GFAP Levels in Specific Rat Brain Areas Following A 13-Week 

Whole-Body Inhalation Exposure to Gasoline DIPE Vapor Condensate 

HLS Study No.: 00-6130 

Sponsor Study No.: 21 1 -DPE-S 

Date: 25June2002 
Summary Report 

Gasoline DIPE Vapor Condensate: 

A 13-Week Whole Body Inhalation Toxicity Study in the Rat 

Huntingdon Life Sciences, Inc. Study No. 00-61 30 

Sponsor Study No 2 1 1 -DIPE-S 

Measurement of Glial Fibrillary Acidic Protein 

I Principal Investigator: 

James P. OYCallaghan, Ph.D. 

Molecular Neurotoxicology Laboratory 

Health Effects Laboratory Divison 

Centers for Disease Control and Prevention 

National Institute for Occupational Safety 

and Health 

1095 Willowdale Road 

Morgantown, WV 26505 

304-285-6079 

304-285-6220 (FAX) 

j do5 acdc. gov 

Study Director: 

Gary M. Hofhan, B.A., DABT 

Huntingdon Life Sciences 

Mettlers Road 

East Millstone, NJ 00875-2360 

732-873-2550 

732-873-3992 (FAX) 

hofhang~~rinceton.huntin~don.com 

STATEMENT OF COMPLIANCE 

This study was conducted in the spirit of compliance with 79.60, CFR Vol. 59, No. 122, 

27 June 1994. This study was performed according to protocol and Standard Operating 

Procedures. 

James P. O'Callaghan, Ph.D. 

Principal Investigator 

- -- 

Date 

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

Study Director 
SIGNATURE PAGE 

SCIENTIST 

The following Scientist was responsible for the overall conduct of this study. 

James P. 07Callaghan, 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 ths 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. Date 

Quality Assurance Auditor 
QUALITY ASSURANCE STATEMENT 

Listed below are the dates that ths 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. 

Type of Date(s) of Reported to 

Inspection Inspection Study Director and Management 

Quality Assurance Senior Auditor Date 
6 

TABLE OF CONTENTS 

Page 

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

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

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

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 

ELISA 

A. Background Information 

B. Critical Parameters 

C. Trouble Shooting 

D. Typical Results 

E. Time Considerations 

Statistics ............................................................................................. 17 

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

References .......................................................................................... 18 

Figure Legends ..................................................................................... 2 1 

Figure 1 : Sample Standard Curve 

Figure 2: pg GFAPImg 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 DIPE Vapor 

Condensate 

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

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

Condensate 

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

a 13-Week Whole-Body Inhalation Exposure to Gasoline DIPE 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 1.3-week inhalation exposure to Gasoline DTPE Vapor Condensate did not elevate GFAP 

levels in any of the nine brain regions examined in either males or females. The data 

suggest that exposure to Gasoline DPE 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; 0 'Callaghan 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, ow 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 (OYCallaghan, 1991a; 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 (OYCallaghan, 

1991a; 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 (0' Callaghan, 199 1 a; 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 DIPE 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 

17 December 2001 (Date Study Director signed the Protocol) 

DATE OF ANIMAL RECEIPT 

24 January 2002 

EXPOSURE INITIATION 

12 February 2002 (Experimental Start Date) 

EXPOSURE TERMINATION 

14 May 2002 

TERMINAL SACRIFICE 

15 May 2002 

EXPERIMENTAL TERMINATION 

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

STUDY COMPLETION 

Day Month Year @ate 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 fiom 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. # 8 1-6721 - 
02,2 mm probe) 

Pipettes 

HotIStir 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. #l72-1063) 

Triton X-100 (e.g. Bio-Rad Cat. #16l-O407) 

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 fi-om a single "pool" of a 1% SDS 

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

be aliquoted and stored fi-ozen 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). Immunoreactivity 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 

mg/ml). to prepare a standard curve in PBS plus 0.5% Triton X-100. For rat 

hippocampal homogenate, the protein values for the standard curve should be 

between approximately 0.25 to 10 pg/lOOpVmicroplate well (i.e. 0.25, 0.5, 1.0, 

2.5, 5 .O, 7.5, 10pg total proteinI100pl). Table 1 shows an example of dilutions 

needed to prepare a standard curve from a homogenate of rat hippocampus 
10 

(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 

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

sample only a single time per tip. 

2. Sample Preparation 

1. Thaw and mix samples: Remove samples fiom the fkeezer, thaw at room 

temperature and vortex or sonify prior to dilution. 

2. Prepare dilutions of the samples: Dilute the samples in PBS + 

0.5% Triton X-100 to a concentration of approximately 10pg total protein/ 

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

diluted to a concentration of 5pg total protein/lOOpl of PBS + 0.5% Triton 

X-100. Samples low in GFAP (e.g. striaturn) may need to be diluted to 20pg 

total protein1100pl 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 Immulon-2 flat bottom plates with rabbit anti-GFAP. Add 1 .Opg total 

immunoglobulin protein 1100 pl PBS /well. (-25 pl of anti-GFAP [Dako] in 10 

ml of PBS is the quantity needed per plate). 

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

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

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

room temperature. 

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

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

carry-over between steps. 

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

each wash. 

Block 1 how with 5% non-fat powdered milk in PBS at 100 p1 

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 pl /well. 

Wash 4X with PBS + 0.5% Triton X-100,200 pl/well. 

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

and incubate for 20 minutes. 

11. Stop reaction with 0.4N NaOH, 100 pllwell. 
11 

12. "Pop" 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. 

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 fashon. 

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 pg 

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

not available, data are expressed as GFAP-like immunoreactivitylper 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 (pg GFAPIgram wet weight or GFAP 

irnmunoreactivity/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: Ths support protocol describes the procedure for preparing brain 

tissue for subsequent analysis of GFAP by Sandwich ELISA (Basic Protocol 1). 

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. # 8 1-6721-02,2 mm probe) 

Pipettes 

HotIStir 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 

peti 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 
12 

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 

large brain areas that don't fit into microhge 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. Wle 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 fi-ozen (-70°C) at time of 

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

retain their GFAP content for at least 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 lut 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 #16 1-0302) 

C. Protocol Steps: 

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

1 % (wlv) SDS. Aliquots of this standard can be stored frozen at - 70 C for 

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

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

1% SDS as follows: 1.0,2.5, 5.0, 7.5, and 10 pgIlOp1 of 1% SDS (no dilution is 

required for last standard). Vortex each tube and add 10 pl of each standard to 
13 

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 

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

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

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

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

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 37OC 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 

NaCV1.O mM KCV2 mM KH2P04/8.0 mM Na2HP04.7H20/pH 7.4 (can be 

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

solutions, determine the total volume that needs to be prepared based on the 

use of 100 pVwell and 96 wells per plate (washes take 200 pVwell). 

2. PBS+O.5% 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-fatt 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 /10ml of PBS. 

The assay is based on the use of ths 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 

1rnmunoResearch)- Make up a stock solution of monoclonal anti-GFAP and 

alkaline phosphatase-conjugated anti-mouse IgG as per the vendors' 

instructions. Store both stocks at 4OC as per the vendor's instructions. Add 

20p1 of the monoclonal antibody solution stock and 3.3 p1 of the alkaline 
14 

phosphatase conjugate stock11 Om1 powdered milk +0.5% Triton X- 100. The 

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 

generate a colored reaction product proportional to the amount of antigen 

(GFAP) present in the samples. Substitution of antibodies fiom other vendors 

may not yield suitable results. Make these solutions fresh on the day 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 fiesh 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 subtype 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; 0' Callaghan, 1993). With the 

development of antibodies to GFAP, irnrnunohistochemical 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; OYCallaghan 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 onimmunohistochemistry 

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

levels of GFAP as a means of quantifying gliosis. 

While GFAP irnmunohistochemistry 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 irnrnunohistochemistry. 

These drawbacks, combined with the need to develop quantitative biomarkers of 

neurotoxicity (OYCallaghan et al., 1995), and to define quantitative aspects of 

toxicant- and disease-induced gliosis, has prompted the development and 

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

to examine gliosis in 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 
15 

of the presence of brain or spinal cord contamination of meat (Schmidt et al., 

1999). 

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 monoor 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 timeconsuming resolution of a protein mixture by SDS-PAGE, followed by 

electrophoretic transfer to a solid support membrane. Anti-GFAP antibodies 

coupled to a variety of detection reagents then can be used for quantification of 

GFAP bound to the membranes. Unfortunately, this approach has been found 

to severely underestimate the concentration of GFAP in the resolved mixture of 

proteins and the effects of treatments known to increase GFAP (O'Callaghan 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; 

OYCallaghan, 1991b). These assays incorporate manual spotting of brain 

homogenates on solid supports, with or without the aid of a template. The 

membranes are then incubated with anti-GFAP polyclonal or monoclonal 

antibodies, which, in turn, are bound by 125~ Protein A. Quantification is 

acheved 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, 

199 1 b; 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 

hgh 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 ths study may have a few additional advantages. Because it is based on 

detergent-solubilized homogenates of a given brain area, any treatment effects 

can be directly related to effects in that brain area, rather than an arbitrarily 

defined extract or sub-fi-action 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 
16 

rule out inter-laboratory differences associated with assaying GFAP content in 

one type of extract in one lab, and another type of extract/fraction 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 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 imrnunohistochemistry 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 

irnrnunohistochemistry 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. 
17 

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

given species, however, should remain stable, if consistent dissections are 

performed. 

E. Time Considerations 

The GFAP assay requires approximately 5 hours for one person to process a 96- 

well microplate. 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 (and 

homogenized) 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 

microplate. 

Statistics: 

Data were subjected to analysis of variance followed bypost hoc comparison of treatment 

means (JMPB, SAS). Differences were considered to be significant at the P < 0.05 level. 

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. 1). The 13-week exposure to Gasoline 

DLPE Vapor Condensate did not elevate (was not associated with any increase in) GFAP 

levels in any brain region in either males or females. These data suggest that under the 

exposure conditions employed, damage-induced gliosis did not occur in the brain regions 

cxamincd. Toxicant-induced gliosis is highly dose-, region-, and time-dependent. It is 

possible, therefore, that exposure to higher concentrations, examination of more brain 

regions, examination of more discrete dissections of a given brain region, or the inclusion 

of more time points, might have resulted in positive findings. We note almost all of the 

pituitaries (males and females) assayed gave results that were below the level of 

sensitivity, therefore, only those values that fell on the curve are presented. This study 

represents one of the first attempts to assay pituitary levels of GFAP and the low levels 

were not unexpected. A recently developed fluorescence-based GFAP ELISA 

(OYCallaghan, unpublished) could be used to assess pituitary data if deemed necessary for 

future studies. In conclusion, exposure to Gasoline DLPE Vapor Condensate did not 

appear to result in gliosis in nine representative brain regions in males or females. 
18 

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459-47 1. 

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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OYCallaghan, 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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Rosengren, L.E., Wikkelss, C. and Hagberg, L. 1994. A sensitive ELISA for glial 

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204. 

Schmidt, G.R., Hossner, K.L., Yemm, R.S., Gould, D.H. and OYCallaghan, J.P. 1999. 

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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. 199 1. Strategies for Assessing Neurotoxicity. Neurosci. Biobehav. Rev. 

15: 89-93. 

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

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20 

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Wang, S., Rosengren, L.E., Karlsson, J.-E., Stigbrand, T. and Haglid, K.G. 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, 

striaturn; Hip, hippocampus; Hypo, hypothalamus; Ctx, cortex; Cbm, cerebellum; BS, 

brain stem. Adapted from Martin and O'Callaghan, 1995. 
Figure 1 

2.0- 

1.5- 

.- E 

V) 

s a3 

n - 1.0- 

0 .- 

CI 

0" 

0.5- 

0.0 

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

A = 0.009 B = 1.349 

C = 7.163 D = 1.964 

?= 1.00 

I I I I I I I . I 

lo-" lo0 10 lo2 

ng GFAP1100 pI 
Figure 2 

OB Str Hip Hypo Ctx Cbm BS 

Brain Region 

Blk= Blank; Std= Standard; ? = Unknowns 
STANDARDS Value Well OD Mean Std Dev CV 

pg total protein 

(ng GFW 

STDOl 1o~g B 1 1.694 1.632 0.087 5.4 

(25. OOng) 

B2 1 .570 

STDOZ 7.5 Pg C1 1.537 1.543 0.008 0.5 

(1 8.75ng) 

C2 1.549 

STD03 5.o~g Dl 1.295 1.314 0.027 2.1 

(12.50ng) 

D2 1.334 

STDO4 2.5~8 El 0.857 0.859 0.002 0.3 

(6.25ng) 

E2 0.861 

STDOS 1.0 Pg F 1 0.386 0.386 0.000 0.1 

(2.50ng) 

F2 0.386 

STD06 0.5 Pg G 1 0.175 0.164 0.016 10.0 

(1.25ng) 

G2 0.152 

STD07 0.25 pg H 1 0.072 0.076 0.006 7.6 

(.625ng) 

H2 0.080 

BL=Blank; STD=Standard; OD=Optical Density; Std Dev=Standard Deviation; 

CV=Coefficient of Variation 
Table 4: Troubleshooting Guide 

Problem 

No color reaction 

Color reaction abnormally low 

Color reaction abnormally high 

Standard curve not sigmoid 

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 37°C 

Repeat assay with correct reagent 

dilutions 

Repeat assay with fresh ht 

Repeat assay and terminate reaction 

with 0.4 N N~OH 

Use correct plate template and reread plate 

Repeat assay with correct standard 

dilution 

Repeat assay with correct dilution 

of standard 

Run multiple dilutions of samples to 

obtain OD values from the linear 

portion of the curve 

Change tips after each use 

Check precision by weighmg 

Check plate washer for even 

dispensing and aspiration 

Pop bubbles and re-read plate 

Read plate at 405 nm 
Table 5: Mean GFAP Levels in Specific Regions of Male Rat Brains Following 

a 13-Week Whole-Body Inhalation Exposure to Gasoline DlPE Vapor Condensate 

Brain Area 

Group I 

Air Control 

0 mglm3 

itriaturn 

iippocampus 

:ortex 

Ilfactory Bulb 

-halamus 

iypothalamus 

:erebellurn 

'ituitary 

test of Brain 

Group ll 

Test Substance 

2,000 mglm3 

Group Ill 

Test Substance 

10,000 mglm3 

Group IV 

Test Substance 

20,000 mglm3 

*Each value represents the mean rt SEM for the concentration of GFAP (pglmg Total Protein) 

n= 5 except for Pituitary; ND, not detected; see Results and Conclusion and Table 7 

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

a 13-Week Whole-Body Inhalation Exposure to Gasoline DlPE Vapor Condensate 

Brain Area 

Group I 

Air Control 

0 mglm3 

Striatum 

Hippocampus 

Cortex 

Olfactory Bulb 

halamus 

Hypothalamus 

Cerebellum 

Pituitary 

Group ll 

Test Substance 

2,000 mglm3 

L 

*Each value represents the mean * SEM for the concentratic 

Group Ill 

Test Substance 

10,000 mglm3 

Group IV 

Test Substance 

20,000 mglm3 

- 

of GFAP (pglmg Total Protein) 

n= 5 except for Pituitary; ND, not detected; see Results and Conclusion and Table 7 
29 

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

Following a 13 Week Whole-Body Inhalation 

Exposure to Gasoline DlPE Vapor Condensate 

o Each value represents the concentration of GFAP(ug1mg Total Protein) * Samples too dilute to detect GFAP 
INTRODUCTION: The following is data generated at Huntingdon Life Sciences, East 

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

the procedures used for test atmosphere generationlcharacterization and animal 

evaluations. 

Huntingdon Life Sciences 00-6 130 Page 1 

2 1 1 -DIPE-S Draft Final Report 

STUDY DATES: Date of Animal Receipt: 24 January 2002 

Experimental Initiation Date: 12 February 2002 (in-life) 

Animal Exposure and Animal Data 

Preface 

Experimental Completion Date: 15 May 2002 (in-life) 

Appendix A 

Draft Report Date: 6 March 2003 

EXPOSURES AND IN-LIFE SUMMARY: The actual measured results during the 

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

and ophthalmoscopic findings. 

TABLE OF CONTENTS 

TABLES 

Chamber Monitoring Results ........................................................................................... 

Summary of Clinical Observations .................................................................................. 

Summary of Ophthalmoscopic Findings 

Ophthalmologist Report ................................................................................................... 

Summary of Ophthalmology Observations ...................................................................... 

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

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

Mean Feed Consumption Values (grams/kg/day) ............................................................ 

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

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

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

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

Individual Feed Consumption Values (grams/kg/day).. ................................................... 

Animal Termination History ............................................................................................ 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Date Exposure 

Number 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group IA - 0 mglm3 (Air Control) 

Nominal 

(mglm3) 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

Mean 

(mg/m3) 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

alvtical Chamber Concentration 

Individual MMAD 

(pm) 

1.209 

4.471 

2.303 

1.520 

Particle Size 

Determinations 

GSD TMC 

(mglm3) 

1.94E-03 

7.37E-03 

3.03E-03 

3.20E-03 

Chamber 

M 

Tem~erature 

vironment 

n 

Humidity 

(%) 

57 

58 

58 

56 

56 

58 

54 

54 

55 

51 

58 

53 

57 

58 

54 

44 

54 

59 

54 

55 

52 

53 

54 

53 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODYINHALATION TOXICITY STUDY IN RATS 

Date Exposure 

Number 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group IA - 0 mglm3 (Air Control) 

Particle Size 

Nominal 

(mglm3) 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

1 

Mean 

(mglm3) 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

~alvtical Chamber Concentration 

Individual MMAD 

(IJm) 

0.9053 

2.495 

2.357 

2.187 

1.413 

Determinai 

GSD 

1s 

TMC 

(mglm3) 

1.20E-02 

4.91 E-03 

7.60E-03 

2.32E-03 

8.01 E-03 

Chamber Environment 

M 

Temperature 

("C) 

27 

25 

24 

24 

24 

24 

24 

25 

25 

25 

25 

25 

24 

23 

24 

23 

24 

25 

25 

24 

24 

25 

24 

24 

in 

Humidity 

(%I 

54 

53 

54 

52 

53 

58 

53 

51 

52 

55 

54 

50 

56 

52 

55 

55 

5 1 

5 1 

53 

52 

50 

47 

54 

52 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Chamber Monitoring Results 

Cumulative Exposure Record 

Day 

65 

66 

69 

70 

71 

72 

73 

76 

77 

78 

79 

80 

83 

84 

85 

86 

87 

89 

90 

91 

92 

Date 

18-Apr-02 

19-Apr-02 

22-Apr-02 

23-Apr-02 

24-Apr-02 

25-Apr-02 

26-Apr-02 

29-Apr-02 

30-Apr-02 

01-May-02 

02-May-02 

03-May-02 

06-May-02 

07-May-02 

08-May-02 

09-May-02 

10-May-02 

12-May-02 

13-May-02 

14-May-02 

15-May-02 

S.D. 

Exposure 

Number 

49 

50 

5 1 

52 

53 

54 

55 

56 

57 

58 

59 

60 

6 1 

62 

63 

64 

65 

66 

67 

68 

69 

Mean 

0 0 

Group IA - 0 mglm3 (Air Control) 

Nominal 

(mglm3) 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0.899 0.9 

Particle Size 

2.8 

Chamber Environment 

Mean 

(mg/m3) 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

MMAD 

(crm) 

1.767 

2.130 

1.325 

1.594 

1.975 

0.392 

Temperature 

("C) 

24 

24 

24 

25 

25 

25 

25 

25 

24 

24 

24 

24 

24 

24 

25 

24 

24 

25 

24 

22 

22 

24.3 

3.13E-03 

Mean 

Humidity 

("A) 

52 

49 

50 

54 

55 

52 

54 

49 

53 

53 

5 1 

54 

5 1 

52 

51 

51 

50 

50 

54 

53 

56 

53.3 

Determinations 

GSD 

2.858 

1.927 

1.605 

2.093 

2.1 32 

Analytical Chamber Concentration 

Individual TMC 

(m91m3) 

5.16E-03 

4.07E-03 

1.29E-03 

2.01 E-03 

4.84E-03 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

(mglm3) 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Chamber Monitoring Results 

Cumulative Exposure Record 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Date Exposure 

Number 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group IB - 0 mg/m3 (Air Control) 

Nominal 

(mg1m3) 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

1 

Mean 

(mg/m3) 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

~alytical Chamber Concentration 

Individual 

(ms 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

MMAD 

(pm) 

0.9933 

3.236 

1.240 

2.023 

1.877 

Particle Size 

?terminations 

GSD 

- - 

- 

TMC 

(mg1m3) 

1.41 E-02 

3.65E-03 

4.86E-03 

2.81 E-03 

9.96E-03 

Chamber 

rb 

Tem~erature 

wironment 

in 

Humiditv 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Chamber Monitoring Results 

Cumulative Exposure Record 

Grouo IB - 0 malm3 (Air Control) 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group HA - 2,000 mg/m3 

Date Exposure 

Number 

Nominal 

(mglm3) 

1940 

1990 

1950 

2150 

2010 

2090 

2060 

21 30 

2000 

2090 

1970 

1990 

2030 

2030 

2050 

1900 

21 00 

2090 

2150 

2020 

201 0 

2010 

2080 

21 60 

Analvtical Chamber Concentration 

Mean 

(mg/m3) 

1985 

1930 

1953 

2085 

1995 

2025 

1985 

I998 

1950 

1995 

I998 

I958 

201 0 

2035 

I998 

I865 

2000 

1915 

I983 

I928 

I883 

I988 

1905 

I968 

Individual 

(ms 

I860 

1910 

1990 

2170 

2000 

2010 

1980 

1940 

1610 

1900 

1910 

1960 

1980 

2050 

1980 

1730 

1990 

1750 

2030 

1820 

21 30 

2020 

1840 

1920 

MMAD 

(IJm) 

1.422 

1.182 

1.832 

1.506 

Particle Size 

Determinations 

Chamber 

l\i 

Temperature 

("C) 

23 

24 

23 

24 

24 

24 

24 

24 

24 

24 

25 

24 

23 

23 

24 

23 

23 

24 

23 

24 

23 

22 

23 

23 

~vironment 

In 

Humidity 

52 

53 

52 

50 

50 

52 

48 

48 

49 

48 

50 

48 

51 

52 

50 

42 

50 

55 

50 

52 

50 

49 

50 

50 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Date Exposure 

Number 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group IIA - 2,000 mg/m3 

Nominal 

(mg1m3) 

21 90 

21 90 

21 60 

21 30 

2180 

2050 

2090 

2030 

2170 

2110 

2110 

2090 

2030 

2130 

2060 

2220 

1980 

2040 

2050 

2060 

2060 

2060 

1910 

2000 

Mean 

(mg/m3) 

2068 

2205 

2095 

2040 

2108 

1913 

1918 

1913 

21 33 

2090 

2028 

I843 

1845 

1875 

2093 

2150 

1950 

I938 

1955 

1960 

1820 

I958 

2040 

2080 

alytical Chamber Concentration 

Individual 

(ms 

2050 

2190 

2060 

2030 

2040 

1940 

1900 

1760 

21 30 

2030 

2040 

1630 

1790 

1890 

21 30 

2030 

1810 

2030 

1950 

2030 

1890 

2110 

2020 

2020 

MMAD 

(IJm) 

0.794 

3.91 1 

0.8422 

1.438 

1.154 

Particle Size 

Determinations 

GSD TMC 

(mglm3) 

1.1 9E-02 

4.72E-03 

4.56E-03 

1.53E-03 

5.62E-03 

Chamber 

rv 

Temperature 

("C) 

26 

23 

23 

23 

23 

23 

23 

24 

24 

24 

24 

24 

23 

23 

23 

23 

23 

24 

24 

23 

23 

23 

23 

23 

ivironment 

an 

Humidity 

(%) 

50 

48 

50 

49 

50 

54 

51 

48 

48 

52 

50 

47 

52 

48 

51 

51 

48 

47 

49 

49 

48 

45 

51 

48 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Date Exposure 

Number 

49 

50 

5 1 

52 

53 

54 

55 

56 

57 

58 

59 

60 

61 

62 

63 

64 

65 

66 

67 

68 

69 

Mean 

S.D. 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group HA - 2,000 mglm3 

Nominal 

(mglm3) 

2010 

2140 

1930 

1970 

2060 

1970 

2060 

2060 

1900 

2090 

2040 

2070 

2060 

21 20 

2190 

1990 

2080 

21 30 

2230 

2330 

21 50 

2068 

83 

Mean 

(mglm3) 

2000 

21 08 

I885 

I963 

2045 

I875 

2020 

I985 

I898 

21 63 

2065 

201 0 

1958 

1935 

1890 

I808 

1915 

1943 

1945 

2015 

2023 

Analvtical Chamber Concentration 

Individual MMAD 

(IJm) 

1.072 

12.34 

1.351 

1.631 

2.344 

3.103 

Particle Size 

Determinations 

GSD TMC 

(mg1m3) 

3.00E-03 

9.96E-03 

1.34E-03 

2.36E-03 

4.51 E-03 

3.13E-03 

Chamber I 

M 

Temoerature 

~vironment 

an 

Humidity 

(%) 

49 

46 

47 

50 

51 

48 

49 

46 

49 

49 

47 

49 

47 

50 

49 

46 

48 

48 

51 

51 

52 

49.4 

2.1 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group IIB - 2,000 mg/m3 

Exposure 

Number 

Nominal I Analvtical Chamber concentration 

Mean 

(mg/m3) 

I963 

1943 

I983 

21 78 

21 35 

2063 

1943 

2035 

1903 

2070 

1938 

1993 

1975 

2050 

2113 

1918 

I998 

2028 

1958 

2023 

I963 

1973 

I985 

2020 

Individual MMAD 

(IJm) 

1.589 

1.378 

3.380 

1.174 

Particle Size 

1s 

TMC 

(mg/m3) 

3.46E-03 

5.03E-03 

3.14E-03 

2.54E-03 

Chamber I 

M 

Temperature 

("C) 

23 

23 

23 

23 

24 

23 

23 

23 

23 

23 

24 

23 

23 

23 

23 

23 

22 

23 

22 

23 

23 

22 

23 

23 

wironment 

In 

Humidity 

('w 

54 

56 

56 

55 

54 

55 

51 

50 

51 

49 

54 

52 

55 

55 

52 

43 

52 

56 

51 

53 

52 

52 

53 

52 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Date Exposure 

Number 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group IIB - 2,000 mglm3 

Nominal 

(mglm3) 

21 90 

2190 

2160 

21 30 

21 80 

2050 

2090 

2030 

21 70 

2110 

2110 

2090 

2030 

21 30 

2060 

2220 

1980 

2040 

2050 

2060 

2060 

2060 

1910 

2000 

Mean 

(mg/m3) 

2098 

2020 

2028 

2040 

2073 

1960 

I968 

1960 

I978 

1915 

I998 

21 30 

1960 

1970 

2098 

2055 

I858 

1868 

1943 

I985 

1970 

2000 

1903 

1920 

alytical Chamber Concentration 

Individual MMAD 

(IJm) 

0.8998 

0.9819 

0.8897 

1 A03 

1.101 

Particle Size 

Determinations 

GSD TMC 

(mg/m3) 

1.47E-02 

1.76E-03 

5.04E-03 

1.74E-03 

6.24E-03 

Chamber 

rv 

Temperature 

("C) 

25 

23 

23 

23 

23 

23 

23 

23 

23 

23 

23 

23 

23 

23 

23 

23 

23 

23 

23 

22 

22 

22 

23 

23 

~vironment 

In 

Humidity 

("A) 

52 

50 

52 

5 1 

5 1 

55 

52 

52 

50 

54 

53 

48 

54 

49 

54 

54 

49 

52 

52 

52 

50 

47 

53 

50 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Date Exposure 

Number 

49 

50 

51 

52 

53 

54 

55 

56 

57 

58 

59 

60 

61 

62 

63 

64 

65 

66 

67 

68 

Mean 

S.D. 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group IIB - 2,000 mg/m3 

Nominal I+ ~alytical Chamber Concentration 

Individual MMAD 

(elm) 

0.91 87 

1.358 

1.314 

1.510 

1.377 

0.647 

Particle Size 

?terminat 

GSD 

1s 

TMC 

(mg/m3) 

2.74E-03 

2.33E-03 

1.16E-03 

2.23E-03 

4.01 E-03 

3.54E-03 

Chamber Environment 

M 

Temperature 

("C) 

22 

23 

23 

23 

23 

22 

23 

23 

23 

23 

23 

23 

23 

22 

23 

22 

22 

22 

22 

22 

22.9 

0.5 

In 

Humidity 

52 

48 

48 

53 

55 

52 

5 1 

49 

52 

51 

50 

53 

48 

53 

51 

50 

50 

50 

53 

53 

51.8 

2.4 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Date Exposure 

Number 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group IIIA - 10,000 mglm3 

Nominal 

(mg/m3) 

9470 

9450 

9540 

9890 

9830 

9740 

10800 

991 0 

9950 

9840 

9990 

9920 

101 00 

101 00 

9200 

9890 

9730 

9860 

9960 

9810 

9890 

9700 

101 00 

9980 

Mean 

(mg/m3) 

9685 

9850 

9295 

10400 

1031 0 

9925 

9680 

lo380 

9758 

lo480 

9823 

9943 

lo650 

10700 

9935 

10200 

lo480 

10420 

10240 

10480 

10280 

9968 

10030 

9973 

~alytical Chamber Concentration 

Individual MMAD 

(IJm) 

0.9889 

1.043 

1.708 

2.245 

Particle Size 

Determinal 

GSD 

1s 

TMC 

(mglm3) 

3.59E-03 

5.74E-03 

2.65E-03 

4.77E-03 

Chamber Environment 

k 

Temperature 

("C) 

24 

24 

24 

24 

24 

24 

24 

24 

24 

24 

25 

24 

24 

24 

24 

23 

23 

24 

23 

24 

23 

22 

24 

23 

In 

Humidity 

(%) 

47 

48 

47 

47 

47 

50 

49 

51 

50 

47 

49 

47 

47 

47 

46 

39 

47 

5 1 

50 

47 

47 

49 

50 

50 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Chamber Monitoring Results 

Date 

Cumulative Exposure Record 

Group IIIA - 10,000 mglm3 

I 

Number Mean 

(mg/m3) 

lo260 

10000 

10320 

10010 

9970 

9475 

9450 

9773 

9670 

9930 

1001 0 

9790 

9970 

9730 

9745 

9098 

10450 

9828 

9945 

10730 

9475 

9743 

10590 

lo830 

Analvtical Chamber Concentration 

Individual MMAD 

Particle Size 

Determinations 

GSD TMC 

(mglm3) 

2.41 E-02 

2.91 E-03 

5.54E-03 

1.78E-03 

7.08E-03 

Chamber Environment 

M 

remperature 

("C) 

26 

24 

23 

23 

23 

23 

23 

24 

24 

24 

24 

24 

23 

23 

23 

23 

23 

24 

24 

24 

24 

24 

23 

23 

an 

Humidity 

("A) 

50 

47 

50 

50 

49 

50 

48 

48 

48 

48 

49 

47 

50 

48 

48 

49 

47 

49 

49 

49 

48 

46 

52 

50 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Date Exposure 

Number 

49 

50 

51 

52 

53 

54 

55 

56 

57 

58 

59 

60 

61 

62 

63 

64 

65 

66 

67 

68 

69 

Mean 

S.D. 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group IIIA - 10,000 mg/m3 

Nominal 

(mg/m3) 

101 00 

9470 

9550 

9780 

9780 

9940 

9680 

9620 

9680 

10200 

9730 

101 00 

10000 

9670 

9460 

9690 

9300 

9860 

981 0 

9710 

9500 

9827 

283 

Mean 

(mg/m3) 

10090 

9633 

lo280 

11100 

9233 

9770 

10090 

9488 

9468 

10010 

9400 

lo380 

9840 

9318 

9658 

9240 

9490 

9940 

9693 

9365 

10790 

alytical Chamber Concentration 

Individual MMAD 

(crm) 

1.618 

1.285 

1.243 

4.537 

1.534 

0.992 

Particle Size 

ns 

TMC 

(mg/m3) 

6.01 E-03 

1.84E-03 

1.17E-03 

3.25E-03 

5.42E-03 

5.91 E-03 

Chamber Environment 

N 

Temperature 

("C) 

23 

23 

23 

24 

24 

24 

24 

24 

23 

24 

23 

24 

24 

23 

24 

23 

23 

23 

23 

22 

22 

23.6 

0.7 

In 

Humidity 

("/.I 

50 

46 

47 

47 

47 

47 

47 

45 

48 

48 

48 

47 

47 

50 

49 

47 

48 

48 

51 

49 

49 

48.2 

1 .a 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Chamber Monitoring Results 

Date Exposure 

Number 

Cumulative Exposure Record 

Group lllB -10,000 mglm3 

Chamber Environment 

Particle Size Mean 

Nominal 

(mglm3) 

9470 

9450 

9540 

9890 

9830 

9740 

10800 

991 0 

9950 

9840 

9990 

9920 

101 00 

101 00 

9200 

9890 

9730 

9860 

9960 

981 0 

9890 

9700 

101 00 

9980 

Mean 

(mglm3) 

9930 

9925 

lo650 

I0270 

9998 

1001 0 

101 50 

101 50 

10240 

9480 

10120 

10740 

lo650 

1001 0 

9955 

10140 

9743 

101 70 

9590 

9655 

9995 

91 68 

10100 

9983 

Analytical Chamber Concentration I 

Individual I MMAD 

?terminat 

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

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Chamber Monitoring Results 

Date 

Cumulative Exposure Record 

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

Number 

Analvtical Chamber Concentration 

Mean I Individual 

Particle Size 

Chamber Environment 

M 

Temperature 

("C) 

26 

23 

24 

24 

24 

24 

24 

23 

23 

23 

24 

24 

24 

24 

24 

24 

24 

24 

23 

23 

23 

23 

24 

24 

In 

Humidity 

(%) 

49 

46 

46 

46 

46 

46 

45 

46 

46 

46 

47 

44 

47 

46 

45 

45 

45 

48 

46 

47 

46 

44 

47 

46 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Date Exposure 

Number 

49 

50 

51 

52 

53 

54 

55 

56 

57 

58 

59 

60 

6 1 

62 

63 

64 

65 

66 

67 

68 

Mean 

S.D. 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group lllB -10,000 mglm3 

Nominal 

(mglm3) 

101 00 

9470 

9550 

9780 

9780 

9940 

9680 

9620 

9680 

10200 

9730 

101 00 

10000 

9670 

9460 

9690 

9300 

9860 

981 0 

971 0 

9832 

282 

Mean 

(mg/m3) 

11000 

10070 

10090 

9758 

10650 

10240 

I0530 

9885 

1 1250 

11130 

10480 

lo650 

11180 

10350 

9923 

10500 

10180 

10550 

9650 

921 8 

alytical Chamber Concentration 

Individual MMAD 

(IJm) 

0.8013 

2.01 3 

1.661 

1.736 

1.547 

0.909 

Particle Size 

Determinations 

GSD TMC 

(mglm3) 

7.49E-03 

5.59E-03 

3.48E-03 

2.83E-03 

6.40E-03 

5.73E-03 

Chamber 

Fv 

Temperature 

("C) 

24 

24 

24 

24 

24 

23 

24 

24 

24 

24 

24 

25 

25 

23 

23 

23 

23 

23 

22 

23 

23.7 

0.7 

wironment 

In 

Humidity 

(%I 

47 

44 

44 

46 

46 

47 

46 

44 

45 

45 

45 

44 

44 

47 

47 

45 

46 

46 

49 

46 

45.9 

1.7 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Exposure 

Number 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group IVA - 20,000 mglm3 

Individual 

Nominal 

MMAD 

(~m) 

1.779 

3.198 

1.192 

1.358 

Analvtical Chamber Concentration 

Mean 

Particle Size 

Determinat 

GSD 

1s 

TMC 

(mg/m3) 

4.97E-03 

1.15E-02 

1.83E-03 

4.54E-03 

Chamber 

M 

Temperature 

("C) 

25 

25 

25 

25 

25 

25 

24 

24 

24 

24 

26 

25 

25 

25 

25 

24 

24 

25 

24 

25 

25 

22 

25 

25 

vironment 

n 

Humidity 

(%I 

48 

50 

50 

51 

49 

50 

50 

53 

5 1 

50 

53 

50 

50 

51 

49 

40 

49 

52 

52 

48 

49 

51 

51 

52 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Chamber Monitoring Results 

Date 

Cumulative Exposure Record 

Group IVA - 20.000 maim3 

Exposure 

Number 

Nominal 

(mg/m3) 

19400 

19000 

19300 

I9800 

19000 

19500 

19300 

19300 

19600 

I8400 

18600 

I8700 

18900 

18800 

18900 

19100 

18800 

I7600 

19700 

I8400 

19400 

I8700 

18900 

18700 

18800 

A 

Mean 

(mg/m3) 

19980 

19880 

19250 

19650 

19350 

20280 

19550 

20330 

20250 

19780 

19480 

20080 

20450 

20300 

19480 

20100 

19830 

19050 

20780 

20330 

20680 

20580 

20900 

20100 

20650 

~alytical Chamber Concentration 

Individual 

(ms 

20500 

20300 

19000 

20400 

19500 

20300 

1 9700 

201 00 

1 9700 

19800 

I8900 

19700 

20200 

19800 

19000 

19500 

19500 

18800 

20800 

20500 

21100 

20400 

21 000 

20000 

20800 

MMAD 

(vm) 

0.7739 

2.346 

0.8053 

1.220 

1.373 

Particle Size 

?terminat1 

GSD 

1s 

TMC 

(mgim3) 

2.44E-02 

4.30E-03 

7.67E-03 

1.09E-03 

1.12E-02 

Chamber Environment 

Mean 

Temperature 

("C) 

28 

25 

24 

24 

25 

25 

25 

25 

25 

25 

26 

26 

25 

24 

24 

24 

25 

25 

26 

25 

25 

25 

24 

24 

24 

Humidity 

(%I 

52 

48 

51 

50 

50 

51 

49 

49 

49 

48 

49 

47 

50 

52 

50 

50 

49 

50 

51 

49 

49 

48 

54 

5 1 

5 1 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Date Exposure 

Number 

15-May-02 I 69 

Mean 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group IVA - 20,000 mglm3 

Nominal 

(mglm3) 

I8400 

18600 

I8900 

I8700 

19000 

18800 

19000 

19000 

I8900 

18800 

18800 

19000 

191 00 

191 00 

18600 

18900 

191 00 

19500 

191 00 

18700 

19070 

51 3 

Mean 

(mglm3) 

20430 

20330 

21200 

20480 

20630 

21080 

19830 

20800 

20350 

20230 

201 00 

20930 

201 00 

201 30 

18550 

191 80 

18830 

19580 

19450 

19900 

~alytical Chamber Concentration 

Individual 

(ms 

21 100 

20500 

21100 

20300 

21 700 

20100 

20300 

21200 

20200 

20300 

1 9700 

20900 

19000 

20000 

191 00 

I8700 

18000 

19300 

19800 

Particle Size 

Determinations 

MMAD GSD TMC 

(elm) (mg/m3) 

0.9267 1.578 2.63E-03 

1.41 3 1.826 1.91 E-03 

3.580 2.863 6.05E-03 

1.597 1.681 1.85E-03 

Chamber I 

M 

Temperature 

("C) 

24 

24 

25 

25 

25 

25 

26 

24 

25 

24 

25 

25 

25 

25 

25 

24 

25 

24 

23 

23 

24.7 

0.8 

In 

Humidity 

(%) 

48 

48 

48 

49 

49 

49 

47 

49 

50 

50 

49 

48 

52 

51 

48 

49 

49 

52 

49 

51 

49.7 

1.9 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Date 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group IVB - 20,000 mg/m3 

, 

Number Mean 

(mg1m3) 

20000 

19200 

19900 

22680 

21 680 

21300 

20400 

20030 

20530 

21 200 

21 380 

20530 

20450 

20900 

21100 

21450 

19950 

21 000 

19650 

19350 

20350 

19400 

21 080 

20430 

alytical Chamber Concentration 

Individual 

Particle Size 

Determinations 

MMAD GSD TMC 

(IJm) 

1.134 1.917 

1.041 2.111 

1.147 1.782 

- 

Chamber 

Fv 

Temperature 

("C) 

24 

24 

24 

24 

24 

25 

25 

25 

25 

25 

25 

24 

24 

24 

24 

25 

25 

25 

25 

25 

24 

23 

24 

24 

wironment 

In 

Humidity 

(%) 

49 

50 

51 

52 

50 

49 

50 

52 

53 

50 

53 

51 

51 

50 

50 

40 

48 

5 1 

51 

47 

48 

51 

5 1 

51 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

Exposure 

Number 

Day 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group IVB - 20,000 mg/m3 

Date Nominal 

(mg/m3) 

19400 

19000 

I9300 

I9800 

I9000 

19500 

I9300 

19300 

19600 

I8400 

18600 

I8700 

18900 

18800 

I8900 

191 00 

18800 

I7600 

I9700 

I8400 

19400 

18700 

18900 

I8700 

Mean 

(mg/m3) 

20680 

20980 

20580 

20800 

20730 

20750 

21 030 

19850 

20380 

I9930 

19650 

18930 

18830 

19250 

19900 

19700 

19880 

18230 

20350 

201 50 

20200 

I9650 

20450 

19300 

Analvtical Chamber Concentration 

Individual 

(mo 

21200 

21 300 

20400 

20800 

20600 

20700 

21200 

20000 

20300 

20400 

20800 

18900 

I9000 

18900 

I9000 

20600 

I9300 

I8200 

20800 

201 00 

20800 

19200 

20800 

19200 

MMAD 

(elm) 

0.7578 

Particle Size F@% 

Determinations 1 Tern~erature 

GSD 

ivironment 

3n 

Humidity 

(%I 

52 

49 

50 

49 

49 

50 

48 

51 

50 

50 

50 

48 

49 

50 

49 

48 

48 

52 

50 

49 

49 

48 

53 

51 
Table A GASOLINE DlPE VAPOR CONDENSATE: A 13-WEEK 00-61 30 

WHOLE-BODY INHALATION TOXICITY STUDY IN RATS 

14-May-02 1 68 

Mean 

Date 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group IVB - 20,000 mglm3 

Exposure 

Number 

Nominal 

(mglm3) 

18800 

I8400 

18600 

18900 

18700 

I9000 

18800 

I9000 

I9000 

18900 

18800 

18800 

I9000 

191 00 

191 00 

18600 

18900 

191 00 

19500 

191 00 

19070 

51 5 

Mean 

(mglm3) 

20080 

18850 

19780 

19850 

19430 

19480 

I9930 

I9300 

I9030 

19900 

18800 

I9200 

I9750 

19680 

20330 

20000 

20000 

20350 

20100 

18880 

~alytical Chamber Concentration 

Individual 

Particle Size 

Chamber Environment 

In 

Humidity 

5 1 

48 

48 

51 

50 

50 

49 

48 

49 

49 

49 

49 

49 

52 

51 

49 

49 

49 

54 

49 

49.8 

1.9 
Table C 

LIONEL F. RUBIN, V.M.D. 

1 1 16 Saint Andrews Road 

Bryn Mawr, PA 1901 0 

(61 0) 520 9430 

February 2, 2002 

Huntingdon Life Sciences, Inc. 

Mettlers Road, Box 2360 

East Millstone, NJ 08875-2360 

Re: study A-1 2 00-61 30F 

Ophthalmoscopic examination of study A-1 2 00-61 30F pretest rats was performed 

January 31,2002. Rats with ocular abnormalities were identified and should be withdrawn 

from inclusion in the study if convenient. 

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

LIONEL F. RUBIN, V.M.D. 

1 1 16 Saint Andrews Road 

Bryn Maw, PA 1 901 0 

(61 0) 520 9430 

May 1 1,2002 

Huntingdon Life Sciences, Inc. 

Mettlers Road, Box 2360 

East Millstone, NJ 08875-2360 

Re: study A-1 2 00-61 30F 

Ophthalmoscopic examination of study A-I 2 00-61 30F rats was performed May 10, 

2002 (terminal examination). I have reviewed the findings of the type and incidence of 

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

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

test compound. 

Lionel F. Rubin, V. M. D. 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 1 

TABLE B 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

MALES SUMMARY OF CLINICAL OBSERVATIONS ................................................................................................................................... 

DAY OF STUDY 

GROUP# -11 92 TOTAL ................................................................................................................................... 

# OF ANIMALS EXAMINED 155 

2 5 5 

355 

4 5 5 

NORMAL 

WITHIN NORMAL LIMITS 

DEAD 

HUMANE SACRIFICE 

TERMINAL SACRIFICE 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 2 

TABLE B 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

FEMALES SUMMARY OF CLINICAL OBSERVATIONS 

DAY OF STUDY 

GROUP# -11 92 TOTAL 

# OF ANIMALS EXAMINED 155 

2 5 5 

3 5 5 

455 

NORMAL 

WITHIN NORMAL LIMITS 

DEAD 

TERMINAL SACRIFICE 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 1 

TABLE C 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

SUMMARY OF OPHTHALMOLOGY OBSERVATIONS 

DAY - 12 

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

DOSE GROUP: 1 2 3 4 

DOSE LEVEL (MG/M3) : 0 2000 10000 20000 ................................................................................................................................... 

MALES total number examined 5 5 5 5 

NO ABNORMALITIES DETECTED 

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

No statistically significant differences 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 2 

TABLE C 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

SUMMARY OF OPHTHALMOLOGY OBSERVATIONS 

DAY -12 

DOSE GROUP: 1 2 3 4 

DOSE LEVEL (MG/M3) : 0 2000 10000 20000 ................................................................................................................................... 

FEMALES total number examined 

NO ABNORMALITIES DETECTED 

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

No statistically significant differences 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 3 

TABLE C 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

DOSE GROUP: 1 2 3 4 

DOSE LEVEL (MG/M3 ) : 0 2000 10000 20000 ................................................................................................................................... 

MALES total number examined 5 4 5 5 

FOCAL RETINOPATHY N 1 0 0 0 

% 20.0 0.0 0.0 0.0 ................................................................................................................................... 

No statistically significant differences 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 4 

TABLE C 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

SUMMARY OF OPHTHALMOLOGY OBSERVATIONS 

DAY 87 

FEMALES total number examined 

NO ABNORMALITIES DETECTED 

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

No statistically significant differences 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 1 

TABLE D 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

MALES MEAN BODY WEIGHTS (GRAMS) ................................................................................................................................... 

DOSE GROUP: 1 2 3 4 

DOSE LEVEL (MG/M3 I : 0 2000 10000 20000 ---------------------------------------------------------------------------------

WEEK 

WEEK 

WEEK 1 

WEEK 2 

WEEK 

WEEK 

WEEK 

WEEK 

WEEK 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N ................................................................... 

No statistically significant differences 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 2 

TABLE D 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

MALES MEAN BODY WEIGHTS (GRAMS) ................................................................................................................................... 

DOSE GROUP: 1 2 3 4 

DOSE LEVEL (MG/M3 ) : 0 2000 10000 20000 

WEEK 8 MEAN 

S.D. 

N 

WEEK 9 MEAN 

S.D. 

N 

WEEK 10 MEAN 

S.D. 

N 

WEEK 11 MEAN 

S.D. 

N 

WEEK 12 MEAN 

S.D. 

N 

WEEK 13 MEAN 

S.D. 

N 

No statistically significant differences 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 3 

TABLE D 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AM) 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

FEMALES MEAN BODY WEIGHTS (GRAMS) 

WEEK -1 MEAN 

S.D. 

N 

WEEK 0 MEAN 

S.D. 

N 

WEEK 1 MEAN 

S.D. 

N 

WEEK 2 MEAN 

S.D. 

N 

WEEK 3 

WEEK 4 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

WEEK 5 

WEEK 6 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

WEEK 7 MEAN 306 289 276 275 

S.D. 27.5 35.2 16.9 13.7 

N 5 5 5 5 ................................................................................................................................... 

No statistically significant differences 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 4 

TABLE D 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

FEMALES MEAN BODY WEIGHTS (GRAMS) ................................................................................................................................... 

DOSE GROUP: 1 2 3 4 

DOSE LEVEL(MG/M3): 0 2000 10000 20000 ................................................................................................................................... 

WEEK 8 MEAN 

S.D. 

N 

WEEK 9 MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

WEEK 10 

WEEK 11 MEAN 

S.D. 

N 

WEEK 12 

WEEK 13 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N .............................................. 

No statistically significant differences 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 1 

TABLE E 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

MALES MEAN BODY WEIGHT CHANGE FROM BASELINE (GRAMS) ................................................................................................................................... 

DOSE GROUP: 1 2 3 4 

DOSE LEVEL (MG/M3 ) : 0 2000 10000 20000 ................................................................................................................................... 

WEEK 

WEEK 

WEEK 0 TO 3 

WEEK 0 TO 4 

WEEK 

WEEK 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N ................................................................................... 

No statistically significant differences 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 2 

TABLE E 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

WEEK 0 TO 7 MEAN 

S.D. 

N 

WEEK 0 TO 8 

WEEK 0 TO 9 

WEEK 0 TO 10 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

WEEK 0 TO 11 MEAN 

S.D. 

N 

WEEK 0 TO 12 

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

MEAN 

S.D. 

N 

No statistically significant differences 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 3 

TABLE E 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

WEEK 0 TO 13 MEAN 259 268 289 256 

S.D. 42.5 17.5 36.3 25.9 

N 5 4 5 5 ................................................................................................................................... 

No statistically significant differences 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 4 

TABLE E 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

FEMALES MEAN BODY WEIGHT CHANGE FROM BASELINE (GRAMS) ................................................................................................................................... 

DOSE GROUP: 1 2 3 4 

DOSE LEVEL (MG/M3 ) : 0 2000 10000 20000 ................................................................................................................................... 

WEEK 

WEEK 

WEEK 0 TO 3 

WEEK 0 TO 4 

WEEK 

WEEK 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N ....................................................................................... 

Statistical key: * = pc0.05 ** = pc0.01 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 5 

TABLE E 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

FEMALES MEAN BODY WEIGHT CHANGE FROM BASELINE (GRAMS) ................................................................................................................................... 

WEEK 0 TO 7 MEAN 

S.D. 

N 

WEEK 0 TO 8 MEAN 

S.D. 

N 

WEEK 0 TO 9 

WEEK 0 TO 10 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

WEEK 0 TO 11 MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 6 

TABLE E 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

WEEK 0 TO 13 MEAN 

S.D. 

N 

No statistically significant differences 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE I 

TABLE F 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

WEEK 4 

WEEK 5 

WEEK 6 

WEEK 7 

WEEK 

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

No statistically significant differences 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 2 

TABLE F 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

WEEK 9 MEAN 

S.D. 

N 

WEEK 10 MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

WEEK 11 

WEEK 12 MEAN 

S.D. 

N 

WEEK 13 MEAN 46 4 4 

S.D. 1.8 1.1 

N 5 4 .................................................................................... 

No statistically significant differences 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 3 

TABLE F 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

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

DOSE GROUP: 1 2 3 4 

DOSE LEVEL (MG/M3 ) : 0 2000 10000 20000 ................................................................................................................................... 

WEEK 

WEEK 

WEEK 

WEEK 

WEEK 4 

WEEK 5 

WEEK 6 

WEEK 7 

WEEK 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

MEAN 

S.D. 

N ................................................................................................................................... 

Statistical key: * = peO.05 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 4 

TABLE F 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

IMiALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

WEEK 9 MEAN 

S.D. 

N 

WEEK 10 MEAN 

S.D. 

N 

MEAN 

S.D. 

N 

WEEK 11 

WEEK 12 MEAN 

S.D. 

N 

WEEK 13 MEAN 

S.D. 

N .................................................. 

No statistically significant differences 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 1 

TABLE G 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL CLINICAL OBSERVATIONS 

MALES GROUP 1 0 MG/M3 

ANIMAL# OBSERVATIONS 

DAY OF 19 

STUDY 12 

1076 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

1077 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

1078 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

1079 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

1080 WITHIN NORMAL LIMITS P 

TERMINAL SACRIFICE P ................................................................................................................................... 

CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

MALES 

----- 

PAGE 2 

TABLE G 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL CLINICAL OBSERVATIONS 

GROUP 2 2000 MG/M3 

DAY OF 19 

ANIMAL# OBSERVATIONS STUDY 12 ................................................................................................................................... 

2066 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

2067 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

2068 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

2069 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

2070 WITHIN NORMAL LIMITS ............................................... 

CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 3 

TABLE G 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

ANIMAL# OBSERVATIONS 

DAY OF 19 

STUDY 12 

3066 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

3067 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

3068 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

3069 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

3070 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE .......................................................... 

CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 4 

TABLE G 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL CLINICAL OBSERVATIONS 

MALES GROUP 4 20000 MG/M3 

4076 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

4077 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

4078 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

4079 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

4080 WITHIN NORMAL LIMITS P 

TERMINAL SACRIFICE P ................................................................................................................................... 

CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 5 

TABLE G 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL CLINICAL OBSERVATIONS 

FEMALES GROUP 1 0 MG/M3 

ANIMAL# OBSERVATIONS 

DAY OF 19 

STUDY 12 

1576 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

1577 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

1578 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

1579 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

1580 WITHIN NORMAL LIMITS P 

TERMINAL SACRIFICE P ................................................................................................................................... 

CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

TABLE G 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

PAGE 6 

INDIVIDUAL CLINICAL OBSERVATIONS 

FEMALES GROUP 2 2000 MG/M3 

ANIMAL# OBSERVATIONS 

DAY OF 19 

STUDY 12 

2566 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

2567 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

2568 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

2569 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

2570 WITHIN NORMAL LIMITS P 

TERMINAL SACRIFICE P ................................................................................................................................... 

CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

FEMALES GROUP 3 ---------------- 

PAGE 7 

TABLE G 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

3566 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

3567 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

3568 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

3569 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

3570 WITHIN NORMAL LIMITS P 

TERMINAL SACRIFICE P ................................................................................................................................... 

CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 8 

TABLE G 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL CLINICAL OBSERVATIONS 

FEMALES GROUP 4 20000 MG/M3 ................................................................................................................................... 

DAY OF 19 

ANIMAL# OBSERVATIONS STUDY 12 ................................................................................................................................... 

4576 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

4577 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

4578 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

4579 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

4580 WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

MALES GROUP 1 0 MG/M~ 

TABLE H 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

PAGE 1 

INDIVIDUAL OPHTHALMOLOGY OBSERVATIONS 

DAY -12 

1076 NO VISIBLE LESIONS 

1077 NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

1079 NO VISIBLE LESIONS 

1080 NO VISIBLE LESIONS ................................................................................................................................... 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 2 

TABLE H 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

2066 NO VISIBLE LESIONS 

2067 NO VISIBLE LESIONS 

2068 NO VISIBLE LESIONS 

2069 NO VISIBLE LESIONS 

2070 NO VISIBLE LESIONS ................................................................................................................................... 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 3 

TABLE H 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 4 

TABLE H 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

4076 NO VISIBLE LESIONS 

4077 NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

4079 NO VISIBLE LESIONS 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

TABLE H 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL OPHTHALMOLOGY OBSERVATIONS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

PAGE 5 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 6 

TABLE H 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

2566 NO VISIBLE LESIONS 

2567 NO VISIBLE LESIONS 

2568 NO VISIBLE LESIONS 

2569 NO VISIBLE LESIONS 

2570 NO VISIBLE LESIONS ................................................................................................................................... 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 7 

TABLE H 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AM) IMMUNOTOXICITY ASSESSMENTS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

TABLE H 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

FEMALES GROUP 4 20000 MG/M3 

INDIVIDUAL OPHTHALMOLOGY OBSERVATIONS 

OBSERVATION ---------------------------------------------------------------

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

PAGE 8 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

MALES GROUP 1 0 MG/M3 

TABLE H 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

PAGE 9 

INDIVIDUAL OPHTHALMOLOGY OBSERVATIONS 

DAY 87 

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

1076 NO VISIBLE LESIONS 

1077 RETINA FOCAL RETINOPATHY; LEFT 

1078 NO VISIBLE LESIONS 

1079 NO VISIBLE LESIONS 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 10 

TABLE H 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

2066 NO VISIBLE LESIONS 

2067 NO VISIBLE LESIONS 

2068 NO VISIBLE LESIONS 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 11 

TABLE H 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

TABLE H 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL OPHTHALMOLOGY OBSERVATIONS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

PAGE 12 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

FEMALES GROUP 1 0 MG/M3 

TABLE H 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

PAGE 13 

INDIVIDUAL OPHTHALMOLOGY OBSERVATIONS 

DAY 87 

1576 NO VISIBLE LESIONS 

1577 NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

1579 NO VISIBLE LESIONS 

1580 NO VISIBLE LESIONS ................................................................................................................................... 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 14 

TABLE H 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

2566 NO VISIBLE LESIONS 

2567 NO VISIBLE LESIONS 

2568 NO VISIBLE LESIONS 

2569 NO VISIBLE LESIONS 

2570 NO VISIBLE LESIONS ................................................................................................................................... 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 15 

TABLE H 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

3566 NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

3568 NO VISIBLE LESIONS 

3569 NO VISIBLE LESIONS 

3570 NO VISIBLE LESIONS ................................................................................................................................... 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 16 

TABLE H 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL OPHTHALMOLOGY OBSERVATIONS 

FEMALES GROUP 4 20000 MG/M3 DAY 87 

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

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 

NO VISIBLE LESIONS 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 1 

TABLE I 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL BODY WEIGHTS (GRAMS) 

MALES GROUP 1 0 MG/M3 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 2 

TABLE I 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL BODY WEIGHTS (GRAMS) 

MALES GROUP 2 2000 MG/M3 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 3 

TABLE I 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL BODY WEIGHTS (GRAMS) 

MALES GROUP 3 10000 MG/M3 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 4 

TABLE I 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL BODY WEIGHTS (GRAMS) 

MALES GROUP 4 20000 MG/M3 

WEEK OF STUDY 

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

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

TABLE I 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL BODY WEIGHTS (GRAMS) 

FEMALES GROUP 1 0 MG/M~ 

WEEK OF STUDY 

ANIMAL# - 1 0 1 2 3 4 5 6 7 8 9 10 ................................................................................................... 

MEAN 

S.D. 

N 

PAGE 5 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

TABLE I 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL BODY WEIGHTS (GRAMS) 

FEMALES GROUP 2 2000 MG/M~ 

WEEK OF STUDY 

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

MEAN 

S.D. 

N 

PAGE 6 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 7 

TABLE I 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL BODY WEIGHTS (GRAMS) 

FEMALES GROUP 3 10000 MG/M3 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

TABLE I 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL BODY WEIGHTS (GRAMS) 

FEMALES GROUP 4 20000 MG/M3 .................................... 

WEEK OF STUDY 

ANIMAL# - 1 0 1 

PAGE 8 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 1 

TABLE J 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL BODY WEIGHT CHANGE FROM BASELINE (GRAMS) 

MALES GROUP 1 0 MG/M3 ................................................................................................................................... 

WEEK OF STUDY 

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

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 2 

TABLE J 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL BODY WEIGHT CHANGE FROM BASELINE (GRAMS) 

MALES GROUP 2 2000 MG/M3 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 3 

TABLE J 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL BODY WEIGHT CHANGE FROM BASELINE (GRAMS) 

MALES GROUP 3 10000 MG/M3 

WEEK OF STUDY 

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

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

MEAN 

S.D. 

N 

TABLE J 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

PAGE 4 

INDIVIDUAL BODY WEIGHT CHANGE FROM BASELINE (GRAMS) 

20000 MG/M3 .................................................................................................................. 

WEEK OF STUDY 

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

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

4 6 82 110 143 160 177 198 215 228 254 269 287 289 

3 5 62 88 110 131 147 168 184 194 206 221 230 241 

36 63 91 119 127 138 165 181 195 215 219 234 229 

3 7 73 105 128 149 176 204 218 242 255 270 279 278 

36 6 6 93 121 144 157 174 193 204 221 230 242 244 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 5 

TABLE J 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL BODY WEIGHT CHANGE FROM BASELINE (GRAMS) 

FEMALES GROUP 1 0 MG/M3 ................................................................................................................................... 

WEEK OF STUDY 

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

1576 23 48 55 6 6 78 78 86 9 1 91 102 103 105 100 

1577 19 3 2 3 7 46 56 53 5 9 67 73 75 75 8 5 88 

1578 15 4 0 5 3 59 7 4 8 0 8 9 83 90 99 107 107 103 

1579 34 57 6 6 95 107 104 117 148 150 149 151 165 154 

1580 17 3 7 5 2 56 6 1 69 87 79 90 8 6 91 93 87 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 6 

TABLE J 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL BODY WEIGHT CHANGE FROM BASELINE (GRAMS) 

FEMALES GROUP 2 2000 MG/M3 ................................................................................................................................... 

WEEK OF STUDY 

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

2566 17 27 3 0 29 3 9 4 0 44 42 5 6 6 4 66 6 1 66 

2567 19 3 1 50 60 68 7 5 79 85 94 93 100 107 101 

2568 22 3 7 64 8 4 87 8 1 95 96 106 102 112 122 117 

2569 25 3 7 4 1 4 9 65 68 66 7 9 86 9 4 88 96 98 

2570 17 3 8 45 55 7 0 72 85 87 90 93 101 110 103 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 7 

TABLE J 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 8 

TABLE J 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL BODY WEIGHT CHANGE FROM BASELINE (GRAMS) 

FEMALES GROUP 4 20000 MG/M~ 

WEEK OF STUDY 

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

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 1 

TABLE K 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 2 

TABLE K 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL FEED CONSUMPTION VALUES (GRAMS/KG/DAY) 

MALES GROUP 2 2000 MG/M3 

WEEK OF STUDY 

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

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 3 

TABLE K 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL FEED CONSUMPTION VALUES (GRAMS/KG/DAY) 

MALES GROUP 3 10000 MG/M3 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 4 

TABLE K 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL FEED CONSUMPTION VALUES (GRAMS/KG/DAY) 

MALES GROUP 4 20000 MG/M3 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 5 

TABLE K 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 6 

TABLE K 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL FEED CONSUMPTION VALUES (GRAMS/KG/DAY) 

FEMALES GROUP 2 2000 MG/M3 ................................................................................................................................... 

WEEK OF STUDY 

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

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 7 

TABLE K 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

INDIVIDUAL FEED CONSUMPTION VALUES (GRAMS/KG/DAY) 

FEMALES GROUP 3 10000 MG/M3 

MEAN 

S.D. 

N 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 8 

TABLE K 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

iLES GROUP 4 2 

INDIVIDUAL FEED CONSUMPTION VALUES (GRAMS/KG/DAY) 

0000 MG/M3 ................................................................................................................................... 

WEEK OF STUDY 

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

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

4576 90 7 9 7 3 70 6 4 6 4 66 63 6 0 62 6 0 6 0 59 61 

4577 SF 9 7 8 7 79 75 7 4 6 9 6 7 68 70 67 6 5 64 6 1 

4578 103 8 1 80 85 75 73 71 71 64 69 77 71 63 56 

4579 SF 84 8 1 7 9 6 9 7 0 7 4 69 67 63 66 6 4 6 5 55 

4580 96 79 72 70 66 65 66 67 63 62 6 1 59 63 56 

MEAN 

S.D. 

N 

SF=Spilled Feeder 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 1 

TABLE L 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

ANIMAL TERMINATION HISTORY 

MALES GROUP 1 0 MG/M~ ................................................................................................................................... 

TYPE OF DATE OF WEEK OF STUDY 

ANIMAL# DEATH DEATH STUDY DAY ................................................................................................................................... 

1076 TERMINAL SACRIFICE 15-MAY-02 13 92 

1077 TERMINAL SACRIFICE 15-MAY-02 13 9 2 

1078 TERMINAL SACRIFICE 15-MAY-02 13 92 

1079 TERMINAL SACRIFICE 15-MAY-02 13 92 

1080 TERMINAL SACRIFICE 15-MAY-02 13 92 ................................................................................................................................... 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 2 

TABLE L 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

ANIMAL TERMINATION HISTORY 

MALES GROUP 2 2000 MG/M3 ................................................................................................................................... 

TYPE OF DATE OF WEEK OF STUDY 

AN I MAL# DEATH DEATH STUDY DAY 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 3 

TABLE L 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

ANIMAL TERMINATION HISTORY 

MALES GROUP 3 10000 MG/M3 ................................................................................................................................... 

TYPE OF DATE OF WEEK OF STUDY 

ANIMAL# DEATH DEATH STUDY DAY ................................................................................................................................... 

3066 TERMINAL SACRIFICE 15-MAY-02 13 92 

3067 TERMINAL SACRIFICE 15-MAY-02 13 9 2 

3068 TERMINAL SACRIFICE 15-MAY-02 13 9 2 

3069 TERMINAL SACRIFICE 15-MAY-02 13 9 2 

3070 TERMINAL SACRIFICE 15 -MAY - 02 13 9 2 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 4 

TABLE L 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

ANIMAL TERMINATION HISTORY 

MALES GROUP 4 20000 MG/M3 

TYPE OF 

DEATH 

DATE OF WEEK OF STUDY 

DEATH STUDY DAY ................................................................................................................................... 

4076 TERMINAL SACRIFICE 15-MAY-02 13 9 2 

4077 TERMINAL SACRIFICE 15-MAY-02 13 9 2 

4078 TERMINAL SACRIFICE 15-MAY-02 13 9 2 

4079 TERMINAL SACRIFICE 15-MAY-02 13 9 2 

4080 TERMINAL SACRIFICE 15-MAY-02 13 9 2 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 5 

TABLE L 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

ANIMAL TERMINATION HISTORY 

FEMALES GROUP 1 0 MG/M3 

TYPE OF DATE OF WEEK OF STUDY 

ANIMAL# DEATH DEATH STUDY DAY ................................................................................................................................... 

1576 TERMINAL SACRIFICE 15-MAY-02 13 92 

1577 TERMINAL SACRIFICE 15-MAY-02 13 92 

1578 TERMINAL SACRIFICE 15 -MAY - 02 13 9 2 

1579 TERMINAL SACRIFICE 15-MAY-02 13 92 

1580 TERMINAL SACRIFICE 15-MAY-02 13 9 2 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 6 

TABLE L 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

ANIMAL TERMINATION HISTORY 

FEMALES GROUP 2 2000 MG/M3 

TYPE OF 

DEATH 

DATE OF WEEK OF STUDY 

DEATH STUDY DAY ................................................................................................................................... 

2566 TERMINAL SACRIFICE 15-MAY-02 13 9 2 

2567 TERMINAL SACRIFICE 15-MAY-02 13 9 2 

2568 TERMINAL SACRIFICE 15-MAY-02 13 9 2 

2569 TERMINAL SACRIFICE 15-MAY-02 13 9 2 

2570 TERMINAL SACRIFICE 15-MAY-02 13 9 2 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 7 

TABLE L 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

ANIMAL TERMINATION HISTORY 

FEMALES GROUP 3 10000 MG/M~ ................................................................................................................................... 

TYPE OF DATE OF WEEK OF STUDY 

ANIMAL# DEATH DEATH STUDY DAY 
Huntingdon Life Sciences 00-6130F 

211-DIPE-S 

PAGE 8 

TABLE L 

GASOLINE DIPE VAPOR CONDENSATE: A 13-WEEK WHOLE-BODY 

INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS 

AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS 

ANIMAL TERMINATION HISTORY 

FEMALES GROUP 4 20000 MG/M~ ................................................................................................................................... 

TYPE OF DATE OF WEEK OF STUDY 

ANIMAL# DEATH DEATH STUDY DAY 

4576 TERMINAL SACRIFICE 

4577 TERMINAL SACRIFICE 

4578 TERMINAL SACRIFICE 

4579 TERMINAL SACRIFICE 

4580 TERMINAL SACRIFICE