Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

i ABSTRACT

In compliance with the Clean Air Act Section 211( b) for fuel and fuel additive registration, the petroleum industry and oxygenate manufactur ers have conducted comparative chronic toxicology testing of evaporative emissions fr om unleaded gasoline alone, and unleaded gasoline containing the additive, methyl tertiary-butyl ether (gasoline MTBE vapor condensate [GMVC]) containing approximately 20% of the additive by mass. Groups of 50 male/50 female CDF(F344)CrlBR rats were expos ed in H2000 whole-body inhalation chambers at GMVC vapor concentrations of 2 g/m 3 (low level), 10 g/m 3 (mid level), and 20 g/m 3 (high level) for 6 hours/day, 5 days/week for 104 weeks (520 exposure da ys). There were no clinical signs of toxicity attributable to GM VC inhalation. Survival of th e GMVC-exposed rats was not significantly different from control rats. Body we ights of high-level GM VC males and high- and mid-level females were significantly below contro l values for most of the study. At the final sacrifice, the body weights of male and fema le high-level rats we re significantly below corresponding control values; weights were 91.5 % and 91.8% of control, respectively. Brain weights of high-level males and females, adrenals of high-level females, and epididymides of mid-level males were significantly less than t hose of the corresponding controls. Significant increases in organ:body weight ratio s were observed in males at final sacrifice; brain and lung in mid- and high-level groups and kidney in high-leve l males. Absolute kidney weight and percent kidney-to-brain weights of males from all GMVC exposure groups euthanized before the scheduled sacrifice were significantly increased co mpared to control values. No effects on total and differential white blood cell (WBC) counts c ould be attributed to GMVC inhalation. Differences in WBC counts and differential cell counts in males at the 12-month sampling time were not found in females at 12 months or at la ter sampling times. These findings suggest the differences may have been due to sampli ng technique than to GMVC inhalation.

The incidence of renal tubule adenomas in mid- level male rats (6/50; 12%) was significantly increased compared to controls (0%) by the Fi sher’s test. There was a non-statistically significant increase in renal tubule carcinomas in males (1/50; 2% in both low- and high-level groups). When incidences of adenomas and carcinomas were combined, a significant trendStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

ii toward increasing incidence with exposure con centration was found with the Cochran-Armitage test. However, only the incidence of combined adenomas and carcinomas in the mid-dose group remained significantly increased compared to th e control group. The increases in combined renal adenomas and carcinomas may be attributed to GMVC treatment, based on similar findings in previous studies with MTBE and wholly va porized gasoline (Bird et al., 1997; MacFarland et al., 1984). In the concurrently conducted st udy on Baseline Gasoline Vapor Condensate (LRRI Protocol FY01-027), the combined incidences of renal adenoma and carcinoma were significantly increased, but significant differences were not noted among individual dose groups. GMVC exposure caused increased severity, but not incidence of chronic progressive nephropathy among mid- and high-level males and high-level females compared to controls. Alpha-2u globulin nephropathy resul ting in a sustained increase in tubule epithe lial cell turnover is a plausible mechanism for GMVC-induced renal tubule adenomas and carcinomas in male rats and is substantiated by other MTBE and light hydrocarbon mechanistic study reports (Cruzan et al., 2007; Borghoff et al., 1991, 1992). However, it should be noted that for compounds inducing renal tumors in male rats through increased alpha-2u globulin accumulation and subsequent increased renal tubule cell tu rnover, alpha-2u globulin overload has been demonstrated to be a male rat specific mechanism with little or no relevan ce to human health risk assessment (Baetcke et al., 1991; Hard, 1998; Hard and Khan, 2004).

In contrast to findings in the parallel study with baseline gaso line vapor condensate, there was no GMVC treatment effect on the incidences of nasal squamous cell carcinoma and thyroid follicular cell adenomas and carcinomas in males.

There was a high background incidence of testicular interstitial ad enomas in control male rats (43/50; 86%). The incidence among high-level males (50/50; 100%) was significantly greater than the control incidence (Fis her’s exact test). The rela tionship between treatment and induction of testicular interstitial adenomas is considered to be equivocal, since background incidence of this tumor is extremely high and hist orical control incidence data from one 2-year bioassay conducted at LRRI for a commercial sponsor was 86% (n = 56). NTP controlStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

iii incidence 1 for air inhalation controls: for NIH-07 diet avg. = 70.1% with range of 46% to 90%; for NTP-2000 diet avg. = 92.1% with range of 82% to 98%. Further, the control incidence in the concurrently run Baseline Gasoline Vapor C ondensate chronic study (LRRI study number FY01- 027) was 48/50 (96%), and no significant increase in the incidence of test icular interstitial adenoma with BGVC exposure was found in that study.

The incidences of mononuclear cell leukemia am ong males and females were high, as expected among a population of aged F344 rats. MCL inci dence in low-level (31/39; 79%) and mid-level (31/38; 82%) males was significan tly greater than cont rols (27/50; 54%), most likely due to protocol driven sampling bias (non-target orga ns in low- and mid-level groups were only examined histologically when gross lesions were observed). Overall, the incidence of MCL appeared unaffected by GMVC inhalation. The onl y other nonproliferative lesion attributable to GMVC exposure was hyaline degeneration of th e olfactory epithelium (males) and respiratory epithelium (females) of the nose.
In summary, chronic GMVC inhalation suppressed body weight in males, and to a greater extent in females, increased the severity of chronic progressive nephropathy in mid- and high-level males and high-level females and caused epithelial (females) and olfactory (males) degeneration in the nasal passages of both sexes. The dege nerative nasal effects were most likely caused by the test material. Chronic exposure to BGVC di d not enhance the development of proliferative lesions (hyperplastic lesions, neopl asms) in female rats. In male rats, chronic exposure to BGVC did enhance the development of renal adenom as and carcinomas, when incidences were combined. Only the mid exposure group had sign ificantly increased in cidence of adenomas alone, or of combined adenomas and carcionomas compared to the control group. There was also an increase in testicular interstitial cell adenoma which was considered to be equivocal with respect to treatment. Conseque ntly, due to treatment-related in creases in renal adenomas and carcinomas in males, chronic inhalation of Ga soline MTBE Vapor Condensate was determined to be carcinogenic in male rats in this study. Gasoline MTBE Vapor Condensate was not determined to be carcinogenic in female rats in this study.

1 Reference - http://ntp.niehs.nih.gov/ntpweb/index.cf m?objectid=92E61F1B-F1F6-975E-7D3BED551F07DC0AStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

iv TABLE OF CONTENTS

Page ABSTRACT ...................................................................................................................... .............. i LIST OF FIGURES ............................................................................................................... ....... vi LIST OF TABLES ................................................................................................................ ........ vi LIST OF APPENDICES ............................................................................................................ .. vii LABORATORY TITLE PAGE .................................................................................................... ix KEY STUDY PERSONNEL ......................................................................................................... x LABORATORY SIGNATURE PAGE ........................................................................................ xi LABORATORY QA STATEMENT ........................................................................................... xii LABORATORY GLP COMPLI ANCE STATEMENT ............................................................. xvi Section SUMMARY ...................................................................................................................... S-1 1. INTRODUCTION ............................................................................................................. 1 -1 2. MATERIALS, METHODS ............................................................................................... 2-1 TEST SUBSTANCE ......................................................................................................... 2-1 Identification, Source, and Storage .......................................................................... 2-1 Analysis ................................................................................................................... 2- 1 Expiration Date ........................................................................................................ 2-1 Reserve Sample ....................................................................................................... 2-2 ANIMALS AND ANIMAL ASSIGNMENTS ................................................................. 2-2 Animal Receipt, Housing, and Quarantine .............................................................. 2-2 Animal Disease Screening Program ........................................................................ 2-2 Justification of Test Animals ................................................................................... 2-3 Environmental Conditions ....................................................................................... 2-3 Diet and Drinking Water ......................................................................................... 2-4 EXPERIMENTAL DESIGN ............................................................................................. 2-4 Group Assignment ................................................................................................... 2-4 Exposure System ..................................................................................................... 2-5 In-Life Endpoints ..................................................................................................... 2-8Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

v Post-Exposure Endpoints ......................................................................................... 2-9 Statistics ................................................................................................................. 2- 12 3. RESULTS .................................................................................................................... ...... 3-1 EXPOSURE ATMOSPHERE ........................................................................................... 3-1 Pre-Exposure Characterization ................................................................................ 3-1 In-Study Data ........................................................................................................... 3-2 Body Weights at Final Sacrifice ............................................................................ 3-10 Absolute Organ Weight at Final Sacrifice ............................................................. 3-10 Percent Organ-to-Body Weight at Final Sacrifice ................................................. 3-12 Percent Organ-to-Brain Weight at Final Sacrifice ................................................. 3-12 Terminal Body Weight and Organ Weight of Euthanized (Moribund) Rats ......... 3-12 Hematology ............................................................................................................ 3-15 Histopathology ....................................................................................................... 3-17 CONCLUSIONS ............................................................................................................. 3-22 COMPARATIVE CHRONIC TOXICI TY AND CARCINOGENICITY BETWEEN GMVC AND BGVC ......................................................................... 3-23 Background ............................................................................................................ 3-23 Results – General ................................................................................................... 3-25 Results – Proliferative Lesions .............................................................................. 3-25 Non-Proliferative Lesions ...................................................................................... 3-28 4. LOCATION OF SPECIMENS, RAW DATA, AND FINAL REPORT .......................... 4-1 PROTOCOL DEVIATIONS ............................................................................................. 4-1 Delayed Sponsor Signature on Amendment 1 ......................................................... 4-1 Personnel .................................................................................................................. 4- 1 Calculation of Nomina l Concentration .................................................................... 4-2 Statistics ................................................................................................................... 4-2 Analysis of Tank 16 (420 pound tank) .................................................................... 4-3 Analysis of Exposure Atmosphere Profiles ............................................................. 4-3 Exhaust System Failure During Conditioning/Quarantine Period Affecting Chamber Pressure .................................................................................................... 4-4 Loss of Supply Air to Exposure System .................................................................. 4-4Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

vi Environmental Conditions ....................................................................................... 4-5 Temporary Loss of Environmental Monitoring System Operation ......................... 4-6 Light and Sound Measurements .............................................................................. 4-7 Gas Chromatographic Analysis ............................................................................... 4-7 Body Weights and Observations Not According to Protocol Defined Schedule ................................................................................................................... 4- 8 Cage Board Usage/Availability ............................................................................... 4-8 Animal Tattoo Requirements for Identification ...................................................... 4-8 Animal Misidentification ......................................................................................... 4-9 Tissue Collection at Necropsy ................................................................................. 4-9 Collection of Organ Wei ghts at Necropsy ............................................................... 4-9 Hematology ............................................................................................................ 4-10 5. LIST OF ABBREVIATIONS ........................................................................................... 5-1 6. REFERENCE LIST ........................................................................................................... 6 -1

LIST OF FIGURES Figure Page 2-1. Vapor Generator for GMVC ............................................................................................ 2-6 3-1. Survival of Male Animals ................................................................................................ 3-4 3-2. Survival of Female Animals ............................................................................................ 3- 4 3-3. Body Weights of Male Ra ts Exposed to GMVC ............................................................. 3-8 3-4. Body Weights of Female Rats Exposed to GMVC ......................................................... 3-8

LIST OF TABLES Table Page 2-1. Experimental Design ...................................................................................................... .. 2-4 2-2. Organs and Representative Sa mples Taken for Examination .......................................... 2-9 2-3. Severity Scoring of Chroni c Progressive Nephropathy ................................................. 2-12 3-1. Results of 3-Day Stability Evaluations ............................................................................ 3-1 3-2. Summary of GMVC Vapor Concentrations .................................................................... 3-2 3-3. Mean Survival Days and Day of 25% Mo rtality for Male and Female Rats ................... 3-5 3-4. Disposition of Rats on Study ........................................................................................... 3 -5Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

vii 3-5. Body Weight Summary for Male Rats ............................................................................. 3-6 3-6. Body Weight Summary in Female Rats ........................................................................... 3-7 3-7. Summary Statistics for Final Sacrifice B ody Weights of Male and Female Rats ......... 3-10 3-8. Absolute Organ Weight at Te rminal Sacrifice (g ± SD) ................................................ 3-11 3-9 Percent Organ-to-Body Weight at Terminal Sacrifice (% ± SD) .................................. 3-13 3-10 Percent Organ-to-Brain Weight at Terminal Sacrifice (% ± SD) .................................. 3-14 3-11. Summary of Hematological Findings in Rats Inhaling GMVC; Median (range) .......... 3-16 3-12. Incidences of Proliferative (Neoplasti c and Hyperplastic) Lesions in Rats .................. 3-18 3-13. Nonproliferative (Non-Neoplastic) Lesion Incidences and Severity Scores ................. 3-21 3-14. Analytical Comparison of Test Materials ...................................................................... 3-24 3-15. Comparison of Incidences of Renal, Nasa l Turbinate, Testicular, and Thyroid Tumors in Male Rats Inha ling GMVC and BGVC ....................................................... 3-26 3-16. Comparison of Leukemia Incidences in Spleens of Rats inhaling GMVC and BGVC .......................................................................................................................... .. 3-28 3-17. Comparison of Chronic Progressive Nephropathy in Rats Inhaling GMVC and BGVC for 2 Years ......................................................................................................... 3-29

LIST OF APPENDICES
Appendix A STUDY PROTOCOL AND AMENDMENTS .......................................................................... A-1 Appendix B ANALYSES OF BULK TEST SUBSTANCE ............................................................................B-1 Appendix C RESULTS OF PERIODIC SERO LOGICAL EVALUATIONS .................................................C-1 Appendix D FEED AND WATER ANALYSES ............................................................................................ D-1 Appendix E SUMMARIES OF GMVC ANALYSES IN EXPOSURE CHAMBERS AND EXPOSURE ROOM AND AEROSOL SCIENTIST’S REPORT .............................................. E-1Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

viii Appendix F WEEKLY GAS CHROMATOGRAPHIC PROFI LES OF CHAMBER ATMOSPHERES ...... F-1 Appendix G ENVIRONMENTAL DATA ...................................................................................................... G-1 Appendix H INDIVIDUAL ANIMAL DISPOSITION .................................................................................. H-1 Appendix I INDIVIDUAL ANIMAL IN-LIF E BODY WEIGHTS .............................................................. I-1 Appendix J INDIVIDUAL ANIMAL SUMMA RIES OF CLINICAL OBSERVATIONS ............................ J-1 Appendix K BODY AND ORGAN WEIGHT S AT NECROPSY ................................................................. K-1 Appendix L DIFFERENTIAL CELL COUNTS AND WHITE BLOOD CELL COUNT ESTIMATES ...... L-1 Appendix M NEOPLASTIC AND NON-NEOPLASTIC LESIONS AND GROSS NECROPSY OBSERVATIONS IN MALE RATS ......................................................................................... M-1 Appendix N NEOPLASTIC AND NON-NEOPLASTIC LESIONS AND GROSS NECROPSY OBSERVATIONS IN FEMALE RATS ..................................................................................... N-1 Appendix O STATISTICIAN’S REPORT...................................................................................................... O-1 Appendix P PATHOLOGIST’S REPORT ...................................................................................................... P-1Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

ix LABORATORY TITLE PAGE

211(b) CHRONIC CARCINOGENICITY STUDY
GASOLINE MTBE VAPOR CONDENSATE (GMVC)

LRRI Study Number: FY01-013

Laboratory: Lovelace Respiratory Res earch Institute (LRRI) 2425 Ridgecrest Dr. SE Albuquerque, NM 87108
Courier Address and Location of Laboratory: Bldg. 9217, Area Y Kirtland Air Force Base Albuquerque, NM 87115

Sponsor: American Petroleum Institute (API) 1220 L Street, NW Washington, DC 20005

September 2009

Total number of pages: ___Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

x KEY STUDY PERSONNEL

Study Director Janet M. Benson, PhD, DABT Lovelace Respiratory Research Institute

Veterinary Pathologist Andrew P. Gigliotti DVM, PhD, DACVP Lovelace Respiratory Research Institute

Director of Quality (Until 2007) Stephanie Taulbee, MSPH Lovelace Respiratory Research Institute

Quality Assurance Manager (Until 2006) Dorothy L. Harris, MS, CRM, RQAP-GLP Lovelace Respiratory Research Institute

Quality Assurance Manager Joan Gallis Lovelace Respiratory Research Institute

Aerosol Scientists Quint H. Powell, MS Lovelace Respiratory Research Institute
Edward B. Barr, MSEE Lovelace Respiratory Research Institute

Laboratory Animal Veterinarian David G. Burt, DVM, DACLAM Lovelace Respiratory Research Institute Roger A. Van Andel, DVM, PhD, DACLAM Lovelace Respiratory Research Institute

SUBCONTRACTOR

Statistician Betty Skipper, PhD Department of Family and Community Medicine, University of New MexicoStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

xi LABORATORY SIGNATURE PAGE

Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC)

Janet M. Benson, PhD, DABT Study Director Lovelace Respiratory Research Institute Date

Edward B. Barr, MSEE Aerosol Scientist Lovelace Respiratory Research Institute Date

Andrew P. Gigliotti, DVM, PhD, DACVP Veterinary Pathologist (Clinical Pathology and Histopathology) Lovelace Respiratory Research Institute Date

Betty Skipper, PhD Director, Biostatistics Department of Family and Community Medicine, University of New Mexico DateStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

xii LABORATORY QA STATEMENT

Study Title 211(b) Chronic Carcinogenicity Study – Gasoline MTBE Vapor Condensate (GMVC) and Baseline Vapor Condensate (BGVC) LRRI Study Number FY01-013 Sponsor Study Number N/A Study Director J. Benson

This study was conducted under 21 CFR Part 58 G ood Laboratory Practice (GLP). This study was inspected by the LRRI Quality Assurance Unit. The final report accurately reflects the raw data. Findings were reported to the Study Director and Test F acility Management as follows:

Study Phase Performed by Date(s) of Inspection or Audit Testing Facility Management Study Director
Afternoon servicing of chambers and animal body weights R. Marr 29-May-01 4-June-01 4-June-01

Gasoline MTBE vapor condensate 20# tank receipt, 420# tank usage log, 20# tank usage and fill log R. Marr 30-May-01 30-May-01 30-May-01

Body weights, clinical observations R. Marr / D. Harris 3-Jul-01 9-Jul-01 9-Jul-01

Environmental data R. Mar / D. Ha rris 7-Aug-01 9-Aug-01 9-Aug-01

Environmental data R. Mar / D. Ha rris 9-Aug-01 9-Aug-01 9-Aug-01 TA management data, clinical observations and body weights, Miran calibration data, GC data R. Marr 22-Oct-01 30-Oct-01 30-Oct-01

Environmental data, clinical observations, body weights, exposure data, GC data, Miran calibration data R. Marr 5-Dec-01 18-Dec-01 18-Dec-01Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

xiii Study Phase Performed by Date(s) of Inspection or Audit Testing Facility Management Study Director
Environmental data, exposure data, GC data, MTBE running exposure concentration summary R. Marr 26-Dec-01 4-Jan-02 4-Jan-02

Environmental data, clinical observations, body weights, 1 point and 8 point performance qualifications R. Marr 7-Mar-02 11-Mar-02 11-Mar-02

Environmental data, clinical observations, body weights, exposure data, GC data, Miran calibration data, TA usage forms R. Marr 1-May-02 13-May-02 13-May-02

Environmental data, clinical observations, body weights, exposure chamber maps, exposure data, GC data, TA usage forms R. Marr 6-Sept-02 12-Sept-02 12-Sept-02

Environmental data, clinical observations, body weights, TA characterization, 1 point and 8 point performance calibrations, chamber atmosphere characterization, individual animal removal records, blood smear/differential morphology, exposure data, TA usage forms R. Marr 6-Nov-02 22-Nov-02 22-Nov-02Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

xiv Study Phase Performed by Date(s) of Inspection or Audit Testing Facility Management Study Director
IANR forms, individual animal removal – chain of custody records, animal room exposure log, sick animal observations records, exposure chamber checklist, exposure chamber maps, 8 point performance calibration, environmental data, exposure data, Miran calibrations R. Marr 17-Dec-02 19-Dec-02 19-Dec-02

Environmental data, GC data, exposure data, chamber profiles, Miran calibrations, body weights and observations R. Marr 7-Jan-03 8-Jan-03 8-Jan-03

Exposure data, TA, body weight and observations, chamber atmosphere, environmental data, Miran calibrations, IANR forms, GC data, animal removal records R. Marr 3-Feb-03 14-Feb-03 14-Feb-03

Exposure data, TA, body weight and observations, chamber atmosphere, environmental data, Miran calibrations, IANR forms, GC data, animal removal records R. Marr 14-Mar-03 27-Mar-03 27-Mar-03

Terminal sacrifices R. Marr 27-May-03 27-May-03 27-May-03 Gasoline MTBE vapor condensate 20# usage and fill log R. Marr 27-May-03 14-Jun-03 14-Jun-03

Organ weight data, gross observations R. Marr 5-Jun-03 6-Jun-03 6-Jun-03Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

xv Study Phase Performed by Date(s) of Inspection or Audit Testing Facility Management Study Director

Animal death dates against IANR R. Marr 2-Jul-03 21-Jul-03 21-Jul-03

Histology specimen preparation D. Harris 22-Jul-03 24-Jul-03 24-Jul-03

Histology lab D. Harris 24-Jul-03 24-Jul-03 24-Jul-03 Exposure data, TA, Path Tox dead animal status list, body weight and observations, chamber atmosphere, environmental data, Miran calibrations, , GC data, 1 point and 8 point performance qualifications, IANR forms, animal removal records and chain of custody, additional animal observations, sick animal observations R. Marr 29-Jul-03 19-Aug-03 19-Aug-03

TA records D. Harris 3-Oct-03 6-Oct-03 6-Oct-03 Draft final report (report text/appendices, all report tabular data, pathologist report, statistics data/report D. Harris 24-Jun-04 16-Jul-04 16-Jul-04

Final Report D. Harris July 2006 July 2006 July 2006 Final Report Text C. Storch June 2009 1-Jul-09 1-Jul-09 Final Report C. Storch 30-Sept-09 30-Sept-09 30-Sept-09

Christa R. Storch, BS, RQAP-GLP Senior Quality Assurance Specialist LRRI Quality Assurance Unit DateStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

xvi LABORATORY GLP COMPLIANCE STATEMENT

Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC)
This study was conducted in compliance with Al ternative Tier 2 testing requirements under Section 211(b) of the Clean Air Act and the EPA Health Effects Test Guidelines OPPTS 870.4200 “Carcinogenicity.” The stipul ations of this protocol were implemented in conformance with EPA regulations as speci fied in 40 CFR 79.60 “Good Laborat ory Practices (GLP) Standards for Inhalation Exposure H ealth Effects Testing.”

It was the Sponsor’s responsibility to maintain records of the met hod of synthesis, fabrication, or derivation of the test substance. The method of fabrication was not available at the time the study was initiated. It is how ever, presently available.

The following were not conducted under GLP Guidelines:

Serological assessments were conducted thr oughout the study. Sera were shipped to BioReliance Corporation, Rockville, MD. None of the assessments were conducted under GLP guidelines due to lack of specification by LRRI.

Light and noise measurements were not c onducted under GLP guidelines. No Standard Operating Procedure was in place for conduct of these measurements.

Janet M. Benson, PhD, DABT Study Director Lovelace Respiratory Research Institute Date

Thomas M. Gray, MS, DABT Sponsor Representative American Petroleum Institute DateStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

S-1 SUMMARY
In compliance with the Clean Air Act Section 211( b) for fuel and fuel additive registration, the petroleum industry and oxygenate manufactur ers have conducted comparative chronic toxicology testing of evaporative emissions fr om unleaded gasoline alone, and unleaded gasoline containing the additive, methyl tertiary-butyl ether (gasoline MTBE vapor condensate [GMVC]) containing approximately 20% of the additive by mass. Groups of 50 male/50 female CDF(F344)CrlBR rats were expos ed in H2000 whole-body inhalation chambers at GMVC vapor concentrations of 2 g/m 3 (low level), 10 g/m 3 (mid level), and 20 g/m 3 (high level) for 6 hours/day, 5 days/week for 104 weeks (520 exposure da ys). There were no clinical signs of toxicity attributable to GM VC inhalation. Survival of the GMVC-exposed rats was not significantly different from control rats. Body we ights of high-level GM VC males and high- and mid-level females were significantly below contro l values for most of the study. At the final sacrifice, the body weights of male and fema le high-level rats we re significantly below corresponding control values; weights were 91.5 % and 91.8% of control, respectively. Brain weights of high-level males and females, adrenals of high-level females, and epididymides of mid-level males were significantly less than t hose of the corresponding controls. Significant increases in organ:body weight ratio s were observed in males at final sacrifice; brain and lung in mid- and high-level groups and kidney in high-leve l males. Absolute kidney weight and percent kidney-to-brain weights of males from all GMVC exposure groups euthanized before the scheduled sacrifice were significantly increased co mpared to control values. No effects on total and differential white blood cell (WBC) counts c ould be attributed to GMVC inhalation. Differences in WBC counts and differential cell counts in males at the 12-month sampling time were not found in females at 12 months or at la ter sampling times. These findings suggest the differences may have been due to sampli ng technique than to GMVC inhalation.

The incidence of renal tubule adenomas in mid- level male rats (6/50; 12%) was significantly increased compared to controls (0%) by the Fi sher’s test. There was a non-statistically significant increase in renal tubule carcinomas in males (1/50; 2% in both low- and high-level groups). When incidences of adenomas and carcinomas were combined, a significant trendStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

S-2 toward increasing incidence with exposure con centration was found with the Cochran-Armitage test. However, only the incidence of combined adenomas and carcinomas in the mid-dose group remained significantly increased compared to th e control group. The increases in combined renal adenomas and carcinomas may be attributed to GMVC treatment, based on similar findings in previous studies with MTBE and wholly va porized gasoline (Bird et al., 1997; MacFarland et al., 1984). In the concurrently conducted st udy on Baseline Gasoline Vapor Condensate (LRRI Protocol FY01-027), the combined incidences of renal adenoma and carcinoma were significantly increased, but significant differences were not noted among individual dose groups. GMVC exposure caused increased severity, but not incidence of chronic progressive nephropathy among mid- and high-level males and high-level females compared to controls. Alpha-2u globulin nephropathy resul ting in a sustained increase in tubule epithe lial cell turnover is a plausible mechanism for GMVC induced renal tubule adenomas and carcinomas in male rats and is substantiated by other MTBE and light hydrocarbon mechanistic study reports (Cruzan et al., 2007; Borghoff et al., 1991, 1992). However, it should be noted that for compounds inducing renal tumors in male rats through increased alpha-2u globulin accumulation and subsequent increased renal tubule cell tu rnover, alpha-2u globulin overload has been demonstrated to be a male rat specific mechanism with little or no relevan ce to human health risk assessment (Baetcke et al., 1991; Hard, 1998; Hard and Khan, 2004).

In contrast to findings in the parallel study with baseline gaso line vapor condensate, there was no GMVC treatment effect on the incidences of nasal squamous cell carcinoma and thyroid follicular cell adenomas and carcinomas in males.

There was a high background incidence of testicular interstitial ad enomas in control male rats (43/50; 86%). The incidence among high-level males (50/50; 100%) was significantly greater than the control incidence (Fishe r’s test). The relationship be tween treatment and induction of testicular interstitial adenomas is considered to be equivocal, since background incidence of this tumor is extremely high and historical contro l incidence data from one 2-year bioassayStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

S-3 conducted at LRRI for a commercial sponsor wa s 86% (n = 56). NTP control incidence 2 for air inhalation controls: for NIH-07 diet avg. = 70.1% with range of 46% to 90%; for NTP-2000 diet avg. = 92.1% with range of 82% to 98%. Further, the control incidence in the concurrently run Baseline Gasoline Vapor Condensate chronic study (LRRI study number FY01-027) was 48/50 (96%), and no significant increase in testicul ar interstitial adenoma among BGVC males was found in that study.

The incidences of mononuclear cell leukemia am ong males and females were high, as expected among a population of aged F344 rats. MCL inci dence in low-level (31/39; 79%) and mid-level (31/38; 82%) males was significan tly greater than cont rols (27/50; 54%), most likely due to protocol driven sampling bias (non-target orga ns in low- and mid-level groups were only examined histologically when gross lesions were observed). Overall, the incidence of MCL appeared unaffected by GMVC inhalation. The onl y other nonproliferative lesion attributable to GMVC exposure was hyaline degeneration of th e olfactory epithelium (males) and respiratory epithelium (females) of the nose.
In summary, chronic GMVC inhalation suppressed body weight in males, and to a greater extent in females, increased the severity of chronic progressive nephropathy in high- and mid-level males and high-level females and caused epithelial (females) and olfactory (males) degeneration in the nasal passages of both sexes. The dege nerative nasal effects were most likely caused by the test material. Chronic exposure to BGVC di d not enhance the development of proliferative lesions (hyperplastic lesions, neopl asms) in female rats. In male rats, chronic exposure to BGVC did enhance the development of renal adenom as and carcinomas, when incidences were combined; only the mid-exposure group had signifi cantly increased incidence of adenomas or combined adenomas and carcionomas compared to the control group. There was also an increase in testicular in terstitial cell ad enoma which was considered to be equivocal with respect to treatment. Consequently, due to treatme nt-related increases in renal adenomas and carcinomas in males chronic inhalation of Gaso line MTBE Vapor Condensate was determined to be carcinogenic in male rats in this study. Gasoline MTBE Vapor Condensate was not determined to be carcinogenic in female rats in this study.

2 Reference - http://ntp.niehs.nih.gov/ntpweb/index.cf m?objectid=92E61F1B-F1F6-975E-7D3BED551F07DC0AStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

1-1 INTRODUCTION
The purpose of this study was to evaluate the chronic toxicity, potenti al carcinogenicity, and exposure concentration-response relationships a ssociated with inhalati on of gasoline vapor condensate containing the fuel a dditive methyl tertiary-butyl ether (gasoline MTBE) vapor condensate (GMVC). The study was conducted in compliance with Alternative Tier 2 testing requirements under Section 211(b) of the Clean Air Act and the EPA Health Effects Test Guidelines OPPTS 870.4200 “Carcinogenicity.” Th e stipulations of th is protocol were implemented in conformance with EPA regulations as specified in 40 CFR 79.60 “Good Laboratory Practices (GLP) Sta ndards for Inhalation Exposure He alth Effects Testing.” The study protocol and amendments are provided in Appendix A.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

2-1 MATERIALS, METHODS

TEST SUBSTANCE Identification, Source, and Storage Gasoline MTBE vapor condensate (GMVC; Lot Nu mber API 00-02) was prepared from gasoline containing 20% MTBE and supplied in 420- pound and 20-pound gas cylinders by Chevron Research and Technology Center (CRTC; Richmond, CA). Original charac terization of the test substance was performed by CRTC and ExxonMob il Biomedical Sciences, Inc. (EMBSI), Annandale, NJ. The reference gas chromatogr aphic profile of the 19 key components was provided by EMBSI.

Twenty-pound cylinders and some 420-pound cylinders were stored at ambient temperature in a storage building dedicated for that purpose at the Lovelace Respiratory Research Institute (LRRI). The remaining 420-pound cylinders were st ored in an outside, controlled area at ambient temperature. The test substance was transferred, as needed, from the 420-pound to the 20-pound cylinders. Only authorized personnel were allowed access to the test substance. Receipt, use, and inventory of this test substance were documented.

Analysis The analytical results ar e provided in Appendix B.

Before dispensing GMVC from each 420-pound tank, a sample was removed from the tank and analyzed by gas chromatography at LRRI usi ng a Shimadzu Model GC-17A/FID (Columbia, MD). The gas chromatographic profile of the 19 major peaks (retention time and relative peak area) was compared with that or iginally determined for the GMVC by EMBSI. This was done to show that all tanks were similar in profile and that the test material was stable throughout the testing period. Results are provided in Appendix B.

Expiration Date An expiration date is not availa ble. The test material is stab le per MSDS. The test substance stability was tested concurrently with the st udy with the analysis of the exposure chamberStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

2-2 atmospheres and each #420 pound tank before its use. Results, provided in Appendix E, showed it to be stable for the duration of the study.

Reserve Sample The Sponsor-contracted archives have reta ined samples of the test substance.

ANIMALS AND ANIMAL ASSIGNMENTS Animal Receipt, Housing, and Quarantine Four hundred-forty CDF(F344)CrlBR rats (5–6 week s old when received) were purchased from Charles River Laboratories (Raleigh, NC). Theywere examined by a veterinarian upon their arrival. All animals were quarantined and acclimated to whole-body inhalation chambers for 23 days. Healthy animals were randomly assigne d by weight to the core exposure groups (400 total; 50 rats/sex/exposure level). The weight range for males the day before exposures began was 134.2–211 g. The weight range for females on the day before exposures began was 108.5– 138.9 g. Following randomization, the rats assigned to the study were id entified by tail tattoo. Five male and five female rats were assigned as sentinels and housed in the control chamber. Five unassigned male and female rats were sacrif iced before exposures began to evaluate their health status as an indicator of the health status of the population on study. The remaining unassigned rats were euthanized. Receipt and initiation of exposures of male and female rats were staggered by one week.

Animal Disease Screening Program Five male and five female rats not assigned to study were sacrificed, bled and received a complete necropsy. No gross lesions were f ound, and tissues were subsequently discarded because there were no subsequent questions rega rding the health stats of the animals on study. Sera from these rats were submitted to BioRelia nce, Gaithersburg, MD, for analysis of antibodies common to rodents. The following tests were run: Cilia-Associated Respiratory Bacillus (CARB), Mycoplasma pulmonis (M. PUI), Pneum onia virus of mice (PVM), Rat Coronavirus/ Sialodacryoadenitis Virus (RCV/SDA), Reovirus (Reo), Sendai Virus (Sendai), Lymphocytic Choriomenengitis Virus (LCM), Parvovirus (Parvo) , Toolan’s H-1 Virus (H-1), and Kilham ratStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

2-3 Virus (KRV). The five male and five female se ntinel rats in the control chamber were bled retroorbitally after 26, 52, and 79 weeks of expos ure. Blood was also obtained from sentinels surviving to 104 weeks. Sera were anal yzed by BioReliance, as described above.

Justification of Test Animals Rats were used in this study because of the larg e database available on th e inhalation toxicity and carcinogenicity of toxic materials in rats. The study design was justif ied because it provided exposure concentration-response information on the possible carcinogenicity associated with repeated inhalation of GMVC.

Environmental Conditions The rats were housed 24 hours per day during qua rantine and exposure in Hazleton 2000 wholebody inhalation chambers. Initially, all rats were housed separately in 3.8-inch wide by 11-inch long by 8-inch high compartments within stainless steel baskets. When male rats reached 400 g they were transferred to bask ets with 5.7-inch by 11-inch by 8-inch compartments. Each chamber contained six baskets.

The chambers and cage racks were washed week ly. The cage racks were rotated clockwise weekly when the rats were transferred from the dirty to the clean chamber.

The chambers were held at appr oximately 1 inch of water negativ e pressure with respect to the exposure room, and the chamber flow rates were maintained at 12 to 15 air changes per hour (400–500 liters per minute [LPM]). Chamber temperatures were maintained at 20 ° to 24 ° C. Temperature, relative humidity, and chamber ai r flow rates were continuously monitored, 24 hours per day. Values for the three parameters were recorded at 30-mi nute intervals. Oxygen concentration in the chambers was maintained at 19%. This parameter was monitored but not recorded.

A 12-hour light cycle was maintained with lights on at 0600. Light levels in the exposure room and noise levels in the chambers were determined periodically.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

2-4 Diet and Drinking Water Unlimited municipal tap water was available at all times. Rats were fed Teklad Certified Rodent Diet (8728C; Harlan Teklad, Madison, WI). F ood was available at all times except during the daily exposure period. Certified diet wa s analyzed for heavy metals, aflatoxin, organophosphates and chlorinated hydrocarbons, and values present were below maximum allowable values established by the manufacturer. Water was analyzed by an independent laboratory for metals, ions, microbes, and pestic ides. Analytes were either present at nondetectable concentrations or sufficiently low con centrations as to not interfere with the outcome of the study. Results of feed and wate r analyses are provided in Appendix D.

EXPERIMENTAL DESIGN Group Assignment The experimental design is shown in Table 2-1.
The Laboratory Animal Veterinarian visually examin ed all rats before they were placed on study. Only animals judged to be of acceptable health were used. Animals were weighed and randomly assigned to a test group using a computer ized data acquisition system (Path-Tox ® ; Xybion, Cedar Knolls, NJ) operated according to LRRI St andard Operating Procedures (SOPs).
Table 2-1. Experimental Design Rat Strain and Sex CDF(F344)CrlBR; 200 males/200 females assigned to core study Animal Source Charles River Laboratories, Raleigh, NC Time Held Before Exposure 23 days Age When Placed on Study 9–10 weeks Study Dates Study Initiation Date May 22, 2001 Initiation of Exposures (Males) May 23, 2001 Initiation of Exposure (Females) May 30, 2001 Last Exposure Day (Males) May 23, 2003 Last Exposure Day (Females) May 30, 2003 Final Sacrifice (Males) May 27–30, 2003 Final Sacrifice (Females) June 2–6, 2003Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

2-5 Table 2-1. Experiment al Design (Concluded) Target Exposure Concentrations Control 0 g/m 3 GMVC Low Level 2 g/m 3 GMVC Mid Level 10 g/m 3 GMVC High Level 20 g/m 3 GMVC Animal Identification Scheme (by tail tattoo) Control E400-E450 (M); E451-E500 (F) Low Level F501-E550 (M); F551-E600 (F) Mid Level G601-G650 (M); G651-G700 (F) High Level H701-H750 (M); H751-H800 (F) Exposure Duration 6 hours/day, 5 days/week for 104 weeks (520 exposure days) In-Life Monitoring Daily observa tions for morbidity, mortality. Weekly body weights for first 13 weeks, then every 4 th week. Weekly detailed clinical observations. Sentinel Animal Bleeds for Serology Prior to study start and at six month intervals thereafter, including prior to final sacrifice. Hematology Evaluations (Control and High Dose groups) At 12 and 18 months and at final sacrifice.

Histopathology All protocol-re quired tissues collected

Exposure System A schematic of the vapor exposure system is shown in Figure 2-1.

The daily supply of GMVC for each exposure chamber was contained in 20-pound gas storage cylinders. Exposure atmospheres were generate d by controlling the flow of pressurized GMVC through a rotameter, into a heated stainless stee l transfer line where the GMVC was completely vaporized. Chamber concentrations were contro lled by adjusting the flow rates of the GMVC and dilution air rate. Chamber exhaust was carri ed to an oxidizer on the roof of the exposure facility where it was burned.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

2-6 Gasoline MTBE Vapor Condensate Vapor Generator

Sight Bowl Bypass To Main Exhaust

Duct GMVC Vapor and Air Mixture To CBR6

Bench Scale

Gasoline MTBE Vapor Condensat
e P

Liquid Pressurizing N 2 (60-80 psig) Nitrogen Carrier Gas

Pneumatic Shutoff Valve Pressure Gauge

GMVC Rotameter Nitrogen Rotameter

Heat Exchanger (Vaporizer) Orifice Meter Low Level Chamber Supply Air

Mid Level Chamber Supply Air

High Level Chamber Supply Air GMVC Vapor and Air Mixture To CBR7

GMVC Vapor and Air Mixture To CBR8 Dilution

Tee Liquid GMVC Line

Nitrogen Line GMVC Vapor Line

Chamber Bypass (3-way Valve) Hot Water Retur

n Hot Water

In

Heat Exchanger

(Vaporizer) Heat Exchanger

(Vaporizer)

Dilution Tee Dilution

Tee

Nitrogen Rotameter Nitrogen Rotameter

Hot Water Return

Hot Water

Return Hot Water In

Hot Water In Sight Bowl Sight Bowl

Orifice Meter Orifice Meter

Chamber Bypass (3-way Valve) Chamber Bypass (3-way Valve)

GMVC Rotameter GMVC Rotameter

Figure 2-1. Vapor Generator for GMVC

Pre-Test Characterization . The exposure system was tested prior to animal exposures. Characterization included the followi ng: 1) uniformity of the dist ribution of total vapor within each chamber; 2) within-day and between-day stabil ity of vapor concentration; 3) within-day and between-day consistency of the hydrocarbon profile as determined by gas chromatography; and 4) determination of the time for vapor concentrat ion to achieve 90% of the equilibrium target value (T90). The exposure atmosphere in the an imals’ breathing zone was also examined for the presence of aerosol particles using a TSI Scanni ng Mobility Particle Sizer (TSI Industries, Shoreview, MN).

Once, prior to the initiation of animal exposures, the concentrati on of the test substance in the generator containment hood and in the exposure room were measured to ensure that the generator containment hood was operating satisfactorily.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

2-7 Chamber Distribution Evaluation During Exposures . During the second week of exposure, the uniformity of vapor distribution wa s re-evaluated to determine the distribution in the presence of the test animals.

Quantitation of Exposure Atmospheres . Vapor concentrations were continuously monitored using Miran 1A infrared analyzers (Foxboro W ilks, Foxboro, CT). The high-, mid-, and lowlevel exposure chambers were each monitored with their own analyzer. The analyzers for the high-, mid-, and low-level chambers underwent w eekly five-point calibrations and daily onepoint calibration checks using test substance. A fourth analyzer was devoted to monitoring the control chamber, the room air, and the hood enclos ing the 20-pound tank of test substance. This Miran underwent a three-point calibration appr oximately quarterly until December 2001, and then once before exposures ended. The Miran 1A analyzers dedicated to the mid- and high-level chambers were calibrated over a range of 6–35 g/m 3 . The Miran 1A analyzers for the control chamber and the low-level chamber were calib rated over a concentration range of 1–7 g/m 3

.

The absorbance in each chamber was monitored continuously. Data were recorded every 3 seconds and the mean of these values was cal culated and saved every 120 minutes. Average values were obtained for the first, second, and third two-hour segments of each exposure day. The mean of the three 120 minute average valu e of values from each exposure chamber was reported as the day’s exposure c oncentration for that chamber.

Qualitative Assessment of Exposure Atmospheres . The qualitative composition of the exposure atmosphere in each chamber was determined w eekly by gas chromatography using a Shimadzu Model GC-17A/FID (Columbia, MD). The pe rcent peak area of each of 19 components was determined and recorded. The retention times of eight representative co mponents were verified against EMBSI values each week, and one-point performance qualifications were performed on any day in which chamber profiles were analyz ed. The one-point check was performed using a certified standard of 840 ppm butane in N 2 (Matheson Tri-Gas, Irving, TX).

Determination of Nominal Concentration . Daily nominal or “anticip ated” usage was calculated by multiplying the average GMVC concentra tion in each chamber (low, mid, high; g/m 3 ) by the total flow through each respective chamber ([L/min * min]/[1000 m 3 ]) and then summing theStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

2-8 values for all three chambers. This value was compared to the actual GMVC usage determined by taking the difference between the weight of the 20-pound cylinder before and after each
exposure.
Concentration of Test Substance in the Exposure Room . Concentration of the test substance in the exposure room was determined periodical ly during the study (at approximately 60-day intervals) using a Miran 1A In frared Spectrometer (Foxboro Wilk s, Foxboro, CT) operated using the same settings as used to monitor the low-level exposure chamber.

In-Life Endpoints Rats were exposed 6 hours/day (plus 14 minutes, th e time for the vapor concentration to reach T90), 5 days/week for 104 weeks (520 exposur e days). The following observations and measurements were made during the dosing phase:

Mortality and Morbidity . Laboratory animal technicians observed the rats twice daily throughout the study. Examinations were orient ed toward identifying dead, weak, or moribund animals and documenting the onset and progression of any abnormal clinical signs. Appropriate actions were taken to minimize the loss of animal s from study (e.g., necropsy or refrigeration of any rats found dead and sacrifi ce of weak or moribund animals).

Body Weight . All animals were individually weighed using the Path-Tox ® data acquisition system (Xybion, Cedar Knolls, NJ). Males and females on this study were initially weighed for randomization on May 7, 2001 and May 14, 2001, respectively. Due to problems with setup and validation of the exposure system, the initiati on of the study was delayed by approximately 10 days. This decision was made by mutual agr eement between LRRI and the Sponsor, after the animals had already been weighed, randomized, and identified. Day -1 weights, obtained on the day prior to initiation of exposures, were take n on May 22 and May 29, for males and females, respectively. Therefore, the Da y -1 weights were obtained 16 da ys after randomization occurred. After initiation of exposures, weights were obtai ned and weekly for 13 weeks and then every 4 weeks thereafter.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

2-9 Clinical Signs of Toxicity . Thorough clinical examinations were made at randomization, on Day 1, and weekly thereafter. Observations we re detailed and carefully recorded using PathTox ® software. Observations includ ed evaluations of skin and fur; eyes and mucous membranes; respiratory and circulatory eff ects; autonomic effects such as salivation and central nervous system effects including tremor s and convulsions; and changes in the level of activity, gait, posture, reactivity to handling or sensory stimuli, altered strength, and stereotypes or bizarre behavior (e.g., self-mutilation, walking backward).
Hematology . At 12 months, 18 months and at final sacrifice, blood smears were prepared and manual differential cell counts were made. Fo r the 12- and 18-month time points, blood was obtained by tail nick. At final sacrifice, bl ood was obtained by cardiac puncture and collected into Vacutainer tubes containing ethylenedi amine tetra-acetic acid (EDTA). Evaluated parameters included total leukocyte estimates (w hite blood cell [WBC]); nucleated red blood cell counts (cells/100 WBC), anisocytosis (0 to 4+ ), polychromasia (1+ to 4+) and; relative differential leukocyte counts for segmented ne utrophils, band neutrophils, lymphocytes, monocytes, eosinophils, basophils, atypical lymphoc ytes and blastocytes. Absolute leukocyte counts were calculated for segmented neutroph ils, band neutrophils, lymphocytes, monocytes, eosinophils, basophils, atypical lymphocytes and blastocytes using the formula WBC * relative percent/100.
Post-Exposure Endpoints

Gross Necropsy . A complete gross examination was perf ormed on all animals at final sacrifice and on those animals that died naturally or were euthanized in a mori bund condition. Sacrifices of rats surviving 520 days of exposure occurred during the week following the last exposure day for each sex. Animals were randomly assigned to a sacrifice day. Samples collected at necropsy are listed in Table 2-2.

Table 2-2. Organs and Representati ve Samples Taken for Examination
Cardiovascular/Hemato p oietic S y stem 1. Aort a 2. Bone marrow ( and/or fresh as p irate ) 3. Heart 4. L y mph nodes (mandibular, mesenteric, bronchial, mediastinal) 5. Splee nStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

2-10 Table 2-2. Organs and Repr esentative Samples Taken for Examination (Concluded)
Di g estive S y stem 1. Cecum 2. Colon 3. Duodenum 4. Eso p ha g us 5. Ileu m 6. Je j unum 7. Live r 8. Pancreas 9. Rectum 10. Salivar y g lands 11. Stomach Glandular S y stem 1. Adrenals 2. Parath y roi d 3. Th y roi d 4. Mammar y g land ( male a and female ) Nervous S y stem 1. Brain ( includin g sections of medulla/ p ons, cerebellum, and cerebrum ) 2. E y es ( retina, o p tic nerve ) 3. Peri p heral nerve ( sciatic or tibial, p referabl y in close p roximit y to the muscle ) 4. Pituitar y 5. S p inal cord ( three levels: ce r vical, mi d -thoracic, and lumbar ) Other 1. All g ross lesions and masses 2. Skin 3. Tail ( for identification ) 4. Skeletal muscle a 5. Femu r a Res p irator y S y stem 1. Lar y nx 2. Lun g ( infused with fixative ) 3. Nose 4. Paranasal sinuses 5. Phar y nx ( reviewed with lary nx section ) 6. Trache a Uro g enital S y stem 1. E p idid y mis 2. Kidne y s a 3. Ovaries 4. Prostate 5. Seminal vesicle ( s ) 6. Testes 7. Urinar y bladder ( infused with fixative ) 8. Uterus

a Examined microsco p icall y but not re q uired b y p rotocol.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

2-11 All study animals received a complete necropsy. Animals were euthanized with an overdose of intraperitoneally injected barbiturate anesthetic (Euthasol ® , Virbac AH Inc., Fort Worth, TX). Body weights and fresh organ weig hts were collected on lungs, liv er, kidneys, adrenals, testes, epididymis, ovaries, uterus, spleen, brain, and heart of final sacrific e and moribund sacrifice animals. Animals found dead received a comple te necropsy with tissue collection, but blood and organ weight data were not rout inely collected. Cardiac blood was collected from animals at final sacrifice for determination of total a nd differential WBC counts. Gross lesions, body weights, and organ weights were entered on pre- prepared forms, and then the information was recorded on the Path-Tox ® database (Version 4.2.2, Module P, Xybion Medical Systems, Cedar Knolls, NJ).

Lungs were gently instilled via the trachea with 10% neutra l buffered formalin (NBF) to approximate normal volume. Organs/tissues were immersion fixed in 10% NBF for subsequent histopathologic examination. Tissues were trimmed, processed routinely, paraffin embedded, sectioned at 5 μm, and stained with hematoxylin and eosin for histopat hologic examination.

Histopathology . All collected tissues and lesions were examined histologically in control (0 g/m 3 ) animals, high-level (20 g/m 3 ) animals, and dead or moribund animals of all groups. Respiratory tissues (lungs, larynx, trachea, and nasal turbinate sect ions), potential target tissues (testes, kidneys of males) and gross lesions we re examined histologically from final sacrifice low- (2 g/m 3 ) and mid- (10 g/m 3 ) level animals. Nomenclature of proliferative lesions was based on the international harmonized nomenclat ure recommended by the Rat Nomenclature Reconciliation Subcommittee of the Soci ety of Toxicologic Pathologists (see http://www.toxpath.org; Standardiz ed Rat Nomenclature). Nomenclature of other lesions was routine, widely understood usage (see B oorman et al., 1990a; G opinath et al., 1987).

Standard, subjective severity scor ing of most nonproliferative lesi ons was based on the extent of tissue affected by the change and the severity of the change within affected areas. Typically, a score of 0 (none) = essentially no tissue affected; 1 (minimal) = 1 to 10% affected, 2 (mild) = 11 to 25% affected, 3 (moderate) = 26 to 50% affecte d, 4 (marked) = 51 to 100% affected. SeverityStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

2-12 scores may have been adjusted up or down (usua lly by 1 point) based on the severity of the change within the affected areas.

The kidney is an expected target tissue for toxici ty in this study, especi ally for development of chronic progressive nephropat hy or hyaline droplet nephropathy. The diagnosis of chronic progressive nephropathy encompasses a large constellation of changes; severity scoring for this diagnosis was somewhat more comple x and is described in Table 2-3.

Table 2-3. Severity Scoring of Chronic Progressive Nephropathy Score Histologic Findings 0 Essentially no changes of chronic pr ogressive nephropathy. May have rare scattered protein casts in tubules, mineral concretions.
1 Increased number and size of tubular protein casts; pr imarily within tubules at corticomedullary junction, some within co llecting ducts. Minor/scattered foci of basophilic, regenerative tubules. Cha nges affect less than 5% of tissue component. 2 Homogeneous protein casts relatively abundant within tubules and collecting ducts (especially at corticomedullary junc tion). Increased size and frequency of tubular basophilia. Accompanying change s present, may include small foci of tubular basement membrane thickening and/or mineralization, interstitial fibrosis, tubular atrophy and dilatation, minor a ssociated mononuclear inflammatory infiltrates, basophilia ch anges (including mesangial proliferation, synechiae, proliferation of parietal epithelium) . Changes may affect up to 25% of component.
3 As 2 with increased tubu lar atrophy/dilatation/regenera tion, interstitial fibrosis, increased inflammatory infiltrates, incr eased thickening of basement membranes of tubules and glomeruli.
4 As 3 with increased seve rity and extent of change s. Senescent glomeruli relatively common. Typically combined ch anges affect well over 50% of kidney.

Statistics Body and Organ Weights . Group mean body weight, organ weight, percent organ-to-body weight data, and percent organ to brain weight data, were tested for statistical significance using Path-Tox ® software. After testing for an overall tr end among test groups by an analysis of variance, Bartlett’s test was used to establish the homogeneity of the data. If the data wereStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

2-13 homogeneous, significant differences between th e control and exposed groups were evaluated using a modified Dunnett’s test. If data were nonhomogeneous, a modified t test was used. Significance levels were set at p ≤ 0.05.

Survival Analysis . The number of days of survival was calculated for each animal in the study. Two female rats that died subsequent to accide ntal nose injuries early in the study were deleted from the analysis. The probability of survival was estimated by the Kaplan-Meier product-limit method using PROC LIFETEST in SAS Version 8.2. Mean numbers of survival days and time to 25% mortality were estimated for each dose gr oup. Log-rank tests were used to test the hypothesis that there are differen ces among the four groups for each sex. The significance level was set at p = 0.05. All reported p-values fo r the survival analysis are two sided.

Analysis of White Blood Cell Total and Differential Cell Counts . For the hematology data, medians and ranges are presented since the distribu tions are highly skewed for some variables. Preliminary analyses were done using the ge neralized estimating e quation approach for longitudinal data analysis since there were three time points. For some variables these analyses showed significant inte ractions between time and group. Th erefore, analyses were done to compare dose groups at each time point and to compare time points within each dose group. The Krusal-Wallis test was used for these comparisons.

Histopathology . The incidences of all neoplastic a nd non-neoplastic lesions are given as the ratio of the number of affected animals to the number of an imals with the site examined microscopically. Three statistical evaluati ons were performed on the histopathology lesion incidence data: 1) Cochran-Armitage test, whic h tests whether the incidence of lesions shows a trend across dose groups; 2) logistic regression that takes death date into account when assessing the presence of a dose-dependent trend; and 3) the Fi sher’s exact test to compare incidences among the four dose groups. The two-sided si gnificance level was se t at p = 0.05. If a significant difference was detected by the Fisher’s test, six possible pairwise comparisons were calculated. Using the Bonferroni correction for pairwise compar isons, each pairwise comparison would be considered significant if p < 0.008.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

2-14 Fisher’s exact test and the Cochran-Armitage test do not use survival information and are appropriate in situations where survival is similar among exposure group as is the case for this study. The Fisher’s exact tests the null hypothesis of equality of prevalences across dose groups against the alternate hypothesis that the preval ences are not equal while the Cochran-Armitage tests the null hypothesis of equality across doses against the alternate hyp othesis of a monotonic increasing or decreasing trend.

In the case of renal tubule nephropathy, statisti cal evaluation of group di fferences in severity scores was performed using the Kolmogorov-Smirnov test within the Path-Tox ® software. The significance level was p ≤ 0.05.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-1 RESULTS

EXPOSURE ATMOSPHERE Pre-Exposure Characterization Three-Day Stability Evaluati on and Nominal GMVC Usage . Before exposures were initiated the system was operated for 6 hours/day for 3 consecutiv e days. The concentration of vapor in each chamber was close to target and constant wi thin each day, and the concentrations were reproducible from day to day (Table 3-1). Gas chromatographic analysis of the chamber atmospheres indicated that the relative percenta ges of the major components were constant over the 3-day period. The percentages of actual GMVC usage/anticipated usage (nominal usage) on the 3 days of stability testing were 95, 97, a nd 90 for days 1, 2, and 3, respectively. Pre-test chamber trial data are pr esented in Appendix E.

Table 3-1. Results of 3- Day Stability Evaluations
Target Exposure Concentration (g/m 3 )/

Chamber Number Achieved Concentration ± SD (n = 3) Day 1 (5/17/01) Day 2 (5/18/01) Day 3 (5/19/01) 2 (Chamber 6) 2.06 ± 0.04 1.97 ± 0.10 1.93 ± 0.05 10 (Chamber 7) 9.45 ± 0.23 9.37 ± 0.15 9.37 ± 0.28 20 (Chamber 8) 18.8 ± 0.28 18.9 ± 1.14 19.5 ± 0.37

Homogeneity of Vapor Concentration . The homogeneity of vapor concentration throughout the exposure chambers was determined by c onducting a chamber distribution study. The concentrations of GMVC were measured from four sampling ports located in front and four sampling ports located in the back of the chambe rs during an exposure day and compared to the concentration obtained in a refe rence location within the chamber. The spatial variations measured in Chambers 6 (2 g/m 3 ), 7 (10 g/m 3 ), and 8 (20 g/m 3 ) were 3.0%, 1.3%, and 0.8%, respectively. These results indicate the vapor was distributed evenly within each of the chambers. Homogeneity of vapor concentr ation data is presented in Appendix E.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-2 T90 Determination . The amounts of time required to reach T90 were 13, 14.1, and 14.2 minutes for Chambers 6, 7, and 8, respectively. A T90 of 14 minutes was chosen fo r use with this study. Daily exposure periods to GMVC we re then 6 hours and 14 minutes.

Confirmation of the Absence of Aerosols . The particle concentrati ons of air inside Chamber 8, operated at a target concentration 20 g GMVC/m 3 , and the control chamber (Chamber 5) were determined using a TSI Aerodynamic Particle Sizer . There was less than 1 particle per cubic centimeter air in the control and high-level chambers, indicating that there were no GMVC aerosols at the highest target vapor concentration used in this study.

In-Study Data Chamber Concentrations and Nominal Usage . The overall study means of the daily vapor concentrations achieved for the 2, 10, and 20 g GMVC/m 3 vapors are provided in Table 3-2. The overall achieved means were within 2% of targ et for each exposure concentration. Thus, all future references to exposure c oncentration are in terms of target concentration. Concentrations of vapor in the control chamber were below the lowest concentration on th e standard curve used to calibrate the control ch amber Miran 1A (1 g GMVC/m 3 ). The overall average of the daily percent nominal usage was 99% ± 4%, indicating excellent agreement between anticipated and actual GMVC usage. Daily vapo r concentrations and nominal us age are provided in Appendix E.

Table 3-2. Summary of GM VC Vapor Concentrations a Target g GMVC/m 3 Achieved Concentrations Percent of Target 0 NA b NA 2 2.02 ± 0.07 101 10 10.0 ± 0.37 100 20 20.3 ± 0.74 101

a Results are the mean ± SD of vapor concentrations obtained in

520 exposure days. b Values were below 1 g GMVC/m 3 , the lowest concentration on

the standard curve used to calibrate the Miran 1A used to make the measurements.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-3 Homogeneity of Vapor Concentration . An evaluation of the homogeneity of vapor concentration in each GMVC chamber was conducted after animal exposures began. The spatial variations measured in Chambers 6 (2 g/m 3 ), 7 (10 g/m 3 ), and 8 (20 g/m 3 ) were 2.8%, 0.86%, and 1.8%, respectively. These results indicate the vapor is distributed evenly within each of the chambers. Homogeneity of vapor concentration data are presented in Appendix E.

Gas Chromatographic Profiles of GMVC Exposure Atmospheres . Gas chromatographic profiles were obtained from each exposure chamber weekly to assess whether the animals were exposed to the relative amounts of the same 19 major components thro ughout the study. The peak areas of the 19 major components were summed to provide a “total peak area” for those components. The peak area of each of the components was di vided by the total peak area for the components to obtain a relative peak area (in percent) for each of the components. These were compared to the reference values provided by EMBSI. Pr ofiles remained acceptably constant throughout the study. Weekly results are provided in Appendix F.

Chamber Environmental Data . The temperature, relative hum idity, chamber flow rate and pressures were maintained within acceptable ranges with occasional excursions outside acceptable limits that are described in Section 4. E nvironmental data are provided in Appendix G.

Survival Analysis . All groups had 50 animals except th e mid-level group for females. Two animals in that group were omitted from the analysis because they were euthanized following accidental nose injuries early in the study. Figures 3-1 and 3-2 show the survival curves for male and female animals, respectively. The diffe rences among the groups are not statistically significant for male (p = 0.46) or female animal s (p = 0.07). Table 3-3 shows means, standard errors, and day of 25% mortality for male and female animals under each experimental condition. For the calculation of mean and standard errors of survival times, the last day of the study was used as the value of the survival tim e for animals that underw ent final sacrifice. Therefore, the means and standard errors may be underestimated since so me of the animals may have lived longer if the protocol had included fo llowing them for a longer period of time. The results indicate that chronic GMVC inhalation di d not shorten the lifespan of the exposed rats compared to the controls.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-4 Figure 3-1. Survival of Male Animals

Figure 3-2. Survival of Female AnimalsStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-5 Table 3-3. Mean Survival Da ys and Day of 25% Mortality for Male and Female Rats

Group Male Female Mean Days of Survival

(SE) Estimate Day of 25% Mortality Mean Days of Survival

(SE) Estimated Day of 25% Mortality Control 652.6 (14.4) 626 665.9 (11.5) 632 Low 651.6 (12.8) 633 685.5 (11.9) 680 Mid 675.3 (9.4) 633 680.9 (15.5) 660 High 674.1 (9.5) 645 697.3 (10.6) 715

Animal Disposition . The disposition of all animals on study is summarized in Table 3-4. A small percentage of animals died naturally. The majority of an imals not surviving 104 weeks on study underwent moribund sacrifices. Individual an imal disposition is provided in Appendix H.

Table 3-4. Disposit ion of Rats on Study

Death Type Control 2 g GMVC/m 3 10 g GMVC/m 3 20 g GMVC/m 3

Male Female Male Female Ma le Female Male Female

Moribund Sacrifice 35 24 29 18 27 17 29 15

Natural Death 1 4 5 2 3 3 2 2

Terminal Sacrifice 14 22 16 30 20 30 19 33

Total 50 50 50 50 50 50 50 50

Body Weight Gain . Mean group body weights of male and female rats are provided in Tables 3-5 and 3-6, respectively. Growth curves are provided in Figures 3-3 and 3-4 for males and females, respectively.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-6 Table 3-5. Body Weight Summary for Male Rats a

Study Day Control 2 g/m 3 10 g/m 3 20 g/m 3 Body Weight (g) Body Weight (g) Percent Control Body Weight (g) Percent Control Body Weight (g) Percent Control Mean SD Mean SD Mean SD Mean SD -1 184.4 7.8 182.1 9.8 98.8 184.1 10.9 99.8 177.9 11.6 96.5 7 188.2 11.5 193.8 11.6 103.0 193.4 12.9 102.8 184.2 20.2 97.9 14 207.5 12.9 207.8 16.2 100.1 208.0 14.3 100.2 199.5 14.5 96.1 21 226.5 14.3 226.1 15.6 99.8 225.3 15.3 99.5 217.4 14.3 96.0 28 239.7 16.1 240.0 15.6 100.1 237.7 16.5 99.2 229.5 14.3 95.7 35 251.2 17.3 250.9 16.4 99.9 250.7 17.4 99.8 241.7 15.5 96.2 42 259.1 18.2 260.7 16.6 100.6 260.5 17.8 100.5 252.2 15.8 97.3 49 272.7 17.8 273.7 15.9 100.4 271.0 17.9 99.4 263.4 16.6 96.6 56 283.1 17.5 282.2 15.7 99.7 281.6 18.3 99.5 275.0 16.2 97.1 63 290.5 18.3 296.5 18.5 102.1 288.6 18.3 99.3 282.0 17.3 97.1 70 297.6 18.4 297.1 16.7 99.8 296.6 18.7 99.7 291.2 17.8 97.8 77 309.1 19.0 306.1 17.0 99.0 305.9 19.6 99.0 299.6 17.6 96.9 84 314.7 18.8 312.5 16.6 99.3 312.3 20.2 99.2 305.8 17.8 97.2 91 319.3 18.9 316.9 16.8 99.2 313.5 18.4 98.2 309.2 17.2 96.8 119 340.8 18.1 338.5 15.8 99.3 338.8 19.8 99.4 329.9 18.5 96.8 147 355.0 17.9 353.3 15.0 99.5 351.1 21.1 98.9 343.8 18.5 96.8 175 368.6 18.3 363.0 15.8 98.5 363.5 21.9 98.6 358.8 18.8 97.3 203 379.4 19.9 375.5 16.2 99.0 377.9 23.0 99.6 369.4 19.7 97.4 231 396.3 21.0 389.5 16.5 98.3 390.2 23.9 98.5 381.4 21.1 96.2 259 397.0 19.4 394.5 16.4 99.4 390.3 22.8 98.3 383.4 19.2 96.6 287 405.3 22.5 400.2 17.7 98.7 398.3 22.7 98.3 389.0 19.6 96.0 315 415.8 22.5 408.6 18.1 98.3 406.5 24.6 97.8 395.5 22.1 95.1 343 426.1 21.9 419.1 19.7 98.4 417.0 26.3 97.9 408.2 23.2 95.8 371 424.8 27.9 420.5 19.5 99.0 419.3 25.5 98.7 406.7 22.2 95.7 399 435.7 25.8 424.4 20.9 97.4 422.9 24.9 97.1 408.1 22.8 93.7 427 437.0 26.2 424.4 20.4 97.1 419.6 24.6 96.0 406.5 23.0 93.0 455 441.8 26.1 424.9 23.8 96.2 424.4 25.0 96.1 407.8 24.9 92.3 487 445.8 26.6 431.8 21.6 96.9 425.2 24.0 95.4 412.3 23.6 92.5 515 444.2 25.0 430.8 22.1 97.0 423.4 24.8 95.3 409.7 23.9 92.2 539 433.6 24.6 420.1 22.9 96.9 415.0 22.3 95.7 397.3 23.1 91.6 567 431.3 25.9 417.8 23.0 96.9 410.2 21.2 95.1 389.7 25.5 90.4 595 425.7 26.1 413.2 19.4 97.1 405.4 19.7 95.2 383.0 29.3 90.0 623 416.6 32.5 408.4 19.7 98.0 392.9 27.0 94.3 381.6 22.6 91.6 651 401.6 34.7 401.1 26.2 99.9 390.5 22.8 97.2 376.0 29.1 93.6 679 402.7 27.6 400.9 26.9 99.6 389.0 15.4 96.6 369.8 23.2 91.8 707 391.8 35.7 383.9 27.6 98.0 381.7 12.6 97.4 362.6 24.7 92.5

a Bolded and italicized values are significantly di fferent from controls at that time point; p ≤ 0.05. Group mean body weight, organ weight, and percent organ-to-body weight data were tested for statistical significance using Path-Tox ® software. After testing for an overall trend among test groups by an analysis of variance, Bartlett’s test was used to establish the homogeneit y of the data. If the data were homogeneous, group differences were evaluated using a modified Dunnett’s test. If data were non-homogeneous, group differences were assessed usin g a modified t -test. Si g nificance levels were set at p ≤ 0.05.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-7 Table 3-6. Body Weight Summary in Female Rats a

Study Day Control 2 g/m 3 10 g/m 3 20 g/m 3 Body Weight (g) Body Weight (g) Percent Control Body Weight (g) Percent Control Body Weight (g) Percent Control Mean SD Mean SD Mean SD Mean SD -1 125.4 5.3 123.6 4.0 98.6 122.8 6.6 97.9 121.8 4.9 97.1 7 135.4 5.9 135.2 4.6 99.9 133.8 7.4 98.8 130.1 5.4 96.1 14 145.6 7.1 144.8 4.9 99.5 141.9 8.4 97.5 136.2 6.3 93.5 21 153.0 8.1 153.1 6.6 100.1 147.5 8.9 96.4 142.3 6.7 93.0 28 156.6 9.6 155.8 7.9 99.5 152.7 9.2 97.5 148.7 7.2 95.0 35 161.3 9.9 160.8 6.7 99.7 156.1 9.7 96.8 151.7 6.9 94.0 42 165.3 8.3 165.3 6.7 100.0 160.9 10.1 97.3 154.5 7.5 93.5 49 170.1 8.8 170.8 6.7 100.4 164.5 10.5 96.7 159.5 7.4 93.8 56 172.1 8.9 173.8 6.9 101.0 168.5 10.4 97.9 162.6 7.6 94.5 63 176.1 9.3 178.9 7.2 101.6 173.7 10.5 98.6 168.1 7.8 95.5 70 181.6 9.2 181.3 7.3 99.8 175.9 10.3 96.9 169.8 7.8 93.5 77 183.7 9.5 183.7 7.3 100.0 177.2 10.4 96.5 170.9 7.8 93.0 84 185.1 9.8 185.2 8.0 100.1 179.5 11.0 97.0 172.9 7.5 93.4 91 186.5 10.1 188.0 7.6 100.8 180.7 11.4 96.9 174.9 7.7 93.8 119 196.2 9.9 197.0 7.8 100.4 189.4 10.6 96.5 182.1 8.2 92.8 147 199.9 10.5 202.4 7.5 101.3 193.1 10.9 96.6 186.0 8.5 93.0 175 207.1 10.6 207.3 7.8 100.1 199.4 10.9 96.3 194.0 9.1 93.7 203 210.5 10.8 209.9 8.8 99.7 204.2 11.3 97.0 196.4 8.4 93.3 231 212.2 10.5 212.8 8.5 100.3 206.9 11.0 97.5 198.1 8.6 93.4 259 214.2 10.2 214.9 9.0 100.3 206.8 11.0 96.5 199.1 8.1 93.0 287 219.0 10.7 220.8 9.5 100.8 212.1 11.5 96.8 201.9 9.2 92.2 315 221.5 12.2 222.0 10.9 100.2 214.7 12.0 96.9 204.7 8.7 92.4 343 225.8 12.9 225.2 11.4 99.7 215.8 13.0 95.6 207.3 8.6 91.8 371 232.6 15.5 232.7 12.8 100.0 220.6 13.8 94.8 208.7 9.8 89.7 399 237.4 17.6 236.5 14.8 99.6 226.4 14.0 95.4 215.0 10.4 90.6 427 243.2 19.6 241.8 16.3 99.4 229.3 15.1 94.3 216.2 11.0 88.9 459 255.9 22.0 255.8 18.1 100.0 239.6 18.3 93.6 226.3 13.0 88.4 487 260.5 22.4 259.7 19.6 99.7 241.8 19.4 92.8 229.6 15.5 88.1 512 258.1 24.4 258.3 17.7 100.1 241.0 17.8 93.4 228.6 12.7 88.6 539 259.5 22.5 261.9 18.6 100.9 243.3 20.9 93.8 229.6 13.2 88.5 567 263.7 21.5 261.5 16.8 99.2 245.5 19.3 93.1 229.4 12.7 87.0 595 266.1 21.6 266.3 15.2 100.1 250.9 18.6 94.3 234.1 12.8 88.0 623 264.1 25.4 269.7 14.8 102.1 249.7 21.2 94.5 233.6 11.7 88.5 651 268.8 26.2 273.3 15.2 101.7 252.5 22.1 93.9 237.3 13.6 88.3 679 269.1 27.6 274.7 15.9 102.1 255.6 24.2 95.0 238.4 13.0 88.6 707 268.4 30.4 273.6 16.7 101.9 256.1 25.2 95.4 239.3 16.1 89.2

a Bolded and italicized values are significantly di fferent from controls at that time point; p ≤ 0.05. Group mean body weight, organ weight, and percent organ-to-body weight data were tested for statistical significance using Path-Tox ® software. After testing for an overall trend among test groups by an analysis of variance, Bartlett’s test was used to establish the homogeneit y of the data. If the data were homogeneous, group differences were evaluated using a modified Dunnett’s test. If data were non-homogeneous, group differences were assessed usin g a modified t -test. Si g nificance levels were set at p ≤ 0.05.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-8 Figure 3-3. Body Weights of Ma le Rats Exposed to GMVC

Figure 3-4. Body Weights of Female Rats Exposed to GMVCStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-9 On Day -1, male rats in the high-dos e group had a group mean body weight (177.9 g) significantly below that of the control rats (184.4g) . Due to recently identif ied problems with the vapor exposure system, the animals had been randomized approximately two weeks before initiation of dosing. Review of the body weight gain from the time of randomization to the day before initiation of exposures (D ay -1) indicated that the contro l males gained an average of 4.99 g/day. The lowest weight gain among control ma les was 3.9 g/day. Six of 50 males in the highdose group gained less than 3.9 g/day, with one of these gaining only 1.9 g/day. The rats gaining less weight were not clustered in one location within the cham ber, suggesting the low weight gain was not due to a basket feeder issue. Clinic al observations on these rats reported them to be normal. Because the rats had already been tattooed, re-randomization was not an option.

At Day -1, mean body weights of the mid- and hi gh-level females at were significantly below controls. Review of the randomization weight and Day -1 weight data indicated the control rats gained an average of 2.27 g/day, with the lowest weight gain in this group being 1.52 g/day. Three of the 50 mid-dose and 5 of 50 high-dose fe males had gains of less than 1.5 g/day. As with the males, there was no obvious clustering of animal with low weight gain, indicating that the lower weight gain wa s not a feeder issue.

The matter of significantly different body wei ghts among males and females before the study start was brought to the attention of the Sponsor and the EP A. Comments from the EPA dated July 6, 2001, indicated the agency did not feel the group differences in body weight were large enough to warrant restarting the study.

All animals gained weight during the study; how ever, body weights were reduced in both males and females exposed to GMVC co mpared to control values. B ody weights of high-level male and female rats remained statistically significan tly below control values for most of the study (with the exception of days 63 and 70 for male s and days 35, 42, and 49 for females). From Days 399–623, body weights of mid-level males and, occasionally, low-level males were also significantly below control valu es. Mid-level female body wei ghts were significantly below control values except on Day 7, Days 28–63, and Day 707. Individual animal in-life body weights are provided in Appendix I.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-10 Clinical Signs of Toxicity . Rats exposed to GMVC displayed no clinical signs of toxicity related to GMVC exposure. As this was a chronic toxicity evaluation, ma ny of the observations observed during the second year of the study were related to agi ng (mammary masses, jaundice). A summary of clinical obse rvations by exposure group is provided in Appendix J.

Body Weights at Final Sacrifice Final sacrifice body weights of male a nd female rats exposed to 20 g GMVC/m 3 were significantly below control values (Table 3-7); weights were 91.5% and 91.8 % of control, respectively. Individual animal data are provided in Appendix K.

Table 3-7. Summary Statistics for Final Sacrifice Body Weight s of Male and Female Rats

Weight in grams Control 2 g GMVC/m 3 10 g GMVC/m 3 20 g GMVC/m 3
Males Mean ± SD 390.0 ± 18.2 375.5 ± 35.2 368.7 ± 28.5 357.0 ± 28.4 a n 14 16 20 19 Females Mean ± SD 265.4 ± 33.3 273.5 ± 20.4 256.3 ± 21.9 243.6 ± 15.7 b
n 22 30 30 33

a Mean significantly different from controls. Data homogeneous by Bartlett’s test. Means

compared using Dunnett’s t test; p ≤ 0.05. b Mean significantly different from controls. Data nonhomogeneous by Bartlett’s test.

Means compared using a Modified t test; p ≤ 0.05.
Absolute Organ Weight at Final Sacrifice Organ weight for adrenal glands, brain, epididymis (males), heart, kidneys (pair), liver, lungs, ovaries (females), spleen, testes (males) and uterus (females) were recorded. At terminal sacrifice, mean group brain weights of high-le vel males and females were significantly below corresponding control values. The epididymis wei ght of mid-level males and adrenal weight of high-level females were significantly below that of the corresponding cont rol values. Summary statistics for males and females are provided in Ta ble 3-8. Individual animal data are provided in Appendix K, pages K-2 through K-16.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-11Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-12 Percent Organ-to-Body Weight at Final Sacrifice Significant change occurred only among male rats . Percent brain-to-body weight and lung-tobody weight were significantly increased in mi d and high GMVC-exposed males compared to controls. The lung results are li kely a result of the treatment- related depression in body weight, rather than effects on the organs weights since there were no cha nges in absolute lung weights in males. For brain there was an apparent tre nd for the effects to be exposure concentration dependent. The percent kidney-to -body weight was increased only in high-level GMVC-exposed males. Summary statistics are provided in Table 3-9 for males and females. Individual animal data are provided in Appendix K pages K-17 through K-31.
Percent Organ-to-Brain Weight at Final Sacrifice There were few effects on organ-to-brain weight s. The percent kidney-to-brain weight was significantly increased for high- level GMVC-exposed male rats. The percent epididymis-tobrain weight was significantly decreased in mi d-level males. The pe rcent adrenal glands-tobrain weight was significantly decreased in hi gh-level GMVC-exposed female rats. Summary statistics are provided in Table 3-10 for males and females. Individual animal data are provided in Appendix K pages K-32 through K-46.
Terminal Body Weight and Organ Wei ght of Euthanized (Moribund) Rats As the rats reached about 20 months of age (18 months on study), mortality due to effects of aging increased. Most animals not surviving to the final sacrifice were euthanized and terminal body and organ weights were recorded. Because the animals were euthanized over a period of several months, interpretation of the statistical significance of differences among exposed and control groups is not as straightforward as with data obtained at the fi nal sacrifice. Summary statistics and individual animal terminal body an d organ weight data ar e provided in Appendix K pages K-47 through K-98. Of particular note, th e absolute kidney and percent kidney-to-brain weight of all GMVC-exposed groups were significantly increased co mpared to control values. Kidney-to-body weight was only si gnificantly increased among ma les exposed to the mid and high levels of GMVC. The epididymis weight and epididymis-to-body weight was significantly decreased among mid-level males. Liver-to-bod y weight ratios for high-level males were statistically significantly increa sed. Among females, the absolute brain weight was significantly decreased in high-level GMVC fe males. The liver-to-body weight was significantly decreased in the low group females and the lung-to-body weig ht was significantly increased among the highlevel females.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-13Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-14Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-15 Hematology A summary of the effects of e xposure concentration and exposure time on WBC estimates and absolute numbers of WBC types is provided in Table 3-11. Individual animal data are located in Appendix L. Statistical analyses were conduc ted on continuous variab les (WBC estimates and percentage and absolute differe ntial cell count data), and are presented in the Statistician’s Report.

Statistically significant differences were found in the WBC estimate, and in absolute numbers of neutrophils, band neutrophils, lymphocytes, monocyt es, and eosinophils at the 12-month point. In all cases, values for the high- level male group were greater th an the controls. The changes were influenced by both differences in absolute numbers of WBCs and in the percentage of the cell types found. These differen ces were not found among females at 12 months, nor were they as pronounced in males or present in females at the 18- and 24-month sacrifices. The results in 12-month males possibly were affected by the tail blood sampling technique, which improved by 18 months. At 12 months we ha d difficulty sampling blood from the tail, especially for the male rats. This may have resulted in artifactually high WBC counts.

By 24 months, when blood was taken by cardiac puncture, few differences in WBC parameters were observed between high-level and control rats of either se x. Only the percentage and absolute numbers of monocytes in high-level males were signif icantly different (lower than controls). In females the percentage and ab solute numbers of atypi cal lymphocytes were significantly lower in high-level animals compared to controls. The presence of atypical lymphocytes is compatible with the high incidence of leukemia in rats at this age. Since controls and high-level females had similar incidences of leukemia, the biological significance of the increased number of atypical lymphocy tes in high-level females is unknown.

The high incidence of leukemia in the aged F 344 rats on study, other phy siologic changes with aging, and the limitations of the estimate of to tal WBC from a blood smear indicate that the hematology results should be interpreted with caution.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-16Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-17 Histopathology Proliferative Lesions Attributable to GMVC Exposure . Proliferative lesions include hyperplasias and neoplasias (benign and malignant). Male rats exposed to 10 g GMVC/m 3 demonstrated significant increases in the incidence of renal tubule cell adenomas (T able 3-12 and Appendices M, N, O, and P). There was also a non-sta tistically significant incr ease in renal tubule carcinomas in the males (1/50; 2% in low and 1/50; 2% in high-level males). Combining adenomas and carcinomas for statistical analysis maintained the statistically significant increase in tumors in the mid-level animals, and additi onally showed a significant trend toward increased incidence with increasing exposure concentration (Table 3-12). It should also be noted that development of renal tumors is a late-life event, and it is possi ble that the incidence of renal tumors in males may have been higher if more than 40% of the males had survived to terminal sacrifice. These renal tumors are considered to be treatment induced based on previous studies with wholly vaporized unleaded ga soline and neat MTBE (MacFarland et al., 1984; Bird et al., 1997).
There was a high background incidence of testicular interstitial ad enomas in control male rats (43/50; 86%). The incidence among high-level males (50/50; 100%) was significantly greater than the control incidence. The relationship between treatment and i nduction of testicular interstitial adenomas is considered to be equi vocal, since background incide nce of this tumor is extremely high (Boorman et al., 1990b; Gopinath et al., 1987) and historical incidence data from one 2-year bioassay conducted at LRRI for a commercial sponsor was 86% (n = 56). NTP control incidence 3 for air inhalation controls: for NIH- 07 diet avg. = 70.1% with range of 46% to 90%; for NTP-2000 diet avg. = 77.2% with range of 66% to 84%). Further, the control incidence in the concurrently run Baseline Gasoline Vapor Condensate chronic study (LRRI study number FY01-027) was 48/50 ( 96%). It has been hypothesi zed that MTBE disruption of hormonal homeostasis could account for increased testicular interstitial cell adenomas (Cruzan et al., 2007). Mechanistic work to date to eval uate this hypothesis has been conducted in in vitro studies with high doses of MTBE; only one in vivo study has been reported and this study found decreases in leuteinizing horm one and testosterone in a single time point measurement. Consequently, this data should be interpreted with caution.

3 Reference - http://ntp.niehs.nih.gov/ntpweb/index. cfm?objectid=92E61F1B-F1F6 -975E-7D3BED551F07DC0AStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-18 Table 3-12. Incidences of Proliferative (Ne oplastic and Hyperplas tic) Lesions in Rats
Control (0 g/m 3 ) Low (2 g/m 3 ) Mid (10 g/m 3 ) High (20 g/m 3 )
MALES Kidney No. examined 50 50 50 50 Adenoma, renal tubule 0 (0%) 0 (0%) 6 (12%) a 2 (4%) Carcinoma, renal tubule 0 (0%) 1 (2%) 0 (0%) 1 (2%) Renal tubule adenoma and carcinoma, combined 0 (0%) 1 (2%) 6 (12%) b 3 (6%)

Nasal Sections (Note that the same tumor may appear in more than
one level.) Turbinate Level 1 No. examined 50 50 50 50 Hyperplasia Avg. severity 3 (6%) 0.2 0 (0%) 0.0 3 (6%) 0.1 4 (8%) 0.2 Turbinate Level 2 No. examined 50 50 50 50 Hyperplasia Avg. severity 0 (0%) 0.0 2 (4%) 0.1 2 (4%) 0.1 3 (6%) 0.2 Carcinoma, squamous cell 1 (2%) 0 (0%) 0 (0%) 0 (0%) Turbinate Level 3 No. examined 50 50 50 50 Carcinoma, squamous cell 0 (0%) 1 (2%) 0 (0%) 1 (2%) Turbinate Level 4 No. examined 50 50 50 50 Carcinoma, squamous cell 0 (0%) 2 (4%) 0 (0%) 2 (4%)
Thyroid No. examined 50 35 31 50 Hyperplasia, follicular cell Avg. severity 0 (0%) 0.0 0 (0%) 0.0 1 (3%) 0.1 0 (0%) 0.0 Adenoma, follicular cell 2 (4%) 0 (0%) 0 (0%) 3 (6%) Carcinoma, follicular cell 1 (2%) 0 (0%) 1 (3%) 3 (6%) Follicular cell adenoma and carcinoma, combined 3 (6%) 0 (0%) 1 (3%) 5 (10%)

Testes No. examined 50 50 50 50 Adenoma, interstitial cell c 43 (86%) 47 (94%) 48 (96%) 50 (100%) d Mesothelioma 0 (0%) 1 (2%) 1 (2%) 2 (4%)
Spleen No. examined 50 39 38 50 Leukemia, mononuclear e 27 (54%) 31 (79%) d 31 (82%) d 31 (62%)Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-19 Table 3-12. Incidences of Proliferative (Ne oplastic and Hyperplastic) Lesions in Rats (Concluded)
Control (0 g/m 3 ) Low (2 g/m 3 ) Mid (10 g/m 3 ) High (20 g/m 3 )
FEMALES Spleen No. examined 50 22 24 50 Leukemia, mononuclear 27 (54% ) 12 (55%) 11 (46%) 23 (46%)
Mammary Gland No. examined 49 20 22 47 Fibroadenoma e 0 (0%) 3 (15%) d 2 (9%) 5 (11%) d

a Incidence is significantly different from controls and from the 2 g/m 3 group (Fisher’s test). b Incidence is significantly diffe rent from controls (Fisher’s test). A positive trend toward

increased incidence with increasing dose was found with the Cochran-Armitage test.

c A positive trend with increasing dose was found with the Cochran-Armitage and Logistic

tests. A trend was also detected with Fish er’s test with the incidence among the 20 g/m 3 group being significantly in creased compared to the control incidence. d Incidence significantly different from controls (Fisher’s test).

e Trend detected with Fisher’s Exact test.
Squamous cell carcinoma occurred in nasal sections with an animal incidence of 1/50 (2%), 2/50 (4%), 0/50 (0%) and 2/50 (4%) for control, low-, mid- and high-dose animals respectively with no statistically significant differe nces or trends. It was not appropriate to combine carcinoma incidence of nasal turbinate levels for statistic al analysis in the lo w- and high-dose groups because the lesion spanned two levels for one of the two animals. By comparison, in the concurrent Baseline Gasoline Vapor Condens ate study, the incidence of squamous cell carcinoma in the nasal pasages (turbinate levels 2–4) was elevated in high-dose males, with tumor incidences of 1/50, 3/50, 3/50 for levels 2, 3, and 4, respectively. The incidences in turbiante level 3 were statistica lly significantly increased with exposure concentration (CochranArmitae test).

There is an increased incidence (see Table 3-12) of thyroid folli cular cell adenoma and carcinoma male rats with combined incidences of 3/50 (6%), 0/35 (0 %), 1/31 (3%) and 5/50 (10% ) for control, low-, mid- and high-level groups, respectivel y. However, the incidences of adenomas, carcinomas and the combined incidences of adenomas and carcinomas was not found to be statistically significant. In the concurrent BGVC study, th ere was a statistically increasedStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-20 incidence of thyroid follicular cell carcinomas in males. However the combined incidences of follicular cell adenomas and carcionomas were not statistically inceased in that study.

Proliferative Lesions Not Attr ibutable to GMVC Exposure . Leukemia, a well-documented feature of old age in F344 rats, did not appear to be enhanced by GMVC exposure. Results for spleen, the defining organ for this disease, are shown for males and females in Table 3-12. In males, the incidences of leukemia in the low- and mid-GMVC levels are greater than controls (Fisher’s test); however, the diagnoses were made on animals where gross lesions in the spleen were present, and therefore some sampling bias was involved. There wa s no overall trend with exposure concentration as determined by the Co chran-Armitage and logistic tests, and the incidence of leukemia in high-level males was not different from controls (Fisher’s test). The incidences of leukemia in GMVC-exposed female s were not different from the incidence among control females.

Mammary gland fibroadenoma was si gnificantly increased in low- and high-level females (Table 3-12). Interpretation of this fi nding is problematic due to the at ypically low control incidence of 0% in control females. Control incidences for this lesion have been reported to range from 27– 40% (Boorman et al., 1990c). The control inci dence in the parallel BGVC study (LRRI study number FY01-027) was 4/49 (8%). Additionally, increases in this lesion have not been observed in previous studies with neat MTBE or w holly vaporized gasolin e (Cruzan et al., 2007; MacFarland et al., 1984). Theref ore, it is unlikely that the in creased incidence of female mammary gland fibroadenoma was treatment-related.

Nonproliferative Lesi ons Attributable to GMVC Exposure . Nonproliferative lesions included degenerative and inflammatory changes. Only lesions showing a positive trend with exposure concentration and lesions in kidney, an e xpected target tissue are discussed here.

Chronic progressive nephr opathy is a lesion of part icular interest to this study, as this lesion is expected to occur in rats, especially male ra ts exposed to hydrocarbon mi xtures. In this study, the incidence of chronic progres sive nephropathy among control ma le and female rats was 44/59 (88%) in males and 42/50 (84%) in females (Table 3-13 and Appe ndices M, N, O, and P). The incidences among male and female rats inhali ng GMVC were not signif icantly increased overStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-21 the incidence in the respective controls. However, the severity of this lesion was significantly increased in mid- and high-level males and in hi gh-level females, compared to the corresponding control values. This finding in males is cons istent with male-rat-specific light hydrocarboninduced alpha-2u globulin overloa d nephropathy that has been re ported previously in studies with wholly vaporized unleaded gasoline and ne at MTBE (MacFarland et al., 1984; Bird et al., 1997). It should be noted that in F344 male rats , after approximately 1 year of age, alpha-2u globulin overload nephropathy is essentially masked by age- related chronic progressive nephropathy, since both forms of nephropathy shar e similar if not identical diagnostic hallmarks. Therefore. the effect of alpha-2u globulin ove rload nephropathy typically manifests itself in increased severity, rather than in cidence, since essentially all male s surviving past 1 year of age will develop CPN. The increased severity of ne phropathy in females at the highest dose must be attributed to a different, likely non-specific mechanism since alpha -2u globulin overload is male rat specific.

Table 3-13. Nonproliferative (Non-Neoplastic) Lesion Incidences and Severity Scores
Control (0 g/m 3 ) Low (2 g/m 3 ) Mid (10 g/m 3 ) High (20 g/m 3 ) Males Kidney No. examined 50 50 50 50 Nephropathy, chronic 44 (88%) a 47 (94%) 50 (100%) 46 (92%) Avg. severity 1.9 2.1 2.6 b 2.7 b

Nose/Turbinate 2 No. examined 50 50 50 50 Degeneration, hyaline - olfactory epithelium 0 (0%) 5 (10%) 7 c (14%) 6 c (12%)
Average severity 0.0 0.2 0.2 0.2 Nose/Turbinate 3 No. examined 50 50 50 50 Degeneration, hyaline - olfactory epithelium 2 (4%) 1 (2%) 7 (14%) 7 (14%) d
Average severity 0 0 0.2 0.3 Nose/Turbinate 4 No. examined 50 50 50 50 Degeneration, hyaline - olfactory epithelium 0 0 1 (2%) 1 (2%)
Average severity 0 0 0 0Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-22 Table 3-13. Nonproliferative (Non-Neoplastic) Lesion Incidences and Severity Scores (Concluded)
Control (0 g/m 3 ) Low (2 g/m 3 ) Mid (10 g/m 3 ) High (20 g/m 3 ) Females Kidney No. examined 50 20 21 50 Nephropathy, chronic 42 (84%) 17 (85%) 15 (71%) 43 (86%) Average severity 1.0 1.0 1.0 1.5 b

Nose/Turbinate 2 No. examined 50 50 50 50 Degeneration, hyaline - respiratory epithelium 0 (0%) 1 (2%) 7 c (14%) 6 c (12%) d
Average severity 0.0 0.0 0.3 0.2

a Percentage affected is in parentheses adjacent to incidence. b Significantly higher than cont rols at 0.05 level using Kolmogorov-Smirnov one-tailed test.

c Significantly different from c ontrols at 0.05 level using Fish er’s exact two-tailed test. d Positive concentration-dependent trend by Coch ran-Armitage and logistic tests. p = 0.054 by

Fisher’s exact (no group comparisons made).
The only other nonproliferative lesion attributab le to GMVC inhalation is hyaline degeneration in the nasal epithelium, with significant change s in the mid- and high-level GMVC animals of both sexes. In males, this lesion occurred in the olfactory epithelium of tu rbinates at levels 2, 3, and 4, while in females, the lesion occurred in the respiratory epithelium of turbinates at level 2. The severity scores for both lesions were quite low, ranging from 0.0 to 0.3 on a scale of 0 to 4.

CONCLUSIONS The overall purpose of the current study was to evaluate the long-term toxicity, including carcinogenicity, of MTBE inhaled as a compone nt of unleaded gasoline vapor for 2 years.

In this study, rats were exposed to 0, 2, 10, and 20 g GMVC/m 3 . The GMVC mixture contained 20% MTBE by mass, so the corresponding MTBE concentrations were 0, 0.4, 2, and 4 g/m 3

. Body weights were affected in males and to a greater extent in females in a concentrationdependent manner, but survival rates were not affected by GMVC exposure in either sex. Exposure to GMVC did not adve rsely affect WBC parameters. Effects seen at 12 months compared to at the 18- and 24-month sampling tim es were more likely due to difficulties inStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-23 blood sampling than to GMVC inha lation. Chronic exposure to GMVC increased the severity of chronic progressive nephropat hy in mid- and high- level ma les and high-level females and caused epithelial (females) and olfactory (males ) degeneration in the nasal passages of both sexes. The degenerative nasal effects were mo st likely caused by toxicant effects of the test material (Boorman et al., 1990d; Gopinath et al., 1987).

Exposure to GMVC resulted in an increase in renal tubule adenomas and combined adenomas and carcinomas in males exposed to GMVC. Tes ticular interstitial cell (Leydig cell) adenomas showed an increasing trend with exposure concentration, with th e incidence in the high-level GMVC group (50/50; 100%) signif icantly greater than controls (43/50; 86%). The relationship of the testicular interstitial cell adenomas with treatment is less clear due to the high background incidence of this lesion in aged male F344 ra ts. However, GMVC induction of testicular interstitial cell adenomas in the 20 g/m 3 group cannot be ruled out, partic ularly in light of similar equivocal observations reporte d in previous studies with neat MTBE (Cruzan et al., 2007). Chronic exposure to GMVC did not enhance the development of pr oliferative lesions (hyperplastic lesions, neoplasms) in female ra ts. Consequently, due to treatment-related increases in renal adenomas and combined andenomas and carcinomas in males, chronic inhalation of Gasoline MTBE Vapor Condensate was determined to be carcinogenic in male rats in this study. Gasoline MTBE Vapor Condensat e was determined not to be carcinogenic in female rats in this study.

COMPARATIVE CHRONIC TOXICITY AN D CARCINOGENICITY BETWEEN GMVC AND BGVC Background The current study on GMVC was conducted in para llel with chronic study on BGVC. Both studies were conducted in compliance with the Cl ean Air Act Section 211(b) for fuel and fuel additive registration. The 211b pr ogram was designed to evaluate the comparative toxicity of evaporative emissions of unleaded gasoline with and without oxygenates, such as MTBE. A comparison of the components of the two test ma terials is presented in Table 3-14. Below, results from the two studies are compared to dete rmine if addition of MTBE affected the chronic toxicity of BGVC alone.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-24 Table 3-14. Analytical Comp arison of Test Materials

Compound BGVC Lot # API 99-01 a (area percent) GMVC Lot # API 00-02 (area percent) Isobutane 3.6 2.2 n-Butane 15.2 11.1 Isopentane 35.1 31.0 n-Pentane 13.2 9.1 Trans-2-pentene 2.5 2.0 2-Methyl-2-butene 3.8 2.9 MTBE -- 21.3 2,3-Dimethylbutane 1.6 0.9 2-Methylpentane 6.3 4.5 3-Methylpentane 3.6 2.6 n-Hexane 3.0 2.1 Methylcyclopentane 1.5 1.1 2,4-Dimethylpentane 1.0 0.9 Benzene 2.1 1.5 2-Methylhexane 1.1 1.0 2,3-Dimethylpentane 1.1 1.0 3-Methylhexane 1.3 1.1 Isooctane 1.3 1.2 Toluene 3.0 2.5

a A second lot of BGVC test material was us ed for the last 10 - 11 weeks of the BGVC

chronic study. See Section 2.1 of the BGVC Chronic Study Report for a comparison of the two lots of BGVC test material.
Methyl tertiary-butyl ether (MTBE) has been used as an oc tane enhancing additive for gasoline and as part of the oxygenated fuels program to reduce carbon monoxide in automotive emissions. Concerns regarding the carcinogeni city of MTBE have been ra ised. In previous chronic inhalation studies, F344 rats we re exposed to 0, 400 (~1.4 g/m 3 ), 3000 (~10.8 g/m 3 ), and 8000 (~28.8 g/m 3 ) ppm MTBE (Bird et al., 1997; Mennear, 1997). An incr eased incidence of renal tubular adenoma and/or carcinoma was observe d in males exposed to 8000 ppm (28.8 g/m 3 ), and an increased incidence in testicular tumors was observe d among rats exposed to 3000 and 8000 ppm (10.8–28.8 g/m 3 ). Belpoggi et al. (1995) reported that 1000 mg MTBE/kg administered by gavage over a 2-year period increa sed the incidences of Leydig cell (testicular) tumors in males and lymphoma and leukemia (combined) in females.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-25 Results – General The effects of treatment on survival (none), clinical observations (none), body weights (decreases), organ weights (few), organ:body weight ratios (increases likely to be an artifact of decreased body weight), orga n:brain weight ratio s (few and not dose dependent), and hematology (changes at 12 months attributable to blood collection technical difficulties) were comparable between the two studies.

Results – Proliferative Lesions The male rat incidences of proliferative lesions are compared in Table 3-15 to help assess the role of MTBE in development of these findings . In both GMVC- and BGVC-exposed males, the incidence of renal tubule adenomas or combined incidences of adenomas and carcinomas peaks at the 10 g vapor/m 3 level and was very similar for th e two studies. At the 20 g vapor/m 3 level, the incidence of adenomas and combined aden omas and carcinomas is higher in the GMVC study.

In testes, there was a high, not unexpected, inci dence of adenomas in males in all dose groups (86%–100% for the GMVC study and 94%–100% for the BGVC study). Inhalation of BGVC did not significantly increase the nu mbers of tumors above control values (48%). However, the incidence of adenomas in the high exposure le vel GMVC males was si gnificantly increased compared to controls, but this is likely attributable to the low incidence among controls in that study (43%). Malignant testicular mesothelioma was observed in both stud ies, but a significant positive trend was only observed in the B GVC study, and only by the Fisher’s test.

Nasal squamous cell carcinoma (turbinate le vels 2–4 combined taking into account tumors spanning two or more levels) was observed in th e low- and high-level groups in both studies. Incidences were generally similar; however th e incidence in the high BGVC exposure group was greater than in the hi gh GMVC exposure group.

Similar to the renal tumor findings, thyroid fo llicular cell adenomas and carcinomas were observed in both studies. However, there was a significant trend toward increase incidence ofStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-26 thyroid follicular carci nomas only in the BGVC study. And in this case, group comparisons showed no significant increases among the individual groups.

Table 3-15. Comparison of Incidences of Renal, Nasal Turbinate, Testicular, and Thyroid Tumors in Male Rats Inhaling GMVC and BGVC

Tissue Diagnosis Control (0 g/m 3 ) Low (2 g/m 3 ) Mid (10 g/m 3 ) High (20 g/m 3 )
GMVC Kidney No. examined 50 50 50 50 Adenoma, renal tubule 0 (0%) 0 (0%) 6 a (12%) 2 (4%) Carcinoma, renal tubule 0 (0%) 1 (2%) 0 (0%) 1 (2%) Renal tubule adenoma and carcinoma, combined 0 (0%) 1 (2%) 6 b (12%) 3 (6%) b

Nasal Turbinates No. examined 50 50 50 50 Carcinoma, squamous cell, animal incidence (any level) 1 (2%) 2 (4%) 0 (0%) 2 (4%)

Testes No. examined 50 50 50 50 Adenoma, interstitial cell c 43 (86%) 47 (94%) 48 (96%) 50 d (100%) Mesothelioma, malignant 0 (0 %) 1 (2%) 1 (2%) 2 (4%) Thyroid No. examined 50 35 31 50 Adenoma, follicular cell 2 (4%) 0 (0%) 0 (0%) 3 (6%) Carcinoma, follicular cell 1 (2%) 0 (0%) 1 (3%) 3 (6%) Follicular cell adenoma and carcinoma, combined 3 (6%) 0 (0%) 1 (3%) 5 (10%)

BGVC Kidney No. examined 50 50 50 50 Adenoma, renal tubule 1 (2%) 1 (2%) 4 (8%) 0 (0%) Carcinoma, renal tubule 0 (0%) 0 (0%) 3 (6%) 0 (0%) Renal tubule adenoma and carcinoma, combined e 1 (2%) 1 (2%) 7 (14%) 0 (0%)

Nasal Turbinates No. examined 50 50 49-50 f 50 Carcinoma, squamous cell, animal incidence (any level) g 0 (0%) 1 (2%) 0 (0%) 3 (6%)

Testes No. examined 50 49 50 50 Adenoma, interstitial cell 48 (96%) 46 (94%) 50 (100%) 49 (98%) Mesothelioma, malignant h 0 (0%) 0 (0%) 4 (8%) 0 (0%)Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-27 Table 3-15. Comparison of Incidences of Renal, Nasal Turbinate, Testicular, and Thyroid Tumo rs in Male Rats Inhaling GMVC and BGVC (Concluded)

Tissue Diagnosis Control (0 g/m 3 ) Low (2 g/m 3 ) Mid (10 g/m 3 ) High (20 g/m 3 )
BGVC (Concluded) Thyroid No. examined 50 29 27 50 Adenoma, follicular cell 0 (0%) 2 (7%) 0 (0%) 2 (4%) Carcinoma, follicular cell i 0 (0%) 0 (0%) 2 (7%) 0 (0%) Follicular cell adenoma and carcinoma, combined j 0 (0%) 2 (7%) 2 (7%) 2 (4%)

a Incidence is significantly different from controls and from the 2 g/m 3 group (Fisher’s test). b Incidence is significantly differe nt from controls (Fisher’s te st). A positive trend toward

increased incidence with increasing dose was found with the Cochran-Armitage test.

c A positive trend with increasing dose was found with the Cochran-Armitage and Logistic

tests. A trend was also detected with Fish er’s test with the incidence among the 20 g/m 3 group being significantly in creased compared to the control incidence. d Incidence significantly different from controls (Fisher’s test).

e A positive trend was found with the Fisher’s ex act test, but pair-wise comparisons are not

significant (p > 0.008). f Forty nine for turbinates 3 and 4; fifty for turbinate 2. g A trend toward increased incidence with expos ure level found for nasal turbinate level 3 only

by the Cochran Armitage test. h Significant trend for increased incidence by FE test (p = 0.014), although pairwise

comparisons are not significant (p > 0.008). i Incidences are different than expected by chan ce (p = 0.029, overall FE test), although pairwise comparisons are not significant (p > 0.008, FE tests). j Incidences are not different than expected by chance (p = 0.14, overall FE test).

The incidences of leukemias in both studies we re high, but incidences did not increase with vapor exposure concentration in ei ther study (Table 3-16). In the cases where incidences in the low- and mid-vapor level groups ar e significantly increased compared to controls, it is possible that some sampling bias occurred because fo r these exposure groups, only tissues with gross abnormalities were examined histologically. Based on the incidences of leukemias in the GMVC and BGVC studies, it does not appear that MT BE-containing unleaded fuel vapors increased the incidence of leukemias over the alrea dy high incidence found in aged rats.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-28 Table 3-16. Comparison of Leukemia Incidences in Spleens of Rats inhaling GMVC and BGVC
Control (0 g/m 3 ) Low (2 g/m 3 ) Mid (10 g/m 3 ) High (20 g/m 3 )
GMVC Males 27/50 (54%) 31/39 (79%) a 31/38 (82%) a 31/50 (62%) Females 27/50 (54%) 12/22 ( 55%) 11/24 (46%) 23/50 (46%)

BGVC Males 32/50 (64%) 23/34 (68 %) 25/38 (66%) 32/50 (64%) Females b 13/50 (26%) 14/25 (56%) 18/32 (56%) 15/50 (30%)

a Significantly different from controls, Fisher’s test; p ≤ 0.05. b Incidences are different than expected by chance (p = 0.007, overall FE test), although pair-wise comparisons are not signif icant (p > 0.008, FE tests).

Overall, it is difficult to dr aw strong conclusions about th e role of MTBE in inducing proliferative lesions, especially in regard to induci ng renal or testicular tumors in male rats. The predictive value of renal tumors and testicular inte rstitial cell adenomas for humans has also been questioned (Baetcke et al., 1991; Hard, 1998; Hard and Khan, 2004; Cook et al., 1999; Prentice and Meikle, 1995).

Non-Proliferative Lesions The incidence of chronic progressive nephropat hy was high among control males and females in both studies and not significantly increased with GMVC or BGVC exposure (Table 3-17). The severity of male chronic progressive nephropat hy was significantly increased in mid- and highlevel males inhaling GMVC and BGVC. The in creased severity of chronic progressive nephropathy in high-level GMVC females is lik ely due to non-specific protein accumulation rather than treatment.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-29 Table 3-17. Comparison of Ch ronic Progressive Nephropathy in Rats Inhaling GMVC and BGVC for 2 Years
Control (0 g/m 3 ) Low (2 g/m 3 ) Mid (10 g/m 3 ) High (20 g/m 3 )
GMVC Males Incidence Avg. severity 44/50 (88%) a 1.9 47/50 (94%) 2.1 50/50 (100%) 2.6 b 46/50 (92%) 2.7 b
Females Incidence Avg. severity 42/50 (84%) 1.0 17/20 (85%) 1.0 15/21 (71%) 1.0 43/50 (86%) 1.5 b

BGVC Males Incidence Avg. severity 49/50 (98%) 2.4 49/50 (98%) 2.5 50/50 (100%) 2.9 c 50/50 (100%) 2.9 c Females Incidence Avg. severity 27/50 (54%) 0.7 12/24 (50%) 1.0 16/25 (64%) 0.9 32/50 (64%) 0.9

a Percentage affected is in parentheses adjacent to incidence. b Significantly higher than cont rols at 0.05 level using Kolm ogorov-Smirnov one-tailed test.

c Overall severity/incidence sign ificantly higher than contro l at 0.05 level using KolmogorovSmirnov one-tailed test.

For both GMVC- and BGVC-treated male rats, th e increased incidence of renal tumors and increased severity is likely due in part to ma le rat specific alpha-2u globulin accumulation and overload in the kidney during the first year of li fe. Other mechanisms may also contribute, as evidenced by increased severity of nephropat hy in high-level GMVC fe males, lacking renal adenomas and carcinomas.

The respiratory epithelial degeneration of the re spiratory and olfactory epithelium is a common, nonspecific finding in aged rats (Boorman et al ., 1990d; Gopinath et al., 1987). Mid- and highlevel males and females exposed to GMVC had an increased incidence of these lesions in nasal turbinate 2. High-level males also had an increa sed incidence of degeneration in turbinate 3. The incidences of this lesion were also signi ficantly increased among male and female rats exposed to the mid and high levels of BGVC . Interestingly, the respiratory epithelialStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

3-30 degeneration in BGVC-exposed male and female rats extended to turbin ate level 3, so it was more extensive than that caused by GMVC. Theref ore, the increased incidence of the epithelial degeneration is not attributable to MTBE, but more likely due to th e irritant propert ies of the fuel vapor mixture.

In summary, we have conducted a side by side co mparison of the toxicity and carcinogenicity of Baseline Gasoline Vapor Condensate and Condens ate containing approximately 20% MTBE. The inhalation exposure systems were identical. Animals were obtained from the same source, were the same age, and housed under identical c onditions. The health effects resulting in F344 rats following 2 years of GMVC or BGVC inha lation were unequivocally comparable in the production of renal adenomas and carcinomas in ma le rats. Methyl tertia ry butyl ether was not identified as enhancing the production of thes e renal adenomas or adenomas and carcinomas compared to those induced by ga soline vapor condensate alone.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

4-1 LOCATION OF SPECIMENS, RA W DATA, AND FINAL REPORT
Specimens were identified by test system, study, natu re, and date of collec tion. All raw data and records and specimens that are required to recons truct the study will be maintained in the LRRI archives for 10 years. The Sponsor will be notif ied and will authorize in writing the destruction or transfer of any specimens, raw data, and st udy records to the Sponsor or to an archive specified by the Sponsor’s C ontracting Representative.

PROTOCOL DEVIATIONS Delayed Sponsor Signature on Amendment 1 Amendment 1 to the study protocol was si gned by the Study Director on August 22, 2001 and submitted to the Sponsor for signature. A signatu re was not obtained after many requests. The operative LRRI SOP at the time stated, “The st udy director signs and dates the amendment prior to implementing the changes and the dated signatu re of the Sponsor is obtained as soon as reasonably possible.” Good Laborat ory Practice guidelines do not require Sponsor signature. Thereafter, LRRI no longer sought Sponsor si gnature on Amendment 1 and considered it approved on August 22, 2001.
Personnel On page 3 of the study protocol, key personnel were listed as Quint H. Powell, MS, as Inhalation Engineer, Thomas H. March, DVM, PhD, DACVP, as Veterinary and Clinical Pathologist, David G. Burt, DVM, DACLAM, as Laboratory Animal Veterinari an, and Justin Kubatko, MS, Statistician. During the study, Mr . Powell left the Inst itute and was replaced by Mr. Edward B. Barr, MSEE, an inhalation engineer with over 25 y ears experience at Lovelace. Dr. Burt left the Institute and was replaced by Roger van Andel, DVM, PhD, DACLAM. Mr. Kubatko also left the Institute. Betty Skipper, PhD, University of New Mexico, was subcont racted to perform the statistics. Dr. Thomas March took over res ponsibilities for the Baseline Gasoline Vapor Condensate carcinogenicity study pathology and was replaced on this study by Dr. Andrew Gigiliotti, DVM, PhD, DACVP.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

4-2 There was no impact on the quality of the study as the above listed personnel were replaced by individuals of equal or greater experience.

Calculation of Nominal Concentration
Section IX.C.6. states, “Nominal concentration will be determin ed daily and will reflect the concentration generated for all three exposure systems combined. Therefore, the total mass of test substance generated will be divided by the total flow distributed to all three exposure chambers. This value will be compared to the sum of the mean concentrations achieved in the three chambers throughout that exposure day.”

The actual and nominal (anticipated) usage were calculated as follows:

Daily nominal or “anticipated” usage was calculated by multiplying the average vapor condensate concentration in each chamber (low, mid, high; g/m 3 ) by the total flow through each respective chamber ([L/min * min/1000L/m 3 ]) and then summing the values for all three chambers. This value was compared to the actual BGVC usage determined by taking the difference between the weight of the 20-pound cylinder before and after the exposure.

There was no adverse impact on the study. The way nominal concentration was calculated and compared to actual gasoline MTBE vapor conde nsate usage provides the same answer as the method described in the protocol.

Statistics Proposed statistical methods were presented in the protocol. In several cases the actual evaluations differed from those orig inally proposed. This occurred in three specific cases. First, the Hoel Walburg test was not used to analyze tumor data although originally listed as a possible statistic in the protocol. Sec ond, hematology results were analyzed as a function of time as well as a function of dose. Finally, in the case of chronic progressive nephropathy, where the incidence of lesions was similar among cont rol and dosed groups, the Kolmogorov-Smirnov test was used to assess differences in severity scor es. This evaluation was done within the P module of the Path-Tox ® software, and not by the Study Statistician.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

4-3 The actual statistics used have been described in the Statistics secti on of the report. The fact that the statistics proposed in the protocol were not always those used for data analysis has no major impact on the quality of the study. In most cases the analysis were more thorough and appropriate than those originally proposed. Since GMVC inhalation di d not affect animal survival, the Hoel Walburg analysis, that assumes that treatment affects animal survival, was not necessary. Since the hematology data did not ha ve normal distributions, alternative statistical approaches were warranted. Since repeated m easurements were made on the same rats over time, evaluation of the effect of time and e xposure concentration on the hematology parameters was appropriate. The use of the Kolm ogorov-Smirnov statistic in the Path-Tox ® database was appropriate because nephropathy was an important endpoint in this study. Statistical analysis of the severity of the lesion as a function of exposure concen tration in the absence of a concentration-response effect on lesion incidence provided more information than would have been gained by analysis of incidence alone.

Analysis of Tank 16 (420 pound tank) The protocol required a gas chromatographi c profile to be performed on all 420 pound tanks prior to use. Tank 16 was inadvertently not an alyzed prior to being used for exposures. However, profiles of the exposure atmosphere we re determined when Tank 16 was used May 17, 2001 through June 28, 2001. Therefore these data are included in Appendix B to the report. Because profiles were obtained on the tank during its use, and there was no indication that the hydrocarbon composition of this tank was different from that of the other tanks, there is no significant impact on the outcome of the study.

Analysis of Exposure Atmosphere Profiles During the weeks of July 23, 2001 and Septem ber 10, 2001, the gas chromatograph used to qualitatively analyze the hydro carbon profiles of the exposure atmospheres was not working properly. Therefore profiles were not obtained dur ing these weeks. The technician responsible for the analyses worked with the Lovelace electr ician and the instrument vendor to troubleshoot and resolve the problems. Profiles were obtaine d on previous and subsequent weeks. Since hydrocarbon profiles in the exposure atmospheres were relatively stable for these lots of fuel, theStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

4-4 loss of the chamber atmosphere profile data dur ing the 2 weeks of the 104-week study did not significantly affect the outcome of th e study or interpretation of results.

Exhaust System Failure During Conditioning/Quarantine Period Affecting Chamber Pressure On August 2, 2001, a subcontractor was at the Institu te to repair a motor that had burned out on the backup exhaust system for the exposure wing. While here, he was asked to change the filters on the main system. To do this, he had to temporar ily shut off the main blower. After the filters had been changed, the blower motor could not be re started. This occurred about 12:30 p.m. The Study Director, Dr. Benson and LRRI Animal Care Operations were notified because the study animals were being quarantined in chambers in that wing. Since air was still being supplied to the chambers, the corks in the door s of the chamber were taken out to allow air flow. During this time the pressure in the chambers changed from negative (approximately -1 inch of water) to positive (about 1 inch of water). The maintena nce crew attempted to restart the motor all afternoon. At 4:00 p.m., the exhaust system was still not working, so the exhaust lines were totally disconnected from the chambers hous ing the study animals. All environmental parameters continued to be monitored by comput er as before the exhaust system went down. Surveillance personnel also checked the conditions in the room twice per sh ift and were directed to open the chamber doors in the event that temperatures exceeded 24 ° C. A new motor was delivered and installed early August 3, 2001. The exhaust system was up and running by
8:30 a.m.

The impact to the study was small because animals had air, and were monitored by personnel throughout the time the exhaust system was down.

Loss of Supply Air to Exposure System On November 27, 2001, at approxim ately 10:30 a.m., supply airflow to the animal chambers dropped, resulting in low exposure chamber flows and pressures. Surveillance personnel were summoned to the room. They monitored th e room closely and made certain Facility Engineeering and Exposure personnel were aware of the situation. It was detemined that the flows and pressures were in a range that would no t adversely affect the health of the animals and therefore the animals remained with in the chambers during the night.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

4-5 During this time, chamber pressures did not ex ceed -3” water (within normal range of LRRI SOP). Chamber flows did not drop below 350 lpm (10 changes per hour), above the minimum stated in EPA OPPTS 870.4200 guide lines, but below those specif ied in the study protocol. Chamber temperatures remained within protocol-defined limits.

On the following morning, November 28, 2001, the Study Engineer and Facility Engineering personnel examined the problem. Flows to th e chamber were adjusted to within normal operating parameters. Facility Engineering pers onnel examined the supply air blower and could not find anything wrong with the supply air system.

At this time, the Study Engineer determined that it was ok to begin exposures. Approximately 30 minutes into the exposure, Facility Engineeri ng discovered that the chamber supply air blower was not working properly. Exposures were term inated after 40 minutes of exposure were completed so that repairs could be made. Exposure concentrations achieved during the 40 minute run were 1.53 g/m 3 , 7.88 g/m 3 and 13.62 g/m 3 for the 2, 10 and 20 g/m 3 chambers, respectively. Flow through the chambers was maintained above 10 air changes per hour and chamber temperatures were withn limits while the blower was being repaired. The environment in the exposure chamber was safe for animal occ upancy at all times. Repairs to the blower were completed aby 12:00 p.m. Exposures were not continued that day. One exposure day was missed but this was not considered significant be cause there were over 500 exposure days during the study.

Environmental Conditions Throughout the gasoline MTBE vapor condensate exposure study, environmental conditions in the animal exposure chambers deviated from prot ocol required ranges, or data were not acquired for short periods because of problems with the monitoring system. Most excursions of temperature and pressure or peri ods of data loss were of short duration (~2% per month). These deviations were judged to have no significan t effect on the outcome of the study. Tables summarizing deviations from pr otocol-specified environmental conditions are included in the Study File.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

4-6 On a few occasions, environmental data were not acquired due to equipment failure or because monitoring devices were not plugged in. These instances are listed below.

On November 23, 2001 RH sensors were not pl ugged into the chamber from 6:59–14:29. Any data acquired were listed as erroneous and not included in the data summary for that day.

The Relative Humidity meter on chamber 6 (2 mg/m 3 ) came loose and data were missing for all or parts of May 24–May 28, 2002. Apparently the sensor became disconnected on Friday (possibly after the chambers were serviced) and this was not noticed until the following Tuesday). Part of the reason for the delay in de tecting this was that RH was not set to alarm when out of the acceptable range and that the disconnect occurred over a weekend, when exposure personnel were not on duty.

A similar occurrence happened July 11–15, 2002, where RH data were not recorded.

Temperature, airflow and relative humidity we re not recorded for approximately 0.5–1 hour on August 30, September 6, and September 12, 2002. Th is was likely due to the fact that the computer monitoring system was shut down and re booted to shorten data backup time. The data would have been logged when the computer was restarted and monitoring resumed, but since the data were not necessarily logged on the half hour, they were not included in the data summaries.

From August 31–September 3, 2002, humidity data for chamber 5 were not recorded because a sensor wire was loose and not repaired.

The impact of the data loss is not great. The RH in the remaining chambers was recorded. The RH in the missed chambers may be inferred fr om the data acquired in the other three.

Temporary Loss of Environmental Monitoring System Operation On May 20, 2003 a major power outage occurred at 12:20 a.m. Normally, in power outage events, emergency backup diesel generators are turned on within 10–15 seconds. During this particular outage, a breaker was tripped a nd emergency power was not provided to the environmental monitoring system and the syst em was on uninterrupted power supply battery power until they were drained. During this outa ge, the supply air fan stopped operating, but dueStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

4-7 to the lack of environmental monitoring capabil ity, this was not detected until morning. All systems were restored by approximately 6:30 a. m. The animals appeared healthy. Estimated flow rates through the chamber were approximately 200–300 L/minute. While supply air was not available, the vacuum system was still oper ational and pulled air from the room into the chambers. Several actions, described in a memo to the study file, were taken to prevent similar situations in the future.

On August 28, 2002 (14:29) to August 29, 2002 (06:29) no environmental data were acquired or saved to file. This was because on August 28, 2002, the LRRI network domain configuration modifications were performed on the monitoring PC in the study control room. The software program controlling and recording environmenta l data was shut down and inadvertently not restarted.

Light and Sound Measurements According to the study protocol, light and sound measurements in the animal exposure room were to be measured quarterly. In 2001, measurements were not made for the July–September quarter. In 2002, no measurement was made between January and March. In 2003, no measurement was made between July and the end of the study in August. The impact on the outcome of the study is considered minimal b ecause light and sound levels were similar among locations and did not vary greatly be tween measurements that were made.

Gas Chromatographic Analysis The original version of the SOP ASP-1166.0, Cali bration of the Shimadzu GC-17A/FID for Analysis of Gasoline Vapor Condensate, indica ted GC calibration for monitoring the exposure atmosphere and test article hydrocarbon compositi on profile may be calibrated daily. However, calibrations were performed only on the days when analyses were pe rformed. The SOP was modified and signed by management on Sept ember 25, 2002, changing the requirement for calibration to only days when analyses are perf ormed. The impact of this SOP deviation is minimal because the GC was calibrated on days when analyses were performed.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

4-8 Body Weights and Observations Not Acco rding to Protocol Defined Schedule Body weights were performed at er ratic intervals. Body weights were generally performed every fourth week on the same day of the week (the da y that chambers are not changed in the exposure room). The body weights obtained between 9/1/02 and 10/14/02 were not performed at exactly weekly intervals. This was because we were in vestigating the feasibility of going to a weekend weigh schedule. This would allow study personne l to perform other task s with the animals on the day chambers are not changed, such as tattoo touch ups after exposure. In trying the new schedule, weights were collected 3–4 days off from the previous schedule. This had minimal effect on the study, since the rats were weighed only a few days later than “normal” and all groups were weighed on the same day, so st atistical comparisons were still valid.

The same explanation holds true for the associated observations. The impact was minimal. The observations were still made, but 3–4 da ys later than the previous schedule.

Cage Board Usage/Availability In December 2002, we ran out of untreated cage board used to line the excreta pans in the H2000 chambers. Although additional cage board wa s ordered, it was on “back order” and was estimated to arrive December 20, 2002. Until we received permission from the Sponsor to switch to treated (with neomycin) cage board, no cage board was used and pans were washed twice daily instead of the usual once per week. Dr. Benson contacted the Sponsor and received permission to use treated cage board on December 13, 2002. Use of untreated cage board continued when it arrived.

There should be no adverse effect on the study associated with not using cage board (with twice daily pan washes to keep ammonia levels down) or associated with use of neomycin-treated cage board. The latter type is used throughout the institute for short-term and long-term inhalation studies.

Animal Tattoo Requirements for Identification During the period between April and August 2002, se veral animals were re peatedly noted as needing tattoo touch ups to aid in identification and fulfill SOP requirements. Tattoos wereStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

4-9 reapplied or touched up from August 2002 on and atte mpts were made to tattoo more frequently. In addition, new equipment was purchased so that tattoos applied were more vivid and lasted longer. There was no adverse effect.

Animal Misidentification Animal S14 (a sentinel in the control chambe r) was designated as moribund and authorized for euthanasia to be conducted on December 20, 2002. A different moribund animal, F514 was incorrectly brought to necropsy, where the sta ff failed to verify th e tattoo number. F514 underwent a complete necropsy that day. The error was noted and S14 was euthanized and underwent necropsy on December 24, 2002. The aff ected Individual Animal Necropsy Record forms were corrected and error coded. Remova l of F514 instead of sentinel S14 did not adversely affect the integrity of the study, as F514 was not healthy when euthanized. Lesions of spleen and testes were observed in both S14 and F514.

Tissue Collection at Necropsy In collecting mediastinal lymph nodes, small porti ons of adjacent thymic tissue was collected because mediastinal lymph nodes are easily mi ssed during sectioning in the absence of a supporting stroma. Data from thymus collected w ith these specific sections were not reported. Thymus was collected and data reported for that particular tissue.

Collection of Organ Weights at Necropsy The protocol states that lung, liv er, kidneys, testes, epididymis, ova ries, uterus, spleen, brain and heart will be weighed at necropsy. It also states that these weights are not to be recorded on animals found dead. The brain weight was not recorded for E402. The seminal vesicle and prostate were weighed for E402, even though these tissues weights are not required by the study protocol. The impact of these deviations wa s minimal. Only one brain weight was lost.

Contrary to protocol, organ wei ghts were recorded for rats found dead. Organ weight summaries have been prepared to exclude natural death animals, so th eir weights do not skew the group means for the final sacrifice and euthanized anim al reports. There is no adverse effect on the outcome of the study.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

4-10 Hematology

Blood smears were to be evaluated for differentia l white blood cell counts, and an estimate of total white blood cells was made. At the 12 month time point, a white blood cell estimate was not made on E419 (control male) because the sm ear was too thick and for high-level female H772. Poor smear quality prevented obtaining a white blood cell estimate for E478 (control female) at the 18 month time point. Because these were the only samples for which a white blood cell estimate were not obtained, the imp act on the outcome of the study is small.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

5-1 LIST OF ABBREVIATIONS
BGVC Baseline gasoline vapor condensate CFR Code of Federal Regulations CPN Chronic progressive nephropathy CRTC Chevron Research and Technology Center EDTA Ethylenediamine tetra-acetic acid EMBSI Exxon Mobile Biomedical Sciences, Inc.
FID Flame ionization detector GC Gas chromatograph GLP Good Laboratory Practices GMVC Gasoline MTBE vapor condensate HN Hydrocarbon nephropathy
KRV/H-1 Kilharn rat virus/H-1 virus LPM Liters per minute LRRI Lovelace Respiratory Research Institute MTBE Methyl tert iary-butyl ether
N BF N eutral buffered formalin
N FPA N ational Fire Protection Associatio n OPPTS Office of Prevention, Pesticides , and Toxic Substances (U.S. EPA)
PVM Pneumonia virus of mice RCV/SDAV Rat corona virus/sialodacryoadenitis SOP Standard operating procedure
SD/Stdev Standard deviation
SE Standard error
WBC White blood cellsStudy Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

6-1 REFERENCE LIST

Baetcke, K. P., Hard, G. C., Rodgers, I. S ., McGaughy, R. E., and Tahan, L. M. (1991). Alpha 2 υ Globulin: Association with Chemically Induced Renal Toxicity and Neoplasia in the Male Rat. (EPA/625/3-91/019F) Risk Assessment Forum, U.S. Environmental Protection Agency (U.S. EPA), Washington, D.C. Belpoggi, F., Soffritti, M., and Maltoni, C. ( 1995). Methyl-Tertiary-Butyl Ether (MTBE)—A Gasoline Additive—Causes Testicular and Lymphohaematopoietic Cancers in Rats. Toxicol. Ind. Health 11(2) : 119-149. Bird, M. G., Burleigh-Flayer, H. D., Chun, J. S., D ouglas, J. F., Kneiss, J. J., and Andrews, L. S. (1997). Oncogenicity Studies of Inhaled Me thyl-Tertiary-Butyl Ether (MTBE) in CD-1 Mice and F-344 Rats. J. Appl. Toxicol. 17(Suppl. 1) : S45-S55. Boorman, G. A., Eustis, S. L., Elwell, M. R., and Leininger, J. R., eds. (1990a). Pathology of the
Fischer Rat , 1 st Ed., Academic Press, San Diego, CA. Boorman, G. A., Chapin, R. E., and Mitsumori, K. (1990b). Testis and Epididymis. In Pathology of the Fischer Rat (Boorman, G. A., Eustis, S. L., Elwell, M. R., and Leininger, J. R., eds.), 1 st Ed., pp. 405-418, Academic Press, San Diego, CA. Boorman, G. A., Wilson, J. T., van Zwieten, M. J., and Eutis, S. L. (1990c). Mammary Gland.
In Pathology of the Fischer Rat (Boorman, G. A., Eustis, S. L., Elwell, M. R., and Leininger, J. R., eds.), 1 st Ed., pp. 295-314, Academic Press, San Diego, CA. Boorman, G. A., Morgan, K. T., and Uriah, L. C. (1990d). Nose, Larynx, and Trachea. In Pathology of the Fischer Rat (Boorman, G. A., Eustis, S. L., Elwell, M. R., and Leininger, J. R., eds.), 1 st Ed., pp. 315-338, Academic Press, San Diego, CA. Borghoff, S. J., Miller, A. B., Bowen, J. P., and Swenbert, J. A. (1991) . Characteristics of Chemical Binding to α 2u-globulin In Vitro – Evaluating Structure-Activity Relationships. Toxicol. Appl. Pharmacol. 107 : 228-238. Borghoff, S. J., Youtsey, N. L., and Swenbert , J. A. (1992). A Comparison of European High Test Gasoline and PS-6 Unleaded Gasoline in Their Abilities to Induce α 2u-Globulin Nephropathy and Renal Cell Proliferation. Toxicol. Lett. 63 : 21-33.Study Number FY01-013 211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate (GMVC) September 2009

6-2 Cruzan, G., Borghoff, S. J., dePeyster, A., Ha rd, G. C., McClain, M., McGregor, D. G., and Thomas, M. G. (2007). Methyl Tertiary -Butyl Ether Mode of Action for Cancer Endpoints in Rodents. Reg. Toxicol. Pharmacol. 47 : 156-165. Cook, J. C., Klinefelter, G. R., Ha rdisty, J. F., Sharpe, R. M., a nd Foster, P. M. (1999). Rodent Leydig Cell Tumorigenesis: A Review of the Physiology, Pathology, Mechanisms, and Relevance to Humans. Crit. Rev. Toxicol. 29(2) : 169-261. Gopinath, C., Prentice, D. E., and Lewis, D. J. (1987). Atlas of Experimental Toxicological Pathology (Gresham, G. A., ed.), MTP Press Limited, Norwell, MA. Hard, G. C. (1998). Mechanisms of Chemically Induced Renal Carcinoge nesis in the Laboratory Rodent. Toxicol. Pathol. 26(1) : 104-112. Hard, G. C. and Khan, K. N. (2004). A Cont emporary Overview of Chronic Progressive Nephropathy in the Laboratory Rat, and its Significance for Human Risk Assessment. Toxicol. Pathol. 32(2) : 171-180. MacFarland, H. N., Ulrich, C. E., Holdsworth, C. E., Ketchen, D. N., Halliwell, W. H., and Blum, S. C. (1984). A Chronic Inhalati on Study wth Unleaded Gasoline Vapor. J. Am. Coll. Toxicol. 3(4): 231-248. Mennear, J. H. (1997). Carcinogenicity Studies on MTBE: Critical Review and Interpretation. Risk Anal. 17(6) : 673-681. Prentice, D. E. and Meikle, A. W. (1995). A Review of Drug-Induced Leydig Cell Hyperplasia and Neoplasia in the Rat and Some Comparisons with Man. Hum. Exp. Toxicol. 14(7) : 562-572.