Protocol No. HLS Study No.004130 IT1Study No. TTI 1001 Abbreviated Title: lrnmunological Evaluationof GasolineDlPEVapor Condensate p 

# 1'' 

# AUDITED DRAFTREPORT lmrnunological Evaluation of GasolineDlPE Vapor Condensatein Female Sprague Dawley Rats Using the Plaque Forming CellAssay

TestSubstance:
Protocol No: Subcontractor's Sponsoc

Contractor'sStudy Director: Sponsor:

Sponsor's Representative: Project Number: Security: Date: PrincipalInvestigator: Studies Conductedat: 

### Gasoline DlPEVapor Condensate HLS 00-6130 

### 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 

### September2002 

### Kimber L. White, Jr., Ph.D. ImmunoTox, Inc. Virginia BioeTechnology Research Park 800 

### East Leigh Street, Suite209 

### 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, Suite209 

### Richmond, VA 23219-1 534 (804) 828-6880 Phone (804) 828-6881 FaxProtocol No. HLS Study No. 005130 Abbreviated Title: Immunological Evaluationof Gasoline DlPEVapor Condensate



### I. GLP COMPLIANCE STATEMENT IT1Study 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 followingexceptions:
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 mixturewere not performed by the Testing Facility. The stability of the positive control article mixture has not been determined bythe Testing Facility.

Kimber 

### L.White, Jr., Ph.D. Principal Investigator IrnmunoTox, Inc. Date

Gary M. Hoffman, B.A., DABT Study Director Huntingdon Life Sciences DateProtocolNo. HLS Study No. 005130 IT1Study No. IT1 1001 AbbreviatedTitle: lrnmunologicalEvaluation of Gasoline DlPEVapor Condensate 

## -

II. QUALITY ASSURANCE STATEMENT

Test Substance: Gasoline DlPEVapor Condensate ReportTitle: lmmunological Evaluationof Gasoline DlPEVapor Condensate in FemaleSprague Dawley Rats Using the Plaque-FormingCellAssay Protocol Title: Gasoline DlPEVapor Condensate: A 13-Week Whole-Body InhalationToxicity Study in Rats with Neurotoxicity Assessments And 4-Week 

### In Wvo Genotoxicity and lmmunotoxicityAssessments
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 conductedthe following inspections and reported to the ImmunoTox, Inc. Principal Investigator, and then has submitted written reports of said inspectionsto the Study Director and Management. InspectionlAuditswere 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 ReportAudit

Approved and submitted by: QualityAssurance Manager Date

Office of Research

### * QualityAssurance Unit

### * Box980568 

### * Richmond, Virginia 23298 0568

### * (804) 828-6587 

### * Fax(804) 828-5604Protocol No. HLS Study No. 008130 IT1Study No. IT1 1001 Abbreviated Title: Immunological Evaluationof Gasoline DlPEVapor Condensate 

# -

HUNTINGDON LIFE SCIENCES QUALITY ASSURANCE STATEMENTProtocd No. HLS StudyNo. 008130 AbbreviatedTitle: lrnrnunologicalEvaluation of Gasoline DIP€Vapor Condensate



### Ill. SIGNATURE 

### OF PRINCIPALS lTlStudv 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 LifeSciences DateProtocolNo. HLS Study No

.006130 AbbreviatedTitle: lmmunologlcalEvaluationof Gasoline DlPEVapor Condensate IT1 Studv No

. IT1 I001



### TABLE OF CONTENTS

GLP Compliance Statement



## ................................................................................................................ 2 QualityAssurance Statements



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

### Huntingdon LifeSciences



## .................................................................................................................... 4 Signature of Principals



## ......................................................................................................................... 5 

### ExecutiveSummary



## ............................................................................................................................. 8 

### Introduction



## ..................................................................................................................................... 10 

### Methods of Procedure



## ....................................................................................................................... I I

ExperimentalDesign



## ...................................................................................................................... I I 

### Variables Assessed



## ........................................................................................................................ 11 

### Terminal Body and Organ Weights



## ............................................................................................. 11 

### Splenocyte Preparation



## ...............................................................................................................11 

### Spleen IgMAntibody Response to theT-dependent Antigen. sRBC



## . Day4'Response



## ............. 12 

### Data



## ................................................................................................................................................ 13



## ..................................................................................... Data Handlingand StatisticalAnalysis 13 

### Data Retention



## ............................................................................................................................ 13 

### Results



## ............................................................................................................................................... 13 

### Terminal Body and Organ Weights



## ................................................................................................ 13 

### Spleen IgMAntibody Response to the T-dependentAntigen, sRBC



## .Day4 Response



## ................ 15



## ......................................................................................................................................... Conclusion 17 

### References



## ...................................................................................................................................... 18 List of Figures

1


## . Absolute (mg) and Relative (%) 

### Spleen Weight in Female Sprague Dawley Rats Exposed to



## ............. Gasoline DlPEVapor Condensate via Inhalationfor 5 

### Days perWeek for 4 

### Weeks 14 

### 2



## . Absolute (mg) and Relative (%) 

### Thymus Weight in Female Sprague Dawley Rats Exposed to



## ............. Gasoline DlPEVapor.Condensatevia Inhalationfor 5 

### Days perWeek for 4 

### Weeks 15 3


## . Spleen Cell Number in 

### Female Sprague Dawley Rats Exposed to Gasoline DlPE Vapor Condensate via Inhalation for 5 

### Days perWeek for 4 

### Weeks



### ................................................. 16 4


## . IgMAntibody-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 DIPEVapor Condensatevia lnhalationfor 5 

### Days perWeek for 4 

### WeeksPmtocd No. HLS StudyNo.005130 IT1Study No. IT1 1001 AbbreviatedTIUe: lmrnunologicelEvaluationof GasolineDlPEVapor 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 perWeek for 

### 4 Weeks


# ............................................................................................ 20 

### APPENDICES A Individual Animal Data B 

### Contracting Sponsor's Exposureand Animal DataProtocolNo. HLS Study No. 008130 Abbreviated Title: immunological Evaluationof Gasdine DlPEVapor 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 immunologicalparameters for HuntingdonLife 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 responseto the T-dependent antigen, sheep erythrocytes. Female Sprague Dawley rats were administered Gasoline DlPEVapor 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 mglm3of the test substancewere 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 relativeweight (% 

### 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 therewas not a dose-dependent effect and the decrease at the low dose is not considered to be biologically significant. Therewas no effect in the thymus relativeweight.
Exposure to Gasoline DlPEVapor Condensate resulted in a statistically significant dose-relateddecrease 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 inthe various parametersevaluated.ProtocolNo. HLS StudyNo. 008130 AbbreviatedTitle: lrnrnunologicalEvaluationof Gasollne DlPEVapor Condensate IT1Studv No. IT1 1001

In conclusion, the results of this immunotoxicological evaluation demonstratethat, under the experimental conditions used, exposure to the Gasoline DlPEVapor Condensatetest substance adverselyaffected the functional ability of the humoral immune component of the immune system.

Table ES-1
SUMMARYTABLE FOR TOXICOLOGYAND IMMUNOLOGY STUDIES


### Parameter Result Maximum Effect ExposureLevel (mglm3) Terminal 

### Body

### Weight

Day 29 No Effect 

### OrganWeights(Absolute 

### and Relatlve)

Spleen (Absolute) No Effect Spleen (Relative) Increase 21% 2,000 Thymus (Absolute) No Effect Thymus (Relative) No Effect 

### Spleen IgMAntibody-Forming Cell Responseto Sheep Erythrocytes

IgM

### AFCI? 

### 0' Spleen Cells Decrease 63% 20,000 IgMAFCISpleen (xlo3) Decrease 64% 20,000Protocol No. HLS Study No. 00-6130 Abbreviated Title: lmmunologlcal Evaluation of Gasoline DlPEVapor Condensate IT1StudvNo. IT1 1001



### V. INTRODUCTION

The purpose of this study was to provide evaluation of immunological parameters for Huntingdon Life Sciences (HLS) Study No. 00-6130. In this study, the ability of the test substance. Gasoline DlPE Vapor Condensate,was evaluatedfor its ability to affect the humoral immune component of the immunesystem, 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 removedthe 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 immunologicalevaluationwas 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 responsiblefor carrying out the IgMantibody-formingcellassay.

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 protocolor standard operating proceduredeviations occurred during the study, which affectedthe quality of the data and the abilityto interpret the data with respect to the immunotoxicologyof Gasoline DlPEVapor Condensate.Protocd No. HLSStudy No. 004130 Abbreviated Title: lrnrnunological Evaluationof Gasoline DlPEVapor 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) personnelto either vehicle (air only) or Gasoline DlPE Vapor Condensate at exposure levels of 2,000, 10,000 or 20,000 mg/m3via 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 concentrationsof 5.0 mglmLfor use as the positive control for this study. The positivecontrol 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 (preparedat PRC), and shipped the spleens on ice in individualshipping containersat 2-8°C by carrier to ImmunoToxfor 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 immunotoxicologicalevaluation.

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.ProtocolNo. HLS Study

### No. 005130 IT1Study

### No. IT1 1001 Abbreviated Title: ImmunologicalEvaluationof GasolineDIP€Vapor Condensate 

# -

Spleen laM Antibody ResDonse to the T-de~endent Antigen, sRBC. Dav 4 

### Res~onse. The primary IgM response to sheep erythrocyteswas 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 cellsfrom each suspensionwas added to separatetest tubes, each containing25 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 approximately36-38°C for 3 hours. Spleen cell counts were performedon 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 plaqueviewer. 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 macrophagesare requiredto function properly in order to obtain an antibody-formingcell (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 immunotoxicologicalassays 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 responseinthe whole animal and, thus, has the potential for immunotoxicity. This assay is one of the Tier I

### assays used bythe NTP~.PmbcolNo. HLS Study No. 008130 Abbreviated Title: immunological Evaluationof Gasoline DIPEVapor Condensate

Data Handlina and StatisticalAnalvsis. The data obtained in this study were analyzed in accordance with standard operating procedure. Datawere first tested for homogeneity of variances usingthe 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 Test7when appropriate. The Jonckheere's ~est' was used to test for exposure levelrelatedtrends across the vehicle and exposed groups. The positive control was comparedto 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.



### Pvalues of 0.05 or less, as compared to the vehicle control group, were considered statisticallysignificant 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 returnedto 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 HuntingdonLife Sciences.

VII. RESULTS

TERMINAL 

### BODY AND 

### ORGAN WEIGHTS.

The terminal body weight data from the study are shown in Table 

### 1for the control and Test Substanceexposed groups. The terminal bodyweights 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 comparedto 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 whenProtoml No. HLS Study No. 008130 Abbreviated Title: lmrnunological Evaluationof Gasdine DlPEVapor 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, comparedto the vehicle control. Inaddition, the positivecontrol, cyclophosphamide, hada significant decrease of 45% on relative spleen weight and a 75% decrease on relative thymus weight, comparedto the vehicle control. Shown graphically in Figures 1 and 

### 2 is the effect on spleen and thymus weights followingexposure to Gasoline 

### DlPEVapor Condensate.
Figure 

### 1
Absolute (mg) and Relative(%}Spleen Weight in FemaleSprague Dawley Rats Exposedto Gasoline DlPEVapor Condensate via Inhalation for 

### 5 Days perWeekfor 

### 4Weeks

Absolute Weight



## *0°

# 1 Relative Weight

Vehicle 2000 10000 20000 50 

# - DlPE 

## CPS (rng/m

## 3) (mg/kg)Protocol No. HLS Study No. 006130 AbbreviatedTitle: ImmunologicalEvaluation of GasolineDlPEVapor Condensate IT1Study No. IT1 1001

Figure 

### 2
Absolute (mg) and Relative

### (%)Thymus Weight in Female Sprague Dawley Rats Exposed to Gasoline DlPEVapor Condensatevia Inhalationfor 

### 5 Days per Week for 

### 4 Weeks

AbsoluteWeight


### 800

# 1

Vehicle 

### 2000 10000 20000 50 

# - DlPE CPS 

### (mg1m3) (mg/kg) RelativeWeight

SPLEEN IGMANTIBODY RESPONSE TO THE T-DEPENDENT ANTIGEN, SRBC. DAY4 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 IgMAFC response in rats. Viabilities were conductedon 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 hada 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, theProtocolNo. HLS StudyNo. 005130 Abbreviated Title: Immunological Evaluation of Gasoline DlPEVapor Condensate IT1Study No. IT1 1001

positive control, cyclophosphamide (CPS), produced an 80% decrease in spleen cell number when compared to the vehicle control group. Figure3 Spleen Cell Number in Female Sprague Dawley Rats Exposedto Gasoline DlPE Vapor Condensatevia Inhalation for 

### 5 Days perWeek for 

### 4Weeks



### Vehicle 

## 2000 10000 20000 50 

## DlPE CPS (mg/m3) (mglkg)

Shown in Table 

### 2 and Figure

### 4 are the functional resultsfrom the IgM antibody-forming cell (AFC) assay. Shown in the left panel are the resultswhen 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 positiveand negative control animal responses are consistentwith 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 responsewas suppressed 63% for the high exposure level. When evaluated as total spleen activity, the responsewas suppressed 64%for the high exposure level.Protocol No. HLS Study No. 00-6130 Abbreviated Title: lmrnunological Evaluationof Gasoline DlPEVapor Condensate IT1Studv 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.
Figure4 IgMAntibody-Forming Cell Response to Sheep Erythrocytes in Female Sprague Dawley Rats Exposed to Gasoline DlPEVapor Condensate via Inhalation for 5 Days perWeek 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 DlPEVapor 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, underthe experimentalconditions of the study, exposure to Gasoline DlPE Vapor Condensate adversely affected the humoral immune response of female Sprague Dawley rats.ProtocolNo. HLS Study No. 004130 Abbreviated Title: immunological Evaluationof Gasoline DlPEVapor Condensate



### IX. REFERENCES IT1Studv 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 8:200-210.


### 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, MunsonAE, Thomas P, Holsapple MP, FentersJ, White KL, Jr., Lauer LD, and DeanJD (1988). 

### Development of a testing battery to assess chemical-induced immunotoxicity. Fund. Appl. Toxicol. 10:2-19. 

### Bartlett MS (1937). 

### Sub-samplingfor 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 BiomedicalSciences.

### AJ Gross and VA Clark, eds. JohnWiley and Sons, NewYork, 

### p. 120-123. 

### Hollander M and Wolfe DA (1975). 

### Jonckheere's Test: Non-parametric Statistics Methods, eds. M. 

### Hollander and D.A. Wolfe, JohnWiley and Sons, New York, p. 124-129. 

### Sokal RR and Rohlf FJ (1981). 

### Biometry. Freeman, San Francisco, p. 226-231.Protocol No. HLS Study No. 005130 Abbreviated Title: lmmundogical Evaluation ofGasoline DlPEVapor Condensate

Table 1 IT1Study No. IT1 1001

BodyWeight (g) and OrganWeights (mg) in Female Sprague Dawley Rats Exposedto

Gasoline DlPEVapor Condensatevia Inhalation for 5 Days perWeek 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 exDosurefor 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 icefor 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-homogeneousdata 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. TheJonckheere'sTest 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 greaterthan 0.05.PmtocdNo. HLS Study No. 008130 AbbreviatedTitle: lmmundogicalEvaluationof Gasoline DIPEVapor Condensate Table TTI Studv No. IT1 1001

Spleen Antibody-Forming Cell Response to T-dependent Antigen Sheep Erythrocytes in Female Sprague Dawley Rats Exposedto Gasoline DlPEVapor Condensate via Inhalation for 5 Days perWeek for 4 Weeks

## - Day4 Response 

### Exposure 

## Body Wgt Spleen

### Wgt Spleen Cells 

### IgM AFCJ IgM AFCISpleen (g) 

### (mg) (XI 07) lo6Spleen 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) 143 377 

## + 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 inhalationvia whole-body exposurefor 5days perweek for 4 weeks. The positive control, cyclophos hamide, was administered i.p. the last

### 4 

# f: days of exposure. Four days priorto 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 numberof IgM sRBC antibody-formingcells was determined. Values representthe 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 groupswere compared to the vehicle control group using the Wilcoxon RankTest. The positive control was compared to the vehicle control using the Student's t Test. Values significantly differentfrom vehicle control at p 

### 5 0.05 are indicated by an asterisk, while those significant at p 

### 50.01 are noted by a double asterisk.The Jonckheerens Testwas 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 0.05Protocol No. HLS Study No. 005130 Abbreviated Title: Immunological Evaluationof GasolineDlPEVapw Condensate



### APPENDIX A

INDIVIDUAL ANIMAL DATA IT1 St udv No. IT1 1001INDIVIDUALANIMAL DATA ORGAN WEIGHTS GASOLINE DlPEVAPOR CONDENSATE 00-6130

ANIMAL NO GROUP DOSE SEXBODY WGT (G SPLEEN WGT (MG THYMUS WGT (MGSPLEENWGT I % BODY WCHYMUS WGT I % BODYWGT 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 10.000MGIM' DlPE 10.000MGIM' DlPE 10.000MGIM' 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 I0MGIKG CYCLOPHOSPHAMIDI F 50 MGIKG CYCLOPHOSPHAMIDI FINDIVIDUAL ANIMAL DATA IGMAFC ASSAY GASOLINE DlPEVAPOR CONDENSATE 00-6130

ANIMAL NO GROUP DOSE SEXIGM ~~~110~ SP.CIGM AFCISPLEEN 10: CELLSISPLEEN ~O~SPLEEN WGT (MG BODYWGT (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 MGIKGCYCLOPHOSPHAMIDI 50 MGIKGCYCLOPHOSPHAMIDI 50 MGIKG CYCLOPHOSPHAMIDI 50 MGIKGCYCLOPHOSPHAMIDI 50 MGIKG CYCLOPHOSPHAMIDI 50 MGIKGCYCLOPHOSPHAMIDI 50 MGIKGCYCLOPHOSPHAMIDI 50 MGIKGCYCLOPHOSPHAMIDI 50 MGlKG CYCLOPHOSPHAMIDI 50 MGIKGCYCLOPHOSPHAMIDIProtocol No. HLS Study No. 00-6130 Abbreviated Title: lmrnunologicalEvaluationof Gasoline DIPEVapor Condensate IT1Study 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 studyreport shouldbe referenced for detailsof the procedures used for test atmosphere generationlcharacterization and animal evaluations.

Huntingdon Life Sciences 00-6130 Page 1 2 11 -DIPE-S Draft Final Report



## STUDY DATES: Date of Animal Receipt: 24 January 2002 Animal Exposure and Animal Data Preface

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

ExperimentalCompletion Date: 14March 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



## ........................................................................................... Summaryof Clinical Observations



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



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



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



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



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



## ................................................................................... IndividualBody Weight Change (grams)



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



## ..................................................... Animal Termination History



## ............................................................................................TableA GASOLINE DlPEVAPOR CONDENSATE:A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS

Chamber Monitoring Results Cumulative Exposure RecordTable A GASOLINE DlPEVAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATIONTOXICITY 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 MeanHumidity

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 0Table A GASOLINE DlPEVAPOR CONDENSATE: A 13-WEEK 00-6130 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 2150 2010 2090 2060 2130 2000 2090 1970 1990 2030 2030 2050 1900 2100 2090 2150 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 2130 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

3.41E-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 2.5Table A GASOLINE DlPEVAPOR CONDENSATE:A 13-WEEK 00-6130 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 2150 2010 2090 2060 2130 2000 2090 1970 1990 2030 2030 2050 1900 2100 2090 2150 2020 2010 2010 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 3.0Table A GASOLINE DlPEVAPOR 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 2.6TableA GASOLINE DlPEVAPOR CONDENSATE: A 13-WEEK 00-6130 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

4.15E-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 InHumidity

47 47 46 46 46 47 45 45 49 46 47 47 46 37 44 47 46 44 45 48 45.8 2.4Table A GASOLINE DlPEVAPOR 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 ParticleSize 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 2.7Table A GASOLINE DlPEVAPOR CONDENSATE:A 13-WEEK 00-6130 WHOLE-BODY INHALATION TOXICITY STUDY IN RATS

Date Chamber Monitoring Results Cumulative Exposure Record

Group IVB - 20,000 mdm3

Exposure Nominal Number (mglm3) 

# I

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 IHuntingdon 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 SACRIFICEHuntingdon 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 differencesHuntingdon 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-PRESENTHuntingdon 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-PRESENTHuntingdon 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 SACRIFICEHuntingdon 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-PRESENTHuntingdon 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-PRESENTHuntingdon 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.

NHuntingdon 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.

NHuntingdon 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.

NHuntingdon 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.

NHuntingdon 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.

NHuntingdon 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.

NHuntingdon 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.

NHuntingdon 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.

NHuntingdon 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.

NHuntingdon 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.

NHuntingdon 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 FeederHuntingdon 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.

NHuntingdon 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.

NHuntingdon 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.

NSF=Spilled FeederHuntingdon 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.

NSF=Spilled FeederHuntingdon 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 28Huntingdon 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 SACRIFICEHuntingdon 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 ANI MAL# DEATH DEATH STUDY DAY

TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICEHuntingdon 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 SACRIFICEHuntingdon 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