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 manufacturers have conducted comparative chronic 

toxicology testing of evaporative emissions from unleaded gasoline (baseline gasoline vapor 

condensate [BGVC]) and unleaded gasoline containing MTBE (gasoline/MTBE vapor 

condensate [GMVC]). This is the final report of the BGVC chronic study. 

Groups of 50 male/50 female CDF(F344)CrlBR rats were exposed in H2000 whole-body 

inhalation chambers at BGVC vapor concentrations of 2 g/m3

 (low level), 10 g/m3 (mid level), 

and 20 g/m3

 (high level) 6 hours/day, 5 days/week for 104 weeks (518 exposure days). 

There were no clinical signs of toxicity attributable to BGVC inhalation. 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 significantly below control values throughout the 

study. Body weights of high-level and mid-level females were below control values after 

approximately 1 and 3 months of exposure, respectively. Body weights of low-level females 

were sporadically affected. At the final sacrifice, body weights of high-level males and highand mid-level female rats were significantly below control values. Among mid-level males, the 

absolute epididymis weight, percent epididymis-to-body and percent epididymis-to-brain 

weights were significantly below 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 females were significantly increased compared to 

controls, however they did not correlate with kidney pathology findings. The spleen weights of 

mid-level females were significantly increased compared to controls, which is likely a result of 

the high incidence of age-related mononuclear cell leukemia (MCL). Among mid-level females, 

the percent organ-to-body weight values for brain, heart, kidney, liver, lungs, and spleen were 

significantly greater than corresponding control values. Among high-level females, the percent 

organ-to-body values for adrenals, brain, heart, liver, kidney, and lungs were significantly greater 

than control values. The percent kidney-to-brain weights for mid- and high-level females and 

the percent spleen-to-brain weight for the mid-level females were significantly greater than the 
Study 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, lymphocytes, 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 lymphocytes 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 cell leukemia in all groups, the relationship of 

treatment to these changes in hematology values 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 nasal passages of both male and 

female rats. The respiratory epithelial degenerative effects were most likely caused by exposure 

to the test material. 

Exposure did not enhance the incidence of chronic progressive nephropathy in male or female 

rats, but did increase the severity 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 BGVC exposure. Consistent with previous studies and the alpha2u globulin overload mechanism of male rat kidney damage, BGVC induced increases in male 

renal adenoma (1/50 in control; 1/50 in 2 g/m3

 group; 4/50 in 10 g/m3 group; and 0/50 in 20 g/m3

group) and carcinoma (0/50 in controls; 0/50 in 2 g/m3 group; 3/50 in 10 g/m3 group; and 0/50 in 

20 g/m3

 group). Although the incidences of renal adenomas and carcinomas taken separately 

were not statistically significant, combined incidences for the control (1/50; 2%), low (1/50, 2%), 

mid (7/50, 14%) and high (0/50; 0%) exposure groups were significant (p = 0.004, Fisher’s 

Exact Test). Pair-wise tests did not demonstrate any significant difference among the individual 

dose groups. These neoplasms may be considered 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 incidence gave a statistically significant trend for 

increased incidence with increased exposure concentration (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 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 comparison, in the concurrent Gasoline MTBE Vapor 

Condensate study (FY01-013), squamous cell carcinoma of oral origin 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 squamous 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, there was an increased incidence of testicular 

mesothelioma in the mid-level group of male rats (4 of 50 examined). 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 incidence in the mid-level group is approximately 3-fold 

greater than the average incidence in male control 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-significant incidence (2 in 50 animals 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 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 influenced the statistical analysis to indicate 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 thyroid 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. Whether the incidence of thyroid follicular carcinoma 

was a causal 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 statistically 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 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 slightly higher than those previously reported for 

control animals, the incidence of thyroid follicular carcinomas and adenomas combined are 

higher than what might be expected (NTP, 1999). However, there are no statistically significant 

differences in the combined incidence of thyroid follicular adenomas and carcinomas among the 

exposure groups. In the concurrent Gasoline MTBE Vapor Condensate study, there were no 

significant increases in thyroid follicular cell adenomas, carcinomas, or combined adenomas and 

carcinomas in males or females. Thyroid follicular carcinoma might be considered to be caused 

by the BGVC exposure despite the lack 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 chronic progressive nephropathy in males and caused 

epithelial degeneration in the nasal passages of both sexes. The degenerative nasal effects were 

most likely caused by the test material. Chronic exposure to BGVC did not enhance the 

development of proliferative lesions (hyperplastic lesions, neoplasms) in female rats. However, 

there was an increased incidence in mononuclear cell leukemia in low- and mid-level exposed 

females, most likely due to protocol-driven sampling bias in these groups. Consistent with 

previous studies and the concurrent GMVC study, chronic exposure 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 enhanced squamous cell carcinoma in the nasal 

specimens, testicular mesothelioma, and thyroid follicular carcinoma. Consequently, due 
Study 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 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 

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 ........................................................................................................ xii 

LABORATORY SIGNATURE PAGE ....................................................................................... xiii 

LABORATORY QA STATEMENT ........................................................................................... xiv 

LABORATORY COMPLIANCE STATEMENT ..................................................................... xvii 

Section

 SUMMARY ....................................................................................................................... S-1

1. INTRODUCTION .............................................................................................................. 1-1

2. MATERIALS, METHODS, AND 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 ........................................................................................ 2-10
Study 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 Euthanized (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 Characterization 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) ................................................................................................................. 4-7
Study 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 Samples 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% Mortality 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 Weights 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-Brain Weight (% ± SD) ..................................................................... 3-14 

3-11 Summary of Hematological Findings in Rats Inhaling BGVC; Median (range) ........... 3-16 

3-12 Summary of Select Proliferative Lesions in Male and Female Rats ............................. 3-18

3-13 Incidence and Severity of Chronic Progressive Nephropathy in Male and Female 

Rats Exposed to BGVC ................................................................................................. 3-23
Study Number FY01-027 

211(b) Chronic Carcinogenicity Study 

Baseline Gasoline Vapor Condensate (BGVC) 

September 2009 

ix 

3-14 Incidence and Severity of Respiratory Epithelial Degeneration in the Nasal 

Passages from Rats Exposed to BGVC ......................................................................... 3-25

3-15 Incidence and Severity of Olfactory Epithelial 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 SEROLOGICAL 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 PROFILES 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 SUMMARIES OF CLINICAL OBSERVATIONS ............................ J-1 

Appendix K 

BODY AND ORGAN WEIGHTS AT NECROPSY ................................................................. K-1 
Study 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-1
Study 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 Research Institute (LRRI)

2425 Ridgecrest Dr. SE 

Albuquerque, NM 87108 

Courier Address and Location of Laboratory: 

Bldg. 9217, Area Y 

Kirtland Air Force Base 

Albuquerque, NM 87115

Sponsor: American Petroleum Institute (API) 

1220 L Street, NW 

Washington, DC 20005

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 Mexico 
Study 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 

 Date 
Study 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 Assurance Unit. The final report accurately 

reflects the raw data. Findings were reported to the Study Director and Test Facility 

Management as follows: 

Study Phase Performed by

Date(s) of Inspection 

or Audit

Testing Facility 

Management

Study 

Director

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-02
Study 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-02
Study 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

Date
Study 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 Alternative Tier 2 testing requirements under 

Section 211(b) of the Clean Air Act and the EPA Health Effects Test Guidelines OPPTS 

870.4200, “Carcinogenicity.” The stipulations of this protocol were implemented in 

conformance with EPA regulations as specified in 40 CFR 79.60, “Good Laboratory Practices 

(GLP) Standards for Inhalation Exposure Health Effects Testing.” 

The following were not conducted under GLP guidelines: 

Serological health assessments were conducted throughout the study. Of the five assessments, 

two were conducted under GLP guidelines, three were not. This was because LRRI did not 

specify the need to run three of the shipments GLP in our communications with BioReliance. 

Light and noise measurements were not conducted under GLP guidelines. No Standard 

Operating Procedure was in place for these endpoints. 

It was the Sponsor’s responsibility to maintain records of the method of synthesis, fabrication, or 

derivation of the test substance. The method of fabrication was not available at the time the 

study was initiated. It is, however, presently available 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 

 Date 
Study 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 manufacturers have conducted comparative chronic 

toxicology testing of evaporative emissions from unleaded gasoline (baseline gasoline vapor 

condensate [BGVC]) and unleaded gasoline containing MTBE (gasoline/MTBE vapor 

condensate [GMVC]) This is the final report of the BGVC chronic study. 

Groups of 50 male/50 female CDF(F344)CrlBR rats were exposed in H2000 whole-body 

inhalation chambers at BGVC vapor concentrations of 2 g/m3

 (low level), 10 g/m3 (mid level), 

and 20 g/m3

 (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 percentages of the major hydrocarbon components of the 

condensate remained relatively constant throughout. 

There were no clinical signs of toxicity attributable to BGVC inhalation. 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 significantly below control values throughout the 

study. Body weights of high-level and mid-level females were below control values after 

approximately 1 and 3 months of exposure, respectively. Body weights of low-level females 

were sporadically affected. At the final sacrifice, body weights of high-level males and highand mid-level female rats were significantly below control values. Among mid-level males, the 

absolute epididymis weight, percent epididymis-to-body and percent epididymis-to-brain 

weights were significantly below 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 females were significantly increased compared to 

controls, however they did not correlate with kidney pathology findings. The spleen weights of 

mid-level females were significantly increased compared to controls, which is likely a result of 

the high incidence of age-related mononuclear cell leukemia (MCL). Among mid-level females, 

the percent organ-to-body weight values for brain, heart, kidney, liver, lungs, and spleen were 

significantly greater than corresponding control values. Among high-level females, the percent 
Study 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, liver, kidney, and lungs were significantly greater 

than control values. The percent kidney-to-brain weights for mid- and high-level females and 

the percent spleen-to-brain weight for the mid-level 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, lymphocytes, 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 lymphocytes 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 cell leukemia in all groups, the relationship of 

treatment to these changes in hematology values 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 nasal passages of both male and 

female rats. The respiratory epithelial degenerative effects were most likely caused by the test 

material. 

Exposure did not enhance the incidence of chronic progressive nephropathy in male or female 

rats, but did increase the severity 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 BGVC exposure. Consistent with previous studies and the alpha2u globulin overload mechanism of male rat kidney damage, BGVC induced increases in male 

renal adenoma (1/50 in control; 1/50 in 2 g/m3

 group; 4/50 in 10 g/m3 group; and 0/50 in 20 g/m3

group) and carcinoma (0/50 in controls; 0/50 in 2 g/m3

 group; 3/50 in 10 g/m3 group; and 0/50 in 

20 g/m3 group). Although the incidences of renal adenomas and carcinomas taken separately 

were not statistically significant, combined incidences for the control (1/50; 2%), low (1/50, 2%), 

mid (7/50, 14%) and high (0/50; 0%) exposure groups were significant (p = 0.004, Fisher’s 

Exact Test). Pair-wise tests did not demonstrate any significant 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/m3 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 incidence gave a statistically significant trend for 

increased incidence with increased exposure concentration (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 comparison, in the concurrent Gasoline MTBE Vapor 

Condensate study (FY01-013), squamous cell carcinoma of oral origin 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 squamous 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, there was an increased incidence of testicular 

mesothelioma in the mid-level group of male rats (4 of 50 examined). 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 incidence in the mid-level group is approximately 3-fold 

greater than the average incidence in male control 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-significant incidence (2 in 50 animals 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 absence of testicular mesothelioma in control 
Study 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 statistical analysis to indicate 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 thyroid 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. Whether the incidence of thyroid follicular carcinoma 

was a causal 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 statistically 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 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 slightly higher than those previously reported for 

control animals, the incidence of thyroid follicular carcinomas and adenomas combined are 

higher than what might be expected (NTP, 1999). However, there are no statistically significant 

differences in the combined incidence of thyroid follicular adenomas and carcinomas among the 

exposure groups. In the concurrent Gasoline MTBE Vapor Condensate study, there were no 

significant increases in thyroid follicular cell adenomas, carcinomas, or combined adenomas and 

carcinomas in males or females. Thyroid follicular carcinoma might be considered to be caused 

by the exposure despite the lack 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 chronic progressive nephropathy in males and caused 

epithelial degeneration in the nasal passages of both sexes. The degenerative nasal effects were 

most likely caused by the test material. Chronic exposure to BGVC did not enhance the 

development of proliferative lesions (hyperplastic lesions, neoplasms) in female rats. However, 

there was an increased incidence in mononuclear cell leukemia in low- and mid-level exposed 

females, most likely due to protocol-driven sampling bias in these groups. Consistent with 
Study Number FY01-027 

211(b) Chronic Carcinogenicity Study 

Baseline Gasoline Vapor Condensate (BGVC) 

September 2009 

S-5 

previous studies and the concurrent GMVC study, chronic exposure 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 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 

1-1 

INTRODUCTION 

The purpose of this study was to evaluate the chronic toxicity, potential carcinogenicity, and 

exposure concentration-response relationships associated with inhalation of baseline gasoline 

vapor condensate (BGVC). The study was conducted in compliance with Alternative Tier 2 

testing requirements under Section 211(b) of the Clean Air Act and the EPA Health Effects Test 

Guidelines OPPTS 870.4200m, “Carcinogenicity.” The stipulations of this protocol were 

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

Laboratory Practices (GLP) Standards for Inhalation Exposure Health Effects Testing.” The 

study protocol and amendments are provided in Appendix A. 
Study Number FY01-027 

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 through 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 inaccurate dose calibrations resulting in chamber 

concentrations up to 40% greater 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 influence 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 Comparison 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 1.6 
Study Number FY01-027 

211(b) Chronic Carcinogenicity Study 

Baseline Gasoline Vapor Condensate (BGVC) 

September 2009 

2-2 

Table 2-1. Analytical Comparison of Test Material 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. The test substance was transferred, 

as needed, from the 420-pound to the 20-pound cylinders. Only authorized personnel were 

allowed access to the test substance. Receipt, use, and inventory of this test substance were 

documented. 

Analysis

Before dispensing BGVC from each 420-pound tank, a sample was removed from the tank and 

analyzed by gas chromatography (GC) with flame ionization detection (FID) at LRRI using a 

Shimadzu Model GC-17A/FID (Shimadzu Scientific Instruments, Columbia, MD). The gas 

chromatographic profile of the 18 major peaks (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. Additionally, there were differences in the relative 

concentrations of various alkanes and isoalkane components between 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 study with the analysis of the exposure chamber 
Study 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 the duration of the study. 

Reserve Sample

The Sponsor-contracted archives have retained samples of the test substance. 

ANIMALS AND ANIMAL ASSIGNMENTS 

Animal Receipt, Housing, and Quarantine

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

Charles River Laboratories (Raleigh, NC). The rats were examined by a veterinarian upon their 

arrival. All animals were quarantined and acclimated to whole-body inhalation chambers 

for20 days. Healthy animals were randomly assigned by weight to the core exposure groups 

(400 total; 50 rats/sex/exposure level). The weight 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 before exposures began to evaluate their health 

status as an indicator of the health status of the population on study. The remaining unassigned 

rats were euthanized. Receipt and initiation of exposures of male and female rats were staggered 

by one week. 

Animal Disease Screening Program

Five male and five female rats not assigned to study were sacrificed, bled and received a 

complete necropsy. No gross lesions were found, and tissues were subsequently discarded 

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

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

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

(CARB), Mycoplasma pulmonis (M. PUI), Pneumonia virus of mice (PVM), Rat Coronavirus/ 

Sialodacryoadenitis Virus (RCV/SDA), Reovirus (Reo), Sendai Virus (Sendai), Lymphocytic 

Choriomenengitis Virus (LCM), Parvovirus (Parvo), Toolan’s H-1 Virus (H-1), and Kilham rat 

Virus (KRV). The five male and five female sentinel 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 large database available on the inhalation toxicity and 

carcinogenicity of toxic materials in rats. The study design was justified because it provided 

exposure concentration-response information on the possible carcinogenicity associated with 

repeated inhalation of BGVC. 

Environmental Conditions

The rats were housed 24 hours per day during quarantine and exposure in Hazleton 2000 wholebody inhalation chambers. Initially, all rats were housed separately in 3.8-inch wide by 11-inch 

long by 8-inch high compartments within stainless steel baskets. When 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 weekly. The cage racks were rotated clockwise 

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

The chambers were held at approximately 1 inch of water negative pressure with respect to the 

exposure room, and the chamber flow rates were maintained at 12 to 15 air changes per hour 

(400–500 liters per minute [LPM]). Chamber temperatures were maintained at 20o to 24oC. 

Temperature, relative humidity, and chamber air flow rates were continuously monitored, 24 

hours per day. Values for the three parameters were recorded at 30-minute intervals. Oxygen 

concentration in the chambers was maintained at 19%. This parameter was monitored, but not 

recorded. 

A 12-hour light/dark cycle was maintained with lights on at 0600. Light levels in the exposure 

room and noise levels in the chambers were determined periodically. 
Study Number FY01-027 

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). Food was available at all times except during the 

daily exposure period. Certified diet was analyzed for heavy metals, aflatoxin, 

organophosphates and chlorinated hydrocarbons, and values present were below maximum 

allowable values established by the manufacturer. Water was analyzed by an independent 

laboratory for metals, ions, microbes, and pesticides. Analytes were either present at nondetectable concentrations or sufficiently low concentrations as to not interfere with the outcome 

of the study. 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/m3

 BGVC 

Low Level 2 g/m3 BGVC 

Mid Level 10 g/m3 BGVC 

High Level 20 g/m3 BGVC 
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211(b) Chronic Carcinogenicity Study 

Baseline Gasoline Vapor Condensate (BGVC) 

September 2009 

2-6 

Table 2-2. Experimental 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 observations for morbidity and 

mortality. Weekly body weights for first 13 

weeks, then every 4th week. Weekly detailed 

clinical observations. 

Sentinel Animal Bleeds for Serology Prior to study start and at six-month intervals 

thereafter, including prior to final sacrifice. 

Hematology Evaluations (Control and 

High-Level Exposure Groups) 

At 12 and 18 months and at final sacrifice. 

Histopathology All protocol-required 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 using 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

N2 (60-80 psig)

Nitrogen

Carrier Gas

Pneumatic

Shutoff

Pressure Valve

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 Generation 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 steel transfer line where the BGVC was completely 

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

and dilution air rate. Chamber exhaust was carried 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) uniformity of the distribution of total vapor within 

each chamber; 2) within-day and between-day stability of vapor concentration; 3) within-day and 

between-day consistency of the hydrocarbon profile as determined by gas chromatography; and 
Study 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 animals’ breathing zone was also examined for the 

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

Shoreview, MN). 

Once, prior to the initiation of animal exposures, the concentrations of the test substance in the 

generator control box and in the exposure room were 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 was 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 were each monitored with their own analyzer. 

The analyzers for the high-, mid-, and low-level chambers underwent weekly five-point 

calibrations and daily one-point calibration checks using test substance. A fourth analyzer was 

devoted to monitoring the control chamber, the room air, and the hood enclosing the 20 pound 

tank of test substance. This Miran 1A was calibrated approximately quarterly until December 

2001, and then once before exposures ended. The Miran 1A analyzers dedicated to the mid- and 

high-level chambers were calibrated over a range of 6–35 g/m3

. The Miran 1A analyzers for the 

control chamber and the low-level chamber were calibrated over a concentration range of 1–7 

g/m3

. 

The absorbance signals were recorded continuously. 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 exposure concentration for that chamber. 

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

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

Model GC-17A/FID. The percent peak area of each of 18 major components was determined 

and recorded. The retention times of eight representative components were verified against 
Study Number FY01-027 

211(b) Chronic Carcinogenicity Study 

Baseline Gasoline Vapor Condensate (BGVC) 

September 2009 

2-9 

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

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

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

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

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

total flow through each respective chamber ([L/min * min]/1000 m3) and then summing the 

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 approximately 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-level exposure chamber. 

In-Life Endpoints

Rats were exposed 6 hours/day (plus 14 minutes, the time for the vapor concentration to reach 

T90), 5 days/week for 104 weeks (518 exposure days). The following observations and 

measurements were made during the dosing phase. 

Mortality and Morbidity. Laboratory animal technicians observed the rats twice daily 

throughout the study. Examinations were oriented toward identifying dead, weak, or moribund 

animals and documenting the onset and progression of any abnormal clinical signs. Appropriate 

actions were taken to minimize the loss of animals from study (e.g., necropsy or refrigeration of 

any rats found dead and sacrifice of weak or moribund animals). 

Body Weight. All animals were individually weighed using the Path-Tox® data acquisition 

system (Xybion, Cedar Knolls, NJ) on study Day -7 (to randomly assign 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 were 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 included evaluations of skin and fur; eyes and mucous membranes; 

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

system effects, including tremors and convulsions, and changes in the level of activity, 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 sacrifice, blood smears were prepared, and 

manual differential cell counts were made. For the 12 and 18 month time points, blood was 

obtained by tail nick from the control and high-level-exposed rats. At final sacrifice, blood was 

obtained by cardiac puncture from all rats and collected into Vacutainer tubes containing 

ethylenediamine tetra-acetic acid (EDTA). Parameters evaluated included total leukocyte 

estimates (WBC), nucleated red blood cell counts (cells/100 WBC), anisocytosis (0 to 4+), 

polychromasia (1+ to 4+), and relative differential leukocyte counts for segmented neutrophils, 

band neutrophils, lymphocytes, monocytes, eosinophils, basophils, atypical lymphocytes and 

blastocytes. Absolute leukocyte counts were calculated for segmented neutrophils, band 

neutrophils, lymphocytes, monocytes, eosinophils, basophils, atypical lymphocytes and 

blastocytes using the formula WBC*relative percent/100. 

Post-Exposure Endpoints

Gross Necropsy. A complete gross examination was performed on all animals at final sacrifice 

and on those animals that died naturally or were 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 Representative Samples Taken for Examination 

Cardiovascular/Hematopoietic System 

1. Aorta 

2. Bone marrow (and/or fresh aspirate) 

3. Heart 

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

5. Spleen 

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 medulla/pons, cerebellum, and cerebrum) 

2. Eyes (retina, optic nerve) 

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

4. Pituitary 

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

Other 

1. All gross lesions and masses 

2. Skin 

3. Tail (for identification) 

Respiratory System 

1. Larynx 

2. Lung (infused with fixative) 

3. Nose 

4. Paranasal sinuses 

5. Pharynx (evaluated with larynx) 

6. Trachea 
Study Number FY01-027 

211(b) Chronic Carcinogenicity Study 

Baseline Gasoline Vapor Condensate (BGVC) 

September 2009 

2-12 

Table 2-3. Organs and Representative Samples 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 weights were collected on lungs, liver, kidneys, adrenals, testes, 

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

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

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

final sacrifice for determination of total and differential white blood cell counts. Gross lesions, 

body weights, and organ weights were entered on pre-prepared forms and then the information 

was recorded on the Path-Tox® database. 

Lungs were gently instilled via the trachea with 10% neutral buffered formalin (NBF) to 

approximate normal volume. Organs and tissues were immersion fixed in 10% NBF for 

subsequent histopathologic examination. Tissues were trimmed, processed routinely, paraffin 

embedded, sectioned at 5 μm and stained with hematoxylin and eosin for histopathologic 

examination. 

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

(0 g/m3

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

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

(testes, kidneys of males) and gross lesions from non-target tissues were examined histologically 

from final sacrifice low-level (2 g/m3

) and mid-level (10 g/m3) animals. Nomenclature of 

proliferative lesions was based on the international harmonized nomenclature recommended by 

the Rat Nomenclature Reconciliation Subcommittee of the Society of Toxicologic Pathologists 
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Baseline Gasoline Vapor Condensate (BGVC) 

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2-13 

(see http://www.toxpath.org; Standardized Rat Nomenclature). 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 statistical significance using PathTox® software. After testing for an overall trend among test groups by an analysis of variance, 

Bartlett’s test was used to establish the homogeneity of the data. If the data were homogeneous, 

group differences were evaluated using a modified Dunnett’s test. If data were nonhomogeneous, group differences were assessed using a modified t-test. Significance levels were 

set at p ≤ 0.05. 

Survival Analysis. The probability of survival was estimated 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 significance 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 distributions are highly skewed for some variables. 

Preliminary analyses were done using the generalized estimating equation approach for 

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

analyses showed significant interactions 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 and non-neoplastic lesions are given as the 

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

microscopically. Three statistical evaluations were performed on the histopathology lesion 

incidence data: 1) Cochran-Armitage test, which tests whether the incidence of lesions shows a 

trend across exposure groups; 2) logistic regression test that takes death date into account when 
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Baseline Gasoline Vapor Condensate (BGVC) 

September 2009 

2-14 

assessing the presence of an exposure-dependent trend; and 3) the Fisher’s exact test, which 

compares incidences among the four exposure groups. The two-sided significance level was set 

at p = 0.05. If a significant difference 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 survival is similar among exposure groups as is the case for this 

study. The Fisher’s exact test tests the null hypothesis of equality of prevalences across exposure 

groups against the alternate hypothesis that the prevalences are not equal while the CochranArmitage test tests the null hypothesis of equality across exposures against the alternate 

hypothesis of a monotonic increasing or decreasing trend. 

Additionally, differences between groups with regard to both the severity and incidence of nonproliferative lesions were analyzed by the Kolmogorov-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 

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Baseline Gasoline Vapor Condensate (BGVC) 

September 2009 

3-1 

RESULTS AND CONCLUSIONS 

EXPOSURE ATMOSPHERE 

Pre-Test Chamber Trials

Three-Day Stability Evaluation and Nominal BGVC Usage. Before exposures were initiated the 

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

chamber was close to target and constant within each day, and the concentrations were 

reproducible from day to day (Table 3-1). Gas chromatographic analysis of the chamber 

atmospheres indicated that the relative percentages of the major components were constant over 

the 3-day period. The percentages of actual 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/m3)/ 

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 conducting a chamber distribution study. The 

concentrations of BGVC were measured from four sampling ports located in front and four 

sampling ports located in the back of the chambers during a pre-test chamber trial day and 

compared to the concentration obtained in a reference location. The spatial variation measured 

in Chambers 2 (2 g/m3

), 3 (10 g/m3) and 4 (20 g/m3) 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. 
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Baseline Gasoline Vapor Condensate (BGVC) 

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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/m3

 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/m3 vapors are provided in Table 3-2. The 

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

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

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

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

). The overall average of the 

daily percent nominal usage is 98% ± 6%, indicating excellent agreement between anticipated and 

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

Table 3-2. Summary of BGVC Vapor Concentrationsa

Target 

g BGVC/m3

 Achieved 

Concentrations 

 Percent of 

Target 

 0 < 1b 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 vapor 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 readings were below the lowest 

concentration on the standard curve used to calibrate the control 

chamber Miran 1A (1 g BGVC/m3

). 

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/m3

), 3 (10 g/m3), and 4 (20 g/m3) were 0.49%, 0.59%, and 5.39%, 

respectively. These results indicate the vapor was distributed evenly within each of the chambers. 
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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 throughout 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 results are provided in Appendix F. 

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

pressures were maintained within acceptable ranges 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 for male and female animals, respectively. The 

differences among the groups are not statistically significant 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 among groups within and between sexes. For the calculation of 

mean and standard errors of survival times, the last day of the study was used as the value of the 

survival time for animals that were sacrificed at the time that the study was terminated. 

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

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

results indicate that chronic BGVC inhalation did not shorten the lifespan of the exposed rats 

compared to the controls. 
Study Number FY01-027 

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Baseline Gasoline Vapor Condensate (BGVC) 

September 2009 

3-4 

Figure 3-1. Survival of Male Animals 

Figure 3-2. Survival of Female Animals 
Study Number FY01-027 

211(b) Chronic Carcinogenicity Study 

Baseline Gasoline Vapor Condensate (BGVC) 

September 2009 

3-5 

Table 3-3. Mean Survival Days 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 majority of males underwent moribund sacrifices. 

Individual animal disposition is provided in Appendix H. 

Table 3-4. Disposition of Rats on Study 

Death Type 

Control 2 g BGVC/m3

 10 g BGVC/m3 20 g BGVC/m3

Male Female Male Female Male 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 2a

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

Total 50 50 50 50 50 50 50 50 

a

Malocclusion. 

b

Missing, presumed dead. 

c

Nose injury. 

d

Accidental removal through misidentification (included in survival analysis). e

Accidental death due to trauma. 
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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 weight during the study. However, 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 were only sporadically below control values. 

At terminal sacrifice, male percent of treated: control weights were 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 toxicity evaluation, many of the observations observed 

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

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

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). Individual animal data are provided in Appendix K. 

Absolute Organ Weights at Final Sacrifice

Only the mean epididymis weights of mid-level males were significantly different (reduced) 

from the mean control values. The kidney weights for mid- and high-level female rats were 

significantly greater than the mean control values, however they did not correlate with kidney 

pathology findings. In addition the spleen weight of mid-level females was significantly 

increased compared to controls. The mean value was strongly influenced by a few large spleens 

(>10 g) from animals having mononuclear leukemia. The effects 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 

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Baseline Gasoline Vapor Condensate (BGVC) 

September 2009 

3-7 

Table 3-5. Body Weight Summary for Male Ratsa

Study 

Day Control 2 g/m3

Percent 

Control 

(2 g/m3) 10 g/m3

Percent 

Control 

(10 g/m3) 20 g/m3

Percent 

Control 

(20 g/m3) 

 -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 different from controls at that time point; p ≤ 0.05. Group 

mean body weight, organ weight, and percent organ-to-body weight data were tested for statistical 

significance using Path-Tox® software. After testing for an overall trend among test groups by an analysis 

of variance, Bartlett’s test was used to establish the homogeneity of the data. If the data were homogeneous, 

group differences were evaluated using a modified Dunnett’s test. If data were non-homogeneous, group 

differences were assessed using a modified t-test. Significance levels were set at p ≤ 0.05. 
Study Number FY01-027 

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Baseline Gasoline Vapor Condensate (BGVC) 

September 2009 

3-8 

Table 3-6. Body Weight Summary for Female Ratsa

Study 

Day Control 2 g/m3

Percent 

Control 

(2 g/m3) 10 g/m3

Percent 

Control 

(10 g/m3) 20 g/m3

Percent 

Control 

(20 g/m3) 

 -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 Dunnett’s test. If data were non-homogeneous, group 

differences were assessed using a modified t-test. Significance levels were set at p ≤ 0.05. 
Study Number FY01-027 

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Baseline Gasoline Vapor Condensate (BGVC) 

September 2009 

3-9 

Figure 3-3. Body Weights of Male Rats Exposed to BGVC 

Figure 3-4. Body Weights of Female Rats Exposed to BGVC 
Study Number FY01-027 

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September 2009 

3-10 

Table 3-7. Summary Statistics for Terminal Body Weights of Male and Female Rats 

 Control 2 g BGVC/m3

 10 g BGVC/m3 20 g BGVC/m3

Males

Mean ± SD 377.5 ± 31.3 388.3 ± 24.0 357.8 ± 36.3 341.9 ± 60.7a 

n 18 22 26 17 

Females

Mean ± SD 273.9 ± 20.5 261.5 ± 24.1 247.8 ± 22.4b

 251.0 ± 19.4b 

n 29 27 27 33 

a

Mean significantly different from control; data nonhomogeneous by Bartlett’s test. Means 

compared using a Modified t test. 

b

Mean significantly different from control; data homogeneous by Bartlett’s test. Means 

compared using Dunnett’s test of significance. 
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Baseline Gasoline Vapor Condensate (BGVC) 

September 2009 

3-11 
Study Number FY01-027 

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3-12 

Percent Organ-to-Body Weight at Final Sacrifice

In males the mean percent epididymis-to-body weight was significantly lower than the control 

value for the mid-level group. The percent adrenal-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, heart, kidney, liver, lungs, and spleen were 

significantly greater than the value for controls. Among high level females, the percents organto-body weight for adrenals, brain, 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 treatment related. Summary statistics are provided 

in Table 3-9. Individual animal 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 lower for the mid-level group than for the controls. 

The percent kidney-to-brain weight for mid- and high-level females and the percent spleen-tobrain weight for the mid-level females were significantly 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 was not treatment related. Summary statistics 

are provided in Table 3-10. Individual animal data are provided in K-3. 

Terminal Body Weight and Organ Weights of Euthanized (Moribund) Rats

As the rats reached about 20 months of age (18 months on study), mortality due to the effects of 

aging increased. Most animals not surviving to the final sacrifice were euthanized and terminal 

body and organ weights were recorded. Because the animals were euthanized over a period of 

several months, interpretation of the statistical significance of differences among exposed and 

control groups is not as straightforward as with data obtained at the final sacrifice. Summary 

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

K-4 through K-6. 
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3-13 
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The absolute kidney weights of low- and high-level 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 was significantly increased for the 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 high-level males. The percent 

epididymis-to-brain weight was significantly decreased in high-level males. There were no 

effects on terminal body or organ weights in females. Since cause of death of euthanized 

animals was not determined, the significance of the 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 are numerous, statistically significant 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 and concomitant development of degenerative, 

inflammatory, and neoplastic (e.g., mononuclear cell leukemia [MCL]) changes. Statistically 

significant interactive effects between time and exposure were not assessed. 

For females, there are significantly fewer numbers of total WBCs, neutrophils, lymphocytes, 

eosinophils, and nucleated red blood cells (RBCs) in the high-level 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 myelosuppression in the high-level exposure 

groups, in that the controls have slightly elevated total WBC 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 had difficulty sampling blood from the tail; that may have resulted 

in artifactual results. At 18 months and at the final sacrifice, there are no significant differences 

between the groups of females in any of the estimated WBC numbers. Overall, the relationship 

of treatment with the changes observed in females at 12 months is unknown. 
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3-17 

Only the absolute number of lymphocytes is significantly elevated in the high-level male group 

at the final sacrifice time-point (p = 0.01, Kruskal-Wallis test) in male rats. The range of 

lymphocyte numbers at this time-point, however, is very large (0.6 × 103 to 346.5 × 103 cells/μL 

for controls and 0.9 × 103

 cells/μL to 189.6 × 103cells/μL for high level) and 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 time-point. Given that BGVC exposure did not 

definitively enhance splenic involvement with MCL (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). Although 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 did not demonstrate any significant differences between groups in 

the combined incidence of renal adenoma + carcinoma (p > 0.008), although this 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 rats is unknown. These 

neoplasms may be considered to be exposure-related. For compounds inducing renal tumors in 

male rats through increased alpha 2u-globulin accumulation and subsequent increased renal 

tubule cell turnover, the human health relevance of the mechanism, and thus, the tumors, is often 

regarded as suspect (Baetcke et al., 1991; Hard, 1998; Hard and 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 exposed to hydrocarbons for 2 years 

or more. Rather, the severity of chronic progressive nephropathy may be correlated more 
Study 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 carcinogenesis bioassays of light hydrocarbon 

exposure (Doi et al., 2007). 

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

Tissue Diagnosis 

Control 

(0 g/m3) 

Low 

(2 g/m3) 

Mid 

(10 g/m3) 

High 

(20 g/m3) 

MALES

Kidney No. examined 50 50 50 50 

Adenoma, renal tubule 1 (2%) 1 (2%) 4 (8%) 0 (0%) 

Carcinoma, renal tubule 0 (0%) 0 (0%) 3 (6%) 0 (0%) 

Renal tubule adenoma and 

carcinoma, combineda 

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 cellb 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, malignantc 0 (0%) 0 (0%) 4 (8%) 0 (0%) 

 Adenoma, interstitial celld

 48 (96%) 46 (94%) 50 (100%) 49 (98%) 

 

Thyroid No. examined 50 29 27 50 

 Hyperplasia, follicular celle,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 cellg 0 (0%) 0 (0%) 2 (7%) 0 (0%) 

 Follicular cell adenoma and 

carcinoma, combinedh

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 Male and Female Rats (Concluded) 

Tissue Diagnosis 

Control 

(0 g/m3

) 

Low 

(2 g/m3) 

Mid 

(10 g/m3) 

High 

(20 g/m3) 

FEMALES

Spleen No. examined 50 25 32 50 

 Leukemia, mononuclear celli 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 increasing exposure level (p < 0.019, CA). Note 

that mid-dose turbinate levels 3 and 4 for one animal (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 significant (p > 0.008, FE tests). 

d

Incidences are not different than expected by chance (p = 0.26, overall FE test). e

Average for all animals examined (both affected and unaffected). 

f

There is a significant increasing trend with exposure 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 significant (p > 0.008, FE tests). 

h

Incidences are not different than expected 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 significant (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 appropriate to combine carcinoma incidence of nasal 

turbinate levels for statistical analysis in the high dose group because the lesion spanned two or 

three levels in the same three animals. The level 3 incidence gave a statistically significant trend 

for increased incidence with increased exposure concentration (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 comparison, in the concurrent Gasoline MTBE Vapor 

Condensate study (FY01-013), squamous cell carcinoma of oral origin 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 
Study Number FY01-027 

211(b) Chronic Carcinogenicity Study 

Baseline Gasoline Vapor Condensate (BGVC) 

September 2009 

3-20 

exposure was not causal, spontaneous oral squamous 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, there 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 control 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 substance. 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. Incidences of mesothelioma in the 

duodenum (2 of 23 examined), jejunum (3 of 23), colon (3 of 24), pancreas (3 of 24), spleen (3 

of 38), epididymis (3 of 25), prostate gland (2 of 25), and seminal vesicle (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 scrotum (Hall, 1990), and those found in this study are 

consistent with an origination from this investment of the testes. 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-significant incidence (2 

in 50 animals 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 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 influenced the statistical 
Study Number FY01-027 

211(b) Chronic Carcinogenicity Study 

Baseline Gasoline Vapor Condensate (BGVC) 

September 2009 

3-21 

analysis to indicate 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 thyroid 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 causal 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 statistically 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 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). 

Thyroid follicular epithelial adenomas 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 previously reported for control animals, the incidence of 

thyroid follicular carcinomas and adenomas combined are higher than what might be expected 

(NTP, 1999). However, there are no statistically significant differences in the combined 

incidence of thyroid follicular adenomas and carcinomas among the exposure groups (Table 

3-12). In the concurrent Gasoline MTBE Vapor Condensate study, there were no significant 

increases in thyroid follicular cell adenomas, carcinomas, or combined adenomas and 

carcinomas in males or females. Thyroid follicular carcinoma might be considered to be caused 

by the exposure despite the lack of an apparent exposure concentration-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 displayed 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 relevance of the test article 

exposure to human risk might be questioned, given that mechanisms of chemical thyroid 

carcinogenesis are believed to be different between humans and rodents (U.S. Environmental 
Study 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 carcinogenic in humans, but rodents are believed to be 

more susceptible to carcinogenic processes involving 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 significant 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 considered 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 significant higher incidence of 

keratoacanthomas in the skin (3 tumors in 29 rats and 3 in 26, respectively). However, there is a 

lack of a dose-response for these incidences, and most likely a result of protocol-driven gross 

sampling bias in non-target organ tissues where only tissues with gross lesions were evaluated in 

the low- and mid-dose groups at terminal sacrifice. In females, there was a statistically 

significantly increased incidence of splenic MCL in the low- and mid-dose groups. This finding 

was considered not to be treatment-related, but rather 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, respectively. Statistical evaluation of lesions is provided in the 

statistician’s report, Appendix O. The pathologist’s report is provided in Appendix P. 

Nonproliferative Changes. Kidneys of nearly all male rats on study have evidence of chronic 

progressive nephropathy, and there is no significant difference in the incidence among the 

groups. However, overall combination of incidence 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 specific light hydrocarbon-induced 

alpha-2u globulin overload nephropathy that has been reported previously in studies with wholly 

vaporized unleaded gasoline. It should be noted that in F344 male rats, after approximately 
Study 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 nephropathy is essentially masked by age-related 

chronic progressive nephropathy, since both forms of nephropathy share similar if not identical 

diagnostic hallmarks. Therefore the effects alpha-2u globulin overload nephropathy typically 

manifests itself in increased severity, rather than incidence, since essentially all males surviving 

past 1 year of age will develop CPN. 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 Chronic Progressive Nephropathy 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 significantly greater than control using the KolmorogovSmirnoff test, p ≤ 0.05. 

The incidence and severity of chronic progressive nephropathy in female rats was much less than 

in males and was not significantly different from that of the control group of female rats (Table 

3-13). Renal findings in females that are part of the constellation of chronic progressive 

nephropathy, including cortical tubular epithelial regeneration and corticomedullary tubular 

protein accumulation, had increased incidence with increasing exposure level (p < 0.05, CA and 

logistics tests; Appendix P). Therefore, even though overall severity scores were not different 

from controls, at least some renal findings in females 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, characterized primarily by accumulation 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 incidence can be enhanced 

by inhalation exposure to irritants or toxicants. The finding is somewhat sporadic, but its 

incidence in BGVC-exposed male rats is significantly greater in the respiratory epithelium of the 

nasal passages at turbinate level 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, Table 3-14). The combination of incidence and severity of the 

lesion at turbinate level 2, however, is not significantly different among the groups. Difference 

in incidence of the degenerative change in respiratory epithelium at turbinate level 3 (second 

palatal ridge) of male rats is also statistically significant (p = 0.007, Fisher’s exact test over all 

groups), but the CA and logistic trend tests 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 degeneration for female rats is somewhat greater 

than that of the males suggesting that this finding is more of a female-specific lesion (Table 

3-14). Higher incidences with significant trends for increases with increasing exposure level of 

the respiratory epithelial lesion were noted at both turbinate levels 2 and 3 of the female rats. 

The incidences at turbinate levels 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 statistically 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 control and low-level groups. This exposureresponse relationship was also present in the females 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 epithelium. 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), there 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 group (p = 0.006, pair-wise Fisher’s exact test). c

Overall severity/incidence is significantly greater than control (p < 0.05, KolmorogovSmirnoff test). 

d

Significantly different from the control and low-level groups (p < 0.008, Fisher’s exact test). e

Significantly different from the low-level 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-level 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 evaluate the long-term toxicity, including 

carcinogenicity of Baseline Gasoline Vapor Condensate. The results of this study provide a 

basis for comparing the results of a companion study in which the vapor condensate contained 

20% by weight of the additive methyl tertiary-butyl 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 passages of both sexes. The degenerative 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 exposed females, most likely due to protocoldriven sampling bias in these groups. Consistent with previous studies and the concurrent 

GMVC study chronic exposure to BGVC did enhance the development of renal adenomas and 

carcinomas (combined) in male rats. In contrast with the concurrent GMVC study, BGVC may 

also have enhanced squamous cell carcinoma in the nasal passages in male rats, testicular 

mesothelioma. Also, in contrast to the GMVC study, there is some evidence that BGVC 

exposure caused thyroid follicular cell carcinoma in males. 

Consequently, due primarily to treatment-related effects in the male kidney, and to possible 

effects on testes, and nose, and thyroid, chronic inhalation of Baseline 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, RAW DATA, AND FINAL REPORT 

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

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

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

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

specified by the Sponsor’s Contracting Representative. 

PROTOCOL DEVIATIONS 

Calculation of Nominal Concentration

Section IX.C.6. of the protocol states “Nominal concentration will be determined daily and will 

reflect the concentration generated for all three exposure systems combined. Therefore, the total 

mass of test substance generated will be divided by the total flow distributed to all three exposure 

chambers. 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 values for all three chambers. This value was 

compared to the actual BGVC usage determined by taking the difference between the weight of 

the 20-pound cylinder before and after the exposure. There is no impact because the way 

nominal concentration was calculated and compared to the actual BGVC usage provides the 

same answer as the method described in the protocol. 

Environmental Conditions

Throughout the BGVC exposure study, environmental conditions in the animal exposure 

chambers deviated periodically from protocol required ranges, or data were not acquired because 

of problems with the monitoring system. Most excursions 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 from protocol-specified environmental 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 Institute to repair a motor that had burned out on 

the backup exhaust system for the exposure wing. While here, he was asked to change the filters 

on the main system. To do this, he had to temporarily shut off the main blower. After the filters 

had been changed, the blower motor could not be restarted. This occurred about 12:30 p.m. The 

Study Director, Dr. Benson and LRRI Animal Care Operations were notified because the study 

animals were being quarantined in chambers in that wing. Since air was still being supplied to 

the chambers, the corks in the doors of the chamber were taken out to allow air flow. During this 

time the pressure in the chambers changed from negative (approximately -1 inch of water) to 

positive (about 1 inch of water). The maintenance crew attempted to restart the motor all 

afternoon. At 4:00 p.m., the exhaust system was still not working, so the exhaust lines were 

totally disconnected from the chambers housing the study animals. All environmental 

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

Surveillance personnel also checked the conditions in the room twice per shift and were directed 

to open the chamber doors in the event that temperatures exceeded 24°C. A new motor was 

delivered and installed early August 3, 2001. The exhaust system was up and running by 

8:30 a.m. 

The impact to the study was small, because animals had air, and were monitored by personnel 

throughout the time the exhaust system was down. 

Temporary Loss of Environmental Monitoring System Operation

On May 20, 2003 a major power outage occurred at 12:20 a.m. Normally, in power outage 

events, emergency backup diesel generators are turned on within 10–15 seconds. During this 

particular outage, a breaker was tripped and emergency power was not provided to the 

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

power until they were drained. During this outage, the supply air fan stopped operating, but due 

to the lack of environmental monitoring capability, this was not detected until morning. All 

systems were restored by approximately 6:30 a.m. The animals appeared healthy. Estimated 

flow rates through the chamber were approximately 200–300 L/minute. While supply air was 

not available, the vacuum system was still operational and pulled air from the room into the 
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chambers. Several actions, described in a memo to the study file, were taken to prevent similar 

situations in the future. 

On August 28, 2002 (14:29) to August 29, 2002 (06:29) no environmental data were acquired or 

saved to file. This was because on August 28, 2002, the LRRI network domain configuration 

modifications were performed on the monitoring PC in the study control room. The software 

program controlling and recording environmental data was shut down and inadvertently not 

restarted. 

Light and Sound Measurements

According to the study protocol, light and sound measurements in the animal exposure room 

were to be measured quarterly. In 2001, measurements were not made for the July–September 

quarter. In 2002, no measurement was made between January and March. In 2003, no 

measurement was made between July and the end of the study in August. The impact on the 

outcome of the study is considered minimal because light and sound levels were similar among 

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

Gas Chromatographic Analysis

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

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

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

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

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

calibration to only days when analyses are performed. The impact of this SOP deviation is 

minimal because the GC was calibrated on days when analyses were performed. 

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 was ordered, it was on “back order” and was 
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estimated to arrive December 20, 2002. Until we received permission from the Sponsor to 

switch to treated (with neomycin) cage board, no cage board was used and pans were washed 

twice daily instead of the usual once per week. Dr. Benson contacted the Sponsor and received 

permission to use treated cage board on December 13, 2002. Use of untreated cage board 

continued when it arrived. 

There should be no adverse effect on the study associated with not using cage board (with twice 

daily pan washes to keep ammonia levels down) or associated with use of neomycin-treated cage 

board. The latter type is used throughout the institute for short-term and long-term inhalation 

studies. 

Collection of Organ Weights at Necropsy

The study protocol states that lung, liver, kidneys, testes, epididymis, ovaries, uterus, spleen, 

brain, and heart will be weighed at necropsy. It also states that these weights are not to be 

recorded on animals found dead. 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 Necropsy 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, L363, I576, K265, K251, and L399). There is no 

impact on the quality or integrity of the study 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 originally proposed. This occurred in three specific cases. First, 
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the Hoel Walburg test was not used to analyze tumor data although originally listed as a possible 

statistic in the protocol. Second, hematology results were analyzed as a function of time as well 

as a function of dose. Finally, in the case of chronic progressive nephropathy, where the 

incidence of lesions was similar among control and dosed groups, the Kolmogorov-Smirnov test 

was used to assess differences in severity scores. This evaluation was done within the P module 

of the Path-Tox® software, and not by the Study Statistician. 

The actual statistics used have been described in the Statistics section of the report. The fact that 

the statistics proposed in the protocol were not always those used for data analysis has no major 

impact on the quality of the study. In most cases the 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 have normal distributions, alternative statistical 

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

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

was appropriate. The use of the Kolmogorov-Smirnov statistic in the Path-Tox® database was 

appropriate because nephropathy was an important endpoint in this study. Statistical analysis of 

the severity of the lesion as a function of exposure concentration in the absence of a 

concentration-response effect on lesion incidence provided more information than would have 

been gained by analysis of incidence alone. 

Neat Test Article Characterization of 420-pound Tanks

Test article lot number API 99-01, tank BG22, was inadvertently not analyzed prior to use 

September 24 thru November 18, 2002. However, the tank was analyzed during use for the 

inhalation exposures and the composition was verified. For this reason, this deviation did not 

affect the validity of the study. 

Personnel

On page 3 of the study protocol, key personnel was 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, 
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PhD, Professor, Department of Family and Community Medicine (MSC 095040, 1 University of 

New Mexico, Albuquerque, NM, 87131), was subcontracted 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 effect 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 month 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 approximately 10:30 a.m., supply airflow to the animal chambers 

dropped, resulting in low exposure chamber flows and pressures. Surveillance personnel were 

summoned to the room. They monitored the room closely and made certain Facility Engineering 

and Exposure personnel were aware of the situation. It was determined that the flows and 

pressures were in a range that would not adversely affect the health of the animals and therefore 

the animals remained within the chambers during the night. 

During this time, chamber pressures did not exceed -3” water (within normal range of LRRI 

SOP). Chamber flows did not drop below 350 lpm (10 changes per hour), above the minimum 

stated in EPA OPPTS 870.4200 guidelines, but below those specified in the study protocol. 

Chamber temperatures remained within protocol-defined limits. 

On the following morning, November 28, 2001, the Study Engineer and Facility Engineering 

personnel examined the problem. Flows to the chamber were adjusted to within normal 
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operating parameters. Facility Engineering personnel examined the supply air blower and could 

not find anything wrong with the supply air system. 

At this time, the Study Engineer determined that it was ok to begin exposures. Approximately 

30 minutes into the exposure, Facility Engineering discovered that the chamber supply air blower 

was not working properly. Exposures were terminated after 40 minutes of exposure were 

completed so that repairs could be made. Exposure concentrations achieved during the 

40-minute run were 1.53 g/m3

, 7.88 g/m3 and 13.62 g/m3 for the 2, 10 and 20 g/m3 chambers, 

respectively. Flow through the chambers was maintained above 10 air changes per hour and 

chamber temperatures were within limits while the blower was being repaired. The environment 

in the exposure chamber was safe for animal occupancy at all times. Repairs to the blower were 

completed by 12:00 p.m. Exposures were not continued that day. One exposure day was missed 

but this was not considered significant because there were over 500 exposure days during the 

study. 

Body Weights of Males Not Collected According to Schedule (two deviations) 

The protocol states that body weights were to be performed weekly for the first 13 weeks and ten 

every 4 weeks thereafter. Observations were to be made weekly. 

During February 2003, the weigh/observation schedule 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 per 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/observations, but on different days than per 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. 
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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 the 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 recalibrated 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, and November 16, 2001, respectively. 
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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 

NBF Neutral buffered formalin 

NFPA National Fire Protection Association

OPPTS Office of Prevention, Pesticides, and Toxic Substances (U.S. EPA) 

PVM Pneumonia virus of mice 

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 cell 
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REFERENCE LIST 

Baetcke, K. P., Hard, G. C., Rodgers, I. S., McGaughy, R. E., and Tahan, L. M. (1991). Alpha2υGlobulin: Association with Chemically Induced Renal Toxicity and Neoplasia in the 

Male Rat. (EPA/625/3-91/019F) Risk Assessment Forum, U.S. Environmental 

Protection Agency (U.S. EPA), Washington, D.C. 

Boorman, G. A., Eustis, S. L., Elwell, M. R., and Leininger, J. R., eds. (1990). Pathology of the 

Fischer Rat, 1st Ed., Academic Press, San Diego, CA. 

Doi, A. M., Hill, G., Seely, J., Hailey, J. R., Kissling, G., Bucher, J. R. (2007). α2u-Globulin 

Nephropathy and Renal Tumors in National Toxicology Program studies. Toxicol. 

Pathol. 35(4): 533-540. 

Hall, W. C. (1990). Peritoneum, Retroperitoneum, 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 Carcinogenesis in the Laboratory 

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

Hard, G. C., and Khan, K. N. (2004). A Contemporary Overview of Chronic Progressive 

Nephropathy in the Laboratory Rat, and its Significance for Human Risk Assessment. 

Toxicol. Pathol. 32(2): 171-180. 

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 Inhalation 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_searches/historical_controls/path/r_inhar.txt 

Rat Nomenclature Reconciliation Subcommittee of the Society of Toxicologic Pathologists, 

http://www.toxpath.org; Standardized Rat Nomenclature. 
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U.S. Environmental Protection Agency (1998). Risk Assessment Forum. Assessment of 

Thyroid Follicular Cell Tumors. EPA/630/R-97/002, Washington, DC.