# GFAP Levels in Specific Rat Brain Areas Following A 

# 13-Week Whole-Body InhalationExposure to Gasoline DIPE Vapor Condensate

HLS Study 

# No.: 00-6130 

## Sponsor StudyNo.: 

# 21 1 

## -DPE-S Date: 25June2002

## Summary Report Gasoline DIPE Vapor Condensate: A 13-Week Whole Body Inhalation Toxicity Study in the Rat

Huntingdon Life Sciences, Inc. Study No. 00-6130 Sponsor StudyNo 2 1 1 -DIPE-S Measurement of Glial FibrillaryAcidic Protein 

# I Principal Investigator:

James P. OYCallaghan, Ph.D. Molecular Neurotoxicology Laboratory Health Effects Laboratory Divison Centers for Disease Control and Prevention National Institute for Occupational Safety and Health 1095Willowdale Road Morgantown, 

# WV 26505 304-285-6079 304-285-6220 (FAX) j

## do5acdc. gov StudyDirector: Gary M. Hofhan, B.A., DABT Huntingdon Life Sciences Mettlers Road East Millstone,NJ 00875-2360 732-873-2550 732-873-3992 (FAX) 

# hofhang~~rinceton.huntin~don.com

STATEMENT OF COMPLIANCE This study was conducted in the spirit of compliance with 79.60, CFR Vol. 59, No. 122, 27 June 1994. This study was performed according to protocol and Standard Operating Procedures.

James P. O'Callaghan, Ph.D. Principal Investigator - -- 

## Date

Gary M. Hofhan, B.A., D.A.B.T. Date StudyDirector

## SIGNATURE PAGE
SCIENTIST


# The following Scientistwas responsible for the overall conduct ofthis study.

James P. 07Callaghan, Ph.D. PrincipalInvestigator

SCIENTIFICREVIEW 

# The following Scientist has reviewed and approvedthis report. Date

Gary

# M. Hoffman, B.A., D.A.B.T. StudyDirector Date

## 4 

## QUALITY ASSURANCE STATEMENT


# Listed below are the dates that ths study was inspected by the QualityAssurance Unit of the Centers for Disease Control-NationalInstitutefor Occupational Safety and Health, Health Effects Laboratory Division, Morgantown, WV, and the dates that findings were reported to the StudyDirector and Management. Thisreport reflects the raw data as far as can be reasonablyestablished. Type of Date(s) of Reported to Inspection Inspection Study Director and Management



# H. Leon Pringle, Ph.D. Date Quality Assurance Auditor

## QUALITY ASSURANCE STATEMENT
Listed below are the dates that ths study was inspected by the QualityAssurance Unit of Huntingdon Life Sciences, East Millstone, New Jersey, and the dates that findings were reported to the StudyDirector and Management. 

## This report reflects the raw data as far as canbe reasonably established. 

## Type of Date(s) of Reportedto Inspection Inspection Study Director and Management

Quality Assurance SeniorAuditor Date

## 6 TABLE OF CONTENTS Page



# ........................................................................................... Summary 7



# ........................................................................................ Introduction 7



# ................................................................. Materials,Methods/Procedures 8 

## Basic Protocol 1: GFAP SandwichELISA A. Introduction B. 'Materials C. Protocol Steps 1. StandardCurve Preparation 2. Sample Preparation 3. GFAP Assay Support Protocol 1: 

## Brain Tissue Preparation A. Introduction B. Materials C. Protocol Steps Support Protocol 2: Assay for Total Protein A. Introduction B. Materials C. Protocol Steps Reagents and Solutions General Commentary on GFAP Assays; Specific Commentary on the GFAP ELISA A. Background Information B. Critical Parameters 

## C. Trouble Shooting D. Typical Results E. Time Considerations



# ............................................................................................. Statistics 17



# .......................................................................... Results and Conclusions 17



# .......................................................................................... References 18



# ..................................................................................... Figure Legends 

## 2 1 Figure 1: 

## Sample StandardCurve Figure 2: pg GFAPImgTotal Protein per Brain Region



# ............................................................................................... Tables 24 Table 1: 

## GFAP Standard Curve Preparation Table 2: Microtiter Plate Template Table 3: SampleValues for GFAP Standard Curve Table 4: Troubleshooting Guide Table 

## 5: Mean GFAP Levels in Specific Regions of Male Rat Brains Following a 13-WeekWhole-Body Inhalation Exposureto Gasoline DIPE Vapor Condensate Table 

## 6: Mean GFAP Levels in Specific Regions of Female Rat Brains Following a 13-WeekWhole-Body Inhalation Exposure to Gasoline DIPE Vapor Condensate Table 7: Individual GFAP Levels in Specific Regions ofRat Brains Following a 13-Week Whole-Body Inhalation Exposureto Gasoline DIPE Vapor Condensate Appendix A =

## Testing Facility Exposure and In-Life Data

## 7 

# Summary
Toxicant-induced injury ofthe adult or developing central nervous system ofthe rat results in hypertrophy of astrocytes at the site ofinjury. Thehallmark of this response is the enhanced expression ofthe major intermediate filament protein of astrocytes,GFAP. A 

## 1.3-weekinhalation exposure to Gasoline 

# DTPE Vapor Condensate did not elevate GFAP levels in any of the nine brain regions examined in either males or females. The data suggest that exposure to Gasoline 

## DPEVapor Condensate under the regimen employed does not result in gliosisin any ofthe nine brain regions examined.



# Introduction
A characteristic feature of chemical-induced damage of the nervous system is selectivity; exposure to different nervous system toxicants results in damage to different brain regions and celltypes (Switzer, 1991;Balaban, 1992;

# 0'Callaghan et al., 1995). The differential susceptibility ofnervous system celltypes to injury often isreferred to as "selective vulnerability"(Spencer and Schuamburg, 1999; Baumgarten and Zimmerman, 1992). 

# An implicit assumption underlying this concept is that intrinsicproperties of individualneural cell types render them susceptible to damageby specific chemical exposures (Baumgarten and Zimmerman, 1992). Unfortunately, owknowledge of the mechanismsthat confer such vulnerabilityto specific toxic insults is limited. Thus, often there is no a

## priori 

## basis for predicting the cell types affectedby toxic exposures of the nervous system. Giventhe extreme cellular and molecular heterogeneity ofthe nervous system (McKay and Hockfield, 1982; Sutcliffe, 1988),the fact that targets of chemical-induced neurotoxicity are diverse and unpredictable should not be surprising. Thisbiologically-based situation does, however, make assessment of neurotoxicity difficultbecause one must face the problem of decidingwhere to look for damage. Overcomingthis obstacle requires a "marker" ofneural injury that can be used to localize (i.e. "mark") sites of damage anywhere in the nervous system.
A universal cellularreaction to damage ofthe central nervous system is hypertrophy of astrocytes. Thehallmark of this response, often termed "reactive gliosis,"is the enhanced expression ofthe major intermediate filament protein of astrocytes, glial fibrillaryacidic protein (GFAP). Thus, an increase in the brain concentration of GFAP serves as a biochemical indicator ofneurotoxicity. To validate the use of GFAP as a biomarker of neurotoxicity,prototypeneurotoxicants have been administered to experimental animals and the effects of these agents on the tissue content of GFAP have been determined by immunoassay (OYCallaghan, 1991a;Norton et al., 1992). Assays of GFAP were found to reveal dose-, time- and region-dependent patterns ofneurotoxicity at toxicant dosages below those that cause light microscopic evidence of cell loss or damage (OYCallaghan, 1991a;Norton et al., 1992). Moreover, the temporal and regional increments in GFAP correspond to the temporal and regional patterns ofneuronal damage, as revealed by sensitivesilver stains (Balaban, 1992). These findings indicate that assays of GFAP represent a sensitive, simple and quantitative approach for evaluation of nervous system damage (0'Callaghan, 199 1 a; Norton et al., 1992).
As part of the U.S. Environmental Protection Agency's testing requirementsunder the Clean Air Act, identificationand characterization of the potential adverse effects of gasoline and various oxygenate-gasoline blends is to be determined. Neurotoxicity

## 8 assessment constitutes a portion ofthese testing activities. Subchronic(13-week) inhalation exposures to gasoline and gasoline plus each of 6 fuel additiveswill be undertaken along with a two-generation reproduction toxicity study that includes a neurotoxicity component. Thepurpose of thepresent study was to use the GFAP assay for assessing the potential neurotoxic effects of Gasoline 

## DIPE Vapor Condensate. A control (air) and three exposure levels to the test condensate (2,000, 10,000and 20,000 mg/m3)were used. Although the EPA Guidelines (CFR 59, No. 122,79.67, 1994) specify six regions to be analyzed, we expanded our analysis to include an additional three areas of the brain to maximize the potential for detecting enhanced expression of GFAP due to exposure to the test substance.



## Materials,Methods/Procedures
STUDY DATES STUDY INITIATION 17December 2001 (Date StudyDirector signed the Protocol) 

## DATE OF ANIMAL RECEIPT 24 January 2002 

## EXPOSURE INITIATION 12February 2002 (Experimental Start Date) 

## EXPOSURE TERMINATION 14May 2002 

## TERMINAL SACRIFICE 15 May 2002 

## EXPERIMENTAL TERMINATION Day Month Year (Date Final Report is signedby the Principal Investigator) 

## STUDY COMPLETION Day Month Year @ate Final Report is signedby the StudyDirector)


## I. Basic Protocol 1: 

## GFAP SandwichELISA
A. 

## Introduction:The GFAP sandwich ELISA is suitable for assaying the concentration of GFAP present in homogenates of brain tissue. This method has successfullybeen applied to analysis of at least the following species: mouse, rat, guinea pig, dog, monkey, man, chicken, pigeon, trout and cod. Because GFAP is evolutionarilyconserved, it is likelythat this assay canbe very broadly applied across many species. The assay does not require preparation of any special materials or reagents; all components are available fiom commercial sources at modest cost. The 96-well microplate format lends itselfto processing largenumbers of samples and it makes the assay suitable for automation with a variety of liquid handling systems. It takes a minimum of 5 hours to process a single 96-well plate after preparation of the tissue homogenates (Support Protocol 1).


## B. Materials:
Vortex Mixer or Ultrasonic Cell Disruptor (e.g.PGC cat. # 

## 81-6721 -

## 02,2 mm probe) Pipettes HotIStir Plate Microplate Reader 96-well Microtiter Plates (Immulon 2, Dynatech) Pipette tips Rabbit anti-Glial FibrillaryAcidic Protein Antibody (DAKO, Cat. #Z0334, Lot #096(401)) Non-Fat Dry Milk (Carnation) Mouse anti-Glial FibrillaryAcidic Protein Antibody (Oncogene Research Products Cat. #IF03L, Lot #D15158-3) Phosphate Buffered Saline (e.g. Pierce Cat. #28374) Alkaline Phosphatase conjugated anti-mouse IgG (Jackson Immuno Research Cat #3 15-055-003) Alkaline Phosphatase Substrate Kit (e.g. Bio-Rad Cat. #l72-1063) TritonX-100 (e.g.Bio-Rad Cat. #16l-O407) Sodium Hydroxide (e.g., 0.4NNaOH, Fisher LC 243204)


## C. Protocol Steps:



# 1. Standard Curve Preparation 1. Prepare a GFAP standard: Thepreferred standard consists of an aliquot of a brain homogenate prepared as described in Support Protocol 1. This standard shouldbe prepared from the same species that was used to prepare the samples to be assayed for GFAP because GFAP immunoreactivity with a given set of immunodetection reagents will differ among different species. A largenumber of standardscan be prepared in advance fi-oma single "pool" of a 1% SDS homogenate prepared as described in SupportProtocol 1. This homogenate can be aliquoted and stored fi-ozenat -70°Cprior to use. 

## Thus, the GFAP standard essentially consists ofa control sample. This is preferable over using apure GFAP standard because using control tissue as a standard obviates any influence ofthe tissue "matrix" on the assay performance. To express the data in units of GFAP per unit oftotal protein, aliquots of a 1% SDShomogenate are stillto be used as a GFAP standard. This is accomplishedby "standardizing the standard" with addition of a known amount ofpure GFAP to the 1% SDS homogenate (i.e. an internal standard). Immunoreactivity values generated from standard curves ofthe GFAP "spiked" homogenate and the homogenate alone then are used to determine the concentration of GFAP in homogenate. For analysis of GFAP in regions ofrat brain, we routinely use aliquots of a hippocampal homogenate as a standard. It contains approximately2.5 pg GFAP per mg of total protein. Other species (e.g.mouse) contain different levels of GFAP in hippocampus. 2. 

## Prepare dilutions of the GFAP standard: Remove a tube ofthe GFAP standard from the freezer, thaw at room temperature and vortex or sonify prior to dilution. Using a rat hippocampal homogenate as a typical standard (-2.5 pg GFAPImgtotal protein), use the total protein value for this homogenate (- 

## 10 mg/ml).to prepare a standard curve in PBS plus 0.5% Triton X-100. For rat hippocampal homogenate, the protein values for the standard curve shouldbe between approximately 0.25 to 10pg/lOOpVmicroplate well (i.e. 0.25, 0.5, 1.0, 2.5, 5.O, 7.5, 10pgtotalproteinI100pl). Table 1 shows an example of dilutions needed to prepare a standard curve from a homogenate ofrat hippocampus

## 10 (hippocampus std.). Typically, standards are run in duplicate. Detergents act as wetting agents, therefore, more than a single use of apipette tip with SDSor Triton X-100-containing samples can lead to carry-over errors. Thus, it is recommended to use only a single pipette tip per sample and to withdraw the sample only a single time per tip.
2. 

## Sample Preparation 1. Thaw and mix samples: Remove samples fiom the fkeezer, thaw at room temperature and vortex or sonify prior to dilution. 2. Prepare dilutions of the samples: Dilute the samples in PBS 

# + 0.5% TritonX-100 to a concentration of approximately 10pgtotal protein/ 100pl. Samples high in GFAP (e. g. cerebellum)may need to be diluted to a concentration of 5pgtotal protein/lOOpl of PBS 

# +0.5% Triton X-100. Samples low in GFAP (e.g. striaturn)may need to be diluted to 20pg total protein1100pl ofPBS 

# + 0.5% Triton X-100. These dilution factors are determined empirically. The best practice is to prepare multiple dilutions ofeach sample to insure that optical density readings for a given sample fall on the linear portion of the standard curve. Typically, samples (like standards) are run in duplicate. Detergents act as wetting agents, therefore, more than a single use of apipette tip with SDS- or Triton-X-100-containing samples can lead to carry-over errors. Thus, it is recommended to use only a single pipette tipper sample and to withdraw the sample only a single time per tip.
3. 

## GFAP Assay Coat Immulon-2 flat bottom plates with rabbit anti-GFAP. Add 1.Opg total immunoglobulin protein 1100 pl PBS /well. (-25 pl of anti-GFAP [Dako]in 10 ml ofPBS is the quantity needed per plate). Incubate the coated plate at 37OC for 1 hour. This step may be done at the beginning of the assay or it may be done the night before with storage overnight at 4OC. Perform all other incubation and reagent addition steps at room temperature. Empty the plate into a sink and tap on absorbent paper to remove excess liquid. This latter procedure is important to eliminate the possibility ofany reagent carry-over between steps. Wash plates 4X with PBS (200 pl per well), tapping and blotting between each wash. Block 1howwith 5% non-fat powdered milk in PBS at 100p1 per well. Empty plate, tap on absorbent paper (upsidedown) to remove excess liquid, load diluted standard curve and samples in a volume of 100p1 per well. Incubate for 1 hour. The template, Table 2, is an example of a typical 96-well microplatelayout for GFAP standards and unknowns. Wash 4X with PBS 

# + 0.5% Triton X-100,200 pllwell. Incubate for 1hour in monoclonal anti-GFAP (1:500)(Chemicon) 

# + alkaline phosphatase conjugated anti-mouse IgG (1:3000)(JacksonImmunoResearch) made up in 5% non-fat drymilk 

# +0.5%Triton X- 100, 100pl /well. Wash 4X with PBS 

# +0.5% Triton X-100,200 pl/well. Add P-nitrophenylphosphate substrate (Bio-Rad) in a volume of 100pVwell and incubate for 20 minutes. 11. Stop reaction with 0.4NNaOH, 100 pllwell.

## 11 12. "Pop" any bubbles in the plate wells with a needle orpipette tip to insure uniform and accurate readings of standard and sample ODs. Read plate at 405 

# nm. 13. Calculate the GFAP concentration in the samplesby comparing their optical density (OD) values to those obtained for the linear portion ofthe GFAP standard curve. Software programs linked to specificplate readers shouldbe programmed to plot OD vs. GFAP values in linear vs. log linear fashon. Typically, we utilize the 4-parameter curve fit equation and generatecurves as shown in Fig. 1 and Table 3. Most programs allow for automatic subtractionof blanks and incorporation of dilution factors. Data are expressed as pg GFAPIpermg totalprotein or, ifthe absolute amount of GFAP in the standard is not available, data are expressed as GFAP-like immunoreactivitylper mg total protein. Totalprotein concentration in the samples is estimated from the total protein assay described in Support Protocol 2. Data also canbe expressed on the basis oftissuewet weight (pg GFAPIgramwet weight or GFAP irnmunoreactivity/gramwet weight). Although this approach permits elimination of the totalprotein assay (Basic Support Protocol 2), we find that the GFAP values obtained are slightlymore variable.

11. 

## SupportProtocol 1: 

## Brain tissue preparation
A. 

## Introduction: Thssupport protocol describes the procedure for preparing brain tissue for subsequent analysis of GFAP by Sandwich ELISA (Basic Protocol 1). This procedure does not describe orrecommend a specific approach for dissecting brain tissue. 

## It is noted, however, that reliable dissections are essential for obtaining reproducible results with the GFAP ELISA (see commentary).
B. Materials: DissectingInstruments Balance Ultrasonic Cell Disruptor (e.g.PGC cat. # 

## 81-6721-02,2mm probe) Pipettes HotIStir Plate Pipette tips Microfuge tubes
C. 

## Protocol Steps: 1. Sacrificeanimals and remove thebrain as rapidly as possible. 2. Dissect brain regions. If a number ofregions are to be dissected,this process can be aidedbykeeping thebrain firm on a cold plate (e.g. Thermoelectrics cold plate, Aldrich Chemical Co. or simply an inverted peti dish placed on ice) maintained at approximately 4°C. Rat or mouse brains canbe dissected into 10-15regions, free hand, within approximately 10 minutes. For this study, nine regions were dissected: Striatum,Hippocampus, Cortex, Olfactory Bulb, Thalamus, Hypothalamus, Cerebellum,Pituitary, and Rest of Brain. 

## This number of regions can be prepared on a coldplate or at room temperature without degradation of GFAP as assessed by immunoblot analysis. All brain

# 12 regions can be stored frozen indefinitely in capped microfuge tubes at this step in the protocol, oryou can proceed to the next step. 3. 

# Weigh and homogenize the dissectedbrain parts. Tare an appropriately labeled microfuge or other suitable storagetube, or weighboat for the large brain areasthat don't fit into microhge tubes, ie; Cerebellum, Cortex, Rest ofBrain and Thalamus). Place individual brain regions in the tube or weigh boat, obtain the weight, transfer large brain areas to large glass homogenization tube, and immerse the tissue in 10volumes of hot (85-95°C) 1% (wlv) SDS. For example, 0.1 grams of tissue would be immersed in 1.0ml of SDS. Wle the SDS is stillhot, homogenize the tissue by sonification with an ultrasonification microprobe. Large brain areas are sonifiedin the glass homogenization vessel with the ultrasonification microprobe, then homogenized using a motor-driven Teflon pestle. Samples should be stored fi-ozen (-70°C) at time of sacrifice prior to assay. Samples prepared and stored in this manner retain their GFAP content for at least 

# 5years. 

## 111. Support Protocol 2: 

## Assay for Total Protein


# A. Introduction: 

# This support protocol describes the procedure for assayingthe concentration of totalprotein in the SDS-homogenates. The procedure described essentially is the bicinchoninic acid (BCA) method described by Smith et al. (1985) which is available in lut form (seematerials). To assay total protein concentration ofthe SDS-homogenatesthe assay must be compatiblewith 1% SDS. Use ofthe BCA assay is not an absolute requirement as other detergent compatible methods are available (e.g. Bio-Rad DC protein assay). Bovine serum albumin isused as theprotein standard in the described procedure. Other protein standards can be substituted.


# B. Materials: 

# Microplate Reader Pipettes Pipette tips 96-well Microtiter Plates Microfuge tubes Incubator Vortex Mixer Miscellaneous Laboratory Glassware BCA Protein Kit (Pierce#23223) Bovine Serum Albumin (BSA) (Sigma A7888) Sodium Dodecyl Sulfate(Bio-Rad#16 1-0302)
C. 

## Protocol Steps:



# 1. Prepare total protein standards. Prepare a 1mglml solution ofBSA in 1

% 

# (wlv) SDS. Aliquots ofthis standard can be stored frozen at - 70 

## C

## for future use. Thaw as needed, but do not re-freeze. 

# 2. Prepare a total protein standard curve. Prepare dilutions of the BSA standard in 1% SDS as follows: 1.0,2.5, 5.0, 7.5, and 10pgIlOp1of 1% SDS (no dilution is required for last standard). Vortex each tube and add 10pl of each standard to

# 13
a well of the microtiterplate; add 10p1 of 1% SDS to serves as a blank. Typically, standards are run in duplicate. Detergents act as wetting agents, therefore, more than a single use of apipette tip with SDS-containing samples
can lead to carry-over errors. Thus, it is recommendedto use only a single pipette tipper sample and to withdraw the sample only a single time per tip. 

# 3. Prepare dilutionsofthe samples. Thaw the samples, vortex and dilute a 10pl aliquot with 190p1 of 1% SDS. Vortex the dilution tube and add a 10pl aliquot into a well of a microtiterplate. 4. Add the protein assay reagent. Add 200 p1 of the BCA reagent (composed of 50:1 ratio of solutionA: solutionB of the Pierce BCA reagent) to each standard and sample. 5. Incubate the plate at 37OC for 30 minutes. Other incubation temperatures are permissible; follow direction provided with the kit. 

# 6. "Pop" any bubbles in the microtiterplate wells with a needle orpipette tip to insureuniform and accurate readings and read the plate at 562 nm. 7. Calculate the concentration of total protein in the samples from the standard curve. Softwareprograms linked to specificplate readers should be programmed to plot OD vs. totalprotein in a linear fashion. Most programs allow for automatic subtraction ofblanks and incorporation of dilution factors. Because the sampleswereprepared in 10 volumes of diluent, typically, totalprotein values are approximately 10mglml.
IV. 

## Reagents and Solutions


# 1.Phosphate Buffered Saline (PBS)- 

# Onepacket ofPBS is mixed thoroughly

with 500 ml of deionized water to give a final concentration of: 137 mM 

# NaCV1.O mM

# KCV2 mM 

# KH2P04/8.0 mM

# Na2HP04.7H20/pH 7.4 (can be stored at 4OC for at least a month). For this and all subsequent reagents and solutions,determine the total volume that needs to be preparedbased on the
use of 100pVwell and 96 wells per plate (washes take 200 pVwell). 2. PBS+O.5% Triton X-100- 

# 2.5ml ofTriton X-100 is added to 500 ml ofPBS (can be stored at 4OC for at least a month). 3. 

## Blocking agent(BLOTT0)- 

# (PBS 

# + 5% powdered milk or PBS 

# + 0.5% Triton X- 100

# + 5% powdered milk). Five grams of

# non-fatt powdered milk is added per 100 ml of PBS orper 100 ml ofPBS

# +Triton X-100. Prepare at least 100 ml of each to facilitatedissolving the powdered milk; PBS may be warmed slightlyto facilitate this process. Make these solutionsup fresh the day of assay and do not save. Also, do not retain thepowdered milk for greater than a month or two (room temperature). The dry 

# milk tends to discolor and will not go into solution at shelftimes longer than 2 months. 4. Polyclonalanti-GFAP 

# (Dako)-Add 25p1of antibody solution/10ml ofPBS. The assay is based on the use ofthsantibody as a "capture" reagent. Substitution of an antibody from another vendor may not yield suitableresults. Make this solutionfresh on the day ofuse and do not save. 5. Monoclonal anti-GFAP 

# (Chemicon; formerlyBoehringer Mannheim) combinedwith Alkaline Phosphatase-conjugatedanti-mouse IgG 

# (Jackson 1rnmunoResearch)-Make up a stock solution ofmonoclonal anti-GFAP and alkalinephosphatase-conjugated anti-mouseIgG asper the vendors' instructions. Storeboth stocks at 4OC asper the vendor's instructions. Add 20p1 of the monoclonal antibody solution stock and 3.3 p1 of the alkaline

## 14 phosphatase conjugate stock11 Om1 powdered milk +0.5% Triton

# X- 100. The assayisbased on the use of the monoclonal antibody as a "detection" reagent and the alkalinephosphatase conjugate to bind to the detection antibody and generate a colored reaction product proportional to the amount of antigen (GFAP) present in the samples. Substitution of antibodies fiom other vendors may not yield suitable results. Make these solutions fresh on the day ofuse and do not save. 6.

## P-nitrophenylphosphatesubstrate (BioRad)- Mix 2ml of diethanolamine buffer on a stirrer with 2 p-nitrophenylphosphatetablets and 8 ml of deionized water. Make this solution fiesh on the day ofuse and do not save.
V: 

## General Commentary on GFAP Assays; Specific Commentary on the GFAP ELISA
A. BackgroundInformation
It has longbeen known that damage to the central nervous system results in astrogliosis (gliosis,reactive gliosis, glial activation), a responseto brain injury characterizedby hypertrophy and, less often, hyperplasia of astrocytes, a subtype of CNS glia (Eng, 1988;Norenberg, 1994). At the electron microscopic level, astrogliosis is characterizedby the accumulation of glial filaments. GFAP was found to be the major protein component of these filaments (Eng, 1988). As such, GFAP serves as a biomarker for filament accumulation and, therefore, of gliosis (Eng, 1988; Norton et al., 1992;

# 0'Callaghan, 1993). With the development of antibodies to GFAP, irnrnunohistochemical analysis ofthis protein soon documented that gliosis occurs in response to diverse insults of the CNS, including trauma, disease, and toxic exposures (Eng, 1988; Norenberg, 1994; Norton et al., 1992;O'Callaghan, 1993; OYCallaghan et al., 1995). Thus, a large body of evidence now has been accumulated demonstrating the ubiquity of the glialresponse to all types of CNS damage based onimmunohistochemistry of GFAP. Onlyrecently, however,have methodsbeen introduced to assay levels of GFAP as a means of quantifying gliosis.
While GFAP irnmunohistochemistry has proven useful for revealing patterns of gliosis afterbrain injury, this approach does not lend itselfto quantification or the analysis of large numbers of samples. Small (25-50%),but toxicologically significant increases, also may be difficult to detect by irnrnunohistochemistry. These drawbacks, combined with the need to develop quantitativebiomarkers of neurotoxicity (OYCallaghan et al., 1995), and to define quantitative aspects of toxicant- and disease-induced gliosis, has prompted the development and implementation of a number of GFAP assays. These assays have been applied to examine gliosis in specificbrain areas already known to be affected by disease or other insult. 

# In addition, they also can be broadly applied in a risk assessment context (U.S.EPA) to screen forpotential sites ofneural damage resulting from toxic exposures of the CNS. Recently, analysis of GFAP has been used to demonstrate that the degree of cortical gliosis in postmortem brain tissue from victims ofAlzheimer's disease correlates with the severityof dementia scoresin these individuals prior to death (G. Webster Ross, submitted). Analysis of GFAP in cerebrospinal fluid (CSF) also has been applied to the human condition as an indicator ofthe severity of traumatic injury to the brain (Rosengren et al., 1994). Finally, analysis of GFAP can be used as an indicator

# 15 of the presence ofbrain or spinal cord contamination ofmeat (Schmidt et al., 1999).
Ofthe number of GFAP assays that have appeared in the literatureover the last 15 years, all essentially fall into two categories: 1) solid phase immunoassays where GFAP is immobilized on a solid support matrix and detected by monoorpolyclonal antibodies or 2) 

# liquid-phase assays where GFAP from brain extracts or solubilizedbrain tissue (or CSF) is "captured" by one antibody and then detectedby another antibody raised in a different host species (Butler et al., 1986). The assay described in this studyis of the secondtype and it has a number of advantages over the solid phase assays. Specifically, solid-phase detection and "quantification" of GFAP most commonly involves the timeconsuming resolution of a protein mixture by SDS-PAGE, followed by electrophoretic transfer to a solid support membrane. Anti-GFAP antibodies coupled to a variety of detection reagents then can be used for quantification of GFAP bound to the membranes. Unfortunately, this approach has been found to severely underestimate the concentration of GFAP in the resolved mixture of proteins and the effects oftreatments known to increase GFAP (O'Callaghan et al., 1999). Other solidphase assays for GFAP have been developed that do not rely on prior resolution ofprotein mixtures by SDS-PAGE (Wang et al., 1990; OYCallaghan, 1991b). These assays incorporate manual spotting of brain homogenates on solid supports,with or without the aid of a template. The membranes are then incubated with anti-GFAPpolyclonal or monoclonal antibodies, which, in turn, are bound by 125~ 

# ProteinA. Quantification is acheved by gamma spectrometry orby densitometry of the autoradiographs. These assays give a linear signal over a fairly large range of spotted protein. However, they require large amounts ofreagents, includingradiolabeled reagents, and they do not have impressive throughput.
The sandwich ELISA for GFAP described in this study or similarELISAs describedpreviously (Eng et al., 1986;Kretzschmar et al., 1985; O'Callaghan, 1991 b; Rosengren et al., 1994),have several advantages in comparison to the other methods for assaying GFAP described above. They are easierto perform because they have fewer steps. They are more sensitive. Although they may require a greater number ofreagents, ELISAs adapted to the microplateformat permit the use of very smallvolumes, which results in a significantoverall cost reduction. The 96-wellmicroplate format also has the advantage of speed and hghthroughput. From sample applicationto data collection, all steps canbe performed in the plate. Moreover,the microplate-based format permits the entire assayto be automated through the use ofrobotic liquid handlingprocessors. Finally, radioactivity isnot involved, making the assay saferto perform and allowing the user to avoid costly and time-consumingradioactivitydisposal procedures. While most ofthe GFAP sandwich ELISAs described to date are similar and share the advantages affordedby this technique, the assay described in ths study may have a few additional advantages. Because it is based on detergent-solubilized homogenates of a given brain area, any treatment effects can be directlyrelated to effects in that brain area, rather than an arbitrarily definedextract or sub-fi-action that may contain only aportion of the total GFAP in that area. Using a solubilized homogenaterather than a subfractionof a given brain area also facilitatescomparisons of quantitative data on GFAP to immunohistochemical staining of GFAP in that area. Moreover, it also helps

# 16 rule out inter-laboratory differences associated with assayingGFAP content in one type of extract in one lab, and another type of extract/fractionin another lab. Finally, the same SDS-denatured homogenate used to assay GFAP can be subjected to multiple assays for additional glial or neuronalproteins, thereby permittingcomparisons to be made among multiple markers of neurotoxicity in a single sample. For example, the dopaminergicneurotoxicant, 1-methyl-4- 

# phenyl-l,2,3,6-tetrahydropyridine, causes a large increasein GFAP that results from damage to dopaminergic nerve terminals, damage that can be quantified by immunoassayoftyrosine hydroxylase (TH), a marker of dopaminecontaining nerve terminals in the target region (O'Callaghan et al., 1990). Both markers can be assayed from aliquots ofthe same tissue sample and, on this basis, we find that larger decreases in TH predict greater increases in GFAP.
B. Critical Parameters 

# The most critical aspect of the GFAP assay is the absoluterequirement for preparation of consistently dissected regions of the brain (see Support Protocol 1). Consistent dissections yield consistent GFAP values with the use ofonly a few animals per dose or time point (seeAnticipated Results). The particular regions to be dissected depend on the questions being addressed. If a target region is known or suspected, dissections canbe limited to the region of interest. Ifthe GFAP assay isbeing applied in a screening context,multiple (10-15)brain regions must be dissected in order to avoid the possibility of diluting localized increases in GFAP. The possibility exists that extremely localized increases in GFAP may fail to be detected with the assay. While GFAP imrnunohistochemistryis relatively insensitive in comparisonto the GFAP assay, and it may not detect small increases in GFAP, it can reveal small "hot spots" of gliosis (e.g. see effects ofMK-801) (Fix et al., 1995). Such discrete astrocyticresponses could escape quantification with the GFAP assay due to dilution of signalby surrounding tissue. No one approach can be broadly applied to detect all toxicant-induced damage ofthe CNS. Therefore, it isprudent to use the GFAP ELISA in conjunctionwith GFAP irnrnunohistochemistryand other sensitivemorphological approaches for detection of neural damage, such as silver degeneration stains (Switzer, 2000), Fluoro-Jade (Schmued et al., 2000), and stainsthat detect activatedmicroglia (Streit et al., 1999).
In terms of the GFAP ELISA itself, the key requirements for optimal performance ofthe assay include: 1) use of the specified antibodies,2) addition of the reagents at room temperature, 

# 3) fresh (daily) preparation of all reagents containing antibodies andlor non-fat 

# dry milk and 4) mixing ofthe standards and samples prior to their dilution or addition to the microplate wells. A troubleshooting guide is provided in Table 4 that covers most problems encountered with the assay.
C. Troubleshooting(Table 4)


## D. Typical Results 

# Typical GFAP assay values obtained for different regions ofthe rat brain are presented in Fig. 2. Absolute values for GFAP (pglmg total protein) may vary depending on the GFAP standard used and the species subjectedto evaluation.

## 17 Region-to-region differences in GFAP values from untreated animals of a given species,however, should remain stable, if consistent dissections are performed.


## E. Time Considerations The GFAP assayrequires approximately 5 hours for one person to process a 96- well microplate. The timerequired to preparebrain samples depends on the number ofbrain areas to be dissected and on whether the areas are stored frozen prior to homogenization With practice, 10brain areas can be prepared (and homogenized) from 50rats in a day. Two people are required: one to dissect the brains and one (or more) to weigh and homogenize the tissue. The total protein assay requires approximately 1hour for one person to process a 96-well microplate.


## Statistics:
Data were subjectedto analysis ofvariance followedbypost hoc comparison oftreatment means (JMPB, 

## SAS). Differences were considered to be significant at the 

## P <

## 0.05 level.


## Results and Conclusions
Theresults of the GFAP analysis arepresented in Tables 5 and 6. Results arereported according to gender,because sex-dependent responses to toxic substances are not uncommon, includingresponses reflected in levels of GFAP. Control levels for GFAP varied markedly according to brain region, consistent with known levels observed for GFAP across different brain regions (see Fig. 1). 

## The 13-week exposureto Gasoline 

## DLPE Vapor Condensate did not elevate (was not associated with any increase in) GFAP levels in anybrain region in either males or females. These data suggestthat under the exposure conditions employed, damage-induced gliosis did not occur in the brain regions cxamincd. Toxicant-induced gliosis is highly dose-, region-, and time-dependent. It is possible, therefore, that exposure to higher concentrations, examination of more brain regions, examination ofmore discrete dissections of a givenbrain region, or the inclusion ofmore timepoints, might have resulted in positive findings. 

## We note almost all of the pituitaries (males and females) assayed gave results that were below the level of sensitivity, therefore, only those values that fell on the curve are presented. This study represents one ofthe first attempts to assay pituitary levels of GFAP and the low levels were not unexpected. A recently developed fluorescence-based GFAP ELISA (OYCallaghan, unpublished) could be used to assess pituitary data if deemed necessaryfor future studies. 

# In conclusion, exposure to Gasoline 

## DLPE Vapor Condensate did not appearto result in gliosis in nine representative brain regions in males or females.

## 18 References



## Balaban, C.D. 1992. The use of selective silver degeneration stains in neurotoxicology: Lessons from studies of selectiveneurotoxicants. In: The Vulnerable Brain and Environmental Risks, Vol 1,Malnutrition and Hazard Assessment. R.L. Issacson and K.F. Jensen. Eds.: 223-238, Plenum. New York, 

# NY. Baurngarten,H.G.& B. Zimmermann. 1992.Cellular and subcellular targets of neurotoxins: the concept of selective vulnerability. In: Handbook ofExperimental Pharmacology, Vol. 102Selective Neurotoxicity. H. Herken and F. Hucho. Eds., pp. 1-28.Springer-Verlag. Heidelberg. Butler, J.E., Spradling,J.E., Suter, M., Dierks, S.E.,Heyermann, H., and Peterman, J.H. 1986. The immunochemistryof sandwichELISAs-I. The binding characteristic of immunoglobulins to monoclonal and polyclonal capture antibodiesadsorbed on plastic and their detection by symmetrical and asymmetrical antibody-enzyme conjugates. Mol. Immunol. 23: 971982. Eng, L.F., Stocklin, E., Lee, Y.-L. Shiurba, R.A., Coria, F., Halks-Miller, M. Mozsgai, C., Fukayama, G. and Gibbs, M. 1986. Astrocyte culture on nitrocellulosemembranes andplastic: Detection of cytoskeletal proteins and mRNAs by immunocytochemistry and in situ hybridization.

# J. Neurosci. Res. 16:239-250. Eng, L.F. 1988. Regulation of glial intermediate filaments in astrogliosis.In Biochemical Pathology ofAstrocytes. (M.D. Norenberg, L. Hertz & 

## A. Schousboe, eds.) pp. 79-90. A.R. Liss, New York. Fix, A.S., Wightman, K.A. and OYCallaghan, J.P. 1995. Reactive gliosis inducedby MIS-801 in the rat posterior cingulate/retrosplenial cortex: GFAP evaluation by sandwich ELISA and irnmunocytochemistry. Neurotoxicology 16:229-237. Kretzschmar, H.A., DeArmond, S.J.,Forno, L.S. 1985. Measurement of GFAP in hepatic encephalopathyby ELISA and transblots. 

# J. Neuropathol.Exp. Neurol44: 459-471. McKay, R.D.G. and S.J. Hockfield. 1982. Monoclonal antibodies distinguish antigenically discrete neuronaltypes in the vertebrate central nervous system.Proc. Natl. Acad. Sci. USA 79: 6747-6751. Martin, P.M. and OYCallaghan, J.P. (1995). Biochemical immunohistology. In: Central Nervous System Trauma: Research Techniques, S.T. Ohnishi and T. Ohnishi, eds., pp. 509-516,CRC Press, Boca Raton. Norenberg, M.D. 1994. Astrocyte responses to CNS injury. 

# J. Neuropathol.Exp. Neurol53: 213-220. Norton, W.T., Aquino, D.A., Hozumi, I., Chiu, F.-C., and Brosnan, C.F. 1992. Quantitative aspects ofreactive gliosis:A review. Neurochem. Res. 17: 877-885.

# OYCallaghan, J.P. 1991a.Assessment ofneurotoxicity: Use of GFAP as a biomarker. Biomed. Environ. Sci. 4: 197-206.


# 0'Callaghan, J.P. 1991b. Quantification of glial firbrillary acidic protein: Comparison of slot-immunobinding assays with a novel sandwich ELISA. Neurotoxicol. Teratol. 

# 13:275-281. OYCallaghan, J.P. 1993.Quantitative features ofreactive gliosis followingtoxicantinduced damage of the CNS. Ann. 

# NYAcad. Sci. 

# 679: 195-210.


# 0'Callaghan, J.P., Imai, H., Miller, D.B. and Minter, A. 1999. Quantitative immunoblots of proteinsresolved from brain homogenates: Underestimation of specific protein concentration and oftreatment effects.Anal. Biochem. 

# 274: 18-26. OYCallaghan, J.P., Miller, D.B. and Reinhard, J.F., Jr. 1990. Characterization ofthe origins of astrocyteresponseto injuryusing the dopaminergic neurotoxicant, 1-methyl

# 4-phenyl-l,2,3,6-tetrahydro-pyridine. Brain Res. 

# 521:

# 73-80. OYCallaghan, J.P., Jensen, K.F. and Miller,D.B. 1995. Quantitative aspects ofdrugand toxicant-induced astrogliosis.Neurochem. Int. 

# 26: 1 15-124. Rosengren, L.E., Wikkelss, C. and Hagberg, L. 1994.A sensitiveELISA for glial fibrillaryacidicprotein: application in CSF of adults.J. Neurosci. Methods 

# 5 1: 

# 197- 204. Schmidt, G.R., Hossner, K.L., Yemm, R.S., Gould, D.H. and OYCallaghan, J.P. 1999. An enzyme-linked immunosorbent assay for glial fibrillary acidicprotein as an indicator of the presence of brain or spinal cored in meat.J. Food Protect. 

# 62: 394-397. Schmued, L.C. and Hopkins, K.J. 2000. Fluoro-Jade: novel fluorochromes for detecting toxicant-induced neuronal degeneration. Toxicol. Pathol. 

# 28: 91-99. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner,F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson,.B.J.Klenk, D.C. 1985.Measurement of protein using bicinchoninic acid.Anal. Biochem. 

# 150:76-85. Spencer,P.S. and H.H. Schaumburg. 1999. Experimental and Clinical Neurotoxicology, 2ndEdition, Oxford Unviversity Press, Oxford, Cary, NC Streit, W.J. 

# ,Walter, S.A., and Pennell, N.A. 1999. Reactive microgliosis. Prog. Neurobiol, 

# 57: 563-581. Sutcliffe, J.G. 1988.mRNA in the mammalian central nervous system.Ann. Rev. Neurosci. 

# 11: 

# 157-198.

Switzer, R.C. 1991. Strategies for Assessing Neurotoxicity. Neurosci. Biobehav. Rev. 

# 15: 89-93. Switzer, R.C. 2000. Application of silverdegeneration stains for neurotoxicitytesting. Toxicol.Pathol. 

# 28: 70-83.

## 20 U.S. Environmental Protection Agency, Pesticide Assessment Guidelines, Subdivision 

## F. Hazard Evaluation: Human and Domestic Animals. Addendum 10, Series 81, 82, 83, Neurotoxicity. EPA-540109-91 - 

## 123,Springfield,VA: National Technical Information Service. Wang, S., Rosengren, 

## L.E., Karlsson, J.-E., Stigbrand, T. and Haglid, K.G. 1990.

# A simple quantitativedot-immunobinding assay for glial and neuronal markerproteins in SDS-solubilized brain tissue extracts.

# J. Neurosci. 

## Methods 33: 219-227.

## Figure Legends Figure 1. 

## Sample GFAP standard curve. GFAP values in nanograms correspond to levels found in .25 -10 

## pg 

## total hippocampal homogenate protein. Dilutions ofthis homogenate were used to construct the GFAP standard curve shown. Figure 

# 2. Levels of GFAP found in differentregions ofrat brain. OB, olfactorybulbs; Str, striaturn;Hip, hippocampus; Hypo, hypothalamus; Ctx, cortex; Cbm, cerebellum; BS, brain stem. Adapted from Martin and O'Callaghan, 1995.

# Figure 1

2.0-

1.5- 

# E 

# .- V) 

## s 

## a3 

# n 

# - 1.0- 0 

# .- CI 

# 0"

0.5-

0.0 y = ((A-D)/(l+(xlC)" B)) + 

## D A = 

### 0.009 B = 

### 1.349 

## C = 

### 7.163 D = 

### 1.964 

# ?= 1.00

I I I I I I I

# .I 

# lo-" lo0 10 

# lo2 ng 

# GFAP1100 pI

# Figure 2

OB Str 

# Hip 

# Hypo Ctx Cbm 

# BS 

# Brain Region

# Blk= Blank; Std= Standard; ? =

# Unknowns

## STANDARDS Value Well OD Mean Std Dev CV 

## pg 

## total protein

(ng GFW STDOl 1o~g B 1 1.694 1.632 0.087 5.4 (25.OOng) B2 1.570 

## STDOZ 7.5 Pg C1 1.537 1.543 0.008 0.5 (18.75ng) C2 1.549 

## STD03 5.o~g Dl 1.295 1.314 0.027 2.1 (12.50ng) D2 1.334 

## STDO4 2.5~8 El 0.857 0.859 0.002 0.3 (6.25ng) E2 0.861 

## STDOS 1.0Pg 

## F1 0.386 0.386 0.000 0.1 (2.50ng) F2 0.386 

## STD06 0.5 Pg G 1 0.175 0.164 0.016 10.0 (1.25ng) G2 0.152 

## STD07 0.25 pg 

## H 1 0.072 0.076 0.006 7.6 (.625ng) H2 0.080
BL=Blank; STD=Standard; OD=OpticalDensity; StdDev=Standard Deviation; CV=Coefficient of Variation

## Table 4: 

## Troubleshooting Guide

Problem No color reaction

Colorreaction abnormally low

Colorreaction abnormally high

Standard curve not sigmoid

Samples not on linear portion of curve

Duplicates are not similar

Colorreaction obtained for standards and samples, but 

## OD values not as expected Possible Cause Incorrect preparation of color reagent

Antibody was not as specified in the protocol
One or more antibodies were omitted
or used at the wrong dilution Incubator was set at less that 37°C

Antibody solution too dilute; incorrect preparation of color reagent P-nitrophenylphosphate substrate kit is too old
Colorreactionwas not terminated
Incorrectplate template set in the plate reader
Incorrect standard dilution
Incorrect standard dilution

Incorrect sample dilution

Carry over fromusing same tip Poor pipetting technique
Plate washer malfunction

Bubbles throughout the plate Plate read at incorrect wavelength Solution If color reactionhas not been terminated, remove reagent, add new color reagent and continue assay Obtain correct antibody and repeat assay Repeat assay with proper reagents used at the correct dilutions Repeat assay with incubator temperature set at 37°C Repeat assay with correct reagent dilutions Repeat assay with fresh ht

Repeat assay and terminate reaction with 0.4 

## N N~OH Use correct plate template and reread plate Repeat assay with correct standard dilution Repeat assay with correct dilution of standard
Run multiple dilutions of samples to obtain OD values fromthe linear portion of the curve Change tips after each use
Checkprecision by weighmg
Checkplate washer for even dispensing and aspiration Pop bubbles and re-read plate Read plate at 405 

## nm

## Table 5: Mean GFAP Levels in Specific Regions of Male Rat Brains Following a 13-Week Whole-Body Inhalation Exposure to Gasoline DlPE Vapor Condensate

Brain 

### Area Group I Air Control

0 mglm3

itriaturn iippocampus :ortex Ilfactory Bulb -halamus iypothalamus :erebellurn 'ituitary test of Brain 

### Group ll Test Substance

2,000 mglm3 Group Ill Test Substance

10,000 mglm3 Group IV Test Substance

20,000 mglm3

*Each value represents the mean rt 

### SEM for the concentration of GFAP (pglmg 

### Total Protein) 

### n= 

### 5 except for Pituitary; ND, not detected; see Results and Conclusion and Table 

### 7



## Table 6: 

## Mean GFAP Levels in Specific Regions of Female Rat Brains Following a 13-Week Whole-Body Inhalation Exposure to Gasoline DlPE Vapor Condensate

Brain Area Group I Air Control

0 mglm3

Striatum Hippocampus Cortex Olfactory Bulb

halamus Hypothalamus Cerebellum Pituitary 

### Group 

## ll Test Substance

2,000 mglm3

L 

### *Each value representsthe mean

# * SEM for the concentratic 

### Group Ill Test Substance

10,000 mglm3 Group IV Test Substance

20,000 mglm3



# - of GFAP (pglmg 

### Total Protein) 

### n= 5 except for Pituitary; ND, not detected; see Results and Conclusion and Table 

### 7

## 29 Table 

### 7: Individual GFAP Levels in Specific Regions of Rat Brains Following a 13

### Week Whole-Body Inhalation Exposureto Gasoline DlPEVapor Condensate



### o Each value represents the concentrationof GFAP(ug1mg Total Protein) 

### * Samples too dilute to detect GFAP

# INTRODUCTION: The following is data generated at Huntingdon Life Sciences, East Millstone,NJ. The separately issued main study report shouldbe referenced for details of the procedures used fortest atmospheregenerationlcharacterization and animal evaluations.

Huntingdon Life Sciences 00-6 130 Page 1 2 1 1 -DIPE-S Draft Final Report



# STUDY DATES: Date of Animal Receipt: 24 January 2002 Experimental Initiation Date: 12February 2002 (in-life) Animal Exposure and Animal Data Preface

Experimental CompletionDate: 15May 2002 (in-life) Appendix A

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, feed consumption and ophthalmoscopic findings.


# TABLE 

# OF CONTENTS
TABLES



# ........................................................................................... Chamber Monitoring Results Summary of Clinical Observations



# .................................................................................. Summary of Ophthalmoscopic Findings Ophthalmologist Report



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

...................................................................... Summary of Ophthalmology Observations Mean Body Weights (grams)



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



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



# ............................................................ Mean Feed Consumption Values (grams/kg/day) Individual Clinical Observations



# ..................................................................................... Individual Ophthalmology Observations



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



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



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



# ................................................... Individual Feed ConsumptionValues (grams/kg/day).. Animal Termination History



# ............................................................................................Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-6130 WHOLE-BODY INHALATION TOXICITY STUDY IN RATS

Date Exposure Number Chamber Monitoring Results Cumulative Exposure Record Group IA 

### - 0 mglm3 (Air Control)

Nominal
(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 Mean (mg/m3) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 alvtical Chamber Concentration Individual MMAD (pm)

1.209

4.471

2.303

1.520 Particle Size Determinations GSD TMC (mglm3)

1.94E-03

7.37E-03

3.03E-03

3.20E-03 Chamber M Tem~erature vironment
n Humidity
(%) 57 58 58 56 56 58 54 54 55 51 58 53 57 58 54 44 54 59 54 55 52 53 54 53

### Table A GASOLINE DlPEVAPOR CONDENSATE: A 13-WEEK 00-6130 WHOLE-BODYINHALATION TOXICITY STUDY IN RATS

Date Exposure Number Chamber Monitoring Results Cumulative Exposure Record Group IA 

### - 0 mglm3 (Air Control)

Particle Size Nominal
(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 1 

### Mean (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 ~alvtical Chamber Concentration Individual MMAD (IJm) 0.9053

2.495

2.357

2.187

1.413 Determinai GSD 1s 

### TMC (mglm3) 1.20E-02

4.91 E-03

7.60E-03

2.32E-03

8.01 E-03 Chamber Environment M Temperature
("C) 27 25 24 24 24 24 24 25 25 25 25 25 24 23 24 23 24 25 25 24 24 25 24 24 in Humidity 

## (%I 54 53 54 52 53 58 53 51 52 55 54 50 56 52 55 55 5 1 51 53 52 50 47 54 52Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS

Chamber Monitoring Results Cumulative Exposure Record

Day

65 66 69 70 71 72 73 76 77 78 79 80 83 84 85 86 87 89 90 91 92 Date

18-Apr-02 19-Apr-02 22-Apr-02 23-Apr-02 24-Apr-02 25-Apr-02 26-Apr-02 29-Apr-02 30-Apr-02 01-May-02 02-May-02 03-May-02 06-May-02 07-May-02 08-May-02 09-May-02 10-May-02 12-May-02 13-May-02 14-May-02 15-May-02
S.D. Exposure Number

49 50 5 1 52 53 54 55 56 57 58 59 60 6 1 62 63 64 65 66 67 68 69 Mean 0 0 Group IA 

### - 0 mglm3 (Air Control)

Nominal
(mglm3) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.899 0.9 Particle Size

2.8 Chamber Environment

Mean (mg/m3) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MMAD (crm)

1.767

2.130

1.325

1.594

1.975 0.392 Temperature
("C) 24 24 24 25 25 25 25 25 24 24 24 24 24 24 25 24 24 25 24 22 22 24.3 3.13E-03 MeanHumidity


### ("A) 52 49 50 54 55 52 54 49 53 53 5 1 54 5 1 52 51 51 50 50 54 53 56 53.3 Determinations GSD

2.858

1.927

1.605

2.093

2.132 Analytical Chamber Concentration Individual TMC (m91m3)

5.16E-03

4.07E-03

1.29E-03

2.01 E-03

4.84E-03 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 (mglm3) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0Table A GASOLINE DlPE VAPOR CONDENSATE:A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS

Chamber Monitoring Results Cumulative Exposure RecordTable A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 WHOLE-BODY INHALATION TOXICITY STUDY IN RATS

Date Exposure Number Chamber Monitoring Results Cumulative Exposure Record Group IB 

## - 0 mg/m3(Air Control)

Nominal
(mg1m3) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 Mean (mg/m3) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ~alytical Chamber Concentration Individual 

### (ms 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MMAD (pm) 0.9933

3.236

1.240

2.023

1.877 Particle Size ?terminations

GSD



## --



# - TMC (mg1m3) 1.41E-02

3.65E-03

4.86E-03

2.81E-03

9.96E-03 Chamber rb Tem~erature wironment in HumiditvTable A GASOLINE DlPE VAPOR CONDENSATE:A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS

Chamber Monitoring Results Cumulative Exposure Record Grouo IB

## - 0 malm3(Air Control)Table A GASOLINE DlPE VAPOR CONDENSATE:A 13-WEEK 00-6130 WHOLE-BODY INHALATIONTOXICITY STUDY IN RATS

Chamber Monitoring Results Cumulative Exposure Record

Group HA

## - 2,000 mg/m3

Date Exposure Number Nominal
(mglm3)

1940 1990 1950 2150 2010 2090 2060 2130 2000 2090 1970 1990 2030 2030 2050 1900 2100 2090 2150 2020 2010 2010 2080 2160 Analvtical Chamber Concentration Mean (mg/m3)

1985 1930 1953 2085 1995 2025 1985 I998 1950 1995 I998 I958 2010 2035 I998 I865 2000 1915 I983 I928 I883 I988 1905 I968 Individual 

### (ms I860 1910 1990 2170 2000 2010 1980 1940 1610 1900 1910 1960 1980 2050 1980 1730 1990 1750 2030 1820 2130 2020 1840 1920 MMAD (IJm)

1.422

1.182

1.832

1.506 Particle Size Determinations Chamber l\i Temperature
("C) 23 24 23 24 24 24 24 24 24 24 25 24 23 23 24 23 23 24 23 24 23 22 23 23 ~vironment In Humidity

52 53 52 50 50 52 48 48 49 48 50 48 51 52 50 42 50 55 50 52 50 49 50 50Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 WHOLE-BODY INHALATIONTOXICITY STUDY IN RATS

Date Exposure Number Chamber Monitoring Results Cumulative Exposure Record

Group IIA 

## - 2,000 mg/m3

Nominal
(mg1m3)

2190 2190 2160 2130 2180 2050 2090 2030 2170 2110 2110 2090 2030 2130 2060 2220 1980 2040 2050 2060 2060 2060 1910 2000 Mean (mg/m3)

2068 2205 2095 2040 2108 1913 1918 1913 2133 2090 2028 I843 1845 1875 2093 2150 1950 I938 1955 1960 1820 I958 2040 2080 alytical Chamber Concentration Individual 

### (ms

2050 2190 2060 2030 2040 1940 1900 1760 2130 2030 2040 1630 1790 1890 2130 2030 1810 2030 1950 2030 1890 2110 2020 2020 MMAD (IJm) 0.794

3.91 1

0.8422

1.438

1.154 Particle Size Determinations GSD TMC (mglm3) 1.1 9E-02

4.72E-03

4.56E-03

1.53E-03

5.62E-03 Chamber 

## rv Temperature
("C) 26 23 23 23 23 23 23 24 24 24 24 24 23 23 23 23 23 24 24 23 23 23 23 23 ivironment an Humidity
(%) 50 48 50 49 50 54 51 48 48 52 50 47 52 48 51 51 48 47 49 49 48 45 51 48Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 WHOLE-BODY INHALATIONTOXICITY STUDY IN RATS

Date Exposure Number

49 50 5 1 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 Mean S.D. Chamber Monitoring Results Cumulative Exposure Record

Group HA

## - 2,000 mglm3

Nominal
(mglm3)

2010 2140 1930 1970 2060 1970 2060 2060 1900 2090 2040 2070 2060 2120 2190 1990 2080 2130 2230 2330 2150 2068

83 Mean (mglm3)

2000 2108 I885 I963 2045 I875 2020 I985 I898 2163 2065 201 0 1958 1935 1890 I808 1915 1943 1945 2015 2023 Analvtical Chamber Concentration Individual MMAD (IJm)

1.072

12.34

1.351

1.631

2.344 3.103 Particle Size Determinations GSD TMC (mg1m3)

3.00E-03

9.96E-03

1.34E-03

2.36E-03

4.51E-03 3.13E-03 Chamber I M Temoerature ~vironment an Humidity
(%) 49 46 47 50 51 48 49 46 49 49 47 49 47 50 49 46 48 48 51 51 52 

### 49.4 2.1Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 WHOLE-BODY INHALATIONTOXICITY STUDY IN RATS

Chamber Monitoring Results Cumulative Exposure Record

Group IIB 

### - 2,000 mg/m3

Exposure Number Nominal 

## I Analvtical Chamber concentration Mean (mg/m3)

I963 1943 I983 2178 2135 2063 1943 2035 1903 2070 1938 1993 1975 2050 2113 1918 I998 2028 1958 2023 I963 1973 I985 2020 Individual MMAD (IJm)

1.589

1.378

3.380

1.174 Particle Size 1s TMC

(mg/m3)

3.46E-03

5.03E-03

3.14E-03

2.54E-03 Chamber I M Temperature
("C) 23 23 23 23 24 23 23 23 23 23 24 23 23 23 23 23 22 23 22 23 23 22 23 23 wironment In Humidity 

# ('w 54 56 56 55 54 55 51 50 51 49 54 52 55 55 52 43 52 56 51 53 52 52 53 52Table A GASOLINE DlPEVAPOR CONDENSATE: A 13-WEEK 00-61 30 WHOLE-BODY INHALATION TOXICITY STUDY IN RATS

Date Exposure Number Chamber MonitoringResults Cumulative Exposure Record

Group IIB

## - 2,000 mglm3

Nominal
(mglm3)

2190 2190 2160 2130 2180 2050 2090 2030 2170 2110 2110 2090 2030 2130 2060 2220 1980 2040 2050 2060 2060 2060 1910 2000 Mean (mg/m3)

2098 2020 2028 2040 2073 1960 I968 1960 I978 1915 I998 2130 1960 1970 2098 2055 I858 1868 1943 I985 1970 2000 1903 1920 alytical Chamber Concentration Individual MMAD (IJm) 0.8998

0.9819

0.8897

1 A03

1.101 Particle Size Determinations GSD TMC (mg/m3) 1.47E-02

1.76E-03

5.04E-03

1.74E-03

6.24E-03 Chamber 

## rv Temperature
("C) 25 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 22 22 22 23 23 ~vironment In Humidity


## ("A) 52 50 52 5 1 5 1 55 52 52 50 54 53 48 54 49 54 54 49 52 52 52 50 47 53 50Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 WHOLE-BODY INHALATION TOXICITY STUDY IN RATS

Date Exposure Number

49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 Mean S.D. Chamber Monitoring Results Cumulative Exposure Record

Group IIB

## - 2,000 mg/m3

Nominal

# I+ ~alytical Chamber Concentration Individual MMAD (elm)

0.91 87

1.358

1.314

1.510

1.377 0.647 Particle Size ?terminat

GSD 1s TMC (mg/m3)

2.74E-03

2.33E-03

1.16E-03

2.23E-03

4.01 E-03 3.54E-03 Chamber Environment M Temperature
("C) 22 23 23 23 23 22 23 23 23 23 23 23 23 22 23 22 22 22 22 22 

### 22.9 0.5 InHumidity

52 48 48 53 55 52 5 1 49 52 51 50 53 48 53 51 50 50 50 53 53 

### 51.8 2.4Table A GASOLINE DlPE VAPOR CONDENSATE:A 13-WEEK 00-61 30 WHOLE-BODY INHALATION TOXICITY STUDY IN RATS

Date Exposure Number Chamber Monitoring Results Cumulative Exposure Record

Group IIIA 

### - 10,000 mglm3

Nominal
(mg/m3)

9470 9450 9540 9890 9830 9740 10800 9910 9950 9840 9990 9920 101 00 101 00 9200 9890 9730 9860 9960 9810 9890 9700 101 00 9980 Mean (mg/m3)

9685 9850 9295 10400 1031 0 9925 9680 lo380 9758 lo480 9823 9943 lo650 10700 9935 10200 lo480 10420 10240 10480 10280 9968 10030 9973 ~alytical Chamber Concentration Individual MMAD (IJm)

0.9889

1.043

1.708

2.245 Particle Size Determinal GSD 1s TMC (mglm3)

3.59E-03

5.74E-03

2.65E-03

4.77E-03 Chamber Environment 

### k Temperature
("C) 24 24 24 24 24 24 24 24 24 24 25 24 24 24 24 23 23 24 23 24 23 22 24 23 In Humidity
(%) 47 48 47 47 47 50 49 51 50 47 49 47 47 47 46 39 47 5 1 50 47 47 49 50 50Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 

### 00-6130 WHOLE-BODY INHALATION TOXICITY STUDY IN RATS

Chamber Monitoring Results

Date Cumulative Exposure Record Group IIIA

## - 10,000 mglm3 

# I

Number Mean (mg/m3) 

### lo260 10000 10320 10010 9970 9475 9450 9773 9670 9930 1001 0 9790 9970 9730 9745 9098 10450 9828 9945 10730 9475 9743 10590 lo830 Analvtical Chamber Concentration Individual MMAD Particle Size Determinations GSD TMC (mglm3) 

### 2.41 E-02

2.91 E-03

5.54E-03

1.78E-03

7.08E-03 Chamber Environment 

### M remperature
("C) 

### 26 24 23 23 23 23 23 24 24 24 24 24 23 23 23 23 23 24 24 24 24 24 23 23 an Humidity


### ("A) 50 47 50 50 49 50 48 48 48 48 49 47 50 48 48 49 47 49 49 49 48 46 52 50Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-6130 WHOLE-BODY INHALATION TOXICITY STUDY IN RATS

Date Exposure Number

49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 Mean 

### S.D. Chamber Monitoring Results Cumulative Exposure Record

Group IIIA

## - 10,000 mg/m3

Nominal
(mg/m3) 101 00 9470 9550 9780 9780 9940 9680 9620 9680 10200 9730 101 00 10000 9670 9460 9690 9300 9860 9810 9710 9500 9827 283 Mean (mg/m3) 10090 9633 lo280 11100 9233 9770 10090 9488 9468 10010 9400 lo380 9840 9318 9658 9240 9490 9940 9693 9365 10790 alytical Chamber Concentration Individual MMAD (crm)

1.618

1.285

1.243

4.537

1.534 0.992 Particle Size ns TMC

(mg/m3)

6.01E-03

1.84E-03

1.17E-03

3.25E-03

5.42E-03 5.91E-03 Chamber Environment N Temperature
("C) 23 23 23 24 24 24 24 24 23 24 23 24 24 23 24 23 23 23 23 22 22 23.6 0.7 In Humidity 

## ("/.I 50 46 47 47 47 47 47 45 48 48 48 47 47 50 49 47 48 48 51 49 49 48.2 1

## .aTable A GASOLINE DlPE VAPOR CONDENSATE:

### A 13-WEEK 00-61 30 WHOLE-BODY INHALATIONTOXICITY STUDY IN RATS

Chamber Monitoring Results

Date Exposure Number Cumulative Exposure Record Group lllB -10,000 mglm3 Chamber Environment Particle Size Mean Nominal
(mglm3)

9470 9450 9540 9890 9830 9740 10800 9910 9950 9840 9990 9920 101 00 101 00 9200 9890 9730 9860 9960 9810 9890 9700 101 00 9980 Mean (mglm3)

9930 9925 lo650 I0270 9998 1001 0 101 50 101 50 10240 9480 10120 10740 lo650 1001 0 9955 10140 9743 101 70 9590 9655 9995 9168 10100 9983 Analytical Chamber Concentration 

# I Individual 

# I MMAD ?terminat GSDTable A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 WHOLE-BODY INHALATIONTOXICITY STUDY IN RATS

Chamber Monitoring Results

Date Cumulative Exposure Record 

# , ~roup 

### IIIB -l~,oOo mg/m3

Number Analvtical Chamber Concentration Mean 

# I Individual Particle Size Chamber Environment M Temperature
("C) 26 23 24 24 24 24 24 23 23 23 24 24 24 24 24 24 24 24 23 23 23 23 24 24 In Humidity
(%) 49 46 46 46 46 46 45 46 46 46 47 44 47 46 45 45 45 48 46 47 46 44 47 46

### Table A GASOLINE DlPE VAPOR CONDENSATE:A 13-WEEK 00-61 30 WHOLE-BODY INHALATION TOXICITY STUDY IN RATS

Date Exposure Number



### 49 50 51 52 53 54 55 56 57 58 59 60 6 1 62 63 64 65 66 67 68 Mean S.D. 

### Chamber Monitoring Results Cumulative Exposure Record

Group lllB-10,000 mglm3

Nominal


### (mglm3) 101 00 9470 9550 9780 9780 9940 9680 9620 9680 10200 9730 101 00 10000 9670 9460 9690 9300 9860 9810 9710 9832 282 

### Mean (mg/m3) 11000 10070 10090 9758 10650 10240 I0530 9885 1 1250 11130 10480 lo650 11180 10350 9923 10500 10180 10550 9650 9218 alytical Chamber Concentration 

### Individual MMAD (IJm)

0.8013

2.013

1.661

1.736

1.547 0.909 

### Particle Size Determinations GSD TMC (mglm3)

7.49E-03

5.59E-03

3.48E-03

2.83E-03

6.40E-03 5.73E-03 Chamber 

### Fv Temperature
("C) 24 24 24 24 24 23 24 24 24 24 24 25 25 23 23 23 23 23 22 23 23.7 0.7 wironment 

### In Humidity 

## (%I 47 44 44 46 46 47 46 44 45 45 45 44 44 47 47 45 46 46 49 46 45.9 1.7Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 WHOLE-BODY INHALATIONTOXICITY STUDY IN RATS

Exposure Number Chamber Monitoring Results Cumulative Exposure Record

Group IVA

## - 20,000 mglm3

Individual Nominal MMAD (~m)

1.779

3.198

1.192

1.358 Analvtical Chamber Concentration Mean Particle Size Determinat GSD 1s TMC (mg/m3)

4.97E-03

1.15E-02

1.83E-03

4.54E-03 Chamber M Temperature
("C) 25 25 25 25 25 25 24 24 24 24 26 25 25 25 25 24 24 25 24 25 25 22 25 25 vironment
n Humidity 

## (%I 48 50 50 51 49 50 50 53 5 1 50 53 50 50 51 49 40 49 52 52 48 49 51 51 52

### Table A GASOLINE DlPEVAPOR CONDENSATE: A 13-WEEK 00-6130 WHOLE-BODY INHALATION TOXICITY STUDY IN RATS

Chamber Monitoring Results

Date Cumulative Exposure Record Group IVA

## - 20.000 maim3

Exposure Number Nominal
(mg/m3) 19400 19000 19300 I9800 19000 19500 19300 19300 19600 I8400 18600 I8700 18900 18800 18900 19100 18800 I7600 19700 I8400 19400 I8700 18900 18700 18800 A 

### Mean (mg/m3) 19980 19880 19250 19650 19350 20280 19550 20330 20250 19780 19480 20080 20450 20300 19480 20100 19830 19050 20780 20330 20680 20580 20900 20100 20650 ~alytical Chamber Concentration Individual 

### (ms

20500 20300 19000 20400 19500 20300 1 9700 201 00 1 9700 19800 I8900 19700 20200 19800 19000 19500 19500 18800 20800 20500 21100 20400 21 000 20000 20800 MMAD (vm) 0.7739

2.346

0.8053

1.220

1.373 Particle Size ?terminat1

GSD 1s 

### TMC (mgim3) 2.44E-02

4.30E-03

7.67E-03

1.09E-03

1.12E-02 Chamber Environment Mean Temperature
("C) 28 25 24 24 25 25 25 25 25 25 26 26 25 24 24 24 25 25 26 25 25 25 24 24 24 Humidity 

## (%I 52 48 51 50 50 51 49 49 49 48 49 47 50 52 50 50 49 50 51 49 49 48 54 5 1 5 1

### Table A GASOLINE DlPE VAPOR CONDENSATE:A 13-WEEK 00-61 30 WHOLE-BODY INHALATION TOXICITY STUDY IN RATS

Date Exposure Number



### 15-May-02 

# I 69 Mean 

### Chamber Monitoring Results Cumulative Exposure Record

Group IVA

## - 20,000 mglm3

Nominal


### (mglm3) I8400 18600 I8900 I8700 19000 18800 19000 19000 I8900 18800 18800 19000 191 00 191 00 18600 18900 191 00 19500 191 00 18700 19070 51

3 

### Mean (mglm3) 20430 20330 21200 20480 20630 21080 19830 20800 20350 20230 20100 20930 201 00 201 30 18550 191 80 18830 19580 19450 19900 ~alytical Chamber Concentration 

### Individual 

### (ms

21100 20500 21100 20300 21700 20100 20300 21200 20200 20300 1 9700 20900 19000 20000 191 00 I8700 18000 19300 19800 Particle Size Determinations MMAD GSD TMC (elm) 

### (mg/m3) 0.9267 1.578 2.63E-03

1.413 1.826 1.91E-03

3.580 2.863 6.05E-03

1.597 1.681 1.85E-03 Chamber I 

### M Temperature
("C) 24 24 25 25 25 25 26 24 25 24 25 25 25 25 25 24 25 24 23 23 24.7 0.8 

### InHumidity
(%) 

### 48 48 48 49 49 49 47 49 50 50 49 48 52 51 48 49 49 52 49 51 49.7 1.9

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

### WHOLE-BODY INHALATION TOXICITY STUDY IN RATS

Date Chamber Monitoring Results Cumulative Exposure Record

Group IVB

## - 20,000 

### mg/m3 

# ,

Number Mean (mg1m3) 20000 19200 19900 22680 21680 21300 20400 20030 20530 21200 21380 20530 20450 20900 21100 21450 19950 21000 19650 19350 20350 19400 21080 20430 

### alytical Chamber Concentration Individual Particle Size Determinations MMAD GSD TMC

(IJm)

1.134 1.917

1.041 2.111

1.147 1.782



# - Chamber Fv Temperature
("C) 24 24 24 24 24 25 25 25 25 25 25 24 24 24 24 25 25 25 25 25 24 23 24 24 

### wironment In Humidity
(%) 49 50 51 52 50 49 50 52 53 50 53 51 51 50 50 40 48 5 1 51 47 48 51 

### 51 51Table A GASOLINE DlPE VAPOR CONDENSATE:

### A 13-WEEK 00-61 30 WHOLE-BODY INHALATIONTOXICITY STUDY IN RATS

Exposure Number Day Chamber Monitoring Results Cumulative Exposure Record

Group IVB 

## - 20,000 mg/m3

Date Nominal
(mg/m3) 19400 19000 I9300 I9800 I9000 19500 I9300 19300 19600 I8400 18600 I8700 18900 18800 I8900 191 00 18800 I7600 I9700 I8400 19400 18700 18900 I8700 Mean (mg/m3) 20680 20980 20580 20800 20730 20750 21030 19850 20380 I9930 19650 18930 18830 19250 19900 19700 19880 18230 20350 20150 20200 I9650 20450 19300 Analvtical Chamber Concentration Individual 

### (mo

21200 21300 20400 20800 20600 20700 21200 20000 20300 20400 20800 18900 I9000 18900 I9000 20600 I9300 I8200 20800 201 00 20800 19200 20800 19200 MMAD (elm) 0.7578 Particle Size 

# F@%

Determinations 

# 1 Tern~erature GSD ivironment 3n Humidity 

## (%I 52 49 50 49 49 50 48 51 50 50 50 48 49 50 49 48 48 52 50 49 49 48 53 51Table A GASOLINE DlPE VAPOR CONDENSATE:A 13-WEEK 00-61 30 WHOLE-BODY INHALATIONTOXICITY STUDY IN RATS

14-May-02 

# 1 68 Mean Date Chamber Monitoring Results Cumulative Exposure Record

Group IVB 

## - 20,000 mglm3

Exposure Number Nominal
(mglm3) 18800 I8400 18600 18900 18700 I9000 18800 I9000 I9000 18900 18800 18800 I9000 191 00 191 00 18600 18900 191 00 19500 191 00 19070 51

5 Mean (mglm3) 20080 18850 19780 19850 19430 19480 I9930 I9300 I9030 19900 18800 I9200 I9750 19680 20330 20000 20000 20350 20100 18880 ~alytical Chamber Concentration Individual Particle Size Chamber Environment In Humidity

5 1 48 48 51 50 50 49 48 49 49 49 49 49 52 51 49 49 49 54 49 49.8 1.9

## Table C 

# LIONEL F. RUBIN, V.M.D. 1 116 Saint Andrews Road Bryn Mawr, PA 1901 0 (61 0) 520 9430 February 2, 2002 Huntingdon Life Sciences, Inc. Mettlers Road, Box 2360 East Millstone, 

# NJ 08875-2360
Re: study A-12 00-61 30F Ophthalmoscopicexaminationof study A-12 00-61 30F pretest ratswas performed January31,2002. Ratswith ocularabnormalitieswere identifiedand should

# bewithdrawn from inclusion 

# in the study if convenient.

Lionel F. Rubin, V. 

# M. D.

## Table C 

## LIONEL F. RUBIN, V.M.D. 1 116 Saint Andrews Road Bryn Maw, PA 1 9010 (61 0) 520 9430 May 11,2002 Huntingdon Life Sciences, Inc. Mettlers Road, Box 2360 East Millstone, NJ 08875-2360
Re: study A-12 00-6130F Ophthalmoscopic examinationof studyA-I2 00-6130F ratswas performed May 10, 2002 (terminal examination). 

## I have reviewed the findings of the type and incidence of ocular abnormalities. There is no indication of dose or compound related ocular disease. In my opinion, none of the ocular abnormalities is attributable to the administrationof the test compound.

Lionel F. Rubin, V. M. 

## D.Huntingdon Life Sciences 00-6130F 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

MALES SUMMARY OF CLINICAL OBSERVATIONS

................................................................................................................................... DAY OF STUDY GROUP# -11 92 TOTAL

................................................................................................................................... # OF ANIMALS EXAMINED 155 2 5 5 355 4 5 5

NORMAL

WITHIN NORMAL LIMITS

DEAD

HUMANE SACRIFICE

TERMINAL SACRIFICEHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 2

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

FEMALES SUMMARY OF CLINICAL OBSERVATIONS

DAY OF STUDY GROUP# -11 92 TOTAL
# OF ANIMALS EXAMINED 155 2 5 5 3 5 5 455

NORMAL

WITHIN NORMAL LIMITS

DEAD

TERMINAL SACRIFICEHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 1

TABLE C

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

SUMMARY OF OPHTHALMOLOGY OBSERVATIONS DAY - 12

................................................................................................................................... DOSE GROUP: 1 2 3 4 DOSE LEVEL (MG/M3) : 0 2000 10000 20000

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

MALES total number examined 5 5 5 5 NO ABNORMALITIES DETECTED

................................................................................................................................... No statistically significant differencesHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 2

TABLE C

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

SUMMARY OF OPHTHALMOLOGY OBSERVATIONS DAY -12

DOSE GROUP: 1 2 3 4 DOSE LEVEL (MG/M3): 0 2000 10000 20000

................................................................................................................................... FEMALES total number examined NO ABNORMALITIES DETECTED

................................................................................................................................... No statistically significant differencesHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 3

TABLE C

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

DOSE GROUP: 1 2 3 4 DOSE LEVEL (MG/M3 ) : 0 2000 10000 20000

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

MALES total number examined 5 4 5 5

FOCAL RETINOPATHY N 1 0 0 0 % 20.0 0.0 0.0 0.0

................................................................................................................................... No statistically significant differencesHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 4

TABLE C

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

SUMMARY OF OPHTHALMOLOGY OBSERVATIONS DAY 87

FEMALES total number examined NO ABNORMALITIES DETECTED

................................................................................................................................... No statistically significant differencesHuntingdon Life Sciences 00-6130F 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

MALES MEAN BODY WEIGHTS (GRAMS)

................................................................................................................................... DOSE GROUP: 1 2 3 4 DOSE LEVEL (MG/M3 

### I : 0 2000 10000 20000

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

WEEK

WEEK

WEEK 1

WEEK 2

WEEK

WEEK

WEEK

WEEK

WEEK MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

................................................................... No statistically significant differencesHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 2

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

MALES MEAN BODY WEIGHTS (GRAMS)

................................................................................................................................... DOSE GROUP: 1 2 3 4 DOSE LEVEL (MG/M3 ) : 0 2000 10000 20000

WEEK 8 MEAN S.D.

N

WEEK 9 MEAN S.D.

N

WEEK 10 MEAN S.D.

N

WEEK 11 MEAN S.D.

N

WEEK 12 MEAN S.D.

N

WEEK 13 MEAN S.D.

N

No statistically significant differencesHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 3

TABLE D

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

FEMALES MEAN BODY WEIGHTS (GRAMS)

WEEK -1 MEAN S.D.

N

WEEK 0 MEAN S.D.

N

WEEK 1 MEAN S.D.

N

WEEK 2 MEAN S.D.

N

WEEK 3

WEEK 4 MEAN S.D.

N

MEAN S.D.

N

WEEK 5

WEEK 6 MEAN S.D.

N

MEAN S.D.

N

WEEK 7 MEAN 306 289 276 275 S.D. 27.5 35.2 16.9 13.7

N 5 5 5 5

................................................................................................................................... No statistically significant differencesHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 4

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 WEIGHTS (GRAMS)

................................................................................................................................... DOSE GROUP: 1 2 3 4 DOSE LEVEL(MG/M3): 0 2000 10000 20000

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

WEEK 8 MEAN S.D.

N

WEEK 9 MEAN S.D.

N

MEAN S.D.

N WEEK 10

WEEK 11 MEAN S.D.

N
WEEK 12

WEEK 13 MEAN S.D.

N

MEAN S.D.

N

.............................................. No statistically significant differencesHuntingdon Life Sciences 00-6130F 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

MALES MEAN BODY WEIGHT CHANGE FROM BASELINE (GRAMS)



### ................................................................................................................................... DOSE GROUP: 1 2 3 4 DOSE LEVEL (MG/M3 ) : 0 2000 10000 20000

...................................................................................................................................
WEEK

WEEK

WEEK 0 TO 3

WEEK 0 TO 4

WEEK

WEEK MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

................................................................................... No statistically significant differencesHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 2

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 TO 7 MEAN S.D.

N

WEEK 0 TO 8

WEEK 0 TO 9

WEEK 0 TO 10 MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

WEEK 0 TO 11 MEAN S.D.

N

WEEK 0 TO 12

.............................. MEAN S.D.

N

No statistically significant differencesHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 3

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 TO 13 MEAN 259 268 289 256 S.D. 42.5 17.5 36.3 25.9

N 5 4 5 5

................................................................................................................................... No statistically significant differencesHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 4

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

FEMALES MEAN BODY WEIGHT CHANGE FROM BASELINE (GRAMS)

................................................................................................................................... DOSE GROUP: 1 2 3 4 DOSE LEVEL (MG/M3 ) : 0 2000 10000 20000

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

WEEK

WEEK

WEEK 0 TO 3

WEEK 0 TO 4

WEEK

WEEK MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

....................................................................................... Statistical key: 

### * = pc0.05 

### ** = pc0.01Huntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 5

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

FEMALES MEAN BODY WEIGHT CHANGE FROM BASELINE (GRAMS)

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

WEEK 0 TO 7 MEAN S.D.

N

WEEK 0 TO 8 MEAN S.D.

N

WEEK 0 TO 9

WEEK 0 TO 10 MEAN S.D.

N

MEAN S.D.

N

WEEK 0 TO 11 MEAN S.D.

NHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 6

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 TO 13 MEAN S.D.

N

No statistically significant differencesHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE I

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

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N WEEK 4

WEEK 5

WEEK 6

WEEK 7

WEEK

................................................ No statistically significant differencesHuntingdon Life Sciences 00-6130F 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

WEEK 9 MEAN S.D.

N

WEEK 10 MEAN S.D.

N

MEAN S.D.

N WEEK 11

WEEK 12 MEAN S.D.

N

WEEK 13 MEAN 46 4 4 S.D. 1.8 1.1

N 5 4

.................................................................................... No statistically significant differencesHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 3

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

FEMALES MEAN FEED CONSUMPTION VALUES (GR&MS/KG/DAY)

................................................................................................................................... DOSE GROUP: 1 2 3 4 DOSE LEVEL (MG/M3 ) : 0 2000 10000 20000

...................................................................................................................................
WEEK

WEEK

WEEK

WEEK

WEEK 4

WEEK 5

WEEK 6

WEEK 7

WEEK MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

................................................................................................................................... Statistical key: 

### * = peO.05Huntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 4

TABLE F

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

WEEK 9 MEAN S.D.

N

WEEK 10 MEAN S.D.

N

MEAN S.D.

N WEEK 11

WEEK 12 MEAN S.D.

N

WEEK 13 MEAN S.D.

N

.................................................. No statistically significant differencesHuntingdon Life Sciences 00-6130F 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

INDIVIDUAL CLINICAL OBSERVATIONS MALES GROUP 1 0 MG/M3

ANIMAL# OBSERVATIONS DAY OF 19 STUDY 12

1076 WITHIN NORMAL LIMITS TERMINAL SACRIFICE
1077 WITHIN NORMAL LIMITS TERMINAL SACRIFICE
1078 WITHIN NORMAL LIMITS TERMINAL SACRIFICE
1079 WITHIN NORMAL LIMITS TERMINAL SACRIFICE
1080 WITHIN NORMAL LIMITS P TERMINAL SACRIFICE P

................................................................................................................................... CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENTHuntingdon Life Sciences 00-6130F 211-DIPE-S

MALES


### ----- 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

INDIVIDUAL CLINICAL OBSERVATIONS GROUP 2 2000 MG/M3

DAY OF 19 ANIMAL# OBSERVATIONS STUDY 12

...................................................................................................................................
2066 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

2067 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

2068 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

2069 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

2070 WITHIN NORMAL LIMITS

............................................... CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENTHuntingdon Life Sciences 00-6130F 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

ANIMAL# OBSERVATIONS DAY OF 19 STUDY 12

3066 WITHIN NORMAL LIMITS TERMINAL SACRIFICE
3067 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

3068 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

3069 WITHIN NORMAL LIMITS TERMINAL SACRIFICE
3070 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

.......................................................... CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENTHuntingdon Life Sciences 00-6130F 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

INDIVIDUAL CLINICAL OBSERVATIONS MALES GROUP 4 20000 MG/M3

4076 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

4077 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

4078 WITHIN NORMAL LIMITS TERMINAL SACRIFICE
4079 WITHIN NORMAL LIMITS TERMINAL SACRIFICE
4080 WITHIN NORMAL LIMITS P TERMINAL SACRIFICE P

................................................................................................................................... CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENTHuntingdon Life Sciences 00-6130F 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

INDIVIDUAL CLINICAL OBSERVATIONS FEMALES GROUP 1 0 MG/M3

ANIMAL# OBSERVATIONS DAY OF 19 STUDY 12

1576 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

1577 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

1578 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

1579 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

1580 WITHIN NORMAL LIMITS P TERMINAL SACRIFICE P

................................................................................................................................... CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENTHuntingdon Life Sciences 00-6130F 211-DIPE-S 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 PAGE 6

INDIVIDUAL CLINICAL OBSERVATIONS FEMALES GROUP 2 2000 MG/M3

ANIMAL# OBSERVATIONS DAY OF 19 STUDY 12

2566 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

2567 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

2568 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

2569 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

2570 WITHIN NORMAL LIMITS P TERMINAL SACRIFICE P

................................................................................................................................... CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENTHuntingdon Life Sciences 00-6130F 211-DIPE-S

FEMALES GROUP 3

---------------- PAGE 7

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

3566 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

3567 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

3568 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

3569 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

3570 WITHIN NORMAL LIMITS P TERMINAL SACRIFICE P

................................................................................................................................... CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENTHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 8

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 CLINICAL OBSERVATIONS FEMALES GROUP 4 20000 MG/M3

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

DAY OF 19 ANIMAL# OBSERVATIONS STUDY 12

...................................................................................................................................
4576 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

4577 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

4578 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

4579 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

4580 WITHIN NORMAL LIMITS TERMINAL SACRIFICE

CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENTHuntingdon Life Sciences 00-6130F 211-DIPE-S

MALES GROUP 1 0 MG/M~ 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 PAGE 1

INDIVIDUAL OPHTHALMOLOGY OBSERVATIONS DAY -12

1076 NO VISIBLE LESIONS
1077 NO VISIBLE LESIONS

NO VISIBLE LESIONS

1079 NO VISIBLE LESIONS
1080 NO VISIBLE LESIONS



### ...................................................................................................................................Huntingdon Life Sciences 00-6130F 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

2066 NO VISIBLE LESIONS

2067 NO VISIBLE LESIONS

2068 NO VISIBLE LESIONS

2069 NO VISIBLE LESIONS

2070 NO VISIBLE LESIONS

...................................................................................................................................Huntingdon Life Sciences 00-6130F 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

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONSHuntingdon Life Sciences 00-6130F 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

4076 NO VISIBLE LESIONS

4077 NO VISIBLE LESIONS

NO VISIBLE LESIONS

4079 NO VISIBLE LESIONSHuntingdon Life Sciences 00-6130F 211-DIPE-S 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 OPHTHALMOLOGY OBSERVATIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONS PAGE 5Huntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 6

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

2566 NO VISIBLE LESIONS

2567 NO VISIBLE LESIONS

2568 NO VISIBLE LESIONS

2569 NO VISIBLE LESIONS

2570 NO VISIBLE LESIONS

...................................................................................................................................Huntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 7

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 AM) IMMUNOTOXICITY ASSESSMENTS

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONSHuntingdon Life Sciences 00-6130F 211-DIPE-S 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

FEMALES GROUP 4 20000 MG/M3 INDIVIDUAL OPHTHALMOLOGY OBSERVATIONS

OBSERVATION

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

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONS PAGE 8Huntingdon Life Sciences 00-6130F 211-DIPE-S

MALES GROUP 1 0 MG/M3 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 PAGE 9

INDIVIDUAL OPHTHALMOLOGY OBSERVATIONS DAY 87
ANIMAL# PART OF EYE OBSERVATION

...................................................................................................................................
1076 NO VISIBLE LESIONS
1077 RETINA FOCAL RETINOPATHY; LEFT

1078 NO VISIBLE LESIONS

1079 NO VISIBLE LESIONSHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 10

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

2066 NO VISIBLE LESIONS

2067 NO VISIBLE LESIONS

2068 NO VISIBLE LESIONSHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 11

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

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONSHuntingdon Life Sciences 00-6130F 211-DIPE-S 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 OPHTHALMOLOGY OBSERVATIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONS PAGE 12Huntingdon Life Sciences 00-6130F 211-DIPE-S

FEMALES GROUP 1 0 MG/M3 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 PAGE 13

INDIVIDUAL OPHTHALMOLOGY OBSERVATIONS DAY 87

1576 NO VISIBLE LESIONS

1577 NO VISIBLE LESIONS

NO VISIBLE LESIONS

1579 NO VISIBLE LESIONS

1580 NO VISIBLE LESIONS

...................................................................................................................................Huntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 14

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

2566 NO VISIBLE LESIONS

2567 NO VISIBLE LESIONS

2568 NO VISIBLE LESIONS

2569 NO VISIBLE LESIONS

2570 NO VISIBLE LESIONS

...................................................................................................................................Huntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 15

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

3566 NO VISIBLE LESIONS

NO VISIBLE LESIONS

3568 NO VISIBLE LESIONS

3569 NO VISIBLE LESIONS

3570 NO VISIBLE LESIONS

...................................................................................................................................Huntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 16

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 OPHTHALMOLOGY OBSERVATIONS FEMALES GROUP 4 20000 MG/M3 DAY 87

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

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONSHuntingdon Life Sciences 00-6130F 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

INDIVIDUAL BODY WEIGHTS (GRAMS) MALES GROUP 1 0 MG/M3

MEAN S.D.

NHuntingdon Life Sciences 00-6130F 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

INDIVIDUAL BODY WEIGHTS (GRAMS) MALES GROUP 2 2000 MG/M3

MEAN S.D.

NHuntingdon Life Sciences 00-6130F 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 BODY WEIGHTS (GRAMS) MALES GROUP 3 10000 MG/M3

MEAN S.D.

NHuntingdon Life Sciences 00-6130F 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 BODY WEIGHTS (GRAMS) MALES GROUP 4 20000 MG/M3

WEEK OF STUDY ANIMAL# 

### - 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13

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

MEAN S.D.

NHuntingdon Life Sciences 00-6130F 211-DIPE-S 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 BODY WEIGHTS (GRAMS) FEMALES GROUP 1 0 MG/M~

WEEK OF STUDY ANIMAL# - 1 0 1 2 3 4 5 6 7 8 9 10

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

MEAN S.D.

N PAGE 5Huntingdon Life Sciences 00-6130F 211-DIPE-S 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 BODY WEIGHTS (GRAMS) FEMALES GROUP 2 2000 MG/M~

WEEK OF STUDY ANIMAL# - 1 0 1 2 3 4 5 6 7 8 9 10

MEAN S.D.

N PAGE 6Huntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 7

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 BODY WEIGHTS (GRAMS) FEMALES GROUP 3 10000 MG/M3

MEAN S.D.

NHuntingdon Life Sciences 00-6130F 211-DIPE-S 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 BODY WEIGHTS (GRAMS) FEMALES GROUP 4 20000 MG/M3

.................................... WEEK OF STUDY ANIMAL# 

### - 1 0 1 PAGE 8

MEAN S.D.

NHuntingdon Life Sciences 00-6130F 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
INDIVIDUAL BODY WEIGHT CHANGE FROM BASELINE (GRAMS) MALES GROUP 1 0 MG/M3

................................................................................................................................... WEEK OF STUDY ANIMAL# 0-1 0-2 0-3 0-4 0-5 0-6 0-7 0-8 0-9 0-10 0-11 0-12 0-13

MEAN S.D.

NHuntingdon Life Sciences 00-6130F 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

INDIVIDUAL BODY WEIGHT CHANGE FROM BASELINE (GRAMS) MALES GROUP 2 2000 MG/M3

MEAN S.D.

NHuntingdon Life Sciences 00-6130F 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
INDIVIDUAL BODY WEIGHT CHANGE FROM BASELINE (GRAMS) MALES GROUP 3 10000 MG/M3

WEEK OF STUDY ANIMAL# 0-1 0-2 0-3 0-4 0-5 0-6 0-7 0-8 0-9 0-10 0-11 0-12 0-13

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

MEAN S.D.

NHuntingdon Life Sciences 00-6130F 211-DIPE-S

MEAN S.D.

N 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 PAGE 4

INDIVIDUAL BODY WEIGHT CHANGE FROM BASELINE (GRAMS) 20000 MG/M3

.................................................................................................................. WEEK OF STUDY 0-1 0-2 0-3 0-4 0-5 0-6 0-7 0-8 0-9 0-10 0-11 0-12 0-13

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

4 6 82 110 143 160 177 198 215 228 254 269 287 289 35 62 88 110 131 147 168 184 194 206 221 230 241 36 63 91 119 127 138 165 181 195 215 219 234 229 37 73 105 128 149 176 204 218 242 255 270 279 278 36 6 6 93 121 144 157 174 193 204 221 230 242 244Huntingdon Life Sciences 00-6130F 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
INDIVIDUAL BODY WEIGHT CHANGE FROM BASELINE (GRAMS) FEMALES GROUP 1 0 MG/M3

................................................................................................................................... WEEK OF STUDY ANIMAL# 0-1 0-2 0-3 0-4 0-5 0-6 0-7 0-8 0-9 0-10 0-11 0-12 0-13

................................................................................................................................... 1576 23 48 55 6 6 78 78 86 9 1 91 102 103 105 100 1577 19 32 37 46 56 53 5 9 67 73 75 75 85 88 1578 15 4 0 5 3 59 7 4 8 0 8 9 83 90 99 107 107 103 1579 34 57 6 6 95 107 104 117 148 150 149 151 165 154 1580 17 3 7 5 2 56 6 1 69 87 79 90 8 6 91 93 87

MEAN S.D.

NHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 6

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

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 0-5 0-6 0-7 0-8 0-9 0-10 0-11 0-12 0-13

................................................................................................................................... 2566 17 27 3 0 29 39 4 0 44 42 5 6 6 4 66 6 1 66 2567 19 3 1 50 60 68 7 5 79 85 94 93 100 107 101 2568 22 3 7 64 84 87 8 1 95 96 106 102 112 122 117 2569 25 37 4 1 49 65 68 66 7 9 86 9 4 88 96 98 2570 17 3 8 45 55 7 0 72 85 87 90 93 101 110 103

MEAN S.D.

NHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 7

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

MEAN S.D.

NHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 8

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

INDIVIDUAL BODY WEIGHT CHANGE FROM BASELINE (GRAMS) FEMALES GROUP 4 20000 MG/M~

WEEK OF STUDY ANIMAL# 0-1 0-2 0-3 0-4 0-5 0-6 0-7 0-8 0-9 0-10 0-11 0-12 0-13

MEAN S.D.

NHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 1

TABLE K

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 00-6130F 211-DIPE-S PAGE 2

TABLE K

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) MALES GROUP 2 2000 MG/M3

WEEK OF STUDY

0 1 2 3 4 5 6 7 8 9 10 11 12 13

MEAN S.D.

NHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 3

TABLE K

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) MALES GROUP 3 10000 MG/M3

MEAN S.D.

NHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 4

TABLE K

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) MALES GROUP 4 20000 MG/M3

MEAN S.D.

NHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 5

TABLE K

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 00-6130F 211-DIPE-S PAGE 6

TABLE K

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 2 2000 MG/M3

................................................................................................................................... WEEK OF STUDY ANIMAL# 0 1 2 3 4 5 6 7 8 9 10 11 12 13

MEAN S.D.

NHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 7

TABLE K

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 00-6130F 211-DIPE-S PAGE 8

TABLE K

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

iLES GROUP 4 2 INDIVIDUAL FEED CONSUMPTION VALUES (GRAMS/KG/DAY) 0000 MG/M3

................................................................................................................................... WEEK OF STUDY ANIMAL# 0 1 2 3 4 5 6 7 8 9 10 11 12 13

................................................................................................................................... 4576 90 7 9 7 3 70 6 4 6 4 66 63 6 0 62 6 0 6 0 59 61 4577 SF 9 7 87 79 75 74 6 9 6 7 68 70 67 6 5 64 6 1 4578 103 81 80 85 75 73 71 71 64 69 77 71 63 56 4579 SF 84 81 7 9 6 9 7 0 7 4 69 67 63 66 64 65 55 4580 96 79 72 70 66 65 66 67 63 62 6 1 59 63 56

MEAN S.D.

NSF=Spilled FeederHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 1

TABLE L

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 MALES GROUP 1 0 MG/M~

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

................................................................................................................................... 1076 TERMINAL SACRIFICE 15-MAY-02 13 92 1077 TERMINAL SACRIFICE 15-MAY-02 13 9 2 1078 TERMINAL SACRIFICE 15-MAY-02 13 92 1079 TERMINAL SACRIFICE 15-MAY-02 13 92 1080 TERMINAL SACRIFICE 15-MAY-02 13 92

...................................................................................................................................Huntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 2

TABLE L

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 MALES GROUP 2 2000 MG/M3

................................................................................................................................... TYPE OF DATE OF WEEK OF STUDY AN I MAL# DEATH DEATH STUDY DAYHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 3

TABLE L

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 MALES GROUP 3 10000 MG/M3

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

................................................................................................................................... 3066 TERMINAL SACRIFICE 15-MAY-02 13 92 3067 TERMINAL SACRIFICE 15-MAY-02 13 9 2 3068 TERMINAL SACRIFICE 13 9 2 15-MAY-02 3069 TERMINAL SACRIFICE 15-MAY-02 13 9 2 3070 TERMINAL SACRIFICE 15-MAY 

### - 02 13 9 2Huntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 4

TABLE L

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 MALES GROUP 4 20000 MG/M3

TYPE OF

DEATH DATE OF WEEK OF STUDY DEATH STUDY DAY

................................................................................................................................... 4076 TERMINAL SACRIFICE 15-MAY-02 13 9 2 4077 TERMINAL SACRIFICE 15-MAY-02 13 9 2 4078 TERMINAL SACRIFICE 15-MAY-02 13 9 2 4079 TERMINAL SACRIFICE 15-MAY-02 13 9 2 4080 TERMINAL SACRIFICE 15-MAY-02 13 9 2Huntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 5

TABLE L

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/M3

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

................................................................................................................................... 1576 TERMINAL SACRIFICE 15-MAY-02 13 92 1577 TERMINAL SACRIFICE 15-MAY-02 13 92 1578 TERMINAL SACRIFICE 15-MAY 

### - 02 13 9 2 1579 TERMINAL SACRIFICE 15-MAY-02 13 92 1580 TERMINAL SACRIFICE 15-MAY-02 13 9 2Huntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 6

TABLE L

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

DEATH DATE OF WEEK OF STUDY DEATH STUDY DAY

................................................................................................................................... 2566 TERMINAL SACRIFICE 15-MAY-02 13 9 2 2567 TERMINAL SACRIFICE 15-MAY-02 13 9 2 2568 TERMINAL SACRIFICE 15-MAY-02 13 9 2 2569 TERMINAL SACRIFICE 15-MAY-02 13 9 2 2570 TERMINAL SACRIFICE 15-MAY-02 13 9 2Huntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 7

TABLE L

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/M~

................................................................................................................................... TYPE OF DATE OF WEEK OF STUDY ANIMAL# DEATH DEATH STUDY DAYHuntingdon Life Sciences 00-6130F 211-DIPE-S PAGE 8

TABLE L

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/M~

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

4576 TERMINAL SACRIFICE 4577 TERMINAL SACRIFICE 4578 TERMINAL SACRIFICE 4579 TERMINAL SACRIFICE 4580 TERMINAL SACRIFICE