Document vBVNdJpxv12QrVkb3gJGqg5Oq

jent: To: Cc: Attachments: McDonald, Jacob Friday, June 30, 2017 2:26 PM 'stuart.johnson@vw.com' 'Michael Spallek' FY14-050_EUGT NHP Diesel Report_Final.pdf; Aerosol Contributing Scientist Report Final.docx.pdf Reports Stuart/Michael, Attached please find the final reports for the EUGT diesel study. We removed the BALF data, as I found some technical issues with it that made it difficult to interpret. The story shows that the new tech diesel decreased the inflammation shown in bronchial brushings and blood. These are the data I will submit to Inhalation Toxicology in a paper next month. We hope that you can submit the final ~70K of payment. Thanks, Jake l *o o Lovelacem Exposures to Diesel or Alternate Technology Emissions to Evaluate Biological Response in Non-Human Primates (NHP) Aerosol and Chemistry Contributing Scientist Report o Lovelace Respiratory Research Institute (LRRI) 2425 Ridgecrest Drive SE Albuquerque, NM 87108 Courier Address and Location of Laboratory: Bldg. 9217, Area Y Kirtland Air Force Base Albuquerque, NM 87115 o Test Facility: Principal Investigator: EUGT NHP Diesel Exposures Page 2 o f 43 Lovelace Respiratory Research Institute (LRRI) 2425 Ridgecrest Drive SE Albuquerque, NM 87108 Courier Address and Location of Laboratory: Bldg. 9217, Area Y Kirtland Air Force Base Albuquerque, NM 87115 Philip Kuehl, PhD LRRI Confidential EUGT NHP Diesel Exposures Page 3 o f 43 T ABLE OF CONTENTS_____________________ ________________________ Page No. 1.0 EXECUTIVE SUMMARY......................................................................................................... 7 2.0 MATERIALS AND METHODS...............................................................................................8 2.1 Test Vehicles.....................................................................................................................8 2.1.1 Old Technology Diesel Emissions Vehicle (1997 Ford F250)...................... 8 2.1.2 New Technology Diesel Emissions Vehicle (20)4 Volkswagen Beetle) .... JO 2.2 Fuel........................... .................................................................... .... 14 2.3 Chassis Dynamometer........................ ......................................... ... 5 2.4 Driving Cycle................................................................................. ... 18 2.5 Exposure System .......................................................................... ... 20 2.6 Characterization of the Exposure Atmosphere............... .......... ., 2.6.1 Gravimetric Analysis for Aerosol Mass Concentration 2.6.2 NOx Analyzers......... ................................. ................ . 2.6.3 C0/CO2 Analyzers.... ..................................................... 2.6.4 Total Hydro Carbon (THC) Analyzers.......................... 2.6.5 DustTrak Real Time Aerosol Concentration Monitor... 2.6.6 Fast Mobility Particle Sizer (FM PS)............................. ...23 ...23 ...23 ... 23 ... 24 ...24 ...25 3.0 RESULTS................................................................................................ 3.1 Aerosol <'haracterization...................................... .............. ......... 3.2 Exposure Gases Concentration.................................................... 3.2.1 Tunnel NOx D ata..... ......................................... ............ 3.2.2 Chamber Gases D ata......................................... ............. 3.3 Exposure Particle Size Distribution,.,......................... ................ ... 26 ...26 ... 30 ...30 ... 34 ... 38 Confidential EUGT NHP Diesel Exposures Page 4 of 43 L IST OF TABLES______________________________________________ Page No. Table I. Average gravimetric aerosol concentration (pgW jmeasured for three atmospheres.,.. 26 fable 2. Comparison ol'NO. N02 and NOx concentration in the dilution tunnel for three test atmospheres........................................................... 31 Table 3. Average o f chamber gases data for air, NTDE and OTDE exposures........................... 35 Table 4. A typical particle size distribution for NTDE exposure........................................ 40 Table 5. A typical particle size distribution for OTDE exposure........................................ 40 LIST OF FIGURES______________________ _________________________________ Page No. Figure 1, 1997 Ford F250 on the chassis dyno in the laboratory......................................... 8 Figure 2. 1997 Ford F250 on the chassis dyno in the laboratory....................................................... 9 Figure 3. OTDF exhaust connections to the dilution tunnel intake................................................. 10 Figure 4. Name plate of the OTDF. Navistar 7.3 L V8 diesel engine.............................................. 10 Figure 5. NTDE 2014/2015 VW Beette Diesel on the chassis dynamometer....................11 Figure 6. NTDF exhaust connection to the exhaust tunnel.... ............................................. 12 Figure 7. NTDE exhaust connection to the exhaust dilution tunnel................................................ 13 Figure 8. NTDE and OTDE vehicles side-by-side .................... 14 Figure 9. Label o f diesel fuel barrel used during the study..............................................................14 Figure 10. Mustang chassis dynamometer........................................................................... 15 Figure 11. Another view o f the dynamometer.............. 16 Figure 12. K.B for the Mustang dynamometer........................................... 16 Figure 13. Variable frequency drive for the cooling fan (left) and the dynamometer (right)....... 17 Figure 14. 32000 CFV1 cooling fan..................................... 17 Figure 15. Computer cart with the desktop computer with PowerDyne software.........................18 Figure 16. FTP driving cycle.............................. 19 Figure 17. Target and actual speed during a FTP cycle for a NTDE vehicle................................19 Figure 18. Target and actual speed during a FTP cycle for a NTDF vehicle....... 20 Figure 19. Schematic o f the exposure system.................................................................................. 21 Figure 20. An H-1000 whole body exposure chamber used in the study......................................22 Confidential EUGT NHP Diesel Exposures Page 5 o f 43 Figure 21. H-1000 exposure chambers used in the study. Associated piping can also be seen in the picture........................................................................................................................... 23 Figure 22. Analyzers used during this study to characterize the exposure atmosphere................ 24 Figure 23. TSI DustTrak Aerosol Monitor Model 8520...................................................................25 Figure 24. TSI Fast Mobility Particle Sizer.......................................................................................26 Figure 25. Graphical representation of the aerosol mass concentration in the exposure atmosphere......................................................................................................................... 27 Figure 26. Graphical representation of the aerosol mass concentration in the exposure atmosphere (DustTrak Data).............................................................................................28 Figure 27. Example of variation of aerosol concentration as a function o f time for NTDE vehicle exposure............................................................................................................................. 28 Figure 28. Example of variation o f aerosol concentration as a function of time for NTDE vehicle exposure............................................................................................................................. 29 Figure 29. Example of variation of aerosol concentration as a function of time for OTDE vehicle exposure............................................................................................................................. 29 Figure 30. Example of variation of aerosol concentration as a function of time for OTDE vehicle exposure............................................................................................................................. 30 Figure 31. Comparison of NO, N 02 and NOx concentration in the dilution tunnel for three test atmospheres........................................................................................................................ 31 Figure 32. NO, N 02 and NOx concentration in the dilution tunnel for an air exposure............... 32 Figure 33. Tunnel NOx, N 02, NO as a function o f time for 5May2015 NTDE Exposure........... 32 Figure 34. Tunnel NOx, N 02, NO as a function o f time for 6 May 2015 NTDE Exposure.........33 Figure 35. Tunnel NOx, N 02, NO as a function o f time for 7 May 2015 NTDE Exposure.........33 Figure 36. Tunnel NOx, N 02, NO as a function o f time for 27May2015 OTDE Exposure.........34 Figure 37. Chamber gases data for three exposure atmospheres.....................................................35 Figure 38. Chamber gases as a function of time for air exposure....................................................36 Figure 39. Chamber gases as a function of time for NTDE exposure o f 05 May 2 0 1 5................ 36 Figure 40. Chamber gases as a function of time for NTDE exposure of 06 May 2 0 1 5................ 37 Figure 41. Chamber gases as a function of time for NTDE exposure of 06 May 2 0 1 5................ 37 Figure 42, Chamber gases as a function of time for OTDE exposure.............................................38 Figure 43. Number size distribution for NTDE exhaust inside the exposure chamber using FMPS ........................................................................................................................................... 39 Confidential EUGT NHP Diesel Exposures Page 6 o f 43 Figure 44. Number size distribution for OTDE exhaust inside the exposure chamber using FMPS Confidential o o EUGT NHP Diesel Exposures Page 7 of 43 1.0 EXECUTIVE SUMMARY The objective o f this work was to evaluate the biological response in non-human primates (NHP) to exhaust o f Old Technology Diesel Emissions (OTDE) vehicles and compare it to that o f New Technology Diesel Emissions (NTDE) vehicle exhaust. The vehicles were operated on a chassis dynamometer on FTP duty cycle. 10 NHPs were exposed to filtered air (control), NTDE and OTDE, in this order. The exposures took place in whole-body exposure chambers where the animals were exposed to 4-hrs to the test atmosphere. The exposure atmosphere was characterized for aerosol mass concentration, carbon monoxide, carbon dioxide, nitrous oxides and hydrocarbons. The particle size distribution o f the test atmosphere was also measured. This report describes the vehicles used in the testing, the parameters of the chassis dynamometer, and NHP exposure conditions during these tests. Confidential EUGT NHP Diesel Exposures Page 8 of 43 2.0 MATERIALS AND METHODS This section describes the test vehicles, the parameters of the chassis dynamometer, and measurement systems used to characterize the exposure conditions during these tests. 2.1 Test Vehicles Following two test vehicles were used during this study: 2.1.1 Old Technology Diesel Emissions Vehicle (1997 Ford F250) OTDE vehicle used during these tests was a 1997 Ford F250 (Super Duty XL / XLT, Supercab 2D) with a 7.3 Liter diesel engine (Power Stroke 7.3 L, Navistar V8 225 BHP@3000 rpm, International Trucks). Figures 1-4 show the OTDE vehicle on the dynamometer. The truck was purchased from a local used-car dealer and went through inspection by a diesel mechanic before purchase. Figure 1 .1997 Ford F250 on the chassis dyno in the laboratory. Confidential EUGT NHP Diesel Exposures Page 9 o f 43 Confidential EUGT NHP Diesel Exposures Page 10 of 43 Figure 3. OTDE exhaust connections to the dilution tunnel intake. Figure 4. Name plate of the OTDE Navistar 7.3 L V8 diesel engine. 2.1.2 New Technology Diesel Emissions Vehicle (2014 Volkswagen Beetle) NTDE vehicle was a 2014/2015 Volkswagen Beetle (2.0 L Diesel) provided by Volkswagen of USA. Figures 5-7 show the vehicle on the chassis dyno. Confidential o EUGT NHP Diesel Exposures Page 11 of 43 Confidential EUGT NHP Diesel Exposures Page 12 of 43 Figure 6. NTDE exhaust connection to the exhaust tunnel Confidential EUGT NHP Diesel Exposures Page 13 of 43 Confidential Figure 7. NTDE exhaust connection to the exhaust dilution tunnel EUGT NHP Diesel Exposures Page 14 of 43 Figure 8. NTDE and OTDE vehicles side-by-side 2.2 Fuel Certified diesel fuel (Diesel .05 5Y5 Cert Fuel) from Chevron Philipps Chemical Company, The Woodlands, Texas) was used during this study. The specifications of the fuel are attached as Appendix A. Figure 9 shows the label from one of the fuel barrel used for the study. Confidential Figure 9. Label of diesel fuel barrel used during the study o EUGT NHP Diesel Exposures Page 15 of 43 2.3 Chassis Dynamometer A chassis dynamometer from Mustang Dynamometer, Twinsburg, Ohio (Figures 10-12) was used during this study. The dynamometer was an AC Hybrid Chassis Dynamometer, Sr. No. 15526. This dynamometer was supplied with an ABB Variable Frequency Drive (Fig. 13, ACS800-U10120-5+L502-P901, ABB). The system also included a cooling fan (Model A65-- 449AV139STFCP3, 32000 CFM, Hartzell, Piqua, Ohio, Figure 14) to simulate the wind velocity during road driving conditions. The cooling fan was controlled by using a variable frequency drive (Emerson Commander SK, Emerson Industrial Automation, Eden Prairie, Minnesota, shown on left side in Figure 13). PowerDyne PC software by Mustang Dynamometer installed on a Desktop computer (Figure 15) was used to control the dynamometer and the cooling fan. Figure 10. Mustang chassis dynamometer Confidential EUGT NHP Diesel Exposures Page 16 o f 43 Figure 11. Another view of the dynamometer Confidential Figure 12. KB for the Mustang dynamometer o EUGT NHP Diesel Exposures Page 17 of 43 Figure 13. Variable frequency drive for the cooling fan (left) and the dynamometer (right) Confidential Figure 14. 32000 CFM cooling fan EUGT NHP Diesel Exposures Page 18 o f 43 Figure 15. Computer cart with the desktop computer with PowerDyne software 2.4 Driving Cycle Both OTDE and NTDE were driven on the chassis dyno using a FTP (Federal Test Procedure) Driving Trace (U. S. Environmental Protection Agency). Figure 16 shows the vehicle speed as a function o f time for the FTP cycle. FTP cycle is 1371 seconds (approx. 23-minutes) cycle with a mean speed o f 19.56 mph and travelling distance of 7.45 miles. Multiple cycles were run successively for the duration o f exposures which were 4-hours long. The vehicles were started in idle before the start of exposures for at least 20-minutes. Figures 17-18 show the output from the Confidential EUGT NHP Diesel Exposures Page 19 o f 43 PowerDyne software with two representative FTP cycles, showing both the target and the actual speed during that particular cycle. 0w) o Time (seconds) T r a c e S p e e d (MPH) HC (PPM) 56-70 Figure 16. FTP driving cycle Mustang Dynamometer Driver's Trace Printout Date/Time Printed; Ql-Jul-15 12;22 PH 51.03 95.36 15-69 14.02 23,35 22.63 17-01 11.34 5.67 o.co 1-00 133.00 275-00 912.00 549.00 686.00 823.00 960.00 1097,001234.001371.00 Test Time (Seconds) Trace Speed (MPH) Figure 17. Target and actual speed during a FTP cycle for a NTDE vehicle Confidential EUGT NHP Diesel Exposures Page 20 of 43 / Tnyil{MPH) 10S9 1223 1361 69 205 341 477 613 749 885 1021 1157 1293 Time (Seconds) Figure 18. Target and actual speed during a FTP cycle for a NTDE vehicle 2.5 Exposure System Figure 19 shows the sketch of the exposure system used during this study. Non-human primates were exposed to the diluted diesel exhaust in whole-body exposure chambers (Figure 20, H-1000, Lab Products, Inc., Maywood, NJ). Each H-1000 whole body exposure chamber can house up to two NHPs. Two H-1000 chambers were setup in parallel for this study. Figure 19 shows the schematic for only one chamber. Figure 21 shows the exposure chambers and the dilution system used in this study. These chambers have internal volume of 1-m3. Exhaust from the test vehicle was connected to the dilution tunnel with about 300-cfm (8.5 mVmin) of filtered atmospheric air flow. A fraction of the diluted vehicle exhaust removed from the dilution tunnel was used as exposure atmosphere. This extracted exhaust from the dilution tunnel was further diluted with the HEPA filtered room air to achieve target 300-pg/m3 aerosol mass concentration in the exposure chamber. Confidential Exhaust to Atmosphere Diluted Vehicle y Exhaust Dilution Tunnel I NOx Analyzer HEPA Filtered Room Air Control Valve T XTb HEPA Filter Vehicle Exhaust Filtered Atmospheric Air Blower Diluted Veh.cle To Exhaust |\ Water Trap Figure 19. Schematic of the exposure system o EUGT NHP Diesel Exposures Page 22 o f 43 Confidential EUGT NHP Diesel Exposures Page 23 o f 43 Figure 21. H-1000 exposure chambers used in the study. Associated piping can also be seen in the picture 2.6 Characterization of the Exposure Atmosphere The exposure atmosphere o f the whole body exposure chambers was characterized using the following instruments: 2.6.1 Gravimetric Analysis for Aerosol Mass Concentration Aerosol concentration was measured inside the exposure chambers by collection o f known volumes o f the aerosol onto Teflo filters (P/N: R2PJ047, 47-mm diameter, Pall Corporation, Ann Arbor, Michigan). Filter samples were collected at a nominal flowrate o f 10.0 L/min throughout the entire duration o f all exposures. For air exposures and NDTE exposures, four samples were collected for 60-min duration each for a 4-hr exposure. For OTDE exposures, eight samples were collected for 30-min duration each. Aerosol concentration was determined by differential weight analysis of the filter samples. 2.6.2 NOx Analyzers Chemiluminescence NOx Analyzers from Teledyne Advanced Pollution Instrumentation (TAPI, San Diego, California) were used to measure the NO, N 02 and NOx concentration in the dilution tunnel and the exposure chambers. The analyzers were calibrated and spanned every day before the start o f the testing using certified calibration gases. 2.6.3 C0/C02 Analyzers Non-Dispersive Infrared (NDIR) analyzers from California Analytical Instruments, Orange, California were used to measure the concentration of carbon monoxide and carbon dioxide in the exposure chambers. The analyzer was zeroed and spanned every day before the start of the testing using certified calibration gases. Confidential EUGT NHP Diesel Exposures Page 24 o f 43 2.6.4 Total Hydro Carbon (THC) Analyzers Model 300M-HFID Flame Ionization Detector (California Analytical Instruments, Orange, California) was used for measurement of total hydro carbon in the exposure atmosphere. The analyzer was zeroed and spanned every day before the start o f the testing using certified calibration gases. Figure 22 shows the NOx, CO/C02 and THC analyzers used in this study. Figure 22. Analyzers used during this study to characterize the exposure atmosphere 2.6.5 DustTrak Real Time Aerosol Concentration Monitor The real time aerosol monitoring was performed by using a DustTrak Aerosol Monitor (Model 8520, TSI, Inc., Shoreview, MN). Figure 23 shows a DustTrak used in this study, DustTrak is a portable, battery-operated laser photometer which gives a real-time digital readout o f the aerosol Confidential EUGT NHP Diesel Exposures Page 25 of 43 concentration. It can be used to measure particle concentrations corresponding to PMto,PM2 s, PMio, or respirable size fractions. The instrument was used in PMio configuration. DustTrak was not used for actual measurement of aerosol mass concentration in the exposure chambers. It was only used as an indicator of change in the aerosol concentration during the transient duty cycles. Figure 23. TSI DustTrak Aerosol Monitor Model 8520. 2.6.6 Fast Mobility Particle Sizer (FMPS) A TSI Fast Mobility Particle Sizer (FMPS, Model 3091, TSI, Inc., Shoreview, Minnesota) was used to measure particulate matter in the ultrafine size range of 5.6 to 560 nm. The FMPS (Fig. 24) uses an electrical mobility technique and low-noise electrometers to determine particle size. The number, surface and mass median diameters and geometric standard deviations (GSD) were calculated with TSI FMPS software. Confidential EUGT NHP Diesel Exposures Page 26 o f 43 Figure 24. TSI Fast Mobility Particle Sizer 3.0 RESULTS 3.1 Aerosol Characterization Table 1 shows the gravimetric aerosol concentration measured during the exposure for three atmospheres. The average results for both chambers are also shown in Figure 25. Table 1. Average gravimetric aerosol concentration (pg/m3) measured for three atmospheres AIR NTDE OTDE Date 21-Apr15 22-Apr15 23-Apr15 Averag e Chambe r1 0.0 5.5 18.2 7.9 Chambe r2 0.0 11.6 0.0 3.9 Date 7-May15 8-May15 9-May15 Averag e Chambe r1 15.8 0.0 13.3 9.7 Chambe r2 2.5 0.8 5.0 2.8 Date 27Mav-15 28May-15 29May-15 Averag e Chambe r1 556.7 402.4 269.2 409.4 Chambe r2 497.0 313.3 328.2 379.5 Std. Dev. 9.3 6.7 Std. Dev, 8.5 2.1 Std. Dev. 143.8 102.1 Confidential EUGT NHP Diesel Exposures Page 27 o f 43 Filter Mass Concnetrations (pg/m3) for Three Exposure Atmospheres 600 500 E 400 0c 1 300 ucOf Uo 200 n 5 otOabo.# 100 < Chamber 1 Chamber 2 -100 Figure 25. Graphical representation of the aerosol mass concentration in the exposure atmosphere Figure 26 shows the aerosol mass concentration as measured by a TSI DustTrak. These are average concentration for NTDE and OTDE vehicles for chamber 1 and chamber 2. As mentioned earlier, this reading was only to get an estimate of the aerosol concentration variation in the test section and to indicate the transient nature of aerosol in the test section. Figure 27 and 28 are two representative graphs for aerosol concentration variation for NTDE vehicle over 4-hr exposure. Figures 29 and 30 show the same for OTDE vehicle. Note the difference in the vertical scale of the graphs for NTDE and OTDE. Minor peaks with NTDE may be caused by the movement o f NHP inside the chamber during the exposure. Confidential EUGT NHP Diesel Exposures Page 28 of 43 DustTrak Mass Concnetrations (mg/m3) for NTDE ___ and OTDE Exposure Atmospheres 0.3 0.25 0.2 E 'S E. >.15 U0.1 (0 16.05 o3 i Chamber 1 l Chamber 2 NTDE OTDE Figure 26. Graphical representation of the aerosol mass concentration in the exposure atmosphere (DustTrak Data) Aerosol mg/m3as a Function of Time for NTDE (DustTrak Data) _ _ 0.016 4 0014 0 .0 1 2 O - w 0.008 <3C 0.006 - O i/ i <4J L 11 1 . 1 i l i JiIAM r wJ m rnI mIjuj _________________ II >. i o t N u _____ itr 6: 14 6:43 7:12 7:40 8:09 8:38 9:07 9:36 10:04 10:33 11:02 Time (hrs) Figure 27. Example of variation of aerosol concentration as a function of time for NTDE vehicle exposure C o n fid en tial o o EUGT NHP Diesel Exposures Page 29 o f 43 Aerosol mg/m3as a Function of Time for NTDE (DustTrak Data) Figure 28. Example of variation of aerosol concentration as a function of time for NTDE vehicle exposure Figure 29. Example of variation of aerosol concentration as a function of time for OTDE vehicle exposure Confidential EUGT NHP Diesel Exposures Page 30 of 43 Figure 30. Example of variation of aerosol concentration as a function of time for OTDE vehicle exposure 3.2 Exposure Gases Concentration The following sections describe the data from the gas analyzers for this study. The first section describes the data from the NOx analyzer measuring the readings in the dilution tunnel (Fig. 12 shows the location of this analyzer). The second section describes the data from NOx, C 0/C 02 and THC analyzers from the whole-body exposure chambers for three atmospheres. 3.2.1 Tunnel NOx Data Figure 31 shows the comparison of NO, NO2 and NO* in the dilution tunnel for the three test atmospheres. The data is also shown in Table 2. It can be seen from the graph and data that concentration of these gases in OTDE vehicle diluted exhaust were significantly higher than those in the air and NTDE vehicle exhaust atmospheres with minimal difference between air and NTDE atmospheres. Figure 32 shows typical NO, N 02 and NOx concentrations in the dilution tunnel for air exposures. Figures 33-35 show the details of these concentrations for NTDE exhaust. All three graphs are reported as we noticed some peak in the concentrations starting 9:20 hrs on 6-May tests which continued for 45-minutes and appears to have spilled over to the earlier part o f the test on 7-May (Figure 35). This is probably due to regeneration cycle o f the exhaust system o f NTDE, however, it will be confirmed once the vehicle data is reviewed by VW of USA. Figure 36 shows the details for a typical OTDE exhaust NO, N 02 and NOx concentrations in the dilution tunnel. Confidential EUGT NHP Diesel Exposures Page 31 of 43 Comparison of NO, N02 and NOx concentration in the dilution tunnel for three test atmospheres AIR NTDE OTDE Figure 31. Comparison of NO, N02 and NOx concentration in the dilution tunnel for three test atmospheres Table 2. Comparison o f NO, N 02 and NOx concentration in the dilution tunnel for three test atmospheres AIR NTDE OTDE Tunnel NO (ppm) 0.06 0.60 146.22 Tunnel NOx (ppm) 0.06 0.92 169.77 Tunnel N 02 (ppm) 0.00 0.31 23.49 Confidential EUGT NHP Diesel Exposures Page 32 of 43 NO, N02 and NOx concentration in the dilution tunnel for an air exposure o EQa . uo Tunnel NO (ppm) x O Tunnel NOx (ppm) PNl Tunnel N02 (ppm) O Figure 32. NO, N 02 and NOx concentration in the dilution tunnel for an air exposure Tunnel NOx, N02, NO as a function of time for 5May2015 NTDE Exposure -LO lb " ~14 uQ. VI u oO 10 - oz 8 ' CM o 4A - \d I 0 6;28 k 740 JL 8:S2 ----Tunnel NO (ppm) ........Tunnel NOx (ppm) -Tunnel N02 (ppm) 1 10f:04Tf T 11:16 Time (hrs) Figure 33. Tunnel NOx, N02, NO as a function of time for 5May2015 NTDE Exposure Confidential o EUGT NHP Diesel Exposures Page 33 o f 43 Tunnel NOx, N02, NO as a function of time for 06May2015 NTDE Exposure 111 Tunnel NO (ppm) 1-"Tunnel NOx (ppm) ^ -- Tunnel N02 (ppm) Figure 34. Tunnel NOx, N02, NO as a function of time for 6May2015 NTDE Exposure Tunnel NOx, N02, NO as a function of time for 07May2015 NTDE Exposure - ^ --Tunnel NO (ppm) Tunnel NOx (ppm) 11 1 Tunnel N02 (ppm) Figure 35. Tunnel NOx, N02, NO as a function of time for 7May2015 NTDE Exposure Confidential EUGT NHP Diesel Exposures Page 34 o f 43 Tunnel NOx, N02, NO as a function of time for 27May2015 OTDE Exposure ...... Tunnel NO (ppm) Tunnel NOx (ppm) -- -Tunnel N02 (ppm) Figure 36. Tunnel NOx, N02, NO as a function of time for 27May2015 OTDE Exposure 3.2.2 Chamber Gases Data Figure 37 shows the chamber gases data for NO, NO2, NOx, CO, CO2 and THC. These values are average values for each test atmosphere in the exposure chamber. The data is also shown in Table 3 for comparison. It can be seen from this data that for most o f the gases, other than THC, there is a large difference between the concentration of gases for NTDE and OTDE atmospheres with a minimal difference between air and NTDE exposures. Figure 38 shows typical chamber gases for a typical air exposure for the duration o f the exposure. Figures 39, 40 and 41 NTDE exposures for 5, 6 and 7 May, respectively. The trend of higher exposure gas concentration towards the end of exposures on 6 May, as seen on dilution tunnel data, can also be seen in Figure 40, although to a lower extent when compared to tunnel data. This is due to air exchange rate of the chamber and time it takes to replace existing exposure atmosphere. Similar data for a typical OTDE exposure is shown in Figure 42. Confidential EUGT NHP Diesel Exposures Page 35 o f 43 Comparison of Chamber Gases for Three Test Atmospheres 12 10 ^8 Ea. Q. C 2 6 rLo. ucj u 4 VO) Vif l AIR INTDE IOTOE fa Chamber NO Chamber NOx Chamber N02 Chamber C02 Chamber CO Chamber HC (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) Figure 37. Chamber gases data for three exposure atmospheres Table 3. Average of chamber gases data for air, NTDE and OTDE exposures AIR NTDE OTDE Chamber NO (ppm) 0.037 0.083 9.348 Chamber NOx (ppm) 0.034 0.093 9.940 Chamber N02 (ppm) -0.003 0.010 0.592 Chamber C02 (ppm) 0.068 0.110 0.146 Chamber CO (ppm) 0.225 0.166 3.804 Chamber HC (ppm) 1.593 1.159 1.548 Confidential EUGT NHP Diesel Exposures Page 36 o f 43 Chamber gases as a function of time for air expsoure EIB Chamber NO (ppm) Time (hrs) Chamber NOx (ppm) Chamber N02 (ppm) Chamber C02 (ppm) Chamber CO (ppm) Chamber HC (ppm) Chamber CO (ppm) 30 sec moving average Figure 38. Chamber gases as a function of time for air exposure Chamber gases as a function of time for NTDE expsoure 05 May 2015 Time (hrs) Chamber NO (ppm) Chamber N02 (ppm) Chamber CO (ppm) -Chamber CO (ppm) 30-sec moving average Chamber NOx (ppm) Chamber C02 (ppm) -Chamber HC (ppm) Confidential Figure 39. Chamber gases as a function of time for NTDE exposure of 05 May 2015 EUGT NHP Diesel Exposures Page 37 of 43 Chamber gases as a function of time for NTDE expsoure 06 May 2015 Time (hrs) Chamber NO (ppm) Chamber N02 (ppm) Chamber CO (ppm) Chamber CO (ppm) 30-sec moving avg. Chamber NOx (ppm) Chamber C02 (ppm) Chamber HC (ppm) Figure 40. Chamber gases as a function of time for NTDE exposure of 06 May 2015 Chamber gases as a function of time for NTDE expsoure 07 May 2015 Confidential Time (hrs) Chamber NO (ppm) Chamber NOx (ppm) Chamber N02 (ppm) ' Chamber C02 (ppm) Chamber CO (ppm) Chamber HC(ppm) Chamber CO (ppm) 30-sec moving avg. Figure 41. Chamber gases as a function of time for NTDE exposure of 07 May 2015 3 2.5 2 1.5 1 0.5 0 -0.5 EUGT NHP Diesel Exposures Page 38 of 43 Chamber gases as a function of time for OTDE expsoure 3.5 2.5 ?a. 23 V 1.5 XI .uEmc 6:15 6:45 7:15 7:45 8:15 8:45 9:15 9:45 10:15 Chamber NO (ppm) Chamber C02 (ppm) Chamber NOx (ppm) Chamber CO (ppm) Chamber N02 (ppm) Chamber HC (ppm) Figure 42. Chamber gases as a function of time for OTDE exposure 3.3 Exposure Particle Size Distribution Figures 43 and 44 show typical particle size distribution for NTDE and OTDE when measured by using a TSI Fast Mobility Particle Sizer. These distributions are for number size distributions. Number size distribution along with surface area, volume and mass size distribution for these two samples are also shown in Table 4 and Table 5 for NTDE and OTDE, respectively. Note the scale of vertical axis for number concentration for the two distributions and the difference in the location of particle size peaks. Confidential EUGT NHP Diesel Exposures Page 39 of 43 Figure 43. Number size distribution for NTDE exhaust inside the exposure ehambcr using FMPS Figure 44. Number size distribution for OTDE exhaust inside the exposure chamber using FMPS Confidential EUGT NHP Diesel Exposures Page 40 o f 43 Table 4. A typical particle size distribution for NTDE exposure dN/dlogDp #/cm3 Median (nm) 11.0 Mean (nm) 21.4 Geo. Mean (nm) 15.4 Mode (nm) 10.75 Geo. Std. Dev. 2.03 Total 741.4 Surface nm2/cm3 87.3 79.4 64.6 124.09 2.07 2.31 e +06 Volume nm3/cm3 112.2 102.2 93.0 124.09 1.63 3.06 e +07 Mass pg/m3 112.2 102.2 93.0 124.09 1.63 3.06 e -02 o Table 5. A typical particle size distribution for OTDE exposure dN dlogDp #/cm3 Median (nm) 148.2 Mean (nm) 158.4 Geo. Mean (nm) 142.6 Mode (nm) 124.09 Geo. Std. Dev. 1.69 Total 2.64 e +06 Surface nm2/cm3 205.7 206.2 195.7 254.83 1.40 2.42 e +11 Volume nnrVcm3 231.0 225.7 216.5 254.83 1.35 8.30 e +12 Mass Mg/m3 231.0 225.7 216.5 254.83 1.35 8.30 e +03 Confidential APPENDIX A EUGT NHP Diesel Exposures Page 41 of 43 DIESEL FUEL SPECIFICATIONS / Confidential Chevron EUGT NHP Diesel Exposures Page 42 of 43 Chevron Phillips Chemical Company Issued Sales Specification Name of Product DIESEL .05 SYS CERT FUEL Chevron Phillips Chemical Company LP 10001 Six Pines Drive "TheWoodlands, TX 77380 800-858-4327 Technical Service: 832 813-1862 Revision Date 12/6/2013 Chevron Phillips Chemicals International N.V Bmsselsesteenweg 355 B-3090 Overuse, Belgium +32 (0) 2 689 12 11 Chevron Philips Chemicals Asia PTE Ltd. 5 Temasele Boulevard 05-01 Suntec Tower Rve Singapore 038985 +65 6337 9700 1 Test API Gravity Aromatics Carbon Carbon Density Cetane Index Cetane Number Cloud Point Corrosion (3 hrs lip 50C) Distillation - 5% Distillation- 10% Distillation - 20% Distillation 30% [Distillation- 40% Distillation - 50% Disdiiation'- '60% 1Disfiiiation - 7 0 % ^ D islatfon-_80% Disbllatfon 90% Distillation 95% Distillation- EP Disdlladon- IBP Disdiiation - Loss Disdiiation - Residue Rash Point, PM Hydrogen Net Heat of Combustion Olefins Particulate Matter Polynuclear Aromatics Pour Point Saturates SEC Aromatics Specific Gravity 60/60 Sulhr Units -- LV% WT% 9/gal -- FAH -- FAH FAH FAH FAH FAH FAH FAH FAH FAH FAH FAH FAH FAH ML ML FAH WT% BTU/LB LV% mg/l WT% FAH LV% WT% ppm Method ASTM D-4052 ASTM D-1319 Calculated Calculated ASTM D-976 ASTM D-613 ASTM D-2500 ASTM D-130 ASTM 0 8 6 ASTM D-86 ASTM 0 86 ASTM 0 86 ASTM 0 86 ASTM 0 86 ASTM D-86 ASTM 0 86 ASTM OS6 ASTM 0 86 ASTM 0 86 ASTM 0 86 ASTM 0 86 ASTM 0 86 ASTM 0 86 ASTM 93 STM 3343 ASTM D-3338 ASTM 01319 ASTM 06217 ASTM 05186 ASTM 0 97 ASTM 01319 [ASTM 05186 [ASTM 04052 [ASTM 05453 Typical -- -- -- -- -- - ... *** -- ... -- -- -- -- -- -- -- -- -- **- -- -- -- ... ... '-- Minimum Maximum Qualitative Mote 32.0 28.0 2750 46.0 37.0 31.0 -- 2806 48.0 -- -- -- -- -- -- -REPORT -- -- 46 48 -- 1 -- ---- ---- 400 460 ... -- '3BMi>r REPORT -- -- ---- ---- 470 540 -- -- ---- 560 630 7 . . i-- 610 690 340 400 -- ---- 130 -- "-- ---- ---- -- ... ... :... -- ;-- -- -- -- -- -- -- -- ... --- REPORT REPORT REPORT -- REPORT REPORT REPORT REPORT -- -- REPORT REPORT -- REPORT REPORT REPORT -- -- -- -- -- 0.8398 300.0 15.0 -- 0 -- -- 0.8654 500.0 ... ... -- ~ ... -- -- REPORT -- REPORT REPORT -- -- * ! Printed Versions of this document are uncontrolled Notice: Before using this product; the tse r Is advised and cautioned to make Jts-am i determination and assessment of it safety and su to b ily of die p u to ct for the spedtic use in que3xxi and is further advised against retying on the information contained Isre in as it may relate to any s p e tft use fm appfcation. R is the uWmate m sponsW ty of the usw to e r a that the product suited and the nfarmatwn 9 apptcsble to the u se 's specific appfecation. Chevron Philip s Chemical Company LP does not make and expressly declaim s, a l warranties, mdudhg warranties of merchantabitty or fitness for a particular puposc regardless of whether oral or w ftten, express or tmpied, or aftegedfy arising from any usaoe o f any trade or from any course of dealng in comectJon wtth the use o f the information contained herein or the product te s t, The user eapro^ y i^ u n s a l rts* and la b fty , w h^he based m contract or otherwise, m connecdon with the use of the Wwmadon contained hedn or the product ts rf- R ather, Information contained herein is oiver w ttfvx* rrference to any rtelectuaJ property issues, as w e i as federal, state or kxal laws which may be encountaed m the use thereof. Such questions sh g iii be nwsbgated by the user Confidential Viscosity @ 40C Notes Dyed Diesel tax exempt. cSt ASTM 0445 EUGT NHP Diesel Exposures Page 43 o f 43 1.1 3.2 Confidential o V O o /_ Lovelace* Exposures to Diesel or Alternate Technology Emissions to Evaluate Biological Response in Non-Human Primates Final Report LRRI Study Number: FY14-050 To EUGT Jake McDonald, PhD Lovelace Respiratory Research Institute (LRRI) 2425 Ridgecrest Drive, SE Albuquerque, NM 87108 Courier Address and Location of Laboratory: Bldg 9217, Area Y Kirtland Air Force Base Albuquerque, NM 87115 o LRRI Draft Final Report F Y 14-050 Page 2 o f 14 SUMMARY The objective of this study was to conduct inhalation studies o f filtered air (FA), old technology diesel (OTD) emissions, and new technology diesel (NTD) emissions in female cynomolgus macaques, and to compare the relative biological responses among different combustion/aftertreatment technologies. The same 10 female cynomolgus macaques were utilized for all three exposures, which were conducted in the order of FA, then NTD, and finally OTD. Whole body inhalation exposures demonstrated substantial differences between the FA, NTD, and OTD exposure atmospheres. OTD exposures contained higher particulate matter concentrations, higher concentrations o f NO, NOx, NO?, CO, and CO2 gasescompared to FA and NTD exposure atmospheres. Hematology analysis showed that some cell types were increased at 1 or 4 hours post exposure for FA and OTD exposures, but not for NTD exposures. While some o f these hematology comparisons reached statistical significance, the biological relevance of the changes observed is unclear, due in part to the finding that some significant changes were observed following the FA exposure, BAL cell counts showed that neutrophils and macrophages were elevated in lavage fluid at 4 hours post exposure for all three exposure atmospheres. This is possibly due to carryover of inflammation resulting from baseline lung sampling procedures. Bronchial brushings were analyzed by qPCR for expression of CC-16, Muc5ac, and SPDEF. FA and NTD exposures induced subtle increases in the expression o f these genes. OTD exposure induced robust, though not statistically significant, increases in the expression of these genes. The increases in Muc5ac (linked to increased mucus production) and SPDEF (linked to differentiation of mucus-producing goblet cells in the lung in response to acute and chronic inflammatory stimuli) could be indicative of a greater degree o f lung tissue damage following this acute exposure. An increase in CC-16 expression has been shown to have a protective role in lung tissue by modulating various mediators of the inflammatory response. CC-16 may be elevated in bronchial brushing samples following OTD exposure due to an increase in the activation of various inflammatory pathways. Based on the results shown here, the OTD showed an increase in inflammation systemically and in the lung, while NTD did not. This suggests that for these endpoints the new technology decreased the biological response. TABLE OF CONTENTS SIGNATURES................. ERRS Draft Final Report F Y 14-050 Page 3 o f 14 ____ _________ Page No. Errorl Bookmark not defined. SUMMARY....................... 1 OBJECTIVE......................................................................................................................6 2 COMPLIANCE.................................................................................................................6 3 KEY STUDY PERSONNEL..................................................................................................... 6 METHODS. 4.1 Test System and Assignment......................................... 7 4.2 Clinical Observations........................................................................................................ 7 4.3 Whole Body Inhalation Exposures.................................................................................. 7 4.4 Body Weights................................. 8 4.5 Blood Collections..................... 8 4.6 Lung Lavage, Bronchial Brushings, and Lung Biopsy Collections .............................. 8 4.7 Hematology........................................................................................................................9 4.8 Statistical Analysis............................................................................................................ 9 4.9 Records Retention.,........................................................................................................... 9 RESULTS AND DISCUSSION...............................................................................................9 5.1 Animal Room Environment................... 9 5.2 Whole Body Inhalation Exposure...................................................................................10 5.3 Clinical Obseivations...................................................................................................... 10 5.4 Body Weights..................... 10 5.5 Hematology Measurements.................... 10 5.6 BAL Cell Counts.................... Error! Bookmark notdefined, 5.7 BAL Cytokines.............................................................. Error! Bookmark notdefined. 5.8 BAL Chemistry.............................................................. Error! Bookmark notdefined. 5.9 Serum Cytokines............................................................................................................11 5.! 0 Bronchial Brushings Gene Expression......................................................................... 12 CONCLUSIONS. .... 13 o LRRI Draft Final Report FY14-050 Page 4 o f 14 LIST OF TABLES Table 1. Experimental Design...............................................................................................................7 LIST OF FIGURES Figure 1. Group average hematology values for all animals prior to, 1 hour post, and 4 hours post each exposure atmosphere. * represents p < 0.05 compared to exposure-matched baseline samples.............................................................................................................. 11 Figure 2. Group average serum cytokine data for all animals prior to and at 4 hours post each exposure............................................................................................................................. 12 Figure 3. Group average fold-change expression levels for CC-16, Muc5ac, and SPDEF gene expression in bronchial brushings samples..................................................................... 13 LIST OF APPENDICES Appendix A: Aerosol Contributing Scientist Report LRRI Draft Final Report FY14-050 Page 5 o f 14 LIST OF ACRONYMS/ABBREVIATIONS AAALAC Association for Assessment and Accreditation o f Laboratory Animal Care AAPM American Association of Physics in Medicine Ave average Bldg building BW body weight C Celsius CFR Code of Federal Regulations cm centimeters EDTA Ethylenediaminetetraacetic acid FA Filtered air FDA Food and Drug Administration GI Gastrointestinal GLP Good Laboratory Practices ID identification IM intramuscular IV intravenous kg kilograms LRRI Lovelace Respiratory Research Institute mg/kg milligram of material per kilogram of body weight mg/mL milligram o f material per milliliter of liquid mg/m3 milligram o f material per cubic meter o f air min minutes mL milliliter NBF Neutral buffered formalin NHP Nonhuman primate NIST National Institute o f Standards and Technology NM New Mexico NTD New technology diesel OTD Old technology diesel SD standard deviation SOP Standard Operating Procedure LRRI Draft Final Report FY14-050 Page 6 of 14 1 OBJECTIVE The objective o f this study was to conduct inhalation studies of filtered air, old technology diesel emissions, and new technology diesel emissions in female cynomolgus macaques, and to compare the relative biological responses among different combustion/after-treatment technologies. 2 COMPLIANCE This study was not conducted in strict accordance with 21 CFR Part 58 (Good Laboratory Practices for Non-Clinical Laboratory Studies) and is not intended to fulfill formal regulatory requirements consistent with Investigational New Drug Applications and/or other FDA regulatory submissions. However, the principles of the regulations were followed including documentation and protocol and SOP adherence. This study complied with all applicable sections o f the Final Rules of the Animal Welfare Act regulations (9 CFR Parts 1,2, and 3), as well as the Guidefo r the Care and Use o fLaboratory Animals (National Research Council, 2011). LRRI is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). 3 KEY STUDY PERSONNEL Study Director: Jeremy Brower, PhD email: ibrovver@lrri.ore phone: 505-348-9662 Sponsor Representative: Michael Spallek, PhD email: xx@ xx.org phone: Contributing Scientists: (Aerosol) Philip Kuehl, PhD email: pkuchl@ lrri.org phone: 505-348-9745 Ha in inad Irshad, MS email: hirshad@ lrri.org phone: 505-348-9623 Attending Veterinarian: Carol Emerson, DVM, MS, ACLAM email: cem erson@ lrri.org phone: 505-348-9611 Contributing Scientist Immunology: Yohannes Tesfaigzi, PhD email: vtesfaig@ lrri.org phone: 505-348-9495 4 METHODS The study was conducted with a crossover study design with a washout period o f approximately 2 weeks between exposures. Each arm of the study involved baseline collections followed by whole body inhalation exposures and post-exposure sample collection. The experimental design is shown in Table 1. Due to limitations o f whole body exposure capacity, animals were exposed in 3 cohorts with all study procedures being conducted on the cohorts on consecutive days. T O Exposure Animals per Group Table 1. Experimental Design Exposure Duration (hours) Blood Sampling Time Points LRRI Draft Final Report F Y 14-050 Page 7 o f 14 Lung (Brushings, Sampling Time Points Filtered Air New Technology Diesel Old Technology Diesel 10 10 10 4 Baseline, 1 hour, 4 Baseline, 4 hours 4 hours post-exposure post-exposure 4 4.1 Test System and Assignment Ten (10) female cynomolgus macaques were ordered and screened for the study from Charles River Laboratories in Houston, TX. Animals ranged in age from 2.6 - 3.6 years at the time of study start. All animals were used for all exposure arms in the study, therefore no randomization took place. 4.2 Clinical Observations Animals were observed twice daily from arrival at LRRI through completion of the in-life portion o f the study. Observations included, but were not limited to the following: reactivity to general stimuli, description of any abnormal behaviors, lesions, or other abnormal appearance. Special attention was paid to observe clinical signs associated with the respiratory tract (such as apnea, labored or rapid breathing, malaise, marked nasal discharge, etc.), lethargy, appetite, dehydration, skin lesions, and grooming. Additional veterinary observations were noted on study specific forms or the LRRI animal management system (AMS). On exposure days animals had documented observations prior to the start o f each exposure, at approximately 1 hour intervals throughout each 4 hour exposure, and again after the end of exposure. 4.3 Whole Body Inhalation Exposures NHPs were exposed to each o f the three exposure atmospheres by whole body inhalation in Hazelton H I000 exposure chambers. Prior to the start of study, all animals were conditioned to being inside of an HI 000 exposure chamber for up to 4.5 hours, Each exposure was 4 hours in duration and were conducted as described in the Aerosol Contributing Scientist report (Appendix A). Briefly, both NTD and OTD vehicles were driven on a chassis dynamometer using a Federal Test Procedure Driving Trace. Vehicle emissions were then added to a dilution tunnel with filtered atmospheric air flowing at 300 cfm. A fraction o f this diluted vehicle exhaust was removed from the dilution tunnel and was further diluted with HEPA filtered room air prior to being used as the exposure atmosphere inside of the H 100 chambers. During method development, the final dilution step was adjusted to achieve a target 300 pg/m3 aerosol mass concentration in the OTD vehicle emissions atmosphere. These same settings were then used for all study exposures for both NTD and OTD emissions. LRRI Draft Final Report F Y 14-050 Page 8 of 14 Based on these fixed settings, aerosols were monitored for particulate mass concentration and particle size, carbon monoxide, carbon dioxide, nitrous oxides, and hydrocarbons. 4.4 Body Weights Animals were weighed prior to the start of the study and again prior to each subsequent exposure for health monitoring. 4.5 Blood Collections NHPs had blood collected at baseline (the day prior to each exposure) and at 1 and 4 hours post the end of each exposure. Blood was collected from either the femoral, cephalic or saphenous veins for hematology (target of 1-2 mL per collection) and collection of serum. Baseline and 4 hour blood samples were collected while the animals were anesthetized for other study procedures. One (1) hour blood samples were collected alert while the animals were restrained in a standard restraint chair. Prior to the first alert blood collection, animals were conditioned to pole and collar restraint as well as chair restraint for up to 25 minutes with 15 minutes of arm restraint. At baseline and 4 hours post exposure, animals had a total of 5 mL of blood collected: 2 mL into a K.3EDTA tube for plasma collection, 1 mL into a K.3EDTA for hematology assessment, and 2 mL into a serum separator tube for serum collection. At 1 hour post exposure, animals had 1 mL of blood collected into a K3EDTA tube for hematology assessment. All blood samples were collected from either the left or right femoral vessels. 4.6 Bronchial Brushings Animals were fasted prior to the start of each exposure, but were allowed juice and water during exposure, due to anesthetic events required post exposure. At baseline and at 4 hours following each exposure, animals were sedated with ketamine (8 mg/kg by intramuscular injection) and were maintained on a flow of isoflurane (1-5%) for anesthesia during procedures. Animals were monitored for pulse, respiratory rate, and SPO2 while anesthetized. After blood collections, animals underwent collection of lung lavage, bronchial brushings, and bronchial biopsy. An Olympus bronchoscope was used to guide the operator to lavage the right and left diaphragmatic lobes with approximately 10 mL o f sterile saline. The returned lavagate was stored on wet ice until processing. Lavage samples were transferred to the LRRI Clinical Pathology department for analysis total protein and lactate dehydrogenase in lavage supernatant and for cell counts and differentials. Bronchial brushings were collected at up to 4 sites per sampling session using cytology brushes designed to fit into the 3 mm sampling port on the bronchoscope. The same cytology brush was used for all 4 collections for a given animal. After the first collection, the brush was swirled in a cryovial containing sterile saline. After the next 3 collections, the brush was swirled in a cryovial containing RNAlater. After the final collection, the cytology brush was cut and placed into the final collection tube. The samples collected into sterile saline were centrifuged, the supernatant discarded, and the dry cell pellet stored frozen at -70 to -90 C. The samples collected into RNAlater were transferred directly to -70 to -90 C for storage for possible future analysis. LRRI Draft Final Report F Y 14-050 Page 9 of 14 4.7 Hematology For hematology analyses, whole blood was collected with syringe and needle or butterfly and placed into blood tubes (vacutainers) containing K3EDTA as an anticoagulant. Hematology samples were analyzed by automated analysis using an ADVIATM 120 Hematology System (Bayer Corporation). Samples were stored for up to 2 days at 2-8 C prior to analysis. 4.8 Statistical Analysis For BALF cell counts and cytokines and serum cytokines, paired t tests were performed to compare endpoint at post 4hr with baseline for each exposure. Then to compare the change at post 4h from baseline between exposures, repeated measurements analyses were performed since one animal went through all three exposures, with baseline and post-exposure data included in the model. The Tukey method was used for multiple comparisons. For Hematology, the measurements were made at baseline, post lh r and 4hr for each exposure. To compare the change after exposure from baseline, repeated ANOVA were performed with adjustment o f baseline and time by exposure. Dunnett's method was used to compare the post exposure measurements with baseline. To assess the exposure effects on the measurement change after exposure from baseline, repeated ANOVA was performed with adjustment of baseline, exposure, time and interactions of exposure and time. If the exposure and time interaction (exposure* time) was significant (p value < 0.05) which indicated that the exposure differences varied across time, the exposure comparison was conducted at each time point. The significant level for pairwise exposure group comparisons was fixed at the 0.05 level for each time using Bonfcrroni correction. If the exposure and time interaction (exposure* time) was not significant, the interaction term was dropped from the model indicating the exposure differences were generally the same across different time point. The data were then refitted with baseline, exposure and time for the designated endpoint. Tukey's method was used for multiple comparisons. Statistical calculations were performed using the SAS software system, Version 9.3 (Cary, NC). All reported p-values are two-sided, and statistical significance was assessed at p < 0.05. 4.9 Records Retention This signed report and all associated specimens and raw data generated as result o f this study are retained in LRRI's controlled document and specimen management systems. After acceptance o f the final report by the Sponsor, the study file will be maintained at LRRI for one year. At that time the Sponsor will be notified to request permission to discard or ship the study file to the Sponsor. The final report, detailing this location, will be amended as necessary. 5 RESULTS AND DISCUSSION 5.1 Animal Room Environment Temperature and relative humidity in animal housing areas were within the target range specified in the protocol throughout the quarantine and dosing periods (see Appendix B). Average daily humidity during the quarantine and study period ranged from 30-60%. Average daily temperature during the quarantine and study period ranged from 21.0-26.1C. LRRI Draft Final Report F Y 14-050 Page 10 of 14 5.2 Whole Body Inhalation Exposure Animals were exposed to single, acute exposures of filtered air (FA), NTD emissions, and OTD emissions, in that order. Each exposure session was 4 hours in duration with exposure atmospheres being characterized for particulate mass concentration and particle size, carbon monoxide, carbon dioxide, nitrous oxides, and hydrocarbons. Complete details of the exposure characterizations are included in the Aerosol Contributing Scientist Report (Appendix A), however average gravimetric particulate mass concentrations o f the 3 atmospheres were 7.9, 9.7, and 409.4 pg/m3 for the FA, NTD, and OTD exposure atmospheres, respectively. Real time DustTrak monitoring confirms temporal variation in aerosol mass concentration throughout the 4 hour exposures of OTD emissions, which coincides with the FTP cycle used to generate the atmosphere. OTD atmospheres had significantly greater concentrations of NO, NOx, NO2, and CO than did either of the FA or NTD atmospheres. Chamber CO2 concentrations were greater in both vehicle emission atmospheres than in FA atmospheres, and were greater in OTD atmospheres than in NTD atmospheres, though the differences were much smaller than other gases. Concentration of total hydrocarbon content was similar among all 3 exposure atmospheres. 5.3 Clinical Observations Animals were observed prior to the start of each exposure, at approximately 1 hour intervals throughout each 4 hour exposure, and again after the end o f exposure. No abnormal observations were noted prior to, during, or after the end o f any of the exposure sessions. 5.4 Body Weights Body weights (BW) were collected prior to each exposure session for determination of drug doages. All BWs remained stable throughout the study period. 5.5 Hematology Measurements Group average hematology results for white blood cells (WBC), platelets (PLT), neutrophils (PMN), lymphocytes (Lymph), hematocrit (HCT), hemoglobin (HGB), and reticulocytes (RET) are shown in Figure 1. WBCs significantly increased at 1 hour post FA and OTD exposures. PMNs significantly increased at 1 hour post OTD exposure. Lymphocytes significantly increased at both 1 and 4 hours post FA exposure and at 4 hours post OTD exposure. NTD exposures did not induce any significant changes from baseline at any time point, though the changes from baseline of PMN and WBC were significantly lower at 1 hour post exposure for NTD than for OTD or FA. Due to the fact that many o f these changes from baseline occurred following the FA control exposure, the biological relevance of these changes is unclear. There were generally no differences between any collection for PLT, HCT, or HGB. LRRI Draft Final Report FY 14-050 Page 11 o f 14 PLT PMN RET HGB Figure 1. Group average hematology values for all animals prior to, 1 hour post, and 4 hours post each exposure atmosphere. * represents p < 0.05 compared to exposure-matched baseline samples. 5.6 Serum Cytokines Group average serum cytokine data is shown in Figure 4. IL-lb, IL-6, IL-8, and TNF-alpha were assayed by a multiplexed Luminex assay, per kit instructions. CC-16 was assayed be ELISA, per kit instructions. None o f the comparisons achieved statistical significance. CC-16 was also measured in serum, though virtually all values were below the limit of detection o f the assay. IL-1b (1/2 LOD) IL-6 (1/2 LOD) LRRI Draft Final Report FY14-050 Page 12 o f 14 Figure 2. Group average serum cytokine data for all animals prior to and at 4 hours post each exposure. 5.7 Bronchial Brushings Gene Expression Group average fold-change expression levels for CC-16, MucSac, and SPDEF genes are shown in Figure 6. Relative to exposure-matched baseline samples, FA and NTD exposures induced subtle increases in the expression of both MucSac and SPDEF. Additionally, NTD exposure induced a subtle increase in the expression of CC-16. The OTD exposure, however, induced a strong increase in the expression o f all 3 genes in these bronchial brushings samples. Though these increases are robust, they did not achieve statistical significance. o CC16 LRRI Draft Final Report FY14-050 Page 13 of 14 Muc5ac Figure 3. Group average fold-change expression levels for CC-16, Muc5ac, and SPDEF gene expression in bronchial brushings samples. 6 CONCLUSIONS The objective of this study was to conduct inhalation studies of filtered air (FA), old technology diesel (OTD) emissions, and new technology diesel (NTD) emissions in female cynomolgus macaques, and to compare the relative biological responses among different combustion/aftertreatment technologies. The same 10 female cynomolgus macaques were utilized for all three exposures, which were conducted in the order o f FA, then NTD, and finally OTD. Whole body inhalation exposures demonstrated substantial differences between the FA, NTD, and OTD exposure atmospheres. OTD exposures contained higher particulate matter concentrations, higher concentrations of NO, NOx, NO:, CO, and CO2 gases compared to FA and NTD exposure atmospheres. Hematology analysis showed that some cell types were increased at l or 4 hours post exposure for FA and OTD exposures, but not for NTD exposures. While some o f these hematology comparisons reached statistical significance, the biological relevance of the changes observed is unclear, due in part to the finding that some significant changes were observed following the FA exposure. BAL cell counts showed that neutrophils and macrophages were elevated in lavage LRRI Draft Final Report F Y 14-050 Page 14 o f 14 fluid at 4 hours post exposure for all three exposure atmospheres. This is possibly due to carryover of inflammation resulting from baseline lung sampling procedures. Bronchial brushings were analyzed by qPCR for expression o f CC-16, Muc5ac, and SPDEF. FA and NTD exposures induced subtle increases in the expression o f these genes. OTD exposure induced robust, though not statistically significant, increases in the expression of these genes. The increases in Muc5ac (linked to increased mucus production) and SPDEF (linked to differentiation of mucus-producing goblet cells in the lung in response to acute and chronic inflammatory stimuli) could be indicative o f a greater degree o f lung tissue damage following this acute exposure. An increase in CC-16 expression has been shown to have a protective role in lung tissue by modulating various mediators of the inflammatory response. CC-16 may be elevated in bronchial brushing samples following OTD exposure due to an increase in the activation of various inflammatory pathways. It is possible that the changes observed in bronchial brushings (samples of actual lung tissue) represent the earliest changes to the inhaled atmospheres in this acute exposure experiment.