## Data Requirement

EPA 79.61 and OPPTS 870.3700 and OECD 414

STUDY NO. 05-4287

SPONSOR STUDY NO. 211-DIPE-DEV



## GASOLINE DIPE VAPOR CONDENSATE:

EMBRYO-FETAL TOXICITY STUDY IN RATS BY

INHALATION EXPOSURE

2nd 

## Draft Final Report

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

Performed by: Huntingdon Life Sciences (HLS)

1 00 Mettlers Road

East Millstone, New Jersey 08875-2360

Submitted to: American Petroleum Institute (API)

1220 L Street, Northwest

Washington, D.C. 20005-4070

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

Date: 2 December 2011

Page 1 ofxxx

### Huntingdon Life Sciences 05-4287

211-DIPE-DEV



## STATEMENT OF COMPLIANCE Page2

Draft Final Report

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

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

Sciences' Standard Operating Procedures.

It 

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

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

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

### addition, the Sponsor

conducted a non-GLP purity analysis.

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

Study Director

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

Sponsor Representative Date

Date

### Huntingdon Life Sciences



## SCIENTISTS 05-4287

211-DIPE-DEV



## SIGNATURE PAGE Page3

Draft Final Rwort

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

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

(Appendix Q).

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

Study Director



## SCIENTIFIC REVIEW

The following Scientist has reviewed and approved this report:

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

Director, Developmental and Reproductive Toxicology Date

Date

## Huntingdon Life Sciences 05-4287

211-DIPE-DEV

QUALITY ASSURANCE STATEMENT Page4

Draft Final Report

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

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

reported to the Study Director and Management.

Type of Inspection

GLP Protocol Review

Exposure Monitoring and

Equipment Records

Formulation Chemistry: Inhalation

Dose Analysis

Terminal Necropsy

Fetal Soft Tissue Evaluations and

Training Records

Exposure Data and Report

Final Report and Study Data

Protocol Amendments 1 and 2

Pathology Associates Facility

Inspection

Protocol and Draft Protocol

Amendment 4a

Final Protocol Amendment 4a

Protocol Amendment 3

Protocol Amendment 4 Date(s) of Inspection

21-25 Apr 05

8 Aug05

16 Aug 05

22 Aug 05

17 Oct 05

19-20 Dec 05

14 Dec 05- 04-Jan-06

12 Jan 06

13 

## Jul 06

03 May 11



## 25 May 11

10Nov 08

01 Dec 11



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

Quality Assurance Group Leader Reported to

Study Director

and Management

25 Apr05

11 Aug05

16 Aug 05

23 Aug05

21 Oct 05

20 Dec 05

06-Jan-06

12 Jan 06

18 Oct 06

05 May 11

25 May 11

10Nov 08

01 Dec 11

Date

a 

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

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

Management on the dates indicated above.

## Huntingdon Life Sciences 05-4287

211-DIPE-DEV PageS

Draft 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 pregnant rat when administered by whole-body

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

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

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

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

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

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

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

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

of the Clean Air Act.

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

CD® female rats (24/group) at target concentrations of2000, 10000 and 20000 mg/m 3 

## for

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

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

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

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

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

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

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

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

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

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

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

and ossification variations.

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

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

19776 ± 961 mg/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 consumption (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 variants. A NOAEL for embryo-fetal effects was

therefore established at 20000 mg/m 3

.

## Huntingdon Life Sciences 05-4287

211-DIPE-DEV

TABLE OF CONTENTS 

## Page6

Draft Final Rc:mort

COVER PAGE

..................................................................................................................... 1

STATEMENT OF COMPLIANCE

..................................................................................... 2

SIGNATURE P AGE

............................................................................................................ 3

QUALITY ASSURANCE STATEMENT

.......................................................................... 4

SUMMARY

......................................................................................................................... 5

TABLE OF CONTENTS

..................................................................................................... 6

1. INTRODUCTION

.................................................................................................. 8

2. MATERIALS AND METHODS



## 2.1. Study Management

...................................................................................... 8

2.2. Study Dates

.................................................................................................. 9

2.3. Experimental Outline

................................................................................. 10

2.4. Justifications

.............................................................................................. 1 0

2.5. Test Substance

........................................................................................... 11

2.6. Test Animals

.............................................................................................. 13

2.7. Animal Assignment

................................................................................... 14

2.8. Animal Identification

................................................................................. 14

2.9. VeterinaryCare

.......................................................................................... 14

2.10. Animal Husbandry During Non-Exposure Periods

.................................... 15

2.11. Test Substance Administration and Chamber Operation

........................... 17

2.12. Experimental Evaluations

.......................................................................... 17

2.13. Postmortem Evaluations

............................................................................ 18

2.14. Fetal Evaluations

........................................................................................ 19

2.15. Statistical Analysis

..................................................................................... 20

2.16. Data Storage

............................................................................................... 21

2.17. Regulatory References

............................................................................... 21

2.18. Protocol Deviations

.................................................................................... 22

3. RESULTS AND DISCUSSION



## 3 .1. Test Substance Analysis

............................................................................. 23

3 .2. Chamber Monitoring

.................................................................................. 23

3.3. Maternal Data

............................................................................................. 24

3.4. Fetal Data

................................................................................................... 26

4. CONCLUSIONS

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

## 27

REFERENCES

.................................................................................................................. 28

CALCULATIONS

............................................................................................................. 29

## Huntingdon Life Sciences 05-4287

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Draft Final Report

GENERAL PREFACE

...................................................................................................... 30

FIGURES



## 1. Mean Gestation Body Weights

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

2. Mean Gestation Feed Consumption

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

TABLES



## 1. Summary of Survival and Pregnancy

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

2. Summary of Clinical Observations During Gestation

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

3. Summary of Gestation Body Weights (grams)

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

4. Summary of Gestation Body Weight Gain (grams)

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

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

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

6. Summary of Maternal Necropsy Observations

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

7. Summary of Cesarean Section Data

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

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

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

9. Summary of Fetal Observations

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

APPENDICES



## A. Analytical Report

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

B. Inhalation Report

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

C. Individual Animal Termination History

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

D. Individual Clinical Observations During Gestation

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

E. Individual Gestation Body Weights (grams)

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

F. Individual Gestation Body Weight Gain (grams)

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

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

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

H. Individual Maternal Necropsy Observations

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

I. 

## Individual Female Reproduction Data and Mean Fetal Weights

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



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

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

K. Individual Fetal Body Weights

....................................................................................... . L. 

## Individual Fetal External, Visceral and Skeletal Observations

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

M. Statistical Analysis Report

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

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

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

0. 

## Protocol and Protocol Amendments

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

P. Historical Control Data

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

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

R. Report Amendments

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

## Huntingdon Life Sciences



## 1. INTRODUCTION 05-4287

211-DIPE-DEV Page 8

Draft Final Report

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

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

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

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

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

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

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

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

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

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

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

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

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

evaluated within the context of the treatment requirements of those proscribed

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



## 2. MATERIALS AND METHODS

2.1. STUDY MANAGEMENT

2.1.1. SPONSOR

American Petroleum Institute (API)

1220 L Street, Northwest

Washington, D.C. 20005-4070



## 2.1.2. SPONSOR REPRESENTATIVE

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



## 2.1.3. TESTING FACILITY

Huntingdon Life Sciences (HLS)

1

00 Mettlers Road

East Millstone, New Jersey 08875-2360

## Huntingdon Life Sciences 05-4287

211-DIPE-DEV



## 2.1.4. STUDY DIRECTOR 1 

## Page9

Draft Final Rcmort

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

Toxicologist

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



## 2.1.5. PRINCIPAL INVESTIGATOR-SKELETAL

ASSESSMENTS

Michael Mercieca, M.S.

Pathology Associates International 

## (P AI)



## 2.1.6. PRINCIPAL INVESTIGATOR- STATISTICS

Graham Healey, BSc, MSc, ARCS

Huntingdon Life Sciences, UK



## 2.2. STUDY DATES

2.2.1. STUDY INITIATION

3 August 2005 (Date Study Director signed the Protocol)



## 2.2.2. DATE OF ANIMAL RECEIPT

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



## 2.2.3. EXPOSURE INITIATION

6 August 2005 (Experimental Start Date

- First GD 5)



## 2.2.4. EXPOSURE TERMINATIONS

24 August 2005 (Last GD 20)



## 2.2.5. TERMINAL SACRIFICE

22-25 August 2005 (GD 21)

1 

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

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

fmalization of the study report.

## Huntingdon Life Sciences 05-4287

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Draft Final Report

Group

1

2



### 3

4 

## 2.2.6. EXPERIMENTAL TERMINATION

01 December 2005 (Date oflast data collection)

2.2.7. STUDY COMPLETION

Day Month Year (Date Final Report IS 

## signed by the Study

Director)

2.3. EXPERIMENTAL OUTLINE

Exposure Treatment Number of Animals

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

Day21 External Soft Tissue Skeletal

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



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



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

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



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

levels are expressed as mg/m 3 

### of test substance. The exposures were conducted by whole-body exposure.

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

treated groups.

The ftrst day of gestation (GD 0

= 

### day of detection of positive sign of mating) was defmed as Day 0 of the

study for each animal.



## 2.4. JUSTIFICATIONS

2.4.1. ROUTE, DURATION AND FREQUENCY OF

ADMINISTRATION

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

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

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

EMBSI study, and as recommended in the relevant EPA and

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

2.4.2. EXPOSURE LEVEL SELECTION

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

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

explosive limit (LEL) of the test substance (the low, intermediate

## Huntingdon Life Sciences 05-4287

211-DIPE-DEV Page 11

Draft Final Report

and high exposure levels were approximately 5, 25, and 50% of the

LEL).

2.4.3. TEST ANIMAL SELECTION



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

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

## is the

rodent species that is recommended by the relevant EPA and

OECD test guidelines. Historical control data are also available

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

2.4.4. NUMBER OF ANIMALS



## The number of animals in this study was considered the minimum

necessary to allow for meaningful interpretation of the data as

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

endpoints of principal interest (eg 

## embryo-fetal death, fetal

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

## 20 litters tends to provide the necessary degree of consistency

between studies. In 

## the expectation of pregnancy rates of

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

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

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

group was comparable to the number in the EMBSI study

(25/group).

2.5. TEST SUBSTANCE



## Gasoline DIPE (di-isopropyl ether) Vapor Condensate

2.5.1. TEST SUBSTANCE CATEGORY



## Gasoline product

2.5.2. SUPPLIER



## ChevronTexaco Research and Technology Company

100 Chevron Way

Richmond, CA 94802

## Huntingdon Life Sciences 05-4287

211-DIPE-DEV Page 12

Draft Final Report

2.5.3.

2.5.4.

2.5.5.

2.5.6.

2.5.7.

2.5.8. LOT NUMBER



## API 01-06

PURITY



## 100% Gasoline DIPE Vapor Condensate

DESCRIPTION



## Colorless liquid

DATES RECEIVED



## 7 December 2001

05 August 2002

EXPIRATION DATE



## Not available, stable per MSDS.

ANALYSIS



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

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

of the test substance was documented and is maintained by the

Sponsor. In 

## addition, the Sponsor conducted a non-GLP purity

analysis and the Testing Facility conducted a GLP stability analysis

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

2.5.9. STORAGE



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

when being used in the inhalation laboratory.

2.5.10. DISPENSING



## The test substance was received from ChevronTexaco Research

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

5-gallon cylinders were practical to be used for exposure

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

Testing Facility from the 100 gallon cylinders into 5-gallon

cylinders using nitrogen pressurization.

## Huntingdon Life Sciences 05-4287

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Draft Final Report

2.6. 2.5.11. ARCHIVAL SAMPLE



## An 

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

the Archives of the Testing Facility under conditions specified for

test article storage. Since multiple studies were conducted with the

same test substance, a common archival sample was taken and

appropriately labeled.

2.5.12. DISPOSITION



## The unused portion of the test substance was returned to the

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

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

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

disposition.

TEST ANIMALS

2.6.1. SPECIES



## Albino Rats (Outbred) VAF/Plus®

Sprague- Dawley Derived (CD®)

Crl:CD® (SD) IGS BR

2.6.2. SUPPLIER



## Charles River Laboratories

Raleigh, NC27610

2.6.3. NUMBER OF ANIMALS



## Ordered and Received:

100 time-mated females

Placed on test:

96 females

2.6.4. AGE AT RECEIPT



## Approximately 10-12 weeks

## Huntingdon Life Sciences 05-4287

211-DIPE-DEV

2.6.5. AGE AT INITIATION OF EXPOSURES



## Approximately 10-12 weeks Page 14

Draft Final Rwort

2.6.6. WEIGHT AT INITIATION OF EXPOSURES (GRAMS)



## Females: Mean



## 244 Range



## 216-280

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

the mean weight.

2.6.7. ACCLIMATION PERIOD



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

initiation of exposures on GD 5.

2.7. ANIMAL ASSIGNMENT



## Animals were placed into study groups using a computerized

randomization program. This ranked Gestation Day 4 body weights into

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

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

maintained in the study file.

2.8. ANIMAL IDENTIFICATION



## Each animal was assigned a temporary identification number upon receipt.

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

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

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

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

exposure level identification and contained the study number and animal

number.

2.9. VETERINARY CARE



## Animals were monitored by the technical staff for any conditions requiring

possible veterinary care, and the staff veterinarian approved the animals

for use in the study.

## Huntingdon Life Sciences 05-4287

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Draft 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 com-cob bedding were employed (in case

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

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

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

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

provided at least every 7 days.

2.10.3. FEED



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

MO) was available without restriction, except during actual

exposures.

2.10.4. FEED ANALYSIS



## Analytical certificates for relevant batches of feed, provided by the

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

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

contaminants in the feed that were expected to interfere with the

objectives of this study.

2.10.5. WATER



## Facility water was supplied by Elizabethtown Water Company,

(Westfield, NJ) and was provided ad libitum 

## to individual animal

cages through an automated watering system, except during actual

exposures.

## Huntingdon Life Sciences 05-4287

211-DIPE-DEV

2.10.6. WATER ANALYSIS 

## Page 16

Draft Final Report

Water analyses are conducted monthly by the Elizabethtown Water

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

water meets standards specified under the EPA Federal Safe

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

## addition,

water samples are collected biannually from representative rooms

in the Testing Facility. Chemical and microbiological water

analyses were conducted on these samples by a subcontract

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

analyses (provided by the supplier) and chemical and

microbiological water analyses (provided by the subcontractor)

results are maintained on file at the Testing Facility and are

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

contaminants in the water that were expected to interfere with this

study.

2.10.7. BEDDING



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

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

no known contaminants in the bedding that were expected to

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

is included in Appendix N.

2.10.8. ENVIRONMENTAL CONDITIONS

Light/Dark Cycle



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

automatic timer.

Temperature



## Temperature was monitored in accordance with Testing Facility

SOPs and was maintained within the specified range:

Desired Range:

Actual Range:

Daily Average Range: 18 to 26°C

23 to 24°C

23 to 24°C

## Huntingdon Life Sciences 05-4287

211-DIPE-DEV

Relative Humidity 

## Page 17

Draft Final Report

Relative humidity was monitored in accordance with Testing

Facility SOPs and was maintained within the specified range:

Desired Range:

Actual Range:

Daily Average Range:

Air Changes 

## 30to 70%

49 to 67%

55 to 60%

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

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

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

these records.

2.11. TEST SUBSTANCE ADMINISTRATION AND CHAMBER

OPERATION



## See Appendix B, Inhalation Report.

2.12. EXPERIMENTAL EVALUATIONS

2.12.1. VIABILITY EXAMINATION (CAGE-SIDE)



## Observations for mortality, morbidity, and signs of severe toxicity

were made at least twice daily.

2.12.2. DETAILED PHYSICAL EXAMINATIONS

In-Chamber: 

## All animals were observed as a group at least once

during each exposure. This was routinely performed near the

middle of each exposure. Any pertinent behavioral changes and all

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

were recorded.

Out-of-Chamber: 

## Animals on study were examined daily from

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

Examinations included observations of general condition, skin and

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

occurrence of secretions and excretions, and autonomic activity

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

pattern). Changes in gait, posture and response to handling as well

## Huntingdon Life Sciences 05-4287

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Draft Final Report

as the presence of clonic or tonic movements, stereotypy (e.g.,

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

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

evaluations of respiration. During the treatment period, these

evaluations were performed after exposure.

2.12.3. BODY WEIGHTS



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

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

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

14-21 and 5-21.

2.12.4. FEED CONSUMPTION



## Feed consumption was recorded and reported for the intervals:

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

2.13. POSTMORTEM EVALUATIONS

2.13.1. METHOD OF EUTHANASIA



## All dams were euthanized by carbon dioxide inhalation, by

approximately noon on each day. All live fetuses were euthanized

with intraperitoneal sodium pentobarbital.

2.13.2. MACROSCOPIC EXAMINATION



## A macroscopic postmortem examination was performed on all the

dams. Macroscopic lesions or tissues with significant findings were

saved in 10% Neutral Buffered Formalin (NBF).

Apparently non-pregnant uteri were stained with ammonium

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

status or detect implantation sites not otherwise visible.

Each dam had the following examinations of the reproductive tract

onGD21:

The intact uterus (ovaries attached) was removed from the

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

weighed. Corpora lutea were counted and the number per ovary

## Huntingdon Life Sciences 05-4287

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Draft Final Report

recorded. The number and location of the following was recorded

for each uterine hom:

• live fetuses

• dead fetuses (no significant degeneration)

• late embryo-fetal deaths (recognizable dead fetus undergoing

degeneration, regardless of size)

• early embryonic deaths (evidence of implantation but no

recognizable fetus)

Each placenta was examined macroscopically. The maternal carcass,

uterus, ovaries and placentas were then discarded.

2.14. FETAL EVALUATIONS

2.14.1. EXTERNAL EVALUATIONS



## All live fetuses were weighed and individually identified within

cassettes. Each live and dead retained fetus was given a

macroscopic external examination for defects, including

observation of the palate.

2.14.2. FETAL SOFT TISSUE (VISCERAL) EVALUATION



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

fetuses within the litter, nominally) were placed in Modified

Davidson's fixative for preservation and decalcification. These

fetuses were subjected to soft tissue examination by gross

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

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

variations were recorded. Particular attention was paid to the

reproductive tract for altered signs of development. During the

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

inspection of the gonads. Following complete dissection of the

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

buffered formalin.

2.14.3. FETAL SKELETAL EVALUATIONS



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

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

process the sex of each fetus was determined by internal inspection

## Huntingdon Life Sciences 05-4287

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and recorded. The eviscerated fetuses were placed in 70%

isopropyl alcohol for preservation and processed for staining of the

skeleton using Alizarin Red S. Subsequently, these fetuses were

evaluated for skeletal malformations and ossification variations by

an approved subcontractor, Pathology Associates International

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

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

with a mold inhibitor.

2.14.4. DEAD FETUSES

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



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

discarded.

2.15. STATISTICAL ANALYSIS

Analysis was performed by the Statistics Department of Huntingdon Life

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

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

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

report.

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

details of the methods and statistical results.

2.15.1. CONTINUOUS DATA

The following mean measures were analyzed:

Maternal body weight values and body weight changes during

gestation

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

ofbody weight/day)

Gravid uterine weight

Corpora lutea

Implantation sites

Pre-implantation loss as%

Early embryonic deaths ('early resorptions')

Late embryo-fetal deaths ('late resorptions')

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

Live fetuses

## Huntingdon Life Sciences 05-4287

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Number of males and females per litter

2.15.2. INCIDENCE DATA



## The following measures were analyzed:

Maternal necropsy findings Page 21

Draft Final Report

Maternal pregnancy data (including corpora lutea, implantation

sites, resorptions, etc.)

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

5 visceral and 7 skeletal findings required analysis

2.15.3. FETAL BODY WEIGHT



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

sexes.

2.16. DATA STORAGE



## All data documenting experimental details and study procedures and

observations were recorded and maintained as raw data.

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

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

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

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

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

of these materials.

2.17. REGULATORY REFERENCES

2.17.1. TEST GUIDELINES



## This study was designed to meet or exceed the pertinent

requirements of:

US EPA Vehicle Emissions Inhalation Exposure Guideline 79.61,

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

Organization for Economic Cooperation and Development (22

January 2001). OECD Guidelines for Testing of Chemicals;

OECD Guideline 414: Prenatal Developmental Toxicity Study

## Huntingdon Life Sciences 05-4287

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Draft Final Report

US EPA OPPTS Health Effects Test Guidelines 870.3700, Prenatal

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

2.17.2. GOOD LABORATORY PRACTICES



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

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

according to protocol and Huntingdon Life Sciences' Standard

Operating Procedures. The Testing Facility was responsible for

the GLP compliance and archiving of raw data produced by

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

2.17.3. ANIMAL WELFARE ACT COMPLIANCE



## This study complied with all appropriate parts of the Animal

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

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

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

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

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

2.18. PROTOCOL DEVIATIONS



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

affect the integrity of the study:

1. 

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

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

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

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

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

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

the fetuses were removed prior to recording necessary weights.

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

examination as per the protocol.

## Huntingdon Life Sciences 05-4287

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3. RESULTS AND CONCLUSIONS

3.1. TEST SUBSTANCE ANALYSIS

(Appendix A) 

## Page 23

Draft Final Report

GC analysis of the test substance showed no significant changes in

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

studies at this Testing Facility.

3.2. CHAMBER MONITORING

(Appendix B)



## Pre-study chamber distribution analyses showed that the test substance

was evenly distributed within each chamber. Chamber monitoring showed

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

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

the sound and light levels were acceptable.

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

concentrations (see Appendix B) are summarized as follows:

Target Analytical Nominal

Group Concentration Concentration Concentration

{mg/m 3 ) 

### (mg/m 3 ) 

### {mg/m 3 )



## 1 Air Control 0 0.00± 0.00 0±0

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

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

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

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

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

targeted exposure levels and to the nominal concentrations. Chamber

environmental conditions averaged 24°C temperature and 41% relative

humidity.

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Mean particle size distribution measurements for the exposures are

summarized as follows:

Mass Median Geometric Total

Aerodynamic Mass Group Standard Diameter Concentration

(J.tm) Deviation (mg/m 3 )



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

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

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

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

These results indicated that the atmospheres were essentially gas/vapor

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

test substance chambers and the air control chamber.

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

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

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

vaporized test substance. These data demonstrated that the test animals

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

substance in their acceptably proper proportions. The data were consistent

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

the atmosphere generation techniques.

3.3. MATERNAL DATA

3.3.1. MORTALITY AND PREGNANCY

(Table 

## 1; Appendices C and M)

All animals survived until the scheduled termination of the study

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

## Huntingdon Life Sciences 05-4287

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## 3.3.2. CLINICAL OBSERVATIONS

(Table 2; Appendices B and D) Page 25

Draft Final Report

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

overt reaction to treatment. There was only an increased incidence

of red nasal discharge among the 10,000 and 20,000 mg/m 3



## exposed animals, as compared with the other groups.

Chromodacryorrhea was also observed at a low incidence at

20,000 mg/m 3

, 

## in animals that did not show the nasal discharge.

These two findings (interpreted as secretion of porphyrin) are

common reactions to mild stress in rats. An 

## animal (#4908)

exposed at 20,000 mg/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 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

•



## 3.3.5. MACROSCOPIC POSTMORTEM EVALUATIONS

(Table 6; Appendices H and M)

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

tissue of one animal (#4908) at 20,000 mg/m 3

. 

## This finding was

considered incidental, and not related to treatment.

## Huntingdon Life Sciences 05-4287

211-DIPE-DEV

3.3.6. UTERINE IMPLANTATION DATA

(Table 7; Appendices I and M) 

## Page 26

Draft Final Report

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

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

groups.

3.3.7. GRAVID UTERINE WEIGHT DATA

(Table 8; Appendices J and M)



## Adjusted gravid uterine weight was reduced in the 20,000 mglm 3



## group but this reduction was not considered adverse.

3.4. FETAL DATA

3.4.1. Fetal Body Weights

(Table 7; Appendices I, K and M)



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

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

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

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

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

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

values are comparable to the historical control range for published

data from Charles River Labs (see Appendix P).

3.4.2. Fetal Observations

(Table 9; Appendices L and M)



## A very small number of major abnormalities occurred sporadically

in the study, with no relationship to the exposures.

Similarly, there were sporadic incidences across the groups, of

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

with the exposures was inferred.

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

was higher at 20,000 mg/m 3 

## than in the other groups, the difference

## Huntingdon Life Sciences 05-4287

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Draft Final Report

from Control did not achieve statistical significance, and remained

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

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

is approximated by the 2000 and 10000 mg/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.

4. CONCLUSIONS



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

20,000 mg/m 3 

## of Gasoline DIPE Vapor Condensate, 6 hours/day on gestation days

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

consumption during the exposure period. These changes were negligible at

2,000 mg/m 3 

## and most noticeable at 20,000 mg/m 3

• 

### It 

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

2,000 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 variants. A NOAEL for embryo-fetal effects was

therefore established at 20,000 mg/m 3

.

## Huntingdon Life Sciences 05-4287

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REFERENCES 

## Page 28

Draft Final Report

Salewski, E. 1964. Farbemethode zum makroskopischen machweis von

implantationsstellen am uterus der ratte. Archiv. Path. Exp. Pharmakol. 

## 247: 367.

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

University of Chicago Press, Page 271.

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

## Huntingdon Life Sciences



## Feed Consumption (glkg/day): 05-4287

211-DIPE-DEV



## CALCULATIONS Page29

Draft Final Report

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

body weights in kilograms ]/no. days



## Preimplantation Loss:

no. of corpora lutea- no. of implantation sites



## Percent Preimplantation Loss:

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



## Postimplantation Loss:

total no. of early and late resorptions and dead fetuses



## Percent Postimplantation Loss:

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



## Mean% Live Fetuses (number per animal):

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



## Mean% Dead Fetuses (number per animal):

'



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



## Mean Percent Resorptions (implants per animal):

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

## Huntingdon Life Sciences

General Notes 

## 05-4287

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General Preface Page 30

Draft Final Report

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

Unrounded individual animal data values are used to calculate the reported

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

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

subsequent calculations may produce minor discrepancies between the

calculated values and those presented in this report.HuntingdonLifeSciences05-4287211-DIPE-DEVPage·1DraftFinalReoortIu=rn--MeanGestationBodyWeights(grams)IFigure1I440-e-Group1 -0mg/m34202 -2000mg/m3400----tr---Group3 -10000mg/m3-380Ill--*-Group4-20000mg/m3Ee360Cl-J!l340.cm·320>-0300m280260--.--.240220200456.789101112131415 161718192021GestationDayHuntingdonLifeSciences05-4287211-DIPE-DEV9489847974i;''Cc,69a,64595449IMeanGestationFeedConsumption(g/kg/day)_/.//'---__·/....:....--8-Group1-0mg/m3--.--Group2-2000mg/m3----&---Group3-10000mg!m3->IE--Group4-20000mg/m3Page2Draft.FinalReoortFigure2I444to5·5to88to1111to14 14to1717to2020to215to21GestationDayInterval10-MAR-201117:37HuntingdonLifesciences05-4287-DOSEGROUP:DOSELEVEL{MG/M3):-------------No.offemalesmatedNPregnantN--Died/sacrificedmoribundN-ElectivesacrificeNNonpregnantN-Died/sacrificedmoribundN-ElectivesacrificeNTotalno.offemalesdied/Nsacrificedmoribund%Examinedatscheduledc-.sectionN-NonpregnantN-WithtotalimplantlossN%-WithviablefetusesN%TABLE1GASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSURESUMMARYOFSURVIVALANDPREGNANCY----------------------------------------------------------------123402000100002000024242424232324220000000011020000000000000.00.00.00.024242424110200000.00.00.00.02323242295.895.8100.091.710-MAR-201117:41HuntingdonLifeSciences05-4287TABLE2GASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSURESUMMARYOFCLINICALOBSERVATIONSDURINGGESTATION-(frequency/animals)DOSEGROUP:DOSELEVEL(MG/M3):DAY4to21NormalWITHINNORMALLIMITSTERMINALSACRIFICEDermal-GeneralALOPECIA-GENERALOcularCHROMODACRYORRHEA-UNILATERALOral/BuccalINCISORSMALOCCLUDEDNASALDISCHARGE-REDPalpablemassesMASS10431/2424/240/00/00/01/10/022000421/2424/240/00/00/011/30/0310000382/2424/240/00/00/050/150/0420000349/2324/248/114/318/143/95/110-MAR-201117:43HuntingdonLifeSciences05-4287TABLE3GASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSURESUMMARYOFGESTATIONBODYWEIGHTS(GRAMS)------------------------------------------------------------------DOSEGROUP:1234DOSELEVEL_(MG/M3):0'20001000020000--------------------------------------------------------------DAY4MEAN234235234235S.D.14.016.615.714.6N23232422DAY5MEAN244245244245S.D.14.616.513.915.2N23'232422DAY8.MEAN259260256256S.D.12.818.014.416.3N23232422DAY11MEAN281279274275S.D.14.919.615.619.5N23232422DAY14MEAN301296292290S.D.17.020.314.819.5N23232422DAY17MEAN332327323321S.D.22.224.318.724.9N23232422DAY20MEAN378370367365S.D.26.329.924.227.1N23232422DAY21MEAN38837937637428.632.725.927.410-MAR-2Di117:44HuntingdonLifeSciences05-4287DOSEGROUP:DOSELEVEL(MG/M3}:TABLE4GASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSURESUMMARYOFGESTATIONBODYWEIGHTGAIN(GRAMS}1230200010000420000----------------------------------------------------------------DAYS4TO5DAYS5TO8DAYS8TO11DAYS11TO14DAYS14TO17DAYS17TO20DAYS20TO21DAYS5TO14DAYS14TO21DAYS5TO21MEANS.D.NMEANS.D.NMEANS.D.NMEANS.D.NMEANS.D.NMEANS.D.NMEANS.D.NMEANS.D.NMEANS.D.NMEANS.D.N106.123154.523224.723204.223317.323467.623105.223578.4238615.52314420.6231110104.75.23.22324221513114.75.54.72324221918.203.63.95.52324221717155.03.74.02324223131316.97.66.82324224444448.99.97.72324229985.04.95.12324225048457.87.79.523242284848316.31?.010.023242213413212921.521.016.0232422'17:45HuntingdonLifeSciences05-4287DOSEGROUP:DOSELEVEL(MG/M3):TABLE5GASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSURESUMMARYOFGESTATIONFEEDCONSUMPTION(GRAMS/KG/DAY)1230200010000420000-----------------------------------------------------------DAYS4TO5DAYS5TO8DAYS8TO11DAYS11TO14DAYS14TO17DAYS17TO20DAYS20TO21DAYS5TO21MEANS.D.NMEANS.D.NMEANS.D.NMEANS.D.NMEANS.D.NMEANS.D.NMEANS.D.NMEANS.D.N7110.522816.022857.122857.122814.522744.523538.123764.72374757311.511.410.223242285807914.16.06.02324218280784.65.55.72324218282784.73.94.02322218181794.56.04.42322217374733.33.74.32322224747467.55.87.82324227574724.03.63.823242210-MAR-201117:46HuntingdonLifeSciences05-4287DOSEGROUP:DOSELEVEL(MG/M3):TABLE6GASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSURESUMMARYOFMATERNALNECROPSYOBSERVATIONS1230200010000420000---------------------------------------------------------------------------------------------FEMALESN2424"2424GROSSEXAMN00a·1----------MASSN0001%0.00.00.04.210-MAR-201117:48HuntingdonLifeSciences05-4287TABLE7GASOLINEDIPEVAPORCONDENSATE:'EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSURESUMMARYOFCESAREANSECTIONDATA·DOSEGROUP:123DOSELEVEL(MG/M3):0200010000------------FetalBodyWeight(g)MEAN5.75.85.7S.D.0.290.300.28N232324MaleFetusesMEAN5.95.95.8S.D.0.320.330.32FemaleFetusesMEAN5.65.65.5S.D.0.280.330.254200005.50.32225.70.335.40.3110-MAR-201117:48HuntingdonLifeSci.ences05-4287TABLE7GASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSUREDOSEGROUP:DOSELEVEL(MG/M3):PregnantN(atscheduledsacrifice)DamswithnoViableFetusesNDamswithViableFetusesNCo.rporaLuteaNo.peranimalImplantationSitesNo.peranimalPreimplantationLossNo.peranimal%peranimalLiveFetusesNo.peranimalMalesFemalesTOTALMEANS.D.TOTALMEANS.D.TOTALMEANS.D.MEAN%S.D.TOTALMEANS.D.·TOTALMEAN%S.D.TOTALMEAN%S.D.SUMMARYOFCESAREANSECTIONDATA1023023.34314.92.7831113.52.71321.41.889.313.4630013.02.6414648.510.9415451.510.94220002302333414.52.0631013.51.50241.01.466.67.9928612.42.6413346.114.7515353.914.753100002402435414.82.5231513.12.85391.62.96'10.418.5830212.63.0615451.212.5014848.812.504200002202233415.22.6330714.02.38271.21.777.69.1429213.32.6013546.813.9215753.213.9210-MAR-201117:·48HuntingdonLifeSciences05-4287TABLE7GASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSURESUMMARYOFCESAREANSECTIONDATADOSEGROUP:123DOSELEVEL{MG/M3):0200010000Postimplantation.Loss.TOTAL112413No.peranimalMEAN0.51.00.5S.D.0.672.080.83%implantsperanimalMEAN%3.47.95.1S.D.4.6614.898.64DeadFetusesTOTAL000No.peranimalMEAN0.00.00.0S.D.0.000.000.00%ofimplantsperanimalMEAN%0.00.00.0S.D.0.000.000.00Resorptions:EarlyTOTAL102313No.peranimalMEAN0.41.00.5S.D.0.662.070.83%ofimplantsperanimalMEAN%3.17.65.1S.D.4.6614.808.64Resorptions:LateTOTAL110No.peranimal.MEAN0.00.00.0S.D.0.210.210.00%ofimplantsperanimalMEAN%0.30.30.0S.D.1.391.600.00420000150.70.655.25.1500.00.000.00.00150.70.655.25.1500.00.000.00.0010-MAR-201117:49.HuntingdonLifeSciences05-4287TABLE8GASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSURESUMMARYOFGRAVIDUTERINEWEIGHTANDNETBODYWEIGHTCHANGE(GRAMS)DOSEGROUP:1.23DOSELEVEL(MG/M3):0200010000420000---------------------------------------------------------------------------------------------UTERUSWEIGHTMEAN101979696S.D.19.419.822.515.7N22232420ADJUSTEDWEIGHTMEAN286283280273S.D.18.119.017.115.9N22232420NETWEIGHTCHANGEFROMDAY5MEAN43373629S.D.11.19.111.710.6N22232420WEIGHTCHANGEFROMDAY5MEAN144134132129S.D.20.621.521.016.0N23232422ADJUSTEDWEIGHT=TERMINALBODYWEIGHTMINUSGRAVIDUTERINEWEIGHTNETWEIGHTCHANGEFROMDAY5=TERMINALCORRECTEDBODYWEIGHTMINUSDAY5BODYWEIGHTHuntingdonLifeSciences05-4287Page1211-DIPE-DEVDraftFemalesSummarvofFetalObservationsTable9FetusesLittersObservationsGroup1 23 412 34{MajorabnormalitiesinCAPITALS)Numberexamined30028630229223232422HYDROCEPHALYa,ANOPHTHALMIA;HYPOPLASTICAPICALLUNG11LOBE,THYMUSANDSPLEENMICROPHTHALMIA/ANOPHTHALMIA,UNILATERAL11EXOCCIPITALFUSEDTO1STCERVICALARCH11MANDIBLESHORTENED,MODERATE11THORACICVERTEBRA13,LUMBARVERTEBRA1:ACENTRIC,11MALFORMEDFORELIMBSHORTENED,RIGHT11ANALATRESIA,ABSENTSACRALANDCAUDALVERTEBRAE11Additionalsubclavianartery11Innominatearteryabsent1 2 11 21Undescendedthymictissue10115116838aNotedasDOMEDCRANIUMinexternalobservations.HuntingdonLifeSciences05-4287Page2211-DIPE-DEVDraftFemalesSummarvofFetalObservationsTable9FetusesLittersObservationsGroup1 2341i34(MajorabnormalitiesinCAPITALS)Numberexamined30028630229223232422Ren.alpapilla(e)notfullydeveloped1 2 1121Ureter(s)distended11111 1Leftumbilicalartery14t2131 2Testisdisplaced,medially,left117thcervicalrib(s)present1 313Sternebra(e)malaligned111113399103Rib(s)rudimentary,13th1311Rib(s)rudimentary,14th2 7512155 925presacralvertebrae13'1127presacralvertebrae11HuntingdonLifeSciences05-4287Page3.z'11-DIPE-DEVDraftFemalesSummarvofFetalObservationsITable9FetusesLittersObservationsGroup12 3 41234(MajorabnormalitiesinCAPITALS)Numberexamined30028630229223232422Hyoidbody/arch(es)unossified1 11 11 1Sternebra5and/or6unossified13 3132Metatarsals:1unossified,bilateral21

### Huntingdon Life Sciences 05-4287

Analytical Report

STUDY TITLE

Analytical Report For: 

### Page 1

Draft Final Report

Appendix A

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

by Inhalation Exposure

AUTHOR

Yonggang Wang

REPORT DATE

08 May 2009

STUDY NUMBER

05-4287

### Huntingdon Life Sciences 05-4287

SIGNATURES

Written by: 

### Analytical Report

Yonggang Wang, M.S.

Laboratory Manager

Formulation Chemistry Services

Reviewed by:

--------------- Kay Saladdin, B.S.

Associate Director

Formulation Chemistry Services

Approved by:

______________ _ Barbara A. Litzenberger

Director

Analytical Services Date

Date

Date 

### Page2

Draft Final Report

Appendix A

### Huntingdon Life Sciences 05-4287

Analytical Report

Table of Contents 

### Page3

Draft Final Report

Appendix A

Study Title Page

............................................................................................................... 1

Signature Page

................................................................................................................ 2

Table of Contents

............................................................................................................. 3

1. Summary

...................................................................................................................... 4

2. Experimental Procedures

............................................................................................. 4

3. Results and Discussion

................................................................................................ 5

Tables

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

........ 6

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

........................ 7

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

........ 8

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

....................... 9

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

................... 10

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

................... 11

Figures

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

............. 12

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

Chamber Sample

................................................................................................... 13

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

Chamber Sample

................................................................................................... 14

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

Chamber Sample

................................................................................................... 15

### Huntingdon Life Sciences 05-4287

Analytical Report

1. Summary 

### Page4

Draft Final Report

Appendix A

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

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

comparison to prior data at this Testing Facility.

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

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

test atmospheres to the neat test substance.

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

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

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

Ionization Detection (FID).

2. Experimental Procedures

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

the analytical method are maintained in this study file.

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

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

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

the charcoal tubes with Carbon Disulfide (CS 2 ). 

### The extracted solutions were analyzed by Gas

Chromatography equipped with a Supelco PetrocolTM DH 150 (150m x 0.25mm, 1.0 

### J.liTl) column

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

computer was used for data collection and processing.

Date of sample analysis is listed as follows:

Interval Date of Exposures Date Received Date Analyzed

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

Pretest /Trials 02 

### & 05Aug 05 02 

### & 05 Aug 05 03-05 Aug 05

Exposure-4 09 

### & 11 Aug 05 09 

### & 11 Aug 05 09-11 Aug 05

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

Exposure-18 23Aug 05 23Aug 05 23- 24Aug 05

### Huntingdon Life Sciences 05-4287

Analytical Report

3. Results and Discussion 

### Page 5

Draft Final Report

Appendix A

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

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

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

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

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

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

Figures I to IV.

## Huntingdon Life Sciences 

## 05-4287

Analytical Report

Table I. Chamber Components Confirmation 

## Page6

Draft Final Report

Appendix A

Area Percent of Gasoline DIPE Vapor Condensate Test Substance Stability

Pretest

Area%

Compound MeanTM Mean TM TM TM

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

00-4205 8 00-4205 8 05-4287b 05-4287b

lsobutane 1.61 1.60 1.34 1.42

N-Butane 9.57 9.59 9.05 9.25

3-Methyl-1-butene 0.35 0.34 0.34 0.34

lsopentane 32.26 32.35 32.58 32.50

N-Pentane 7.38 7.46 9.99 9.93

Trans-2-pentene 1.11 1.14 2.16 2.16

2,3-Dimethylbutane 1.76 1.78 1.10 1.08

2-Methylpentane 5.31 5.29 5.00 5.45

3-Methylpentane 3.21 3.20 3.23 3.16

N-hexane & DIPE 22.24 22.16 22.17 21.78

Methylcyclopentane 1.44 1.43 1.44 1.39

2,4-Dimethylpentane 1.16 1.17 0.93 1.15

Benzene 2.29 2.28 2.20 2.18

2-Methylhexane 1.33 1.32 1.33 1.26

2,3-Dimethylpentane 1.38 1.37 1.38 1.35

3-Methylhexane 1.54 1.53 1.49 1.48

lsooctane 1.71 1.69 1.65 1.58

Toluene 3.08 3.09 2.43 2.34

Total 98.73 98.79 99.81 99.80

ND 

### = none detected. <LOQ 

### = Less than the limit of quantification 

### = less than

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

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

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

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

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

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

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

pentene but the column used for this study separated the two peaks entirely.

## Huntingdon Life Sciences 

## 05-4287

Compound

lsobutane

N-Butane

3-Methyl-1-butene

I so pentane

N-Pentane

Trans-2-pentene

2,3-Dimethylbutane

2-Methylpentane

3-Methylpentane

N-hexane & DIPE

Methylcyclopentane

2,4-Dimethylpentane

Benzene

2-Methylhexane

2,3-Dimethylpentane

3-Methylhexane

lsooctane

Toluene

Total 

## Analytical Report

Table II. Chamber Components Confirmation

Area Percent of Gasoline DIPE Vapor Condensate

Pretest /Trials

Area%

DIPESTD DIPE STD Spiked Spiked Sample Sample

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

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

1.40 1.59 1.44 1.45 ND 1.52

9.30 9.70 9.41 9.52 ND 9.62

0.36 0.35 0.36 0.35 ND 0.36

32.61 32.29 32.74 33.00 ND 32.08

10.15 9.81 10.19 10.27 ND 9.99

2.35 2.18 2.43 2.41 ND 2.30

1.70 1.63 1.42 1.40 ND 1.66

5.23 5.28 5.24 5.14 ND 5.48

3.24 3.19 3.34 3.34 ND 3.24

19.12 20.13 19.87 19.25 ND 19.64

1.46 1.44 1.15 1.23 ND 1.47

1.22 1.19 1.27 1.20 ND 1.20

2.32 2.23 2.38 2.32 ND 2.42

1.28 1.27 1.34 1.28 ND 1.32

1.28 1.24 1.37 1.32 ND 1.29

1.52 1.48 1.59 1.55 ND 1.54

1.65 1.50 0.50 1.09 ND 1.58

3.11 2.93 3.09 3.02 ND 3.22

99.30 99.43 99.13 99.14 0.00 99.93 

## Page 7

Draft Final Report

Appendix A

Sample Sample

301 401

002_003 002_004

1.73 1.64

10.55 10.19

0.35 0.36

32.85 32.85

10.08 10.13

2.25 2.28

1.34 1.37

5.17 5.03

3.14 3.23

18.65 19.66

1.41 1.30

1.17 1.22

2.24 2.31

1.21 1.29

1.24 1.26

1.49 1.55

1.24 0.65

2.91 2.98

99.02 99.30

ND 

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

substance standard.

### Huntingdon Life Sciences 05-4287

Analytical Report PageS

Draft Final Report

Appendix A

Table Ill. Summary of Chamber Components Confirmation

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

Area%

Compound Spiked TM Samples Samples Samples Samples

Control-1 Standard-1 Group 1 Group2 Group3 Group4

(Tank#5)



### I so butane 0.95 1.21 ND 1.59 1.71 1.44

N-Butane 6.92 7.70 ND 10.43 10.56 9.32

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

lsopentane 30.72 31.26 ND 32.88 32.67 32.50

N-Pentanea 9.74a 8.92 ND 9.35 9.29 9.67a

Trans-2-pentene a 1.89 ND 2.11 2.08 a

2,3-Dimethylbutane 1.54 1.46 ND 1.47 1.46 1.34

2-Methylpentane 5.72 5.38 ND 5.26 5.06 5.00

3-Methylpentane 3.63 3.55 ND 3.21 3.17 3.20

N-hexane & DIPE 22.50 21.71 ND 19.27 19.62 20.39

Methylcyclopentane 1.67 1.68 ND 1.46 1.43 1.47

2,4-Dimethylpentane 1.44 1.41 ND 1.21 1.18 1.24

Benzene 2.87 2.50 ND 2.31 2.25 2.31

2-Methylhexane 1.66 1.58 ND 1.46 1.27 1.34

2,3-Dimethylpentane 1.58 1.52 ND 1.30 1.27 1.36

3-M ethyl hexane 1.79 1.73 ND 1.56 1.48 1.54

lsooctane 1.51 1.65 ND 1.31 1.48 1.58

Toluene 3.74 3.70 ND 3.16 2.94 2.91

Total 98.35 99.18 ND 99.70 99.28 96.95

ND 

### = none detected.

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

baverage ofGroups II, Ill and IV only All Samples 0 Spiked TM

Control-2 Standard-2

(Tank#5)



### 1.58 1.30 1.48

10.10 8.67 9.40

0.35 0.37 0.37

32.68 32.94 32.07

9.44a 9.05a 9.26a

a a a

1.42 1.52 1.67

5.11 5.56 5.37

3.19 3.42 3.26

19.76 20.90 20.64

1.45 1.59 1.50

1.21 1.34 1.25

2.29 2.42 2.35

1.36 1.48 1.38

1.31 1.51 1.38

1.53 1.70 1.56

1.46 1.82 1.61

3.00 3.57 3.23

97.24 99.16 97.78

## Huntingdon Life Sciences 

## 05-4287

Analytical Report Page9

Draft Final Report

Appendix A

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

Compound

lsobutane

N-Butane

3-Methyl-1-butene

lsopentane

N-Pentane

Trans-2-pentene

2,3-Dimethylbutane

2-Methylpentane

3-Methylpentane

N-hexane & DIPE

Methylcyclopentane

2,4-Dimethylpentane

Benzene

2-Methylhexane

2,3-Dimethylpentane

3-Methylhexane

lsooctane

Toluene

Total

NO 

### = none detected.

a Resample results DIPE DIPE

STD1 STD2

(Tank#S) (Tank#S)

003_002 003_009

1.50 1.45

9.07 8.68

0.37 0.34

32.26 32.02

9.62 9.56

2.17 2.13

1.65 1.54

5.28 5.42

3.29 3.40

20.26 20.25

1.50 1.58

1.28 1.28

2.34 2.41

1.36 1.40

1.36 1.43

1.60 1.64

1.43 1.66

3.19 3.36

99.53 99.55 

### Area%

Spiked Spiked Sample Sample Sample Sample

Control1 Control2 1001 2001 3001 4001

003_006 003_007 003_008 003_003 003_010 3 003_005

1.18 1.03 ND 1.98 1.66 1.67

7.55 6.26 ND 12.46 10.22 10.32

0.32 0.30 ND 0.38 0.37 0.36

30.69 30.24 ND 32.72 32.80 32.45

9.36 8.99 ND 9.51 10.14 9.99

2.14 2.07 ND 2.23 2.32 2.28

1.53 1.57 ND 1.36 1.39 1.50

5.52 5.85 ND 4.36 5.09 5.11

3.43 3.71 ND 2.80 3.21 3.11

21.94 20.90 ND 18.42 19.25 19.67

1.62 1.85 ND 1.28 1.47 1.41

1.32 1.50 ND 1.16 1.20 1.17

2.54 2.88 ND 2.18 2.32 2.24

1.48 1.65 ND 1.33 1.25 1.22

1.46 1.75 ND 1.30 1.25 1.26

1.73 2.03 ND 1.57 1.49 1.45

1.82 2.23 ND 1.37 1.50 1.41

3.73 4.70 ND 3.39 3.05 2.87

99.36 99.51 0.00 99.80 99.98 99.49

## Huntingdon Life Sciences 

## 05-4287

Analytical Report Page 10

Draft Final Report

Appendix A

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

Area%

DIPE DIPE Spiked Spiked Sample Sample Sample Sample

STD 1 STD2 Control1 Control2 1002 2002 3002 4002

{Tank#5) (Tank #5)

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

lsobutane 1.18 1.50 0.90 1.36 ND 0.99 1.86 1.21

N-Butane 6.84 9.32 7.17 9.65 ND 7.21 10.76 8.24

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

lsopentane 30.09 31.63 30.61 32.93 ND 32.12 32.39 31.99

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

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

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

2-Methylpentane 5.67 5.33 5.76 5.16 ND 6.03 5.04 4.96

3-Methylpentane 3.59 3.21 3.78 3.39 ND 3.77 3.09 3.34

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

Methylcyclopentane 1.75 1.48 1.76 1.56 ND 1.72 1.42 1.61

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

Benzene 2.69 2.39 2.85 2.38 ND 2.63 2.25 2.54

2-Methylhexane 1.62 1.37 1.63 1.47 ND 1.53 1.23 1.51

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

3-Methylhexane 1.91 1.55 1.91 1.64 ND 1.75 1.46 1.68

lsooctane 1.88 1.60 1.16 1.65 ND 1.18 1.42 1.75

Toluene 4.17 3.33 3.75 3.01 ND 3.67 3.03 2.96

Total 99.67 98.82 99.96 99.88 0.00 99.85 99.59 97.42

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

..

NO 

### = None Detected

### Huntingdon Life Sciences 05-4287

Analytical Report Page 11

Draft Final Report

Appendix A



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

DIPE DIPE

STD1 STD2

(Tank#5) (Tank#5)

Compound 005_002 005_009

lsobutane 0.95 1.50

N-Butane 7.20 10.19

3-Methyl-1-butene 0.31 0.32

lsopentane 31.42 32.57

N-Pentane 8.32 8.10

Trans-2-pentene 1.55 2.15

2,3-Dimethylbutane 1.17 1.75

2-Methylpentane 5.19 5.35

3-Methylpentane 3.76 3.17

N-hexane & DIPE 22.38 20.49

Methylcyclopentane 1.80 1.43

2,4-Dimethylpentane 1.50 1.21

Benzene 2.48 2.26

2-Methylhexane 1.73 1.36

2,3-Dimethylpentane 1.52 1.33

3-Methylhexane 1.68 1.48

lsooctane 1.65 1.58

Toluene 3.73 2.99

Total 98.34 99.23

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

ND 

### = None Detected 

## Area%

Spiked Spiked

Control1 Control2

005_001 005_007

0.77 1.52

6.03 10.11

0.41 0.36

30.87 32.65

8.31 7.92

1.95 1.66

1.28 1.51

5.87 5.68

3.68 3.17

23.71 21.04

1.64 1.36

1.49 1.21

3.23 2.01

1.88 1.31

1.66 1.32

1.74 1.44

1.54 1.59

3.73 3.00

99.79 99.86 Sample Sample Sample Sample

1003 2003 3003 4003

005_008 005_004 005_005 005_006

ND 1.79 1.60 1.44

ND 11.62 10.69 9.41

ND 0.36 0.34 0.35

ND 33.79 32.83 33.07

ND 7.97 7.92 8.24

ND 1.73 1.72 1.91

ND 1.45 1.66 1.37

ND 5.38 5.05 4.94

ND 3.07 3.20 3.16

ND 19.75 20.09 20.94

ND 1.37 1.41 1.38

ND 1.12 1.19 1.20

ND 2.11 2.19 2.15

ND 1.22 1.32 1.30

ND 1.21 1.30 1.32

ND 1.35 1.48 1.49

ND 1.37 1.53 1.59

ND 2.41 2.74 2.90

0.00 99.07 98.26 98.16

### Huntingdon Life Sciences 05-4287

Analytical Report Page 12

Draft Final Report

Appendix A

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

(File# 003_008.rst)

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

Response (mV) vs Time (minutes)

### Huntingdon Life Sciences 05-4287

Analytical .Report Page 13

Draft Final Report

Appendix A

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



### (File# 003_003.rst)**~ ~**  f8 8!

r; ,;**~ ~** 

II II II II I II II I



# i**~~** 

# j

i**~** il' 

# u j t,**~** 

# I i5



### ...**~ ~·** **:;:;~ ~** **~~** 

# I .g.! 

# Hi j 

# J 

# I 

# I j**~** 

# i!



# ..

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

Response (mV) vs Time (minutes)

### Huntingdon Life Sciences 05-4287 Page 14

Draft Final Report

Figure Ill.



### im 

## i

10 

### Analytical Report Appendix A

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

(File# 003_010.rst)**~:g** N**~ ~** **~** g Si**~~ ~**  rj :R g :8**~**



# .**~ ~~** 



# I ij :g**~** 

# ll**~ ~~~** **~** 

# I 15 

# if 

# I HI**~** 

# J j**~ :j!!.~**  

# ! -N

### ~ .2**~~** 

# ""**~~-** 

## "'

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

Response (mV) vs Time (minutes)

### Huntingdon Life Sciences 05-4287



### Analytical Report Page 15

Draft Final Report

Appendix A

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

(File# 003_005)

0 

## g

II II II II I II II I



# I 

# .. 

# i**~** co**~** w 

# ll**~ ~**  

# .. a.

!=**~~ ~**  

# j a 

# !f**~ ~**  

# .**~ ]~** **q~** 

# I**~** 

# i

..**~**

### ~**!~ ~**  

### :i**~** 

## j**~** .;,**~"'** ,1: <W 

# "'**~**

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

Response (mV) vs Time (minutes)

## Huntingdon Life Sciences 05-4287



## 211-DIPE-DEV

Inhalation Report

TABLE OF CONTENTS 

## Page 1

Draft Final Report

AppendixB

1. IN"TRODUCTION

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

2. MATERIALS AND METHODS



## 2.1. Husbandry During Exposure Periods

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

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

............... :

...................**~** 

## .................... .

3. 

## FIGURES



## 1. Chamber Generation System and

Whole-Body Exposure Chamber (Group 1)

........................................ . 2. Chamber Generation System and

Whole-Body Exposure Chambers (Groups 2- 4)

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

4. TABLES



## I. Summary ofln-Chamber Observations

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

II. Chamber Monitoring Results

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

III. Equipment List

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

IV. Chamber Distribution Records

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

V. Miran Calibration

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

VI. Testing Room and Chambers Environmental Monitoring

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

## Huntingdon Life Sciences

1. INTRODUCTION 

## 05-4287

211-DIPE-DEV

Inhalation Report Page2

Draft Final Report

AppendixB

This appendix presents the methodology for exposure generation, monitoring and

results.

2. MATERIALS AND METHODS

2.1. HUSBANDRY DURING EXPOSURE PERIODS

2.1.1. HOUSING



## Animals were individually housed in stainless steel wire mesh

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

exposure chamber. The placement of the animals in the

whole-body exposure chamber was rotated daily to ensure uniform

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

included in the raw data.

2.1.2. FEED



## None was provided during exposure.

2.1.3. WATER



## None was provided during exposure.

2.1.4. ENVIRONMENTALCONDITIONS



## Chamber temperature and relative humidity were recorded every

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

possible, within the ranges presented below. Excursions outside

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

Temperature



## Desired:

Actual: 20 to 24°C

20 to 25°C

## Huntingdon Life Sciences 05-4287

211-DIPE-DEV

Inhalation Report Page3

Draft Final Report



# I

AppendixB

The daily average temperature within the chamber was outside the

-

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

respectively.

Relative Humidity



## Desired:

Actual: 40 to 60%

36 to 54%

The daily average relative humidity within the chamber was

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

and III, respectively.

2.2. TEST SUBSTANCE ADMINISTRATION AND CHAMBER

OPERATIONS

2.2.1. ROUTE OF ADMINISTRATION



## Inhalation via whole-body exposures.

2.2.2. TEST SUBSTANCE ADMINISTRATION



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

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

an appropriate procedure determined during pre-study trials. The

trials were performed to evaluate the optimal set of conditions and

equipment to generate a stable and uniform atmosphere at the

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

of the study and is outlined in this report.

2.2.3. TARGET EXPOSURE LEVELS



## Group 1 - 0 mg/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

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Inhalation Report Page4

Draft Final Report

AppendixB

2.2.4. DURATION AND FREQUENCY OF ADMINISTRATION



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

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

2.2.5. CHAMBER OPERATIONS



## The whole-body exposure chambers each had a volume of

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

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

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

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

changes/hour) and a T

99 

## equilibrium time (calculated by

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

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

Group Airflow Rate Air Change 

## T99

(Lpm) 

### (min) (min)



## 1 210 4.7 22

2 208 4.8 22

3 216 4.6 21

4 210 4.7 22

The chamber atmospheres were exhausted through the in-house

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

-and an activated charcoal bed.

· Chamber static pressure was recorded every half-hour during

exposure. The chambers were operated for a minumum of

23 minutes (approximate 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

211-DIPE-DEV

Inhalation Report

2.2.6. EXPOSURE PROCEDURE

Group 1 

## Page5

Draft Final Report

AppendixB

Houseline nitrogen was delivered from a regulator with a

backpressure gauge via W' 

## tubing to a flowmeter regulated by a

metering valve. This nitrogen flow was then directed into the

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

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

chamber. This nitrogen flow simulated the generation nitrogen

flow for Groups 2, 3 and 4.

Groups 2-4



## Houseline nitrogen was delivered from a regulator with a

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

flow systems: the test substance pressurization flow, the purge

flow and the volatilization flow.

The test substance pressurization flow was directed via**~"tubing** 

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

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

valve was used to adjust and maintain the pressure within the

cylinder. From the pressurized cylinder, the test substance flowed

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

through a filter to prevent equipment contamination. From the

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

/ 8 " 

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

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

substance flowed via 1 

## /g" tubing onto the glass helix of a counter

current volatilization chamber. The glass helix was heated by a

nichrome wire, which was controlled by an autotransformer and

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

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

## tubing

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

was delivered via 1

/ 8 " 

## tubing to the bottom of the tube containing

the nichrome wire. This nitrogen flow continuously purged the

# I Huntingdon Life Sciences 05-4287

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# I Page6

Draft Final Report

AppendixB

area surrounding the nichrome wire within the tube, thereby

protecting the wire from oxidation.

The nitrogen for the volatilization system was directed via 

## W'

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

flowmeter, the volatilization nitrogen flowed via 

## W' tubing to a

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

This nitrogen flowed up through the volatilization chamber passing

over the coil and volatilizing the test substance. The pressure

within the counter-current volatilization chamber was maintained

slightly negative to the room and was monitored with a pressure

gauge.

This test substance laden nitrogen exited the top of the

volatilization chamber through a glass elbow, which directed the

flow, via W' 

## tubing, to the turret of 1000 Liter glass and stainless

steel exposure chamber. As the test substance laden nitrogen was

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

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

2.3. EXPOSURE CONCENTRATION DETERMINATION

2.3.1. NOMINAL CONCENTRATION



## A nominal exposure concentration was calculated daily. The flow

of air through the chambers was monitored using appropriate

calibrated equipment. The nominal concentrations in mg/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



## Cone (mg/m 3

) 

## = amount consumed (g) 

## x 1000 mglg 

## x 1000 L/m 3



## exposure duration (min) x airflow (Lpm)

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2.3.2. CHAMBER SAMPLING 

## Page?

Draft Final Report

AppendixB

Determinations of the exposure levels were made using a MIRAN®

Ambient Air analyzer equipped with a strip chart recorder. The

test atmosphere was drawn from the normal sampling portal

through the MIRAN® and measurements were recorded at least

4 times during each exposure. The exposure levels were

determined by comparison of the measured absorbance to a

calibrated response curve constructed using the same instrument

settings.

Also, one charcoal tube sample per chamber per week was

analyzed by gas chromatography (GC) to characterized at least

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

test substance) to show test substance stability and comparison

between the neat liquid test substance and the vaporized test

atmospheres.

See Table III for equipment list.

2.3.3. PARTICLE SIZE DISTRIBUTION



## Particle size distribution measurements were performed once

during each week of exposure using a TSI Aerodynamic Particle

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

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

appropriate settings prior to sampling. The particle size

distributions were calculated by the computer and printed out. The

mass median aerodynamic diameter, geometric standard deviation

and total mass concentration were calculated. For comparison,

room air samples were similarly collected at least once weekly.

2.3.4. CHAMBER AND EXPOSURE ROOM ENVIRONMENT



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

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

the study (Day 19).

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Draft Final Report

AppendixB

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

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

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

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

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

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

### Huntingdon Life Sciences

Group 1

Note: 05-4287

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Figure I

Chamber Generation System

And Whole Body Exposure Chamber

1 000 L Exposure Chamber

H-9

H-7

Drain 

### Page9

Draft Final Report

AppendixB

To Exhaust System



# '

Sampling

Ports



## /

1. Animals were individually housed on two levels within the exposure chamber.

## Huntingdon Life Sciences 05,-4287

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Groups 2-4 Inhalation Report

Figure II

Chamber Generation System

And Whole Body Exposure Chamber Page 

## 10

Draft Final Report

AppendixB

To Exhaust System

1 000 L Exposure Chamber

NUIOFO Valve 

# ' Backpressure

Gauge



# \

Plastic

tubing Test Material

Cylinder

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



## Notes: Nupro

Valve Volatilization

Chamber

1-- Temp.

H anCidit

um1 y Gauge

H-8

H-9

H-7

Drain



## 1. Animals were individually housed on two levels within the exposure chamber.HuntingdonLifeSciences05-4287211-DIPE-DEVPage11DraftFinalReoortrIInh:ti:-R:rt_"____------I--1TableISummaryofIn-ChamberObservationsExposureDay12 3 4 5 6 7 8 9101112131415 16 171819Group1 -0mg/m3WithinNormalLimits6121824 24 24 24 24 24 24 24 242424242418126Group2 -2000mg/m3WithinNormalLimits6121824 24 24 2424 24 24242424 24 242418126Group3 -10000mg/m3WithinNormalLimits612182424 24 242424242424 2424 24 2418126Group4 -20000mg/m3WithinNormalLimits6.121824 2424 242424 24 24 24 24 24 24 2418126HuntingdonLifeSciencesDayDateExposureNumberNominalMean(mglm3)(mglm3)06-Aug-05100.0017-Aug-05200.0028-Aug-05300.0039-Aug-05400.004.10-Aug-05500.00511-Aug-05600.00612-Aug-05700.00713-Aug-05800.00814-Aug-05900.00915-Aug-051000.001016-Aug-05ll00.00ll17-Aug-051200.001218-Aug-051300.001319-Aug-051400.001420-Aug-051500.001521-Aug-051600.001622-Aug-051700.001723-Aug-051800.001824-Aug-051900.00Mean0S.D.0--05-4287211-DIPE-DEVAppendixB -TableIIChamberMonitoringResultsCumulativeExposureRecordGroup1-0mglmJ(Aironly)ChamberConcentrationIndividual(mglm3)0.000.000.000.000.000.000.000.000.000.000.000.000.000.00-0.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.000.00---·ParticleSizeDeterminationsMMADGSDTMC(J.lm)(mglm3)0.80ll2.0314.25E-030.76751.7924.78E-038.22102.276l.64E-033.2632.0333.56E-034.30.20.0Page12DrnftFinalReportChamberEnvironmentMeanTemperatureHumidity(OC)(%)234223412439244024412440254025422443253924402540254025402440254024402441234124400.71.0HuntingdonLifeSciencesDayDateExposureNumberNominalMean(mg/m3)(mg/m3)06-Aug-05I20001900I7-Aug-0522000190028-Aug-0532000200039-Aug-05421002100410-Aug-05520002000511-Aug-05620002000612-Aug-05720002000713-Aug-05820002000814-Aug-05920002000915-Aug-0510200020001016-Aug-0511190020001117-Aug-0512200020001218-Aug-0513200020001319-Aug-0514200020001420-Aug-0515190020001521-Aug-0516200020001622-Aug-0517200019001723-Aug-0518200020001824-Aug-051920002000Mean1995S.D.4005-4287211-DIPE-DEVAppendixB -TableIIChamberMonitoringResultsCumulativeExposureRecordGroup2-2000mg!m3ChamberConcentrationIndividual(mg/m3)1800180019001900200019001800190022001800200020002200200020002000210020001900210020002200210018002100200020002000200019002000200019002000200020002100200020002000200020002000190020001900210021001900190020002000190021002000200020001800200020002000190021002000190019001900200020001900200020002100180020002100198293MMAD(!lm)3.8820.79357.5084.0613.4ParticleSizeDeterminationsGSDTMC(mg/m3)2.9547.23E-032.2946.14E-032.0572.08E-032.4355.15E-030.50.013DraftFinalReportChamberEnvironmentMeanTemperatureHumidity(OC)(%)234323422340244124432442244124432445244124422441244224422442244224422343234224420.51.1HuntingdonLifeSciencesDayDateExposureNumberNominalMean(mglm3)(mglm3)06-Aug-051110001000017-Aug-05211000980028-Aug-05311000990039-Aug-054110009600410-Aug-0551200010000511-Aug-0561200010000612-Aug-057110009700713-Aug-0581200010000814-Aug-059120009800915-Aug-05101200098001016-Aug-051112000100001117-Aug-051212000100001218-Aug-051311000100001319-Aug-05141100098001420-Aug-051512000100001521-Aug-051612000110001622-Aug-051711000100001723-Aug-0518110001000018·24-Aug-05191100011000Mean11474S.D.51305-4287211-DIPE-DEVAppendixB-TableIIChamberMonitoringResultsCumulativeExposureRecordGroup3-10000mglm3ChamberConcentrationIndividual(mglm3)100009600100001100094009700100009900100001000098009900930095009700.99001100010000100001000097009900940011000990096009400970010000100001000010000970010000100009400960099009700100009900980011000110001000010000100001000010000100001000010000960097009900990011000970097001000011000110001100010000110001000010000100001100091009600110001100012000100001100010072546ParticleSizeDeterminationsMMADGSDTMC(!1m)(mglm3)0.81122.4505.75E-034.9523.3341.07E-02'5.5232.2461.91E-033.7622.6776.12E-032.60.60.0Page14DraftFinalReportChamberEnvironmentMeanTemperatureHumidity("C)(%)234224412441244125412541254025422544254025412540I2539II2542I2542254224402441234125410.71.1HuntingdonLifeSciencesDayDateExposureNumberNominalMean(mg/m3)(mg/m3)06-Aug-051200001900017-Aug-052210002100028-Aug-053190002000039-Aug-0542100020000410-Aug-05520000200005ll-Aug-0562000020000612-Aug-0572000020000713-Aug-0582000020000814-Aug-0591900019000915-Aug-0510200002000010.16-Aug-05111900020000II17-Aug-051220000210001218-Aug-051320000200001319-Aug-05.1420000200001420-Aug-051519000200001521-Aug-051619000200001622-Aug-051720000200001723-Aug-051820000200001824-Aug-05192000019000Mean19842S.D.60205-4287211-DIPE-DEVAppendixB -TableIIChamberMonitoringResultsCumulativeExposureRecordGroup4 -20000mg/m3ChamberConcentrationIndividual(mg/m3)1800020000200001900019000200002200021000210002000019000190002100020000200002000018000180002200020000180002000021000200001900020000200002000019000190001900021000210002000018000180002000020000190001900020000200001900019000210002100021000200001900020000200002000020000210001800021000190002000020000200002000019000200001900021000200002000020000200002000020000200001700020000200002000019776961ParticleSizeDeterminationsMMADGSDTMC{l.tm)(mg/m3)0.78112.1374.84E-030.76402.2609.80E-037.8102.2724.24E-033.1182.2236.29E-033.30.10.0---Page15DraftFinalReportChamberEnvironmentMeanTemperatureHumidityeq(%)234223412440244024412441244124442445244124412441244124422442.244224402341234024411.3--

### Huntingdon Life Sciences

Exposure Chamber 

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Table III

Equipment List

1000 Liter glass and stainless steel chamber (Wahmann).

Compound Reservoir 

### Page 16

Draft Final Report

AppendixB

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

cylinder and used on study.

Compound Generator



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

heated by a nichrome wire).

Flowmeters



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

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

calibrated prestudy with a Gilibrator®Bubble Generator, 

## PIN D800286, SIN 569-S,

flow cell assembly 

## PIN D80026B, BD #1860).

Flowmeter, size 0- 65 mm, 

## PIN G6-02-R3, G6-03-R3, G6-04-R3 (Key Instruments).

PressureNacuum Gauges



### Union Carbide backpressure gauge, 

## PIN SG 6383.

Norgreen backpressure gauge, 

## PIN 9892K23.

U.S. Gauge backpressure gauge, 

## PIN 12672-1.

Ashcroft backpressure gauge, 

## PIN 733-47.

Matheson® backpressure gauge, 

## PIN 63-3161.

Dwyer® Magnehelic® gauge.

Variable Auto Transformer



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

Chamber Air Flow



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

Gas Meter, Model8586799, (Singer).

Chamber Static Pressure



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

Dwyer® Mark ll 

### Manometer, Model25 (Dwyer® Instruments, Inc.).

### Huntingdon Life Sciences

Regulators



### Norgreen, 

## PIN 9892K23

Union Carbide, 

## PIN SG 3800 30.

Metering Valve 

### 05-4287

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Table III

Equipment List

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

Timers



### Gralab Universal Timer, Model171.

Absorbent Tube Sampling



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

Vacuum Pumps



### Thomas Industries Inc., Model707CM50.

Neptune Dyna-pump®, Model4K.

Tubing



### Clear plastic, sizes 3/16", W', 

## W' (Norton, Baxter).



## W' stainless steel.

Stainless steel cross(Swage).

Teflon®, size 1/8", 

### \4", 

## Yz".

Plastic "T".

Air 

### .f\nalyzer 

### Page 17

Draft Final Report

AppendixB**~** 

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

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

Mode1No.22-195

Balston® MicrofibreTM Disposable Filter Unit was attachedin-line.

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

Particle Sizer



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

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

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Table III

Equipment List Page 18

Draft Final Report

AppendixB

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

Hygrometer!fhermometer.

Digital Sound Meter 840029 (SPER Scientific)

Quantum Instruments Photo Meter 1.

Oxygen/Gas Analyzer, Model1214S (Gastech)

Thermometer

T 0 

### Sentry Digital Alarm Module, ModelllO.

Balance

Pelouze, No. 4040

Mettler PM30000K (Mettler-Instrument Corporation).

Miscellaneous

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

DFlf' filter, Grade DQ (Balston).

## Huntingdon Life Sciences 05-4287

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Table V

Miran Calibration

Methodology for Gasoline DIPE Vapor Condensate 

## Page20

Draft Final Report

AppendixB

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

wavelength, microns 10.2

pathlength, dial setting 5.30

slit width, mm 1

range, absorbance 1A

response, seconds 1

gam High

chart speed, em/min 1

chart volts 1

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

1

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

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

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

separate syringe for calibration.

.

Injection Calculated Absorbance

Volume Concentration I 

## Operator 1 Operator 2 Average

(IlL) (mg/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 Cone. (mg/m 3

) 

### = 

## Injection volume CuL) x Density ( 0.68 mg/uL) x 1000 Llm 3



## 5.64 L (Volume ofMiran cell)

### Huntingdon Life Sciences 05-4287

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Table V

Miran Calibration Page 21

Draft Final Report

AppendixB

30000**~------------------------------------~**



### 25000

## ~ {(0000



### ("

n



### t15000

# ~ n

.8JOOOO



### 5000 y 

# = -12213x 4 

### + 

### 16436x 3

- 

### 751. 71x 2 

### + 

### 30139x

R

2 

# = 0.9997



## 0**+----+----+----+----+----+----+----+--~**



### 0.0000 0.1000 0.2000 0.3000 0.4000 0.5000 0.6000 0.7000 0.8000

Absorbance (Volts)

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Table V

Miran Calibration Page22

Draft Final Report

AppendixB

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

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

Expected Acceptable

Injection Calculated Absorbance Absorbance

Volume Concentration Reading Range

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

17 2050 0.0686 0.0583

- 0.0789

85 10248 0.328 0.2788- 0.377

175 21099 0.637 0.541-0.733

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

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

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

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

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

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

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

## Huntingdon Life Sciences 05-4287

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Table VI Page 23

Draft Final Report

AppendixB

Testing Room and Chambers Environmental Monitoring

Test Particle

Interval Location Substance Light Noise Oxygen Sizing

(mg/m 3

) 

### I 

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



## Pretest Room 813

- Front 0 32.3 62.8



## -

Room 813

- Back 0 38.6 59.5



# - Group 1- Chamber



# - - - 20 2.23 X 

## 10- 2



## Group 2 Chamber



## - 

# - 

# - 20 1.62 X 

## 10- 2



## Group 3 Chamber



# - - - 20 3.29 x 

# w- 2



## Group 4 Chamber



## - - 

# - 20 3.30 x 

# w- 2



## Day3 Room 813

- Front 0 30.0 64.1



# -

Room 813

- Back 0 38.0 61.2



## - Group 1 Chamber



## - - 

## - 20

Group 2 Chamber



# - 

# - - 20

Group 3 Chamber



## - - - 20

Group 4 Chamber



# - - - 20

Day 12 Room 811

- Front 0 30.4 63.8



# -

Room 811- Back 0 38.7 59.3



# -

Group 1 Chamber



# - - 

# - 20

Group 2 Chamber



## - 

# - - 20

Group 3 Chamber



## - - 

# - 20

Group 4 Chamber



# - - - 20

Day 19 Room 811 -Front 0 31.1 63.9



# -

Room 811- Back 0 38.0 60.1



## -

Group 1 Chamber



## - - 

# - 20

Group 2 Chamber



## - 

# - - 20

Group 3 Chamber



## - 

# - - 20

Group 4 Chamber



# - - 

# - 20

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

Appendix B).10-MAR-201117:08HuntingdonLifeSciences05-4287FEMALESGROUP1ANIMAL#----190119021903190419051906190719081909191019111912191319141915191619171918191919201921192219231924APPENDIXCGASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSUREINDIVIDUALANIMALTERMINATIONHISTORY0MG/M3-------------------------------------------------------TYPEOFDATEOFGESTATIONPREGNANCYDEATHDEATHDAYSTATUSTERMINALSACRIFICE22-AUG-0521pTERMINALSACRIFICE22-AUG-0521pTERMINALSACRIFICE.22-AUG-0521pTERMINALSACRIFICE22-AUG-0521pTERMINAL·SACRIFICE22-AUG-0521pTERMINALSACRIFICE22-AUG-0521pTERMINALSACRIFICE23-'AUG-0521pTERMINALSACRIFICE23-AUG-0521pTERMINALSACRIFICE23-AUG-0521pTERMINALSACRIFICE23-AUG-0521pTERMINALSACRIFICE23-AUG-0521pTERMINALSACRIFICE23-AUG-0521pTERMINALSACRIFICE24-AUG-0521pTERMINALSACRIFICE24-AUG-0521pTERMINALSACRIFICE24-AUG-0521NPTERMINALSACRIFICE24-AUG-0521pTERMINALSACRIFICE24-AUG-0521pTERMINALSACRIFICE24-AUG-0521pTERMINALSACRIFICE25-AUG-0521pTERMINALSACRIFICE25-AUG-0521pTERMINALSACRIFICE25-AUG-0521pTERMINALSACRIFICE25-AUG-0521pTERMINALSACRIFICE25-AUG-0521pTERMINALSACRIFICE25-AUG-0521p-------------------NP-NOTPREGNANT,P-PREGNANT10-MAR-201117:08HuntingdonLifeSciences05-4287FEMALESGROUP2ANIMAL#2000MG/M3APPENDIXCGASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSUREINDIVIDUALANIMALTERMINATIONHISTORY--------------------------------------------------TYPEOFDATEOFGESTATIONPREGNANCYDEATHDEATHDAYSTATUS---------------------------------------------------------------2901TERMINALSACRIFICE22-AUG-0521p2902TERMINALSACRIFICE22-AUG-0521p2903TERMINALSACRIFICE22-AUG-0521p2904TERMINALSACRIFICE22-AUG-0521p2905TERMINALSACRIFICE22-AUG-0521p2906TERMINALSACRIFICE22-AUG-0521p2907TERMINALSACRIFICE23-AUG-0521p2908TERMINALSACRIFICE23-AUG-0521p2909TERMINALSACRIFICE23-AUG-0521p2910TERMINALSACRIFICE23-AUG-0521p2911TERMINALSACRIFICE23-AUG-0521p2912TERMINALSACRIFICE23-AUG-0521p2913TERMINALSACRIFICE24-AUG-0521p2914TERMINALSACRIFICE24-AUG-0521p2915TERMINALSACRIF.ICE24-AUG-0521p2916TERMINALSACRIFICE24-AUG-0521p2917TERMINALSACRIFICE24-AUG-0521p2918TERMINALSACRIFICE24-AUG-0521p2919TERMINALSACRIFICE25-AUG-0521NP2920TERMINALSACRIFICE25-AUG-0521p2921TERMINALSACRIFICE25-AUG.-0521p2922TERMINALSACRIFICE25-AUG-0521p2923TERMINALSACRIFICE25-AUG-0521p2924TERMINALSACRIFICE25-AUG-0521p--------------------------------NP-NOTPREGNANT,P-PREGNANT10-MAR-201117:08HuntingdonLifeSciences05-4287FEMALESGROUP3ANIMALll10000MG/M3APPENDIXCGASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSUREINDIVIDUALANIMAL·TERMINATIONHISTORY-------------------------------------------------------TYPEOFDATEOFGESTATIONPREGNANCYDEATHDEATHDAYSTATUS----------------------------------------------------------------------------3901TERMINALSACRIFICE22-AUG-0521p3902TERMINALSACRIFICE22-AUG-0521p3903TERMINALSACRIFICE22-AUG-0521p3904TERMINALSACRIFICE22-AUG-0521p3905TERMINALSACRIFICE22-AUG-0521p3906TERMINALSACRIFICE22-AUG-0521p3907TERMINALSACRIFICE23-AUG-0521p3908TERMINALSACRIFICE23'-AUG-0521p3909TERMINALSACRIFICE23-AUG-0521p3910TERMINALSACRIFICE23-AUG-0521p3911TERMINALSACRIFICE23-AUG-0521p3912TERMINALSACRIFICE23-AUG-0521p3913TERMINALSACRIFICE24-AUG-0521p3914TERMINALSACRIFICE24-AUG-0521p3915TERMINALSACRIFICE24-AUG-0521p3916TERMINALSACRIFICE24-AUG-0521p3917TERMINALSACRIFICE24-AUG-0521p3918TERMINALSACRIFICE24-AUG-0521p3919TERMINALSACRIFICE25-AUG-0521p3920TERMINALSACRIFICE25-AUG-0521p3921TERMINALSACRIFICE25-AUG-0521p3922TERMINALSACRIFICE25-AUG-0521p3923TERMINALSACRIFICE25-AUG-0521p3924TERMINALSACRIFICE25-AUG-0521p-------------------------NP-NOTPREGNANT,P-PREGNANT10-MAR-201117:08HuntingdonLifeSciences05-4287FEMALESGROUP4ANIMALll20000MG/M3APPENDIXCGASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSUREINDIVIDUALANIMALTERMINATIONHISTORY--------------------------------------------------------TYPEOFDATEOFGESTATIONPREGNANCYDEATHDEATHDAYSTATUS-------------------------------------------------------------------------4901TERMINALSACRIFICE22-AUG-0521p4902TERMINALSACRIFICE22-AUG-0521p4903TERMINALSACRIFICE22-AUG-0521p4904TERMINALSACRIFICE22-AUG-0521p4905TERMINALSACRIFICE22-AUG-0521p·4906TERMINALSACRIFICE22-AUG-0521p4907TERMINALSACRIFICE23-AUG-0521p4908TERMINALSACRIFICE23-AUG-0521NP4909TERMINALSACRIFICE23-AUG-0521p4910TERMINALSACRIFICE23-AUG-0521p4911TERMINALSACRIFICE23-AUG-0521p4912TERMINAL23-AUG-0521p4913TERMINALSACRIFICE24-AUG-0521p4914TERMINALSACRIFICE24-AUG-0521p4915TERMINALSACRIFICE24-AUG-0521p4916TERMINALSACRIFICE,24-AUG-0521p4917TERMINALSACRI.FICE24-AUG-0521p4918TERMINALSACRIFICE24-AUG-0521p4919TERMINALSACRIFICE25-AUG-0521NP4920TERMINALSACRIFICE25-AUG-0521p49;21TERMINALSACRIFICE25-AUG-0521p4922TERMINALSACRIFICE25-AUG-:-0521p4923TERMINALSACRIFICE25-AUG-0521p4924TERMINALSACRIFICE25-AUG-0521p----------------------------------------------NP-NOTPREGNANT,P-PREGNANT10-MAR-201117:13HuntingdonLifeSciences05-4287APPENDIXDGASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSUREGROUP10MG/M3FEMALEJIOBSERVATIONS----1901TERMINALSACRIFICEWITHINNORMALLIMITS1902TERMINALSACRIFICEWITHINNORMALLIMITS1903TERMINALSACRIFICEWITHINNORMALLIMITS1904TERMINALSACRIFICEWITHINNORMALLIMITS1905TERMINALSACRiFICEWITHINNORMALLIMITS1906TERMINALSACRIFICEWITHINNORMALLIMITS1907TERMINALSACRIFICEWITHINNORMALLIMITS1908TERMINALSACRIFICEWITHINNORMALLIMITS1909TERMINALSACRIFICENASALDISCHARGE-REDWITHINNORMALLIMITS1910TERMINALSACRIFICEWITHINNORMALLIMITS1911TERMINALSACRIFICEWITHINNORMALLIMITSINDIVIDUALCLINICALOB.SERVATIONSDURINGGESTATIONDAYOFGESTATION11 1 1 1 1 1 1 1 1 2 2012 3 4 5 6 7 8 9012 3 4 5 6 7 8 901pp p p pp.pp p p p p p p p p p p ppp p p p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p p pppp p p p p p p pp p p p p p p ppp p p p p p p p p p p p p p p p p p ppp p p p p p p p p p pppp p p p p pCODE:1-SLIGHT·2-MODERATEP-PRESENT10-MAR-201117:13HuntingdonLifeSciences05-4287APPENDIXDGASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSUREGROUP10MG/M3OBSERVATIONS---------------------'1912TERMINALSACRIFICEWITHINNORMALLIMITS1913TERMINALSACRIFICEWITHINNORMALLIMITS1914TERMINALSACRIFICEWITHINNORMALLIMITS1915TERMINALSACRIFICEWITHINNORMALLIMITS1916TERMINALSACRIFICEWITHINNORMALLIMITS1917TERMINALSACRIFICEWITHINNORMALLIMITS1918TERMINALSACRIFICEWITHINNORMALLIMITS1919TERMINALSACRIFICEWITHINNORMALLIMITS1920TERMINALSACRIFICEWITHINNORMALLIMITS1921TERMINALSACRIFICEWITHINNORMALLIMITS1922TERMINALSACRIFICEWITHINNORMALLIMITSINDIVIDUALCLINICALOBSERVATIONSDURINGGESTATIONDAYOFGESTATION11 1 1 1 1 1 1 1 1 2 2012 3 4 5 6 7 8 9012 3 4 5 6 7 8 901pp p p p p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p p p ppp p p p p pp"pp p p p p p p p p p p ppp p p p p p p p p p pp,pp p p p p p ppp p p p p p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p p p ppp p p p p p p p p pp.pp p p p p p p p ppp p p p p p p p p p p p p p p p p p pppp p p p p p p p p p p p p p p p p p p p ppp p pp.pp p p p p p p p p p p pp?p pCODE:1-SLIGHT2-MODERATE3-MARKED,P-PRESENTi10-MAR-201117:13HuntingdonLifeSciences05-4287APPENDIXDGASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSUREGROUP1FEMALE#192319240MG/M3OBSERVATIONSTERMINALSACRIFICEWITHINNORMALLIMITSTERM!NALSACRIFICEWITHINNORMALLIMITSINDIVIDUALCLINICALOBSERVATIONSDURINGGESTATIONDAYOFGESTATION11 1 1 1 1 1 1 1 1 2 2012 3 4 5 6 7 8 9012 3 4 5 6 7 8 901pp p p p p p p p p p p p p p p p p p p pP.pp p p p p p p p p p p p p pp p p p pCODE:1-SLIGHT2-MODERATE3-MARKEDP-PRESENT10-MAR-201117:13HuntingdonLifeSciences05-4287APPENDIXDGASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSUREGROUP2FEMALE#290129022903290429052906290729082909291020.00MG/M3OBSERVATIONSTERMINALSACRIFICENASALDISCHARGE-REDWITHINNORMALLIMITSTERMINALSACRIFICEWITHINNORMALLIMITSTERMINALSACRIFICEWITHINNORMALLIMITSTERMINALSACRIFICEWITHINNORMALLIMITSTERMINALSACRIFICEWITHINNORMALLIMITSTERMINALSACRIFICEWITHINNORMALLIMITSTERMINALSACRIFICEWITHINNORMALLIMITSTERMINALSACRIFICENASALDISCHARGE-REDWITHINNORMALLIMITSTERMINALSACRIFICENORMALLIMITSTERMINALSACRIFICEWITHINNORMALLIMITSINDIVIDUALCLINICALOBSERVATIONSDURINGGESTATIONDAYOFGESTATION11 1 1 1 1 1 1 1 1 2 2012 3 4 5 6 7 8 9012 3 4 5 6 7 8 901pp pp p p p p p p p p p p p pppppp p p p p p p ppp p p p p p p p ppp p p p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p ppp p p p p p p p p p p pPcp p p p ppp p p p p p p p p p p p p p p p p p ppp p pp p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p p pCODE:1-SLIGHT2-MODERATE3-MARKEDP-PRESENT10-MAR-201117:13HuntingdonLifeSciences05-4287APPENDIXDGASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSUREGROUP22000MG/M3FEMALE*OBSERVATIONS---------------------2911TERMINALSACRIFICEWITHINNORMAL'LIMITS2912TERMINALSACRIFICEWITHINNORMALLIMITS2913TERMINALSACRIFICEWITHINNORMALLIMITS2914TERMINALSACRIFICEWITHINNORMALLIMITS2915TERMINALSACRIFICEWITHINNORMALLIMITS2916TERMINALSACRIFICEWITHINNORMALLIMITS2917TERMINALSACRIFICEWITHINNORMALLIMITS2918TERMINALSACRIFICENASALDISCHARGE-REDWITHINNORMALLIMITS2919TERMINALSACRIFICEWITHINNORMALLIMITS2920SACRIFICEWITHINNORMALLIMITS2921TERMINALSACRIFICEWITHINNORMALLIMITSINDIVIDUALCLINICALOBSERVATIONSDURINGGESTATIONDAYOFGESTATION11 1 1 1 1 1 1 1 1 2 20 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1pP P PP.PP P P P P P P P P P P P P Ppp p p p p p p p p p p p p p p p p p ppp p p p p p pp-pp p p p p p p p p p ppp p p p p p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p p p ppp p pp.pp p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p p p ppp p p pp pp p p pp p p p p p p p p ppp p p p p p p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p p p p pCODE:1-SLIGHT2-MODERATE3-MARKEDP-PRESENT10-MAR-201117:13HuntingdonLifeSciences05-4287APPENDIXDGASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSUREGROUP2FEMALE#2922.2923.29242000MG/M3OBSERVATIONSTERMINALSACRIFICEWITHINNORMALLIMITSTERMINALSACRIFICEWITHINNORMALLIMITSTERMINALSACRIFICEWITHINNORMALLIMITSINDIVIDUALCLINICALOBSERVATIONSDURINGGESTATIONDAYOFGESTATION11 1 1 1 1 111 1 2 2012 3 4 5 6 7 8 9012 3 4 5 6 7 8 901pp p p p p p p p p p p p p p p p ppp p ppp p p P ppp ppP p.Pp p p p p p p p Ppp p p p p p p p p p p p p p p ppp p p pCODE:1-SLIGHT2-MODERATE3-MARKEDP-PRESENT10-MAR-201'117:13HuntingdonLifeSciences05-4287APPENDIXDGASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSUREINDIVIDUALCLINICALOBSERVATIONSDURINGGESTATIONGROUP3FEMALE#39013902390339043905390639073908390910000MG/M3OBSERVATIONSTERMINALSACRIFICEWITHINNORMALLIMITSTERMINALSACRIFICENASALDISCHARGE-REDWITHINNORMALLIMITS·TERMINALSACRIFICENASALDISCHARGE-REDWITHINNORMALLIMITSTERMINALSACRIFICEWITHINNORMALLIMITSTERMINALSACRIFICEWITHINNORMALLIMITSTERMINALSACRIFICENASALDISCHARGE-REDWITHINNORMALLIMITSTERMINALSACRIFICENASALDISCHARGE-REDWITHINNORMALTERMINALSACRIFICENASALDISCHARGE-REDWITHINNORMALLIMITSTERMINALSACRIFICENASALDISCHARGE-REDWITHINNORMALLIMITSCODE:1-SLIGHT2-MODERATE3-MARKEDP-PRESENTDAYOFGESTATION11'11 1 1l1 1 1 2 2012 3 4 5 6 7 8 9012 3 4 5 6 7 8 901pp p p p p p p p p p p p p p p p p pppp p p p p p p p p p p p p p p p ppp pp p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p pppp p p p p p p_pp pp p p p p p ppp ppp p p p p p p p p pp p p p p ppp p p pp p p p p p p p p p p p p ppppp p p p p p pp p p p p p p p p p p10-MAR-201117:13LifeSciences05-4287APPENDIXDGASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSUREfNDIVIDUALCLINICALOBSERVATIONSDURINGGESTATIONGROUP3FEMALElf39103911391239133914391539163917391810000MG/M3OBSERVATIONSTERMINALSACRIFICENASALDISCHARGE-REDWITHINNORMALLIMITSTERMINALSACRIFICENASALDISCHARGE-REDWITHINNORMALLIMITSTERMINALSACRIFICENORMALLIMITSTERMINALSACRIFICEWITHINNORMALLIMITSTERMINALSACRIFICEWITHINNORMALLIMITSTERMINALSACRIFICENASALDISCHARGE-REDWITHINNORMALLIMITSTERMINALSACRIFICENASALDISCHARGE-REDWITHINNORMALLIMITSTERMINALSACRIFICENASALDISCHARGE-REDWITHINNORMALLIMITSTERMINALSACRIFICEWITHINNORMALLIMITSCODE:1-SLIGHT3-MARKEDP-PRESENTDAYOFGESTATION11 1 1 1 1 1 1 1 1 2 2012 3 4 5 6 7 8 9o.1 2 3 4 5 6 7 8 901ppp p p p p p p p p p p p p p p p p pppp p p p p p p p ppp pp pp p pppp p p p p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p p p ppp p p p p pp p p p p p pp p p p p p ppp pP,p p p p p p p p p p p p p p p p pp.ppp p p p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p p p p10-MAR-201117:13HuntingdonLifeSciences05-4287APPENDIXDGASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSUREINDIVIDUALCLINICALOBSERVATIONSDURINGGESTATIONGROUP3FEMALElf391939203921·39223923392410000MG/M3OBSERVATIONSTERMINALSACRIFICEWITHINNORMALLIMITSTERMINALSACRIFICENASALDISCHARGE-REDWITHINNORMALLIMITSTERMINALSACRIFICENASALDISCHARGE-REDWITHIN.NORMALLIMITSTERMINALSACRIFICENASALDISCHARGE-REDWITHINNORMALLIMITSTERMINALSACRIFICEWITHINNORMALLIMITSTERMINALSACRIFICENASALDISCHARGE-REDWITHINNORMALLIMITSCODE:1-SLIGHT2-MODERATE3-MARKEDP-PRESENTDAYOFGESTATION11 1 1 1 1 1 1 1 1 2 2012 3 4 56789012 3 4 567890.1pp p p p p p p p p p p p p p p p p p p p ppppp p p p p p p p p p p p p p p p p p pppp p0p p p p p p p p p p p p p p p p p ppp pp p p p p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p p p p ppp p p p pp p pp p p p pp p p p p p p p10-MAR-201117:13HuntingdonLifeSciences05-4287APPENDIXDGASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSUREINDIVIDUALCLINICALOBSERVATIONSDURINGGROUP4FEMALE;#490149024903490449054906490749084909491020000MG/M3OBSERVATIONSTERMINALSACRIFICEWITHINNORMALLIMITSTERMINALSACRIFICEALOPECIA-GENERALCHROMODACRYORRHEA-UNILATERALWITHINNORMALLIMITSTERMINALSACRIFICENASALDISCHARGE-REDWITHINNORMALLIMITSTERMINALSACRIFICEWITHINNORMALLIMITSTERMINALSACRIFICEWITHINNORMALLIMITSTERMINALSACRIFICEWITHINNORMALLIMITSTERMINALSACRIFICEWITHINNORMALLIMITSTERMINALSACRIFICEINCISORSMALOCCLUDEDMASSTERMINALSACRIFICEWITHINNORMALLIMITSTERMINALSACRIFICENASALDISCHARGE-REDWITHINNORMALLIMITSCODE:1-SLIGHT2-MODERATE3-MARKEDP-PRESENTDAYOFGESTATION11 1 1 1 1 1 1 1 1 2 2012 3 4 5 6 7 8 9012 3 4 5 6 7 8 901pp p p p p p p p p p p p p p p p p pp p p p pp p p p ppp p p pp p pp2 2 2 2 2 2 2 2ppp p p p p p pp ppPPPPRPPPPPPPPPPPPpp p p p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p p p2 2 2 2 2pp p p p p p p p p p p p p p p p p p pppp pp p p p p p p p p p p p ppp p10-MAR-201117:13HuntingdonLifeSciences05-4287APPENDIXDGASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSUREINDIVIDUALCLINICALOBSERVATIONSDURINGGESTATIONGROUP4FEMALE#491149124913.491449154916491749184919492020000MG/M3OBSERVATIONSTERMINALSACRIFICEWITHINNORMALLIMITSTERMINALSACRIFICEWITHINNORMALLIMITSTERMINALSACRIFICEWITHINNORMALLIMITSTERMINALSACRIFICENASALDISCHARGE-REDWITHINNORMALLIMITSTERMINALSACRIFICEWITHINNORMALLIMITSTERMINALSACRifiCENASALDISCHARGEREDWITHINNORMALLIMITSTERMINALSACRIFICENASALDISCHARGE-REDWITHINNORMALLIMITSTERMINALSACRIFICECHROMODACRYORRHEA-UNILATERALWITHINNORMALLIMITSTERMINALSACRIFICEWITHIN.NORMALLIMITSTERMINALSACRIFICENASALDISCHARGE-REDWITHINNORMALLIMITSCODE:1-SLIGHT2-MODERATE3-MARKEDP-PRESENTDAYOFGESTATION11 1 1 1 1 1 1 1 1 2 2012,34567 8 9012 3 4567.8901pp p p p p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p p p ppp p p pp p p p p p p p p p p p p p p ppp p p p p p p p p p p p p p p p p p p ppp p p p pp p p p p p p p p p p p p p ppp p pp p ppp p p p ppp pp pp p pppp p p p p p p p pppp p p p p p p ppp p p p p p p p p p p p p pp.pp p p p ppp p p pp p p p ppp p p p p p p p ppp10-MAR-201117:13HuntingdonLifeSciences05-42S7APPENDIXDGASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSUREINDIVIDUALCLINICALOBSERVATIONSDURINGGESTATIONGROUP4FEMALElf492149224923492420000MG/M3OBSERVATIONSTERMINALSACRIFICE,NASALDISCHARGE-REDWITHINNORMALLIMITSTERMINALSACRIFICENASALDISCHARGE-REDWITHINNORMALLIMITSTERMINALSACRIFICENASALDISCHARGE-REDWITHINNORMALLIMITSTERMINALSACRIFICECHROMODACRYORRHEA-BILATERALCHROMODACRYORRHEA-,UNILATERALWITHINNORMALLIMITSCODE:1-SLIGHT2-MODERATE3-MARKEDP-PRESENTDAYOFGESTATION11 1 1 1 1 1 1 1 1 2 2012 3 4 5 6 7 8 9012 3 4 5 6 7g901ppp p p p p p p p p p pp p p p p p p p pppp p pp p p p p p pp p pp p p p p p ppp pp p p p p p p ppp p p p pp ppp pppp p p p p p p p p p p p p 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23AVERAGEFETALBODYWEIGHTMALESFEMALESLITTER5.65.95.86.46.16.05.55.46.36.36.16.05.76.66.05.55.55.95.45.75.96.26.35.90.33235.35.55.56.05.75.95.25.15.85.75.65.95.66.25.95.35.25.85.05.85.75.95.95.60.33235.55.75.76.15.85.95.45.26.05.95.86.05.66.35.95.45.35.95.35.75.86.06.15.80.302310-MAR-201117:22HuntingdonLifeSciences05-4287APPENDIXIGASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSUREINDIVIDUALFEMALEREPRODUCTIONDATAANDMEANFETALWEIGHTS(GRAMS}GROUP310000MG/M3CORPORA%PREIMPL.IMPLANTLUTEALOSSSITES390139023903390439053906390739083909391039113912391339143915391639173918391939203921392239233924MEANS.D.N11131413151413131416161420181016211513141317161514.82.52249.10.07.10.00.00.00.00.07.112.50.07.160.016.770.012.533.30.00.00.07.70.00.06.710.418.5824101313131514131313141613815314141513141217161413.12.8524FETUSESLIVEDEAD(n}%1000.01200.0,1300.01300.01500.01400.01100.01000.01200.01400.01600.01200.0800.01400.0200.01400.01400.01300.01300.014'00.01100.01700.01600.01400.012.60.03.060.0024 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24AVERAGEFETALBODYWEIGHTMALESFEMALESLITTER5.95.65.26.15.35.55.85.75.96.15.96.16.55.56.45.75.55.85.66.15.86.05.65.95.80.32245.65.55.15.54.95.65.55.45.65.75.35.95.95.25.35.65.25.45.35.85.85.65.55.65.50.25245.85.65.15.85.15.65.65.55.85.95.56.06.45.35.85.75.35.55.56.05.85.85.65.85.70.282410-MAR-201117:22HuntingdonLifeSciences05-4287APPENDIXIGASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSUREINDIVIDUALFEMALEREPRODUCTIONDATAANDMEANFETALWEIGHTS(GRAMS)GROUP420000MG/M3CORPORA%PREIMPL.IMPLANTANIMAL#LUTEALOSSSITES49014902490349044905151117.18164906194907134908NP49094910491149124913491449154916491749184919NP492049214<:l2249234924111417111514.161616211413161318MEANS.D.15.22.6322NNP=NOTPREGNANT6.70.011.80.00.015.815.49.10.00.00.06.714.36.30.06.338.114.315.46.30.00.07.69.14221411151816161110141711141215161513121115131814.02.3822FETUSESLIVEDEAD1310141816161091316111410141614131111151117(n)%00000000.00.00.00.00.00.00.000.000.000.000.000.000.000.000.000.000.0000000.00.00.00.00.013.30.00.02.600.000.00222222RESORPTIONSEARLYLATETOTAL11100011110021010100210000000000000000000000(n)%17.119.116.700.000.0010.09.1110.017.115.900.000.0216.716.700.016.700.018.300.000.0215.415.60.70.00.75.20.650.000.655.1522222222SEXMALEFEMALE6391097468664631064565757758796351051041168965104126.17.12.052.492222AVERAGEFETALBODYWEIGHTMALESFEMALESLITTER5.65.85.85.45.45.56.06.05.75.66.56.06.35.65.06.05.95.45.75.45.75.65.70.33225.35.35.65.05.05.35.65.75.55.35.75.75.95.14.75.85.35.15.45.25.25.45.40.31225.55.55.75.25.25.45.75.95.65.46.25.86.15.24.95.95.55.25.65.25.55.45.50.322210-MAR-201117:23HuntingdonLifeSciences05-4287APPENDIXJGASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSUREINDIVIDUALGRAVIDUTERINEWEIGHTANDNETBODYWEIGHTCHANGE(GRAMS)GROUP1 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=TERMINALCORRECTEDBODYWEIGHTMINUSDAY5BODYWEIGHTNP=NOTPREGNANTx=EXCLUDEDFROMMEAN10-MAR-201117:23HuntingdonLifeSciences05-4287APPENDIXJGASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSUREINDIVIDUALGRAVIDUTERINEWEIGHTANDNETBODYWEIGHTCHANGE(GRAMS)GROUP310000MG/M3--UTERUSADJUSTEDNETWEIGHTCHANGEANIMALlfWEIGHTWEIGHTFROMDAY53901802461639028827533390395280493904103276313905107281333906104288433907832614339087526338390990280363910108269233911117283293912963032839136830267391410028939391519305473916110298413917100308513918101293303919932754839201142593339218624819392213627233392312126819392411228628MEAN96.280.36.S.D.22.517.111.7N242424ADJUSTEDWEIGHT=TERMINALBODYWEIGHTMINUSGRAVIDUTERINEWEIGHTWEIGHTCHANGEFROMDAY59612114413314014612611312613114612413513967151151131141147105169140140132.21.024NETWEIGHTCHANGEFROMDAY5=TERMINALCORRECTEDBODYWEIGHTMINUSDAY5BODYWEIGHT10-MAR-201117:23HuntingdonLifeSciences05-4287APPENDIXJGASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSUREINDIVIDUALGRAVIDUTERINEWEIGHTANDNETBODYWEIGHTCHANGE(GRAMS)GROUP420000MG/M3------UTERUSADJUSTEDNETWEIGHTCHANGEWEIGHTCHANGEANIMALl!WEIGHTWEIGHTFROMDAY5FROMDAY54901912371710849027827449127490310327229132490412325721144490511027321131490611927020139490(79261451234908xNP144909.7025423934910NT144491111327420133491294292351294913105267211274914842964212649159727522118491610828127135491711430334148491894300351294919xNP14920762724712449218427440124492210426216120492381270231044924PART124x318x47x171MEAN96.273.29.129.S.D.15.715.910.616.0N20202022ADJUSTEDWEIGHTTERMINALBODYWEIGHTMINUSGRAVIDUTERINEWEIGHTNETWEIGHTCHANGEFROMDAY5=TERMINALCORRECTEDBODYWEIGHTMINUSDAY5BODYWEIGHTNP=NOTPREGNANTNT=NOTTAKENPART=PARTIALDELIVERY;5LIVEPUPSDELIVEREDx=EXCLUDEDFROMMEAN10-MAR-201117:24HuntingdonLifeSciences05-4287APPENDIXKGASOLINEDIPEVAP.ORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSUREGROUP10MG/M3FEMALE#MEAN1901190219031904190519061907190819091910191119121913191419151916191719181919192019211922192319245.96.05.95.55.55.56.35.76.26.05.65.25.56.0NP5.55.55.55.86.15.46.05.95.6MEAN5.7S.D.0.29N23FETUS#1 25.65.65.75.33.25.66.46.06.35.95.85.65.06.0.5.35.55.45.66.25.15.86.15.75.96.66.35.15.75.36.35.86.66.05.85.05.96.35.45.15.36.26.05.36.55.9E35.96.05.35.66.45.26.06.46.15.25.55.3E6.45.55.75.66.06.15.15.65.55.945.85.75.65.4E5.66.2/5.66.46.05.55.05.26.1/5.25.45.66.26.35.65.95.65.6E-EarlyresorptionL-Lateresorption/-DenotespositionofcervixNP=NOTPREGNANTINDIVIDUALFETALBODYWEIGHTS(GRAMS}56.0/6.36.25.7/5.65.46.25.46.3/6.3E4.75.56.15.65.15.75.3676.35.35.9/6.36.45.65.4 5.95.6/6.15.4EI6.16.65.7/5.56.26.05.7 5.95.65.4/5.05.25.9EI5.45.85.75.45.75.25.4/6.05.05.88 95.4E5.9 5.65.56.2/6.05.75.9 5.86.15.46.5 6.3E6.05.76.76.15.85.6 5.45.25.4/5.65.65.86.26.0/E5.25.55.75.15.7/5.9106.05.96.25.45.85.86.16.26.25.75.75.55.95.65.75.56.65.25.55.8/6.56.05.55.35.85.3/5.55.9 5.96.26.06.05.2 5.76.1/6.25.35.6/5.35.4116.26.06.05.35.35.25.36.46.2/5.45.15.46.15.66.1/L5.64.85.75.85.7126.56.05.45.65.75.35.86.46.45.75.26.15.95.05.35.55.75.35.46.15.6136.25.55.25.85.35.76.35.65.25.35.65.75.46.05.26.05.85.5146.55.75.35.45.9E5.35.35.45.96.15.26.45.6155.36.16.25.65.55.95.25.46.15.316 17 18195.65.25.55.710-MAR-201117:24HuntingdonLifeSciences05-4287APPENDIXKGASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSUREINDIVIDUALFETALBODYWEIGHTS{GRAMS)GROUP22000MG/M3FEMALE#MEAN29012902290329042905290629072908290929102911291229132914291529.16291729182919292029212922292329245.55.75.76.15.85.95.45.26.05.95.86.05.66.35.95.45.35.9NP5.35.75.86.06.1MEAN5.8S.D.0.30N23FETUS#1 25.4/5.25.15.96.16.04.95.35.75.75.56.45.6E5.45.55.25.75.5E5.45.45.95.65.75.85.96.25.85.84.9E5.56.05.95.6EE5.3E6.05.7E6.05.96.535.36.05.36.45.96.1/5.14.96.56.05.45;95.56.16.25.35.05.9L5:75.95.85.945.35.75.56.05.66.06.05.76.45.75.76.455.75.55.55.85.65.55.45.65.8/5.35.7/6.05.7 5.6E EI6.06.25.1 5.65.55.0/6.05.35.4/5.16.05.45.45.86.1 6.36.5 6.1E-EarlyresorptionL-Lateresorption/-DenotespositionofcervixNP=NOTPREGNANT65.16.1E5.75.9E5.15.1/6.45.8/5.45.875.65.9/5.9/6.45.95.55.5/5.16.26.15.66.085.35.35.9E5.75.95.75.65.86.45.45.85.75.55.7E E E5.76.25.3/5.34.95.8/5.6 5.6 5.45.46.15.45.3 5.24.95.8 5.65.75.6/6.05.96.3/6.5 6.46.05.3/5.99E6.25.9EI6.36.25.34.85.76.05.76.110116.15.9E5.55.9 5.96.85.95.6/6.26.16.25.4 5.2E5.35.9 5.86.25.96.36.16.1/6.15.1/5.65.66.66.4E5.96.26.15.4 5.75.45.65.44.95.96.0/6.1E5.14.56.0/5.85.65.86.05.85.76.06.25.76.1 6.5125.96.16.45.86.15.25.46.55.75.96.0E5.65.65.36.55.66.05.86.36.1136.35.55.85.34.95.26.26.15.6E5.75.36.35.35.66.06.3146.25.85.16.46.16.35.86.25.25.46.2156.15.25.55.56.1161718195.25.95.810-MAR-201117:24HuntingdonLifeSciences05-428.7GROUP310000MG/M3FEMALEllMEAN39013902"3903390439053906390739083909391039113912391339143915391"6391739183919392039213922392339245.85.615.15.85.15.65.65.55.85.95.56.06.4I5.35.8I5.75.35.55.56.05.85.85.65.8MEAN5.7S.D.0.28N24E-EarlyresorptionFETUSll1 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.5.35.95.4E6.l,.6.03.35.94.95.64.85.55.65.85.85.75.55.8125.84.85.85.95.95.7E6.16.46.26.05.35.75.55.45.76.14.95.25..45.8135.65.35.95.06.15.85.85.75.95.55.75.05.85.75.15.35.55.56.16.0145.55.86.45.75.35.85.34.85.86.25.35.6155.25.65.95.75.75.7166.06.35.51718 196.110-MAR-201117:24HuntingdonLifeSciences05-4287GROUP420000MG/M3FEMALE#MEAN4901490249034904490549064907490849094910491149124913491449154916491749184919492049214922492349245.55.55.75.25.25.45.7NP5.95.65.46.25.86.15.24.95.5NP5.25.65.25.55.4MEAN5.5S.D.0.32N22E-EarlyresorptionFETUS#125.76.05.64.85.25.56.06.15.55.16.76.05.95.55.06.05.25.25.45.05.34.75.95.55.75.15.35.45.66.05.75.66.75.8E4.75.45.95.35.05.84.95.25.7/-DenotespositionofcervixNP=NOTPREGNANT35.6.E5.75.85.15.35.6E5.25.15.35.66.55.34.85.85.3E5.25.85.65.3APPENDIXKGASOLINEDIPEVAPORCONDENSATE:EMBRYO-FETALTOXICITYSTUDYINRATSBYINHALATIONEXPOSURE45.3/5.65.75.15.55.46.15.86.05.66.05.75.95.24.95.65.45.25.65.05.95.9INDIVIDUALFETALBODYWEIGHTS(GRAMS)5675.5 5.45.25.4/5.45.3E5.9/5.44.94.85.1/5.25.6 5.15.1 5.9 5.55.65.6/E6.35.46.25.8 5.85.75.84.45.36.36.6/5.65.8/6.15.7.6.3E5.95.4E5.34.7 4.55.3/6.05.75.75.2/5.55.45.15.1/5.36.45.6/5.35.1 5.65.34.85.5 5.65.25.6 5.689105.3 5.4 5.65.36.05.15.9 5.25.85.55.1 5.15.04.54.9/5.4/5.55.05.76.05.46.1/6,05.45.7/E5.55.4 5.45.55.85.76.05.4 5.36.16.2/5.76.45.14.35.2/5.15.05.1E6.2/6.06.25.35.35.4 5.85.05.35.8 5.65.35.3/5.35.8/5.25.95.45.35.8115.45.15.55.85.25.05.95.05.66.96.06.14.84.95.85.95.25.05.2EE12 13E5.26.16.05.2 5.35.2 5.75.35.45.8 5.85.4/E5.95.86.66.15.44.84.25.86.1.5.85.75.35.34.95.8E5.5/5.6145.45.84.95.55.45.85.15.65.55.26.05.25.1155.75.35.15.65.65.04.76.15.55.6164.95.45.35.64.85.21718 195.15.65.95.85.4HuntingdonLifeSciences05-4287211-DIPE-DEVPage1DraftreportFemalesINDIVIDUALFETALEXTERNALVISCERALANDSKELETALOBSERVATIONSAPPENDIXLAnimalNumberGroup1 -0MG/M3·19016s,5v1219037s,7v3101119047s,8v61119057s,6v1219086s,6v619098s,7v89MFMMFFMF.FFM6.55.36.26.05.45.33.25.75.75.76.7bnormalities(onlyfetuseswithabnormalitiesarelisted)FateIMaiorabnormalitiesinCAPITALSsSternebra(e)malaligned,#4and#5,slightsRib(s)14thrudimentary,leftvAdditionalsubclavianartery,leftsRib(s)14thrudimentary,bilateralsHyoidbody/arch(es),unossifiedbodyvRenalpapilla(e)notfullydeveloped(grade1),leftsANALATRESIAANDFILAMENTOUSTAILVERTEBRALAGENESIS:SACRALVERTEBRAE#1AND#2SMALLINSIZE;SACRALVERTEBRAE#3AND#4ANDALLCAUDALVERTEBRAEABSENT;ILIAANDISCHIACLOSELYAPPROXIMATEDSternebra(e)5and/or6unossified,#5vUndescendedthymictissue,rights25presacralvertebra(e)Rib(s)13thrudimentary,rightvUndescendedthymictissue,leftsSternebra(e)malaligned,#2through#4,slights =skeletalexamination,v =visceralexamination,M=Male,F=Female,g=gramsHuntingdonLifeSciences05-4287211-DIPE-DEVFemalesINDIVIDUALFETALEXTERNALVISCERALANDSKELETALOBSERVATIONSAnimalNumberGroup1 -0MG/M319106s,7v2919118s,7v4919127s,8v·391219136s,5v619146s,6v819167s,7v1;3FFMMFFFMFFM6.05.85.55.45.35.45.25.95.85.35.5!Abnormalities(onlyfetuseswithabnormalitiesarelisted)FateIMaiorabnormalitiesinCAPITALSsSternebra(e)malaligned,#4,slightvLeftumbilicalarteryvUndescendedthymictissue,leftvUndescendedthymictissue,rightvUndescendedthymictissue,leftvUndescendedthymictissue,rightUreter(s)distended,bilateralsSternebra(e)malaligned,#4and#5,slightsSternebra(e)malaligned,#3through#5,slighttomoderatesSternebra(e)malalfgned,#4and#5,slightvUndescendedthymictissue,rightvUndescendedthymictissue,lefts =skeletalexamination,v =visceralexamination,M=Male,F=Female,g=gramsPage2DraftreportAPPENDIXLHuntingdonLifeSciences05-4287211-DIPE-DEVFemalesINDIVIDUALFETALEXTERNALVISCERALANDSKELETALOBSERVATIONSAnimalNumberGroup1 -0MG/M319187s,7v1119197s,7v9111319218s,7v261219238s,7v7-19247s,7v9LF5.9F5.6F6.0F5.3M5.5M5.3F5.2F5.3bnormalities(onlyfetuseswithabnormalitiesarelisted)FateIMaiorabnormalitiesinCAPITALSCrown-rumpdistance2.4emsSternebra(e)malaligned,#4and#5,slightsSternebra(e)malaligned,#4and#5,slightsSternebra(e)malaligned,#4and#5,slightvInnominatearteryabsentvUndescendedthymictissue,leftvUndescendedthymictissue,rightDisplacedtestismedially,leftsSternebra(e)malaligned,#3and#4,slighttomoderate.sSternebra(e)malaligned,#3through#5,slights =skeletalexamination,v =visceralexamination,M=Male,F=Female,L=LateResorption,g=gramsPage3DraftreportAPPENDIXLHuntingdonLifeSciences05-4287211-DIPE-DEVFemalesIINDIVIDUALFETALEXTERNAL,VISCERALANDSKELETALOBSERVATIONSAnimalbnormalities(onlyfetuseswithabnormalitiesarelisted)NumberFateMa·orabnormalitiesinCAPITALSGroup2-2000MG/M329015s,5v2M5.6vUndescendedthymictissue,left3 F5.3sSternebra(e)malqligned,#3through#4,slight8 M5.3vUndescendedthymictissue,bilateral2902.5s,6v2 F5.7sSternebra(e)malaligned,#3and#4,slight29036s,5v2M5.8vUndescendedthymictissue,left3 M5.3sSternebra(e)malaligned,#4and#5Rib(s)14thrudimentary,right29046s,6v14F6.2sRIGHTFORELIMBSHORTENED29058s,8v1M6.1sSternebra(e)malaligned,#3through#5,slight7 F5.9sSternebra(e)malaligned,#4and#5,slight10F5.6vUndescendedthymictissue,right29087s,7v14M5.1vLeftumbilicalartery29095s,5v6 M6.4sRib(s)14thrudimentary,right11M5.8vLeftumbilicalarterys =skeletalexamination,v =visceralexamination,M=Male,F=Female,g=gramsPage4DraftreportAPPENDIX-LHuntingdonLifeSciences05-4287211-DIPE-DEVFemalesIINDIVIDUALFETALEXTERNAL,VISCERALANDSKELETALOBSERVATIONSAnimalbnormalities(onlyfetuseswithabnormalitiesarelisted)NumberFateMa'orabnormalitiesinCAPITALSGroup2-2000MG/M329107s,7v3 F6.0vUndescendedthymictissue,bilateral4F5.7sRib(s)14thrudimentary,bilateral5F5.3vUndescendedthymictissue,left6 F5.8sRib(s)14thrudimentary,bilateral8F6.4sRib(s)14thrudimentary,left13F5.2v.Undescendedthymictissue,right29128s,9v4 M6.4s7thcervicalrib(s)present,left10.F6.1sSHORTMANDIBLE,moderate15M5.5vUndescendedthymictissue,leftLeftumbilicalartery17F5.8vLeftumbilicalartery29137s,7v1 M5.6sSternebra(e)malaligned,#4and#5,slightRib(s)14thrudimentary,bilateral29176s,6v4 M5.5sRib(s)14th·rudimentary,bilateral13F5.3vUndescendedthymictissue,bilateral29186s,7v2M6.0sSternebra(e)malaligned,#4and#5,slights =skeletalexamination,v =visceralexamination,M=Male,F=Female,g=gramsPage5DraftreportAPPENDIXLHuntingdonLifeSciences05-4287211-DIPE-DEVFemalesINDIVIDUALFETALEXTERNALVISCERALANDSKELETALOBSERVATIONSAnimalIIFetusAbnormalities(onlyfetuseswithabnormalitiesarelisted)NumberExamNumberSexFateMa·orabnormalitie,sinCAPITALSGroup2-2000MG/M329205s,6v3LCrown-rumpdistance2.9em12M5.6sSternebra(e)malaligned,#4and#5,slight29215s,5v3M5.7sSternebra(e)malaligned,#3,slight29238s,7v1F5.4sSternebra(e)malaligned,#3through#5,slight7F6.5sSternebra(e)malaligned,#3and#4,slight14F5.4vUndescendedthymictissue,left29247s,7v11F6.5vUndescendedthymictissue,lefts=skeletalexamination,v=visceralexamination,M=Male,F=Female,L=LateResorption,g=gramsPage6DraftreportAPPENDIXLHuntingdonLifeSciences05-4287211-DIPE-DEVFemalesIINDIVIDUALFETALEXTERNAL,VISCERALANDSKELETALOBSERVATIONSIAnimalbnormalities(onlyfetuseswithabnormalitiesare·listed)NumberFateMa'orabnormalitiesinCAPITALSGroup3 -10000MG/M339015s,5v1M5.5sSternebra(e)malaligned,#4,slight39037s,6v9 F5.0sSternebra(e)malaligned,#3and#4,slight39058s,7v1F5.1sSternebra(e)malaligned,#5,slightRib(s)14thrudimentary,right5F4.8sSternebra(e)malaligned,#5,slight39067s,7v1M5.4vInnominatearteryabsent9F5.6vRenalpapilla(e)notfullydevelopedEgrade1),leftUreter(s)distendedleft39076s,5v6F5.3sSternebra(e)malaligned,#5,slight8F5.5sHyoidbody/arch(es),unossifiedbody39107s,7v6 M6.0sRib(s)14thrudimentary,right7 F5.7vInnominatearteryabsents =skeletalexamination,v =visceralexamination,M=Male,F=Female,g=gramsPage7DraftreportAPPENDIXLHuntingdonLifeSciences05-4287211-DIPE-DEVFemalesIINDIVIDUALFETALEXTERNAL1VISCERALANDSKELETALOBSERVATIONSAnimalFetusIWeightI!Abnormalities(onlyfetuseswithabnormalitiesarelisted)Number NumberSex(a)FateMaiorabnormalitiesinCAPITALSGroup3 -10000MG/M339117s,9v8F3.7vMICROPHTHALMIA/ANOPHTHALMIA,leftUndescendedthymictissue,rightLeftumbilicalartery11F3.3vDOMED·cRANIUM,moderate;HYDROCEPHALYANOPHTHALMIA,bilateralHypoplasticlunglobe(s),apicallobeHypoplasticthymus,bilaterallobesHypoplasticspleen13F5.5sRib(s)14thrudimentary,bilateral39126s,6v6M6.3vUndescendedthymictissue,bilateral8M5.8vUndescendedthymictissue,bilateral12F6.0vUndescendedthymictissue,right39134s,4v1M7.0sRib(s)14thrudimentary,right5M6.2sSternebra(e)malaligned,#4,slight39147s,7v5M5.0vUndescendedthymictissue,left15M5.9sRib(s)14thrudimentary,left39167s,7v2M5.5sSternebra(e)5and/or6unossified,#58M5.7sSternebra(e)malaligned,#3and#4,slights =skeletalexamination,v =visceralexamination,M=Male,F=Female,g=gramsPage8DraftreportAPPENDIXLHuntingdonLifeSciences05-4287211-DIPE-DEVFemalesINDIVIDUALFETALEXTERNALVISCERALANDSKELETALOBSERVATIONSAnimalNumberGroup3-10000MG/M339177s,7v11F39186s,7v14F39216s,5v6M8F39228s,9v2F10F39238s,8v16M39247s,7v4 M6F4.84.86.26.06.05.85.56.05.2.,Abnormalities(onlyfetuseswithabnormalitiesarelisted)FateMaiorabnormalitiesinCAPITALSsSternebra(e)5and/or6unossified,#5sSternebra(e)malaligned,#3and#4,slightsSternebra(e)malaligned,#3through#5,slightssternebra(e)malaligned,#3through#5,slightsSternebra(e)malaligned,#4and#5sSternebra(e)malaligned,#3through#5,slightvRenalpapilla(e)notfullydeveloped(grade1),leftsSternebra(e)malaligned,#4,slightsSternebra(e)5and/or6unossified,#5s =skeletalexamination,v =visceralexamination,M=Male,F=Female,g=gramsPage9DraftreportAPPENDIXLHuntingdonLifeSciences05-4287211-DIPE-DEVFemalesINDIVIDUALFETALEXTERNALVISCERALANDSKELETALOBSERVATIONSAnimalIIFetusIIWeightI!Abnormalities(onlyfetuseswithabnormalitiesarelisted)NumberExamNumberSex(a)FateMaiorabnormalitiesinCAPITALSGroup4 -20.000MG/M349017s,6v2 M5.9vUndescendedthymictissue,left5F5.5sHyoidbody/arch(es)unossifiedbody10M5.6vUndescendedthymictissue,bilateral49025s,5v5 F5.4s7thcervicalrib(s)rightRib(s)14thrudimentary,bilateral11F5.1sSternebra(e)5and/or6unossified,#549049s,9v3 M5.8vUndescendedthymictissue,left49058s,8v12F5.2vUndescendedthymictissue,left14M5.5vUndescendedthymictissue,left15M5.1s.Sternebra(e)malaligned,#2through#4,slighttomoderate49068s,8v5F5.1vUndescendedthymictissue,right11F5.0vUndescendedthymictissue,leftPage10DraftreportAPPENDIXL49075s,5v1M6.0sVERTEBRALANOMALY:THORACICCENTRUM#13ANDLUMBARCENTRUM#1ABSENT;THORACICARCHES#13ANDLUMBARARCHES#1MALFORMEDANDCLOSELYAPPROXIMATED11MS.9vUndescendedthymictissue,lefts =skeletalexamination,v =visceralexamination,M=Male,F=Female,g=gramsHuntingdonLifeSciences05-4287211-DIPE-DEVFemalesINDIVIDUALFETALEXTERNAL.VISCERALANDSKELETALOBSERVATIONSAnimalbnormalities(onlyfetuseswithabnormalitiesarelisted)NumberFateMa·orabnormalitiesinCAPITALSGroup4 -20000MG/M3.49095s,4v4F5.8sRib(s)14thrudimentary,right6M5.4sEXOCCIPITALANDCERVICALARCH.#1FUSED,rightSternebra(e)malaligned,#4and#5,slight49106s,7v13F5.8s27presacralvertebra(e)Rib(s)14thrudimentary,bilateral49118s,8v5M5.8sRib(s)14thrudimentary,right16F5.6sRib(s)14thrudimentary,left49125s,6v2 M6.7sRib(s)14thrudimentary,right8F5.8sSternebra(e)malaligned,#3through#5,slighttomoderate49157s,7v3F5.3sRib(s)14thrudimentary,right8F5.1sRib(s)14thrudimentary,left15F5.0vInnominatearteryabsent49168s,8v13F4.2vLeftumbilicalarterys =skeletalexamination,v =visceralexamination,M=Male,F=Female,g=gramsPage11DraftreportAPPENDIXLHuntingdonLifeSciences05-4287211-DIPE-DEVFemalesIINDIVIDUALFETALEXTERNAL,VISCERALANDSKELETALOBSERVATIONSAnimalAbnormalities(onlyfetuseswithabnormalitiesarelisted)NumberFateMa'orabnormalitiesinCAPITALSGroup4 -20000MG/M349177s,7v4 F5.6vUndescendedthymictissue,left10F6.0s7thcervicalrib(s)present,right12M5.8sRib(s)14thrudimentary,right14F6.0sRib(s)14thru-dimentary,left49216s,5v1 M5.4s7thcervicalrib(s)present,right25presacralvertebra(e)Sternebra(e)5and/or6unossified,#5Rib(s)13thrudimentary,right2 M5.8vUndescendedthymictissue,right3 M5.2s25presacralvertebra(e)Sternebra(e)5and/or6unossified,#5Rib(s)13thrudimentary,bilateralMetatarsals,1unossified,bilateral11F5.0s25presacralvertebra(e)Rib(s)13thrudimentary,bilateralMetatarsals,1unossified,bilateral49236s,5v4 M5.9vUndescendedthymictissue,left12M5.8sRib(s)14thrudimentary,lefts =skeletalexamination,v =visceralexamination,M=Male,F=Female,g=gramsPage12DraftreportAPPENDIXLHuntingdonLifeSciences05-4287211-DIPE-DEVFemalesINDIVIDUALFETALEXTERNALVISCERALANDSKELETALOBSERVATIONSAnimalNumberGroup4-20000MG/M349248s,9v11417FFF4.75.1.5.8!Abnormalities(onlyfetuseswithabnormalitiesarelisted)FateMaiorabnormalitiesinCAPITALSvRenalpapilla(e)notfullydeveloped(grade1),bilateralUreter(s)distended,leftvLeftumbilicalarterysRib(s)14thrudimentary,lefts =skeletalexamination,v =visceralexamination,M=Male,F=Female,g=gramsPage13DraftreportAPPENDIXL

### 05-4287



### GASOLINE DIPE VAPOR CONDENSATE

EMBRYO-FETAL TOXICITY STUDY IN RATS BY INHALATION EXPOSURE

Statistical Analysis

Page 1 of 18 Authors

Graham 

### F Healey

Simon Bate

Statistics Department

Statistical analysis

### 05-4287



## COMPLIANCE STATEMENT

This phase of the study was conducted in compliance with the following Good Laboratory

Practice standards and I consider the data generated to be valid.

The UK Good Laboratory Practice Regulations (Statutory Instrument 1999 No 3106

as amended by Statutory Instrument 2004 No. 994).

OECD Principles of Good Laboratory Practice (as revised in 1997),

ENV /MC/CHEM(98) 17.

EC Commission Directive 2004/10/EC of 11 February 2004 (Official Journal No L

50/44).

US EPA 79.60, CFR Vol. 59, No. 122,27 June 1994.

These principles of Good Laboratory Practice are accepted by the regulatory authorities of

the United States of America and Japan on the basis of intergovernmental agreements.

Principal Investigator

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



### :2:

Statistical analysis

### 05-4287

INTRODUCTION AND EXPERIMENTAL DESIGN

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

, 

### I 0000 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, dll, d14, d17, d20, d21)

Maternal body weight change (d4-d5, d5-d8, d8-dll, dll-dl4, d14-d17, d17-d20, d5-dl4,

d14-d21, d5-d21)

Maternal feed consumption {d4-d5, d5-d8, d8-dll, dll-dl4, d14-d17, d17-d20, d20-d21,

d5-d21)

Maternal uterus weight (absolute and adjusted)

Maternal net bodyweight change from d5 minus gravid uterine weight

Reproductive performance

Corpora lutea

%Preimplantation loss

Number of implant sites

Fetuses alive

Early resorptions

Late resorptions

Total resorptions

%Total resorptions/implant sites

Number of males

Number of females

There were no dead fetuses, therefore the "Fetuses dead" and "%Fetuses dead/total" data

were not analyzed.

Fetal body weight

Mean fetal body weight: males

Mean fetal body weight: females

Mean fetal body weight: litter (pooled males and females)

Discrete fmdings

Maternal pregnancy summary

Maternal necropsy fmdings

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

required statistical analysis).



### :3:

Statistical analysis

### 05-4287



## METHODOLOGY

Continuous maternal parameters

The analysis was performed at each timepoint separately.

Bartlett's test (Bartlett, 1937) was first applied (Proc GLM, SAS Institute 1999) to determine

if the groups had equal variances. If the test was not significant at the 1% level, parametric

procedures were used. If the test was significant then non-parametric procedures were used.

For the parametric choice, a one-way analysis of variance (Armitage, Berry and Matthews

2002) was initially applied (Proc GLM, SAS Institute 1999). If this was significant at the 5%

level, then each treatment group was compared with the control using Dunnett's test (Dunnett

1955, 1964).

For the non-parametric choice, a Kruskal-Wallis test (Kruskal and Wallis 1952, 1953) was

initially applied (Proc Npar1way, SAS Institute 1999). If this was significant at the 5% level,

then each treatment group was compared with the control using Steel's test (Steel1959).

Reproductive performance

All parameters except fetal bodyweight were analyzed using the same methods as for

continuous maternal parameters, described above.

Fetal body weight

The fetal body weight was analyzed by separate and combined sexes using mixed models

(Proc Mixed, SAS Institute 1999). The separate-sex analysis had dose group as explanatory

variable, a random animal effect and litter size as covariate. The combined analysis had dose

group and fetal sex and their interaction as explanatory variables, with litter size as covariate.

For both sets of analysis, the Satterthwaite method was used for the fixed effects denominator

degrees of freedom. If the dose group effect was significant at the 5% level, then each

treatment group was compared with the control using Dunnett's test (Dunnett 1955, 1964),

with the means being adjusted for litter size.



## : 4 :

Statistical analysis

### Discrete findings

Maternal pregnancy summary

Maternal necropsy fmdings

Fetal findings: external, visceral and skeletal malformations and variations

For maternal parameters: 05-4287

These parameters were analyzed using non-parametric methods. If the dose group effect in

was statistically significant, each dose group was compared with the control group using

pairwise Wilcoxon rank sum tests (Wilcoxon 1945).

For fetal fmdings:

Only fmdings having an incidence of greater than one were included in the statistical

analysis, since for less frequent parameters there was no chance of statistical significance.

These parameters were analyzed by generalized estimating equations (Proc Genmod, SAS

Institute 1999) using a log link function and assuming Poisson distributed data. The litter

was the basis for analysis where the correlation among littermates was modelled using a

compound symmetric structure. Results are presented using the model based standard errors.

If the dose group effect in the model was statistically significant, each dose group was

compared with the control group using pairwise tests associated with least squares means, in

this case being the mean incidence adjusted for litter size. For very low frequency fmdings

this method did not converge. Therefore non-parametric methods, the Kruskal-Wallis test

(Kruskal and Wallis 1952, 1953) were applied to the total number of affected foetuses in the

litter instead. If this was significant at the 5% level then each treatment group was compared

with the control using Wilcoxon rank sum tests (Wilcoxon 1945).

DATA HANDLING



### Data were received as text files and Excel spreadsheets and re-formatted for software input.

The software used for all the analyses was SAS 8.2 (SAS Institute 1999). For the analysis of

uterine weight, adjusted bodyweight and the reanalysis of fetal fmdings conducted in this

updated report, SAS 9.1.3 (SAS Institute 2002) and Quasar 1.1 (Quasar 1.1 2009) were used for

the statistical analyses.

RESULTS



### The data for animals 1915,2919,4908 and 4919 were excluded, where necessary, since the

animals did not become pregnant. For maternal net bodyweight change from d5 minus

gravid uterine weight, animals 1912 and 4924 were also excluded.



### : 5:

Statistical analysis

### 05-4287

For food consumption, the animal2908 value for the d5-d8 period was an extreme statistical

outlier, being about 11 standard deviations higher than the mean. This value was excluded

from the analysis.

Continuous maternal parameters

Summary statistics are presented in Tables 1 to 4 respectively. Bartlett's test was not

significant for any parameter at any timepoint. The results can be summarised as follows:

Bodyweight

No significant differences at any timepoint.

Bodyweight change

d5-d8 significant difference at 20000 mg/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.

Uterus weight

Absolute: no significant differences.

Adjusted: no significant differences.



### :6:

Statistical analysis

### 05-4287



### Reproductive performance

Summary statistics are presented in Table 5. Bartlett's test was significant for several

parameters. The subsequent analysis was thus either parametric (P) or non-parametric (NP).

The methods and results can be summarised as follows:

Corpora lutea

%Preimplantation loss

Number of implant sites

Fetuses alive

Early resorptions

Late resorptions

Total resorptions

%Total resorptions/implant sites

Number of males

Number of females



### Fetal bodyweight p



### NP

p



### p

NP

p



### NP

NP

p

p 

### No significant differences.

No significant differences.

No significant differences.

No significant differences.

No significant differences.

No significant differences.

No significant differences.

No significant differences.

No significant differences.

No significant differences.

Summary statistics, for means adjusted for litter size, are presented in Table 6.

For the pooled sex (litter) analysis there was a strong sex difference and evidence for a

difference between treatment groups (p=0.0487). The subsequent Dunnett's test was

significant at the high dose (p=O.Ol6).

For males, there was a tendency for reduced mean bodyweight at higher doses, but the overall

comparison did not attain statistical significance.

For females, there was a significant difference between groups (p=0.016). The subsequent

Dunnett's test was significant at the high dose (p=0.007, a reduction from control).

There was a strong effect of litter size in all three analyses.

These results can be summarised as follows:

No significant differences. Male fetal body weight

Female fetal body weight

Litter body weight Significant difference at 20000 mg/m 3 

### (p=0.007).

Significant difference at 20000 mg/m 3 

### (p=0.016).



### :7:

Statistical analysis

### 05-4287



### Discrete findings

For the Maternal pregnancy summary, only one parameter, the number of non-pregnant

females, required analysis. This is summarised in Table 7.

The incidence of fetal fmdings, for those fmdings with a total incidence of greater than one is

summarised in Table 8. Only five Visceral and seven Skeletal fmdings, all of them

variations, required statistical analysis. The analysis was either generalised linear modelling

(GL) or non-parametric (NP).

The results can be summarised as follows:

Maternal parameters

Non-pregnant females NP No significant differences

Visceral fmdings

Heart, V Innominate artery absent

Torso, V Left umbilical artery NP No significant differences

GL No significant differences

Torso, V Renal papilla (E) not fully developed

Torso, V Undescended thymus NP No significant differences

GL No significant differences

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

Skeletal fmdings

Cervical vertebrae, V 7th cervical rib(s) present NP

Ribs, V Rib( s) 13th Rudimentary NP

Ribs, V Rib(s) 14 1 

### h Rudimentary GL

Sacral vertebrae, V 25 Presacral vertebra( e) NP

Skull, V Hyoid body/arch(es) unossified NP

Sternebrae, V Sternebra( e) 5 and/or 6 unossified. NP

Sternebrae, V Sternebra( e) malaligned GL No significant differences

No significant differences

No significant differencest

No significant differences

No significant differences

No significant differences

Significant difference at

20000 mg/m 3 

### (p=0.047).

t There was some evidence of a statistically significant difference at 20000 mg/m 3 

### (p=0.067).



### : 8:

Statistical analysis

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## REFERENCES

ARMITAGE, P., BERRY, G. and MATTHEWS, J.N.S. (2002) 

### Statistical Methods in Medical

Research, 4th 

### edition. Blackwell Science, Oxford, UK, pp208-215.

BARTLETT, M.S. (1937) Properties of sufficiency and statistical tests. 

### Proceedings of the

Royal Society, Series A, 160: 268-282.

DUNNETT, C.W. (1955) A multiple comparison procedure for comparing several

treatments with a control. 

### Journal of the American Statistical Association 50: 1096-1121.

DUNNETT, C.W. (1964) New tables for multiple comparisons with a control. 

### Biometrics

20: 482-491.

KRUSKAL, W.H. and WALLIS, W.A. (1952) Use ofRanks in one-criterion variance

analysis. 

### Journal of the American Statistical Association 47: 583-621.

KRUSKAL, W.H. and WALLIS, W.A. (1953) Errata for Kruskal-Wallis (1952) 

### Journal of

the American Statistical Association 48: 907-911.

QUASAR 1.1 User Guide (Stand-Alone) version 1.1 (2009). Huntingdon Life Sciences

Internal Document.

SAS INSTITUTE (1999) SAS OnlineDoc® 

### Version Eight. SAS Institute Inc., Cary, NC,

USA

SAS INSTITUTE (2002) SAS OnlineDoc® 

### Version Nine. SAS Institute Inc., Cary, NC, USA.

STEEL, R.G.D. (1959) A multiple comparison rank sum test: treatment versus control.



### Biometrics 15, 560-572.

WILCOXON, F. (1945) Individual comparisons by ranking methods. 

### Biometrics Bulletin,

1, 80-83.



### :9:

Statistical analysis

### TABLEt

Maternal body 

### weight (g)

Omg/m 3 2000 mg/m 3

(n=23) (n=23)

Day of mean (sd) mean (sd)

gestation

4 233.8 (14.1) 234.7 (16.6)

5 244.0 (14.6) 245.3 (16.6)

8 259.3 (12.8) 260.4 (18.1)

11 281.5 (14.9) 279.0 (19.7)

14 301.5 (17.1) 295.5 (20.2)

17 332.1 (22.3) 326.7 (24.2)

20 378.1 (26.3) 370.3 (29.9)

21 387.8 (28.5) 379.4 (32.7)

Analysis performed= Parametric throughout.

No significant differences found for any parameter.

n = number oflitters, sd = standard deviation



### :10: 10000 mg/m 3

(n=24)

mean (sd)

233.9 (15.7)

243.9 (13.9)

256.6 (14.4)

274.4 (15.6)

292.0 (14.8)

322.8 (18.7)

366.8 (24.1)

375.7 (26.0) 

### 05-4287

20000mglm'

(n=22)

mean (sd)

235.4 (14.7)

245.0 (15.3)

255.6 (16.2)

275.4 (19.6)

290.2 (19.6)

321.1 (24.9)

365.5 (27.1)

373.6 (27.4)

Statistical analysis

### TABLE2

Maternal body weight change 

### (g)

Days of Omg/m, 2000 mg!m'

gestation (n=23) (n=23)

Period mean (sd) mean (sd)

4-5 10.3 (6.1) 10.7 (4.7)

5-8 15.5 (4.4) 15.0 (4.8)

8-11 22.2 (4.7) 18.6 (3.7) 

## *

11-14 20.0 (4.2) 16.8 (5.1) 

## *

14-17 30.7 (7.3) 31.2 (6.9)

17-20 46.2 (7.5) 43.5 (8.9)

5-14 57.6 (8.4) 50.2 (7.9) 

## *

14-21 86.4 (15.3) 84.0 (16.2)

5-21 143.9 (20.6) 134.2 (21.5)

Analysis performed= Parametric throughout. 

## *

= p<0.05, 

## **

= p<0.01

n = number of litters, sd = standard deviation 10000 mg/m'

(n=24)

mean (sd)

10.2 (5.3)

12.5 (5.4)

17.9 (4.0) 

## **

17.5 (3.8)

31.0 (7.5)

44.1 (9.8)

47.8 (7.6) 

## **

83.8 (17.0)

131.8 (20.9) 

### 05-4287

20000mg/mj

(n=22)

mean (sd)

9.6 (3.2)

10.6 (4.7) 

## **

19.7 (5.6)

15.0 (4.0) 

## **

31.0 (6.7)

44.5 (7.7)

45.2 (9.4) 

## **

83.5 (10.1)

128.6 (16.0)



### Maternal net bodyweight change from d5 minus gravid uterine weight 

### (g)

Omg!m' 2000 mg!m'

(n=22) (n=23)

Parameter mean(sd) mean (sd)

Absolute 42.95 (11.08) 37.39 (9.16)

Analysis performed= Parametric throughout.

n = number oflitters, sd = standard deviation 

## ***

= p<0.001



### :11: 10000 mg/m' 20000 mg/mj

(n=24) (n=20)

mean (sd) mean (sd)

35.71 (11.75) 29.35 (10.65) 

## ***

Statistical analysis

### TABLE3

Maternal feed consumption (glkg/day)

Days of Omg/mj 2000 mg/mj

gestation (n=22-23) (n=22-23)

Period mean (sd) mean (sd)

4-5 71.2 (10.5) 74.2 (11.5)

5-8 80.8 (5.9) 82.7 (5.5)

8-11 85.1 (7.1) 81.8 (4.6)

11-14 85.0 (7.1) 81.6 (4.6)

14-17 80.9 (4.5) 80.8 (4.5)

17-20 74.5 (4.5) 73.4 (3.3)

20-21 53.3 (8.1) 46.8 (7.6) 

## *

5-21 76.2 (4.8) 75.0 (4.1)

Analysis performed= Parametric throughout. 

## *

= p<0.05, 

## **

= p<0.01

n = number of litters, sd

= standard deviation

Some variation in group size due to missing values.



### :12: 10000 mg/mj

(n=22-24)

mean (sd)

75.0 (11.4)

79.7 (5.9)

80.3 (5.5) 

## *

82.4 (3.9)

80.6 (6.0)

73.6 (3.8)

47.0 (5.8) 

## *

73.6 (3.5) 

### 05-4287

20000 mg/m 3

(n=21-22)

mean (sd)

73.1 (10.3)

78.8 (5.9)

77.4 (5.7) 

## **

77.8 (3.9) 

## **

79.2 (4.4)

73.1 (4.3)

45.9 (7.8) 

## **

71.5 (3.9) 

## **

Statistical analysis

### TABLE4

Maternal uterus weights 

### (g)

Omg/mj 2000 mg/mj

(n=22) (n=23)

Parameter mean (sd) mean (sd)

Absolute 101.3 (19.3) 96.5 (19.9)

Adjusted 286.0 (18.1) 282.7 (19.0)

Analysis performed= Parametric throughout.

No significant differences found for any parameter.

n = number of litters, sd = standard deviation



### :13: 10000 mg/mj

(n=24)

mean (sd)

96.1 (22.5)

279.5 (17.2) 

### 05-4287

20000mg/mj

(n=20)

mean (sd)

96.4 (15.6)

275.3 (18.4)

Statistical analysis

### TABLES

Reproductive performance

Omg/mj 2000mg/m 5

(n=23) (n=23)

Parameter mean(sd) mean (sd)

corpora lutea ( 1) 14.9 (2.8) 14.5 (2.1)

%preimplantation loss (2) 9.3 (13.5) 6.6 (8.0)

number of implant sites (1) 13.5 (2.7) 13.5 (1.5)

fetuses alive (1) 13.0 (2.6) 12.4 (2.6)

early resorptions (2) 0.4 (0.7) 1.0 (2.1)

late resorpti ons ( 1) 0.0 (0.2) 0.0 (0.2)

total resorpti ons ( 2) 0.5 (0.7) 1.0(2.1)

%total resorp./implant sites (2) 3.4 (4.7) 7.9 (14.9)

number of males (1) 6.3 (1.7) 5.8 (2.2)

number of females (1) 6.7 (2.0) 6.7 (2.3)

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

No significant differences found for any parameter.

n = number oflitters, sd = standard deviation

Male and female bw n= 11



### :14: 05-4287

10000 mg/m 5 20000 mg!m 5

(n=24) (n=22)

mean (sd) mean(sd)

14.8 (2.5) 15.2 (2.6)

10.4 (18.6) 7.6 (9.1)

13.1 (2.8) 14.0 (2.4)

12.6 (3.1) 13.3 (2.6)

0.5 (0.8) 0.7 (0.6)

0.0 (0.0) 0.0 (0.0)

0.5 (0.8) 0.7 (0.6)

5.1 (8.6) 5.2 (5.2)

6.4 (2.3) 6.1 (2.1)

6.2 (2.3) 7.1 (2.5)

Statistical analysis

### TABLE6

Mean (adjusted) fetal bodyweight (g)

Omg/mj 2000 mg/mj 10000 mg/m 3 20000 mg/m 3

(n=23) {n=23) (n=24) {n=22)

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

male 5.88 (0.06) 5.87 (0.06) 5.78 (0.06) 5.71 (0.06)

female 5.58 (0.06) 5.59 (0.06) 5.47 (0.06) 5.36 (0.06) 

## **

litter 5.73 (0.06) 5.73 (0.06) 5.62 (0.06) 5.54 (0.06) 

## *

*

= 

### p<0.05, 

## **

= p<0.01, 

## ***

= p<0.001

p values vs control using Dunnett's test following mixed model 

### ANOV A significant at 5%.

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



### :15: 05-4287

Statistical analysis

### 05-4287



### TABLE7

Maternal pregnancy summary and necropsy f"mdings

Parameter Finding 0 mg/mj 2000mg/mj 10000 mg/mj 20000 mglm

(n=24) (n=24) (n=24) (n=24)

Pregnancy Non-pregnant 

### 1 1 0 2

Only findings where the total incidence was greater than one are included.

Analysis performed= Generalized linear modelling or Non-parametric tests (see text).

No significant differences found for any parameter.

n = number of litters



### :16:

Statistical analysis

### TABLES

Fetal findings: Visceral

Tissue Organ Finding Omg/mj 2000 

### mg!m'

(n=23) (n=23)

Visceral HEART V INNOMINATE ARTERY 1 0

ABSENT

TORSO V LEFT UMBILICAL 1 4

ARTERY

TORSO V RENAL PAPILLA (E) NOT 1 0

FULLY DEVELOPED

TORSO V UNDESCENDED 10 11

THYMUS

TORSO V URETER(S) DISTENDED 1 0

Only findings where the total incidence was greater than one are included.

Analysis performed= Generalized linear modelling or Non-parametric tests (see text).

No significant differences found for any parameter.

n = number of litters



### :17: 05-4287

lOOOOmg/m 20000mg/m

(n=24) (n=22)

2 1

1 2

2 1



### 5 11

1 1

Statistical analysis

### TABLE 8 (cont'd)

Fetal findings: Skeletal

Tissue Organ Finding Omg!nl 

### 2000mglm 5

(n=23) (n=23)

Skeletal CERVICAL V 7TH CERVICAL RIB(S) 0 1

VERT PRESENT

RIBS 

# IV RIB(S) 13TH 1 0

RUDIMENTARY

RIBS V RIB(S) 14TH 2 7

RUDIMENTARY

SACRAL VERT 

# IV 25 PRESACRAL 1 0

IVERTEBRA(E)

SKULL 

## jv HYOID BODY/ARCH(ES) 1 0

iUNOSSIFIED BODY

STERNEBRAE 

## jv STERNEBRA(E) 5 1 0

lANDfOR 6 UN OSSIFIED

STERNEBRAE 

## jv STERNEBRA(E) 11 11**~LALIGNED**

Only findings where the total incidence was greater than one are included.

Analysis performed= Generalized linear modelling or Non-parametric tests (see text).

*= 

### p<0.05

n = number oflitters



### :18: 05-4287

10000mg/m 20000 mg/m 3

(n=24) (n=22)

0 3

0 3

5 12

0 3

1 1

3 3

13 3*

Statistical analysis

### Huntingdon Life Sciences

Table of Contents 05-4287

211-DIPE-DEV

Certificates of Analysis Page 1

Draft Final Report

AwendixN

Feed Certificates of Analysis

.................. _ ............................................................................. . Water Certificates of Analysis:

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

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

### Certified Papers Retrieval

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Product Desc:

Lab Number: CERTIFIED RODENT DIET MEAL

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Entered: 5/31/2005

!Assay 

# II Analysisi!Units

!PROTEIN II 2o.all%

IFAT ACID (HYDRO.) II 6.34,,%

!FIBER (CRUDE) II 3.64,,%

!ARSENIC II LESS THAN 0.2,,PPM

!cADMIUM II LESS THAN 0.05,,PPM

!cALCIUM II 0.943,,%

I LEAD II 0.17oiiPPM

!MERCURY II LESS THAN 0.025,,PPM

!PHOSPHORUS II 0.69811%

!sELENIUM 

# II 0.21411PPM

iORGANOPHOSPHATESi!PPM !iORGANOPHOSPHATES!iPPM

IDiazinon

# ~~~~~S THAN IIDisulfoton

# ~~~~~STHAN

IEthion

# ~~~~~STHAN 

# II Malathion

# ~~~~~S THAN

!Methyl Parathion

# ~~~~~STHAN 

# II Parathion

# ~~~~~S THAN

IThimet

# ~~~~~S THAN IIThiodan

# ~~~~~S THAN

ITrithion

# II~~~STHAN II II

PESTICIDES AND

I PPM

# II~~~TICIDES AND IIPPM PCB

jAidrin IILESS THAN 0.02 liAipha-BHC IILESS THAN 0.02

jseta-BHC IILESS THAN 0.02 

# !I Chlordane IILESS THAN 0.02

IDDE IILESS THAN 0.02 II DDT IILESS THAN 0.02

iDelta-BHC IILESS THAN 0.02 II Dieldrin IILESS THAN 0.02

jEndrin IILESS THAN 0.02 IIHCB IILESS THAN 0.02

!Heptachlor IILESS THAN 0.02 !!Heptachlor Epoxide IILESS THAN 0.02

!Lindane IILESS THAN 0.02 !!Methoxychlor IILESS THAN 0.02

IMirex IILESS THAN 0.02 II PCB IILESS THAN 0.15

IIIAFLATOXINS i!Aflatoxins IILESS THAN 5 PPB



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_ spc002.asp Page 1 of2

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### 12/8/2005

### Certified 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



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# JJ

12/8/2005

### Certified Papers Retrieval

Product Code: 5002M

Product Desc:

Lab Number: CERTIFIED RODENT DIET MEAL

L0517995-3

Lot Code: JUN 07 05 3C

Entered: 6/24/2005

jAssay 

# II Analysisjjunits

jPROTEIN 

# II 20.911%

jFAT ACID (HYDRO.) 

# II 5.8711%

jFIBER (CRUDE) 

# II 3.7511%

jARSENIC 

# II LESS THAN 0.2jjPPM

jcADMIUM 

# I 0.0933jjPPM

jcALCIUM 1.0111%

jLEAD 0.209jjPPM

jMERCURY LESS THAN 0.025jjPPM

jPHOSPHORUS 0.72311%

jsELENIUM 0.30911PPM

jORGANOPHOSPHATESi!PPM lioRGANOPHOSPHATESi!PPM

IDiazinon

# II6~~STHAN IIDisulfoton

# II6~~STHAN

IEthion

# II6~~STHAN II Malathion

# II6~~STHAN

!Methyl Parathion

# II6~~STHAN II Parathion

# II6~~STHAN

IThimet

# II6~~STHAN IIThiodan

# II6~~STHAN

ITrithion

# II6~~STHAN II II

lliESTICIDES AND

PCB II PPM

# II~~~TICIDES AND IIPPM

jAidrin IILESS THAN 0.02 i!Aipha-BHC IILESS THAN 0.02

jseta-BHC IILESS THAN 0.02 II Chlordane IILESS THAN 0.02

jDDE IILESS THAN 0.02 II DDT IILESS THAN 0.02

jDelta-BHC IILESS THAN 0.02 II Dieldrin IILESS THAN 0.02

jEndrin IILESS THAN 0.02 IIHCB IILESS THAN 0.02

!Heptachlor IILESS THAN 0.02 i!Heptachlor Epoxide IILESS THAN 0.02

jlindane IILESS THAN 0.02 

# II Methoxychlor IILESS THAN 0.02

jMirex IILESS THAN 0.02 

# II PCB IILESS THAN 0.15

IIIAFLA TOXINS i!Aflatoxins IILESS THAN 5 PPB



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### 12/8/2005

### LAB 10# 

### 3!;)-401 

## BENCHMARK ANAL YTICS

4777 SAUCON CREEK ROAD

CENTER VALLEY, PA 18034-9004

PHONE (610) 974-8100

FAX (610) 974-8104

SEND DATA TO: INV #: 383492-383494

NAME: Terry Kusznir 383496-383498

COMPANY:

ADDRESS:

PHONE:



### FAX: Huntingdon Life Sciences

PO Box 2360, Mettlers Road

East Millstone, NJ 08875-2360

(732) 873-2550 x2100

(732) 873-3992 TEST REPORT PAGE: 1 OF6

PO#:

PWSID#:

. .**=====::::;=========-==.::===========::::======.===·===================~====================================~======** PROJECT NAME: ANIMAL DRINKING WATER GRAB SAMPLES: 383492-383494

SAMPLED BY: CARAKEIPER 383496-383498

LOCATION: SEE BELOW COMPOSITE SAMPLES: NONE

DATE: 07/13/05 DATE RECEIVED BY LAB: 07114/05

TIME: SEE BELOW TIME RECEIVED BY LAB: 0819

RECEIVED FOR LAB BY: 

### R SMOLENAK

SAMPLE: SITE #1, F-WING ROOM 546 TIME: 1608

TEST RESULT UNITS METHOD**p~c;:UDOMONAS** *

. 

### _,;**~~~~~(** <1 PER 100 ML SM#19 9215 D 383492 07114/05 1505 07/15/05

STANDARD PLATE <1 CFUIML SM#20 9215 B 383494 07/14/05 1105 07/16/05 JD

COUNT

TOTAL COLIFORM 0 PER 100 ML EPA 600-R-00-013 383493 07/14/05 1308 07/15/05 MJG



### E. COLI 0 PER 100 ML EPA 600-R-00-013 383493 07/14/05 1308 07/15/05 MJG

SAMPLE: SITE #2, V-1 ROOM 725 

# I 

# I

TEST RESULT UNITS METHOD

PSEUDOMONAS * <1 PER 100 ML SM#19 9215 D 07/14/05 1505 07/15/05

STANDARD PLATE <1 CFUIML SM#20 9215 B 383498 07/14/05 1105 07/16/05 JD

COUNT

TOTAL COLIFORM 0 PER 100 ML EPA 600-R-00-013 383497 07114/05 1308 07115/05 MJG

E. COLI 0 PER 100 ML EPA 600-R-00-013 383497 07/14/05 1308 07/15/05 MJG

REMARKS:

fl--It=: ABOVE TEST RESULTS MEET ALL THE REQUIREMENTS OF NELAC.



### )s PARAMETER IS NOT CURRENTLY UNDER THE NELAC CERTIFICATION PROGRAM.

MANAGER:

___

# _,~=->-~~pc..<....:;...:;..:::.__;_;==·**'----l,a,_£-.,..£.,~..::..=:.=------** DATE: 

# 94**d~** )'nt--

### LAB 10# 

### :}9-401 

## BENCHMARK ANAL YTICS

4777 SAUCON CREEK ROAD

CENTER VALLEY, PA 18034-9004

PHONE (610) 974-8100

FAX (610) 974-8104

SEND DATA TO:

NAME:

COMPANY:

ADDRESS:

PHONE:

FAX: Terry Kusznir

Huntingdon Life Sciences

PO Box 2360, Mettlers Road

East Millstone, NJ 08875-2360

(732) 873-2550 X 2100

(732) 873-3992 TEST REPORT INV#: 383495

383499,503

PAGE: 20F6

PO#:

PWSID#:



# ==============================================================================================

PROJECT NAME: ANIMAL DRINKING WATER

SAMPLED BY: CARA KEIPER

LOCATION: SEE BELOW

DATE: 07/13/05

TIME: SEE BELOW

SAMPLE: SITE #1, F-WING ROOM 546

TEST

COPPER

·'

# ~=~D**~** !\./>!' (

1**'r'\~** 

### "

SAMPLE:

TEST

COPPER

LEAD

SAMPLE:

TEST

COPPER

LEAD

REMARKS: RESULT

0.0660

0.0017 UNITS

MG/L

MG/L

SITE #2, V-1 ROOM 725

RESULT

0.0177

<0.0005 UNITS

MG/L

MG/L

SITE #3, V-2 ROOM 910

RESULT

0.0548

0.0007 UNITS

MG/L

MG/L METHOD

EPA200.8

EPA200.8

METHOD

EPA200.8

EPA200.8

METHOD

EPA200.8

EPA200.8 GRAB SAMPLES:

COMPOSITE SAMPLES:

DATE RECEIVED BY LAB:

TIME RECEIVED BY LAB:

INV# 383495

383499,503

NONE

07/14/05

0819

R. SMOLENAK

383495 07/15/05 0735 07/15/05 KP

383495 07/15/05 0735 07/15/05 KP

TIME:

INV#

383499 07115/05 07115/05 KP

383499 07/15/05 0735 07/15/05 KP

INV#

383503 07/15/05 0735 07/15/05

383503 07/15/05 0735 07/15/05 KP



# REVIEWED

TYE ABOVE TEST RESULTS MEET ALL THE REQUIREMENTS OF NELAC ANDRE LATE ONLY TO THESE SAMPLES.

# MANAGER:---.,='~'-"'=='=-=--~==.:..::=-=-=-·**----'==G.~· .c...k¥v-=-=~-=-----**  DATE:

### LAB ID# 39-401

SEND DATA TO:

NAME:

COMPANY:

ADDRESS:

PHONE:

FAX: 

## BENCHMARK ANAL YTICS

4777 SAUCON CREEK ROAD

CENTER VALLEY, PA 18034-9004

PHONE (610) 974-8100

FAX (610) 974-8104

INV #: 383500-383502

Terry Kusznir

Huntingdon Life Sciences

PO Box 2360, Mettlers Road

East Millstone, NJ 08875-2360

(732) 873-2550 x21 00

(732) 873-3992 TEST REPORT 383504-383506

PAGE: 30F6

PO#:

PWSID#:



# =====================================================================================================

PROJECT NAME:

SAMPLED BY:

LOCATION:

DATE:

TIME:

SAMPLE:

TEST**P~EUDOMONAS** *

·,

STANDARD PLATE

COUNT

TOTAL COLIFORM

E. COLI

SAMPLE:

TEST

PSEUDOMONAS *

STANDARD PLATE

COUNT

TOTAL COLIFORM

E. COLI

REMARKS: ANIMAL DRINKING WATER

CARAKEIPER

SEE BELOW

07/13/05

SEE BELOW

SITE #3, V-2 ROOM 910

RESULT UNITS METHOD

<1 PER 100 ML SM#19 9215 D

9 CFUIML SM#20 9215 B GRAB SAMPLES: 383500-383502

383504-383506

COMPOSITE SAMPLES: NONE

DATE RECEIVED BY LAB: 07/14/05

TIME RECEIVED BY LAB: 0819

RECEIVED FOR LAB BY: R. SMOLENAK

383500 07114/05 1505 07/15/05

383502 07/14/05 11 05 07116/05 JD



### 0

0 PER 100 ML EPA 600-R-00-013 383501 07/14/05 1308 07115/05 MJG

PER 100 ML EPA 600-R-00-013 383501 07/14/05 1308 07115/05 MJG

SITE 

### #4, V-3 ROOM 950

RESULT UNITS METHOD

<1 PER 100 ML SM#19 9215 D 383504 07114/05 1505 07/15/05

4 CFU/ML SM#20 9215 B 383506 07/14/05 1105 07/16/05 JD

0 PER 100 ML EPA 600-R-00-013 383505 07114/05 1308 07/15/05 MJG

0 PER 100 ML EPA 600-R-00-013 383505 07/14/05 1308 07/15/05 MJG

THE ABOVE TEST RESULTS MEET ALL THE REQUIREMENTS OF NELAC. 

# REVIEWED

!IS PARAMETER IS NOT CURRENTLY UNDER THE NELAC CERTIFICATION PROGRAM.

DATE:

# ~ 

# aC:**~**

### LAB ID# 39-401

SEND DATA TO:

NAME:

COMPANY:

ADDRESS:

PHONE:

FAX: 

## BENCHMARK ANAL YTICS

4777 SAUCON CREEK ROAD

CENTER VALLEY, PA 18034-9004

PHONE (610) 974-8100

FAX (610) 974-8104

Terry Kusznir INV #: 383507-383511

Huntingdon Life Sciences

PO Box 2360, Mettlers Road

East Millstone, NJ 08875-2360

{732) 873-2550 X 

### 2100

(732) 873-3992 TEST REPORT 

### 383515

PAGE: 40F6

PO#:

PWSID#:



# ==============================================================================================

)AMPLE:

"EST**~OPPER** 

### .EAD

tEMARKS: L-WING ROOM 458

RESULT

0.0166

<0.0005 UNITS

MG/L

MG/L METHOD

EPA200.7

EPA200.9 INV#

383515 07/15/05 0735 07/15/05 KP

383515 07/15/05 0735 07/15/05 KP

HE ABOVE TEST RESULTS MEET ALL THE REQUIREMENTS OF NELAC AND RELATE ONLY TO THESE SAMPLES.



# REVIEWED

ANAGER:

___

# ___,....~~~-=~-=·**=~=·· _{l-=..;,..c......;k±~-'--·**  __ DATE:

# --~"21=--...-...,...--=d~S.~r~;;_t/}r_

### LAB ID# 39-401

SEND DATA TO:

NAME:

COMPANY:

ADDRESS:

PHONE:

FAX: 

## BENCHMARK ANAL YTICS

4777 SAUCON CREEK ROAD

CENTER VALLEY, PA 18034-9004

PHONE (610) 974-8100

FAX (610) 974-8104

INV #: 383508-383510

Terry Kusznir

Huntingdon Life Sciences

PO Box 2360, Mettlers Road

East Millstone, NJ 08875-2360

(732} 873-2550 x2100

(732) 873-3992 TEST REPORT 383512-383514

PAGE: 50F6

PO#:

PWSID#:**==================================================================~==~==============================:** PROJECT NAME: ANIMAL DRINKING WATER GRAB SAMPLES: 383508-383510

SAMPLED BY: CARAKEIPER 383512-383514

LOCATION: SEE BELOW COMPOSITE SAMPLES: NONE

DATE: 07113/05 DATE RECEIVED BY LAB: 07114/05

TIME: SEE BELOW TIME RECEIVED BY LAB: 0819

RECEIVED FOR LAB BY: C. PASSOW

SAMPLE: SITE #5, INHALATION ROOM 812

TEST RESULT UNITS METHOD

PSEUDOMONAS * <1 PER 100 ML SM#19 9215 D 383508 07114/05 1505 07/15/05 JD

i

b I ANDARD PLATE <1 CFU/ML SM#20 9215 B 383510 07/14/05 1105 07116/05 JD

COUNT

TOTAL COLIFORM 0 PER 100 ML EPA 600-R-00-013 383509 07/14/05 1308 07/15/05 MJG

E. COLI 0 PER 100 ML EPA 600-R-00-013 383509 07/14/05 1308 07/15/05 MJG

SAMPLE: SITE 

### #6, L-WING ROOM 458

TEST RESULT UNITS METHOD

PSEUDOMONAS * <1 PER 100 ML SM#19 9215 D .07/14/05 1505 07/15/05

STANDARD PLATE <1 CFU/ML SM#20 9215 B 383514 07/14/05 1105 07/16/05 JD

COUNT

TOTAL COLIFORM 0 PER 100 ML EPA 600-R-00-013 383513 07/14/05 1308 07/15/05 MJG

E. COLI 0 PER 100 ML EPA 600-R-00-013 383513 07/14/05 1308 07/15/05 MJG

REMARKS:

THE ABOVE TEST RESULTS MEET ALL THE REQUIREMENTS OF NELAC AND RELATE ONLY TO THESE SAMPLES

!IS PARAMETER IS NOT CURRENTLY UNDER THE NELAC CERTIFICATION PROGRAM. 

# REVIEWED

MANAGER:

# ----r,~'""""""-=->o.<Jl~i""'==. -=>-===-

__

# 0~~'---------- DATE:

# ~-9---P-"'~~J-«=....<..S:~M-;..;,__

### LAB 10# 

### 39-401

SEND DATA TO:

NAME:

COMPANY:

ADDRESS:

PHONE:

FAX: 

## BENCHMARK ANAL YTICS

4777 SAUCON CREEK ROAD

CENTER VALLEY, PA 18034-9004

PHONE (610) 974-8100

FAX (610) 974-8104

Terry Kusznir

Huntingdon Life Sciences

PO Box 2360, Mettlers Road

East Millstone, NJ 08875-2360

(732) 873-2550 x2100

(732) 873-3992 TEST REPORT INV #: 383516-383518

PAGE: 60F6

PO#:

PWSID#:



# =====================================================================================================

PROJECT NAME: ANIMAL DRINKING WATER GRAB SAMPLES: 383516-383518

SAMPLED BY: CARAKEIPER COMPOSITE SAMPLES: NONE

LOCATION: SEE BELOW DATE RECEIVED BY LAB: 07/14/05

DATE: 07/13/05 TIME RECEIVED BY LAB: 0819

TIME: SEE BELOW RECEIVED FOR LAB BY: C. PASSOW

SAMPLE: SITE #7 PHARMACY 1

TEST RESULT UNITS METHOD

PSEUDOMONAS * <1 PER 100 ML SM#19 9215 B 383516 07/14/05 1505 07/15/05 JD

·,\

I



# s I ANDARD PLATE 1,140 CFU/ML SM#20 9215 8 383518 07114/05 1105 07/16/05 JD

COUNT

TOTAL COLIFORM 0 PER 100 ML EPA 600-R-00-013 383517 07/14/05 1308 07/15/05 MJG

E. COLI 0 PER 100 ML EPA 600-R-00-013 383517 07/14/05 1308 07/15/05 MJG

REMARKS:**TH~)** ABOVE TEST RESULTS MEET ALL THE REQUIREMENTS OF NELAC AND RELATE ONLY TO HTESE**SA~LES**



# · EVI£Vvco

MANAGER:

___ 

### ,.._,xf

.....

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

## Ott_Lf dS", 

## J?tr: 7(/ 

## l...b 

## ¥

,

### LAB ID# 39-401

SEND DATA TO:

NAME:

COMPANY:

ADDRESS:

PHONE:



### FAX: 

## BENCHMARK ANAL YTICS

4777 SAUCON CREEK ROAD

CENTER VALLEY, PA 18034-9004

PHONE (610) 974-8100

FAX (610) 974-8104

Terry Kusznir

Huntingdon Life Sciences

PO Box 2360, Mettlers Road

East Millstone, NJ 08875-2360

(732) 873-2550 x2100

(732) 873-3992 TEST REPORT INV #: 387033

PAGE: 1 OF 1

PO#:

PWSID#:**==:===============-=========================-:-======================~:;::=========================·=============** PROJECT NAME:

SAMPLED BY:

LOCATION:

DATE:

TIME:

SAMPLE:

TEST

STANDARD PLATE 

## cOuNT'· '·

· ·**~EMARKS:** ANIMAL DRINKING WATER

TERRY KUSZNIR

SEE BELOW

08/04/05

1455 GRAB SAMPLES: NONE.

COMPOSITE SAMPLES: NONE

DATE RECEIVED BY LAB: 08/05/05

TIME RECEIVED BY LAB: 0838

RECEIVED FOR LAB BY: C. PASSOW

"HE ABOVE TEST RESULTS MEET ALL THE REQUIREMENTS OF NELAC AND RELATE ONLY TO THESE SAMPLES.

. ,··

.. ·· '

. . . . . . . .

DATE:

### Elizabethtown Water Company

Physical & 

### Chemical Analyses

General Source Raritan-Millstone Plant

Sample No.1 Plant Delivered Water-8:10a.m. 8-9-05 MT

Sample No.2

Sample No.3

Sample No.4

Sample No.5

Sample No.6

Sample No.7

Sample No.8

Parameter MCL (mgll) 1.0 2.0 3.0 4.0

Temperature • F



# - 81

Turbidit}'_iNTUl 0.3 NTU 0.13

Color 10* 1.0

Threshold I Odor 40 • 

### c 3TON* 2.6/3cc

Threshold Taste 3 TTN* 3.0

Conductivity (micromhos I em)



# - 308

Hardness, Total (as mg /I CaC03) 250 mg II* 94

Alkalinity



# - 36

IPH 6.5-8.5 * 6.7

Chlorine, Free I Total (mg /I Cl)



## - 0.67/.74

Calcium (as mg /I CaC03)



# - 66

Magnesium (as mg /I CaC03)



# - 28

. Iron,**Tot~l** (mg II Fe) 0."3 ing1 I* NO

Sulfates (mg 1 I S04) 250 mg11* 46.3

Chlorides (mg 1 I Cl) 250 mg11 * 31.0

Fluoride (mg 1 I F) 2 mgfl* <0.1

Total Dissolved Solids (mg 1 I) 500 mg II* 204

Total Suspended Solids (mg 1 I)



# - 0.0

Ammonia Nitrogen (mg /IN)



# - <0.05

Nitrate Nitrogen (mg 1 I N) 10 mgfl 0.65

Dissolved Oxygen (mg 1 I 02)



# - 7.5

BOD 5 (mg 1 I 02)



## - -

Langelier Index +1-1.0 -2.10

Surfactants (mg 1 I LAS) 0.5 mgll* <0.05

1-fydrog_en Sulfide (m_g_( I H2S as S)



# - 0

Nitrite Nitrogen (mg 1 I N) 1 mgfl <.01

Phosphate (mg 1 I P03)



# - <.05

Manganese (mg /I MN) 0.05 mg 1 I* NO

. Zinc 5 mgfl* 0.54

T.O.C. (mg /I) 5mgfl* 1.58

* Secondary or Recommended MCL

Anv.Questions please call Water Quality Specialist- (732) 302-3192 Date August9,2005

5.0 6.0 7.0 8.0

·

...

Laboratory Analyst DSD



### I



# REVIEWED/**\l~~J**

# !i Sterigenics.

Facility:

Customer Name:

Customer PO #:



## Run 

## Date:

Run Number:

Total Pallets: 

## Westerville

The Anderson Inc.

05/09/05

ANDOl AND 6050006

21



## MATERIALS PROCESSED:

Qty Product

1,680 

## BED'O COB 1/4" BAG·

l 

## SAMPLE-506

1,681 Bags Total

Minimum Dose:

Maximum Dose: sterigenics

.. 305 Enterprise Drive 

## * Westerville, Ohio 43081 " USA *

614-8884077

.. FAX 614-888-4223 

## * www.sterigenics.com



## Description

28.4 kGy

52.1 kGy 

### Certificate of Processing**C~~::** 

## Production A

Date Format; MM/DD/YY

CUstomer 

### Ref:



## Lot Number LOT 

## #

. .



## DC295

DC295 425RB

425RB



## Sterigenics certifies that the materials listed above (as described by 

## its

Manufacturer} received the indicated doses**within~** 

## th precision and accuracy**~** 

## the dosimetry system employed,

.**~}, (/,I~** 



## certified By:

# fl/~

Date: 05/10/05

ISO 

### 9001:2000 REGISTERED

l?age 1 

### of l



### Original

## ·--""' 

## NAMSA

PEOPLE > SCIENCE > SOLliTIONS



### Confidential

MS005_00P

Ted Weaver

Andersons

P.O. Box 119

Maumee. OH 43537

Test Article:

IDNo.

'?rocedure/Test Method:



### Test Article R.eceived:



### Test Start 

### Date:

Test 

### Termination Date:

·

Number of Products Tested:

Articles Tested

Product 

### Section

Product Section STERILITY TEST 

### Lab No.

P.O. No.



### Test Facility:

"42SURB

..**~Bed** 

### 0' Cobs; Lot 

### #DC295

Lot: DC295



### S-20431-0 1-00/Immersion

May 19,2005

May20,2005

June 

### 3,2005

1

STERILITY TEST RESULTS



### Te:n Article Identity 

### Maintained as Submitted by 

### Client

Number of Type Incubation

Articles of Media 

### Temperature

Tested (Degrees 

### C)

I 

### SCDB400ml 20-25



### 1 FTM-400ml 

### 30-35 05T_38013_0I



### TED WEAVER

NAMSA



### 6750 Wales 

### Road

Northwood, OH 43619

Number of 

### Number of



### Days Positive

Incubated Articles



### 14 0

14 

### 0

SCDB

= 

### Soybean 

### Casein Digest Broth 

### FTM

= 

### Fluid Thioglycollate Medium



### Results and conclusions apply oruy to 

### the test 

### article tested. No 

### further evaluation of these 

### results is 

### made by

NAMSA 

### Any extrapolation of these data to 

### other samples 

### is the responsibility of the sponsor. All procedures were

conducted 

### in conformance with good laboratory practice and ISO 17025.

Record Storage: All raw data pertaining 

### to this study and a copy of the final report are to be retained in designated

NAMSA archive files for a period of 

### S years.

cal Date 

### Completed

## ~..-3 -t}s-- Approved By

Michelle R... Pierce

Manager. Microbiology

Authorization for dupliC"-31oon of tl1i:; report, except in whole. is reso.-vcd pending NAMSA's written**approv<~l.** 

### Page 1 ofl

## PROTOCOL

GASOLINE DIPE VAPOR CONDENSATE

EMBRYO-FETAL TOXICITY STUDY IN RATS BY INHALATION EXPOSURE

HLS Study No.:



## Protocol Version:

Date: 05-4287



## Final Protocol

3 

## August 2005

### Huntingdon Life Sciences

Study No. 05-4287 Final Protocol**PROTOCOLSIGNATURES~REFACE**



### Study Title: Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study



## in 

## Rats by Inhalation Exposure

HLS Study No.: 05-4287

Sponsor Study No.: 211-DIPE-DEV

This is the Final Protocol. It 

### has been reviewed and approved by:

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

Sponsor Representative

American Petroleum Institute (API)



### Address: 1220 L 

### Street, Northwest

Washington, D.C. 20005-4070

Phone No.: 202-682-8480

Fax No.: 202-682-8270

Email: grayt@api.com

Ship Unused Test Substance and

Empty Test Substance Containers to: Name:

Supplier:

Address:

Phone No.:

Fax No.:

Email: Michael C. Henley

ChevronTexaco Energy

Technology Company

100 Chevron Way

Richmond, CA 94802-0627

510-242-3062

510-242-5542

mche@chevron.com

## Huntingdon Life Sciences

Study No. 05-4287

.

TABLE OF CONTENTS Final Protocol



## 1. INTRODUCTION

.................................................................................................... l

2. STUDY PERSONNEL

............................................................................................. !



## 3. PROPOSED STUDY DATES

................................................................................. !



## 4. EXPERIMENTAL DESIGN

................................................................................... 3

4.1. JUSTIFICATION FOR ROUTE, DURATION AND FREQUENCY OF

ADMINISTRATION

........................................................................................................ 4

4.2. IDSTIFICATION FOR TEST ANIMAL SELECTION

........................................ .4



## 4.3. IDSTIFICATION FOR NUMBER OF ANIMALS

.............................................. .4



## 4.4. EXPOSURE LEVEL SELECTION AND IDSTIFICATION

............................... .4



## 5. TEST SUBSTANCE

................................................................................................ 4

5.1. TESTSUBSTANCE

.............................................................................................. 4

5.2. IDENTIFICATION OF TEST SUBSTANCE

........................................................ 5

5.3. ARClllVALSAMPLES

......................................................................................... 5

5.4. UNUSED TEST SUBSTANCE

............................................................................. 5



## 6. TEST ANIMALS

..................................................................................................... 5

6.1. SPEClES

................................................................................................................. 5

6.2. SUPPLlER

.............................................................................................................. 6

6.3. ANIMAL REQUIREMENTS/SPECIFICATIONS

................................................ 6

6.4. ACCLIMATION PERIOD

..................................................................................... 6

6.5. ANIMAL CARE AND HUSBANDRY

................................................................. 6

6.6. ANIMAL HUSBANDRY DURING EXPOSURE

................................................ 8

6.7. ANIMALIDENTIFICATION

................................................................................ 9

6.8. ASSIGNMENT OF ANIMALS INTO GROUPS

.................................................. 9

7. TEST SUBSTANCE ADMINISTRATION

........................................................... 9

7.1. ROUTE OF ADMINISTRATION

......................................................................... 9

7 .2. FREQUENCY OF ADMINISTRATION

............................................................... 9

7.3. DURATION OF ADMINISTRATION

.................................................................. 9

7.4. ADMINISTRATION OF TEST SUBSTANCE

..................................................... 9

7.5. EXPOSURE CONCENTRATION DETERMINATION

..................................... 10

7.6. PARTICLE SIZE DISTRIBUTION ANALYSIS

................................................. lO

7.7. CHAMBER AND EXPOSURE ROOM ENVIRONMENT

................................ 11

7.8. SUMMARY OF CHAMBER ACTIVITY

........................................................... 11

8. IN- LIFE EXPERIMENTAL OBSERVATIONS

................................................ 11

8.1. CLINICALOBSERVATIONS

............................................................................. 12

8.2. BODYWEIGIITS

................................................................................................ 12

## Huntingdon Life Sciences

Study No. 05-4287 Final Protocol

8.3. FEED CONSUMP'I'ION

...................................................................................... 12

9. TERMINAL OBSERV ATIONS

........................................................................... 13

9.1. METHOD OF EUTHANASIA

............................................................................ 13

9.2. MACROSCOPIC POSTMORTEM EXAMINATIONS

...................................... 13

9.3. FETALEVALUATIONS

..................................................................................... 14



## 10. ARCHIVING OF RECORDS AND SPECIMENS

............................................. 15

11. STATISTICAL EVALUATIONS

........................................................................ 15

11.1. CONTINUOUS DATA

..................................................................................... 15

11.2. INCIDENCE DATA

......................................................................................... 18

12. REPORTING

......................................................................................................... 18

12.1. STATUS REPORTS

......................................................................................... 18

12.2. FINALREPORT

............................................................................................... 19

12.3. ROBUST SUMMARY

..................................................................................... 20

13. REGULATORY REFERENCES

......................................................................... 20

13.1. TEST GUIDEI.JNES

......................................................................................... 20

13.2. GOOD LABORATORY PRACTICES

............................................................. 21

13.3. ANIMAL WELFARE ACT COMPLIANCE

................................................... 21

13.4. INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC)

..... 22



## 14. QUALITY ASSURANCE MONITORING

......................................................... 22

15. ALTERATION OF PROTOCOL AND STUDY DESIGN

................................ 22

## Huntingdon Life Sciences

Study No. 05-4287

1. INTRODUCTION

HLS Study No.:

Study Title:

Test Substance:

Testing Facility:

Purpose: 05-4287 

## Pagel

Final Protocol

Gasoline DIPE Vapor Condensate:

Embryo-Fetal Toxicity Study in Rats

by Inhalation Exposure

Gasoline DIPE Vapor Condensate

Huntingdon Life Sciences

100 Mettlers Road

East Millstone, NJ 08875-2360

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

toxicity of Gasoline DIPE Vapor Condensate in the pregnant rat when

administered by whole-body inhalation exposure. The study should permit

detection of gross maternal organ changes and effects on the developing

conceptus, including death, structural abnormalities or altered growth.

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

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

previous study is incomplete due to technical errors made in evaluating the

fetuses. The Sponsor needs to repeat the previous study to satisfy the

Environmental Protection Agency's Alternative Tier 2 Testing Requirements

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



## 2. STUDY PERSONNEL

Study Director

Alternate Contact Keith P. Hazelden, BSc, CBiol, MIBiol

Gary M. Hoffman, B.A., DABT

Inhalation Toxicologist

Tel.: 732-873-2550 x2920

Email: hoffmang@princeton.huntingdon.com

Additional key personnel will be documented in the project file and presented in the final

report.

## Huntingdon Life Sciences

Study No. 05-4287

3. PROPOSED STUDY DATES

Study Initiation Date

Receipt of test animals:

Initiation of exposures:

Termination of exposures:

Necropsy:

Experimental Termination:

Submission of Draft Final Report:

Study Completion Date: 

## Page2

Final Protocol

Date Study Director Signs Protocol

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

6 August 2005 (Experimental Start

Date) (First GD 5)

24 August 2005 (Last GD 20)

22-25 August 2005 (GD 21)

November 2005 (Date of last data

collection)

30 December 2005

Date Final Report signed by Study

DirectorHuntingdonLifeSciencesStudvNo.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.

## Huntingdon Life Sciences

Study No. 05-4287 Page4

Final Protocol

4.1. JUSTIFICATION FOR ROUTE, DURATION AND FREQUENCY

OF ADMINISTRATION

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

potential routes of human exposure to this test substance. The duration and

frequency of the exposures are as for the EMBSI study and as

recommended in the relevant OECD and EPA test guidelines, ensuring

adequate exposure of post-implantation embryo-fetal development.



## 4.2. JUSTIFICATION FOR TEST ANIMAL SELECTION

The rat was used for the EMBSI study and is used as a surrogate for

humans in the detection of embryo-fetal toxicity and is the rodent species

that is recommended by the relevant OECD and EPA test guidelines.

Historical control data are also available with this strain of rat for

comparative evaluation, if necessary.



## 4.3. JUSTIFICATION FOR NUMBER OF ANIMALS

The number of animals in this study is considered the minimum necessary

to allow for meaningful interpretation of the data as required by OECD

and EPA test guidelines. Some of the endpoints of principal interest (eg



## embryo-fetal death, fetal malformations) are low-frequency events and a

group size of -20 litters tends to provide the necessary degree of

consistency between studies. 

## In the expectation of pregnancy rates of 80-

100%, the group size of 24 mated female rats is considered appropriate for

this study and should provide a sufficient number of litters for evaluation

in each group. Also, the number of animals is comparable to the number

in the EMBSI study (25/group).



## 4.4. EXPOSURE LEVEL SELECTION AND JUSTIFICATION

The exposure levels were as used in the EMBSI study and as proscribed by

the Sponsor based on the lower explosive limit of the test substance (low,

intermediate and high levels were

- 5, 25, and 50% of the LEL).



## 5. TEST SUBSTANCE

5.1. TEST SUBSTANCE

Gasoline DIPE (di-isopropyl ether) Vapor Condensate



## TEST SUBSTANCE CATEGORY: gasoline product

Description, lot number, storage, expiration date (if available) and

handling procedures, as well as other pertinent information will be

## Huntingdon Life Sciences

Study No. 05-4287 PageS

Final Protocol

documented in the study data and final report. The test substance is

supplied by the Sponsor and will be stored (ambient conditions) in an

outside solvent shed except when in use in the inhalation laboratory.



## 5.2. IDENTIFICATION OF TEST SUBSTANCE

Unless otherwise noted, the identity, strength, composition, stability and

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

substance will be documented by the Sponsor before its use in the study.

This documentation will be maintained by the Sponsor at the address

indicated on the signature page of this protocol. The Sponsor has conducted

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

The Testing Facility will conduct a stability analysis of the test substance

by GC prior to the start of this study.



## 5.3. ARCIDVAL SAMPLES

An archival sample from each lot of test substance will be taken and stored

in the Archives of the Testing Facility. If 

## multiple studies are conducted

with the same substance, a common archival sample may be taken and

appropriately labeled.



## 5.4. UNUSED TEST SUBSTANCE

The unused portion of the test substance as well as any empty test

substance containers will be returned to the Sponsor's designee following

completion of the study. Empty test substance containers will be returned

to the Sponsor's designee on an as needed basis. The Sponsor will be

responsible for tracking their disposition. In 

## the event the Sponsor wishes

the Testing Facility to arrange for disposal, a cost for this service will be

provided.



## 6. TEST ANIMALS

6.1. SPECIES

Albino Rats (Outbred), Sprague Dawley- derived

[Crl: CD® IGS BR, V AF/Plus®]

## Huntingdon Life Sciences

Study No. 05-4287

6.2. SUPPLIER Page6

Final Protocol

Charles River Laboratories (Raleigh, NC): Documentation will be

maintained in the study file and included in the final report. This supplier

and facility are the same as in the EMBSI study.



## 6.3. ANIMAL REQUIREMENTS/SPECIFICATIONS

6.3.1. NUMBER

Order: 100 time-mated rats.

Place on study: 96 time-mated rats.



## 6.3.2. AGE AND WEIGHT

Time-mated female rats 70-84 days in age and weighing 210-250

grams on Gestation Day 

## 0 will be purchased. Animals will be

received on GD 0, 1, 2 or 3.



## 6.4. ACCLIMATION PERIOD

Animals will be acclimated for a minimum of 2 days from time of receipt

until initiation of exposures (GD 5).



## 6.5. ANIMAL CARE AND HUSBANDRY

6.5.1. FACILITIES MANAGEMENT/ANIMAL HUSBANDRY

Currently acceptable practices of good animal husbandry will be

followed, e.g., 

## Guide for the Care and Use of Laboratory Animals;

National Academy Press, 1996. Huntingdon Life Sciences Inc. is

fully accredited by the Association for Assessment and

Accreditation of Laboratory Animal Care International

(AAALAC).



## 6.5.2. VETERINARY CARE

Animals are monitored by the technical staff for any conditions

requiring possible veterinary care. If any such conditions are

identified, a staff veterinarian will be notified for an examination

and evaluation. Animals will be treated as outlined in the Animal

Welfare Act Compliance section of this protocol. The staff

veterinarian will approve the animals for inclusion on test.

## Huntingdon Life Sciences

Study No. 05-4287

6.5.3. ENVIRONMENTAL CONDITIONS

Light/Dark Cycle Page7

Final Protocol



## Twelve-hour light/dark cycle provided by an automatic timer.

Temperature



## Temperature will be monitored in accordance with HLS SOPs to

ensure that the desired range of 18-26°C is maintained.

Temperature will be maintained within this range to the maximum

extent possible. The actual temperature range will be included in

the final report.

Humidity



## Humidity will be monitored in accordance with HLS SOPs to

ensure that the desired range of 30 to 70% is maintained.

Humidity will be maintained within this range to the maximum

extent possible. The actual humidity range will be included in the

final report.

Air Changes



## Animal quarters will have 10-15 air changes per hour. The actual

number of air changes per hour in each animal room is recorded at

least twice each year and the Testing Facility retains these records.

6.5.4. HOUSING



## All animals will be housed individually in stainless steel suspended

cages with wire mesh floors and fronts. Cages will be arranged in

such a way that possible effects due to placement are minimized

(as per EPA guideline). Each cage will be fitted to secure a glass

feeder jar with a stainless steel lid. Clean feed jars and fresh feed

will be provided at least every 7 days for periods when feed

consumption is not being recorded and at least every 7 days when

feed consumption will be recorded.

All dams after exposure on GD20 will be transferred to solid

bottom cages with bedding material (ground corncobs).

6.5.5. FEED



## Certified Rodent Diet, No. 5002; (Meal) (PMI 

## Feeds, Inc., St. Louis,

MO) ad libitum 

## except during exposures.

## Huntingdon Life Sciences

Study No. 05-4287 PageS

Final Protocol

6.5.6. FEED ANALYSIS

Analytical certification of batches of feed provided by the

manufacturer will be maintained on file at the Testing Facility and

included in the final report. There are no known contaminants in

the feed that are expected to interfere with the objectives of this

study.



## 6.5.7. WATER

Facility water is supplied by Elizabethtown Water Company,

(Westfield, NJ) and will be provided ad libitum 

## except during

exposures to individual animal cages through an automated

watering system.



## 6.5.8. WATER ANALYSIS

Water analyses are conducted monthly by the Elizabethtown Water

Company to ensure that water meets standards specified under the

EPA Federal Safe Drinking Water Act Regulations (40 CFR part

141). Water analyses, provided by the supplier, will be maintained

on file at the Testing Facility and included in the final report. In

addition, water samples are collected biannually from

representative rooms in the Testing Facility. Chemical and

microbiological water analyses are conducted on these samples by

a subcontract laboratory. Results are maintained on file at the

Testing Facility and included in the final report. There are no

known contaminants in the water that are expected to interfere with

this study.



## 6.6. ANIMAL HUSBANDRY DURING EXPOSURE

Housing:

Feed:

Water: Individually in stainless steel wire mesh cages.

None.

None.

## Huntingdon Life Sciences

Study No. 05-4287

6.7. ANIMAL IDENTIFICATION Page9

Final Protocol

Each animal will be assigned a temporary identification number upon

receipt. Mter selection for study, each animal will be ear-tagged by the

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

assigned by the Testing Facility plus the study number will comprise the

unique animal number for each animal. If 

## the tag is lost, it will be replaced

or the animal will be tattooed for identification. Each cage will be provided

with a cage card that will be color-coded for exposure level identification

and will contain the study number and animal number.



## 6.8. ASSIGNMENT OF ANIMALS INTO GROUPS

Animals will be placed into study groups using a computerized

randomization program. This ranks Gestation Day 4 body weights into

blocks and randomly assigns each animal within each block into one of the

study groups. Animals may be replaced based on physical examinations

and/or body weight changes up to GD 5, prior to exposures. Disposition

of all animals not utilized in the study will be maintained in the study file.



## 7. TEST SUBSTANCE ADMINISTRATION

7.1. ROUTE OF ADMINISTRATION

Inhalation by whole-body exposures.



## 7.2. FREQUENCY OF ADMINISTRATION

Once daily.



## 7.3. DURATION OF ADMINISTRATION

The test substance will be administered for 6 hours/day, 7 days per week

for Gestation Days (GD) 5-20. The duration of administration was chosen

to be the same as in the EMBSI study.



## 7.4. ADMINISTRATION OF TEST SUBSTANCE

The test substance will be administered as a 

## vapor in the breathing air of

the animals. The test atmosphere will be generated by an appropriate

procedure determined during pre-study trials. The trials will be performed

(at least three 6-hour periods) to evaluate the optimal set of conditions and

equipment to generate a stable and uniform atmosphere at the target

exposure levels. The method will be described in the raw data of the study

and in the report.

## Huntingdon Life Sciences

Study No. 05-4287 Page 10

Final Protocol

The whole-body exposure chambers will each have a volume of

approximately 1000 liters. Each chamber will be operated at a minimum

flow rate of 200 liters per minute. The final airflow will be set to provide

at least one air change in 5.0 minutes (12 air changes/hour) and a T99

equilibrium time of at most 23 minutes. This chamber size and airflow rate

is considered adequate to maintain the oxygen level at least 19% and the

animal loading factor below 5%. At the end of the exposure, all animals

will remain in the chamber for a minimum of the T 99 

## equilibrium time.

During this time, the chamber will be operated at approximately the same

flow rate using clean air only.



## 7.5. EXPOSURE CONCENTRATION DETERMINATION

A nominal exposure concentration will be calculated. The flow of air

through the chamber will be monitored using appropriate calibrated

equipment. The test substance consumed during the exposure will be

divided by the total volume of air passing through the chamber (volumetric

flow rate times total exposure time) to give the nominal concentration.

During each exposure, measurements of airborne concentrations will be

performed in the animals' breathing zone at least 4 times using an

appropriate sampling procedure and IR analytical procedure. Also, one

sample per chamber per week will be analyzed by gas chromatography

(GC) to characterize at least 10 major components (comprising at least

80% by weight of the test substance) to show test substance stability and

comparison between the neat liquid test substance and the vaporized test

atmospheres.

If 

## more than the normal amount of trials is required because of test

substance generation or monitoring problems (80 technician hours), the

Sponsor will be consulted prior to additional trials (additional cost).



## 7.6. PARTICLE SIZE DISTRIBUTION ANALYSIS

During each week of exposure, particle size determinations will be

performed using a TSI Aerodynamic Particle Sizer to demonstrate the

absence of any condensation aerosol.

## Huntingdon Life Sciences

Study No. 05-4287 Page 11

Final Protocol

7.7. CHAMBER AND EXPOSURE ROOM ENVIRONMENT

Chamber temperature, humidity, airflow rate and static pressure will be

monitored continuously and recorded every 30 minutes during exposure.

Chamber temperature and relative humidity will be maintained, to the

maximum extent possible, between 20 to 24°C and 40 to 60%,

respectively. Chamber oxygen levels (maintained at least 19%) will be

measured pretest and at the beginning, middle and end of the study.

Air samples will be taken in the vapor generation area pretest and at the

beginning, middle and end of the study. Light (maintained approximately

30-40 foot-candles at 1.0 meter above the floor) and noise levels

(maintained below 85 decibels) in the exposure room will be measured

pretest and at the beginning, middle and end of the study.



## 7.8. SUMMARY OF CHAMBER ACTIVITY

The minimum frequency of chamber activity is summarized below:

Activity Frequency/chamber

Measured Test Substance Concentration

Measured Test Substance Characterization

Particle Size

Temperature

Relative Humidity

Airflow Rate

Static Pressure

Nominal Test Substance Concentration

(excluding the air control chamber)

Rotation Pattern of Exposure Cages

Loading/Unloading Verification



## 8. IN-LIFE EXPERIMENTAL OBSERVATIONS 4X/day

lX/week

lX/week

13X/day

13X/day

13X/day

13X/day

IX/ day

IX/ day

IX/ day

## Huntingdon Life Sciences

Study No. 05-4287

8.1. CLINICAL OBSERVATIONS

8.1.1. VIABILITY OBSERVATIONS (cage-side) Page 12

Final Protocol

Observations for mortality, morbidity, and signs of severe toxicity

will be made at least twice daily. Animals in extremely poor health

or in a possibly moribund condition will be identified for further

monitoring and possible euthanasia.



## 8.1.2. DETAILED PHYSICAL EXAMINATIONS

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

during each exposure. This will routinely be performed near the

middle of each exposure and may be performed more frequently if

significant signs of toxicity are noted. Pertinent behavioral

changes and all signs of toxicity, including mortality, should be

recorded. These signs should include time of onset, degree and

duration.

Out-of-Chamber: Animals on study will be examined daily from

day after receipt through to terminal euthanasia on GD 21.

Examinations will include observations of general condition, skin

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

occurrence of secretions and excretions, and autonomic activity

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

pattern). Changes in gait, posture and response to handling as well

as the presence of clonic or tonic movements, stereotypy (e.g.,

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

self-mutilation, walking backward) will be recorded as well as

evaluations of respiration. During the treatment period, these

evaluations will be performed after exposures.



## 8.2. BODY WEIGHTS

Body weights will be recorded on GD 4 (for randomization into test groups),

5, 8, 11, 14, 17, 20 and 21 (day of scheduled sacrifice). Weight gain will be

calculated for intervals as found appropriate, including (but not limited to)

GD 5-14, GD 14-21 and 5-21.



## 8.3. FEED CONSUMPTION

Feed consumption will be recorded and reported for these intervals:

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

## Huntingdon Life Sciences

Study No. 05-4287



## 9. TERMINAL OBSERVATIONS

9.1. METHOD OF EUTHANASIA Page 13

Final Protocol



## All dams will be euthanized (by noon) by an overdose of carbon dioxide

inhalation. All live fetuses will be euthanized by an intraperitoneal

overdose of sodium pentobarbital.



## 9.2. MACROSCOPIC POSTMORTEM EXAMINATIONS

A macroscopic postmortem examination will be performed on all the dams,

including those dying spontaneously or euthanized for humane reasons or in

either a moribund condition or after prematurely delivering a litter.

Macroscopic lesions or tissues with significant findings will be saved (10%

Neutral Buffered Formalin).

Females that are found dead or sacrificed prior to scheduled termination

will have implantation data (ie 

## number of fetuses and early/late embryofetal deaths) determined at necropsy. Corpora lutea will be counted for

these animals.

Females that do not have fetuses when caesarean-sectioned will have their

uterus' examined for implantation sites and resorptions, including

confirmation of apparent non-pregnant status, by means of staining with

ammonium sulphide (modified Salewsk:i test).

Dams showing signs of premature delivery will be euthanized on the day

such evidence is observed. Reproductive tracts will be examined for

implantation data (implantation sites, live/dead fetuses and early/late

embryo-fetal deaths). Fetuses obtained earlier than Gestation Day (GD) 21

will be counted, sexed externally, evaluated for external malformations,

euthanized and discarded. Corpora lutea will be counted for these animals.

Any dams initiating delivery on GD 21 will be immediately euthanized

and the fetuses processed with the other GD 21 fetuses.

Dams surviving on GD 21 will have the following examinations of their

pregnancies:

## Huntingdon Life Sciences

Study No. 05-4287

9.2.1. Reproductive System Page 14

Final Protocol

The intact uteri (ovaries attached) will be removed from the

abdominal cavity. The gravid uteri, including the cervix, will be

weighed. Corpora lutea will be counted and the number per ovary

recorded. The number and location of the following will be recorded

for each uterine hom:

• live fetuses

• dead fetuses (no significant degeneration)

• late embryo-fetal deaths (recognizable dead fetus undergoing

degeneration, regardless of size)

• early embryonic deaths (evidence of implantation but no

recognizable fetus)

Each placenta will be examined macroscopically. The maternal

carcass, uterus, ovaries and placenta will then be discarded.

9.3. FETAL EVALUATIONS

9.3.1. External Evaluations

All live fetuses will be weighed and individually identified within

cassettes. Each live and dead retained fetus will be given a

macroscopic external examination for defects including

observation of the palate.

9.3.2. Fetal Soft Tissue (Visceral) Evaluation

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

fetuses within the litter, nominally) will be placed in Modified

Davidson's fixative for preservation and decalcification. These

fetuses will be subjected to soft tissue examination by gross

dissection of the torso and a razor blade sectioning technique for

the head (derived from that of Wilson, J.S., Warkany, J. 

## (1965),

Teratology: Principles and Techniques. The University of Chicago

Press. Chicago and London. Page 271). All malformations and

variations will be recorded. Particular attention will be paid to the

reproductive tract for altered signs of development. Unusual

malformations or developmental findings will be photographed.

During the dissection process, the sex of each fetus will be

confirmed by internal inspection of the gonads. Following

complete dissection of the fetuses, all carcasses and sections will

be preserved in 10% neutral buffered formalin.

## Huntingdon Life Sciences

Study No. 05-4287

9.3.3. Fetal Skeletal Evaluations Page 15

Final Protocol

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

fetuses within the litter, nominally) will be eviscerated, placed in



## 70% isopropyl alcohol for preservation and processed for staining

of the skeleton using Alizarin RedS. Subsequently, these fetuses

will be evaluated for skeletal malformations and ossification

variations. These skeletons will then be stored in 100% glycerin

with a mold inhibitor. During the dissection process, the sex of

each fetus will be confirmed by internal inspection of the gonads.



## 9.3.4. Dead Fetuses

These will be removed from the uterus and examined for external

malformations. Only those with obvious external malformations will

be preserved (10% neutral buffered formalin) for possible future

examination. All other dead fetuses, and early embryonic deaths,

will be discarded.



## 10. ARCHIVING OF RECORDS AND SPECIMENS

All data documenting experimental details and study procedures and observations

will be recorded and maintained as raw data.

At the completion of the study, all reports, raw data, preserved specimens and

retained samples will be maintained in the Testing Facility's Archives for a period

of one year after submission of the signed final report.

The Sponsor will be contacted in order to determine the final disposition of these

materials. The Sponsor is responsible for all costs associated with the storage of

these materials beyond one year from the issuance of the final report and for any

costs associated with the shipment of these materials to the Sponsor or to any

other facility designated by the Sponsor.



## 11. STATISTICAL EVALUATIONS

Analysis will be performed by Graham Healey (Principal Investigator) of

Huntingdon Life Sciences Ltd, Woolley Road, Alconbury, Huntingdon,

Cambridgeshire, PE28 4HS, England. The Testing Facility will be responsible for

the GLP compliance and the archiving of the raw data and original final report.



## 11.1 CONTINUOUS DATA

The following mean measures will be analyzed as described below:

## Huntingdon Life Sciences

Study No. 05-4287 Page 16

Final Protocol



## maternal body weight values and body weight changes during gestation

(Note: body weight measurements will only be compared to GD 

## 5 values

or subsequent relevant values)

maternal feed consumption values (presented as grams of feed/kg of body

weight/day)

gravid uterine weights

corpora lutea

implantation data

pre-implantation loss

early embryonic deaths (evidence of implantation but no recognizable

fetus)

live fetuses

dead fetuses (no significant degeneration)

late embryo-fetal deaths (recognizable dead fetus undergoing

degeneration, regardless of size)

total embryo-fetal deaths and as % 

## of implant sites

number of males and females per litter

Mean values of all exposure groups will be compared to the mean value

for the control group at each time interval. Evaluation of equality of group

means will be made by the appropriate statistical method (either

parametric or non-parametric methods), followed by a multiple

comparison test if needed. Bartlett's test (Bartlett, 1937; Sokal and Rohlf,

1995; Snedecor and Cochran, 1967) will be performed to determine if

groups have equal variances. For all parameters if the variances are equal,

parametric procedures will be used; if not, nonparametric procedures will

be used.

The parametric method will be the standard one-way analysis of variance

(ANOVA) using the F ratio to assess significance (Armitage, 1971;

Dunlap and Duffy, 1975). If 

## significant differences among the means are

indicated Dunnett's (Dunlap et al., 1981; Dunnett, 1955, 1964) test will be

used to determine which means are significantly different from the control.

The nonparametric method will be the Kruskal-Wallis test (Kruskal and

Wallis, 1952, 1953) and if differences are indicated, Steel's test (Steel,

1959) will be used to determine which means differ from control.

Bartlett's test for equality of variance will be conducted at the 1%

significance level; all other statistical tests will be conducted at the 5% 

## and

1% significance levels.

References for these procedures are:

## Huntingdon Life Sciences

Study No. 05-4287 Page 17

Final Protocol

Armitage, P. 1971. Statistical Methods in Medical Research. Oxford, UK:

Blackwell Scientific Publications.

Bartlett, M.S. 1937. Properties of sufficiency and statistical tests.

Proceedings of the Royal Society, Series A 160:268-282.

Dunlap, W.P. and Duffy, J.A. 1975. Fortran IV functions for calculating

exact probabilities associated with z, chi-Square, t and F Values. Behav.

Res. Methods and Instrumentations 7:59-60.

Dunlap, W.P., Marx, M.S. and Agamy, G.G. 1981. Fortran IV functions

for calculating probabilities associated with Dunnett's test. Behav. Res.

Methods and Instrumentation 13: 363-366.

Dunnett, C.W. 1955. A multiple comparison procedure for comparing

several treatments with a control. Journal of the American Statistical

Association 50: 1096-1121.

Dunnett, C.W. 1964. New tables for multiple comparisons with a control.

Biometrics 20: 482-491.

Kruskal, W.H. and Wallis, W.A. 1952. Use of ranks in one-criterion

variance analysis. Journal of the American Statistical Association 47: 583-

621.

Kruskal, W.H. and Wallis, W.A. 1953. Errata for Kruskal-Wallis (1952)

Journal of the American Statistical Association 48: 907-911.

Snedecor, G.W. and Cochran, W.G. (1989) Statistical Methods. 8th

edition Iowa State University Press, Iowa, USA.

Sokal, R.R. and Rohlf, F.J. 1995. Biometry. 3rd Edition. San Francisco:

W.H. Freeman, pp. 369-371.

Steel, R.G.D. 1959. A multiple comparison rank sum test: treatment

versus control. Biometrics 15: 560-572.



## 11.1.1 FETAL BODY WEIGHT

The fetal body weight (by sex and as a composite for both sexes) will be

analyzed by a mixed model analysis of variance that provides an accurate

statistical model of the biology. The analysis uses the litter as the basis for

analysis and effectively uses the litter size as a covariate. The model

considers dose group, litter size, and fetal sex as explanatory variables. If



## the dose group effect in the model is statistically significant, the dose

group least squares means will be tested pairwise vs the control group

using t-tests associated with least squares means. The least squares means

allows comparisons that account for differences in litter size and sex. The

mathematical model is based on a paper by Chen et. al. (1996). Statistical

## Huntingdon Life Sciences

Study No. 05-4287 Page 18

Final Protocol



## significance of differences from control will be recognized at the 5% or

1%, two-sided levels.

Reference:

Chen, J.J., Gaylor, D.W. and Laborde, J.B. 1996. Dose-response

modeling of growth for developmental toxicity. Environmetrics 7:135-

144.

11.2 INCIDENCE DATA



## premature deliveries

total pregnancy loss (no live fetuses)

maternal necropsy findings

external fetal defects

skeletal malformations and variations

soft tissue malformations and variations

These data will be analyzed based on a Generalized Estimating Equation

(GEE) application of the linearized model (Ryan, 1992). For litter

endpoints, the model uses the litter as the basis for analysis and considers

correlation among littermates by incorporating an estimated constant

correlation and the litter size as a covariate. If 

## the dose group effect in the

model is statistically significantly the dose group least squares means will

be tested pairwise vs. the control group using t-tests associated with least

squares means. The least squares means allows comparisons that account

for differences in litter size. Statistical significance of differences from

control will be recognized at the 5% or 1%, two-sided levels.

Reference:

Ryan, L. 

## 1992. The use of generalized estimating equations for risk

assessment in developmental toxicity. Risk Analysis, 12:439-447.

12. REPORTING

12.1. STATUS REPORTS



## Periodic verbal and written updates on study progress will be provided by

the Study Director. In 

## general, a written status report will be submitted

weekly and following completion of the GD21 terminations.

·'.''·

## Huntingdon Life Sciences

Study No. 05-4287

12.2. FINAL REPORT Page 19

Final Protocol

One unbound hard copy and one electronic copy of an audited draft report

will be submitted following termination of the study. After receipt and

review of the Sponsor's comments, appropriate changes will be made and

an unbound hard copy and electronic copy of the revised report will be

forwarded to the Sponsor for a final review. Upon receipt of the Sponsor's

final comments, appropriate changes will be made and two hard copies

and one electronic copy of a signed, final report will be issued. (Additional

copies will be provided at additional cost). The report will minimally

include:



## 12.2.1. Body of Report

• Compliance Statement (including Sponsor signature line)

• Quality Assurance Statement

• Abstract

• Introduction

• Experimental Design (including justifications for exposure

levels)

• Materials and Methods

• Results and Discussion

• Conclusion and No-Observed-Effect-Level (NOEL) or NoObserved-Adverse-Effect-Level (NOAEL) Statement

• Statistical Procedures

• Protocol Deviations and Study Impact Statements



## 12.2.2. Summary Tables

• Exposure data

• Mortality data

• Pregnancy data

• Summary of maternal physical in-life observations

• Maternal body weight and weight change data during gestation

• Maternal feed consumption data

• Mean number of corpora lutea and implantation sites

• Mean number and percent of resorptions and live fetuses

• Summary of maternal macroscopic postmortem examination data

including gravid uterine weights

• Pre-implantation loss

• Post-implantation loss

• Mean fetal weight

• Total number of male and female fetuses per group and mean

percent male/percent female per group

## Huntingdon Life Sciences

Study No. 05-4287 Page20

Final Protocol

• Incidence of litters and fetuses with observations (external,

visceral and skeletal)



## 12.2.3. Appendix Tables (Individual data)

• Individual animal termination history

• Individual clinical observations

• Individual maternal body weight data and weight change data

• Individual maternal feed consumption data

• Individual uterine implantation data and corpora lutea data

• Maternal macroscopic postmortem observations

• Individual fetal observations (external, visceral and skeletal)



## 12.2.4. Appendices

• Inhalation report

• Analytical results

• Feed and water analysis

• Personnel involved in the study

• Protocol and Amendment(s)



## 12.3. ROBUST SUMMARY

This report will be separately provided by the Testing Facility's UK office

in both IUCLID and WORD® electronic formats.



## 13. REGULATORY REFERENCES

13.1. TEST GillDELINES

This study is designed to meet or exceed the pertinent requirements of:

US EPA Vehicle Emissions Inhalation Exposure Guideline 79.61, CFR

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

Organization for Economic Cooperation and Development (22 January

2001). OECD Guidelines for Testing of Chemicals; OECD Guideline

414: Prenatal Developmental Toxicity Study.

US EPA OPPTS Health Effects Test Guidelines 870.3700, Prenatal

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

## Huntingdon Life Sciences

Study No. 05-4287

13.2. GOOD LABORATORY PRACTICES Page21

Final Protocol



## This study will be conducted in accordance with US EPA 79.60, CFR Vol.

59, No. 122, 27 June 1994. This study will be performed according to

protocol and Huntingdon Life Sciences' Standard Operating Procedures.

13.3. ANIMAL WELFARE ACT COMPLIANCE



## This study will comply with all appropriate parts of the Animal Welfare

Act regulations: 9 CFR Parts 1 and 2 Final Rules, Federal Register,

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

30, 1989 and 9 CFR Part 3 Animal Welfare Standards; Final Rule, Federal

Register, Volume 56, No. 32, February 15, 1991, pp. 6426-6505 effective

March 18, 1991. The Sponsor should make particular note of the

following:

1. The Sponsor's signature on this protocol documents for the study

described, there are no generally accepted non-animal alternatives and

the study does not unnecessarily duplicate previous experiments.

2. All procedures used in this study have been designed to avoid

discomfort, distress and pain to the animals. All methods are described

in this study protocol or in written laboratory standard operating

procedures.

3. Any procedures outlined in this study protocol which are expected to

cause more than momentary or slight pain or distress to the animals

will be performed with appropriate sedatives, analgesics or

anaesthetics unless the withholding of these agents is justified for

scientific reasons, in writing, by the Sponsor and the Study Director

and approved by the IACUC; in which case the procedure will

continue for the minimum time necessary. Documentation of the

justification for withholding treatment for pain or distress and IACUC

approval of the procedures will be made prior to study initiation on the

IACUC Protocol Review form.

4. Animals experiencing more than momentary or slight pain or distress

due to the test substance, injury or illness may be treated by the Testing

Facility's veterinary staff with approved analgesics or agents to relieve

pain after consultation with the Study Director/Sponsor. However, in

emergency situations, the veterinary staff is authorized to administer

emergency treatment as necessary. Any subsequent treatment or

euthanasia will be administered after consultation with the Study

## Huntingdon Life Sciences

Study No. 05-4287 Page 22

Final Protocol

Director. The Sponsor will be advised by the Study Director of all

emergency situations in as timely a manner as possible.

5. Methods of euthanasia used during this study are in conformance with

the above referenced regulations.



## 13.4. INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC)

The IACUC Protocol Review subcommittee has reviewed this protocol

and found it to be in compliance with all appropriate animal welfare

regulations.



## 14. QUALITY ASSURANCE MONITORING

The Quality Assurance Unit of Huntingdon Life Sciences (East Millstone, NJ)

will monitor the facilities, equipment, personnel, methods, practices, records,

protocols, study conduct, raw data, draft and final reports and controls

used/produced at the Testing Facility in this study, to assure that they are in

conformance with this protocol, company Standard Operating Procedures, and the

appropriate Good Laboratory Practice regulations.



## 15. ALTERATION OF PROTOCOL AND STUDY DESIGN

Alterations to this protocol may be made as the study progresses. No changes in

the protocol will be made without the consent of the Sponsor. In the event that

the Sponsor authorizes a protocol change verbally, such changes will be honored

by the Testing Facility and will be followed by a written verification. The Study

Director and a Sponsor representative will sign all protocol modifications. Two

members of the Institutional Animal Care and Use Committee prior to the

modification's implementation will also sign any modifications potentially

affecting animal welfare.

### Huntingdon Life Sciences 05-4287 Page 1

Protocol Amendment No.: 1



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

Inhalation Exposure

No. of Pages in Amendment: 1

Change(s)



### Protocol Page 2:

The date for experimental termination (date of last data collection) is amended from November to

December 2005.

The date for issue of the Draft Final Report (audited) is amended from 30 December 2005 to

12 January 2006.

Reason(s) for Change(s)

To assist with the logistics of fetal examinations, and report production, audit and review, prior to

issue of the draft report.

Amendment approved by:

Keith P Hazelden

Study Director

Huntingdon Life Sciences

East Millstone, New Jersey

# 'ts~or

Date

### Huntingdon Life Sciences 05-4287 Page 1



### Protocol Amendment No.: 2

Study Title: Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study 

### in Rats by

Inhalation Exposure

No. of Pages in Amendment: 2



### Changes (inserted text underlined)

Protocol Page 15:

9.3.3. Fetal Skeletal Evaluations

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

litter, nominally) will" be eviscerated, placed in 70% isopropyl alcohol for

preservation and processed for staining of the skeleton using Alizarin Red S.

Subsequently, these fetuses will be evaluated for skeletal malformations and

ossification variations by an approved subcontractor, Michael Mercieca (Principal

Investigator) of Pathology Associates International (PAl), 15 Worman's Mill

Court. Suite l, 

### Frederick, MD 21701. USA. The skeletons will then be stored in

100% glycerin with a mold inhibitor. During the dissection process, the sex of

each fetus will be confirmed by internal inspection of the gonads.

The Testing Facility will be responsible for the GLP compliance and archiving of

raw data produced by the subcontractor.



### Reason for Changes

To assist with the logistics of fetal examinations and ensure timely report production, the

skeletal evaluations are being subcontracted to an approved contractor.

There are no additional cost implications associated with this change.

## Huntingdon Life Sciences

Amendment approved by:

Keith P Hazelden

Study Director

Huntingdon Life Sciences

East Millstone, New Jersey 05-4287 Page2

Protocol Amendment No.: 2



## 28 October 2005

Date

# ~l,2t)P)

Date

## Huntingdon Life Sciences 05-4287

211-DIPE-DEV



## Protocol Amendment 

## No.: 3

Study Title: Gasoline DIPE Vapor Condensate:

Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure

Change(s)

Protocol Signatures/Preface Page and Page**1~** 

## Section 2 Study Personnel,

update Study Director

From:

Keith P. 

## Hazelden, BSc, CBiol, Mffiiol

To:

Gary 

## M.**Hoffin~** 

## BA, DABT

Tel: 732-873-2550 x 2920

Email: HoffinanG@Princeton.Huntingdon.com

Reason(s) for Change(s) Page30

Keith P. Hazelden was responsible for the conduct of the study and submission of the 

## draft

report although is no longer employed with Huntingdon Life Sciences. Gary 

## Hoffinan will

assume Study Director responsibility for finalization of the report. Robert M. Parker will be

Alternate Contact (x2389) and will provide reproductive toxicology consultation.

Amendment approved by:

Gary 

## M.**Ho~** 

## BA, DABT

Study Director

## Huntingdon Life Sciences '!\

/,



## 05-4287

211-DIPE-DEV

Protocol Amendment No.: 4

Study Title: Gasoline DIPE Vapor Condensate:

Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure

Change(s)

Statistical Evaluations, page 15: Page 31

Revise from: Analysis will be performed by Graham Healey (Principal Investigator) of

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

PE28 4HS, England. The Testing Facility will be responsible for the GLP compliance and

the archiving of the raw data and original final report.

Revise to: Analysis will be initially performed by Graham Healey (Principal Investigator)

of Huntingdon Life Sciences Ltd, Woolley Road, Alconbury, Huntingdon,

Cambridgeshire, PE28 4HS, England. The Testing Facility will be responsible for the GLP

compliance and the archiving of the raw data and original final report. Further statistical

analysis (gravid uterus weight and net body weight change minus gravid uterine

weight) will 

## be performed by Gareth Thomas (Principal Investigator) and expert

report prepared for Testing Facility auditing. This additional phase of the study will

also be conducted in compliance with principles of Good Laboratory Practice

Standards as set forth in: The UK Good Laboratory Practice Regulations

(Statutory Instrument 1999 No 3106 as amended by Statutory Instrument 2004 No.

994). OECD Principles of Good Laboratory Practice (as revised in 1997),

ENV/MC/CHEM(98)17. EC Commission Directive 2004/10/EC of 11 February 2004

(Official Journal No L 50/44). Test Site-UK QA will audit the study protocol and

any subsequent protocol amendments pertaining to the statistical phase of work

conducted by the Principal Investigator. The dates of inspection .will be specified on

the QA Statement of the f"mal report. The SAS 9.1.3 [SAS INSTITUTE (2002) SAS

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.

### Huntingdon Life Sciences

Amendment approved by:

Gary M. Hoffinan, BA, DABT

Study Director 

## 05.;.4287

211-DIPE-DEV

Protocol Amendment No.: 4

# c~tr

Date Page32HuntingdonLifeSciencesRatStrain:Sprague-Dawley[Crl:CDIGSBR]Supplier:CharlesRiverLaboratoriesFetalVariation:Rib(s)14thRudimentaryStudyRoute1stGD6LastGD20ALLSep-02Jul-0502-4235OralgavageSep-02Oct-0203-4253InhalationFeb-05Mar-0503-4255OralgavageJun-03Sep-0304-4261InhalationAug-04Sep-0404-4267SubcutaneousNov-04Dec-0405-4292IntravenousJun-05Jul-0505-4287211-DIPE-DEVHistoricalControlDataFetalSkeletalObservationsFetusesNo.No.with%withExaminedFinding Finding902798.76160116.88167127.191482315.541502214.6713564.4414253.52LittersNo. No.withExaminedFinding134452262472215229224224Page1DraftFinalReportAppendixP%withFinding33.5827.2729.1768.1840.9118.1818.18

## ~ 

# 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 -CHARLES RIVER LABORATORIES, PRECLINICAL SERVICES, PENNSYLVANIA

SUMMARY OF REPRODUCTIVE INDICES

Crl:CD(SD) RATS

DAY 21 CAESAREAN-SECTION

PERIOD: JUNE 2008- JUNE 2010

FULL

STUDIES

NUMBER OF STUDIES

NUMBER OF RATS:

TESTED

PREGNANT

FOUND DEAD

ABORTED

DELIVERED

NUMBER OF RATS PREGNANT AT

CAESAREAN-SECTIONING

NUMBER OF RATS WITH SINGLE

CONCEPTUS LITTER:

LIVE

RESORBED

ABORTED 48

1195

1106

7*

1

13

1089

1

0

0

RANGE/STUDY

MEANor% MEANor%

%PREGNANT

AVERAGENO.CORPORALUTEA

AVERAGE NO. IMPLANTATIONS

AVERAGE % PREIMPLANTATION

LOSS

AVERAGE LITTER SIZE

AVERAGE NO. LIVE FETUSES

AVERAGE NO. DEAD FETUSES 92.9

15.8

14.8

5.4

14.1

0.0

AVERAGE NO. RESORPTIONS 0.7

AVERAGE NO. EARLY RESORPTIONS 0. 7

AVERAGE NO. LATE RESORPTIONS 0.0 (76.0-100)

(13.6-17.9)

(13.0-17.0)

(1.8-11.3)

(12.5-16.1)

(0.3-1.2)

(0.3-1.2) DOSAGE-RANGE

STUDIES

MEANor%

93.6

15.6

14.5

5.1

14.0

0.0

0.6

0.6

0.0 11

86

79

1

0

0

78

0

0

0

RANGE/STUDY

MEANor%

(62.5-100)

(14.0-16.5)

(13.3-15.5)

(3.0-10.2)

(12.8-15.2)

(0.1-1.0)

(0.1-1.0)

(0-0.2)



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

-20-

### CHARLES RIVER LABORATORIES, PRECLINICAL SERVICES, PENNSYLVANIA

SUMMARY OF REPRODUCTIVE INDICES

Crl:CD(SD) RATS

DAY 21 CAESAREAN-SECTION

FULL DOSAGE-RANGE

STUDIES STUDIES

RANGE/STUDY RANGE/STUDY

MEANor% MEANor% MEANor% MEANor%

AVERAGE % POSTIMPLANTATION

LOSS 4.7 (2.1-8.8) 4.6 (1.4-7.4)

AVERAGE% DAMS WITH ANY

RESORPTIONS 46.1 (24.0-75.0) 44.9 (12.5-80.0)

AVERAGE% DAMS WITH ALL

CONCEPTUSES RESORBED 0.0 0.0

AVERAGE% DAMS WITH ONE OR

MORE LIVE FETUSES 100.0 100.0

AVERAGE SEX RATIO

(%MALES/LITTER) 49.8 ( 42.6-55.9) 49.3 (42.0-55.4)

AVERAGE FETAL BODY WEIGHT (G) 5.47 (5.16-5.75) 5.39 (5.04-5.65)

AVERAGE FOR MALES (G) 5.61 (5.27-5.91) 5.53 (5.19-5.86)

AVERAGE FOR FEMALES (G) 5.32 (4.97-5.57) 5.25 ( 4.92-5.52)

AVERAGE% DEAD OR RESORBED

CONCEPTUSES/LITTER 4.8 (2.1-8.8) 4.2 (1.2-7 .4)

-21

-

## Huntingdon Life Sciences 05-4287

211-DIPE-DEV Page 1

Draft Final Report

Testing Facility Personnel Appendix Q

TITLE/DEPARTMENT

SENIOR VICE PRESIDENT,

SAFETY ASSESSMENT

DIRECTOR, TOXICOLOGY

OPERATIONS

DIRECTOR, QUALITY

ASSURANCE

STUDY DIRECTORa

INHALATION TOXICOLOGIST

VETERINARIAN

REPORT PRODUCTION

MANAGER/SUPERVISOR

Reproductive Toxicology

Inhalation Toxicology

Analytical Services

Pharmacy

Necropsy and Fetal Pathology

STATISTICIAN

SUBCONTRACTOR NAME/DEGREE

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

Diplomate A.C.V.P.

Ian Vanterpool, F.I.A.T.

Nicki S. Iacono, B.S.

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

GaryM. Hoffman, B.A., D.A.B.T.

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

Teresa S. Kusznir, V.M.D.

Janet E. Scimone, B.A.

Sally Wilcox, B.Sc.

Stuart Cracknell, CBiol, MIBiol.

Kay Saladdin, B.S.

MichaelS. McCarthy

G. Elizabeth Baxter, B.S.

Graham F Healey, B.Sc., M.Sc, ARCS.,

Prinicipal Investigator

Michael Mercieca, Principal Investigator

Pathology Associates International (P AI)

aKeith Hazeldon was responsible for the conduct ofthe study and submission ofthe draft

report; however he is no longer employed with Huntingdon Life Sciences. Gary

Hoffman assumes responsibility for finalization of the study report.

### Huntingdon Life Sciences 05-4287

211-DIPE-DEV

Report Amendments

There are no amendments for this report at this time. Page 1

Draft Final Report

AppendixR