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TITLE: METABOLISM, DISPOSITION, AND PHARMACOKINETICS 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: Rt10-

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.
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Table of Contents

Page

1.0 INTRODUCTION..............................................................................................................................4

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

3.0 STUDY ORGANIZATION ................................................................................................................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 COMPLIANCE.....................................................................................................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 Assessments ............................................................. 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 .................................................................. 21
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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 CALCULATIONS......................................................................................23

12.0 DATA FROM “EXTRA” ANIMALS ...............................................................................................24

13.0 STATISTICAL ANALYSIS.............................................................................................................25

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

15.0 MAINTENANCE OF RECORDS AND RAW DATA......................................................................27

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

17.0 PROTOCOL AMENDMENTS AND DEVIATIONS........................................................................28

18.0 REFERENCES...............................................................................................................................28
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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 the 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 inhalation 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 14C DIPE in male Fischer 344 rats

following a 6 hr 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 verified prior to exposure of animals. The study found that DIPE is 

absorbed by inhalation, and rapidly eliminated as exhaled volatiles (approximately 70 %), and as 14CO2

(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 CO2 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. Metabolites 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 14C DIPE/DIPE (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 14C DIPE and 

evaluate the disposition during and following exposure. 
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3. Conduct a single gavage administration (low and high dose) of 14C 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 Exposures ADME, Low Concentration

1) Conduct a single nose-only exposure of male (Group 1) and female rats (Group 2) to 600 ppm 

14C DIPE/DIPE (8 per sex) for 6 hours. Analyze the amount of 14C in whole body digests from 

rats exposed to 14C DIPE/DIPE (4 rats per sex).

2) Collect excreta from 4 male and female rats exposed once to 14C DIPE/DIPE for 24 hr

following exposure. 

3) Analyze 14C in tissue and carcass digests, urine, feces, exhaled CO2, and exhaled volatiles in 

rats exposed to 14C 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 14C DIPE/DIPE (8 per sex) for 6 hours. Analyze the amount of 14C in whole body digests

from rats exposed to 14C DIPE/DIPE (4 rats per group).

2) Collect excreta from 4 male and female rats exposed once to 14C DIPE/DIPE for 24 h following 

exposure.

3) Analyze 14C in tissue and carcass digests, urine, feces, exhaled CO2, and exhaled volatiles in 

rats exposed to 14C 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 14C 

DIPE/DIPE (8 per sex) for 6 hours. Analyze the amount of 14C in whole body digests from rats 

exposed to 14C DIPE/DIPE (4 rats per sex).

3) Collect excreta from 4 male and female rats exposed to DIPE (4 days) and 14C DIPE/DIPE (1 

day) for 24 h following the last inhalation exposure.
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4) Analyze 14C in tissue and carcass digests, urine, feces, exhaled CO2, and exhaled volatiles in 

rats exposed to DIPE and 14C DIPE/DIPE.

Aim 3

Gavage Administration, ADME

1) 14C 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) Collect excreta for 24 h following dosing and analyze 14C in tissue and carcass digests, urine, 

feces, exhaled CO2, and exhaled volatiles in rats administered 14C 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 

rats (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 3600 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 President 
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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: (919) 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 validity 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 McIntyre, 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:
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The Hamner Institutes 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: kroberts@thehamner.org

The Hamner Institutes Quality Assurance Contact Patricia O’Brien Pomerleau

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).
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Table 1. Inhalation and Oral ADME Study Groups

GroupaAnimals Exposure 

Route

Exposure N

f

Sample Collection End

points

Animal 

Numbers

1

a Male 

Rats

Inhalatione600 ppm 14C 

DIPE/DIPE, 6 

hours

8 Urine, feces, blood, 

tissues, volatiles, CO2

ADME 1-01 –

1-08

2

a

Female 

Rats

Inhalatione600 ppm 14C 

DIPE/DIPE, 6 

hours

8 Urine, feces, blood, 

tissues, volatiles, CO2

ADME 2-01 –

2-08

3

b Male 

Rats

Inhalatione3600 ppm 14C 

DIPE/DIPE, 6 

hours

8 Urine, feces, blood, 

tissues, volatiles, CO2

ADME 3-01 –

3-08

4

b

Female 

Rats

Inhalatione3600 ppm 14C 

DIPE/DIPE, 6 

hours

8 Urine, feces, blood, 

tissues, volatiles, CO2

ADME 4-01 –

4-08

5

c Male 

Rats

Inhalatione3600 ppm

unlabeled 

DIPE for 4 

days, and 14C 

DIPE/DIPE 6 

hours

8 Urine, feces, blood, 

tissues, volatiles, CO2

ADME 5-01 –

5-08

6

c

Female 

Rats

Inhalatione3600 ppm

unlabeled 

DIPE for 4 

days, and 14C 

DIPE/DIPE 6 

hours

8 Urine, feces, blood, 

tissues, volatiles, CO2

ADME 6-01 –

6-08

7

d Male 

Rats

Gavage 50 mg/kg 14C 

DIPE/DIPE

4 Urine, feces, blood, 

tissues, volatiles, CO2

ADME 7-01 –

7-08

8

d

Female 

Rats

Gavage 50 mg/kg 14C 

DIPE/DIPE

4 Urine, feces, blood, 

tissues, volatiles, CO2

ADME 8-01 –

8-08

9

d Male 

Rats

Gavage 200 mg/kg 14C 

DIPE/DIPE

4 Urine, feces, blood, 

tissues, volatiles, CO2

ADME 9-01 –

9-08

10dFemale 

Rats

Gavage 200 mg/kg 14C 

DIPE/DIPE

4 Urine, feces, blood, 

tissues, volatiles, CO2

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-only.

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.
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Table 2. Inhalation Pharmacokinetic Study Groups

Group Animals Exposure 

Route

Exposure N

c

Sample Collection End

points

Animal 

Numbers

11

a Male 

Rats

Inhalation 600 ppm 

DIPE, 6 

hours

8 Blood PK 11-01 –

11-08

12

a

Female 

Rats

Inhalation 600 ppm 

DIPE, 6 

hours

8 Blood PK 12-01 –

12-08

13

b Male 

Rats

Inhalation 3600 ppm 

DIPE, 6 

hours

8 Blood PK 13-01 –

13-08

14

b

Female 

Rats

Inhalation 3600 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 without rebreathing (Cannon et al., 1983). The exposure 

atmosphere generation has been designed to safely generate a reproducible 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 The Hamner Institutes PI, and provided to the Sponsor for 

review. Verification of the performance of the exposure system and conduct 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 report 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. 
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Metabolism and Distribution – Inhalation (single exposure, low concentration)

Eight male (Group 1) and eight female F344 rats (Group 2) will be exposed for approximately 6 hr 

to 600 ppm 14C DIPE/DIPE via nose only inhalation. At the end of exposure, four male and four female

rats will be immediately euthanized by CO2 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 CO2. 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 CO2 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 CO2, 

and tissues and carcass will be collected. The radioactivity in these samples will be determined 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 concentration)

Eight male (Group 3) and eight female F344 rats (Group 4) will be exposed for approximately 6 hr 

to 3600 ppm 14C DIPE/DIPE via nose only inhalation. At the end of exposure, four male and four female 

rats will be immediately euthanized by CO2 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 CO2. 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 CO2 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 CO2, and tissues and carcass will be collected. The radioactivity in 

these samples will be determined via 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 3600 ppm unlabeled DIPE via nose only inhalation for 4 days. On the fifth day, they will be 

exposed for approximately 6 hr to 3600 ppm 14C DIPE/DIPE. At the end of exposure, four male and four 

female rats will be immediately euthanized by CO2 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 volatiles, and expired CO2. Urine and feces will be collected 
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over dry ice for approximately at 24 hr. Expired volatiles and CO2 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 female rats (Groups 8 and 10) will each be administered a single 

dose of 14C DIPE/DIPE by gavage at a low dose (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 20 mg/kg would be feasible as an aqueous solution. Alternative dose vehicles 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 vehicle 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 CO2. Urine and feces will be collected over dry ice approximately at 24 hr. Expired volatiles 

and CO2 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. CO2 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 point, 

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 validated 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 blood sample based on the peak area response, and the regression equation for 

the standard curve.
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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 

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 point, making a total of 64 

samples following initiation of the exposure. The samples will be collected from a group of four animals 

during 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 validated 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 blood 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 information 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 species 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 generation system for further study.

5.3 Routes of Administration and Dose Levels

The route of administration is an expected potential exposure route in humans (inhalation) and

has been used in toxicity and safety assessment studies. The exposure concentration is similar to the 

mid range concentration from an inhalation study conducted on the subchronic toxicity of DIPE (Dalbey 
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and Feuston, 1996), and is expected to be without significant toxicity. Proposed gavage 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 COMPLIANCE

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 prestudy exposure system method development was completed at The Hamner Institutes and was not 

conducted under GLPs. 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 Diisopropyl 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 

inspections 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 Institutes 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: C6H14O

MOLECULAR WEIGHT: 102.18

STRUCTURE: 

H3C

CH

O

CH3

CH

CH3

CH3

SOURCE OF NON-LABELED TEST SUBSTANCE: The non-labeled DIPE will be purchased from 

Sigma-Aldrich, Milwaukee, WI. 

PRODUCT NUMBER: 398276
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LOT NUMBER: To be added by amendment

SOURCE OF RADIOLABELED TEST SUBSTANCE: [2-

14C]-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 1H and 13C 

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-

14C] DIPE will be confirmed by 1H NMR spectroscopy, and by coelution of radioactivity with the 

unlabeled DIPE standard on HPLC with radioactivity detection. The chemical and radiochemical 

purity of the test substance will be verified by RTI using GC methods based upon those available in 

the literature, or developed by RTI. For safety concerns in working 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 disposed of immediately in accordance with RTI safety standards, and an 

additional batch will be procured. 

STORAGE CONDITIONS: [2-

14C]DIPE) DIPE will be stored in the dark at approximately -20C. 

Nonradiolabeled DIPE will be stored in the dark at room temperature. 

STABILITY: DIPE presents particular problems with storage, since the recommended length 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 the study to confirm stability. Radiochemical purity of 

the [2-

14C]- DIPE will be confirmed by HPLC before and after the animal exposures, 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 125 – 150 g (female)

4. Number/Sex: 

ADME, Inhalation Exposure to 600 ppm 14C DIPE: 8 Males rats and 8 female rats

ADME, Inhalation Exposure to 3600 ppm 14C DIPE: 8 Male rats and 8 female rats

ADME, Repeated Exposure to 3600 ppm DIPE and 14C DIPE: 8 Male rats and 8 female rats
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ADME, Gavage Dosing 14C 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 Exposure 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 needed 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 protocol 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 individually 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 animals 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 absence of clinical signs will be 

used. During the quarantine period and prior to initiation of the experiments detailed in Section 9.0, rats 
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will be housed (maximum of 3 per cage) in polycarbonate cages with stainless steel bar lids 

accommodating a water bottle. 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, NJ). 

Cannulated animals will be housed individually, and will be used within 1-2 days 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. Water 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, approximately 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 ether, 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 conclusions 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 using a computerized HVAC Monitoring and Control System.

8.1.5 Acclimation and Housing during 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. Animals will each be 

assigned an eartag number, and then assigned unique study designations using a series of computer 

generated numbers as described in SOP 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. 
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8.3 Body Weights

Individual body weights will be measured during the quarantine 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 CO2 exposure.

9.0 STUDY PROCEDURES

9.1 Cannulation of Rats for Blood 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 heparinized 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 14C-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 through the exposure tower. 

For inhalation exposure to [2-

14C]DIPE/DIPE (Groups 1-6), [2-14C]DIPE will be weighed into a 

tared flask with a Teflon faced screw cap. The weight of the labeled 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 by 
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liquid 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 spectrophotomete. Concentrations of DIPE will be monitored at strategic locations 

to document the exposure concentrations. 

The dose solution for the gavage administration of 14C DIPE will be prepared by sequentially 

weighing [2-

14C]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 Inhalation 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 system 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 the 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 placed 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 supply. 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 14C DIPE. The inhalation 

system will be strategically placed in a chemical hood to prevent any DIPE from entering the laboratory.

Data 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 adverse 
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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 the 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 after 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 min 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-hr period will not be more than 20% of blood volume from each rat. Heparinized saline will be 

injected into each cannula as 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 after 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 collected over dry ice at 024 after the end of inhalation exposure to 

14C DIPE, or after gavage dosing with 14C 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 14CO2 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 measured. Urine and feces will be 
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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 14C DIPE by inhalation, the amount of 14C 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, into a closed chamber for exposure to CO2 to euthanize the 

rat. The 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 animals: 

Blood

femur cecum + large intestine + rectum

skin (hair shaved) small intestine

subcutaneous fat liver

abdominal fat muscle (gastrocnemius)

brain heart

testes (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 (

14C) content by LSS. The 

remaining carcass of the animal from which the tissues listed above have been taken will be analyzed for 

total radioactivity. Tissues will be prepared as described in section 9.6.3 below.

9.6 Analysis of Biological Samples for Total Radioactivity

9.6.1 Blood

Whole blood samples will be assayed for total radioactivity (14C) in duplicate by LSS. For LSS

analysis, blood aliquots (approximately 50 μl) will be placed in tared vials containing approximately 1 mL 

Soluene-350TM for solubilization and then weighed. After solubilization, samples will be bleached (by 

adding approximately 125 μl of 70% perchloric acid, and then adding approximately 0.3 ml of 30% H2O2)

prior to addition of scintillation cocktail and analysis by LSS. 
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9.6.2 Excreta

Duplicate aliquots of urine will be analyzed directly (without solubilization 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-350TM (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 μl of 70% 

perchloric acid, and then adding approximately 0.3 ml of 30% H2O2) prior to addition of scintillation 

cocktail, if necessary. The requirement for bleaching will be determined by the intensity of the color of the 

samples. 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 solubilization in Soluene350TM (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 μl of 70% 

perchloric acid, and then adding approximately 0.3 ml of 30% H2O2) 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 for individual samples will be recorded in the study records.

9.6.4 Exhaled Breath Traps

Aliquots of 1.0 N KOH from the exhaled breath trap for CO2 will be analyzed by LSS after addition 

of scintillation cocktail. 

9.6.5 Exhaled Volatiles Traps

Each charcoal 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 quantitation 
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(LLOQ) for the analytical method were determined. 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 to 14C DIPE, only low levels of radioactivity 

were recovered in urine, with <5% of the activity excreted over 7 days in urine. Since urine samples 

collected over the 24 hour period following DIPE administration will contain extremely low levels of the 

administered 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 temperature and humidity data will be collected using a computerized HVAC Monitoring 

and Control System. All other data, such as animal receipt and quarantine records, will be manually 

recorded unless noted otherwise in the study records.

11.0 PHARMACOKINETIC CALCULATIONS

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 analyzed, 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 blood 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 (T1/2), area under the 

blood concentration-time curve extrapolated from time zero to infinity (AUC), maximum concentration 

achieved (Cmax), time to maximum concentration (Tmax). 

Occasionally, a data point (i.e., a concentration) that cannot be predicted by a PK method of 

analysis may be encountered. The following procedure will be used to evaluate such data points as 

outliers. 
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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 unable 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 Grubbs 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 the 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 

raw 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 group. 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 will 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, samples from “extra” animals will not normally be 
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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 

(not 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 calculations, if the data are from samples collected prior to 

elimination. The fate of all “extra” animals will be documented in the study records.

13.0 STATISTICAL ANALYSIS

All blood concentration data, and tissue and excreta 14C-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 concentration 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 warranted, to further compare exposure 

concentration or gender. Any additional tests will be documented in the study records and described in 

the report.

 

14.0 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:
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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 methods used.

d. The lot number(s) of the test substances, 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 tissues.

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 Assurance 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

Regulatory Analysis and Scientific Affairs 

American Petroleum Institute 

1220 L Street NW 

Washington, DC 20005 

p: (202)682-8344

f: (202)682-8031 
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POST OFFICE BOX 12194

RESEARCH TRIANGLE PARK, NC 27709

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15.0 MAINTENANCE OF RECORDS 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 International 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 location 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 preparation 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 substance, DIPE, is known to differ markedly in stability and quality 

during storage, producing dangerous 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 continue 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 

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. 

16.0 SAFETY PRECAUTIONS

This study will be conducted in accordance with Nuclear Regulatory 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

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DIPE is a volatile liquid that represents a severe 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 

substance 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 material 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. 

17.0 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, facsimile, or telephone) as soon as feasible (no more than

24 hr 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 protocol 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.

18.0 REFERENCES

Belpoggi, F., Soffritti, 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 rats. 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-

7.

US Food and Drug Administration (2001). Guidance for Industry: Bioanalytical Method Validation.