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

Final Report

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

Performed by: Huntingdon Life Sciences (HLS) 

100 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: 8 November 2012 

Page 1 of 219 



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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 of 2000, 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 

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

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

SIGNATURE PAGE ...........................................................................................................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 ..............................................................................................10 

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

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

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

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

2.9. Veterinary Care ..........................................................................................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 ...................................................................................................27 

4. CONCLUSIONS ..................................................................................................28

REFERENCES ..................................................................................................................29

CALCULATIONS .............................................................................................................30 
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GENERAL PREFACE ......................................................................................................31 

FIGURES 

1. Mean Gestation Body Weights ....................................................................................32 

2. Mean Gestation Feed Consumption .............................................................................33 

TABLES 

1. Summary of Survival and Pregnancy ..........................................................................34 

2. Summary of Clinical Observations During Gestation .................................................35 

3. Summary of Gestation Body Weights (grams) ............................................................36 

4. Summary of Gestation Body Weight Gain (grams) .....................................................37 

5. Summary of Gestation Feed Consumption (grams/kg/day) .........................................38 

6. Summary of Maternal Necropsy Observations ............................................................39 

7. Summary of Cesarean Section Data ............................................................................40 

8. Summary of Gravid Uterine Weight and Net Body Weight Change (grams) .............43 

9. Summary of Fetal Observations ...................................................................................44 

APPENDICES 

A. Analytical Report .........................................................................................................47 

B. Inhalation Report .........................................................................................................62 

C. Individual Animal Termination History ......................................................................85 

D. Individual Clinical Observations During Gestation ....................................................89 

E. Individual Gestation Body Weights (grams) .............................................................101 

F. Individual Gestation Body Weight Gain (grams) ......................................................105 

G. Individual Gestation Feed Consumption (grams/kg/day) ..........................................109 

H. Individual Maternal Necropsy Observations .............................................................113 

I. Individual Female Reproduction Data and Mean Fetal Weights ...............................121 

J. Individual Gravid Uterine Weight and Net Body Weight Change (grams) ..............125 

K. Individual Fetal Body Weights ..................................................................................129 

L. Individual Fetal External, Visceral and Skeletal Observations .................................133 

M. Statistical Analysis Report .........................................................................................146 

N. Certificates of Analysis – (Feed, Water and Bedding) ..............................................164 

O. Protocol and Protocol Amendments ..........................................................................178 

P. Historical Control Data ..............................................................................................214 

Q. Testing Facility Personnel .........................................................................................218 

R. Report Amendments ..................................................................................................219 
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1. INTRODUCTION 

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) 

100 Mettlers Road 

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

Keith P. Hazelden, BSc, CBiol, MIBiol, 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 (PAI) 

2.1.6. PRINCIPAL INVESTIGATOR – STATISTICS 

Gareth D. Thomas, B.Sc. (Hons). 

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 

finalization of the study report. 
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2.2.6. EXPERIMENTAL TERMINATION 

01 December 2005 (Date of last data collection) 

2.2.7. STUDY COMPLETION 

8 November 2012 (Date Study Director signed the Final Report) 

2.3. EXPERIMENTAL OUTLINE 

Group Group 

Designation 

Exposure 

Levels 

(mg/m3)a

Treatment

Gestation

Days 

Number of Animals 

Time-Mated

Females 

Euthanized

Gestation 

Day 21 

Fetal Evaluations 

(Fetuses/Litter) 

External Soft Tissue Skeletal

1 Control 0 (air only) 5-20 24 All All ~1/2 ~1/2 

2 Low 2000 5-20 24 All All ~1/2 ~1/2 

3 Mid 10000 5-20 24 All All ~1/2 ~1/2 

4 High 20000 5-20 24 All All ~1/2 ~1/2 

a

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 first day of gestation (GD 0 = day of detection of positive sign of mating) was defined 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 

80-100%, 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. LOT NUMBER 

API 01-06 

2.5.4. PURITY 

100% Gasoline DIPE Vapor Condensate 

2.5.5. DESCRIPTION 

Colorless liquid 

2.5.6. DATES RECEIVED 

7 December 2001 

05 August 2002 

2.5.7. EXPIRATION DATE 

Not available, stable per MSDS. 

2.5.8. 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 100-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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2.5.11. 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. 

2.6. 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 

2.6.6. WEIGHT AT INITIATION OF EXPOSURES (GRAMS) 

Mean Range

Females: 244 216-280 

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 corn-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 

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, PA). 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 

Corn-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: 18 to 26°C 

Actual Range: 23 to 24°C 

Daily Average Range: 23 to 24°C 
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Relative Humidity 

Relative humidity was monitored in accordance with Testing 

Facility SOPs and was maintained within the specified range: 

Desired Range: 30 to 70% 

Actual Range: 49 to 67% 

Daily Average Range: 55 to 60% 

Air Changes 

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 

on GD 21:

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 horn: 

• 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 Warkany, 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 10% 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 

and recorded. The eviscerated fetuses were placed in 70% 
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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 

(PAI), 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 

of body 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 

Number of males and females per litter 
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2.15.2. INCIDENCE DATA 

The following measures were analyzed: 

Maternal necropsy findings 

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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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, CFR 

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 (PAI, 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 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. 

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) 

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: 

Group 

Target 

Concentration

(mg/m3

) 

Analytical 

Concentration 

(mg/m3) 

Nominal 

Concentration 

(mg/m3) 

1 Air Control 0 0.00 ± 0.00 0 ± 0 

2 Gasoline DIPE 

Vapor Condensate 2000 1982 ± 93 1995 ± 40 

3 Gasoline DIPE 

Vapor Condensate 10000 10072 ± 546 11474 ± 513 

4 Gasoline DIPE 

Vapor Condensate 20000 19776 ± 961 19842 ± 602 

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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Mean particle size distribution measurements for the exposures are 

summarized as follows: 

Group 

Mass Median 

Aerodynamic 

Diameter 

(μm) 

Geometric 

Standard 

Deviation 

Total 

Mass 

Concentration 

(mg/m3

) 

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

2 Gasoline DIPE 

Vapor Condensate 

4.061 ± 3.4 2.435 ± 0.5 5.15E-03 

3 Gasoline DIPE 

Vapor Condensate 

3.762 ± 2.6 2.677 ± 0.6 6.12E-03 

4 Gasoline DIPE 

Vapor Condensate 3.118 ± 3.3 2.223 ± 0.1 6.29E-03 

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 92-100% across all the groups. 
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3.3.2. CLINICAL OBSERVATIONS 

(Table 2; Appendices B and D) 

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 10000 and 20000 mg/m3 exposed 

animals, as compared with the other groups. Chromodacryorrhea 

was also observed at a low incidence at 20000 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 20000 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 minor, non-statistically 

significant reductions in maternal body weight (97.6%, 96.9% and 

96.2% of the Control group value on GD 21 for the 2000, 10000 

and 20000 mg/m3 groups, respectively). 

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. 
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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 20000 mg/m3. This finding was 

considered incidental, and not related to treatment. 

3.3.6. UTERINE IMPLANTATION DATA 

(Table 7; Appendices I and M) 

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) 

The maternal net body weight change from Day 5 minus the gravid 

uterine weight (“net weight change from Day 5” in Table 8 and 

Appendix J) was significantly reduced (95% of the control group 

value in the 20000 mg/m3 group); however, this reduction was not 

considered adverse because the reduction was minimal and within 

normal animal variation. 

Uterine weight was reduced (96% of the control group value) but 

this reduction was not considered adverse because the differences 

were slight, within normal animal variation and there were no 

statistically significant differences between the treated group and 

control group. 

The percent reduction in uterine weight and in maternal net body 

weight change from Day 5 minus the gravid uterine weight in the 

exposed groups was comparable with the minor reduction in 

maternal body weights and body weight changes observed in these 

groups. 
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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 20000 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 20000 mg/m3 than in the other groups, the difference 

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. 
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4. CONCLUSIONS 

Exposure of pregnant rats to atmospheres of 0 (Air Control), 2000, 10000 or 

20000 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 

2000 mg/m3 and most noticeable at 20000 mg/m3. It was considered that a noobserved-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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REFERENCES 

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.
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CALCULATIONS 

Feed Consumption (g/kg/day): 

[(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 of live 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 05-4287 Page 31 

 211-DIPE-DEV Final Report 

 

General Preface 

General Notes

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. 
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 211-DIPE-DEV 

Mean Gestation Body Weights (grams) F

200220240260280300320340360380400420440

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 

Body Weights (grams)

Gestation Day

Group 1 - 0 mg/m3Group 2 - 2000 mg/m3Group 3 - 10000 mg/m3Group 4 - 20000 mg/m3
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 211-DIPE-DEV 

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

4449545964697479848994

4 to 5 5 to 8 8 to 11 11 to 14 14 to 17 17 to 20 20 to 2

g/kg/day

Gestation Day Interval

Group 1 - 0 mg/m3Group 2 - 2000 mg/m3Group 3 - 10000 mg/m3Group 4 - 20000 mg/m3
10-MAR-2011 17:37 

Huntingdon Life Sciences 05-4287  TABLE 1  GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY  IN RATS BY INHALATION EXPOSURE  SUMMARY OF SURVIVAL AND PREGNANCY ------------------------------------------------------------------------------------------------------------------ DOSE GROUP: 1 2 3 4  DOSE LEVEL (MG/M3): 0 2000 10000 200------------------------------------------------------------------------------------------------------------------No. of females mated N 24 24 24 Pregnant N 23 23 24  - Died/sacrificed moribund N 0 0 0  - Elective sacrifice N 0 0 0 Nonpregnant N 1 1 0  - Died/sacrificed moribund N 0 0 0  - Elective sacrifice N 0 0 0 Total no. of females died/ N 0 0 0  sacrificed moribund % 0.0 0.0 0.0 Examined at scheduled c-section N 24 24 24  - Nonpregnant N 1 1 0  - With total implant loss N 0 0 0  % 0.0 0.0 0.0  - With viable fetuses N 23 23 24  % 95.8 95.8 100.0 9------------------------------------------------------------------------------------------------------------------
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: 1 2 3 4  DOSE LEVEL (MG/M3): 0 2000 10000 200------------------------------------------------------------------------------------------------------------------ DAY 4 to 21  Normal ---------- WITHIN NORMAL LIMITS 431/24 421/24 382/24 3TERMINAL SACRIFICE 24/24 24/24 24/24  Dermal-General ------------------ ALOPECIA - GENERAL 0/ 0 0/ 0 0/ 0  Ocular ---------- CHROMODACRYORRHEA - UNILATERAL 0/ 0 0/ 0 0/ 0  Oral/Buccal --------------- INCISORS MALOCCLUDED 0/ 0 0/ 0 0/ 0 NASAL DISCHARGE - RED 1/ 1 11/ 3 50/15  Palpable masses ------------------- MASS 0/ 0 0/ 0 0/ 0 
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 4  DOSE LEVEL (MG/M3): 0 2000 10000 200------------------------------------------------------------------------------------------------------------------ DAY 4 MEAN 234 235 234  S.D. 14.0 16.6 15.7 1 N 23 23 24  DAY 5 MEAN 244 245 244  S.D. 14.6 16.5 13.9 1 N 23 23 24  DAY 8 MEAN 259 260 256  S.D. 12.8 18.0 14.4 1 N 23 23 24  DAY 11 MEAN 281 279 274  S.D. 14.9 19.6 15.6 1 N 23 23 24  DAY 14 MEAN 301 296 292  S.D. 17.0 20.3 14.8 1 N 23 23 24  DAY 17 MEAN 332 327 323  S.D. 22.2 24.3 18.7 2 N 23 23 24  DAY 20 MEAN 378 370 367  S.D. 26.3 29.9 24.2 2 N 23 23 24  DAY 21 MEAN 388 379 376  S.D. 28.6 32.7 25.9 2 N 23 23 24 ------------------------------------------------------------------------------------------------------------------
10-MAR-2011 17:44 

Huntingdon Life Sciences 05-4287  TABLE 4  GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY  IN RATS BY INHALATION EXPOSURE  SUMMARY OF GESTATION BODY WEIGHT GAIN (GRAMS) ------------------------------------------------------------------------------------------------------------------ DOSE GROUP: 1 2 3 4  DOSE LEVEL (MG/M3): 0 2000 10000 200------------------------------------------------------------------------------------------------------------------ DAYS 4 TO 5 MEAN 10 11 10  S.D. 6.1 4.7 5.2  N 23 23 24  DAYS 5 TO 8 MEAN 15 15 13  S.D. 4.5 4.7 5.5  N 23 23 24  DAYS 8 TO 11 MEAN 22 19 18  S.D. 4.7 3.6 3.9  N 23 23 24  DAYS 11 TO 14 MEAN 20 17 17  S.D. 4.2 5.0 3.7  N 23 23 24  DAYS 14 TO 17 MEAN 31 31 31  S.D. 7.3 6.9 7.6  N 23 23 24  DAYS 17 TO 20 MEAN 46 44 44  S.D. 7.6 8.9 9.9  N 23 23 24  DAYS 20 TO 21 MEAN 10 9 9  S.D. 5.2 5.0 4.9  N 23 23 24  DAYS 5 TO 14 MEAN 57 50 48  S.D. 8.4 7.8 7.7  N 23 23 24  DAYS 14 TO 21 MEAN 86 84 84  S.D. 15.5 16.3 17.0 1 N 23 23 24  DAYS 5 TO 21 MEAN 144 134 132  S.D. 20.6 21.5 21.0 1 N 23 23 24 ------------------------------------------------------------------------------------------------------------------
10-MAR-2011 17:45 

Huntingdon Life Sciences 05-4287  TABLE 5  GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY  IN RATS BY INHALATION EXPOSURE  SUMMARY OF GESTATION FEED CONSUMPTION (GRAMS/KG/DAY) ------------------------------------------------------------------------------------------------------------------ DOSE GROUP: 1 2 3 4  DOSE LEVEL (MG/M3): 0 2000 10000 200------------------------------------------------------------------------------------------------------------------ DAYS 4 TO 5 MEAN 71 74 75  S.D. 10.5 11.5 11.4 1 N 22 23 24  DAYS 5 TO 8 MEAN 81 85 80  S.D. 6.0 14.1 6.0  N 22 23 24  DAYS 8 TO 11 MEAN 85 82 80  S.D. 7.1 4.6 5.5  N 22 23 24  DAYS 11 TO 14 MEAN 85 82 82  S.D. 7.1 4.7 3.9  N 22 23 22  DAYS 14 TO 17 MEAN 81 81 81  S.D. 4.5 4.5 6.0  N 22 23 22  DAYS 17 TO 20 MEAN 74 73 74  S.D. 4.5 3.3 3.7  N 23 23 22  DAYS 20 TO 21 MEAN 53 47 47  S.D. 8.1 7.5 5.8  N 23 23 24  DAYS 5 TO 21 MEAN 76 75 74  S.D. 4.7 4.0 3.6  N 23 23 24 ------------------------------------------------------------------------------------------------------------------
10-MAR-2011 17:46 

Huntingdon Life Sciences 05-4287  TABLE 6  GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY  IN RATS BY INHALATION EXPOSURE  SUMMARY OF MATERNAL NECROPSY OBSERVATIONS ------------------------------------------------------------------------------------------------------------------ DOSE GROUP: 1 2 3 4  DOSE LEVEL (MG/M3): 0 2000 10000 200------------------------------------------------------------------------------------------------------------------FEMALES N 24 24 24 GROSS EXAM N 0 0 0 ---------- 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 4  DOSE LEVEL (MG/M3): 0 2000 10000 200------------------------------------------------------------------------------------------------------------------Pregnant N 23 23 24  (at scheduled sacrifice)  Dams with no Viable Fetuses N 0 0 0  Dams with Viable Fetuses N 23 23 24 Corpora Lutea TOTAL 343 334 354  No. per animal MEAN 14.9 14.5 14.8 1 S.D. 2.78 2.06 2.52 2Implantation Sites TOTAL 311 310 315  No. per animal MEAN 13.5 13.5 13.1 1 S.D. 2.71 1.50 2.85 2Preimplantation Loss TOTAL 32 24 39  No. per animal MEAN 1.4 1.0 1.6  S.D. 1.88 1.46 2.96 1 % per animal MEAN% 9.3 6.6 10.4  S.D. 13.46 7.99 18.58 9Live Fetuses TOTAL 300 286 302  No. per animal MEAN 13.0 12.4 12.6 1 S.D. 2.64 2.64 3.06 2 Males TOTAL 146 133 154  MEAN% 48.5 46.1 51.2 4 S.D. 10.94 14.75 12.50 13 Females TOTAL 154 153 148  MEAN% 51.5 53.9 48.8 5 S.D. 10.94 14.75 12.50 13------------------------------------------------------------------------------------------------------------------
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 4  DOSE LEVEL (MG/M3): 0 2000 10000 200------------------------------------------------------------------------------------------------------------------Postimplantation Loss TOTAL 11 24 13  No. per animal MEAN 0.5 1.0 0.5  S.D. 0.67 2.08 0.83 0 % implants per animal MEAN% 3.4 7.9 5.1  S.D. 4.66 14.89 8.64 5Dead Fetuses TOTAL 0 0 0  No. per animal MEAN 0.0 0.0 0.0  S.D. 0.00 0.00 0.00 0 % of implants per animal MEAN% 0.0 0.0 0.0  S.D. 0.00 0.00 0.00 0Resorptions: Early TOTAL 10 23 13  No. per animal MEAN 0.4 1.0 0.5  S.D. 0.66 2.07 0.83 0 % of implants per animal MEAN% 3.1 7.6 5.1  S.D. 4.66 14.80 8.64 5Resorptions: Late TOTAL 1 1 0  No. per animal MEAN 0.0 0.0 0.0  S.D. 0.21 0.21 0.00 0 % of implants per animal MEAN% 0.3 0.3 0.0  S.D. 1.39 1.60 0.00 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 4  DOSE LEVEL (MG/M3): 0 2000 10000 200------------------------------------------------------------------------------------------------------------------Fetal Body Weight (g) MEAN 5.7 5.8 5.7  S.D. 0.29 0.30 0.28 0 N 23 23 24  Male Fetuses MEAN 5.9 5.9 5.8  S.D. 0.32 0.33 0.32 0 Female Fetuses MEAN 5.6 5.6 5.5  S.D. 0.28 0.33 0.25 0------------------------------------------------------------------------------------------------------------------
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 4  DOSE LEVEL (MG/M3): 0 2000 10000 200------------------------------------------------------------------------------------------------------------------UTERUS WEIGHT MEAN 101 97 96  S.D. 19.4 19.8 22.5 1 N 22 23 24 ADJUSTED WEIGHT MEAN 286 283 280  S.D. 18.1 19.0 17.1 1 N 22 23 24 NET WEIGHT CHANGE FROM DAY 5 MEAN 43 37 36  S.D. 11.1 9.1 11.7 1 N 22 23 24 WEIGHT CHANGE FROM DAY 5 MEAN 144 134 132  S.D. 20.6 21.5 21.0 1 N 23 23 24 ------------------------------------------------------------------------------------------------------------------UTERUS WEIGHT = ABSOLUTE GRAVID UTERINE WEIGHT ADJUSTED WEIGHT = TERMINAL BODY WEIGHT - UTERUS WEIGHT NET WEIGHT CHANGE FROM DAY 5 = ADJUSTED WEIGHT – DAY 5 BODY WEIGHT WEIGHT CHANGE FROM DAY 5 = TERMINAL BODY WEIGHT – DAY 5 BODY WEIGHT 
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Females Summary of Fetal Observations 

 Fetuses Litters 

Observations Group 1 2 3 4 1 2 (Major abnormalities in CAPITALS) Number examined 300 286 302 292 23 23 HYDROCEPHALYa, ANOPHTHALMIA; HYPOPLASTIC APICAL LUNG 

LOBE, THYMUS AND SPLEEN 

1 

MICROPHTHALMIA/ANOPHTHALMIA, UNILATERAL 1 EXOCCIPITAL FUSED TO 1ST CERVICAL ARCH 1 MANDIBLE SHORTENED, MODERATE 1 1THORACIC VERTEBRA 13, LUMBAR VERTEBRA 1: ACENTRIC, MALFORMED 

1 

FORELIMB SHORTENED, RIGHT 1 1ANAL ATRESIA, ABSENT SACRAL AND CAUDAL VERTEBRAE 1 1Additional subclavian artery 1 1Innominate artery absent 1 2 1 1 Undescended thymic tissue 10 11 5 11 6 8 

aNoted as DOMED CRANIUM in external observations. 
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Females Summary of Fetal Observations 

 Fetuses Litters 

Observations Group 1 2 3 4 1 2 (Major abnormalities in CAPITALS) Number examined 300 286 302 292 23 23 Renal papilla (e) not fully developed 1 2 1 1 Ureter(s) distended 1 1 1 1 

Left umbilical artery 1412 13Testis displaced, medially, left 1 1

7th cervical rib(s) present 1 3 1 

Sternebra(e) malaligned 11 11 13 3 9 9 Rib(s) rudimentary, 13th 1 3 1 Rib(s) rudimentary, 14th 2 7 5 12 1 5 25 presacral vertebrae 1 3 1 27 presacral vertebrae 1 
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Females Summary of Fetal Observations 

 Fetuses Litters 

Observations Group 1 2 3 4 1 2 (Major abnormalities in CAPITALS) Number examined 300 286 302 292 23 23 Hyoid body/arch(es) unossified 1 1 1 1 

Stern

e

bra 5 a

n

d/or 6 u

n

o

s

sifie

d 1 33 1 

Metatarsals: 1 unossified, bilateral 

2 















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Inhalation Report Appendix B 

TABLE OF CONTENTS 

1. INTRODUCTION ................................................................................................63 

2. MATERIALS AND METHODS 

2.1. Husbandry During Exposure Periods ........................................................63 

2.2. Test Substance Administration and Chamber Operations .........................64 

2.3. Exposure Concentration Determination .....................................................67 

3. FIGURES 

1. Chamber Generation System and 

Whole-Body Exposure Chamber (Group 1) .....................................70 

2. Chamber Generation System and 

Whole-Body Exposure Chambers (Groups 2 – 4) ............................71 

4. TABLES 

I. Summary of In-Chamber Observations .....................................................72 

II. Chamber Monitoring Results .....................................................................73 

III. Equipment List ...........................................................................................77 

IV. Chamber Distribution Records ..................................................................80 

V. Miran Calibration .......................................................................................81 

VI. Testing Room and Chambers Environmental Monitoring .........................84 
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Inhalation Report Appendix B 

1. INTRODUCTION 

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. ENVIRONMENTAL CONDITIONS 

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: 20 to 24°C 

Actual: 20 to 25°C 
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Inhalation Report Appendix B 

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: 40 to 60% 

Actual: 36 to 54% 

The daily average relative humidity within the chamber was 

outside the desired range on 2/19 days and 1/19 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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Inhalation Report Appendix B 

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 1000 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 for each 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 HEPA 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 Appendix B 

2.2.6. EXPOSURE PROCEDURE 

Group 1 

Houseline nitrogen was delivered from a regulator with a 

backpressure gauge via 1⁄4” tubing to a flowmeter regulated by a 

metering valve. This nitrogen flow was then directed into the 

turret of the 1000 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 gauge 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 1⁄4” 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

/8” 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 1⁄4” 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 
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Inhalation Report Appendix B 

area surrounding the nichrome wire within the tube, thereby 

protecting the wire from oxidation. 

The nitrogen for the volatilization system was directed via 1⁄4” 

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

flowmeter, the volatilization nitrogen flowed via 1⁄4” 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 1⁄2” 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 

Conc (mg/m3

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

 exposure duration (min) x airflow (Lpm) 
Huntingdon Life Sciences 05-4287 Page 68 

 211-DIPE-DEV Final Report 

Inhalation Report Appendix B 

2.3.2. CHAMBER SAMPLING 

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). 
Huntingdon Life Sciences 05-4287 Page 69 

 211-DIPE-DEV Final Report 

Inhalation Report Appendix B 

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 05-4287 Page 70 

 211-DIPE-DEV Final Report 

Group 1 Inhalation Report Appendix B 

Figure I 

Chamber Generation System 

And Whole Body Exposure Chamber 

Note: 

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

Groups 2 - 4 Inhalation Report Appendix B 

Figure II 

Chamber Generation System 

And Whole Body Exposure Chamber 

Notes: 

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

Inhalation Report Ap

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/m3Within Normal Limits 6 12 18 24 24 24 24 24 24 24 24 24 24 24 24 24 18 12 
Huntingdon Life Sciences 05-4287

211-DIPE-DEV

Appendix B - Table II

Chamber Monitoring Results

Cumulative Exposure Record

Group 1 - 0 mg/m3 (Air only)

 Chamber Env

Day Date Exposure Chamber Concentration Determinations Temperature 

Number Nominal Mean Individual MMAD GSD TMC

(mg/m3) (mg/m3) (μm) (mg/m3) (°C) 

0 6-Aug-05 1 0 0.00 0.00 0.00 0.00 0.00 23 1 7-Aug-05 2 0 0.00 0.00 0.00 0.00 0.00 23 2 8-Aug-05 3 0 0.00 0.00 0.00 0.00 0.00 24 3 9-Aug-05 4 0 0.00 0.00 0.00 0.00 0.00 0.8011 2.031 4.25E-03 24 4 10-Aug-05 5 0 0.00 0.00 0.00 0.00 0.00 24 5 11-Aug-05 6 0 0.00 0.00 0.00 0.00 0.00 24 6 12-Aug-05 7 0 0.00 0.00 0.00 0.00 0.00 25 7 13-Aug-05 8 0 0.00 0.00 0.00 0.00 0.00 25 8 14-Aug-05 9 0 0.00 0.00 0.00 0.00 0.00 24 9 15-Aug-05 10 0 0.00 0.00 0.00 0.00 0.00 25 

10 16-Aug-05 11 0 0.00 0.00 0.00 0.00 0.00 0.7675 1.792 4.78E-03 24 11 17-Aug-05 12 0 0.00 0.00 0.00 0.00 0.00 25 12 18-Aug-05 13 0 0.00 0.00 0.00 0.00 0.00 25 13 19-Aug-05 14 0 0.00 0.00 0.00 0.00 0.00 25 14 20-Aug-05 15 0 0.00 0.00 0.00 0.00 0.00 24 15 21-Aug-05 16 0 0.00 0.00 0.00 0.00 0.00 25 16 22-Aug-05 17 0 0.00 0.00 0.00 0.00 0.00 24 17 23-Aug-05 18 0 0.00 0.00 0.00 0.00 0.00 8.2210 2.276 1.64E-03 24 18 24-Aug-05 19 0 0.00 0.00 0.00 0.00 0.00 23 

Mean 0 0.00 3.263 2.033 3.56E-03 24 

S.D. 0 0.00 4.3 0.2 0.0 0.7 

Particle Size Mean

(mg/m3)
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211-DIPE-DEV

Appendix B - Table II

Chamber Monitoring Results

Cumulative Exposure Record

Group 2 - 2000 mg/m3

 Chamber En

Day Date Exposure Chamber Concentration Temperature 

Number Nominal Mean Individual MMAD GSD TMC

(mg/m3) (mg/m3) (μm) (mg/m3) (°C) 

0 6-Aug-05 1 2000 1900 1800 1800 1900 1900 23 1 7-Aug-05 2 2000 1900 2000 1900 1800 1900 23 2 8-Aug-05 3 2000 2000 2200 1800 2000 2000 23 3 9-Aug-05 4 2100 2100 2200 2000 2000 2000 3.882 2.954 7.23E-03 24 4 10-Aug-05 5 2000 2000 2100 2000 1900 2100 24 5 11-Aug-05 6 2000 2000 2000 2200 2100 1800 24 6 12-Aug-05 7 2000 2000 2100 2000 2000 2000 24 7 13-Aug-05 8 2000 2000 2000 1900 2000 2000 24 8 14-Aug-05 9 2000 2000 1900 2000 2000 2000 24 9 15-Aug-05 10 2000 2000 2100 2000 2000 2000 24 

10 16-Aug-05 11 1900 2000 2000 2000 2000 1900 0.7935 2.294 6.14E-03 24 11 17-Aug-05 12 2000 2000 2000 1900 2100 2100 24 12 18-Aug-05 13 2000 2000 1900 1900 2000 2000 24 13 19-Aug-05 14 2000 2000 1900 2100 2000 2000 24 14 20-Aug-05 15 1900 2000 2000 1800 2000 2000 24 15 21-Aug-05 16 2000 2000 2000 1900 2100 2000 24 16 22-Aug-05 17 2000 1900 1900 1900 1900 2000 24 17 23-Aug-05 18 2000 2000 2000 1900 2000 2000 7.508 2.057 2.08E-03 23 18 24-Aug-05 19 2000 2000 2100 1800 2000 2100 23 

Mean 1995 1982 4.061 2.435 5.15E-03 24 

S.D. 40 93 3.4 0.5 0.0 0.5 

Mea

(mg/m3)

Particle Size

Determinations
Huntingdon Life Sciences 05-4287

211-DIPE-DEV

Appendix B - Table II

Chamber Monitoring Results

Cumulative Exposure Record

Group 3 - 10000 mg/m3

 Chamber 

Day Date Exposure Chamber Concentration Temperatur

Number Nominal Mean Individual MMAD GSD TMC

(mg/m3) (mg/m3) (μm) (mg/m3) (°C) 

0 6-Aug-05 1 11000 10000 10000 9600 10000 11000 23 1 7-Aug-05 2 11000 9800 9400 9700 10000 9900 24 2 8-Aug-05 3 11000 9900 10000 10000 9800 9900 24 3 9-Aug-05 4 11000 9600 9300 9500 9700 9900 0.8112 2.450 5.75E-03 24 4 10-Aug-05 5 12000 10000 11000 10000 10000 10000 25 5 11-Aug-05 6 12000 10000 9700 9900 9400 11000 25 6 12-Aug-05 7 11000 9700 9900 9600 9400 9700 25 7 13-Aug-05 8 12000 10000 10000 10000 10000 10000 25 8 14-Aug-05 9 12000 9800 9700 10000 10000 9400 25 9 15-Aug-05 10 12000 9800 9600 9900 9700 10000 25 

10 16-Aug-05 11 12000 10000 9900 9800 11000 11000 4.952 3.334 1.07E-02 25 11 17-Aug-05 12 12000 10000 10000 10000 10000 10000 25 12 18-Aug-05 13 11000 10000 10000 10000 10000 10000 25 13 19-Aug-05 14 11000 9800 9600 9700 9900 9900 25 14 20-Aug-05 15 12000 10000 11000 9700 9700 10000 25 15 21-Aug-05 16 12000 11000 11000 11000 11000 10000 25 16 22-Aug-05 17 11000 10000 11000 10000 10000 10000 24 17 23-Aug-05 18 11000 10000 11000 9100 9600 11000 5.523 2.246 1.91E-03 24 18 24-Aug-05 19 11000 11000 11000 12000 10000 11000 23 

Mean 11474 10072 3.762 2.677 6.12E-03 25 

S.D. 513 546 2.6 0.6 0.0 0.7 

M

(mg/m3)

Particle Size

Determinations
Huntingdon Life Sciences 05-4287

211-DIPE-DEV

Appendix B - Table II

Chamber Monitoring Results

Cumulative Exposure Record

Group 4 - 20000 mg/m3

 Chamber 

Day Date Exposure Chamber Concentration Temperatur

Number Nominal Mean Individual MMAD GSD TMC

(mg/m3) (mg/m3) (μm) (mg/m3) (°C) 

0 6-Aug-05 1 20000 19000 18000 20000 20000 19000 23 1 7-Aug-05 2 21000 21000 19000 20000 22000 21000 23 2 8-Aug-05 3 19000 20000 21000 20000 19000 19000 24 3 9-Aug-05 4 21000 20000 21000 20000 20000 20000 0.7811 2.137 4.84E-03 24 4 10-Aug-05 5 20000 20000 18000 18000 22000 20000 24 5 11-Aug-05 6 20000 20000 18000 20000 21000 20000 24 6 12-Aug-05 7 20000 20000 19000 20000 20000 20000 24 7 13-Aug-05 8 20000 20000 19000 19000 19000 21000 24 8 14-Aug-05 9 19000 19000 21000 20000 18000 18000 24 9 15-Aug-05 10 20000 20000 20000 20000 19000 19000 24 

10 16-Aug-05 11 19000 20000 20000 20000 19000 19000 0.7640 2.260 9.80E-03 24 11 17-Aug-05 12 20000 21000 21000 21000 21000 20000 24 12 18-Aug-05 13 20000 20000 19000 20000 20000 20000 24 13 19-Aug-05 14 20000 20000 20000 21000 18000 21000 24 14 20-Aug-05 15 19000 20000 19000 20000 20000 20000 24 15 21-Aug-05 16 19000 20000 20000 19000 20000 19000 24 16 22-Aug-05 17 20000 20000 21000 20000 20000 20000 24 17 23-Aug-05 18 20000 20000 20000 20000 20000 20000 7.810 2.272 4.24E-03 23 18 24-Aug-05 19 20000 19000 17000 20000 20000 20000 23 

Mean 19842 19776 3.118 2.223 6.29E-03 24 

S.D. 602 961 3.3 0.1 0.0 0.4 

M

(mg/m3)

Particle Size

Determinations
Huntingdon Life Sciences 05-4287 Page 77 

 211-DIPE-DEV Final Report 

Inhalation Report Appendix B 

Table III 

Equipment List 

Exposure Chamber 

1000 Liter glass and stainless steel chamber (Wahmann). 

Compound Reservoir 

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, P/N D800286, S/N 569-S, 

flow cell assembly P/N D80026B, BD #1860). 

Flowmeter, size 0 – 65 mm, P/N G6-02-R3, G6-03-R3, G6-04-R3 (Key Instruments). 

Pressure/Vacuum Gauges 

Union Carbide backpressure gauge, P/N SG 6383. 

Norgreen backpressure gauge, P/N 9892K23. 

U.S. Gauge backpressure gauge, P/N 12672-1. 

Ashcroft backpressure gauge, P/N 733-47. 

Matheson® backpressure gauge, P/N 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, Model 8586799, (Singer). 

Chamber Static Pressure 

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

Dwyer® Mark II Manometer, Model 25 (Dwyer® Instruments, Inc.). 
Huntingdon Life Sciences 05-4287 Page 78 

 211-DIPE-DEV Final Report 

Inhalation Report Appendix B 

Table III 

Equipment List 

Regulators 

Norgreen, P/N 9892K23 

Union Carbide, P/N SG 3800 30. 

Metering Valve 

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

Timers 

Gralab Universal Timer, Model 171. 

Absorbent Tube Sampling 

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

Vacuum Pumps 

Thomas Industries Inc., Model 707CM50. 

Neptune Dyna-pump®, Model 4K. 

Tubing 

Clear plastic, sizes 3/16”, 1⁄4”, 1⁄2” (Norton, Baxter). 

1⁄2” stainless steel. 

Stainless steel cross(Swage). 

Teflon®, size 1/8”, 1⁄4”, 1⁄2”. 

Plastic “T”. 

Air Analyzer 

MIRAN® 1A-CVF Ambient Air Analyzer (Wilks) with a Cole Parmer Strip Chart 

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

Model No. 22-195 

Balston® MicrofibreTM Disposable Filter Unit was attached in-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 05-4287 Page 79 

 211-DIPE-DEV Final Report 

Inhalation Report Appendix B 

Table III 

Equipment List 

Environmental Monitoring 

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

Hygrometer/Thermometer. 

Digital Sound Meter 840029 (SPER Scientific) 

Quantum Instruments Photo Meter 1. 

Oxygen/Gas Analyzer, Model 1214S (Gastech) 

Thermometer 

T° Sentry Digital Alarm Module, Model 110. 

Balance 

Pelouze, No. 4040 

Mettler PM30000K (Mettler Instrument Corporation). 

Miscellaneous 

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

DFU® filter, Grade DQ (Balston). 
Huntingdon Life Sciences 05-4287 Page 80 

 211-DIPE-DEV Final Report 

Inhalation Report Appendix B 

Table IV 

Chamber Distribution Records 

Group 

(target) Date Port 

Analytical (IR) 

Concentration 

(mg/m3) Ratio to H-1

 

2 (2000 mg/m3) 02 August 2005 H-1 1900 1.00 

 H-2 2000 1.05 

 H-1 2100 1.00 

 H-7 2100 1.00 

 H-8 2000 0.95 

 

3 (10000 mg/m3) 02 August 2005 H-1 11000 1.00 

 H-2 9900 0.90 

 H-1 10000 1.00 

 H-7 10000 1.00 

 H-8 9900 0.99 

 

4 (20000 mg/m3) 02 August 2005 H-1 19000 1.00 

 H-2 19000 1.00 

 H-1 19000 1.00 

 H-7 19000 1.00 

 H-1 20000 1.00 

 H-8 20000 1.00 
Huntingdon Life Sciences 05-4287 Page 81 

 211-DIPE-DEV Final Report 

Inhalation Report Appendix B 

Table V 

Miran Calibration 

Methodology for Gasoline DIPE Vapor Condensate 

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 

 gain High 

 chart speed, cm/min 1 

 chart volts 1 

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

10 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 Concentration1 Operator 1 Operator 2 Average 

 (μL) (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 Conc. (mg/m3) = Injection volume (μL) x Density ( 0.68 mg/μL) x 1000 L/m3

 5.64 L (Volume of Miran cell) 
Huntingdon Life Sciences 05-4287 Page 82 

 211-DIPE-DEV Final Report 

Inhalation Report Appendix B 

Table V 

Miran Calibration 

y = -12213x4 + 16436x3 - 751.71x2 + 30139x

R2 = 0.9997

0

5000

10000

15000

20000

25000

30000

0.0000 0.1000 0.2000 0.3000 0.4000 0.5000 0.6000 0.7000 0.8000

C

o

n

c

e

n

t

r

a

t

i

o

n

(

m

g

/

m

3

)

Absorbance (Volts)
Huntingdon Life Sciences 05-4287 Page 83 

 211-DIPE-DEV Final Report 

Inhalation Report Appendix B 

Table V 

Miran Calibration 

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 

 (μL) (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. 
Huntingdon Life Sciences 05-4287 Page 84 

 211-DIPE-DEV Final Report 

Inhalation Report Appendix B 

Table VI 

Testing Room and Chambers Environmental Monitoring 

Interval Location 

Test 

Substance 

(mg/m3

) 

Light 

(Ft Candles)

Noise 

(dB) 

Oxygen 

(%) 

Particle 

Sizing 

(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 10-2

 Group 4 Chamber - - - 20 3.30 x 10-2

 

Day 3 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 a

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  APPENDIX C  GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY  IN RATS BY INHALATION EXPOSURE  INDIVIDUAL ANIMAL TERMINATION HISTORY FEMALES GROUP 1 0 MG/M3 ------------------------------------------------------------------------------------------------------------------ TYPE OF DATE OF GESTATION PREGNANCY  ANIMAL# DEATH DEATH DAY STATUS ------------------------------------------------------------------------------------------------------------------ 1901 TERMINAL SACRIFICE 22-AUG-05 21 P  1902 TERMINAL SACRIFICE 22-AUG-05 21 P  1903 TERMINAL SACRIFICE 22-AUG-05 21 P  1904 TERMINAL SACRIFICE 22-AUG-05 21 P  1905 TERMINAL SACRIFICE 22-AUG-05 21 P  1906 TERMINAL SACRIFICE 22-AUG-05 21 P  1907 TERMINAL SACRIFICE 23-AUG-05 21 P  1908 TERMINAL SACRIFICE 23-AUG-05 21 P  1909 TERMINAL SACRIFICE 23-AUG-05 21 P  1910 TERMINAL SACRIFICE 23-AUG-05 21 P  1911 TERMINAL SACRIFICE 23-AUG-05 21 P  1912 TERMINAL SACRIFICE 23-AUG-05 21 P  1913 TERMINAL SACRIFICE 24-AUG-05 21 P  1914 TERMINAL SACRIFICE 24-AUG-05 21 P  1915 TERMINAL SACRIFICE 24-AUG-05 21 NP  1916 TERMINAL SACRIFICE 24-AUG-05 21 P  1917 TERMINAL SACRIFICE 24-AUG-05 21 P  1918 TERMINAL SACRIFICE 24-AUG-05 21 P  1919 TERMINAL SACRIFICE 25-AUG-05 21 P  1920 TERMINAL SACRIFICE 25-AUG-05 21 P  1921 TERMINAL SACRIFICE 25-AUG-05 21 P  1922 TERMINAL SACRIFICE 25-AUG-05 21 P  1923 TERMINAL SACRIFICE 25-AUG-05 21 P  1924 TERMINAL SACRIFICE 25-AUG-05 21 P ------------------------------------------------------------------------------------------------------------------NP-NOT PREGNANT, P-PREGNANT 
10-MAR-2011 17:08 

Huntingdon Life Sciences 05-4287  APPENDIX C  GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY  IN RATS BY INHALATION EXPOSURE  INDIVIDUAL ANIMAL TERMINATION HISTORY FEMALES GROUP 2 2000 MG/M3 ------------------------------------------------------------------------------------------------------------------ TYPE OF DATE OF GESTATION PREGNANCY  ANIMAL# 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 SACRIFICE 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  APPENDIX C  GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY  IN RATS BY INHALATION EXPOSURE  INDIVIDUAL ANIMAL TERMINATION HISTORY FEMALES GROUP 3 10000 MG/M3 ------------------------------------------------------------------------------------------------------------------ TYPE OF DATE OF GESTATION PREGNANCY  ANIMAL# 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  APPENDIX C  GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY  IN RATS BY INHALATION EXPOSURE  INDIVIDUAL ANIMAL TERMINATION HISTORY FEMALES GROUP 4 20000 MG/M3 ------------------------------------------------------------------------------------------------------------------ TYPE OF DATE OF GESTATION PREGNANCY  ANIMAL# 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 SACRIFICE 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 SACRIFICE 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  4921 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  INDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATION GROUP 1 0 MG/M3 ------------------------------------------------------------------------------------------------------------------ DAY OF 1 1 1 1 1 1 1 1 1 1 2 2  FEMALE# OBSERVATIONS GESTATION 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 ------------------------------------------------------------------------------------------------------------------ 1901 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P  1902 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P  1903 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P  1904 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P  1905 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P  1906 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P  1907 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P  1908 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P  1909 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P  1910 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P  1911 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS 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  INDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATION GROUP 1 0 MG/M3 ------------------------------------------------------------------------------------------------------------------ DAY OF 1 1 1 1 1 1 1 1 1 1 2 2  FEMALE# OBSERVATIONS GESTATION 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 ------------------------------------------------------------------------------------------------------------------ 1912 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P  1913 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P  1914 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P  1915 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P  1916 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P  1917 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P  1918 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P  1919 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P P  1920 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P P  1921 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P P  1922 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS 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  INDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATION GROUP 1 0 MG/M3 ------------------------------------------------------------------------------------------------------------------ DAY OF 1 1 1 1 1 1 1 1 1 1 2 2  FEMALE# OBSERVATIONS GESTATION 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 ------------------------------------------------------------------------------------------------------------------ 1923 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P P  1924 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS 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  INDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATION GROUP 2 2000 MG/M3 ------------------------------------------------------------------------------------------------------------------ DAY OF 1 1 1 1 1 1 1 1 1 1 2 2  FEMALE# OBSERVATIONS GESTATION 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 ------------------------------------------------------------------------------------------------------------------ 2901 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P  2902 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P  2903 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P  2904 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P  2905 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P  2906 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P  2907 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P  2908 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P P P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P  2909 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P  2910 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS 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  INDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATION GROUP 2 2000 MG/M3 ------------------------------------------------------------------------------------------------------------------ DAY OF 1 1 1 1 1 1 1 1 1 1 2 2  FEMALE# OBSERVATIONS GESTATION 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 ------------------------------------------------------------------------------------------------------------------ 2911 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P  2912 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P  2913 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P  2914 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P  2915 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P  2916 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P  2917 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P  2918 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P P P P P P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P  2919 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P P  2920 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P P  2921 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS 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  INDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATION GROUP 2 2000 MG/M3 ------------------------------------------------------------------------------------------------------------------ DAY OF 1 1 1 1 1 1 1 1 1 1 2 2  FEMALE# OBSERVATIONS GESTATION 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 ------------------------------------------------------------------------------------------------------------------ 2922 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P P  2923 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P P  2924 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS 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  INDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATION GROUP 3 10000 MG/M3 ------------------------------------------------------------------------------------------------------------------ DAY OF 1 1 1 1 1 1 1 1 1 1 2 2  FEMALE# OBSERVATIONS GESTATION 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 ------------------------------------------------------------------------------------------------------------------ 3901 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P  3902 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P  3903 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P  3904 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P  3905 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P  3906 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P  3907 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P P P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P  3908 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P P P P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P  3909 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P  WITHIN NORMAL LIMITS 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  INDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATION GROUP 3 10000 MG/M3 ------------------------------------------------------------------------------------------------------------------ DAY OF 1 1 1 1 1 1 1 1 1 1 2 2  FEMALE# OBSERVATIONS GESTATION 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 ------------------------------------------------------------------------------------------------------------------ 3910 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P  3911 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P P P P P P P P P P P  WITHIN NORMAL LIMITS P P P P P P P P  3912 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P  3913 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P  3914 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P  3915 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P P P P P P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P  3916 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P P P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P  3917 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P  3918 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS 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  INDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATION GROUP 3 10000 MG/M3 ------------------------------------------------------------------------------------------------------------------ DAY OF 1 1 1 1 1 1 1 1 1 1 2 2  FEMALE# OBSERVATIONS GESTATION 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 ------------------------------------------------------------------------------------------------------------------ 3919 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P P  3920 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P  3921 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P P P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P  3922 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P  3923 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P P  3924 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P P P P P P P P  WITHIN NORMAL LIMITS 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  INDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATION GROUP 4 20000 MG/M3 ------------------------------------------------------------------------------------------------------------------ DAY OF 1 1 1 1 1 1 1 1 1 1 2 2  FEMALE# OBSERVATIONS GESTATION 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 ------------------------------------------------------------------------------------------------------------------ 4901 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P  4902 TERMINAL SACRIFICE P  ALOPECIA - GENERAL 2 2 2 2 2 2 2 2  CHROMODACRYORRHEA - UNILATERAL P  WITHIN NORMAL LIMITS P P P P P P P P P  4903 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P P P P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P  4904 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P  4905 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P  4906 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P  4907 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P  4908 TERMINAL SACRIFICE P  INCISORS MALOCCLUDED P P P P P P P P P P P P P P P P P P P  MASS 2 2 2 2 2  4909 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P  4910 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P P P  WITHIN NORMAL LIMITS 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  INDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATION GROUP 4 20000 MG/M3 ------------------------------------------------------------------------------------------------------------------ DAY OF 1 1 1 1 1 1 1 1 1 1 2 2  FEMALE# OBSERVATIONS GESTATION 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 ------------------------------------------------------------------------------------------------------------------ 4911 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P  4912 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P  4913 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P  4914 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P P P P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P  4915 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P  4916 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P P P P P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P  4917 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P P P P P P P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P  4918 TERMINAL SACRIFICE P  CHROMODACRYORRHEA - UNILATERAL P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P  4919 TERMINAL SACRIFICE P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P P  4920 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P P P P  WITHIN NORMAL LIMITS 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  INDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATION GROUP 4 20000 MG/M3 ------------------------------------------------------------------------------------------------------------------ DAY OF 1 1 1 1 1 1 1 1 1 1 2 2  FEMALE# OBSERVATIONS GESTATION 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 ------------------------------------------------------------------------------------------------------------------ 4921 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P  4922 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P P P P  WITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P  4923 TERMINAL SACRIFICE P  NASAL DISCHARGE - RED P P P P P P P P P P P  WITHIN NORMAL LIMITS P P P P P P P P P P  4924 TERMINAL SACRIFICE P  CHROMODACRYORRHEA - BILATERAL P  CHROMODACRYORRHEA - UNILATERAL P P P P P P P P P P P P P P  WITHIN NORMAL LIMITS P P P P P P ------------------------------------------------------------------------------------------------------------------CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENT 
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  ANIMAL# 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 222 229 237 254 270 298 335 347  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  4910 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 220 224 237 258 275 306 338 348  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  APPENDIX F  GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDY  IN RATS BY INHALATION EXPOSURE  INDIVIDUAL GESTATION BODY WEIGHT GAIN (GRAMS) GROUP 1 0 MG/M3 ------------------------------------------------------------------------------------------------------------------ DAY OF GESTATION  ANIMAL# 4 - 5 5 - 8 8 - 11 11 - 14 14 - 17 17 - 20 20 - 21 5 - 14 14 - 21 5 - 21 ------------------------------------------------------------------------------------------------------------------ 1901 18 29 16 28 29 48 9 73 86 159  1902 15 14 19 20 28 47 5 53 80 132  1903 -6 22 19 22 35 46 17 63 98 161  1904 19 12 15 24 29 59 10 51 98 149  1905 21 17 24 22 27 44 6 63 77 139  1906 14 15 18 22 24 51 14 55 88 143  1907 8 18 21 10 25 37 6 49 68 116  1908 19 17 32 23 31 46 12 72 89 161  1909 8 16 22 21 36 57 8 60 101 161  1910 7 15 15 14 28 50 11 44 88 132  1911 12 14 22 20 37 47 13 56 97 153  1912 11 10 21 17 33 51 1 49 85 133  1913 8 11 20 16 29 37 2 46 68 114  1914 4 9 25 18 28 50 10 51 88 139  1915x NP -2 14 1 -6 11 -1 1 10 11 21  1916 15 9 28 20 39 53 4 57 96 153  1917 5 15 29 19 41 48 10 62 99 161  1918 10 15 27 27 40 43 14 69 96 165  1919 9 21 24 20 18 45 17 65 80 145  1920 9 14 16 19 12 21 0 48 33 81  1921 6 17 22 20 29 49 9 59 87 146  1922 7 15 25 14 28 45 14 54 87 141  1923 5 17 28 25 42 46 21 69 109 179  1924 14 14 23 19 37 43 10 56 89 146 MEAN 10 15 22 20 31 46 10 57 86 144 S.D. 6.1 4.5 4.7 4.2 7.3 7.6 5.2 8.4 15.5 20.6  N 23 23 23 23 23 23 23 23 23 23  NP=NOT PREGNANT x=EXCLUDED FROM MEAN 
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  ANIMAL# 4 - 5 5 - 8 8 - 11 11 - 14 14 - 17 17 - 20 20 - 21 5 - 14 14 - 21 5 - 21 ------------------------------------------------------------------------------------------------------------------ 2901 12 11 13 17 23 37 4 41 64 105  2902 14 3 11 17 25 43 3 32 70 102  2903 12 10 22 13 22 48 5 44 74 119  2904 12 27 17 26 28 43 12 70 84 153  2905 17 16 17 22 33 54 11 55 98 153  2906 16 18 15 24 24 44 4 57 72 128  2907 19 13 21 18 34 46 13 52 93 145  2908 11 16 16 18 25 43 13 50 81 130  2909 7 17 18 17 26 41 11 51 78 129  2910 13 16 16 23 41 46 7 54 94 148  2911 12 16 23 14 32 51 8 53 90 143  2912 10 14 24 17 41 59 19 55 119 175  2913 5 17 22 19 31 46 3 57 81 138  2914 5 11 24 3 21 25 0 37 47 84  2915 12 14 18 18 36 46 10 50 92 143  2916 9 19 17 20 45 48 13 55 105 161  2917 8 19 15 11 28 32 15 45 76 120  2918 11 13 25 11 29 42 8 49 79 128  2919x NP 6 -1 11 7 2 -5 9 17 6 23  2920 8 8 17 16 27 38 12 41 77 118  2921 11 17 22 17 31 40 4 56 75 132  2922 -4 21 18 9 40 21 11 48 73 121  2923 15 16 19 20 41 55 18 55 114 168  2924 10 14 18 16 34 52 9 48 95 143 MEAN 11 15 19 17 31 44 9 50 84 134 S.D. 4.7 4.7 3.6 5.0 6.9 8.9 5.0 7.8 16.3 21.5  N 23 23 23 23 23 23 23 23 23 23  NP=NOT PREGNANT x=EXCLUDED FROM MEAN 
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 3 10000 MG/M3 ------------------------------------------------------------------------------------------------------------------ DAY OF GESTATION  ANIMAL# 4 - 5 5 - 8 8 - 11 11 - 14 14 - 17 17 - 20 20 - 21 5 - 14 14 - 21 5 - 21 ------------------------------------------------------------------------------------------------------------------ 3901 19 3 13 16 22 36 6 32 64 96  3902 24 11 20 21 19 43 7 52 69 121  3903 12 17 18 24 20 52 13 59 84 144  3904 13 17 22 14 28 52 -1 53 80 133  3905 11 8 12 18 37 57 8 38 102 140  3906 -1 21 13 16 43 48 6 49 97 146  3907 13 10 16 22 34 35 8 49 77 126  3908 14 10 19 18 21 39 6 47 66 113  3909 14 10 23 18 30 45 0 51 75 126  3910 5 11 17 13 31 49 10 41 90 131  3911 12 9 22 17 33 57 8 48 98 146  3912 10 6 18 16 30 45 8 40 84 124  3913 3 25 15 17 36 26 16 57 77 135  3914 10 16 24 14 28 42 15 54 85 139  3915 12 10 15 9 17 15 1 34 32 67  3916 10 13 22 17 39 51 10 51 100 151  3917 4 18 22 22 39 44 6 62 89 151  3918 5 18 14 10 32 41 16 42 89 131  3919 12 10 17 19 36 45 14 46 95 141  3920 9 15 21 18 32 52 9 54 93 147  3921 6 13 12 17 24 34 5 42 63 105  3922 6 14 19 22 44 55 15 54 114 169  3923 8 14 12 18 31 50 16 44 96 140  3924 14 2 23 23 38 45 10 47 93 140 MEAN 10 13 18 17 31 44 9 48 84 132 S.D. 5.2 5.5 3.9 3.7 7.6 9.9 4.9 7.7 17.0 21.0  N 24 24 24 24 24 24 24 24 24 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  INDIVIDUAL GESTATION BODY WEIGHT GAIN (GRAMS) GROUP 4 20000 MG/M3 ------------------------------------------------------------------------------------------------------------------ DAY OF GESTATION  ANIMAL# 4 - 5 5 - 8 8 - 11 11 - 14 14 - 17 17 - 20 20 - 21 5 - 14 14 - 21 5 - 21 ------------------------------------------------------------------------------------------------------------------ 4901 12 5 18 17 21 48 -2 40 68 108  4902 12 14 16 20 23 45 11 50 78 127  4903 16 4 20 11 28 63 7 35 98 132  4904 5 13 21 15 34 52 10 49 95 144  4905 15 8 15 18 34 51 4 42 90 131  4906 11 12 16 23 27 49 12 52 87 139  4907 5 12 19 19 25 44 4 50 73 123  4908x NP 5 4 8 -9 6 10 -5 3 11 14  4909 9 2 9 13 24 33 11 24 69 93  4910 7 22 22 15 27 45 13 59 85 144  4911 13 10 18 13 32 52 9 40 93 133  4912 10 14 20 15 26 47 7 49 80 129  4913 10 11 15 17 32 44 7 44 83 127  4914 10 8 23 13 30 42 12 43 83 126  4915 8 8 18 12 26 40 14 38 80 118  4916 8 7 18 17 31 48 16 41 94 135  4917 11 14 20 15 44 46 8 49 99 148  4918 6 11 24 8 42 32 13 43 86 129  4919x NP 6 -3 2 8 -2 -2 -3 7 -7 1  4920 5 13 21 16 31 32 10 50 73 124  4921 5 13 21 12 36 33 10 45 79 124  4922 12 15 15 18 34 46 -6 48 73 120  4923 11 3 25 5 28 37 6 33 71 104  4924 11 15 39 18 47 49 4 71 100 171 MEAN 10 11 20 15 31 44 8 45 83 129 S.D. 3.2 4.7 5.5 4.0 6.8 7.7 5.1 9.5 10.0 16.0  N 22 22 22 22 22 22 22 22 22 22  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 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  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 4 20000 MG/M3 ------------------------------------------------------------------------------------------------------------------ DAY OF GESTATION  ANIMAL# 4 - 5 5 - 8 8 - 11 11 - 14 14 - 17 17 - 20 20 - 21 5 - 21 ------------------------------------------------------------------------------------------------------------------ 4901 71 CF CF CF CF 77 47 62  4902 60 70 73 77 73 76 52 70  4903 83 78 75 72 71 71 43 68  4904 58 71 76 76 76 67 42 68  4905 83 80 82 81 79 72 39 72  4906 81 76 77 81 75 65 42 69  4907 84 81 84 84 83 79 50 77  4908x NP 63 67 66 60 66 69 46 62  4909 73 67 67 75 77 77 40 67  4910 69 92 82 79 78 73 57 77  4911 68 74 75 75 80 72 47 70  4912 71 81 77 76 74 72 45 71  4913 85 83 78 78 77 73 49 73  4914 68 76 75 79 84 77 45 73  4915 88 90 80 77 75 69 42 72  4916 74 83 81 80 83 80 46 76  4917 76 79 74 76 81 72 47 71  4918 68 75 72 71 83 66 59 71  4919x NP 74 68 70 72 66 66 54 66  4920 73 82 84 82 88 78 55 78  4921 72 82 72 72 81 70 54 72  4922 59 77 70 79 78 79 21 67  4923 92 77 80 77 81 71 45 72  4924 53 80 92 87 86 73 42 77 MEAN 73 79 78 78 79 73 46 72 S.D. 10.2 6.0 5.7 4.0 4.4 4.3 7.8 3.8  N 22 21 21 21 21 22 22 22  NP=NOT PREGNANT x=EXCLUDED FROM MEAN  CF=Contaminated Feeder 
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 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 ------------------------------------------------------------------------------------------------------------------ 1917 NO REMARKABLE OBSERVATIONS  1918 NO REMARKABLE OBSERVATIONS  1919 NO REMARKABLE OBSERVATIONS  1920 NO REMARKABLE OBSERVATIONS  1921 NO REMARKABLE OBSERVATIONS  1922 NO REMARKABLE OBSERVATIONS  1923 NO REMARKABLE OBSERVATIONS  1924 NO REMARKABLE OBSERVATIONS ------------------------------------------------------------------------------------------------------------------
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 2 2000 MG/M3 ------------------------------------------------------------------------------------------------------------------ ANIMAL# ORGAN OBSERVATION ------------------------------------------------------------------------------------------------------------------ 2901 NO REMARKABLE OBSERVATIONS  2902 NO REMARKABLE OBSERVATIONS  2903 NO REMARKABLE OBSERVATIONS  2904 NO REMARKABLE OBSERVATIONS  2905 NO REMARKABLE OBSERVATIONS  2906 NO REMARKABLE OBSERVATIONS  2907 NO REMARKABLE OBSERVATIONS  2908 NO REMARKABLE OBSERVATIONS  2909 NO REMARKABLE OBSERVATIONS  2910 NO REMARKABLE OBSERVATIONS  2911 NO REMARKABLE OBSERVATIONS  2912 NO REMARKABLE OBSERVATIONS  2913 NO REMARKABLE OBSERVATIONS  2914 NO REMARKABLE OBSERVATIONS  2915 NO REMARKABLE OBSERVATIONS  2916 NO REMARKABLE OBSERVATIONS ------------------------------------------------------------------------------------------------------------------
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 2 2000 MG/M3 ------------------------------------------------------------------------------------------------------------------ ANIMAL# ORGAN OBSERVATION ------------------------------------------------------------------------------------------------------------------ 2917 NO REMARKABLE OBSERVATIONS  2918 NO REMARKABLE OBSERVATIONS  2919 NP SALEWSKI'S TEST NO FOCI PRESENT  2920 NO REMARKABLE OBSERVATIONS  2921 NO REMARKABLE OBSERVATIONS  2922 NO REMARKABLE OBSERVATIONS  2923 NO REMARKABLE OBSERVATIONS  2924 NO REMARKABLE OBSERVATIONS ------------------------------------------------------------------------------------------------------------------ NP=NOT PREGNANT 
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 3 10000 MG/M3 ------------------------------------------------------------------------------------------------------------------ ANIMAL# ORGAN OBSERVATION ------------------------------------------------------------------------------------------------------------------ 3901 NO REMARKABLE OBSERVATIONS  3902 NO REMARKABLE OBSERVATIONS  3903 NO REMARKABLE OBSERVATIONS  3904 NO REMARKABLE OBSERVATIONS  3905 NO REMARKABLE OBSERVATIONS  3906 NO REMARKABLE OBSERVATIONS  3907 NO REMARKABLE OBSERVATIONS  3908 NO REMARKABLE OBSERVATIONS  3909 NO REMARKABLE OBSERVATIONS  3910 NO REMARKABLE OBSERVATIONS  3911 NO REMARKABLE OBSERVATIONS  3912 NO REMARKABLE OBSERVATIONS  3913 NO REMARKABLE OBSERVATIONS  3914 NO REMARKABLE OBSERVATIONS  3915 NO REMARKABLE OBSERVATIONS  3916 NO REMARKABLE OBSERVATIONS ------------------------------------------------------------------------------------------------------------------
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 3 10000 MG/M3 ------------------------------------------------------------------------------------------------------------------ ANIMAL# ORGAN OBSERVATION ------------------------------------------------------------------------------------------------------------------ 3917 NO REMARKABLE OBSERVATIONS  3918 NO REMARKABLE OBSERVATIONS  3919 NO REMARKABLE OBSERVATIONS  3920 NO REMARKABLE OBSERVATIONS  3921 NO REMARKABLE OBSERVATIONS  3922 NO REMARKABLE OBSERVATIONS  3923 NO REMARKABLE OBSERVATIONS  3924 NO REMARKABLE OBSERVATIONS ------------------------------------------------------------------------------------------------------------------
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 4 20000 MG/M3 ------------------------------------------------------------------------------------------------------------------ ANIMAL# ORGAN OBSERVATION ------------------------------------------------------------------------------------------------------------------ 4901 NO REMARKABLE OBSERVATIONS  4902 EXTERNAL EXAM ALOPECIA- GENERAL  EXTERNAL FINDING CONFIRMED  4903 NO REMARKABLE OBSERVATIONS  4904 NO REMARKABLE OBSERVATIONS  4905 NO REMARKABLE OBSERVATIONS  4906 NO REMARKABLE OBSERVATIONS  4907 NO REMARKABLE OBSERVATIONS  4908 NP GROSS EXAM 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 
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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 4 20000 MG/M3 ------------------------------------------------------------------------------------------------------------------ ANIMAL# ORGAN OBSERVATION ------------------------------------------------------------------------------------------------------------------ 4914 NO REMARKABLE OBSERVATIONS  4915 NO REMARKABLE OBSERVATIONS  4916 NO REMARKABLE OBSERVATIONS  4917 NO REMARKABLE OBSERVATIONS  4918 NO REMARKABLE OBSERVATIONS  4919 NP SALEWSKI'S TEST NO FOCI PRESENT  4920 NO REMARKABLE OBSERVATIONS  4921 NO REMARKABLE OBSERVATIONS  4922 NO REMARKABLE OBSERVATIONS  4923 NO REMARKABLE OBSERVATIONS  4924 NO REMARKABLE OBSERVATIONS ------------------------------------------------------------------------------------------------------------------ NP=NOT PREGNANT 
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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 FETUSES RESORPTIONS SEX AVERAGE  ANIMAL# LUTEA LOSS SITES LIVE DEAD EARLY LATE TOTAL MALE FEMALE MALES F (n) % (n) % ------------------------------------------------------------------------------------------------------------------ 1901 16 25.0 12 11 0 0.0 1 0 1 8.3 6 5 6.1  1902 16 25.0 12 12 0 0.0 0 0 0 0.0 8 4 6.1  1903 15 6.7 14 14 0 0.0 0 0 0 0.0 7 7 6.3  1904 22 31.8 15 15 0 0.0 0 0 0 0.0 8 7 5.7  1905 14 0.0 14 13 0 0.0 1 0 1 7.1 6 7 5.4  1906 15 6.7 14 13 0 0.0 1 0 1 7.1 6 7 5.6  1907 9 0.0 9 9 0 0.0 0 0 0 0.0 6 3 6.4  1908 14 7.1 13 12 0 0.0 1 0 1 7.7 7 5 5.9  1909 15 0.0 15 15 0 0.0 0 0 0 0.0 7 8 6.4  1910 14 7.1 13 13 0 0.0 0 0 0 0.0 6 7 6.0  1911 19 10.5 17 15 0 0.0 2 0 2 11.8 6 9 5.7  1912 15 0.0 15 15 0 0.0 0 0 0 0.0 5 10 5.4  1913 13 0.0 13 11 0 0.0 2 0 2 15.4 5 6 5.8  1914 12 0.0 12 12 0 0.0 0 0 0 0.0 5 7 6.1  1915 NP  1916 17 11.8 15 14 0 0.0 1 0 1 6.7 6 8 5.6  1917 18 5.6 17 17 0 0.0 0 0 0 0.0 7 10 5.7  1918 15 0.0 15 14 0 0.0 0 1 1 6.7 10 4 5.6  1919 15 6.7 14 14 0 0.0 0 0 0 0.0 5 9 6.1  1920 9 55.6 4 4 0 0.0 0 0 0 0.0 1 3 6.3  1921 15 0.0 15 15 0 0.0 0 0 0 0.0 8 7 5.4  1922 14 7.1 13 13 0 0.0 0 0 0 0.0 7 6 6.1  1923 16 6.3 15 15 0 0.0 0 0 0 0.0 8 7 6.1  1924 15 0.0 15 14 0 0.0 1 0 1 6.7 6 8 5.7 MEAN 14.9 9.3 13.5 13.0 0.0 0.0 0.4 0.0 0.5 3.4 6.3 6.7 5.9 S.D. 2.78 13.46 2.71 2.64 0.00 0.00 0.66 0.21 0.67 4.66 1.70 1.99 0.32  N 23 23 23 23 23 23 23 23 23 23 23 23 23  NP=NOT PREGNANT 
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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 FETUSES RESORPTIONS SEX AVERAGE  ANIMAL# LUTEA LOSS SITES LIVE DEAD EARLY LATE TOTAL MALE FEMALE MALES F (n) % (n) % ------------------------------------------------------------------------------------------------------------------ 2901 13 15.4 11 10 0 0.0 1 0 1 9.1 7 3 5.6  2902 13 7.7 12 11 0 0.0 1 0 1 8.3 6 5 5.9  2903 13 7.7 12 11 0 0.0 1 0 1 8.3 7 4 5.8  2904 14 0.0 14 12 0 0.0 2 0 2 14.3 5 7 6.4  2905 18 11.1 16 16 0 0.0 0 0 0 0.0 6 10 6.1  2906 15 13.3 13 12 0 0.0 1 0 1 7.7 3 9 6.0  2907 13 0.0 13 13 0 0.0 0 0 0 0.0 8 5 5.5  2908 21 28.6 15 14 0 0.0 1 0 1 6.7 6 8 5.4  2909 11 0.0 11 10 0 0.0 1 0 1 9.1 5 5 6.3  2910 14 0.0 14 14 0 0.0 0 0 0 0.0 4 10 6.3  2911 15 6.7 14 14 0 0.0 0 0 0 0.0 5 9 6.1  2912 17 0.0 17 17 0 0.0 0 0 0 0.0 12 5 6.0  2913 14 0.0 14 14 0 0.0 0 0 0 0.0 6 8 5.7  2914 14 0.0 14 4 0 0.0 10 0 10 71.4 1 3 6.6  2915 13 7.7 12 11 0 0.0 1 0 1 8.3 3 8 6.0  2916 16 6.3 15 15 0 0.0 0 0 0 0.0 8 7 5.5  2917 14 7.1 13 12 0 0.0 1 0 1 7.7 5 7 5.5  2918 14 7.1 13 13 0 0.0 0 0 0 0.0 9 4 5.9  2919 NP  2920 13 0.0 13 11 0 0.0 1 1 2 15.4 7 4 5.4  2921 14 7.1 13 11 0 0.0 2 0 2 15.4 5 6 5.7  2922 16 25.0 12 12 0 0.0 0 0 0 0.0 5 7 5.9  2923 15 0.0 15 15 0 0.0 0 0 0 0.0 5 10 6.2  2924 14 0.0 14 14 0 0.0 0 0 0 0.0 5 9 6.3 MEAN 14.5 6.6 13.5 12.4 0.0 0.0 1.0 0.0 1.0 7.9 5.8 6.7 5.9 S.D. 2.06 7.99 1.50 2.64 0.00 0.00 2.07 0.21 2.08 14.89 2.24 2.29 0.33  N 23 23 23 23 23 23 23 23 23 23 23 23 23  NP=NOT PREGNANT 
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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 FETUSES RESORPTIONS SEX AVERAGE  ANIMAL# LUTEA LOSS SITES LIVE DEAD EARLY LATE TOTAL MALE FEMALE MALES F (n) % (n) % ------------------------------------------------------------------------------------------------------------------ 3901 11 9.1 10 10 0 0.0 0 0 0 0.0 6 4 5.9  3902 13 0.0 13 12 0 0.0 1 0 1 7.7 6 6 5.6  3903 14 7.1 13 13 0 0.0 0 0 0 0.0 5 8 5.2  3904 13 0.0 13 13 0 0.0 0 0 0 0.0 8 5 6.1  3905 15 0.0 15 15 0 0.0 0 0 0 0.0 8 7 5.3  3906 14 0.0 14 14 0 0.0 0 0 0 0.0 7 7 5.5  3907 13 0.0 13 11 0 0.0 2 0 2 15.4 6 5 5.8  3908 13 0.0 13 10 0 0.0 3 0 3 23.1 4 6 5.7  3909 14 7.1 13 12 0 0.0 1 0 1 7.7 6 6 5.9  3910 16 12.5 14 14 0 0.0 0 0 0 0.0 7 7 6.1  3911 16 0.0 16 16 0 0.0 0 0 0 0.0 5 11 5.9  3912 14 7.1 13 12 0 0.0 1 0 1 7.7 5 7 6.1  3913 20 60.0 8 8 0 0.0 0 0 0 0.0 6 2 6.5  3914 18 16.7 15 14 0 0.0 1 0 1 6.7 6 8 5.5  3915 10 70.0 3 2 0 0.0 1 0 1 33.3 1 1 6.4  3916 16 12.5 14 14 0 0.0 0 0 0 0.0 9 5 5.7  3917 21 33.3 14 14 0 0.0 0 0 0 0.0 4 10 5.5  3918 15 0.0 15 13 0 0.0 2 0 2 13.3 5 8 5.8  3919 13 0.0 13 13 0 0.0 0 0 0 0.0 8 5 5.6  3920 14 0.0 14 14 0 0.0 0 0 0 0.0 8 6 6.1  3921 13 7.7 12 11 0 0.0 1 0 1 8.3 5 6 5.8  3922 17 0.0 17 17 0 0.0 0 0 0 0.0 8 9 6.0  3923 16 0.0 16 16 0 0.0 0 0 0 0.0 13 3 5.6  3924 15 6.7 14 14 0 0.0 0 0 0 0.0 8 6 5.9 MEAN 14.8 10.4 13.1 12.6 0.0 0.0 0.5 0.0 0.5 5.1 6.4 6.2 5.8 S.D. 2.52 18.58 2.85 3.06 0.00 0.00 0.83 0.00 0.83 8.64 2.26 2.32 0.32  N 24 24 24 24 24 24 24 24 24 24 24 24 24 
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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 FETUSES RESORPTIONS SEX AVERAGE  ANIMAL# LUTEA LOSS SITES LIVE DEAD EARLY LATE TOTAL MALE FEMALE MALES F (n) % (n) % ------------------------------------------------------------------------------------------------------------------ 4901 15 6.7 14 13 0 0.0 1 0 1 7.1 6 7 5.6  4902 11 0.0 11 10 0 0.0 1 0 1 9.1 3 7 5.8  4903 17 11.8 15 14 0 0.0 1 0 1 6.7 9 5 5.8  4904 18 0.0 18 18 0 0.0 0 0 0 0.0 10 8 5.4  4905 16 0.0 16 16 0 0.0 0 0 0 0.0 9 7 5.4  4906 19 15.8 16 16 0 0.0 0 0 0 0.0 7 9 5.5  4907 13 15.4 11 10 0 0.0 1 0 1 9.1 4 6 6.0  4908 NP  4909 11 9.1 10 9 0 0.0 1 0 1 10.0 6 3 6.0  4910 14 0.0 14 13 0 0.0 1 0 1 7.1 8 5 5.7  4911 17 0.0 17 16 0 0.0 1 0 1 5.9 6 10 5.6  4912 11 0.0 11 11 0 0.0 0 0 0 0.0 6 5 6.5  4913 15 6.7 14 14 0 0.0 0 0 0 0.0 4 10 6.0  4914 14 14.3 12 10 0 0.0 2 0 2 16.7 6 4 6.3  4915 16 6.3 15 14 0 0.0 1 0 1 6.7 3 11 5.6  4916 16 0.0 16 16 0 0.0 0 0 0 0.0 10 6 5.0  4917 16 6.3 15 14 0 0.0 1 0 1 6.7 6 8 6.0  4918 21 38.1 13 13 0 0.0 0 0 0 0.0 4 9 5.9  4919 NP  4920 14 14.3 12 11 0 0.0 1 0 1 8.3 5 6 5.4  4921 13 15.4 11 11 0 0.0 0 0 0 0.0 6 5 5.7  4922 16 6.3 15 15 0 0.0 0 0 0 0.0 5 10 5.4  4923 13 0.0 13 11 0 0.0 2 0 2 15.4 7 4 5.7  4924 18 0.0 18 17 0 0.0 1 0 1 5.6 5 12 5.6 MEAN 15.2 7.6 14.0 13.3 0.0 0.0 0.7 0.0 0.7 5.2 6.1 7.1 5.7 S.D. 2.63 9.14 2.38 2.60 0.00 0.00 0.65 0.00 0.65 5.15 2.05 2.49 0.33  N 22 22 22 22 22 22 22 22 22 22 22 22 22  NP=NOT PREGNANT 
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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 UTERUS WEIGHT = ABSOLUTE GRAVID UTERINE WEIGHT ADJUSTED WEIGHT = TERMINAL BODY WEIGHT - UTERUS WEIGHT NET WEIGHT CHANGE FROM DAY 5 = ADJUSTED WEIGHT – DAY 5 BODY WEIGHT WEIGHT CHANGE FROM DAY 5 = TERMINAL BODY WEIGHT – 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 UTERUS WEIGHT = ABSOLUTE GRAVID UTERINE WEIGHT ADJUSTED WEIGHT = TERMINAL BODY WEIGHT - UTERUS WEIGHT NET WEIGHT CHANGE FROM DAY 5 = ADJUSTED WEIGHT – DAY 5 BODY WEIGHT WEIGHT CHANGE FROM DAY 5 = TERMINAL BODY WEIGHT – 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 WEIGHT CHANGE  ANIMAL# WEIGHT WEIGHT FROM DAY 5 FROM DAY 5  3901 80 246 16 96  3902 88 275 33 121  3903 95 280 49 144  3904 103 276 31 133  3905 107 281 33 140  3906 104 288 43 146  3907 83 261 43 126  3908 75 263 38 113  3909 90 280 36 126  3910 108 269 23 131  3911 117 283 29 146  3912 96 303 28 124  3913 68 302 67 135  3914 100 289 39 139  3915 19 305 47 67  3916 110 298 41 151  3917 100 308 51 151  3918 101 293 30 131  3919 93 275 48 141  3920 114 259 33 147  3921 86 248 19 105  3922 136 272 33 169  3923 121 268 19 140  3924 112 286 28 140 MEAN 96. 280. 36. 132. S.D. 22.5 17.1 11.7 21.0  N 24 24 24 24 UTERUS WEIGHT = ABSOLUTE GRAVID UTERINE WEIGHT ADJUSTED WEIGHT = TERMINAL BODY WEIGHT - UTERUS WEIGHT NET WEIGHT CHANGE FROM DAY 5 = ADJUSTED WEIGHT – DAY 5 BODY WEIGHT WEIGHT CHANGE FROM DAY 5 = TERMINAL BODY WEIGHT – DAY 5 BODY WEIGHT 
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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  ANIMAL# 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  4907 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 UTERUS WEIGHT = ABSOLUTE GRAVID UTERINE WEIGHT ADJUSTED WEIGHT = TERMINAL BODY WEIGHT - UTERUS WEIGHT NET WEIGHT CHANGE FROM DAY 5 = ADJUSTED WEIGHT – DAY 5 BODY WEIGHT WEIGHT CHANGE FROM DAY 5 = TERMINAL BODY WEIGHT – DAY 5 BODY WEIGHT  NP=NOT PREGNANT NT=NOT TAKEN PART=PARTIAL DELIVERY; 5 LIVE PUPS DELIVERED x=EXCLUDED FROM MEAN 
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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 1 0 MG/M3  FEMALE# MEAN FETUS#  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16  1901 5.9 5.6 5.9 5.9 5.8 6.0/ 6.3 5.3 5.4 E 6.0 6.2 6.5  1902 6.0 5.6 6.6 6.0 5.7 6.3 5.9/ 6.3 5.9 5.6 5.9 6.0 6.0  1903 5.9 5.7 6.3 5.3 5.6 6.2 6.4 5.6 5.5 6.2/ 6.2 6.0 5.4 6.2 6.5  1904 5.5 5.3 5.1 5.6 5.4 5.7/ 5.4 5.9 6.0 5.7 5.4 5.3 5.6 5.5 5.7 5.3  1905 5.5 3.2 5.7 6.4 E 5.6 5.6/ 6.1 5.9 5.8 5.8 5.3 5.7 5.2 5.3  1906 5.5 5.6 5.3 5.2 5.6 5.4 5.4 E / 6.1 5.4 5.8 5.2 5.3 5.8 5.4  1907 6.3 6.4 6.3 6.0 6.2/ 6.2 6.1 6.6 6.5 6.3  1908 5.7 6.0 5.8 6.4 5.6 5.4 5.7/ 5.5 E 6.0 6.1 5.3 5.8 5.3  1909 6.2 6.3 6.6 6.1 6.4 6.3/ 6.2 6.0 5.7 6.7 6.2 6.4 6.4 5.7 5.9 6.1  1910 6.0 5.9 6.0 5.2 6.0 6.3 5.7 5.9 6.1 5.8 6.2 6.2/ 6.4 6.3  1911 5.6 5.8 5.8 5.5 5.5 E 5.6 5.4/ 5.6 5.4 5.7 5.4 5.7 5.6 E 6.2 5.6  1912 5.2 5.6 5.0 5.3 5.0 4.7 5.0 5.2 5.2 5.4/ 5.7 5.1 5.2 5.2 5.3 5.6  1913 5.5 5.0 5.9 E 5.2 5.5 5.9 E / 5.6 5.6 5.5 5.4 6.1 5.3  1914 6.0 6.0 6.3 6.4 6.1/ 6.1 5.4 5.8 5.8 6.2 5.9 6.1 5.9  1915 NP  1916 5.5 5.3 5.4 5.5 5.2 5.6 5.7 5.4 6.0/ E 5.6 5.6 5.0 5.6 5.3 5.5  1917 5.5 5.5 5.1 5.7 5.4 5.1 5.7 5.2 5.2 5.5 5.7 6.1/ 5.3 5.7 5.4 5.9 5.5  1918 5.5 5.4 5.3 5.6 5.6 5.7 5.4/ 6.0 5.7 5.1 5.5 L 5.5 5.4 5.9 5.2  1919 5.8 5.6 6.2 6.0 6.2 5.3 5.0 5.8 5.7/ 5.9 6.6 5.6 5.7 6.0 6.1  1920 6.1 / 6.2 6.0 6.1 6.3  1921 5.4 5.1 5.3 5.1 5.6 5.2 5.5 5.3/ 5.5 5.9 5.9 4.8 5.3 5.2 5.2 5.4  1922 6.0 5.8 6.5 5.6 5.9 5.8/ 6.5 6.2 6.0 6.0 6.1 5.7 5.4 6.0  1923 5.9 6.1 5.9 5.5 5.6 6.0 5.5 5.2 5.7 6.1/ 6.2 5.8 6.1 5.8 6.4 6.1  1924 5.6 5.7 E 5.9 5.6 5.3 5.8 5.3 5.6/ 5.3 5.4 5.7 5.6 5.5 5.6 5.3 MEAN 5.7 S.D. 0.29  N 23 E-Early resorption L-Late resorption  /-Denotes position of cervix  NP=NOT PREGNANT 
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 FETUS#  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16  2901 5.5 5.4/ 5.6 5.3 5.3 5.7 5.1 5.6 5.3 E 6.1 5.9  2902 5.7 5.2 5.7 6.0 5.7 5.5 6.1 5.9/ 5.3 6.2 E 5.5 5.9  2903 5.7 5.1 5.8 5.3 5.5 5.5 E 5.9/ 5.9 5.9 5.9 5.9 6.1  2904 6.1 5.9 5.9 6.4 6.0 5.8 5.7 6.4 E E / 6.8 5.9 6.4 6.3 6.2  2905 5.8 6.1 6.2 5.9 5.6 5.6 5.9 5.9 5.7 6.3 5.6/ 6.2 5.8 5.5 5.8 6.1 5.2  2906 5.9 6.0 5.8 6.1/ 6.0 5.5 E 5.5 5.9 6.2 6.1 6.2 6.1 5.8  2907 5.4 4.9 5.8 5.1 6.0 5.4 5.1 5.5/ 5.7 5.3 5.4 5.2 5.2 5.3  2908 5.2 5.3 4.9 4.9 5.7 5.6 5.1/ 5.1 5.6 4.8 E 5.3 5.4 4.9 5.1 5.2  2909 6.0 5.7 E 6.5 6.4 5.8/ 6.4 6.2 5.8 5.7 5.9 5.8  2910 5.9 5.7 5.5 6.0 5.7 5.3 5.8/ 6.1 6.4 6.0 6.2 5.9 6.5 5.2 6.4  2911 5.8 5.5 6.0 5.4 5.7 5.7/ 5.4 5.6 5.4 5.7 6.3 6.1 5.7 6.2 6.1  2912 6.0 6.4 5.9 5.9 6.4 6.0 5.8 6.0 5.8 6.1 6.1/ 6.1 5.9 6.1 6.3 5.5 5.9  2913 5.6 5.6 5.6 5.5 5.7 5.6 5.7 5.5 5.7 5.1/ 5.6 5.6 6.0 5.6 5.8  2914 6.3 E E 6.1 E E / E E E 6.6 6.4 E E E 6.2  2915 5.9 5.4 E 6.2 6.0 6.2 5.7 6.2 5.3/ 5.9 6.2 6.1 5.6  2916 5.4 5.5 5.3 5.3 5.1 5.6 5.3 4.9 5.8/ 5.4 5.7 5.4 5.6 5.7 5.2 5.5  2917 5.3 5.2 E 5.0 5.5 5.0/ 5.6 5.6 5.4 5.6 5.4 4.9 5.3 5.3  2918 5.9 5.7 6.0 5.9 6.0 5.3 5.4 6.1 5.4 5.9 6.0/ 6.1 6.5 6.3  2919 NP  2920 5.3 5.5 5.7 L 5.4/ 5.1 5.3 5.2 4.9 E 5.1 4.5 5.6 5.3  2921 5.7 E E 5.7 6.0 5.4 5.8 5.6 5.7 6.0/ 5.8 5.6 6.0 5.6  2922 5.8 5.4 6.0 5.9 5.4 5.8 5.6/ 6.0 5.9 5.8 6.0 5.8 5.8  2923 6.0 5.4 5.9 5.8 6.1 6.3 6.3/ 6.5 6.4 5.7 6.0 6.2 6.3 6.0 5.4 6.1  2924 6.1 5.9 6.5 5.9 6.5 6.1 6.0 5.3/ 5.9 5.7 6.1 6.5 6.1 6.3 6.2 MEAN 5.8 S.D. 0.30  N 23 E-Early resorption L-Late resorption  /-Denotes position of cervix  NP=NOT PREGNANT 
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 3 10000 MG/M3  FEMALE# MEAN FETUS#  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16  3901 5.8 5.5 6.0 5.9 5.7 5.7 6.2 5.7/ 6.0 5.1 5.9  3902 5.6 5.6 5.9 5.3 E 5.2 5.5 5.2 5.4 5.8/ 5.6 5.8 5.8 5.6  3903 5.1 5.4 5.2 5.6 5.2 5.3/ 5.0 5.1 4.9 5.0 4.8 5.1 4.8 5.3  3904 5.8 5.4 5.9 6.1 4.9 6.0 6.2 5.8/ 6.1 5.8 6.3 5.6 5.8 5.9  3905 5.1 5.1 5.4 4.7 4.9 4.8 4.9 4.8 5.2 4.7 5.4/ 5.3 5.9 5.0 5.5 5.2  3906 5.6 5.4 5.7 4.6 5.2 5.5 5.6 5.5 5.5 5.6 5.9/ 5.9 5.9 6.1 5.8  3907 5.6 5.7 E 6.0 5.6 5.6 5.3/ 6.0 5.5 E 5.6 5.4 5.7 5.8  3908 5.5 5.6 5.7 E 6.1 5.1 5.2 5.4/ 5.6 5.3 5.1 E E 5.8  3909 5.8 6.0 5.8 5.6 E 5.8 5.9 5.6 5.3 5.4 5.7/ 6.1 6.1 5.7  3910 5.9 5.9 6.0 6.2 5.8 6.0 6.0/ 5.7 6.0 6.2 4.3 6.0 6.4 5.9 6.4  3911 5.5 5.7 6.3 5.9 6.0 6.2 5.5 5.6 3.7 5.7 5.4/ 3.3 6.2 5.5 5.7 5.6 6.0  3912 6.0 6.1 E 5.9 6.4 6.3/ 6.3 5.8 5.8 5.9 6.0 5.9 6.0 5.7  3913 6.4 / 7.0 6.3 5.8 6.7 6.2 6.4 6.4 5.9  3914 5.3 5.2 5.3 5.3 5.8 5.0 E / 5.6 5.4 5.2 5.4 4.9 5.3 5.0 5.3 5.9  3915 5.8 / E 6.4 5.3  3916 5.7 5.6 5.5 5.7 5.5 5.8 5.4 5.9/ 5.7 5.7 5.6 5.6 5.7 5.8 5.8  3917 5.3 4.6 5.3 5.2 5.6 5.5/ 5.4 5.0 5.4 5.7 4.9 4.8 5.5 5.7 5.3  3918 5.5 5.7 E 5.6 5.8 5.8 5.9/ E 5.5 5.3 5.7 5.5 5.4 5.1 4.8 5.7  3919 5.5 5.6 5.8 5.5 5.8 5.1 5.5/ 5.9 5.7 4.9 5.3 5.6 5.7 5.3  3920 6.0 6.1 5.7 6.7 6.4 6.0 5.5 6.2/ 6.2 5.8 5.8 5.8 6.1 5.5 5.8  3921 5.8 5.6 6.3 6.0 E 5.6 6.2 5.5/ 6.0 6.1 5.9 5.8 4.9  3922 5.8 6.2 6.0 5.8 5.5 5.8 5.3 5.7 5.6 5.8/ 5.8 5.7 5.2 5.5 6.2 5.7 6.3  3923 5.6 5.5 5.5 5.1 5.5 5.5 5.8 5.8 5.7/ 5.6 5.7 5.5 5.4 6.1 5.3 5.7 5.5  3924 5.8 5.6 5.9 5.8 6.0 6.0 5.2 5.6 5.8/ 6.3 5.8 5.8 5.8 6.0 5.6 MEAN 5.7 S.D. 0.28  N 24 E-Early resorption  /-Denotes position of cervix 
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 4 20000 MG/M3  FEMALE# MEAN FETUS#  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16  4901 5.5 5.7 5.9 5.6 5.3/ 5.5 5.4 5.2 5.3 5.4 5.6 5.4 E 5.2 5.4  4902 5.5 6.0 5.5 E 5.6 5.4/ 5.4 5.3 5.3 6.0 5.1 5.1  4903 5.7 5.6 5.7 5.7 5.7 E 5.9/ 5.4 5.9 5.2 5.8 5.5 6.1 6.0 5.8 5.7  4904 5.2 4.8 5.1 5.8 5.1 4.9 4.8 5.1/ 5.5 5.1 5.1 5.8 5.2 5.3 4.9 5.3 4.9  4905 5.2 5.2 5.3 5.1 5.5 5.2 5.6 5.1 5.0 4.5 4.9/ 5.2 5.2 5.7 5.5 5.1 5.4  4906 5.4 5.5 5.4 5.3 5.4 5.1 5.9 5.5 5.4/ 5.5 5.0 5.0 5.3 5.4 5.4 5.6 5.3  4907 5.7 6.0 5.6 5.6 6.1 5.6 5.6/ E 5.7 6.0 5.4 5.9  4908 NP  4909 5.9 6.1 6.0 E 5.8 6.3 5.4 6.2 6.1/ 6.0 5.4  4910 5.6 5.5 5.7 5.2 6.0 5.8 5.8 5.7 5.7/ E 5.5 5.0 5.8 5.8 5.8  4911 5.4 5.1 5.6 5.1 5.6 5.8 4.4 5.3 5.4 5.4 5.5 5.6 5.4/ E 5.1 5.6 5.6  4912 6.2 6.7 6.7 5.3 6.0 6.3 6.6/ 5.6 5.8 5.7 6.0 6.9  4913 5.8 6.0 5.8 5.6 5.7 5.8/ 6.1 5.7 5.4 5.3 6.1 6.0 5.9 5.8 5.6  4914 6.1 5.9 E 6.5 5.9 6.3 E 5.9 6.2/ 5.7 6.4 6.1 6.6  4915 5.2 5.5 4.7 5.3 5.2 5.4 E 5.3 5.1 4.3 5.2/ 4.8 6.1 5.4 5.5 5.0  4916 4.9 5.0 5.4 4.8 4.9 4.7 4.5 5.3/ 5.1 5.0 5.1 4.9 4.8 4.2 5.2 4.7 4.8  4917 5.9 6.0 5.9 5.8 5.6 6.0 5.7 5.7 E 6.2/ 6.0 5.8 5.8 6.1 6.0 6.1  4918 5.5 5.2 5.3 5.3 5.4 5.2/ 5.5 5.4 6.2 5.3 5.3 5.9 5.8 5.7  4919 NP  4920 5.2 5.2 5.0 E 5.2 5.1 5.1/ 5.3 5.4 5.8 5.0 5.2 5.3  4921 5.6 5.4 5.8 5.2 5.6 6.4 5.6/ 5.3 5.3 5.8 5.6 5.0  4922 5.2 5.0 4.9 5.8 5.0 5.1 5.6 5.3 5.3 5.3/ 5.3 5.2 5.3 4.9 5.2 5.5  4923 5.5 5.3 5.2 5.6 5.9 4.8 5.5 5.6 5.8/ 5.2 5.9 E 5.8 E  4924 5.4 4.7 5.7 5.3 5.9 5.2 5.6 5.6 5.4 5.3 5.8 E 5.5/ 5.6 5.1 5.6 5.2 MEAN 5.5 S.D. 0.32  N 22 E-Early resorption  /-Denotes position of cervix  NP=NOT PREGNANT 
Huntingdon Life Sciences 05-4287 

 211-DIPE-DEV 

Females INDIVIDUAL FETAL EXTERNAL, VISCERAL AND SKELETAL OBSERVATIONS 

Animal Fetus Weight Abnormalities (only fetuses with abnormalities are listed) 

Number Exam Number Sex (g) Fate Major abnormalities in CAPITALS 

Group 1 - 0 MG/M3

 1901 6s, 5v 12 M 6.5 s Sternebra(e) malaligned, #4 and #5, slight  1903 7s, 7v 3 F 5.3 s Rib(s) 14th rudimentary, left 

 10 M 6.2 v Additional subclavian artery, left  11 M 6.0 s Rib(s) 14th rudimentary, bilateral 

 1904 7s, 8v 6 F 5.4 s Hyoid body/arch(es), unossified body 

 11 F 5.3 v Renal papilla (e) not fully developed (grade 1), left 

 1905 7s, 6v 1 M 3.2 s ANAL ATRESIA AND FILAMENTOUS TAIL 

 VERTEBRAL AGENESIS: SACRAL VERTEBRAE #1 AND #2 S

SACRAL VERTEBRAE #3 AND #4 AND ALL CAUDAL VERTEBILIA AND ISCHIA CLOSELY APPROXIMATED 

 Sternebra(e) 5 and/or 6 unossified, #5 

 2 F 5.7 v Undescended thymic tissue, right 

 1908 6s, 6v 6 F 5.7 s 25 presacral vertebra(e) 

 Rib(s) 13th rudimentary, right 

 1909 8s, 7v 8 F 5.7 v Undescended thymic tissue, left 

 9 M 6.7 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 Fetus Weight Abnormalities (only fetuses with abnormalities are listed) 

Number Exam Number Sex (g) Fate Major abnormalities in CAPITALS 

Group 1 - 0 MG/M3

 1910 6s, 7v 2 F 6.0 s Sternebra(e) malaligned, #4, slight  9 F 5.8 v Left umbilical artery  1911 8s, 7v 4 M 5.5 v Undescended thymic tissue, left  9 M 5.4 v Undescended thymic tissue, right  1912 7s, 8v 3 F 5.3 v Undescended thymic tissue, left  9 F 5.4 v Undescended thymic tissue, right  Ureter(s) distended, bilateral  12 F 5.2 s Sternebra(e) malaligned, #4 and #5, slight  1913 6s, 5v 6 M 5.9 s Sternebra(e) malaligned, #3 through #5, slight to moderat 1914 6s, 6v 8 F 5.8 s Sternebra(e) malaligned, #4 and #5, slight  1916 7s, 7v 1 F 5.3 v Undescended thymic tissue, right  3 M 5.5 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 Fetus Weight Abnormalities (only fetuses with abnormalities are listed) 

Number Exam Number Sex (g) Fate Major abnormalities in CAPITALS 

Group 1 - 0 MG/M3

 1918 7s, 7v 11 L Crown-rump distance 2.4 cm  1919 7s, 7v 9 F 5.9 s Sternebra(e) malaligned, #4 and #5, slight  11 F 5.6 s Sternebra(e) malaligned, #4 and #5, slight  13 F 6.0 s Sternebra(e) malaligned, #4 and #5, slight  1921 8s, 7v 2 F 5.3 v Innominate artery absent  6 M 5.5 v Undescended thymic tissue, left  12 M 5.3 v Undescended thymic tissue, right  Displaced testis medially, left  1923 8s, 7v 7 F 5.2 s Sternebra(e) malaligned,#3 and #4, slight to moderate  1924 7s, 7v 9 F 5.3 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 INDIVIDUAL FETAL EXTERNAL, VISCERAL AND SKELETAL OBSERVATIONS 

Animal Fetus Weight Abnormalities (only fetuses with abnormalities are listed) 

Number Exam Number Sex (g) Fate Major 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) malaligned, #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 INDIVIDUAL FETAL EXTERNAL, VISCERAL AND SKELETAL OBSERVATIONS 

Animal Fetus Weight Abnormalities (only fetuses with abnormalities are listed) 

Number Exam Number Sex (g) Fate Major 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 Fetus Weight Abnormalities (only fetuses with abnormalities are listed) 

Number Exam Number Sex (g) Fate Major abnormalities in CAPITALS 

Group 2 - 2000 MG/M3

 2920 5s, 6v 3 L Crown-rump distance 2.9 cm  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 INDIVIDUAL FETAL EXTERNAL, VISCERAL AND SKELETAL OBSERVATIONS 

Animal Fetus Weight Abnormalities (only fetuses with abnormalities are listed) 

Number Exam Number Sex (g) Fate Major 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 (grade 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 INDIVIDUAL FETAL EXTERNAL, VISCERAL AND SKELETAL OBSERVATIONS 

Animal Fetus Weight Abnormalities (only fetuses with abnormalities are listed) 

Number Exam Number Sex (g) Fate Major 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 Fetus Weight Abnormalities (only fetuses with abnormalities are listed) 

Number Exam Number Sex (g) Fate Major abnormalities in CAPITALS 

Group 3 - 10000 MG/M3

 3917 7s, 7v 11 F 4.8 s Sternebra(e) 5 and/or 6 unossified, #5  3918 6s, 7v 14 F 4.8 s Sternebra(e) malaligned, #3 and #4, slight  3921 6s, 5v 6 M 6.2 s Sternebra(e) malaligned, #3 through #5, slight 

 8 F 6.0 s Sternebra(e) malaligned, #3 through #5, slight  3922 8s, 9v 2 F 6.0 s Sternebra(e) malaligned, #4 and #5  10 F 5.8 s Sternebra(e) malaligned, #3 through #5, slight  3923 8s, 8v 16 M 5.5 v Renal papilla (e) not fully developed (grade 1), left 

 3924 7s, 7v 4 M 6.0 s Sternebra(e) malaligned, #4, slight  6 F 5.2 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 Fetus Weight Abnormalities (only fetuses with abnormalities are listed) 

Number Exam Number Sex (g) Fate Major abnormalities in CAPITALS 

Group 4 - 20000 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) present, 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 moderat 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 LUM

#1 ABSENT; THORACIC ARCHES #13 AND LUMBAR ARCHE#1 MALFORMED AND CLOSELY APPROXIMATED 

 11 M 5.9 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 Fetus Weight Abnormalities (only fetuses with abnormalities are listed) 

Number Exam Number Sex (g) Fate Major 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 moderat 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 INDIVIDUAL FETAL EXTERNAL, VISCERAL AND SKELETAL OBSERVATIONS 

Animal Fetus Weight Abnormalities (only fetuses with abnormalities are listed) 

Number Exam Number Sex (g) Fate Major 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 rudimentary, 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 Fetus Weight Abnormalities (only fetuses with abnormalities are listed) 

Number Exam Number Sex (g) Fate Major abnormalities in CAPITALS 

Group 4 - 20000 MG/M3

 4924 8s, 9v 1 F 4.7 v Renal papilla (e) not fully developed (grade 1), bilateral 

 Ureter(s) distended, left  14 F 5.1 v Left umbilical artery  17 F 5.8 s Rib(s) 14th rudimentary, left 

s = skeletal examination, v = visceral examination, M=Male, F=Female, g=grams 
05-4287 

Page 1 of 18 

Statistical analysis

GASOLINE DIPE VAPOR CONDENSATE

EMBRYO-FETAL TOXICITY STUDY IN RATS BY INHALATION EXPOSURE 

Statistical Analysis 

Authors 

Graham F Healey 

Simon Bate 

Gareth Thomas 

Statistics Department 

Appendix M Page 146
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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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INTRODUCTION AND EXPERIMENTAL DESIGN 

This rat study had a control and three groups treated at 2000 mg/m3

, 10000 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, d11, d14, d17, d20, d21) 

Maternal body weight change (d4-d5, d5-d8, d8-d11, d11-d14, d14-d17, d17-d20, d5-d14, 

d14-d21, d5-d21) 

Maternal feed consumption (d4-d5, d5-d8, d8-d11, d11-d14, 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 findings

Maternal pregnancy summary 

Maternal necropsy findings 

Fetal findings: external, visceral and skeletal (only certain visceral and skeletal findings 

required statistical analysis). 

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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 (Steel 1959). 

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. 

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Discrete findings

Maternal pregnancy summary 

Maternal necropsy findings 

Fetal findings: external, visceral and skeletal malformations and variations 

For maternal parameters: 

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 findings: 

Only findings 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 findings 

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 findings 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. 

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For food consumption, the animal 2908 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. 

Maternal uterus weight and maternal bodyweight minus gravid uterine weight 

 Maternal uterus weight: no significant differences. 

 Maternal bodyweight minus gravid uterine weight: no significant differences. 

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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 P No significant differences. 

%Preimplantation loss NP No significant differences. 

Number of implant sites P No significant differences. 

Fetuses alive P No significant differences. 

Early resorptions NP No significant differences. 

Late resorptions P No significant differences. 

Total resorptions NP No significant differences. 

%Total resorptions/implant sites NP No significant differences. 

Number of males P No significant differences. 

Number of females P No significant differences. 

Fetal bodyweight

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=0.016, a reduction from control). 

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 Significant decrease at 20000 mg/m3

 (p=0.007). 

Litter body weight Significant decrease at 20000 mg/m3

 (p=0.016). 

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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 findings, for those findings with a total incidence of greater than one is 

summarised in Table 8. Only five Visceral and seven Skeletal findings, 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 findings

Heart, V Innominate artery absent NP No significant differences 

Torso, V Left umbilical artery GL No significant differences 

Torso, V Renal papilla (E) not fully developed NP No significant differences 

Torso, V Undescended thymus GL No significant differences 

Torso, V Ureter(s) distended NP No significant differences 

Skeletal findings

Cervical vertebrae, V 7th cervical rib(s) present NP No significant differences 

Ribs, V Rib(s) 13th Rudimentary NP No significant differences 

Ribs, V Rib(s) 14th Rudimentary GL No significant differences† 

Sacral vertebrae, V 25 Presacral vertebra(e) NP No significant differences 

Skull, V Hyoid body/arch(es) unossified NP No significant differences 

Sternebrae, V Sternebra(e) 5 and/or 6 unossified. NP No significant differences 

Sternebrae, V Sternebra(e) malaligned GL Significant decrease from control 

at 20000 mg/m3

 (p=0.047). 

† There was some evidence of a statistically significant increase from control at 20000 mg/m3

(p=0.067). 

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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 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. 

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. 

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TABLE 1 

Maternal body weight (g) 

 0 mg/m3

(n=23) 

2000 mg/m3

(n=23) 

10000 mg/m3

(n=24) 

20000 mg/m3

(n=22) 

Day of 

gestation 

mean (sd) mean (sd) mean (sd) mean (sd) 

4 

5 

8 

11 

14 

17 

20 

21 

233.8 (14.1) 

244.0 (14.6) 

259.3 (12.8) 

281.5 (14.9) 

301.5 (17.1) 

332.1 (22.3) 

378.1 (26.3) 

387.8 (28.5) 

234.7 (16.6) 

245.3 (16.6) 

260.4 (18.1) 

279.0 (19.7) 

295.5 (20.2) 

326.7 (24.2) 

370.3 (29.9) 

379.4 (32.7) 

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) 

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) 

Analysis performed = Parametric throughout. 

No significant differences found for any parameter. 

n = number of litters, sd = standard deviation 

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TABLE 2 

Maternal body weight change (g) 

Days of 

gestation 

0 mg/m3

(n=23) 

2000 mg/m3

(n=23) 

10000 mg/m3

(n=24) 

20000 mg/m3

(n=22) 

Period mean (sd) mean (sd) mean (sd) mean (sd) 

4-5 

5-8 

8-11 

11-14 

14-17 

17-20 

5-14 

14-21 

5-21 

10.3 (6.1) 

15.5 (4.4) 

22.2 (4.7) 

20.0 (4.2) 

30.7 (7.3) 

46.2 (7.5) 

57.6 (8.4) 

86.4 (15.3) 

143.9 (20.6) 

10.7 (4.7) 

15.0 (4.8) 

18.6 (3.7) * 

16.8 (5.1) * 

31.2 (6.9) 

43.5 (8.9) 

50.2 (7.9) * 

84.0 (16.2) 

134.2 (21.5) 

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) 

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) 

Analysis performed = Parametric throughout. 

* = p<0.05, ** = p<0.01 

n = number of litters, sd = standard deviation 

Maternal net bodyweight change from d5 minus gravid uterine weight (g) 

 0 mg/m3

(n=22) 

2000 mg/m3

(n=23) 

10000 mg/m3

(n=24) 

20000 mg/m3

(n=20) 

Parameter mean (sd) mean (sd) mean (sd) mean (sd) 

Absolute 42.95 (11.08) 37.39 (9.16) 35.71 (11.75) 29.35 (10.65) *** 

Analysis performed = Parametric throughout. 

n = number of litters, sd = standard deviation 

*** = p<0.001 

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TABLE 3 

Maternal feed consumption (g/kg/day) 

Days of 

gestation 

0 mg/m3

(n=22-23) 

2000 mg/m3

(n=22-23) 

10000 mg/m3

(n=22-24) 

20000 mg/m3

(n=21-22) 

Period mean (sd) mean (sd) mean (sd) mean (sd) 

4-5 

5-8 

8-11 

11-14 

14-17 

17-20 

20-21 

5-21 

71.2 (10.5) 

80.8 (5.9) 

85.1 (7.1) 

85.0 (7.1) 

80.9 (4.5) 

74.5 (4.5) 

53.3 (8.1) 

76.2 (4.8) 

74.2 (11.5) 

82.7 (5.5) 

81.8 (4.6) 

81.6 (4.6) 

80.8 (4.5) 

73.4 (3.3) 

46.8 (7.6) * 

75.0 (4.1) 

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) 

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) ** 

 

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.

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TABLE 4 

Maternal uterus weights and maternal bodyweight minus gravid uterine weight (g) 

 0 mg/m3

(n=22) 

2000 mg/m3

(n=23) 

10000 mg/m3

(n=24) 

20000 mg/m3

(n=20) 

Parameter mean (sd) mean (sd) mean (sd) mean (sd) 

Absolute 

AdjustedA

101.3 (19.3) 

286.0 (18.1) 

96.5 (19.9) 

282.7 (19.0) 

96.1 (22.5) 

279.5 (17.2) 

96.4 (15.6) 

275.3 (18.4) 

Analysis performed = Parametric throughout. 

A = Maternal bodyweight minus gravid uterine weight 

No significant differences found for any parameter. 

n = number of litters, sd = standard deviation 

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TABLE 5 

Reproductive performance 

0 mg/m3

(n=23) 

2000 mg/m3

(n=23) 

10000 mg/m3

(n=24) 

20000 mg/m3

(n=22) 

Parameter mean (sd) mean (sd) mean (sd) mean (sd) 

corpora lutea (1) 

%preimplantation loss (2) 

number of implant sites (1) 

fetuses alive (1) 

early resorptions (2) 

late resorptions (1) 

total resorptions (2) 

%total resorp./implant sites (2)

number of males (1) 

number of females (1) 

14.9 (2.8) 

9.3 (13.5) 

13.5 (2.7) 

13.0 (2.6) 

0.4 (0.7) 

0.0 (0.2) 

0.5 (0.7) 

3.4 (4.7) 

6.3 (1.7) 

6.7 (2.0) 

14.5 (2.1) 

6.6 (8.0) 

13.5 (1.5) 

12.4 (2.6) 

1.0 (2.1) 

0.0 (0.2) 

1.0 (2.1) 

7.9 (14.9) 

5.8 (2.2) 

6.7 (2.3) 

14.8 (2.5) 

10.4 (18.6) 

13.1 (2.8) 

12.6 (3.1) 

0.5 (0.8) 

0.0 (0.0) 

0.5 (0.8) 

5.1 (8.6) 

6.4 (2.3) 

6.2 (2.3) 

15.2 (2.6) 

7.6 (9.1) 

14.0 (2.4) 

13.3 (2.6) 

0.7 (0.6) 

0.0 (0.0) 

0.7 (0.6) 

5.2 (5.2) 

6.1 (2.1) 

7.1 (2.5) 

Analysis performed = (1) Parametric or (2) Non-parametric 

No significant differences found for any parameter. 

n = number of litters, sd = standard deviation 

Male and female bw n=11 

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TABLE 6 

Mean (adjusted) fetal bodyweight (g) 

0 mg/m3

(n=23) 

2000 mg/m3

(n=23) 

10000 mg/m3

(n=24) 

20000 mg/m3

(n=22) 

 mean (se) mean (se) mean (se) mean (se) 

male 

 

female 

litter 

5.88 (0.06) 

5.58 (0.06) 

5.73 (0.06) 

5.87 (0.06) 

5.59 (0.06) 

5.73 (0.06) 

5.78 (0.06) 

5.47 (0.06) 

5.62 (0.06) 

5.71 (0.06) 

5.36 (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 ANOVA significant at 5%. 

n = number of litters, se =standard error of the adjusted mean 

Page 160
05-4287 

: : 

Statistical analysis

16

TABLE 7 

Maternal pregnancy summary and necropsy findings 

Parameter Finding 0 mg/m3

(n=24) 

2000 mg/m3

(n=24) 

10000 mg/m3

(n=24) 

20000 mg/m3

(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 

Page 161
05-4287 

: : 

Statistical analysis

17

TABLE 8 

Fetal findings: Visceral 

Tissue Organ Finding 0 mg/m3

(n=23) 

2000 mg/m3

(n=23) 

10000 mg/m3

(n=24) 

20000 mg/m3

(n=22) 

Visceral HEART 

TORSO 

TORSO 

TORSO 

TORSO 

V INNOMINATE ARTERY 

ABSENT 

V LEFT UMBILICAL 

ARTERY 

V RENAL PAPILLA (E) NOT 

FULLY DEVELOPED 

V UNDESCENDED 

THYMUS 

V URETER(S) DISTENDED 

1 

1 

1 

10 

1 

0 

4 

0 

11 

0 

2 

1 

2 

5 

1 

1 

2 

1 

11 

1 

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 

Page 162
05-4287 

: : 

Statistical analysis

18

TABLE 8 (cont'd) 

Fetal findings: Skeletal 

Tissue Organ Finding 0 mg/m3

(n=23) 

2000 mg/m3

(n=23) 

10000 mg/m3

(n=24) 

20000 mg/m3

(n=22) 

Skeletal CERVICAL 

VERT 

RIBS 

RIBS 

SACRAL VERT 

SKULL 

STERNEBRAE 

STERNEBRAE 

V 7TH CERVICAL RIB(S) 

PRESENT 

V RIB(S) 13TH

RUDIMENTARY 

V RIB(S) 14TH

RUDIMENTARY 

V 25 PRESACRAL 

VERTEBRA(E) 

V HYOID BODY/ARCH(ES) 

UNOSSIFIED BODY 

V STERNEBRA(E) 5 

AND/OR 6 UNOSSIFIED 

V STERNEBRA(E) 

MALALIGNED 

0 

1 

2 

1 

1 

1 

11 

1 

0 

7 

0 

0 

0 

11 

0 

0 

5 

0 

1 

3 

13 

3 

3 

12 

3 

1 

3 

3* 

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 of litters 

Page 163
Huntingdon Life Sciences 05-4287 Page 164 

211-DIPE-DEV Final Report 

 

Certificates of Analysis Appendix N 

Table of Contents 

Feed Certificates of Analysis ..........................................................................................165 

Water Certificates of Analysis .........................................................................................168 

Bedding Certificates of Analysis .....................................................................................176 
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 

http:/ /www.labdiet.com/certified/pwa _ spc002.asp 

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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 

http:/ /www.labdiet.com/certified/pwa _ spc002.asp 

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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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Page 167
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--

Page 168
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: 

Page 169
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: ~ 

Page 170
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_ 

Page 171
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-;..;,__ 

Page 172
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 ¥ , 

Page 173
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: 

Page 174
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 

Page 175
!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 

Page 176
·--""' 

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 

Page 177
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 

Appendix O Page 178
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 

Page 179
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 

Page 180
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 

Page 181
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. 

Page 182
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 

Page 183
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 

F

Number of Animals 

Fetal Eval

(Fetuses/L

Euthanized 

Gestation Soft 

Day 21b External Tissue 

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 expressed as 

test substance. The exposures will be conducted via whole-body exposure. Control animals will be treated with air only with 

treatment regimen as the treated groups. 

bPostmortem evaluations will also be performed on animals which are found dead or euthanized for humane reasons or in a m

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 the study 

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 

Page 185
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®] 

Page 186
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. 

Page 187
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. 

Page 188
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. 

Page 189
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. 

Page 190
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. 

Page 191
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 

Page 192
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. 

Page 193
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: 

Page 194
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. 

Page 195
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: 

Page 196
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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: 

Page 197
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 

Page 198
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. 

·'.''· 

Page 199
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 

Page 200
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. 

Page 201
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 

Page 202
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. 

Page 203
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 

Page 204
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. 

Page 205
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 

Page 206
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 

Page 207
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. 

Page 208
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 

Page 209
Huntingdon Life Sciences 05-4287 Page 33 

 211-DIPE-DEV 

Protocol Amendment No.: 5

Study Title: Gasoline DIPE Vapor Condensate: 

 Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure 

Change(s) 

Statistical Evaluations, page 15: 

CONTINUOUS DATA 

The following mean measures will be analyzed as described below: 

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, Berry and Matthews 2002 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 

Page 210
Huntingdon Life Sciences 05-4287 Page 34 

 211-DIPE-DEV 

Protocol Amendment No.: 5

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: 

Armitage, P., Berry, G. and Matthews, J.N.S. (2002) Statistical Methods in Medical 

Research, 4th edition. Blackwell Science, Oxford, UK, pp 208-215. 

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. 

Page 211
Huntingdon Life Sciences 05-4287 Page 35 

 211-DIPE-DEV 

Protocol Amendment No.: 5

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 (Proc Mixed, SAS Institute 1999) 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 and will include a random 

animal effect. The Satterthwaite method will be used for the fixed effects 

denominator degrees of freedom. If the dose group effect in the model is statistically 

significant, the dose group least squares means will be tested pairwise vs compared 

with the control group using t-tests associated with least squares means Dunnett’s test 

with the means being adjusted for the litter size. 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 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.

SAS INSTITUTE (1999) SAS OnlineDoc Version Eight. SAS Institute Inc., Cary, 

NC, USA. 

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 

 For maternal parameters: 

These parameters will be analyzed using non-parametric methods. If the dose 

group effect is statistically significant, each dose group will be compared with the 

control group using pairwise Wilcoxon rank sum tests (Wilcoxon 1945). 

For fetal findings: 

Page 212

Huntingdon Life Sciences 05-4287 

 211-DIPE-DEV 

Historical Control Data 

Fetal Skeletal Observations 

Rat Strain: Sprague-Dawley [Crl: CD IGS BR] Supplier: Charles River Laboratories Fetal Variation: Rib(s) 14th Rudimentary 

Fetuses Litters 

Study Route 1st GD 6 Last GD 20 

No. 

Examined 

No. with 

Finding 

% with 

Finding 

No. 

Examined 

No. with 

Finding 

ALL Sep-02 Jul-05 902 79 8.76 134 45 02-4235 Oral gavage Sep-02 Oct-02 160 11 6.88 22 6 03-4253 Inhalation Feb-05 Mar-05 167 12 7.19 24 7 03-4255 Oral gavage Jun-03 Sep-03 148 23 15.54 22 15 04-4261 Inhalation Aug-04 Sep-04 150 22 14.67 22 9 04-4267 Subcutaneous Nov-04 Dec-04 135 6 4.44 22 4 05-4292 Intravenous Jun-05 Jul-05 142 5 3.52 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 -

Page 215
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 

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Page 216
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 -

Page 217
Huntingdon Life Sciences 05-4287 Page 218 

211-DIPE-DEV Final Report 

 

Testing Facility Personnel Appendix Q 

a

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 finalization of the study report. 

TITLE/DEPARTMENT NAME/DEGREE 

SENIOR VICE PRESIDENT, 

 SAFETY ASSESSMENT 

Sylvie J. Gosselin, D.V.M., Ph.D., 

 Diplomate A.C.V.P. 

 

DIRECTOR, TOXICOLOGY 

 OPERATIONS Ian Vanterpool, F.I.A.T. 

 

DIRECTOR, QUALITY 

 ASSURANCE Nicki S. Iacono, B.S. 

 

STUDY DIRECTORa Keith P. Hazelden, B.Sc., C.Biol., M.I.Biol. 

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

 

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

 

VETERINARIAN Teresa S. Kusznir, V.M.D. 

 

REPORT PRODUCTION Janet E. Scimone, B.A. 

 

MANAGER/SUPERVISOR 

 Reproductive Toxicology 

 Inhalation Toxicology 

 Analytical Services 

 Pharmacy 

 Necropsy and Fetal Pathology 

Sally Wilcox, B.Sc. 

Stuart Cracknell, CBiol, MIBiol. 

Yonggang Wang, M.A. 

Michael S. McCarthy 

G. Elizabeth Baxter, B.S. 

 

STATISTICIAN Gareth D. Thomas, B.Sc. (Hons).

Principal Investigator 

 

SUBCONTRACTOR Michael Mercieca, Principal Investigator 

Pathology Associates International (PAI) 
Huntingdon Life Sciences 05-4287 Page 219 

211-DIPE-DEV Final Report 

 

Report Amendments Appendix R 

There are no amendments for this report at this time.