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. Study No. 00-613 1 

Sponsor Study No 21 1-TBA-S 

Measurement of Glial Fibrillary Acidic Protein 

Principal Investigator: 

James P. O7Callaghan, Ph.D. 

Molecular Neurotoxicology Laboratory 

Health Effects Laboratory Divison 

Centers for Disease Control and Prevention 

National Institute for Occupational Safety 

and Health 

1095 Willowdale Road 

Morgantown, WV 26505 

304-285-6079 

304-285-6220 (FAX) 

jdo5@cdc.gov 

Study Director: 

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 

SCIENTIFIC REVIEW 

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-National Institute for Occupational Safety and Health, 

Health Effects Laboratory Division, Morgantown, WV, and the dates that findings were 

reported to the Study Director and Management. This report 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 the raw 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 

GFAP Necropsy 25 Sep 02 25 Sep 02 

Fran Jannone, B.A., RQAP-GLP Date 

Quality Assurance Senior Auditor 
6 

TABLE OF CONTENTS 

Page 

Summary ........................................................................................... 7 

Introduction ........................................................................................ 7 

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

I. 

11. 

111. 

IV. 

v. 

Basic Protocol 1 : GFAP Sandwich ELISA 

A. Introduction 

B. Materials 

C. Protocol Steps 

1. Standard Curve Preparation 

2. Sample Preparation 

3. GFAP Assay 

Support Protocol 1 : Brain Tissue Preparation 

A. Introduction 

B. Materials 

C. Protocol Steps 

Support Protocol 2: Assay for Total Protein 

A. Introduction 

B. Materials 

C. Protocol Steps 

Reagents and Solutions 

General Commentary on GFAP Assays; Specific Commentary on the GFAP 

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 Standard Curve 

Figure 2: pg GFAP/mg Total Protein per Brain Region 

Tables ............................................................................................... 24 

Table 1 : GFAP Standard Curve Preparation 

Table 2: Microtiter Plate 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-Week Whole-Body Inhalation Exposure to Gasoline TBA Vapor 

Condensate 

Table 6: Mean GFAP Levels in Specific Regions of Female Rat Brains Following 

a 13-Week Whole-Body Inhalation Exposure to Gasoline TBA Vapor 

Condensate 

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

13-Week Whole-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 filament protein of astrocytes, GFAP. 

A 13-week inhalation exposure to Gasoline TBA Vapor Condensate caused a slight 

increase in the cortex of the lowest exposure group males and a slight decrease in the 

hypothalamus of the high exposure group males. Overall, the data suggest that exposure to 

Gasoline TBA Vapor Condensate under the regimen employed does not result in gliosis in 

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 

susceptibility of nervous system cell types to injury often is referred to as "selective 

vulnerability"(Spencer and Schuamburg, 1999; Baumgarten and Zimmerman, 1992). An 

implicit assumption underlying this concept is that intrinsic properties of individual neural 

cell types render them susceptible to damage by specific chemical exposures (Baumgarten 

and Zimmerman, 1992). Unfortunately, our knowledge of the mechanisms that confer 

such vulnerability to specific toxic 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 heterogeneity of the nervous system (McKay and 

Hockfield, 1982; Sutcliffe, 1988), the fact that targets of chemical-induced neurotoxicity 

are diverse and unpredictable should not be surprising. This biologically-based situation 

does, however, make assessment of 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. 

A universal cellular reaction to damage of the central nervous system is hypertrophy of 

astrocytes. The hallmark of this response, often termed "reactive gliosis," is the enhanced 

expression of the major intermediate filament protein of astrocytes, glial fibrillary acidic 

protein (GFAP). Thus, an increase in the brain concentration of GFAP serves as a 

biochemical indicator of neurotoxicity. To validate the use of GFAP as a biomarker of 

neurotoxicity, prototype neurotoxicants have been administered to experimental animals 

and the effects of these agents on the tissue content of GFAP have been determined by 

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 revealed by 

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 characterization of 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 

assessing the potential neurotoxic effects of Gasoline TBA 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 

exposure to 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 September 2002 

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 Sandwich ELISA 

A. Introduction: The GFAP sandwich ELISA is suitable for assaying the 

concentration of GFAP present in homogenates of brain tissue. This 

method has successfully been 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 

HotIS tir Plate 
Microplate Reader 

96-well Microtiter Plates (Immulon 2, Dynatech) 

Pipette tips 

Rabbit anti-Glial Fibrillary Acidic Protein Antibody (DAKO, Cat. 

#Z0334, Lot #096(401)) 

Non-Fat Dry Milk (Carnation) 

Mouse anti-Glial Fibrillary Acidic Protein Antibody (Oncogene Research 

Products Cat. #IF03L, Lot #D15158-3) 

Phosphate Buffered Saline (e.g. Pierce Cat. #28374) 

Alkaline Phosphatase conjugated anti-mouse IgG (Jackson Immuno 

Research Cat #3 15-055-003) 

Alkaline Phosphatase Substrate Kit (e.g. Bio-Rad Cat. #172-1063) 

Triton X-100 (e.g. Bio-Rad Cat. #161-0407) 

Sodium Hydroxide (e.g., 0.4N NaOH, 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 species that was used to prepare the samples 

to be assayed for GFAP because GFAP immunoreactivity with a given set of 

immunodetection reagents will differ among different species. A large number 

of standards can be prepared in advance from a single "pool" of a 1 % SDS 

homogenate prepared as described in Support Protocol 1. This homogenate can 

be aliquoted and stored frozen at -70°C prior to use. Thus, the GFAP standard 

essentially consists of a control sample. This is preferable over using a pure 

GFAP standard because using control tissue as a standard obviates any 

influence of the tissue "matrix" on the assay performance. To express the data 

in units of GFAP per unit of total protein, aliquots of a 1% SDS homogenate 

are still to be used as a GFAP standard. This is accomplished by "standardizing 

the standard" with addition of a known amount of pure GFAP to the 1% SDS 

homogenate (i.e. an internal standard). Imrnunoreactivity values generated 

from standard curves of the GFAP "spiked" homogenate and the homogenate 

alone then are used to determine the concentration of 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 temperature and vortex or sonify prior to 

dilution. Using a rat hippocampal homogenate as a typical standard (-2.5 pg 

GFAPImg total 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 should be 

between approximately 0.25 to 10 pg/100pl/microplate well (i.e. 0.25,0.5, 1.0, 

2.5,5.0,7.5, 1Opg total protein/lOOpl). Table 1 shows an example of dilutions 

needed to prepare a standard curve from a homogenate of rat hippocampus 

(hippocampus std.). Typically, standards are run in duplicate. Detergents 

act as wetting agents, therefore, more than a single use of a pipette tip with SDSor Triton X-100-containing samples can lead to carry-over errors. Thus, it is 
10 

recommended to use only a single pipette tip per sample and to withdraw the 

sample only a single time per tip. 

2. Sample Preparation 

1. Thaw and mix samples: Remove samples 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 concentration of approximately 10pg total protein/ 

100p1. Samples high in GFAP (e. g. cerebellum) may need to be 

diluted to a concentration of 5pg total proteinI100pl of PBS + 0.5% Triton 

X-100. Samples low 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 to prepare multiple dilutions 

of each sample to insure that optical density readings for a given sample fall 

on the linear portion of the standard curve. Typically, samples (like 

standards) are run in duplicate. Detergents act as wetting agents, therefore, 

more than a single use of a pipette 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 tip per sample and to withdraw the sample only a single time 

per tip. 

3. GFAP Assay 

Coat Irnmulon-2 flat bottom plates with rabbit anti-GFAP. Add 1.0pg total 

immunoglobulin protein I100 p1 PBS /well. (-25p1 of anti-GFAP [Dako] in 10 

ml of PBS is the quantity needed per plate). 

Incubate the coated plate at 37°C for 1 hour. This step may be done at the 

beginning of the assay or it may be done the night before with storage 

overnight at 4OC. Perform all other incubation and reagent addition steps at 

room temperature. 

Empty the plate into a sink and tap on absorbent paper to remove excess liquid. 

This latter procedure is important to eliminate the possibility of any reagent 

carry-over between steps. 

Wash plates 4X with PBS (200 pl per well), tapping and blotting between 

each wash. 

Block 1 hour with 5 % non-fat powdered milk in PBS at 100 pl 

per well. 

Empty plate, tap on absorbent paper (upside down) to remove excess liquid, 

load diluted standard curve and samples in a volume of 100 p1 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-mouse IgG (1 :3000)(Jackson ImmunoResearch) 

made up in 5% non-fat dry milk + 0.5% Triton X-100, 100 p1 /well. 

Wash 4X with PBS + 0.5% Triton X-100,200 pllwell. 

Add P-nitrophenylphosphate substrate (Bio-Rad) in a volume of 100 pYwell 

and incubate for 20 minutes. 

Stop reaction with 0.4N NaOH, 100 pYwell. 

12. "PO$' any bubbles 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. Calculate the GFAP concentration in the samples by comparing their optical 

density (OD) values to those obtained for the linear portion of the GFAP 

standard curve. Software programs linked to specific plate readers should be 

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 subtraction of 

blanks and incorporation of dilution factors. Data are expressed as yg 

GFAP/per mg total protein or, if the absolute amount of GFAP in the standard is 

not available, data are expressed as GFAP-like immunoreactivity/per mg total 

protein. Total protein concentration in the samples is estimated from the total 

protein assay described in Support Protocol 2. Data also can be expressed on 

the basis of tissue wet weight (yg GFAPIgram wet weight or GFAP 

irnrnunoreactivity/gram wet weight). Although this approach permits 

elimination of 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 procedure for preparing brain 

tissue for subsequent analysis of GFAP by 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 (see commentary). 

B. Materials: 

Dissecting Instruments 

Balance 

Ultrasonic Cell Disruptor (e.g. PGC cat. # 81-6721-02,2 mm probe) 

Pipettes 

Hods tir Plate 

Pipette tips 

Microfuge tubes 

C. Protocol Steps: 

1. Sacrifice animals and remove the brain as rapidly as possible. 

2. Dissect brain regions. If a number of regions are to be dissected, this 

process can be aided by 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. This number of 

regions can be prepared on a cold plate or at room temperature without 

degradation of GFAP as assessed by immunoblot analysis. All brain 

regions can be stored frozen indefinitely in capped microfuge tubes at 

this step in the protocol, or you can proceed to the next step. 

3. Weigh and homogenize the dissected brain parts. Tare an appropriately 

labeled microfuge or other suitable storage tube, or weigh boat for the 
12 

large brain 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 homogenization tube, and immerse the tissue in 10 volumes of 

hot (85-95°C) 1% (wlv) SDS. For example, 0.1 grams of tissue would be 

immersed in 1.0 ml of SDS. While the SDS is still hot, homogenize the 

tissue by sonification with an ultrasonification microprobe. Large brain 

areas are sonified in the glass homogenization vessel with the 

ultrasonification microprobe, then homogenized using a motor-driven 

Teflon pestle. Samples should be stored frozen (-70°C) at time of 

sacrifice prior to assay. Samples prepared and stored in this manner 

retain their GFAP content for at least 5 years. 

111. Support Protocol 2: Assay for Total Protein 

A. Introduction: This support protocol describes the procedure for assaying the 

concentration of total protein in the SDS-homogenates. The procedure described 

essentially is the bicinchoninic acid (BCA) method described by Smith et al. 

(1985) which is available in kit form (see materials). To assay total protein 

concentration of the SDS-homogenates the assay must be compatible with 1% 

SDS. Use of the BCA assay is not an absolute requirement as other detergent 

compatible methods are available (e.g. Bio-Rad DC protein assay). Bovine 

serum albumin is used as the protein standard in the described procedure. Other 

protein standards can be substituted. 

B. Materials: 

Microplate Reader 

Pipettes 

Pipette tips 

96-well Microtiter Plates 

Microfuge tubes 

Incubator 

Vortex Mixer 

Miscellaneous Laboratory Glassware 

BCA Protein Kit (Pierce #23223) 

Bovine Serum Albumin (BSA) (Sigma A7888) 

Sodium Dodecyl Sulfate (Bio-Rad #l6 1-0302) 

C. Protocol Steps: 

1. Prepare total protein standards. Prepare a 1 mglml solution of BSA in 

1% (w/v) SDS. Aliquots of this standard can be stored frozen at -70 OC for 

fiture use. Thaw as needed, but do not re-freeze. 

2. Prepare a total protein standard curve. Prepare dilutions of the BSA standard in 

1 % SDS as follows: 1 .O, 2.5, 5.0,7.5, and 10 pgIlOp1 of 1 % SDS (no dilution is 

required for last standard). Vortex each tube and add 10 pl of each standard to 

a well of the microtiter plate; add 10 p1 of 1 % SDS to serves as a blank. 

Typically, standards are run in duplicate. Detergents act as wetting agents, 

therefore, more than a single use of a pipette tip with SDS-containing samples 
13 

can lead to carry-over errors. Thus, it is recommended to use only a single 

pipette tip per sample and to withdraw the sample only a single time per tip. 

3. Prepare dilutions of the samples. Thaw the samples, vortex and dilute a 10 pl 

aliquot with 190 p1 of 1% SDS. Vortex the dilution tube and add a 10 p1 

aliquot into a well of a microtiter plate. 

4. Add the protein assay reagent. Add 200 p1 of the BCA reagent (composed of 

50: 1 ratio of solution A: solution B of the Pierce BCA reagent) to each standard 

and sample. 

5. Incubate the plate at 37°C for 30 minutes. Other incubation temperatures are 

permissible; follow direction provided with the kit. 

6. "Pop" any bubbles in the microtiter plate wells with a needle or pipette tip to 

insure uniform and accurate readings and read the plate at 562 nm. 

7. Calculate the concentration of total protein in the samples from the standard 

curve. Software programs linked to specific plate readers should be 

programmed to plot OD vs. total protein in a linear fashion. Most 

programs allow for automatic subtraction of blanks and incorporation of 

dilution factors. Because the samples were prepared in 10 volumes of 

diluent, typically, total protein values are approximately 10 mglml. 

IV. Reagents and Solutions 

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

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

NaCll1.0 mM KC112 rnM KH2P04/8.0 mM Na2HP04.7H20/pH 7.4 (can be 

stored at 4OC for at least a month). For this and all subsequent reagents and 

solutions, determine the total volume that needs to be 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 100 ml of PBS or per 100 ml 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 greater than a month 

or two (room temperature). The dry milk tends to discolor and will not go into 

solution at shelf times longer than 2 months. 

4. Polyclonal anti-GFAP (Dako)- Add 25p1 of antibody solution 110ml of PBS. 

The assay is based on the use of this antibody as a "capture" reagent. 

Substitution of 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) 

combined with Alkaline Phosphatase-conjugated anti-mouse IgG (Jackson 

hnmunoResearch)- Make up a stock solution of monoclonal anti-GFAP and 

alkaline phosphatase-conjugated anti-mouse IgG as per the vendors7 

instructions. Store both 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 conjugate to bind to the detection antibody and 
14 

generate a colored reaction product proportional to the amount of antigen 

(GFAP) present in the samples. Substitution of 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 GFAP Assays; Specific Commentary on the GFAP 

ELISA 

A. Background Information 

It has long been known that damage to the central nervous system results in 

astrogliosis (gliosis, reactive gliosis, glial activation), a response to brain injury 

characterized by hypertrophy and, less often, hyperplasia of astrocytes, a sub-type 

of CNS glia (Eng, 1988; Norenberg, 1994). At the electron microscopic level, 

astrogliosis is characterized by the accumulation of glial filaments. GFAP 

was found to be the major protein component of these filaments (Eng, 1988). As 

such, GFAP serves as a biomarker for filament accumulation and, therefore, of 

gliosis (Eng, 1988; Norton et al., 1992; O'Callaghan, 1993). With the development 

of antibodies to GFAP, immunohistochemical analysis of 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 

evidence now has been accumulated demonstrating the ubiquity of the glial 

response to all types of CNS damage based onimrnunohistochemistry of GFAP. 

Only recently, however, have methods been introduced to assay levels of GFAP as 

a means of quantifying gliosis. 

While GFAP immunohistochemistry has proven useful for revealing patterns of 

gliosis after brain injury, this approach does not lend itself to quantification or the 

analysis of large numbers of samples. Small (25-50%), but toxicologically 

significant increases, also may be difficult to detect by 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 

implementation of a number of GFAP assays. These assays have been applied to 

examine gliosis in specific brain areas already known to be affected by disease or 

other insult. In addition, they also can be broadly applied in a risk assessment 

context (U.S. EPA) to screen for potential sites of neural damage resulting from 

toxic exposures of the CNS. Recently, analysis of GFAP has been used to 

demonstrate that the degree of cortical gliosis in postmortem brain tissue from 

victims of Alzheimer's disease correlates with the severity of dementia scores in 

these individuals prior to death (G. Webster Ross, submitted). Analysis of GFAP in 

cerebrospinal fluid (CSF) also has been applied to the human condition as an 

indicator 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 contamination of meat (Schmidt et al., 1999). 
15 

Of the number of GFAP assays that have appeared in the literature over the last 

15 years, all essentially fall into two categories: 1) solid phase immunoassays 

where GFAP is immobilized on a solid support matrix and detected by mono- or 

polyclonal antibodies or 2) liquid-phase assays where GFAP from brain extracts or 

solubilized brain tissue (or CSF) is "captured" by one antibody and then detected 

by another antibody raised in a different host species (Butler et al., 1986). The 

assay described in 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 mixture by SDS-PAGE, followed by electrophoretic transfer to a solid 

support membrane. Anti-GFAP antibodies coupled to a variety of detection 

reagents then can be used for quantification of GFAP bound to the membranes. 

Unfortunately, this approach has been found to severely underestimate the 

concentration of GFAP in the resolved mixture of proteins and the effects of 

treatments known to increase GFAP (07Callaghan et.al., 1999). Other solid phase 

assays for GFAP have been developed that do not rely on prior resolution of 

protein mixtures by SDS-PAGE (Wang et al., 1990; 07Callaghan, 1991.b). These 

assays incorporate manual spotting of brain homogenates on solid supports, with or 

without the aid of a template. The membranes are then incubated with anti-GFAP 

polyclonal or monoclonal antibodies, which, in turn, are bound by lZ51 Protein A. 

Quantification is achieved by gamma spectrometry or by densitometry of 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 described in this study or similar ELISAs 

described previously (Eng et al., 1986; Kretzschmar et al., 1985; O'Callaghan, 

1991 ; Rosengren et al., 1994), have several advantages in comparison to the other 

methods for assaying GFAP described above. They are easier to perform because 

they have fewer steps. They are more sensitive. Although they may require a 

greater number 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 advantage of speed and high 

throughput. From sample application to data collection, all steps can be performed 

in the plate. Moreover, the microplate-based format permits 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 allowing the 

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 

advantages afforded by this technique, the assay described in this study may have a 

few additional advantages. Because it is based on detergent-solubilized 

homogenates of a given brain area, any treatment effects can be 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 

comparisons of quantitative data on GFAP to immunohistochemical staining of 

GFAP in that area. Moreover, it also helps rule out inter-laboratory differences 

associated with assaying GFAP content in one type of extract in one lab, and 

another type of extradfraction in another lab. Finally, the same SDS-denatured 

homogenate used to assay GFAP can be subjected to multiple assays for additional 

glial or neuronal proteins, thereby permitting comparisons to be made among 
16 

multiple markers of neurotoxicity in 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 quantified by immunoassay of tyrosine hydroxylase 

(TH), a marker of dopamine containing 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 decreases in TH predict greater 

increases in GFAP. 

B. Critical Parameters 

The most critical aspect of the GFAP assay is the absolute requirement for 

preparation of consistently dissected regions of the brain (see Support Protocol 1). 

Consistent dissections yield consistent GFAP values with the use of only a few 

animals per dose or time point (see Anticipated Results). The particular regions to 

be dissected depend on the questions being 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 order to avoid the possibility of diluting localized increases in GFAP. 

The possibility exists that extremely localized increases in GFAP may fail to be 

detected with the assay. While GFAP immunohistochemistry is relatively 

insensitive in 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 quantification with 

the GFAP assay due to dilution of signal by 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 sensitive morphological approaches for detection 

of neural damage, such as silver degeneration stains (Switzer, 2000), Fluoro-Jade 

(Schmued et al., 2000), and stains that detect activated microglia (Streit et 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 

antibodies andlor non-fat dry milk and 4) mixing of the standards and samples prior 

to their dilution or addition to the microplate wells. A troubleshooting guide is 

provided in Table 4 that covers most problems encountered with the assay. 

C. Troubleshooting (Table 4) 

D. Typical Results 

Typical GFAP assay values obtained for different regions of the rat brain are 

presented in Fig. 2. Absolute values for GFAP (pglmg total protein) may vary 

depending on the GFAP standard used and the species subjected to evaluation. 

Region-to-region differences in GFAP values from untreated animals of a given 

species, however, should remain stable, if consistent dissections are performed. 
17 

E. Time Considerations 

The GFAP assay requires approximately 5 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, 10 brain areas can be prepared (andhomogenized) 

from 50 rats in a day. Two people are required: one to dissect the brains and one 

(or more) to weigh and homogenize the tissue. The total protein assay requires 

approximately 1 hour for one person to process a 96-well rnicroplate. 

Statistics: 

The effect of treatment on GFAP concentration was determined by separate one-way 

ANOVAs for each of the nine brain areas from males and females utilizing the JMP@ 

statistical package (SAS, 1995). The significance level was set a P<0.05 and, to ensure 

detection of between group treatment effects, The Least Significant-Difference test 

(Keppel, 1973) was used in post-hoc analyses. 

Results and Conclusions 

The results of the GFAP analysis are presented in Tables 5 and 6. Results are reported 

according to gender, because sex-dependent responses to toxic substances are not 

uncommon, including responses reflected in levels of GFAP. Control levels for GFAP 

varied markedly according to brain region, consistent with known levels observed for 

GFAP across different brain regions (see Fig. 2). The 13-week exposure to Gasoline TBA 

Vapor Condensate was associated with a slight increase in GFAP in the cortex from group 

I1 males and a slight decrease in GFAP in the hypothalamus of group IV males (see 

Table 5). These statistically significant changes in GFAP were not likely to be adverse 

because the increase in GFAP was small in magnitude and not concentration-related and 

the decrease in GFAP cannot be interpreted as adverse because the neurotoxicological 

significance of 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 affected in 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 dissections of 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) could be 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 representative brain regions in males or females. 
18 

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Eng, L.F., Stocklin, E., Lee, Y.-L. Shiurba, R.A., Coria, F., Halks-Miller, M. Mozsgai, 

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Fix, A.S., Wightman, K.A. and O'Callaghan, J.P. 1995. Reactive gliosis induced by 

MK-801 in the rat posterior cingulate/retrosplenial cortex: GFAP evaluation by 

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Kretzschmar, H.A., DeArmond, S.J., Forno, L.S. 1985. Measurement of GFAP in 

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McKay, R.D.G. and S.J. Hockfield. 1982. Monoclonal antibodies distinguish 

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Norton, W.T., Aquino, D.A., Hozumi, I., Chiu, F.-C., and Brosnan, C.F. 1992. 

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07Callaghan, J.P. 1993. Quantitative features of reactive gliosis following toxicantinduced damage of the CNS. Ann. NY Acad. Sci. 679: 195-210. 

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immunoblots of proteins resolved from brain homogenates: Underestimation of 

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Rosengren, L.E., Wikkels~, C. and Hagberg, L. 1994. A sensitive ELISA for glial 

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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, 2nd Edition, Oxford Unviversity Press, Oxford, Cary, NC 

Streit, W.J. , Walter, S.A., and Pennell, N.A. 1999. Reactive microgliosis. Prog. 

Neurobiol, 57: 563-581. 

Sutcliffe, J.G. 1988. mRNA in the mammalian central nervous system. Ann. Rev. 

Neurosci. 11: 157-198. 

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

15: 89-93. 

Switzer, R.C. 2000. Application of silver degeneration stains for neurotoxicity testing. 

Toxicol. Pathol. 28: 70-83. 
20 

U.S. Environmental Protection Agency, Pesticide Assessment Guidelines, 

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Wang, S., Rosengren, L.E., Karlsson, J.-E., Stigbrand, T. and Haglid, KG. 1990. A 

simple quantitative dot-immunobinding assay for glial and neuronal marker proteins in 

SDS-solubilized brain tissue extracts. J. Neurosci. Methods 33: 219-227. 
Figure Legends 

Figure 1. Sample GFAP standard curve. GFAP values in nanograms correspond to levels 

found in .25 -10 pg total hippocampal homogenate protein. Dilutions 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 GFAP Standard 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=Optical Density; Std Dev=Standard Deviation; 

CV=Coefficient of Variation 
Table 4: Troubleshooting Guide 

1 Problem 

I No color reaction 

c Color reaction abnormally low 

c Color reaction abnormally high 

w Standard curve not sigmoid 

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 dilutions of samples to 

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 Gasoline TBA 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 

*Each value represents the mean k SEM for the concentration of 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 Exposure to Gasoline TBA 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 * 0.1 2 

6.05 * 0.47 

4.81 * 0.22 

ND 

5.1 8 * 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 

5.1 8 * 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 

2.1 6 t 0.1 5 

7.01 + 0.72 

3.96 * 0.1 2 

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 * 0.65 
Table 7: Individual GFAP Levels in Specific Regions of Rat Brains 

Following a 13 Week 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 concentration of GFAP(ug1mg Total Protein) 

* Samples too dilute to detect GFAP 
Huntingdon Life Sciences 00-6131 Page 1 

21 1-TBA-S Draft Final Report 

INTRODUCTION: The following is data generated at Huntingdon Life Sciences, East 

Millstone, NJ. The separately issued main study report should be referenced for details of 

the procedures used for test atmosphere generationlcharacterization and animal 

evaluations. 

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 during the 

exposures were comparable to the targeted exposure levels. There were no exposurerelated effects seen in the test animals with regards to body weights, feed consumption 

and ophthalmoscopic findings. 

TABLE OF CONTENTS 

TABLES 

Chamber Monitoring Results ........................................................................................... Summary of Clinical Observations (pretest only) ............................................................ Summary of Ophthalmoscopic Findings 

Ophthalmologist Report ................................................................................................... Summary of Ophthalmology Observations ...................................................................... Mean Body Weights (grams) ........................................................................................... Mean Body Weight Change from Baseline (grams) ........................................................ Mean Feed Consumption Values (gramskglday) ............................................................ Individual Clinical Observations (pretest only) ............................................................... Individual Ophthalmology Observations ......................................................................... 

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

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

Individual Feed Consumption Values (gramskglday) ..................................................... Animal Termination History ............................................................................................ 
Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-6131 
Table 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 

I 

Group IA - 0 (air only) mg/m3 

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 

2.1 9E-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 

53 
Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-6131 
Table A GASOLINE TEA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY 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 

55 
Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-6131 

Chamber Monitoring Results 

Cumulative Exposure Record 
Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-6131 

Chamber Monitoring Results 

Cumulative Exposure 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 

S.D. 5.6 

TMC 

(mg/m3) 

5.32E-03 

6.1 8E-03 

3.13E-03 

1.1 6E-02 

9.01 E-03 

MMAD 

Olm) 

1.613 

5.507 

0.8693 

2.1 57 

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 

0 
Table A GASOLINE TBA 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 

21 13 

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 GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-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 

21 50 

21 10 

2080 

1950 

2020 

21 60 

2020 

2020 

21 90 

Mean 

(mglm3) 

2000 

1935 

1933 

1953 

1975 

2005 

1970 

1963 

21 35 

21 88 

1953 

1845 

2043 

2023 

201 3 

2070 

2090 

2068 

21 83 

2033 

2065 

21 05 

2213 

2020 

alytical Chamber Concentration 

Individual MMAD 

Olm) 

2.312 

4.144 

0.7525 

0.8197 

1.124 

Particle Size 

?termha 

GSD 

ms 

TMC 

(mg/m3) 

4.69E-03 

9.38E-03 

2.35E-02 

2.61 E-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 

50 
Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY 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 

21 40 

21 50 

21 80 

2070 

1950 

2025 

105 

Mean 

(ms/m3) 

21 08 

1840 

2000 

1900 

2070 

2030 

21 03 

2028 

1898 

1975 

2113 

201 8 

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 

3.71 E-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 

5.0 
Table 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) 

0.81 83 

0.7358 

14.68 

1.010 

0.8253 

Particle Size 

!terminat 

GSD 

ns 

TMC 

(ms/m3) 

8.49E-03 

2.1 8E-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 

5 1 

5 1 

49 

52 

49 

45 

52 
Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-6131 

Chamber Monitoring Results 

Date Exposure 

Number 

Nominal 1 

Mean 

(mg/m3) (mg/m3) 

2090 2105 

2070 2060 

1990 1935 

2010 1830 

21 50 2048 

2110 2123 

2080 2020 

1950 2018 

2020 1970 

21 60 2203 

2020 1968 

2020 2088 

21 90 21 65 

2090 2008 

2090 1995 

21 40 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 

Nominal I 

(ms/m3) 

1990 

1510 

1890 

1920 

2080 

1940 

2010 

1980 

1990 

2040 

1990 

1910 

1950 

1980 

21 40 

21 40 

21 50 

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 

aGauge fell prior to the 30 minutes exposure reading on Test Day 78. Therefore, the readings could not be recorded for the remainder of the exposure on that day. 
Table A GASOLINE TEA VAPOR CONDENSATE: A 13-WEEK WHOLE-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 Monitoring Results 

Cumulative Exposure 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 GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY 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 GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-61 31 

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 

7 Nominal ~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 

5.1 
Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-6131 

Chamber Monitoring Results 

Cumulative Exposure 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 

991 0 

10100 

10100 

9920 

10100 

10200 

10300 

9320 

10100 

Mean 

mm3) 

10040 

101 40 

961 5 

9763 

10040 

9853 

9898 

101 00 

lOO7O 

991 8 

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 

tions I 

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 GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-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 

971 5 

9973 

alytical Chamber Concentration 

Individual 

Particle Size 

MMAD 

Olm) 

1.403 

2.459 

0.7601 

0.9700 

0.8294 

etermina 

GSD 

ms 

TMC 

(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 GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-61 31 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group lllB - 10,000 mglm3 -- - - - -- - 

Chamber Environment 

Particle Size Mean 

Analytical Chamber Concentration 

A 

ms 

TMC 

(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 

991 0 

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 

981 5 

9878 

991 0 

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 GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-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 

201 80 

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 

51 
Table A GASOLINE TBA VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 00-61 31 

Chamber Monitoring Results 

Cumulative Exposure 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 

1.81 E-02 

4.37E-03 

2.71 E-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 

52 
Table A GASOLINE TBA 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 

201 50 

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 INHALATION TOXICITY 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 

lalytical Chamber Concentration 

Individual 

t 

MMAD 

Olm) 

0.8428 

0.7643 

0.7572 

1.277 

0.8255 

Particle Size 

ns 

TMC 

(ms/m3) 

8.1 7E-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 

48 
Table 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 INHALATION TOXICITY STUDY IN RATS 00-6131 

Chamber Monitoring Results 

Cumulative Exposure 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 

Nominal Analytical Chamber Concentration 

(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 

Mean I Individual MMAD GSD 

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 LIMITS 
Huntingdon 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 Saint Andrews 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-1 2 00-61 31 F 

Ophthalmoscopic examination of study A-1 2 00-61 31 F pretest rats was performed 

June 13, 2002. No abnormality was seen. All rats are suitable for inclusion in the study. n 

Lionel F. Rubin, V. M. D. 
Table C - Termination 

LIONEL F. RUBIN, V.M.D. 

1 1 16 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-1 2 00-61 31 F 

Ophthalmoscopic examination of study A-1 2 00-61 31 F rats was performed 

September 17, 2002 (terminal examination). I have reviewed the findings of the type and 

incidence of ocular abnormalities. There is no indication of dose or compound related 

ocular disease. In my opinion, none of the ocular abnormalities is attributable to the 

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 DETECTED 
Huntingdon 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 DETECTED 
Huntingdon 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 DETECTED 
Huntingdon 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 differences 
Huntingdon 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 differences 
Huntingdon 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 differences 
Huntingdon 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 

9 E 

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

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 3NITOSY3 
Huntingdon 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 differences 
Huntingdon 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 differences 
Huntingdon 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 3NI10SV3 
Huntingdon 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 differences 
Huntingdon 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 differences 
Huntingdon 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: * = pc0.05 
Huntingdon 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: ** = pc0.01 
Huntingdon 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 differences 
Huntingdon 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-PRESENT 
Huntingdon 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-PRESENT 
Huntingdon 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-PRESENT 
Huntingdon 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-PRESENT 
Huntingdon 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 5 
Huntingdon 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-PRESENT 
Huntingdon 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 7 
Huntingdon 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-PRESENT 
Huntingdon 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 LESIONS 
Huntingdon 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 LESIONS 
Huntingdon 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 LESIONS 
Huntingdon 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 LESIONS 
Huntingdon 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 LESIONS 
Huntingdon 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 LESIONS 
Huntingdon 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 LESIONS 
Huntingdon 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 LESIONS 
Huntingdon 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 LESIONS 
Huntingdon 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. 

N 
Huntingdon 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. 

N 
Huntingdon 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. 

N 
Huntingdon 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. 

N 
Huntingdon 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. 

N 
Huntingdon 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. 

N 
Huntingdon 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. 

N 
Huntingdon 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. 

N 
Huntingdon 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. 

N 
Huntingdon 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. 

N 
Huntingdon 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. 

N 
Huntingdon 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. 

N 
Huntingdon 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. 

N 
Huntingdon 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. 

N 
Huntingdon 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. 

N 
Huntingdon 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. 

N 
Huntingdon 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 6 1 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 6 1 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 6 1 62 60 5 4 56 5 4 52 50 44 

MEAN 

S.D. 

N 
Huntingdon 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. 

N 
Huntingdon 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. 

N 
Huntingdon 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. 

N 
Huntingdon 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. 

N 

SF=Spilled Feeder 
Huntingdon 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. 

N 

SF=Spilled Feeder 
Huntingdon 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. 

N 

SF-Spilled Feeder 
Huntingdon 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. 

N 

SF=Spilled Feeder 
Huntingdon 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 DAY 
Huntingdon 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 DAY 
Huntingdon 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 DAY 
Huntingdon 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 9 2 
Huntingdon 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 9 2 

1577 TERMINAL SACRIFICE 25-SEP-02 13 9 2 

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 2 
Huntingdon 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/M3 
Huntingdon 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 SACRIFICE 
Huntingdon 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