DRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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TITLE: METABOLISM, DISPOSITION , AND PHARMACOKINETI CS OF DIISOPROPYL ETHER IN

MALE AND FEMALE RATS

SPONSOR : Section 211(b) Research Group American Petroleum Institute 1220 L Street NW Washington, DC 20005

TESTING FACILITY: RTI International*

3040 Cornwallis Road Post Office Box 12194 Research Triangle Park, NC 27709 RTI PROJECT NO.: 0212608 .000.002 and 0212608 .000.003 RTI Study Code: Rt 10

- RTI STUDY DIRECTOR: Timothy R. Fennell
PROPOSED EXPERIMENTAL START DATE: To be added by Amendment
PROPOSED EXPERIMENTAL TERMINATION DATE: To be added by Amendment
AMENDMENTS:

No. Date Section Pages

1
2
3
4

Russell White Sponsor’s Representative Date Timothy R. Fennell, Ph.D. Study Director RTI International Date

Kay Roberts Principal Investigator The Hamner Institutes for Health Sciences Date

* RTI International is a trade name of Research Triangle Institute.DRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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Table of Contents
Page

1.0 INTRODUCTION

................................ ................................ ................................ .............................. 4
2.0 OBJECTIVES

................................ ................................ ................................ ................................ .. 4
3.0 STUDY ORGANIZAT ION

................................ ................................ ................................ ................ 6
4.0 STUDY DESIGN

................................ ................................ ................................ .............................. 8
5.0 JUSTIFICATIONS

................................ ................................ ................................ .......................... 13
5.1 Animal Species

................................ ................................ ................................ .............................. 13
5.2 Numbers of Animals

................................ ................................ ................................ ....................... 13
5.3 Routes of Administration and Dose Levels

................................ ................................ .................... 13
6.0 REGULATORY COMPLIANC E

................................ ................................ ................................ ..... 14
7.0 TEST SUBSTANCE

................................ ................................ ................................ ....................... 14
8.0 ANIMALS

................................ ................................ ................................ ................................ ....... 15
8.1 Husbandry

................................ ................................ ................................ ................................ ...... 16
8.1.1 Identification

................................ ................................ ................................ ...................... 16
8.1.2 Quarantine

................................ ................................ ................................ ........................ 16
8.1.3 Feed and Water

................................ ................................ ................................ ................ 17
8.1.4 Environmental

................................ ................................ ................................ ................... 17
8.1.5 Acclimation and Housing during Studies

................................ ................................ .......... 17
8.2 Randomization and Assignment of Animals to Treatment Groups

................................ ................ 17
8.3 Body Weights

................................ ................................ ................................ ................................ . 18
8.4 Found Dead/Moribund Animals

................................ ................................ ................................ ..... 18
8.5 Euthanasia

................................ ................................ ................................ ................................ ..... 18
9.0 STUDY PROCEDURES

................................ ................................ ................................ ................. 18
9.1 Cannulation of Rats for Blood Collection

................................ ................................ ....................... 18
9.2 Test Chemical Preparation and Analysis

................................ ................................ ....................... 18
9.3 Inhalation Exposure

................................ ................................ ................................ ....................... 19
9.4 Ante mortem Observations and Functional A ssessments

................................ ............................. 19
9.5 Collection and Storage of Biological Samples

................................ ................................ ............... 20
9.5 .1 Blood

................................ ................................ ................................ ................................ . 20
9.5.2 Excreta

................................ ................................ ................................ .............................. 20
9.5.3 Carcass

................................ ................................ ................................ ............................. 21
9.5.3.1 Tissues

................................ ................................ ................................ ........................... 21
9.6 Analysis of Biological Samples for Total Radioactivity

................................ ................................ .. 21DRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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9.6.1 Blood

................................ ................................ ................................ ................................ . 21
9.6.2 Excreta

................................ ................................ ................................ .............................. 22
9.6.3 Tissues and Carcass

................................ ................................ ................................ ......... 22
9.6.4 Exhaled Breath Traps

................................ ................................ ................................ ....... 22
9.6.5 Exhaled Volatiles Traps

................................ ................................ ................................ .... 22
9.7 Analysis of Biological Samples for DIPE, Isopropanol and Acetone

................................ ............. 22
9.8 Analysis of Urine Samples for Radioactive Metabolites

................................ ................................ 23
10.0 DATA COLLECTION

................................ ................................ ................................ ..................... 23
11.0 PHARMACOKINETIC CALC ULATIONS

................................ ................................ ...................... 23
12.0 DATA FROM “EXTRA” AN IMALS

................................ ................................ ............................... 24
13.0 STATISTICAL ANALYSIS

................................ ................................ ................................ ............. 25
14.0 RECORDS AND REPORT

................................ ................................ ................................ ............ 25
15.0 MAINTENANCE OF RECOR DS AND RAW DATA

................................ ................................ ...... 27
16.0 SAFETY PRECAUTIONS

................................ ................................ ................................ .............. 27
17.0 PROTOCOL AMENDMENTS AND DEVIATIONS

................................ ................................ ........ 28
18.0 REFERENCES

................................ ................................ ................................ ............................... 28DRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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1.0 INTRODUCTION The Environmental Protection Agency under the Fuel and Fuel Additives health effects testing program regulations codified at 40 CFR part 79

. 58(c) t has indicated the need for data on t he toxicity of oxygenated fuel additives, including diisopropyl ether (DIPE). A component of the data required to
evaluate the potential toxicity of DIPE is the investigation of the uptake, pharmacokinetics and metabolism of DIPE following inhalation exposure in rats (the expected primary routes of exposure)

. Investigation of the kinetics of DIPE following single and repeated in halation exposure will provide an assessment of the effects of exposure on the capability of DIPE to alter the capacity for its metabolism by enzyme induction or inhibition. A pilot study has been conducted to evaluate the disposition of 14 C DIPE in mal e Fischer 344 rats following a 6 h r inhalation exposure to 3600 ppm DIPE ( RTI

- 934, Metabolism and Pharmacokinetics of Diisopropyl Ether in Male and Female Rats: Pilot Study, in preparation )

. A nose

- only inhalation system was set up and its performance ver ified prior to exposure of animals. The study found that DIPE is absorbed by inhalation, and rapidly eliminated as exhaled volatiles (approximately 70 %) , and as 14 CO 2 (approximately 20%)

. The total amount excreted in urine over 7 days was approximately 5% of the recovered radioactivity, and feces contained 1%. Approximately 3% was recovered in the carcass at 7 days. Greater than 90% of the radioactivity recovered was obtained in the first 24 hours following the end of the exposure
, so urine and feces will only be collected for 24

- hours during the definitive studies described in this protocol

. The loss of radioactivity during euthanasia by exposure to CO 2 was determined to be small, so no special precautions will be taken to avoid loss of radioactivity during
euthanasia. On exposure to unlabeled DIPE by inhalation, DIPE, and metabolites acetone and isopropanol were detected and quantitated by gas chromatography

- mass spectrometry

. Metabolit es in
urine will not be characterized because of the low amount of radioactivity excreted by this route.

2 .0 OBJECTIVES The objectives of this study are to evaluate the absorption, distribution, metabolism and excretion (ADME) and pharmacokinetic properties of DIPE, with the following aims : 1. Conduct single nose

- only inhalation exposures of male and female rats to a low and a high
concentration of 14 C DIPE/DIP E ( 8 per sex) for 6 hours, and evaluate the disposition during and following exposure.
2. Conduct repeated (four daily nose
- only ) inhalation exposures of male and female rats to a high concentration of DIPE, followed by a single nose

- only exposure to 14 C DI PE and evaluate the disposition during and following exposure.DRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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3. Conduct a single gavage administration (low and high dose) of 14 C DIPE/DIPE to male and female rats and evaluate the disposition of DIPE.
4. Conduct a single nose

- only inhalation exposure , and measure the kinetics of elimination of DIPE, isopropanol and acetone from blood.
These aims can be further subdivided into specific exposures and study groups, outlined in Tables 1 and 2, and described in more detail below.
Aim 1 Inhalation Exposure s ADME, Low Concentration
1 ) Conduct a single nose
- only exposure of male (Group 1) and female rats (Group 2) to 600 ppm

14 C DIPE/DIPE ( 8 per sex) for 6 hours. A naly ze the amount of 14 C in whole body digest s from rats exposed to 14 C DIPE/DIPE (4 rats per s ex).
2 ) C ollect excreta from 4 male and female rats exposed once to 14 C DIPE / DIPE for 24 h r following exposure. 3 ) A nalyze 14 C in tissue and carcass digest s , urine, feces, exhaled C O 2 , and exhaled volatiles in rats exposed to 14 C DIPE / DIPE

. Inhalation Exposures ADME, High Concentration
1 ) Conduct a single nose
- only exposure of male (Group 3) and female rats (Group 4) to 3600
ppm 14 C DIPE/DIPE ( 8 per sex) for 6 hours. A naly ze the amount of 14 C in whole body digest s from rats exposed to 14 C DIPE/DIPE (4 r ats per group).
2 ) C ollect excreta from 4 male and female rats exposed once to 14 C DIPE / DIPE for 24 h following
exposure

. 3 ) A nalyze 14 C in tissue and carcass digest s , urine, feces, exhaled C O 2 , and exhaled volatiles in rats exposed to 14 C DIPE / DIPE

.

Aim 2 Inhalation Exposures ADME, Repeat Exposure
1 ) Conduct 4 days of nose

- only exposure of male (Group 5) and female rats (Group 6) to 3600
ppm DIPE ( 8 per sex) for 6 hours per day

. 2 ) Conduct a nose

- only exposure of male (Group 5) and female rats (Group 6) to 3600 ppm 14 C DIPE/DIPE ( 8 per sex) for 6 hours. A naly ze the amount of 14 C in whole body digest s from rats exposed to 14 C DIPE/DIPE (4 rats per sex).
3 ) C ollect excreta from 4 male and female rats exposed to DIPE (4 days) and 14 C DIPE / DIPE (1 day) for 24 h following the last inhalation exposure

.DRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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4 ) A nalyze 14 C in tissue and carcass digest s , urine, feces, exhaled C O 2 , and exhaled volatiles in rats exposed to DIPE and 14 C DIPE / DIPE

.

Aim 3 Gavage Administration, ADME
1 ) 14 C DIPE/DIPE will be administered once by gavage to male (Groups 7 and 9) and female rats (Groups 8 and 10) at one of two doses (50 mg/kg and 200 mg/kg) ( 4 rats per sex and dose).
2 ) C ollect excreta for 24 h following dosing and a nalyze 14 C in tissue and carcass digest s , urine, feces, exhaled C O 2 , and exhaled volatiles in rats administered 14 C DIPE / DIPE by gavage

.

Aim 4 Inhalation Exposure , Pharmacokinetics , Low Concentration
1 ) Conduct a single nose
- only exposure of jugular vein cannulated male (Group 11) and female

ra ts (Group 12) to 600 ppm DIPE ( 8 per sex) for 6 hours. Collect blood at timepoints during and following exposure, and analyze by GC

- MS for DIPE, isopropanol and acetone. Inhalation Exposure , Pharmacokinetics , High Concentration
1 ) Conduct a single nose
- only exposure of jugular vein cannulated male (Group 13) and female
rats (Group 14) to 3 600 ppm DIPE ( 8 per sex) for 6 hours. Collect blood at timepoints during and following exposure, and analyze by GC

- MS for DIPE, isopropanol and acetone.

3 .0 STUDY ORGANIZATION

Testing Facility: RTI International Discovery and Analytical Sciences
3040 Cornwallis Road
PO Box 12194 Research Triangle Park, NC 27709
For the purposes of this study, the terms “RTI”, “RTI International”, and “Research Triangle Institute” are synonymous.

Test Facility Management: Elizabeth Hill , Vice PresidentDRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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Discovery and Analytical Sciences
RTI International
Phone: ( 919 ) 485 5674

Study Director: Timothy R. Fennell, Ph.D. Discovery Sciences
RTI International
Phone: ( 9 19 ) 485 2781
FAX: ( 919 ) 541 6499
Email: fennell@rti.org

The Study Director has overall responsibility for the conduct of the entire study and will sign the final report to indicate acceptance of responsibility for the va lidity of the data.

Lead Quality Assurance Contact: Celia Keller, M.S. Discovery and Analytical Sciences QA Unit
RTI International
Phone: ( 919 ) 541 7272
Email: cdk@rti.org

The lead Quality Assurance contact has overall responsibility for quality assurance for the entire study.

RTI Study Personnel Timothy Fennell , Ph.D.

– Study Director Norman Gaudette , B.S.

– Research Chemist Rodney Snyder, M.S.

– Research Chemist Yan Hong, M.S

. – Research Chemist Alyssa Mc Intyre , DVM

– Veterinarian Melody Gower

– Biologist
Other personnel will be used as required. A full list of study participants will be included in the study report.

Inhalation exposures will be conducted at RTI by staff from :DRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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The Hamner Institut es for Health Sciences
6 Davis Drive
P.O. Box 12137 Research Triangle Park
NC 27709 For the purposes of this study, the terms and “ The Hamner Institutes for Health Sciences ”
, and

“ The Hamner Institutes ” are synonymous.

The Hamner Institutes Principal Investigator Kay Roberts
The Hamner Institutes Phone (919) 558 1306 Fax (919) 558 1300
Email: k roberts@thehamner.org

The Hamner Institutes Quality Assurance Contact Patricia O’Brien Pom erleau
The Hamner Institutes Phone (919) 558 1341 Fax (919) 558 1300
Email: pomerleau@thehamner.org

The Hamner Institutes Study Personnel Kay C. Roberts, A.S.

– Principal Investigator
Research Associate Carl U. Parkinson, LATg

– Research Associate

4 .0 STUDY DESIGN To achieve the objectives described above, the following studies/exposures will be conducted as described in Table 1 (ADME studies) and Table 2 (pharmacokinetic studies)

.DRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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Table 1. Inhalation and Oral ADME Study Groups

Group a Animals Exposure

Route Exposure N f Sample Collection End points Animal

Numbers 1 a Male

Rats Inhalation e 600 ppm 14 C DIPE /DIPE, 6 hours 8 Urine, feces, blood, tissues, volatiles, CO 2 ADME 1

- 01

– 1

- 08

2 a Female

Rats Inhalation e 600 ppm 14 C DIPE /DIPE, 6 hours 8 Urine, feces, blood, tissues, volatiles, CO 2 ADME 2

- 01

– 2

- 08

3 b Male

Rats Inhalation e 3 600 ppm 14 C DIPE /DIPE, 6 hours 8 Urine, feces, blood, tissues, volatiles, CO 2 ADME 3

- 01

– 3

- 08

4 b Female

Rats Inhalation e 3 600 ppm 14 C DIPE /DIPE, 6 hours 8 Urine, feces, blood, tissues, volatiles, CO 2 ADME 4

- 01

– 4

- 08

5 c Male

Rats Inhalation e 3 600 ppm unlabeled DIPE for 4 days, and 14 C DIPE /DIPE 6 hours 8 Urine, feces, blood, tissues, volatiles, CO 2 ADME 5

- 01

– 5

- 08

6 c Female

Rats Inhalation e 3 600 ppm unlabeled DIPE for 4 days, and 14 C DIPE /DIPE 6 hours 8 Urine, feces, blood, tissues, volatiles, CO 2 ADME 6

- 01

– 6

- 08

7 d Male

Rats Gavage 50 mg/kg 14 C DIPE /DIPE 4 Urine, feces, blood, tissues, volatiles, CO 2 ADME 7

- 01

– 7

- 08 8 d Female

Rats Gavage 50 mg/kg 14 C DIPE /DIPE 4 Urine, feces, blood, tissues, volatiles, CO 2 ADME 8

- 01

– 8

- 08 9 d Male

Rats Gavage 200 mg/kg 14 C DIPE /DIPE 4 Urine, feces, blood, tissues, volatiles, CO 2 ADME 9

- 01

– 9

- 08 10 d Female

Rats Gavage 200 mg/kg 14 C DIPE /DIPE 4 Urine, feces, blood, tissues, volatiles, CO 2 ADME 10

- 01

– 10

- 08

a Inhalation exposures for Groups 1 and 2 will be conducted concurrently.

b Inhalation exposures for Groups 3 and 4 will be conducted concurrently.

c Inhalation exposures for Groups 5 and 6 will be conducted concurrently.

d Gavage administration for Groups 7

- 10 will be conducted concurrently.

e Inhalation exposures are nose

- onl y. f Additional animals, designated as “extra” will be ordered for each exposure group. If not used on the study for exposure to DIPE, they will be used to provide blank tissues, carcass, and urine and feces for determination of background radioactivity. The fate of the extra animals will be documented in the study record.DRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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Table 2. Inhalation Pharmacokinetic Study Groups

Group Animals Exposure

Route Exposure N c Sample Collection End points Animal

Numbers 1 1 a Male Rats Inhalation 600 ppm DIPE
, 6 hours 8 Blood PK 11

- 01

– 11

- 08

1 2 a Female Rats Inhalation 600 ppm DIPE
, 6 hours 8 Blood PK 12

- 01

– 12

- 08

1 3 b Male Rats Inhalation 3 600 ppm DIPE
, 6 hours 8 Blood PK 13

- 01

– 13

- 08

1 4 b Female Rats Inhalation 3 600 ppm DIPE
, 6 hours 8 Blood PK 14

- 01

– 14

- 08

a Inhalation exposures for Groups 11 and 12 will be conducted concurrently.

b Inhalation exposures for Groups 13 and 14 will be conducted concurrently.

c Additional animals, designated as “extra” will be ordered for each exposure group. . If not used

on the study for exposure to DIPE, they will be used to provide blood for calibrations for sample analysis. The fate of the extra animals will be documented in the study record.

All work will be conducted at RTI International.
The exposure system will consist of a Cannon nose

- only inhalation tower, which is designed for

nose
- only inhalation exposure of rodents withou t rebreathing (Cannon et al., 1983 )

. The exposure atmosphere generation has been designed to safely generate a repro ducible and stable atmosphere for exposure of rodents to DIPE for 6 hrs. The concentration of DIPE on the exposure tower will be monitored using a Miran infrared analyzer (Foxboro, MA) or by GC. The system used will be documented
in the raw data and will be described in the final report. A report documenting the setup and performance of the exposure system has been provided by T he Hamner Institutes PI, and provi ded to the Sponsor for
review. Verification of the performance of the exposure system and con duct of pilot ADME and pharmacokinetic studies has been conducted under GLPs and will be reported in the final report RTI

- 934 Metabolism and Pharmacokinetics of Diisopropyl Ether in Male and Female Rats. The Hamner Institutes PI will be responsible for a r eport describing the performance of the inhalation system during the exposures conducted in this protocol . It will be included as an appendix in the final report.DRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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Metabolism and Distribution

– Inhalation (single exposure , low concentration ) Eight mal e (Group 1) and eight female F344 rats (Group 2) will be exposed for approximately 6 hr
to 6 00 ppm 14 C DIPE/DIPE via nose only inhalation. At the end of exposure, four male and four female rats will be immediately euthanized by CO 2 asphyxiation. Radioactivity will be determined in the whole body digest. The dose administered will be determined by the total radioactivity measured in the whole body digest. The remaining four rats will be transferred to glass metabolism cages for the collection of urine, feces, expired volatiles, and expired CO 2 . Urine and feces will be collected over dry ice for 24 h following exposure, because the pilot study demonstrated greater than 90% of the administered radioactivity excreted in 24 h . Expired volatiles and CO 2 will be collected at approximately 1, 3, 6, and 24 hr after the end of exposure

. At 24 hours after exposure the rats will be euthanized by exposure to CO 2 , and tissues and carcass will be collected. The radioactivity in these samples will be determine d via scintillation counting. One unexposed rat will be used for the collection of urine, feces, and tissue samples that will be used for determination of background radioactivity.
Metabolism and Distribution

– Inhalation (single exposure , high concentr ation ) Eight male (Group 3) and eight female F344 rats (Group 4) will be exposed for approximately 6 hr to 3600 ppm 14 C DIPE/DIPE via nose only inhalation. At the end of exposure, four male and four female rats will be immediately euthanized by CO 2 asphyx iation. Radioactivity will be determined in the whole body digest. The dose administered will be determined by the total radioactivity measured in the whole body digest. The remaining four rats will be transferred to glass metabolism cages for the colle ction of urine, feces, expired volatiles, and expired CO 2 . Urine and feces will be collected over dry ice for 24
hours because the pilot study demonstrated over 90% excretion in 24 h . Expired volatiles and CO 2 will be collected at approximately 1, 3, 6, and 24 hr after the end of exposure. At 24 hours after exposure the rats will be euthanized by exposure to CO 2 , and tissues and carcass will be collected. The radioactivity in these samples will be determined v ia scintillation counting.
Metabolism and Distribution

– Inhalation (repeated exposure , high concentration ) Eight male (Group 5) and eight female F344 rats (Group 6) will be exposed for approximately 6
hr /day to 36 00 ppm unlabeled DIPE via nose only inha lation for 4 days

. On the fifth day, they will be exposed for approximately 6 hr to 3600 ppm 14 C DIPE /DIPE . At the end of exposure, four male and four female rats will be immediately euthanized by CO 2 asphyxiation. Radioactivity will be determined in the whole body digest. The dose administered will be determined by the total radioactivity measured in the whole body digest. The remaining 4 male and 4 female rats will be transferred to glass metabolism cages for the collection of urine, feces, expired vo latiles, and expired CO 2 . Urine and feces will be collectedDRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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over dry ice for approximately at 24 hr. Expired volatiles and CO 2 will be collected at approximately 1, 3, 6, and 24 hr after the end of exposure

. The radioactivity in these samples will be determined via scintillation counting.
Metabolism and Distribution

– Gavage
Male (Groups 7 and 9) and f emale rats (Groups 8 and 10) will each be administered a single
dose of 14 C DIPE /DIPE by gavage at a low d ose (50 mg/kg) and a high dose (200 mg/kg) (4 male and 4 female rats at each dose) . Based on water solubility, a solution of 2 mg/mL in water may be achieved
, and a high dose of only 2 0 mg/kg would be feasible as an aqueous solution

. Alternative dose ve hicles to achieve higher doses include corn oil, or 10 % Cremophor:water. Tertiary butyl methyl ether has been administered by gavage at high doses (up to 1000 mg/kg) using a solution in 10 % Emulphor:water (Prescott

- Mathews et al., 1999). The dose vehic le to be used will be added by amendment. After dosing, the rats will be transferred to glass metabolism cages for the collection of urine, feces, expired volatiles, and expired CO 2 . Urine and feces will be collected over dry ice approximately at 24 hr. Expired volatiles
and CO 2 will be collected at approximately 1, 3, 6, and 24 hr after dosing . The radioactivity in these samples will be determined via scintillation counting. The radioactivity will be measured in the carcass via scintillation counting after whole body digestion. All urine, feces, and expired volatile trap samples will be stored at approximately

- 20°C or below until analyzed. CO 2 trap samples will be stored at room temperature.
Pharmacokinetics

– Inhalation (single exposure , low concentration ) Eight male and eight female F344 rats with jugular vein cannulas will be exposed for approximately 6 hr to 600 ppm unlabeled DIPE via nose only inhalation . Blood samples (approximately 100 μL) will be collected via the cannulas (ca. 5, 10, 15, 30, 60, 120, 240, and 360 minutes following initiation of exposure) and after exposure (at ca. 375, 390, 420, 440, 480, 600, 960, and 1440 minutes following the initiation of the exposure). Blood samples will be collected from four rats at each time p oint, making a total of 64 samples following initiation of the exposure. Analysis of DIPE, isopropanol, and acetone will be conducted with a GC equipped with a mass spectrometer, and a headspace autosampler. The analysis will be conducted using a validat ed analysis method. A standard curve will be prepared in triplicate for DIPE, isopropanol, and acetone. Blood will be obtained from control rats. A standard curve will be generated by preparing a stock solution of DIPE, isopropanol, and acetone, and ser ially diluting to give the appropriate concentrations . Aliquots of blood (90 μL) and standard (10 μL) will be mixed in GC vials to generate the standard blood samples. The concentration of DIPE, isopropanol, and acetone will
be determined for each blood sample based on the peak area response, and the regression equation for
the standard curve.DRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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Pharmacokinetics

– Inhalation (single exposure , high concentration ) Eight male and eight female F344 rats will be exposed for approximately 6 hr to 3600 ppm
unlabe led DIPE via nose only inhalation. Blood samples (approximately 100 μL) will be collected via the
cannulas (ca. 5, 10, 15, 30, 60, 120, 240, and 360 minutes following initiation of exposure) and after exposure (at ca. 375, 390, 420, 440, 480, 600, 960, and 1440 minutes following the initiation of the exposure). Blood samples will be collected from four rats at each time point, making a total of 64 samples following initiation of the exposure. The samples will be collected from a group of four animals
duri ng the exposure, and a second group of four animals following the exposure. Analysis of DIPE, isopropanol, and acetone will be conducted with a GC equipped with a mass spectrometer, and a headspace autosampler. The analysis will be conducted using a vali dated analysis method. A standard curve will be prepared in triplicate for DIPE, isopropanol, and acetone. Blood will be obtained from control rats. A standard curve will be generated by preparing a stock solution of DIPE, isopropanol, and acetone, and serially diluting to give the appropriate concentrations . Aliquots of blood (90 μL) and standard (10 μL) will be mixed in GC vials to generate the standard blood samples. The concentration of DIPE, isopropanol, and acetone will be determined for each blo od sample based on the peak area response, and the regression equation for the standard curve.

5 .0 JUSTIFICATIONS

5 .1 Animal Species The present studies are designed to evaluate the uptake, distribution, and excretion of a chemical entity to provide info rmation that will be used for safety assessments to humans. No in vitro techniques are available that allow for adequate determination of pharmacokinetics, distribution, and excretion of chemicals by mammals. Fischer 344 rats are an established animal sp ecies and strain for toxicological testing, and pharmacokinetic studies

.
5 .2 Numbers of Animals The numbers of animals used in this study are considered acceptable to develop the analytical procedures, and to evaluate the appropriateness of the exposure ge neration system for further study

.
5 .3 Routes of Administration and Dose Levels The route of administration is a n expected potential exposure route in humans ( inhalation ) and
ha s been used in toxicity and safety assessment studies. The exposure concentration is similar to the mid range concentration from a n inhalation study conducted on the subchronic toxicity of DIPE (DalbeyDRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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and Feuston, 1996) , and is expected to b e without significant toxicity. Proposed g avage doses of 50 and
250 mg/kg are at or below the doses of 250 and 1000 mg/kg DIPE administered by gavage in a carcinogenicity bioassay conducted in male and female Sprague Dawley rats (Belpoggi et al., 2002)

6 .0 REGULATORY COMPLIANC E This study will be carried out in compliance with the EPA Good Laboratory Practices (GLP) Standards for Inhalation Exposure Health Effects Testing, 40 CFR part 79, subpart F § 79.60

. The pre

study exposure system method development was completed at The Hamner Institutes and was not conducted under G LPs. Verification of the performance of the exposure system and conduct of pilot ADME and pharmacokinetic studies has been conducted under GLPs by The Hamner Institutes and will be reported in the final report RTI

- 934 Metabolism and Pharmacokinetics of Di isopropyl Ether in Male and
Female Rats. The Quality Assurance Unit at the testing facility will prepare and sign a QA Statement to be included in the final report. It will specify the phases of the study that were inspected, the dates on which inspectio ns were made, and the dates on which results of the inspections were reported to the Study
Director and the Study Director’s management. The Quality Assurance Unit at the test site will prepare and sign a QA Statement to be included in
The Hamner Institu tes report on the inhalation exposure. It will specify: the phases of the study that were inspected by The Hamner Institutes Quality Assurance Unit; the dates on which inspections were made; the dates on which the results of the inspections were reported to The Hamner Institutes Principal Investigator and the Principal Investigator’s Management; and the dates on which the results of the inspections were reported to the Study Director and the Study Director’s Management.

7 .0 TEST SUBSTANCE
NAME : Diisopropyl ether ( DIPE ; CAS No. 108

- 20

- 3 )
MOLECULAR FORMULA : C 6 H 14 O
MOLECULAR WEIGHT : 102.18
STRUCTURE:

SOURCE OF NON

- LABELED TEST SUBSTANCE : The non

- labeled DIPE will be purchased from Sigma

- Aldrich, Milwaukee, W I

. PRODUCT NUMBER: 398276DRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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LOT NUMBER: To be added by amendment
SOURCE OF R

ADIOLABELED TEST SUBSTANCE : [2

- 14 C]

- labeled DIPE will be obtained by custom synthesis from American Radiolabeled Chemicals (St. Louis, MO). The target specific activity will
be approximately 2 mCi/mmol. LOT NUMBER(S): To be listed in the final report

. IDENTITY AND PURITY : The identity of the unlabeled DIPE will be confirmed at RTI by 1 H and 13 C nuclear magnetic resonance ( NMR ) spectroscopy , and by mass spectrometry. The purity of the test chemical will be determined by GC with flame ionization detector ( FID ) , or by GC

- MS. The identity of [2

- 14 C] DIPE will be confirmed by 1 H NMR spectroscopy, and by coelution of radioactivity with the
unla beled DIPE standard on HPLC with radioactivity detection. The chemical and radiochemical purity of the test substance will be verified by RTI using G C methods based upon those available in the literature, or developed by RTI. For safety concerns in worki ng with DIPE, which can form explosive peroxides on storage, the unlabeled DIPE will be monitored semi

- quantitatively for buildup of peroxides using commercially available peroxide test strips. If the levels of peroxide exceed 80 ppm, the DIPE will be dis posed of immediately in accordance with RTI safety standards , and an additional batch will be procured.
STORAGE CONDITIONS : [ 2

- 14 C] DIPE ) DIPE will be stored in the dark at approximately

- 20  C.
Nonradiolabeled DIPE will be stored in the dark at room tem perature. STABILITY

: DIPE presents particular problems with storage, since the recommended leng th of storage to avoid the buildup of peroxides is 3 months. At the conclusion of the study, all unused DIPE will be disposed of. A sample of the unlabeled DIPE will be analyzed before the start of this study
, periodically throughout the study, and after th e study to confirm stability. Radiochemical purity of
the [2

- 14 C]

- DIPE will be confirmed by HPLC before and after the animal ex posures , and after the study to confirm stability

.

8 .0 ANIMALS

1. Species and Strains: Fischer 344 rats
2. Approximate Age: 8

- 9 weeks old at time of exposure 3. Approximate Weight: 150

- 200 g (male) and 1 25

– 150 g (female)
4. Number/Sex:
ADME, Inhalation Exposure to 600 ppm 14 C DIPE : 8 Males rats and 8 female rats
ADME, Inhalation Exposure to 3600 ppm 14 C DIPE : 8 Male rats and 8 female rats
ADME, Repeated Exposure to 3600 ppm DIPE and 14 C DIPE : 8 Male rats and 8 female ratsDRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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ADME, Gavage Dosing 14 C DIPE : 4 Male rats and 4 female rats for the low dose and 4 male rats and 4 female rats for the high dose.
Pharmacokinetics, Inhalation Exposure to 600 ppm DIPE: 8 Male and 8 female jugular vein cannulated rats
Pharmacokinetics, Inhalation Exp osure to 3600 ppm DIPE: 8 Male and 8 female jugular vein cannulated rats

At least 96 rats will be ordered to ensure sufficient animals for the completion of the studies. It is expected that additional rats will be ordered to provide control blood as need ed for calibration curves, and that extra cannulated animals may be needed to ensure cannula patency in the pharmacokinetic studies . The number of animals received and used in the studies will be documented in the study record and in the Final Report.

5. Sources: Charles River ( Kingston , NY , or Raleigh, NC ) will be the primary source of animals. In the event that suitable animals cannot be provided from the primary source, acceptable alternate sources are Charles River Laboratories, Inc. ( Portage, MI) and Harlan (Indianapolis, IN). The source(s) of all animals will be documented in the raw data, and
included in the Final Report.

8 .1 Husbandry Research Triangle Institute is accredited by AAALAC International . Animal procedures detailed in this protoco l are in accordance with the Animal Welfare Act, "Guide for the Care and Use of Laboratory Animals" (NRC, 1996), and the Office of Laboratory Animal Welfare (NIH) . All animal procedures will be reviewed by RTI’s Institutional Animal Care and Use Committee (IACUC) before initiation of the studies. In the opinion of the Sponsor and Study Director , the study does not unnecessarily duplicate any previous
work.

8 .1.1 Identification Rats will be identified by individual eartags. Metabolism cages will be indivi dually coded by number and color that are related to dose and treatment groups. All individual animal data will be referenced to either eartag number or to treatment group and animal number or to both.

8 .1.2 Quarantine Uncannulated a nimals will be quarantined for a minimum of seven days before use on a study. Animals will be examined by a veterinarian prior to their release from quarantine, and only animals determined to be in good health as indicated by body weight gain and the abse nce of clinical signs will be used. During the quarantine period and prior to initiation of the experiments detailed in Section 9 .0, ratsDRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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will be housed (maximum of 3 per cage) in polycarbonate cages with stainless steel bar lids accommodating a water bot tle. Cage sizes are approximately 19" x 10 1/2" x 8" high ( ca. 143 sq. in. floor space). Contact bedding will be Sani

- Chips (P.J. Murphy Forest Products Corp , Montville, N J ). Cannulated animals will be housed individually , and will be used within 1

- 2 day s of arrival at RTI.

8 .1.3 Feed and Water Animals will be provided Certified Purina Rodent Chow (5002) ad libitum , except during the periods of inhalation exposure

. W ater will be provided ad libitum except during the period of inhalation
exposure. The source of the water is the City of Durham, NC. The analysis of water and analysis of the rodent chow for chemical composition and possible chemical contamination will be provided by the suppliers and maintained in the Study Records. In addition, appr oximately once per year, RTI conducts an analysis of drinking water contaminants using an outside laboratory. Samples of water will be collected for analysis for tertiary butyl alcohol, tertiary amyl methyl ether, ethyl tertiary butyl ether, diisopropyl e ther, and methyl tertiary butyl ether. The samples will be sent to Kiff Analytical ( Davis, CA) for analysis. It is anticipated that contaminant levels will be below those permitted in the certified feed and will not affect the design, conduct, or conclus ions of this study

. It is anticipated that contaminant
levels measured in water will not affect the design, conduct, or conclusions of this study

.
8 .1.4 Environmental Air circulation will be 100% fresh air. Room temperature will be maintained at 64

 79  F and relative humidity at 30

 70% and monitored at least once a day. Light/darkness will be cycled at 12

- h intervals. Any deviations from these conditions shall be included in the study records. Environmental parameters will be recorded automatically usin g a computerized HVAC Monitoring and Control System.

8 .1.5 Acclimation and Housing d uring Studies During the course of the studies , rats will be placed in glass Roth

- type metabolism cages for the
collection of urine and feces

. There will be no acclimation period

.
8 .2 Randomization and Assignment of Animals to Treatment Groups Animals will be specifically purchased for inhalation exposure studies, and will not be assigned randomly to specific study groups

. Animals will be specifically purchased for gavage studies at two dose
levels , to be run concurrently, and will be randomly assigned to study groups. A nimals will each be assigned an eartag number, and then assigned unique study designations using a series of computer generated numbers as described in SO P DPK

- HUS

- 001 Assignment of Animals into Groups , using the procedure for Assignment of Animals within a Single Group or Multiple Groups without Regard to Weight Mean and Range.DRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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8 .3 Body Weights Individual body weights will be measured during the quarant ine period, the day of exposure , and
at sacrifice.

8 .4 Found Dead/Moribund Animals The Study Director or the veterinarian with the approval of the Study Director will authorize
euthanasia of animals with life

- threatening clinical signs that indicate that they are unlikely to survive until the next scheduled observation. The time of death will be estimated as precisely as possible and
recorded.

8 .5 Euthanasia Rats will be euthanized with CO 2 exposure

.

9 .0 STUDY PROCEDURES

9 .1 Cannulation of Rats for Blo od Collection Rats with indwelling jugular vein cannulae will be purchased from Charles River Laboratories, Inc. ( Raleigh NC, or Kingston, NY) or other approved vendor. Animals will be cannulated by the vendor, and shipped to RTI the day after surgery, or as soon as practical. The rats will be exposed to DIPE 1 to 2 days after arrival at RTI. Cannula patency will be maintained as per the vendors recommendations, or with daily flushings with hepa rinized saline ( sterile saline containing 20 IU/mL of sodium heparin ), and will be documented in the study record

.
9 .2 Test Chemical Preparation and Analysis For inhalation exposure to unlabeled DIPE ( Groups 5, 6, and 11

- 14 ) , an exposure atmosphere
will be generated using unlabeled DIPE.
For inhalation exposure to 14 C

- labeled DIPE ( Groups 1

- 6 ) , the amount of material required will be calculated based on the exposure concentration, the duration of exposure, the number of animals, and
the flow rate of air t hrough the exposure tower. For inhalation exposure to [2

- 14 C] DIPE /DIPE ( Groups 1

- 6 ) , [2

- 14 C]DIPE will be weighed into a
tared flask with a Teflon faced screw cap. The weight of the label ed chemical added will be recorded. Unlabeled DIPE will be added, and the weight added will be recorded. The nominal specific activity of the DIPE mixture will then be calculated. This will be verified by weighing an aliquot into a sealed flask, recording the weight added, and adding solvent, and recording the weight added. Aliquots of the solution of DIPE will be placed in scintillation vials, and the weight added will be recorded. The amount of solution
added will be calculated based on the density of the solvent . Ultima GoldTM scintillation cocktail (Perkin Elmer) will be added to the scintillation vials, and the amount of radioactivity added will be determined byDRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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l iquid scintillation spectroscopy (LSS) . The specific activity of the labeled DIPE will then be calculated
from the data obtained. The exposure atmosphere concentration will be monitored using a calibrated analytical
instrument MIRAN IR spectro photo mete. Concentrations of DIPE will be monitored at strategic locations to document the exposure concentrations. The dose solution for the gavage administration of 14 C DIPE will be prepared by sequentially weighing [2

- 14 C] DIPE, unlabeled DIPE and water into a closed vial. The weight of each component added will be used to calculate a nominal concentration. HPLC analysis of the dose solution will be conducted to verify the radiochemical purity of the dose. The concentration of radioactivity in the dose solution will be determined by scintillation counting. The concentration of DIPE in the dose solution will be determined by GC analysis

.
9 .3 Inh alation Exposure The inhalation system including the generation and exposure systems will be constructed with materials that are chemically compatible with DIPE to minimize chemical losses. The generation system will include a generator and delivery syste m to deliver a steady flow of DIPE to the exposure tower air supply at appropriate flow rates to maintain the target concentration of 600 or 3600 ppm. The generation system will consist of a syringe containing the chemical with a syringe pump to deliver t he chemical to the air supply of the exposure chamber. The air supply will be controlled with an electronic mass flow meter to maintain a total air flow that will assure at least 12 air changes per hour. The generation and delivery system will be place d in a chemical hood to contain any DIPE that may leak from the system. The exposure system will be a flow

- past nose only exposure system. The incoming air for the exposure system will be filtered to eliminate the possibility of contamination in the air sup ply. The air supply temperature and relative humidity will be maintained between 64 to 79 degrees Fahrenheit and 30 to 70%, respectively. The chamber exhaust flow will be adjusted to maintain a slight negative pressure during the exposure to prevent DIPE from entering the laboratory area. The chamber exhaust will be filtered through a disposable charcoal filter and disposed at the end of the exposure. Closed nose

- only
tubes will be used to hold the test animals during inhalation exposures to 14 C DIPE

. The inhalation system will be strategically placed in a chemical hood to prevent any DIPE from entering the laboratory.
D ata necessary to recreate the inhalation exposure will be documented in a study notebook and will be reviewed by The Hamner Institutes Quality Assurance Unit.

9 .4 Ante mortem Observations and Functional Assessments Animals will be observed twice per day for mortality, morbidity, signs of toxicity, and for any acute distress that might be related to the test procedure or test substances . Animals exhibiting adverseDRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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reactions will be closely monitored. All signs of poor health or abnormal behavior will be recorded. Samples collected from dead or moribund animals will be included in the analysis if the animal was not
moribund/dead at t he time of collection. The Sponsor will be notified as soon as possible if it is anticipated that the sacrifice may affect the integrity of the study

.
9 .5 Collection and Storage of Biological Samples

9 .5.1 Blood Except at sacrifice, blood will be sampled through indwelling jugular cannulae from Groups 11

- 14

. For pharmacokinetic determinations, blood will be sampled from each animal pre

- exposure and from four rats each at approximately 5 min, 10 min, 15 min, 30 min, and at approximately 1, 2, 4 and 6 hr af ter the exposure initiation. At the end of the approximate 6 hr exposure, all rats will be removed from the inhalation exposure tower, and placed in cages. Blood will be drawn from four rats at approximately 375, 390, 420, 440, 480, 600, 960, and 1440 mi n after the beginning of the exposure (at approximately 15, 30, 60, 80 min, and 2 hr, 4 hr, 10 hr, and 18 hr after the end of the exposure). To facilitate the withdrawal of blood samples, animals will be stagger

- started on the exposure tower. At each time point approximately
100  L of blood will be withdrawn using a heparinized syringe. The total volume of blood collected over
the 24

- h r period will not be more than 20% of blood volume from each rat. Heparinized saline will be injected into each cannula a s necessary to help prevent blood clots from forming in the cannula. If a vascular cannula is determined to no longer be patent before exposure on Day 1, that animal will not be used in the study. If the vascular cannula is determined not to be patent af ter exposure, the animal will be replaced by a designated “extra” animal if it is available. If an “extra” animal is not available, blood will be sampled from the tail vein for the remainder of the study and recorded appropriately. Each blood sample will be placed immediately in a preweighed headspace vial, and the vial will be capped with a crimp seal. The blood samples will be analyzed within 24 h of collection for DIPE, acetone and isopropanol by GC/MS.

9 .5.2 Excreta
In Groups 1

- 10
, urine will be co llected over dry ice at 0

 24 after the end of inhalation exposure to

14 C DIPE , or after gavage dosing with 14 C DIPE . Feces will be collected over dry ice at 0

 24 hours after termination of exposure. Exhaled volatile organics will be collected on a series of two charcoal traps, and expired 14 CO 2 will be collected in 1.0 N KOH at 0

 1, 1

 3, 3

 6, 6

 8, 8

 24 hours after termination of exposure or gavage dosing

. At the end of excreta collection, the cage will be rinsed with water, and with
ethanol. The rinses will be analyzed for total radioactivity as described for urine (Section 9 .6.2). The weight of urine and/or feces collected for each sample interval will be meas ured. Urine and feces will beDRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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analyzed for total radioactivity. Excreta not assayed within a day of collection will be stored at approximately

- 20  C in the dark

.
9 .5.3 Carcass For Groups 1

- 6 exposed to 14 C DIPE by inhalation
, t he amount of 14 C retained will be determined by directly euthanizing 4 rats from each group at the end of the inhalation exposure. Each rat will be placed, still in the nose

- only restraint tube
, in to a closed chamber for exposure to CO 2 to euthanize the
rat. T he carcass will be digested with 2N ethanolic NaOH . After digestion , the amount of radioactivity in the carcass will be determined by LSS of aliquots

.
9 .5.3.1 Tissues
In Groups 1

- 10 , following sacrifice, selected tissues will be collected from four ani mal s : Blood
femur cecum + large intestine + rectum skin (hair shaved) small intestine
subcutaneous fat liver
abdominal fat muscle (gastrocnemius)
brain heart
teste s (epididymis removed) kidneys
stomach contents small intestine contents
stomach large intestine + cecum contents lungs spleen
residual carcass ovaries and uterus

The harvested tissues will be weighed and analyzed for radiochemical ( 14 C) content by LSS. The remaining carcass of the animal from which the tissues li sted above have been taken will be analyzed for total radioactivity. T issues will be prepared as described in section 9 .6.3 below

.
9 .6 Analysis of Biological Samples for Total Radioactivity

9 .6.1 Blood
W hole b lood samples will be assayed for total radioactivity ( 14 C ) in duplicate by LSS. For LSS analysis , blood aliquots (approximately 50 μ l) will be placed in tared vials containing approximately 1 mL
Soluene

- 350 TM for solubilization and then weighed

. After solubilization, samples will be bleached ( by adding approximately 125 μ l of 70% perchloric acid, and then adding approximately 0.3 ml of 30% H 2 O 2 ) prior to addition of scintillation cocktail and analysis by LSS

.DRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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9 .6.2 Excreta
D uplicate aliquots of urine will be analyzed directly (without solubili zation or bleaching) for radiochemical content. Feces will be homogenized with an approximately equal mass of water. The weight of the feces homogenate will be determined, and duplicate homogenate aliquots will be weighed
into scintillation vials. After solubilization of the homogenate aliquots with Soluene

- 350 TM (normally about 2 mL per sample) , scintillation cocktail will be added to the vials
, and the samples will be analyzed for total radioactivity by LSS. Samples may be bleached ( by adding approximately 125 μ l of 70 % perchloric acid, and then adding approximately 0.3 ml of 30% H 2 O 2 ) prior to addition of scintillation
cocktail , if necessary

. The requirement for bleaching will be determined by the intensity of the color of the sampl es. Control samples of urine and feces will be collected from unexposed rats, and analyzed for radiochemical content to determine background counts.

9 .6.3 Tissues and Carcass Tissues and carcass will be analyzed for total radioactivity following solubiliz ation in Soluene

350 TM (normally about 2 mL per tissue sample) or 2N ethanolic sodium hydroxide

. Duplicate samples of the solubilized carcass will be analyzed. Liver will be homogenized
, and duplicate aliquots of the homogenate solubilized

. Other tissues will be solubilized in their entirety or after being divided into multiple pieces. Solubilized tissue samples may be bleached ( by adding approximately 125 μ l of 70 % perchloric acid , and then adding approximately 0.3 ml of 30% H 2 O 2 ) prior to addition of scintillation
cocktail and analysis by LSS. The requirement for bleaching will be determined by the intensity of the color of the samples. Dark tissues, including blood, liver, lung, heart, muscle and kidneys will require bleaching. The procedure used f or individual samples will be recorded in the study record s

.
9 .6.4 Exhaled Breath Traps Aliquots of 1.0 N KOH from the exhaled breath trap for CO 2 will be analyzed by LSS after addition
of scintillation cocktail.

9 .6.5 Exhaled Volatiles Traps Each ch arcoal trap will be extracted by elution with 4 .0 ml of dimethylformamide (DMF). The radioactivity in the DMF wash will be measured by LSS of duplicate aliquots.

9

. 7 Analysis of Biological Samples for DIPE, Isopropanol and Acetone Quantitative analysis of DIPE, acetone and isopropanol will be conducted in blood collected in Groups 11

- 14

. A quantitative method for the analysis of DIPE, acetone, and isopropanol in blood has been developed by GC with headspace analysis. The method has been validated with acceptance criteria based on those outlined in the US Food and Drug Administration’s Guidance for Industry: Bioanalytical Method Validation (2001). The accuracy, precision, range, and lower limit of quantitationDRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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(LLOQ) for the analytical method were deter mined. The method will be documented in a Project Specific Analytical Method, and will be documented in the raw data, and in the final report.

9.8 Analysis of Urine Samples for Radioactive Metabolites In a pilot study conducted with inhalation exposure t o 14 C DIPE, only low levels of radioactivity were recovered in urine, with <5% of the activity excreted over 7 days in urine. Since urine samples
col lected over the 24 hour period following DIPE administration will contain extremely low levels of the
admi nistered dose, the nature of the radioactivity present will not be characterized.

10 .0 DATA COLLECTION
The DebraTM laboratory information management system (Lablogic Systems Limited, Sheffield (United Kingdom) will be used for collection of body weights, animal observations, tissue and sample weights , and radioactivity data. Therefore, the raw data for these measurements will be the electronic
data collected in Debra unless otherwise noted in the study records. The Debra system will be used to
calculate and report radioactivity recovered in each aliquot analyzed, in each sample, and in each animal. The Debra system will be used to calculate and report summary data for tissues and excreta.
Room t emperature and humidity data will be collected using a computerized HV AC Monitoring and Control System . All other data, such as animal receipt and quarantine records, will be manually recorded unless noted otherwise in the study record s

.

1 1 .0 PHARMACOKINETIC CALC ULATIONS
Blood concentration data for DIPE and if applicable, acetone and isopropanol, will be presented graphically. Data obtained following the end of exposure (at 360 min and greater time points) will be used
to derive terminal elimination kinetic data. Mean blood concentration

- time data will be a nalyzed, as appropriate, by noncompartmental (model

- independent) methods using the least

- squares fitting program
WinNonlin  (Statistical Consulting Inc., Cary, NC). Pharmacokinetic analysis will be conducted for
individual animals if possible
, using the b lood concentrations from animals sampled following exposure

. After the best

- fit model is selected, the following pharmacokinetic parameters will be determined as appropriate: terminal elimination rate constant, terminal elimination half

- life (T 1/2 ), area under the
blood concentration

- time curve extrapolated from time zero to infinity (AUC), maximum concentration achieved (C max ), time to maximum concentration (T max ). Occasionally, a data point (i.e., a concentration) that cannot be predicted by a PK meth od of analysis may be encountered. The following procedure will be used to evaluate such data points as
outliers.DRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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1. Identify suspected outliers by visual inspection of the data. Types of data points that should be considered as suspected outliers include non
- zero concentrations prior to dosing, an individual concentration that is much different from that predicted by the PK method of analysis (such as lone high concentration preceded and followed by much lower concentrations).
2. Rule out physiological or other processes which may explain the suspected outlier. Certain processes, such as enterohepatic circulation or absorption from more than one site in the gastrointestinal tract, may result in unusual C

- T profiles, which a PK method of analysis would be u nable to approximate. Furthermore, non

- zero concentrations at time zero may be possible if the analyte is present endogenously, or if some endogenous material interferes with the assay for the target analyte. If a concentration is deemed to
be an outlier solely because it cannot be explained by PK methods of analysis, the possibility of some process which might explain it such as sample analysis should be
considered.
3. Once a concentration that is suspected as a PK outlier cannot be explained by PK
methods of analysis or physiologic processes, the Grubb s method (Grubbs 1969) will be
used to test whether it is an outlier.
4. If a concentration cannot be explained by PK methods of analysis and is substantially
different from that seen in the other animals at th e same time (based on the above criteria), it is considered a PK outlier and will not be included in the calculation of PK parameters or mean concentrations. Reasons for exclusion will be documented in the

ra w data and in the final report.

12.0 DATA FROM “EXTRA” ANIMALS To better ensure that complete data from the required number of animals will be obtained, particularly in Groups 5, 6, and 11

- 14 , one or more additional animals, predesignated as “extras”, will start the study as part of each treatment grou p. If a “core” study animal fails to complete the study due to loss of samples, misdosing, morbidity, or other accidents, it will be replaced with a designated “extra” animal
and data will cease to be obtained from the “core” animal. The “extra” animal w ill then become part of the “core” group and the original animal will be removed from the “core” group. All such substitutions will be documented as to reason and approved in writing by the Study Director. An “extra” animal may also be
substituted for a “core” animal as described in Section 9 .5.1. Terminal blood samples or blood samples obtained via the tail vein may be included in the analysis in the event that cannulas are no longer patent. Except for animals that become part of a “core” group, sample s from “extra” animals will not normally beDRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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analyzed. However, all data obtained in the study will be reported. Data used to construct group means and to obtain pharmacokinetic parameters will consist of the data obtained from the “core” study animals (no t the “extras”) except in cases where an “extra” has taken the place of a “core” animal. In that case,
the data from the “extra” animal will be used instead. Data from the “core” animal that was eliminated from the study will be used in these calculation s, if the data are from samples collected prior to
elimination. The fate of all “extra” animals will be documented in the study records.

1 3 .0 STATISTICAL ANALYSIS
All blood concentration data, and tissue and excreta 14 C

- content will be reported in tables as the

mean  standard deviation (SD). Statistical analysis of pharmacokinetic data will be conducted to compare values between gender and exposure concentrations. Statistical analysis of the percentage of dose in excreta and tissue samples and concentra tion of radioactivity in tissues will be conducted to compare values between gender and exposure concentrations. Values will be compared between groups using Student’s t

- test for continuous variables. The alpha level for each statistical comparison will be 0.05, and the significance level (one

- or two

- tailed) for each statistical comparison will be reported as p<0.05, p<0.01 or p<0.001. Additional analysis may be done, if wa rranted, to further compare exposure concentration or gender. Any additional tests will be documented in the study records and described in the report.

1 4 .0 RECORDS AND REPORT The following will be maintained in the record:

a. Protocol and any amendments
b. Animal receipt records

c. Quarantine records
d. Temperature and humidity records for the treatment rooms

e. Animal research facility room logs
f. Feed and water analysis for contaminants
g. Test chemical receipt, storage and use records
h. Balance calibration log references
i. Correspondences j. All other raw data and documentation.

Results of the studies will be described in an audited draft report, which will be submitted to the Sponsor for approval. This report will include but not be limited to:DRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

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a. Name and address of the facility performing the study, dates of study initiation and completion, and RTI study number.
b. A copy of the signed, dated and approved protocol and all deviations and authorized amendments to the original protocol.

c. A detailed description of all metho ds used.
d. The lot number(s) of the test substance s , reference and analytical standards and details of
the formulation of doses.

e. Animal information to include: supply source, species, strain or substrain, sex, individual animal weights (randomization through sacrifice), approximate age at initiation of dosing, and procedure used for individual animal identification and assignment to the treatment group.
f. Tabulated individual results for blood, urine and tissues.
g. Tabulated mean results for blood, urine, and tis sues. h. Graphical presentation of disposition data for radioactivity.
i. Inhalation exposure report , which will include a description of the exposure system and the atmosphere generation system used, a description of the procedure for analysis of test
chemical concentration, and the analysis data from each day of exposure, including a table of individual measurements of test chemical concentration at the exposure port, together with
mean and standard deviation. j. A statement prepared and signed by the Quality Assu rance Unit that specifies the dates audits and inspections were made and findings reported to the Study Director and to management.
k. A compliance statement signed by the Study Director.

Upon acceptance of the audited draft report by the Sponsor, a final report will be issued. Four (unbound) copies of the audited draft report and four copies (unbound) of the final report will be shipped
to:

Russell White

Reg ulatory Analysis and Scientific Affairs American Petroleum Institute 1220 L Street NW Washington, DC 20005 p: (202)682

- 834 4 f: (202)682

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1 5 .0 MAINTENANCE OF RECOR DS AND RAW DATA Records will be maintained in the laboratories of the study personnel while the studies are being conducted. Copies of raw data generated while conducting the study and any transformations, calculations or operations performed on the data will be recorded in the study file. All original study records, protocols , amendments, and the final report will be stored in the RTI Inter national Archives. The applicable record retention requirements for this study are the Good Laboratory Practices (GLPs)
Standards for Inhalation Exposure Health Effects Testing, 40 CFR part 79, subpart F § 79.60

. Facility
data will be maintained in the archives of RTI International and The Hamner Institutes , respectively. Documentation and raw data will be maintained in the Archives for a period of ten years following issuance of the final report

. The storage loc ation of biological samples will be documented in the final report. Chemical and biological samples, or aliquots thereof, will be maintained for a minimum of ten
years following issuance of the final report , or for as long as the quality of the preparatio n affords evaluation, whichever is less. Wet specimens of blood, urine and feces will be disposed of after quality
assurance verification ( when the QAU assures that discarding the samples does not negatively impact the integrity of the study)

. The test s ubstance , DIPE, is known to differ markedly in stability and quality during storage, producing danger ous explosive peroxides. Therefore, for reasons of safety, no samples of the test substance will be retained after the end of the study.
Materials will be maintained in the RTI Archive for a period of one year after the signature of the final report as part of the initial study cost. At that point, the Sponsor will be contacted to determine the final disposition of these materials. The Sponsor may continu e to store these materials in the RTI Archive, have RTI ship them to the Sponsor or an alternative archive facility, or have RTI dispose of them. The Sponsor will be responsible for all costs associated with the storage of these materials beyond 1 year
fr om 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.

1 6 .0 SAFETY PRECAUTIONS This study will be conducted in accordance with Nuclear Regu latory Commission (NRC) regulations, North Carolina License #032

- 0131

- 1.

a. Precautions for laboratory personnel:
All work will be done in well

- ventilated areas properly designated for use of radiolabeled compounds. Work will be carried out in accordance with RTI safety standards for work with radiolabeled compounds.
b. All radioactive wastes will be disposed of in accordance with standard RTI safety policies.DRAFT PROTOCOL RTI INTERNATIONAL POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK, NC 27709 RTI

- 1117 Page 28 of 29

DIPE is a volatile liquid that represents a s evere peroxide forming hazard on exposure to air. Peroxides are potentially explosive. The container should be tightly closed when not in use. Storage under a nitrogen atmosphere is recommended to avoid the generation of peroxides. The unlabeled test

s ubstance should be tested for peroxides periodically after opening, using a peroxide test strip. Testing should be conducted at two months after opening a container, and monthly thereafter. If peroxide levels of greater than 80 ppm are detected, the mate rial should be disposed of. Use personal protective equipment, including safety glasses, lab coat, and chemical resistant gloves. The inhalation exposure system will be placed in or in close proximity to a fume hood. The exposure generation system will be grounded.

1 7 .0 PROTOCOL AMENDMENTS AND DEVIATIONS This protocol may be amended by the Study Director with agreement of the Sponsor as the study progresses. Normally, a formal amendment will be prepared and signed by the Study Director and the Sponsor’s Representative prior to the change. If instances arise where a change is urgent, the change may become effective upon approval by the Study Director. A notification of the urgent change will be sent to the Sponsor’s Representative (email, facsi mile, or telephone) as soon as feasible (no more than
24 h r after the Study Director’s approval ) . Subsequently, a formal protocol amendment will be prepared for approval by the Study Director and the Sponsor’s Representative. Any deviations from the prot ocol that occur in the course of the conduct of the study will be
documented. The cause for the deviation and its effect if any on the outcome of the study will be explained and the Study Director will sign the document.

1 8 .0 REFERENCES

Belpoggi, F., S offritti, M., Minardi, F., Bua, L., Cattin, E., and Maltoni, C. (2002) Results of long term carcinogenicity bioassays on tert

- amyl

- methyl

- ether (TAME) and di

- isopropyl

- ether (DIPE) in rats.
Ann. N.Y. Acad. Sci. 982: 70

- 86. Cannon, W. C., Blanton, E. and McDonald, K. E. (1983). The flow

- past chamber: an improved nose

- only exposure system for rodents. Am. Ind. Hyg. Assoc. J. 44 , 923

- 928. Dalbey
, W . and Feuston
, M

. (1996)

. Subchronic and developmental toxicity studies of vaporized diisopropyl ether in r ats. J Toxicol Environ Health 49, 29

- 43. Grubbs FE (1969). Procedure for detecting outlying observations in samples. Technometrics, 11 :1

– 21

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National Research Council ( 2010 ). Guide for the Care and Use of Laboratory Animals. National Academy Press: Washington, DC.
Prescott

- Mathews, J. S., Poet, T. S. and Borghoff, S. J. (1999). Evaluation of the in vivo interaction of methyl tert

- butyl ether with alpha2u

- globulin in male F

- 344 rats. Toxicol Appl Pharmacol 157 , 60

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. US Food and Drug Administration (2 001)

. Guidance for Industry: Bioanalytical Method Validation

.