Protocol No. HLS Study No. 00-6131 IT1 Study No. IT1 1101 

Abbr~.ed.Title: immunological Evaluation of Gasoline TBA Vapor Condensate v " " 

Pd AUDITED DRAFT REPORT 

lrnmunological Evaluation of Gasoline TBA 

Vapor Condensate in Female Sprague Dawley Rats 

Using the Plaque Forming Cell Assay 

Test Substance: 

Protocol No: 

Subcontractor's Sponsor: 

Contractor's Study Director: 

Sponsor: 

Sponsor's Representative: 

Immunotox's Project Number: 

Security: 

Date: 

Principal Investigator: 

Studies Conducted at: 

Gasoline TBA Vapor Condensate 

HLS 00-61 31 

Huntingdon Life Sciences 

Mettlers Road 

East Millstone, NJ 08875 

(732) 873-2550 Phone 

(732) 873-3992 Fax 

Gary M. Hoffman, B.A., DABT 

American Petroleum Institute 

1200 L Street, NW 

Washington, DC 20005 

Thomas Gray, M.S., DABT 

30 January 2004 

Kimber L. White, Jr., Ph.D. 

ImmunoTox, Inc. 

Virginia BioaTechnology Research Park 

800 East Leigh Street, Suite 209 

Richmond, VA 2321 9-1 534 

(804) 828-6880 Phone 

(804) 828-6881 Fax 

ImmunoTox, Inc. 

Virginia Bio~Technology Research Park 

800 East Leigh Street, Suite 209 

Richmond, VA 2321 9-1 534 

(804) 828-6880 Phone 

(804) 828-6881 Fax 
Protocol No. HLS Study No. 00-6131 

Abbreviated Title: lmrnunological Evaluation of Gasoline TEA Vapor Condensate 

I. GLP COMPLIANCE STATEMENT 

IT1 Study No. IT1 1101 

This study was conducted in compliance with EPA Good Laboratory Practices as set forth in 79.60, CFR 

Vol. 59, No. 122, 27 June 1994 with the following exceptions: 

Kimber L. White, Jr., Ph.D. 

Principal Investigator 

IrnmunoTox, Inc. 

Date 

Gary M. Hoffman, B.A., DABT 

Study Director 

Huntingdon Life Sciences 

Date 
Protocol No. HLS Study No. 00-6131 IT1 Study No. IT1 1101 

Abbreviated Title: immunological Evaluation of Gasoline TBA Vapor Condensate .. 

II. QUALITY ASSURANCE STATEMENT - IMMUNOTOX 

Test Substance: Gasoline TBA Vapor Condensate 

Report Title: lrnrnunological Evaluation of Gasoline TBA Vapor Condensate 

in Female Sprague Dawley Rats Using the Plaque-Forming Cell Assay 

Protocol Title: Gasoline TBA Vapor Condensate: A 13-Week Whole-Body 

Inhalation Toxicity Study in Rats with Neurotoxicity Assessments 

And 4-Week In Vivo Genotoxicity and lmmunotoxicity Assessments 

Huntingdon Life Sciences, Inc. Study No. 00-6131 

Sponsor Study No. 21 1 -TBA-S 

The final report for the indicated protocol has been reviewed by the Quality Assurance Unit of 

Virginia Commonwealth University. Furthermore, the Quality Assurance Unit has conducted the following 

inspections and reported to the ImmunoTox, Inc. Principal Investigator, and then has submitted written 

reports of said inspections to the Study Director and Management via the Principal Investigator. 

Inspection/Audits were performed and reported on the following dates: 

I Performed I Reported to ( Reported to S.D. and I Activity 

Approved and 

submitted by: 

Quality Assurance Manager Date 

Office of Research * Quality Assurance Unit * Box 980568 * Richmond, Virginia 23298 0568 * (804) 828-6587 * Fax (804) 828-5604 

HUNTINGDON LIFE SCIENCES QUALITY ASSURANCE STATEMENT 
Protocol No. HLS Study No. 00-6131 

Abbreviated Title: immunological Evaluation of Gasoline TEA Vapor Condensate 

IT1 Study No. IT1 11 01 

HUNTINGDON LIFE SCIENCES QUALITY ASSURANCE STATEMENT 

Listed below are the dates that this study was inspected by the Quality Assurance Unit of 

Huntingdon Life Sciences, East Millstone, New Jersey, and the dates that findings were 

reported to the Study Director and Management. This report reflects the raw data as far 

as can be reasonably established. 

Reported to 

Study Director and 

Type of Inspection Date(s) of Inspection Management 

GLP Protocol Review 9 Apr 02 

Positive Control Immuno 19 Jul02 

- Toxicity Dose Adm. 

Immunotoxicity Blood 23 JulO2 

Collection & Necropsy 

9 Apr 02 

19 Jul02 

Fran Jannone, B. A., RQAP-GLP 

Quality Assurance Senior Auditor 

Date 
Protocol No. HLS Study No. 00-61 31 

Abbreviated Title: lmmunological Evaluation of Gasoline TEA Vapor Condensate 

. IT1 Study No. IT1 1101 

(I 

Ill. SIGNATURE OF PRINCIPALS 

This report describes the results used to evaluate the relative immunotoxicological potential of the test 

substance, Gasoline TBA Vapor Condensate, which was administered by inhalation via whole-body 

exposure to female Sprague Dawley rats. 

Kimber L. White, Jr., Ph.D., Principal Investigator, was responsible for the overall conduct of the 

immunotoxicity evaluations in this study. Vanessa L. Peachee, M.S., served as the Assistant Principal 

lnvestigator and was responsible for the day-to-day activities of the immunotoxicity evaluations in this 

study. 

Kimber L. White, Jr., Ph.D. 

Principal lnvestigator 

ImmunoTox, Inc. 

Vanessa L. Peachee, M.S. 

Assistant Principal lnvestigator 

ImmunoTox, Inc. 

Approved: 

Gary M. Hoffman, B.A., DABT 

Study Director 

Huntingdon Life Sciences 

Date 

Date 

Date 
Protocol No . HLS Study No . 00-6131 

Abbreviated Title: immunological Evaluation of Gasoline TBA Vapor Condensate 

TABLE OF CONTENTS 

IT1 Studv No . IT1 11 01 

I . 

II . 

111 . 

IV . 

v . 

VI . 

VII . 

VIII . 

IX . 

X . 

XI . 

GLP Compliance Statement ................................................................................................................ 2

Quality Assurance Statements 

ImmunoTox. Inc ................................................................................................................................... 3 

Huntingdon Life Sciences .................................................................................................................... 4 

Signature of Principals ......................................................................................................................... 5 

Executive Summary ............................................................................................................................ 8 

Introduction ....................................................................................................................................... 10 

Methods of Procedure ...................................................................................................................... 11 

Experimental Design ..................................................................................................................... 11 

Variables Assessed ........................................................................................................................ 11 

Terminal Body and Organ Weights ............................................................................................. 11 

Splenocyte Preparation .................. : ........................................................................................ 12 

Spleen IgM Antibody Response to the T-dependent Antigen, sRBC . Day 4 Response ............. 12 

Data ............................................................................................................................................... 13 

Data Handling and Statistical Analysis ....................................................................................... 13 

Data Retention ............................................................................................................................ 13 

Results .......................................................................................................................................... 13 

Terminal Body and Organ Weights ................................................................................................ 13 

Spleen IgM Antibody Response to the T-dependent Antigen. sRBC . Day 4 Response ................ 15 

Conclusion ......................................................................................................................................... 17 

References ........................................................................................................................................ 18 

List of Figures 

1 . Absolute (mg) and Relative (%) Spleen Weight in Female Sprague Dawley Rats Exposed to 

Gasoline TBA Vapor Condensate via Inhalation for 5 Days per Week for 4 Weeks ............... 14 

2 . Absolute (mg) and Relative (%) Thymus Weight in ema ale Sprague Dawley Rats Exposed to 

Gasoline TBA Vapor Condensate via Inhalation for 5 Days per Week for 4 Weeks ............... 15 

3 . Total Spleen Cell Numbers in Female Sprague Dawley Rats Exposed to Gasoline TBA Vapor 

Condensate via Inhalation for 5 Days per Week for 4 Weeks ................................................. 16 

4 . IgM Antibody-Forming Cell Response to Sheep Erythrocytes in Female Sprague Dawley Rats 

Exposed to Gasoline TBA Vapor Condensate via lnhalation for 5 Days per Week for 4 Weeks17 

List of Tables 

ESI Summary Table for Toxicology and Immunology Studies ......................................................... 9 

1 Body Weight (g) and Organ Weights (mg) in Female Sprague Dawley Rats Exposed to 

Gasoline TBA Vapor Condensate via lnhalation for 5 Days per Week for 4 Weeks 
Protocol No. HLS Study No. 00-6131 

Abbreviated Title: immunological Evaluation ot Gasoline TBA Vapor Condensate 

IT1 Study No. IT1 1101 

2. Spleen Antibody-Forming Cell Response to T-dependent Antigen Sheep Erythrocytes in 

Female Sprague Dawley Rats Exposed to Gasoline TBA Vapor Condensate via Inhalation for 

5 Days per Week for 4 Weeks .... .....................................................,.,..................................... 20 

APPENDICES 

A Individual Animal Data 

B Contracting Sponsor's Exposure and Animal Data 
Protocol No. HLS Study No. 00-6131 

Abbreviated Title: Immunological Evaluation of Gasoline TBA Vapor Condensate 

IT1 Study No. IT1 1101 

IV. EXECUTIVE SUMMARY 

The study was conducted as part of Huntingdon Life Sciences (HLS) Study No. 00-6131 at ImmunoTox, 

Inc., Richmond, Virginia. The Principal Investigator was Kimber L. White, Jr., Ph.D., and Vanessa L. 

Peachee, MS., served as the Assistant Principal Investigator. The study was conducted to provide 

evaluation of immunological parameters for Huntingdon Life Sciences. 

The objective of the study was to determine the potential effects of Gasoline TBA Vapor Condensate for 

its ability to affect the humoral immune component of the immune system, when evaluated in the 

antibody-forming cell response to the T-dependent antigen, sheep erythrocytes. Female Sprague Dawley 

rats were administered Gasoline TBA Vapor Condensate for 5 days per week for 4 weeks by inhalation 

via whole body exposure by Huntingdon Life Sciences (HLS) Princeton Research Center (PRC) 

personnel. Three exposure levels of 2000, 10000 and 20000 mg/m3 of the test substance were used in 

the study. The in-life phase of the study was conducted by HLS, East Millstone, NJ, and the 

immunological evaluation was conducted by ImmunoTox, Inc., Richmond, VA. On the. day of sacrifice, 

spleens were placed in tubes containing media, placed on wet ice, and shipped to ImmunoTox, Inc. in 

Richmond, VA, for assay evaluation on the following day. 

Executive Summary Table ES-1 shows a summary of the selected toxicology and immunology 

parameters evaluated. Exposure to Gasoline TBA Vapor Condensate resulted in'no statistically significant 

changes in terminal body weight for any exposure level. Furthermore, there were no statistically 

significant effects observed in either thymus or spleen weight following exposure to Gasoline TBA Vapor 

Condensate, when evaluated either as absolute weight or relative weight, as compared to the air control. 

Exposure to Gasoline TBA Vapor Condensate did not result in any statistically significant effect in the IgM 

antibody-forming cell (AFC) response to the T-dependent antigen, sheep erythrocytes, when evaluated as 

either specific activity (AFCII 0' spleen cells) or as total spleen activity (AFCIspleen). The positive control, 

CPS, produced the anticipated results in the various parameters evaluated. 

In conclusion, the results of this imrnunotoxicological evaluation demonstrate that, under the experimental 

conditions used, exposure to the Gasoline TBA Vapor Condensate test substance did not adversely affect 

the functional ability of the humoral immune component of the immune system. 
Protocol No. HLS Study No. 00-6131 

Abbreviated Title: immunological Evaluation of Gasoline TBA Vapor Condensate 

Table ES-1 

IT1 Studv No. IT1 1101 

SUMMARY TABLE FOR TOXICOLOGY AND IMMUNOLOGY STUDIES 

Parameter Result Maximum Effect Exposure Level 

(ms/m3) 

Terminal Body Weight 

Day 29 No Effect 

Organ Weights (Absolute and Relative) 

Spleen (Absolute) No Effect 

Spleen (Relative) No Effect 

Thymus (Absolute) No Effect 

Thymus (Relative) No Effect 

Spleen IgM Antibody-Forming Cell Response to Sheep Erythrocytes 

IgM AFC/~ 0' Spleen Cells No Effect 

IgM AFCISpleen (XI 03) No Effect 
Protocol No. HLS Study No. 00-6131 

Abbreviated Title: immunological Evaluation of Gasoline TEA Vapor Condensate 

V. INTRODUCTION 

IT1 Study No. IT1 1101 

The purpose of this study was to provide evaluation of immunological parameters fo Ir Huntin gdon Life 

Sciences (HLS) Study No. 00-61 31. In this study, the test substance, Gasoline TBA Vapor Condensate, 

was evaluated for its ability to affect the humoral immune component of the immune system, when 

evaluated in the antibody-forming cell response to the T-dependent antigen sheep erythrocytes. The 

study was conducted in female animals because female rats have a more robust immune response than 

do the male animal of the species. Accordingly, female rats have a greater sensitivity for detecting an 

adverse effect of a compound should one occur. Routinely, irnmunotoxicology evaluations conducted by 

the National Toxicology Program (NTP) evaluate compounds only in female animals. Four days prior to 

sacrifice, lmmunoTox personnel sensitized the rats by intravenous administration of sheep erythrocytes at 

the HLS facility. On the day of sacrifice, HLS Princeton Research Center (PRC) personnel aseptically 

removed the spleen from each animal. The spleens were weighed, placed in tubes containing media, and 

sent to ImmunoTox, Inc. in Richmond, VA, on wet ice for evaluation the following day. Spleens were 

received on 24 July 2002 and the immunological evaluation was conducted on the same day. The IgM 

antibody-forming cell (AFC) response to the T-dependent antigen sheep erythrocytes, also referred to as 

the plaque assay, was the immunological assay conducted to evaluate the effect of Gasoline TBA Vapor 

Condensate on the immune response. This assay has been shown to be the most predictive functional 

assay for determining the immunotoxicological potential of a compound (Luster eta/.'). 

Kimber L. White, Jr., Ph.D., was the Principal lnvestigator for the immunological evaluation conducted by 

ImmunoTox, Inc., and Gary M. Hoffman, B.A., DABT, was the HLS Study Director. Vanessa L. Peachee, 

M.S., served as the Assistant Principal lnvestigator for ImmunoTox, Inc. and was responsible for carrying 

out the IgM antibody-forming cell assay. 

In evaluating the effects of Gasoline TBA Vapor Condensate on the immune system, the immunologic 

and toxicologic parameters evaluated were spleen and thymus weights, and the spleen IgM antibody 

response to the T-dependent antigen (sheep erythrocytes, sRBC). 

To the best of our knowledge, no significant protocol or standard operating procedure deviations occurred 

during the study, which affected the quality of the data and the ability to interpret the data with respect to 

the immunotoxicology of Gasoline TBA Vapor Condensate. 
Protocol No. HLS Study No. 00-6131 

Abbreviated Title: immunological Evaluation of Gasoline TEA Vapor Condensate 

VI. METHODS OF PROCEDURE 

IT1 Study No. IT1 1101 

The immunotoxicological satellite study consisted of a vehicle group, three exposure levels of Gasoline 

TBA Vapor Condensate, and a positive control group. There were 10 female Sprague Dawley rats in each 

of the groups. Animals were exposed by Huntingdon Life Sciences Princeton Research Center (PRC) 

personnel to either vehicle (air only) or Gasoline TBA Vapor Condensate at exposure levels of 2000, 

10000 or 20000 mg/m3 via inhalation for 4 weeks (5 days per week). Cyclophosphamide (CPS) was given 

as the positive control. Cyclophosphamide (CAS #6055-19-2, lot number 091K1176, received 12 July 

2002, expiration September 2004, white powder, storage 2-8*C, purity 99.5%), was obtained from the 

Sigma Chemical Company (responsible for its characterization), and was dissolved and diluted in 

phosphate buffered saline at Huntingdon Life Sciences to stock concentrations of 5.0 mgImL for use as 

the positive control for this study. The positive control animals received 50 mglkg Q 10 mUkg of CPS, a 

known immunosuppressive agent, administered intraperitoneally (i.p.) once on the last 4 days of 

exposure. These animals were not chamber exposed. On the day of sacrifice, one day after the last 

exposure, PRC personnel aseptically removed the spleen from each animal, weighed it, placed it in a 

collecting tube containing Earle's Balanced Salt Solution (EBSS) with HEPES and Gentamicin solution 

(prepared at PRC), and shipped the spleens on wet ice in individual shipping containers at 2-8°C by 

carrier to ImmunoTox for overnight delivery. Upon receipt, spleens were further processed for 

determination of IgM antibody response. 

Terminal Bodv and Oraan Weiahts. The terminal body weights were obtained by Huntingdon Life 

Sciences PRC personnel. Huntingdon Life Sciences PRC personnel collected blood (serum) samples 

(orbital collection anesthetized via carbon dioxideloxygen inhalation) and then sacrificed (carbon dioxide 

inhalation) the animals on the day after the final exposure. The serum samples were frozen (920°C). 

The thymuses were removed, weighed and preserved (formalin) for possible histopathology. Spleens 

were removed, weighed, and shipped at the time of sacrifice by PRC personnel to ImmunoTox, Inc. for 

immunotoxicological evaluation. 
Protocol No. HLS Study No. 00-61 31 

Abbreviated Title: immunological Evaluation of Gasoline TBA Vapor Condensate 

IT1 Study No. IT1 1101 

S~lenocvte Preparation. Upon arrival at the ImmunoTox testing facility, spleens were accessioned in 

accordance with the SOP for receipt of biological samples. Single-cell suspensions were prepared from 

each spleen using a stomachera 80 Lab Blender in accordance with the SOP for rat spleens. Cell 

suspensions were then centrifuged and resuspended in Earle's Balanced Salt Solution with HEPES. 

Viability of splenocytes was determined using propidium iodide (PI) and the Coulter EPICS XL-MCL Flow 

Cytometer. 

Spleen laM Antibodv Res~onse to the T-de~endent Antisen. sRBC. Dav 4 Response. As background, 

sheep erythrocytes (sRBC) are a T-dependent antigen and, thus, T cells, B cells, and macrophages are 

required to function properly in order to obtain an antibody-forming cell (AFC) response. If the test article 

affects any of these cell types to a significant degree, an altered response will be observed. As a result, 

the T-dependent IgM response to sRBC is one of the most sensitive immunotoxicological assays currently 

in use. A significant modulation in the IgM AFC response, when appropriately compared to vehicle 

controls, indicates that the test agent is capable of modifying the hurnoral immune response in the whole 

animal and, thus, has the potential for immunotoxicity. This assay is one of the Tier I assays used by the 

NTP~. 

The primary IgM response to sheep erythrocytes was measured using a modified hemolytic plaque assay 

of ~erne~. Rats were exposed to the test article for 5 days per week for 4 weeks. Rats were sensitized by 

ImmunoTox, Inc. personnel with 2x10' sRBC i.~. four days prior to sacrifice and, on the day after the last 

exposure, animals were sacrificed by PRC personnel. Spleen cell suspensions were prepared as 

described above. The cells were centrifuged and resuspended in a 6-ml volume, and 1:50 and 1:150 

dilutions were prepared. An 0.1-mI aliquot of spleen cells from each suspension was added to separate 

test tubes, each containing 25 p1 guinea pig complement, 25 p1 sRBC, and 0.5 ml of warm agar (0.5%). 

After thoroughly mixing, each test tube mixture was plated onto a separate petri dish, covered with a 

microscope cover slip, and incubated at approximately 36-38°C for 3 hours. Spleen cell counts were 

performed on the 6-rnl samples using a Model Z1 Coulter Counter. The spleen weight, cellslspleen, 

AFCl10' spleen cells, and AFCIspleen were determined. The plaques, which developed, were counted 

using a Bellco plaque viewer. A plaque, occurring from the lysis of sRBC, is elicited as a result of the 

interaction of complement and antibodies (produced in response to the i.v. sensitization) directed against 

sRBC. Each plaque is generated from a single IgM antibody-producing B cell, permitting the number of 

AFC present in the whole spleen to be calculated. The data are expressed as specific activity (AFCII o6 

spleen cells) and total spleen activity (AFCIspleen). 
Protocol No. HLS Study No. 00-6131 

Abbreviated Title: lmmunological Evaluation of Gasoline TBA Vapor Condensate 7' 

IT1 Study No. IT1 11 01 

Data Handlina and Statistical Analysis. The data obtained in this study were analyzed in accordance with 

standard operating procedure. Data were first tested for homogeneity of variances using the Bartlett's Chi 

Square ~est~. Homogeneous data were evaluated by a parametric one-way analysis of variance5. When 

significant differences occur, exposed groups were compared to the vehicle control group using the 

Dunnett's t ~est'. Non-homogeneous data were evaluated using a non-parametric analysis of variance5. 

When significant differences occur, exposed groups were compared to vehicle control group using the 

Gehan-Wilcoxon ~est' when appropriate. The Jonckheere's ~est' was used to test for exposure levelrelated trends across the vehicle and exposed groups. The positive control was compared to the vehicle 

control group using the Student t ~est~. Thecriteria for accepting the results of the positive control in the 

assay was a statistically significant (p 5 0.05) decrease in the response as compared to the vehicle 

control group. 

P values of 0.05 or less, as compared to the vehicle control group, were considered statistically significant 

and are indicated in the tables and in the figures with a single asterisk ('). A double asterisk (*') was 

used to indicate.a p value of 0.01 or less. In the text, the word significant indicates that the response was 

statistically significant at p 5 0.05. In the tables the abbreviation NS is used to indicate "Not Significant" 

for p values greater than 0.05. 

Data Retention. All data and records were returned to the Contracting Sponsor following acceptance of 

the final report. Upon completion of this study, the report and raw data for this study will be maintained in 

the archives of Huntingdon Life Sciences. 

VII. RESULTS 

TERMINAL BODY AND ORGAN WEIGHTS. 

The terminal body weight data from the study are shown in Table 1 for the control and Test Substanceexposed groups. The mean ending weight for the vehicle-exposed rats was 262.1 * 5.1 grams. No 

statistically significant differences were observed in terminal body weights of the Gasoline TBA Vapor 

Condensate-exposed animals at any exposure level as compared to the vehicle (air only) controls. 

The organ weights of the control and Test Substance-exposed rats are shown in Table 1. No effect was 

observed, following exposure to Gasoline TBA Vapor Condensate, on spleen or thymus weights when 

evaluated as absolute weight or as relative weight. Treatment with the positive control, 

cyclophosphamide, had a significant decrease of 55% on absolute spleen weight and a significant 

decrease of 77% on absolute thymus weight, compared to the vehicle control. In addition, the positive 
Protocol No. HLS Study No. 00-6131 IT1 Study No. IT1 1101 

Abbreviated Title: lrnrnunological Evaluation of Gasoline TBA Vapor Condensate - 

control, cyclophosphamide, had a significant decrease of 52% on relative spleen weight and a 75% 

decrease on relative thymus weight, compared to the vehicle control. Shown graphically in Figures 1 and 

2 is the lack of effect on spleen and thymus weights following exposure to Gasoline TBA Vapor 

Condensate. 

Figure 1 

Absolute (mg) and Relative (%) Spleen Weight in Female Sprague Dawley Rats Exposed to Gasoline 

TBA Vapor Condensate via Inhalation for 5 Days per Week for 4 Weeks 

Absolute Weight Relative Weight 

Vehicle 2000 10000 20000 - 50 Vehicle 2000 10000 20000 - 50 

TBA CPS TBA CPS 

(ms/m3) (mgfkg) (mglm3) (mgfkg) 
Protocol No. HLS Study No. 00-61 31 

Abbreviated Title: lmmunological Evaluation of Gasoline TBA Vapor Condensate 

IT1 Studv No. IT1 1101 

Figure 2 

Absolute (mg) and Relative (%) Thymus Weight in Female Sprague Dawley Rats Exposed to Gasoline 

TBA Vapor Condensate via Inhalation for 5 Days per Week for 4 Weeks 

Absolute Weight Relative Weight 

Vehicle 2000 10000 20000 - 50 

TBA CPS 

(mg1m3) (mgh) 

vehicle 2000 10600 20000 50 

The spleen IgM antibody-forming cell response, i.e. plaque assay, was evaluated on spleens removed 1 

day after the last exposure, which was Day 4 after antigen sensitization. Day 4 after antigen sensitization 

is the peak day for the sRBC IgM AFC response in rats. Viabilities were conducted on all cell suspensions 

using propidium iodide (PI) and the Coulter EPICS XL-MCL Flow Cytometer. The viabilities from all 

samples were greater than 71% with the exception of four samples. 

The results of the AFC response are shown in Table 2 and in Figures 3 and 4. As indicated above, 

exposure to Gasoline TBA Vapor Condensate did not result in spleen weights that were significantly 

different from the vehicle control group. Furthermore, as shown graphically in Figure 3, there were no 

significant differences in spleen cell number following exposure to Gasoline TBA Vapor Condensate as 

compared to the vehicle control group. As expected, the positive control, cyclophosphamide (CPS), 

produced an 83% decrease in spleen cell number when compared to the vehicle control group. 
Protocol No. HLS Study No. 00-61 31 

Abbreviated Title: lrnrnunological Evaluation of Gasoline TBA Vapor Condensate - IT1 Study No. IT1 11 01 

Figure 3 

Total Spleen Cell Numbers in Female Sprague Dawley Rats Exposed to Gasoline TBA 

Vapor Condensate via Inhalation for 5 Days per Week for 4 Weeks 

Vehicle 2000 10000 20000 50 

TBA CPS 

(mg1m3) (mglkg) 

Shown in Table 2 and Figure 4 are the functional results from the IgM antibody-forming cell (AFC) assay. 

Shown in the left panel are the results when the data are expressed as specific activity and the results of 

the total spleen activity are shown in the right panel. No effect was observed, following exposure to 

Gasoline TBA Vapor Condensate, when evaluated as either specific activity (AFCIIO' spleen) or as total 

spleen activity (AFCIspleen). 

As anticipated, the positive control, CPS, produced a significant decrease in specific activity (99%) and 

total spleen cell activity (1 00%) when compared to the vehicle control animals. 
Protocol No. HLS Study No. 00-6131 

Abbreviated Title: lmrnunological Evaluation of Gasoline TEA Vapor Condensate 

IT1 Study No. IT1 1101 

Figure 4 

IgM Antibody-Forming Cell Response to Sheep Erythrocytes in Female Sprague Dawley Rats Exposed to 

Gasoline TBA Vapor Condensate via Inhalation for 5 Days per Week for 4 Weeks 

Specific Activity Total Spleen Activity 

vehicle 2000 10000 20000 50 7 

TBA CPS 

(mg~m3) (mg/kg) 

VIII. CONCLUSION 

Exposure of female Sprague Dawley rats to Gasoline TBA Vapor Condensate for a period of 5 days per 

week for 4 weeks resulted in no statistically significant effects in the humoral immune response to the Tdependent antigen, sheep erythrocytes. There were no statistically significant effects on body weight, 

spleen or thymus weights (absolute or relative), spleen cell number, specific activity (~~~110~ spleen), or 

total spleen activity (AFCIspleen). Based on the immunological parameters evaluated, under the 

experimental conditions of the study, exposure to Gasoline TBA Vapor Condensate did not adversely 

affect the hurnoral immune response of female Sprague Dawley rats. 
Protocol No. HLS Study No. 00-6131 

Abbreviated Title: lrnrnunological Evaluation of Gasoline TBA Vapor Condensate 

IX. REFERENCES 

IT1 Studv No. IT1 1101 

Luster MI, Pottier C, Pait DG, White KL, Jr., Gennings C, Munson AE and Rosenthal GJ (1992) 

Risk assessment in immunotoxicology. I. Sensitivity and predictability of immune tests. Fund. Appl. 

Toxicol. l8:2OO-2lO. 

Jerne NK, Henry C, Nordin AA, Fun H, Koros MC, and Lefkovits 1 (1974). Plaque-forming cells: 

Methodology and theory. Trnspl. Rev. 18:130-191. 

Luster MI, Munson AE, Thomas P, Holsapple MP, Fenters J, White KL, Jr., Lauer LD, and Dean JD 

(1 988). Development of a testing battery to assess chemical-induced immunotoxicity. Fund. Appl. 

Toxicol. 1 0:2-19. 

Bartlett MS (1 937). Sub-sampling for attributes. J. Roy. Stat. Soc. Suppl. 4:131-135. 

Kruskal WH and Wallis WA (1952). Use of ranks in one-criterion variance analysis. J. Amer. Stat. 

ASSOC. 471583-621. 

Dunnett CW (1955). A multiple comparison procedure for comparing several treatments with a 

control. J. Amer. Stat. Assoc. 5O:lO96-ll2l, 

Gross AJ and Clark VA (1 975). Gehan-W ilcoxon Test. In Survival Distribution: Reliability 

Applications in Biomedical Sciences. AJ Gross and VA Clark, eds. John Wiley and Sons, New York, 

p. 120-123. 

Hollander M and Wolfe DA (1975). Jonckheere's Test: Non-parametric Statistics Methods, eds. M. 

Hollander and D.A. Wolfe, John Wiley and Sons, New York, p. 124-1 29. 

Sokal RR and Rohlf FJ (1981). Biometry. Freeman, San Francisco, p. 226-231. 
Protocol No. HLS Study No. 00-6131 

Abbreviated Title: lrnrnunological Evaluation of Gasoline TBA Vapor Condensate 

Table t 

IT1 Study No. IT1 1101 

Body Weight (g) and Organ Weights (mg) in Female Sprague Dawley Rats Exposed to 

Gasoline TBA Vapor Condensate via Inhalation for 5 Days per Week for 4 Weeks 

Parameter Vehicle Gasoline TBA Vapor (rndm3) Cvclophosphamide WNH Trend 

2000 10000 20000 50 mglkg Analysis 

(10) (10) (10) (10) (10) 

Body Wgt (g) 262.1 * 5.1 255.4 i 6.7 252.6 + 6.8 254.2 + 5.2 243.4 + 5.0* H NS 

Spleen (mg) 676 * 31 630 * 27 614 * 27 611 + 24 304 + 12** H NS 

% Body Wgt 0.259 & 0.009 0.247 * 0.008 0.242 * 0.011 0.242 -t 0.010 0.124 * 0.004** H p 0.05 

Thymus (mg) 571 & 33 490 i 33 513 rt 32 537 2 33 134 + 9** H NS 

% Body Wgt 0.217 * 0.012 0.194 k 0.014 0.202 +- 0.012 0.212 k 0.012 0.054 +- 0.003** H NS 

Female Sprague Dawley rats were administered vehicle control (air only) or gasoline TBA vapor condensate by inhalation via whole-body 

exposure for 5 days per week for 4 weeks. The positive control, cyclophosphamide, was administered i.p. on the last 4 days of exposure. On 

the day of sacrifice, spleens were placed in tubes containing media and sent to Richmond, VA, on ice for next day cell preparation. The rats 

were necropsied and indicated organs weighed. Values represent the mean * SE derived from the number of animals indicated in 

parentheses. H = homogeneous data and NH = non-homogeneous data using the Bartlett's Test for homogeneity. Homogeneous data were 

evaluated using a parametric analysis of variance. When significant differences occurred, exposed groups were compared to the vehicle 

control group using the Dunnett's t Test. The positive control was compared to the vehicle control using the Student's t Test. Values 

significantly different from vehicle control at p 5 0.05 are indicated by an asterisk, while those significant at p 5 0.01 are noted by a double 

asterisk. The Jonckheere's Test was used to test for dose-related trends among the vehicle and exposed groups. 

Key: g = grams; mg = milligrams; m3 = cubic meter of air; kg = kilograms; Wgt = weight; NS = not significant for p values greater than 0.05. 
Protocol No. HLS Study No. 00-61 31 

Abbreviated Title: immunological Evaluation of Gasoline TBA Vapor Condensate 

Table 2 

t IT1 Study No. IT1 1101 

Spleen Antibody-Forming Cell Response to T-dependent Antigen Sheep Erythrocytes in Female Sprague Dawley Rats Exposed to 

Gasoline TBA Vapor Condensate via Inhalation for 5 Days per Week for 4 Weeks - Day 4 Response 

Exposure Body Wgt Spleen Wgt Spleen Cells IgM AFCI IgM AFCfSpleen 

(g) (mg) (x lo7) lo6 Spleen Cells (X lo3) 

Vehicle 262.1 * 5.1 

(10) 

Gasoline TBA Vapor Condensate 

2000 mg/m3 255.4 + 6.7 

(10) 

10000 mg/m3 252.6 * 6.8 

(10) 

20000 mg/m3 254.2 2 5.2 

(10 

Cyclophosphamide 

50 mg/kg 243.4 * 5.0* 

(10 

H/NH H 

Trend Analysis NS 

Female Sprague Dawley rats were administered vehicle control (air only) or gasoline TBA vapor condensate by inhalation via 

whole-body exposure for 5 days per week for 4 weeks. The positive control, cyclophospham~de, was administered i.p. the last 

4 days of exposure. Four days prior to sacrifice, the rats were immunized (iv) with 2x10 sRBC. On the day of sacrifice, 

spleens were placed in tubes containing media and sent to Richmond, VA, on ice for next day cell preparation. Spleens were 

prepared into single cell suspensions and the number of IgM sRBC antibody-forming cells was determined. Values represent 

the mean * SE derived from the number of animals indicated in parentheses. H, = homogeneous data and NH = nonhomogeneous data using the Bartlett's Test for homogeneity. Homogeneous data were evaluated using a parametric analysis 

of variance. When significant differences occurred, exposed groups were compared to the vehicle control group using the 

Dunnett's t Test. The positive control was compared to the vehicle control using the Student's t Test. Values significantly 

different from vehicle control at p 5 0.05 are indicated by an asterisk, while those significant at p 5 0.01 are noted by a double 

asterisk. The Jonckheere's Test was used to test for dose-related trends among the vehicle and exposed groups. 

Key: g = grams; mg = milligrams; m3 = cubic meter of air; kg = kilograms; Wgt = weight; NS = not significant for p values 

greater than 0.05. 
Protocol No. HLS Study No. 00-61 31 

Abbreviated Title: immunological Evaluation of Gasolirle TBA Vapor Condensate 

APPENDIX A 

IT1 Study No. IT1 1101 

k. b 

INDIVIDUAL ANIMAL DATA 
INlDlVlDUAL ANIMAL DATA 

ORGAN WEIGHTS 

GASOLINE TBA VAPOR CONDENSATE 

00-61 31 

ANIMAL NO GROUP DOSE SEX BODY WGT SPLEEN WGT THYMUS WGT SPLEEN WGTI THYMUS WGTI 

(G) (MG) (MG) % BODY WGT % BODY WGT 

GI 

GI 

GI 

GI 

GI 

GI 

GI 

GI 

GI 

GI 

GI1 

GI1 

GI1 

GI1 

GI1 

GI1 

GI1 

GI1 

GI1 

GI1 

Glll 

Glll 

Glll 

Glll 

Glll 

Glll 

Glll 

Glll 

Glll 

Glll 

GIV 

GlV 

GIV 

GIV 

GIV 

GIV 

GIV 

GIV 

GIV 

GIV 

GV 

GV 

GV 

GV 

GV 

GV 

GV 

GV 

GV 

GV 

AIR ONLY 

AIR ONLY 

AIR ONLY 

AIR ONLY 

AIR ONLY 

AIR ONLY 

AIR ONLY 

AIR ONLY 

AIR ONLY 

AIR ONLY 

2.000 MG/M~ TBA 

2,000 MGIM~ TBA 

2.000 MGIM~ TBA 

2,000 MGIM~ TEA 

2,000 MGIM~ TBA 

2,000 MG/M~ TBA 

2.000 MWM3 TBA 

2,000 MG/M~ TEA 

2,000 MGIM~ TEA 

2,000 MWM3 TBA 

10,000 MGIM~ TBA 

10,000 MWM3 TBA 

10,000 MGIM~ TEA 

10.000 MGIM~ TBA 

10,000 MWM3 TBA 

10.000 MG/M~ TBA 

10,000 MWM3 TEA 

10.000 MGIM~ TBA 

10,000 MWM3 TBA 

10.000 MGIM~ TBA 

20,000 MWM3 TBA 

20.000 MG/M~ TBA 

20,000 MG/M~ TBA 

20,000 MWM3 TBA 

20,000 MGIM~ TBA 

20,000 MWM3 TEA 

20,000 MGIM~ TBA 

20.000 MGi/M3 TBA 

20,000 MWM3 TBA 

20,000 MGIM~ TBA 

50 MWKG CYCLOPHOSPHAMIDE 

50 MWKG CYCLOPHOSPHAMIDE 

50 MWKG CYCLOPHOSPHAMIDE 

50 MWKG CYCLOPHOSPHAMIDE 

50 MGIKG CYCLOPHOSPHAMIDE 

50 MWKG CYCLOPHOSPHAMIDE 

50 MGlKG CYCLOPHOSPHAMIDE 

50 MGlKG CYCLOPHOSPHAMIDE 

50 MGlKG CYCLOPHOSPHAMIDE 

50 MGlKG CYCLOPHOSPHAMIDE 
INDIVIDUAL ANIMAL DATA 

IGM AFC ASSAY 

GASOLINE TBA VAPOR CONDENSATE 

00-6131 

ANIMAL NO GROUP DOSE SEX IGM AFCl IGM AFCI CELLS1 SPLEEN WGT BODY WGT 

1 o6 SP.C. SPLEEN lo3 SPLEEN lo7 (MG) (G) 

GI 

GI 

GI 

GI 

GI 

GI 

GI 

GI 

GI 

GI 

GI1 

GI1 

GI1 

GI1 

GI1 

GI1 

GI1 

GI1 

GI1 

GI1 

Glll 

Glll 

Glll 

Glll 

Glll 

Glll 

Glll 

Glll 

Glll 

Glll 

GIV 

G IV 

GIV 

GIV 

GIV 

GIV 

GIV 

GIV 

GIV 

GIV 

GV 

GV 

GV 

GV 

GV 

GV 

GV 

GV 

GV 

GV 

AIR ONLY 

AIR ONLY 

AIR ONLY 

AIR ONLY 

AIR ONLY 

AIR ONLY 

AIR ONLY 

AIR ONLY 

AIR ONLY 

AIR ONLY 

2,000 MG1M3 TEA 

2.000 MGIM~ TEA 

2,000 MGIM~ TEA 

2.000 MGIM~ TEA 

2,000 MG/M~ TEA 

2,000 MGIM~ TEA 

2.000 MG1M3 TEA 

2,000 MGIM~ TEA 

2,000 MG1M3 TBA 

2,000 MGIM~ TBA 

10,000 MGIM~ TBA 

10,000 MG/M~ TBA 

10,000 MGIM~ TBA 

10.000 MGIM~ TBA 

10,000 MGIM~ TEA 

10,000 MG1M3 TBA 

10,000 MG/M~ TBA 

10,000 MG/M~ TBA 

10.000 MG/M~ TBA 

10,000 MGIM~ TBA 

20.000 MGIM~ TBA 

20,000 MGIM~ TBA 

20,000 MGIM~ TBA 

20,000 MG/M~ TBA 

20,000 MGIM~ TBA 

20,000 MG1M3 TBA 

20,000 MGIM~ TEA 

20,000 MGIM~ TBA 

20,000 MG/M3 TBA 

20.000 MGIM~ TBA 

50 MGlKG CYCLOPHOSPHAMIDE 

50 MGlKG CYCLOPHOSPHAMIDE 

50 MGlKG CYCLOPHOSPHAMIDE 

50 MGIKG CYCLOPHOSPHAMIDE 

50 MGlKG CYCLOPHOSPHAMIDE 

50 MGIKG CYCLOPHOSPHAMIDE 

50 MGlKG CYCLOPHOSPHAMIDE 

50 MGKG CYCLOPHOSPHAMIDE 

50 MGlKG CYCLOPHOSPHAMIDE 

50 MGKG CYCLOPHOSPHAMIDE 
Protocol NO. HLS Study NO. 00-6131 

Abbreviated Title: immunological Evaluation of Gasoline TBA Vapor Condensate 

APPENDIX B 

CONTRACTING SPONSOR'S EXPOSURE AND ANIMAL DATA 
INTRODUCTION: The following is data generated at Huntingdon Life Sciences, East 

Millstone, NJ. The separately issued main study report should be referenced for details of 

the procedures used for test atmosphere generationkharacterization and animal 

evaluations. 

Huntingdon Life Sciences 00-6131 Page 1 

21 1-TBA-S Draft Final Report 

STUDY DATES: Date of Animal Receipt: 6 June 2002 

Animal Exposure and Animal Data 

Preface 

Experimental Initiation Date: 25 June 2002 (in-life) 

Appendix B 

Experimental Completion Date: 23 July 2002 (in-life) 

Draft Report Date: 2 June 2004 

EXPOSURES AND IN-LIFE SUMMARY: The actual measured results during the 

exposures were comparable to the targeted exposure levels. There were no exposurerelated effects seen in the test animals with regards to body weights and feed 

consumption. 

TABLE OF CONTENTS 

TABLES 

A. Chamber Monitoring Results ........................................................................................... B. Summary of Clinical Observations (pretest only) ............................................................ C. Mean Body Weights (grams) ........................................................................................... D. Mean Body Weight Change from Baseline (grams) ........................................................ E. Mean Feed Consumption Values (grams/kg/day) ............................................................ F. Individual Clinical Observations (pretest only) ............................................................... G. Individual Body Weights (grams) .................................................................................... H. Individual Body Weight Change from Baseline (grams) ................................................. 

I. Individual Feed Consumption Values (grams/kg/day) ..................................................... 

J. Animal Termination History ............................................................................................ 
Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-61 31 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group IA - 0 (air only) mg/m3 

Chamber Environment 

Particle Size I Me: 

Nominal alytical Chamber Concentration 

(mg/m3) 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

P 

Mean 

(mg1m3) 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

1s 

TMC 

(mg1m3) 

1.19E-02 

2.1 1 E-02 

2.03E-02 

7.41 E-03 

Temperature 

("C) 

25 

26 

25 

25 

25 

24 

24 

24 

24 

24 

25 

25 

25 

25 

25 

24 

24 

24 

24 

24 

Date Exposure 

Number 

Humidity 

("4 

50 

68 

52 

7 1 

5 1 

54 

53 

54 

52 

52 

5 1 

52 

38 

47 

50 

49 

56 

52 

53 

5 1 

eterminati 

Individual MMAD GSD 

Olm) 

0.9138 

0.8260 

0.7595 

5.014 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

Mean 

S.D. 
Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-6131 

Chamber Monitoring Results 

Cumulative Exposure Record 

Day 

0 

1 

2 

3 

6 

7 

8 

9 

10 

13 

14 

15 

16 

17 

20 

21 

22 

23 

24 

27 

Date 

25-Jun-02 

26-Jun-02 

27-Jun-02 

28-Jun-02 

1 -Jul-02 

2-JuI-02 

3-Jul-02 

4-Jul-02 

5-Jul-02 

8-Jul-02 

9-Jul-02 

10-Jul-02 

1 1-JuI-02 

12-Jul-02 

15-Jul-02 

16-Jul-02 

17-Jul-02 

18-JuI-02 

19-Jul-02 

22-Jul-02 

S.D. 

Exposure 

Number 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

Mean 

0.4 7.4 

Chamber Environment 

Mean 

Temperature 

("C) 

24 

25 

24 

24 

24 

25 

25 

24 

25 

24 

24 

24 

24 

24 

24 

24 

24 

24 

24 

25 

24.3 

0 

Humidity 

("4 

51 

70 

54 

76 

52 

56 

54 

55 

55 

53 

54 

55 

39 

49 

5 1 

5 1 

58 

55 

54 

53 

54.8 

0 

Group IB - 0 (air only) mg/m3 

0.686 

Nominal 

(mglm3) 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

Particle Size 

MMAD 

Olm) 

1.905 

0.8133 

0.8024 

2.076 

1.399 

0.458 

Mean 

(mglm3) 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

8.1 6E-03 

Determinations 

GSD 

2.407 

3.176 

2.148 

2.298 

2.507 

TMC 

(ms/m3) 

1.36E-02 

2.72E-02 

1.92E-02 

4.83E-03 

1.62E-02 

Analytical Chamber Concentration 

Individual 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

(mglm3) 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 
Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-6131 

Chamber Monitoring Results 

Date Exposure 

Number 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

Mean 

S.D. 

Cumulative Exposure Record 

Group llA - 2,000 mg/m3 

Nominal 

(mglm3) 

1990 

1950 

2070 

2140 

2070 

21 20 

2010 

2030 

1990 

1930 

1890 

1920 

2020 

1880 

2010 

21 20 

2050 

2090 

2030 

2030 

201 7 

75 

Mean 

(mg/m4 

1998 

1963 

2063 

21 20 

2063 

2095 

2050 

2070 

1998 

1963 

1905 

2058 

2078 

1958 

2033 

1903 

1935 

1890 

1933 

1925 

alytical Chamber Concentration 

Individual MMAD 

Olm) 

0.8293 

0.7460 

0.7361 

1.612 

Particle Size 

Ins 

TMC 

(mg/m3) 

7.72E-03 

1.96E-02 

1.67E-02 

3.35E-03 

Chamber Environment 

Mean 

Temperature 

("C) 

24 

25 

23 

24 

23 

24 

23 

24 

24 

24 

24 

24 

24 

24 

24 

23 

23 

23 

23 

23 

23.7 

0.6 

Humidity 

("4 

45 

63 

47 

61 

46 

49 

48 

49 

48 

48 

47 

47 

34 

42 

45 

47 

53 

48 

50 

48 

48.3 

6.0 
Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-6131 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group IIB - 2,000 mg/m3 

Chamber Environment 

Particle Size Mean 

Humidity 

("A) 

50 

68 

53 

71 

50 

51 

51 

53 

52 

50 

52 

52 

37 

46 

49 

49 

56 

51 

5 1 

49 

Nominal 

(mg/m3) 

1990 

1950 

2070 

21 40 

2070 

21 20 

201 0 

2030 

1990 

1930 

1890 

1920 

2020 

1880 

2010 

21 20 

2050 

2090 

2030 

2030 

201 7 

75 

1s 

TMC 

(mg/m3) 

8.49E-03 

2.1 8E-02 

7.02E-02 

2.96E-03 

Temperature 

("C) 

23 

24 

23 

24 

23 

23 

23 

23 

23 

23 

23 

23 

23 

23 

23 

22 

22 

22 

23 

23 

23.0 

0.5 

Date Ixposure 

Number 

- --- 

Analvtical Chamber Concentration I 

MMAD 

Olm) 

0.8183 

0.7358 

14.68 

1.010 

terminat 

Mean GSD 

(mglm3) 

2090 

2090 

2038 

21 28 

1965 

2068 

2020 

2073 

1995 

18% 

1750 

1990 

2033 

1930 

2010 

2058 

1843 

2118 

2095 

1928 

Individual 

20 

Mean 

S.D. 
Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-6131 

Chamber Monitoring Results 

Date Exposure 

Number 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

Mean 

S.D. 

Cumulative Exposure Record 

Group IIIA - 10,000 mg/m3 

Nominal 

(mg/m3) 

9760 

10200 

9440 

9850 

9970 

10200 

10100 

10100 

10300 

9970 

10300 

10300 

10300 

9590 

9840 

991 0 

101 00 

101 00 

9920 

101 00 

10020 

239 

1 

Mean 

(mglm3 

10430 

10330 

9878 

9943 

9763 

101 80 

9665 

1031 0 

9980 

10070 

9975 

10300 

10060 

9808 

9778 

lo360 

10390 

9895 

10980 

10680 

~alytical Chamber Concentration 

Individual 

Particle Size 

C 

MMAD 

Olm) 

0.8742 

0.7422 

0.7415 

1.168 

terminati 

GSD 

S 

TMC 

(mg/m3) 

8.64E-03 

2.39E-02 

1.89E-02 

2.78E-03 

Chamber El 

Me, 

Tem~erature 

ironment 

Humidity 

("w 

47 

64 

49 

61 

47 

48 

50 

51 

49 

48 

48 

48 

34 

42 

46 

47 

53 

51 

52 

49 

49.2 

6.1 
Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-6131 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group lllB - 10,000 mglm3 

Day Date Exposure Nominal L 

0 25-Jun-02 

1 26-Jun-02 

2 27-Jun-02 

3 28-Jun-02 

6 1 -Jul-02 

7 2-Jul-02 

8 3-Jul-02 

9 4-Jul-02 

10 5-JuI-02 

13 8-Jul-02 

14 9-Jul-02 

15 10-Jul-02 

16 11-Jul-02 

17 12-JuI-02 

20 15-Jul-02 

21 16-Jul-02 

22 17-Jul-02 

23 18-Jul-02 

24 19-Jul-02 I 

27 22-Jul-02 20 10100 

Mean 10020 

S.D. 239 

Analytical Chamber Concentration 

Mean I Individual 

Particle Size 

MMAD 

Olm) 

0.8268 

0.7424 

0.7374 

0.8683 

etermina 

GSD 

Chamber Environment 

Mear 

I 

1 

Humidity 

(W 

45 

64 

47 

60 

46 

46 

47 

48 

46 

46 

47 

48 

33 

41 

45 

44 

49 

47 

48 

45 

47.1 

6.2 

ms 

TMC 

(ms/m3) 

7.73E-03 

2.36E-02 

2.03E-02 

2.96E-03 

1.36E-02 

8.55E-03 

Temperatlire 

("'7 

23 

23.8 

0.5 
Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-6131 

Chamber Monitoring Results 

Date Exposure 

Number 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

Mean 

Cumulative Exposure Record 

Group IVA - 20,000 mglm3 

Nominal 

(mg/m3) 

18900 

20600 

19200 

181 00 

19600 

19300 

19600 

19600 

19400 

19200 

19700 

19400 

18900 

191 00 

18900 

19300 

19200 

191 00 

18900 

18600 

19230 

499 

1 

Mean 

(mg/m3) 

20400 

21000 

19900 

20000 

20380 

20050 

19450 

19950 

20080 

1 9700 

201 80 

20850 

19430 

20680 

19380 

19550 

20100 

19880 

20930 

20980 

~alytical Chamber Concentration 

Individual 

Determinat 

Particle Size 

MMAD 

Olm) 

0.8760 

0.7496 

0.7553 

1.008 

Chamber Environment 

Mean 

GSD 

ns Temperature Humidity 

TMC 

(mg/m3) ("C) ("4 
Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-61 31 

Chamber Monitoring Results 

Exposure 

Number 

Day 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

Mean 

Date 

S.D. 

Cumulative ~x~osuie Rcord 

Group IVB - 20,000 mg/m3 

I 

Nominal 

(mg/m3) 

18900 

20600 

19200 

181 00 

19600 

19300 

19600 

19600 

19400 

19200 

19700 

19400 

18900 

19100 

18900 

19300 

19200 

19100 

18900 

18600 

19230 

499 

Particle Size 

Determinations 

Individual I MMAD I GSD I TMC 

Analvtical Chamber Concentration 

Mean 

- 

- 

Chamber En 

Mea 

Tem~erature 

ronment 

Humidity 

("4 

48 

67 

53 

68 

5 1 

50 

53 

54 

52 

5 1 

5 1 

52 

36 

45 

50 

47 

54 

53 

5 1 

5 1 

51.9 

6.7 
Huntingdon Life Sciences 00-61311 

Irnrnunotoxicity Sub-Group 

PAGE 1 

TABLE B 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

NORMAL 

WITHIN NORMAL LIMITS 
Huntingdon Life Sciences 00-61311 

Immunotoxicity Sub-Group 

PAGE I 

TABLE C 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

FEMALES MEAN BODY WEIGHTS (GRAMS) ................................................................................................................................... 

DOSE GROUP: I I I I11 IV V 

DOSE LEVEL (MG/M3) : 0 2000 10000 20000 POSITIVE CONTROL ................................................................................................................................... 

WEEK 0 

WEEK 1 

WEEK 2 

WEEK 3 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

WEEK 4 MEAN 

S.D. 

N ......................................... 

No statistically significant differences 
Huntingdon Life Sciences 00-61311 

Irnmunotoxicity Sub-Group 

PAGE 1 

TABLE D 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

WEEK 0 TO 1 MEAN 

S.D. 

N 

WEEK 0 TO 2 MEAN 

S.D. 

N 

WEEK 0 TO 3 MEAN 

S.D. 

N 

WEEK 0 TO 4 MEAN 

S.D. 

N ...................................... 

Statistical key: ** = pc0.01 
Huntingdon Life Sciences 00-61311 

Immunotoxicity Sub-Group 

PAGE 1 

TABLE E 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

FEMALES 

.....-----.--.-.-------------------- 

DOSE GROUP: 

DOSE LEVEL (MG/M3) : 

MEAN 

S.D. 

N 

WEEK 1 MEAN 

S.D. 

N 

WEEK 2 MEAN 

S.D. 

N 

WEEK 3 
Huntingdon Life Sciences 00-61311 

Immunotoxicity Sub-Group 

PAGE 1 

TABLE F 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL CLINICAL OBSERVATIONS 

FEMALES GROUP I 0 MG/M~ 

ANIMAL# OBSERVATIONS 

DAY OF 1 

STUDY 1 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 
Huntingdon Life Sciences 00-61311 

Irnrnunotoxicity Sub-Group 

PAGE 2 

TABLE F 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL CLINICAL OBSERVATIONS 

FEMALES GROUP I1 2000 MG/M3 

ANIMAL# OBSERVATIONS 

DAY OF 1 

STUDY 1 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 
Huntingdon Life Sciences 00-61311 

Irnrnunotoxicity Sub-Group 

PAGE 3 

TABLE F 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS ................................................................................................................................... 

CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT 
Huntingdon Life Sciences 00-61311 

Immunotoxicity Sub-Group 

PAGE 4 

TABLE F 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL CLINICAL OBSERVATIONS 

FEMALES GROUP IV 20000 MG/M~ 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS ------------------------...---.----------------- 

CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT 

DAY OF 
Huntingdon Life Sciences 00-61311 

Imrnunotoxicity Sub-Group 

TABLE F 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL CLINICAL OBSERVATIONS 

FEMALES GROUP V POSITIVE CONTROL 

PAGE 5 

DAY OF 1 

ANIMAL# OBSERVATIONS STUDY 1 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS 

WITHIN NORMAL LIMITS --------------.-.----------------------..-----------------------------...----.-----

CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT 
Huntingdon Life Sciences 00-61311 

Irnrnunotoxicity Sub-Group 

PAGE 1 

TABLE G 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

1581 

1582 

1583 

1584 

1585 

1586 

1587 

1588 

1589 

1590 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-61311 

Imrnunotoxicity Sub-Group 

PAGE 2 

TABLE G 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

2571 

2572 

2573 

2574 

2575 

2576 

2577 

2578 

2579 

2580 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-61311 

Immunotoxicity Sub-Group 

PAGE 3 

TABLE G 

GASOLINE TEA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

3571 

3572 

3573 

3574 

3575 

3576 

3577 

3578 

3579 

3580 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-61311 

Irnrnunotoxicity Sub-Group 

PAGE 4 

TABLE G 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

4581 

4582 

4583 

4584 

4585 

4586 

4587 

4588 

4589 

4590 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-61331 

Imrnunotoxicity Sub-Group 

PAGE 5 

TABLE G 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL BODY WEIGHTS (GRAMS) 

FEMALES GROUP V POSITIVE CONTROL 

WEEK OF STUDY 

ANIMAL# - 1 0 1 2 3 4 

5551 

5552 

5553 

5554 

5555 

5556 

5557 

5558 

5559 

5560 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-61311 

Immunotoxicity Sub-Group 

PAGE 1 

TABLE H 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL BODY WEIGHT CHANGE FROM BASELINE (GRAMS) 

FEMALES GROUP I 0 MG/M3 ................................................................................................................................... 

WEEK OF STUDY 

ANIMAL# 0-1 0-2 0-3 0-4 ................................................................................................................................... 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-61311 

Irnrnunotoxicity Sub-Group 

PAGE 2 

TABLE H 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL BODY WEIGHT CHANGE FROM BASELINE (GRAMS) 

FEMALES GROUP I1 2000 MG/M~ ................................................................................................................................... 

WEEK OF STUDY 

ANIMAL# 0-1 0-2 0-3 0-4 ................................................................................................................................... 

2571 

2572 

2573 

2574 

2575 

2576 

2577 

2578 

2579 

2580 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-61311 

Immunotoxicity Sub-Group 

PAGE 3 

TABLE H 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

3571 

3572 

3573 

3574 

3575 

3576 

3577 

3578 

3579 

3580 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-61311 

Immunotoxicity Sub-Group 

PAGE 4 

TABLE H 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-61311 

Immunotoxicity Sub-Group 

PAGE 5 

TABLE H 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL BODY WEIGHT CHANGE FROM BASELINE (GRAMS) 

FEMALES GROUP V POSITIVE CONTROL ................................................................................................................................... 

WEEK OF STUDY 

ANIMAL# 0-1 0-2 0-3 0-4 ................................................................................................................................... 

5551 

5552 

5553 

5554 

5555 

5556 

5557 

5558 

5559 

5560 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-61311 

Immunotoxicity Sub-Group 

PAGE 1 

TABLE I 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-61311 

Irnrnunotoxicity Sub-Group 

PAGE 2 

TABLE I 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL FEED CONSUMPTION VALUES (GRAMS/KG/DAY) 

FEMALES GROUP I1 2000 MG/M3 

MEAN 

S.D. 

N 

SF=Spilled Feeder 
Huntingdon Life Sciences 00-61311 

Irnrnunotoxicity Sub-Group 

PAGE 3 

TABLE I 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL FEED CONSUMPTION VALUES (GRAMS/KG/DAY) 

FEMALES GROUP I11 10000 MG/M3 

WEEK OF STUDY 

ANIMAL# 0 1 2 3 

MEAN 

S.D. 

N 

SF=Spilled Feeder 
Huntingdon Life Sciences 00-61311 

Irnrnunotoxicity Sub-Group 

PAGE 4 

TABLE I 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

MEAN 

S.D. 

N 

SF=Spilled Feeder 
Huntingdon Life Sciences 00-61311 

Irnrnunotoxicity Sub-Group 

PAGE 5 

TABLE I 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL FEED CONSUMPTION VALUES (GRAMS/KG/DAY) 

FEMALES GROUP V POSITIVE CONTROL 

WEEK OF STUDY 

ANIMAL# 0 1 2 3 

--..--------------------------------------------------------------------------.--

MEAN 

S.D. 

N 

SF=Spilled Feeder 
Huntingdon Life Sciences 00-61311 

Irnrnunotoxicity Sub-Group 

PAGE 1 

TABLE J 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

ANIMAL TERMINATION HISTORY 

FEMALES GROUP I 0 MG/M3 

TYPE OF 

DEATH 

DATE OF WEEK OF STUDY 

DEATH STUDY DAY 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 
Huntingdon Life Sciences 00-61311 

Irnrnunotoxicity Sub-Group 

PAGE 2 

TABLE J 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

ANIMAL TERMINATION HISTORY 

FEMALES GROUP I1 2000 MG/M3 ---~---~~..-----.-~~~------~~.~~~~~.~----~~-~.~---~~.--..~-.~~~

TYPE OF DATE OF WEEK OF STUDY 

ANIMAL# DEATH DEATH STUDY DAY 
Huntingdon Life Sciences 00-61311 

Immunotoxicity Sub-Group 

PAGE 3 

TABLE J 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

TYPE OF 

DEATH ........................... 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 

DATE OF 

DEATH 

23-JUL-02 

23-JUL-02 

23-JUL-02 

23-JUL-02 

23-JUL-02 

23-JUL-02 

23-JUL-02 

23-JUL-02 

23-JUL-02 

23-JUL-02 

STUDY 

DAY 

2 8 

28 

28 

2 8 

2 8 

28 

28 

2 8 

2 8 

28 
Huntingdon Life Sciences 00-61311 

Irnrnunotoxicity Sub-Group 

PAGE 4 

TABLE J 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

TYPE OF DATE OF WEEK OF STUDY 

ANIMAL# DEATH DEATH STUDY DAY ----------------.----.-..------------------..--------------.------------.-----..---

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 

TERMINAL SACRIFICE 
Huntingdon Life Sciences 00-61311 

Immunotoxicity Sub-Group 

PAGE 5 

TABLE J 

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS