PB81-230955 Respiratory Carcinogenicity of Diesel Fuel Emissions IIT Research Inst. Chicago , IL Jul 81 U.S. DEPARTMENT OF COMMERCE National Technical Information Service NTIS ------- PB8 1-230*955 EPA-buu/i-oi-U34 July 1981 RESPIRATORY CARCINOGENICITY OF DIESEL FUEL EMISSIONS by Alan M. Shefner IIT Research Institute Life Sciences Research Division 10 West 35th Street Chicago, Illinois 60616 Grant No. R806326-01-1 Project Officer Donald E. Gardner Environmental Toxicology Division Health Effects Research Laboratory US Environmental Protection Agency Research Triangle Park, NC 27711 OFFICE OF RESEARCH AND DEVELOPMENT HEALTH EFFECTS RESEARCH LABORATORY US ENVIRONMENTAL PROTECTION AGENCY RESEARCH TRIANGLE PARK, NC 27711 ------- TECHNICAL REPORT DATA iPleaie read Insxmctions oh the reverie be fort completing) 1. SSPORT NO. EPA-600/1-81-054 2. ORD Report 3- AGCp|jg|IT-S 4. -tTLS ANO SUBTITLS RESPIRATORY CARCINOGENICITY OF DIESEL FUEL EMISSIONS S. REPORT OATS ¦ llllv 1Q81 8. PERFORMING ORGANIZATION CODE 7. AUTWCRISI Alan M. Shefner 3. PERFORMING ORGANIZATION REPORT NO. 9. PERFORMING ORGANIZATION NAME ANO ADDRESS IIT Research Institute Life Sciences Research Division 10 West 35th Street Chicago, IL 60616 10. PROGRAM ELEMENT NO. A9GA1A 11. CONTRACT/GRANT NO. R806326-01-1 12. SPONSORING AGENCY NAME AND ADDRESS Health Effects Research Laboratory RTP, NC 13. TYPE OP REPORT ANO PSRIOO COVERED Office of Research and Development U.S. Environmental Protection Agency Research Triangle Park, NC 27711 14. SPONSORING AGENCY CODE EPA-600/11 15. SUPPLEMENTARY NOTES Project Officer: Donald E. Gardner 18. ABSTRACT The objective of this program was to evaluate the possible respiratory carcinogenic effects of diesel fuel emission particles and organic extracts of these particles in suitable animal models. Because of our previous experience in the use of the Schreiber method for localized tumor induction and the rapid response time observed in this model with certain known direct acting carcinogens it was planned-to initiate our studies with this model. Subsequently studies were to be initiated using the Saffiotti technique for intratracheal instillation for evaluation of life-time effects. This report also describes certain studies which were initiated but not completed due to decisions concerning program relevance and to choices made concering the expenditure of available program funds. 17. KEY WORDS ANO DOCUMENT ANALYSIS a. DESCRIPTORS b.IOENTIFIERS/OP6N SNOED TERMS c COSAT1 Field/Group 13. ;isr^iaur:aN 57atsm6:MT RELEASE TO PUBLIC 19. SECURITY CLASS (This Report) UNCLASSIFIED 21. n°2§,: ?AGes 20. SECURITY CLASS (This pagt) UNCLASSIFIED 22. PRICE SPA form 2230-1 (9-7J) ------- DISCLAIMER This report has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the U.S. Environmental Protection Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. 11 ------- FOREWORD The many benefits of our modern, developing, industrial society are accompanied by certain hazards. Careful assessment of the relative risk of existing and new man-made environmental hazards is necessary for the establishment of sound regulatory policy. These regulations serve to enhance the quality of our environment in order to promote the public health and welfare and the productive capacity of our Nation's population. The Health Effects Research Laboratory, Research Triangle Park, conducts a coordinated environmental health research program in toxi- cology, epidemiology, and clinical studies using human volunteer subj- ects. These studies address problems in air pollution, non-ionizing radiation, environmental carcinogenesis and the toxicology of pesticides as well as other chemical pollutants. The Laboratory participates in the development and revision of air quality criteria documents on pollu- tants for which national ambient air quality standards exist or are proposed, provides the data for registration of new pesticides or proposed suspension of those already in use, conducts research on hazardous and toxic materials, and is primarily responsible for provi- ding the health basis for non-ionizing radiation standards. Direct support to the regulatory function of the Agency is provided in the form of expert testimony and preparation of affidavits as well as expert advice to the Administrator to assure the adequacy of health care and surveillance of persons having suffered imminent and substantial endanger- ment of their health.. This grant was initiated to provide information relating to the potential respiratory carcinogenicity of diesel emission products. F. Gordon Hueter Director Health Effects Research Laboratory iii ------- ABSTRACT The objective of this program was to evaluate the possible respiratory carcinogenic effects of diesel fuel emission particles and organic extracts of these particles in suitable animal models. Because of our previous ex- perience in the use of the Schreiber method for localized tumor induction and the rapid response time observed in this model with certain known direct acting carcinogens 1t was planned to initiate our studies with this model. Subsequently studies were to be initiated using the Saffiottl technique for intratracheal instillation for evaluation of life-time effects. The program was initially planned for a three-year period with the major emphasis during the first year to be placed on short-term studies with the Schreiber model. Shortly after program initiation a scientific review meet- ing was held on the Diesel Emission Health Effects Research Program (December 12, 13, 1978 at Arlington, Virginia). As a result of this meeting and other program considerations concerning risk assessment utility, emphasis under this grant was gradually shifted to the utilization of the Saffiotti intra- tracheal instillation model. In addition, program plans were modified to include the assessment of coke oven extract, roofing tar extract and cigarette smoke condensate on which a human epidemiologic data base existed. As a result of those considerations this grant was terminated after one year and an extensive study utilizing the Saffiotti technique was initiated under Cooperative Agreement No. R806929-01-0 on September 1, 1979. Some of the initial dose range studies which were started on the original program were continued and completed under the successor program and have been reported upon as required. This report also describes certain studies which were initiated but not completed due to decisions concerning program relevance and to choices made concerning the expenditure of available program funds. This report was submitted in fulfillment of Grant No. R806326-01-1 under the sponsorship of the U.S. Environmental Protection Agency. This report covers the period from October 2, 1978 to September 30, 1979, and work under this grant was completed as of September 30, 1979. 1 v ------- CONTENTS Page Disclaimer ii Foreword i i i Abstract iv Tables vi Figures vi Abbreviations vii Acknowledgments vi i 1 1. Introduction, Conclusions and Recommendations 1 2. Materials and Methods 2 3. Experimental Procedures . 10 Localized Treatment with DP Extracts 10 Intratracheal Instillation Experiments with the Saffiotti Method 10 Lung Clearance of Diesel Particles 10 4. Results and Discussion 13 Localized Treatment with DFE (Schreiber Method) ... 13 Dose-Range Study of Diesel Emission Particles ... 15 Lung Clearance of Diesel Particles 17 References 20 Appendi ces A. Safety Plan for Handling of Carcinogens and Test Materials 21 B. Protocol for the Collection and Submission of Necropsy Specimens (L6109) 24 v ------- TABLES Number Page 1 Particle Size Distribution of the "As Received" DP (Cumulative Number % Greater Than the Stated Diameter 3 2 Particle Size Distribution of Diesel Particle and Ferric Oxide Suspensions (Cumulative Number % Greater Than the Stated Diameter, .... 6 3 Assay of 1.0 ml Aliquots of Typical Suspensions 7 4 Percent Increase in Body Weight and Percent Death in Hamsters Administered Diesel Fuel Extract by Localized Intratracheal Instillation 14 5 Dose-Range Study to Determine Toxicity of Diesel Fuel Particles Administered by the Saffiotti Technique 16 FIGURES Number Page 1 Experimental Design for Dose-Range Study to Determine Toxicity of Diesel Fuel Extract Using the Localized Intratracheal Instillation Technique 11 2 Experimental Design for Dose-Range Study to Determine Toxicity of Diesel Fuel Extract Using the Saffiotti Intratracheal Instillation Technique 12 iv ------- LIST OF ABBREVIATIONS BA — Benz(a)anthracene BP — Benzo(a)pyrene DFE — Diesel Fuel Emission Extract DMBA — Dimethylbenz(a)anthracene DMSO Dimethyl Sulfoxide DP — Diesel Fuel Emission Particles ETOH » «• Ethyl Alcohol Fe2°3 — Ferric Oxide GEL — Gelatin MNU — Methyl nitrosourea PG Propylene Glycol vii ------- ACKNOWLEDGMENTS The diesel fuel exhaust particles and the dichloromethane extract of exhaust particles used in this program were supplied by the Environmental Sciences Research Laboratory, EPA, Research Triangle Park, North Carolina through the courtesy of Drs. Ronald Bradow, Roy B. Zweidinger and SHvestre Tejade. viii ------- SECTION 1 INTRODUCTION, CONCLUSIONS, AND RECOMMENDATIONS This grant was initiated to provide information relating to the poten- tial respiratory carcinogenicity of diesel emission products. Emphasis was placed on the Schreiber model for localized tumor induction since it offered the possibility of a more rapid determination of adverse effects. Following a presentation at a Diesel Emission Health Effects Program meeting 1n Dec- ember of 1978 and subsequent program discussions it was decided to deempha- size work using the Schreiber model and to initiate dose range toxicity studies by the Safflotti intratracheal instillation technique. This decision was reached for three primary reasons. 1. The Safflotti method offers a proven and widely accepted model for Inhalation effects. 2. Total dose administered 1s known with the Safflotti technique but not for the Schreiber method. 3. The Saffiottl method is ideal for the evaluation of diesel emission particles which cannot be tested using the Schreiber technique. Since EPA's program for the evaluation of the health effects of diesel fuel emissions was aimed at developing a series of potency comparisons between diesel emissions and other materials on which there existed an epidemiologic data base, it was recoiranended thatthis respiratory carcino- genesis study also Include an evaluation of these additional materials. Therefore the remainder of the first year of this grant was devoted to initiating dose range studies with diesel emission particles using the Safflotti method. This grant was terminated after the first year and the dose range studies were incorporated into and completed under a Cooperative Agreement (R806929-01-0) which was entered into on September 1, 1979. Results of these studies were summarized in a series of quarterly progress reports (IITRI Report Nos. L6109-1 through 3). The results of studies which were 1n progress at the time this grant was terminated were summarized in reports on the successor program (IITRI Report Nos. L6119-1 and L6119-2). 1 ------- SECTION 2 MATERIALS AND METHODS TEST MATERIALS Diesel Particle and Diesel Particle-Ferric Oxide Suspensions Diesel fuel exhaust particles (DP) were supplied to IITRI as dry loose powders packed under nitrogen, in dry ice. Exhaust from several runs of the EPA's 350 Oldsmobile diesel test engine were collected on a single teflon coated glass fiber filter. The exhaust particles were then scraped from the filter surface to produce the loose powders supplied to IITRI. Characteristics— Optical microscopical examination of the powder indicated no significant contamination of the OP by teflon and glass fibers. Microscopical examin- ation also indicated geometric particle sizes were too large for intra- tracheal instillation. Particle morphologies indicated that aggregation of the individual submlcrometer carbon particles comprising the exhaust had occurred both on the collection filter and before particles reached the filter. The presence of large diameter (up to 70 ym) hollow carbon spheres indicated some aggregation occurred before deposition on the filter. The predominant aggregate morphology — flakes ranging in thickness from 0 to 0.5 ym - - could have been formed both before and after deposition on the filter. The size distribution of the DP aggregates "as received" is shown in Table 1. Attempts to disperse the powder into its constituent submicrometer particles by conventional techniques for deagglomeration further confirmed the conclusion that the DP observed microscopically were indeed aggregates rather than agglomerates. Further microscopical examination revealed-that the only way the powder could be dispersed into the individual exhaust particles was by dissolving the condensed organic species (extractable materials) which formed a cementing agent that held the ultra fine carbon particles in sheets and hollow spheres. Since microscopical analysis In- dicated that ;ome of the aggregates had been formed before the exhaust was captured on the filter the most logical approach to reducing the particle size distribution of the powder to a range suitable for intratracheal in- stillation was to crush the aggregates. 2 ------- TABLE 1. PARTICLE SIZE DISTRIBUTION OF THE "AS RECEIVED" DP (CUMULATIVE NUMBER % GREATER THAN THE STATED DIAMETER) Geometri c Diameter ym 2 Aerodynami c Di ameter um Greater Than Stated Diameter (No. %) 0 0 100 1.0 0.4 83.9 3.0 2.0 54.3 • 5.5 2.6 21.9 8.0 3.4 10.2 10.5 4.1 5.4 13.0 4.6 2.3 15.5 5.3 1.0 18.0 5.8 0.5 20.5 7.3 0.2 ^"Geometric Diameter is the diameter of a circle having the same projected area as the DP. 2 Aerodynamic Diameter is the diameter of a sphere having the same volume as the DP, assuming an average thickness of 0.3 ysn for the DP aggregates. 3 ------- Suspension Preparation— Diesel exhaust particles (DP) were to be prepared as suspensions in gelatin-saline, with end without ferric oxide (Fe203). Concentrations of DP were to be 5, 3 and 1 mg per 0.2 ml of suspension. DP suspensions with FepOg contained equal mass concentrations (5, 3 and 1 mg/0.2 ml) of DP and Fe^O-; Particle sizes below 10 ym were required. The low density of the DP powder, its wide particle size range (0-150 ym), its hydrophobic and electrostatic natures, and sterility requirements precluded the possibility of dry grinding the powder to the desired size range, before suspension in gelatin-saline. Rather, size reduction and sus- pension of the reduced particles were best accomplished simultaneously. This was achieved by use of a ball milling technique. Milling apparatus consisted of wide mouth, cylindrical, pyrex glass jars with silicon-rubber lined bakelite caps; 3-5 mm solid pyrex glass beads; and a variable speed roller. The milling jars were half-filled with glass beads before the appropriate volume of saline and masses of gelatin and iron oxide were added. The jars were then loosely covered with the bakelite caps and autoclaved. After the jars had cooled, the DP were added. Because of the hydrophobic nature of the DP, they were first mixed with a wetting agent, propylene glycol (PG) (7% by volume of the total suspension). The resulting paste was then quickly transferred to the sterilized milling jar. The control suspensions also contained propylene glycol and were ball milled. A milling time of 10 days was required to reduce 95% by mass of the DP aggregates below 10 ym. The completed suspension was separated from the milling balls with a buchner funnel. The funnel as well as the collecting vessels were all auto- claved before use. The DP tended to agglomerate in the suspension. The agglomerates in fresh suspensions could be redispersed by simple shaking. However, aged suspensions required more vigorous deagglomeration methods, and suspensions over 4 weeks old could not be deagglomerated. Therefore, suspension volumes sufficient for three weeks of instillation were prepared at one time. To prevent particle size distribution changes with repeated deagglomerations, suspensions were supplied in vials containing one week's worth of material which was stored at -70 C until administered. Suspension Characterizations- Suspensions supplied for intratracheal Instillations were checked for sterility, particle size distribution and mass concentrations of suspended particles. Standard culture techniques were used to check each vial supplied for sterility. Optical microscopical examination of a small aliquot of each suspension consisted of determining the largest aggregate size present and estimating the per cent of oversized particles present. Previous work with benzo(a)pyrene ball milled suspensions has indicated that particle size distributions are consistent from batch to batch. Therefore, detailed 4 ------- particle size distribution analyses were performed by image analysis on only three of the batches prepared. Table 2 lists typical particle size distri- butions for DP and Fe-O., suspensions. Note that the additions of the Fe-03 served to further reduce DP particle sizes. No significant differences fn particle size distributions were noted between the various concentrations prepared. Assays of suspended particle concentrations were performed by several different methods. Total suspended particle concentrations were determined by filtering aliquots of suspension through tared 0.05 ym pore size membrane filters. Aliquot size used was 0.5 to 1.0 ml, depending upon the suspended particle concentration. For the DP suspensions, alone, this assay method provided the concentration of the DP particles in suspension. For the DP- Fe203 suspensions, however, further assaying was required to provide mass concentrations of each particle type. Iron concentration was determined by digestion of a 0.2 ml aliquot of suspension in hydrochloric acid; the dis- solved iron was separated from the insoluble carbon particles by filtration. Iron concentration in the filtrate was determined spectrophotometrically. This assay method was specifically used to determine delivered dose concen- trations. DP concentrations in the DP-Fe^ suspensions were determined by low temperature ashing the filter containing both the DP and Fe^Oj particles (from the total suspended particle assay). The total mass lost during ash- ing was the mass of the filter plus the mass of the carbonaceous diesel ex- haust particles. Aliquots of each batch prepared were assayed by filtration and ashing techniques. Table 3 presents the typical results of these two assay methods. Reproducibility of the filtration technique was determined to be ± 3%. The two different assaying methods produced DP concentrations with ± 52 of each other. Actual DP concentrations were consistently lower than the theoretical concentrations, by 7 to 10%. The low concentrations can be attributed to two factors: solvation of some components of the DP (up to 52 of the total DP mass according to the literature); and the difficulty in completely transferring a suspension from one vessel to another, particularly during separation of the grinding media from the suspension. Known Carcinogens Certain experimental protocols required the administration of known carcinogens to test hamsters. BP, BA, DMBA and MNU were handled with the precautions described in the appended program Safety Plan (Appendix A). 5 ------- TABLE 2. PARTICLE SIZE DISTRIBUTION OF DP AND DP-FE-O, SUSPENSIONS (CUMULATIVE NUMBER % GREATER THAN THE STATED DIAMETER Geometric Diameter urn Aerodynamic Diameter um 0 0 1.0 0.4 3.0 2.0 5.5 2.6 8.0 3.4 10.5 4.1 13.0 4.6 15.5 5.3 18.0 5.8 20.5 7.3 Greater Than Stated Diameter No. % of DP No. % of DP + Fe„i 100 to 100 83.7 84.9 48.2 44.7 20.5 19.7 7.9 8.2 2.6 2.1 1.0 0.7 0.3 0.2 0.1 0.1 0 0 Geometric Diameter 1s the diameter of a circle having the same projected area. 2 Aerodynamic Diameter is the diameter of a sphere having the same volume as the particles, assuming a thickness of 0.3 ym for the DP. 6 ------- TABLE 3. ASSAY OF 1.0 ML ALIQUOTS OF TYPICAL SUSPENSIONS Theoretical Masses,mg Assay By Filtration Assay By Masses, mg Ashing Suspension* DP Fe^Oo Total DP Feo0, Total DP Feo0* Total DP-H1 25 25 22.8 22.8 23.8 23.8 DP+FE-Hi 25 25 50 - 50.7 24.4 26.3 50.7 DP-Med 15 - 15 13.9 13.9 13.3 - 13.3 OP+Fe-Med 15 15 30 - 29.6 13.8 15.8 29.6 DP-Lo 5 - 5 5.3 5.3 5.3 - 5.3 DP+Fe-Lo 5 - 10 • 9.4 4.4 5.0 9.4 *Ht = 5 mg/0,2 ml Med = 3 mg/0.2 ml Lo = 1 mg/0.2 ml 7 ------- ANIMALS Male Syrian Golden hamsters used in these studies were purchased from ARS Sprague-Dawley, Madison, Wisconsin or Engle Labs, Farmersburg, Indiana at 4 to 5 weeks of age. Upon arrival and inspection for general state of health, animals were housed three per cage in polycarbonate cages (18 1/2 x 10 x 8 1/2 in.). Cages, bedding, water bottles and food were changed twice weekly. Bedding consisted of Ab-Sorb-Dr1 or Pine-Dri and food (Wayne Blox) and tap water were provided ad libitum. Animal rooms were lighted with fluorescent lights on a 12-hour light/dark cycle. Animal room temperature was maintained at 20°C to 24°C with ambient relative humidity of 30 to 70%. All animals were quarantined for a period of at least two weeks prior to study initiation. During this time the health status of the hamsters was evaluated by physical examinations, gross necropsy and routine diagnostic microbiological workup. No abnormal pathology was observed in the quarantine hamsters. Animal Identification Animals were randomized into group sizes required by the experimental design. Hamsters were identified uniquely within each experiment by group, cage and individual animal number. Individual identification was achieved by ear punching of the hamsters. Individual cage cards contained group, cage and individual animal information. Observations Animals were observed twice daily for signs of toxicity or morbidity. Once weekly hamsters were weighed and given a physical examination. Animals that were moribund were Isolated and observed more closely. Hamsters were allowed to die spontaneously, killed when moribund or sacrificed as specified by protocol. Individual necropsy forms were prepared with results of gross examination. Selected tissues and any lesions were submitted for histopath- ologic evaluation. A typical protocol for necropsy 1s presented in Appendix B. INTRATRACHEAL INSTILLATION (SAFFIOTTI TECHNIQUE) The method used is that previously described by Saffiotti and co-worker (1,2). Before each intratracheal instillation the hamster is anesthetized with halothane using an Airco Veterinary Anesthesia Machine, Heedbrink Model 960 (3). When the righting reflex is lost the animal is hung on a slanted board, its back on the board and its mouth kept open by hanging the lower incisor teeth on a wire hook, while the upper incisors are retained by a tight rubber band. A volume of 0.2 ml of the test material 1s drawn into a 0.25 tuberculin syringe. The syringe is fitted with a blunt 19 gauge needle about 3 in. long and bent at a 135° angle at 45 mm from the tip. A clear view of the pharynx is provided by a headlight worn by the operator The tongue is pulled outward with a forceps and the rhythmic opening and 8 ------- closing of the vocal cords observed. The blunt end of the needle is inserted into the tracheal lumen past the open vocal cords the moment they are open. The needle is pushed almost to the bottom of the trachea and the suspension gently injected. The hamster is kept on the board for a minute to make certain no suspension is regurgitated. LOCALIZED INTRATRACHEAL INSTILLATION The device used for localized treatment of the hamster trachea was originally reported by Schreiber et at C4,5) and modified In our laboratory (6). A catheter fabricated of stainless steel is introduced into the trachea and 0.5 ml of the material under study is expressed from the outer tubes of the catheter. Simultaneously the inner tube of the catheter assembly is extended an additional 5 mm further into the trachea. The inner tube is attached to a vacuum system and the test solution is reabsorbed through the tip of the inner catheter. The test solution is applied for a period of five seconds during which period the vacuum exhaust is also in operation. The vacuum continues for an additional two seconds following which the catheter is withdrawn. 9 ------- SECTION 3 EXPERIMENTAL PROCEDURES GENERAL CONSIDERATIONS Two general classes of experiments were carried out under this program. One series utilized a procedure for localized treatment of the trachea (6) and the other employed the Saffiotti model for intratracheal instillation. In addition a pilot study to explore the respiratory tract retention of a single dose of diesel particles was carried out. Localized Treatment with Diesel Particle Extracts Three initial experiments were carried out using this technique in order to determine dose ranges of diesel extract suitable for future experi- ments. A number of solvent mixtures including dimethyl sulfoxide, propylene glycol, ethanol and saline were investigated for their ability to solubilize the extract at desired concentrations. A typical experimental design is illustrated in Figure 1. Intratracheal Instillation Experiments with the Saffiotti Method A dose-range study of DP to determine suitable doses for the conduct of the chronic studies was carried out as shown in Figure 2. The diesel part- icles were prepared as suspensions both with and without ferric oxide as described under Materials and Methods. Hamsters received 0.2 ml containing the indicated levels of DP or DP-Fe203 with the exception of the 10 mg dose groups which received two 5 mg treatments weekly. Surviving hamsters were sacrificed five weeks following the 15-week treatment period and selected tissues taken for histologic processing. Lung Clearance of Diesel Particles A limited study of lung clearance of diesel particles was carried out on a small group of hamsters. Each animal was administered a single dose of 5 mg DP by intratracheal instillation and pairs of animals were killed at 1 hour, 8 days, 30 days and 60 days post treatment. The lungs and trachea of one animal at each time period were photographed and lung and trachea from the second animal were processed for histopathologic examination. 10 ------- Figure 1. Experimental Design for Dose-Range Study to Determine Toxicity of Diesel Fuel Extract Using the Localized Intratracheal Instillation Technique. Group No. of Hamsters Treatment 1 25 Solvent (10% DMS0, 10% ET0H, 20% PG, 60% saline) 2 50 1.00% (5.00 mg) DFE 3 50 0.50% (2.50 mg) DFE 4 50 0.25% (1.25 mg) DFE 5 10 Saline 6 10 Shelf Control Male Syrian Golden hamsters (Engle Labs.) received the initial instillations at 7 wks of age. Volume of solution delivered at each instillation was 0.5 ml. Animals were treated lX/wk for 10 wks. All animals which survived the treatment period were sacrificed at 12, 15 and 25 wks after the initial treatment. 11 ------- Figure 2. Experimental Design for Dose-Range Study to Determine Toxicity of Diesel Fuel Particles Using the Saffiotti Intratracheal Instillation Technique. Group No. of Hamsters Treatment Frequency 1 50 5 mg DP 2X/wk 2 50 5 mg DP lX/wk 3 50 3 mg DP lX/wk 4 50 1 mg DP lX/wk 5 50 5 mg DP + 5 mg FegOj 2X/wk 6 50 5 mg DP + 5 mg FegOg IX/wk 7 50 3 mg DP + 3 mg Fe20j lX/wk 8 50 1 mg DP + 1 mg Fe£®3 lX/wk 9 50 3 mg BP + 3 mg Fe203 lX/wk 10 50 1.5 mg BP + 1.5 mg Fe20j lX/wk 11 50 5 mg FegOj in solvent 2X/wk 12 50 Solvent, 0.5% gel-saline, 7% PG 2X/wk 13 25 Shelf Controls NONE Male Syrian Golden hamsters (Engle Labs.) received the initial instillation at 12 wks of age. Volume of suspension delivered at each instillation was 0.2 ml. All animals received 15 wks of treatment and survivors were sacrificed 5 wks after the final treatment. 12 ------- SECTION 4 RESULTS AND DISCUSSION LOCALIZED TREATMENT WITH DFE (SCHREIBER METHOD) A series of preliminary experiments were carried out on the dichloro- methane extract of diesel emission particles. The catheter design used in this technique requires that the test material be in solution and of relat- ively low viscosity. A number of solvent mixtures containing varying quant- ities of PG, DMSO, ethanol, and saline were tried as appropriate solvents in this system. Suitable dosage forms containing up to 1% DFE were prepared and evaluated for toxicity. Three preliminary experiments were carried out in which weekly treatments were carried out for ten weeks (Experiments 1 and 2) and for fifteen weeks (Experiment 3). The results of these experiments, suiranarized in Table 4, Indicate no untoward effect on weight gain or on mortality that can be attributed to treatment with DFE. Although it appeared possible therefore to prepare and test extracts by this technique it was decided not to continue with studies using this method of treatment. The localized treatment method cannot be used with particle suspensions so no direct comparison of the carcinogenic potential of DP and DFE can be carried out. In addition this technique has not been widely used and it has not been demonstrated that the trachea 1s responsive to the classes of combustion products that are present in DFE. Thirdly the total dose of test material delivered to the hamster cannot be readily determined in this procedure. The concentration of test material to which the test animals are exposed is known, but how much of the test material is retained by the animal following the reabsorbtion phase of the Interbation cycle is uncertain. Thus it was concluded that while the method is of potential Interest as a research tool, program emphasis was to be placed on carcinogenicity trials using the better established intratracheal Instillation method of Saffiotti. 13 ------- TABLE 4. PERCENT INCREASE IN BODY WEIGHT AND PERCENT DEATH IN HAMSTERS ADMINISTERED DIESEL FUEL EXTRACT BY LOCALIZED INTRATRACHEAL INSTILLATION Experiment 1 Experiment 2 Experiment "3 % Increase % Increase % Increase Treatment in Body Weight % Death in Body Uelqht % Death in Body Weight % Death Solventa 66 Qb 81 8 88 0 1.0%, DFE 66 8 76 6 85 4 0.5%, DFE 50 Q 59 10 87 6 0.25t, DFE 35 12 63 2 64 6 Saline N.A. N.A. 63 10 96 0 Shelf Control 49 0 62 0 92 0 aSolvent varied for each experiment. ^Percent of spontaneous death during 6 months of holding. ------- DOSE RANGE STUDY OF DIESEL EMISSION PARTICLES An experiment was carried out to provide information for selection of doses for the chronic study of diesel particles by the Saffiotti method. Diesel particles and DP-Fe203 mixtures were prepared by ball milling as de- tailed in Materials and Methods. Hamsters were treated at weekly doses of 1, 3, 5, and 10 mg. Animals at the 10 mg weekly level were treated twice weekly with doses of 5 mg each. Hamsters were held for 5 weeks following the 15 weeks of treatment, survivors were sacrificed, and tissues were processed for histopathologic examination. Table 5 shows the results obtained in this experiment in terms of mortality and percent increase in body weight over the twenty weeks of experiment. No treatment associated increased mortality was seen with the exception of the 3 mg benzo(a)pyrene positive control group which produced a high degree of lethality and a decreased weight gain. Twice weekly treat- ments with 5 mg DP or with 5 mg DP admixed with 5 mg of Fe203 led to a decreased weight gain as compared to control animals also receiving twice weekly instillations. Microscopic examination of histologic sections of hamsters on this study led to the following conclusions. 1. There was no significant non-respiratory tract pathology 1n treated animals. 2. Lesions of the lungs were common in treated animals and Included these findings: a. Adenomatous hyperplasia is more severe and extensive in DP treated animals than in vehicle control animals. b. Adenomas are more numerous and larger 1n the DP treated animals than in the vehicle control. c. Metaplasia to ciliated epithelium and squamous metaplasia occurred 1n some DP treated animals but not in vehicle controls. d. Severe multifocal reactive pneumonitis with hyperplasia and evidence of atypia occurred in DP treated animals but did not occur in vehicle controls. 15 ------- TABLt 5. DOSE-RANGE STUDY TO DETERMINE THE TOXICITY OF DIESEL FUEL PARTICLES ADMINISTERED BY THE SAFFIOTTI TECHNIQUE TREATMENT FREQUENCY ONCE WEEKLY TWICE WEEKLY Treatment No. of Animals Percent Mortality Percent Increase In Body Weight Percent MortalIty Percent Increase 1n Body Weight 5 mg DP 50 6 14 6 8 3 mg DP 50 2 14 - — 1 mg DP 50 6 14 - — 5 mg DP:5 mg Fe203 50 6 11 10 10 3 mg DP:3 mg Fe203 50 4 10 - — 1 mg DP:1 mg Fe20j 50 4 10 - — 3 mg BP:3 mg Fe20.j 50 38 7 - — 1.5 mg BP:1.5 mg Fe20 3 50 12 10 - — 5 mg Fe20-j 50 - — 6 13 Solvent 50 t - — 10 13 None 25' 0 9 ------- The degree of severity and frequency of occurance of hyperplastic, metaplastic, and dysplastic changes in lung tissue of hamsters given twice weekly treatments of 5 mg DP each plus the decreased weight gain observed in these treatment groups led to the conclusion that the high dose selected for the chronic study would be 5 mg of diesel particles, once weekly, for 15 weeks. LUNG CLEARANCE OF DIESEL PARTICLES Hamsters were treated with a single instillation of 5 mg DP and pairs of animals were sacrificed at time periods of 1 hour, 8 days, 30 days and 60 days post treatment. Color photographs were taken of en bloc and exploded views of the lungs and trachea from one animal of each pair. The respiratory tract of the other ani»nal was processed for histopathology. Photographic reproductions of the lungs and trachea of animals in this study were included in IITRI Reports L6109-1 and L6109-2. These photographs showed that the particles were dispersed throughout the lung tissue as well as the trachea at one hour post instillation. Eight days after the instill- ation the particles were still very prevalent in the lungs, but appeared to be mostly cleared from the trachea. At 30 days post instillation, the part- icles remained heavily concentrated in the lungs. Gross observation of a hamster sacrificed 60 days post instillation showed particles still remaining in the lung but in apparently reduced amounts. Lungs and tracheas of additional hamsters killed at these four time periods following a single administration of 5 mg DP were also processed for histopathologic examination. A summary of the histopathologic findings on these animals follows. 79-393 DP 1 Hr. • Lung— There were numerous regions with diesel particles within the alveoli, bronchioles and bronchi. The diesel particles varied in form and in size and were found in spaces and upon the surfaces of the structures of the lung. There was no reaction to the diesel particles by the lung tissues. • Trachea— Diesel particles were found upon the mucosal surface. 17 ------- 79-395 DP 8 Days • Lung— Macrophages were present in numerous places within alveolar spaces and within septa. Within the macrophages there were diesel particles and extracellularly also there were diesel particles. Some of the diesel particles were on the epithelial surface of the alveolar septa. The internal structure of most of the macrophages was not visible because of the density of the diesel particles, however some macrophages did have one to several nuclei visible. The alveolar and bronchiolar epethelium was hyperplastic and proliferating in several places where there were diesel particles and macrophages. These lesions, focally, combined to form adenomatoid and papillomatoid structures. • Trachea— The diesel particle material had penetrated, in small amounts, into the submucosa. 79-397 DP 30 Days • Lung— In many places there were macrophages within alveolar spaces and septa; within the macrophages there were diesel particles. The alveolar and bronchiolar epithelium, in association with the macrophages bearing particles was hyperplastic and proliferating 1n several loci. The macrophages were less numerous than in the lung tissue from case 79-395 tsee above). • Trachea— Similar to that seen in case 79-395 (see above). 79-520 DP 60 Days • Lung— Similar to some extent to case 79-397 except that there was more- hyperplasia and proliferation of the alveolar and bronchiolar epithelium. There was also hyperplasia and proliferation of the bronchial epithelium. t Trachea— Similar to that seen in case 79-395. 18 ------- The study demonstrated that the Saffiotti method of intratracheal instillation led to good dispersion of the diesel particles throughout alveoli, bronchioles, and bronchi. Although no quantitative estimates of clearance rates can be obtained the photographic representations and tissue sections both showed that diesel particles were still present in good number even 60 days after a single instillation of 5 mg of DP. 19 ------- REFERENCES 1. Saffiottl, U.: Experimental respiratory tract carcinogenesis; in Prog, exp. Tumor Res., vol. 11, pp. 302-333 (Karger, Basel 1969). 2. Saffiotti, U.: CEFIS, F., and KOLB, L.H.: A method for the experimental induction of bronchogenic carcinoma. Cancer Res. 28: 104-124 (1968). 3. Smith, D. M., Goddard, K. M., Wilson, R. B. and Newberne, P. M.: An apparatus for anesthetizing small laboratory rodents. Lab. Animal Science 23: 869-871, 1973. 4. Schreiber, H. and Nettesheim, P.: A new method for pulmonary cytology in rats and hamsters. Cancer Res. 32: 737-745 (1972). 5. Schreiber, H.; Schreiber, K., and Martin, D. H.: Experimental tumor induction in a circumscribed region of the hamster trachea: correlation of histology and exfoliative cytology. J. Natn. Cancer Inst. 54: 187-197 (1975). 6. Grubbs, C. J., Moon, R. C., Norikane, K., Thompson, H. J. and Becci, P.J.: 1-Methyl-l-nitrosourea induction of cancer in a localized area of the Syrian golden hamster trachea; in Prog. exp. Tumor Res., vol. 24, pp. 345-355 (Karger, Basel 1979). 20 ------- APPENDIX A SAFETY PLAN FOR HANDLING OF CARCINOGENS AND TEST MATERIALS General safety precautions for personnel handling the carcinogens benzo- (a)pyrene (BP) and methyl nitrosourea (MNU) will be in accord with the overall IITRI safety program which meets or exceeds Federal (OSHA) and State Stan- dards. This program is under the overall jurisdiction of the IITRI Safety Officer. The IITRI medical center, which is staffed by an industrial health R.N., will be available for health problems and will keep complete records on all staff members involved in handling the carcinogens. Preparing and Handling Methyl nitrosourea MNU is highly toxic, explosive, and a very potent carcinogen. It is sensitive to light, heat, high pH, and moisture. 1. The chemical, will be kept at -70° 1n a sealed container until subdivided. 2. Before being opened and weighed, the chemical will be removed from the freezer and defrosted at room temperature. 3. The container will be opened in a safety hood maintained under negative pressure and subdivided into smaller containers. 4. Multiples of four bottles, housed in a larger container with desiccant, will be refrozen at -70 . 5. In preparation for intratracheal injection, the following steps will be carried out in a negative pressure hood: a. Weigh Erlenmeyer flask. b. Weigh MNU. c. Add MNU to flask and weigh both as a check on the weight of MNU. d. Add vehicle (steroid suspending solution) to achieve stock concentration. e. Cover flask with parafilm. f. Heat to 30°C in a water bath for 15 min to solubillze MNU. g. Dilute stock to achieve injecting concentration. 21 ------- 6. In all instances, two buckets of sodium carbonate solution (pH >_ 10) will be present and, should any portion of the body or personal attire be contaminated, the worker will IMMEDIATELY flood himself with sodium carbonate solution. Preparing and Handling of Dimethylbenz(a)anthracene DMBA 1s a potent carcinogen and will be handled using the same pre- cautions as described for MNU. Preparing and Handling Benzo(a)pyrene in Ferric Oxide BP is a potent carcinogen and is relatively Insensitive to light, temperature, moisture, or pH. It is solubel in nonpolar solvents and in- soluble in water. For administration to hamsters, the BP will be combined with FegOj as a nucTeated preparation. 1. The dry chemical will be stored in the refrigerator for not longer than six weeks. 2. To prepare for intratracheal injection, the following steps will be carried out in a negative pressure hood: a. Weigh Erlenmeyer flask. b. Weigh BP-Fe203 c. Add carcinogen to flask and reweigh as a check on the weight of BP-Fe-O,. d. Add gelatin-saline venicle and stopper flask. e. Vortex for 10 min. Intratracheal (IT) Administration of Carcinogens 1. During all IT procedures, suspenion and solutions will be covered at all times except when supplemental vortexing is performed Ce.g., with BP-FegOj suspensions). 2. Laboratory counter tops will be covered with absorbant paper backed with a plastic sheet. 3. The cover from the flask containing the carcinogen will be removed and an aliquot withdrawn into a 0.25 ml syringe. 4. After the carcinogen has been administered to the animal, the IT needle will be replaced and the next animal treated. Both syringe and needel will be replaced after six hamsters have been treated. 5. All treated animals and their cages will be handled only by persons wearing gloves, masks, and laboratory coats. 22 ------- Cleanup Procedures 1. MNU a. All instruments and glassware used in preparing and administering MNU will be placed in sodium carbonate solution (pH > 10) for at least 18 hrs, washed with soap and dried far reuse. b. All used gloves, masks, contamination mats, and all other disposable material will be placed in sodium carbonate solution overnight. These will then be bagged in plastic and incinerated. 2. BP-Fe203 a. All instruments and glassware will be soaked in 1% Alconox* solution for at least 1 hr, rinsed and sonicated in fresh Alconox solution, rinsed in distilled water and dried for reuse. b. All gloves, masks, contamination mats, and other disposable material will be bagged in plastic and disposed of by incineration at 1800°F. 3. DMBA a. Bag and incinerate all waste and disposables. Personnel Considerations 1. All personnel will wear surgical gloves, face masks, and laboratory coats. 2. All project team members will be instructed in <1 handling and safety precautions. 3. Procedures and Precautions will be posted 1n appropriate places 1n the laboratory. 4. New personnel will be given safety training before joining the program. 5. The Principal Investigator will initially and periodically monitor operations to ensure adherence to all protocols. 23 ------- APPENDIX B PROTOCOL FOR THE COLLECTION AND SUBMISSION OF NECROPSY SPECIMENS (L6109) INTRODUCTION Prior to sacrifice of animals on project L6109 the following procedures will be used: 1. Final weighing of animal. 2. Animal necropsy forms completed with accession number. 3. Specimen jar labeled with experiment, animal group data, and accession number written on a small piece of card, and placed in the jar and on specimen jar tape. PROCEDURES FOR NECROPSY EXAMINATION Anesthetic and Verification Station (.1 Person) Animals will be anesthetized in a CO? chamber. After the animal is anesthetized, it is removed from the chamber. This animal with its necropsy form and specimen jar 1s carried to the necropsy examination station. After the necropsy has been completed the necropsy form and specimen jar is returned to verifier, for verification, before the animal carcass is dis- carded. Necropsy Examination Station (2 to 3 Persons) Person performing the necropsy examination will record the gross- observation findings on the necropsy form. . All pertinent information con- cerning the animal must be recorded 1n the information block at the top of the form. No pathologic diagnosis will be made to gross; all abnormalities will be described as to location, color, consistency and size. The path- ologist will be called to view all unusual lesions. Pin the animal to the dissecting board exposing the ventral surface. Disinfect the area with 705! alcohol. Using scissors rapidly make a "V" shaped incision through the skin and muscle layers extending from the pubis to each axilla (.Observe all organs). 24 ------- Dissect the trachea, larynx, esophagus and lungs en mass. Inflate the lungs via intratracheal perfusion with 10% formalin, attach a paper clip to trachea before placing the lungs and accompanying tissue into formalin. Turn the animal over and remove the skin from the head including the fleshy tip of the nose, examine the nasal region for bulges or deviation, submit the intact maxillary region to histology for sectioning. When the gross necropsy examination is completed, the form should be signed, dated, and returned with the specimen jar to verification station. 1. Anesthetic and Verification (Charlotte Nicholes) 2. Necropsy Examination (Edward Clchocki) (Mary Ann Phee) (Lawrence Dooley) 25 ------- ORGAN CHECK LIST Accession No. 79- Project No. L6109 IT/N NK 1 V.N NN 1 ItTn NN EXTERNAL SURFACE/ORIFICES LIVER * ADRENALS AXILLARY LYMPH NODE NR NF GALL BLADDER * KIDNEYS • SKIN - ABDOMINAL URETERS SALIVARY GLAND-SMAX. SPLEEN * MUSCLE LYMPH NODE - MANDIB. PANCREAS SCIATIC NERVE MAMMARY GLAND PUBLIC SYMPHYSIS EAR/TAG NR NR PERITONEAL CAVITY OVARIES EAR CANALS Nft NR STERNUM UTERUS NR NR BRAIN * MARROW SMEAR -FD1UR VAGINA NR NR PITUITARY COSTO-CHONDRAL JUNCTION NR NR TESTES * EYES PLEURAL CAVITY VAS DEFERENS NR NR NASAL TURB. NR NR TONGUE NR NR PROSTATE SPINAL CORD - Thoracic with ribs THYMUS URETHRA SPINAL CORD-LUMBAR THYROIDS — BLADDER BLOOD SMEAR HEART * STOMACH ESOPHAGUS DUODENUM PARATHYROIDS JEJUNUM LUNGS * ILEUM L1 LARYNX CECUM L2 TRACHEA COLON L3 RESPIRATORY NODES RECTUM NR NR L4 AORTA MESENTERIC LYMPH NODE L5 T/N ¦ Tissue taken at necropsy and is normal NR ¦ Not Required NN « Tissue taken at necropsy and is not normal Lymph Node Organs are to be weighed Verified = Fixed tissues are checked to confirm the presence of the indicated organs or tissues. 26 L.N. * Tissues collected by: Date Read and reviewed by: Date Verified by: Date ------- OBSERVATIONS I WEIGHTS Species Hamster (m) F Study D Project No. 16109 Dose_ Age wk days Project Leader Dr. Clinton Srubbs Accession No. 79- Animal No. Duration wk Di ed/ft a c| Date days Testes Ki dneys Spleen Liver Heart Lungs Brain OBSERVATIONS Balance Operator Prosector Wet sectioner, call Pathologist Read and reviewed by: 27 Date ------- |