Protocol No. HLS Study No. 004130 IT1 Study No. TTI 1001 

Abbreviated Title: lrnmunological Evaluation of Gasoline DlPE Vapor Condensate p 

1'' AUDITED DRAFT REPORT 

lmrnunological Evaluation of Gasoline DlPE 

Vapor Condensate in Female Sprague Dawley Rats 

Using the Plaque Forming Cell Assay 

Test Substance: 

Protocol No: 

Subcontractor's Sponsoc 

Contractor's Study Director: 

Sponsor: 

Sponsor's Representative: 

Project Number: 

Security: 

Date: 

Principal Investigator: 

Studies Conducted at: 

Gasoline DlPE Vapor Condensate 

HLS 00-61 30 

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 

lTl 1001 

1- . . 

10 September 2002 

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

ImmunoTox, Inc. 

Virginia BioeTechnology 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 Bi~~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. 005130 

Abbreviated Title: Immunological Evaluation of Gasoline DlPE Vapor Condensate 

I. GLP COMPLIANCE STATEMENT 

IT1 Study No. IT1 1001 

P ". . ' "' I 

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: 

The identity, strength, purity and composition or other characteristics to define the positive control article 

have not been determined by the Testing Facility. The positive control article has been characterized as 

per the Certificate of Analysis on file with the Testing Facility. The stability of the positive control article 

has not been determined by the Testing Facility. Analyses to determine the uniformity (as applicable) or 

concentration of the positive control mixture were not performed by the Testing Facility. The stability of 

the positive control article mixture has not been determined by the Testing Facility. 

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. 005130 IT1 Study No. IT1 1001 

Abbreviated Title: lrnmunological Evaluation of Gasoline DlPE Vapor Condensate - 

II. QUALITY ASSURANCE STATEMENT 

Test Substance: Gasoline DlPE Vapor Condensate 

Report Title: lmmunological Evaluation of Gasoline DlPE Vapor Condensate 

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

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

Inhalation Toxicity Study in Rats with Neurotoxicity Assessments 

And 4-Week In Wvo Genotoxicity and lmmunotoxicity Assessments 

Protocol No.: HLS Study No. 00-61 30 

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. 

InspectionlAudits were performed and reported on the following dates: 

Performed Reported Activity 

15 Mar 02 15 Mar 02 AFC Assay 

01 Sep 02 

07 Sep 02 

09 Sep 02 Data Audit 

09 Sep 02 1 " Draft Report Audit 

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 
Protocol No. HLS Study No. 008130 IT1 Study No. IT1 1001 

Abbreviated Title: Immunological Evaluation of Gasoline DlPE Vapor Condensate - 

HUNTINGDON LIFE SCIENCES QUALITY ASSURANCE STATEMENT 
Protocd No. HLS Study No. 008130 

Abbreviated Title: lrnrnunological Evaluation of Gasoline DIP€ Vapor Condensate 

Ill. SIGNATURE OF PRINCIPALS 

lTl Studv No. IT1 1001 

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

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

exposure to female Sprague Dawley rats. 

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

imrnunotoxicity 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 imrnunotoxicity evaluations in this 

study. 

Kirnber L. White, Jr., Ph.D. Date 

Principal lnvestigator 

ImrnunoTox, Inc. 

Vanessa L. Peachee, M.S. Date 

Assistant Principal lnvestigator 

IrnmunoTox. Inc. 

Approved: 

Gary M. Hoffman, B.A., DABT 

Study Director 

Huntingdon Life Sciences 

Date 
Protocol No. HLS Study No . 006130 

Abbreviated Title: lmmunologlcal Evaluation of Gasoline DlPE Vapor Condensate 

IT1 Studv No . IT1 I001 

TABLE OF CONTENTS 

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 ....................................................................................................................... I I 

Experimental Design ...................................................................................................................... I I 

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

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

Splenocyte Preparation ............................................................................................................... 11 

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

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

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

3 . Spleen Cell Number in Female Sprague Dawley Rats Exposed to Gasoline DlPE 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 DIPE Vapor Condensate via lnhalation for 5 Days per Week for 4 

Weeks .................................................................................................................................... 17 

List of Tables 

ES1 Summary Table for Toxicology and Immunology Studies ......................................................... 9 

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

Gasoline DIPE Vapor Condensate via lnhalation for 5 Days per Week for 4 Weeks 
Pmtocd No. HLS Study No. 005130 IT1 Study No. IT1 1001 

Abbreviated TIUe: lmrnunologicel 

................................................................................................................................................. 

Evaluation of Gasoline DlPE Vapor Condensate - 

19 

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

Female Sprague Dawley Rats Exposed to Gasoline DlPE 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. 008130 

Abbreviated Title: immunological Evaluation of Gasdine DlPE Vapor Condensate 

IT1 Study No. IT1 1001 

IV. EXECUTIVE SUMMARY 

The study was conducted as part of Huntingdon Life Sciences (HLS) Study No. 00-6130 at ImmunoTox, 

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

Peachee, M.S., 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 DlPE 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 DlPE 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 2,000, 10,000 and 20,000 mglm3 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 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 DlPE 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 

DlPE Vapor Condensate, when evaluated as absolute weight, as compared to the air control. When 

evaluated as relative weight (% body weight) in the spleen, there was a statistically significant increase at 

the low (2000 mg/m3) dose exposure level of Gasoline DlPE Vapor Condensate, however there was not a 

dose-dependent effect and the decrease at the low dose is not considered to be biologically significant. 

There was no effect in the thymus relative weight. 

Exposure to Gasoline DlPE Vapor Condensate resulted in a statistically significant dose-related decrease 

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

evaluated as either specific activity (AFCIO~ spleen cells) or as total spleen activity (AFCIspleen). The 

decrease reached the level of statistical significance at the high (20,000 mglm3) exposure level. The 

positive control, CPS, produced the anticipated results in the various parameters evaluated. 
Protocol No. HLS Study No. 008130 

Abbreviated Title: lrnrnunological Evaluation of Gasollne DlPE Vapor Condensate 

IT1 Studv No. IT1 1001 

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

conditions used, exposure to the Gasoline DlPE Vapor Condensate test substance adversely affected the 

functional ability of the humoral immune component of the immune system. 

Table ES-1 

SUMMARY TABLE FOR TOXICOLOGY AND IMMUNOLOGY STUDIES 

Parameter Result Maximum Effect Exposure Level 

(mglm3) 

Terminal Body Weight 

Day 29 No Effect 

Organ Weights (Absolute and Relatlve) 

Spleen (Absolute) No Effect 

Spleen (Relative) Increase 21 % 2,000 

Thymus (Absolute) No Effect 

Thymus (Relative) No Effect 

Spleen IgM Antibody-Forming Cell Response to Sheep Erythrocytes 

IgM AFCI? 0' Spleen Cells Decrease 63% 20,000 

IgM AFCISpleen (xlo3) Decrease 64% 20,000 
Protocol No. HLS Study No. 00-6130 

Abbreviated Title: lmmunologlcal Evaluation of Gasoline DlPE Vapor Condensate 

IT1 Studv No. IT1 1001 

V. INTRODUCTION 

The purpose of this study was to provide evaluation of immunological parameters for Huntingdon Life 

Sciences (HLS) Study No. 00-61 30. In this study, the ability of the test substance. Gasoline DlPE 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 Tdependent antigen sheep erythrocytes. 

Four days prior to sacrifice, ImmunoTox 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 ice for evaluation the following day. 

Spleens were received on 15 March 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 DlPE 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 et 

a/.'). 

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., sewed as the Assistant Principal Investigator for ImmunoTox, Inc. and was responsible for carrying 

out the IgM antibody-forming cell assay. 

In evaluating the effects of Gasoline DlPE 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 DlPE Vapor Condensate. 
Protocd No. HLS Study No. 004130 

Abbreviated Title: lrnrnunological Evaluation of Gasoline DlPE Vapor Condensate 

IT1 Study No. TTI 1001 

VI. METHODS OF PROCEDURE 

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

DlPE 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 DlPE Vapor Condensate at exposure levels of 

2,000, 10,000 or 20,000 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 108H0568, received 

28 February 2001, expiration 30 June 2002, white powder, storage 2-8OC, purity 99.2%), 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 mglmL for use as 

the positive control for this study. The positive control animals received 50 mglkg @ 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 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. 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 (5- 

20°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. 

S~lenocvte Pre~aration. 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. 
Protocol No. HLS Study No. 005130 IT1 Study No. IT1 1001 

Abbreviated Title: Immunological Evaluation of Gasoline DIP€ Vapor Condensate - 

Spleen laM Antibody ResDonse to the T-de~endent Antigen, sRBC. Dav 4 Res~onse. 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.v. 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 150 and 1:150 dilutions were 

prepared. An 0.1-ml aliquot of spleen cells from each suspension was added to separate test tubes, each 

containing 25 pl 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, ~~~110' spleen cells, and 

AFClspleen 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 (AFCI~O' spleen cells) and 

total spleen activity (AFClspleen). 

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 

humoral 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~. 
Pmbcol No. HLS Study No. 008130 

Abbreviated Title: immunological Evaluation of Gasoline DIPE Vapor Condensate 

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

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

Square Test4. 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 Test7 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'. The criteria 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 terminal body weights were obtained by Huntingdon Life Sciences PRC personnel. 

The mean ending weight for the vehicle-exposed rats was 251.6 + 4.3 grams. No statistically significant 

differences were observed in terminal body weights of the Gasoline DlPE 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 DlPE Vapor Condensate, on spleen or thymus weights when 
Protoml No. HLS Study No. 008130 

Abbreviated Title: lmrnunological Evaluation of Gasdine DlPE Vapor Condensate 

evaluated as absolute weight. When evaluated as relative weight, there was a statistically significant 

increase in the spleen of 21% at the low dose exposure level when compared to the vehicle control. 

However, the effect on relative weight was not dose dependent and is not considered to be biologically 

significant. Furthermore, there was no effect on the relative weight of the thymus when exposed to 

Gasoline DlPE Vapor Condensate. Treatment with the positive control, cyclophosphamide, had a 

significant decrease of 51% on absolute spleen weight and a significant decrease of 77% on absolute 

thymus weight, compared to the vehicle control. In addition, the positive control, cyclophosphamide, had a 

significant decrease of 45% 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 effect on spleen and thymus 

weights following exposure to Gasoline DlPE Vapor Condensate. 

Figure 1 

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

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

Absolute Weight 

*0° 1 

Relative Weight 

Vehicle 2000 10000 20000 - 50 

DlPE CPS 

(rng/m 3) (mg/kg) 
Protocol No. HLS Study No. 006130 

Abbreviated Title: Immunological Evaluation of Gasoline DlPE Vapor Condensate 

IT1 Study No. IT1 1001 

Figure 2 

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

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

Absolute Weight 

800 1 

Vehicle 2000 10000 20000 - 50 

DlPE CPS 

(mg1m3) (mg/kg) 

Relative Weight 

SPLEEN IGM ANTIBODY RESPONSE TO THE T-DEPENDENT ANTIGEN, SRBC. DAY 4 RESPONSE. 

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 72% with the exception of one sample, which had a viability of 69.8%. 

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

exposure to Gasoline DlPE 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 DlPE Vapor Condensate as 

compared to the vehicle control group. There was a slight, albeit not statistically significant increase in 

spleen cell number in the low dose group consistent with effects seen with spleen weights. The effect of 

DlPE on spleen weights and spleen cell number can not be appropriately evaluated since the animals in 

this study were sensitized with sheep erythrocytes. A more appropriate approach to evaluate the effects 

of DlPE on spleen parameters would be to evaluate unsensitized exposed animals. As expected, the 
Protocol No. HLS Study No. 005130 

Abbreviated Title: Immunological Evaluation of Gasoline DlPE Vapor Condensate 

IT1 Study No. IT1 1001 

positive control, cyclophosphamide (CPS), produced an 80% decrease in spleen cell number when 

compared to the vehicle control group. 

Figure 3 

Spleen Cell Number in Female Sprague Dawley Rats Exposed to Gasoline DlPE 

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

Vehicle 2000 10000 20000 50 

DlPE CPS 

(mg/m3) (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. While the response of the vehicle animals was 

somewhat lower than had been observed in the past, the positive and negative control animal responses 

are consistent with the range of responses observed with the Sprague Dawley outbred strain of rats. As 

can be seen, a dose-related decrease in the IgM-antibody-forming cell response to the T-dependent 

antigen, sRBC, was observed when the data were evaluated as either specific activity (AFCIO' spleen) 

or as total spleen activity (AFCIspleen). For both parameters, exposure to Gasoline DlPE Vapor 

Condensate reached the level of statistical significance at the high exposure level. When evaluated as 

specific activity, the response was suppressed 63% for the high exposure level. When evaluated as total 

spleen activity, the response was suppressed 64% for the high exposure level. 
Protocol No. HLS Study No. 00-6130 

Abbreviated Title: lmrnunological Evaluation of Gasoline DlPE Vapor Condensate 

IT1 Studv No. IT1 1001 

As anticipated, the positive control, CPS, produced a significant decrease in specific activity (1 00%) and 

total spleen cell activity (1 00%) when compared to the vehicle control animals. 

Figure 4 

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

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

Specific Activity Total Spleen Activity 

vehicle 2000 10000 20000 - 50 

DlPE CPS 

(mg/m3) (mglkg) 

Vehicle 2000 10000 20000 50 - 

DlPE CPS 

(mg/m3) (mg/kg) 

VIII. CONCLUSION 

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

week for 4 weeks resulted in a dose-related decrease in the humoral immune response to the Tdependent antigen, sheep erythrocytes. Although the functional ability of the animals was reduced, there 

was no statistically significant effect on body weight, absolute spleen weight, thymus weight, or spleen 

cell number. Based on the immunological parameters evaluated, under the experimental conditions of the 

study, exposure to Gasoline DlPE Vapor Condensate adversely affected the humoral immune response 

of female Sprague Dawley rats. 
Protocol No. HLS Study No. 004130 

Abbreviated Title: immunological Evaluation of Gasoline DlPE Vapor Condensate 

IX. REFERENCES 

IT1 Studv No. IT1 1001 

Luster MI, Portier 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. 1 8:200-2 10. 

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

Methodology and theory. Trnspl. Rev. 18:13O-I9l. 

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

(1988). Development of a testing battery to assess chemical-induced immunotoxicity. Fund. Appl. 

Toxicol. 10:2-19. 

Bartlett MS (1937). Sub-sampling for attributes. J. Roy. Stat. Soc. Suppl. 4:131-135. 

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

ASSOC. 47:583-621. 

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

co.ntrol. J. Amer. Stat. Assoc. 50:1096-1121. 

Gross AJ and Clark VA (1975). Gehan-Wilcoxon 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. 124-129. 

Sokal RR and Rohlf FJ (1 981). Biometry. Freeman, San Francisco, p. 226-231. 
Protocol No. HLS Study No. 005130 

Abbreviated Title: lmmundogical Evaluation of Gasoline DlPE Vapor Condensate 

Table 1 

IT1 Study No. IT1 1001 

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

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

Parameter Vehicle Gasoline DIPE Vavor (mdm3) Cvclovhos~hamide WNH Trend 

2000 10000 20000 50mg/kg Analysis 

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

Body Wgt (g) 251.6 + 4.3 254.5 + 4.9 249.8 + 3.5 247.9 + 5.4 228.7 + 4.6** H NS 

Spleen (mg) 544 + 24 667 rt: 30 579 rt: 33 604 + 39 267 + 14** H NS 

% Body Wgt 0.215 + 0.010 0.261 f 0.010* 0.232 f 0.010 0.242 f 0.013 0.118 + 0.005** H NS 

Thymus (mg) 526 + 24 540 + 33 528 21 550 f 45 122 + 9** H NS 

% Body Wgt 0.209 + 0.008 0.214 + 0.015 0.211 + 0.007 0.220 + 0.016 0.052 f 0.004** NH NS 

Female Sprague Dawley rats 

exDosure for 5 days per week 

were administered vehicle control (air only) or gasoline DlPE vapor condensate by inhalation via whole-body 

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: mg = milligrams; m3 = cubic meter of air; kg = kilograms; Wgt = weight; NS = not significant for p values greater than 0.05. 
Pmtocd No. HLS Study No. 008130 

Abbreviated Title: lmmundogical Evaluation of Gasoline DIPE Vapor Condensate 

Table 

TTI Studv No. IT1 1001 

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

Exposed to Gasoline DlPE Vapor Condensate via Inhalation for 5 Days per Week for 4 Weeks - Day 4 Response 

Exposure Body Wgt Spleen Wgt Spleen Cells IgM AFCJ IgM AFCISpleen 

(g) (mg) (XI 07) lo6 Spleen Cells (X lo3) 

Vehicle 251.6 2 4.3 544 f 24 62.65 * 2.50 688 + 128 440 k 95 

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

Gasoline DIPE Vapor Condensate 

2000 mg/m3 254.5 k 4.9 667 * 30 75.17 * 4.34 657 + 166 503 + 137 

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

14 3 377 + 106 

1 (10) 

44** 159 26** 

1 (10) 

10000 mg/m3 249.8 + 3.5 579 + 33 65.87 * 3.29 553 k 

(10) (10) (10) (10 

20000 mg/m3 247.9 + 5.4 604 + 39 64.82 * 3.92 252 + 

(10) (10) (10) (10 

Cyclophosphamide 

228.7 + 4.6** 267 f 14** 12.52 f 0.73** 0 + 

H/NH H 

Trend Analysis NS 

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

whole-body exposure for 5 days per week for 4 weeks. The positive control, cyclophos hamide, was administered i.p. the last 4 f: 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 = 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. Nonhomogeneous data were evaluated using a non-parametric analysis of variance. When significant differences occurred, 

exposed groups were compared to the vehicle control group using the Wilcoxon Rank 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 Jonckheerens Test was used to test for doserelated 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. 005130 

Abbreviated Title: Immunological Evaluation of Gasoline DlPE Vapw Condensate 

APPENDIX A 

INDIVIDUAL ANIMAL DATA 

IT1 St udv No. IT1 1001 
INDIVIDUAL ANIMAL DATA 

ORGAN WEIGHTS 

GASOLINE DlPE VAPOR CONDENSATE 

00-6130 

ANIMAL NO GROUP DOSE SEXBODY WGT (G SPLEEN WGT (MG THYMUS WGT (MGSPLEEN WGT I % BODY WCHYMUS WGT I % BODY WGT 

1581 GI AIR ONLY F 281.3 564 534 0.200 0.190 

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 

GIV 

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 

2.000 MGIM' DlPE 

2,000 MGIM' DlPE 

2,000 MGIM' DlPE 

2.000 MGIM' DlPE 

2.000 MGIM' DlPE 

2.000 MGIM' DlPE 

2,000 MGIM' DlPE 

2.000 MGIM' DlPE 

2.000 MGIM' DlPE 

2.000 MG/M~ DlPE 

10.000 MGIM' DlPE 

10.000 MGIM' DlPE 

10.000 MGIM' DlPE 

10.000 MG/M~ DlPE 

10,000 MGIM' DlPE 

10.000 MGIM' DlPE 

10,ooo MGIM%IPE 

10,ooo MGIM%IPE 

10,ooo MGIM~IPE 

IO,OOO MGIM%IPE 

20.000 MGIM' DlPE 

20.000 MGIM' DlPE 

20.000 MGIM~IPE 

20.000 MGIM' DlPE 

20.000 MGIM' DlPE 

20.000 MGIM' DlPE 

20.000 MGIM' DlPE 

20.000 MGIM' DlPE 

20.000 MGIM' DlPE 

20.000 MGIM' DlPE 

50 MGIKG CYCLOPHOSPHAMIDI F 

50 MGIKG CYCLOPHOSPHAMIDI F 

50 MGIKG CYCLOPHOSPHAMIDI F 

50 MGIKG CYCLOPHOSPHAMIDI F 

50 MGIKG CYCLOPHOSPHAMIDI F 

50 MGIKG CYCLOPHOSPHAMIDI F 

50 MGIKG CYCLOPHOSPHAMIDI F 

iO MGIKG CYCLOPHOSPHAMIDI F 

I0 MGIKG CYCLOPHOSPHAMIDI F 

50 MGIKG CYCLOPHOSPHAMIDI F 
INDIVIDUAL ANIMAL DATA 

IGM AFC ASSAY 

GASOLINE DlPE VAPOR CONDENSATE 

00-6130 

ANIMAL NO GROUP DOSE SEXIGM ~~~110~ SP.CIGM AFCISPLEEN 10: CELLSISPLEEN ~O~SPLEEN WGT (MG BODY WGT (G) 

1581 GI AIR ONLY F 814 528 64.86 564 281.3 

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 

GIV 

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 

2,000 MGIM' DlPE 

2,000 MGIM' DlPE 

2.000 MGIM' DlPE 

2.000 MGIM%IPE 

2.000 MGIM~ DlPE 

2.000 MGIM~ DlPE 

2.000 MGIM' DlPE 

2.000 MGIM' DlPE 

2.000 MGIM' DlPE 

2.000 MGIM%IPE 

10.000 MGIM' DlPE 

10.000 MGIM' DIPE 

10,000 MGIM' DIPE 

10.000 MGIM' DlPE 

10.000 MGIM' DlPE 

10.000 MGIM' DlPE 

10,000 MGIM' DlPE 

10,000 MGIM' DlPE 

io.ooo MGIM%IPE 

10.000 MGIM' DlPE 

20.000 MGIM' DlPE 

20,000 MGIM' DlPE 

20,000 MGIM' DIPE 

20.000 MGIM' DIPE 

20.000 MGIM' DIPE 

20.000 MGIM' DlPE 

20.000 MGIM' DlPE 

20.000 MGIM' DlPE 

20.000 MG/M~ DlPE 

20.000 MGIM' DlPE 

50 MGIKG CYCLOPHOSPHAMIDI 

50 MGIKG CYCLOPHOSPHAMIDI 

50 MGIKG CYCLOPHOSPHAMIDI 

50 MGIKG CYCLOPHOSPHAMIDI 

50 MGIKG CYCLOPHOSPHAMIDI 

50 MGIKG CYCLOPHOSPHAMIDI 

50 MGIKG CYCLOPHOSPHAMIDI 

50 MGIKG CYCLOPHOSPHAMIDI 

50 MGlKG CYCLOPHOSPHAMIDI 

50 MGIKG CYCLOPHOSPHAMIDI 
Protocol No. HLS Study No. 00-6130 

Abbreviated Title: lmrnunological Evaluation of Gasoline DIPE Vapor Condensate - IT1 Study No. IT1 1001 

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 generationlcharacterization and animal 

evaluations. 

Huntingdon Life Sciences 00-6 130 Page 1 

2 1 1 -DIPE-S Draft Final Report 

STUDY DATES: Date of Animal Receipt: 24 January 2002 

Animal Exposure and Animal Data 

Preface 

Experimental Initiation Date: 14 February 2002 (in-life) 

Appendix B 

Experimental Completion Date: 14 March 2002 (in-life) 

Draft Report Date: 6 March 2003 

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 

Chamber Monitoring Results ........................................................................................... 

Summary of Clinical Observations .................................................................................. 

Mean Body Weights (grams) ........................................................................................... 

Mean Body Weight Change (grams) ................................................................................ 

Mean Feed Consumption Values (gramslkglday) ............................................................ 

Individual Clinical Observations ..................................................................................... 

Individual Body Weights (grams) ................................................................................... 

Individual Body Weight Change (grams) ........................................................................ 

Individual Feed Consumption Values (gramslkglday) ..................................................... 

Animal Termination History ............................................................................................ 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Chamber Monitoring Results 

Cumulative Exposure Record 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Chamber Monitoring Results 

Cumulative Exposure Record 

Day 

0 

1 

4 

5 

6 

7 

8 

11 

12 

13 

14 

15 

18 

19 

20 

21 

22 

25 

26 

27 

Date 

14-Feb-02 

15-Feb-02 

18-Feb-02 

19-Feb-02 

20-Feb-02 

21-Feb-02 

22-Feb-02 

25-Feb-02 

26-Feb-02 

27-Feb-02 

28-Feb-02 

01-Mar-02 

04-Mar-02 

05-Mar-02 

06-Mar-02 

07-Mar-02 

08-Mar-02 

11-Mar-02 

12-Mar-02 

13-Mar-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 0 

Group IB - 0 rnglm3 (Air Control) 

Nominal 

(mg/m3) 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0.891 0.6 

Particle Size 

4.3 

Chamber Environment 

Mean 

(rnglm3) 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

MMAD 

(clrn) 

1.230 

3.222 

2.090 

1.465 

2.002 

0.290 

Temperature 

("C) 

24 

24 

24 

24 

24 

24 

24 

24 

25 

24 

23 

24 

24 

24 

24 

25 

24 

24 

23 

22 

23.9 

2.59E-03 

Mean 

Humidity 

60 

58 

57 

59 

55 

54 

57 

54 

59 

56 

69 

58 

55 

45 

55 

59 

55 

55 

55 

56 

56.6 

Determinations 

GSD 

1.627 

2.309 

1.807 

1.966 

1.927 

Analytical Chamber Concentration 

Individual TMC 

(m91m3) 

1.72E-03 

7.63E-03 

3.69E-03 

2.72E-03 

3.94E-03 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

(rnglm3) 

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 DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Date Exposure 

Number 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

Mean 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group llA - 2,000 mg/m3 

Particle Size 

Nominal 

(mg1m3) 

1950 

21 50 

2010 

2090 

2060 

21 30 

2000 

2090 

1970 

1990 

2030 

2030 

2050 

1900 

21 00 

2090 

21 50 

2020 

201 0 

2010 

2042 

66 

Mean 

(mg1m3) 

1953 

2085 

1995 

2025 

I985 

1998 

1950 

1995 

I998 

I958 

201 0 

2035 

I998 

I865 

2000 

1915 

I983 

I928 

I883 

I988 

alytical Chamber Concentration 

Individual 

(ms 

1990 

2170 

2000 

2010 

1980 

1940 

1610 

1900 

1910 

1960 

1980 

2050 

1980 

1730 

1990 

1750 

2030 

1820 

21 30 

2020 

1977 

102 

MMAD 

(pm) 

1.422 

1.182 

1.832 

1.506 

1 A86 

0.269 

1s 

TMC 

(mg/m3) 

3.09E-03 

4.38E-03 

2.65E-03 

3.51 E-03 

3.41 E-03 

7.37E-04 

Chamber 

M 

remperature 

("C) 

23 

24 

24 

24 

24 

24 

24 

24 

25 

24 

23 

23 

24 

23 

23 

24 

23 

24 

23 

22 

23.6 

0.7 

vironment 

n 

Humidity 

52 

50 

50 

52 

48 

48 

4-9 

48 

50 

48 

51 

52 

50 

42 

50 

55 

50 

52 

50 

49 

49.8 

2.5 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Date Exposure 

Number 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

Mean 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group IIB - 2,000 mg/m3 

Nominal 

(mg/m3) 

1950 

21 50 

201 0 

2090 

2060 

21 30 

2000 

2090 

1970 

1990 

2030 

2030 

2050 

1900 

2100 

2090 

2150 

2020 

2010 

201 0 

2042 

66 

Mean 

(mg/m3) 

I983 

2178 

2135 

2063 

1943 

2035 

1903 

2070 

I938 

1993 

1975 

2050 

2113 

1918 

I998 

2028 

1958 

2023 

I963 

1973 

alytical Chamber Concentration 

Individual MMAD 

(w) 

1.589 

1.378 

3.380 

1.174 

1.880 

1.014 

Particle Size 

ns 

TMC 

(mg/m3) 

3.46E-03 

5.03E-03 

3.14E-03 

2.54E-03 

3.54E-03 

I .06E-03 

Chamber Environment 

N 

Temperature 

("C) 

23 

23 

24 

23 

23 

23 

23 

23 

24 

23 

23 

23 

23 

23 

22 

23 

22 

23 

23 

22 

23.0 

0.5 

In 

Humidity 

(%) 

56 

55 

54 

55 

5 1 

50 

51 

49 

54 

52 

55 

55 

52 

43 

52 

56 

51 

53 

52 

52 

52.4 

3.0 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Exposure 

Number 

Day 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

Mean 

S.D. 

Date 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group lllA - 10,000 mglm3 

Nominal 1 Mean , ~alytical Chamber Concentration 

Individual MMAD 

(elm) 

0.9889 

1.043 

1.708 

2.245 

1.496 

0.597 

Particle Size 

Determinat 

GSD 

ivironment 

an 

Humidity 

47 

47 

47 

50 

49 

51 

50 

47 

49 

47 

47 

47 

46 

39 

47 

51 

50 

47 

47 

49 

47.7 

2.6 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Date Exposure 

Number 

13-Mar-02 1 20 

Mean 

S.D. 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group lllB -10,000 mglm3 

Nominal 

(mglm3) 

9540 

9890 

9830 

9740 

10800 

9910 

9950 

9840 

9990 

9920 

101 00 

10100 

9200 

9890 

9730 

9860 

9960 

9810 

9890 

9700 

9883 

293 

Mean 

(mglm3) 

10650 

10270 

9998 

1001 0 

10150 

101 50 

10240 

9480 

10120 

10740 

10650 

1001 0 

9955 

10140 

9743 

101 70 

9590 

9655 

9995 

9168 

~alytical Chamber Concentration 

Individual MMAD 

(IJ~) 

1.351 

1.013 

1.729 

1.443 

1.384 

0.295 

Particle Size 

:terminal 

GSD 

1s 

TMC 

(mglm3) 

4.54E-03 

6.20E-03 

2.53E-03 

3.34E-03 

4.1 5E-03 

1.60E-03 

Chamber Environment 

Me2 

Temperature 

("C) 

24 

24 

24 

24 

24 

24 

24 

24 

25 

24 

24 

24 

24 

23 

23 

24 

23 

23 

24 

23 

23.8 

0.5 

In 

Humidity 

47 

47 

46 

46 

46 

47 

45 

45 

49 

46 

47 

47 

46 

37 

44 

47 

46 

44 

45 

48 

45.8 

2.4 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Chamber Monitoring Results 

Date 

Cumulative Exposure Record 

Group IVA - 20,000 mg/m3 

, 

Number 

S.D. 1 538 

Mean 

(mg/m3) 

19480 

20430 

20600 

201 30 

20650 

20550 

21400 

20480 

20500 

20600 

20030 

20300 

20750 

20950 

20500 

19900 

20050 

19680 

20130 

20950 

Analvtical Chamber Concentration 

Individual 

Particle Size 

IS 

TMC 

(mg/m3) 

4.97E-03 

1.15E-02 

1.83E-03 

4.54E-03 

5.71 E-03 

4.10E-03 

MMAD 

(1.m 

1.779 

3.198 

1.192 

1.358 

1.882 

0.912 

Chamber 

rv 

Temperature 

("C) 

25 

25 

25 

25 

24 

24 

24 

24 

26 

25 

25 

25 

25 

24 

24 

25 

24 

25 

25 

22 

24.6 

0.8 

Determinat 

GSD 

2.630 

3.812 

1.779 

2.17 

2.598 

0.881 

~vironment 

In 

Humidity 

(%) 

50 

5 1 

49 

50 

50 

53 

51 

50 

53 

50 

50 

5 1 

49 

40 

49 

52 

52 

48 

49 

51 

49.9 

2.7 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Date 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group IVB - 20,000 mdm3 

Exposure Nominal 

Number I (mglm3) 

Mean 1 19430 

S.D. 1 538 

Analytical Chamber Concentration 

Mean I Individual MMAD 

I Particle Size 

?terminat 

GSD 

Chamber Environment 

Mean 

IS Temperature I Humidity 

TMC I I 
Huntingdon Life Sciences 00-61301 

211-DIPE-S 

PAGE 1 

TABLE B 

GASOLINE DIPE 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 

DEAD 

TERMINAL SACRIFICE 

Huntingdon Life Sciences 00-61301 

211-DIPE-S 

PAGE 1 

TABLE D 

GASOLINE DIPE 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 WEIGHT CHANGE FROM BASELINE (GRAMS) ................................................................................................................................... 

DOSE GROUP: 1 2 3 4 5 

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

WEEK 0 TO 1 

WEEK 0 TO 2 

WEEK 0 TO 3 

WEEK 0 TO 4 

------------------ 

Statistical key: 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N ---------------- 
Huntingdon Life Sciences 00-61301 

211-DIPE-S 

PAGE 1 

TABLE E 

GASOLINE DIPE 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 MEAN 

S.D. 

N 

WEEK 1 MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

WEEK 2 

WEEK 3 MEAN 

S.D. 

N 

WEEK 4 MEAN 

S.D. 

N 

No statistically significant differences 
Huntingdon Life Sciences 00-61301 

211-DIPE-S 

PAGE 1 

TABLE F 

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

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMLTNOTOXICITY ASSESSMENTS 

INDIVIDUAL CLINICAL OBSERVATIONS 

FEMALES GROUP 1 0 MG/M3 ................................................................................................................................... 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE ................................................................................................................................... 

CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT 
Huntingdon Life Sciences 00-61301 

211-DIPE-S 

PAGE 2 

TABLE F 

GASOLINE DIPE 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 2 2000 MG/M3 ................................................................................................................................... 

ANIMAL# OBSERVATIONS 

DAY OF 12 

STUDY 3 8 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE ...................................................... 

CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT 
Huntingdon Life Sciences 00-61301 

211-DIPE-S 

TABLE F 

PAGE 3 

GASOLINE DIPE 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 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 
Huntingdon Life Sciences 00-61301 

211-DIPE-S 

PAGE 4 

TABLE F 

GASOLINE DIPE 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 4 20000 MG/M3 ................................................................................................................................... 

OBSERVATIONS 

............................. 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE ................................................................................................................................... 

CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT 
Huntingdon Life Sciences 00-61301 

211-DIPE-S 

TABLE F 

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

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

PAGE 5 

LLES GROUP 5 POS 

INDIVIDUAL CLINICAL OBSERVATIONS 

IVE CONTROL 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS P 

TERMINAL SACRIFICE P 

CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT 
Huntingdon Life Sciences 00-61301 

211-DIPE-S 

PAGE 1 

TABLE G 

GASOLINE DIPE 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 00-61301 

211-DIPE-S 

PAGE 2 

TABLE G 

GASOLINE DIPE 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 00-61301 

211-DIPE-S 

PAGE 3 

TABLE G 

GASOLINE DIPE 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 3 10000 MG/M3 ................................................................................................................................... 

WEEK OF STUDY 

AN I MAL# - 1 0 1 2 

-------------------------------------+---- 

3571 

3572 

3573 

3574 

3575 

3576 

3577 

3578 

3579 

3580 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-61301 

211-DIPE-S 

PAGE 4 

TABLE G 

GASOLINE DIPE 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 00-61301 

211-DIPE-S 

PAGE 5 

TABLE G 

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

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

5551 

5552 

5553 

5554 

5555 

5556 

5557 

5558 

5559 

5560 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-61301 

211-DIPE-S 

PAGE 1 

TABLE H 

GASOLINE DIPE 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 1 0 MG/M3 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-61301 

211-DIPE-S 

PAGE 2 

TABLE H 

GASOLINE DIPE 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 2 2000 MG/M3 

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 00-61301 

211-DIPE-S 

PAGE 3 

TABLE H 

GASOLINE DIPE 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 00-61301 

211-DIPE-S 

PAGE 4 

TABLE H 

GASOLINE DIPE 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 00-61301 

211-DIPE-S 

PAGE 5 

TABLE H 

GASOLINE DIPE 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 00-61301 

211-DIPE-S 

PAGE 1 

TABLE I 

GASOLINE DIPE 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 

SF=Spilled Feeder 
Huntingdon Life Sciences 00-61301 

211-DIPE-S 

PAGE 2 

TABLE I 

GASOLINE DIPE 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 00-61301 

211-DIPE-S 

PAGE 3 

TABLE I 

GASOLINE DIPE 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 3 10000 MG/M3 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-61301 

211-DIPE-S 

PAGE 4 

TABLE I 

GASOLINE DIPE 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 4 20000 MG/M~ 

MEAN 

S.D. 

N 

SF=Spilled Feeder 
Huntingdon Life Sciences 00-61301 

211-DIPE-S 

PAGE 5 

TABLE I 

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

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

5551 

5552 

5553 

5554 

5555 

5556 

5557 

5558 

5559 

5560 

MEAN 

S.D. 

N 

SF=Spilled Feeder 
Huntingdon Life Sciences 00-61301 

211-DIPE-S 

PAGE 1 

TABLE J 

GASOLINE DIPE 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 1 0 MG/M~ 

................................................................................................................................... 

TYPE OF DATE OF WEEK OF STUDY 

AN I MAL# DEATH DEATH STUDY DAY 

................................................................................................................................... 

1581 TERMINAL SACRIFICE 14 -MAR- 02 4 28 

1582 TERMINAL SACRIFICE 14-MAR-02 4 28 

1583 TERMINAL SACRIFICE 14 -MAR-02 4 28 

1584 TERMINAL SACRIFICE 14-MAR-02 4 28 

1585 TERMINAL SACRIFICE 14-MAR-02 4 2 8 

1586 TERMINAL SACRIFICE 14-MAR-02 4 28 

1587 TERMINAL SACRIFICE 14-MAR-02 4 2 8 

1588 TERMINAL SACRIFICE 14-MAR-02 4 2 8 

1589 TERMINAL SACRIFICE 14-MAR-02 4 2 8 

1590 TERMINAL SACRIFICE 14-MAR-02 4 28 
Huntingdon Life Sciences 00-61301 

211-DIPE-S 

PAGE 2 

TABLE J 

GASOLINE DIPE 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 2 2000 MG/M3 ................................................................................................................................... 

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 00-61301 

211-DIPE-S 

PAGE 3 

TABLE J 

GASOLINE DIPE 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 3 10000 MG/M3 

TYPE OF DATE OF WEEK OF STUDY 

AN I MAL# 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 00-61301 

211-DIPE-S 

PAGE 4 

TABLE J 

GASOLINE DIPE 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 4 20000 MG/M3 ................................................................................................................................... 

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 00-61301 

211-DIPE-S 

PAGE 5 

TABLE J 

GASOLINE DIPE 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 5 POSITIVE CONTROL 

TYPE OF 

DEATH 

DATE OF WEEK OF STUDY 

DEATH STUDY DAY ................................................................................................................................... 

5551 TERMINAL SACRIFICE 14-MAR-02 4 28 

5552 TERMINAL SACRIFICE 14-MAR-02 4 28 

5553 TERMINAL SACRIFICE 14 -MAR- 02 4 28 

5554 TERMINAL SACRIFICE 14-MAR-02 4 28 

5555 TERMINAL SACRIFICE 14-MAR-02 4 28 

5556 TERMINAL SACRIFICE 14 -MAR- 02 4 28 

5557 TERMINAL SACRIFICE 14-MAR-02 4 28 

5558 TERMINAL SACRIFICE 14 -MAR-02 4 28 

5559 TERMINAL SACRIFICE 14-MAR-02 4 28 

5560 TERMINAL SACRIFICE 14-MAR-02 4 2 8