# GFAP Levels in Specific Rat Brain Areas Following A 13-Week Whole-Body Inhalation Exposure to Gasoline 

# TBA Vapor Condensate

HLS Study 

# No.: 00-6131 Sponsor Study No.: 21

## 1-TBA-S Date: 

# 6 November 2002

## Summary Report Gasoline TBA Vapor Condensate: A 13-Week Whole Body Inhalation Toxicity Study in the Rat
Huntingdon Life Sciences,Inc. StudyNo. 00-613

# 1 Sponsor Study No 211-TBA-S Measurement of Glial FibrillaryAcidic Protein


## Principal Investigator:
James P. O7Callaghan, Ph.D. Molecular Neurotoxicology Laboratory Health EffectsLaboratory 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) jdo5@cdc.gov StudyDirector: Gary M. Hoffman, B.A., DABT Huntingdon Life Sciences Mettlers Road East Millstone,NJ 00875-2360 732-873-2550 732-873-3992 (FAX) hoffmang@princeton.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. O7Callaghan, Ph.D. Date Principal Investigator

Gary M. Hoffman, B.A.,

## D.A.B.T. Date Study Director

# SIGNATURE PAGE SCIENTIST The following Scientist was responsible for the overall conduct of this study.

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



# SCIENTIFICREVIEW The following Scientist has reviewed and approved this report. Date

Gary 

# M. Hoffman, B.A., D.A.B.T. Study Director Date

## 4 

# QUALITY ASSURANCE STATEMENT
Listed below are the dates that this study was inspected by the Quality Assurance Unit of the Centers for Disease Control-NationalInstitute for Occupational Safety and Health, Health EffectsLaboratory Division,Morgantown,WV, and the dates that findingswere reported to the Study Director and Management. Thisreport reflects the raw data as far as can be reasonably established. 

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



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

# QUALITY ASSURANCE STATEMENT Listed below are the dates that this study was inspected by the Quality Assurance Unit of Huntingdon Life Sciences, East Millstone, New Jersey, and the dates that findings were reported to the Study Director and Management. This report reflects theraw data as far as can be reasonably established. 

# Reported to Study Director and Type of Inspection Date(s)of Inspection Management GLP Protocol Review 9 

## Apr 02 

## 9 Apr 02 GFAPNecropsy 25 Sep02 25 Sep02

Fran Jannone, B.A., RQAP-GLP Date Quality Assurance Senior Auditor

## 6 TABLE OFCONTENTS Page



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



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



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

11.

111.



# IV. 

# v. Basic Protocol 1:

## GFAP SandwichELISA A. Introduction B. Materials C. Protocol Steps 1. Standard Curve Preparation 2. Sample Preparation 3. GFAP Assay SupportProtocol 1:

## Brain Tissue Preparation A. Introduction B. Materials C. Protocol Steps SupportProtocol 

## 2: Assay for Total Protein A. Introduction B. Materials C. Protocol Steps Reagents and Solutions General Commentary on GFAPAssays; Specific Commentary on the GFAP ELIS A 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 21 Figure 1:

## Sample StandardCurve Figure 2: pg GFAP/mg Total Protein per Brain Region Tables



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

## GFAP Standard Curve Preparation Table 2: MicrotiterPlate Template Table 

## 3: Sample Values 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 TBA Vapor

Condensate Table 

## 6: Mean GFAP Levels in SpecificRegions of Female Rat Brains Following a 13-Week Whole-Body Inhalation Exposureto Gasoline TBA Vapor

Condensate Table 7: Individual GFAP Levels in SpecificRegions of Rat Brains Following a 13-WeekWhole-Body Inhalation Exposure to Gasoline TBA Vapor Condensate Appendix A =

## Testing Facility Exposure and In-Life Data

## 7 

# Summary Toxicant-induced injury of the adult or developing central nervous system of the rat results in hypertrophy of astrocytes at the site of injury. The hallmark of this response is the enhanced expression of the major intermediate filamentprotein of astrocytes, GFAP. A 13-week inhalation exposure to Gasoline TBA Vapor Condensate caused a slight increasein the cortex of the lowest exposure group males and a slight decreasein the hypothalamusof the high exposure group males. Overall,the data suggest that exposure to Gasoline TBA Vapor Condensate under the regimen employed does not result in gliosisin any of the 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 cell types (Switzer, 1991; Balaban, 1992;07Callaghan et al., 1995). The differential susceptibilityof nervous system cell types to injury often is referred to as "selective vulnerability"(Spencerand Schuamburg, 1999;Baumgarten and Zimmerman, 1992). An implicit assumption underlying this concept is that intrinsic properties of individualneural cell types render them susceptible to damageby specific chemical exposures (Baumgarten and Zimmerman, 1992). Unfortunately, our knowledge of the mechanisms that confer such vulnerabilityto specifictoxic insults is limited. Thus, often there is no a

## priori basis for predicting the cell types affected by toxic exposures of the nervous system. Given the extreme cellular and molecular heterogeneityof the nervous system (McKay and Hockfield, 1982; Sutcliffe, 1988),the fact that targets of chemical-induced neurotoxicity are diverse and unpredictable shouldnot be surprising. This biologically-based situation does, however, make assessmentof neurotoxicity difficult because one must face the problem of deciding where to look for damage. Overcoming this obstacle requires a "marker"of neural injury that can be used to localize (i.e., "mark") sites of damage anywhere in the nervous system.
Auniversal cellular reaction to damage of the centralnervous systemis hypertrophy of astrocytes. The hallmark of this response, often termed "reactive gliosis," is the enhanced expression of the major intermediate filamentprotein of astrocytes, glial fibrillary acidic protein (GFAP). Thus, an increase in the brain concentrationof GFAP serves as a biochemical indicator of neurotoxicity. To validatethe use of GFAP as a biomarker of neurotoxicity, prototype neurotoxicantshave been administered to experimental animals and the effects of these agentson the tissue content of GFAP have been determinedby immunoassay (07Callaghan,1991;Norton et al., 1992). Assays of GFAP were found to reveal dose-, time- and region-dependent patterns of neurotoxicity at toxicant dosages below those that cause light microscopic evidence of cell loss or damage (07Callaghan, 1988;Norton et al., 1992). Moreover,the temporal and regional increments in GFAP correspond to the temporal and regional patterns of neuronal damage, as revealedby sensitive silver stains (Balaban, 1992). These findings indicate that assays of GFAP represent a sensitive, simple and quantitative approach for evaluation of nervous system damage (07Callaghan, 1991;Norton et al., 1992).
As part of the U.S. Environmental Protection Agency's testing requirements under the Clean Air Act, identification and characterizationof the potential adverse effects of gasoline and various oxygenate-gasoline blends is to be determined. Neurotoxicity

## 8 assessment constitutes a portion of these testing activities. Subchronic (13-week) inhalation exposures to gasoline and gasoline plus each of 6 fuel additives will be undertaken along with a two-generation reproduction toxicity study that includes a neurotoxicity component. The purpose of the present study was to use the GFAP assay for assessingthe potential neurotoxic effects of GasolineTBA Vapor Condensate. A control (air) and three exposure levels to the test condensate (2,000, 10,000 and 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 exposureto the test substance. 

# Materials, Methods/Procedures STUDY DATES STUDY INITIATION 6 May 2002 (Date Study Director signed the Protocol) 

# DATE OF ANIMAL RECEIPT 6 June 2002 

# EXPOSURE INITIATION 25 June 2002 (Experimental Start Date) 

# EXPOSURE TERMINATION 24 September2002 

# TERMINAL SACRIFICE 25 September 2002 

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

# STUDY COMPLETION Day Month Year (Date Final Report is signed by the Study Director)


# I. Basic Protocol 1: 

# GFAP SandwichELISA
A. 

# Introduction:The GFAP sandwich ELISA is suitable for assaying the concentrationof GFAPpresent in homogenatesof 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 evolutionarily conserved, it is likely that this assay can be very broadly applied across many species. The assay does not require preparation of any special materials or reagents; all components are available from commercial sources at modest cost. The 96-well microplate format lends itself to processing large numbers 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

## rnm 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 Fibrillary Acidic Protein Antibody (OncogeneResearch Products Cat.#IF03L,Lot #D15158-3) Phosphate Buffered Saline (e.g.Pierce Cat.#28374) Alkaline Phosphatase conjugated anti-mouseIgG (Jackson Immuno Research Cat #315-055-003) Alkaline Phosphatase SubstrateKit (e.g.Bio-Rad Cat.#172-1063) Triton X-100 (e.g. Bio-Rad Cat.#161-0407) Sodium Hydroxide (e.g., 0.4NNaOH, Fisher LC 243204) 

# C.Protocol Steps:

1. 

# Standard Curve Preparation 1. Prepare a GFAP standard: The preferred standard consists of an aliquot of a brain homogenate prepared as described in Support Protocol 1. This standard should be prepared from the same speciesthat was used to prepare the samples to be assayed for GFAP because GFAP immunoreactivitywith a given set of immunodetection reagents will differ among different species. A large number of standardscan be prepared in advance from a single "pool" of a 1% 

## SDS homogenate prepared as described in Support Protocol 1. This homogenatecan be aliquoted and stored frozen at -70°C prior to use. Thus, the GFAP standard essentially consists ofa control sample. This ispreferable over using apure GFAPstandard because using control tissue as a standard obviates any influence ofthe tissue "matrix" on the assayperformance. 

## To express the data in units of GFAPper unit of total protein, aliquots of a 1% SDS homogenate are still to be used as a GFAP standard. This is accomplishedby "standardizing the standard" with addition of a known amount of pure GFAP to the 1% SDS homogenate (i.e. an internal standard). Imrnunoreactivityvalues generated from standard curves of the GFAP "spiked" homogenate and the homogenate alone then are used to determinethe concentrationof GFAP in homogenate. For analysis of GFAP in regions of rat brain, we routinely use aliquots of a hippocampal homogenate as a standard. It contains approximately 2.5 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 of the GFAP standard from the freezer,thaw at room temperatureand 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 mglml) 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/100pl/microplate well (i.e. 0.25,0.5, 1.0, 2.5,5.0,7.5, 1Opgtotal protein/lOOpl). Table 1 shows an example of dilutions needed to prepare a standard curve from a homogenateof rat hippocampus (hippocampus std.). Typically, standards are run induplicate. Detergents act as wetting agents, therefore, more than a single use ofapipette tip with SDSor TritonX-100-containing samples can lead to carry-overerrors. Thus, it is

## 10 recommended to use only a single pipette tipper sample and to withdraw the sample only 

# a single time per tip.


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

# + 0.5% Triton X-100 to a concentrationof approximately 10pgtotal protein/ 100p1. Sampleshigh in GFAP (e. g. cerebellum)may need to be diluted to a concentrationof 5pg total proteinI100pl of PBS 

# +0.5% Triton X-100. Sampleslow in GFAP (e.g. striatum)may need to be diluted to 20pg total protein/lOOpl of PBS

# +0.5% Triton X-100. These dilution factors are determined empirically. The best practice is toprepare multiple dilutions ofeach sample to insure that optical density readings for a given sample fall on the linear portion ofthe standard curve. Typically, samples (like standards)are run in duplicate. Detergents act as wetting agents, therefore, more than a single use ofapipette 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. 

# GFAPAssay Coat Irnmulon-2 flat bottom plates with rabbit anti-GFAP. Add 1.0pgtotal immunoglobulin protein I100 p1 PBS /well. (-25p1 of anti-GFAP [Dako]in 10 ml 

## of PBS is the quantityneeded per plate). Incubatethe coated plate at 37°C for 1hour. 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-overbetween steps. 

## Wash plates 4X with PBS (200pl per well), tapping and blotting between each wash. Block 1hour with 5% 

## non-fat powdered milk in PBS at 100pl per well. Empty plate, tap on absorbent paper (upside down) to remove excess liquid, load diluted standard curve and samplesin a volume of 100p1 per well. Incubate for 1 hour. The template, Table 2, is an example of a typical 96-well microplate layout for GFAP standards and unknowns. Wash 4X with PBS 

# +0.5% Triton X-100,200 pllwell. Incubate for 1

## hour in monoclonal anti-GFAP (1:500)(Chemicon)

# + alkaline phosphatase conjugated anti-mouseIgG (1:3000)(JacksonImmunoResearch) made up in 5% non-fat dry milk 

# + 0.5% Triton X-100, 100p1 /well. Wash 4X with PBS 

# +0.5% Triton X-100,200 pllwell. Add P-nitrophenylphosphate substrate (Bio-Rad)in a volume of 100pYwell and incubatefor 20 minutes. Stop reaction with 0.4NNaOH, 100pYwell. 12. "PO$' 

## anybubbles in the plate wells-with a needle or pipette tip to insure uniform and accurate readings of standard and sample ODs. Read plate at 405 nm.

## 11 13. Calculatethe GFAP concentrationin the samplesby comparing their optical density (OD) values to those obtained for the linear portion of the GFAP standard curve. Softwareprograms linked to specific plate readers shouldbe programmed to plot OD vs. GFAP values in linear vs. log linear fashion. Typically, we utilize the 4-parameter curve fit equation and generate curves as shown in Fig. 1 and Table 3. Most programs allow for automatic subtractionof blanks and incorporationof dilution factors. Data are expressed as yg GFAP/permg total protein or, if the absolute amount of GFAP in the standard is not available, data are expressed as GFAP-likeimmunoreactivity/permg total protein. Total protein concentrationin the samples is estimated from the total protein assay describedin Support Protocol 2. Data also can be expressed on the basis of tissue wet weight (yg GFAPIgramwet weight or GFAP irnrnunoreactivity/gram wet weight). Although this approach permits eliminationof the total protein assay (Basic Support Protocol 2), we find that the GFAP values obtained are slightly more variable.
11. 

# Support Protocol 1: Brain tissue preparation
A. Introduction: This support protocol describes the procedurefor preparingbrain tissue for subsequent analysisof GFAPby Sandwich ELISA (Basic Protocol I). This procedure does not describe or recommend a specific approach for dissecting brain tissue. 

## It is noted, however, that reliable dissections are essential for obtaining reproducible results with the GFAP ELISA (seecommentary).


# B. Materials: Dissecting Instruments Balance Ultrasonic Cell Disruptor (e.g.PGC cat. # 

## 81-6721-02,2 mm probe) Pipettes Hods tir Plate Pipette tips Microfugetubes 

# C. Protocol Steps: 1. Sacrifice animals andremove the brain as rapidly as possible. 2. Dissect brain regions. If a number of regions are to be dissected, this process can be aidedby keeping the brain firm on a cold plate (e.g. Thermoelectrics cold plate, Aldrich Chemical Co. or simply an inverted petri dish placed on ice) maintained at approximately 4°C. Rat or mouse brains can be dissected into 10-15 regions, 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. 

## Thisnumber of regions can be prepared on a cold plate or at room temperature without degradation ofGFAP as assessed by immunoblot analysis. All brain 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 storage tube, or weigh boat for the

## 12 largebrain areas that don't fit into microfuge tubes, ie; Cerebellum, Cortex, Rest of Brain and Thalamus). Place individual brain regions in the tube or weigh boat, obtain the weight, transfer large brain areas to large glass homogenizationtube, and immersethe tissue in 10volumes of hot (85-95°C) 1% (wlv) SDS. For example,0.1 grams of tissue would be immersedin 1.0ml of SDS. While the SDS is still hot, homogenizethe tissueby sonificationwith 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 shouldbe stored frozen (-70°C) at time of sacrificeprior to assay. 

## Samplesprepared 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 describesthe procedure for assaying the concentrationof total protein in the SDS-homogenates. The procedure described essentiallyis the bicinchoninic acid (BCA) method describedby Smith et al. (1985) which is availablein kit form (see materials). To assay total protein concentrationof the SDS-homogenates the assay must be compatible with 1% SDS. Use of the BCA assay is not an absolute requirement as other detergent compatiblemethods are available (e.g.Bio-Rad DC protein assay). Bovine serum albumin is used as the protein standardin the described procedure. Other protein standards can be substituted.


# B. Materials: Microplate Reader Pipettes Pipette tips 96-well Microtiter Plates Microfugetubes Incubator Vortex Mixer MiscellaneousLaboratory Glassware BCA Protein Kit (Pierce#23223) Bovine SerumAlbumin (BSA) (SigmaA7888) SodiumDodecyl Sulfate (Bio-Rad #l61-0302) 

# C. Protocol Steps:

1. Prepare total protein standards. Prepare a 1mglml solution of BSA in 1% (w/v)SDS. 

## Aliquots ofthis standard can be stored frozen at 

## -70OC

## for fiture use. Thaw as needed, but do not re-freeze. 2. Prepare a total protein standard curve. Prepare dilutions of the BSA standardin 1

% 

## SDS as follows: 1.O, 2.5, 5.0,7.5, and 10pgIlOp1 of 1 % 

## SDS (no dilution is required for last standard). Vortex each tube and add 10pl of each standard to a well of the microtiter plate; add 10p1 of 1 % 

## SDS to serves as a blank. 

## Typically, standards are run in duplicate. Detergents act aswetting agents, therefore, more than a single use ofapipette tip with SDS-containing samples

## 13
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. Prepare dilutions of the samples. Thaw the samples,vortex and dilute a 10pl aliquot with 190p1 of 1% SDS. Vortex the dilution tube and add a 10p1 aliquot into a well of a microtiter plate. 4. Add the protein assay reagent. Add 200 p1 of the BCA reagent (composedof 50: 1 ratio of solution A: solution B of the Pierce BCA reagent) to each standard and sample. 5. Incubatethe plate at 37°C for 30 minutes. Other incubation temperatures are permissible; follow directionprovided with the kit. 

## 6. "Pop" any bubbles in the microtiterplate wells with a needle or pipette tip to insure uniform and accurate readings and read the plate at 562 nm. 7. Calculatethe concentrationof total protein in the samples from the standard curve. Softwareprograms linked to specific plate readers should be programmed to plot OD vs. total protein in a linear fashion. Most programs allow for automatic subtractionof blanks and incorporation of dilution factors. Because the sampleswere prepared in 10volumes of diluent, typically, total protein values are approximately 10mglml.


## IV. 

# Reagents and Solutions
1.

# Phosphate Buffered Saline (PBS)- One packet of PBS is mixed thoroughly

with 500 

## ml of deionizedwater to give a final concentration of: 137

## mM NaCll1.0 mM 

## KC112 

## rnM KH2P04/8.0mM

## Na2HP04.7H20/pH 7.4 (can be stored at 4OC for at least a month). For this and all subsequent reagents and solutions, determinethe total volume that needs to be prepared based on the use of 100 pllwell and 96 wells per plate (washes take 200 pllwell). 2. 

# PBS+O.S%Triton X-100-2.5ml of Triton X-100 is added to 500 ml of PBS (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-fat powdered milk is added per 100ml of PBS or per 100ml of PBS 

# +Triton X-100. Prepare at least 100 ml of each to facilitate dissolving the powdered milk; PBS may be warmed slightly to facilitate this process. Make these solutions up fresh the day of assay and do not save. Also, do not retain the powdered milk for greaterthan 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 110mlof PBS. The assay is based on the use of this antibody as a "capture" reagent. Substitutionof an antibody from another vendor may not yield suitable results. Make this solution fresh on the day of use and do not save. 

# 5. 

# Monoclonal anti-GFAP (Chemicon; formerly Boehringer Mannheim) 

# combinedwith Alkaline Phosphatase-conjugatedanti-mouseIgG (Jackson hnmunoResearch)- Make up a stock solution of monoclonal anti-GFAPand alkaline phosphatase-conjugated anti-mouseIgG as per the vendors7 instructions. Storeboth stocks at 4°C as per the vendor's instructions. Add 201.11 of the monoclonal antibody solution stock and 3.3 pl of the alkaline phosphatase conjugate stock/lOml powdered milk +0.5% Triton X-100. The assay is based on the use of the monoclonal antibody as a "detection" reagent and the alkaline phosphatase conjugateto bind to the detection antibody and

## 14 generate a colored reactionproduct proportional to the amount of antigen (GFAP)present in the samples. Substitutionof antibodies from other vendors may not yield suitable results. Make these solutions fresh on the day of use and do not save. 

# 6.P-nitrophenylphosphate substrate (BioRad)- Mix 2ml 

## of diethanolamine buffer on a stirrer with 2 

## p-nitrophenylphosphate tablets and 8 ml 

## of deionized water. Make this solution fresh on the day of use and do not save. 

# V: General Commentary on GFAPAssays; Specific Commentary on the GFAP ELISA
A. Background Information
It has longbeen known that damage to the centralnervous system results in astrogliosis (gliosis, reactive gliosis, glial activation), aresponse to brain injury characterizedby hypertrophy and, less often, hyperplasia of astrocytes, a sub-type of CNS glia (Eng, 1988; Norenberg, 1994). At the electron microscopic level, astrogliosisis characterizedby the accumulation of glial filaments. GFAP was found to be the majorprotein componentof these filaments (Eng, 1988). As such, GFAP serves as abiomarker for filament accumulation and, therefore, of gliosis (Eng, 1988;Norton et al., 1992; O'Callaghan, 1993). With the development of antibodies to GFAP, immunohistochemical analysisof this 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; O'Callaghan et al., 1995). Thus, a large body of evidencenow has been accumulated demonstrating the ubiquity of the glial response to all types of CNS damagebased onimrnunohistochemistryof GFAP. Only recently, however, have methods been introducedto assay levels of GFAP as a means of quantifying gliosis.
While GFAP immunohistochemistry has proven useful for revealing patterns of gliosis afterbrain injury, this approach does not lend itself to quantification or the analysisof large numbers of samples. Small (25-50%),but toxicologically significant increases, also may be difficult to detectby immunohistochemistry. These drawbacks, combined with the need to develop quantitative biomarkers of neurotoxicity (O'Callaghan et al., 1995),and to define quantitative aspects of toxicant- and disease-induced gliosis,has prompted the development and implementationof a number of GFAP assays. These assays have been applied to examine gliosis in specificbrain areas already known to be affectedby disease or other insult. In addition,they also can be broadly applied in arisk assessment context (U.S. EPA) to screen for potential sites of neural damage resulting from toxic exposures of the CNS. Recently, analysisof GFAPhas been used to demonstratethat the degree of cortical gliosis in postmortembrain tissue from victims of Alzheimer's disease correlates with the severity of dementia scores in these individualsprior to death (G.Webster Ross, submitted). Analysis of GFAP in cerebrospinal fluid (CSF) also has been applied to the human condition as an indicator of the severity of traumatic injury to the brain (Rosengren et al., 1994). Finally, analysis of GFAP can be used as an indicator of the presence of brain or spinal cord contaminationof meat (Schmidt et al., 1999).

## 15 Of the number of GFAP assays that have appearedin the literature over the last 15years, all essentially fall into two categories: 1) solid phase immunoassays where GFAP is immobilizedon a solid support matrix and detectedby mono- or polyclonal antibodiesor 2) liquid-phase assays where GFAP frombrain extracts or solubilizedbrain tissue (or CSF) is "captured" by one antibody and then detected by another antibody raised in a differenthost species (Butleret al., 1986). The assay describedin this study is of the second type and it has a number of advantages over the solid phase assays. Specifically, solid-phase detection and "quantification" of GFAP most commonly involves the time-consuming resolution of a protein mixtureby SDS-PAGE, followedby electrophoretic transfer to a solid supportmembrane. Anti-GFAP antibodies coupled to a variety of detection reagents then can be used for quantificationof GFAP bound to the membranes. Unfortunately, this approach has been found to severely underestimate the concentrationof GFAP in the resolved mixture of proteins and the effects of treatmentsknown to increase GFAP (07Callaghan et.al., 1999). Other solidphase assays for GFAP have been developedthat do not rely on prior resolution of protein mixturesby SDS-PAGE (Wang et al., 1990;07Callaghan,1991.b). These assays incorporate manual spottingof brain homogenateson solid supports,with or without the aid of a template. The membranes are then incubated with anti-GFAP polyclonal or monoclonal antibodies,which, in turn, are bound by lZ51 Protein A. Quantification is achievedby gamma spectrometry or by densitometryof the autoradiographs. These assays give a linear signal over a fairly large range of spotted protein. However,they require large amounts of reagents, including radiolabeled reagents, and they do not have impressive throughput.
The sandwich ELISA for GFAP describedin this study or similar ELISAs describedpreviously (Eng et al., 1986; Kretzschmar et al., 1985;O'Callaghan, 1991;

## Rosengrenet al., 1994),have several advantagesin comparison to the other methods for assaying GFAP described above. They are easier to performbecause they have fewer steps. They are more sensitive. Although they may require a greaternumber of reagents, ELISAs adapted to the microplate format permit the use of very small volumes, which results in a significant overall cost reduction. The 96-well microplate format also has the advantageof speed and high throughput. From sample application to data collection, all steps can be performed in the plate. Moreover,the microplate-based formatpermits the entire assay to be automated through the use of robotic liquid handling processors. Finally, radioactivity is not involved, making the assay safer to perform and allowingthe user to avoid costly and time-consuming radioactivity disposal procedures. While most of the GFAP sandwich ELISAs described to date are similar and share the advantagesafforded by this technique,the assay describedin this study may have a few additional advantages. Because it is based on detergent-solubilized homogenatesof a given brain area, any treatment effects can be directly related to effects in that brain area, rather than an arbitrarily defined extract or sub-fraction that may contain only a portion of the total GFAP in that area. Using a solubilized homogenate rather than a subfraction of a given brain area also facilitates comparisonsof quantitative data on GFAP to immunohistochemical stainingof GFAP in that area. Moreover, it alsohelps rule out inter-laboratory differences associatedwith assaying GFAP content in one type of extract in one lab, and another type of extradfraction in another lab. Finally, the same SDS-denatured homogenateused to assay GFAP can be subjected to multiple assays for additional glial or neuronal proteins, thereby permitting comparisonsto be made among

## 16 multiple markers of neurotoxicityin a single sample. For example, the dopaminergic neurotoxicant, 

# 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine, causes a large increase in GFAP that results from damage to dopaminergic nerve terminals, damage that can be quantifiedby immunoassayof tyrosinehydroxylase (TH), a marker of dopaminecontaining nerve terminals in the target region (O'Callaghan et al., 1990). Both markers can be assayed from aliquots of the same tissue sample and, on this basis, we find that larger decreasesin THpredict greater increasesin GFAP. 

# B. Critical Parameters
The most critical aspect of the GFAP assay is the absolute requirement for preparation of consistently dissectedregions of the brain (see Support Protocol 1). Consistent dissectionsyield consistent GFAP values with the use of only a few animalsper dose or time point (seeAnticipated Results). The particular regions to be dissected depend on the questionsbeing addressed. If a target region is known or suspected, dissections can be limited to the region of interest. If the GFAP assay is being applied in a screening context, multiple (10-15) brain regions must be dissected in orderto avoid the possibility of diluting localized increases in GFAP. The possibility exists that extremely localized increasesin GFAP may fail to be detected with the assay. While GFAP immunohistochemistryis relatively insensitivein comparison to the GFAP assay, and it may not detect small increases in GFAP, it can reveal small "hot spots" of gliosis (e.g. see effects of MK-801) (Fix et al., 1995). Such discrete astrocytic responses could escape quantificationwith the GFAP assay due to dilution of signalby surrounding tissue. No one approach can be broadly applied to detect all toxicant-induced damage of the CNS. Therefore, it is prudent to use the GFAP ELISA in conjunction with GFAP imrnunohistochemistry and other sensitivemorphological approaches for detection of neural damage, such as silver degeneration stains (Switzer,2000),Fluoro-Jade (Schmued et al., 2000), and stainsthat detect activated microglia (Streitet al., 1999).
In terms of the GFAP ELISA itself, the key requirements for optimal performance of the assay include: 1)use of the specified antibodies,

# 2) addition of the reagents at room temperature,

## 3) fresh (daily)preparation of all reagents containing antibodiesandlornon-fat dry milk and 4) mixing of the standards and samplesprior to their dilution or addition to the microplate wells. A troubleshooting guide is provided in Table 4 that coversmost problems encounteredwith the assay. 

# C. Troubleshooting (Table

# 4)
D. Typical Results Typical GFAP assay values obtained for different regions of the rat brain are presented in Fig. 2. Absolute values for GFAP (pglmg total protein) may vary dependingon the GFAP standardused and the species subjected to evaluation. Region-to-region differencesin GFAP values from untreated animals of a given species, however, shouldremain stable,if consistent dissections are performed.

## 17 

# E. Time Considerations
The GFAP assay requires approximately5 hours for one person to process a 96- wellmicroplate.The time required to prepare brain samples depends on the number of brain areas to be dissected and on whether the areas are stored frozen prior to homogenization With practice, 10brain areas can be prepared (andhomogenized) from 50 rats in a day. Two people arerequired: one to dissect the brains and one (or more) to weigh and homogenizethe tissue. The total protein assay requires approximately 1hour for one person to process a 96-well rnicroplate. 

# Statistics:
The effect of treatment on GFAP concentrationwas determinedby separate one-way ANOVAs for each of the nine brain areas from males and females utilizing the 

## JMP@ statisticalpackage (SAS, 1995). The significancelevel was set a P<0.05 and, to ensure detection of between group treatment effects,The Least Significant-Difference test (Keppel, 1973)was used inpost-hoc analyses.



# Results and Conclusions
The results of the GFAP analysis arepresented in Tables 5 and 6. Results are reported according to gender, because sex-dependent responses to toxic substances are not uncommon, including responsesreflected in levels of GFAP. Control levels for GFAP varied markedly according to brain region, consistentwith known levels observedfor GFAP across different brain regions (see Fig. 2). The 13-week exposure to Gasoline TBA Vapor Condensatewas associatedwith a slight increase in GFAP in the cortex from group I1males and a slight decreasein GFAP in the hypothalamusof group 

# IV males (see Table 5). These statistically significantchanges in GFAP were not likely to be adverse because the increase in GFAP was small in magnitude and not concentration-relatedand the decreasein GFAP cannot be interpreted as adverse because the neurotoxicological significanceof decreases in GFAP are unknown. Moreover,the changes observed occurred in brain regions that are dfficult to reliably dissect. None of the values for females were affectedin any exposure group. Overall,these data suggest that under the exposure conditions employed, damage-induced gliosis did not occur in the brain regions examined. 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 of more discrete dissectionsof a given brain region, or the inclusion of more time points, might have resulted in positive findings. We note that most 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 of the first attempts to assay pituitary levels of GFAP and the low levels were not unexpected. A recently developed fluorescence-based GFAP ELISA (O'Callaghan, unpublished) couldbe used to assess pituitary data if deemed necessary for future studies. In conclusion, exposure to Gasoline TBA Vapor Condensate did not appear to result in gliosis in nine representativebrain regions in males or females.

## 18 

## References

Balaban, C.D. 1992. The use of selective silverdegeneration stainsin neurotoxicology: Lessons from studiesof selectiveneurotoxicants. In: The VulnerableBrain and Environmental Risks, Vol 1,Malnutrition and Hazard Assessment. R.L. Issacson and K.F.Jensen. Eds.: 223-238, Plenum. New York, NY. Baumgarten, H.G.&B. Zimmermann. 1992. Cellularand subcellular targets of neurotoxins: the concept of selectivevulnerability. In: Handbook of Experimental Pharmacology, Vol. 102 SelectiveNeurotoxicity. 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 immunochemistry of sandwichELISAs-I.Thebinding characteristicof immunoglobulins to monoclonal and polyclonal capture antibodies adsorbed on plastic and their detection by symmetrical and asymmetrical antibody-enzyme conjugates. Mol. Zmmunol. 23: 971982. Eng, L.F., Stocklin, E., Lee, Y.-L. Shiurba, R.A., Coria,F., Halks-Miller, M. Mozsgai, C., Fukayarna, G. and Gibbs,

## M. 1986.Astrocyte culture on nitrocellulosemembranes and plastic: 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 filamentsin astrogliosis. In Biochemical Pathology of Astrocytes. (M.D. Norenberg, L. Hertz & 

## A. Schousboe, eds.) pp. 79-90.A.R. Liss, New York. Fix, A.S.,Wightman, K.A. and O'Callaghan, J.P. 1995. Reactive gliosisinduced by MK-801 in the rat posterior cingulate/retrosplenial cortex: GFAP evaluationby sandwich ELISA and immunocytochemistry.Neurotoxicology 16: 229-237. Keppel, G. Design and Analysis: A Researcher's Handbook, Prentice-Hall,Inc., Englewood Cliffs, N.J., 1973. 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 neuronal types in the vertebrate central nervous system. Proc. Natl. Acad. Sci. USA 79: 6747-6751. 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 of reactive gliosis: A review. Neurochem. Res. 17: 877-885. O'Callaghan, J.P. 1988.Neurotypic and gliotypic proteins as biochemical markers of neurotoxicity. Neurotoxicol. Tet-ntol. 

## 10:445-452.

## O'Callaghan, J.P. 1991. Quantification of glial firbrillary acidic protein: Comparison of slot-irnmunobinding assays with a novel sandwich ELISA. 

## Neurotoxicol. Teratol. 13: 275-281. 07Callaghan, J.P. 1993. Quantitative features of reactive gliosis following toxicantinduced damage of the CNS. 

## Ann. 

# NYAcad. Sci. 679: 195-210. 07Callaghan, J.P., Imai, H., Miller, D.B. and Minter, A. 1999. Quantitative immunoblots of proteins resolved from brain homogenates: Underestimation of specific protein concentration and of treatment effects.

## Anal. Biochem. 274: 18-26. O'Callaghan, J.P., Wller,D.B. and Reinhard, J.F., Jr. 1990. Characterization of the origins of astrocyte response to injury using the dopaminergic neurotoxicant, l-methyl

# 4-phenyl-l,2,3,6-tetrahydro-pyridine. Brain Res. 521: 73-80. 07Callaghan, J.P., Jensen, K.F. and Miller, D.B. 1995. Quantitative aspects of drugand toxicant-induced astrogliosis.

## Neurochem. Int. 26: 1 15-124. Rosengren, L.E.,Wikkels~, C. and Hagberg,

## L. 1994. A sensitive ELISA for glial fibrillary acidic protein: application in CSF of adults.

# J. Neurosci. Methods 

# 5 1: 

## 197- 204. SAS Institute, Inc., JMP, Statistics and Graphics Guide, Version 3, 1995 Schmidt, G.R.,Hossner, K.L., Yemm, R.S., Gould,D.H. and 07Callaghan, J.P. 1999. An enzyme-linked immunosorbent assay for glial fibrillary acidic protein 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. Strategiesfor Assessing Neurotoxicity. 

## Neurosci. Biobehav. Rev. 15: 89-93. Switzer, R.C. 2000. Application of silver degeneration stains for neurotoxicity testing. 

## Toxicol. Pathol. 28: 70-83.

## 20 U.S. Environmental ProtectionAgency, Pesticide Assessment Guidelines, Subdivision 

## F. Hazard Evaluation:Human and Domestic Animals. Addendum 10, Series 8 1, 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, 

## KG. 1990. A simple quantitative dot-immunobindingassay 

## for glial and neuronal marker proteins in SDS-solubilizedbrain 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 of this homogenate were used to construct the GFAP standard curve shown. Figure 2. Levels of GFAP found in different regions of rat brain. OB, olfactory bulbs; Str, striatum;Hip, hippocampus;Hypo, hypothalamus; Ctx, cortex; Cbm, cerebellum; BS, brain stem. Values are mean 

# + SE.

# Figure 

# 1

Y = ((A-D)/(l

## +(xlC)^ B))

# + D A = 

### 0.009 B = 

### 1.349

# Figure 2

" 

# OB Str 

# Hip Hypo Ctx Cbm BS 

# Brain 

# Region

# I TABLE 1: GFAP Standard Curve Pre~aration

7

- , 

## 0.25 pg(0.625ng) 

## tube #5 700pl 

## from

tube #6 700 p1

## Table 2: 

## Microtiter Plate Terndate

Blk= Blank; Std= Standard; ? 

## =

## Unknowns

# Table 3: 

# Sample Values for 

# GFAPStandard Curve PLATE'BLANK 

## Well 

## OD Mean ' 

## ~td~ev 

## *‘cV' 

## BL A1 0.001 0.0 0.001 0.0 

### A2 -0.001



## STANDARDS Value Well OD Mean Std Dev CV 

## pg total protein

(ng GFAP) STDOl

BL=Blank; STD=Standard; OD=OpticalDensity; Std Dev=Standard Deviation; CV=Coefficient of Variation

# Table 4: Troubleshooting Guide 

# 1 Problem 

# I No color reaction

Color reaction abnormally low



# c

Color reaction abnormallyhigh



# c

Standard curve not sigmoid 

# w

I Samples not on linear portion of

curve

Duplicates are not similar

Color reaction 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
Color reaction was not terminated Incorrect plate template set in the plate reader
Incorrect standard dilution
Incorrect standard dilution
Incorrect sample dilution
Carry over from using same tip Poor pipetting technique
Plate washer malfunction
Bubbles throughout the plate Plate read at incorrect wavelength 

## Solution If color reaction has 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 37OC Repeat assay with correct reagent dilutions Repeat assay with fresh lut

Repeat assay and terminate reaction with 0.4 N NaOH Use correct plate template and reread plate Repeat assay with correct standard dilution - 

### Repeat assay with correct dilution of standard Run multiple dilutionsof samplesto obtain OD values from the linear portion of the curve Change tips after each use Check precision by weighing Check plate washer for even dispensing and aspiration
POD bubbles and re-read date 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 GasolineTBA Vapor Condensate

Brain Area

Striatum Hippocampus Cortex Olfactory Bulb Thalamus Hypothalamus Cerebellum Pituitary 

### Group I Air Control



# o mg/m3 Group ll Test Substance

2,000 mg/m3 Group Ill Test Substance

10,000 mg/m3 Group IV Test Substance

20,000 mg/m3

*Eachvalue represents the mean k 

### SEM for the concentrationof GFAP (pg/rng

### Total Protein) n= 5 except for Pituitary; 

### ND, not detected; see Results and Conclusion and Table 

### 7 +Statistically different from Air Control, Pc0.05



## Table 6: 

## Mean GFAP Levels in Specific Regions of Female Rat Brains Following a 13

## Week Whole-Body Inhalation Exposureto GasolineTBA Vapor Condensate

Brain Area 

### Group I Air Control

0 mg/m3 Group ll Test Substance

2,000 mg/m3 Group Ill Test Substance

10,000 mg/m3 Group IV Test Substance

20,000 mg/m3

2.49 

# * 0.12 6.05 

# * 0.47 4.81 

# * 0.22 ND 5.18

# * 0.33 n of GFAP (pglrng

### Toti ;onclusion and Table Striaturn Hippocampus Cortex Olfactory Bulb Thalamus 

# ' Hypothalamus

Cerebellum Pituitary Rest of Brain 1.96 20.12 5.18 

# *0.53 3.96 

# *0.22 ND 3.95 

# *0.12 I Protein) 

### 7 *Each value represents the mean & 

### SEM for the concentrati n= 5 except for Pituitary; 

### ND, not detected; see Results and 1.32

# *0.20* 2.72 

# * 0.12 1.17 t 

### 0.06 1.80

# * 0.24 2.16 t 

### 0.15 7.01 

# +0.72 3.96 

# * 0.12 ND 4.51 0.32 1.47

# * 0.12 2.74 t 

### 0.07 1.24 

# * 0.07 1.91

# *0.10 2.08 0.07 6.27 +: 

### 0.75 4.42 t 

### 0.34 0.07

# * 0.00 5.00

# * 0.65Table 7: Individual GFAP Levels in Specific Regions of Rat Brains Following a 13Week Whole-Body Inhalation Exposure to Gasoline TBA Vapor Condensate

1.675' 2.954 1.238 2.757 2.378 6.246 6.430 

## * 5.786 1.121 2.468 1.185 1.593 2.615 6.352 4.244 

## * 5.310 1.085 2.604 1.280 2.376 2.255 7.812 4.798 0.053 4.177 1.053 2.638 1.242 2.096 2.239 5.527 6.269 

## * 4.100 1.158 1.931 0.921 1.777 1.947 9.453 4.321 c 5.184 1.012 2.653 1.129 1.579 2.099 5.337 3.692 

## * 4.058 1.405 2.832 1.146 1.773 2.274 8.462 4.31 8 .L 5.588 0.986 2.266 1.037 1.440 1.726 8.257 3.681 .i 3.673 2.076 2.889 1.393 2.735 2.658 7.815 4.082 c 4.544 1.116 2.947 1.158 1.449 2.040 5.181 4.044 

## * 4.669 1.009 2.910 1.510 1.685 2.229 7.020 4.392 

## * 3.925 1.079 2.633 1.229 1.846 2.280 10.708 4.008 

## * 5.784 1.602 3.373 1.776 2.588 4.222 7.805 5.128 c 5.662 1.509 2.572 1.245 2.469 2.618 6.595 4.625 

## * 6.110 1.073 2.330 1.355 1.982 2.313 4.853 5.119 

## * 4.371 1.764 2.731 1.253 2.258 2.197 8.602 5.628 

## * 7.438 1.480 2.769 1.281 1.772 2.227 5.121 4.487 

## * 5.052 1.050 2.610 1.410 1.983 1.892 4.723 3.926 

## * 3.623 1.529 2.616 1.000 1.748 1.949 7.483 4.415 0.072 4.425 1.517 2.966 1.266 1.762 2.126 5.442 3.639 

## * 4.448 1.346 3.104 1.354 1.654 2.294 5.280 5.129 0.061 4.141 0.910 2.637 1.084 1.995 2.033 7.206 4.539 

## * 4.069 1.263 3.329 1.399 2.388 2.162 7.470 5.094 

## * 4.527 1.215 3.858 1.520 2.192 2.885 6.910 6.286 

## * 5.172 1.159 2.076 0.795 1.345 1.546 5.526 3.607 

## * 4.335 1.153 2.674 1.309 1.607 2.305 6.444 4.631 

## * 4.765 1.628 3.036 1.413 2.418 2.695 4.457 5.054 

## * 5.964 1.292 2.462 1.139 1.865 2.209 5.785 5.504 

## * 4.545 1.362 2.665 1.446 1.961 2.379 7.284 4.580 

## * 5.984 1.635 3.491 1.543 2.472 2.851 6.286 4.259 

## * 4.655 1.630 2.791 1.488 2.505 2.708 7.442 5.193 

## * 4.657 1.027 2.662 1.174 2.051 2.274 5.180 4.069 

## * 3.729 1.405 2.708 1.406 2.052 2.421 3.850 3.636 .i 4.123 1.230 2.419 1.015 2.966 1.814 3.403 5.059 

## * 4.093 1.306 2.502 1.277 1.844 2.296 4.985 5.366 c 3.844 0.953 2.643 1.249 1.973 1.989 4.357 4.361 

## * 3.744 0.866 1.929 0.993 1.646 1.871 4.051 3.643 c 4.279 1.462 2.582 1.014 1.774 1.647 5.962 4.610 c 3.799 1.042 2.701 1.265 1.957 2.376 6.836 3.494 c 4.201 0.907 1.742 1.026 1.667 1.913 4.713 3.680 c 3.741

Each value represents the concentrationof GFAP(ug1mg Total Protein) * Samples too dilute to detect GFAP

## Huntingdon Life Sciences 00-6131 Page 

# 1 211-TBA-S Draft Final Report



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

## generationlcharacterizationand animal evaluations. Animal Exposure and Animal Data Preface



# STUDY DATES: Date of Animal Receipt: 6 June 2002 Appendix A

Experimental Initiation Date: 25 June 2002 (in-life) Experimental Completion Date: 25 September 2002 (in-life) Draft Report Date: 2 June 2004



# EXPOSURES AND IN-LIFE SUMMARY: The actual measured results duringthe exposures were comparableto the targeted exposure levels. There were no exposurerelated effectsseen in the test animals with regards to body weights, feed consumption and ophthalmoscopic findings. 

# TABLE OF CONTENTS
TABLES
ChamberMonitoring Results



# ........................................................................................... Summary of Clinical Observations(pretest only)



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



# ................................................................................................... Summaryof Ophthalmology Observations



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



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



# ........................................................ Mean Feed ConsumptionValues (gramskglday)



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



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

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



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



# ................................................. Individual Body Weight Change from Baseline (grams) Individual Feed ConsumptionValues (gramskglday)



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



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

Chamber Monitoring Results

Date Exposure Number CumulativeExposure Record Group IA

# - 0 (air only) mg/m3 

# I

Nominal
(ms/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 0 

### A Mean (m9/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 0 ilytical Chamber Concentration Individual MMAD Olm)

2.423

3.669

0.7598

10.81

1.075 Particle Size Determinations GSD TMC (ms/m3)

4.95E-03

7.68E-03

2.19E-02

6.53E-03

2.46E-03 Chamber 

### En Mea Temperature
("C) 25 25 24 24 24 24 24 25 26 25 25 26 25 25 24 24 25 25 25 25 25 24 25 24 24 .onment Humidity 

## (%I 53 51 5 1 54 54 55 5 1 43 47 48 5 1 50 5 1 52 55 54 5 1 52 55 55 62 53 53 54 53Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-6131Table A GASOLINETEA VAPOR CONDENSATE:A 13-WEEKWHOLE-BODY INHALATIONTOXICITY STUDY IN RATS 00-6131

Chamber Monitoring Results

Date Exposure Number Cumulative Exposure Record Group IB

# - 0 (air only) mglm3 

# I 

# I Particle Size Nominal

# 1 Mean 1 alytical Chamber Concentration 

# 1 I Individual 

# I MMAD Chamber Environment
terminations

GSD

2.407

3.176

2.148

2.298

3.100 Mean
TMC (mg/m3)

1.36E-02

2.72E-02

1.92E-02

4.83E-03

2.72E-02 Temperature


### ("C) 24 25 24 24 24 25 25 24 25 24 24 24 24 24 24 24 24 24 24 25 23 21 23 23 Humidity
("/') 51 70 54 76 52 56 54 55 55 53 54 55 39 49 5 1 5 1 58 55 54 53 55 53 48 55Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATIONTOXICITY STUDY IN RATS 00-6131

Chamber Monitoring Results Cumulative Exposure RecordTable A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEKWHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-6131

Chamber Monitoring Results CumulativeExposure Record
Chamber Environment
Day

65 66 69 70 71 72 73 76 77 78 79 80 83 84 85 86 87 89 90 Mean Temperature
W) 24 25 25 24 24 23 24 24 24 25 25 25 25 23 23 23 23 24 23 24.1 0.8 Particle Size Exposure Number

49 50 5 1 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 Mean Date

29-Aug-02 30-Aug-02 2-Sep-02 3-Sep-02 4-Sep-02 5-Sep-02 6-Sep-02 9-Sep-02 10-Sep-02 11-Sep-02 12-Sep-02 13-Sep-02 16-Sep-02 17-Sep-02 18-Sep-02 19-Sep-02 20-Sep-02 22-Sep-02 23-Sep-02 Humidity 

## (%I 54 54 53 56 66 56 47 5 1 54 51 35 53 61 51 58 6 1 57 54 56 54.3 5.6 S.D. TMC (mg/m3)

5.32E-03

6.18E-03

3.13E-03

1.1 6E-02 9.01E-03 MMAD Olm)

1.613

5.507

0.8693

2.157 1.389 Group I6

# - 0 (air only) mg/m3

Determinations GSD

2.554

2.062

2.131

2.330 0.470 Nominal (mg/m3) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Mean (mg/m3) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Analytical Chamber Concentration Individual

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

Chamber Monitoring Results Cumulative Exposure Record

Mean (m@m3)

1998 1963 2063 2120 2063 2095 2050 2070 1998 1963 1905 2058 2078 1958 2033 1903 1935 1890 1933 1925 2080 1923 2113 1840 alytical Chamber Concentration Determinations 

# I Temperature 

# 1 

# I Particle Size 

## individual 

# I MMAD 

# I GSD 

# I TMC 

# I 

# I Chamber Environment Mean Humidity 

## ("/.I 45 63 47 61 46 49 48 49 48 48 47 47 34 42 45 47 53 48 50 48 50 48 44 50 Group HA

# - 2,000 mglm3

Day

0 Date

25-Jun-02 Exposure Number Nominal (mglm3) 

# -Table A GASOLINETBA VAPOR CONDENSATE: A 13-WEEKWHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-6131

Chamber Monitoring Results

Date Cumulative Exposure Record Grow HA

# - 2.000 ma/m3

:xposure Number Nominal (mglm3)

2090 2070 1990 2010 2150 2110 2080 1950 2020 2160 2020 2020 2190 Mean (mglm3)

2000 1935 1933 1953 1975 2005 1970 1963 2135 2188 1953 1845 2043 2023 2013 2070 2090 2068 2183 2033 2065 2105 2213 2020 alytical Chamber Concentration Individual MMAD Olm)

2.312

4.144

0.7525

0.8197

1.124 Particle Size ?termha

GSD msTMC (mg/m3)

4.69E-03

9.38E-03

2.35E-02

2.61E-03

3.03E-03 Chamber Environment Temperature ("C) 23 24 23 23 23 23 23 24 24 24 24 24 23 23 23 23 23 24 24 23 24 23 24 23 Humidity 

# ("w 50 48 49 5 1 5 1 50 48 40 44 44 46 47 47 48 50 49 48 48 52 50 59 52 48 50Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEKWHOLE-BODY INHALATIONTOXICITY STUDY 

### IN RATS 00-6131

Date Exposure Number

49 50 5 1 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 llA

# - 2,000 mg/m3

Nominal (mg/m3)

1990 1510 1890 1920 2080 1940 201 0 1980 1990 2040 1990 1910 1950 1980 2140 2140 2150 2180 2070 1950 2025 105 Mean (ms/m3)

21 08 1840 2000 1900 2070 2030 2103 2028 1898 1975 2113 2018 1863 1880 2040 201 3 2053 21 53 2000 2088 alytical Chamber Concentration Individual MMAD Olm)

3.784

3.475

0.8395 Particle Size Ins TMC

(mg/m3)

8.03E-03

5.22E-03

3.71E-03 Chamber Environment Mean Temperature ("C) 23 23 23 24 24 24 24 24 23 23 23 24 23 23 23 23 23 23 23 24 23.4 0.6 Humidity 

## ("4 50 50 47 49 60 49 40 46 49 47 32 49 57 46 51 52 52 48 49 41 48.4 5.0Table A GASOLINE TBA VAPOR CONDENSATE:A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-6131

Chamber Monitoring Results

Date Number Cumulative Exposure Record Grou~ 

### HB

# - 2.000 ma/m3

Analytical Chamber Concentration Mean 

# I Individual MMAD Olm)

0.8183

0.7358

14.68

1.010

0.8253 Particle Size !terminat

GSD ns TMC (ms/m3)

8.49E-03

2.18E-02

7.02E-02

2.96E-03

1.75E-02 Chamber Environment Temperature
("C) 23 24 23 24 23 23 23 23 23 23 23 23 23 23 23 22 22 22 23 23 22 21 22 22 Humidity 

# ('w 50 68 53 71 50 51 5 1 53 52 50 52 52 37 46 49 49 56 51 51 49 52 49 45 52Table A GASOLINE TBA VAPOR CONDENSATE:A 13-WEEKWHOLE-BODY INHALATIONTOXICITY STUDY IN RATS 00-6131

Chamber MonitoringResults

Date Exposure Number Nominal 1 Mean (mg/m3) (mg/m3)

2090 2105 2070 2060 1990 1935 2010 1830 2150 2048 2110 2123 2080 2020 1950 2018 2020 1970 2160 2203 2020 1968 2020 2088 2190 2165 2090 2008 2090 1995 2140 2028 2200 2073 2070 2030 2130 2025 2030 2123 1950 1855 1990 1993 1930 1958 1950 2010 Individual

### Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK

### WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-6131



### Chamber Monitoring Results Cumulative Exposure Record

Number

30-Aug-02 

# - 

# t Chamber Environment Particle Size Mean 

## I Nominal (ms/m3)

1990 1510 1890 1920 2080 1940 2010 1980 1990 2040 1990 1910 1950 1980 21 40 2140 2150 2180 2070 2026 106 

### Mean 

# 1 Analytical Chamber Concentration I terminat GSD

2280 1650 2030 2240 1890 2080 1940 2010 2050 1990 1800 2030 2090 1930 1900 2030 2020 2110 1790 

### Temperature (mg/m3) MMAD Olm)

0.8647

2.964

1.148

2.542 

### Humidity 

# ("w 53 53 49 56 65 54 44 49 52


### a 34 53 58 49 54 56 55 50 52



### aGaugefell priorto the 30 

### minutesexposure reading on Test Day 78. 

### Therefore, the readings could not be recorded for the remainderof the exposure on that day.Table A GASOLINE TEA VAPOR CONDENSATE: A 13-WEEKWHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-61 31



### Mean (ms/m3)

10430 10330 9878 9943 9763 10180 9665 10310 9980 lOO7O 9975 10300 10060 9808 9778 lo360 10390 9895 10980 10680 10300 10450 10500 10490 

### Chamber MonitoringResults CumulativeExposure Record

Particle Size alytical Chamber Concentration Del Individual 

## I MMAD 

# I Group MA

# - 10,000 

### mglm3

IS 

### TMC (mglmJ)

8.64E-03

2.39E-02

1.89E-02

2.78E-03

1.69E-02 

### Day Chamber Environment Mean Date Exposure Number 

# I
Nominal (mglmJ) 

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

Chamber Monitoring Results Cumulative Exposure Record

Grou~ IIIA

# - 10.000 ma/m3

Exposure 

## 1~omln.l Particle Size
Number 

# I Chamber Environment Mean Analytical Chamber Concentration Mean 

# I Individual C MMAD Olm)

8.779

2.937

0.7457

0.8931

0.8287 erminati GSD Humidity
(%) 50 50 50 52 52 52 49 40 44 44 47 47 49 5

### 1 52 52 48 50 5 1 52 59 52 5 1 5 1

### Table A GASOLINETBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATIONTOXICITY STUDY IN RATS 00-6131



### 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

Grow MA

# - 10.000 

### ma/m3

Nominal 

# 7 ~alytical Chamber Concentration Individual C 

### MMAD bm)

1.870

11.81

0.8990 

### Particle Size Chamber Environment
terminations Mean

GSD

2.517 

### Temperature ("C) 23 23 23 24 24 24 24 24 23 23 23 24 23 23 23 23 23 23 23 24 23.5 0.6 

### TMC (mg/m3)

5.24E-03 

### Humidity 

## ("4 50 50 48 51 60 50 40 47 50 47 32 49 59 46 51 53 53 49 50 42 49.3 5.1Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATIONTOXICITY STUDY IN RATS 

### 00-6131

Chamber Monitoring Results CumulativeExposure Record

Grou~ 

### HIB

# - 10.000 ma/m3

Date Exposure Number Nominal (mg1m3)



### 9760 10200 9440 9850 9970 10200 10100 10100 10300 9970 10300 10300 10300 9590 9840 9910 10100 10100 9920 10100 10200 10300 9320 10100 Mean mm3)



### 10040 101 40 9615 9763 10040 9853 9898 101 00 lOO7O 9918 10230 10370 10380 10250 9243 10190 9518 9518 10060 lo650 9600 9885 9240 10010 alytical Chamber Concentration Individual MMAD Olm)



### 0.8268

0.7424

0.7374

0.8683

0.7874 Particle Size etermina

GSD Chamber Environment Mear 

## I tions 1 Humidity


### ("A) 45 64 47 60 46 46 47 48 46 46 47 48 33 41 45 44 49 47 48 45 49 46 42 47

### Table A GASOLINETBA VAPOR CONDENSATE: A 13-WEEKWHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-6131

Date Zxposure Number Chamber Monitoring Results Cumulative Exposure Record

Grou~ IIIB 

## - 10.000 maim3

Nominal (ms/m3)

9960 10200 10400 10300 10400 9790 10000 9580 10500 9910 9770 9770 10000 9560 9660 9850 10100 10600 10000 9820 10100 10000 9830 9080 Mean (mslm3)

10500 10020 10000 10090 10160 10100 10240 lo620 10750 11150 10260 10680 lo280 10240 9605 10330 9880 10380 9955 10200 10020 9960 9715 9973 alytical Chamber Concentration Individual Particle Size
MMAD Olm)

1.403

2.459

0.7601

0.9700

0.8294 etermina GSD msTMC (mgim3)

2.32E-03

2.80E-03

2.68E-02

3.03E-03

2.64E-03 Chamber Environment Mear Temperature
("C) 23 23 24 24 24 24 24 24 24 24 23 24 24 24 24 24 24 24 23 23 23 22 24 24 1 

### Humidity 

# ("w 48 49 47 48 48 48 46 39 42 41 44 45 45 46 47 48 45 47 48 48 57 5

### 1 47 47

### Table A GASOLINETBA VAPOR CONDENSATE: A 13-WEEKWHOLE-BODY INHALATIONTOXICITY STUDY IN RATS 00-6131

Chamber Monitoring Results Cumulative Exposure Record

Group lllB

# - 10,000 

### mglm3

-- - - - -- - 

### Chamber Environment ParticleSize Mean Analytical Chamber Concentration

A 

### msTMC (mglm3)

4.93E-03

1.26E-03

1.62E-03 Humidity 

## ("'4 46 46 44 50 59 48 38 45 47 45 30 45 53 45 49 5 1 50 47 48 46.8 5.0 

### Date Exposure Number Nominal (mg/m3)

10300 9600 9850 10000 10300 10400 9790 10100 10200 10500 9910 9480 9870 10400 9960 10000 9620 9960 101 00 9997 297 

### Temperature 

## ("'3 24 24 24 23 23 23 24 23 24 24 24 25 24 23 23 23 23 23 22 23.6 0.6 

### MMAD Olm)

1.726

2.221

0.9092 Mean (mg/m3)

10390 9490 9815 9878 9910 10180 9235 9655 101 70 10500 10170 10570 10520 10080 9620 9635 lo480 9870 9633 Individual

49 50 5 1 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 Mean S.D.Table A GASOLINETBA VAPOR CONDENSATE: A 13-WEEKWHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-6131

Chamber Monitoring Results Cumulative Exposure Record

Grou~ IVA 

# - 20.000 ma/m3

Date Exposure Number Nominal
(ms/m3)

18900 20600 19200 181 00 19600 19300 19600 19600 19400 19200 19700 19400 18900 191 00 18900 19300 19200 191 00 18900 18600 18700 18600 18600 18900 1 Mean (mslm3) 20400 21000 19900 20000 20380 20050 19450 19950 20080 19700 20180 20850 19430 20680 19380 19550 20100 19880 20930 20980 19500 17750 20830 20500 alytical Chamber Concentration Individual Particle Size

MMAD Olm)

0.8760

0.7496

0.7553

1.008

0.8458 terminat1 GSD ns TMC (ms/m3)

9.70E-03

1.50E-02

1.34E-02

3.29E-03

1.82E-02 Chamber Environment Temperature
("C) 26 26 26 26 25 25 25 24 25 25 25 26 26 26 26 25 24 24 25 25 25 22 25 26 Humidity 

## (%I 49 67 5 1 65 50 5 1 53 56 52 51 51 5 1 37 45 49 49 55 52 50 50 53 52 47 51Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEKWHOLE-BODY INHALATIONTOXICITY STUDY IN RATS 00-61 31

Chamber Monitoring Results CumulativeExposure Record

Grou~ 

### IVA

# - 20,000 ma/m3

Date Exposure Number Nominal
(mslm3)

191 00 18400 18500 18900 191 00 18900 18800 191 00 19700 18800 18700 18800 19000 19500 19400 191 00 19300 19200 18800 18800 181 00 19100 19200 18700 Mean (mg/m3) 20650 19800 20380 19500 19480 19250 19530 19950 21380 20530 19480 19200 18500 19000 19430 20630 19500 20250 20300 20580 19830 20280 20230 19980 alytical Chamber Concentration Individual Particle Size

MMAD Olm)

1.689

2.505

0.7685

1.301

0.8524 !terminat GSD ns TMC (mg1m3)

2.75E-03

3.68E-03

1.81E-02

4.37E-03

2.71E-03 Chamber Environment 

# I Mea

Temperature
("'3 26 26 25 25 25 25 25 26 26 26 26 26 25 25 25 25 25 26 26 25 26 24 24 24 Humidity 

# ("w 52 52 51 52 52 53 51 43 45 46 49 49 50 53 55 53 50 5 1 54 53 61 53 53 52Table A GASOLINETBA VAPOR CONDENSATE:A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-6131

Chamber Monitoring Results Cumulative Exposure Record

Group IVA

# - 20,000 mg/m3 Chamber Environment Particle Size ns TMC

(ms/m3)

3.33E-03

4.25E-03

1.90E-03 Date Exposure Number Nominal
(mglm3)

19400 1 9900 19700 19700 18900 19800 19200 18700 19100 19800 18800 191 00 191 00 19500 19000 19000 19800 18200 19000 1 9700 19115 452 Mean (mglm3) 20080 20030 20280 20330 20050 20850 20580 20430 20400 20600 19600 201 80 20150 20250 20480 19850 19500 19830 20680 20200 alytical Chamber Concentration Individual

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

### Table A GASOLINE TEA VAPOR CONDENSATE: A 13-WEEK

### WHOLE-BODY INHALATIONTOXICITY STUDY IN RATS 00-6131



### Date Exposure Number Chamber Monitoring Results Cumulative Exposure Record

Grou~ IVB

# - 20.000 

### malm3

Nominal
(mg/m3)

18900 20600 19200 18100 19600 19300 19600 19600 19400 19200 19700 19400 18900 19100 18900 19300 19200 191 00 18900 18600 18700 18600 18600 

### Mean (mslm3) 20830 22030 18680 19930 19750 19530 201 00 19580 19300 19830 19830 19200 20230 20350 20080 20450 19980 19930 19230 19400 20080 19400 20150 

### lalyticalChamber Concentration Individual t MMAD Olm)

0.8428

0.7643

0.7572

1.277

0.8255 

### Particle Size ns TMC (ms/m3)

8.17E-03

2.07E-02

1.47E-02

2.92E-03

1.37E-02 

### Chamber Environment Mean Temperature
("C) 25 25 24 25 24 25 25 25 25 25 24 24 24 24 24 25 25 25 25 25 24 22 24 

### Humidity 

# ("w 48 67 53 68 5 1 50 53 54 52 51 5 1 52 36 45 50 47 54 53 5 1 51 53 5 1 48Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-6131

Chamber Monitoring Results

Date Exposure Number Cumulative Exposure Record Group IVB

# - 20,000 mglm3

Nominal 

## I Analvtical Chamber Concentration Individual (mglm3)

18900 19100 18400 18500 18900 19100 18900 18800 19100 19700 18800 18700 18800 19000 19500 19400 19100 19300 19200 18800 18800 18100 19100 Mean (mglm3)

19550 19650 20080 19400 20250 20180 19600 19880 19880 20650 19330 20300 20050 19750 19980 20580 20650 20330 20030 20500 20830 18730 19000 Determinat Particle Size
MMAD Olm)

1.032

1.793

0.7982

0.9155 Chamber Environment Mean
GSD ior Humidity 

# ("w
52 54 52 51 51 53 52 5 1 43 47 46 48 50 50 51 55 53 50 52 55 54 62 55

### Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK 

### WHOLE-BODY INHALATIONTOXICITY STUDY IN RATS 00-6131



### Chamber Monitoring Results CumulativeExposure Record

Grou~ IVB

# - 20.000 maim3 Chamber Environment Day Particle Size Date 

# I Mea Humidity 

## ("4 52 52 51 51 49 54 63 52 42 50 5 1 49 33 49 58 47 54 56 56 51 52 51.5 

### 5.3 Analytical Chamber Concentration Nominal
(mg/m3)

19200 18700 19400 19900 19700 19700 18900 19800 19200 18700 191 00 19800 18800 19100 19100 19500 19000 19000 19800 18200 19000 

### Temperature
("C) 25 25 25 25 25 24 24 24 24 24 26 25 25 26 26 24 24 24 23 24 24 

### 24.6 0.8 Ixposure Number :erminati GSD Mean 

# I Individual MMAD Olm) 0.8423

2.275

3.337

0.8639 

### TMC (mglm3) 2.57E-03

6.20E-03

2.26E-03

1.07E-03



### Mean S.D.Huntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 1

TABLE B

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

MALES SUMMARY OF CLINICAL OBSERVATIONS

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

NORMAL

WITHIN NORMAL LIMITSHuntingdon Life Sciences 00-6131F GFAP Sub-Group

FEMALES TABLE B

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

SUMMARY OF CLINICAL OBSERVATIONS

NORMAL

WITHIN NORMAL LIMITS DAY OF STUDY GROUP# -11 TOTAL

................................ 15 2 5 3 5 4 5 PAGE 2

## Table 

## C - 

## Pretest 

## LIONEL 

## F. RUBIN, V.M.D. 1 I 

## 16 SaintAndrews Road Bryn Maw, PA 1901 0 (61 0) 520 9430 June 14,2002 Huntingdon Life Sciences, Inc. Mettlers Road, Box 2360 East Millstone, NJ 08875-2360
Re: study A-12 00-61 31F 

## Ophthalmoscopic examination of studyA-1

## 2 00-6131

## F pretest ratswas performed June 13, 2002. No abnormalitywas seen. All rats are suitablefor inclusion in the study. 

# n

Lionel 

## F. Rubin, V. M. D.

## Table C - 

## Termination 

# LIONEL F. 

# RUBIN, V.M.D. 1 116 

# Saint Andrews Road Bryn Mawr, PA 1901 0 (61 

# 0) 520 9430 

# September 18,2002 

# Huntingdon Life Sciences, Inc. Mettlers Road, Box 2360 

# East Millstone, N 

# J 08875-2360 

# Re: study A-12 00-6131

# F Ophthalmoscopic examination of study A-12 00-6131F 

# rats was performed September 17, 2002 

# (terminalexamination). 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 administration of the test compound.

Lionel F. 

# Rubin, V. M. D.Huntingdon Life Sciences 00-6131F SPONSOR STUDY NO.: 211-TBA-S PAGE 1

TABLE C

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

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 DETECTEDHuntingdon Life Sciences 00-6131F SPONSOR STUDY NO.: 211-TBA-S PAGE 2

TABLE C

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

SUMMARY OF OPHTHALMOLOGY OBSERVATIONS DAY - 12

FEMALES total number examined NO ABNORMALITIES DETECTEDHuntingdon Life Sciences 00-6131F SPONSOR STUDY NO.: 211-TBA-S PAGE 3

TABLE C

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

CONJUNCTIVITIS

MALES total number examined

-.Huntingdon Life Sciences 00-6131F SPONSOR STUDY NO.: 211-TBA-S PAGE 4

TABLE C

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

SUMMARY OF OPHTHALMOLOGY OBSERVATIONS DAY 84

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

................................................................................................................................... FEMALES total number examined 5 5 5 5 NO ABNORMALITIES DETECTEDHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 1

TABLE D
GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS
MALES MEAN BODY WEIGHTS (GRAMS)

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

...................................................................................................................................
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 S.D.

N No statistically significant differencesHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 2

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

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 00-6131F GFAP Sub-Group PAGE 3

TABLE D

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

FEMALES MEAN BODY WEIGHTS (GRAMS)

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

------------------------.--.------------------------.--..--.-----..------.---------------------------------------------------------
WEEK -1 MEAN S.D.

N

WEEK 0

WEEK 1

WEEK 2 MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N WEEK 3

WEEK 4 MEAN S.D.

N

WEEK 5

WEEK 6 MEAN S.D.

N

MEAN S.D.

N

WEEK 7 MEAN S.D.

N

......................................... No statistically significant differencesHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 4

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

FEMALES MEAN BODY WEIGHTS (GRAMS)

................................................................................................................................... DOSE GROUP: 1 2 3 4 DOSE LEVEL (MG/M~) : 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 differences

### s t.tz PLT
5 L'OZ 6PT 

### s Z'EZ ZST 

### s E'ZE SST:



### s E'LZ ZET: 

### s 6'02 ZET:



### s P'8I LOT 

### s L'OZ SOT



### s O'EI EL



### s Z'L 9E



### ............................................................. OOOOT 

## oooz 0

E Z T

SLNBWSSBSSY ALI3IXOLONllWWI LUN ALI3IXOLON33 OAIA NI Y33M-P W SLN3WSSBSSY hLI3IXOLOBfl3N HLIM SLW NI ACUlLS T613IXOL NOILWVHNI Aa08-3TOHM X33M-ET Y :3LYSNBCIN03 BOdYA Y88.L 3NITOSY3Huntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 2

TABLE E

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

MALES MEAN BODY WEIGHT CHANGE FROM BASELINE (GRAMS)

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

...................................................................................................................................
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 271 S.D. 65.9

N 5



### ...................................................................... No statistically significant differencesHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 3

TABLE E

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

WEEK 0 TO 13 MEAN 280 269 249 269 S.D. 62.8 47.3 30.5 31.6

N 5 5 5 5

................................................................................................................................... No statistically significant differencesHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 4

TABLE E

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

FEMALES MEAN BODY WEIGHT CHANGE FROM BASELINE (GRAMS)



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



### ...................................................................................................................................
WEEK 0 TO 1 MEAN S.D.

N

WEEK 0 TO 2

WEEK 0 TO 3

WEEK 0 TO 4 MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

WEEK 0 TO 5 MEAN S.D.

N

WEEK 0 TO 6 MEAN 55 56 55 44 S.D. 7.6 9.9 9.1 12.3

N 5 5 5 5



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

### * = pe0.05

### s Z'L 6s



### s L'ET L

### s

........................................
: (EW/~W) ~3~31 asoa :dnom 3soa

SLNBWSSBSSV ALI3IXO.LO~I ALI3IXOLONa3 OAIA NI X33M-t W SLN3WSS3SSV ALI3IXOLOXl3N HLIM S.LW NI AallLS XLI3IXO.L NOILWVHNI Aa08-310HM X33M-ET V :3LVSN3aN03 FlOdVA VKL 3NI10SV3Huntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 6

TABLE E

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

WEEK 0 TO 13 MEAN S.D.

N

................................................. No statistically significant differencesHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 1

TABLE F
GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS
MALES MEAN FEED CONSUMPTION VALUES (GRAMS/KG/DAY)

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

...................................................................................................................................
WEEK 0 MEAN S.D.

N

WEEK 1

WEEK 2

WEEK 3 MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N

MEAN S.D.

N WEEK 4

WEEK 5 MEAN S.D.

N
WEEK 6

WEEK 7 MEAN S.D.

N

MEAN S.D.

N

WEEK 8 MEAN S.D.

N

----------------------------. No statistically significant differencesHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 2

TABLE F

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

MALES MEAN FEED CONSUMPTION VALUES (GRAMS/KG/DAY)

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

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



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

### * = pc0.05Huntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 3

TABLE F

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

WEEK 0 MEAN S.D.

N

WEEK 1 MEAN S.D.

N

WEEK 2 MEAN S.D.

N

WEEK 3 MEAN S.D.

N

WEEK 4

WEEK 5 MEAN S.D.

N

MEAN S.D.

N
WEEK 6

WEEK 7 MEAN S.D.

N

MEAN S.D.

N

WEEK 8 MEAN S.D.

N
Statistical key: 

### ** = pc0.01Huntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 4

TABLE F

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

FEMALES MEAN FEED CONSUMPTION VALUES (GRAMS/KG/DAY)

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

...................................................................................................................................
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 00-6131F GFAP Sub-Group PAGE 1

TABLE G

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

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

................................................................................................................................... CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENTHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 2

TABLE G

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

INDIVIDUAL CLINICAL OBSERVATIONS MALES GROUP 2 2000 MG/M3

ANIMAL# OBSERVATIONS DAY OF 1 STUDY 1

2066 WITHIN NORMAL LIMITS P
2067 WITHIN NORMAL LIMITS P
2068 WITHIN NORMAL LIMITS P
2069 WITHIN NORMAL LIMITS P
2070 WITHIN NORMAL LIMITS P

................................................................................................................................... CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENTHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 3

TABLE G

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

ANIMAL# OBSERVATIONS DAY OF 1 STUDY 1

3066 WITHIN NORMAL LIMITS P
3067 WITHIN NORMAL LIMITS P
3068 WITHIN NORMAL LIMITS P
3069 WITHIN NORMAL LIMITS P
3070 WITHIN NORMAL LIMITS P

................................................................................................................................... CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENTHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 4

TABLE G

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

DAY OF 1 ANIMAL# OBSERVATIONS STUDY 1

...................................................................................................................................
4076 WITHIN NORMAL LIMITS P
4077 WITHIN NORMAL LIMITS P
4078 WITHIN NORMAL LIMITS P
4086 WITHIN NORMAL LIMITS P
4080 WITHIN NORMAL LIMITS P

................................................................................................................................... CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENTHuntingdon Life Sciences 00-6131F GFAP Sub-Group TABLE G

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

1576 WITHIN NORMAL LIMITS P
1577 WITHIN NORMAL LIMITS P
1578 WITHIN NORMAL LIMITS P
1579 WITHIN NORMAL LIMITS P
1596 WITHIN NORMAL LIMITS P

........................................................................................... CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT PAGE 5Huntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 6

TABLE G

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

INDIVIDUAL CLINICAL OBSERVATIONS FEMALES GROUP 2 2000 MG/M3

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

2566 WITHIN NORMAL LIMITS P
2567 WITHIN NORMAL LIMITS P
2568 WITHIN NORMAL LIMITS P
2569 WITHIN NORMAL LIMITS P
2570 WITHIN NORMAL LIMITS P



### ................................................................................................................................... CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENTHuntingdon Life Sciences 00-6131F GFAP Sub-Group

FEMALES GROUP 3 10000 MG/M3



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

ANIMAL# OBSERVATIONS

.................................
3566 WITHIN NORMAL LIMITS
3567 WITHIN NORMAL LIMITS
3568 WITHIN NORMAL LIMITS
3569 WITHIN NORMAL LIMITS
3570 WITHIN NORMAL LIMITS TABLE G

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS PAGE 7Huntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 8

TABLE G

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

INDIVIDUAL CLINICAL OBSERVATIONS FEMALES GROUP 4 20000 MG/M3

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

ANIMAL# OBSERVATIONS DAY OF 1 STUDY 1

4576 WITHIN NORMAL LIMITS P
4577 WITHIN NORMAL LIMITS P
4578 WITHIN NORMAL LIMITS P
4579 WITHIN NORMAL LIMITS P
4596 WITHIN NORMAL LIMITS P



### ................................................................................................................................... CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENTHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 1

TABLE H

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

1076 NO VISIBLE LESIONS
1077 NO VISIBLE LESIONS

1078 NO VISIBLE LESIONS
1079 NO VISIBLE LESIONSHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 2

TABLE H

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

2066 NO VISIBLE LESIONS
2067 NO VISIBLE LESIONS
2068 NO VISIBLE LESIONS

NO VISIBLE LESIONS
2070 NO VISIBLE LESIONS

...................................................................................................................................Huntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 3

TABLE H

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

INDIVIDUAL OPHTHALMOLOGY OBSERVATIONS MALES GROUP 3 10000 MG/M3 DAY -12
ANIMAL# PART OF EYE OBSERVATION

3066 NO VISIBLE LESIONS
3067 NO VISIBLE LESIONS
3068 NO VISIBLE LESIONS

NO VISIBLE LESIONS
3070 NO VISIBLE LESIONS

...................................................................................................................................Huntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 4

TABLE H

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

INDIVIDUAL OPHTHALMOLOGY OBSERVATIONS MALES GROUP 4 20000 MG/M3 DAY -12

................................................................................................................................... ANIMAL# PART OF EYE OBSERVATION

4076 NO VISIBLE LESIONS
4077 NO VISIBLE LESIONS
4078 NO VISIBLE LESIONS
4086 NO VISIBLE LESIONSHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 5

TABLE H

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

1576 NO VISIBLE LESIONS
1577 NO VISIBLE LESIONS
1578 NO VISIBLE LESIONS
1579 NO VISIBLE LESIONS
1596 NO VISIBLE LESIONS

...................................................................................................................................Huntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 6

TABLE H

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

INDIVIDUAL OPHTHALMOLOGY OBSERVATIONS FEMALES GROUP 2 2000 MG/M3 DAY 

### - 12

................................................................................................................................... AN I MAL# PART OF EYE OBSERVATION

-----------------------------------------------------------------------------------------.
2566 NO VISIBLE LESIONS
2567 NO VISIBLE LESIONS
2568 NO VISIBLE LESIONS
2569 NO VISIBLE LESIONS
2570 NO VISIBLE LESIONSHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 7

TABLE H

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

INDIVIDUAL OPHTHALMOLOGY OBSERVATIONS FEMALES GROUP 3 10000 MG/M3 DAY - 12

................................................................................................................................... ANIMAL# PART OF EYE OBSERVATION

...................................................................................................................................
NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONSHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 8

TABLE H

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

INDIVIDUAL OPHTHALMOLOGY OBSERVATIONS FEMALES GROUP 4 20000 MG/M3 DAY -12

................................................................................................................................... ANIMAL# PART OF EYE OBSERVATION

4576 NO VISIBLE LESIONS
4577 NO VISIBLE LESIONS
4578 NO VISIBLE LESIONS
4579 NO VISIBLE LESIONS
4596 NO VISIBLE LESIONS

...................................................................................................................................Huntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 9

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

INDIVIDUAL OPHTHALMOLOGY OBSERVATIONS MALES GROUP 1 0 MG/M3 DAY 84

................................................................................................................................... ANIMAL# PART OF EYE OBSERVATION

...................................................................................................................................
1076
1077 CONJUNCTIVA
1078
1079
1080 NO VISIBLE LESIONS

CONJUNCTIVITIS; RIGHT

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONSHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 10

TABLE H

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

INDIVIDUAL OPHTHALMOLOGY OBSERVATIONS MALES GROUP 2 2000 MG/M3 DAY 84

................................................................................................................................... ANIMAL# PART OF EYE OBSERVATION

2066 NO VISIBLE LESIONS
2067 NO VISIBLE LESIONS
2068 NO VISIBLE LESIONS
2069 NO VISIBLE LESIONSHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 11

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

INDIVIDUAL OPHTHALMOLOGY OBSERVATIONS MALES GROUP 3 10000 MG/M3 DAY 84



### ................................................................................................................................... ANIMAL# PART OF EYE OBSERVATION

3066 NO VISIBLE LESIONS

NO VISIBLE LESIONS
3068 CONJUNCTIVA CONJUNCTIVITIS; LEFT
3069 NO VISIBLE LESIONSHuntingdon Life Sciences 00-6131~ GFAP Sub-Group PAGE 12

TABLE H

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

INDIVIDUAL OPHTHALMOLOGY OBSERVATIONS MALES GROUP 4 20000 MG/M3 DAY 84

ANIMAL# PART OF EYE OBSERVATION

4076 NO VISIBLE LESIONS

4077 NO VISIBLE LESIONS

4078 NO VISIBLE LESIONS

4086 NO VISIBLE LESIONS

4080 NO VISIBLE LESIONS

...................................................................................................................................Huntingdon Life Sciences 00-6131F GFAP Sub-Group

FEMALES GROUP 1 0 MG/M3 TABLE H

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

INDIVIDUAL OPHTHALMOLOGY OBSERVATIONS DAY 84

1576 NO VISIBLE LESIONS
1577 NO VISIBLE LESIONS
1578 NO VISIBLE LESIONS
1579 NO VISIBLE LESIONS
1596 NO VISIBLE LESIONS

...................................................................................................................................Huntingdon Life Sciences 00-6131F GFAP Sub-Group TABLE H

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS PAGE 14

INDIVIDUAL OPHTHALMOLOGY OBSERVATIONS FEMALES GROUP 2 2000 MG/M3 DAY 84

................................................................................................................................... ANIMAL# PART OF EYE OBSERVATION

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONSHuntingdon Life Sciences 00-6131F GFAP Sub-Group

FEMALES GROUP 3 10000 MG/M3 TABLE H

GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS PAGE 15

INDIVIDUAL OPHTHALMOLOGY OBSERVATIONS DAY 84

3566 NO VISIBLE LESIONS
3567 NO VISIBLE LESIONS
3568 NO VISIBLE LESIONS
3569 NO VISIBLE LESIONS
3570 NO VISIBLE LESIONS

...................................................................................................................................Huntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 16

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

INDIVIDUAL OPHTHALMOLOGY OBSERVATIONS FEMALES GROUP 4 20000 MG/M~ DAY 84

................................................................................................................................... ANIMAL# PART OF EYE OBSERVATION



### ...................................................................................................................................
NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONS

NO VISIBLE LESIONSHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 1

TABLE I

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

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

MEAN S.D.

NHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 2

TABLE I

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

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

MEAN S.D.

NHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 3

TABLE I

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

INDIVIDUAL BODY WEIGHTS (GRAMS) MALES GROUP 3 10000 MG/M3

MEAN S.D.

NHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 4

TABLE I

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

INDIVIDUAL BODY WEIGHTS (GRAMS) MALES GROUP 4 20000 MG/M3

WEEK OF STUDY

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

MEAN S.D.

NHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 5

TABLE I

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

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

MEAN S.D.

NHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 6

TABLE I

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

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

MEAN S.D.

NHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 7

TABLE I

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

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

MEAN S.D.

NHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 8

TABLE I

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

INDIVIDUAL BODY WEIGHTS (GRAMS) FEMALES GROUP 4 20000 MG/M~

MEAN S.D.

NHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 1

TABLE J

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

INDIVIDUAL BODY WEIGHT CHANGE FROM BASELINE (GRAMS) MALES GROUP 1 0 MG/M3

MEAN S.D.

NHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 2

TABLE J

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

................................................................................................................................... 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 00-6131F GFAP Sub-Group PAGE 3

TABLE J

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

INDIVIDUAL BODY WEIGHT CHANGE FROM BASELINE (GRAMS) MALES GROUP 3 10000 MG/M3

MEAN S.D.

NHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 4

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

INDIVIDUAL BODY WEIGHT CHANGE FROM BASELINE (GRAMS) MALES GROUP 4 20000 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

................................................................................................................................... 4076 25 50 79 100 118 138 155 164 172 187 204 214 218 4077 3 9 80 108 138 148 169 192 217 225 244 257 266 269 4078 41 77 108 130 148 172 198 215 231 245 261 283 277 4086 5 4 99 136 159 176 205 233 243 258 274 292 304 305 4080 4 0 79 107 132 155 184 205 217 228 245 269 273 277

MEAN S.D.

NHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 5

TABLE J

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

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

MEAN S.D.

NHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 6

TABLE J

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

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

MEAN S.D.

NHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 7

TABLE J
GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS
INDIVIDUAL BODY WEIGHT CHANGE FROM BASELINE (GRAMS) FEMALES 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 00-6131F GFAP Sub-Group PAGE 8

TABLE J

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

INDIVIDUAL BODY WEIGHT CHANGE FROM BASELINE (GRAMS) FEMALES GROUP 4 20000 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 00-6131F GFAP Sub-Group PAGE 1

TABLE K

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

INDIVIDUAL FEED CONSUMPTION VALUES (GRAMS/KG/DAY) MALES GROUP 1 0 MG/M3



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

................................................................................................................................... 1076 87 70 6 7 60 5 8 61 58 56 5 0 5 3 5 2 5 1 4 8 48 1077 92 73 6 9 65 5 6 6 0 4 6 53 52 4 8 51 48 48 50 1078 96 79 78 73 6 4 61 6 2 6 1 56 57 59 58 55 5 7 1079 9 9 7 9 7 4 70 6 1 5 8 57 5 4 52 5 2 5 2 5 0 47 4 5 1080 102 83 7 4 69 63 61 62 60 5 4 56 54 52 50 44

MEAN S.D.

NHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 2

TABLE K

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

INDIVIDUAL FEED CONSUMPTION VALUES (GRAMS/KG/DAY) MALES GROUP 2 2000 MG/M3

MEAN S.D.

NHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 3

TABLE K

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

INDIVIDUAL FEED CONSUMPTION VALUES (GRAMS/KG/DAY) MALES GROUP 3 10000 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 00-6131F GFAP Sub-Group PAGE 4

TABLE K

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

INDIVIDUAL FEED CONSUMPTION VALUES (GRAMS/KG/DAY) MALES GROUP 4 20000 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 00-6131F GFAP Sub-Group PAGE 5

TABLE K

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

INDIVIDUAL FEED CONSUMPTION VALUES (GRAMS/KG/DAY) FEMALES GROUP 1 0 MG/M3

MEAN S.D.

NSF=Spilled FeederHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 6

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

INDIVIDUAL FEED CONSUMPTION VALUES (GRAMS/KG/DAY) FEMALES GROUP 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.

NSF=Spilled FeederHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 7

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

INDIVIDUAL FEED CONSUMPTION VALUES (GRAMS/KG/DAY) FEMALES GROUP 3 10000 MG/M~

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

MEAN S.D.

NSF-Spilled FeederHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 8

TABLE K

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

INDIVIDUAL FEED CONSUMPTION VALUES (GRAMS/KG/DAY) FEMALES GROUP 4 20000 MG/M3

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

MEAN S.D.

NSF=Spilled FeederHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 1

TABLE L

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

ANIMAL TERMINATION HISTORY MALES GROUP 1 0 MG/M3

................................................................................................................................... TYPE OF DATE OF WEEK OF STUDY ANIMAL# DEATH DEATH STUDY DAYHuntingdon Life Sciences 00-6131F GFAP Sub-Group TABLE L PAGE 2

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

ANIMAL TERMINATION HISTORY MALES GROUP 2 2000 MG/M3

................................................................................................................................... TYPE OF DATE OF WEEK OF STUDY ANIMAL# DEATH DEATH STUDY DAYHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 3

TABLE L

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

ANIMAL TERMINATION HISTORY MALES GROUP 3 10000 MG/M3

................................................................................................................................... TYPE OF DATE OF WEEK OF STUDY ANIMAL# DEATH DEATH STUDY DAYHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 4

TABLE L

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

ANIMAL TERMINATION HISTORY MALES GROUP 4 20000 MG/M3

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

................................................................................................................................... 4076 TERMINAL SACRIFICE 25-SEP-02 13 92 4077 TERMINAL SACRIFICE 25-SEP-02 13 92 4078 TERMINAL SACRIFICE 25-SEP-02 13 9 2 4086 TERMINAL SACRIFICE 25-SEP-02 13 92 4080 TERMINAL SACRIFICE 25-SEP-02 13 92Huntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 5

TABLE L

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

ANIMAL TERMINATION HISTORY FEMALES GROUP 1 0 MG/M3



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



### ................................................................................................................................... 1576 TERMINAL SACRIFICE 25-SEP-02 13 92 1577 TERMINAL SACRIFICE 25-SEP-02 13 92 1578 TERMINAL SACRIFICE 25-SEP-02 13 9 2 1579 TERMINAL SACRIFICE 25-SEP-02 13 9 2 1596 TERMINAL SACRIFICE 25-SEP-02 13 9 2Huntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 6

TABLE L

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

ANIMAL TERMINATION HISTORY FEMALES GROUP 2 2000 MG/M3Huntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 7

TABLE L

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

ANIMAL TERMINATION HISTORY FEMALES GROUP 3 10000 MG/M3

................................................................................................................................... TYPE OF DATE OF WEEK OF STUDY ANIMAL# DEATH DEATH STUDY DAY
3566 TERMINAL SACRIFICE 3567 TERMINAL SACRIFICE 3568 TERMINAL SACRIFICE 3569 TERMINAL SACRIFICE 3570 TERMINAL SACRIFICEHuntingdon Life Sciences 00-6131F GFAP Sub-Group PAGE 8

TABLE L

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

ANIMAL TERMINATION HISTORY FEMALES GROUP 4 20000 MG/M3

TYPE OF

DEATH DATE OF WEEK OF STUDY DEATH STUDY DAY
TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE