DRAFT Final Report
METABOLISM, DISPOSITION, AND PHARMACOKINETICS OF DIISOPROPYL ETHER IN MALE AND FEMALE RATS
SUBMITTED TO:
Section 211(b) Research Group
American Petroleum Institute
1220 L Street NW
Washington, DC 20005

TESTING FACILITY:
Discovery Sciences
RTI International*
3040 Cornwallis Road
P.O. Box 12194
Research Triangle Park, NC 27709-2194

RTI Protocol:	RTI-1117

RTI Report No.	RTI/0212608

August 15, 2015

                           Timothy R. Fennell, Ph.D.
                                Study Director

*RTI International is a trade name of Research Triangle Institute.

SUMMARY
This study evaluated the disposition of diisopropyl ether (DIPE) in male and female Fischer 344 rats following nose only exposure and gavage administration, and pharmacokinetics following nose only exposure.    Prior to these studies a pilot study (reported separately) was conducted to develop the nose-only inhalation system, to evaluate the disposition of DIPE in male rats and pharmacokinetics of DIPE in male and female rats.  In this report are the results of nose only inhalation studies to evaluate the disposition of [2-[14]C]DIPE in male and female Fischer 344 rats exposed to 600 or 3600 ppm DIPE for 6 h, and the disposition of [2-[14]C]DIPE at 3600 ppm, following 4 consecutive daily exposures to unlabeled DIPE for 6 h.  The kinetics of DIPE in blood, together with two metabolites, isopropanol and acetone, were determined in blood samples from male and female rats exposed to 600 or 3600 ppm DIPE by nose only inhalation for 6 h.
In Groups 1 and 2, eight male and eight female Fischer 344 rats were exposed to [2-[14]C] DIPE in a nose-only inhalation system at a target concentration of 600 ppm for 6 hours (actual concentration 605  12 ppm).  Following exposure, 4 male and 4 female rats were euthanized immediately and the radioactivity in the carcass was determined by digestion and scintillation counting.  The estimated dose recovered was 100 +- 5 mg/kg for males, and 96.8 +- 2.4 mg/kg for females.   Four male and four female rats were transferred to all-glass metabolism cages, and urine, feces, exhaled CO2 and exhaled volatiles were collected for 24 hr.  After 1 day, the rats were euthanized and the radioactivity remaining in the tissues and carcass was determined by digestion and scintillation counting.  Radioactivity was eliminated primarily as exhaled volatiles (42 and 41 % of the recovered radioactivity, in males and females, respectively), and [14]CO2 (38 and 41%) . Small amounts of radioactivity were recovered in urine (5.1 and 4.2 % of the recovered radioactivity).  A lesser amount was recovered (<1%) in feces, and 7.9 and 8.2 % in carcass at 1 day.   Based on the recovered radioactivity at 24 hr, the estimated dose for male rats was 80.3  2.4 mg/kg, and for female rats was 77.5  1.5 mg/kg.
In Groups 3 and 4, eight male and eight female Fischer 344 rats were exposed to [2-[14]C] DIPE in a nose-only inhalation system at a target concentration of 3600 ppm for 6 hours (actual concentration 3600  48 ppm).  Following exposure, 4 male and 4 female rats were euthanized immediately and the radioactivity in the carcass was determined by digestion and scintillation counting.  The estimated dose recovered was 425 +- 14 mg/kg for males, and 395 +- 47 mg/kg for females.   Four male and four female rats were transferred to all-glass metabolism cages, and urine, feces, exhaled CO2 and exhaled volatiles were collected for 24 hr.  After 1 day, the rats were euthanized and the radioactivity remaining in the tissues and carcass was determined by digestion and scintillation counting.  Radioactivity was eliminated primarily as exhaled volatiles (64 and 71 % of the recovered radioactivity in males and females, respectively), and [14]CO2 (22 and 18 %). Small amounts of radioactivity were recovered in urine (4.2 and 3.6 % of the recovered radioactivity).  A lesser amount was recovered (<1%) in feces, and approximately 5 % in carcass at 1 day.   Based on the recovered radioactivity at 24 hr, the estimated dose for male rats was 301.1  18 mg/kg, and for female rats was 286  22 mg/kg.
In Groups 5 and 6, eight male and eight female Fischer 344 rats were exposed to 4 consecutive days of unlabeled DIPE at 3600 ppm, for 6 hr/day (actual concentration 3555 +- 55 ppm).  This was followed by a single 6-hr nose only exposure to [2-[14]C]DIPE on day 5 (actual concentration 3621  86 ppm).  Following exposure, 4 male and 4 female rats were euthanized immediately and the radioactivity in the carcass was determined by digestion and scintillation counting.  The estimated dose recovered was 786 +- 9 mg/kg for males, and 480 +- 39 mg/kg for females.   Four male and four female rats were transferred to all-glass metabolism cages, and urine, feces, exhaled CO2 and exhaled volatiles were collected for 24 hr.  At 1 day following the end of the last exposure, the rats were euthanized and the radioactivity remaining in the tissues and carcass was determined by digestion and scintillation counting.  Radioactivity was eliminated primarily as exhaled volatiles (45 and 53 % of the recovered radioactivity in males and females, respectively), and [14]CO2 (34 and 26 %). Small amounts of radioactivity were recovered in urine (8.5 and 6.8 % of the recovered radioactivity).  A lesser amount was recovered (<1%) in feces, and approximately 7 and  8 % in carcass at 1 day.   Based on the recovered radioactivity at 24 hr, the estimated dose for male rats was 543.9  11.2 mg/kg, and for female rats was 401.8   59.2 mg/kg.
In Groups 7 and 8, 4 male and 4 female Fischer 344 rats were administered [2-[14]C]DIPE by gavage at a dose of 50 mg/kg.   The rats were transferred to all-glass metabolism cages, and urine, feces, exhaled CO2 and exhaled volatiles were collected for 24 hr.  At 1 day, the rats were euthanized and the radioactivity remaining in the tissues and carcass was determined by digestion and scintillation counting.  Radioactivity was eliminated primarily as exhaled volatiles (52 and 56 % of the administered dose in males and females, respectively), and [14]CO2 (17 and 18 %). Small amounts of radioactivity were recovered in urine (2.5 and 2.2 % of the administered dose).  A lesser amount was recovered (<1%) in feces, and approximately 5 % in the tissues and carcass at 1 day.
In Groups 9 and 10, 4 male and 4 female Fischer 344 rats were administered [2-[14]C]DIPE by gavage at a dose of 200 mg/kg.   The rats were transferred to all-glass metabolism cages, and urine, feces, exhaled CO2 and exhaled volatiles were collected for 24 hr.  At 1 day, the rats were euthanized and the radioactivity remaining in the tissues and carcass was determined by digestion and scintillation counting.  Radioactivity was eliminated primarily as exhaled volatiles (56 and 61 % of the administered dose in males and females, respectively), and [14]CO2 (9 and 9 %). Small amounts of radioactivity were recovered in urine (2 % of the administered dose).  A lesser amount was recovered (<1%) in feces, and approximately 3 % in the tissues and carcass at 1 day.
In Groups 11, 12, 15 and 16, nose-only inhalation exposure of jugular-vein cannulated male and female Fischer 344 rats to 600 ppm (Groups 11 and 12, actual concentration 677 +- 15 ppm) or 3600 ppm DIPE (Groups 15 and 16, actual concentration 3594 +- 198 ppm) for 6 hr was conducted, and serial blood samples were obtained during exposure and up to 18 h following exposure.  Headspace GC-MS analysis of DIPE, isopropanol and acetone was conducted on the blood samples.  In all groups, DIPE was less than the limit of quantitation (LOQ) in preexposure samples.  At both exposure concentrations, DIPE accumulated rapidly during the 6 hours of exposure, and rapidly declined after the exposure.  There was little difference in blood levels at peak concentration between males and females.  All samples were below LOQ by 24 hr. In male and female rats, isopropanol was close to or below LOQ in preexposure samples.  The concentration of isopropanol rose steadily during the exposure, and then declined gradually.  In male rats, blood concentrations of isopropanol reached peak concentrations at approximately twice those of female rats.  All samples were below LOQ by 24 hr.  In male and female rats, acetone was above LOQ in the preexposure samples.  Acetone levels in both male and female rats rose steadily during the exposure, and reaching a plateau following exposure until 7 hr following the initiation of exposure and then declined rapidly reaching preexposure levels by 16 hrs.  The concentration achieved in males exceeded the range of the calibration curve, and for samples obtained between 6 hours and 10 hours are estimated by linear extrapolation of the calibration curve.  The peak concentrations of acetone achieved in male rats were approximately 50% higher than those in female rats.   Non-compartmental pharmacokinetic analysis of DIPE indicted a half-life of approximately 1 hr at both exposure concentrations in male and female rats.  The AUC for DIPE was similar between male and female rats at equivalent exposure concentrations.  The half-life for isopropanol was similar to DIPE and among the exposure concentrations and genders, with values ranging from 1-1.35 hr.  However, the AUC and Cmax values were higher in male rats than in female rats at both exposure concentrations.   The half-lives observed for acetone were longer than DIPE and isopropanol, ranging from 2.4  -  3.0 hr.  The Cmax and AUC values were highest for acetone compared with DIPE and isopropanol, and were higher in male rats than female rats.
This study has shown the rapid uptake and elimination of DIPE on inhalation exposure, reflected in excretion and exhalation of radioactivity, and the determination of DIPE, acetone and isopropanol in blood.   The majority of inhaled DIPE was eliminated as CO2 and exhaled volatiles.  Oral administration of DIPE indicated extensive elimination of radioactivity by exhalation as CO2 and volatiles.
Quality Assurance Statement

Laboratory GLP Compliance Statement

This study was 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 method of synthesis of the test chemical diisopropyl ether and [2-[14]C]-diisopropyl ether, or their location was not available at the time of conduct of the study.   Information on the method of synthesis of [2-[14]C]-diisopropyl ether was not obtained from the vendor.  The supplier of unlabeled diisopropyl ether has been unable to provide information on the method of synthesis.
There were no significant deviations (Appendix A) that would affect the integrity or quality of the study or the interpretation of the results.  Raw data, copies of the inhalation data, and the final report generated as a result of this study are archived at RTI International until they are no longer needed for compliance. Since diisopropyl ether is a volatile and reactive ether, samples of the test chemical or the samples collected during the study will not be retained.

Timothy R. Fennell, Ph.D.		Date
Study Director

                               Table of Contents
                                                                           Page
1.0  	Introduction	1
2.0  	Objectives and Protocol	1
3.0  	Materials and Methods	2
3.1  	Test Substance	2
3.1.1  	Nonradiolabeled diisopropyl ether	2
3.1.2  	[2-[14]C]-labeled diisopropyl ether	2
3.2  	Reference Substances	3
3.3  	Test System	3
3.4  	Test Chemical Preparation and Analysis	7
3.5  	Inhalation Exposures	8
3.5.1	Instrumentation	8
3.5.3	Single Exposures  -  Metabolism and Distribution	8
3.5.4	Single Exposures  -  Pharmacokinetics	8
3.5.5	Repeat Exposures  -  Metabolism and Distribution	8
3.6  	Oral Gavage Administration	8
3.7 	Collection of Samples	9
3.7.1	Blood Sample Collection	9
3.7.2	Excreta	10
3.7.3	Nose Only Tube Contents	11
3.7.4	Carcass	11
3.7.5	Tissues	11
3.8  	Analysis of Samples for Total Radioactivity	12
3.8.1	Excreta and Nose-Only Tube Contents	12
3.8.2	Tissues and Carcass	12
3.8.3	  Exhaled Breath Traps	12
3.8.4	  Exhaled Volatiles Traps	12
3.9  	Analysis of Biological Samples for DIPE, Isopropanol and Acetone	13
3.10  	Data Collection and Reporting	13
3.11  	Pharmacokinetic Analysis	13
3.12  	Statistical Analysis	14
4.0  	Results	14
4.1  	Analysis of Test Substance Identity and Purity	14
4.2  	Test System	15
4.3  	Inhalation ADME Groups	15
4.3.1	Test Article Formulation	15
4.3.2	Inhalation exposure	15
4.3.3  	Radioactivity Retained Following Exposure	15
4.3.4  	Disposition of Radioactivity	15
4.4	Gavage ADME Groups	17
4.4.1	Test Article Formulation	17
4.4.2	Test Article Administration	18
4.4.3  	Disposition of Radioactivity	18
4.5	Pharmacokinetics Study Groups	18
4.4.1	Test Chemical Formulation	18
4.5.2	Inhalation exposure	19
4.5.3  	Concentrations of DIPE, isopropanol and acetone in plasma following DIPE exposure.	19
5.0  	Discussion	20
6.0  	References	22
7.0  	RECORDS and Reports	22
8.0  	Storage of Records and Biological Samples	23
9.0  	REGULATORY COMPLIANCE	23
10.0  	STUDY PARTICIPANTS	23

List of Tables

                                                                           Page
Table 1.  Body Weights of Male and Female Fischer 344 Rats in Inhalation Exposure Groups	25
Table 2.  Body Weights[a] and Dose Administered to Male and Female Fischer 344 Rats in Oral Gavage Groups	26
Table 3.  Body Weights of Male and Female Fischer 344 Rats in Pharmacokinetics Groups	27
Table 4.  Specific Activity of Dose Formulations	28
Table 5.  Summary of DIPE Concentrations Determined During Inhalation Exposure	29
Table 6.  Radioactivity Retained in Carcass following Inhalation Exposure to [2-[14]C]DIPE	30
Table 7.  Total Recovery of Radioactivity following Inhalation Exposure to [2-[14]C]DIPE	32
Table 8.  Disposition of Radioactivity (% of Recovered Radioactivity) Following Inhalation Exposure of Male F344 Rats to 600 ppm [2-[14]C]DIPE  (Group 1)	34
Table 9.  Concentration of DIPE equivalents (ug/g) in Tissues Following Inhalation Exposure of Male F344 Rats to 600 ppm (Group 1)	35
Table 10.  Disposition of Radioactivity (% of Recovered Radioactivity) Following Inhalation Exposure of Female F344 Rats to 600 ppm [2-[14]C]DIPE  (Group 2).	36
Table 11.  Concentration of DIPE equivalents (ug/g) in Tissues Following Inhalation Exposure of Female F344 Rats to 600 ppm [2-[14]C]DIPE  (Group 2).	37
Table 12.  Disposition of Radioactivity (% of Recovered Radioactivity) Following Inhalation Exposure of Male F344 Rats to 3600 ppm [2-[14]C]DIPE  (Group 3)	38
Table 13.  Concentration of DIPE equivalents (ug/g) in Tissues Following Inhalation Exposure of Male F344 Rats to 3600 ppm [2-[14]C]DIPE  (Group 3).	39
Table 14.  Disposition of Radioactivity (% of recovered radioactivity) Following Inhalation Exposure of Female F344 Rats to 3600 ppm [2-[14]C]DIPE  (Group 4)	40
Table 15.  Concentration of DIPE equivalents (ug/g) in Tissues Following Inhalation Exposure of Female F344 Rats to 3600 ppm [2-[14]C]DIPE  (Group 4).	41
Table 16.  Disposition of Radioactivity (Percent of recovered radioactivity) Following Repeat Inhalation Exposure of Male F344 Rats to 3600 ppm DIPE with [2-[14]C]DIPE Exposure on Last Day (Group 5)	42
Table 17.  Concentration of DIPE equivalents (ug/g) in Tissues Following Repeat Inhalation Exposure of Male F344 Rats to 3600 ppm DIPE with [2-[14]C]DIPE Exposure on Last Day (Group 5)	44
Table 18.  Disposition of Radioactivity (% of Recovered Radioactivity) Following Repeat Inhalation Exposure of Female F344 Rats to 3600 ppm DIPE with [2-[14]C]DIPE Exposure on Last Day (Group 6)	45
Table 19.  Concentration of DIPE equivalents (ug/g) in Tissues Following Repeat Inhalation Exposure of Female F344 Rats to 3600 ppm DIPE with [2-[14]C]DIPE Exposure on Last Day (Group 6)	47
Table 20.  Percent of Administered Dose Recovered in Excreta and Tissues 24 h Following Gavage Administration of [2-[14]C]DIPE (50 mg/kg) to Male F344 Rats (Group 7)	48
Table 21.  Concentration of DIPE equivalents (ug/g) in Tissues 24 h Following Gavage Administration of  [2-[14]C]DIPE (50 mg/kg) to Male F344 Rats (Group 7)	49
Table 22.  Percent of Administered Dose in Excreta and Tissues 24 h Following Gavage Administration of [2-[14]C]DIPE (50 mg/kg) to Female F344 Rats (Group 8)	50
Table 23.  Concentration of DIPE equivalents (ug/g) in Tissues 24 h Following Gavage Administration of [2-[14]C]DIPE (50 mg/kg) to Female F344 Rats (Group 8)	51
Table 24.  Percent of Radioactivity Recovered in Excreta and Tissues 24 h Following Gavage Administration of [2-[14]C]DIPE (200 mg/kg) to Male F344 Rats (Group 9)	52
Table 25.  Concentration of DIPE equivalents (ug/g) in Tissues 24 h Following Gavage Administration of [2-[14]C]DIPE (200 mg/kg) to Male F344 Rats (Group 9)	53
Table 26.  Percent of Radioactivity Recovered in Excreta and Tissues 24 h Following Oral Gavage Administration of [2-[14]C]DIPE (200 mg/kg) to Female F344 Rats (Group 10)	54
Table 27.  Concentration of DIPE equivalents (ug/g) in Tissues 24 h Following Oral Gavage Administration of [2-[14]C]DIPE (200 mg/kg) to Female F344 Rats (Group 10)	55
Table 28.  Concentration of DIPE in blood from male and female rats exposed to 600 ppm DIPE.	56
Table 29.  Concentration of Isopropanol in blood from male and female rats exposed to 600 ppm DIPE.	57
Table 30.  Concentration of Acetone in blood from male and female rats exposed to 600 ppm DIPE.	58
Table 31.  Concentration of DIPE in blood from male and female rats exposed to 3600 ppm DIPE.	59
Table 32.  Concentration of Isopropanol in blood from male and female rats exposed to 3600 ppm DIPE.	60
Table 33.  Concentration of Acetone in blood from male and female rats exposed to 3600 ppm DIPE.	61
Table 34.	  Pharmacokinetic Analysis of DIPE, Isopropanol and Acetone in male and female rats exposed to 600 ppm DIPE.	62
Table 35.	  Pharmacokinetic Analysis of DIPE, Isopropanol and Acetone in male and female rats exposed to 3600 ppm DIPE.	62
Table 36.	  Recovery of DIPE in Excreta and Tissues Following Inhalation Exposure of Male and Female F344 Rats to 600 ppm or 3600 ppm	63
Table 37.  Recovery of DIPE in Excreta and Tissues Following Inhalation Exposure of Male and Female F344 Rats to a Single or Repeated Exposure to 3600 ppm DIPE	64
Table 38.  Concentration of DIPE equivalents (ug/g) in Tissues Following Inhalation Exposure of Male and Female F344 Rats to 600 ppm or 3600 ppm	65
Table 39.  Concentration of DIPE equivalents (ug/g) in Tissues Following Inhalation Exposure of Male and Female F344 Rats to single or repeat exposures to 3600 ppm DIPE	66
Table 40.  Recovery of DIPE Following Gavage Administration to Male and Female F344 Rats of 50 or 200 mg/kg DIPE	67
Table 41.  Concentration of DIPE equivalents (ug/g) in Tissues Following Gavage Administration to Male and Female F344 Rats of 50 or 200 mg/kg DIPE	68
Table 42.  Urine, Feces and Cage Rinse from Groups 1-10.	5
Table 43.  CO2 Trap Data from Groups 1-10.	12
Table 44.  Exhaled VOC Trap Data from Groups 1-10	27
Table 45.  NO Tube Contents from Groups 1-6.	42
Table 46.  GI Tract and Contents from Groups 1-6.	44
Table 47.  Tissues and Carcass Data from Groups 1-10.	58
Table 48.  Carcass Digests from Group 1-10.	92
Table 49.  Recovery of Radioactivity in Samples from Group 1	94
Table 50.  Recovery of Radioactivity in Samples from Group 2	95
Table 51.  Recovery of Radioactivity in Samples from Group 3	96
Table 52.  Recovery of Radioactivity in Samples from Group 4	97
Table 53.  Recovery of Radioactivity in Samples from Group 5	98
Table 54.  Recovery of Radioactivity in Samples from Group 6	99
Table 55.  Recovery of Radioactivity in Samples from Group 7	100
Table 56.  Recovery of Radioactivity in Samples from Group 8	101
Table 57.  Recovery of Radioactivity in Samples from Group 9	102
Table 58.  Recovery of Radioactivity in Samples from Group 10	103
Table 59.  Blood Sample Collection Times for Groups 11 and 12	104
Table 60.  Blood Sample Collection Times for Groups 15 and 16	108
Table 61.  Blood Sample Weights for Groups 11 and 12.	112
Table 62.  Blood Sample Weights for Groups 11 and 12.	116
Table 63.  Low Calibration curve for DIPE in blood for 600 ppm exposures.	120
Table 64.  High Calibration curve for DIPE in blood for 600 ppm exposures.	121
Table 65.  Low Calibration curve for Isopropanol in blood for 600 ppm exposures.	122
Table 66.  High Calibration curve for Isopropanol in blood for 600 ppm exposures.	123
Table 67.  Low Calibration curve for Acetone in blood for 600 ppm exposures.	124
Table 68.  High Calibration curve for Acetone in blood for 600 ppm exposures.	125
Table 69.  Sample Analysis for DIPE in blood from male rats exposed to 600 ppm DIPE.	126
Table 70.  Sample Analysis for DIPE in blood from female rats exposed to 600 ppm DIPE.	128
Table 71.  Sample Analysis for Isopropanol in blood from male rats exposed to 600 ppm DIPE.	130
Table 72.  Sample Analysis for Isopropanol in blood from female rats exposed to 600 ppm DIPE.	132
Table 73.  Sample Analysis for Acetone in blood from male rats exposed to 600 ppm DIPE.	134
Table 74.  Sample Analysis for Acetone in blood from female rats exposed to 600 ppm DIPE.	136
Table 75.  Sample Analysis for DIPE in blood from male rats exposed to 3600 ppm DIPE.	138
Table 81.  Sample Analysis for DIPE in blood from female rats exposed to 3600 ppm DIPE.	145
Table 82.  Sample Analysis for Isopropanol in blood from male rats exposed to 3600 ppm DIPE.	147
Table 83.  Sample Analysis for Isopropanol in blood from female rats exposed to 3600 ppm DIPE.	149
Table 84.  Sample Analysis for Acetone in blood from male rats exposed to 3600 ppm DIPE.	151
Table 85.  Sample Analysis for Acetone in blood from female rats exposed to 3600 ppm DIPE.	153

List of Figures

                                                                           Page
Figure 1.  Cumulative Excretion of Radioactivity in Urine and Feces, CO2 and Exhaled VOC Traps for Male (Group 1) and Female (Group 2) Rats Exposed to 600 ppm [2-[14]C]DIPE	69
Figure 2.  Cumulative Excretion of Radioactivity in Urine and Feces, CO2 and Exhaled VOC Traps for Male (Group 3) and Female (Group 4) Rats Exposed to 3600 ppm [2-14C]DIPE	70
Figure 3.  Cumulative Excretion of Radioactivity in Urine and Feces, CO2 and Exhaled VOC Traps for Male (Group 5) and Female (Group 6) Rats Exposed to 3600 ppm DIPE for 4 Days Followed by Exposure to 3600 ppm [2-14C]DIPE on Day 5	71
Figure 4.  Cumulative Excretion of Radioactivity in Urine and Feces, CO2 and Exhaled VOC Traps for Male (Group 7) and Female (Group 8) Rats Administered 50 mg/kg [2-14C]DIPE by Gavage	72
Figure 5.  Cumulative Excretion of Radioactivity in Urine and Feces, CO2 and Exhaled VOC Traps for Male (Group 9) and Female (Group 10) Rats Administered 200 mg/kg [2-14C]DIPE by Gavage	73
Figure 6.  Blood Concentrations (Mean +- SD) of DIPE in Male and Female Rats Exposed to 600 ppm DIPE for 6 hr.	74
Figure 7.  Blood Concentrations (Mean +- SD) of Isopropanol in Male and Female Rats Exposed to 600 ppm DIPE for 6 hr.	75
Figure 8.  Blood Concentrations of Acetone (Mean +- SD) in Male and Female Rats Exposed to 600 ppm DIPE for 6 hr.	76
Figure 9.  Blood Concentrations of DIPE (Mean +- SD) in Male and Female Rats Exposed to 3600 ppm DIPE for 6 hr.	77
Figure 10.  Blood Concentrations of Isopropanol (Mean +- SD) in Male and Female Rats Exposed to 3600 ppm DIPE for 6 hr.	78
Figure 11.  Blood Concentrations of Acetone (Mean +- SD) in Male and Female Rats Exposed to 3600 ppm DIPE for 6 hr.	79

                              List of Appendices

Appendix A:	Approved Study Protocol, Amendments, and Deviations

Appendix B:	Test Chemical Analysis Report Diisopropyl Ether

Appendix C:	Test Chemical Analysis Report [2-[14]C] Diisopropyl Ether

Appendix D:	Method Validation for Analysis of DIPE, Isopropanol and Acetone

Appendix E:	Inhalation Summary Report

Appendix F:	Pharmacokinetic Analysis

Appendix G	Equations Used in Calculations

Appendix H 	Raw Data Tables

Appendix I	Analysis of Oral Dose Formulations

Appendix J	RTI-934, Metabolism and Pharmacokinetics of Diisopropyl Ether in Male and Female Rats: Pilot Study

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) 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 health risks of DIPE is the investigation of the uptake, pharmacokinetics and metabolism of DIPE following inhalation (the expected primary routes of exposure) and oral dosing in rats.  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 own metabolism by enzyme induction or inhibition.
A pilot study has been conducted to evaluate the disposition of [2-[14]C]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, contained in Appendix J).  A nose-only inhalation system was set up and its performance verified prior to exposure of animals.  The pilot study found that DIPE is absorbed by inhalation, and rapidly eliminated as exhaled volatiles (approximately 70 %), and as [14]CO2 (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 in the pilot study to be small, so no special precautions will be taken to avoid loss of radioactivity during euthanasia on the definitive studies. On exposure to unlabeled DIPE by inhalation in the pilot study, DIPE, and metabolites acetone and isopropanol were detected and quantitated by gas chromatography-mass spectrometry.  Metabolites in urine will not be characterized in these definitive studies because of the low amount of radioactivity excreted by this route during the pilot study.

2.0  	Objectives and Protocol
The objectives of this study were 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 [2-[14]C]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 [2-[14]C]DIPE/DIPE and evaluate the disposition during and following exposure.
      3. Conduct a single gavage administration (low and high dose) of [2-[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.
      The study protocol is presented in Appendix A.
3.0  	Materials and Methods
3.1  	Test Substance
    NAME:  Diisopropyl ether (DIPE; CAS No. 108-20-3)
    MOLECULAR FORMULA:  C6H14O
    MOLECULAR WEIGHT:  102.17
STRUCTURE:

3.1.1  	Nonradiolabeled diisopropyl ether
      Source:  	The non-labeled DIPE was purchased from Sigma-Aldrich, Milwaukee, WI.  A certificate of analysis from the vendor indicated purity of 99.3% by GLC, with 7 PPM BHT
      Product Number:  	398276
      Lot Number:  	52396DM

      The unlabeled test material was characterized at RTI by gas chromatographic (GC) analysis for purity and stability, and for identify by [1]H and [13]C Nuclear Magnetic Resonance (NMR) spectroscopy and by GC-mass spectroscopy.  Details of the analysis are included in Appendix B.
3.1.2  	[2-[14]C]-labeled diisopropyl ether
      Position of radiolabel:	[2-[14]C]-
      Source:	American Radiolabeled Chemicals, Inc. St. Louis MO, prepared as a custom synthesis
      Specific Activity:	2 mCi/mmol
      Product No.:	ARC-3099
      Lot No.:	120308
      Identity:	The identity of [2-[14]C]DIPE ether was confirmed by chromatographic comparison with unlabeled DIPE using HPLC coupled with radioactivity detection.
      Purity:	The radiochemical purity of [2-[14]C]DIPE was determined chromatographically to be 97.3 %, using the HPLC system described in Appendix C.
      The labeled test material was characterized at RTI by HPLC coupled with radioactivity and uv (195 nm) detection for purity and stability, and for identify by [1]H NMR spectroscopy and by GC/MS..  Details of the analysis are included in Appendix C.   Analysis of the radiolabeled material indicated that it contained approximately 50 % of unlabeled pentane.  It is most likely that this arose from the purification of the radiolabel by the vendor, and could not be removed.  Since substantial dilution of the radiolabel with unlabeled DIPE is needed to achieve the appropriate mass and specific activity, estimation of the amount of contamination was conducted prior to conducting the in-life investigations reported.  Estimates of the % pentane in each formulation are included in Table 4.
3.2  	Reference Substances
Isopropanol (2-propanol, product number 34863, lot number SHBB1344V) was obtained from Sigma-Aldrich.  The purity specified by the Vendor's certificate of analysis was 99.98%.  Acetone, (product number 270725, lot number SHBB6647V) was obtained from Sigma-Aldrich.  Purity from the Vendor Certificate of Analysis was 99.9%.

3.3  	Test System
Source:  Male and female Fischer 344 rats were purchased from Charles River (Kingston, NY).  Animal weights at the time they were used in studies are shown in Table 1.  Male and female Fischer 344 rats with jugular catheters were purchased from the same source.
Diet/Water:  Animals were fed Certified Purina Rodent Chow #5002 and were furnished tap water ad libitum.  The analysis of each feed batch for nutrient levels and possible contaminants was performed by the supplier, examined by the Study Director, and maintained in the study records.  The feed was stored at approximately 6070 F, and the period of use did not exceed six months from the milling date.  The source of the water was the City of Durham, NC.  Approximately once a year, the City of Durham provides analyses of the drinking water for potential contaminants.  Documentation of these analyses were inspected by the Study Director and maintained in the study records.  In addition, samples of water were collected during the study for analysis of diisopropyl ether, tertiary amyl methyl ether, ethyl tertiary butyl ether, diisopropyl ether, and methyl tertiary butyl ether.  The samples were sent to Kiff Analytical (Davis, CA 95616) for analysis.  The results were examined by the Study Director and maintained in the study records.
Identification:  Individual ear tags were used to uniquely identify animals used.
Housing:  Rats were housed (maximum of three per cage) in polycarbonate cages with stainless steel bar lids accommodating a water bottle until they were used in an experiment.  Cage sizes are 19" x 10.5" x 8" high (143 sq. in. floor space).  Contact bedding was Sani-Chips hardwood chips (P. J. Murphy Forest Products Co.; Montville, NJ).  Rats with jugular cannulas were singly housed.
When necessary for sample collection rats were housed post exposure in individual glass metabolism chambers for collection of urine, feces, exhaled CO2, and exhaled volatiles.  Three unexposed rats (2 male and 1 female) were housed in individual glass metabolism chambers for 1 day for collection of urine and feces for determination of radioactivity background.
Quarantine:  Rats were quarantined for a minimum of three days before use on a study, except for rats with jugular cannulas, for which the quarantine period was shortened to 1-2 days.  A veterinarian or qualified designee examined the animals prior to their release from quarantine.
Assignment of animals to Groups:  Animal exposures were conducted in separate groups, separated in time. Group designations are shown in the table below for ADME endpoints for Groups 1-10, which included the collection of urine, feces, blood, tissues, exhaled volatiles, and CO2.  For inhalation exposures where male and female rats were exposed simultaneously to the same exposure atmosphere, a single group of males and a single group of females were obtained at the same time.

Study Design:  Inhalation and Oral ADME Study Groups
Group[a]
Animals
Exposure Route
Exposure
                                     N[f]
Sample Collection
End
points
1[a]
Male Rats
Inhalation[e]
600 ppm [14]C DIPE/DIPE, 6 hours
                                       8
Urine, feces, blood, tissues, volatiles, CO2
ADME
2 a
Female Rats
Inhalation[e]
600 ppm [14]C DIPE/DIPE, 6 hours
                                       8
Urine, feces, blood, tissues, volatiles, CO2
ADME
3 b
Male Rats
Inhalation[e]
3600 ppm [14]C DIPE/DIPE, 6 hours
                                       8
Urine, feces, blood, tissues, volatiles, CO2
ADME
4 b
Female Rats
Inhalation[e]
3600 ppm [14]C DIPE/DIPE, 6 hours
                                       8
Urine, feces, blood, tissues, volatiles, CO2
ADME
5 c
Male Rats
Inhalation[e]
3600 ppm unlabeled DIPE for 4 days, and [14]C DIPE/DIPE 6 hours
                                       8
Urine, feces, blood, tissues, volatiles, CO2
ADME
6 c
Female Rats
Inhalation[e]
3600 ppm unlabeled DIPE for 4 days, and [14]C DIPE/DIPE 6 hours
                                       8
Urine, feces, blood, tissues, volatiles, CO2
ADME
7 d
Male Rats
Gavage
50 mg/kg [14]C DIPE/DIPE
                                       4
Urine, feces, blood, tissues, volatiles, CO2
ADME
8 d
Female Rats
Gavage
50 mg/kg [14]C DIPE/DIPE
                                       4
Urine, feces, blood, tissues, volatiles, CO2
ADME
9 d
Male Rats
Gavage
200 mg/kg [14]C DIPE/DIPE
                                       4
Urine, feces, blood, tissues, volatiles, CO2
ADME
10[d]
Female Rats
Gavage
200 mg/kg [14]C DIPE/DIPE
                                       4
Urine, feces, blood, tissues, volatiles, CO2
ADME
[a] Inhalation exposures for Groups 1 and 2 were conducted concurrently.
[b] Inhalation exposures for Groups 3 and 4 were conducted concurrently.
[c] Inhalation exposures for Groups 5 and 6 were conducted concurrently.
[d] Gavage administration for Groups 7 - 10 were conducted concurrently.
[e] Inhalation exposures were nose-only.
      f Additional animals, designated as "extra" were ordered for each exposure group.

Study Design.  Inhalation and Oral Pharmacokinetic Study Groups
Group
Animals
Exposure Route
Exposure
                                     N[c]
Sample Collection
End
points
11[a]
Male Rats
Inhalation
600 ppm DIPE, 6 hours
                                       8
Blood
PK
12 a
Female Rats
Inhalation
600 ppm  DIPE, 6 hours
                                       8
Blood
PK
15 b
Male Rats
Inhalation
3600 ppm  DIPE, 6 hours
                                       8
Blood
PK
16[b]
Female Rats
Inhalation
3600 ppm  DIPE, 6 hours
                                       8
Blood
PK
[a] Inhalation exposures for Groups 11 and 12 were conducted concurrently.
[b] Inhalation exposures for Groups 15 and 16 were conducted concurrently. Note that inhalation exposure of Groups 13 and 14 was conducted, but because of analytical issues, these exposures were repeated with Groups 15 and 16.  Data are not reported for Groups 13 and 14.
      [c] Additional animals, designated as "extra" were ordered for each exposure group.

Separate shipments of animals were obtained for Groups 5/6, 1/2, 3/4, 7-10, 11/12 and 15/16.  Eleven rats per sex for Groups 5/6 were weighed and exposed to nonradiolabeled DIPE by nose only inhalation for 4 days.  Body weights of the rats were reviewed as were observations made during exposures and rats that appeared to acclimate well and showed the least percentage decrease in body weight for each sex were assigned to the last exposure to [2-[14]C]DIPE .  Prior to the start of the exposures, animals were acclimated to nose only tubes for approximately 30 minutes.  Within a Group and sex for Groups 1-4 and 15/16, animals were assigned study IDs in order of eartag numbers from lowest to highest after animals that did not acclimate well to the nose only tubes prestudy were eliminated.  For Groups 11 and 12, animals were assigned study IDs in order of eartag numbers (lowest to highest) in the order subject 5-8, then subject 1-4 and then any extras as may have been exposed.  .   For Groups 7-10, the ear tag numbers were assigned in numerical order to sequential values using a series of computer generated numbers (one series per sex) 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.
Environmental:  Temperature and relative humidity in RTI animal rooms were continuously monitored, controlled, and recorded using an automated system (Siebe/Barber-Colman Network 8000 System with Revision 4.4.1 for Signal software [Siebe Environmental Controls (SEC)/Barber-Colman Company; Loves Park, IL]).  The target environmental ranges were 6479 F (18 C - 26 C) for temperature and 3070% relative humidity, with a 12-h light cycle per day.
Euthanasia:  At the end of the in-life phase, the rats were euthanized by overexposure to carbon dioxide.
3.4  	Test Chemical Preparation and Analysis
For inhalation exposures to unlabeled DIPE (Groups 5/ 6, 11/12 and 15/16), an appropriate amount of unlabeled DIPE was prepared for generation of the exposure atmosphere, 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-[14]C]DIPE/DIPE (Groups 1-4 and Day 5 of exposure for Groups 5 and 6), [2-[14]C]DIPE was added to an appropriate amount of unlabeled DIPE for generation of the exposure atmosphere.   The amount of labeled and unlabeled DIPE required was 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-[14]C]DIPE was weighed into a tared flask with a Teflon faced screw cap.  The weight of the labeled chemical added was recorded.   Unlabeled DIPE was added and the weight added was recorded.  The specific activity of the DIPE mixture was calculated based on the recorded weights and LSS analysis of aliquots as follows.  Weighed aliquots were added to known (weighed) amounts of DMF in sealed vials and then weighed aliquots of these dilution samples were added to scintillation vials containing Ultima Gold(TM) scintillation cocktail (Perkin Elmer) and were assayed by liquid scintillation spectroscopy (LSS) for radiochemical content.  The specific activity of the [2-[14]C]DIPE/DIPE formulation was then calculated from the data obtained.
The exposure atmosphere concentration was monitored using  calibrated Miran IR detectors.   The concentration of DIPE was monitored at the inlet to the tower, and at the outlet as described in the Inhalation Summary Report: Nose Only Exposures at RTI International (Appendix E).
The oral dose formulation was prepared by combining nonradiolabled DIPE, [2-[14]C]DIPE, Cremophor and water appropriate to administration of 200 mg/kg DIPE in a dose volume of 10 mL/kg.  The same formulation was used for both dose levels and was administered at the rate of 10 mL/kg for the 200 mg/kg dose and 2.5 mL/kg for the 50 mg/kg dose.  The dose vehicle was 10 % Cremophor (Sigma Lot BCBG5391V ) in distilled deionized water.  The formulation was prepared in a flask with a Teflon face screw cap;  the tare weight of the container and the weights of each individual component added were recorded.  The container was chilled on dry ice prior to opening for each addition, then was capped tightly and allowed to warm to room temperature before weighing.  Components were added in the order listed above and the contents of the container were mixed after addition of the Cremophor and again after addition of water until the formulation appeared homogenous.  Weighed aliquots of the formulation were added to weighed samples of 1:1 acetonitrile:water in vials with Teflon lined screw caps.  Weighed aliquots of these dose formulation dilution samples were assayed for radiochemical content by LSS.  The specific activity of the [2-[14]C]DIPE/DIPE in the dose formulation was calculated from the weight and LSS data.  HPLC and GC analysis was conducted for the oral dose formulations to verify concentration of DIPE in the solution.
3.5  	Inhalation Exposures
3.5.1	Instrumentation
The generation, exposure and monitoring systems are described in the `Inhalation Summary Report: Nose Only Exposures at RTI International'  AppendixE.
3.5.3	Single Exposures  -  Metabolism and Distribution
Eight male (Group 1) and eight female (Group 2) F344 rats were exposed for approximately 6 hr to a target concentration of 600 ppm [2-[14]C]DIPE/DIPE via nose only inhalation.  Eight male (Group 3) and eight female (Group 4) F344 rats were exposed for approximately 6 hr to 3600 ppm.
3.5.4	Single Exposures  -  Pharmacokinetics
Eight male (Group 11) and eight female (Group 12) F344 rats with jugular vein cannulas were exposed for approximately 6 hr to 600 ppm DIPE via nose only inhalation.  Note that groups 13 and 14 were removed from the analysis.  Eight male (Group 15) and eight female (Group 16) F344 rats were exposed for approximately 6 hr to 3600 ppm DIPE via nose only inhalation.
3.5.5	Repeat Exposures  -  Metabolism and Distribution
Eleven male (Group 5) and eleven female (Group 6) F344 rats were exposed for approximately 6 hr/day to 3600 ppm unlabeled DIPE via nose only inhalation for 4 days.  On the fifth day, eight rats per sex were exposed for approximately 6 hr to 3600 ppm [2-[14]C]DIPE /DIPE.
3.6  	Oral Gavage Administration
Male (Group 7, N=4) and female rats (Group 8, N=4) were administered a single dose of [2-[14]C]DIPE/DIPE by gavage at 50 mg/kg.  Male (Group 9, N=4) and female (Group 10, N=4) rats were administered a single dose of [2-[14]C]DIPE/DIPE by gavage at 200 mg/kg. The formulation was prepared at a single concentration of 20 mg/mL in 10% Cremophor EL in water.  Analysis was conducted by headspace GC-MS using a method similar to that use for the analysis of blood.  The formulation was diluted in 10% dimethylformamide in water (10 uL diluted in a total volume of 2 mL).  An aliquot was added to a 10-mL headspace vial and crimp sealed.  To the sealed vial was added through the septum with a syringe a 10 uL aliquot  of internal standard (16  uL of MTBE in a total of 50 mL of water).  The samples were analyzed with the instrument conditions outlined below.  A calibration curve was prepared over the range of 5  -  250 ug/mL.

Conditions for Dose Formulation Analysis of DIPE
6890 GC

Injection port
split/splitless

Temperature
150 ºC

Split ratio
15:1 (calculated by adding flow rate of HSS )
Carrier gas
Helium

Flow rate
2.5 ml/min

Column
DB-624 30m x 0.32 mm i.d. 1.8 um film thickness

(J&W, Agilent technologies, Wilmington, DE)

GC oven program

Initial temperature
30 ºC

Initial time
2.5 min

Temperature rate
5 ºC/min

Final temperature
35ºC

Final time
1.25 min

Temperature rate A
90 ºC/min

Final temperature A
150 ºC

Run time
6.03 min

G1888A headspace sampler

Loop size
1 ml

Vial Pressure
15 psig

Carrier pressure
6.0 psig

Headspace oven
65 ºC

loop temperature
90 ºC

Transfer line temperature
110 ºC

Equilibration time
10 min

GC cycle time
10 min

Pressurization
0.2 min

Loop Fill
0.2 min

Loop Equilibration
0.05 min

Inject
0.5 min

Shake
low

5973 MSD

SIM
DIPE(m/z 87); MTBE (m/z 73)
Quad temperature
150 ºC

Source temperature
230ºC

Transfer line
230 ºC

Tune
Atune.u

Solvent delay
2.5 min

3.7 	Collection of Samples
3.7.1	Blood Sample Collection
<IP_ADDRESS>	ADME Groups
For Groups 1-10, blood was collected from 4 rats per group at termination (24 h following termination of inhalation exposure or 24 h following gavage administration) by cardiac puncture into heparinized syringes.
<IP_ADDRESS>	PK Groups
For Groups 11, 12, 15 and 16, blood samples were collected by jugular vein cannula prior to dosing from each rat (time = 0), and from 3 or 4 rats per group at approximately 5, 10, 15, and 30 min, and at approximately 1, 2, 4 and 6 hr after the exposure initiation.  At the end of the 6 hr exposure, all rats were removed from the inhalation exposure tower, and at least 4/group were placed in cages metabolism cages for continued blood and excreta collection, while 4/group were euthanized immediately (see Section 3.6.4).  Blood was drawn from 3 or 4 rats per group at approximately 375, 390, 420, 440, 480, 600, 960, and 1440 min after the beginning of the exposure (at approximately 15, 30, 60, and 80 min, and 2, 4, 10, and 18 hr after the end of the exposure).  To facilitate the withdrawal of blood samples, animals were stagger-started on the exposure tower.
Blood samples (approximately 100 L each) were drawn from the jugular vein cannula into a heparinized 1 mL syringe, and the sample was immediately placed in a preweighed headspace vial.  The vial was immediately crimped and weighed, and the weight of the blood aliquot was determined.  The blood samples were maintained on ice, until placed in the headspace autosampler for analysis.  All blood samples were analyzed by GC-MS within 24 hours of collection.
Blood samples from unexposed rats were used for preparation of standard curves.  Blood samples were collected by cardiac puncture under CO2 anesthesia, and the rats were euthanized.

3.7.2	Excreta
In Groups 1-10, 4 rats per group were placed in all-glass metabolism cages for the separate collection of urine, feces, exhaled volatiles, and exhaled CO2 at the end of inhalation exposure to [2-[14]C]DIPE or immediately following gavage dosing.  Urine and feces were collected over dry ice at 024 hours after termination of exposure.  Exhaled volatile organics were collected on a series of two charcoal traps (Product Number 226-16, SKC Sorbent Tube, Anasorb CSC, Coconut Charcoal, 10 X 110 mm size, 2-section, 200/800 mg sorbent, SKC Inc, Eighty Four, PA) over the intervals 01, 13, 36 and 624 hours after termination of exposure.   Expired [14]CO2 was collected in 1.0 N KOH over the intervals 01, 13, 36 and 624 hours after termination of exposure.   At the end of excreta collection, the cages were rinsed with water and with ethanol, and these rinses were combined.   The rinses were analyzed for total radioactivity.  The weight of urine and/or feces collected for each sample interval was recorded.  Urine and feces were analyzed for total radioactivity.  Excreta were stored at approximately -20 C in the dark.
Three unexposed rats were housed in all-glass metabolism cages for 24 hours, as described above, for the collection of urine and feces.  The samples collected were used to supply background samples for the analysis of radioactivity.
3.7.3	Nose Only Tube Contents
For each of the [2-[14]C]DIPE inhalation exposure groups (1-6), four rats per group  were transferred immediately at the end of the exposure period to glass metabolism cages for the separate collection of excreta, expired VOC and expired [14]CO2.  Any excreta from the nose-only exposure tube was transferred to a vial, weighed and stored at -20 °C.
3.7.4	Carcass
For each of the [2-[14]C]DIPE inhalation exposure groups (1-6), four rats per group (still in the nose-only restraint tubes) were euthanized by CO2 exposure immediately following termination of DIPE exposure.   The carcass and any urine and feces in the restraint tube was immediately  transferred to a Nalgene bottle, weighed and then digested with 2N ethanolic NaOH.
Following collection of tissues at necropsy for Groups 1-10, remaining carcass and excess blood was added to a Nalgene bottle, weighed and then digested in 2N ethanolic NaOH.
Carcasses from Groups 11, 12, 15 and 16 were not analyzed.
3.7.5	Tissues
At the end of each disposition experiment, the animals were euthanized and blood was withdrawn into a heparinized syringe by cardiac puncture.  Tissues listed in the table below were harvested and weighed.  GI tract tissues and contents were weighed and digested in 2 N ethanolic NaOH.  The remaining tissues (in their entirely, except liver and dispersed tissues such as skin, fat and muscle) were divided into suitable aliquots for digestion and weighed into scintillation vials containing approximately 2 mL Soluene-350.  Liver was homogenized prior to digestion of aliquots in Soluene-350.  A sample of blood (about 1 mL) was reserved and stored frozen at -20 °C.  The remaining carcass and blood from each rat were combined and digested as described in 3.6.3.

skin (hair shaved)
lungs
subcutaneous fat
brain
abdominal fat
testes (epididymis removed) or ovaries and uterus
muscle (gastrocnemius)
stomach
femur
stomach contents
liver
small intestine
kidneys
small intestine contents
spleen
cecum + large intestine + rectum
heart
cecum + large intestine contents
blood

3.8  	Analysis of Samples for Total Radioactivity
Ultima Gold(TM) scintillation cocktail was used in all determinations of radiochemical content.  Radioactivity was determined using a Packard 1900CA Liquid Scintillation Counter.
3.8.1	Excreta and Nose-Only Tube Contents
Duplicate weighed aliquots of urine (approximately 0.05 g) and cage rinse (approximately 0.5 g) and the nose-only (NO) tube contents samples were analyzed directly (without solubilization or bleaching) for radiochemical content.  Duplicate aliquots of urine were weighed into scintillation vials containing scintillation cocktail.  Feces were homogenized with an approximately equal mass of water.  The weight of the feces homogenate was determined, and duplicate homogenate aliquots were weighed into scintillation vials.  After solubilization of the homogenate aliquots with Soluene-350(TM) (about 2 mL per sample), samples were bleached (by adding approximately 125 ul of 70% perchloric acid, and then approximately 0.3 ml of 30% H2O2) then scintillation cocktail was added to the vials, and the samples were analyzed for total radioactivity by LSS.  Control samples of urine and feces were collected from unexposed animals and analyzed as described above to determine background counts.
3.8.2	Tissues and Carcass
Carcasses and GI tract tissues and contents were analyzed for total radioactivity following solubilization in 2N ethanolic NaOH.  Duplicate or triplicate weighed aliquots of the solubilized carcass and GI tract samples were analyzed.  Tissues were analyzed for total radioactivity following solubilization in Soluene-350(TM) (about 2 mL per tissue sample).  Solubilized tissue samples were bleached (by adding approximately 125 uL of 70% perchloric acid, and then approximately 0.3 mL of 30% H2O2) prior to addition of scintillation cocktail and analysis by LSS.
Control samples of carcass and tissues were collected from unexposed animals and analyzed as described above to determine background counts.
3.8.3	  Exhaled Breath Traps
Duplicate aliquots of 1.0 N KOH from the exhaled breath trap for CO2 were analyzed by LSS after addition of scintillation cocktail.  Control samples of 1 N KOH were analyzed as described above to determine background counts.
3.8.4	  Exhaled Volatiles Traps
The contents of each charcoal trap were extracted with dimethylformamide (DMF) to determine absorbed radioactivity.  For a single extraction, charcoal was transferred to a centrifuge tube, DMF (8.0 mL) was added, and the sample weighed.  After vortexing for approximately 30 seconds, the samples were centrifuged, and the supernatant was removed.  Radioactivity was determined in duplicate weighed aliquots of the DMF extract by LSS. Control samples of DMF were analyzed as described above to determine background counts.
3.9  	Analysis of Biological Samples for DIPE, Isopropanol and Acetone
Blood collected in Groups 11, 12, 15 and 16 was analyzed for DIPE, acetone and isopropanol using the validated Project Specific Method for Analysis of Diisopropyl Ether in Blood Samples (AM-0209408.001) described in Appendix D.  The method consists of GC/MS analysis of DIPE, isopropanol and acetone with injection of headspace from sample vials, and detection using a mass-selective detector.  The method incorporated calibration standards for DIPE, acetone and isopropanol in order to measure amounts of these compounds in blood samples.

3.10  	Data Collection and Reporting
Study data was collected and reported in the Debra(TM) system version <IP_ADDRESS>.  This includes data for pot weights, sample weights, homogenate weights and aliquot weights, and scintillation counting data.  Calculations of sample data are described in Appendix G, and were reported with the Debra(TM) system.  Individual values for sample weights and radioactivity determinations are presented in Appendix H.  Data for radioactivity where the sample aliquot dpm values were below the background for the sample were considered to be less than the limit of detection, and are set to 0 dpm per aliquot.  Data for all samples that were above the limit of detection are reported, and included in calculations.  However, where the sample aliquot dpm values were less than three times the background for the sample, the sample has been flagged with an asterisk in the Summary Tables and appendix tables.
3.11  	Pharmacokinetic Analysis
Non-compartmental pharmacokinetic analysis of DIPE, isopropanol and acetone was conducted using WinNonlin version 1.5 (Pharsight Corporation, Cary NC).  The analysis of kinetic data was conducted using mean values for each timepoint, because samples for all of the timepoints could not be collected from individual animals.  As a result, a single value was obtained for each parameter, and statistical analysis was not conducted on the various parameters calculated.  The complete data analysis is shown in the Appendix F.
The following parameters were estimated for concentration vs. time data: maximum observed concentration and time at which the maximum concentration was observed (Cmax and Tmax, respectively), terminal elimination rate constant (λz) and half-life (T1/2λz), area under the concentration vs. time curve extrapolated to infinity (AUCinf) and area under the concentration vs. time curve extrapolated to infinity divided by dose administered (AUCinf/D).
Glossary of Kinetic Parameters
                               WinNonlin Output
                                 Table/Report
                                  Definition
                                     Cmax
                                     Cmax
Maximum observed concentration, occurring at Tmax
                                     Tmax
                                     Tmax
Time at which Cmax occurs
                                   Lambda_z
                                   Lambda z
Terminal elimination rate constant
                                 t1/2_Lamda_z
                                     T1/2
Terminal elimination half-life
                                    AUCall

                                    AUCall

Area under the plasma concentration-time curve

3.12  	Statistical Analysis
Statistical analysis was conducted in SAS version 9, with Student's t-Test was used for all comparisons.  A test of equality of variances was included in the analysis.  If the equality of variances test was not significant then the t-Test method reported was the pooled method, if it was significant then the Satterthwaite method was reported.  For recovery of dose, and tissue concentrations, comparisons were made between gender at the same exposure, between exposure concentration or dose for the same gender, and between single and repeated exposures.  Significance is reported for values with P<0.05, 0.01, or 0.001.

4.0  	Results
4.1  	Analysis of Test Substance Identity and Purity
Information on the identity, purity, and stability of the test chemical is included in Appendix B.  Prior to study initiation, the test chemical purity was determined by GC to be 99.18  0.19 %.  Following completion of the study, test chemical purity was determined to be 99.43  0.01 %.  The radiochemical purity of [2-[14]C]DIPE specified by the vendor was 96.18 % by HPLC, and was verified by HPLC at RTI prior to the inhalation exposure of rats as 97.3  0.53% (Appendix C).  The radiochemical purity of the [2-[14]C]DIPE/DIPE oral dose formulation was 92.8 +- 0.15% on the day of dose administration.
4.2  	Test System
Male and female Fischer 344 rats were used for the inhalation exposures to [2-[14]C]DIPE (Table 1), gavage administration of [2-[14]C]DIPE/DIPE (Table 2), and inhalation exposure to unlabeled DIPE (Table 3).

4.3  	Inhalation ADME Groups
4.3.1	Test Article Formulation
For Groups 1-4 and for the last day of exposure for Groups 5 and 6, the test substance was prepared by mixing unlabeled DIPE with [2-[14]C]DIPE, and used without further dilution.  The test chemical formulation for the first 4 days of exposure for Groups 5 and 6 consisted of unlabeled DIPE alone.  The specific activity of each formulation is reported in Table 4.  Estimates of the percentage of pentane contaminant in the inhalation exposures are also included, and were approximately 0.5  -  0.7 %.
4.3.2	Inhalation exposure
The exposure concentration data for each six-hour exposure of rats to target concentrations of 600 or 3600 ppm DIPE is shown in Table 5.  The details of the actual exposure are reported in Appendix E, Inhalation Summary Report:  Nose-Only Inhalation Exposures at RTI International.
4.3.3  	Radioactivity Retained Following Exposure
Four rats in each group were euthanized immediately following inhalation exposure to [2-[14]C]DIPE/DIPE (Groups 1-6).  The amount of radioactivity retained in the carcass was determined by solubilization followed by scintillation counting.  The total radioactivity retained and the estimated doses in mg/kg are presented in Table 6.  Following exposure to 600 and 3600 ppm, the mean amount of radioactivity recovered ranged from 4.6 -7.0 uCi and 9.9-14 uCi, respectively.  For each dose level, the calculated dose for male rats was higher than for female. When converted to mg DIPE, and normalized to body weight, the single exposures produced similar doses in male and females.  Calculated mean doses (mg/kg) were 96.8 mg/kg in females and 100 mg/kg in males at 600 ppm.  After a single exposure to 3600 ppm, males retained 425 mg/kg, compared with 395 mg/kg in females.  A larger amount (11.8-23.7 uCi) of radioactivity was retained following repeat exposure to 3600 ppm DIPE.  For the last radiolabeled exposure, the retained dose was higher in males than the single exposure.
4.3.4  	Disposition of Radioactivity
Four rats from each Group 1-6 were placed in metabolism cages for collection of urine and feces and exhaled breath immediately following the inhalation exposure to [2-[14]C]DIPE.  Transfer of the rats from the inhalation tower to the metabolism cage was conducted with the rat retained in the nose-only tube.  Following transfer of the rat to the metabolism cage, the nose-only tube contents were collected for analysis for radiochemical content.  The rats were maintained in the metabolism cages for 24 h following the end of the exposure.  The total recovery of radioactivity from all of the samples collected from rats Groups 1-6 is presented in Table 7, and the group means ranged from approximately 3.7 to 15.8 uCi.  The estimated doses based on the total activity recovered at between the single exposure and 24 hr following the exposure were 80.3  2.4 (male, 600 ppm), 77.5  1.5 (female, 600 ppm), 301.1  18.4 (male, 3600 ppm), and 286.5  22.1 (female, 3600 ppm) mg/kg (Table 7).  Following repeated exposures to 3600 ppm, followed by a single exposure to [2-[14]C]DIPE, the estimated doses based on recovered radioactivity were 543.9  11.2 (male) and  401.8  59.2 (female)  mg/kg.
The amount of radioactivity in urine, feces, exhaled VOCs, CO2, and tissues and carcass expressed as a percentage of the total radioactivity recovered for Groups 1-6 is presented in Tables 8, 10, 12, 14, 16, and 18.  Some observations that were made in the pilot study were also borne out in this study.  The majority of the radioactivity derived from CO2 was recovered in the first CO2 trap.  Little radioactivity was recovered in the second CO2 trap.  Many of the samples collected from the second trap contained levels of radioactivity that were below 3 times background.  The majority of the radioactivity retained on the charcoal traps for exhaled volatiles was retained on the first charcoal trap of two in series.
In Groups 1 and 2, 4 male and 4 female rats were transferred to all-glass metabolism cages following a single exposure to 600 ppm [2-14C]DIPE, and urine, feces, exhaled CO2 and exhaled volatiles were collected for 24 hr.  After 1 day, the rats were euthanized and the radioactivity remaining in the tissues and carcass was determined by digestion and scintillation counting.  Radioactivity was eliminated primarily as exhaled volatiles (Table 8 and Table 10) (42 and 41 % of the recovered radioactivity, in males and females, respectively), and [14]CO2 (39 and 41%). Small amounts of radioactivity were recovered in urine (5.1 and 4.2 % of the recovered radioactivity).  A lesser amount was recovered (<1%) in feces, and 7.9 and 8.2 % in carcass at 1 day.  The cumulative recovery of radioactivity is presented in Figure 1.   Based on the recovered radioactivity at 24 hr, the estimated dose for male rats was 80.3  2.4 mg/kg, and female rats was 77.5  1.5 mg/kg.  The concentrations of radioactivity in tissues at 24 hr are presented in Table 9 and Table 11.  Highest concentrations were observed in liver, kidney, small intestine, spleen and lung.
In Groups 3 and 4, following a single exposure to 3600 ppm [2-[14]C]DIPE, the radioactivity recovered is presented in Table 12 and Table 14.  Radioactivity was eliminated primarily as exhaled volatiles (64 and 71 % of the recovered radioactivity in males and females, respectively), and [14]CO2 (22 and 18 %). Small amounts of radioactivity were recovered in urine (4.2 and 3.6 % of the recovered radioactivity).  A lesser amount was recovered (<1%) in feces, and approximately 5 % in carcass at 1 day.  The cumulative recovery of radioactivity is presented in Figure 2.    Based on the recovered radioactivity at 24 hr, the estimated dose for male rats was 301.1  18 mg/kg, and female rats was 286  22 mg/kg.  The concentration of radioactivity in tissues at 24 hr is presented in Table 13 and Table 15.  As with the 600 ppm exposure, highest concentrations were observed in liver, kidney, small intestine, spleen and lung.
In Groups 5 and 6, eight male and female Fischer 344 rats were exposed to 4 consecutive days of unlabeled DIPE at 3600 ppm, for 6 hr/day (3555 +- 55 ppm).  This was followed by a single 6-hr nose only exposure to [2-[14]C]DIPE on day 5 (actual concentration 3621  86 ppm).  Four male and four female rats were transferred to all-glass metabolism cages, and urine, feces, exhaled CO2 and exhaled volatiles were collected for 24 hr.  At 1 day following the end of the last exposure, the rats were euthanized and the radioactivity remaining in the tissues and carcass was determined by digestion and scintillation counting (Table 16 and Table 18).   Radioactivity was eliminated primarily as exhaled volatiles (45 and 53% of the recovered radioactivity in males and females, respectively), and [14]CO2 (34 and 26%). For one set of CO2 trap samples (rat 5-02, 24 hr, trap 1 and trap 2), the higher amount of radioactivity was recorded in trap 2, and the lower value in trap 1.  This was inconsistent with the values of all of the other groups.  The samples were realiquoted and recounted, and confirmed that the analysis was correct.  It is suspected that the samples were switched at collection, but this cannot be confirmed with certainty.   For the purpose of reporting summary statistics, the value recorded for trap 2 has been switched with that recorded for trap 1.   Small amounts of radioactivity were recovered in urine (8.5 and 6.8% of the recovered radioactivity).  A lesser amount was recovered (<1%) in feces, and approximately 7 and 8 % in carcass at 1 day.   The cumulative recovery of radioactivity is presented in Figure 3.   Based on the recovered radioactivity at 24 hr, the estimated dose for male rats was 543.9  11.2 mg/kg, and female rats was 401.8   59.2 mg/kg.  The concentrations of radioactivity in tissues at 24 hr are presented in Table 17 and Table 19. As with the single 3600 ppm exposure, highest concentrations were observed in liver, kidney, small intestine and lung.  However, subcutaneous and abdominal adipose tissues were considerably increased compared with a single exposure, and abdominal adipose was the highest tissue concentration in both males and females.
4.4	Gavage ADME Groups
4.4.1	Test Article Formulation
For gavage administration, unlabeled DIPE was mixed with [2-[14]C] labeled DIPE and prepared at a concentration of approximately 20 mg/mL in a vehicle consisting of 10% Cremophor EL in water.  The concentration of DIPE in the formulation was verified by dilution and headspace GC-MS analysis.  Prior to dosing, stability of the dose solution was verified for 6 hr at room temperature and for 24 hours at refrigerator temperature.  The specific activity of the formulation is reported in Table 4. The estimated content of pentane from the 14C DIPE is also reported as approximately 4.15 % of the DIPE, and 0.083 % of the formulation.
4.4.2	Test Article Administration
The body weights and doses administered to the animals on the oral disposition groups (Groups 7-10) are shown in Table 2.
4.4.3  	Disposition of Radioactivity
In Groups 7 and 8, 4 male and female Fischer 344 rats were administered [2-[14]C]DIPE by gavage at a dose of 50 mg/kg.   The rats were transferred to all-glass metabolism cages, and urine, feces, exhaled CO2 and exhaled volatiles were collected for 24 hr.  At 1 day, the rats were euthanized and the radioactivity remaining in the tissues and carcass was determined by digestion and scintillation counting (Table 20 and Table 22).  Radioactivity was eliminated primarily as exhaled volatiles (52 and 56 % of the administered dose in males and females, respectively), and [14]CO2 (17 and 18%). Small amounts of radioactivity were recovered in urine (2.5 and 2.2% of the administered dose).  A lesser amount was recovered (<1%) in feces, and approximately 5 % in the tissues and carcass at 1 day.   The cumulative recovery of radioactivity is presented in Figure 4.   The concentrations of radioactivity in tissues at 24 hr are presented in Table 21 and Table 23. As with the inhalation exposures, highest concentrations were observed in liver, kidney, small intestine and lung.  Additional tissues with similar levels were stomach, large intestine, spleen, and skin.
In Groups 9 and 10, 4 male and female Fischer 344 rats were administered [2-[14]C]DIPE/DIPE by gavage at a dose of 200 mg/kg.   The rats were transferred to all-glass metabolism cages, and urine, feces, exhaled CO2 and exhaled volatiles were collected for 24 hr.  At 1 day, the rats were euthanized and the radioactivity remaining in the tissues and carcass was determined by digestion and scintillation counting (Table 24 and Table 26).  Radioactivity was eliminated primarily as exhaled volatiles (56 and 61% of the administered dose in males and females, respectively), and [14]CO2 (9 and 9%). Small amounts of radioactivity were recovered in urine (2% of the administered dose).  A lesser amount was recovered (<1%) in feces, and approximately 3% in the tissues and carcass at 1 day.  The cumulative recovery of radioactivity is presented in Figure 5.   The concentrations determined in the tissues showed a pattern similar to the low dose gavage groups, with highest concentrations were observed in liver, kidney, small intestine and lung (Error! Reference source not found. and Table 27).  Additional tissues with similar levels were stomach, spleen, and skin.

4.5	Pharmacokinetics Study Groups
4.4.1	Test Chemical Formulation
The inhalation pharmacokinetics groups (Groups 11, 12, 15 and 16) were exposed to atmospheres generated using unlabeled DIPE alone.
4.5.2	Inhalation exposure
The body weights of the animals used during the course of the inhalation pharmacokinetic groups (Groups 11, 12, 15 and 16) are shown in Table 3.  The exposure concentration data for each six-hour exposure of rats to target concentrations of 600 or 3600 ppm DIPE is shown in Table 5.  The details of the inhalation exposure system are described in the report for the pilot study, (RTI-934, Metabolism and Pharmacokinetics of Diisopropyl Ether in Male and Female Rats: Pilot Study Appendix E, Inhalation Summary Report for Setup and Evaluation of Inhalation System at RTI International) and the details of the actual exposure are reported in Appendix E, Inhalation Summary Report: Nose-Only Exposures at RTI International.  The actual exposure concentrations were 677 +- 15 ppm (Groups 11 and 12) and 3594  +- 198  ppm (Groups 15 and 16) (Table 5) .
4.5.3  	Concentrations of DIPE, isopropanol and acetone in plasma following DIPE exposure.
DIPE, isopropanol and acetone were determined by headspace GC-MS analysis in blood samples obtained from jugular vein cannulated male and female rats exposed to 600 or 3600 ppm DIPE for 6 h. The concentrations of DIPE determined on exposure to 600 ppm are presented in Table 28  and in Figure 6.  DIPE was less than the limit of quantitation (LOQ) in preexposure samples.  DIPE accumulated rapidly during the 6 hours of exposure, and rapidly declined after the exposure.  There was little difference in blood levels at peak concentration between males and females.  All samples were below LOQ by 24 hr. In the male rats, isopropanol was below LOQ in preexposure samples.  The concentration of isopropanol rose steadily during the exposure, and then declined gradually (Table 29 and Figure 7).  In male rats, blood concentrations of isopropanol reached peak concentrations at approximately twice those of female rats.  All samples were below LOQ by 24 hr.  In the female rats, isopropanol was below LOQ in preexposure samples.  In male and female rats, acetone was above LOQ in all of the preexposure samples, with levels ranging from 3- 4 ug/mL.  Acetone levels in both male and female rats rose steadily during the exposure, and in males continued to rise following exposure until 7 hr following the initiation of exposure (Figure 8 and Table 30). The concentration achieved in males exceeded the range of the calibration curve, and for samples obtained between 6 hours and 10 hours are estimated by linear extrapolation of the calibration curve. In females, the acetone levels were approximately the same between 6 and 7.33 hours following the exposure, and then declined rapidly reaching preexposure levels by 16 hrs.  The peak concentrations of acetone achieved in male rats were approximately 50% higher than those in female rats.   Male rats reached preexposure levels by 16 hours.
On exposure of rats to 3600 ppm DIPE, similar results were obtained.  DIPE accumulated rapidly during the 6 hours of exposure, and rapidly declined after the exposure (Table 31 and Error! Reference source not found.).  There was little difference in blood levels at peak concentration between males and females.  All samples were below LOQ by 16 hr. In the male rats, isopropanol was below LOQ in preexposure samples.  The concentration of isopropanol rose steadily during the exposure, and then declined gradually (Table 32 and Figure 10).  In male rats, blood concentrations of isopropanol reached peak concentrations at approximately twice those of female rats.  All samples were below LOQ by 24 hr.  In the female rats, isopropanol was below LOQ in preexposure samples, and were below LOQ by 16 hr.  In male and female rats, acetone was above LOQ in all of the preexposure samples, with levels ranging from 3- 4 ug/mL.  Acetone levels in both male and female rats rose steadily during the exposure, and in males continued to rise following exposure until 7 hr following the initiation of exposure (and Table 33).  In females, the acetone levels plateaued between 6 and 8 hours following the exposure, and then declined rapidly reaching preexposure levels by 16 hrs.  The peak concentrations of acetone achieved in male rats were approximately 50% higher than those in female rats.   Male rats reached preexposure levels by 24 hours.
Non-compartmental pharmacokinetic analysis of DIPE indicated a half-life of approximately 1 hr at both exposure concentrations in male and female rats (Table 34 and Table 35).  The AUC for DIPE was similar between male and female rats at equivalent exposure concentrations.  The half-life for isopropanol was similar to DIPE and among the exposure concentrations and genders, with values ranging from 1-1.35 hr.  However, the AUC and C max values were higher in male rats than in female rats at both exposure concentrations.   The half-lives observed for acetone were longer than DIPE and isopropanol, ranging from 2.4  -  3.0 hr.  The Cmax and AUC values were highest for acetone compared with DIPE and isopropanol, and were higher in male rats than female rats.
5.0  	Discussion
In conjunction with the pilot study reported separately, the  objectives of the study were accomplished.  An inhalation exposure system was developed, and used to conduct inhalation exposures of male and female rats to 600 and 3600 ppm DIPE.  Exposures were conducted with [2-[14]C]DIPE to determine the disposition of DIPE following single exposures to 600 or 3600 ppm, and with a single exposure  to [2-[14]C] DIPE following four daily exposures to unlabeled DIPE.   [2-[14]C]DIPE was also administered by gavage dosing, to determine the disposition of orally administered material.   A headspace GC-MS method for analysis of DIPE, isopropanol, and acetone was developed, and used for the analysis of blood samples from male and female rats exposed to DIPE to determine the pharmacokinetics of DIPE, and its two metabolites.
 In the pilot study, comparison of the exposure of rats to 3600 ppm [2-[14]C]DIPE/DIPE euthanized immediately following exposure with that of rats euthanized following 7 day of collection of excreta and exhaled breath yielded values that were similar.  This suggested that there were no significant losses of radioactivity from the various sample types during processing of radioactivity.  In this study, in each group exposed to [14]C DIPE, 4 rats were euthanized immediately following exposure for determination of total radioactivity taken up, and 4 rats were euthanized following collection of urine, feces, exhaled breath VOCs and CO2 for 24 h.   The dose dependence was similar in male and female rats, with approximately 100 mg/kg taken up at 600 ppm in both sexes, and 425 and 395 mg/kg in males and females, respectively, at 3600 ppm.  The comparison of these two groups provides a means of estimating the recovery of dose from the inhalation groups.  For compounds such as DIPE which is extremely volatile, and its volatile metabolites, acetone and isopropanol, trapping and analyzing the radioactivity can be challenging.   At 600 ppm, the recovery of radioactivity in the animals in metabolism cages was 80.3 and 80.1 % in males and females, respectively, compared with the rats euthanized immediately.  Similarly at 3600 ppm, the recovery was 70.8 and 72.5 % in males and females, respectively.  In the repeat dose groups there was a substantial difference between male (786 mg/kg) and females (480 mg/kg) in the amount present immediately following exposure, with an apparent increase in males compared to a single exposure.   On repeat exposure to 3600 ppm, recovery was 69.2 % and 83.7 % in males and females respectively.   The difference in yield between the dose estimated from the total carcass and the collection of excreta and exhaled radioactivity suggests the possible loss of a portion of radioactivity, presumably volatile radioactivity during the transfer of animals in and out of metabolism cages, or during the collection and processing of samples.
It was previously observed in the pilot study that radioactivity was rapidly excreted by exhalation as volatile components (71% of the recovered radioactivity) and as [14]CO2 (25% of the recovered radioactivity) on exposure of male rats to 3600 ppm DIPE.  The cumulative excretion of radioactivity was 94% and 95% within 24 and 48 hours, respectively, of the end of the exposure.  At seven days, only 0.6% was recovered in the carcass.  This supported the shortening of the collection of excreta for 24 hr following the end of the exposure.  In the present studies, approximately 12-13% of the radioactivity recovered was in the carcass and tissues in the male and female rats exposed to 600 ppm, and approximately 8 and 7% of the radioactivity was recovered in the male and female rats, respectively, exposed to 3600 ppm.  With repeated exposures to 3600 ppm DIPE, approximately 11 and 12% of the recovered radioactivity was found in the tissues and carcass in male and female rats respectively. With gavage administration at 24 hr after dosing, approximately 70 -80% of the administered dose was recovered in excreta and exhaled breath, with approximately 3-6% in the tissues and carcass.
There appear to be qualitative differences between male and female rats with respect to the metabolism of DIPE. The AUC and Cmax values for acetone and isopropanol were higher in male rats than in female rats.  This implies either greater oxidation of DIPE in male rats to produce these metabolites, or that their subsequent metabolism and/or elimination from the blood of male rats is slower than in female rats.  Statistical comparison of the data from each set of exposures was conducted, and values are compared in Tables 36-42.  While many of the values were statistically significantly different between groups, they are not highlighted for discussion because the differences were to be expected (concentration differences between low and high dose exposures) or occur in samples with very low concentrations or percent recovery, where the differences are not biologically significant.  In Table 36, the CO2 Trap 1 values at 3600 ppm are lower in both the male and female rats compared with the same sex exposed to 600 ppm (means of 38.42 and 41.21 % in males and females, respectively at 600 ppm, compared with 20.51 and 17.74 % at 3600 ppm).    Exhaled VOC traps were higher at 3600 ppm in both males and females than at 600 ppm.  Both of these observations imply a saturation of the metabolism of DIPE to CO2, with a corresponding increase in exhaled volatiles.  In Table 37, there were significant increases with repeated inhalation exposure in both male and female rats in the recovery of radioactivity in urine and feces, a decrease in exhaled VOCs, and an increase in the recovery of radioactivity in the carcass digest.  The differences between groups for tissue concentrations were most profound for the single vs. repeated exposures (Table 39), with increased concentrations in adipose, muscle, blood, spleen, liver, kidney, heart, lung, brain, testis and ovary, small intestine, cecum + large intestine and rectum, and carcass digest.
6.0  	References

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.
National Research Council (1996).  Guide for the Care and Use of Laboratory Animals.  National Academy Press: Washington, DC.
US Food and Drug Administration (2001).  Guidance for Industry: Bioanalytical Method Validation.

7.0  	RECORDS and Reports
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)          Randomization records
    h)          Test chemical receipt, storage, and use records
    i)          Balance calibration log references
    j)          Correspondences
    k)          All other raw data and documentation
Upon acceptance of the audited draft report by the Sponsor, a final report will be issued.
8.0  	Storage of Records and Biological Samples
A copy of the final report and the records for this study, including all raw data, will be retained in the RTI Archives, for the length of time specified in the appropriate regulations.
Biological specimens that remain stable for reanalysis and samples of nonradiolabeled and radiolabeled DIPE will be stored at RTI in a secured area by the RTI project number for up to two years after the date of submission of the final report.
The Sponsor will be notified in writing when the RTI retention time has expired.
9.0  	REGULATORY COMPLIANCE
These studies were performed in compliance with the EPA Good Laboratory Practices (GLP) Standards for Inhalation Exposure Health Effects Testing, 40 CFR part 79, subpart F § 79.60.  Deviations from the approved protocol are documented in Appendix A.
10.0  	STUDY PARTICIPANTS
RTI International
Study Director: 	Timothy R. Fennell, Ph.D.
Veterinarian: 	A. McIntyre, D.V.M.
Animal Facility Supervisor:
Laboratory Staff:  	N. F. Gaudette, BS
	S. R. Black, BS
            	Y. Hong, BS
            	P. R. Patel, BS
            	S. L Watson, MS
            	R. W. Snyder, MS
            	M. P. Gower
Secretary: 	K. G. Ancheta

The Hamner Institutes for Health Research
Principal Investigator:: 	K. Roberts.
Laboratory Staff:  	K. Roberts

Quality Assurance Reviewer:  Jeanne deWard, BS, LATG, RQAP-GLP

Table 1.  Body Weights of Male and Female Fischer 344 Rats in Inhalation Exposure Groups
                                    Subject
                                     Route
                                   Treatment
                                      Sex
                                Subject Wt (g)
                                     1-01
                                  Inhalation
                                   13426-51A
                                     Male
                                      177
                                     1-02
                                  Inhalation
                                   13426-51A
                                     Male
                                      199
                                     1-03
                                  Inhalation
                                   13426-51A
                                     Male
                                      191
                                     1-04
                                  Inhalation
                                   13426-51A
                                     Male
                                      189
                                     1-05
                                  Inhalation
                                   13426-51A
                                     Male
                                      186
                                     1-06
                                  Inhalation
                                   13426-51A
                                     Male
                                      178
                                     1-07
                                  Inhalation
                                   13426-51A
                                     Male
                                      196
                                     1-08
                                  Inhalation
                                   13426-51A
                                     Male
                                      186
                                     2-01
                                  Inhalation
                                   13426-51A
                                    Female
                                      129
                                     2-02
                                  Inhalation
                                   13426-51A
                                    Female
                                      130
                                     2-03
                                  Inhalation
                                   13426-51A
                                    Female
                                      127
                                     2-04
                                  Inhalation
                                   13426-51A
                                    Female
                                      122
                                     2-05
                                  Inhalation
                                   13426-51A
                                    Female
                                      132
                                     2-06
                                  Inhalation
                                   13426-51A
                                    Female
                                      120
                                     2-07
                                  Inhalation
                                   13426-51A
                                    Female
                                      131
                                     2-08
                                  Inhalation
                                   13426-51A
                                    Female
                                      124
                                     3-01
                                  Inhalation
                                   13426-39B
                                     Male
                                      177
                                     3-02
                                  Inhalation
                                   13426-39B
                                     Male
                                      184
                                     3-03
                                  Inhalation
                                   13426-39B
                                     Male
                                      200
                                     3-04
                                  Inhalation
                                   13426-39B
                                     Male
                                      186
                                     3-05
                                  Inhalation
                                   13426-39B
                                     Male
                                      206
                                     3-06
                                  Inhalation
                                   13426-39B
                                     Male
                                      188
                                     3-07
                                  Inhalation
                                   13426-39B
                                     Male
                                      198
                                     3-08
                                  Inhalation
                                   13426-39B
                                     Male
                                      189
                                     4-01
                                  Inhalation
                                   13426-39B
                                    Female
                                      149
                                     4-02
                                  Inhalation
                                   13426-39B
                                    Female
                                      149
                                     4-03
                                  Inhalation
                                   13426-39B
                                    Female
                                      152
                                     4-04
                                  Inhalation
                                   13426-39B
                                    Female
                                      148
                                     4-05
                                  Inhalation
                                   13426-39B
                                    Female
                                      148
                                     4-06
                                  Inhalation
                                   13426-39B
                                    Female
                                      143
                                     4-07
                                  Inhalation
                                   13426-39B
                                    Female
                                      151
                                     4-08
                                  Inhalation
                                   13426-39B
                                    Female
                                      150
                                     5-01
                                  Inhalation
                                   13426-21A
                                     Male
                                      170
                                     5-02
                                  Inhalation
                                   13426-21A
                                     Male
                                      174
                                     5-03
                                  Inhalation
                                   13426-21A
                                     Male
                                      170
                                     5-04
                                  Inhalation
                                   13426-21A
                                     Male
                                      169
                                     5-05
                                  Inhalation
                                   13426-21A
                                     Male
                                      170
                                     5-06
                                  Inhalation
                                   13426-21A
                                     Male
                                      183
                                     5-07
                                  Inhalation
                                   13426-21A
                                     Male
                                      178
                                     5-08
                                  Inhalation
                                   13426-21A
                                     Male
                                      177
                                     6-01
                                  Inhalation
                                   13426-21A
                                    Female
                                      151
                                     6-02
                                  Inhalation
                                   13426-21A
                                    Female
                                      144
                                     6-03
                                  Inhalation
                                   13426-21A
                                    Female
                                      147
                                     6-04
                                  Inhalation
                                   13426-21A
                                    Female
                                      147
                                     6-05
                                  Inhalation
                                   13426-21A
                                    Female
                                      140
                                     6-06
                                  Inhalation
                                   13426-21A
                                    Female
                                      146
                                     6-07
                                  Inhalation
                                   13426-21A
                                    Female
                                      145
                                     6-08
                                  Inhalation
                                   13426-21A
                                    Female
                                      143

Table 2.  Body Weights[a] and Dose Administered to Male and Female Fischer 344 Rats in Oral Gavage Groups
                                    Subject
                                     Route
                                   Treatment
                                      Sex
                           Concentration (mg/g dose)
                                Subject Wt (g)
                           Actual Dose Rate (mg/kg)
                                     7-01
                                    Gavage
                                   13426-99a
                                     Male
                                     18.4
                                    197.84
                                     47.1
                                     7-02
                                    Gavage
                                   13426-99a
                                     Male
                                     18.4
                                    194.25
                                     48.7
                                     7-03
                                    Gavage
                                   13426-99a
                                     Male
                                     18.4
                                    206.47
                                     46.6
                                     7-04
                                    Gavage
                                   13426-99a
                                     Male
                                     18.4
                                    203.72
                                     48.9
                                     8-01
                                    Gavage
                                   13426-99a
                                    Female
                                     18.4
                                    144.44
                                     48.7
                                     8-02
                                    Gavage
                                   13426-99a
                                    Female
                                     18.4
                                    143.71
                                     49.6
                                     8-03
                                    Gavage
                                   13426-99a
                                    Female
                                     18.4
                                    145.15
                                     49.3
                                     8-04
                                    Gavage
                                   13426-99a
                                    Female
                                     18.4
                                    150.85
                                     48.7
                                     9-01
                                    Gavage
                                   13426-99a
                                     Male
                                     18.4
                                    186.02
                                     201.3
                                     9-02
                                    Gavage
                                   13426-99a
                                     Male
                                     18.4
                                    200.42
                                     197.4
                                     9-03
                                    Gavage
                                   13426-99a
                                     Male
                                     18.4
                                    198.02
                                     198.8
                                     9-04
                                    Gavage
                                   13426-99a
                                     Male
                                     18.4
                                    196.27
                                     196.8
                                     10-01
                                    Gavage
                                   13426-99a
                                    Female
                                     18.4
                                    133.04
                                     193.7
                                     10-02
                                    Gavage
                                   13426-99a
                                    Female
                                     18.4
                                    144.39
                                     194.8
                                     10-03
                                    Gavage
                                   13426-99a
                                    Female
                                     18.4
                                    142.75
                                     193.7
                                     10-04
                                    Gavage
                                   13426-99a
                                    Female
                                     18.4
                                    143.21
                                     195.8

[a]Body weight on the day of exposure

Table 3.  Body Weights of Male and Female Fischer 344 Rats in Pharmacokinetics Groups
Subject
Route
Treatment
Subject  Wt (g)
11-01
Inhalation
DIPE Aliq 13307-07E (600 ppm Exp)
180
11-02
Inhalation
DIPE Aliq 13307-07E (600 ppm Exp)
174
11-03
Inhalation
DIPE Aliq 13307-07E (600 ppm Exp)
186
11-04
Inhalation
DIPE Aliq 13307-07E (600 ppm Exp)
189
11-05
Inhalation
DIPE Aliq 13307-07E (600 ppm Exp)
174
11-06
Inhalation
DIPE Aliq 13307-07E (600 ppm Exp)
168
11-07
Inhalation
DIPE Aliq 13307-07E (600 ppm Exp)
169
11-08
Inhalation
DIPE Aliq 13307-07E (600 ppm Exp)
173
11-09
Inhalation
DIPE Aliq 13307-07E (600 ppm Exp)
178
11-10
Inhalation
DIPE Aliq 13307-07E (600 ppm Exp)
172
12-01
Inhalation
DIPE Aliq 13307-07E (600 ppm Exp)
137
12-02
Inhalation
DIPE Aliq 13307-07E (600 ppm Exp)
130
12-03
Inhalation
DIPE Aliq 13307-07E (600 ppm Exp)
128
12-04
Inhalation
DIPE Aliq 13307-07E (600 ppm Exp)
129
12-05
Inhalation
DIPE Aliq 13307-07E (600 ppm Exp)
134
12-06
Inhalation
DIPE Aliq 13307-07E (600 ppm Exp)
129
12-07
Inhalation
DIPE Aliq 13307-07E (600 ppm Exp)
131
12-08
Inhalation
DIPE Aliq 13307-07E (600 ppm Exp)
131
15-01
Inhalation
DIPE Aliq 13307-07C
179.97
15-02
Inhalation
DIPE Aliq 13307-07C
182.81
15-03
Inhalation
DIPE Aliq 13307-07C
184.54
15-04
Inhalation
DIPE Aliq 13307-07C
185.09
15-05
Inhalation
DIPE Aliq 13307-07C
186.70
15-06
Inhalation
DIPE Aliq 13307-07C
180.10
15-07
Inhalation
DIPE Aliq 13307-07C
187.57
15-08
Inhalation
DIPE Aliq 13307-07C
183.10
15-09
Inhalation
DIPE Aliq 13307-07C
169.53
16-01
Inhalation
DIPE Aliq 13307-07C
138.70
16-02
Inhalation
DIPE Aliq 13307-07C
154.71
16-03
Inhalation
DIPE Aliq 13307-07C
138.75
16-04
Inhalation
DIPE Aliq 13307-07C
148.74
16-05
Inhalation
DIPE Aliq 13307-07C
136.20
16-06
Inhalation
DIPE Aliq 13307-07C
153.55
16-07
Inhalation
DIPE Aliq 13307-07C
149.50
16-08
Inhalation
DIPE Aliq 13307-07C
137.02
16-09
Inhalation
DIPE Aliq 13307-07C
140.12

Table 4.  Specific Activity of Dose Formulations
                                 DOSE GROUP(S)
                                   Exposure
                                TREATMENT NAME
                                DPM/G DOSE PREP
                          SPECIFIC ACTIVITY (uCi/mg)
                           % Pentane from14C DIPE[a]
                                     1, 2
                              600 ppm Inhalation
                              14C DIPE 13426-51A
                                   833799338
                                    0.3756
                                     0.71
                                     3, 4
                              3600 ppm Inhalation
                              14C DIPE 13426-39B
                                   374959659
                                    0.1689
                                     0.48
                                     5, 6
                              3600 ppm Inhalation
                              14C DIPE 13426-21A
                                   378754628
                                    0.1706
                                     0.52
                                     7-10
                                    Gavage
                                   13426-99A
                                   66949847
                                    1.6401
                                   4.15[b]

a % of mass in total DIPE as pentane contaminant from 14C DIPE preparation
b  Pentane was 4.15 % of total DIPE, and of the total gavage formulation represented 0.08 %.

Table 5.  Summary of DIPE Concentrations Determined During Inhalation Exposure

                                 Animal Group
                                  Assignment
                               Groups 1 & 2
                               Groups 3 & 4
                              Groups 5 & 6[1]
                              Groups 5 & 6[2]
                               Groups 11 &12
                               Groups 15 &16

ACC -Inlet

Exposure mean

                                      605

                                     3600
                                     3555
                                     3621

                                      677

                                     3594
(ppm)
Std Dev
                                      12
                                      48
                                      55
                                      86
                                      15
                                      198

No. of Data Points
                                      13
                                      13
                                       4
                                      13
                                      15
                                      13

ACC - Exhaust

Exposure mean

                                      521

                                     3502
                                     3033
                                     3365

                                      596

                                     2401
(ppm)
Std Dev
                                      21
                                      122
                                      327
                                      37
                                      81
                                      373

No. of Data Points
                                      13
                                      13
                                       4
                                      13
                                      15
                                      14

Exposure

Exposure mean

                                     71.0

                                     72.7
                                     69.9
                                     68.5

                                     70.4

                                     71.0
Temperature
Std Dev
                                     ' 0.9
                                      1.7
                                      0.6
                                      1.0
                                      0.6
                                      0.5
(°F)
No. of Data Points
                                      14
                                      14
                                       4
                                      14
                                      16
                                      14

Exposure

Exposure mean

                                     46.8

                                     44.2
                                     45.6
                                     48.1

                                     46.4

                                     44.8
Relative Humidity
Std Dev
                                      1.7
                                      2.3
                                      0.6
                                      1.1
                                      1.1
                                      0.6
(%)
No. of Data Points
                                      14
                                      14
                                       4
                                      14
                                      16
                                      14

Exposure

Exposure mean

                                     -0.13

                                     -0.14
                                     -0.7
                                     -0.3

                                     -0.35

                                     -0.25
Static Pressure
Std Dev
                                     0.33
                                      0.2
                                      N/A
                                      0.1
                                      N/A
                                      N/A
(in H20)
No. of Data Points
                                      14
                                      14
                                       1
                                      14
                                       1
                                       1

 ACC: Analytical Chamber Concentration
[1]Mean data from 4 consecutive nonradiolabeled Daily Exposures
[2]Data from radiolabeled exposure (Day 5)
Much of the data in this table is from the Inhalation Report (Table 3)

Table 6.  Radioactivity Retained in Carcass following Inhalation Exposure to [2-[14]C]DIPE

                                    Subject
                                uCi recovered
                                 Treatment ID
                     Treatment Specific Activity (uCi/mg)
                              Subject Weight (g)
                               Calculated mg/kg
                                    600 ppm
                                     1-05
                                     6.70
                                   13426-51A
                                    0.3756
                                      186
                                     95.9
                                     Male
                                     1-06
                                     6.84
                                   13426-51A
                                    0.3756
                                      178
                                      102
                                     Rats
                                     1-07
                                     7.13
                                   13426-51A
                                    0.3756
                                      196
                                     96.9

                                     1-08
                                     7.38
                                   13426-51A
                                    0.3756
                                      186
                                      106

                                     Mean
                                     7.01

                                      100

                                      SD
                                     0.30

                                       5
                                    600 ppm
                                     2-05
                                     4.69
                                   13426-51A
                                    0.3756
                                      132
                                     94.6
                                    Female
                                     2-06
                                     4.50
                                   13426-51A
                                    0.3756
                                      120
                                     99.8
                                     Rats
                                     2-07
                                     4.68
                                   13426-51A
                                    0.3756
                                      131
                                     95.2

                                     2-08
                                     4.54
                                   13426-51A
                                    0.3756
                                      124
                                     97.4

                                     Mean
                                     4.60

                                     96.8

                                      SD
                                     0.10

                                      2.4
                                   3600 ppm
                                     3-05
                                     14.26
                                   13426-39B
                                    0.1689
                                      206
                                      410
                                     Male
                                     3-06
                                     14.05
                                   13426-39B
                                    0.1689
                                      188
                                      442
                                     Rats
                                     3-07
                                     13.98
                                   13426-39B
                                    0.1689
                                      198
                                      418

                                     3-08
                                     13.71
                                   13426-39B
                                    0.1689
                                      189
                                      430

                                     Mean
                                     14.00

                                      425

                                      SD
                                     0.23

                                      14
                                   3600 ppm
                                     4-05
                                     11.06
                                   13426-39B
                                    0.1689
                                      148
                                      443
                                    Female
                                     4-06
                                     8.87
                                   13426-39B
                                    0.1689
                                      143
                                      367
                                     Rats
                                     4-07
                                     8.79
                                   13426-39B
                                    0.1689
                                      151
                                      345

                                     4-08
                                     10.83
                                   13426-39B
                                    0.1689
                                      150
                                      428

                                     Mean
                                     9.89

                                      395

                                      SD
                                     1.23

                                      47

Table 6 (contd).  Radioactivity Retained in Carcass following Inhalation Exposure to [2-[14]C]DIPE.

Subject
uCi recovered
Treatment ID
Treatment Specific Activity (uCi/mg)
Subject Weight (g)
Calculated mg/kg

3600 ppm
5-05
23.12
13426-21A
0.1706
170
797
Male
5-06
24.24
13426-21A
0.1706
183
776
Rats
5-07
23.81
13426-21A
0.1706
178
784
Repeat Dose
5-08
23.79
13426-21A
0.1706
177
788

Mean
23.74

786

SD
0.46

9
3600 ppm
6-05
11.72
13426-21A
0.1706
140
491
Female
6-06
10.67
13426-21A
0.1706
146
428
Rats
6-07
12.88
13426-21A
0.1706
145
521
Repeat Dose
6-08
11.75
13426-21A
0.1706
143
481

Mean
11.75

480

SD
0.90

39

Table 7.  Total Recovery of Radioactivity following Inhalation Exposure to [2-[14]C]DIPE

Subject
Total DPM Recovered[a]
Total uCi Recovered
Treatment Specific Activity (uCi/mg)
Subject Weight (g)
Calculated mg/kg
Recovery[b]
600 ppm
1-01
11918767.56
5.4
0.3756
177
80.8

Male
1-02
12762521.02
5.7
0.3756
199
76.9

Rats
1-03
13179424.36
5.9
0.3756
191
82.8

1-04
12742726.24
5.7
0.3756
189
80.9

Mean
12650860
5.7

80.3
80.3%

SD
527967
0.2

2.4

600 ppm
2-01
8360051.832
3.8
0.3756
129
77.7

Female
2-02
8171814.887
3.7
0.3756
130
75.4

Rats
2-03
8253144.187
3.7
0.3756
127
77.9

2-04
8034082.091
3.6
0.3756
122
79.0

Mean
8204773
3.7

77.5
80.1%

SD
137444
0.1

1.5

3600 ppm
3-01
21702607.88
9.8
0.1689
177
327.0

Male
3-02
19577062.13
8.8
0.1689
184
283.8

Rats
3-03
22186752.17
10.0
0.1689
200
295.9

3-04
20758698.38
9.4
0.1689
186
297.6

Mean
21056280
9.5

301.1
70.8%

SD
1150702
0.5

18.4

3600 ppm
4-01
14689278.77
6.6
0.1689
149
262.9

Female
4-02
17057828.46
7.7
0.1689
149
305.3

Rats
4-03
15524299.92
7.0
0.1689
152
272.4

4-04
16940807.22
7.6
0.1689
148
305.3

Mean
16053054
7.2

286.5
72.5%

SD
1145592
0.5

22.1

[a] Total recovery calculated from the total DPM recovered in all samples at 24 hours.
[b]Comparison of dose determined from recovery of radioactivity in metabolism cages vs. rats euthanized immediately after exposure  in Table 6.

Table 7 (contd).  Total Recovery of Radioactivity following Inhalation Exposure to [2-[14]C]DIPE

Subject
Total DPM Recovered[a]
Total uCi Recovered
Treatment Specific Activity (uCi/mg)
Subject Weight (g)
Calculated mg/kg
Recovery[b]
3600 ppm
5-01
34229059.1
15.4
0.1706
170
531.6

Male
5-02
36445421.36
16.4
0.1706
174
553.0

Rats
5-03
35655993.45
16.1
0.1706
170
553.8

5-04
34371714.37
15.5
0.1706
169
537.0

Mean
35175547
15.8

543.9
69.2%

SD
1062292
0.5

11.2

3600 ppm
16-01
25641124.49
11.6
0.1706
151
448.4

Female
6-02
24737526.38
11.1
0.1706
144
453.6

Rats
6-03
20783471.41
9.4
0.1706
147
373.3

6-04
18488482.63
8.3
0.1706
147
332.1

Mean
22412651
10.1

401.8
83.7%

SD
3360625
1.5

59.2

  [a] Total recovery calculated from the total DPM recovered in all samples
  [b]Comparison of dose determined from recovery of radioactivity in metabolism cages vs. rats euthanized immediately after exposure
Table 8.  Disposition of Radioactivity (% of Recovered Radioactivity) Following Inhalation Exposure of Male F344 Rats to 600 ppm [2-[14]C]DIPE  (Group 1)
Sample
Time point
                                     1-01
                                     1-02
                                     1-03
                                     1-04
                                     Mean
                                      SD
Urine
24 h
                                     4.62
                                     6.76
                                     3.95
                                     5.02
                                     5.09
                                      1.2

Subtotal
                                     4.62
                                     6.76
                                     3.95
                                     5.02
                                     5.09
                                      1.2
Feces
24 h
                                     0.73
                                     0.48
                                     2.00
                                     0.55
                                     0.94
                                     0.71

Subtotal
                                     0.73
                                     0.48
                                     2.00
                                     0.55
                                     0.94
                                     0.71
CO2 Trap 1
1 h
                                     2.15
                                     1.68
                                     1.86
                                     1.88
                                     1.89
                                     0.20
CO2 Trap 1
3 h
                                     7.85
                                     6.50
                                     7.12
                                     8.16
                                     7.41
                                     0.75
CO2 Trap 1
6 h
                                     16.95
                                     14.75
                                     16.76
                                     17.93
                                     16.6
                                     1.33
CO2 Trap 1
24 h
                                     10.39
                                     14.15
                                     13.09
                                     12.47
                                     12.52
                                     1.58

SUBTOTAL
                                     37.34
                                     37.07
                                     38.83
                                     40.43
                                     38.42
                                     1.55
CO2 Trap 2
1 h
                                    **0.00
                                    **0.00
                                     *0.01
                                     *0.02
                                     0.01
                                     0.01
CO2 Trap 2
3 h
                                    **0.01
                                     *0.00
                                    **0.00
                                     *0.01
                                     0.01
                                     0.01
CO2 Trap 2
6 h
                                     *0.01
                                     *0.01
                                    **0.01
                                    **0.00
                                     0.01
                                     0.00
CO2 Trap 2
24 h
                                     0.98
                                     2.02
                                     0.69
                                     *0.12
                                     0.95
                                     0.79

Subtotal
                                     1.00
                                     2.03
                                     0.71
                                     0.15
                                     0.98
                                     0.79
Exhaled VOC Trap 1
1 h
                                     21.80
                                     16.59
                                     16.68
                                     18.97
                                     18.51
                                     2.45
Exhaled VOC Trap 1
3 h
                                     15.01
                                     13.56
                                     13.49
                                     16.04
                                     14.53
                                     1.23
Exhaled VOC Trap 1
6 h
                                     7.40
                                     8.98
                                     7.98
                                     7.67
                                     8.01
                                     0.69
Exhaled VOC Trap 1
24 h
                                     0.50
                                     0.88
                                     0.78
                                     0.24
                                     0.60
                                     0.29

SUBTOTAL
                                     44.71
                                     40.01
                                     38.94
                                     42.92
                                     41.65
                                     2.65
Exhaled VOC Trap 2
1 h
                                     *0.00
                                     0.01
                                     *0.00
                                     0.01
                                     0.01
                                     0.01
Exhaled VOC Trap 2
3 h
                                     0.02
                                     0.02
                                     0.01
                                     0.01
                                     0.02
                                     0.01
Exhaled VOC Trap 2
6 h
                                     0.02
                                     N.C.
                                     0.03
                                     0.03
                                     0.02
                                     0.01
Exhaled VOC Trap 2
24 h
                                     0.12
                                     0.51
                                     0.09
                                     0.03
                                     0.19
                                     0.22

Subtotal
                                     0.17
                                     0.54
                                     0.12
                                     0.07
                                     0.23
                                     0.21
Cage Rinse
24 h
                                     0.45
                                     0.43
                                     1.03
                                     0.43
                                     0.59
                                     0.34
NO Tube Contents
24 h
                                     0.37
                                     1.01
                                     2.13
                                     0.18
                                     0.92
                                     0.88
Adipose subcutaneous
24 h
                                     0.01
                                     0.01
                                     0.02
                                     0.00
                                     0.01
                                     0.01
Adip (Abdominal)
24 h
                                     0.01
                                     0.01
                                     0.02
                                     0.01
                                     0.01
                                     0.01
Muscle Gastrocnemius
24 h
                                     0.01
                                     0.01
                                     0.01
                                     0.01
                                     0.01
                                     0.00
Skin (neck)
24 h
                                     0.01
                                     0.02
                                     0.03
                                     0.01