### 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 Pe troleum 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

### Huntingdon Life Sciences 05-4287 Page 5 211-DIPE-DEV Final Report

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 pre gnant 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 re peat the previous study to satisfy the Environmental Protection Agency’s Altern ative 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/m 3 

### for 6 hours/day, 7 days per week for Gestation Da ys (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. Th e following parameters were evaluated: viability, clinical observations, body weights a nd 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 ex ternally visible abnormalities and weighed. Approximately one-half of the fetuses (alternating fetuses) in each litter were examined for soft-tissue abnormalities using a microdissec tion 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/m 3 

### . 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 consum ption (up to approximately 10%) during the exposure period. These changes were negligible at 2000 mg/m 3 

### and most noticeable at 20000 mg/m 3 

### . It was considered that a no-observed-adverse-effect level (NOAEL) for maternal effects was evident at 2000 mg/m 3 

### .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 vari ants. A NOAEL for embryo-fetal effects was therefore established at 20000 mg/m 3



### .

### Huntingdon Life Sciences 05-4287 Page 6 211-DIPE-DEV Final Report

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 Ev aluations ..........................................................................17 2.13. Postmortem Evaluations ............................................................................18 2.14. Fetal Evaluations ........................................................................................19 2.15. Statistical An alysis .....................................................................................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

### Huntingdon Life Sciences 05-4287 Page 7 211-DIPE-DEV Final Report

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 Term ination History ......................................................................85 D. Individual Clinical Observa tions During Gestation ....................................................89 E. Individual Gestation B ody Weights (grams) .............................................................101 F. Individual Gestation Body We ight Gain (grams) ......................................................105 G. Individual Gestation Feed Cons umption (grams/kg/day) ..........................................109 H. Individual Maternal Necr opsy Observations .............................................................113 I. Individual Female Reproduction Da ta 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 Anal ysis 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

### Huntingdon Life Sciences 05-4287 Page 8 211-DIPE-DEV Final Report

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 n ecropsy 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 t echnical errors made in evaluating the male reproductive system of the fetuses. The Sponsor needed to repeat the previous study to satisfy the Environmenta l 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

### Huntingdon Life Sciences 05-4287 Page 9 211-DIPE-DEV Final Report

2.1.4. STUDY DIRECTOR 1



### 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 Hu ntingdon Life Sciences. Gary Ho ffman assumes responsibility for finalization of the study report.

### Huntingdon Life Sciences 05-4287 Page 10 211-DIPE-DEV Final Report

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 pe r week, for gestation days (GD) 5-20. Exposure

levels are expressed as mg/m 3 of test substance. The exposures were conducted by whole-body exposure. Control animals were exposed to air enriched with n itrogen 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 exposur e to this test substance. The duration and frequency of the expos ures were the same as for the EMBSI study, and as recommended in the relevant EPA andOECD 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

### Huntingdon Life Sciences 05-4287 Page 11 211-DIPE-DEV Final Report

and high exposure levels were appr oximately 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 reco mmended 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 te st 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 expect ation of pregnancy rates of 80-100%, the group size of 24 mated female rats was considered appropriate for this study, to pr ovide 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

### Huntingdon Life Sciences 05-4287 Page 12 211-DIPE-DEV Final Report

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 id entity, 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 S ponsor 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.

### Huntingdon Life Sciences 05-4287 Page 13 211-DIPE-DEV Final Report

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 s ubstance 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

### Huntingdon Life Sciences 05-4287 Page 14 211-DIPE-DEV Final Report

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 da ys 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 anim al 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 anim al 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 t echnical staff for any conditions requiring possible veterinary care, and the staff veterinarian approved the animals for use in the study.

### Huntingdon Life Sciences 05-4287 Page 15 211-DIPE-DEV Final Report

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.

### Huntingdon Life Sciences 05-4287 Page 16 211-DIPE-DEV Final Report

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 an alysis 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

### Huntingdon Life Sciences 05-4287 Page 17 211-DIPE-DEV Final Report

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 tim e 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

### Huntingdon Life Sciences 05-4287 Page 18 211-DIPE-DEV Final Report

as the presence of clonic or t onic movements, stereotypy (e.g., excessive grooming, repetitive circli ng) 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 tissu es 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 ut erus, including the cervix, was weighed. Corpora lutea were counted and the number per ovary

### Huntingdon Life Sciences 05-4287 Page 19 211-DIPE-DEV Final Report

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 r azor 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. Followi ng 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%

### Huntingdon Life Sciences 05-4287 Page 20 211-DIPE-DEV Final Report

isopropyl alcohol for preservation a nd processed for staining of the skeleton using Alizarin Red S. Subsequently, these fetuses were evaluated for skeletal malformati ons 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 we re 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/feta l and early embryonic deaths were discarded.

2.15. STATISTICAL ANALYSIS
Analysis was performed by the Statis tics 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

### Huntingdon Life Sciences 05-4287 Page 21 211-DIPE-DEV Final Report

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 skelet al 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 Inha lation 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

### Huntingdon Life Sciences 05-4287 Page 22 211-DIPE-DEV Final Report

US EPA OPPTS Health Effects Test Guidelines 870.3700, Prenatal Developmental Toxicity Study EPA 712-C-98-207, August 1998.

2.17.2. GOOD LABORATORY PRACTICES
This study was conducted in accordance with US EPA 79.60, 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 o ccurred 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.

### Huntingdon Life Sciences 05-4287 Page 23 211-DIPE-DEV Final Report

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 resu lts 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/m 3 

### ) Analytical Concentration (mg/m 3 

### ) Nominal Concentration (mg/m 3 

### )

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) expos ure 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.

### Huntingdon Life Sciences 05-4287 Page 24 211-DIPE-DEV Final Report

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/m 3 

### )

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 sc heduled termination of the study and pregnancy rates were 92-100% across all the groups.

### Huntingdon Life Sciences 05-4287 Page 25 211-DIPE-DEV Final Report

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/m 3 

### exposed animals, as compared with the other groups. Chromodacryorrhea was also observed at a low incidence at 20000 mg/m 3 

### , in animals that did not show the nasal di scharge. These two findings (interpreted as secretion of porphyrin) are common reactions to mild stress in rats. An animal (#4908) exposed at 20000 mg/m 3


### 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/m 3 

### 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/m 3 

### and most noticeable at 20000 mg/m 3



### .

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/m 3 

### and most noticeable at 20000 mg/m 3



### .

### Huntingdon Life Sciences 05-4287 Page 26 211-DIPE-DEV Final Report

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/m 3 

### . 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 cha nge from Day 5” in Table 8 and Appendix J) was significantly reduced (95% of the control group value in the 20000 mg/m 3 

### 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 differen ces between the treated group and control group.

The percent reduction in uterine weight and in maternal net body weight change from Day 5 minus th e gravid uterine weight in the exposed groups was comparable with the minor reduction in maternal body weights and body wei ght changes observed in these
groups.

### Huntingdon Life Sciences 05-4287 Page 27 211-DIPE-DEV Final Report

3.4. FETAL DATA

3.4.1. Fetal Body Weights
(Table 7; Appendices I, K and M)

There was no effect of the exposur es 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, particular ly 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-tissu e and skeletal. No association with the exposures was inferred.

Although the incidence of 14 th 

### (supernumerary, rudimentary) ribs was higher at 20000 mg/m 3 

### than in the other groups, the difference from Control did not achieve sta tistical significance, and remained consistent with the range of incidences that are seen in this strain of animal. The mean group incide nce for this strain in this laboratory is approximated by the 2000 and 10000 mg/m 3 

### group mean incidences (7 and 5 fetuses in 5 litters, respectively), while the 20000 mg/m 3 

### 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.

### Huntingdon Life Sciences 05-4287 Page 28 211-DIPE-DEV Final Report

4. CONCLUSIONS Exposure of pregnant rats to atmos pheres of 0 (Air Control), 2000, 10000 or 20000 mg/m 3 

### of Gasoline DIPE Vapor Condensat e, 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/m 3 

### and most noticeable at 20000 mg/m 3 

### . It was considered that a noobserved-adverse-effect level (NOAEL) for maternal effects was evident at 2000 mg/m 3 

### . There was no effect of the exposures at any level on pregnancy outcome, in terms of pre- or post-implanta tion loss, fetal weight or the incidence of fetal abnormalities and variants. A NOAEL for embryo-fetal effects was therefore established at 20000 mg/m 3



### .

### Huntingdon Life Sciences 05-4287 Page 29 211-DIPE-DEV Final Report

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.

### Huntingdon Life Sciences 05-4287 Page 30 211-DIPE-DEV Final Report

CALCULATIONS

Feed Consumption (g/kg/day):

[(grams of feed presented – grams of feed re maining)/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 present ed in this report may be rounded. Unrounded individual animal data values ar e 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 discr epancies between the calculated values and those presented in this report.Huntingdon Life Sciences05-4287Page 32211-DIPE-DEVFinalReportMean Gestation Body Weights (grams)Figure 12002202402602803003203403603804004204404 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Body Weights (grams)Gestation DayGroup 1 - 0 mg/m3Group 2 - 2000 mg/m3Group 3 - 10000 mg/m3Group 4 - 20000 mg/m3Huntingdon Life Sciences05-4287Page 33211-DIPE-DEVFinalReportMean Gestation Feed Consumption (g/kg/day)Figure 244495459646974798489944 to 5 5 to 8 8 to 11 11 to 14 14 to 17 17 to 20 20 to 21 5 to 21

g/kg/dayGestation Day IntervalGroup 1 - 0 mg/m3Group 2 - 2000 mg/m3Group 3 - 10000 mg/m3Group 4 - 20000 mg/m310-MAR-2011 17:37Huntingdon Life Sciences 05-4287TABLE 1GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSURESUMMARY OF SURVIVAL AND PREGNANCY-----------------------------------------------------------------------------------------------------------------------------------DOSE GROUP: 1 2 3 4DOSE LEVEL (MG/M3): 0 2000 10000 20000-----------------------------------------------------------------------------------------------------------------------------------No. of females mated N 24 24 24 24Pregnant N 23 23 24 22- Died/sacrificed moribund N 0 0 0 0- Elective sacrifice N 0 0 0 0Nonpregnant N 1 1 0 2- Died/sacrificed moribund N 0 0 0 0- Elective sacrifice N 0 0 0 0Total no. of females died/ N 0 0 0 0sacrificed moribund % 0.0 0.0 0.0 0.0Examined at scheduled c-section N 24 24 24 24- Nonpregnant N 1 1 0 2- With total implant loss N 0 0 0 0% 0.0 0.0 0.0 0.0- With viable fetuses N 23 23 24 22% 95.8 95.8 100.0 91.7-----------------------------------------------------------------------------------------------------------------------------------Page 3410-MAR-2011 17:41Huntingdon Life Sciences 05-4287TABLE 2GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSURESUMMARY OF CLINICAL OBSERVATIONS DURING GESTATION - (frequency/animals)-----------------------------------------------------------------------------------------------------------------------------------DOSE GROUP: 1 2 3 4DOSE LEVEL (MG/M3): 0 2000 10000 20000-----------------------------------------------------------------------------------------------------------------------------------DAY 4 to 21Normal----------WITHIN NORMAL LIMITS 431/24 421/24 382/24 349/23TERMINAL SACRIFICE 24/24 24/24 24/24 24/24Dermal-General------------------ALOPECIA - GENERAL 0/ 0 0/ 0 0/ 0 8/ 1Ocular----------CHROMODACRYORRHEA - UNILATERAL 0/ 0 0/ 0 0/ 0 14/ 3Oral/Buccal---------------INCISORS MALOCCLUDED 0/ 0 0/ 0 0/ 0 18/ 1NASAL DISCHARGE - RED 1/ 1 11/ 3 50/15 43/ 9Palpable masses-------------------MASS 0/ 0 0/ 0 0/ 0 5/ 1Page 3510-MAR-2011 17:43Huntingdon Life Sciences 05-4287TABLE 3GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSURESUMMARY OF GESTATION BODY WEIGHTS (GRAMS)-----------------------------------------------------------------------------------------------------------------------------------DOSE GROUP: 1 2 3 4DOSE LEVEL (MG/M3): 0 2000 10000 20000-----------------------------------------------------------------------------------------------------------------------------------DAY 4 MEAN 234 235 234 235S.D. 14.0 16.6 15.7 14.6N 23 23 24 22DAY 5 MEAN 244 245 244 245S.D. 14.6 16.5 13.9 15.2N 23 23 24 22DAY 8 MEAN 259 260 256 256S.D. 12.8 18.0 14.4 16.3N 23 23 24 22DAY 11 MEAN 281 279 274 275S.D. 14.9 19.6 15.6 19.5N 23 23 24 22DAY 14 MEAN 301 296 292 290S.D. 17.0 20.3 14.8 19.5N 23 23 24 22DAY 17 MEAN 332 327 323 321S.D. 22.2 24.3 18.7 24.9N 23 23 24 22DAY 20 MEAN 378 370 367 365S.D. 26.3 29.9 24.2 27.1N 23 23 24 22DAY 21 MEAN 388 379 376 374S.D. 28.6 32.7 25.9 27.4N 23 23 24 22-----------------------------------------------------------------------------------------------------------------------------------Page 3610-MAR-2011 17:44Huntingdon Life Sciences 05-4287TABLE 4GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSURESUMMARY OF GESTATION BODY WEIGHT GAIN (GRAMS)-----------------------------------------------------------------------------------------------------------------------------------DOSE GROUP: 1 2 3 4DOSE LEVEL (MG/M3): 0 2000 10000 20000-----------------------------------------------------------------------------------------------------------------------------------DAYS 4 TO 5 MEAN 10 11 10 10S.D. 6.1 4.7 5.2 3.2N 23 23 24 22DAYS 5 TO 8 MEAN 15 15 13 11S.D. 4.5 4.7 5.5 4.7N 23 23 24 22DAYS 8 TO 11 MEAN 22 19 18 20S.D. 4.7 3.6 3.9 5.5N 23 23 24 22DAYS 11 TO 14 MEAN 20 17 17 15S.D. 4.2 5.0 3.7 4.0N 23 23 24 22DAYS 14 TO 17 MEAN 31 31 31 31S.D. 7.3 6.9 7.6 6.8N 23 23 24 22DAYS 17 TO 20 MEAN 46 44 44 44S.D. 7.6 8.9 9.9 7.7N 23 23 24 22DAYS 20 TO 21 MEAN 10 9 9 8S.D. 5.2 5.0 4.9 5.1N 23 23 24 22DAYS 5 TO 14 MEAN 57 50 48 45S.D. 8.4 7.8 7.7 9.5N 23 23 24 22DAYS 14 TO 21 MEAN 86 84 84 83S.D. 15.5 16.3 17.0 10.0N 23 23 24 22DAYS 5 TO 21 MEAN 144 134 132 129S.D. 20.6 21.5 21.0 16.0N 23 23 24 22-----------------------------------------------------------------------------------------------------------------------------------Page 3710-MAR-2011 17:45Huntingdon Life Sciences 05-4287TABLE 5GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSURESUMMARY OF GESTATION FEED CONSUMPTION (GRAMS/KG/DAY)-----------------------------------------------------------------------------------------------------------------------------------DOSE GROUP: 1 2 3 4DOSE LEVEL (MG/M3): 0 2000 10000 20000-----------------------------------------------------------------------------------------------------------------------------------DAYS 4 TO 5 MEAN 71 74 75 73S.D. 10.5 11.5 11.4 10.2N 22 23 24 22DAYS 5 TO 8 MEAN 81 85 80 79S.D. 6.0 14.1 6.0 6.0N 22 23 24 21DAYS 8 TO 11 MEAN 85 82 80 78S.D. 7.1 4.6 5.5 5.7N 22 23 24 21DAYS 11 TO 14 MEAN 85 82 82 78S.D. 7.1 4.7 3.9 4.0N 22 23 22 21DAYS 14 TO 17 MEAN 81 81 81 79S.D. 4.5 4.5 6.0 4.4N 22 23 22 21DAYS 17 TO 20 MEAN 74 73 74 73S.D. 4.5 3.3 3.7 4.3N 23 23 22 22DAYS 20 TO 21 MEAN 53 47 47 46S.D. 8.1 7.5 5.8 7.8N 23 23 24 22DAYS 5 TO 21 MEAN 76 75 74 72S.D. 4.7 4.0 3.6 3.8N 23 23 24 22-----------------------------------------------------------------------------------------------------------------------------------Page 3810-MAR-2011 17:46Huntingdon Life Sciences 05-4287TABLE 6GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSURESUMMARY OF MATERNAL NECROPSY OBSERVATIONS-----------------------------------------------------------------------------------------------------------------------------------DOSE GROUP: 1 2 3 4DOSE LEVEL (MG/M3): 0 2000 10000 20000-----------------------------------------------------------------------------------------------------------------------------------FEMALES N 24 24 24 24GROSS EXAM N 0 0 0 1----------MASS N 0 0 0 1% 0.0 0.0 0.0 4.2-----------------------------------------------------------------------------------------------------------------------------------Page 3910-MAR-2011 17:48Huntingdon Life Sciences 05-4287TABLE 7GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSURESUMMARY OF CESAREAN SECTION DATA-----------------------------------------------------------------------------------------------------------------------------------DOSE GROUP: 1 2 3 4DOSE LEVEL (MG/M3): 0 2000 10000 20000-----------------------------------------------------------------------------------------------------------------------------------Pregnant N 23 23 24 22(at scheduled sacrifice)Dams with no Viable Fetuses N 0 0 0 0Dams with Viable Fetuses N 23 23 24 22Corpora Lutea TOTAL 343 334 354 334No. per animal MEAN 14.9 14.5 14.8 15.2S.D. 2.78 2.06 2.52 2.63Implantation Sites TOTAL 311 310 315 307No. per animal MEAN 13.5 13.5 13.1 14.0S.D. 2.71 1.50 2.85 2.38Preimplantation Loss TOTAL 32 24 39 27No. per animal MEAN 1.4 1.0 1.6 1.2S.D. 1.88 1.46 2.96 1.77% per animal MEAN% 9.3 6.6 10.4 7.6S.D. 13.46 7.99 18.58 9.14Live Fetuses TOTAL 300 286 302 292No. per animal MEAN 13.0 12.4 12.6 13.3S.D. 2.64 2.64 3.06 2.60Males TOTAL 146 133 154 135MEAN% 48.5 46.1 51.2 46.8S.D. 10.94 14.75 12.50 13.92Females TOTAL 154 153 148 157MEAN% 51.5 53.9 48.8 53.2S.D. 10.94 14.75 12.50 13.92-----------------------------------------------------------------------------------------------------------------------------------Page 4010-MAR-2011 17:48Huntingdon Life Sciences 05-4287TABLE 7GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSURESUMMARY OF CESAREAN SECTION DATA-----------------------------------------------------------------------------------------------------------------------------------DOSE GROUP: 1 2 3 4DOSE LEVEL (MG/M3): 0 2000 10000 20000-----------------------------------------------------------------------------------------------------------------------------------Postimplantation Loss TOTAL 11 24 13 15No. per animal MEAN 0.5 1.0 0.5 0.7S.D. 0.67 2.08 0.83 0.65% implants per animal MEAN% 3.4 7.9 5.1 5.2S.D. 4.66 14.89 8.64 5.15Dead Fetuses TOTAL 0 0 0 0No. per animal MEAN 0.0 0.0 0.0 0.0S.D. 0.00 0.00 0.00 0.00% of implants per animal MEAN% 0.0 0.0 0.0 0.0S.D. 0.00 0.00 0.00 0.00Resorptions: Early TOTAL 10 23 13 15No. per animal MEAN 0.4 1.0 0.5 0.7S.D. 0.66 2.07 0.83 0.65% of implants per animal MEAN% 3.1 7.6 5.1 5.2S.D. 4.66 14.80 8.64 5.15Resorptions: Late TOTAL 1 1 0 0No. per animal MEAN 0.0 0.0 0.0 0.0S.D. 0.21 0.21 0.00 0.00% of implants per animal MEAN% 0.3 0.3 0.0 0.0S.D. 1.39 1.60 0.00 0.00-----------------------------------------------------------------------------------------------------------------------------------Page 4110-MAR-2011 17:48Huntingdon Life Sciences 05-4287TABLE 7GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSURESUMMARY OF CESAREAN SECTION DATA-----------------------------------------------------------------------------------------------------------------------------------DOSE GROUP: 1 2 3 4DOSE LEVEL (MG/M3): 0 2000 10000 20000-----------------------------------------------------------------------------------------------------------------------------------Fetal Body Weight (g) MEAN 5.7 5.8 5.7 5.5S.D. 0.29 0.30 0.28 0.32N 23 23 24 22Male Fetuses MEAN 5.9 5.9 5.8 5.7S.D. 0.32 0.33 0.32 0.33Female Fetuses MEAN 5.6 5.6 5.5 5.4S.D. 0.28 0.33 0.25 0.31-----------------------------------------------------------------------------------------------------------------------------------Page 4210-MAR-2011 17:49Huntingdon Life Sciences 05-4287TABLE 8GASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSURESUMMARY OF GRAVID UTERINE WEIGHT AND NET BODY WEIGHT CHANGE (GRAMS)-----------------------------------------------------------------------------------------------------------------------------------DOSE GROUP: 1 2 3 4DOSE LEVEL (MG/M3): 0 2000 10000 20000-----------------------------------------------------------------------------------------------------------------------------------UTERUS WEIGHT MEAN 101 97 96 96S.D. 19.4 19.8 22.5 15.7N 22 23 24 20ADJUSTED WEIGHT MEAN 286 283 280 273S.D. 18.1 19.0 17.1 15.9N 22 23 24 20NET WEIGHT CHANGE FROM DAY 5 MEAN 43 37 36 29S.D. 11.1 9.1 11.7 10.6N 22 23 24 20WEIGHT CHANGE FROM DAY 5 MEAN 144 134 132 129S.D. 20.6 21.5 21.0 16.0N 23 23 24 22-----------------------------------------------------------------------------------------------------------------------------------UTERUS WEIGHT = ABSOLUTE GRAVID UTERINE WEIGHTADJUSTED WEIGHT = TERMINAL BODY WEIGHT - UTERUS WEIGHTNET WEIGHT CHANGE FROM DAY 5 = ADJUSTED WEIGHT – DAY 5 BODY WEIGHTWEIGHT CHANGE FROM DAY 5 = TERMINAL BODY WEIGHT – DAY 5 BODY WEIGHTPage 43Huntingdon Life Sciences 05-4287 Page 44211-DIPE-DEV Final ReportFemalesSummary of Fetal ObservationsTable 9Fetuses LittersObservations Group 1 2 3 4 1 2 3 4(Major abnormalities in CAPITALS) Number examined 300 286 302 292 23 23 24 22HYDROCEPHALYa, ANOPHTHALMIA; HYPOPLASTIC APICAL LUNGLOBE, THYMUS AND SPLEEN1 1MICROPHTHALMIA/ANOPHTHALMIA, UNILATERAL 1 1EXOCCIPITAL FUSED TO 1ST CERVICAL ARCH 1 1MANDIBLE SHORTENED, MODERATE 1 1THORACIC VERTEBRA 13, LUMBAR VERTEBRA 1: ACENTRIC,MALFORMED1 1FORELIMB SHORTENED, RIGHT 1 1ANAL ATRESIA, ABSENT SACRAL AND CAUDAL VERTEBRAE 1 1Additional subclavian artery 1 1Innominate artery absent 1 2 1 1 2 1Undescended thymic tissue 10 11 5 11 6 8 3 8aNoted as DOMED CRANIUM in external observations.Huntingdon Life Sciences 05-4287 Page 45211-DIPE-DEV Final ReportFemalesSummary of Fetal ObservationsTable 9Fetuses LittersObservations Group 1 2 3 4 1 2 3 4(Major abnormalities in CAPITALS) Number examined 300 286 302 292 23 23 24 22Renal papilla (e) not fully developed12 112 1Ureter(s) distended11 111 1Left umbilical artery1412 1312Testis displaced, medially, left117th cervical rib(s) present1313Sternebra(e) malaligned11 11 13 39 9 10 3Rib(s) rudimentary, 13th1311Rib(s) rudimentary, 14th2 7 5 12 1 5 5 925 presacral vertebrae131127 presacral vertebrae11Huntingdon Life Sciences 05-4287 Page 46211-DIPE-DEV Final ReportFemalesSummary of Fetal ObservationsTable 9Fetuses LittersObservations Group 1 2 3 4 1 2 3 4(Major abnormalities in CAPITALS) Number examined 300 286 302 292 23 23 24 22Hyoid body/arch(es) unossified11 111 1Sternebra5and/or6unossified1 33 1 32Metatarsals: 1 unossified, bilateral21Page 47Page 48Page 49Page 50Page 51Page 52Page 53Page 54Page 55Page 56Page 57Page 58Page 59Page 60Page 61

### Huntingdon Life Sciences 05-4287 Page 62 211-DIPE-DEV Final Report

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 Concentrati on 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 Monitori ng Results .....................................................................73 III. Equipment List ...........................................................................................77 IV. Chamber Distribution Records ..................................................................80 V. Miran Calibration .......................................................................................81 VI. Testing Room and Chambers Environmental Monitoring .........................84

### Huntingdon Life Sciences 05-4287 Page 63 211-DIPE-DEV Final Report

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 main tained, 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

### Huntingdon Life Sciences 05-4287 Page 64 211-DIPE-DEV Final Report

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. Th e 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 met hod 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/m 3 

### Group 2 - 2000 mg/m 3 

### Group 3 - 10000 mg/m 3 

### Group 4 - 20000 mg/m 3

### Huntingdon Life Sciences 05-4287 Page 65 211-DIPE-DEV Final Report

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 T 99 

### equilibrium time (calculated by multiplying the air change by the expone ntial factor 4.6) of at most 21-22 minutes. Initial settings for each group were as follows:

Grou p Airflow Rate Air Chan g e T 99 

### (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 T 99 

### ) 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.

### Huntingdon Life Sciences 05-4287 Page 66 211-DIPE-DEV Final Report

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 fl ow 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 ma intain 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 equipm ent contamination. From the filter tube, the test substance fl owed to a liquid flowmeter via 1 

### / 8 

### ” tubing. The outlet of the flowmeter was regulated by a built-in metering valve. From this flowme ter 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 nitr ogen flow continuously purged the

### Huntingdon Life Sciences 05-4287 Page 67 211-DIPE-DEV Final Report

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 nitr ogen flowed via 1⁄4” tubing to a ball and socket joint at the botto m 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 turre t 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/m 3 

### 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/m 3 

### ) = amount consumed (g) x 1000 mg/g x 1000 L/m 3 

### 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 (D ay 12) and end of the study (Day 19). Light (maintained appr oximately 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.

### Huntingdon Life Sciences 05-4287 Page 71 211-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.Huntingdon Life Sciences05-4287Page 72211-DIPE-DEV FinalReportInhalation ReportAppendix BTable ISummary of In-Chamber ObservationsExposure Day1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19Group 1 – 0 mg/m3Within Normal Limits 6 12 18 24 24 24 24 24 24 24 24 24 24 24 24 24 18 12 6Group 2 – 2000 mg/m3Within Normal Limits 6 12 18 24 24 24 24 24 24 24 24 24 24 24 24 24 18 12 6Group 3 – 10000 mg/m3Within Normal Limits 6 12 18 24 24 24 24 24 24 24 24 24 24 24 24 24 18 12 6Group 4 – 20000 mg/m3Within Normal Limits 6 12 18 24 24 24 24 24 24 24 24 24 24 24 24 24 18 12 6Huntingdon Life Sciences05-4287211-DIPE-DEVAppendix B - Table IIPage 73Final ReportChamber Monitoring ResultsCumulative Exposure RecordGroup 1 - 0 mg/m3(Air only)Chamber EnvironmentDay Date Exposure Chamber Concentration Determinations Temperature HumidityNumber 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 23 421 7-Aug-05 2 0 0.00 0.00 0.00 0.00 0.00 23 412 8-Aug-05 3 0 0.00 0.00 0.00 0.00 0.00 24 393 9-Aug-05 4 0 0.00 0.00 0.00 0.00 0.00 0.8011 2.031 4.25E-03 24 404 10-Aug-05 5 0 0.00 0.00 0.00 0.00 0.00 24 415 11-Aug-05 6 0 0.00 0.00 0.00 0.00 0.00 24 406 12-Aug-05 7 0 0.00 0.00 0.00 0.00 0.00 25 407 13-Aug-05 8 0 0.00 0.00 0.00 0.00 0.00 25 428 14-Aug-05 9 0 0.00 0.00 0.00 0.00 0.00 24 439 15-Aug-05 10 0 0.00 0.00 0.00 0.00 0.00 25 3910 16-Aug-05 11 0 0.00 0.00 0.00 0.00 0.00 0.7675 1.792 4.78E-03 24 4011 17-Aug-05 12 0 0.00 0.00 0.00 0.00 0.00 25 4012 18-Aug-05 13 0 0.00 0.00 0.00 0.00 0.00 25 4013 19-Aug-05 14 0 0.00 0.00 0.00 0.00 0.00 25 4014 20-Aug-05 15 0 0.00 0.00 0.00 0.00 0.00 24 4015 21-Aug-05 16 0 0.00 0.00 0.00 0.00 0.00 25 4016 22-Aug-05 17 0 0.00 0.00 0.00 0.00 0.00 24 4017 23-Aug-05 18 0 0.00 0.00 0.00 0.00 0.00 8.2210 2.276 1.64E-03 24 4118 24-Aug-05 19 0 0.00 0.00 0.00 0.00 0.00 23 41Mean 0 0.00 3.263 2.033 3.56E-03 24 40S.D. 0 0.00 4.3 0.2 0.0 0.7 1.0Particle Size Mean(mg/m3)Huntingdon Life Sciences05-4287211-DIPE-DEVAppendix B - Table IIPage 74Final ReportChamber Monitoring ResultsCumulative Exposure RecordGroup 2 - 2000 mg/m3Chamber EnvironmentDay Date Exposure Chamber Concentration Temperature HumidityNumber 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 23 431 7-Aug-05 2 2000 1900 2000 1900 1800 1900 23 422 8-Aug-05 3 2000 2000 2200 1800 2000 2000 23 403 9-Aug-05 4 2100 2100 2200 2000 2000 2000 3.882 2.954 7.23E-03 24 414 10-Aug-05 5 2000 2000 2100 2000 1900 2100 24 435 11-Aug-05 6 2000 2000 2000 2200 2100 1800 24 426 12-Aug-05 7 2000 2000 2100 2000 2000 2000 24 417 13-Aug-05 8 2000 2000 2000 1900 2000 2000 24 438 14-Aug-05 9 2000 2000 1900 2000 2000 2000 24 459 15-Aug-05 10 2000 2000 2100 2000 2000 2000 24 4110 16-Aug-05 11 1900 2000 2000 2000 2000 1900 0.7935 2.294 6.14E-03 24 4211 17-Aug-05 12 2000 2000 2000 1900 2100 2100 24 4112 18-Aug-05 13 2000 2000 1900 1900 2000 2000 24 4213 19-Aug-05 14 2000 2000 1900 2100 2000 2000 24 4214 20-Aug-05 15 1900 2000 2000 1800 2000 2000 24 4215 21-Aug-05 16 2000 2000 2000 1900 2100 2000 24 4216 22-Aug-05 17 2000 1900 1900 1900 1900 2000 24 4217 23-Aug-05 18 2000 2000 2000 1900 2000 2000 7.508 2.057 2.08E-03 23 4318 24-Aug-05 19 2000 2000 2100 1800 2000 2100 23 42Mean 1995 1982 4.061 2.435 5.15E-03 24 42S.D. 40 93 3.4 0.5 0.0 0.5 1.1Mean(mg/m3)Particle SizeDeterminationsHuntingdon Life Sciences05-4287211-DIPE-DEVAppendix B - Table IIPage 75Final ReportChamber Monitoring ResultsCumulative Exposure RecordGroup 3 - 10000 mg/m3Chamber EnvironmentDay Date Exposure Chamber Concentration Temperature HumidityNumber 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 23 421 7-Aug-05 2 11000 9800 9400 9700 10000 9900 24 412 8-Aug-05 3 11000 9900 10000 10000 9800 9900 24 413 9-Aug-05 4 11000 9600 9300 9500 9700 9900 0.8112 2.450 5.75E-03 24 414 10-Aug-05 5 12000 10000 11000 10000 10000 10000 25 415 11-Aug-05 6 12000 10000 9700 9900 9400 11000 25 416 12-Aug-05 7 11000 9700 9900 9600 9400 9700 25 407 13-Aug-05 8 12000 10000 10000 10000 10000 10000 25 428 14-Aug-05 9 12000 9800 9700 10000 10000 9400 25 449 15-Aug-05 10 12000 9800 9600 9900 9700 10000 25 4010 16-Aug-05 11 12000 10000 9900 9800 11000 11000 4.952 3.334 1.07E-02 25 4111 17-Aug-05 12 12000 10000 10000 10000 10000 10000 25 4012 18-Aug-05 13 11000 10000 10000 10000 10000 10000 25 3913 19-Aug-05 14 11000 9800 9600 9700 9900 9900 25 4214 20-Aug-05 15 12000 10000 11000 9700 9700 10000 25 4215 21-Aug-05 16 12000 11000 11000 11000 11000 10000 25 4216 22-Aug-05 17 11000 10000 11000 10000 10000 10000 24 4017 23-Aug-05 18 11000 10000 11000 9100 9600 11000 5.523 2.246 1.91E-03 24 4118 24-Aug-05 19 11000 11000 11000 12000 10000 11000 23 41Mean 11474 10072 3.762 2.677 6.12E-03 25 41S.D. 513 546 2.6 0.6 0.0 0.7 1.1Mean(mg/m3)Particle SizeDeterminationsHuntingdon Life Sciences05-4287211-DIPE-DEVAppendix B - Table IIPage 76Final ReportChamber Monitoring ResultsCumulative Exposure RecordGroup 4 - 20000 mg/m3Chamber EnvironmentDay Date Exposure Chamber Concentration Temperature HumidityNumber 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 23 421 7-Aug-05 2 21000 21000 19000 20000 22000 21000 23 412 8-Aug-05 3 19000 20000 21000 20000 19000 19000 24 403 9-Aug-05 4 21000 20000 21000 20000 20000 20000 0.7811 2.137 4.84E-03 24 404 10-Aug-05 5 20000 20000 18000 18000 22000 20000 24 415 11-Aug-05 6 20000 20000 18000 20000 21000 20000 24 416 12-Aug-05 7 20000 20000 19000 20000 20000 20000 24 417 13-Aug-05 8 20000 20000 19000 19000 19000 21000 24 448 14-Aug-05 9 19000 19000 21000 20000 18000 18000 24 459 15-Aug-05 10 20000 20000 20000 20000 19000 19000 24 4110 16-Aug-05 11 19000 20000 20000 20000 19000 19000 0.7640 2.260 9.80E-03 24 4111 17-Aug-05 12 20000 21000 21000 21000 21000 20000 24 4112 18-Aug-05 13 20000 20000 19000 20000 20000 20000 24 4113 19-Aug-05 14 20000 20000 20000 21000 18000 21000 24 4214 20-Aug-05 15 19000 20000 19000 20000 20000 20000 24 4215 21-Aug-05 16 19000 20000 20000 19000 20000 19000 24 4216 22-Aug-05 17 20000 20000 21000 20000 20000 20000 24 4017 23-Aug-05 18 20000 20000 20000 20000 20000 20000 7.810 2.272 4.24E-03 23 4118 24-Aug-05 19 20000 19000 17000 20000 20000 20000 23 40Mean 19842 19776 3.118 2.223 6.29E-03 24 41S.D. 602 961 3.3 0.1 0.0 0.4 1.3Mean(mg/m3)Particle SizeDeterminations

### 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 stainles s steel chamber (Wahmann).

Compound Reservoir Sponsor cylinder (100 gallons) and smaller cyli nders (5 gallons) filled from Sponsor

cylinder and used on study.

Compound Generator Counter-Current Volatilizati on 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 Trak TM 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 backpressu re gauge, P/N 12672-1. Ashcroft backpressure gauge, P/N 733-47. Matheson ® 

### backpressure ga uge, 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.), ca librated 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 ® 

### Microfibre TM 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 Eps on 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 In strument 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/m 3 

### ) 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/m 3 

### ) 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/m 3 

### ) 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 calibra tion series was performed as shown below. The resultant data were plotted to obtain a calibration curve. Each observer used a separate syringe for calibration.

Injection Calculated Absorbance Volume Concentration 1 

### Operator 1 Operator 2 Average (μL) (mg/m 3 

### ) (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/m 3 

### ) = Injection volume (μL) x Density ( 0.68 mg/μL) x 1000 L/m 3 

### 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 = -12213x 4 

### + 16436x 3 

### - 751.71x 2 

### + 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 inj ection. 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 tw ice. 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/m 3 

### ) Light (Ft Candles) Noise (dB) Oxygen (%) Particle Sizing (mg/m 3 

### ) 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 17:08Huntingdon Life Sciences 05-4287APPENDIX CGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL ANIMAL TERMINATION HISTORYFEMALES GROUP 1 0 MG/M3-----------------------------------------------------------------------------------------------------------------------------------TYPE OF DATE OF GESTATION PREGNANCYANIMAL# DEATH DEATH DAY STATUS-----------------------------------------------------------------------------------------------------------------------------------1901 TERMINAL SACRIFICE 22-AUG-05 21 P1902 TERMINAL SACRIFICE 22-AUG-05 21 P1903 TERMINAL SACRIFICE 22-AUG-05 21 P1904 TERMINAL SACRIFICE 22-AUG-05 21 P1905 TERMINAL SACRIFICE 22-AUG-05 21 P1906 TERMINAL SACRIFICE 22-AUG-05 21 P1907 TERMINAL SACRIFICE 23-AUG-05 21 P1908 TERMINAL SACRIFICE 23-AUG-05 21 P1909 TERMINAL SACRIFICE 23-AUG-05 21 P1910 TERMINAL SACRIFICE 23-AUG-05 21 P1911 TERMINAL SACRIFICE 23-AUG-05 21 P1912 TERMINAL SACRIFICE 23-AUG-05 21 P1913 TERMINAL SACRIFICE 24-AUG-05 21 P1914 TERMINAL SACRIFICE 24-AUG-05 21 P1915 TERMINAL SACRIFICE 24-AUG-05 21 NP1916 TERMINAL SACRIFICE 24-AUG-05 21 P1917 TERMINAL SACRIFICE 24-AUG-05 21 P1918 TERMINAL SACRIFICE 24-AUG-05 21 P1919 TERMINAL SACRIFICE 25-AUG-05 21 P1920 TERMINAL SACRIFICE 25-AUG-05 21 P1921 TERMINAL SACRIFICE 25-AUG-05 21 P1922 TERMINAL SACRIFICE 25-AUG-05 21 P1923 TERMINAL SACRIFICE 25-AUG-05 21 P1924 TERMINAL SACRIFICE 25-AUG-05 21 P-----------------------------------------------------------------------------------------------------------------------------------NP-NOT PREGNANT, P-PREGNANTPage 8510-MAR-2011 17:08Huntingdon Life Sciences 05-4287APPENDIX CGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL ANIMAL TERMINATION HISTORYFEMALES GROUP 2 2000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------TYPE OF DATE OF GESTATION PREGNANCYANIMAL# DEATH DEATH DAY STATUS-----------------------------------------------------------------------------------------------------------------------------------2901 TERMINAL SACRIFICE 22-AUG-05 21 P2902 TERMINAL SACRIFICE 22-AUG-05 21 P2903 TERMINAL SACRIFICE 22-AUG-05 21 P2904 TERMINAL SACRIFICE 22-AUG-05 21 P2905 TERMINAL SACRIFICE 22-AUG-05 21 P2906 TERMINAL SACRIFICE 22-AUG-05 21 P2907 TERMINAL SACRIFICE 23-AUG-05 21 P2908 TERMINAL SACRIFICE 23-AUG-05 21 P2909 TERMINAL SACRIFICE 23-AUG-05 21 P2910 TERMINAL SACRIFICE 23-AUG-05 21 P2911 TERMINAL SACRIFICE 23-AUG-05 21 P2912 TERMINAL SACRIFICE 23-AUG-05 21 P2913 TERMINAL SACRIFICE 24-AUG-05 21 P2914 TERMINAL SACRIFICE 24-AUG-05 21 P2915 TERMINAL SACRIFICE 24-AUG-05 21 P2916 TERMINAL SACRIFICE 24-AUG-05 21 P2917 TERMINAL SACRIFICE 24-AUG-05 21 P2918 TERMINAL SACRIFICE 24-AUG-05 21 P2919 TERMINAL SACRIFICE 25-AUG-05 21 NP2920 TERMINAL SACRIFICE 25-AUG-05 21 P2921 TERMINAL SACRIFICE 25-AUG-05 21 P2922 TERMINAL SACRIFICE 25-AUG-05 21 P2923 TERMINAL SACRIFICE 25-AUG-05 21 P2924 TERMINAL SACRIFICE 25-AUG-05 21 P-----------------------------------------------------------------------------------------------------------------------------------NP-NOT PREGNANT, P-PREGNANTPage 8610-MAR-2011 17:08Huntingdon Life Sciences 05-4287APPENDIX CGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL ANIMAL TERMINATION HISTORYFEMALES GROUP 3 10000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------TYPE OF DATE OF GESTATION PREGNANCYANIMAL# DEATH DEATH DAY STATUS-----------------------------------------------------------------------------------------------------------------------------------3901 TERMINAL SACRIFICE 22-AUG-05 21 P3902 TERMINAL SACRIFICE 22-AUG-05 21 P3903 TERMINAL SACRIFICE 22-AUG-05 21 P3904 TERMINAL SACRIFICE 22-AUG-05 21 P3905 TERMINAL SACRIFICE 22-AUG-05 21 P3906 TERMINAL SACRIFICE 22-AUG-05 21 P3907 TERMINAL SACRIFICE 23-AUG-05 21 P3908 TERMINAL SACRIFICE 23-AUG-05 21 P3909 TERMINAL SACRIFICE 23-AUG-05 21 P3910 TERMINAL SACRIFICE 23-AUG-05 21 P3911 TERMINAL SACRIFICE 23-AUG-05 21 P3912 TERMINAL SACRIFICE 23-AUG-05 21 P3913 TERMINAL SACRIFICE 24-AUG-05 21 P3914 TERMINAL SACRIFICE 24-AUG-05 21 P3915 TERMINAL SACRIFICE 24-AUG-05 21 P3916 TERMINAL SACRIFICE 24-AUG-05 21 P3917 TERMINAL SACRIFICE 24-AUG-05 21 P3918 TERMINAL SACRIFICE 24-AUG-05 21 P3919 TERMINAL SACRIFICE 25-AUG-05 21 P3920 TERMINAL SACRIFICE 25-AUG-05 21 P3921 TERMINAL SACRIFICE 25-AUG-05 21 P3922 TERMINAL SACRIFICE 25-AUG-05 21 P3923 TERMINAL SACRIFICE 25-AUG-05 21 P3924 TERMINAL SACRIFICE 25-AUG-05 21 P-----------------------------------------------------------------------------------------------------------------------------------NP-NOT PREGNANT, P-PREGNANTPage 8710-MAR-2011 17:08Huntingdon Life Sciences 05-4287APPENDIX CGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL ANIMAL TERMINATION HISTORYFEMALES GROUP 4 20000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------TYPE OF DATE OF GESTATION PREGNANCYANIMAL# DEATH DEATH DAY STATUS-----------------------------------------------------------------------------------------------------------------------------------4901 TERMINAL SACRIFICE 22-AUG-05 21 P4902 TERMINAL SACRIFICE 22-AUG-05 21 P4903 TERMINAL SACRIFICE 22-AUG-05 21 P4904 TERMINAL SACRIFICE 22-AUG-05 21 P4905 TERMINAL SACRIFICE 22-AUG-05 21 P4906 TERMINAL SACRIFICE 22-AUG-05 21 P4907 TERMINAL SACRIFICE 23-AUG-05 21 P4908 TERMINAL SACRIFICE 23-AUG-05 21 NP4909 TERMINAL SACRIFICE 23-AUG-05 21 P4910 TERMINAL SACRIFICE 23-AUG-05 21 P4911 TERMINAL SACRIFICE 23-AUG-05 21 P4912 TERMINAL SACRIFICE 23-AUG-05 21 P4913 TERMINAL SACRIFICE 24-AUG-05 21 P4914 TERMINAL SACRIFICE 24-AUG-05 21 P4915 TERMINAL SACRIFICE 24-AUG-05 21 P4916 TERMINAL SACRIFICE 24-AUG-05 21 P4917 TERMINAL SACRIFICE 24-AUG-05 21 P4918 TERMINAL SACRIFICE 24-AUG-05 21 P4919 TERMINAL SACRIFICE 25-AUG-05 21 NP4920 TERMINAL SACRIFICE 25-AUG-05 21 P4921 TERMINAL SACRIFICE 25-AUG-05 21 P4922 TERMINAL SACRIFICE 25-AUG-05 21 P4923 TERMINAL SACRIFICE 25-AUG-05 21 P4924 TERMINAL SACRIFICE 25-AUG-05 21 P-----------------------------------------------------------------------------------------------------------------------------------NP-NOT PREGNANT, P-PREGNANTPage 8810-MAR-2011 17:13Huntingdon Life Sciences 05-4287APPENDIX DGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATIONGROUP 1 0 MG/M3-----------------------------------------------------------------------------------------------------------------------------------DAY OF 1 1 1 1 1 1 1 1 1 1 2 2FEMALE# 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 PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P1902 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P1903 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P1904 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P1905 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P1906 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P1907 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P1908 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P1909 TERMINAL SACRIFICE PNASAL DISCHARGE - RED PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P1910 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P1911 TERMINAL SACRIFICE PWITHIN 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-PRESENTPage 8910-MAR-2011 17:13Huntingdon Life Sciences 05-4287APPENDIX DGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATIONGROUP 1 0 MG/M3-----------------------------------------------------------------------------------------------------------------------------------DAY OF 1 1 1 1 1 1 1 1 1 1 2 2FEMALE# 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 PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P1913 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P1914 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P1915 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P1916 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P1917 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P1918 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P1919 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P P1920 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P P1921 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P P1922 TERMINAL SACRIFICE PWITHIN 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-PRESENTPage 9010-MAR-2011 17:13Huntingdon Life Sciences 05-4287APPENDIX DGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATIONGROUP 1 0 MG/M3-----------------------------------------------------------------------------------------------------------------------------------DAY OF 1 1 1 1 1 1 1 1 1 1 2 2FEMALE# 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 PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P P1924 TERMINAL SACRIFICE PWITHIN 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-PRESENTPage 9110-MAR-2011 17:13Huntingdon Life Sciences 05-4287APPENDIX DGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATIONGROUP 2 2000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------DAY OF 1 1 1 1 1 1 1 1 1 1 2 2FEMALE# 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 PNASAL DISCHARGE - RED P PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P2902 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P2903 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P2904 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P2905 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P2906 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P2907 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P2908 TERMINAL SACRIFICE PNASAL DISCHARGE - RED P P PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P2909 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P2910 TERMINAL SACRIFICE PWITHIN 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-PRESENTPage 9210-MAR-2011 17:13Huntingdon Life Sciences 05-4287APPENDIX DGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATIONGROUP 2 2000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------DAY OF 1 1 1 1 1 1 1 1 1 1 2 2FEMALE# 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 PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P2912 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P2913 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P2914 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P2915 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P2916 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P2917 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P2918 TERMINAL SACRIFICE PNASAL DISCHARGE - RED P P P P P PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P2919 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P P2920 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P P2921 TERMINAL SACRIFICE PWITHIN 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-PRESENTPage 9310-MAR-2011 17:13Huntingdon Life Sciences 05-4287APPENDIX DGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATIONGROUP 2 2000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------DAY OF 1 1 1 1 1 1 1 1 1 1 2 2FEMALE# 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 PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P P2923 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P P2924 TERMINAL SACRIFICE PWITHIN 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-PRESENTPage 9410-MAR-2011 17:13Huntingdon Life Sciences 05-4287APPENDIX DGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATIONGROUP 3 10000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------DAY OF 1 1 1 1 1 1 1 1 1 1 2 2FEMALE# 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 PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P3902 TERMINAL SACRIFICE PNASAL DISCHARGE - RED PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P3903 TERMINAL SACRIFICE PNASAL DISCHARGE - RED P PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P3904 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P3905 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P3906 TERMINAL SACRIFICE PNASAL DISCHARGE - RED PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P3907 TERMINAL SACRIFICE PNASAL DISCHARGE - RED P P PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P3908 TERMINAL SACRIFICE PNASAL DISCHARGE - RED P P P PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P3909 TERMINAL SACRIFICE PNASAL DISCHARGE - RED PWITHIN 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-PRESENTPage 9510-MAR-2011 17:13Huntingdon Life Sciences 05-4287APPENDIX DGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATIONGROUP 3 10000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------DAY OF 1 1 1 1 1 1 1 1 1 1 2 2FEMALE# 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 PNASAL DISCHARGE - RED PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P3911 TERMINAL SACRIFICE PNASAL DISCHARGE - RED P P P P P P P P P P PWITHIN NORMAL LIMITS P P P P P P P P3912 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P3913 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P3914 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P3915 TERMINAL SACRIFICE PNASAL DISCHARGE - RED P P P P P PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P3916 TERMINAL SACRIFICE PNASAL DISCHARGE - RED P P PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P3917 TERMINAL SACRIFICE PNASAL DISCHARGE - RED P PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P3918 TERMINAL SACRIFICE PWITHIN 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-PRESENTPage 9610-MAR-2011 17:13Huntingdon Life Sciences 05-4287APPENDIX DGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATIONGROUP 3 10000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------DAY OF 1 1 1 1 1 1 1 1 1 1 2 2FEMALE# 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 PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P P3920 TERMINAL SACRIFICE PNASAL DISCHARGE - RED P PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P3921 TERMINAL SACRIFICE PNASAL DISCHARGE - RED P P PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P3922 TERMINAL SACRIFICE PNASAL DISCHARGE - RED P PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P3923 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P P3924 TERMINAL SACRIFICE PNASAL DISCHARGE - RED P P P P P P P PWITHIN NORMAL LIMITS P P P P P P P P P P P P P-----------------------------------------------------------------------------------------------------------------------------------CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENTPage 9710-MAR-2011 17:13Huntingdon Life Sciences 05-4287APPENDIX DGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATIONGROUP 4 20000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------DAY OF 1 1 1 1 1 1 1 1 1 1 2 2FEMALE# 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 PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P4902 TERMINAL SACRIFICE PALOPECIA - GENERAL 2 2 2 2 2 2 2 2CHROMODACRYORRHEA - UNILATERAL PWITHIN NORMAL LIMITS P P P P P P P P P4903 TERMINAL SACRIFICE PNASAL DISCHARGE - RED P P P PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P4904 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P4905 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P4906 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P4907 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P4908 TERMINAL SACRIFICE PINCISORS MALOCCLUDED P P P P P P P P P P P P P P P P P P PMASS 2 2 2 2 24909 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P4910 TERMINAL SACRIFICE PNASAL DISCHARGE - RED P P PWITHIN 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-PRESENTPage 9810-MAR-2011 17:13Huntingdon Life Sciences 05-4287APPENDIX DGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATIONGROUP 4 20000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------DAY OF 1 1 1 1 1 1 1 1 1 1 2 2FEMALE# 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 PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P4912 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P4913 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P4914 TERMINAL SACRIFICE PNASAL DISCHARGE - RED P P P PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P4915 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P4916 TERMINAL SACRIFICE PNASAL DISCHARGE - RED P P P P PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P4917 TERMINAL SACRIFICE PNASAL DISCHARGE - RED P P P P P P PWITHIN NORMAL LIMITS P P P P P P P P P P P P P4918 TERMINAL SACRIFICE PCHROMODACRYORRHEA - UNILATERAL PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P4919 TERMINAL SACRIFICE PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P P4920 TERMINAL SACRIFICE PNASAL DISCHARGE - RED P P P PWITHIN 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-PRESENTPage 9910-MAR-2011 17:13Huntingdon Life Sciences 05-4287APPENDIX DGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL CLINICAL OBSERVATIONS DURING GESTATIONGROUP 4 20000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------DAY OF 1 1 1 1 1 1 1 1 1 1 2 2FEMALE# 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 PNASAL DISCHARGE - RED PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P P P P4922 TERMINAL SACRIFICE PNASAL DISCHARGE - RED P P P PWITHIN NORMAL LIMITS P P P P P P P P P P P P P P P P P4923 TERMINAL SACRIFICE PNASAL DISCHARGE - RED P P P P P P P P P P PWITHIN NORMAL LIMITS P P P P P P P P P P4924 TERMINAL SACRIFICE PCHROMODACRYORRHEA - BILATERAL PCHROMODACRYORRHEA - UNILATERAL P P P P P P P P P P P P P PWITHIN NORMAL LIMITS P P P P P P-----------------------------------------------------------------------------------------------------------------------------------CODE: 1-SLIGHT 2-MODERATE 3-MARKED P-PRESENTPage 10010-MAR-2011 17:16Huntingdon Life Sciences 05-4287APPENDIX EGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL GESTATION BODY WEIGHTS (GRAMS)GROUP 1 0 MG/M3-----------------------------------------------------------------------------------------------------------------------------------DAY OF GESTATIONANIMAL# 4 5 8 11 14 17 20 21-----------------------------------------------------------------------------------------------------------------------------------1901 212 230 259 275 303 332 380 3891902 213 228 241 261 280 308 354 3601903 229 223 245 264 286 322 368 3841904 230 248 260 275 299 328 387 3971905 233 254 271 295 316 343 387 3931906 249 264 278 296 318 342 393 4061907 209 218 235 256 266 291 328 3341908 220 239 256 288 311 342 388 4001909 232 239 255 278 299 335 392 4001910 233 240 255 269 284 312 361 3721911 247 258 272 294 314 351 398 4111912 253 264 274 295 313 345 396 3971913 232 239 250 270 286 314 351 3531914 241 245 253 278 296 324 374 3841915x NP 245 243 257 258 253 263 263 2641916 246 261 270 298 318 357 410 4141917 249 254 269 297 316 357 405 4151918 259 269 283 310 338 378 420 4341919 214 223 245 268 288 306 351 3681920 222 232 246 261 280 292 313 3131921 231 236 253 275 295 324 373 3821922 235 242 257 282 295 324 368 3821923 243 247 264 292 317 359 405 4261924 245 259 274 297 316 353 395 405MEAN 234 244 259 281 301 332 378 388S.D. 14.0 14.6 12.8 14.9 17.0 22.2 26.3 28.6N 23 23 23 23 23 23 23 23NP=NOT PREGNANT x=EXCLUDED FROM MEANPage 10110-MAR-2011 17:16Huntingdon Life Sciences 05-4287APPENDIX EGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL GESTATION BODY WEIGHTS (GRAMS)GROUP 2 2000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------DAY OF GESTATIONANIMAL# 4 5 8 11 14 17 20 21-----------------------------------------------------------------------------------------------------------------------------------2901 209 221 233 245 263 286 323 3272902 216 230 234 245 262 287 330 3332903 224 236 246 267 280 302 350 3542904 232 244 271 288 313 342 385 3972905 235 252 268 285 306 339 393 4042906 241 257 275 290 313 337 381 3852907 202 221 233 255 273 307 353 3652908 214 224 240 256 274 299 342 3552909 228 235 252 270 286 312 353 3642910 234 248 263 279 302 342 389 3952911 246 258 274 297 310 342 393 4012912 270 280 294 318 335 376 435 4542913 229 234 251 273 291 322 369 3722914 239 244 255 279 281 302 327 3282915 244 257 271 290 307 343 389 3992916 248 257 276 293 312 357 405 4182917 254 261 280 295 306 334 366 3812918 266 277 290 315 326 355 398 4052919x NP 211 218 217 228 235 237 232 2402920 222 229 237 254 270 298 335 3472921 228 239 256 278 295 326 366 3702922 233 229 250 269 278 318 339 3502923 242 257 274 292 312 353 408 4262924 243 253 267 285 301 335 387 396MEAN 235 245 260 279 296 327 370 379S.D. 16.6 16.5 18.0 19.6 20.3 24.3 29.9 32.7N 23 23 23 23 23 23 23 23NP=NOT PREGNANT x=EXCLUDED FROM MEANPage 10210-MAR-2011 17:16Huntingdon Life Sciences 05-4287APPENDIX EGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL GESTATION BODY WEIGHTS (GRAMS)GROUP 3 10000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------DAY OF GESTATIONANIMAL# 4 5 8 11 14 17 20 21-----------------------------------------------------------------------------------------------------------------------------------3901 210 230 232 245 262 284 320 3263902 219 242 253 273 295 313 356 3633903 219 231 249 267 291 310 362 3753904 233 245 263 285 299 327 379 3793905 237 248 256 268 286 322 379 3883906 246 245 267 279 295 338 386 3923907 206 218 229 245 267 302 337 3443908 212 226 236 255 273 294 333 3383909 231 245 255 278 296 325 370 3703910 241 246 257 274 287 318 367 3773911 243 254 263 285 302 336 393 4013912 264 275 280 299 315 345 390 3993913 232 235 260 276 293 328 354 3703914 241 250 267 290 305 333 375 3903915 246 258 268 283 292 309 324 3243916 248 257 270 292 309 348 399 4083917 252 256 275 297 319 357 401 4083918 258 263 280 294 304 337 377 3943919 216 227 237 254 273 309 354 3683920 218 227 242 262 281 313 365 3743921 223 230 243 255 272 295 329 3343922 233 239 253 271 294 338 393 4083923 241 249 263 275 293 323 373 3893924 245 258 260 283 305 344 388 398MEAN 234 244 256 274 292 323 367 376S.D. 15.7 13.9 14.4 15.6 14.8 18.7 24.2 25.9N 24 24 24 24 24 24 24 24Page 10310-MAR-2011 17:16Huntingdon Life Sciences 05-4287APPENDIX EGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL GESTATION BODY WEIGHTS (GRAMS)GROUP 4 20000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------DAY OF GESTATIONANIMAL# 4 5 8 11 14 17 20 21-----------------------------------------------------------------------------------------------------------------------------------4901 209 220 226 243 260 281 330 3284902 213 225 239 255 274 297 342 3524903 228 244 248 267 278 306 369 3764904 231 236 249 270 285 319 370 3804905 237 252 260 275 293 327 379 3834906 239 250 262 279 302 329 378 3894907 211 216 229 248 267 291 335 3404908x NP 214 218 222 230 221 228 237 2334909 222 231 233 243 255 279 313 3244910 235 242 264 286 301 328 373 3864911 241 254 264 282 294 326 378 3874912 247 257 271 291 306 332 379 3864913 235 246 257 272 289 322 365 3724914 244 254 262 285 297 327 368 3804915 245 253 261 279 291 317 357 3714916 246 254 261 278 295 326 374 3894917 258 269 282 303 318 362 408 4174918 259 265 275 300 308 350 381 3944919x NP 212 217 215 217 225 223 221 2184920 220 224 237 258 275 306 338 3484921 229 234 247 268 279 315 348 3584922 233 246 261 276 294 327 373 3664923 236 247 250 275 280 308 345 3514924 261 271 286 325 343 389 439 442MEAN 235 245 256 275 290 321 365 374S.D. 14.6 15.2 16.3 19.5 19.5 24.9 27.1 27.4N 22 22 22 22 22 22 22 22NP=NOT PREGNANT x=EXCLUDED FROM MEANPage 10410-MAR-2011 17:17Huntingdon Life Sciences 05-4287APPENDIX FGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL GESTATION BODY WEIGHT GAIN (GRAMS)GROUP 1 0 MG/M3-----------------------------------------------------------------------------------------------------------------------------------DAY OF GESTATIONANIMAL# 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 1591902 15 14 19 20 28 47 5 53 80 1321903 -6 22 19 22 35 46 17 63 98 1611904 19 12 15 24 29 59 10 51 98 1491905 21 17 24 22 27 44 6 63 77 1391906 14 15 18 22 24 51 14 55 88 1431907 8 18 21 10 25 37 6 49 68 1161908 19 17 32 23 31 46 12 72 89 1611909 8 16 22 21 36 57 8 60 101 1611910 7 15 15 14 28 50 11 44 88 1321911 12 14 22 20 37 47 13 56 97 1531912 11 10 21 17 33 51 1 49 85 1331913 8 11 20 16 29 37 2 46 68 1141914 4 9 25 18 28 50 10 51 88 1391915x NP -2 14 1 -6 11 -1 1 10 11 211916 15 9 28 20 39 53 4 57 96 1531917 5 15 29 19 41 48 10 62 99 1611918 10 15 27 27 40 43 14 69 96 1651919 9 21 24 20 18 45 17 65 80 1451920 9 14 16 19 12 21 0 48 33 811921 6 17 22 20 29 49 9 59 87 1461922 7 15 25 14 28 45 14 54 87 1411923 5 17 28 25 42 46 21 69 109 1791924 14 14 23 19 37 43 10 56 89 146MEAN 10 15 22 20 31 46 10 57 86 144S.D. 6.1 4.5 4.7 4.2 7.3 7.6 5.2 8.4 15.5 20.6N 23 23 23 23 23 23 23 23 23 23NP=NOT PREGNANT x=EXCLUDED FROM MEANPage 10510-MAR-2011 17:17Huntingdon Life Sciences 05-4287APPENDIX FGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL GESTATION BODY WEIGHT GAIN (GRAMS)GROUP 2 2000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------DAY OF GESTATIONANIMAL# 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 1052902 14 3 11 17 25 43 3 32 70 1022903 12 10 22 13 22 48 5 44 74 1192904 12 27 17 26 28 43 12 70 84 1532905 17 16 17 22 33 54 11 55 98 1532906 16 18 15 24 24 44 4 57 72 1282907 19 13 21 18 34 46 13 52 93 1452908 11 16 16 18 25 43 13 50 81 1302909 7 17 18 17 26 41 11 51 78 1292910 13 16 16 23 41 46 7 54 94 1482911 12 16 23 14 32 51 8 53 90 1432912 10 14 24 17 41 59 19 55 119 1752913 5 17 22 19 31 46 3 57 81 1382914 5 11 24 3 21 25 0 37 47 842915 12 14 18 18 36 46 10 50 92 1432916 9 19 17 20 45 48 13 55 105 1612917 8 19 15 11 28 32 15 45 76 1202918 11 13 25 11 29 42 8 49 79 1282919x NP 6 -1 11 7 2 -5 9 17 6 232920 8 8 17 16 27 38 12 41 77 1182921 11 17 22 17 31 40 4 56 75 1322922 -4 21 18 9 40 21 11 48 73 1212923 15 16 19 20 41 55 18 55 114 1682924 10 14 18 16 34 52 9 48 95 143MEAN 11 15 19 17 31 44 9 50 84 134S.D. 4.7 4.7 3.6 5.0 6.9 8.9 5.0 7.8 16.3 21.5N 23 23 23 23 23 23 23 23 23 23NP=NOT PREGNANT x=EXCLUDED FROM MEANPage 10610-MAR-2011 17:17Huntingdon Life Sciences 05-4287APPENDIX FGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL GESTATION BODY WEIGHT GAIN (GRAMS)GROUP 3 10000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------DAY OF GESTATIONANIMAL# 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 963902 24 11 20 21 19 43 7 52 69 1213903 12 17 18 24 20 52 13 59 84 1443904 13 17 22 14 28 52 -1 53 80 1333905 11 8 12 18 37 57 8 38 102 1403906 -1 21 13 16 43 48 6 49 97 1463907 13 10 16 22 34 35 8 49 77 1263908 14 10 19 18 21 39 6 47 66 1133909 14 10 23 18 30 45 0 51 75 1263910 5 11 17 13 31 49 10 41 90 1313911 12 9 22 17 33 57 8 48 98 1463912 10 6 18 16 30 45 8 40 84 1243913 3 25 15 17 36 26 16 57 77 1353914 10 16 24 14 28 42 15 54 85 1393915 12 10 15 9 17 15 1 34 32 673916 10 13 22 17 39 51 10 51 100 1513917 4 18 22 22 39 44 6 62 89 1513918 5 18 14 10 32 41 16 42 89 1313919 12 10 17 19 36 45 14 46 95 1413920 9 15 21 18 32 52 9 54 93 1473921 6 13 12 17 24 34 5 42 63 1053922 6 14 19 22 44 55 15 54 114 1693923 8 14 12 18 31 50 16 44 96 1403924 14 2 23 23 38 45 10 47 93 140MEAN 10 13 18 17 31 44 9 48 84 132S.D. 5.2 5.5 3.9 3.7 7.6 9.9 4.9 7.7 17.0 21.0N 24 24 24 24 24 24 24 24 24 24Page 10710-MAR-2011 17:17Huntingdon Life Sciences 05-4287APPENDIX FGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL GESTATION BODY WEIGHT GAIN (GRAMS)GROUP 4 20000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------DAY OF GESTATIONANIMAL# 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 1084902 12 14 16 20 23 45 11 50 78 1274903 16 4 20 11 28 63 7 35 98 1324904 5 13 21 15 34 52 10 49 95 1444905 15 8 15 18 34 51 4 42 90 1314906 11 12 16 23 27 49 12 52 87 1394907 5 12 19 19 25 44 4 50 73 1234908x NP 5 4 8 -9 6 10 -5 3 11 144909 9 2 9 13 24 33 11 24 69 934910 7 22 22 15 27 45 13 59 85 1444911 13 10 18 13 32 52 9 40 93 1334912 10 14 20 15 26 47 7 49 80 1294913 10 11 15 17 32 44 7 44 83 1274914 10 8 23 13 30 42 12 43 83 1264915 8 8 18 12 26 40 14 38 80 1184916 8 7 18 17 31 48 16 41 94 1354917 11 14 20 15 44 46 8 49 99 1484918 6 11 24 8 42 32 13 43 86 1294919x NP 6 -3 2 8 -2 -2 -3 7 -7 14920 5 13 21 16 31 32 10 50 73 1244921 5 13 21 12 36 33 10 45 79 1244922 12 15 15 18 34 46 -6 48 73 1204923 11 3 25 5 28 37 6 33 71 1044924 11 15 39 18 47 49 4 71 100 171MEAN 10 11 20 15 31 44 8 45 83 129S.D. 3.2 4.7 5.5 4.0 6.8 7.7 5.1 9.5 10.0 16.0N 22 22 22 22 22 22 22 22 22 22NP=NOT PREGNANT x=EXCLUDED FROM MEANPage 10810-MAR-2011 17:19Huntingdon Life Sciences 05-4287APPENDIX GGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL GESTATION FEED CONSUMPTION -- (GRAMS/KG/DAY)GROUP 1 0 MG/M3-----------------------------------------------------------------------------------------------------------------------------------DAY OF GESTATIONANIMAL# 4 - 5 5 - 8 8 - 11 11 - 14 14 - 17 17 - 20 20 - 21 5 - 21-----------------------------------------------------------------------------------------------------------------------------------1901 66 89 79 91 79 75 48 771902 83 82 83 88 81 77 44 761903 43 66 93 90 84 72 58 771904 76 82 79 83 78 71 46 731905 72 79 87 84 81 72 43 741906 63 71 72 78 77 76 58 721907 63 80 82 77 82 73 44 731908 95 89 96 94 90 79 58 841909 77 86 85 87 79 72 58 781910 75 79 76 82 82 72 55 741911 76 79 81 79 78 73 43 721912 70 78 76 72 72 72 36 671913 81 80 81 75 81 72 51 731914 66 77 80 82 82 78 58 761915x NP 56 75 67 67 69 63 72 691916 76 78 84 84 83 74 46 751917 73 86 91 89 83 75 54 801918 73 74 86 84 83 73 66 781919 54 85 92 92 75 69 58 781920 78 92 100 104 93 91 65 911921 70 83 92 89 81 74 54 791922 71 81 91 82 77 73 63 781923 CF CF CF CF CF 79 62 711924 65 81 87 83 78 71 57 76MEAN 71 81 85 85 81 74 53 76S.D. 10.5 6.0 7.1 7.1 4.5 4.5 8.1 4.7N 22 22 22 22 22 23 23 23NP=NOT PREGNANT x=EXCLUDED FROM MEANCF=Contaminated FeederPage 10910-MAR-2011 17:19Huntingdon Life Sciences 05-4287APPENDIX GGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL GESTATION FEED CONSUMPTION -- (GRAMS/KG/DAY)GROUP 2 2000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------DAY OF GESTATIONANIMAL# 4 - 5 5 - 8 8 - 11 11 - 14 14 - 17 17 - 20 20 - 21 5 - 21-----------------------------------------------------------------------------------------------------------------------------------2901 84 89 83 82 79 76 39 752902 83 81 79 76 79 79 52 742903 70 73 79 76 74 72 50 712904 44 80 78 87 78 75 52 752905 73 82 78 82 82 75 35 722906 79 80 75 80 73 65 41 692907 78 89 90 81 83 72 43 762908 86 145 86 91 87 76 47 892909 94 80 90 86 83 77 55 792910 85 88 82 84 87 76 35 762911 74 80 83 76 75 71 38 712912 76 84 84 82 82 75 44 752913 76 86 88 83 82 75 48 772914 72 74 82 72 78 73 45 712915 73 75 76 79 89 72 52 742916 72 83 82 82 86 72 47 752917 75 90 83 82 78 71 50 762918 77 80 81 83 76 70 40 722919x NP 76 78 81 79 72 74 65 752920 59 79 74 78 79 76 68 762921 76 87 84 84 82 73 53 772922 45 78 76 76 86 67 47 712923 78 87 80 86 76 74 54 762924 77 94 88 89 84 77 41 79MEAN 74 85 82 82 81 73 47 75S.D. 11.5 14.1 4.6 4.7 4.5 3.3 7.5 4.0N 23 23 23 23 23 23 23 23NP=NOT PREGNANT x=EXCLUDED FROM MEANPage 11010-MAR-2011 17:19Huntingdon Life Sciences 05-4287APPENDIX GGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL GESTATION FEED CONSUMPTION -- (GRAMS/KG/DAY)GROUP 3 10000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------DAY OF GESTATIONANIMAL# 4 - 5 5 - 8 8 - 11 11 - 14 14 - 17 17 - 20 20 - 21 5 - 21-----------------------------------------------------------------------------------------------------------------------------------3901 76 70 67 75 73 70 52 683902 74 72 74 83 75 70 40 693903 72 84 85 86 74 72 58 773904 81 88 89 88 82 74 42 773905 71 77 77 81 80 74 47 733906 47 71 79 80 86 75 39 723907 92 87 85 90 94 78 47 803908 88 88 90 86 85 78 50 793909 69 75 77 80 76 69 39 693910 74 77 80 78 73 65 43 693911 84 90 85 78 80 74 48 763912 73 75 76 82 84 74 54 743913 55 85 79 87 86 77 50 773914 66 78 81 79 73 67 49 713915 84 81 84 84 86 76 42 753916 87 78 75 CF 88 CF 38 703917 66 78 83 86 87 72 47 753918 81 82 79 85 80 76 56 763919 83 82 83 84 84 79 55 783920 69 80 83 80 78 75 42 733921 97 78 71 77 73 72 42 693922 64 85 87 CF CF CF 51 743923 71 83 79 82 77 73 48 743924 75 69 78 82 CF 79 50 72MEAN 75 80 80 82 81 74 47 74S.D. 11.4 6.0 5.5 3.9 6.0 3.7 5.8 3.6N 24 24 24 22 22 22 24 24CF=Contaminated FeederPage 11110-MAR-2011 17:19Huntingdon Life Sciences 05-4287APPENDIX GGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL GESTATION FEED CONSUMPTION -- (GRAMS/KG/DAY)GROUP 4 20000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------DAY OF GESTATIONANIMAL# 4 - 5 5 - 8 8 - 11 11 - 14 14 - 17 17 - 20 20 - 21 5 - 21-----------------------------------------------------------------------------------------------------------------------------------4901 71 CF CF CF CF 77 47 624902 60 70 73 77 73 76 52 704903 83 78 75 72 71 71 43 684904 58 71 76 76 76 67 42 684905 83 80 82 81 79 72 39 724906 81 76 77 81 75 65 42 694907 84 81 84 84 83 79 50 774908x NP 63 67 66 60 66 69 46 624909 73 67 67 75 77 77 40 674910 69 92 82 79 78 73 57 774911 68 74 75 75 80 72 47 704912 71 81 77 76 74 72 45 714913 85 83 78 78 77 73 49 734914 68 76 75 79 84 77 45 734915 88 90 80 77 75 69 42 724916 74 83 81 80 83 80 46 764917 76 79 74 76 81 72 47 714918 68 75 72 71 83 66 59 714919x NP 74 68 70 72 66 66 54 664920 73 82 84 82 88 78 55 784921 72 82 72 72 81 70 54 724922 59 77 70 79 78 79 21 674923 92 77 80 77 81 71 45 724924 53 80 92 87 86 73 42 77MEAN 73 79 78 78 79 73 46 72S.D. 10.2 6.0 5.7 4.0 4.4 4.3 7.8 3.8N 22 21 21 21 21 22 22 22NP=NOT PREGNANT x=EXCLUDED FROM MEANCF=Contaminated FeederPage 11210-MAR-2011 17:21Huntingdon Life Sciences 05-4287APPENDIX HGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL MATERNAL NECROPSY OBSERVATIONSGROUP 1 0 MG/M3-----------------------------------------------------------------------------------------------------------------------------------ANIMAL# ORGAN OBSERVATION-----------------------------------------------------------------------------------------------------------------------------------1901 NO REMARKABLE OBSERVATIONS1902 NO REMARKABLE OBSERVATIONS1903 NO REMARKABLE OBSERVATIONS1904 NO REMARKABLE OBSERVATIONS1905 NO REMARKABLE OBSERVATIONS1906 NO REMARKABLE OBSERVATIONS1907 NO REMARKABLE OBSERVATIONS1908 NO REMARKABLE OBSERVATIONS1909 NO REMARKABLE OBSERVATIONS1910 NO REMARKABLE OBSERVATIONS1911 NO REMARKABLE OBSERVATIONS1912 NO REMARKABLE OBSERVATIONS1913 NO REMARKABLE OBSERVATIONS1914 NO REMARKABLE OBSERVATIONS1915 NP SALEWSKI'S TEST NO FOCI PRESENT1916 NO REMARKABLE OBSERVATIONS-----------------------------------------------------------------------------------------------------------------------------------NP=NOT PREGNANTPage 11310-MAR-2011 17:21Huntingdon Life Sciences 05-4287APPENDIX HGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL MATERNAL NECROPSY OBSERVATIONSGROUP 1 0 MG/M3-----------------------------------------------------------------------------------------------------------------------------------ANIMAL# ORGAN OBSERVATION-----------------------------------------------------------------------------------------------------------------------------------1917 NO REMARKABLE OBSERVATIONS1918 NO REMARKABLE OBSERVATIONS1919 NO REMARKABLE OBSERVATIONS1920 NO REMARKABLE OBSERVATIONS1921 NO REMARKABLE OBSERVATIONS1922 NO REMARKABLE OBSERVATIONS1923 NO REMARKABLE OBSERVATIONS1924 NO REMARKABLE OBSERVATIONS-----------------------------------------------------------------------------------------------------------------------------------Page 11410-MAR-2011 17:21Huntingdon Life Sciences 05-4287APPENDIX HGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL MATERNAL NECROPSY OBSERVATIONSGROUP 2 2000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------ANIMAL# ORGAN OBSERVATION-----------------------------------------------------------------------------------------------------------------------------------2901 NO REMARKABLE OBSERVATIONS2902 NO REMARKABLE OBSERVATIONS2903 NO REMARKABLE OBSERVATIONS2904 NO REMARKABLE OBSERVATIONS2905 NO REMARKABLE OBSERVATIONS2906 NO REMARKABLE OBSERVATIONS2907 NO REMARKABLE OBSERVATIONS2908 NO REMARKABLE OBSERVATIONS2909 NO REMARKABLE OBSERVATIONS2910 NO REMARKABLE OBSERVATIONS2911 NO REMARKABLE OBSERVATIONS2912 NO REMARKABLE OBSERVATIONS2913 NO REMARKABLE OBSERVATIONS2914 NO REMARKABLE OBSERVATIONS2915 NO REMARKABLE OBSERVATIONS2916 NO REMARKABLE OBSERVATIONS-----------------------------------------------------------------------------------------------------------------------------------Page 11510-MAR-2011 17:21Huntingdon Life Sciences 05-4287APPENDIX HGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL MATERNAL NECROPSY OBSERVATIONSGROUP 2 2000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------ANIMAL# ORGAN OBSERVATION-----------------------------------------------------------------------------------------------------------------------------------2917 NO REMARKABLE OBSERVATIONS2918 NO REMARKABLE OBSERVATIONS2919 NP SALEWSKI'S TEST NO FOCI PRESENT2920 NO REMARKABLE OBSERVATIONS2921 NO REMARKABLE OBSERVATIONS2922 NO REMARKABLE OBSERVATIONS2923 NO REMARKABLE OBSERVATIONS2924 NO REMARKABLE OBSERVATIONS-----------------------------------------------------------------------------------------------------------------------------------NP=NOT PREGNANTPage 11610-MAR-2011 17:21Huntingdon Life Sciences 05-4287APPENDIX HGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL MATERNAL NECROPSY OBSERVATIONSGROUP 3 10000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------ANIMAL# ORGAN OBSERVATION-----------------------------------------------------------------------------------------------------------------------------------3901 NO REMARKABLE OBSERVATIONS3902 NO REMARKABLE OBSERVATIONS3903 NO REMARKABLE OBSERVATIONS3904 NO REMARKABLE OBSERVATIONS3905 NO REMARKABLE OBSERVATIONS3906 NO REMARKABLE OBSERVATIONS3907 NO REMARKABLE OBSERVATIONS3908 NO REMARKABLE OBSERVATIONS3909 NO REMARKABLE OBSERVATIONS3910 NO REMARKABLE OBSERVATIONS3911 NO REMARKABLE OBSERVATIONS3912 NO REMARKABLE OBSERVATIONS3913 NO REMARKABLE OBSERVATIONS3914 NO REMARKABLE OBSERVATIONS3915 NO REMARKABLE OBSERVATIONS3916 NO REMARKABLE OBSERVATIONS-----------------------------------------------------------------------------------------------------------------------------------Page 11710-MAR-2011 17:21Huntingdon Life Sciences 05-4287APPENDIX HGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL MATERNAL NECROPSY OBSERVATIONSGROUP 3 10000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------ANIMAL# ORGAN OBSERVATION-----------------------------------------------------------------------------------------------------------------------------------3917 NO REMARKABLE OBSERVATIONS3918 NO REMARKABLE OBSERVATIONS3919 NO REMARKABLE OBSERVATIONS3920 NO REMARKABLE OBSERVATIONS3921 NO REMARKABLE OBSERVATIONS3922 NO REMARKABLE OBSERVATIONS3923 NO REMARKABLE OBSERVATIONS3924 NO REMARKABLE OBSERVATIONS-----------------------------------------------------------------------------------------------------------------------------------Page 11810-MAR-2011 17:21Huntingdon Life Sciences 05-4287APPENDIX HGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL MATERNAL NECROPSY OBSERVATIONSGROUP 4 20000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------ANIMAL# ORGAN OBSERVATION-----------------------------------------------------------------------------------------------------------------------------------4901 NO REMARKABLE OBSERVATIONS4902 EXTERNAL EXAM ALOPECIA- GENERALEXTERNAL FINDING CONFIRMED4903 NO REMARKABLE OBSERVATIONS4904 NO REMARKABLE OBSERVATIONS4905 NO REMARKABLE OBSERVATIONS4906 NO REMARKABLE OBSERVATIONS4907 NO REMARKABLE OBSERVATIONS4908 NP GROSS EXAM MASS; LEFTMAMMARY GLAND - LOWER LATERAL, 2.0 X 2.5 CM.,TAN, FIRMEXTERNAL FINDING CONFIRMEDTISSUE SAVEDEXTERNAL EXAM INCISOR MALOCLUDEDEXTERNAL FINDING CONFIRMEDSALEWSKI'S TEST NO FOCI PRESENT4909 NO REMARKABLE OBSERVATIONS4910 NO REMARKABLE OBSERVATIONS4911 NO REMARKABLE OBSERVATIONS4912 NO REMARKABLE OBSERVATIONS4913 NO REMARKABLE OBSERVATIONS-----------------------------------------------------------------------------------------------------------------------------------NP=NOT PREGNANTPage 11910-MAR-2011 17:21Huntingdon Life Sciences 05-4287APPENDIX HGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL MATERNAL NECROPSY OBSERVATIONSGROUP 4 20000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------ANIMAL# ORGAN OBSERVATION-----------------------------------------------------------------------------------------------------------------------------------4914 NO REMARKABLE OBSERVATIONS4915 NO REMARKABLE OBSERVATIONS4916 NO REMARKABLE OBSERVATIONS4917 NO REMARKABLE OBSERVATIONS4918 NO REMARKABLE OBSERVATIONS4919 NP SALEWSKI'S TEST NO FOCI PRESENT4920 NO REMARKABLE OBSERVATIONS4921 NO REMARKABLE OBSERVATIONS4922 NO REMARKABLE OBSERVATIONS4923 NO REMARKABLE OBSERVATIONS4924 NO REMARKABLE OBSERVATIONS-----------------------------------------------------------------------------------------------------------------------------------NP=NOT PREGNANTPage 12010-MAR-2011 17:22Huntingdon Life Sciences 05-4287APPENDIX IGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL FEMALE REPRODUCTION DATA AND MEAN FETAL WEIGHTS (GRAMS)GROUP 1 0 MG/M3-----------------------------------------------------------------------------------------------------------------------------------CORPORA %PREIMPL. IMPLANT FETUSES RESORPTIONS SEX AVERAGE FETAL BODY WEIGHTANIMAL# LUTEA LOSS SITES LIVE DEAD EARLY LATE TOTAL MALE FEMALE MALES FEMALES LITTER(n) % (n) %-----------------------------------------------------------------------------------------------------------------------------------1901 16 25.0 12 11 0 0.0 1 0 1 8.3 6 5 6.1 5.7 5.91902 16 25.0 12 12 0 0.0 0 0 0 0.0 8 4 6.1 5.7 6.01903 15 6.7 14 14 0 0.0 0 0 0 0.0 7 7 6.3 5.6 5.91904 22 31.8 15 15 0 0.0 0 0 0 0.0 8 7 5.7 5.4 5.51905 14 0.0 14 13 0 0.0 1 0 1 7.1 6 7 5.4 5.6 5.51906 15 6.7 14 13 0 0.0 1 0 1 7.1 6 7 5.6 5.4 5.51907 9 0.0 9 9 0 0.0 0 0 0 0.0 6 3 6.4 6.1 6.31908 14 7.1 13 12 0 0.0 1 0 1 7.7 7 5 5.9 5.5 5.71909 15 0.0 15 15 0 0.0 0 0 0 0.0 7 8 6.4 6.0 6.21910 14 7.1 13 13 0 0.0 0 0 0 0.0 6 7 6.0 6.0 6.01911 19 10.5 17 15 0 0.0 2 0 2 11.8 6 9 5.7 5.5 5.61912 15 0.0 15 15 0 0.0 0 0 0 0.0 5 10 5.4 5.1 5.21913 13 0.0 13 11 0 0.0 2 0 2 15.4 5 6 5.8 5.3 5.51914 12 0.0 12 12 0 0.0 0 0 0 0.0 5 7 6.1 5.9 6.01915 NP1916 17 11.8 15 14 0 0.0 1 0 1 6.7 6 8 5.6 5.4 5.51917 18 5.6 17 17 0 0.0 0 0 0 0.0 7 10 5.7 5.4 5.51918 15 0.0 15 14 0 0.0 0 1 1 6.7 10 4 5.6 5.3 5.51919 15 6.7 14 14 0 0.0 0 0 0 0.0 5 9 6.1 5.7 5.81920 9 55.6 4 4 0 0.0 0 0 0 0.0 1 3 6.3 6.1 6.11921 15 0.0 15 15 0 0.0 0 0 0 0.0 8 7 5.4 5.3 5.41922 14 7.1 13 13 0 0.0 0 0 0 0.0 7 6 6.1 5.8 6.01923 16 6.3 15 15 0 0.0 0 0 0 0.0 8 7 6.1 5.6 5.91924 15 0.0 15 14 0 0.0 1 0 1 6.7 6 8 5.7 5.4 5.6MEAN 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 5.6 5.7S.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 0.28 0.29N 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23NP=NOT PREGNANTPage 12110-MAR-2011 17:22Huntingdon Life Sciences 05-4287APPENDIX IGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL FEMALE REPRODUCTION DATA AND MEAN FETAL WEIGHTS (GRAMS)GROUP 2 2000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------CORPORA %PREIMPL. IMPLANT FETUSES RESORPTIONS SEX AVERAGE FETAL BODY WEIGHTANIMAL# LUTEA LOSS SITES LIVE DEAD EARLY LATE TOTAL MALE FEMALE MALES FEMALES LITTER(n) % (n) %-----------------------------------------------------------------------------------------------------------------------------------2901 13 15.4 11 10 0 0.0 1 0 1 9.1 7 3 5.6 5.3 5.52902 13 7.7 12 11 0 0.0 1 0 1 8.3 6 5 5.9 5.5 5.72903 13 7.7 12 11 0 0.0 1 0 1 8.3 7 4 5.8 5.5 5.72904 14 0.0 14 12 0 0.0 2 0 2 14.3 5 7 6.4 6.0 6.12905 18 11.1 16 16 0 0.0 0 0 0 0.0 6 10 6.1 5.7 5.82906 15 13.3 13 12 0 0.0 1 0 1 7.7 3 9 6.0 5.9 5.92907 13 0.0 13 13 0 0.0 0 0 0 0.0 8 5 5.5 5.2 5.42908 21 28.6 15 14 0 0.0 1 0 1 6.7 6 8 5.4 5.1 5.22909 11 0.0 11 10 0 0.0 1 0 1 9.1 5 5 6.3 5.8 6.02910 14 0.0 14 14 0 0.0 0 0 0 0.0 4 10 6.3 5.7 5.92911 15 6.7 14 14 0 0.0 0 0 0 0.0 5 9 6.1 5.6 5.82912 17 0.0 17 17 0 0.0 0 0 0 0.0 12 5 6.0 5.9 6.02913 14 0.0 14 14 0 0.0 0 0 0 0.0 6 8 5.7 5.6 5.62914 14 0.0 14 4 0 0.0 10 0 10 71.4 1 3 6.6 6.2 6.32915 13 7.7 12 11 0 0.0 1 0 1 8.3 3 8 6.0 5.9 5.92916 16 6.3 15 15 0 0.0 0 0 0 0.0 8 7 5.5 5.3 5.42917 14 7.1 13 12 0 0.0 1 0 1 7.7 5 7 5.5 5.2 5.32918 14 7.1 13 13 0 0.0 0 0 0 0.0 9 4 5.9 5.8 5.92919 NP2920 13 0.0 13 11 0 0.0 1 1 2 15.4 7 4 5.4 5.0 5.32921 14 7.1 13 11 0 0.0 2 0 2 15.4 5 6 5.7 5.8 5.72922 16 25.0 12 12 0 0.0 0 0 0 0.0 5 7 5.9 5.7 5.82923 15 0.0 15 15 0 0.0 0 0 0 0.0 5 10 6.2 5.9 6.02924 14 0.0 14 14 0 0.0 0 0 0 0.0 5 9 6.3 5.9 6.1MEAN 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 5.6 5.8S.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 0.33 0.30N 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23NP=NOT PREGNANTPage 12210-MAR-2011 17:22Huntingdon Life Sciences 05-4287APPENDIX IGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL FEMALE REPRODUCTION DATA AND MEAN FETAL WEIGHTS (GRAMS)GROUP 3 10000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------CORPORA %PREIMPL. IMPLANT FETUSES RESORPTIONS SEX AVERAGE FETAL BODY WEIGHTANIMAL# LUTEA LOSS SITES LIVE DEAD EARLY LATE TOTAL MALE FEMALE MALES FEMALES LITTER(n) % (n) %-----------------------------------------------------------------------------------------------------------------------------------3901 11 9.1 10 10 0 0.0 0 0 0 0.0 6 4 5.9 5.6 5.83902 13 0.0 13 12 0 0.0 1 0 1 7.7 6 6 5.6 5.5 5.63903 14 7.1 13 13 0 0.0 0 0 0 0.0 5 8 5.2 5.1 5.13904 13 0.0 13 13 0 0.0 0 0 0 0.0 8 5 6.1 5.5 5.83905 15 0.0 15 15 0 0.0 0 0 0 0.0 8 7 5.3 4.9 5.13906 14 0.0 14 14 0 0.0 0 0 0 0.0 7 7 5.5 5.6 5.63907 13 0.0 13 11 0 0.0 2 0 2 15.4 6 5 5.8 5.5 5.63908 13 0.0 13 10 0 0.0 3 0 3 23.1 4 6 5.7 5.4 5.53909 14 7.1 13 12 0 0.0 1 0 1 7.7 6 6 5.9 5.6 5.83910 16 12.5 14 14 0 0.0 0 0 0 0.0 7 7 6.1 5.7 5.93911 16 0.0 16 16 0 0.0 0 0 0 0.0 5 11 5.9 5.3 5.53912 14 7.1 13 12 0 0.0 1 0 1 7.7 5 7 6.1 5.9 6.03913 20 60.0 8 8 0 0.0 0 0 0 0.0 6 2 6.5 5.9 6.43914 18 16.7 15 14 0 0.0 1 0 1 6.7 6 8 5.5 5.2 5.33915 10 70.0 3 2 0 0.0 1 0 1 33.3 1 1 6.4 5.3 5.83916 16 12.5 14 14 0 0.0 0 0 0 0.0 9 5 5.7 5.6 5.73917 21 33.3 14 14 0 0.0 0 0 0 0.0 4 10 5.5 5.2 5.33918 15 0.0 15 13 0 0.0 2 0 2 13.3 5 8 5.8 5.4 5.53919 13 0.0 13 13 0 0.0 0 0 0 0.0 8 5 5.6 5.3 5.53920 14 0.0 14 14 0 0.0 0 0 0 0.0 8 6 6.1 5.8 6.03921 13 7.7 12 11 0 0.0 1 0 1 8.3 5 6 5.8 5.8 5.83922 17 0.0 17 17 0 0.0 0 0 0 0.0 8 9 6.0 5.6 5.83923 16 0.0 16 16 0 0.0 0 0 0 0.0 13 3 5.6 5.5 5.63924 15 6.7 14 14 0 0.0 0 0 0 0.0 8 6 5.9 5.6 5.8MEAN 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 5.5 5.7S.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 0.25 0.28N 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24Page 12310-MAR-2011 17:22Huntingdon Life Sciences 05-4287APPENDIX IGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL FEMALE REPRODUCTION DATA AND MEAN FETAL WEIGHTS (GRAMS)GROUP 4 20000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------CORPORA %PREIMPL. IMPLANT FETUSES RESORPTIONS SEX AVERAGE FETAL BODY WEIGHTANIMAL# LUTEA LOSS SITES LIVE DEAD EARLY LATE TOTAL MALE FEMALE MALES FEMALES LITTER(n) % (n) %-----------------------------------------------------------------------------------------------------------------------------------4901 15 6.7 14 13 0 0.0 1 0 1 7.1 6 7 5.6 5.3 5.54902 11 0.0 11 10 0 0.0 1 0 1 9.1 3 7 5.8 5.3 5.54903 17 11.8 15 14 0 0.0 1 0 1 6.7 9 5 5.8 5.6 5.74904 18 0.0 18 18 0 0.0 0 0 0 0.0 10 8 5.4 5.0 5.24905 16 0.0 16 16 0 0.0 0 0 0 0.0 9 7 5.4 5.0 5.24906 19 15.8 16 16 0 0.0 0 0 0 0.0 7 9 5.5 5.3 5.44907 13 15.4 11 10 0 0.0 1 0 1 9.1 4 6 6.0 5.6 5.74908 NP4909 11 9.1 10 9 0 0.0 1 0 1 10.0 6 3 6.0 5.7 5.94910 14 0.0 14 13 0 0.0 1 0 1 7.1 8 5 5.7 5.5 5.64911 17 0.0 17 16 0 0.0 1 0 1 5.9 6 10 5.6 5.3 5.44912 11 0.0 11 11 0 0.0 0 0 0 0.0 6 5 6.5 5.7 6.24913 15 6.7 14 14 0 0.0 0 0 0 0.0 4 10 6.0 5.7 5.84914 14 14.3 12 10 0 0.0 2 0 2 16.7 6 4 6.3 5.9 6.14915 16 6.3 15 14 0 0.0 1 0 1 6.7 3 11 5.6 5.1 5.24916 16 0.0 16 16 0 0.0 0 0 0 0.0 10 6 5.0 4.7 4.94917 16 6.3 15 14 0 0.0 1 0 1 6.7 6 8 6.0 5.8 5.94918 21 38.1 13 13 0 0.0 0 0 0 0.0 4 9 5.9 5.3 5.54919 NP4920 14 14.3 12 11 0 0.0 1 0 1 8.3 5 6 5.4 5.1 5.24921 13 15.4 11 11 0 0.0 0 0 0 0.0 6 5 5.7 5.4 5.64922 16 6.3 15 15 0 0.0 0 0 0 0.0 5 10 5.4 5.2 5.24923 13 0.0 13 11 0 0.0 2 0 2 15.4 7 4 5.7 5.2 5.54924 18 0.0 18 17 0 0.0 1 0 1 5.6 5 12 5.6 5.4 5.4MEAN 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 5.4 5.5S.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 0.31 0.32N 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22NP=NOT PREGNANTPage 12410-MAR-2011 17:23Huntingdon Life Sciences 05-4287APPENDIX JGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL GRAVID UTERINE WEIGHT AND NET BODY WEIGHT CHANGE (GRAMS)GROUP 1 0 MG/M3-----------------------------------------------------------------------------------------------------------------------------------UTERUS ADJUSTED NET WEIGHT CHANGE WEIGHT CHANGEANIMAL# WEIGHT WEIGHT FROM DAY 5 FROM DAY 51901 91 298 68 1591902 94 265 38 1321903 112 272 49 1611904 111 286 38 1491905 98 295 41 1391906 98 308 44 1431907 72 262 44 1161908 92 308 69 1611909 119 281 42 1611910 104 268 29 1321911 117 294 35 1531912 NT 1331913 84 269 30 1141914 103 281 36 1391915x NP 211916 108 306 45 1531917 126 289 35 1611918 108 326 57 1651919 113 255 32 1451920 36 277 45 811921 108 274 38 1461922 106 276 34 1411923 121 304 57 1791924 107 298 39 146MEAN 101. 286. 43. 144.S.D. 19.4 18.1 11.1 20.6N 22 22 22 23UTERUS WEIGHT = ABSOLUTE GRAVID UTERINE WEIGHTADJUSTED WEIGHT = TERMINAL BODY WEIGHT - UTERUS WEIGHTNET WEIGHT CHANGE FROM DAY 5 = ADJUSTED WEIGHT – DAY 5 BODY WEIGHTWEIGHT CHANGE FROM DAY 5 = TERMINAL BODY WEIGHT – DAY 5 BODY WEIGHTNP=NOT PREGNANT NT=NOT TAKEN x=EXCLUDED FROM MEANPage 12510-MAR-2011 17:23Huntingdon Life Sciences 05-4287APPENDIX JGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL GRAVID UTERINE WEIGHT AND NET BODY WEIGHT CHANGE (GRAMS)GROUP 2 2000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------UTERUS ADJUSTED NET WEIGHT CHANGE WEIGHT CHANGEANIMAL# WEIGHT WEIGHT FROM DAY 5 FROM DAY 52901 73 253 32 1052902 85 247 17 1022903 86 268 32 1192904 98 299 55 1532905 120 284 33 1532906 95 290 33 1282907 95 270 50 1452908 95 259 35 1302909 86 278 43 1292910 109 286 38 1482911 109 292 35 1432912 134 320 40 1752913 101 271 37 1382914 37 290 46 842915 91 308 52 1432916 113 304 47 1612917 86 295 34 1202918 105 300 23 1282919x NP 232920 81 266 36 1182921 88 282 43 1322922 93 257 27 1212923 126 300 42 1682924 113 283 30 143MEAN 97. 283. 37. 134.S.D. 19.8 19.0 9.1 21.5N 23 23 23 23UTERUS WEIGHT = ABSOLUTE GRAVID UTERINE WEIGHTADJUSTED WEIGHT = TERMINAL BODY WEIGHT - UTERUS WEIGHTNET WEIGHT CHANGE FROM DAY 5 = ADJUSTED WEIGHT – DAY 5 BODY WEIGHTWEIGHT CHANGE FROM DAY 5 = TERMINAL BODY WEIGHT – DAY 5 BODY WEIGHTNP=NOT PREGNANT x=EXCLUDED FROM MEANPage 12610-MAR-2011 17:23Huntingdon Life Sciences 05-4287APPENDIX JGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL GRAVID UTERINE WEIGHT AND NET BODY WEIGHT CHANGE (GRAMS)GROUP 3 10000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------UTERUS ADJUSTED NET WEIGHT CHANGE WEIGHT CHANGEANIMAL# WEIGHT WEIGHT FROM DAY 5 FROM DAY 53901 80 246 16 963902 88 275 33 1213903 95 280 49 1443904 103 276 31 1333905 107 281 33 1403906 104 288 43 1463907 83 261 43 1263908 75 263 38 1133909 90 280 36 1263910 108 269 23 1313911 117 283 29 1463912 96 303 28 1243913 68 302 67 1353914 100 289 39 1393915 19 305 47 673916 110 298 41 1513917 100 308 51 1513918 101 293 30 1313919 93 275 48 1413920 114 259 33 1473921 86 248 19 1053922 136 272 33 1693923 121 268 19 1403924 112 286 28 140MEAN 96. 280. 36. 132.S.D. 22.5 17.1 11.7 21.0N 24 24 24 24UTERUS WEIGHT = ABSOLUTE GRAVID UTERINE WEIGHTADJUSTED WEIGHT = TERMINAL BODY WEIGHT - UTERUS WEIGHTNET WEIGHT CHANGE FROM DAY 5 = ADJUSTED WEIGHT – DAY 5 BODY WEIGHTWEIGHT CHANGE FROM DAY 5 = TERMINAL BODY WEIGHT – DAY 5 BODY WEIGHTPage 12710-MAR-2011 17:23Huntingdon Life Sciences 05-4287APPENDIX JGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL GRAVID UTERINE WEIGHT AND NET BODY WEIGHT CHANGE (GRAMS)GROUP 4 20000 MG/M3-----------------------------------------------------------------------------------------------------------------------------------UTERUS ADJUSTED NET WEIGHT CHANGE WEIGHT CHANGEANIMAL# WEIGHT WEIGHT FROM DAY 5 FROM DAY 54901 91 237 17 1084902 78 274 49 1274903 103 272 29 1324904 123 257 21 1444905 110 273 21 1314906 119 270 20 1394907 79 261 45 1234908x NP 144909 70 254 23 934910 NT 1444911 113 274 20 1334912 94 292 35 1294913 105 267 21 1274914 84 296 42 1264915 97 275 22 1184916 108 281 27 1354917 114 303 34 1484918 94 300 35 1294919x NP 14920 76 272 47 1244921 84 274 40 1244922 104 262 16 1204923 81 270 23 1044924 PART 124x 318x 47x 171MEAN 96. 273. 29. 129.S.D. 15.7 15.9 10.6 16.0N 20 20 20 22UTERUS WEIGHT = ABSOLUTE GRAVID UTERINE WEIGHTADJUSTED WEIGHT = TERMINAL BODY WEIGHT - UTERUS WEIGHTNET WEIGHT CHANGE FROM DAY 5 = ADJUSTED WEIGHT – DAY 5 BODY WEIGHTWEIGHT CHANGE FROM DAY 5 = TERMINAL BODY WEIGHT – DAY 5 BODY WEIGHTNP=NOT PREGNANT NT=NOT TAKEN PART=PARTIAL DELIVERY; 5 LIVE PUPS DELIVERED x=EXCLUDED FROM MEANPage 12810-MAR-2011 17:24Huntingdon Life Sciences 05-4287APPENDIX KGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL FETAL BODY WEIGHTS (GRAMS)GROUP 1 0 MG/M3FEMALE# MEAN FETUS#1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 191901 5.9 5.6 5.9 5.9 5.8 6.0/ 6.3 5.3 5.4 E 6.0 6.2 6.51902 6.0 5.6 6.6 6.0 5.7 6.3 5.9/ 6.3 5.9 5.6 5.9 6.0 6.01903 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 6.51904 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 5.31905 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 5.31906 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 5.41907 6.3 6.4 6.3 6.0 6.2/ 6.2 6.1 6.6 6.5 6.31908 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 5.31909 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 6.11910 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 6.31911 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 5.21912 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 5.61913 5.5 5.0 5.9 E 5.2 5.5 5.9 E / 5.6 5.6 5.5 5.4 6.1 5.31914 6.0 6.0 6.3 6.4 6.1/ 6.1 5.4 5.8 5.8 6.2 5.9 6.1 5.91915 NP1916 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 5.51917 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 5.71918 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 5.21919 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 6.11920 6.1 / 6.2 6.0 6.1 6.31921 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 5.41922 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 6.01923 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 6.11924 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 5.3MEAN 5.7S.D. 0.29N 23E-Early resorption L-Late resorption/-Denotes position of cervixNP=NOT PREGNANTPage 12910-MAR-2011 17:24Huntingdon Life Sciences 05-4287APPENDIX KGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL FETAL BODY WEIGHTS (GRAMS)GROUP 2 2000 MG/M3FEMALE# MEAN FETUS#1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 192901 5.5 5.4/ 5.6 5.3 5.3 5.7 5.1 5.6 5.3 E 6.1 5.92902 5.7 5.2 5.7 6.0 5.7 5.5 6.1 5.9/ 5.3 6.2 E 5.5 5.92903 5.7 5.1 5.8 5.3 5.5 5.5 E 5.9/ 5.9 5.9 5.9 5.9 6.12904 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 6.22905 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 5.22906 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 5.82907 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 5.32908 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 5.22909 6.0 5.7 E 6.5 6.4 5.8/ 6.4 6.2 5.8 5.7 5.9 5.82910 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 6.42911 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 6.12912 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 5.82913 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 5.82914 6.3 E E 6.1 E E / E E E 6.6 6.4 E E E 6.22915 5.9 5.4 E 6.2 6.0 6.2 5.7 6.2 5.3/ 5.9 6.2 6.1 5.62916 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 5.52917 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 5.32918 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 6.32919 NP2920 5.3 5.5 5.7 L 5.4/ 5.1 5.3 5.2 4.9 E 5.1 4.5 5.6 5.32921 5.7 E E 5.7 6.0 5.4 5.8 5.6 5.7 6.0/ 5.8 5.6 6.0 5.62922 5.8 5.4 6.0 5.9 5.4 5.8 5.6/ 6.0 5.9 5.8 6.0 5.8 5.82923 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 6.12924 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 6.2MEAN 5.8S.D. 0.30N 23E-Early resorption L-Late resorption/-Denotes position of cervixNP=NOT PREGNANTPage 13010-MAR-2011 17:24Huntingdon Life Sciences 05-4287APPENDIX KGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL FETAL BODY WEIGHTS (GRAMS)GROUP 3 10000 MG/M3FEMALE# MEAN FETUS#1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 193901 5.8 5.5 6.0 5.9 5.7 5.7 6.2 5.7/ 6.0 5.1 5.93902 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 5.63903 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 5.33904 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 5.93905 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 5.23906 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 5.83907 5.6 5.7 E 6.0 5.6 5.6 5.3/ 6.0 5.5 E 5.6 5.4 5.7 5.83908 5.5 5.6 5.7 E 6.1 5.1 5.2 5.4/ 5.6 5.3 5.1 E E 5.83909 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 5.73910 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 6.43911 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 6.03912 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 5.73913 6.4 / 7.0 6.3 5.8 6.7 6.2 6.4 6.4 5.93914 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 5.93915 5.8 / E 6.4 5.33916 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 5.83917 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 5.33918 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 5.73919 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 5.33920 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 5.83921 5.8 5.6 6.3 6.0 E 5.6 6.2 5.5/ 6.0 6.1 5.9 5.8 4.93922 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 6.13923 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 5.53924 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 5.6MEAN 5.7S.D. 0.28N 24E-Early resorption/-Denotes position of cervixPage 13110-MAR-2011 17:24Huntingdon Life Sciences 05-4287APPENDIX KGASOLINE DIPE VAPOR CONDENSATE: EMBRYO-FETAL TOXICITY STUDYIN RATS BY INHALATION EXPOSUREINDIVIDUAL FETAL BODY WEIGHTS (GRAMS)GROUP 4 20000 MG/M3FEMALE# MEAN FETUS#1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 194901 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 5.44902 5.5 6.0 5.5 E 5.6 5.4/ 5.4 5.3 5.3 6.0 5.1 5.14903 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 5.74904 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 5.1 5.64905 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 5.44906 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 5.34907 5.7 6.0 5.6 5.6 6.1 5.6 5.6/ E 5.7 6.0 5.4 5.94908 NP4909 5.9 6.1 6.0 E 5.8 6.3 5.4 6.2 6.1/ 6.0 5.44910 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 5.84911 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 5.94912 6.2 6.7 6.7 5.3 6.0 6.3 6.6/ 5.6 5.8 5.7 6.0 6.94913 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 5.64914 6.1 5.9 E 6.5 5.9 6.3 E 5.9 6.2/ 5.7 6.4 6.1 6.64915 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 5.04916 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 4.84917 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 6.14918 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 5.74919 NP4920 5.2 5.2 5.0 E 5.2 5.1 5.1/ 5.3 5.4 5.8 5.0 5.2 5.34921 5.6 5.4 5.8 5.2 5.6 6.4 5.6/ 5.3 5.3 5.8 5.6 5.04922 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 5.54923 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 E4924 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 5.8 5.4MEAN 5.5S.D. 0.32N 22E-Early resorption/-Denotes position of cervixNP=NOT PREGNANTPage 132Huntingdon Life Sciences 05-4287 Page 133211-DIPE-DEV Final ReportFemales INDIVIDUAL FETAL EXTERNAL, VISCERAL AND SKELETAL OBSERVATIONS APPENDIX LAnimal Fetus Weight Abnormalities (only fetuses with abnormalities are listed)Number Exam Number Sex (g) Fate Major abnormalities in CAPITALSGroup 1 - 0 MG/M31901 6s, 5v12 M 6.5 s Sternebra(e) malaligned, #4 and #5, slight1903 7s, 7v3 F 5.3 s Rib(s)14th rudimentary, left10 M 6.2 v Additional subclavian artery, left11 M 6.0 s Rib(s) 14th rudimentary, bilateral1904 7s, 8v6 F 5.4 s Hyoid body/arch(es), unossified body11 F 5.3 v Renal papilla (e) not fully developed (grade 1), left1905 7s, 6v1 M 3.2 s ANAL ATRESIA AND FILAMENTOUS TAILVERTEBRAL AGENESIS: SACRAL VERTEBRAE #1 AND #2 SMALL IN SIZE;SACRAL VERTEBRAE #3 AND #4 AND ALL CAUDAL VERTEBRAE ABSENT;ILIA AND ISCHIA CLOSELY APPROXIMATEDSternebra(e) 5 and/or 6 unossified, #52 F 5.7 v Undescended thymic tissue, right1908 6s, 6v6 F 5.7 s 25 presacral vertebra(e)Rib(s) 13th rudimentary, right1909 8s, 7v8 F 5.7 v Undescended thymic tissue, left9 M 6.7 s Sternebra(e)malaligned, #2 through #4, slights = skeletal examination, v = visceral examination , M=Male, F=Female, g=gramsHuntingdon Life Sciences 05-4287 Page 134211-DIPE-DEV Final ReportFemales INDIVIDUAL FETAL EXTERNAL, VISCERAL AND SKELETAL OBSERVATIONS APPENDIX LAnimal Fetus Weight Abnormalities (only fetuses with abnormalities are listed)Number Exam Number Sex (g) Fate Major abnormalities in CAPITALSGroup 1 - 0 MG/M31910 6s, 7v2 F 6.0 s Sternebra(e) malaligned, #4, slight9 F 5.8 v Left umbilical artery1911 8s, 7v4 M 5.5 v Undescended thymic tissue, left9 M 5.4 v Undescended thymic tissue, right1912 7s, 8v3 F 5.3 v Undescended thymic tissue, left9 F 5.4 v Undescended thymic tissue, rightUreter(s) distended, bilateral12 F 5.2 s Sternebra(e) malaligned, #4 and #5, slight1913 6s, 5v6 M 5.9 s Sternebra(e) malaligned, #3 through #5, slight to moderate1914 6s, 6v8 F 5.8 s Sternebra(e) malaligned, #4 and #5, slight1916 7s, 7v1 F 5.3 v Undescended thymic tissue, right3 M 5.5 v Undescended thymic tissue, lefts = skeletal examination, v = visceral examination, M=Male, F=Female, g=gramsHuntingdon Life Sciences 05-4287 Page 135211-DIPE-DEV Final ReportFemales INDIVIDUAL FETAL EXTERNAL, VISCERAL AND SKELETAL OBSERVATIONS APPENDIX LAnimal Fetus Weight Abnormalities (only fetuses with abnormalities are listed)Number Exam Number Sex (g) Fate Major abnormalities in CAPITALSGroup 1 - 0 MG/M31918 7s, 7v11 L Crown-rump distance 2.4 cm1919 7s, 7v9 F 5.9 s Sternebra(e) malaligned, #4 and #5, slight11 F 5.6 s Sternebra(e) malaligned, #4 and #5, slight13 F 6.0 s Sternebra(e) malaligned, #4 and #5, slight1921 8s, 7v2 F 5.3 v Innominate artery absent6 M 5.5 v Undescended thymic tissue, left12 M 5.3 v Undescended thymic tissue, rightDisplaced testis medially, left1923 8s, 7v7 F 5.2 s Sternebra(e) malaligned,#3 and #4, slight to moderate1924 7s, 7v9 F 5.3 s Sternebra(e) malaligned, #3 through #5, slights = skeletal examination, v = visceral examination, M=Male, F=Female, L=Late Resorption, g=gramsHuntingdon Life Sciences 05-4287 Page 136211-DIPE-DEV Final ReportFemales INDIVIDUAL FETAL EXTERNAL, VISCERAL AND SKELETAL OBSERVATIONS APPENDIX LAnimal Fetus Weight Abnormalities (only fetuses with abnormalities are listed)Number Exam Number Sex (g) Fate Major abnormalities in CAPITALSGroup 2 - 2000 MG/M32901 5s, 5v2 M 5.6 v Undescended thymic tissue, left3 F 5.3 s Sternebra(e)malaligned, #3 through #4, slight8 M 5.3 v Undescended thymic tissue, bilateral2902 5s, 6v2 F 5.7 s Sternebra(e) malaligned, #3 and #4, slight2903 6s, 5v2 M 5.8 v Undescended thymic tissue, left3 M 5.3 s Sternebra(e) malaligned, #4 and #5Rib(s) 14th rudimentary, right2904 6s, 6v14 F 6.2 s RIGHT FORELIMB SHORTENED2905 8s, 8v1 M 6.1 s Sternebra(e) malaligned, #3 through #5, slight7 F 5.9 s Sternebra(e) malaligned, #4 and #5, slight10 F 5.6 v Undescended thymic tissue, right2908 7s, 7v14 M 5.1 v Left umbilical artery2909 5s, 5v6 M 6.4 s Rib(s) 14th rudimentary, right11 M 5.8 v Left umbilical arterys = skeletal examination, v = visceral examination, M=Male, F=Female, g=gramsHuntingdon Life Sciences 05-4287 Page 137211-DIPE-DEV Final ReportFemales INDIVIDUAL FETAL EXTERNAL, VISCERAL AND SKELETAL OBSERVATIONS APPENDIX LAnimal Fetus Weight Abnormalities (only fetuses with abnormalities are listed)Number Exam Number Sex (g) Fate Major abnormalities in CAPITALSGroup 2 - 2000 MG/M32910 7s, 7v3 F 6.0 v Undescendedthymic tissue, bilateral4 F 5.7 s Rib(s) 14th rudimentary, bilateral5 F 5.3 v Undescended thymic tissue, left6 F 5.8 s Rib(s)14th rudimentary, bilateral8 F 6.4 s Rib(s)14th rudimentary, left13 F 5.2 v Undescended thymic tissue, right2912 8s, 9v 4 M 6.4 s 7th cervical rib(s) present, left10 F 6.1 s SHORT MANDIBLE, moderate15 M 5.5 v Undescended thymic tissue, leftLeft umbilical artery17 F 5.8 v Left umbilical artery2913 7s, 7v1 M 5.6 s Sternebra(e) malaligned, #4 and #5, slightRib(s) 14th rudimentary, bilateral2917 6s, 6v4 M 5.5 s Rib(s) 14th rudimentary, bilateral13 F 5.3 v Undescended thymic tissue, bilateral2918 6s, 7v2 M 6.0 s Sternebra(e) malaligned, #4 and #5, slights = skeletal examination, v = visceral examination, M=Male, F=Female, g=gramsHuntingdon Life Sciences 05-4287 Page 138211-DIPE-DEV Final ReportFemales INDIVIDUAL FETAL EXTERNAL, VISCERAL AND SKELETAL OBSERVATIONS APPENDIX LAnimal Fetus Weight Abnormalities (only fetuses with abnormalities are listed)Number Exam Number Sex (g) Fate Major abnormalities in CAPITALSGroup 2 - 2000 MG/M32920 5s, 6v 3 L Crown-rump distance 2.9 cm12 M 5.6 s Sternebra(e) malaligned, #4 and #5, slight2921 5s, 5v3 M 5.7 s Sternebra(e) malaligned, #3, slight2923 8s, 7v1 F 5.4 s Sternebra(e) malaligned, #3 through #5, slight7 F 6.5 s Sternebra(e) malaligned, #3 and #4, slight14 F 5.4 v Undescended thymic tissue, left2924 7s, 7v11 F 6.5 v Undescended thymic tissue, lefts = skeletal examination, v = visceral examination, M=Male, F=Female, L=Late Resorption, g=gramsHuntingdon Life Sciences 05-4287 Page 139211-DIPE-DEV Final ReportFemales INDIVIDUAL FETAL EXTERNAL, VISCERAL AND SKELETAL OBSERVATIONS APPENDIX LAnimal Fetus Weight Abnormalities (only fetuses with abnormalities are listed)Number Exam Number Sex (g) Fate Major abnormalities in CAPITALSGroup 3 - 10000 MG/M33901 5s, 5v1 M 5.5 s Sternebra(e) malaligned, #4, slight3903 7s, 6v9 F 5.0 s Sternebra(e) malaligned, #3 and #4, slight3905 8s, 7v1 F 5.1 s Sternebra(e) malaligned, #5, slightRib(s) 14th rudimentary, right5 F 4.8 s Sternebra(e) malaligned, #5, slight3906 7s, 7v1 M 5.4 v Innominate artery absent9 F 5.6 v Renal papilla (e)not fully developed (grade 1), leftUreter(s) distended left3907 6s, 5v6 F 5.3 s Sternebra(e) malaligned, #5, slight8 F 5.5 s Hyoid body/arch(es), unossified body3910 7s, 7v6 M 6.0 s Rib(s) 14th rudimentary, right7 F 5.7 v Innominate artery absents = skeletal examination, v = visceral examination, M=Male, F=Female, g=gramsHuntingdon Life Sciences 05-4287 Page 140211-DIPE-DEV Final ReportFemales INDIVIDUAL FETAL EXTERNAL, VISCERAL AND SKELETAL OBSERVATIONS APPENDIX LAnimal Fetus Weight Abnormalities (only fetuses with abnormalities are listed)Number Exam Number Sex (g) Fate Major abnormalities in CAPITALSGroup 3 - 10000 MG/M33911 7s, 9v8 F 3.7 v MICROPHTHALMIA/ANOPHTHALMIA, leftUndescended thymic tissue, rightLeft umbilical artery11 F 3.3 v DOMED CRANIUM, moderate; HYDROCEPHALYANOPHTHALMIA, bilateralHypoplastic lung lobe(s), apical lobeHypoplastic thymus, bilateral lobesHypoplastic spleen13 F 5.5 s Rib(s)14th rudimentary, bilateral3912 6s, 6v6 M 6.3 v Undescended thymic tissue, bilateral8 M 5.8 v Undescended thymic tissue, bilateral12 F 6.0 v Undescended thymic tissue, right3913 4s, 4v1 M 7.0 s Rib(s) 14th rudimentary, right5 M 6.2 s Sternebra(e) malaligned, #4, slight3914 7s, 7v5 M 5.0 v Undescended thymic tissue, left15 M 5.9 s Rib(s) 14th rudimentary, left3916 7s, 7v2 M 5.5 s Sternebra(e) 5 and/or 6 unossified, #58 M 5.7 s Sternebra(e) malaligned, #3 and #4, slights = skeletal examination, v = visceral examination, M=Male, F=Female, g=gramsHuntingdon Life Sciences 05-4287 Page 141211-DIPE-DEV Final ReportFemales INDIVIDUAL FETAL EXTERNAL, VISCERAL AND SKELETAL OBSERVATIONS APPENDIX LAnimal Fetus Weight Abnormalities (only fetuses with abnormalities are listed)Number Exam Number Sex (g) Fate Major abnormalities in CAPITALSGroup 3 - 10000 MG/M33917 7s, 7v11 F 4.8 s Sternebra(e) 5 and/or 6 unossified, #53918 6s, 7v14 F 4.8 s Sternebra(e) malaligned, #3 and #4, slight3921 6s, 5v6 M 6.2 s Sternebra(e) malaligned, #3 through #5, slight8 F 6.0 s Sternebra(e)malaligned, #3 through #5, slight3922 8s, 9v2 F 6.0 s Sternebra(e) malaligned, #4 and #510 F 5.8 s Sternebra(e) malaligned, #3 through #5, slight3923 8s, 8v16 M 5.5 v Renal papilla (e)not fully developed (grade 1), left3924 7s, 7v4 M 6.0 s Sternebra(e) malaligned, #4, slight6 F 5.2 s Sternebra(e) 5 and/or 6 unossified, #5s = skeletal examination, v = visceral examination, M=Male, F=Female, g=gramsHuntingdon Life Sciences 05-4287 Page 142211-DIPE-DEV Final ReportFemales INDIVIDUAL FETAL EXTERNAL, VISCERAL AND SKELETAL OBSERVATIONS APPENDIX LAnimal Fetus Weight Abnormalities (only fetuses with abnormalities are listed)Number Exam Number Sex (g) Fate Major abnormalities in CAPITALSGroup 4 - 20000 MG/M34901 7s, 6v2 M 5.9 v Undescended thymic tissue, left5 F 5.5 s Hyoid body/arch(es) unossified body10 M 5.6 v Undescended thymic tissue, bilateral4902 5s, 5v5 F 5.4 s 7th cervical rib(s) present, rightRib(s) 14th rudimentary, bilateral11 F 5.1 s Sternebra(e) 5 and/or 6 unossified, #54904 9s, 9v3 M 5.8 v Undescended thymic tissue, left4905 8s, 8v12 F 5.2 v Undescended thymic tissue, left14 M 5.5 v Undescended thymic tissue, left15 M 5.1 s Sternebra(e) malaligned, #2 through #4, slight to moderate4906 8s, 8v5 F 5.1 v Undescended thymic tissue, right11 F 5.0 v Undescended thymic tissue, left4907 5s, 5v1 M 6.0 s VERTEBRAL ANOMALY: THORACIC CENTRUM #13 AND LUMBAR CENTRUM#1 ABSENT; THORACIC ARCHES #13 AND LUMBAR ARCHES#1 MALFORMED AND CLOSELY APPROXIMATED11 M 5.9 v Undescended thymic tissue, lefts = skeletal examination, v = visceral examination, M=Male, F=Female, g=gramsHuntingdon Life Sciences 05-4287 Page 143211-DIPE-DEV Final ReportFemales INDIVIDUAL FETAL EXTERNAL, VISCERAL AND SKELETAL OBSERVATIONS APPENDIX LAnimal Fetus Weight Abnormalities (only fetuses with abnormalities are listed)Number Exam Number Sex (g) Fate Major abnormalities in CAPITALSGroup 4 - 20000 MG/M34909 5s, 4v4 F 5.8 s Rib(s) 14th rudimentary, right6 M 5.4 s EXOCCIPITAL AND CERVICAL ARCH #1 FUSED, rightSternebra(e) malaligned, #4 and #5, slight4910 6s, 7v13 F 5.8 s 27 presacral vertebra(e)Rib(s) 14th rudimentary, bilateral4911 8s, 8v5 M 5.8 s Rib(s) 14th rudimentary, right16 F 5.6 s Rib(s) 14th rudimentary, left4912 5s, 6v2 M 6.7 s Rib(s) 14th rudimentary, right8 F 5.8 s Sternebra(e) malaligned, #3 through #5, slight to moderate4915 7s, 7v3 F 5.3 s Rib(s) 14th rudimentary, right8 F 5.1 s Rib(s)14th rudimentary, left15 F 5.0 v Innominate artery absent4916 8s, 8v13 F 4.2 v Left umbilical arterys = skeletal examination, v = visceral examination, M=Male, F=Female, g=gramsHuntingdon Life Sciences 05-4287 Page 144211-DIPE-DEV Final ReportFemales INDIVIDUAL FETAL EXTERNAL, VISCERAL AND SKELETAL OBSERVATIONS APPENDIX LAnimal Fetus Weight Abnormalities (only fetuses with abnormalities are listed)Number Exam Number Sex (g) Fate Major abnormalities in CAPITALSGroup 4 - 20000 MG/M34917 7s, 7v4 F 5.6 v Undescended thymic tissue, left10 F 6.0 s 7th cervical rib(s) present, right12 M 5.8 s Rib(s) 14th rudimentary, right14 F 6.0 s Rib(s) 14th rudimentary, left4921 6s, 5v1 M 5.4 s 7th cervical rib(s) present, right25 presacral vertebra(e)Sternebra(e) 5 and/or 6 unossified, #5Rib(s) 13th rudimentary, right2 M 5.8 v Undescended thymic tissue, right3 M 5.2 s 25 presacral vertebra(e)Sternebra(e) 5 and/or 6 unossified, #5Rib(s) 13th rudimentary, bilateralMetatarsals, 1 unossified, bilateral11 F 5.0 s 25 presacral vertebra(e)Rib(s) 13th rudimentary, bilateralMetatarsals, 1 unossified, bilateral4923 6s, 5v4 M 5.9 v Undescended thymic tissue, left12 M 5.8 s Rib(s) 14th rudimentary, lefts = skeletal examination, v = visceral examination, M=Male, F=Female, g=gramsHuntingdon Life Sciences 05-4287 Page 145211-DIPE-DEV Final ReportFemales INDIVIDUAL FETAL EXTERNAL, VISCERAL AND SKELETAL OBSERVATIONS APPENDIX LAnimal Fetus Weight Abnormalities (only fetuses with abnormalities are listed)Number Exam Number Sex (g) Fate Major abnormalities in CAPITALSGroup 4 - 20000 MG/M34924 8s, 9v1 F 4.7 v Renal papilla (e) notfully developed (grade 1), bilateralUreter(s) distended, left14 F 5.1 v Left umbilical artery17 F 5.8 s Rib(s) 14th rudimentary, lefts = 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

### 05-4287

: : Statistical analysis



### 2 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). 

## Page 147

### 05-4287

: : Statistical analysis



### 3 INTRODUCTION AND EXPERIMENTAL DESIGN

This rat study had a control and three groups treated at 2000 mg/m 3 

### , 10000 mg/m 3 

### and 20000 mg/m 3 

### . 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). 

## Page 148

### 05-4287

: : Statistical analysis



### 4 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. 

## Page 149

### 05-4287

: : Statistical analysis



### 5 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. 

## Page 150

### 05-4287

: : Statistical analysis



### 6 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/m 3 

### ( p <0.01). d8-d11 significant difference at 2000 mg/m 3 

### ( p <0.05) and 10000 mg/m 3 

### ( p <0.01). d11-d14 significant difference at 2000 mg/m 3 

### ( p <0.05) and 20000 mg/m 3 

### ( p <0.01). d5-d14 significant difference at 2000 mg/m 3 

### ( p <0.05), 10000 mg/m 3 

### ( p <0.01) and 20000 mg/m 3 

### ( p <0.01).

Maternal net bodyweight change from d5 minus gravid uterine weight Significant difference at 20000 mg/m 3 

### ( p <0.001).

Maternal feed consumption d8-d11 significant difference at 10000 mg/m 3 

### ( p <0.05) and 20000 mg/m 3 

### ( p <0.01). d11-d14 significant difference at 20000 mg/m 3 

### ( p <0.01). d20-d21 significant difference at 2000 mg/m 3 

### ( p <0.05), 10000 mg/m 3 

### ( p <0.05) and 20000 mg/m 3 

### ( p <0.01). d5-d21 significant difference at 20000 mg/m 3 

### ( 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. 

## Page 151

### 05-4287

: : Statistical analysis



### 7 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/m 3 

### ( p =0.007). Litter body weight Significant decrease at 20000 mg/m 3 

### ( p =0.016). 

## Page 152

### 05-4287

: : Statistical analysis



### 8 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) 13 th 

### Rudimentary NP No significant differences Ribs, V Rib(s) 14 th 

### 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/m 3 

### ( p =0.047).

† There was some evidence of a statistically significant increase from control at 20000 mg/m 3 

### ( p =0.067). 

## Page 153

### 05-4287

: : Statistical analysis



### 9 REFERENCES

ARMITAGE, P., BERRY, G. and MATTHEWS, J.N.S. (2002) Statistical Methods in Medical Research, 4 th 

### 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. 

## Page 154

### 05-4287

: : Statistical analysis 

### 10 TABLE 1

Maternal body weight (g)

0 mg/m 3 (n=23) 2000 mg/m 3 (n=23) 10000 mg/m 3 (n=24) 20000 mg/m 3 (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 

## Page 155

### 05-4287

: : Statistical analysis 

### 11 TABLE 2

Maternal body weight change (g)

Days of gestation 0 mg/m 3 (n=23) 2000 mg/m 3 (n=23) 10000 mg/m 3 (n=24) 20000 mg/m 3 (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/m 3 (n=22) 2000 mg/m 3 (n=23) 10000 mg/m 3 (n=24) 20000 mg/m 3 (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 

## Page 156

### 05-4287

: : Statistical analysis 

### 12 TABLE 3

Maternal feed consumption (g/kg/day)

Days of gestation 0 mg/m 3 (n=22-23) 2000 mg/m 3 (n=22-23) 10000 mg/m 3 (n=22-24) 20000 mg/m 3 (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. 

## Page 157

### 05-4287

: : Statistical analysis 

### 13 TABLE 4

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

0 mg/m 3 (n=22) 2000 mg/m 3 (n=23) 10000 mg/m 3 (n=24) 20000 mg/m 3 (n=20) Parameter mean (sd) mean (sd) mean (sd) mean (sd)

Absolute

Adjusted A 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 

## Page 158

### 05-4287

: : Statistical analysis 

### 14 TABLE 5

Reproductive performance

0 mg/m 3 (n=23) 2000 mg/m 3 (n=23) 10000 mg/m 3 (n=24) 20000 mg/m 3 (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 

## Page 159

### 05-4287

: : Statistical analysis 

### 15 TABLE 6

Mean (adjusted) fetal bodyweight (g)

0 mg/m 3 (n=23) 2000 mg/m 3 (n=23) 10000 mg/m 3 (n=24) 20000 mg/m 3 (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/m 3 (n=24) 2000 mg/m 3 (n=24) 10000 mg/m 3 (n=24) 20000 mg/m 3 (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/m 3 (n=23) 2000 mg/m 3 (n=23) 10000 mg/m 3 (n=24) 20000 mg/m 3 (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/m 3 (n=23) 2000 mg/m 3 (n=23) 10000 mg/m 3 (n=24) 20000 mg/m 3 (n=22)

Skeletal CERVICAL VERT

RIBS

RIBS

SACRAL VERT

SKULL

STERNEBRAE

STERNEBRAE V 7TH CERVICAL RIB(S) PRESENT

V RIB(S) 13 TH
RUDIMENTARY

V RIB(S) 14 TH
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 .....................................................................................176Certified 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 Page 1 of2

I 

# I

I

I 

# I I

I

I

I

I

I

I



# I II

12/8/2005 

## Page 165Certified Papers Retrieval 

# l.ablJ 

# ·er**~** IJ

, . ·
. PMI NUTRITION**,~** .• 11111'·1 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 Page 1 of2



# I

I



# JJ

12/8/2005 

## Page 166Certified 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

http:/ /www.labdiet.com/certifiedlpwa

_ spc002.asp Page 1 of2

I 

## I I 

# I

I

I

I

I

I

I

I

12/8/2005 

## Page 167LAB 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 168LAB 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 169LAB 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 170LAB 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 171LAB 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 172LAB 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:

___ ,.._,xf

.....

## :zta:::.>.G.b,,.,..lQJJ!1""'"~=::=..;;_-'----_....~~fv<""""""4...=->-=------ DATE: 

### Ott_Lf dS", 

### J?tr: 7(/ l...b 

### ¥

, 

## Page 173LAB 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 174Elizabethtown 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 178Huntingdon 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 183HuntingdonLifeSciencesStudvNo.05-42874.EXPERIMENTALDESIGNGroupGroupDesi2nationExposureLevels(mg/m3)a1Control0(aironly)2Low20003Mid100004High20000TreatmentGestationDays5-205-205-205-20TimeMatedFemales24242424Page3FinalProtocolNumberofAnimalsFetalEvaluations(Fetuses/Litter)EuthanizedGestationSoftDay21bExternalTissueSkeletalAllAllAllAllAllAllAllAllaExposureswillbe6hoursperdayfor7consecutivedaysperweekforgestationdays5-20.Exposurelevelsareexpressedasmg/m3oftestsubstance.Theexposureswillbeconductedviawhole-bodyexposure.Controlanimalswillbetreatedwithaironlywiththesametreatmentregimenasthetreatedgroups.bPostmortemevaluationswillalsobeperformedonanimalswhicharefounddeadoreuthanizedforhumanereasonsorinamoribundconditionduringthecourseofthestudy.Thefirstdayofgestation(GestationDay0=dayofdetectionofpositivesignofmating)willbedefinedasDay0ofthestudyforeachanimal.Page 184

### 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 187Huntingdon 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 193Huntingdon 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 196Huntingdon Life Sciences Study No. 05-4287 Page 16 Final Protocol



### maternal body weight values and body weight changes during gestation (Note: body weight measurements will only be compared to GD 

### 5 values

or subsequent relevant values)

maternal feed consumption values (presented as grams of feed/kg of body weight/day) gravid uterine weights

corpora lutea implantation data pre-implantation loss early embryonic deaths (evidence of implantation but no recognizable fetus)

live fetuses

dead fetuses (no significant degeneration)

late embryo-fetal deaths (recognizable dead fetus undergoing degeneration, regardless of size)

total embryo-fetal deaths and as % 

### of implant sites

number of males and females per litter

Mean values of all exposure groups will be compared to the mean value for the control group at each time interval. Evaluation of equality of group

means will be made by the appropriate statistical method (either parametric or non-parametric methods), followed by a multiple comparison test if needed. Bartlett's test (Bartlett, 1937; Sokal and Rohlf, 1995; Snedecor and Cochran, 1967) will be performed to determine if
groups have equal variances. For all parameters if the variances are equal, parametric procedures will be used; if not, nonparametric procedures will be used.

The parametric method will be the standard one-way analysis of variance (ANOVA) using the F ratio to assess significance (Armitage, 1971; Dunlap and Duffy, 1975). If 

### significant differences among the means are indicated Dunnett's (Dunlap et al., 1981; Dunnett, 1955, 1964) test will be used to determine which means are significantly different from the control. The nonparametric method will be the Kruskal-Wallis test (Kruskal and Wallis, 1952, 1953) and if differences are indicated, Steel's test (Steel, 1959) will be used to determine which means differ from control. Bartlett's test for equality of variance will be conducted at the 1% significance level; all other statistical tests will be conducted at the 5% 

### and 1% significance levels.

References for these procedures are: 

## 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 198Huntingdon 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 199Huntingdon 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 201Huntingdon 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 203Huntingdon 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 204Huntingdon 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 205Huntingdon 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 206Huntingdon 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 207Huntingdon 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 OnlineDoc 0 

### 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 208Huntingdon 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 wei ght changes during gestation (Note: body weight measurements will only be comp ared 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 co mpared to the mean value for the control group at each tim e interval. Evaluation of equality of group means will be made by the appropriate statistical method (either parame tric 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 de termine 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 va riance 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, 4 th 

### 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. Fortra n IV functions for calculating exact probabilities associated with z, chi

- Square, t and F Values. Behav. Res. M ethods 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 th e American Statistical Association 50: 1096- 1121.
Dunnett, C.W. 1964. New tables for m ultiple 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 Wa llis, W.A. 1953. Errata for Kruskal-Wallis (1952) Journal of the American Statistical Association 48: 907-911.

Snedeco r, G.W. and Cochran, W.G. (1989) Statis tical Methods. 8th edition Iowa State University Press, Iowa, USA.
Sokal, R.R. and Rohlf, F.J. 1995. Biomet ry. 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 compos ite 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. Th e analysis uses the litter as the basis for analysis and ef fectively 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 p airwise vs compared with the control group using t

- tests associated with least squares means Dunnett’s test with the means being adju sted for the litter size . The least squares means allows comparisons that account for differences in litter size and sex. The mathematical model i s based on a paper by Chen et. al. (1996). Statistical significanc e 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 devel opmental 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 pairw ise Wilcoxon rank sum tests (Wilcoxon 1945).

For fetal findings: 

## Page 212Page 213Huntingdon Life Sciences05-4287Page 214211-DIPE-DEVFinalReportHistorical Control DataFetal Skeletal ObservationsAppendix PRat Strain: Sprague-Dawley [Crl: CD IGS BR]Supplier: Charles River LaboratoriesFetal Variation: Rib(s) 14th RudimentaryFetuses LittersStudy Route 1st GD 6 Last GD 20No.ExaminedNo. withFinding% withFindingNo.ExaminedNo. withFinding% withFindingALLSep-02 Jul-05 902 79 8.76 134 45 33.5802-4235 Oral gavage Sep-02 Oct-02 160 11 6.88 22 6 27.2703-4253 Inhalation Feb-05 Mar-05 167 12 7.19 24 7 29.1703-4255 Oral gavage Jun-03 Sep-03 148 23 15.54 22 15 68.1804-4261 Inhalation Aug-04 Sep-04 150 22 14.67 22 9 40.9104-4267 Subcutaneous Nov-04 Dec-04 135 6 4.44 22 4 18.1805-4292 Intravenous Jun-05 Jul-05 142 5 3.52 22 4 18.18

### ~ 

# 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 215CHARLES RIVER LABORATORIES, PRECLINICAL SERVICES, PENNSYLVANIA SUMMARY OF REPRODUCTIVE INDICES Crl:CD(SD) RATS DAY 21 CAESAREAN-SECTION

PERIOD: JUNE 2008- JUNE 2010

FULL STUDIES

NUMBER OF STUDIES

NUMBER OF RATS: TESTED PREGNANT FOUND DEAD ABORTED
DELIVERED

NUMBER OF RATS PREGNANT AT

CAESAREAN-SECTIONING

NUMBER OF RATS WITH SINGLE

CONCEPTUS LITTER: LIVE RESORBED ABORTED 48

1195 1106

7*

1 13

1089

1 0 0

RANGE/STUDY MEANor% MEANor%

%PREGNANT

AVERAGENO.CORPORALUTEA

AVERAGE NO. IMPLANTATIONS

AVERAGE % PREIMPLANTATION

LOSS

AVERAGE LITTER SIZE

AVERAGE NO. LIVE FETUSES

AVERAGE NO. DEAD FETUSES 92.9

15.8

14.8

5.4

14.1

0.0

AVERAGE NO. RESORPTIONS 0.7

AVERAGE NO. EARLY RESORPTIONS 0. 7

AVERAGE NO. LATE RESORPTIONS 0.0 (76.0-100)

(13.6-17.9)

(13.0-17.0)

(1.8-11.3)

(12.5-16.1)

(0.3-1.2)

(0.3-1.2) DOSAGE-RANGE STUDIES

MEANor%

93.6

15.6

14.5

5.1

14.0

0.0

0.6

0.6

0.0 11

86 79

1 0 0

78

0 0 0

RANGE/STUDY MEANor%

(62.5-100)

(14.0-16.5)

(13.3-15.5)

(3.0-10.2)

(12.8-15.2)

(0.1-1.0)

(0.1-1.0)

(0-0.2)



## * Two were unscheduled sacrifices and one was euthanized due to an intubation accident

-20- 

## Page 216CHARLES 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 DIRECTOR a 

### 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.