ProtocolNo. HLSStudyNo. 00-6131 IT1 Study No. IT1 1101 Abbr~.ed.Title: immunological Evaluationof GasolineTBA Vapor Condensate 

### v " " 

# Pd AUDITED DRAFTREPORT 

# lrnmunological Evaluation of Gasoline TBA 

# Vapor Condensate

# in Female Sprague Dawley Rats Using the Plaque Forming CellAssay

TestSubstance: ProtocolNo: Subcontractor's Sponsor:

Contractor's Study Director: Sponsor:

Sponsor's Representative:
Immunotox's Project Number: Security: Date: Principal Investigator: Studies Conductedat: GasolineTBA Vapor Condensate


### HLS 00-61 31 HuntingdonLife Sciences Mettlers Road East Millstone, NJ 

### 08875 (732)873-2550Phone 

### (732)873-3992Fax 

### Gary 

### M. Hoffman, B.A., 

### DABT American Petroleum Institute 

### 1200 L 

### Street, NW Washington, DC 

### 20005 Thomas Gray, 

### M.S., DABT



### 30 January 

### 2004 Kimber L. White, Jr., Ph.D. ImmunoTox, Inc. Virginia BioaTechnology Research Park 

### 800 East Leigh Street, Suite 

### 209 Richmond,VA 

### 23219-1 534 (804) 828-6880Phone 

### (804)828-6881Fax



## ImmunoTox, Inc. Virginia 

### Bio~Technology Research Park 800 East Leigh Street, Suite 

### 209 Richmond,VA 

### 23219-1 534 (804) 828-6880Phone 

### (804) 828-6881FaxProtocol No. HLS Study No. 00-6131 AbbreviatedTitle: lmrnunologicalEvaluationof GasolineTEA Vapor Condensate


### I. GLP COMPLIANCE 

### STATEMENT IT1 Study No. IT1 1101

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

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

Kimber L. White, 

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

Gary 

### M. Hoffman, B.A., DABT Study Director Huntingdon Life Sciences DateProtocol No. HLS Study No. 00-6131 IT1 Study No. IT1 1101 AbbreviatedTitle: immunological Evaluation of GasolineTBA Vapor Condensate 

### ..

II. QUALITY ASSURANCE STATEMENT

# - IMMUNOTOX

Test Substance: Gasoline TBA Vapor Condensate Report Title: lrnrnunological Evaluation of Gasoline TBA Vapor Condensate in FemaleSprague Dawley Rats Usingthe Plaque-Forming Cell Assay ProtocolTitle: Gasoline TBA Vapor Condensate: A 13-Week Whole-Body Inhalation Toxicity Study in Rats with Neurotoxicity Assessments And 4-Week 

### In Vivo Genotoxicity and lmmunotoxicity Assessments Huntingdon Life Sciences, Inc. Study No. 00-6131 Sponsor Study No. 211 -TBA-S The final report for the indicated protocol has been reviewed by the Quality Assurance Unit of Virginia Commonwealth University. Furthermore,the Quality Assurance Unit has conductedthe following inspections and reported to the ImmunoTox, Inc. Principal Investigator, and then has submitted written reports of said inspectionstothe Study Director and Managementvia the Principal Investigator. Inspection/Audits were performedand reported on the following dates: 

## I Performed 

## I Reportedto ( Reportedto S.D. and 

## I Activity



### Approved and submittedby: QualityAssurance Manager Date

OfficeofResearch 

### * QualityAssurance Unit 

### * Box 980568 

### * Richmond, Virginia 23298 0568

### * (804) 828-6587 

### * Fax (804) 828-5604



### HUNTINGDONLIFE SCIENCES QUALITY ASSURANCE STATEMENTProtocol No. HLSStudy No. 00-6131 AbbreviatedTitle: immunological Evaluationof GasolineTEA Vapor Condensate IT1 Study No. IT1 1101



## HUNTINGDON LIFE

## SCIENCES QUALITY ASSURANCE STATEMENT
Listedbelow are the dates that this studywas inspectedby the Quality AssuranceUnit of Huntingdon Life Sciences,East Millstone,New Jersey, and the dates that findings were reported to the Study Director and Management. Thisreport reflects the raw data as 

### far as can be reasonably established. 

### Reported to StudyDirector and Type ofInspection Date(s) ofInspection Management 

## GLP Protocol Review 9 Apr 02 PositiveControlImmuno 19Jul02



## -Toxicity Dose Adm. Immunotoxicity Blood 23 

### JulO2 Collection &

### Necropsy 9 Apr 02 19Jul02

Fran Jannone, B.A., RQAP-GLP Quality Assurance SeniorAuditor DateProtocol No. HLS Study No. 00-6131 Abbreviated Title: lmmunological Evaluation of GasolineTEA Vapor Condensate 

# . IT1Study No. IT1 1101 (I



### Ill. SIGNATUREOF PRINCIPALS

This report describes the results used to evaluate the relative immunotoxicological potential of the test substance, Gasoline TBA Vapor Condensate, which was administered by inhalation via whole-body exposureto female Sprague Dawley rats.

Kimber 

### L. White, Jr., Ph.D., Principal Investigator, was responsible for the overall conduct of the immunotoxicity evaluations in this study. Vanessa L. Peachee, M.S., served as the Assistant Principal lnvestigator and was responsible for the day-to-day activities of the immunotoxicity evaluations in this study.

Kimber L. White, Jr., Ph.D. Principal lnvestigator ImmunoTox, Inc.

Vanessa L. Peachee, M.S. Assistant Principal lnvestigator ImmunoTox, Inc.

Approved:

Gary M. Hoffman, B.A., DABT Study Director Huntingdon Life Sciences Date

Date

DateProtocol No

. HLS Study No

.00-6131 Abbreviated Title: immunological Evaluationof GasolineTBA Vapor Condensate



### TABLE OF CONTENTS IT1Studv No

.IT1 11 01

I



### . II



## .

111



## . IV



## . 

## v

. VI



## .

VII



## .

VIII



## . IX



## . X


# .

XI


# . GLP Compliance Statement



## ................................................................................................................ 2 Quality Assurance Statements ImmunoTox. Inc



## ................................................................................................................................... 3 Huntingdon Life Sciences



## ....................................................................................................................4 

### Signature of Principals



## .........................................................................................................................5 Executive Summary



## ............................................................................................................................ 8 Introduction



## ....................................................................................................................................... 10 Methods of Procedure



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



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



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

Terminal Body and OrganWeights



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



## ..................:


## ........................................................................................ 12 Spleen IgMAntibody Response to the T-dependent Antigen, sRBC



## . Day 4 Response



## ............. 12 Data



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

Data Handling and StatisticalAnalysis



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



## ............................................................................................................................ 13 Results



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



### Terminal Body andOrganWeights



## ................................................................................................ 13 Spleen IgMAntibody Response to the T-dependentAntigen. sRBC



## . Day 4 

### Response



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



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



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

1



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

### Spleen Weight in Female Sprague Dawley Rats Exposed to Gasoline TBA Vapor Condensatevia Inhalation for 5 Days perWeek for 4 

### Weeks



## ............... 14 2



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

### Thymus Weight in

## ema ale Sprague Dawley Rats Exposedto Gasoline TBA Vapor Condensatevia Inhalation for 5 

### Days perWeek for 4 

### Weeks



## ............... 15 3



## . Total Spleen Cell Numbers in Female Sprague Dawley Rats Exposedto GasolineTBA Vapor Condensatevia Inhalationfor 5 Days per Week for 4 

### Weeks



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


# . IgMAntibody-FormingCell Responseto Sheep Erythrocytes in Female Sprague Dawley Rats Exposedto GasolineTBA Vapor Condensatevia lnhalationfor 

### 5 Days per Week for 4Weeks17 List of Tables ESI SummaryTable for Toxicology and Immunology Studies



## .........................................................9

1 

### Body Weight (g) and Organ Weights (mg) in Female Sprague Dawley Rats Exposed to Gasoline TBA Vapor Condensatevia lnhalationfor 5 

### Days perWeek for 4

### WeeksProtocolNo. HLS Study No. 00-6131 AbbreviatedTitle: immunological Evaluationot GasolineTBA Vapor Condensate IT1Study No. IT1 1101

2. 

### Spleen Antibody-Forming Cell Response to T-dependent Antigen Sheep Erythrocytes in Female Sprague Dawley Rats Exposedto GasolineTBA Vapor Condensate via Inhalationfor 5 Days per Week for 4 

### Weeks



## .... .

## ....................................................,.,..................................... 20 APPENDICES A Individual Animal Data B Contracting Sponsor's Exposureand Animal DataProtocol No. HLS Study No. 00-6131 AbbreviatedTitle: ImmunologicalEvaluationof GasolineTBA Vapor Condensate IT1Study No. IT1 1101



### IV. EXECUTIVE SUMMARY

The study was conducted as part of Huntingdon Life Sciences (HLS) Study No. 00-6131 at ImmunoTox, Inc., Richmond, Virginia. The Principal Investigator was Kimber L. White, Jr., Ph.D., and Vanessa 

### L. Peachee, MS., served as the Assistant Principal Investigator. The study was conducted to provide evaluation of immunological parametersfor Huntingdon Life Sciences.

The objective of the study was to determinethe potential effects of GasolineTBA Vapor Condensate for its ability to affect the humoral immune component of the immune system, when evaluated in the antibody-forming cell response to the T-dependent antigen, sheep erythrocytes. Female Sprague Dawley rats were administered Gasoline TBA Vapor Condensate for 

### 5 days per week for 

### 4 weeks by inhalation via whole body exposure by Huntingdon Life Sciences (HLS) Princeton Research Center (PRC) personnel. Three exposure levels of 2000, 10000 and 20000 mg/m3 of the test substance were used in the study. The in-life phase of the study was conducted by HLS, East Millstone, NJ, 

### and the immunological evaluation was conducted by ImmunoTox, Inc., Richmond, VA. On the.day of sacrifice, spleens were placed in tubes containing media, placed on wet ice, and shipped to ImmunoTox, Inc. in Richmond, VA, for assay evaluationon the following day.
Executive Summary Table ES-1 shows a summary of the selected toxicology and immunology parameters evaluated. Exposureto Gasoline TBA Vapor Condensate resulted in'no statisticallysignificant changes in terminal body weight for any exposure level. Furthermore, there were no statistically significant effects observed in either thymus or spleen weight following exposure to Gasoline TBA Vapor Condensate, when evaluatedeither as absolute weight or relative weight, as comparedto the air control.
Exposure to GasolineTBA Vapor Condensate did not result in any statistically significant effect inthe IgM antibody-forming cell (AFC) response to the T-dependent antigen, sheep erythrocytes,when evaluated as either specific activity (AFCII0' spleen cells) or as total spleen activity (AFCIspleen). The positivecontrol, CPS, produced the anticipated results inthe various parameters evaluated.

In conclusion, the results of this imrnunotoxicological evaluation demonstratethat, underthe experimental conditions used, exposure to the GasolineTBA Vapor Condensatetest substance did not adversely affect the functional ability of the humoral immune componentof the immune system.Protocol No. HLS Study No. 00-6131 Abbreviated Title: immunological Evaluation of GasolineTBA Vapor Condensate

Table ES-1 IT1 Studv No. IT1 1101

SUMMARY TABLE FOR TOXICOLOGY AND IMMUNOLOGY STUDIES
Parameter Result Maximum Effect Exposure Level (ms/m3) Terminal BodyWeight Day 29 No Effect Organ Weights (Absoluteand Relative)

Spleen (Absolute) No Effect Spleen (Relative) No Effect Thymus (Absolute) No Effect Thymus (Relative) No Effect Spleen 

### IgM Antibody-Forming Cell Response to Sheep Erythrocytes IgM AFC/~ 

### 0' Spleen Cells No Effect IgM AFCISpleen (XI 

### 03) No EffectProtocol No. HLS Study No. 00-6131 AbbreviatedTitle: immunological Evaluation of GasolineTEA Vapor Condensate


### V. INTRODUCTION IT1 Study No. IT1 1101



### The purpose of this study was to provide evaluation of immunological parameters foIr Huntin gdon Life Sciences (HLS) Study No. 00-6131. In this study, the test substance, Gasoline TBA Vapor Condensate,
was evaluated for its ability to affect the humoral immune component of the immune system, when evaluated in the antibody-forming cell response to the T-dependent antigen sheep erythrocytes. The study was conducted in female animals because female rats have a more robust immune responsethan do the male animal of the species. Accordingly, female rats have a greater sensitivity for detecting an adverse effect of a compound should one occur. Routinely, irnmunotoxicology evaluations conducted by the National Toxicology Program (NTP) evaluate compounds only in female animals. Four days prior to sacrifice, lmmunoTox personnel sensitized the rats by intravenousadministrationof 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 wet ice for evaluation the following day. Spleens were received on 24 July 2002 and the immunological evaluation was conducted on the same day. The IgM antibody-forming cell (AFC) response to the T-dependent antigen sheep erythrocytes, also referred to as the plaque assay, was the immunological assay conducted to evaluate the effect of GasolineTBA Vapor Condensate on the immune response. This assay has been shown to be the most predictivefunctional assayfor determiningthe immunotoxicologicalpotentialof a compound (Luster eta/.').



### Kimber 

### L. White, Jr., Ph.D., was the Principal lnvestigator for the immunological evaluation conducted by ImmunoTox, Inc., and Gary M. Hoffman, B.A., DABT, was the HLS Study Director. Vanessa L. Peachee, 

### M.S., served as the Assistant Principal lnvestigator for ImmunoTox, Inc. andwas responsible for carrying out the IgM antibody-formingcell assay.

In evaluating the effects of Gasoline TBA Vapor Condensate on the immune system, the immunologic and toxicologic parameters evaluated were spleen and thymus weights, and the spleen IgM antibody responseto the T-dependent antigen (sheep erythrocytes, sRBC).

To the best of our knowledge, no significant protocol or standard operating proceduredeviations occurred during the study, which affected the quality of the data and the ability to interpret the data with respect to the immunotoxicologyof Gasoline TBA Vapor Condensate.ProtocolNo. HLSStudy No. 00-6131 Abbreviated Title: immunological Evaluationof GasolineTEA Vapor Condensate



### VI. METHODSOF PROCEDURE IT1Study No. IT1 1101



### The immunotoxicological satellite study consisted of a vehicle group, three exposure levels of Gasoline TBA Vapor Condensate, and a positive controlgroup. There were 10female Sprague Dawley rats in each of the groups. Animals were exposed by Huntingdon Life Sciences Princeton Research Center (PRC) personnel to either vehicle (air only) or Gasoline TBA Vapor Condensate at exposure levels of 2000, 10000 or 20000 mg/m3via inhalation for 

### 4 weeks 

### (5 days per week). Cyclophosphamide (CPS) was given
as the positive control. Cyclophosphamide (CAS #6055-19-2, lot number 091K1176, received 12 July 2002, expiration September 2004, white powder, storage 2-8*C, purity 99.5%), was obtained from the Sigma Chemical Company (responsible for its characterization), and was dissolved and diluted in phosphate buffered saline at Huntingdon Life Sciences to stock concentrations of 5.0 mgImL for use as the positive control for this study. The positive control animals received 

### 50 mglkg 

### Q 10 mUkg of CPS, 

### a known immunosuppressive agent, administered intraperitoneally (i.p.) once on the last 

### 4 days of exposure. These animals were not chamber exposed. 

### On the day of sacrifice, one day after the last exposure, PRC personnel aseptically removed the spleen from each animal, weighed it, placed it in a collecting tube containing Earle's Balanced Salt Solution (EBSS) with HEPES and Gentamicin solution (prepared at PRC), and shipped the spleens on wet ice in individual shipping containers at 2-8°C by carrier to ImmunoTox for overnight delivery. Upon receipt, spleens were further processed for determination of IgM antibody response.

Terminal Bodv and Oraan Weiahts. The terminal body weights were obtained by Huntingdon Life Sciences PRC personnel. Huntingdon Life Sciences PRC personnel collected blood (serum) samples (orbital collection anesthetized via carbon dioxideloxygen inhalation) and then sacrificed (carbon dioxide inhalation) the animals on the day after the final exposure. The serum samples were frozen (920°C). The thymuses were removed, weighed and preserved (formalin) for possible histopathology. Spleens
were removed, weighed, and shipped at the time of sacrifice by PRC personnel to ImmunoTox, Inc. for immunotoxicological evaluation.Protocol No. HLS Study No. 00-61 31 AbbreviatedTitle: immunological Evaluation of GasolineTBA Vapor Condensate IT1 Study No. IT1 1101



### S~lenocvte Preparation. Upon arrival at the ImmunoTox testing facility, spleens were accessioned in accordance with the SOP for receipt of biological samples. Single-cell suspensions were prepared from each spleen using a stomachera 

### 80 Lab Blender in accordance with the SOP for rat spleens. Cell suspensions were then centrifuged and resuspended in Earle's Balanced Salt Solution with HEPES. Viability of splenocytes was determined using propidium iodide (PI) andthe Coulter EPICS XL-MCL Flow Cytometer.

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

### Response. As background, sheep erythrocytes (sRBC) are a T-dependent antigen and, thus, T cells, B cells, and macrophages are required to function properly in order to obtain an antibody-forming cell (AFC) response. If the test article affects any of these cell types to a significant degree, an altered response will be observed. As a result, the T-dependent IgM responseto 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, indicatesthat the test agent is capable of modifyingthe hurnoral immune response in the whole animal and, thus, has the potentialfor immunotoxicity. This assay is one of the Tier I

### assays used by the NTP~.

The primary IgM responseto 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. personnelwith 2x10' sRBC i.~. four days prior to sacrifice and, on the day after the last exposure, animals were sacrificed by PRC personnel. Spleen cell suspensions were prepared as described above. The cells were centrifuged and resuspended in a 6-ml volume, and 1:50 and 1:150 dilutions were prepared. An 0.1-mI aliquot of spleen cells from each suspension was added to separate test tubes, each containing25 p1 

### guinea pig complement, 25 p1 

### sRBC, and 0.5 ml of warm agar (0.5%). After thoroughly mixing, each test tube mixture was plated onto a separate petri dish, covered with a microscope cover slip, and incubated at approximately 

### 36-38°C for 

### 3 hours. Spleen cell counts were performed on the 6-rnl samples using a Model 

### Z1 Coulter Counter. The spleen weight, cellslspleen, AFCl10' spleen cells, and AFCIspleen were determined. The plaques, which developed, were counted using a Bellco plaque viewer. A plaque, occurring from the lysis of sRBC, is elicited as a result of the interactionof complement and antibodies (produced in responseto the i.v. sensitization) directedagainst sRBC. Each plaque is generated from a single IgM antibody-producing B cell, permitting the number of AFC present in the whole spleen to be calculated. The data are expressed as specific activity(AFCII 

## o6 spleen cells) andtotal spleen activity (AFCIspleen).Protocol No. HLSStudy No. 00-6131 AbbreviatedTitle: lmmunologicalEvaluation of GasolineTBA Vapor Condensate IT1Study No. IT1 1101 7'



### Data Handlina and StatisticalAnalysis. The data obtained in this study were analyzed in accordance with standard operating procedure. Data were first testedfor homogeneity of variances using the Bartlett's Chi Square ~est~. Homogeneous data were evaluated by a parametric one-way analysis of variance5. When significant differences occur, exposed groups were compared to the vehicle control group using the Dunnett's t ~est'.Non-homogeneousdata were evaluated using a non-parametric analysis of variance5. When significant differences occur, exposed groups were compared to vehicle control group using the Gehan-Wilcoxon ~est' when appropriate. The Jonckheere's ~est' was used to test for exposure levelrelated trends across the vehicle and exposed groups. The positive control was compared to the vehicle control group using the Student t ~est~. Thecriteria for accepting the results of the positive control in the assay was a statistically significant (p 5 

### 0.05) decrease in the response as compared to the vehicle controlgroup.

Pvalues of 0.05 or less, as comparedto 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 usedto indicate.ap value of 0.01 or less. Inthe text, the word significant indicatesthat the responsewas statistically significant at p 5 

### 0.05. In the tables the abbreviation NS is usedto indicate "Not Significant" for p values greater than 0.05.

Data Retention. All data and records were returned to the Contracting Sponsor following acceptance of the final report. Upon completion of this study, the report and raw data for this study will be maintained in the archives of Huntingdon Life Sciences.

VII. RESULTS

TERMINAL BODY AND 

### ORGAN WEIGHTS.



### The terminal body weight data from the study are shown in Table 1 for the control and Test Substanceexposed groups. The mean ending weight for the vehicle-exposed rats was 262.1 

# * 5.1 grams. No statistically significant differences were observed in terminal body weights of the Gasoline TBA Vapor Condensate-exposed animals at any exposure levelas comparedtothe vehicle (air only) controls.
The organ weights of the control and Test Substance-exposed rats are shown in Table 1. 

### No effect was observed, following exposure to Gasoline TBA Vapor Condensate, on spleen or thymus weights when evaluated as absolute weight or as relative weight. Treatment with the positive control, cyclophosphamide, had a significant decrease of 55% on absolute spleen weight and a significant decrease of 77% 

### on absolute thymus weight, compared to the vehicle control. In addition, the positiveProtocol No. HLSStudy No. 00-6131 IT1Study No. IT1 1101 Abbreviated Title: lrnrnunologicalEvaluationof Gasoline TBA Vapor Condensate 

# -

control, cyclophosphamide, had a significant decrease of 52% 

### 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 lack of effect on spleen and thymus weights following exposure to Gasoline 

### TBA Vapor Condensate. Figure 1



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

### Spleen Weight in Female Sprague Dawley Rats Exposedto Gasoline TBA 

### Vapor Condensatevia Inhalationfor 5 Days perWeek for 4 

### Weeks

Absolute Weight RelativeWeight

Vehicle 

### 2000 10000 20000 50 

# - Vehicle 

### 2000 10000 20000 50 

# - TBA 

### CPS TBA 

### CPS 

### (ms/m3) (mgfkg) (mglm3) (mgfkg)ProtocolNo. HLSStudy No. 00-6131 AbbreviatedTitle: lmmunological Evaluationof GasolineTBA Vapor Condensate IT1 Studv No. IT1 1101



### Figure 

### 2
Absolute (mg) and Relative (%) 

### Thymus Weight in Female Sprague Dawley Rats Exposedto Gasoline TBA Vapor Condensatevia Inhalationfor 

### 5 Days per Week for 

### 4Weeks

Absolute Weight RelativeWeight

Vehicle 

### 2000 10000 20000 50 

# - TBA CPS (mg1m3) (mgh) vehicle 

### 2000 10600 20000 50

The spleen IgM antibody-formingcell 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. Viabilitieswere conductedon all cell suspensions using propidium iodide (PI) and the Coulter EPICS XL-MCL 

### Flow Cytometer. The viabilities from all samples were greater than 71% 

### with the exceptionof four samples.

The results of the AFC response are shown 

### in Table 

### 2 and in Figures 3 and 4. 

### As indicated above, exposure to Gasoline TBA Vapor Condensate did not result in spleen weights that were significantly different from the vehicle control group. Furthermore, as shown graphically in Figure 3, there were no significant differences in spleen cell number following exposure to Gasoline TBA Vapor Condensate as compared to the vehicle control group. As expected, the positive control, cyclophosphamide (CPS), produced an 83% 

### decrease in spleencell numberwhen compared to the vehiclecontrol group.Protocol No. HLS Study No. 00-61 31 AbbreviatedTitle: lrnrnunologicalEvaluation of GasolineTBA Vapor Condensate IT1 Study No. IT1 11 01 

# -

Figure 3 

### Total Spleen Cell Numbers in Female Sprague Dawley Rats Exposedto GasolineTBA Vapor Condensatevia Inhalationfor 5 

### Days perWeek for 4 

### Weeks



### Vehicle 

## 2000 10000 

## 20000 50 

## TBA 

## CPS 

## (mg1m3) 

## (mglkg)

Shown in Table 2 

### and Figure4 

### are the functional results from the IgM antibody-formingcell (AFC) assay. Shown in the left panel are the results when the data are expressedas specific activity andthe results of the total spleen activity are shown in the right panel. No effect was observed, following exposure to Gasoline TBA Vapor Condensate, when evaluated as either specific activity (AFCIIO' spleen) or as total spleen activity (AFCIspleen).

As anticipated, the positive control, CPS, produced a significant decrease in specific activity (99%) and total spleen cell activity (100%) when compared to the vehiclecontrolanimals.ProtocolNo. HLS Study No. 00-6131 Abbreviated Title: lmrnunological Evaluation of Gasoline TEA Vapor Condensate IT1 Study No. IT1 1101



### Figure4 IgMAntibody-Forming Cell Responseto Sheep Erythrocytes in Female Sprague Dawley Rats Exposedto GasolineTBA Vapor Condensatevia Inhalationfor 5 Days per Week for 4 

### Weeks Specific Activity Total SpleenActivity

vehicle 2000 10000 20000 50

7 

### TBA 

## CPS (mg~m3) (mg/kg)

VIII. CONCLUSION Exposure of female Sprague Dawley rats to GasolineTBA Vapor Condensate for a period of 5 days per week for 4 weeks resulted in no statistically significant effects in the humoral immune responseto the 

### Tdependent antigen, sheep erythrocytes. There were no statistically significant effects on body weight, spleen or thymus weights (absolute or relative), spleencell number, specific activity (~~~110~ spleen), or total spleen activity (AFCIspleen). Based on the immunological parameters evaluated, under the experimental conditions of the study, exposure to Gasoline TBA Vapor Condensate did not adversely affect the hurnoral immune response of female Sprague Dawley rats.ProtocolNo. HLS Study No. 00-6131 AbbreviatedTitle: lrnrnunologicalEvaluationof GasolineTBA Vapor Condensate


### IX. REFERENCES IT1Studv No. IT1 1101



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

### (1992) Risk assessment in immunotoxicology. I. Sensitivity and predictabilityof immune tests. Fund. Appl. Toxicol. 

### l8:2OO-2lO. Jerne NK, Henry C, Nordin AA, Fun H, Koros 

### MC, and Lefkovits 

### 1 (1974). Plaque-forming cells: Methodology andtheory. Trnspl. 

### Rev. 18:130-191.
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. KruskalWH and Wallis WA 

### (1952). Use of ranks in one-criterion variance analysis. J. 

### Amer. Stat. ASSOC. 

### 471583-621. Dunnett CW 

### (1955). 

### A multiple comparison procedure for comparing several treatments with a control. 

### J. Amer. Stat. Assoc. 

### 5O:lO96-ll2l, Gross AJ 

### and Clark VA 

### (1975). Gehan-Wilcoxon Test. In Survival Distribution: Reliability Applications in Biomedical Sciences. AJ Gross and VA Clark, eds. John Wiley and Sons, NewYork, p. 

### 120-123. Hollander M and Wolfe DA 

### (1975).Jonckheere's Test: Non-parametric Statistics Methods, eds. M. Hollanderand D.A. 

### Wolfe, John Wiley and Sons, NewYork, 

### p. 

### 124-129. Sokal RR and Rohlf FJ

### (1981). Biometry. Freeman, San Francisco, p. 

### 226-231.Protocol No. HLSStudy No. 00-6131 Abbreviated Title: lrnrnunological Evaluation of GasolineTBA Vapor Condensate 

### Table t IT1 Study No. IT1 1101



### BodyWeight (g) and Organ Weights (mg) in Female Sprague Dawley Rats Exposedto GasolineTBA Vapor Condensate via Inhalationfor 

### 5 Days perWeek for 4 

### Weeks

Parameter Vehicle Gasoline TBA Vapor 

### (rndm3) Cvclophosphamide 

### WNH Trend 

### 2000 10000 20000 50 

### mglkg Analysis 

### (10) (10) (10) (10) 

### (10)

Body Wgt (g) 262.1 

# * 5.1 255.4 i 6.7 252.6 

## + 6.8 254.2 

## + 5.2 243.4 

## + 5.0* H 

### NS Spleen (mg) 676 

# * 31 630 

# * 27 614 

# * 27 611 

## + 24 304 

## + 12** H 

### NS

% Body Wgt 0.259 & 0.009 0.247 

# * 0.008 0.242 

# * 0.011 0.242 -t 0.010 0.124 

# * 0.004** H p 0.05 Thymus (mg) 571 & 33 490 i 33 513 rt 32 537 2 33 134 

## + 9** H 

### NS

% Body Wgt 0.217 

# * 0.012 0.194 k 0.014 0.202 +- 0.012 0.212 k 0.012 0.054 +- 0.003** H 

### NS Female Sprague Dawley rats were administered vehicle control (air only) or gasoline TBA vapor condensate by inhalation via whole-body exposure for 5 days per week for 4 

### weeks. The positive control, cyclophosphamide, was administered i.p. on the last 4 

### days 

### of exposure. On the day of sacrifice, spleens were placed in tubes containing media and sent to Richmond, VA, on ice for next day cell preparation. The rats were necropsied and indicated organs weighed. Values represent the mean 

# * SE derived from the number of animals indicated in parentheses. 

### H 

### = homogeneous data and 

### NH 

### = non-homogeneous data using the Bartlett'sTest 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 usedto test for dose-relatedtrends among the vehicle and exposed groups. Key: 

### g 

### = grams; mg 

### = milligrams; m3

### = cubic meter of air; kg 

### = kilograms;Wgt =

### weight; 

### NS = not significant for p values greater than 0.05.Protocol No. HLS Study No. 00-61 31 Abbreviated Title: immunological Evaluationof GasolineTBA Vapor Condensate


### Table 2 t IT1 Study No. IT1 1101



### Spleen Antibody-Forming Cell Response to T-dependent Antigen Sheep Erythrocytes in Female Sprague Dawley Rats Exposed to GasolineTBA Vapor Condensatevia Inhalationfor 5 

### Days perWeek for 4 

### Weeks 

## - Day4 

### Response 

### Exposure Body

### Wgt Spleen

### Wgt Spleen Cells 

## IgM AFCI 

### IgM AFCfSpleen (g) 

### (mg) (x 

### lo7) lo6

### Spleen Cells (X 

## lo3)
Vehicle 262.1 

# * 5.1 (10) Gasoline TBA Vapor Condensate 2000 mg/m3 255.4 

# + 6.7 (10) 10000 mg/m3 252.6 

# * 6.8 (10) 20000 mg/m3 254.2 2 5.2 (10 Cyclophosphamide

50 mg/kg 243.4 

# * 5.0* (10 

### H/NH H Trend Analysis 

### NS Female Sprague Dawley rats were administeredvehicle control (air only) or gasoline TBA vapor condensate by inhalationvia whole-body exposure for 

### 5 days per week for 

### 4 weeks. The positive control, cyclophospham~de, was administered i.p. the last 4 

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

### On the day of sacrifice, spleens were placed in tubes containing media and sent to Richmond, 

### VA, on ice for next day cell preparation. Spleens were prepared into single cell suspensions and the number of IgM sRBC antibody-forming cells was determined. Values represent the mean 

# * SE derived from the number of animals indicated in parentheses. 

### H, = homogeneous data and 

### NH 

### = nonhomogeneousdata 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 usedto test for dose-related trends among the vehicle and exposed groups. Key: g = 

### grams; mg 

### = milligrams; m3

### = cubic meter of air; kg = 

### kilograms; Wgt = 

### weight; 

### NS = 

### not significant for p values greater than 0.05.Protocol No. HLS Study No. 00-61 31 Abbreviated Title: immunological Evaluationof GasolirleTBA Vapor Condensate



### APPENDIX A IT1 Study No. IT1 1101 

### k. b



### INDIVIDUAL ANIMAL DATAINlDlVlDUAL ANIMAL DATA ORGAN WEIGHTS GASOLINE TBA VAPOR CONDENSATE 00-6131

ANIMAL NO GROUP DOSE SEX BODYWGT SPLEEN WGT THYMUS WGT SPLEEN WGTI THYMUS WGTI (G) (MG) (MG) % BODYWGT % BODY WGT
GI GI GI GI GI GI GI GI GI GI GI1 GI1 GI1 GI1 GI1 GI1 GI1 GI1 GI1 GI1 Glll Glll Glll Glll Glll Glll Glll Glll Glll Glll GIV GlV GIV GIV GIV GIV GIV GIV GIV GIV GV GV GV GV GV GV GV GV GV GV AIR ONLY AIR ONLY AIR ONLY AIR ONLY AIR ONLY AIR ONLY AIR ONLY AIR ONLY AIR ONLY AIR ONLY 2.000 MG/M~ TBA 2,000 MGIM~ TBA 2.000 MGIM~TBA 2,000 MGIM~ TEA 2,000 MGIM~TBA 2,000 MG/M~ TBA 2.000 MWM3 TBA 2,000 MG/M~ TEA 2,000 MGIM~TEA 2,000 MWM3TBA 10,000 MGIM~ TBA 10,000 MWM3TBA 10,000 MGIM~ TEA 10.000 MGIM~ TBA 10,000 MWM3 TBA 10.000 MG/M~ TBA 10,000 MWM3TEA 10.000 MGIM~ TBA 10,000 MWM3TBA 10.000 MGIM~ TBA 20,000 MWM3TBA 20.000 MG/M~ TBA 20,000 MG/M~ TBA 20,000 MWM3TBA 20,000 MGIM~ TBA 20,000 MWM3 TEA 20,000 MGIM~ TBA 20.000 MGi/M3TBA 20,000 MWM3TBA 20,000 MGIM~ TBA 50 MWKGCYCLOPHOSPHAMIDE 50 MWKG CYCLOPHOSPHAMIDE 50 MWKG CYCLOPHOSPHAMIDE 50 MWKG CYCLOPHOSPHAMIDE 50 MGIKG CYCLOPHOSPHAMIDE 50 MWKG CYCLOPHOSPHAMIDE 50 MGlKG CYCLOPHOSPHAMIDE 50 MGlKG CYCLOPHOSPHAMIDE 50 MGlKG CYCLOPHOSPHAMIDE 50 MGlKG CYCLOPHOSPHAMIDEINDIVIDUALANIMAL DATA IGMAFC ASSAY GASOLINE TBA VAPOR CONDENSATE 00-6131

ANIMAL NO GROUP DOSE SEX IGMAFCl IGMAFCI CELLS1 SPLEENWGT BODY WGT 1o6SP.C. SPLEEN lo3 SPLEEN lo7 (MG) (G)
GI GI GI GI GI GI GI GI GI GI GI1 GI1 GI1 GI1 GI1 GI1 GI1 GI1 GI1 GI1 Glll Glll Glll Glll Glll Glll Glll Glll Glll Glll GIV 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 AIR ONLY 2,000 MG1M3TEA 2.000 MGIM~ TEA 2,000 MGIM~TEA 2.000 MGIM~ TEA 2,000 MG/M~ TEA 2,000 MGIM~TEA 2.000 MG1M3TEA 2,000 MGIM~ TEA 2,000 MG1M3TBA 2,000 MGIM~TBA 10,000 MGIM~ TBA 10,000MG/M~ TBA 10,000 MGIM~ TBA 10.000 MGIM~TBA 10,000 MGIM~TEA 10,000MG1M3TBA 10,000MG/M~ TBA 10,000 MG/M~ TBA 10.000 MG/M~ TBA 10,000 MGIM~TBA 20.000 MGIM~ TBA 20,000 MGIM~TBA 20,000 MGIM~TBA 20,000 MG/M~ TBA 20,000 MGIM~ TBA 20,000 MG1M3TBA 20,000 MGIM~TEA 20,000 MGIM~TBA 20,000 MG/M3TBA 20.000 MGIM~ TBA 50 MGlKG CYCLOPHOSPHAMIDE 50 MGlKG CYCLOPHOSPHAMIDE 50 MGlKG CYCLOPHOSPHAMIDE 50 MGIKG CYCLOPHOSPHAMIDE 50 MGlKG CYCLOPHOSPHAMIDE 50 MGIKG CYCLOPHOSPHAMIDE 50 MGlKG CYCLOPHOSPHAMIDE 50 MGKGCYCLOPHOSPHAMIDE 50 MGlKG CYCLOPHOSPHAMIDE 50 MGKGCYCLOPHOSPHAMIDEProtocolNO.HLS StudyNO.00-6131 AbbreviatedTitle: immunological Evaluationof GasolineTBA Vapor Condensate



### APPENDIX B



### CONTRACTING SPONSOR'S EXPOSUREAND ANIMAL DATA

### INTRODUCTION: The following is data generated at Huntingdon Life Sciences, East Millstone, NJ. The separately issued main study report should be referenced for details of the procedures used for test atmosphere 

### generationkharacterization and animal evaluations.

Huntingdon Life Sciences 00-6131 Page 1 21 1-TBA-S Draft Final Report



### STUDY DATES: Date of Animal Receipt: 6 June 2002 Animal Exposure and Animal Data Preface

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

Experimental Completion Date: 23 July 2002 (in-life) Draft Report Date: 2 June 2004



### EXPOSURES AND IN-LIFE SUMMARY: The actual measured results during the exposures were comparable to the targeted exposure levels. There were no exposurerelated effects seen in the test animals with regards to body weights and feed consumption.


### TABLE 

### OF CONTENTS
TABLES A. Chamber Monitoring Results



## ........................................................................................... B. Summaryof Clinical Observations (pretest only)



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



## ........................................................................................... D. Mean Body Weight Change from Baseline (grams)



## ........................................................ E. Mean Feed ConsumptionValues (grams/kg/day)



## ............................................................ F. Individual Clinical Observations (pretest only)



## ............................................................... G. IndividualBody Weights (grams)



## ....................................................................................

................................................. H. IndividualBody Weight Change from Baseline (grams) 

### I. Individual Feed ConsumptionValues (grams/kg/day)



## ..................................................... J. Animal TerminationHistory



## ............................................................................................Table A GASOLINETBA VAPOR CONDENSATE: A 13-WEEKWHOLE-BODY INHALATIONTOXICITY STUDY IN RATS 00-61 31

Chamber Monitoring Results Cumulative Exposure Record Group IA

# - 0 (air only) mg/m3 Chamber Environment Particle Size 

# I Me: alytical Chamber Concentration 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 P Mean (mg1m3) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1s TMC (mg1m3)

1.19E-02

2.1 1E-02

2.03E-02

7.41E-03 Temperature
("C) 25 26 25 25 25 24 24 24 24 24 25 25 25 25 25 24 24 24 24 24 Date Exposure Number Humidity 

### ("4 50 68 52 7 1 5 1 54 53 54 52 52 51 52 38 47 50 49 56 52 53 5 1 eterminati GSD Individual MMAD Olm)

0.9138

0.8260

0.7595

5.014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Mean S.D.Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEKWHOLE-BODY INHALATIONTOXICITY STUDY IN RATS 00-6131

Chamber Monitoring Results Cumulative Exposure Record

Day

0 1 2 3 6 7 8 9 10 13 14 15 16 17 20 21 22 23 24 27 Date

25-Jun-02 26-Jun-02 27-Jun-02 28-Jun-02

1 -Jul-02 2-JuI-02 3-Jul-02 4-Jul-02 5-Jul-02 8-Jul-02 9-Jul-02 10-Jul-02 11-JuI-02 12-Jul-02 15-Jul-02 16-Jul-02 17-Jul-02 18-JuI-02 19-Jul-02 22-Jul-02

S.D. Exposure Number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Mean 0.4 7.4 Chamber Environment Mean Temperature
("C) 24 25 24 24 24 25 25 24 25 24 24 24 24 24 24 24 24 24 24 25 24.3 0 Humidity 

### ("4 51 70 54 76 52 56 54 55 55 53 54 55 39 49 51 5 1 58 55 54 53 54.8 0 Group IB

# - 0 (air only) mg/m3

0.686 Nominal
(mglm3) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Particle Size

MMAD Olm)

1.905

0.8133

0.8024

2.076

1.399 0.458 Mean (mglm3) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

8.16E-03 Determinations GSD

2.407

3.176

2.148

2.298

2.507 TMC (ms/m3)

1.36E-02

2.72E-02

1.92E-02

4.83E-03

1.62E-02 Analytical Chamber Concentration Individual

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 (mglm3) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0TableA GASOLINETBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-6131

Chamber Monitoring Results

Date Exposure Number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Mean S.D. Cumulative Exposure Record Group llA

# - 2,000 mg/m3

Nominal (mglm3)

1990 1950 2070 2140 2070 2120 2010 2030 1990 1930 1890 1920 2020 1880 2010 2120 2050 2090 2030 2030 2017

75 Mean (mg/m4

1998 1963 2063 2120 2063 2095 2050 2070 1998 1963 1905 2058 2078 1958 2033 1903 1935 1890 1933 1925 alytical Chamber Concentration Individual MMAD Olm)

0.8293

0.7460

0.7361

1.612 Particle Size Ins TMC

(mg/m3)

7.72E-03

1.96E-02

1.67E-02

3.35E-03 Chamber Environment Mean Temperature
("C) 24 25 23 24 23 24 23 24 24 24 24 24 24 24 24 23 23 23 23 23 23.7 0.6 Humidity 

### ("4 45 63 47 61 46 49 48 49 48 48 47 47 34 42 45 47 53 48 50 48 48.3 6.0Table A GASOLINETBA VAPOR CONDENSATE:A 13-WEEK WHOLE-BODY INHALATIONTOXICITY STUDY IN RATS 00-6131

Chamber Monitoring Results Cumulative Exposure Record Group IIB

## - 2,000 mg/m3 Chamber Environment Mean Particle Size Humidity
("A) 50 68 53 71 50 51 51 53 52 50 52 52 37 46 49 49 56 51 5 1 49 Nominal
(mg/m3)

1990 1950 2070 2140 2070 21 20 2010 2030 1990 1930 1890 1920 2020 1880 2010 21 20 2050 2090 2030 2030 2017

75 1sTMC (mg/m3)

8.49E-03

2.18E-02

7.02E-02

2.96E-03 Temperature
("C) 23 24 23 24 23 23 23 23 23 23 23 23 23 23 23 22 22 22 23 23 23.0 0.5 Date Ixposure Number - --- Analvtical Chamber Concentration I MMAD Olm)

0.8183

0.7358

14.68

1.010 terminat GSD Mean (mglm3)

2090 2090 2038 2128 1965 2068 2020 2073 1995 18% 1750 1990 2033 1930 2010 2058 1843 2118 2095 1928 Individual

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

Chamber Monitoring Results

Date Exposure Number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Mean S.D. Cumulative Exposure Record Group IIIA

## - 10,000 mg/m3

Nominal (mg/m3)

9760 10200 9440 9850 9970 10200 10100 10100 10300 9970 10300 10300 10300 9590 9840 9910 101 00 101 00 9920 101 00 10020 239 1 Mean (mglm3

10430 10330 9878 9943 9763 101 80 9665 1031 0 9980 10070 9975 10300 10060 9808 9778 lo360 10390 9895 10980 10680 ~alytical Chamber Concentration Individual Particle Size C MMAD Olm)

0.8742

0.7422

0.7415

1.168 terminati GSD S TMC (mg/m3)

8.64E-03

2.39E-02

1.89E-02

2.78E-03 Chamber El Me, Tem~erature ironment Humidity 

## ("w 47 64 49 61 47 48 50 51 49 48 48 48 34 42 46 47 53 51 52 49 49.2 6.1Table A GASOLINETBA VAPOR CONDENSATE:A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-6131

Chamber Monitoring Results Cumulative Exposure Record

Group lllB

## - 10,000 mglm3

Day Date Exposure Nominal 

## L

0 25-Jun-02 1 26-Jun-02 2 27-Jun-02 3 28-Jun-02 6 1 -Jul-02 7 2-Jul-02 8 3-Jul-02 9 4-Jul-02 10 5-JuI-02 13 8-Jul-02 14 9-Jul-02 15 10-Jul-02 16 11-Jul-02 17 12-JuI-02 20 15-Jul-02 21 16-Jul-02 22 17-Jul-02 23 18-Jul-02 24 19-Jul-02 I 27 22-Jul-02 20 10100 Mean 10020 S.D. 239 Analytical Chamber Concentration Mean 

### I Individual ParticleSize

MMAD Olm)

0.8268

0.7424

0.7374

0.8683 etermina GSD Chamber Environment Mear 

### I 1 Humidity 

### (W 45 64 47 60 46 46 47 48 46 46 47 48 33 41 45 44 49 47 48 45 47.1 6.2 msTMC

(ms/m3)

7.73E-03

2.36E-02

2.03E-02

2.96E-03

1.36E-02 8.55E-03 Temperatlire 

### ("'7

23 23.8 0.5Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEKWHOLE-BODY INHALATIONTOXICITY STUDY IN RATS 00-6131

Chamber Monitoring Results

Date Exposure Number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Mean CumulativeExposure Record Group IVA

## - 20,000 mglm3

Nominal
(mg/m3)

18900 20600 19200 181 00 19600 19300 19600 19600 19400 19200 19700 19400 18900 191 00 18900 19300 19200 191 00 18900 18600 19230 499 1 Mean (mg/m3) 20400 21000 19900 20000 20380 20050 19450 19950 20080 1 9700 20180 20850 19430 20680 19380 19550 20100 19880 20930 20980 ~alytical Chamber Concentration Individual Determinat Particle Size

MMAD Olm)

0.8760

0.7496

0.7553

1.008 Chamber Environment Mean
GSD ns Temperature Humidity TMC (mg/m3) ("C) 

### ("4Table A GASOLINETBA VAPOR CONDENSATE: A 13-WEEKWHOLE-BODY INHALATIONTOXICITY STUDY IN RATS 00-61 31

Chamber Monitoring Results

Exposure Number Day

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Mean Date

S.D. Cumulative~x~osuie Rcord Group IVB

## - 20,000 mg/m3 

## I

Nominal
(mg/m3)

18900 20600 19200 181 00 19600 19300 19600 19600 19400 19200 19700 19400 18900 19100 18900 19300 19200 19100 18900 18600 19230 499 Particle Size Determinations Individual 

## I MMAD 

## I GSD 

## I TMC Analvtical Chamber Concentration Mean


# -

- Chamber En Mea Tem~erature ronment Humidity 

### ("4 48 67 53 68 5 1 50 53 54 52 5 1 5 1 52 36 45 50 47 54 53 51 5 1 51.9 6.7Huntingdon Life Sciences 00-61311 Irnrnunotoxicity Sub-Group PAGE 1

TABLE B

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

NORMAL

WITHIN NORMAL LIMITSHuntingdon Life Sciences 00-61311 Immunotoxicity Sub-Group PAGE I

TABLE C

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

FEMALES MEAN BODY WEIGHTS (GRAMS)

................................................................................................................................... DOSE GROUP: I I I I11 IV V DOSE LEVEL (MG/M3): 0 2000 10000 20000 POSITIVE CONTROL

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

WEEK 0

WEEK 1

WEEK 2

WEEK 3 MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

WEEK 4 MEAN S.D.

N

......................................... No statistically significant differencesHuntingdon Life Sciences 00-61311 Irnmunotoxicity Sub-Group PAGE 1

TABLE D

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

WEEK 0 TO 1 MEAN S.D.

N

WEEK 0 TO 2 MEAN S.D.

N

WEEK 0 TO 3 MEAN S.D.

N

WEEK 0 TO 4 MEAN S.D.

N

...................................... Statistical key: ** = pc0.01Huntingdon Life Sciences 00-61311 Immunotoxicity Sub-Group PAGE 1

TABLE E

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

FEMALES

.....-----.--.-.-------------------- DOSE GROUP: DOSE LEVEL (MG/M3):

MEAN S.D.

N

WEEK 1 MEAN S.D.

N

WEEK 2 MEAN S.D.

N

WEEK 3Huntingdon Life Sciences 00-61311 Immunotoxicity Sub-Group PAGE 1

TABLE F

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

INDIVIDUAL CLINICAL OBSERVATIONS FEMALES GROUP I 0 MG/M~

ANIMAL# OBSERVATIONS DAY OF 1 STUDY 1

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITSHuntingdon Life Sciences 00-61311 Irnrnunotoxicity Sub-Group PAGE 2

TABLE F

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

INDIVIDUAL CLINICAL OBSERVATIONS FEMALES GROUP I1 2000 MG/M3

ANIMAL# OBSERVATIONS DAY OF 1 STUDY 1

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITSHuntingdon Life Sciences 00-61311 Irnrnunotoxicity Sub-Group PAGE 3

TABLE F

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

................................................................................................................................... CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENTHuntingdon Life Sciences 00-61311 Immunotoxicity Sub-Group PAGE 4

TABLE F

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

INDIVIDUAL CLINICAL OBSERVATIONS FEMALES GROUP IV 20000 MG/M~

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

------------------------...---.----------------- CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT DAY OFHuntingdon Life Sciences 00-61311 Imrnunotoxicity Sub-Group TABLE F

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

INDIVIDUAL CLINICAL OBSERVATIONS FEMALES GROUP V POSITIVE CONTROL PAGE 5

DAY OF 1 ANIMAL# OBSERVATIONS STUDY 1

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

WITHIN NORMAL LIMITS

--------------.-.----------------------..-----------------------------...----.------------.-.--..---------------------------------- CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENTHuntingdon Life Sciences 00-61311 Irnrnunotoxicity Sub-Group PAGE 1

TABLE G

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

1581 1582 1583 1584 1585 1586 1587 1588 1589 1590

MEAN S.D.

NHuntingdon Life Sciences 00-61311 Imrnunotoxicity Sub-Group PAGE 2

TABLE G

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

2571 2572 2573 2574 2575 2576 2577 2578 2579 2580

MEAN S.D.

NHuntingdon Life Sciences 00-61311 Immunotoxicity Sub-Group PAGE 3

TABLE G

GASOLINE TEA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

3571 3572 3573 3574 3575 3576 3577 3578 3579 3580

MEAN S.D.

NHuntingdon Life Sciences 00-61311 Irnrnunotoxicity Sub-Group PAGE 4

TABLE G

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

4581 4582 4583 4584 4585 4586 4587 4588 4589 4590

MEAN S.D.

NHuntingdon Life Sciences 00-61331 Imrnunotoxicity Sub-Group PAGE 5

TABLE G

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

INDIVIDUAL BODY WEIGHTS (GRAMS) FEMALES GROUP V POSITIVE CONTROL

WEEK OF STUDY ANIMAL# - 1 0 1 2 3 4

5551 5552 5553 5554 5555 5556 5557 5558 5559 5560

MEAN S.D.

NHuntingdon Life Sciences 00-61311 Immunotoxicity Sub-Group PAGE 1

TABLE H

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

INDIVIDUAL BODY WEIGHT CHANGE FROM BASELINE (GRAMS) FEMALES GROUP I 0 MG/M3

................................................................................................................................... WEEK OF STUDY ANIMAL# 0-1 0-2 0-3 0-4

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

MEAN S.D.

NHuntingdon Life Sciences 00-61311 Irnrnunotoxicity Sub-Group PAGE 2

TABLE H

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

INDIVIDUAL BODY WEIGHT CHANGE FROM BASELINE (GRAMS) FEMALES GROUP I1 2000 MG/M~

................................................................................................................................... WEEK OF STUDY ANIMAL# 0-1 0-2 0-3 0-4

................................................................................................................................... 2571 2572 2573 2574 2575 2576 2577 2578 2579 2580

MEAN S.D.

NHuntingdon Life Sciences 00-61311 Immunotoxicity Sub-Group PAGE 3

TABLE H

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

3571 3572 3573 3574 3575 3576 3577 3578 3579 3580

MEAN S.D.

NHuntingdon Life Sciences 00-61311 Immunotoxicity Sub-Group PAGE 4

TABLE H

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

MEAN S.D.

NHuntingdon Life Sciences 00-61311 Immunotoxicity Sub-Group PAGE 5

TABLE H

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

INDIVIDUAL BODY WEIGHT CHANGE FROM BASELINE (GRAMS) FEMALES GROUP V POSITIVE CONTROL

................................................................................................................................... WEEK OF STUDY ANIMAL# 0-1 0-2 0-3 0-4

................................................................................................................................... 5551 5552 5553 5554 5555 5556 5557 5558 5559 5560

MEAN S.D.

NHuntingdon Life Sciences 00-61311 Immunotoxicity Sub-Group PAGE 1

TABLE I

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

MEAN S.D.

NHuntingdon Life Sciences 00-61311 Irnrnunotoxicity Sub-Group PAGE 2

TABLE I

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

INDIVIDUAL FEED CONSUMPTION VALUES (GRAMS/KG/DAY) FEMALES GROUP I1 2000 MG/M3

MEAN S.D.

NSF=Spilled FeederHuntingdon Life Sciences 00-61311 Irnrnunotoxicity Sub-Group PAGE 3

TABLE I

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

INDIVIDUAL FEED CONSUMPTION VALUES (GRAMS/KG/DAY) FEMALES GROUP I11 10000 MG/M3

WEEK OF STUDY ANIMAL# 0 1 2 3

MEAN S.D.

NSF=Spilled FeederHuntingdon Life Sciences 00-61311 Irnrnunotoxicity Sub-Group PAGE 4

TABLE I

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

MEAN S.D.

NSF=Spilled FeederHuntingdon Life Sciences 00-61311 Irnrnunotoxicity Sub-Group PAGE 5

TABLE I

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

INDIVIDUAL FEED CONSUMPTION VALUES (GRAMS/KG/DAY) FEMALES GROUP V POSITIVE CONTROL

WEEK OF STUDY ANIMAL# 0 1 2 3

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

MEAN S.D.

NSF=Spilled FeederHuntingdon Life Sciences 00-61311 Irnrnunotoxicity Sub-Group PAGE 1

TABLE J

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

ANIMAL TERMINATION HISTORY FEMALES GROUP I 0 MG/M3

TYPE OF

DEATH DATE OF WEEK OF STUDY DEATH STUDY DAY

TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICEHuntingdon Life Sciences 00-61311 Irnrnunotoxicity Sub-Group PAGE 2

TABLE J

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

ANIMAL TERMINATION HISTORY FEMALES GROUP I1 2000 MG/M3

---~---~~..-----.-~~~------~~.~~~~~.~----~~-~.~---~~.--..~-.~~~------~-..------..--------.--------..-------..---------------------- TYPE OF DATE OF WEEK OF STUDY ANIMAL# DEATH DEATH STUDY DAYHuntingdon Life Sciences 00-61311 Immunotoxicity Sub-Group PAGE 3

TABLE J

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

TYPE OF

DEATH

........................... TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE DATE OF DEATH
23-JUL-02 23-JUL-02 23-JUL-02 23-JUL-02 23-JUL-02 23-JUL-02 23-JUL-02 23-JUL-02 23-JUL-02 23-JUL-02 STUDY DAY

2 8 28 28 2 8 2 8 28 28 2 8 2 8 28Huntingdon Life Sciences 00-61311 Irnrnunotoxicity Sub-Group PAGE 4

TABLE J

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

TYPE OF DATE OF WEEK OF STUDY ANIMAL# DEATH DEATH STUDY DAY

----------------.----.-..------------------..--------------.------------.-----..----.------.--------------------------------------- TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICEHuntingdon Life Sciences 00-61311 Immunotoxicity Sub-Group PAGE 5

TABLE J

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS