Data Requirement 

EPA 79.61 and OPPTS 870.3700 and OECD 414 

STUDY NO. 05-4287 

SPONSOR STUDY NO. 211-DIPE-DEV 

GASOLINE DIPE VAPOR CONDENSATE: 

EMBRYO-FETAL TOXICITY STUDY IN RATS BY 

INHALATION EXPOSURE 

2nd Draft Final Report 

Author: Gary M. Hoffman, B.A., D.A.B.T. 

Performed by: Huntingdon Life Sciences (HLS) 

1 00 Mettlers Road 

East Millstone, New Jersey 08875-2360 

Submitted to: American Petroleum Institute (API) 

1220 L Street, Northwest 

Washington, D.C. 20005-4070 

Attn: Thomas M. Gray, M.S., D.A.B.T. 

Date: 2 December 2011 

Page 1 ofxxx 
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STATEMENT OF COMPLIANCE 

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Draft Final Report 

This study was conducted in accordance with US EPA 79.60, CFR Vol. 59, No. 122, 27 

June 1994. This study was performed according to protocol and Huntingdon Life 

Sciences' Standard Operating Procedures. 

It was the Sponsor's responsibility to maintain the method of synthesis, fabrication, or 

derivation of the test fuel, and this was not completed at the time of the study conduct but 

has been completed since and is on file with the Sponsor. In addition, the Sponsor 

conducted a non-GLP purity analysis. 

Gary M. Hoffman, B.A., D.A.B.T. 

Study Director 

Thomas M. Gray, M.S., D.A.B.T. 

Sponsor Representative 

Date 

Date 
Huntingdon Life Sciences 

SCIENTISTS 

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SIGNATURE PAGE 

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Draft Final Rwort 

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

Departmental supervisory personnel are listed on the personnel page of this report 

(Appendix Q). 

Gary M. Hoffman, B.A., D.A.B.T. 

Study Director 

SCIENTIFIC REVIEW 

The following Scientist has reviewed and approved this report: 

Robert M. Parker, Ph.D., D.A.B.T. 

Director, Developmental and Reproductive Toxicology 

Date 

Date 
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QUALITY ASSURANCE STATEMENT 

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Listed below are the dates that this study was inspected by the Quality Assurance Unit of 

Huntingdon Life Sciences, East Millstone, New Jersey, and the dates that findings were 

reported to the Study Director and Management. 

Type of Inspection 

GLP Protocol Review 

Exposure Monitoring and 

Equipment Records 

Formulation Chemistry: Inhalation 

Dose Analysis 

Terminal Necropsy 

Fetal Soft Tissue Evaluations and 

Training Records 

Exposure Data and Report 

Final Report and Study Data 

Protocol Amendments 1 and 2 

Pathology Associates Facility 

Inspection 

Protocol and Draft Protocol 

Amendment 4a 

Final Protocol Amendment 4a 

Protocol Amendment 3 

Protocol Amendment 4 

Date(s) of Inspection 

21-25 Apr 05 

8 Aug05 

16 Aug 05 

22 Aug 05 

17 Oct 05 

19-20 Dec 05 

14 Dec 05- 04-Jan-06 

12 Jan 06 

13 Jul 06 

03 May 11 

25 May 11 

10Nov 08 

01 Dec 11 

Fran Jannone, B.A., RQAP-GLP 

Quality Assurance Group Leader 

Reported to 

Study Director 

and Management 

25 Apr05 

11 Aug05 

16 Aug 05 

23 Aug05 

21 Oct 05 

20 Dec 05 

06-Jan-06 

12 Jan 06 

18 Oct 06 

05 May 11 

25 May 11 

10Nov 08 

01 Dec 11 

Date 

a Audits performed by Huntingdon Life Sciences-UK Quality Assurance and reported to 

Principal Investigator and Management on the date of inspection and Study Director and 

Management on the dates indicated above. 
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GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY 

STUDY IN RATS VIA INHALATION EXPOSURES 

SUMMARY 

This study was designed to assess the potential maternal and/or developmental toxicity of 

Gasoline DIPE Vapor Condensate in the pregnant rat when administered by whole-body 

inhalation exposure. The study design permitted evaluation of maternal toxicity as 

revealed by clinical, body weight and food consumption changes and by gross necropsy 

findings, and evaluation of the developing conceptus, including death, structural 

abnormalities, or altered growth. This particular study is a near replicate of a previous 

study conducted by ExxonMobil Biomedical Sciences, Inc. (EMBSI Study Number 

171734). The previous study was incomplete due to technical errors made in evaluating 

the fetuses. The Sponsor needed to repeat the previous study to satisfy the Environmental 

Protection Agency's Alternative Tier 2 Testing Requirements under Sections 211(b) (2) 

of the Clean Air Act. 

The test substance was administered once daily as a vapor to pregnant Sprague Dawley 

CD® female rats (24/group) at target concentrations of2000, 10000 and 20000 mg/m3 for 

6 hours/day, 7 days per week for Gestation Days (GD) 5-20. In addition, a control group 

(24/sex) received nitrogen enriched air only while in chamber. Exposure levels were 

determined 4 times per chamber per day. Particle size distribution measurements were 

also made once per chamber per week. The following parameters were evaluated: 

viability, clinical observations, body weights and feed consumption. A cesarean section 

was performed on GD 21. The animals were subjected to a macroscopic postmortem 

evaluation and corpora lutea/implantation data were recorded and the gravid uterus was 

weighed. The fetuses were examined for externally visible abnormalities and weighed. 

Approximately one-half of the fetuses (alternating fetuses) in each litter were examined 

for soft-tissue abnormalities using a microdissection procedure. The other half of the 

fetuses were then stained with Alizarin Red S and examined for skeletal abnormalities 

and ossification variations. 

The overall mean(± standard deviation) analytical exposure concentrations for the control 

and the respective exposure groups were as follows: 0 ± 0, 1982 ± 93, 10072 ± 546, 

19776 ± 961 mg/m3

. The exposure regimen resulted in only transient reductions in 

maternal weight gain (approximately 12-25% reduced from Control at GD 11-14 interval, 

partly resolving by GD 21) and feed consumption (up to approximately 10%) during the 

exposure period. These changes were negligible at 2000 mg/m3 and most noticeable at 

20000 mg/m3

. It was considered that a no-observed-adverse-effect level (NOAEL) for 

maternal effects was evident at 2000 mg/m3.There was no effect of the exposures at any 

level on pregnancy outcome, in terms of pre- or post-implantation loss, fetal weight or the 

incidence of fetal abnormalities and variants. A NOAEL for embryo-fetal effects was 

therefore established at 20000 mg/m3

. 
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TABLE OF CONTENTS 

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COVER PAGE ..................................................................................................................... 1 

STATEMENT OF COMPLIANCE ..................................................................................... 2 

SIGNATURE P AGE ............................................................................................................ 3 

QUALITY ASSURANCE STATEMENT .......................................................................... 4 

SUMMARY ......................................................................................................................... 5 

TABLE OF CONTENTS ..................................................................................................... 6 

1. INTRODUCTION .................................................................................................. 8 

2. MATERIALS AND METHODS 

2.1. Study Management ...................................................................................... 8 

2.2. Study Dates .................................................................................................. 9 

2.3. Experimental Outline ................................................................................. 10 

2.4. Justifications .............................................................................................. 1 0 

2.5. Test Substance ........................................................................................... 11 

2.6. Test Animals .............................................................................................. 13 

2.7. Animal Assignment ................................................................................... 14 

2.8. Animal Identification ................................................................................. 14 

2.9. VeterinaryCare .......................................................................................... 14 

2.10. Animal Husbandry During Non-Exposure Periods .................................... 15 

2.11. Test Substance Administration and Chamber Operation ........................... 17 

2.12. Experimental Evaluations .......................................................................... 17 

2.13. Postmortem Evaluations ............................................................................ 18 

2.14. Fetal Evaluations ........................................................................................ 19 

2.15. Statistical Analysis ..................................................................................... 20 

2.16. Data Storage ............................................................................................... 21 

2.17. Regulatory References ............................................................................... 21 

2.18. Protocol Deviations .................................................................................... 22 

3. RESULTS AND DISCUSSION 

3 .1. Test Substance Analysis ............................................................................. 23 

3 .2. Chamber Monitoring .................................................................................. 23 

3.3. Maternal Data ............................................................................................. 24 

3.4. Fetal Data ................................................................................................... 26 

4. CONCLUSIONS .................................................................................................. 27 

REFERENCES .................................................................................................................. 28 

CALCULATIONS ............................................................................................................. 29 
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GENERAL PREFACE ...................................................................................................... 30 

FIGURES 

1. Mean Gestation Body Weights ....................................................................................... . 

2. Mean Gestation Feed Consumption ................................................................................ . 

TABLES 

1. Summary of Survival and Pregnancy .............................................................................. . 

2. Summary of Clinical Observations During Gestation .................................................... . 

3. Summary of Gestation Body Weights (grams) ............................................................... . 

4. Summary of Gestation Body Weight Gain (grams) ........................................................ . 

5. Summary of Gestation Feed Consumption (grams/kg/day) ............................................ . 

6. Summary of Maternal Necropsy Observations ............................................................... . 

7. Summary of Cesarean Section Data ................................................................................ . 

8. Summary of Gravid Uterine Weight and Net Body Weight Change (grams) ................. . 

9. Summary of Fetal Observations ...................................................................................... . 

APPENDICES 

A. Analytical Report ............................................................................................................ . 

B. Inhalation Report ............................................................................................................. . 

C. Individual Animal Termination History .......................................................................... . 

D. Individual Clinical Observations During Gestation ........................................................ . 

E. Individual Gestation Body Weights (grams) ................................................................... . 

F. Individual Gestation Body Weight Gain (grams) ........................................................... . 

G. Individual Gestation Feed Consumption (grams/kg/day) ............................................... . 

H. Individual Maternal Necropsy Observations ................................................................... . 

I. Individual Female Reproduction Data and Mean Fetal Weights .................................... . 

J. Individual Gravid Uterine Weight and Net Body Weight Change (grams) .................... . 

K. Individual Fetal Body Weights ....................................................................................... . 

L. Individual Fetal External, Visceral and Skeletal Observations ....................................... . 

M. Statistical Analysis Report .............................................................................................. . 

N. Certificates of Analysis- (Feed, Water and Bedding) .................................................... . 

0. Protocol and Protocol Amendments ............................................................................... . 

P. Historical Control Data ................................................................................................... . 

Q. Testing Facility Personnel ............................................................................................... . 

R. Report Amendments ....................................................................................................... . 
Huntingdon Life Sciences 

1. INTRODUCTION 

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The study was designed to assess the potential maternal and/or developmental 

toxicity of Gasoline DIPE (di-isopropyl ether) Vapor Condensate in the pregnant 

rat when administered by whole-body inhalation exposure. The study design 

permitted evaluation of maternal toxicity as revealed by clinical, body weight and 

food consumption changes and by gross necropsy findings, and evaluation of the 

developing conceptus, including death, structural abnormalities, or altered growth. 

This particular study is a near replicate of a previous study conducted by 

ExxonMobil Biomedical Sciences, Inc. (EMBSI Study Number 171734). The 

previous study was incomplete due to technical errors made in evaluating the male 

reproductive system of the fetuses. The Sponsor needed to repeat the previous 

study to satisfy the Environmental Protection Agency's Alternative Tier 2 Testing 

Requirements under Sections 211(b) (2) of the Clean Air Act. The potential 

impact of the test substance on the fetal male reproductive system must still be 

evaluated within the context of the treatment requirements of those proscribed 

under the 211 (b) Alternative Tier 2 Testing Requirements. 

2. MATERIALS AND METHODS 

2.1. STUDY MANAGEMENT 

2.1.1. SPONSOR 

American Petroleum Institute (API) 

1220 L Street, Northwest 

Washington, D.C. 20005-4070 

2.1.2. SPONSOR REPRESENTATIVE 

Thomas M. Gray, M.S., D.A.B.T. 

2.1.3. TESTING FACILITY 

Huntingdon Life Sciences (HLS) 

1 00 Mettlers Road 

East Millstone, New Jersey 08875-2360 
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2.1.4. STUDY DIRECTOR1 

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Draft Final Rcmort 

Keith P. Hazel den, BSc, CBiol, Mffiiol, Eurotox Registered 

Toxicologist 

Gary M. Hoffman, B.A., D.A.B.T. 

2.1.5. PRINCIPAL INVESTIGATOR-SKELETAL 

ASSESSMENTS 

Michael Mercieca, M.S. 

Pathology Associates International (P AI) 

2.1.6. PRINCIPAL INVESTIGATOR- STATISTICS 

Graham Healey, BSc, MSc, ARCS 

Huntingdon Life Sciences, UK 

2.2. STUDY DATES 

2.2.1. STUDY INITIATION 

3 August 2005 (Date Study Director signed the Protocol) 

2.2.2. DATE OF ANIMAL RECEIPT 

4 August 2005 (GD 0, 1, 2 or 3) 

2.2.3. EXPOSURE INITIATION 

6 August 2005 (Experimental Start Date - First GD 5) 

2.2.4. EXPOSURE TERMINATIONS 

24 August 2005 (Last GD 20) 

2.2.5. TERMINAL SACRIFICE 

22-25 August 2005 (GD 21) 

1

Keith Hazeldon was responsible for the conduct of the study and submission of the draft report; however 

he is no longer employed with Huntingdon Life Sciences. Gary Hoffman assumes responsibility for 

fmalization of the study report. 
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Group 

1 

2 

3 

4 

2.2.6. EXPERIMENTAL TERMINATION 

01 December 2005 (Date oflast data collection) 

2.2.7. STUDY COMPLETION 

Day Month Year (Date Final Report IS signed by the Study 

Director) 

2.3. EXPERIMENTAL OUTLINE 

Exposure Treatment 

Number of Animals 

Group Euthanized Fetal Evaluations Levels Gestation Time-Mated Designation (mg/m3t Days Females Gestation (Fetuses/Litter) 

Day21 External Soft Tissue Skeletal 

Control 0 (air only) 5-20 24 All All ~112 ~112 

Low 2000 5-20 24 All All ~112 ~112 

Mid 10000 5-20 24 All All ~112 ~112 

High 20000 5-20 24 All All ~112 ~112 

"Exposures were 6 hours per day for 7 consecutive days per week, for gestation days (GD) 5-20. Exposure 

levels are expressed as mg/m3 of test substance. The exposures were conducted by whole-body exposure. 

Control animals were exposed to air enriched with nitrogen only, using the same treatment regimen as the 

treated groups. 

The ftrst day of gestation (GD 0 = day of detection of positive sign of mating) was defmed as Day 0 of the 

study for each animal. 

2.4. JUSTIFICATIONS 

2.4.1. ROUTE, DURATION AND FREQUENCY OF 

ADMINISTRATION 

The inhalation route was used for the EMBSI study and is one of 

the potential routes of human exposure to this test substance. The 

duration and frequency of the exposures were the same as for the 

EMBSI study, and as recommended in the relevant EPA and 

OECD test guidelines, ensuring adequate exposure of postimplantation embryo-fetal development. 

2.4.2. EXPOSURE LEVEL SELECTION 

The exposure levels were the same as those used in the EMBSI 

study, and as prescribed by the Sponsor based on the lower 

explosive limit (LEL) of the test substance (the low, intermediate 
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and high exposure levels were approximately 5, 25, and 50% of the 

LEL). 

2.4.3. TEST ANIMAL SELECTION 

The rat was used for the EMBSI study and is an accepted surrogate 

for humans in the detection of embryo-fetal toxicity. It is the 

rodent species that is recommended by the relevant EPA and 

OECD test guidelines. Historical control data are also available 

with this strain of rat for comparative evaluation, if necessary. 

2.4.4. NUMBER OF ANIMALS 

The number of animals in this study was considered the minimum 

necessary to allow for meaningful interpretation of the data as 

required by the OECD and EPA test guidelines. Some of the 

endpoints of principal interest (eg embryo-fetal death, fetal 

malformations) are low-frequency events and a group size of 

~ 20 litters tends to provide the necessary degree of consistency 

between studies. In the expectation of pregnancy rates of 

80-1 00%, the group size of 24 mated female rats was considered 

appropriate for this study, to provide a sufficient number of litters 

for evaluation in each group. Also, the number of animals in each 

group was comparable to the number in the EMBSI study 

(25/group). 

2.5. TEST SUBSTANCE 

Gasoline DIPE (di-isopropyl ether) Vapor Condensate 

2.5.1. TEST SUBSTANCE CATEGORY 

Gasoline product 

2.5.2. SUPPLIER 

ChevronTexaco Research and Technology Company 

100 Chevron Way 

Richmond, CA 94802 
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2.5.3. 

2.5.4. 

2.5.5. 

2.5.6. 

2.5.7. 

2.5.8. 

LOT NUMBER 

API 01-06 

PURITY 

100% Gasoline DIPE Vapor Condensate 

DESCRIPTION 

Colorless liquid 

DATES RECEIVED 

7 December 2001 

05 August 2002 

EXPIRATION DATE 

Not available, stable per MSDS. 

ANALYSIS 

Prior to use in this study, the identity, strength, composition, stability 

and method of synthesis, fabrication and/or derivation of each batch 

of the test substance was documented and is maintained by the 

Sponsor. In addition, the Sponsor conducted a non-GLP purity 

analysis and the Testing Facility conducted a GLP stability analysis 

of the test substance by GC prior to the start of this study. 

2.5.9. STORAGE 

Room temperature (ambient) in an outside solvent shed, except 

when being used in the inhalation laboratory. 

2.5.10. DISPENSING 

The test substance was received from ChevronTexaco Research 

and Technology Company in 1 00-gallon cylinders. Since only 

5-gallon cylinders were practical to be used for exposure 

operations, the test substance was dispensed, as needed, at the 

Testing Facility from the 100 gallon cylinders into 5-gallon 

cylinders using nitrogen pressurization. 
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2.6. 

2.5.11. ARCHIVAL SAMPLE 

An archival sample of the test substance was taken and is stored in 

the Archives of the Testing Facility under conditions specified for 

test article storage. Since multiple studies were conducted with the 

same test substance, a common archival sample was taken and 

appropriately labeled. 

2.5.12. DISPOSITION 

The unused portion of the test substance was returned to the 

Sponsor's designee following completion of the study. Empty test 

substance containers were returned to the Sponsor's designee on an 

as-needed basis. The Sponsor was responsible for tracking their 

disposition. 

TEST ANIMALS 

2.6.1. SPECIES 

Albino Rats (Outbred) VAF/Plus® 

Sprague-Dawley Derived (CD®) 

Crl:CD® (SD) IGS BR 

2.6.2. SUPPLIER 

Charles River Laboratories 

Raleigh, NC27610 

2.6.3. NUMBER OF ANIMALS 

Ordered and Received: 

100 time-mated females 

Placed on test: 

96 females 

2.6.4. AGE AT RECEIPT 

Approximately 10-12 weeks 
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2.6.5. AGE AT INITIATION OF EXPOSURES 

Approximately 10-12 weeks 

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2.6.6. WEIGHT AT INITIATION OF EXPOSURES (GRAMS) 

Females: 

Mean 

244 

Range 

216-280 

Individual weights of animals placed on test were within ±20% of 

the mean weight. 

2.6.7. ACCLIMATION PERIOD 

Animals were acclimated for 2-5 days from the day of receipt until 

initiation of exposures on GD 5. 

2.7. ANIMAL ASSIGNMENT 

Animals were placed into study groups using a computerized 

randomization program. This ranked Gestation Day 4 body weights into 

blocks and randomly assigned each animal within each block into one of 

the study groups. Disposition of all animals not utilized in the study is 

maintained in the study file. 

2.8. ANIMAL IDENTIFICATION 

Each animal was assigned a temporary identification number upon receipt. 

After selection for study, each animal was ear-tagged by the Testing 

Facility with a number assigned by the Facility. The number assigned plus 

the study number comprised the unique animal number for each animal. 

Each cage was provided with a cage card that was color-coded for 

exposure level identification and contained the study number and animal 

number. 

2.9. VETERINARY CARE 

Animals were monitored by the technical staff for any conditions requiring 

possible veterinary care, and the staff veterinarian approved the animals 

for use in the study. 
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2.10. ANIMAL HUSBANDRY DURING NON-EXPOSURE PERIODS 

2.10.1. FACILITIES MANAGEMENT/ANIMAL HUSBANDRY 

Currently acceptable practices of good animal husbandry were 

followed e.g., Guide for the Care and Use of Laboratory Animals; 

National Academy Press, 1996. Huntingdon Life Sciences, East 

Millstone, New Jersey is fully accredited by the Association for 

Assessment and Accreditation of Laboratory Animal Care 

International (AAALAC). 

2.10.2. HOUSING 

All animals were housed individually in stainless steel suspended 

cages with wire mesh floors and fronts, except for GD 20-21, when 

solid-bottom cages with com-cob bedding were employed (in case 

any animal littered prematurely). Cage rack positions in the room 

were rotated each week, so that possible effects due to placement 

were minimized. Each cage was fitted to secure a glass feeder jar 

with a stainless steel lid. Clean feed jars and fresh feed were 

provided at least every 7 days. 

2.10.3. FEED 

Certified Rodent Diet, No. 5002; (Meal) (PMI Feeds, Inc., St. Louis, 

MO) was available without restriction, except during actual 

exposures. 

2.10.4. FEED ANALYSIS 

Analytical certificates for relevant batches of feed, provided by the 

manufacturer, are maintained on file at the Testing Facility and are 

included in this report (Appendix N). There were no known 

contaminants in the feed that were expected to interfere with the 

objectives of this study. 

2.10.5. WATER 

Facility water was supplied by Elizabethtown Water Company, 

(Westfield, NJ) and was provided ad libitum to individual animal 

cages through an automated watering system, except during actual 

exposures. 
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2.10.6. WATER ANALYSIS 

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Water analyses are conducted monthly by the Elizabethtown Water 

Company, Westfield, NJ (Raritan-Millstone plant) to ensure that 

water meets standards specified under the EPA Federal Safe 

Drinking Water Act Regulations ( 40 CFR part 141 ). In addition, 

water samples are collected biannually from representative rooms 

in the Testing Facility. Chemical and microbiological water 

analyses were conducted on these samples by a subcontract 

laboratory (Benchmark Analytics, Center Valley, P A). Water 

analyses (provided by the supplier) and chemical and 

microbiological water analyses (provided by the subcontractor) 

results are maintained on file at the Testing Facility and are 

included in this report (Appendix N). There were no known 

contaminants in the water that were expected to interfere with this 

study. 

2.10.7. BEDDING 

Com-cob bedding (supplier) was provided in the solid-bottomed 

plastic cages used from GD 20 (lot number DC295). There were 

no known contaminants in the bedding that were expected to 

interfere with this study. An analysis of a representative batch used 

is included in Appendix N. 

2.10.8. ENVIRONMENTAL CONDITIONS 

Light/Dark Cycle 

A twelve-hour light/dark cycle was provided, controlled by an 

automatic timer. 

Temperature 

Temperature was monitored in accordance with Testing Facility 

SOPs and was maintained within the specified range: 

Desired Range: 

Actual Range: 

Daily Average Range: 

18 to 26°C 

23 to 24°C 

23 to 24°C 
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Relative Humidity 

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Relative humidity was monitored in accordance with Testing 

Facility SOPs and was maintained within the specified range: 

Desired Range: 

Actual Range: 

Daily Average Range: 

Air Changes 

30to 70% 

49 to 67% 

55 to 60% 

The animal room was set up to provide 10-15 air changes per hour. 

The actual number of air changes per hour in each animal room is 

recorded at least twice each year, and the Testing Facility retains 

these records. 

2.11. TEST SUBSTANCE ADMINISTRATION AND CHAMBER 

OPERATION 

See Appendix B, Inhalation Report. 

2.12. EXPERIMENTAL EVALUATIONS 

2.12.1. VIABILITY EXAMINATION (CAGE-SIDE) 

Observations for mortality, morbidity, and signs of severe toxicity 

were made at least twice daily. 

2.12.2. DETAILED PHYSICAL EXAMINATIONS 

In-Chamber: All animals were observed as a group at least once 

during each exposure. This was routinely performed near the 

middle of each exposure. Any pertinent behavioral changes and all 

signs of toxicity (including time of onset, degree and duration) 

were recorded. 

Out-of-Chamber: Animals on study were examined daily from 

the day after receipt through to terminal euthanasia on GD 21. 

Examinations included observations of general condition, skin and 

fur, eyes, nose, oral cavity, abdomen and external genitalia, 

occurrence of secretions and excretions, and autonomic activity 

(e.g., lacrimation, piloerection, pupil size, unusual respiratory 

pattern). Changes in gait, posture and response to handling as well 
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as the presence of clonic or tonic movements, stereotypy (e.g., 

excessive grooming, repetitive circling) or bizarre behavior (e.g., 

self-mutilation, walking backward) were recorded, as well as 

evaluations of respiration. During the treatment period, these 

evaluations were performed after exposure. 

2.12.3. BODY WEIGHTS 

Body weights were recorded on GD 4 (for randomization into test 

groups), 5, 8, 11, 14, 17, 20 and 21 (day of scheduled sacrifice). 

Weight gain was calculated for these intervals, and also GD 5-14, 

14-21 and 5-21. 

2.12.4. FEED CONSUMPTION 

Feed consumption was recorded and reported for the intervals: 

GD 4-5, 5-8, 8-11, 11-14, 14-17, 17-20, 20-21 and 5-21. 

2.13. POSTMORTEM EVALUATIONS 

2.13.1. METHOD OF EUTHANASIA 

All dams were euthanized by carbon dioxide inhalation, by 

approximately noon on each day. All live fetuses were euthanized 

with intraperitoneal sodium pentobarbital. 

2.13.2. MACROSCOPIC EXAMINATION 

A macroscopic postmortem examination was performed on all the 

dams. Macroscopic lesions or tissues with significant findings were 

saved in 10% Neutral Buffered Formalin (NBF). 

Apparently non-pregnant uteri were stained with ammonium 

sulphide (modified Salewski test) to confirm the non-pregnant 

status or detect implantation sites not otherwise visible. 

Each dam had the following examinations of the reproductive tract 

onGD21: 

The intact uterus (ovaries attached) was removed from the 

abdominal cavity. The gravid uterus, including the cervix, was 

weighed. Corpora lutea were counted and the number per ovary 
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recorded. The number and location of the following was recorded 

for each uterine hom: 

• live fetuses 

• dead fetuses (no significant degeneration) 

• late embryo-fetal deaths (recognizable dead fetus undergoing 

degeneration, regardless of size) 

• early embryonic deaths (evidence of implantation but no 

recognizable fetus) 

Each placenta was examined macroscopically. The maternal carcass, 

uterus, ovaries and placentas were then discarded. 

2.14. FETAL EVALUATIONS 

2.14.1. EXTERNAL EVALUATIONS 

All live fetuses were weighed and individually identified within 

cassettes. Each live and dead retained fetus was given a 

macroscopic external examination for defects, including 

observation of the palate. 

2.14.2. FETAL SOFT TISSUE (VISCERAL) EVALUATION 

Approximately one-half of the fetuses in each litter (alternating 

fetuses within the litter, nominally) were placed in Modified 

Davidson's fixative for preservation and decalcification. These 

fetuses were subjected to soft tissue examination by gross 

dissection of the torso, and a razor blade sectioning technique for 

the head (Wilson and W arkany, 1965). All malformations and 

variations were recorded. Particular attention was paid to the 

reproductive tract for altered signs of development. During the 

dissection process, the sex of each fetus was confirmed by internal 

inspection of the gonads. Following complete dissection of the 

fetuses, all carcasses and sections were preserved in 1 0% neutral 

buffered formalin. 

2.14.3. FETAL SKELETAL EVALUATIONS 

Approximately one-half of the fetuses in each litter (alternating 

fetuses within the litter, nominally) were eviscerated, during which 

process the sex of each fetus was determined by internal inspection 
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and recorded. The eviscerated fetuses were placed in 70% 

isopropyl alcohol for preservation and processed for staining of the 

skeleton using Alizarin Red S. Subsequently, these fetuses were 

evaluated for skeletal malformations and ossification variations by 

an approved subcontractor, Pathology Associates International 

(PAl), 15 Worman's Mill Court, Suite 1, Fredrick, MD 21701, 

USA. These skeletal specimens were then stored in 100% glycerin 

with a mold inhibitor. 

2.14.4. DEAD FETUSES 

No conceptuses in this category (recently dead fetuses) occurred in 

this study. All late embryonic/fetal and early embryonic deaths were 

discarded. 

2.15. STATISTICAL ANALYSIS 

Analysis was performed by the Statistics Department of Huntingdon Life 

Sciences Ltd, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, 

PE28 4HS, England. The Testing Facility was responsible for GLP 

compliance and the archiving of the raw data and original final statistical 

report. 

A copy of the statistical report is provided as Appendix M. This provides 

details of the methods and statistical results. 

2.15.1. CONTINUOUS DATA 

The following mean measures were analyzed: 

Maternal body weight values and body weight changes during 

gestation 

Maternal feed consumption values (presented as grams of feed/kg 

ofbody weight/day) 

Gravid uterine weight 

Corpora lutea 

Implantation sites 

Pre-implantation loss as% 

Early embryonic deaths ('early resorptions') 

Late embryo-fetal deaths ('late resorptions') 

Total embryo-fetal deaths, and as% of implantation sites 

Live fetuses 
Huntingdon Life Sciences 05-4287 

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Number of males and females per litter 

2.15.2. INCIDENCE DATA 

The following measures were analyzed: 

Maternal necropsy findings 

Page 21 

Draft Final Report 

Maternal pregnancy data (including corpora lutea, implantation 

sites, resorptions, etc.) 

Soft tissue (visceral) and skeletal fetal findings, of which only 

5 visceral and 7 skeletal findings required analysis 

2.15.3. FETAL BODY WEIGHT 

Fetal body weight was analyzed by sex and as a composite for both 

sexes. 

2.16. DATA STORAGE 

All data documenting experimental details and study procedures and 

observations were recorded and maintained as raw data. 

At the completion of the study (submission of the signed final report), all 

raw data, preserved specimens and retained samples, as well as the original 

study protocol (and any amendments) and the original final report will be 

maintained in the Archives of the Testing Facility for one year following 

issue of the Final report. The Sponsor will determine the final disposition 

of these materials. 

2.17. REGULATORY REFERENCES 

2.17.1. TEST GUIDELINES 

This study was designed to meet or exceed the pertinent 

requirements of: 

US EPA Vehicle Emissions Inhalation Exposure Guideline 79.61, 

CFR Vol. 59, No. 122, 27 June 1994. 

Organization for Economic Cooperation and Development (22 

January 2001). OECD Guidelines for Testing of Chemicals; 

OECD Guideline 414: Prenatal Developmental Toxicity Study 
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Draft Final Report 

US EPA OPPTS Health Effects Test Guidelines 870.3700, Prenatal 

Developmental Toxicity Study EPA 712-C-98-207, August 1998. 

2.17.2. GOOD LABORATORY PRACTICES 

This study was conducted in accordance with US EPA 79.60, CPR 

Vol. 59, No. 122, 27 June 1994. This study was performed 

according to protocol and Huntingdon Life Sciences' Standard 

Operating Procedures. The Testing Facility was responsible for 

the GLP compliance and archiving of raw data produced by 

subcontractors (PAl, and HLS Dept. of Statistics). 

2.17.3. ANIMAL WELFARE ACT COMPLIANCE 

This study complied with all appropriate parts of the Animal 

Welfare Act regulations: 9 CPR Parts 1 and 2 Final Rules, Federal 

Register, Volume 54, No. 168, August 31, 1989, pp. 36112-36163, 

effective October 30, 1989 and 9 CPR Part 3 Animal Welfare 

Standards; Final Rule, Federal Register, Volume 56, No. 32, 

February 15, 1991, pp. 6426-6505, effective March 18, 1991. 

2.18. PROTOCOL DEVIATIONS 

The following protocol deviations occurred during the study, but did not 

affect the integrity of the study: 

1. Due to technician oversight, the Set B animals (1907-1912, 2907-

2912, 3907-3912 and 4907-4912) were not given clean feeders and 

fresh feed after seven days of use. They were fed on Gestation Day 

2 but were not given new feeders until Gestation Day 10. 

2. Due to technician oversight, gravid uterine weights for Animal 

Nos. 1912 and 4910 were not taken. The uteri were opened and all 

the fetuses were removed prior to recording necessary weights. 

3. Fetus number 13 from dam number 2921 did not have an external 

examination as per the protocol. 
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3. RESULTS AND CONCLUSIONS 

3.1. TEST SUBSTANCE ANALYSIS 

(Appendix A) 

Page 23 

Draft Final Report 

GC analysis of the test substance showed no significant changes in 

composition, comparing pre-study results to those obtained during prior 

studies at this Testing Facility. 

3.2. CHAMBER MONITORING 

(Appendix B) 

Pre-study chamber distribution analyses showed that the test substance 

was evenly distributed within each chamber. Chamber monitoring showed 

that the chamber oxygen levels were at least 19%. Chamber room 

monitoring showed that no test substance was present in the room and that 

the sound and light levels were acceptable. 

The target and mean (± standard deviation) analytical (IR) and nominal 

concentrations (see Appendix B) are summarized as follows: 

Target Analytical Nominal 

Group Concentration Concentration Concentration 

{mg/m3

) (mg/m3) {mg/m3

) 

1 Air Control 0 0.00± 0.00 0±0 

2 Gasoline DIPE 2000 1982± 93 1995 ± 40 Vapor Condensate 

3 Gasoline DIPE 10000 10072± 546 11474± 513 Vapor Condensate 

4 Gasoline DIPE 20000 19776 ± 961 19842± 602 Vapor Condensate 

The analytically measured (IR) exposure levels of the airborne test 

substance were acceptably close, in the opinion of the study director, to the 

targeted exposure levels and to the nominal concentrations. Chamber 

environmental conditions averaged 24°C temperature and 41% relative 

humidity. 
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Draft Final Report 

Mean particle size distribution measurements for the exposures are 

summarized as follows: 

Mass Median Geometric Total 

Aerodynamic Mass Group Standard Diameter Concentration 

(J.tm) Deviation (mg/m3

) 

1 Air Control 3.263 ± 4.3 2.033 ± 0.2 3.56E-03 

2 Gasoline DIPE 4.061 ± 3.4 2.435 ± 0.5 5.15E-03 

Vapor Condensate 

3 Gasoline DIPE 3.762 ± 2.6 2.677 ± 0.6 6.12E-03 

Vapor Condensate 

4 Gasoline DIPE 3.118 ± 3.3 2.223 ± 0.1 6.29E-03 

Vapor Condensate 

These results indicated that the atmospheres were essentially gas/vapor 

only, as expected, since there was no substantial difference between the 

test substance chambers and the air control chamber. 

Analysis of the major components in the neat test substance and the test 

atmospheres (see Appendix A) showed an acceptably close comparison, in 

the opinion of the study director, between the neat test substance and the 

vaporized test substance. These data demonstrated that the test animals 

were exposed, as expected, to all of the major components of the test 

substance in their acceptably proper proportions. The data were consistent 

pretest and during the study indicating stability of the test substance and 

the atmosphere generation techniques. 

3.3. MATERNAL DATA 

3.3.1. MORTALITY AND PREGNANCY 

(Table 1; Appendices C and M) 

All animals survived until the scheduled termination of the study 

and pregnancy rates were 92-1 00% across all the groups. 
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3.3.2. CLINICAL OBSERVATIONS 

(Table 2; Appendices B and D) 

Page 25 

Draft Final Report 

During the exposure and non-exposure periods, there was little 

overt reaction to treatment. There was only an increased incidence 

of red nasal discharge among the 10,000 and 20,000 mg/m3 

exposed animals, as compared with the other groups. 

Chromodacryorrhea was also observed at a low incidence at 

20,000 mg/m3

, in animals that did not show the nasal discharge. 

These two findings (interpreted as secretion of porphyrin) are 

common reactions to mild stress in rats. An animal (#4908) 

exposed at 20,000 mg/m3 was noted with an abdominal mass on 

GD 17 -21 and confirmed as a mammary gland mass at necropsy 

(see 3.3.5.). 

3.3.3. BODY WEIGHTS 

(Figure 1; Tables 3, 4 and 8; Appendices E, F, J and M) 

The exposure regimen resulted in only transient reductions in 

maternal weight gain (up to approximately 25% reduced from 

Control at GD 11-14 interval, partly resolving by GD 21) during 

the exposure period. These changes were negligible at 

2000 mg/m3 and most noticeable at 20000 mg/m3

• 

3.3.4. FEED CONSUMPTION 

(Figure 2; Table 5; Appendices G and M) 

The exposure regimen resulted in only transient reductions in feed 

consumption (up to approximately 10% reduced) during the 

exposure period. These changes were negligible at 2000 mg/m3 

and most noticeable at 20000 mg/m3

• 

3.3.5. MACROSCOPIC POSTMORTEM EVALUATIONS 

(Table 6; Appendices H and M) 

The only maternal necropsy finding was of a mass in the mammary 

tissue of one animal (#4908) at 20,000 mg/m3

. This finding was 

considered incidental, and not related to treatment. 
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3.3.6. UTERINE IMPLANTATION DATA 

(Table 7; Appendices I and M) 

Page 26 

Draft Final Report 

There was no effect of the exposures on the pregnancies. Pre- and 

post-implantation losses and litter size were similar in all the 

groups. 

3.3.7. GRAVID UTERINE WEIGHT DATA 

(Table 8; Appendices J and M) 

Adjusted gravid uterine weight was reduced in the 20,000 mglm3 

group but this reduction was not considered adverse. 

3.4. FETAL DATA 

3.4.1. Fetal Body Weights 

(Table 7; Appendices I, K and M) 

There was no effect of the exposures on fetal weight at GD 21. 

The very marginal (3-4%) difference from Control that occurred in 

the 20,000 mg/m3 group, while achieving statistical significance in 

total fetal and female weights, was considered not to represent an 

effect of the exposures, particularly in context with the lack of 

other signs of effect on pregnancy outcome in the study and the 

values are comparable to the historical control range for published 

data from Charles River Labs (see Appendix P). 

3.4.2. Fetal Observations 

(Table 9; Appendices L and M) 

A very small number of major abnormalities occurred sporadically 

in the study, with no relationship to the exposures. 

Similarly, there were sporadic incidences across the groups, of 

structural variants, both soft-tissue and skeletal. No association 

with the exposures was inferred. 

Although the incidence of 14th (supernumerary, rudimentary) ribs 

was higher at 20,000 mg/m3 than in the other groups, the difference 
Huntingdon Life Sciences 05-4287 

211-DIPE-DEV 

Page27 

Draft Final Report 

from Control did not achieve statistical significance, and remained 

consistent with the range of incidences that are seen in this strain of 

animal. The mean group incidence for this strain in this laboratory 

is approximated by the 2000 and 10000 mg/m3 group mean 

incidences (7 and 5 fetuses in 5 litters, respectively), while the 

20000 mg/m3 exposure group incidence (12 fetuses in 9 litters) is 

nearer the upper limit of the range seen previously (14 fetuses in 

10 litters) (see Appendix P). 

There was no evidence of any effect of the exposures on the 

general state of skeletal ossification. 

4. CONCLUSIONS 

Exposure of pregnant rats to atmospheres of 0 (Air Control), 2,000, 10,000 or 

20,000 mg/m3 of Gasoline DIPE Vapor Condensate, 6 hours/day on gestation days 

5-20, resulted in only transient reductions in maternal weight gain and feed 

consumption during the exposure period. These changes were negligible at 

2,000 mg/m3 and most noticeable at 20,000 mg/m3

• It was considered that a noobserved-adverse-effect level (NOAEL) for maternal effects was evident at 

2,000 mg/m3

. There was no effect of the exposures at any level on pregnancy 

outcome, in terms of pre- or post-implantation loss, fetal weight or the incidence 

of fetal abnormalities and variants. A NOAEL for embryo-fetal effects was 

therefore established at 20,000 mg/m3

. 
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REFERENCES 

Page 28 

Draft Final Report 

Salewski, E. 1964. Farbemethode zum makroskopischen machweis von 

implantationsstellen am uterus der ratte. Archiv. Path. Exp. Pharmakol. 247: 367. 

Wilson, J.S. and Warkany, J.1965, Teratology: Principles and Techniques, Chicago: The 

University of Chicago Press, Page 271. 

Note: Appendix M provides details of the methods and statistical results. 
Huntingdon Life Sciences 

Feed Consumption (glkg/day): 

05-4287 

211-DIPE-DEV 

CALCULATIONS 

Page29 

Draft Final Report 

[(grams of feed presented- grams of feed remaining)/average of the previous and current 

body weights in kilograms ]/no. days 

Preimplantation Loss: 

no. of corpora lutea- no. of implantation sites 

Percent Preimplantation Loss: 

[(no. of corpora lutea- no. of implantation sites)/no. of corpora lutea] X 100 

Postimplantation Loss: 

total no. of early and late resorptions and dead fetuses 

Percent Postimplantation Loss: 

(no. of early and late resorptions and dead fetuses/no. of implantation sites) X 100 

Mean% Live Fetuses (number per animal): 

(no. of male or female live fetuses/ total number oflive fetuses) X 100 

Mean% Dead Fetuses (number per animal): 

' 

(no. of dead fetuses/ total number of implantation sites) X 100 

Mean Percent Resorptions (implants per animal): 

total no. of early or late resorptions/ total number of implantation sites) X 100 
Huntingdon Life Sciences 

General Notes 

05-4287 

211-DIPE-DEV 

General Preface 

Page 30 

Draft Final Report 

Individual animal data values presented in this report may be rounded. 

Unrounded individual animal data values are used to calculate the reported 

mean and standard deviation values. Therefore, use of the reported individual 

values to reproduce means, standard deviations and/or to perform any 

subsequent calculations may produce minor discrepancies between the 

calculated values and those presented in this report. 
Huntingdon Life Sciences 05-4287 

211-DIPE-DEV 

I u=r n--Mean Gestation Body Weights (grams) I 

440 

-e- Group 1 - 0 mg/m3 

420 2 - 2000 mg/m3 

400 ----tr--- Group 3 - 10000 mg/m3 

- 380 Ill --*-Group 4 -20000 mg/m3 

E 

e 360 Cl -

J!l 340 .c m· 

320 

>-

0 300 m 

280 260 

--.--. 

240 

220 

200 

4 5 6 .7 8 9 10 11 12 13 14 15 16 17 1

Gestation Day 
Huntingdon Life Sciences 05-4287 

211-DIPE-DEV 

94 

89 

84 

79 

74 

i;' 

'C c,69 

a, 

64 

59 

54 

49 

I Mean Gestation Feed Consumption (g/kg/day) 

_/.//' ---

__ 

·/ 

.. .. : .. .. 

--8- Group 1 -0 mg/m3 

--.-- Group2 -2000 mg/m3 

----&--- Group 3 - 1 0000 m g!m 3 

->IE-- Group 4 - 20000 mg/m3 

44 

4to 5 · 5to 8 8 to 11 11 to 14 14 to 17 17 to 20 20 to 

Gestation Day Interval 
10-MAR-2011 17:37 

Huntingdon Life sciences 05-4287 

- DOSE GROUP : 

DOSE LEVEL {MG/M3): 

-------------

No. of females mated N 

Pregnant N-

- Died/sacrificed moribund N 

- Elective sacrifice N 

Nonpregnant N 

- Died/sacrificed moribund N - Elective sacrifice N 

Total no. of females died/ N 

sacrificed moribund % 

Examined at scheduled c-.section N 

- Nonpregnant N 

- With total implant loss N 

% 

- With viable fetuses N 

% 

TABLE 1 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

SUMMARY OF SURVIVAL AND PREGNANCY 

---------------------------------------------------------------

1 2 3 

0 2000 10000 20

24 24 24 

23 23 24 

0 0 0 

0 0 0 

1 1 0 

0 0 0 

0 0 0 

0 0 0 

0.0 0.0 0.0 

24 24 24 

1 1 0 

0 0 0 

0.0 0.0 0.0 

23 23 24 

95.8 95.8 100.0 
10-MAR-2011 17:41 

Huntingdon Life Sciences 05-4287 

TABLE 2 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

SUMMARY OF CLINICAL OBSERVATIONS DURING GESTATION - (frequency/animals) 

DOSE GROUP: 

DOSE LEVEL (MG/M3): 

DAY 4 to 21 

Normal 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

Dermal-General 

ALOPECIA - GENERAL 

Ocular 

CHROMODACRYORRHEA - UNILATERAL 

Oral/Buccal 

INCISORS MALOCCLUDED 

NASAL DISCHARGE - RED 

Palpable masses 

MASS 

1 

0 

431/24 

24/24 

0/ 0 

0/ 0 

0/ 0 

1/ 1 

0/ 0 

2 

2000 

421/24 

24/24 

0/ 0 

0/ 0 

0/ 0 

11/ 3 

0/ 0 

3 

10000 

382/24 

24/24 

0/ 0 

0/ 0 

0/ 0 

50/15 

0/ 0 

20
10-MAR-2011 17:43 

Huntingdon Life Sciences 05-4287 

TABLE 3 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

SUMMARY OF GESTATION BODY WEIGHTS (GRAMS) 

------------------------------------------------------------------

DOSE GROUP: 1 2 3 

DOSE LEVEL_ (MG/M3): 0 ' 2000 10000 20

--------------------------------------------------------------

DAY 4 MEAN 234 235 234 

S.D. 14.0 16.6 15.7 

N 23 23 24 

DAY 5 MEAN 244 245 244 

S.D. 14.6 16.5 13.9 

N 23 ' 23 24 

DAY 8. MEAN 259 260 256 

S.D. 12.8 18.0 14.4 

N 23 23 24 

DAY 11 MEAN 281 279 274 

S.D. 14.9 19.6 15.6 

N 23 23 24 

DAY 14 MEAN 301 296 292 

S.D. 17.0 20.3 14.8 

N 23 23 24 

DAY 17 MEAN 332 327 323 

S.D. 22.2 24.3 18.7 

N 23 23 24 

DAY 20 MEAN 378 370 367 

S.D. 26.3 29.9 24.2 

N 23 23 24 

DAY 21 MEAN 388 379 376 

28.6 32.7 25.9 
10-MAR-2Di1 17:44 

Huntingdon Life Sciences 05-4287 

DOSE GROUP: 

DOSE LEVEL (MG/M3}: 

TABLE 4 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

SUMMARY OF GESTATION BODY WEIGHT GAIN (GRAMS} 

1 2 3 

0 2000 10000 

20

-------------------------------------------------------------

DAYS 4 TO 5 

DAYS 5 TO 8 

DAYS 8 TO 11 

DAYS 11 TO 14 

DAYS 14 TO 17 

DAYS 17 TO 20 

DAYS 20 TO 21 

DAYS 5 TO 14 

DAYS 14 TO 21 

DAYS 5 TO 21 

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 

MEAN 

S.D. 

N 

10 

6.1 

23 

15 

4.5 

23 

22 

4.7 

23 

20 

4.2 

23 

31 

7.3 

23 

46 

7.6 

23 10 

5.2 

23 

57 

8.4 

23 

86 

15.5 

23 

144 

20.6 

23 

11 10 

4.7 5.2 

23 24 

15 13 

4.7 5.5 

23 24 

19 18 

3.6 3.9 

23 24 

17 17 

5.0 3.7 

23 24 

31 31 

6.9 7.6 

23 24 

44 44 

8.9 9.9 

23 24 

9 9 

5.0 4.9 

23 24 

50 48 

7.8 7.7 

23 24 

84 84 

16.3 1?.0 

23 24 

134 132 

21.5 21.0 

23 24 
' 17:45 

Huntingdon Life Sciences 05-4287 

DOSE GROUP: 

DOSE LEVEL (MG/M3): 

TABLE 5 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

SUMMARY OF GESTATION FEED CONSUMPTION (GRAMS/KG/DAY) 

1 2 3 

0 2000 10000 

20

-----------------------------------------------------------

DAYS 4 TO 5 

DAYS 5 TO 8 

DAYS 8 TO 11 

DAYS 11 TO 14 

DAYS 14 TO 17 

DAYS 17 TO 20 

DAYS 20 TO 21 DAYS 5 TO 21 

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 

71 

10.5 

22 

81 

6.0 

22 

85 

7.1 

22 

85 

7.1 

22 

81 

4.5 

22 

74 

4.5 

23 

53 

8.1 

23 

76 

4.7 

23 

74 75 

11.5 11.4 

23 24 

85 80 

14.1 6.0 

23 24 

82 80 

4.6 5.5 

23 24 

82 82 

4.7 3.9 

23 22 

81 81 

4.5 6.0 

23 22 

73 74 

3.3 3.7 

23 22 

47 47 

7.5 5.8 

23 24 

75 74 

4.0 3.6 

23 24 
10-MAR-2011 17:46 

Huntingdon Life Sciences 05-4287 

DOSE GROUP: 

DOSE LEVEL (MG/M3): 

TABLE 6 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

SUMMARY OF MATERNAL NECROPSY OBSERVATIONS 

1 2 3 

0 2000 10000 

20

-----------------------------------------------------------------------------------------

FEMALES N 24 24 "24 

GROSS EXAM N 0 0 a· 

----------

MASS N 0 0 0 

% 0.0 0.0 0.0 
10-MAR-2011 17:48 

Huntingdon Life Sciences 05-4287 

TABLE 7 

GASOLINE DIPE VAPOR CONDENSATE:' EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

SUMMARY OF CESAREAN SECTION DATA 

·DOSE GROUP: 1 2 3 

DOSE LEVEL (MG/M3): 0 2000 10000 

------------

Fetal Body Weight (g) MEAN 5.7 5.8 5.7 

S.D. 0.29 0.30 0.28 

N 23 23 24 

Male Fetuses MEAN 5.9 5.9 5.8 

S.D. 0.32 0.33 0.32 

Female Fetuses MEAN 5.6 5.6 5.5 

S.D. 0.28 0.33 0.25 

20
10-MAR-2011 17:48 

Huntingdon Life Sci.ences 05-4287 

TABLE 7 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

DOSE GROUP: 

DOSE LEVEL (MG/M3): 

Pregnant N 

(at scheduled sacrifice) 

Dams with no Viable Fetuses N 

Dams with Viable Fetuses N 

Co.rpora Lutea 

No. per animal 

Implantation Sites 

No. per animal 

Preimplantation Loss 

No . per animal 

% per animal 

Live Fetuses 

No. per animal 

Males 

Females 

TOTAL 

MEAN 

S.D. 

TOTAL 

MEAN 

S.D. 

TOTAL 

MEAN 

S.D. 

MEAN% 

S.D. 

TOTAL 

MEAN 

S.D. 

·TOTAL 

MEAN% 

S.D. 

TOTAL 

MEAN% 

S.D. 

SUMMARY OF CESAREAN SECTION DATA 

1 

0 

23 

0 

23. 

343 

14.9 

2.78 

311 

13.5 

2. 71 

32 

1.4 

1. 88 

9.3 

13.46 

300 

13.0 

2.64 

146 

48.5 

10.94 

154 

51.5 

10.94 

2 

2000 

23 

0 

23 

334 

14.5 

2.06 

310 

13.5 

1. 50 

24 

1.0 

1.46 

6.6 

7.99 

286 

12.4 

2.64 

133 

46.1 

14.75 

153 

53.9 

14.75 

3 

10000 

24 

0 

24 

354 

14.8 

2.52 

315 

13.1 

2.85 

39 

1.6 

2. 96 ' 

10.4 

18.58 

302 

12.6 

3.06 

154 

51.2 

12.50 

148 

48.8 

12.50 

20
10-MAR-2011 17 :·48 

Huntingdon Life Sciences 05-4287 

TABLE 7 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

SUMMARY OF CESAREAN SECTION DATA 

DOSE GROUP: 1 2 3 

DOSE LEVEL {MG/M3): 0 2000 10000 

Postimplantation .Loss. TOTAL 11 24 13 

No. per animal MEAN 0.5 1.0 0.5 

S.D. 0.67 2.08 0.83 

% implants per animal MEAN% 3.4 7.9 5.1 

S.D. 4.66 14.89 8.64 

Dead Fetuses TOTAL 0 0 0 

No . per animal MEAN 0.0 0.0 0.0 

S.D. 0.00 0.00 0.00 

% of implants per animal MEAN% 0.0 0.0 0.0 

S.D. 0.00 0.00 0.00 

Resorptions: Early TOTAL 10 23 13 

No. per animal MEAN 0.4 1.0 0.5 

S.D. 0.66 2.07 0.83 

% of implants per animal MEAN% 3.1 7.6 5.1 

S.D. 4.66 14.80 8.64 

Resorptions: Late TOTAL 1 1 0 

No . per animal . MEAN 0.0 0.0 0.0 

S.D. 0.21 0.21 0.00 

% of implants per animal MEAN% 0.3 0.3 0.0 

S.D. 1. 39 1. 60 0.00 

20
10-MAR-2011 17:49 

.Huntingdon Life Sciences 05-4287 

TABLE 8 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

SUMMARY OF GRAVID UTERINE WEIGHT AND NET BODY WEIGHT CHANGE (GRAMS) 

DOSE GROUP: 1. 2 3 

DOSE LEVEL (MG/M3): 0 2000 10000 

20

-----------------------------------------------------------------------------------------

UTERUS WEIGHT MEAN 101 97 96 

S.D. 19.4 19.8 22.5 

N 22 23 24 

ADJUSTED WEIGHT MEAN 286 283 280 

S.D. 18.1 19.0 17.1 

N 22 23 24 

NET WEIGHT CHANGE FROM DAY 5 MEAN 43 37 36 

S.D. 11.1 9.1 11.7 

N 22 23 24 

WEIGHT CHANGE FROM DAY 5 MEAN 144 134 132 

S.D. 20.6 21.5 21.0 

N 23 23 24 

ADJUSTED WEIGHT = TERMINAL BODY WEIGHT MINUS GRAVID UTERINE WEIGHT 

NET WEIGHT CHANGE FROM DAY 5 = TERMINAL CORRECTED BODY WEIGHT MINUS DAY 5 BODY WEIGHT 
Huntingdon Life Sciences 05-4287 

211-DIPE-DEV 

Females Summarv of Fetal Observations 

Fetuses Litters 

Observations Group 1 2 3 4 1 

{Major abnormalities in CAPITALS) Number examined 300 286 302 292 23 2

HYDROCEPHALYa, ANOPHTHALMIA; HYPOPLASTIC APICAL LUNG 1 

LOBE, THYMUS AND SPLEEN 

MICROPHTHALMIA/ANOPHTHALMIA, UNILATERAL 1 

EXOCCIPITAL FUSED TO 1ST CERVICAL ARCH 1 

MANDIBLE SHORTENED, MODERATE 1 

THORACIC VERTEBRA 13, LUMBAR VERTEBRA 1: ACENTRIC, 1 

MALFORMED 

FORELIMB SHORTENED, RIGHT 1 

ANAL ATRESIA, ABSENT SACRAL AND CAUDAL VERTEBRAE 1 1 

Additional subclavian artery 1 1 

Innominate artery absent 1 2 1 1 

Undescended thymic tissue 10 11 5 11 6 

aNoted as DOMED CRANIUM in external observations. 
Huntingdon Life Sciences 05-4287 

211-DIPE-DEV 

Females Summarv of Fetal Observations 

Fetuses Litters 

Observations Group 1 2 3 4 1 

(Major abnormalities in CAPITALS) Number examined 300 286 302 292 23 2

Ren.al papilla (e) not fully developed 1 2 1 1 

Ureter(s) distended 1 1 1 1 

Left umbilical artery 1 4 t 2 1 

Testis displaced, medially, left 1 1 

7th cervical rib(s) present 1 3 

Sternebra(e) malaligned 11 11 13 3 9 

Rib(s) rudimentary, 13th 1 3 1 

Rib(s) rudimentary, 14th 2 7 5 12 1 

25 presacral vertebrae 1 3 '1 

27 presacral vertebrae 1 
Huntingdon Life Sciences 05-4287 

z'11-DIPE-DEV 

Females Summarv of Fetal Observations I 

Fetuses Litters 

Observations Group 1 2 3 4 1 

(Major abnormalities in CAPITALS) Number examined 300 286 302 292 23 2

Hyoid body/arch(es) unossified 1 1 1 1 

Sternebra 5 and/or 6 unossified 1 3 3 1 

Metatarsals: 1 unossified, bilateral 2 
Huntingdon Life Sciences 05-4287 

Analytical Report 

STUDY TITLE 

Analytical Report For: 

Page 1 

Draft Final Report 

Appendix A 

Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats 

by Inhalation Exposure 

AUTHOR 

Yonggang Wang 

REPORT DATE 

08 May 2009 

STUDY NUMBER 

05-4287 
Huntingdon Life Sciences 05-4287 

SIGNATURES 

Written by: 

Analytical Report 

Yonggang Wang, M.S. 

Laboratory Manager 

Formulation Chemistry Services 

Reviewed by: ---------------

Kay Saladdin, B.S. 

Associate Director 

Formulation Chemistry Services 

Approved by: ______________ _ 

Barbara A. Litzenberger 

Director 

Analytical Services 

Date 

Date 

Date 

Page2 

Draft Final Report 

Appendix A 
Huntingdon Life Sciences 05-4287 

Analytical Report 

Table of Contents 

Page3 

Draft Final Report 

Appendix A 

Study Title Page ............................................................................................................... 1 

Signature Page ................................................................................................................ 2 

Table of Contents ............................................................................................................. 3 

1. Summary ...................................................................................................................... 4 

2. Experimental Procedures ............................................................................................. 4 

3. Results and Discussion ................................................................................................ 5 

Tables 

I Gasoline DIPE Vapor Condensate Characterization ("Test Substance Stability") ........ 6 

II Gasoline DIPE Vapor Condensate Characterization ("Pretest/Trials") ........................ 7 

Ill Gasoline DIPE Vapor Condensate Characterization ("Summary of Exposures") ........ 8 

IV-A Gasoline DIPE Vapor Condensate Characterization ("Exposure 4") ....................... 9 

IV-B Gasoline DIPE Vapor Condensate Characterization ("Exposure 11 ") ................... 10 

IV-C Gasoline DIPE Vapor Condensate Characterization ("Exposure 18") ................... 11 

Figures 

I A Typical Gas Chromatogram of Group 1 (Air Control) Chamber Sample ............. 12 

II A Typical Gas Chromatogram of Group 2 (Gasoline DIPE Vapor Condensate) 

Chamber Sample ................................................................................................... 13 

Ill A Typical Gas Chromatogram of Group 3 (Gasoline DIPE Vapor Condensate) 

Chamber Sample ................................................................................................... 14 

IV A Typical Gas Chromatogram of Group 4 (Gasoline DIPE Vapor Condensate) 

Chamber Sample ................................................................................................... 15 
Huntingdon Life Sciences 05-4287 

Analytical Report 

1. Summary 

Page4 

Draft Final Report 

Appendix A 

A neat sample of the Gasoline DIPE (Diisopropyl Ether) Vapor Condensate (G/DIPE VC) test 

substance was assayed before initiation of animal exposures to demonstrate stability by 

comparison to prior data at this Testing Facility. 

Inhalation chamber samples of the Gasoline DIPE Vapor Condensate were analyzed during each 

week of the study to demonstrate the stability of the test substance and the comparability of the 

test atmospheres to the neat test substance. 

The analytical method (HLS-001-02R2) was validated at Huntingdon Life Sciences (HLS). The 

analytical method involves, when appropriate, the collection of chamber air samples onto charcoal 

tubes, extraction with carbon disulfide, and quantification using Gas Chromatography with Flame 

Ionization Detection (FID). 

2. Experimental Procedures 

The analytical method (HLS-001-02R2) was validated by Formulation Chemistry at HLS. Details of 

the analytical method are maintained in this study file. 

The neat test substance and the charcoal tube samples containing the test substance were 

received from the Inhalation Department at HLS. Samples analyzed to determine the relative 

concentration of the major components of Gasoline DIPE Vapor Condensate were extracted from 

the charcoal tubes with Carbon Disulfide (CS2). The extracted solutions were analyzed by Gas 

Chromatography equipped with a Supelco PetrocolTM DH 150 (150m x 0.25mm, 1.0 J.liTl) column 

and Flame Ionization Detector (FID). PE Nelson TotaiChrom 6.2.1 installed on a personal 

computer was used for data collection and processing. 

Date of sample analysis is listed as follows: 

Interval Date of Exposures Date Received Date Analyzed 

Pretest/Stability N/AV 29 Mar 05 30-31 Mar 05 

Pretest /Trials 02 & 05Aug 05 02 & 05 Aug 05 03-05 Aug 05 

Exposure-4 09 & 11 Aug 05 09 & 11 Aug 05 09-11 Aug 05 

Exposure-11 16 Aug 05 16 Aug 05 16 -18 Aug 05 

Exposure-18 23Aug 05 23Aug 05 23- 24Aug 05 
Huntingdon Life Sciences 05-4287 

Analytical Report 

3. Results and Discussion 

Page 5 

Draft Final Report 

Appendix A 

Before the study, a Gasoline DIPE Vapor Condensate sample was analyzed to determine the 

stability of the area percent of the test substance's major components. During the trials and 

exposures, Gasoline DIPE Vapor Condensate air samples were analyzed to determine the area 

percent of the test substance's major components in the chamber. The results of the stability 

analysis, pretest, summary of animal exposures and individual animal exposures are presented in 

Tables I, II, Ill and IVA-IVC. Typical chromatograms of Groups 1, 2, 3, and 4 are presented in 

Figures I to IV. 
Huntingdon Life Sciences 05-4287 

Analytical Report 

Table I. Chamber Components Confirmation 

Page6 

Draft Final Report 

Appendix A 

Area Percent of Gasoline DIPE Vapor Condensate Test Substance Stability 

Pretest 

Area% 

Compound MeanTM Mean TM TM TM 

Std-1 for Std-2 Std-1 Std-2 

00-42058 00-42058 05-4287b 05-4287b 

lsobutane 1.61 1.60 1.34 1.42 

N-Butane 9.57 9.59 9.05 9.25 

3-Methyl-1-butene 0.35 0.34 0.34 0.34 

lsopentane 32.26 32.35 32.58 32.50 

N-Pentane 7.38 7.46 9.99 9.93 

Trans-2-pentene 1.11 1.14 2.16 2.16 

2,3-Dimethylbutane 1.76 1.78 1.10 1.08 

2-Methylpentane 5.31 5.29 5.00 5.45 

3-Methylpentane 3.21 3.20 3.23 3.16 

N-hexane & DIPE 22.24 22.16 22.17 21.78 

Methylcyclopentane 1.44 1.43 1.44 1.39 

2,4-Dimethylpentane 1.16 1.17 0.93 1.15 

Benzene 2.29 2.28 2.20 2.18 

2-Methylhexane 1.33 1.32 1.33 1.26 

2,3-Dimethylpentane 1.38 1.37 1.38 1.35 

3-Methylhexane 1.54 1.53 1.49 1.48 

lsooctane 1.71 1.69 1.65 1.58 

Toluene 3.08 3.09 2.43 2.34 

Total 98.73 98.79 99.81 99.80 

ND = none detected. <LOQ = Less than the limit of quantification = less than 

25% of the area count of the component in the test substance standard. 

a These values are obtained from the Summary report for Study 00-4205. 

b These values are obtained from standard injections made on 31 March 2005 for DIPE test substance 

stability (see Sequence# 001 in 05-4287). 

Note: The discrepancy in the N-Pentane and Trans-2-pentene values are due to 

the analytical columns used for each study analyses. The analytical column used 

for Study 00-4205 analyses produced a split peak for N-Pentane and Trans-2-

pentene but the column used for this study separated the two peaks entirely. 
Huntingdon Life Sciences 05-4287 

Compound 

lsobutane 

N-Butane 

3-Methyl-1-butene 

I so pentane 

N-Pentane 

Trans-2-pentene 

2,3-Dimethylbutane 

2-Methylpentane 

3-Methylpentane 

N-hexane & DIPE 

Methylcyclopentane 

2,4-Dimethylpentane 

Benzene 

2-Methylhexane 

2,3-Dimethylpentane 

3-Methylhexane 

lsooctane 

Toluene 

Total 

Analytical Report 

Table II. Chamber Components Confirmation 

Area Percent of Gasoline DIPE Vapor Condensate 

Pretest /Trials 

Area% 

DIPESTD DIPE STD Spiked Spiked Sample Sample 

(Tank#4) (Tank#S) Control1 Control2 101 201 

002_008 002_009 002_005 002_006 002_007 002_002 

1.40 1.59 1.44 1.45 ND 1.52 

9.30 9.70 9.41 9.52 ND 9.62 

0.36 0.35 0.36 0.35 ND 0.36 

32.61 32.29 32.74 33.00 ND 32.08 

10.15 9.81 10.19 10.27 ND 9.99 

2.35 2.18 2.43 2.41 ND 2.30 

1.70 1.63 1.42 1.40 ND 1.66 

5.23 5.28 5.24 5.14 ND 5.48 

3.24 3.19 3.34 3.34 ND 3.24 

19.12 20.13 19.87 19.25 ND 19.64 

1.46 1.44 1.15 1.23 ND 1.47 

1.22 1.19 1.27 1.20 ND 1.20 

2.32 2.23 2.38 2.32 ND 2.42 

1.28 1.27 1.34 1.28 ND 1.32 

1.28 1.24 1.37 1.32 ND 1.29 

1.52 1.48 1.59 1.55 ND 1.54 

1.65 1.50 0.50 1.09 ND 1.58 

3.11 2.93 3.09 3.02 ND 3.22 

99.30 99.43 99.13 99.14 0.00 99.93 

Page 7 

Draft Final Report 

Appendix A 

Sample Sample 

301 401 

002_003 002_004 

1.73 1.64 

10.55 10.19 

0.35 0.36 

32.85 32.85 

10.08 10.13 

2.25 2.28 

1.34 1.37 

5.17 5.03 

3.14 3.23 

18.65 19.66 

1.41 1.30 

1.17 1.22 

2.24 2.31 

1.21 1.29 

1.24 1.26 

1.49 1.55 

1.24 0.65 

2.91 2.98 

99.02 99.30 

ND = none detected. <LOQ = Less than the limit of quantification = less than 25% of the area count of the component in the test 

substance standard. 
Huntingdon Life Sciences 05-4287 

Analytical Report 

PageS 

Draft Final Report 

Appendix A 

Table Ill. Summary of Chamber Components Confirmation 

Mean Area Percent of Gasoline DIPE Vapor Condensate (Exposures 4, 11, and 18} 

Area% 

Compound Spiked TM Samples Samples Samples Samples 

Control-1 Standard-1 Group 1 Group2 Group3 Group4 

(Tank#5) 

I so butane 0.95 1.21 ND 1.59 1.71 1.44 

N-Butane 6.92 7.70 ND 10.43 10.56 9.32 

3-Methyl-1-butene 0.37 0.33 ND 0.36 0.36 0.34 

lsopentane 30.72 31.26 ND 32.88 32.67 32.50 

N-Pentanea 9.74a 8.92 ND 9.35 9.29 9.67a 

Trans-2-pentene a 1.89 ND 2.11 2.08 a 

2,3-Dimethylbutane 1.54 1.46 ND 1.47 1.46 1.34 

2-Methylpentane 5.72 5.38 ND 5.26 5.06 5.00 

3-Methylpentane 3.63 3.55 ND 3.21 3.17 3.20 

N-hexane & DIPE 22.50 21.71 ND 19.27 19.62 20.39 

Methylcyclopentane 1.67 1.68 ND 1.46 1.43 1.47 

2,4-Dimethylpentane 1.44 1.41 ND 1.21 1.18 1.24 

Benzene 2.87 2.50 ND 2.31 2.25 2.31 

2-Methylhexane 1.66 1.58 ND 1.46 1.27 1.34 

2,3-Dimethylpentane 1.58 1.52 ND 1.30 1.27 1.36 

3-M ethyl hexane 1.79 1.73 ND 1.56 1.48 1.54 

lsooctane 1.51 1.65 ND 1.31 1.48 1.58 

Toluene 3.74 3.70 ND 3.16 2.94 2.91 

Total 98.35 99.18 ND 99.70 99.28 96.95 

ND = none detected. 

a N-Pentane co-eluted with Trans-2-pentene during the analysis for exposure 11. 

baverage ofGroups II, Ill and IV only 

All Samples 0 Spiked TM 

Control-2 Standard-2 

(Tank#5) 

1.58 1.30 1.48 

10.10 8.67 9.40 

0.35 0.37 0.37 

32.68 32.94 32.07 

9.44a 9.05a 9.26a 

a a a 

1.42 1.52 1.67 

5.11 5.56 5.37 

3.19 3.42 3.26 

19.76 20.90 20.64 

1.45 1.59 1.50 

1.21 1.34 1.25 

2.29 2.42 2.35 

1.36 1.48 1.38 

1.31 1.51 1.38 

1.53 1.70 1.56 

1.46 1.82 1.61 

3.00 3.57 3.23 

97.24 99.16 97.78 
Huntingdon Life Sciences 05-4287 

Analytical Report 

Page9 

Draft Final Report 

Appendix A 

Table IV-A: Gasoline DIPE Vapor Condensate Characterization ("Exposure 4") 

Compound 

lsobutane 

N-Butane 

3-Methyl-1-butene 

lsopentane 

N-Pentane 

Trans-2-pentene 

2,3-Dimethylbutane 

2-Methylpentane 

3-Methylpentane 

N-hexane & DIPE 

Methylcyclopentane 

2,4-Dimethylpentane 

Benzene 

2-Methylhexane 

2,3-Dimethylpentane 

3-Methylhexane 

lsooctane 

Toluene 

Total 

NO = none detected. 

a Resample results 

DIPE DIPE 

STD1 STD2 

(Tank#S) (Tank#S) 

003_002 003_009 

1.50 1.45 

9.07 8.68 

0.37 0.34 

32.26 32.02 

9.62 9.56 

2.17 2.13 

1.65 1.54 

5.28 5.42 

3.29 3.40 

20.26 20.25 

1.50 1.58 

1.28 1.28 

2.34 2.41 

1.36 1.40 

1.36 1.43 

1.60 1.64 

1.43 1.66 

3.19 3.36 

99.53 99.55 

Area% 

Spiked Spiked Sample Sample Sample Sample 

Control1 Control2 1001 2001 3001 4001 

003_006 003_007 003_008 003_003 003_0103 003_005 

1.18 1.03 ND 1.98 1.66 1.67 

7.55 6.26 ND 12.46 10.22 10.32 

0.32 0.30 ND 0.38 0.37 0.36 

30.69 30.24 ND 32.72 32.80 32.45 

9.36 8.99 ND 9.51 10.14 9.99 

2.14 2.07 ND 2.23 2.32 2.28 

1.53 1.57 ND 1.36 1.39 1.50 

5.52 5.85 ND 4.36 5.09 5.11 

3.43 3.71 ND 2.80 3.21 3.11 

21.94 20.90 ND 18.42 19.25 19.67 

1.62 1.85 ND 1.28 1.47 1.41 

1.32 1.50 ND 1.16 1.20 1.17 

2.54 2.88 ND 2.18 2.32 2.24 

1.48 1.65 ND 1.33 1.25 1.22 

1.46 1.75 ND 1.30 1.25 1.26 

1.73 2.03 ND 1.57 1.49 1.45 

1.82 2.23 ND 1.37 1.50 1.41 

3.73 4.70 ND 3.39 3.05 2.87 

99.36 99.51 0.00 99.80 99.98 99.49 
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Analytical Report 

Page 10 

Draft Final Report 

Appendix A 

Table IV-B: Gasoline DIPE Vapor Condensate Characterization ("Exposure 11 ") 

Area% 

DIPE DIPE Spiked Spiked Sample Sample Sample Sample 

STD 1 STD2 Control1 Control2 1002 2002 3002 4002 

{Tank#5) (Tank #5) 

Compound 004_001 004_008 004_005 004_006 004_007 004_002 004_003 004_004 

lsobutane 1.18 1.50 0.90 1.36 ND 0.99 1.86 1.21 

N-Butane 6.84 9.32 7.17 9.65 ND 7.21 10.76 8.24 

3-Methyl-1-butene 0.32 0.45 0.37 0.44 ND 0.33 0.36 0.31 

lsopentane 30.09 31.63 30.61 32.93 ND 32.12 32.39 31.99 

N-Pentane 8.81 1 0.11a 11.54a 10.23a ND 10.57 9.81 10.78a 

Trans-2-pentene 1.96 a a a ND 2.37 2.19 a 

2,3-Dimethylbutane 1.57 1.73 1.80 1.48 ND 1.61 1.34 1.15 

2-Methylpentane 5.67 5.33 5.76 5.16 ND 6.03 5.04 4.96 

3-Methylpentane 3.59 3.21 3.78 3.39 ND 3.77 3.09 3.34 

N-hexane & DIPE 22.48 21.18 21.85 20.75 ND 19.63 19.52 20.55 

Methylcyclopentane 1.75 1.48 1.76 1.56 ND 1.72 1.42 1.61 

2,4-Dimethylpentane 1.46 1.25 1.50 1.32 ND 1.34 1.15 1.34 

Benzene 2.69 2.39 2.85 2.38 ND 2.63 2.25 2.54 

2-Methylhexane 1.62 1.37 1.63 1.47 ND 1.53 1.23 1.51 

2,3-Dimethylpentane 1.68 1.39 1.62 1.46 ND 1.40 1.27 1.50 

3-Methylhexane 1.91 1.55 1.91 1.64 ND 1.75 1.46 1.68 

lsooctane 1.88 1.60 1.16 1.65 ND 1.18 1.42 1.75 

Toluene 4.17 3.33 3.75 3.01 ND 3.67 3.03 2.96 

Total 99.67 98.82 99.96 99.88 0.00 99.85 99.59 97.42 

a N-pentane and Trans-2-pentene are coeluted .. 

NO = None Detected 
Huntingdon Life Sciences 05-4287 

Analytical Report 

Page 11 

Draft Final Report 

Appendix A 

Table IV-C: Gasoline DIPE Vapor Condensate Characterization ("Exposure 18") 

DIPE DIPE 

STD1 STD2 

(Tank#5) (Tank#5) 

Compound 005_002 005_009 

lsobutane 0.95 1.50 

N-Butane 7.20 10.19 

3-Methyl-1-butene 0.31 0.32 

lsopentane 31.42 32.57 

N-Pentane 8.32 8.10 

Trans-2-pentene 1.55 2.15 

2,3-Dimethylbutane 1.17 1.75 

2-Methylpentane 5.19 5.35 

3-Methylpentane 3.76 3.17 

N-hexane & DIPE 22.38 20.49 

Methylcyclopentane 1.80 1.43 

2,4-Dimethylpentane 1.50 1.21 

Benzene 2.48 2.26 

2-Methylhexane 1.73 1.36 

2,3-Dimethylpentane 1.52 1.33 

3-Methylhexane 1.68 1.48 

lsooctane 1.65 1.58 

Toluene 3.73 2.99 

Total 98.34 99.23 

a N-pentane and Trans-2-pentene are co-eluted. 

ND = None Detected 

Area% 

Spiked Spiked 

Control1 Control2 

005_001 005_007 

0.77 1.52 

6.03 10.11 

0.41 0.36 

30.87 32.65 

8.31 7.92 

1.95 1.66 

1.28 1.51 

5.87 5.68 

3.68 3.17 

23.71 21.04 

1.64 1.36 

1.49 1.21 

3.23 2.01 

1.88 1.31 

1.66 1.32 

1.74 1.44 

1.54 1.59 

3.73 3.00 

99.79 99.86 

Sample Sample Sample Sample 

1003 2003 3003 4003 

005_008 005_004 005_005 005_006 

ND 1.79 1.60 1.44 

ND 11.62 10.69 9.41 

ND 0.36 0.34 0.35 

ND 33.79 32.83 33.07 

ND 7.97 7.92 8.24 

ND 1.73 1.72 1.91 

ND 1.45 1.66 1.37 

ND 5.38 5.05 4.94 

ND 3.07 3.20 3.16 

ND 19.75 20.09 20.94 

ND 1.37 1.41 1.38 

ND 1.12 1.19 1.20 

ND 2.11 2.19 2.15 

ND 1.22 1.32 1.30 

ND 1.21 1.30 1.32 

ND 1.35 1.48 1.49 

ND 1.37 1.53 1.59 

ND 2.41 2.74 2.90 

0.00 99.07 98.26 98.16 
Huntingdon Life Sciences 05-4287 

Analytical Report 

Page 12 

Draft Final Report 

Appendix A 

Figure I. A Typical Gas Chromatogram of Group 1 (Air Control} Chamber Sample 

(File# 003_008.rst) 

10 20 30 40 50 60 70 60 90 100 110 120 130 140 150 160 

Response (mV) vs Time (minutes) 
Huntingdon Life Sciences 05-4287 

Analytical .Report 

Page 13 

Draft Final Report 

Appendix A 

Figure II. A Typical Gas Chromatogram of Group 2 (G/DIPE VC) Chamber Sample 

(File# 003_003.rst) 

~ ~ f8 8! r; ,; ~ ~ 

II II II II I II II I 

i ~~ j t, i 

~ 

~ il' u j I i5 ... ~ 

~· :;:;~ ~ ~~ I .g.! Hi j J I I j ~ i! .. 10 20 30 40 50 60 70 60 90 100 110 120 130 140 150 160 

Response (mV) vs Time (minutes) 
Huntingdon Life Sciences 05-4287 Page 14 

Draft Final Report 

Figure Ill. 

im 

i 

10 

Analytical Report Appendix A 

A Typical Gas Chromatogram of Group 3 (G/DIPE VC) Chamber Sample 

(File# 003_010.rst) 

~:g N ~ ~ ~ g Si 

~~ ~ rj :R g :8 ~ 

. ~ ~~ 

I 

ij :g ~ ll ~ 

~~~ ~ I 15 HI ~ if I J j ~ :j!!.~ ! -N ~ .2 

~~ "" ~~- "' 20 30 40 50 60 70 60 90 100 110 120 130 140 150 160 

Response (mV) vs Time (minutes) 
Huntingdon Life Sciences 05-4287 

Analytical Report 

Page 15 

Draft Final Report 

Appendix A 

Figure IV. A Typical Gas Chromatogram of Group 4 {G/DIPE VC) Chamber Sample 

(File# 003_005) 

0 

g 

II II II II I II II I 

I .. i ~ co 

~ w 

~ .. a. ll ~ 

!= ~~ ~ j a !f ~ ~ . ~ 

]~ q~ I ~ i .. ~ 

~ !~ ~ :i ~ j ~ 

.;, ~"' ,1: <W "' ~ 

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 

Response (mV) vs Time (minutes) 
Huntingdon Life Sciences 05-4287 

211-DIPE-DEV 

Inhalation Report 

TABLE OF CONTENTS 

Page 1 

Draft Final Report 

AppendixB 

1. IN"TRODUCTION ................................................................................................... . 

2. MATERIALS AND METHODS 

2.1. Husbandry During Exposure Periods ............................................................ .. 

2.2. Test Substance Administration and Chamber Operations ............................ . 

2.3. Exposure Concentration Determination ............... : ................... ~ .................... . 

3. FIGURES 

1. Chamber Generation System and 

Whole-Body Exposure Chamber (Group 1) ........................................ . 

2. Chamber Generation System and 

Whole-Body Exposure Chambers (Groups 2- 4) .............................. .. 

4. TABLES 

I. Summary ofln-Chamber Observations ........................................................ . 

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

III. Equipment List .............................................................................................. . 

IV. Chamber Distribution Records .................................................................... .. 

V. Miran Calibration .......................................................................................... . 

VI. Testing Room and Chambers Environmental Monitoring ............................ . 
Huntingdon Life Sciences 

1. INTRODUCTION 

05-4287 

211-DIPE-DEV 

Inhalation Report 

Page2 

Draft Final Report 

AppendixB 

This appendix presents the methodology for exposure generation, monitoring and 

results. 

2. MATERIALS AND METHODS 

2.1. HUSBANDRY DURING EXPOSURE PERIODS 

2.1.1. HOUSING 

Animals were individually housed in stainless steel wire mesh 

cages within a 1000 Liter glass and stainless steel whole-body 

exposure chamber. The placement of the animals in the 

whole-body exposure chamber was rotated daily to ensure uniform 

exposure of the animals. A description of the animal rotation is 

included in the raw data. 

2.1.2. FEED 

None was provided during exposure. 

2.1.3. WATER 

None was provided during exposure. 

2.1.4. ENVIRONMENTALCONDITIONS 

Chamber temperature and relative humidity were recorded every 

half-hour during exposure and maintained, to the maximum extent 

possible, within the ranges presented below. Excursions outside 

the specified range did not affect the integrity of the study. 

Temperature 

Desired: 

Actual: 

20 to 24°C 

20 to 25°C 
Huntingdon Life Sciences 05-4287 

211-DIPE-DEV 

Inhalation Report 

Page3 

Draft Final Report 

I AppendixB 

The daily average temperature within the chamber was outside the -

desired range on 7/19 days and 12/19 days for Groups I and III, 

respectively. 

Relative Humidity 

Desired: 

Actual: 

40 to 60% 

36 to 54% 

The daily average relative humidity within the chamber was 

outside the desired range on 2/19 days and 1119 days for Groups I 

and III, respectively. 

2.2. TEST SUBSTANCE ADMINISTRATION AND CHAMBER 

OPERATIONS 

2.2.1. ROUTE OF ADMINISTRATION 

Inhalation via whole-body exposures. 

2.2.2. TEST SUBSTANCE ADMINISTRATION 

The test substance was administered once daily as a vapor in the 

breathing air of the animals. The test atmosphere was generated by 

an appropriate procedure determined during pre-study trials. The 

trials were performed to evaluate the optimal set of conditions and 

equipment to generate a stable and uniform atmosphere at the 

target exposure levels. The method was described in the raw data 

of the study and is outlined in this report. 

2.2.3. TARGET EXPOSURE LEVELS 

Group 1 - 0 mg/m3 

Group 2 - 2000 mg/m3 

Group 3 - 10000 mg/m3 

Group 4 - 20000 mg/m3 
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AppendixB 

2.2.4. DURATION AND FREQUENCY OF ADMINISTRATION 

The test substance was administered once daily for 6 hours/day, 

7 days per week for Gestation Days (GD) 5-20. 

2.2.5. CHAMBER OPERATIONS 

The whole-body exposure chambers each had a volume of 

approximately 1 000 Liters. The chambers were operated. at a 

minimum flow rate of 200 Liters per minute. The final airflow was 

set to provide at least one air change (calculated by dividing the 

chamber volume by the airflow rate) in 5.0 minutes (12 air 

changes/hour) and a T99 equilibrium time (calculated by 

multiplying the air change by the exponential factor 4.6) of at most 

21-22 minutes. Initial settings foreach group were as follows: 

Group Airflow Rate Air Change T99 

(Lpm) (min) (min) 

1 210 4.7 22 

2 208 4.8 22 

3 216 4.6 21 

4 210 4.7 22 

The chamber atmospheres were exhausted through the in-house 

filtering system, which consisted of a coarse filter, a HEP A filter, 

-and an activated charcoal bed. 

· Chamber static pressure was recorded every half-hour during 

exposure. The chambers were operated for a minumum of 

23 minutes (approximate T99) following the exposure with clean air 

only to allow the atmosphere to clear. 

Refer to Figures 1 and 2, and Table III for equipment details. 
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Inhalation Report 

2.2.6. EXPOSURE PROCEDURE 

Group 1 

Page5 

Draft Final Report 

AppendixB 

Houseline nitrogen was delivered from a regulator with a 

backpressure gauge via W' tubing to a flowmeter regulated by a 

metering valve. This nitrogen flow was then directed into the 

turret of the 1 000 Liter glass and stainless steel exposure chamber 

where it was mixed with room air as it was drawn into the 

chamber. This nitrogen flow simulated the generation nitrogen 

flow for Groups 2, 3 and 4. 

Groups 2-4 

Houseline nitrogen was delivered from a regulator with a 

backpressure gal[ge through a stainless steel cross to create three 

flow systems: the test substance pressurization flow, the purge 

flow and the volatilization flow. 

The test substance pressurization flow was directed via ~"tubing 

through a metering valve, attached to a backpressure gauge, into 

the vapor inlet valve of the test substance cylinder. The metering 

valve was used to adjust and maintain the pressure within the 

cylinder. From the pressurized cylinder, the test substance flowed 

from the liquid outlet valve through a quick-disconnect fitting and 

through a filter to prevent equipment contamination. From the 

filter tube, the test substance flowed to a liquid flowmeter via 1

/ 8" 

tubing. The outlet of the flowmeter was regulated by a built-in 

metering valve. From this flowmeter and metering valve, the test 

substance flowed via 1/g" tubing onto the glass helix of a counter 

current volatilization chamber. The glass helix was heated by a 

nichrome wire, which was controlled by an autotransformer and 

was inserted in the center of the glass tube that supported the helix. 

The nitrogen for the purge flow system was directed, via ~" tubing 

to a flowmeter regulated by a metering valve. The purge nitrogen 

was delivered via 1

/ 8" tubing to the bottom of the tube containing 

the nichrome wire. This nitrogen flow continuously purged the 
I 

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Inhalation Report I 

Page6 

Draft Final Report 

AppendixB 

area surrounding the nichrome wire within the tube, thereby 

protecting the wire from oxidation. 

The nitrogen for the volatilization system was directed via W' 

tubing to a flowmeter regulated by a metering valve. From the 

flowmeter, the volatilization nitrogen flowed via W' tubing to a 

ball and socket joint at the bottom of the volatilization chamber. 

This nitrogen flowed up through the volatilization chamber passing 

over the coil and volatilizing the test substance. The pressure 

within the counter-current volatilization chamber was maintained 

slightly negative to the room and was monitored with a pressure 

gauge. 

This test substance laden nitrogen exited the top of the 

volatilization chamber through a glass elbow, which directed the 

flow, via W' tubing, to the turret of 1000 Liter glass and stainless 

steel exposure chamber. As the test substance laden nitrogen was 

drawn into the chamber, it was mixed with room air. 

Refer to Figures 1 and 2, and Table III for equipment details. 

2.3. EXPOSURE CONCENTRATION DETERMINATION 

2.3.1. NOMINAL CONCENTRATION 

A nominal exposure concentration was calculated daily. The flow 

of air through the chambers was monitored using appropriate 

calibrated equipment. The nominal concentrations in mg/m3 were 

determined by weighing the generation apparatus containing the 

test substance before and after the exposure and dividing the 

difference in these weights by the total volume of air used during 

the exposure (flowrate multiplied by total exposure time). 

Calculation 

Cone (mg/m3

) = amount consumed (g) x 1000 mglg x 1000 L/m3 

exposure duration (min) x airflow (Lpm) 
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Inhalation Report 

2.3.2. CHAMBER SAMPLING 

Page? 

Draft Final Report 

AppendixB 

Determinations of the exposure levels were made using a MIRAN® 

Ambient Air analyzer equipped with a strip chart recorder. The 

test atmosphere was drawn from the normal sampling portal 

through the MIRAN® and measurements were recorded at least 

4 times during each exposure. The exposure levels were 

determined by comparison of the measured absorbance to a 

calibrated response curve constructed using the same instrument 

settings. 

Also, one charcoal tube sample per chamber per week was 

analyzed by gas chromatography (GC) to characterized at least 

10 major components (comprising at least 80% by weight of the 

test substance) to show test substance stability and comparison 

between the neat liquid test substance and the vaporized test 

atmospheres. 

See Table III for equipment list. 

2.3.3. PARTICLE SIZE DISTRIBUTION 

Particle size distribution measurements were performed once 

during each week of exposure using a TSI Aerodynamic Particle 

Sizer. The samples were drawn for 20 seconds at a rate of 

5.0 Lpm. A computer was used to program the system to the 

appropriate settings prior to sampling. The particle size 

distributions were calculated by the computer and printed out. The 

mass median aerodynamic diameter, geometric standard deviation 

and total mass concentration were calculated. For comparison, 

room air samples were similarly collected at least once weekly. 

2.3.4. CHAMBER AND EXPOSURE ROOM ENVIRONMENT 

Chamber oxygen levels (maintained at least 19%) were measured 

pretest and at the beginning (Day 3), middle (Day 12) and end of 

the study (Day 19). 
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Page 8 

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AppendixB 

Air samples were taken in the vapor generation area pretest and at 

the beginning (Day 3), middle (Day 12) and end of the study 

(Day 19). Light (maintained approximately 30 to 40 foot-candles 

at 1.0 meters above the floor) and noise levels (maintained below 

85 decibels) in the exposure room were measured pretest and at the 

beginning (Day 3), middle (Day 12) and end of the study (Day 19). 
Huntingdon Life Sciences 

Group 1 

Note: 

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Inhalation Report 

Figure I 

Chamber Generation System 

And Whole Body Exposure Chamber 

1 000 L Exposure Chamber 

H-9 

H-7 

Drain 

Page9 

Draft Final Report 

AppendixB 

To Exhaust System 

' Sampling 

Ports 

/ 

1. Animals were individually housed on two levels within the exposure chamber. 
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Groups 2-4 Inhalation Report 

Figure II 

Chamber Generation System 

And Whole Body Exposure Chamber 

Page 10 

Draft Final Report 

AppendixB 

To Exhaust System 

1 000 L Exposure Chamber 

NUIOFO 

Valve 

Backpressure ' Gauge 

\ 

Plastic 

tubing 

Test Material 

Cylinder 

1!:::::===='\=====®:=lfl Generation Flowmeter 

Notes: 

Nupro 

Valve 

Volatilization 

Chamber 1--

Temp. 

HanCidit um1 y Gauge 

H-8 

H-9 

H-7 

Drain 

1. Animals were individually housed on two levels within the exposure chamber. 
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211-DIPE-DEV 

r I Inh:ti:-R:rt_" ____ ------I 

Table I 

Summary of In-Chamber Observations 

Exposure Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 

Group 1 - 0 mg/m3 

Within Normal Limits 6 12 18 24 24 24 24 24 24 24 24 24 24 24 24 24 18 12 

Group 2 - 2000 mg/m3 

Within Normal Limits 6 12 18 24 24 24 24 24 24 24 24 24 24 24 24 24 18 12 

Group 3 - 10000 mg/m3 

Within Normal Limits 6 12 18 24 24 24 24 24 24 24 24 24 24 24 24 24 18 12 

Group 4 - 20000 mg/m3 

Within Normal Limits 6 . 12 18 24 24 24 24 24 24 24 24 24 24 24 24 24 18 12 
Huntingdon Life Sciences 

Day Date Exposure 

Number Nominal Mean 

(mglm3) (mglm3) 

0 6-Aug-05 1 0 0.00 

1 7-Aug-05 2 0 0.00 

2 8-Aug-05 3 0 0.00 

3 9-Aug-05 4 0 0.00 

4 .10-Aug-05 5 0 0.00 

5 11-Aug-05 6 0 0.00 6 12-Aug-05 7 0 0.00 

7 13-Aug-05 8 0 0.00 

8 14-Aug-05 9 0 0.00 

9 15-Aug-05 10 0 0.00 

10 16-Aug-05 ll 0 0.00 

ll 17-Aug-05 12 0 0.00 

12 18-Aug-05 13 0 0.00 

13 19-Aug-05 14 0 0.00 

14 20-Aug-05 15 0 0.00 

15 21-Aug-05 16 0 0.00 

16 22-Aug-05 17 0 0.00 

17 23-Aug-05 18 0 0.00 

18 24-Aug-05 19 0 0.00 

Mean 0 

S.D. 0 

- -

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Appendix B - Table II 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group 1-0 mglmJ(Air only) 

Chamber Concentration 

Individual 

(mglm3) 

0.00 0.00 0.00 0.00 

0.00 0.00 0.00 0.00 

0.00 0.00 0.00 0.00 

0.00 0.00 - 0.00 0.00 

0.00 0.00 0.00 0.00 

0.00 0.00 0.00 0.00 

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 

0.00 0.00 0.00 0.00 

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 

0.00 0.00 0.00 0.00 

0.00 0.00 0.00 0.00 

0.00 0.00 0.00 0.00 

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 

0.00 0.00 0.00 0.00 

0.00 0.00 0.00 0.00 

0.00 0.00 0.00 0.00 

0.00 0.00 

---· 

Particle Size 

Determinations 

MMAD GSD TMC 

(J.lm) (mglm3) 

0.80ll 2.031 4.25E-03 

0.7675 1.792 4.78E-03 

8.2210 2.276 l.64E-03 

3.263 2.033 3.56E-03 

4.3 0.2 0.0 

Tem
Huntingdon Life Sciences 

Day Date Exposure 

Number Nominal Mean 

(mg/m3) (mg/m3) 

0 6-Aug-05 I 2000 1900 

I 7-Aug-05 2 2000 1900 

2 8-Aug-05 3 2000 2000 

3 9-Aug-05 4 2100 2100 

4 10-Aug-05 5 2000 2000 

5 11-Aug-05 6 2000 2000 

6 12-Aug-05 7 2000 2000 

7 13-Aug-05 8 2000 2000 

8 14-Aug-05 9 2000 2000 

9 15-Aug-05 10 2000 2000 

10 16-Aug-05 11 1900 2000 

11 17-Aug-05 12 2000 2000 

12 18-Aug-05 13 2000 2000 

13 19-Aug-05 14 2000 2000 

14 20-Aug-05 15 1900 2000 

15 21-Aug-05 16 2000 2000 

16 22-Aug-05 17 2000 1900 

17 23-Aug-05 18 2000 2000 

18 24-Aug-05 19 2000 2000 

Mean 1995 

S.D. 40 

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Appendix B - Table II 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group 2-2000 mg!m3 

Chamber Concentration 

Individual 

(mg/m3) 

1800 1800 1900 1900 

2000 1900 1800 1900 2200 1800 2000 2000 

2200 2000 2000 2000 2100 2000 1900 2100 

2000 2200 2100 1800 

2100 2000 2000 2000 2000 1900 2000 2000 

1900 2000 2000 2000 

2100 2000 2000 2000 2000 2000 2000 1900 2000 1900 2100 2100 

1900 1900 2000 2000 

1900 2100 2000 2000 

2000 1800 2000 2000 

2000 1900 2100 2000 

1900 1900 1900 2000 

2000 1900 2000 2000 

2100 1800 2000 2100 

1982 

93 

MMAD 

(!lm) 

3.882 

0.7935 

7.508 

4.061 

3.4 

Particle Size 

Determinations 

GSD TMC 

(mg/m3) 

2.954 7.23E-03 

2.294 6.14E-03 

2.057 2.08E-03 2.435 5.15E-03 

0.5 0.0 

Tem
Huntingdon Life Sciences 

Day Date Exposure 

Number Nominal Mean 

(mglm3) (mglm3) 

0 6-Aug-05 1 11000 10000 

1 7-Aug-05 2 11000 9800 

2 8-Aug-05 3 11000 9900 

3 9-Aug-05 4 11000 9600 

4 10-Aug-05 5 12000 10000 

5 11-Aug-05 6 12000 10000 

6 12-Aug-05 7 11000 9700 

7 13-Aug-05 8 12000 10000 

8 14-Aug-05 9 12000 9800 

9 15-Aug-05 10 12000 9800 

10 16-Aug-05 11 12000 10000 

11 17-Aug-05 12 12000 10000 

12 18-Aug-05 13 11000 10000 

13 19-Aug-05 14 11000 9800 

14 20-Aug-05 15 12000 10000 

15 21-Aug-05 16 12000 11000 

16 22-Aug-05 17 11000 10000 17 23-Aug-05 18 11000 10000 

18 · 24-Aug-05 19 11000 11000 

Mean 11474 

S.D. 513 

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Appendix B - Table II 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group 3- 10000 mglm3 

Chamber Concentration 

Individual 

(mglm3) 

10000 9600 10000 11000 

9400 9700 10000 9900 

10000 10000 9800 9900 

9300 9500 9700 .9900 

11000 10000 10000 10000 

9700 9900 9400 11000 

9900 9600 9400 9700 

10000 10000 10000 10000 

9700 10000 10000 9400 9600 9900 9700 10000 

9900 9800 11000 11000 

10000 10000 10000 10000 10000 10000 10000 10000 

9600 9700 9900 9900 

11000 9700 9700 10000 

11000 11000 11000 10000 

11000 10000 10000 10000 11000 9100 9600 11000 

11000 12000 10000 11000 

10072 

546 

Particle Size 

Determinations 

MMAD GSD TMC 

(!1m) (mglm3) 

0.8112 2.450 5.75E-03 

4.952 3.334 1.07E-02 

' 

5.523 2.246 1.91E-03 

3.762 2.677 6.12E-03 

2.6 0.6 0.0 

Tem
Huntingdon Life Sciences 

Day Date Exposure 

Number Nominal Mean 

(mg/m3) (mg/m3) 

0 6-Aug-05 1 20000 19000 

1 7-Aug-05 2 21000 21000 

2 8-Aug-05 3 19000 20000 

3 9-Aug-05 4 21000 20000 

4 10-Aug-05 5 20000 20000 

5 ll-Aug-05 6 20000 20000 

6 12-Aug-05 7 20000 20000 

7 13-Aug-05 8 20000 20000 

8 14-Aug-05 9 19000 19000 

9 15-Aug-05 10 20000 20000 

10 . 16-Aug-05 11 19000 20000 

II 17-Aug-05 12 20000 21000 

12 18-Aug-05 13 20000 20000 

13 19-Aug-05 . 14 20000 20000 

14 20-Aug-05 15 19000 20000 

15 21-Aug-05 16 19000 20000 

16 22-Aug-05 17 20000 20000 17 23-Aug-05 18 20000 20000 

18 24-Aug-05 19 20000 19000 

Mean 19842 

S.D. 602 

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Appendix B - Table II 

Chamber Monitoring Results 

Cumulative Exposure Record 

Group 4 - 20000 mg/m3 

Chamber Concentration 

Individual 

(mg/m3) 

18000 20000 20000 19000 

19000 20000 22000 21000 

21000 20000 19000 19000 21000 20000 20000 20000 

18000 18000 22000 20000 

18000 20000 21000 20000 19000 20000 20000 20000 19000 19000 19000 21000 

21000 20000 18000 18000 

20000 20000 19000 19000 

20000 20000 19000 19000 

21000 21000 21000 20000 

19000 20000 20000 20000 

20000 21000 18000 21000 

19000 20000 20000 20000 

20000 19000 20000 19000 

21000 20000 20000 20000 20000 20000 20000 20000 

17000 20000 20000 20000 

19776 

961 

Particle Size 

Determinations 

MMAD GSD TMC 

{l.tm) (mg/m3) 

0.7811 2.137 4.84E-03 

0.7640 2.260 9.80E-03 

7.810 2.272 4.24E-03 

3.118 2.223 6.29E-03 

3.3 0.1 0.0 

---

Tem
Huntingdon Life Sciences 

Exposure Chamber 

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Table III 

Equipment List 

1000 Liter glass and stainless steel chamber (Wahmann). 

Compound Reservoir 

Page 16 

Draft Final Report 

AppendixB 

Sponsor cylinder (100 gallons) and smaller cylinders (5 gallons) filled from Sponsor 

cylinder and used on study. 

Compound Generator 

Counter-Current Volatilization Unit, coiled glass rod insert, (Crown Glass Co., Inc. 

heated by a nichrome wire). 

Flowmeters 

Flowmeter, size 0-5,0-20, 0-40 Lpm (Dwyer® Instruments Inc.). 

Top TrakTM Mass Flow Meter size 0-1 Lpm, Model 821-1 (Sierra Instruments), 

calibrated prestudy with a Gilibrator®Bubble Generator, PIN D800286, SIN 569-S, 

flow cell assembly PIN D80026B, BD #1860). 

Flowmeter, size 0- 65 mm, PIN G6-02-R3, G6-03-R3, G6-04-R3 (Key Instruments). 

PressureNacuum Gauges 

Union Carbide backpressure gauge, PIN SG 6383. 

Norgreen backpressure gauge, PIN 9892K23. 

U.S. Gauge backpressure gauge, PIN 12672-1. 

Ashcroft backpressure gauge, PIN 733-47. 

Matheson® backpressure gauge, PIN 63-3161. 

Dwyer® Magnehelic® gauge. 

Variable Auto Transformer 

Variable Autotransformer, Type 3PN 1010 (Staco Energy Products Company). 

Chamber Air Flow 

Dwyer® Magnehelic® gauge (Dwyer® Instruments Inc.), calibrated prestudy with a Dry 

Gas Meter, Model8586799, (Singer). 

Chamber Static Pressure 

Dwyer® Magnehelic® gauge (Dwyer® Instruments Inc.), calibrated prestudy with 

Dwyer® Mark ll Manometer, Model25 (Dwyer® Instruments, Inc.). 
Huntingdon Life Sciences 

Regulators 

Norgreen, PIN 9892K23 

Union Carbide, PIN SG 3800 30. 

Metering Valve 

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Table III 

Equipment List 

Metering Valve, Model SS-4L (Nupro® Co.). 

Timers 

Gralab Universal Timer, Model171. 

Absorbent Tube Sampling 

Charcoal Tubes, ORB0-32, Lot #2000 (Supelco ). 

Vacuum Pumps 

Thomas Industries Inc., Model707CM50. 

Neptune Dyna-pump®, Model4K. 

Tubing 

Clear plastic, sizes 3/16", W', W' (Norton, Baxter). 

W' stainless steel. 

Stainless steel cross(Swage). 

Teflon®, size 1/8", \4", Yz". 

Plastic "T". 

Air .f\nalyzer 

Page 17 

Draft Final Report 

AppendixB 

~ lA-CVF Ambient Air Analyzer (Wilks) with a Cole Parmer Strip Chart 

Recorder, Model 201, and a Micronta® LCD Benchtop Digital Multimeter, 

Mode1No.22-195 

Balston® MicrofibreTM Disposable Filter Unit was attachedin-line. 

Syringe, size 0-25 mL, Gas-tight No. 1025 (Hamilton). 

Particle Sizer 

TSI Aerodynamic Particle. Sizer, Model 331001, and a DELL computer, Model 

486P/25, equipped with an Epson LQ-5707 printer, Model P630B. 
Huntingdon Life Sciences 

Environmental Monitoring 

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Table III 

Equipment List 

Page 18 

Draft Final Report 

AppendixB 

VWR Temperature and Humidity Gauge, tested prestudy with a Big Digit Traceable 

Hygrometer!fhermometer. 

Digital Sound Meter 840029 (SPER Scientific) 

Quantum Instruments Photo Meter 1. 

Oxygen/Gas Analyzer, Model1214S (Gastech) 

Thermometer 

T0 Sentry Digital Alarm Module, ModelllO. 

Balance 

Pelouze, No. 4040 

Mettler PM30000K (Mettler-Instrument Corporation). 

Miscellaneous 

Quick-disconnect fitting with toggle valve (REGO®). 

DFlf' filter, Grade DQ (Balston). 

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Inhalation Report 

Table V 

Miran Calibration 

Methodology for Gasoline DIPE Vapor Condensate 

Page20 

Draft Final Report 

AppendixB 

Settings: The instrument settings for the Miran 2225 Unit are summarized below: 

wavelength, microns 10.2 

pathlength, dial setting 5.30 

slit width, mm 1 

range, absorbance 1A 

response, seconds 1 

gam High 

chart speed, em/min 1 

chart volts 1 

Calibrations: The Miran® was turned on and allowed to warm up for approximately 

1 0 minutes. The cell was flushed with room air for approximately one minute. The loop 

was closed, the unit was zeroed and the calibration series was performed as shown below. 

The resultant data were plotted to obtain a calibration curve. Each observer used a 

separate syringe for calibration. . 

Injection Calculated Absorbance 

Volume Concentration I Operator 1 Operator 2 Average 

(IlL) (mg/m3

) (volts) (volts) (volts) 

8.5 1025 0.0338 0.0331 0.0335 

17 2050 0.0681 0.0691 0.0686 

85 10248 0.323 0.333 0.328 

175 21099 0.630 0.644 0.637 

210 25319 0.744 0.758 0.751 

1

Calculated Cone. (mg/m3

) = Injection volume CuL) x Density ( 0.68 mg/uL) x 1000 Llm3 

5.64 L (Volume ofMiran cell) 
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Table V 

Miran Calibration 

Page 21 

Draft Final Report 

AppendixB 

30000 ~------------------------------------~ 

25000 

~ 

{(0000 

(" 

n 

t15000 

~ n 

.8JOOOO 

5000 

y = -12213x4 + 16436x3 - 751. 71x2 + 30139x 

R2 = 0.9997 

0 +----+----+----+----+----+----+----+--~ 

0.0000 0.1000 0.2000 0.3000 0.4000 0.5000 0.6000 0.7000 0.8000 

Absorbance (Volts) 
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Table V 

Miran Calibration 

Page22 

Draft Final Report 

AppendixB 

Calibration Checks: A three-point calibration check of the Miran® was performed for each 

exposure prior to sampling the chambers. The parameters are shown below: 

Expected Acceptable 

Injection Calculated Absorbance Absorbance 

Volume Concentration Reading Range 

(IlL) (mg/m3) (volts) (volts) 

17 2050 0.0686 0.0583 - 0.0789 

85 10248 0.328 0.2788- 0.377 

175 21099 0.637 0.541-0.733 

The absorbance was recorded after each injection. The, absorbance was considered 

satisfactory if it was within 15% of the original calibration series. If any of the absorbance 

values fell outside the 15% range, the injection was rechecked as follows. The volume for 

the value that was out of range was reinjected twice. The closer pair of the three injections 

were averaged and the results were compared to the original curve. If the average of the 

pair was within the 15% range, the original was accepted. If the value of the average was 

outside the 15% range, the Study Director decided if a new graph was to be prepared. 
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Inhalation Report 

Table VI 

Page 23 

Draft Final Report 

AppendixB 

Testing Room and Chambers Environmental Monitoring 

Test Particle 

Interval Location Substance Light Noise Oxygen Sizing 

(mg/m3

) I (Ft Candles) (dB) (%) (mg/m3)a 

Pretest Room 813 - Front 0 32.3 62.8 -

Room 813 - Back 0 38.6 59.5 - Group 1-Chamber - - - 20 2.23 X 10-2 

Group 2 Chamber - - - 20 1.62 X 10-2 

Group 3 Chamber - - - 20 3.29 x w-2 

Group 4 Chamber - - - 20 3.30 x w-2 

Day3 Room 813 - Front 0 30.0 64.1 - Room 813 - Back 0 38.0 61.2 -

Group 1 Chamber - - - 20 

Group 2 Chamber - - - 20 

Group 3 Chamber - - - 20 

Group 4 Chamber - - - 20 

Day 12 Room 811 - Front 0 30.4 63.8 - Room 811- Back 0 38.7 59.3 - Group 1 Chamber - - - 20 

Group 2 Chamber - - - 20 

Group 3 Chamber - - - 20 

Group 4 Chamber - - - 20 

Day 19 Room 811 -Front 0 31.1 63.9 - Room 811- Back 0 38.0 60.1 -

Group 1 Chamber - - - 20 

Group 2 Chamber - - - 20 

Group 3 Chamber - - - 20 

Group 4 Chamber - - - 20 

·Pretest results presented above. For Weeks 1, 2 and 3 results, see CMR (Table II of 

Appendix B). 
10-MAR-2011 17:08 

Huntingdon Life Sciences 05-4287 

FEMALES GROUP 1 

ANIMAL# 

----

1901 1902 1903 

1904 1905 

1906 1907 

1908 1909 1910 

1911 

1912 

1913 

1914 1915 1916 1917 

1918 1919 

1920 

1921 

1922 1923 1924 

APPENDIX C 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL ANIMAL TERMINATION HISTORY 

0 MG/M3 

-------------------------------------------------------

TYPE OF DATE OF GESTATION PREGNANCY 

DEATH DEATH DAY STATUS 

TERMINAL SACRIFICE 22-AUG-05 21 p TERMINAL SACRIFICE 22-AUG-05 21 p 

TERMINAL SACRIFICE. 22-AUG-05 21 p 

TERMINAL SACRIFICE 22-AUG-05 21 p TERMINAL ·SACRIFICE 22-AUG-05 21 p TERMINAL SACRIFICE 22-AUG-05 21 p TERMINAL SACRIFICE 23-'AUG-05 21 p 

TERMINAL SACRIFICE 23-AUG-05 21 p TERMINAL SACRIFICE 23-AUG-05 21 p TERMINAL SACRIFICE 23-AUG-05 21 p 

TERMINAL SACRIFICE 23-AUG-05 21 p 

TERMINAL SACRIFICE 23-AUG-05 21 p TERMINAL SACRIFICE 24-AUG-05 21 p 

TERMINAL SACRIFICE 24-AUG-05 21 p TERMINAL SACRIFICE 24-AUG-05 21 NP TERMINAL SACRIFICE 24-AUG-05 21 p TERMINAL SACRIFICE 24-AUG-05 21 p 

TERMINAL SACRIFICE 24-AUG-05 21 p TERMINAL SACRIFICE 25-AUG-05 21 p TERMINAL SACRIFICE 25-AUG-05 21 p 

TERMINAL SACRIFICE 25-AUG-05 21 p TERMINAL SACRIFICE 25-AUG-05 21 p TERMINAL SACRIFICE 25-AUG-05 21 p TERMINAL SACRIFICE 25-AUG-05 21 p 

-------------------

NP-NOT PREGNANT, P-PREGNANT 
10-MAR-2011 17:08 

Huntingdon Life Sciences 05-4287 

FEMALES GROUP 2 

ANIMAL# 

2000 MG/M3 

APPENDIX C 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL ANIMAL TERMINATION HISTORY 

--------------------------------------------------

TYPE OF DATE OF GESTATION PREGNANCY 

DEATH DEATH DAY STATUS 

---------------------------------------------------------------

2901 TERMINAL SACRIFICE 22-AUG-05 21 p 

2902 TERMINAL SACRIFICE 22-AUG-05 21 p 2903 TERMINAL SACRIFICE 22-AUG-05 21 p 2904 TERMINAL SACRIFICE 22-AUG-05 21 p 2905 TERMINAL SACRIFICE 22-AUG-05 21 p 

2906 TERMINAL SACRIFICE 22-AUG-05 21 p 2907 TERMINAL SACRIFICE 23-AUG-05 21 p 2908 TERMINAL SACRIFICE 23-AUG-05 21 p 

2909 TERMINAL SACRIFICE 23-AUG-05 21 p 

2910 TERMINAL SACRIFICE 23-AUG-05 21 p 

2911 TERMINAL SACRIFICE 23-AUG-05 21 p 2912 TERMINAL SACRIFICE 23-AUG-05 21 p 2913 TERMINAL SACRIFICE 24-AUG-05 21 p 

2914 TERMINAL SACRIFICE 24-AUG-05 21 p 2915 TERMINAL SACRIF.ICE 24-AUG-05 21 p 

2916 TERMINAL SACRIFICE 24-AUG-05 21 p 

2917 TERMINAL SACRIFICE 24-AUG-05 21 p 2918 TERMINAL SACRIFICE 24-AUG-05 21 p 

2919 TERMINAL SACRIFICE 25-AUG-05 21 NP 2920 TERMINAL SACRIFICE 25-AUG-05 21 p 2921 TERMINAL SACRIFICE 25-AUG.-05 21 p 

2922 TERMINAL SACRIFICE 25-AUG-05 21 p 2923 TERMINAL SACRIFICE 25-AUG-05 21 p 2924 TERMINAL SACRIFICE 25-AUG-05 21 p 

--------------------------------

NP-NOT PREGNANT, P-PREGNANT 
10-MAR-2011 17:08 

Huntingdon Life Sciences 05-4287 

FEMALES GROUP 3 

ANIMALll 

10000 MG/M3 

APPENDIX C 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL ANIMAL·TERMINATION HISTORY 

-------------------------------------------------------

TYPE OF DATE OF GESTATION PREGNANCY 

DEATH DEATH DAY STATUS 

----------------------------------------------------------------------------

3901 TERMINAL SACRIFICE 22-AUG-05 21 p 3902 TERMINAL SACRIFICE 22-AUG-05 21 p 

3903 TERMINAL SACRIFICE 22-AUG-05 21 p 3904 TERMINAL SACRIFICE 22-AUG-05 21 p 3905 TERMINAL SACRIFICE 22-AUG-05 21 p 3906 TERMINAL SACRIFICE 22-AUG-05 21 p 

3907 TERMINAL SACRIFICE 23-AUG-05 21 p 

3908 TERMINAL SACRIFICE 23'-AUG-05 21 p 3909 TERMINAL SACRIFICE 23-AUG-05 21 p 3910 TERMINAL SACRIFICE 23-AUG-05 21 p 3911 TERMINAL SACRIFICE 23-AUG-05 21 p 3912 TERMINAL SACRIFICE 23-AUG-05 21 p 

3913 TERMINAL SACRIFICE 24-AUG-05 21 p 3914 TERMINAL SACRIFICE 24-AUG-05 21 p 3915 TERMINAL SACRIFICE 24-AUG-05 21 p 3916 TERMINAL SACRIFICE 24-AUG-05 21 p 

3917 TERMINAL SACRIFICE 24-AUG-05 21 p 3918 TERMINAL SACRIFICE 24-AUG-05 21 p 3919 TERMINAL SACRIFICE 25-AUG-05 21 p 

3920 TERMINAL SACRIFICE 25-AUG-05 21 p 3921 TERMINAL SACRIFICE 25-AUG-05 21 p 3922 TERMINAL SACRIFICE 25-AUG-05 21 p 3923 TERMINAL SACRIFICE 25-AUG-05 21 p 

3924 TERMINAL SACRIFICE 25-AUG-05 21 p 

-------------------------

NP-NOT PREGNANT, P-PREGNANT 
10-MAR-2011 17:08 

Huntingdon Life Sciences 05-4287 

FEMALES GROUP 4 

ANIMALll 

20000 MG/M3 

APPENDIX C 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL ANIMAL TERMINATION HISTORY 

--------------------------------------------------------

TYPE OF DATE OF GESTATION PREGNANCY 

DEATH DEATH DAY STATUS 

-------------------------------------------------------------------------

4901 TERMINAL SACRIFICE 22-AUG-05 21 p 4902 TERMINAL SACRIFICE 22-AUG-05 21 p 

4903 TERMINAL SACRIFICE 22-AUG-05 21 p 4904 TERMINAL SACRIFICE 22-AUG-05 21 p 4905 TERMINAL SACRIFICE 22-AUG-05 21 p· 4906 TERMINAL SACRIFICE 22-AUG-05 21 p 

4907 TERMINAL SACRIFICE 23-AUG-05 21 p 4908 TERMINAL SACRIFICE 23-AUG-05 21 NP 4909 TERMINAL SACRIFICE 23-AUG-05 21 p 

4910 TERMINAL SACRIFICE 23-AUG-05 21 p 4911 TERMINAL SACRIFICE 23-AUG-05 21 p 4912 TERMINAL 23-AUG-05 21 p 

4913 TERMINAL SACRIFICE 24-AUG-05 21 p 4914 TERMINAL SACRIFICE 24-AUG-05 21 p 4915 TERMINAL SACRIFICE 24-AUG-05 21 p 4916 TERMINAL SACRIFICE ,24-AUG-05 21 p 

4917 TERMINAL SACRI.FICE 24-AUG-05 21 p 4918 TERMINAL SACRIFICE 24-AUG-05 21 p 4919 TERMINAL SACRIFICE 25-AUG-05 21 NP 

4920 TERMINAL SACRIFICE 25-AUG-05 21 p 49;21 TERMINAL SACRIFICE 25-AUG-05 21 p 4922 TERMINAL SACRIFICE 25-AUG-:-05 21 p 4923 TERMINAL SACRIFICE 25-AUG-05 21 p 

4924 TERMINAL SACRIFICE 25-AUG-05 21 p 

----------------------------------------------

NP-NOT PREGNANT, P-PREGNANT 
10-MAR-2011 17:13 

Huntingdon Life Sciences 05-4287 

APPENDIX D 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

GROUP 1 0 MG/M3 

FEMALEJI OBSERVATIONS 

----

1901 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

1902 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

1903 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

1904 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

1905 TERMINAL SACRiFICE 

WITHIN NORMAL LIMITS 

1906 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

1907 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

1908 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

1909 TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN NORMAL LIMITS 

1910 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

1911 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

INDIVIDUAL CLINICAL OB.SERVATIONS DURING GESTATION 

DAY OF 

GESTATION 

1 1 1 1 1 1 1 1 1 1 2 2 

0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 

p 

p p p p p.p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p 

p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 

CODE: 1-SLIGHT ·2-MODERATE P-PRESENT 
10-MAR-2011 17:13 

Huntingdon Life Sciences 05-4287 

APPENDIX D 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

GROUP 1 0 MG/M3 

OBSERVATIONS 

---------------------

'1912 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

1913 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

1914 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

1915 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

1916 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

1917 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

1918 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

1919 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

1920 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

1921 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

1922 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

INDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATION 

DAY OF 

GESTATION 

1 1 1 1 1 1 1 1 1 1 2 2 

0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 

p 

p p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p"p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p,p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p.p p p p p p p p p p 

p p p p p p p p p p p p p p p p p p p 

p p p p p p p p p p p p p p p p p p p p 

p p p p.p p p p p p p p p p p p p p? p 

CODE: 1-SLIGHT 2-MODERATE 3-MARKED,P-PRESENT 
10-MAR-2011 17:13 

Huntingdon Life Sciences 05-4287 

APPENDIX D 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

GROUP 1 

FEMALE# 

1923 

1924 

0 MG/M3 

OBSERVATIONS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERM!NAL SACRIFICE 

WITHIN NORMAL LIMITS 

INDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATION 

DAY OF 

GESTATION 

1 1 1 1 1 1 1 1 1 1 2 2 

0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 

p 

p p p p p p p p p p p p p p p p p p p p P

p 

p p p p p p p p p p p p p p p p p p p 

CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT 
10-MAR-2011 17:13 

Huntingdon Life Sciences 05-4287 

APPENDIX D 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

GROUP 2 

FEMALE# 

2901 

2902 

2903 

2904 

2905 

2906 

2907 

2908 

2909 

2910 

20.00 MG/M3 

OBSERVATIONS 

TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

INDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATION 

DAY OF 

GESTATION 

1 1 1 1 1 1 1 1 1 1 2 2 

0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 

p 

p p 

p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p Pc p p p p p 

p 

p p p p p p p p p p p p p p p p p p p 

p p p 

p p p p p p p p p p p p p p p 

p p p p p p p p p p p p p p p p p p 

p p p p p p p p p p p p p p p p p p 

CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT 
10-MAR-2011 17:13 

Huntingdon Life Sciences 05-4287 

APPENDIX D 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

GROUP 2 2000 MG/M3 

FEMALE* OBSERVATIONS 

---------------------

2911 TERMINAL SACRIFICE 

WITHIN NORMAL'LIMITS 

2912 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

2913 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

2914 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

2915 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

2916 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

2917 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

2918 TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN NORMAL LIMITS 

2919 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

2920 SACRIFICE 

WITHIN NORMAL LIMITS 

2921 TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

INDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATION 

DAY OF 

GESTATION 

1 1 1 1 1 1 1 1 1 1 2 2 

0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 

p 

P P P P.P P P P P P P P P P P P P P P 

p 

p p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p-p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p p p 

p 

p p p p. p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p p p 

p 

p p p p p p 

p p p p p p p p p p p p p p 

p p p p p p p p p p p p p p p p p p p p p 

p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 

CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT 
10-MAR-2011 17:13 

Huntingdon Life Sciences 05-4287 

APPENDIX D 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

GROUP 2 

FEMALE# 

2922 

.2923 

.2924 

2000 MG/M3 

OBSERVATIONS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

INDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATION 

DAY OF 

GESTATION 

1 1 1 1 1 1 1 1 1 1 2 2 

0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 

p 

p p p p p p p p p p p p p p p p p p p p p 

p 

p p p P p p p p p P p .P p p p p p p p p P 

p 

p p p p p p p p p p p p p p p p p p p p p 

CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT 
10-MAR-201'1 17:13 

Huntingdon Life Sciences 05-4287 

APPENDIX D 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATION 

GROUP 3 

FEMALE# 

3901 

3902 

3903 

3904 

3905 

3906 

3907 

3908 

3909 

10000 MG/M3 

OBSERVATIONS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN NORMAL LIMITS 

·TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN NORMAL 

TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN NORMAL LIMITS 

CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT 

DAY OF 

GESTATION 

1 1'1 1 1 1 l 1 1 1 2 2 

0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 

p 

p p p p p p p p p p p p p p p p p p 

p 

p 

p p p p p p p p p p p p p p p p p 

p 

p p 

p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p 

p 

p 

p p p p p p p _p p p p p p p p p p 

p 

p p p 

p p p p p p p p p p p p p p p p 

p 

p p p p 

p p p p p p p p p p p p p p p 

p 

p 

p p p p p p p p p p p p p p p p p p 
10-MAR-2011 17:13 

Life Sciences 05-4287 

APPENDIX D 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

fNDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATION 

GROUP 3 

FEMALElf 

3910 

3911 3912 

3913 

3914 

3915 

3916 

3917 

3918 

10000 MG/M3 

OBSERVATIONS 

TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

CODE: 1-SLIGHT 3-MARKED P-PRESENT 

DAY OF 

GESTATION 

1 1 1 1 1 1 1 1 1 1 2 2 

0 1 2 3 4 5 6 7 8 9 o. 1 2 3 4 5 6 7 8 9 0 1 

p 

p 

p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p 

p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p p p 

p 

p p p p p p 

p p p p p p p p p p p p p p 

p 

p p P, 

p p p p p p p p p p p p p p p p p 

p 

. p p 

p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p p p 
10-MAR-2011 17:13 

Huntingdon Life Sciences 05-4287 

APPENDIX D 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATION 

GROUP 3 

FEMALElf 

3919 

3920 

3921· 

3922 

3923 

3924 

10000 MG/M3 

OBSERVATIONS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN.NORMAL LIMITS 

TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN NORMAL LIMITS 

CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT 

DAY OF 

GESTATION 

1 1 1 1 1 1 1 1 1 1 2 2 

0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0.1 

p 

p p p p p p p p p p p p p p p p p p p p p 

p 

p p 

p p p p p p p p p p p p p p p p p p p 

p 

p p p 0 

p p p p p p p p p p p p p p p p p p 

p 

p p 

p p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p 

p p p p p p p p p p p p p 
10-MAR-2011 17:13 

Huntingdon Life Sciences 05-4287 

APPENDIX D 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL CLINICAL OBSERVATIONS DURING 

GROUP 4 

FEMALE;# 

4901 

4902 

4903 

4904 4905 

4906 

4907 4908 

4909 

4910 

20000 MG/M3 

OBSERVATIONS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

ALOPECIA - GENERAL 

CHROMODACRYORRHEA - UNILATERAL 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

INCISORS MALOCCLUDED 

MASS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN NORMAL LIMITS 

CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT 

DAY OF 

GESTATION 

1 1 1 1 1 1 1 1 1 1 2 2 

0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 

p 

p p p p p p p p p p p p p p p p p p 

p p p p p 

p p p p p 

p 

p p p p 

p p p 

p 

2 2 2 2 2 2 2 2 

p 

p 

p p p p p p p p p 

p 

P P P P R P P P P P P P P P P P P 

p 

p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p p 

2 2 2 2 2 

p 

p p p p p p p p p p p p p p p p p p p 

p 

p p p 

p p p p p p p p p p p p p p p p 
10-MAR-2011 17:13 

Huntingdon Life Sciences 05-4287 

APPENDIX D 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATION 

GROUP 4 

FEMALE# 

4911 

4912 

4913. 

4914 

4915 

4916 

4917 

4918 

4919 

4920 

20000 MG/M3 

OBSERVATIONS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN NORMAL LIMITS 

TERMINAL SACRifiCE 

NASAL DISCHARGE RED 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

CHROMODACRYORRHEA - UNILATERAL 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

WITHIN. NORMAL LIMITS 

TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN NORMAL LIMITS 

CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT 

DAY OF 

GESTATION 

1 1 1 1 1 1 1 1 1 1 2 2 

0 1 2,3 4 56 7 8 9 0 1 2 3 4 56 7.8 9 0 1 

p 

p p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p p p 

p 

p p p p 

p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p p p p p p 

p 

p p p p p 

p p p p p p p p p p p p p p p 

p 

p p p p p p p 

p p p p p p p p p p p p p 

p 

p 

p p p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p p p p p.p p p p p p 

p 

p p p p 

p p p p p p p p p p p p p p p p p 
10-MAR-2011 17:13 

Huntingdon Life Sciences 05-42S7 

APPENDIX D 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATION 

GROUP 4 

FEMALElf 

4921 

4922 

4923 

4924 

20000 MG/M3 

OBSERVATIONS 

TERMINAL SACRIFICE , 

NASAL DISCHARGE - RED 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

NASAL DISCHARGE - RED 

WITHIN NORMAL LIMITS 

TERMINAL SACRIFICE 

CHROMODACRYORRHEA - BILATERAL CHROMODACRYORRHEA -,UNILATERAL 

WITHIN NORMAL LIMITS 

CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT 

DAY OF 

GESTATION 

1 1 1 1 1 1 1 1 1 1 2 2 

0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 g 9 0 1 

p 

p 

p p p p p p p p p p p p p p p p p p p p 

p 

p p p p 

p p p p p p p p p p p p p p p p p 

p 

p p p p p p p p p p p 

p p p p p p p p p p 

p 

p 

p p p p p p p p p p p p p p 

P PPPPP 
10-MAR-2011 17:16 

Huntingdon Life Sciences 05-4287 

APPENDIX E 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL GESTATION BODY WEIGHTS (GRAMS) 

GROUP 1 0 MG/M3 

---------------------------------------------

DAY OF GESTATION 

ANIMALlf 4 5 8 11 14 17 20 21 

------------------------------------------------------------------------------------

1901 212 230 259 275 303 332 380 389 1902 213 228 241 261 280 308 354 360 1903 229 223 245 264 286 322 368 384 

1904 230 248 260 275 299 328 387 397 1905 233 254 271 295 316 343 387 393 1906 249 264 278 296 318 342 393 406 1907 209 218 235 256 266 291 328 334 

1908 220 239 256 288 311 342 388 400 1909 232 239 255 278 299 335 392 400 

1910 233 240 255 269 284 312 361 372 

1911 247 258 272 294 314 351 398 411 1912 253 264 274 295 313 345 396 397 

1913 232 239 250 270 286 314 351 353 

1914 241 245 253 278 296 324 374 384 

1915x NP 245 243 257 258 253 263 263 264 

1916 246 261 270 298 318 357 410 414 1917 249 254 269 297 316 357 405 415 

1918 259 269 283 310 338 378 420 434 1919 214 223 245 268 288 306 351 368 1920 222 232 246 261 280 292 313 313 

1921 231 236 253 275 295 324 373 382 1922 235 242 257 282 295 324 368 382 

1923 243 247 264 292 317 359 405 426 1924 245 259 274 297 316 353 395 405 

MEAN 234 244 259 281 301 332 378 388 

S.D. 14.0 14.6 12.8 14.9 17.0 22.2 26.3 28.6 

N 23 23 23 23 23 23 23 23 NP=NOT PREGNANT x=EXCLUDED FROM MEAN 
10-MAR-2011 17:16 

Huntingdon Life Sciences 05-4287 

APPENDIX E 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL GESTATION BODY WEIGHTS (GRAMS) 

GROUP 2 2000 MG/M3 

--------------------------------------

DAY OF GESTATION 

ANIMAL# 4 5 8 11 14 17 20 21 

----------------------------------------------------------------------

2901 209 221 233 245 263 286 323 327 2902 216 230 234 245 262 287 330 333 2903 224 236 246 267 280 302 350 354 

2904 232 244 271 288 313 342 385 397 2905 235 252 268 285 306 339 393 404 2906 241 257 275 290 313 337 381 385 

2907 202 221 233 255 273 307 353 365 2908 '214 224 240 256 274 299 342 355 2909 228 235 252 270 286 312 353 364 

2910 234 248 263 279 302 342 389 395 2911 246 258 274 297 310 342 393 401 

2912 270 280 294 318 335 376 435 454 2913 229 234 251 273 291 322 369 372 2914 239 244 255 279 281 302 327 328 

2915 244 257 271 290 307 343 389 399 2916 248 257 276 293 312 357 405 418 2917 254 261 280 295 306 334 366 381 

' 2918 266 277 290 315 326 355 398 405 

2919x NP 211 218 217 228 235 237 232 240 2920 222 2i9 237 254 270 298 335 347 

2921 228 239 256 278 295 326 366 370 2922 233 229 250 269 278 318 339 350 

2923 242 257 274 292 312 353 408 426 2924 243 253 267 285 301 335 387 396 

MEAN 235 245 260 279 296 327 370 379 

S.D. 16.6 16.5 18.0 19.6 20.3 24.3 29.9 32.7 

N 23 23 23 23 23 23 23 23 

NP=NOT PREGNANT X=EXCLUDED FROM MEAN 
10-MAR-2011 17:16 

Huntingdon Life Sciences 05-4287 

APPENDIX E 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL GESTATION BODY WEIGHTS (GRAMS) 

GROUP 3 10000 MG/M3 

----------------------------------------

DAY OF GESTATION 

ANIMAL# 4 5 8 11 14 17 20 .21 

---------------------------------------------------------------------------------

3901 210 230 232 245 262 284 320 326 3902 219 242 253 273 295 313 356 363 3903 219 231 249 267 291 310 . 362 375 

3904 233 245 263 285 299 327 379 379 3905 237 248 256 268 286 322 379 388 3906 246 245 267 279 295 338 386 392 3907 206 218 229 245 267 302 337 344 

3908 212 226 236 255 273 294 333 338 3909 231 245 255 278 296 325 370 370 3910 241 246 257 274 287 318 367 377 

3911 243 254 263 285 302 336 393 401 3912 264 275 280 299 315 345 390 399 

3913 232 235 260 276 293 328 354 370 

3914 241 250 267 290 305 333 375 390 3915 246 258 268 283 292 309 324 324 3916 248 257 270 292 309 348 399 408 3917 252 256 275 297 319 357 401 408 

3918 258 263 280 294 304 337 377 394 3919 216 227 237 254 273 309 354 368 3920 218 227 242 262 281 313 365 374 

3921 223 230 243 255 272 295 329 334 3922 . 233 239 253 271 294 338 393 408 3923 241 249 263 275 293 323 373 .389 3924 245 258 260 283 305 344 388 398 

MEAN 234 244 256 274 292 323 367 376 

S.D. 15.7 13.9 14.4 15.6 14.8 18.7 24.2 25.9 

N 24 24 24 24 24 24 24 24 
10-MAR-2011 17:16 

Huntingdon Life Sciences 05-4287 

APPENDIX E 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY' 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL GESTATION BODY WEIGHTS (GRAMS) 

GROUP 4 20000 MG/M3 

-----------------------------------

DAY OF GESTATION 

ANIMAL# 4 5 8 11 14 17 20 21 

-----------------------------------------------------------------------------

4901 209 220 226 243 260 281 330 328 4902 213 225 239 255 274 297 342 352 

4903 228 244 248 267 278 306 369 376 

4904 231 236 249 270 285 319 370 380 4905 237 252 260 275 293 327 379 383 4906 239 250 262 279 302 329 378 389 4907· 211 216 229 248 267 291 335 340 

4908x NP 214 218 222 230 221 228 237 233 

4909 222 231 233 243 255 279 313 324 4Q10 235 242 264 286 301 328 373 386 4911 241 254 264 282 294 326 378 387 4912 247 257 271 291 306 332 379 386 

4913 235 246 257 272 289 322 365 372 4914 244 254 262 285 297 327 368 380 

4915 245 253 261 279 291 317 357 371 4916 246 254 261 278 295 326 374 389 4917 258 269 282 303 318 362 408 417 

4918 259 265 275 300 308 350 381 394 4919x NP 212 217 215 217 225 223 221 218 4920 220 224 237 258 275 306 3'38 348 4921 229 234 247 268 279 315 348 358 4922 233 246 261 276 294 327 373 366 

4923 236 247 250 275 280 308 345 351 4924 261 271 286 325 343 389 439 442 

MEAN 235 245 256 275 290 321 365 374 

S.D. 14.6 15.2 16.3 19.5 19.5 24.9 27.1 27.4 

N 22 22 22 22 22 22 22 22 NP=NOT PREGNANT x=EXCLUDED FROM MEAN 
10-MAR-2011 17:17 

Huntingdon Life Sciences 05-4287 

GROUP 1 0 MG/M3 

APPENDIX F 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL GESTATION BODY WEIGHT GAIN (GRAMS) 

DAY OF GESTATION 

ANIMAL# 4 - 5 5 - 8 8 - 11 11 - 14 14 - 17 17 - 20 20 - 21 5 - 14 14 - 21 

1901 1902 1903 

1904 1905 1906 

1907 1908. 

1909 1910 1911 

1912 1913 1914 

1915x NP 

1916 1917 

1918 1919 

1920 1921 

1922 

1923 

1924 

MEAN 

S.D. 

N 

NP=NOT PREGNANT 

18 15 

-6 

19 

21 

14 

8 

19 

8 

7 

12 

11 

8 

4 

-2 

15 

5 

10 

9 

9 

6 

7 

5 

14 

10 

6.1 

23 

29 14 22 

12 

17 15 

18 17 

16 15 

14 

10 11 

9 

14 

9 

15 

15 

21 

14 

17 

15 

17 

14 

15 

4.5 

23 

16 19 19 

15 

24 

18 

21 

32 

22 

15 

22 

21 20 25 

1 

28 29 

27 

24 

16 

22 25 

28 23 

22 

4.7 

23 

x=EXCLUDED FROM MEAN 

28 

.20 

22 

24 

22 22 

10 

23 

21 14 

20 

17 16 18 

-6 

20 

19 

27 

20 

19 

20 14 

25 

19 

20 

4.2 

23 

29 28 

35 

29 27 24 

25 

31 

36 

28 

37 

33 

29 28 

11 

39 

41 40 18 

12 

29 28 

42 

37 

31 

7.3 

23 

48 47 46 

59 

44 

51 

37 

46 

57 50 

47 

51 

37 

50 

-1 

53 

48 

43 45 

21 

49 45 

46 

43 

46 

7.6 

23 

9 

5 

17 

10 

6 

14 

6 

12 

8 

11 13 

1 

2 

10 

1 

4 

10 

14 

17 

0 

9 

,14 

21 

10 

10 

5.2 

23 

73 

53 

63 

51 

63 

55 

49 

72 

60 

44 

56 

49 46 

51 

10 

57 

62 

69 65 

48 

59 54 

69 

56 

57 

8.4 

23 

86 80 

98 

98 

77 

88 

68 

89 

101 

88 

97 

85 

68 

88 

11 

96 99 

96 

80 

33 

87 

87 

109 

89 

86 

15.5 

23 

5 - 21 

159 132 161 

149 

139 

143 

116 

161 161 

132 

153 

133 

114 139 

21 

153 

161 

165 145 

81 

146 141 

179 146 

144 

20.6 

23 
10-MAR-2011 17:17 

Huntingdon Life Sciences 05-4287 

APPENDIX F 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL GESTATION BODY WEIGHT GAIN (GRAMS) 

GROUP 2 2000 MG/M3 

DAY OF GESTATION 

ANIMALll 4 - 5 5 - 8 8 - 11 11 - 14 14 - 17 17 - 20 20 - 21 5 - 14 14 - 21 

2901 2902 2903 

2904 2905 2906 2907 

2908 2909 2910 

2911 2912 2913 

2914 

2915 

2916 2917 

2918 

2919x NP 

2920 

2921 

2922 2923 2924 

MEAN 

S.D. 

N 

NP=NOT PREGNANT 

12 

14 

12 

12 17 16 

19 

11 

7 

13 

12 

10 

5 

5 

12 

9 

.8 

11 

6 

8 

11 

-4 

15 10 

11 

4.7 

23 

11 

3 

10 

27 

16 18 13 

16 

17 

16 

16 

14 

17 

11 

14 

19 19 

13 

-1 

8 

17 

21 

16 

14 

15 

4.T 

23 

13 11 

22 

17 17 

15 

21 

16 18 16 

23 

24 

22 

24 

18 

17 

15 

25 11 

17 

22 

18 

19 18 

19 

3.6 

23 

x=EXCLUDED FROM MEAN 

17 17 

13 

26 22 24 

18 

18 

17 

23 

14 

17 

19 

3 

18 

20 

11 

11 

7 

16 

17 

9 

20 

16 

17 

5.0 

23 

23 

25 

22 

28 

33 

24 

34 

25 26 

41 

32 

41 

31 

21 

36 

45 

28 

29 

2 

27 

31 

40 41 

34 

31 

6.9 

23 

37 

43 

48 

43 

54 

44 46 

43 41 46 

51 59 

46 

25 

46 48 

32 

42 

-5 

38 

40 

21 

55 52 

44 

8.9 

23 

4 

3 

5 

12 

11 

4 

13 

13 

11 

7 

8 

19 

3 

0 

10 

13 

15 

8 

9 

12 

4 

11 

18 

9 

9 

5.0 

23 

41 

32 

44 

70 

55 57 52 

50 

51 

54 

53 55 57 

37 

50 55 

45 

49 

17 

41 

56 

48 

55 

48 

50 

7.8 

23 

64 

70 

74 

84 

98 

72 

93 

81 

78 

94 

90 

119 

81 

47 

92 

105 

76 

79 

6 

77 

75 73 

114 

95 

84 

16.3 

23 

5 - 21 

105 

102 

119 

153 153 128 145 

130 129 148 

143 

175 138 

84. 

143 161 120 

128 

23 

118 

132 

121 168 143 

134 

21.5 

23 
10-MAR-2011 17:17 

Huntingdon Life Sciences 05-4287 

GROUP 3 10000 MG/:M3 

APPENDIX F 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL GESTATION BODY WEIGHT GAIN (GRAMS) 

DAY OF GESTATION 

ANIMALJI 

3901 3902 3903 

3904 3905 3906 3907 

3908 3909 3910 

3911 

3912 

' 3913 

3914 

3915 3916 3917 

3918 3919 3920 

3921 

3922 

3923 

3924 

MEAN 

S.D. 

N 

4 - 5 5 - 8 8 - 11 11 - 14 14 - 17 17 - 20 20 - 21 

19 

24 

12 

13 

11 

-1 

13 

14 14 

5 

12 

10 

3 

10 

12 

10 

4 

5 

12 

9 

6 

6 

8 

14 

10 

5.2 

24 

3 

11 17 

17 

8 

21 

10 

10 10 

11 

9 

6 

25 

16 

10 

B 

18 

18 

10 15 

13 

14 14 

2 

13 

5.5 

24 

13 

20 

18 

22 

12 

13 16 

19 

23 

17 

22 

18 15 

24 

15 

22 22 

14 

17 

21 

12 

19 

12 

23 

18 

3.9 

24 

16 

21 

24 

14 

18 16 

22 

18 18 13 

17 

16 

17 

14 

9 

17 

22 

10 19 18 

17 

22 

18 

23 

17 

3.7 

24 

22 

19 

20 

28 

37 

43 

34 

21 

30 31 

33 

30 36 

28 

17 

39 39 

32 

36 

32 

24 

44 

31 38 

31 

7.6 

24 

36 

43 

52 

52 57 

48 

35 

39 

45 49 

57 

45 

26 

42 15 

51 

44 

41 

45 

52 

34 

55 

50 

45 

44 

9.9 

24 

6 

7 

13 

-1 

8 

6 

8 

6 

0 

10 

8 8 

16 15 

1 

10 

6 

16 

14 

9 

5 

15 

16 10 

9 

4.9 

24 

5 - 14 14 - 21 

32 

52 

59 

53 

38 

49 49 

47 

51 

41 

48 

40 

57 54 

34 

51 

62 

42 

46 

54 

42 

54 

44 47 

48 

7.7 

24 

64 

69 

84 

80 

102 

97 

77 

66 

75 

90 

98 

84 

77 

85 

32 

100 

89 

89' 

95 93 

63 

114 

96 

93 

84 

17.0 

24 

5 - 21 

96 

121 

144, 

133 

140 146 126 

113 

126 131 

146 

124 135 139 

67 

151 151 

131 

141 147 

105 

169 140 

140 

132 

21.0 

24 
10-MAR-2011 17:17 

Huntingdon Life Sciences 05-4287 

APPENDIX F 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

. IN RATS BY INHALATION EXPOSURE 

INVIVIDUAL GESTATION BODY WEIGHT GAIN (GRAMS) 

GROUP 4 20000 MG/M:3 

DAY OF GESTATION 

ANIMAL# 4 - 5 5 - 8 8 - 11 11 - 14 14 - 17 17 - 20 20 - 21 5 - 14 14 - 21 

4901 4902 4903 

4904 4905 4906 4907 

4908x NP 

4909 

4910 

4911 4912 4913 4914 

4915 

4916 4917 

4918 4919x NP 4920 

4921 

4922 4923 4924 

MEAN 

S.D. 

N 

NP=NOT PREGNANT 

12 

12 

16 

5 

15 11 

5 

5 

9 

7 

13 

10 10 

10 

8 8 

11 

6 6 

5 

5 

12 

11 

11 

10 

3.2 

22 

5 

14 

4 

13 

8 

12 12 

4 

2 

22 

10 

14 

11 

8 

8 

7 

14 

11 

-3 

13 

13 15 

3 

15 

11 

4.7 

.22 

18 16 

20 

21 

15 16 19 

8 

9 

22 

18 

20 

15 

23 

18 

18 

20 

24 

2 

21 

21 

15 

25 

39 

20 

5.5 

22 

x=EXCLUDED FROM MEAN 

17 

20 

11 

15 18 

23 

19 

-9 

13 

15 

13 

15 

17 

13 12 

17 15 

8 8 

16 

12 

18 

5 

18 

15 

4.0 

22 

21 23 28 

34 34 

27 25 

6 

24 27 

32 

26 

32 

30 

26 

31 

44 

42 

-2 

31 

36 

34 

28 

47. 

31 

6.8 

22 

48 45 

63 

52 

51 

49 44 

10 

33 

45 

52 

47 44 42 

40 48 46 

32 

-2 

32 

33 

46 

37 

49 

44 

7.7 

22 

-2 

11 

7 

10 

4 

12 

4 

-5 

11 

13 

9 

7 

7 

12 

14 16 

8 

13 

-3 

10 

10 

-6 

6 

4 

8 

5.1 

22 

40 

50 

35 

49 42 

52 50 

3 

24 

59 

40 49 44 43 

38 

41 49 

43 

7. 

50 

45 

48 

33 

71 

45 

9.5 

22 

68 

·78 

98 

95 

90 

87 

73 

11 

69 

85 

93 

80 

83 83 

80 

94 99 

86 

-7 

73 

79 73 71 

100 

83 

10.0 

22 

5 - 21 

108 

127 132 

144 131 139 123 

14 

93 

144 

133 129 127 126 

118 

135 148 

129 

1 

124 

124 

120 104 171 

129 

16.0 

22 
10-MAR-2011 17:19 

Huntingdon Life Sciences 05-4287 

APPENDIX G 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL GESTATION FEED CONSUMPTION -- (GRAMS/KG/DAY) 

GROUP 1 0 MG/M3 

------------------

DAY OF GESTATION 

ANIMAL# 4 - 5 5 - 8 8 - 11 11 - 14 14 - 17 17 - 20 20 - 21 5 - 21 

------------------------------------------------------------------------------------

1901 66 89 79 91 79 75 48 77 

1902 83 82 83 88 81 77 44 76 1903 43 66 93 90 84 72 58 77 

1904 76 82 79 83 78 71 46 73 1905 72 79 87 84 81 72 43 74 1906 63 71 72 78 77 76 58 72 

1907 63 80 82 77 82 73 44 73 

1908 95 89 96 94 90 79 58 84 1909 77 86 85 87 79 72 58 78 1910 75 79 76 82 82 72 55 74 

1911 76 79 81 79 78 73 43 72 1912 70 78 76 72 72 72 36 67 1913 81 80 81 75 81 72 51 73 

1914 66 77 80 82 82 78 58 76 

1915x NP 56 75 67 67 69 63 72 69 

1916 76 78 84 84 83 74 46 75 1917 73 86 91 89 83 75 54 80 

1918 73 74 86 84 83 73 66 78 1919 54 85 92 92 75 69 58 78 1920 78 92 100 104 93 91 65 91 

1921 70 83 92 89 81 74 54 79 1922 71 81 91 82 77 73 63 78 1923 CF CF CF CF CF 79 62 71 1924 65 81 87 83 78 71 57 76 

MEAN 71 81 85 '85 81 74 53 76 

S.D. 10.5 6.0 7.1 7.1 4.5 4.5 8.1 4.7 

N 22 22 22 22 22 23 23 23 

NP=NOT PREGNANT x=EXCLUDED FROM MEAN 

CF=Contaminated Feeder 
10-MAR-2011 17:19 

Huntingdon Life Sciences 05-4287 

APPENDIX G 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL GESTATION FEED CONSUMPTION -- (GRAMS/KG/DAY) 

GROUP 2 2000 MG/M3 

----------------

DAY OF GESTATION 

ANIMAL# 4 - 5 5 - 8 8 - 11 11 - 14 14 - 17 17 - 20 20 - 21 5 - 21 

-------------------------------------------------------------------------

2901 84 89 83 82 79 76 39 75 2902 83 81 79 76 79 79 52 74 2903 70 73 79 76 74 72 50 71 

2904 44 80 78 87 78 75 52 75 2905 73 82 78 82 82 75 35 72 2906 79 80 75 80 73 65 41 69 

2907 78 89 90 81 83 72 43 76 2908 86 145 86 91 87 76 47 89 2909 94 80 90 86 83 77 55 79 2910 85 88 82 84 87 76 35 76 

2911 74 80 83 76 75 71 38 71 2912 76 84 84 82 82 75 44 75 2913 76 86 88 83 82 75 48 77 

2914 72. 74 82 72 78 73 45 71 2915 73 75 76 79 89 72 52 74 2916 72 83 82 82 86 72 47 75 2917 .75 90 83 82 78 71 50 76 

2918 77 80 81 83 76 70 40 72 2919x NP 76 78 81 79 72 74 65 75 2920 59 79 74 78 79 76 68 76 

2921 76 87 84 84 82 73 53 77 2922 45 78 76 76 86 67 47 71 2923 78 87 80 86, 76 74 54 76 

2924 77 94 88 89 84 77 41 79 

MEAN 74 85 82 82 81 73 47 75 

S.D. 11.5 14.1 4.6 4.7 4.5 3.3 7.5 4.0 

N 23 23 23 23 23 23 23 23 NP=NOT PREGNANT x=EXCLUDED FROM MEAN 
10-MAR-2011 17:19 

Huntingdon Life Sciences 05-4287 

APPENDIX G 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS By INHALATION EXPOSURE 

INDIVIDUAL GESTATION FEED CONSUMPTION -- {GRAMS/KG/DAY) 

GROUP 3 10000 MG/M3 

----------

DAY OF GESTATION 

ANIMAL# 4 - 5 5 - 8 8 - 11 11 - 14 14 - 17 17 - 20 20 - 21 5 - 21 

-------------------------------------------------------------------------------

3901 76 70 67 75 73 70 52 68 3902 74 72 74 83 75 70 40 69 3903 72 84 85 86 74 72 58 77 

3904 81 88 89 88 82 74 42 77 3905 71 77 77 81 80 74 47 73 3906 47 71 79 80 86 75 39 72 3907 92 87 85 90 94 78 47 80 3908 88 88 90 86 85 78 50 79 

3909 69 75 77 80 76 69 39 69 3910 74 77 80 78 73 65 43 69 3911 84 90, 85 78 80 74 48 76 3912 73 75 76 82 84 74 54 74 

3913 55 85 79 87 86 77 50 77 3914 66 78 81 79 73 67 49 71 3915 84 81 84 84 86 76 42 75 3916 87 78 75 CF 88 CF 38 70 

3917 66 78 83 86 87 72 47 75 

3918 81 82 79 85 80 76 56 76 3919 83 82 83 84 84 79 55 78 3920 69 80 83 80 78 75 42 73 3921 97 78 71 77 73 72 42 69 

3922 64 85 87 CF CF CF 51 74 

3923 71 83 79 82 77 73 48 74 3924 75 69 78 82 CF 79 50 72 

MEAN 75 80 80 82 81 74 47 74 

S.D. 11.4 6.0 5.5 3.9 6.0 3.7 5.8 3.6 

N 24 24 24 22 22 22 24 24 

Feeder 
10-MAR-2011 17:19 
10-MAR-2011 17:21 

Huntingdon Life Sciences 05-4287 

APPENDIX H 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL MATERNAL NECROPSY OBSERVATIONS 

GROUP 1 0 MG/M3 

---------

ANIMAL# ORGAN OBSERVATION 

----------------------------------------------------------

1901 NO REMARKABLE OBSERVATIONS 1902 NO REMARKABLE OBSERVATIONS 

1903 NO REMARKABLE OBSERVATIONS 1904 NO REMARKABLE OBSERVATIONS 

1905 NO REMARKABLE OBSERVATIONS 

1906 NO REMARKABLE OBSERVATIONS 

. 1907 NO REMARKABLE OBSERVATIONS 

1908 NO REMARKABLE OBSERVATIONS 1909 NO REMARKABLE OBSERVATIONS 

1910 NO REMARKABLE OBSERVATIONS 

1911 NO REMARKABLE OBSERVATIONS 1912 NO REMARKABLE OBSERVATIONS 

1913 NO REMARKABLE OBSERVATIONS 1914 NO REMARKABLE OBSERVATIONS 

1915 NP SALEWSKI'S TEST NO FOCI PRESENT 

1916 NO REMARKABLE OBSERVATIONS 

---------------

NP=NOT PREGNANT 
10-MAR-2011 17:21 

Huntingdon Life Sciences 

GROUP 1 0 MG/M3 

ANIMAL# ORGAN 

1917 1918 

1919 1920 

1921 

1922 

1923 

1924 

APPENDIX H 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL MATERNAL NECROPSY OBSERVATIONS 

OBSERVATION 

NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS 
10-MAR-2011 17:21 

Huntingdon Life Sciences 05-4287 

GROUP 2 2000 MG/M3 

------------

ANIMALlf ORGAN 

----

2901 2902 

2903 

2904 

2905 

2906 

2907 

2908 

2909 

2910 

2911 2912 

2913 

2914 

2915 

2916 

APPENDIX H 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL MATERNAL NECROPSY OBSERVATIONS 

OBSERVATION 

--------------------------

NO REMARKABLE OBSERVATIONS NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS NO OBSERVATIONS 

NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS 
10-MAR-2011 17:21 

Huntingdon Life Sciences 05-4287 

GROUP 2 2000 MG/M3 

------------

ANIMAL# 

-------

2917 

2918 

2919 NP 

2920 

2921 

2922 

2923 

2924 

NP=NOT PREGNANT 

ORGAN 

APPENDIX H 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL MATERNAL NECROPSY OBSERVATIONS 

OBSERVATION 

NO REMARKABLE OBSERVATIONS NO REMARKABLE OBSERVATIONS 

SALEWSKI'S TEST NO FOCI PRESENT 

NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS NO REMARKABLE OBSERVATIONS 
10-MAR-2011 17:21 

Huntingdon Life Sciences 05-4287 

GROUP 3 10000 MG/M3 

-------------

ANIMALlt ORGAN 

-------

3901 3902 

3903 

3904 

3905 3906 

3907 3908 

3909 

3910 

3911 

3912 3913 

3914 

3915 3916 

APPENDIX H 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL MATERNAL NECROPSY OBSERVATIONS 

OBSERVATION 

NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS NO REMARKABLE OBSERVATIONS NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS NO REMARKABLE OBSERVATIONS NO REMARKABLE OBSERVATIONS 
10-MAR-2011 17:21 

Huntingdon Life Sciences 05-4287 

GROUP 3 10000 MG/M3 

ANIMALlf ORGAN 

3917 3918 

3919 

3920 

3921 

3922 

3923 

3924 

APPENDIX H 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL MATERNAL NECROPSY OBSERVATIONS 

OBSERVATION 

NO REMARKABLE OBSERVATIONS NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS 
10-MAR-2011 17:21 

Huntingdon Life Sciences 05-4287 

GROUP 4 20000 MG/M3 

-------------

ANIMAL# 

-------

4901 

4902 

4903 

4904 4905 

4906 

4907 4908 NP 

APPENDIX H 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

ORGAN 

EXTERNAL EXAM 

GROSS EXAM 

INDIVIDUAL MATERNAL NECROPSY OBSERVATIONS 

OBSERVATION 

NO REMARKABLE OBSERVATIONS 

ALOPECIA- GENERAL 

EXTERNAL FINDING CONFIRMED 

NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS 

MASS; LEFT 

MAMMARY GLAND- LOWER LATERAL, 2.0 X 2.5 CM.,TAN, FIRM 

EXTERNAL FINDING CONFIRMED 

TISSUE SAVED 

EXTERNAL EXAM INCISOR MALOCLUDED 

EXTERNAL FINDING CONFIRMED 

SALEWSKI'S TEST NO FOCI PRESENT 

4909 NO REMARKABLE OBSERVATIONS 

4910 NO REMARKABLE OBSERVATIONS 

4911 NO REMARKABLE OBSERVATIONS 4912 NO REMARKABLE OBSERVATIONS 

4913 NO REMARKABLE OBSERVATIONS 

NP=NOT PREGNANT 
10-MAR-2011 17:21 

Huntingdon Life Sciences 05-4287 

GROUP 4 2oooo MG/M3 

-------------

ANIMALJI 

-------

4914 4915 

4916 4917 

4918 

4919 NP 

4920 

4921 

4922 

4923 

4924 

NP=NOT PREGNANT 

ORGAN 

APPENDIX H 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL MATERNAL NECROPSY OBSERVATIONS 

OBSERVATION 

NO REMARKABLE OBSERVATIONS NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS 

SALEWSKI'S TEST NO FOCI PRESENT 

NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS 

NO REMARKABLE OBSERVATIONS 
10-MAR-2011 17:22 

Huntingdon Life Sciences 05-4287 

APPENDIX I 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL FEMALE REPRODUCTION DATA AND MEAN FETAL WEIGHTS {GRAMS) 

GROUP 1 0 MG/M3 

CORPORA %PREIMPL. IMPLANT 

ANIMAL# LUTEA LOSS SITES 

1901 1902 

1903 

1904 1905 1906 

1907 

1908 1909 

1910 

1911 

1912 1913 

1·914 

1915 NP 

16 16 

15 

22 

14 15 

9 

14 

15 

14 19 15 

13 

12 

1916 17 

1917 18 1918 15 1919 15 

1920 9 1921 15 1922 14 1923 16 

1924 15 

MEAN 

S.D. 

14.9 

2.78 

N 23 

NP=NOT PREGNANT 

25.0 25.0 

6.7 

31.8 

0.0 

6.7 

0.0 

7.1 

0.0 

7.1 

10.5 

0.0 

0.0 

0.0 

11.8 

5.6 

0.0 

6.7 

55.6 

0.0 

7.1 

6.3 

0.0 

9.3 

13.46 

23 

12 12 

14 

15 

14 

14 

9 

13 

15 

13 17 15 

13 

12 

15 

17 

15 

14 

4 

15 13 15 

15 

13.5 

2. 71 

23 

FETUSES 

LIVE DEAD 

11 

12 

14 15 

13 

13 

9 

12 

15 

13 15 

15 

11 

12 

14 

17 

14 14 

4 

15 13 

15 

14 

{n) % 

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 0. 0 

0 0. 0 

13.0 0.0 

2.64 0.00 

23 23 

0.0 

0.00 

23 

RESORPTIONS 

EARLY LATE 

1 

0 

0 0 

1 

1 

0 

1 

0 

0 

2 

0 

2 

0 

1 

0 0 0 

0 0 0 0 

1 

0.4 

0.66 

23 

0 0 

0 

0 

0 0 

0 0 0 

0 0 0 0 

0 0 

0 

1 

0 

0 0 0 0 

0 

0.0 

0.21 

23 

TOTAL 

{n) % 

1 8.3 

0 0. 0 

0 0. 0 

0 0. 0 

1 7.1 

1 7.1 

0 0. 0 

1 7.7 

0 0.0 

0 0.0 

2 11.8 

0 0. 0 

2 15.4 

0 0.0 

1 6. 7 

0 0.0 

1 6. 7 

0 0.0 

0 0.0 

0 0. 0 

0 0. 0 

0 0. 0 

1 6. 7 

0.5 3.4 

0.67 4.66 

23 23 

SEX 

MALE FEMALE 

6 

8 

7 

8 

6 

6 

6 

7 7 

6 6 

5 

5 

5 

6 

7 

10 

5 

1 

8 

7 

8 

6 

6.3 

1. 70 

23 

5 

4 

7 

7 

7' 

7 

3 

5 

8 

7 

9 

10 

6 

7 

8 

10 

4 

9 

3 

7 

6 

7 

8 

6.7 

1. 99 

23 

AVERAGE 

MALES 

6.1 6.1 

6.3 

5.7 5.4 5.6 

6.4 

5.9 

6.4 

6.0 

5.7 5.4 5.8 

6.1 

5.6 

5.7 

5.6 

6.1 

6.3 

5.4 

6.1 6.1 

5.7 5.9 

0.32 

23 
10-MAR-2011 17:22 

Huntingdon Life sciences 05-4287 

APPENDIX I 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL FEMALE REPRODUCTION DATA AND MEAN FETAL WEIGHTS (GRAMS) 

GROUP 2 2000 MG/M3 

CORPORA %PREIMPL. IMPLANT 

ANIMAL* LUTEA LOSS SITES 

2901 2902 

2903 

2904 2905 2906 2907 

2908 2909 2910 

2911 

2912 2913 

;l914 

2915 2916 

2917 

2918 

13 13 

13 

14 18 15 13 

2'1 

11 

14 

15 17 

14 14 13 16 

14 14 

2919 NP 2920 13 

14 16 15 

14 

2921 2922 2923 2924 

MEAN 

S.D. 

N 

14.5 

2.06 

23 

NP=NOT PREGNANT 

15.4 

7.7 

7.7 

0.0 

11.1 13.3 

0.0 

28.6 

0.0 0.0 

6.7 

0.0 

0.0 0.0 

7.7 

6.3 

7.1 7.1 

0.0 

7.1 

25.0 

0.0 0.0 

6.6 

7.99 

23 

11 

12 

12 

14 

16 

13 13 

15 11 

14 14 

17 

14 

14 

12 

15 

13 13 

13 

13 12 

15 

14 

13.5 1. 50 

23 

FETUSES 

LIVE DEAD 

10 11 

11 

12 

16 

12 

13 

14 

10 

14 14 

17 

14 

4 

11 

15 

12 13 

(n) % 

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 

11 0 0.0 

0.0 

0.0 0.0 0.0 

11 0 

12 0 

15 0 14 0 

12.4 0.0 0.0 

2.64 0.00 0.00 

23 23 23 

RESORPTIONS 

EARLY LATE 

1 

1 

1 

2 

0 

1 

0 

1 1 

0 0 

0 0 

10 

1 

0 

1 

0 

1 

2 

0 0 0 

1.0 

0 

0 

0 

0 0 0 0 

0 

0 

0 

0 

0 

0 0 0 0 

0 

0 

1 

0 

0 0 

0 

0.0 

TOTAL 

(n) % 

1 9.1 

1 8.3 

1 8.3 

2 14.3 

0 0. 0 

1 7. 7 

0 0. 0 

1 6. 7 

1 9.1 

0 0.0 

0 0. 0 

0 0. 0 

0 0. 0 

10 71.4 

1 8. 3 

0 0.0 

1 7. 7 

0 0. 0 

2 15.4 

2 15.4 

0 0. 0 

0 0. 0 0 0. 0 

1.0 7.9 

2.07 0.21 2.08 14.89 

23 23 23 23 

-\ 

SEX 

MALE FEMALE 

7 

6 

7 

5 

6 

3 

8 

6 

5 

4 

5 

12 

6 

1 

3 

8 

5 

9 

7 

5 

5 

5 

5 

5.8 

3 

5 

4 

7 

10 

9 

5 

8 

5 

10 

9 5 

8 

3 

8 

7 

7 

4 

4 

6 

7 

10 

9 

6.7 

2.24 2.29 

23 23 

AVERAGE 

MALES 

5.6 

5.9 

5.8 

6.4 6.1 6.0 

5.5 

5.4 

6.3 6.3 6.1 

6.0 

5.7 

6.6 6.0 

5.5 

5.5 

5.9 

5.4 5.7 

5.9 

6.2 6.3 

5.9 

0.33 

23 
10-MAR-2011 17:22 

Huntingdon Life Sciences 05-4287 

APPENDIX I 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL FEMALE REPRODUCTION DATA AND MEAN FETAL WEIGHTS (GRAMS} 

GROUP 3 10000 MG/M3 

CORPORA %PREIMPL. IMPLANT 

LUTEA LOSS SITES 

3901 3902 

3903 3904 3905 

3906 

3907 

3908 3909 3910 3911 

3912 3913 3914 

3915 

3916 

3917 3918 3919 3920 

3921 

3922 3923 

3924 

MEAN 

S.D. 

N 

11 13 

14 

13 

15 

14 

13 

13 

14 16 16 

14 

20 

18 10 16 

21 

15 13 

14 

13 

17 

16 15 

14.8 

2. 52 

24 

9.1 

0.0 

7.1 

0.0 0.0 0.0 0.0 

0.0 

7.1 

12.5 

0.0 

7.1 

60.0 

16.7 

70.0 

12.5 

33.3 

0.0 0.0 0.0 

7.7 

0.0 0.0 

6.7 

10.4 

18.58 

24 

10 13 

13 

13 

15 

14 

13 

13 

13 

14 

16 

13 

8 

15 

3 

14 

14 

15 

13 

14 

12 

17 

16 14 

13.1 

2.85 

24 

FETUSES 

LIVE DEAD 

(n} % 

10 0 0.0 12 0 0. 0, 

13 0 0. 0 

13 0 0. 0 

15 0 0.0 

14 0 0.0 

11 0 0.0 

10 0 0.0 

12 0 0. 0 

14 0 0. 0 

16 0 0. 0 

12 0 0.0 

8 0 0. 0 

14 0 0.0 

2 0 0. 0 

14 0 0.0 

14 0 0. 0 

13 0 0.0 

13 0 0.0 

14 '0 0.0 

11 0 0.0 

17 0 0.0 

16 0 0. 0 

14 0 0. 0 

12.6 0.0 

3.06 0.00 

24 24 

0.0 

0.00 

24 

RESORPTIONS 

EARLY LATE 

0 

1 

0 0 0 

0 

2 

3 

1 

0 0 

1 

0 

1 1 

0 

0 

2 

0 0 

1 

0 0 

0 

0.5 

0.83 

24 

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 

0.00 

24 

TOTAL 

(n} % 

0 0.0 

1 7. 7 

0 0. 0 

0 0. 0 0 0. 0 

0 0. 0 

2, 15.4 

3 23.1 

1 7. 7 

0 0.0 

0 0.0 

1 7. 7 

0 0.0 

1 6. 7 

1 33.3 

0 0. 0 

0 0. 0 

2 13.3 

0 0.0 0 0.0 

1 8.3 

0 0. 0 

0 0. 0 

0 0. 0 

0.5 5.1 

0.83 8.64 

24 24 

SEX 

MALE FEMALE 

6 

6 

5 

8 8 

7 

6 

4 

6 

7 

5 5 

6 

6 

1 

9 

4 

5 

8 8 

5 

8 

13 

8 

4 

6 

8 

5 

7 7 

5 

6 

6 

7 

11 

7 

2 

8 

1 

5 

10 

8 

5 

6 

6 

9 

3 

6 

6. 4 6. 2 ' 

2.26 2.32 

24 24 

AVERAGE 

MALES 

5.9 

5.6 

5.2 

6.1 

5.3 5.5 5.8 

5.7 5.9 

6.1 

5.9 

6.1 6.5 

5.5 

6.4 

5.7 

5.5 5.8 5.6 

6.1 

5.8 

6.0 

5.6 5.9 

5.8 

0.32 

24 
10-MAR-2011 17:22 

Huntingdon Life Sciences 05-4287 

APPENDIX I 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL FEMALE REPRODUCTION DATA AND MEAN FETAL WEIGHTS (GRAMS) 

GROUP 4 20000 MG/M3 

CORPORA %PREIMPL. IMPLANT 

ANIMAL# LUTEA LOSS SITES 

4901 4902 

4903 4904 4905 

15 

11 

17. 

18 16 

4906 19 

4907 13 4908 NP 4909 

4910 

4911 4912 

4913 4914 4915 4916 

4917 

4918 4919 NP 

4920 4921 4<:l22 4923 

4924 

11 

14 

17 

11 

15 

14. 

16 16 

16 

21 

14 

13 

16 13 

18 

MEAN 

S.D. 

15.2 

2.63 

N 22 

NP=NOT PREGNANT 

6.7 

0.0 

11.8 

0.0 0.0 

15.8 

15.4 

9.1 

0.0 0.0 0.0 

6.7 

14.3 

6.3 

0.0 

6.3 

38.1 

14.3 

15.4 

6.3 

0.0 

0.0 

7.6 

9.14 

22 

14 

11 

15 18 

16 16 

11 10 

14 

17 11 

14 

12 

15 16 

15 

13 

12 

11 15 

13 

18 

14.0 

2.38 

22 

FETUSES 

LIVE DEAD 

13 10 

14 

18 

16 16 

10 

9 

13 

16 11 

14 10 14 16 

14 

13 

11 

11 15 

11 

17 

(n) % 

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 

0.0 

13.3 0.0 0.0 

2.60 0.00 0.00 

22 22 22 

RESORPTIONS 

EARLY LATE TOTAL 

1 1 

1 

0 0 0 

1 

1 

1 1 

0 

0 

2 

1 

0 

1 

0 

1 

0 0 

2 

1 

0 

0 

0 0 

0 

0 

0 

0 

0 

0 0 

0 0 

0 0 

0 0 

0 0 0 0 

0 

(n) % 

1 7.1 

1 9.1 

1 6. 7 

0 0.0 

0 0. 0 

0 

1 

0.0 

9.1 

1 10.0 

1 7.1 1 5.9 

0 0.0 

0 0.0 

2 16.7 

1 6. 7 

0 0. 0 

1 6.7 

0 0. 0 

1 8.3 

0 0. 0 

0 0. 0 

2 15.4 

1 5. 6 

0.7 0.0 0.7 5.2 

0.65 0.00 0.65 5.15 

22 22 22 22 

SEX 

MALE FEMALE 

6 3 

9 

10 

9 

7 

4 

6 

8 

6 6 

4 

6 

3 

10 

6 

4 

5 

6 

5 

7 

5 

7 7 

5 

8 

7 

9 

6 

3 

5 

10 

5 

10 

4 

11 

6 

8 

9 

6 

5 

10 

4 

12 

6.1 7.1 

2.05 2.49 

22 22 

AVERAGE 

MALES 

5.6 5.8 

5.8 

5.4 5.4 

5.5 

6.0 

6.0 

5.7 5.6 

6.5 

6.0 6.3 

5.6 5.0 

6.0 

5.9 

5.4 

5.7 5.4 5.7 

5.6 5.7 

0.33 

22 
10-MAR-2011 17:23 

Huntingdon Life Sciences 05-4287 

APPENDIX J 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL GRAVID UTERINE WEIGHT AND NET BODY WEIGHT CHANGE (GRAMS) 

GROUP 1 0 MG/M3 

----------------------------------------

UTERUS ADJUSTED NET WEIGHT CHANGE WEIGHT CHANGE 

ANIMAL# WEIGHT WEIGHT FROM DAY 5 FROM DAY 5 

1901 91 298 68 159 1902 94 265 38 132 1903 112 272 49 161 

1904 111 286 38 149 1905 98 295 41 139 1906 98 308 44 143 

1907 72 262 44 116 

1908 92 308 69 161 

1909 119 281 42 161 1910 104 268 29 132 

1911 117 294 35 153 1912 NT 133 1913 84 269 30 114 

1914 103 281 36 139 

1915x NP 21 

1916 108 306 45 153 1917 126 289 35 161 

1918 108 326 57 165 1919 113 255 32 145 1920 36 277 45 81 

1921 108 274 38 146 1922 106 276 34 141 1923 121 304 57 179 1924 107 298 39 146 

MEAN 101. 286. 43. 144. 

S.D. 19.4 18.1 11.1 20.6 

N 22 22 22 23 

ADJUSTED WEIGHT TERMINAL BODY WEIGHT MINUS GRAVID UTERINE WEIGHT 

NET WEIGHT CHANGE FROM DAY 5 = TERMINAL CORRECTED BODY WEIGHT MINUS DAY 5 BODY WEIGHT 

NP=NOT PREGNANT NT=NOT TAKEN x=EXCLUDED FROM MEAN 
10-MAR-2011 17:23 

Huntingdon Life Sciences 05-4287 

APPENDIX J 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL GRAVID UTERINE WEIGHT AND NET BODY WEIGHT CHANGE (GRAMS) 

GROUP 2 2000 MG/M3 

----------------------------------------------------------------

UTERUS ADJUSTED NET WEIGHT CHANGE WEIGHT CHANGE 

ANIMAL# WEIGHT WEIGHT FROM DAY 5 FROM DAY 5 

2901 73 253 32 105 2902 85 247 17 102 2903 86 268 32 119 

2904 98 299 55 153 

2905 120 284 33 153 

2906 95 290 33 128 

2907 95 270 50 145 

2908 95 259 35 130 2909 86 278 43 129 2910 109 286 38 148 

2911 109 292 35 143 2912 134 320 40 175 2913 101 271 37 138 

2914 37 290 46 84 2915 91 308 52 143 2916 113 304 47 161 2917 86 295 34 120 

2918 105 300 23 128 2919x NP 23 2920 81 266 36 118 

2921 88 282 43 132 2922 93 257 27 121 2923 126 300 42 168 2924 113 283 30 143 

MEAN 97. 283. 37. 134. 

S.D. 19.8 19.0 9.1 21.5 

N 23 23 23 23 

ADJUSTED WEIGHT = TERMINAL BODY WEIGHT MINUS GRAVID UTERINE WEIGHT 

NET WEIGHT CHANGE FROM DAY 5 = TERMINAL CORRECTED BODY WEIGHT MINUS DAY 5 BODY WEIGHT 

NP=NOT PREGNANT x=EXCLUDED FROM MEAN 
10-MAR-2011 17:23 

Huntingdon Life Sciences 05-4287 

APPENDIX J 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL GRAVID UTERINE WEIGHT AND NET BODY WEIGHT CHANGE (GRAMS) 

GROUP 3 10000 MG/M3 

--

UTERUS ADJUSTED NET WEIGHT CHANGE 

ANIMALlf WEIGHT WEIGHT FROM DAY 5 

3901 80 246 16 3902 88 275 33 3903 95 280 49 

3904 103 276 31 

3905 107 281 33 3906 104 288 43 3907 83 261 43 

3908 75 263 38 3909 90 280 36 3910 108 269 23 

3911 117 283 29 3912 96 303 28 3913 68 302 67 

3914 100 289 39 3915 19 305 47 3916 110 298 41 3917 100 308 51 

3918 101 293 30 3919 93 275 48 3920 114 259 33 

3921 86 248 19 3922 136 272 33 3923 121 268 19 3924 112 286 28 

MEAN 96. 280. 36. 

S.D. 22.5 17.1 11.7 

N 24 24 24 

ADJUSTED WEIGHT = TERMINAL BODY WEIGHT MINUS GRAVID UTERINE WEIGHT 

WEIGHT CHANGE 

FROM DAY 5 

96 

121 144 

133 

140 146 126 

113 

126 131 

146 

124 135 

139 

67 

151 151 

131 141 147 

105 169 140 140 

132. 

21.0 

24 

NET WEIGHT CHANGE FROM DAY 5 = TERMINAL CORRECTED BODY WEIGHT MINUS DAY 5 BODY WEIGHT 
10-MAR-2011 17:23 

Huntingdon Life Sciences 05-4287 

APPENDIX J 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL GRAVID UTERINE WEIGHT AND NET BODY WEIGHT CHANGE (GRAMS) 

GROUP 4 20000 MG/M3 

------

UTERUS ADJUSTED NET WEIGHT CHANGE WEIGHT CHANGE 

ANI MALl! WEIGHT WEIGHT FROM DAY 5 FROM DAY 5 

4901 91 237 17 108 4902 78 274 49 127 4903 103 272 29 132 

4904 123 257 21 144 4905 110 273 21 131 4906 119 270 20 139 

490( 79 261 45 123 

4908x NP 14 4909. 70 254 23 93 4910 NT 144 4911 113 274 20 133 4912 94 292 35 129 

4913 105 267 21 127 

4914 84 296 42 126 4915 97 275 22 118 4916 108 281 27 135 4917 114 303 34 148 

4918 94 300 35 129 4919x NP 1 4920 76 272 47 124 

4921 84 274 40 124 4922 104 262 16 120 

4923 81 270 23 104 4924 PART 124x 318x 47x 171 

MEAN 96. 273. 29. 129. 

S.D. 15.7 15.9 10.6 16.0 

N 20 20 20 22 

ADJUSTED WEIGHT TERMINAL BODY WEIGHT MINUS GRAVID UTERINE WEIGHT 

NET WEIGHT CHANGE FROM DAY 5 = TERMINAL CORRECTED BODY WEIGHT MINUS DAY 5 BODY WEIGHT 

NP=NOT PREGNANT NT=NOT TAKEN PART=PARTIAL DELIVERY; 5 LIVE PUPS DELIVERED x=EXCLUDED FROM MEAN 
10-MAR-2011 17:24 

Huntingdon Life Sciences 05-4287 

APPENDIX K 

GASOLINE DIPE VAP.OR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

GROUP 1 0 MG/M3 

FEMALE# MEAN 

1901 1902 

1903 

1904 1905 

1906 1907 1908 1909 

1910 

1911 1912 1913 1914 1915 

1916 1917 

1918 1919 1920 

1921 

1922 1923 

1924 

5.9 

6.0 

5.9 

5.5 5.5 5.5 

6.3 

5.7 

6.2 6.0 

5.6 5.2 5.5 

6.0 

NP 

5.5 5.5 

5.5 

5.8 

6.1 

5.4 

6.0 

5.9 5.6 

MEAN 5.7 

S.D. 0.29 

N 23 

FETUS# 

1 2 

5.6 

5.6 

5.7 

5.3 

3.2 

5.6 

6.4 6.0 6.3 

5.9 

5.8 

5.6 

5.0 

6.0 

.5. 3 

5.5 

5.4 5.6 

6.2 

5.1 5.8 

6.1 

5.7 

5.9 

6.6 6.3 

5.1 5.7 5.3 

6.3 

5.8 

6.6 

6.0 

5.8 

5.0 5.9 

6.3 

5.4 

5.1 

5.3 

6.2 

6.0 

5.3 

6.5 

5.9 

E 

3 

5.9 

6.0 

5.3 

5.6 

6.4 

5.2 

6.0 

6.4 6.1 

5.2 

5.5 5.3 

E 

6.4 

5.5 5.7 

5.6 

6.0 6.1 

5.1 5.6 

5.5 5.9 

4 

5.8 5.7 5.6 

5.4 

E 

5.6 

6.2/ 

5.6 

6.4 6.0 

5.5 5.0 5.2 

6.1/ 

5.2 5.4 

5.6 

6.2 6.3 

5.6 5.9 

5.6 5.6 

E-Early resorption L-Late resorption 

/-Denotes position of cervix 

NP=NOT PREGNANT 

INDIVIDUAL FETAL BODY WEIGHTS (GRAMS} 

5 

6.0/ 

6.3 6.2 

5.7/ 

5.6 

5.4 

6.2 

5.4 

6.3/ 

6.3 

E 

4.7 

5.5 

6.1 

5.6 5.1 

5.7 

5.3 

6 7 

6.3 5.3 

5.9/ 6.3 

6.4 5.6 

5.4 5.9 

5.6/ 6.1 

5.4 E I 

6.1 6.6 

5.7/ 5.5 

6.2 6.0 

5.7 5.9 

5.6 5.4/ 5.0 5.2 5.9 E I 5.4 5.8 

5.7 5.4 

5. 7 5.2 

5.4/ 6.0 

5.0 5.8 

8 9 

5.4 E 5.9 5.6 5.5 6.2/ 

6.0 5.7 

5.9 5.8 

6.1 5.4 

6.5 6.3 

E 6. 0 

5. 7 6. 7 

6.1 5.8 

5.6 5.4 

5.2 5.4/ 

5.6 5.6 

5. 8 6.2 

6.0/ E 5.2 5. 5 

5.7 5.1 

5.7/ 5.9 

10 

6.0 

5.9 

6.2 

5.4 5.8 

5.8 

6.1 6.2 

6.2 

5.7 5.7 5.5 5.9 

5.6 

5.7 

5.5 

6.6 

5.2 5.5 

5.8/ 6.5 

6.0 5.5 

5.3 5.8 

5.3/ 5.5 5.9 5.9 

6.2 6.0 6.0 

5.2 5.7 6.1/ 6.2 5.3 5.6/ 5.3 5.4 

11 

6.2 6.0 6.0 

5.3 5.3 5.2 

5.3 

6.4 

6.2/ 

5.4 

5.1 5.4 

6.1 

5.6 

6.1/ 

L 

5.6 

4.8 

5.7 5.8 5.7 

12 

6.5 6.0 

5.4 

5.6 5.7 

5.3 5.8 

6.4 6.4 

5.7 5.2 

6.1 

5.9 

5.0 5.3 

5.5 

5.7 5.3 5.4 

6.1 

5.6 

13 

6.2 

5.5 5.2 5.8 

5.3 5.7 

6.3 

5.6 5.2 5.3 

5.6 5.7 

5.4 

6.0 

5.2 

6.0 

5.8 

5.5 

14 

6.5 

5.7 5.3 5.4 

5.9 

E 

5.3 

5.3 

5.4 

5.9 

6.1 

5.2 

6.4 

5.6 

15 

5.3 

6.1 

6.2 

5.6 

5.5 

5.9 

5.2 

5.4 

6.1 

5.3 

16 

5.6 

5.5 
10-MAR-2011 17:24 

Huntingdon Life Sciences 05-4287 

APPENDIX K 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

INDIVIDUAL FETAL BODY WEIGHTS {GRAMS) 

GROUP 2 2000 MG/M3 

FEMALE# MEAN 

2901 

2902 

2903 

2904 2905 2906 2907 2908 

2909 2910 2911 

2912 

2913 

2914 2915 

29.16 

2917 2918 

2919 2920 2921 2922 

2923 2924 

5.5 5.7 5.7 

6.1 

5.8 5.9 5.4 5.2 

6.0 

5.9 5.8 

6.0 

5.6 

6.3 

5.9 

5.4 5.3 

5.9 

NP 

5.3 5.7 5.8 

6.0 6.1 

MEAN 5.8 

S.D. 0.30 

N 23 

FETUS# 

1 2 

5.4/ 

5.2 5.1 

5.9 

6.1 6.0 

4.9 

5.3 

5.7 5.7 5.5 

6.4 

5.6 

E 

5.4 5.5 5.2 

5.7 

5.5 

E 

5.4 

5.4 5.9 

5.6 

5.7 5.8 

5.9 

6.2 

5.8 5.8 

4.9 

E 

5.5 

6.0 

5.9 

5.6 

E 

E 

5.3 

E 

6.0 

5.7 

E 

6.0 

5.9 

6.5 

3 

5.3 

6.0 

5.3 

6.4 

5.9 

6.1/ 

5.1 

4.9 

6.5 6.0 

5.4 5;9 

5.5 

6.1 

6.2 

5.3 

5.0 

5.9 

L 

5:7 

5.9 5.8 

5.9 

4 

5.3 5.7 5.5 

6.0 

5.6 

6.0 6.0 

5.7 

6.4 

5.7 5.7 

6.4 

5 

5.7 5.5 5.5 

5.8 

5.6 5.5 5.4 5.6 

5.8/ 

5.3 

5.7/ 

6.0 

5.7 5.6 

E E I 

6.0 6.2 

5.1 5.6 

5.5 5.0/ 

6.0 5.3 

5.4/ 5.1 

6.0 5.4 

5.4 5.8 

6.1 6.3 6.5 6.1 

E-Early resorption L-Late resorption 

/-Denotes position of cervix 

NP=NOT PREGNANT 

6 

5.1 

6.1 

E 

5.7 

5.9 

E 

5.1 

5.1/ 

6.4 

5.8/ 

5.4 5.8 

7 

5.6 

5.9/ 5.9/ 

6.4 

5.9 

5.5 

5.5/ 

5.1 

6.2 6.1 

5.6 

6.0 

8 

5.3 5.3 5.9 

E 

5.7 5.9 5.7 5.6 

5.8 

6.4 

5.4 5.8 

5.7 5.5 5.7 

E E E 

5.7 6.2 5.3/ 5.3 4.9 5.8/ 5.6 5.6 5.4 

5.4 6.1 5.4 

5.3 5.2 4.9 5.8 5.6 5.7 

5.6/ 6.0 5.9 

6.3/ 6.5 6.4 

6.0 5.3/ 5.9 

9 

E 

6.2 

5.9 

E I 

6.3 6.2 

5.3 

4.8 

5.7 

6.0 

5.7 

6.1 

10 11 

6.1 5.9 

E 5. 5 

5.9 5.9 

6.8 5.9 

5.6/ 6.2 

6.1 6.2 

5.4 5.2 

E 5.3 

5.9 5.8 

6.2 5.9 

6.3 6.1 

6.1/ 6.1 

5.1/ 5.6 5.6 

6.6 6.4 E 

5.9 6.2 6.1 

5.4 5.7 5.4 5.6 5.4 4.9 

5.9 6.0/ 6.1 

E 5.1 4.5 

6.0/ 5.8 5.6 

5.8 6.0 5.8 

5.7 6.0 6.2 

5.7 6.1 6.5 

12 

5.9 

6.1 

6.4 

5.8 

6.1 

5.2 5.4 

6.5 

5.7 

5.9 

6.0 

E 

5.6 5.6 

5.3 

6.5 

5.6 

6.0 

5.8 

6.3 

6.1 

13 

6.3 

5.5 5.8 

5.3 

4.9 

5.2 

6.2 6.1 

5.6 

E 

5.7 5.3 

6.3 

5.3 5.6 

6.0 6.3 

14 

6.2 

5.8 5.1 

6.4 6.1 

6.3 

5.8 

6.2 

5.2 

5.4 

6.2 

15 

6.1 

5.2 

5.5 

5.5 

6.1 

16 

5.2 

5.9 
10-MAR-2011 17:24 

Huntingdon Life Sciences 05-428.7 

GROUP 3 10000 MG/M3 

FEMALEll MEAN 

3901 3902 

"3903 

3904 

3905 

3906 

3907 3908 3909 

3910 

3911 3912 3913 

3914 3915 391"6 3917 

3918 3919 3920 

3921 

3922 3923 3924 

5.8 

5. 61 

5.1 

5.8 5.1 5.6 5.6 

5.5 5.8 5.9 

5.5 

6.0 

6. 4 I 

5.3 

5.8 I 

5.7 5.3 

5.5 5.5 

6.0 

5.8 5.8 5.6 5.8 

MEAN 5.7 

S.D. 0.28 

N 24 

E-Early resorption 

FETUSll 1 2 

5.5 

5.6 

5.4 5.4 

5.1 5.4 5.7 

5.6 

6.0 

5.9 

5.7 

6.1 

7.0 

5.2 

E 

5.6 

4.6 

5.7 5.6 

6.1 

5.6 

6.2 

5.5 

5.6 

6.0 

5.9 5.2 

.5. 9 

5.4 

5.7 

E 

5.7 5.8 

6.0 

6.3 

E 

6.3 

5.3 

6.4 

5.5 5.3 

E 

5.8 5.7 

6.3 6.0 

5.5 

5.9 

/-Denotes position of cervix 

3 

5.9 5.3 5.6 

6.1 

4.7 

4.6 

6.0 

E 

5.6 

6.2 

5.9 5.9 5.8 

5.3 5.3 5.7 5.2 

5.6 5.5 

6.7 

6.0 

5.8 5.1 5.8 

APPENDIX K 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

4 

5.7 

E 

5.2 

4.9 4.9 

5.2 5.6 

6.1 

E 

5.8 

6.0 

6.4 

6.7 

5.8 5.5 5.6 

5.8 5.8 

6.4 

E 

5.5 5.5 

6.0 

INDIVIDUAL FETAL BODY WEIGHTS (GRAMS} 

5 

5.7 5.2 

5.3/ 

6.0 

4.8 

5.5 5.6 

5.1 5.8 

6.0 

6.2 

6.3/ 

6.2 

5.0 5.8 

5.5/ 

5.8 5.1 

6.0 

5.6 5.8 5.5 

6.0 

6 

6.2 

5.5 

5.0 

6.2 

4.9 

5.6 

5.3/ 

5.2 5.9 

6.0/ 

5.5 

6.3 6.4 

E I 

5.4 5.4 

5.9/ 5.5/ 

5.5 

6.2 

5.3 5.8 5.2 

7 8 9 

5.7/ 6.0 5.1 

5.2 5.4 5.8/ 

5.1 4.9 5.0 

5.8/ 6.1 5.8 

4.8 5.2 4. 7 

5.5 5.5 5.6 

6.0 5.5 E 

5.4/ 5.6 5.3 

5.6 5.3 5.4 5.7 6.0 6.2 

5.6 3.7 5.7 

5.8 5.8 5.9 

6.4 5.9 

5.6 5.4 5.2 

5.9/ 5.7 5.7 

5.0 5.4 5.7 

E 5.5 5.3 

5.9 5.7 4.9 

6.2/ 6.2 5.8 

5.5/ 6.0 6.1 

5.7 5.6 5.8/ 5.8 5.7/ 5.6 5.6 5.8/ 6.3 

10 

5.9 5.6 

4.8 

6.3 

5.4/ 5.9/ 

5.6 

5.1 

5.7/ 

4.3 

5.4/ 

6.0 

5.4 5.6 

4.9 

5.7 5.3 5.8 

5.9 5.8 

5.7 5.8 

11 

5.8 

5.1 

5. 6 . 5.3 5.9 

5.4 

E 

6.l,. 

6.0 3.3 

5.9 

4.9 

5.6 

4.8 

5.5 5.6 5.8 

5.8 5.7 5.5 

5.8 

12 

5.8 

4.8 

5.8 5.9 5.9 5.7 

E 

6.1 6.4 

6.2 

6.0 

5.3 5.7 5.5 

5.4 

5.7 

6.1 

4.9 

5.2 

5 .. 4 

5.8 

13 

5.6 

5.3 

5.9 

5.0 

6.1 

5.8 

5.8 

5.7 5.9 

5.5 

5.7 

5.0 5.8 5.7 

5.1 5.3 

5.5 

5.5 

6.1 6.0 

14 

5.5 5.8 

6.4 

5.7 

5.3 5.8 5.3 

4.8 

5.8 

6.2 

5.3 5.6 

15 

5.2 

5.6 

5.9 

5.7 

5.7 5.7 

16 

6.0 

6.3 

5.5 
10-MAR-2011 17:24 

Huntingdon Life Sciences 05-4287 

GROUP 4 20000 MG/M3 

FEMALE# MEAN 

4901 4902 4903 

4904 4905 4906 4907 4908 

4909 

4910 

4911 4912 

4913 

4914 4915 4916 4917 

4918 

4919 

4920 4921 4922 

4923 4924 

5.5 5.5 5.7 

5.2 5.2 5.4 5.7 

NP 

5.9 5.6 5.4 

6.2 

5.8 

6.1 

5.2 

4.9 

5.5 

NP 

5.2 5.6 5.2 

5.5 5.4 

MEAN 5.5 

S.D. 0.32 

N 22 

E-Early resorption 

FETUS# 

1 2 

5.7 

6.0 

5.6 

4.8 

5.2 5.5 

6.0 

6.1 

5.5 5.1 

6.7 

6.0 

5.9 5.5 5.0 

6.0 

5.2 5.2 5.4 5.0 

5.3 

4.7 

5.9 5.5 5.7 

5.1 5.3 5.4 5.6 

6.0 

5.7 5.6 

6.7 

5.8 

E 

4.7 

5.4 5.9 

5.3 5.0 5.8 

4.9 

5.2 5.7 

/-Denotes position of cervix 

NP=NOT PREGNANT 

3 

5.6 

.E 

5.7 

5.8 

5.1 

5.3 5.6 

E 

5.2 5.1 5.3 

5.6 

6.5 

5.3 

4.8 

5.8 

5.3 

E 

5.2 5.8 

5.6 5.3 

APPENDIX K 

GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY 

IN RATS BY INHALATION EXPOSURE 

4 

5.3/ 

5.6 5.7 

5.1 

5.5 5.4 

6.1 

5.8 

6.0 

5.6 

6.0 

5.7 5.9 

5.2 

4.9 

5.6 

5.4 

5.2 5.6 5.0 

5.9 5.9 

INDIVIDUAL FETAL BODY WEIGHTS (GRAMS) 

5 6 7 

5.5 5.4 5.2 

5.4/ 5.4 5.3 

E 5.9/ 5.4 

4.9 4.8 5.1/ 

5.2 5.6 5.1 

5.1 5.9 5.5 5.6 5.6/ E 

6.3 5.4 6.2 

5.8 5.8 5.7 5.8 4.4 5.3 

6.3 6.6/ 5.6 

5.8/ 6.1 5.7. 

6.3 E 5.9 

5.4 E 5.3 

4.7 4.5 5.3/ 

6.0 5.7 5.7 

5.2/ 5.5 5.4 

5.1 5.1/ 5.3 

6.4 5.6/ 5.3 

5.1 5.6 5.3 

4.8 5.5 5.6 

5.2 5.6 5.6 

8 9 10 

5.3 5.4 5.6 5.3 6.0 5.1 

5.9 5.2 5.8 

5.5 5.1 5.1 

5.0 4.5 4.9/ 

5.4/ 5.5 5.0 

5.7 6.0 5.4 

6.1/ 6,0 5.4 

5.7/ E 5.5 

5.4 5.4 5.5 

5.8 5.7 6.0 

5.4 5.3 6.1 

6.2/ 5.7 6.4 

5.1 4.3 5.2/ 5.1 5.0 5.1 

E 6.2/ 6.0 

6.2 5.3 5.3 

5.4 5.8 5.0 

5.3 5.8 5.6 5.3 5.3/ 5.3 

5.8/ 5.2 5.9 

5.4 5.3 5.8 

11 

5.4 

5.1 5.5 

5.8 5.2 5.0 5.9 

5.0 5.6 

6.9 

6.0 6.1 

4.8 4.9 

5.8 5.9 

5.2 5.0 5.2 

E E 

12 13 

E 5.2 

6.1 6.0 

5.2 5.3 5.2 5.7 5.3 5.4 

5.8 5.8 

5.4/ E 

5.9 5.8 

6.6 6.1 5.4 

4.8 4.2 

5.8 6.1 

.5. 8 5. 7 

5.3 5.3 4.9 

5.8 E 

5.5/ 5.6 

14 

5.4 

5.8 

4.9 

5.5 5.4 

5.8 

5.1 

5.6 

5.5 5.2 

6.0 

5.2 

5.1 

15 

5.7 

5.3 

5.1 5.6 

5.6 

5.0 

4.7 

6.1 

5.5 

5.6 

16 

4.9 

5.4 5.3 

5.6 

4.8 

5.2 
Huntingdon Life Sciences 05-4287 

211-DIPE-DEV 

Females INDIVIDUAL FETAL EXTERNAL VISCERAL AND SKELETAL OBSERVATIONS 

Animal 

Number 

Group 1 - 0 MG/M3 · 

1901 6s, 5v 12 

1903 7s, 7v 3 

10 11 

1904 7s, 8v 6 

11 

1905 7s, 6v 1 

2 

1908 6s, 6v 6 

1909 8s, 7v 8 

9 

M 

F 

M M 

F F 

M 

F 

.F 

F 

M 

6.5 

5.3 

6.2 6.0 

5.4 

5.3 

3.2 

5.7 

5.7 

5.7 

6.7 

bnormalities (only fetuses with abnormalities are listed) 

Fate IMaior abnormalities in CAPITALS 

s Sternebra(e) malaligned, #4 and #5, slight 

s Rib(s) 14th rudimentary, left 

v Additional subclavian artery, left 

s Rib(s) 14th rudimentary, bilateral 

s Hyoid body/arch(es), unossified body 

v Renal papilla (e) not fully developed (grade 1), left 

s ANAL ATRESIA AND FILAMENTOUS TAIL 

VERTEBRAL AGENESIS: SACRAL VERTEBRAE #1 AND #2 

SACRAL VERTEBRAE #3 AND #4 AND ALL CAUDAL VERTE

ILIA AND ISCHIA CLOSELY APPROXIMATED 

Sternebra(e) 5 and/or 6 unossified, #5 

v Undescended thymic tissue, right 

s 25 presacral vertebra(e) 

Rib(s) 13th rudimentary, right 

v Undescended thymic tissue, left 

s Sternebra(e) malaligned, #2 through #4, slight 

s = skeletal examination, v = visceral examination , M=Male, F=Female, g=grams 
Huntingdon Life Sciences 05-4287 

211-DIPE-DEV 

Females INDIVIDUAL FETAL EXTERNAL VISCERAL AND SKELETAL OBSERVATIONS 

Animal 

Number 

Group 1 - 0 MG/M3 

1910 6s, 7v 2 

9 

1911 8s, 7v 4 

9 

1912 7s, 8v· 3 

9 

12 

1913 6s, 5v 6 

1914 6s, 6v 8 

1916 7s, 7v 1 

;3 

F 

F 

M 

M 

F 

F 

F 

M 

F 

F 

M 

6.0 

5.8 

5.5 

5.4 

5.3 

5.4 5.2 

5.9 

5.8 

5.3 5.5 

!Abnormalities (only fetuses with abnormalities are listed) 

Fate IMaior abnormalities in CAPITALS 

s Sternebra(e) malaligned, #4, slight 

v Left umbilical artery v Undescended thymic tissue, left 

v Undescended thymic tissue, right v Undescended thymic tissue, left 

v Undescended thymic tissue, right 

Ureter(s) distended, bilateral 

s Sternebra(e) malaligned, #4 and #5, slight 

s Sternebra(e) malaligned, #3 through #5, slight to modera

s Sternebra(e) malalfgned, #4 and #5, slight 

v Undescended thymic tissue, right v Undescended thymic tissue, left 

s = skeletal examination, v = visceral examination, M=Male, F=Female, g=grams 
Huntingdon Life Sciences 05-4287 

211-DIPE-DEV 

Females INDIVIDUAL FETAL EXTERNAL VISCERAL AND SKELETAL OBSERVATIONS 

Animal 

Number 

Group 1 - 0 MG/M3 

1918 7s, 7v 11 

1919 7s, 7v 9 

11 13 

1921 8s, 7v 2 

6 

12 

1923 8s, 7v 7 

-1924 7s, 7v 9 

L 

F 5.9 

F 5.6 F 6.0 

F 5.3 

M 5.5 M 5.3 

F 5.2 F 5.3 

bnormalities (only fetuses with abnormalities are listed) 

Fate IMaior abnormalities in CAPITALS 

Crown-rump distance 2.4 em 

s Sternebra(e) malaligned, #4 and #5, slight s Sternebra(e) malaligned, #4 and #5, slight 

s Sternebra(e) malaligned, #4 and #5, slight 

v Innominate artery absent 

v Undescended thymic tissue, left v Undescended thymic tissue, right 

Displaced testis medially, left 

s Sternebra(e) malaligned,#3 and #4, slight to moderate . 

s Sternebra(e) malaligned, #3 through #5, slight 

s = skeletal examination, v = visceral examination, M=Male, F=Female, L=Late Resorption, g=grams 
Huntingdon Life Sciences 05-4287 

211-DIPE-DEV 

Females I INDIVIDUAL FETAL EXTERNAL, VISCERAL AND SKELETAL OBSERVATIONS 

Animal bnormalities (only fetuses with abnormalities are listed) 

Number Fate Ma ·or abnormalities in CAPITALS 

Group 2- 2000 MG/M3 

2901 5s, 5v 2 M 5.6 v Undescended thymic tissue, left 

3 F 5.3 s Sternebra(e) malqligned, #3 through #4, slight 

8 M 5.3 v Undescended thymic tissue, bilateral 

2902. 5s, 6v 2 F 5.7 s Sternebra(e) malaligned, #3 and #4, slight 

2903 6s, 5v 2 M 5.8 v Undescended thymic tissue, left 

3 M 5.3 s Sternebra(e) malaligned, #4 and #5 

Rib(s) 14th rudimentary, right 

2904 6s,6v 14 F 6.2 s RIGHT FORELIMB SHORTENED 

2905 8s, 8v 1 M 6.1 s Sternebra(e) malaligned, #3 through #5, slight 

7 F 5.9 s Sternebra(e) malaligned, #4 and #5, slight 

10 F 5.6 v Undescended thymic tissue, right 

2908 7s, 7v 14 M 5.1 v Left umbilical artery 

2909 5s, 5v 6 M 6.4 s Rib(s) 14th rudimentary, right 

11 M 5.8 v Left umbilical artery 

s = skeletal examination, v = visceral examination, M=Male, F=Female, g=grams 
Huntingdon Life Sciences 05-4287 

211-DIPE-DEV 

Females I INDIVIDUAL FETAL EXTERNAL, VISCERAL AND SKELETAL OBSERVATIONS 

Animal bnormalities (only fetuses with abnormalities are listed) 

Number Fate Ma'or abnormalities in CAPITALS 

Group 2- 2000 MG/M3 

2910 7s, 7v 3 F 6.0 v Undescended thymic tissue, bilateral 

4 F 5.7 s Rib(s) 14th rudimentary, bilateral 

5 F 5.3 v Undescended thymic tissue, left 

6 F 5.8 s Rib(s) 14th rudimentary, bilateral 

8 F 6.4 s Rib(s) 14th rudimentary, left 

13 F 5.2 v . Undescended thymic tissue, right 

2912 8s, 9v 4 M 6.4 s 7th cervical rib(s) present, left 

10 . F 6.1 s SHORT MANDIBLE, moderate 15 M 5.5 v Undescended thymic tissue, left 

Left umbilical artery 

17 F 5.8 v Left umbilical artery 

2913 7s, 7v 1 M 5.6 s Sternebra(e) malaligned, #4 and #5, slight 

Rib(s) 14th rudimentary, bilateral 

2917 6s, 6v 4 M 5.5 s Rib(s) 14th ·rudimentary, bilateral 

13 F 5.3 v Undescended thymic tissue, bilateral 

2918 6s, 7v 2 M 6.0 s Sternebra(e) malaligned, #4 and #5, slight 

s = skeletal examination, v = visceral examination, M=Male, F=Female, g=grams 
Huntingdon Life Sciences 05-4287 

211-DIPE-DEV 

Females INDIVIDUAL FETAL EXTERNAL VISCERAL AND SKELETAL OBSERVATIONS 

Animal I I Fetus Abnormalities (only fetuses with abnormalities are listed) 

Number Exam Number Sex Fate Ma·or abnormalitie,s in CAPITALS 

Group 2- 2000 MG/M3 

2920 5s, 6v 3 L Crown-rump distance 2.9 em 

12 M 5.6 s Sternebra(e) malaligned, #4 and #5, slight 

2921 5s, 5v 3 M 5.7 s Sternebra(e) malaligned, #3, slight 

2923 8s, 7v 1 F 5.4 s Sternebra(e) malaligned, #3 through #5, slight 

7 F 6.5 s Sternebra(e) malaligned, #3 and #4, slight 

14 F 5.4 v Undescended thymic tissue, left 

2924 7s, 7v 11 F 6.5 v Undescended thymic tissue, left 

s = skeletal examination, v = visceral examination, M=Male, F=Female, L=Late Resorption, g=grams 
Huntingdon Life Sciences 05-4287 

211-DIPE-DEV 

Females I INDIVIDUAL FETAL EXTERNAL, VISCERAL AND SKELETAL OBSERVATIONS I 

Animal bnormalities (only fetuses with abnormalities are· listed) 

Number Fate Ma'or abnormalities in CAPITALS 

Group 3 - 10000 MG/M3 

3901 5s, 5v 1 M 5.5 s Sternebra(e) malaligned, #4, slight 

3903 7s, 6v 9 F 5.0 s Sternebra(e) malaligned, #3 and #4, slight 

3905 8s, 7v 1 F 5.1 s Sternebra(e) malaligned, #5, slight 

Rib(s) 14th rudimentary, right 

5 F 4.8 s Sternebra(e) malaligned, #5, slight 

3906 7s, 7v 1 M 5.4 v Innominate artery absent 

9 F 5.6 v Renal papilla (e) not fully developed Egrade 1), left 

Ureter(s) distended left 

3907 6s, 5v 6 F 5.3 s Sternebra(e) malaligned, #5, slight 

8 F 5.5 s Hyoid body/arch(es), unossified body 

3910 7s, 7v 6 M 6.0 s Rib(s) 14th rudimentary, right 

7 F 5.7 v Innominate artery absent 

s = skeletal examination, v = visceral examination, M=Male, F=Female, g=grams 
Huntingdon Life Sciences 05-4287 

211-DIPE-DEV 

Females I INDIVIDUAL FETAL EXTERNAL1 VISCERAL AND SKELETAL OBSERVATIONS 

Animal Fetus I Weight I !Abnormalities (only fetuses with abnormalities are listed) 

Number Number Sex (a) Fate Maior abnormalities in CAPITALS 

Group 3 - 10000 MG/M3 

3911 7s, 9v 8 F 3.7 v MICROPHTHALMIA/ ANOPHTHALMIA, left 

Undescended thymic tissue, right 

Left umbilical artery 

11 F 3.3 v DOMED ·cRANIUM, moderate; HYDROCEPHALY 

ANOPHTHALMIA, bilateral 

Hypoplastic lung lobe(s), apical lobe 

Hypoplastic thymus, bilateral lobes 

Hypoplastic spleen 

13 F 5.5 s Rib(s) 14th rudimentary, bilateral 

3912 6s, 6v 6 M 6.3 v Undescended thymic tissue, bilateral 

8 M 5.8 v Undescended thymic tissue, bilateral 

12 F 6.0 v Undescended thymic tissue, right 

3913 4s, 4v 1 M 7.0 s Rib(s) 14th rudimentary, right 

5 M 6.2 s Sternebra(e) malaligned, #4, slight 

3914 7s, 7v 5 M 5.0 v Undescended thymic tissue, left 

15 M 5.9 s Rib(s) 14th rudimentary, left 

3916 7s, 7v 2 M 5.5 s Sternebra(e) 5 and/or 6 unossified, #5 

8 M 5.7 s Sternebra(e) malaligned, #3 and #4, slight 

s = skeletal examination, v = visceral examination, M=Male, F=Female, g=grams 
Huntingdon Life Sciences 05-4287 

211-DIPE-DEV 

Females INDIVIDUAL FETAL EXTERNAL VISCERAL AND SKELETAL OBSERVATIONS 

Animal 

Number 

Group 3- 10000 MG/M3 

3917 7s, 7v 11 F 

3918 6s, 7v 14 F 

3921 6s, 5v 6 M 

8 F 

3922 8s, 9v 2 F 

10 F 

3923 8s,8v 16 M 

3924 7s, 7v 4 M 

6 F 

4.8 4.8 

6.2 

6.0 

6.0 

5.8 

5.5 

6.0 

5.2 

.,Abnormalities (only fetuses with abnormalities are listed) 

Fate Maior abnormalities in CAPITALS 

s Sternebra( e) 5 and/or 6 unossified, #5 

s Sternebra(e) malaligned, #3 and #4, slight 

s Sternebra(e) malaligned, #3 through #5, slight s sternebra(e) malaligned, #3 through #5, slight 

s Sternebra(e) malaligned, #4 and #5 s Sternebra(e) malaligned, #3 through #5, slight 

v Renal papilla (e) not fully developed (grade 1), left 

s Sternebra(e) malaligned, #4, slight 

s Sternebra(e) 5 and/or 6 unossified, #5 

s = skeletal examination, v = visceral examination, M=Male, F=Female, g=grams 
Huntingdon Life Sciences 05-4287 

211-DIPE-DEV 

Females INDIVIDUAL FETAL EXTERNAL VISCERAL AND SKELETAL OBSERVATIONS 

Animal I I Fetus I I Weight I !Abnormalities (only fetuses with abnormalities are listed) 

Number Exam Number Sex (a) Fate Maior abnormalities in CAPITALS 

Group 4 - 20.000 MG/M3 

4901 7s, 6v 2 M 5.9 v Undescended thymic tissue, left 

5 F 5.5 s Hyoid body/arch(es) unossified body 

10 M 5.6 v Undescended thymic tissue, bilateral 

4902 5s, 5v 5 F 5.4 s 7th cervical rib(s) right 

Rib(s) 14th rudimentary, bilateral 

11 F 5.1 s Sternebra(e) 5 and/or 6 unossified, #5 

4904 9s, 9v 3 M 5.8 v Undescended thymic tissue, left 

4905 8s, 8v 12 F 5.2 v Undescended thymic tissue, left 

14 M 5.5 v Undescended thymic tissue, left 

15 M 5.1 s. Sternebra(e) malaligned, #2 through #4, slight to moder

4906 8s, 8v 5 F 5.1 v Undescended thymic tissue, right 

11 F 5.0 v Undescended thymic tissue, left 

4907 5s, 5v 1 M 6.0 s VERTEBRAL ANOMALY: THORACIC CENTRUM #13 AND LU

#1 ABSENT; THORACIC ARCHES #13 AND LUMBAR ARCH

#1 MALFORMED AND CLOSELY APPROXIMATED 

11 M S.9 v Undescended thymic tissue, left 

s = skeletal examination, v = visceral examination, M=Male, F=Female, g=grams 
Huntingdon Life Sciences 05-4287 

211-DIPE-DEV 

Females INDIVIDUAL FETAL EXTERNAL. VISCERAL AND SKELETAL OBSERVATIONS 

Animal bnormalities (only fetuses with abnormalities are listed) 

Number Fate Ma ·or abnormalities in CAPITALS 

Group 4 - 20000 MG/M3 

.4909 5s, 4v 4 F 5.8 s Rib(s) 14th rudimentary, right 

6 M 5.4 s EXOCCIPITAL AND CERVICAL ARCH .#1 FUSED, right 

Sternebra(e) malaligned, #4 and #5, slight 

4910 6s, 7v 13 F 5.8 s 27 presacral vertebra(e) 

Rib(s) 14th rudimentary, bilateral 

4911 8s, 8v 5 M 5.8 s Rib(s) 14th rudimentary, right 

16 F 5.6 s Rib(s) 14th rudimentary, left 

4912 5s, 6v 2 M 6.7 s Rib(s) 14th rudimentary, right 

8 F 5.8 s Sternebra(e) malaligned, #3 through #5, slight to modera

4915 7s, 7v 3 F 5.3 s Rib(s) 14th rudimentary, right 

8 F 5.1 s Rib(s) 14th rudimentary, left 

15 F 5.0 v Innominate artery absent 

4916 8s, 8v 13 F 4.2 v Left umbilical artery 

s = skeletal examination, v = visceral examination, M=Male, F=Female, g=grams 
Huntingdon Life Sciences 05-4287 

211-DIPE-DEV 

Females I INDIVIDUAL FETAL EXTERNAL, VISCERAL AND SKELETAL OBSERVATIONS 

Animal Abnormalities (only fetuses with abnormalities are listed) 

Number Fate Ma'or abnormalities in CAPITALS 

Group 4 - 20000 MG/M3 

4917 7s, 7v 4 F 5.6 v Undescended thymic tissue, left 

10 F 6.0 s 7th cervical rib(s) present, right 

12 M 5.8 s Rib(s) 14th rudimentary, right 14 F 6.0 s Rib(s) 14th ru-dimentary, left 

4921 6s, 5v 1 M 5.4 s 7th cervical rib(s) present, right 

25 presacral vertebra(e) 

Sternebra(e) 5 and/or 6 unossified, #5 

Rib(s) 13th rudimentary, right 

2 M 5.8 v Undescended thymic tissue, right 

3 M 5.2 s 25 presacral vertebra(e) 

Sternebra(e) 5 and/or 6 unossified, #5 

Rib(s) 13th rudimentary, bilateral 

Metatarsals, 1 unossified, bilateral 

11 F 5.0 s 25 presacral vertebra(e) 

Rib(s) 13th rudimentary, bilateral 

Metatarsals, 1 unossified, bilateral 

4923 6s, 5v 4 M 5.9 v Undescended thymic tissue, left 

12 M 5.8 s Rib(s) 14th rudimentary, left 

s = skeletal examination, v = visceral examination, M=Male, F=Female, g=grams 
Huntingdon Life Sciences 05-4287 

211-DIPE-DEV 

Females INDIVIDUAL FETAL EXTERNAL VISCERAL AND SKELETAL OBSERVATIONS 

Animal 

Number 

Group 4- 20000 MG/M3 

4924 8s, 9v 1 

14 17 

F 

F F 

4.7 

5.1 

.5.8 

!

Abnormalities (only fetuses with abnormalities are listed) 

Fate Maior abnormalities in CAPITALS 

v Renal papilla (e) not fully developed (grade 1), bilateral 

Ureter(s) distended, left 

v Left umbilical artery 

s Rib(s) 14th rudimentary, left 

s = skeletal examination, v = visceral examination, M=Male, F=Female, g=grams 
05-4287 

GASOLINE DIPE VAPOR CONDENSATE 

EMBRYO-FETAL TOXICITY STUDY IN RATS BY INHALATION EXPOSURE 

Statistical Analysis 

Page 1 of 18 

Authors 

Graham F Healey 

Simon Bate 

Statistics Department 

Statistical analysis 
05-4287 

COMPLIANCE STATEMENT 

This phase of the study was conducted in compliance with the following Good Laboratory 

Practice standards and I consider the data generated to be valid. 

The UK Good Laboratory Practice Regulations (Statutory Instrument 1999 No 3106 

as amended by Statutory Instrument 2004 No. 994). 

OECD Principles of Good Laboratory Practice (as revised in 1997), 

ENV /MC/CHEM(98) 17. 

EC Commission Directive 2004/10/EC of 11 February 2004 (Official Journal No L 

50/44). 

US EPA 79.60, CFR Vol. 59, No. 122,27 June 1994. 

These principles of Good Laboratory Practice are accepted by the regulatory authorities of 

the United States of America and Japan on the basis of intergovernmental agreements. 

Principal Investigator 

Gareth D. Thomas, B.Sc. (Hons). 

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Statistical analysis 
05-4287 

INTRODUCTION AND EXPERIMENTAL DESIGN 

This rat study had a control and three groups treated at 2000 mg/m3

, I 0000 mg/m3 and 

20000 mg/m3

. There were up to 24 dams per group. 

The following parameters were subjected to statistical analysis: 

Continuous maternal parameters 

Maternal body weight (d4, d5, d8, dll, d14, d17, d20, d21) 

Maternal body weight change (d4-d5, d5-d8, d8-dll, dll-dl4, d14-d17, d17-d20, d5-dl4, 

d14-d21, d5-d21) 

Maternal feed consumption {d4-d5, d5-d8, d8-dll, dll-dl4, d14-d17, d17-d20, d20-d21, 

d5-d21) 

Maternal uterus weight (absolute and adjusted) 

Maternal net bodyweight change from d5 minus gravid uterine weight 

Reproductive performance 

Corpora lutea 

%Preimplantation loss 

Number of implant sites 

Fetuses alive 

Early resorptions 

Late resorptions 

Total resorptions 

%Total resorptions/implant sites 

Number of males 

Number of females 

There were no dead fetuses, therefore the "Fetuses dead" and "%Fetuses dead/total" data 

were not analyzed. 

Fetal body weight 

Mean fetal body weight: males 

Mean fetal body weight: females 

Mean fetal body weight: litter (pooled males and females) 

Discrete fmdings 

Maternal pregnancy summary 

Maternal necropsy fmdings 

Fetal fmdings: external, visceral and skeletal (only certain visceral and skeletal fmdings 

required statistical analysis). 

:3: 

Statistical analysis 
05-4287 

METHODOLOGY 

Continuous maternal parameters 

The analysis was performed at each timepoint separately. 

Bartlett's test (Bartlett, 1937) was first applied (Proc GLM, SAS Institute 1999) to determine 

if the groups had equal variances. If the test was not significant at the 1% level, parametric 

procedures were used. If the test was significant then non-parametric procedures were used. 

For the parametric choice, a one-way analysis of variance (Armitage, Berry and Matthews 

2002) was initially applied (Proc GLM, SAS Institute 1999). If this was significant at the 5% 

level, then each treatment group was compared with the control using Dunnett's test (Dunnett 

1955, 1964). 

For the non-parametric choice, a Kruskal-Wallis test (Kruskal and Wallis 1952, 1953) was 

initially applied (Proc Npar1way, SAS Institute 1999). If this was significant at the 5% level, 

then each treatment group was compared with the control using Steel's test (Steel1959). 

Reproductive performance 

All parameters except fetal bodyweight were analyzed using the same methods as for 

continuous maternal parameters, described above. 

Fetal body weight 

The fetal body weight was analyzed by separate and combined sexes using mixed models 

(Proc Mixed, SAS Institute 1999). The separate-sex analysis had dose group as explanatory 

variable, a random animal effect and litter size as covariate. The combined analysis had dose 

group and fetal sex and their interaction as explanatory variables, with litter size as covariate. 

For both sets of analysis, the Satterthwaite method was used for the fixed effects denominator 

degrees of freedom. If the dose group effect was significant at the 5% level, then each 

treatment group was compared with the control using Dunnett's test (Dunnett 1955, 1964), 

with the means being adjusted for litter size. 

: 4 : 

Statistical analysis 
Discrete findings 

Maternal pregnancy summary 

Maternal necropsy fmdings 

Fetal findings: external, visceral and skeletal malformations and variations 

For maternal parameters: 

05-4287 

These parameters were analyzed using non-parametric methods. If the dose group effect in 

was statistically significant, each dose group was compared with the control group using 

pairwise Wilcoxon rank sum tests (Wilcoxon 1945). 

For fetal fmdings: 

Only fmdings having an incidence of greater than one were included in the statistical 

analysis, since for less frequent parameters there was no chance of statistical significance. 

These parameters were analyzed by generalized estimating equations (Proc Genmod, SAS 

Institute 1999) using a log link function and assuming Poisson distributed data. The litter 

was the basis for analysis where the correlation among littermates was modelled using a 

compound symmetric structure. Results are presented using the model based standard errors. 

If the dose group effect in the model was statistically significant, each dose group was 

compared with the control group using pairwise tests associated with least squares means, in 

this case being the mean incidence adjusted for litter size. For very low frequency fmdings 

this method did not converge. Therefore non-parametric methods, the Kruskal-Wallis test 

(Kruskal and Wallis 1952, 1953) were applied to the total number of affected foetuses in the 

litter instead. If this was significant at the 5% level then each treatment group was compared 

with the control using Wilcoxon rank sum tests (Wilcoxon 1945). 

DATA HANDLING 

Data were received as text files and Excel spreadsheets and re-formatted for software input. 

The software used for all the analyses was SAS 8.2 (SAS Institute 1999). For the analysis of 

uterine weight, adjusted bodyweight and the reanalysis of fetal fmdings conducted in this 

updated report, SAS 9.1.3 (SAS Institute 2002) and Quasar 1.1 (Quasar 1.1 2009) were used for 

the statistical analyses. 

RESULTS 

The data for animals 1915,2919,4908 and 4919 were excluded, where necessary, since the 

animals did not become pregnant. For maternal net bodyweight change from d5 minus 

gravid uterine weight, animals 1912 and 4924 were also excluded. 

: 5: 

Statistical analysis 
05-4287 

For food consumption, the animal2908 value for the d5-d8 period was an extreme statistical 

outlier, being about 11 standard deviations higher than the mean. This value was excluded 

from the analysis. 

Continuous maternal parameters 

Summary statistics are presented in Tables 1 to 4 respectively. Bartlett's test was not 

significant for any parameter at any timepoint. The results can be summarised as follows: 

Bodyweight 

No significant differences at any timepoint. 

Bodyweight change 

d5-d8 significant difference at 20000 mg/m3 {p<0.01). 

d8-d11 significant difference at 2000 mg/m3 (p<0.05) and 10000 mg/m3 {p<0.01). 

d11-d14 significant difference at 2000 mg/m3 {p<0.05) and 20000 mg/m3 (p<0.01). 

d5-d14 significant difference at 2000 mg/m3 {p<0.05), 10000 mg/m3 {p<0.01) and 

20000 mg/m3 (p<0.01). 

Maternal net bodyweight change from d5 minus gravid uterine weight 

Significant difference at 20000 mg/m3 {p<0.001). 

Maternal feed consumption 

d8-d11 significant difference at 10000 mg/m3 (p<0.05) and 20000 mg/m3 {p<0.01). 

d11-d14 significant difference at 20000 mg/m3 {p<0.01). 

d20-d21 significant difference at 2000 mg/m3 {p<0.05), 10000 mg/m3 {p<0.05) and 

20000 mg/m3 {p<0.01). 

d5-d21 significant difference at 20000 mg/m3 {p<0.01). 

All changes were reductions from the Control. 

Uterus weight 

Absolute: no significant differences. 

Adjusted: no significant differences. 

:6: 

Statistical analysis 
05-4287 

Reproductive performance 

Summary statistics are presented in Table 5. Bartlett's test was significant for several 

parameters. The subsequent analysis was thus either parametric (P) or non-parametric (NP). 

The methods and results can be summarised as follows: 

Corpora lutea 

%Preimplantation loss 

Number of implant sites 

Fetuses alive 

Early resorptions 

Late resorptions 

Total resorptions 

%Total resorptions/implant sites 

Number of males 

Number of females 

Fetal bodyweight 

p 

NP 

p 

p 

NP 

p 

NP 

NP 

p 

p 

No significant differences. 

No significant differences. 

No significant differences. 

No significant differences. 

No significant differences. 

No significant differences. 

No significant differences. 

No significant differences. 

No significant differences. 

No significant differences. 

Summary statistics, for means adjusted for litter size, are presented in Table 6. 

For the pooled sex (litter) analysis there was a strong sex difference and evidence for a 

difference between treatment groups (p=0.0487). The subsequent Dunnett's test was 

significant at the high dose (p=O.Ol6). 

For males, there was a tendency for reduced mean bodyweight at higher doses, but the overall 

comparison did not attain statistical significance. 

For females, there was a significant difference between groups (p=0.016). The subsequent 

Dunnett's test was significant at the high dose (p=0.007, a reduction from control). 

There was a strong effect of litter size in all three analyses. 

These results can be summarised as follows: 

Male fetal body weight No significant differences. 

Female fetal body weight 

Litter body weight 

Significant difference at 20000 mg/m3 (p=0.007). 

Significant difference at 20000 mg/m3 (p=0.016). 

:7: 

Statistical analysis 
05-4287 

Discrete findings 

For the Maternal pregnancy summary, only one parameter, the number of non-pregnant 

females, required analysis. This is summarised in Table 7. 

The incidence of fetal fmdings, for those fmdings with a total incidence of greater than one is 

summarised in Table 8. Only five Visceral and seven Skeletal fmdings, all of them 

variations, required statistical analysis. The analysis was either generalised linear modelling 

(GL) or non-parametric (NP). 

The results can be summarised as follows: 

Maternal parameters 

Non-pregnant females NP No significant differences 

Visceral fmdings 

Heart, V Innominate artery absent 

Torso, V Left umbilical artery 

NP No significant differences 

GL No significant differences 

Torso, V Renal papilla (E) not fully developed 

Torso, V Undescended thymus 

NP No significant differences 

GL No significant differences 

Torso, V Ureter(s) distended NP No significant differences 

Skeletal fmdings 

Cervical vertebrae, V 7th cervical rib(s) present NP 

Ribs, V Rib( s) 13th Rudimentary NP 

Ribs, V Rib(s) 141h Rudimentary GL 

Sacral vertebrae, V 25 Presacral vertebra( e) NP 

Skull, V Hyoid body/arch(es) unossified NP 

Sternebrae, V Sternebra( e) 5 and/or 6 unossified. NP 

Sternebrae, V Sternebra( e) malaligned GL 

No significant differences 

No significant differences 

No significant differencest 

No significant differences 

No significant differences 

No significant differences 

Significant difference at 

20000 mg/m3 (p=0.047). 

t There was some evidence of a statistically significant difference at 20000 mg/m3 (p=0.067). 

: 8: 

Statistical analysis 
05-4287 

REFERENCES 

ARMITAGE, P., BERRY, G. and MATTHEWS, J.N.S. (2002) Statistical Methods in Medical 

Research, 4th edition. Blackwell Science, Oxford, UK, pp208-215. 

BARTLETT, M.S. (1937) Properties of sufficiency and statistical tests. Proceedings of the 

Royal Society, Series A, 160: 268-282. 

DUNNETT, C.W. (1955) A multiple comparison procedure for comparing several 

treatments with a control. Journal of the American Statistical Association 50: 1096-1121. 

DUNNETT, C.W. (1964) New tables for multiple comparisons with a control. Biometrics 

20: 482-491. 

KRUSKAL, W.H. and WALLIS, W.A. (1952) Use ofRanks in one-criterion variance 

analysis. Journal of the American Statistical Association 47: 583-621. 

KRUSKAL, W.H. and WALLIS, W.A. (1953) Errata for Kruskal-Wallis (1952) Journal of 

the American Statistical Association 48: 907-911. 

QUASAR 1.1 User Guide (Stand-Alone) version 1.1 (2009). Huntingdon Life Sciences 

Internal Document. 

SAS INSTITUTE (1999) SAS OnlineDoc® Version Eight. SAS Institute Inc., Cary, NC, 

USA 

SAS INSTITUTE (2002) SAS OnlineDoc® Version Nine. SAS Institute Inc., Cary, NC, USA. 

STEEL, R.G.D. (1959) A multiple comparison rank sum test: treatment versus control. 

Biometrics 15, 560-572. 

WILCOXON, F. (1945) Individual comparisons by ranking methods. Biometrics Bulletin, 

1, 80-83. 

:9: 

Statistical analysis 
TABLEt 

Maternal body weight (g) 

Omg/m3 2000 mg/m3 

(n=23) (n=23) 

Day of mean (sd) mean (sd) 

gestation 

4 233.8 (14.1) 234.7 (16.6) 

5 244.0 (14.6) 245.3 (16.6) 

8 259.3 (12.8) 260.4 (18.1) 

11 281.5 (14.9) 279.0 (19.7) 

14 301.5 (17.1) 295.5 (20.2) 

17 332.1 (22.3) 326.7 (24.2) 

20 378.1 (26.3) 370.3 (29.9) 

21 387.8 (28.5) 379.4 (32.7) 

Analysis performed= Parametric throughout. 

No significant differences found for any parameter. 

n = number oflitters, sd = standard deviation 

:10: 

10000 mg/m3 

(n=24) 

mean (sd) 

233.9 (15.7) 

243.9 (13.9) 

256.6 (14.4) 

274.4 (15.6) 

292.0 (14.8) 

322.8 (18.7) 

366.8 (24.1) 

375.7 (26.0) 

05-4287 

20000mglm' 

(n=22) 

mean (sd) 

235.4 (14.7) 

245.0 (15.3) 

255.6 (16.2) 

275.4 (19.6) 

290.2 (19.6) 

321.1 (24.9) 

365.5 (27.1) 

373.6 (27.4) 

Statistical analysis 
TABLE2 

Maternal body weight change (g) 

Days of Omg/m, 2000 mg!m' 

gestation (n=23) (n=23) 

Period mean (sd) mean (sd) 

4-5 10.3 (6.1) 10.7 (4.7) 

5-8 15.5 (4.4) 15.0 (4.8) 

8-11 22.2 (4.7) 18.6 (3.7) * 

11-14 20.0 (4.2) 16.8 (5.1) * 

14-17 30.7 (7.3) 31.2 (6.9) 

17-20 46.2 (7.5) 43.5 (8.9) 

5-14 57.6 (8.4) 50.2 (7.9) * 

14-21 86.4 (15.3) 84.0 (16.2) 

5-21 143.9 (20.6) 134.2 (21.5) 

Analysis performed= Parametric throughout. 

* = p<0.05, ** = p<0.01 

n = number of litters, sd = standard deviation 

10000 mg/m' 

(n=24) 

mean (sd) 

10.2 (5.3) 

12.5 (5.4) 

17.9 (4.0) ** 

17.5 (3.8) 

31.0 (7.5) 

44.1 (9.8) 

47.8 (7.6) ** 

83.8 (17.0) 

131.8 (20.9) 

05-4287 

20000mg/mj 

(n=22) 

mean (sd) 

9.6 (3.2) 

10.6 (4.7) ** 

19.7 (5.6) 

15.0 (4.0) ** 

31.0 (6.7) 

44.5 (7.7) 

45.2 (9.4) ** 

83.5 (10.1) 

128.6 (16.0) 

Maternal net bodyweight change from d5 minus gravid uterine weight (g) 

Omg!m' 2000 mg!m' 

(n=22) (n=23) 

Parameter mean(sd) mean (sd) 

Absolute 42.95 (11.08) 37.39 (9.16) 

Analysis performed= Parametric throughout. 

n = number oflitters, sd = standard deviation 

*** = p<0.001 

:11: 

10000 mg/m' 20000 mg/mj 

(n=24) (n=20) 

mean (sd) mean (sd) 

35.71 (11.75) 29.35 (10.65) *** 

Statistical analysis 
TABLE3 

Maternal feed consumption (glkg/day) 

Days of Omg/mj 2000 mg/mj 

gestation (n=22-23) (n=22-23) 

Period mean (sd) mean (sd) 

4-5 71.2 (10.5) 74.2 (11.5) 

5-8 80.8 (5.9) 82.7 (5.5) 

8-11 85.1 (7.1) 81.8 (4.6) 

11-14 85.0 (7.1) 81.6 (4.6) 

14-17 80.9 (4.5) 80.8 (4.5) 

17-20 74.5 (4.5) 73.4 (3.3) 

20-21 53.3 (8.1) 46.8 (7.6) * 

5-21 76.2 (4.8) 75.0 (4.1) 

Analysis performed= Parametric throughout. 

* = p<0.05, ** = p<0.01 

n = number of litters, sd = standard deviation 

Some variation in group size due to missing values. 

:12: 

10000 mg/mj 

(n=22-24) 

mean (sd) 

75.0 (11.4) 

79.7 (5.9) 

80.3 (5.5) * 

82.4 (3.9) 

80.6 (6.0) 

73.6 (3.8) 

47.0 (5.8) * 

73.6 (3.5) 

05-4287 

20000 mg/m3 

(n=21-22) 

mean (sd) 

73.1 (10.3) 

78.8 (5.9) 

77.4 (5.7) ** 

77.8 (3.9) ** 

79.2 (4.4) 

73.1 (4.3) 

45.9 (7.8) ** 

71.5 (3.9) ** 

Statistical analysis 
TABLE4 

Maternal uterus weights (g) 

Omg/mj 2000 mg/mj 

(n=22) (n=23) 

Parameter mean (sd) mean (sd) 

Absolute 101.3 (19.3) 96.5 (19.9) 

Adjusted 286.0 (18.1) 282.7 (19.0) 

Analysis performed= Parametric throughout. 

No significant differences found for any parameter. 

n = number of litters, sd = standard deviation 

:13: 

10000 mg/mj 

(n=24) 

mean (sd) 

96.1 (22.5) 

279.5 (17.2) 

05-4287 

20000mg/mj 

(n=20) 

mean (sd) 

96.4 (15.6) 

275.3 (18.4) 

Statistical analysis 
TABLES 

Reproductive performance 

Omg/mj 2000mg/m5 

(n=23) (n=23) 

Parameter mean(sd) mean (sd) 

corpora lutea ( 1) 14.9 (2.8) 14.5 (2.1) 

%preimplantation loss (2) 9.3 (13.5) 6.6 (8.0) 

number of implant sites (1) 13.5 (2.7) 13.5 (1.5) 

fetuses alive (1) 13.0 (2.6) 12.4 (2.6) 

early resorptions (2) 0.4 (0.7) 1.0 (2.1) 

late resorpti ons ( 1) 0.0 (0.2) 0.0 (0.2) 

total resorpti ons ( 2) 0.5 (0.7) 1.0(2.1) 

%total resorp./implant sites (2) 3.4 (4.7) 7.9 (14.9) 

number of males (1) 6.3 (1.7) 5.8 (2.2) 

number of females (1) 6.7 (2.0) 6.7 (2.3) 

Analysis performed= (1) Parametric or (2) Non-parametric 

No significant differences found for any parameter. 

n = number oflitters, sd = standard deviation 

Male and female bw n= 11 

:14: 

05-4287 

10000 mg/m5 20000 mg!m5 

(n=24) (n=22) 

mean (sd) mean(sd) 

14.8 (2.5) 15.2 (2.6) 

10.4 (18.6) 7.6 (9.1) 

13.1 (2.8) 14.0 (2.4) 

12.6 (3.1) 13.3 (2.6) 

0.5 (0.8) 0.7 (0.6) 

0.0 (0.0) 0.0 (0.0) 

0.5 (0.8) 0.7 (0.6) 

5.1 (8.6) 5.2 (5.2) 

6.4 (2.3) 6.1 (2.1) 

6.2 (2.3) 7.1 (2.5) 

Statistical analysis 
TABLE6 

Mean (adjusted) fetal bodyweight (g) 

Omg/mj 2000 mg/mj 10000 mg/m3 20000 mg/m3 

(n=23) {n=23) (n=24) {n=22) 

mean (se) mean (se) mean (se) mean (se) 

male 5.88 (0.06) 5.87 (0.06) 5.78 (0.06) 5.71 (0.06) 

female 5.58 (0.06) 5.59 (0.06) 5.47 (0.06) 5.36 (0.06) ** 

litter 5.73 (0.06) 5.73 (0.06) 5.62 (0.06) 5.54 (0.06) * 

* = p<0.05, ** = p<0.01, *** = p<0.001 

p values vs control using Dunnett's test following mixed model ANOV A significant at 5%. 

n =number oflitters, se =standard error of the adjusted mean 

:15: 

05-4287 

Statistical analysis 
05-4287 

TABLE7 

Maternal pregnancy summary and necropsy f"mdings 

Parameter Finding 0 mg/mj 2000mg/mj 10000 mg/mj 20000 mglm 

(n=24) (n=24) (n=24) (n=24) 

Pregnancy Non-pregnant 1 1 0 2 

Only findings where the total incidence was greater than one are included. 

Analysis performed= Generalized linear modelling or Non-parametric tests (see text). 

No significant differences found for any parameter. 

n = number of litters 

:16: 

Statistical analysis 
TABLES 

Fetal findings: Visceral 

Tissue Organ Finding Omg/mj 2000 mg!m' 

(n=23) (n=23) 

Visceral HEART V INNOMINATE ARTERY 1 0 

ABSENT 

TORSO V LEFT UMBILICAL 1 4 

ARTERY 

TORSO V RENAL PAPILLA (E) NOT 1 0 

FULLY DEVELOPED 

TORSO V UNDESCENDED 10 11 

THYMUS 

TORSO V URETER(S) DISTENDED 1 0 

Only findings where the total incidence was greater than one are included. 

Analysis performed= Generalized linear modelling or Non-parametric tests (see text). 

No significant differences found for any parameter. 

n = number of litters 

:17: 

05-4287 

lOOOOmg/m 20000mg/m 

(n=24) (n=22) 

2 1 

1 2 

2 1 

5 11 

1 1 

Statistical analysis 
TABLE 8 (cont'd) 

Fetal findings: Skeletal 

Tissue Organ Finding Omg!nl 2000mglm5 

(n=23) (n=23) 

Skeletal CERVICAL V 7TH CERVICAL RIB(S) 0 1 

VERT PRESENT 

RIBS IV RIB(S) 13TH 1 0 

RUDIMENTARY 

RIBS V RIB(S) 14TH 2 7 

RUDIMENTARY 

SACRAL VERT IV 25 PRESACRAL 1 0 

IVERTEBRA(E) 

SKULL jv HYOID BODY/ARCH(ES) 1 0 

iUNOSSIFIED BODY 

STERNEBRAE jv STERNEBRA(E) 5 1 0 

lANDfOR 6 UN OSSIFIED 

STERNEBRAE jv STERNEBRA(E) 11 11 

~LALIGNED 

Only findings where the total incidence was greater than one are included. 

Analysis performed= Generalized linear modelling or Non-parametric tests (see text). 

*= p<0.05 

n = number oflitters 

:18: 

05-4287 

10000mg/m 20000 mg/m3 

(n=24) (n=22) 

0 3 

0 3 

5 12 

0 3 

1 1 

3 3 

13 3* 

Statistical analysis 
Huntingdon Life Sciences 

Table of Contents 

05-4287 

211-DIPE-DEV 

Certificates of Analysis 

Page 1 

Draft Final Report 

AwendixN 

Feed Certificates of Analysis .................. _ ............................................................................. . 

Water Certificates of Analysis: ............................................................................................. . 

Bedding Certificates of Analysis .......................................................................................... . 
Certified Papers Retrieval 

Product Code: 5002M 

Product Desc: 

Lab Number: 

CERTIFIED RODENT DIET MEAL 

L0516825-2 

Lot Code: MAY 12 05 18 

Entered: 5/31/2005 

!Assay II Analysisi!Units 

!PROTEIN II 2o.all% 

IFAT ACID (HYDRO.) II 6.34,,% 

!FIBER (CRUDE) II 3.64,,% 

!ARSENIC II LESS THAN 0.2,,PPM 

!cADMIUM II LESS THAN 0.05,,PPM 

!cALCIUM II 0.943,,% 

I LEAD II 0.17oiiPPM 

!MERCURY II LESS THAN 0.025,,PPM 

!PHOSPHORUS II 0.69811% 

!sELENIUM II 0.21411PPM 

iORGANOPHOSPHATESi!PPM !iORGANOPHOSPHATES!iPPM 

IDiazinon ~~~~~S THAN IIDisulfoton ~~~~~STHAN 

IEthion ~~~~~STHAN II Malathion ~~~~~S THAN 

!Methyl Parathion ~~~~~STHAN II Parathion ~~~~~S THAN 

IThimet ~~~~~S THAN IIThiodan ~~~~~S THAN 

ITrithion II~~~STHAN II II 

PESTICIDES AND 

I PPM II~~~TICIDES AND IIPPM PCB 

jAidrin IILESS THAN 0.02 liAipha-BHC IILESS THAN 0.02 

jseta-BHC IILESS THAN 0.02 !I Chlordane IILESS THAN 0.02 

IDDE IILESS THAN 0.02 II DDT IILESS THAN 0.02 

iDelta-BHC IILESS THAN 0.02 II Dieldrin IILESS THAN 0.02 

jEndrin IILESS THAN 0.02 IIHCB IILESS THAN 0.02 

!Heptachlor IILESS THAN 0.02 !!Heptachlor Epoxide IILESS THAN 0.02 

!Lindane IILESS THAN 0.02 !!Methoxychlor IILESS THAN 0.02 

IMirex IILESS THAN 0.02 II PCB IILESS THAN 0.15 

IIIAFLATOXINS i!Aflatoxins IILESS THAN 5 PPB 

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Certified Papers Retrieval 

l.ablJIJ 

,~ 

·er , 

.• 

. · . 11111'·1 PMI NUTRITION 

iN: 

~ .!I.! ~-~··i 

Product Code: 5002M 

Product Desc: 

Lab Number: 

CERTIFIED RODENT DIET MEAL 

L0517995-2 

Lot Code: JUN 07 05 38 

Entered: 6/24/2005 

!Assay Analysisllunits 

!PROTEIN 2111% 

IFAT ACID (HYDRO.) 5.6211% 

IFIBER (CRUDE) 4.0111% 

!ARSENIC LESS THAN 0.211PPM 

!cADMIUM 0.088211PPM 

!cALCIUM I 1.0311% 

I LEAD 0.21311PPM 

!MERCURY LESS THAN 0.02511PPM 

!PHOSPHORUS 0.74411% 

!sELENIUM 0.31611PPM 

IORGANOPHOSPHATESIIPPM lloRGANOPHOSPHATESIIPPM 

IDiazinon II6~~STHAN IIDisulfoton II6~~STHAN 

IEthion II6~~STHAN IIMalathion 110.03 

!Methyl Parathion II6~~STHAN II Parathion II6~~STHAN 

IThimet II6~~STHAN IIThiodan II6~~STHAN 

ITrithion II6~~STHAN II II 

~~~TICIDES AND IIPPM II~~~TICIDES AND II PPM 

!Aldrin IILESS THAN 0.02 IIAipha-BHC IILESS THAN 0.02 

IBeta-BHC IILESS THAN 0.02 llchlordane IILESS THAN 0.02 

IDDE IILESS THAN 0.02 II DDT IILESS THAN 0.02 

IDelta-BHC IILESS THAN 0.02 II Dieldrin IILESS THAN 0.02 

IEndrin IILESS THAN 0.02 IIHCB IILESS THAN 0.02 

!Heptachlor IILESS THAN 0.02 IIHeptachlor Epoxide IILESS THAN 0.02 

I Lindane IILESS THAN 0.02 II Methoxychlor IILESS THAN 0.02 

IMirex IILESS THAN 0.02 II PCB IILESS THAN 0.15 

IAFLATOXINS IIAflatoxins IILESS THAN 5 PPB 

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Certified Papers Retrieval 

Product Code: 5002M 

Product Desc: 

Lab Number: 

CERTIFIED RODENT DIET MEAL 

L0517995-3 

Lot Code: JUN 07 05 3C 

Entered: 6/24/2005 

jAssay II Analysisjjunits 

jPROTEIN II 20.911% 

jFAT ACID (HYDRO.) II 5.8711% 

jFIBER (CRUDE) II 3.7511% 

jARSENIC II LESS THAN 0.2jjPPM 

jcADMIUM I 0.0933jjPPM 

jcALCIUM 1.0111% 

jLEAD 0.209jjPPM 

jMERCURY LESS THAN 0.025jjPPM 

jPHOSPHORUS 0.72311% 

jsELENIUM 0.30911PPM 

jORGANOPHOSPHATESi!PPM lioRGANOPHOSPHATESi!PPM 

IDiazinon II6~~STHAN IIDisulfoton II6~~STHAN 

IEthion II6~~STHAN II Malathion II6~~STHAN 

!Methyl Parathion II6~~STHAN II Parathion II6~~STHAN 

IThimet II6~~STHAN IIThiodan II6~~STHAN 

ITrithion II6~~STHAN II II 

lliESTICIDES AND 

PCB II PPM II~~~TICIDES AND IIPPM 

jAidrin IILESS THAN 0.02 i!Aipha-BHC IILESS THAN 0.02 

jseta-BHC IILESS THAN 0.02 II Chlordane IILESS THAN 0.02 

jDDE IILESS THAN 0.02 II DDT IILESS THAN 0.02 

jDelta-BHC IILESS THAN 0.02 II Dieldrin IILESS THAN 0.02 

jEndrin IILESS THAN 0.02 IIHCB IILESS THAN 0.02 

!Heptachlor IILESS THAN 0.02 i!Heptachlor Epoxide IILESS THAN 0.02 

jlindane IILESS THAN 0.02 II Methoxychlor IILESS THAN 0.02 

jMirex IILESS THAN 0.02 II PCB IILESS THAN 0.15 

IIIAFLA TOXINS i!Aflatoxins IILESS THAN 5 PPB 

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LAB 10# 3!;)-401 BENCHMARK ANAL YTICS 

4777 SAUCON CREEK ROAD 

CENTER VALLEY, PA 18034-9004 

PHONE (610) 974-8100 

FAX (610) 974-8104 

SEND DATA TO: INV #: 383492-383494 

NAME: Terry Kusznir 383496-383498 

COMPANY: 

ADDRESS: 

PHONE: 

FAX: 

Huntingdon Life Sciences 

PO Box 2360, Mettlers Road 

East Millstone, NJ 08875-2360 

(732) 873-2550 x2100 

(732) 873-3992 TEST REPORT 

PAGE: 1 OF6 

PO#: 

PWSID#: . . 

=====::::;=========-==.::===========::::======.===·===================~====================================~====== 

PROJECT NAME: ANIMAL DRINKING WATER GRAB SAMPLES: 383492-383494 

SAMPLED BY: CARAKEIPER 383496-383498 

LOCATION: SEE BELOW COMPOSITE SAMPLES: NONE 

DATE: 07/13/05 DATE RECEIVED BY LAB: 07114/05 

TIME: SEE BELOW TIME RECEIVED BY LAB: 0819 

RECEIVED FOR LAB BY: R SMOLENAK 

SAMPLE: SITE #1, F-WING ROOM 546 TIME: 1608 

TEST RESULT UNITS METHOD 

p~c;:UDOMONAS * 

. _,; ~~~~~( <1 PER 100 ML SM#19 9215 D 383492 07114/05 1505 07/15/05 

STANDARD PLATE <1 CFUIML SM#20 9215 B 383494 07/14/05 1105 07/16/05 JD 

COUNT 

TOTAL COLIFORM 0 PER 100 ML EPA 600-R-00-013 383493 07/14/05 1308 07/15/05 MJG 

E. COLI 0 PER 100 ML EPA 600-R-00-013 383493 07/14/05 1308 07/15/05 MJG 

SAMPLE: SITE #2, V-1 ROOM 725 

I I 

TEST RESULT UNITS METHOD 

PSEUDOMONAS * <1 PER 100 ML SM#19 9215 D 07/14/05 1505 07/15/05 

STANDARD PLATE <1 CFUIML SM#20 9215 B 383498 07/14/05 1105 07/16/05 JD 

COUNT 

TOTAL COLIFORM 0 PER 100 ML EPA 600-R-00-013 383497 07114/05 1308 07115/05 MJG 

E. COLI 0 PER 100 ML EPA 600-R-00-013 383497 07/14/05 1308 07/15/05 MJG 

REMARKS: 

fl--It=: ABOVE TEST RESULTS MEET ALL THE REQUIREMENTS OF NELAC. 

)s PARAMETER IS NOT CURRENTLY UNDER THE NELAC CERTIFICATION PROGRAM. 

MANAGER: ___ _,~=->-~~pc..<....:;...:;..:::.__;_;==· '----l,a,_£-.,..£.,~..::..=:.=------ DATE: 94 d~ )'nt--
LAB 10# :}9-401 BENCHMARK ANAL YTICS 

4777 SAUCON CREEK ROAD 

CENTER VALLEY, PA 18034-9004 

PHONE (610) 974-8100 

FAX (610) 974-8104 

SEND DATA TO: 

NAME: 

COMPANY: 

ADDRESS: 

PHONE: 

FAX: 

Terry Kusznir 

Huntingdon Life Sciences 

PO Box 2360, Mettlers Road 

East Millstone, NJ 08875-2360 

(732) 873-2550 X 2100 

(732) 873-3992 TEST REPORT 

INV#: 383495 

383499,503 

PAGE: 20F6 

PO#: 

PWSID#: 

============================================================================================== 

PROJECT NAME: ANIMAL DRINKING WATER 

SAMPLED BY: CARA KEIPER 

LOCATION: SEE BELOW 

DATE: 07/13/05 

TIME: SEE BELOW 

SAMPLE: SITE #1, F-WING ROOM 546 

TEST 

COPPER 

·' ~=~D ~ !\./>!' ( 

1 'r'\~ " 

SAMPLE: 

TEST 

COPPER 

LEAD 

SAMPLE: 

TEST 

COPPER 

LEAD 

REMARKS: 

RESULT 

0.0660 

0.0017 

UNITS 

MG/L 

MG/L 

SITE #2, V-1 ROOM 725 

RESULT 

0.0177 

<0.0005 

UNITS 

MG/L 

MG/L 

SITE #3, V-2 ROOM 910 

RESULT 

0.0548 

0.0007 

UNITS 

MG/L 

MG/L 

METHOD 

EPA200.8 

EPA200.8 

METHOD 

EPA200.8 

EPA200.8 

METHOD 

EPA200.8 

EPA200.8 

GRAB SAMPLES: 

COMPOSITE SAMPLES: 

DATE RECEIVED BY LAB: 

TIME RECEIVED BY LAB: 

INV# 

383495 

383499,503 

NONE 

07/14/05 

0819 

R. SMOLENAK 

383495 07/15/05 0735 07/15/05 KP 

383495 07/15/05 0735 07/15/05 KP 

TIME: 

INV# 

383499 07115/05 07115/05 KP 

383499 07/15/05 0735 07/15/05 KP 

INV# 

383503 07/15/05 0735 07/15/05 

383503 07/15/05 0735 07/15/05 KP 

REVIEWED 

TYE ABOVE TEST RESULTS MEET ALL THE REQUIREMENTS OF NELAC ANDRE LATE ONLY TO THESE SAMPLES. 

MANAGER:---.,='~'-"'=='=-=--~==.:..::=-=-=-· ----'==G.~· .c...k¥v-=-=~-=----- DATE: 
LAB ID# 39-401 

SEND DATA TO: 

NAME: 

COMPANY: 

ADDRESS: 

PHONE: 

FAX: 

BENCHMARK ANAL YTICS 

4777 SAUCON CREEK ROAD 

CENTER VALLEY, PA 18034-9004 

PHONE (610) 974-8100 

FAX (610) 974-8104 

INV #: 383500-383502 

Terry Kusznir 

Huntingdon Life Sciences 

PO Box 2360, Mettlers Road 

East Millstone, NJ 08875-2360 

(732) 873-2550 x21 00 

(732) 873-3992 TEST REPORT 

383504-383506 

PAGE: 30F6 

PO#: 

PWSID#: 

===================================================================================================== 

PROJECT NAME: 

SAMPLED BY: 

LOCATION: 

DATE: 

TIME: 

SAMPLE: 

TEST 

P~EUDOMONAS * ·, 

STANDARD PLATE 

COUNT 

TOTAL COLIFORM 

E. COLI 

SAMPLE: 

TEST 

PSEUDOMONAS * 

STANDARD PLATE 

COUNT 

TOTAL COLIFORM 

E. COLI 

REMARKS: 

ANIMAL DRINKING WATER 

CARAKEIPER 

SEE BELOW 

07/13/05 

SEE BELOW 

SITE #3, V-2 ROOM 910 

RESULT UNITS METHOD 

<1 PER 100 ML SM#19 9215 D 

9 CFUIML SM#20 9215 B 

GRAB SAMPLES: 383500-383502 

383504-383506 

COMPOSITE SAMPLES: NONE 

DATE RECEIVED BY LAB: 07/14/05 

TIME RECEIVED BY LAB: 0819 

RECEIVED FOR LAB BY: R. SMOLENAK 

383500 07114/05 1505 07/15/05 

383502 07/14/05 11 05 07116/05 JD 

0 

0 

PER 100 ML EPA 600-R-00-013 383501 07/14/05 1308 07115/05 MJG 

PER 100 ML EPA 600-R-00-013 383501 07/14/05 1308 07115/05 MJG 

SITE #4, V-3 ROOM 950 

RESULT UNITS METHOD 

<1 PER 100 ML SM#19 9215 D 383504 07114/05 1505 07/15/05 

4 CFU/ML SM#20 9215 B 383506 07/14/05 1105 07/16/05 JD 

0 PER 100 ML EPA 600-R-00-013 383505 07114/05 1308 07/15/05 MJG 

0 PER 100 ML EPA 600-R-00-013 383505 07/14/05 1308 07/15/05 MJG 

THE ABOVE TEST RESULTS MEET ALL THE REQUIREMENTS OF NELAC. REVIEWED !IS PARAMETER IS NOT CURRENTLY UNDER THE NELAC CERTIFICATION PROGRAM. 

DATE: ~ aC: ~ 
LAB ID# 39-401 

SEND DATA TO: 

NAME: 

COMPANY: 

ADDRESS: 

PHONE: 

FAX: 

BENCHMARK ANAL YTICS 

4777 SAUCON CREEK ROAD 

CENTER VALLEY, PA 18034-9004 

PHONE (610) 974-8100 

FAX (610) 974-8104 

Terry Kusznir INV #: 383507-383511 

Huntingdon Life Sciences 

PO Box 2360, Mettlers Road 

East Millstone, NJ 08875-2360 

{732) 873-2550 X 2100 

(732) 873-3992 TEST REPORT 

383515 

PAGE: 40F6 

PO#: 

PWSID#: 

============================================================================================== 

)AMPLE: 

"EST 

~OPPER 

.EAD 

tEMARKS: 

L-WING ROOM 458 

RESULT 

0.0166 

<0.0005 

UNITS 

MG/L 

MG/L 

METHOD 

EPA200.7 

EPA200.9 

INV# 

383515 07/15/05 0735 07/15/05 KP 

383515 07/15/05 0735 07/15/05 KP 

HE ABOVE TEST RESULTS MEET ALL THE REQUIREMENTS OF NELAC AND RELATE ONLY TO THESE SAMPLES. 

REVIEWED 

ANAGER: ___ ___,....~~~-=~-=· =~=·· _{l-=..;,..c......;k±~-'--· __ DATE: --~"21=--...-...,...--=d~S.~r~;;_t/}r_ 
LAB ID# 39-401 

SEND DATA TO: 

NAME: 

COMPANY: 

ADDRESS: 

PHONE: 

FAX: 

BENCHMARK ANAL YTICS 

4777 SAUCON CREEK ROAD 

CENTER VALLEY, PA 18034-9004 

PHONE (610) 974-8100 

FAX (610) 974-8104 

INV #: 383508-383510 

Terry Kusznir 

Huntingdon Life Sciences 

PO Box 2360, Mettlers Road 

East Millstone, NJ 08875-2360 

(732} 873-2550 x2100 

(732) 873-3992 TEST REPORT 

383512-383514 

PAGE: 50F6 

PO#: 

PWSID#: 

==================================================================~==~==============================: 

PROJECT NAME: ANIMAL DRINKING WATER GRAB SAMPLES: 383508-383510 

SAMPLED BY: CARAKEIPER 383512-383514 

LOCATION: SEE BELOW COMPOSITE SAMPLES: NONE 

DATE: 07113/05 DATE RECEIVED BY LAB: 07114/05 

TIME: SEE BELOW TIME RECEIVED BY LAB: 0819 

RECEIVED FOR LAB BY: C. PASSOW 

SAMPLE: SITE #5, INHALATION ROOM 812 

TEST RESULT UNITS METHOD 

PSEUDOMONAS * <1 PER 100 ML SM#19 9215 D 383508 07114/05 1505 07/15/05 JD 

i 

b I ANDARD PLATE <1 CFU/ML SM#20 9215 B 383510 07/14/05 1105 07116/05 JD 

COUNT 

TOTAL COLIFORM 0 PER 100 ML EPA 600-R-00-013 383509 07/14/05 1308 07/15/05 MJG 

E. COLI 0 PER 100 ML EPA 600-R-00-013 383509 07/14/05 1308 07/15/05 MJG 

SAMPLE: SITE #6, L-WING ROOM 458 

TEST RESULT UNITS METHOD 

PSEUDOMONAS * <1 PER 100 ML SM#19 9215 D .07/14/05 1505 07/15/05 

STANDARD PLATE <1 CFU/ML SM#20 9215 B 383514 07/14/05 1105 07/16/05 JD 

COUNT 

TOTAL COLIFORM 0 PER 100 ML EPA 600-R-00-013 383513 07/14/05 1308 07/15/05 MJG 

E. COLI 0 PER 100 ML EPA 600-R-00-013 383513 07/14/05 1308 07/15/05 MJG 

REMARKS: 

THE ABOVE TEST RESULTS MEET ALL THE REQUIREMENTS OF NELAC AND RELATE ONLY TO THESE SAMPLES 

!IS PARAMETER IS NOT CURRENTLY UNDER THE NELAC CERTIFICATION PROGRAM. 

REVIEWED 

MANAGER: ----r,~'""""""-=->o.<Jl~i""'==. -=>-===-__ 0~~'---------- DATE: ~-9---P-"'~~J-«=....<..S:~M-;..;,__ 
LAB 10# 39-401 

SEND DATA TO: 

NAME: 

COMPANY: 

ADDRESS: 

PHONE: 

FAX: 

BENCHMARK ANAL YTICS 

4777 SAUCON CREEK ROAD 

CENTER VALLEY, PA 18034-9004 

PHONE (610) 974-8100 

FAX (610) 974-8104 

Terry Kusznir 

Huntingdon Life Sciences 

PO Box 2360, Mettlers Road 

East Millstone, NJ 08875-2360 

(732) 873-2550 x2100 

(732) 873-3992 TEST REPORT 

INV #: 383516-383518 

PAGE: 60F6 

PO#: 

PWSID#: 

===================================================================================================== 

PROJECT NAME: ANIMAL DRINKING WATER GRAB SAMPLES: 383516-383518 

SAMPLED BY: CARAKEIPER COMPOSITE SAMPLES: NONE 

LOCATION: SEE BELOW DATE RECEIVED BY LAB: 07/14/05 

DATE: 07/13/05 TIME RECEIVED BY LAB: 0819 

TIME: SEE BELOW RECEIVED FOR LAB BY: C. PASSOW 

SAMPLE: SITE #7 PHARMACY 1 

TEST RESULT UNITS METHOD 

PSEUDOMONAS * <1 PER 100 ML SM#19 9215 B 383516 07/14/05 1505 07/15/05 JD ·,\ 

I 

s I ANDARD PLATE 1,140 CFU/ML SM#20 9215 8 383518 07114/05 1105 07/16/05 JD 

COUNT 

TOTAL COLIFORM 0 PER 100 ML EPA 600-R-00-013 383517 07/14/05 1308 07/15/05 MJG 

E. COLI 0 PER 100 ML EPA 600-R-00-013 383517 07/14/05 1308 07/15/05 MJG 

REMARKS: 

TH~) ABOVE TEST RESULTS MEET ALL THE REQUIREMENTS OF NELAC AND RELATE ONLY TO HTESE SA~LES 

· EVI£Vvco 

MANAGER: ___ 7(/ ,.._,xf ..... :zta:::.>.G.b,,.,..lQJJ!1""'"~=::=..;;_-'----_....~~fv<""""""4...=->-=------ DATE: Ott_Lf dS", J?tr: l...b ¥ , 
LAB ID# 39-401 

SEND DATA TO: 

NAME: 

COMPANY: 

ADDRESS: 

PHONE: 

FAX: 

BENCHMARK ANAL YTICS 

4777 SAUCON CREEK ROAD 

CENTER VALLEY, PA 18034-9004 

PHONE (610) 974-8100 

FAX (610) 974-8104 

Terry Kusznir 

Huntingdon Life Sciences 

PO Box 2360, Mettlers Road 

East Millstone, NJ 08875-2360 

(732) 873-2550 x2100 

(732) 873-3992 TEST REPORT 

INV #: 387033 

PAGE: 1 OF 1 

PO#: 

PWSID#: 

==:===============-=========================-:-======================~:;::=========================·============= 

PROJECT NAME: 

SAMPLED BY: 

LOCATION: 

DATE: 

TIME: 

SAMPLE: 

TEST 

STANDARD PLATE 

cOuNT'· '· · · 

~EMARKS: 

ANIMAL DRINKING WATER 

TERRY KUSZNIR 

SEE BELOW 

08/04/05 

1455 

GRAB SAMPLES: NONE. 

COMPOSITE SAMPLES: NONE 

DATE RECEIVED BY LAB: 08/05/05 

TIME RECEIVED BY LAB: 0838 

RECEIVED FOR LAB BY: C. PASSOW 

"HE ABOVE TEST RESULTS MEET ALL THE REQUIREMENTS OF NELAC AND RELATE ONLY TO THESE SAMPLES. . ,·· .. ·· ' . . . . . . . . 

DATE: 
Elizabethtown Water Company 

Physical & Chemical Analyses 

General Source Raritan-Millstone Plant 

Sample No.1 Plant Delivered Water-8:10a.m. 8-9-05 MT 

Sample No.2 

Sample No.3 

Sample No.4 

Sample No.5 

Sample No.6 

Sample No.7 

Sample No.8 

Parameter MCL (mgll) 1.0 2.0 3.0 4.0 

Temperature • F - 81 

Turbidit}'_iNTUl 0.3 NTU 0.13 

Color 10* 1.0 

Threshold I Odor 40 • c 3TON* 2.6/3cc 

Threshold Taste 3 TTN* 3.0 

Conductivity (micromhos I em) - 308 

Hardness, Total (as mg /I CaC03) 250 mg II* 94 

Alkalinity - 36 

IPH 6.5-8.5 * 6.7 

Chlorine, Free I Total (mg /I Cl) - 0.67/.74 

Calcium (as mg /I CaC03) - 66 

Magnesium (as mg /I CaC03) - 28 

. Iron, Tot~l (mg II Fe) 0."3 ing1 I* NO 

Sulfates (mg 1 I S04) 250 mg11* 46.3 

Chlorides (mg 1 I Cl) 250 mg11 * 31.0 

Fluoride (mg 1 I F) 2 mgfl* <0.1 

Total Dissolved Solids (mg 1 I) 500 mg II* 204 

Total Suspended Solids (mg 1 I) - 0.0 

Ammonia Nitrogen (mg /IN) - <0.05 

Nitrate Nitrogen (mg 1 I N) 10 mgfl 0.65 

Dissolved Oxygen (mg 1 I 02) - 7.5 

BOD 5 (mg 1 I 02) - -

Langelier Index +1-1.0 -2.10 

Surfactants (mg 1 I LAS) 0.5 mgll* <0.05 

1-fydrog_en Sulfide (m_g_( I H2S as S) - 0 

Nitrite Nitrogen (mg 1 I N) 1 mgfl <.01 

Phosphate (mg 1 I P03) - <.05 

Manganese (mg /I MN) 0.05 mg 1 I* NO 

. Zinc 5 mgfl* 0.54 

T.O.C. (mg /I) 5mgfl* 1.58 

* Secondary or Recommended MCL 

Anv.Questions please call Water Quality Specialist- (732) 302-3192 

Date August9,2005 

5.0 6.0 7.0 8.0 

· ... 

Laboratory Analyst DSD 

I 

REVIEWED/ \l~~J 
!i Sterigenics. 

Facility: 

Customer Name: 

Customer PO #: 

Run Date: 

Run Number: 

Total Pallets: 

Westerville 

The Anderson Inc. 

05/09/05 

ANDOl AND 6050006 

21 

MATERIALS PROCESSED: 

Qty Product 

1,680 BED'O COB 1/4" BAG· 

l SAMPLE-506 

1,681 Bags Total 

Minimum Dose: 

Maximum Dose: 

sterigenics .. 305 Enterprise Drive * Westerville, Ohio 43081 " USA * 

614-8884077 .. FAX 614-888-4223 * www.sterigenics.com 

Description 

28.4 kGy 

52.1 kGy 

Certificate of Processing 

C~~:: Production A 

Date Format; MM/DD/YY 

CUstomer Ref: 

Lot Number LOT # . . 

DC295 

DC295 

425RB 

425RB 

Sterigenics certifies that the materials listed above (as described by its 

Manufacturer} received the indicated doses within~ th precision and accuracy 

~ the dosimetry system employed, . ~}, (/,I~ 

certified By: fl/~ 

Date: 05/10/05 

ISO 9001:2000 REGISTERED 

l?age 1 of l 

Original 
·--""' 

NAMSA 

PEOPLE > SCIENCE > SOLliTIONS 

Confidential 

MS005_00P 

Ted Weaver 

Andersons 

P.O. Box 119 

Maumee. OH 43537 

Test Article: 

IDNo. 

'?rocedure/Test Method: 

Test Article R.eceived: 

Test Start Date: 

Test Termination Date: · 

Number of Products Tested: 

Articles Tested 

Product Section 

Product Section 

STERILITY TEST 

Lab No. 

P.O. No. 

Test Facility: 

"42SURB .. ~Bed 0' Cobs; Lot #DC295 

Lot: DC295 

S-20431-0 1-00/Immersion 

May 19,2005 

May20,2005 

June 3,2005 

1 

STERILITY TEST RESULTS 

Te:n Article Identity Maintained as Submitted by Client 

Number of Type Incubation 

Articles of Media Temperature 

Tested (Degrees C) 

I SCDB400ml 20-25 

1 FTM-400ml 30-35 

05T_38013_0I 

TED WEAVER 

NAMSA 

6750 Wales Road 

Northwood, OH 43619 

Number of Number of 

Days Positive 

Incubated Articles 

14 0 

14 0 

SCDB = Soybean Casein Digest Broth FTM = Fluid Thioglycollate Medium 

Results and conclusions apply oruy to the test article tested. No further evaluation of these results is made by 

NAMSA Any extrapolation of these data to other samples is the responsibility of the sponsor. All procedures were 

conducted in conformance with good laboratory practice and ISO 17025. 

Record Storage: All raw data pertaining to this study and a copy of the final report are to be retained in designated 

NAMSA archive files for a period of S years. 

cal Date Completed ~..-3 -t}s-- Approved By 

Michelle R... Pierce 

Manager. Microbiology 

Authorization for dupliC"-31oon of tl1i:; report, except in whole. is reso.-vcd pending NAMSA's written approv<~l. Page 1 ofl 
PROTOCOL 

GASOLINE DIPE VAPOR CONDENSATE 

EMBRYO-FETAL TOXICITY STUDY IN RATS BY INHALATION EXPOSURE 

HLS Study No.: 

Protocol Version: 

Date: 

05-4287 

Final Protocol 

3 August 2005 
Huntingdon Life Sciences 

Study No. 05-4287 Final Protocol 

PROTOCOLSIGNATURES~REFACE 

Study Title: Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study 

in Rats by Inhalation Exposure 

HLS Study No.: 05-4287 

Sponsor Study No.: 211-DIPE-DEV 

This is the Final Protocol. It has been reviewed and approved by: 

Thomas M. Gray, M.S., D.A.B.T. 

Sponsor Representative 

American Petroleum Institute (API) 

Address: 1220 L Street, Northwest 

Washington, D.C. 20005-4070 

Phone No.: 202-682-8480 

Fax No.: 202-682-8270 

Email: grayt@api.com 

Ship Unused Test Substance and 

Empty Test Substance Containers to: 

Name: 

Supplier: 

Address: 

Phone No.: 

Fax No.: 

Email: 

Michael C. Henley 

ChevronTexaco Energy 

Technology Company 

100 Chevron Way 

Richmond, CA 94802-0627 

510-242-3062 

510-242-5542 

mche@chevron.com 
Huntingdon Life Sciences 

Study No. 05-4287 . 

TABLE OF CONTENTS 

Final Protocol 

1. INTRODUCTION .................................................................................................... l 

2. STUDY PERSONNEL ............................................................................................. ! 

3. PROPOSED STUDY DATES ................................................................................. ! 

4. EXPERIMENTAL DESIGN ................................................................................... 3 

4.1. JUSTIFICATION FOR ROUTE, DURATION AND FREQUENCY OF 

ADMINISTRATION ........................................................................................................ 4 

4.2. IDSTIFICATION FOR TEST ANIMAL SELECTION ........................................ .4 

4.3. IDSTIFICATION FOR NUMBER OF ANIMALS .............................................. .4 

4.4. EXPOSURE LEVEL SELECTION AND IDSTIFICATION ............................... .4 

5. TEST SUBSTANCE ................................................................................................ 4 

5.1. TESTSUBSTANCE .............................................................................................. 4 

5.2. IDENTIFICATION OF TEST SUBSTANCE ........................................................ 5 

5.3. ARClllVALSAMPLES ......................................................................................... 5 

5.4. UNUSED TEST SUBSTANCE ............................................................................. 5 

6. TEST ANIMALS ..................................................................................................... 5 

6.1. SPEClES ................................................................................................................. 5 

6.2. SUPPLlER .............................................................................................................. 6 

6.3. ANIMAL REQUIREMENTS/SPECIFICATIONS ................................................ 6 

6.4. ACCLIMATION PERIOD ..................................................................................... 6 

6.5. ANIMAL CARE AND HUSBANDRY ................................................................. 6 

6.6. ANIMAL HUSBANDRY DURING EXPOSURE ................................................ 8 

6.7. ANIMALIDENTIFICATION ................................................................................ 9 

6.8. ASSIGNMENT OF ANIMALS INTO GROUPS .................................................. 9 

7. TEST SUBSTANCE ADMINISTRATION ........................................................... 9 

7.1. ROUTE OF ADMINISTRATION ......................................................................... 9 

7 .2. FREQUENCY OF ADMINISTRATION ............................................................... 9 

7.3. DURATION OF ADMINISTRATION .................................................................. 9 

7.4. ADMINISTRATION OF TEST SUBSTANCE ..................................................... 9 

7.5. EXPOSURE CONCENTRATION DETERMINATION ..................................... 10 

7.6. PARTICLE SIZE DISTRIBUTION ANALYSIS ................................................. lO 

7.7. CHAMBER AND EXPOSURE ROOM ENVIRONMENT ................................ 11 

7.8. SUMMARY OF CHAMBER ACTIVITY ........................................................... 11 

8. IN-LIFE EXPERIMENTAL OBSERVATIONS ................................................ 11 

8.1. CLINICALOBSERVATIONS ............................................................................. 12 

8.2. BODYWEIGIITS ................................................................................................ 12 
Huntingdon Life Sciences 

Study No. 05-4287 Final Protocol 

8.3. FEED CONSUMP'I'ION ...................................................................................... 12 

9. TERMINAL OBSERV ATIONS ........................................................................... 13 

9.1. METHOD OF EUTHANASIA ............................................................................ 13 

9.2. MACROSCOPIC POSTMORTEM EXAMINATIONS ...................................... 13 

9.3. FETALEVALUATIONS ..................................................................................... 14 

10. ARCHIVING OF RECORDS AND SPECIMENS ............................................. 15 

11. STATISTICAL EVALUATIONS ........................................................................ 15 

11.1. CONTINUOUS DATA ..................................................................................... 15 

11.2. INCIDENCE DATA ......................................................................................... 18 

12. REPORTING ......................................................................................................... 18 

12.1. STATUS REPORTS ......................................................................................... 18 

12.2. FINALREPORT ............................................................................................... 19 

12.3. ROBUST SUMMARY ..................................................................................... 20 

13. REGULATORY REFERENCES ......................................................................... 20 

13.1. TEST GUIDEI.JNES ......................................................................................... 20 

13.2. GOOD LABORATORY PRACTICES ............................................................. 21 

13.3. ANIMAL WELFARE ACT COMPLIANCE ................................................... 21 

13.4. INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) ..... 22 

14. QUALITY ASSURANCE MONITORING ......................................................... 22 

15. ALTERATION OF PROTOCOL AND STUDY DESIGN ................................ 22 
Huntingdon Life Sciences 

Study No. 05-4287 

1. INTRODUCTION 

HLS Study No.: 

Study Title: 

Test Substance: 

Testing Facility: 

Purpose: 

05-4287 

Pagel 

Final Protocol 

Gasoline DIPE Vapor Condensate: 

Embryo-Fetal Toxicity Study in Rats 

by Inhalation Exposure 

Gasoline DIPE Vapor Condensate 

Huntingdon Life Sciences 

100 Mettlers Road 

East Millstone, NJ 08875-2360 

The study is designed to assess the potential maternal and/or developmental 

toxicity of Gasoline DIPE Vapor Condensate in the pregnant rat when 

administered by whole-body inhalation exposure. The study should permit 

detection of gross maternal organ changes and effects on the developing 

conceptus, including death, structural abnormalities or altered growth. 

This particular study is a near replicate of a previous study conducted by 

ExxonMobil Biomedical Sciences, Inc. (EMBSI Study Number 171734). The 

previous study is incomplete due to technical errors made in evaluating the 

fetuses. The Sponsor needs to repeat the previous study to satisfy the 

Environmental Protection Agency's Alternative Tier 2 Testing Requirements 

under Sections 211 (b) (2) of the Clean Air Act. 

2. STUDY PERSONNEL 

Study Director 

Alternate Contact 

Keith P. Hazelden, BSc, CBiol, MIBiol 

Gary M. Hoffman, B.A., DABT 

Inhalation Toxicologist 

Tel.: 732-873-2550 x2920 

Email: hoffmang@princeton.huntingdon.com 

Additional key personnel will be documented in the project file and presented in the final 

report. 
Huntingdon Life Sciences 

Study No. 05-4287 

3. PROPOSED STUDY DATES 

Study Initiation Date 

Receipt of test animals: 

Initiation of exposures: 

Termination of exposures: 

Necropsy: 

Experimental Termination: 

Submission of Draft Final Report: 

Study Completion Date: 

Page2 

Final Protocol 

Date Study Director Signs Protocol 

4 August 2005 (GD 0, 1, 2 or 3) 

6 August 2005 (Experimental Start 

Date) (First GD 5) 

24 August 2005 (Last GD 20) 

22-25 August 2005 (GD 21) 

November 2005 (Date of last data 

collection) 

30 December 2005 

Date Final Report signed by Study 

Director 
Huntingdon Life Sciences 

Studv No. 05-4287 

4. EXPERIMENTALDESIGN 

Group 

Group Desi2nation Exposure Levels (mg/m3)a 

1 Control 0 (air only) 

2 Low 2000 

3 Mid 10000 

4 High 20000 

Treatment 

Gestation 

Days 

5-20 5-20 

5-20 5-20 

TimeMated 

Females 

24 24 

24 24 

Number of Anim

Fet

(F

Euthanized 

Gestation 

Day 21b External 

All All 

All All All All All All 

aExposures will be 6 hours per day for 7 consecutive days per week for gestation days 5-20. Exposure levels are expr

test substance. The exposures will be conducted via whole-body exposure. Control animals will be treated with air o

treatment regimen as the treated groups. 

bPostmortem evaluations will also be performed on animals which are found dead or euthanized for humane reasons o

condition during the course of the study. 

The first day of gestation (Gestation Day 0 =day of detection of positive sign of mating) will be defined as Day 0 of t

animal. 
Huntingdon Life Sciences 

Study No. 05-4287 

Page4 

Final Protocol 

4.1. JUSTIFICATION FOR ROUTE, DURATION AND FREQUENCY 

OF ADMINISTRATION 

The inhalation route was used for the EMBSI study and is one of the 

potential routes of human exposure to this test substance. The duration and 

frequency of the exposures are as for the EMBSI study and as 

recommended in the relevant OECD and EPA test guidelines, ensuring 

adequate exposure of post-implantation embryo-fetal development. 

4.2. JUSTIFICATION FOR TEST ANIMAL SELECTION 

The rat was used for the EMBSI study and is used as a surrogate for 

humans in the detection of embryo-fetal toxicity and is the rodent species 

that is recommended by the relevant OECD and EPA test guidelines. 

Historical control data are also available with this strain of rat for 

comparative evaluation, if necessary. 

4.3. JUSTIFICATION FOR NUMBER OF ANIMALS 

The number of animals in this study is considered the minimum necessary 

to allow for meaningful interpretation of the data as required by OECD 

and EPA test guidelines. Some of the endpoints of principal interest (eg 

embryo-fetal death, fetal malformations) are low-frequency events and a 

group size of -20 litters tends to provide the necessary degree of 

consistency between studies. In the expectation of pregnancy rates of 80-

100%, the group size of 24 mated female rats is considered appropriate for 

this study and should provide a sufficient number of litters for evaluation 

in each group. Also, the number of animals is comparable to the number 

in the EMBSI study (25/group). 

4.4. EXPOSURE LEVEL SELECTION AND JUSTIFICATION 

The exposure levels were as used in the EMBSI study and as proscribed by 

the Sponsor based on the lower explosive limit of the test substance (low, 

intermediate and high levels were - 5, 25, and 50% of the LEL). 

5. TEST SUBSTANCE 

5.1. TEST SUBSTANCE 

Gasoline DIPE (di-isopropyl ether) Vapor Condensate 

TEST SUBSTANCE CATEGORY: gasoline product 

Description, lot number, storage, expiration date (if available) and 

handling procedures, as well as other pertinent information will be 
Huntingdon Life Sciences 

Study No. 05-4287 

PageS 

Final Protocol 

documented in the study data and final report. The test substance is 

supplied by the Sponsor and will be stored (ambient conditions) in an 

outside solvent shed except when in use in the inhalation laboratory. 

5.2. IDENTIFICATION OF TEST SUBSTANCE 

Unless otherwise noted, the identity, strength, composition, stability and 

method of synthesis, fabrication and/or derivation of each batch of the test 

substance will be documented by the Sponsor before its use in the study. 

This documentation will be maintained by the Sponsor at the address 

indicated on the signature page of this protocol. The Sponsor has conducted 

a purity analysis of the test substance by GC prior to the start of this study. 

The Testing Facility will conduct a stability analysis of the test substance 

by GC prior to the start of this study. 

5.3. ARCIDVAL SAMPLES 

An archival sample from each lot of test substance will be taken and stored 

in the Archives of the Testing Facility. If multiple studies are conducted 

with the same substance, a common archival sample may be taken and 

appropriately labeled. 

5.4. UNUSED TEST SUBSTANCE 

The unused portion of the test substance as well as any empty test 

substance containers will be returned to the Sponsor's designee following 

completion of the study. Empty test substance containers will be returned 

to the Sponsor's designee on an as needed basis. The Sponsor will be 

responsible for tracking their disposition. In the event the Sponsor wishes 

the Testing Facility to arrange for disposal, a cost for this service will be 

provided. 

6. TEST ANIMALS 

6.1. SPECIES 

Albino Rats (Outbred), Sprague Dawley- derived 

[Crl: CD® IGS BR, V AF/Plus®] 
Huntingdon Life Sciences 

Study No. 05-4287 

6.2. SUPPLIER 

Page6 

Final Protocol 

Charles River Laboratories (Raleigh, NC): Documentation will be 

maintained in the study file and included in the final report. This supplier 

and facility are the same as in the EMBSI study. 

6.3. ANIMAL REQUIREMENTS/SPECIFICATIONS 

6.3.1. NUMBER 

Order: 100 time-mated rats. 

Place on study: 96 time-mated rats. 

6.3.2. AGE AND WEIGHT 

Time-mated female rats 70-84 days in age and weighing 210-250 

grams on Gestation Day 0 will be purchased. Animals will be 

received on GD 0, 1, 2 or 3. 

6.4. ACCLIMATION PERIOD 

Animals will be acclimated for a minimum of 2 days from time of receipt 

until initiation of exposures (GD 5). 

6.5. ANIMAL CARE AND HUSBANDRY 

6.5.1. FACILITIES MANAGEMENT/ANIMAL HUSBANDRY 

Currently acceptable practices of good animal husbandry will be 

followed, e.g., Guide for the Care and Use of Laboratory Animals; 

National Academy Press, 1996. Huntingdon Life Sciences Inc. is 

fully accredited by the Association for Assessment and 

Accreditation of Laboratory Animal Care International 

(AAALAC). 

6.5.2. VETERINARY CARE 

Animals are monitored by the technical staff for any conditions 

requiring possible veterinary care. If any such conditions are 

identified, a staff veterinarian will be notified for an examination 

and evaluation. Animals will be treated as outlined in the Animal 

Welfare Act Compliance section of this protocol. The staff 

veterinarian will approve the animals for inclusion on test. 
Huntingdon Life Sciences 

Study No. 05-4287 

6.5.3. ENVIRONMENTAL CONDITIONS 

Light/Dark Cycle 

Page7 

Final Protocol 

Twelve-hour light/dark cycle provided by an automatic timer. 

Temperature 

Temperature will be monitored in accordance with HLS SOPs to 

ensure that the desired range of 18-26°C is maintained. 

Temperature will be maintained within this range to the maximum 

extent possible. The actual temperature range will be included in 

the final report. 

Humidity 

Humidity will be monitored in accordance with HLS SOPs to 

ensure that the desired range of 30 to 70% is maintained. 

Humidity will be maintained within this range to the maximum 

extent possible. The actual humidity range will be included in the 

final report. 

Air Changes 

Animal quarters will have 10-15 air changes per hour. The actual 

number of air changes per hour in each animal room is recorded at 

least twice each year and the Testing Facility retains these records. 

6.5.4. HOUSING 

All animals will be housed individually in stainless steel suspended 

cages with wire mesh floors and fronts. Cages will be arranged in 

such a way that possible effects due to placement are minimized 

(as per EPA guideline). Each cage will be fitted to secure a glass 

feeder jar with a stainless steel lid. Clean feed jars and fresh feed 

will be provided at least every 7 days for periods when feed 

consumption is not being recorded and at least every 7 days when 

feed consumption will be recorded. 

All dams after exposure on GD20 will be transferred to solid 

bottom cages with bedding material (ground corncobs). 

6.5.5. FEED 

Certified Rodent Diet, No. 5002; (Meal) (PMI Feeds, Inc., St. Louis, 

MO) ad libitum except during exposures. 
Huntingdon Life Sciences 

Study No. 05-4287 

PageS 

Final Protocol 

6.5.6. FEED ANALYSIS 

Analytical certification of batches of feed provided by the 

manufacturer will be maintained on file at the Testing Facility and 

included in the final report. There are no known contaminants in 

the feed that are expected to interfere with the objectives of this 

study. 

6.5.7. WATER 

Facility water is supplied by Elizabethtown Water Company, 

(Westfield, NJ) and will be provided ad libitum except during 

exposures to individual animal cages through an automated 

watering system. 

6.5.8. WATER ANALYSIS 

Water analyses are conducted monthly by the Elizabethtown Water 

Company to ensure that water meets standards specified under the 

EPA Federal Safe Drinking Water Act Regulations (40 CFR part 

141). Water analyses, provided by the supplier, will be maintained 

on file at the Testing Facility and included in the final report. In 

addition, water samples are collected biannually from 

representative rooms in the Testing Facility. Chemical and 

microbiological water analyses are conducted on these samples by 

a subcontract laboratory. Results are maintained on file at the 

Testing Facility and included in the final report. There are no 

known contaminants in the water that are expected to interfere with 

this study. 

6.6. ANIMAL HUSBANDRY DURING EXPOSURE 

Housing: 

Feed: 

Water: 

Individually in stainless steel wire mesh cages. 

None. 

None. 
Huntingdon Life Sciences 

Study No. 05-4287 

6.7. ANIMAL IDENTIFICATION 

Page9 

Final Protocol 

Each animal will be assigned a temporary identification number upon 

receipt. Mter selection for study, each animal will be ear-tagged by the 

Testing Facility with a number assigned by the Testing Facility. The number 

assigned by the Testing Facility plus the study number will comprise the 

unique animal number for each animal. If the tag is lost, it will be replaced 

or the animal will be tattooed for identification. Each cage will be provided 

with a cage card that will be color-coded for exposure level identification 

and will contain the study number and animal number. 

6.8. ASSIGNMENT OF ANIMALS INTO GROUPS 

Animals will be placed into study groups using a computerized 

randomization program. This ranks Gestation Day 4 body weights into 

blocks and randomly assigns each animal within each block into one of the 

study groups. Animals may be replaced based on physical examinations 

and/or body weight changes up to GD 5, prior to exposures. Disposition 

of all animals not utilized in the study will be maintained in the study file. 

7. TEST SUBSTANCE ADMINISTRATION 

7.1. ROUTE OF ADMINISTRATION 

Inhalation by whole-body exposures. 

7.2. FREQUENCY OF ADMINISTRATION 

Once daily. 

7.3. DURATION OF ADMINISTRATION 

The test substance will be administered for 6 hours/day, 7 days per week 

for Gestation Days (GD) 5-20. The duration of administration was chosen 

to be the same as in the EMBSI study. 

7.4. ADMINISTRATION OF TEST SUBSTANCE 

The test substance will be administered as a vapor in the breathing air of 

the animals. The test atmosphere will be generated by an appropriate 

procedure determined during pre-study trials. The trials will be performed 

(at least three 6-hour periods) to evaluate the optimal set of conditions and 

equipment to generate a stable and uniform atmosphere at the target 

exposure levels. The method will be described in the raw data of the study 

and in the report. 
Huntingdon Life Sciences 

Study No. 05-4287 

Page 10 

Final Protocol 

The whole-body exposure chambers will each have a volume of 

approximately 1000 liters. Each chamber will be operated at a minimum 

flow rate of 200 liters per minute. The final airflow will be set to provide 

at least one air change in 5.0 minutes (12 air changes/hour) and a T99 

equilibrium time of at most 23 minutes. This chamber size and airflow rate 

is considered adequate to maintain the oxygen level at least 19% and the 

animal loading factor below 5%. At the end of the exposure, all animals 

will remain in the chamber for a minimum of the T 99 equilibrium time. 

During this time, the chamber will be operated at approximately the same 

flow rate using clean air only. 

7.5. EXPOSURE CONCENTRATION DETERMINATION 

A nominal exposure concentration will be calculated. The flow of air 

through the chamber will be monitored using appropriate calibrated 

equipment. The test substance consumed during the exposure will be 

divided by the total volume of air passing through the chamber (volumetric 

flow rate times total exposure time) to give the nominal concentration. 

During each exposure, measurements of airborne concentrations will be 

performed in the animals' breathing zone at least 4 times using an 

appropriate sampling procedure and IR analytical procedure. Also, one 

sample per chamber per week will be analyzed by gas chromatography 

(GC) to characterize at least 10 major components (comprising at least 

80% by weight of the test substance) to show test substance stability and 

comparison between the neat liquid test substance and the vaporized test 

atmospheres. 

If more than the normal amount of trials is required because of test 

substance generation or monitoring problems (80 technician hours), the 

Sponsor will be consulted prior to additional trials (additional cost). 

7.6. PARTICLE SIZE DISTRIBUTION ANALYSIS 

During each week of exposure, particle size determinations will be 

performed using a TSI Aerodynamic Particle Sizer to demonstrate the 

absence of any condensation aerosol. 
Huntingdon Life Sciences 

Study No. 05-4287 

Page 11 

Final Protocol 

7.7. CHAMBER AND EXPOSURE ROOM ENVIRONMENT 

Chamber temperature, humidity, airflow rate and static pressure will be 

monitored continuously and recorded every 30 minutes during exposure. 

Chamber temperature and relative humidity will be maintained, to the 

maximum extent possible, between 20 to 24°C and 40 to 60%, 

respectively. Chamber oxygen levels (maintained at least 19%) will be 

measured pretest and at the beginning, middle and end of the study. 

Air samples will be taken in the vapor generation area pretest and at the 

beginning, middle and end of the study. Light (maintained approximately 

30-40 foot-candles at 1.0 meter above the floor) and noise levels 

(maintained below 85 decibels) in the exposure room will be measured 

pretest and at the beginning, middle and end of the study. 

7.8. SUMMARY OF CHAMBER ACTIVITY 

The minimum frequency of chamber activity is summarized below: 

Activity Frequency/chamber 

Measured Test Substance Concentration 

Measured Test Substance Characterization 

Particle Size 

Temperature 

Relative Humidity 

Airflow Rate 

Static Pressure 

Nominal Test Substance Concentration 

(excluding the air control chamber) 

Rotation Pattern of Exposure Cages 

Loading/Unloading Verification 

8. IN-LIFE EXPERIMENTAL OBSERVATIONS 

4X/day 

lX/week 

lX/week 

13X/day 

13X/day 

13X/day 

13X/day 

IX/ day 

IX/ day 

IX/ day 
Huntingdon Life Sciences 

Study No. 05-4287 

8.1. CLINICAL OBSERVATIONS 

8.1.1. VIABILITY OBSERVATIONS (cage-side) 

Page 12 

Final Protocol 

Observations for mortality, morbidity, and signs of severe toxicity 

will be made at least twice daily. Animals in extremely poor health 

or in a possibly moribund condition will be identified for further 

monitoring and possible euthanasia. 

8.1.2. DETAILED PHYSICAL EXAMINATIONS 

In-Chamber: All animals will be observed as a group at least once 

during each exposure. This will routinely be performed near the 

middle of each exposure and may be performed more frequently if 

significant signs of toxicity are noted. Pertinent behavioral 

changes and all signs of toxicity, including mortality, should be 

recorded. These signs should include time of onset, degree and 

duration. 

Out-of-Chamber: Animals on study will be examined daily from 

day after receipt through to terminal euthanasia on GD 21. 

Examinations will include observations of general condition, skin 

and fur, eyes, nose, oral cavity, abdomen and external genitalia, 

occurrence of secretions and excretions, and autonomic activity 

(e.g., lacrimation, piloerection, pupil size, unusual respiratory 

pattern). Changes in gait, posture and response to handling as well 

as the presence of clonic or tonic movements, stereotypy (e.g., 

excessive grooming, repetitive circling) or bizarre behavior (e.g., 

self-mutilation, walking backward) will be recorded as well as 

evaluations of respiration. During the treatment period, these 

evaluations will be performed after exposures. 

8.2. BODY WEIGHTS 

Body weights will be recorded on GD 4 (for randomization into test groups), 

5, 8, 11, 14, 17, 20 and 21 (day of scheduled sacrifice). Weight gain will be 

calculated for intervals as found appropriate, including (but not limited to) 

GD 5-14, GD 14-21 and 5-21. 

8.3. FEED CONSUMPTION 

Feed consumption will be recorded and reported for these intervals: 

GD 4-5,5-8, 8-11, 11-14, 14-17, 17-20,20-21 and 5-21. 
Huntingdon Life Sciences 

Study No. 05-4287 

9. TERMINAL OBSERVATIONS 

9.1. METHOD OF EUTHANASIA 

Page 13 

Final Protocol 

All dams will be euthanized (by noon) by an overdose of carbon dioxide 

inhalation. All live fetuses will be euthanized by an intraperitoneal 

overdose of sodium pentobarbital. 

9.2. MACROSCOPIC POSTMORTEM EXAMINATIONS 

A macroscopic postmortem examination will be performed on all the dams, 

including those dying spontaneously or euthanized for humane reasons or in 

either a moribund condition or after prematurely delivering a litter. 

Macroscopic lesions or tissues with significant findings will be saved (10% 

Neutral Buffered Formalin). 

Females that are found dead or sacrificed prior to scheduled termination 

will have implantation data (ie number of fetuses and early/late embryofetal deaths) determined at necropsy. Corpora lutea will be counted for 

these animals. 

Females that do not have fetuses when caesarean-sectioned will have their 

uterus' examined for implantation sites and resorptions, including 

confirmation of apparent non-pregnant status, by means of staining with 

ammonium sulphide (modified Salewsk:i test). 

Dams showing signs of premature delivery will be euthanized on the day 

such evidence is observed. Reproductive tracts will be examined for 

implantation data (implantation sites, live/dead fetuses and early/late 

embryo-fetal deaths). Fetuses obtained earlier than Gestation Day (GD) 21 

will be counted, sexed externally, evaluated for external malformations, 

euthanized and discarded. Corpora lutea will be counted for these animals. 

Any dams initiating delivery on GD 21 will be immediately euthanized 

and the fetuses processed with the other GD 21 fetuses. 

Dams surviving on GD 21 will have the following examinations of their 

pregnancies: 
Huntingdon Life Sciences 

Study No. 05-4287 

9.2.1. Reproductive System 

Page 14 

Final Protocol 

The intact uteri (ovaries attached) will be removed from the 

abdominal cavity. The gravid uteri, including the cervix, will be 

weighed. Corpora lutea will be counted and the number per ovary 

recorded. The number and location of the following will be recorded 

for each uterine hom: 

• live fetuses 

• dead fetuses (no significant degeneration) 

• late embryo-fetal deaths (recognizable dead fetus undergoing 

degeneration, regardless of size) 

• early embryonic deaths (evidence of implantation but no 

recognizable fetus) 

Each placenta will be examined macroscopically. The maternal 

carcass, uterus, ovaries and placenta will then be discarded. 

9.3. FETAL EVALUATIONS 

9.3.1. External Evaluations 

All live fetuses will be weighed and individually identified within 

cassettes. Each live and dead retained fetus will be given a 

macroscopic external examination for defects including 

observation of the palate. 

9.3.2. Fetal Soft Tissue (Visceral) Evaluation 

Approximately one-half of the fetuses in each litter (alternating 

fetuses within the litter, nominally) will be placed in Modified 

Davidson's fixative for preservation and decalcification. These 

fetuses will be subjected to soft tissue examination by gross 

dissection of the torso and a razor blade sectioning technique for 

the head (derived from that of Wilson, J.S., Warkany, J. (1965), 

Teratology: Principles and Techniques. The University of Chicago 

Press. Chicago and London. Page 271). All malformations and 

variations will be recorded. Particular attention will be paid to the 

reproductive tract for altered signs of development. Unusual 

malformations or developmental findings will be photographed. 

During the dissection process, the sex of each fetus will be 

confirmed by internal inspection of the gonads. Following 

complete dissection of the fetuses, all carcasses and sections will 

be preserved in 10% neutral buffered formalin. 
Huntingdon Life Sciences 

Study No. 05-4287 

9.3.3. Fetal Skeletal Evaluations 

Page 15 

Final Protocol 

Approximately one-half of the fetuses in each litter (alternating 

fetuses within the litter, nominally) will be eviscerated, placed in 

70% isopropyl alcohol for preservation and processed for staining 

of the skeleton using Alizarin RedS. Subsequently, these fetuses 

will be evaluated for skeletal malformations and ossification 

variations. These skeletons will then be stored in 100% glycerin 

with a mold inhibitor. During the dissection process, the sex of 

each fetus will be confirmed by internal inspection of the gonads. 

9.3.4. Dead Fetuses 

These will be removed from the uterus and examined for external 

malformations. Only those with obvious external malformations will 

be preserved (10% neutral buffered formalin) for possible future 

examination. All other dead fetuses, and early embryonic deaths, 

will be discarded. 

10. ARCHIVING OF RECORDS AND SPECIMENS 

All data documenting experimental details and study procedures and observations 

will be recorded and maintained as raw data. 

At the completion of the study, all reports, raw data, preserved specimens and 

retained samples will be maintained in the Testing Facility's Archives for a period 

of one year after submission of the signed final report. 

The Sponsor will be contacted in order to determine the final disposition of these 

materials. The Sponsor is responsible for all costs associated with the storage of 

these materials beyond one year from the issuance of the final report and for any 

costs associated with the shipment of these materials to the Sponsor or to any 

other facility designated by the Sponsor. 

11. STATISTICAL EVALUATIONS 

Analysis will be performed by Graham Healey (Principal Investigator) of 

Huntingdon Life Sciences Ltd, Woolley Road, Alconbury, Huntingdon, 

Cambridgeshire, PE28 4HS, England. The Testing Facility will be responsible for 

the GLP compliance and the archiving of the raw data and original final report. 

11.1 CONTINUOUS DATA 

The following mean measures will be analyzed as described below: 
Huntingdon Life Sciences 

Study No. 05-4287 

Page 16 

Final Protocol 

maternal body weight values and body weight changes during gestation 

(Note: body weight measurements will only be compared to GD 5 values 

or subsequent relevant values) 

maternal feed consumption values (presented as grams of feed/kg of body 

weight/day) 

gravid uterine weights 

corpora lutea 

implantation data 

pre-implantation loss 

early embryonic deaths (evidence of implantation but no recognizable 

fetus) 

live fetuses 

dead fetuses (no significant degeneration) 

late embryo-fetal deaths (recognizable dead fetus undergoing 

degeneration, regardless of size) 

total embryo-fetal deaths and as % of implant sites 

number of males and females per litter 

Mean values of all exposure groups will be compared to the mean value 

for the control group at each time interval. Evaluation of equality of group 

means will be made by the appropriate statistical method (either 

parametric or non-parametric methods), followed by a multiple 

comparison test if needed. Bartlett's test (Bartlett, 1937; Sokal and Rohlf, 

1995; Snedecor and Cochran, 1967) will be performed to determine if 

groups have equal variances. For all parameters if the variances are equal, 

parametric procedures will be used; if not, nonparametric procedures will 

be used. 

The parametric method will be the standard one-way analysis of variance 

(ANOVA) using the F ratio to assess significance (Armitage, 1971; 

Dunlap and Duffy, 1975). If significant differences among the means are 

indicated Dunnett's (Dunlap et al., 1981; Dunnett, 1955, 1964) test will be 

used to determine which means are significantly different from the control. 

The nonparametric method will be the Kruskal-Wallis test (Kruskal and 

Wallis, 1952, 1953) and if differences are indicated, Steel's test (Steel, 

1959) will be used to determine which means differ from control. 

Bartlett's test for equality of variance will be conducted at the 1% 

significance level; all other statistical tests will be conducted at the 5% and 

1% significance levels. 

References for these procedures are: 
Huntingdon Life Sciences 

Study No. 05-4287 

Page 17 

Final Protocol 

Armitage, P. 1971. Statistical Methods in Medical Research. Oxford, UK: 

Blackwell Scientific Publications. 

Bartlett, M.S. 1937. Properties of sufficiency and statistical tests. 

Proceedings of the Royal Society, Series A 160:268-282. 

Dunlap, W.P. and Duffy, J.A. 1975. Fortran IV functions for calculating 

exact probabilities associated with z, chi-Square, t and F Values. Behav. 

Res. Methods and Instrumentations 7:59-60. 

Dunlap, W.P., Marx, M.S. and Agamy, G.G. 1981. Fortran IV functions 

for calculating probabilities associated with Dunnett's test. Behav. Res. 

Methods and Instrumentation 13: 363-366. 

Dunnett, C.W. 1955. A multiple comparison procedure for comparing 

several treatments with a control. Journal of the American Statistical 

Association 50: 1096-1121. 

Dunnett, C.W. 1964. New tables for multiple comparisons with a control. 

Biometrics 20: 482-491. 

Kruskal, W.H. and Wallis, W.A. 1952. Use of ranks in one-criterion 

variance analysis. Journal of the American Statistical Association 47: 583-

621. 

Kruskal, W.H. and Wallis, W.A. 1953. Errata for Kruskal-Wallis (1952) 

Journal of the American Statistical Association 48: 907-911. 

Snedecor, G.W. and Cochran, W.G. (1989) Statistical Methods. 8th 

edition Iowa State University Press, Iowa, USA. 

Sokal, R.R. and Rohlf, F.J. 1995. Biometry. 3rd Edition. San Francisco: 

W.H. Freeman, pp. 369-371. 

Steel, R.G.D. 1959. A multiple comparison rank sum test: treatment 

versus control. Biometrics 15: 560-572. 

11.1.1 FETAL BODY WEIGHT 

The fetal body weight (by sex and as a composite for both sexes) will be 

analyzed by a mixed model analysis of variance that provides an accurate 

statistical model of the biology. The analysis uses the litter as the basis for 

analysis and effectively uses the litter size as a covariate. The model 

considers dose group, litter size, and fetal sex as explanatory variables. If 

the dose group effect in the model is statistically significant, the dose 

group least squares means will be tested pairwise vs the control group 

using t-tests associated with least squares means. The least squares means 

allows comparisons that account for differences in litter size and sex. The 

mathematical model is based on a paper by Chen et. al. (1996). Statistical 
Huntingdon Life Sciences 

Study No. 05-4287 

Page 18 

Final Protocol 

significance of differences from control will be recognized at the 5% or 

1%, two-sided levels. 

Reference: 

Chen, J.J., Gaylor, D.W. and Laborde, J.B. 1996. Dose-response 

modeling of growth for developmental toxicity. Environmetrics 7:135-

144. 

11.2 INCIDENCE DATA 

premature deliveries 

total pregnancy loss (no live fetuses) 

maternal necropsy findings 

external fetal defects 

skeletal malformations and variations 

soft tissue malformations and variations 

These data will be analyzed based on a Generalized Estimating Equation 

(GEE) application of the linearized model (Ryan, 1992). For litter 

endpoints, the model uses the litter as the basis for analysis and considers 

correlation among littermates by incorporating an estimated constant 

correlation and the litter size as a covariate. If the dose group effect in the 

model is statistically significantly the dose group least squares means will 

be tested pairwise vs. the control group using t-tests associated with least 

squares means. The least squares means allows comparisons that account 

for differences in litter size. Statistical significance of differences from 

control will be recognized at the 5% or 1%, two-sided levels. 

Reference: 

Ryan, L. 1992. The use of generalized estimating equations for risk 

assessment in developmental toxicity. Risk Analysis, 12:439-447. 

12. REPORTING 

12.1. STATUS REPORTS 

Periodic verbal and written updates on study progress will be provided by 

the Study Director. In general, a written status report will be submitted 

weekly and following completion of the GD21 terminations. 

·'.''· 
Huntingdon Life Sciences 

Study No. 05-4287 

12.2. FINAL REPORT 

Page 19 

Final Protocol 

One unbound hard copy and one electronic copy of an audited draft report 

will be submitted following termination of the study. After receipt and 

review of the Sponsor's comments, appropriate changes will be made and 

an unbound hard copy and electronic copy of the revised report will be 

forwarded to the Sponsor for a final review. Upon receipt of the Sponsor's 

final comments, appropriate changes will be made and two hard copies 

and one electronic copy of a signed, final report will be issued. (Additional 

copies will be provided at additional cost). The report will minimally 

include: 

12.2.1. Body of Report 

• Compliance Statement (including Sponsor signature line) 

• Quality Assurance Statement 

• Abstract 

• Introduction 

• Experimental Design (including justifications for exposure 

levels) 

• Materials and Methods 

• Results and Discussion 

• Conclusion and No-Observed-Effect-Level (NOEL) or NoObserved-Adverse-Effect-Level (NOAEL) Statement 

• Statistical Procedures 

• Protocol Deviations and Study Impact Statements 

12.2.2. Summary Tables 

• Exposure data 

• Mortality data 

• Pregnancy data 

• Summary of maternal physical in-life observations 

• Maternal body weight and weight change data during gestation 

• Maternal feed consumption data 

• Mean number of corpora lutea and implantation sites 

• Mean number and percent of resorptions and live fetuses 

• Summary of maternal macroscopic postmortem examination data 

including gravid uterine weights 

• Pre-implantation loss 

• Post-implantation loss 

• Mean fetal weight 

• Total number of male and female fetuses per group and mean 

percent male/percent female per group 
Huntingdon Life Sciences 

Study No. 05-4287 

Page20 

Final Protocol 

• Incidence of litters and fetuses with observations (external, 

visceral and skeletal) 

12.2.3. Appendix Tables (Individual data) 

• Individual animal termination history 

• Individual clinical observations 

• Individual maternal body weight data and weight change data 

• Individual maternal feed consumption data 

• Individual uterine implantation data and corpora lutea data 

• Maternal macroscopic postmortem observations 

• Individual fetal observations (external, visceral and skeletal) 

12.2.4. Appendices 

• Inhalation report 

• Analytical results 

• Feed and water analysis 

• Personnel involved in the study 

• Protocol and Amendment(s) 

12.3. ROBUST SUMMARY 

This report will be separately provided by the Testing Facility's UK office 

in both IUCLID and WORD® electronic formats. 

13. REGULATORY REFERENCES 

13.1. TEST GillDELINES 

This study is designed to meet or exceed the pertinent requirements of: 

US EPA Vehicle Emissions Inhalation Exposure Guideline 79.61, CFR 

Vol. 59, No. 122, 27 June 1994. 

Organization for Economic Cooperation and Development (22 January 

2001). OECD Guidelines for Testing of Chemicals; OECD Guideline 

414: Prenatal Developmental Toxicity Study. 

US EPA OPPTS Health Effects Test Guidelines 870.3700, Prenatal 

Developmental Toxicity Study EPA 712-C-98-207, August 1998. 
Huntingdon Life Sciences 

Study No. 05-4287 

13.2. GOOD LABORATORY PRACTICES 

Page21 

Final Protocol 

This study will be conducted in accordance with US EPA 79.60, CFR Vol. 

59, No. 122, 27 June 1994. This study will be performed according to 

protocol and Huntingdon Life Sciences' Standard Operating Procedures. 

13.3. ANIMAL WELFARE ACT COMPLIANCE 

This study will comply with all appropriate parts of the Animal Welfare 

Act regulations: 9 CFR Parts 1 and 2 Final Rules, Federal Register, 

Volume 54, No. 168, August 31, 1989, pp. 36112-36163 effective October 

30, 1989 and 9 CFR Part 3 Animal Welfare Standards; Final Rule, Federal 

Register, Volume 56, No. 32, February 15, 1991, pp. 6426-6505 effective 

March 18, 1991. The Sponsor should make particular note of the 

following: 

1. The Sponsor's signature on this protocol documents for the study 

described, there are no generally accepted non-animal alternatives and 

the study does not unnecessarily duplicate previous experiments. 

2. All procedures used in this study have been designed to avoid 

discomfort, distress and pain to the animals. All methods are described 

in this study protocol or in written laboratory standard operating 

procedures. 

3. Any procedures outlined in this study protocol which are expected to 

cause more than momentary or slight pain or distress to the animals 

will be performed with appropriate sedatives, analgesics or 

anaesthetics unless the withholding of these agents is justified for 

scientific reasons, in writing, by the Sponsor and the Study Director 

and approved by the IACUC; in which case the procedure will 

continue for the minimum time necessary. Documentation of the 

justification for withholding treatment for pain or distress and IACUC 

approval of the procedures will be made prior to study initiation on the 

IACUC Protocol Review form. 

4. Animals experiencing more than momentary or slight pain or distress 

due to the test substance, injury or illness may be treated by the Testing 

Facility's veterinary staff with approved analgesics or agents to relieve 

pain after consultation with the Study Director/Sponsor. However, in 

emergency situations, the veterinary staff is authorized to administer 

emergency treatment as necessary. Any subsequent treatment or 

euthanasia will be administered after consultation with the Study 
Huntingdon Life Sciences 

Study No. 05-4287 

Page 22 

Final Protocol 

Director. The Sponsor will be advised by the Study Director of all 

emergency situations in as timely a manner as possible. 

5. Methods of euthanasia used during this study are in conformance with 

the above referenced regulations. 

13.4. INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) 

The IACUC Protocol Review subcommittee has reviewed this protocol 

and found it to be in compliance with all appropriate animal welfare 

regulations. 

14. QUALITY ASSURANCE MONITORING 

The Quality Assurance Unit of Huntingdon Life Sciences (East Millstone, NJ) 

will monitor the facilities, equipment, personnel, methods, practices, records, 

protocols, study conduct, raw data, draft and final reports and controls 

used/produced at the Testing Facility in this study, to assure that they are in 

conformance with this protocol, company Standard Operating Procedures, and the 

appropriate Good Laboratory Practice regulations. 

15. ALTERATION OF PROTOCOL AND STUDY DESIGN 

Alterations to this protocol may be made as the study progresses. No changes in 

the protocol will be made without the consent of the Sponsor. In the event that 

the Sponsor authorizes a protocol change verbally, such changes will be honored 

by the Testing Facility and will be followed by a written verification. The Study 

Director and a Sponsor representative will sign all protocol modifications. Two 

members of the Institutional Animal Care and Use Committee prior to the 

modification's implementation will also sign any modifications potentially 

affecting animal welfare. 
Huntingdon Life Sciences 05-4287 Page 1 

Protocol Amendment No.: 1 

Study Title: Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by 

Inhalation Exposure 

No. of Pages in Amendment: 1 

Change(s) 

Protocol Page 2: 

The date for experimental termination (date of last data collection) is amended from November to 

December 2005. 

The date for issue of the Draft Final Report (audited) is amended from 30 December 2005 to 

12 January 2006. 

Reason(s) for Change(s) 

To assist with the logistics of fetal examinations, and report production, audit and review, prior to 

issue of the draft report. 

Amendment approved by: 

Keith P Hazelden 

Study Director 

Huntingdon Life Sciences 

East Millstone, New Jersey 

'ts~or 

Date 
Huntingdon Life Sciences 05-4287 Page 1 

Protocol Amendment No.: 2 

Study Title: Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by 

Inhalation Exposure 

No. of Pages in Amendment: 2 

Changes (inserted text underlined) 

Protocol Page 15: 

9.3.3. Fetal Skeletal Evaluations 

Approximately one-half of the fetuses in each litter (alternating fetuses within the 

litter, nominally) will" be eviscerated, placed in 70% isopropyl alcohol for 

preservation and processed for staining of the skeleton using Alizarin Red S. 

Subsequently, these fetuses will be evaluated for skeletal malformations and 

ossification variations by an approved subcontractor, Michael Mercieca (Principal 

Investigator) of Pathology Associates International (PAl), 15 Worman's Mill 

Court. Suite l, Frederick, MD 21701. USA. The skeletons will then be stored in 

100% glycerin with a mold inhibitor. During the dissection process, the sex of 

each fetus will be confirmed by internal inspection of the gonads. 

The Testing Facility will be responsible for the GLP compliance and archiving of 

raw data produced by the subcontractor. 

Reason for Changes 

To assist with the logistics of fetal examinations and ensure timely report production, the 

skeletal evaluations are being subcontracted to an approved contractor. 

There are no additional cost implications associated with this change. 
Huntingdon Life Sciences 

Amendment approved by: 

Keith P Hazelden 

Study Director 

Huntingdon Life Sciences 

East Millstone, New Jersey 

05-4287 Page2 

Protocol Amendment No.: 2 

28 October 2005 

Date 

~l,2t)P) 

Date 
Huntingdon Life Sciences 05-4287 

211-DIPE-DEV 

Protocol Amendment No.: 3 

Study Title: Gasoline DIPE Vapor Condensate: 

Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure 

Change(s) 

Protocol Signatures/Preface Page and Page 1~ Section 2 Study Personnel, 

update Study Director 

From: 

Keith P. Hazelden, BSc, CBiol, Mffiiol 

To: 

Gary M. Hoffin~ BA, DABT 

Tel: 732-873-2550 x 2920 

Email: HoffinanG@Princeton.Huntingdon.com 

Reason(s) for Change(s) 

Page30 

Keith P. Hazelden was responsible for the conduct of the study and submission of the draft 

report although is no longer employed with Huntingdon Life Sciences. Gary Hoffinan will 

assume Study Director responsibility for finalization of the report. Robert M. Parker will be 

Alternate Contact (x2389) and will provide reproductive toxicology consultation. 

Amendment approved by: 

Gary M. Ho~ BA, DABT 

Study Director 
Huntingdon Life Sciences 

'!\ 

/, 

05-4287 

211-DIPE-DEV 

Protocol Amendment No.: 4 

Study Title: Gasoline DIPE Vapor Condensate: 

Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure 

Change(s) 

Statistical Evaluations, page 15: 

Page 31 

Revise from: Analysis will be performed by Graham Healey (Principal Investigator) of 

Huntingdon Life Sciences Ltd, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, 

PE28 4HS, England. The Testing Facility will be responsible for the GLP compliance and 

the archiving of the raw data and original final report. 

Revise to: Analysis will be initially performed by Graham Healey (Principal Investigator) 

of Huntingdon Life Sciences Ltd, Woolley Road, Alconbury, Huntingdon, 

Cambridgeshire, PE28 4HS, England. The Testing Facility will be responsible for the GLP 

compliance and the archiving of the raw data and original final report. Further statistical 

analysis (gravid uterus weight and net body weight change minus gravid uterine 

weight) will be performed by Gareth Thomas (Principal Investigator) and expert 

report prepared for Testing Facility auditing. This additional phase of the study will 

also be conducted in compliance with principles of Good Laboratory Practice 

Standards as set forth in: The UK Good Laboratory Practice Regulations 

(Statutory Instrument 1999 No 3106 as amended by Statutory Instrument 2004 No. 

994). OECD Principles of Good Laboratory Practice (as revised in 1997), 

ENV/MC/CHEM(98)17. EC Commission Directive 2004/10/EC of 11 February 2004 

(Official Journal No L 50/44). Test Site-UK QA will audit the study protocol and 

any subsequent protocol amendments pertaining to the statistical phase of work 

conducted by the Principal Investigator. The dates of inspection .will be specified on 

the QA Statement of the f"mal report. The SAS 9.1.3 [SAS INSTITUTE (2002) SAS 

OnlineDoc0 Version Nine. SAS Institute Inc., Cary, NC, USA.] or Quasar 1.1. 

[QUASAR 1.1 User Guide (Stand-Alone) version 1.1 (2009). Huntingdon Life 

Sciences Internal Document.] computer system will be used for the statistical 

analysis. 

Reason( s) for Change( s) 

Graham Healey has retired from HLS and this study was reassigned to Gareth Thomas to 

assume the responsibilities of Principal Investigator that had belonged to Graham Healey. 

Also, added clarification of the GLP regulations, auditing and computer systems relevant 

to the statistical evaluations. 
Huntingdon Life Sciences 

Amendment approved by: 

Gary M. Hoffinan, BA, DABT 

Study Director 

05.;.4287 

211-DIPE-DEV 

Protocol Amendment No.: 4 

c~tr 

Date 

Page32 
Huntingdon Life Sciences 

Rat Strain: Sprague-Dawley [Crl: CD IGS BR] 

Supplier: Charles River Laboratories 

Fetal Variation: Rib( s) 14th Rudimentary 

Study Route 1stGD 6 LastGD 20 

ALL Sep-02 Jul-05 

02-4235 Oral gavage Sep-02 Oct-02 

03-4253 Inhalation Feb-05 Mar-05 

03-4255 Oral gavage Jun-03 Sep-03 

04-4261 Inhalation Aug-04 Sep-04 

04-4267 Subcutaneous Nov-04 Dec-04 

05-4292 Intravenous Jun-05 Jul-05 

05-4287 

211-DIPE-DEV 

Historical Control Data 

Fetal Skeletal Observations 

Fetuses 

No. No. with %with 

Examined Finding Finding 

902 79 8.76 

160 11 6.88 

167 12 7.19 

148 23 15.54 

150 22 14.67 135 6 4.44 

142 5 3.52 

Litters 

No. No. with 

Examined Finding 

134 45 

22 6 24 7 22 15 

22 9 22 4 22 4 
~ 

charles river 

accelerating drug development exactly. 

Reproductive Toxicology 

Historical Control Data in Rats 

905 Sheehy Drive, Bldg. A, Horsham, PA 19044 • 215.443.8710 • FAX 215.443.8587 

- 1 -
CHARLES RIVER LABORATORIES, PRECLINICAL SERVICES, PENNSYLVANIA 

SUMMARY OF REPRODUCTIVE INDICES 

Crl:CD(SD) RATS 

DAY 21 CAESAREAN-SECTION 

PERIOD: JUNE 2008- JUNE 2010 

FULL 

STUDIES 

NUMBER OF STUDIES 

NUMBER OF RATS: 

TESTED 

PREGNANT 

FOUND DEAD 

ABORTED 

DELIVERED 

NUMBER OF RATS PREGNANT AT 

CAESAREAN-SECTIONING 

NUMBER OF RATS WITH SINGLE 

CONCEPTUS LITTER: 

LIVE 

RESORBED 

ABORTED 

48 

1195 

1106 

7* 

1 

13 

1089 

1 

0 

0 

RANGE/STUDY 

MEANor% MEANor% 

%PREGNANT 

AVERAGENO.CORPORALUTEA 

AVERAGE NO. IMPLANTATIONS 

AVERAGE % PREIMPLANTATION 

LOSS 

AVERAGE LITTER SIZE 

AVERAGE NO. LIVE FETUSES 

AVERAGE NO. DEAD FETUSES 

92.9 

15.8 

14.8 

5.4 

14.1 

0.0 

AVERAGE NO. RESORPTIONS 0.7 

AVERAGE NO. EARLY RESORPTIONS 0. 7 

AVERAGE NO. LATE RESORPTIONS 0.0 

(76.0-100) 

(13.6-17.9) 

(13.0-17.0) 

(1.8-11.3) 

(12.5-16.1) 

(0.3-1.2) 

(0.3-1.2) 

DOSAGE-RANGE 

STUDIES 

MEANor% 

93.6 

15.6 

14.5 

5.1 

14.0 

0.0 

0.6 

0.6 

0.0 

11 

86 

79 

1 

0 

0 

78 

0 

0 

0 

RANGE/STUDY 

MEANor% 

(62.5-100) 

(14.0-16.5) 

(13.3-15.5) 

(3.0-10.2) 

(12.8-15.2) 

(0.1-1.0) 

(0.1-1.0) 

(0-0.2) 

* Two were unscheduled sacrifices and one was euthanized due to an intubation accident 

-20-
CHARLES RIVER LABORATORIES, PRECLINICAL SERVICES, PENNSYLVANIA 

SUMMARY OF REPRODUCTIVE INDICES 

Crl:CD(SD) RATS 

DAY 21 CAESAREAN-SECTION 

FULL DOSAGE-RANGE 

STUDIES STUDIES 

RANGE/STUDY RANGE/STUDY 

MEANor% MEANor% MEANor% MEANor% 

AVERAGE % POSTIMPLANTATION 

LOSS 4.7 (2.1-8.8) 4.6 (1.4-7.4) 

AVERAGE% DAMS WITH ANY 

RESORPTIONS 46.1 (24.0-75.0) 44.9 (12.5-80.0) 

AVERAGE% DAMS WITH ALL 

CONCEPTUSES RESORBED 0.0 0.0 

AVERAGE% DAMS WITH ONE OR 

MORE LIVE FETUSES 100.0 100.0 

AVERAGE SEX RATIO 

(%MALES/LITTER) 49.8 ( 42.6-55.9) 49.3 (42.0-55.4) 

AVERAGE FETAL BODY WEIGHT (G) 5.47 (5.16-5.75) 5.39 (5.04-5.65) 

AVERAGE FOR MALES (G) 5.61 (5.27-5.91) 5.53 (5.19-5.86) 

AVERAGE FOR FEMALES (G) 5.32 (4.97-5.57) 5.25 ( 4.92-5.52) 

AVERAGE% DEAD OR RESORBED 

CONCEPTUSES/LITTER 4.8 (2.1-8.8) 4.2 (1.2-7 .4) 

-21 -
Huntingdon Life Sciences 05-4287 

211-DIPE-DEV 

Page 1 

Draft Final Report 

Testing Facility Personnel Appendix Q 

TITLE/DEPARTMENT 

SENIOR VICE PRESIDENT, 

SAFETY ASSESSMENT 

DIRECTOR, TOXICOLOGY 

OPERATIONS 

DIRECTOR, QUALITY 

ASSURANCE 

STUDY DIRECTORa 

INHALATION TOXICOLOGIST 

VETERINARIAN 

REPORT PRODUCTION 

MANAGER/SUPERVISOR 

Reproductive Toxicology 

Inhalation Toxicology 

Analytical Services 

Pharmacy 

Necropsy and Fetal Pathology 

STATISTICIAN 

SUBCONTRACTOR 

NAME/DEGREE 

Sylvie J. Gosselin, D.V.M., Ph.D., 

Diplomate A.C.V.P. 

Ian Vanterpool, F.I.A.T. 

Nicki S. Iacono, B.S. 

Keith P. Hazelden, B.Sc., C.Biol., M.I.Biol. 

GaryM. Hoffman, B.A., D.A.B.T. 

Gary M. Hoffman, B.A., D.A.B.T. 

Teresa S. Kusznir, V.M.D. 

Janet E. Scimone, B.A. 

Sally Wilcox, B.Sc. 

Stuart Cracknell, CBiol, MIBiol. 

Kay Saladdin, B.S. 

MichaelS. McCarthy 

G. Elizabeth Baxter, B.S. 

Graham F Healey, B.Sc., M.Sc, ARCS., 

Prinicipal Investigator 

Michael Mercieca, Principal Investigator 

Pathology Associates International (P AI) 

aKeith Hazeldon was responsible for the conduct ofthe study and submission ofthe draft 

report; however he is no longer employed with Huntingdon Life Sciences. Gary 

Hoffman assumes responsibility for finalization of the study report. 
Huntingdon Life Sciences 05-4287 

211-DIPE-DEV 

Report Amendments 

There are no amendments for this report at this time. 

Page 1 

Draft Final Report 

AppendixR