Final Report Protocol FY98-056 FINAL REPORT TIER 2 TESTING OF BIODIESEL EXHAUST EMISSIONS Study Report Number FY98-056 Submitted to: National Biodiesel Board (NBB) 1907 Williams Street - P.O. Box 104898 Jefferson City, Missouri 65110 Submitted by: Lovelace Respiratory Research Institute (LRRI) P.O. Box 5890 Albuquerque, NM 87185-5890 May 22, 2000 Total numbers of pages 1005 1 Final Report Protocol FY98-056 TABLE OF CONTENTS Page TABLE OF CONTENTS.................................................................................................................2 COMPLIANCE STATEMENT.......................................................................................................7 LIST OF ACRONYMS ...................................................................................................................8 CONTRIBUTING LRRI PERSONNEL .........................................................................................9 SUBCONTRACTING CONTRIBUTORS .....................................................................................9 REPORT SIGNATURES ..............................................................................................................10 QUALITY ASSURANCE STATEMENT ....................................................................................11 TEST MATERIALS CHARACTERIZATION/STABILITY.......................................................12 ARCHIVAL STORAGE ...............................................................................................................12 SUMMARY...................................................................................................................................13 I. INTRODUCTION .............................................................................................................14 II. MATERIALS AND METHODS.......................................................................................14 A. Study Dates ............................................................................................................14 B. Protocol and Experimental Design ........................................................................14 C. Test Fuel Characterization .....................................................................................14 D. Exposure Generation System.................................................................................14 E. Animals and Animal Husbandry............................................................................17 F. Animal Exposures..................................................................................................18 G. Body Weights.........................................................................................................18 H. Mortality and Clinical Observations......................................................................19 I. Feed Consumption .................................................................................................19 J. Ophthalmologic Evaluation ...................................................................................20 K. Positive Control Study of Neuropathologic Lesions in Acrylamide-Treated Rats ........................................................................................................................20 L. General Histology Group.......................................................................................22 M. Special Histology Group........................................................................................22 N. Clinical Pathology..................................................................................................22 O. Glial Fibrillary Acidic Protein Assay ....................................................................26 P. Micronucleus Assay...............................................................................................26 Q. Sister Chromatid Exchange Assay.........................................................................27 R. Individual Animal Fertility ....................................................................................28 S. Reproductive Toxicology and Teratology .............................................................29 T. Salmonella Typhimurium Reverse Mutation Assay...............................................29 III. RESULTS AND DISCUSSION........................................................................................31 A. Test Fuel Characterization .....................................................................................31 B. Animals and Animal Husbandry............................................................................31 C. Animal Exposures..................................................................................................31 D. Body Weights.........................................................................................................35 E. Mortality and Clinical Observations......................................................................35 2 Final Report Protocol FY98-056 F. Feed Consumption .................................................................................................36 G. Ophthalmologic Evaluation ...................................................................................36 H. General Histology Group.......................................................................................37 I. Special Histology Group........................................................................................42 J. Histology Discussion .............................................................................................44 K. Clinical Chemistry .................................................................................................45 L. Hematology............................................................................................................46 M. Glial Fibrillary Acidic Protein Assay ....................................................................47 N. Micronucleus Assay...............................................................................................48 O. Sister Chromatid Exchange Assay.........................................................................50 P. Individual Animal Fertility ....................................................................................52 Q. Reproductive Toxicology and Teratology .............................................................53 R. Mutagenicity Testing .............................................................................................53 IV. CONCLUSIONS................................................................................................................54 V. REFERENCES ..................................................................................................................58 LIST OF TABLES AND FIGURES Table Page 1 Experimental design for Tier 2 study.................................................................................15 2 Animal numbers and assignments for Tier 2 study. ..........................................................16 3 Cerebellar lesions found in acrylamide-treated rats...........................................................21 4 Criteria for severity grading of lung lesions. .....................................................................23 5 Clinical chemistry analytical methods...............................................................................25 6 Hematology tests................................................................................................................25 7 Summary of NOx concentration data. ................................................................................32 8 Summary of particulate and gas analyte concentration data..............................................32 9 Particle size distribution of biodiesel exhaust particulates measured at two times during the study..................................................................................................................34 10 Organs examined at histology............................................................................................38 11 Incidence and severity of lung lesions after 13 weeks of exposure...................................38 12 Comparison of lung lesions after 13 weeks of high-level exposure and 13 weeks of high-level exposure with 28 days recovery. ..................................................................42 13 Clinical chemistry data analysis.........................................................................................46 14 Summary data for glial fibrillary acidic protein assay.......................................................47 15 Summary data for micronucleus evaluation from bone marrow cells from biodiesel study rats.............................................................................................................49 16 Summary data for sister chromatid exchange assay. .........................................................51 3 Final Report Protocol FY98-056 Figure Page 1 An increased number of AMs containing black particles was characteristic of the lung lesions in the rats exposed to the high concentration.................................................39 2 AMs in the rats in the highest exposure concentration contained many black particles that obscured the cellular details and nucleus of the cell (arrows)......................40 3 Alveolar bronchiolarization (arrows), lining of ADs and alveoli adjacent to TBs with ciliated and Clara cells, was seen in a few airways of rats in the highest exposure