used at the ITRI in well-defined studies to identify the health risks to man from exposure to potentially toxic agents in the absence of an adequate-understanding of their effects on 23 ,.--------- ~ BPJ 35572 BP-00019227 man. Other methods such as cell cu1ture are used .whenever possible to reduce the number of animals needed in the studies. However, adequate alternatives to the use of laboratory animals for the studie.s proposed here are not currently available. .As such, the use of laboratory animals for- these studies is necessary. We recognize that the use of animals in studies such as these is a privilege, not a right. Thus, policies have been instituted to insure ihe ethical and humane care and treatment of animals at the rmn. This is accomplished nhrough review and approval of protocols using animals and the establishment of standard operating procedures for -their care. l!TIU has a current National Hnstitutes of Health assurance number for animal care and, use; that number is A3083-0l. nru is also fully accredited by the American Association for Accreditation of Laboratory Animal Care; our accreditation number is 000200. HTRI has an officially constituted "Animal Car-e and Use Committee"; the responsibilities of the Committee include reviewing all animal care and usage at HTRX. The Committee is responsible for assuring that all animals at ITRI are kept, treated, and used in a humane and lawful manner. This draft proposal is currently under review by the IT!RI Animal Research Committee and will be approved prior -to fcinal submission to API. ITRI is a registered research facility with the USDA; our registration number 85-R-003. HTRI is in full compliance with the Animal Welfare Act as administered by the USDA. ITRI is in full compliance with all other relevant local, state,- and federal regulations concemi!!g animal care and use. 24 /---~-- BPJ 35573 BP-00019228 XVI. PROJECf COSTS A General The costs given in Table 2 are our best estimates of the costs for conducting the research outlined in the proposal. Because of uncertainties of abe exact protocols that will be used, the budget may require modification as the protocols are finalized or as the direction of the research is modified fr-om the findings in early stages of the research. The IT!lU is operated by the Lovelace Biomedical and Environmental Research Institute (LBERJI) for the DOE. The LlBERH is a non-profit corporation which operates on a cost-reimbursable no-fee basis. Therefore, the costs for perfonning the research will in all cases be the actuaLcosts (labor, supplies, services, etc.) of the effort expended i<Y do the work. The HRI cannot exceed the amount of the agreement without amending the agreement through the DOE and with the sponsor. B. Basis for Cost Estimates The cost estimates in Table 2 are for the Jevel of effort stated and our best estimate of the cost of supplies required to do the studies outlined in the proposal. The salaries used are those projected to be in effect for the period 1 Jan 1993 through 31 Dec 1996 (3 years). As work progresses, we ~serve the right to change personnel assignments and to execute procurement actions based-on research results obtained, the nature of proposed research, and our considered scientific and managerial judgment. Procurement activities will be carried out in accordance with DOE procurement regulations in effect at the-time purchases are made. 25 ,-- --- - BP3 35574 BP-00019229 Table 2 Projected Cost !Estimate lfor Biomarkers of Exposure to Benzene PERSONNEL . R. Henderson, Principal Investigator W. Bechtold, Chemist I. Chang, ~tatistician To be Assigned, Research Technologists Total Salary Frirtge Benefits Supplies/Services Animal Purchase Miscellaneous suppli~s/glassware Subtotal Travel Other Expenses Animal maintenance Word prQcessing service Illustratidn service Computer service Subtotal Total Direct Costs Indirect Costs Total Costs Year 1 Effort $ Year2 Effort $ Year 3 Effort $ Total !Effort $ .10 .10 .10 .30 .10 .20 .10 .40 .20 .00 .li.O .30 .10 .30 .20 .60 24,900 29,200 25,400 6,000 7,000 6,100 79,500 19,100 1,000 10.000 11,000 1,000 1,000 ~ 11,500 1,500 0 11.000 11,000 2,000 2,000 31.500 33,500 4,500 1,200 500 500 ~ 7,200 50,100 35,100 85.200 1,200 500 500 5.300 7,500 56,700 39,700 96.400 0 500 500 ~ 6,6i00 51,100 35,800 86.91[]10 2,400 1,500 1,500 15.900 21,300 157,990 110,600 268.500 BP3 35575 ) ttl '"0 I 0 0 0...... \0 N C,N 0 The fringe benefit rate is calculated as a percent of total salaries and includes the following: FHCA Medical insurance Retirement Benefits Life linsmance IDisabiKity Insurance Unemp!oyment Compensation Wor!anen's Compensation This rate has had an increasing trend, attributable to increases in the employer- paid FICA rate as well as .medical insurance. The 24 percent rate used is based on the last three years' operating experience. The overhead costs include those costs associated with functions such as general management and administration, business office, procurement, property management, personnel, facilities operation and maintenance, library, quality assurance, and health protection operations. The Institute's overhead cost rate is calculated and adjusted monthly with an , annual rate set at the end of each fiscal year. All costs-incurred are audited nonnally by U. S. Government auditors, and all overhead rates are verified. The last fiscal-year audited was FY-=1991 and, as in previous years-, no exceptions in rates were_j)oted. The Institute's overhead-expenses are distributed as a percentage rate on all modifted total direct costs. Although the rate in FY-1992-was lower than what we are projecting for FY-1993, it is a-pr-ovisional rate and actual charges will be made. 27 / --------- BPJ 35576 BP-00019231 I lin-house support services include the costs of animals, animal care, histopathology preparation, shop, computer, word processing and illustration services that are provided within the Institute on a centralized basis. The costs of these services are charged to direct projects on a pro rata basis dependent upon the !evel of service used by each individual project. The number of animals used and the days of animal maintenance have been based on the proposed work plan. The level of shop, computer, word processing and illustration services required for this project were estimated based on our ex~rience with similar projects. Travel expenses are for investigators to present results at national meetings and to report results at meetings called by the API. The estimated budget is written for a 3-year period even though we realized that the initial contract will be for one year only. 28 ------------------ BP3 355 77 BP-00019232 Draft Proposal for the Study of -DOSIMETRY OF BENZENE iN TillE BONJE MARROW Submitted to the American Petroleum Institute by the Inhalation Toxicology Research llnstitute P. 0. Box 5890 Albuquerque, NM 87185 Operated by the Lovelace Biomedical and Environmental Research Institute for the U. S. Department of Energy under Contract No. DE-AC04-76EV01013 November 1992 ITRI Contact: C. H. Hobbs, lDVM ITRI Assistant Director 505-845-1 045 or R. F. Henderson, PhD Principal Investigator 505-845-1154 ,...----------BP3 35578 BP-00019233 I. INTRODUCriON -Benzene is a known hematotoxin and leukemogen in humans. To determine the health risks associated with benzene exposure in a quantitative fashion, it is essential to determine dose/response relationships. The dose of greatest interest is the biologically effective dose, i.e., the dose that causes a change in the biological system that leads to an adverse health effect. Bn the case of benzene, the mechanism by which benzene and/or its metabolites induces acute myelogenous leukemia in humans is not known. In the absence of a mechanism, one cannot determine the true biologically effective dose, but the dose to the target tissue, the bone marrow, can be determined. Recent work by Dr.lRichard Irons implicates the benzene metabolite, hydroquinone, as a major toxic benzene metabolite in the bone marrow (Irons et al., 1992)~ Other work implicates free radical fonns of benzene metabolites as potential bone marrow toxins (Subrahmanyam et al., 1991). The following proposal outlines research to be conducted in collaboration with Dr. Irons to determine the dosimetry of benzene and its metabolites in the bone marrow that is associated with specific toxic endpoints. II. BACKGROUND . R-eports by Irons (1992) indicate that in vitro pretreatment of mouse bone marrow cells with pmolar levels of hydr-oquinone greatly enhances the response of the cells to granulocyte/macrophage colony-stimulating factor (rGM-CSF). No stimulation occurred with phenol, catechol, or muconaldehyde. The bone marrow cells affected by the hydroquinone and rGM-CSF were nonadherent and lineage-depleted bone marrow cells, suggesting an .---------2 --------~ BPJ 35579 BP-00019234 intrinsic effect on 'recruitment of myeloid progenitor cells, cells not normally responsive to xGM-CSf. In in vivo experimen5, Eastmond, Smith and Irons reported a synergistic effect of co- administration of phenol and hydroquinone in inducing hematotoxic effects in mice. The authors suggested that the effect was due to phenol stimulation of peroxidase-dependent metabolism of hydroquioone to form the highly reactive benzoquinone. Growing evidence supports the contention that free radicals may play a role- in inducing the toxic effects of benzene (Subrahmanyam et al., 1991). Kt has been suggested that either myeloperoxidase, prostaglandin (H) synthas~. or eosinophil peroxidase enzymes react with the phenolic metabolites of benzene to produce free radical intermediates. These free radical intermediates can then initiate lipid peroxidation, depiete cellular antioxidants such as g]utathione, or cause damage to DNA and proteins. Evidence from both in vivo and in vitro studies suggests that these events do occur as a result of benzene metabolism. For instance, Lewis et al. (1988) showed that of the quinone metabolites of benzene, only hydroquinone and benzenetriol were capable of auto-oxidizing to produce-superoxide anjon free radicals, and only these tw-o metabolites were effective at causing DNA singlestrand breaks. However, no in vivo data has been reported that links the dose of the metabolite to the target cells with the subsequent induction of DNA strand breaks. In an in vivo study, Anwar et al. (1989)-showed that dimethyl sulfoxide (DMSO, a hydroxyl radical scavenger) could reduce by 95% the micronuclei frequency in bone marrow of mice dosed orally with benzene when administered-by gavage 1 hr after the benzene exposure. This reauction in micronuclei frequency was dependent on the DMSO concentration. However, the treatment with DMSO also reduced the levels of urinary 3 BPJ 35580 BP-00019235 benzene metabolites. This finding raises the question as to whether the DMSO altered the normal uptake of benzene to yield a lower dose of benzene and its metabolites to the target tissue, bone marrow, or whether DMSO protected the target tissue by scavenging free rndicals. When only one free radical scavenger is used, it is difficult to establish that ibis agent acted only as a free radical scavenger, and that its effect was not due to some other property of the agent. it is important to establish that the concentration of the scavenger in the target tissue was adequate to perform a free radical scavenging function. Ht is equally important to establish that the scavenger did not alter the normal metabolism and distribution of ihe benzene metabolites. The IT!RI has had extensive experience in determining the effect of exposure concentration, species and route of exposure on theJDetabolism of benzene. Work completeciin B6C3F1 mice, F344/N rats and cynomolgus monkeys indicates that the formation of hydroquinone and muconaldehyde metabolites are favored at low doses or exposure concentrations (Henderson et aL, 1989; Sabourin et al., 1989; Medinsky et al, 1989; Sabourin et aL, 1992). For inhalation exposures, the- foP.nation of hydroquinone metabolites or muconic acid is-linearly related to exposure concentration up to 200 ppm benzene (6-hr exposures), but decreases at higher concentrations. The relation -of exposure concentration to blood and urine levels of the major benzene metabolites has been determined based on the "area under the curve" for each metabolite during and after the 6-hr exposure. However, equivalent values have not been obtained for bone marrow, for which only pooled samples (pooled samples from all time_points during and after the exposure) were analyzed. 4 BP3 35581 BP-00019236 m. SPECIFIC AIMS The specific aims of ibis project are: 1. 'fo determine the relationship between exposure concentration and ilie dosimetry of benzene and its metabolites in the bone marrow of B6C3F1 mice and to correlate the dosimetry with toxic changes in the marrow. 2. To determine the role of free radicals in producing toxicity in lhe bone marrow of benzene-exposed mice. XV. PROPOSED EXPERIMENTS A. Specific Aim 1 A series of studies designed to link in vivo dosimetry data for benzene and its metabolites to the potential biological effects described above are planned. l?rior work at tlte ITRI has indicated that the pathways leading to putative toxic metabolites of benzene (represented by hydroquinone metabolites and muconic acid) are saturated at low exposure concentrations, so that a higher fraction of the metabolites are related to putative toxic metabolites at low doses than at high doses (Henderson et aL, 1989; Sabourin et aL, 1989). The formation of hydroquinone metabolites and muconic acid are linearly related to exposure concentration_up to about 200 ppm-,6-hr expesure), but decrease as a fraction of &be total metabolites formed above that concentration. We wish to choo~e two exposure concentrations below this breakpoint and one above it, to see the effect of concentration on .dosimetry to the bone marrow and on the induced toxic effects. B6C3F1 miee will be exposed for 6 hr to either filtered air or 6, 60, or 600 ppm benzene. The levels of benzene and benzene metabolites will be determined in the blood, bone marrow, and urine during (at 2 and at 4 hr) ,.-- --- -- ------.... 5 8P3 35582 BP-00019237 and after the exposure (up to 12 hr) to obtain an "area under the curve" dosimetry for each metabolite. The dosimetry of the metaboiites will be compared to those required to inducetoxic changes in the bone marrow in !rons' in vitro studies. Toxic responses in ihe bone marrow will be observed by Dr. Hrons. .The effects of benzene -orposure in vivo will be examined on colony-forming response of murine bone marrow cells stimulated with recombinant cytokines. Mice will be killed by cervical dislocation and bone marrow flushed from femora using phosphate-buffered saline containing 1% bovine serum albumin. Bone marrow cells will be purified over a discontinuous gradient and nonadherent cells obtained. -Purified cell suspensions will be transported on ice to Dr. Irons' laboratory where they will be cultured as previously described (Irons et aL, 1992). Kt is recognized-that the optimum time of exposure for inducing toxic effects is not known. Therefore, the exposure time may be varied to achieve a maximal r-esponse if that is necessary. The exposure time of 6 hr is based on a large data base at !TRI based on 6-hr exposures. This data base indicates that oiood metabolites have essentially come to equilibrium by 6 hr of exposure of mice to as Jow as 5 ppm benzene. B. Specific Aim 2 In order to assess whether DMSO acts as a scavenger of benzene-induced free radicals or by altering benzene metabolism or distribution, one set of animals will be dosed with DMSO one hour after benzene exposure. !n addition, we will examine mice that have been treated with buthionine sulfoximine-(BSO), a_glutathione synthesis inhibitor. if free radicals are involved in benzene toxicity, depleted glutatliione induced by BSO treatment should-e~acerbate benzene toxicity. Gtutathione depletion should not significantly affect benzene metabolite levels as-glutathione conjugation is only a minor pathway for benzene 6 ---------- - - - - ...... BPJ 35583 BP-00019238 metabolism in mice (Sabourin et al., 1989). Thus, if free radical intermediates are involved in benzene-induced toxicity, DMSO should mitigate -the toxicity and BSO should exacerbate the toxicity. Both dosimetry (BTRI) and bone marrow toxicity (Irons) will be evaluated in &he mice. Besides being a scavenger of free radicals, DMSO may also alter membrane characteristics, especially at the high concentrations of DMSO used in many in vitro studies. Such alterations of membranes would complicate interpretation of results in our studies. We anticipate that tissue levels of DMSO will be too low in vivo to cause such complications. We plan to assay for DMSn in the tissues of treated mice to address thi,s issue. For DMSO-treated mice, one hr after exposure DMSO (12.5% at a volume of 0.01 ml/g body weight) will be administered by gavage to one set of mice in eacl:LeJCPOSure group (controls and exposed groups). For BSO-treated mice, one set of both controls and exposed mice will be administered BSO in their drinking water prior to exposure. The glutathione levels in the bone marrow of these mice will be examined immediately before and after the benzene exposure to determine whether-the BSO treatment did indeed-deplete bone marrow glutathione levels. After exposure, mice from all groups will be placed in metabolism cages and urine will be collected for 24 hr. At the end-of this period, the mice will be sacrificed. The pooled urine, tht blood, and the bone marrow will be analyzed for hydroquinone, phenol, and catechol by the method of Ric!cert et al. (1979), for conjugated benzene metabolites by the method of Sabourin et al. (1989}, and for DMSO using a GC/MS technique. The bone marrow tissue will also be assayed for-micronuclei formation as described in the aforementioned p~per by Anwar et al. (1989) and for-DNA single strand breaks using standard techniques. 7 ..------------ ~ BP3 35584 BP-00019239 C. Figure 1. Time Line The time line for the expected completion of these studies is shown in V. EXPECflED RESULTS These studies will integrate dosimetry studies with studies that determine the effects of- that dosimetry on the bone marrow. The results will provide information on which metabolites are the likely biologically effective compounds and if free radicals are involved in producing the toxic responses. VI. FUToliE PLANS U desired by Al?I, a similar approach can be used to study the effect of co-exposure to toluene on the dosimetry of benzene and its metabolites in bone marrow. Ail inhibitory effect of toluene on the metabolism of benzene has been reported by Kkeda et aL (1972). VII. REFERENCES Anwar, W. A., W. W. Au, M.S. Legator and V. M.S. Ramanujam (1989) Effect of dimethyl sulfoxide on the genotoxicity and metabolism of benzene in vivo. Garcinogenesis 10: 441-445. Eastmond, D. A., M. T. Smith and R. D. nrons (1987) An interaction of benzene metabolites reproduces the myelotoxicity observed with-benzene exposure. Toxicol. Appl. l?harmacol. 91 :85-95. 8 BP-00019240 Years 1 2 3 Specific Aim 1. !Dosimetry in bone marrow a. Exposure of mice 6 ppm 60 ppm 600 pp H ~H b. Analysis of metabolites funher exposures as needed based on early findings c. Assessment of toxicity 2. Role of free radicals HHH a. Studies with DMSO b. Studies with BSO 3. Reports Figure 1. Time LineJor Proposed Study ------- --- --.. 9 BP3 35586 BP-00019241 Henderson, R. f., P. J. Sabourin, W. E. Bechtold, W. C. Griffith, M. A. Medinsky, L S. Birnbaum and G. W. Lucier (19"89) The effect of dose, dose rate, route of administration and species on tissue and blood levels of benzene metaboJites. Environ. Health Perspect. 82:9-17. Ikeda, M., H. Ohtsuji and T. Imamura (1972) In vivo suppression of benzene and styrene oxidation bycoadministered toluene in rats and effects of phenobarbital. Xenobiotica 2:101-106. Irons, R. D., W. S. Stillman, D. lB. Colagiovanni and V. A. Henry (1992) Synergistic action of the benzene metabolite hydroquinone on myelepbietic.-stimulating activity of granulocyte/macrophage colony-stimulating factor in vitro. Proc. Natl. Acad. Sci. 89:3691-3695. JLewis, J. G., W. Stewart and D. 0. Adams (1988) Role of oxygen radicals in induction of DNA damage by metabolites of benzene. Cancer Res. 48:4762-4765. Medinsky, M.A., P.-J. Sabourin, R. F. Henderson, G. Lucier and IL. S. Birnbaum (1989) Differences jn the pathways for metabolism of benzene in rats and mice simulated by a physiological model. Environ.-Health Perspect. 82:43-49. Rickert, D. E.,M. S. Baker, J. S. Bus, et a1 (1979) Benzene disposition in the rat after . exposure by inhalation. Toxicol. Appl. Pharmacol. 49:4-17-423. Sabourin, P. J., W. E. Bechtold, W. C. Griffith, et al. {1989) Effect of exposure concentration, exposure rate, and route of administration on metabolism of benzene by F344 rats and B6C3F1 mice. Toxicol. Appl. Pharmacol. 99:421-444. 10 , . - - - - ---~ BP3 35587 BP-00019242 Sabourin, lP'. J., B. A. Muggenburg, R. C. Couch, D. lLefler, G. Lucier, L S. Birnbaum and R. F. Henderson (1992) Metabolism of 14C-benzene by cynomolgus monkeys and chimpanzees. Toxicol. Appl. Pharmacal. 114:277-284.. Subrahmanyam, V. V., D. Ross, D. A., !Eastmond and M. T. Smith (19>91) Potential role of free radicals in benzene-induced myelotoxicity and leukemia. free Rad. Bioi. Mecl. 11:495-515. VIU. RESOURCE AND RESEARCH MANAGEMENT The Institute's-resource management structure is shown in Figure 2. HTRH'sDirector,_ Dr. JoeL. Mauderly, serves also as President of LBERR. Dr. Robert K. .lfones is the .Associate Director of ITRI and Vice-President of LBE-RI. Dr. Charles lHL Hobbs, Dr. .l!ohn F. Lechner and Dr. Bruce B. Boecker act as Assistant Directors of ITRH. Dr. Hobbs is responsible for management of the chemical toxicology projects at XTRll, Dr. Lechner is responsible for molecular biology projects, and Dr. Boecker is in charge of project-s in radiation toxicology. These five members 'of the-Directorate provide overall institutional resource management and each-has responsibility for people, space and equipment included within their specific research- groups and support service units. The research project described in this proposal will be under the overall supervision of Dr. C. H. Hobbs. _Each project also has a project coordinator or study director. In this case it will be Dr. R. IF. Henderson. Since the Institute is operated under contract with the DOE, we must and do -oomply with all federal regulations relative to personnel activities, procurement, property management, accounting standards, facility maintenance, security and health and safety procedures. We have developed and utilize a computerized management information system 11 ........~----------- BP3 35588 BP-00019243 mNJ [FU~~u~ U\fJ IFU3l lUJIRlIE ~ uCD~~~~[L,~W [RHE~~~~CC[}{] I CQJ U'~ CID lfD 0~@J~0IT'D CID ~ ~lCU'QJJ tcGJJ [J@ DlNl~u~ulVlu~ - I Joe L. Mauderly Director I I Aerosol Sci:mce r- Group Exposure Section Chemistry & Biochemical - Toxicology Group - Analytical Chemistry Section Pathology Group - Clinical Pathology Section ~ - Histopathology Section - Necropsy Seciion \ Pathophysiology ..... Group - Chronic Studies Section - Clinical Section 1 PJoooiil I(. Joroa!Jss Assoc!mi!lf IDI~C!!Idor . -1 IHeall~ f'roteciion Unii I ~ Computer Unii ~ Qu~lity Assurance Unii H Educational Program 4 ~~tll!rnllli Audliaov ' I -i EI nglnearIlnllJ & f"scilntes Unii HFinancial 1\/lanagemsmt IIJniQ I HHrwfliaro IPiesourcas J Unill H UI'DrmU)f urln H ProcrwwsmenR U,i'lill HPropaoily ~BIU'ISlg011l11l0riJil Uril~ - 'VGchnlcl!l!l ..._ Comm~nnic~lons IUn~ rl O~~lUiualreisJsilH.CliHucoalcl'>rlmln ~ ~~ l~a$lrlunsofotm\l. ~rcltC811' lOirll!lCI!C!T H l iAnimal CSJuc& UIT1i1l 91oB'Mlihram8l~ics SW!o1111 H IAGrosoD IP'mgr!BJm H j'' I Chsmlcai "ii"ollicoiogv PJadidloo Do~-RIG$poi"'Sill IP'u~usm H iF'rogreJm Chronic !E){posUJrs ~ RladJIQIJOOJJi!:llrc1~ ~lmraU11!7 IP'ro@usrn fP'rog)UBIM , HCIEoltmf)~XPlsann~~s IP'mijrsm II ~ !l".l!lfthCt!Jcmes!s fP'tl[)llJtSlM II H IJohii'11 IF. ledmau Ass!3~BJI"DU IOiratctor I H IMolecular til Cailular Tolllcology Group ,j~Chamic.m! Cancrsr Mechanisms Program tl~ AMedmisl:dunmi~mSC!anPcreorg' r8lm BP3 35589 ) ttl ""0 I 0 00.... \0 ++N---- to account for all financial transactions and manpower data as well as for activities relating to property management and other support services activities. Monthly printed reports on curreni period and year-to-date transactions are provided to all appropriate personnel for management purPoses. finally, it shoula be noted that a year-end financial audit is conducted on the Institute's prior fiscal year financial transactions--by U. S. Government auditors to assure compliance with federal regulations and to verify an approiJ"iate indirect cost basis which is applicable to all sponsors for whom research has been conducted. Kn summary, we feel that the Institute has the effective and experienced management team required to conduct both cost-effective and high-quality research for sponsors. KX. RESEARCH PROJECf MANAGEMENT AND PERSONNEL A multidisciplinary team of scientists has been chosen for work on this project. Table 1 lists the members of the research team. Curriculum vitae of all professional staff are included in the Appendix. The ITRI scientists with primary responsibility for the management and conduct of these proposed stuaies are-Dr. C. H. Hobbs and Dr. R. f. Henderson. In the context of the Good Laboratory Practice Regulations, Dr. Henderson will be the Study Director (Principal Xnvestigator) for the studies and as such she-is responsible for ove-rall conduct of the studies and will serve as the contact at ITRI for API for conducting the studies. -Dr. :Henderson is trained as a biochemical toxicologist and has--extensive experience in the_ toxicokinetics of inhaled vapors. Dr. Kirk Maples is a chemist-aAd will be in charge of monitoring the exposures and analyzing samples for benzene and its meiabolites. ------------- 13 BP3 35590 BP-00019245 Table 1 Summary of Scientific l?ersoimel with Substantial Hnvolvement in the l?roject Name Henderson, R. f. Maples, K. JR. Chang, l-Y. Degree/Discipline Ph.D., Biochemical Tw:icologist Ph.D., Chemist, Toxicologist M.S., Statistician Role in Sti!dy Study Director (Principle investigator) Co-Investigator, Metabolite -Analysis Co-Investigator, Statistical Analyses 14 ~------- BP3 35591 BP-00019246 Dr. n-Yiin Chang is a statistician with experience in analysis of animal toxicology data. She will be responsible for all statistical analyses of data on the effect of exposure onconcentration the level of benzene metabolites, tissues, and excreta samples. Appropriate technical and other supponing personnel wm be assigned ao the project to meet project objectives. The professional, technical and other supporting personnel will-work as a team. Should the need arise for technical expertise not available from personnel on the aeam, assistance-of other members of the Institute staff will be enlisted. The HnstiMe retains the authority to assign equivalent personnel or otherwise use Institute personnel, within the limits of the authorized budget, to best achieve the project's objectives. X. R-EPORTS At 6-mo intervals, a letter report on the progress of the project will be submitted to API by LIBERI. The LBERI will immediately notify API of any private or govemmentaJ_request for information on ongoing or completed r-esearch conducted under this Agreement, including any subpoena or other legal instrument requesting information. The Contractor will cooperatefully with any effort by API to narrow the scope of any such request, to obtain a protective order limitingJhe use or disclosure of irfformation. XI. PUBUCATIONS API and the LBERI agree to full public disclosure of scientific information developed through this Agreement. The LBERI shall not release such information without prior API knowledge and review. 15 , . - - - - - - -------....... BP3 35592 BP-00019247 The scientific conclusions and professional judgments arising out of performance of the -project-Will be the responsibility of the LlBlERL The LlBlER!, however, will not publish or othetwise release data, conclusions, or manuscripts in a citable or quotable form without prior APE knowledge and opportunity to comment. API opportunity to comment is for the purpose of clarification and format or editorial comments, but not for abe purpose of substituting API's opinion for that of the LBERn. XII. liNSTITUTIONAL RESOURCES A General The Inhalation Toxicology Research institute (ITJR.ll) has been operated by the Lovelace organization for the U. S. -Department of Energy (DOlE) or its predecessor agencies since the early 1960's. Ht is currently operated under a DOE five-year, cost-reimbursable, no-fee contract by the Lovelace Biomedical and Environmental Research Institute (LBERI), a subsidiary- of the Lovelace Medical Foundation (LMF). LBERI's only mission is to operate ITRI for DOE and to conduct research for DOE and other sponsors where appropriate. LBERI has no financial assets other than those iris responsible for managing for the United States government or its other sponsors. ITJR.I facilities are totally government owned and are located on Kirtland Air Force Base East in Albuquerque, New Mexico. These facilities contain approximately 271,000 ft2-of space for laboratories, animal breeding and maintenance activities, offices and essential support services. Sincel:lrst occupied in 1963, new additions or modifications have been added on nearly an annual basis to meet the ever-changing nature of the institute's research program. These facilities represent one of the most modern and complete 16 - - - - - - - - - - ..... BP3 35593 BP-00019248 laboratories for the conduct of inhalation toxicology research that are available anywhere in the world. Pertinent details are provided below. lB. facilities The. Institute's government-owned facilities consist of approximately 271,000 ft2 of space. They include a central laboratory, office and small animal and primate maintenance complex, dog kennels on either side of the centrallaboraiory and spedalized facilities for machine shop, electronics shop, analytical chemistry, rodent quarantine and rodent breeding, primate quarantine, receiving and warehouse, health protection and waste handling located more peripheral to the central complex. lFrom the standpoint of this proposal, the following facilities are considered to be of principal importance: 1. Modern analytical chemistry laboratories containing state-of-the-art instruments. The instrumentation includes liquid scintillation counters, spectrotluorometers, high-pressure Hquid chromatographs with auto . injectors and auto sample collectors, UV and VJ[S spectrophotometers, gas chromatograph/mass spectrometers with a computer data system, gas chromatographs, a laboratory data system for quantitation of GC and HPLC data. infrared spectrometer with a computerized data system, and a nuclear magnetic resonance spectrometer. 2. Laboratories for safely developing and-testing generation procedures for inhalation exposure environments using either radiolabeled or unlabeled materials, including potential carcinogens. These laboratories are ---... 17 BP3 35594 BP-00019249 equipped with instruments for physicochemical characterization of the exposure environments. 3. Hazardous material storage and preparation area designed and constructed for storage of hazardous materials in a controlled access area. The facility has storage capabilities adjacent to a preparation area. This area provides for storage of radioactive materials in a shielded vault and storage of nonradioactive materialS- in a cabinet or refrigerator. The preparation room is equipped with a hot cell and three hoods for preparing aerosol generator. solutions and other operations related to preparing materials for. use in inhalation exposures. 4. Inhalation exposure facilities specifically designed to allow controlled access to an area fully equipped for acute inhalation exposures. Each of two exposure complexes within the facility has its own control room; two exposure rooms and a pr-eparation room-. Associated equipment in the facility includes an ultrasonic unit for decontamination of exposure apparatus and an electron microscope. A variety of laboratory animals have been exposed nose only in the facility, ranging in size from mice to dogs. Materials aerosolized for animal expesures nave included chemical carcinogens, a variety of radioactive compounds and wpors. Exposures have included respiratory monitoring by whole-body plethysmography in some cases and the facility is equipped to make these respiratory measurements for small laboratory animals, dogs and monkeys. 18 , r - - - - - - - - BP3 35595 BP-00019250 S. Quarantine facilities are available for isolation and stabilization of animals received from commercial suppliers. Rigid standard operating procedures are followed during the quarantine period while the health status of newly acquired animals is evaluated. 6. A barrier-sustained, rodent housing facility is operated on a clean-dirty corrido~ system. All personnel must shower before entering the facility and must wear clean laboratory-supplied clothing, face mask and gloves. All materials entering the facility are sanitized (i.e., cages, lids, caps, personnel clothing). Feed is pasteurized by suppliers and all bedding is sterilized prior to use. All items that cannot be entered through the tunnel washer or autoclaved are chemically sterilized. The animal rooms are maintained at 20 to 23C and at 30 to 50% relative humidity under a 12 hr light and dark cycle. ITRI is a registered research facility, in .compliance with Section 6 of P.L. 89-544 (as amended), the Laboratory Animal W-elfare Act, and has been fully accredited by- the American Association for Accreditation of-Laboratory Animal Care since 1910. The last accreditation inspection was conducted in June, 1988. Animals are maintained in accordance with the recommendations in NIH Publication No. 85-23, entitle-d~ "Guide for the Care and-Use of Laboratory_ Animals" and in compliance with the Laboratory Animal Welfare Act. - 7. A cellular toxicology faci-lity is specifically designed and staffed to test the effects of chemical toxicants, carcinogens and radiation on ..,..------.----- 19 BP3 35596 BP-00019251 eukaryotic and prokaryotic cells in culture. JEqually important are the capabilities in this facility to determine the in vitro functional capacities of cells exposed to toxicants in vivo. A FAC:STAR PLUS (Becton!Dickinson) flow cytometer is available. 8. A comprehensive program for hazardous waste handling and disposal. Radioactive and hazardous wastes generated at the Institute are collected, treated, packa-ged and shipped in sccordance with-~U EPA, DOT and DOE regulations, and in-a manner that minimizes hazard to personnel or the environment. Hazardous chemical wastes-are collected and handied in-a manner consistent with the requirements of the Resource Conservation and Recovery AcL(RCRA). As w1th radioactive wastes, all hazardous chemical wastes are shipped to EPA-approved disposal sites for final disposal. 9. In-house computer capability consisting of a network of Digital Equipment Corporation VAX series computers. lit currently includes a VAX11n8o, three M-icro:VAX H computers and five PDP-11 series computers. The latter are required for r~al-time data acquisition. lExtensive use is also made of personal computers for automated-data collection, scientific calculations, word processing and graphics. These computer resources pro.V-ide facilities for collection, storage and processing of both scientific and business m management data for the total Institute. 20 BPJ 35597 BP-00019252 /... 10. Emergency electrical generation capability is provided to all essential areas of the facilities. This capability includes two diesel-powered 750 KW and two 100 KW generators. They receive routine testing on a weekly basis and require only six seconds for full activation. AJI of the facilities described above are incorporated into ~his emergency power grid. 11. A complete in-house shop capability including a machine shop, electronics shop and carpenter shop. The personnel in each shop area continue to provide the necessary experience and skills for abe nnitial design and fabrication or subsequent modification of prototype exposure chambers, characterization devices and other instruments required at ITRI. 12. An excellent research library which provides all services required to support our research program. These include current subscription to 300 journals, over 9,500 books-and approximately 19,000 documents. Local and regional interlibrary loan services are available through the University of New Mexico and a consortium of New Mexico Medical Libraries. On-line- literature search services are maintained with over 100 data bases through 4 major systems. These services will be essential for providing available literature on specific chemicals which HRI is requested to study. Although-this by no means describes the-Institute'"s total facility and equipment resources, it does desG:-ibe tbose-which would be of 21 ---------- ----. BPJ 35598 BP-00019253 major use in the conduct of the proposed research and demonstrates that nTRI has all critical resources necessary ro meet the needs of this research proposal. Further, the essential resources will be available on a noninterference basis relative uo our commitments to JDOE and other federal agencies currently supporting research &~ rnu. Xm. QUALITY ASSURANCE The research described in this proposal is ex:ploratory in nature and will be conducted in the spirit of the Good laboratory Practice (GLIP) regulations. Specific quality requirements will be addressed as integral parts of project-specific protocol(s) and standard opexating procedures-that might be needed. The approval of the ITRli Quality .Assurance Unit is required on all protocols and standard operating procedures at the HTRI. General requirements for research conducted in the spirit of GLPs at iTR.I are that study personnel will h~ve documented training and experience; that work will be done following ITRIapproved protocols and applicable RTR.I-approved standard operating procedures; that study personnel follow good recordkeeping procedures-and verify the accuracy of data collected; that personnel maintain calibration and maintenance documentation for equipment and instruments; and that'study personnel ensure test chemicals, reagents, and specimens integrity throughout collection, analysis' and storage processes. Jt has been our elCperience that research such as tQis is difficult to conduct within strict GLJP guidelines due to the many adjustments that occur in the research plan as protocols are developed and Conducted. ----22 - ------------ BP3 35599 BP-00019254 API staff, representatives of sponsoring companies, and consultants retained by API will have reasonable access to LBEJRY personnel and facilities engaged in the work coveredby this Agreement. API may designate one or more consultants who may observe or audit the conduct of the work by this proposal. The UBERI shaJI cooperate fully with such observations and audits. All records, new data or other documentation relating to the studies covered by this Agreement shall be retained by LlBlERI according to GLP regulations for a period of at least ten (10) years from the date of conclusion of the studies. The materials retained pursuant to the above paragraph shall be stored in an archive which ensures that they will be maintained in a safe and secure manner and allows for their expeditious retrieval when needed. Material retained in the archive shall be indexed by test substance, date of study, test system and nature ofstudy. lln the event that the Contractor or its archiving facility discontinue operations or for other reasons are not able to archive the material, all raw-data, specimens and other documentation pertaining to the testing covered by this Agreement shall be transferred to such other facility as API may direct. XIV. !HEALTH PR0TECfKON All applicable ITRI health and safety requirement will be adhered to. These cover general laboratory practices. Specific safety requirements will be included as an integral part of the protocols and standard operat4ng procedures required for the studies. The approval of the ITRI Health Protection Operations Unit is required on all protocols and standard operatingprocedures at the XTRL 23 r - - - - - --- ----- ... BP3 35600 BP-00019255 XV. CARE AND USE OF LABORATORY ANIMALS The use of laboratory anima-ls in w.eJI-designed biomedi~al research studies has resulted in significant advances in health care and prevention of disease in humans. Animals are used at the XTl!U in well-defined studies ao identify the health risks ao man from exposure to potentially toxic agents in the absence of an adequate understanding of their effects on man. Other methods such as cell culture are used whenever possible to reduce the number of animals needed in the studies. JH[owever,~adequate alternatives to the use of laboratory animals for the studies proposed here are not currently available. IV. such, the use of laboratory animals for these studies is necessary. We recognize that the use of animals in studies such as these is a privilege, not a righl Thus, policies have been instituted to insure the ethical and~humane care and treatment of animals at the XTIU. This is accomplished through review and approval of protocols using animals and the establishment of standard operating procedures for their care. liTRI has a current National Institutes of Health assurance number for anima-l care and use;~that number is A3083-01. XTRI is also fully accredited by 1he American Association for Accreditation of laboratory Animal Care; our accreditation number is 000200. ITRI has an officially constituted "Animal Care and Use Committee"; the responsibilities of the Committee include reviewing all animal care and usage at lTRI. The Committee is responsible for assuring that all animals at ITRX are kept, treated, and used in a humane and lawful manner. This draft proposal is currently under review by the I1RI Animal Research Committee and will be approved prior to final submission to API. i1RUs a registered-research facility with the USDA; our registration~number 85-R-003. ITRI is in full compliance with the Animal 24 BPJ 35601 BP-00019256 Welfare Act as administered by the USDA. JITRK is in full compliance with all other relevant local, state, and federal regulations concerning animal care and use. . XVl!. l?ROJECf COSTS A. General The costs given in Table 2 are our best estimates of the costs for conducting ahe research outlined in the proposal. Because -Of uncertainties of the exact protocols that will be used, the budget may require modification as the protocols are finalized or as the direction of the research is modified from the findings in early stages of the research. The XTRI is operated by the lovelace Biomedical and Environmental Research Xnsiliute-(LBER!) for ihe DOE. The LBERU is a non-profit corporation which operates on a cost-reimbursable no-fee basis. Therefore, the costs for performing the research will in-all thecases be the actual costs {labor, supplies, services, etc.) of the effort expended to do work. B. Basis for Cost Estimates The cost estimates in Table 2 are for -the level of effort stated and our best estimate of the cost of supplies required to do the studies outlined in the proposal. The -salaries used are those projected ta be in effect for the period 1 Jan 199l-through 31 Dec 1996 (3 years). As work progresses, we reserve the right to change personnel assignments and to execute procurement actions based on. research results obtained, the nature of proposed research, and our considered scientific and managerial judgment. !Procurement activities will be carried out in accor-dance with DOE procurement regulations in-effect at the time _purchases are made. ,-- - ----- . ----- -- 25 BP3 35602 BP-00019257 Table 2 Projected Cost !Estimate !for Dosimetll1' of Benzene nn .the Bone Marrow PERSONNEL R. Henderson, Princip'al llnvestigator K. Maples, Chemist I. Chang, Statistician To be Assigned, Re~earch Technologists Total Salary Fringe Benefits Supplies/Services Animal Purchase Miscellaneous supplie~/glassware Subtotal Travel Other Expenses Animal maintenance Word processing service Illustration service Computer service Subtotal Total Direcf Costs !ndirect Costs Total Costs Year 1 Effort $ Year 2 Effort $ Year 3 Effort $ Total Effort $ I .15 .15 .15 .45 tO .20 .20 .60 .05 .05 .05 .15 .40 .40 AO 11..20 36,800 38,600 40,500 9,200 9,700 10,100 115,900 29,000 1,000 Ua.QQQ 16,000 3,000 l,WO ~ 16,600 3,200 1,li.OO ~ Jl.7,ll00 3,400 3,200 46.500 49,700 9,600 600 500 500 lJ100 2,600 67,600 47,300 114.900 600 1,100 !,100 1...1.00 3,900 72,000 50,400 122.400 600 1,200 1,200 1.200 4,200 75,300 52,700 128.000 1,800 2,800 i:~cig 10,700 214,900 150,400 365.3QQ ... 'SP3 35&03 ) ttl '"0 I 0 0 0...... \0 N (J1 00 The fringe benefit rate is calculated as a percent of total salaries and includes the following: FICA Medical insurance Retirement Benefits JLife linsurance Disability linsurance Unemployment Compensation Workmen's Compensation This rate has had an increasing trend, attributable to increases in the employerpaid FICA rate as well as medical insurance. The 24 percent rate-used is based on the last three years' operating experience. The overhead costs inc1ude those costs associated with functions such as general management and administration, business office, pr-ocurement, property management, personnel, facilities operation and ma~ntenance, library, quality assurance, and health protection operations. The Institute's overhead cost rate is calculated and adjusted monthly with an annual rate set at the end of each fiscal ~ear. All costs incurred are audited 11ormally by U. S. Government auditors, and all overhead rates are verified. The last fiscal year audited was lFY-1991 and, as in previous years, no exceptiOns in- rates were noted. The Institute's overhead e)(penses are distributed as a percentage rate on all modified total direct costs. AJthough the rate-in FY-1992 was lower than what we are projecting for FY-1993, it is a provjsional rate-and actual charges will be made. 27 ,.---- - ------------ ....._ BP3 35604 BP-00019259 . In-house support services include the costs of animals, animal care, histopathology preparation, shop, computer, word processing and illustration services that are provided within the Institute on a centralized basis. The costs of these services are charged to direct projects on a pro rata basis dependent upon the level of service used by each individual project The number of animals used and the days of animal maintenance have been based on the proposed work plan. The level of shop, computer, word processing and illustration services required for this project were estimated based on our experience with similar projects. Travel expenses are -f-ar investigators to present results at national meetings and to report results at meetings called by the AlP!. The estimated budget is written for a 3-year period-even though we realized that the initial contract will be for one year only. ----28 ,------BP3 35605 BP-00019260