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 manufacturers have conducted comparative chronic 

toxicology testing of evaporative emissions from 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 exposed in H2000 whole-body inhalation chambers at GMVC vapor 

concentrations of 2 g/m3

 (low level), 10 g/m3 (mid level), and 20 g/m3 (high level) for 6 

hours/day, 5 days/week for 104 weeks (520 exposure days). There were no clinical signs of 

toxicity attributable to GMVC inhalation. Survival of the GMVC-exposed rats was not 

significantly different from control rats. Body weights of high-level GMVC males and high- and 

mid-level females were significantly below control values for most of the study. At the final 

sacrifice, the body weights of male and female high-level rats were 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 those of the corresponding controls. Significant 

increases in organ:body weight ratios were observed in males at final sacrifice; brain and lung in 

mid- and high-level groups and kidney in high-level 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 compared to control values. No effects on total 

and differential white blood cell (WBC) counts could 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 later sampling times. These findings suggest the 

differences may have been due to sampling 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 Fisher’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 trend 
Study Number FY01-013 

211(b) Chronic Carcinogenicity Study 

Gasoline MTBE Vapor Condensate (GMVC) 

September 2009 

ii 

toward increasing incidence with exposure concentration 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 the 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 vaporized gasoline (Bird et al., 1997; MacFarland et 

al., 1984). In the concurrently conducted study 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 resulting in a sustained increase in tubule epithelial 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 turnover, alpha-2u globulin overload has been 

demonstrated to be a male rat specific mechanism with little or no relevance 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 gasoline 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 adenomas in control male rats 

(43/50; 86%). The incidence among high-level males (50/50; 100%) was significantly greater 

than the control incidence (Fisher’s exact test). The relationship between treatment and 

induction of testicular interstitial adenomas is considered to be equivocal, since background 

incidence of this tumor is extremely high and historical control incidence data from one 2-year 

bioassay conducted at LRRI for a commercial sponsor was 86% (n = 56). NTP control 
Study Number FY01-013 

211(b) Chronic Carcinogenicity Study 

Gasoline MTBE Vapor Condensate (GMVC) 

September 2009 

iii 

incidence1

 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 the incidence of testicular interstitial 

adenoma with BGVC exposure was found in that study. 

The incidences of mononuclear cell leukemia among males and females were high, as expected 

among a population of aged F344 rats. MCL incidence in low-level (31/39; 79%) and mid-level 

(31/38; 82%) males was significantly greater than controls (27/50; 54%), most likely due to 

protocol driven sampling bias (non-target organs 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 only other nonproliferative lesion attributable to 

GMVC exposure was hyaline degeneration of the 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 degenerative nasal effects were most likely caused by 

the test material. Chronic exposure to BGVC did not enhance the development of proliferative 

lesions (hyperplastic lesions, neoplasms) in female rats. In male rats, chronic exposure to BGVC 

did enhance the development of renal adenomas and carcinomas, when incidences were 

combined. Only the mid exposure group had significantly increased incidence 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. Consequently, due to treatment-related increases in renal adenomas 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 Condensate was not 

determined to be carcinogenic in female rats in this study. 

 

1 Reference - http://ntp.niehs.nih.gov/ntpweb/index.cfm?objectid=92E61F1B-F1F6-975E-7D3BED551F07DC0A 
Study 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 COMPLIANCE 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 ..................................................................................................... 2-8
Study 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 TOXICITY 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 Nominal 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 .................................................................. 4-4
Study 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 Weights 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 Rats 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 Samples Taken for Examination .......................................... 2-9

2-3. Severity Scoring of Chronic 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% Mortality for Male and Female Rats ................... 3-5

3-4. Disposition of Rats on Study ........................................................................................... 3-5
Study 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 Body Weights of Male and Female Rats ......... 3-10

3-8. Absolute Organ Weight at Terminal 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 (Neoplastic 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, Nasal Turbinate, Testicular, and Thyroid 

Tumors in Male Rats Inhaling 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 SEROLOGICAL 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-1 
Study Number FY01-013 

211(b) Chronic Carcinogenicity Study 

Gasoline MTBE Vapor Condensate (GMVC) 

September 2009 

viii 

Appendix F 

WEEKLY GAS CHROMATOGRAPHIC PROFILES OF CHAMBER ATMOSPHERES ...... F-1 

Appendix G 

ENVIRONMENTAL DATA ...................................................................................................... G-1 

Appendix H 

INDIVIDUAL ANIMAL DISPOSITION .................................................................................. H-1 

Appendix I 

INDIVIDUAL ANIMAL IN-LIFE BODY WEIGHTS .............................................................. I-1 

Appendix J 

INDIVIDUAL ANIMAL SUMMARIES OF CLINICAL OBSERVATIONS ............................ J-1 

Appendix K 

BODY AND ORGAN WEIGHTS 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-1
Study 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 Research 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 Mexico 
Study 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 

 Date 
Study 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 Good 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 Facility 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. Harris 7-Aug-01 9-Aug-01 9-Aug-01 

Environmental data R. Mar / D. Harris 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-01 
Study 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-02 
Study 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-03 
Study 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 

 Date 
Study 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 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.” The stipulations of this protocol were implemented in conformance 

with EPA regulations as specified in 40 CFR 79.60 “Good Laboratory Practices (GLP) Standards 

for Inhalation Exposure Health Effects Testing.” 

It was the Sponsor’s responsibility to maintain records of the method 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 however, presently available. 

The following were not conducted under GLP Guidelines: 

Serological assessments were conducted throughout 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 conducted 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 

 Date 
Study 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 manufacturers have conducted comparative chronic 

toxicology testing of evaporative emissions from 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 exposed in H2000 whole-body inhalation chambers at GMVC vapor 

concentrations of 2 g/m3

 (low level), 10 g/m3 (mid level), and 20 g/m3 (high level) for 6 

hours/day, 5 days/week for 104 weeks (520 exposure days). There were no clinical signs of 

toxicity attributable to GMVC inhalation. Survival of the GMVC-exposed rats was not 

significantly different from control rats. Body weights of high-level GMVC males and high- and 

mid-level females were significantly below control values for most of the study. At the final 

sacrifice, the body weights of male and female high-level rats were 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 those of the corresponding controls. Significant 

increases in organ:body weight ratios were observed in males at final sacrifice; brain and lung in 

mid- and high-level groups and kidney in high-level 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 compared to control values. No effects on total 

and differential white blood cell (WBC) counts could 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 later sampling times. These findings suggest the 

differences may have been due to sampling 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 Fisher’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 trend 
Study Number FY01-013 

211(b) Chronic Carcinogenicity Study 

Gasoline MTBE Vapor Condensate (GMVC) 

September 2009 

S-2 

toward increasing incidence with exposure concentration 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 the 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 vaporized gasoline (Bird et al., 1997; MacFarland et 

al., 1984). In the concurrently conducted study 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 resulting in a sustained increase in tubule epithelial 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 turnover, alpha-2u globulin overload has been 

demonstrated to be a male rat specific mechanism with little or no relevance 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 gasoline 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 adenomas in control male rats 

(43/50; 86%). The incidence among high-level males (50/50; 100%) was significantly greater 

than the control incidence (Fisher’s test). The relationship between treatment and induction of 

testicular interstitial adenomas is considered to be equivocal, since background incidence of this 

tumor is extremely high and historical control incidence data from one 2-year bioassay 
Study Number FY01-013 

211(b) Chronic Carcinogenicity Study 

Gasoline MTBE Vapor Condensate (GMVC) 

September 2009 

S-3 

conducted at LRRI for a commercial sponsor was 86% (n = 56). NTP control incidence2

 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 testicular interstitial adenoma among BGVC males was 

found in that study. 

The incidences of mononuclear cell leukemia among males and females were high, as expected 

among a population of aged F344 rats. MCL incidence in low-level (31/39; 79%) and mid-level 

(31/38; 82%) males was significantly greater than controls (27/50; 54%), most likely due to 

protocol driven sampling bias (non-target organs 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 only other nonproliferative lesion attributable to 

GMVC exposure was hyaline degeneration of the 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 degenerative nasal effects were most likely caused by 

the test material. Chronic exposure to BGVC did not enhance the development of proliferative 

lesions (hyperplastic lesions, neoplasms) in female rats. In male rats, chronic exposure to BGVC 

did enhance the development of renal adenomas and carcinomas, when incidences were 

combined; only the mid-exposure group had significantly increased incidence of adenomas or 

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. Consequently, due to treatment-related increases in renal adenomas 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 Condensate was not 

determined to be carcinogenic in female rats in this study. 

 

2 Reference - http://ntp.niehs.nih.gov/ntpweb/index.cfm?objectid=92E61F1B-F1F6-975E-7D3BED551F07DC0A 
Study 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, potential carcinogenicity, and 

exposure concentration-response relationships associated with inhalation of gasoline vapor 

condensate containing the fuel additive 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.” The stipulations of this protocol were 

implemented in conformance with EPA regulations as specified in 40 CFR 79.60 “Good 

Laboratory Practices (GLP) Standards for Inhalation Exposure Health 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 Number 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 characterization of the test 

substance was performed by CRTC and ExxonMobil Biomedical Sciences, Inc. (EMBSI), 

Annandale, NJ. The reference gas chromatographic 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 stored 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 are 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 using 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 originally 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 available. The test material is stable per MSDS. The test substance 

stability was tested concurrently with the study with the analysis of the exposure chamber 
Study 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 retained samples of the test substance. 

ANIMALS AND ANIMAL ASSIGNMENTS 

Animal Receipt, Housing, and Quarantine

Four hundred-forty CDF(F344)CrlBR rats (5–6 weeks old when received) were purchased from 

Charles River Laboratories (Raleigh, NC). They were 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 assigned 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 identified 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 sacrificed 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 found, and tissues were subsequently discarded 

because there were no subsequent questions regarding the health stats of the animals on study. 

Sera from these rats were submitted to BioReliance, Gaithersburg, MD, for analysis of antibodies 

common to rodents. The following tests were run: Cilia-Associated Respiratory Bacillus 

(CARB), Mycoplasma pulmonis (M. PUI), Pneumonia 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 rat 
Study 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 sentinel rats in the control chamber were bled 

retroorbitally after 26, 52, and 79 weeks of exposure. Blood was also obtained from sentinels 

surviving to 104 weeks. Sera were analyzed by BioReliance, as described above. 

Justification of Test Animals

Rats were used in this study because of the large database available on the inhalation toxicity and 

carcinogenicity of toxic materials in rats. The study design was justified 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 quarantine 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 baskets with 5.7-inch by 11-inch by 8-inch compartments. Each 

chamber contained six baskets. 

The chambers and cage racks were washed weekly. The cage racks were rotated clockwise 

weekly when the rats were transferred from the dirty to the clean chamber. 

The chambers were held at approximately 1 inch of water negative 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 air flow rates were continuously monitored, 24 

hours per day. Values for the three parameters were recorded at 30-minute 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). Food was available at all times except during the 

daily exposure period. Certified diet was 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 pesticides. Analytes were either present at nondetectable concentrations or sufficiently low concentrations as to not interfere with the outcome 

of the study. Results of feed and water analyses are provided in Appendix D. 

EXPERIMENTAL DESIGN 

Group Assignment

The experimental design is shown in Table 2-1. 

The Laboratory Animal Veterinarian visually examined 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 computerized data acquisition system (Path-Tox®; Xybion, Cedar 

Knolls, NJ) operated according to LRRI Standard 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, 2003 
Study Number FY01-013 

211(b) Chronic Carcinogenicity Study 

Gasoline MTBE Vapor Condensate (GMVC) 

September 2009 

2-5 

Table 2-1. Experimental Design (Concluded) 

Target Exposure Concentrations 

Control 0 g/m3 GMVC 

Low Level 2 g/m3 GMVC 

Mid Level 10 g/m3 GMVC 

High Level 20 g/m3

 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 observations for morbidity, mortality. 

Weekly body weights for first 13 weeks, then 

every 4th 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-required 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 generated by controlling the flow of pressurized GMVC 

through a rotameter, into a heated stainless steel transfer line where the GMVC was completely 

vaporized. Chamber concentrations were controlled by adjusting the flow rates of the GMVC 

and dilution air rate. Chamber exhaust was carried 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

N2 (60-80 psig)

Nitrogen

Carrier Gas

Pneumatic

Shutoff

Pressure Valve

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 following: 1) uniformity of the distribution of total vapor within 

each chamber; 2) within-day and between-day stability 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 concentration to achieve 90% of the equilibrium target 

value (T90). The exposure atmosphere in the animals’ breathing zone was also examined for the 

presence of aerosol particles using a TSI Scanning Mobility Particle Sizer (TSI Industries, 

Shoreview, MN). 

Once, prior to the initiation of animal exposures, the concentration 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 was 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 Wilks, 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 weekly 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 enclosing the 20-pound tank of test substance. This 

Miran underwent a three-point calibration approximately 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/m3

. The Miran 1A analyzers for the control 

chamber and the low-level chamber were calibrated over a concentration range of 1–7 g/m3. 

The absorbance in each chamber was monitored continuously. Data were recorded every 3 

seconds and the mean of these values was calculated 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 value of values from each exposure chamber was 

reported as the day’s exposure concentration for that chamber. 

Qualitative Assessment of Exposure Atmospheres. The qualitative composition of the exposure 

atmosphere in each chamber was determined weekly by gas chromatography using a Shimadzu 

Model GC-17A/FID (Columbia, MD). The percent peak area of each of 19 components was 

determined and recorded. The retention times of eight representative components were verified 

against EMBSI values each week, and one-point performance qualifications were performed on 

any day in which chamber profiles were analyzed. The one-point check was performed using a 

certified standard of 840 ppm butane in N2 (Matheson Tri-Gas, Irving, TX). 

Determination of Nominal Concentration. Daily nominal or “anticipated” usage was calculated 

by multiplying the average GMVC concentration in each chamber (low, mid, high; g/m3

) by the 

total flow through each respective chamber ([L/min * min]/[1000 m3

]) and then summing the 
Study 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 periodically during the study (at approximately 60-day 

intervals) using a Miran 1A Infrared Spectrometer (Foxboro Wilks, 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, the time for the vapor concentration to reach 

T90), 5 days/week for 104 weeks (520 exposure 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 oriented 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 animals from study (e.g., necropsy or refrigeration of 

any rats found dead and sacrifice 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 initiation of the study was delayed by approximately 10 

days. This decision was made by mutual agreement 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 taken on May 22 and May 29, for males and females, 

respectively. Therefore, the Day -1 weights were obtained 16 days after randomization occurred. 

After initiation of exposures, weights were obtained 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 were detailed and carefully recorded using PathTox® software. Observations included evaluations of skin and fur; eyes and mucous membranes; 

respiratory and circulatory effects; autonomic effects such as salivation and central nervous 

system effects including tremors 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. For the 12- and 18-month time points, blood was 

obtained by tail nick. At final sacrifice, blood was obtained by cardiac puncture and collected 

into Vacutainer tubes containing ethylenediamine tetra-acetic acid (EDTA). Evaluated 

parameters included total leukocyte estimates (white 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 neutrophils, band neutrophils, lymphocytes, 

monocytes, eosinophils, basophils, atypical lymphocytes and blastocytes. Absolute leukocyte 

counts were calculated for segmented neutrophils, 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 performed on all animals at final sacrifice 

and on those animals that died naturally or were euthanized in a moribund 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 Representative Samples Taken for Examination 

Cardiovascular/Hematopoietic System

1. Aorta

2. Bone marrow (and/or fresh aspirate)

3. Heart 

4. Lymph nodes (mandibular, mesenteric, bronchial, mediastinal) 

5. Spleen
Study Number FY01-013 

211(b) Chronic Carcinogenicity Study 

Gasoline MTBE Vapor Condensate (GMVC) 

September 2009 

2-10 

Table 2-2. Organs and Representative Samples Taken for Examination (Concluded)

Digestive System 

1. Cecum 

2. Colon 

3. Duodenum

4. Esophagus 

5. Ileum

6. Jejunum 

7. Liver

8. Pancreas 

9. Rectum 

10. Salivary glands 

11. Stomach 

Glandular System 

1. Adrenals 

2. Parathyroid

3. Thyroid

4. Mammary gland (malea

 and female)

Nervous System

1. Brain (including sections of medulla/pons, cerebellum, and cerebrum)

2. Eyes (retina, optic nerve)

3. Peripheral nerve (sciatic or tibial, preferably in close proximity to the muscle)

4. Pituitary

5. Spinal cord (three levels: cervical, mid-thoracic, and lumbar)

Other 

1. All gross lesions and masses

2. Skin 

3. Tail (for identification)

4. Skeletal musclea

5. Femur

a

Respiratory System 

1. Larynx 

2. Lung (infused with fixative)

3. Nose 

4. Paranasal sinuses 

5. Pharynx (reviewed with larynx section)

6. Trachea

Urogenital System 

1. Epididymis 

2. Kidneys

a

3. Ovaries 

4. Prostate 

5. Seminal vesicle(s)

6. Testes 

7. Urinary bladder (infused with fixative)

8. Uterus 

a

Examined microscopically but not required by protocol.
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 weights were collected on lungs, liver, kidneys, adrenals, testes, 

epididymis, ovaries, uterus, spleen, brain, and heart of final sacrifice and moribund sacrifice 

animals. Animals found dead received a complete necropsy with tissue collection, but blood and 

organ weight data were not routinely collected. Cardiac blood was collected from animals at 

final sacrifice for determination of total and 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% neutral 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 histopathologic examination. 

Histopathology. All collected tissues and lesions were examined histologically in control 

(0 g/m3

) animals, high-level (20 g/m3) animals, and dead or moribund animals of all groups. 

Respiratory tissues (lungs, larynx, trachea, and nasal turbinate sections), potential target tissues 

(testes, kidneys of males) and gross lesions were examined histologically from final sacrifice 

low- (2 g/m3

) and mid- (10 g/m3) level animals. Nomenclature of proliferative lesions was based 

on the international harmonized nomenclature recommended by the Rat Nomenclature 

Reconciliation Subcommittee of the Society of Toxicologic Pathologists (see 

http://www.toxpath.org; Standardized Rat Nomenclature). Nomenclature of other lesions was 

routine, widely understood usage (see Boorman et al., 1990a; Gopinath et al., 1987). 

Standard, subjective severity scoring of most nonproliferative lesions 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% affected, 4 (marked) = 51 to 100% affected. Severity 
Study 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 (usually by 1 point) based on the severity of the 

change within the affected areas. 

The kidney is an expected target tissue for toxicity in this study, especially for development of 

chronic progressive nephropathy or hyaline droplet nephropathy. The diagnosis of chronic 

progressive nephropathy encompasses a large constellation of changes; severity scoring for this 

diagnosis was somewhat more complex 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 progressive nephropathy. May have rare 

scattered protein casts in tubules, mineral concretions. 

1 Increased number and size of tubular protein casts; primarily within tubules at 

corticomedullary junction, some within collecting ducts. Minor/scattered foci of 

basophilic, regenerative tubules. Changes affect less than 5% of tissue 

component. 

2 Homogeneous protein casts relatively abundant within tubules and collecting 

ducts (especially at corticomedullary junction). Increased size and frequency of 

tubular basophilia. Accompanying changes present, may include small foci of 

tubular basement membrane thickening and/or mineralization, interstitial fibrosis, 

tubular atrophy and dilatation, minor associated mononuclear inflammatory 

infiltrates, basophilia changes (including mesangial proliferation, synechiae, 

proliferation of parietal epithelium). Changes may affect up to 25% of 

component. 

3 As 2 with increased tubular atrophy/dilatation/regeneration, interstitial fibrosis, 

increased inflammatory infiltrates, increased thickening of basement membranes 

of tubules and glomeruli. 

4 As 3 with increased severity and extent of changes. Senescent glomeruli 

relatively common. Typically combined changes 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 trend among test groups by an analysis of 

variance, Bartlett’s test was used to establish the homogeneity of the data. If the data were 
Study Number FY01-013 

211(b) Chronic Carcinogenicity Study 

Gasoline MTBE Vapor Condensate (GMVC) 

September 2009 

2-13 

homogeneous, significant differences between the 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 accidental 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 group. Log-rank tests were used to test the 

hypothesis that there are differences among the four groups for each sex. The significance level 

was set at p = 0.05. All reported p-values for 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 distributions are highly skewed for some variables. 

Preliminary analyses were done using the generalized estimating equation approach for 

longitudinal data analysis since there were three time points. For some variables these analyses 

showed significant interactions between time and group. Therefore, 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 and non-neoplastic lesions are given as the 

ratio of the number of affected animals to the number of animals with the site examined 

microscopically. Three statistical evaluations were performed on the histopathology lesion 

incidence data: 1) Cochran-Armitage test, which 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 Fisher’s exact test to compare incidences 

among the four dose groups. The two-sided significance level was set 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 comparisons, 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 prevalences are not equal while the Cochran-Armitage 

tests the null hypothesis of equality across doses against the alternate hypothesis of a monotonic 

increasing or decreasing trend. 

In the case of renal tubule nephropathy, statistical evaluation of group differences 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 Evaluation and Nominal GMVC Usage. Before exposures were initiated the 

system was operated for 6 hours/day for 3 consecutive days. The concentration of vapor in each 

chamber was close to target and constant within 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 percentages 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, and 90 for days 1, 2, and 3, respectively. Pre-test 

chamber trial data are presented in Appendix E. 

Table 3-1. Results of 3-Day Stability Evaluations 

Target Exposure 

Concentration (g/m3

)/ 

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 conducting 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 chambers during an exposure day and compared to the 

concentration obtained in a reference location within the chamber. The spatial variations 

measured in Chambers 6 (2 g/m3), 7 (10 g/m3), and 8 (20 g/m3) 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 concentration 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 for use with this study. 

Daily exposure periods to GMVC were then 6 hours and 14 minutes. 

Confirmation of the Absence of Aerosols. The particle concentrations of air inside Chamber 8, 

operated at a target concentration 20 g GMVC/m3

, 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/m3

 vapors are provided in Table 3-2. The 

overall achieved means were within 2% of target for each exposure concentration. Thus, all 

future references to exposure concentration are in terms of target concentration. Concentrations 

of vapor in the control chamber were below the lowest concentration on the standard curve used 

to calibrate the control chamber Miran 1A (1 g GMVC/m3

). The overall average of the daily 

percent nominal usage was 99% ± 4%, indicating excellent agreement between anticipated and 

actual GMVC usage. Daily vapor concentrations and nominal usage are provided in Appendix E. 

Table 3-2. Summary of GMVC Vapor Concentrationsa

Target 

g GMVC/m3

 

Achieved 

Concentrations 

Percent of 

Target 

 0 NAb 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/m3, 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/m3), 7 (10 g/m3), and 8 (20 g/m3) 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 throughout 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 divided 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. Profiles remained acceptably constant throughout the 

study. Weekly results are provided in Appendix F. 

Chamber Environmental Data. The temperature, relative humidity, chamber flow rate and 

pressures were maintained within acceptable ranges with occasional excursions outside acceptable 

limits that are described in Section 4. Environmental data are provided in Appendix G. 

Survival Analysis. All groups had 50 animals except the 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 differences among the groups are not statistically 

significant for male (p = 0.46) or female animals (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 time for animals that underwent final sacrifice. 

Therefore, the means and standard errors may be underestimated since some of the animals may 

have lived longer if the protocol had included following them for a longer period of time. The 

results indicate that chronic GMVC inhalation did 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 Animals 
Study Number FY01-013 

211(b) Chronic Carcinogenicity Study 

Gasoline MTBE Vapor Condensate (GMVC) 

September 2009 

3-5 

Table 3-3. Mean Survival Days 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 animals not surviving 104 weeks on 

study underwent moribund sacrifices. Individual animal disposition is provided in Appendix H. 

Table 3-4. Disposition of Rats on Study 

Death Type 

Control 2 g GMVC/m3

 10 g GMVC/m3 20 g GMVC/m3

Male Female Male Female Male 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 Ratsa

Study 

Day 

Control 2 g/m3

 10 g/m3 20 g/m3

Body Weight (g) Body Weight (g) 

Percent 

Control

Body Weight (g) 

Percent 

Control

Body Weight (g) 

Percent 

Mean SD Mean SD Mean SD Mean SD Control

 -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 different 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 homogeneity 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 using a modified t-test. Significance 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 Ratsa

Study 

Day 

Control 2 g/m3

 10 g/m3 20 g/m3

Body Weight (g) Body Weight (g) Percent 

Control

Body Weight (g) Percent 

Control

Body Weight (g) Percent 

Mean SD Mean SD Mean SD Mean SD Control

 -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 different 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 homogeneity 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 using a modified t-test. Significance 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 Male Rats Exposed to GMVC 

Figure 3-4. Body Weights of Female Rats Exposed to GMVC 
Study 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-dose group had a group mean body weight (177.9 g) 

significantly below that of the control rats (184.4g). Due to recently identified 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 (Day -1) indicated that the control males gained an average of 4.99 

g/day. The lowest weight gain among control males 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 chamber, suggesting the low weight 

gain was not due to a basket feeder issue. Clinical 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 high-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 females 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 was not a feeder issue. 

The matter of significantly different body weights among males and females before the study 

start was brought to the attention of the Sponsor and the EPA. 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; however, body weights were reduced in both males 

and females exposed to GMVC compared to control values. Body weights of high-level male 

and female rats remained statistically significantly below control values for most of the study 

(with the exception of days 63 and 70 for males 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 values. Mid-level female body weights 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, many of the observations 

observed during the second year of the study were related to aging (mammary masses, jaundice). 

A summary of clinical observations by exposure group is provided in Appendix J. 

Body Weights at Final Sacrifice

Final sacrifice body weights of male and female rats exposed to 20 g GMVC/m3 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 Weights of Male and Female Rats 

Weight in grams 

Control 2 g GMVC/m3

 10 g GMVC/m3 20 g GMVC/m3

Males 

Mean ± SD 390.0 ± 18.2 375.5 ± 35.2 368.7 ± 28.5 357.0 ± 28.4a 

n 14 16 20 19 

 Females 

Mean ± SD 265.4 ± 33.3 273.5 ± 20.4 256.3 ± 21.9 243.6 ± 15.7b 

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-level males and females were significantly below 

corresponding control values. The epididymis weight of mid-level males and adrenal weight of 

high-level females were significantly below that of the corresponding control values. Summary 

statistics for males and females are provided in Table 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 

3-11 
Study 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 mid and high GMVC-exposed males compared to 

controls. The lung results are likely a result of the treatment-related depression in body weight, 

rather than effects on the organs weights since there were no changes in absolute lung weights in 

males. For brain there was an apparent trend 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 weights. 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 mid-level males. The percent adrenal glands-tobrain weight was significantly decreased in high-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 Weight 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 final sacrifice. Summary 

statistics and individual animal terminal body and organ weight data are provided in Appendix K 

pages K-47 through K-98. Of particular note, the absolute kidney and percent kidney-to-brain 

weight of all GMVC-exposed groups were significantly increased compared to control values. 

Kidney-to-body weight was only significantly increased among males 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-body weight ratios for high-level males were 

statistically significantly increased. Among females, the absolute brain weight was significantly 

decreased in high-level GMVC females. The liver-to-body weight was significantly decreased in 

the low group females and the lung-to-body weight was significantly increased among the highlevel females. 
Study Number FY01-013 

211(b) Chronic Carcinogenicity Study 

Gasoline MTBE Vapor Condensate (GMVC) 

September 2009 

3-13 
Study Number FY01-013 

211(b) Chronic Carcinogenicity Study 

Gasoline MTBE Vapor Condensate (GMVC) 

September 2009 

3-14 
Study Number FY01-013 

211(b) Chronic Carcinogenicity Study 

Gasoline MTBE Vapor Condensate (GMVC) 

September 2009 

3-15 

Hematology

A summary of the effects of exposure 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 conducted on continuous variables (WBC estimates and 

percentage and absolute differential 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, monocytes, and eosinophils at the 12-month point. 

In all cases, values for the high-level male group were greater than the controls. The changes 

were influenced by both differences in absolute numbers of WBCs and in the percentage of the 

cell types found. These differences 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 had 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 sex. Only the percentage and 

absolute numbers of monocytes in high-level males were significantly different (lower than 

controls). In females the percentage and absolute numbers of atypical 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 lymphocytes in high-level females is unknown. 

The high incidence of leukemia in the aged F344 rats on study, other physiologic changes with 

aging, and the limitations of the estimate of total 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 

3-16 
Study 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/m3

 demonstrated 

significant increases in the incidence of renal tubule cell adenomas (Table 3-12 and Appendices 

M, N, O, and P). There was also a non-statistically significant increase 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 additionally 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 possible 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 gasoline and neat MTBE (MacFarland et al., 1984; Bird et al., 1997). 

There was a high background incidence of testicular interstitial adenomas 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 induction of testicular 

interstitial adenomas is considered to be equivocal, since background incidence 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 incidence3

 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 hypothesized that MTBE disruption of 

hormonal homeostasis could account for increased testicular interstitial cell adenomas (Cruzan et 

al., 2007). Mechanistic work to date to evaluate 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 hormone 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-7D3BED551F07DC0A 
Study Number FY01-013 

211(b) Chronic Carcinogenicity Study 

Gasoline MTBE Vapor Condensate (GMVC) 

September 2009 

3-18 

Table 3-12. Incidences of Proliferative (Neoplastic and Hyperplastic) Lesions in Rats 

 

Control 

(0 g/m3

) 

Low 

(2 g/m3) 

Mid 

(10 g/m3) 

High 

(20 g/m3) 

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 cellc 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 (Neoplastic and Hyperplastic) Lesions in Rats 

(Concluded) 

 

Control 

(0 g/m3) 

Low 

(2 g/m3) 

Mid 

(10 g/m3) 

High 

(20 g/m3) 

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 

Fibroadenomae 0 (0%) 3 (15%)d 2 (9%) 5 (11%)d

a

Incidence is significantly different from controls and from the 2 g/m3 group (Fisher’s test). 

b

Incidence is significantly different 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 Fisher’s test with the incidence among the 20 g/m3

group being significantly increased 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 differences or trends. It was not appropriate to combine carcinoma 

incidence of nasal turbinate levels for statistical analysis in the low- and high-dose groups 

because the lesion spanned two levels for one of the two animals. By comparison, in the 

concurrent Baseline Gasoline Vapor Condensate 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 statistically significantly increased with exposure concentration (CochranArmitae test). 

There is an increased incidence (see Table 3-12) of thyroid follicular 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, respectively. 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, there was a statistically increased 
Study 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 Attributable 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 was no overall trend with 

exposure concentration as determined by the Cochran-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 females were not different from the incidence among 

control females. 

Mammary gland fibroadenoma was significantly increased in low- and high-level females (Table 

3-12). Interpretation of this finding is problematic due to the atypically 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 incidence 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 wholly vaporized gasoline (Cruzan et al., 2007; 

MacFarland et al., 1984). Therefore, it is unlikely that the increased incidence of female 

mammary gland fibroadenoma was treatment-related.

Nonproliferative Lesions 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 expected target tissue are discussed here. 

Chronic progressive nephropathy is a lesion of particular interest to this study, as this lesion is 

expected to occur in rats, especially male rats exposed to hydrocarbon mixtures. In this study, 

the incidence of chronic progressive nephropathy among control male and female rats was 44/59 

(88%) in males and 42/50 (84%) in females (Table 3-13 and Appendices M, N, O, and P). The 

incidences among male and female rats inhaling GMVC were not significantly increased over 
Study 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 high-level females, compared to the corresponding 

control values. This finding in males is consistent with male-rat-specific light hydrocarboninduced alpha-2u globulin overload nephropathy that has been reported previously in studies 

with wholly vaporized unleaded gasoline and neat 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 share similar if not identical diagnostic hallmarks. 

Therefore. the effect of alpha-2u globulin overload nephropathy typically manifests itself in 

increased severity, rather than incidence, since essentially all males surviving past 1 year of age 

will develop CPN. The increased severity of nephropathy 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/m3

) 

Low 

(2 g/m3) 

Mid 

(10 g/m3) 

High 

(20 g/m3) 

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 2.6b

 2.7b

Nose/Turbinate 2 No. examined 50 50 50 50 

Degeneration, hyaline - 

olfactory epithelium 

0 (0%) 5 (10%) 7c

 (14%) 6c (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 0 
Study 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/m3) 

Low 

(2 g/m3) 

Mid 

(10 g/m3) 

High 

(20 g/m3) 

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 1.5b

Nose/Turbinate 2 No. examined 50 50 50 50 

Degeneration, hyaline - 

respiratory epithelium 

0 (0%) 1 (2%) 7c

 (14%) 6c (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 controls at 0.05 level using Kolmogorov-Smirnov one-tailed test. c

Significantly different from controls at 0.05 level using Fisher’s exact two-tailed test. 

d

Positive concentration-dependent trend by Cochran-Armitage and logistic tests. p = 0.054 by 

Fisher’s exact (no group comparisons made). 

The only other nonproliferative lesion attributable to GMVC inhalation is hyaline degeneration 

in the nasal epithelium, with significant changes in the mid- and high-level GMVC animals of 

both sexes. In males, this lesion occurred in the olfactory epithelium of turbinates 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 component of unleaded gasoline vapor for 2 years. 

In this study, rats were exposed to 0, 2, 10, and 20 g GMVC/m3

. The GMVC mixture contained 

20% MTBE by mass, so the corresponding MTBE concentrations were 0, 0.4, 2, and 4 g/m3

. 

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 adversely affect WBC parameters. Effects seen at 12 months 

compared to at the 18- and 24-month sampling times were more likely due to difficulties in 
Study Number FY01-013 

211(b) Chronic Carcinogenicity Study 

Gasoline MTBE Vapor Condensate (GMVC) 

September 2009 

3-23 

blood sampling than to GMVC inhalation. Chronic exposure to GMVC 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 degenerative nasal effects were most 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. Testicular interstitial cell (Leydig cell) adenomas 

showed an increasing trend with exposure concentration, with the incidence in the high-level 

GMVC group (50/50; 100%) significantly 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 rats. However, GMVC induction of testicular 

interstitial cell adenomas in the 20 g/m3

 group cannot be ruled out, particularly in light of similar 

equivocal observations reported in previous studies with neat MTBE (Cruzan et al., 2007). 

Chronic exposure to GMVC did not enhance the development of proliferative lesions 

(hyperplastic lesions, neoplasms) in female rats. 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 Condensate was determined not to be carcinogenic in 

female rats in this study. 

COMPARATIVE CHRONIC TOXICITY AND CARCINOGENICITY BETWEEN GMVC 

AND BGVC 

Background

The current study on GMVC was conducted in parallel with chronic study on BGVC. Both 

studies were conducted in compliance with the Clean Air Act Section 211(b) for fuel and fuel 

additive registration. The 211b program 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 materials is presented in Table 3-14. Below, 

results from the two studies are compared to determine 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 Comparison of Test Materials 

Compound 

BGVC Lot # API 99-01a

(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 used 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 octane enhancing additive for gasoline 

and as part of the oxygenated fuels program to reduce carbon monoxide in automotive emissions. 

Concerns regarding the carcinogenicity of MTBE have been raised. In previous chronic 

inhalation studies, F344 rats were exposed to 0, 400 (~1.4 g/m3), 3000 (~10.8 g/m3), and 8000 

(~28.8 g/m3) ppm MTBE (Bird et al., 1997; Mennear, 1997). An increased incidence of renal 

tubular adenoma and/or carcinoma was observed in males exposed to 8000 ppm (28.8 g/m3), and 

an increased incidence in testicular tumors was observed among rats exposed to 3000 and 8000 

ppm (10.8–28.8 g/m3

). Belpoggi et al. (1995) reported that 1000 mg MTBE/kg administered by 

gavage over a 2-year period increased 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), organ:brain weight ratios (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/m3

 level and was very similar for the two studies. At the 20 g vapor/m3 level, 

the incidence of adenomas and combined adenomas and carcinomas is higher in the GMVC 

study. 

In testes, there was a high, not unexpected, incidence 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 numbers of tumors above control values (48%). However, the 

incidence of adenomas in the high exposure level GMVC males was significantly 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 studies, but a significant 

positive trend was only observed in the BGVC study, and only by the Fisher’s test. 

Nasal squamous cell carcinoma (turbinate levels 2–4 combined taking into account tumors 

spanning two or more levels) was observed in the low- and high-level groups in both studies. 

Incidences were generally similar; however the incidence in the high BGVC exposure group was 

greater than in the high GMVC exposure group. 

Similar to the renal tumor findings, thyroid follicular cell adenomas and carcinomas were 

observed in both studies. However, there was a significant trend toward increase incidence of 
Study Number FY01-013 

211(b) Chronic Carcinogenicity Study 

Gasoline MTBE Vapor Condensate (GMVC) 

September 2009 

3-26 

thyroid follicular carcinomas 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/m3

) 

Low 

(2 g/m3) 

Mid 

(10 g/m3) 

High 

(20 g/m3) 

GMVC

Kidney No. examined 50 50 50 50 

Adenoma, renal tubule 0 (0%) 0 (0%) 6a

 (12%) 2 (4%) 

Carcinoma, renal tubule 0 (0%) 1 (2%) 0 (0%) 1 (2%) 

Renal tubule adenoma and 

carcinoma, combined 

0 (0%) 1 (2%) 6b (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 cellc 43 (86%) 47 (94%) 48 (96%) 50d (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, combinede

1 (2%) 1 (2%) 7 (14%) 0 (0%) 

Nasal 

Turbinates 

No. examined 50 50 49-50f

 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, malignanth

 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 Tumors in Male Rats Inhaling GMVC and BGVC (Concluded) 

Tissue Diagnosis 

Control 

(0 g/m3) 

Low 

(2 g/m3) 

Mid 

(10 g/m3) 

High 

(20 g/m3) 

BGVC (Concluded) 

Thyroid No. examined 50 29 27 50 

 Adenoma, follicular cell 0 (0%) 2 (7%) 0 (0%) 2 (4%)

 Carcinoma, follicular celli 0 (0%) 0 (0%) 2 (7%) 0 (0%)

 Follicular cell adenoma and 

carcinoma, combinedj

0 (0%) 2 (7%) 2 (7%) 2 (4%) 

a

Incidence is significantly different from controls and from the 2 g/m3 group (Fisher’s test). 

b

Incidence is significantly different 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 Fisher’s test with the incidence among the 20 g/m3

group being significantly increased compared to the control incidence. 

d

Incidence significantly different from controls (Fisher’s test). e

A positive trend was found with the Fisher’s exact 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 exposure 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 chance (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 were high, but incidences did not increase with 

vapor exposure concentration in either study (Table 3-16). In the cases where incidences in the 

low- and mid-vapor level groups are significantly increased compared to controls, it is possible 

that some sampling bias occurred because for 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 MTBE-containing unleaded fuel vapors increased the 

incidence of leukemias over the already 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/m3

) 

Low 

(2 g/m3) 

Mid 

(10 g/m3) 

High 

(20 g/m3) 

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%) 

Femalesb 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 significant (p > 0.008, FE tests). 

Overall, it is difficult to draw strong conclusions about the role of MTBE in inducing 

proliferative lesions, especially in regard to inducing renal or testicular tumors in male rats. The 

predictive value of renal tumors and testicular interstitial 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 nephropathy 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 nephropathy was significantly increased in mid- and highlevel males inhaling GMVC and BGVC. The increased severity of chronic progressive 

nephropathy in high-level GMVC females is likely 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 Chronic Progressive Nephropathy in Rats Inhaling GMVC and 

BGVC for 2 Years 

Control 

(0 g/m3) 

Low 

(2 g/m3) 

Mid 

(10 g/m3) 

High 

(20 g/m3) 

GMVC

Males 

 Incidence 

 Avg. severity 

44/50 (88%)a

1.9 

47/50 (94%) 

2.1 

50/50 (100%) 

2.6b 

46/50 (92%) 

2.7b 

Females 

 Incidence 

 Avg. severity 

42/50 (84%) 

1.0 

17/20 (85%) 

1.0 

15/21 (71%) 

1.0 

43/50 (86%) 

1.5b 

BGVC

Males 

 Incidence 

 Avg. severity 

49/50 (98%) 

2.4 

49/50 (98%) 

2.5 

50/50 (100%) 

2.9c 

50/50 (100%) 

2.9c 

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 controls at 0.05 level using Kolmogorov-Smirnov one-tailed test. c

Overall severity/incidence significantly higher than control at 0.05 level using KolmogorovSmirnov one-tailed test. 

For both GMVC- and BGVC-treated male rats, the increased incidence of renal tumors and 

increased severity is likely due in part to male rat specific alpha-2u globulin accumulation and 

overload in the kidney during the first year of life. Other mechanisms may also contribute, as 

evidenced by increased severity of nephropathy in high-level GMVC females, lacking renal 

adenomas and carcinomas. 

The respiratory epithelial degeneration of the respiratory 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 increased incidence of degeneration in turbinate 3. 

The incidences of this lesion were also significantly increased among male and female rats 

exposed to the mid and high levels of BGVC. Interestingly, the respiratory epithelial 
Study 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 turbinate level 3, so it was 

more extensive than that caused by GMVC. Therefore, the increased incidence of the epithelial 

degeneration is not attributable to MTBE, but more likely due to the irritant properties of the fuel 

vapor mixture. 

In summary, we have conducted a side by side comparison of the toxicity and carcinogenicity of 

Baseline Gasoline Vapor Condensate and Condensate 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 conditions. The health effects resulting in F344 

rats following 2 years of GMVC or BGVC inhalation were unequivocally comparable in the 

production of renal adenomas and carcinomas in male rats. Methyl tertiary butyl ether was not 

identified as enhancing the production of these renal adenomas or adenomas and carcinomas 

compared to those induced by gasoline vapor condensate alone. 
Study Number FY01-013 

211(b) Chronic Carcinogenicity Study 

Gasoline MTBE Vapor Condensate (GMVC) 

September 2009 

4-1 

LOCATION OF SPECIMENS, RAW DATA, AND FINAL REPORT 

Specimens were identified by test system, study, nature, and date of collection. All raw data and 

records and specimens that are required to reconstruct the study will be maintained in the LRRI 

archives for 10 years. The Sponsor will be notified and will authorize in writing the destruction 

or transfer of any specimens, raw data, and study records to the Sponsor or to an archive 

specified by the Sponsor’s Contracting Representative. 

PROTOCOL DEVIATIONS 

Delayed Sponsor Signature on Amendment 1

Amendment 1 to the study protocol was signed by the Study Director on August 22, 2001 and 

submitted to the Sponsor for signature. A signature was not obtained after many requests. The 

operative LRRI SOP at the time stated, “The study director signs and dates the amendment prior 

to implementing the changes and the dated signature of the Sponsor is obtained as soon as 

reasonably possible.” Good Laboratory Practice guidelines do not require Sponsor signature. 

Thereafter, LRRI no longer sought Sponsor signature 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 Veterinarian, and Justin Kubatko, MS, 

Statistician. During the study, Mr. Powell left the Institute and was replaced by Mr. Edward B. 

Barr, MSEE, an inhalation engineer with over 25 years 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 subcontracted to perform the 

statistics. Dr. Thomas March took over responsibilities 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 determined 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/m3

) by the total flow through each 

respective chamber ([L/min * min/1000L/m3]) 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 condensate 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 originally 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. Second, 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 control and dosed groups, the Kolmogorov-Smirnov test 

was used to assess differences in severity scores. 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 section 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 did not affect animal 

survival, the Hoel Walburg analysis, that assumes that treatment affects animal survival, was not 

necessary. Since the hematology data did not have normal distributions, alternative statistical 

approaches were warranted. Since repeated measurements were made on the same rats over 

time, evaluation of the effect of time and exposure concentration on the hematology parameters 

was appropriate. The use of the Kolmogorov-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 concentration 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 chromatographic profile to be performed on all 420 pound tanks 

prior to use. Tank 16 was inadvertently not analyzed prior to being used for exposures. 

However, profiles of the exposure atmosphere were 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 September 10, 2001, the gas chromatograph used to 

qualitatively analyze the hydrocarbon profiles of the exposure atmospheres was not working 

properly. Therefore profiles were not obtained during these weeks. The technician responsible 

for the analyses worked with the Lovelace electrician and the instrument vendor to troubleshoot 

and resolve the problems. Profiles were obtained on previous and subsequent weeks. Since 

hydrocarbon profiles in the exposure atmospheres were relatively stable for these lots of fuel, the 
Study Number FY01-013 

211(b) Chronic Carcinogenicity Study 

Gasoline MTBE Vapor Condensate (GMVC) 

September 2009 

4-4 

loss of the chamber atmosphere profile data during the 2 weeks of the 104-week study did not 

significantly affect the outcome of the study or interpretation of results. 

Exhaust System Failure During Conditioning/Quarantine Period Affecting Chamber Pressure

On August 2, 2001, a subcontractor was at the Institute 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 temporarily shut off the main blower. After the filters 

had been changed, the blower motor could not be restarted. 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 doors 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 maintenance 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 housing the study animals. All environmental 

parameters continued to be monitored by computer as before the exhaust system went down. 

Surveillance personnel also checked the conditions in the room twice per shift 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 approximately 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 the 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 not adversely affect the health of the animals and 

therefore the animals remained within 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 exceed -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 guidelines, but below those specified 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 the chamber were adjusted to within normal 

operating parameters. Facility Engineering personnel 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 Engineering discovered that the chamber supply air blower 

was not working properly. Exposures were terminated 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/m3

, 7.88 g/m3 and 13.62 g/m3 for the 2, 10 and 20 g/m3 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 occupancy 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 because 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 protocol required ranges, or data were not acquired 

for short periods because of problems with the monitoring system. Most excursions of 

temperature and pressure or periods of data loss were of short duration (~2% per month). These 

deviations were judged to have no significant effect on the outcome of the study. Tables 

summarizing deviations from protocol-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 plugged 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/m3

) 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 detecting 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 were not recorded for approximately 0.5–1 hour on 

August 30, September 6, and September 12, 2002. This was likely due to the fact that the 

computer monitoring system was shut down and rebooted 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 from 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 and emergency power was not provided to the 

environmental monitoring system and the system was on uninterrupted power supply battery 

power until they were drained. During this outage, the supply air fan stopped operating, but due 
Study Number FY01-013 

211(b) Chronic Carcinogenicity Study 

Gasoline MTBE Vapor Condensate (GMVC) 

September 2009 

4-7 

to the lack of environmental monitoring capability, 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 operational 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 environmental 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 because light and sound levels were similar among 

locations and did not vary greatly between measurements that were made. 

Gas Chromatographic Analysis

The original version of the SOP ASP-1166.0, Calibration of the Shimadzu GC-17A/FID for 

Analysis of Gasoline Vapor Condensate, indicated GC calibration for monitoring the exposure 

atmosphere and test article hydrocarbon composition profile may be calibrated daily. However, 

calibrations were performed only on the days when analyses were performed. The SOP was 

modified and signed by management on September 25, 2002, changing the requirement for 

calibration to only days when analyses are performed. 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 According to Protocol Defined Schedule

Body weights were performed at erratic intervals. Body weights were generally performed every 

fourth week on the same day of the week (the day 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 investigating the feasibility of going to a weekend 

weigh schedule. This would allow study personnel to perform other tasks 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 statistical 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 days 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 was 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, several animals were repeatedly noted as 

needing tattoo touch ups to aid in identification and fulfill SOP requirements. Tattoos were 
Study 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 attempts 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 chamber) 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 staff failed to verify the 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 affected Individual Animal Necropsy Record 

forms were corrected and error coded. Removal 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 portions of adjacent thymic tissue was collected 

because mediastinal lymph nodes are easily missed during sectioning in the absence of a 

supporting stroma. Data from thymus collected with 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, liver, kidneys, testes, epididymis, ovaries, 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 was minimal. Only one brain weight was lost. 

Contrary to protocol, organ weights were recorded for rats found dead. Organ weight summaries 

have been prepared to exclude natural death animals, so their weights do not skew the group 

means for the final sacrifice and euthanized animal 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 differential 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 smear 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 impact 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 tertiary-butyl ether 

NBF Neutral buffered formalin 

NFPA National Fire Protection Association

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 cells 
Study 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). Alpha2υGlobulin: 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., Douglas, J. F., Kneiss, J. J., and Andrews, L. S. 

(1997). Oncogenicity Studies of Inhaled Methyl-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, 1st 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.), 1st 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.), 1st 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.), 1st 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 α2u-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 α2u-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., Hard, 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., Hardisty, J. F., Sharpe, R. M., and 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 Carcinogenesis in the Laboratory 

Rodent. Toxicol. Pathol. 26(1): 104-112. 

Hard, G. C. and Khan, K. N. (2004). A Contemporary 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 Inhalation 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.