Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

i ABSTRACT

In compliance with the Clean Air Act Section 211( b) for fuel and fuel additive registration, the petroleum industry and oxygenate manufactur ers have conducted comparative chronic toxicology testing of evaporative emissions fr om unleaded gasoline (baseline gasoline vapor condensate [BGVC]) and unleaded gasoline containing MTBE (gasoline/MTBE vapor condensate [GMVC]). This is the fina l report of the BGVC chronic study.

Groups of 50 male/50 female CDF(F344)Crl BR rats were exposed in H2000 whole-body inhalation chambers at BGVC vapor concentrations of 2 g/m 3 (low level), 10 g/m 3 (mid level), and 20 g/m 3 (high level) 6 hours/day, 5 days/wee k for 104 weeks (518 exposure days).

There were no clinical signs of t oxicity attributable to BGVC i nhalation. Survival of the BGVCexposed rats was not significantly different from that of control rats. Body weights of males in the mid- and high-level BGVC groups were sign ificantly below control values throughout the study. Body weights of high-level and mid-leve l females were below control values after approximately 1 and 3 months of exposure, re spectively. Body weights of low-level females were sporadically affected. At the final sacr ifice, body weights of high-level males and highand mid-level female rats were significantly be low control values. Among mid-level males, the absolute epididymis weight, percent epidi dymis-to-body and percent epididymis-to-brain weights were significantly belo w the corresponding control values. The adrenal-to-body and brain-to-body weight values of high-level males were significantly greater than control values. The kidney weights of high- and mid-level female s were significantly increased compared to controls, however they did not co rrelate with kidney pathology fi ndings. The spleen weights of mid-level females were significantly increased comp ared to controls, which is likely a result of the high incidence of age-related mononuclear cel l leukemia (MCL). Among mid-level females, the percent organ-to-body weight values for brai n, heart, kidney, liver, lungs, and spleen were significantly greater than corres ponding control values. Among hi gh-level females, the percent organ-to-body values for adrenals, brain, heart, li ver, kidney, and lungs were significantly greater than control values. The percent kidney-to-br ain weights for mid- and high-level females and the percent spleen-to-brain weight for the mid-le vel females were significantly greater than theStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

ii corresponding control values. It is likely that many of the organ-to-body weights increases were artifacts of the treatment-related depression in body weight. For females, there were significantly fewer numbers of total WBCs, neutrophils, lymphoc ytes, eosinophils, and nucleated red blood cells (RBCs) in the high-level group at the 12 month time-point, only. In males, only the absolute number of lymphocyt es was significantly elevated in the high-level group at the final sacrifice time-point. Due to blood sampling difficulties at the 12 month time point and the high, age-related incidence of mononuclear cel l leukemia in all groups, the relationship of treatment to these changes in hematology valu es is unknown. BGVC exposure was associated with an increase in the incidence of respiratory epithelial degeneration and a significant decrease in the incidence of olfactory epithelial degeneration in the na sal passages of both male and female rats. The respiratory epithelial degenera tive effects were most likely caused by exposure
to the test material.

Exposure did not enhance the incidence of chroni c progressive nephropathy in male or female rats, but did increase the severi ty of this lesion among mid- and high-level groups of males. The increased severity of chronic progressive nephropathy in males is consistent with previous studies on wholly vaporized unleaded gasoline, and is attributable to male rat specific alpha-2u globulin overload as a result of BG VC exposure. Consistent with previous studies and the alpha2u globulin overload mechanism of male rat kidn ey damage, BGVC induced increases in male renal adenoma (1/50 in control; 1/50 in 2 g/m 3 group; 4/50 in 10 g/m 3 group; and 0/50 in 20 g/m 3 group) and carcinoma (0/50 in controls; 0/50 in 2 g/m 3 group; 3/50 in 10 g/m 3 group; and 0/50 in 20 g/m 3 group). Although the incidences of renal adenomas and carcinomas taken separately were not statistically sign ificant, combined incidences for the control (1/50; 2%), low (1/50, 2%), mid (7/50, 14%) and high (0/50; 0%) exposur e groups were significant (p = 0.004, Fisher’s Exact Test). Pair-wise tests did not demonstrate any significa nt difference among the individual dose groups. These neoplasms may be c onsidered to be exposure related.

The incidence of squamous cell carcinoma in the nasal passages (turbinate levels 2-4) was elevated in the high-level group of male rats, with tumor incidence of 1/50, 3/50, and 3/50 for levels 2, 3 and 4, respectively. The level 3 inci dence gave a statistically significant trend for
increased incidence with incr eased exposure concen tration (p = 0.019, Cochran-Armitage [CA]Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

iii test). Squamous cell carcinoma was noted in th e nasal specimens of only one female control rat of the current study. The squamous cell carcinom as in all of the male nasal specimens had morphologies consistent with or igination from the oral mucosa and had varying degrees of invasion into the nasal passages. By compar ison, in the concurrent Gasoline MTBE Vapor Condensate study (FY01-013), squam ous cell carcinoma of oral or igin occurred in one male control, two male GMVC low dose and two male high GMVC dose, but in no female rats.
While the fact that the nasal tumors were of oral origin might suggest that the inhalation exposure was not causal, spontaneous oral squam ous cell carcinomas in other studies have been nearly as rare as those originating from the nasal mucosa (~0.6%; Haseman et al., 1990; NTP, 1999). The historical incidence of squamous cell carcinoma of oral origin in a large cigarette smoke study conducted at LRRI in the 1990s was 0/118 (0%) in males and 3/119 (2.5% in females). Taken together, the data provide some evidence that the development of nasal squamous cell carcinomas in male rats may have resulted from test article exposure.

Similar to the incidence of renal neoplasms, th ere was an increased incidence of testicular mesothelioma in the mid-level group of male ra ts (4 of 50 examined). Fisher’s exact test demonstrated that this incidence was greater th an expected, but pair-w ise testing between the groups and trend tests for exposure concentration -response effects did not indicate statistically significant effects of exposure. The incidence in the mid-level group is approximately 3-fold greater than the average incidence in male c ontrol F344 rats exposed to air in inhalation toxicology studies (28 tumors in 1,055 rats; NTP, 1999) and may be regarded as an effect of exposure to the test substance. As with this study, there were no mesotheliomas found in the
testes or other tissues of the control male animals of the GMVC study (LRRI protocol FY01- 013), although there was a statistically non-significan t incidence (2 in 50 an imals or 4%) of the tumor in the testes of the high-level GMVC-exposed rats and sporadic incidence of the tumor in other tissues of GMVC-exposed male rats. The ab sence of testicular mesothelioma in control animals of the present study, where 1 or 2 tumors might normally be expected to be found in 50 animals (NTP, 1999), may have influenced the sta tistical analysis to i ndicate a significant exposure-related incidence in the mid-level BGVC -exposed male rats, when no causal increase is actually present.Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

iv There was a trend for increased incidence of t hyroid follicular cell carcinoma in male animals (p < 0.05, FE test over all groups; see Table 3-12 ) but there were no significant differences among the groups. There were two tumors in the 27 mid-dose glands examined and no tumors in any other male control or exposed group. Whethe r the incidence of thyroid follicular carcinoma was a causal effect of exposure to the test substa nce or a sporadic occurrence remains a question, especially given that there is no apparent e xposure concentration-response effect. Pair-wise testing suggests no statistically significant di fferences existed among the groups (p > 0.008, FE test). However, the incidence (7%) in the mid- level group of males is co nsiderably higher than that reported in control F344 rats exposed to air (average incidence of 1% in 21 studies, range of 0 carcinomas in 52 rats to 1 in 45; NTP, 1999) . While the incidence of thyroid follicular adenomas reported here are similar to or sligh tly higher than those pr eviously reported for control animals, the incidence of thyroid fo llicular carcinomas and adenomas combined are higher than what might be expect ed (NTP, 1999). However, there are no statistically significant differences in the combined incidence of thyr oid follicular adenomas and carcinomas among the exposure groups. In the concurrent Gasolin e MTBE Vapor Condensate study, there were no significant increases in thyroid follicular cell ad enomas, carcinomas, or combined adenomas and carcinomas in males or females. Thyroid follicu lar carcinoma might be considered to be caused by the BGVC exposure despite the l ack of an apparent exposure concentration-response effect.

In summary, chronic BGVC inhalation suppressed body weight in males, and to a greater extent in females, increased the severity of chroni c progressive nephropathy in males and caused epithelial degeneration in the nasa l passages of both sexes. The degenerative nasal effects were most likely caused by the test material. Ch ronic exposure to BGVC did not enhance the development of proliferative lesi ons (hyperplastic lesions, neoplasm s) in female rats. However,
there was an increased inciden ce in mononuclear cell leukemia in low- and mid-level exposed females, most likely due to protocol-driven sa mpling bias in these groups. Consistent with previous studies and the conc urrent GMVC study, chronic expos ure to BGVC did enhance the development of renal adenomas and carcinomas in male rats. In contrast with the concurrent GMVC chronic study, BGVC may also have enha nced squamous cell carcinoma in the nasal specimens, testicular mesothelioma, and thyroid follicular carcinoma. Consequently, dueStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

v primarily to treatment-related effects in the male kidney, nose, testes, and thyroid, chronic
inhalation of Baseline Gasoline Va por Condensate was determined to be carcinogenic in male rats in this study. Chronic inhalation of Ba seline Gasoline Vapor Condensate was determined not to be carcinogenic in fe male rats in this study.Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

vi TABLE OF CONTENTS Page
ABSTRACT ...................................................................................................................... ............... i
TABLE OF CONTENTS ............................................................................................................. .. vi
LIST OF FIGURES ............................................................................................................... ...... viii
LIST OF TABLES ................................................................................................................ ....... viii
LIST OF APPENDICES ............................................................................................................ .... ix LABORATORY TITLE PAGE ..................................................................................................... xi KEY STUDY PERSONNEL ........................................................................................................ xi i LABORATORY SIGNATURE PAGE ....................................................................................... xiii LABORATORY QA STATEMENT ........................................................................................... xiv LABORATORY COMPLIANCE STATEMENT ..................................................................... xvii Section
SUMMARY ...................................................................................................................... . S-1
1. INTRODUCTION .............................................................................................................. 1-1 2. MATERIALS, METHODS, A ND PROCEDURES .......................................................... 2-1
TEST SUBSTANCE .......................................................................................................... 2-1 Identification, Source, and Storage ........................................................................... 2-1 Analysis .................................................................................................................... 2 -2 Expiration Date ......................................................................................................... 2-2 Reserve Sample ........................................................................................................ 2-3
ANIMALS AND ANIMAL ASSIGNMENTS .................................................................. 2-3 Animal Receipt, Housing, and Quarantine ............................................................... 2-3 Animal Disease Screening Program ......................................................................... 2-3 Justification of Test Animals .................................................................................... 2-4 Environmental Conditions ........................................................................................ 2-4 Diet and Drinking Water .......................................................................................... 2-5
EXPERIMENTAL DESIGN .............................................................................................. 2-5 Group Assignment .................................................................................................... 2-5 Exposure System ...................................................................................................... 2-6 In-Life Endpoints ...................................................................................................... 2-9 Post-Exposure Endpoints ........................................................................................ 2-10Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

vii Statistics .................................................................................................................. 2 -13
3. RESULTS AND CONCLUSIONS .................................................................................... 3-1
EXPOSURE ATMOSPHERE ............................................................................................ 3-1 Pre-Test Chamber Trials ........................................................................................... 3-1 In-Study Data ............................................................................................................ 3-2 Final Body Weights .................................................................................................. 3-6 Absolute Organ Weights at Final Sacrifice .............................................................. 3-6 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 Weights of Euthani zed (Moribund) Rats ........ 3-12 Hematology ............................................................................................................. 3-15 Histopathology ........................................................................................................ 3-17
CONCLUSIONS .............................................................................................................. 3-27 4. LOCATION OF SPECIMENS, RAW DATA, AND FINAL REPORT ........................... 4-1
PROTOCOL DEVIATIONS .............................................................................................. 4-1 Calculation of Nominal Concentration ..................................................................... 4-1 Environmental Conditions ........................................................................................ 4-1 Exhaust System Failure During Conditioning/Quarantine Period Affecting Chamber Pressure ..................................................................................................... 4-2 Temporary Loss of Environmental Monitoring System Operation .......................... 4-2 Light and Sound Measurements ............................................................................... 4-3 Gas Chromatographic Analysis ................................................................................ 4-3 Cage Board Usage/Availability ................................................................................ 4-3 Collection of Organ Weights at Necropsy ................................................................ 4-4 Assignment of Severity Scores ................................................................................. 4-4 Statistics .................................................................................................................... 4-4 Neat Test Article Characte rization of 420-pound Tanks .......................................... 4-5 Personnel ................................................................................................................... 4 -5 Tissue Collection ...................................................................................................... 4-6 Hematology ............................................................................................................... 4-6 Loss of Supply Air to Exposure System ................................................................... 4-6 Body Weights of Males Not Collected According to Schedule (two deviations) ................................................................................................................. 4 -7Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

viii Quantitation of Exposure Atmosphere ..................................................................... 4-8
5. LIST OF ABBREVIATIONS ............................................................................................ 5-1
6. REFERENCE LIST ............................................................................................................ 6-1

LIST OF FIGURES Figure Page 2-1 Vapor Generation System for BGVC .............................................................................. 2-7 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 BGVC .............................................................. 3-9 3-4 Body Weights of Female Rats Exposed to BGVC .......................................................... 3-9

LIST OF TABLES Table Page 2-1 Analytical Comparison of Test Material Lots ................................................................. 2-1 2-2 Experimental Design ....................................................................................................... . 2-5 2-3 Organs and Representative Sa mples Taken for Examination ........................................ 2-11 3-1 Results of 3-Day Stability Evaluations ............................................................................ 3-1 3-2 Summary of BGVC Vapor Concentrations ..................................................................... 3-2 3-3 Mean Survival Days and Day of 25% Mo rtality for Male and Female Rats ................... 3-5 3-4 Disposition of Rats on Study ........................................................................................... 3- 5 3-5 Body Weight Summary for Male Rats............................................................................. 3-7 3-6 Body Weight Summary for Female Rats ......................................................................... 3-8 3-7 Summary Statistics for Terminal Body We ights of Male and Female Rats .................. 3-10 3-8 Absolute Organ Weight (g ± SD) .................................................................................. 3-11 3-9 Percent Organ-to-Body Weight (% ± SD) ..................................................................... 3-13 3-10 Percent Organ-to-BrainWeight (% ± SD) ..................................................................... 3-14 3-11 Summary of Hematological Findings in Rats Inhaling BGVC; Median (range) ........... 3-16 3-12 Summary of Select Proliferative Le sions in Male and Female Rats ............................. 3-18 3-13 Incidence and Severity of Chronic Pr ogressive Nephropathy in Male and Female Rats Exposed to BGVC ................................................................................................. 3-23Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

ix 3-14 Incidence and Severity of Respirator y Epithelial Degeneration in the Nasal Passages from Rats Exposed to BGVC ......................................................................... 3-25 3-15 Incidence and Severity of Olfactory Ep ithelial Degeneration in the Nasal Passages from Rats Exposed to BGVC ......................................................................................... 3-26

LIST OF APPENDICES Appendix A
STUDY PROTOCOL AND AMENDMENTS .......................................................................... A-1
Appendix B
ANALYSES OF BULK TEST SUBSTANCE ............................................................................B-1
Appendix C
RESULTS OF PERIODIC SERO LOGICAL EVALUATIONS .................................................C-1
Appendix D
RESULTS OF FEED AND WATER ANALYSES ................................................................... D-1 Appendix E
SUMMARIES OF BGVC ANALYSES IN EXPOSURE CHAMBERS AND EXPOSURE ROOM AND AEROSOL SCIENTIST’S REPORT .............................................. E-1
Appendix F
WEEKLY GAS CHROMATOGRAPHIC PROFI LES OF CHAMBER ATMOSPHERES ...... F-1 Appendix G
ENVIRONMENTAL DATA ...................................................................................................... G-1
Appendix H
INDIVIDUAL ANIMAL DISPOSITION .................................................................................. H-1
Appendix I
IN-LIFE BODY WEIGHTS ......................................................................................................... I-1
Appendix J
INDIVIDUAL ANIMAL SUMMA RIES OF CLINICAL OBSERVATIONS ............................ J-1
Appendix K
BODY AND ORGAN WEIGHT S AT NECROPSY ................................................................. K-1Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

x Appendix L
DIFFERENTIAL CELL COUNTS AND WHITE BLOOD CELL COUNT ESTIMATES ...... L-1
Appendix M
NEOPLASTIC AND NON-NEOPLASTIC LESIONS IN MALE RATS ................................. M-1 Appendix N
NEOPLASTIC AND NON-NEOPLASTIC LESIONS IN FEMALE RATS ............................ N-1
Appendix O
STATISTICIAN’S REPORT...................................................................................................... O-1
Appendix P
PATHOLOGIST’S REPORT ...................................................................................................... P-1Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

xi LABORATORY TITLE PAGE

211(b) CHRONIC CARCINOGENICITY STUDY
BASELINE GASOLINE VAPOR CONDENSATE (BGVC)

LRRI Study Number: FY01-027

Laboratory: Lovelace Respiratory Re search 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

Study Initiation Date: August 12, 2001

Study Completion Date: TBD

September 2009

Total number of pages: ___Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

xii KEY STUDY PERSONNEL

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

Veterinary Pathologist Thomas F. March, DVM, PhD, DACVP Lovelace Respiratory Research Institute

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

Laboratory Animal Veterinarian David G. Burt, DVM, ACLAM 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, BS Lovelace Respiratory Research Institute

SUBCONTRACTOR

Statistician Betty Skipper, PhD Department of Family and Community Medicine, University of New MexicoStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

xiii LABORATORY SIGNATURE PAGE

Study Number FY01-027
211(b) Chronic Carcinogenicity Study
Baseline Gasoline Vapor Condensate (BGVC)

Janet M. Benson, PhD, DABT Study Director Date

Edward B. Barr, MSEE Aerosol Scientist Date

Thomas H. March, DVM, PhD, DACVP Veterinary Pathologist (Clinical Pathology and Histopathology) Date

Betty Skipper, PhD Director, Biostatistics Department of Family and Community Medicine University of New Mexico DateStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

xiv LABORATORY QA STATEMENT

Study Title 211(b) Chronic Carcinogenicity Study – Baseline Gasoline Vapor Condensate (BGVC) LRRI Study Number FY01-027 Sponsor Study Number N/A Study Director J. Benson

This study was inspected by the LRRI Quality A ssurance 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
Miran spectrophotometer calibration R. Marr 17-Jul-01 18-Jul-01 18-Jul-01

Animal Conditioning, Chamber maintenance, Husbandry process D. Harris 3-Aug-01 12-Aug-01 12-Aug-01

Day -7 randomization. Body weights and observations D. Harris 6-Aug-01 12-Aug-01 12-Aug-01

Data inspection for conditioning room R. Marr 8-Aug-01 8-Aug-01 8-Aug-01

Protocol D. Harris 12-Aug-01 12-Aug-01 12-Aug-01
Day -1 body weights and observations D. Harris 12-Aug-01 12-Aug-01 12-Aug-01

TA logbook data audit R. Marr 18-Oct-01 18-Oct-01 18-Oct-01
Environmental, Miran and GC data audit R. Marr / D. Harris 18-Oct-01 30-Oct-01 30-Oct-01

Environmental data audit, Body weight and observation data, Miran calibrations R. Marr 4-March-02 7-Mar-02 7-Mar-02Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

xv Study Phase Performed by Date(s) of Inspection or Audit Testing Facility Management Study Director
Environmental data, exposure data, body weight and observation printouts R. Marr 2-May-02 6-May-02 6-May-02

Environmental data, exposure data, TA characterization, chamber atmosphere, TA use/fill logs, body weight and observation data, exposure chamber maps and checklists R. Marr 14-Aug-02 6-Sept-02 6-Sept-02

Body weight, observation and blood smear procedures R. Marr 16-Aug-02 21-Aug-02 21-Aug-02

IANR forms, body weight and observation data, environmental data, chamber atmosphere, exposure data, TA use/fill logs, Miran calibrations R. Marr 6-Nov-02 25-Nov-02 25-Nov-02

IANR forms, animal removal, chain of custody records, animal exposure room log, sick animal observations, exposure chamber checklist, chamber maps, environmental data, exposure data, body weight and observation data, chamber atmospheres R. Marr / D. Harris 19-Dec-02 20-Dec-02 20-Dec-02

Exposure data, TA, body weight and observations, chamber atmosphere, environmental data, Miran calibrations, IANR forms, GC data, animal removal records R. Marr 18-Mar-03 27-Mar-02 27-Mar-02Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

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

Terminal sacrifices D. Harris 12-Aug-03 19-Aug-03 19-Aug-03

Terminal sacrifices D. Harris 18-Aug-03 19-Aug-03 19-Aug-03

IANR and N module data J. Godbe 13-Nov-03 17-Nov-03 17-Nov-03
Draft final report D. Harris 17-Aug-04 2-Sept-04 2-Sept-04

Statistical Data C. Storch 28-Jan-09 9-Feb-09 9-Feb-09
Pathology Report C. Storch 27-Apr-09 13-May-09 13-May-09
Final Report Text C. Storch 4-May-09 13-May-09 13-May-09
Final Report C. Storch 30-Sept-09 30-Sept-09 30-Sept-09

Christa R. Storch, BS, RQAP-GLP Senior Quality Assurance Specialist LRRI Quality Assurance Unit DateStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

xvii LABORATORY COMPLIANCE STATEMENT

Study Number FY01-027
211(b) Chronic Carcinogenicity Study
Baseline Gasoline Vapor Condensate (BGVC)

This study was conducted in compliance with Al ternative Tier 2 testing requirements under Section 211(b) of the Clean Air Act and the EPA Health Effects Test Guidelines OPPTS 870.4200, “Carcinogenicity.” The stipulations of this protocol were implemented in conformance with EPA regulations as specifi ed in 40 CFR 79.60, “Good Laboratory Practices (GLP) Standards for Inhalation E xposure Health Effects Testing.”

The following were not conducted under GLP guidelines:

Serological health assessments were conducted throughout the study. Of the five assessments, two were conducted under GLP gui delines, three were not. This was because LRRI did not specify the need to run three of the shipment s GLP in our communications with BioReliance.

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

It was the Sponsor’s responsibility to maintain r ecords of the method of s ynthesis, fabrication, or
derivation of the test substance. The method of fabrication was not available at the time the study was initiated. It is, however, pr esently available with the sponsor.

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

Thomas M. Gray, MS, DABT Sponsor Representative American Petroleum Institute DateStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

S-1 SUMMARY

In compliance with the Clean Air Act Section 211( b) for fuel and fuel additive registration, the petroleum industry and oxygenate manufactur ers have conducted comparative chronic toxicology testing of evaporative emissions fr om unleaded gasoline (baseline gasoline vapor condensate [BGVC]) and unleaded gasoline containing MTBE (gasoline/MTBE vapor condensate [GMVC]) This is the fina l report of the BGVC chronic study.

Groups of 50 male/50 female CDF(F344)Crl BR rats were exposed in H2000 whole-body inhalation chambers at BGVC vapor concentrations of 2 g/m 3 (low level), 10 g/m 3 (mid level), and 20 g/m 3 (high level) 6 hours/day, 5 days/week for 104 weeks (518 exposure days). The overall mean exposure concentrations in each BGVC chamber were within 2% of target throughout the study, and the relative percentage s of the major hydrocarbon components of the condensate remained relatively constant throughout.

There were no clinical signs of t oxicity attributable to BGVC i nhalation. Survival of the BGVCexposed rats was not significantly different from that of control rats. Body weights of males in the mid- and high-level BGVC groups were sign ificantly below control values throughout the study. Body weights of high-level and mid-leve l females were below control values after approximately 1 and 3 months of exposure, re spectively. Body weights of low-level females were sporadically affected. At the final sacr ifice, body weights of high-level males and highand mid-level female rats were significantly be low control values. Among mid-level males, the absolute epididymis weight, percent epidi dymis-to-body and percent epididymis-to-brain weights were significantly belo w the corresponding control values. The adrenal-to-body and brain-to body weight values of high-level males were significantly greater than control values. The kidney weights of high- and mid-level female s were significantly increased compared to controls, however they did not co rrelate with kidney pathology fi ndings. The spleen weights of mid-level females were significantly increased comp ared to controls, which is likely a result of the high incidence of age-related mononuclear cel l leukemia (MCL). Among mid-level females, the percent organ-to-body weight values for brai n, heart, kidney, liver, lungs, and spleen were significantly greater than corres ponding control values. Among hi gh-level females, the percentStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

S-2 organ-to-body values for adrenals, brain, heart, li ver, kidney, and lungs were significantly greater than control values. The percent kidney-to-br ain weights for mid- and high-level females and the percent spleen-to-brain weight for the mid-le vel females were significantly greater than the corresponding control values. It is likely that many of the organ-to-body weights increases were artifacts of the treatment-related depression in body weight. For females, there were significantly fewer numbers of total WBCs, neutrophils, lymphoc ytes, eosinophils, and nucleated red blood cells (RBCs) in the high-level group at the 12 month time-point, only. In males, only the absolute number of lymphocyt es was significantly elevated in the high-level group at the final sacrifice time-point. Due to blood sampling difficulties at the 12 month time point and the high, age-related incidence of mononuclear cel l leukemia in all groups, the relationship of treatment to these changes in hematology valu es is unknown. BGVC exposure was associated with an increase in the incidence of respiratory epithelial degeneration and a significant decrease in the incidence of olfactory epithelial degeneration in the na sal passages of both male and female rats. The respiratory epithelial degenerati ve effects were most likely caused by the test
material.

Exposure did not enhance the incidence of chroni c progressive nephropathy in male or female rats, but did increase the severi ty of this lesion among mid- and high-level groups of males. The increased severity of chronic progressive nephropathy in males is consistent with previous studies on wholly vaporized unleaded gasoline, and is attributable to male rat specific alpha-2u globulin overload as a result of BG VC exposure. Consistent with previous studies and the alpha2u globulin overload mechanism of male rat kidn ey damage, BGVC induced increases in male renal adenoma (1/50 in control; 1/50 in 2 g/m 3 group; 4/50 in 10 g/m 3 group; and 0/50 in 20 g/m 3 group) and carcinoma (0/50 in controls; 0/50 in 2 g/m 3 group; 3/50 in 10 g/m 3 group; and 0/50 in 20 g/m 3 group). Although the incidences of renal adenomas and carcinomas taken separately were not statistically sign ificant, combined incidences for the control (1/50; 2%), low (1/50, 2%), mid (7/50, 14%) and high (0/50; 0%) exposur e groups were significant (p = 0.004, Fisher’s Exact Test). Pair-wise tests did not demonstrate any significa nt difference among the individual dose groups. These neoplasms may be considered to be exposure related. The highest incidence of renal neoplasms in males was in the 10 g/m 3 exposure group.Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

S-3 The incidence of squamous cell carcinoma in the nasal passages (turbinate levels 2-4) was elevated in the high-level group of male rats, with tumor incidence of 1/50, 3/50, and 3/50 for levels 2, 3 and 4, respectively. The level 3 inci dence gave a statistically significant trend for
increased incidence with incr eased exposure concen tration (p = 0.019, Cochran-Armitage [CA] test). Squamous cell carcinoma was noted in th e nasal specimens of only one female control rat of the current study. The squamous cell carcinom as in all of the male nasal specimens had morphologies consistent with or igination from the oral mucosa and had varying degrees of invasion into the nasal passages. By compar ison, in the concurrent Gasoline MTBE Vapor Condensate study (FY01-013), squam ous cell carcinoma of oral or igin occurred in one male control, two male GMVC low dose and two male high GMVC dose, but in no female rats.
While the fact that the nasal tumors were of oral origin might suggest that the inhalation exposure was not causal, spontaneous oral squam ous cell carcinomas in other studies have been nearly as rare as those originating from the nasal mucosa (~0.6%; Haseman et al., 1990; NTP, 1999). The historical incidence of squamous cell carcinoma of oral origin in a large cigarette smoke study conducted at LRRI in the 1990s was 0/118 (0%) in males and 3/119 (2.5% in females). Taken together, the data provide some evidence that the development of nasal squamous cell carcinomas in male rats may have resulted from test article exposure.

Similar to the incidence of renal neoplasms, th ere was an increased incidence of testicular mesothelioma in the mid-level group of male ra ts (4 of 50 examined). Fisher’s exact test demonstrated that this incidence was greater th an expected, but pair-w ise testing between the groups and trend tests for exposure concentration -response effects did not indicate statistically significant effects of exposure. The incidence in the mid-level group is approximately 3-fold greater than the average incidence in male c ontrol F344 rats exposed to air in inhalation toxicology studies (28 tumors in 1,055 rats; NTP, 1999) and may be regarded as an effect of exposure to the test substance. As with this study, there were no mesotheliomas found in the
testes or other tissues of the control male animals of the GMVC study (LRRI protocol FY01- 013), although there was a statistically non-significan t incidence (2 in 50 an imals or 4%) of the tumor in the testes of the high-level GMVC-exposed rats and sporadic incidence of the tumor in other tissues of GMVC-exposed male rats. The ab sence of testicular mesothelioma in controlStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

S-4 animals of the present study, where 1 or 2 tumors might normally be expected to be found in 50 animals (NTP, 1999), may have influenced the sta tistical analysis to i ndicate a significant exposure-related incidence in the mid-level BGVC -exposed male rats, when no causal increase is actually present.

There was a trend for increased incidence of t hyroid follicular cell carcinoma in male animals (p < 0.05, FE test over all groups; see Table 3-12 ), but there were no significant differences among the groups. There were two tumors in the 27 mid-dose glands examined and no tumors in any other male control or exposed group. Whethe r the incidence of thyroid follicular carcinoma was a causal effect of exposure to the test substa nce or a sporadic occurrence remains a question, especially given that there is no apparent e xposure concentration-response effect. Pair-wise testing suggests no statistically significant di fferences existed among the groups (p > 0.008, FE test). However, the incidence (7%) in the mid- level group of males is co nsiderably higher than that reported in control F344 rats exposed to air (average incidence of 1% in 21 studies, range of 0 carcinomas in 52 rats to 1 in 45; NTP, 1999) . While the incidence of thyroid follicular adenomas reported here are similar to or sligh tly higher than those pr eviously reported for control animals, the incidence of thyroid fo llicular carcinomas and adenomas combined are higher than what might be exp ected (NTP, 1999). However, ther e are no statistically significant differences in the combined incidence of thyr oid follicular adenomas and carcinomas among the exposure groups. In the concurrent Gasolin e MTBE Vapor Condensate study, there were no significant increases in thyroid follicular cell ad enomas, carcinomas, or combined adenomas and carcinomas in males or females. Thyroid follicu lar carcinoma might be considered to be caused by the exposure despite the lack of an appa rent exposure concentr ation-response effect.

In summary, chronic BGVC inhalation suppressed body weight in males, and to a greater extent in females, increased the severity of chroni c progressive nephropathy in males and caused epithelial degeneration in the nasa l passages of both sexes. The degenerative nasal effects were most likely caused by the test material. Ch ronic exposure to BGVC did not enhance the development of proliferative lesi ons (hyperplastic lesions, neoplasm s) in female rats. However,
there was an increased inciden ce in mononuclear cell leukemia in low- and mid-level exposed females, most likely due to protocol-driven sa mpling bias in these groups. Consistent withStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

S-5 previous studies and the conc urrent GMVC study, chronic expos ure to BGVC did enhance the development of renal adenomas and carcinomas in male rats. In contrast with the concurrent GMVC chronic study, BGVC may have enhanced squamous cell carcinoma in the nasal specimens, testicular mesothelioma, and thyroid follicular cell carcinoma. Consequently, due primarily to treatment-related effects in the male kidney, nose, testes, and thyroid, chronic
inhalation of Baseline Gasoline Va por Condensate was determined to be carcinogenic in male rats in this study. Chronic inhalation of Ba seline Gasoline Vapor Condensate was determined not to be carcinogenic in fe male rats in this study.Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

1-1 INTRODUCTION

The purpose of this study was to evaluate the chronic toxicity, potential carcinogenicity, and exposure concentration-response relationships a ssociated with inhalation of baseline gasoline vapor condensate (BGVC). The study was conduc ted 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 870.4200m, “Car cinogenicity.” The stipulatio ns of this protocol were implemented in conformance with EPA re gulations as specified in 40CFR 79.60, “Good Laboratory Practices (GLP) Standards for Inhala tion Exposure Health Effects Testing.” The study protocol and amendments are provided in Appendix A.Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

2-1 MATERIALS, METHODS, AND PROCEDURES

TEST SUBSTANCE
Identification, Source, and Storage
BGVC was prepared and supplied in 420- pound and 20-pound gas cylinders by Chevron Research and Technology Center (CRTC; Richmond, CA). Two lots of BGVC were used in the study. API 99-01 was used from the first day of exposures through the week of May 19, 2003. Starting the week of May 27, 2003 and running thr ough the last day of exposures, API 02-08 was used. It was necessary to prepare a second lot of test material because the first attempt to conduct this study was aborted due to inaccura te dose calibrations resulting in chamber concentrations up to 40% greate r than target concentrations. While there were some minor
differences in the relative concentrations of various alkanes and isoalkane components between the lots, these differences did not influen ce the outcome of the study (see Table 2-1). Original
characterization of both lots of the test substance was performed by CRTC and ExxonMobil Biomedical Sciences, Inc. (EMBSI), Annandale, NJ.

Table 2-1. Analytical Compar ison of Test Material Lots

Compound BGVC Lot # API 99-01 (area percent) BGVC Lot # API 02-08 (area percent) Isobutane 3.6 2.1 n-Butane 15.2 19.9 Isopentane 35.1 32.0 n-Pentane 13.2 5.4 Trans-2-pentene 2.5 2.4 2-Methyl-2-butene 3.8 3.3 2,3-Dimethylbutane 1.6 6.2 2-Methylpentane 6.3 9.6 3-Methylpentane 3.6 6.8 n-Hexane 3.0 1.2 Methylcyclopentane 1.5 0.7 2,4-Dimethylpentane 1.0 0.6 Benzene 2.1 1.9 2-Methylhexane 1.1 1.6Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

2-2 Table 2-1. Analytical Comparison of Test Mate rial Lots (Concluded)

Compound BGVC Lot # API 99-01 (area percent) BGVC Lot # API 02-08 (area percent) 2,3-Dimethylpentane 1.1 1.0 3-Methylhexane 1.3 2.2 Isooctane 1.3 0.7 Toluene 3.0 2.6

Twenty-pound cylinders and some 420-pound cylinders were stored at ambient temperature in a storage building dedicated for that purpose at LRRI. The remaining 420-pound cylinders were stored in an outside, controlled area at ambient temperature. Th e test substance was transferred, as needed, from the 420-pound to the 20-pound cyli nders. Only authorized personnel were allowed access to the test substance. Receipt, us e, and inventory of this test substance were
documented.

Analysis
Before dispensing BGVC from each 420-pound tank, a sample was removed from the tank and analyzed by gas chromatography (GC) with flam e ionization detection (FID) at LRRI using a Shimadzu Model GC-17A/FID (Shimadzu Scientif ic Instruments, Columbia, MD). The gas chromatographic profile of the 18 major peak s (retention time and relative peak area) was compared with that originally determined for the BGVC by ExxonMobil Biomedical Sciences, Inc. (EMBSI, Annandale, NJ). Tank BG22, used September 24 thru November 18, 2002, was inadvertently not analyzed prior to use. Add itionally, there were differences in the relative
concentrations of various alka nes and isoalkane components be tween the two lots of test material. The absence of analysis of tank BG22 and test material lot API 99-01 vs. lot API 02-08 were considered minor issues and were judged to have no significant effect on the outcome of the study. 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 st udy with the analysis of the exposure chamberStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

2-3 atmospheres and each #420 pound tank before its use. Results, provided in Appendix E, showed
it to be stable for th e duration of the study.

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

ANIMALS AND ANIMAL ASSIGNMENTS
Animal Receipt, Housing, and Quarantine
Four hundred-forty CDF(F344)CrlBR rats (5–6 week s old when received) were purchased from Charles River Laboratories (Raleigh, NC). The rats were examined by a veterinarian upon their arrival. All animals were quarantined a nd acclimated to whole-body inhalation chambers for20 days. Healthy animals were randomly a ssigned by weight to the core exposure groups (400 total; 50 rats/sex/exposure level). The weig ht range of male rats assigned to study at randomization 152.0–194.4 g. For females, the weight range at randomization was 112.2– 138.7 g. Following randomization, the rats assigned to 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 be fore exposures began to evaluate their health
status as an indicator of the health status of the populati on on study. The remaining unassigned rats were euthanized. Receipt and initiation of e xposures of male and female rats were staggered by one week.

Animal Disease Screening Program
Five male and five female rats not assigned to study were sacrificed, bled and received a complete necropsy. No gross lesions were f ound, and tissues were subsequently discarded because there were no subsequent questions rega rding the health stats of the animals on study.
Sera from these rats were submitted to BioRelia nce, Rockville, MD, for analysis of antibodies common to rodents. The following tests were run: Cilia-Associated Respiratory Bacillus (CARB), Mycoplasma pulmonis (M. PUI), Pneum onia virus of mice (PVM), Rat Coronavirus/ Sialodacryoadenitis Virus (RCV/SDA), Reovirus (Reo), Sendai Virus (Sendai), Lymphocytic Choriomenengitis Virus (LCM), Parvovirus (Parvo) , Toolan’s H-1 Virus (H-1), and Kilham rat Virus (KRV). The five male and five female sen tinel rats in the control chamber were bled retro-Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

2-4 orbitally after 26, 52, and 78 weeks of exposure. Blood was also obtained from sentinels surviving to 104 weeks. Sera were analyzed by BioReliance, as described above. The data are provided in Appendix C.

Justification of Test Animals
Rats were used in this study because of the larg e database available on th e inhalation toxicity and carcinogenicity of toxic material s in rats. The study design was justified because it provided exposure concentration-response information on the possible carcinogenic ity associated with repeated inhalation of BGVC.

Environmental Conditions
The rats were housed 24 hours per day during qua rantine and exposure in Hazleton 2000 wholebody inhalation chambers. Initially, all rats were housed separately in 3.8-inch wide by 11-inch long by 8-inch high compartments within stainless steel baskets. When the 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 week ly. The cage racks were rotated clockwise weekly when the rats were transferred from the dirt y to the clean chambers.

The chambers were held at appr oximately 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 temper atures were maintained at 20o to 24oC. Temperature, relative humidity, and chamber ai r flow rates were continuously monitored, 24 hours per day. Values for the three parameters were recorded at 30-mi nute intervals. Oxygen
concentration in the chambers was maintained at 19%. This parameter was monitored, but not
recorded.

A 12-hour light/dark cycle was maintained with li ghts on at 0600. Light levels in the exposure
room and noise levels in the cham bers were determined periodically.Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

2-5 Diet and Drinking Water
Unlimited municipal tap water was available at all times. Rats were fed Teklad Certified Rodent Diet (8728C; Harlan Teklad, Madison, WI). F ood was available at all times except during the daily exposure period. Certif ied 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 pestic ides. Analytes were either present at nondetectable concentrations or sufficiently low con centrations as to not interfere with the outcome of the study. Feed and water analyses results are provided in Appendix D.

EXPERIMENTAL DESIGN
Group Assignment
The experimental design is shown in Table 2-2.

Table 2-2. 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 14 days (males and females) Age When Placed on Study 7 to 8 weeks Study Dates Study Initiation Date August 12, 2001 Initiation of Exposures (Males) August 13, 2001 Initiation of Exposure (Females) August 20, 2001 Last Exposure Day (Males) August 8, 2003 Last Exposure Day (Females) August 15, 2003 Final Sacrifice (Males) August 12–15, 2003 Final Sacrifice (Females) August 18–22, 2003 Target Exposure Concentrations Control 0 mg/m 3 BGVC Low Level 2 g/m 3 BGVC Mid Level 10 g/m 3 BGVC High Level 20 g/m 3 BGVCStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

2-6 Table 2-2. Experiment al Design (Concluded) Animal Identification Scheme(by tail tattoo) Control I001-I050 (M); I051-I100 (F) Low Level J101-J150 (M); J151-J200 (F) Mid Level K201-K250 (M); K251-K300 (F) High Level L301-L350 (M); L351-L400 (F) Exposure Duration 6 hours/day, 5 days/week for 104 weeks (518 exposure days) In-Life Monitoring Daily obs ervations for morbidity and mortality. Weekly body weights for first 13 weeks, then every 4 th week. Weekly detailed clinical observations. Sentinel Animal Bleeds for Serology Prior to study start and at six-month intervals thereafter, including prior to final sacrifice. Hematology Evaluations (Control and High-Level Exposure Groups) At 12 and 18 months and at final sacrifice.

Histopathology All protocol-re quired tissues collected.

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 usi ng a computerized data acquisition system (Path-Tox ® ; Xybion, Cedar Knolls, NJ) operated according to LRRI Standard Operating Procedures (SOPs).

Exposure System
A schematic of the vapor exposure system is shown in Figure 2-1.Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

2-7 Gasoline Baseline Vapor Condensate Vapor Generator

Sight Bowl Bypass To Main Exhaust

Duct BGVC Vapor

and Air Mixture To CBR2

Bench Scale

Gasoline Baseline Vapor Condensate P

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

Pneumatic Shutoff Valve Pressure Gauge

BGVC Rotameter Nitrogen Rotameter

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

Mid Level Chamber Supply Air

High Level Chamber Supply Air BGVC Vapor and Air Mixture To CBR3

BGVC Vapor

and Air Mixture To CBR4 Dilution

Tee Liquid BGVC Line

Nitrogen Line BGVC 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)

BGVC Rotameter BGVC Rotameter

Figure 2-1. Vapor Genera tion System for BGVC

The daily supply of BGVC for each exposure chamber was contained in 20-pound gas storage cylinders. Exposure atmospheres were generated by controlling the flow of pressurized BGVC through a rotameter, into a heated stainless stee l transfer line where the BGVC was completely vaporized. Chamber concentrations were contro lled by adjusting the flow rates of the BGVC
and dilution air rate. Chamber exhaust was carri ed to an oxidizer on the roof of the exposure facility where it was burned.

Pre-Test Characterization . The exposure system was tested prior to animal exposures. Characterization included the following: 1) uniform ity of the distribution of total vapor within each chamber; 2) within-day and between-day stabili ty of vapor concentration; 3) within-day and between-day consistency of the hydrocarbon profile as determined by gas chromatography; andStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

2-8 4) determination of the time for vapor concentration to achieve 90% of the equilibrium target value (T90). The exposure atmosphere in the anim als’ breathing zone was also examined for the presence of aerosol particles us ing a TSI Scanning Mobility Pa rticle Sizer (TSI Industries, Shoreview, MN).

Once, prior to the initiation of an imal exposures, the concentrations of the test substance in the generator control box and in the exposure room we re determined to ensure that the generator containment hood was operating satisfactorily.

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

Quantitation of Exposure Atmospheres . Vapor concentrations in the exposure chambers were continuously monitored using Miran 1A infrared analyzers (Foxboro Wilks, Foxboro, CT). The high-, mid-, and low- level exposure chambers we re each monitored with their own analyzer. The analyzers for the high-, mid-, and low-le vel chambers underwent weekly five-point calibrations and daily one-point cal ibration checks using te st 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 1A was calibrated approximately quarterly until December 2001, and then once before exposures ended. The Mi ran 1A analyzers dedicated to the mid- and high-level chambers were calibrated over a range of 6–35 g/m 3 . The Miran 1A analyzers for the
control chamber and the low-le vel chamber were calibrated ove r a concentration range of 1–7 g/m 3

. The absorbance signals were recorded continuousl y. Average values were obtained for the first, second and third 2-hour segments of each exposure day. The mean of the three values from each exposure chamber was reported as the day’s e xposure concentration for that chamber.

Qualitative Assessment of Exposure Atmospheres . The qualitative composition of the exposure atmosphere in each chamber was determined w eekly by gas chromatography using a Shimadzu Model GC-17A/FID. The percen t peak area of each of 18 ma jor components was determined and recorded. The retention times of eight re presentative components were verified againstStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

2-9 EMBSI values each week and, one-point performa nce qualifications were performed on any day in which chamber profiles were analyzed. The one-point check was perf ormed using a certified standard of 840 ppm butane in N 2 (Matheson Tri-Gas, Irving, TX).

Determination of Nominal Concentration . Daily nominal or “anticip ated” usage was calculated by multiplying the average BGVC concentra tion in each chamber (low, mid, high; g/m 3 ) by the total flow through each respective chamber ([L/min * min]/1000 m 3 ) and then summing 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 each

exposure.
Concentration of Test Substance in the Exposure Room . Concentration of the test substance in the exposure room was determined at approximate ly 60-day intervals using a Miran 1A Infrared Spectrometer (Foxboro Wilks, Foxboro, CT) operated using the same settings as were used to
monitor the low-leve l 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 (518 exposur e days). The following observations and measurements were made during the dosing phase.

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

Body Weight . All animals were individually weighed using the Path-Tox ® data acquisition system (Xybion, Cedar Knolls, NJ) on study Day -7 (to randomly assi gn rats to groups by weight), Day -1, weekly for 13 weeks, and then every 4 weeks thereafter.

Clinical Signs of Toxicity . Thorough clinical examinations were made at randomization, on Day -1, and weekly thereafter. Observations we re detailed and carefully recorded using Path-Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

2-10 Tox ® software. Observations includ ed evaluations of skin and fur; eyes and mucous membranes; respiratory and circulatory eff ects; autonomic effects such as salivation and central nervous system effects, including tremors and convulsions, and changes in the level of activity, gate, and 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 sa crifice, 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 from the cont rol and high-level-expos ed rats. At final sacrifice, blood was obtained by cardiac puncture from all rats and collected into Vacuta iner tubes containing ethylenediamine tetra-acetic acid (EDTA). Pa rameters evaluated included total leukocyte estimates (WBC), nucleated red blood cell counts (cells/100 WBC), anisocytosis (0 to 4+), polychromasia (1+ to 4+), and relative differe ntial 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, eosinophi ls, basophils, atypical lymphocytes and blastocytes using the formul a WBC*relative percent/100.

Post-Exposure Endpoints

Gross Necropsy . A complete gross examination was perf ormed on all animals at final sacrifice and on those animals that died naturally or were sacrificed in a moribund condition. Sacrifices of rats surviving 518 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 2-3.Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

2-11 Table 2-3. Organs and Representati ve Samples Taken for Examination Cardiovascular/Hematopoietic System 1. Aorta 2. Bone marrow (and/or fresh aspirate) 3. Heart 4. Lymph nodes (mandibular, mesent eric, bronchial, mediastinal) 5. Spleen 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 Nervous System 1. Brain (including sections of medu lla/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) Respiratory System 1. Larynx 2. Lung (infused with fixative) 3. Nose 4. Paranasal sinuses 5. Pharynx (evaluated with larynx) 6. TracheaStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

2-12 Table 2-3. Organs and Representative Sa mples Taken for Examination (Concluded) Urogenital System 1. Epididymides 2. Female mammary gland 3. Kidneys 4. Ovaries 5. Prostate 6. Seminal vesicle(s) 7. Testes 8. Urinary bladder (infused with fixative) 9. Uterus

All study animals received a complete necropsy. Animals were euthanized with an overdose of intraperitoneally injected barbiturate anesthetic (Euthasol ® , Virbac AH Inc., Fort Worth, TX). Body weights and fresh organ weig hts were collected on lungs, liv er, kidneys, adrenals, testes, epididymides, ovaries, uterus, spleen, brain and heart of final sacrific e and moribund sacrifice animals. Animals found dead received a comple te necropsy with tissue collection, but blood and organ weight data were not rout inely collected. Cardiac blood was collected from animals at
final sacrifice for determination of total and di fferential white blood cell counts. Gross lesions, body weights, and organ weights we re entered on pre-prepared forms and then the information
was recorded on the Path-Tox ® database.

Lungs were gently instilled via the trachea w ith 10% neutral buffered formalin (NBF) to approximate normal volume. Organs and tissu es were immersion fixed in 10% NBF for subsequent histopathologic exam ination. 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/m 3 ) animals, high-level (20 g/m 3 ) animals, and dead or moribund animals of all groups. Respiratory tissues (lungs, larynx, trachea and nasal turbinate sectio ns), potential target tissues (testes, kidneys of males) and gross lesions from non-target tissues were examined histologically from final sacrifice low-level (2 g/m 3 ) and mid-level (10 g/m 3 ) animals. Nomenclature of proliferative lesions was based on the intern ational harmonized nomenclature recommended by
the Rat Nomenclature Reconciliation Subcommitt ee of the Society of Toxicologic PathologistsStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

2-13 (see http://www.toxpath.org; Standardized Rat Nome nclature). Nomenclature of other lesions was routine, widely understood usage (see Boorman et al., 1990).

Statistics
Body and Organ Weights . Group mean body weight, organ weight, percent organ-to-body weight, and percent organ-to-brain weight data were tested for st atistical significance using PathTox ® software. After testing for an overall tre nd 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 m odified Dunnett’s test. If data were nonhomogeneous, group differences were assessed usi ng a modified t-test. Significance levels were set at p ≤ 0.05.

Survival Analysis . The probability of survival was es timated by the Kaplan-Meier product-limit method using PROC LIFETEST in SAS Version 8.2 (SAS Institute, Cary, NC). Mean numbers of survival days and time to 25% mortality were estimated for each exposure group by the PROC LIFETEST program. Log-rank tests were used to test the hypothesis that there are differences among the four groups for each sex. The significa nce level was set at p = 0.05. All reported pvalues 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 because the distri butions are highly skewed for some variables. Preliminary analyses were done using the ge neralized estimating equation approach for longitudinal data analysis because there were three time points. For some variables these analyses showed significant in teractions between time and group. Therefore, analyses were performed to compare exposure groups at each time point and to compare time points within each exposure group. The Krusal-Wallis test was used for these comparisons.

Histopathology . The incidences of all neoplastic a nd non-neoplastic lesions are given as the
ratio of the number of affected animals to the number of an imals with the site examined microscopically. Three statis tical evaluations were perfor med on the histopathology lesion incidence data: 1) Cochran-Armitage test, whic h tests whether the incide nce of lesions shows a trend across exposure groups; 2) logi stic regression test that take s death date into account whenStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

2-14 assessing the presence of an exposure-dependent trend; and 3) the Fish er’s exact test, which compares incidences among the four exposure gr oups. The two-sided significance level was set at p = 0.05. If a significant diffe rence was detected by the Fisher’s exact test, six possible pairwise comparisons were calculated. Using the Bonferroni correction for pair-wise comparisons, each pair-wise comparison would be considered significant if p < 0.008.

Fisher’s exact test and the Cochran-Armitage test do not use survival information and are appropriate in situations where su rvival is similar among exposure groups as is the case for this study. The Fisher’s exact test tests the null hypoth esis of equality of prevalences across exposure groups against the alternate hypot hesis that the prev alences are not equa l while the CochranArmitage test tests the null hypothesis of equa lity across exposures against the alternate hypothesis of a monotonic increa sing or decreasing trend.

Additionally, differences between groups with re gard to both the severity and incidence of nonproliferative lesions were analyzed by the Ko lmogorov-Smirnov two-sample , one-tailed test as performed by the Path-Tox system ( c.f. , Algorithms used in Path/Tox System 4.2.2; Vol. I, Chap. 7, pp. 24-25). These analyses were performed by the pathologist. The significance level was set at p = 0.05.Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

3-1 RESULTS AND CONCLUSIONS

EXPOSURE ATMOSPHERE

Pre-Test Chamber Trials
Three-Day Stability Evaluati on and Nominal BGVC Usage . Before exposures were initiated the system was operated for 6 hours/day for 3 consecu tive days. The concentration of vapor in each chamber was close to target and constant w ithin 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 BGVC usage/anticipated usage (nominal usage) on the 3 days of stability testing were 96, 95, and 96% for Days 1, 2, and 3, respectively (see Appendix E).

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

Target Exposure Concentration (g/m 3 )/

Chamber Number Achieved Concentration ± SD (n = 3) Day 1 (7/23/01) Day 2 (7/24/01) Day 3 (7/25/01) 2 (Chamber 2) 2.03 ± 0.03 2.07 ± 0.04 2.09 ± 0.09 10 (Chamber 3) 9.64 ± 0.47 9.21 ± 0.13 10.0 ± 0.29 20 (Chamber 4) 20.3 ± 0.26 20.7 ± 0.96 20.6 ± 0.89

Homogeneity of Vapor Concentration . The homogeneity of vapor concentration throughout the exposure chambers was determined by c onducting a chamber distribution study. The
concentrations of BGVC were measured from four sampling ports locat ed in front and four sampling ports located in the back of the cham bers during a pre-test chamber trial day and compared to the concentration obtained in a re ference location. The spatial variation measured in Chambers 2 (2 g/m 3 ), 3 (10 g/m 3 ) and 4 (20 g/m 3 ) was 1.6%, 1.0%, and 0.2%, respectively. These results indicate the vapor is distributed evenly within each of the chambers (Appendix E).

T90 Determination . The amounts of time required to reach T90 were 14.2, 12.1, and 13.7 minutes for Chambers 2, 3, and 4, respectively. A T90 of 14 minutes was chosen for use with this study. Daily exposures periods to BGVC were then 6 hours and 14 minutes.Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

3-2 Confirmation of the Absence of Aerosols . The particle concentrations of air inside Chamber 4, operated at a target concentration of 20 g BGVC/m 3 and the control chamber (Chamber 1) were determined using a TSI Aerodynamic Particle Sizer . There was less than one particle per cubic centimeter air in the control and high-level chambers, indicating that there were no BGVC 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 BGVC/m 3 vapors are provided in Table 3-2. The overall achieved means were within 2% of targ et for each exposure concentration. Thus, all future references to exposure c oncentration are in terms of target concentration. Concentrations of vapor in the control chamber were below the lowest concentration on th e standard curve used
to calibrate the control chamber Miran 1A analyzer (1 g BGVC/m 3 ). The overall average of the daily percent nominal usage is 98% ± 6%, indicat ing excellent agreement between anticipated and actual BGVC usage. Daily vapo r concentrations and nominal us age are provided in Appendix E.

Table 3-2. Summary of BG VC Vapor Concentrations a Target g BGVC/m 3 Achieved Concentrations Percent of Target
0 < 1 b NA
2 2.0 ± 0.07 101
10 10.1 ± 0.34 101
20 20.3 ± 0.74 101

a Results are the mean ± SD of va por concentrations obtained in 518 exposure days (combined days for males and females). b Concentrations of “zero” were obtained on all but 13 exposure

days. On these 13 days, the r eadings were below the lowest concentration on the standard curv e used to calibrate the control chamber Miran 1A (1 g BGVC/m 3 ).
Homogeneity of Vapor Concentration . An evaluation of the homogeneity of vapor concentration in each BGVC chamber was conducted after animal exposures began. The spatial variations measured in Chambers 2 (2 g/m 3 ), 3 (10 g/m 3 ), and 4 (20 g/m 3 ) were 0.49%, 0.59%, and 5.39%, respectively. These results indicate the vapor was di stributed evenly within each of the chambers.Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

3-3 Gas Chromatographic Profiles of BGVC 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 18 major components thro ughout the study. The peak areas of the 18 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 values were compared to the reference values provided by EMBSI. Profiles remained acceptably constant throughout the study. Weekly result s are provided in Appendix F.

Chamber Environmental Data . The temperature, relative humidity, chamber flow rate, and pressures were maintained within acceptable ra nges with only occasional excursions outside acceptable limits. Environmental data are provided in Appendix G.

Survival Analysis . There were 50 animals per group for males and 48 animals per group for the females. The following eight females were omitted from the survival analysis because they were removed from the study for reasons not related to the BGVC exposure: I055 (malocclusion); I074 (malocclusion), J176 (missing), J184 (nose injury), K274 (nose injury), K298 (accidental trauma), L356 (nose injury), and L357 (nose injury).

Figures 3-1 and 3-2 show the survival curves fo r male and female animals, respectively. The differences among the groups are no t statistically signifi cant for male (p = 0.24) or female (p = 0.53) animals. Table 3-3 shows means, standard errors, and day of 25% mortality for male and female animals under each experimental condition. The day of 25% mortality is the day of the study at which 25% of the animals had died in each group. This provides a way of comparing mortality at a single time point am ong groups within and between se xes. For the calculation of mean and standard errors of survival times, the la st day of the study was used as the value of the
survival time for animals that were sacrificed at the time that the study was terminated. Therefore, the means and standard errors may be underestimated since so me of the animals may have lived longer if the protocol had included following them fo r a longer period of time. The
results indicate that chronic BGVC inhalation di d not shorten the lifespan of the exposed rats compared to the controls.Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

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

Figure 3-2. Survival of Female AnimalsStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

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

Male Female

Group Mean Days of Survival (SE) Estimated Day of 25% Mortality Mean Days of Survival (SE) Estimated Day of 25% Mortality
Control 660.5 (10.2) 613 686.8 (11.4) 680

Low 666.5 (12.1) 607 642.9 (24.1) 619
Mid 679.9 (11.6) 642 674.8 (12.2) 640
High 669.6 (8.4) 621 678.9 (12.6) 708

Animal Disposition . The disposition of all animals on study is provided in Tables 3-4. A small percentage of animals died naturally. A ma jority of males underwen t moribund sacrifices. Individual animal disposition is provided in Appendix H.

Table 3-4. Disposit ion of Rats on Study

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

Male Female Male Female Ma le Female Male Female

Moribund Sacrifice 30 17 25 18 19 17 26 12

Natural Death 2 2 3 3 4 4 7 3

Final Sacrifice 18 29 22 27 26 27 17 33

Other 0 2 a 0 2 b,c 1 d 2 c,e 0 2 c

Total 50 50 50 50 50 50 50 50

a Malocclusion. b Missing, presumed dead.

c Nose injury. d Accidental removal through misidentificat ion (included in survival analysis).

e Accidental death due to trauma.Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

3-6 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. All animals gained we ight during the study. Ho wever, body weights of males in the mid- and high-level BGVC exposure groups were significantly below control values throughout the study. Body weights of high-level females were below control weights from approximately Day 31 to the end of the study. Body weights of mid-level females were significantly below control values from Day 122 to the end of the study (except days 290, 318 and 378), while body weights of low-level females we re only sporadically below control values. At terminal sacrifice, male percent of trea ted: control weights we re 99.3%, 93.4% and 91.7% for low-, mid- and high-level groups, respectively. At terminal sacrifice, female percent treated:control weights were 97.4%, 91.2% and 89.6% for low-, mid- and high-level groups, respectively. Individual animal body weights are provided in Appendix I.

Clinical Signs of Toxicity . Rats exposed to BGVC displayed no clinical signs of toxicity-related BGVC exposure. As this was a chronic toxic ity evaluation, many of the observations observed during the second year of the study were relate d to aging (mammary masses, jaundice). A summary of clinical observations by e xposure group is provided in Appendix J.

Final Body Weights
Body weights at final sacrifice of high-level male and mid- and high- level female rats were significantly below control values (Table 3-7). Indi vidual animal data are provided in Appendix K.

Absolute Organ Weights at Final Sacrifice
Only the mean epididymis weights of mid-leve l males were significantly different (reduced) from the mean control values. The kidney weig hts for mid- and high-level female rats were significantly greater than the mean control valu es, however they did not correlate with kidney pathology findings. In addition the spleen weig ht of mid-level females was significantly increased compared to controls. The mean valu e was strongly influenced by a few large spleens (>10 g) from animals having m ononuclear leukemia. The eff ects on absolute organ weights
were not considered to be treatment related. Summary statistics are provided in Table 3-8. Individual animal data are provided in Appendix K-1.Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

3-7 Table 3-5. Body Weight Summary for Male Rats a

Study Day Control 2 g/m 3 Percent Control (2 g/m 3 ) 10 g/m 3 Percent Control (10 g/m 3 ) 20 g/m 3 Percent Control (20 g/m 3 ) -1 174.5 173.1 99.2 172.1 98.6 173.1 99.2 10 207.0 206.0 99.5 203.6 98.4 202.3 97.7 17 221.8 223.6 100.8 215.4 97.1 213.4 96.2 24 233.9 234.9 100.4 226.4 96.8 222.6 95.2 31 237.4 241.6 101.8 230.8 97.2 227.2 95.7 38 246.7 249.6 101.2 238.3 96.6 233.6 94.7 45 257.0 262.4 102.1 249.1 96.9 242.6 94.4 52 266.3 271.8 102.1 258.2 97.0 249.3 93.6 59 273.3 278.3 101.8 264.3 96.7 254.1 93.0 66 281.8 283.1 100.5 271.4 96.3 261.7 92.9 73 291.3 293.2 100.7 281.8 96.7 271.4 93.2 80 302.7 302.3 99.9 293.1 96.8 281.8 93.1 87 311.8 310.8 99.7 300.0 96.2 288.6 92.6 94 316.3 316.3 100.0 304.6 96.3 294.5 93.1 122 340.5 336.5 98.8 325.7 95.7 314.7 92.4 150 358.0 355.0 99.2 341.1 95.3 331.6 92.6 178 369.5 367.0 99.3 354.6 96.0 342.0 92.6 206 379.1 375.1 98.9 366.4 96.6 355.3 93.7 234 393.7 390.8 99.3 380.4 96.6 367.9 93.4 262 406.3 398.3 98.0 389.7 95.9 376.9 92.8 290 411.8 407.9 99.1 395.8 96.1 384.7 93.4 318 422.8 416.1 98.4 400.8 94.8 393.9 93.2 346 425.0 417.0 98.1 406.6 95.7 392.6 92.4 374 428.0 422.2 98.6 409.8 95.7 393.4 91.9 405 437.1 433.0 99.1 416.7 95.3 400.8 91.7 433 440.9 437.3 99.2 418.6 94.9 404.3 91.7 458 427.9 425.3 99.4 405.6 94.8 393.6 92.0 486 428.7 421.4 98.3 402.9 94.0 390.3 91.0 514 431.5 424.9 98.5 404.2 93.7 389.8 90.3 542 424.1 418.8 98.8 400.8 94.5 386.0 91.0 570 419.5 415.4 99.0 397.1 94.7 382.5 91.2 598 422.7 414.9 98.2 396.7 93.8 382.8 90.6 626 418.2 411.7 98.4 395.4 94.5 377.5 90.3 661 402.9 398.8 99.0 381.6 94.7 369.6 91.7 682 400.3 395.9 98.9 377.8 94.4 359.9 89.9 710 392.2 389.6 99.3 366.4 93.4 359.5 91.7

a Bolded and italicized values are significantly di fferent from controls at that time point; p ≤ 0.05. Group mean body weight, organ weight, and percent organ-to -body weight data were tested for statistical significance using Path-Tox ® software. After testing for an overall trend among test groups by an analysis of variance, Bartlett’s test was used to establish the homogeneity of the data. If the data were homogeneous, group differences were evaluated using a modified D unnett’s test. If data we re non-homogeneous, group differences were assessed using a modified t-test. Significance levels were set at p ≤ 0.05.Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

3-8 Table 3-6. Body Weight Summary for Female Rats a

Study Day Control 2 g/m 3 Percent Control (2 g/m 3 ) 10 g/m 3 Percent Control (10 g/m 3 ) 20 g/m 3 Percent Control (20 g/m 3 ) -1 126.8 126.2 99.5 125.0 98.6 124.5 98.2 10 135.8 136.4 100.4 135.6 99.9 133.6 98.4 17 141.5 143.5 101.4 141.0 99.7 138.5 97.9 24 144.8 148.1 102.3 144.5 99.8 143.0 98.8 31 149.9 152.0 101.4 148.7 99.2 145.7 97.2 38 153.7 157.1 102.2 151.9 98.8 150.0 97.6 45 157.1 161.1 102.5 155.3 98.9 153.7 97.8 52 161.4 163.9 101.5 156.2 96.8 153.8 95.3 59 163.0 167.0 102.5 160.8 98.7 158.7 97.4 66 168.0 173.0 103.0 166.7 99.2 163.5 97.3 73 171.8 173.6 101.1 169.7 98.8 167.5 97.5 80 174.2 177.1 101.7 171.9 98.7 169.0 97.0 87 177.7 180.5 101.6 174.6 98.3 171.1 96.3 94 179.0 182.5 102.0 176.8 98.8 173.1 96.7 122 187.3 187.7 100.2 182.8 97.6 179.0 95.6 150 192.6 193.5 100.5 188.5 97.9 184.9 96.0 178 197.5 196.9 99.7 192.2 97.3 187.3 94.8 206 204.9 206.7 100.9 200.2 97.7 193.7 94.5 234 206.8 204.2 98.7 200.3 96.9 194.6 94.1 262 206.4 206.6 100.1 196.8 95.5 196.6 95.3 290 211.4 211.2 99.9 206.8 97.8 199.9 94.6 318 215.1 214.2 99.6 209.9 97.6 205.3 95.4 346 221.2 219.7 99.3 213.0 96.3 207.4 93.8 378 228.2 227.2 99.6 222.0 97.3 216.8 95.0 405 236.6 235.9 99.7 226.7 95.8 222.0 93.8 431 237.6 233.0 98.1 227.1 95.6 221.3 93.1 458 241.0 235.2 97.6 225.4 93.5 220.5 91.5 486 245.6 237.5 96.7 229.2 93.3 218.7 89.0 514 252.7 241.6 95.6 232.9 92.2 227.3 89.9 542 260.0 247.0 95.0 238.4 91.7 231.2 88.9 570 260.6 252.0 96.7 244.1 93.7 233.8 89.7 598 269.1 255.9 95.1 250.7 93.2 240.4 89.3 626 270.5 259.0 95.7 249.8 92.3 240.0 88.7 654 270.5 259.7 96.0 249.4 92.2 243.6 90.1 682 273.1 263.4 96.4 251.6 92.1 242.4 88.8 710 274.3 267.2 97.4 250.1 91.2 245.8 89.6

a Bolded & 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 D unnett’s test. If data we re non-homogeneous, group differences were assessed using a modified t-test. Significance levels were set at p ≤ 0.05.Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

3-9 Figure 3-3. Body Weights of Ma le Rats Exposed to BGVC

Figure 3-4. Body Weights of Female Rats Exposed to BGVCStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

3-10 Table 3-7. Summary Statistics for Terminal Body Weights of Male and Female Rats Control 2 g BGVC/m 3 10 g BGVC/m 3 20 g BGVC/m 3
Males
Mean ± SD 377.5 ± 31.3 388.3 ± 24.0 357.8 ± 36.3 341.9 ± 60.7 a
n 18 22 26 17 Females
Mean ± SD 273.9 ± 20.5 261.5 ± 24.1 247.8 ± 22.4 b 251.0 ± 19.4 b
n 29 27 27 33

a Mean significantly different from control; da ta nonhomogeneous by Bartlett’s test. Means

compared using a Modified t test. b Mean significantly different from control; da ta homogeneous by Bar tlett’s test. Means

compared using Dunnett’s test of significance.Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

3-11Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

3-12 Percent Organ-to-Body Weight at Final Sacrifice
In males the mean percent epididymis-to-body we ight was significantly lower than the control value for the mid-level group. The percent adre nal-to-body and brain-to- body weight values of high-level males were significantly greater than control values. Among mid-level females, the percent organ-to-body weight values for brain, he art, kidney, liver, l ungs, and spleen were significantly greater than the valu e for controls. Among high leve l females, the percents organto-body weight for adrenals, brai n, heart, liver, kidney, and lungs were significantly greater than controls. However, there was not a trend for increasing organ:body weight ratio with increasing exposure level, suggesting the effect was not trea tment related. Summary statistics are provided
in Table 3-9. Individual anim al data are provided in K-2.

Percent Organ-to-Brain Weight at Final Sacrifice
There were few effects on percent organ weights since there were no differences in absolute brain weights between control and treated animals. The patterns of effects were similar to those reported above for the absolute organ weights. For males, the percent epididymis-to-brain weight was significantly lo wer for the mid-level group than for the controls.

The percent kidney-to-brain weight for mid- a nd high-level females and the percent spleen-tobrain weight for the mid-level females were sign ificantly greater than the corresponding control values. However, there was not a trend for increasing organ-to-brain weight ratio with increasing exposure level, suggesting the effect wa s not treatment related. Summary statistics are provided in Table 3-10. Individual animal data are provided in K-3.

Terminal Body Weight and Organ Wei ghts of Euthanized (Moribund) Rats
As the rats reached about 20 months of age (18 m onths on study), mortality due to the effects of aging increased. Most animals not surviving to th e 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 statisti cal significance of differences among exposed and control groups is not as straightforward as with data obtained at the fi nal sacrifice. Summary statistics and individual animal terminal body an d organ weight data ar e provided in Appendices K-4 through K-6.Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

3-13Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

3-14Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

3-15 The absolute kidney weights of low- and high-leve l males were significantly greater than control, while the epididymis weight for high-level males was significantly below control. Similarly, the percent kidney-to-body weight wa s significantly increased for th e high-level males, and the percent testes-to-body weight was significantly increased among mid- and high-level males. The percent kidney-to-brain weight was significantly increased for hi gh-level males. The percent epididymis-to-brain weight was significantly decr eased in high-level males. There were no effects on terminal body or organ weights in fema les. Since cause of death of euthanized animals was not determined, the significance of th e changes that did not correlate with terminal
sacrifice data is unknown.

Hematology
Percentage and estimated absolute numbers of white blood cells are presented in Appendix L. As might be expected, there ar e numerous, statistically signifi cant changes over time in WBC counts in the control and high- level groups (Table 3-11). These may be regarded as less important than differences between the groups at a given time-point, in that such time-related changes are expected to occur with aging a nd concomitant development of degenerative, inflammatory, and neoplastic (e.g., mononuclear cel l leukemia [MCL]) cha nges. Statistically significant interactive e ffects between time and exposure were not assessed.

For females, there are significantly fewer numbers of total WBCs, neutrophils, lymphocytes, eosinophils, and nucleated red bl ood cells (RBCs) in the high-lev el group at the 12 month timepoint (estimated absolute numbers; p ≤ 0.05, Kruskal-Wallis test). The significantly decreased numbers at 12 months may not be an indication of myelosuppre ssion in the high-level exposure groups, in that the controls ha ve slightly elevated total W BC and absolute lymphocyte counts from what might be expected in a group of normal female F344 rats of this age. The results in the 12 animals possibly were affected by the tail blood sampling technique, which improved by
18 months. At 12 months we ha d difficulty sampling blood from the tail; that may have resulted
in artifactual results. At 18 m onths and at the final sacrifice, there are no significant differences between the groups of females in any of the es timated WBC numbers. Overall, the relationship of treatment with the changes observed in females at 12 months is unknown.Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

3-16Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

3-17 Only the absolute number of lymphocytes is sign ificantly elevated in the high-level male group at the final sacrifice time-point (p = 0.01, Krus kal-Wallis test) in male rats. The range of lymphocyte numbers at this time-point , however, is very large (0.6 × 10 3 to 346.5 × 10 3 cells/μL
for controls and 0.9 × 10 3 cells/μL to 189.6 × 10 3 cells/μL for high level) a nd may be attributable
to the incidence of MCL in these aged animals. This is corroborated by the enlarged number of atypical lymphocytes at the final sacrifice tim e-point. Given that BGVC exposure did not definitively enhance splenic involvement with MC L (below), the relationship of treatment to lymphocyte differences is unknown.

Individual animal data are provided in Appendix L.

Histopathology
Proliferative Changes . There was an increased incidence of renal tubule adenoma and carcinoma in males that peaked at the mid-dose level with incidences of 4/50 and 3/ 50, respectively. These results, presented in Table 3-12, are consistent with previous studies on wholly vaporized unleaded gasoline (MacFarland et al., 1984). Al though these results taken individually are not statistically different from control values in the present study, the combined adenoma + carcinoma incidences are significant (p = 0.004, Fisher’s Exact [FE] test, Table 3-12, and Appendix O). Pair-wise tests di d not demonstrate any significant differences between groups in
the combined incidence of renal adenoma + carci noma (p > 0.008), although th is incidence in the mid-level exposure group compared to the in the high-level exposure group approached the significance level (p = 0.01, FE test with Bonferroni’s correction: c.f. Appendix O). The reason for the lack of renal neoplasms in the high- level group of male ra ts is unknown. These neoplasms may be considered to be exposure-re lated. For compounds inducing renal tumors in male rats through increased alpha 2u-globulin accumulation and subsequent increased renal tubule cell turnover, the human heal th relevance of the mechanism, and thus, the tumors, is often regarded as suspect (Baetcke et al., 1991; Hard, 1998; Hard a nd Khan, 2004). Of an ancillary note, however, is that increased severity of the hydrocarbon-induced alpha 2u-globulin nephropathy, which is assessable only at early time-points in an exposure, is not always predictive of increased incidence of renal neoplasia in rats ex posed to hydrocarbons for 2 years or more. Rather, the severity of chronic progressive nephropathy may be correlated moreStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

3-18 reliably with the incidence of renal neoplasms in carcinogenesi s bioassays of light hydrocarbon exposure (Doi et al., 2007).

Table 3-12. Summary of Sel ect Proliferative Lesions in Male and Female Rats

Tissue Diagnosis Control (0 g/m 3 ) Low (2 g/m 3 ) Mid (10 g/m 3 ) High (20 g/m 3 )
MALES Kidney No. examined 50 50 50 50 Adenoma, renal tubule 1 (2%) 1 (2%) 4 (8%) 0 (0%) Carcinoma, renal tubule 0 (0 %) 0 (0%) 3 (6%) 0 (0%) Renal tubule adenoma and carcinoma, combined a 1 (2%) 1 (2%) 7 (14%) 0 (0%)

Nasal Passages (Note that the same tumor may occur at more than one level.) Turbinate Level 2 No. examined 50 50 50 50 Carcinoma, squamous cell 0 (0%) 0 (0%) 0 (0%) 1 (2%) Turbinate Level 3 No. examined 50 50 49 50 Carcinoma, squamous cell b 0 (0%) 0 (0%) 0 (0%) 3 (6%) Turbinate Level 4 No. examined 50 50 49 50 Carcinoma, squamous cell 0 (0%) 1 (2%) 0 (0%) 3 (6%)

Testes No. examined 50 49 50 50 Mesothelioma, malignant c 0 (0%) 0 (0%) 4 (8%) 0 (0%) Adenoma, interstitial cell d 48 (96%) 46 (94%) 50 (100%) 49 (98%)
Thyroid No. examined 50 29 27 50 Hyperplasia, follicular cell e, f Avg. severity 1 (2%) 0.0 0 (0%) 0.0 2 (7%) 0.1 6 (12%) 0.2 Adenoma, follicular cell 0 (0%) 2 (7%) 0 (0%) 2 (4%) Carcinoma, follicular cell g 0 (0%) 0 (0%) 2 (7%) 0 (0%) Follicular cell adenoma and carcinoma, combined h 0 (0%) 2 (7%) 2 (7%) 2 (4%)

Spleen No. examined 50 34 38 50 Leukemia, mononuclear cell 32 ( 64%) 23 (68%) 25 (66%) 32 (64%)Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

3-19 Table 3-12. Summary of Select Proliferative Lesions in Ma le and Female Rats (Concluded)

Tissue Diagnosis Control (0 g/m 3 ) Low (2 g/m 3 ) Mid (10 g/m 3 ) High (20 g/m 3 ) FEMALES Spleen No. examined 50 25 32 50 Leukemia, mononuclear cell i 13 (26%) 14 (56%) 18 (56%) 15 (30%)

a Incidences are different than expected by chance (p = 0.004, overall FE test), although pair-wise

comparisons are not significant (p > 0.008, FE tests with Bonferroni’s correction; c.f. , Appendix O). b Significant trend for increased incidence with in creasing exposure level (p < 0.019, CA). Note

that mid-dose turbinate levels 3 and 4 for one anim al (K221) were autolytic, resulting in an n of 49.

c Incidences are different than expected by chance (p = 0.014, overall FE test), although pair-wise

comparisons are not signif icant (p > 0.008, FE tests). d Incidences are not different than expect ed by chance (p = 0.26, overall FE test).

e Average for all animals examined (both affected and unaffected). f There is a significant increasi ng trend with exposur e concentration (CA; p = 0.018; Logistic regression p = 0.040).
g Incidences are different than expected by chance (p = 0.029, overall FE test), although pair-wise

comparisons are not signif icant (p > 0.008, FE tests). h Incidences are not different than expect ed by chance (p = 0.14, overall FE test). i Incidences are different than expected by chance (p = 0.007, overall FE test), although pair-wise

comparisons are not signif icant (p > 0.008, FE tests).

The incidence of squamous cell carcinoma in the nasal passages (turbinate levels 2-4) was elevated in the high-level group of male rats, with tumor incidence of 1/50, 3/50, and 3/50 for levels 2, 3 and 4, respectively. It was not appr opriate to combine carcinoma incidence of nasal turbinate levels for statistical analysis in th e high dose group because the lesion spanned two or
three levels in the same three animals. The leve l 3 incidence gave a sta tistically significant trend
for increased incidence with increased exposure concentratio n (p = 0.019, Cochran-Armitage [CA] test). Squamous cell carcinoma was noted in the nasal specimens of only one female control rat of the current study. The squamous cell carcinomas in all of the male nasal specimens had morphologies consistent with origination from the oral mucosa and had varying degrees of invasion into the nasal passages. By compar ison, in the concurrent Gasoline MTBE Vapor Condensate study (FY01-013), squam ous cell carcinoma of oral or igin occurred in one male control, two male GMVC low dose and two male high GMVC dose, but in no female rats.
While the fact that the nasal tumors were of oral origin might suggest that the inhalationStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

3-20 exposure was not causal, spontaneous oral squam ous cell carcinomas in other studies have been nearly as rare as those originating from the nasal mucosa (~ 0.6%; Haseman et al., 1990; NTP, 1999). The historical incidence of squamous cell carcinoma of oral origin in a large cigarette smoke study conducted at LRRI in the 1990s was 0/118 (0%) in males and 3/119 (2.5% in females). Taken together, the data provide some evidence that the development of nasal squamous cell carcinomas in male rats may have resulted from test article exposure.

Similar to the incidence of renal neoplasms, th ere was an increased incidence of testicular mesothelioma in the mid-level group of male rats (4 of 50 examined; Table 3-12). Fisher’s exact test demonstrated that this incidence was greater than expected, but pair-wise testing between the groups and trend tests for exposure concentration -response effects did not indicate statistically significant effects of exposure. The reason for the lack of an exposure concentration-response effect are not known. However, the incidence in the mid-level group is approximately 3-fold greater than the average incidence in male c ontrol F344 rats exposed to air in inhalation toxicology studies (28 tumors in 1,055 rats or 2.7%; NTP, 1999) and may be regarded as an effect of exposure to the test su bstance. Mesothelioma was also evident in sections of a variety of abdominal organs from male rats and was regarded to be the result of metastasis, in that all but one of these (epididymidal mesothelioma; one of 28 epididymides examined in the low-level group) occurred in the mid-level group of male rats. Inciden ces of mesothelioma in the duodenum (2 of 23 examined), jejunum (3 of 23), co lon (3 of 24), pancreas (3 of 24), spleen (3 of 38), epididymis (3 of 25), prostate gland (2 of 25), and seminal vesicl e (3 of 25) of the midlevel male rats were statistically significant by FE tests. Most mesotheliomas are thought to originate in the tunica vaginalis of the scrotu m (Hall, 1990), and those found in this study are consistent with an origination from this investme nt of the testes. As with this study, there were
no mesotheliomas found in the testes or other ti ssues of the control male animals of the GMVC study (LRRI protocol FY01-013), a lthough there was a statistically non-significant incidence (2 in 50 animals or 4%) of the tumor in the te stes of the high-level GMVC-exposed rats and sporadic incidence of the tumor in other tissues of GMVC-exposed male rats. The absence of
testicular mesothelioma in control animals of the present study, where 1 or 2 tumors might normally be expected to be found in 50 animals (NTP, 1999), may have infl uenced the statisticalStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

3-21 analysis to indicate a significant exposure-rel ated incidence in the mid-level BGVC-exposed male rats, when no causal increase is actually present.

There was a trend for increased incidence of t hyroid follicular cell carcinoma in male animals (p < 0.05, FE test over all groups; see Table 3-12). There were two tumors in the 27 mid-dose glands examined and no tumors in any other male control or exposed group. Whether the incidence of thyroid follicular carcinoma was a cau sal effect of exposure to the test substance or a sporadic occurrence remains a question, especially given that there is no apparent exposure concentration-response effect. Pair-wise testing suggests no stat istically significant differences existed among the groups (p > 0.008, FE test). However, the incidence (7%) in the mid-level group of males is considerably higher than that reported in co ntrol F344 rats exposed to air (average incidence of 1% in 21 studies, range of 0 carcinomas in 52 rats to 1 in 45; NTP, 1999). Thyroid follicular epithelial ade nomas were also present in the low-level (2 of 29 examined) and high-level groups of male rats (2 of 50 examined ; see Table 3-12), although the incidences were not statistically significant. While the incidence of thyroid follicular adenomas reported here are similar to or slightly higher than those previous ly reported for control an imals, the incidence of thyroid follicular carcinomas and adenomas comb ined are higher than what might be expected (NTP, 1999). However, there are no statistical ly significant differences in the combined incidence of thyroid follicular adenomas a nd carcinomas among the exposure groups (Table 3-12). In the concurrent Gasoline MTBE Va por Condensate study, ther e were no significant increases in thyroid follicul ar cell adenomas, carcinomas, or combined adenomas and carcinomas in males or females. Thyroid follicu lar carcinoma might be considered to be caused by the exposure despite the lack of an apparent exposure concentra tion-response effect. Further, follicular epithelial hyperplasia at minimal to mild severity was noted in male rats of the control, mid-, and high-level groups (2–12% incidence) and also displaye d an increased incidence with increasing exposure level (CA p- value = 0.018; logistic p-value = 0.040). This also suggested that thyroid follicular proliferative lesions in the male animals were caused by the exposure.

Despite the proliferative lesions in the thyroid glands of male rats, the releva nce of the test article exposure to human risk might be questioned, given that mech anisms of chemical thyroid carcinogenesis are believed to be different between humans and rodents (U.S. EnvironmentalStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

3-22 Protection Agency, 1998). Mutation of thyroid follicular cell DNA may lead directly to cancer and is the only mechanism verified to be carcino genic in humans, but rodents are believed to be more susceptible to carcinogenic processes involvi ng stimulation of thyroid follicular cell growth through disruption of pituitary-thyroid hormonal physiology. Mutagenesis of thyroid follicular cells and hormone disruption were not evaluated in the current study. Other proliferative (and nonproliferative) lesions occurred which were not clearly signifi cant or were judged to have confounding factors which must be considered in interpretation. In males these included mononuclear cell leukemia (MCL) in several tissues with the spleen consid ered to be the tissue of origin where there was no difference between control and treatment group incidence and mesothelioma affecting the spleen and segments of the gastrointestinal and genitourinary tract.
Male rats of the low- and mid-dose levels had a statistically significan t higher incidence of keratoacanthomas in the skin (3 tumors in 29 rats and 3 in 26, resp ectively). However, there is a lack of a dose-response for thes e incidences, and most likely a re sult of protocol-driven gross sampling bias in non-target organ tissues where onl y tissues with gross lesi ons were evaluated in the low- and mid-dose groups at terminal sacrif ice. In females, th ere was a statistically significantly increased in cidence of splenic MCL in the low- and mid-dose groups. This finding was considered not to be treatment-related, but ra ther attributable to gross sampling bias in the low- and mid-dose groups. Additionally, some findings were decreased in exposed animals compared to controls, these are not generally discussed, as their significance with respect to
treatment is unknown.

Histopathology summaries and individual animal data for males and females are provided in Appendices M and N, respectivel y. Statistical evaluation of lesions is provided in the statistician’s report, Appendix O. The pat hologist’s report is pr ovided in Appendix P.

Nonproliferative Changes . Kidneys of nearly all male rats on study have evidence of chronic progressive nephropathy, and th ere is no significant differenc e in the incidence among the groups. However, overall combination of inciden ce and severity of the lesion is significantly worse in the mid- and high-level exposure groups (p ≤ 0.05, Kolmogorov-Smirnoff test, Table 3-13). This finding in males is consistent with male-rat sp ecific light hydrocarbon-induced alpha-2u globulin overload nephropat hy that has been reported previ ously in studies with wholly vaporized unleaded gasoline. It should be noted that in F344 male ra ts, after approximatelyStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

3-23 1 year of age, alpha-2u globulin overload neph ropathy is essentially masked by age-related chronic progressive nephropathy, since both forms of nephropathy sh are similar if not identical diagnostic hallmarks. Therefore the effects alpha-2u globulin overloa d nephropathy typically manifests itself in increased severity, rather than incidence, since essentially all males surviving past 1 year of age will develop CPN. Thus, the statistically significant worsening of chronic progressive nephropathy in male rats with higher levels of exposure is consistent with an effect induced by the test substance.

Table 3-13. Incidence and Severity of Ch ronic Progressive Nephr opathy in Male and Female Rats Exposed to BGVC Control Low Level Mid Level High Level Males No. examined 50 50 50 50 No. with lesion (%) 49 (98) 49 (98) 50 (100) a 50 (100) a No. with minimal grade 1 (%) 6 (12) 4 (8) 3 (6) 2 (4) No. with mild grade 2 (%) 20 (40) 18 (36) 9 (18) 10 (20) No. with moderate grade 3 (%) 20 (40) 24 (48) 28 (56) 27 (54) No. with marked grade 4 (%) 3 (6) 3 (6) 10 (20) 11 (22) Average severity grade 2.4 2.5 2.9 2.9

Females No. examined 50 24 25 50 No. with lesion (%) 27 (54) 12 (50) 16 (64) 32 (64) No. with minimal grade 1 (%) 21 (42) 3 (12.5) 10 (40) 22 (44) No. with mild grade 2 (%) 6 (12) 6 (25) 5 (20) 9 (18) No. with moderate grade 3 (%) 0 2 (8.3) 1 (4) 1 (2) No. with marked grade 4 (%) 0 1 (4.2) 0 0 Average severity grade 0.7 1.0 0.9 0.9

a Overall severity/incidence is significantlygreater than control using the KolmorogovSmirnoff test, p ≤ 0.05.

The incidence and severity of ch ronic progressive nephropathy in female rats was much less than in males and was not significantly different from th at of the control group of female rats (Table 3-13). Renal findings in female s that are part of the conste llation of chronic progressive nephropathy, including cortical t ubular epithelial regeneration and corticomedullary tubular protein accumulation, had increased incidence with increasing exposure level (p < 0.05, CA and logistics tests; Appendix P). Th erefore, even though overall seve rity scores were not different from controls, at least some renal findings in fe males are consistent with enhancement of chronic progressive nephropathy with increasing exposure level.Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

3-24 Degeneration of the nasal epithelium, charac terized primarily by accum ulation of globular, homogeneous, brightly eosinophilic material in the cytoplasm of respiratory and/or olfactory epithelial cells, is present at minimal-to-moderate severity in all groups of animals (Tables 3-14 and 3-15). This is a common finding in aged rats, but its severity or inci dence can be enhanced by inhalation exposure to irritant s or toxicants. The finding is somewhat sporadic, but its incidence in BGVC-exposed male ra ts is significantly greater in the respiratory epithelium of the nasal passages at turbinate leve l 2 (approximately at the level of the incisive papilla) with a significant trend for increased incidence with increasing level of exposure (p < 0.005, CochranArmitage and logistics tests, Ta ble 3-14). The combination of incidence and severity of the lesion at turbinate level 2, however, is not si gnificantly different among the groups. Difference in incidence of the degenerative change in re spiratory epithelium at turbinate level 3 (second palatal ridge) of male rats is al so statistically significant (p = 0.007, Fisher’s exact test over all groups), but the CA and logistic trend test s do not demonstrate any significance among the groups. The incidence of the lesion in the mid- level group is significantly greater than in the low-level group (Table 3-14).

The incidence of nasal respiratory epithelial dege neration for female rats is somewhat greater than that of the males suggesting that this find ing is more of a female-specific lesion (Table 3-14). Higher incidences with significant trends for increases with increas ing exposure level of the respiratory epithelial lesion were noted at both turbinate levels 2 and 3 of the female rats. The incidences at turbinate leve ls 2 and 3 in the high-level group were significantly greater than the control and low-level groups. The incidence at turbinate level 3 in the mid-level group was significantly greater than in the low-level group. The combination of incidence and severity of the respiratory epithelial lesion was significantly greater only in the female high-level exposure group and only at turbinate level 2. The incidence and/or severity of the lesion in both sexes are likely induced by exposure to the test substance.

Interestingly, BGVC exposure caused a statistica lly significant exposure- related decrease in the degeneration of olfactory epithelium in turbinate levels 3 and 4 (level of the first molar) in the nasal passages from male rats (Table 3-15). At turbinate level 4, the incidence in the high-level male group was significantly less than in the co ntrol and low-level groups. This exposureresponse relationship was also present in the fema les at turbinate level 4 (Table 3-15), where the incidence in the mid- and high-level rats was less than in controls. This suggests that the BGVC exposure was associated with some protection of the olfactory epith elium. Given that olfactory epithelium contains relatively large amounts of inducible toxicant metabolizing enzymes (e.g.,Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

3-25 CYP450, glutathione- S -transferases, carboxyl esterases), ther e is a possibility that these cells over time became relatively resistant to toxic or irritant effects of BGVC exposure because of metabolizing enzyme induction and enhanced detoxification capacity.

Table 3-14. Incidence and Severity of Respiratory Epithelial Degeneration in the Nasal Passages from Rats Exposed to BGVC Control Low Level Mid Level High Level Males, Turbinate Level 2 No. examined 50 50 50 50 No. with lesion (%) 2 (4) 3 (6) 7 (14) 11 (22) a No. with minimal grade 1 (%) 2 (4) 3 (6) 4 (8) 10 (20) No. with mild grade 2 (%) 0 0 1 (2) 1 (2) No. with moderate grade 3 (%) 0 0 1 (2) 0 Average severity grade 0 0.1 0.2 0.2 Males, Turbinate Level 3 No. examined 50 50 49 50 No. with lesion (%) 1 (2) 0 7 (14) b 2 (4) No. with minimal grade 1 (%) 0 0 5 (10) 2 (4) No. with mild grade 2 (%) 1 (2) 0 2 (4) 0 Average severity grade 0 -- 0.2 0 Females, Turbinate Level 2 No. examined 49 49 50 50 No. with lesion (%) 8 (16) 11 (22) 18 (36) 26 (52) a,c, d No. with minimal grade 1 (%) 6 (12) 8 (16) 10 (20) 15 (30) No. with mild grade 2 (%) 2 (4) 3 (6) 7 (14) 10 (20) No. with moderate grade 3 (%) 0 0 1 (2) 1 (2) Average severity grade 0.2 0.3 0.5 0.8 Females, Turbinate Level 3 No. examined 49 49 50 50 No. with lesion (%) 2 (4) 0 8 (16) e 12 (24) a, d No. with minimal grade 1 (%) 2 (4) 0 6 (12) 5 (10) No. with mild grade 2 (%) 0 0 2 (4) 7 (14) Average severity grade 0 -- 0.2 0.4

a Significant trend for increased incidence with increasing exposure level (p < 0.005,

Cochran-Armitage and logistics tests). b Significantly different from the low-level gr oup (p = 0.006, pair-wise Fi sher’s exact test).

c Overall severity/incidence is significantlygreater than control (p < 0.05, KolmorogovSmirnoff test). d Significantly different from the control and lo w-level groups (p < 0.008, Fi sher’s exact test).

e Significantly different from the low-leve l group (p < 0.008, Fisher’s exact test).Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

3-26 Table 3-15. Incidence and Severity of Olfactory Epithelial Degeneration in the Nasal Passages from Rats Exposed to BGVC Control Low Level Mid Level High Level Males, Turbinate Level 3 No. examined 50 50 49 50 No. with lesion (%) 15 (30) 3 (6) 7 (14) a 4 (8) b No. with minimal grade 1 (%) 14 (28) 1 (2) 6 (12) 4 (8) No. with mild grade 2 (%) 1 (2) 2 (4) 1 (2) 0 Average severity grade 0.3 0.1 0.2 0.1
Males, Turbinate Level 4 No. examined 50 50 49 50 No. with lesion (%) 18 (36) 10 (20) 6 (12) 1 (2) b ,c, d No. with minimal grade 1 (%) 17 (34) 10 (20) 2 (4) 1 (2) No. with mild grade 2 (%) 1 (2) 0 4 (8) 0 Average severity grade 0.4 0.2 0.2 0
Females, Turbinate Level 3 No. examined 49 49 50 50 No. with lesion (%) 15 (31) 13 (27) 11 (22) 13 (26) No. with minimal grade 1 (%) 15 (31) 11 (23) 9 (18) 11 (22) No. with mild grade 2 (%) 0 2 (4) 2 (4) 2 (4) Average severity grade 0.3 0.3 0.3 0.3
Females, Turbinate Level 4 No. examined 49 49 50 49 No. with lesion (%) 21 (43) 10 (20) 7 (14) e 2 (4) b ,c,e No. with minimal grade 1 (%) 20 (41) 10(20) 6 (12) 2 (4) No. with mild grade 2 (%) 1 (2) 0 1 (2) 0 Average severity grade 0.4 0.2 0.2 0

a Significantly greater than the low-leve l group (p < 0.008, Fisher’s exact test). b Significant trend for decreased incidence with increasing exposure level (p < 0.005, CochranArmitage and logistics tests).

c Overall severity/incidence is significantly less than control (p < 0.05, Kolmorogov-Smirnoff

test). d Significantly less than control and low-level groups (p < 0.008, Fisher’s exact test).

e Significantly less than control (p < 0.008, Fisher’s exact test).Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

3-27 CONCLUSIONS
The overall purpose of this study was to ev aluate the long-term toxicity, including carcinogenicity of Baseline Gaso line Vapor Condensate. The re sults of this study provide a basis for comparing the results of a compani on study in which the vapor condensate contained 20% by weight of the additive methyl tertiary-b utyl ether, “211(b) Chronic Carcinogenicity Study Gasoline MTBE Vapor Condensate.”

Chronic BGVC inhalation suppressed body weight in males, and to a greater extent in females, increased the severity of chronic progressive nephropathy in males and caused epithelial degeneration in the nasal passage s of both sexes. The degenera tive nasal effects were most likely caused by effects of the test material.

Chronic exposure to BGVC did not enhance the development of proliferative lesions (hyperplastic lesions, neoplasms) in female rats. However, there was an increased incidence in
mononuclear cell leukemia in low- and mid-level e xposed females, most likely due to protocoldriven sampling bias in these groups. Consiste nt with previous studies and the concurrent GMVC study chronic exposure to BGVC did enha nce the development of renal adenomas and carcinomas (combined) in male rats. In cont rast with the concurre nt GMVC study, BGVC may also have enhanced squamous cell carcinoma in the nasal passages in male rats, testicular mesothelioma. Also, in contrast to the GM VC study, there is some evidence that BGVC exposure caused thyroid follicular cell carcinoma in males.

Consequently, due primarily to treatment-relate d effects in the male kidney, and to possible effects on testes, and nose, and thyroid, ch ronic inhalation of Ba seline Gasoline Vapor Condensate was determined to be carcinogenic in male rats in this study. Chronic inhalation of Baseline Gasoline Vapor Condensate was determined not to be carcinogenic in female rats in this study.Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

4-1 LOCATION OF SPECIMENS, RA W DATA, AND FINAL REPORT

Specimens were identified by test system, study, na ture, and date of collec tion. All raw data and records and specimens that are required to recons truct the study will be maintained in the LRRI archives for 10 years. The Sponsor will be notif ied and will authorize in writing the destruction or transfer of any specimens, raw data, and st udy records to the Sponsor or to an archive specified by the Sponsor’s C ontracting Representative.

PROTOCOL DEVIATIONS

Calculation of Nominal Concentration
Section IX.C.6. of the protocol states “Nominal concentration will be determined daily and will reflect the concentration generate d for all three exposure systems co mbined. Therefore, the total mass of test substance generated wi ll be divided by the total flow distributed to all three exposure chambers. The 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: The daily nominal usage was calculated by multiplying the average BGVC concentration in each chamber by the total flow through each chamber and then summing the valu es 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 expo sure. There is no impact because the way nominal concentration was calculated and compar ed to the actual B GVC usage provides the same answer as the method described in the protocol.

Environmental Conditions
Throughout the BGVC exposure study, environmen tal conditions in the animal exposure chambers deviated periodically fr om protocol required ranges, or data were not acquired because of problems with the monitoring system. Most excu rsions or periods of data loss were of short duration. These deviations are judged to have no significant effect on the outcome of the study. Tables summarizing deviations fr om protocol-specified environm ental conditions are included in the Study File.Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

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

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

Temporary Loss of Environmental Monitoring System Operation
On May 20, 2003 a major power outage occurred at 12:20 a.m. Normally, in power outage events, emergency backup diesel generators are turned on within 10–15 seconds. During this particular outage, a breaker was tripped a nd emergency power was not provided to the environmental monitoring system and the syst em was on uninterrupted power supply battery power until they were drained. During this outag e, the supply air fan stopped operating, but due 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 ope rational and pulled air from the room into theStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

4-3 chambers. Several actions, described in a memo to the study file, were taken to prevent similar
situations in the future.

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

Light and Sound Measurements
According to the study protocol, light and sound measurements in the animal exposure room were to be measured quarterly. In 2001, measur ements 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 be tween measurements that were made.

Gas Chromatographic Analysis
The original version of LRRI SOP ASP-1166.0, “C alibration of the Shim adzu GC-17A/FID for Analyses of Gasoline Vapor Condensate,” indica ted GC calibration for monitoring the exposure atmosphere and test article hydrocarbon compos ition profile be performed daily. However, calibrations were performed only on the days when analyses were conducted. The SOP was modified and signed by management Sept ember 25, 2002, changing the requirement for calibration to only days when analyses are perf ormed. The impact of this SOP deviation is minimal because the GC was calibrated on days when analyses were performed.

No chamber composition sample was analyzed during the week of September 10, 2001 due to problems with the gas chromatograph. The impact of this deviation was judged to be minimal.

Cage Board Usage/Availability
In December 2002, we ran out of untreated cage board used to line the excreta pans in the H2000 chambers. Although additional cage board wa s ordered, it was on “back order” and wasStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

4-4 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 as sociated 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 in stitute for short-term and long-term inhalation
studies.

Collection of Organ Weights at Necropsy
The study protocol states that lung, liver, kidneys, testes, epid idymis, 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. Organ weights were recorded, however, for the following rats found dead: J165, J179, J19, K259, K271, and K285. 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. The lung weight was not obtained on one female, L380, at final sacrifice. The impact of these deviations are minimal.

Assignment of Severity Scores
LRRI SOP PAN-0455.7, “Rodent Necr opsy Procedure,” Section 6.2, states, “For animals that are entered into the Path/Tox computer system, the lesions are described and graded on a scale of 1 to 4....” There were several instances of severity codes not assigned to lesions that were masses or nodules (examples are K54, L390, L383, L 363, I576, K265, K251, and L399). There is no impact on the quality or integrity of the st udy because other descriptors of the masses are provided.

Statistics
Proposed statistical methods were presented in the protocol. In several cases the actual evaluations differed from those orig inally proposed. This occurred in three specific cases. First,Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

4-5 the Hoel Walburg test was not used to analyze tu mor data although originally listed as a possible statistic in the protocol. Sec ond, hematology results were analyzed as a function of time as well as a function of dose. Finally, in the case of chronic progressive nephropathy, where the incidence of lesions was similar among cont rol and dosed groups, the Kolmogorov-Smirnov test
was used to assess differences in severity scores. This evalua tion was done within the P module
of the Path-Tox ® software, and not by the Study Statistician.

The actual statistics used have been described in the Statistics section of the report. The fact that the statistics proposed in the prot ocol were not always those used for data analysis has no major impact on the quality of the study. In most cases the analyses were more thorough and appropriate than those originally proposed. Since BGVC 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 ha ve normal distributions, alternative statistical approaches were warranted. Since repeated m easurements were made on the same rats over time, evaluation of the effect of time and e xposure concentration on the hematology parameters was appropriate. The use of the Kolm ogorov-Smirnov statistic in the Path-Tox ® database was appropriate because nephropathy was an important endpoint in this study. St atistical analysis of the severity of the lesion as a function of exposure concen tration in the absence of a concentration-response effect on lesion incidence provided more information than would have been gained by analysis of incidence alone.

Neat Test Article Characterization of 420-pound Tanks
Test article lot number API 99-01, tank BG22, was inadvertently not anal yzed prior to use September 24 thru November 18, 2002. However, the tank was analyzed during use for the inhalation exposures and the composition was verifi ed. For this reason, this deviation did not affect the validity of the study.

Personnel
On page 3 of the study protocol, key personnel wa s listed as Quint H. Powell, MS, as Inhalation Engineer. 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. Betty J. Skipper,Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

4-6 PhD, Professor, Department of Family and Community Medicine (MSC 095040, 1 University of New Mexico, Albuquerque, NM, 87131), was subc ontracted to perform the statistics.

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

Tissue Collection
Nasal tissue and pituitary (protocol required tissues ) were inadvertently not collected for animal I093 (female control). Tissue was not present at trim, so likely not collected at necropsy tissue
harvest. This omission had little eff ect on the overall outcome of the study.

Hematology
Due to blood sampling difficulties at the 12 month sampling time, blood smears were not
obtained for male animals I011 and L321 or for female animals I072, I074, L353, L355, L356, L358, L362, L371, L3673, and L378. At the 18 mont h time point, no blood smear was collected
from males I011 and L321 or from females I072 and I074.

Loss of Supply Air to Exposure System
On November 27, 2001, at approxim ately 10:30 a.m., supply airflow to the animal chambers dropped, resulting in low exposure chamber flows and pressures. Surveillance personnel were summoned to the room. They monitored the room closely and made certain Facility Engineering and Exposure personnel were aware of the situation. It was de termined that the flows and pressures were in a range that would not adversel y affect the health of the animals and therefore the animals remained within the chambers during the night.

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

On the following morning, November 28, 2001, the Study Engineer and Facility Engineering personnel examined the problem. Flows to th e chamber were adjusted to within normalStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

4-7 operating parameters. Facility Engineering pers onnel examined the supply air blower and could not find anything wrong with the supply air system.

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

Body Weights of Males Not Collected A ccording to Schedule (two deviations)
The protocol states that body wei ghts were to be performed weekly for the first 13 weeks and ten every 4 weeks thereafter. Observat ions were to be made weekly.

During February 2003, the weigh/observation sche dule was modified. Sessions were moved to the weekends (February 9 and 16) from weekdays so that we could collect the 18 month blood samples from control and high-level rats as pe r study protocol. Therefore the time between sessions was altered from that prescribed in the protocol. The impact on the study was limited, as we did perform weigh/observatio ns, but on different days than pe r the original schedule. The normal schedule was resumed once the blood collections were complete.

Male body weights were not collected on May 28, 2003 due to a scheduling error. Weights were collected on June 4, 2003. Therefore, the interval was 5 weeks instead of 4. The next scheduled day for weighing was June 25. This resulted in a smaller interval (3 233ks) between weigh
sessions.Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

4-8 The impact was that we did not have data assessing the health status of the animals as scheduled. However, weighing occurred during the very next week, and the schedule was followed
thereafter.

Quantitation of Exposure Atmosphere
On October 25, 2001 and November 16, 2001, one point checks of the Miran calibration curves were compared to the wrong calibrations curves. The low-level Mirans would not have passed the one-point checks if the current calibration curves had been used.

For both days, MIR 2 would not have passed th e one-point check and should have been recalibrated. However, MIR 3 and 4 would have passed and therefore do not deviate from the standard system operation. This error was not noted until well after the exposures had been completed, therefore the MIR 2 was not recalib rated and the existing calibration curve (not updated) was used. The daily run sheets were corrected.

The impact of using the wrong standard curve was minimal because the extent of deviation from the acceptable range was only 3% from each curve (i.e., 0.082 instead for 0.085 for MIR 2 and 1.18 instead of 1.15 on October 25, a nd November 16, 2001, respectively.Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

5-1 LIST OF ABBREVIATIONS

API American Petroleum Institute BGVC Baseline gasoline vapor condensate CA Cochran-Armitage test CFR Code of Federal Regulations CRTC Chevron Research and Technology Center EDTA Ethylenediamine tetra-acetic acid EMBSI ExxonMobil Biomedical Sciences, Inc.
FE Fisher’s exact test
FID Flame ionization detector GC Gas chromatograph GLP Good Laboratory Practices
KRV/H-1 Kilharn rat virus/H-1 virus LPM Liters per minute LRRI Lovelace Respiratory Research Institute
MCL Mononuclear cell leukemia
N BF N eutral buffered formalin
N FPA N ational Fire Protection Associatio n OPPTS Office of Prevention, Pesticides , and Toxic Substances (U.S. EPA)
PVM Pneumonia virus of mice
RBC Red blood cell RCV/SDAV Rat corona virus/sialodacryoadenitis SOP Standard operating procedure
SD/Stdev Standard deviation
SE Standard error
T90 Time for chamber concentration to reach 90% of equilibrium value
WBC White blood cellStudy Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

6-1 REFERENCE LIST

Baetcke, K. P., Hard, G. C., Rodgers, I. S ., McGaughy, R. E., and Tahan, L. M. (1991). Alpha 2 υ Globulin: Association with Chemically Induc ed 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. Boorman, G. A., Eustis, S. L., Elwell, M. R., and Leininger, J. R., eds. (1990). Pathology of the
Fischer Rat , 1 st Ed., Academic Press, San Diego, CA. Doi, A. M., Hill, G., Seely, J., Hailey, J. R., Kissling, G., Bucher, J. R. (2007). α 2u-Globulin Nephropathy and Renal Tumors in Na tional Toxicology Program studies. Toxicol. Pathol. 35(4) : 533-540. Hall, W. C. (1990). Peritoneum, Retroperit oneum, Mesentery, and Abdominal Cavity. In Pathology of the Fischer Rat (Boorman, G. A. et al., eds.), pp. 63-69, Academic Press, Inc., San Diego, CA. Hard, G. C. (1998). Mechanisms of Chemically Induced Renal Carcinogen esis in the Laboratory
Rodent. Toxicol. Pathol. 26(1) : 104-112. Hard, G. C., and Khan, K. N. (2004). A C ontemporary Overview of Chronic Progressive Nephropathy in the Laboratory Rat, and its Significance for Human Risk Assessment. Toxicol. Pathol. 32(2) : 171-180. Haseman, J. K., Arnold, J., and Eustis, S. L. ( 1990). Tumor Incidences in Fischer 344 Rats:
NTP Historical Data. In Pathology of the Fischer Rat (Boorman, G. A. et al., eds.), pp. 555-564, Academic Press, Inc., San Diego, CA. MacFarland, H. N., Ulrich, C. E., Holdsworth, C. E., Ketchen, D. N., Halliwell, W. H., and Blum, S. C. (1984). A Chronic Inhalati on Study with Unleaded Gasoline Vapor. J. Am. Coll. Toxicol. 3(4): 231-48. National Toxicology Program (NTP) (1999). c.f. , http://ntpserver.niehs.nih.gov/ntp/research/database_sear ches/historical_contro ls/path/r_inhar.txt
Rat Nomenclature Reconciliation Subcommittee of the Society of Toxicologic Pathologists, http://www.toxpath.org; Standa rdized Rat Nomenclature.Study Number FY01-027 211(b) Chronic Carcinogenicity Study Baseline Gasoline Vapor Condensate (BGVC) September 2009

6-2 U.S. Environmental Protection Agency (1998). Risk Assessment Forum. Assessment of Thyroid Follicular Cell Tumors . EPA/630/R-97/002, Washington, DC.